NITROGEN-CONTAINING HETEROCYCLES: THEIR APPLICATION IN TOTAL SYNTHESIS OF BIS- AND TRIS-INODLE NATURAL PRODUCTS AND 20S PROTEASOME MODULATION By Kyra Rose Dvorak A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Chemistry – Master of Science 2024 ABSTRACT Herein, the syntheses of nitrogen-containing heterocycles are applied to the areas of total synthesis and small molecule design for proteasome modulation. Indole alkaloids have been shown to display diverse biological activities. Moreover, it has been reported that the vast majority of biologically active compounds bare one or more nitrogen-containing heterocyclic moieties. Therefore, the synthesis of indole alkaloids baring nitrogen-containing heterocycles are of particular interest. Progress toward the first total synthesis of Tulongicin and its related natural product analogues is described. Particular emphasis is placed on the exploration and optimization of the key imidazoline cyclization and Friedel-Crafts alkylation steps. In addition, the design and synthesis of small molecule acyl Astemizole analogues containing various N-heterocyclic cores is discussed. These acyl Astemizole analogues were explored for their use in proteasome modulation and several active scaffolds were identified. ACKNOWLEDGEMENTS First, I would like to thank my advisor Dr. Jetze Tepe. Thank you for supporting me and allowing me to follow my research passions. Additionally, your patience in teaching me, yet allowing me the freedom to explore my own ideas along the way has helped me build my confidence and provided me the tools to become an independent scientist. Thank you for fostering a great lab environment. I would also like to thank my committee members, Dr. Huang, Dr. Borhan, and Dr. Draths for your guidance, feedback, and willingness to answer all my questions along the way. I would especially like to thank Dr. Draths. I am grateful to have had such a welcoming and encouraging fellow Belle as a mentor. To my labmates, thank you for providing such a fun, supportive, and encouraging work environment. Grace, Konika, and Dare, thank you for guiding me as I started graduate school and always being there along the way whenever I had questions. Thank you to my co-workers and friends Sophie, Allison, Konika, Dare, Charles, Daniel, Evan, Bahareh, Shannon, Sydney, and Miracle. I truly felt as if I was working with my best friends every day and I couldn’t have made it through the tumultuous life of graduate school without you. I especially would like to thank Daniel and Evan. Ever since we found each other during recruitment weekend, we have been through every step of graduate school together and your support, countless discussions, and friendship have meant the world. I would also like to thank my friend, roommate, and fellow graduate student, Elizabeth. I will always be grateful for our countless chats after long days in lab and weekend brunches at Anna’s House. iii Lastly, I would like to thank my family. To my parents and my brother, thank you for your endless love and support. I would not be where I am today without you. You all inspire me every day. To my girlfriend, Hannah, thank you for your love and support. Thank you for always being there to celebrate the wins and bounce back from the losses. I could not have made it through the highs and lows of graduate school without you. iv TABLE OF CONTENTS LIST OF ABBREVIATIONS.........................................................................................................vi Chapter 1: Recent Advances in the Total Synthesis of Bis- and Tris-Indole Alkaloids Containing N-Heterocyclic Linker Moieties1* ...................................................................................................1 REFERENCES.....................................................................................................................77 Chapter 2: Efforts Toward the Total Synthesis of Tulongicin and Its Related Natural Product Analogues ......................................................................................................................................88 REFERENCES ..................................................................................................................175 APPENDIX........................................................................................................................179 Chapter 3: Synthesis of Small Molecule Acyl-Astemizole Analogues as Activators of the 20S Proteasome ..................................................................................................................................216 REFERENCES ..................................................................................................................268 APPENDIX........................................................................................................................270 Chapter 4: Conclusions and Future Works..................................................................................296 v AcOH AIBN Aq AMC Ar BBB BBN BEMP Bn Boc Bop LIST OF ABBREVIATIONS Acetic acid Azobisisobutyronitrile Aqueous 7-amino-4-methylcoumarin Aryl Blood brain barrier Borabicyclo[3.3.1]nonane 1,3,2-Diazaphosphorin-2-amine Benzyl Tert-butyloxycarbonyl Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophospate Bpin Bis(pinacolato)diboron Bu ºC Butyl- Degrees Celsius Casp-L Caspase-like CD CDI CT-L dba DBU DCC DCM Circular dichroism Carbonyldiimidazole Chymotrypsin-like Dibenzylideneacetone 1,8-Diazabicyclo[5.4.0]undec-7-ene N,N’-Dicyclohexylcarbodiimide Dichloromethane vi DDQ DEAD 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone Diethyl azodicarboxylate DIBAL-H Diisobutylaluminum hydride DIPEA DMA DMAP DMF DMP DMSO DPPA dppp EC200 EA Equiv Et IBX IDP IPA iPr LDA N,N-Diisopropylethylamine Dimethylacetamide 4-Dimethylaminopyridine Dimethylformamide Dess-Martin periodinane Dimethylsulfoxide Diphenylphosphoryl azide 1,3-Bis(diphenylphosphino)propane 200% efficacy concentration Ethyl acetate Equivalents Ethyl- 2-iodoxybenzoic acid Intrinsically disordered proteins Isopropyl alcohol Isopropyl- Lithium diisopropylamide LiHMDS Lithium bis(trimethylsilyl)amide mCPBA m-chlorobenzoic acid Me Methyl- vii MeCN Acetonitrile MP MS NBS NCS NMR Ph PIDA Pr rt Sat. Soln. TBAB TBAF TBAI TBS tBu TCDI TFA THF T-L TLC TMG Melting point Mass spectrometry N-bromosuccinimide N-chlorosuccinimide Nuclear magnetic resonance Phenyl- (Diacetoxyiodo)benzene Propyl- Room temperature Saturated Solution Tetrabutylammonium bromide Tetrabutylammonium fluoride Tetrabutylammonium iodide Tert-butyldimethylsilyl chloride Tert-butyl 1,1’-thiocarbonyldiimidazole Trifluoroacetic acid Tetrahydrofuran Trypsin-like Thin layer chromatography 1,1,3,3-Tetramethylguanidine viii TMS Ts VioB Tetramethylsilane Tosyl- Violacein biosynthesis protein ix Chapter 1: Advances in the Total Synthesis of Bis- and Tris-Indole Alkaloids Containing N- Heterocyclic Linker Moieties1* 1.1 Introduction As their name suggests, indole alkaloids are natural products that contain one or more indole structural moieties. They are commonly isolated from a variety of marine sources, including sponges, tunicates, red algae, acorn worms, and symbiotic bacteria, and they represent the largest, and among the most complicated, class of the marine alkaloids.1 Indole alkaloids have been shown to display diverse biological activities, including cytotoxic, antitumor, antiviral, antibacterial, and anti-inflammatory activities.2 The structure, activity, and synthesis of indole alkaloids have been discussed in several reviews over the years.3 The goal of this review is to provide a detailed overview on recent advances in isolation and total synthesis of bis- and tris- indole alkaloid natural products that contain N-heterocyclic linker moieties. These heterocyclic moieties are of particular interest as it has been reported that more than 85% of biologically active compounds contain one or more heterocyclic moieties with N-heterocycles being the most prevalent.4 These heterocyclic moieties are of particular interest as it has been reported that more than 85% of biologically active compounds contain one or more heterocyclic moieties.5 The biological activities of these natural products will also be discussed. 1 * This chapter was reproduced from the recently published review with permission from the Royal Society of Chemistry (RSC), Natural Product Reports. (Dvorak, K.; Tepe, J., Advances in the Synthesis of Bis- and Tris-Indole Alkaloids containing N-Heterocyclic Linker Moieties, Natural Product Reports, 2024, DOI: 10.1039/D4NP0). 1 1.2 Bis-indole alkaloids 1.2.1. Imidazole and imidazoline linker moieties 1.2.1.1. Topsentins and Spongotines Topsentins and Spongotines are classes of marine alkaloid bis-indole natural products that contain characteristic 2-carbonylimidazole or 2-carbonylimidazoline linker moieties between their two indole fragments. The first few natural products that were discovered in these classes were Deoxytopsentin (Topsentin A) (1-1a), Topsentin (Topsentin B1) (1-1b), and Bromotopsentin (Topsentin B2) (1-1c). These three natural products were first isolated from Mediterranean marine sponge Topsentia Genitrix.6 The structures of these natural products were elucidated via spectroscopic methods, as shown in Scheme 1.1, and 1-1a, 1-1b, and 1-1c were identified as weakly cytotoxic for fish and for dissociated cells of the freshwater sponge Ephydatia Fluviatilis.5 After these initial discoveries, the structurally related analogues 1-1d-h and 1-2a-e, were isolated from various marine sponges, including Spongosorites,2,5,7,8 Hexadella,9,10 Discodermia Calyx,11 Rhaphisia Lacezie,12 and Topsentia.5,13 Natural products of the Topsentin and Spongotine classes have been shown to possess cytotoxic, anticancer, antibacterial, antiviral, antifungal and anti-inflammatory activities.3,14 2 Scheme 1.1. Chemical structures of Topsentin and Spongotine natural products (1-1a-h, 1-2a-e, 3) and the synthetic analogue Hydroxytopsentin (1-1i). O N NH N H R3 N H 1-1a-i R1 Deoxytopsentin (Topsentin A) (1-1a): R1=R2=R3=H Topsentin (Topsentin B1) (1-1b): R1=R3=H, R2=OH Bromotopsentin (Topsentin B2) (1-1c): R1=Br, R2=OH, R3=H Isotopsentin (1-1d): R1=OH, R2=R3=H Isobromotopsentin (1-1e): R1=H, R2=Br, R3=OH Bromodeoxytopsentin (1-1f): R1=Br, R2=R3=H Isobromodeoxytopsentin (1-1g): R1=R3=H, R2=Br Dibromodeoxytopsentin (1-1h): R1=R2=Br, R3=H Hydroxytopsentin (1-1i): R1=R2=OH, R3=H R2 R1 O N NR4 N H N H 1-2a-e R2 Spongotine A (1-2a): R1=Br, R2=R3=H Spongotine B (1-2b): R1=R3=H, R2=Br Spongotine C (1-2c): R1=R2=Br, R3=H Topsentin C (1-2d): R1=R2=Br, R3=Me Topsentin D (1-2e): R1=R2=R3=H OH N NH N H Br Br N H Dihydrospongotine C (1-3) In addition to these previously discussed natural products, the most recently isolated natural product in the Spongotine class was Dihydrospongotine C (1-3), which was isolated as a single enantiomer in 2017 from the Topsentia sp. marine sponge. Its structure was elucidated via spectroscopic methods. Both stereocenters were determined to have the S-stereochemistry configurations consistent with experimental and calculated circular dichroism (CD) data (MPW1PW91/6-31G(d,p).13 In addition, 1-3 displayed antibacterial activity toward S. aureus (MIC: 3.7 μg/mL), anti-HIV activity (IC50 (YU2): 3.5 μM; IC50 (HxB2): 4.5 μM), and displayed no evidence of cytotoxic activity toward mammalian cells.13 Dihydrospongotine C (1-3) has yet to be accessed via total synthesis. The first natural product of the Topsentin and Spongotine classes to be accessed via total synthesis was Deoxytopsentin (Topsentin A) (1-1a) by Braekman, J., et. al. in 1988.15 As shown in Scheme 1.2, 3-(bromoacetyl)indole (1-4) was reacted with 1,1-dimethylhydrazine in acidic conditions to afford the amine salt (1-5). Then, 1-5 was refluxed in isopropanol (IPA) to prompt 3 migration of the methyl groups and elimination of dimethylamine to afford the imine intermediate 1-6, which immediately dimerized to afford 1-1a in 27% yield from 1-5.15 Scheme 1.2. The first total synthesis of Deoxytopsentin (1-1a). Br O N H 1-4 NH2NMe2 HBr Me Me N NH2 Br O IPA, heat N H 1-5 O NH H N H 1-6 dimerization 27% (from 1-5) O N NH N H N H Deoxytopsentin (1-1a) Additional progress was then made toward the synthesis of the Topsentin natural products 1-1a, 1-1b, 1-1d, and the natural product analogue 1-1i. The synthetic route began with the synthesis of key indole-3-keto-aldehyde fragments 1-7a and 1-7b via a subsequent oxidative step that was carried out on the indole-3-α-chloro-ketone starting material. The penultimate step of this synthesis was the condensation and cyclization of 1-7a and 1-7b to ideally afford Topsentin (1b), as shown in Scheme 1.3. However, this cyclization proved to be unselective, affording a mixture of 1-1a (26%), 1-8b (9%), 1-8c (10%), and 1-8d (8%). Each of these intermediates were isolated before undergoing quantitative hydrogenolysis to remove the benzyl group, affording 1- 1b, 1-1d, and the natural product analogue 1-1i. The same condensation/cyclization reaction was carried out to dimerize and condense two equivalents of 1-7a, rather than the mixed keto- aldehyde intermediates, and the desired product 1-1a was accessed in 63% yield. This emphasized that the selectivity was the major issue with this approach.16 Over the years, additional dimerization-cyclization approaches toward the total synthesis of 1-1a have also been completed, such as the dimerization of an indolic α-amino-ketone fragment by Miyake et. al.17 4 Scheme 1.3. Total synthesis of Topsentins 1-1a, 1-1b, 1-1d, and the natural product analogue 1- 1i. O O H O O H N H 1-7a + BnO N H 1-7b NH3, EtOH/H2O (3:1), rt to reflux, 30 min O O H N H 1-7a NH3, EtOH/H2O (3:1), rt to reflux, 30 min 63% O N NH N H R2 R1 N H Topsentin (1-1b): R1=R2=OH (quant) Hydroxytopsentin (1-1i): R1=H, R2=OH (quant) Isotopsentin (1-1d): R1=OH, R2=H (quant) H2, Pd/C (10 wt%), EtOH, rt, 4 h Topsentin A (1-1a): R1=R2=H (26%) 1-8b: R1=R2=OBn (9%) 1-8c: R1=H, R2=OBn (10%) 1-8d: R1=OBn, R2=H (8%) Considering the selectivity issues of these dimerization-like approaches toward the Topsentin natural products, additional synthetic approaches toward these scaffolds were developed to improve this selectivity, such as the use of lithiation and subsequent cross coupling reactions. For example, in this approach, the imidazole core was the starting point, and the indole-3-aldehyde and indole moieties were subsequently added to the imidazole ring (Scheme 1.4).18,19 There were two lithiation and subsequent nucleophilic addition approaches to access the key imidazoline intermediate (1-13). In the first synthetic strategy, di-iodo-imidazole (1-9) was first lithiated with nBuLi, which subsequently underwent nucleophilic addition to indole-3- aldehydes (1-10) to access 1-11 in high yields. In the second approach, the electronics of the reaction were reversed in which the 3-iodo-indoles (1-11) underwent lithium-halogen exchange and were subsequently added to the imidazole-2-aldehydes (1-12) to afford the key intermediate 1-13 in good yield. To access the Topsentin natural products 1-1a, 1-1b, 1-1c, and 1-1f, the 5 alcohol 1-13 was oxidized to the ketone 1-14 using MnO2 and coupled with the tributyltin indole 1-15 via the Stille method in high yield. After removal of protecting groups, the desired products 1-1a, 1-1b, 1-1c, and 1-1f were isolated in 57-92% yield.18,19 This approach proved to be a much more efficient and highly selective approach toward installing two indole fragments on the imidazole core that bear different substituents. Scheme 1.4. Lithiation and cross-coupling synthetic approaches toward 1-1a, 1-1b, 1-1c, and 1- 1f. I I N N PG BuLi, THF, -78 ºC, 45 min then, O H PG=BOM 1-9 R2 N SO2Ph 1-10 R2=H, OBn 74-85% HO I N N PG R2 N SO2Ph 1-13 MnO2, DCM, rt, 3h 87-98% O I N N PG R2 N SO2Ph 1-14 I R2 O 1-11 N SO2Ph R2=H, OMOM PG=BOM, SEM 1. BuLi, THF, -78ºC I 2. N N PG 1-12 44-64% SnBu3 R1 N SO2Ph 1-15 R1=H, Br PdCl2(PPh3)2 (8-10 mol%), CuI (14-24 mol%), DMF, 120ºC, 2 h 51-79% Deoxytopsentin (1-1a) Topsentin (1-1b) Bromotopsentin (1-1c) Bromodeoxytopsentin (1-1f) Protecting group removal 57-92% R1 PG N O N 1-16 N SO2Ph R2 N SO2Ph Considering the efficiency of cross-coupling, additional cross-coupling approaches toward these natural products were completed. For example, Kawasaki et al. coupled a borylated 6 indole 1-17 with N-SEM-di-iodoimidazole 1-18 and subsequently de-iodinated to afford the imidazole intermediate 1-19 in high yields (Scheme 1.5). A late-stage lithiation of 1-19 was then carried out and subsequently reacted with the indole-3-amide intermediate 1-20. Lastly, the protecting groups were removed using BBr3, to render Topsentin (1-1b) in 69% yield.20 Scheme 1.5. Cross-coupling and late-stage lithiation approach to Topsentin (1-1b). B(OH)2 I + I N TBS 1-17 1. Pd(PPh3)4, Na2CO3, benzene, MeOH, H2O 75% 2. EtMgBr, THF, then H2O 84% N N SEM 1-18 1. BuLi, THF, -78ºC 2. O OMe N Me SEM MeO N 1-20 TBS N N N TBS 1-19 3. HCl (20%) 48% (over 3 steps) Topsentin (1-1b) BBr3 69% O N NH N H N H 1-21 OMe The newer isolated Spongotine natural products were accessed via total synthesis more recently. The first total syntheses of several Spongotine and Topsentin natural products were achieved via a common key cyclization approach toward the imidazoline core, in which a keto- thioimidate (1-25a-c) fragment and diamine (1-29a-b) fragment were cyclized.21,22 Intermediates 1-25a-c were synthesized in four steps from commercially available indole starting materials (1- 22a-c) in good yields (Scheme 1.6). There were two different approaches for accessing the indolic diamine fragments (1-29a and 1-29b). The first approach was used to synthesize the indolic diamine (1-29a). In this approach, an indolic hydroxylamine intermediate (1-26a) underwent reductions and hydrogenolysis via hydrogen and Pearlman’s catalyst to access the 7 debenzylated intermediate in 90% yield. Then, the Boc protecting group was quantitatively removed under acidic conditions to access 1-29a.23 The second approach was a little more step- intensive to access the indolic diamine intermediate 1-29b as it could not be synthesized via the hydrogenative method of 1-29a due to problematic dehydrohalogenation of indoles under hydrogenative conditions.23 In order to avoid hydrogenation conditions, the 6-bromo-indolic hydroxylamine intermediate 1-26b was oxidized to 1-27 via MnO2 and subsequently reacted with hydroxylamine hydrochloride to remove the benzyl group and reduced via TiCl3 to achieve 1-28 in high yields. From there, 1-28 was subjected to acidic conditions to remove the Boc group and access 1-29b in 99% yield.22 The desired natural products were then accessed via the key base-catalyzed imidazoline cyclization between 1-25a-c and 1-29a-b. This cyclization was carried out with either Et3N or Amberlyst A21 resin to access the desired natural products (1-2a (74%), 1-2b (46%), 1-2c (65%), 1-2e (72%)) and the natural product analogue (1-2f (63%)) in good yields. Then, the imidazoline cores of 1-2a-c, 1-2e, and 1-2f were oxidized via IBX to the imidazole of Topsentins 1-1f (78%), 1-1g (34%), 1-1h (90%), 1-1a (91%), and Topsentin analogue 1-1j (quant). This was the first total synthesis of Spongotine A-C (1-2a-c), Bromodeoxytopsentin (1-1f), Isobromodeoxytopsentin (1-1g), and Dibromodeoxytopsentin (1- 1h).21,22 The natural product analogue 1-1j was also synthesized and could be de-methylated in the future with BBr3 to access Hydroxytopsentin (Scheme 1.6). 8 Scheme 1.6. Total synthesis of 1-1a, 1-1f-h, 1-2a-c, and 1-2e. O Cl O N H (COCl)2 Et2O, 0ºC, 4 h R2 1. n-Bu3SnH, EtOAc, 0ºC to rt, 18 h 2. S8, piperidine, pyridine, 80ºC, 5 h 1-23a: R2=H: 92% 1-23b: R2=Br: 82% 1-23c: R2=OMe: 71% R2 N H 1-22a: R2=H 1-22b: R2=Br 1-22c: R2=OMe S N O N H R2 1-24a: R2=H: 30% 1-24b: R2=Br: 31% 1-24c: R2=OMe: 33% R2 MeI reflux, 5 h I S N O N H 1-25a: R2=H: 98% 1-25b: R2=Br: quant 1-25c: R2=OMe: quant Ph HO N NHBoc R1 N H 1-26a: R1=H 1-26b: R1=Br 1. H2, Pd(OH)2, MeOH/AcOH (96:4), rt, 50 h 90% 2. HCl, MeOH, rt, 1 h quant MnO2, toluene, 100ºC, 10 min 77% Br Ph N O 1-27 N H N 1. NH2OH•HCl, MeOH, rt, 1.5 h 72% 2. TiCl3, HCl, MeOH, rt, 10 min 92% NHBoc O HCl•H2N NH2•HCl R1 N H 1-29a: R1=H 1-29b: R1=Br HCl, MeOH, rt, 10 min 92% HCl•H2N NHBoc Br N H 1-28 IBX DMSO, rt, 15 h Bromodeoxytopsentin (1-1f): R1=Br; R2=H (78%) Isobromodeoxytopsentin (1-1g): R1=H; R2=Br (34%) Dibromodeoxytopsentin (1-1h): R1=Br; R2=Br (90%) Deoxytopsentin (1-1a): R1=H; R2=H (91%) 1-1j: R1=H; R2=OMe (quant) 1-25a, 1-25b, or 1-25c, Amberlyst A 21 R1 or Et3N, 1-29a or 1-29b MeOH, rt, 48 h NH R2 N H N H Spongotine A (1-2a): R1=Br; R2=H (74%) Spongotine B (1-2b): R1=H; R2=Br (46%) Spongotine C (1-2c): R1=Br; R2=Br (65%) Topsentin D (1-2e): R1=H; R2=H (72%) 1-2f: R1=H; R2=OMe (63%) 9 The first enantioselective total synthesis of Spongotine A (1-2a) was achieved via a key imidazoline cyclization between a keto-aldehyde fragment (1-34) and an optically active diamine fragment (1-33), as shown in Scheme 1.7.24 First, 1-34 was synthesized in three steps from 1H- indole, via a keto-acyl chloride intermediate, according to literature procedures.25 Fragment 1-33 was synthesized via Sharpless dihydroxylation of a 3-vinyl indole intermediate (1-30) to access (R)-1-31 in high yield and enantioselectivity. Then, (R)-1-31 underwent a stereospecific Mitsunobu reaction to access the diazide intermediate (S)-1-32 in 95% yield, in which the (S)- stereochemistry was set via inversion of the chiral center. The diazide (S)-1-32 was then reduced to access the optically active indolic diamine intermediate (S)-1-33 in high yields with 98% ee. To synthesize the imidazoline core and achieve Spongotine A (1-2a), (S)-1-33 and 1-34 underwent condensation, cyclization, and oxidation to the imidazoline via NCS. Subsequent removal of the tosyl protecting group with base achieved Spongotine A (1-2a) in 51% yield. Through these final steps, the stereochemistry was retained, allowing for the first enantioselective total synthesis of Spongotine A (1-2a).24 In addition, the specific optical rotation of the synthesized (S)-Spongotine A (1-2a) matched that of the natural Spongotine A (1-2a), allowing for the establishment of its previously unknown absolute configuration as (S)- Spongotine A. 10 Scheme 1.7. The first enantioselective total synthesis of (S)-Spongotine A (1-2a). AD-mix-β K2OsO4•2H2O (DHQD)2 PHAL OH HO N3 N3 Br N Ts 1-30 t-BuOH/H2O (1:1) rt, 12 h 99%, 98%ee Br N Ts 1-31 Ph3P, DEAD, DPPA, toluene, 0ºC to rt, 18 h 92% O O H Br N Ts 1-32 Ph3P, H2O, toluene, reflux, 18 h 89% H2N NH2 Br N Ts 1-33 N Ts 1-34 then NCS, MeCN, 0ºC to rt, 18 h then, 2N NaOH, MeOH, reflux, 15 min 51% O N NH N H Br N H Spongotine A (1-2a) 1.2.1.2. Nortopsentins The Nortopsentin class of bis-indole natural products are structurally similar to those of the Topsentin class, however, they lack a carbonyl moiety on the imidazole linker moiety, as shown in Scheme 1.8, Nortopsentin A (1-35a), B (1-35b), and C (1-35c) were first isolated from Spongosorites ruetzleri in 1987.26 Nortopsentins A-C (1-35a-c) were found to possess cytotoxic and antifungal activities. Interestingly, the methylated derivatives of 1-35a-c also displayed enhanced cytotoxic activity in P388 cells compared to the isolated natural products.26 In addition, the unnatural synthetic Nortopsentin analogue 1-35d, unfortunately also referred to as Nortopsentin D in several early literature reports, was accessed via hydrogenation of 1-35a-c, as indole readily undergo de-halogenation under hydrogenation conditions.27 Several years later, in 1996, the more complex structural variant of this class, Nortopsentin D (1-36), composed of a tri- substituted imidazolinone ((4H)-imidazol-4-one) core, was first isolated from the axinellid 11 sponge, Dragmacidon sp.28 Later, 1-36 was also isolated from the sponge Agelas dendromorpha.29 It is interesting to mention that the methylated derivative of 1-36 was also shown to have antifungal activity against yeast and high cytotoxicity toward tumoral cells.28,30 Scheme 1.8. Chemical structures of Nortopsentins A-D (1-35a-c, 1-36) and Nortopsentin synthetic analogue (1-35d). R1 HN R2 N N H NH Nortopsentin A (1-35a): R1=R2=Br Nortopsentin B (1-35b): R1=H, R2=Br Nortopsentin C (1-35c): R1=Br, R2=H Nortopsentin Synthetic Analogue (1-35d): R1=R2=H Br HN O NH N HN N NH2 Br N H Nortopsentin D (1-36) The first total syntheses of Nortopsentins A-C (1-35a-c), as well as the synthetic analogue 1-35d, were achieved via a successive Pd-catalyzed cross-couplings of indole fragments to the imidazole core, as shown in Scheme 1.9.30 The synthetic analogue 1-35d was the first of these to be accessed via successive cross coupling reactions of a tri-brominated imidazole (1-38) with an N-TBS-protected indole-3-boronic acid (1-37a), using Pd(PPh3)4 as a catalyst. The protecting groups and remaining bromide were then removed to access 1-35d in high yields. During route development, SEM- and MOM-protecting groups were also explored for 1-37a, yet the TBS- protecting group resulted in the highest yield of the coupling reaction.30 More recently, an adapted method for palladium-catalyzed cross coupling of unprotected imidazoles was developed and utilized in the synthesis of 1-35d, as shown in Scheme 1.9.31 This Suzuki-Miyaura cross- coupling method allowed for a significantly expedited approach toward 1-35d, as compared to previous reports. However, this method is likely only efficient for natural products bearing 12 symmetrical indole moieties. In a similar manner, the first total syntheses of Nortopsentin A (1- 35a) and C (1-35c) were achieved, as shown in Scheme 1.9. The efficient synthesis of 1-35c was very similar to that of 1-35d. Iodide was utilized as a coupling partner here, rather than bromide. Iodide was likely used here to prevent potential selectivity issues given the bromide substituents on the boronic acid coupling partners. The other major difference was the removal of the additional iodide before the second indole coupling (Scheme 1.9, synthesis of 1-35c). It is noteworthy to mention that the selective de-iodination to access intermediate 1-43 was confirmed via a NOESY correlation between the SEM group on the nitrogen and the C5-H of the imidazole.30 The approach toward the first total synthesis of Nortopsentin A (1-35a) and Nortopsentin B (1-35b), which also bear bromide substituents on their indoles, implemented a later stage iodination approach in the synthesis of 1-46 before the second cross coupling reaction to access 1-35a and 1-35b in moderate and good yield, respectively (Scheme 1.9).30 13 Scheme 1.9. The first total synthesis of Nortopsentin A-C (1-35a-c) and the Nortopsentin synthetic analogue (1-35d). I N I I N SEM I N I N SEM 1-41 N 1-42 TBS Pd(PPh3)4, Na2CO3 45% B(OH)2 R N PG PG = SEM, Boc N I N SEM 1-44 Pd(PPh3)4, Na2CO3 58% R N N SEM N TBS N 1-37a: R= H 1-37b: R= Br Br Br N H 1-38 Pd(OAc)2 (2.5 mol%), A-taPhos (6 mol%), Br Br N Br N SEM 1-38 Br N 1-45a (R=H) 1-45b (R=Br) Br N SEM NIS (PhICO2)2 K2CO3, dioxane/H2O, 125ºC 68% Pd(PPh3)4, Na2CO3 89% N TBS 39 N I N SEM 37a, Pd(PPh3)4, Na2CO3 68% N N SEM 40 N TBS 1. t-BuLi 2. H2O 3. HCl (20%) 70% 1. 1-37b, Pd(PPh3)4, Na2CO3 2. HCl (20%) 46% R N TBS 1-46a (R=H): 65% 1-46b (R=Br): 43% 1. 1-37b, Pd(PPh3)4, Na2CO3 2. TBAF 3. HCl (20%) 11% 1. EtMgBr 2. H2O 74% 1-35d I N N SEM Br N TBS 43 N TBS 1. 1-37b, Pd(PPh3)4, Na2CO3 2. TBAF 3. HCl (20%) 50% Nortopsentin C (1-35c) Nortopsentin Synthetic Analogue (1-35d) Nortopsentin B (1-35b) Nortopsentin A (1-35a) In addition to these cross-coupling approaches, a different non-cross-coupling approach was used to access the Nortopsentin natural products 1-35b and 1-35d. This method utilized the condensation of a nitrile with an α-amino-ketone fragment and subsequent cyclization and aromatization of the imidazole at high temperatures to achieve Nortopsentin B (1-35b) and the Nortopsentin D synthetic analogue 1-35d in good yields (Scheme 1.10).17 This method was not viable for the total synthesis of Nortopsentins A (1-35a) and C (1-35c). This was likely due to the necessary hydrogenation step for the synthesis of intermediate 1-48, which would not tolerate a bromide substituent on the indole (1-48), as has been previously discussed. 14 Scheme 1.10. Synthesis of Nortopsentin B (1-35b) and its synthetic analogue (1-35d) via thermal cyclization. O NH2 CN N H N H 1-48 190-210ºC R2 1-47a: R2 = H 1-47b: R2 = Br Nortopsentin B (1-35b): 60% Nortopsentin Synthetic Analogue (1-35d): 65% The first total synthesis of the more structurally complex Nortopsentin D (1-36) was completed by K. Keel, et. al., in 2021, as shown in Scheme 1.11.32 The complex tri-substituted imidazolinone ((4H)-imidazol-4-one) core was constructed via a late-stage Pinacol-like rearrangement. Two key fragments were utilized in this approach; an alkyne fragment (1-49) and amidine fragment (1-51). The alkyne (1-49) and amidine (1-51) were accessed in two and five steps, respectively from the commercially available 6-bromo-1H-indole.32 The alkyne fragment (1-49) was oxidized to the di-ketone intermediate (1-50) via mercuric nitrate monohydrate. The key condensation-cyclization and subsequent Pinacol-like rearrangement steps were then carried out to form the tri-substituted imidazolinone core of Nortopsentin D (1-36), accessing 1-52 in 52% yield. The N-tosyl and N-boc protecting groups were deprotected during this reaction. Precise deprotection was key for optimization, as successful cyclization was dependent upon electronics. It was found that the N-boc protecting group of 1-51 had to first be deprotected to allow for sufficient nucleophilicity of the amidine (1-51) to condense and cyclize with the di- ketone (1-50). The presence of the N-tosyl protecting group of 1-50 was also found to contribute to increased electrophilicity of the ketone. Therefore, initial acidic conditions were implemented to remove the N-boc group of 1-51, followed by the strategic use of a mildly nucleophilic base 15 and solvent to allow for cyclization to occur prior to de-tosylation of 1-50 (Scheme 1.11). After removal of the remaining protecting group to access the 2-amino-imidazole substituent, Nortopsentin D (1-36) was accessed in 70% yield.32 Scheme 1.11. First total synthesis of Nortopsentin D (1-36). HCl•HN NH2 Br Hg(NO3)2•H2O N Ts DMF, rt 49% Br N O N N O 1-50 N Ts N N N 1-49 Br HN O NH N H N NH2 N H2NNH2•H2O EtOH, rt, 30 h 70% N Boc Br 1-51 1. TFA, DCM/H2O, rt, 16 h 2. KHCO3, NaOH, IPA, reflux, 4 h 52% Br HN O NH N N N N Br N H Nortopsentin D (1-36) Br N H 1-52 1.2.2. Imidazolinone linker moieties 1.2.2.1. Rhopaladins Natural products of the Rhopaladin class are bis-indole alkaloids comprised of two indole fragments connected by an imidazolinone ((4H)-imidazol-4-one) linker moiety, similar to Nortopsentin D (1-36). In addition, the indolylcarbonyl substitution at the C-2 position of the imidazolinone core is similar to the Spongotine and Topsentin natural product classes (Scheme 1.12). However, unlike any of the previously discussed natural product classes, the second indole moiety is connected at the 5-position of the imidazolinone core by a unique vinyl chain. 16 Rhopaladins A-D (1-53a-d) were first isolated in 1998 by Sato et. al. from the marine tunicate Rhopalaea sp.33 The geometry of the alkene present in these natural products (1-53a-d) was identified as (Z) by a NOESY experiment that was run on Rhopaladin C (1-53d). The studies identified various NOESY correlations, such as a NOESY correlation between the two C-2 hydrogens of the indole moieties, indicating a (Z) geometry of the alkene. In terms of biological activities, these natural products were reported to demonstrate antibacterial activity against Sarcina lutea and Corynebacterium xerosis and inhibitory activity against cyclin dependent kinase 4 and c-erbB-2 kinase. However, their broader biological activity has yet to be explored.33 Scheme 1.12. Chemical structure of Rhopaladins A-D (1-53a-d). O NH O N R2 R1 NH HN Rhopaladin A (1-53a): R1=OH, R2=Br Rhopaladin B (1-53b): R1=OH, R2=H Rhopaladin C (1-53c): R1=H, R2=Br Rhopaladin D (1-53d): R1=H, R2=H The first total synthesis of Rhopaladin D (1-53d) was reported in 2000, in which a key intermolecular aza-Wittig reaction was utilized, followed by subsequent condensation and cyclization to form the imidazolinone core of 1-53d.34 To perform the aza- Wittig reaction, the azide intermediate (1-56) was first accessed in three steps in high yields from indolyl-3-aldehyde 1-54 (Scheme 1.13). Then, the aza-Wittig reaction was performed on azide intermediate 1-56 to access intermediate 1-57, which was immediately condensed and cyclized with keto-acyl chloride 1-23a to access imidazolinone 1-58 in 56% yield over two steps. Lastly, the protecting group was removed to afford Rhopaladin D (1-53d) in 60% yield. However, 1-58 was isolated as a 6:4 mixture of E/Z isomers and after chromatographic separation, the (Z) isomer isomerized to 17 the (E) isomer upon sunlight irradiation. Therefore, after the last step, Rhopaladin D (1-53d) was also isolated as a mixture of E/Z isomers.34 Scheme 1.13. First total synthesis of Rhopaladin D (53d). O H N SEM 1-54 N3CH2COOEt, NaOEt, EtOH, -15ºC 80% CO2Et N3 1. LiOH, THF, H2O 90% 2. CDI, NH3, DMF 92% N SEM 1-55 CO2NH2 N3 N SEM 1-56 PPh2Me, THF O NH O N N SEM HN 1-58 23a, polymer-bound BEMP, THF 56% CO2NH2 N PPh2Me N SEM 1-57 TBAF, THF, reflux 60% O NH O N NH HN Rhopaladin D (1-53d) A couple years later, the total syntheses of all four Rhopaladins A-D (1-53a-d) were achieved via a key imidate-based cyclization with tryptophan esters to form the imidazolinone core of these natural products, as shown in Scheme 1.14.35 First, the carbonyl nitrile intermediate (1-60a-b) was synthesized via a TMS-cyanohydrin intermediate from aldehyde 1-59a-b, followed by subsequent oxidation to the carbonyl nitrile 1-60a-b in high yields. Then, 1-60a-b underwent a Pinner reaction with gaseous hydrochloric acid and ethanol to form the imidate intermediate (1-61a-b). Compounds 1-61a-b were immediately condensed and cyclized with tryptophan methyl ester hydrochloride (1-62a) in the presence of triethylamine to access Rhopaladin C (1-53b) and D (1-53d) in 38% and 35% yield, respectively. Interestingly, the 18 dehydrogenation to form the alkene of the Rhopaladins occurs spontaneously after the cyclization has occurred. This is likely due to the highly conjugated nature of the Rhopaladins, contributing to high stability. Interestingly, this cyclization and spontaneous dehydrogenation specifically produced (Z)-isomers of the Rhopaladins, as was confirmed via NOESY correlations, including a NOESY correlation between the C2-H’s of the indole moieties. The presence of the (E)-isomers could not be detected.35 Thus, this method was proven to be very selective to the (Z)-isomer, which is a major advantage compared to the previous synthesis of Rhopaladin D (1-53d). Rhopaladins A and B (1-53a and 1-53b) were synthesized in the same manner as Rhopaladin C and D (1-53c and 1-53d), as shown in Scheme 1.14. The only difference was the identity of the tryptophan methyl ester (1-62b). In addition, after the key cyclization reaction and spontaneous dehydrogenation to access 1-63a and 1-63b, an additional de-methylation step via BBr3 was required to access Rhopaladins A (1-53a) and B (1-53b) in 81% and 62% yields, respectively. In addition, similarly to 1-53c and 1-53d, the Rhopaladins A (1-53a) and B (1-53b) were confirmed with NOESY as the desired (Z)-isomer.35 19 Scheme 1.14. Total synthesis of Rhopaladins A-D (1-53a-d). R2 O H N H 1-59a: R2=H 1-59b: R2=Br 1. TMSCN, DME or MeCN, reflux, 1.5-3 h 67-88% 2. DDQ, dioxane, rt, 0.5-8 h 91-97% O CN R2 HCl(g), EtOH, R2 NH•HCl O OEt Et2O, 0ºC, 8 h N H 1-60a: R2=H 1-60b: R2=Br N H 1-61a: R2=H: 74% 1-61b: R2=Br: 59% BBr3 DCM, 4.5 days R1 O NH O N R1 R2 NH HN Rhopaladin D (1-53d): 35% Rhopaladin C (1-53c): 38% 1-63a (R1=OMe, R2=H) 1-63b (R1=OMe, R2=Br) NH2•HCl OMe O N H 1-62a: R1=H 1-62b: R1=OMe Et3N, dioxane, 60ºC, 72 h Rhopaladin A (53a)(81%) Rhopaladin B (53b)(62%) 1.2.2.2. Spongosoritins Recently, the first of the Spongosoritins were isolated, which marks another natural product class to contain an imidazolinone (2,3- dihydro-4H-imidazol-4-one) core. Spongosoritins A-D (1-64a-d) were first isolated in 2021 from Spongosorites sp. by Park, et. al.36 As shown in Scheme 1.15, the chemical structure of the Spongosoritins contains one indole moiety and one indolyl-3-ketone moiety connected by a 2-methoxy-1-imidazole-5-one linker. These chemical structures were elucidated via spectroscopic methods. In addition, these Spongosoritins (1-64a- d) contain one stereocenter at the C-2 position of the imidazolinone. The absolute configuration of this position was determined via a density functional theory (DFT)-based computational method and electronic circular dichroism (ECD). Comparing measured data with the calculated data, the stereochemistry was assigned as a (2R) configuration.36 20 Scheme 1.15. Chemical structures of Spongosoritins A-D (1-64a-d). O H N O R2 R1 N O NH N H Spongosoritin A (1-64a): (R1=H, R2=OH) Spongosoritin B (1-64b): (R1=H, R2=H) Spongosoritin C (1-64c): (R1=Br, R2=H) Spongosoritin D (1-64d): (R1=Br, R2=OH) Initial biological activity exploration of the Spongosoritins (1-64a-d) was conducted and it was found that they exhibited moderate inhibition against transpeptidase sortase A and weak inhibition against human pathogenic bacteria and A549 and K562 cancer cell lines. The biological activity findings are detailed in Table 1.1, including the known antibiotic, ampicillin, for comparison.36 Though some information is now known, much of the Spongosoritins’ biological activities remain elusive. Furthermore, Spongosoritins A-D (1-64a-d) have yet to be accessed via total synthesis. Table 1.1. Biological activity of Spongosoritins A-D (1-64a-d).36 Gram (+) MIC (μg/mL) Gram (-) MIC (μg/mL) # 1-64a 1-64b 1-64c 1-64d ampicillin S. aureus >128 64 32 16 0.13 Enterococcus faecalis >128 >128 128 120 0.5 Enterococcus faecium >128 >128 >128 120 1 Klebsiella pneumonia >128 128 >128 >128 --- Salmonella enterica >128 128 64 64 0.25 E. coli >128 >329.8 >128 >128 >128 >274.7 62.7 43.9 8 --- 77.3 55.7 61.2 70.9 --- 24.2 28.2 37.7 54.2 --- IC50 (μM) Srt A A549 K562 1.2.2.3. Violaceins Natural products of the Violacein class are purple-blue pigments produced from bacteria, unlike many other bis-indole natural products which come from marine sponges and tunicates. Violacein (1-65a) was first discovered in 1882 by Boisbaudran, et. al.37 Natural products of the Violacein class (1-65a-g) have been isolated from several Gram-negative bacteria, including 21 Chromobacterium violaceum, Janthionobacterium lividum, Pseudoalteromonas luteoviolacea, Psudomonas sp., Collimonas sp., Dunganella. etc.38,39,40,41,42 As shown in Scheme 1.16, the structure of the Violacein natural products consist of three main sub-classes, the Violaceins (1- 65a-c), whose two indole moieties are connected by a 1,3-dihydro-2H-pyrrol-2-one core linker, the Protoviolaceinic Acids (1-65d-e), whose two indole moieties are connected by a pyrrole-2- carboxylic acid linker, and the Proviolaceins (1-65f-g), which contain a 2H-pyrrol-2-one core. These various sub-classes are produced from the same biosynthetic pathway, either as related paths or as intermediates toward one another.43 In addition, these natural products exhibit significant biological activity, such as antibacterial, antifungal, anticancer, antiviral, and antiparasitic activity.44 Scheme 1.16. Chemical structures of Violacein (1-65a) and related natural product analogues (1- 65b-g). R1 HN R2 R1 R2 R1 R2 O HN CO2H HN O N NH O Violacein (1-65a) (R1=OH, R2=H) Deoxyviolacein (1-65b) (R1=H, R2=H) Oxyviolacein (1-65c) (R1=OH, R2=OH) HN NH HN NH Protoviolaceinic Acid (1-65d) (R1=OH, R2=H) Protodeoxyviolaceinic Acid (1-65e) (R1=H, R2=H) Proviolacein (1-65f) (R1=OH, R2=H) Prodeoxyviolacein (1-65g) (R1=H, R2=H) Natural products of the Violacein class (1-65a-g) have been most widely accessed via exploration of biosynthetic pathways.43 However, these biosynthetic pathways fall outside the scope of this review. Progress has also been made toward the total synthesis of these compounds. The first total synthesis of Violacein and Deoxyviolacein was achieved by Ballantine, et. al.45 via a key reaction of the lactone intermediates 1-66a or 1-66b with ammonia under heating to replace the oxygen atom of the lactone with a nitrogen atom to afford the desired 1,3-dihydro- 2H-pyrrol-2-one core of Deoxyviolacein (1-65b) and imidazolinone intermediate (1-67) in good 22 yields, as is shown in Scheme 1.17. Violacein was then accessed after an additional de- methylation step of 1-67 to complete the first total synthesis of Violacein (1-65a).45 Scheme 1.17. Total synthesis of Violacein (1-65a) and Deoxyviolacein (1-65b). NH O NH3, EtOH, reflux, 4 h R1 O HN N H NH O R1 O O N O 1-66a (R1=H) 1-66b (R1=OMe) Deoxyviolacien (1-65b) 1-67 (R1=OMe) HBr, AcOH (5%), 3 h, then, sat. aq. HCO3 - Violacein (1-65a) Violacein and Deoxyviolacein were also accessed via total synthesis in 2001, in which Steglich, et. al. implemented a strategy where the two indole groups were attached stepwise to an already formed pyrrolinone core, as shown in Scheme 1.18.46 First, pyrrolinone 1-68 underwent an acid-catalyzed reaction with 1-22a or 1-22d, and subsequent protection of both the nitrogens via Boc-anhydride, to access intermediates 1-69a-b in high yields. Then, 1-69a-b were converted to their corresponding enolates using LiHMDS as the base, followed by subsequent reaction with N-Boc-isatin (1-70) to afford the aldol condensation intermediates (1-71a-b). Then, 1-71a-b were immediately heated to afford Deoxyviolacein (1-65b) and intermediate 1-72 in 30% and 24%, respectively. It is noteworthy to mention that upon adsorption to silica gel and heating for 15-20 minutes, dehydration, dehydrogenation, and cleavage of the Boc-protecting groups all occurred simultaneously. Lastly, 1-72 underwent debenzylation to access Violacein (1-65a) in 82% yield. The configurations of 1-65a-b were confirmed as (E)-isomers via NMR.46 This 23 approach was advantageous as it was significantly higher yielding than the first reported total synthesis of these natural products (1-65a-b). Scheme 1.18. Total synthesis of Violacein (1-65a) and Deoxyviolacein (1-65b). O HN 1-68 1. 1-22a or 1-22d, HCl (cat.), THF, reflux 80-85% R1 O BocN 2. Boc2O, DMAP, THF, 0ºC 83-85% N Boc 1-69a (R1=H) 1-69b (R1=OBn) 1. LiHMDS, THF, -78ºC 2. O O O N Boc 1-70 BocN R1 NBoc HO O -78ºC to rt, then H2O N Boc 1-71a (R1=H) 1-71b (R1=OBn) R1 O HN N H NH SiO2, O 180ºC-210ºC Deoxyviolacien (1-65b) (R1=H): 30% 1-72 (R1=OBn): 24% Violacein (1-65a) Pd/C, cyclohexa-1,4-diene EtOH 82% A more recent approach used a tandem ring-closing metathesis (RCM)/ isomerization/ nucleophilic addition sequence toward the total synthesis of Violacein, as shown in Scheme 1.19.47 This tandem RCM/ isomerization/ nucleophilic addition was the first step used in the total synthesis, followed by elimination to intermediate 1-74 in good yields. The oxindole moiety was then attached via a Ti-catalyzed tautomerization/aldol condensation with isatin to access 1-75 in high yield. After acid-mediated removal of protecting groups, Violacein (1-65a) was achieved in 49% yield. The authors mentioned that the final product 1-65a was very difficult to handle and had very minimal solubility in common solvents. Therefore, it was suspected that the crude yield was significantly higher than the isolated yield.47 24 Scheme 1.19. Total synthesis of Violacein (1-65a) via a tandem RCM/isomerization/nucleophilic addition sequence. O PMBN 1. (o-Tolyl)-Hoveyda-Grubbs 2nd generation (5 mol%) (5-OBn)-indole, THF, reflux, 13 h 70% BnO O PMBN 2. LDA, PhSeBr THF, -78ºC, 1 h, then, H2O2 (35%), DCM, rt, 0.5 h 44% 1-73 N H 1-74 HO O N H O BnO O PMBN Ti(Oi-Pr)4, Toluene, reflux, 0.5 h 88% N H 1-75 NH O O HN NH TFA O reflux, 47 h 49% N H Violacein (1-65a) 1.2.3. Pyrrole and pyrrole-dione linker moieties 1.2.3.1. Arcyriarubins Arcyriarubins A-C (1-76a-c) are bisindolylmaleimides first isolated by Steglich, et. al. in 1980 from the red sporangia of the slime mold Arcyria denudate.48 Arcyriarubins B (1-76b) and C (1-76c) were also isolated from Tubifera casparyi in 2003 by Ishibashi, et. al.49 The chemical structure (Scheme 1.20) of these compounds was elucidated by spectroscopic means. Arcyriarubins A-C (1-76a-c) contain two indole moieties linked by a pyrrole-2,5-dione (maleimide) core. These natural products display moderate antibiotic, antifungal, cytotoxic, and kinase inhibition activities.48,49 These natural products are also of great interest, as many structurally related bisindolylmaleimides are selective protein kinase C (PKC) inhibitors, which show promise as novel potential therapeutics for autoimmune diseases.50,51 25 Scheme 1.20. Chemical structures of Arcyriarubins A-C (1-76a-c) and Dihydroarcyriarubins A-B (1-77a-b). H N O R1 N H O N H R2 HO Arcyriarubin A (1-76a): R1=R2=H Arcyriarubin B (1-76b): R1=OH, R2=H Arcyriarubin C (1-76c): R1=R2=OH H N O N H O N H Dihydroarcyriarubin B (1-77a): R2=H Dihydroarcyriarubin C (1-77b): R2=OH R2 In addition, two structurally related natural products that have also been isolated include Dihydroarcyriarubins B (1-77a) and C (1-77b). As shown in Scheme 1.20, these natural products are incredibly similar to the Arcyriarubins (1-76a-c), however, the reduced pyrrolidine-2,5- dione (succinimide) core of the Dihydroarcyriarubins (1-77a-b) sets them apart. Dihydroarcyriarubin B (1-77a) was first isolated by Steglich, et. al. from the slime mold, Arcyria denudate, alongside the Arcyriarubins (1-76a-b).48 Dihydroarcyriarubin C (1-77b) was first isolated in 2003 by Ishibashi, et. al. from Arcyria ferruginae, alongside Arcyriarubin C (1-76b).49 The biological activities of the Dihydroarcyriarubins B (1-77a) and C (1-77b) are very similar to that of 1-76a- c, including antibiotic, antifungal, cytotoxic, and kinase inhibition activities.48,49,52 The first natural products of the Arcyriarubin class to be accessed via total synthesis were Arcyriarubin A (1-76a) and Arcyriarubin B (1-76b) in 1988 by Steglich, et. al.53 These total syntheses were accomplished using Grignard reactions to install the indole moieties on the maleimide core, as shown in Scheme 1.21. In the first total synthesis of Arcyriarubin A (1-76a), a Grignard reaction was carried out via an indole Grignard reagent and 3,4-dibromo-N- methylmaleimide to access bisindolylmaleimide intermediate (1-78) in high yield. To remove the methyl group of 1-78, a maleic anhydride intermediate (1-79) was accessed and then 26 subsequently reacted with ammonium acetate at high temperatures to access Arcyriarubin A (1- 76a) in high yields.53 Scheme 1.21. Total syntheses of Arcyriarubins A-B (1-76a-b). Me N O O Br 1-80 N Boc THF, reflux, 1 h 89% CH3 N O O N Boc N H 1-81 (R = OTHP) ∆ (180ºC), R 45 min 94% CH3 N O O OH N H 1-82 N H 1. 10% KOH (aq.) 2. NH4AcO, 140ºC, 30 min 81% Arcyriarubin B (1-76b) 1. 10% KOH (aq.), reflux 2. 2N HCl 80% O O O N H N H 1-79 R N MgBr 1-77a (R = H) 1-77b (R = OTHP) Me N O toluene, reflux, 2 h, O Br Br O N H 70% CH3 N O N H 1-78 NH4AcO, 140ºC, 10 min 80% Arcyriarubin A (1-76a) H N O O X X 1-84a (X=Cl) 1-84b (X=Br) MgBr N H 1-83 THF/Et2O or toluene, 60ºC-120º, 8-24 h 72-78% Due to the asymmetric nature of the indole moieties in Arcyriarubin B (1-76b), the synthetic approach to this natural product was slightly different. As shown in Scheme 1.21, the first total synthesis of 1-76b was achieved via a reaction of an indole Grignard reagent (1-77b) with a mono-indolic maleimide intermediate (1-80) to access intermediate 1-81 in 89% yield. 27 This approach allowed for the synthesis of a maleimide intermediate with asymmetric indole moieties (1-81) in a stepwise manner. After de-methylation and removal of protecting groups, Arcyriarubin B (1-76b) was accessed in high yield.53 Several years later, a similar Grignard approach to the total synthesis of Arcyriarubin A (1-76a) was utilized in 1995 (Scheme 1.21).54 Here, an indole Grignard (1-83) was reacted with 3,4-dichloromaleimide (1-84a) at high temperature to access Arcyriarubin A (1-76a) in 72% yield. This approach was advantageous in that it allowed for the synthesis of Arcyriarubin A (1- 76a) without the need for protecting groups, which greatly expediting its synthesis.54 However, the intolerance for asymmetric indole moieties in this method makes it unsuitable for application to other Arcyriarubins. This Grignard strategy was again slightly improved upon in 2017 for the total synthesis of Arcyriarubin A (1-76a), as shown in Scheme 1.21.55 Utilizing the same indole Grignard (1-83) and 3,4-dibromomaleimide (1-84b) at a higher temperature, for a shorter reaction time, Arcyriarubin A (1-76a) was synthesized in 78% yield.55 It is also noteworthy to mention that Arcyriarubin C (1-76c) has yet to be accessed via total synthesis. In addition to this Grignard approach, a more biomimetic approach has also been explored toward the total synthesis of Arcyriarubin A (1-76a), as shown in Scheme 1.22. This approach consisted of a base-mediated condensation-cyclization reaction between an indole-3- acetamide intermediate (1-85) and an indole-3-oxo-acyl chloride intermediate (1-86a) to access Arcyriarubin A (1-76a), although in a low 11% yield. Recently, an adapted and improved biomimetic total synthesis of Arcyriarubin A (1-76a) was developed, as shown in Scheme 1.22. For this approach, intermediate 1-85 was condensed and cyclized with an indole-3-oxo-ester intermediate (1-86b) to access Arcyriarubin A (1-76a) in an excellent 96% yield.56 28 Scheme 1.22. Total synthesis of Arcyriarubin A (1-76a). O O R N H 1-86a (R=Cl) 1-86b (R=OEt) KOt-Bu, THF, 0ºC to rt, 48 h 96% O NH2 N H 1-85 H N O N H O N H Arcyriarubin A (1-76a) In terms of the Dihydroarcyriarubins (1-77a-b), Dihydroarcyriarubin B (1-77a) has yet to be accessed via total synthesis. On the other hand, the first total synthesis of Dihydroarcyriarubin C (1-77b) was completed in 2007 by Ishibashi, et. al., as shown below in Scheme 1.23.52 First, the bis-indolyl-N-methylmaleimide intermediate (1-87) was synthesized in four steps from 3,4- dibromomaleimide and 6-benzyloxyindole, following the same Grignard approach as was previously published by Steglich, et. al.53 To remove the N-methyl group, intermediate 1-87 was converted to the maleic anhydride intermediate (1-88), which was subsequently reacted with ammonium acetate at high temperatures to access the bis- indolylmaleimide intermediate (1-89) in very high yields. Then, 1-89 underwent hydrogenation to reduce the maleimide core of 1-89 to the succinimide core of Dihydroarcyriarubin C (77b). In addition to the desired reduction of 1- 89, both benzyl groups were also removed. While this was beneficial for the total synthesis of Dihydroarcyriarubin C (1-77b); it was also the reason why this approach was not viable for synthesizing Arcyriarubin C (1-76c). It is noteworthy to mention that at a 12-hour hydrogenation reaction time, cis- Dihydroarcyriarubin C (1-77b) was accessed in 95% yield, but at a 5-hour reaction time, trans- Dihydroarcyriarubin C (1-77b) was accessed in 98% yield. In addition, cis- Dihydroarcyriarubin C (1-77b) could be isomerized to trans- Dihydroarcyriarubin C (1-77b) either by magnesium in methanol or DBU in tetrahydrofuran in 81% and 93% yield, respectively. 29 To accomplish the first total synthesis of Dihydroarcyriarubin C (1-77b), both the cis-(1-77b) and trans-(1-77b) isomers were synthesized to allow for the stereochemical determination of the isolated Dihydroarcyriarubin C (1-77b). Comparison of their NMR data allowed for confirmation of the isolated 1-77b as trans-Dihydroarcyriarubin C (1-77b).52 Scheme 1.23. Total synthesis of Dihydroarcyriarubin C (1-77b). Me N 1-87 O N H O N H BnO KOH OBn EtOH/H2O (5:1), reflux, 4.5 h 89% BnO Cis- Dihydroarcyriarubin C (1-77b) Mg, MeOH, reflux, 7 h 81% OR DBU, THF, rt to reflux, 15 h 93% Pd/C, H2 EtOH, rt, 12 h 95% Trans- Dihydroarcyriarubin C (1-77b) BnO Pd/C, H2, EtOH, rt, 5 h 98% O O O N H 1-88 N H NH4OAc, 140ºC, 3 h 97% H N O N H 1-89 O N H OBn OBn 1.2.3.2. Lycogarubins Lycogarubins A-C (1-90a-c) and Lycagolic acid (1-90d) were first isolated independently by Steglich, et. al. and Asakawa, et. al. from Lycogala epidendrum, a slime mold (Myxomycetes).57,58 Lycagolic Acid (1-90d) has also been referred to as chromopyrrolic acid (CPA). The chemical structures of these natural products were elucidated via spectroscopic analysis. As shown below in Scheme 1.24, the two indole moieties of these bis-indole natural products are linked by a pyrrole-2,5-methyl ester core in Lycograubins A-C (1-90a-c) or a 30 pyrrole-2,5-carboxylic acid core in Lycagolic acid (1-90d). These natural products closely resemble the previously discussed Arcyriarubin natural products (1-76a-c), differing mainly in the aromatic pyrrole core of 1-90a-d rather than the maleimide core of 1-76a-c. Much of the biological activity of these natural products is unexplored, though Lycogarubin C displayed moderate anti-HSV-I virus activity.58 In addition, these compounds are of interest due to their structural similarity to potent kinase inhibitors.52 Scheme 1.24. Chemical structures of Lycogarubins A-C (1-90a-c). R1O2C R2 H N CO2R1 R3 N H N H Lycogarubin A (1-90a): R1=Me, R2=OH, R3=OH Lycogarubin B (1-90b): R1=Me, R2=H, R3=OH Lycogarubin C (1-90c): R1=Me, R2=R3=H Lycogalic Acid (1-90d): R1=R2=R3=H Many of the early syntheses of the Lycogarubins (1-90a-c) and Lycagolic acid (1-90d) were carried out via the exploration of the biosynthetic pathway to these natural products.59 However, the first of these natural products to be accessed via total synthesis was Lycogarubin C (1-90c) using a palladium-catalyzed Suzuki coupling reaction of dimethyl 3,4-dibromo-1H- pyrrole-2,5-dicarboxylate (1-91) with an indole-3-boronic acid (1-92) and subsequent removal of the protecting group via TBAF to access Lycogarubin C (1-90c) in 81% yield, as shown in Scheme 1.25.35 31 Scheme 1.25. Total synthesis of Lycogarubin C (1-90C). 1. B(OH)2 N TBS 1-92 Br Br Pd(PPh3)4 cat., Na2CO3, DMF, 150ºC MeO2C N H 1-91 CO2Me 2. TBAF•3H2O 81% H N MeO2C CO2Me N H N H Lycogarubin C (1-90c) This Suzuki method has been implemented in recent literature as well in the synthesis of 1-90c and similar structures.60 Considering the ongoing study of the biosynthesis of the Lycogarubin natural products, several biomimetic syntheses have been developed as well. One of the earliest of these was completed in 2006 in which a methyl 3-(1H-indol-3-yl)-2- oxopropanoate intermediate (1-93) underwent a dimerization/cyclization sequence over three steps to access Lycogarubin C (1-90c) in 42% yield (Scheme 1.26).59,61 Scheme 1.26. Biomimetic total syntheses of Lycogarubin C (1-90C) and Lycagolic acid (1-90d). O O O N H 1-93 1. NaOMe, MeOH, 0ºC, 15 min 2. I2, MeOH, 0ºC to rt, 30 min 3. NH4OH, MeOH, 1 h 42% MeO2C H N CO2Me HO2C H N CO2H KOH 95% N H N H Lycogarubin C (1-90c) TMSCHN2, MeOH, rt 69% (over two steps) N H N H Lycagolic Acid (1-90d) 1. Cu(OAc)2, Mn(OAc)3, NaOAc, xylene, reflux 54% 2. Mg, MeOH 93% CO2Me NH2 N Ts 1-94 32 LaStaO, VioB, (NH4)SO4, Na2HPO4-NaH2PO4, pH 8.0, 28ºC CO2H NH2 N H L-Tryptophan Another biomimetic synthesis of Lycogarubin C (1-90c), as well as Lycagolic acid (1- 90d), was carried out via a Cu/Mn co-oxidized dimerization/cyclization of a Tosyl-tryptophan methyl ester (1-94) with another molecule of itself (Scheme 1.26). It is noteworthy to mention that biosynthesis of the Lycogarubins also begins with tryptophan starting materials. After removal of the protecting groups, Lycogarubin C (1-90c) was accessed in high yields. Lycogarubin C (1-90c) was then converted to Lycagolic acid (1-90d) in 95% yield via base- catalyzed ester hydrolysis.62 Recently, a semi-total synthesis of Lycogarubin C (1-90c) was completed via Lycagolic acid (1-90d) as an intermediate (Scheme 1.26). First, enzymatic biological reactions were conducted to accomplish the dimerization/cyclization of the L-Tryptophan (L-Trp) starting material (1-94) to access Lycagolic acid (1-90d). Then, 1-90d was immediately reacted with TMSCHN2 in methanol to access Lycogarubin C (1-90c) in 69% over two steps from the simple and readily accessible L-Trp (1-94) starting material.63 Another synthetic method that has been used to synthesize Lycogarubin C (1-90c) and Lycagolic acid (1-90d) was the use of key Diels-Alder and Kornfeld-Boger ring contraction reactions. One example utilizing this method, as shown in Scheme 1.27, began with the synthesis of the di-protected-bis-indole alkene intermediate (1-97) via a Wittig reaction between the bromide intermediate (1-95) and the aldehyde intermediate (1-96). Then, 1-97 underwent a Diels-Alder reaction with the tetrazine intermediate (1-98), followed by a subsequent Kornfeld- Boger ring contraction to access the pyrrole intermediate (1-99) in 54% yield over two steps. After removal of the protecting groups, Lycogarubin C (1-90c) was accessed in 91% yield. Protection of both indole moieties was necessary to access intermediate 1-99 in good yields and to avoid side product formation via the Diels-Alder/ Kornfeld-Boger reaction sequence.64 33 Scheme 1.27. Total synthesis of Lycogarubin C (1-90c). 1. PPh3, THF 2. KOt-Bu O H N SO2Ph 1-96 65% Br N SO2Ph 1-95 1. MeO2C N N CO2Me N N 1-98 toluene, reflux 2. Zn, AcOH 54% (over 2 steps) N SO2Ph N SO2Ph 1-97 MeO2C H N CO2Me N SO2Ph N SO2Ph 1-99 Mg, NH4Cl, MeOH 91% Lycogarubin C (1-90c) Another example in which this method was implemented, as shown in Scheme 1.28, began with the Diels-Alder reaction between 1-98 and 1,2-bis-(tributylstannyl)-acetylene to access diazine intermediate (1-100) in very high yield. Stille coupling of 1-100 with the 3-iodo- indole intermediate (1-101) and a subsequent Kornfeld-Boger ring contraction of the diazine core led to the desired pyrrole core of 1-103 in good yields. Then, 1-103 could be subjected to LiOH or KOH to access Lycogarubin C (1-90c) or Lycagolic acid (1-90d), respectively, in very high yields. Furthermore, Lycogarubin C (1-90c) could also be converted to Lycogolic acid (1-90d) via KOH in very high yields.65 This approach (Scheme 1.28) is favorable in comparison to the previously discussed Diels-Alder/Kornfeld Boger approach (Scheme 1.27), as it not only allowed for the total synthesis of Lycogarubin C (1-90c) in good yields, but also allowed for the total synthesis of Lycagolic acid (1-90d) in good yields. 34 Scheme 1.28. Total syntheses of Lycogarubin C (1-90c) and Lycagolic acid (1-90d). Bu3Sn SnBu3 I 1-98, dioxane, 45ºC 97% N N MeO2C CO2Me Bu3Sn SnBu3 1-100 N CO2Me 1-101 (Ph3P)2PdCl2, CuCl2, LiCl, MeO2C N N CO2Me DMF/Toluene, 60ºC 70% N MeO2C N CO2Me 1-102 Zn, AcOH 68% Lycogolic acid (1-90d) KOH > 95% KOH > 95% Lycogarubin C (1-90c) MeO2C H N CO2Me N N CO2Me CO2Me 1-103 LiOH 89% 1.2.3.3. Lynamicins More recently, a class of natural products that is structurally related to the Lycogarubins (1-90a-d) was discovered. In 2008, Lynamicns A-E (1-104a-e) were isolated by Potts, et. al. from a novel marine actinomycete of the proposed genus Marinispora. The chlorinated bis- indole chemical structures linked by a pyrrole moiety, as shown in Scheme 1.29, was confirmed in these natural products via spectroscopic methods.66 Not only were these natural products (1- 104a-e) the first examples of bis-indole pyrrole alkaloids bearing chloride substituents, but the Lynamicins A-E (1-104a-e) have also been found to display broad-spectrum antibiotic activity against both Gram-positive and Gram-negative bacteria, such as Staphylococcus aureus and Enterococcus faecium.66 The Lynamicins have also been shown to possess anticancer and kinase inhibition activity.67 35 Scheme 1.29. Chemical structures of Lynamicins A-G (1-104a-g). R3 R4 R6 N R2 N H R1 N H Cl R5 Lynamycin A (1-104a): R1=H, R2=COOMe, R3=Cl, R4=H, R5=H Lynamicin B (1-104b): R1=H, R2=COOMe, R3=Cl, R4=Cl, R5=H Lynamicin C (1-104c): R1=H, R2=H, R3=Cl, R4=Cl, R5=Cl Lynamycin D (1-104d): R1=COOMe, R2=COOMe, R3=Cl, R4=H, R5=H Lynamicin E (1-104e): R1=COOMe, R2=COOMe, R3=H, R4=H, R5=Cl Lynamicin F (1-104f): R1=H, R2=COOMe, R3=Cl, R4=H, R5=H, R6=Me Lynamicin G (1-104g): R1=R2=COOMe, R3=Cl, R4=H, R5=H, R6=Me Two additional natural products of the Lynamicin class, Lynamicin F (1-104f) and Lynamicin G (1-104g), were isolated in 2014 by Zhang, et. al.68 from the deep-sea-derived actinomycete, Streptomyces sp. The chemical structures of Lynamicin F (1-104f) and Lynamicin G (1-104g), as shown in Scheme 1.29, were elucidated via spectroscopic methods. The characteristic N-methyl group in 1-104f and 1-104g was confirmed via key HMBC correlations between the hydrogens on the methyl group and the C-2 and C-5 carbons of the pyrrole ring. Interestingly, the presence of this methyl group seems to have a significant effect on biological activity because, unlike Lyncamicins A-E (1-104a-e), Lynamicin F (1-104f) and Lynamicin G (1- 104g) showed no significant antibiotic activity against Staphylococcus aureus, Escherichia coli, etc. and no significant cytotoxic activity toward cancer cell lines.68 The first of the Lycogarubin class to be accessed via total synthesis was Lynamicin D (1- 104d) by Nikolakaki, et. al. in 2017, which utilized a Suzuki coupling approach to install the two indole moieties to the pyrrole core.68 As shown in Scheme 1.30, first, the 3,4-dibromopyrrole intermediate 1-106 was initially synthesized in two steps from the corresponding pyrrole-2- methylester in good yields. An indolic boronic ester intermediate (1-105) then underwent a 36 palladium-catalyzed Suzuki reaction with 1-106 to access intermediate 1-107 in 76% yield. After removal of the Boc protecting groups via acidic conditions, Lynamicin D (1-104d) was achieved in 97% yield.68 Recently, a semi-total synthesis of Lynamicin D (1-104d), resembling that of Lycogarubin C (1-90c), was also completed (Scheme 1.30).63 First, enzymatic biological reactions were conducted to accomplish the dimerization/cyclization of the 5-chloro-L- Tryptophan (5-Cl-L-Trp) starting material (1-108), following the biosynthetic path, to access the carboxylic acid analogue of Lynamicin D (1-109). Then, 1-109 was immediately reacted with TMSCHN2 in MeOH to access Lynamicin D (1-104d) in 37% over two steps from the simple 5- Cl-L-Trp (1-108) starting material.63 Scheme 1.30. Total synthesis of Lynamicin D (1-104d). Br Br O B O Cl N Boc 1-105 MeO2C N H 1-106 CO2Me MeO2C Cl H N CO2Me Cl Pd(OAc)2, PPh3, Na2CO3, H2O/THF, 60ºC 76% N Boc N Boc 1-107 TFA, DCM 97% Lynamicin D (1-104d) TMSCHN2, MeOH, rt, 37% (over two steps) CO2H NH2 LzrO, VioB, (NH4)2SO4 Na2HPO4- NaH2PO4, pH 8.0, 28ºC Cl N H 1-108 HO2C Cl H N CO2H Cl N H 1-109 N H The first total synthesis of Lynamicin A (1-104a) was completed in 2021 by Smith, et. al., as well as a slightly adapted total synthesis of Lynamicin D (1-104d).69 As shown in Scheme 37 1.31, these total syntheses follow a very similar Suzuki cross-coupling to that of Nikolakaki’s previously discussed method to install the indole moieties onto the pyrrole core of these natural products.67 However, the Suzuki cross coupling step between the boronic ester intermediate (1- 110) and the 3,4-dibromo pyrrole intermediate (1-106) utilized different palladium catalysts, as shown in Scheme 1.31, and featured a subsequent deprotection of the protecting groups to access Lynamicin D (1-104d) in 67% yield. Then, Lynamicin D (1-104d) was subjected to a basic environment, at high temperatures, which led to the removal of one of the ester groups on the pyrrole ring, accessing Lynamicin A (1-104a) in 67% yield.69 Lynamicins B, C, and E-G (1- 104b, c, e-g) have yet to be accessed via total synthesis. Scheme 1.31. Total synthesis of Lynamicin A (1-104a). Cl Bpin N Boc 1. 1-106, Pd(OAc)2/SPhos, K2CO3, THF, H2O, 60ºC 2. NaOMe, MeOH, THF, 0ºC to rt Cl 1-110 MeO2C H N CO2Me N H N H Lynamicin D (1-104d) Cl 1. KOH, H2O, MeOH, ∆ Cl 2. neat, 180ºC 27% (over two steps) H N MeO2C Cl N H N H Lynamicin A (1-104a) 1.2.4. Piperazine, piperazinone, and pyridine linker moieties 1.2.4.1. Dragmacidins The Dragmacidin class of natural products consists of two main structural sub-classes. The first sub-class consists of Dragmacidin (1-111) and Dragmacidins A-C (1-111a-c), which are bis-indole alkaloids whose two indole moieties are linked by a piperazine core, as shown in Scheme 1.32. Dragmacidin was first isolated in 1988, by Koehn et. al., from Dragmacidon marine sponge.70 A couple years later, Dragmacidin A (1-111a) and Dragmacidin B (1-111b) were isolated from Hexadella marine sponge.10 The chemical structure of these natural products was elucidated via spectroscopic methods. Dragmacidin (1-111) and Dragmacidins A-B (1-111a- 38 b) were confirmed to have trans configurations.10,71 Dragmacidin C (1-111c) was the last of this sub-class to be discovered and was first isolated in 1991, by Smith, et. al., from the sea squirt Didenium candidum.71 Dragmacidin C (1-111c) was initially thought to have trans configuration, but it was later shown to be of cis configuration.72 The biological activities of Dragmacidin (1- 111) and Dragmacidins A-C (1-111a-c) are relatively unexplored; however, Dragmacidin (1-111) and Dragmacidin A (1-111a) have been found to display significant cytotoxicity against a variety of cancer cell lines.70 Scheme 1.32. Chemical structures of Dragmacidin (1-111) and Dragmacidins A-C (1-111a-c). NH R4 Br R3 R1 N N R2 Br HN (Cis)-Dragmacidin (1-111): R1=H, R2=Me, R3=OH, R4=Br Trans-Dragmacidin A (1-111a): R1=Me, R2=R3=R4=H Trans-Dragmacidin B (1-111b): R1=R2=Me, R3=R4=H Cis-Dragmacidin C (1-111c): R1=R2=R3=R4=H The second sub-class of the Dragmacidins is comprised of more structurally complex natural products, including Dragmacidins D (1-111d), E, (1-111e), and F (1-111f). These bis- indole natural products (1-111d-f) contain a 2-pyridone core that links their two indole, or modified indole, moieties, as shown in Scheme 1.33. Another structural distinction of this sub- class is the presence of a guanidine moiety. Dragmacidin D (1-111d) and Dragmacidin E (1- 111e) were isolated in 1988 by Carroll, et. al. from Spongosorites and Hexadella marine sponges and their structures were elucidated via spectroscopic means.73 In addition, Dragmacidin D (1- 111d) and Dragmacidin E (1-111e) were found to be potent inhibitors of serine-threonine protein phosphatases.73 Dragmacidin F (1-111f) was isolated a couple years later, in 2000, by Riccio, et. al. from a Halicortex marine sponge. The complex ring structure of Dragmacidin F (1-111f) was 39 elucidated via extensive spectroscopic analysis and its unprecedented carbon skeleton was proposed to result from the cyclization of a partially oxidized form of Dragmacidin D (1-111d). Dragmacidin F (1-111f) was found to display antiviral activity against HSV-1 and HIV-1.74 Dragmacidin E (1-111e) and Dragmacidin F (1-111f) have both been accessed via total synthesis; however, these syntheses will not be discussed in detail as these chemical structures fall outside the scope of this review.75,76 Scheme 1.33. Chemical structures of Dragmacidins D-G (1-111d-g). H N Br H2N HN N N O NH H2N HN N NH N N H O Br N H OH HO N H Dragmacidin D (1-111d) Dragmacidin E (1-111e) H2N N HN O HN NH H N N H O Br Br Br S NH N H NH2 H N N N N H Dragmacidin F (1-111f) Dragmacidin G (1-111g) Recently, Dragmacidin G (1-111g) was isolated by Wright, et. al. from Spongosorites marine sponge. As is shown in Scheme 1.33, the chemical structure of Dragmacidin G (1-111g) does not exactly fit into the second sub-class of the Dragmacidins, as it is the only natural product in its class to contain a piperazine (1,4-diazine) core linking the two indole moieties. However, Dragmacidin G (1-111g) does contain a characteristic guanidine moiety, thus enabling It to still be considered part of the second structural sub-class of the Dragmacidins. In addition, 40 Dragmacidin G (1-111g) was found to exhibit antibacterial activity against Stapphylococcus aureus, Mycobacterium tuberculosis, Plasmodium falciparum, etc., and cytotoxic activity against a panel of pancreatic cell lines.77 Dragmacidin G (1-111g) has yet to be accessed via total synthesis. Dragmacidin (1-111) was the first natural product of this class to be accessed via total synthesis. In 1994, Wuonola et. al. completed the first racemic total synthesis of Dragmacidin (1- 111) via an initial condensation of an oxo-acyl chloride (1-112) and an amino nitrile (1-113) (synthesized via a Strecker method), followed by subsequent oxidation and cyclization, as shown in Scheme 1.34. These transformations proceeded in high yields. The cyclized intermediate was then subjected to reductive conditions to access 1-115a and 1-115b. This reduction, specifically the loss of the hydroxyl group, was not stereoselective at room temperature, resulting in a ratio of (1-115a:1-115b = 1:1). However, significant selectivity in favor of the desired trans isomer (1- 115b) was achieved by lowering the temperature to 0°C for this reduction reaction, as shown in Scheme 1.34, resulting in 37% of the trans isomer (1-115b) compared to 9% of the undesired cis isomer (1-115a). Then, 1-115b was de-methylated to afford Dragmacidin (1-111).78 41 Scheme 1.34. First total synthesis of Dragmacidin (1-111). MeHN CN Br Br H N 1. Br O OMe N H Br 1-112 Br O Cl N H 1-113 Et3N, DCM, rt, 4 h 91% 2. NH4OH, H2O2, Bu3NHSO4, dioxane, rt, 3 h 92% 3. NH4OH, EtOH, ∆, 24 h 62% Br HN O OH O OMe N Me BH3•THF, 0ºC, 48 h N H 1-114 NH Br Br + Br MeO H N N Me Br HN 1-115b (37%) BBr3, DCM, rt, 12 h Dragmacidin (1-111) NH Br Br H N N Me MeO 1-115a (9%) HN The first total synthesis of Dragmacidin B (1-111b) was achieved in the same year as Dragmacidin (1-111). However, considering its less substituted indole moieties, the approach toward Dragmacidin B (1-111b) was less step intensive, as shown in Scheme 1.35. First, an N- dimethylated piperazinedione (1-116) was brominated before undergoing a double nucleophilic attack with 6-bromo-1H-indole to access intermediate 1-117 in 57% yield. The bromination and nucleophilic attacks were conducted in a one-pot manner and no acid or base was necessary for the nucleophilic step. Then, 1-117 was reduced to access Dragmacidin B (1-111b) in moderate yield. This made for a very expeditious two-step approach to complete the total synthesis of Dragmacidin B (1-111b) in 14% yield overall (Scheme 1.35).79 42 Scheme 1.35. First total synthesis of Dragmacidin B (1-111b). O Me N N Me 1-116 1. NBS, AIBN, CCl4 Br O 2. Br N H DMF 57% O HN Me N N Me 1-117 O BH3•THF 25% NH Br Dragmacidin B (1-111b) The first total synthesis of Dragmacidin A (1-111a) was completed in 2000 via a condensation/cyclization sequence of methylated (1-118) and free (1-119) indolic α-amino carboxylic acids to access the piperazinedione intermediate (1-121) in good yields, as shown in Scheme 1.36. The condensation step was diastereoselective and the desired isomer (1-120b) predominated. After the cyclization, the carbonyl groups of intermediate (1-121a) were removed via reductive conditions to access Dragmacidin A (1-111a) in 45% yield. The total yield of Dragmacidin A (1-111a) after this efficient three-step approach was 21% overall.80 43 Scheme 1.36. Total syntheses of Dragmacidins A-C (111a-c). H2N CO2Me MeO2C Me NBoc O NH Me BocN Br CO2H Br N Ac 1-118 N Ac 1-119 BOP, DIPEA THF O Br HN H N N R NH Br O 1-121a (R=H) (66%) 1-121b (R=Me) (70%) BH3•THF, 0ºC to rt Br Br N H N H Br 1-120a (minor) Me NBoc + O NH N H Br MeO2C N H 1-120b (major) 1. HCO2H, rt 2. NH3, MeOH, 0ºC 70% Dragmacidin A (1-111a) (45%) OR H N N H 122 (42%) Br HN NH HCO2H, NaBH3CN, Br 70ºC 22% Dragmacidin B (1-111b) HCO2H, rt, then, NH3, MeOH, Br 1-120a 0ºC to rt 66% NH O H N N H 1-123 O HN Br BH3•THF, 0ºC to rt 20% Dragmacidin C (1-111c) The first total synthesis of Dragmacidin C (1-111c) was completed in 2002 via the same three step method (condensation/cyclization/oxidation) that was previously discussed for the first 44 total synthesis of Dragmacidin A (1-111a), and the relative cis configuration of Dragmacidin C (1-111c) was confirmed (Scheme 1.36). As shown in Scheme 1.36, Dragmacidin B (1-111b) was also synthesized using this method, though incomplete reduction of 1-121b resulted in a prominent side product in which one carbonyl remained on the ring. This accounts for the lower yield of 1-122. In the synthesis of Dragmacidin B (1-111b) a late-stage methylation step was carried out on the piperazine intermediate (1-122) to install the two methyl groups on the piperazine ring of Dragmacidin B (1-111b).72 Optically active Dragmacidin A (1-111a) was synthesized via a Sharpless asymmetric aminohydroxylation of 6-bromo-3-vinylindole (1-30) to access 1-124 in 65% yield and 94% enantiomeric excess, as shown in Scheme 1.37. The hydroxyl group of 1-124 was then tosylated and subsequently substituted by an azido group in high yield over two steps. The resulting amino azide intermediate (1-125) was then deprotected and acylated with 1-23b, followed by reduction of the azide and subsequent cyclization to afford 1-127. After Boc-protection, 1-127 was selectively and quantitatively methylated and subsequently deprotected to afford 1-128. The reduction of intermediate 1-128 proved to be problematic, as it was nonselective. In fact, the desired isomer (1-129) was the minor product in just 17% isolated yield (82% of cis isomer isolated). Despite this low yield, 1-129 was de-tosylated via L-selectride and the piperazinone was reduced to the desired piperazine core to access (2S, 5R)-Dragmacidin A (1-111a) in 42% yield over two steps.81 45 Scheme 1.37. First total synthesis of optically active Dragmacidin (1-111). BocHN OH BocHN N3 1-30 Boc N Na Cl K2OsO4•H2O, (DHQ)2PHAL, PrOH, H2O 65%, 94% ee Br 1. TsCl, DMAP 2. NaN3, DMF, 76% (over 2 steps) N Ts Br N Ts 1-125 1-124 Ts N 1. TFA, DCM 2. 23b, Et3N, DMF 85% Ts N Br Bu3P, PhMe 94% Br HN O N3 O 1-126 Br Br O H N N HN 1-127 O Me N N HN 1-128 1. Boc2O, DMAP 96% 2. MeI, K2CO3, then TFA quant Br N H Ts N Br NaBH4, MeOH 17% Dragmacidin A (1-111a) 1. L-Selectride, THF, reflux 80% Br 2. BH3•THF 42% Ts N Br O Me N (S) (R) N H 1-129 HN An expedited method of synthesizing optically active Dragmacidin A (1-111a) was developed in which a bis-indolylpyrazine intermediate (1-131) was protected and subsequently reduced to the corresponding piperazine intermediate (1-132) in high yields, as shown in Scheme 1.38. Then, de-symmetrization of the piperazine (1-132) was completed via enantioselective formylation using a chiral formylating reagent. This method ultimately proved to be only moderately stereoselective, affording the dextrorotatory isomer of Dragmacidin A (1-111a) in 46 44% enantiomeric excess after transformation of the aldehyde to a methyl group and subsequent deprotection.82 Using a similar reductive approach to that used on the bis-indolylpyrazine intermediate (1-131), an efficient synthesis of Dragmacidin B (1-111b) was achieved in 53% overall yield over two steps, as shown in Scheme 1.38. It should be noted that this was not an efficient method for synthesizing Dragmacidin C (1-111c), as the undesired isomer predominated and less than 5% yield of Dragmacidin C (1-111c) was isolated.83,84 Scheme 1.38. Total synthesis of Dragmacidin A-C (1-111a-c). 1. H O N N H SO2C6F5 SO2C6F5 1-133 Dragmacidin A (1-111a) TBAF, THF, ∆ 70% Br 2. BH3•THF, ∆ 51% SEMN NSEM H N N H 1-132 1. NaH, SEMCl, DMF 75% 2. NaBH3CN, AcOH 71% NH NH2 O xylene, EtOH Br Ar, 130ºC, 3 days 75% N N HN 1-131 Br N H 130 Dragmacidin B (1-111b) NaBH3CN (25 equiv.), AcOH 70% Br Br Br Br NaBH3CN (50 equiv.), HCO2H NH H N N H 1-134 (61%) + HN Dragmacidin C (1-111c) (>5%) 47 Recently, the first total synthesis of the more structurally complex Dragmacidin D (111d) was completed, as shown in Scheme 1.39.85 First, the iodinated indolic diamine intermediate (1- 135) was synthesized in 11 linear steps from commercially available starting materials. Here, the desired stereoisomer of 1-135 was accessed via the use of Evan’s oxazolidinone chiral auxiliary ((S)-3-acryloyl-4-phenyloxazolidin-2-one) and column chromatography.85 After the key diamine intermediate (1-135) was synthesized, it was condensed with 6-bromo-1H-indole-3-oxo- acyl chloride and subsequently cyclized and oxidized to access 2-piperazinone intermediate (1- 137) in 50% yield over multiple steps, as shown in Scheme 1.39. Then, 1-137 was protected to allow for selective α-bromination, followed by a Staudinger reaction to access the α-amino ester (1-139) in high yields. Treatment of this α-amino ester (1-139) with pyrazole-1-carboxamidine afforded a guanidine intermediate, which underwent subsequent reduction and cyclization to construct the 2-aminoimidazole moiety of Dragmacidin D in good yields. Deprotection then afforded Dragmacidin D (1-111d) in 50% yield. This synthesis revised the earlier stereochemical assignment that was based on biosynthetic considerations, assigning the absolute configuration as (R)-(+)-Dregmacidin D (1-111d). Furthermore, chiral HPLC-DAD methodology was developed and utilized to confirm, for the first time, that naturally occurring Dragmacidin D (1- 111d) was isolated as either a racemate or a scalemic mixture (39% ee), which has prompted questions regarding the absolute configurations of Dragmacidins D-F (1-111d-f).85 48 Scheme 1.39. First total synthesis of Dragmacidin D (1-111d). O OMe NHBoc NH2 O 1-23b, Et3N, DCM, rt, 8 h OMe N Boc OBn 1-135 H N Br O O O NH NHBoc 1. TFA, DCM 2. NaHCO3 (aq), MeOH, DCM MeO 3. DDQ, dioxane N H OBn 1-136 50% BnO HN NH Br N N H 1-137 O O MeO 1. Boc2O, DMAP, DCM, rt 85% 2. SEMCl, DIPEA, AgOTf, DCM 90% NBoc N N Br OSEM BnO BocN 1-138 O NH2 MeO NBoc N N Br OSEM BnO BocN 1-139 NBoc N N 1. BocHN 1-140 Et3N, DMAP, MeCN, 40ºC 80% 2. DIBAL-H, Et2O, -78ºC, then TFA, DCM 39% 3. BBr3, DCM, 0ºC 50% 1. LDA, THF, then CBr4, -78ºC to 0ºC 60% 2. NaN3DMF, rt, then PPh3, THF/H2O quant NH2 NH OTf HN NH Br N N H O HO HN 1.2.4.2. Hamacanthins Dragmacidin D (1-111d) Another class of bis-indole alkaloids that are structurally related to the Dragmacidins are the Hamacanthins (1-141a-d, 1-142a-d, 1-143a-d, 1-144a-d), which contain a 5,6-dihydro-2- pyridone or 2-piperazinone core connecting the two indole moieties, as shown in Scheme 1.40. The first of these natural products to be isolated were Hamacanthin A (1-141a) and Hamacanthin B (1-143a), which were isolated from Hamacantha marine sponge in 1994 by Gunasekera, et. al. These natural products were found to display antifungal activity.86 49 Scheme 1.40. Chemical structures of Hamacanthins A-B (1-141a and 143a), Dihydrohamacanthins A-B (142a and 144a) and related Debrominated natural products. O H N N R1 HN NH R2 R1 NH R2 O H N N H HN Hamacanthin A (1-141a): (R1 = R2 = Br) 6’-Debromohamacanthin A (1-141b): (R1 = H, R2 = Br) 6’’-Debromohamacanthin A (1-141c): (R1 = Br, R2 = H) 6’,6’’-Debromohamacanthin A (1-141d): (R1 = R2 = H) cis-3,4-dihydroamacanthin A (1-142a): (R1 = R2 = Br) trans-3,4-dihydroamacanthin A (1-142b): (R1 = R2 = Br) 6’-Debromo-trans-3,4-dihydrohamacanthin A (1-142c): (R1 = H, R2 = Br) 6’’-Debromo-trans-3,4-dihydrohamacanthin A (1-142d): (R1 = Br, R2 = H) R1 O H N N R2 R1 HN NH HN O H N N H R2 NH Hamacanthin B (1-143a): (R1 = R2 = Br) 6’-Debromohamacanthin B (1-143b): (R1 = H, R2 = Br) 6’’-Debromohamacanthin B (1-143c): (R1 = Br, R2 = H) 6’,6’’-Debromohamacanthin B (143d): (R1 = R2 = H) cis-3,4-dihydroamacanthin B (1-144a): (R1 = R2 = Br) 6’-Debromo-cis-3,4-dihydrohamacanthin A (1-144b): (R1 = H, R2 = Br) 6’’-Debromo-cis-3,4-dihydrohamacanthin A (1-144c): (R1 = Br, R2 = H) 6’,6’’-Debromo-cis-3,4-dihydrohamacanthin A (1-144d): (R1 = H, R2 = H) In more recent years, the de-bromo and dihydro Hamacanthin analogues (1-141b-d, 1- 142a-d, 1-143b-d, 1-144a-d) have been isolated from various natural marine sources, as shown in Table 1.2. The configurations of these natural products are also indicated in Table 2, and many were later confirmed via total syntheses. Despite their structural similarities, the configurations of these Hamacanthins vary. For example, Hamacanthin A (1-141a) exists as the (S)-isomer, while its analogous mono de-bromo natural products (1-141b and 1-141c) exist as the (R)- isomers, and its bis de-bromo analogue (1-141d) exists as the (S)-isomer (Table 1.2). Overall, 50 natural products of the Hamacanthin class (1-141a-d, 1-142a-d, 1-143a-d, 1-143a-d) have been shown to display antifungal, antibacterial, and cytotoxic activity.86 Table 1.2. Hamacanthin natural products – Isolation data.2, 7, 8, 12, 86, 11 Compound Configuration 1-141a 1-141b 1-141c 1-141d 1-143a 1-143b 1-143c 1-143d 1-142a 1-142b 1-142c 1-142d 1-144a 1-144b 1-144c 1-144d S R R S S R R R 3R, 6R 3S, 6R 3S, 6R 3S, 6R 3S, 5R 3S, 5R 3S, 5R 3S, 5R Natural Source Hamacantha, Spongosorites, Disodermia calyx Spongosorites Spongosorites, Discodermia calyx Spongosorites Hamacantha, Spongosorites, Disodermia calyx Spongosorites, Discodermia calyx Spongosorites Spongosorites Rhaphizia lacazei Rhaphizia lacazei, Spongosorites Rhaphizia lacazei, Spongosorites Rhaphizia lacazei, Spongosorites Rhaphizia lacazei, Spongosorites, Discodermia calyx Rhaphizia lacazei, Spongosorites Rhaphizia lacazei, Spongosorites Spongosorites The first enantioselective total synthesis of the unnatural (R)-Hamacanthin A (1-141a) was completed in 2001 by Jiang, et. al. via the coupling of a 3-indolyl-α-oxo-acetyl chloride intermediate (1-23b) and 3-indolyl azidoethylamine intermediate (R)-(1-146), prior to an intramolecular aza-Wittig type cyclization to access (R)-Hamacanthin A (1-141a) in high yields, as shown in Scheme 1.41.87 The 3-indolyl azidoethylamine intermediate was synthesized in four steps from N-tosylated-6-bromo-3-vinyl indole in high yields. The stereocenter of (1-141a) was established via a Sharpless asymmetric dihydroxylation reaction, followed by a stereospecific azidation. In spectroscopic comparison of the naturally isolated Hamacanthin A (1-141a) and the synthesized (R)-Hamacanthin A (141a), it was confirmed that the natural Hamacanthin A (1- 141a) existed as the (S)-isomer.87, 88 51 Scheme 1.41. Enantioselective total syntheses of Hamacanthins A and B (141a and 143a). 1-30 AD-mix-α i-BuOH/H2O (1:1) Br 92%, ee 99% N (S)-1-31 Ts (R)-1-31 1. TsCl, Bu2SnO, then, NaN3, DMF 60% 2. SnCl2•H2O, then Boc2O, then PPh3, DEAD, DPPA, PhMe 85% NHBoc N3 Br N Ts (S)-1-148 1. TFA, DCM 2. 1-23b, Et3N, DMF 93% Br N3 O HN O (S)-1-149 N Ts 1. Bu3P, PhMe 2. L-Selectride 62% OH HO 1. TBDMSCl, imidazole 94% OH N3 2. Ph3P, DEAD, DPPA 87% 3. TBAF, THF, rt 98% Br N Ts (R)-1-145 1. LiAlH4 2. Boc2O, Et3N 3. TsCl, Et3N, DMAP 4. NaN3, DMF 64% BocHN N3 Br N Ts (R)-1-146 1. TFA, DCM 2. 1-23b, Et3N, DMF 74% Br NH Br NH O H N O N Ts (R)-1-147 N3 Br 1. Bu3P, PhMe 2. NaOH, MeOH, ∆ 85% (S)-Hamacanthin B (1-143a) (R)-Hamacanthin A (1-141a) A year later, the same research team completed the first enantioselective total synthesis of Hamacanthin B (1-143a). As shown in Scheme 1.41, this synthesis was completed via a similar approach as was previously discussed for (R)-Hamacanthin A (1-141a). Considering the established (S)-configuration of (1-141a), (S)-Hamacanthin B (1-143a) was synthesized. When 52 compared to the naturally isolated Hamacanthin B (1-143a), the synthesized (S)- 1-143a confirmed the configuration of the natural 1-143a as the (S)- isomer.88 With the elucidation of the absolute configuration of (S)-Hamacanthin A (1-141a), the first enantioselective total synthesis of the desired (S)-Hamacanthin A (1-141a) was completed via deprotection and simultaneous inversion of the stereocenter of (R)-1-150 to access (S)-1-141a in 78% yield, as shown in Scheme 1.42. This allowed for additional confirmation that this was the correct configuration of the naturally occurring (S)-Hamacanthin A (1-141a). The synthetic approach toward (R)-1-150 was previously discussed in the Dragmacidin section (Scheme 1.37).82 Also, using intermediate (R)-1-150, the first total syntheses of the unnatural (3S, 6S)-cis- 3,4-dihydrohamacanthin A (1-142a) and (3R, 6S)-trans-3,4-dihydrohamacanthin A (1-142b) were both completed. The reduction of (R)-1-150 afforded 62% yield of (3S,6S)-cis-1-151 and 36% yield of (3R,6S)-trans-1-151. These 2-piperidone intermediates (1-151) were then deprotected via L-selectride to access (3S,6S)-cis-3,4-dihydrohamacanthin A (1-142a) and (3R,6S)-trans-3,4-dihydrohamacanthin A (1-142b) in 87% and 88% yields, respectively (Scheme 1.42). Via comparison of the naturally isolated cis-3,4-dihydrohamacanthin A (1-142a) and trans-3,4-dihydrohamacanthin A (1-142b) with the synthesized (3S,6S)-cis-1-142a and (3S, 6S)- trans-1-142b, it was found that the configurations of these synthesized natural products did not match that of the isolated optical rotation data. Therefore, it was concluded that the absolute configurations of cis-3,4-dihydrohamacanthin A (1-142a) and cis-3,4-dihydrohamacanthin A (1- 142a) are (3R,6R) and (3S,6R), respectively.81 Additionally, racemic cis- and trans-3,4- dihydrohamacanthins can be accessed, albeit in low yields, via partial reduction of cyclized dipeptides (121 and 123), as was also discussed in the Dragmacidin section (Scheme 1.36).71 53 Scheme 1.42. Enantioselective total syntheses of Hamacanthin A (1-141a) and Dihydrohamacanthin A-B (1-141a-b). Ts N Ts N Ts N Br Br Br O H N N HN (R)-1-150 NaBH4, MeOH O H N N H (3S,6S)-1-151 (62%) O + H N N H (3R,6S)-1-151 (36%) HN HN Br NaOH, MeOH, ∆ 78% (S)- Hamacanthin A (1-141a) L-Selectride, Br THF, ∆ 87% (3S,6S)-cis-3,4- dihydrohamacanthin A (1-142a) L-Selectride, Br THF, ∆ 88% (3R,6S)-trans-3,4- dihydrohamacanthin A (1-142b) The first total syntheses of racemic 6’-debromo-trans-3,4-dihydrohamacanthin A (1- 142c) and the unnatural 6’’-debromo-cis-3,4-dihydrohamacanthin A (1-142e) were carried out via the condensation and subsequent cyclization of an indolic α-amino ketone intermediate (1- 48) with an indolic keto-amide intermediate (1-153). After formation of this bis- indolylpyrazinone intermediate, it underwent reduction to access 6’-debromo-trans-3,4- dihydrohamacanthin A (1-142c) and the unnatural 6’’-debromo-cis-3,4-dihydrohamacanthin A (1-142e) in 70% yield as a 1:1 mixture (Scheme 1.43). While it was not enantioselective, this method proved to be a very expeditious synthesis of these natural products.89 This method also allowed for the first racemic total synthesis of 6’’-debromo-cis-3,4-dihydrohamacanthin B (1- 54 144c) and 6’,6’’-debromo-cis-3,4-dihydrohamacanthin B (1-144d). As shown in Scheme 1.43, this method was adapted to achieve the 3,5-disubstituted cyclized product by condensing and cyclizing an indolic oxo-acyl chloride intermediate (1-23a-b) with an indolic α-amino ketone intermediate (1-48). In addition to being an efficient and high yielding route, this method was selective for the desired cis-isomers of 6’’-debromo-cis-3,4-dihydrohamacanthin B (1-144c) and 6’,6’’-debromo-cis-3,4-dihydrohamacanthin B (1-144d), respectively.89 Scheme 1.43. Enantioselective total syntheses of Debromo-Dihydrohamacanthins (1-142c and 1- 141c-d). NH2 O 1-23a or 1-23b Et3N, THF 90% R1 N H 1-48 O NH O N H O N H 1-155a (R1 = H) 1-155b (R1 = Br) R1 NH4OH, 100ºC, 2 days 85% O NH2 O Br N H 1-153 MeSO3H, PhCl, 130ºC, 3 days 30% NH Br O H N N HN 1-154 NaBH3CN, i-PrCO2H, rt, 16 h 70% (1:1 trans:cis) 6’-Debromo-trans-3,4- dihydrohamacanthin A (1-142c) 6’’-Debromo-cis-3,4- dihydrohamacanthin A (1-142e) A similar bio-mimetic cyclization of a ketoamide (1-157) was proven efficient for the synthesis of both racemic Hamacanthin A (1-141a) and Hamacanthin B (1-143a). As shown in Scheme 1.44, to access Hamacanthin B (1-143a), nucleophilic attack of the primary amine on the indolic (C3) ketone and subsequent dehydration led to intermediate (II), which was 55 O H N N HN NH 1-156a (R1 = H) 1-156b (R1 = Br) NaBH3CN, AcOH, rt, 16 h 70-71% 6’’-Debromo- cis-3,4dihydrohamacanthin B (1-144c) (R1 = H) 6’,6’’-Debromo-cis-3,4- dihydrohamacanthin B (1-144d) (R1 = Br) deprotected to access 1-143a. On the other hand, nucleophilic attack of the primary amine on the amide carbonyl to form a five-membered ring intermediate (III), followed by a ring opening that resulted in a rearrangement of the original ketoamide to intermediate (IV). Cyclization of intermediate (IV) then led to Hamacanthin A (1-141a) after removal of protecting groups.89 Scheme 1.44. Total synthesis of Hamacanthins A (1-141a) and B (1-141b). Br BocHN N H O HN O 1-157 Br N R’’ 1. HCO2H, DCM, rt 2. Solvent, pH 4, 80ºC, 1 h BocHN Br HN N R‘ O OH H N NH N R’ III Br N R’’ Br N R‘ Br N R’’ O Br N R’’ O O I H N N II NH2 H N O O Br N R’’ Removal of protecting groups Hamacanthin B (1-143a) IV N N H V R’’ N O Removal of protecting groups Br Hamacanthin A (1-141a) N R‘ N R‘ Br Br Br 56 Through mechanistic study, it was determined that the ratio of 1-141a:1-143a was heavily dependent on the identity of the solvent, and the protecting groups (R’ and R’’), as indicated by the results shown in Table 1.3. In this case, more polar, protic solvents, such as ethanol, led to predominant formation of intermediate (II) while more non-polar solvents, such as dichloroethane (DCE), led to predominant formation of intermediate (V). In addition, the presence of stronger electron-withdrawing groups, such as a tosyl group, on the indole nitrogen led to predominant formation of intermediate (II) over intermediate (V) due to the resulting increased electrophilicity of the carbonyl group adjacent to the indole, making nucleophilic attack on this carbonyl preferable.89 Table 1.3. Cyclization conditions of keto-amide (1-157). R’ R’’ Solvent Yield (V) Yield (II) Ts H Ts H Ts H Ts H Ts Ac Ts Ts DCE 1,4-Dioxane EtOH DMF DCE DCE 42% 30% 15% 7% 42% 18% 35% 38% 75% 46% 55% 68% In 2007, along with enantioselective total synthesis of (S)-Hamacanthins A and B (1-141a and 1-143a), the first enantioselective total synthesis of cis-3,4-dihydrohamacanthin B (1-144a) was also completed.90 As shown in Scheme 1.45, this synthesis proceeded via the condensation of an indolic amino- alcohol intermediate (1-162) and an indolic oxo-acyl chloride intermediate (1-23b) to access a similar keto-amide intermediate (1-163), as was previously discussed. Subsequent cyclization of the keto-amide intermediate (1-163) and stereospecific reduction afforded (3S-,5R)-cis-3,4-dihydrohamacanthin B (1-144a) in high yields. Here, the 57 stereochemistry was set via the installation of an (S)-phenyloxazolidone chiral auxiliary (S)-(1- 159).90 Scheme 1.45. First enantioselective total synthesis of Dihydrohamacanthin B (1-144a). O N3 O N O Ph N H 1-161a (52%) + O O N3 N Ph O O N H 1-158 + O N Br Ph3P OMe PhH, ∆ 96% TMSN3, MeSO3H, DCM, mol. sieves, 0ºC to rt Ph Br O O N O OMe O Br N H 1-160 O Ph (S)-1-159 1-23b steps BocHN O HN O N Ac 1-163 Br Br N H 1-161b (43%) BocHN OH Br N H 1-162 Br NAc 1. HCO2H, DCM, rt, then EtOH, ∆ 71% 2. NaBH3CN, MeOH, rt 67% 1. Bu3P, 10% HCl, THF, 0ºC 2. Boc2O, DCM 3. NaBH4, THF, H2O 4. 10% LiOH 48% (3S,5R)-cis-3,4- dihydrohamacanthin B (1-144a) In a slightly adapted, but very similar condensation of an indolic diamine intermediate (1- 164) and an indolic oxo-acyl chloride intermediate (1-23a-b) and following cyclization, racemic Hamacanthin A (1-141a) and its related Debromohamacanthins (1-141b-d) were accessed in moderate to high yields, as shown in Scheme 1.46.22 The indolic diamine intermediates (164a-b) were synthesized using the same method discussed previously in the Spongotine Section 58 (Scheme 5). At present, several debromo analogues of Hamacanthin B and Dihydrohamacanthin B have yet to be accessed via total synthesis. Scheme 1.46. Total synthesis of Hamacanthin A (1-141a) and related Debromohamacanthins (1- 141b-d). NHBoc NH2 R1 N H 1-164a (R=H) 1-164b (R=Br) R1 1-23a or 1-23b, Et3N, THF BocHN H N O N H O N H 1-165 1.2.4.3. Fellutanines R2 HCO2H, ClCH2Ch2Cl, ∆, 40 h 36-90% Hamacanthins A and related Debromohamacanthins (3R,6S)-1-141a-d Fellutanines A-C (1-166a-c) were first isolated in 2000 by Kozlovsky et. al. from the fungi Penicillium fellutanum. The 3,6-di-substituted-bis-indolyl-piperazine-2,5-dione structures of these natural products was elucidated via spectroscopic methods (Scheme 1.47). In addition, it was discovered that Fellutanine B (1-166b) contained an isopentenyl group on the 2-position of one of the indole moieties and Fellutanine C (1-166c) contained two isopentenyl substituents, one on the 2-position of each indole moiety.91 The configuration of these Fellutanines A-C (1- 166a-c) was initially reported as the cis-isomer, but this structure was later revised to the correct trans-isomer of the piperazine-2,5-dione core.91 Much of the biological activity of these compounds is unknown, though some studies have shown they possess antibacterial activity, but no significant cytotoxic activity.91, 92 59 Scheme 1.47. Chemical structures of Fellutanines A-C (1-166a-c). R2 NH NH O O HN HN R1 Fellutanine A (1-166a): R1=R2=H Fellutanine B (1-166b): R1=3-isopentenyl, R2=H Fellutanine C (1-166c): R1=R2=3-isopentenyl Interestingly, the first total synthesis of Fellutanine C (1-166c) was completed before its first isolation, as it was an intermediate that was accessed in the 1995 total synthesis of another related fused bicyclic natural product, Gypsetin.93 As is shown in Scheme 1.48, this total synthesis began with indolic C-2 reverse prenylation of N-phthaloyl-L-tryptophan methyl ester (1-167) via prenyl-9-BBN in high yield, followed by hydrazinolysis and subsequent Boc- protection and saponification to access intermediate (1-170) in high yields. Then, 1-170 and 1- 168 were coupled to access the desired cis-Fellutanine C (1-166c) in a very high 87% yield over three steps.93 60 Scheme 1.48. First total synthesis of Fellutanine C (1-166c). CO2Me NPhth 1. (CH3)3COCl, Et3N, THF, -78ºC, 30 min then, prenyl-9-BBN, -78ºC, 6 h 95% 2. N2H4, EtOH, rt, 3 days 65% N H 1-167 CO2Me NH2 N H 1-168 CO2H NHBoc Boc2O, Et3N, THF, 1 h quant LiOH/THF/ MeOH/H2O rt, 3 h 94% N H 1-169 N H 1-170 CO2Me NHBoc 1-168, 1. BopCl, DCM, -78ºC, 6 h 2. THF, DCM, rt, 1 h 3. NH3, MeOH, reflux, 12h 87% Fellutanine C (1-166c) 1-168 ∆ 140ºC 30% Fellutanine C (1-166c) In a similar approach using L-tryptophan analogues as starting materials, Fellutanine C (1-166c) was synthesized via the same intermediate (1-168) used previously, though here undergoing an expeditious dimerization and cyclization via pyrolysis conditions to access cis- Fellutanine C (1-166c) in 30% yield.94 This proved to be an advantageous process in terms of step count by saving four steps compared to the previous synthesis, yet the yield decreased significantly in spite of this. The first total synthesis of Fellutanine A (1-166a) was completed in 2008 in a similar method to that of Fellutanine C (1-166c). Here, a tryptophan analogue (1-169) and TrpOMe•HCl were condensed and subsequently cyclized to access the desired cis-Fellutanine A (1-166a) in 76% yield (Scheme 1.49).95 61 Scheme 1.49. First total synthesis of Fellutanine A-B (1-166a-b). H N BocHN CO2H NHBoc 1. HOBT, DCC, DMF/ DCM, 1 h, 0ºC N H 1-169 2. TrpOMe•HCl, Et3N, N2, rt, overnight 85% H N O 1-171 O O NH 1. HCOOH, Anisole, 3 h 2. Sec-butyl alcohol, toluene, reflux, 3 h 76% Fellutanine A (1-166a) Fellutanine B (1-166b) (>70%) + Fellutanine C (1-166c) (9%) O O P OH O P O OH OH MgCl2 (aq. buffer), rt, pH=7.9, enzymatic reaction In addition to the previously discussed synthetic methods of accessing these Fellutanine natural products, many enzymatic biosynthetic methods have also been utilized over the years, using L-tryptophan as the starting material to access these molecules.96, 97 Recently, a semi-total synthesis was developed and conducted to achieve all three natural products, Fellutanines A-C (166a-c), via the same route. As shown in Scheme 1.49, the same condensation and subsequent cyclization of tryptophan derivatives (1-169) and TrpOMe•HCl was conducted to access cis- Fellutanine A (1-166a) in 76% yield. Then, the enzymatic C2-reverse-prenylation of indole was performed via DMAPP prenyltransferase and aqueous MgCl2 under slightly basic conditions, to access the mono-reverse prenylated cis-Fellutanine B (1-166b) as the major product in >70% yield. It is noteworthy to mention that, while not the major product, cis-Fellutanine C (1-166c) was also accessed via this method in 9% yield. The most significant achievement of this work was the first total synthesis of Fellutanine B (1-166b) and the development of a late stage selective reverse prenylation methodology of indole. It was also a significant that all three Fellutanines A-C (1-166a-c) could be accessed via this same route (Scheme 1.49).98 62 1.2.5. Pyridine and diazine linker moieties 1.2.5.1. Hyrtinadine/Alocasin/Scalaridine Three structurally related bis-indole natural products with heterocyclic aromatic linker moieties were discovered between 2007 and 2013. The first of these was Hyrtinadine, A (1- 172a), which was isolated in 2007 by Endo, et. al. from the Okinawan marine sponge Hyrtios sp.99 This bis-indolyl-2,5-di-substituted pyrimidine structure was elucidated via spectroscopic methods (Scheme 1.50). Hyrtiniadine A (1-172a) was also found to be highly cytotoxic towards murine Leukemia L1210 and human epidermoid carcinoma KB cell lines.99 The next of these related natural products to be discovered was Alocasin A (1-172b), which was isolated in 2012 by Zhu, et. al. from the dried rhizomes of the herbaceous plant Alocasia macrorrhiza.100 Alocasin A (1-172b) was reportedly the first heterocycle-linked bis-indole alkaloid isolated from a terrestrial source. As shown in Scheme 1.50, the chemical structure of (1-172b) has remarkable structural similarity to that of Hyrtinidine A (1-172a). However, the two indole moieties of Alocasin A (1-172b) are connected by a pyrazine linker moiety, rather than the pyrimidine of (1- 172a). Much is unknown regarding the biological activity of Alocasin A (1-172b); however, it has been shown to display weak antiproliferative activity against Hep-2 and Hep-G2 cell lines.100 The most recent of these to be discovered was Scalaridine A (1-173), which was isolated in 2013 by Lee et. al. from the marine sponge Scalarispongia sp., along with the previously discussed Hyrtinidine A (1-172a).101 This natural product (1-173) was determined to have a bis-indolyl-2,5- di-substituted pyridine structure. Interestingly, this is the only bis-indole alkaloid of its kind to contain a pyridine linker moiety. Much is also unknown regarding the biological activity of Scalaridine A (1-173), but much like its two aforementioned structural relatives, it also displayed significant cytotoxicity.101 63 Scheme 1.50. Chemical structures of Hyrtinidine A (1-172a), Alocasin A (1-172b) and Scalaridine A (1-173). H N X Y N N H HO OH HO N OH HN NH Scalaridine A (1-173) Hyrtinadine A (1-172a) (X=C, Y=N) Alocasin A (1-172b) (X=N, Y=C) The first of these natural products to be accessed via total synthesis was Hyrtinadine A (1-172b) in 2011, by Müller, et. al. As shown in Scheme 1.51, the first total synthesis of Hyrtinadine A (1-173) was achieved via sequential palladium-catalyzed Masuda borylation and double Suzuki cross-coupling reactions to install the two indole moieties on the 2- and 5- positions of the pyrimidine core. Subsequent de-methylathylation via BBr3 achieved Hyrtinadine A (1-172a) in 78% yield.102 64 Scheme 1.51. First total synthesis of Hyrtinadine A (1-172a), Alocasin (1-172b), and Scalaridine A (1-173). 1. Pd(PPh3)4 (3 mol%), HBpin, Et3N, 1,4-dioxane, 80ºC, 3 h then, N Br MeO I N Boc 1-174 N Br Cs2CO3, MeOH, 100ºC, o.n. 64% 2. BBr3, DCM, -78ºC to rt, 20 h 78% N Br Br N Pd(PPh3)4, Cs2CO3 1,4-dioxane, 100ºC, 18 h 69% MeO Bpin N Boc 1-175 MeO N Boc 1-176 Hyrtinadine A (1-172a) Boc N N N OMe Pd(PPh3)4 (10 mol%), Cs2CO3, dioxane, 100ºC, 18 h N Br Br 89% MeO N OMe BocN 1-177 NBoc 1. TFA, DCM 90% 2. HBr (aq.), AcOH, reflux, 18 h 43% Alocasin A (1-172b) 1. TFA 91% BBr3, DCM 81% Scalaridine (1-173) In 2015, the first total syntheses of Alocasin A (1-172b) and Scalaridine A (1-173) were completed via a similar borylation and subsequent double Suzuki coupling route as was used in the first total synthesis of Hyrtinadine A (1-172a), followed by de-methylation of the indolic hydroxyl groups to access Alocasin A (1-172b) and Scalaridine A (1-173) in high yields.103, 104 One year later, a different approach to the total synthesis of all three natural products (1- 172a, 1-172b, and 1-173), was conducted via successive palladium-catalyzed reactions; a Kosugi-Migita-Stille cross coupling reaction to install the aromatic substituents on the 65 heterocyclic linker, a reductive N- heterocyclization to form the indole moieties, and a final hydrogenolysis to access 1-172a, 1-172b, and 1-173 in high yields, as shown in Scheme 1.52.105 Scheme 1.52. Total synthesis of Hyrtinadine A (1-172a), Alocasin (1-172b), and Scalaridine A (1-173). BnO SnBu3 Br N N Br BnO NO2 Pd(dba)2, CuI, PPh3, DMF, 90ºC, 41 h 1-178 39% BnO NO2 X Y N NO2 1-180a (X=N, Y=C) 1-180b (X=C, Y=N) Bu3Sn BnO I N N NO2 Pd(dba)2, CuI, PPh3, DMF, 120ºC, 48 h Bu3Sn 53% 1-179 Pd(dba)2, dppp, 1,10-phen, DMF, CO (6 atm), 120ºC, 72 h 68-89% 10% Pd/C, H2 (4 atm), EtOH, 60ºC, 12 h 86-87% Alocasin A (1-172b) (X=N, Y=C) Hyrtinadine A (1-172a) (X=C, Y=N) BnO I Bu3Sn SnBu3 NO2 NO2 N 1-181 Pd(dba)2, CuI, PPh3, DMF, 120ºC, 48 h 35% OBn N 1-182 1. Pd(dba)2, dppp, 1,10-phen, DMF, CO (6 atm), 120ºC, 68 h 67% 2. 10% Pd/C, H2 (4 atm), EtOH, 60ºC, 12 h 79% NO2 OBn Scalaridine A (1-173) As can be concluded from the previously discussed syntheses of Hyrtinidine A (1-172a), Alocasin A (1-172b), and Scalaridine A (1-173), the common method of synthesis for these natural products is the use of palladium-catalyzed cross-coupling reactions to install the indole moieties on the aromatic N-heterocyclic cores. In recent years, additional total syntheses of 1- 172a-b and 1-173 have been reported using similar palladium-catalyzed cross coupling approaches.106, 107, 108 A couple examples of these are shown in Scheme 1.53, such as the 2022 total synthesis of Scalaridine A (1-173) and the significantly expedited one-pot approach using a 66 similar palladium-catalyzed methodology in the total synthesis of Alocasin A (1-172b). The latter achieved Alocasin A (1-172b) in 81% from the iodoindole intermediate (1-183), using a three phase, one-pot method.109 Scheme 1.53. Total synthesis of Alocasin A (1-172b) and Scalaridine A (1-173). MeO I N Ts 1-183 Pd(PPh3)4 (5 mol%), Et3N, HBpin MeO 1,4-dioxane, 80ºC, 4 h Br O B O N Ts 1-184 Br 1. Pd(PPh3)4 (5 mol%), HBpin, Et3N, 1,4-dioxane, 80ºC, 3 h 2. N Br Br N Cs2CO3, MeOH, 100ºC 3. KOH, 100ºC, 3 h 81% Alocasin A (1-172b) N Cs2CO3, MeOH, 60ºC, 18 h MeO N then, KOH, 100ºC, 3 h 64% OMe HN 1-185 NH HBr (aq. 48%), HCOOH, 120ºC, 16 h then, NH3 (aq. 25%), rt, 1 h Scalaridine A (1-173) 1.3. Tris-indole Alkaloids Compared to their previously discussed bis-indole alkaloid counterparts, tris-indole alkaloids are rarer and more elusive in nature. However, in recent years, discovery and synthesis of these natural products has become more prevalent. 1.3.1. Aromatic N-heterocyclic linker moieties 1.3.1.1. Gelliusines Gelliusines A (1-186a) and B (1-186b) were first isolated as enantiomeric pairs in 1994 by Bifulco, et. al. from the deep-water marine sponge Gellius sp. (also known as Orina sp.).110 67 The unique tris-indole chemical structure of these natural products, in which an indole core connects two additional indole moieties at the C-2 and C-6 positions, was elucidated via spectroscopic methods, as shown in Scheme 1.54. In addition, Gelliusines A (1-186a) and B (1- 186b) were determined to also contain three amine substituents. These amine substituents were of particular interest as it is hypothesized that they could contribute to favorable water-solubility of these natural products, which could be beneficial in bioactive applications. The absolute stereochemistry of the chiral centers of 1-186a and 1-186b was inconclusive via obtained data and, because Gelliusines A (1-186a) and B (1-186b) have yet to be accessed via total synthesis, their absolute configurations remain unknown. In terms of their biological activity, Gelliusines A (1-186a) and B (1-186b) were found to display cytotoxic activity, as well as anti-serotonin activity. The latter activity is unsurprising considering the structural similarities of Gelliusines A (1-186a) and B (1-186b) and serotonin.110 Scheme 1.54. Chemical structures of Gelliusines A-C (1-186a-c). Br HN HO NH2 NH2 N H NH2 N H Gelliusine A (1-186a) Gelliusine B (1-186b) Br HN HO NH2 NH2 Br N H Gelliusine C (1-186c) NH2 Br N H One year later, an additional structurally similar natural product of this class, Gelliusine C (1-186c) was isolated by the same group from the deep-water marine sponge Gellius sp. (also known as Orina sp.). As shown in Scheme 1.54, the chemical structure of Gelliusine C (1-186c) was determined to be a similar tris-indole alkaloid containing in indole core connecting two 68 additional indole moieties, as well as containing three amine substituents. The main difference was, instead of the 2,6-substituted indole core of 1-186a-b, the indole core of Gelliusine C (1- 186c) had the two additional indole moieties connected at the C2 and C4 positions. Similar to 1- 186a-b, the absolute configuration of Gelliusine C (1-186c) was inconclusive via obtained data and, because Gelliusines C (1-186c) have yet to be accessed via total synthesis, its absolute configuration also remains unknown. In terms of their biological activity, Gelliusine C (1-186c) exhibited cytotoxic activity, as well as anti-serotonin activity.111 1.3.1.2. Tricepyridinium Tricepyridinium (1-187) is a tris-indole alkaloid with a quaternary pyridinium core that was first isolated in 2017 by Abe et. al. from a culture of Escheichia coli clone incorporating metagenomic libraries from the marine sponge Discodermia calyx. The 1,3,5-tri-substituted pyridinium core connecting the three indole moieties of Tricepyridinium (1-187) was elucidated via spectroscopic methods, as shown in Scheme 1.55.112 Scheme 1.55. Chemical structure of Tricepyridinium (1-187). HN NH N NH Tricepyridinium (1-187) Tricepyridinium (1-187) was found to display potent antibacterial activity, which is summarized in Table 1.4. Several bis-indole analogues of Tricepyridinium (1-187) were also synthesized and tested in this initial exploration of biological activity and 1-187 displayed more 69 potent antibacterial activity than any of these analogues. Therefore, it was concluded that all three indole moieties of 1-187 were necessary for its potent antibacterial activity. In contrast, 1- 187 did not display any cytotoxic activity against murine leukemic P388 cell line, while a bis- indole analogue of 1-187 lacking the indole moiety on the pyridinium nitrogen exhibited significant cytotoxic activity against this same cell line. This indicated that the cytotoxic activity of Tricepyridinium (1-187) was improved via removal of the indole in this position.112 Table 1.4. Known biological activity of Tricepyridinium (1-187).112 Bacteria MIC (µg/mL) B. cerus S. aureus (MSSA) C. albicans E. coli (W3110) 0.78 1.56 12.5 >100 The first total synthesis of Tricepyridinium (1-187) was completed in 2017 by Abe, et. al. at the time of its isolation and structural elucidation to confirm its structure. As shown in Scheme 1.56, a bis-borylation of 3,5-dibromopyridine was completed to access intermediate 1-188, followed by a subsequent palladium-catalyzed double Suzuki coupling to install two of the indole moieties on the pyridine ring. Boc deprotection and late-stage alkylation of the pyridine nitrogen via 1-191 to install the third indole moiety afforded Tricepyridinium (1-187) in high yields.112 70 Scheme 1.56. Total syntheses of Tricepyridinium (1-187). Br N Boc PdCl2(dppf)2, K2CO3 DMSO, 90ºC, 2 h 73% O B O O B O N 1-188 BocN NBoc N 1-189 TFA, DCM, 0ºC, 1 h 97% HN NH N 1-190 Br Br Br N Cs2CO3, Pd(PPh3)4, 1,4-dioxane 88% O B O N Boc 1-192 63% N H 1-191 1,4-dioxane, 90ºC, 42 h Tricepyridinium (1-187) In 2021, the total synthesis of Tricepyridinium (1-187) was completed via a similar approach as previously described. However, the major difference in this approach was the palladium catalyzed Suzuki coupling reaction, in which the electronics of the coupling partners were swapped. In this case, an indolic boronic ester (1-192) was coupled with 3,5- dibromopyridine, similar to the previously discussed approaches toward Scalaridine A (1-173). This approach led to a slightly higher yield of intermediate (1-189) of 88% compared to the previous 73% yield. Then, 1-189 was deprotected and acylated via alkyl bromide 1-191 to access Tricepyridinium (1-187) in 59% yield over two steps (Scheme 1.56).113 1.3.2. Non-aromatic N-heterocyclic linker moieties 1.3.2.1. Tulongicin Tulongicin (1-193) is a tri-indole alkaloid that was first isolated in 2017 by Liu et. al. from Topsentia sp., along with its previously discussed bis-indole alkaloid analogues, 71 Dibromodeoxytopsentin (1-1h), Spongotine C (1-2c), and Dihydrospongotine C (1-3). Tulongicin (1-193) was structurally elucidated via spectroscopic methods. As shown in Scheme 1.57, Tulongicin (1-193) is comprised of an imidazoline core that links three brominated indole moieties. It is noteworthy to mention, 1-193 was the first marine alkaloid of its kind to contain the structurally complex bis-indole methane moiety connected to an imidazoline core. The absolute configuration of (4S)-1-193 was determined via comparison of experimental and calculated circular dichroism (CD) data (MPW1PW91/6-31G(d,p). The only known biological activity of 1-193 is its strong antibacterial activity toward S. aureus (MIC 1.2 mg/mL), its moderate anti-HIV activity (YU2: IC50 3.9 mM, HxB2: IC50 2.7 mM), and its lack of cytotoxicity in mammalian cells. It is also noteworthy to mention that Tulongicin (1-193) has yet to be accessed via total synthesis.13 Scheme 1.57. Chemical structure of Tulongicin (1-193). Br NH Br N H N HN (S) Br N H Tulongicin (1-193) 1.3.2.2. Araiosamines The Araiosamines are arguably the most structurally complex of N-heterocyclic linked tris-indole alkaloid natural products that have been isolated to date. Araiosamines A-C (1-194a- c) were isolated in 2011 by Wei et. al. from the marine sponge Clathria (Thalysias) araiosa.114 As shown in Scheme 1.58, Araiosamines A and B (1-194a-b) contain three brominated indole moieties that are connected by cyclic guanidine and 2- imidazolinone linker moieties. The even 72 more complex Araiosamine C (1-194c) contain three brominated indole moieties that are connected by a very complex fused and bridged cyclic guanidine core. These complex structures were elucidated via careful spectroscopic analysis. It is noteworthy to mention that the absolute configuration of these natural products is not known. The chemical structure of Araiosamine D falls outside the scope of this review.114 Interestingly, at the time of their isolation, Araiosamines A-C (1-194a-c) were not reported to exhibit any significant biological activity. However, several years later when some of these natural products were accessed via total synthesis, the Araiosamines were reported to display significant antibacterial activity against Gram-positive and Gram-negative bacteria, such as S. aureus and E. coli.115 Scheme 1.58. Chemical structures of Araiosamines A-C (1-194a-c). HN H N HN OR NH Br O H N N H Br HN HN Br H N NH HN Br N NH N H NH Br HN HN Br Araiosamine A (1-194a) (R = H) Araiosamine B (1-194b) (R = Me) Araiosamine C (1-194c) Of these Araiosamine natural products, Araiosamine C (1-194c) is the only one to have been accessed via total synthesis. In 2016, Baran, et. al. completed the first total synthesis via key guanidine installation and selective C-H functionalization steps driving toward a biomimetic final step to construct the complex core of 1-194c. As is shown in Scheme 1.59, the first total synthesis of Araiosamine C (1-194c) began with formation of 1-195, followed by deprotection and subsequent guanidinylation. Many guanidinylation reagents that were screened led to no reaction and it was hypothesized that the TFA group would impart adequate reactivity for the 73 guanidinylation reagent to react with hindered or electron deficient amines. Reduction of the ester and subsequent cyclization led to 1-198 in 36% yield over three steps. The oxime intermediate (1-199) was then synthesized and subsequently reduced to the primary amine intermediate via SmI2. The reduction of the oxime (1-199) was very challenging because the oxime moiety is sterically encumbered by two adjacent indole groups and other functionalities such as the N,O-acetal or three aryl bromides are likely more prone toward reduction. After reaction of the resulting amine with N,N-di-Boc-S-methylisothiourea, the guanidine intermediate (1-200) was accessed in 53% over two steps.115 As shown in Scheme 1.59, after deprotection and elimination of the methoxy group via acidic conditions and high temperatures, 1-201 was setup for the pivotal cyclization of the guanidine nitrogen and the fused cyclic imine/enamine to form the characteristic bridgehead of 1-194c. After this sequence, Araiosamine C (1-194c) was accessed in a very high 81% yield.115 74 Scheme 1.59. First total synthesis of Araiosamine C (1-194c). 1. BocHN NTFA NH2 OH CO2Me N N 1-196 THF, rt, BocN NH OH R’ R’’ 1-195 R’’ 2. DDQ, MeCN, rt, R’ R’ = 6-bromo-indol-3-yl R’’=N-Boc-6-bromo-indol-3-yl R’’ R’’ 1-197 CO2Me DIBAL-H, Br DCM, -78ºC 36% (over 3 steps) HN HN Br Br HN N HO OH HN BocN 1-198 PPTS, MeOH/HC(OMe)3 (2:1), rt, then DMP, THF, rt, then NH2OH•HCl, NaOAc, EtOH, 50ºC 63% Br Br 1. SmI2, THF/H2O (6:1), rt 2. SMe BocN NHBoc HgCl2, DMF, rt 53% (over 2 steps) Br HN HN HO N N HN HN BocN OMe 1-199 Araiosamine C (1-194c) (81%) Br Br Br Br HN H N Boc N R HN HN N HN HN BocN OMe 1-200 (R=Boc) TFA, DCM, rt to 90ºC Br Br HN HN NH H N H2N HN N HN HN 1-201 Since the absolute configuration of Araiosamine C (1-194c) was not known, the researchers adapted the previously discussed racemic path to 1-194c to gain access to a stereoselective synthesis. As shown in Scheme 1.60, this was done by utilizing Ellman’s Auxiliary to synthesize the optically active 1-203 in 76% yield as the favored diastereomer (d.r. = 7:1:2:0.5). This stereochemistry was then retained through to 1-205, and using the same methodology as was previously discussed, the optically active (+)-Araiosamine C (1-194c) was 75 synthesized, which based on optical rotation data.115 It is noteworthy to mention that Araiosamines A and B (1-194a-b) have yet to be accessed via total synthesis. Scheme 1.60. Enantioselective total synthesis of Araiosamine C (1-194c). O S N t-Bu NC R’’ 1-175 LiHMDS O S NH t-Bu 76% (d.r. = 7:1:2:0.5) R’ 1-202 R’ R’’ 1-203 CN TMSCl MeOH Boc2O, NaHCO3 (aq.) 78% (over 2 steps) NH2 CN R’ R’’ 1-204 NHBoc (S) CN (S) R’ R’’ 1-205 steps (+)-Araiosamine C (1-194c) 1.4. Conclusions To conclude, the examples discussed in this review summarize the wide range of bis- and tris-indole alkaloids containing N-heterocyclic linker moieties that have been identified and isolated in recent years. These molecules exhibit potent biological activities, such as antibacterial, antiviral, cytotoxic, and anti-inflammatory activities. Natural products of this kind seem to share significant antibacterial activity overall. This could prove to be very advantageous in the future, as antibiotic resistance continues to be a major global issue. However, many of these previously discussed natural products have yet to be accessed via total synthesis, preventing their detailed biological evaluations. Development of synthetic methodologies to access these natural products and explore their promising biological activities will likely be of significant interest to the field in the future. 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Soc., 2016, 138 (43), 14234–14237. https://doi.org/10.1021/jacs.6b09701. 87 Chapter 2: Efforts Toward the Total Synthesis of Tulongicin and Its Related Natural Product Analogues 2.1 Introduction Indole alkaloids are commonly found in a variety of marine sources, including sponges, tunicates, red alga, acorn worms, and symbiotic bacteria, and they represent the largest, and among the most complicated, class of the marine alkaloids.1 Indole alkaloids have been shown to display diverse biological activities, including cytotoxic, antitumor, antiviral, antibacterial, and anti-inflammatory activities.2 Tulongicin 2-1 is a tri-indole alkaloid that was first isolated from Topsentia sp. by Hong- Bing Liu in 2017 near the Ulong Channel in Koror, Palau, along with its bis-indole alkaloids Spongotine C 2-2, Dibromodeoxytopsentin 2-3, and Dihydrospongotine C (2-4).3 This constituted the first isolation of 2-1 and 2-4, however, 2-2 and 2-3 have both been previously isolated. Dibromodeoxytopsentin (2-3) was first isolated in 2005 from Spongosorites sp. and Spongotine C (2-2) was first isolated in 2007 from the same Spongosorites sp.2,4 In the isolation process of these natural products, a 1g sample of Topsentia sp. was extracted with MeOH/DCM (1:1) and compounds were separated via column chromatography to afford Tulongicin (1.6 mg), Spongotine C (7.3 mg), Dibromodeoxytopsentin (1.9 mg), and Dihydrospongotine (134.9 mg). The chemical structures of these natural products are shown in Scheme 2.1. As shown, the chemical structure of Tulongicin (2-1) is comprised of three indole moieties connected by an imidazoline linker. In addition, Tulongicin (2-1) was the first example of a natural product containing a bis-indolemethane moiety linked to an imidazoline ring, making 2-1 an attractive target for total synthesis.3 88 Scheme 2.1. Chemical structures, and absolute stereochemistry of Tulongicin, Spongotine C, Dibromodeoxytopsentin, and Dihydrospongotine C. Br NH Br Br N HN (S) N H Br N H Br O N HN HN (S) N H Br Tulongicin (2-1) Spongotine C (2-2) O N HN HN Br OH (S) N HN HN (S) N H Br N H Br Dibromodeoxytopsentin (2-3) Dihydrospongotine C (2-4) Tulongicin (2-1) was characterized and structurally elucidated via mass spectrometry (HRESIMS) and various nuclear magnetic resonance (NMR) experiments, including 1H, 13C, 1H- 1H COSY, HSQC, and HMBC.3 The absolute configuration of (4S)-2-1 was determined via comparison of experimental and calculated circular dichroism (CD) data (MPW1PW91/6- 31G(d,p). Using similar methods, the absolute stereochemistry of 2-4 was also determined to be (4S,6S)-2-4b.3 The biological properties of Tulongicin (2-1) have not been fully explored. As shown in Table 2.1, the only known biological activity of 2-1 is its strong antibacterial activity toward S. aureus, its moderate anti-HIV activity, and its lack of cytotoxicity in mammalian cells. Compounds 2-2 and 2-4 have been shown to display similar strong antibacterial and moderate 89 anti-HIV activity. Compound 2-2 exhibited the strongest antibacterial activity, while 2-3 displayed significantly decreased activity (Table 2.1).3 Table 2.1. Biological activity of Tulongicin (2-1) and its related natural product analogues. Natural Product 2-1 2-2 2-3 2-4 Antibacterial activity S. aureus (MIC) 1.2 mg/mL 1.1 mg/mL 11.0 mg/mL 3.7mg/mL Anti-HIV activity YU2 (IC50) 3.9 mM 9.5 mM 57.0 mM 3.5 mM HxB2 (IC50) 2.7 mM 12.0 mM N/A 4.5 mM It is notable to mention that both 2-2 and 2-3 have been previously accessed via total synthesis. The first total syntheses of both 2-2 and 2-3 were completed by Guinchard, et. al., in 2007.5 This first total synthesis of 2-2 and 2-3 is shown in in Scheme 2.2. As shown, 2-2 was accessed via cyclization of a diamine fragment and a keto-thioimidate fragment to form the imidazoline core. Then, compound 2-3 was accessed via oxidation of 2-2. The keto-thioimidate fragment was synthesized in four steps from the commercially available 6-bromo-1H-indole. However, the diamine fragment was synthesized in 5 steps from the non-commercially available brominated indolic N-hydroxylamine (Scheme 2.2). Therefore, it is important to note that two additional steps, nitrone formation and subsequent indolic hydroxylamine synthesis, were necessary to access this hydroxylamine staring material from commercially available tert-butyl (2-oxoethyl) carbamate, N-benzylhydroxylamine, and 6-bromo-1H-indole.6 Thus, 2-2 and 2-3 were accessed in seven and eight linear steps, respectively, from commercially available starting materials. 90 Scheme 2.2. First total syntheses of Spongotine C (2-2) and Dibromodeoxytopsentin (2-3). 2.2a. Synthesis of 1-(2-(6-bromo-1H-indol-3-yl)-1-(methylthio)-2-oxoethylidene)piperidin-1-ium fragment: O Cl O N H n-Bu3SnH EtOAc, 0ºC to r.t., 18h Br (COCl)2 Br N H Et2O, 0ºC, 4h, 82% Br S8, piperidine pyridine, 80ºC, 5h 35% (over 2 steps) Br S N O N H MeI, Reflux, 5h Quant. Br O N H O H O N H I S N 2.2b. Synthesis of 1-(6-bromo-1H-indol-3-yl)ethane-1,2-diamine dihydrochloride fragment: NHBoc OH N Ph Br N H Accessed in 2 steps from commercially available materials MnO2 Toluene, 100ºC, 10 min, 77% Br NHBoc O N Ph N H NHOH•HCl MeOH r.t., 1.5h 72% Br NHBoc NHOH N H TiCl3, HCl MeOH, r.t., 10 min, 92% Br NHBoc NH2 HCl NH2•HCl NH2•HCl N H MeOH, 0ºC 2h, 99% Br N H 2.2c. Synthesis of Spongotine C (2-2) and Dibromodeoxytopsentin (2-3): I S N Et3N MeOH r.t., 24h 65% Br NH2•HCl NH2•HCl N H O N NH N H Br IBX Br DMSO, 15h, r.t. 90% N H O N NH N H Br Spongotine C (2-2) Dibromodeoxytopsentin (2-3) O N H + N H Br Br 91 Tulongicin (2-1) and Dihydrospongotine C (2-4) are some of the known tris- and bis- indole alkaloids that have yet to be accessed via total synthesis, as highlighted in chapter 1 of this thesis. Chapter 2 of this thesis will discuss efforts in the first total syntheses of Tulongicin (2-1) and Dihydrospongotine C (2-4), as well as expedited total syntheses of Spongotine C (2-2) and Dibromodeoxytopsentin (2-3). 2.2 Results & Discussion Although a considerable amount is known about the biosynthesis of indole alkaloids7,8, studies of the specific chemotypes in Tulongicin (2-1) and Dihydrospongotine C (2-4) have yet to be published.3 However, the fact that 2-1, 2-2, 2-3, and 2-4 were all isolated from Topsentia sp. can give some insight into potential transformations that could lead to these compounds from one another. For instance, it is conceivable that 2-1 could be accessed via a Friedel Craft’s alkylation of 2-2 or 2-4 and that 2-3 and 2-4 could be accessed via oxidation and reduction of 2- 2, respectively. This potential interconversion of these related natural products was taken into consideration when planning the first total synthesis of Tulongicin 2-1 and its analogues (2-2, 2- 3, and 2-4). In addition, particular consideration was given to the Friedel Craft’s alkylation step in planning the first total synthesis of 2-1, as, to the best of my knowledge, a reaction such as this has never been utilized to access a similar complex bis-indole methane moiety attached to an imidazoline ring. 2.2.1. Initial retrosynthetic analysis for the total synthesis of Tulongicin (2-1) via Spongotine C (2-2) as an intermediate (Route A) My initial retrosynthesis for the first total synthesis of Tulongicin (2-1) (Route A) is shown in Scheme 2.3. It was hypothesized that 2-1 could be accessed via a reductive Friedel Craft’s alkylation of 2-2. In turn, 2-2 could be accessed through a [3+2] cyclization of 6-bromo- 92 1H-indole-3-carbonyl cyanide (2-5) and 6-bromo-3-(1-tosylaziridin-2-yl)-1H-indole (2-6). The cyclization of cyanide and aziridine moieties to form imidazoline rings is precedented in literature, however the use of a carbonyl cyanide moiety would be an expansion upon previous work and has yet to be attempted to the best of my knowledge.9 As shown below, 2-5 and 2-6 could be accessed in 2 and 3 steps, respectively from the same, commercially available 6-bromo- 1H-indole starting material.10,11,12 This approach would also be an expedited total synthesis of 2- 2 compared to its aforementioned previous first total synthesis (Scheme 2.2). In addition, as is shown in Scheme 2.3, it is hypothesized that Dibromodeoxytopsentin (2-3) and Dihydrospongotine C (2-4) could also be accessed via Spongotine C (2-2) as an intermediate via a single oxidative or reductive step, respectively. 93 Scheme 2.3. Initial retrosynthesis of Tulongicin (2-1), Spongotine C (2-2), Dibromodeoxytopsentin (2-3), Dihydrospongotine C (2-4) – Route A. Br NH N HN N H Tulongicin (2-1) Br N H Reductive FC Br Br N H N N H O N H Br N H Spongotine C (2-2) Br Br O N H N N H 2-3 Br N H Oxidation Spongotine C (2-2) Reduction HO N N H N H 2-4 N H Br Br Imidazoline cyclization ([3+2] cycloaddition) Ts N N H 2-6 + Br Aziridination O N Br N H 2-5 Amide dehydration to nitrile O O NH2 Br N H 2-7 Oxoacet- amidation Br N H 2-8 Wittig O H Vilsmeier- Hack Br N H (Commercially Available) Br N H 2-9 The transformation of 2-2 to 2-1 via a reductive Friedel Craft’s alkylation is based off previous work by Savela et. al. As shown in Scheme 2.4, Savela and co-workers showed that 94 triaryl products could be accessed via a one-pot FeCl3-catalyzed, silane-mediated reductive Friedel Craft’s alkylation.13 However, Savela’s methodology had only been applied to substituted benzaldehydes or benzyl ketones with substituted benzene nucleophiles. Therefore, applying a similar approach for the transformation of 2-2 to 2-1 to form the synthetically challenging bis indole methane moiety linked to the imidazoline ring would be a significant expansion of this methodology. Figure 2.4. Literature precedence for reductive Friedel Craft’s alkylation, as well as the proposed expansion of this methodology in the first total synthesis of (2-1). Previous Work: O R1 + R2 (R1 = H or Me) (R2 = alkyl or aryl) FeCl3 (cat.), Me3SiCl, Et3SiH 50-90ºC R1 R2 Br This Work: O N HN HN 2-2 N H N H Br Br Br Br NH N N H HN 2-1 Br N H 2.2.1.1. Indole carbonyl cyanide fragment synthesis The indole carbonyl cyanide (2-10) was synthesized from 1H-indole in two steps, as shown in Scheme 2.5.10 It is important to note that, for cost purposes, 1H-indole was used as a model starting material in this synthesis rather than 6-bromo-1H-indole in progress toward 2-5 to allow for preliminary optimization. The first step involved oxoacetamidation of 1H-indole to 95 afford the 2-(1H-indol-3-yl)-2-oxoacetamide intermediate (2-9) (97%). Subsequent amide dehydration to the nitrile afforded 2-10 in high yield (79%). Scheme 2.5. Synthesis of the indole carbonyl cyanide fragment (2-10). N H (COCl)2 (1.5 equiv) Et2O, 0 ºC, 2 h then, sat. aq. NH4Cl, sat. aq. KOH, r.t., 1 h 97% O O NH2 SOCl2 (2.5 equiv) O N N H 2-9 DMF, 0 ºC, 3 h 81% N H 2-10 2.2.1.2. Indolic aziridine fragment synthesis The synthesis of 2-6 was identified as a key step for optimization. This was because previous reports have highlighted aziridination of electron dense systems, such as this indole system, to be significantly more difficult than that of more electron poor systems.14,15,16,17 Therefore, model systems were used for initial optimization of this aziridination toward the synthesis of 2-6. First, a styrene model system was used. As shown in Scheme 2.6, the 2-phenyl- 1-tosylaziridine product (2-11a) was accessed in high yield (70%) using Chloramine-T•H2O as the nitrogen source (Table 2.1). Next, in order to more closely represent the electron rich indole ring system of 2-6, a para-methoxy styrene model system was used to further optimize reaction conditions. As shown by the data in Table 2.1, the yields were significantly lower for the 2-(4- methoxyphenyl)-1- tosylaziridine product (2-11b) (8-16%) compared to that of the (2-11a) (70%) utilizing a variety of reaction conditions. 96 Scheme 2.6. Aziridination of styrene model systems to afford (2-11a) and (2-11b). (See Table 2.1) R Ts N R 2-11a (R = H) 2-11b (R = OMe) Table 2.1. Reaction condition screening for aziridination of styrene model systems. R Nitrogen Source Catalysts/Additives* Solvent H Chloramine-T• H2O (1 equiv) OMe Chloramine-T• H2O (1 equiv) OMe Chloramine-T• H2O (1 equiv.) OMe PhINTs (1 equiv) OMe PhINTs (1 equiv) I2/ TBAB DCM/H2O (2:1) I2/ TBAB DCM/H2O (2:1) CuI/ TBAB DCM/H2O (2:1) I2/ TBAI I2/ TBAI CH3CN CH3CN Temp Rxn Time 24 h rt Yield 70% rt rt rt rt 24 h 8% 24 h 15% 3 h 18 h 15% 10% *Catalytic amount = 10. mol% As shown in Table 2.1, this decreased yield can likely be attributed to the increased electron density of the para-methoxy styrene model system. It is also evident from the data that copper aids in activating this alkene toward aziridination, as shown by the slightly higher yields of the CuI catalyzed reactions (Table 2.1). Also, as shown by the data, it appears that Chloramine-T•H2O and N-tosyliminobenzyliodinane (PhINTs) were similar in their effectiveness as a nitrogen source. However, it is not clear if PhINTs was allowed to reach its full potential as it suffered significant degradation issues, evidenced by isolation of significant amounts of p- toluenesulfonamide and iodobenzene degradation products after purification. This degradation can also explain why the yields of the PhINTs-mediated aziridinations decreased as time increased (Table 2.1). If this degradation issue were to be solved or mitigated, PhINTs may be a superior nitrogen source for aziridinations of electron dense alkene systems, as has been reported in literature.14,15 97 Though the yields of (2-11b) were relatively low, the focus was shifted to the target compound, 2-6 in the interest of time. It is noteworthy to mention that, for cost purposes, 1H- indole was used as a model starting material for initial optimization. In the synthesis of 2-15, first, the 1-tosyl-3-vinyl-1H-indole intermediate (2-14) was synthesized in three steps from 1H- indole (Scheme 2.7).11,12,18 The first step toward (2-14) was the Vilsmeier-Haack formylation of 1H-indole at the C-3 position to afford 2-12 in good yield (53%).12 Next, an N-tosylation of 2-12 was completed to access 2-13 in quantitative yield, followed by a subsequent Wittig reaction to afford the vinyl indole intermediate 2-14 in good yield (70%).11,18 Once 2-14 was accessed, the aziridination of this compound was explored (Scheme 2.8). Table 2.2 details the various conditions that were explored in the synthesis of 2-15 via aziridination of 2-14. Scheme 2.7. Stynthesis of the 1-tosyl-3-vinyl-1H-indole intermediate (2-14). Vilsmeier Haack 1. POCl3 (3.0 equiv) 2. 20% NaOH soln. DMF, 0 ºC to rt, 24 h 53% O H N H 2-12 NaH (2.5 equiv), TsCl (1.6 equiv) THF, 0 ºC to r.t., 18 h quant O H N Ts 2-13 N H Wittig Ts N N Ts 2-15 CH3PPh3Br (1.5 equiv), nBuLi (1.5 equiv) THF, -30º C, 1 h 70% N Ts 2-14 (See Table 2.2) 98 Table 2.2. Aziridination of 3-vinyl indole reaction conditions. Nitrogen Source Catalysts/Additives Solvent Temp Chloramine-T• H2O (1 equiv) PhINTs (1 equiv) TsNH2 (2.0 equiv) CuI (0.1 equiv)/ TBAB (0.1 equiv) CuI (0.1 equiv)/ TBAI (0.05 equiv) PhI(OAc)2 (1 equiv)/ Cu(CH3CN)4PF6 (0.05 equiv) DCM/H2O (2:1) MeCN DCM rt rt rt Rxn Time 24 h 24 h 24 h Yield No pure product isolated* No pure product isolated* No pure product isolated* *2-15 identified via crude NMR and mass spectrometry (HRMS-ESI (m/z): Calculated for C24H22N2O4S2 (M+H)+: 467.1099, Found: 467.1110). As shown by the results outlined in Table 2.2, the aziridination of 2-14 proved to be very difficult. Though a variety of reaction conditions were explored, no desired product 2-15 was ever isolated (Table 2.2). As shown in Table 2.2, conditions to form PhINTs in situ were also explored utilizing p-toluensulfonamide and PhI(OAc)2 in efforts to avoid the aforementioned problematic degradation of PhINTs.19 However, 2-15 was detected via crude NMR and mass spectrometry in all trials detailed in Table 2.2. Therefore, it is believed that the problem with this aziridination was not the efficiency of the reaction, but the stability of the product, leading to difficulty isolating 2-15. This instability is likely because the indole ring system of 2-14 is more electron dense than that of the previous para-methoxy styrene model system. In fact, the presence of its heteroatomic nitrogen atom allows for potential intramolecular nucleophilic ring opening of the strained aziridine ring of 2-15, especially in the presence of any mild acid upon workup. Specifically, cascade of the indole nitrogen lone pair of electrons through the ring could result in formation of an exocyclic alkene, opening the aziridine. The N-tosyl moiety was strategically present in (2-14) to mitigate this issue. However, it is evident that, while this N-tosylation may have allowed for the aziridination to occur, it did not allow for product stability. This was the first problem that indicated this initial synthetic route to access 2-2 may needed to be adapted. 99 2.2.1.3. Exploration of the [3+2] imidazoline cyclization Another unforeseen problem with this initial synthetic approach (Route A) was identified in the initial explorations of the [3+2] imidazoline cyclization step. Preliminary exploration was conducted via a model system in which 2-11a was cyclized with 2-10, as shown in Scheme 2.8. Notably, there was no desired product formation, rather, an unexpected major side product was isolated instead. Scheme 2.8. Initial exploration of the [3+2] imidazoline cyclization – side product formation. Ts N + O N N H BF3OEt2 (10 mol%) DCM, rt, 4 h O N HN HN + 2-11a 2-10 2-16 (No evidence of product) Ts N N Ts 2-17 (76%) As shown in Scheme 2.8, the desired [3+2] cycloaddition reaction to form the imidazoline core did not occur in this model system. Instead, the unexpected 2,5-diphenyl-1,4- ditosylpiperazine side product 2-17 was isolated in significant yield (76%) as the only product formed. It was hypothesized that 2-17 was afforded by the cyclization of 2-11a with another molecule of itself. This was confirmed by subsequent control reactions in which 2-11a was reacted with and without Lewis acid (10 mol% BF3OEt2), as shown in Scheme 2.9. The results and specific reaction conditions of these control reactions are outlined in Table 2.3. Scheme 2.9. Side product formation – control experiments. (See Table 2.3) Ts N 2-11a Ts N N Ts 2-17 100 Table 2.3. Exploration of side product (2-17) formation – reaction conditions. Trial Lewis Acid Solvent Temp. (ºC) Time Yield of (2-17) 1 2 BF3OEt2 (10 mol%) None DCM 20 ºC DCM 20 ºC 4 h 4 h Major product in crude NMR No product formation identified As shown, the data indicated not only that 2-11a can cyclize with itself, but also that the Lewis acid is necessary in the cyclization of the piperazine ring, which supported the hypothesized mechanism. Therefore, a potential mechanism for this cyclization was determined, as shown in Scheme 2.10, in which the BF3OEt2 coordinates to, and activates, the nitrogen atom of 2-11a. This facilitates nucleophilic attack by the nitrogen atom of another 2-11a molecule. Once this intermediate is formed, the now activated remaining aziridine can then open via nucleophilic attack of the other N atom and undergo subsequent cyclization. This likely arises from the combination of the very reactive activated aziridine and the weakly nucleophilic cyanide (2-10), leading to a faster reaction between 2-11a and itself than the desired reaction between 2-11a and 2-10. Similar Lewis acid-catalyzed cyclizations of aziridines to form piperazine rings have been reported in literature.20 Considering these problems that arose with Route A, adaptations were made in order to mitigate these issues. 101 Scheme 2.10. Proposed mechanism of piperazine side product (2-17) formation. Ts N 2-11a BF3OEt2 BF3OEt2 Ts BF3OEt2 N Ts N 2-11a BF3OEt2 N Ts N Ts Ts N N Ts 2-17 2.2.2. Adapted retrosynthetic analysis for the total synthesis of tulongicin (2-1) via Spongotine C (2-2) as an intermediate (Route B) To address both major problems associated with Route A, a newly adapted route Route B was developed, as shown in Scheme 2.11. This route was hypothesized to solve the problems of aziridine instability (2-6) and its problematic dimerization by utilizing a more stable diamine fragment (2-18) to cyclize with 2-5 to form the imidazoline core of Spongotine C (2-2) (Scheme 2.11). Despite the use of the same cyanide fragment (2-5), the mechanism of this imidazoline cyclization would likely be more favorable than Route A because 2-5 would now act as the electrophile rather than the nucleophile. Similar cyclizations have been previously reported via indolic carbonyl cyanides and cysteine methyl esters to form thiazolines.21 Therefore, this approach would be an expansion upon this previous work to utilize a diamine fragment instead (2-18). As shown in Scheme 2.11, it was hypothesized that 2-18 could be synthesized in two steps from the commercially available 6-bromo-1H-indole via di-amination and subsequent bis- deprotection. This adapted route would also be a more expeditious way to access (2-2) with a longest linear sequence (LLS) of three steps as opposed to the five steps in Route A. 102 Scheme 2.11. Adapted retrosynthesis of Tulongicin (2-1) via Spongotine C (2-2) as an intermediate – Route B. Br N N H O N H N H Spongotine C (2-2) Br N H Reductive FC Tulongicin (2-1) Br Imidazoline cyclization O N HCl•H2N NH2•HCl Br N H 2-5 + Br N H 2-18 Amide dehydration to nitrile O NH2 O Br N H 2-7 Oxoacet- amidation Deprotection BnHN NHBoc Br N H 2-19 Di-amination (Mannich-like) Br N H (Commercially Available) 2.2.2.1. Initial exploration of imidazoline cyclization using a model system To explore the feasibility of this approach, a model system was first investigated using commercially available starting materials. As in Scheme 2.12, propane-1,2-diamine and benzoyl cyanide underwent cyclization catalyzed by 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). However, rather than the desired cyclized product (2-20), the unexpected imidazoline side product (2-21) was isolated in good yield (65%). This likely occurred due to initial attack of the more electrophilic carbonyl moiety, rather than the cyanide, leading to loss of cyanide as a leaving group and subsequent cyclization to the imidazoline (2-21). 103 Scheme 2.12. Exploration of model imidazoline cyclization via a model system to access 2-21. H2N NH2 (1.0 equiv) DBU (10 mol%) DMF, rt, 4 h O N O N HN 2-20 N N H 2-21 (65%) 2.2.2.2. Indolic diamine fragment synthesis Despite the identification of this side product, the focus was moved to the target compound since this carbonyl cyanide was electronically different than the model benzoyl cyanide, containing a less electrophilic ketone. First, the synthesis of a model diamine was caried out, as shown in Scheme 2.13, in 2 steps from 1H-indole.22 Again, for initial optimization 1H- indole was used as a model starting material for cost purposes. First, the di-protected diamine (2- 23) was accessed via a Mannich-like di-amination of 1H-indole in good yields (50%). Subsequently, 2-23 underwent Boc-deprotection via ethanolic hydrochloric acid to remove the Boc protecting group, as shown in Scheme 2.13, to access 2-24. It is noteworthy to mention the debenzylation was postponed to a later stage to prevent rotamers, allowing for more facile analysis of the cyclization step. The debenzylation remains a point of future optimization. Scheme 2.13. Synthesis of diamine fragment (2-24). BnNH2 (1.0 equiv), O BnHN NHBoc BnHN NH2•HCl BocHN H 2-22 (1.0 equiv) TMG (10 mol%) N H H2O, 50 ºC, 12 h, 50% N H 2-23 104 HCl-EtOH rt, 18 h, quant N H 2-24 2.2.2.3. Exploration of imidazoline cyclization via indolic diamine and carbonyl cyanide fragments Next, the imidazoline cyclization of the diamine fragment (2-24) and the carbonyl cyanide fragment (2-10) was investigated (Scheme 2.14). However, as shown in Table 2.4, no evidence of the desired product (2-25) was identified among several different reaction conditions. Due to the fact that this cyclization of 2-24 and 2-10 was not leading to any desired product formation, the synthetic route toward Spongotine C (2-2) was again adapted. Scheme 2.14. Imidazoline cyclization of the indolic diamine fragment (2-24) and the carbonyl cyanide fragment (2-10). BnHN NH2•HCl O N + N H 2-24 N H 2-10 DBU (10 mol%) DMF, rt, Time O N HN BnN 2-25 N H Table 2.4. Reaction condition screening for the imidazoline cyclization of 2-24 and 2-10. Trial Time Yield 1 2 3 4 h No desired product isolated 24 h No desired product isolated 48 h No desired product isolated 2.2.3. Adapted retrosynthetic analysis for the total synthesis of Tulongicin (2-1) via Spongotine C (2-2) as an intermediate (Route C) Due to this lack of reactivity in (Scheme 2.14), an adapted synthetic route (Route C) was developed. In Route C, the same diamine fragment (2-24) was used in the key cyclization to form the imidazoline core of Spongotine C (2-2), as well as a new keto-thioimidate fragment (2- 105 26) (Scheme 2.15). This approach is similar to that of the first total synthesis of Spongotine C (2-2) (Scheme 2.2). However, this approach is more expedited (LLS: 5), particularly in the synthesis of the diamine fragment (2-18). As is shown in Scheme 2.15, this keto-thioimidate fragment (2-26) could be synthesized in three steps from the commercially available 6-bromo- 1H-indole, following the same reaction sequence as the first total synthesis of Spongotine C (2- 2) (Scheme 2.2).5 It was hypothesized that the keto-thioimidate fragment (2-26) is essentially a more activated version of that of the previously explored carbonyl cyanide fragment (2-5). The more electrophilic imidate moiety and a good sulfur leaving group will, ideally, drive the reaction to occur. Scheme 2.15. Adapted retrosynthesis of Tulongicin (2-1) via Spongotine C (2-2) as an intermediate – Route C. Br N N H O N H N H Spongotine C (2-2) Br N H Reductive FC Tulongicin (2-1) Imidazoline cyclization Br O S N I HCl•H2N NH2•HCl Br N H 2-26 + Br N H 2-18 Methylation Deprotection O S N BnHN NHBoc Br N H 2-27 Thioamination Br N H 2-19 Di-amination (Mannich-like) O Cl oxo-acyl chloride formation O N H 2-28 Br N H (Commercially Available) Br 106 2.2.3.1. Indolic keto-thioimidate fragment synthesis In exploration of Route C, the keto-thioimidate fragment (2-32) was synthesized in three isolated steps from the commercially available 1H-indole (used as a model starting material) (Scheme 2.16). First, 1H-indole was treated with oxalyl chloride to afford 2-29 in excellent yield (87%). Next, 2-29 was reduced using n-Bu3SnH to the ketoaldehyde intermediate (2-30), which immediately underwent a reaction with S8 and piperidine to afford 2-31 in good yield (45% over two steps). Lastly, 2-31 was S-methylated to access 2-32 in quantitatively. Scheme 2.16. Synthesis of the keto-thioimidate fragment (2-26). (COCl)2 N H Et2O, 0 ºC, 4 h, 87% S8 (1.2 equiv), piperidine (1.5 equiv) pyridine, 80 ºC, 5 h 45% (Over 2 steps) O O Cl N H 2-29 S N O N H 2-31 n-Bu3SnH (1.0 equiv) EtOAc, 0 ºC to rt, 18 h MeI, Reflux, 5 h quant O O H N H 2-30 O N H 2-32 I S N 2.2.3.2. Exploration of the base mediated imidazoline cyclization via the keto-thioimidate and diamine fragments Next, the base-mediated imidazoline cyclization of the keto-thioimidate fragment (2-32) and the diamine fragment (2-24) was explored, as shown in Scheme 2.17. As is detailed in Table 2.5, of the reaction conditions screened, the highest yield of the desired imidazoline product (2- 25) was achieved using triethylamine (Et3N) as a base and a 48-hour reaction time (53%). This marked exciting progress toward the first total synthesis of Tulongicin and its related analogues, as this was the first successful imidazoline cyclization. As shown in Table 2.5, overall, yields of 107 this cyclization were lower when Amberlyst-A21was used as the base. It is notable to mention that this is likely due to the difficult workup, rather than efficiency of the reaction. Upon workup, the resin proved difficult to sufficiently wash and extract the product. Scheme 2.17. Base-mediated imidazoline cyclization of the keto-thioimidate fragment (2-32) and the diamine fragment (2-24). O S N I + N H 2-32 BnHN NH2•HCl Base MeOH, rt, Time N H 2-24 O N HN BnN 2-25 N H N Amberlyst A-21 Table 2.5. Reaction time optimization for the base-mediated imidazoline cyclization of the keto- thioimidate fragment (2-32) and the diamine fragment (2-24). Base Time Yield Amberlyst A-21 Amberlyst A-21 Amberlyst A-21 Et3N Et3N Et3N 24h 36h 48h 24h 48h 60h 26% 32% 32% 49% 53% 52% 2.2.4. Adapted retrosynthetic analysis for the total synthesis of Tulongicin (2-1) via Spongotine C (2-2) as an intermediate (Route D) In the search of a more optimized and novel synthetic route toward Tulongicin (2-1) and its related analogues, a more expedited synthetic route was developed (Route D). As shown by the retrosynthetic detailed in Scheme 2.18, it was hypothesized that the imidazoline core could be formed via a cyclization of a diamine fragment (2-18) and a keto aldehyde fragment (2-33). 108 Such cyclizations have been precedented in literature.23, 24 It was hypothesized that 2-33 could be accessed in two steps from commercially available 1-(6-bromo-1H-indol-3-yl)ethan-1-one (Scheme 2.18). This route to access Spongotine C (2-2) would be significantly more expeditious (LLS = 3), compared to Route C (LLS = 5) and the first total synthesis of Spongotine C (2-2) (LLS =7) (Scheme 2.2). This approach would also allow for the first total synthesis of Tulongicin (2-1) in four linear steps. Scheme 2.18. Adapted retrosynthesis of Tulongicin (2-1) via Spongotine C (2-2) as an Imidazoline cyclization Br intermediate – Route D. Br N N H O N H N H Spongotine C (2-2) Br N H Reductive FC Tulongicin (2-1) O O H HCl•H2N NH2•HCl Br N H 2-33 Kornblum Oxidation + Br N H 2-18 Deprotection O BnHN NHBoc Br N H 2-34 Acylation Br N H 2-19 Di-amination (Mannich-like) Br N H (Commercially Available) 2.2.4.1. Indolic keto-aldehyde fragment synthesis via Kornblum oxidation and subsequent imidazoline cyclization – non-brominated model system The indolic keto-aldehyde intermediate (2-35) was accessed in one step from 1H-indol-3- yl)ethan-1-one as a model staring material for initial optimization. As shown in Scheme 2.19, 109 1H-indol-3-yl)ethan-1-one underwent iodination and subsequent Kornblum Oxidation25 to access 2-35. This was then directly carried forward to the imidazoline cyclization with the diamine fragment (2-24) in a one-pot method, followed by subsequent oxidation via NCS to the afford the imidazoline product (2-25) in good yield over two steps (45%) (Scheme 2.19). This was a very significant result toward the first total synthesis of Tulongicin (2-1) and its related analogues in that it constituted the most expeditious route to access 2-25 thus far. In addition, intermediate 2- 25 is only one debenzylation step away from a non-brominated Spongotine C analogue. Considering the previously reported problematic de-halogenation of heterocycles when subjected to hydrogenative debenzylation conditions,22 it was hypothesized that the final debenzylation reaction would be better optimized using the target brominated scaffold, rather than the non- brominated model system (2-25). This would allow for monitoring of any potential de- halogenation when screening debenzylation conditions. Therefore, the final debenzylation step remains a point of exploration and optimization for the future. Scheme 2.19. Synthesis of imidazoline intermediate (2-25) via Kornblum oxidation and subsequent oxidative imidazoline cyclization. Kornblum Oxidation O I2 (1.1 equiv) N H DMSO, 100 ºC, 2 h O H O N H 2-35 1. NaHCO3 (4.0 equiv), NH2•HCl BnHN 2-24 N H MeCN/MeOH (1:1), 0 ºC, 1 h 2. NCS (1.0 equiv) 0 ºC to rt, 12 h 45% (over 2 steps) O N H N N Bn 2-25 NH 110 2.2.5. Optimization of a one-pot reductive Friedel Craft’s methodology 2.2.5.1. Reductive Friedel Craft’s alkylation of benzophenone with anisole – model system. In the exploration and optimization of the one-pot reductive Friedel Craft’s alkylation, model systems were first explored. The first model system explored was the FeCl3-catalyzed reductive alkylation of benzophenone with anisole (2-36) as the nucleophile, as in Scheme 2.20. This model system afforded the desired product (2-37) in high yields and regioselectivity (91% (98:2 para:ortho)). Scheme 2.20. Reductive Friedel Craft’s alkylation of benzophenone with anisole – model system. O O O (2-36) (excess), FeCl3 (4 mol%) Me3SiCl (1.1 equiv) Et3SiH (1.05 equiv) rt, 2 h, then 50 ºC 1 h 91% (98:2 2-37a:2-37b) + O 2-37a 2-37b 2.2.5.2. Reductive Friedel Craft’s alkylation of benzophenone with indole nucleophiles – model system. Another reductive Friedel Craft’s model system was then explored. The FeCl3-catalyzed reductive alkylation of benzophenone (B) with indole nucleophiles was explored, as it was more representative of the target methodology for the total synthesis of Tulongicin (2-1). Both 1H- indole (A) and N-tosyl-1H-indole (2-38) were investigated as shown in Scheme 2.21. 111 Scheme 2.21. Reductive Friedel Craft’s alkylation of benzophenone with indole nucleophiles – model system. O (B) FeCl3 (X mol%) Me3SiCl (1.1 equiv) Et3SiH (1.05 equiv) Solvent, Temp. 1, Time 1 Then, N R (R = H (A), Ts (2-38)) Solvent, Temp. 2, Time 2 R N (2-39) (R = H) (2-40) (R = Ts) N R N R (2-42) (R = H) (2-43) (R = Ts) + + (2-41) R N RN NHR (2-44) (R = H) (2-45) (R = Ts) This second model system of the reductive Friedel Craft’s alkylation (Scheme 2.21) proved to be more difficult than the first model system. This was due to competitive side reactions. One side product that was isolated was diphenylmethane (2-41). This was likely a result of competitive over-reduction of benzophenone via the triethyl silane reducing agent due to the slower alkylation reaction times for this model system. However, this over-reduction occurred before complete consumption of the benzophenone starting material (B), as significant amounts of (B) were recovered in all trials. This increased reaction time correlated to the tendency of these indole nucleophiles, 1H- indole (A) or N-tosyl-1H-indole (2-38), to polymerization in Lewis acidic conditions.26,27,28 From these model reactions, indole dimer (2-42 or 2-43) and trimer side products (2-44 or 2-45) were identified and/or isolated, as shown in Table 2.6. In several of these trials, varying amounts of a brown/black insoluble precipitate was produced. This was likely correlated to further, more complex indole polymerization.26,27 In order to mitigate this problem of indole polymerization, 112 several different parameters were investigated including temperature, time, solvent, etc. as shown in Table 2.6. Table 2.6. Reductive Friedel Craft’s Alkylation Optimization. Trial R 1* H Solvent Mol% FeCl3 (X) 4 DCE Equiv (B) 1.0 Equiv (A) or (2-38) 5.0 Temp 1 Temp 2 Time 1 Time 2 Yield 2* H DCE 3* H DCE 4 Ts DCE 4 4 4 rt à 50 ºC rt à 50 ºC 50 ºC 1 h 3 h 50 ºC 1 h 3 h 1.0 1.0 1.0 2.0 rt à 50 ºC 0 ºC à rt 1 h 2 h (0 ºC), 10 h (rt) 1.0 2.0 rt à 50 ºC rt à 60º C 1 h 12 h 5 Ts DCE 10 1.0 2.0 rt à 50 ºC rt à 60 ºC 1 h 12 h 6 H NO2Me 4 1.0 2.0 rt rt 1 h 12 h 7 H NO2Me 10 1.0 2.0 rt 8 Ts NO2Me 10 1.0 2.0 rt 9 H NO2Me 10 1.0 2.0 rt 10 H NO2Me 10 1.0 2.0 rt 0 ºC à rt 1 h rt à 60 ºC 1 h -15 ºC à rt 1 h -15 ºC à rt 0 h 2 h (0 ºC), 12 h (rt) 2 h (rt), 12 h (60 ºC) 2 h (-15 ºC), 12 h (rt) 2 h (-15 ºC), 12 h (rt) 2-39 (0%) 2-41 (19%) 2-42 (23%) 2-44 (10%) 2-39 (0%) 2-41 (18%) 2-42 (22%) 2-44 (10%) 2-39 (0%) 2-41 (15%) 2-42 (20%) 2-44 (7%) 2-40 (0%) 2-41 (19%) 2-43 (9%) 2-45 (Trace)** 2-40 (0%) 2-41 (18%) 2-43 (8%) 2-45 (Trace)** 2-39 (11%) 2-41 (15%) 2-42 (15%) 2-44 (5%) 2-39 (14%) 2-41 (11%)*** 2-42 (12%) 2-44 (6%) 2-40 (0%) 2-41 (17%) 2-43 (4%) 2-45 (0%) 2-39 (17%) 2-41 (14%) 2-42 (10%) 2-44 (5%) 2-39 (16%) 2-41 (12%) 2-42 (14%) 2-44 (6%) *black insoluble precipitate formed (likely indole polymer) **(2-45) identified via crude mass spec (HRMS-ESI (m/z): Calculated for C45H38N3O6S3 (M+H)+: 814.2079, Found: 814.2099.) ***Data Outlier due to minor spill 113 It was found that, as temperature was lowered during addition of the indole nucleophile, (A) or (2-38), the dimerization slightly decreased. In addition, N-tosylation of the indole also significantly lowered dimerization. However, N-tosylation also decreased the reactivity of the indole nucleophile, resulting in no alkylation. As shown in Table 2.6, it was found that solvent and slow addition of (A) or (2-38) had the most significant impact on the reaction. The only trials in which the desired alkylated product (2-39) was isolated in moderate yield (11-17%) were trials 6, 7, 9 and 10 (Table 2.6). The highest yielding was trial 9 in which the solvent was nitromethane (NO2Me) and (A) was added dropwise at cold temperatures (-15ºC). Nitromethane has been reported to selectively solubilize the activated Lewis acid coordinated indole intermediate, facilitating alkylation to form the desired product. NO2Me does not solubilize the indole species which are not coordinated to the Lewis acid, preventing excessive indole polymerization in solution.29 The cold temperatures also seem help in slowing this fast polymerization to some extent. Trial 10 (Table 2.6) was an attempt to additionally mitigate the over-reduction of benzophenone by decreasing Time 1. Though this led to a slight decrease in the yield of the over- reduction side product (2-41), this earlier addition of indole also contributed to a slight increase in polymerization. Overall, it was found that the slower indole alkylation reaction was unable to compete with the faster polymerization and over-reduction side reactions, leading to relatively low yields of the desired product (2-39) using the most optimized conditions. To further study this indole polymerization, a few control reactions were run, as shown in Scheme 2.22, in which 1H-indole was allowed to react with only the Lewis acid (FeCl3). As shown in Table 2.7, in the presence of the Lewis acid, the indole polymerization was fast. In fact, this polymerization was observed after only one hour of reacting with the Lewis acid (Table 114 2.7). However, in the case where no Lewis acid was present, no reaction was observed, confirming that the Lewis acid catalyzes this polymerization reaction. Scheme 2.22. Indole polymerization control reactions. FeCl3 (X mol%) N H DCM, rt, Time HN NH + N H 2-42 H N NH2 2-44 Table 2.7. Indole polymerization control reactions – reaction condition screening. R H H H X (mol%) Time 0 10 10 24 h 1 h 14 h Observations No reaction (2-42) and (2-44) identified via NMR (2-42) and (2-44) identified via NMR 2.2.5.3. Exploration of Friedel Craft’s alkylation step Due to the previously discussed low-yielding indole model systems, the one-pot reductive Friedel Craft’s alkylation was then broken up into separate steps to analyze each individually. To analyze the Friedel Craft’s alkylation step alone, similar conditions were run to those previously discussed, yet the reduction conditions were omitted. The Friedel Craft’s alkylation conditions were run with diphenyl methanol as the substrate and 1H-indole as the nucleophile (Scheme 2.23). The desired alkylated product (2-39) was accessed in high yields (78%) with minor amounts of the indole dimer side product (2-42) isolated. 115 Scheme 2.23. Friedel Craft’s alkylation of diphenylmethanol with 1H-indole as the nucleophile. OH N H FeCl3 (10 mol%) MeNO2, rt, 3 h (11% recovered) NH 2-39 (79%) NH + N H 2-42 (9%) 2.2.5.4. Exploration of ketone reduction step Since the Friedel Craft’s alkylation of diphenylmethanol was high yielding, the focus was transitioned from these simpler model systems to the target imidazoline intermediate (2-25) for exploration of the reduction step (Scheme 2.24). As outlined in Table 2.8, several reducing agents, from moderate to strong, were screened in the reduction of (2-25). However, none of these reducing agents afforded the desired product (2-46). Scheme 2.24. Ketone reduction of the imidazoline intermediate (2-25). O N H N N Bn 2-25 (See Table 2.8) NH HO N N Bn N H 2-46 NH 116 Table 2.8. Ketone reduction of the imidazoline intermediate (2-25) – reaction condition screening. Reducing Agent Solvent Temp Time Yield Et3SiH (1.1 equiv) NaBH4 (1.5 equiv) NaBH4 (1.5 equiv) LiBH4 (1.1 equiv) LiBH4 (1.1 equiv) MeOH MeOH EtOH EtOH EtOH rt à 60 ºC 0 ºC à rt 0 ºC à reflux 0 ºC à rt 0 ºC à reflux DIBAL-H (1.1 equiv) MeOH 0 ºC à rt DIBAL-H (1.1 equiv) EtOH rt à 60 ºC LiAlH4 (1.1 equiv) MeOH 0 ºC à rt * Only starting material recovered ** Evidence of reductive imidazoline ring opening identified 24 h 24 h 24 h 24 h 24 h 24 h 24 h 24 h No evidence of desired product* No evidence of desired product* No evidence of desired product* No evidence of desired product* No evidence of desired product* No evidence of desired product* No evidence of desired product** No evidence of desired product** The non-brominated, benzylated Spongotine C intermediate (2-25) was difficult to reduce, even with relatively strong reducing agents, such as DIBAL-H and LiAlH4. This is likely due to the significant electron donation from the indole, contributing to the decreased electrophilicity of the carbonyl. It is also noteworthy to mention that evidence of reductive imidazoline ring opening was identified when using stronger reducing agents, such as DIBAL-H and LiAlH4. Similar reductive imidazoline ring openings have been reported in literature.30 In order to mitigate this issue, it was hypothesized that protecting the indole nitrogen, thus significantly decreasing its electron donation, could help to push this reaction forward and potentially allow for the use of milder reducing agents. To test this hypothesis, a model system was explored in which tosylated (2-47) and un- tosylated 3-acyl-1H-indoles underwent reduction with relatively mild NaBH4 as a reducing agent, following literature procedures (Scheme 2.25).31 As shown, in the case of (2-47) this reduction proceeded in excellent yields to access the reduced product (2-48) (94%). However, in the case of 3-acyl-1H-indole, no reaction was observed. These results supported the hypothesis 117 that N-protection of the indole could allow for more facile reduction of this ketone. Therefore, this approach was identified as a promising path forward for the reduction of 2-25. Scheme 2.25. Ketone reduction of 3-acyl-1H-indole model system – effect of N-tosylation. O N H NaH (2.5 equiv), TsCl (1.6 equiv) THF, 0 ºC to rt, 18 h quant O N Ts 2-47 NaBH4 (1.0 equiv) MeOH, rt, 3 h 94% HO N Ts 2-48 O N H NaBH4 (1.0 equiv) MeOH, rt, 3 h HO N H 2-49 Considering these findings, the imidazoline intermediate (2-25) was tosylated to access the bis-protected intermediate (2-50) in 70% yield, as shown in Scheme 2.26. Scheme 2.26. Bis-tosylation of the imidazoline intermediate (2-25). O N N Bn N H 2-25 NH NaH (3.5 equiv), TsCl (3.0 equiv), THF, 0 ºC, to rt, 18 h 70% O N N Bn N Ts 2-50 NTs Once the bis-tosylated imidazoline intermediate (2-50) was accessed, it subsequently was subjected to various reductive conditions in attempts to access intermediate 2-51 (Scheme 2.27). NaBH4 was initially used as the reducing agent, similar to the reduction in Scheme 2.25 (Table 2.9). Unfortunately, no desired product was identified among several different NaBH4-mediated reduction reactions. As is shown in Table 2.9, parameters such as temperature and equivalents of reducing agent were screened. In addition, CeCl3 (Luche conditions)32 was explored as a catalyst to coordinate to the ketone and facilitate hydride attack. However, despite screening various equivalents of reducing agent and catalyst loadings, no desired product was isolated (Table 2.9). 118 Notably, with increased equivalents of the reducing agent, evidence of reductive imidazoline ring opening was identified. This evidence indicates that the imidazoline is sensitive to reductive conditions. Moreover, the reduction of the ketone and the reductive ring opening of the imidazoline are likely competitive reactions. Due to this issue, the synthetic approach toward the first total synthesis of Tulongicin (2-1) was adapted. Scheme 2.27. Ketone reduction of the bis-tosylated imidazoline intermediate (2-50). O N N Bn N Ts 2-50 NaBH4 (equiv), Catalyst (equiv), Solvent, temp, time NTs HO N N Bn N Ts 2-51 NTs Table 2.9. Ketone reduction of the bis-tosylated imidazoline intermediate (2-50) – reaction condition screening. Reducing Agent Catalyst Solvent Temp Time NaBH4 (1.0 equiv) None NaBH4 (1.5 equiv) NaBH4 (1.5 equiv) NaBH4 (1.5 equiv) NaBH4 (5.0 equiv) NaBH4 (5.0 equiv) NaBH4 (10.0 equiv) None CeCl3 (10 mol%) CeCl3 (10 mol%) CeCl3 (10 mol%) CeCl3 (10 mol%) CeCl3 (10 mol%) MeOH MeOH MeOH rt rt rt MeOH reflux MeOH rt MeOH reflux MeOH reflux 24 h 24 h 24 h 24 h 24 h 24 h 24 h Yield Trace a Trace a Trace a Trace a Trace a, b Trace a, b Trace a, b a Evidence of desired product identified in mass spectrometry (HRMS-ESI (m/z): Calculated for C41H36N4O5S2 (M+H)+: 729.2205, Found: 729.2230) bEvidence of reductive imidazoline ring opening identified 2.2.6. Adapted retrosynthetic analysis for the total synthesis of Tulongicin (2-1) via Dihydrospongotine C (2-4) as an intermediate (Route E) Considering these previously discussed issues, an updated retrosynthetic approach for the first total synthesis of Tulongicin (2-1) was completed, as shown in Scheme 2.28. It was 119 hypothesized that 2-1 could be accessed via a key Friedel Craft’s alkylation of Dihydrospongotine C (2-4) and that this could circumvent the problems associated with the previous reductive conditions. It was also hypothesized that Dihydrospongotine (2-4) could be accessed via an imidazoline cyclization of (S)-2-(6-bromo-1H-indol-3-yl)-2- hydroxyacetaldehyde (2-52) and 1-(6-bromo-1H-indol-3-yl)ethane-1,2-diamine (2-18). Similar transformations have been reported in literature.33 However, though various aldehyde fragments have been explored in literature, to the best of my knowledge, this would be the first use of an alpha hydroxyaldehyde fragment in such cyclizations. The diamine fragment (2-18) was proposed to be synthesized via the same aforementioned Mannich-like di-amination and subsequent bis-deprotection of commercially available 6-bromo-1H-indole.22 In addition, it was hypothesized that the hydroxyacetaldehyde intermediate (2-52) could be accessed via a selective primary alcohol oxidation of the diol intermediate (2-53), as similar transformations have been reported in literature.34 Synthesis of the diol intermediate (2-53) was proposed to follow subsequent Vilsmeier Haack Formylation, Wittig, and Sharpless dihydroxylation steps of the commercially available 6-bromo-1H-indole. This adapted route would not only allow for the synthesis of Tulongicin (2-1) and Dihydrospongotine C (2-4) in a longest linear sequence (LLS) of 6 steps and 5 steps, respectively, but it would also allow for the potential of an enantioselective synthesis. 120 Scheme 2.28. Adapted retrosynthetic analysis for the total synthesis of Tulongicin (2-1) via Dihydrospongotine C (2-4) as an intermediate – Route E. HO N N H NH Br Imidazoline Cyclization Br N H Dihydrospongotine C (2-4) Br N H FC Alkylation Tulongicin (2-1) HO O H H2N NH2 Br N H 2-52 + Br N H 2-18 Primary Alcohol Oxidation Bis- Deprotection OH HO BnHN NHBoc Dihydrospongotine C (2-4) Br N H 2-53 Br N H 2-19 Oxidation Sharpless Dihydroxylation Mannich-Like Diamination O N H O N H N N H 2-2 N N H 2-3 N H Oxidation N H Br Br Br Br 2.2.6.1. Synthesis of diol intermediate Br N 2-54 Ts Br N H Vilsmeier Haack Formylation Wittig O H Br N H 2-9 It is worth noting that, for cost purposes, 1H-indole was used as a model system instead of 6-bromo-1H-indole for preliminary optimization. As shown in Scheme 2.29, in the synthesis 121 of the model diol intermediate), commercially available 1H-indole-3-carboxaldehyde was quantitatively N-protected with a tosyl group to access 2-13. Then, 2-13 was subjected to a Wittig reaction to afford the 3-vinyl indole intermediate (2-14) in 70% yield, which subsequently underwent a Sharpless dihydroxylation to access (S)-2-55 in high yield. Scheme 2.29. Synthesis of diol intermediate (S)-(2-55). O H N H NaH (2.2 equiv), TsCl (1.2 equiv), THF, 0 ºC to rt, 18 h quant O H CH3(Ph)3PBr (1.5 equiv), nBuLi (1.5 equiv) THF, -30 ºC, 2 h 70% N Ts 2-13 OH HO AD-mix-α (2.0 equiv) H2O/tBuOH (1:1) 0 ºC to rt, 18 h 78% N Ts 2-14 N Ts 2-55 2.2.6.2. Exploration of the selective primary alcohol oxidation and subsequent imidazoline cyclization As is shown in Scheme 2.30, the alcohol intermediate ((S)-2-55) was then subjected to a selective primary alcohol oxidation via 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) as the catalytic oxidant and phenyliodine (III) diacetate (PIDA) as the co-oxidant. Similar selective primary alcohol oxidation such as this have been carried out in literature.35 As shown in Table 2.10, despite screening various reaction times and equivalents of PIDA, no evidence of the desired product was found in this reaction. Instead of isolation of the desired product, across multiple trials, four different side products were isolated: (2-58), (2-59), (2-13), and (2-60). As is shown in Table 2.10, in the first trial with 1.75 equivalents of PIDA and an 18-hour reaction time, 48% yield of 2-58, 33% yield of 2-59, 21% yield of 2-13, and 76% yield of 2-60 were isolated. When this reaction was closely monitored via thin layer chromatography (TLC), it was discovered that this reaction was very fast. In fact, the starting material was consumed in one hour. Therefore, a 1-hour reaction time was used in subsequent trials. As shown in Table 2.10, with 1.75 equivalents of PIDA and a 1-hour reaction time, 46% yield of 2-58, 30% yield of 2-59, 122 32% yield of 2-13, and 71% yield of 2-60 were isolated. In attempts to slow this reaction, less equivalents of PIDA (1.2 equiv) were used, along with the same 1-hour reaction time. However, comparable yields of the aforementioned side products were isolated, as shown in Table 2.10. Overall, despite screening different reaction times and equivalents of PIDA, similar results were obtained across trials. Scheme 2.30. Exploration of the selective primary alcohol oxidation and subsequent imidazoline cyclization. OH HO O HO TEMPO (0.18 equiv), PIDA (X equiv) DCM, rt, Time N Ts 2-55 N Ts 2-56 I2 (1.25 equiv), K2CO3 (3.0 equiv), EtOH, rt, 18 h BnHN NH2 N H (2-24) HO N Ts TsN N N Bn 2-57 N N Bn 2-58 O NH NH H NH 2-59 N Ts 2-13 2-60 I N N Bn 123 Table 2.10. Exploration of the selective primary alcohol oxidation and subsequent imidazoline cyclization – conditions screened. PIDA (equiv) Time Yield (2-57) 1.75 1.75 1.2 18 h No evidence of desired product 1 h 1 h No evidence of desired product No evidence of desired product Yield (2-58) 48% 46% 45% Yield (2-59) 33% 30% 28% Yield (2-13) 21% 32% 34% Yield (2-60) 76% 71% 70% Considering the significant formation of these side products, it was important to determine a potential mechanism by which they could be formed. Though not as common as other oxidants, it has been reported in literature that when PIDA is used in the oxidation of 1,2- diols it leads to oxidative cleavage, following a similar mechanism to that of sodium periodate ( Scheme 2.31).36 As shown, the first step of the mechanism likely involves sequential nucleophilic displacements of the acetate groups of PIDA and proton transfer steps to afford the 5-membered ring intermediate (II) and two equivalents of acetic acid. This 5-membered ring intermediate would then readily undergo oxidative cleavage to form the aldehyde intermediate (2-13), formaldehyde, and iodobenzene (2-60). As shown, this oxidative cleavage mechanism can explain the formation of the aldehyde (2-13) and the iodobenzene (2-60) side products. However, when considering the presence of the diamine intermediate (2-24) in the reaction, the remaining imidazoline side products can be explained by cyclization of the diamine fragment (2- 24) with the aldehyde side product (2-13) or formaldehyde to access side products 2-58 or 2-59, respectively. 124 Scheme 2.31. Oxidative cleavage mechanism of the diol intermediate (2-55). O O I OH O O HO (PIDA) O O I O HO N Ts 2-55 PT O OH I N Ts O PT OH O + I + H H 2-60 O H N Ts 2-13 OI O N Ts II To test this theory, a control reaction was run in which the 1,2-diol intermediate (2-55) was reacted with PIDA alone for two hours, as is shown in Scheme 2.32. When 2-55 was subjected to PIDA, the aldehyde intermediate (2-13) was isolated in 81% yield. In addition, iodobenzene (2-60) was also isolated in 78% yield. Formaldehyde was not isolated, but considering its volatility, it was likely evaporated upon workup. These results confirmed this issue of the oxidative cleavage side reaction. Therefore, exploration of other co-oxidants for the TEMPO-catalyzed oxidation of (2-55) remains a focus for future optimization of this approach. Scheme 2.32. Oxidative cleavage of diol intermediate (2-55) with PIDA. OH HO N Ts 2-55 PIDA (1.2 equiv) DCM, rt, 2 h O H N Ts 2-13 (81%) + I 2-60 (78%) 125 2.2.6.3. Adapted synthesis of (S)-2-(6-bromo-1H-indol-3-yl)-2-hydroxyacetaldehyde (2-52) Along with the continuing optimization of the aforementioned reduction of the alcohol intermediate (2-55), an expedited synthetic route toward the hydroxyacetaldehyde intermediate (2-52) was explored. As shown in Scheme 2.33, this expedited approach would allow 2-52 to be accessed in three steps opposed to the previous four steps necessary in the previous approach. As shown in Scheme 2.33, it was proposed that 2-52 could be accessed via an alpha hydroxylation of the aldehyde intermediate (2-61). Similar alpha hydroxylation reactions have been reported for various alkyl and aryl aldehydes, however, aldehydes with complex indole substituents, such as (2-61) have yet to be explored.37,38 It was hypothesized that this aldehyde (2-61) could be accessed by oxidation of the alcohol intermediate (2-62). It is notable to mention that there is significantly more literature precedence for alpha hydroxylation of ketones, yet aldehyde substrates tend to be more elusive. The proposed synthesis of the alcohol intermediate (2-62) includes oxo-ester synthesis and subsequent oxidation of the commercially available 1H-indole starting material.39 Scheme 2.33. Expedited retrosynthetic analysis of hydroxyacetaldehyde intermediate (2-52). HO O Alpha Hydroxylation O Oxidation OH Oxo-ester Synthesis and Reduction Br N H 2-52 Br N H 2-61 Br N H 2-62 Br N H Progress in the forward direction has been made toward this expedited synthesis of 2-52 using commercially available 2-(1H-indol-3-yl)ethan-1-ol as a model starting material for initial optimization. As shown in Scheme 2.34, the aldehyde intermediate (2-63) was synthesized via an IBX oxidation of 2-(1H-indol-3-yl)ethan-1-ol in high yields (69%). Then, the aldehyde intermediate (2-63) was subjected to the mild hydroxylamine alpha oxidation reagent, O- 126 benzoyl-N-(tert-butyl)hydroxylamine in efforts to access the alpha-oxidized intermediate (2-64) (Scheme 2.34). This reaction proceeds via an imine intermediate that subsequently tautomerizes to the enamine, which undergoes a heat-induced sigmatropic rearrangement to access the alpha- oxidized product (2-64).37 However, this reaction did not afford the desired product (2-64). Instead, an unexpected dimer side product (2-65) was identified via crude mass spectrometry (HRMS-ESI (m/z): Calculated for C20H18N2O2 (M+H)+: 319.1447, Found: 319.1461) (Scheme 2.34). The only other compound that was recovered was the hydroxylamine starting material (57%). This dimer side product (2-65) was likely the result of an aldol condensation of 2-63 with another molecule of itself. Considering this hypothesis, along with the recovery of the hydroxylamine starting material, it is likely that this aldol reaction is faster than the desired alpha hydroxylation reaction, especially at elevated temperatures. Therefore, the prevention of the aldol condensation remains the focus of future optimization. One proposed solution includes the protection of the indole moiety with an electron withdrawing group, such as a tosyl group, to decrease the reactivity of 2-63. Scheme 2.34. Synthesis of intermediate (2-64). OH N H IBX (1.1 equiv) MeCN, 80 ºC, 3 h 69% O N H 2-63 N H O Ph O (1.0 equiv) THF/H2O (1:1) rt to 50 ºC O BzO O OH NH N H 2-65 N H 2-64 (0%) 2.2.7. Adapted retrosynthetic analysis for the total synthesis of Tulongicin (2-1) via a bis- indole intermediate (Route F) Though additional exploration and optimization remains a priority for Route E, simultaneously, an additional bis-indole approach (Route F), was developed. While this approach 127 would not allow for the first total synthesis of Dihydrospongotine C (2-4) as an intermediate, it would have the benefit of being the most expedited route yet (LLS: 3steps) (Scheme 2.35). As shown in Scheme 2.35, it is hypothesized that Tulongicin (2-1) could be accessed via an imidazoline cyclization of a bis-indole aldehyde fragment (2-66) and a diamine fragment (2-18). Similar imidazoline cyclizations have been reported in literature, however, to the best of my knowledge, there has been no reported imidazoline cyclization resulting in the synthesis of an imidazoline bearing a complex 2-bis-indole methane moiety. The diamine fragment (2-18) was proposed to follow the same Mannich-like di-amination and deprotection procedure, as was previously discussed.22 It was hypothesized that the bis-indole aldehyde fragment (2-66) could be accessed via reduction of the bis-indole ester intermediate (2-67). The proposed synthesis of 2-67 included a methine synthesis using the commercially available ethyl-2-oxoacetate and 6- bromo-1H-indole, as has been reported in literature.40 128 Scheme 2.35. Adapted retrosynthetic analysis for the total synthesis of Tulongicin (2-1) via a bis-indole intermediate – Route F. Br Br H N N H N N Bn Tulongicin (2-1) Br Imidazoline Cyclization Br N H Br H N N H 2-66 O H + Br H2N NH2 N H 2-18 Bis- Deprotection BnHN NHBoc Reduction Br Br H N N H 2-67 H O O O Methine Synthesis O O Br N H 2-19 Mannich-Like Diamination Br N H 2.2.7.1. Synthesis of brominated bis indole aldehyde fragment First, 6-bromo-1H-indole was subjected to a methine synthesis with ethyl 2-oxoacetate, using scandium triflate (Sc(OTF)2) as a catalyst, to afford the desired bis-indole ester intermediate (2-67) in quantitative yield (Scheme 2.36). Then, 2-67 underwent a DIBAL-H reduction at -78ºC to afford the desired bis-indole aldehyde product (2-66). As is shown in Table 2.11, various equivalents of DIBAL-H reducing agent were explored with the optimal conditions being 1.8 equivalents of DIBAL-H to access 2-66 in 72% yield. The quench temperature of this 129 reaction was also found to have a great impact on yield. For instance, a 20ºC quench temperature suffered from a low yield of 2-66 and an increased formation of an over-reduced alcohol product (Table 2.11). Scheme 2.36. Synthesis of bis-indole aldehyde intermediate (2-66). O O O H (1.0 equiv), Sc(OTf)3 (0.2 equiv), Br Br N H DCM, 0 ºC to rt 18 h quant Br O O DIBAL-H (X equiv) DCM, -78 ºC, 1 h Br Br H N N H 2-67 O H H N N H 2-66 Table 2.11. Ester reduction of 2-67 to access the aldehyde fragment (2-66) – Reaction conditions screened. DIBAL-H (equiv) Quench Temperature 1.0 1.0 1.8 20 ºC (rt) -78 ºC -78 ºC -78 ºC *over-reduced alcohol product was present 2.0 Yield (2-66) 15%* 48% 81% 67%* 2.2.7.2. Synthesis of brominated indolic diamine fragment As shown in Scheme 2.37, the brominated indolic diamine fragment was synthesized in the same manner as the aforementioned indolic diamine model system (Scheme 2.13). First, commercially available 6-bromo-1H-indole underwent di-amination to access the di-protected diamine (2-19) in 52% yield. Then, as shown in Scheme 2.37, the di-protected diamine (2-19) was quantitatively Boc-deprotected to access the mono-protected diamine (2-68). 130 Scheme 2.37. Synthesis of brominated indolic diamine fragment (2-68). BocHN O (1.0 equiv), BnNH2 (1.0 equiv), TMG (10 mol%), BnHN NHBoc BnHN NH2•HCl Br N H H2O, 45 ºC, 18 h 52% Br N H 2-19 EtOH-HCl rt, 24 h quant Br N H 2-68 2.2.7.3. Exploration of imidazoline cyclization via bis-indole aldehyde and diamine fragments As shown in Scheme 2.38, the base-mediated imidazoline cyclization between the bis- indole aldehyde fragment (2-66) and the indolic diamine fragment (2-68) was explored using potassium carbonate as the base and iodine to oxidize the cyclized intermediate to the imidazoline product (2-69). As shown in Table 2.12, despite screening a variety of reaction times and temperatures, the desired product was only found in trace amounts at best, as determined by crude mass spectrometry. At elevated temperatures, messy NMRs and large masses in mass spectrometry were found (Table 2.12). This data could likely correspond to some sort of dimer or polymer of the aldehyde fragment, as one could envision the alpha proton of this intermediate to be very sensitive to basic conditions and elevated temperatures. For instance, if 2-66 were to form an enolate, a myriad of problematic side reactions could occur, such as aldol condensations, etc. Therefore, since elevated temperatures and base are necessary for this imidazoline formation, the bis-indole aldehyde (2-66) may not be a viable fragment for this reaction. 131 Scheme 2.38. Imidazoline cyclization via bis-indole aldehyde (2-66) and diamine fragments (2- 68). BnHN NH2•HCl Br N H 2-68 (1.0 equiv), K2CO3 (3.0 equiv), I2 (1.2 equiv), EtOH, Temp., Time Br Br O H H N N H 2-66 Br Br H N N H Br N N Bn 2-69 N H Table 2.12. Imidazoline cyclization – reaction condition screening. Temperature Time rt rt rt to 50ºC rt to 50ºC 5 h 18 h 18 h 3 h Yield (2-69) No evidence of product No evidence of product Tracea, b Trace a, b a. Mass of desired product (2-69) identified in crude mass spectrometry (HRMS-ESI (m/z): Calculated for C35H26Br3N5 (M+H)+, (Br79, Br79, Br79); (Br81, Br79, Br79); (Br81, Br81, Br79); (Br81, Br81, Br81): 753.9817; 755.9796; 757.9776; 759.9755, Found: 753.9833; 755.9807; 757.9791; 759.9774). b. Messy crude NMRs and large masses in crude MS (potential polymerization). 2.2.7.4. Synthesis of bis-indole cyanide To address the previously discussed problems with the aldehyde intermediate, it was hypothesized that bis-indole cyanide intermediate (2-70) could be cyclized with the diamine fragment (2-18) to form the imidazoline core of Tulongicin (2-1) (Scheme 2.39). Similar cyclizations have been reported for less complex substrates in literature.41 This bis-indole cyanide intermediate would be less reactive and, therefore, less susceptible to polymerization or other side reactions. It was proposed that the bis-indole cyanide (2-70) could be accessed in one step via an adapted Strecker reaction from the commercially available 6-bromo-1H-indole-3- 132 carbaldehyde, trimethylsilanecarbonitrile (TMSCN), and 6-bromo-1H-indole, as shown in Scheme 2.39.42 Scheme 2.39. Retrosynthesis of the bis indole cyanide intermediate (2-70). Br Br H N N H 2-70 Strecker N O H TMS + + Br Br N H N N H As shown in Scheme 2.40, the bis-indole cyanide intermediate (2-70) was synthesized via an acid-mediated Strecker reaction with the commercially available 6-bromo-1H-indole-3- carbaldehyde, trimethylsilanecarbonitrile (TMSCN) in 75% yield. This reaction was reproducible at multi-gram scale. Scheme 2.40. Synthesis of the bis indole cyanide intermediate (2-70). O H Br N H (1.0 equiv), TMSCN (1.1 equiv), Br N H AcOH, 60 ºC, 18 h 75% Br Br H N N H 2-70 N 2.2.7.5. Exploration of imidazoline cyclization via bis-indole cyanide and diamine fragments Once 2-70 was accessed, the imidazoline cyclization between the bis-indole cyanide fragment (2-70) and the indolic diamine fragment (2-68) was explored. As was previously 133 mentioned, the bis-indole cyanide (2-70) is less reactive than the previously used aldehyde intermediate (2-66). Therefore, a copper catalyst was used in this cyclization reaction, as well as heat. As shown in Scheme 2.41, 2-70 was reacted with the diamine (2-68) and CuCl2 as a catalyst at 120ºC for 24 hours. Notably, no side reactions were identified during this cyclization. The only compounds identified after this reaction was run were the starting materials (2-70 and 2-68) and the product (2-69). Unfortunately, the diamine starting material (2-68) and the desired imidazoline product co-eluted, contributing to difficult purification. Due to this difficult purification, the desired cyclized product has yet to be isolated. However, 2-69 has been identified via mass spectrometry (HRMS-ESI (m/z): Calculated for C35H26Br3N5 (M+H)+, (Br79, Br79, Br79); (Br81, Br79, Br79); (Br81, Br81, Br79); (Br81, Br81, Br81): 753.9817; 755.9796; 757.9776; 759.9755, Found: 753.9833; 755.9807; 757.9791; 759.9774). This was very exciting progress toward the first total synthesis of Tulongicin, as intermediate 2-69 is only one deprotection step away from Tulongicin (2-1). Additional optimization of this reaction, as well as its final deprotection, remain the focus for the future of this project, as Route F has shown the most promise toward the first total synthesis of Tulongicin (2-1). Scheme 2.41. Imidazoline cyclization via bis-indole cyanide (2-70) and diamine fragments (2- 68). Br Br H N N H 2-70 2-68 (1.1 equiv), CuCl2 (20 mol%), DMF, 120 ºC, 24 h N Br Br H N N H N N Bn 2-69 Br N H 134 2.3. Conclusions In conclusion, this chapter summarizes the synthetic efforts toward the total synthesis of Tulongicin (2-1) and its related natural product analogues Spongotine C (2-2), Dibromodeoxytopsentin (2-3), and Dihydrospongotine C (2-4). As has been discussed in this chapter, key challenges in these syntheses were identified which hindered their completion at the present time. However, despite the fact that these total syntheses of Tulongicin (2-1) and its related natural product analogues have not yet been achieved, these studies have provided valuable insights to, hopefully, allow for future achievement in their syntheses. Currently, further research building upon these findings toward the total synthesis of Tulongicin (2-1) is underway in the Tepe lab. 135 2.4. Experimental 14 O 12 O 11 10 NH213 6 5 1 4 2 3 8 7 NH 9 O O NH2 N H 15 2-(1H-indol-3-yl)-2-oxoacetamide (2-9) Oxalyl chloride (0.5 mL, 6.4 mmol, 1.5 equiv.) was added dropwise to a stirred solution of indole (0.5 g, 4.3 mmol, 1.0 equiv.) in anhydrous Et2O (10 mL) at 0ºC under an atmosphere of argon gas. The mixture was then stirred for 2 hours at 0ºC under argon gas. A fresh NH3 solution was then prepared by mixing equal parts saturated aqueous NH4Cl solution and saturated aqueous KOH solution. After consumption of starting material by TLC, the freshly prepped saturated ammonia solution was added to the mixture (5 mL). This was allowed to stir for 1 hour at room temperature. The resulting yellow precipitate was then vacuum filtered, using a Buchner funnel, and washed with large amounts of chloroform to yield the 2-(1H-indol-3-yl)-2-oxoacetamide product as a yellow solid. This crude product was used in the next step without any additional purification (97% by NMR). Mp: 220ºC (decomp.). 1H NMR (500 MHz, DMSO) δ 12.27 (s, 1H, (H9)), 8.68 (d, J = 3.2 Hz, 1H, (H8)), 8.22 (d, J = 6.3 Hz, 1H, (H1)), 8.08 (s, 1H, (H13)), 7.72 (s, 1H, (H13)), 7.53 (d, J = 5.8 Hz, 1H, (H4)), 7.28 – 7.22 (m, 2H, (H5, H6)). 13C NMR (126 MHz, DMSO) δ 183.42 (C10), 166.47 (C11), 138.70 (C8), 136.73 (C3), 126.58 (C2), 123.81 (C5), 122.91 (C6), 121.67 (C1), 112.99 (C4), 112.50 (C7). FTIR (cm-1): 3419, 3381, 3301, 3066, 1741, 1661, 1554. HRMS-ESI (m/z), calculated for C10H8N2O2 (M+H)+: 189.0664, Found: 189.0681. 136 12 O 11 N 13 10 6 5 1 4 2 3 8 7 NH 9 O N N H 16 1H-Indole Carbonyl Cyanide (2-10) SOCl2 (0.57 mL, 7.8 mmol, 2.5 equiv) was added dropwise to a stirred solution of 2-(1H-indol- 3-yl)-2-oxoacetamide (0.45 g, 3.9 mmol, 1.0 equiv.) in anhydrous DMF at 0ºC under an atmosphere of argon gas. This solution was allowed to stir at 0ºC for 2 hours. The reaction mixture was then quenched with a few drops of water and the organic layer was extracted with EtOAc (3x30mL). The combined organic layers were then washed with saturated aqueous LiCl solution (3x30mL) to ensure the removal of DMF from the organic layer. The combined organic layers were then dried with MgSO4, filtered, and the solvent was removed in vacuo to afford the 1H-indole-3-carbonyl cyanide product as a yellow-tan solid (0.54 g, 81%). Mp: 167ºC (decomp.). 1H NMR (500 MHz, DMSO) δ 12.92 (s, 1H, (H9)), 8.65 (d, J = 3.5 Hz, 1H, (H8)), 8.04 (d, J = 7.5 Hz, 1H, (H1)), 7.59 (d, J = 7.6 Hz, 1H, (H4)), 7.37 (td, J = 7.5, 7.3, 1.4 Hz, 1H, (H5)), 7.33 (td, J = 7.4, 7.3, 1.3 Hz, 2H, (H6)). 13C NMR (126 MHz, DMSO) δ 158.60 (C10), 141.49 (C8), 137.58 (C3), 124.98 (C5), 124.23 (C2), 123.87 (C6), 121.00 (C1), 116.21 (C7), 114.39 (C11), 113.35 (C4). FTIR (cm-1): 3401, 3072, 2900, 2249, 1724, 1512. HRMS-ESI (m/z), calculated for C10H6N2O (M+H)+: 171.0558, Found: 171.0573. 137 19 CH3 15 16 14 17 12 O 13 18 S 10 O 11 7 N 9 8 Ts N 6 5 1 4 2 3 18a 2-phenyl-1-tosylaziridine (2-11a) To a mixture of I2 (0.13 g, 0.5 mmol, 0.1 equiv.), TBAB (0.16 g, 0.5 mmol, 0.1 equiv.), and chloramine-T monohydrate (1.2 g, 5.0 mmol, 1.0 equiv.) in DCM/H2O (2:1, 50:25 mL) was added styrene (0.58 mL, 5.0 mmol, 1.0 equiv.,) at 20ºC, under ambient conditions. This mixture was stirred vigorously overnight under ambient conditions. The reaction mixture was then treated with saturated aqueous Na2S2O3 (5 mL) and extracted with DCM (3x30 mL). The combined organic extracts were then dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was purified via silica gel flash column chromatography (EtOAc/Hexanes) to afford the desired product as a white crystalline solid (0.95 g, 70%). Mp: 87-89ºC. 1H NMR (500 MHz, CDCl3) δ 7.87 (d, J = 8.3 Hz, 2H, (H14, H18)), 7.33 (d, J = 8.2 Hz, 2H, (H15, H17)), 7.30 – 7.26 (m, 3H, (H4, H5, H6)), 7.24 – 7.19 (m, 2H, (H1, H3)), 3.78 (dd, J = 7.2, 4.5 Hz, 1H, (H7)), 2.99 (d, J = 7.2 Hz, 1H, (H8)), 2.43 (s, 3H, (H19)), 2.39 (d, J = 4.5 Hz, 1H, (H8)). 13C NMR (126 MHz, CDCl3) δ 144.77 (C16), 135.14 (C2), 135.06 (C13), 129.87 (C15, C17), 128.66 (C4, C6), 128.41 (C5), 128.07 (C14, C18), 126.66 (C1, C3), 41.13 (C7), 36.05 (C8), 21.76 (C19). FTIR (cm-1): 3041, 2878, 1551, 1356, 1155. HRMS-ESI (m/z), calculated for C15H15NO2S (M+H)+: 274.0902, Found: 274.0904. 138 Ts N O 18b 2-(4-methoxyphenyl)-1-tosylaziridine (2-11b) To a mixture of TBAB (64 mg, 0.2 mmol, 0.1 equiv.), CuI (38 mg, 0.2 mmol, 0.1 equiv.), chloramine-T•H2O (0.5 mg, 2.0 mmol, 1.0 equiv.), in DCM/H2O (2:1), was added 1-methoxy-4- vinylbenzene (0.54 mL, 4.0 mmol, 2.0 equiv.) dropwise at 20ºC under ambient conditions. The reaction mixture was stirred vigorously overnight under ambient conditions. The reaction mixture was then treated with saturated aqueous Na2S2O3 (3 mL) and extracted with DCM (3x30 mL). The combined organic extracts were then dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was purified via silica gel flash column chromatography (EtOAc/Hexanes) to afford the desired product as a white crystalline solid (95 mg, 15%). Mp: 89-91ºC. 1H NMR (500 MHz, CDCl3) δ 7.85 (d, J = 8.3 Hz, 2H, (H16, H20)), 7.31 (d, J = 8.4 Hz, 2H, (H17, H19)), 7.12 (d, J = 8.7 Hz, 2H, (H1, H3)), 6.81 (d, J = 8.7 Hz, 2H, (H4, H6)), 3.75 (s, 3H, (H11)), 3.73 (dd, J = 7.2, 4.6 Hz, 1H, (H7)), 2.95 (d, J = 7.2 Hz, 1H, (H9)), 2.42 (s, 3H, (H21)), 2.37 (d, J = 4.6 Hz, 1H, (H9)). 13C NMR (126 MHz, CDCl3) δ 159.74 (C5), 144.65 (C18), 135.14 (C15), 129.80 (C17, C19), 127.97 (C16, C20), 127.88 (C4, C6), 127.01(C2), 114.06 (C1, C3), 55.32 (C11), 40.97 (C7), 35.81 (C9), 21.68 (C21). FTIR (cm-1): 3013, 2875, 1514, 1351, 1252, 1153, 1039. HRMS-ESI (m/z), calculated for C16H17NO3S (M+H)+: 304.1007, Found: 304.1070, calculated for C16H18NO3SNa (M+H)+: 327.0905, Found: 327.0925. 139 12 O 10 8 7 NH 9 6 5 1 4 2 3 O H N H 19 1H-indole-3-carbaldehyde (2-12) Anhydrous DMF (3 mL) was added to an oven-dried flask. This was then cooled to 0ºC under an atmosphere of nitrogen gas. Then, POCl3 (1.2 mL, 12.4 mmol, 3.0 equiv) was added dropwise and the reaction was stirred for 10 minutes at 0ºC to allow formation of the Vilsmeier reagent. After 10 minutes, 1H-indole (0.57 g, 4.9 mmol, 1.0 equiv) was added in a minimal amount of anhydrous DMF. The reaction mixture was then placed in a pre-heated oil bath at 80ºC. The reaction was then allowed to stir overnight at 80ºC under nitrogen gas. The reaction was then slowly cooled to 0ºC, quenched with deionized H2O (2 mL), and treated with 20% Aqueous NaOH solution until the reaction mixture reaches a pH of ~9-11. The resulting solution was then extracted with EtOAc (3x40 mL) and the combined organic layers were washed with LiCl (3x40mL) to ensure removal of DMF from the organic layers. The combined organic fractions were then dried over Na2SO4, filtered, and concentrated in vacuo. The crude residue was then purified via silica gel flash column chromatography (EtOAc/Hexanes) to yield the desired product as a yellow solid. This yellow solid was then recrystallized (Et2O) to yield yellow crystals (0.37 g, 53%). Mp: 198-199ºC. 1H NMR (500 MHz, DMSO) δ 12.12 (s, 1H, (H9)), 9.92 (s, 1H, (H10)), 8.27 (s, 1H, (H8)), 8.07 (d, J = 7.6 Hz, 1H, (H1)), 7.49 (d, J = 8.0 Hz, 1H, (H4)), 7.24 (td, J = 8.0, 7.1, 1.3 Hz, 1H, (H5)), 7.20 (td, J = 7.7, 7.2, 1.2 Hz, 1H, (H6)). 13C NMR (126 MHz, DMSO) δ 185.41 (C10), 138.94 (C3), 137.49 (C8), 124.55 (C2), 123.91 (C5), 122.57 (C6), 121.27 (C1), 118.60 (C7), 112.87 (C4). FTIR (cm- 140 1): 3401, 3036, 2908, 1712, 1520. HRMS-ESI (m/z), calculated for C9H7NO (M+H)+: 146.0606, Found: 146.0621. 11 O 10 8 7 N 9 14 O 6 5 1 4 2 3 S 12 O 13 15 16 20 17 19 18 CH321 O H N Ts 20 1-tosyl-1H-indole-3-carbaldehyde (2-13) To an oven-dried flask, charged with N2 gas, was added anhydrous THF (20mL). Then, 60% NaH solution in mineral oil (0.30 g, 7.5 mmol, 2.5 equiv) was added and this mixture was cooled to 0ºC. Next, a solution of 1H-indole-3-carbaldehyde (0.44 g, 3.0 mmol, 1.0 equiv) in anhydrous THF (10mL) was added dropwise at 0ºC under an atmosphere of N2 gas. This pink reaction mixture was allowed to stir for 20 minutes at 0ºC under N2. After 20 minutes, Tosyl Chloride (0.92 g, 4.8 mmol, 1.6 equiv) was added at 0ºC, resulting in an immediate yellow color change. The reaction mixture was allowed to warm to 20ºC and was stirred for 12 hours. When TLC showed complete consumption of starting material, the reaction was quenched slowly with deionized water (3mL) dropwise. The reaction mixture was then extracted with EtOAc (3x40mL). The combined organic layers were then dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was purified via silica gel flash column chromatography (EtOAc/Hexanes) to yield the desired product as an off-white solid (0.90 g, Quantitative). Mp: 147-149ºC. 1H NMR (500 MHz, CDCl3) δ 10.10 (s, 1H, (H10)), 8.26 (d, J = 7.8 Hz, 1H, (H1)), 141 8.23 (s, 1H, (H8)), 7.95 (d, J = 8.3 Hz, 1H, (H4)), 7.85 (d, J = 8.4 Hz, 2H, (H16, H20)), 7.41 (td, J = 8.4, 7.2, 1.3 Hz, 1H, (H5)), 7.36 (td, J = 7.8, 7.3, 1.3 Hz, 1H, (H6)), 7.30 (d, J = 8.2 Hz, 2H, (H17, H19)), 2.38 (s, 3H, (H21)). 13C NMR (126 MHz, CDCl3) δ 185.23 (C10), 146.05 (C18), 136.11 (C8), 135.45 (C3), 134.42 (C15), 130.22 (C17, C19), 127.13 (C16, C20), 126.20 (C5), 126.17 (C2), 124.94 (C6), 122.50 (C1), 122.25 (C7), 113.13 (C4), 21.57 (C21). FTIR (cm-1): 3118, 3069, 1701, 1537, 1245, 1037. HRMS-ESI (m/z), calculated for C16H13NO3S (M+H)+: 300.0694, Found: 300.0709. 6 5 1 4 2 3 O 14 10 CH211 7 N 9 8 S 12 13 O 15 16 20 17 19 18 CH321 N Ts 21 1-tosyl-3-vinyl-1H-indole (2-14) To an oven-dried flask was added methyltriphenylphosphonium bromide (1.3 g, 3.6 mmol, 1.5 equiv) and anhydrous THF (20mL). This was cooled to -30ºC and stirred under an atmosphere of N2 gas. After the mixture reached -30ºC, nBuLi (2.5M in Hexanes) (1.44 mL, 3.6 mmol, 1.5 equiv) was added dropwise. This was stirred for 1 hour at -30ºC under N2. After 1 hour, 1-tosyl- 1H-indole-3-carbaldehyde (720 mg, 2.4 mmol, 1 equiv) was added in a solution of anhydrous THF (10 mL) dropwise. The reaction mixture was then stirred for an additional 1 hour at -30ºC under N2. Once TLC showed consumption of starting material, the reaction was quenched with DI H2O (0.5 mL), poured into Et2O (150 mL), dried with Na2SO4, filtered, and concentrated in 142 vacuo. The crude residue was then purified via silica gel flash column chromatography (EtOAc/Hexanes) to yield the desired product as a white solid. (0.51 g, 70%). Mp: 99-101ºC. 1H NMR (500 MHz, CDCl3) δ 7.99 (d, J = 8.3 Hz, 1H, (H1)), 7.77 (d, J = 8.4 Hz, 2H, (H16, H20)), 7.74 (d, J = 7.9 Hz, 1H, (H4)), 7.60 (s, 1H, (H8)), 7.33 (td, J = 8.3, 7.3, 1.1 Hz, 1H, (H6)), 7.27 (td, J = 7.9, 7.3, 1.1 Hz, 1H, (H5)), 7.22 (d, J = 8.2 Hz, 2H, (H17, H19)), 6.77 (dd, J = 17.8, 11.3 Hz, 1H, (H10)), 5.79 (d, J = 17.9 Hz, 1H, (H11)), 5.35 (d, J = 11.3 Hz, 1H, (H11)), 2.34 (s, 3H, (H21)). 13C NMR (126 MHz, CDCl3) δ 145.18 (C18), 135.61 (C3), 135.23 (C15), 130.06 (C17, C19), 129.13 (C2), 127.69 (C10), 127.00 (C16, C20), 125.04 (C8), 124.21 (C1), 123.64 (C6), 121.06 (C7), 120.55 (C5), 115.47 (C11), 113.86 (C4), 21.72 (C21). FTIR (cm-1): 3082, 3007, 2895, 1636, 1596, 1251, 1043. HRMS-ESI (m/z), calculated for C17H15NO2S (M+H)+: 298.0902, Found: 298.0923. 143 Ts N BF3OEt2 Ts BF3OEt2 N Ts N BF3OEt2 N Ts N Ts 18a BF3OEt2 Ts N N Ts 25 9 12 10 11 25 24 21 O 6 5 13 31 O 14 37 36 29 CH3 26 23 S 19 N 1 N 4 S 20 32 35 CH338 27 28 22 O 8 7 2 3 30 O 33 34 17 16 18 15 2,5-diphenyl-1,4-ditosylpiperazine (2-17) This side product was isolated as a clear, colorless oil. 1H NMR (500 MHz, CDCl3) δ 7.47 (d, J = 8.3 Hz, 4H, (H24, H28, H33, H37)), 7.31 – 7.22 (m, 6H, (H8, H12, H10, H18, H14, H16)), 7.20 (d, J = 8.2 Hz, 4H, (H25, H27, H34, H36)), 7.18 – 7.14 (m, 4H, (H9, H11, H14, H18)), 4.83 (dd, J = 10.3, 5.8 Hz, 2H, (H2, H5)), 3.93 (dd, J = 14.9, 5.8 Hz, 2H, (H3, H6)), 3.54 (dd, J = 14.9, 10.4 Hz, 2H, (H3, H6)), 2.41 (s, 6H, (H29, H38)). 13C NMR (126 MHz, CDCl3) δ 143.75 (C23, C32), 138.52 (C7, C13), 136.83 (C26, C35), 129.75 (C25, C27, C34, C36), 128.78 (C8, C12, C18, C14), 128.25 (C10, C16), 127.29 (C24, C28, C33, C37), 126.71 (C9, C11, C15, C17), 58.33 (C2, C5), 47.72 (C3, C6), 21.68 (C29, C38). FTIR (cm-1): 3032, 2890, 1522, 1349, 1159. HRMS-ESI (m/z): calculated for C30H30N2O4S2 (M+H)+: 547.1725, Found: 547.1742. 144 8 N 9 7 NH 11 10 CH312 N N H 6 5 1 4 2 3 5-methyl-2-phenyl-4,5-dihydro-1H-imidazole (2-21) Benzoyl cyanide (0.12 g, 1.0 mmol, 1.0 equiv), was dissolved in minimal DMF. Methyl diamine (0.9 mL, 1.0 mmol, 1.0 equiv) was then added, followed by DBU (0.015 mL, 0.1 mmol, 0.1 equiv). This was then placed in a pre-heated oil bath and was stirred at 40ºC under N2 for 4 hours. After 4 hours, the reaction was allowed to cool to 20ºC, was extracted with ethyl acetate (3x20 mL), and concentrated in vacuo. The crude residue was then purified via silica gel flash column chromatography (EtOAc/Hexanes) to yield this side product as white solid. (0.10 g, 65%). Mp: 102-103ºC. 1H NMR (500 MHz, CDCl3) Mixture of tautomers δ 7.83 – 7.77 (m, 2H, (H1, H3)), 7.52 – 7.44 (m, 1H, (H5), 7.44 – 7.37 (m, 2H, (H4, H6)), 7.36 – 7.28 (m, 1H, (H11 or H8)), 7.10 – 7.05 (m, 1H, (H11 or H8)), 4.46 – 4.34 (m, 1H, (H10)), 3.76 – 3.66 (m, 1H, (H9)), 3.53 – 3.44 (m, 1H, (H9)), 1.32 (d, J = 6.7 Hz, 3H, (H12)). 13C NMR (126 MHz, CDCl3) Mixture of tautomers δ 168.94 (C7) and 168.35 (C7), 134.16 (C2) and 134.02 (C2), 131.76 (C5) and 131.72 (C5), 128.73 (C4, C6) and 128.71 (C4, C6), 127.16 (C1, C3) and 127.15 (C1, C3), 47.41 (C10), 46.75 (C9), 18.58 (C12). FTIR (cm-1): 3385, 3071, 2983, 1637, 1542. HRMS-ESI (m/z): calculated for C10H12N2 (M+H)+: 161.1073, Found: 161.1082. 145 10 CH3 8 11 CH3 CH39 O 7 O 5 2 O4 O 6 NH 1 3 O O N H tert-butyl (2-oxoethyl)carbamate (2-22) In a round-bottom flask tert-butyl (2,3-dihydroxypropyl)carbamate (1.0 g, 5.2 mmol, 1.0 equiv) was dissolved in DI water at room temperature under an atmosphere of N2 gas. The flask was then covered in aluminum foil to protect the reaction mixture from light. Then, NaIO4 (1.3 g, 6.2 mmol, 1.2 equiv) was added at room temperature under an atmosphere of N2 gas. This mixture was allowed to stir for 1 hour under N2 gas, covered in aluminum foil. After 1 hour, a white precipitate had formed. This white precipitate was then filtered out by filtration via a buchner funnel. The filtrate was then extracted with DCM (3x30 mL), dried over Na2SO4, and concentrated in vacuo to afford the desired product as a yellow oil, which afforded a white crystalline solid upon recrystallization in the freezer. (0.66 g, 80%). Mp: 37-38ºC. 1H NMR (500 MHz, CDCl3) δ 9.65 (s, 1H, (H3)), 5.20 (s, 1H, (H1)), 4.07 (d, J = 4.8 Hz, 2H, (H2)), 1.45 (s, 9H, (H9, H10, H11)).13C NMR (126 MHz, CDCl3) δ 197.81 (C3), 155.90 (C5), 80.17 (C8), 51.28 (C2), 28.27 (C9, C10, C11). FTIR (cm-1): 3321, 2981, 2878, 1738, 1685, 1523, 1252. HRMS-ESI (m/z): calculated for C7H13NO3 (M+H)+: 160.0974, Found: 160.0998. 146 26 25 27 24 22 23 14 18 CH3 CH320 17 CH319 O 16 15 13 NH NH 12 11 10 O 21 6 5 1 4 2 3 8 7 NH 9 NHBoc HN N H 29 tert-butyl (2-(benzylamino)-2-(1H-indol-3-yl)ethyl)carbamate (2-23) Indole (0.70g, 6.0 mmol, 2.0 equiv), benzyl amine (0.33 mL, 3.0 mmol, 1.0 equiv), N-Boc Glycinal (0.48 g, 3.0 mmol, 1.0 equiv), and 1,1,3,3-tetramethyl guanidine (0.033 mL, 0.3 mmol, 0.1 equiv) were added sequentially to DI H2O (8 mL) at 20ºC. The reaction mixture was then added to a pre-heated oil bath (45ºC). The reaction was then stirred for 12 hours sat 45ºC under an atmosphere of N2 gas. After completion of the reaction, the reaction mixture was diluted with EtOAc (50 mL) and water (20 mL). The organic layer was then extracted with EtOAc (3x30 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The crude residue was then purified via silica gel flash column chromatography (EtOAc/Hexanes) to yield the desired product as an orange-brown oil. (0.42 g, 50%). 1H NMR (500 MHz, CDCl3) δ 7.67 (d, J = 7.8 Hz, 1H, (H1)), 7.40 (d, J = 8.1 Hz, 1H, (H4)), 7.36 – 7.17 (m, 7H, (H5, H8, H22, H23, H24, H25, H26)), 7.14 (t, J = 7.3 Hz, 1H, (H6)), 5.31 – 5.25 (m, 1H, (H10)), 4.63 (S, 1H. (H13)), 3.73 – 3.66 (m, 1H. (H11)), 3.64 (d, J = 13.1 Hz, 1H, (H14)), 3.54 (d, J = 13.3 Hz, 1H, (H14)), 3.49 – 3.38 (m, 1H, (H11)), 2.24 (s, 1H, (H12)), 1.41 (s, 9H, (H18, H19, H20)). 13C NMR (126 MHz, CDCl3) δ 156.22 (C15), 139.34 (C22), 136.16 (C3), 129.16 (C2), 128.57 (C23, C27), 128.32 (C24 C26), 127.34 (C25), 124.27 (C8), 121.83 (C5), 121.17 (C1), 119.91 (C6), 109.97 (C4), 102.64, (C7), 79.90, (C17), 69.71 (C10), 49.83 (C14), 45.36 (C11), 28.42 (C18, C19, C20). FTIR (cm-1): 147 3413, 3341, 3313, 3054, 2886, 1690, 1508, 1245. HRMS-ESI (m/z), calculated for C22H27N3O2 (M+H)+: 366.2181, Found: 366.2199. 19 18 20 17 15 16 14 13 NH2 NH 12 11 10 6 5 1 4 2 3 8 7 NH 9 HN NH2•HCl N H 1-(1H-indol-3-yl)ethane- N-Benzyl-1,2-diamine hydrochloride (2-24) To an oven-dried flask was added tert-butyl (2-(benzylamino)-2-(1H-indol-3-yl)ethyl)carbamate (0.26 g, 1.0 mmol, 1.0 equiv) and ethanolic HCl (3.3M in EtOH, 5.0 mL). This was stirred at 20ºC under an atmosphere of N2 gas for 18 hours. After completion, as indicated by TLC, the reaction mixture was concentrated in vacuo to obtain a semi-solid. This semi-solid was triturated with Et2O (5.0 mL) and concentrated in vacuo to afford the crude product as a reddish-brown crystalline foam. This was used in subsequent steps without any further purification (>99% determined by NMR, quantitative). 1H NMR (500 MHz, CD3OD) δ 7.80 (s, 1H, (H8)), 7.78 (d, J = 7.9 Hz, 1H, (H1)), 7.53 (d, J = 8.2 Hz, 1H, (H4)), 7.51 – 7.36 (m, 5H, (H16, H17, H18, H19, H20)), 7.27 (t, J = 7.7 Hz, 1H, (H5)), 7.20 (t, J = 7.6 Hz, 1H, (H6)), 5.12 (dd, J = 10.1, 5.1 Hz, 1H, (H10)), 4.27 (d, J = 12.9 Hz, 1H, (H14)), 3.96 (d, J = 12.9 Hz, 1H, (H14)), 3.90 – 3.78 (m, 2H, (H11)). 13C NMR (126 MHz, CD3OD) δ 138.24 (C3), 132.02 (C15), 131.12 (C16, C20), 130.57 (C18), 130.11 (C17, C19), 128.10 (C8), 127.54 (C2), 123.94 (C5), 121.66 (C6), 118.95 (C1), 113.30 (C4), 104.22 (C7), 53.63 (C10), 50.59 (C11), 41.54 (C14). FTIR (cm-1): 3412, 148 3345, 3299, 3012, 2892, 1552. HRMS-ESI (m/z), calculated for C17H19N3 (M+H)+: 266.1657, Found: 266.1681. 14 O 12 O 11 10 Cl 13 6 5 1 4 2 3 8 7 NH 9 O O Cl N H 39 2-(1H-indol-3-yl)-2-oxoacetyl chloride (2-29) To an oven-dried flask, charged with N2 gas, was added 1H-indole (0.5 g, 4.3 mmol, 1.0 equiv). This was dissolved in anhydrous Et2O (10 mL). This solution was cooled to 0ºC and switched to an atmosphere of Argon gas. After 3 hours, the reaction was warmed to 20ºC and stirred an additional 1 hour. The resulting yellow precipitate was isolated by vacuum filtration, via a Buchner funnel, and was washed with minimal Et2O. The resulting yellow solid was dried on vacuum overnight to afford the desired product as a yellow crystalline solid (0.77 g, 87%). Mp: 128-130ºC. 1H NMR (500 MHz, DMSO) δ 12.39 (s, 1H, (H9)), 8.42 (d, J = 3.3 Hz, 1H, (H8)), 8.17 (d, J = 7.0 Hz, 1H, (H1)), 7.54 (d, J = 7.1 Hz, 1H, (H4)), 7.29 (ddd, J = 7.2, 5.7, 1.6 Hz, 1H, (H5)), 7.26 (ddd, J = 7.1, 5.4, 1.5 Hz, 1H, (H6)). 13C NMR (126 MHz, DMSO) δ 180.89 (C10), 165.37 (C11), 138.19 (C8), 136.87 (C3), 125.75 (C2), 123.87 (C5), 122.89 (C6), 121.30 (C1), 112.89 (C4), 112.47 (C7). FTIR (cm-1): 3405, 3015, 2869, 1771, 1702, 1511. HRMS-ESI (m/z), calculated for C10H6ClNO2 (M+H)+: 208.0165, 210.0138, Found: 208.0180, 210.0153. 149 14 S 12 O 11 10 6 5 1 4 2 3 8 7 NH 9 16 17 N 15 20 18 19 O S N N H 40 1-(1H-indol-3-yl)-2-(piperidin-1-yl)-2-thioxoethan-1-one (2-31) 2-(1H-indol-3-yl)-2-oxoacetyl chloride (0.21 g, 1.0 mmol, 1.0 equiv), was dissolved in anhydrous EtOAc (2.0 mL). To this was added a solution of n-Bu3SnH (0.30 mL, 1.0 mmol, 1.0 equiv) in anhydrous EtOAc (1.0 mL) at 0ºC under an atmosphere of N2 gas. The reaction mixture was then stirred for 30 minutes at 0ºC. The reaction was then allowed to warm to 20ºC and was stirred for 16 hours at 20ºC under N2. After 16 hours, hexanes was added and the resulting yellow precipitate was washed with large amounts of hexanes and collected by vacuum filtration. This crude product was then dissolved in a minimal amount of piperidine. To this was added S8 (0.33 g, 1.2 mmol, 1.2 equiv), followed by piperidine (0.15 mL, 1.5 mmol, 1.5 equiv) at 20ºC under N2. The reaction mixture was then warmed to 80ºC and was stirred for 5 hours under N2. After 5 hours, the reaction was allowed to cool to 20ºC, then DI H2O was added (15 mL). The organic layer was extracted with EtOAc (3x30 mL), the combined organic layers were then washed with brine (30 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The crude residue was then purified via silica gel flash column chromatography (EtOAc/Hexanes) to yield the desired product as a brown oil. (0.14 g, 45% over 2 steps). 1H NMR (500 MHz, CD3OD) δ 8.17 (d, J = 7.1 Hz, 1H, (H1)), 8.01 (s, 1H, (H8)), 7.48 (d, J = 7.1, 2.0 Hz, 1H, (H4)), 7.27 (td, J = 7.3, 5.5, 1.4 Hz, 1H, (H5)), 7.24 (td, J = 7.2, 5.6, 1.3 Hz, 1H, (H6)), 4.27 – 4.21 (m, 2H, (H16, H20)), 3.62 – 3.55 (m, 2H, (H16, H20)), 1.81 – 1.68 (m, 4H, (H17, H19)), 1.58 – 1.54 (m, 2H, (H18)). 13C NMR (126 MHz, CD3OD) δ 196.45 (C11), 187.66 (C10), 138.60 (C3), 137.44 (C8), 150 126.82 (C2), 124.83 (C5), 123.78 (C6), 122.49 (C1), 114.35 (C7), 113.30 (C4), 53.97 (C16, C20), 49.21 (C16, C20), 27.67 (C18), 26.39 (C17, C19), 25.04 (C17, C19). FTIR (cm-1): 3437, 3065, 2873 1695, 1542, 1132. HRMS-ESI (m/z), calculated for C15H17N2OS (M+H)+: 273.1062, Found: 273.1100, C15H17N2OSNa (M+H)+: 296.0959, Found: 296.0972. 13 S CH314 12 O O I S N N H 38 11 10 16 N 17 20 8 18 19 6 5 1 4 2 3 7 NH 9 1-(2-(1H-indol-3-yl)-1-(methylthio)-2-oxoethylidene)piperidin-1-ium iodide (2-32) 1-(1H-indol-3-yl)-2-(piperidin-1-yl)-2-thioxoethan-1-one (0.13 g, 0.46 mmol, 1.0 equiv) was dissolved in iodomethane (1.3 mL, 9.2 mmol, 20.0 equiv) in an oven-dried flask under N2 gas. This solution was refluxed for 5 hours under N2. After completion of the reaction (indicated by TLC), the reaction mixture was allowed to cool to 20ºC. Then, any excess iodomethane was evaporated in vacuo to afford the desired product as an off-white semi-solid (0.19 g, Quantitative). This was used in subsequent steps without any further purification. 1H NMR (500 MHz, CD3OD) δ 8.53 (s, 1H, (H8)), 8.27 (d, J = 7.0 Hz, 1H, (H1)), 7.60 (d, J = 7.1 Hz, 1H, (H4)), 7.41 (pd, J = 7.3, 7.2, 7.2, 7.1, 1.4 Hz, 2H, (H5, H6)), 4.26 – 4.18 (m, 1H, (H16, H20)), 4.16 – 4.09 (m, 1H, (H16 or H20)), 4.09 – 4.01 (m, 1H, (H16 or H20)), 3.94 – 3.85 (m, 1H, (H16 or H20)), 2.66 (s, 3H, (H14)), 2.15 – 2.09 (m, 1H, (H18)), 2.05 – 1.94 (m, 1H, (H18)), 1.92 – 1.77 (m, 4H, (H17, H19)). 13C NMR (126 MHz, CD3OD) δ 186.03 (C10), 176.79 (C11), 140.23 (C8), 139.28 (C3), 126.52 (C5), 125.79 (C2), 125.35 (C6), 122.66 (C1), 114.32 (C7), 114.08 (C4), 58.68 (C20, C16), 55.78 (C20, C16), 28.15 (C17, C19), 26.77 (C18), 23.49 (C17, 151 C19), 16.77 (C14). FTIR (cm-1): 3403, 3068, 2885, 1699, 1628, 1520. HRMS-ESI (m/z): Calculated for C16H19NOS+: 287.1218, Found: 287.1232. 16 O 10 11 1 2 7 N N 15 13 14 22 23 24 19 25 18 17 3 4 NH 9 8 26 27 32 21 28 29 NH 20 30 31 6 5 NH N N Bn O N H (1-benzyl-5-(1H-indol-3-yl)-4,5-dihydro-1H-imidazol-2-yl)(1H-indol-3-yl)methanone (2-25) Procedure A: To a stirred solution of N1-benzyl-1-(1H-indol-3-yl)ethane-1,2-diamine hydrochloride (2-24) (150.7 mg, 0.5 mmol, 1 equiv) and the thioimidate fragment 2-32 (0.21 g, 0.5 mmol, 1.0 equiv) in anhydrous MeOH (3 mL) was added Amberlyst A-21 (0.35 g). This was allowed to stir at 20ºC under N2 for 36 hours. Then the MeOH was concentrated in vacuo and ethyl acetate (25 mL) and water (10 mL) were added. The organic layer was then extracted with EtOAc (3x25 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The crude residue was then purified via silica gel flash column chromatography (EtOAc/Hexanes) to yield the desired product as a tan-brown semi-solid. (0.067 g, 32%). Procedure B: To a stirred solution of N1-benzyl-1-(1H-indol-3-yl)ethane-1,2-diamine hydrochloride (2-24) (0.15 g, 0.5 mmol, 1.0 equiv) and the thioimidate fragment 2-32 (0.21 g, 0.5 mmol, 1.0 equiv) in anhydrous MeOH (5.0 mL) was added triethylamine (0.18 mL, 2.5 equiv). This was allowed to stir at 20ºC under N2 for 48 hours. Then the MeOH was concentrated in vacuo and ethyl acetate 152 (25 mL) and water (10 mL) were added. The organic layer was then extracted with EtOAc (3x25 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The crude residue was then purified via silica gel flash column chromatography (EtOAc/Hexanes) to yield the desired product as a tan-brown semi-solid. (0.11 g, 53%). Procedure C: To an oven-dried flask purged with N2 gas was added I2 (0.28 g, 1.1 mmol, 1.1 equiv). To this was added 1-(1H-indol-3-yl)ethan-1-one (0.16 g, 1.0 mmol, 1.0 equiv) dissolved in DMSO (2.5 mL) at 20ºC under an atmosphere of N2 gas. This reaction mixture was then heated to 100ºC and was stirred for 2h. The resulting solution was then slowly cooled to 0ºC and the reaction mixture was treated with NaHCO3 (0.34 g, 4.0 mmol, 4.0 equiv). N1-benzyl-1-(1H-indol-3-yl)ethane-1,2- diamine hydrochloride (2-24) (0.30 g, 1.0 mmol, 1.0 equiv) in MeCN/MeOH (1:1) (5.0 mL/5.0 mL) was also added and the reaction was allowed to stir for 1 hour at 0ºC under N2. After 1h, NCS (0.13 g, 1.0 mmol, 1.0 equiv) was added at 0ºC and the reaction was allowed to stir for 20 minutes at 0ºC. Then, the reaction mixture was allowed to warm to room temperature and was stirred for an additional 12 hours at 20ºC under N2 gas. The solvent was then removed in vacuo and the crude residue was dissolved in chloroform (30 mL). This solution was then washed was saturated aqueous, NaHCO3 (50 mL), saturated aqueous Na2S2O3 (50 mL) and ice cold saturated aqueous NaCl (3x50mL). The organic layer was then dried over Na2SO4, filtered, and concentrated in vacuo. The crude residue was then purified via silica gel flash column chromatography (EtOAc/Hexanes) to yield the desired product as a tan-brown semi-solid. (0.19 g, 45%). 1H NMR (500 MHz, CD3OD) δ 8.33 (d, J = 6.6 Hz, 1H, (H1)), 8.31 (s, 1H, (H8)), 7.65 (d, J = 6.9 Hz, 1H, (H22)), 7.50 (d, J = 6.5, Hz, 1H, (H4)), 7.42 (d, J = 8.2 Hz, 1H, (H25)), 7.32 – 7.26 (m, 2H, (H5, H6)), 7.21 – 7.13 (m, 5H, (H21, H24, H28, H30, H32)), 7.15 – 7.00 (m, 3H, 153 (H23, H29, H31)), 5.04 (dd, J = 11.8, 9.5 Hz, 1H, (H14)), 4.66 (d, J = 15.6 Hz, 1H, (H26)), 4.29 (dd, J = 14.7, 11.9 Hz, 1H, (H13)), 4.03 (dd, J = 14.7, 9.5 Hz, 1H, (H13)), 3.90 (d, J = 15.6 Hz, 1H, (H26)).13C NMR (126 MHz, CD3OD) δ 184.12 (C10), 164.16 (C11), 139.42 (C8), 138.99 (C19), 138.67 (C3), 138.30 (C27), 129.42 (C29, C31), 129.28 (C28, C32), 128.40 (C30), 127.01 (C2), 126.63 (C18), 125.52 (C21), 125.13 (C5), 124.07 (C6), 122.98 (C1), 122.93 (C24), 120.32 (C23), 120.11 (C22), 117.17 (C7), 114.38 (C17), 113.28 (C4), 112.88 (C25), 60.77 (C13), 58.78 (C14), 48.23 (C26). FTIR (cm-1): 3405, 3399, 3052, 2877, 1709, 1637, 1536. HRMS-ESI (m/z): Calculated for C27H22N4O (M+H)+: 419.1872, Found: 419.1891. O 20 O 17 14 7 16 15 2 3 21 CH3 18 19 8 13 9 12 10 11 6 5 1 4 ((4-methoxyphenyl)methylene)dibenzene (2-37) To a flame-dried r.b. flask was added anisole (1 mL, 18.4 mmol, 10 equiv). To this was added FeCl3 (11.0 mg, 0.07 mmol, 0.04 equiv), followed by benzophenone (0.32 g, 1.8 mmol, 1.0 equiv) at room temperature, under an atmosphere of N2 gas. Next Me3SiCl (0.25 mL, 1.9 mmol, 1.1 equiv) was added to the mixture, which dissolved the FeCl3. Then, Et3SiH (0.30 mL, 1.9 mmol, 1.05 equiv) was diluted to a total volume of 1 mL with anisole. This was then added dropwise (0.1 mL/ 5 min). The resulting solution was then stirred for 2 hours at room temperature under an atmosphere of N2 gas. Then, the reaction mixture was heated to 50ºC until full conversion was observed via TLC (~1 hour). After completion, the solution was allowed to 154 cool to room temperature and the solution was then diluted with EtOAc (30 mL) and washed with aqueous sodium bicarbonate solution (2x 3 mL), extracted with EtOAc (3 x 30 mL), dried over Na2SO4, filtered, and concentrated in vacuo the crude residue was then purified via silica gel flash column chromatography (EtOAc/Hexanes) to yield the desired product as a clear, colorless oil. (0.44 g, 91% [98:2 para:ortho]). 1H NMR (500 MHz, CDCl3) δ 7.29 (t, J = 7.5 Hz, 4H, (H4, H6, H8, H12)), 7.21 (t, J = 7.2 Hz, 2H, (H5, H13)), 7.12 (d, J = 7.7 Hz, 4H, (H1, H3, H9, H13)), 7.04 (d, J = 8.5 Hz, 2H, (H15, H19)), 6.84 (d, J = 8.8 Hz, 2H, (H16, H18)), 5.52 (s, 1H, (H7)), 3.79 (s, 3H, (H21)). 13C NMR (126 MHz, CDCl3) δ 158.13, (C17), 144.37, (C2, C8), 136.21, (C14), 130.49, (C15, C19), 129.50, (C1, C3, C9, C13), 128.40, (C4, C6, C10, C12), 126.35, (C5, C11), 113.77, (C16, C18), 56.12, (C7), 55.35, (C21). FTIR (cm-1): 3083, 3061, 2954, 1564, 1250, 1041. HRMS-ESI (m/z): Calculated for C20H18O (M+H)+: 275.1436, Found: 275.1447. 6 5 1 4 2 3 7 N 9 8 S 10 O 12 O11 13 14 18 15 17 16 CH319 N S O O 1-tosyl-1H-indole (2-38) To an oven-dried flask, charged with N2 gas, was added anhydrous THF (20mL). Then, 60% NaH solution in mineral oil (0.3 g, 7.5 mmol, 2.5 equiv) was added and this mixture was cooled to 0ºC. Next, a solution of 1H-indole (0.35 g, 3.0 mmol, 1.0 equiv) in anhydrous THF (10 mL) was added dropwise at 0ºC under an atmosphere of N2 gas. This gray reaction mixture was 155 allowed to stir for 20 minutes at 0ºC under N2. After 20 minutes, tosyl-chloride (0.92 g, 4.8 mmol, 1.6 equiv) was added at 0ºC, resulting in an immediate gray-blue color change. The reaction mixture was allowed to warm to 20ºC and was stirred for 12 hours. When TLC showed complete consumption of starting material, the reaction was quenched slowly with deionized water (3mL) dropwise. The reaction mixture was then extracted with EtOAc (3x40mL). The combined organic layers were then dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was purified via silica gel flash column chromatography (EtOAc/Hexanes) to yield the desired product as a clear, colorless oil (0.82 g, Quantitative). 1H NMR (500 MHz, CDCl3) δ 7.84 (s, 1H, (H17)), 7.40 – 7.26 (m, 12H, (H1, H3, H4, H5, H6, H9, H10, H11, H12, H13, H19, H22)), 7.23 (t, J = 7.4 Hz, 1H, (H21)), 7.06 (t, J = 7.5 Hz, 1H, (H20)), 6.56 (d, J = 1.4 Hz, 1H, (H18)), 5.75 (s, 1H, (H7)). 13C NMR (126 MHz, CDCl3) δ 145.02, (C16), 135.34, (C13), 134.89, (C3), 130.83, (C2), 129.95, (C15, C17), 126.88, (C14, C18), 126.42, (C8), 124.63, (C5), 123.36, (C6), 121.46, (C4), 113.61, (C1), 109.12, (C7), 21.62, (C19). FTIR (cm-1): 3100, 3067, 3047, 2917, 1582, 1355, 1156. HRMS-ESI (m/z): Calculated for C15H13NO2S (M+H)+: 272.0745, Found: 272.0766. 156 NH 22 21 16 17 NH 20 6 5 15 18 19 14 7 1 4 2 3 8 13 9 12 10 11 3-benzhydryl-1H-indole (2-39) To an oven-dried round-bottom flask was added FeCl3 (16 mg, 0.10 mmol, 0.1 equiv) at room temperature under an atmosphere of nitrogen gas. Next, benzophenone (0.18 g, 1.0 mmol, 1.0 equiv) was added in 0.5 mL NO2Me, followed by Me3SiCl (0.14 mL, 1.1 mmol, 1.1 equiv). Then, Et3SiH (0.17 mL, 1.05 mmol, 1.05 equiv) (diluted to 1.0 mL with NO2Me) dropwise and in increments of 0.1 mL every 5 minutes. Lastly, indole (0.23 g, 2.0 mmol, 2.0 equiv) was added dropwise in minimal NO2Me at -15ºC. The resulting solution was allowed to stir at -15ºC for 2 hours and then was allowed to warm to room temperature and was stirred for another 12 hours at rt under an atmosphere of nitrogen gas. The reaction mixture was then concentrated in vacuo and the resulting crude residue was purified via silica gel flash column chromatography (EtOAc/Hexanes) to afford the desired product as an off-white crystalline solid (54 mg, 17%). Mp: 118-120ºC. 1H NMR (500 MHz, CDCl3) δ 7.84 (s, 1H, (H17)), 7.40 – 7.26 (m, 12H, (H1, H3, H4, H5, H6, H9, H10, H11, H12, H13, H19, H22)), 7.23 (t, J = 7.4 Hz, 1H, (H21)), 7.06 (t, J = 7.5 Hz, 1H, (H20)), 6.56 (d, J = 1.4 Hz, 1H, (H18)), 5.75 (s, 1H, (H7)). 13C NMR (126 MHz, CDCl3) δ 144.04 (C2, C8), 136.75 (C16), 129.11 (C1, C3, C9, C13), 128.39 (C4, C6, C10, C12), 127.05 (C15), 126.34 (C5, C11), 124.19 (C18), 122.18 (C21), 120.00 (C19), 119.90 (C14), 119.48 (C20), 111.18 (C22), 48.90 (C7). FTIR (cm-1): 3456, 3083, 2926, 1592. HRMS- ESI (m/z): Calculated for C21H17N (M+H)+: 284.1403, Found: 284.1425. 157 Diphenyl methane (2-41) This side product was isolated as a clear, colorless oil. 1H NMR (500 MHz, CDCl3) δ 7.46 – 7.37 (m, 1H, (H4, H6, H10, H12)), 7.35 – 7.29 (m, 1H, (H1, H3, H5, H9, H11)), 4.11 (s, 1H, (H7)). 13C NMR (126 MHz, CDCl3) δ 141.24 (C2, C8), 129.06 (C4, C6, C10, C12), 128.57 (C1, C3, C9, C13), 126.18 (C5, C11), 42.08 (C7). FTIR (cm-1): 3059, 2836, 1512. GC-MS: Product mass identified via GC-MS (See Appendix (2-41)). 17 18 16 15 11 12 10 13 NH 14 6 5 1 4 2 3 8 7 NH 9 NH N H 3-(indolin-2-yl)-1H-indole (2-42) This side Product was isolated as a light purple-brown colored semi-solid. 1H NMR (500 MHz, CDCl3) δ 7.99 (s, 1H, (H14)), 7.62 (d, J = 7.9 Hz, 1H, (H1)), 7.34 (d, J = 8.0 Hz, 1H, (H4)), 7.24 (t, J = 7.6 Hz, 1H, (H5)), 7.20 (d, J = 7.4 Hz, 1H, (H15)), 7.15 – 7.09 (m, 3H, (H6, H8, H17)), 6.81 (t, J = 7.4 Hz, 1H, (H16)), 6.70 (d, J = 7.7 Hz, 1H, (H18)), 5.26 (t, J = 8.7 Hz, 1H, (H10)), 3.50 (dd, J = 15.6, 9.1 Hz, 1H, (H11)), 3.24 (dd, J = 15.6, 8.3 Hz, 1H, (H11)). 13C NMR 158 (126 MHz, CDCl3) δ 150.99, (C13), 136.81, (C3), 129.05, (C12), 127.56, (C17), 125.84, (C2), 124.80, (C15), 122.36, (C5), 121.32, (C8), 119.66, (C6), 119.55, (C1), 119.33, (C7), 118.90, (C16), 111.45, (C4), 109.39, (C18), 56.42, (C10), 37.70, (C11). FTIR (cm-1): 3452, 3383, 3082, 2869, 1540. HRMS-ESI (m/z): Calculated for C16H14N2 (M+H)+: 235.1235, Found: 235.1253. 30 O 32 37 33 36 34 35 CH338 6 5 1 4 2 3 O 22 16 15 11 12 10 7 N 9 8 S 19 17 18 13 N 14 S 20 O 31 O21 23 24 28 25 27 26 CH329 NTs N Ts 1-tosyl-3-(1-tosylindolin-2-yl)-1H-indole (2-43) This side Product was isolated as a pinkish off-white semi-solid. 1H NMR (500 MHz, CDCl3) δ 7.88 (d, J = 8.4 Hz, 1H, (H1)), 7.76 – 7.71 (m, 3H, (H4, H24, H28)), 7.58 (s, 1H, (H8)), 7.45 (d, J = 8.3 Hz, 2H, (H33, H37)), 7.32 – 7.22 (m, 3H, (H5, H17, H18)), 7.20 (d, J = 8.1 Hz, 2H, (H25, H27)), 7.15 (d, J = 7.8 Hz, 1H, (H15)), 7.09 – 7.05 (m, 2H, (H6, H16)), 7.02 (d, J = 8.2 Hz, 2H, (H34, H36)), 5.53 (dd, J = 10.1, 3.1 Hz, 1H, (H10)), 3.23 (dd, J = 16.1, 10.1 Hz, 1H, (H11)), 2.95 (dd, J = 16.2, 3.2 Hz, 1H, (H11)), 2.32 (s, 3H, (H29)), 2.30 (s, 3H, (H38)). 13C NMR (126 MHz, CDCl3) δ 144.97, (C26), 143.86, (C35), 141.56, (C13), 135.60, (C3), 135.07, (C32), 135.01, (C23), 131.24, (C2), 129.91, (C25, C27), 129.42, (C34, C36), 128.80, (C12), 128.14, (C5), 126.93, (C33, C37), 126.91, (C24, C28), 125.07, (C17), 124.75, (C6), 124.69, 159 (C18), 124.40, (C8), 123.34, (C7), 123.20, (C16), 119.95, (C15), 116.85, (C4), 113.77, (C1), 58.57, (C10), 35.68, (C11), 21.58, (C29), 21.49, (C38). FTIR (cm-1): 3072, 2926, 1526, 1362, 1345, 1174, 1157. HRMS-ESI (m/z): Calculated for C30H36N2O4S2 (M+H)+: 543.1412, Found: 543.1435. 21 22 25 26 27 12 NH 13 14 11 10 20 18 NH 17 16 1 4 6 5 19 15 2 3 23 24 8 7 NH29 H N HN NH2 2-(2,2-di(1H-indol-3-yl)ethyl)aniline (2-44) This side product was isolated as a brown semi-solid. 1H NMR (500 MHz, CDCl3) δ 7.89 (s, 2H, (H9)), 7.48 (d, J = 7.9 Hz, 2H, (H23, H24)), 7.28 (d, J = 8.0 Hz, 2H, (H20, H27)), 7.14 (td, J = 7.8, 7.5, 1.0 Hz, 2H, (H21, H26)), 7.03 – 6.94 (m, 4H, (H1, H5, H22, H25)), 6.92 (d, J = 1.5 Hz, 2H, (H14, H16)), 6.63 (td, J = 7.5, 7.4, 1.2 Hz, 1H, (H6)), 6.55 (d, J = 7.3 Hz, 1H, (H4)), 4.85 (t, J = 7.3 Hz, 1H, (H8)), 3.41 (d, J = 7.3 Hz, 1H, (H7)). 13C NMR (126 MHz, CDCl3) δ 144.78, (C3), 136.64, (C12, C18), 130.42, (C1), 126.96, (C5), 126.10, (C2), 122.01, (C14, C16), 121.90, (C21, C26), 119.71, (C23, C24), 119.59, (C11, C19) 119.19, (C22, C25), 118.87, (C4), 115.81, (C6), 111.20, (C20, C27), 111.15, (C10, C15), 37.18, (C7), 34.48, (C8). FTIR (cm-1): 3438, 3315, 3056, 2884, 1512. HRMS-ESI (m/z): Calculated for C24H21N3 (M+H)+: 352.1814, Found: 352.1835. 160 6 5 1 4 O N S O O 12 O 10 CH311 2 3 O 15 7 N 9 8 S 13 16 21 14 O 17 18 20 19 CH322 1-(1-tosyl-1H-indol-3-yl)ethan-1-one (2-47) To an oven-dried flask, charged with N2 gas, was added anhydrous THF (20mL). Then, 60% NaH solution in mineral oil (0.10 g, 2.5 mmol, 2.5 equiv) was added and this mixture was cooled to 0ºC. Next, a solution of 1-(1H-indol-3-yl)ethan-1-one (0.16 g, 1.0 mmol, 1.0 equiv) in anhydrous THF (10mL) was added dropwise at 0ºC under an atmosphere of N2 gas. This gray reaction mixture was allowed to stir for 20 minutes at 0ºC under N2. After 20 minutes, Tosyl Chloride (0.31 g, 1.6 mmol, 1.6 equiv) was added at 0ºC. The reaction mixture was allowed to warm to 20ºC and was stirred for 12 hours. When TLC showed complete consumption of starting material, the reaction was quenched dropwise with deionized water (3mL) dropwise until bubbles subsided and the reaction mixture became clear. This reaction mixture was then extracted with EtOAc (3x30mL). The combined organic layers were then dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was purified via silica gel flash column chromatography (EtOAc/Hexanes) to yield the desired product as a white solid (0.31 g, Quantitative). M.p.: 150-151ºC. 1H NMR (500 MHz, CDCl3) δ 8.33 (d, J = 7.5 Hz, 1H, (H1)), 8.21 (s, 1H, (H8)), 7.92 (d, J = 8.1 Hz, 1H, (H4)), 7.84 (d, J = 8.5 Hz, 2H, (H17, H21)), 7.37 (td, J = 8.0, 7.3, 1.4 Hz, 1H, (H5)), 7.33 (td, J = 7.7, 1.2 Hz, 1H, (H6)), 7.28 (d, J = 8.1 Hz, 2H, 161 (H18, H20)), 2.57 (s, 3H, (H11)), 2.36 (s, 3H, (H22)). 13C NMR (126 MHz, CDCl3) δ 193.61, (C10), 146.07, (C19), 135.00, (C3), 134.61, (C16), 132.33, (C8), 130.37, (C18, C20), 127.63, (C2), 127.27, (C17, C21), 125.86, (C5), 124.95, (C6), 123.24, (C1), 121.70, (C7), 113.16, (C4), 27.93, (C22), 21.78, (C11). FTIR (cm-1): 3060, 2853, 1692, 1564, 1346, 1157. HRMS-ESI (m/z): Calculated for C17H15NO3S (M+H)+: 314.0851, Found: 314.0881. HO N S O O 1-(1-tosyl-1H-indol-3-yl)ethan-1-ol (2-48) To an oven-dried flask charged with N2 gas was added 1-(1-tosyl-1H-indol-3-yl)ethan-1-ol (0.16 g, 0.5 mmol, 1.0 equiv) in MeOH (5.0 mL). At 20ºC, under an atmosphere of N2 gas, was then added NaBH4 (18.9 mg, 0.5 mmol, 1.0 equiv). This reaction mixture was then allowed to stir for 3 hours at 20ºC under an atmosphere of N2 gas. Once the reaction had reached complete conversion as indicated by TLC, the solvent was removed in vacuo. The crude residue was then dissolved in EtOAc (25 mL) and water (20mL), extracted with EtOAc (3x25mL), dried over Na2SO4, filtered, and concentrated in vacuo. The resulting crude residue was then purified via silica gel flash column chromatography (EtOAc/Hexanes) to yield the desired product as a white solid (0.15 g, 94%). M.p.: 113-115ºC. 1H NMR (500 MHz, CDCl3) δ 7.97 (d, J = 8.3 Hz, 1H, 162 (H1)), 7.76 (d, J = 8.4 Hz, 2H, (H17, H21)), 7.65 (d, J = 7.8 Hz, 1H, (H4)), 7.50 (s, 1H, (H8)), 7.32 (t, J = 7.8 Hz, 1H, (H5)), 7.27 – 7.19 (m, 3H, (H6, H18, H20)), 5.12 (q, J = 12.9, 6.4 Hz, 1H, (H10)), 2.33 (s, 3H, (H22)), 1.61 (d, J = 6.5 Hz, 3H, (H11)). 13C NMR (126 MHz, CDCl3) δ 145.10, (C19), 135.63, (C3), 135.33, (C16), 130.02, (C18, C20), 129.04, (C2), 127.14, (C7), 126.96, (C17, C21), 124.95, (C5), 123.27, (C6), 122.20, (C8), 120.50, (C4), 113.83, (C1), 63.97, (C10), 23.56, (C22), 21.69, (C11). FTIR (cm-1): 3321, 3191, 2905, 1574, 1348, 1201, 1166. HRMS-ESI (m/z): Calculated for C17H17NO3S (M+H)+: 316.1007, Found: 316.1027. O N N N S O O O N S O 2 3 1 4 6 5 9 N 7 8 S 33 O 36 18 19 N 35 O O 38 31 30 29 27 28 32 26 11 O N 16 15 12 10 14 17 N 13 39 45 41 44 42 43 CH351 24 25 21 23 22 37 O 20 S 34 46 40 50 47 48 49 CH352 (1-benzyl-4-(1-tosyl-1H-indol-3-yl)-4,5-dihydro-1H-imidazol-2-yl)(1-tosyl-1H-indol-3- yl)methanone (2-50) To an oven-dried flask was added anhydrous THF (5 mL). This was then cooled to 0ºC and NaH (42 mg (60% in mineral oil), 3.5 equiv) was added. Then, the imidazoline intermediate (0.13 g, 1.0 equiv) was added dropwise in THF (5 mL) at 0ºC under an atmosphere of N2 gas. This mixture was then allowed to stir for 1 hour at 0ºC. After 1 hr, TsCl (0.17 g, 3.0 equiv.) was 163 added and the reaction was allowed to warm to room temperature and was stirred overnight under an atmosphere of N2 gas. After the reaction had reached complete conversion, as indicated by TLC, it was slowly (dropwise) quenched with DI water. The reaction was then extracted with ethyl acetate (3 X 25mL), the organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. The resulting crude residue was then purified via silica gel flash column chromatography (EtOAc/Hexanes) to yield the desired product as an off-white/ yellow semi-solid (155 mg, 70%). 1H NMR (500 MHz, CDCl3) δ 8.93 (s, 1H, (H8)), 8.42 (d, J = 7.0 Hz, 1H, (H1)), 8.04 (d, J = 8.4 Hz, 1H, (H25)), 7.94 (d, J = 7.6 Hz, 1H, (H4)), 7.89 (d, J = 8.4 Hz, 2H, (H41, H45)), 7.79 (d, J = 8.4 Hz, 2H, (H46, H50)), 7.59 (d, J = 7.8 Hz, 1H, (H22)), 7.48 (s, 1H, (H18)), 7.42 – 7.33 (m, 4H, (H2, H3, H23, H24)), 7.32 – 7.17 (m, 7H, (H28, H30, H32, H42, H44, H47, H49)), 7.07 – 7.01 (m, 2H, (H29, H31)), 4.96 (d, J = 15.8 Hz, 1H, (H26)), 4.85 (dd, J = 11.9, 9.9 Hz, 1H, (H14)), 4.38 (dd, J = 16.0, 11.9 Hz, 1H, (H15)), 4.01 (dd, J = 16.0, 9.9 Hz, 1H, (H15)), 3.82 (d, J = 15.8 Hz, 1H, (H26)), 2.36 (s, 3H, (H51)), 2.34 (s, 3H, (H52)). 13C NMR (126 MHz, CDCl3) δ 182.55 (C10), 160.86 (C12), 146.14 (C43), 145.29 (C48), 137.28 (C27), 136.75 (C5), 135.97 (C39), 135.11 (C40), 134.70 (C20), 130.37 (C42, C44), 130.04 (C47, C49), 128.67 (C29, C31), 128.42 (C6), 128.09 (C41, C45), 127.91 (C21), 127.62 (C30), 127.47 (C46, C50), 126.92 (C28, C32), 125.97 (C8), 125.28 (C3), 125.14 (C23), 124.67 (C2), 123.64 (C24), 123.10 (C22), 121.27 (C17), 120.42 (C18), 119.23 (C9), 114.05 (C25), 113.19 (C4), 60.45 (C14), 55.91 (C15), 47.86 (C26), 21.72 (C51), 21.65 (C52). FTIR (cm-1): 3001, 2946, 1709, 1521, 1442, 1373, 1179. HRMS-ESI (m/z): Calculated for C41H34N4O5S2 (M+H)+: 727.2049, Found: 727.2061. 164 2 3 1 4 13 OH 12 OH 11 10 6 5 O 16 9 N 7 18 8 S 14 17 15 O 22 21 19 20 CH323 OH HO N Ts (S)-1-(1-tosyl-1H-indol-3-yl)ethane-1,2-diol (2-55) To an oven-dried round bottom flask was added a solution of AD-mix- α (1.25 g, 1.6 mmol, 2.0 equiv) in t-BuOH/H2O (1:1, 10 mL:10 mL). This solution was then stirred vigorously at 20ºC under an atmosphere of N2 gas for 1 hour. After 1 hour, the reaction mixture was then cooled to 0ºC and then 3-vinyl indole (0.24 g, 0.80 mmol, 1.0 equiv) was then added under an atmosphere of N2 gas. The resulting reaction mixture was then allowed to stir for 3 hours at 0ºC. After 3 hours, the reaction was allowed to warm to 20ºC and was then stirred overnight at 20ºC, under an atmosphere of N2. After 18 hours, the reaction was quenched at 20ºC with Na2SO3 (1.50 g). This was then allowed to stir at 20ºC for an additional 2 hours. After 2 hours, the aqueous layer was then extracted with EtOAc (3x20 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The crude residue was then purified via silica gel column chromatography (EtOAc/Hexanes) to yield the desired product as a clear, pale yellow oil (0.20 g, 78%). Mp: 118-120ºC. 1H NMR (500 MHz, CDCl3) δ 7.94 (d, J = 8.4 Hz, 1H, (H1)), 7.70 (d, J = 8.4 Hz, 2H, (H18, H22)), 7.59 (s, 1H, (H8)), 7.47 (d, J = 7.8 Hz, 1H, (H4)), 7.27 (t, J = 7.9 Hz, 1H, (H3)), 7.12 (t, J = 7.6 Hz, 1H, (H2)), 7.05 (d, J = 8.2 Hz, 2H, (H19, H21)), 4.99 (dd, J = 7.4, 2.4 Hz, 1H, (H10)), 3.80 (dd, J = 11.4, 2.2 Hz, 165 1H, (H11)), 3.73 (dd, J = 11.3, 7.9 Hz, 1H, (H11)), 3.67 (s, 1H, (H12 or H13)), 2.19 (s, 3H, (H23)). 13C NMR (126 MHz, CDCl3) δ 145.06 (C20), 135.18 (C5), 134.91 (C17), 129.87 (C19, C21), 128.89 (C6), 126.81 (C18, C22), 124.86 (C3), 123.48 (C8), 123.29 (C2), 122.19 (C9), 120.12 (C4), 113.65 (C1), 68.56 (C10), 66.48 (C11), 21.44 (C23). FTIR (cm-1): 3321, 3043, 2897, 1596, 1356, 1167, 1098, 1056. HRMS-ESI (m/z): calculated for C17H17NO4S (M+H)+: 332.0957, Found: 332.0971. 28 29 30 24 N 25 O 33 S 31 27 23 22 1 5 N 26 6 N 2 7 17 32 O 36 37 35 38 34 39 CH340 4 3 16 15 12 8 14 11 13 9 21 20 NH 10 18 19 O S N O N N N H 3-(1-benzyl-5-(1H-indol-3-yl)-4,5-dihydro-1H-imidazol-2-yl)-1-tosyl-1H-indole (2-58) This side product was isolated as a light brown semi-solid. 1H NMR (500 MHz, CD3OD) δ 8.31 (s, 1H, (H26)), 8.08 (d, J = 8.4 Hz, 1H, (H27)), 7.84 (d, J = 8.6 Hz, 2H, (H35, H39)), 7.64 (d, J = 8.1 Hz, 1H, (H30)), 7.55 (d, J = 8.1 Hz, 1H, (H16)), 7.49 – 7.44 (m, 2H, (H13, H29)), 7.37 – 7.31 (m, 2H, (H9, H28)), 7.29 – 7.23 (m, 5H, (H18, H19, H20, H36, H38)), 7.21 (t, J = 8.2 Hz, 1H, (H14)), 7.08 (t, J = 7.5 Hz, 1H, (H15)), 6.99 – 6.93 (m, 2H, (H17, H21)), 5.38 (dd, J = 12.7, 8.7 Hz, 1H, (H3)), 4.52 (d, J = 16.2 Hz, 1H, (H6)), 4.41 (t, J = 12.7 Hz, 1H, (H4)), 4.16 (dd, J = 13.1, 8.7 Hz, 1H, (H4)), 4.08 (d, J = 16.2 Hz, 1H, (H6)), 2.28 (s, 3H, (H40)). 13C NMR (126 MHz, CD3OD) δ 161.51 (C1), 147.73 (C37), 139.11 (C11), 136.45 (C34), 136.03 (C24), 135.53 166 (C7), 131.36 (C36, C38), 131.06 (C23), 130.77 (C26), 129.96 (C18, C20), 129.12 (C19), 128.61 (C17, C21), 128.32 (C35, C39), 127.31 (C29), 126.60 (C9), 126.15 (C12), 125.87 (C28), 123.41 (C14), 121.58 (C30), 120.88 (C15), 119.34 (C16), 115.25 (C27), 113.36 (C13), 112.54 (C8), 109.67 (C22), 59.66 (C3), 54.81 (C4), 49.85 (C6), 21.50 (C40). FTIR (cm-1): 3404, 3091, 2905, 1640, 1344, 1166. HRMS-ESI (m/z): Calculated for C33H28N4O2S (M+H)+: 545.2011, Found: 545.2029. N N N H 5 N N 2 7 1 6 17 4 3 16 15 12 8 14 11 13 9 21 20 NH 10 18 19 3-(1-benzyl-4,5-dihydro-1H-imidazol-5-yl)-1H-indole (2-59) This side product was isolated as a reddish-brown semi-solid. 1H NMR (500 MHz, CD3OD) δ 7.46 (d, J = 8.2 Hz, 1H, (H16)), 7.44 (d, J = 8.5 Hz, 1H, (H13)), 7.38 (s, 1H, (H1)), 7.34 – 7.28 (m, 3H, (H17, H21, H19)), 7.24 (s, 1H, (H9)), 7.21 – 7.17 (m, 1H, (H14)), 7.18 – 7.13 (m, 2H, (H18, H20)), 7.06 (t, J = 7.5 Hz, 1H, (H15)), 5.12 (dd, J = 12.1, 9.3 Hz, 1H, (H3)), 4.55 (d, J = 15.0 Hz, 1H, (H6)), 4.25 (t, J = 12.6 Hz, 1H, (H4)), 4.04 – 3.96 (m, 2H, (H4, H6)). 13C NMR (126 MHz, CD3OD) δ 156.91 (C1), 135.00 (C7), 128.49 (C17, C21), 128.06 (C18, C20), 127.83 (C11), 127.74 (C19), 127.59 (C12), 124.77 (C8), 121.87 (C9), 119.29 (C14), 118.07 (C15), 116.84 (C16), 111.78 (C13), 57.73 (C3), 56.97 (C4), 44.28 (C6). FTIR (cm-1): 3427, 3048, 2899, 1639, 1556. HRMS-ESI (m/z): Calculated for C18H17N3 (M+H)+: 276.1501, Found: 276.1523. 167 6 2 1 I 5 3 4 I 7 Iodobenzene (2-60) This side product was isolated as a clear, colorless oil. 1H NMR (500 MHz, CDCl3) δ 7.72 (d, J = 7.9 Hz, 1H, (H3, H5)), 7.34 (t, J = 7.5 Hz, 1H, (H1)), 7.12 (t, J = 7.8 Hz, 1H, (H2, H6)). 13C NMR (126 MHz, CDCl3) δ 137.62 (C3, C5), 130.38 (C2, C6), 127.60 (C1), 94.52 (C4). FTIR (cm-1): 3081, 2898, 1541. 12 O O 11 10 6 5 1 4 2 3 8 7 NH 9 N H 2-(1H-indol-3-yl)acetaldehyde (2-63) To an oven-dried round bottom flask purged with N2 gas was added 2-(1H-indol-3-yl)ethan-1-ol (0.24 g, 1.5 mmol, 1.0 equiv) dissolved in anhydrous DMSO (3.0 mL) at 20ºC. Then IBX (0.46 g, 1.7 mmol, 1.1 equiv) was added at 20ºC, under an atmosphere of N2 gas. The resulting solution was then heated to 80ºC and allowed to stir for 3 hours under N2 gas. After 2 hours, the reaction was allowed to cool to room temperature and was diluted with ice cold H2O (20 mL), extracted with EtOAc (3x30 mL), dried over Na2SO4, filtered, and concentrated in vacuo to afford the desired product as a orange-red oil (0.16 g, 69%). 1H NMR (500 MHz, CDCl3) δ 9.77 (t, J = 2.5 Hz, 1H, (H11)), 8.19 (s, 1H, (H9)), 7.55 (d, J = 8.0 Hz, 1H, (H1)), 7.41 (d, J = 8.1 Hz, 168 1H, (H4)), 7.26 – 7.21 (m, 1H, (H5)), 7.20 – 7.13 (m, 2H, (H6, H8)), 3.82 (d, J = 2.5 Hz, 2H, (H10)). 13C NMR (126 MHz, CDCl3) δ 199.76 (C11), 136.00 (C3), 127.51 (C2), 123.52 (C8), 122.73 (C5), 120.13 (C6), 118.66 (C1), 111.47 (C4), 106.30 (C7), 40.50 (C10). FTIR (cm-1): 3399, 3051, 2890, 1725, 1596. HRMS-ESI (m/z), calculated for C10H9NO (M+H)+: 160.0762, Found: 160.0769. Br Br H N N H O O 16 14 13 NH 12 Br 20 17 18 2 3 19 1 4 Br 21 15 11 24 O 10 22 6 5 9 8 NH 7 25 O 23 CH326 ethyl 2,2-bis(6-bromo-1H-indol-3-yl)acetate (2-67) To an oven-dried round bottom flask was added 6-bromo-1-H-indole (1.18 g, 6.0 mmol, 1.0 equiv) dissolved in anhydrous DCM (10 mL) at 20ºC under an atmosphere of N2 gas. The resulting solution was then cooled to 0ºC. To this was then added ScOTf3 (0.30 g, 0.6 mmol, 0.2 equiv) and ethyl glyoxylate solution (50 wt% in Toluene) (0.61 g, 3.0 mmol, 1.0 equiv) at 0ºC under an atmosphere of N2. The resulting solution was then allowed to warm to 20ºC and was stirred for 18 hours under N2. After 18 hours, the reaction was quenched with DI H2O (20 mL), extracted with DCM (3x30mL), dried over Na2SO4, filtered, and concentrated in vacuo to yield the desired product as a red-orange foaming solid (>98% purity by NMR). 1H NMR (500 MHz, CDCl3) δ 8.11 (s, 2H, (H7, H13)), 7.46 – 7.42 (m, 4H, (H1, H4, H16, H19)), 7.17 (dd, J = 8.5, 1.8 Hz, 2H, (H2, H18)), 6.98 (d, J = 1.9 Hz, 2H, (H8, H12)), 5.40 (s, 1H, (H10)), 4.22 (q, J = 7.1 Hz, 2H, (H25)), 1.26 (t, J = 7.1 Hz, 3H, (H26)). 13C NMR (126 169 MHz, CDCl3) δ 173.38 (C22), 137.21(C5, C14), 129.17 (C6, C15), 125.51 (C3, C17), 124.01 (C2, C18), 123.08 (C1, C19), 120.63 (C8, C12), 115.90 (C9, C11), 114.37 (C4, C16), 61.58 (C25), 40.63 (C10), 14.35 (C26). FTIR (cm-1): 3398, 3061, 2902, 1698, 1596, 1197, 1102. HRMS-ESI (m/z): Calculated for C20H16Br2N2O2 (M+H)+, (Br79, Br79); (Br79, Br81); (Br81, Br81): 474.9657; 476.9636; 478.9616, Found: 474.9675; 476.9657; 478.9631. Br Br Br 23 1 4 17 2 3 18 9 NH 8 7 6 5 10 16 12 11 21 O H 22 19 Br 24 13 20 15 NH 14 O H H N N H 2,2-bis(6-bromo-1H-indol-3-yl)acetaldehyde (2-66) To an oven-dried flask was added ethyl 2,2-di(6-bromo-1H-indol-3-yl)acetate (1.4 g, 3.0 mmol, 1.0 equiv), dissolved in anhydrous DCM (10 mL). The resulting solution was then cooled to - 78ºC, under an atmosphere of N2 gas. At -78ºC, under N2, DIBAL-H solution (1.0M in hexane, 5.4 mL, 5.4 mmol, 1.8 equiv) was added dropwise, slowly. The resulting solution was then allowed to stir for 1 hour at -78ºC, under N2. After 1h, the reaction was allowed to warm to room temperature and was quenched with 2M HCl solution (20mL) and was allowed to stir at rt for 30 minutes. After 30 minutes, the reaction was then extracted with DCM (3x30mL), dried over Na2SO4, filtered, and concentrated in vacuo. The resulting crude residue was then purified via silica gel column chromatography (hexanes/ethyl acetate) to yield the desired product as a red/brown oil. (1.05 g, 81%). 1H NMR (500 MHz, CDCl3) δ 9.90 (d, J = 2.6 Hz, 1H, (H22)), 8.21 (s, 2H, (H9, H14)), 7.55 (d, J = 1.5 Hz, 2H, (H1, H20)), 7.36 (d, J = 8.5 Hz, 2H, (H4, 170 H17)), 7.19 (dd, J = 8.4, 1.5 Hz, 2H, (H3, H18)), 7.10 (d, J = 2.3 Hz, 2H, (H8, H15)), 5.30 (d, J = 2.6 Hz, 2H, (H10)). 13C NMR (126 MHz, CDCl3) δ 197.02 (C16), 137.35 (C6, C13), 125.67 (C5, C12), 124.12 (C8, C15), 123.43 (C3, C18), 120.67 (C4, C17), 116.31 (C2, C19), 114.48 (C1, C20), 110.93 (C7, C11), 47.94 (C10). FTIR (cm-1): 3413, 3085, 2887, 1730, 1533. HRMS-ESI (m/z): Calculated for C18H12Br2N2O (M+H)+, (Br79, Br79); (Br79, Br81); (Br81, Br81): 430.9395; 432.9374; 434.9354, Found: 430.948; 432.9389; 434.9368. 24 CH3 25 CH3 16 17 15 18 14 19 20 1 4 6 5 2 3 Br 12 23 CH326 O 28 13 NH O 22 27 NH 21 11 10 8 9 NH 7 BnHN NHBoc Br N H tert-butyl (2-(benzylamino)-2-(6-bromo-1H-indol-3-yl)ethyl)carbamate (2-19) 6-bromo-1H-indole (1.18 g, 6.0 mmol, 2.0 equiv), benzyl amine (0.33 mL, 3.0 mmol, 1.0 equiv), N-Boc Glycinal (0.48 g, 3.0 mmol, 1.0 equiv), and 1,1,3,3-tetramethyl guanidine (0.033 mL, 0.3 mmol, 0.1 equiv) were added sequentially to DI H2O (8 mL) at 20ºC. The reaction mixture was then added to a pre-heated oil bath (45ºC). The reaction was then stirred for 12 hours sat 45ºC under an atmosphere of N2 gas. After completion of the reaction, the reaction mixture was diluted with EtOAc (50 mL) and water (20 mL). The organic layer was then extracted with EtOAc (3x30 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The crude residue was then purified via silica gel flash column chromatography (EtOAc/Hexanes) to yield the desired product as an yellow-brown oil. (1.39 g, 52%).1H NMR (500 MHz, CDCl3) δ 7.53 (s, 171 1H, (H4)), 7.48 (d, J = 8.5 Hz, 1H, (H1)), 7.36 – 7.26 (m, 4H, (H2, H16, H17, H18)), 7.25 – 7.17 (m, 3H, (H8, H15, H19)), 5.18 (t, J = 5.8 Hz, 1H, (H10)), 4.58 (s, 1H, (H13)), 3.71 – 3.60 (m, 2H, (H11, H20)), 3.51 (d, J = 13.3 Hz, 1H, (H20)), 3.42 – 3.31 (m, 1H, (H11)), 2.24 (s, 1H, (H21)), 1.41 (s, 9H, (H24, H25, H26)). 13C NMR (126 MHz, CDCl3) δ 156.20 (C27), 139.08 (C14), 137.14 (C5), 128.65 (C16, C18), 128.34 (C15, C19), 127.94 (C6), 127.66 (C9), 127.47 (C17), 124.93 (C2), 123.20 (C8), 122.32 (C1), 115.49 (C3), 113.07 (C4), 79.90 (C23), 69.99 (C10), 49.91 (C20), 45.48 (C11), 28.43 (C24, C25, C26). FTIR (cm-1): 3405, 3355, 3052, 2925, 1689, 1525, 1221. HRMS-ESI (m/z): Calculated for C22H26BrN3O2 (M+H)+, (Br79); (Br81): 444.1287; 446.1266, Found: 444.1305; 446.1290. 16 17 15 18 14 19 13 NH 20 ClH 22 21 NH2 11 10 2 3 1 4 6 5 8 9 NH 7 Br 12 BnHN NH2•HCl Br N H N1-benzyl-1-(6-bromo-1H-indol-3-yl)ethane-1,2-diamine hydrochloride (2-68) To an oven-dried flask was added tert-butyl (2-(benzylamino)-2-(6-bromo-1H-indol-3- yl)ethyl)carbamate (0.44 g, 1.0 mmol, 1.0 equiv) and ethanolic HCl (3.3M in EtOH, 5.0 mL). This was stirred at 20ºC under an atmosphere of N2 gas for 18 hours. After completion, as indicated by TLC, the reaction mixture was concentrated in vacuo to obtain a semi-solid. This semi-solid was triturated with Et2O (5.0 mL) and concentrated in vacuo to afford the crude product as a reddish-purple foaming solid. This was used in subsequent steps without any further purification (>99% determined by NMR, quantitative). 1H NMR (500 MHz, CD3OD) δ 7.81 (s, 172 1H, (H4)), 7.71 (d, J = 1.4 Hz, 1H, (H8)), 7.68 (d, J = 8.5 Hz, 1H, (H1)), 7.51 – 7.41 (m, 5H, (H15, H16, H17, H18, H19)), 7.31 (d, J = 8.6 Hz, 1H, (H2)), 5.09 (dd, J = 10.3, 4.6 Hz, 1H, (H10)), 4.27 (d, J = 12.9 Hz, 1H, (H20)), 3.97 (d, J = 12.9 Hz, 1H, (H20)), 3.88 – 3.71 (m, 2H, (H11)). 13C NMR (126 MHz, CD3OD) δ 141.26 (C14), 139.15 (C5), 131.15 (C16, C18), 130.73 (C2), 130.25 (C15, C19), 130.22 (C17), 129.97 (C8), 126.67 (C6), 124.91 (C1), 122.54 (C9), 120.61 (C4), 116.09 (C3), 53.35 (C10), 50.37 (C20), 41.41 (C11). FTIR (cm-1): 3407, 3350, 3318, 3055, 2919, 1515. HRMS-ESI (m/z): Calculated for C17H18BrN3 (M+H)+, (Br79); (Br81): 344.0762; 346.0742, Found: 344.0781; 346.0760. Br Br Br 21 1 4 17 2 3 18 C N 9 NH 8 7 6 5 10 16 N 23 12 11 19 Br 22 13 20 15 NH 14 H N N H 2,2-bis(6-bromo-1H-indol-3-yl)acetonitrile (2-70) To an oven-dried rb flask was added 6-bromo-1H-indole (0.19 g, 1.0 mmol, 1.0 equiv) and 6- bromo-1H-indole-3-aldehyde (0.22 g, 1.0 mmol, 1.0 equiv) in glacial acetic acid (2.5 mL) at room temperature, under an atmosphere of N2 gas. To this was then added TMS-CN (0.15 mL, 1.2 mmol, 1.2 equiv) at room temperature, under N2 gas. The resulting mixture was then heated to 60ºC and was stirred for 18 hours at 60ºC under N2 gas. After 18 hours, the reaction mixture was diluted with ethyl acetate (30 mL), then quenched with saturated aqueous NaHCO3 and extracted with ethyl acetate (3x30mL). The organic extracts were then dried over Na2SO4, filtered, and concentrated in vacuo. The resulting crude residue was then purified via silica gel 173 column chromatography (hexanes/ ethyl acetate) to afford the desired product as an orange- brown semi-solid (0.18 g, 68%). 1H NMR (500 MHz, CDCl3) δ 8.19 (s, 2H, (H9, H14)), 7.56 (d, J = 1.6 Hz, 2H, (H1, H20)), 7.40 (d, J = 8.4 Hz, 2H, (H4, H17)), 7.21 (dd, J = 8.5, 1.6 Hz, 2H, (H3, H18)), 7.17 (dd, J = 2.6, 0.9 Hz, 2H, (H8, H15)), 5.59 (s, 1H, (H10)). 13C NMR (126 MHz, CDCl3) δ 137.46 (C6, C13), 124.42 (C5, C12), 123.90 (C8, C15), 123.83 (C3, C18), 120.25 (C4, C17), 119.36 (C16), 116.71 (C2, C19), 114.67 (C1, C20), 110.54 (C7, C11), 26.29 (C10). FTIR (cm-1): 3422, 3077, 2876, 2250, 1526. 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KD_1_38_PROTON_01 7 2 2 1 . 8 6 8 . 8 6 8 . 2 2 8 . 1 2 8 . 8 0 8 . 2 7 7 . 3 5 7 . 2 5 7 . 6 2 7 . 6 2 7 . 6 2 7 . 5 2 7 . 5 2 7 . 4 2 7 . 4 2 7 . 6 d - O S M D 0 5 2 . 14 O 12 O 11 10 NH213 6 5 1 4 2 3 8 7 NH 9 8 6 8 . 8 6 8 . 3 2 8 . 1 2 8 . 8 0 8 . 2 7 7 . 3 5 7 . 2 5 7 . 1500 1000 500 0 1000 500 0 1 0 . 1 8.68 f1 (ppm) 8 0 . 1 0 0 . 1 8.2 8.1 8.0 7.9 f1 (ppm) 7.8 7 0 . 1 1 1 . 1 7.7 7.6 7.5 6 0 . 1 1 0 . 1 8 0 . 1 0 0 . 1 7 0 . 1 1 1 . 1 7 5 2 . 12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 f1 (ppm) KD_1_38_CARBON_01 . 2 4 3 8 1 . 7 4 6 6 1 . 0 7 8 3 1 . 3 7 6 3 1 . 8 5 6 2 1 . 1 8 3 2 1 . 1 9 2 2 1 7 6 . 1 2 1 . 9 9 2 1 1 . 0 5 2 1 1 o s m d 5 4 0 4 . o s m d 8 2 0 4 . o s m d 1 2 0 4 . o s m d 2 1 . 0 4 o s m d 4 0 0 4 . o s m d 5 9 9 3 . o s m d 7 8 9 3 . o s m d 2 6 9 3 . o s m d 5 4 9 3 . 14 O 12 O 11 10 NH213 6 5 1 4 2 3 8 7 NH 9 2400 2300 2200 2100 2000 1900 1800 1700 1600 1500 1400 1300 1200 1100 1000 900 800 700 600 500 400 300 200 100 0 -100 -200 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 -5 200 190 180 170 160 150 140 130 120 110 100 f1 (ppm) 90 80 70 60 50 40 30 20 10 0 179 Figure 2.2. 1H NMR and 13C NMR spectra of compound 2-10. KD_2_18_PROTON_01 2 9 2 1 . 5 6 8 . 4 6 8 . 5 0 8 . 4 0 8 . 9 5 7 . 8 5 7 . 8 3 7 . 8 3 7 . 7 3 7 . 7 3 7 . 5 3 7 . 5 3 7 . 5 3 7 . 4 3 7 . 3 3 7 . 2 3 7 . 2 3 7 . 5 6 8 . 4 6 8 . 5 0 8 . 4 0 8 . 9 5 7 . 8 5 7 . 400 8 3 7 . 8 3 7 . 7 3 7 . 7 3 7 . 5 3 7 . 5 3 7 . 5 3 7 . 4 3 7 . 3 3 7 . 2 3 7 . 2 3 7 . 300 200 100 0 0 0 . 1 8.7 8.6 8.5 8.4 8.3 1 0 . 1 8.2 8.1 f1 (ppm) 4 0 . 1 8.0 7.9 7.8 7.7 7.6 5 2 2 . 7.40 7.35 f1 (ppm) 500 400 300 200 100 0 6 d - O S M D 0 5 2 . 1 4 6 5 12 O 11 N 13 10 2 3 8 7 NH 9 8 0 . 1 0 0 . 1 1 0 . 1 4 0 . 1 5 2 2 . 13.5 13.0 12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 f1 (ppm) KD_2_18_CARBON_01 . 0 6 8 5 1 9 4 . 1 4 1 . 8 5 7 3 1 . 8 9 4 2 1 . 3 2 4 2 1 . 7 8 3 2 1 0 0 . 1 2 1 . 1 2 6 1 1 . 9 3 4 1 1 . 5 3 3 1 1 . 8 9 4 2 1 . 3 2 4 2 1 . 7 8 3 2 1 0 0 . 1 2 1 . 1 2 6 1 1 . 9 3 4 1 1 . 5 3 3 1 1 5 4 3 2 1 0 126 124 122 120 118 116 114 112 f1 (ppm) 6 d - O S M D 2 5 9 3 . 12 O 11 N 13 10 6 5 1 4 2 3 8 7 NH 9 1500 1400 1300 1200 1100 1000 900 800 700 600 500 400 300 200 100 0 -100 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 200 190 180 170 160 150 140 130 120 110 100 f1 (ppm) 90 80 70 60 50 40 30 20 10 0 180 Figure 2.3. 1H NMR and 13C NMR spectra of compound 2-11a. phenylaziridine_2D_PROTON_01 8 8 7 . 7 8 7 . 4 3 7 . 2 3 7 . 0 3 7 . 8 2 7 . 8 2 7 . 7 2 7 . 7 2 7 . l 3 C D C 6 2 7 . 3 2 7 . 2 2 7 . 1 2 7 . 1 2 7 . 9 7 3 . 8 7 3 . 8 7 3 . 7 7 3 . 9 9 2 . 8 9 2 . 3 4 2 . 9 3 2 . 8 3 2 . 8 8 7 . 7 8 7 . 4 3 7 . 2 3 7 . 0 3 7 . 8 2 7 . 8 2 7 . 7 2 7 . 7 2 7 . l 3 C D C 6 2 7 . 3 2 7 . 2 2 7 . 1 2 7 . 1 2 7 . 1000 9 7 3 . 8 7 3 . 8 7 3 . 7 7 3 . 800 600 400 200 0 600 500 400 300 200 100 0 9 9 2 . 8 9 2 . 3 4 2 . 9 3 2 . 8 3 2 . 1500 19 CH3 15 16 14 17 12 O 13 18 S 10 O 11 7 N 9 8 1000 500 0 6 5 1 4 2 3 4 9 . 1 8 0 2 . 2 9 2 . 0 0 2 . 0 0 . 1 4 1 . 1 4 3 3 . 2 1 . 1 7.9 7.8 7.7 7.6 7.5 f1 (ppm) 7.4 7.3 7.2 3.85 3.78 f1 (ppm) 3.71 3.0 2.9 2.8 2.7 f1 (ppm) 2.6 2.5 2.4 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 f1 (ppm) 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 4 9 . 1 8 0 2 . 2 9 2 . 0 0 2 . 0 0 . 1 4 1 . 1 4 3 3 . 2 1 . 1 phenylaziridine_2D_CARBON_01 . 7 7 4 4 1 4 1 . 5 3 1 . 6 0 5 3 1 . 7 8 9 2 1 . 6 6 8 2 1 . 1 4 8 2 1 . 7 0 8 2 1 . 6 6 6 2 1 4 1 . 5 3 1 . 6 0 5 3 1 . 7 8 9 2 1 . 6 6 8 2 1 . 1 4 8 2 1 . 4 0 8 2 1 . 6 6 6 2 1 3 1 . 1 4 5 0 6 3 . 6 7 . 1 2 19 CH3 15 16 14 17 12 O 13 18 S 10 O 11 7 N 9 8 6 5 1 4 2 3 l 3 C D C 6 1 . 7 7 300 250 200 150 100 50 0 135 134 133 132 131 f1 (ppm) 130 129 128 127 200 190 180 170 160 150 140 130 120 110 100 f1 (ppm) 90 80 70 60 50 40 30 20 10 0 181 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0 -200 500 450 400 350 300 250 200 150 100 50 0 -50 Figure 2.4. 1H NMR and 13C NMR spectra of compound 2-11b. KD_2_35_F16-18_PROTON_01 6 8 7 . 5 8 7 . 2 3 7 . 1 3 7 . l 3 C D C 6 2 7 . 3 1 . 7 1 1 . 7 2 8 6 . 0 8 6 . 5 7 3 . 4 7 3 . 3 7 3 . 3 7 3 . 2 7 3 . 5 9 2 . 4 9 2 . 2 4 2 . 7 3 2 . 6 3 2 . 6 8 7 . 5 8 7 . 2 3 7 . 1 3 7 . l 3 C D C 6 2 7 . 3 1 . 7 1 1 . 7 2 8 6 . 0 8 6 . 1500 5 7 3 . 4 7 3 . 3 7 3 . 3 7 3 . 2 7 3 . 5 9 2 . 4 9 2 . 2 4 2 . 7 3 2 . 6 3 2 . 1000 500 0 1000 500 0 21 CH3 18 15 S 12 8 N 17 16 O 14 7 9 19 20 13 O 6 5 1 4 2 3 11 CH3 O 10 1000 500 0 0 7 . 1 9 7 . 1 2 8 . 1 7.8 7.6 7.4 f1 (ppm) 7.2 7.0 3 8 . 1 6.8 1 9 2 . 6 0 . 1 3.75 f1 (ppm) 3.70 0 0 . 1 0 9 2 . 0 0 . 1 3.0 2.9 2.8 2.7 f1 (ppm) 2.6 2.5 2.4 0 7 . 1 9 7 . 1 2 8 . 1 3 8 . 1 1 9 2 . 6 0 . 1 0 0 . 1 0 9 2 . 0 0 . 1 12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 f1 (ppm) KD_2_35_F16-18_CARBON_01 . 4 7 9 5 1 . 5 6 4 4 1 4 1 . 5 3 1 . 0 8 9 2 1 . 7 9 7 2 1 . 8 8 7 2 1 . 1 0 7 2 1 . 6 0 4 1 1 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 l 3 C D C 6 1 . 7 7 l 3 c d c 6 1 . 7 7 l 3 c d c 1 4 7 7 . l 3 c d c 1 9 6 7 . 2 3 5 5 . . 7 9 0 4 . 1 8 5 3 8 6 . 1 2 21 CH3 18 15 S 12 8 N 17 16 O 14 7 9 19 20 13 O 6 5 1 4 2 3 11 CH3 O 10 . 0 8 9 2 1 . 7 9 7 2 1 . 8 8 7 2 1 . 1 0 7 2 1 400 300 200 100 0 130 129 128 127 f1 (ppm) 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 182 4000 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0 -200 900 800 700 600 500 400 300 200 100 0 -100 Figure 2.5. 1H NMR and 13C NMR spectra of compound 2-12. KD_1_48_PROTON_01 2 1 . 2 1 2 1 . 2 1 2 9 9 . 7 2 8 . 8 0 8 . 7 0 8 . 0 5 7 . 8 4 7 . 6 2 7 . 6 2 7 . 4 2 7 . 4 2 7 . 3 2 7 . 3 2 7 . 1 2 7 . 1 2 7 . 0 2 7 . 0 2 7 . 8 1 . 7 8 1 . 7 O 2 H 3 3 3 . o s m d 9 4 2 . o s m d 9 4 2 . o s m d 8 4 2 . o s m d 8 4 2 . o s m d 8 4 2 . 12 O 10 8 7 NH 9 6 5 1 4 2 3 8 0 8 . 7 0 8 . 0 0 . 1 8.1 8.0 7.9 7.8 f1 (ppm) 7.7 7.6 6 2 7 . 6 2 7 . 4 2 7 . 4 2 7 . 3 2 7 . 3 2 7 . 1 2 7 . 1 2 7 . 0 2 7 . 0 2 7 . 8 1 . 7 8 1 . 7 0 5 7 . 8 4 7 . 1000 500 0 3 0 . 1 7.5 1 1 . 1 7.25 0 1 . 1 7.20 f1 (ppm) 800 600 400 200 0 0 1 . 1 4 9 0 . 5 0 . 1 0 0 . 1 3 0 . 1 1 1 . 1 0 1 . 1 13.5 13.0 12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 f1 (ppm) KD_1_48_CARBON_01 . 1 4 5 8 1 . 4 9 8 3 1 . 9 4 7 3 1 . 5 5 4 2 1 . 1 9 3 2 1 . 7 5 2 2 1 7 2 . 1 2 1 . 0 6 8 1 1 . 7 8 2 1 1 o s m d 5 4 0 4 . o s m d 8 3 0 4 . o s m d 9 2 0 4 . o s m d 1 2 0 4 . o s m d 2 1 . 0 4 o s m d 4 0 0 4 . o s m d 5 9 9 3 . o s m d 9 7 9 3 . o s m d 2 6 9 3 . o s m d 5 4 9 3 . 12 O 10 8 7 NH 9 6 5 1 4 2 3 4200 4000 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0 -200 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 -5 200 190 180 170 160 150 140 130 120 110 100 f1 (ppm) 90 80 70 60 50 40 30 20 10 0 183 Figure 2.6. 1H NMR and 13C NMR spectra of compound 2-13. KD_1_76_PROTON_01 0 1 . 0 1 6 2 8 . 5 2 8 . 3 2 8 . 5 9 7 . 4 9 7 . 6 8 7 . 4 8 7 . 3 4 7 . 1 4 7 . 0 4 7 . 8 3 7 . 6 3 7 . 5 3 7 . 1 3 7 . 9 2 7 . l 3 C D C 6 2 7 . 6 2 8 . 5 2 8 . 3 2 8 . 5 9 7 . 4 9 7 . 6 8 7 . 4 8 7 . 3 4 7 . 2 4 7 . 1 4 7 . 1 4 7 . 0 4 7 . 9 3 7 . 8 3 7 . 8 3 7 . 6 3 7 . 6 3 7 . 5 3 7 . 1 3 7 . 9 2 7 . 500 400 300 200 100 0 800 600 400 200 0 l 3 C D C 6 2 7 . 3 9 0 . 7 9 0 . 0 0 . 1 2 0 2 . 8.3 8.2 8.1 8.0 7.9 7.8 f1 (ppm) 4 0 . 1 6 0 . 1 7.40 7.35 f1 (ppm) 7 0 2 . 7.3 f1 (ppm) 4 9 0 . 3 9 0 . 7 9 0 . 0 0 . 1 2 0 2 . 4 0 . 1 6 0 . 1 7 0 2 . 1500 1000 500 0 11 O 10 8 7 N 9 14 O 6 5 1 4 2 3 S 12 O 13 15 16 20 17 19 18 CH321 8 3 2 . 7 3 3 . 12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 KD_1_76_CARBON_01 . 3 2 5 8 1 . 5 0 6 4 1 1 1 . 6 3 1 . 5 4 5 3 1 . 2 4 4 3 1 . 2 2 0 3 1 3 1 . 7 2 1 . 0 2 6 2 1 7 1 . 6 2 1 . 4 9 4 2 1 . 0 5 2 2 1 . 5 2 2 2 1 3 1 . 3 1 1 f1 (ppm) l 3 C D C 6 1 . 7 7 3 1 . 7 2 1 . 0 2 6 2 1 7 1 . 6 2 1 . 4 9 4 2 1 . 0 5 2 2 1 . 5 2 2 2 1 20 15 10 5 0 127.0 126.5 126.0 125.5 125.0 124.5 124.0 123.5 123.0 122.5 122.0 f1 (ppm) 7 5 . 1 2 10 8 11 O 6 5 1 4 2 3 7 N 9 14 O S 12 O 13 15 16 20 17 19 18 CH321 200 190 180 170 160 150 140 130 120 110 100 f1 (ppm) 90 80 70 60 50 40 30 20 10 0 184 1800 1700 1600 1500 1400 1300 1200 1100 1000 900 800 700 600 500 400 300 200 100 0 -100 32 30 28 26 24 22 20 18 16 14 12 10 8 6 4 2 0 -2 Figure 2.7. 1H NMR and 13C NMR spectra of compound 2-14. KD_1_74_PROTON_01 0 0 8 . 8 9 7 . 8 7 7 . 6 7 7 . 5 7 7 . 3 7 7 . 0 6 7 . 5 3 7 . 3 3 7 . 2 3 7 . 8 2 7 . 7 2 7 . l 3 C D C 6 2 7 . 5 2 7 . 3 2 7 . 1 2 7 . 0 8 6 . 8 7 6 . 6 7 6 . 4 7 6 . 1 8 5 . 8 7 5 . 6 3 5 . 4 3 5 . 4 3 2 . 6 5 1 4 2 3 O 14 10 CH211 7 N 9 8 S 12 13 O 15 16 20 17 19 18 CH321 5 2 7 . 3 2 7 . 1 2 7 . 1200 1000 800 600 400 200 0 6 2 7 . 5 2 7 . 600 500 400 300 200 100 0 0 8 6 . 8 7 6 . 6 7 6 . 4 7 6 . 400 300 200 100 0 l 3 C D C 6 2 7 . 5 3 7 . 5 3 7 . 4 3 7 . 3 3 7 . 2 3 7 . 2 3 7 . 9 2 7 . 8 2 7 . 7 2 7 . 7 2 7 . 8 7 7 . 6 7 7 . 5 7 7 . 3 7 7 . 0 6 7 . 1000 800 600 400 200 0 6 8 . 1 9 9 0 . 6 8 0 . 7.6 7.7 f1 (ppm) 4 9 0 . 7.35 7.30 f1 (ppm) 3 3 . 1 4 8 . 1 7.25 3 3 . 1 4 8 . 1 7.24 7.20 f1 (ppm) 0 9 0 . 6.9 6.8 f1 (ppm) 6.7 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 9 8 0 . 6 8 . 1 9 9 0 . 6 8 0 . 4 9 0 . 3 3 . 1 4 8 . 1 0 9 0 . 0 0 . 1 9 9 0 . 9 9 2 . KD_1_74_CARBON_01 f1 (ppm) 8 1 . 5 4 1 . 1 6 5 3 1 . 3 2 5 3 1 . 6 0 0 3 1 3 1 . 9 2 1 . 9 6 7 2 1 . 0 0 7 2 1 . 4 0 5 2 1 . 1 2 4 2 1 . 4 6 3 2 1 6 0 . 1 2 1 . 5 5 0 2 1 . 7 4 5 1 1 . 6 8 3 1 1 l 3 C D C 6 1 . 7 7 . 1 6 5 3 1 . 3 2 5 3 1 40 30 20 10 0 . 6 0 0 3 1 3 1 . 9 2 1 . 9 6 7 2 1 . 0 0 7 2 1 . 4 0 5 2 1 . 1 2 4 2 1 . 4 6 3 2 1 6 0 . 1 2 1 . 5 5 0 2 1 70 60 50 40 30 20 10 0 135.0 135.5 f1 (ppm) 130 128 126 124 122 120 f1 (ppm) 2 7 . 1 2 2 3 O 14 6 5 1 4 10 CH211 7 N 9 8 S 12 13 O 15 16 20 17 19 18 CH321 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 185 2000 1900 1800 1700 1600 1500 1400 1300 1200 1100 1000 900 800 700 600 500 400 300 200 100 0 -100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.8. 1H NMR and 13C NMR spectra of compound 2-17. KD_1_70-71_repurify_F10-12_PROTON_01 8 4 7 . 6 4 7 . 7 2 7 . 7 2 7 . 6 2 7 . 6 2 7 . 1 2 7 . 9 1 . 7 8 1 . 7 7 1 . 7 6 1 . 7 6 1 . 7 5 8 4 . 3 8 4 . 3 8 4 . 1 8 4 . 5 9 3 . 4 9 3 . 2 9 3 . 1 9 3 . 6 5 3 . 4 5 3 . 3 5 3 . 1 5 3 . 1 4 2 . 9 12 10 11 25 24 21 O 6 5 13 31 O 14 37 36 29 CH3 26 23 S 19 N 1 N 4 S 20 32 35 CH338 27 28 22 O 8 7 2 3 30 O 33 34 17 16 18 15 500 0 8 4 7 . 6 4 7 . 7 2 7 . 7 2 7 . 6 2 7 . 6 2 7 . 1 2 7 . 9 1 . 7 8 1 . 7 7 1 . 7 6 1 . 7 6 1 . 7 1000 5 8 4 . 3 8 4 . 3 8 4 . 1 8 4 . 5 9 3 . 4 9 3 . 2 9 3 . 1 9 3 . 6 5 3 . 4 5 3 . 3 5 3 . 1 5 3 . 800 600 400 200 0 600 500 400 300 200 100 0 8 9 . 1 2 0 3 . 5 9 . 1 4 9 . 1 7.5 7.4 7.3 f1 (ppm) 7.2 6 9 0 . 4.8 f1 (ppm) 9 9 0 . 9 9 0 . 3.8 3.6 f1 (ppm) 2 0 4 . 1 1 . 6 6 9 3 . 3 9 3 . 5 9 . 1 0 0 2 . 1 0 2 . 7 0 6 . 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 f1 (ppm) KD_1_70-71_repurify_F10-12_CARBON_01 . 5 7 3 4 1 . 2 5 8 3 1 . 3 8 6 3 1 . 5 7 9 2 1 . 8 7 8 2 1 . 5 2 8 2 1 . 9 2 7 2 1 . 1 7 6 2 1 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 l 3 C D C 6 1 . 7 7 3 3 8 5 . 2 7 7 4 . 8 6 . 1 2 29 CH3 26 23 S 19 N 1 N 4 S 20 32 35 CH338 27 28 22 O 8 7 2 3 30 O 33 34 25 24 21 O 6 5 13 31 O 14 37 36 17 16 18 15 9 12 10 11 . 5 7 9 2 1 . 8 7 8 2 1 . 5 2 8 2 1 . 9 2 7 2 1 . 1 7 6 2 1 100 50 0 130 129 128 f1 (ppm) 127 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0 -200 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 -20 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 186 Figure 2.9. 1H NMR and 13C NMR spectra of compound 2-21. KD_2_22_rerun_PROTON_01 1 8 7 . 0 8 7 . 9 7 7 . 9 7 7 . 8 7 7 . 9 4 7 . 9 4 7 . 9 4 7 . 9 4 7 . 8 4 7 . 8 4 7 . 7 4 7 . 7 4 7 . 6 4 7 . 6 4 7 . 6 4 7 . 6 4 7 . 2 4 7 . 2 4 7 . 1 4 7 . 0 4 7 . 9 3 7 . 9 3 7 . l 3 C D C 6 2 7 . 1 4 4 . 1 4 4 . 0 4 4 . 9 3 4 . 9 3 4 . 8 3 4 . 7 3 4 . 6 3 4 . 6 3 4 . 4 7 3 . 2 7 3 . 2 7 3 . 1 7 3 . 0 7 3 . 0 7 3 . 9 6 3 . 8 6 3 . 6 6 3 . 2 5 3 . 1 5 3 . 1 5 3 . 0 5 3 . 8 4 3 . 8 4 3 . 7 4 3 . 6 4 3 . 3 3 . 1 2 3 . 1 1 8 7 . 0 8 7 . 9 7 7 . 9 7 7 . 8 7 7 . 9 4 7 . 9 4 7 . 9 4 7 . 9 4 7 . 8 4 7 . 8 4 7 . 7 4 7 . 7 4 7 . 6 4 7 . 6 4 7 . 6 4 7 . 6 4 7 . 2 4 7 . 2 4 7 . 1 4 7 . 0 4 7 . 9 3 7 . 9 3 7 . l 3 C D C 6 2 7 . 8 N 9 7 NH 11 10 CH312 6 5 1 4 2 3 3 3 . 1 2 3 . 1 3000 2000 1000 0 800 600 400 200 0 2 4 4 . 1 4 4 . 1 4 4 . 0 4 4 . 9 3 4 . 9 3 4 . 8 3 4 . 7 3 4 . 6 3 4 . 6 3 4 . 5 3 4 . 4 7 3 . 2 7 3 . 2 7 3 . 0 7 3 . 0 7 3 . 9 6 3 . 8 6 3 . 6 6 3 . 2 5 3 . 1 5 3 . 1 5 3 . 0 5 3 . 9 4 3 . 8 4 3 . 8 4 3 . 7 4 3 . 7 4 3 . 1500 1000 500 0 7 3 3 . 7.8 2 5 . 1 0 2 3 . 7 8 0 . 9 8 0 . 0 0 . 1 7.7 7.6 7.5 7.4 f1 (ppm) 7.3 7.2 7.1 7.0 4.4 4.3 4.2 4.1 4.0 3.9 3.8 f1 (ppm) 7 4 . 1 3.7 4 3 . 1 3.6 3.5 2 0 3 . 1.3 f1 (ppm) 7 3 3 . 2 5 . 1 0 2 3 . 7 8 0 . 9 8 0 . 0 0 . 1 7 4 . 1 4 3 . 1 7 5 0 . 2 0 3 . 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 f1 (ppm) 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 KD_2_22_rerun_CARBON_01 . 4 9 8 6 1 . 5 3 8 6 1 6 1 . 4 3 1 . 2 0 4 3 1 6 7 . 1 3 1 2 7 . 1 3 1 . 3 7 8 2 1 . 1 7 8 2 1 6 1 . 7 2 1 5 1 . 7 2 1 l 3 C D C 6 1 . 7 7 1 4 7 4 . . 5 7 6 4 8 5 8 1 . 8 N 9 7 NH 11 10 CH312 6 5 1 4 2 3 6 1 . 4 3 1 . 2 0 4 3 1 6 7 . 1 3 1 2 7 . 1 3 1 60 50 40 30 20 10 0 . 3 7 8 2 1 . 1 7 8 2 1 100 80 60 40 20 0 6 1 . 7 2 1 5 1 . 7 2 1 100 80 60 40 20 0 134 133 f1 (ppm) 132 131 128.8 128.6 f1 (ppm) 127.2 127.1 f1 (ppm) 200 190 180 170 160 150 140 130 120 110 100 f1 (ppm) 90 80 70 60 50 40 30 20 10 0 187 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0 -200 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.10. 1H NMR and 13C NMR spectra of compound 2-22. KD_2_59_PROTON_01 5 6 9 . 0 0 . 1 l 3 C D C 6 2 7 . 0 2 5 . 7 0 4 . 6 0 4 . 5 4 . 1 10 CH3 8 11 CH3 CH39 7 O 5 2 O4 O 6 NH 1 3 2000 1000 0 7 0 4 . 6 0 4 . 1 0 2 . 4.10 4.05 f1 (ppm) 5 9 0 . 1 0 2 . 8 0 9 . 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 f1 (ppm) 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 KD_3_16_CARBON_01 . 1 8 7 9 1 . 0 9 5 5 1 l 3 C D C 6 1 . 7 7 7 1 . 0 8 8 2 . 1 5 . 7 2 8 2 10 CH3 8 11 CH3 CH39 7 O 5 2 O4 O 6 NH 1 3 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 f1 (ppm) 188 34000 32000 30000 28000 26000 24000 22000 20000 18000 16000 14000 12000 10000 8000 6000 4000 2000 0 -2000 300 280 260 240 220 200 180 160 140 120 100 80 60 40 20 0 -20 Figure 2.11. 1H NMR and 13C NMR spectra of compound 2-23. diprotected_diamine_PROTON_01 7 6 7 . 6 6 7 . 1 4 7 . 0 4 7 . 3 3 7 . 2 3 7 . 1 3 7 . 9 2 7 . 7 2 7 . l 3 C D C 6 2 7 . 3 2 7 . 3 2 7 . 1 2 7 . 0 2 7 . 8 1 . 7 6 1 . 7 4 1 . 7 3 1 . 7 0 3 5 . 8 2 5 . 7 2 5 . 3 6 4 . 0 7 3 . 8 6 3 . 7 6 3 . 6 6 3 . 3 6 3 . 5 5 3 . 3 5 3 . 5 4 3 . 4 4 3 . 3 4 3 . 1 4 3 . 0 4 3 . 4 2 2 . 2500 26 25 27 24 22 23 14 18 CH3 CH320 17 CH319 O 16 15 13 NH NH 12 11 10 O 21 2000 6 5 1 4 2 3 8 7 NH 9 7 6 7 . 6 6 7 . 1 4 7 . 0 4 7 . 3 3 7 . 2 3 7 . 1 3 7 . 9 2 7 . 7 2 7 . l 3 C D C 6 2 7 . 3 2 7 . 3 2 7 . 1 2 7 . 0 2 7 . 8 1 . 7 6 1 . 7 4 1 . 7 3 1 . 7 800 600 400 200 0 8 6 3 . 7 6 3 . 6 6 3 . 3 6 3 . 5 5 3 . 3 5 3 . 4 4 3 . 3 4 3 . 1 4 3 . 600 500 400 300 200 100 0 1500 1000 500 0 2500 0 0 . 1 7.7 7.6 7.5 5 1 . 1 7.4 f1 (ppm) 8 6 7 . 4 1 . 1 7.3 7.2 7.1 4 9 0 . 5 1 . 1 9 1 . 1 2 2 . 1 3.6 f1 (ppm) 3.4 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 f1 (ppm) 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 0 . 1 5 1 . 1 8 6 7 . 4 1 . 1 2 0 . 1 8 0 . 1 4 9 0 . 5 1 . 1 9 1 . 1 2 2 . 1 8 0 . 1 9 3 9 . diprotected_diamine_PROTON_01 7 6 7 . 6 6 7 . 1 4 7 . 0 4 7 . 3 3 7 . 2 3 7 . 1 3 7 . 9 2 7 . 7 2 7 . l 3 C D C 6 2 7 . 3 2 7 . 3 2 7 . 1 2 7 . 0 2 7 . 8 1 . 7 6 1 . 7 4 1 . 7 3 1 . 7 0 3 5 . 8 2 5 . 7 2 5 . 3 6 4 . 0 7 3 . 8 6 3 . 7 6 3 . 6 6 3 . 3 6 3 . 5 5 3 . 3 5 3 . 5 4 3 . 4 4 3 . 3 4 3 . 1 4 3 . 0 4 3 . 4 2 2 . 7 6 7 . 6 6 7 . 1 4 7 . 0 4 7 . 3 3 7 . 2 3 7 . 1 3 7 . 9 2 7 . 7 2 7 . l 3 C D C 6 2 7 . 3 2 7 . 3 2 7 . 1 2 7 . 0 2 7 . 8 1 . 7 6 1 . 7 4 1 . 7 3 1 . 7 800 600 400 200 0 8 6 3 . 7 6 3 . 6 6 3 . 3 6 3 . 5 5 3 . 3 5 3 . 4 4 3 . 3 4 3 . 1 4 3 . 600 500 400 300 200 100 0 0 0 . 1 7.7 7.6 7.5 5 1 . 1 7.4 f1 (ppm) 8 6 7 . 4 1 . 1 7.3 7.2 7.1 4 9 0 . 5 1 . 1 9 1 . 1 2 2 . 1 3.6 f1 (ppm) 3.4 26 25 27 24 22 23 14 18 CH3 CH320 17 CH319 O 16 15 13 NH NH 12 11 10 O 21 2000 6 5 1 4 2 3 8 7 NH 9 1500 1000 500 0 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 f1 (ppm) 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 0 . 1 5 1 . 1 8 6 7 . 4 1 . 1 2 0 . 1 8 0 . 1 4 9 0 . 5 1 . 1 9 1 . 1 2 2 . 1 8 0 . 1 9 3 9 . 189 Figure 2.12. 1H NMR and 13C NMR spectra of compound 2-24. 19 18 20 17 15 16 14 13 NH2 NH 12 11 10 6 5 1 4 2 3 8 7 NH 9 D O 3 D C 1 3 3 . 300 200 100 0 KD_2_50_PROTON_01 0 8 7 . 9 7 7 . 7 7 7 . 4 5 7 . 3 5 7 . 6 4 7 . 4 4 7 . 3 4 7 . 2 4 7 . 1 4 7 . 1 4 7 . 0 4 7 . 9 3 7 . 9 2 7 . 7 2 7 . 6 2 7 . 2 2 7 . 0 2 7 . 9 1 . 7 4 1 . 5 2 1 . 5 1 1 . 5 0 1 . 5 9 2 4 . 6 2 4 . 7 9 3 . 5 9 3 . 8 8 3 . 7 8 3 . 5 8 3 . 4 8 3 . 2 8 3 . 0 8 3 . 0 8 7 . 9 7 7 . 7 7 7 . 4 5 7 . 3 5 7 . 6 4 7 . 4 4 7 . 3 4 7 . 2 4 7 . 1 4 7 . 1 4 7 . 0 4 7 . 9 3 7 . 9 2 7 . 7 2 7 . 6 2 7 . 2 2 7 . 0 2 7 . 9 1 . 7 1000 800 600 400 200 0 4 1 . 5 2 1 . 5 1 1 . 5 0 1 . 5 400 9 2 4 . 6 2 4 . 7 9 3 . 5 9 3 . 7 8 3 . 5 8 3 . 4 8 3 . 2 8 3 . 0 8 3 . 300 200 100 0 300 200 100 0 1 9 0 . 1 0 . 1 9 0 . 1 4 4 6 . 7.8 7.7 7.6 7.5 f1 (ppm) 7.4 7.3 3 1 . 1 0 1 . 1 7.2 4 6 . 1 5.16 5.12 f1 (ppm) 0 0 . 1 4.3 f1 (ppm) 1 0 . 1 2 0 2 . 4.0 3.9 f1 (ppm) 3.8 1 9 0 . 1 0 . 1 9 0 . 1 9 4 5 . 3 1 . 1 0 1 . 1 4 6 . 1 0 0 . 1 1 0 . 1 2 0 2 . 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 f1 (ppm) KD_2_50_PROTON_01 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 8 7 . 9 7 7 . 7 7 7 . 4 5 7 . 3 5 7 . 6 4 7 . 4 4 7 . 3 4 7 . 2 4 7 . 1 4 7 . 1 4 7 . 0 4 7 . 9 3 7 . 9 2 7 . 7 2 7 . 6 2 7 . 2 2 7 . 0 2 7 . 9 1 . 7 4 1 . 5 2 1 . 5 1 1 . 5 0 1 . 5 9 2 4 . 6 2 4 . 7 9 3 . 5 9 3 . 8 8 3 . 7 8 3 . 5 8 3 . 4 8 3 . 2 8 3 . 0 8 3 . 0 8 7 . 9 7 7 . 7 7 7 . 4 5 7 . 3 5 7 . 6 4 7 . 4 4 7 . 3 4 7 . 2 4 7 . 1 4 7 . 1 4 7 . 0 4 7 . 9 3 7 . 9 2 7 . 7 2 7 . 6 2 7 . 2 2 7 . 0 2 7 . 9 1 . 7 1000 800 600 400 200 0 4 1 . 5 2 1 . 5 1 1 . 5 0 1 . 5 400 9 2 4 . 6 2 4 . 7 9 3 . 5 9 3 . 7 8 3 . 5 8 3 . 4 8 3 . 2 8 3 . 0 8 3 . 300 200 100 0 300 200 100 0 1 9 0 . 1 0 . 1 9 0 . 1 4 4 6 . 7.8 7.7 7.6 7.5 f1 (ppm) 7.4 7.3 3 1 . 1 0 1 . 1 7.2 4 6 . 1 5.16 5.12 f1 (ppm) 0 0 . 1 4.3 f1 (ppm) 1 0 . 1 2 0 2 . 4.0 3.9 f1 (ppm) 3.8 19 18 20 17 15 16 14 13 NH2 NH 12 11 10 6 5 1 4 2 3 8 7 NH 9 D O 3 D C 1 3 3 . 300 200 100 0 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 f1 (ppm) 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 1 9 0 . 1 0 . 1 9 0 . 1 9 4 5 . 3 1 . 1 0 1 . 1 4 6 . 1 0 0 . 1 1 0 . 1 2 0 2 . 190 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0 -200 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0 -200 Figure 2.13. 1H NMR and 13C NMR spectra of compound 2-29. KD_2_38_PROTON_01 9 3 2 1 . 2 4 8 . 2 4 8 . 8 1 . 8 7 1 . 8 5 5 7 . 4 5 7 . 0 3 7 . 0 3 7 . 9 2 7 . 8 2 7 . 7 2 7 . 7 2 7 . 7 2 7 . 7 2 7 . 6 2 7 . 6 2 7 . 4 2 7 . 4 2 7 . 6 d - O S M D 0 5 2 . 6 5 14 O 12 O 11 10 Cl 13 1 4 2 3 8 7 NH 9 2 4 8 . 2 4 8 . 8 1 . 8 7 1 . 8 5 5 7 . 4 5 7 . 0 3 7 . 0 3 7 . 9 2 7 . 8 2 7 . 7 2 7 . 7 2 7 . 7 2 7 . 7 2 7 . 6 2 7 . 6 2 7 . 4 2 7 . 4 2 7 . 2000 1000 0 5 0 . 1 0 0 . 1 1 0 . 1 8.4 8.3 8.2 8.1 8.0 f1 (ppm) 7.9 7.8 7.7 7.6 7.5 1500 1000 500 0 6 1 . 1 2 0 . 1 7.28 f1 (ppm) 7.24 0 0 . 1 0 0 . 1 1 0 . 1 5 0 . 1 6 1 . 1 2 0 . 1 13.0 12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 f1 (ppm) 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 KD_2_38_CARBON_01 . 9 8 0 8 1 . 7 3 5 6 1 9 1 . 8 3 1 . 7 8 6 3 1 . 5 7 5 2 1 . 7 8 3 2 1 . 9 8 2 2 1 0 3 . 1 2 1 . 9 8 2 1 1 . 7 4 2 1 1 6 d - O S M D 2 5 9 3 . 14 O 12 O 11 10 Cl 13 6 5 1 4 2 3 8 7 NH 9 8500 8000 7500 7000 6500 6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 -500 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 -5 200 190 180 170 160 150 140 130 120 110 100 f1 (ppm) 90 80 70 60 50 40 30 20 10 0 191 Figure 2.14. 1H NMR and 13C NMR spectra of compound 2-31. KD_4_6_PROTON_01 7 1 . 8 6 1 . 8 1 0 8 . 8 4 7 . 7 4 7 . 8 2 7 . 8 2 7 . 7 2 7 . 7 2 7 . 6 2 7 . 5 2 7 . 5 2 7 . 4 2 7 . 4 2 7 . 3 2 7 . 3 2 7 . 5 2 4 . 4 2 4 . 4 2 4 . 3 2 4 . 0 6 3 . 9 5 3 . 8 5 3 . 7 5 3 . D O 3 D C 1 3 3 . 9 7 . 1 9 7 . 1 7 7 . 1 6 7 . 1 5 7 . 1 4 7 . 1 3 7 . 1 1 7 . 1 0 7 . 1 6 5 . 1 14 S 12 O 11 10 16 17 N 15 20 18 19 6 5 1 4 2 3 8 7 NH 9 7 1 . 8 6 1 . 8 1 0 8 . 8 4 7 . 7 4 7 . 8 2 7 . 8 2 7 . 7 2 7 . 7 2 7 . 6 2 7 . 5 2 7 . 5 2 7 . 4 2 7 . 4 2 7 . 3 2 7 . 3 2 7 . 600 500 400 300 200 100 0 500 400 300 200 100 0 0 0 . 1 3 9 0 . 8.2 8.1 8.0 2 0 . 1 7.9 7.8 f1 (ppm) 7.7 7.6 7.5 7.30 2 2 2 . 7.25 f1 (ppm) 7.20 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 f1 (ppm) 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 0 . 1 3 9 0 . 2 0 . 1 2 2 2 . 2 2 2 . 8 2 2 . 5 5 4 . 7 0 2 . indole_thioamine_2D_CARBON_01 . 5 4 6 9 1 . 6 6 7 8 1 . 0 6 8 3 1 . 4 4 7 3 1 . 2 8 6 2 1 . 3 8 4 2 1 . 8 7 3 2 1 . 9 4 2 2 1 . 5 3 4 1 1 . 0 3 3 1 1 D O 3 D C 0 0 9 4 . . 1 2 9 4 7 9 3 5 . 7 6 7 2 . 9 3 6 2 . 4 0 5 2 . 14 S 12 O 11 10 6 5 1 4 2 3 8 7 NH 9 16 17 N 15 20 18 19 D O 3 D C 0 0 9 4 . . 1 2 9 4 80 60 40 20 0 49.5 49.0 f1 (ppm) 48.5 200 190 180 170 160 150 140 130 120 110 100 f1 (ppm) 90 80 70 60 50 40 30 20 10 0 192 1400 1300 1200 1100 1000 900 800 700 600 500 400 300 200 100 0 -100 110 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.15. 1H NMR and 13C NMR spectra of compound 2-32. KD_2_53_PROTON_01 3 5 8 . 8 2 8 . 7 2 8 . 6 2 8 . 6 2 8 . 0 6 7 . 9 5 7 . 4 4 7 . 4 4 7 . 3 4 7 . 2 4 7 . 1 4 7 . 1 4 7 . 0 4 7 . 9 3 7 . 8 3 7 . 8 3 7 . 4 2 4 . 3 2 4 . 1 2 4 . 1 2 4 . 9 1 . 4 4 1 . 4 3 1 . 4 2 1 . 4 1 1 . 4 0 1 . 4 7 0 4 . 5 0 4 . 4 0 4 . 1 9 3 . 0 9 3 . 9 8 3 . 7 8 3 . 7 8 3 . D O 3 D C 1 3 3 . 6 6 2 . 5 1 . 2 4 1 . 2 3 1 . 2 2 1 . 2 2 1 . 2 0 1 . 2 1 0 2 . 9 9 . 1 7 9 . 1 7 9 . 1 9 8 . 1 8 8 . 1 7 8 . 1 6 8 . 1 5 8 . 1 3 8 . 1 13 S CH314 12 O 11 10 16 N 17 20 8 18 19 6 5 1 4 2 3 7 NH 9 3 5 8 . 8 2 8 . 6 2 8 . 0 6 7 . 9 5 7 . 600 500 400 300 200 100 0 4 4 7 . 4 4 7 . 3 4 7 . 2 4 7 . 1 4 7 . 1 4 7 . 0 4 7 . 9 3 7 . 8 3 7 . 8 3 7 . 4 2 4 . 3 2 4 . 1 2 4 . 1 2 4 . 9 1 . 4 4 1 . 4 3 1 . 4 2 1 . 4 1 1 . 4 0 1 . 4 7 0 4 . 5 0 4 . 4 0 4 . 1 9 3 . 0 9 3 . 9 8 3 . 7 8 3 . 7 8 3 . 300 500 400 300 200 100 0 200 100 0 4 8 0 . 0 0 . 1 8.6 8.4 8.2 8.0 f1 (ppm) 7.8 2 0 . 1 7.6 0 2 2 . 7.45 7.40 f1 (ppm) 1 2 . 1 0 1 . 1 6 1 . 1 4.2 4.1 4.0 f1 (ppm) 1 1 . 1 3.9 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 f1 (ppm) 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 4 8 0 . 0 0 . 1 2 0 . 1 0 2 2 . 1 2 . 1 0 1 . 1 6 1 . 1 1 1 . 1 6 2 3 . 6 9 0 . 4 3 . 1 3 0 4 . KD_thioimidate_2D_CARBON_01 . 3 0 6 8 1 . 9 7 6 7 1 . 3 2 0 4 1 . 8 2 9 3 1 . 2 5 6 2 1 . 9 7 5 2 1 . 5 3 5 2 1 . 6 6 2 2 1 . 2 3 4 1 1 . 8 0 4 1 1 D O 3 D C 0 0 9 4 . 8 6 8 5 . . 8 7 5 5 5 1 . 8 2 . 7 7 6 2 9 4 3 2 . 7 7 6 1 . 19 S CH320 12 O 11 10 14 15 + N 13 18 16 17 . 2 5 6 2 1 . 9 7 5 2 1 . 5 3 5 2 1 . 6 6 2 2 1 . 2 3 4 1 1 . 8 0 4 1 1 6 5 1 4 2 3 8 7 NH 9 100 50 0 . 3 0 6 8 1 . 9 7 6 7 1 . 3 2 0 4 1 . 8 2 9 3 1 30 20 10 0 80 60 40 20 0 185 180 f1 (ppm) 141 140 f1 (ppm) 139 126 124 122 120 f1 (ppm) 118 116 114 2400 2300 2200 2100 2000 1900 1800 1700 1600 1500 1400 1300 1200 1100 1000 900 800 700 600 500 400 300 200 100 0 -100 -200 600 550 500 450 400 350 300 250 200 150 100 50 0 -50 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 193 Figure 2.16. 1H NMR and 13C NMR spectra of compound 2-25. KD_2_54_2D_PROTON_01 4 3 8 . 3 3 8 . 1 3 8 . 6 6 7 . 5 6 7 . 1 5 7 . 9 4 7 . 3 4 7 . 1 4 7 . 2 3 7 . 1 3 7 . 0 3 7 . 0 3 7 . 9 2 7 . 9 2 7 . 8 2 7 . 8 2 7 . 8 2 7 . 7 2 7 . 6 2 7 . 9 1 . 7 8 1 . 7 8 1 . 7 7 1 . 7 7 1 . 7 7 1 . 7 6 1 . 7 6 1 . 7 5 1 . 7 5 1 . 7 0 1 . 7 0 1 . 7 8 0 7 . 7 0 7 . 5 0 7 . 6 0 5 . 4 0 5 . 3 0 5 . 1 0 5 . 8 6 4 . 5 6 4 . 2 3 4 . 9 2 4 . 9 2 4 . 6 2 4 . 6 0 4 . 4 0 4 . 3 0 4 . 1 0 4 . 1 9 3 . 8 8 3 . D O 3 D C 1 3 3 . 16 O 10 11 1 2 7 N N 15 13 14 6 5 3 4 NH 9 8 26 27 32 21 28 29 NH 20 22 23 24 19 25 18 17 4 3 8 . 3 3 8 . 1 3 8 . 6 6 7 . 5 6 7 . 1 5 7 . 9 4 7 . 3 4 7 . 1 4 7 . 1200 1000 800 600 400 200 0 6 0 5 . 4 0 5 . 3 0 5 . 1 0 5 . 8 6 4 . 5 6 4 . 2 3 4 . 9 2 4 . 9 2 4 . 6 2 4 . 6 0 4 . 4 0 4 . 3 0 4 . 1 0 4 . 1 9 3 . 8 8 3 . 1500 30 31 1000 500 0 7 9 0 . 1 0 . 1 0 0 . 1 9 9 0 . 1 0 . 1 4 0 . 1 9 9 0 . 8.4 8.2 8.0 7.8 7.6 7.4 5.0 4.9 4.8 4.7 4.6 f1 (ppm) 4 0 . 1 3 0 . 1 8 0 . 1 4.3 4.2 4.1 4.0 3.9 4.5 f1 (ppm) 4.4 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 f1 (ppm) 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 7 9 0 . 1 0 . 1 0 0 . 1 9 9 0 . 1 0 . 1 3 4 2 . 2 2 5 . 1 1 . 3 4 0 . 1 9 9 0 . 4 0 . 1 3 0 . 1 8 0 . 1 KD_2_54_2D_CARBON_01 2 1 . 4 8 1 6 1 . 4 6 1 . 2 4 9 3 1 . 9 9 8 3 1 . 7 6 8 3 1 . 0 3 8 3 1 . 2 4 9 2 1 . 8 2 9 2 1 . 0 4 8 2 1 . 1 0 7 2 1 . 3 6 6 2 1 . 2 5 5 2 1 3 1 . 5 2 1 . 7 0 4 2 1 . 8 9 2 2 1 . 3 9 2 2 1 . 2 3 0 2 1 1 1 . 0 2 1 7 1 . 7 1 1 . 8 3 4 1 1 . 8 2 3 1 1 . 8 8 2 1 1 6 5 D O 3 D C 0 0 9 4 . . 3 2 8 4 . 7 7 0 6 . 8 7 8 5 16 O 10 11 1 2 7 N N 15 13 14 22 23 24 19 25 18 17 3 4 NH 9 8 26 27 32 21 28 29 NH 20 . 2 4 9 2 1 . 8 2 9 2 1 . 0 4 8 2 1 . 1 0 7 2 1 . 3 6 6 2 1 . 2 5 5 2 1 3 1 . 5 2 1 . 7 0 4 2 1 . 8 9 2 2 1 . 3 9 2 2 1 . 2 3 0 2 1 1 1 . 0 2 1 . 2 4 9 3 1 . 9 9 8 3 1 . 7 6 8 3 1 . 0 3 8 3 1 15 10 5 0 140 139 f1 (ppm) 138 129 128 127 126 125 f1 (ppm) 124 123 122 121 120 30 31 30 20 10 0 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 -500 100 90 80 70 60 50 40 30 20 10 0 200 190 180 170 160 150 140 130 120 110 100 f1 (ppm) 90 80 70 60 50 40 30 20 10 0 194 Figure 2.17. 1H NMR and 13C NMR spectra of compound 2-37. KD_3_54_Repurified_F7-8_PROTON_01 1 3 7 . 9 2 7 . 8 2 7 . l 3 C D C 6 2 7 . 3 2 7 . 2 2 7 . 0 2 7 . 3 1 . 7 2 1 . 7 5 0 7 . 3 0 7 . 5 8 6 . 3 8 6 . 2 5 5 . 9 7 3 . l 3 C D C 6 2 7 . 1 3 7 . 9 2 7 . 8 2 7 . 20 O 17 14 7 16 15 2 3 21 CH3 18 19 8 13 9 12 10 11 6 5 1 4 3 2 7 . 2 2 7 . 0 2 7 . 3 1 . 7 2 1 . 7 5 0 7 . 3 0 7 . 5 8 6 . 3 8 6 . 4000 3000 2000 1000 0 8 9 3 . 7 9 . 1 9 9 3 . 2 0 2 . 5 0 2 . 7.3 7.2 7.0 7.1 f1 (ppm) 6.9 6.8 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 8 9 3 . 7 9 . 1 9 9 3 . 2 0 2 . 5 0 2 . 0 0 . 1 5.5 f1 (ppm) 1 2 3 . 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 KD_3_54_Repurified_F7-8_CARBON_01 3 1 . 8 5 1 . 7 3 4 4 1 . 1 2 6 3 1 . 9 4 0 3 1 . 0 5 9 2 1 . 0 4 8 2 1 . 5 3 6 2 1 . 7 7 3 1 1 l 3 C D C 6 1 . 7 7 2 1 . 6 5 5 3 5 5 . 20 O 17 14 7 16 15 2 3 21 CH3 18 19 8 13 9 12 10 11 6 5 1 4 200 190 180 170 160 150 140 130 120 110 100 f1 (ppm) 90 80 70 60 50 40 30 20 10 0 195 8500 8000 7500 7000 6500 6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 -500 400 380 360 340 320 300 280 260 240 220 200 180 160 140 120 100 80 60 40 20 0 -20 Figure 2.18. 1H NMR and 13C NMR spectra of compound 2-38. l 3 C D C 6 2 7 . KD_3_53_Repurified_PROTON_01 2 0 8 . 2 0 8 . 0 0 8 . 0 0 8 . 8 7 7 . 6 7 7 . 8 5 7 . 7 5 7 . 4 5 7 . 2 5 7 . 3 3 7 . 3 3 7 . 2 3 7 . 2 3 7 . 1 3 7 . 0 3 7 . 0 3 7 . 8 7 7 . 6 7 7 . 8 5 7 . 7 5 7 . 4 5 7 . 2 5 7 . 3 3 7 . 3 3 7 . 2 3 7 . 2 3 7 . 1 3 7 . 0 3 7 . 0 3 7 . 2 0 8 . 2 0 8 . 0 0 8 . 0 0 8 . 500 400 300 200 100 0 l 3 C D C 6 2 7 . 4 2 7 . 4 2 7 . 3 2 7 . 1 2 7 . 1 2 7 . 9 1 . 7 1000 800 600 400 200 0 3 0 . 1 8.02 8.00 f1 (ppm) 5 1 . 2 1 0 . 1 2 0 . 1 9 0 . 1 8 2 3 . 7.8 7.7 7.6 7.5 f1 (ppm) 7.4 7.3 7.2 4 2 7 . 4 2 7 . 3 2 7 . 1 2 7 . 1 2 7 . 9 1 . 7 6 6 6 . 6 6 6 . 6 6 6 . 5 6 6 . 6 6 6 . 6 6 6 . 6 6 6 . 5 6 6 . 0 0 . 1 6.67 6.65 6.66 f1 (ppm) 500 0 3 0 . 1 5 1 . 2 1 0 . 1 2 0 . 1 9 0 . 1 8 2 3 . 0 0 . 1 2 3 2 . 6 5 6 5 3 . 3500 3000 2500 1 4 2 3 7 N 9 8 S 10 O 12 O11 2000 13 14 18 15 17 16 CH319 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 f1 (ppm) 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 l 3 C D C 6 2 7 . KD_3_53_Repurified_PROTON_01 2 0 8 . 2 0 8 . 0 0 8 . 0 0 8 . 8 7 7 . 6 7 7 . 8 5 7 . 7 5 7 . 4 5 7 . 2 5 7 . 3 3 7 . 3 3 7 . 2 3 7 . 2 3 7 . 1 3 7 . 0 3 7 . 0 3 7 . 8 7 7 . 6 7 7 . 8 5 7 . 7 5 7 . 4 5 7 . 2 5 7 . 3 3 7 . 3 3 7 . 2 3 7 . 2 3 7 . 1 3 7 . 0 3 7 . 0 3 7 . 2 0 8 . 2 0 8 . 0 0 8 . 0 0 8 . 500 400 300 200 100 0 l 3 C D C 6 2 7 . 4 2 7 . 4 2 7 . 3 2 7 . 1 2 7 . 1 2 7 . 9 1 . 7 1000 800 600 400 200 0 3 0 . 1 8.02 8.00 f1 (ppm) 5 1 . 2 1 0 . 1 2 0 . 1 9 0 . 1 8 2 3 . 7.8 7.7 7.6 7.5 f1 (ppm) 7.4 7.3 7.2 4 2 7 . 4 2 7 . 3 2 7 . 1 2 7 . 1 2 7 . 9 1 . 7 6 6 6 . 6 6 6 . 6 6 6 . 5 6 6 . 6 6 6 . 6 6 6 . 6 6 6 . 5 6 6 . 0 0 . 1 6.67 6.65 6.66 f1 (ppm) 500 0 3 0 . 1 5 1 . 2 1 0 . 1 2 0 . 1 9 0 . 1 8 2 3 . 0 0 . 1 2 3 2 . 6 5 6 5 3 . 1 4 2 3 7 N 9 8 S 10 O 12 O11 2000 13 14 18 15 17 16 CH319 1500 1000 500 0 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 f1 (ppm) 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 196 1500 1000 500 0 3500 3000 2500 Figure 2.19. 1H NMR and 13C NMR spectra of compound 2-39. diphenyl_indole_PROTON_01 4 8 7 . 7 3 7 . 7 3 7 . 5 3 7 . 4 3 7 . 2 3 7 . 0 3 7 . 9 2 7 . 7 2 7 . l 3 C D C 6 2 7 . 7 3 7 . 7 3 7 . 5 3 7 . 4 3 7 . 2 3 7 . 0 3 7 . 9 2 7 . 7 2 7 . l 3 C D C 6 2 7 . 5 2 7 . 4 2 7 . 2 2 7 . 8 0 7 . 6 0 7 . 5 0 7 . 2500 2000 1500 1000 500 0 6 6 2 1 . 9 1 . 1 7.4 7.3 7.2 f1 (ppm) 7 9 0 . 7.1 5 2 7 . 4 2 7 . 2 2 7 . 8 0 7 . 6 0 7 . 5 0 7 . 7 5 6 . 6 5 6 . 5 7 5 . 7 5 6 . 6 5 6 . 300 200 100 0 0 0 . 1 6.58 6.57 6.56 6.55 f1 (ppm) 22 21 16 17 NH 20 6 5 15 18 19 14 7 1 4 2 3 8 13 9 12 10 11 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 2 1 . 1 6 6 2 1 . 9 1 . 1 7 9 0 . 0 0 . 1 5 0 . 1 5.5 f1 (ppm) 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 Diphenyl_Indole_2D_CARBON_01 . 4 0 4 4 1 . 5 7 6 3 1 1 1 . 9 2 1 . 9 3 8 2 1 . 5 0 7 2 1 . 4 3 6 2 1 9 1 . 4 2 1 8 1 . 2 2 1 . 0 0 0 2 1 . 0 9 9 1 1 . 8 4 9 1 1 8 1 . 1 1 1 l 3 C D C 6 1 . 7 7 0 9 8 4 . 22 21 16 17 NH 20 6 5 15 18 19 14 7 1 4 2 3 8 13 9 12 10 11 7 1 . 5 2 1 . 5 4 4 2 1 1 1 . 3 2 1 . 0 4 2 2 1 . 5 2 0 2 1 . 4 2 8 1 1 . 6 0 6 1 1 . 6 9 5 1 1 . 3 5 5 1 1 700 600 500 400 300 200 100 0 125 124 123 122 121 120 119 118 117 116 f1 (ppm) 200 190 180 170 160 150 140 130 120 110 100 f1 (ppm) 90 80 70 60 50 40 30 20 10 0 197 4200 4000 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0 -200 1400 1300 1200 1100 1000 900 800 700 600 500 400 300 200 100 0 -100 Figure 2.20. 1H NMR and 13C NMR spectra of compound 2-41. l 3 C D C 6 2 7 . KD_2_71_F2-5_PROTON_01 2 4 7 . 2 4 7 . 1 4 7 . 0 4 7 . 0 4 7 . 9 3 7 . 3 3 7 . 2 3 7 . 2 3 7 . 1 3 7 . 1 3 7 . 1 3 7 . 0 3 7 . 0 3 7 . 2 4 7 . 2 4 7 . 1 4 7 . 0 4 7 . 0 4 7 . 9 3 7 . 3 3 7 . 2 3 7 . 2 3 7 . 1 3 7 . 1 3 7 . 1 3 7 . 0 3 7 . 0 3 7 . l 3 C D C 6 2 7 . 0 1 . 4 6 5 1 4 2 3 7 9 8 13 12 10 11 4000 3000 2000 1000 0 0 0 2 . 7.4 f1 (ppm) 8 9 2 . 7.3 7.2 7.5 0 0 2 . 8 9 2 . 0 1 . 1 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 f1 (ppm) 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 KD_2_71_F2-5_CARBON_01 7 2 . 1 4 1 . 7 0 9 2 1 . 9 5 8 2 1 . 3 2 6 2 1 l 3 C D C 6 1 . 7 7 0 1 . 2 4 6 5 1 4 2 3 7 9 8 13 12 10 11 190 180 170 160 150 140 130 120 110 100 f1 (ppm) 90 80 70 60 50 40 30 20 10 0 198 15000 14000 13000 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 -1000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0 -200 Figure 2.21. Gas-Chromatography Mass Spectrometry (GC-MS) spectrum of compound 2-41 (top) vs reported GC-MS spectrum (bottom, NIST database). 199 Figure 2.22. 1H NMR and 13C NMR spectra of compound 2-42. indole_dimer_cdcl3_2D_PROTON_01 9 9 7 . 3 6 7 . 1 6 7 . 5 3 7 . 4 3 7 . l 3 C D C 6 2 7 . 5 2 7 . 4 2 7 . 2 2 7 . 0 2 7 . 9 1 . 7 5 1 . 7 4 1 . 7 3 1 . 7 3 1 . 7 3 1 . 7 2 1 . 7 1 1 . 7 1 1 . 7 0 1 . 7 9 0 7 . 3 8 6 . 2 8 6 . 0 8 6 . 1 7 6 . 0 7 6 . 8 2 5 . 6 2 5 . 5 2 5 . 2 5 3 . 1 5 3 . 9 4 3 . 7 4 3 . 6 2 3 . 5 2 3 . 3 2 3 . 1 2 3 . 17 18 16 15 11 12 10 13 NH 14 6 5 1 4 2 3 8 7 NH 9 3 6 7 . 1 6 7 . 5 3 7 . 4 3 7 . l 3 C D C 6 2 7 . 3 8 6 . 2 8 6 . 0 8 6 . 1 7 6 . 0 7 6 . 8 2 5 . 6 2 5 . 5 2 5 . 5 2 7 . 4 2 7 . 2 2 7 . 0 2 7 . 9 1 . 7 800 600 400 200 0 800 600 400 200 0 700 600 500 400 300 200 100 0 2 5 3 . 1 5 3 . 9 4 3 . 7 4 3 . 6 2 3 . 5 2 3 . 3 2 3 . 1 2 3 . 600 500 400 300 200 100 0 0 0 . 1 7.6 7.5 7.4 f1 (ppm) 4 0 . 1 8 2 . 1 0 2 . 1 7.3 7.2 5 0 . 1 0 9 0 . 6.8 6.7 f1 (ppm) 7 9 0 . -100 5.25 f1 (ppm) 3 4 . 1 3.5 0 1 . 1 3.2 3.4 f1 (ppm) 3.3 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 f1 (ppm) 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 9 0 . 0 0 . 1 4 0 . 1 8 2 . 1 0 2 . 1 7 8 2 . 5 0 . 1 0 9 0 . 7 9 0 . 3 4 . 1 0 1 . 1 indole_dimer_cdcl3_2D_PROTON_01 9 9 7 . 3 6 7 . 1 6 7 . 5 3 7 . 4 3 7 . l 3 C D C 6 2 7 . 5 2 7 . 4 2 7 . 2 2 7 . 0 2 7 . 9 1 . 7 5 1 . 7 4 1 . 7 3 1 . 7 3 1 . 7 3 1 . 7 2 1 . 7 1 1 . 7 1 1 . 7 0 1 . 7 9 0 7 . 3 8 6 . 2 8 6 . 0 8 6 . 1 7 6 . 0 7 6 . 8 2 5 . 6 2 5 . 5 2 5 . 2 5 3 . 1 5 3 . 9 4 3 . 7 4 3 . 6 2 3 . 5 2 3 . 3 2 3 . 1 2 3 . 17 18 16 15 11 12 10 13 NH 14 6 5 1 4 2 3 8 7 NH 9 3 6 7 . 1 6 7 . 5 3 7 . 4 3 7 . l 3 C D C 6 2 7 . 3 8 6 . 2 8 6 . 0 8 6 . 1 7 6 . 0 7 6 . 8 2 5 . 6 2 5 . 5 2 5 . 5 2 7 . 4 2 7 . 2 2 7 . 0 2 7 . 9 1 . 7 800 600 400 200 0 800 600 400 200 0 700 600 500 400 300 200 100 0 2 5 3 . 1 5 3 . 9 4 3 . 7 4 3 . 6 2 3 . 5 2 3 . 3 2 3 . 1 2 3 . 600 500 400 300 200 100 0 0 0 . 1 7.6 7.5 7.4 f1 (ppm) 4 0 . 1 8 2 . 1 0 2 . 1 7.3 7.2 5 0 . 1 0 9 0 . 6.8 6.7 f1 (ppm) 7 9 0 . -100 5.25 f1 (ppm) 3 4 . 1 3.5 0 1 . 1 3.2 3.4 f1 (ppm) 3.3 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 f1 (ppm) 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 9 0 . 0 0 . 1 4 0 . 1 8 2 . 1 0 2 . 1 7 8 2 . 5 0 . 1 0 9 0 . 7 9 0 . 3 4 . 1 0 1 . 1 200 3000 2500 2000 1500 1000 500 0 3000 2500 2000 1500 1000 500 0 Figure 2.23. 1H NMR and 13C NMR spectra of compound 2-43. KD_Ts_Indole_Dimer_PROTON_01 9 8 7 . 8 8 7 . 4 7 7 . 3 7 7 . 2 7 7 . 2 7 7 . 8 5 7 . 8 5 7 . 6 4 7 . 4 4 7 . 2 3 7 . 1 3 7 . 0 3 7 . 9 2 7 . l 3 C D C 6 2 7 . 5 2 7 . 5 2 7 . 4 2 7 . 4 2 7 . 3 2 7 . 2 2 7 . 2 2 7 . 1 2 7 . 9 1 . 7 6 1 . 7 4 1 . 7 8 0 7 . 8 0 7 . 7 0 7 . 7 0 7 . 3 0 7 . 1 0 7 . 4 5 5 . 4 5 5 . 2 5 5 . 2 5 5 . 6 2 3 . 3 2 3 . 2 2 3 . 0 2 3 . 7 9 2 . 6 9 2 . 4 9 2 . 3 9 2 . 2 3 2 . 0 3 2 . 9 8 7 . 8 8 7 . 4 7 7 . 3 7 7 . 2 7 7 . 2 7 7 . 8 5 7 . 6 4 7 . 4 4 7 . 2 3 7 . 1 3 7 . 0 3 7 . 9 2 7 . l 3 C D C 6 2 7 . 5 2 7 . 5 2 7 . 4 2 7 . 4 2 7 . 3 2 7 . 2 2 7 . 2 2 7 . 1 2 7 . 9 1 . 7 6 1 . 7 4 1 . 7 8 0 7 . 8 0 7 . 7 0 7 . 7 0 7 . 3 0 7 . 1 0 7 . 2500 2000 1500 1000 500 0 4 5 5 . 4 5 5 . 2 5 5 . 2 5 5 . 600 500 400 300 200 100 0 6 2 3 . 3 2 3 . 2 2 3 . 0 2 3 . 7 9 2 . 6 9 2 . 4 9 2 . 3 9 2 . 500 6 5 1 4 2 3 O 22 400 300 200 100 0 30 O 32 37 33 36 34 35 CH338 16 15 11 12 10 7 N 9 8 S 19 17 18 13 N 14 S 20 O 31 O21 23 24 28 25 27 26 CH329 5 8 0 . 3 8 2 . 5 9 0 . 5 9 . 1 7.9 7.8 7.7 7.6 7.5 7.4 f1 (ppm) 3 1 . 1 7.3 0 0 2 . 6 2 2 . 8 1 . 1 1 3 2 . 1 9 . 1 7.2 7.1 7.0 0 1 . 1 -100 5.55 5.50 f1 (ppm) 3 0 . 1 3.2 7 0 . 1 3.1 f1 (ppm) 3.0 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5 8 0 . 3 8 2 . 5 9 0 . 5 9 . 1 3 1 . 1 1 0 5 . 0 0 2 . 6 2 2 . 8 1 . 1 1 3 2 . 1 9 . 1 4 0 . 1 5.5 f1 (ppm) 8 9 0 . 1 0 . 1 0 9 2 . 1 8 2 . 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 -500 KD_Ts_Indole_Dimer_CARBON_01 . 7 9 4 4 1 . 6 8 3 4 1 6 5 . 1 4 1 . 0 6 5 3 1 . 7 0 5 3 1 . 1 0 5 3 1 4 2 . 1 3 1 . 1 9 9 2 1 . 2 4 9 2 1 . 0 8 8 2 1 4 1 . 8 2 1 . 3 9 6 2 1 . 7 0 5 2 1 . 5 7 4 2 1 . 9 6 4 2 1 . 0 4 4 2 1 . 4 3 3 2 1 . 0 2 3 2 1 . 5 9 9 1 1 . 5 8 6 1 1 . 7 7 3 1 1 . 7 5 8 5 8 6 5 3 . 8 5 . 1 2 9 4 . 1 2 . 0 6 5 3 1 . 7 0 5 3 1 . 1 0 5 3 1 4 2 . 1 3 1 . 1 9 9 2 1 . 2 4 9 2 1 . 0 8 8 2 1 4 1 . 8 2 1 . 3 9 6 2 1 . 1 9 6 2 1 . 7 0 5 2 1 . 5 7 4 2 1 . 9 6 4 2 1 . 0 4 4 2 1 . 4 3 3 2 1 . 0 2 3 2 1 50 40 30 20 10 0 6 5 1 4 2 3 O 22 16 15 11 12 10 7 N 9 8 S 19 17 18 13 N 14 S 20 O 31 O21 23 24 28 25 27 26 CH329 30 O 32 37 33 36 34 35 8 5 . 1 2 9 4 . 1 2 CH338 50 40 30 20 10 0 135 134 133 132 f1 (ppm) 131 130 129 128 127.0 126.5 126.0 125.5 125.0 f1 (ppm) 124.5 124.0 123.5 21.3 21.6 f1 (ppm) 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 -5 70 60 50 40 30 20 10 0 200 190 180 170 160 150 140 130 120 110 100 f1 (ppm) 90 80 70 60 50 40 30 20 10 0 201 Figure 2.24. 1H NMR and 13C NMR spectra of compound 2-44. Indole_trimer_cdcl3_2D_PROTON_01 9 8 7 . 9 4 7 . 8 4 7 . 9 2 7 . 8 2 7 . l 3 C D C 6 2 7 . 5 1 . 7 5 1 . 7 4 1 . 7 4 1 . 7 2 1 . 7 2 1 . 7 0 0 7 . 0 0 7 . 9 9 6 . 9 9 6 . 9 9 6 . 8 9 6 . 7 9 6 . 7 9 6 . 6 9 6 . 6 9 6 . 2 9 6 . 2 9 6 . 5 6 6 . 5 6 6 . 4 6 6 . 3 6 6 . 2 6 6 . 2 6 6 . 6 5 6 . 4 5 6 . 7 8 4 . 5 8 4 . 4 8 4 . 1 4 3 . 0 4 3 . 21 22 25 26 27 12 NH 13 14 11 10 20 18 NH 17 16 1 4 6 5 19 15 2 3 23 24 8 7 NH29 9 4 7 . 8 4 7 . 9 2 7 . 8 2 7 . l 3 C D C 6 2 7 . 6 0 2 . 7.5 1 0 2 . 7.3 7.4 f1 (ppm) 700 600 500 400 300 200 100 0 -100 5 1 . 7 5 1 . 7 4 1 . 7 4 1 . 7 2 1 . 7 2 1 . 7 0 0 7 . 0 0 7 . 9 9 6 . 9 9 6 . 9 9 6 . 8 9 6 . 7 9 6 . 2 9 6 . 2 9 6 . 5 6 6 . 5 6 6 . 4 6 6 . 3 6 6 . 2 6 6 . 2 6 6 . 6 5 6 . 4 5 6 . 1000 500 0 600 400 200 0 400 300 200 100 0 7 8 4 . 5 8 4 . 4 8 4 . 1 4 3 . 0 4 3 . 200 100 0 600 500 400 300 200 100 0 6 1 . 2 7.15 f1 (ppm) 7.10 9 0 4 . 6 9 . 1 7.00 6.93 f1 (ppm) 0 0 . 1 6.65 6 9 0 . 6.60 f1 (ppm) 6.55 4 0 . 1 4.83 f1 (ppm) 0 1 . 2 -100 3.39 f1 (ppm) 14 13 12 11 10 9 8 7 3 8 . 1 6 0 2 . 1 0 2 . 6 1 . 2 9 0 4 . 6 9 . 1 0 0 . 1 6 9 0 . 4 0 . 1 5 6 f1 (ppm) 0 1 . 2 4 3 2 1 0 -1 -2 Indole_trimer_cdcl3_2D_CARBON_01 . 8 7 4 4 1 . 4 6 6 3 1 . 2 4 0 3 1 . 6 9 6 2 1 0 1 . 6 2 1 . 1 0 2 2 1 0 9 . 1 2 1 . 1 7 9 1 1 . 9 5 9 1 1 9 1 . 9 1 1 . 7 8 8 1 1 . 1 8 5 1 1 0 2 . 1 1 1 5 1 . 1 1 1 l 3 C D C 6 1 . 7 7 8 1 . 7 3 8 4 4 3 . 21 22 25 26 27 12 NH 13 14 11 10 20 18 NH 17 16 1 4 6 5 19 15 2 3 23 24 8 7 NH29 . 6 9 6 2 1 0 1 . 6 2 1 . 1 0 2 2 1 0 9 . 1 2 1 . 1 7 9 1 1 . 9 5 9 1 1 9 1 . 9 1 1 . 7 8 8 1 1 150 0 2 . 1 1 1 5 1 . 1 1 1 150 100 50 0 100 50 0 127 126 125 124 123 f1 (ppm) 122 121 120 119 111.0 f1 (ppm) 200 190 180 170 160 150 140 130 120 110 100 f1 (ppm) 90 80 70 60 50 40 30 20 10 0 202 1500 1400 1300 1200 1100 1000 900 800 700 600 500 400 300 200 100 0 -100 260 250 240 230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.25. 1H NMR and 13C NMR spectra of compound 2-47. KD_3_acyl_indole_PROTON_01 3 3 8 . 2 3 8 . 1 2 8 . 3 9 7 . 1 9 7 . 4 8 7 . 3 8 7 . 8 3 7 . 8 3 7 . 7 3 7 . 7 3 7 . 5 3 7 . 5 3 7 . 5 3 7 . 4 3 7 . 3 3 7 . 2 3 7 . 2 3 7 . 9 2 7 . 7 2 7 . l 3 C D C 6 2 7 . 3 3 8 . 2 3 8 . 1 2 8 . 3 9 7 . 1 9 7 . 4 8 7 . 3 8 7 . 3000 2000 1000 0 8 3 7 . 8 3 7 . 7 3 7 . 7 3 7 . 5 3 7 . 5 3 7 . 5 3 7 . 4 3 7 . 3 3 7 . 2 3 7 . 2 3 7 . 2000 4000 9 2 7 . 7 2 7 . 1500 3000 1000 2000 500 0 1000 0 0 0 . 1 1 0 . 1 6 0 . 1 1 1 . 2 8.4 8.3 8.2 8.1 f1 (ppm) 8.0 7.9 7.8 7 0 . 1 6 1 . 1 4 3 2 . 7.36 f1 (ppm) 7.32 4 3 2 . 7.26 f1 (ppm) 7 5 2 . 6 3 2 . 12 O 10 CH311 10000 6 5 1 4 2 3 O 15 7 N 9 8 S 13 16 21 14 O 17 18 20 19 CH322 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 f1 (ppm) 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 0 . 1 1 0 . 1 6 0 . 1 1 1 . 2 7 0 . 1 6 1 . 1 4 3 2 . 9 3 3 . 3 4 3 . KD_3_acyl_indole_PROTON_01 3 3 8 . 2 3 8 . 1 2 8 . 3 9 7 . 1 9 7 . 4 8 7 . 3 8 7 . 8 3 7 . 8 3 7 . 7 3 7 . 7 3 7 . 5 3 7 . 5 3 7 . 5 3 7 . 4 3 7 . 3 3 7 . 2 3 7 . 2 3 7 . 9 2 7 . 7 2 7 . l 3 C D C 6 2 7 . 3 3 8 . 2 3 8 . 1 2 8 . 3 9 7 . 1 9 7 . 4 8 7 . 3 8 7 . 3000 2000 1000 0 8 3 7 . 8 3 7 . 7 3 7 . 7 3 7 . 5 3 7 . 5 3 7 . 5 3 7 . 4 3 7 . 3 3 7 . 2 3 7 . 2 3 7 . 2000 4000 9 2 7 . 7 2 7 . 1500 3000 1000 2000 500 0 1000 0 0 0 . 1 1 0 . 1 6 0 . 1 1 1 . 2 8.4 8.3 8.2 8.1 f1 (ppm) 8.0 7.9 7.8 7 0 . 1 6 1 . 1 4 3 2 . 7.36 f1 (ppm) 7.32 4 3 2 . 7.26 f1 (ppm) 7 5 2 . 6 3 2 . 12 O 10 CH311 10000 6 5 1 4 2 3 O 15 7 N 9 8 S 13 16 21 14 O 17 18 20 19 CH322 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 f1 (ppm) 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 0 . 1 1 0 . 1 6 0 . 1 1 1 . 2 7 0 . 1 6 1 . 1 4 3 2 . 9 3 3 . 3 4 3 . 203 Figure 2.26. 1H NMR and 13C NMR spectra of compound 2-48. KD_4_23_PROTON_01 8 9 7 . 6 9 7 . 7 7 7 . 6 7 7 . 6 6 7 . 4 6 7 . 0 5 7 . 3 3 7 . 2 3 7 . 0 3 7 . l 3 C D C 6 2 7 . 5 2 7 . 3 2 7 . 2 2 7 . 0 2 7 . 4 1 . 5 3 1 . 5 1 1 . 5 0 1 . 5 8 9 7 . 6 9 7 . 7 7 7 . 6 7 7 . 6 6 7 . 4 6 7 . 0 5 7 . 3 3 7 . 2 3 7 . 0 3 7 . l 3 C D C 6 2 7 . 5 2 7 . 3 2 7 . 2 2 7 . 0 2 7 . 3000 4 1 . 5 3 1 . 5 1 1 . 5 0 1 . 5 2000 1000 0 1000 500 0 0 0 . 1 9 9 . 1 1 0 . 1 8.0 7.9 7.8 7.7 7.6 f1 (ppm) 5 9 0 . 7.5 8 0 . 1 6 0 3 . 7.4 7.3 7.2 3 0 . 1 5.1 f1 (ppm) 6 5 1 4 12 OH 10 CH311 2 3 O 15 7 N 9 8 S 13 14 O 16 17 21 18 20 19 CH322 3 3 2 . 2 6 . 1 1 6 . 1 2 6 . 1 1 6 . 1 7000 6000 5000 4000 3000 2000 1000 0 5 4 3 . 1.59 f1 (ppm) 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 f1 (ppm) 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 0 . 1 9 9 . 1 1 0 . 1 5 9 0 . 8 0 . 1 6 0 3 . 3 0 . 1 4 2 3 . 5 4 3 . 9 8 . 1 KD_4_23_CARBON_01 0 1 . 5 4 1 . 3 6 5 3 1 . 3 3 5 3 1 . 2 0 0 3 1 . 4 0 9 2 1 4 1 . 7 2 1 . 6 9 6 2 1 . 5 9 4 2 1 . 7 2 3 2 1 . 0 2 2 2 1 . 0 5 0 2 1 . 3 8 3 1 1 l 3 C D C 6 1 . 7 7 . 7 9 3 6 . 3 6 5 3 1 . 3 3 5 3 1 . 2 0 0 3 1 . 4 0 9 2 1 4 1 . 7 2 1 . 6 9 6 2 1 . 5 9 4 2 1 . 7 2 3 2 1 . 0 2 2 2 1 . 0 5 0 2 1 150 100 50 0 136 134 132 130 128 f1 (ppm) 126 124 122 120 6 5 3 2 . 9 6 . 1 2 12 OH 6 5 1 4 2 3 O 15 10 CH311 7 N 9 8 S 13 14 O 16 17 21 18 20 19 CH322 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 204 7000 6500 6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 -500 300 250 200 150 100 50 0 Figure 2.27. 1H NMR and 13C NMR spectra of compound 2-50. KD_4_126_F14-17_PROTON_01 31 30 29 27 28 32 26 11 O N 16 15 12 10 14 17 N 13 18 19 N 35 O O 38 2 3 1 4 6 5 9 N 7 8 S 33 O 36 24 25 21 20 S 34 46 40 50 47 48 49 CH352 39 45 41 44 42 43 CH351 3 9 8 . 3 4 8 . 1 4 8 . 5 0 8 . 3 0 8 . 4 9 7 . 3 9 7 . 0 9 7 . 8 8 7 . 0 8 7 . 8 7 7 . 0 6 7 . 8 5 7 . 8 4 7 . 1 4 7 . 0 4 7 . 0 4 7 . 0 4 7 . 9 3 7 . 9 3 7 . 9 3 7 . 8 3 7 . 8 3 7 . 8 3 7 . 7 3 7 . 7 3 7 . 7 3 7 . 7 3 7 . 7 3 7 . 6 3 7 . 6 3 7 . 5 3 7 . 5 3 7 . 5 3 7 . 4 3 7 . 4 3 7 . 0 3 7 . 8 2 7 . 6 2 7 . 6 2 7 . 5 2 7 . 2 2 7 . 2 2 7 . 2 2 7 . 1 2 7 . 4 0 7 . 4 0 7 . 3 0 7 . 3 0 7 . 7 9 4 . 4 9 4 . 7 8 4 . 5 8 4 . 5 8 4 . 3 8 4 . 1 4 4 . 9 3 4 . 8 3 4 . 5 3 4 . 4 0 4 . 2 0 4 . 1 0 4 . 9 9 3 . 4 8 3 . 0 8 3 . 6 3 2 . 4 3 2 . 23 22 37 O 1600 1500 1400 1300 1200 1100 3 4 8 . 1 4 8 . 5 0 8 . 3 0 8 . 4 9 7 . 3 9 7 . 0 9 7 . 8 8 7 . 0 8 7 . 8 7 7 . 0 6 7 . 8 5 7 . 8 4 7 . 9 3 7 . 9 3 7 . 8 3 7 . 8 3 7 . 7 3 7 . 7 3 7 . 7 3 7 . 7 3 7 . 7 3 7 . 6 3 7 . 6 3 7 . 5 3 7 . 0 3 7 . 8 2 7 . 6 2 7 . 6 2 7 . 5 2 7 . 2 2 7 . 2 2 7 . 2 2 7 . 1 2 7 . 4 0 7 . 4 0 7 . 3 0 7 . 3 0 7 . 7 9 4 . 4 9 4 . 7 8 4 . 5 8 4 . 5 8 4 . 3 8 4 . 1 4 4 . 9 3 4 . 8 3 4 . 5 3 4 . 4 0 4 . 2 0 4 . 1 0 4 . 9 9 3 . 4 8 3 . 0 8 3 . 600 6 3 2 . 4 3 2 . 1500 1000 500 0 500 400 300 200 100 0 1000 500 0 0 0 . 1 9 0 . 1 1 1 . 1 2 3 2 . 0 3 2 . 8.4 8.2 8.0 7.8 9 0 . 1 7.6 f1 (ppm) 7 0 . 1 9 4 3 . 1 7 7 . 5 2 2 . 2 1 . 1 1 1 . 1 4 1 . 1 7.4 7.2 7.0 5.0 4.8 4.6 4.4 f1 (ppm) 4.2 6 1 . 1 4.0 4 1 . 1 3.8 0 3 3 . 1 3 3 . 2.4 2.3 f1 (ppm) 14 13 12 11 10 8 9 0 . 9 0 0 . 1 9 0 . 1 1 1 . 1 2 3 2 . 0 3 2 . 9 0 . 1 7 0 . 1 9 4 3 . 1 7 7 . 5 2 2 . 2 1 . 1 1 1 . 1 4 1 . 1 6 1 . 1 4 1 . 1 0 3 3 . 1 3 3 . 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 -2 . 6 8 0 6 1 4 1 . 6 4 1 . 9 2 5 4 1 . 8 2 7 3 1 . 5 7 6 3 1 . 7 9 5 3 1 1 1 . 5 3 1 . 0 7 4 3 1 . 7 3 0 3 1 . 4 0 0 3 1 . 7 6 8 2 1 . 2 4 8 2 1 . 9 0 8 2 1 . 1 9 7 2 1 . 2 6 7 2 1 . 7 4 7 2 1 . 2 9 6 2 1 . 7 9 5 2 1 . 8 2 5 2 1 4 1 . 5 2 1 . 7 6 4 2 1 . 4 6 3 2 1 0 1 . 3 2 1 7 2 . 1 2 1 . 2 4 0 2 1 . 3 2 9 1 1 . 5 0 4 1 1 9 1 . 3 1 1 l 3 C D C 6 1 . 7 7 5 4 0 6 . . 1 9 6 5 6 8 7 4 . 2 7 . 1 2 5 6 . 1 2 KD_4_126_F14-17_CARBON_01 . 5 5 2 8 1 31 30 29 27 28 32 26 11 O N 16 15 12 10 14 17 N 13 18 19 N 35 O O 38 2 3 1 4 6 5 9 N 7 8 S 33 O 36 24 25 21 23 22 37 O 20 S 34 46 40 50 47 48 49 CH352 39 45 41 44 42 43 CH351 4 1 . 6 4 1 . 9 2 5 4 1 . 8 2 7 3 1 . 5 7 6 3 1 . 7 9 5 3 1 1 1 . 5 3 1 . 0 7 4 3 1 . 7 3 0 3 1 . 4 0 0 3 1 . 7 6 8 2 1 . 2 4 8 2 1 . 9 0 8 2 1 . 1 9 7 2 1 . 2 6 7 2 1 . 7 4 7 2 1 . 2 9 6 2 1 . 7 9 5 2 1 . 8 2 5 2 1 4 1 . 5 2 1 . 7 6 4 2 1 . 4 6 3 2 1 0 1 . 3 2 1 7 2 . 1 2 1 . 2 4 0 2 1 . 3 2 9 1 1 . 5 0 4 1 1 9 1 . 3 1 1 2 7 . 1 2 5 6 . 1 2 300 200 100 0 300 200 100 0 145 140 135 130 f1 (ppm) 125 120 115 22 21 f1 (ppm) 900 800 700 600 500 400 300 200 100 0 -100 360 340 320 300 280 260 240 220 200 180 160 140 120 100 80 60 40 20 0 -20 -40 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 205 Figure 2.28. 1H NMR and 13C NMR spectra of compound 2-55. KD_6_48_F22-29_PROTON_01 5 9 7 . 3 9 7 . 1 7 7 . 9 6 7 . 9 5 7 . 8 4 7 . 7 4 7 . 8 2 7 . 7 2 7 . 5 2 7 . 4 1 . 7 2 1 . 7 1 1 . 7 6 0 7 . 4 0 7 . 0 0 5 . 0 0 5 . 9 9 4 . 8 9 4 . 1 8 3 . 1 8 3 . 9 7 3 . 8 7 3 . 5 7 3 . 3 7 3 . 3 7 3 . 1 7 3 . 7 6 3 . 9 1 . 2 13 OH 12 OH 11 10 5000 8 4500 2 3 1 4 6 5 O 16 9 N 7 18 S 14 17 15 O 22 21 5 9 7 . 3 9 7 . 1 7 7 . 9 6 7 . 9 5 7 . 8 4 7 . 7 4 7 . 8 2 7 . 7 2 7 . 5 2 7 . 4 1 . 7 2 1 . 7 1 1 . 7 6 0 7 . 4 0 7 . 0 0 5 . 0 0 5 . 9 9 4 . 8 9 4 . 1 8 3 . 1 8 3 . 9 7 3 . 8 7 3 . 5 7 3 . 3 7 3 . 3 7 3 . 1 7 3 . 7 6 3 . 19 20 CH323 500 400 300 200 100 0 400 300 200 100 0 3000 2000 1000 0 0 0 . 1 8.0 7.9 7.8 7 9 . 1 7.7 0 0 . 1 7.6 9 9 0 . 7.5 f1 (ppm) 0 1 . 1 6 0 . 1 8 9 . 1 7.4 7.3 7.2 7.1 7.0 3 0 . 1 5.00 4.95 f1 (ppm) 7 0 . 1 3.8 9 1 . 1 2 9 0 . 3.7 f1 (ppm) 3.6 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 f1 (ppm) 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 0 . 1 7 9 . 1 0 0 . 1 9 9 0 . 0 1 . 1 6 0 . 1 8 9 . 1 3 0 . 1 7 0 . 1 9 1 . 1 2 9 0 . 4 0 3 . KD_6_48_F22-29_CARBON_01 . 6 0 5 4 1 8 1 . 5 3 1 . 1 9 4 3 1 . 7 8 9 2 1 . 9 8 8 2 1 . 1 8 6 2 1 . 6 8 4 2 1 . 8 4 3 2 1 . 9 2 3 2 1 9 1 . 2 2 1 2 1 . 0 2 1 . 5 6 3 1 1 l 3 C D C 6 1 . 7 7 6 5 8 6 . 8 4 6 6 . 8 1 . 5 3 1 . 1 9 4 3 1 . 7 8 9 2 1 . 9 8 8 2 1 . 1 8 6 2 1 . 6 8 4 2 1 . 8 4 3 2 1 . 9 2 3 2 1 500 0 136 134 132 130 f1 (ppm) 128 126 124 2 3 1 4 4 4 . 1 2 12 OH 13 OH 11 10 6 5 O 16 9 N 7 18 8 S 14 17 15 O 22 21 19 20 CH323 200 190 180 170 160 150 140 130 120 110 100 f1 (ppm) 90 80 70 60 50 40 30 20 10 0 206 4000 3500 3000 2500 2000 1500 1000 500 0 950 900 850 800 750 700 650 600 550 500 450 400 350 300 250 200 150 100 50 0 -50 Figure 2.29. 1H NMR and 13C NMR spectra of compound 2-58 KD_6_53_F31-37_PROTON_01 1 3 8 . 9 0 8 . 8 0 8 . 5 8 7 . 3 8 7 . 5 6 7 . 3 6 7 . 6 5 7 . 5 5 7 . 9 4 7 . 7 4 7 . 6 4 7 . 4 4 7 . 5 3 7 . 4 3 7 . 2 3 7 . 8 2 7 . 6 2 7 . 6 2 7 . 5 2 7 . 4 2 7 . 2 2 7 . 1 2 7 . 9 1 . 7 9 0 7 . 8 0 7 . 6 0 7 . 7 9 6 . 7 9 6 . 6 9 6 . 5 9 6 . 0 4 5 . 8 3 5 . 7 3 5 . 5 3 5 . 4 5 4 . 1 5 4 . 3 4 4 . 1 4 4 . 8 3 4 . 8 1 . 4 6 1 . 4 5 1 . 4 4 1 . 4 0 1 . 4 6 0 4 . 28 29 D O 3 D C 1 3 3 . 30 27 8 2 2 . 24 N 25 23 26 O 33 32 O S 31 36 37 35 38 34 39 CH340 5 N 22 1 6 N 2 7 17 4 3 16 15 12 8 14 11 13 9 21 20 NH 10 18 19 1 3 8 . 9 0 8 . 8 0 8 . 5 8 7 . 3 8 7 . 5 6 7 . 3 6 7 . 6 5 7 . 5 5 7 . 9 4 7 . 7 4 7 . 6 4 7 . 4 4 7 . 5 3 7 . 4 3 7 . 2 3 7 . 8 2 7 . 6 2 7 . 6 2 7 . 5 2 7 . 4 2 7 . 2 2 7 . 1 2 7 . 9 1 . 7 9 0 7 . 8 0 7 . 6 0 7 . 7 9 6 . 7 9 6 . 6 9 6 . 5 9 6 . 0 4 5 . 8 3 5 . 7 3 5 . 5 3 5 . 4 5 4 . 1 5 4 . 3 4 4 . 1 4 4 . 8 3 4 . 8 1 . 4 6 1 . 4 5 1 . 4 4 1 . 4 0 1 . 4 6 0 4 . 1000 500 0 400 300 200 100 0 7 9 0 . 0 0 . 1 9 9 . 1 5 1 . 1 4 2 . 1 2 2 2 . 8 2 2 . 1 6 5 . 4 3 . 1 0 3 . 1 9 0 2 . 5 0 . 1 9 0 . 1 0 1 . 1 6 1 . 1 9 3 . 1 8.3 8.2 8.1 8.0 7.9 7.8 7.7 7.6 f1 (ppm) 7.5 7.4 7.3 7.2 7.1 7.0 5.4 5.3 5.2 5.1 5.0 4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.0 f1 (ppm) 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 f1 (ppm) 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 7 9 0 . 0 0 . 1 9 9 . 1 5 1 . 1 4 2 . 1 2 2 2 . 8 2 2 . 1 6 5 . 4 3 . 1 0 3 . 1 9 0 2 . 5 0 . 1 9 0 . 1 0 1 . 1 6 1 . 1 9 3 . 1 0 8 3 . KD_6_53_F31-37_CARBON_01 1 5 . 1 6 1 . 3 7 7 4 1 1 1 . 9 3 1 . 5 4 6 3 1 . 3 0 6 3 1 . 3 5 5 3 1 6 3 . 1 3 1 6 0 . 1 3 1 . 7 7 0 3 1 . 6 9 9 2 1 2 1 . 9 2 1 . 1 6 8 2 1 . 2 3 8 2 1 . 1 3 7 2 1 . 0 6 6 2 1 5 1 . 6 2 1 . 7 8 5 2 1 . 1 4 3 2 1 8 5 . 1 2 1 . 8 8 0 2 1 . 4 3 9 1 1 . 5 2 5 1 1 . 6 3 3 1 1 . 4 5 2 1 1 . 7 6 9 0 1 . 5 4 6 3 1 . 3 0 6 3 1 . 3 5 5 3 1 6 3 . 1 3 1 6 0 . 1 3 1 . 7 7 0 3 1 . 6 9 9 2 1 2 1 . 9 2 1 . 1 6 8 2 1 . 2 3 8 2 1 . 1 3 7 2 1 . 0 6 6 2 1 5 1 . 6 2 1 . 7 8 5 2 1 6 2 8 5 . D O 3 D C 0 0 9 4 . 5 8 9 4 . 6 6 9 5 . . 1 8 4 5 28 29 0 5 . 1 2 30 24 N 25 O 33 S 31 32 O 36 37 35 38 34 39 27 23 22 1 5 N 26 6 N 2 7 17 4 3 16 15 12 8 14 11 13 9 21 20 NH 10 5 4 8 4 . 18 19 CH340 . 1 4 3 5 200 150 100 50 0 100 80 60 40 20 0 136 135 134 133 132 131 f1 (ppm) 130 129 128 127 126 60 55 50 f1 (ppm) 200 190 180 170 160 150 140 130 120 110 100 f1 (ppm) 90 80 70 60 50 40 30 20 10 0 207 1900 1800 1700 1600 1500 1400 1300 1200 1100 1000 900 800 700 600 500 400 300 200 100 0 -100 250 240 230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 -20 Figure 2.30. 1H NMR and 13C NMR spectra of compound 2-59. KD_6_60_F52-60_PROTON_01 7 4 7 . 5 4 7 . 4 4 7 . 8 3 7 . 2 3 7 . 1 3 7 . 1 3 7 . 0 3 7 . 9 2 7 . 4 2 7 . 0 2 7 . 8 1 . 7 7 1 . 7 6 1 . 7 5 1 . 7 4 1 . 7 8 0 7 . 6 0 7 . 5 0 7 . 4 1 . 5 2 1 . 5 1 1 . 5 9 0 5 . 7 5 4 . 4 5 4 . 7 2 4 . 5 2 4 . 2 2 4 . 2 0 4 . 2 0 4 . 1 0 4 . 0 0 4 . 9 9 3 . 8 9 3 . D O 3 D C 1 3 3 . 5 N N 2 7 1 6 17 4 3 16 15 12 8 14 11 13 9 21 20 NH 10 18 19 7 4 7 . 5 4 7 . 4 4 7 . 8 3 7 . 2 3 7 . 1 3 7 . 1 3 7 . 0 3 7 . 9 2 7 . 4 2 7 . 0 2 7 . 8 1 . 7 7 1 . 7 6 1 . 7 5 1 . 7 4 1 . 7 8 0 7 . 6 0 7 . 5 0 7 . 4 1 . 5 2 1 . 5 1 1 . 5 9 0 5 . 40 30 20 10 0 15 10 5 0 7 5 4 . 4 5 4 . 7 2 4 . 5 2 4 . 2 2 4 . 2 0 4 . 2 0 4 . 1 0 4 . 0 0 4 . 9 9 3 . 8 9 3 . 15 10 5 0 8 9 0 . 0 0 . 1 8 0 . 1 7.5 7.4 9 1 . 3 7.3 7 9 0 . 1 2 . 1 5 9 . 1 6 9 0 . 7.2 7.1 f1 (ppm) 1 3 . 1 5.15 5.10 f1 (ppm) 2 5 . 1 0 0 . 1 4.6 4.5 4.4 4.3 f1 (ppm) 4.2 4.1 7 8 . 1 4.0 8 9 0 . 0 0 . 1 8 0 . 1 9 1 . 3 7 9 0 . 1 2 . 1 5 9 . 1 6 9 0 . 1 3 . 1 2 5 . 1 0 0 . 1 7 8 . 1 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 f1 (ppm) 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 KD_6_60_F52-60_CARBON_01 . 1 9 6 5 1 . 0 0 5 3 1 . 9 4 8 2 1 . 6 0 8 2 1 . 3 8 7 2 1 . 4 7 7 2 1 . 9 5 7 2 1 . 7 7 4 2 1 7 8 . 1 2 1 . 9 2 9 1 1 . 7 0 8 1 1 . 4 8 6 1 1 8 7 . 1 1 1 3 7 7 5 . . 7 9 6 5 . 8 2 4 4 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 -5 . 9 4 8 2 1 . 6 0 8 2 1 . 3 8 7 2 1 . 4 7 7 2 1 . 9 5 7 2 1 40 30 20 10 0 128.8 128.0 f1 (ppm) 5 N N 2 7 1 6 17 4 3 16 15 12 8 14 11 13 9 21 20 NH 10 18 19 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 -5 200 190 180 170 160 150 140 130 120 110 100 f1 (ppm) 90 80 70 60 50 40 30 20 10 0 208 Figure 2.31. 1H NMR and 13C NMR spectra of compound 2-60. l 3 C D C 6 2 7 . KD_1_21_3-4_PROTON_01 3 7 7 . 1 7 7 . 6 3 7 . 4 3 7 . 3 3 7 . 3 7 7 . 1 7 7 . 6 3 7 . 4 3 7 . 3 3 7 . 3 1 . 7 2 1 . 7 0 1 . 7 1 6 2 5 3 4 I 7 l 3 C D C 6 2 7 . 3 1 . 7 2 1 . 7 0 1 . 7 2500 2000 1500 1000 500 0 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 f1 (ppm) 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 f1 (ppm) 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 KD_1_73_F2-3_CARBON_01 . 2 6 7 3 1 . 8 3 0 3 1 . 0 6 7 2 1 . 2 5 4 9 l 3 C D C 6 1 . 7 7 1 6 2 5 3 4 I 7 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 209 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0 -200 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.32. 1H NMR and 13C NMR spectra of compound 2-63. KD_6_97_F5-6_PROTON_01 8 7 9 . 7 7 9 . 7 7 9 . 9 1 . 8 6 5 7 . 4 5 7 . 2 4 7 . 0 4 7 . 6 2 7 . l 3 C D C 6 2 7 . 4 2 7 . 3 2 7 . 8 1 . 7 8 1 . 7 7 1 . 7 6 1 . 7 4 1 . 7 2 8 3 . 2 8 3 . 12 O 11 10 6 5 1 4 2 3 8 7 NH 9 l 3 C D C 6 2 7 . 6 5 7 . 4 5 7 . 2 4 7 . 0 4 7 . 6 2 7 . 8 7 9 . 7 7 9 . 7 7 9 . 800 600 400 200 0 4 2 7 . 3 2 7 . 8 1 . 7 8 1 . 7 7 1 . 7 6 1 . 7 4 1 . 7 800 600 400 200 0 0 0 . 1 9.76 9.78 f1 (ppm) 1 1 . 1 7.6 7.5 0 1 . 1 7.4 4 0 . 1 3 3 2 . 7.3 7.2 7.1 f1 (ppm) 800 600 400 200 0 2 8 3 . 2 8 3 . 7 3 2 . 3.82 f1 (ppm) 0 0 . 1 4 0 . 1 1 1 . 1 0 1 . 1 4 0 . 1 3 3 2 . 7 3 2 . 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 f1 (ppm) 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 2500 2400 2300 2200 2100 2000 1900 1800 1700 1600 1500 1400 1300 1200 1100 1000 900 800 700 600 500 400 300 200 100 0 -100 -200 KD_6_97_F5-6_CARBON_01 . 6 7 9 9 1 . 0 0 6 3 1 . 1 5 7 2 1 . 2 5 3 2 1 . 3 7 2 2 1 3 1 . 0 2 1 . 6 6 8 1 1 7 4 . 1 1 1 . 0 3 6 0 1 l 3 C D C 6 1 . 7 7 0 5 0 4 . 12 O 11 10 6 5 1 4 2 3 8 7 NH 9 55 50 45 40 35 30 25 20 15 10 5 0 -5 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 f1 (ppm) 210 Figure 2.33. 1H NMR and 13C NMR spectra of compound 2-67. KD_6_111C_PROTON_01 1 1 . 8 5 4 7 . 4 4 7 . 3 4 7 . l 3 C D C 6 2 7 . 8 1 . 7 8 1 . 7 7 1 . 7 6 1 . 7 8 9 6 . 8 9 6 . 0 4 5 . 4 2 4 . 3 2 4 . 1 2 4 . 0 2 4 . 7 2 . 1 6 2 . 1 5 2 . 1 16 14 13 NH 12 Br 20 17 18 2 3 19 1 4 Br 21 15 11 24 O 10 22 6 5 9 8 NH 7 25 O 23 CH326 5 4 7 . 4 4 7 . 3 4 7 . l 3 C D C 6 2 7 . 8 1 . 7 8 1 . 7 7 1 . 7 6 1 . 7 8 9 6 . 8 9 6 . 3000 2000 1000 0 4 2 4 . 3 2 4 . 1 2 4 . 0 2 4 . 1500 1000 500 0 7 2 . 1 6 2 . 1 5 2 . 1 3000 2000 3500 1000 0 1 6 3 . 7.45 7.40 7.35 7.30 7.25 6 7 2 . 7.20 f1 (ppm) 7.15 7.10 7.05 7.00 6.95 0 9 . 1 1 0 2 . 4.24 f1 (ppm) 4.16 1 3 3 . 1.30 1.25 1.20 f1 (ppm) 5 0 2 . 1 6 3 . 6 7 2 . 0 9 . 1 0 0 . 1 1 0 2 . 1 3 3 . 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 f1 (ppm) 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 KD_6_111C_CARBON_01 . 8 3 3 7 1 . 1 2 7 3 1 7 1 . 9 2 1 . 1 5 5 2 1 . 1 0 4 2 1 . 8 0 3 2 1 . 3 6 0 2 1 . 0 9 5 1 1 . 7 3 4 1 1 l 3 C D C 6 1 . 7 7 8 5 . 1 6 3 6 0 4 . 5 3 4 1 . 16 14 13 NH 12 Br 20 17 18 2 3 19 1 4 Br 21 15 11 24 O 10 22 6 5 9 8 NH 7 25 O 23 CH326 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 211 7000 6500 6000 5500 5000 4500 4000 3000 2500 2000 1500 1000 500 0 -500 550 500 450 400 350 300 250 200 150 100 50 0 -50 Figure 2.34. 1H NMR and 13C NMR spectra of compound 2-66. KD_7_12C_PROTON_01 0 9 9 . 0 9 9 . 1 2 8 . 5 5 7 . 5 5 7 . 7 3 7 . 5 3 7 . l 3 C D C 6 2 7 . 0 2 7 . 0 2 7 . 9 1 . 7 8 1 . 7 0 1 . 7 9 0 7 . 0 3 5 . 9 2 5 . Br 23 1 4 17 2 3 18 9 NH 8 7 6 5 10 16 12 11 21 O H 22 19 Br 24 13 20 15 NH 14 0 9 9 . 0 9 9 . 250 200 150 100 50 0 7 3 7 . 5 3 7 . 5 5 7 . 5 5 7 . 500 400 300 200 100 0 l 3 C D C 6 2 7 . 0 2 7 . 0 2 7 . 9 1 . 7 8 1 . 7 0 1 . 7 9 0 7 . 600 500 400 300 200 100 0 0 0 . 1 9.93 9.90 f1 (ppm) 7 3 2 . 7.54 7.56 f1 (ppm) 5 5 2 . 4 6 2 . 3 1 . 2 -100 7.35 7.30 7.25 7.20 7.15 7.10 f1 (ppm) 0 0 . 1 2 5 2 . 7 3 2 . 5 5 2 . 4 6 2 . 3 1 . 2 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5 1 . 1 5.5 f1 (ppm) 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 KD_6_133C_CARBON_01 . 2 0 7 9 1 . 5 3 7 3 1 . 7 6 5 2 1 2 1 . 4 2 1 . 3 4 3 2 1 . 7 6 0 2 1 . 1 3 6 1 1 . 8 4 4 1 1 . 3 9 0 1 1 l 3 C D C 6 1 . 7 7 4 9 7 4 . Br 23 1 4 17 2 3 18 9 NH 8 7 6 5 10 16 12 11 21 O H 22 19 Br 24 13 20 15 NH 14 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 212 2000 1900 1800 1700 1600 1500 1400 1300 1200 1100 1000 900 800 700 600 500 400 300 200 100 0 -100 360 340 320 300 280 260 240 220 200 180 160 140 120 100 80 60 40 20 0 -20 Figure 2.35. 1H NMR and 13C NMR spectra of compound 2-19. KD_7_16_PROTON_01 3 5 7 . 0 5 7 . 8 4 7 . 4 3 7 . 2 3 7 . 1 3 7 . 0 3 7 . 7 2 7 . l 3 C D C 6 2 7 . 3 2 7 . 2 2 7 . 1 2 7 . 9 1 . 7 0 2 5 . 9 1 . 5 7 1 . 5 8 5 4 . 9 6 3 . 8 6 3 . 6 6 3 . 5 6 3 . 2 6 3 . 3 5 3 . 0 5 3 . 9 3 3 . 8 3 3 . 7 3 3 . 5 3 3 . 4 3 3 . 4 2 2 . 25 CH3 CH326 O 28 13 NH 16 17 15 18 14 19 20 1 4 . 1 24 CH3 23 O 22 27 NH 21 11 3 5 7 . 0 5 7 . 8 4 7 . 4 3 7 . 2 3 7 . 1 3 7 . 0 3 7 . 7 2 7 . l 3 C D C 6 2 7 . 3 2 7 . 2 2 7 . 1 2 7 . 9 1 . 7 1500 1000 500 0 0 2 5 . 9 1 . 5 7 1 . 5 600 500 400 300 200 100 0 9 6 3 . 8 6 3 . 6 6 3 . 5 6 3 . 2 6 3 . 3 5 3 . 0 5 3 . 9 3 3 . 8 3 3 . 7 3 3 . 5 3 3 . 4 3 3 . 500 400 300 200 100 0 2 3 1 4 6 5 Br 12 10 8 9 NH 7 0 0 . 1 3 4 . 1 7.6 7.5 7.4 f1 (ppm) 2 5 4 . 7.3 6 4 3 . 7.2 7.1 9 1 . 1 -100 5.3 5.1 5.2 f1 (ppm) 3 6 2 . 6 5 . 1 6 6 . 1 3.6 3.4 f1 (ppm) 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 f1 (ppm) 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 KD_7_16_CARBON_01 . 0 2 6 5 1 . 8 0 9 3 1 4 1 . 7 3 1 . 5 6 8 2 1 . 4 3 8 2 1 . 4 9 7 2 1 . 6 6 7 2 1 . 7 4 7 2 1 . 3 9 4 2 1 . 0 2 3 2 1 . 2 3 2 2 1 . 9 4 5 1 1 . 7 0 3 1 1 l 3 C D C 6 1 . 7 7 0 9 9 7 . 9 9 9 6 . . 1 9 9 4 8 4 5 4 . 3 4 8 2 . 0 0 . 1 3 4 . 1 2 5 4 . 6 4 3 . 9 1 . 1 9 2 . 1 3 6 2 . 6 5 . 1 6 6 . 1 8 0 . 1 9 8 . 1 1 24 CH3 25 CH3 16 17 15 18 14 19 23 CH326 O 28 13 NH O 22 27 NH 21 11 20 1 4 6 5 2 3 Br 12 . 5 6 8 2 1 . 4 3 8 2 1 . 4 9 7 2 1 . 6 6 7 2 1 . 7 4 7 2 1 40 30 20 10 0 130 129 128 f1 (ppm) 127 126 10 8 9 NH 7 200 190 180 170 160 150 140 130 120 110 100 f1 (ppm) 90 80 70 60 50 40 30 20 10 0 213 1900 1800 1700 1600 1500 1400 1300 1200 1100 1000 900 800 700 600 500 400 300 200 100 0 -100 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.36. 1H NMR and 13C NMR spectra of compound 2-68. KD_6_124C_PROTON_01 1 8 7 . 1 7 7 . 1 7 7 . 9 6 7 . 8 6 7 . 7 4 7 . 5 4 7 . 5 4 7 . 4 4 7 . 3 4 7 . 3 4 7 . 2 3 7 . 0 3 7 . 0 1 . 5 9 0 5 . 8 0 5 . 7 0 5 . 9 2 4 . 6 2 4 . 8 9 3 . 5 9 3 . 6 8 3 . 5 8 3 . 4 8 3 . 3 8 3 . 2 8 3 . 0 8 3 . 7 7 3 . 6 7 3 . D O 3 D C 1 3 3 . 16 17 15 18 14 19 13 NH 20 ClH 22 21 NH2 11 10 2 3 1 4 6 5 8 9 NH 7 Br 12 200 150 100 50 0 0 8 7 . 1 7 7 . 0 7 7 . 8 6 7 . 7 6 7 . 6 4 7 . 5 4 7 . 4 4 7 . 3 4 7 . 2 4 7 . 2 4 7 . 1 3 7 . 9 2 7 . 0 1 . 5 9 0 5 . 8 0 5 . 7 0 5 . 8 2 4 . 5 2 4 . 7 9 3 . 5 9 3 . 6 8 3 . 5 8 3 . 3 8 3 . 2 8 3 . 1 8 3 . 9 7 3 . 6 7 3 . 5 7 3 . 400 300 200 100 0 150 100 50 0 0 0 . 1 0 0 . 1 6 1 . 1 7.9 7.8 7.7 7.6 f1 (ppm) 7.5 8 2 5 . 6 6 . 1 7.4 7.3 5 4 . 1 5.04 f1 (ppm) 5 2 . 1 8 0 . 1 4.2 4.0 f1 (ppm) 1 4 2 . 3.8 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 f1 (ppm) 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 0 . 1 0 0 . 1 6 1 . 1 8 2 5 . 6 6 . 1 5 4 . 1 5 2 . 1 8 0 . 1 1 4 2 . KD_6_124C_CARBON_01 6 2 . 1 4 1 5 1 . 9 3 1 5 1 . 1 3 1 . 3 7 0 3 1 . 5 2 0 3 1 . 2 2 0 3 1 . 7 9 9 2 1 . 7 6 6 2 1 . 1 9 4 2 1 . 4 5 2 2 1 . 1 6 0 2 1 . 9 0 6 1 1 D O 3 D C 0 0 9 4 . 5 3 3 5 . 7 3 0 5 . 1 4 . 1 4 16 17 15 18 14 19 13 NH 20 ClH 22 21 NH2 11 10 2 3 1 4 6 5 8 9 NH 7 Br 12 5 1 . 1 3 1 . 3 7 0 3 1 . 5 2 0 3 1 . 2 2 0 3 1 . 7 9 9 2 1 30 20 10 0 131.5 131.0 130.5 130.0 f1 (ppm) 1800 1700 1600 1500 1400 1300 1200 1100 1000 900 800 700 600 500 400 300 200 100 0 -100 28 26 24 22 20 18 16 14 12 10 8 6 4 2 0 -2 200 190 180 170 160 150 140 130 120 110 100 f1 (ppm) 90 80 70 60 50 40 30 20 10 0 214 Figure 2.37. 1H NMR and 13C NMR spectra of compound 2-70. l 3 C D C 6 2 7 . KD_7_44_dry_PROTON_01 9 1 . 8 6 5 7 . 6 5 7 . 0 4 7 . 9 3 7 . 6 5 7 . 6 5 7 . 0 4 7 . 9 3 7 . 2 2 7 . 1 2 7 . 0 2 7 . 0 2 7 . 8 1 . 7 8 1 . 7 7 1 . 7 7 1 . 7 9 5 5 . Br 21 1 4 17 2 3 18 9 NH 8 7 6 5 10 16 N 23 12 11 19 Br 22 13 20 15 NH 14 l 3 C D C 6 2 7 . 2 2 7 . 1 2 7 . 0 2 7 . 0 2 7 . 8 1 . 7 8 1 . 7 7 1 . 7 7 1 . 7 3000 2000 1000 0 5 9 . 1 3 2 2 . 3 2 2 . 4 1 . 2 7.55 7.50 7.45 7.40 7.35 f1 (ppm) 7.30 7.25 7.20 7.15 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 0 2 2 . 5 9 . 1 3 2 2 . 3 2 2 . 4 1 . 2 0 0 . 1 5.5 f1 (ppm) 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 KD_7_44_dry_CARBON_01 . 6 4 7 3 1 . 2 4 4 2 1 . 0 9 3 2 1 . 3 8 3 2 1 . 5 2 0 2 1 . 6 3 9 1 1 . 1 7 6 1 1 . 7 6 4 1 1 . 4 5 0 1 1 l 3 C D C 6 1 . 7 7 9 2 6 2 . 9 NH 8 7 6 5 10 16 N 23 12 11 Br 21 1 4 17 2 3 18 19 Br 22 13 20 15 NH 14 80 60 40 20 0 . 2 4 4 2 1 . 0 9 3 2 1 . 3 8 3 2 1 124.5 124.0 f1 (ppm) 200 190 180 170 160 150 140 130 120 110 100 f1 (ppm) 90 80 70 60 50 40 30 20 10 0 215 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 -1000 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 Chapter 3: Synthesis of Small Molecule Acyl-Astemizole Analogues as Activators of the 20S Proteasome 3.1 Introduction The 26S proteasome is a protein complex in the body that is responsible for the proteolysis of redundant proteins into recyclable peptide fragments.1,2,3 As shown in Figure 3.1, the 26S proteasome is comprised of one or two 19S regulatory particles which associate with the barrel-shaped 20S core particle. This 20S core particle (also referred to as the 20S proteasome) is comprised of two stacked heptameric beta rings, which are sandwiched between two heptameric alpha rings.1 The beta rings are responsible for the catalytic proteolytic activity of the proteasome, as they contain the three unique catalytic sites (the chymotrypsin-like (CT-L), trypsin-like (T-L), and caspase-like (Casp-L) sites), and the alpha rings serve as a “gate,” which opens and is activated upon association with the 19S regulatory particle.1,2 The 19S regulatory particle not only activates the proteasome by opening the gate, but it also facilitates recognition of “tagged” (ubiquitinated) proteins followed by their subsequent, de-ubiquitination, unfolding, and translocation of the substrate through the gate of the 20S core particle.4 The 20S proteasome lacks this 19S regulatory particle and is, therefore, considered the latent state of the proteasome with predominantly closed gates, preventing the degradation of folded proteins. However, it has been shown that the 20S proteasome is able to degrade intrinsically disordered proteins (IDPs) via a ubiquitin-independent mechanism 5,6 Unfortunately, this IDP proteolysis via the 20S proteasome is hindered by the fact that the 20S proteasome exists primarily in the closed-gate conformation. 216 Figure 3.1. General diagram of 26S and 20S proteasomes and a ubiquitinated protein substrate. Enhancing the IDP proteolysis would have significant therapeutic implications, as the accumulation of IDPs in the body has been associated with the progression of many neurodegenerative diseases, such as Alzheimer's and Parkinson’s disease.7 As many neurodegenerative diseases, such as Alzheimer's and Parkinson’s disease, have very few FDA approved therapeutics, the development of novel 20S proteasome activators that can facilitate the open-gate conformation of the 20S proteasome and enhance IDP proteolysis has been a primary focus of the Tepe lab. A potent 20S proteasome activator that has been identified in the Tepe lab is acyl Astemizole, whose structure is shown in Scheme 3.1. Dr. Allison Vanecek found that acyl Astemizole displayed an EC200 value of 4.6 µM when probed for enhancement of the 20S proteasome. In addition, acyl Astemizole has drug-like physical properties, with a molecular weight just under 500 g/mol (472.6 g/mol) and a cLogP value of 3.32, in accordance with Lipinski’s Rule of Five.8,9 Although acyl Astemizole has been shown to display potent proteasome activity, its structure-activity relationship remains largely unexplored. Considering this, acyl Astemizole was identified as a promising target for analogue development. My goal in 217 this project, in collaboration with Dr. Allison Vanecek, was to design and synthesize acyl Astemizole analogues with improved potency, while maintaining drug-like properties. This chapter will discuss the proteosome activation studies of a small library of synthesized acyl Astemizole analogues. Scheme 3.1. Chemical structure of acyl Astemizole. F N N NH N O OMe Acyl Astemizole (3-1) MW: 472.56 g/mol cLog P: 3.32 3.2. Results and Discussion It was hypothesized that ideal analogues would have comparable or lower molecular weights and comparable cLogP values to that of acyl Astemizole (3-1). This would allow for additional modifications to be made to analogues to increase potency, without violating the Lipiniski’s rule of five.9 To begin exploring which structural moieties of acyl Astemizole are necessary for 20S proteasome activation and/or where other structural modifications could be incorporated without decreasing the 20S proteasome activity, initial acyl Astemizole analogues were designed in which portions of the compound were removed to see how the activity was affected. In the first step of analogue design, several key moieties of acyl Astemizole’s chemical 218 structure were identified as potential areas to probe in structure activity relationship studies. As shown in Scheme 3.2, the 4-fluoro benzyl protecting group, the fused benzene ring of the benzimidazole core, the nitrogen atom of the benzimidazole core, and the amino linker group were identified as chemical moieties to be probed. Scheme 3.2 Chemical structure of acyl Astemizole and highlighted functionality to be probed during analogue exploration. F N N NH N O OMe Acyl Astemizole (3-1) 3.2.1. Acyl Astemizole analogue development 3.2.1.1. Class one analogues The first moiety of acyl Astemizole’s chemical structure that was probed via analogue development was the fused benzene ring of the benzimidazole core (green, Scheme 3.2). This is a very interesting moiety to investigate because if analogues lacking this fused benzene ring are found to maintain 20S proteasome modulation activity, this would allow for active, significantly lower molecular weight acyl Astemizole analogues. As shown in Scheme 3.3, in this first class of analogues, two initial acyl Astemizole imidazoline analogues (3-2 and 3-3) were designed for synthesis and biological analysis. To explore the importance of the fused benzene ring of acyl 219 Astemizole (3-1) with respect to its 20S proteasome activity, this moiety was analyzed in stages. The first imidazoline analogue (3-2) maintains the benzene ring as a substituent, rather than a fused ring, and the second imidazoline analogue (3-3) does not contain the benzene moiety (Scheme 3.3). Scheme 3.3. Initial class one acyl Astemizole analogues (3-2 and 3-3). F F N N NH N N NH N O N O 3-2 3-3 OMe OMe 3.2.1.2. Class two analogues The second class of acyl Astemizole analogues that was developed was focused on probing acyl Astemizole’s nitrogen linker atom (yellow, Scheme 3.2) and the nitrogen atom of the benzimidazole core (red, Scheme 3.2). These analogues were designed to explore the scope of the structure-activity relationship of the linker atom and heterocyclic core of acyl Astemizole. As shown in Scheme 3.4, initially, two thio-linker analogues were developed. To study these structural changes in a stepwise manner, a thio-linker acyl Astemizole was designed (3-4), as well as a thio-linker analogue with a benzimidazole core (3-5) (Scheme 3.4). Analogue 3-5 was particularly interesting as its synthesis would not only allow for investigation into the scope of the heterocyclic core of acyl Astemizole (3-1), but it would also allow for initial exploration of the importance of the 4-fluorobezyl moiety. 220 Scheme 3.4. Initial class two acyl Astemizole analogues (3-4 and 3-5). F N N S N S S N O N O 3-4 3-5 OMe OMe 3.2.1.3. Class three analogues The third class of acyl Astemizole analogues that was developed was aimed at probing the 4-fluorobenzyl group (blue, Scheme 3.2) of acyl Astemizole (3-2). Similar to class one, this aryl moiety would be interesting to probe because if analogues lacking this aryl ring are found to maintain 20S proteasome modulation activity, this would allow for active, significantly lower molecular weight acyl Astemizole analogues. Therefore, as shown in Scheme 3.5, the first class three analogue that was designed was debenzylated acyl Astemizole (3-6) to determine if this aryl ring was necessary for 20S proteasome activity. On the other hand, in exploration of different aryl groups, three additional analogues were designed. As shown in Scheme 3.5, the first was an analogue baring a benzyl group (3-7) in order to explore the importance of the fluoro-substituent on biological activity. Then, two analogues exploring aryl moiety linker length were also designed: one with one additional methylene (3-8) and one with one less methylene (3- 9) (Scheme 3.5). 221 Scheme 3.5. Initial class three acyl Astemizole analogues (3-6, 3-7, 3-8, and 3-9). F N N NH F N N NH N N NH N O N O N O N O 3-7 3-8 3-9 OMe OMe OMe OMe NH N N H 3-6 3.2.2. Acyl Astemizole analogue synthesis 3.2.2.1. Class one acyl Astemizole analogue syntheses The first class one acyl Astemizole analogue to be synthesized was the imidazoline analogue baring the phenyl substituent (3-2). It was hypothesized that this imidazoline analogue could be accessed via a late stage imidazoline cyclization of a diamine fragment with an isothiocyanate intermediate. Similar cyclizations have been reported in literature.10 As shown in Scheme 3.6, first, the diamine intermediate (3-11) was synthesized in two steps from the commercially available 4-fluorobenzylamine via a Strecker reaction and a subsequent cyanide reduction in high yields. Then, the commercially available 2-(4-methoxyphenyl)acetic acid was quantitatively converted to the respective acyl chloride (3-13) and this acyl chloride intermediate (3-13) was used to acylate the commercially available tert-butyl piperidin-4-ylcarbamate to access the protected amino-piperidine intermediate (3-14) in 81% yield. This piperidine intermediate (3-14) was then quantitatively deprotected and subsequently underwent isothiocyanate formation via 1,1’-thiocarbonyldiimidazole (TCDI) to access the isothiocyanate 222 intermediate (3-16) in 73% yield. The last step of this synthesis was the imidazoline cyclization in which the diamine intermediate (3-11) was condensed with the isothiocyanate intermediate (3- 16) to the corresponding thiourea, followed by methylation, cyclization, and loss of a thiol group to form the desired imidazoline analogue (3-2) in 73% yield. Scheme 3.6. Synthesis of initial class one acyl Astemizole imidazoline analogues (3-3 and 3-4). F O HO NH2 PhCHO (1.0 equiv), HO CN (1.0 equiv), Et3N (0.1 equiv) DCM, rt, 18 h 65% NC H N Ph 3-10 F LiAlH4 (3.0 equiv), AlCl3 (1.0 equiv), THF, 0 ºC to rt, 24 h 87% H2N OMe SOCl2 (1.0 equiv), DMF/DCM, rt, 20 h quant O Cl F 3-13 H2N NH2 Br (1.0 equiv), K2CO3 (1.0 equiv), DMF, rt, 18 h 58% H2N H N 3-12 BocHN NH OMe (1.1 equiv), Et3N (1.25 equiv) BocHN N DCM, 0 ºC to rt, 18 h 81% 3-14 HCl-EtOH, rt, 18 h quant F H2N N O 3-15 H N Ph 3-11 O F OMe OMe OMe S N N N N (1.0 equiv) DCM, -10 ºC to rt, 18 h 73% N N C S O 3-16 F N O R N N NH R = Ph (3-2) (73%) R = H (3-3) (68%) OMe 3-11 or 3-12 (1.0 equiv), EtOH, rt, 18h then, MeI (3.0 equiv), EtOH, reflux, 3 h 223 As shown in Scheme 3.6, the imidazoline analogue lacking the phenyl substituent (3-3) was synthesized in the same manner as 3-2. The only difference was the structure of the diamine intermediate (3-12). This diamine intermediate (3-12) was synthesized in one step via mono-4- fluorobenzylation of the commercially available ethylenediamine to access 3-12 in 58% yield. This diamine intermediate then underwent the same condensation/methylation/cyclization sequence with the isothiocyanate intermediate (3-16) to access the desired acyl Astemizole imidazoline analogue (3-3) in 68% yield. 3.2.2.2. Class two acyl Astemizole analogue syntheses 3.2.2.2.1. Thio-linker analogue synthesis It was hypothesized that the thio-linker analogue (3-4) could be accessed via a late-stage oxidative nucleophilic displacement of a sulfone group as a good leaving group. Similar transformations have been previously reported in literature for the synthesis of 2-amino and 2- thio benzimidazoles from 2-methylsulfonyl benzimidazole, as shown in Scheme 3.7.11 The 2- methylsulfonyl benzimidazole starting material can be readily synthesized via a mild oxidation of 2-methylthio-benzimidazole via meta-chloroperoxybenzoic acid (m-CPBA) following literature procedures.12 Scheme 3.7. Previous work reported for the synthesis of 2-amino and 2-thio benzimidazoles from 2-methylsulfonyl benzimidazole. N N H SO2Me X R heat, EtOH or DMF 69-95% (R = alkyl, aryl) (X = NH2, SH) N N H X R Application of this methodology was used for the synthesis of the thio-linker acyl Astemizole analogue (3-4) using a complex sulfur nucleophile (3-20), as shown in Scheme 3.8. 224 In the synthesis of 3-4, the 4-benzylthiopiperidine intermediate (3-18) was first achieved via benzylation and subsequent acid-mediated Boc-deprotection in very high yields. Next, the N- acylated 4-thio piperidine nucleophile (3-20) was synthesized in three steps from the commercially available 2-(4-methoxyphenyl)acetic acid via acid chloride formation, acylation of the piperidine intermediate (3-18), and subsequent de-benzylation in high yields (Scheme 3.8). With the sulfur nucleophile (3-20) in hand, the key late-stage oxidative nucleophilic displacement of the sulfone group could be explored. After 4-fluorobenzylation of 2- (methylthio)-1H-benzo[d]imidazole to access 3-21, the resulting protected 2-methylthio- benzimidazole intermediate (3-21) was subjected to oxidative conditions via m-CPBA to install the sulfone moiety. Immediately after oxidation, the recently formed sulfone intermediate was subjected to nucleophilic attack by 3-20 to displace the sulfone group, accessing the desired thio- linker analogue (3-4) in 67% yield (Scheme 3.8). 225 Scheme 3.8. Synthesis of thio-linker acyl Astemizole analogue (3-4). O HO SOCl2 (1.0 equiv), DMF/DCM, rt, 20 h quant O Cl 3-13 OMe HS NBoc K2CO3 (1.0 equiv), BnBr (1.0 equiv) DMF, 70 ºC, 18 h 91% 3-18 (1.1 equiv), Et3N (1.25 equiv), OMe DCM, 0 ºC to rt, 18 h 79% BnHS N O 3-19 BnS NBoc 3-17 HCl-EtOH, rt, 18 h quant. BnS NH•HCl 3-18 OMe H2, Pd(OH)2/C EtOH, rt, 24 h quant HS N O 3-20 OMe F F F Br (1.0 equiv), K2CO3 (1.0 equiv) DMF, rt, 24 h 75% SMe N N H SMe N N 3-21 m-CPBA (2.5 equiv), DCM, 0 ºC, 1 h then, 3-20 (5.0 equiv), Toluene, rt to reflux, 18 h 67% N N S 3-4 N O OMe To design a more expeditious synthetic route toward acyl Astemizole analogues that structurally resemble 3-4, an additional approach was explored. Instead of going through step- intensive synthesis of the N-acylated 4-thiopiperidine nucleophile (3-20), this process was expedited by installing the piperidine moiety at an earlier stage. As shown in Scheme 3.9, 2- thiobenzimidazole underwent base-mediated nucleophilic attack on the commercially available N-Boc-4-mesyloxypiperidine, as has been reported in literature for similar transformations.13 After this nucleophilic attack to displace the mesyloxy moiety, the piperidine substituted intermediate (3-22) was accessed in 46% yield. Intermediate 3-22 then underwent 4- fluorobenzylation and subsequent Boc deprotection to access intermediate 3-24 in very high yields. The last step was acylation of the piperidine intermediate (3-24) via the same acyl 226 chloride intermediate (3-13) to access the thio-linker analogue (3-4) in 91% yield (Scheme 3.9). This adapted approach, including the aforementioned single step to access the acyl chloride intermediate (3-13), was five steps overall. This was significantly more expedited than the previous approach, which consisted of seven steps overall (Scheme 3.8). Scheme 3.9. Expedited synthesis of the acyl Astemizole thio-linker analogue (3-4). O S O O N Boc (1.0 equiv), K2CO3 (1.0 equiv), EtOH, rt to reflux, 18 h 46% SH N N H F N N S 3-4 N O OMe K2CO3 (1.0 equiv), F Br S N N H 3-22 F (1.0 equiv), DMF, rt, 18 h 92% NBoc S N N 3-23 NBoc HCl-EtOH, rt, 18 h quant F OMe O Cl 3-13 (1.0 equiv) Et3N (1.25 equiv), DCM, 0 ºC to rt, 18 h 91% N N S 3-24 NH•HCl 3.2.2.2.1. Thio-linker benzothiazole analogue synthesis The thio-linker benzothiazole analogue (3-5) was synthesized via the same procedure as was previously discussed for the expedited synthesis of 3-4. As shown in Scheme 3.10, the commercially available 2-mercaptobenzothiazole was reacted with the commercially available N-Boc-4-mesyloxypiperidine to undergo nucleophilic displacement of the mesyloxy group, accessing the desired piperidine-substituted intermediate (3-25) in 88% yield. This yield was 227 significantly higher than that of the previously discussed 3-22 (Scheme 3.9). It is noteworthy to mention that the conversion in both nucleophilic displacement reactions was high. Therefore, the isolation method was likely the cause of these significantly different yields, as 3-22 was isolated via filtration of the formed precipitate and 3-25 was isolated via column chromatography. Column chromatography appeared to be the superior form of isolation for this reaction. The protected piperidine intermediate (3-25) was quantitatively Boc-deprotected and subsequently underwent acylation via the acyl chloride intermediate (3-13) to access the desired thio-linker benzothiazole analogue (3-5) in 72% yield. Scheme 3.10. Synthesis of the thio-linker benzothiazole analogue (3-5). O S O O N S SH N Boc (1.0 equiv), K2CO3 (1.0 equiv), EtOH, rt to reflux, 18 h 88% S N S 3-25 HCl-EtOH rt, 18 h quant NBoc S N S 3-26 NH•HCl 3-13 (1.0 equiv), Et3N (1.25 equiv), DCM, 0 ºC to rt, 18 h 72% N S S 3-5 N O OMe 228 3.2.2.3. Class three acyl Astemizole analogue syntheses 3.2.2.3.1. Debenzylated acyl Astemizole analogue synthesis In the synthesis of the debenzylated acyl Astemizole analogue (3-6), a different synthetic route was devised in order to allow for the synthesis of the free benzimidazole core. It was hypothesized that this analogue (3-6) could be expeditiously synthesized via a thermal coupling of the commercially available 2-chlorobenzimidazole and the previously synthesized N-acylated 4-amino piperidine tail (3-15). Similar thermal couplings have been reported in literature in the synthesis of Astemizole and related analogues.14 As shown in Scheme 3.11, commercially available 2-chlorobenzimidazole underwent thermal coupling with an excess of the N-acylated 4- aminopiperidine tail to afford the debenzylated acyl Astemizole analogue (3-6) in 54% yield. Scheme 3.11. Synthesis of the debenzylated acyl Astemizole analogue (3-6). O H2N N OMe 3-15 (5.0 equiv) DMA, 140 ºC, 48 h 54% Cl N N H NH N N H 3-6 N O OMe 3.2.2.3.2. Progress toward synthesis of the benzylated analogue It was proposed that the benzylated acyl Astemizole analogue (3-7) could be synthesized via the same thermal coupling approach as was previously used in the synthesis of 3-6. Contrary to this, the previously discussed approach to the thio-linker analogues (3-4 and 3-5, Scheme 3.9 and 3.10) was identified as potentially problematic for the nitrogen linker analogues due to possible regioselectivity issues that could arise with the late-stage acylation. Therefore, the thermal coupling method was identified as the most favorable approach for synthesis of acyl 229 Astemizole analogues containing nitrogen linker atoms. As shown in Scheme 3.12, in the synthesis of 3-7, commercially available 2-chlorobenzimidazole was benzylated to access the benzylated 2-chlorobenzimidazole intermediate (3-27) in 88% yield. The next proposed step in this synthesis was the thermal coupling of 3-27 and 3-15 to access the benzylated acyl Astemizole analogue (3-7). This last thermal coupling step remains a point of focus for the future. Scheme 3.12. Synthesis of the benzylated acyl Astemizole analogue (3-7). Br Cl N N H (1.0 equiv), NaH (2.0 equiv), DMSO, rt, 1 h then, BnBr (1.3 equiv), 18 h 88% N N Cl 3-15 (5.0 equiv) DMA, 140 ºC, 48 h NH N N 3-7 N O 3-27 OMe 3.2.2.3.3. Progress toward synthesis of the two methylene aryl linker analogue It was proposed that the acyl Astemizole analogue containing an extra aryl linker methylene (3- 8) could be synthesized via the same thermal coupling approach as was previously used in the synthesis of 3-6 and 3-7. As shown in Scheme 3.13, the commercially available 2- chlorobenzimidazole was first alkylated with the commercially available 1-(2-bromoethyl)-4- fluorobenzene to access the desired alkylated intermediate (3-28) in 74% yield. The next proposed step in this synthesis was the thermal coupling of 3-28 and 3-15 to access the acyl Astemizole analogue containing an extra aryl linker methylene (3-8). This last thermal coupling step remains a point of focus for the future. 230 Scheme 3.13. Synthesis of the acyl Astemizole analogue containing an extra aryl linker methylene (3-8). Br Cl N N H (1.0 equiv), F K2CO3 (1.0 equiv), DMF, rt, 24 h 74% N N Cl 3-15 (5.0 equiv) DMA, 140 ºC, 48 h F NH N N 3-8 N O F 3-28 OMe 3.2.2.3.4. Progress toward synthesis of the 4-fluorophenyl substituted analogue Due to synthetic limitations in the formation of 2-chloro-1-(4-fluorophenyl)- benzimidazole as an intermediate, unfortunately, the thermal coupling approach was not viable for the synthesis of the 4-fluorophenyl substituted analogue (3-9). Therefore, despite the aforementioned potential regioselectivity issues with the late-stage acylation, the same method that was used in the synthesis of the thio-linker analogues (3-4 and 3-5, Scheme 3.9) was proposed for the synthesis of 3-9. As shown in Scheme 3.14, first, commercially available 2- aminobenzimidazole underwent a Chan-Lam coupling reaction to afford the 4-fluorophenyl intermediate (3-29) in 73% yield, following literature procedures.15 Then, 3-29 was reacted with the commercially available N-Boc-4-mesyloxypiperidine to undergo nucleophilic displacement of the mesyloxy group, accessing the desired piperidine-substituted intermediate (3-30) in 65% yield. The last two proposed steps for the synthesis of 3-9 include acid-mediated Boc- deprotection and subsequent late-stage acylation of the piperidine (3-31) with the acyl chloride 231 intermediate (3-13) to access the 4-fluorophenyl substituted analogue (3-9). These last two steps remain a point of future exploration and optimization. Scheme 3.14. Synthesis of the 4-fluorophenyl substituted acyl Astemizole analogue (3-9). HO B HO F N N H NH2 (1.0 equiv), air, Cu(OAc)2 (20 mol%), EtOH, rt, 24 h 73% O S O O N N NH2 3-29 N Boc (1.0 equiv), K2CO3 (1.0 equiv), EtOH, rt to reflux, 18 h 65% N N NH 3-30 NBoc F F F NH N N 3-9 HCl-EtOH, rt, 18h OMe N N NH O Cl 3-13 (1.0 equiv), Et3N (1.25 equiv), DCM, 0 ºC to rt, 18 h 3-31 NH•HCl F N O OMe 3.2.3. Initial acyl Astemizole analogue 20S proteasome activity studies The 20S proteasome activity of these synthesized acyl Astemizole analogues was explored in collaboration with Dr. Allison Vanecek. Dr. Vanecek tested these analogues via a fluorogenic peptide degradation assay which utilized short peptide substrates conjugated to the fluorophore 7-amino-4-methylcoumarin (AMC). The three fluorogenic substrate probes Suc- LLVY-AMC, Boc-LRR-AMC, and Z-LLE-AMC were used to test for the activity of the 20S proteasome’s three unique catalytic sites, the chymotrypsin-like (CT-L), trypsin-like (T-L), and 232 caspase-like (Casp-L) sites, respectively. To explore the ability of these synthesized acyl Astemizole analogues to enhance 20S proteasome activity, purified human 20S proteasome was pretreated with varying concentrations of the acyl Astemizole analogue, followed by addition of the fluorogenic substrate probe(s). Proteolytic cleavage of the peptide results in the release of AMC. The resulting fluorescence from the release of this AMC is measured as a rate over time to quantify 20S proteolytic activity. As shown in Figure 3.2, the calculated max fold increase and EC200 values were calculated. All biological data was collected in triplicate (n=3). Figure 3.2. Chemical structures of acyl Astemizole analogues and exploration of their 20S proteasome modulation activity. F F F F N N NH N N NH N N NH N N S N S S NH N N H N O N O N O N O N O N O Acyl Astemizole (3-1) 3-2 3-3 3-4 3-5 3-6 OMe OMe OMe OMe OMe OMe MW: 472.56 g/mol cLog P: 3.32 MW: 500.62 g/mol cLog P: 4.44 MW: 424.52 g/mol cLog P: 2.69 MW: 489.61 g/mol cLog P: 4.78 MW: 398.54 g/mol cLog P: 3.69 MW: 364.45 g/mol cLog P: 1.19 Enhancement of 20S Proteasome Activity Max Fold Increase EC200 (µM) Acyl Astemizole (3-1) 3-2 3-3 3-4 3-5 3-6 8.3 ± 1.0 2.9 ± 0.5 5.5 ± 1.4 4.2 ± 1.1 6.2 ± 0.7 1.8 ± 0.5 4.6 ± 1.2 2.7 ± 0.7 8.4 ± 1.1 3.7 ± 0.6 8.1 ± 1.2 >80.0 The imidazoline acyl Astemizole analogues (3-2 and 3-3) were studied to explore the importance of the fused benzene ring of acyl Astemizole’s benzimidazole core. As shown in Figure 3.2, the 20S proteasome activity assay results for the phenyl imidazoline analogue (3-2) 233 showed maximum fold enhancement of 2.9-fold (i.e. 290%) over the vehicle at the highest concentration tested and an EC200 value of 2.7 µM. It is notable to mention that the EC200 value represents the concentration of the drug, or analogue, required to increase the activity by 200%. Compared to acyl Astemizole (3-1), 3-2 showed a large decrease in the maximum fold increase compared to acyl Astemizole, but an almost two-fold increase in potency, emphasizing the importance of this aryl ring for 20S proteasome modulation activity. This also indicated that the phenyl ring did not have to be fused to the benzimidazole core to allow for activity but could also be present as a substituent. To further study the relationship of this benzene moiety to the 20S proteasome activity, the imidazoline analogue lacking the phenyl substituent (3-3) was also analyzed. The results showed lower maximum fold increase and an almost two-fold loss in potency, with an EC200 of 8.5 µM, compared to acyl Astemizole (3-1) (Figure 3.2). Overall, these findings emphasize the importance of this benzene ring moiety to the 20S proteasome activity. To explore if the incorporation of a thio-linker, rather than an amino-linker, in the 2- postion of the benzimidazole core would maintain activity, analogue 3-4 was studied. As shown in Figure 3.2, although the maximum fold increase in activity over the vehicle was decreased, the EC200 value was comparable to that of acyl Astemizole, suggesting replacement of the amine with a sulfur atom maintains 20S proteasome activity and is a feasible option for future analogues. Another thio-linker analogue with a benzothiazole core (3-5) was also analyzed and was found to be moderately active (EC200 = 8.1 μM). However, since analogue 3-5 had two scaffold changes compared to 3-4, additional analogues are required to determine whether the leap this moderately active is due to the benzothiazole core, or loss of the 4-fluorobenzyl moiety. To explore the effect of the 4-fluorobenzyl group on 20S proteasome activity, the debenzylated acyl Astemizole analogue (3-6) was studied. As shown in Figure 3.2, 3-6 was 234 shown to be unable to enhance 20S proteasome activity, demonstrating the great importance of the 4-fluorobenzyl group. Considering the debenzylated analogue’s lack of reactivity, the previously discussed moderate activity (EC200 = 8.1 μM) of 3-5 was even more intriguing, as it lacks the 4-fluorobenzyl moiety but still maintains a some 20S proteasome activity. The design and synthesis of additional analogues, such as a benzothiazole with an amino linker atom or additional 4-fluoroaryl moieties, is needed to study this activity further and remains a focus for the future. 3.3. Conclusions Scheme 3.15. Future acyl Astemizole analogues to be synthesized and studied. F N N NH N N NH F N N NH F N S N N S NH N O N O N O N O N O 3-7 3-8 3-9 3-32 3-33 OMe OMe OMe OMe OMe In summary, several acyl Astemizole analogues have been synthesized which have given valuable insights into the relationship between several structural moieties of acyl Astemizole and its 20S proteasome modulation activity. However, some structure activity relationship questions remain regarding these aforementioned synthesized analogues, specifically focusing on the activities of 3-5 and 3-6. As was previously mentioned, the 4-fluorobenzyl group of acyl Astemizole (3-1) was identified as very important for biological activity as it was found that the debenzylated acyl Astemizole analogue (3-6) lacked 20S proteasome activity. Interestingly, 235 though, 3-5 was found to have moderate 20S proteasome activity even though it lacked the 4- fluorobenzyl moiety. The synthesis of additional analogues to explore these findings, such as a benzothiazole analogue with an amino linker (3-32) and a benzothiazole analogue with the 4- fluorobenzyl group on the amino linker (3-33) were identified as a point of focus for future studies (Scheme 3.15). In addition, as shown in Scheme 3.15, to further explore the scope of the 4-fluorobenzyl aryl moiety with respect to 20S proteasome activity, the completion of the previously discussed aryl analogue syntheses (3-7, 3-8, and 3-9) were also identified as a point of focus for future studies. Overall, the design, synthesis, and biological studies of these acyl Astemizole analogues have provided valuable insights into the structure-activity relationship of acyl Astemizole (3-1). As acyl Astemizole (3-1) remains a promising target in the Tepe lab, and these findings will be of great use for future analogue development. 236 3.4 Experimental 18 F 15 12 11 6 5 14 13 9 NH 7 16 17 1 4 F NH 2 3 N 8 N 10 2-((4-fluorobenzyl)amino)-2-phenylacetonitrile (3-10) To a mixture of benzaldehyde (0.20 mL, 2.0 mmol, 1.0 equiv), 4-fluorobenzylamine (0.23 mL, 2.0 mmol, 1.0 equiv), and acetone cyanohydrin (0.18 mL, 2.0 mmol, 1.0 equiv) in anhydrous MeCN (10 mL) in an oven dried flask, was added Et3N (0.01 mL, 0.1 mmol, 0.05 equiv) at room temperature, under an atmosphere of N2 gas. This resulting solution was then allowed to stir for 18 hours at room temperature under N2. After 18 hours, the reaction was quenched with H2O (5 mL), extracted with ethyl acetate (3x30mL), dried over Na2SO4, filtered, and concentrated in vacuo. The resulting crude residue was then purified via silica gel column chromatography (hexanes/ethyl acetate) to yield the desired product as a yellow oil that crystallized to a yellowish, clear, crystalline solid (0.31 g, 65%). M.P.: 39-40ºC, 1H NMR (500 MHz, CDCl3) δ 7.54 (d, J = 7.0 Hz, 2H, (H1, H5)), 7.46 – 7.33 (m, 5H, (H2, H3, H4, H13, H17)), 7.04 (t, J = 8.7 Hz, 2H, (H14, H16)), 4.74 (s, 1H, (H7)), 4.03 (d, J = 13.0 Hz, 1H, (H11)), 3.93 (d, J = 13.0 Hz, 1H, (H11)). 13C NMR (126 MHz, CDCl3) δ 162.38 (d, J = 245.6 Hz, (C15)), 134.67 (C6), 133.90 (d, J = 2.9 Hz, (C12)), 130.13 (d, J = 8.1 Hz, (C13, C17)), 129.21 (C3), 129.11 (C2, C4), 127.38 (C1, C5), 118.74 (C8), 115.58 (d, J = 21.0 Hz, (C14, C16)), 53.49 (C7), 50.60 (C11). 237 FTIR (cm-1): 3314, 3069, 2896, 2254, 1503. HRMS-ESI (m/z): Calculated for C15H13FN2 (M+H)+: 241.1141, Found: 241.1147. 17 F 14 11 10 6 5 13 12 9 NH 7 15 16 1 4 18 NH2 8 F NH NH2 2 3 N1-(4-fluorobenzyl)-1-phenylethane-1,2-diamine (3-11) An oven-dried rb flask was purged with Argon gas for 5 min. To this was then added LiAlH4 (0.23 g, 6.0 mmol, 3.0 equiv), which was then purged again with Ar gas for another 5 min. This rb was then cooled to 0ºC and then anhydrous THF (3 mL) was then added. After 10 minutes, a solution of AlCl3 (0.27 g, 2.0 mmol, 1.0 equiv) in THF (10 mL) was added at 0ºC, under Ar. This was allowed to stir for 15 minutes to allow for formation of the active catalyst. Then, the mono-p-fluoro-benzylated amino nitrile intermediate (3-10) (0.48 g, 2.0 mmol, 1.0 equiv) was added dropwise in anhydrous THF (3 mL) at 0ºC. The reaction was then allowed to warm to rt and was stirred for 24 hours under an atmosphere of Ar gas. After completion, the reaction was then quenched slowly (dropwise) with 2M aqueous NaOH solution until the bubbling stopped. The organic layer was then extracted with ethyl acetate (3x30mL), dried over Na2SO4, filtered, and concentrated in vacuo. The resulting crude residue was then purified via silica gel column chromatography (MeOH (1% Et3N)/DCM) to yield the desired product as a clear, colorless oil (0.43 g, 87%). 1H NMR (500 MHz, CD3OD) δ 7.44 – 7.35 (m, 4H, (H1, H2, H4, H5)), 7.34 – 238 7.26 (m, 3H, (H3, H12, H16)), 7.01 (t, J = 8.8 Hz, 2H, (H13, H15)), 3.73 (dd, J = 8.4, 5.6 Hz, 1H, (H7)), 3.64 (d, J = 13.2 Hz, 1H, (H10)), 3.49 (d, J = 13.2 Hz, 1H, (H10)), 2.98 (dd, J = 12.8, 5.5 Hz, 1H, (H8)), 2.92 (dd, J = 12.8, 5.5 Hz, 1H, (H8)). 13C NMR (126 MHz, CD3OD) δ 163.25 (d, J = 243.2 Hz, (C14)), 141.76 (C6), 137.04 (d, J = 2.9 Hz, (C11)), 131.23 (d, J = 8.1 Hz, (C12, C16)), 129.89 (C2, C4), 128.92 (C3), 128.47 (C1, C5), 115.89 (d, J = 21.5 Hz, (C13, C15)), 63.17 (C7), 50.93 (C10), 47.35 (C8). FTIR (cm-1): 3374, 3352, 3311, 3055, 2886, 1508. HRMS-ESI (m/z): Calculated for C15H17FN2 (M+H)+: 245.1454, Found: 245.1475. H N H2N NH25 1 F 2 4 11 NH 3 6 7 10 9 8 F 12 N1-(4-fluorobenzyl)ethane-1,2-diamine (3-12) To an oven-dried round bottom flask was added ethylenediamine (0.67 mL, 10.0 mmol, 1.0 equiv) dissolved in anhydrous DMF (15 mL) at 20ºC under an atmosphere of N2 gas. To this was then added K2CO3 (1.38 g, 10.0 mmol, 1.0 equiv) and 4-fluoro benzyl bromide (1.24 mL, 10.0 mmol, 1.0 equiv) sequentially at 20ºC under N2 gas. The resulting reaction mixture was then allowed to stir at 20ºC for 18 hours under N2 gas. After 18 hours, the reaction was quenched with deionized H2O (20 mL), washed with saturated aqueous LiCl solution (3x50 mL), extracted with EtOAc (3x50 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The resulting crude residue was then purified via silica gel column chromatography (DCM/MeOH (2% TEA)) to yield the desired product as a clear pale-yellow oil. (0.97 g, 58%). 1H NMR (500 MHz, CD3OD) δ 7.36 (dd, J = 8.6, 5.4 Hz, 2H, (H7, H11)), 7.05 (t, J = 8.8 Hz, 2H, (H8, H10)), 3.74 (s, 2H, (H4)), 2.75 (t, J = 6.3 Hz, 2H, (H2)), 2.65 (t, J = 6.3 Hz, 2H, (H1)). 13C NMR (126 MHz, 239 CD3OD) δ 163.48 (d, J = 243.7 Hz, (C9)), 136.82 (d, J = 2.9 Hz, (C6)), 131.37 (d, J = 8.1 Hz, (C7, C11)), 116.04 (d, J = 21.5 Hz, (C8, C10)), 53.64 (C4), 52.07 (C2), 41.75 (C1). FTIR (cm- 1): 3356, 3301, 3279, 3021, 2891, 1515. HRMS-ESI (m/z): Calculated for C9H13FN2 (M+H)+: 169.1441, Found: 169.1467. O Cl OMe 4 O 2 Cl 1 3 10 5 9 6 8 7 12 CH3 O 11 2-(4-methoxyphenyl)acetyl chloride (3-13) 4-methoxyphenyl acetic acid (1.66 g, 10.0 mmol, 1.0 equiv) was first dissolved in anhydrous DCM (20mL) in an oven-dried flask at room temperature under an atmosphere of N2 gas. SOCl2 was then added (1.1 mL, 15.0 mmol, 1.5 equiv), followed by DMF (4 drops, 0.01 mmol, 0.001 eqiuv). The resulting solution was then allowed to stir at room temperature, under an atmosphere of N2 gas for 20 hours. After 20 hours, the reaction was then concentrated in vacuo to afford the desired product as an orange oil (1.85 g, Quantitative). 1H NMR (500 MHz, DMSO) δ 7.16 (d, J = 8.7 Hz, 2H, (H6, H10)), 6.86 (d, J = 8.7 Hz, 2H, (H7, H9)), 3.72 (s, 3H, (H12)), 3.48 (s, 2H, (H3)). 13C NMR (126 MHz, DMSO) δ 173.00 (C2), 158.06 (C8), 130.42 (C6, C10), 126.99 (C5), 113.69 (C7, C9), 55.06 (C12), 39.81 (C3). FTIR (cm-1): 3037, 2836, 1779, 1503, 1249, 1041. HRMS-ESI (m/z): Calculated for C9H9ClO2 (M+H)+; (M+2)2+: 185.0369; 187.0377, Found: 185.0372; 187.0381. 240 25 CH3 O 24 9 8 10 7 4 O 5 6 2 3 O OMe 22 CH3 11 N1 12 15 20 O 13 14 17 NH 16 O N O N H CH323 19 O 18 CH321 tert-butyl (1-(2-(4-methoxyphenyl)acetyl)piperidin-4-yl)carbamate (3-14) To an oven-dried flask was added acyl chloride intermediate (0.55 g, 3.0 mmol, 1.0 equiv) in anhydrous DCM (2mL). (N-Boc-4-amino)piperidine (0.66 g, 3.3 mmol, 1.1 equiv) in anhydrous DCM (3mL) was then added dropwise to the solution at 0ºC under an atmosphere of N2 gas. Et3N (0.41 mL, 2.75 mmol, 1.25 equiv) was then also added dropwise at 0ºC under N2. The reaction was then allowed to warm to room temperature and was stirred for 18 hours at room temperature under N2 gas. The reaction was then quenched with H2O (10 mL), washed with saturated NaHCO3 solution (10 mL), extracted with DCM (3x30mL), dried over Na2SO4, filtered, and concentrated in vacuo. The resulting crude residue was then purified via silica gel column chromatography (DCM/MeOH) to yield the desired product as a pale yellow, crystalline solid (0.85 g, 81%). M.P.: 128-129ºC. 1H NMR (500 MHz, CD3OD) δ 7.16 (d, J = 8.6 Hz, 2H, (H7, H9)), 6.88 (d, J = 8.7 Hz, 2H, (H6, H10)), 4.43 – 4.34 (m, 1H, (H11 or H15(A))), 3.97 – 3.89 (m, 1H, (H11 or H15(B))), 3.77 (s, 3H, (H25)), 3.70 (d, J = 4.6 Hz, 2H, (H3)), 3.57 – 3.47 (m, 1H, (H13)), 3.12 (t, J = 12.9 Hz, 1H, (H11 or H15(B))), 2.82 (t, J = 12.9 Hz, 1H, (H11 or 241 H15(A))), 1.87 – 1.80 (m, 1H, (H12 or H14(A))), 1.79 – 1.68 (m, 1H, (H12 or H14(B))), 1.42 (s, 9H, (H21, H22, H23)), 1.33 – 1.21 (m, 1H, (H12 or H14(A))), 1.16 – 1.05 (m, 1H, (H12 or H14(B))). 13C NMR (126 MHz, CD3OD) δ 172.33 (C2), 160.03 (C17), 157.71 (C8), 130.67 (C7, C9), 128.27 (C5), 115.18 (C6, C10), 80.11 (C19), 55.69 (C25), 54.82 (C13), 46.23 (C11 or 15(B)), 42.02 (C11 or 15(A)), 40.59 (C3), 33.37 (C12 or 14(B)), 32.65 (C12 or 14(A)), 28.73 (C21, C22, C23). FTIR (cm-1): 3321, 3022, 2875, 1686, 1662, 1503, 1250, 1225, 1041. HRMS-ESI (m/z): Calculated for C19H28N2O4 (M+H)+: 349.2127, Found: 349.2132. 17 CH3 O 16 4 O 2 3 10 5 9 6 8 7 O N HCl•H2N 12 13 11 14 N 1 15 OMe NH218 ClH 19 1-(4-aminopiperidin-1-yl)-2-(4-methoxyphenyl)ethan-1-one hydrochloride (3-15) To an oven-dried flask was added the acylated Boc-amino piperidine intermediate (0.35 g, 1.0 mmol, 1.0 equiv) dissolved in anhydrous EtOH (3 mL). To this was then added HCl solution (4M in EtOH, 3.3 mL) at room temperature, under an atmosphere of N2 gas. The resulting reaction mixture was then stirred for 18 hours at rt under N2 gas. After 18 hours, the reaction was then concentrated in vacuo to afford the desired product as a yellow foaming solid (0.28 g, Quantitative). 1H NMR (500 MHz, CD3OD) δ 7.19 (d, J = 7.0 Hz, 2H, (H7, H9)), 6.89 (d, J = 6.9 Hz, 2H, (H6, H10)), 4.60 (d, J = 11.7 Hz, 1H, (H11 or H15(A))), 4.15 – 4.07 (m, 1H, (H11 or H15(B))), 3.79 – 3.75 (m, 5H, (H3, H17)), 3.38 – 3.35 (m, 1H, (H13)), 3.17 – 3.14 (m, 1H, (H11 242 or H15(B))), 2.81 – 2.73 (m, 1H, (H11 or H15(A))), 2.08 – 2.02 (m, 1H, (H12 or H14(A))), 2.01 – 1.94 (m, 1H, (H12 or H14(B))), 1.49 – 1.42 (m, 1H, (H12 or H14(A))), 1.35 – 1.30 (m, 1H, (H12 or H14(B))). 13C NMR (126 MHz, CD3OD) δ 172.57 (C2), 160.02 (C8), 130.75 (C7, C9), 127.79 (C5), 115.24 (C6, C10), 55.78 (C17), 49.40 (C13), 45.65 (H11 or H15(B)), 41.42 (H11 or H15(A)), 40.38 (C3), 31.20 (H12 or H14(B)), 30.58 (H12 or H14(A)). FTIR (cm-1): 3445, 3362, 3023, 2879, 1659, 1522, 1253, 1042. HRMS-ESI (m/z): Calculated for C17H26N2O2 (M+H)+: 249.1603, Found: 249.1629. O N N C S 7 N 1 6 2 5 3 16 O N 4 8 15 14 20 CH3 9 10 13 O 19 11 12 OMe 17 S 18 1-(4-isothiocyanatopiperidin-1-yl)-2-(4-methoxyphenyl)ethan-1-one (3-16) To an oven-dried round bottom flask was added the neutralized 2-amino piperidine intermediate (3-15) (0.57 g, 2.0 mmol, 1.0 equiv) dissolved in anhydrous DCM (15 mL) at room temperature under an atmosphere of N2 gas. The resulting solution was then cooled to -10ºC. Then, di(1H- imidazol-1-yl)methanethione (392.1 mg, 2.2 mmol, 1.1 equiv) was added at -10ºC under N2 gas. The resulting solution was then allowed to warm to room temperature and was stirred for 18 hours. After 18 hours, the reaction was quenched with H2O (25 mL), extracted with EtOAc (3x30mL), dried over Na2SO4, filtered, and concentrated in vacuo to yield the desired product as a pale-yellow oil (0.42 g, 73%). 1H NMR (500 MHz, CDCl3) δ 7.15 (d, J = 8.7 Hz, 2H, (H11, H15)), 6.86 (d, J = 8.7 Hz, 2H, (H12, H14)), 3.95 – 3.88 (m, 1H, (H1)), 3.80 (s, 3H, (H20)), 3.71 – 3.68 (m, 2H, (H3 or H5(A))), 3.67 (s, 2H, (H9)), 3.61 – 3.53 (m, 1H, (H3 or H5(B))), 3.44 243 – 3.30 (m, 1H, (H3 or H5(B))), 1.91 – 1.86 (m, 1H, (H2 or H6(A))), 1.81 – 1.70 (m, 1H, (H2 or H6(A))), 1.68 – 1.61 (m, 1H, (H2 or H6(B))), 1.60 – 1.52 (m, 1H, (H2 or H6(B))). 13C NMR (126 MHz, CDCl3) δ 169.85 (C8), 158.65 (C13), 137.09 (C17), 129.60 (C11, C15), 126.85 (C10), 114.40 (C12, C14), 55.42 (C20), 53.06 (C1), 43.03 (C3 or C5(B)), 40.42 (C9), 38.69 (C3 or C5(A)), 32.49 (C2 or C6(B)), 31.93 (C2 or C6(A)). FTIR (cm-1): 3023, 2887, 2101, 1685, 1255, 1224, 1045. HRMS-ESI (m/z): Calculated for C15H18N2O2S (M+H)+: 291.1167, Found 291.1179. N O H N N N Ph F 3 2 N 4 NH 6 7 8 1 5 N 33 32 34 15 37 35 36 16 13 14 19 18 17 F 20 OMe 12 9 11 N 10 21 22 29 O 23 24 28 25 27 26 31 CH3 O 30 1-(4-((1-(4-fluorobenzyl)-5-phenyl-4,5-dihydro-1H-imidazol-2-yl)amino)piperidin-1-yl)-2- (4-methoxyphenyl)ethan-1-one (3-2) To an oven-dried round bottom flask was added the diamine intermediate (3-11) (0.48 g, 2.0 mmol, 1.0 equiv) dissolved in anhydrous EtOH (10mL). At 20ºC, under an atmosphere of N2, was then added the isothiocyanate intermediate (3-16) (0.58 g, 2.0 mmol, 1.0 equiv). The resulting solution was then allowed to stir for 18 hours at room temperature, under N2 gas. After 18 hours, the reaction mixture was heated to reflux (80ºC) and was stirred at reflux for 3 hours under an atmosphere of N2 gas. Upon heating, methyl iodide (0.37 mL, 6.0 mmol, 3.0 equiv) was added in three batches (one per hour). After the 3 hours, the reaction mixture was allowed to cool to room temperature and then the solvent was removed in vacuo. The resulting crude residue was 244 then purified via silica gel column chromatography (DCM/MeOH) to yield the desired product as a yellow oil. (0.73 g, 73%). 1H NMR (500 MHz, CD3OD) δ 7.44 – 7.38 (m, 3H, (H33, H35, H37)), 7.32 – 7.25 (m, 2H, (H15, H19)), 7.20 (d, J = 8.6 Hz, 2H, (H24, H28)), 7.11 – 7.06 (m, 4H, (H16, H18, H34, H36)), 6.89 (d, J = 8.8 Hz, 2H, (H25, H27)), 4.85 (dd, J = 9.8, 8.2 Hz, 1H, (H4)), 4.74 (dd, J = 16.3, 4.3 Hz, 1H, (H3)), 4.69 – 4.61 (m, 1H, (H9 or 11(A))), 4.18 – 4.07 (m, 2H, (H8 or 12(A) , H9 or 11(B))), 3.94 (dd, J = 16.3, 3.3 Hz, 1H, (H3)), 3.82 – 3.77 (m, 1H, (H13)), 3.77 (s, 3H, (H31)), 3.76 (s, 2H, (H22)), 3.73 – 3.65 (m, 2H, (H7, H13)), 3.62 – 3.57 (m, 1H, (H9 or 11(B))), 3.20 – 3.10 (m, 1H, (H8 or 12(A))), 2.79 – 2.70 (m,1H, (H9 or 11(A))), 2.17 – 2.01 (m, 1H, (H8 or 12(B)), 1.56 – 1.43 (m, 1H, (H8 or 12(B))). 13C NMR (126 MHz, CD3OD) δ 174.03 (C21), 166.49 (C17), 160.15 (C26), 158.26 (C1), 143.02 (C32), 138.22 (C14), 130.81 (C34, C36), 130.70 (C24, C28), 130.45 (C33, C37), 130.07 (C35), 128.69 (C15, C19), 128.50 (C23), 116.76 (C16, C18), 115.26 (C25, C27), 64.04 (C4), 59.99 (C22), 55.67 (C31), 52.91 (C7), 51.00 (C9 or C11(B)), 46.36 (C3), 46.16 (C8 or C12(A)), 41.90 (C9 or C11(A)), 40.66 (C13), 33.06 (C8 or C12(B)). FTIR (cm-1): 3352, 3009, 2886, 1655, 1639, 1512, 1248, 1039. HRMS-ESI (m/z): Calculated for C30H33FN4O2 (M+H)+: 501.2666, Found: 501.2690. 245 3 2 N 4 NH 6 7 8 1 5 N 13 12 9 11 N 10 N O 14 15 19 16 18 17 OMe F 20 H N N N F 21 22 29 O 23 24 28 25 27 26 31 CH3 O 30 1-(4-((1-(4-fluorobenzyl)-4,5-dihydro-1H-imidazol-2-yl)amino)piperidin-1-yl)-2-(4- methoxyphenyl)ethan-1-one (3-3) To an oven-dried round bottom flask was added mono 4-fluorobenzylated ethylenediamine intermediate (3-12) (0.34 g, 2.0 mmol, 1.0 equiv) dissolved in anhydrous EtOH (10mL). At 20ºC, under an atmosphere of N2, was then added the isothiocyanate intermediate (3-16) (0.58 g, 2.0 mmol, 1.0 equiv). The resulting solution was then allowed to stir for 18 hours at room temperature, under N2 gas. After 18 hours, the reaction mixture was heated to reflux (80ºC) and was stirred at reflux for 3 hours under an atmosphere of N2 gas. Upon heating, methyl iodide (0.37 mL, 6.0 mmol, 3.0 equiv) was added in three batches (one per hour). After the 3 hours, the reaction mixture was allowed to cool to room temperature and then the solvent was removed in vacuo. The resulting crude residue was then purified via silica gel column chromatography (DCM/MeOH) to yield the desired product as a clear, colorless oil. (0.58 g, 68%). 1H NMR (500 MHz, CD3OD) δ 7.52 – 7.43 (m, 2H, (H15, H19)), 7.30 (d, J = 8.5 Hz, 2H, (H24, H28)), 7.24 (d, J = 8.6 Hz, 2H, (H25, H27)), 6.97 (t, J = 8.7 Hz, 2H, (H16, H18)), 5.47 (s, 2H, (H13)), 5.08 – 4.95 (m, 1H, (H7)), 4.87 – 4.78 (m, 1H, (H9 or H11(A))), 4.29 – 4.23 (m, 1H, (H9 or H11(B))), 3.88 – 3.77 (m, 2H, (H4)), 3.76 (s, 3H, (H31)), 3.75 – 3.69 (m, 4H, (H3, H22)), 3.30 – 3.23 (m, 1H, (H9 or H11(B))), 2.90 – 2.81 (m, 1H, (H9 or H11(A))), 2.54 – 2.43 (m, 1H, (H8 or H12(A))), 246 2.35 – 2.25 (m, 1H, (H8 or H12(B))), 2.11 – 2.05 (m, 1H, (H8 or H12(B))), 1.99 – 1.93 (m, 1H, (H8 or H12(A))). 13C NMR (126 MHz, CD3OD) δ 166.85 (C21), 162.49 (d, J = 54.3 Hz, (C17)), 160.15 (C26), 153.50 (C1), 131.66 (d, J = 3.4 Hz, (C14)), 130.79 (C24, C28), 130.14 (d, J = 8.1 Hz, (C15, C19)), 128.16 (C23), 116.72 (d, J = 22.4 Hz, (C16, C18)), 115.33 (C25, C27), 55.72 (C31), 54.55 (C4), 53.36 (C13), 50.93 (C3), 49.28 (C9 or C11(B)), 46.76 (C9 or C11(A)), 42.59 (C7), 40.75 (C22), 35.23 (C8 or C12(A)), 31.17 (C8 or C12(B)). FTIR (cm-1): 3344, 3011, 2882, 1659, 1638, 1506, 1255, 1042. HRMS-ESI (m/z): Calculated for C24H29FN4O2 (M+H)+: 425.2347, Found: 425.2361. O O N S 10 9 S 6 1 11 8 7 12 13 5 2 4 3 21 O N 14 20 17 CH3 O 15 16 CH318 CH319 tert-butyl 4-(benzylthio)piperidine-1-carboxylate (3-17) To an oven-dried flask purged with N2 gas was added tert-butyl 4-mercaptopiperidine-1- carboxylate (0.65 g, 3.0 mmol, 1.0 equiv) dissolved in anhydrous DMF (5mL). To this was then added K2CO3 (0.41 g, 3.0 mmol, 1.0 equiv) and benzyl bromide (0.36 mL, 3.0 mmol, 1.0 equiv) sequentially at room temperature under an atmosphere of N2 gas. The resulting mixture was then heated to 70ºC and was allowed to stir under N2 gas for 18 hours. After 18 hours, the reaction was cooled to room temperature, then diluted with H2O (10mL) and ethyl acetate (30mL) and was washed with saturated aqueous LiCl solution (3x30mL). Then organic layer was then extracted with ethyl acetate (3x30mL), dried over Na2SO4, filtered, and concentrated in vacuo. The resulting crude residue was then purified via silica gel column chromatography 247 (Hexanes/ethyl acetate) to yield the desired product as a clear, colorless oil (0.84 g, 91%). 1H NMR (500 MHz, CDCl3) δ 7.34 – 7.27 (m, 4H, (H9, H10, H12, H13)), 7.26 – 7.20 (m, 1H, (H11)), 4.00 – 3.84 (m, 2H, (H3 or H4)), 3.75 (s, 2H, (H7)), 2.86 (t, J = 11.3 Hz, 2H, (H3 or H4)), 2.88-2.65 (m, 1H, (H1)), 1.95 – 1.79 (m, 2H, (H2 or H5)), 1.57 – 1.45 (m, 2H, (H2 or H5)), 1.44 (s, 9H, (H17, H18, H19)). 13C NMR (126 MHz, CDCl3) δ 154.80 (C20), 138.50 (C8), 128.84 (C9, C13), 128.66 (C10, C12), 127.12 (C11), 79.64 (C16), 43.27 (C3, C4), 40.60 (C1), 34.69 (C7), 32.29 (C2, C5), 28.54 (C17, C18, C19). FTIR (cm-1): 3004, 2878, 1678, 1556, 1255, 1052. HRMS-ESI (m/z): Calculated for C17H25NO2S (M+H)+: 309.1762, Found: 309.1789. HCl•HN S ClH 15 5 1 NH 14 4 2 13 12 7 8 11 3 S 6 9 10 4-(benzylthio)piperidine hydrochloride (3-18) To an oven-dried flask was added tert-butyl 4-(benzylthio)piperidine-1-carboxylate intermediate (0.84 g, 2.7 mmol, 1.0 equiv) dissolved in anhydrous EtOH (5 mL). To this was then added HCl solution (4M in EtOH, 4.5 mL) at room temperature, under an atmosphere of N2 gas. The resulting reaction mixture was then stirred for 18 hours at rt under N2 gas. After 18 hours, the reaction was then concentrated in vacuo to afford the desired product as a pink crystalline solid (0.66 g, Quantitative). M.P.: 145-146ºC, 1H NMR (500 MHz, CD3OD) δ 7.38-7.33 (m, 2H, (H9, H13)), 7.30 (t, J = 7.5 Hz, 2H, (H10, H12)), 7.23 (t, J = 7.3 Hz, 1H, (H11)), 3.83 (s, 2H, (H7)), 3.40 – 3.35 (m, 2H, (H1 or H5)), 3.06 – 2.98 (m, 2H, (H1 or H5)), 2.93 – 2.84 (m, 1H, (H3)), 2.18 – 2.10 (m, 2H, (H2 or H4)), 1.81 – 1.70 (m, 2H, (H2 or H4)). 13C NMR (126 MHz, 248 CD3OD) δ 139.67 (C8), 129.92 (C9, C13), 129.57 (C10, C12), 128.11 (C11), 49.85 (C1 or C5), 45.18 (C1 or C5), 44.26 (C3), 38.75 (C7), 35.59 (C2 or C4), 30.25 (C2 or C4). FTIR (cm-1): 3332, 3032, 2836, 1509. HRMS-ESI (m/z): Calculated for C12H17NS (M+H)+: 208.1160, Found: 208.1180. O N S MeO O 24 21 17 16 CH325 22 23 18 O 20 19 15 N1 14 13 8 9 12 4 S 7 10 11 6 2 5 3 1-(4-(benzylthio)piperidin-1-yl)-2-(4-methoxyphenyl)ethan-1-one (3-19) To an oven-dried flask was added acyl chloride intermediate (0.18 g, 1.0 mmol, 1.0 equiv) in anhydrous DCM (2 mL). 4-(benzylthio)piperidine (0.23 g, 1.1 mmol, 1.1 equiv) in anhydrous DCM (3 mL) was then added dropwise to the solution at 0ºC under an atmosphere of N2 gas. Et3N (0.18 mL, 1.25 mmol, 1.25 equiv) was then also added dropwise at 0ºC under N2. The reaction was then allowed to warm to room temperature and was stirred for 18h at room temperature under N2 gas. The reaction was then quenched with H2O (10 mL), washed with saturated NaHCO3 solution (10 mL), extracted with DCM (3x30mL), dried over Na2SO4, filtered, and concentrated in vacuo. The resulting crude residue was then purified via silica gel column chromatography (DCM/MeOH) to yield the desired product as a clear, colorless oil (0.28 g, 79%). 1H NMR (500 MHz, CD3OD) δ 7.33 – 7.24 (m, 4H, (H10, H11, H13, H14)), 7.23 – 7.17 (m, 1H, (H12)), 7.14 (d, J = 8.7 Hz, 2H, (H19, H23)), 6.87 (d, J = 8.7 Hz, 2H, (H20, H22)), 4.26 – 4.18 (m, 1H, (H2 or H6(A))), 3.88 – 3.80 (m, 1H, (H2 or H6(B))), 3.75 (s, 3H, (H25)), 3.73 249 (s, 2H, (H8)), 3.67 (s, 2H, (H16)), 3.09 (td, J = 12.2, 3.1 Hz, 1H, (H2 or H6(B))), 2.89 (td, J = 12.1, 3.1 Hz, 1H, (H2 or H6(B))), 2.78 – 2.69 (m, 1H, (H4)), 1.93 – 1.84 (m, 1H, (H3 or H5(A))), 1.79 – 1.70 (m, 1H, (H3 or H5(B))), 1.43 – 1.31 (m, 1H, (H3 or H5(A))), 1.24 – 1.13 (m, 1H, (H3 or H5(B))). 13C NMR (126 MHz, CD3OD) δ 172.18 (C15), 160.04 (C21), 140.04 (C9), 130.64 (C19, C23), 129.89 (C11, C13), 129.47 (C10, C14), 128.25 (C17), 127.94 (C12), 115.16 (C20, C23), 55.67 (C25), 46.83 (C2 or C6(B)), 42.56 (C2 or C6(A)), 41.24 (C4), 40.62 (C16), 35.26 (C8), 33.69 (C3 or C5(B)), 33.16 (C3 or C5(A)). FTIR (cm-1): 3013, 2882, 1654, 1515, 1252, 1039. HRMS-ESI (m/z): Calculated for C21H25NO2S (M+H)+: 356.1684, Found: 356.1701. O 11 O 13 12 8 N1 MeO N SH O 17 14 10 9 CH318 15 16 6 2 5 3 4 SH 7 1-(4-mercaptopiperidin-1-yl)-2-(4-methoxyphenyl)ethan-1-one (3-20) To an oven-dried flask was added 1-(4-(benzylthio)piperidin-1-yl)-2-(4-methoxyphenyl)ethan-1- one (0.37 g, 1.0 mmol, 1.0 equiv) dissolved in EtOH (5mL). To this was then added Pd(OH)2/C (10 wt%, 1 scoop) at rt, under an atmosphere of H2 gas (utilizing an H2 balloon). The reaction was then allowed to stir for 24 hours under H2 gas. After 24 hours, the reaction was then worked up via filtration through a celite pad and washed with MeOH (400mL) to remove the excess Pd(OH)2/C. The resulting filtrate was then concentrated in vacuo to afford the desired product as a clear, pale-yellow oil (0.27 g, Quantitative). 1H NMR (500 MHz, CD3OD) δ 7.17 (d, J = 7.8 Hz, 2H, (H12, H16)), 6.88 (d, J = 7.6 Hz, 2H, (H13, H15)), 3.77 (s, 3H, (H18)), 3.70 (s, 2H, (H9)), 3.57 – 3.52 (m, 2H, (H2 or H6)), 3.53 – 3.43 (m, 2H, (H2 or H6)), 3.22 – 3.18 (m, 1H, 250 (H4)), 1.65 – 1.44 (m, 2H, (H3 or H5)), 1.39 – 1.27 (m, 2H, (H3 or H5)). 13C NMR (126 MHz, CD3OD) δ 172.15 (C8), 159.96 (C14), 130.61 (C12, C16), 128.39 (C10), 115.09 (C13, C15), 58.30 (C25), 55.69 (C2 or C6), 48.52 (C2 or C6), 44.11 (C4), 40.68 (C9), 27.20 (C3 or C5), 26.62 (C3 or C5). FTIR (cm-1): 3050, 2885, 2575, 1653, 1511, 1244, 1048. HRMS-ESI (m/z): Calculated for C14H19NO2S (M+H)+: 266.1215, Found: 266.1233. 5 4 F 17 18 16 19 1 2 3 N N 12 13 8 F 11 7 9 10 6 14 S CH315 N N S 1-(4-fluorobenzyl)-2-(methylthio)-1H-benzo[d]imidazole (3-21) To an oven-dried flask purged with N2 gas was added 2-(methylthio)-1λ2-benzo[d]imidazole (0.82 g, 5.0 mmol, 1.0 equiv) dissolved in anhydrous DMF (5mL). To this was then added K2CO3 (0.69 g, 5.0 mmol, 1.0 equiv) and 4-fluorobenzyl bromide (0.62 mL, 5.0 mmol, 1.0 equiv) sequentially at room temperature under an atmosphere of N2 gas. The resulting mixture was then allowed to stir at room temperature under N2 gas for 24 hours. After 24 hours, the reaction was then diluted with H2O (10 mL) and ethyl acetate (30 mL) and was washed with saturated aqueous LiCl solution (3x30mL). Then organic layer was then extracted with ethyl acetate (3x30mL), dried over Na2SO4, filtered, and concentrated in vacuo. The resulting crude residue was then purified via silica gel column chromatography (MeOH (1% Et3N)/DCM) to yield the desired product as a white, crystalline solid (1.02 g, 75%). M.P.: 99-100ºC, 1H NMR (500 MHz, CD3OD) δ 7.59 (d, J = 6.8 Hz, 1H, (H16)), 7.33 (d, J = 6.9 Hz, 1H, (H19)), 7.25 – 251 7.15 (m, 4H, (H6, H10, H17, H18)), 7.03 (t, J = 8.8 Hz, 2H, (H7, H9)), 5.34 (s, 2H, (H4)), 2.75 (s, 3H, (H15)). 13C NMR (126 MHz, CD3OD) δ 163.75 (d, J = 245.6 Hz, (C8)), 154.71 (C13), 144.21 (C2), 137.27 (C1), 133.29 (d, J = 2.9 Hz, (C5)), 130.10 (d, J = 8.1 Hz, (C6, C10)), 123.58 (C17), 123.40 (C18), 118.44 (C19), 116.58 (d, J = 21.9 Hz, (C7, C9)), 110.65 (C16), 47.62 (C4), 14.98 (C15). FTIR (cm-1): 3052, 2867, 1501. HRMS-ESI (m/z): Calculated for C15H13FN2S (M+H)+: 273.0862, Found: 273.0887. N N S N F 2 3 1 4 6 5 9 N N 7 8 10 S 11 16 17 12 15 19 18 20 23 21 22 O F 24 14 N 13 26 28 25 O 33 27 32 31 29 30 OMe O 34 CH335 1-(4-((1-(4-fluorobenzyl)-1H-benzo[d]imidazol-2-yl)thio)piperidin-1-yl)-2-(4- methoxyphenyl)ethan-1-one (3-4) Procedure A To an oven-dried flask was added the 1-(4-fluorobenzyl)-2-(methylthio)-1H-benzo[d]imidazole intermediate (3-21) (54.5 mg, 0.2 mmol, 1.0 equiv) dissolved in anhydrous DCM (3mL). This solution was then cooled to 0ºC. At 0ºC, under an atmosphere of N2 gas was added m-CPBA (75% mixture, 0.11 g, 0.50 mmol, 2.5 equiv), portion-wise. This was then allowed to stir for one hour at 0ºC under N2 gas. After 1h, the reaction mixture was then quenched with saturated 252 aqueous NaHCO3 solution (5mL), extracted with DCM (3x20mL), dried over Na2SO4, filtered, and concentrated in vacuo. The resulting yellowish crude product was then immediately dissolved in a mixture of anhydrous toluene (5mL). To this was then added the acylated thio- piperidine tail intermediate (3-20) (0.27 g, 1.0 mmol, 5.0 equiv) at room temperature, under an atmosphere of N2 gas. The resulting solution was then heated to reflux (~110ºC) and was allowed to stir for 18 hours under N2 gas. After 18 hours, the reaction was allowed to cool to room temperature and was quenched with H2O (1mL), then was diluted in MeOH (100mL) and concentrated in vacuo. The resulting crude residue was then purified via silica gel column chromatography (DCM/MeOH) to yield the desired product as a pale yellowish/off-white semi- solid (0.066 g, 67%). Procedure B To an oven dried round bottom flask was added 1-(4-fluorobenzyl)-2-(piperidin-4-ylthio)-1H- benzo[d]imidazole hydrochloride intermediate (3-24) (0.38 g, 1.0 mmol, 1.0 equiv) dissolved in anhydrous DCM (10 mL). This solution was first neutralized to pH~7 using anhydrous Et3N. The resulting solution was then cooled to 0ºC. Then, at 0ºC under an atmosphere of N2 gas, was added Et3N (0.17 mL, 1.25 mmol, 1.25 equiv). Next, at 0ºC, under N2 gas, was added 2-(4- methoxyphenyl)acetyl chloride (3-13) (0.18 g, 1.0 mmol, 1.0 equiv) dropwise in anhydrous DCM (3 mL). The resulting solution was then allowed to warm to 20ºC and was stirred for 18 hours at 20ºC under N2 gas. After 18 hours, the reaction was then quenched with H2O (20 mL), extracted with DCM (3x30mL), dried over Na2SO4, filtered, and concentrated in vacuo. The resulting crude residue was then purified via silica gel column chromatography (DCM/MeOH) to yield the desired product as a yellowish semi-solid. (0.45 g, 91%). 1H NMR (500 MHz, CD3OD) δ 7.60 (dd, J = 7.2, 1.6 Hz, 1H, (H4)), 7.34 (dd, J = 7.2, 1.6 Hz, 1H, (H1)), 7.23 (ddd, J = 7.4, 253 6.2, 1.5 Hz, 1H, (H3)), 7.20 (ddd, J = 7.4, 6.2, 1.5 Hz, 1H, (H2)), 7.17 – 7.10 (m, 4H, (H19, H23, H28, H32)), 7.00 (t, J = 8.7 Hz, 2H, (H20, H22)), 6.84 (d, J = 8.7 Hz, 2H, (H29, H31)), 5.35 (s, 2H, (H17)), 4.33 – 4.25 (m, 1H, (H13, H15(A))), 3.97 – 3.82 (m, 2H, (H11, H13, H15(B))), 3.73 (s, 3H, (H35)), 3.67 (d, J = 5.7 Hz, 2H, (H26)), 3.26 – 3.17 (m, 1H, (H13, H15(B))), 3.04 – 2.95 (m, 1H, (H13, H15(A))), 2.12 – 2.03 (m, 1H, (H12, H16(A))), 1.99 – 1.90 (m, 1H, (H12, H16(B))), 1.60 – 1.49 (m, 1H, (H12, H16(A))), 1.39 – 1.25 (m, 1H, (H12, H16(B))). 13C NMR (126 MHz, CD3OD) δ 172.21 (C25), 163.66 (d, J = 245.1 Hz, (C21)), 160.00 (C30), 151.22 (C8), 144.14 (C6), 136.83 (C5), 133.38 (d, J = 3.3 Hz, (C18)), 130.65 (C28, C32), 129.97 (d, J = 8.1 Hz, (C19, C23)), 128.12 (C27), 124.01 (C3), 123.70 (C2), 118.85 (C4), 116.58 (d, J = 21.9 Hz, (C20, C22)), 115.04 (C29, C31), 111.07 (C1), 55.66 (C35), 47.74 (C17), 46.76 (C13, C15(B)), 44.98 (C11), 42.55 (C13, C15(A)), 40.60 (C26), 33.67 (C12, C16(B)), 33.04 (C12, C16(A)). FTIR (cm-1): 3027, 2893, 1658, 1517, 1243, 1042. HRMS-ESI (m/z): Calculated for C28H28FN3O2S (M+H)+: 490.1964, Found: 490.1979. 2 3 1 4 6 5 9 N NH 7 8 10 S 11 15 12 14 16 N 13 S N N H NBoc 22 O 23 17 O 18 CH319 CH321 CH320 tert-butyl 4-((1H-benzo[d]imidazol-2-yl)thio)piperidine-1-carboxylate (3-22) 254 To an oven-dried round bottom flask was added containing a solution of tert-butyl 4- ((methylsulfonyl)oxy)piperidine-1-carboxylate (0.56 g, 2.0 mmol, 1.0 equiv) and 2-mercapto- benzimidazole (0.30 g, 2.0 mmol, 1.0 equiv) in anhydrous EtOH (10 mL) was added K2CO3 (0.28 g, 2.0 mmol, 1.0 equiv) at room temperature, under an atmosphere of N2 gas. The resulting reaction mixture was then heated to reflux (80ºC) and was allowed to stir for 18 hours under an atmosphere of N2 gas. After 18 hours, the reaction was allowed to cool to room temperature and was poured into saturated aqueous NH4Cl solution (30 mL), followed by extraction with EtOAc (3x30mL). The extract was then washed with brine (25 mL), dried over Na2SO4 and concentrated in vacuo. The resulting precipitate was then collected via filtration after suspension in a mixed solvent of EtOAc/MeOH to afford the desired product as a white solid. (0.31 g, 46%). M.P.: 240ºC (decomp.), 1H NMR (500 MHz, CDCl3) δ 9.08 (s, 1H, (H7)), 7.69 (d, J = 7.2 Hz, 1H, (H1)), 7.35 (d, J = 7.4 Hz, 1H, (H4)), 7.26 – 7.17 (m, 2H, (H2, H3)), 4.06 – 3.99 (m, 1H, (H11)), 3.97 – 3.94 (m, 2H, (H13, H14)), 3.10 – 3.01 (m, 2H, (H13, H14)), 2.19 – 2.08 (m, 2H, (H12, H15)), 1.74 – 1.63 (m, 2H, (H12, H15)), 1.46 (s, 9H, (H19, H20, H21)). 13C NMR (126 MHz, CDCl3) δ 159.35 (C22), 154.83 (C8), 148.41 (C6), 145.52 (C5), 122.96 (C2), 122.30 (C3), 118.70 (C1), 114.91 (C2), 79.92 (C18), 44.17 (C13, C14), 43.84 (C11), 32.56 (C12, C15), 28.57 (C19, C20, C21). FTIR (cm-1): 3423, 3051, 2890, 1688, 1244. HRMS-ESI (m/z): Calculated for C17H23N3O2S (M+H)+: 334.1589, Found: 334.1595. 255 2 3 1 4 6 5 9 N N 7 8 10 S 11 15 16 12 14 N N S 18 17 19 22 20 21 NBoc F 23 24 N 13 30 O 31 25 O 26 CH327 CH329 CH328 F tert-butyl 4-((1-(4-fluorobenzyl)-1H-benzo[d]imidazol-2-yl)thio)piperidine-1-carboxylate (3- 23) To an oven-dried round bottom flask was added tert-butyl 4-((1H-benzo[d]imidazol-2- yl)thio)piperidine-1-carboxylate (0.67 g, 1.5 mmol, 1.0 equiv) dissolved in anhydrous DMF (10 mL) at 20ºC under an atmosphere of N2 gas. To this was then added K2CO3 (0.21 g, 1.5 mmol, 1.0 equiv) and 4-fluoro benzyl bromide (0.18 mL, 1.5 mmol, 1.0 equiv) sequentially at 20ºC under N2 gas. The resulting reaction mixture was then allowed to stir at 20ºC for 18 hours under N2 gas. After 18 hours, the reaction was quenched with DI H2O (20 mL), washed with saturated aqueous LiCl solution (3x30 mL), extracted with EtOAc (3x30 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The resulting crude residue was then purified via silica gel column chromatography (DCM/MeOH) to yield the desired product as a clear pale-yellow oil. (0.81 g, 92%).1H NMR (500 MHz, CD3OD) δ 7.61 (d, J = 6.6 Hz, 1H, (H4)), 7.40 (d, J = 6.6 Hz, 1H, (H1)), 7.27 – 7.22 (m, 2H, (H2, H3)), 7.20 (dd, J = 8.8, 5.3 Hz, 2H, (H19, H21)), 7.04 (t, J = 8.7 Hz, 2H, (H18, H22)), 5.43 (s, 2H, (H16)), 3.99 – 3.90 (m, 3H, (H11, H13, H14)), 3.16 – 2.94 (m, 2H, (H13, H14)), 2.13 – 2.05 (m, 2H, (H12, H15)), 1.65 – 1.56 (m, 2H, (, H12, H15)), 1.45 (s, 9H, (H27, H28, H29)). 13C NMR (126 MHz, CD3OD) δ 163.73 (d, J = 245.1 Hz, (C20)), 256 156.26 (C30), 151.46 (C8), 144.20 (C6), 136.91 (C5), 133.56 (d, J = 3.3 Hz, (C17)), 130.05 (d, J = 8.1 Hz, (C19, C21)), 124.01 (C3), 123.70 (C2), 118.85 (C1), 116.61 (d, J = 21.9 Hz, (C18, C22)), 111.11 (C4), 81.22 (C26), 54.84 (C16), 47.78 (C11), 45.33 (C13, C14), 33.08 (C12, C15), 28.64 (C27, C28, C29). FTIR (cm-1): 3076, 2899, 1685, 1237. HRMS-ESI (m/z): Calculated for C24H28FN3O2S (M+H)+: 442.1964, Found: 442.1978. N N S 2 3 1 4 6 5 9 N N 7 8 10 S 11 15 16 12 14 NH•HCl 18 17 19 22 20 21 F 23 13 NH 24 ClH 25 F 1-(4-fluorobenzyl)-2-(piperidin-4-ylthio)-1H-benzo[d]imidazole hydrochloride (3-24) To an oven-dried flask was added tert-butyl 4-((1-(4-fluorobenzyl)-1H-benzo[d]imidazol-2- yl)thio)piperidine-1-carboxylate (0.44 g, 1.0 mmol, 1.0 equiv) and ethanolic HCl (3.3M in EtOH, 5 mL). This was stirred at 20ºC under an atmosphere of N2 gas for 18 hours. After completion, as indicated by TLC, the reaction mixture was concentrated in vacuo to obtain a semi-solid. This semi-solid was triturated with Et2O (5 mL) and concentrated in vacuo to afford the crude product as a yellow-red foaming solid. This was used in subsequent steps without any further purification (>99% determined by NMR, Quantitative). 1H NMR (500 MHz, CD3OD) δ 7.72 – 7.64 (m, 1H, (H4)), 7.51 – 7.39 (m, 1H, (H1)), 7.26 – 7.18 (m, 2H, (H2, H3)), 7.02 – 6.99 (m, 2H, (H19, H21)), 6.79 – 6.69 (m, 2H, (H18, H22)), 5.37 (s, 2H, (H16)), 4.03 – 3.76 (m, 1H, (H11)), 3.13 – 3.01 (m, 2H, (H13, H14)), 2.88 – 2.77 (m, 2H, (H13, H14)), 2.03 – 1.99 (m, 2H, (H12, H15)), 257 1.71 – 1.68 (m, 2H, (H12, H15)). 13C NMR (126 MHz, CD3OD) δ 164.21 (d, J = 240.3 Hz, (C20)), 149.63 (C8), 134.28 (C6), 133.33 (C5), 131.09 (d, J = 8.6 Hz, (C17)), 129.77 (C3), 128.14 (d, J = 31.9 Hz, (C19, C21)), 126.91 (C2), 117.20 (d, J = 21.9 Hz, (C18, C22)), 115.17 (C1), 114.12 (C4), 50.49 (C16), 44.76 (C13, C14), 37.85 (C11), 32.36 (C12, C15). FTIR (cm- 1): 3331, 3032, 2883, 1517. HRMS-ESI (m/z): Calculated for C19H20FN3S (M+H)+: 342.1440, Found: 342.1466. 2 3 1 4 6 5 9 N S 7 8 10 S 11 15 12 14 16 N 13 N S S NBoc 22 O 23 17 O 18 CH319 CH321 CH320 tert-butyl 4-(benzo[d]thiazol-2-ylthio)piperidine-1-carboxylate (3-25) To an oven-dried round bottom flask was added containing a solution of tert-butyl 4- ((methylsulfonyl)oxy)piperidine-1-carboxylate (0.56 g, 2.0 mmol, 1.0 equiv) and 2-mercapto- benzothiazole (0.33 g, 2.0 mmol, 1.0 equiv) in anhydrous EtOH (15 mL) was added K2CO3 (0.28 g, 2.0 mmol, 1.0 equiv) at room temperature, under an atmosphere of N2 gas. The resulting reaction mixture was then heated to reflux (80ºC) and was allowed to stir for 18 hours under an atmosphere of N2 gas. After 18 hours, the reaction was allowed to cool to room temperature and was poured into saturated aqueous NH4Cl solution (30 mL), followed by extraction with EtOAc (3x30mL). The extract was then washed with brine (20 mL), dried over Na2SO4 and concentrated 258 in vacuo. The resulting crude residue was then purified via silica gel column chromatography (DCM/MeOH) to yield the desired product as a clear pale orange-pink solid. (0.62 g, 88%). M.P.: 148 – 150ºC. 1H NMR (500 MHz, CDCl3) δ 7.88 (d, J = 8.1 Hz, 1H, (H4)), 7.76 (d, J = 8.0 Hz, 1H, (H1)), 7.42 (td, J = 8.3, 7.2, 1.3 Hz, 1H, (H2)), 7.31 (td, J = 8.2, 7.2, 1.2 Hz, 1H, (H3)), 4.16 – 4.05 (m, 1H, (H11)), 3.96 (s, 2H, (H13, H14)), 3.16 – 3.08 (m, 2H, (H13, H14)), 2.23 – 2.14 (m, 2H, (H12, H15)), 1.79 – 1.68 (m, 2H, (H12, H15)), 1.47 (s, 9H, (H19, H20, H21)). 13C NMR (126 MHz, CDCl3) δ 165.26 (C8), 154.80 (C22), 153.41 (C6), 135.43 (C5), 126.21 (C2), 124.56 (C3), 121.79 (C1), 121.13 (C4), 79.93 (C18), 44.74 (C13, C14), 32.20 (C11), 28.63 (C12, C15), 28.57 (C19, C20, C21). FTIR (cm-1): 3021, 2893, 1672, 1505, 1221. HRMS-ESI (m/z): Calculated for C12H22N2O2S2Na (M+H)+: 374.1099, Found: 374.1102. 2 3 1 4 6 5 9 N S 7 8 10 S 11 15 12 14 13 NH 16 N S S NH•HCl ClH 17 2-(piperidin-4-ylthio)benzo[d]thiazole hydrochloride (3-26) To an oven-dried flask was added tert-butyl 4-(benzo[d]thiazol-2-ylthio)piperidine-1-carboxylate (0.60 g, 1.8 mmol, 1.0 equiv) and ethanolic HCl (3.3M in EtOH, 5 mL). This was stirred at 20ºC under an atmosphere of N2 gas for 18 hours. After completion, as indicated by TLC, the reaction mixture was concentrated in vacuo to obtain a semi-solid. This semi-solid was triturated with Et2O (5 mL) and concentrated in vacuo to afford the crude product quantitatively as an off-white foaming solid. This was used in subsequent steps without any further purification (>99% 259 determined by NMR, Quantitative). 1H NMR (500 MHz, CD3OD) δ 7.90 (d, J = 8.0 Hz, 1H, (H4)), 7.86 (d, J = 8.1 Hz, 1H, (H1)), 7.48 (td, J = 8.2, 7.2, 1.3 Hz, 1H, (H2)), 7.38 (td, J = 8.3, 7.4, 1.3 Hz, 1H, (H3)), 4.32 – 4.16 (m, 1H, (H11)), 3.54 – 3.44 (m, 2H, (H13, H14)), 3.29 – 3.25 (m, 2H, (H13, H14)), 2.54 – 2.45 (m, 2H, (H12, H15)), 2.15 – 2.03 (m, 2H, (H12, H15)). 13C NMR (126 MHz, CD3OD) δ 165.88 (C8), 154.35 (C6), 136.44 (C5), 127.53 (C2), 126.03 (C3), 122.51 (C1), 122.48 (C4), 44.50 (C13, 14), 42.54 (C11), 30.03 (C12, C15). FTIR (cm-1): 3311, 3002, 2875, 1505. HRMS-ESI (m/z): Calculated for C12H14N2S2 (M+H)+: 251.0677, Found: 251.0686. N S S N O 2 3 1 4 6 5 9 N S 7 8 10 S 11 16 12 15 14 N 13 18 20 17 O 25 19 24 23 21 22 OMe O 26 CH327 1-(4-(benzo[d]thiazol-2-ylthio)piperidin-1-yl)-2-(4-methoxyphenyl)ethan-1-one (3-5) To an oven dried round bottom flask was added 2-(piperidin-4-ylthio)benzo[d]thiazole hydrochloride intermediate (3-26) (0.49 g, 1.8 mmol, 1.0 equiv) dissolved in anhydrous DCM (10 mL). This solution was first neutralized to pH~7 using anhydrous Et3N. The resulting solution was then cooled to 0ºC. Then, at 0ºC under an atmosphere of N2 gas, was added Et3N (0.31 mL, 2.3 mmol, 1.25 equiv). Next, at 0ºC, under N2 gas, was added 2-(4- 260 methoxyphenyl)acetyl chloride (3-13) (0.32 g, 1.8 mmol, 1.0 equiv) dropwise in anhydrous DCM (1 mL). The resulting solution was then allowed to warm to 20ºC and was stirred for 18 hours at 20ºC under N2 gas. After 18 hours, the reaction was then quenched with H2O (20 mL), extracted with DCM (3x30mL), dried over Na2SO4, filtered, and concentrated in vacuo. The resulting crude residue was then purified via silica gel column chromatography (DCM/MeOH) to yield the desired product as a white semi-solid. (0.49 g, 72%). 1H NMR (500 MHz, CD3OD) δ 7.85 (d, J = 8.0 Hz, 1H, (H4)), 7.82 (d, J = 8.1 Hz, 1H, (H1)), 7.45 (t, J = 7.6 Hz, 1H, (H2)), 7.35 (t, J = 7.6 Hz, 1H, (H3)), 7.17 (d, J = 8.3 Hz, 2H, (H21, H23)), 6.88 (d, J = 8.4 Hz, 2H, (H20, H24)), 4.38 – 4.29 (m, 1H, (H13 or H15(A))), 4.13 – 4.05 (m, 1H, (H11)), 3.99 – 3.92 (m, 1H, (H13 or H15(B))), 3.76 (s, 3H, (H27)), 3.72 (d, J = 2.6 Hz, 2H, (H18)), 3.35 – 3.29 (m, 1H, (H13 or H15(B))), 3.15 – 3.01 (m, 1H, (H13 or H15(A))), 2.25 – 2.16 (m, 1H, (H12 or H16(A))), 2.12 – 2.04 (m, 1H, (H12 or H16(B))), 1.72 – 1.59 (m, 1H, (H12 or H16(A))), 1.52 – 1.41 (m, 1H, (H12 or H16(B))). 13C NMR (126 MHz, CD3OD) δ 172.38 (C17), 166.83 (C8), 160.12 (C22), 154.37 (C6), 136.37 (C5), 130.70 (C21, C23), 128.19 (C19), 127.45 (C2), 125.86 (C3), 122.38 (C1), 122.36 (C4), 115.21 (C20, C24), 55.68 (C27), 46.87 (C13 or C15(B)), 45.35 (C11), 42.64 (C13 or C15(A)), 40.62 (C18), 33.53 (C12 or C16(B)), 32.97 (C12 or C16(A)). FTIR (cm-1): 3076, 2893, 1656, 1510, 1255, 1044. HRMS-ESI (m/z): Calculated for C21H22N2O2S2 (M+H)+: 399.1201, Found: 399.1221. 261 NH N N H N O 2 3 1 4 6 5 9 N NH 7 10 NH 8 11 16 12 15 14 N 13 18 20 17 O 25 19 24 23 21 22 OMe O 26 CH327 1-(4-((1H-benzo[d]imidazol-2-yl)amino)piperidin-1-yl)-2-(4-methoxyphenyl)ethan-1-one – (3-6) In an oven-dried flask, 2-chloro-1H-benzo[d]imidazole (0.15 g, 1.0 mmol, 1.0 equiv) was dissolved in anhydrous dimethylacetamide (DMA) (5 mL). To this was then added the neutralized acylated 4-amino piperidine tail (3-15) (1.24 g, 5.0 mmol, 5.0 equiv). The resulting mixture was then heated to 140ºC and was allowed to stir for 48 hours under an atmosphere of nitrogen gas. After 48 hours, the solution was allowed to cool to room temperature and was diluted with EtOAc (30 mL), washed with saturated aqueous LiCl solution (2x30mL), extracted with EtOAc (3x30mL), dried over Na2SO4, filtered, and concentrated in vacuo. The resulting crude residue was then purified via silica gel column chromatography (DCM/MeOH) to yield the desired product as a clear, colorless oil. (0.20 g, 54%). 1H NMR (500 MHz, CD3OD) δ 7.40 – 7.33 (m, 2H, (H1, H4)), 7.27 – 7.22 (m, 2H, (H2, H3)), 7.20 (d, J = 8.0 Hz, 2H, (H21, H23)), 6.89 (d, J = 8.1 Hz, 2H, (H20, H24)), 4.56 – 4.50 (m, 1H, (H13 or H15(A))), 4.15 – 4.00 (m, 1H, (H13 or H15(B))), 3.91 – 3.80 (m, 1H, (H11)), 3.79 – 3.74 (m, 5H, (H18, H27)), 3.31 – 3.23 (m, 1H, (H13 or H15(B))), 2.98 – 2.86 (m, 1H, (H13 or H15(A)), 2.02 – 1.95 (m, 1H, (H12 or 262 H16(A))), 1.89 – 1.80 (m, 1H, (H12 or H16(B))), 1.53 – 1.41 (m, 1H, (H12 or H16(A))), 1.36 – 1.32 (m, 1H, (H12 or H16(B))). 13C NMR (126 MHz, CD3OD) δ 172.42 (C17), 160.09 (C22), 150.80 (C8), 131.75 (C5, C6), 130.74 (C21, C23), 128.21 (C19), 124.50 (C2, C3), 115.22 (C20, C24), 112.42 (C1, C4), 55.70 (C27), 51.91 (C11), 45.97 (C13 or C15(B)), 41.59 (C13 or C15(A)), 40.64 (C18), 33.02 (C12 or C16(B)), 32.44 (C12 or C16(A)). FTIR (cm-1): 3422, 3348, 3025, 2879, 1661, 1530, 1252, 1040. HRMS-ESI (m/z): Calculated for C21H24N4O2 (M+H)+: 365.1978, Found: 365.1998. N N Cl 2 3 1 4 6 5 9 N N 7 8 Cl 10 11 13 12 14 17 15 16 1-benzyl-2-chloro-1H-benzo[d]imidazole (3-27) To an oven-dried round bottom flask was added 2-chloro-1H-benzimidazole (0.50 g, 3.3 mmol, 1.0 equiv) dissolved in anhydrous DMSO (3 mL). Then NaH (0.25 g (60 wt% in mineral oil), 6.6 mmol, 2.0 equiv) at room temperature. The resulting reaction mixture was then allowed to stir for one hour at room temperature under an atmosphere of N2 gas. After one hour, benzyl bromide (0.51mL, 4.3 mmol, 1.3 equiv) was added at room temperature under N2 gas and the reaction was allowed to stir for 18 hours. After 18 hours the reaction was then quenched slowly with ice cold H2O (20 mL) and a white precipitate then formed. The white precipitate was then collected via vacuum filtration to afford the desired product as a white solid (0.70 g, 88%). M.P.: 106- 263 108ºC, 1H NMR (500 MHz, CD3OD) δ 7.61 (dd, J = 6.2, 3.1 Hz, 1H, (H4)), 7.44 (dd, J = 6.1, 3.2 Hz, 1H, (H1)), 7.37 – 7.23 (m, 5H, (H2, H3, H14, H15, H16)), 7.19 (d, J = 7.1 Hz, 2H, (H13, H17)), 5.49 (s, 2H, (H11)). 13C NMR (126 MHz, CD3OD) δ 142.26 (C6), 141.98 (C8), 136.83 (C12), 136.34 (C5), 130.01 (C14, C16), 129.17 (C15), 127.96 (C13, C17), 124.85 (C3), 124.33 (C2), 119.51 (C4), 111.74 (C1), 48.73 (C11). FTIR (cm-1): 3009, 2875, 1508. HRMS- ESI (m/z): Calculated for C14H11ClN2 (M+H)+, (Cl35); (Cl37): 243.0689; 245.0663, Found: 243.0705; 245.0683. 2 3 1 4 6 5 9 N N 7 8 Cl 10 11 12 13 18 14 17 15 16 F 19 N N Cl F 2-chloro-1-(4-fluorophenethyl)-1H-benzo[d]imidazole (3-28) In an oven dried flask, 2-chloro-1H-benzimidazole (0.31 g, 2.0 mmol, 1.0 equiv) was dissolved in anhydrous DMF (5 mL). To this was then sequentially added K2CO3 (0.28 g, 2.0 mmol, 1.0 equiv) and 1-(2-bromoethyl)-4-fluorobenzene (0.41 g, 2.0 mmol, 1.0 equiv) at room temperature. This resulting solution was allowed to stir for 24 hours at room temperature, under an atmosphere of nitrogen gas. After 24 hours, the reaction mixture was diluted in EtOAc (30 mL), washed with saturated aqueous LiCl solution (2x30mL), extracted with EtOAc (3x30mL), dried over Na2SO4, filtered, and concentrated in vacuo. The resulting crude residue was then purified 264 via silica gel column chromatography (DCM/MeOH) to yield the desired product as a white solid. (0.41 g, 74%). M.P.: 70 - 71ºC, 1H NMR (500 MHz, CDCl3) δ 7.72 – 7.67 (m, 1H, (H4)), 7.30 – 7.24 (m, 2H, (H2, H3)), 7.20 – 7.17 (m, 1H, (H1)), 7.01 (dd, J = 8.5, 5.5 Hz, 2H, (H15, H17)), 6.94 (t, J = 8.6 Hz, 2H, (H14, H18)), 4.38 (t, J = 7.2 Hz, 2H, (H11)), 3.08 (t, J = 7.2 Hz, 2H, (H12)). 13C NMR (126 MHz, CDCl3) δ 162.12 (d, J = 245.6 Hz, (C16)), 141.77 (C6), 140.61 (C8), 134.84 (C5), 132.89 (d, J = 3.3 Hz, (C13)), 130.40 (d, J = 8.1 Hz, (C15, C17)), 123.30 (C3), 122.84 (C2), 119.68 (C4), 115.80 (d, J = 21.5 Hz, (C14, C18)), 109.36 (C1), 46.09 (C11), 34.90 (C12). FTIR (cm-1): 3022, 2884, 1514. HRMS-ESI (m/z): Calculated for C15H12ClFN2 (M+H)+, (Cl35); (Cl37): 275.0751; 277.0762, Found: 275.0758; 277.0770. 2 3 1 4 6 5 9 N N 7 8 NH210 11 16 12 15 13 14 F 17 N N NH2 F 1-(4-fluorophenyl)-1H-benzo[d]imidazol-2-amine (3-29) To an oven-dried round bottom flask was added 1H-benzo[d]imidazol-2-amine (0.40 g, 3.0 mmol, 1.0 equiv) at room temperature in anhydrous EtOH (5 mL). To this was then added (4- fluorophenyl)boronic acid (0.42 g, 3.0 mmol, 1.0 equiv) and Cu(OTf)2 (0.22 g, 0.6 mmol, 0.2 equiv) under an atmosphere of air (air balloon). The resulting reaction mixture was then allowed to stir for 24 hours at room temperature under an atmosphere of air. After 24 hours, the reaction mixture was concentrated in vacuo and then was immediately purified via silica gel column 265 chromatography (DCM/MeOH (2% Et3N)) to yield the desired product an off-white solid (0.50 g, 73%). M.P.: 125-127ºC, 1H NMR (500 MHz, CD3OD) δ 7.52 (dd, J = 8.9, 4.9 Hz, 2H, (H13, H15)), 7.38 (t, J = 8.7 Hz, 2H, (H12, H16)), 7.30 (d, J = 7.8 Hz, 1H, (H4)), 7.10 (t, J = 7.6 Hz, 1H, (H2)), 6.99 (t, J = 7.6 Hz, 1H, (H3)), 6.87 (d, J = 7.9 Hz, 1H, (H1)). 13C NMR (126 MHz, CD3OD) δ 164.04 (d, J = 247.5 Hz, (C14)), 155.57 (C8), 143.58 (C6), 141.91 (C5), 132.03 (d, J = 3.0 Hz, (C11)), 130.60 (d, J = 9.1 Hz, (C13, C15)), 123.32 (C2), 121.41 (C3), 118.25 (d, J = 22.9 Hz, (C12, C16)), 115.86 (C4), 109.27 (C1). FTIR (cm-1): 3377, 3315, 3041, 2881, 1524. HRMS-ESI (m/z): Calculated for C13H10FN3 (M+H)+: 228.0937, Found: 228.0954. 29 CH3 30 CH3 27 CH328 25 O 24 O 26 21 N 20 19 22 18 23 8 NH 10 NBoc 2 3 1 4 6 5 9 N N 7 11 16 12 15 13 14 F 17 N N NH F tert-butyl 4-((1-(4-fluorophenyl)-1H-benzo[d]imidazol-2-yl)amino)piperidine-1-carboxylate (3-30) To an oven-dried round bottom flask was added containing a solution of tert-butyl 4- ((methylsulfonyl)oxy)piperidine-1-carboxylate (0.14 g, 0.5 mmol, 1.0 equiv) and 1-(4- fluorophenyl)-1H-benzo[d]imidazol-2-amine (3-29) (0.11 g, 0.5 mmol, 1.0 equiv) in anhydrous 266 EtOH (10 mL) was added K2CO3 (69.1 mg, 0.5 mmol, 1.0 equiv) at room temperature, under an atmosphere of N2 gas. The resulting reaction mixture was then heated to reflux (80ºC) and was allowed to stir for 18 hours under an atmosphere of N2 gas. After 18 hours, the reaction was allowed to cool to room temperature and was poured into saturated aqueous NH4Cl solution (30 mL), followed by extraction with EtOAc (3x30mL). The extract was then washed with brine (20 mL), dried over Na2SO4 and concentrated in vacuo. The resulting crude residue was then purified via silica gel column chromatography (DCM/MeOH) to yield the desired product as an off-white foaming solid. (0.27 g, 65%). 1H NMR (500 MHz, CD3OD) δ 7.53 (dd, J = 8.9, 4.8 Hz, 2H, (H13, H15)), 7.39 (t, J = 8.7 Hz, 2H, (H12, H16)), 7.31 (d, J = 7.8 Hz, 1H, (H4)), 7.13 (td, J = 7.6, 1.2 Hz, 1H, (H2)), 7.01 (td, J = 7.6, 1.2 Hz, 1H, (H3)), 6.89 (d, J = 7.9 Hz, 1H, (H1)), 4.89 – 4.85 (m, 1H, (H18)), 3.76 – 3.61 (m, 2H, (H20, H22)), 3.40 – 3.26 (m, 2H, (H20, H22)), 2.03 – 1.92 (m, 2H, (H19, H23)), 1.88 – 1.70 (m, 2H, (H19, H23)), 1.46 (s, 9H, (H28, H29, H30)). 13C NMR (126 MHz, CD3OD) δ 164.14 (d, J = 248.0 Hz, (C14)), 156.33 (C24), 155.25 (C8), 140.90 (C6), 135.85 (C5), 131.72 (d, J = 3.2 Hz, (C11)), 130.67 (d, J = 9.1 Hz, (C13, C15)), 123.54 (C2), 121.73 (C3), 118.31 (d, J = 23.4 Hz, (C12, C16)), 115.59 (C4), 109.44 (C1), 81.35 (C27), 78.96 (C18), 46.45 (C20, C22), 32.64 (C19, C23), 28.61 (C28, C29, C30). FTIR (cm-1): 3339, 3021, 2883, 1680, 1528, 1233. HRMS-ESI (m/z): Calculated for C23H27FN4O2 (M+H)+: 411.2196, Found: 411.2221. 267 REFERENCES (1) Tanaka, K., The Proteasome: Overview of Structure and Functions, Proc. Jpn. Acad., Ser. B, 2009, 85 (1), 12–36. https://doi.org/10.2183/pjab.85.12. (2) Jones, C. L.; Tepe, J. J., Proteasome Activation to Combat Proteotoxicity, Molecules, 2019, 24 (15), 2841. https://doi.org/10.3390/molecules24152841. (3) George, D. E.; Tepe, J. J., Advances in Proteasome Enhancement by Small Molecules, Biomolecules, 2021, 11 (12), 1789. https://doi.org/10.3390/biom11121789. (4) Da Fonseca, P. C. A.; Morris, E. P., Structure of the Human 26S Proteasome, Journal of Biological Chemistry, 2008, 283 (34), 23305–23314. https://doi.org/10.1074/jbc.M802716200. (5) Ben-Nissan, G.; Sharon, M., Regulating the 20S Proteasome Ubiquitin-Independent Degradation Pathway, Biomolecules, 2014, 4 (3), 862–884. https://doi.org/10.3390/biom4030862. 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Commun., 2014, 50 (85), 12911– 12914. https://doi.org/10.1039/C4CC05628K. 269 Figure 3.3. 1H NMR and 13C NMR spectra of compound 3-10. APPENDIX KD_5_39_2D_PROTON_01 4 5 7 . 3 5 7 . 4 4 7 . 3 4 7 . 3 4 7 . 2 4 7 . 0 4 7 . 0 4 7 . 9 3 7 . 8 3 7 . 7 3 7 . 6 3 7 . 4 5 7 . 3 5 7 . 4 4 7 . 3 4 7 . 3 4 7 . 2 4 7 . 0 4 7 . 0 4 7 . 9 3 7 . 8 3 7 . 7 3 7 . 6 3 7 . l 3 C D C 6 2 7 . l 3 C D C 6 2 7 . 18 F 15 12 11 6 5 14 13 9 NH 7 16 17 1 4 2 3 8 N 10 6 0 7 . 4 0 7 . 2 0 7 . 4 7 4 . 4 0 4 . 1 0 4 . 4 9 3 . 2 9 3 . 6 0 7 . 4 0 7 . 2 0 7 . 3000 4 0 4 . 1 0 4 . 4 9 3 . 2 9 3 . 2000 1000 0 3000 2000 1000 0 6 0 2 . 7.6 7.5 8 1 . 5 7.4 3 1 . 2 7.3 f1 (ppm) 7.2 7.1 7.0 1 2 . 1 9 1 . 1 4.1 3.9 4.0 f1 (ppm) 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 f1 (ppm) 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 6 0 2 . 8 1 . 5 3 1 . 2 0 0 . 1 1 2 . 1 9 1 . 1 KD_5_39_2D_CARBON_01 . 5 3 3 6 1 0 4 . 1 6 1 . 7 6 4 3 1 . 2 9 3 3 1 . 9 8 3 3 1 6 1 . 0 3 1 . 9 0 0 3 1 . 1 2 9 2 1 1 1 . 9 2 1 . 8 3 7 2 1 . 4 7 8 1 1 . 6 6 5 1 1 . 9 4 5 1 1 l 3 C D C 6 1 . 7 7 9 4 3 5 . 0 6 0 5 . . 2 9 3 3 1 . 9 8 3 3 1 150 6 1 . 0 3 1 . 9 0 0 3 1 . 1 2 9 2 1 1 1 . 9 2 1 . 8 3 7 2 1 . 6 6 5 1 1 . 9 4 5 1 1 1000 1000 100 50 0 800 600 400 200 0 800 600 400 200 0 133.8 f1 (ppm) 130.5 130.0 129.5 129.0 128.5 128.0 127.5 127.0 f1 (ppm) 115.2 115.6 f1 (ppm) 18 F 15 12 11 6 5 14 13 9 NH 7 16 17 1 4 2 3 8 N 10 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 f1 (ppm) 270 8500 8000 7500 7000 6500 6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 -500 1100 1000 900 800 700 600 500 400 300 200 100 0 -100 Figure 3.4. 1H NMR and 13C NMR spectra of compound 3-11. KD_5_52_F22-27_PROTON_01 1 4 7 . 0 4 7 . 9 3 7 . 8 3 7 . 7 3 7 . 6 3 7 . 2 3 7 . 2 3 7 . 1 3 7 . 0 3 7 . 9 2 7 . 8 2 7 . 7 2 7 . 2 0 7 . 0 0 7 . 8 9 6 . D O 3 D C 1 3 3 . 4 7 3 . 3 7 3 . 3 7 3 . 1 7 3 . 5 6 3 . 2 6 3 . 9 4 3 . 7 4 3 . 9 9 2 . 8 9 2 . 7 9 2 . 5 9 2 . 3 9 2 . 2 9 2 . 1 9 2 . 0 9 2 . 2 0 7 . 0 0 7 . 8 9 6 . 1200 1000 800 600 400 200 0 4 7 3 . 3 7 3 . 3 7 3 . 1 7 3 . 5 6 3 . 2 6 3 . 9 4 3 . 7 4 3 . D O 3 D C 1 3 3 . 9 9 2 . 8 9 2 . 7 9 2 . 5 9 2 . 3 9 2 . 2 9 2 . 1 9 2 . 0 9 2 . 1000 800 600 400 200 0 9 7 . 1 7.0 f1 (ppm) 7 1 . 1 0 0 . 1 3.8 3.7 3.6 5 0 . 1 3.5 5 9 0 . 7 9 0 . 3.4 3.3 f1 (ppm) 3.2 3.1 3.0 2.9 2.8 17 F 14 11 10 6 5 13 12 9 NH 7 15 16 1 4 2 3 18 NH2 8 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 f1 (ppm) 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 1 7 3 . 9 7 2 . 9 7 . 1 7 1 . 1 0 0 . 1 5 0 . 1 5 9 0 . 7 9 0 . KD_5_52_F22-27_CARBON_01 . 2 2 4 6 1 . 8 2 2 6 1 6 7 . 1 4 1 . 5 0 7 3 1 . 3 0 7 3 1 6 2 . 1 3 1 0 2 . 1 3 1 . 9 8 9 2 1 . 2 9 8 2 1 . 7 4 8 2 1 . 8 9 5 1 1 . 1 8 5 1 1 D O 3 D C 0 0 9 4 . 3 9 0 5 . 5 3 7 4 . 7 1 . 3 6 . 5 0 7 3 1 . 3 0 7 3 1 6 2 . 1 3 1 0 2 . 1 3 1 . 9 8 9 2 1 . 2 9 8 2 1 . 7 4 8 2 1 300 200 100 0 . 8 9 5 1 1 . 1 8 5 1 1 2500 2000 1500 1000 500 0 136.8 137.1 f1 (ppm) 131 130 129 128 127 126 125 124 123 122 121 120 119 118 117 116 f1 (ppm) 17 F 14 11 10 6 5 13 12 9 NH 7 15 16 1 4 2 3 18 NH2 8 2000 1900 1800 1700 1600 1500 1400 1300 1200 1100 1000 900 800 700 600 500 400 300 200 100 0 -100 4500 4000 3500 3000 2500 2000 1500 1000 500 0 200 190 180 170 160 150 140 130 120 110 100 f1 (ppm) 90 80 70 60 50 40 30 20 10 0 271 Figure 3.5. 1H NMR and 13C NMR spectra of compound 3-12. KD_7_46_F44-46_PROTON_01 7 3 7 . 6 3 7 . 6 3 7 . 4 3 7 . 6 0 7 . 5 0 7 . 3 0 7 . D O 3 D C 1 3 3 . 4 7 3 . 7 7 2 . 5 7 2 . 4 7 2 . 6 6 2 . 5 6 2 . 4 6 2 . NH25 1 2 4 11 NH 3 6 7 10 9 8 F 12 8 3 7 . 7 3 7 . 6 3 7 . 5 3 7 . 7 0 7 . 5 0 7 . 3 0 7 . 7 7 2 . 6 7 2 . 5 7 2 . 7 6 2 . 5 6 2 . 4 6 2 . 2000 1000 0 8 0 2 . 7 9 . 1 7.4 7.3 7.2 f1 (ppm) 7.1 7.0 1 3 2 . 1 3 2 . 2.8 2.7 f1 (ppm) 4000 3000 2000 1000 0 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 f1 (ppm) 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 8 0 2 . 7 9 . 1 4 3 2 . 1 3 2 . 1 3 2 . KD_7_46_F44-46_CARBON_01 . 5 4 4 6 1 . 1 5 2 6 1 . 2 8 6 3 1 0 4 . 1 3 1 4 3 . 1 3 1 2 1 . 6 1 1 . 5 9 5 1 1 D O 3 D C 0 0 9 4 . 4 6 3 5 . 7 0 2 5 . 5 7 . 1 4 NH25 1 2 4 11 NH 3 6 7 10 9 8 F 12 . 2 8 6 3 1 0 4 . 1 3 1 4 3 . 1 3 1 20 15 10 5 0 80 60 40 20 0 2 1 . 6 1 1 . 5 9 5 1 1 80 60 40 20 0 137.0 136.6 f1 (ppm) 131.6 131.2 f1 (ppm) 116 f1 (ppm) 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 f1 (ppm) 272 7500 7000 6500 6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 -500 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 -5 Figure 3.6. 1H NMR and 13C NMR spectra of compound 3-13. KD_5_45_C_PROTON_01 7 1 . 7 5 1 . 7 7 8 6 . 5 8 6 . 2 7 3 . 8 4 3 . 6 d - O S M D 0 5 2 . 12 CH3 O 11 4 O 2 Cl 1 3 10 5 9 6 8 7 7 1 . 7 5 1 . 7 7 8 6 . 5 8 6 . 5000 4000 3000 2000 1000 0 6 2 2 . 0 0 2 . 7.2 7.1 7.0 f1 (ppm) 6.9 6.8 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 f1 (ppm) 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 6 2 2 . 0 0 2 . 6 0 3 . 9 0 2 . KD_5_45_C_CARBON_01 . 0 0 3 7 1 . 6 0 8 5 1 . 2 4 0 3 1 . 9 9 6 2 1 . 9 6 3 1 1 6 d - O S M D 2 5 9 3 . . 1 8 9 3 6 0 5 5 . 12 CH3 O 11 4 O 2 Cl 1 3 10 5 9 6 8 7 . 1 2 0 4 100 50 0 40.5 40.0 f1 (ppm) 39.5 200 190 180 170 160 150 140 130 120 110 100 f1 (ppm) 90 80 70 60 50 40 30 20 10 0 273 17000 16000 15000 14000 13000 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 -1000 240 230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 -20 Figure 3.7. 1H NMR and 13C NMR spectra of compound 3-14. KD_6_129_F27-29_PROTON_01 25 CH3 O 24 9 8 10 7 4 O 5 6 2 3 11 N1 12 15 20 O 13 14 22 CH3 17 NH 16 CH323 19 O 18 CH321 7 1 . 7 5 1 . 7 9 8 6 . 7 8 6 . 1 4 4 . 0 4 4 . 0 4 4 . 0 4 4 . 9 3 4 . 9 3 4 . 8 3 4 . 8 3 4 . 7 3 4 . 7 3 4 . 7 3 4 . 6 3 4 . 6 9 3 . 5 9 3 . 4 9 3 . 4 9 3 . 4 9 3 . 3 9 3 . 2 9 3 . 2 9 3 . 2 9 3 . 1 9 3 . 1 9 3 . 7 7 3 . 1 7 3 . 0 7 3 . 5 5 3 . 4 5 3 . 3 5 3 . 2 5 3 . 1 5 3 . 0 5 3 . 9 4 3 . 9 4 3 . D O 3 D C 1 3 3 . 4 1 . 3 2 1 . 3 9 0 3 . 4 8 2 . 2 8 2 . 9 7 2 . 5 8 . 1 4 8 . 1 4 8 . 1 3 8 . 1 2 8 . 1 1 8 . 1 0 8 . 1 7 7 . 1 6 7 . 1 5 7 . 1 4 7 . 1 4 7 . 1 3 7 . 1 2 4 . 1 1 3 . 1 0 3 . 1 9 2 . 1 8 2 . 1 6 2 . 1 3 2 . 1 4 1 . 1 4 1 . 1 2 1 . 1 1 1 . 1 0 1 . 1 9 0 . 1 7 0 . 1 6 0 . 1 7 1 . 7 5 1 . 7 9 8 6 . 7 8 6 . 0 0 2 . 7.2 7.1 7.0 f1 (ppm) 0 0 2 . 6.9 600 500 400 300 200 100 0 -100 1 4 4 . 0 4 4 . 0 4 4 . 0 4 4 . 9 3 4 . 9 3 4 . 8 3 4 . 8 3 4 . 7 3 4 . 7 3 4 . 7 3 4 . 6 3 4 . 6 9 3 . 5 9 3 . 4 9 3 . 4 9 3 . 4 9 3 . 3 9 3 . 2 9 3 . 2 9 3 . 2 9 3 . 1 9 3 . 1 9 3 . 7 7 3 . 1 7 3 . 0 7 3 . 5 5 3 . 4 5 3 . 3 5 3 . 2 5 3 . 1 5 3 . 0 5 3 . 9 4 3 . 9 4 3 . D O 3 D C 1 3 3 . 4 1 . 3 2 1 . 3 9 0 3 . 4 8 2 . 2 8 2 . 9 7 2 . 300 200 100 0 4 2 . 1 4.4 2 1 . 1 3 1 . 3 4 9 . 1 5 2 . 1 9 0 . 1 4.2 4.0 3.8 3.6 f1 (ppm) 3.4 3.2 3.0 6 3 . 1 2.8 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 f1 (ppm) 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 0 2 . 0 0 2 . 4 2 . 1 2 1 . 1 3 1 . 3 4 9 . 1 5 2 . 1 9 0 . 1 6 3 . 1 4 4 2 . 7 1 . 9 7 1 . 1 8 2 . 1 . 7 6 0 3 1 . 7 2 8 2 1 8 1 . 5 1 1 1 1 . 0 8 D O 3 D C 0 0 9 4 . 9 6 5 5 . 2 8 4 5 . . 3 2 6 4 2 0 2 4 . 9 5 0 4 . . 7 3 3 3 5 6 2 3 . . 3 7 8 2 KD_6_129_F27-29_CARBON_01 . 3 3 2 7 1 . 3 0 0 6 1 . 1 7 7 5 1 25 CH3 O 24 9 8 10 7 4 O 5 6 2 3 11 N1 12 15 20 O 13 14 22 CH3 17 NH 16 CH323 19 O 18 CH321 1100 1000 900 800 700 600 500 400 300 200 100 0 -100 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 -5 200 190 180 170 160 150 140 130 120 110 100 f1 (ppm) 90 80 70 60 50 40 30 20 10 0 274 Figure 3.8. 1H NMR and 13C NMR spectra of compound 3-15. KD_6_135C_PROTON_01 0 2 7 . 8 1 . 7 0 9 6 . 8 8 6 . 1 6 4 . 9 5 4 . 3 1 . 4 2 1 . 4 1 1 . 4 0 1 . 4 9 7 3 . 7 7 3 . 7 3 3 . 5 3 3 . 4 3 3 . 6 1 . 3 9 7 2 . 7 7 2 . 4 7 2 . 6 0 2 . 4 0 2 . 9 9 . 1 9 9 . 1 6 9 . 1 7 4 . 1 5 4 . 1 3 3 . 1 1 3 . 1 0 3 . 1 9 2 . 1 8 2 . 1 17 CH3 O 16 4 O 2 3 10 5 9 6 8 7 12 13 11 14 N 1 15 NH218 ClH 19 0 2 7 . 8 1 . 7 0 9 6 . 8 8 6 . 300 200 100 0 0 0 2 . 7.2 9 1 . 2 7.1 7.0 f1 (ppm) 6.9 6.8 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 f1 (ppm) 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 0 2 . 9 1 . 2 0 0 . 1 5 9 0 . 7 8 4 . 2 3 . 1 5 0 . 1 3 9 0 . 2 1 . 1 5 0 . 1 0 0 . 1 8 0 . 1 KD_6_135C_CARBON_01 . 7 5 2 7 1 12 13 11 14 N 1 15 NH218 ClH 19 . 2 0 0 6 1 3 4 O 2 17 CH3 O 16 10 5 9 6 8 7 . 5 7 0 3 1 . 9 7 7 2 1 . 4 2 5 1 1 D O 3 D C 0 0 9 4 . 0 4 9 4 . . 8 7 5 5 5 6 5 4 . 2 4 . 1 4 8 3 0 4 . 0 2 . 1 3 8 5 0 3 . D O 3 D C 0 0 9 4 . 0 4 9 4 . 49.6 48.8 f1 (ppm) 100 50 0 2100 2000 1900 1800 1700 1600 1500 1400 1300 1200 1100 1000 900 800 700 600 500 400 300 200 100 0 -100 550 500 450 400 350 300 250 200 150 100 50 0 -50 200 190 180 170 160 150 140 130 120 110 100 f1 (ppm) 90 80 70 60 50 40 30 20 10 0 275 Figure 3.9. 1H NMR and 13C NMR spectra of compound 3-16. 6 1 . 7 4 1 . 7 7 8 6 . 5 8 6 . 4 9 3 . 3 9 3 . 3 9 3 . 2 9 3 . 1 9 3 . 1 9 3 . 0 8 3 . 1 7 3 . 0 7 3 . 9 6 3 . 8 6 3 . 7 6 3 . 0 6 3 . 0 6 3 . 9 5 3 . 8 5 3 . 7 5 3 . 7 5 3 . 6 5 3 . 5 5 3 . 2 4 3 . 1 4 3 . 1 4 3 . 0 4 3 . 9 3 3 . 9 3 3 . 8 3 3 . 7 3 3 . 0 9 . 1 8 8 . 1 7 8 . 1 7 8 . 1 9 7 . 1 8 7 . 1 8 7 . 1 7 7 . 1 6 7 . 1 6 7 . 1 5 7 . 1 4 7 . 1 3 7 . 1 7 6 . 1 6 6 . 1 5 6 . 1 4 6 . 1 3 6 . 1 3 6 . 1 2 6 . 1 9 5 . 1 8 5 . 1 8 5 . 1 7 5 . 1 6 5 . 1 6 5 . 1 5 5 . 1 4 5 . 1 4500 KD_7_50_PROTON_01 7 N 1 6 2 5 3 17 S 18 16 O N 4 8 15 14 20 CH3 9 10 13 O 19 11 12 l 3 C D C 6 2 7 . 6 1 . 7 4 1 . 7 7 8 6 . 5 8 6 . 3 9 3 . 3 9 3 . 2 9 3 . 1 9 3 . 1 9 3 . 0 8 3 . 1 7 3 . 0 7 3 . 9 6 3 . 8 6 3 . 7 6 3 . 0 6 3 . 8 5 3 . 7 5 3 . 7 5 3 . 6 5 3 . 5 5 3 . 2 4 3 . 1 4 3 . 1 4 3 . 0 4 3 . 9 3 3 . 9 3 3 . 8 3 3 . 2000 1500 1000 500 0 1500 1000 500 0 7 8 . 1 7 8 . 1 8 7 . 1 7 7 . 1 6 7 . 1 6 7 . 1 5 6 . 1 4 6 . 1 3 6 . 1 3 6 . 1 2 6 . 1 9 5 . 1 8 5 . 1 8 5 . 1 7 5 . 1 600 500 400 300 200 100 0 5 0 2 . 0 0 2 . 7.1 7.0 f1 (ppm) 6.9 1 2 . 1 4 1 . 3 4 0 2 . 0 6 2 . 4 1 . 1 8 0 . 1 4 0 . 1 6 2 . 1 2 2 . 1 2 2 . 1 3.9 3.8 3.7 3.6 3.5 3.4 f1 (ppm) 1.9 1.8 1.7 f1 (ppm) 1.6 1.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 f1 (ppm) 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 5 0 2 . 0 0 2 . 1 2 . 1 4 1 . 3 4 0 2 . 0 6 2 . 4 1 . 1 8 0 . 1 4 0 . 1 6 2 . 1 2 2 . 1 2 2 . 1 KD_7_50_CARBON_01 . 2 7 9 6 1 . 2 5 8 5 1 . 5 9 6 3 1 . 7 4 9 2 1 . 2 7 6 2 1 . 7 2 4 1 1 8 2 5 5 . 2 9 2 5 . 9 8 2 4 . 9 2 0 4 . 5 5 8 3 . 5 3 2 3 . 0 8 . 1 3 7 N 1 6 2 5 3 17 S 18 16 O N 4 8 15 14 20 CH3 9 10 13 O 19 11 12 200 190 180 170 160 150 140 130 120 110 100 f1 (ppm) 90 80 70 60 50 40 30 20 10 0 276 4000 3500 3000 2500 2000 1500 1000 500 0 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 3.10. 1H NMR and 13C NMR spectra of compound 3-2. KD_7_53_ppt1_PROTON_01 D O 3 D C 1 3 3 . 8 1 . 3 7 1 . 3 5 1 . 3 4 1 . 3 3 1 . 3 2 1 . 3 2 1 . 3 7 7 2 . 7 7 2 . 6 7 2 . 5 7 2 . 4 7 2 . 4 7 2 . 2 7 2 . 2 7 2 . 1 1 . 2 7 0 2 . 4 0 2 . 2 0 2 . 3 5 . 1 2 5 . 1 1 5 . 1 0 5 . 1 9 4 . 1 8 4 . 1 7 4 . 1 3 4 7 . 2 4 7 . 1 4 7 . 0 4 7 . 0 4 7 . 0 3 7 . 9 2 7 . 9 2 7 . 8 2 7 . 8 2 7 . 7 2 7 . 1 2 7 . 0 2 7 . 9 0 7 . 9 0 7 . 8 0 7 . 8 0 7 . 7 0 7 . 0 9 6 . 9 8 6 . 7 8 4 . 5 8 4 . 5 8 4 . 3 8 4 . 6 7 4 . 5 7 4 . 2 7 4 . 2 7 4 . 8 6 4 . 7 6 4 . 6 6 4 . 6 6 4 . 5 6 4 . 4 6 4 . 4 6 4 . 6 1 . 4 3 1 . 4 2 1 . 4 1 1 . 4 1 1 . 4 9 0 4 . 6 9 3 . 6 9 3 . 3 9 3 . 2 9 3 . 1 8 3 . 8 7 3 . 7 7 3 . 6 7 3 . 3 7 3 . 2 7 3 . 9 6 3 . 9 6 3 . 7 6 3 . 6 6 3 . 2 6 3 . 1 6 3 . 0 6 3 . 0 6 3 . 9 5 3 . 8 5 3 . 8 5 3 . 28 25 27 26 31 CH3 O 30 7 8 4 . 5 8 4 . 5 8 4 . 3 8 4 . 6 7 4 . 5 7 4 . 2 7 4 . 2 7 4 . 8 6 4 . 7 6 4 . 6 6 4 . 6 6 4 . 5 6 4 . 4 6 4 . 4 6 4 . 6 1 . 4 3 1 . 4 2 1 . 4 1 1 . 4 1 1 . 4 9 0 4 . 8 0 4 . 6 9 3 . 6 9 3 . 3 9 3 . 2 9 3 . 1 8 3 . 8 7 3 . 7 7 3 . 6 7 3 . 3 7 3 . 2 7 3 . 9 6 3 . 9 6 3 . 7 6 3 . 6 6 3 . 2 6 3 . 1 6 3 . 0 6 3 . 0 6 3 . 9 5 3 . 8 5 3 . 8 5 3 . D O 3 D C 1 3 3 . 8 1 . 3 7 1 . 3 6 1 . 3 5 1 . 3 4 1 . 3 3 1 . 3 2 1 . 3 2 1 . 3 7 7 2 . 7 7 2 . 6 7 2 . 5 7 2 . 4 7 2 . 4 7 2 . 2 7 2 . 2 7 2 . 400 3 2 N 4 NH 6 7 8 1 5 N 33 32 34 15 37 35 36 16 13 14 19 18 17 F 20 12 9 11 N 10 21 22 29 O 23 24 3 4 7 . 2 4 7 . 1 4 7 . 0 4 7 . 0 4 7 . 0 3 7 . 9 2 7 . 9 2 7 . 8 2 7 . 8 2 7 . 7 2 7 . 1 2 7 . 0 2 7 . 9 0 7 . 9 0 7 . 8 0 7 . 8 0 7 . 7 0 7 . 0 9 6 . 9 8 6 . 1500 1000 500 0 0 0 3 . 4 0 2 . 7.4 7.3 2 1 . 2 7.2 f1 (ppm) 3 0 4 . 6 0 2 . 6 9 0 . 0 0 . 1 1 1 . 1 3 1 . 2 5 2 . 1 2 2 . 1 0 9 2 . 2 0 2 . 9 2 2 . 7 1 . 1 5 2 . 1 8 1 . 1 7.1 7.0 6.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 f1 (ppm) 3.7 3.6 3.5 3.4 3.3 3.2 3.1 3.0 2.9 2.8 2.7 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 f1 (ppm) 5.0 4.5 4.0 3.5 3.0 2.5 2.0 0 0 3 . 4 0 2 . 2 1 . 2 3 0 4 . 6 0 2 . 6 9 0 . 0 0 . 1 1 1 . 1 3 1 . 2 5 2 . 1 2 2 . 1 0 9 2 . 2 0 2 . 9 2 2 . 7 1 . 1 5 2 . 1 8 1 . 1 5 5 . 1 3 3 . 1 1.5 1.0 0.5 0.0 KD_7_53_ppt1_CARBON_01 . 3 0 4 7 1 NH 6 3 2 N 4 1 5 N 33 32 34 15 37 35 36 16 13 14 19 18 17 F 20 . 9 4 6 6 1 5 1 . 0 6 1 . 2 0 3 4 1 . 2 2 8 3 1 . 0 7 0 3 1 . 5 4 0 3 1 . 9 6 8 2 1 . 0 5 8 2 1 . 6 7 6 1 1 . 6 2 5 1 1 D O 3 D C 0 0 9 4 . 4 0 4 6 . 9 9 9 5 . 7 6 5 5 . . 1 9 2 5 0 0 . 1 5 0 9 . 1 4 6 6 0 4 . 6 0 3 3 . 9 4 4 2 . 7 8 12 9 11 N 10 21 22 29 O 23 24 . 0 7 0 3 1 . 5 4 0 3 1 28 25 . 9 6 8 2 1 . 0 5 8 2 1 27 26 31 CH3 O 30 . 6 7 6 1 1 . 6 2 5 1 1 15 10 5 0 130 128 126 124 122 120 118 116 f1 (ppm) 200 190 180 170 160 150 140 130 120 110 100 f1 (ppm) 90 80 70 60 50 40 30 20 10 0 277 300 1000 200 100 0 2000 1900 1800 1700 1600 1500 1400 1300 1200 1100 900 800 700 600 500 400 300 200 100 0 -100 35 30 25 20 15 10 5 0 Figure 3.11. 1H NMR and 13C NMR spectra of compound 3-3. KD_7_52r_F23-30_PROTON_01 3 2 N 4 NH 6 7 8 1 5 N 13 12 9 11 N 10 14 15 19 16 18 17 F 20 0 5 7 . 9 4 7 . 8 4 7 . 7 4 7 . 6 4 7 . 1 3 7 . 9 2 7 . 5 2 7 . 3 2 7 . 9 9 6 . 7 9 6 . 5 9 6 . 7 4 5 . 9 9 4 . 5 8 4 . 4 8 4 . 1 8 4 . 7 2 4 . 5 2 4 . 5 8 3 . 2 8 3 . 8 7 3 . 6 7 3 . 4 7 3 . 3 7 3 . 2 7 3 . 2 7 3 . D O 3 D C 1 3 3 . 8 2 3 . 5 2 3 . 5 2 3 . 8 8 2 . 7 8 2 . 5 8 2 . 3 8 2 . 2 5 2 . 0 5 2 . 9 4 2 . 8 4 2 . 7 4 2 . 5 4 2 . 5 4 2 . 3 3 2 . 0 3 2 . 7 2 2 . 0 1 . 2 9 0 2 . 7 0 2 . 7 9 . 1 7 9 . 1 5 9 . 1 21 22 29 O 23 24 28 25 27 26 31 CH3 O 30 0 5 7 . 9 4 7 . 8 4 7 . 7 4 7 . 6 4 7 . 1 3 7 . 9 2 7 . 5 2 7 . 3 2 7 . 9 9 6 . 7 9 6 . 5 9 6 . 2 8 3 . 8 7 3 . 6 7 3 . 4 7 3 . 3 7 3 . 2 7 3 . 2 7 3 . 150 100 50 0 6 2 2 . 7.5 7.4 5 3 2 . 9 2 2 . 7.3 7.2 f1 (ppm) 0 0 2 . 7.1 7.0 5 0 2 . 0 4 3 . 0 6 3 . 3 0 4 . 3.7 3.8 f1 (ppm) 3.6 200 150 100 50 0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 f1 (ppm) 6 2 2 . 5 3 2 . 9 2 2 . 0 0 2 . 2 1 . 2 5 1 . 1 5 3 . 1 8 1 . 1 5 0 2 . 0 4 3 . 0 6 3 . 3 0 4 . 8 0 . 1 8 1 . 1 3 3 . 1 3 6 . 1 4 9 . 1 6 8 . 1 KD_7_52_F23_CARBON_01 3 2 N 4 NH 6 7 8 1 5 N 13 12 9 11 N 10 14 15 19 16 18 17 F 20 . 5 8 6 6 1 . 9 4 2 6 1 5 1 . 0 6 1 . 0 5 3 5 1 6 6 . 1 3 1 . 9 7 0 3 1 7 1 . 0 3 1 1 1 . 0 3 1 6 1 . 8 2 1 . 1 8 6 1 1 . 3 6 6 1 1 . 3 3 5 1 1 D O 3 D C 0 0 9 4 . . 6 7 6 4 . 2 7 5 5 5 5 4 5 . 6 3 3 5 . 3 9 0 5 . 8 2 9 4 . 9 5 2 4 . . 5 7 0 4 3 2 5 3 . 7 1 . 1 3 21 22 29 O 23 24 28 25 27 26 . 9 4 2 6 1 30 6 6 . 1 3 1 20 10 0 31 CH3 7 1 . 0 3 1 1 1 . 0 3 1 O 30 30 20 10 0 40 30 20 10 0 . 1 8 6 1 1 . 3 6 6 1 1 . 3 3 5 1 1 60 50 40 30 20 10 0 D O 3 D C 0 0 9 4 . 8 2 9 4 . 60 50 40 30 20 10 0 163.2 162.8 f1 (ppm) 131.6 f1 (ppm) 130.2 129.8 f1 (ppm) 117 116 f1 (ppm) 49.5 49.0 f1 (ppm) 48.5 200 190 180 170 160 150 140 130 120 110 100 f1 (ppm) 90 80 70 60 50 40 30 20 10 0 278 340 320 300 280 260 240 220 200 180 160 140 120 100 80 60 40 20 0 -20 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 -5 Figure 3.12. 1H NMR and 13C NMR spectra of compound 3-17. l 3 C D C 6 2 7 . KD_5_67_F8-10_PROTON_01 2 3 7 . 1 3 7 . 1 3 7 . 10 9 S 6 1 11 8 7 12 13 5 2 4 3 21 O N 14 20 17 CH3 O 15 16 CH318 CH319 6 2 7 . 5 2 7 . 4 2 7 . 4 2 7 . 4 2 7 . 3 2 7 . 3 2 7 . 2 2 7 . 6 9 3 . 2 9 3 . 9 8 3 . 5 7 3 . 8 8 2 . 6 8 2 . 4 8 2 . 1 7 2 . 0 7 2 . 9 6 2 . 9 6 2 . 8 6 2 . 7 6 2 . 7 6 2 . 6 6 2 . 5 6 2 . 1 9 . 1 7 8 . 1 5 8 . 1 3 5 . 1 2 5 . 1 0 5 . 1 8 4 . 1 4 4 . 1 8 8 2 . 6 8 2 . 4 8 2 . 1 7 2 . 0 7 2 . 9 6 2 . 9 6 2 . 8 6 2 . 7 6 2 . 7 6 2 . 6 6 2 . 5 6 2 . 1 9 . 1 7 8 . 1 5 8 . 1 3 5 . 1 2 5 . 1 0 5 . 1 8 4 . 1 4 4 . 1 400 300 200 100 0 9 2 2 . 2 1 . 1 3 3 2 . 3.0 2.5 2.0 f1 (ppm) 7 4 2 . 6 9 9 . 1.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 f1 (ppm) KD_5_67_F8-10_CARBON_01 . 0 8 4 5 1 . 0 5 8 3 1 . 4 8 8 2 1 . 6 6 8 2 1 2 1 . 7 2 1 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 l 3 C D C 6 1 . 7 7 4 6 9 7 . . 7 2 3 4 0 6 0 4 . 9 6 4 3 . 9 2 2 3 . 4 5 8 2 . 0 0 4 . 2 1 . 1 4 2 2 . 9 2 2 . 9 2 2 . 2 1 . 1 3 3 2 . 7 4 2 . 6 9 9 . 10 9 S 6 1 11 8 7 12 13 5 2 4 3 21 O N 14 20 17 CH3 O 15 16 CH318 CH319 1000 . 4 8 8 2 1 . 6 6 8 2 1 800 600 400 200 0 128.4 f1 (ppm) 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 279 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0 -200 550 500 450 400 350 300 250 200 150 100 50 0 -50 Figure 3.13. 1H NMR and 13C NMR spectra of compound 3-18. KD_5_71C_PROTON_01 6 3 7 . 5 3 7 . 2 3 7 . 0 3 7 . 9 2 7 . 4 2 7 . 3 2 7 . 2 2 7 . D O 3 D C 1 3 3 . 3 8 3 . 8 3 3 . 7 3 3 . 5 0 3 . 2 0 3 . 0 0 3 . 1 9 2 . 9 8 2 . 7 8 2 . 6 1 . 2 3 1 . 2 8 7 . 1 6 7 . 1 4 7 . 1 1 7 . 1 ClH 15 5 1 NH 14 4 2 13 12 7 8 11 3 S 6 9 10 6 3 7 . 5 3 7 . 2 3 7 . 0 3 7 . 9 2 7 . 4 2 7 . 3 2 7 . 2 2 7 . 5 0 3 . 2 0 3 . 0 0 3 . 1 9 2 . 9 8 2 . 7 8 2 . 6 1 . 2 3 1 . 2 8 7 . 1 6 7 . 1 4 7 . 1 1 7 . 1 500 0 1 0 2 . 7 0 2 . 2 0 . 1 7.4 7.3 f1 (ppm) 7.2 0 0 . 1 9 9 . 1 3.0 0 0 2 . 1 0 2 . 2.5 f1 (ppm) 2.0 1000 900 800 700 600 400 500 300 200 100 400 0 300 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 f1 (ppm) 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 KD_5_71C_CARBON_01 . 7 6 9 3 1 . 2 9 9 2 1 . 7 5 9 2 1 1 1 . 8 2 1 D O 3 D C 0 0 9 4 . 5 8 9 4 . 8 1 . 5 4 . 6 2 4 4 . 5 7 8 3 9 5 5 3 . 5 2 0 3 . 1 0 2 . 7 0 2 . 2 0 . 1 0 0 2 . 3 3 2 . 9 9 . 1 0 0 . 1 0 0 2 . 1 0 2 . ClH 15 5 1 NH 14 4 2 13 12 7 8 11 3 S 6 9 10 . 7 5 8 2 1 . 2 2 8 2 1 . 6 7 6 2 1 200 150 100 50 0 D O 3 D C 0 0 9 4 . 5 8 9 4 . 100 80 60 40 20 0 129 128 f1 (ppm) 127 49.5 48.6 f1 (ppm) 200 190 180 170 160 150 140 130 120 110 100 f1 (ppm) 90 80 70 60 50 40 30 20 10 0 280 200 100 0 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 -20 Figure 3.14. 1H NMR and 13C NMR spectra of compound 3-19. KD_5_73_2D_PROTON_01 1 3 7 . 1 3 7 . 0 3 7 . 9 2 7 . 9 2 7 . 9 2 7 . 8 2 7 . 7 2 7 . 7 2 7 . 6 2 7 . 6 2 7 . 2 2 7 . 1 2 7 . 1 2 7 . 1 2 7 . 0 2 7 . 0 2 7 . 9 1 . 7 9 1 . 7 8 1 . 7 5 1 . 7 3 1 . 7 8 8 6 . 6 8 6 . 4 2 4 . 4 2 4 . 4 2 4 . 3 2 4 . 3 2 4 . 2 2 4 . 2 2 4 . 1 2 4 . 1 2 4 . 0 2 4 . 0 2 4 . 0 2 4 . 6 8 3 . 6 8 3 . 5 8 3 . 5 8 3 . 5 8 3 . 4 8 3 . 3 8 3 . 3 8 3 . 3 8 3 . 2 8 3 . 2 8 3 . 1 8 3 . 5 7 3 . 3 7 3 . 7 6 3 . D O 3 D C 1 3 3 . 2 1 . 3 1 1 . 3 0 1 . 3 9 0 3 . 8 0 3 . 7 0 3 . 6 0 3 . 1 9 2 . 1 9 2 . 9 8 2 . 9 8 2 . 8 8 2 . 7 8 2 . 6 8 2 . 5 7 2 . 5 7 2 . 4 7 2 . 3 7 2 . 3 7 2 . 2 7 2 . 1 7 2 . 1 7 2 . 0 9 . 1 9 8 . 1 8 8 . 1 7 8 . 1 6 8 . 1 6 7 . 1 6 7 . 1 4 7 . 1 3 7 . 1 3 7 . 1 1 4 . 1 0 4 . 1 9 3 . 1 8 3 . 1 7 3 . 1 7 3 . 1 6 3 . 1 5 3 . 1 4 3 . 1 3 3 . 1 3 2 . 1 2 2 . 1 1 2 . 1 0 2 . 1 9 1 . 1 8 1 . 1 8 1 . 1 7 1 . 1 6 1 . 1 18 O 20 19 15 N1 O 24 21 17 16 CH325 22 23 14 13 8 9 12 4 S 7 10 11 6 2 5 3 1 3 7 . 1 3 7 . 0 3 7 . 9 2 7 . 9 2 7 . 9 2 7 . 8 2 7 . 7 2 7 . 7 2 7 . 6 2 7 . 6 2 7 . 2 2 7 . 1 2 7 . 1 2 7 . 1 2 7 . 0 2 7 . 0 2 7 . 9 1 . 7 9 1 . 7 8 1 . 7 5 1 . 7 3 1 . 7 8 8 6 . 6 8 6 . 1200 1000 800 600 400 200 0 4 2 4 . 3 2 4 . 1 2 4 . 1 2 4 . 0 2 4 . 5 8 3 . 5 8 3 . 5 8 3 . 3 8 3 . 3 8 3 . 2 8 3 . 5 7 3 . 3 7 3 . 7 6 3 . 700 600 500 400 300 200 100 0 2800 2600 2400 2200 2000 1800 1600 2 1 . 3 1 1 . 3 0 1 . 3 9 0 3 . 8 0 3 . 7 0 3 . 6 0 3 . 1 9 2 . 1 9 2 . 9 8 2 . 9 8 2 . 8 8 2 . 7 8 2 . 6 8 2 . 6 7 2 . 5 7 2 . 5 7 2 . 4 7 2 . 3 7 2 . 3 7 2 . 2 7 2 . 1 7 2 . 1 7 2 . 400 0 9 . 1 9 8 . 1 8 8 . 1 7 8 . 1 6 8 . 1 6 7 . 1 6 7 . 1 3 7 . 1 3 7 . 1 9 3 . 1 8 3 . 1 6 3 . 1 5 3 . 1 1 2 . 1 0 2 . 1 8 1 . 1 7 1 . 1 300 300 200 100 0 200 1400 100 1200 1000 0 9 1 . 4 7.4 7.3 0 2 2 . 0 0 . 1 7.2 8 1 . 2 0 2 . 1 7 2 . 1 1 5 3 . 0 2 2 . 7 1 . 2 -100 7.1 f1 (ppm) 7.0 6.9 6.8 4.0 f1 (ppm) 3.6 0 2 . 1 3.1 3.0 8 1 . 1 2.9 f1 (ppm) 7 1 . 1 2.8 2.7 2 2 . 1 1 2 . 1 9 2 . 1 4 2 . 1 1.5 f1 (ppm) 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 f1 (ppm) 9 1 . 4 0 0 . 1 0 2 2 . 8 1 . 2 0 2 . 1 7 2 . 1 1 5 3 . 0 2 2 . 7 1 . 2 0 2 . 1 8 1 . 1 7 1 . 1 2 2 . 1 1 2 . 1 9 2 . 1 4 2 . 1 KD_5_73_2D_CARBON_01 8 1 . 2 7 1 . 4 0 0 6 1 . 4 0 0 4 1 . 4 6 0 3 1 . 9 8 9 2 1 . 7 4 9 2 1 . 5 2 8 2 1 . 4 9 7 2 1 6 1 . 5 1 1 D O 3 D C 0 0 9 4 . 7 6 5 5 . 3 8 6 4 . 6 5 2 4 . 4 2 . 1 4 2 6 0 4 . 6 2 5 3 . 9 6 3 3 . 6 1 . 3 3 18 O 20 19 15 N1 O 24 21 17 16 CH325 22 23 14 13 8 9 12 4 S 7 10 11 6 2 5 3 500 400 300 200 100 0 . 4 6 0 3 1 . 9 8 9 2 1 . 7 4 9 2 1 . 5 2 8 2 1 . 4 9 7 2 1 130 129 f1 (ppm) 128 800 600 400 200 0 -200 1200 1100 1000 900 800 700 600 500 400 300 200 100 0 -100 200 190 180 170 160 150 140 130 120 110 100 f1 (ppm) 90 80 70 60 50 40 30 20 10 0 281 Figure 3.15. 1H NMR and 13C NMR spectra of compound 3-20. KD_5_118C_PROTON_01 8 1 . 7 6 1 . 7 9 8 6 . 7 8 6 . 7 7 3 . 0 7 3 . 6 5 3 . 5 5 3 . 4 5 3 . 2 5 3 . 0 5 3 . 9 4 3 . 7 4 3 . 6 4 3 . 1 2 3 . 8 1 . 3 4 5 . 1 2 5 . 1 0 5 . 1 8 4 . 1 7 3 . 1 5 3 . 1 2 3 . 1 1 3 . 1 11 O 13 12 8 N1 O 17 14 10 9 CH318 15 16 6 2 5 3 4 SH 7 8 1 . 7 6 1 . 7 9 8 6 . 7 8 6 . 100 80 60 40 20 0 0 0 2 . 7.2 0 9 . 1 7.1 7.0 f1 (ppm) 6.9 6.8 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 f1 (ppm) 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 0 2 . 0 9 . 1 7 3 3 . 8 4 2 . 2 6 2 . 1 2 2 . 5 5 . 1 2 9 2 . 3 6 2 . KD_5_118C_CARBON_01 5 1 . 2 7 1 . 6 9 9 5 1 . 1 6 0 3 1 . 9 3 8 2 1 . 9 0 5 1 1 D O 3 D C 0 0 9 4 . 0 3 8 5 . 9 6 5 5 . . 2 5 8 4 1 1 . 4 4 8 6 0 4 . 0 2 7 2 . 2 6 6 2 . 11 O 13 12 8 N1 O 17 14 10 9 CH318 15 16 6 2 5 3 4 SH 7 D O 3 D C 0 0 9 4 . . 2 5 8 4 50 40 30 20 10 0 49.5 49.0 f1 (ppm) 48.5 1000 900 800 700 600 500 400 300 200 100 0 60 55 50 45 40 35 30 25 20 15 10 5 0 -5 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 282 8 F 11 10000 Figure 3.16. 1H NMR and 13C NMR spectra of compound 3-21. KD_5_63_F8-16_PROTON_01 0 6 7 . 8 5 7 . 4 3 7 . 3 3 7 . 3 2 7 . 2 2 7 . 2 2 7 . 1 2 7 . 1 2 7 . 0 2 7 . 0 2 7 . 9 1 . 7 8 1 . 7 8 1 . 7 7 1 . 7 6 1 . 7 4 0 7 . 3 0 7 . 1 0 7 . 4 3 5 . D O 3 D C 1 3 3 . 5 7 2 . 5 4 17 18 16 19 1 2 3 N N 12 13 9 10 7 6 14 S CH315 0 6 7 . 8 5 7 . 4 3 7 . 3 3 7 . 3 2 7 . 2 2 7 . 2 2 7 . 1 2 7 . 1 2 7 . 0 2 7 . 0 2 7 . 9 1 . 7 8 1 . 7 8 1 . 7 7 1 . 7 6 1 . 7 4 0 7 . 3 0 7 . 1 0 7 . 2000 1000 0 0 0 . 1 5 1 . 1 2 3 4 . 8 1 . 2 7.6 7.5 7.4 7.3 f1 (ppm) 7.2 7.1 7.0 0 0 . 1 5 1 . 1 2 3 4 . 8 1 . 2 1 4 2 . 8 3 3 . 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 f1 (ppm) KD_5_63_F8-16_CARBON_01 . 2 7 4 6 1 . 7 7 2 6 1 . 1 7 4 5 1 . 1 2 4 4 1 . 7 2 7 3 1 . 0 3 3 3 1 . 8 2 3 3 1 4 1 . 0 3 1 . 7 0 0 3 1 . 8 5 3 2 1 . 0 4 3 2 1 . 4 4 8 1 1 . 6 6 6 1 1 . 9 4 6 1 1 . 5 6 0 1 1 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 D O 3 D C 0 0 9 4 . 2 6 7 4 . 5 4 17 18 16 19 1 2 3 N N 12 13 8 F 11 8 9 4 1 . 7 9 10 6 14 S CH315 500 400 300 200 100 0 . 8 2 3 3 1 4 1 . 0 3 1 . 7 0 0 3 1 . 8 5 3 2 1 . 0 4 3 2 1 . 4 4 8 1 1 . 6 6 6 1 1 . 9 4 6 1 1 100 50 0 133.3 f1 (ppm) 130 129 128 127 126 125 124 123 f1 (ppm) 122 121 120 119 118 117 116 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 -1000 550 500 450 400 350 300 250 200 150 100 50 0 -50 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 f1 (ppm) 283 Figure 3.17. 1H NMR and 13C NMR spectra of compound 3-4. KD_6_31_F21-22_PROTON_01 2 3 1 4 6 5 9 N N 7 8 10 S 11 16 17 12 15 1 6 7 . 1 6 7 . 0 6 7 . 9 5 7 . 5 3 7 . 5 3 7 . 4 3 7 . 4 3 7 . 4 2 7 . 4 2 7 . 3 2 7 . 3 2 7 . 2 2 7 . 1 2 7 . 1 2 7 . 0 2 7 . 0 2 7 . 9 1 . 7 8 1 . 7 6 1 . 7 4 1 . 7 3 1 . 7 3 1 . 7 2 1 . 7 2 0 7 . 0 0 7 . 8 9 6 . 5 8 6 . 3 8 6 . 5 3 5 . 1 3 4 . 1 3 4 . 0 3 4 . 8 2 4 . 7 2 4 . 5 9 3 . 4 9 3 . 3 9 3 . 2 9 3 . 2 9 3 . 1 9 3 . 0 9 3 . 0 9 3 . 9 8 3 . 8 8 3 . 7 8 3 . 6 8 3 . 5 8 3 . 3 7 3 . 8 6 3 . 7 6 3 . D O 3 D C 1 3 3 . 5 2 3 . 4 2 3 . 2 2 3 . 2 2 3 . 1 2 3 . 0 2 3 . 9 1 . 3 2 0 3 . 2 0 3 . 0 0 3 . 0 0 3 . 9 9 2 . 8 9 2 . 7 9 2 . 9 0 2 . 8 0 2 . 7 0 2 . 6 0 2 . 5 0 2 . 7 9 . 1 6 9 . 1 5 9 . 1 4 9 . 1 3 9 . 1 7 5 . 1 6 5 . 1 4 5 . 1 3 5 . 1 5 3 . 1 4 3 . 1 3 3 . 1 3 3 . 1 2 3 . 1 1 3 . 1 0 3 . 1 19 18 20 23 21 22 F 24 14 N 13 26 28 25 O 33 27 32 31 29 30 O 34 CH335 1 6 7 . 1 6 7 . 0 6 7 . 9 5 7 . 5 3 7 . 5 3 7 . 4 3 7 . 4 3 7 . 4 2 7 . 4 2 7 . 3 2 7 . 3 2 7 . 2 2 7 . 1 2 7 . 1 2 7 . 0 2 7 . 0 2 7 . 9 1 . 7 8 1 . 7 6 1 . 7 4 1 . 7 3 1 . 7 3 1 . 7 2 1 . 7 2 0 7 . 0 0 7 . 8 9 6 . 5 8 6 . 3 8 6 . 1000 800 600 400 200 0 1 3 4 . 0 3 4 . 8 2 4 . 5 9 3 . 4 9 3 . 3 9 3 . 2 9 3 . 2 9 3 . 1 9 3 . 0 9 3 . 0 9 3 . 9 8 3 . 8 8 3 . 7 8 3 . 6 8 3 . 5 8 3 . 3 7 3 . 8 6 3 . 7 6 3 . D O 3 D C 1 3 3 . 5 2 3 . 4 2 3 . 2 2 3 . 2 2 3 . 1 2 3 . 0 2 3 . 9 1 . 3 2 0 3 . 2 0 3 . 0 0 3 . 0 0 3 . 9 9 2 . 8 9 2 . 7 9 2 . 9 0 2 . 8 0 2 . 7 0 2 . 6 0 2 . 5 0 2 . 7 9 . 1 6 9 . 1 5 9 . 1 4 9 . 1 3 9 . 1 7 5 . 1 6 5 . 1 4 5 . 1 3 5 . 1 5 3 . 1 4 3 . 1 3 3 . 1 3 3 . 1 2 3 . 1 1 3 . 1 0 3 . 1 500 0 0 0 . 1 7.6 7.7 6 0 . 1 3 1 . 1 5 0 . 1 0 2 4 . 3 0 2 . 5 2 2 . 7.5 7.4 7.3 7.2 f1 (ppm) 7.1 7.0 6.9 6.8 0 1 . 1 7 2 2 . 1 7 3 . 0 9 . 1 9 1 . 1 4.0 3.5 3 1 . 1 3.0 2.5 f1 (ppm) 4 1 . 1 1 1 . 1 2.0 4 2 . 1 9 4 . 1 1.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 f1 (ppm) 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 0 . 1 6 0 . 1 3 1 . 1 5 0 . 1 0 2 4 . 3 0 2 . 5 2 2 . 2 1 . 2 0 1 . 1 7 2 2 . 1 7 3 . 0 9 . 1 9 1 . 1 3 1 . 1 4 1 . 1 1 1 . 1 4 2 . 1 9 4 . 1 KD_6_31_F21-22_CARBON_01 . 1 2 2 7 1 . 4 6 4 6 1 . 9 6 2 6 1 . 0 0 0 6 1 2 2 . 1 5 1 4 1 . 4 4 1 . 3 8 6 3 1 . 9 3 3 3 1 . 7 3 3 3 1 . 5 6 0 3 1 . 0 0 0 3 1 . 3 9 9 2 1 2 1 . 8 2 1 . 1 0 4 2 1 . 0 7 3 2 1 . 5 8 8 1 1 . 7 6 6 1 1 . 0 5 6 1 1 6 1 . 5 1 1 7 0 . 1 1 1 . 9 3 3 3 1 . 7 3 3 3 1 . 0 0 0 3 1 . 3 9 9 2 1 2 1 . 8 2 1 . 1 0 4 2 1 . 0 7 3 2 1 . 5 8 8 1 1 . 7 6 6 1 1 . 0 5 6 1 1 100 50 0 133.3 f1 (ppm) 130 129 128 127 126 125 124 123 f1 (ppm) 122 121 120 119 118 117 116 D O 3 D C 0 0 9 4 . 6 6 5 5 . 4 7 7 4 . . 6 7 6 4 8 9 4 4 . 5 5 2 4 . 0 6 0 4 . 7 6 3 3 . 4 0 3 3 . 2 3 1 4 6 5 9 N N 7 8 10 S 11 16 17 12 15 19 18 20 23 21 22 F 24 400 300 200 100 0 14 N 13 26 28 25 O 33 27 32 31 29 30 O 34 CH335 2300 2200 2100 2000 1900 1800 1700 1600 1500 1400 1300 1200 1100 1000 900 800 700 600 500 400 300 200 100 0 -100 -200 450 400 350 300 250 200 150 100 50 0 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 f1 (ppm) 284 Figure 3.18. 1H NMR and 13C NMR spectra of compound 3-22. 3 2 7 . 3 2 7 . 2 2 7 . 2 2 7 . 1 2 7 . 5 0 4 . 4 0 4 . 3 0 4 . 2 0 4 . 1 0 4 . 1 0 4 . 0 0 4 . 9 9 3 . 6 9 3 . 8 8 3 . 8 0 3 . 6 0 3 . 3 0 3 . 5 1 . 2 5 1 . 2 4 1 . 2 3 1 . 2 2 1 . 2 1 1 . 2 0 1 . 2 2 7 . 1 1 7 . 1 0 7 . 1 9 6 . 1 7 6 . 1 7 6 . 1 6 4 . 1 l 3 C D C 6 2 7 . KD_6_17_ppt_PROTON_01 2 3 1 4 6 5 9 N NH 7 8 0 9 . 9 6 7 . 8 6 7 . 6 3 7 . 5 3 7 . 8 10 S 11 15 12 14 16 N 13 22 O 23 17 O 18 CH319 CH321 CH320 9 6 7 . 8 6 7 . 6 3 7 . 5 3 7 . l 3 C D C 6 2 7 . 3 2 7 . 3 2 7 . 2 2 7 . 2 2 7 . 1 2 7 . 3000 2000 1000 0 0 0 . 1 7.7 7.6 0 0 . 1 8 0 2 . 7.5 f1 (ppm) 7.4 7.3 7.2 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 f1 (ppm) 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 4 9 0 . 0 0 . 1 0 0 . 1 8 0 2 . 1 3 . 1 9 3 2 . 5 4 2 . 0 5 2 . 0 4 2 . 5 7 9 . KD_6_17_ppt_CARBON_01 . 5 3 9 5 1 . 3 8 4 5 1 . 1 4 8 4 1 . 2 5 5 4 1 . 6 9 2 2 1 . 0 3 2 2 1 . 0 7 8 1 1 . 1 9 4 1 1 l 3 C D C 6 1 . 7 7 2 9 9 7 . 7 1 . 4 4 4 8 3 4 . 6 5 2 3 . 7 5 8 2 . 2 3 1 4 6 5 9 N NH 7 8 10 S 11 15 12 14 16 N 13 22 O 23 17 O 18 CH319 CH321 CH320 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 285 3500 3000 2500 2000 1500 1000 500 0 55 50 45 40 35 30 25 20 15 10 5 0 -5 Figure 3.19. 1H NMR and 13C NMR spectra of compound 3-23. KD_6_21r_F17-23_PROTON_01 2 3 1 4 6 5 9 N N 7 8 10 S 11 15 16 12 14 18 17 19 22 20 21 F 23 24 N 13 30 O 31 25 O 26 CH327 CH329 CH328 2 6 7 . 1 6 7 . 1 4 7 . 9 3 7 . 6 2 7 . 5 2 7 . 5 2 7 . 4 2 7 . 4 2 7 . 3 2 7 . 3 2 7 . 2 2 7 . 2 2 7 . 1 2 7 . 0 2 7 . 9 1 . 7 6 0 7 . 4 0 7 . 3 0 7 . 2 6 7 . 1 6 7 . 1 4 7 . 9 3 7 . 6 2 7 . 5 2 7 . 5 2 7 . 4 2 7 . 4 2 7 . 3 2 7 . 3 2 7 . 2 2 7 . 2 2 7 . 1 2 7 . 0 2 7 . 9 1 . 7 6 0 7 . 4 0 7 . 3 0 7 . 3 4 5 . 8 9 3 . 7 9 3 . 6 9 3 . 5 9 3 . 4 9 3 . 3 9 3 . 2 9 3 . D O 3 D C 1 3 3 . 1 1 . 3 6 0 3 . 5 0 3 . 1 1 . 2 0 1 . 2 9 0 2 . 7 0 2 . 7 0 2 . 6 0 2 . 4 6 . 1 3 6 . 1 2 6 . 1 1 6 . 1 9 5 . 1 8 5 . 1 7 5 . 1 6 5 . 1 5 4 . 1 1000 800 600 400 200 0 8 9 3 . 7 9 3 . 6 9 3 . 5 9 3 . 4 9 3 . 3 9 3 . 2 9 3 . D O 3 D C 1 3 3 . 1 1 . 2 0 1 . 2 9 0 2 . 7 0 2 . 7 0 2 . 6 0 2 . 4 6 . 1 3 6 . 1 2 6 . 1 1 6 . 1 9 5 . 1 8 5 . 1 7 5 . 1 6 5 . 1 5 4 . 1 5 3 . 1 3 3 . 1 2 3 . 1 1 3 . 1 1500 1000 500 0 0 0 . 1 7 0 . 1 3 1 . 2 8 0 2 . 0 0 2 . 7.7 7.6 7.5 7.4 7.3 f1 (ppm) 7.2 7.1 7.0 6.9 3 3 3 . 4.0 3.5 3.0 2.5 f1 (ppm) 5 2 2 . 2.0 6 3 2 . 7 5 9 . 1 5 2 . 1.5 14 13 12 11 10 9 8 7 6 f1 (ppm) 5 0 0 . 1 7 0 . 1 3 1 . 2 8 0 2 . 0 0 2 . 1 2 2 . 3 3 3 . 4 1 2 2 . 3 5 2 2 . 6 3 2 . 7 5 9 . 2 1 0 -1 -2 KD_6_21r_F17-23_CARBON_01 2 3 1 4 6 5 9 N N 7 . 1 7 4 6 1 . 6 7 2 6 1 . 6 2 6 5 1 6 4 . 1 5 1 . 0 2 4 4 1 . 1 9 6 3 1 . 7 5 3 3 1 . 4 5 3 3 1 . 9 0 0 3 1 . 2 0 0 3 1 . 1 0 4 2 1 . 0 7 3 2 1 . 5 8 8 1 1 . 0 7 6 1 1 . 2 5 6 1 1 1 1 . 1 1 1 2 2 . 1 8 D O 3 D C 0 0 9 4 . 8 7 7 4 . 3 3 5 4 . 4 8 4 5 . 8 0 3 3 . 4 6 8 2 . 8 10 S 11 15 16 12 14 18 17 19 22 20 21 F 23 . 7 5 3 3 1 . 4 5 3 3 1 24 N 13 30 O 31 25 O 26 CH327 CH329 CH328 . 9 0 0 3 1 . 2 0 0 3 1 . 0 7 6 1 1 . 2 5 6 1 1 150 100 50 0 300 200 100 0 133.5 133.0 132.5 132.0 131.5 131.0 130.5 130.0 f1 (ppm) 117 116 f1 (ppm) 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 f1 (ppm) 286 2000 1900 1800 1700 1600 1500 1400 1300 1200 1100 1000 900 800 700 600 500 400 300 200 100 0 -100 380 360 340 320 300 280 260 240 220 200 180 160 140 120 100 80 60 40 20 0 -20 Figure 3.20. 1H NMR and 13C NMR spectra of compound 3-24. KD_6_22_PROTON_01 1 7 7 . 8 6 7 . 9 4 7 . 7 4 7 . 4 4 7 . 2 4 7 . 4 2 7 . 2 2 7 . 0 2 7 . 0 0 7 . 7 7 6 . 4 7 6 . 3 7 6 . 1 7 6 . 9 6 6 . 7 3 5 . D O 3 D C 1 3 3 . 0 9 3 . 7 0 3 . 5 0 3 . 6 8 2 . 4 8 2 . 9 7 2 . 1 0 2 . 3 7 . 1 0 7 . 1 2 6 . 1 2 3 1 4 6 5 9 N N 7 8 10 S 11 15 16 12 14 18 17 19 22 20 21 F 23 13 NH 24 ClH 25 13 12 11 10 9 8 7 6 f1 (ppm) 5 6 2 . 1 8 3 . 1 4 1 . 2 4 9 . 1 4 1 . 2 5 9 . 1 1 0 . 1 4 5 0 2 . 2 5 2 . 3 3 1 . 2 2 0 2 . 2 1 0 -1 -2 KD_6_22_CARBON_01 7 1 . 5 6 1 . 6 2 3 6 1 . 3 6 9 4 1 . 8 2 4 3 1 . 3 3 3 3 1 2 1 . 1 3 1 5 0 . 1 3 1 . 7 7 9 2 1 . 7 2 8 2 1 . 2 0 8 2 1 . 1 9 6 2 1 . 8 2 7 1 1 1 1 . 7 1 1 7 1 . 5 1 1 2 1 . 4 1 1 D O 3 D C 0 0 9 4 . 9 4 0 5 . . 6 7 4 4 5 8 7 3 . 6 3 2 3 . 2 3 1 4 6 5 9 N N 7 8 10 S 11 15 16 12 14 18 17 19 22 20 21 F 23 13 NH 24 ClH 25 2 1 . 1 3 1 5 0 . 1 3 1 . 7 7 9 2 1 . 7 2 8 2 1 . 2 0 8 2 1 . 1 9 6 2 1 80 60 40 20 0 . 8 2 7 1 1 1 1 . 7 1 1 70 60 50 40 30 20 10 0 131 130 129 f1 (ppm) 128 127 118 117 f1 (ppm) 200 190 180 170 160 150 140 130 120 110 100 f1 (ppm) 90 80 70 60 50 40 30 20 10 0 287 2500 2000 1500 1000 500 0 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 3.21. 1H NMR and 13C NMR spectra of compound 3-25. KD_6_90_F2-4_PROTON_01 2 3 1 4 6 5 9 N S 7 8 8 7 . 7 8 7 . 7 7 7 . 5 7 7 . 8 10 S 11 15 12 14 16 N 13 22 O 23 17 O 18 CH319 CH321 CH320 800 600 400 200 0 8 8 7 . 7 8 7 . 7 7 7 . 5 7 7 . 4 4 7 . 3 4 7 . 2 4 7 . 2 4 7 . 2 4 7 . 1 4 7 . 0 4 7 . 2 3 7 . 2 3 7 . 1 3 7 . 1 3 7 . 1 3 7 . 9 2 7 . 9 2 7 . l 3 C D C 6 2 7 . 2 1 . 4 2 1 . 4 1 1 . 4 0 1 . 4 0 1 . 4 9 0 4 . 8 0 4 . 8 0 4 . 7 0 4 . 6 9 3 . 4 1 . 3 2 1 . 3 0 1 . 3 1 2 2 . 0 2 2 . 0 2 2 . 9 1 . 2 8 1 . 2 7 1 . 2 6 1 . 2 8 7 . 1 7 7 . 1 6 7 . 1 5 7 . 1 3 7 . 1 2 7 . 1 1 7 . 1 0 7 . 1 7 4 . 1 4 4 7 . 3 4 7 . 2 4 7 . 2 4 7 . 2 4 7 . 1 4 7 . 0 4 7 . 2 3 7 . 2 3 7 . 1 3 7 . 1 3 7 . 1 3 7 . 9 2 7 . 9 2 7 . 2 1 . 4 2 1 . 4 1 1 . 4 0 1 . 4 0 1 . 4 9 0 4 . 8 0 4 . 8 0 4 . 7 0 4 . 6 9 3 . 4 1 . 3 2 1 . 3 0 1 . 3 1 2 2 . 0 2 2 . 0 2 2 . 9 1 . 2 8 1 . 2 7 1 . 2 6 1 . 2 8 7 . 1 7 7 . 1 6 7 . 1 5 7 . 1 3 7 . 1 2 7 . 1 1 7 . 1 0 7 . 1 800 600 400 200 0 3 8 0 . 7.9 3 8 0 . 7.8 f1 (ppm) 3 9 0 . 7.45 7.40 7.35 f1 (ppm) 8 8 0 . 7.30 0 0 . 1 0 9 . 1 4.0 3.5 8 9 . 1 3.0 f1 (ppm) 5 0 2 . 5 0 2 . 2.5 2.0 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 f1 (ppm) 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 KD_6_90_F2-4_CARBON_01 . 6 2 5 6 1 . 0 8 4 5 1 . 1 4 3 5 1 . 3 4 5 3 1 . 1 2 6 2 1 . 6 5 4 2 1 9 7 . 1 2 1 3 1 . 1 2 1 l 3 C D C 6 1 . 7 7 3 9 9 7 . . 4 7 4 4 0 2 2 3 . 3 6 8 2 . 7 5 8 2 . 3 8 0 . 3 8 0 . 3 9 0 . 8 8 0 . 0 0 . 1 0 9 . 1 8 9 . 1 5 0 2 . 5 0 2 . 8 1 . 0 1 2 3 1 4 6 5 9 N S 7 8 10 S 11 15 12 14 16 N 13 22 O 23 17 O 18 CH319 CH321 CH320 100 80 60 40 20 0 3 6 8 2 . 7 5 8 2 . 28.8 28.4 f1 (ppm) 2000 1900 1800 1700 1600 1500 1400 1300 1200 1100 1000 900 600 500 400 300 200 100 800 0 700 600 500 400 300 200 100 0 -100 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 -5 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 288 Figure 3.22. 1H NMR and 13C NMR spectra of compound 3-26. KD_6_95_F17-19_PROTON_01 1 9 7 . 9 8 7 . 7 8 7 . 5 8 7 . 9 4 7 . 9 4 7 . 8 4 7 . 8 4 7 . 8 4 7 . 6 4 7 . 6 4 7 . 9 3 7 . 9 3 7 . 8 3 7 . 8 3 7 . 7 3 7 . 6 3 7 . 6 3 7 . 6 2 4 . 5 2 4 . 5 2 4 . 4 2 4 . 3 2 4 . 3 2 4 . 2 2 4 . 1 2 4 . 0 2 4 . 0 5 3 . 9 4 3 . 9 4 3 . 7 4 3 . 7 4 3 . 6 4 3 . D O 3 D C 1 3 3 . 8 2 3 . 7 2 3 . 6 2 3 . 5 2 3 . 1 5 2 . 1 5 2 . 0 5 2 . 9 4 2 . 8 4 2 . 7 4 2 . 6 4 2 . 2 1 . 2 1 1 . 2 0 1 . 2 9 0 2 . 8 0 2 . 8 0 2 . 7 0 2 . 6 0 2 . 5 0 2 . 4 0 2 . 2 3 1 4 6 5 9 N S 7 8 10 S 11 15 12 14 13 NH 16 ClH 17 1 9 7 . 0 9 7 . 7 8 7 . 6 8 7 . 0 5 7 . 0 5 7 . 8 4 7 . 8 4 7 . 8 4 7 . 7 4 7 . 7 4 7 . 0 4 7 . 9 3 7 . 8 3 7 . 8 3 7 . 8 3 7 . 7 3 7 . 6 3 7 . 400 300 200 100 0 200 150 100 50 0 6 2 4 . 5 2 4 . 5 2 4 . 4 2 4 . 3 2 4 . 3 2 4 . 2 2 4 . 1 2 4 . 0 2 4 . D O 3 D C 1 3 3 . 8 2 3 . 7 2 3 . 6 2 3 . 5 2 3 . 0 5 3 . 9 4 3 . 9 4 3 . 7 4 3 . 7 4 3 . 6 4 3 . 1 5 2 . 1 5 2 . 0 5 2 . 9 4 2 . 8 4 2 . 7 4 2 . 6 4 2 . 2 1 . 2 1 1 . 2 0 1 . 2 9 0 2 . 8 0 2 . 8 0 2 . 7 0 2 . 6 0 2 . 5 0 2 . 4 0 2 . 4 9 0 . 0 0 . 1 7.85 7.90 f1 (ppm) 5 0 . 1 4 0 . 1 7.50 7.45 7.40 f1 (ppm) 7 1 . 1 4.2 0 5 2 . 9 1 . 2 6 2 2 . 6 2 2 . 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 f1 (ppm) 750 700 650 600 550 500 250 200 450 150 400 100 350 50 0 4 9 0 . 0 0 . 1 5 0 . 1 4 0 . 1 7 1 . 1 0 5 2 . 9 1 . 2 6 2 2 . 6 2 2 . 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 f1 (ppm) KD_6_95_F17-19_CARBON_01 . 8 8 5 6 1 . 5 3 4 5 1 . 4 4 6 3 1 . 3 5 7 2 1 . 3 0 6 2 1 . 1 5 2 2 1 . 8 4 2 2 1 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 D O 3 D C 0 0 9 4 . 0 5 4 4 . 5 5 2 4 . 3 0 0 3 . 2 3 1 4 6 5 9 N S 7 8 10 S 11 15 12 14 13 NH 16 ClH 17 3 1 . 1 2 1 0 1 . 1 2 1 30 20 10 0 121.2 121.0 f1 (ppm) 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 289 300 250 200 150 100 50 0 -50 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 -5 Figure 3.23. 1H NMR and 13C NMR spectra of compound 3-5. 6 8 7 . 5 8 7 . 3 8 7 . 1 8 7 . 7 4 7 . 5 4 7 . 3 4 7 . 6 3 7 . 4 3 7 . 3 3 7 . 8 1 . 7 6 1 . 7 9 8 6 . 7 8 6 . 6 3 4 . 5 3 4 . 5 3 4 . 4 3 4 . 4 3 4 . 3 3 4 . 3 3 4 . 2 3 4 . 2 3 4 . 1 3 4 . 1 3 4 . 2 1 . 4 2 1 . 4 1 1 . 4 0 1 . 4 0 1 . 4 9 0 4 . 8 0 4 . 8 0 4 . 7 0 4 . 8 9 3 . 8 9 3 . 7 9 3 . 7 9 3 . 6 9 3 . 6 9 3 . 5 9 3 . 5 9 3 . 4 9 3 . 4 9 3 . 4 9 3 . 3 9 3 . 6 7 3 . 3 7 3 . 2 7 3 . 5 3 3 . 4 3 3 . 3 3 3 . 2 3 3 . D O 3 D C 1 3 3 . 3 1 . 3 2 1 . 3 1 1 . 3 0 1 . 3 0 1 . 3 8 0 3 . 7 0 3 . 3 2 2 . 2 2 2 . 1 2 2 . 0 2 2 . 9 1 . 2 8 1 . 2 0 1 . 2 9 0 2 . 8 0 2 . 7 0 2 . 6 0 2 . 0 7 . 1 9 6 . 1 8 6 . 1 7 6 . 1 7 6 . 1 6 6 . 1 5 6 . 1 5 6 . 1 3 6 . 1 2 6 . 1 0 5 . 1 0 5 . 1 8 4 . 1 8 4 . 1 7 4 . 1 6 4 . 1 6 4 . 1 5 4 . 1 4 4 . 1 3 4 . 1 3200 KD_6_101_dry_PROTON_01 2 3 1 4 6 5 9 N S 7 8 10 S 11 16 12 15 14 N 13 18 20 17 O 25 19 24 23 21 22 O 26 CH327 6 8 7 . 5 8 7 . 3 8 7 . 1 8 7 . 7 4 7 . 5 4 7 . 3 4 7 . 6 3 7 . 4 3 7 . 3 3 7 . 8 1 . 7 6 1 . 7 9 8 6 . 7 8 6 . 2000 3 7 3 . 2 7 3 . D O 3 D C 1 3 3 . 7 3 3 . 5 3 3 . 4 3 3 . 3 3 3 . 2 3 3 . 3 1 . 3 2 1 . 3 1 1 . 3 0 1 . 3 0 1 . 3 8 0 3 . 7 0 3 . 1500 1000 500 0 0 0 . 1 0 0 . 1 2 1 . 1 2 1 . 1 1 3 2 . 8 3 2 . 7.8 7.6 7.4 f1 (ppm) 7.2 7.0 6.8 7 9 . 1 8 7 . 1 3.7 3.6 3.5 3.4 f1 (ppm) 3.3 3.2 2 0 . 1 3.1 1200 1000 800 600 400 200 0 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 f1 (ppm) 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 0 . 1 0 0 . 1 2 1 . 1 2 1 . 1 1 3 2 . 8 3 2 . 5 0 . 1 5 1 . 1 7 1 . 1 2 4 3 . 7 9 . 1 8 7 . 1 2 0 . 1 2 1 . 1 5 0 . 1 9 1 . 1 0 2 . 1 . 8 3 2 7 1 . 3 8 6 6 1 2 1 . 0 6 1 . 7 3 4 5 1 . 7 3 6 3 1 . 0 7 0 3 1 9 1 . 8 2 1 . 5 4 7 2 1 . 6 8 5 2 1 . 8 3 2 2 1 . 6 3 2 2 1 . 1 2 5 1 1 D O 3 D C 0 0 9 4 . 8 6 5 5 . . 7 8 6 4 5 3 5 4 . 4 6 2 4 . 2 6 0 4 . 3 5 3 3 . 7 9 2 3 . KD_6_101_dry_CARBON_01 2 3 1 4 6 5 9 N S 7 8 10 S 11 16 12 15 14 N 13 18 20 17 O 25 19 24 23 21 22 O 26 CH327 . 8 3 2 2 1 . 6 3 2 2 1 50 0 122.4 122.1 f1 (ppm) 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 290 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0 -200 650 600 550 500 450 400 350 300 250 200 150 100 50 0 -50 Figure 3.24. 1H NMR and 13C NMR spectra of compound 3-6. KD_6_136_dry_PROTON_01 2 3 1 4 6 5 9 N NH 7 10 NH 8 11 16 12 15 14 N 13 8 3 7 . 7 3 7 . 7 3 7 . 6 3 7 . 5 3 7 . 4 2 7 . 1 2 7 . 9 1 . 7 0 9 6 . 8 8 6 . 1000 17 O 25 18 19 24 800 20 23 21 22 O 26 CH327 600 400 200 0 8 3 7 . 7 3 7 . 7 3 7 . 6 3 7 . 5 3 7 . 4 2 7 . 1 2 7 . 9 1 . 7 0 9 6 . 8 8 6 . 4 5 4 . 2 5 4 . 2 1 . 4 0 1 . 4 0 1 . 4 9 0 4 . 9 0 4 . 7 0 4 . 7 0 4 . 6 0 4 . 6 0 4 . 4 0 4 . 3 0 4 . 9 9 3 . 7 9 3 . 8 8 3 . 7 8 3 . 6 8 3 . 6 8 3 . 5 8 3 . 5 8 3 . 4 8 3 . 3 8 3 . 2 8 3 . 1 8 3 . 9 7 3 . 7 7 3 . 6 7 3 . D O 3 D C 1 3 3 . 9 2 3 . 8 2 3 . 6 2 3 . 6 9 2 . 3 9 2 . 0 9 2 . 0 0 2 . 0 0 2 . 7 9 . 1 7 9 . 1 7 8 . 1 7 8 . 1 5 8 . 1 5 8 . 1 0 5 . 1 8 4 . 1 7 4 . 1 6 4 . 1 5 4 . 1 5 4 . 1 3 4 . 1 7 3 . 1 6 3 . 1 2 1 . 4 0 1 . 4 0 1 . 4 9 0 4 . 9 0 4 . 7 0 4 . 7 0 4 . 6 0 4 . 6 0 4 . 4 0 4 . 3 0 4 . 9 9 3 . 7 9 3 . 8 8 3 . 7 8 3 . 6 8 3 . 6 8 3 . 5 8 3 . 5 8 3 . 4 8 3 . 3 8 3 . 2 8 3 . 1 8 3 . 9 7 3 . 7 7 3 . 6 7 3 . D O 3 D C 1 3 3 . 9 2 3 . 8 2 3 . 6 2 3 . 300 D O 3 D C 1 3 3 . 9 2 3 . 8 2 3 . 6 2 3 . 6 9 2 . 3 9 2 . 0 9 2 . 0 0 2 . 0 0 2 . 7 9 . 1 7 9 . 1 7 8 . 1 7 8 . 1 5 8 . 1 5 8 . 1 0 5 . 1 8 4 . 1 7 4 . 1 6 4 . 1 5 4 . 1 5 4 . 1 3 4 . 1 7 3 . 1 6 3 . 1 200 100 0 1000 900 800 700 600 400 300 200 100 500 0 5 2 2 . 7.4 8 0 2 . 9 3 2 . 7.2 0 0 2 . 7.0 6.8 f1 (ppm) 9 3 . 1 3 1 . 1 2 7 4 . 2 2 . 1 4.1 4.0 3.9 3.8 3.7 f1 (ppm) 3.6 3.5 3.4 3.3 2 2 . 1 8 1 . 1 3.0 5 1 . 1 1 2 . 1 2.0 9 4 . 1 7 1 . 1 1.5 2.5 f1 (ppm) 14 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 -2 5 2 2 . 8 0 2 . 9 3 2 . 0 0 2 . 9 9 0 . 9 3 . 1 3 1 . 1 2 7 4 . 2 2 . 1 8 1 . 1 5 1 . 1 1 2 . 1 9 4 . 1 7 1 . 1 KD_6_136_dry_CARBON_01 2 3 1 4 6 5 9 N NH 7 . 2 4 2 7 1 . 9 0 0 6 1 . 0 8 0 5 1 5 7 . 1 3 1 . 4 7 0 3 1 . 1 2 8 2 1 . 0 5 4 2 1 . 2 2 5 1 1 . 2 4 2 1 1 D O 3 D C 0 0 9 4 . . 7 9 5 4 1 9 . 1 5 . 0 7 5 5 9 5 . 1 4 4 6 0 4 . 2 0 3 3 . 4 4 2 3 . 10 NH 8 11 16 12 15 14 N 13 18 20 17 O 25 19 24 23 21 22 O 26 CH327 5 7 . 1 3 1 . 4 7 0 3 1 . 1 2 8 2 1 . 0 5 4 2 1 . 2 2 5 1 1 . 2 4 2 1 1 70 60 50 40 30 20 10 0 130 125 120 115 f1 (ppm) 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 f1 (ppm) 291 400 300 200 100 0 650 600 550 500 450 400 350 300 250 200 150 100 50 0 -50 Figure 3.25. 1H NMR and 13C NMR spectra of compound 3-27. KD_6_106C_PROTON_01 2 6 7 . 1 6 7 . 1 6 7 . 0 6 7 . 5 4 7 . 4 4 7 . 3 4 7 . 3 4 7 . 3 3 7 . 2 3 7 . 0 3 7 . 0 3 7 . 9 2 7 . 8 2 7 . 8 2 7 . 7 2 7 . 6 2 7 . 0 2 7 . 8 1 . 7 9 4 5 . D O 3 D C 1 3 3 . 2 3 1 4 6 5 9 N N 7 8 Cl 10 11 13 12 14 17 15 16 2 6 7 . 2 6 7 . 1 6 7 . 0 6 7 . 5 4 7 . 4 4 7 . 4 4 7 . 3 4 7 . 3 3 7 . 2 3 7 . 1 3 7 . 0 3 7 . 9 2 7 . 9 2 7 . 8 2 7 . 7 2 7 . 6 2 7 . 0 2 7 . 9 1 . 7 0 0 . 1 7.6 7.7 0 1 . 1 7.5 7.4 f1 (ppm) 5 5 5 . 7.3 8 0 2 . 7.2 1200 1000 800 600 400 200 0 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 f1 (ppm) 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 0 0 . 1 0 1 . 1 5 5 5 . 8 0 2 . 6 4 2 . KD_6_106C_CARBON_01 . 6 2 2 4 1 8 9 . 1 4 1 . 3 8 6 3 1 . 4 3 6 3 1 . 1 0 0 3 1 7 1 . 9 2 1 . 6 9 7 2 1 . 5 8 4 2 1 . 3 3 4 2 1 . 1 5 9 1 1 4 7 . 1 1 1 2 3 1 4 6 5 9 N N 7 8 Cl 10 11 13 12 14 17 15 16 . 6 2 2 4 1 8 9 . 1 4 1 . 3 8 6 3 1 . 4 3 6 3 1 150 100 50 0 D O 3 D C 0 0 9 4 . . 3 7 8 4 300 200 100 0 D O 3 D C 0 0 9 4 . . 3 7 8 4 142 140 138 136 f1 (ppm) 50.0 49.5 49.0 f1 (ppm) 48.5 48.0 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 f1 (ppm) 292 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0 -200 450 400 350 300 250 200 150 100 50 0 Figure 3.26. 1H NMR and 13C NMR spectra of compound 3-28. 5 2 7 . 9 1 . 7 9 1 . 7 8 1 . 7 8 1 . 7 7 1 . 7 2 0 7 . 1 0 7 . 0 0 7 . 9 9 6 . 6 9 6 . 4 9 6 . 3 9 6 . 0 4 4 . 8 3 4 . 7 3 4 . 9 0 3 . 8 0 3 . 6 0 3 . l 3 C D C 6 2 7 . 0 7 7 . 0 7 7 . 9 6 7 . 9 6 7 . 9 2 7 . 9 2 7 . 8 2 7 . 8 2 7 . 7 2 7 . 6 2 7 . 6 2 7 . KD_7_5_F15-18_PROTON_01 2 3 1 4 6 5 9 N N 7 8 Cl 10 11 12 13 18 14 17 15 16 F 19 0 7 7 . 0 7 7 . 9 6 7 . 9 6 7 . 9 2 7 . 8 2 7 . 8 2 7 . 7 2 7 . 6 2 7 . 6 2 7 . l 3 C D C 6 2 7 . 0 4 4 . 8 3 4 . 7 3 4 . 9 0 3 . 8 0 3 . 6 0 3 . 5 2 7 . 9 1 . 7 9 1 . 7 8 1 . 7 8 1 . 7 7 1 . 7 2 0 7 . 1 0 7 . 0 0 7 . 9 9 6 . 6 9 6 . 4 9 6 . 3 9 6 . 5000 7000 6000 5000 4000 3000 2000 1000 0 0 0 . 1 7.7 9 4 2 . 4 1 . 1 7.6 7.5 7.4 7.3 7.2 7.1 f1 (ppm) 0 3 1 . 2 9 1 . 2 7.0 6.9 0 0 . 1 9 4 2 . 4 1 . 1 9 1 . 2 3 1 . 2 3 5 2 . 3 5 2 . 14 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 KD_7_5_F15-18_CARBON_01 . 9 0 3 6 1 4 1 . 1 6 1 7 7 . 1 4 1 . 1 6 0 4 1 . 4 8 4 3 1 . 0 9 2 3 1 . 7 8 2 3 1 . 3 4 0 3 1 . 7 3 0 3 1 . 0 3 3 2 1 . 4 8 2 2 1 . 8 6 9 1 1 . 9 8 5 1 1 . 2 7 5 1 1 . 6 3 9 0 1 . 0 9 2 3 1 . 7 8 2 3 1 . 3 4 0 3 1 . 7 3 0 3 1 . 9 8 5 1 1 . 2 7 5 1 1 150 100 50 0 250 200 150 100 50 0 133.0 132.5 132.0 131.5 f1 (ppm) 131.0 130.5 116.1 115.5 f1 (ppm) 4.0 3.5 3.0 f1 (ppm) 2 5 2 . -1000 2 5 2 . 3 l 3 C D C 6 1 . 7 7 2 1 0 -1 -2 9 0 6 4 . 0 9 4 3 . 1 4 2 3 6 5 9 N N 7 8 Cl 10 11 12 13 18 14 17 15 16 F 19 8500 8000 7500 7000 6500 6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 -500 400 350 300 250 200 150 100 50 0 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 f1 (ppm) 293 Figure 3.27. 1H NMR and 13C NMR spectra of compound 3-29. KD_6_104_F11-15_2_PROTON_01 3 5 7 . 2 5 7 . 1 5 7 . 0 5 7 . 9 3 7 . 8 3 7 . 6 3 7 . 1 3 7 . 9 2 7 . 2 1 . 7 1 1 . 7 9 0 7 . 0 0 7 . 9 9 6 . 7 9 6 . 8 8 6 . 7 8 6 . D O 3 D C 1 3 3 . 2 3 1 4 6 5 9 N N 7 8 NH210 11 16 12 15 13 14 F 17 3 5 7 . 2 5 7 . 1 5 7 . 0 5 7 . 9 3 7 . 8 3 7 . 6 3 7 . 1 3 7 . 9 2 7 . 2 1 . 7 1 1 . 7 9 0 7 . 0 0 7 . 9 9 6 . 7 9 6 . 8 8 6 . 7 8 6 . 2500 2000 1500 1000 500 0 5 0 2 . 4 0 2 . 7.6 7.5 7.4 4 1 . 1 7.3 8 0 . 1 7.1 7 0 . 1 7.0 0 0 . 1 6.9 6.8 7.2 f1 (ppm) 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 f1 (ppm) 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 5 0 2 . 4 0 2 . 4 1 . 1 8 0 . 1 7 0 . 1 0 0 . 1 KD_6_104_F11-15_2_CARBON_01 . 2 0 5 6 1 . 5 0 3 6 1 . 7 5 5 5 1 . 8 5 3 4 1 1 9 . 1 4 1 . 3 0 2 3 1 . 3 6 0 3 1 . 6 5 0 3 1 . 2 3 3 2 1 1 4 . 1 2 1 . 4 3 8 1 1 6 1 . 8 1 1 . 6 8 5 1 1 . 7 2 9 0 1 D O 3 D C 0 0 9 4 . 2 3 1 4 6 5 9 N N 7 8 NH210 11 16 12 15 13 14 F 17 . 3 0 2 3 1 . 3 6 0 3 1 . 6 5 0 3 1 40 30 20 10 0 . 4 3 8 1 1 6 1 . 8 1 1 120 100 80 60 40 20 0 132.0 131.5 131.0 130.5 f1 (ppm) 117.9 f1 (ppm) 200 190 180 170 160 150 140 130 120 110 100 f1 (ppm) 90 80 70 60 50 40 30 20 10 0 294 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0 -200 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 3.28. 1H NMR and 13C NMR spectra of compound 3-30. KD_6_110_F14-17_PROTON_01 5 5 7 . 4 5 7 . 3 5 7 . 2 5 7 . 0 4 7 . 9 3 7 . 7 3 7 . 2 3 7 . 1 3 7 . 4 1 . 7 4 1 . 7 3 1 . 7 3 1 . 7 1 1 . 7 1 1 . 7 3 0 7 . 3 0 7 . 1 0 7 . 1 0 7 . 0 0 7 . 0 0 7 . 0 9 6 . 8 8 6 . 8 8 4 . 7 8 4 . 6 8 4 . 6 8 4 . 0 7 3 . 9 6 3 . 8 6 3 . 7 6 3 . 6 6 3 . 6 6 3 . 5 6 3 . 3 3 3 . D O 3 D C 1 3 3 . 0 0 2 . 9 9 . 1 8 9 . 1 7 9 . 1 7 9 . 1 6 9 . 1 6 9 . 1 5 9 . 1 4 9 . 1 9 7 . 1 8 7 . 1 7 7 . 1 7 7 . 1 6 7 . 1 5 7 . 1 4 7 . 1 3 7 . 1 2 7 . 1 6 4 . 1 2 3 1 4 6 5 9 N N 7 11 16 12 15 13 14 F 17 29 CH3 30 CH3 27 CH328 25 O 24 O 26 21 N 20 19 22 18 23 8 NH 10 5 5 7 . 4 5 7 . 3 5 7 . 2 5 7 . 0 4 7 . 9 3 7 . 7 3 7 . 2 3 7 . 1 3 7 . 4 1 . 7 4 1 . 7 3 1 . 7 3 1 . 7 1 1 . 7 1 1 . 7 3 0 7 . 3 0 7 . 1 0 7 . 1 0 7 . 0 0 7 . 0 0 7 . 0 9 6 . 8 8 6 . 8 8 4 . 7 8 4 . 6 8 4 . 6 8 4 . 1000 800 600 400 200 0 7 5 2 . 7.5 0 6 2 . 7.4 6 3 . 1 7.3 7.2 f1 (ppm) 7 3 . 1 7 3 . 1 7.1 7.0 0 3 . 1 6.9 500 400 300 200 100 0 D O 3 D C 1 3 3 . 0 7 3 . 9 6 3 . 8 6 3 . 7 6 3 . 6 6 3 . 6 6 3 . 5 6 3 . 3 3 3 . 0 0 2 . 9 9 . 1 8 9 . 1 7 9 . 1 7 9 . 1 6 9 . 1 6 9 . 1 5 9 . 1 4 9 . 1 9 7 . 1 8 7 . 1 7 7 . 1 7 7 . 1 6 7 . 1 5 7 . 1 4 7 . 1 3 7 . 1 2 7 . 1 500 400 300 2500 200 100 2000 0 7 9 . 1 0 0 2 . 1500 6 5 . 1 -100 0 9 . 1 9 0 2 . 4.92 4.86 f1 (ppm) 3.5 3.0 2.5 2.0 f1 (ppm) 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 KD_6_110_F14-17_2D_CARBON_01 7 5 2 . 0 6 2 . 6 3 . 1 7 3 . 1 7 3 . 1 0 3 . 1 6 5 . 1 5.0 f1 (ppm) 0 9 . 1 9 0 2 . 7 9 . 1 0 0 2 . 7 5 9 . 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 D O 3 D C 0 0 9 4 . 5 4 6 4 . 4 6 2 3 . . 1 6 8 2 2 1 . 5 6 1 5 1 . 3 6 1 . 3 3 6 5 1 . 5 2 5 5 1 . 0 9 0 4 1 . 5 8 5 3 1 2 7 . 1 3 1 . 0 7 0 3 1 . 3 6 0 3 1 . 4 5 3 2 1 3 7 . 1 2 1 . 1 4 8 1 1 . 2 2 8 1 1 . 9 5 5 1 1 . 4 4 9 0 1 5 3 . 1 8 6 9 8 7 . 29 CH3 30 CH3 27 CH328 25 O 24 O 26 21 N 20 19 22 18 23 8 NH 10 2 3 1 4 6 5 9 N N 7 11 16 12 15 13 14 F 17 2 7 . 1 3 1 . 0 7 0 3 1 . 3 6 0 3 1 60 50 40 30 20 10 0 . 1 4 8 1 1 . 2 2 8 1 1 80 60 40 20 0 131.8 131.6 131.4 131.2 f1 (ppm) 131.0 130.8 130.6 118.5 118.2 f1 (ppm) 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 295 5000 4500 4000 3500 3000 1000 500 0 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 Chapter 4: Conclusions and Future Works To conclude, this thesis covers the work that I completed while in the graduate program in Dr. Jetze Tepe’s lab at Michigan State University. My work has focused on the application of nitrogen-containing heterocycles in the total synthesis of bis- and tris-indole natural products and in the design and synthesis of small molecule acyl Astemizole analogues to be explored as 20S proteasome modulators. Progress toward the total synthesis of Tulogicin and its related bis-indole natural product analogues, including the associated challenges of various synthetic approaches, were discussed. Though incomplete, the results of these studies have provided valuable insights to, hopefully, allow for future achievement in the total syntheses of Tulongicin and its related analogues. Currently, further research building upon these findings toward the first total synthesis of Tulongicin is underway in the Tepe lab. Future work for the potential continuation of this project would not only include the synthesis of Tulongicin and its related Bis-indole natural products but would also include further exploration of their biological activities, which remain largely unknown. Lastly, the design and synthesis of a small library of acyl Astemizole analogues, in collaboration with Dr. Allison Vanecek, was discussed. Several of these analogues displayed potent 20S proteasome activity, which has provided valuable insight into the relationship between multiple structural moieties of acyl Astemizole and its biological activity. The findings of these studies will be useful in future analogue development, as acyl Astemizole remains a promising target in the Tepe lab. In particular, the 4-fluorobenzyl group of acyl Astemizole was identified as very important for its biological activity. These findings inspired the design of several novel acyl Astemizole analogues. As future work for this project, these novel analogues 296 could be synthesized and tested for 20S proteasome activity to further explore the structure- activity relationship of acyl Astemizole. 297