FIRST TOTAL SYNTHESIS OF NAGELAMIDE W AND DEVELOPMENT OF NITROGEN CONTAINING HETEROCYCLIC COMPOUNDS AS 20S PROTEASOME MODULATORS By Dare E. George A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Chemistry – Doctor of Philosophy 2024 ABSTRACT Nitrogen-containing heterocyclic compounds are vital in organic chemistry due to their unique properties and reactivity, playing key roles in pharmaceuticals, agrochemicals, and functional materials. Over 75% of FDA-approved drugs include these heterocycles, underscoring their importance in modern medicine. The discovery of novel N-heterocyclic compounds with significant biological activities is rapidly expanding, driven by advances in synthetic methodologies like transition metal-catalysis and biocatalysis. Looking ahead, the importance of N-heterocyclic compounds is expected to grow. The advent of new technologies and an increasing understanding of disease mechanisms will likely drive the demand for novel pharmaceuticals incorporating nitrogen heterocycles. This trend is anticipated to lead to the discovery of new drugs with improved efficacy and safety profiles, addressing unmet medical needs and enhancing patient outcomes. This dissertation focuses on developing new methods for synthesizing N-heterocyclic compounds, specifically pyrazoles and pyridines, through reactions of epoxides with hydrazine and ammonia, respectively. Both established and novel methodologies were also utilized to synthesize the nitrogen-containing natural product nagelamide W in 11 linear steps, resulting in an overall yield of 6%. Additionally, N-heterocyclic compounds were evaluated as 20S proteasome modulators and used as chemical probes to elucidate their binding sites on the 20S proteasome. These efforts aim to enhance the understanding and application of N-heterocycles in medicinal and biological sciences. ACKNOWLEDGEMENTS First, I would like to thank my advisor, Dr. Jetze Tepe for your unwavering support throughout my PhD journey. Thank you for believing in me, constantly motivating me, pushing me to my full potential, and giving me the space to explore ideas and grow to be the independent scientist that I am today. Your mentorship and support have made my PhD journey a memorable one. I am also grateful for the fellowship and program opportunities you forwarded my way and encouraged me to apply for. Lastly, thank you for assembling such an amazing, brilliant, and supportive group of people with whom I have had the pleasure of working over the past five years. It has been a great honor to be part of your group, and I appreciate the opportunity immensely. To my lab mates, current and past, thank you for your support and being part of my PhD experience. To Shafaat – thank you for training me when I first joined the lab, even though I have little to no experience, your willingness to train me and answer my questions is one of the foundations of the chemist I am today. To Katarina, Grace, Taylor, Sophie, and Allison – thank you all for your suggestion, guidance, and always checking how things are going with me in the lab. To Charles and Konika, thank you for walking this journey with me every step of the way. From, first committee meeting, to second year oral, and to seminar. Thank you for being such good and supportive friends. To Evan, you became my lab buddy; brainstorming and troubleshooting reactions with you were invaluable experiences that greatly enriched my research and made the lab an enjoyable and collaborative environment. To Allison, Sydney, and Shannon, thank you for your collaboration and being such a wonderful teammate. To Daniel, Kyra, Bahar, Miracle, Ayoob, Christi, and Linh – thank you for supports and for being wonderful lab mates. It’s being an honor to have worked alongside every one of you. iii To my committee members, Dr. Huang, Dr. Draths, and Dr. Maleczka, thank you for your supports, guidance and invaluable feedback over the years. I have learned a great deal from each of you, whether through our meetings or by taking your class. To Dr. Wulff, it has been a pleasure learning from you during my time at MSU. Taking your classes and working on the natural product database with you have been invaluable sources of knowledge. Thank you for sharing your expertise in chemistry, as well as insights and stories beyond the field. I would also like to thank Dr. Tony Schilmiller for helping me with mass spectrometry over the years, and Dr. Dan Holmes and Dr. Li Xie for their help with my NMR problems. I would also like to extend my gratitude to the people from my undergraduate university. To my mentors, Dr. Tomide Fadare and Dr. Feyisola Olatunji, thank you for your constant motivation and support. You both gave me the confidence to pursue a PhD, and without your guidance, my journey might have taken a different path. To my OAU friends, Fiyinfoluwa, Felix, Glory, and Raheemat, I appreciate you and thank you for being such great friends. To my childhood friend, Emmanuel, thank you for being such an amazing friend that I could always rely on. To my parents and siblings, I love you all and hope I have made you proud. Thank you for your endless support, encouragement, and for constantly checking up on me. Mom, I love you so much. I couldn't have done this without you – your prayers and motivation kept me going. To all my friends within the department and outside, thank you for being such wonderful friends. Bismarck, Ryan, and Konika – we call ourselves the “best four”. Thank you for being there throughout this PhD journey. To the Nigerian community and my close friends – Michael, Chidiogo, Femi, Ebenezer, Daniel, Rofiat, Omolade, Sodiq, Paninga, Kunle, Mark, Emmanuel, John, and many others – you all became my family in the U.S. and have been people I could always iv rely on. To Miracle, thank you for being a wonderful friend and partner. Your constant support means the world to me, and I am grateful for your love and for having you in my life. v TABLE OF CONTENTS LIST OF ABBREVIATIONS ....................................................................................................... vii Chapter 1: Synthesis of 3,4-disubstituted 1H-pyrazole and 3,5-disubstituted pyridines from epoxides* ........................................................................................................................................ 1 REFERENCES ......................................................................................................................... 28 APPENDIX ............................................................................................................................... 31 Chapter 2: First total synthesis of nagelamide W* ....................................................................... 48 REFERENCES ......................................................................................................................... 89 APPENDIX ............................................................................................................................... 92 Chapter 3: Advances in proteasome enhancement by small molecules ………………………..148 REFERENCES ....................................................................................................................... 179 Chapter 4: Synthesis of pyrazolo[1,5-b]pyridazines analogs as potential 20S proteasome activator ....................................................................................................................................... 195 REFERENCES ....................................................................................................................... 232 APPENDIX ............................................................................................................................. 234 Chapter 5: Progress towards the design and synthesis of molecular proteasome staples and probes .......................................................................................................................................... 266 REFERENCES ....................................................................................................................... 315 APPENDIX ............................................................................................................................. 317 vi LIST OF ABBREVIATIONS AD Alzheimer's disease AFM Atomic force microscopy ALS Amyotrophic lateral sclerosis AMC Amino methyl coumarin ATP Adenosine Triphosphate Boc Bn Tert-butoxycarbonyl Benzyl BTZ Bortezomib Calcd Calculated C-L Caspase-Like CaMKII Ca 2+ /Calmodulin-dependent protein kinase II cAMP Cyclic adenosine monophosphate CAS CDI Chemical abstracts service Carbonyldiimidazole CDK Cyclin-dependent kinase cGMP Cyclic guanosine monophosphate CP Core particle CT-L Chymotrypsin-Like Da Dalton DBDMH 1,3-dibromo-5,5-dimethylhydantoin DCM Dichloromethane DEAD Diethyl azodicarboxylate vii DIBAL-H di-isobutylaluminum hydride DIPEA N,N-diisopropylethylamine DMAP 4-dimethylaminopyridine DMF dimethylformamide DMF-DMA N,N-Dimethylformamide dimethylacetal DMSO dimethylsulfoxide dppf 1,1,-bis(diphenylphosphino)ferrocene DUBs deubiquitinating enzymes DYRK2 Dual specificity tyrosine-phosphorylation-regulated kinase 2 EC200 200% Effective Concentration EDC/EDCI 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Equiv Equivalent ESI FDA FTIR GST Electrospray ionization U.S. Food and Drug Administration Fourier transform infrared Glutathione S-transferase HBTU Hexafluorophosphate benzotriazole tetramethyl uronium HD Huntington’s disease HFIP Hexafluoro-2-propanol HOSA Hydroxylamine-O-sulfonic acid Htt Huntingtin HWE Horner–Wadsworth–Emmons IC50 half-maximal inhibitory concentration viii IDPs Intrisically disordered proteins LUMO Lowest unoccupied molecular orbital MAPK Mitogen-activated protein kinase MAPT Microtubule-associated protein tau MDa Mega dalton MeOH Methanol MHz Megahertz MP MW NBA NBS ND NDP Melting point Molecular weight N-bromoacetamide N-bromosuccinimide Not detected No detected product NFTs Neurofibrillary tangles NIH National Institutes of Health NMR Nuclear magnetic resonance NOE Nuclear overhauser effect NR No reaction NRF2 Nuclear factor (erythroid-derived 2)-like 2 NSAID Non-steroidal anti-inflammatory drug PAL PD Photo affirnity labelling Parkinson’s disease PDE3 Phosphodiesterase type-3 ix PDE4 Phosphodiesterase type-4 PDE5 Phosphodiesterase type-5 PEG Polyethylene glycol PGPH peptidylglutamyl-peptide hydrolyzing PKA PPh3 Protein kinase A Triphenylphosphine PySeSePy Dipyridyl diselenide RNA Ribonucleic acid ROESY Rotating frame overhause effect spectroscopy RP RT RU SAR SDS Regulatory particle Room temperature Response unit Structure activity relationship Sodium dodecyl sulfate siRNA Small interfering ribonucleic acid SNc Substantia nigra pars compacta SOCl2 Thionylchloride SOD1 superoxide dismutase 1 SPR Surface plasma resonance t-BHQ tert-Butylhydroquinone T-L TEA TFA Trypsin-like Triethylamine Trifluoroacetic acid x THF TLC TMS TOF Ub UPS UV Tetrahydrofuran Thin-layer chromatography Trimethylsilane Time of flight Ubiquitin Ubiquitin-proteasome system Ultraviolet xi Chapter 1: Synthesis of 3,4-disubstituted 1H-pyrazole and 3,5-disubstituted pyridines from epoxides* 1.1 Introduction Pyrazole and pyridine are essential nitrogen-containing heterocycles present in wide varieties of important biological molecules, pharmaceuticals, and other chemical compounds.1-3 Pyrazole, with its five-membered ring containing two adjacent nitrogen atoms, is a fundamental scaffold in medicinal chemistry. It serves as the core structure for numerous drugs, including FDA- approved nonsteroidal anti-inflammatory drugs (NSAIDs) lonazolac and celecoxib (Figure 1.1). Additionally, pyrazole derivatives exhibit a wide range of biological activities such as antitumor, antimicrobial, and antiviral properties, making them valuable targets in drug discovery and development.4 Figure 1.1: some FDA-approved drugs with pyrazole or pyridine scaffolds Cl OH O N N FF F N N Cl O N N H N N Cl O S O H2N Cl Lonazolac nonsteroidal anti-inflammatory drug celecoxib nonsteroidal anti-inflammatory drug Rimonabant inverse agonist of cannabinoid receptor NH OH F F N F Enpiroline (malaria treatment) F OH O N S O O N H N Piroxicam (arthritis) F F F F F N O N N N netupitant (antiemetic) F F F N N H pyrazole N pyridine * This chapter is reproduced in part with permission from reference 35. Copyright 2022 American Chemical Society 1 Pyridine, a six-membered aromatic ring containing one nitrogen atom, is another essential heterocycle in both natural and synthetic chemistry.5 It is a key component in various biologically active molecules, pharmaceuticals, and agrochemicals. Pyridine derivatives are also extensively used as building blocks in drug synthesis and as solvents in chemical reactions due to their unique properties. The diverse functionalities and reactivities of pyrazole and pyridine derivatives contribute significantly to their widespread presence in biology and chemistry, making them indispensable in various scientific fields. Several classical approaches to synthesize pyrazoles and pyridines include the Knorr pyrazole synthesis,6 Boger pyridine synthesis,7 Chichibabin pyridine synthesis,8 Kröhnke pyridine synthesis,9 and the Hantzsch pyridine synthesis,10 which have been well documented in the literature. Recent literature reviews have properly documented the synthesis of pyrazoles11-14 and pyridines15-18 via several approaches. However, some of these existing methods suffer from one or more limitations including low yields, high temperatures, a high proportion of byproducts, harsh reaction conditions, limited availability of substrates, and tedious multistep procedures, which require isolation of intermediates. Several recent modifications have been made to address some of these limitations.19-20 However, the synthesis of 3,4-disubstituted 1H-pyrazoles21-26 and 3,5- disubstituted pyridines27-30 using readily available starting materials such as aldehydes, ketones, alkenes, alkynes, is still limited to a few reports. In this chapter, Shafaat Mehedi and I developed a new approach to access pyrazoles and pyridines through the utilization of the readily available epoxides with hydrazine or ammonia. In an initial study, Mehedi et al31 reported the synthesis of 2,3-disubstituted quinolines from the reaction of aliphatic or aromatic epoxides and aromatic amines in the presence of scandium(III) triflate as Lewis acid (Scheme 1.1a). Inspired by a reported study where the authors in-situ 2 generated aldehydes from epoxides and other similar reported conversions,32-34 we report the one- pot synthesis of 3,4-disubstitued 1H-pyrazoles from epoxides and hydrazine using scandium(III) triflate as Lewis acid and N-bromosuccinimide (NBS) as oxidant (Scheme 1.1c). Similar to a Chichibabin-type pyridine synthesis (Scheme 1.1b), we also expanded the reaction scope to one- pot 3,5-disubstituted pyridine synthesis from the reaction of ammonia and epoxides using scandium(III) triflate and iron(III) chloride (Scheme 1.1d). Scheme 1.1: Previous work and our approach to access pyrazoles and pyridines Previous work: (a) Mehedi et al: Synthesis of quinoline from epoxides and anilines R1 + R2 NH2 O Sc(OTf)3 TEMPO THF, molecular sieves 65 °C R1 R2 R2 N (b) Chichibabin pyridine syntheis NH3 + Ph 400 - 500 °C O Ph Ph Ph N This work: O R (c) Pyrazoles synthesis NH2NH2 Sc(OTf)3 then, NBS R NH N (d) Pyridines synthesis NH3 Sc(OTf)3 then, FeCl3 R N R R 1.2 Results and Discussion 1.2.1 Synthesis of 3,4-disubstituted 1H-pyrazole We started our study by first investigating the synthesis of pyrazoles by treating 2 equivalents of styrene oxide (1-1a) with a single equivalent of hydrazine in the presence of 0.25 3 equivalent of Sc(OTf)3 as Lewis acid in tetrahydrofuran (THF) at 65 °C (Table 1.1, entry 1).35 This reaction resulted in mixtures of cis and trans pyrazoline (1-2) in 1:4 cis/trans ratio in 69% yield after 15 hours. Increasing the reaction time to 24 hours led to no significant change in reaction yield (Table 1.1, entry 2). When Sc(OTf)3 was increased to 0.5 equivalent, the formation of product reduced to 62% (Table 1.1, entry 3). Increasing the equivalent of styrene epoxide from 2 to 3 equivalents also led to a decrease in the desired product yield to 57% (Table 1.1, entry 4). Running the reaction in dichloromethane at 37 °C or room temperature also led to decrease in reaction yield to 31% and 23% respectively (Table 1.1, entry 5 & 6). In addition to screening of temperature and equivalent, we tried substituting Sc(OTf)3 with iron(III) chloride (FeCl3) or triflic acid (TfOH), FeCl3 gave lower yield relative to Sc(OTf)3 and only trace amount of product was observed when TfOH was used (Table 1.1, entry 7&8). It is critical to mention that no product was isolated in the absence of the acid nor under basic conditions (Table 1.1, entry 9&10). Table 1.1: Optimization of pyrazoline synthesis NH2 + H2N O 1-1a Lewis acid (X equiv) THF, temp, 15 h N NH 1-2 entry equiv of 1-1a acid X equiv temp (°C) Yield of 1-2 (%)a 1 2b 3 4 5c 6d 7 8 9 10e 2 2 2 3 2 2 2 2 2 2 65 65 65 65 37 rt 65 65 65 65 Sc(OTf)3 0.25 Sc(OTf)3 0.25 Sc(OTf)3 0.5 Sc(OTf)3 0.25 Sc(OTf)3 0.25 Sc(OTf)3 0.25 FeCl3 TfOH 0.25 0.25 - - - - 4 69 68 62 57 31 23 33 trace N/A N/A amixture of cis and trans (1-2). breaction ran for 24 h. creaction was ran in dichloromethane. drt = roomtemperature. ereaction ran with 1 equivalent of triethylamine. Following the optimization of reaction condition, we evaluated various oxidants for the conversion of the pyrazoline to the pyrazole final product. Among the various oxidants we evaluated, diacetoxyiodobenzene (PhI(OAc)2, potassium peroxymonosulfate (oxone), ferric oxide (Fe2O3), manganese dioxide (MnO2), iodine (I2), bromine (Br2), N-bromosuccinimide (NBS), 1,3- dibromo-5,5-dimethylhydantoin (DBDMH), N-bromoacetamide (NBA), and N-bromosaccharin, only DBDMH and NBS showed significant conversion of the pyrazoline to the desired 1H- pyrazole (1-3a) product, with NBS (2 equivalents) offering the best yield of 64% (Table 1.2, Entry 8). When the amount of NBS was increased to 2.5 equivalent, there was no significant improvement in isolated product yield. Also, decreasing the amount of NBS to 1 equivalent resulted in reduction of the product yield to 37%. Given that over 90% of the intermediate pyrazoline converted to pyrazole, the 69% of pyrazoline formed in the first step led to 64% of pyrazole in the second step using N-bromosuccinimide (NBS). From this screen and optimization, we moved to explore the substrate scope of this methodology using various epoxides. Table 1.2: Optimization of pyrazole synthesis NH2 + H2N O 1-1a Sc(OTf)3 (0.25 equiv) THF, temp, 15 h then, oxidant (X equiv) 3 h NH N 1-3a Entry Oxidant X equiv temp oxidant (°C) Yield of 1-3a (%) 1 2 PhI(OAc)2 PhI(OAc)2 1 2 65 65 NDP NDP 5 Table 1.2 (cont’d) 3 4 5 6a 7 8 9 10 11 12 13 Oxone Fe2O3 MnO2 I2 NBS NBS NBS DBDMH N-bromoacetamide 1 1 1 1 1 2 2.5 2 2 N-bromosaccharine 2 Br2 2 65 65 65 NDP NDP NDP 110 NDP 65 65 65 65 65 65 65 37 64 63 48 trace 13 trace NDP: No desired product. areaction was ran in DMSO Using different substituted styrene epoxides (1-1) as shown in Scheme 1.2, both electron withdrawing and electron donating groups are well tolerated and leading to the corresponding pyrazoles in 45-67% yields (1-3b – 1-3g). Epoxides with electron donation group such as 4- methoxy styrene oxide and 4-ethyl styrene oxide gave good yields, 62% and 67% respectively (1- 3b – 1-3c). This is possibly due to the increased in rate of the in-situ formed aldehydes from the epoxides. Epoxide with poor electron withdrawing groups gave lower yields (1-3d – 1-3g) and when strong electron withdrawing groups like 4-NO2 (1-3h) and 4-CF3 (1-3i) were used, the reaction did not provide any desired product. It is also important to note that alkyl epoxides did not provide any desired product as well. 6 Scheme 1.2: Substrate scope for pyrazole synthesis NH2 H2N + R O Sc(OTf)3 (0.25 equiv) 15 h then, NBS (2.5 equiv) 3h THF, 65 °C R NH N R 1-3 1-1 H N N H N N H N N OMe H N N Cl 1-3a 64% MeO 1-3b 62% a 1-3c 67% Cl 1-3d 49% H N N H N N H N N H N N Cl F Br NO2 Cl 1-3e 53% F 1-3f 61% Br 1-3g 45% O2N 1-3h 0% H N N CF3 F3C 1-3i 0% a1.2 equiv of NBS was used. While earlier study31 suggests that the reaction begins with the transformation of epoxides into aldehydes, the complete mechanism remains unclear. In the attempt to determine the plausible mechanism of the reaction, we have found that when phenylhydrazine (1-4) was treated with 1- 1a, the reaction provided the Fischer indole36 product 1-5 (Scheme 1.3) in excellent yield (83%).37 This result indicates the possible formation of hydrazone, as a key intermediate in the reaction mechanism. To further explore this, we found that treatment of cinnamaldehyde 1-6 with hydrazine under the optimized reaction condition afforded 97% of the pyrazoline product 1-7. 7 Scheme 1.3: Synthesis of indole and pyrazoline NH2 + H N 1-4 NH2 + H2N O 1-1a O H 1-6 Sc(OTf)3 (0.25 equiv) THF, 65 °C, 12 h 83% N H 1-5 Sc(OTf)3 (0.25 equiv) THF, 65 °C, 15 h 97% N NH 1-7 Therefore, based on the observed results (See mechanistic experiment 1 – 4 in experimental section), as well as from previous report (See reference 31: supplemental, mechanistic experiment 3) two plausible mechanisms are proposed in Scheme 1.4. The reaction is initiated following the rapid in situ generations of aldehyde I, which further converts to hydrazone II in the proposed pathway 1.38 The intermediate II and its tautomer or enol III could then undergo a Mannich-type reaction to generate intermediate IV. Further cyclization (a 5-exo-trig cyclization) and dehydration or elimination of hydrazine in IV produced the intermediate pyrazoline. Due to the pyrazole formation being observed only with the N-Br reagents, the reaction may form the intermediate VI upon treatment with NBS. Finally, deprotonation and isomerization of VI generate the pyrazole. Alternatively, pathway 2, the hydrazine could undergo double condensation with two molecules of aldehyde I to generate intermediate VIII. This intermediate could undergo cyclization to give the pyrazoline intermediate. However, such cyclization would invoke an unfavored 5-endo-trig cyclization. However, if the cyclization of intermediate VIII proceeds through carbocation formation, then pathway 2 could be another possible mechanism to access the pyrazole (Scheme 1.3 eq 2, see also mechanistic experiment 1 and 2 in experimental). 8 Scheme 1.4: Proposed mechanisms for pyrazole synthesis Pathway 1: O Sc(OTf)3 R Meinwald rearrangement R O H Sc(OTf)3 O NH2NH2 R I NH2NH2 ZH R Z = O, NNH2 O N NH2 II R R ZH III R HN NH2 R Z IV HN NH R -H2Z R HN NH O N Br NBS HN N R ZH R V Pathway 2: R pyrazoline Br O H R VI N O R H N N R pyrazole H N N R R O Sc(OTf)3 O Sc(OTf)3 Meinwald rearrangement R O H O NH2NH2 N NH2 R I R I R II N NH VIII R N NH R R NBS mediated oxidation R N N H R pyrazoline R R NH N pyrazole R R 5-endo-trig cyclization unfavored 1.2.2 Synthesis of 3,5-disubstituted pyridines To further extend the scope of this reaction, we expanded the methodology to a Chichibabin-type pyridine synthesis (Scheme 1.1d) using epoxides and ammonia instead of hydrazine. We started by treating three equivalents of epoxides with ammonia using the optimized condition as in the pyrazoline synthesis. This reaction provided 52% of the desired pyridine 1-4a (Table 1.3, entry 3). Further optimization gave 83% of desired product when two equivalent of ammonia solution was used with 0.125 equiv of Sc(OTf)3 at 110 °C (Table 1.3, entry 8). Previous study reported FeCl3 to facilitate debenzylation at the C-2 position of the quinolines31-32, based on 9 these reports, addition of 0.5 equivalent of FeCl3 was used to increase the in situ dealkylation at the C-2 position of the pyridine leading to increased reaction yield, up to 94% (Table 1.3, entry 10). Table 1.3: Optimization of pyridine synthesis NH3 + O Lewis acid (x equiv) solvent, temp, 24 h a equiv b equiv entry a b solvent Lewis Acid x N 1-4a temp (°C) 65 65 65 yield % 43 50 52 1 2 3 4 5 6 7 1 1 1 3 4 4 1 4 THF THF THF Sc(OTf)3 Sc(OTf)3 0.5 0.5 Sc(OTf)3 0.25 1,4-Dioxane + Sc(OTf)3 0.25 110 39 THF 1,4-Dioxane + 1 1 Sc(OTf)3 0.25 110 THF 2 1 THF Sc(OTf)3 0.25 65 53 54 1,4-Dioxane + Sc(OTf)3 2 1 THF 1,4-Dioxane + Sc(OTf)3 0.25 110 70 8 2 1 THF 0.125 110 83 9 2 1 110 91 1,4-Dioxane + Sc(OTf)3 + 0.125 + THF FeCl3 0.25 10 Table 1.3 (cont’d) 10 2 1 1,4-Dioxane + Sc(OTf)3 + 0.125 + 110 94 THF FeCl3 0.5 1,4-Dioxane + 11 2 1 FeCl3 0.5 110 57 THF 1,4-Dioxane + 12 2 1 FeCl3 1 110 45 THF After the optimization of the reaction condition, we explored the reaction scope (Scheme 1.5) by treating various epoxides 1-1 with ammonia solution under the optimized condition. Similar to the result obtained from the pyrazole reaction scope, electron-donating group on phenyl ring gave good yields (1-4b) while electron withdrawing group gave lower yields (1-4c-g). It is important to mention that a moderate yield of 55% of the pyridine was isolated with 2- chlorostyrene oxide was used, likely due to the steric effect of chlorine at the ortho position. 11 Scheme 1.5: Substrate scope of the pyridine synthesis NH3 + R O Sc(OTf)3 (0.125 equiv) 15 min rt then, FeCl3 (0.5 equiv) 1,4-Dioxane:THF, 24 h, 110 °C pressure tube 2 equiv 1 equiv R R N 1-4 Br Br Cl Cl N 1-4a 94% N 1-4b 94% N 1-4c 67% N 1-4d 85% Cl Cl N 1-4e 77% Cl Cl N 1-4f 55% N 1-4g 87% F F Based on the observed results (See mechanistic experiment 5 in experimental) we proposed a plausible mechanism in Scheme 1.6. Similar to the pyrazole synthesis, at first, the reaction initiated by rapid formation of aldehyde I, which undergoes condensation with ammonia to give imine II. A Mannich-type reaction between the generated imine II and the enol tautomer or enamine III could generate intermediate IV. After that, the condensation of another aldehyde I to the intermediate IV, followed by tautomerism and cyclization generates the intermediate VI. Elimination of ammonia or dehydration of VI forms the intermediate VII. Finally, debenzylation27,32,39 at the C-2 position provides the final pyridine product. 12 Scheme 1.6: Proposed mechanism for the synthesis of pyridine O Sc(OTf)3 R Meinwald rearrangement R O H Sc(OTf)3 O NH3 NH R I NH3 R II R NH2 ZH R R Z = O, NH III ZH Z R IV O R - H2O H R R N R Z 1.3 Conclusions HN R R Z R V N R R ZH - H2Z N R R VI R oxidative debenzylation N R ref. 27, 32, & 38 R pyridine R VII In conclusion, we have established a new approach to synthesis 3,4-disubstituted 1H- pyrazoles and 3,5-disubstituted pyridines using easily accessible epoxides as the starting material. These reactions accommodate a range of aromatic epoxides, producing the corresponding pyrazole and pyridine product with moderate to excellent yields. 1.4 Experimental section General information Commercially available reagents were used without additional purification. All reactions were performed under an argon atmosphere with commercial-grade reagents. All chemicals and solvents were purchased from commercial sources and used without further purification. Styrene oxide (1a) (CAS no. 96-09-3), phenylhydrazine (5) (CAS no. 100-63-0), and trans-cinnamaldehyde (7) (CAS no. 14371-10-9) were purchased from Sigma Aldrich. Scandium(III) triflate (CAS no. 144026-79- 9), N-bromosuccinimide (CAS no. 128-08-5), and iron(III) chloride anhydrous (CAS no. 7705-08- 0) were purchased from Oakwood Chemicals. Other epoxides were purchased from Enamine. N- bromosuccinimide was recrystallized before use and stored in the dark. THF was dried under 3Å molecular sieve and checked for any water content before using it in the reaction. All flasks were 13 oven-dried overnight and cooled under argon. Hydrazine monohydrate (N2H4 64-65 %, reagent grade, 98%) was purchased from Sigma Aldrich and used in all pyrazole reactions. A 0.4 M solution of ammonia in THF was purchased from Sigma Aldrich and used in all pyridine reactions. All NMR spectra were recorded on a 500 MHz spectrometer. Signal multiplicities are given as s (singlet), bs (broad singlet), d (doublet), t (triplet), dd (doublet of doublet), and m (multiplet). The mass spectrometer ionization method was ESI with a Quadrupole detector and infrared spectra were recorded on a Jasco Series 6600 FTIR spectrometer. General method for the synthesize of 1H-pyrazoles (1-3) To a solution of dry tetrahydrofuran (THF) (5 mL) in a 50 mL dry round bottom flask, different substituted epoxides (2 equiv, 1 mmol) were added, followed by Sc(OTf)3 (0.25 equiv, 0.125 mmol) at room temperature. After 15 minutes, hydrazine monohydrate (1 equiv, 0.5 mmol) was added at room temperature. The reaction was placed under argon gas and was refluxed for 15 hours at 65 °C in oil bath. Then, N-bromosuccinimide (NBS) (2.5 equiv, 1.25 mmol) was added and stirred for 3 hours at 65 °C. After that, the reaction mixture was cooled to room temperature and the solvent was evaporated under reduced pressure. Then 10 mL of dichloromethane and 10 mL saturated NaHCO3 solution was added, and the solution was extracted with 3´10 mL of dichloromethane. The combined organic layer was collected and evaporated under reduced pressure and crude products were purified by column chromatography. H N N 3-benzyl-4-phenyl-1H-pyrazole (1-3a): Purified with automated CombiFlash chromatography (silica gel, 20-40 microns, gradient 25% ethyl acetate in hexane) to give yellow solid (75 mg, 14 64%). mp = 135 – 137 °C. 1H NMR (500 MHz, CD2Cl2) δ 7.56 (s, 1H), 7.30 – 7.26 (m, 4H), 7.23 – 7.08 (m, 6H), 4.09 (s, 2H). 13C{1H} NMR (125 MHz, CD2Cl2) δ 141.2, 137.8, 133.8, 132.5, 127.91, 127.85, 127.8, 126.9, 125.8, 125.6, 119.5, 31.0. IR (neat, cm-1): 3125, 2964, 1606, 1521, 1259. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C16H15N2 +) 235.1230; Found 235.1239. H N N OMe MeO 3-(4-methoxybenzyl)-4-(4-methoxyphenyl)-1H-pyrazole (1-3b): Purified with automated CombiFlash chromatography (silica gel, 20-40 microns, gradient 40% ethyl acetate in hexane) to give yellow oil (91 mg, 62%). 1H NMR (500 MHz, CD2Cl2) δ 7.60 (s, 1H), 7.31 (d, J = 8.7 Hz, 2H), 7.10 (d, J = 8.7 Hz, 2H), 6.92 (d, J = 8.7 Hz, 2H), 6.83 (d, J = 8.7 Hz, 2H), 4.09 (s, 2H), 3.82 (s, 3H), 3.77 (s, 3H). 13C{1H} NMR (125 MHz, CD2Cl2) δ 158.3, 142.0, 134.3, 130.6, 129.5, 128.8, 126.7, 125.7, 119.7, 113.98, 113.96, 55.20, 55.15, 30.8. IR (neat, cm-1): 3143, 2907, 1610, 1509, 1241. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C18H19N2O2 +) 295.1441; found 295.1449. H N N 3-(4-ethylbenzyl)-4-(4-ethylphenyl)-1H-pyrazole (1-3c): Purified with automated CombiFlash chromatography (silica gel, 20-40 microns, gradient 35% ethyl acetate in hexane) to give a clear oil (97 mg, 67%). 1H NMR (500 MHz, CD2Cl2) δ 7.45 (s, 1H), 7.19 (d, J = 8.1 Hz, 2H), 7.10 (d, J = 8.1 Hz, 2H), 7.02 (d, J = 8.1 Hz, 2H), 6.98 (d, J = 8.1 Hz, 2H), 4.02 (s, 2H), 2.55 (q, J = 7.6 Hz, 2H), 2.50 (q, J = 7.6 Hz, 2H), 1.14 (t, J = 7.6 Hz, 3H), 1.09 (t, J = 7.6 Hz, 3H). 13C{1H} NMR (125 MHz, CD2Cl2) δ 141.8, 141.7, 141.0, 135.1, 133.8, 129.8, 127.7, 127.32, 127.31, 126.8, 15 119.3, 30.4, 27.7, 27.6, 14.68, 14.66. IR (neat, cm-1): 3019, 2922, 1614, 1513. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C20H23N2 +) 291.1856; Found 291.1863. H N N Cl Cl 3-(3-chlorobenzyl)-4-(3-chlorophenyl)-1H-pyrazole (1-3d): Purified with automated CombiFlash chromatography (silica gel, 20-40 microns, gradient 30% ethyl acetate in hexane) to give a clear oil (74 mg, 49%). 1H NMR (500 MHz, CD2Cl2) δ 7.57 (s, 1H), 7.25 – 7.19 (m, 2H), 7.18 – 7.10 (m, 4H), 7.08 (s, 1H), 6.98 (d, J = 6.5 Hz, 1H), 4.04 (s, 2H). 13C{1H} NMR (125 MHz, CD2Cl2) δ 142.2, 139.9, 134.2, 133.5, 132.3, 129.19, 129.17, 127.8, 126.9, 126.1, 125.9, 125.7, 125.6, 125.2, 118.6, 30.9. IR (neat, cm-1): 3153, 2961, 1599, 1260. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C16H13Cl2N2 +) 303.0451, 305.0521; Found 303.0459, 305.0432. H N N Cl Cl 3-(4-chlorobenzyl)-4-(4-chlorophenyl)-1H-pyrazole (1-3e): Purified with automated CombiFlash chromatography (silica gel, 20-40 microns, gradient 30% ethyl acetate in hexane) to give a light yellow oil (80 mg, 53%). 1H NMR (500 MHz, CD2Cl2) δ 7.48 (s, 1H), 7.24 – 7.21 (m, 2H), 7.17 – 7.10 (m, 4H), 6.98 – 6.95 (m, 2H), 3.99 (s, 2H). 13C{1H} NMR (125 MHz, CD2Cl2) δ 141.9, 136.5, 132.4, 131.4, 130.9, 129.0, 128.2, 128.04, 127.96, 127.9, 118.7, 30.4. IR (neat, cm- 1): 3133, 2920, 1601, 1489. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C16H13Cl2N2 +) 303.0451, 305.0521; Found 303.0458, 305.0431. 16 H N N F F 3-(4-fluorobenzyl)-4-(4-fluorophenyl)-1H-pyrazole (1-3f): Purified with automated CombiFlash chromatography (silica gel, 20-40 microns, gradient 30% ethyl acetate in hexane) to give a white solid (82 mg, 61%). mp = 88 – 90 °C. 1H NMR (500 MHz, CD2Cl2) δ 7.50 (s, 1H), 7.21 – 7.17 (m, 2H), 7.04 – 6.92 (m, 4H), 6.90 – 6.82 (m, 2H), 4.00 (s, 2H). 13C{1H} NMR (125 MHz, CD2Cl2) δ161.9 (d, J = 244.0 Hz), 161.8 (d, J = 243.0 Hz), 133.7, 132.7, 129.2 (d, J = 8.0 Hz), 128.7 (d, J = 8.0 Hz), 128.49, 128.46, 118.8, 114.7 (d, J = 21.0 Hz), 114.6 (d, J = 21.0 Hz), 30.2. IR (neat, cm-1): 3036, 2916, 1607, 1506, 1220. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C16H13F2N2 +) 271.1041; Found 271.1049. H N N Br Br 3-(4-bromobenzyl)-4-(4-bromophenyl)-1H-pyrazole (1-3g): Purified with automated CombiFlash chromatography (silica gel, 20-40 microns, gradient 30% ethyl acetate in hexane) to give a white solid (88 mg, 45%). mp: 152 – 154 °C. 1H NMR (500 MHz, CD2Cl2) δ 7.51 (s, 1H), 7.40 (d, J = 8.5 Hz, 2H), 7.30 (d, J = 8.4 Hz, 2H), 7.12 (d, J = 8.5 Hz, 2H), 6.94 (d, J = 8.4 Hz, 2H), 3.99 (s, 2H). 13C{1H} NMR (125 MHz, CD2Cl2) δ 142.1, 137.0, 132.4, 131.3, 131.0, 130.9, 129.5, 128.6, 119.5, 118.8, 30.6. IR (neat, cm-1): 3127, 2922, 1517, 1486, 1259. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C16H13Br2N2 +) 390.9440, 392.9420; Found 390.9450, 392.9427. 17 Mechanistic experiments for pyrazole Experiment 1 O H + H2N NH2 H N N 3-benzyl-4-phenyl-1H-pyrazole (1-3a): To a solution of dry tetrahydrofuran (THF) (5 mL) in a 50 mL dry round bottom flask, phenylacetaldehyde (2 equiv, 2 mmol) was added, followed by Sc(OTf)3 (0.25 equiv, 0.25 mmol) at room temperature. After 15 minutes, hydrazine monohydrate (1 equiv, 1 mmol) was added at room temperature. The reaction was placed under argon gas and was refluxed for 15 hours at 65 °C in oil bath. Then, N-bromosuccinimide (NBS) (2.5 equiv, 2.5 mmol) was added and stirred for 3 hours at 65 °C. After that, the reaction mixture was cooled to room temperature and the solvent was evaporated under reduced pressure. Then 15 mL of dichloromethane and 15 mL saturated NaHCO3 solution was added, and the solution was extracted with 3´15 mL of dichloromethane. The combined organic layer was collected and evaporated under reduced pressure and the crude product was purified using automated CombiFlash chromatography (silica gel, 20-40 microns, gradient 25% ethyl acetate in hexane) to yield the desired product (117 mg, 50%). Experiment 2 H N NH2 + O N H 1-5 3-phenyl-1H-indole (1-5): To a solution of dry tetrahydrofuran (THF) (5 mL) in a 50 mL dry round bottom flask, styrene oxide 1-1a (2 equiv, 1 mmol) was added, followed by Sc(OTf)3 (0.25 18 equiv, 0.125 mmol) at room temperature. After 15 minutes, phenylhydrazine 1-4 (1 equiv, 0.5 mmol) was added at room temperature. The reaction was placed under argon gas and was refluxed for 12 hours at 65 °C under air in the oil bath. After that, the reaction mixture was cooled to room temperature and the solvent was evaporated under reduced pressure. Then 10 mL of dichloromethane and 10 mL saturated NaHCO3 solution was added, and the solution was extracted with 3´10 mL of dichloromethane. The combined organic layer was collected and evaporated under reduced pressure and the crude product 3-phenylindole (1-5) was purified using automated CombiFlash chromatography (silica gel, 20-40 microns, gradient 10% ethyl acetate in hexane) to give a light brown solid (80 mg, 83%). mp = 85 – 86 °C. 1H NMR (500 MHz, CD2Cl2) δ 8.13 (bs, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.60 – 7.52 (m, 2H), 7.35 – 7.30 (m, 2H), 7.28 (d, J = 8.0 Hz, 1H), 7.22 (d, J = 2.6 Hz, 1H), 7.18 – 7.14 (m, 1H), 7.14 – 7.10 (m, 1H), 7.08 – 7.05 (m, 1H). 13C{1H} NMR (125 MHz, CD2Cl2) δ 135.9, 134.8, 128.0, 126.5, 125.1, 124.7, 121.5, 121.2, 119.4, 118.8, 117.0, 110.7. IR (neat, cm-1): 3398, 3051, 1594, 1453, 1260. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C14H12N+) 194.0964; Found 194.0966. The spectroscopic data are consistence with previous literature reports.40 NH2 + H2N O H NHN 1-7 5-phenyl-4,5-dihydro-1H-pyrazole (1-7): To a solution of dry tetrahydrofuran (THF) (5 mL) in a 50 mL dry round bottom flask, cinnamaldehyde (1-6) (2 equiv, 1 mmol) was added, followed by Sc(OTf)3 (0.25 equiv, 0.125 mmol) at room temperature. After 15 minutes, hydrazine monohydrate (1 equiv, 0.5 mmol) was added at room temperature. The reaction was placed under argon gas and was refluxed for 15 hours at 65 °C in oil bath. After that, the reaction mixture was cooled to room temperature and the solvent was evaporated under reduced pressure. Then 10 mL of 19 dichloromethane and 10 mL saturated NaHCO3 solution was added, and the solution was extracted with 3´10 mL of dichloromethane. The combined organic layer was evaporated under reduced pressure and the crude product was purified using automated CombiFlash chromatography (silica gel, 20-40 microns, gradient 35% ethyl acetate in hexane) to give a yellow oil (71 mg, 97%). 1H NMR (500 MHz, CD2Cl2) δ 7.23 – 7.18 (m, 4H), 7.17 – 7.15 (m, 1H), 6.65 (t, J = 1.6 Hz, 1H), 5.70 (s, 1H), 4.59 (dd, J = 10.5, 9.5 Hz, 1H), 2.99 (ddd, J = 17.1, 10.5, 1.6 Hz, 1H), 2.54 (ddd, J = 17.1, 9.5, 1.6 Hz, 1H). 13C{1H} NMR (125 MHz, CD2Cl2) δ 142.3, 141.7, 127.8, 126.6, 125.5, 61.6, 41.9. IR (neat, cm-1): 3032, 2961, 1645, 1586. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C9H11N2 +) 147.0917; Found 147.0923. Experiment 3 N NH 1-2 NH N 1-3a 3-benzyl-4-phenyl-1H-pyrazole (1-3a): To a solution of dry tetrahydrofuran (THF) (5 mL) in a 50 mL dry round bottom flask, a mixture of cis and trans 3-benzyl-4-phenyl-4,5-dihydro-1H- pyrazole (1-2) (1 equiv, 0.5 mmol) was added. Then, N-bromosuccinimide (NBS) (2 equiv, 1 mmol) was added and stirred for 3 hours at 65 °C. After that, the reaction mixture was cooled to room temperature and the solvent was evaporated under reduced pressure. Then 15 mL of dichloromethane and 15 mL water was added, and the solution was extracted with 3´15 mL of dichloromethane. The combined organic layer was collected and evaporated under reduced pressure and the crude product (1-3a) was purified using automated CombiFlash chromatography (silica gel, 20-40 microns, gradient 25% ethyl acetate in hexane) to yield the desired product (109 mg, 93%). 20 Experiment 4 To a solution of dry tetrahydrofuran (THF) (1 mL) in a 4 mL vial, styrene oxide (2 equiv, 0.1 mmol) were added, followed by Sc(OTf)3 (0.25 equiv, 0.0125 mmol) at room temperature. After that, hydrazine monohydrate (1 equiv, 0.05 mmol) was added at room temperature. The reaction was placed under argon gas and was refluxed for 2 hours at 65 °C in oil bath. Then, a crude mass was checked using a mass spectrometer by ESI (+) method with a Quadrupole detector (Figure 1.2) Then, N-bromosuccinimide (NBS) (2.5 equiv, 0.125 mmol) was added and another crude mass spec was checked after 15 mins (Figure 1.3). Figure 1.2: Mass spec after 2h of addition of hydrazine 21 Figure 1.3: Mass spec after addition of NBS General method for the synthesize of pyridines (1-4) To a solution of dry 1,4-dioxane (2 mL) in a 50 mL dry pressure tube, different substituted epoxides (1 equiv, 1 mmol) were added, followed by Sc(OTf)3 (0.125 equiv, 0.125 mmol) at room temperature. After 15 minutes, 0.4 M ammonia in tetrahydrofuran (THF) solution (2 equiv, 2 mmol, 5 mL) was added dropwise, followed by FeCl3 (0.5 equiv, 0.5 mmol) was added at room temperature. The pressure tube was then sealed and placed in an oil bath. Then, the temperature of the oil bath was slowly (in 30 minutes) raised to 110 °C and the reaction was run for 24 hours at 110 °C. After that, the reaction mixture was cooled to room temperature and the solvent was evaporated under reduced pressure. Then 10 mL of dichloromethane and 10 mL saturated NaHCO3 solution was added, and the solution was extracted with 3´10 mL of dichloromethane. The combined organic layer was collected and evaporated under reduced pressure and crude products 22 were purified using automated CombiFlash chromatography (silica gel, 20-40 microns, gradient 7.5-12.5% ethyl acetate in hexane). N 3,5-diphenylpyridine (1-4a)26: Purified with automated CombiFlash chromatography (silica gel, 20-40 microns, gradient 7.5% ethyl acetate in hexane) to give a light yellow solid (72 mg, 94%). mp = 132 – 135 °C. 1H NMR (500 MHz, CDCl3) δ 8.84 (d, J = 1.8 Hz, 2H), 8.06 (t, J = 1.8 Hz, 1H), 7.65 (d, J = 7.5 Hz, 4H), 7.51 (t, J = 7.5 Hz, 4H), 7.44 (t, J = 7.5 Hz, 2H). 13C{1H} NMR (125 MHz, CDCl3) δ 146.9, 137.6, 136.5, 132.8, 129.0, 128.1, 127.1. IR (neat, cm-1): 3056, 3034, 2957, 1576. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C17H14N+) 232.1121; Found 232.1129. N 3,5-bis(4-ethylphenyl)pyridine (1-4b): Purified with automated CombiFlash chromatography (silica gel, 20-40 microns, gradient 12.5% ethyl acetate in hexane) to give a white solid (90 mg, 94%). mp = 130 – 132 °C. 1H NMR (500 MHz, CD2Cl2) δ 8.69 (s, 2H), 7.99 – 7.95 (m, 1H), 7.51 (d, J = 7.7 Hz, 4H), 7.26 (d, J = 7.7 Hz, 4H), 2.63 (q, J = 7.6 Hz, 4H), 1.19 (t, J = 7.6 Hz, 6H). 13C{1H} NMR (125 MHz, CD2Cl2) δ 145.8, 143.8, 135.6, 134.4, 131.4, 127.8, 126.3, 27.8, 14.7. IR (neat, cm-1): 3027, 2961, 1513, 1259. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C21H22N+) 288.1747; Found 288.1763. 23 Br Br N 3,5-bis(4-bromophenyl)pyridine (1-4c): Purified with automated CombiFlash chromatography (silica gel, 20-40 microns, gradient 10% ethyl acetate in hexane) to give a white solid (87 mg, 67%). mp = 165 – 168 °C. 1H NMR (500 MHz, CD2Cl2) δ 8.72 (d, J = 2.2 Hz, 2H), 7.93 (t, J = 2.2 Hz, 1H), 7.59 – 7.55 (m, 4H), 7.48 – 7.45 (m, 4H). 13C{1H} NMR (125 MHz, CD2Cl2) δ 146.3, 135.9, 134.7, 131.52, 131.47, 128.1, 121.8. IR (neat, cm-1): 3069, 2962, 1590, 1486, 1258. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C17H12Br2N+) 387.9331, 389.9311; Found 387.9341, 389.9326. Cl Cl N 3,5-bis(4-chlorophenyl)pyridine (1-4d): Purified with automated CombiFlash chromatography (silica gel, 20-40 microns, gradient 10% ethyl acetate in hexane) to give a white solid (85 mg, 85%). mp = 170 – 172 °C. 1H NMR (500 MHz, CD2Cl2) δ 8.71 (d, J = 2.2 Hz, 2H), 7.93 (t, J = 2.2 Hz, 1H), 7.55 – 7.51 (m, 4H), 7.46 – 7.37 (m, 4H). 13C{1H} NMR (125 MHz, CD2Cl2) δ 146.4, 135.5, 134.6, 133.6, 131.6, 128.5, 127.8. IR (neat, cm-1): 3027, 2964, 1595, 1494, 1386. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C17H12Cl2N+) 300.0341, 302.0312; Found 300.0350, 302.0323. Cl Cl N 3,5-bis(3-chlorophenyl)pyridine (1-4e): Purified with automated CombiFlash chromatography (silica gel, 20-40 microns, gradient 10% ethyl acetate in hexane) to give a light yellow solid (77 mg, 77%). mp = 153 – 155 °C. 1H NMR (500 MHz, CD2Cl2) δ 8.73 (t, J = 2.5 Hz, 2H), 7.95 – 7.94 (m, 1H), 7.63 – 7.54 (m, 2H), 7.50 – 7.46 (m, 2H), 7.42 – 7.30 (m, 4H). 13C{1H} NMR (125 24 MHz, CD2Cl2) δ 146.7, 138.7, 134.5, 134.1, 131.9, 129.7, 127.5, 126.5, 124.7. IR (neat, cm-1): 3052, 2959, 1565, 1259. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C17H12Cl2N+) 300.0352, 302.0326; Found 300.0350, 302.0323. Cl N Cl 3,5-bis(2-chlorophenyl)pyridine (1-4f): Purified with automated CombiFlash chromatography (silica gel, 20-40 microns, gradient 10% ethyl acetate in hexane) to give a pale yellow solid (55 mg, 55%). mp = 132 – 134 °C. 1H NMR (500 MHz, CD2Cl2) δ 8.60 (d, J = 2.1 Hz, 2H), 7.84 (t, J = 2.1 Hz, 1H), 7.49 – 7.41 (m, 2H), 7.36 – 7.34 (m, 2H), 7.33 – 7.30 (m, 3H), 7.29 – 7.27 (m, 1H). 13C{1H} NMR (125 MHz, CD2Cl2) δ 148.2, 136.8, 135.9, 133.5, 131.9, 130.7, 129.3, 128.8, 126.5. IR (neat, cm-1): 3007, 2962, 1563, 1258. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C17H12Cl2N+) 300.0341, 302.0312; Found 300.0349, 302.0322. F F N 3,5-bis(4-fluorophenyl)pyridine (1-4g): Purified with automated CombiFlash chromatography (silica gel, 20-40 microns, gradient 10% ethyl acetate in hexane) to give a white solid (77 mg, 87%). mp = 170 – 171 °C. 1H NMR (500 MHz, CD2Cl2)) δ 8.68 (d, J = 2.2 Hz, 2H), 7.91 (t, J = 2.2 Hz, 1H), 7.57 – 7.54 (m, 4H), 7.15 – 7.11 (m, 4H). 13C{1H} NMR (125 MHz, CD2Cl2) δ 163.2 (d, J = 246.0 Hz), 146.1, 135.0, 133.2, 131.6, 128.3 (d, J = 8.5 Hz), 115.3 (d, J = 21.5 Hz). IR (neat, cm-1): 3087, 1603, 1512, 1223. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C17H12F2N+) 268.0932; Found 268.0939. 25 Mechanistic experiments for pyridines Experiment-5 O H + NH3 N 1-4a 3,5-diphenylpyridine (1-4a): To a solution of dry 1,4-dioxane (2 mL) in a 50 mL dry pressure tube, phenylacetaldehyde (1 equiv, 1 mmol) was added, followed by Sc(OTf)3 (0.125 equiv, 0.125 mmol) at room temperature After 15 minutes, 0.4 M ammonia in THF solution (2 equiv, 2 mmol, 5 mL) was added dropwise, followed by FeCl3 (0.5 equiv, 0.5 mmol) was added at room temperature. The pressure tube was then sealed and placed in an oil bath. Then, the temperature of the oil bath was slowly (in 30 minutes) raised to 110 °C and the reaction was run for 24 hours at 110 °C. After that, the reaction mixture was cooled to room temperature and the solvent was evaporated under reduced pressure. Then 10 mL of dichloromethane and 10 mL saturated NaHCO3 solution was added, and the solution was extracted with 3´10 mL of dichloromethane. The combined organic layer was collected and evaporated under reduced pressure and the crude product (1-4a) was purified using automated CombiFlash chromatography (silica gel, 20-40 microns, gradient 7.5% ethyl acetate in hexane) to give the desired product (55 mg, 72%). Experiment 6 To a solution of dry 1,4-dioxane (2.0 mL) in a 15 mL pressure tube, styrene oxide (1 equiv, 1.0 mmol) were added, followed by Sc(OTf)3 (0.125 equiv, 0.125 mmol) at room temperature After 15 minutes, 0.4 M ammonia in tetrahydrofuran (THF) solution (2 equiv, 2 mmol, 5 mL) was added dropwise, followed by FeCl3 (0.5 equiv, 0.5 mmol) was added at room temperature. The pressure tube was then sealed and placed in an oil bath. Then, the temperature of the oil bath was slowly 26 (in 30 minutes) raised to 110 °C and the reaction was run for 1 hours at 110 °C. After that, a crude mass was checked using a mass spectrometer by ESI (+) method with a Quadrupole detector (Figure 1.4). 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Chem. Int. Ed. 2008, 47, 5056-5059. 30 APPENDIX Figure 1.5: 1H and 13C{1H} NMR spectra of 1-3a 6 5 7 . 2 3 7 . 0 3 7 . 9 2 7 . 8 2 7 . 6 2 7 . 2 2 7 . 0 2 7 . 9 1 7 . 7 1 7 . 5 1 7 . 3 1 7 . 2 1 7 . 0 1 7 . 9 0 7 . l 2 c 2 d c 3 2 . 5 l 2 c 2 d c 3 2 . 5 9 0 4 . 5 6 5 7 . 0 0 . 1 7.7 7.6 7.5 7.4 3 0 3 7 . 0 9 2 7 . 7 7 2 7 . 1 6 2 7 . 7 1 2 7 . 2 0 2 7 . 6 8 1 7 . 1 7 1 7 . 6 4 1 7 . 1 3 1 7 . 6 1 1 7 . 4 0 1 7 . 9 8 0 7 . 3 0 4 . 7.3 f1 (ppm) 6 0 6 . 7.2 7.1 7.0 6.9 3 2 1 0 -1 -2 l 2 c 2 d c 1 . 3 5 l 2 c 2 d c 9 . 2 5 l 2 c 2 d c 7 . 2 5 l 2 c 2 d c 5 . 2 5 l 2 c 2 d c 3 . 2 5 0 . 1 3 13 12 11 10 9 8 7 6 f1 (ppm) 5 8 9 . 0 6 9 . 3 6 9 . 5 2 . 1 4 1 8 . 7 3 1 8 . 3 3 1 5 . 2 3 1 9 . 7 2 1 8 . 7 2 1 8 . 7 2 1 9 . 6 2 1 8 . 5 2 1 6 . 5 2 1 5 . 9 1 1 2 . 1 4 1 8 . 7 3 1 8 . 3 3 1 5 . 2 3 1 9 0 . 2 4 30 20 10 0 142 140 138 136 134 132 f1 (ppm) 7000 6500 6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 -500 260 250 240 230 220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 31 Figure 1.6: 1H and 13C{1H} NMR spectra of 1-3b 0 6 7 . 1 3 7 . 0 3 7 . 1 1 . 7 9 0 7 . 9 0 7 . 3 9 6 . 2 9 6 . 4 8 . 6 2 8 . 6 9 0 4 . 2 8 . 3 7 7 . 3 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 0 0 1 . 3 9 1 . 4 9 1 . 0 2 2 . 5 1 2 . 8 0 2 . 4 9 2 . 1 0 . 3 3 . 8 5 1 . 0 2 4 1 . 3 4 3 1 . 6 0 3 1 . 5 9 2 1 8 . 8 2 1 . 7 6 2 1 . 7 5 2 1 . 7 9 1 1 . 0 4 1 1 . 0 4 1 1 2 . 5 5 2 . 5 5 l 2 c 2 d c 9 3 5 . l 2 c 2 d c 7 . l 2 c 2 d c 4 . 3 5 3 5 l 2 c 2 d c 2 . 3 5 l 2 c 2 d c 0 . 3 5 8 . 0 3 100000 90000 80000 70000 60000 50000 40000 30000 20000 10000 0 90 85 80 75 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 32 Figure 1.7: 1H and 13C{1H} NMR spectra of 1-3c 5 4 7 . 0 2 7 . 8 1 . 7 1 1 . 7 9 0 7 . 2 0 7 . 1 0 7 . 9 9 6 . 7 9 6 . l 2 c 2 d c 0 2 . 5 l 2 c 2 d c 0 2 . 5 l 2 c 2 d c 0 2 . 5 2 0 4 . 7 5 2 . 6 5 2 . 4 5 2 . 3 5 2 . 2 5 2 . 0 5 2 . 9 4 2 . 7 4 2 . 5 1 . 1 4 1 . 1 2 1 . 1 0 1 . 1 9 0 . 1 7 0 . 1 7 5 2 . 6 5 2 . 4 5 2 . 3 5 2 . 2 5 2 . 0 5 2 . 9 4 2 . 7 4 2 . 5 1 . 1 4 1 . 1 2 1 . 1 0 1 . 1 9 0 . 1 7 0 . 1 10000 8000 6000 4000 2000 0 0 1 2 . 6 0 2 . 2.5 1 0 . 3 5 9 2 . 2.0 1.5 1.0 f1 (ppm) 13 12 11 10 9 8 7 6 f1 (ppm) 5 2 9 0 . 9 9 . 1 4 0 2 . 9 8 . 3 5 0 2 . 4 0 1 . 2 6 0 2 . 3 2 1 0 . 3 5 9 2 . 1 0 -1 -2 8 . 1 4 1 7 . 1 4 1 0 . 1 4 1 1 . 5 3 1 8 . 3 3 1 8 . 9 2 1 . 7 7 2 1 . 6 7 2 1 . 3 7 2 1 . 3 7 2 1 1 . 7 2 1 8 . 6 2 1 . 7 6 2 1 3 . 9 1 1 l 2 c 2 d c 1 . 3 5 l 2 c 2 d c 9 2 5 . l 2 c 2 d c 7 2 5 . l 2 c 2 d c 4 2 5 . l 2 c 2 d c 2 2 5 . . 4 0 3 . 7 7 2 . 6 7 2 . 7 4 1 . 7 4 1 8 . 1 4 1 7 . 1 4 1 0 . 1 4 1 1 . 5 3 1 8 . 3 3 1 145 140 135 f1 (ppm) . 7 4 1 . 7 4 1 15.0 14. 8 14.6 14.4 f1 (ppm) . 7 7 2 . 6 7 2 10 8 6 4 2 0 28 .0 27. 8 27.6 f1 (ppm) 27.4 27.2 200 100 0 500 400 300 200 100 0 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 33 50000 45000 40000 35000 30000 25000 20000 15000 10000 5000 0 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 1.8: 1H and 13C{1H} NMR spectra of 1-3d 7 5 7 . 4 2 7 . 3 2 7 . 1 2 7 . 0 2 7 . 7 1 . 7 7 1 . 7 6 1 . 7 5 1 . 7 5 1 . 7 4 1 . 7 4 1 . 7 2 1 . 7 2 1 . 7 0 1 . 7 8 0 7 . 8 9 6 . 7 9 6 . l 2 c 2 d c 3 2 . 5 l 2 c 2 d c 3 2 . 5 4 0 4 . 4 2 7 . 3 2 7 . 1 2 7 . 0 2 7 . 7 1 . 7 7 1 . 7 6 1 . 7 5 1 . 7 5 1 . 7 4 1 . 7 4 1 . 7 2 1 . 7 2 1 . 7 0 1 . 7 8 0 7 . 8 9 6 . 7 9 6 . 45000 40000 35000 15000 10000 30000 5000 0 1 0 2 . 1 0 4 . 9 9 0 . 3 0 . 1 7.3 7.2 7.1 7.0 f1 (ppm) 13 12 11 10 9 8 7 6 f1 (ppm) 5 8 9 0 . 8 9 . 1 4 9 3 . 8 9 0 . 1 0 . 1 0 1 . 2 4 3 2 1 0 -1 -2 l 2 c 2 d c 7 2 5 . l 2 c 2 d c 5 2 5 . l 2 c 2 d c 3 2 5 . . 9 0 3 l 2 c 2 d c 9 2 5 . . 2 2 4 1 . 9 9 3 1 . 2 4 3 1 5 . 3 3 1 . 3 2 3 1 . 2 9 2 1 . 2 9 2 1 . 8 7 2 1 . 9 6 2 1 1 . 6 2 1 . 9 5 2 1 . 7 5 2 1 . 6 5 2 1 2 . 5 2 1 6 . 8 1 1 . 2 2 4 1 . 9 9 3 1 . 2 4 3 1 5 . 3 3 1 . 3 2 3 1 . 2 9 2 1 . 2 9 2 1 . 8 7 2 1 l 2 c 2 d c 1 . 3 5 20 10 0 145 140 f1 (ppm) 135 130 Apodization = 3.0Hz 25000 20000 15000 10000 5000 0 450 400 350 300 250 200 150 100 50 0 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 34 Figure 1.9: 1H and 13C{1H} NMR spectra of 1-3e 8 4 7 . 4 2 7 . 3 2 7 . 3 2 7 . 2 2 7 . 2 2 7 . 1 2 7 . 6 1 . 7 6 1 . 7 5 1 . 7 4 1 . 7 4 1 . 7 3 1 . 7 3 1 . 7 3 1 . 7 2 1 . 7 1 1 . 7 1 1 . 7 8 9 6 . 8 9 6 . 7 9 6 . 6 9 6 . 6 9 6 . 5 9 6 . l 2 c 2 d c 2 2 . 5 l 2 c 2 d c 1 2 . 5 l 2 c 2 d c 1 2 . 5 9 9 3 . 8 4 7 . 3 2 7 . 3 2 7 . 2 2 7 . 2 2 7 . 1 2 7 . 6 1 . 7 6 1 . 7 5 1 . 7 4 1 . 7 4 1 . 7 3 1 . 7 3 1 . 7 3 1 . 7 2 1 . 7 1 1 . 7 1 1 . 7 8 9 6 . 8 9 6 . 7 9 6 . 6 9 6 . 6 9 6 . l 2 c 2 d c 1 2 . 5 9 9 3 . 5000 0 7 9 0 . 3 9 . 1 7 9 3 . 4 0 2 . 12 10 8 6 f1 (ppm) 9 0 2 . 4 2 0 -2 13 12 11 10 9 8 7 6 f1 (ppm) 5 7 9 0 . 3 9 . 1 7 9 3 . 4 0 2 . 9 0 2 . 4 3 2 1 0 -1 -2 . 9 1 4 1 . 5 6 3 1 . 4 2 3 1 . 4 1 3 1 . 9 0 3 1 . 0 9 2 1 2 . 8 2 1 0 . 8 2 1 0 . 8 2 1 . 9 7 2 1 7 . 8 1 1 l 2 c 2 d c 1 . 3 5 l 2 c 2 d c 9 2 5 . l 2 c 2 d c 7 2 5 . l 2 c 2 d c 5 2 5 . l 2 c 2 d c 2 2 5 . . 4 0 3 . 9 1 4 1 . 5 6 3 1 . 4 2 3 1 . 4 1 3 1 . 9 0 3 1 5 0 146 144 142 140 138 f1 (ppm) 136 134 132 130 Apodization = 3.0Hz 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 35 9500 9000 8500 8000 7500 7000 6500 6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 -500 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 1.10: 1H and 13C{1H} NMR spectra of 1-3f 0 5 7 . 1 2 7 . 1 2 7 . 0 2 7 . 9 1 7 . 9 1 7 . 8 1 7 . 8 1 7 . 7 1 7 . 3 0 7 . 2 0 7 . 2 0 7 . 1 0 7 . 0 0 7 . 8 9 6 . 8 9 6 . 6 9 6 . 6 9 6 . 5 9 6 . 5 9 6 . 4 9 6 . 8 8 . 6 7 8 . 6 6 8 . 6 5 8 . 6 4 8 . 6 4 8 . 6 l 2 c 2 d c 2 2 . 5 l 2 c 2 d c 2 2 . 5 l 2 c 2 d c 2 2 . 5 0 0 4 . 1 2 7 . 0 2 7 . 9 1 7 . 9 1 7 . 8 1 7 . 8 1 7 . 3 0 7 . 2 0 7 . 2 0 7 . 1 0 7 . 0 0 7 . 8 9 6 . 8 9 6 . 6 9 6 . 6 9 6 . 5 9 6 . 5 9 6 . 4 9 6 . 8 8 . 6 7 8 . 6 6 8 . 6 5 8 . 6 4 8 . 6 4 8 . 6 10000 8000 6000 4000 2000 0 8 9 1 . 7.2 7.3 8 9 3 . 4 8 . 1 7.1 7.0 6.9 6. 8 f1 (ppm) 13 12 11 10 9 8 7 6 f1 (ppm) 5 1 0 . 1 9 9 . 1 1 0 4 . 5 8 . 1 4 1 . 2 4 3 2 1 0 -1 -2 l 2 c 2 d c 2 2 5 . l 2 c 2 d c 9 2 5 . l 2 c 2 d c 7 2 5 . l 2 c 2 d c 5 2 5 . 9 . 1 6 1 8 . 1 6 1 . 9 9 5 1 8 . 9 5 1 7 . 3 3 1 . 7 2 3 1 . 2 9 2 1 1 . 9 2 1 7 . 8 2 1 6 . 8 2 1 5 . 8 2 1 5 . 8 2 1 8 . 8 1 1 . 7 4 1 1 . 6 4 1 1 . 5 4 1 1 . 4 4 1 1 . 2 9 2 1 1 . 9 2 1 7 . 8 2 1 6 . 8 2 1 5 . 8 2 1 5 . 8 2 1 l 2 c 2 d c 1 . 3 5 100 50 0 . 2 0 3 9 . 1 6 1 8 . 1 6 1 . 9 9 5 1 8 . 9 5 1 80 60 40 20 0 129.2 129.0 128 . 8 128 .6 f1 (ppm) 164 163 162 161 160 159 f1 (ppm) . 7 4 1 1 . 6 4 1 1 . 5 4 1 1 . 4 4 1 1 150 100 50 0 114. 8 114.7 114.6 f1 (ppm) 114.5 114.4 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 36 34000 32000 30000 28000 26000 24000 22000 20000 18000 16000 14000 12000 10000 8000 6000 4000 2000 0 -2000 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 1.11: 1H and 13C{1H} NMR spectra of 1-3g 1 5 7 . 0 4 7 . 8 3 7 . 0 3 7 . 8 2 7 . 1 1 . 7 0 1 . 7 4 9 6 . 2 9 6 . l 2 c 2 d c 3 2 . 5 l 2 c 2 d c 3 2 . 5 l 2 c 2 d c 2 2 . 5 9 9 3 . 13 12 11 10 9 8 7 6 f1 (ppm) 5 0 9 0 . 2 9 . 1 2 8 . 1 7 0 2 . 9 9 . 1 0 3 2 . 4 3 2 1 0 -1 -2 1 . 2 4 1 1 . 2 4 1 . 0 7 3 1 . 4 2 3 1 3 . 1 3 1 0 . 1 3 1 . 9 0 3 1 . 5 9 2 1 6 . 8 2 1 . 5 9 1 1 8 . 8 1 1 l 2 C 2 D C 1 . 3 5 l 2 C 2 D C 9 2 5 . l 2 C 2 D C 7 2 5 . l 2 C 2 D C 5 2 5 . l 2 C 2 D C 3 2 5 . . 6 0 3 1 . 2 4 1 . 0 7 3 1 . 4 2 3 1 3 . 1 3 1 0 . 1 3 1 . 9 0 3 1 35000 30000 25000 20000 15000 10000 5000 0 4.0×107 3 .5×107 3 .0×107 2000000 1000000 2.5×107 Apodization = 3.0Hz 144 142 140 138 f1 (ppm) 136 134 132 0 2.0×107 1.5×107 1.0×107 5.0×106 0.0 210 200 190 180 170 160 150 140 130 120 110 100 f1 (ppm) 90 80 70 60 50 40 30 20 10 0 -10 37 Figure 1.12: 1H and 13C{1H} NMR spectra of 1-2 4 2 7 . 4 2 7 . 3 2 7 . 3 2 7 . 2 2 7 . 1 2 7 . 1 2 7 . 0 2 7 . 9 1 . 7 9 1 . 7 8 1 . 7 8 1 . 7 7 1 . 7 5 1 . 7 4 1 . 7 3 1 . 7 1 1 . 7 0 1 . 7 0 1 . 7 0 1 . 7 6 0 7 . 6 0 7 . 5 0 7 . 5 0 7 . 6 6 6 . 1 4 5 . 5 7 . 3 3 7 . 3 2 7 . 3 1 7 . 3 1 7 . 3 1 7 . 3 0 7 . 3 9 6 . 3 8 6 . 3 6 8 2 . 5 8 2 . 3 8 2 . 2 8 2 . 9 7 2 . 7 7 2 . 6 7 2 . 5 7 2 . 4 2 7 . 4 2 7 . 3 2 7 . 3 2 7 . 2 2 7 . 1 2 7 . 1 2 7 . 0 2 7 . 9 1 . 7 9 1 . 7 8 1 . 7 8 1 . 7 7 1 . 7 5 1 . 7 4 1 . 7 3 1 . 7 1 1 . 7 0 1 . 7 0 1 . 7 0 1 . 7 6 0 7 . 6 0 7 . 5 0 7 . 5 0 7 . 1500 1000 500 0 7.4 7.3 8 0 6 . 7.2 f1 (ppm) 5 0 2 . 7 1 . 2 7.1 7.0 6.9 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 -2 8 0 6 . 7 1 . 2 5 0 2 . 0 9 0 . 2 0 . 1 7 2 2 . 4 2 . 1 7 2 . 1 . 7 4 4 1 1 . 9 3 1 . 6 7 3 1 4 . 8 2 1 0 . 8 2 1 . 8 7 2 1 1 . 7 2 1 . 3 6 2 1 . 7 5 2 1 l 2 c 2 d c 1 . 3 5 7 . 8 6 1 . 7 5 l 2 c 2 d c 9 2 5 . l 2 c 2 d c 7 2 5 . l 2 c 2 d c 5 2 5 . l 2 c 2 d c 2 2 5 . . 2 9 3 4 . 8 2 1 0 . 8 2 1 . 8 7 2 1 1 . 7 2 1 . 3 6 2 1 . 7 5 2 1 150 100 50 0 130 129 128 127 f1 (ppm) 126 125 124 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 38 1600 1500 1400 1300 1200 1100 1000 900 800 700 600 500 400 300 200 100 0 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 Figure 1.13: 1H and 13C{1H} NMR spectra of 1-5 3 1 . 8 3 8 7 . 1 8 7 . 7 5 7 . 7 5 7 . 5 5 7 . 5 5 7 . 4 3 7 . 4 3 7 . 3 3 7 . 1 3 7 . 1 3 7 . 9 2 7 . 8 2 7 . 3 2 7 . 2 2 7 . 8 1 . 7 8 1 . 7 8 1 . 7 6 1 . 7 5 1 . 7 5 1 . 7 5 1 . 7 3 1 . 7 3 1 . 7 2 1 . 7 2 1 . 7 0 1 . 7 0 1 . 7 8 0 7 . 8 0 7 . 7 0 7 . 6 0 7 . 5 0 7 . 5 0 7 . l 2 c 2 d c 5 1 . 5 l 2 c 2 d c 4 1 . 5 l 2 c 2 d c 4 1 . 5 4 3 7 . 4 3 7 . 3 3 7 . 1 3 7 . 1 3 7 . 9 2 7 . 8 2 7 . 3 2 7 . 2 2 7 . 8 1 . 7 6 1 . 7 5 1 . 7 3 1 . 7 3 1 . 7 2 1 . 7 2 1 . 7 0 1 . 7 0 1 . 7 8 0 7 . 8 0 7 . 7 0 7 . 6 0 7 . 5 0 7 . 5 0 7 . 4000 3000 2000 1000 0 6 9 . 1 3 9 0 . 4 9 0 . 9 0 . 1 3 1 . 1 5 1 . 1 7.35 7.30 7.25 7.20 f1 (ppm) 7.15 7.10 7.05 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 -2 0 8 . 0 1 0 1 . 9 9 1 . 6 9 1 . 3 9 0 . 4 9 0 . 9 0 1 . 3 1 . 1 5 1 . 1 . 9 5 3 1 . 8 4 3 1 0 . 8 2 1 . 5 6 2 1 1 . 5 2 1 . 7 4 2 1 5 . 1 2 1 2 . 1 2 1 . 4 9 1 1 8 . 8 1 1 . 0 7 1 1 . 7 0 1 1 l 2 c 2 d c 1 . 3 5 l 2 c 2 d c 9 2 5 . l 2 c 2 d c 6 2 5 . l 2 c 2 d c 4 2 5 . l 2 c 2 d c 2 2 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 39 6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 650 600 550 500 450 400 350 300 250 200 150 100 50 0 Figure 1.14: 1H and 13C{1H} NMR spectra of 1-7 3 2 7 . 3 2 7 . 9 1 . 7 8 1 . 7 7 1 . 7 7 1 . 7 6 1 . 7 6 1 . 7 6 1 . 7 5 1 . 7 5 6 6 . 5 6 6 . 5 6 6 . l 2 c 2 d c 1 2 . 5 l 2 c 2 d c 1 2 . 5 l 2 c 2 d c 1 2 . 5 1 6 4 . 9 5 4 . 8 5 4 . 7 5 4 . 2 0 . 3 2 0 . 3 0 0 . 3 0 0 . 3 9 9 2 . 9 9 2 . 7 9 2 . 6 9 2 . 7 5 2 . 7 5 2 . 5 5 2 . O 2 H 5 5 2 . 4 5 2 . 3 5 2 . 2 5 2 . 2 5 2 . 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 0 8 . 3 4 9 0 . 1 8 . 0 7 8 . 0 7 9 0 . 5 1 . 1 5 1 . 1 3 2 1 0 -1 -2 . 3 2 4 1 . 7 1 4 1 . 8 7 2 1 . 6 6 2 1 . 5 5 2 1 . 6 1 6 l 2 c 2 d c 1 . 3 5 l 2 c 2 d c 9 2 5 . l 2 c 2 d c 7 2 5 . l 2 c 2 d c 5 2 5 . l 2 c 2 d c 3 2 5 . . 9 1 4 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 450 400 350 300 250 200 150 100 50 0 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 40 Figure 1.15: 1H and 13C{1H} NMR spectra of 1-4a 5 8 4 8 . . 8 8 7 0 . 8 7 0 . 8 6 0 . 8 7 6 7 . 6 6 7 . 4 5 7 . 2 5 7 . 1 5 7 . 6 4 7 . 5 4 7 . 3 4 7 . 3 4 7 . l 3 c d c 7 2 7 . 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 8 9 . 1 9 9 0 . 8 0 4 . 1 0 4 . 3 9 . 1 . 9 6 4 1 . 6 7 3 1 . 5 6 3 1 . 8 2 3 1 . 0 9 2 1 1 . 8 2 1 1 . 7 2 1 l 3 c d c 2 7 7 . l 3 c d c 9 6 7 . l 3 c d c 7 6 7 . 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 750 700 650 600 550 500 450 400 350 300 250 200 150 100 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 41 Figure 1.16: 1H and 13C{1H} NMR spectra of 1-4b 9 6 . 8 7 9 7 . 7 9 7 . 2 5 7 . 0 5 7 . 7 2 7 . 6 2 7 . l 2 c 2 d c 4 2 . 5 l 2 c 2 d c 3 2 . 5 6 6 2 . 4 6 2 . 3 6 2 . 1 6 2 . 1 2 . 1 9 1 . 1 8 1 . 1 13 12 11 10 9 1 0 2 . 9 9 3 . 0 0 4 . 9 9 0 . 8 7 6 f1 (ppm) 2 0 4 . 8 9 5 . 5 4 3 2 1 0 -1 -2 8 . 5 4 1 8 . 3 4 1 . 6 5 3 1 . 4 4 3 1 4 . 1 3 1 . 8 7 2 1 . 3 6 2 1 l 2 c 2 d c 1 . 3 5 l 2 c 2 d c 9 2 5 . l 2 c 2 d c 7 2 5 . l 2 c 2 d c 5 2 5 . l 2 c 2 d c 2 2 5 . . 8 7 2 . 7 4 1 26000 24000 22000 20000 18000 16000 14000 12000 10000 8000 6000 4000 2000 0 260 240 220 200 180 160 140 120 100 80 60 40 20 0 -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 42 Figure 1.17: 1H and 13C{1H} NMR spectra of 1-4c 2 7 . 8 1 7 . 8 4 9 7 . 3 9 7 . 3 9 7 . 8 5 7 . 8 5 7 . 8 5 7 . 7 5 7 . 6 5 7 . 6 5 7 . 8 4 7 . 7 4 7 . 7 4 7 . 6 4 7 . 6 4 7 . 5 4 7 . 5 4 7 . l 2 c 2 d c 4 2 . 5 l 2 c 2 d c 4 2 . 5 l 2 c 2 d c 4 2 . 5 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 -2 1 9 1 . 3 0 1 . 3 9 3 . 3 1 4 . . 3 6 4 1 . 9 5 3 1 . 7 4 3 1 5 . 1 3 1 5 . 1 3 1 1 . 8 2 1 8 . 1 2 1 l 2 c 2 d c 1 . 3 5 l 2 c 2 d c 9 2 5 . l 2 c 2 d c 7 2 5 . l 2 c 2 d c 5 2 5 . l 2 c 2 d c 3 2 5 . 160000 150000 140000 130000 120000 110000 100000 90000 80000 70000 60000 50000 40000 30000 20000 10000 0 100 90 80 70 60 50 40 30 20 10 0 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 43 Figure 1.18: 1H and 13C{1H} NMR spectra of 1-4d 1 7 . 8 1 7 . 8 4 9 7 . 3 9 7 . 3 9 7 . 5 5 7 . 4 5 7 . 4 5 7 . 3 5 7 . 2 5 7 . 2 5 7 . 2 5 7 . 1 5 7 . 3 4 7 . 2 4 7 . 2 4 7 . 1 4 7 . 0 4 7 . 0 4 7 . l 2 c 2 d c 4 2 . 5 l 2 c 2 d c 4 2 . 5 l 2 c 2 d c 4 2 . 5 13 12 11 10 9 9 7 1 . 8 2 4 . 9 8 . 3 3 0 1 . 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 -2 . 4 6 4 1 . 5 5 3 1 . 6 4 3 1 6 . 3 3 1 6 . 1 3 1 5 . 8 2 1 . 8 7 2 1 l 2 c 2 d c 1 . 3 5 l 2 c 2 d c 9 2 5 . l 2 c 2 d c 7 2 5 . l 2 c 2 d c 5 2 5 . l 2 c 2 d c 3 2 5 . 45000 40000 35000 30000 25000 20000 15000 10000 5000 0 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 44 Figure 1.19: 1H and 13C{1H} NMR spectra of 1-4e 4 7 . 8 4 7 . 8 3 7 . 8 6 9 7 . 6 9 7 . 5 9 7 . 5 9 7 . 9 5 7 . 8 5 7 . 8 5 7 . 0 5 7 . 0 5 7 . 9 4 7 . 9 4 7 . 8 4 7 . 8 4 7 . 8 4 7 . 7 4 7 . 0 4 7 . 0 4 7 . 9 3 7 . 8 3 7 . 7 3 7 . 7 3 7 . 6 3 7 . 5 3 7 . 5 3 7 . 4 3 7 . 4 3 7 . 3 3 7 . l 2 c 2 d c 4 2 . 5 l 2 c 2 d c 4 2 . 5 l 2 c 2 d c 4 2 . 5 l 2 c 2 d c 3 2 . 5 l 2 c 2 d c 3 2 . 5 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 -2 8 8 . 1 7 0 . 1 1 9 . 1 4 1 . 2 1 0 4 . . 7 6 4 1 7 . 8 3 1 . 5 4 3 1 1 . 4 3 1 9 . 1 3 1 . 7 9 2 1 . 5 7 2 1 . 5 6 2 1 . 7 4 2 1 l 2 c 2 d c 1 . 3 5 l 2 c 2 d c 9 2 5 . l 2 c 2 d c 7 2 5 . l 2 c 2 d c 5 2 5 . l 2 c 2 d c 3 2 5 . 20000 19000 18000 17000 16000 15000 14000 13000 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 -1000 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 -20 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 45 Figure 1.20: 1H and 13C{1H} NMR spectra of 1-4f 0 6 . 8 9 5 . 8 5 8 7 . 4 8 7 . 4 8 7 . 6 4 7 . 6 4 7 . 6 4 7 . 5 4 7 . 4 4 7 . 6 3 7 . 6 3 7 . 5 3 7 . 5 3 7 . 4 3 7 . 3 3 7 . 3 3 7 . 2 3 7 . 1 3 7 . 1 3 7 . 0 3 7 . 0 3 7 . 0 3 7 . 9 2 7 . 9 2 7 . 8 2 7 . 7 2 7 . l 2 c 2 d c 4 2 . 5 l 2 c 2 d c 3 2 . 5 l 2 c 2 d c 3 2 . 5 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 -2 8 9 1 . 8 9 0 . 5 0 2 . 9 9 6 . 2 . 8 4 1 8 . 6 3 1 . 9 5 3 1 5 . 3 3 1 . 9 1 3 1 . 7 0 3 1 3 . 9 2 1 8 . 8 2 1 . 5 6 2 1 l 2 c 2 d c 1 . 3 5 l 2 c 2 d c 9 2 5 . l 2 c 2 d c 7 2 5 . l 2 c 2 d c 5 2 5 . l 2 c 2 d c 3 2 5 . 30000 28000 26000 24000 22000 20000 18000 16000 14000 12000 10000 8000 6000 4000 2000 0 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 46 Figure 1.21: 1H and 13C{1H} NMR spectra of 1-4g 6 8 6 . 8 4 8 6 . 8 4 1 9 7 . 9 0 9 7 . 2 0 9 7 . 0 7 5 7 . 0 6 5 7 . 5 5 5 7 . 4 4 5 7 . 6 4 1 . 7 0 3 1 . 7 3 1 1 . 7 l 2 c 2 d c 5 3 2 . 5 l 2 c 2 d c 3 3 2 . 5 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 -2 6 9 1 . 0 0 1 . 8 0 4 . 7 9 3 . 2 . 3 6 1 2 . 1 6 1 . 1 6 4 1 2 . 3 3 1 2 . 3 3 1 . 6 1 3 1 3 . 8 2 1 2 . 8 2 1 3 . 5 1 1 . 1 5 1 1 l 2 c 2 d c 1 . 3 5 l 2 c 2 d c 9 2 5 . l 2 c 2 d c 7 2 5 . l 2 c 2 d c 5 2 5 . l 2 c 2 d c 3 2 5 . 19000 18000 17000 16000 15000 14000 13000 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 -1000 90 80 70 60 50 40 30 20 10 0 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 47 Chapter 2: First total synthesis of nagelamide W* 2.1 Introduction Pyrrole imidazole alkaloids are marine metabolites with highly diverse structures ranging from the simplest member of the family, oroidin, to the most complex members - stylissadines A and B isolated till date.1 These alkaloids have a high N to C ratios (∼ 1:2) and show a diverse range of pharmacological properties including antibacterial, antifungal and anticancer activities.2-5 The intriguing biological properties and unique structural features of these compounds have attracted several chemists to target them for total synthesis, aiming to further investigate their biological properties. The pyrrole imidazole alkaloids are often classified as cyclic or acyclic as either monomeric, dimeric, or tetrameric pyrrole imidazole alkaloids based on the number of oroidin units in the alkaloid structure (Figure 2.1) .4,6 The biosynthesis of pyrrole imidazole alkaloids, also known as oroidin alkaloids are hypothesized to be from oroidin and its congeners: hymenidin and clathrodin (Figure 2.1).7-8 This biosynthesis is believed to occur through a variety of oxidation, cyclization, or dimerization, but only a few specifics are known.9-11 * This chapter is reproduced in part with permission from reference 40. Copyright 2024 American Chemical Society 48 Figure 2.1: Structure of oroidin, its congeners, and selected examples of different classes of oroidin alkaloids H2N HN N R2 R1 N H H2N N 10 H N O oroidin: R1 = R2 = Br hymenidin: R1 = H, R2 = Br clathrodin: R1 = R2 = H 12 N ACYCLIC & CYCLIC MONOMERIC Br Br Br Br O N N H N N NH2 HN 7 NH O NH 9 NH nagelamide W (2-1) (acyclic monomeric) NH2 dibromophakelin (cyclic monomeric) ACYCLIC &CYCLIC DIMERIC Br Br NH O HN O R 9 10 Br HN Br NH 10' 9' HN NH2 N N H N NH2 nagelamide A: R = H; 9,10-dihydro nagelamide B: R= OH; 9',10'-dihydro nagelamide C: R = H; ∆9(10),9'(10') nagelamide D: R = H; 9,9',10,10'-tetrahydro (acyclic dimeric) H2N HN N N N H NH2 O HN NH Br X HN Y N H O Br nagelamide E: X = Y= H nagelamide F: X = Br, Y= H nagelamide G: X = Y= Br (cyclic dimeric) CYCLIC TETRAMERIC Br Br NH HN Br Br Br O HN O NH HN O HN H N OH H HN O NH HN NH O HN HN N H Br NH O NH H N N H H HO O stylissadine A H N Br Br NH The nagelamides are a class of over 30 pyrrole imidazole alkaloids with remarkable structural diversity. These alkaloids were isolated predominantly by the Kobayashi lab from marine sponges found mostly from Pacific and Caribbean variants of sponges.12 The nagelamides first appeared in the literature in 2004 when nagelamides A-H were reported by the Kobayashi lab 49 (Figure 2.1).13 Since then, additional members of this family of natural products have been isolated and reported. Over the decades, several groups have developed different strategies to access these natural products but only nagelamides D14-15 and E16 have succumbed to total synthesis and no other total synthesis has appeared in the literature till date.17 Additionally, de novo synthesis of nagelamide H and didebromonagelamide A has been reported.18 Nagelamide W (2-1) (Figure 2.1) was isolated from marine sponges Agelas sp. collected from Kerama Island, Okinawa in 2013.19 The natural product was isolated alongside nagelamide U and V. During isolation, the relative stereochemistry of nagelamide W at the imidazoline core was assigned based on the ROESY correlation of H-9/H-12 and 7-NH/H-10, which suggests an anti- relationship for H-9 and H-10, and a trans configuration was assigned but the absolute stereochemistry was not determined. Nagelamide W was isolated as a colorless solid and was found to be optically active [𝛼]! "# = – 1.2 (c 0.25, MeOH). Nagelamide W is hypothesized to be biologically made by guanidination of the olefine in oroidin as depicted in Scheme 2.1. The biological properties of nagelamide W is underexplored and it is only known to have moderate antimicrobial activity against Candida albicans (IC50 4µg/mL).19 This furthers strengthen why nagelamide W should be synthesized to further explore its biological activity. Scheme 2.1: Proposed biological pathway for the synthesis of Nagelamide W H2N HN N H N O oroidin R2 R1 N H NH H2N NH2 H N N H2N N NH2 NH O N H nagelamide W (1-1) Br H N Br 50 2.2 Results and Discussion 2.2.1 Unsuccessful attempt to synthesize nagelamide W The initial approach to access nagelamide W (2-1) is shown in Scheme 2.2. In this retrosynthesis, nagelamide W could be accessed by hydrazine-mediated double deprotection of the pyrimidine part of the imidazo[1,2-a]pyrimidine core in 2-2, using the heterocycle as a masked amino imidazolyl group.20-21 Compound 2-2 can be obtained by series of chemical transformation of 2-5. The key approach in this retrosynthesis was the proposed Friedel-Crafts like 1,4-addition of imidazo[1,2-a]pyrimidine and aldehyde 2-7. At this step the two chiral centers in the natural product would be created, as well as the key imidazoline core of the natural product. Scheme 2.2: Restrosynthesis of nagelamide W H N N H2N N NH2 NH O N H nagelamide W (2-1) deprotection Br H N Br N N N 2-4 NH2 reductive amination N N N N Br Br N H O 2-3 Br X Br N H H N O 2-2 N N N imidazo[1,2-a]pyrimidine 1,4-addition H N N N O H N N N 2-7 N N N O N N N N N 2-5 The closest example for the proposed Friedel-Craft like 1,4-addition step is based on the previous work done by the MacMillan group on the enantioselective alkylation of pyrrole and indole using MacMillan catalyst and α,β-unsaturated aldehydes (Scheme 2.3).22-23 The reaction proceeds through the reversible formation of iminium ions with the chiral catalyst, which lowers the LUMO of the α,β-unsaturated aldehyde. Then, the conjugate addition of pyrrole or indole follows, resulting in the formation of the product. The reactions show to be efficient with yields 51 up to 90% and enantioselectivity up to 99% ee. While pyrrole and indole have been extensively studied, the reactivity of imidazo[1,2-a]pyrimidine remains unexplored. The first aim of the current approach to access nagelamide W became to first investigate the reactivity of imidazo[1,2- a]pyrimidine and aldehyde 2-7 in the Friedel-Crafts like 1,4-addition reaction that was pioneered by the MacMillan lab. Scheme 2.3: Literature precedence on asymmetric Friedel-Craft alkylation with pyrrole and indole O Me N N H •TFA (20 mol%) O THF-H2O 68-90% yield, 87 - 97 %ee N Me R O + R N Me O Me N N H •TFA + R O 1-8 (20 mol%) CH2Cl2/i-PrOH 70-94% yield, 89 - 97 %ee N Me O R N Me Following the proposed retrosynthesis, Vilsmeier-Haack formylation of imidazole[1,2- a]pyrimidine gave aldehyde 2-7 in moderate yield24 and was used to explore the Friedel-Crafts like 1,4-addition. Initially, the reaction of imidazo[1,2-a]pyrimidine with aldehyde 2-7 in the presence of MacMillan catalyst gave no product (Scheme 2.4). This preliminary result led to the investigation of the nucleophilicity and electrophilicity of imidazo[1,2-a]pyrimidine and aldehyde 2-7 respectively to understand the underlying cause of the reactivity failure. It is also apparent that the electronics of the reactants, especially the aldehyde, is quite different from the one utilized by the MacMillan group. Exploring the limits of these reactants could reveal their limitation and perhaps how those limitation could be overcome. 52 Scheme 2.4: Attempt on Friedel-Craft like 1,4-addition O H N N N (1.0 equiv) 2-8 (20 mol%) N POCl3 (3.2 equiv) N N N DMF, 0°C - 90 °C, 12 h 43% N N 2-7 DCE/i-PrOH (85:15) – 40°C or 0 °C - reflux O H N N N N N N In order to understand the reactivity of imidazo[1,2-a]pyrimidine towards conjugate addition, crotonaldehyde and cyclohexenone were reacted with imidazo[1,2-a]pyrimidine using iminium catalyst as shown in Scheme 2.5. No products were obtained in any of the reactions, and in some instances, the starting materials decomposed upon heating to reflux. It was concluded that imidazo[1,2-a]pyrimidine is a weak nucleophile and does not undergo conjugate addition under the explored conditions, even when using highly reactive electrophiles such as crotonaldehyde. Scheme 2.5: Investigation of the nucleophilicity of imidazo[1,2-a]pyrimidine O Me N N H •TFA catalyst electrophile (1.0 equiv) catalyst (20 mol%) N DCM/i-PrOH (85:15) N N E N N N entry E O O 1 2 3 4 temp –40 0 – rt –40 yield 0% 0% 0% 0 – rt 0% Concurrent investigation of the electrophilicity of aldehyde 2-7 toward conjugate addition did not show promising result as well. Initially, the aldehyde's electrophilicity was assessed using the MacMillan catalyst and anisole as the nucleophile (Scheme 2.6a). The reaction gave no product even after being stirred at room temperature for up to 3 days. Increasing the temperature of the reaction also did not lead to the formation of any product. After this, stronger nucleophiles such as Gilman reagents were investigated (Scheme 2.6b), and they all gave no product. In addition, 53 attempt to increase the electrophilicity of the aldehyde by using different Lewis’s acid did not facilitate the formation of any product. The aldehyde's lack of reactivity in conjugate addition was attributed to the anticipated loss of aromaticity in the imidazole ring upon the conjugate addition process. Scheme 2.6: Investigation of the electrophilicity of aldehyde 2-7 (a) O H N N N 2-7 (b) O H N N N 2-7 + + O Me N N H •TFA 20 mol% DCM/i-PrOH (85:15) 0 °C to rt, 3 days OMe OMe O H N N N O N H R R2CuLi 1.1 equiv R = Me or Ph THF, –78 °C, 3.5 h N N w/wo Lewis acid (1.0 equiv) AlCl3, Al(O-iPr)3, or BF3•OEt2 After encountering failure due to the lack of reactivity of both imidazo[1,2-a]pyrimidine and aldehyde 2-7 towards conjugate addition, it became imperative to reevaluate the proposed approach for the total synthesis of nagelamide W, leading to the conclusion that a different approach was warranted. 2.2.2 Alternate approach for the synthesis of nagelamide W Based on the revised retrosynthetic analysis (Scheme 2.7), the proposed method for accessing the 2-aminoimidazole moiety of nagelamide W through a late-stage hydrazine-mediate deprotection20-21 and amidation25 of the amine (2-10) with dibromopyrrole carboxylate derivative remained unchanged. The 2-aminoimidazoline core in 2-10 can be obtained from 2- alkoxyimidazoline 2-11 through a nucleophilic displacement of the alkoxy group similarly to the approached used in the synthesis of dibromophakellin26 and dragmacidin E27 and the primary 54 amine can also be obtained through alcohol transformation. The key intermediate 2-11 in this total synthesis could be obtained through the functionalization of alkene 2-12 to access the key imidazoline core of the natural product.28 Alkene 2-12 could be accessed from imidazo[1,2- a]pyrimidine which is readily available from commercial sources. Scheme 2.7: Revised retrosynthetic analysis of nagelamide W H2N HN N N H N O Br Br N H H2N NH nagelamide W (2-1) H2N HN N N H N O N H X Br O Br N N 2-9 Br Br N H 2-3 H2N HN N NH2 N N N 2-10 alkene functionalization RO HN N N OPHG N N 2-11 alcohol tranformation to amine OPG N H2N + N N N 2-12 N N N commercially available Following the new approach, alkene 2-13 was synthesized using the Horner–Wadsworth– Emmons (HWE) reaction from aldehyde 2-7 to give the desired product 2-13 in up to 89% yield (Scheme 2.8). The reduction of the conjugated ester 2-13 to allylic alcohol 2-14 was first carried out using diisobutylaluminium hydride (DIBAL-H). This reduction gave the desired product in 5- 7% yield (Table 2.1). Mass spectrometry analysis of the crude reaction showed several other products which appeared to be over reduction product including the reduction of imidizo[1,2- a]pyrimidine ring and reduction of both ester and alkene. However, isolation of any these side products via column chromatography was unsuccessful. Different reaction conditions and hydride reagents were also investigated even though DIBAL-H is often used for this type of reduction. Other hydride sources such as LiAlH4 (Table 2.1, entry 5 – 7), NaBH4 (Table 2.1, entry 8), and LiBH4 (Table 2.1, entry 9) failed to give the desired product. In some instances, starting materials 55 were recovered, while in others, complete decomposition occurring at higher equivalent of reducing agent and room temperature. Scheme 2.8: Synthesis of alkene 2-13 and reduction attempts to allylic alcohol 2-14 O O EtO P EtO (1.0 equiv) OEt NaH (1.02 equiv) DCM, 0oC - rt, 2.5 h 89% O H N N N 2-7 O OEt see Table 2.1 N N N 2-13 OH N N N 2-14 Table 2.1: Attempts on the reduction of 2-13 to 2-14 Entry Reagent (equiv.) Conditions Result 1 2 3 4 5 6 7 8 9 DIBAL-H (2.2 equiv) THF, - 78 °C – rt, 2 h 5% product DIBAL-H (4.2 equiv) THF, - 78 °C, 3 h ND, 45% RSM DIBAL-H (3.0 equiv) DCM, - 78 °C – rt, 2 h 7% product, 0% RSM DIBAL-H (4.4 equiv) DCM, - 78 °C – rt, 3 h ND, 0% RSM LiAlH4 (1.1 equiv) THF/DCM (1:1), 0 °C – rt, 3 h ND, 75% RSM LiAlH4 (1.1 equiv) THF, 0 °C – rt, 45 min LiAlH4 (1.5 equiv) THF, rt, 2 h BnCl (1.5 equiv) NaBH4 (2.2 equiv) THF, 0 °C – rt, 24 h LiBH4 THF, 0 °C – rt, 0.5h ND ND NR NR Based on the evidence of imidazo[1,2-a]pyrimidine ring reduction observed through mass spectrometry, it was hypothesized that these side products could be prevented by initially removing the pyrimidine ring to obtain aminoimidazole. This aminoimidazole could then be protected before proceeding with the reduction of the conjugated ester to yield the allylic alcohol. The deprotection of the imidazoline ring was achieved using hydrazine hydrate and boc-protection of the free 56 nitrogens gave the desired triboc-product 2-15 in 64% yield over two steps. With the 2-15 at hand, the reduction of the ester to allylic alcohol was achieved using DIBAL-H in THF at – 78 °C in 84% yield (Scheme 2.9). While this approach diverged from the proposed method outlined in Scheme 2.7 for accessing nagelamide W, it offered insights into the reductive behavior of imidazo[1,2-a]pyrimidine different from what is available in the literature20,29 and provided a strategy for circumventing hydride reduction chemistry when this core is present. Scheme 2.9: Alternate strategy for the reduction of ester 2-13 to allylic alcohol O O OEt N N N 2-13 OEt NH2NH2 • H2O (1.1 equiv) Ethanol, 75 oC, 12 h Boc2O (8 equiv) Et3N (5 equiv) DMAP (20 mol%) THF, rt, 12 h 64% (2 steps) N Boc2N N Boc 2-15 OH DIBAL-H N THF, -78 oC - 0 oC, 2.5 h 84% Boc2N N Boc 2-16 While we successfully obtained the allylic alcohol 2-16, the absence of the imidazo[1,2- a]pyrimidine core as a masked amino imidazolyl group, along with the choice of protecting group, presents future challenges such as protecting group cleavage during alkene functionalization with acid. Therefore, it is necessary to reconsider an alternate approach to access the allylic alcohol while retaining the imidazo[1,2-a]pyrimidine core. We envisioned using Suzuki coupling to access alkene 2-12. Following previously reported procedures,30 propargyl alcohol was benzylated to give benzyl propargyl ether 2-17. The ether then underwent zirconium-mediated hydroboration using Schwartz's reagent and pinacolborane to give organoborane 2-18 in 93% yield.31-32 Iodination of imidazo[1,2-a]pyrimidine with N- iodosuccinimide in anhydrous DMF gave 2-19 in good yield. We then utilized a palladium- 57 catalyzed Suzuki reaction, using aryl iodide 2-19 and organoborane 2-18 to give the desired alkene 2-12 in 75% yield. Scheme 2.10: Synthesis of alkene 2-12 via Suzuki coupling BnBr (1.02 equiv) NaH (1.5 equiv) DMF, 0 °C - rt, 16 h 89% OH propargyl alcohol N NIS (1.02 equiv) N N imidazo[1,2-a] pyrimidine DMF, rt, 3 h 91% HPBin (1.1 equiv) ZrCp2HCl (10 mol%) Et3N (10 mol%) Neat, 60 °C, 24 h 93% 1-18 (1.1 equiv) Pd(PPh3)4 (5 mol%) aq K2CO3 (2M, 2.0 equiv) 1,4-dioxane, 80 °C, 24 h 75% OBn 2-17 I N N N 2-19 O B O 2-18 OBn OBn N N N 2-12 With alkene 2-12 at hand, we proceeded to synthesize the imidazoline core of nagelamide W. First, we treated compound 2-12 with N-bromosuccinimide (NBS) and cyanamide in dichloromethane (DCM) at room temperature to access cyanamide bromide 2-20, but no desired product was obtained (Table 2.2, Entry 1).28 When the solvent was changed to tetrahydrofuran (THF) and the reaction cooled to –78 °C, the desired cyanamide bromide product 2-20 was obtained but only 27% yield (dr = 3:1) (Table 2.2, Entry 2). To optimize the reaction, different conditions were investigated. Implementation of different bromonium sources such as 1,3- dibromo-5,5-dimethylhydantoin (DBDMH) and N-bromophthalimide gave similar results as NBS (Table 2.2, Entries 3-4). Then, the role of solvent was studied using NBS as the bromonium source. When DCM was used as solvent at –70 °C, no desired product was isolated probably due to the low solubility of cyanamide in DCM at this temperature (Table 2.2, Entry 5). We found that the use of DCM and hexafluoroisopropanol (HFIP) as co-solvent in a 5:1 ratio, gave the desired product 2-20 in 31% but also HFIP-adduct 2-21 as a side product in 37% yield (Table 2.2, Entry 6). Previous studies have shown that HFIP activates NBS by forming intermolecular hydrogen bonding with the carbonyl of NBS.33-34 Additionally, HFIP helps solubilize cyanamide at this 58 cryogenic temperature. The combination of THF and HFIP did not seem to increase the yield of the desired product (Table 2.2, Entry 9), and we hypothesized that THF sequesters HFIP and prevents hydrogen bond formation between HFIP and NBS. The yield of 2-20 can be increased up to 63% when the ratio of DCM/HFIP was changed to 9:1 (Table 2.2, Entry 7) and the side product 2-21 can be minimized. A gram scale synthesis afforded 61% of the product 2-20 at –10 °C (Table 2.2, Entry 8). Table 2.2: Synthesis and optimization of cyanamide bromide 2-20 OBn Br+ source N H2N (4.0 equiv) solvent, condition N N N 2-12 N HN N OBn Br F3C O N CF3 OBn Br N N 2-20 (anti & syn mixtures) N N 2-21 side productd entry Br+ source (1.1 equiv) solvent conditions yield of 10b 1 2 3 4 5 6d 7e 8e,f 9 NBS NBS NBDMH N-bromophthalimde NBS NBS NBS NBS NBS DCM THF THF THF DCM rt, 72 h –78 °C, 2 h –78 °C, 3 h –78 °C, 1 h –78 °C - rt, 24 h NDc 27% (dr = 3:1, anti:syn) 29% (dr = 3:1, anti:syn) 28% (dr = 3:1, anti:syn) NDc DCM/HFIP (5:1) 0 °C, 3 h 31% (dr = 2:1, anti:syn) DCM/HFIP (9:1) 0 °C, 2 h 63% (dr = 2:1, anti:syn) DCM/HFIP (9:1) –10 °C, 2 h 61% (dr = 2:1, anti:syn) THF/HFIP (9:1) 0 °C, 3 h 34% (dr = 3:1, anti:syn) art = room temperature, all reported yields are isolate yields. bDiastereomeric ratio (dr) was assigned based on NMR coupling constant. cND = no product isolated. dSide product 1-21 was also isolated in 37% yield. eReaction was ran at 0.075 M concentration. fGram scale. After the synthesis of 2-20 was optimized, we constructed the imidazoline core of nagelamide W in two steps by treating 2-20 with anhydrous ethanolic hydrogen chloride to give the isourea intermediate 2-22. Treating intermediate 2-22 with triethylamine under reflux provided 59 imidazoline 2-23 as a single diastereomer (Scheme 2.11). Notably, the trans-imidazoline 2-23 was only obtained from anti-isomer of 2-22. Hydrogenation of the benzyl ether group in 2-23 to give alcohol 2-24 was not feasible due to the potential hydrogenation of the imidazo[1,2-a]pyrimidine core.29,35 We were able to achieve the debenzylation of 2-23 by treating the compound with 2.2 equivalents of boron trichloride to give the desired alcohol 2-24 in 80% yield. This debenzylation is concentration dependent and the best yield was obtained at [0.50 M]. At lower concentrations yields reduced significantly due to additional C-2 dealkylation of the ethyl group of the imidazoline to give the corresponding imidazolinone. Scheme 2.11: Synthesis of imidazoline core and alcohol 2-24 N HN N OBn Br N N 2-20 dr = (2:1, anti:syn) HCl (4.0 M in EtOH) (10.0 equiv) EtOH, rt, 48 h 2 N O NH BCl3 (1.0 M, 2.2 equiv) N N N OH 2-24 DCM, 0 °C, 2 h [0.5 M] 80% Cl NH2 OBn Br O HN N N N 2-22 Et3N (15.0 equiv) EtOH, reflux, 3 h 51% (2 steps) 2 N O NH N N N OBn 2-23 After obtaining alcohol 2-24, we attempted the conversion of the hydroxyl group to primary amine, which proved to be more challenging than anticipated. A recent manuscript by Herath and Lovely25 is a beautiful review that discuss various strategies that is used for the incorporation of pyrrole carboxamide in the total synthesis of pyrrole-imidazole alkaloids. We started by doing a Mitsunobu reaction using phthalimide, triphenylphosphine, and diethylazodicarboxylate with the aim of obtaining 2-25, which we envisioned using to access 2-26 60 upon treating with hydrazine (Scheme 2.12).36 Using this approach, compound 2-24 was treated under Mitsunobu condition, however the desired imide product 2-25 was only observed in mass spectrometry and the isolated product was the [5,3]-fused imidazoline-aziridine 2-27 with 68% of alcohol starting material recovered after 16 hours (scheme 2.12). Due to incomplete consumption of the alcohol 2-24, the equivalent of phthalimide was increased to 2.3 and triphenylphosphine (PPh3) and diethyl azodicarboxylate (DEAD) equivalents were increased to 2.2 each. Under the condition above, only the aziridine (2-27), was isolated in 28% yield. Also, an attempt to convert the hydroxyl group of 2-24 to chloride using thionyl chloride (SOCl2) did not give the desired product. It was hypothesized that the formation of aziridine is due to the present of free N-H in 2- 24 and protection of the N-H should not result in the formation of the aziridine product, and the desired imide should be obtained in good yield. Scheme 2.12: Unsuccessful attempt to access imide 2-25 O HN O (2.3 equiv) PPh3 (2.2 equiv) DEAD (2.2 equiv) THF, rt, 16 h O N NH N N N OH 2-24 O N NH O 2-25 N O N N N O N N R 28% 2-27 NH2NH2 O N NH NH2 HN H2N N 2-26 To test this theory, N-H protected imidazole was synthesized. The N-tosylated protected ether (2-28) was synthesized in 66% as a mixture of regioisomers from 2-24. Treatment of 61 compound 2-28 with boron trichloride did not result the desired debenzylated product but instead gave the dealkylated imidazolone product (2-29) in 51% yield (Scheme 2.13). The failure to obtain the desired debenzylated product 2-30 led to the investigation of a different route to access the amine from the alcohol 2-24. Scheme 2.13: Unsuccessful attempt to access N-protected free alcohol 2-30 O N NH N N N 2-23 OBn + Et3N (1.5 equiv) DMAP (20 mol%) RSO2Cl DCM, rt, 12 h R = 2,4,6-triisopropylbenzene 66% O N O N S R O OBn N N 2-28 N (mixtures of regioisomers) BCl3 (1.02 equiv) DCM, 0 °C, 1h 51% O HN O N S R O OBn N N 2-29 N mixture of regioisomers O N O N S R O OH N N 2-30 N not isolated Alternatively, mesylating 2-24 provided 2-31 in 75% yield which then underwent a substitution reaction with potassium phthalimide to give 2-25 in good yield. We were able to obtain a crystal structure for compound 2-25 to confirm the trans stereochemistry originally assigned by NMR studies as shown in the scheme above. Treating 2-25 with hydrazine hydrate gave a complex mixture of products with 2-26 only being observed by mass spectrometry (Scheme 2.14). 62 Scheme 2.14: Unsuccessful attempt to access amine 2-26 O N NH N N N OH 2-24 MsCl (1.05 equiv) Et3N (2.0 equiv) DCM, rt, 4 h 75% O N NH N N N OMs 2-31 O K N O (2.0 equiv) DMF, rt, 48 h 77% O N NH O 2-25 N O N N N NH2NH2 complex mixture O N NH NH2 HN H2N N 2-26 only observed in mass spec 2-25 X-ray CCDC 2194100 Following the failed attempts to convert alcohol 2-24 to amine using the Gabriel amine approach, we explored the Staudinger approach.37 First, we converted 2-31 to azide 2-32 followed by Staudinger reduction with triphenylphosphine since hydrogenation was not feasible in the presence of imidazo[1,2-a]pyrimidine.29 The desired amine 2-33 was obtained alongside imidazolone 2-34 as side product (Scheme 2.15a). We hypothesize that the imidazolone 2-34 was formed because of water as co-solvent in the reaction and at high temperature. When equivalent amount of water was used, there was no selectivity towards amine 2-33. We also attempted Staudinger reduction at room temperature and the reaction resulted in no product formation. However, when catalytic amounts of triphenylphosphine and diphenyldisiloxane was used as reducing agent,38 the desired product was obtained in low yield (Scheme 2.15b). Furthermore, direct ligation of azide 2-32 and carboxylic acid 2-37 using 2,2-Dipyridyl diselenide (PySeSePy) as catalyst and that resulted in no product formation (Scheme 2.15c).39 63 Scheme 2.15: Synthesis of compound 2-35 (a) O O O N NH NaN3 (3.0 equiv) N NH PPh3 (1.2 equiv) N NH + N N N 2-31 DMF, 60 °C, 16 h OMs 79% N N N 2-32 THF/H2O (4:1), 70 °C, 4 h N N3 N N N NH2 2-33 2-34 O HN NH N N NH2 Cl3C O Br Br N H Et3N (2.0 equiv) N DMF, rt, 16 h, 50 °C, 2h O NH N NH N N 2-35 O 27% (2 steps) + Br HN Br O NH HN NH N N N 2-36 O 23% (2 steps) HN Br Br O N NH N3 2-32 PPh3 (0.1 equiv) (PhSiH2)2O (1.5 equiv) THF, rt, 24 days then, H2O (10 equiv) Cl3C O Br Br N H Et3N (2.0 equiv) O N NH N N N 2-33 DMF, rt, 16 h, 50 °C, 2h 20% (2 steps) NH2 N N N (b) N N N (c) O N NH + 2-32 N3 HO O Br Br N H 2-37 N N N Me3P (1.0M in THF, 2.4 equiv) PySeSePy (20 mol%) DMF-Toluene (1:2), rt,16h N N N O NH N NH 2-35 O HN Br Br O NH N NH 2-35 O HN Br Br Since we planned on accessing the 2-aminoimidazoline group of the natural product from the 2-ethoxyimidazoline through nucleophilic displacement with ammonia, we thought of an alternate approach to access both 2-aminoimidazoline and primary amine from intermediate 2-31. In this approach, 2-31 underwent two nucleophilic displacements with ammonia to give 2-38 as a 64 mesylate salt (Scheme 2.16). Amidation of the primary amine with 4,5- dibromotrichloroacetylpyrrole in the presence of triethylamine resulted in 2-39 as our final intermediate to nagelamide W. Scheme 2.16: Completion of nagelamide W synthesis O N NH N N N OMs 2-31 NH3 (7.0 M in MeOH) sealed tube, 150 °C, 4.5 h N OMs N NH2 NH 2-38 NH2 N N Cl3C O N H (1.1 equiv) Br Br Et3N (1.5 equiv) DMF, rt, 16 h 52% (2 steps) OMs NH2 HN NH O H N N H 2-39 N N N NH2NH2 • H2O EtOH, rt, 6.5 h 98% Br Br OMs HN H2N H N N NH2 NH O N H nagelamide W (2-1) H N Br Br Once the synthesis of 2-39 was completed, we moved forward to the last step of the total synthesis. Herein, the imidazo[1,2-a]pyrimidine core of 2-39 was deprotected using hydrazine monohydrate affording the natural product, nagelamide W (2-1), as a mono-mesylated salt in 98% yield after column chromatography. To compare the NMR data for the synthetic sample with the isolated sample, bis-trifluoroacetate salt of 2-1 was prepared. Spectroscopic data obtained from the synthesized nagelamide W is in full agreement with the spectra reported by Kobayashi et. al’s (see Table 2.3 and Table 2.4 for comparison of nagelamide W to isolated natural product).19 Slight changes in N-H ppm shift was observed and this is typically seen with exchangeable protons in in NMR. Additionally, the x-ray crystallographic of 2-25 also confirmed the trans stereochemistry that was previously assigned by NOE experiment. 65 Table 2.3: Comparison of 1H-NMR data for isolated19 and synthesized Nagelamide W (2-1)40 •2CF3COO 1H-# 1H-NMR of Isolated, DMSO-d63 1H-NMR of synthesized, 500 Δ1H-NMR MHz, DMSO-d6 1'-NH2 7.70 (2H, br s) 7.69 (2H, br s) 2 6.83 (1H, s) 6.84 (1H, s) 2'-NH 12.27 (1H, br s) 12.16 (1H br s) 3'-NH 12.86 (1H, br s) 12.82 (1H, br s) 4 4.78 (1H, d, J = 7.0) 4.80 (1H, d, J = 7.4) 4'-NH 8.75 (1H, br s) 8.75 (1H, br s) 5'-NH2 8.14 (2H, br s) 8.13 (2H br s) 6 3.98 (1H, q, J = 7.0) 3.98 (1H, q, J = 6.5) 6'-NH 8.79 (1H, br s) 8.61 (1H, br s) 7 3.43 (2H, m) 3.45 (2H, m) 7'-NH 8.37 (1H, t, J = 6.0) 8.37 (1H, t, J = 6.0) 9'-NH 12.72 (1H, br s) 12.74 (1H, d, J = 2.8) -0.01 0.01 -0.11 -0.04 0.02 0.00 -0.01 0.00 -0.18 0.02 0.00 0.02 10 6.94 (1H, d, J = 1.8) 6.91 (1H, d, J = 2.4) -0.03 66 Tables 2.4: Comparison of 13C-NMR data for isolated19 and synthesized Nagelamide W (2-1)40 13C-# 13C-NMR of Isolated, DMSO- 13C-NMR of synthesized, 500 Δ13C-NMR d63 148.3 113.2 124.5 53.2 158.8 60.0 41.3 159.5 127.7 111.6 98.0 105.0 1 2 3 4 5 6 7 8 9 10 11 12 MHz, DMSO-d6 148.2 113.2 124.6 53.3 158.7 60.1 41.4 159.6 127.8 111.7 98.1 105.2 -0.1 0.0 0.1 0.1 -0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.2 2.3 Conclusions In conclusion, we have accomplished the first total synthesis of (±)-nagelamide W in 11 longest linear sequence with an overall yield of 6.0%. The structure described by Kobayashi et. al’s in the isolation report19 has been confirmed by NMR spectroscopy and the trans stereochemistry at the imidazoline core also confirmed by x-ray crystallography. 67 2.4 Experimental section General Information All flasks were oven-dried overnight and cooled under nitrogen. Reactions were carried out under a nitrogen atmosphere unless stated otherwise. All chemicals and solvents were purchased from commercial sources and used without further purification, unless otherwise mentioned. THF and ethanolic hydrogen chloride solution were dried over molecular sieves before use in the reaction. Dichloromethane was dried by passing it through packed column of alumina, and triethylamine was distilled over calcium hydride. N-bromosuccinimide was recrystallized in hot water before use and stored in the dark. Infrared spectra were recorded on a Jasco Series 6600 FTIR spectrometer. The mass spectrometer ionization method was ESI with a quadrupole detector. 1H and 13C NMR spectra were recorded on a 500 MHz spectrometer. Chemical shifts are reported relative to the residue peaks of the solvent: CDCl3: 7.26 ppm for 1H and 77.0 ppm for 13C, CD3OD: 3.31 ppm for 1H and 47.6 ppm for 13C, and DMSO-d6: 2.50 ppm for 1H and 39.5 ppm for 13C. Signal multiplicities are given as s (singlet), br (broad), d (doublet), t (triplet), dd (doublet of doublet), and m (multiplet). Reaction carried out at room temperature (rt) implies temperature range usually between 20 – 22 °C. Column chromatography was performed using a Teledyne ISCO CombiFlash® NextGen system with prepacked columns (RediSep® Normal-phase silica, 20-40 microns and RediSep® Rf Gold Reversed-Phase C18 silica, 20-40 microns). TLCs were performed on pre-coated 0.25 mm thick silica gel 60 F254 plates and pre-coated 150 um thick, C18 reverse phase F254 plates, visualized using UV light and iodine staining. 68 N N N POCl3 DMF, 0 – 90 °C, 12 h O H N N N 2-7 imidazo[1,2-a]pyrimidine-3-carbaldehyde (2-7): To a 50 mL round bottom flask equipped with a magnetic stirrer was added dimethylformamide (15 mL) and was cooled to 0 °C. Then, phosphorus oxychloride (5 mL, 53.7 mmol) was added and stirred at 0 °C for 30 mins. After that, imidazo-[1,2-a]-pyrimidine (2.0 g, 16.8 mmol) was added and the reaction was heated to 90 °C while stirring overnight. The reaction was cooled to room temperature and quenched with lithium bromide (15 mL). The pH of the reaction was adjusted to approximately 11 using 20% sodium hydroxide solution. The product was extracted with ethyl acetate (5 x 150 mL), and the organic layers were combined, dried over sodium sulfate, decanted, and concentrated. The crude was then purified using CombiFlash chromatography (silica gel, 20-40 microns, gradient 3% methanol/dichloromethane) to give 2-7 as yellow solid (1.07 g, 43%). mp = 196 – 198 °C. 1H NMR (500 MHz, CDCl3) δ 9.99 (s, 1H), 9.76 (dd, J = 6.8, 2.1 Hz, 1H), 8.86 (dd, J = 4.2, 2.1 Hz, 1H), 8.54 (s, 1H), 7.23 (dd, J = 6.8, 4.2 Hz, 1H). 13C{1H} NMR (125 MHz, CDCl3) δ 178.8, 154.4, 151.7, 147.6, 136.3, 123.1, 111.6. IR (neat, cm–1): 3109, 2847, 1646, 1613. HRMS (ESI-TOF) m/z: [M + H]+ calcd for (C7H6N3O+) 148.0505; found 148.0536. EtO P EtO O O NaH OEt DCM, 0 °C - rt, 2.5 h O H N N N 2-7 O OEt N N N 2-13 ethyl (E)-3-(imidazo[1,2-a]pyrimidin-3-yl)acrylate (2-13): Triethyl phosphonoacetate (1.40 mL, 6.79 mmol) was added dropwise under argon to a suspension of sodium hydride (NaH) (0.28 g, 6.93 mmol) in anhydrous dichloromethane (DCM) (8 mL) at 0 °C. The resulting solution was 69 stirred for 15 minutes until no hydrogen gas was released from the reaction. Then, a solution of imidazo[1,2-a]pyrimidine-3-carbaldehyde (1.0 g, 6.79 mmol) in anhydrous DCM (30 mL) was added dropwise over 20 minutes. The resulting solution was warmed slowly to room temperature and stirred for 2.5 h. The reaction was then quenched with ammonium chloride (20 mL), extracted with dichloromethane (3 x 40 mL). The organics were combined and washed with brine (50 mL). The organics was dried over sodium sulfate, concentrated, and titurated with cold diethyl ether to give the product as a yellow solid (1.31 g, 89%). mp = 192 – 194 °C. 1H NMR (500 MHz, CDCl3) δ 8.66 (dd, J = 6.9, 2.0 Hz, 1H), 8.63 (dd, J = 4.1, 2.0 Hz, 1H), 8.19 (s, 1H), 7.80 (d, J = 16.0 Hz, 1H), 7.08 (dd, J = 6.9, 4.1 Hz, 1H), 6.43 (d, J = 16.0 Hz, 1H), 4.26 (q, J = 7.1 Hz, 2H), 1.32 (t, J = 7.1 Hz, 3H). 13C{1H} NMR (125 MHz, CDCl3) δ 166.7, 150.8, 150.4, 138.1, 132.0, 127.6, 120.2, 116.0, 110.0, 60.8, 14.3. FTIR (neat, cm-1): 3063, 2977, 1699, 1626, 1516. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C11H12N3O2 +) 218.0924; found 218.0982. O OEt OH N N N 2-13 DIBAL-H DCM, –78 °C – rt, 2 h N N N 2-14 (E)-3-(imidazo[1,2-a]pyrimidin-3-yl)prop-2-en-1-ol (2-14): An oven-dried 25 mL round bottom flask equipped with stirrer bar was charged with 2-13 (0.060 g, 0.28 mmol) an anhydrous dichloromethane (8 mL). The solution was cooled to – 78 °C and diisobutylalluminium hydride DIBAL-H (1 M in hexane, 0.85 mL, 0.85 mmol) was added slowly via syringe. After that, the reaction was warmed to room temperature and stirred for 2 hours. The reaction was quenched with Rochelle’s salt (5 mL) and the product extracted with DCM. The product was purified with CombiFlash chromatography (silica gel, 20-40 microns, gradient 0-5% methanol / dichloromethane) to give the product as a yellowish oil (3.5 mg, 7%). 1H NMR (500 MHz, 70 CD3OD) δ 8.92 (dd, J = 6.9, 1.7 Hz, 1H), 8.57 (dd, J = 4.1, 1.9 Hz, 1H), 7.92 (s, 1H), 7.13 (dd, J = 6.9, 4.1 Hz, 1H), 6.88 (d, J = 16.0 Hz, 1H), 6.54 (dt, J = 16.0, 5.2 Hz, 1H), 4.33 (dd, J = 5.2, 1.7 Hz, 2H). 13C{1H} NMR (125 MHz, CDCl3) δ 149.3, 148.8, 133.3, 131.2, 130.9, 121.9, 114.4, 109.1, 63.2. FTIR (neat, cm-1): 3351, 3061, 2970, 1685. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C11H12N3O2 +) 176.0818; found 176.0824. O OEt O OEt N N N 2-13 NH2NH2 • H2O Boc2O Et3N DMAP Ethanol, 75 °C, 12 h THF, rt, 12 h N Boc2N N Boc 2-15 tert-butyl (E)-2-(bis(tert-butoxycarbonyl)amino)-4-(3-ethoxy-3-oxoprop-1-en-1-yl)-1H- imidazole-1-carboxylate (2-15): To a suspension of 2-13 (0.22 g, 1.0 mmol) in ethanol (10 mL) was added hydrazine hydrate (50 µL, 1.1 mmol). The reaction was heated to 75 °C for 5 hours when TLC showed complete conversion of starting material. The reaction was stopped and cooled down to room temperature. The solvent was removed with rotary evaporator and tetrahydrofuran (15 mL), di-tert-butyl decarbonate Boc2O (1.84 mL, 8.0 mmol), triethylamine (0.70 mL, 5.0 mmol), and dimethylaminopyridine DMAP (0.025 g, 0.2 mmol) were added. The reaction was the stirred overnight at room temperature. The reaction was quenched with ammonium chloride (20 mL) and the product was extracted with ethyl acetate (2 x 20 mL). The organic layers were combined and concentrated. The organics was dried over sodium sulfate and product was purified using CombiFlash chromatography (silica gel, 20-40 microns, gradient 20% ethyl acetate/hexane) to give the product has a yellow oil (0.31 g, 64%). 1H NMR (500 MHz, CDCl3) δ 7.50 (s, 1H), 7.42 (d, J = 15.6 Hz, 1H), 6.57 (d, J = 15.6 Hz, 1H), 4.20 (q, J = 7.1 Hz, 2H), 1.56 (s, 9H), 1.39 (s, 18H), 1.27 (t, J = 7.1 Hz, 3H). 13C{1H} NMR (125 MHz, CDCl3) δ 166.8, 149.1, 145.8, 143.8, 139.2, 135.2, 134.3, 130.5, 119.3, 108.5, 86.5, 83.7, 60.2, 27.7, 27.6, 14.0. FTIR (neat, cm-1): 3082, 71 2991, 1698, 1623. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C23H36N3O8 +) 482.2497; found 482.2481. O OEt OH N Boc2N N Boc 2-15 DIBAL-H THF, -78 °C - 0 °C, 2.5 h N Boc2N N Boc 2-16 tert-butyl (E)-2-(bis(tert-butoxycarbonyl)amino)-4-(3-hydroxyprop-1-en-1-yl)-1H-imidazole- 1-carboxylate (2-16): To a solution of ester 2-15 (1.32 g, 2.74 mmol) in tetrahydrofuran (10 mL) at –78 °C was added diisobutylalluminium hydride DIBAL-H (1 M in hexane, 6.86 mL, 6.86 mmol). The reaction was stirred at –78 °C for 20 mins, then the reaction was stirred at 0 °C for 1 hour. The reaction was then cooled back down to –78 °C and 2 mL of methanol was added to destroy the excess DIBAL-H. The reaction was brought back to room temperature and diluted with ethyl acetate (20 mL) and Rochelle’s salt (20 mL). The mixture was stirred for 3 hours, and organic layer was collected. The aqueous layer was extracted ethyl acetate (2 x 20 mL). The organics were combined, washed with brine, and dried with sodium sulfate. The product was then purified with CombiFlash chromatography (silica gel, 20-40 microns, gradient 40% ethyl acetate/hexane) to give the product has clear oil (1.01 g, 84%). 1H NMR (500 MHz, CDCl3) δ 7.26 (s, 1H), 6.53 (dt, J = 15.7, 5.3 Hz, 1H), 6.42 (dt, J = 15.7, 1.4 Hz, 1H), 4.30 (dd, J = 5.3, 1.4 Hz, 2H), 1.58 (s, 9H), 1.42 (s, 18H). 13C{1H} NMR (125 MHz, CDCl3) δ 149.4, 146.3, 138.5, 136.9, 129.9, 121.1, 114.8, 86.0, 83.6, 63.2, 27.9, 27.8. FTIR (neat, cm-1): 3348, 3021, 1621. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C21H34N3O7 +) 440.2391; found 440.2484. 72 BnBr NaH DMF, 0 °C - rt, 16 h OH propargyl alcohol OBn 2-17 ((prop-2-yn-1-yloxy)methyl)benzene (2-17): To a suspension of sodium hydride (60% in mineral oil, 4.40 g, 110 mmol) in anhydrous DMF (60 mL) at 0 °C was added propargyl alcohol (5.82 mL, 100 mmol) slowly via a syringe. After 15 mins, benzyl bromide (13.0 mL, 110 mmol) was added, and the reaction was stirred at room temperature overnight for 12 hours. The reaction was then diluted with ethyl acetate (200 mL) and washed with aqueous 1 M hydrochloric acid (200 mL). The organic layer was dried over sodium sulfate, decanted, and then concentrated and purified with CombiFlash chromatography (silica gel, 20-40 microns, gradient 0-5% ethyl acetate/hexane) to give the product as a clear oil (13.0 g, 89% yield). 1H NMR (500 MHz, CDCl3) δ 7.28 – 7.18 (m, 5H), 4.49 (s, 2H), 4.04 (d, J = 2.4 Hz, 2H), 2.40 (t, J = 2.4 Hz, 1H). 13C{1H} NMR (125 MHz, CDCl3) δ 137.2, 128.2, 127.8, 127.6, 79.6, 74.6, 71.1, 56.7. FTIR (neat, cm-1): 3289, 2854, 2150, 1495, 1072. Spectroscopy data is consistent with previous literature report.30 HPBin ZrCp2HCl Et3N Neat, 60 °C, 24 h OBn 2-17 OBn O B O 2-18 (E)-2-(3-(benzyloxy)prop-1-en-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (2-18): A dry 250 mL round bottom flask equipped with magnetic stirrer bar was charged with alkyne 2-17 (9.0 g, 61.6 mmol), then zirconocene hydrochloride (1.59 g, 6.16 mmol) was added. The reaction was then purged with argon, and anhydrous triethylamine (0.86 mL, 6.16 mmol) was added followed by dropwise addition of pinacol borane (9.83 mL, 67.7 mmol). The reaction was then refluxed in a preheated aluminum beads at 60 °C for 24 hours. The reaction mixture was then cooled to room temperature and filtered through a short silica plug with diethyl ether/petroleum ether (1:4) as eluent. The filtrate was concentrated to give the crude product 2-18 as a clear oil (15.7 g, 93%). 73 The crude was used without further purification. 1H NMR (500 MHz, CDCl3) δ 7.38 – 7.25 (m, 5H), 6.69 (dt, J = 4.6, 9.2, 18.1 Hz, 1H), 5.82 – 5.74 (dt, J = 18.1, 3.5, 1.8 Hz, 1H), 4.54 (s, 2H), 4.12 (dd, J = 4.6, 1.8 Hz, 2H), 1.28 (s, 12H). 13C{1H} NMR (125 MHz, CDCl3) δ 149.2, 138.3, 128.3, 127.6, 127.6, 83.3, 72.3, 71.7, 24.8 (the alkene CH next to boron was not observed due to quadrupolar coupling effect of the boron). FTIR (neat, cm-1): 3085, 2977, 1643, 1322. Spectroscopy data is consistent with previous literature report.31 N N N imidazo[1,2-a] pyrimidine NIS DMF, rt, 3 h I N N N 2-19 3-iodoimidazo[1,2-a]pyrimidine (2-19): To a solution of imidazo[1,2-a]pyrimidine (3.0 g, 25.18 mmol) in dimethylformamide (20 mL) at room temperature was added N-iodosuccinimide (5.78 g, 25.67 mmol) in the dark. The reaction was stirred in the dark for 3 hours then ethyl acetate (30 mL) was added. The product was filtered and washed with ethyl acetate (100 mL) to give a brown solid (5.6 g, 91% yield). mp = 187 °C (decomposed). 1H NMR (500 MHz, DMSO-d6) δ 8.78 (dd, J = 6.8, 1.9 Hz, 1H), 8.54 (dd, J = 4.1, 1.9 Hz, 1H), 7.90 (s, 1H), 7.18 (dd, J = 6.8, 4.1 Hz, 1H). 13C{1H} NMR (125 MHz, DMSO-d6) δ 150.5, 150.0, 141.0, 135.0, 110.0, 64.1. FTIR (neat, cm- 1): 3115, 1669, 1495. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C6H5IN3 +) 245.9523; found: 245.9535. 74 O B O (2-18) OBn Pd(PPh3)4 aq K2CO3 1,4-dioxane, 80 °C, 24 h I N N N 2-19 OBn N N N 2-12 (E)-3-(3-(benzyloxy)prop-1-en-1-yl)imidazo[1,2-a]pyrimidine (2-12): 3-iodoimidazo[1,2- a]pyrimidine 2-19 (10.0 g, 40.81 mmol) was suspended in degassed 1,4-dioxane (100 mL) and 2- 18 (15.4 g, 44.89 mmol) was added. Then, tetrakis(triphenylphosphine)palladium(0) (2.36 g, 2.04 mmol) was added followed by degassed aqueous potassium carbonate solution (2 M, 41.0 mL, 81.62 mmol). The reaction was purged with argon and refluxed in a preheated oil bath at 80 °C for 24 hours. The reaction was cooled to room temperature, diluted with water (100 mL) and then extracted with ethyl acetate (4 x 200 mL). The organics were combined, dried over sodium sulfate, and decanted. The organic was then concentrated and purified with CombiFlash chromatography (silica gel, 20-40 microns, gradient 0-3% methanol/dichloromethane) to give the product as a yellowish solid (8.1 g, 75% yield). mp = 95 °C. 1H NMR (500 MHz, DMSO-d6) δ 9.06 (dd, J = 6.8, 1.9 Hz, 1H), 8.53 (dd, J = 4.1, 1.9 Hz, 1H), 8.06 (s, 1H), 7.40 – 7.33 (m, 4H), 7.32 – 7.26 (m, 1H), 7.11 (dd, J = 6.8, 4.1 Hz, 1H), 6.98 (d, J = 16.0 Hz, 1H), 6.49 (dt, J = 16.0, 6.0 Hz, 1H), 4.56 (s, 2H), 4.22 (dd, J = 6.0, 1.5 Hz, 2H). 13C{1H} NMR (125 MHz, DMSO-d6) δ 149.5, 148.1, 138.4, 133.0, 132.7, 128.3, 127.6, 127.5, 126.6, 121.5, 116.8, 109.0, 71.4, 70.2. FTIR (neat, cm-1): 3085, 2891, 1654, 1263. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C16H16N3O+) 266.1288; found 266.1292. 75 OBn NBS N H2N DCM/HFIP (9:1), – 10°C, 2 h N N N 2-12 N HN N OBn Br N N 2-20 (dr 2:1) N-(3-(benzyloxy)-2-bromo-1-(imidazo[1,2-a]pyrimidin-3-yl)propyl)cyanamide (2-20). Alkene 2-12 (1.28 g, 4.83 mmol) was dissolved in dichloromethane (58 mL) and hexafluoro-2- propanol (HFIP) (6.4 mL). Then, cyanamide (0.81 g, 19.32 mmol) was added and the solution was cooled to –10 °C. N-bromosuccinimide (0.95 g, 5.31 mmol) was added slowly at –10 °C and then stirred at that temperature for 2 hours in the dark. The reaction was warmed to room temperature and quenched with sodium thiosulfate (25 mL) and sodium bicarbonate (25 mL). The dichloromethane layer was collected, and the aqueous layer was further extracted with dichloromethane (2 x 50 mL). The organics were combined, dried over sodium sulfate and decanted. The organic was concentrated and purified with CombiFlash chromatography (silica gel, 20-40 microns, gradient 0-5% methanol/dichloromethane) to give the product as a foamy yellowish semi-solid (1.14 g, 61% yield (dr = 2:1 (anti : syn)). NMR data for major isomer (anti): 1H NMR (500 MHz, CD3OD) δ 8.90 (dd, J = 7.0, 1.9 Hz, 1H), 8.64 (m, 1H peak overlap with minor isomer), 7.97 (s, 1H), 7.30 – 7.21 (m, 5H, peak overlap with minor isomer), 7.14-7.10 (m, 1H, peak overlap with minor isomer), 5.39 (d, J = 6.6 Hz, 1H), 4.86 (m, 1H, peak overlap with minor isomer), 4.55 – 4.43 (m, 2H), 4.03 (dd, J = 10.8, 4.4 Hz, 1H), 3.73 (dd, J = 10.8, 6.2 Hz, 1H). 13C{1H} NMR (125 MHz, CD3OD) δ 152.6, 149.9, 138.8, 134.9, 134.5, 129.4, 129.1, 129.0, 120.7, 116.0, 110.7, 74.3, 71.8, 52.8, 52.5. FTIR (neat, cm-1): 3081, 3028, 2860, 1618, 1361. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C17H17BrN5O+): 386.0611 & 388.0591; found 386.0609 & 388.0581. 76 N HN N OBn Br HCl EtOH, rt, 48 h Cl NH2 OBn Et3N Br EtOH, reflux, 3 h O HN N N N 2-20 dr = (2:1, anti:syn) N N 2-22 O N NH OBn 2-23 N N N 3-(5-((benzyloxy)methyl)-2-ethoxy-4,5-dihydro-1H-imidazol-4-yl)imidazo[1,2-a]pyrimidine (2-23). Cyanamide bromide 2-20 (2.88 g, 7.46 mmol) was dissolved in anhydrous ethanol (15 mL) and anhydrous ethanolic hydrochloric acid (4.0 M, 18.7 mL, 74.61 mmol) was added. The reaction was stirred at room temperature for 48 hours to give intermediate 2-22, then it was diluted with more anhydrous ethanol (15 mL) and cooled in an ice bath to 0 °C. Anhydrous triethylamine (15.6 mL, 111.92 mmol) was added slowly at 0 °C. Upon complete addition, the reaction was refluxed in a preheated aluminum beads at 80 °C for 3 hours and then cooled to room temperature. The solvent was then removed under reduced pressure. To the crude solid compound was added tetrahydrofuran (THF) (40 mL) and ethyl acetate (10 mL). The insoluble triethylammonium chloride salt was filtered and washed with THF (40 mL), and the filtrate was concentrated and purified with CombiFlash chromatography (silica gel, 20-40 microns, gradient 0-5% methanol/dichloromethane) to give the product as a yellowish foamy semi-solid (1.33 g, 51% yield). The relative trans configuration was assigned based on a 1D NOE NMR spectroscopy experiment which was further confirmed by X-ray crystallography of compound 2-25. 1H NMR (500 MHz, CDCl3) δ 8.58 (dd, J = 7.0, 2.0 Hz, 1H), 8.41 (dd, J = 4.1, 2.0 Hz, 1H), 7.56 (s, 1H), 7.33 – 7.21 (m, 5H), 6.65 (dd, J = 7.0, 4.1 Hz, 1H), 5.0 (d, J = 6.0 Hz, 1H), 4.56 – 4.46 (m, 2H), 4.22 (m, 2H), 4.03 (q, J = 6.0 Hz, 1H), 3.56 (m, 2H), 1.26 (t, J = 7.1 Hz, 3H). 13C{1H} NMR (125 MHz, CDCl3) δ 164.4, 149.4, 149.2, 137.6, 133.0, 132.5, 128.6, 128.1, 128.0, 123.7, 108.3, 73.6, 72.5, 65.4, 14.6 (the imidazoline methine carbons are not present in 13C NMR due to rapid 77 tautomerism of the imidazoline). FTIR (neat, cm-1): 3162, 3028, 2979, 1615, 1308. HRMS (ESI- TOF) m/z: [M+H]+ calcd for (C19H22N5O2 +) 352.1768; found 352.1769. O N NH OBn 2-23 N N N BCl3 DCM, 0 °C, 2 h O N NH OH 2-24 N N N 2-ethoxy-4-(imidazo[1,2-a]pyrimidin-3-yl)-4,5-dihydro-1H-imidazol-5-yl)methanol (2-24). To a solution of 2-23 (0.73 g, 2.07 mmol) in dichloromethane (4 mL) at 0 °C was added boron trichloride solution (1.0 M in dichloromethane, 4.60 mL, 4.55 mmol) dropwise. The reaction mixture was stirred at 0 °C for 2 hours. The reaction was quenched with methanol (5 mL) and basify with triethylamine until pH ≈ 7. The solvent was then removed at reduced pressure and the crude was purified with CombiFlash chromatography (basic alumina, 40-60 microns, gradient 3- 10% methanol/dichloromethane) to give a foamy yellowish semi-solid (0.432g, 80% yield). 1H NMR (500 MHz, CD3OD) δ 8.86 (dd, J = 6.9, 2.0 Hz, 1H), 8.58 (dd, J = 4.2, 2.0 Hz, 1H), 7.68 (s, 1H), 7.10 (dd, J = 6.9, 4.2 Hz, 1H), 5.22 (d, J = 6.0 Hz, 1H), 4.25 (q, J = 7.1 Hz, 2H), 4.01 – 3.94 (m, 1H), 3.71 – 3.61 (m, 2H), 1.33 (t, J = 7.1 Hz, 3H). 13C{1H} NMR (125 MHz, CD3OD) δ 167.1, 151.7, 150.2, 134.7, 132.3, 126.3, 110.2, 66.4, 64.9, 14.8 (the imidazoline methine carbons are not observed in 13C NMR due to rapid tautomerism of the imidazoline). FTIR (cm-1): 3162(br), 2975, 1614, 1494, 1263. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C12H16N5O2 +) 262.1299; found 262.1304. 78 O HN O N NH OH O PPh3 DEAD THF, 0 °C - rt, 16 h N N N 2-24 O N O NH N O + N 2-25 not isolated N N O N N N 2-27 N N isolated 3-(2-ethoxy-1,3-diazabicyclo[3.1.0]hex-2-en-4-yl)imidazo[1,2-a]pyrimidine (2-27): To a solution of alcohol 2-24 (0.045 g, 0.17 mmol) in anhydrous tetrahydrofuran (THF) (4 mL) under argon at 0 °C was added triphenylphosphine (PPh3) (0.097 g, 0.37 mmol) and phthalimide (0.057 g, 0.39 mmol). Then, diethyl azodicarboxylate (DEAD) (40% wt in toluene, 0.17 mL, 0.37 mmol) was added dropwise at 0 °C. The reaction was stirred at 0 °C for an hour and then room temperature overnight. The reaction was concentrated and purified with CombiFlash chromatography (basic alumina gel, gradient 0 - 5% methanol/dichloromethane) to give compound 2-27 as a clear oil (0.012 g, 28%). 1H NMR (500 MHz, CD3OD) δ 8.88 (dd, J = 6.9, 1.9 Hz, 1H), 8.62 (dd, J = 4.1, 1.9 Hz, 1H), 7.77 (s, 1H), 7.14 (dd, J = 6.9, 4.1 Hz, 1H), 5.65 – 5.61 (m, 1H), 4.25 – 4.15 (m, 2H), 3.50 (ddd, J = 5.4, 4.1, 1.6 Hz, 1H), 2.53 (d, J = 5.4 Hz, 1H), 2.15 (d, J = 4.1 Hz, 1H), 1.32 (t, J = 7.1 Hz, 3H). 13C NMR (125 MHz, CD3OD) δ 174.0, 150.7, 148.8, 133.3, 130.7, 124.2, 108.9, 66.7, 59.9, 43.0, 37.4, 13.0. FTIR (neat, cm-1): 2961, 1713, 1618. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C12H14N5O+) 244.1193; found: 244.1194. 79 O N NH N N N 2-23 SO2Cl i-Pr i-Pr Et3N DMAP DCM, rt, 12 h OBn + i-Pr i-Pr i-Pr O N S O i-Pr OBn O N N N N 2-28 mixture of regioisomers 3-(5-((benzyloxy)methyl)-2-ethoxy-1-((2,4,6-triisopropylphenyl)sulfonyl)-4,5-dihydro-1H- imidazol-4-yl)imidazo[1,2-a]pyrimidine (2-28): To a solution of compound 2-23 (0.46 g, 1.32 mol) in dichloromethane (10 mL) was added 2,4,6-triisopropylbenzenesulfonyl chloride (0.44 g, 1.45 mmol), triethylamine (0.28 mL, 1.98 mmol) and 4-dimethylaminopyridine (0.032 g, 0.26 mmol). The reaction was stirred at room temperature overnight for 16 h. The reaction was quenched with ammonium chloride (10 mL) and extracted with dichloromethane (3 x 10 mL). The organic layers were combined, dried over sodium sulfate, concentrated under reduced pressure and then purified with CombiFlash chromatography (silica, 20-40 microns, gradient 1 – 2.5% methanol/dichloromethane) to give the titled compound as inseparable mixture of regioisomers as a foamy solid (0.54 g, 66%). 1H NMR (500 MHz, CDCl3) δ 8.76 (dd, J = 6.9, 2.0 Hz, 1H), 8.60 – 8.51 (m, 3H), 7.85 (s, 1H), 7.58 (d, J = 0.8 Hz, 1H), 7.37 (qd, J = 7.0, 3.6 Hz, 5H), 7.33 – 7.22 (m, 5H), 7.16 (s, 2H), 7.06 (s, 2H), 6.81 (dd, J = 6.9, 4.1 Hz, 1H), 6.65 (dd, J = 6.8, 4.0 Hz, 1H), 5.76 (d, J = 4.4 Hz, 1H), 5.32 – 5.27 (m, 1H), 4.71 – 4.58 (m, 4H), 4.49 (d, J = 11.9 Hz, 1H), 4.27 – 4.13 (m, 5H), 4.10 – 3.97 (m, 3H), 3.94 (dd, J = 9.2, 8.1 Hz, 1H), 3.76 – 3.65 (m, 3H), 3.55 (dd, J = 9.7, 8.2 Hz, 1H), 2.94 – 2.82 (m, 2H), 1.27 – 1.17 (m, 20H), 1.14 (d, J = 6.7 Hz, 7H), 1.08 (t, J = 7.1 Hz, 3H), 1.02 – 0.94 (m, 14H). 13C NMR (125 MHz, CDCl3) δ 157.2, 157.0, 154.4, 154.3, 151.4, 151.3, 149.8, 149.7, 148.9, 137.6, 137.2, 134.9, 132.8, 132.6, 131.4, 130.9, 128.6, 128.5, 128.2, 128.2, 127.9, 127.8, 123.8, 123.7, 122.4, 122.2, 108.5, 108.4, 73.9, 73.5, 71.7, 69.6, 67.2, 80 66.8, 66.8, 63.3, 58.7, 55.8, 34.3, 34.2, 29.4, 29.0, 24.8, 24.7, 24.1, 23.6, 23.5, 23.4, 13.8, 13.8. FTIR (cm-1): 2957, 1656, 1325, 1152; HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C34H44N5O4S+) 618.3109; found: 618.3110 i-Pr i-Pr O N S O i-Pr OBn O N N N N 2-28 mixture of regioisomers i-Pr O i-Pr OBn + i-Pr O N S O HN N O N N i-Pr i-Pr O N S O i-Pr OH N N 2-29 isolated mixture of regioisomers N N 2-30 desired, not formed 5-((benzyloxy)methyl)-4-(imidazo[1,2-a]pyrimidin-3-yl)-1-((2,4,6- triisopropylphenyl)sulfonyl)imidazolidin-2-one (2-29): To a solution of benzyl ether 2-28 (0.20 g, 0.32 mmol) in dichloromethane (8 mL) at 0 °C was added boron trichloride solution (1.0M in heptane, 0.33 mL, 0.33 mmol) slowly over 5 minutes. The reaction mixture was stirred at 0 °C for 3 hours. The reaction was quenched with methanol (5 mL) and triethylamine (1.5 mL). The solvent was then removed at high pressure and the crude was purified with CombiFlash chromatography (silica, 40-60 microns, gradient 3-10% methanol/dichloromethane) to give the titled compound as a yellowish oil (0.097 g, 51%). Major isomer: 1H NMR (500 MHz, CDCl3) δ 8.63 – 8.59 (m, 1H), 8.42 (dd, J = 4.1, 1.9 Hz, 1H), 7.51 (s, 1H), 7.43 – 7.35 (m, 5H), 7.12 (s, 2H), 6.49 (dd, J = 6.8, 4.1 Hz, 1H), 5.24 (t, J = 1.9 Hz, 1H), 4.66 (d, J = 1.8 Hz, 2H), 4.36 (dt, J = 9.5, 3.2 Hz, 1H), 4.23 (dd, J = 8.9, 3.7 Hz, 1H), 4.04 – 3.83 (m, 4H), 1.26 – 1.10 (m, 17H). 13C NMR (125 MHz, CDCl3) Messy carbon. FTIR (cm-1): 3030, 2957, 2867, 1738. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C32H40N5O4S+) 590.2796; found: 590.2814. 81 O N NH N N N OH 2-24 MsCl Et3N DCM, rt, 4 h O N NH N N N OMs 2-31 (2-ethoxy-4-(imidazo[1,2-a]pyrimidin-3-yl)-4,5-dihydro-1H-imidazol-5-yl)methyl methanesulfonate (2-31). Alcohol 2-24 (0.43 g, 1.65 mmol) was dissolved in dichloromethane (18 mL) in a 50 mL round bottom flask and then triethylamine (0.46 mL, 3.30 mmol) was added followed by dropwise addition of methanesulfonyl chloride (134 µL, 1.73 mmol). The reaction was then stirred at room temperature for 4 hours. The reaction was concentrated and purified with CombiFlash chromatography (basic alumina gel, gradient 0 - 4% methanol/dichloromethane) to give the desired product 2-31 as a foamy white semi-solid (0.422 g, 75%). 1H NMR (500 MHz, CD3OD) δ 8.77 (dd, J = 7.0, 2.0 Hz, 1H), 8.60 (dd, J = 4.2, 2.0 Hz, 1H), 7.73 (s, 1H), 7.13 (dd, J = 7.0, 4.2 Hz, 1H), 5.25 (d, J = 6.0 Hz, 1H), 4.38 – 4.29 (m, 3H), 4.29 – 4.24 (m, 2H), 3.14 (s, 3H), 1.34 (t, J = 7.1 Hz, 3H). 13C{1H} NMR (125 MHz, CD3OD) δ 167.1, 152.0, 150.4, 134.8, 132.9, 125.1, 110.3, 72.0, 66.6, 37.3, 14.8 (the imidazoline methine carbons were not observed in 13C-NMR due to rapid tautomerism of the imidazoline). FTIR (neat, cm-1): 3170, 2981, 1617, 1345. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C13H18N5O4S+) 340.1074; found: 340.1071. O N NH N N N OMs 2-31 O K N O DMF, rt, 48 h N O N NH O 2-25 N O N N 2-((2-ethoxy-4-(imidazo[1,2-a]pyrimidin-3-yl)-4,5-dihydro-1H-imidazol-5- yl)methyl)isoindoline-1,3-dione (2-25). To a solution of 2-31 (137 mg, 0.404 mmol) in dimethylformamide (3 mL) was added potassium phthalimide (150 mg, 0.808 mmol). The reaction 82 was stirred at room temperature for 48 hours after which TLC showed complete conversion of starting material. The solvent was removed under reduced pressure and the crude was purified with CombiFlash chromatography (basic alumina gel, gradient 0 - 5% methanol/dichloromethane) to give the desired product as white solid (122 mg, 77% yield). Crystallization from methanol via slow evaporation from 5ml vial over a period of 5 days gave a colorless crystal which was used for X-ray crystal structure. mp = 213 °C (decomposed). 1H NMR (500 MHz, CD3OD) δ 8.78 (dd, J = 7.0, 2.0 Hz, 1H), 8.55 (dd, J = 4.2, 2.0 Hz, 1H), 7.78 – 7.71 (m, 4H), 7.62 (s, 1H), 7.09 (dd, J = 7.0, 4.2 Hz, 1H), 5.22 (d, J = 7.2 Hz, 1H), 4.43 (q, J = 6.4 Hz, 1H), 4.18 (q, J = 7.2 Hz, 2H), 4.00 (dd, J = 14.1, 6.0 Hz, 1H), 3.90 (dd, J = 14.1, 6.0 Hz, 1H), 1.27 (t, J = 7.2 Hz, 3H). 13C{1H} NMR (125 MHz, CD3OD): δ 169.9, 167.0, 164.8, 152.0, 150.2, 135.5, 134.8, 133.1, 132.6, 124.2, 110.3, 66.5, 42.9, 14.7 (the imidazoline methine carbons were not observed in 13C-NMR due to rapid tautomerism of the imidazoline). FTIR (neat, cm-1): 3144, 2934, 1769, 1715, 1613. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C20H19N6O3 +) 391.1513; found: 391.1520. O N NH NaN3 DMF, 60 °C, 16 h OMs 2-31 N N N N N N O N NH N3 2-32 3-(5-(azidomethyl)-2-ethoxy-4,5-dihydro-1H-imidazol-4-yl)imidazo[1,2-a]pyrimidine (2-32): Compound 2-31 (0.18 g, 0.52 mmol) was dissolved in DMF (2.5 mL) in a 7.5 mL reaction vial and to the solution was added sodium azide (0.051 g, 0.78 mmol). The reaction was heated to 60 °C and stirred overnight. The DMF was removed under reduced pressure and the product was purified with CombiFlash chromatography (basic alumina gel, gradient 0 - 2% methanol/dichloromethane) to give the desired product as a foamy white solid (0.12 g, 79%). 1H NMR (500 MHz, CD3OD) δ 8.76 (d, J = 6.8 Hz, 1H), 8.59 (dd, J = 4.1, 1.9 Hz, 1H), 7.70 (s, 1H), 83 7.12 (dd, J = 6.8, 4.1 Hz, 1H), 5.18 (d, J = 6.4 Hz, 1H), 4.27 (q, J = 7.0 Hz, 2H), 4.18 (dd, J = 6.4, 4.9 Hz, 1H), 3.63 (dd, J = 12.6, 4.9 Hz, 1H), 3.45 (dd, J = 12.6, 4.5 Hz, 1H), 1.34 (t, J = 7.0 Hz, 3H). 13C{1H} NMR (125 MHz, CD3OD) δ 165.6, 150.5, 149.0, 133.4, 131.4, 124.0, 108.9, 65.1, 54.2, 13.5 (the imidazoline methine carbons were not observed in 13C-NMR due to rapid tautomerism of the imidazoline). FTIR (neat, cm-1): 3140, 2931, 2103, 1615, 1345. HRMS (ESI- TOF) m/z: [M+H]+ calcd for (C12H15N8O+) 287.1363; found: 287.1360. O N NH PPh3 Br Br Cl3C O N H Et3N N N N N3 2-32 THF/H2O (4:1), 70 °C, 4 h DMF, 50 °C, 16h N O NH N N N NH 2-35 O HN O NH HN N N N + Br Br NH 2-36 O HN Br Br 4,5-dibromo-N-((2-ethoxy-4-(imidazo[1,2-a]pyrimidin-3-yl)-4,5-dihydro-1H-imidazol-5- yl)methyl)-1H-pyrrole-2-carboxamide (2-35) and 4,5-dibromo-N-((5-(imidazo[1,2- a]pyrimidin-3-yl)-2-oxoimidazolidin-4-yl)methyl)-1H-pyrrole-2-carboxamide (2-36): Azide 2-32 (0.032 g, 0.11 mmol) was dissolved in THF (1 mL) and H2O (0.25 mL), triphenylphosphine (0.045 g, 0.17 mmol) was added and the reaction was refluxed for 4 hours to give the corresponding amines. The reaction was concentrated, and the crude was dissolved in DMF (1 mL). Triethylamine (24 µL, 0.17 mmol) and 2,2,2-trichloro-1-(4,5-dibromo-1H-pyrrol-2-yl)ethan-1-one (0.063 g, 0.17 mmol) were added. The reaction was stirred at 50 °C overnight and then concentrated under reduced pressure. The crude was purified with CombiFlash chromatography (basic alumina gel, gradient 0 - 5% methanol/dichloromethane) to give 2-35 as white foamy solid (0.015 g, 27%) and 2-36 as white foamy solid (0.012 g, 23%). 2-35: 1H NMR (500 MHz, CD3OD) δ 9.29 (d, J = 6.8 Hz, 1H), 8.97 (d, J = 4.1 Hz, 1H), 8.34 (s, 1H), 7.53 (dd, J = 6.8, 4.1 Hz, 1H), 6.82 (s, 1H), 5.76 (d, J = 7.1 Hz, 1H), 4.72 – 4.63 (m, 1H), 84 4.55 (q, J = 7.0 Hz, 2H), 3.81 (dd, J = 14.4, 4.9 Hz, 1H), 3.72 (dd, J = 14.4, 4.9 Hz, 1H), 1.48 (t, J = 7.0 Hz, 3H). 13C{1H} NMR (126 MHz, CD3OD) δ 163.5, 161.1, 155.9, 135.0, 133.4, 126.6, 113.4, 112.4, 105.6, 98.7, 71.1, 60.9, 51.8, 41.0, 13.0. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C17H17Br2N7O2 +) 509.9884, 511.9863 & 513.9843; found: 509.9825, 511.9867 & 513.9823. 2-36: 1H NMR (500 MHz, cd3od) δ 9.18 (d, J = 6.8 Hz, 1H), 8.95 (d, J = 4.1 Hz, 1H), 8.13 (s, 1H), 7.51 (dd, J = 6.8, 4.1 Hz, 1H), 6.81 (s, 1H), 5.23 (d, J = 6.1 Hz, 1H), 4.16 (m, 1H), 3.70 – 3.50 (m, 2H). HRMS (ESI-TOF) m/z: [M + H]+ calcd for (C17H17Br2N7O2 +) 481.9571, 483.9550, & 485.9530; found: 481.9597, 483.9584, & 485.9568. O Cl CCl3 Cl3C N H Et2O, rt, 4 h N H O S2-1 2,2,2-trichloro-1-(1H-pyrrol-2-yl)ethan-1-one (S2-1). A 50 mL round bottom flask equipped with magnetic stirrer was charged with diethyl ether (20 mL) and trichloroacetyl chloride (2.50 mL, 22.0 mmol). Pyrrole (1.40 mL, 20.0 mmol) was added in portions every 10 minutes over 1 hour. Then, the reaction was further stirred at room temperature under nitrogen for 3 hours. The reaction was quenched with 5 mL of water followed by addition of 15 mL saturated aqueous potassium carbonate. The organic later was then collected and stirred with activated charcoal for 15 minutes. The mixture was filtered with celite plug and the resulting filtrate was concentrated under reduced pressure. The product was then recrystallized in hexane to give 2,2,2-trichloro-1- (1H-pyrrol-2-yl)ethan-1-one as a white solid (3.6 g, 85% yield). mp = 75 °C. 1H NMR (500 MHz, CDCl3) δ 9.69 (s, br, 1H), 7.40 (ddd, J = 4.0, 2.5, 1.3 Hz, 1H), 7.21 – 7.16 (m, 1H), 6.39 (dt, J = 4.0, 2.5 Hz, 1H). 13C{1H} NMR (125 MHz, CDCl3) δ 173.3, 127.3, 122.9, 121.3, 111.9, 94.9. FTIR (neat, cm-1): 3317, 1649, 1535. HRMS (ESI-TOF) m/z: [M–H]- calcd for (C6H3Cl3NO-): 209.9285, 211.9256, & 213.9226; found: 209.9286, 211.9244, & 213.9215. 85 Cl3C Br2 Cl3C AcOH, 60 °C, 2 h N H O S2-1 O N H S2-2 Br Br 2,2,2-trichloro-1-(4,5-dibromo-1H-pyrrol-2-yl)ethan-1-one (S2-2): To a solution of S2-1 (1.20 g, 5.65 mmol) in acetic acid (15 mL) was added bromine (0.58 mL, 11.30 mmol). The reaction was placed in a preheated aluminum beads at 60 °C and stirred for 2 hours. The reaction was then cooled to room temperature and stirred overnight. Then, water (10 mL) was added, and the pH of the solution was adjusted to 5 with 2M NaOH. The reaction was extracted with ethyl acetate (3 x 30 mL), and the organic layers were combined, dried over sodium sulfate, and filtered. The volatiles were removed, and the pure product was obtained by recrystallization with ethanol/water as a beige solid (1.7g 81%). mp: 138 °C. 1H NMR (500 MHz, DMSO-d6) δ 13.75 (s, 1H), 7.39 (s, 1H). 13C{1H} NMR (125 MHz, DMSO-d6) δ 171.4, 123.8, 122.9, 115.1, 101.3, 94.5. FTIR (neat, cm-1): 3284, 1654, 1365. HRMS (ESI-TOF) m/z: [M–H]- calcd for (C6HBr2Cl3NO-): 365.7495, 367.7475, 369.7446, 371.7425, & 373.7396; found: 365.7489, 367.7473, 369.7451, 371.7417, & 373.7385. O N NH N N N OMs 2-31 NH3 (7.0 M in MeOH) sealed tube, 150 °C, 4.5 h N OMs N NH2 NH 2-38 NH2 N N Br Br Cl3C O N H Et3N DMF, rt, 16 h OMs NH2 HN NH O H N N H 2-39 Br Br N N N 2-amino-5-((4,5-dibromo-1H-pyrrole-2-carboxamido)methyl)-4-(imidazo[1,2-a]pyrimidin- 3-yl)-4,5-dihydro-1H-imidazol-3-ium methanesulfonate (2-39). Compound 2-31 (60.0 mg, 0.177 mmol) was dissolved in ammonia solution (7.0 M in methanol) (5 mL) in a sealed tube and was heated to 150 °C for 4.5 hours. The reaction was cooled to room temperature and the solvent evaporated to give 2-38. The crude compound was redissolved in DMF (1.5 mL) and charged with 86 2,2,2-trichloro-1-(4,5-dibromo-1H-pyrrol-2-yl)ethan-1-one S2-2 (79.0 mg, 0.212 mmol) and triethylamine (49.0 µL, 0.354 mmol) and the reaction was stirred for 24 hours at room temperature. The solvent was removed under reduced pressure and the product purified with CombiFlash chromatography (C18-derived rp silica, gradient 0 - 100% methanol/water) to give the desired product as white solid (53 mg, 52% yield). mp: 189 °C (decompose) 1H NMR (500 MHz, CD3OD) δ 8.82 (dd, J = 6.9, 2.0 Hz, 1H), 8.58 (dd, J = 4.2, 2.0 Hz, 1H), 7.86 (s, 1H), 7.06 (dd, J = 6.9, 4.2 Hz, 1H), 6.78 (s, 1H), 5.11 (d, J = 5.7 Hz, 1H), 4.33 (q, J = 5.7 Hz, 1H), 3.66 (dd, J = 5.7, 2.2 Hz, 2H), 2.70 (s, 3H) (mesylate proton). 13C{1H} NMR (125 MHz, CD3OD) δ 162.2, 160.6, 153.0, 150.0, 146.4, 136.8, 128.4, 114.5, 110.80, 110.76, 106.5, 100.0, 63.3, 58.1, 43.0, 39.5 (mesylate carbon). FTIR (neat, cm-1): 3107, 2924, 1685, 1618, 1561. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C15H15Br2N8O+) 480.9731, 482.9710, & 484.9690; found: 480.9743, 482.9726, & 484.9706. OMs NH2 HN NH O H N N H 2-39 Br Br N N N NH2NH2 • H2O EtOH, rt, 6.5 h H2N H N N OMs HN NH2 NH O N H nagelamide W (2-1) H N Br Br nagelamide W (2-1): Compound 2-39 (21.0 mg, 0.037 mmol) was dissolved in ethanol (2 mL) and hydrazine hydrate (1 mL) and stirred at room temperature for 6.5 hours. The solvent was then removed under reduced pressure and the product was purified with CombiFlash chromatography (C18-derived rp silica, gradient 0 - 100% methanol/water) to give nagelamide W as an off-white solid (19.7 mg, 98% yield). mp = 154-155°C. 1H NMR (500 MHz, CD3OD) δ 6.83 (s, 1H), 6.72 (s, 1H), 4.81 (d, J = 7.0 Hz, 1H), 4.15 (dt, J = 7.0, 5.5 Hz, 1H), 3.58 (t, J = 5.1 Hz, 2H), 2.72 (s, 2H). 13C{1H} NMR (125 MHz, CD3OD) δ 160.8, 159.1, 149.7, 128.2, 127.0, 113.2, 110.9, 105.2, 98.6, 61.0, 54.9, 41.3, 38.1. FTIR (neat, cm-1): 3175, 2928, 1682, 1559. HRMS (ESI-TOF) m/z: 87 [M+H]+ calcd for (C12H15Br2N8O+) 444.9731, 446.9710, & 448.9690; found: 444.9745, 446.9726, & 448.9708. OMs HN H2N H N N NH2 NH O N H nagelamide W (2-1) H N Br Br TFA H2N • 2TFA N H N N NH2 NH O N H H N Br nagelamide W (2-1•2TFA) Br nagelamide W (2-1 • 2TFA): Nagelamide W (2-1) was dissolved in DMSO-d6 and 2 equivalent of trifluoroacetic acid (TFA) was added to give the TFA salt which was directly used to obtain 1H and 13C-NMR. 1H NMR (500 MHz, DMSO-d6) δ 12.82 (s, 1H), 12.74 (d, J = 2.8 Hz, 1H), 12.16 (s, 1H), 8.75 (s, 1H), 8.61 (s, 1H), 8.37 (t, J = 6.0 Hz, 1H), 8.13 (s, 2H), 7.69 (s, 2H), 6.91 (d, J = 2.4 Hz, 1H), 6.84 (s, 1H), 4.80 (d, J = 7.4 Hz, 1H), 3.98 (q, J = 6.5 Hz, 1H), 3.50 – 3.41 (m, 2H). 13C{1H} NMR (125 MHz, DMSO-d6) δ 159.6, 158.7, 148.2, 127.8, 124.6, 113.2, 111.7, 105.2, 98.1, 60.1, 53.3, 41.4. 88 REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. Buchanan, M. S.; Carroll, A. R.; Addepalli, R.; Avery, V. M.; Hooper, J. N. A.; Quinn, R. J., J. Org. Chem. 2007, 72, 2309-2317. Rane, R. A.; Nandave, M.; Nayak, S.; Naik, A.; Shah, D.; Alwan, W. S.; Sahu, N. U.; Naphade, S. 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Staudinger, H.; Meyer, J., Helv. Chim. Acta 1919, 2, 635-646. 38. Lenstra, D. C.; Wolf, J. J.; Mecinovic, J., J. Org. Chem. 2019, 84, 6536-6545. 39. Bures, J.; Martin, M.; Urpi, F.; Vilarrasa, J., J. Org. Chem. 2009, 74, 2203-2206. 40. George, D. E.; Tepe, J. J., J. Org. Chem. 2023, 88, 9306-9312. 91 Figure 2.2: 1H and 13C{1H} NMR spectra of 2-7 APPENDIX DG-1-17-Product-CDCl3 _PROTON_01 O H 7 9 9 . 4 7 9 . 4 7 9 . 3 7 9 . 3 7 9 . 4 8 8 . 4 8 8 . 4 8 8 . 3 8 8 . 2 5 8 . N N N l 3 c d c 6 2 7 . 3 2 7 . 2 2 7 . 1 2 7 . 1 2 7 . 5 9 0 . 8 9 0 . 9 9 0 . 1 0 1 . 6 0 1 . 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 . 8 8 7 1 . 4 4 5 1 . 7 1 5 1 . 6 7 4 1 . 3 6 3 1 . 1 3 2 1 . 6 1 1 1 DG-1-17-Product-CDCl3 _ CARBON_01 O H N N N l 3 c d c 3 7 7 . l 3 c d c 0 7 7 . l 3 c d c 8 6 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 92 25000 20000 15000 10000 5000 0 280 260 240 220 200 180 160 140 120 100 80 60 40 20 0 -20 Figure 2.3: 1H and 13C{1H} NMR spectra of 2-13 l 3 c d c 6 2 7 . DG-1-56-tt27Product_PROTON_01 O OEt N N N 7 6 8 . 7 6 8 . 6 6 8 . 5 6 8 . 4 6 8 . 4 6 8 . 3 6 8 . 3 6 8 . 9 1 8 . 2 8 7 . 9 7 7 . 13 12 11 10 9 8 DG-1-56-tt27Product_ CARBON_01 4 0 1 . 2 9 0 . 7 9 0 . 6 9 0 . 9 0 7 . 8 0 7 . 7 0 7 . 6 0 7 . 4 4 6 . 1 4 6 . 8 2 4 . 7 2 4 . 6 2 4 . 4 2 4 . 4 3 1 . 2 3 1 . 1 3 1 . 7 9 0 . 9 9 0 . 7 6 f1 (ppm) 6 0 2 . 9 0 3 . 5 4 3 2 1 0 -1 l 3 c d c 3 7 7 . l 3 c d c 1 7 7 . l 3 c d c 8 6 7 . . 8 0 6 . 3 4 1 O OEt . 7 6 6 1 . 8 0 5 1 . 4 0 5 1 . 1 8 3 1 . 0 2 3 1 . 6 7 2 1 . 2 0 2 1 . 0 6 1 1 . 0 0 1 1 N N N 6500 6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 -500 85 80 75 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 93 Figure 2.4: 1H and 13C{1H} NMR spectra of 2-14 DG-2-68-tt14-MeOH_PROTON_01 OH N N N 3 9 8 . 3 9 8 . 2 9 8 . 1 9 8 . 7 5 8 . 7 5 8 . 7 5 8 . 6 5 8 . 2 9 7 . 4 1 7 . 3 1 7 . 3 1 7 . 2 1 7 . 0 9 6 . 7 8 6 . 7 5 6 . 6 5 6 . 5 5 6 . 4 5 6 . 3 5 6 . 2 5 6 . O D H 0 9 4 . 4 3 4 . 3 3 4 . 3 3 4 . 2 3 4 . 5 3 3 . d o 3 d c 2 3 3 . d o 3 d c 2 3 3 . d o 3 d c 1 3 3 . d o 3 d c 1 3 3 . d o 3 d c 1 3 3 . 13 12 11 10 9 0 0 1 . 3 0 1 . 1 8 0 . 8 4 0 1 . 1 0 1 . 1 0 1 . 7 6 f1 (ppm) 0 1 2 . 5 4 3 2 1 0 -1 DG-2-51-tt12_ CARBON_01 OH N N N . 3 9 4 1 . 8 8 4 1 . 3 3 3 1 . 2 1 3 1 . 9 0 3 1 . 9 1 2 1 . 4 4 1 1 . 1 9 0 1 l 3 c d c 4 7 7 . l 3 c d c 2 7 7 . l 3 c d c 9 6 7 . . 2 3 6 22000 21000 20000 19000 18000 17000 16000 15000 14000 13000 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 -1000 28 26 24 22 20 18 16 14 12 10 8 6 4 2 0 -2 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 94 Figure 2.5: 1H and 13C{1H} NMR spectra of 2-15 DG-2-81-tt10_PROTON_01 O OEt N Boc2N N Boc 0 5 7 . 3 4 7 . 0 4 7 . l 3 c d c 7 2 7 . 9 5 6 . 6 5 6 . 2 2 4 . 1 2 4 . 9 1 4 . 8 1 4 . t c A O E 0 1 4 . t c A O E 9 0 4 . t c A O E 8 0 4 . t c A O E 6 0 4 . t c A O E 1 0 2 . O 2 H 6 5 1 . 3 6 1 . 7 5 1 . 9 3 1 . 8 2 1 . 7 2 1 . 6 2 1 . t c A O E 4 2 1 . t c A O E 2 2 1 . t c A O E 1 2 1 . 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 4 0 1 . 7 0 1 . 2 0 1 . 2 2 2 . 9 0 0 1 . 4 1 0 2 . 5 6 3 . 7 7 0 . DG-2-121-triboc-cdcl3 _ CARBON_01 O OEt N Boc2N N Boc t c A O E 9 0 7 1 . . 8 6 6 1 . 1 9 4 1 . 8 5 4 1 . 8 3 4 1 . 2 9 3 1 . 2 5 3 1 . 3 4 3 1 . 5 0 3 1 . 3 9 1 1 . 1 9 1 1 . 5 8 0 1 . 5 6 8 . 7 3 8 l 3 c d c 4 7 7 . l 3 c d c 2 7 7 . l 3 c d c 9 6 7 . . 2 0 6 . 7 7 2 . 6 7 2 t c A O E 9 0 2 . t c A O E 2 4 1 . . 0 4 1 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 95 21000 20000 19000 18000 17000 16000 15000 14000 13000 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 -1000 1400 1300 1200 1100 1000 900 800 700 600 500 400 300 200 100 0 -100 Figure 2.6: 1H and 13C{1H} NMR spectra of 2-16 DG-2-147 _PROTON_01 OH N Boc2N N Boc l 3 c d c 7 2 7 . 6 2 7 . 6 5 6 . 5 5 6 . 4 5 6 . 2 5 6 . 3 5 6 . 1 5 6 . 3 4 6 . 4 4 6 . 3 4 6 . 0 4 6 . 0 4 6 . 0 4 6 . 1 3 4 . 0 3 4 . 0 3 4 . 9 2 4 . E C D 3 7 3 . 8 5 1 . 2 4 1 . 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 7 7 0 . 0 9 0 . 2 9 0 . 4 9 1 . 5 2 8 1 . 1 2 9 . DG-2-147 _ CARBON_01 OH N Boc2N N Boc . 4 9 4 1 . 3 6 4 1 . 5 8 3 1 . 9 6 3 1 . 9 9 2 1 . 1 1 2 1 . 8 4 1 1 . 0 6 8 . 6 3 8 l 3 c d c 3 7 7 . l 3 c d c 0 7 7 . l 3 c d c 8 6 7 . E C D 5 3 4 . . 2 3 6 . 9 7 2 . 8 7 2 0 1 . 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 96 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 -5 Figure 2.7: 1H and 13C{1H} NMR spectra of 2-17 DG-3-17-tt13-CDCl3 _PROTON_01 8 2 7 . 7 2 7 . O l 3 c d c 6 2 7 . 6 2 7 . 4 2 7 . 4 2 7 . 4 2 7 . 3 2 7 . 3 2 7 . 3 2 7 . 2 2 7 . 1 2 7 . 0 2 7 . 0 2 7 . 9 1 . 7 9 1 . 7 9 1 . 7 8 1 . 7 8 1 . 7 8 1 . 7 9 4 4 . 4 0 4 . 3 0 4 . 0 4 2 . 9 3 2 . 9 3 2 . 13 12 11 10 9 8 7 6 f1 (ppm) 5 DG-3-17-tt13-CDCl3 _ CARBON_01 . 2 7 3 1 2 . 8 2 1 . 8 7 2 1 . 6 7 2 1 0 9 4 . 3 1 . 2 2 1 . 2 4 . 6 9 7 O l 3 c d c 4 7 7 . 5 8 . 0 3 2 1 0 -1 -2 . 6 4 7 1 . 1 7 . 7 6 5 4000 3500 3000 2500 2000 1500 1000 500 0 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0 -200 l 3 c d c 2 7 7 . l 3 c d c 9 6 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 97 Figure 2.8: 1H and 13C{1H} NMR spectra of 2-18 DG-3-31-cdcl3 _PROTON_01 7 2 7 . 5 2 7 . 5 2 7 . 5 2 7 . 4 2 7 . 4 2 7 . 3 2 7 . 3 2 7 . 0 2 7 . 1 2 7 . 9 1 7 . 9 1 7 . 9 1 7 . 8 1 7 . 8 1 7 . 7 1 7 . 4 6 6 . 3 6 6 . 2 6 6 . 0 6 6 . 9 5 6 . 8 5 6 . 1 7 5 . 7 6 5 . l 2 C 2 H C 9 1 5 . 5 4 4 . 3 0 4 . 3 0 4 . 3 0 4 . 2 0 4 . 9 1 1 . l 3 c d c 6 2 7 . O B O OBn 7 2 7 . l 3 c d c 6 2 7 . 5 2 7 . 5 2 7 . 5 2 7 . 4 2 7 . 4 2 7 . 3 2 7 . 3 2 7 . 0 2 7 . 1 2 7 . 9 1 7 . 9 1 7 . 9 1 7 . 8 1 7 . 8 1 7 . 7 1 7 . 7.30 7.25 9 8 4 . 7.20 7.15 f1 (ppm) 6000 5000 4000 3000 2000 1000 0 -1000 13 12 11 10 9 8 7 6 f1 (ppm) 5 DG-3-31-product_ CARBON_01 9 8 4 . 8 9 0 . 8 9 0 . 2 1 2 . 7 1 2 . 4 4 8 2 1 . 3 2 1 0 -1 -2 O B O OBn . 2 9 4 1 . 3 8 3 1 . 3 8 2 1 . 6 7 2 1 . 6 7 2 1 . 3 3 8 l 3 c d c 3 7 7 . l 3 c d c 1 7 7 . l 3 c d c 8 6 7 . . 3 2 7 . 7 1 7 . 8 4 2 . 3 8 2 1 . 6 7 2 1 . 6 7 2 1 400 300 200 100 0 128 .4 128 .2 128 .0 127. 8 127.6 127.4 127.2 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 98 40000 38000 36000 34000 32000 30000 28000 26000 24000 22000 20000 18000 16000 14000 12000 10000 8000 6000 4000 2000 0 -2000 800 750 700 650 600 550 500 450 400 350 300 250 200 150 100 50 0 -50 Figure 2.9: 1H and 13C{1H} NMR spectra of 2-19 DG-2-80-DMSO-product_PROTON_01 I N N N 8 7 8 . 7 7 8 . 6 7 8 . 6 7 8 . 4 5 8 . 4 5 8 . 3 5 8 . 3 5 8 . 0 9 7 . 8 1 7 . 8 1 7 . 7 1 7 . 6 1 7 . O 2 H 4 3 3 . o s m d 1 5 2 . o s m d 0 5 2 . o s m d 0 5 2 . o s m d 0 5 2 . o s m d 9 4 2 . 13 12 11 10 9 8 7 DG-2-80-DMSO-product_ CARBON_01 I N N N . 5 0 5 1 . 0 0 5 1 . 0 1 4 1 . 0 5 3 1 6 f1 (ppm) . 0 0 1 1 5 4 3 2 1 0 -1 o s m d 0 0 4 . o s m d 9 9 3 . o s m d 7 9 3 . o s m d 5 9 3 . o s m d 4 9 3 . o s m d 2 9 3 . o s m d 0 9 3 . . 1 4 6 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 99 15000 14000 13000 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 -1000 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 2.10: 1H and 13C{1H} NMR spectra of 2-12 7 0 9 . 7 0 9 . 6 0 9 . 6 0 9 . 3 5 8 . 3 5 8 . 3 5 8 . 2 5 8 . 6 0 8 . 8 3 7 . 7 3 7 . 7 3 7 . 6 3 7 . 6 3 7 . 5 3 7 . 5 3 7 . 4 3 7 . 4 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 . 2 1 7 . 2 1 7 . 1 1 7 . 0 1 7 . 0 0 7 . 7 9 6 . 2 5 6 . 1 5 6 . 0 5 6 . 9 4 6 . 8 4 6 . 6 4 6 . 6 5 4 . 2 2 4 . 2 2 4 . 1 2 4 . 1 2 4 . o s m d 1 5 2 . o s m d 0 5 2 . o s m d 0 5 2 . o s m d 0 5 2 . o s m d 9 4 2 . DG-3-55-recrystallized_PROTON_01 OBn N N N 13 12 11 10 DG-3-55-recrystallized_ CARBON_01 3 0 1 . 9 6 9 0 . 3 9 0 . 8 1 9 3 . 7 9 0 . 3 0 1 . 3 0 1 . 2 0 1 . 5 0 2 . 7 0 2 . 7 6 f1 (ppm) 5 4 3 2 1 0 -1 OBn . 5 9 4 1 . 1 8 4 1 . 4 8 3 1 . 7 2 3 1 . 0 3 3 1 . 3 8 2 1 . 5 7 2 1 . 6 7 2 1 . 6 6 2 1 . 5 1 2 1 . 8 6 1 1 . 0 9 0 1 . 4 1 7 . 2 0 7 N N N o s m d 0 0 4 . o s m d 9 9 3 . o s m d 7 9 3 . o s m d 5 9 3 . o s m d 4 9 3 . o s m d 2 9 3 . o s m d 0 9 3 . 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 -10 100 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.11: 1H and 13C{1H} NMR spectra of 2-20 N HN N N N OBn Br . 6 2 5 1 . 9 9 4 1 8 . 8 3 1 . 9 4 3 1 . 5 4 3 1 . 4 9 2 1 1 . 9 2 1 . 0 9 2 1 . 7 0 2 1 . 0 6 1 1 . 7 0 1 1 d o 3 d c 5 9 4 . d o 3 d c 3 . 9 4 d o 3 d c 2 9 4 . d o 3 d c 0 9 4 . d o 3 d c 8 . 8 4 d o 3 d c 7 . 8 4 d o 3 d c 5 . 8 4 . 8 2 5 . 5 2 5 . 3 4 7 8 . 1 7 N HN N N N OBn Br 45 40 35 30 25 20 15 10 5 0 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 101 Figure 2.12: 1H and 13C{1H} NMR spectra of 2-23 O N NH OBn N N N DG-3-67-tt18-29_ CARBON_01 . 4 4 6 1 . 4 9 4 1 . 2 9 4 1 . 6 7 3 1 0 . 3 3 1 . 5 2 3 1 6 . 8 2 1 1 . 8 2 1 0 . 8 2 1 7 . 3 2 1 3 . 8 0 1 O N NH OBn N N N l 3 c d c 4 7 7 . l 3 c d c 2 7 7 . l 3 c d c 9 6 7 . 6 . 3 7 . 5 2 7 . 4 5 6 M C D 5 . 3 5 . 6 4 1 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 102 Figure 2.13: NOE spectra for compound 2-23 1H NMR (CDCl3, 500MHz) of compound 2-23 1D-NOESY exciting proton at δ 5.01 1D-NOESY exciting proton at δ 4.05 1D-NOESY exciting proton at δ 3.57 103 Figure 2.14: 1H and 13C{1H} NMR spectra of 2-24 DG-3-99_PROTON_01 7 8 6 8 5 8 5 8 . . . . 8 8 8 8 9 5 . 8 8 5 . 8 8 5 . 8 7 5 . 8 8 6 7 . 1 1 . 7 2 1 . 7 0 1 . 7 9 0 7 . O D H 3 9 4 . 2 2 . 5 1 2 . 5 7 2 4 . 5 2 4 . 4 2 4 . 2 2 4 . 9 9 3 . 8 9 3 . 7 9 3 . 7 9 3 . 7 6 . 3 7 6 . 3 6 6 . 3 4 6 . 3 5 6 . 3 3 6 . 3 d o 3 d c 2 3 d o 3 d c 1 3 d o 3 d c 1 3 d o 3 d c 1 3 d o 3 d c 0 3 . . . . . 3 3 3 3 3 5 3 . 1 3 3 . 1 2 3 . 1 O N NH OH N N N 13 12 11 10 9 8 7 9 0 . 2 9 0 . 1 9 0 . 9 9 0 . 7 6 f1 (ppm) 9 9 0 . 1 0 2 . 8 9 0 . 8 9 . 1 4 0 . 3 5 4 3 2 1 0 -1 DG-3-99_ CARBON_01 . 1 7 6 1 . 7 1 5 1 . 2 0 5 1 . 7 4 3 1 . 3 2 3 1 . 3 6 2 1 . 2 0 1 1 d o 3 d c 5 9 4 . d o 3 d c 3 . 9 4 d o 3 d c 2 9 4 . d o 3 d c 0 9 4 . d o 3 d c 8 . 8 4 d o 3 d c 7 . 8 4 d o 3 d c 5 . 8 4 . 4 6 6 . 9 4 6 . 8 4 1 O N NH OH N N N 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 -500 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 104 Figure 2.15: 1H and 13C{1H} NMR spectra of 2-27 9 8 8 . 9 8 8 . 8 8 8 . 7 8 8 . 2 6 8 . 3 6 8 . 2 6 8 . 2 6 8 . 7 7 7 . 5 1 7 . 4 1 7 . 4 1 7 . 3 1 7 . 3 6 5 . 3 6 5 . O D H 9 8 4 . 3 2 4 . 3 2 4 . 2 2 4 . 1 2 4 . 1 2 4 . 0 2 4 . 0 2 4 . 9 1 4 . 8 1 4 . 8 1 4 . 7 1 4 . 7 1 4 . 1 5 3 . 1 5 3 . 0 5 3 . 0 5 3 . 0 5 3 . 9 4 3 . 9 4 3 . d o 3 d c 1 3 3 . d o 3 d c 1 3 3 . d o 3 d c 1 3 3 . d o 3 d c 0 3 3 . d o 3 d c 0 3 3 . 4 5 2 . 3 5 2 . 5 1 2 . 4 1 2 . 3 3 1 . 2 3 1 . 0 3 1 . 45000 DG-3-111-tt36-39-MeOH_PROTON_01 O N N N N N 13 12 11 10 9 8 9 8 0 . 7 8 0 . 3 9 0 . 7 9 0 . 7 8 8 0 . 6 f1 (ppm) 1 2 2 . 3 9 0 . 1 0 1 . 0 0 1 . 0 3 3 . 5 4 3 2 1 0 -1 . 0 4 7 1 . 7 0 5 1 . 8 8 4 1 . 3 3 3 1 . 7 0 3 1 . 2 4 2 1 . 9 8 0 1 d o 3 d c 1 8 4 . d o 3 d c 9 7 4 . d o 3 d c 8 7 4 . d o 3 d c 6 7 4 . d o 3 d c 4 7 4 . d o 3 d c 2 7 4 . d o 3 d c 1 7 4 . . 0 3 4 . 4 7 3 . 7 6 6 . 9 9 5 . 0 3 1 DG-3-111-tt36-39_ CARBON_01 O N N N N N 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 -10 105 40000 35000 30000 25000 20000 15000 10000 5000 0 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.16: 1H and 13C{1H} NMR spectra of 2-28 5 2 7 . 5 2 7 . 4 2 7 . 6 1 . 7 6 0 7 . 2 8 6 . 1 8 6 . 1 8 6 . 0 8 6 . 7 6 6 . 6 6 6 . 5 6 6 . 4 6 6 . 6 7 5 . 5 7 5 . 1 3 5 . 0 3 5 . 0 3 5 . 0 3 5 . 9 2 5 . 9 6 4 . 7 6 4 . 5 6 4 . 4 6 4 . 3 6 4 . 2 6 4 . 1 6 4 . 0 6 4 . 0 5 4 . 8 4 4 . 5 2 4 . 4 2 4 . 3 2 4 . 3 2 4 . 2 2 4 . 1 2 4 . 1 2 4 . 9 1 . 4 9 1 . 4 8 1 . 4 8 1 . 4 7 1 . 4 8 0 4 . 7 0 4 . 6 0 4 . 3 0 4 . 2 0 4 . 0 0 4 . 6 9 3 . 4 9 3 . 4 9 3 . 3 7 3 . 2 7 3 . 9 6 3 . 8 6 3 . 7 6 3 . 7 5 3 . 5 5 3 . 5 5 3 . 1 9 2 . 5 2 . 1 4 2 . 1 2 2 . 1 1 2 . 1 1 2 . 1 9 1 . 1 9 1 . 1 4 1 . 1 3 1 . 1 9 0 . 1 8 0 . 1 7 0 . 1 0 0 . 1 0 0 . 1 9 9 0 . 8 9 0 . 7 9 0 . 7 9 0 . 6 9 0 . 6500 l 3 c d c 6 2 7 . DG-4-39-tt18-25-CDCl3_PROTON_01 7 7 8 . 7 7 8 . 6 7 8 . 5 7 8 . 8 5 8 . 8 5 8 . 7 5 8 . 7 5 8 . 5 5 8 . 4 5 8 . 3 5 8 . 3 5 8 . 2 5 8 . 5 8 7 . 8 5 7 . 8 5 7 . 9 3 7 . 9 3 7 . 8 3 7 . 7 3 7 . 7 3 7 . 6 3 7 . 6 3 7 . 5 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 . i-Pr i-Pr O N S O N O i-Pr OBn N N N 14 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 0 0 1 . 3 5 2 . 7 7 0 . 5 9 0 . 9 7 4 . 3 6 4 . 6 0 2 . 5 6 1 . 1 8 0 . 0 0 1 . 1 8 0 . 8 1 1 . 1 0 4 . 5 8 0 . 9 5 4 . 0 2 3 . 7 0 1 . 7 4 2 . 5 8 0 . 1 8 1 . 3 1 5 8 1 . 6 3 6 . 5 7 2 . . 8 1 3 1 2 1 0 -1 -2 DG-4-39-tt18-25-CDCl3_CARBON_01 . 2 7 5 1 . 0 7 5 1 . 4 4 5 1 . 3 4 5 1 4 . 1 5 1 3 . 1 5 1 . 8 9 4 1 . 7 9 4 1 . 9 8 4 1 . 6 7 3 1 . 2 7 3 1 . 9 4 3 1 . 8 2 3 1 . 6 2 3 1 4 . 1 3 1 . 9 0 3 1 . 6 8 2 1 . 5 8 2 1 . 2 8 2 1 . 2 8 2 1 . 9 7 2 1 . 8 7 2 1 . 8 3 2 1 . 7 3 2 1 . 4 2 2 1 . 2 2 2 1 . 5 8 0 1 . 4 8 0 1 . 9 3 7 . 5 3 7 7 . 1 7 . 6 9 6 . 2 7 6 . 8 6 6 . 8 6 6 . 3 3 6 . 7 8 5 . 8 5 5 . 3 4 3 . 2 4 3 . 4 9 2 . 0 9 2 . 8 4 2 . 7 4 2 1 . 4 2 . 6 3 2 . 5 3 2 . 4 3 2 . 8 3 1 . 8 3 1 i-Pr i-Pr O N S O N O i-Pr OBn N N N 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 -10 106 6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 -500 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.17: 1H and 13C{1H} NMR spectra of 2-29 6 2 7 . 5 2 7 . 4 2 7 . 4 2 7 . 5 1 . 7 2 1 . 7 4 0 7 . 2 0 7 . 8 7 6 . 7 7 6 . 0 5 6 . 9 4 6 . 9 4 6 . 8 4 6 . 1 6 5 . 0 6 5 . 6 2 5 . 4 2 5 . 4 2 5 . 4 2 5 . 7 6 4 . 6 6 4 . 7 5 4 . 5 5 4 . 0 5 4 . 8 4 4 . 7 3 4 . 7 3 4 . 6 3 4 . 5 3 4 . 5 3 4 . 4 3 4 . 4 2 4 . 4 2 4 . 3 2 4 . 2 2 4 . 9 9 3 . 7 9 3 . 5 9 3 . 2 9 3 . 2 9 3 . 0 9 3 . 9 8 3 . 8 8 3 . 6 8 3 . 0 7 3 . 9 6 3 . 7 6 3 . 0 6 3 . 9 5 3 . 8 8 2 . 6 8 2 . 5 8 2 . 4 2 . 1 3 2 . 1 2 2 . 1 1 2 . 1 0 2 . 1 0 2 . 1 9 1 . 1 8 1 . 1 6 1 . 1 6 1 . 1 5 1 . 1 3 1 . 1 1 1 . 1 4 0 . 1 3 0 . 1 0 0 . 1 9 9 0 . 8 9 0 . 7 9 0 . 6 9 0 . 4 9 0 . 7 0 0 . l 3 c d c 6 2 7 . DG-4-44-CDCl3_PROTON_01 2 6 8 . 2 6 8 . 1 6 8 . 0 6 8 . 0 6 8 . 9 5 8 . 8 5 8 . 8 4 8 . 8 4 8 . 7 4 8 . 7 4 8 . 3 4 8 . 2 4 8 . 2 4 8 . 1 4 8 . 8 8 7 . 1 5 7 . 1 5 7 . 0 4 7 . 0 4 7 . 9 3 7 . 8 3 7 . 8 3 7 . 7 3 7 . 7 3 7 . 7 3 7 . 6 3 7 . 5 3 7 . 2 3 7 . 1 3 7 . 0 3 7 . 0 3 7 . 9 2 7 . 9 2 7 . 8 2 7 . 7 2 7 . i-Pr i-Pr O N S O HN O i-Pr OBn N N N 14 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 DG-4-44-CDCl3_CARBON_01 1 . 5 5 1 . 2 4 5 1 . 2 2 5 1 . 0 2 5 1 . 0 0 5 1 1 . 9 4 1 . 0 7 3 1 . 7 2 3 1 . 2 2 3 1 2 . 1 3 1 . 7 8 2 1 . 6 8 2 1 . 6 8 2 1 . 5 8 2 1 . 2 8 2 1 . 2 8 2 1 . 9 7 2 1 . 0 4 2 1 . 0 4 2 1 1 . 1 2 1 . 7 8 0 1 . 2 4 7 . 7 3 7 . 5 9 6 . 9 0 6 2 . 2 8 4 1 0 -1 -2 . 2 4 3 1 . 4 3 . 6 9 2 . 3 9 2 . 9 4 2 . 7 4 2 . 6 4 2 . 4 4 2 . 4 4 2 . 5 3 2 . 5 3 2 . 4 3 2 8 1 . 1 0 0 . 1 3 1 . 1 9 3 5 . 4 1 . 2 3 0 . 1 4 1 . 1 8 2 2 . 9 0 . 1 8 0 . 1 7 9 3 . 6 2 7 1 . i-Pr i-Pr O N S O HN O i-Pr OBn N N N 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 -10 107 2100 2000 1900 1800 1700 1600 1500 1400 1300 1200 1100 1000 900 800 700 600 500 400 300 200 100 0 -100 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.18: 1H and 13C{1H} NMR spectra of 2-31 DG-5-15-tt24-28-MeOD _PROTON_01 7 7 . 8 7 7 . 8 6 7 . 8 5 7 . 8 0 6 . 8 0 6 . 8 9 5 . 8 9 5 . 8 3 7 7 . 3 1 . 7 2 1 . 7 2 1 . 7 1 1 . 7 O D H 0 9 4 . 5 2 . 5 4 2 . 5 6 3 4 . 5 3 4 . 5 3 4 . 4 3 4 . 4 3 4 . 3 3 4 . 2 3 4 . 2 3 4 . 2 3 4 . 2 3 4 . 1 3 4 . 0 3 4 . 7 2 4 . 6 2 4 . 5 2 4 . d o 3 d c 1 3 d o 3 d c 0 3 d o 3 d c 0 3 4 3 . . . . 3 3 3 3 4 1 . 3 5 3 . 1 3 3 . 1 2 3 . 1 O N NH OMs N N N 13 12 11 10 9 8 7 9 0 . 7 9 0 . 6 9 0 . 1 0 1 . 7 0 0 1 . 0 1 . 3 6 8 . 1 6 f1 (ppm) 5 4 7 9 2 . 3 6 0 . 3 2 1 0 -1 DG-5-15-tt24-28-MeOD _ CARBON_01 1 . 7 6 1 . 0 2 5 1 . 4 0 5 1 . 8 4 3 1 . 9 2 3 1 1 . 5 2 1 3 . 0 1 1 d o 3 d c 5 9 4 . d o 3 d c 3 . 9 4 d o 3 d c 2 9 4 . d o 3 d c 0 9 4 . d o 3 d c 8 . 8 4 d o 3 d c 7 . 8 4 d o 3 d c 5 . 8 4 . 3 7 3 . 0 2 7 . 6 6 6 . 8 4 1 O N NH OMs N N N 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 -10 108 36000 34000 32000 30000 28000 26000 24000 22000 20000 18000 16000 14000 12000 10000 8000 6000 4000 2000 0 -2000 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 2.19: 1H and 13C{1H} NMR spectra of 2-25 O N NH O N O N N N O N NH O N O N N N 109 Figure 2.20: 1H and 13C{1H} NMR spectra of 2-32 DG-5-121-MeOD _PROTON_01 O N NH N3 N N N 7 7 8 . 6 7 8 . 9 5 8 . 9 5 8 . 8 5 8 . 8 5 8 . 0 7 7 . 3 1 7 . 2 1 7 . 2 1 7 . 1 1 7 . 8 1 5 . 7 1 5 . O D H 4 9 4 . 9 2 4 . 8 2 4 . 5 2 4 . 6 2 4 . 9 1 4 . 8 1 4 . 7 1 4 . 6 1 4 . 5 6 3 . 4 6 3 . 2 6 3 . 1 6 3 . 6 4 3 . 5 4 3 . 4 4 3 . 3 4 3 . 4 3 3 . d o 3 d c 2 3 3 . d o 3 d c 2 3 3 . d o 3 d c 1 3 3 . d o 3 d c 1 3 3 . 9 9 2 . 5 8 2 . 6 3 1 . 4 3 1 . 3 3 1 . 13 12 11 10 9 8 0 9 0 . 3 0 1 . 4 9 0 . 8 0 1 . 7 6 f1 (ppm) 8 9 0 . 4 9 1 . 2 0 1 . 6 0 1 . 3 0 1 . 1 0 3 . 5 4 3 2 1 0 -1 DG-5-121-MeOD _ CARBON_01 O N NH N3 N N N . 6 5 6 1 . 5 0 5 1 . 0 9 4 1 . 4 3 3 1 . 4 1 3 1 . 0 4 2 1 . 9 8 0 1 d o 3 d c 1 8 4 . d o 3 d c 0 8 4 . d o 3 d c 8 7 4 . d o 3 d c 6 7 4 . d o 3 d c 5 7 4 . d o 3 d c 3 7 4 . d o 3 d c 1 7 4 . . 1 5 6 . 2 4 5 . 5 3 1 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 110 6500 6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 -500 600 550 500 450 400 350 300 250 200 150 100 50 0 -50 Figure 2.21: 1H and 13C{1H} NMR spectra of 2-35 0 3 9 . 9 2 9 . 8 9 8 . 7 9 8 . 4 3 8 . 4 5 7 . 3 5 7 . 3 5 7 . 2 5 7 . 2 8 6 . 6 7 5 . 5 7 5 . O D H 2 9 4 . 7 6 4 . 6 6 4 . 5 6 4 . 4 6 4 . 8 5 4 . 6 5 4 . 5 5 4 . 3 5 4 . 3 8 3 . 2 8 3 . 0 8 3 . 9 7 3 . 4 7 3 . 3 7 3 . 1 7 3 . 0 7 3 . d o 3 d c 1 3 3 . d o 3 d c 0 3 3 . d o 3 d c 0 3 3 . 9 4 1 . 8 4 1 . 6 4 1 . DG-5-124-tt20-21-MeOD _PROTON_01 N N N O NH N NH O HN Br Br 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 9 9 0 . 0 1 1 . 2 9 0 . 8 9 0 . 9 9 0 . 5 9 0 . 6 9 0 . 0 1 2 . 8 9 0 . 8 9 0 . 0 1 3 . . 5 3 6 1 . 1 1 6 1 . 9 5 5 1 . 0 5 3 1 . 4 3 3 1 . 6 6 2 1 . 4 3 1 1 . 4 2 1 1 . 6 5 0 1 . 7 8 9 d o 3 d c 1 8 4 . d o 3 d c 9 7 4 . d o 3 d c 8 7 4 . d o 3 d c 6 7 4 . d o 3 d c 4 7 4 . d o 3 d c 2 7 4 . d o 3 d c 1 7 4 . . 0 1 4 . 1 1 7 . 9 0 6 . 8 1 5 . 0 3 1 DG-5-124-tt20-21-MeOD _ CARBON_01 N N N O NH N NH O HN Br Br 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 -10 111 34000 32000 30000 28000 26000 24000 22000 20000 18000 16000 14000 12000 10000 8000 6000 4000 2000 0 -2000 60 55 50 45 40 35 30 25 20 15 10 5 0 -5 Figure 2.22: 1H NMR spectra of 2-36 8 1 9 . 7 1 9 . 5 9 8 . 5 9 8 . 3 1 8 . 3 5 7 . 2 5 7 . 1 5 7 . 0 5 7 . 1 8 6 . O D H 2 2 5 . 4 2 5 . 8 1 4 . 7 1 4 . 6 1 4 . 5 1 4 . 5 6 3 . 4 6 3 . 2 6 3 . 1 6 3 . 0 6 3 . 8 5 3 . 7 5 3 . d o 3 d c 2 3 3 . d o 3 d c 1 3 3 . d o 3 d c 1 3 3 . DG-5-69-tt22-25-MeOD _PROTON_01 O NH HN N N N NH O HN Br Br 13 12 11 10 9 2 9 0 . 8 9 0 . 6 9 0 . 5 1 1 . 8 0 9 0 . 7 6 f1 (ppm) 5 0 1 . 0 0 1 . 4 0 2 . 5 4 3 2 1 0 -1 30000 28000 26000 24000 22000 20000 18000 16000 14000 12000 10000 8000 6000 4000 2000 0 -2000 112 Figure 2.23: 1H and 13C{1H} NMR spectra of S2-1 Cl3C O N H DG-4-70-CDCl3 _ CARBON_01 Cl3C O N H 3 . 3 7 1 . 3 7 2 1 . 9 2 2 1 3 . 1 2 1 9 . 1 1 1 . 9 4 9 l 3 c d c 3 7 7 . l 3 c d c 0 7 7 . l 3 c d c 8 . 6 7 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 113 O 2 H 4 3 . 3 o s m d 0 5 2 . o s m d 9 4 2 . o s m d 9 4 2 . Figure 2.24: 1H and 13C{1H} NMR spectra of S2-2 DG-4-71-DMSO _PROTON_01 5 7 . 3 1 Cl3C O N H Br Br 6 1 . 1 9 3 7 . 1 1 . 1 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 DG-4-71-DMSO _ CARBON_01 Br Br Cl3C O N H 4 . 1 7 1 8 . 3 2 1 . 9 2 2 1 1 . 5 1 1 3 . 1 0 1 . 5 4 9 o s m d 5 0 4 . o s m d 3 . 0 4 o s m d 1 . 0 4 o s m d 0 0 4 . o s m d 8 . 9 3 o s m d 6 9 3 . o s m d 5 9 3 . 55000 50000 45000 40000 35000 30000 25000 20000 15000 10000 5000 0 320 300 280 260 240 220 200 180 160 140 120 100 80 60 40 20 0 -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 -10 114 Figure 2.25: 1H and 13C{1H} NMR spectra of 2-39 2 8 2 8 1 8 1 8 . . . . 8 8 8 8 8 5 . 8 9 5 . 8 8 5 . 8 7 5 . 8 6 8 7 . 8 0 7 . 7 0 7 . 6 0 7 . 5 0 7 . 8 7 6 . O D H 9 8 4 . 1 1 . 5 0 1 . 5 5 3 4 . 4 3 4 . 3 3 4 . 2 3 4 . 7 6 . 3 6 6 . 3 6 6 . 3 5 6 . 3 d o 3 d c 1 3 d o 3 d c 1 3 d o 3 d c 1 3 d o 3 d c 0 3 d o 3 d c 0 3 . . . . . 3 3 3 3 3 0 7 2 . DG-5-41-tt23-MeOD _PROTON_01 OMs NH2 HN NH O H N N H N N N Br Br 13 12 11 10 9 6 0 . 1 4 0 . 1 5 0 . 1 8 8 0 . 1 6 9 0 . 7 7 0 . 1 5 6 f1 (ppm) 6 0 . 1 5 0 2 . 9 9 2 . 4 3 2 1 0 -1 DG-5-41-tt23-MeOD _ CARBON_01 . 2 2 6 1 . 6 0 6 1 0 . 3 5 1 . 0 0 5 1 . 4 6 4 1 8 . 6 3 1 4 . 8 2 1 . 5 4 1 1 8 8 . . 0 1 1 0 1 1 . 5 6 0 1 . 0 0 0 1 d o 3 d c 5 9 4 . d o 3 d c 3 . 9 4 d o 3 d c 2 9 4 . d o 3 d c 0 9 4 . d o 3 d c 8 . 8 4 d o 3 d c 7 . 8 4 d o 3 d c 5 . 8 4 3 . 3 6 1 . 8 5 0 . 3 4 . 5 9 3 OMs NH2 HN NH O H N N H N N N Br Br 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 -10 115 75000 70000 65000 60000 55000 50000 45000 40000 35000 30000 25000 20000 15000 10000 5000 0 -5000 50 45 40 35 30 25 20 15 10 5 0 Figure 2.26: 1H and 13C{1H} NMR spectra of 2-1 DG-5-102-MeOD _PROTON_01 OMs HN H2N H N N NH2 NH O N H H N Br Br 3 8 . 6 2 7 6 . O D H 4 9 4 . 0 8 4 . 2 8 4 . 7 1 . 4 5 1 . 4 4 1 . 4 4 1 . 4 3 1 . 4 9 5 . 3 8 5 . 3 7 5 . 3 d o 3 d c 2 3 d o 3 d c 1 3 d o 3 d c 1 3 d o 3 d c 1 3 d o 3 d c 0 3 . . . . . 3 3 3 3 3 2 7 2 . 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 1 0 . 1 6 0 . 1 6 1 . 1 4 1 . 1 0 0 2 . 0 5 2 . . 2 2 6 1 . 5 0 6 1 1 . 1 5 1 . 6 9 2 1 4 . 8 2 1 . 6 4 1 1 . 3 2 1 1 . 6 6 0 1 . 0 0 0 1 d o 3 d c 5 9 4 . d o 3 d c 3 . 9 4 d o 3 d c 2 9 4 . d o 3 d c 0 9 4 . d o 3 d c 8 . 8 4 d o 3 d c 7 . 8 4 d o 3 d c 5 . 8 4 . 4 2 6 . 3 6 5 . 7 2 4 . 5 9 3 DG-5-102-MeOD _ CARBON_01 OMs HN H2N H N N NH2 NH O N H H N Br Br 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 -10 116 60000 55000 50000 45000 40000 35000 30000 25000 20000 15000 10000 5000 0 -5000 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 Figure 2.27: 1H and 13C{1H} NMR spectra of 2-1 • 2TFA DG-5-136-TFA-DMSO _ CARBON_01 . 6 9 5 1 7 . 8 5 1 2 . 8 4 1 . 8 7 2 1 . 6 4 2 1 2 . 3 1 1 7 . 1 1 1 2 . 5 0 1 1 . 8 9 o s m d 0 0 4 . o s m d 9 9 3 . o s m d 7 9 3 . o s m d 5 9 3 . o s m d 4 9 3 . o s m d 2 9 3 . o s m d 0 9 3 . 1 . 0 6 3 . 3 5 4 . 1 4 100 90 80 70 60 50 40 30 20 10 0 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 117 Crystal Data and Experimental X-ray crystallographic analysis was performed by Dr. Richard Staples. Figure 2.28: X-ray crystal structure of compound 2-25 Experimental. Single colorless plate-shaped crystals of JJT822A_twin1_hklf5 used as received. A suitable crystal with dimensions 0.19 × 0.12 × 0.07 mm3 was selected and mounted on a nylon loop with paratone oil on a XtaLAB Synergy, Dualflex, HyPix diffractometer. The crystal was kept at a steady T = 100.01(12) K during data collection. The structure was solved with the ShelXT 2018/2 (Sheldrick, 2018) solution program using dual methods and by using O. V. Dolomanov, L. J. Bourhis, R. J. Gildea, J. A. K. Howard and H. Puschmann,Olex2: a complete structure solution, refinement and analysis program.J. Appl. Cryst. (2009). 42, 339-341. as the graphical interface. The model was refined with ShelXL 2018/3 (Sheldrick, 2015) using full matrix least squares minimisation on F2. Crystal Data. C20H18N6O3, Mr = 390.40, monoclinic, P21/c (No. 14), a = 15.6910(3) Å, b = 15.0668(2) Å, c = 15.2190(2) Å, b = 93.496(2)°, a = g = 90°, V = 3591.28(10) Å3, T = 100.01(12) K, Z = 8, Z' = 2, m(Cu Ka) = 0.838, 16075 reflections measured, 16075 unique (Rint = .) which were used in all calculations. The final wR2 was 0.2340 (all data) and R1 was 0.0820 (I≥2 s(I)). 118 Table 2.5: Crystal data Compound JJT822A_twin1_hklf5 Formula CCDC Dcalc./ g cm-3 m/mm-1 C20H18N6O3 2194100 1.444 0.838 Formula Weight 390.40 Color Shape colorless plate-shaped Size/mm3 0.19×0.12×0.07 T/K 100.01(12) Crystal System monoclinic Space Group P21/c a/Å b/Å c/Å a/° b/° g/° V/Å3 Z Z' 15.6910(3) 15.0668(2) 15.2190(2) 90 93.496(2) 90 3591.28(10) 8 2 119 Table 2.5 (cont’d) Wavelength/Å 1.54184 Radiation type Cu Ka Qmin/° Qmax/° 2.821 80.341 Measured Refl's. 16075 Indep't Refl's Refl's I≥2 s(I) Rint Parameters Restraints 16075 14464 . 534 0 Largest Peak 0.495 Deepest Hole -0.434 GooF 1.109 wR2 (all data) 0.2340 wR2 R1 (all data) R1 0.2311 0.0867 0.0820 Table 2.6: Structure Quality Indicators Reflections: Refinement: A colorless plate-shaped-shaped crystal with dimensions 0.19×0.12×0.07 mm3 was mounted on a nylon loop with paratone oil. Data were collected using a XtaLAB Synergy, Dualflex, HyPix 120 diffractometer equipped with an Oxford Cryosystems low-temperature device, operating at T = 100.01(12) K. MSU Data were measured using w scans using Cu Ka radiation (micro-focus sealed X-ray tube, 50 kV, 1 mA). The total number of runs and images was based on the strategy calculation from the program CrysAlisPro 1.171.42.64a (Rigaku OD, 2022). The achieved resolution was Q = 80.341. Cell parameters were retrieved using the CrysAlisPro 1.171.42.64a (Rigaku OD, 2022) software and refined using CrysAlisPro 1.171.42.64a (Rigaku OD, 2022) on 23311 reflections, 145 % of the observed reflections. Data reduction was performed using the CrysAlisPro 1.171.42.64a (Rigaku OD, 2022) software which corrects for Lorentz polarization. The final completeness is 100.00 out to 80.341 in Q CrysAlisPro 1.171.42.64a (Rigaku Oxford Diffraction, 2022) Empirical absorption correction using spherical harmonics, implemented in SCALE3 ABSPACK scaling algorithm. The structure was solved in the space group P21/c (# 14) by using dual methods using the ShelXT (Sheldrick, 2015) structure solution program. The structure was refined by Least Squares ShelXL incorporated in Olex2 software program. All non-hydrogen atoms were refined anisotropically. Hydrogen atom positions were calculated geometrically and refined using the riding model, except for the hydrogen atom on the non-carbon atom(s) which were found by difference Fourier methods and refined isotropically when data permits. CCDC 2194100 contains the supplementary crystallographic data for this paper. The data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/structures. _twin_special_details: Component 2 rotated by -179.8461° around [1.00 -0.00 -0.03] (reciprocal) 121 or [1.00 -0.00 0.03] (direct). The value of Z' is 2. This means that there are two independent molecules in the asymmetric unit. Figure 2.29: Drawing at 50% ellipsoids showing the labeling scheme for compound 2-25, where the model has Chirality at C2 (Centro SPGR) S, and Chirality at C3 (Centro SPGR) R Figure 2.30: Drawing at 50% ellipsoids showing the labeling scheme, where the model has Chirality at C2A (Centro SPGR) S, and Chirality at C3A (Centro SPGR) R Figure 2.31: Packing diagram of JJT822A_twin1_hklf5 122 Figure 2.32: Data plots: Diffraction data Figure 2.33: Data plots: Refinement and data 123 Table 2.7: Reflection Statistics Total reflections 18680 Unique reflections 7775 (after filtering) Completeness 0.988 Mean I/s 16.8 hklmax collected (19, 19, 19) hklmin collected (-20, -19, -19) hklmax used (19, 19, 19) hklmin used (-20, 0, 0) Lim dmax collected 100.0 Lim dmin collected 0.77 dmax used 15.66 dmin used 0.78 Friedel pairs 3083 Friedel pairs merged 1 Inconsistent 0 Rint 0.0 equivalents Rsigma 0.0156 Intensity transformed 0 Omitted reflections 0 Omitted by user 0 (OMIT hkl) Multiplicity (13016, 1513, 11) Maximum 0 multiplicity Removed systematic 0 Filtered off 0 absences (Shel/OMIT) 124 Figure 2.34: Selected Crystal Pictures Table 2.8: Fractional Atomic Coordinates (×104) and Equivalent Isotropic Displacement Parameters (Å2×103) for JJT822A_twin1_hklf5. Ueq is defined as 1/3 of the trace of the orthogonalised Uij Atom x y z Ueq O1 O2 O3 N1 N2 N3 N4 N5 N6 C1 C2 C3 C4 1087.0(18) 3924.2(16) 619.5(17) 28.2(6) 886.5(18) 2685.6(17) 3996.5(19) 31.5(6) -784.7(18) 5152.9(16) 4208.2(18) 30.7(6) 739(2) 4103.0(19) 2040(2) 25.3(6) 1481(2) 5210.8(19) 1409(2) 26.4(6) 172(2) 4033.6(19) 3959(2) 25.2(6) 631(2) 7778.9(19) 2768(2) 28.5(7) 1770(2) 7072.6(18) 2288(2) 23.7(6) 1817(2) 8666.8(19) 2386(2) 28.2(7) 1111(2) 4445(2) 1329(2) 23.6(7) 667(2) 4836(2) 2657(2) 24.8(7) 1367(2) 5459(2) 2335(2) 24.4(7) 1564(3) 4228(3) -117(3) 30.3(8) 125 Table 2.8 (cont’d) C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 C17 C18 C19 C20 980(3) 4660(3) -805(3) 33.6(9) 842(3) 4577(2) 3612(2) 27.9(8) 263(3) 3125(2) 4145(2) 26.0(7) -524(2) 2864(2) 4567(2) 25.1(7) -786(3) 2040(2) 4859(3) 28.4(8) -1571(3) 1999(2) 5237(3) 32.1(8) -2070(3) 2750(3) 5326(3) 30.5(8) -1809(3) 3574(2) 5026(3) 29.1(8) -1033(2) 3614(2) 4646(2) 25.7(7) -575(2) 4375(2) 4256(2) 25.7(7) 1177(2) 6424(2) 2454(2) 24.1(7) 498(3) 6877(2) 2746(3) 29.3(8) 1400(3) 7888(2) 2495(2) 25.6(7) 2569(3) 8613(2) 2060(3) 29.5(8) 2972(3) 7804(3) 1833(3) 31.3(8) 2552(2) 7027(2) 1954(3) 27.5(8) O1A 5938.6(18) 10621.9(16) 4614.8(17) 27.9(6) O2A 6072.8(18) 11698.3(17) 979.5(19) 32.7(6) O3A 4122.7(18) 9442.4(17) 1055.6(18) 30.8(6) N1A N2A N3A 5780(2) 10462.1(19) 3119(2) 25.8(6) 6350(2) 9305.5(19) 3939(2) 25.1(6) 5245(2) 10438.9(19) 1128(2) 24.7(6) 126 Table 2.8 (cont’d) N4A 5619(2) 6782(2) 2296(2) 29.4(7) N5A 6788.1(19) 7465.0(19) 2875(2) 23.2(6) N6A 6898(2) 5914(2) 2534(2) 31.4(7) C1A C2A C3A C4A C5A C6A C7A C8A C9A 6030(2) 10096(2) 3919(2) 24.2(7) 5698(2) 9693(2) 2525(2) 25.3(7) 6345(2) 9064(2) 2998(2) 24.3(7) 6185(3) 10234(2) 5464(2) 28.9(8) 6065(3) 10932(3) 6138(3) 32.5(8) 5890(2) 9878(2) 1587(2) 26.7(7) 5400(2) 11318(2) 861(2) 25.4(7) 4577(2) 11624(2) 421(2) 24.7(7) 4379(3) 12421(2) 13(3) 29.5(8) C10A 3544(3) 12531(3) -333(3) 30.6(8) C11A 2931(3) 11869(3) -271(3) 31.4(8) C12A 3144(3) 11059(2) 146(3) 28.4(8) C13A 3974(2) 10953(2) 480(2) 25.1(7) C14A 4409(2) 10180(2) 919(2) 24.6(7) C15A 6160(2) 8107(2) 2793(2) 23.2(7) C16A 5463(3) 7664(2) 2444(3) 29.1(8) C17A 6430(3) 6667(2) 2558(2) 26.2(7) C18A 7700(3) 5963(3) 2849(3) 35.5(9) 127 Table 2.8 (cont’d) C19A 8086(3) 6742(3) 3209(3) 35.3(9) C20A 7618(3) 7495(2) 3219(3) 30.0(8) Table 2.9: Anisotropic Displacement Parameters (×104) for JJT822A_twin1_hklf5. The anisotropic displacement factor exponent takes the form: -2p2[h2a*2 × U11+ ... +2hka* × b* × U12] Atom U11 U22 U33 U23 U13 U12 O1 O2 O3 N1 N2 N3 N4 N5 N6 C1 C2 C3 C4 C5 C6 C7 37.6(15) 18.5(12) 28.9(13) -1.2(10) 5.5(11) -1.5(10) 33.3(15) 20.7(13) 41.2(16) 3.3(11) 6.9(12) 2.7(11) 42.6(16) 13.5(12) 35.8(15) 0.0(10) 0.7(12) 2.1(10) 34.6(17) 13.3(13) 28.2(16) 0.3(11) 3.2(12) -4.3(12) 30.7(16) 13.7(13) 35.2(17) 1.8(11) 4.6(13) -1.6(11) 33.4(17) 14.0(13) 28.5(15) 0.9(11) 4.4(12) -1.8(12) 34.1(18) 14.0(14) 37.9(18) -2.4(12) 6.4(14) 4.5(12) 30.5(16) 11.9(13) 28.7(15) 0.6(11) 1.4(12) 0.8(11) 40.0(19) 13.2(14) 31.0(16) 0.7(11) -1.6(13) -1.9(12) 26.8(18) 15.2(15) 28.9(18) 1.8(13) 2.3(14) 3.4(13) 30.2(19) 13.0(15) 31.5(19) -0.1(13) 4.1(14) -0.1(13) 26.7(18) 13.1(15) 33.7(19) 0.7(13) 2.8(14) -1.0(13) 34(2) 26.7(19) 30.7(19) -2.3(15) 8.2(15) 2.4(15) 41(2) 23.5(18) 36(2) 1.7(15) 5.2(17) -0.3(16) 36(2) 17.3(16) 30.8(19) 1.4(13) 3.8(15) -5.7(14) 34(2) 17.8(16) 25.9(18) 0.8(13) 0.8(14) -1.0(14) 128 Table 2.9 (cont’d) C8 C9 32.1(19) 16.8(16) 26.2(18) 0.9(13) -0.4(14) -1.7(14) 34(2) 15.8(16) 36(2) 2.2(14) 2.2(15) 0.7(14) C10 38(2) 17.9(17) 41(2) 4.2(15) 5.1(17) -2.5(15) C11 30(2) 25.2(18) 36(2) 0.5(15) 5.4(15) 1.2(15) C12 32(2) 19.2(17) 36(2) -2.1(14) 1.1(15) 2.8(14) C13 34(2) 14.3(15) 28.5(18) -0.9(13) -1.2(14) -0.5(13) C14 35(2) 17.4(16) 24.7(17) -1.5(13) -0.8(14) -0.2(14) C15 28.8(18) 13.5(15) 30.2(18) 0.7(13) 4.1(14) -2.6(13) C16 31(2) 17.5(17) 40(2) 0.7(14) 7.5(16) 0.3(14) C17 38(2) 12.4(15) 26.5(18) -0.5(12) 1.8(15) -0.2(13) C18 35(2) 18.0(17) 35(2) 2.8(14) -1.7(16) -6.2(14) C19 32(2) 23.6(18) 39(2) 2.0(15) 2.8(16) -3.9(15) C20 28.7(19) 19.7(17) 34(2) 1.7(14) 1.6(15) -0.2(14) O1A 37.5(15) 17.9(12) 28.1(13) -1.6(9) 0.9(11) 2.7(10) O2A 33.6(15) 22.1(13) 42.2(16) 4.0(11) 1.9(12) -5.8(11) O3A 38.2(16) 16.2(12) 38.6(15) 0.8(10) 6.8(12) -5.1(10) N1A 35.1(18) 14.2(13) 28.1(16) 0.1(11) 2.5(13) 2.4(12) N2A 29.0(16) 15.6(13) 30.5(16) -0.9(11) -0.6(12) 0.4(11) N3A 31.5(16) 16.1(13) 26.7(15) 3.5(11) 3.1(12) -0.7(11) N4A 40.2(19) 17.5(14) 29.8(17) -1.0(12) -1.8(13) -4.5(13) N5A 26.3(15) 13.7(13) 29.9(16) -0.8(11) 3.3(12) -0.4(11) N6A 47(2) 12.1(14) 35.3(18) -1.2(12) 7.0(15) -0.2(13) 129 Table 2.9 (cont’d) C1A 27.9(18) 16.7(15) 28.1(18) -1.3(13) 1.7(14) -0.2(13) C2A 34(2) 15.5(15) 27.1(18) -0.2(13) 3.2(14) 1.3(13) C3A 30.4(19) 13.9(15) 28.8(18) 0.5(13) 2.6(14) 0.3(13) C4A 34(2) 23.9(18) 28.5(19) 0.7(14) 1.2(15) 2.6(15) C5A 40(2) 27.4(19) 31(2) -1.6(15) 4.2(16) 1.2(16) C6A 33(2) 17.8(16) 29.8(19) 2.6(13) 2.4(15) 3.2(14) C7A 31.8(19) 18.6(16) 26.1(18) -0.4(13) 3.8(14) -3.2(14) C8A 29.4(19) 17.6(16) 27.2(18) -1.1(13) 3.3(14) -1.4(13) C9A 39(2) 18.1(16) 32(2) -1.2(14) 3.9(16) -0.6(14) C10A 41(2) 19.1(17) 32(2) -1.2(14) 1.8(16) 3.3(15) C11A 35(2) 26.6(19) 33(2) -5.1(15) -0.3(16) 6.2(15) C12A 32(2) 19.6(17) 34(2) -5.7(14) 2.9(15) 0.4(14) C13A 33(2) 16.0(15) 26.8(18) -1.6(13) 5.0(14) -0.8(14) C14A 35(2) 15.9(15) 23.3(17) -1.7(12) 4.7(14) -0.1(14) C15A 28.5(18) 14.3(15) 26.9(18) 1.6(12) 2.4(14) 0.2(13) C16A 34(2) 20.6(17) 32(2) 1.3(14) 0.6(15) -2.5(14) C17A 38(2) 13.7(15) 26.9(18) -1.3(12) 2.6(15) -2.7(14) C18A 44(2) 17.8(17) 45(2) 1.0(15) 10.9(18) 5.9(16) C19A 30(2) 25.9(19) 51(3) 2.6(17) 4.5(17) 5.7(16) C20A 30(2) 17.7(17) 43(2) -0.4(15) 3.6(16) -3.2(14) 130 Table 2.10: Bond Lengths in Å for JJT822A_twin1_hklf5 Atom Atom Length/Å O1 O1 O2 O3 N1 N1 N2 N2 N3 N3 N3 N4 N4 N5 N5 N5 N6 N6 C2 C2 C3 C1 C4 C7 1.334(4) 1.459(5) 1.214(5) C14 1.218(4) C1 C2 C1 C3 C6 C7 C14 C16 C17 C15 C17 C20 C17 C18 C3 C6 1.362(5) 1.458(4) 1.294(5) 1.479(5) 1.456(5) 1.404(4) 1.383(5) 1.375(5) 1.311(5) 1.382(4) 1.403(4) 1.359(5) 1.358(5) 1.309(5) 1.547(5) 1.513(5) C15 1.497(5) 131 Table 2.10 (cont’d) C4 C7 C8 C8 C9 C5 C8 C9 C13 C10 1.498(6) 1.478(5) 1.390(5) 1.393(5) 1.392(6) C10 C11 1.386(5) C11 C12 1.394(5) C12 C13 1.380(6) C13 C14 1.495(5) C15 C16 1.362(5) C18 C19 1.425(5) C19 C20 1.363(5) O1A C1A 1.337(4) O1A C4A 1.449(4) O2A C7A 1.205(5) O3A C14A 1.222(4) N1A C1A 1.372(5) N1A C2A 1.470(4) N2A C1A 1.292(5) N2A C3A 1.478(5) N3A C6A 1.463(5) N3A C7A 1.410(4) 132 Table 2.10 (cont’d) N3A C14A 1.387(5) N4A C16A 1.372(5) N4A C17A 1.321(5) N5A C15A 1.381(4) N5A C17A 1.401(4) N5A C20A 1.374(5) N6A C17A 1.353(5) N6A C18A 1.321(6) C2A C3A 1.535(5) C2A C6A 1.503(5) C3A C15A 1.501(5) C4A C5A 1.489(5) C7A C8A 1.491(5) C8A C9A 1.380(5) C8A C13A 1.392(5) C9A C10A 1.392(6) C10A C11A 1.392(6) C11A C12A 1.407(5) C12A C13A 1.377(5) C13A C14A 1.487(5) C15A C16A 1.361(5) C18A C19A 1.416(6) 133 Table 2.10 (cont’d) C19A C20A 1.352(5) Table 2.11: Bond Angles in ° for JJT822A_twin1_hklf5 Atom Atom Atom Angle/° C1 C1 C1 C7 O1 C4 116.6(3) N1 C2 106.2(3) N2 C3 103.6(3) N3 C6 123.7(3) C14 N3 C6 123.7(3) C14 N3 C7 112.0(3) C17 N4 C16 104.9(3) C15 N5 C17 106.6(3) C20 N5 C15 131.7(3) C20 N5 C17 121.6(3) C18 N6 C17 116.4(3) O1 C1 N1 115.4(3) N2 C1 O1 126.2(3) N2 C1 N1 118.3(3) N1 C2 N1 C2 C6 C2 N2 C3 C3 C6 C3 C2 99.9(3) 114.0(3) 111.8(3) 106.1(3) N2 C3 C15 113.5(3) 134 Table 2.11 (cont’d) C15 C3 O1 C4 N3 C6 C2 C5 C2 113.6(3) 110.7(3) 113.6(3) O2 C7 N3 124.5(4) O2 C7 N3 C7 C9 C9 C8 C8 C8 C8 C7 129.8(3) 105.7(3) 130.4(3) C13 120.9(4) C13 C8 C7 108.7(3) C8 C9 C10 117.3(3) C11 C10 C9 121.6(3) C10 C11 C12 121.0(4) C13 C12 C11 117.4(3) C8 C13 C14 107.2(3) C12 C13 C8 121.8(3) C12 C13 C14 131.0(3) O3 C14 N3 124.7(4) O3 C14 C13 128.9(4) N3 C14 C13 106.4(3) N5 C15 C3 121.5(3) C16 C15 N5 104.7(3) C16 C15 C3 133.7(3) 135 Table 2.11 (cont’d) C15 C16 N4 112.5(3) N4 C17 N5 111.2(3) N4 C17 N6 127.4(3) N6 C17 N5 121.4(3) N6 C18 C19 124.6(3) C20 C19 C18 118.5(4) N5 C20 C19 117.5(3) C1A O1A C4A 115.6(3) C1A N1A C2A 103.8(3) C1A N2A C3A 103.0(3) C7A N3A C6A 123.6(3) C14A N3A C6A 124.4(3) C14A N3A C7A 112.0(3) C17A N4A C16A 104.9(3) C15A N5A C17A 107.5(3) C20A N5A C15A 131.7(3) C20A N5A C17A 120.7(3) C18A N6A C17A 116.8(3) O1A C1A N1A 115.2(3) N2A C1A O1A 126.0(3) N2A C1A N1A 118.7(3) N1A C2A C3A 99.7(3) 136 Table 2.11 (cont’d) N1A C2A C6A 115.0(3) C6A C2A C3A 113.2(3) N2A C3A C2A 105.5(3) N2A C3A C15A 115.3(3) C15A C3A C2A 112.5(3) O1A C4A C5A 106.9(3) N3A C6A C2A 112.8(3) O2A C7A N3A 124.7(4) O2A C7A C8A 130.3(3) N3A C7A C8A 105.1(3) C9A C8A C7A 129.6(3) C9A C8A C13A 121.8(4) C13A C8A C7A 108.7(3) C8A C9A C10A 117.0(4) C9A C10A C11A 121.8(4) C10A C11A C12A 120.4(4) C13A C12A C11A 117.4(4) C8A C13A C14A 107.5(3) C12A C13A C8A 121.5(3) C12A C13A C14A 130.9(3) O3A C14A N3A 124.7(3) O3A C14A C13A 128.7(4) 137 Table 2.11 (cont’d) N3A C14A C13A 106.6(3) N5A C15A C3A 121.7(3) C16A C15A N5A 104.1(3) C16A C15A C3A 133.9(3) C15A C16A N4A 113.0(4) N4A C17A N5A 110.4(3) N4A C17A N6A 128.1(3) N6A C17A N5A 121.4(3) N6A C18A C19A 124.2(4) C20A C19A C18A 118.7(4) C19A C20A N5A 118.1(3) Table 2.12: Torsion Angles in ° for JJT822A_twin1_hklf5 Atom Atom Atom Atom Angle/° O2 O2 N1 N1 N1 N2 N2 N3 N3 C7 C7 C2 C2 C2 C3 C3 C7 C7 C8 C8 C3 C3 C6 C15 C9 -4.5(7) C13 176.0(4) N2 23.0(3) C15 148.4(3) N3 N5 -71.2(4) -65.4(5) C15 C16 117.2(5) C8 C8 C9 177.5(4) C13 -2.0(4) 138 Table 2.12 (cont’d) N5 N6 C1 C1 C1 C1 C1 C2 C2 C2 C2 C3 C3 C3 C3 C4 C4 C6 C6 C6 C15 C16 N4 0.3(5) C18 C19 C20 0.4(6) C4 C2 C2 C3 C3 C1 C1 C15 C5 C3 C6 C2 -99.5(4) -22.1(4) -141.4(3) -15.5(4) C15 -140.9(3) O1 N2 N5 -167.8(3) 15.0(5) 173.2(3) C15 C16 -4.1(6) O1 N1 N1 N2 N2 N1 N1 C3 C3 N2 N2 C2 C1 C1 C6 C15 C16 O1 O1 N3 N3 N3 C1 C1 C7 C7 C14 -175.9(3) 0.9(4) 176.5(3) 177.9(4) -173.2(3) 3.7(5) -4.0(6) 174.1(3) 5.2(6) O1 N1 N3 N4 N1 N2 O2 C8 O3 139 Table 2.12 (cont’d) C6 C6 C6 C7 C7 C7 C7 C7 C7 C8 C8 C8 C9 C9 C9 N3 C2 C2 N3 N3 N3 C8 C8 C8 C9 C14 C13 -173.7(3) C3 C3 C6 C14 N2 143.9(3) C15 -90.7(4) C2 O3 107.8(4) 176.8(4) C14 C13 -2.2(4) C9 C10 179.9(4) C13 C12 -179.3(3) C13 C14 0.7(4) C10 C11 -0.4(6) C13 C14 C13 C14 O3 N3 -178.1(4) 0.8(4) C8 C8 C13 C12 1.1(6) C13 C14 -178.8(3) C10 C11 C12 1.0(6) C10 C11 C12 C13 -0.6(6) C11 C12 C13 C8 -0.5(6) C11 C12 C13 C14 179.4(4) C12 C13 C14 C12 C13 C14 C13 C14 C8 N3 C9 C6 O3 N3 C10 C2 2.0(7) -179.1(4) -0.6(6) -81.6(4) 140 Table 2.12 (cont’d) C14 C14 C15 C15 C15 C16 C16 C17 C17 C17 C17 C17 C18 C18 N3 N3 N5 N5 N5 N4 N4 N4 N5 N5 N5 N6 N6 N6 C7 C7 C17 C17 O2 C8 N4 N6 -175.5(4) 2.6(4) -0.3(4) -178.7(3) C20 C19 177.2(4) C17 C17 N5 N6 0.4(4) 178.7(4) C16 C15 -0.4(5) C15 C3 -178.0(3) C15 C16 0.0(4) C20 C19 0.6(5) C18 C19 -1.0(6) C17 C17 C15 N4 N5 N5 C3 -176.6(4) 1.5(5) -0.1(6) 5.1(6) C15 C16 -176.9(4) C17 C17 N4 N6 177.0(3) -1.3(5) C18 C19 C20 C20 C20 C20 C20 N5 N5 N5 N5 O2A C7A C8A C9A 1.8(7) O2A C7A C8A C13A -177.7(4) N1A C2A C3A N2A 29.1(3) 141 Table 2.12 (cont’d) N1A C2A C3A C15A 155.6(3) N1A C2A C6A N3A -69.4(4) N2A C3A C15A N5A -82.5(4) N2A C3A C15A C16A 104.2(5) N3A C7A C8A C9A -177.7(4) N3A C7A C8A C13A 2.8(4) N5A C15A C16A N4A -0.4(4) N6A C18A C19A C20A -1.5(7) C1A O1A C4A C5A 178.0(3) C1A N1A C2A C3A -27.5(4) C1A N1A C2A C6A -148.9(3) C1A N2A C3A C2A -19.6(4) C1A N2A C3A C15A -144.3(3) C2A N1A C1A O1A -163.6(3) C2A N1A C1A N2A 18.5(5) C2A C3A C15A N5A 156.6(3) C2A C3A C15A C16A -16.8(6) C3A N2A C1A O1A -176.5(3) C3A N2A C1A N1A 1.2(4) C3A C2A C6A N3A 176.8(3) C3A C15A C16A N4A 173.8(4) C4A O1A C1A N1A 178.8(3) 142 Table 2.12 (cont’d) C4A O1A C1A N2A -3.4(5) C6A N3A C7A O2A 0.1(6) C6A N3A C7A C8A 179.6(3) C6A N3A C14A O3A -3.5(6) C6A N3A C14A C13A 178.6(3) C6A C2A C3A N2A 151.8(3) C6A C2A C3A C15A -81.8(4) C7A N3A C6A C2A 111.5(4) C7A N3A C14A O3A 177.6(3) C7A N3A C14A C13A -0.2(4) C7A C8A C9A C10A -178.9(4) C7A C8A C13A C12A 178.4(3) C7A C8A C13A C14A -3.0(4) C8A C9A C10A C11A 0.2(6) C8A C13A C14A O3A -175.7(4) C8A C13A C14A N3A 2.1(4) C9A C8A C13A C12A -1.1(6) C9A C8A C13A C14A 177.5(3) C9A C10A C11A C12A -0.3(6) C10A C11A C12A C13A -0.2(6) C11A C12A C13A C8A 0.9(5) C11A C12A C13A C14A -177.3(4) 143 Table 2.12 (cont’d) C12A C13A C14A O3A 2.7(7) C12A C13A C14A N3A -179.6(4) C13A C8A C9A C10A 0.5(6) C14A N3A C6A C2A -67.2(4) C14A N3A C7A O2A 179.0(4) C14A N3A C7A C8A -1.5(4) C15A N5A C17A N4A -0.6(4) C15A N5A C17A N6A 178.5(3) C15A N5A C20A C19A 179.7(4) C16A N4A C17A N5A 0.3(4) C16A N4A C17A N6A -178.7(4) C17A N4A C16A C15A 0.1(4) C17A N5A C15A C3A -174.5(3) C17A N5A C15A C16A 0.6(4) C17A N5A C20A C19A 2.6(6) C17A N6A C18A C19A 0.5(6) C18A N6A C17A N4A -179.0(4) C18A N6A C17A N5A 2.1(5) C18A C19A C20A N5A -0.1(6) C20A N5A C15A C3A 8.1(6) C20A N5A C15A C16A -176.8(4) C20A N5A C17A N4A 177.1(3) 144 Table 2.12 (cont’d) C20A N5A C17A N6A -3.7(5) Table 2.13: Hydrogen Fractional Atomic Coordinates (×104) and Equivalent Isotropic Displacement Parameters (Å2×103) for JJT822A_twin1_hklf5. Ueq is defined as 1/3 of the trace of the orthogonalised Uij Atom x y z Ueq 270(40) 3710(40) 1960(40) 54(16) H1 H2 H3 93.8 5122.91 2570.71 1911.61 5322.13 2684.07 H4A 1854.07 3716.59 -377.8 H4B 2007.04 4657.02 98.01 H5A 1315.51 4885.9 -1279.99 H5B 678.4 5153.34 -541.75 H5C 564.68 4224.28 -1044.94 H6A 1387.47 4245.9 3671.58 H6B 910.03 5122.72 3971.13 H9 -444.63 1524.95 4802.15 H10 -1769.77 1444.37 5438.46 H11 -2598.21 2701.16 5595.95 H12 -2151.03 4089.2 5081.03 H16 -10.53 6600.11 2914.4 H18 2866.89 9151.21 1969.57 145 30 29 36 36 50 50 50 34 34 34 38 37 35 35 35 Table 2.13 (cont’d) H19 3521.09 7807.47 1601.5 H20 2796.64 6472.56 1809.94 38 33 H1A 5250(50) 10780(60) 3050(60) 110(30) H2A 5111.25 9437.7 2541.83 H3A 6921.53 9205.01 2788.7 H4AA 6788.93 10041.32 5483.13 H4AB 5823.85 9711.75 5574.13 H5AA 6442.8 11435.05 6034.88 H5AB 6204.36 10687.91 6725.87 H5AC 5470.32 11133.33 6096.06 H6AA 6453.01 10174.34 1579.06 H6AB 5927 9308.47 1268.13 H9A 4793.83 12876.04 -30.28 H10A 3387.17 13071.76 -620.45 H11A 2365.47 11965.06 -510.84 H12A 2732.04 10602.16 195.49 H16A 4923.69 7936.97 2316.47 H18A 8039.7 5442.39 2832.59 H19A 8662.74 6736.18 3439.76 H20A 7855.58 8030.05 3456.91 30 29 35 35 49 49 49 32 32 35 37 38 34 35 43 42 36 146 Citations CrysAlisPro (Rigaku, V1.171.42.64a, 2022) CrysAlisPro (ROD), Rigaku Oxford Diffraction, Poland (?). O.V. Dolomanov and L.J. Bourhis and R.J. Gildea and J.A.K. Howard and H. Puschmann, Olex2: A complete structure solution, refinement and analysis program, J. Appl. Cryst., (2009), 42, 339-341. Sheldrick, G.M., Crystal structure refinement with ShelXL, Acta Cryst., (2015), C71, 3-8. The Rigaku Synergy S Diffractometer was purchased with Support from the MRI program by the National Science Foundation under Grant No. 1919565 147 Chapter 3: Advances in proteasome enhancement by small molecules 3.1 Introduction The degradation of proteins is a continual process that is highly regulated by the two major proteolysis systems, the lysosomal degradation pathway, and the proteasome-mediated pathway. Protein degradation helps maintain biological homeostasis in cells which are needed for all cell functions and for maintaining optimal conditions for enzyme function1. The proteasome pathway is the major pathway for the degradation of misfolded, oxidatively damaged, and redundant proteins. Dysregulation of proteasome function has been identified in the pathogenesis of several neurodegenerative diseases including Parkinson’s disease (PD)2, Alzheimer’s disease (AD), and other neurodegenerative diseases3. The proteasome pathway is also involved in the regulation of several other cellular processes such as cell cycle, stress signaling, gene expression regulation, inflammatory response, cell differentiation, and apoptosis, which makes it an appealing target in the treatment of other types of diseases, including cancer4. Due to the critical role of the proteasome-mediated degradation pathway in cell regulation, the modulation of proteasome proteolytic activity has become a valuable strategy in the pursuit of new therapeutics to treat several neurodegenerative diseases5-8. 3.1.1 The Human Proteasome The human proteasome is a large complex protein responsible for the intracellular degradation of unwanted and damaged proteins via a ubiquitin-dependent and ubiquitin- independent degradation pathway. The most common proteolytic clearance of proteins proceeds by tagging the protein with polyubiquitin, after which it is degraded into small peptides of seven to eight amino acids by the 26S proteasome9. Highly disordered proteins can also be degraded in a ubiquitin-independent manner by the 20S proteasome10. In this chapter, I will cover the use of 148 small molecules to enhance the proteolytic activity of both the 26S proteasome and the 20S proteasome. 3.1.2 Ubiquitin-Proteasome System 3.1.2.1 Ubiquitin Ubiquitin (Ub) is a small protein (approximately 8600 Da) with 76 amino acid residues responsible for tagging a wide range of cellular proteins for proteolytic degradation. In the ubiquitin-proteasome system (UPS) (Figure 3.1), proteins are tagged for proteolysis by covalent ligation to ubiquitin11. Ubiquitination of proteins requires three enzymes in chronological order (Figure 3.1a). The E1 ubiquitin-activating enzyme, just like its name, activates the C-terminal glycine residue of the ubiquitin in an ATP-dependent manner. The binding of the ubiquitin to a cysteine residue of E1 forms a Ub-E1 complex via a thioester linkage. The E2 ubiquitin- conjugating enzymes transfer the ubiquitin from the Ub-E1 complex to itself via a trans- thioesterification to form the Ub-E2 complex and release the E1 enzyme from the system. Lastly, the ubiquitin ligases E3s are responsible for selecting proteins for ubiquitin-mediated proteolysis. Humans have two E1 enzymes, about 40 E2 enzymes, and are estimated to have about 500–1000 E3s12. 149 Figure 3.1: Ubiquitin-proteasome system13. (a) Protein polyubiquitination process using the ubiquitin-activating enzyme E1, conjugating enzymes E2 and the E3 ligase; (b) Polyubiquitinated proteins are degraded by 26S proteasome into small peptides following its deubiquitination After monoubiquitination of the targeted protein, the C-terminus of each ubiquitin molecule can be linked to any of the other seven lysine residues (K6, K11, K27, K29, K33, K48, and K63) on the previous ubiquitin to extend the ubiquitin chain and form the polyubiquitinated tagged protein14-15. However, the signal for protein degradation by the proteasome usually involves the linking of Ub to the K48 of the previous Ub on the protein16-17. In addition, K11, K29, and K63 linked chains have also been shown to play a role in proteasomal degradation17-18. The 26S proteasome degrades polyubiquitinated proteins (Figure 3.1b), and a previous study shows that proteins marked for degradation must be tagged with at least four ubiquitin molecules to be recognized by the 26S proteasome16,19. However, shorter chains, monoubiquitinated and multiple monoubiquitinated proteins can also be targeted for degradation by the proteasome20-23. It is also important to note that the ubiquitination process is reversible, and the deubiquitinating enzymes (see Section 3.3.1.1) are present in the cell to remove ubiquitin-tagged proteins24. 150 3.1.2.2 The 26S Proteasome The 26S proteasome has a molecular weight of approximately 2.5MDa and it is made up of the 20S core particle (CP), and one or two 19S regulatory particle(s) (RP) attached to one or both end(s) of the CP25. The 19S RP (also known as PA700) binds to the 20S CP and facilitates the gate opening of the CP for proteolytic degradation of polyubiquitinated proteins26. The 19S RP is also responsible for recognizing, unfolding, and translocating polyubiquitinated protein into the 20S CP27. Cryo-EM studies have shown many conformation states of the 26S proteasome when engaged with substrate28-35. Some of these studies showed the processes by which substrate is engaged, deubiquitylated, unfolded, and translocated by the proteasome28-29. The proteasome is also referred to as the 30S proteasome when the 20S CP is capped at both ends with the 19S RP36. However, in this chapter, I will refer to the 26S proteasome without distinguishing between the singly or doubly capped CP. 3.1.3 The 20S Proteasome or Core Particle The 20S proteasome is a 700 kDa protein with a cylindrical-like structure. The CP contains four heptameric rings stacked on each other in an α1-7β1-7β1-7α1-7 fashion. The outer α-rings form a gate, and they recognize regulatory particles that allow the opening and closing of the gate37. The inner β-rings contains six proteolytic sites, three on each β-ring (β1, β2, and β5), and are responsible for the proteolytic activity of the proteasome. The three different proteolytic sites of the 20S CP exhibit different substrate preferences even though they all use N-terminal nucleophilic threonine to carry out their proteolytic activities. The β1 exhibits a caspase-like (C-L)/PGPH (peptidylglutamyl-peptide hydrolyzing) activity and preferentially cleaves after acidic residues. The β2 and β5 exhibit trypsin-like (T-L) and chymotrypsin-like (CT-L) activities, and they preferentially cleave after basic and hydrophobic 151 residues, respectively38. The 20S proteasome on its own degrades unstructured proteins using a ubiquitin-independent pathway. 3.1.4 Small Molecule Regulation of Proteasome Function Due to the role of the proteasome in cellular functions, the regulation of proteasome has become a valuable target for the development of therapeutic molecules39. Proteasome inhibition is a therapeutic approach for the treatment of cancer. For example, bortezomib, a dipeptide boronate, was approved by the FDA in 2003 as an anticancer drug to treat mantle cell lymphoma and multiple myeloma40-41. Bortezomib inhibits the 26S proteasome by forming a covalent bond between its boron atom and threonine oxygen in the CT-L catalytic site of the 20S CP40. Molecules that inhibit the proteasome have also been shown to induce apoptosis in cell cultures and murine models of cancer. One of the proposed mechanisms is that proteasome inhibition prevents the degradation of the IκB, an NF-κB inhibitor, which prevents NF-κB nuclear translocation and consequently NF- κB mediated gene expression42. Proteasome inhibition results in the accumulation of IκB43-48, cyclin-dependent kinase (CDK) inhibitor p21 43,49-50, tumor suppressor p53, and other pro- apoptotic proteins51-53. The exceptional increase in apoptosis of certain multiple myeloma cells when treated with proteasome inhibitors has also been linked to an increase in protein unfolding and increasing substrate load on the proteasomes54-55. In addition, proteasome inhibition leads to lethal shortage of amino acids in the cells, which are the building blocks for cells to make new proteins. This amino acid scarcity caused by proteasome inhibition results in increasing ER stress and cell apoptosis56. Many reviews on proteasome inhibition have recently been published57-66, including a recent review by our group on natural products scaffolds as inhibitors of the proteasome67. 152 Proteasome activation by small molecules is a proposed strategy for the treatment of age- related diseases and several neurodegenerative diseases such as Parkinson’s disease (PD), Alzheimer’s Disease (AD), Huntington’s Disease (HD), and Amyotrophic Lateral Sclerosis8,68-72. Increasing the proteolytic activity of proteasome enhances the degradation of specific intrinsically disordered proteins (IDPs) such as α-synuclein, β-amyloid, and tau, to mention a few, which are associated with the pathogenesis of these neurodegenerative diseases. This review will focus on the use of small molecule enhancers of proteasome-mediated proteolysis as a potential strategy for the treatment of various neurodegenerative diseases. 3.2 Proteasome Activity and Diseases As humans age, there is a decline in proteasome function3,73. This reduction could be due to the reduction in the expression of proteasome subunits74, oxidative damage of the protein75-76, and disassembly of the 26S proteasome holocomplex77-78. The decrease in proteasome proteolytic function leads to lower rates of unwanted protein degradation which can induce toxic signaling upon accumulation and aggregation (Figure 3.2). In particular, the accumulation of specific intrinsically disordered proteins (IDPs), such as amyloid-β and α-synuclein, have been identified as a driving cause of many neurodegenerative diseases79-98. The exact mechanism by which these oligomers induce neurotoxicity is complex and still debated, but it is widely accepted that dysregulated IDPs accumulate, and the resulting soluble oligomeric forms of these protein aggregates are likely toxic species in disease pathogenesis79,93,99-102. These soluble oligomeric forms are also responsible for impairing proteasome function, which further drives disease progression 94,103-120. Multiple studies have indicated that enhancing proteasome proteolytic activity prevents the accumulation of these IDPs, reduces brain damage and improves cognitive performance in mouse models , and may be a new therapeutic strategy to treat neurodegenerative 153 diseases8,68-70,72,119,121-132. More recently, it has been recognized that the 20S proteasome of the proteasome plays a critical role in maintaining proteostasis by the direct degradation of oxidatively damaged and highly disordered proteins10,133-138. The 20S proteasome, therefore, serves as the default protease to unremittently maintain low levels of these unwanted IDPs without the need for post-translation modifications, including protein ubiquitination10,133. Highly disordered proteins appear to be the main target of the 20S proteasome139. IDPs are also naturally short-lived, but basal levels are secured by forming proteolytically stable structured complexes with “nannies”, chaperones, or other protein complexes140. However, when these IDPs production outpaces their degradation, they accumulate, oligomerize, and aggregate, resulting in the induction of downstream cytotoxic signaling events. 154 Figure 3.2: Accumulation of partially unfolded, misfolded, and dysregulated intrinsically disordered proteins (IDPs) such as amyloid-β and α-synuclein leads to neurotoxicity and neuronal cell death. The 20S proteasome degrades unwanted IDPs; however, small molecules can enhance the rate of proteasome-mediated degradation of these IDPs and prevent their accumulation 3.2.1 Aging During aging, proteins are more susceptible to several types of modification, such as oxidation, glycoxidation, glycation, conjugation with peroxidation products, etc. These protein modifications can lead to decreased enzyme activity and thermodynamic stability141-142, resulting in the accumulation of damaged proteins in the cell. Several models used to study proteasome proteolytic activity showed a decline in proteasomal activity as we age 143-144 and decreased degradation of oxidized proteins in cell cultures145-146. Reactive oxygen species accumulate during aging, resulting in an increase of oxidatively damaged proteins and an increased demand on the proteasome degradation system to eliminate these pathogenic aggregation-prone proteins113. 155 Unfortunately, proteasome proteolytic activity declines as we age 3,73,75, resulting in the accumulation of oxidatively damaged proteins147. These unwanted protein aggregates interact with the proteasome and further reduce its proteolytic capacity104-107,111,113. This inhibition of proteasome leads to a compounding accumulation of more unwanted proteins and a vicious cycle of progressively worsening aggregation of oxidatively damaged proteins 94,104-107,116-119,148-150. 3.2.2 Neurodegenerative Diseases 3.2.2.1 Parkinson’s Disease (PD) Approximately 10 million people worldwide are affected by PD, making it the second most prevalent neurodegenerative disorder151. PD is characterized pathologically by the loss of dopaminergic neurons as a result of the accumulation of Lewy bodies in the substantia nigra pars compacta (SNc)152. Lewy bodies are the defining pathological hallmark of PD, and its major components are α-synuclein, ubiquitin, parkin, proteasomal components, and other UPS-related proteins. PD has been linked to various UPS proteins such as parkin and UCH-L1. Additionally, the expression of mutant α-synuclein in rat cells inhibits proteasome proteolytic activity, causing essential features common to PD such as inclusion body formation, accumulation of undegraded ubiquitinated protein, and cell death. Dysregulation of proteasome-mediated protein degradation has been associated with both familial and sporadic PD153. Different approaches have been used to determine the role of the proteasome in the pathology of PD. Rat models have been developed to display characteristics such as bradykinesia, tremor, and abnormal posture, which are similar to PD when treated with proteasome regulators154. In addition, α-synuclein and ubiquitin-containing inclusion resembling Lewy bodies were also present at the neurodegenerative sites of the rat neurons. However, other studies could not 156 reproduce similar output155, which became controversial and indicated that proteasome inhibition is not a reliable model to study PD. As an alternative approach to study the development of PD, mouse models of proteasome subunits knock-out were generated. However, the removal of most proteasome genes causes embryonic lethality except for a few immune-related subunits109,156. The deletion of the proteasomal ATPase subunit Psmc1/Rpt2 in the dopaminergic neurons leads to intraneuronal α-synuclein and ubiquitin-positive inclusion, which resulted in neurodegeneration and thus resembling the human PD. This study provided direct support for the involvement of neuronal proteasome and Lewy-like inclusion seen in PD157. 3.2.2.2 Alzheimer’s Disease (AD) AD is the most common cause of dementia, and it is ranked as the sixth leading cause of death in the United State as of 2019158. AD is associated with loss of cognitive functioning such as memory, thinking, and reasoning. It also impacts behavioral activities such as the ability to carry out daily life activities159. The pathogenesis of AD has been attributed to protein misfolding and aggregation80-82,84,89. It is characterized by the aggregation of extracellular β-amyloid plaques and intracellular accumulation of neurofibrillary tangles160. The neurofibrillary tangles (NFTs) are mostly composed of hyperphosphorylated microtubule-associated tau. Filamentous tau formation is triggered due to changes in the concentration of β-amyloid160. Although β-amyloid appears to be more specific to AD, tau is also associated with other neurodegenerative diseases such as corticobasal degeneration, chronic traumatic encephalopathy, argyrophilic grain disease, and progressive supranuclear palsy161. Different experimental and clinical data have shown that the main drivers of synaptic dysfunction, cognitive decline, and neuronal loss in AD patients are associated with soluble toxic 157 β-amyloid oligomers which impair proteasome proteolytic activity, rather than insoluble β- amyloid plaques111,162-165. The ubiquitin-dependent proteasome system is associated with AD and degradation of β- amyloid120,166-168. Studies showed that the activity of the proteasome decreases in some parts of the brain in AD patients169. Similarly, inhibition of the 26S proteasome by lactacystin resulted in the accumulation of β-amyloid in both astrocytes and neurons, suggesting that β-amyloid could be a substrate for proteasomal degradation166. These results indicate that enhancing proteasome proteolytic activity may alleviate some of the factors that drive the pathogenesis of AD. 3.2.2.3 Huntington’s Disease (HD) HD is a brain disorder caused by the mutations of the huntingtin (Htt) gene. HD affects mood, movement and also leads to progressive cognitive deterioration and psychosis as a result of changes in the central part of the brain. The disease is dominant, which implies that it is inheritable by children from their parents. In HD the disorder of polyglutamine in the Htt protein results in toxic functions of mutant Htt, which consequently leads to neurodegeneration. The HD mutation is an unstable expansion of trinucleotide CAG repeats within the Htt gene, which causes polyglutamine stretch in the N- terminal of the protein and results in the formation of fibril and aggregates170-171. Remarkably, the mutant Htt still retains some of the functions of a normal Htt. The number of CAG repeats correlates to the progression of HD and the symptoms. Individuals with 36–40 CAG repeats may or may not develop HD symptoms. However, those with CAG repeats above 40 will eventually develop HD172-173. Greater than 50 long CAG repeats cause early onset of the disease (juvenile HD)174. Several studies suggest that mutant Htt aggregates impair the ubiquitin-proteasome system 104,116,175. However, the actual mechanism of interaction between the mutant Htt aggregate and the 158 proteasome remains unclear. Interestingly, unlike the soluble Htt, the Htt aggregates have been found to be ubiquitinated, and those insoluble aggregates have also been shown not to impair the activity of the 26S proteasome176-177. The inclusions associated with HD have been proposed to be toxic and lead to neuronal death. But the exact mechanism of toxicity remains unsolved. Increasing proteasome-mediated substrate degradation has been shown to increase survival in HD patients’ mutant huntingtin-expressing striatal and skin fibroblasts neurons. Over expression of PA28γ, a proteasome activator subunit also improved cell viability178. 3.2.2.4 Amyotrophic Lateral Sclerosis (ALS) ALS is another progressive neurodegenerative disease that affects the motoneurons in the brain and spinal cord. ALS is characterized by spasticity, muscle weakness, atrophy, and paralysis. The disease is often lethal within three to five years after diagnosis179-181. Like other neurodegenerative diseases, most ALS cases are sporadic (sALS), while about 10% could be familial (fALS) and result as a mutation in multiple genes179. Also, both sALS and fALS are clinically indistinguishable. Efforts at determining the mechanism underlying different fALS forms are thought to give insight into target identification and therapeutics development for both forms of diseases. The first ALS-linked genetic mutation was found in 1993, and it was located in the gene coding of Cu-Zn superoxide dismutase 1 (SOD1)182. Since then, several ALS mutant genes have been identified179,183-185. The recently discovered C9orf72 gene mutation has been identified as the most common cause of fALS and frontotemporal dementia186-187. An expansion of a GGGGCC repeat in the C9orf72 gene translates into five dipeptide-repeats proteins: poly-GA, poly-GP, poly- GR, poly-PR, and poly-PA188-192. A study showed that poly-GA aggregates recruit numerous 26S 159 proteasome complexes which may affect neuronal proteasome-mediated proteostasis and the protein degradation process193. Both the sALS and fALS are often considered proteinopathies since they both tend to aggregate and accumulate misfolded and abnormal proteins generated in the damaged neurons194- 195. The presence of ubiquitinated rich protein inclusions in motor neurons is a feature considered a common hallmark of not only human ALS but also in cellular and animal models of the disease196-197. The abundant accumulation of these ubiquitinated proteins suggests a significant contribution of the ubiquitin-proteasome system in these neuropathological features. In addition, the use of different cellular and animal models of ALS has provided substantial evidence of the involvement of the ubiquitin-proteasome system in the formation of inclusion and neuronal death198. 3.3 Small Molecule Enhancers of 26S Proteasome Activity The role of the proteasome in the regulation of cellular functions has made it an important target for the development of new treatments for cancer and neurodegenerative diseases. In addition, understanding proteasome regulation has allowed scientists to probe the mechanism of different cellular processes that involve the proteasome. Small molecules that directly activate the 26S proteasome are rare and most of the well- studied approaches to enhance the 26S proteasome involve indirect activation by modulation of post-translational modification and by genetic manipulation. A recent review highlights some of the cellular mechanisms that activate 26S proteasomes199. In this review, we will focus on small molecules that activate the 26S proteasome. 160 3.3.1 Indirect Activation of 26S Proteasome 3.3.1.1 Inhibition of Deubiquitinase Deubiquitinating enzymes (DUBs) play a critical role in the ubiquitin-proteasome system (UPS). The 19S RP of the proteasome uses deubiquitinase activity to remove and recycle polyubiquitin from protein substrates that are condemned for proteolysis200. There are three essential DUBs: RPN11, UCH37, and USP14 that are associated with the 19S RP of human proteasome 124,201-202. The main function of these DUBs is to remove monoubiquitin and polyubiquitin chains from substrates tagged for proteasomal degradation203-206. USP14, a DUB of the cysteine protease class, interacts reversibly with the proteasome124,207-209 and it cleaves the ubiquitin chain off the targeted protein before degradation by the proteasome210-211, thereby inhibiting the degradation of ubiquitin-protein conjugates in vitro and in vivo124. Unlike USP14, the RPN11, a DUB of the metalloprotease class, is part of the 19S RP and cleaves the ubiquitin chain after degradation has been initiated by the proteasome201. The mechanism of action of UCH37 is not completely understood yet but this DUB could edit the ubiquitin chains and either prevent the protein from being degraded or enhance its degradation depending on the proteasome needs 201,212-213. A study conducted in 2010 showed that USP14 inhibits protein degradation by the proteasome in murine embryonic fibroblasts. In the same study, the authors showed that inhibition of USP14 by 1-[1-(4-fluorophenyl)-2,5-dimethylpyrrol-3-yl]-2-pyrrolidin-1-ylethanone (IU1, Figure 3.3, compound 3-1) drastically stimulate the degradation of oxidized proteins by the proteasome124. IU1 was identified as a USP14 inhibitor from high-throughput screening (HTS) of over sixty-three thousand compounds for their ability to inhibit USP14. From the HTS, 215 compounds were identified as true inhibitors of USP14, however, screening of the hit compounds 161 against several DUBs only provided three compounds as selective inhibitors of USP14. IU1 was found to be the most active of the three with IC50 of 4–5 µM124. Further optimization of IU1 has led to the discovery of more potent analogues such as IU1-47 (IC50 of 0.6 µM) (Figure 3.3, compound 3-2)214, IU1-248 (IC50 of 0.83 µM) (Figure 3.3, compound 3-3)215, and 1B10 and 1D18 (Figure 3.3, compound 3-4 & 3-5 respectively) which have better membrane permeability216. A recent review by Moon et al.211, is focused on small molecules that inhibit proteasome-associated deubiquitinase and can be consulted for more information on DUB inhibitors. Figure 3.3: Small molecules inhibitors of deubiquitinase enzymes (DUBs) O N O N OH O N F N Cl N NC N IU1 3-1 IU1-47 3-2 IU1-248 3-3 O N O N F N F N Cl Cl 1B10 3-4 1D18 3-5 In a recent study by Kim et al.217, proteasome-mediated proteolysis was increased by knocking down USP14 with small interfering RNA (siRNA) which led to a significant impairment of autophagic flux. This proteasome activation led to an increase in the microtubule-associated protein tau (MAPT) degradation and a decrease in the concentration of its oligomeric forms. This result is also consistent with Boselli et al.’s observation that USP14 inhibition enhances tau degradation in cultured neurons214. 162 3.3.1.2 Modulation of cAMP-Dependent Protein Kinase A (PKA) and cGMP-Dependent Protein Kinase G Phosphorylation of proteasome subunits was recently established as a promising way to proteasome regulation218. The phosphorylation of Ser-14 of Rpn6, a subunit of 19S regulatory particle, by cAMP-dependent PKA has been shown to enhance the hydrolysis of polyubiquitinated proteins and small peptides in cells and in vivo studies219-222. In addition, impeding the phosphorylation of Thr-25 of Rpt3 by dual-specificity tyrosine-regulated kinase 2 (DYRK2)223-224 and Ser-120 of Rpt6 by calcium/calmodulin-dependent protein kinase II (CaMKII)225-226 or PKA119 have been shown to impair proteasome proteolytic capacity and impedes cell proliferation. Small molecules that raise cAMP have therapeutical promise because they enhance the capacity of cell cultures221 and mouse brains119,227 to degrade misfolded proteins such as tau, which has been implicated in the pathogenesis of Alzheimer’s disease. Small molecule inhibitors of phosphodiesterase have been found to increase proteasome function by cAMP/PKA-mediated phosphorylation. Rolipram (Figure 3.4, compound 3-6) is an example of phosphodiesterase type-4 inhibitor (PDE4) that was developed as an antidepressant drug in the early 1990s228. A study shows that Rolipram decreases the level of insoluble tau and improves cognitive performance in mice by increasing proteasome function through activating cAMP-PKA signaling119. Cilostazol (Figure 3.4, compound 3-7) is another phosphodiesterase type-3 inhibitor (PDE3). Administration of Cilostazol in rTg4510 mice also showed improved cognitive performance and increased proteasome function through the cAMP/PKA pathway227. This small molecule was approved by the FDA to treat intermittent claudication and can also be used for secondary stroke prevention229. In late 2020, the FDA completed the clinical trial to determine the therapeutical use of cilostazol for patients with mild cognitive impairment230. 163 O O H N HN O Rolipram 3-6 O O O O N H N Figure 3.4: Rolipram (3-6) and Cilostazol (3-7) are examples of phosphodiesterase type-4 (PDE4) inhibitors. Molecules that inhibit PDE4 raise cAMP/PKA-mediated phosphorylation which increases the rate of degradation of IDPs and misfolded proteins in cellular assays and animal models. Sildenafil (3-8), Tadalafil (3-9), BAY41-2272 (3-10), and Cinaciguat (3-11) are molecules that raise cGMP level and induce cGMP-mediated proteasome activation N N N N O Cilostazol 3-7 N H N O N O S O N N O N HN O Sildenafil 3-8 F NN N N N NH2 Tadalafil 3-9 BAY41-2272 3-10 O O OH HO N O Cinaciguat 3-11 Like cAMP-mediated modulation of 26S proteasome, small molecules that raise cGMP and activate PKG were recently shown to enhance proteasome proteolytic activity without affecting lysosomal degradation and increase the rate of degradation of both short-lived and long- lived proteins, including tau and mutant Htt.199,231. In the study conducted by VerPlank et al.231, treatment of human neuroblast cells (SH-SY5Y) with molecules that raises cGMP such as sildenafil (Figure 3.4, compound 3-8) or tadalafil (Figure 3.4, compound 3-9) which are phosphodiesterase type-5 inhibitors (PDE5), or BAY41-2272 (Figure 3.4, compound 3-10) and cinaciguat, (Figure 3.4, compound 3-11) which are stimulators of soluble guanylyl cyclases, led to a rapid increase in proteasomal activity in cell lysates. However, unlike phosphorylation of Rpn6 by PKA221-222, Rpt3 by DYRK2223-224, or Rpt6 by CaMKII)225-226 or PKA119, phosphorylation of Rpn6, Rpt3, or Rpt6 subunit was not observed in the PKG pathway231. Overexpression of PKG in SH-SY5Y and HEK293 cells led to an increase in the level of phosphorylated proteins compared 164 to cells that were transfected with empty vectors during proteasome preparations. Thus, the 26S proteasome subunit or an associated protein that is phosphorylated in the cGMP-mediated proteasome activation is still unknown and the mechanism of action remains unclear. 3.3.1.3 Inhibition of p38 Mitogen-Activated Protein Kinase (MAPK) MAPKs are enzymes that phosphorylate the hydroxyl group of threonine and serine residues in proteins. These kinases play an important role in the control of cell proliferation and apoptosis. The p38 MAPK is involved in a signaling pathway that regulates various biological functions including biosynthesis of cytokinesis such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α)232-233. The activation of the p38 MAPK pathway as a defense to osmotic stress has been shown to lead to phosphorylation of 19S RP at Thr-273 of the Rpn2 subunit, which resulted in the inhibition of the 26S proteasome proteolytic activity234. In ALS and AD, the over-activation of the p38 MAPK pathway has been reported in animal models and postmortem brains of AD patients196,235-237. The activation of the p38 MAPK pathway in cell lines and animal models has led to tau phosphorylation, neuroinflammation, neurotoxicity, and synaptic dysfunction, which are events associated with Alzheimer’s disease. Therefore, the search for p38 MAPK inhibitors became a novel approach for targeting neurodegenerative diseases238. In 2017, Leestemaker et al.122 discovered imidazole inhibitors of p38 MAPK as enhancers of 26S proteasome proteolytic activity. The compounds were identified from high-throughput screening of over 2750 compounds using a proteasome activity-based probe (Me4BodipyFLAhx3L3VS) that covalently binds to proteasome catalytic sites in an activity- dependent manner in living cells. The group found that PD169316 (Figure 3.5, compound 3-12), a known inhibitor of p38 MAPK and its structural analogues, SB202190 (Figure 3.5, compound 165 3-13), and SB203580 (Figure 3.5, compound 3-14), increases the proteolytic activity of the proteasome in a dose-dependent manner in MelJuSo cells. Further characterization of these compounds showed that they increase proteasome proteolytic activity by inhibiting the p38 MAPK pathway without affecting cell viability, subunits abundance, and the overall level of ubiquitinated proteins122. Similarly, Huang et al.239, demonstrated that treatment of HAP 40 depleted cells with p38 MAPK inhibitor, PD169316, increases the CT-L activity of the proteasome and enhances degradation of both soluble and aggregated forms of mutant Htt in a Huntington’s disease model. These data suggest that the regulation of the p38 MAPK pathway could be a potential way of modulating proteasome-mediated proteolytic activity. Figure 3.5: Imidazole inhibitors of p38 MAPK that enhances proteasome activities F F F N NH NO2 N NH OH O S N NH N PD169316 3-12 N SB202190 3-13 N SB203580 3-14 3.3.1.4 Proteasome Activation by Genetic Manipulation Another approach to enhancing proteasome proteolytic activity is by proteasome subunit overexpression. Overexpression of β5i subunit in HeLa cells and lymphoblasts has led to an increase in the CT-L and T-L activities of the proteasome240-241. Previous studies also showed that stable overexpression of the β5 subunit in human fibroblast cell lines increased the level of other β subunits, increasing the overall proteolytic activity of the three catalytic sites242. Furthermore, overexpression of the 19S RP subunit PSMD11/Rpn6 increases proteasome assembly and proteolytic activity in human embryonic stem cells243. Small molecule activation of the transcription factor NRF2, nuclear factor erythroid 2- related factor 2, enhances the expression of the 20S and 19S proteasome particles and increases 166 the proteolytic activity of the proteasome in cells containing NRF2. 18α-glycyrrhetinic acid (18α- GA) has been shown to increase proteasome proteolytic activities from 1.5- to 1.8-fold, with the activity of the caspase-like site being the most affected in wide-type HFL-1 human fibroblasts. In addition, the increase in the proteasome proteolytic activity was not observed when NRF2 was knockdown using siRNA in HFL-1 cells and the cells were treated with 18α-GA, further confirming the upregulation of proteasome proteolytic activity through NRF2 activation244. The activation of NRF2 increases the level of 20S proteasome subunits: α4, β1, β2, and β5 in both human fibroblasts244 and mice liver245, and the 19S subunits: Rpt2, Rpt5, and Rpn11 in mice liver245. The expression of antioxidant enzymes such as UDP-glucuronosyltransferase (UGT)246, glutathione S-transferase (GST)247, and NAD(P)H quinone oxidoreductase 1248, to name a few, are also controlled by this transcription factor. Activation of NRF2 by tert-Butylhydroquinone (t- BHQ) and sulforaphane increases proteasome proteolytic activity in human embryonic stem cells (hESCs)249 and also protects against oxidative stress250. 3.4 Small Molecule Enhancers of 20S Proteasome Activity 3.4.1 Sodium Dodecyl Sulfate (SDS) SDS (sodium dodecyl sulfate), also known as SLS (sodium lauryl sulfate), is a synthetic organosulfate salt used in cleaning, pharmaceutical, and food products. In 1988, Tanaka et al.251 showed that 20S proteasome proteolytic activity could be enhanced at a low concentration of SDS (0.04–0.08%) in biochemical assays. However, at higher SDS concentrations, the activity of the proteasome is lost, and the SDS inhibits the proteasome252. SDS is an invaluable in vitro tool that is used by most researchers to activate the proteasome as means to test compounds for subsequent proteasome inhibition. It is believed that SDS induces gate opening of the proteasome by partial denaturation of the 20S to facilitate substrate entrance into the catalytic core. However, the actual 167 mechanism of SDS proteasome activation is still unclear and considering that SDS is a detergent, it should not really be considered as a small molecule activator of the 20S proteasome. 3.4.2 Natural Product-Based Activators Several natural products have been identified as 20S proteasome activators, some of which include betulinic acid (Figure 3.6, compound 3-15)253, ursolic acid (Figure 3.6, compound 3- 16)127, and oleuropein (Figure 3.6, compound 3-17)254. Betulinic acid is a triterpene isolated from the bark of Betula pubescens (commonly known as white birch). It was reported as a selective inhibitor of human melanoma and it has been demonstrated to induce programmed cell death in human neuroblastoma and neuroectodermal tumor cells255. Betulinic acid is one of the first reported enhancers of the 20S proteasome. A small peptide assay using Suc-Leu-Leu-Val-Tyr- AMC (used to determine the CT-L activity of the proteasome) showed that’s betulinic acid enhances the CT-L activity of the proteasome with EC50 of approximately 2.5 μg/mL. Unfortunately, several chemical modifications to enhance the activity of betulinic acid resulted in compounds that inhibit the proteasome253. Like betulinic acid, ursolic acid is another triterpenoid that enhances the activity of the 20S proteasome. Ursolic acid is similar in structurally to betulinic acid, and they both enhance the CT-L activity of the 20S proteasome127,253. Although both betulinic acid and ursolic acid showed good activity in small peptide assay, unfortunately, betulinic acid did not show any activity for the turnover of misfolded proteins in vitro and in vivo 72. 168 Figure 3.6: Natural product-based activators of 20S proteasome H H OH O H OH O H HO HO H H Betulinic acid 3-15 Ursolic acid 3-16 HO HO O O O H O O O OH OH HO O HO Oleuropein 3-17 In addition, other natural compounds have been identified as 20S proteasome agonists. Some of these compounds include lipids256 and fatty acids257. In 1993, Ruiz de Mena et al.256 studied the effect of phospholipids on the T-L, CT-L, and C-L activities of the proteasome in rat liver. In the study, the team identified cardiolipin (diphosphatidylglycerol) as a strong CT-L enhancer (up to 60-fold enhancement) and C-L enhancer (up to 30-fold enhancement). SDS and cardiolipin activation was shown to be additive and at either optimal or suboptimal concentrations of both compounds. Furthermore, fatty acids such as oleic, linoleic, and linolenic acids isolated from spinach leaves were found to increase proteasome-mediated substrate degradation by enhancing CT-L and C-L activities at about one-third to one-sixth the required concentration of SDS. Unlike SDS, at extremely low concentration (0.0007–0.0025%, ∼25–90 µM), the T-L catalytic site is inhibited and the degradation of Boc-L-R-R-AMC is prevented257. 3.4.3 AM-404 and MK-886 Although many compounds show increased peptide cleavage activities using the standard aminomethyl coumarin tagged small peptide substrates, most have failed to demonstrate an increase in proteolytic activity under physiological conditions. One likely explanation is that the small peptide probes, used for detection of in vitro proteasome proteolytic activity, may be small enough to inadvertently enter the CP-proteolytic cavity following minor conformational changes 169 to the gate. Trader and Kodadek developed a follow-up assay that uses larger peptides with a single cleavage site and uses LC-MS to monitor proteasome proteolytic activity over time. They also validated molecules from LC-MS assay for their ability to turnover of α-synuclein in cells by monitoring the appearance of free GFP which correlates to the number of α-synuclein that was degraded. Using these assays, the lab was able to identify small molecules capable of increasing 20S mediated proteolytic activity 72. The authors screened 726 compounds in the NIH Clinical Collection and identified AM-404 (Figure 3.7, compound 3-18) and MK-886 (Figure 3.7, compound 3-19) as “true” proteasome enhancers. The study showed both compounds increase the proteolytic activity of the 20S proteasome by 3- to 4-folds with an EC50 of 32 µM, and they also enhance the degradation of α-synuclein in cell culture72. Figure 3.7: Structure of AM-404 and MK-886 OH HN O AM-404 3-18 Cl O O- N S MK-886 3-19 Recently, the Trader’s lab investigated the structural component of AM-404 needed to enhance the proteasome proteolytic activity. In the study, they synthesized various derivatives of AM-404 by varying the aliphatic chain length, degree of unsaturation, and substitutions. They illustrated the importance of the aliphatic chain length and the cis-alkene at C8 of the aliphatic chain in stimulating the 20S proteasome126. 170 3.4.4 Imidazolines Imidazolines are an important class of compounds that are found in various natural and synthetic bioactive molecules258-259. This class of compounds displays a wide range of biological activities including proteasome and NF-κB modulation260-262, and therapeutic significance such as antifungi263, antitumor264, antihelminthics265, antihyperglycemic266, and antihypertensive activity267. Our lab reported the imidazoline, TCH-165 (Figure 3.8, compound 3-20), as a 20S proteasome enhancer as a low (1.5 μM) activator of the 20S proteasome 70. TCH-165 enhanced 20S mediated degradation of IDPs such as α-synuclein, tau, ornithine decarboxylase, and c-Fos in cell cultures. However, it does not affect the degradation of structured proteins such as GAPDH. Treatment of HEK293T cells with TCH-165 showed a time-dependent disassembling of both the singly and doubly capped 26S proteasome and showed an increase in the free 20S CP. TCH-165 prevents the binding of the 19S RP to the 20S proteasome suggesting that the molecule binds directly on the α-ring of the 20S CP and shifts the equilibrium between 26S and 20S proteasomes towards an activated 20S CP. To gain insight into the mechanism of 20S proteasome activation, atomic force microscopy (AFM) imaging revealed that the ratio of open to closed 20S proteasome increases in a dose-dependent manner when treated with TCH-165 at concentrations as low as 200nM70. This further supports that TCH-165 induces the open gate conformation of the 20S CP. It is also important to note that this is the only small molecule with biophysical data (atomic force microscopy (AFM) imaging) that support gate opening of the 20S proteasome70. 171 Figure 3.8: Imidazoline TCH-165 enhances proteasome activities and degrades intrinsically disordered proteins HN O O N N TCH-165 3-20 O 3.4.5 Chlorpromazines During the search for proteasome activators, our lab screened the NIH Clinical Collection and Prestwick libraries, where we identified chlorpromazine (Figure 3.9, compound 3-21) and related phenothiazines as 20S proteasome activators inducing up to 20-fold activity69. Chlorpromazine is an FDA-approved drug that is used in the treatment of schizophrenia or manic- depression in adults. Chlorpromazine is believed to be a dopamine antagonist with some antiserotonergic and antihistaminergic properties268. Figure 3.9: Structure of chlorpromazine and a chlorpromazine analogue 8 S N Cl N Chlorpromazine 3-21 S N Cl CO2CH3 Analogue 8 3-22 Chlorpromazine and related phenothiazines preferentially enhance the CT-L activity of the proteasome and promote degradation of IDPs, such as α-synuclein and tau but not structured proteins in in vitro assays. Chemical modification of chlorpromazine abrogated its dopamine D2R receptor activity while preserving its ability to enhance the 20S proteolytic activity. Analogue 8 172 (Figure 3.9, compound 3-22), an analogue of chlorpromazine with physiological insignificant potency for dopamine receptor (Ki ≥ 250 µM) showed better efficacy with about 10-fold maximum enhancement and EC200 (concentration where the 20S mediated proteolysis is increased by 2-fold or 200%) of 13.5 µM. Interestingly, a structural analogue of chlorpromazine, methylene blue, was also found to enhance the CT-L and T-L activity of the 20S proteasome. Methylene blue was also found to decrease the level of β-amyloid and increase learning and memory in 3xTg-AD mouse model but does not affect tau phosphorylation in vivo269. A recent study also showed that methylene blue inhibits caspase-6-induced neurodegeneration, decreases neuroinflammation, and prevents cognitive impairment in mice270. 3.4.6 Dihydroquinazolines The 3,4-dihroquinazoline compounds are found in several natural products and synthetic compounds with various biological properties. Members of this class of compounds have biological properties that includes antifungal271, antiparasitic272, antitumor273-279, and antiviral activities280-281. In 2021, Mosey et al. synthesized and evaluated several dihydroquinoline analogues as 20S proteasome enhancers125,282. In this study, they were able to identify several promising 20S activators with the most potent being dihydroquinazoline 18 (Figure 3.10, compound 3-23), doubling proteasome proteolytic activity at 1.3 µM (EC200 1.3 μM). The dihydroquinazolines enhance the three catalytic sites activity of the 20S proteasome and increase the degradation of α- synuclein, the IDP identified in the pathogenesis of Parkinson’s disease. 173 Figure 3.10: Structure of dihydroquinoline scaffold and dihydroquinoline 18 (compound 3-23) R4 N N R3 R2 R1 Ph N N O N Ph Dihydroquinazoline 18 3-23 3.4.7 Fluspirilene and Acylfluspirilene In 2021, the Tepe group identified fluspirilene (Figure 3.11, compound 3-24) and its synthetic analogues which were capable of enhancing 20S proteasome proteolytic activity and even restoring the proteolytic activity of 20S proteasome impaired by IDP oligomers132. Fluspirilene and its amide derivative, acylfluspirilene (Figure 3.11, compound 3-25) activate the three catalytic sites of 20S CP and prevent IDP aggregation and oligomerization. Interestingly, acylfluspirilene exhibits more potency (EC200 1.9 µM) compared to fluspirilene and a better maximum fold enhancement of greater than 20-fold. Furthermore, molecular docking shows that fluspirilene and acylfluspirilene bind to the α2-3 intersubunit pocket of the 20S CP, which is different from the previously reported 20S enhancers, TCH-165, dihydroquinoline, and chlorpromazine, which bind in the α1-2 pocket of the proteasome. In silico and in vitro structure- activity relationship (SAR) studies indicated the importance of the in-pocket binding interactions of these molecules with the 20S proteasome. This group of molecules does not enhance the proteolytic activity of the 26S proteasome and may therefore be used to selectively prevent the accumulation of dysregulated intrinsically disordered proteins without affecting regular ubiquitin- dependent protein degradation 132. 174 Figure 3.11: Structure of Fluspirilene and Acylfluspirilene F F O HN N N F Fluspirilene 3-24 O HN N N O Acylfluspirilene 3-25 F 3.4.8 Pyrazolones Pyrazolones are a rare class of compounds that enhance proteasome activation. This class of molecule was first discovered as proteasome activator in 2014 by the Silverman group and as potential compounds for the treatment of ALS71. The pyrazolones (Figure 3.12, compound 3-26– 3-28) were shown to protect neurons in PC12-SOD1G93A cells in cellular models of ALS. The compounds also increased ALS transgenic mouse survival by 13%, further confirming their potential in the development of ALS therapeutics. During the mechanistic investigation of CMB- 087229 (Figure 12, compound 3-27), the compound was found to antagonize G protein-coupled receptor metabotropic glutamate receptor 5 (mGluR5), a previously identified target in ALS therapeutic283, to about 65% at 10 µM concentration. The group investigated if mGluR5 was the target of the pyrazolones by screening known mGluR5 antagonists in their cell-based assay. However, the screened mGluR5 receptor antagonists (including MPEP and fenobam) showed no activity in the assay. Based on the result, it was concluded that the antagonism of the mGluR5 is unlikely to be the mode of action of the pyrazolones. Pull down experiments indicated several 26S proteasome regulatory subunits as a possible target for the pyrazolones. The pyrazolones were able to reverse bortezomib-induced cytotoxicity in the PC12 cells, further supporting evidence that their mechanism of action involved proteasome activation284. 175 Figure 3.12: Structure of pyrazolones that have been shown to increase proteasome activities O NH N H S Cl CMB-3299 3-26 Cl O Cl CMB-087229 3-27 O NH N H O NH N H S Cl Cl Pyrazolone 3 3-28 N N O N Aminophenazone (Aminopyrine) 3-29 O N H N N O Nifenazone 3-30 N Following up on Silverman’s discovery, Santoro et al.285 screened a small library of structurally-related pyrazolones for proteasome enhancement and neuroprotection against amyloid-induced toxicity in neuroblastoma SH-SY5Y cells. The group reported that the aminopyrine analogue (Figure 12, compound 3-29) and nifenazone (Figure 12, compound 3-30) displayed up to twofold induction of 26S proteasome proteolytic activity in cells. Using docking studies coupled with Saturation Transfer Difference (STD) NMR experiments, the group proposed that aminopyrine enhances the 20S proteasome by a mechanism involving binding to the α-ring surfaces of the proteasome; however, only a marginal increase in activity was observed (<30% increase at 10 μM) in a purified proteasome assay. 3.5 Conclusions Efficient proteasome function is critical in maintaining healthy cellular homeostasis. Dysregulation of protein or proteasome impairment can result in a toxic accumulation of unwanted proteins, which is observed in the pathogenesis of different neurodegenerative diseases and aging. Enhancing the proteolytic activity of the proteasome by increasing its capacity, accessibility, or the rate at which it degrades has long been hypothesized as a means to prevent the accumulation 176 of dysregulated IDPs. More recently, researchers from various labs have explored the use of small molecules to induce protein proteolysis. Small molecule proteasome agonists can enhance the proteolytic clearance of unwanted proteins and restore homeostasis. Small molecule enhancers of the 26S proteasome are described herein which mainly induce enhanced 26S-mediated proteolysis of ubiquitinated proteins via an indirect mechanism of proteasome activation. Small molecule inhibitors of deubiquitinases prevent proteins marked for ubiquitin- dependent degradation fromescaping their fate. Even though there are no approved therapies yet based on deubiquitinating enzyme (DUB) inhibitors, this is an emerging field with great significance. Small molecule regulation of upstream signaling pathways, including cAMP- depending protein kinase A and c-GMP-dependent protein kinase G, affect the phosphorylation of the proteasome regulatory particles. As a result, small molecule regulators of phosphodiesterase type-3 (PDE3) can therefore indirectly increase the rate of substrate degradation by the proteasome. Small molecules that directly interact with the 26S proteasome and enhance the rate of 26S proteasome-mediated protein degradation are less known and likely a fruitful field for exploration. Whereas the 26S proteasome targets ubiquitinylated protein substrates, the 20S proteasome is limited to the degradation of only disordered proteins. Several small molecule enhancers of 20S proteasome-mediated protein degradation have been identified in the literature. We summarized herein several different classes of small molecule 20S proteasome enhancers that induce 20S— mediated degradation of dysregulated intrinsically disordered proteins by direct interaction with the 20S core particle. The activation of the proteasome by small molecules is a relatively new field in science. Its potential as a therapeutic approach is still unknown and the consequences of chronic exposure 177 to proteasome enhancers are not known. However, considering the possibility of treating multiple disorders for which there are currently no treatment options available, this approach has enormous potential. However, as in all new fields, the approach still needs further validation, in vivo studies in particular, to fully understand its therapeutic potential and limitation. In addition, more studies are needed to elucidate the mechanistic details of small molecule proteasome activation and its overall cellular consequences. 178 REFERENCES Hetz, C.; Glimcher, L. H., Curr. 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M.; Lanza, V.; Bellia, F.; Sbardella, D.; Tundo, G. R.; Cannizzo, A.; Grasso, G.; Arizzi, M.; Nicoletti, V. G.; Alcaro, S.; et al., ChemMedChem 2020, 15, 302-316. 194 Chapter 4: Synthesis of pyrazolo[1,5-b]pyridazines analogs as potential 20S proteasome activator 4.1 Introduction Protein structures span a broad spectrum, from those with well-defined shapes to proteins that lack a define tertiary structure. Proteins with no tertiary structure are often referred to as intrinsically disordered proteins (IDPs).1 Intrinsically disordered proteins (IDPs) serve a range of critical functions in biological systems and their flexibility and adaptability enable them to participate in diverse cellular processes. IDPs play key roles in signal transduction, gene expression, scaffolding large protein complexes, and driving cell cycle progression.2-3 Although, IDPs are essential in several cellular process, their accumulation and aggregation have been shown to induce toxic signaling.4-5 Accumulation of IDPs such as α-synuclein, amyloid-β and tau, are classic hallmarks of Parkinson’s and Alzheimer’s disease respectively.6 The build-up of these proteins has been shown to disrupt cellular proteostasis, ultimately leading to neuronal death and the further progression of the disease.7-8 Small molecule activation of the proteasome has been proposed as a potential strategy to treat neurodegenerative diseases by targeted degradation of these IDPs.9-14 In Chapter 3, I discussed the structure of the proteasome and examine the link between its proteolytic activity, aging, and several neurodegenerative conditions. Additionally, I provided an overview of the different classes of compounds that are capable of enhancing the proteasome-mediated protein degradation, either directly or indirectly.15 The Tepe lab and other labs have identified various molecules that can enhance the proteolytic activity of the 20S proteasome.9-14 Some of the identified activators from the Tepe’s lab are shown in Figure 4.1. It is hypothesized that these small molecule activators binds to the 𝛼- 195 ring of the 20S proteasome to induce a ring-opening conformation and enhance the rate of proteolytic degradation of intrinsically disordered protein (IDPs) by increasing access to the catalytic core of the proteasome. Figure 4.1: Structure of some known 20S proteasome activators from the Tepe’s lab F NHBn S N N O Cl F O H N N N O Chlorpromazine analog 19 F Acyl fluspirilene F O CO2Et BnN N TCH-165 N N F H N N N O N O N O Dihydroquinazoline 18 Acyl astemizole While some small molecule proteasome activators have been discovered, many exhibit only moderate potency, typically in the micromolar range, therefore limiting the clinical potential of these molecules. To thoroughly assess the feasibility of targeting the proteasome in vivo and to explore the clinical potential of this therapeutic strategy, more potent 20S proteasome enhancers are needed. These would enable a deeper investigation into the utility of proteasome activation in treating various neurodegenerative diseases. Lipinski's Rule of Five is a set of guidelines derived from the observation that most orally available small-molecule drugs exhibit common chemical and physical traits. According to Lipinski, a typical small-molecule drug has a molecular weight (MW) under 500 g/mol, an octanol- water partition coefficient (cLog P) not exceeding 5, and a maximum of 5 hydrogen bond donors and 10 hydrogen bond acceptors.16 These characteristics are associated with small molecules that can cross biological membranes to interact with target receptors. In the search for more potent 20S 196 proteasome enhancers that also adhere to Lipinski’s rule of five, the Tepe lab conducted a cell- based high throughput screen of the Prestwick and NIH Clinical library.17 From the 25,000 compounds screened in this library, compound 4-1 (also called MSU-7) was identified as a 20S proteasome enhancer (Figure 4.2). Figure 4.2: Structure of compound 4-1 (MSU-7) N N N N N N H Cl Compound 4-1 and its analogs has been previously identified as human kinase inhibitor, specifically GSK-3β, CDK-2, and CDK-4 with the potential of using this class of compounds for the treatment of solid tumors.18 Several compounds of this class has also been studied with the goal of repurposing them for the treatment of human African trypanosomiasis.19 We embark on the study of pyrazolo[1,5-b]pyridazine scaffolds with the goal of evaluating them as 20S proteasome enhancers and also discovering a more potent analog of this class of compound. 4.2 Results and Discussion We started by first synthesizing compound 4-1 to verify its 20S proteasome activity. Using a previously establish approach19-20, 4-1 was synthesized in 5 steps starting from dichloropyrimidine 4-2 (Scheme 4.1). Sonogashira coupling of 4-2 with trimethylsilylacetylene yielded the desired TMS-protected alkyne 4-3 which then underwent a TMS deprotection with potassium hydroxide to give the free alkyne 4-4 in good yield. Pyridazine 4-5 was also reacted with hydroxylamine-O-sulfonic acid (HOSA) to generate 1-aminopyridazin-1-ium iodide 4-6 in 87% yield. A [3+2] cycloaddition reaction between 4-6 and alkyne 4-3 provided the pyrazolo[1,5- b]pyridazines scaffold 4-7 as the final intermediate to the desired analog. Nucleophilic aromatic substitution with 4-chlorobenzylamine gave compound 4-1 (MSU-7) in 82% yield. 197 Scheme 4.1: Synthesis of compound 4-1 (MSU-7) TMS H Cl N 4-2 N + TMS Pd(PPh3)2Cl2 (1 mol%) CuI (2 mol%) KOH (0.1M in MeOH, 1 mol%) THF/TEA (5:1), 55℃, 3 h N MeOH, rt, 15 mins Cl 1.10 equiv 88% Cl N 4-3 88% N Cl N 4-4 O S O O HO NH2 N N (1.5 equiv) I KHCO3 (2.4 M) NH2 N N H2O, 70℃, 2 h then, KI (1.0 equiv) 87% 4-6 4-5 N Cl N (1.0 equiv) KOH (2.5 equiv) H2O/DCM (1:2), rt, 16 h 47% N NN N N 4-7 Cl N NN H2N (3.0 equiv) n-BuOH, 110 ℃, 48 h Cl 82% N N N H 4-1 (MSU-7) Cl After the synthesis of 4-1 was achieved, its 20S proteasomal activity was validated by Sophia Staerz and Shannon Cartwright using western blot to measure the impact of 4-1 on purified IDP degradation by the 20S proteasome, in this case, 𝛼-synuclein degradation (Figure 4.3a). The cytotoxicity of compound 4-1 in H929 cells, a multiple myeloma cell line was also performed by Shannon Cartwright and the CC50 of compound 4-1 was determined to be 2.4 µM (Figure 4.3b). CC50 is the concentration at which 50% of the cancer cells were dead. From these preliminary data, I started the synthesis of different fragments of compound 4-1 to identify the fragment(s) that is important for the compound to maintain its 20S proteasomal activities. 198 Figure 4.3: (a) In vitro 𝛼-synuclein degradation with 20S proteasome using TCH as control N=3. (b) Cell viability assay of compound 4-1 in H929 cells. N=3 (a) (b) i g n n i a m e r n i e l c u n y s - 𝛼 % 100 75 50 25 0 y t i l i b a i V % -6.0 -5.5 -5.0 -4.5 -4.0 Log(M) 4.2.1 Investigation of different fragments of compound 4-1 for proteasome activity 4.2.1.1 Synthesis of fragments of compound 4-1 (MSU-7) Following the synthesis and validation of 4-1 as 20S proteasome enhancer, I embarked on the synthesis of fragments of 4-1 with the goal of identifying the fragment needed to maintain proteolytic activity or if the entire structure of 4-1 is needed for activity. Compound 4-7 appears to be a good fragment that can be used to explore the effect of the 4-chlorobenzylamine on the activity of the hit compound. In addition to intermediate 4-7, compounds 4-8 – 4-10 were also synthesized to investigate the important of the 4-chlorobenzyl fragment of 4-1. Compounds 4-8 and 4-9 were synthesized using the approach outlined in Scheme 4.2. Compound 4-8 was accessed in 36% yield by heating 4-7 in excess ethanolic ammonia solution and 4-9 was synthesized by refluxing 4-7 with three equivalents of dimethylamine in n-butanol at 110 °C for 16 hours (Scheme 4.2). Unlike compounds 4-8 and 4-9, accessing 4-10 was a little bit challenging. My initial approach to access 4-10 in a similar fashion for the synthesis of 4-7 using compound 4-6 and phenylacetylene gave no desired product. I hypothesize that the reaction failed because 199 phenylacetylene is an electron-rich alkyne; previous literature on the [3+2] cycloaddition has demonstrated success only with electron-deficient alkynes. Alternatively, following a slightly modified procedure from the literature (Scheme 4.3),19 4-10 was accessed starting from [3+2] cycloaddition of 4-6 with methyl propiolate to give ester 4-11. Base hydrolysis of 4-11 gave carboxylic acid 4-12 in 96% yield, this was then followed by NBS-mediated Hunsdiecker reaction to give aryl bromide 4-13 in 77%. Suzuki coupling of 4-13 with phenylboronic acid gave the desired analog 4-10 in 86% yield. Scheme 4.2: Synthesis of compound 4-8 and 4-9 N N NN NN N Cl N 4-7 N N R N R 4-8: R = H, excess NH3 (2.0 M in ethanol) 70 °C, 16 h, 36% 4-9: R = Me, NH(CH3)2 (3.0 equiv) n-BuOH, 110 °C, 16 h, 81% 200 Scheme 4.3: Synthesis of 4-10 (1.0 equiv) KOH (3.0 equiv) H2O, DCM, rt, 16 h N NN 4-10 I NH2 N N 2.0 equiv 4-6 O I NH2 N N 2.0 equiv 4-6 O (1.0 equiv) K2CO3 (2.0 equiv) H2O/DCM, rt, 16 h 64% N NN O O 4-11 LiOH (1.5 equiv) then, HCl (aq) MeOH/H2O (1:1), rt, 16 h 96% N NN O OH 4-12 NBS (2.0 equiv) DMF, rt, 3 h 77% B(OH)2 (1.2 equiv) Pd(dppf)Cl2 (5 mol%) K2CO3 (3.0 equiv) 1,4-dioxane/H2O (3:1) 80 °C, 16 h 86% N NN 4-10 N NN Br 4-13 After the synthesis of 4-8 – 4-10 were achieved, I set out to synthesize molecules to investigate the importance of the triazole core in the proteasomal activity of 4-1. Initially, the triazole core was replaced with a phenyl or indole to give 4-15 and 4-17, respectively. Compound 4-15 was synthesized in two steps, first by a Suzuki coupling of 4-2 with phenylboronic acid to give 4-14 in 66% yield, followed by nucleophilic aromatic substitution with 4-chlorobenzylamine to give the desired analog 4-15 in 82% yield (Scheme 4.4). Using a slightly different approach, the indole analog 4-17 was synthesized by deprotonating 1H-indole using phenylmagnesium bromide. The deprotonated indole was then reacted with 4-2 under reflux for 48 hours, resulting in 4-16. Nucleophilic aromatic substitution of 4-16 with 4-chlorobenzylamine yielded 4-17 in up 201 to 74% yield. The final analog 4-19 was also synthesized to probe the effect of the absence of any aromatic substituent at the triazole position in compound 4-1. Scheme 4.4: Synthesis of analog 4-15, 4-17, and 4-19 B(OH)2 Pd(dppf)Cl2 (5 mol%) K2CO3 (3.0 equiv) Cl H2N (3.0 equiv) 1,4-dioxane/H2O (3:1) 80 °C, 16 h 66% N n-BuOH, 110 ℃, 16 h 82% N Cl 4-14 Cl N 4-2 N + Cl 1.0 equiv Cl N + N Cl PhMgBr (2.5 equiv) N H THF, 0 °C to reflux, 48 h 33% 1.0 equiv 2.5 equiv Cl NH H2N (3.0 equiv) N n-BuOH, 110 °C, 16 h 74% N Cl 4-16 Cl H2N (2.0 equiv) n-BuOH, 110 °C, 16 h 72% NH2 N N Cl 4-18 NH2 N N N H 4-19 Cl N N N H 4-15 NH N N N H 4-17 Cl Cl 4.2.1.2 Evaluation of compound 4-1 (MSU-7) fragments towards the 20S proteasome The activity of compound 4-7 – 4-10, 4-15, 4-17, and 4-19 towards the 20S proteasome were determined by Shannon Cartwright. The assay utilizes fluorogenic 7-amino-methylcoumarin (AMC) conjugated small peptides that correspond to each of the three catalytic sites of the 20S proteasome. The three fluorogenic small peptides that were used are Suc-LLVY-AMC, Boc-LRR- AMC, and Z-LLE-AMC corresponding to the chymotrypsin-like (CT-L), trypsin-like (T-L), and caspase-like (Casp-L) sites of the 20S proteasome, respectively. Typically, purified human 20S proteasome is pretreated with varying concentration of compounds followed by the three 202 fluorogenic probes. Cleavage of the peptides by 20S proteasome results in the release of AMC which is then measured over time to quantify the proteolytic activity of the 20S proteasome. From this experiment, the concentration required to increase the activity of 20S proteasome by 200% (EC200) and maximum fold enhancement (Max fold increase) over vehicle are usually determined. Table 4.1: Small peptide activity of compound 4-1 (MSU-7) fragments Compound Molecular Weight (g/mol) cLogP EC200 Max Fold Increase at 80 µM 4-8 4-9 4-10 4-15 4-17 4-19 210.24 240.27 195.23 295.77 334.81 2.19 0.79 3.41 2.91 2.90 - ND - ND - 234.69 -0.42 ND - 1.2 - 1.8 - 1.1 From the table shown above, fragments of compound 4-1 that were tested in the small peptide assay showed no proteasome activity with no EC200 due to the compound not attaining 200% activity over vehicle. Since the elimination the benzyl group (4-9) or the triazole core (4- 19) resulted in inactive compounds, we hypothesized that both the triazole core and the benzyl fragment of compound 4-1 are necessary to maintain its proteasomal activity. 4.2.2 Synthesis of analogs of compound 4-1 (MSU-7) with focus on the tail piece After using a fragment-based approach to determine which fragment of 4-1 is necessary to maintain proteasomal activity, the results showed that the entire structure might be needed for activity. I focused on synthesizing new analogs of 4-1 by first focusing on the tail end of the molecule. Analogs 4-20 to 4-28 were synthesized by three different types of chemistries. Compound 4-20 – 4-25 were accessed by nucleophilic aromatic substitution of 4-7 with 203 benzylamines or anilines (Scheme 4.5). While this approach is robust with various amines and anilines, anilines containing tertiary amines did not give the desired product due to the higher reactivity of the tertiary amine over the aniline group (Scheme 4.5, a1 & a2). Scheme 4.5: Synthesis of analog 4-20 – 4-45 by nucleophilic aromatic substitution and limitations N N N N N N H MW: 302.34 CLogP: 1.576 4-20 N N N amine (3.0 equiv) n-BuOH, 110 °C, 16 - 48 h 4-20 to 4-25 N N N N N N 4-7 N N N N N N H N N N H O O OMe MW: 332.37 CLogP: 1.495 4-21 MW: 346.35 CLogP: 1.861 4-22 H2N N N N H2N N N N CF3 N F3C N N a1 MW: 440.43 CLogP: 1.733 4-24 NH2 N N N MW: 268.32 CLogP: 1.852 4-25 N a2 N N N H OMe OMe MW: 348.37 CLogP: 1.945 4-23 unreactive anilines NH2 CN a3 NH2 Br CF3 a4 Additionally, nucleophilic aromatic substitution failed for electron deficient aniline (Scheme 4.5, compound a3 & a4) and resulted in no product formation. Electron deficient aniline analogs such as a3 can be accessed using Buchward-Hartwig coupling of the a3 with 4-7 (Scheme 4.6). While this approach afforded analog 4-26, anilines containing tertiary amine such as a1 and a2 did not give the desired product. 204 Scheme 4.6: Synthesis of analog 4-26 by Buchward-Hartwig coupling N NN N Cl N 4-7 a3 (1.0 equiv) Pd(OAc)2 (5 mol%) Xantphos (10 mol%) Cs2CO3 (3.0 equiv) N N N 1,4-dioxane, 100℃, 24 h N CN N N H MW: 313.32 CLogP: 1.54 4-26 unreactive anilines H2N H2N F3C N N a1 N a2 Analogs of anilines containing tertiary amines that could not be synthesized through either the nucleophilic substitution reaction or Buchward-Hartwig coupling were synthesized using a different approach shown in Scheme 4.7.18,21 The reaction of 1-aminopyridazin-1-ium iodide with 3-butyn-2-one afforded compound 4-29 in 86%. The subsequent reaction of 4-29 with N,N- dimethylformamide dimethyl acetal (DMF-DMA) gave β-amino-α,β-enone 4-30 in 77% yield. Analogs 4-27 and 4-28 were then access by reacting 4-30 with the corresponding guanidine. Scheme 4.7: Synthesis of analogs 4-27 and 4-28 O I NH2 N N (2.0 equiv) 4-6 (1.0 equiv) KOH (3.0 equiv) H2O, DCM, rt, 16 h 86% N NN O 4-29 DMF-DMA (7.0 equiv) neat, 100℃, 24 h 77% N NN O 4-30 N NH R N H H2N N N N N N H N 4-27 or N N N N N N 4-28 N H Following the synthesis of these analogs, their proteasomal activity were explored using the small peptide assay described in section 4.2.1.2 above by Shannon Cartwright. From this 205 experiment, inconsistent results were obtained for the compounds indicating that something else was affecting the activity. It was hypothesized that the pyrazolo[1,5-b]pyridazines exhibit inherent fluorescence that might be interfering with the fluorescence in the assay read-out. To test the hypothesis, Shannon took fluorescence readings comparing compound 4-1 (MSU-7) with TCH- 165. TCH-165 is a known proteasome activator from our lab and has been used as control in most of our lab assays. Figure 4.4 showed the fluorescence reading of TCH-165 + 20S proteasome, MSU-7 + 20S proteasome, Blank + MSU-7, and vehicle. TCH-165 + 20S proteasome and MSU- 7 + 20S proteasome samples contains the compound, 20S proteasome, and substrate. Blank + MSU-7 contains buffer, compound, and substrate. From the data shown in Figure 4.4, we could conclude that MSU-7 has inherent fluorescence that affect the fluorescence readings in the assay. Figure 4.4: Fluorescent readings (time = 0) of small peptide assay comparing MSU-7 and TCH- 165 at different concentration. Data obtained by Shannon Cartwright ) U F R ( e c n e c s e r o u F l 1500 1000 500 100 80 60 40 20 0 80 40 TCH-165 MSU-7 + 20S Proteasome Blank + MSU-7 Vehicle 20 10 Concentration (μM) 5 2.5 1.25 To further confirm the hypothesis of the fluorescence of MSU-7 interfering the small peptide assay, the absorption and emission of MSU-7 compared to free, and peptide bound AMC were determined. The data showed that MSU-7 absorbs and emits at a wavelength that overlaps 206 with both free and peptide bound AMC probe (suc-LLVY-AMC) making it difficult to accurately determine the 20S proteasome activity of MSU-7 or it’s analogs using small peptide AMC probes. Figure 4.5: Absorbance and emission measurements of MSU-7, suc-LLVY-AMC, and free AMC. Data obtained by Shannon Cartwright ) D O ( e c n a b r o s b A 2.0 1.5 1.0 0.5 0.0 MSU-7 Absorbance Free AMC Absorbance 100000 Suc-LLVY-AMC Absorbance i n o s s m E i 75000 50000 25000 0 350 MSU-7 Emission Free AMC Emission Suc-LLVY-AMC Emission 400 450 500 550 600 Wavelength (nm) 200 250 400 350 300 Wavelength (nm) 450 500 From a structural perspective, I hypothesized that the inherent fluorescence of MSU-7 could be attributed to the conjugation of the triazole core to the pyrimidine and the planarity of the molecule. To address this fluorescence problem from a synthesis perspective, I designed new scaffold similar to that of MSU-7. In the new scaffold, the triazole core was replaced with indole and the indole was conjugated with the pyrimidine through the nitrogen (Figure 4.6). The N- conjugated indole pyrimidine would prevent electron donation from the indole to the pyrimidine and ultimately reducing the fluorescence of the new analogs. It is also important to mention that C-3 conjugated indole pyrimidine did not eliminate the fluorescence of the molecule as expected. 207 Figure 4.6: Proposed approach proposed to reduce fluorescence of the analogs extending conjugation N N N N N R N H breaking conjuagtion extending conjugation N N N R N H HN N N R N H high flourescence low flourescence high flourescence To access the N-conjugated indole pyrimidine analog of MSU-7, indole was deprotonated with sodium hydride and treated with 2,3-dichloropyrimidine to give compound 4-31. Nucleophilic addition of 4-chlorobenzylamine to 4-31 gave analog 4-32. As hypothesized, compound 4-32 showed low fluorescence and was able to be tested in the small peptide assay. Unfortunately, the compound showed no proteasomal activity in the assay. Scheme 4.8: Synthesis of low fluorescence MSU-7 analog 4-32 Cl + N N H N Cl NaH (1.0 equiv) DMF, -5 to -10 °C, 0.5 h 23% H2N Cl amine (3.0 equiv) n-BuOH, 110 °C, 16 h 84% N N N Cl 4-31 N N N N H 4-32 Cl 4.3 Conclusion Although several analogs of pyrazolo[1,5-b]pyridazines were synthesized, the potential of these compounds as 20S proteasome enhancers still remains unexplored. The compounds could not be studied for 20S proteasome activation due to their inherent fluorescence that interfere with the fluorescence being measured in the small peptide assay. One potential solution to study these molecules would involve the utilization of non-fluorescence assay. This study is still ongoing and the proteasomal activity of the synthesized analogs would be evaluated when suitable assay becomes available in the lab. 208 4.4 Experimental General information All flasks were oven-dried overnight and cooled under nitrogen. Reactions were carried out under a nitrogen atmosphere unless stated otherwise. All chemicals and solvents were purchased from commercial sources and used without further purification, unless otherwise mentioned. THF and DCM were dried by passing it through packed column of alumina or over activated 3Å molecular sieves. Infrared spectra were recorded on a Jasco Series 6600 FTIR spectrometer. The mass spectrometer ionization method was ESI with a quadrupole detector. 1H and 13C NMR spectra were recorded on a 500 MHz spectrometer. Chemical shifts are reported relative to the residue peaks of the solvent: CDCl3: 7.26 ppm for 1H and 77.0 ppm for 13C, CD3OD: 3.31 ppm for 1H and 47.6 ppm for 13C, and DMSO-d6: 2.50 ppm for 1H and 39.5 ppm for 13C. Signal multiplicities are given as s (singlet), br (broad), d (doublet), t (triplet), dd (doublet of doublet), and m (multiplet). Reaction carried out at room temperature (rt) implies temperature range usually between 20 – 22 °C. Column chromatography was performed using a Teledyne ISCO CombiFlash® NextGen system with prepacked columns (RediSep® Normal-phase silica, 20-40 microns). TLCs were performed on pre-coated 0.25 mm thick silica gel 60 F254 plates and pre-coated 150 um thick, visualized using UV light and iodine staining. Cl N 4-2 N + Cl TMS TMS Pd(PPh3)2Cl2 CuI THF/TEA , 55℃, 3 h N Cl N 4-3 2-chloro-4-((trimethylsilyl)ethynyl)pyrimidine (4-3): To a solution of 2,4-dichloropyrimidine 4- 2 (4.47 g, 30.0 mmol) in degassed tetrahydrofuran (50 mL) and degassed triethylamine (10 mL) was added bis(triphenylphosphine)palladium(II) dichloride PdCl2(PPh3)2 (1 mol%, 0.21 g, 0.30 209 mmol). Then, copper(I) iodide (2 mol%, 0.114 g, 0.60 mmol) and trimethylsilylacetylene (4.38 mL, 33.0 mmol) was added. A color change from orange to dark brown was observed after adding the alkyne. The reaction was then heated to 55 °C for 3 hours and then cooled to room temperature. The triethylammonium salt was filtered out and washed with ethyl acetate. The filtrate was concentrated and purified with CombiFlash chromatography (silica gel, 20-40 microns, gradient 0-10% ethyl acetate/hexane) to give the product as a brown solid (5.56 g, 88%). 1H NMR (500 MHz, CDCl3) δ 8.54 (d, J = 5.0 Hz, 1H), 7.27 (d, J = 5.0 Hz, 1H), 0.20 (s, 9H). 13C{1H} NMR (125 MHz, CDCl3) δ 161.4, 159.5, 152.6, 121.9, 103.4, 100.0, -0.7. The spectroscopy data are consistent with previous literature reports.19 TMS H KOH (0.1M in MeOH) N MeOH, rt, 15 mins Cl N 4-3 N Cl N 4-4 2-chloro-4-ethynylpyrimidine (4-4): A 50 mL round bottom flask equipped with stirrer bar was charged with 4-3 (4.0 g, 18.98 mmol), then methanol (20 mL) was added. Potassium hydroxide (0.1M in MeOH, 0.99 mL, 0.095 mmol) was added and the reaction was stirred for 15 minutes at room temperature after which TLC shows complete conversion. The reaction was stopped, and the solvent was evaporated. The crude was then purified with CombiFlash chromatography (silica gel, 20-40 microns, gradient 5% ethyl acetate/hexane) to give the product as off-white solid (2.31 g, 88%). 1H NMR (500 MHz, CDCl3) δ 8.63 (d, J = 5.0 Hz, 1H), 7.37 (d, J = 5.0 Hz, 1H), 3.47 (s, 1H). 13C{1H} NMR (125 MHz, CDCl3) δ 161.6, 159.8, 152.2, 122.2, 83.8, 79.6. The spectroscopy data are consistent with previous literature reports.19 210 O S O NH2 HO O KHCO3 (2.4 M) H2O, 70℃, 2 h then, KI N N 4-5 I NH2 N N 4-6 1-aminopyridazin-1-ium iodide (4-6): To a solution of hydroxylamine-O-sulfonic acid, HOSA, (7.01 g, 61.98 mmol) in water (15 mL) was added aqueous potassium bicarbonate KHCO3 (2.4M) until the pH is approximately 5. Then, pyridazine 4-5 (3.0 g, 41.32 mmol) was added. The reaction was placed in pre-heated oil bath at 70 °C and stirred for 1.5 hours. The reaction was neutralized with 2.4 M aqueous KHCO3 until pH is approximately 7. The reaction was cooled to 60 °C and potassium iodide (6.87 g, 41.32 mmol) in water (15 mL) was added and the reaction was further stirred for 1 hour. The reaction was concentrated to remove the water and a solution of 5% MeOH in EtOH (50 mL) was then added to the residue. The product was filtered and dried under vacuum to give the desired product as a yellow solid (8.05 g, 87%). 1H NMR (500 MHz, DMSO-d6) δ 9.80 (s, 2H), 9.26 – 9.21 (m, 1H), 9.09 (dt, J = 6.2, 1.0 Hz, 1H), 8.46 (ddd, J = 8.2, 6.2, 2.1 Hz, 1H), 8.10 (ddd, J = 8.2, 5.2, 1.0 Hz, 1H). 13C{1H} NMR (125 MHz, DMSO-d6) δ 153.3, 137.4, 135.3, 129.4. The spectroscopy data are consistent with previous literature reports.19 I NH2 N N N + N Cl 4-4 4-6 KOH H2O/DCM (1:2), rt, 16 h N NN N Cl N 4-7 3-(2-chloropyrimidin-4-yl)pyrazolo[1,5-b]pyridazine (4-7): Alkyne 4-4 (0.91 g, 6.60 mmol) was added to a slurry of 4-6 (2.94 g, 13.20 mmol) in DCM/H2O (2:1) (39 mL) followed by potassium hydroxide KOH (0.93 g, 16.50 mmol). The reaction was then stirred at room temperature overnight for 16 hours. The reaction was diluted with dichloromethane (15 mL) and 211 water (20 mL) and the organic layer was collected. The aqueous layer was further extracted with dichloromethane (3 x 30 mL) and the organics were combined, dried over sodium sulfate, filtered, concentrated, and purified with CombiFlash chromatography (silica gel, 20-40 microns, gradient 2% methanol/dichloromethane) to give the product as off-white solid (0.72 g, 47%). 1H NMR (500 MHz, DMSO-d6) δ 9.02 (s, 1H), 8.88 (d, J = 9.0 Hz, 1H), 8.74 – 8.65 (m, 2H), 8.05 (d, J = 5.4 Hz, 1H), 7.56 (dd, J = 9.0, 4.5 Hz, 1H). 13C{1H} NMR (125 MHz, DMSO-d6) δ 160.2, 144.6, 140.7, 128.9, 120.3, 115.4 (Quaternary carbons were not observed in the C-13 NMR). HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C10H7ClN5 +) 232.0385 & 234.0355; found 232.0387 & 234.0358. The spectroscopy data are consistent with previous literature reports.19 General procedure for nucleophilic aromatic substitution N NN amine (3.0 equiv) N n-BuOH, 110 °C, 16 - 48 h Cl N 4-7 N NN N N R N H To a suspension of compound 4-7 (1.0 equiv) in n-butanol (3 mL) in a sealed tube was added amine (3.0 equiv). The mixture was placed in a pre-heated oil bath at 110 °C and stirred for 16 – 48 hours. The reaction was cooled to room temperature and the solvent was removed under pressure. The mixture was purified with CombiFlash chromatography to give the product. N NN N N N H Cl N-(4-chlorobenzyl)-4-(pyrazolo[1,5-b]pyridazin-3-yl)pyrimidin-2-amine (4-1): Synthesized according to the general procedure for nucleophilic aromatic substitution with 4-7 (0.18 g, 0.78 212 mmol), 4-chlorobenzylamine (0.29 mL, 2.34 mmol) and n-butanol (5 mL) for 48 hours. Purified with CombiFlash chromatography (silica gel, 20-40 microns, gradient 2% methanol/dichloromethane) to give a white solid (0.22 g, 82%). mp = 182 – 183 °C. 1H NMR (500 MHz, CDCl3) δ 8.45 (s, 1H), 8.34 (dd, J = 4.5, 1.9 Hz, 1H), 8.29 (d, J = 5.2 Hz, 1H), 7.38 – 7.30 (m, 4H), 7.02 (s, 1H), 6.93 (d, J = 5.3 Hz, 1H), 5.68 (s, 1H), 4.69 (d, J = 5.9 Hz, 2H). 13C{1H} NMR (125 MHz, CDCl3) δ 162.2, 160.0, 158.1, 142.9, 139.3, 137.8, 133.0, 132.9, 129.5, 128.7, 128.5, 117.6, 110.8, 106.7, 45.1. FTIR (neat, cm-1): 3243, 3071, 2974, 1620. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C17H14ClN6 +) 337.0963 & 339.0934 ; found 337.0964 & 339.0939. NH3 (2.0 M in EtOH) 70 °C, 24 h N NN N Cl N 4-7 N NN N NH2 N 4-8 4-(pyrazolo[1,5-b]pyridazin-3-yl)pyrimidin-2-amine (4-8): Compound 4-7 (0.05 g, 0.22 mmol) was suspended in ethanolic ammonia solution (2.0 M, 2.5 mL) and the reaction was stirred for 24 hours. After 24 hours, TLC showed incomplete conversion and addition ethanolic ammonia solution (2.0 M, 2.0 mL) was added. The reaction was further stirred for 24 hours and the product was then concentrated and purified with CombiFlash chromatography (silica gel, 20-40 microns, gradient 0-5% methanol/dichloromethane) to give a white solid (0.017 g, 36%). 1H NMR (500 MHz, DMSO-d6) δ 9.15 (dd, J = 9.0, 1.8 Hz, 1H), 8.79 (s, 1H), 8.57 (dd, J = 4.3, 1.8 Hz, 1H), 8.23 (d, J = 5.2 Hz, 1H), 7.41 (dd, J = 9.0, 4.4 Hz, 1H), 7.10 (d, J = 5.2 Hz, 1H), 6.67 (s, 2H). 13C{1H} NMR (125 MHz, DMSO-d6) δ 164.0, 159.7, 158.8, 144.4, 139.9, 132.9, 130.3, 118.9, 110.6, 105.8. The spectroscopy data are consistent with previous literature reports.19 213 N NN N N N N,N-dimethyl-4-(pyrazolo[1,5-b]pyridazin-3-yl)pyrimidin-2-amine (4-9): Synthesized according to the general procedure for nucleophilic aromatic substitution with 4-7 (0.40 g, 1.73 mmol), dimethylamine (2.6 mL, 5.17 mmol) and n-butanol (1 mL) for 16 hours. Purified with CombiFlash chromatography (silica gel, 20-40 microns, gradient 0–3% methanol/dichloromethane) to give a white solid (0.34 g, 81%). 1H NMR (500 MHz, DMSO-d6) δ 8.90 (d, J = 8.9 Hz, 1H), 8.80 (s, 1H), 8.58 – 8.54 (m, 1H), 8.31 (d, J = 5.0 Hz, 1H), 7.41 (dd, J = 8.9, 4.3 Hz, 1H), 7.09 (d, J = 5.0 Hz, 1H), 3.17 (s, 6H). 13C{1H} NMR (125 MHz, DMSO-d6) δ 162.3, 159.3, 158.4, 144.3, 140.1, 132.7, 129.6, 119.4, 110.9, 105.0, 37.4. The spectroscopy data are consistent with previous literature reports.19 I NH2 N N + 4-6 O KOH O H2O/DCM, rt, 16 h N NN O O 4-11 methyl pyrazolo[1,5-b]pyridazine-3-carboxylate (4-11): To a slurry of 4-6 (1.0 g, 4.48 mmol) in DCM (10 mL) was added methyl propiolate (0.20 mL, 2.24 mmol) and cooled to 0 °C. Then, K2CO3 (6.19 g, 4.48 mmol) in H2O (5 mL) was added. The reaction was warmed to room temperature and stirred for 16 hours. The reaction was diluted with dichloromethane (5 mL) and water (10 mL) and the organic layer was collected. The aqueous layer was further extracted with dichloromethane (3 x 15 mL) and the organics were combined, dried over sodium sulfate, filtered, concentrated, and purified with CombiFlash chromatography (silica gel, 20-40 microns, gradient 0-100% ethyl acetate/hexane) to give the product as light-yellow solid (0.51 g, 64%). 1H NMR 214 (500 MHz, CDCl3) δ 8.52 (dd, J = 9.0, 1.9 Hz, 1H), 8.45 (s, 1H), 8.41 (dd, J = 4.4, 1.9 Hz, 1H), 7.25 (dd, J = 9.0, 4.4 Hz, 1H), 3.91 (s, 3H). 13C{1H} NMR (125 MHz, CDCl3) δ 163.1, 143.2, 142.4, 135.0, 128.1, 119.2, 104.2, 51.6. The spectroscopy data are consistent with previous literature reports.22 N NN O O 4-11 LiOH then, HCl (aq) MeOH/H2O (1:1), rt, 16 h N NN O OH 4-12 pyrazolo[1,5-b]pyridazine-3-carboxylic acid (4-12): To a solution of ester 4-11 (0.50 g, 2.82 mmol) in methanol (3 mL) at room temperature was added lithium hydroxide (0.10 g, 4.28 mmol) in H2O (3 mL). The reaction was stirred at room temperature for 16 hours and then diluted with H2O (10 mL). The reaction was cooled to 0 °C and quenched with 1.0 M HCl solution (10 mL), the product precipitated out and was filtered and washed with water to give a white solid (0.44 g, 96%). 1H NMR (500 MHz, DMSO-d6) δ 12.85 (s, 1H), 8.64 (dd, J = 4.4, 1.7 Hz, 1H), 8.53 (dd, J = 9.0, 1.7 Hz, 1H), 8.49 (s, 1H), 7.50 (dd, J = 9.0, 4.4 Hz, 1H). 13C{1H} NMR (125 MHz, DMSO- d6) δ 164.0, 144.8, 142.5, 135.0, 128.3, 120.7, 104.9. The spectroscopy data are consistent with previous literature reports.22 N NN O OH 4-12 NBS DMF, rt, 3 h N NN Br 4-13 3-bromopyrazolo[1,5-b]pyridazine (4-13): Carboxylic acid 4-12 (0.36 g, 2.21 mmol) was dissolved in DMF (4 mL) and NBS (0.79 g, 4.42 mmol) was added. The reaction was stirred at room temperature for 3 hours and the solvent was removed under reduced pressure. The crude was purified with CombiFlash chromatography (silica gel, 20-40 microns, gradient 0-20% ethyl 215 acetate/hexane) to give the product as white solid (0.34 g, 77%). 1H NMR (500 MHz, CDCl3) δ 8.29 (dd, J = 4.4, 1.7 Hz, 1H), 8.01 (s, 1H), 7.92 (dd, J = 9.0, 1.7 Hz, 1H), 7.06 (dd, J = 9.0, 4.4 Hz, 1H). 13C{1H} NMR (125 MHz, CDCl3) δ 142.6, 139.8, 131.9, 125.8, 116.5, 84.8. The spectroscopy data are consistent with previous literature reports.22 B(OH)2 Pd(dppf)Cl2 K2CO3 + 1,4-dioxane/H2O (3:1) 80 °C, 16 h N NN Br 4-13 N NN 4-10 3-phenylpyrazolo[1,5-b]pyridazine (4-10): To a solution of arylbromide (0.10 g, 0.50 mmol) in degassed 1,4-dioxane/H2O (3:1) (8 mL) was added phenylboronic acid (0.073 g, 0.60 mmol), Pd(dppf)Cl2 (5 mol%, 0.018 g, 0.025 mmol), and K2CO3 (0.15 g, 1.50 mmol). The solution was purge with argon and stirred in preheated oil bath at 80 °C for 16 hours. The reaction was cooled to room temperature, diluted with water (8 mL) and then extracted with ethyl acetate (3 x 10 mL). The organics were combined, dried over sodium sulfate, concentrated, and purified with CombiFlash chromatography (silica gel, 20-40 microns, gradient 0-20% ethyl acetate/hexane) to give the product as white solid (0.084 g, 86%). 1H NMR (500 MHz, CDCl3) δ 8.30 (dd, J = 4.2, 1.7 Hz, 1H), 8.23 (s, 1H), 8.21 – 8.14 (m, 1H), 7.64 – 7.52 (m, 2H), 7.48 (t, J = 7.5 Hz, 2H), 7.35 (t, J = 7.5 Hz, 1H), 7.04 (dd, J = 9.0, 4.2 Hz, 1H). FTIR (neat, cm-1): 3094, 1601, 1541. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C12H10N3 +) 196.0870; found 196.0886. The spectroscopy data are consistent with previous literature reports.19 Cl N 4-2 N + Cl B(OH)2 Pd(dppf)Cl2 K2CO3 1,4-dioxane/H2O (3:1) 80 °C, 16 h N N Cl 4-14 216 2-chloro-4-phenylpyrimidine (4-14): To a solution of 4-2 (0.50 g, 3.36 mmol) in degassed 1,4- dioxane/H2O (3:1) (12 mL) was added phenylboronic acid (0.41 g, 3.36 mmol), Pd(dppf)Cl2 (5 mol%, 0.12 g, 0.17 mmol), and K2CO3 (0.99 g, 10.08 mmol). The solution was purge with argon and stirred in preheated oil bath at 80 °C for 16 hours. The reaction was cooled to room temperature, diluted with water (10 mL) and then extracted with ethyl acetate (3 x 10 mL). The organics were combined, dried over sodium sulfate, concentrated, and purified with CombiFlash chromatography (silica gel, 20-40 microns, gradient 0-20% ethyl acetate/hexane) to give the product as white solid (0.43 g, 66%). 1H NMR (500 MHz, CDCl3) δ 8.63 (d, J = 5.2 Hz, 1H), 8.12 – 8.06 (m, 2H), 7.65 (d, J = 5.2 Hz, 1H), 7.59 – 7.48 (m, 3H). 13C{1H} NMR (125 MHz, CDCl3) δ 167.2, 161.9, 159.9, 135.0, 131.9, 129.1, 127.4, 115.2. The spectroscopy data are consistent with previous literature reports.23 N N N H 4-15 Cl N-(4-chlorobenzyl)-4-phenylpyrimidin-2-amine (4-15): Synthesized according to the general procedure for nucleophilic aromatic substitution with 4-14 (0.03 g, 0.16 mmol), 4- chlorobenzylamine (57 µL, 0.47 mmol) and 2-methoxylethanol (2 mL) for 16 hours. Purified with CombiFlash chromatography (silica gel, 20-40 microns, gradient 0-100% ethylacetate/hexane) to give a white solid (0.041 g, 89%). 1H NMR (500 MHz, CDCl3) δ 8.27 (s, 1H), 8.01 (dd, J = 6.5, 2.9 Hz, 2H), 7.46 (dd, J = 5.2, 1.8 Hz, 3H), 7.34 – 7.26 (m, 4H), 6.99 (d, J = 5.2 Hz, 1H), 6.07 (s, 1H), 4.68 (d, J = 6.0 Hz, 2H). 13C{1H} NMR (125 MHz, CDCl3) δ 164.9, 162.5, 158.6, 138.1, 137.3, 132.8, 130.6, 128.9, 128.7, 128.6, 127.0, 106.9, 44.8. FTIR (neat, cm-1): 3260, 3065, 2916,1561. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C17H15ClN3 +) 296.0950; found 296.0977. 217 Cl N + N Cl 4-2 PhMgBr THF, 0 °C to reflux, 48 h N H NH N Cl N 4-16 3-(2-chloropyrimidin-4-yl)-1H-indole (4-16): 1H-Indole (0.88 g, 7.5 mmol) was dissolved in anhydrous THF (20 mL) and cooled to 0 °C. Then, phenylmagnesium bromide (3M in Et2O, 2.5 mL, 7.5 mmol) was added slowly and stirred for 15 minutes. A solution of 2,4-dichloropyrimidine (0.45 g, 3.0 mmol) in anhydrous THF (10 mL) was added slowly and the solution was reflux for 48 hours. The reaction was then cooled to room temperature and quenched with 1 M HCl (25 mL). The reaction was extracted with ethyl acetate (3 x 30 mL). The organics were combined, dried over sodium sulfate, concentrated, and purified with CombiFlash chromatography (silica gel, 20- 40 microns, gradient 20-50% ethyl acetate/hexane) to give the product as yellow solid (0.23 g, 33%). mp = 215 – 217 °C. 1H NMR (500 MHz, DMSO-d6) δ 12.09 (s, 1H), 8.52 (d, J = 5.3 Hz, 2H), 8.43 – 8.41 (m, 1H), 7.91 (d, J = 5.3 Hz, 1H), 7.51 – 7.49 (m, 1H), 7.25 – 7.21 (m, 2H). 13C{1H} NMR (125 MHz, DMSO-d6) δ 165.5, 160.7, 159.0, 137.7, 131.6, 125.5, 123.2, 122.1, 121.8, 115.0, 112.8, 112.3. FTIR (neat, cm-1): 3144, 3047, 2915, 1573. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C12H9ClN3 +) 230.0480; found: 230.0485. NH N N N H 4-17 Cl N-(4-chlorobenzyl)-4-(1H-indol-3-yl)pyrimidin-2-amine (4-17): Synthesized according to the general procedure for nucleophilic aromatic substitution with 4-16 (0.05 g, 0.22 mmol), 4- chlorobenzylamine (80 µL, 0.65 mmol) and n-butanol (3 mL) for 16 hours. Purified with 218 CombiFlash chromatography (silica gel, 20-40 microns, gradient 0–30% ethyl acetate/hexane) to give a yellow solid (0.054 g, 74%). mp = 190 – 191 °C. 1H NMR (500 MHz, DMSO-d6) δ 11.67 (s, 1H), 8.19 (s, 1H), 8.13 (d, J = 5.3 Hz, 1H), 7.58 (t, J = 5.9 Hz, 1H), 7.44 – 7.32 (m, 5H), 7.13 (t, J = 7.2 Hz, 1H), 7.02 (d, J = 5.3 Hz, 2H), 4.58 (s, 2H). 13C{1H} NMR (125 MHz, DMSO-d6) δ 162.6, 162.3, 156.9, 140.1, 137.0, 130.9, 128.8, 128.5, 128.1, 125.3, 122.2, 122.0, 120.3, 113.7, 111.8, 105.5, 43.6. FTIR (neat, cm-1): 3407, 3215, 3032, 2990, 1594. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C19H16ClN4 +) 335.1058; found: 335.1067 Cl H2N n-BuOH, 110 °C, 16 h NH2 N N Cl 4-18 NH2 N N N H 4-19 Cl N2-(4-chlorobenzyl)pyrimidine-2,4-diamine (4-19): Synthesized according to the general procedure for nucleophilic aromatic substitution with 4-18 (0.50 g, 3.86 mmol), 4- chlorobenzylamine (0.94 mL, 7.72 mmol) and n-butanol (5 mL) for 16 hours. Purified with CombiFlash chromatography (silica gel, 20-40 microns, gradient 20–100% ethyl acetate/hexane) to give a yellow solid (0.65g, 72%). 1H NMR (500 MHz, DMSO-d6) δ 7.63 (d, J = 5.7 Hz, 1H), 7.34 – 7.25 (m, 4H), 6.91 (s, 1H), 6.30 (s, 2H), 5.68 (d, J = 5.7 Hz, 1H), 4.39 (d, J = 6.5 Hz, 2H). 13C{1H} NMR (125 MHz, DMSO-d6) δ 164.4, 162.0, 155.1, 140.6, 131.3, 129.3, 128.4, 95.9, 43.5. FTIR (neat, cm-1): 3466, 3170, 2952, 1638, 1572. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C11H12ClN4 +) 234.0745 & 237.0716; found: 235.0688 & 237.0660. 219 N N N N N N H 4-20 N-benzyl-4-(pyrazolo[1,5-b]pyridazin-3-yl)pyrimidin-2-amine (4-20): Synthesized according to the general procedure for nucleophilic aromatic substitution with 4-7 (0.060 g, 0.26 mmol), benzylamine (85 µL, 0.78 mmol) and n-butanol (3 mL) for 16 hours. Purified with CombiFlash chromatography (silica gel, 20-40 microns, gradient 2% methanol/dichloromethane) to give a white solid (0.061 g, 78%). mp = 211 – 212 °C. 1H NMR (500 MHz, DMSO-d6) δ 8.78 (s, 1H), 8.53 (s, 1H), 8.28 (d, J = 5.2 Hz, 1H), 7.80 (t, J = 6.2 Hz, 1H), 7.37 (d, J = 7.1 Hz, 2H), 7.31 (m, 2H), 7.20 (t, J = 7.1 Hz, 1H), 7.11 (d, J = 5.2 Hz, 1H), 4.57 (d, J = 4.6 Hz, 2H). 13C{1H} NMR (125 MHz, DMSO-d6) δ 162.3, 159.1, 158.3, 143.9, 140.4, 139.5, 132.4, 128.3, 127.3, 126.9, 126.6, 118.5, 110.2, 105.6, 44.3. FTIR (neat, cm-1): 3240, 3032, 2879, 1623, 1565. HRMS (ESI- TOF) m/z: [M+H]+ calcd for (C17H15N6 +) 303.1353; found: 303.1362. N N N N N N H 4-21 OMe N-(4-methoxybenzyl)-4-(pyrazolo[1,5-b]pyridazin-3-yl)pyrimidin-2-amin(4-21): Synthesized according to the general procedure for nucleophilic aromatic substitution with 4-7 (0.060 g, 0.26 mmol), 4-methoxybenzylamine (0.10 mL, 0.78 mmol) and n-butanol (3 mL) for 16 hours. Purified with CombiFlash chromatography (silica gel, 20-40 microns, gradient 2% methanol/dichloromethane) to give a yellow solid (0.074 g, 85%). mp = 188 – 189 °C. 1H NMR 220 (500 MHz, DMSO-d6) δ 8.78 (s, 1H), 8.54 (s, 1H), 8.27 (d, J = 5.2 Hz, 1H), 7.72 (t, J = 6.0 Hz, 1H), 7.29 (d, J = 7.9 Hz, 3H), 7.10 (d, J = 5.2 Hz, 1H), 6.87 (d, J = 8.6 Hz, 2H), 4.49 (s, 2H), 3.68 (s, 3H). 13C{1H} NMR (125 MHz, DMSO-d6) δ 162.3, 158.4, 158.1, 143.9, 139.5, 132.4, 132.3, 128.7, 128.0, 118.5, 113.7, 110.2, 105.5, 55.0, 43.7. FTIR (neat, cm-1): 3224, 3032, 2956, 1584. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C18H17N6O+) 333.1458; found: 333.1467. N N N N N N H 4-22 O O N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4-(pyrazolo[1,5-b]pyridazin-3-yl)pyrimidin-2- amine (4-22): Synthesized according to the general procedure for nucleophilic aromatic substitution with 4-7 (0.040 g, 0.17 mmol), 2,3-dihydrobenzo[b][1,4]dioxin-6-amine (64 µL, 0.51 mmol) and n-butanol (3 mL) for 16 hours. Purified with CombiFlash chromatography (silica gel, 20-40 microns, gradient 0-5% methanol/dichloromethane) to give a yellow solid (0.020 g, 34%). mp = 215 – 217 °C. 1H NMR (500 MHz, DMSO-d6) δ 9.37 (s, 1H), 9.13 (d, J = 7.2 Hz, 1H), 8.86 (s, 1H), 8.59 (dd, J = 4.4, 2.0 Hz, 1H), 8.41 (d, J = 5.2 Hz, 1H), 7.42 (dd, J = 9.0, 4.4 Hz, 1H), 7.36 (d, J = 2.0 Hz, 1H), 7.30 (d, J = 5.2 Hz, 1H), 7.09 (dd, J = 8.7, 2.4 Hz, 1H), 6.80 (d, J = 8.7 Hz, 1H), 4.26 – 4.18 (m, 4H). 13C{1H} NMR (125 MHz, DMSO-d6) δ 160.5, 159.6, 158.5, 144.5, 143.4, 140.2, 138.8, 134.5, 132.9, 130.0, 119.2, 117.1, 113.3, 110.5, 109.2, 107.7, 64.7, 64.4. FTIR (neat, cm-1): 3257, 3081, 2942, 1625, 1573. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C18H15N6O2 +) 347.1251; found: 347.1257. 221 N N N N N N H 4-23 OMe OMe N-(3,5-dimethoxyphenyl)-4-(pyrazolo[1,5-b]pyridazin-3-yl)pyrimidin-2-amine (4-23): Synthesized according to the general procedure for nucleophilic aromatic substitution with 4-7 (0.030 g, 0.13 mmol), 3,5-dimethoxyaniline (60 mg, 0.39 mmol) and n-butanol (3 mL) for 48 hours. Purified with CombiFlash chromatography (silica gel, 20-40 microns, gradient 0-5% methanol/dichloromethane) to give a yellow solid (0.026 g, 57%). mp = 196 – 197 °C. 1H NMR (500 MHz, DMSO-d6) δ 9.53 (s, 1H), 9.21 (d, J = 8.9 Hz, 1H), 8.89 (s, 1H), 8.60 (dd, J = 4.4, 1.8 Hz, 1H), 8.47 (d, J = 5.2 Hz, 1H), 7.43 (dd, J = 8.9, 4.4 Hz, 1H), 7.37 (d, J = 5.2 Hz, 1H), 7.04 (t, J = 2.2 Hz, 2H), 6.15 (t, J = 2.2 Hz, 1H), 3.73 (s, 6H). FTIR (neat, cm-1): 3333, 3055, 2993, 1612, 1587. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C18H17N6O2 +) 349.1408; found 349.1407. The spectroscopy data are consistent with previous literature reports.21 N N N N N N CF3 N 4-24 NH2 4-(4-(4-(pyrazolo[1,5-b]pyridazin-3-yl)pyrimidin-2-yl)piperazin-1-yl)-3- (trifluoromethyl)aniline (4-24): Synthesized according to the general procedure for nucleophilic aromatic substitution with 4-7 (0.030 g, 0.13 mmol), 4-(4-methylpiperazin-1-yl)-3- (trifluoromethyl)aniline (0.10 mg, 0.39 mmol) and n-butanol (3 mL) for 48 hours. Purified with 222 CombiFlash chromatography (silica gel, 20-40 microns, gradient 0-10% methanol/dichloromethane) to give a white solid (0.036 g, 63%). mp = 80 – 83 °C. 1H NMR (500 MHz, CD3OD) δ 8.71 (dd, J = 9.1, 1.7 Hz, 1H), 8.49 (s, 1H), 8.37 (dd, J = 4.4, 1.7 Hz, 1H), 8.20 (d, J = 5.2 Hz, 1H), 7.23 (dd, J = 9.1, 4.4 Hz, 1H), 7.18 (d, J = 8.5 Hz, 1H), 6.94 (d, J = 2.5 Hz, 1H), 6.89 (d, J = 5.2 Hz, 1H), 6.83 (dd, J = 8.5, 2.5 Hz, 1H), 4.15 – 3.53 (m, 4H), 2.85 (t, J = 4.8 Hz, 4H). 13C{1H} NMR (125 MHz, CD3OD) δ 163.0, 160.9, 158.8, 147.0, 144.7, 140.3, 133.9, 130.3, 129.4 (q, J = 27.8 Hz), 126.7 (q, J = 270.7 Hz), 126.f4, 119.8, 119.8, 113.6 (q, J = 5.4 Hz), 112.3, 106.5, 68.6, 45.9. FTIR (neat, cm-1): 3341, 3102, 2846, 1618, 1568. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C21H20F3N8 +) 441.1758; found 441.1759. N N N N N N 4-25 N,N-diethyl-4-(pyrazolo[1,5-b]pyridazin-3-yl)pyrimidin-2-amine (4-25): Synthesized according to the general procedure for nucleophilic aromatic substitution with 4-7 (0.030 g, 0.13 mmol), 4-((diethylamino)methyl)aniline (0.041 mg, 0.23 mmol) and n-butanol (2 mL) for 24 hours. Purified with CombiFlash chromatography (silica gel, 20-40 microns, gradient 0-5% methanol/dichloromethane) to give a white solid (0.020 g, 57%). mp = 82 – 84 °C. 1H NMR (500 MHz, cd3od) δ 8.83 (dd, J = 9.1, 2.0 Hz, 1H), 8.54 (s, 1H), 8.41 (dd, J = 4.4, 1.9 Hz, 1H), 8.19 (d, J = 5.2 Hz, 1H), 7.27 (dd, J = 9.1, 4.4 Hz, 1H), 6.90 (d, J = 5.2 Hz, 1H), 3.65 (q, J = 7.1 Hz, 4H), 1.22 (t, J = 7.1 Hz, 6H). 13C{1H} NMR (125 MHz, CD3OD) δ 160.6, 159.5, 157.3, 143.4, 138.8, 132.5, 128.8, 118.2, 111.1, 104.0, 42.0, 12.1. FTIR (neat, cm-1): 3100, 2973, 1619. HRMS (ESI- TOF) m/z: [M+H]+ calcd for (C14H17N6 +) 269.1509; found 269.1511. 223 N NN N + Cl N 4-7 NH2 a3 Pd(OAc)2 Xantphos Cs2CO3 1,4-dioxane, 100℃, 24 h CN N N N N N N H 4-26 CN 3-((4-(pyrazolo[1,5-b]pyridazin-3-yl)pyrimidin-2-yl)amino)benzonitrile (4-26): To a suspension of 4-7 (0.070 g, 0.30 mmol) in degassed anhydrous dioxane (5 mL) was added cesium carbonate (0.29 g, 0.90 mmol), palladium acetate (5 mol%, 3.4 mg, 0.015 mmol), xantphos (10 mol%, 0.058 g, 0.030 mmol) and 3-aminobenzonitrile (0.035 g, 0.30 mmol). The reaction was purged with argon and refluxed at 100 °C for 18 hours. The reaction was then cooled to room temperature and the solvent was removed under pressure. The reaction was purified with CombiFlash chromatography (silica gel, 20-40 microns, gradient 0 - 10% methanol/dichloromethane) to give a yellow solid (0.053 g, 56%). mp = >250 °C. 1H NMR (500 MHz, DMSO-d6) δ 9.95 (s, 1H), 9.13 (d, J = 8.8 Hz, 1H), 8.91 (s, 1H), 8.63 (d, J = 2.7 Hz, 1H), 8.54 (d, J = 5.2 Hz, 1H), 8.37 (s, 1H), 7.93 (d, J = 8.2 Hz, 1H), 7.53 (t, J = 7.9 Hz, 1H), 7.54 – 7.40 (m, 2H), 7.41 (d, J = 7.5 Hz, 1H). 13C{1H} NMR (125 MHz, DMSO-d6) δ 160.0, 160.0, 158.6, 144.6, 141.9, 140.5, 133.0, 130.5, 129.8, 125.2, 123.9, 121.7, 119.6, 119.5, 111.8, 110.3, 109.1. FTIR (neat, cm-1): 3109, 2920, 2255, 1618. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C17H12N7 +) 314.1149; found 314.1143. O KOH H2O, DCM, rt, 16 h I NH2 N N 4-6 N NN O 4-29 1-(pyrazolo[1,5-b]pyridazin-3-yl)ethan-1-one (4-29): To a slurry of 1-aminopyridazin-1-ium iodide 4-6 (2.0 g, 8.97 mmol) in dichloromethane (25 mL), 3-butyn-2-one (0.35 mL, 4.48 mmol) 224 was added. The reaction was cooled to 0 °C and potassium hydroxide (0.63 g, 11.20 mmol) in water (12.7 mL) was added. The reaction was brought to room temperature and stirred for 16 hours. The organic layer was separated and collected, and the aqueous layer was further extracted with dichloromethane (2 x 25 mL). The organics were combined, dried over sodium sulfate and purified with CombiFlash chromatography (silica gel, 20-40 microns, gradient 40 – 100% ethyl acetate/hexane) to give reddish black solid (0.62 g, 86%). 1H NMR (500 MHz, CDCl3) δ 8.73 (dd, J = 9.0, 2.0 Hz, 1H), 8.46 (dd, J = 4.4, 1.9 Hz, 1H), 8.42 (s, 1H), 7.30 (dd, J = 9.0, 4.4 Hz, 1H), 2.58 (s, 3H). 13C{1H} NMR (125 MHz, CDCl3) δ 192.3, 143.8, 142.3, 134.4, 129.0, 120.0, 113.1, 27.9. The spectroscopic data are consistent with previous literature reports.22 N NN O 4-29 DMF-DMA neat, 100℃, 24 h N NN O 4-30 N (E)-3-(dimethylamino)-1-(pyrazolo[1,5-b]pyridazin-3-yl)prop-2-en-1-one (4-30) A 15 mL sealed tube equipped with magnetic stirrer was charged with ketone 4-29 (0.61 g, 3.80 mmol) and N,N-dimethylformamide dimethyl acetal DMF-DMA (3.54 mL, 26.58 mmol) was added. The reaction was placed in pre-heated oil bath at 100 °C and stirred for 16 hours overnight. The reaction was concentrated and purified with CombiFlash chromatography (silica gel, 20-40 microns, gradient 2 – 5% methanol/dichloromethane) to give yellowish solid (0.63 g, 77%). 1H NMR (500 MHz, CDCl3) δ 8.78 (dd, J = 9.0, 2.0 Hz, 1H), 8.32 (q, J = 2.0 Hz, 2H), 7.75 (d, J = 12.3 Hz, 1H), 7.14 (dd, J = 9.0, 4.5 Hz, 1H), 5.59 (d, J = 12.3 Hz, 1H), 3.10 (s, 3H), 2.90 (s, 3H). 13C{1H} NMR (125 MHz, CDCl3) δ 182.4, 152.8, 143.1, 139.9, 134.4, 129.7, 118.3, 114.5, 92.9, 45.0, 37.3. The spectroscopic data are consistent with previous literature reports.22 225 Br Br S1 Et2NH, rt, 0.5 h Br N S2 N-(4-bromobenzyl)-N-ethylethanamine (S2): A 50 mL round bottom flask equipped with magnetic stirrer was charged with 4-bromobenzylbromide S1 (1.0 g, 4.0 mmol) and diethylamine (5 mL) was added. The reaction was stirred at room temperature for 30 minutes (TLC shows complete conversion) and the reaction was stopped, diluted with 30 mL ethyl acetate. The organics was washed with 1 M potassium hydroxide (2 x 10 mL) and brine (10 mL). The organic was concentrated, dried over sodium sulfate, and concentrated to give the desired product as a colorless oil (0.96 g, 99%). 1H NMR (500 MHz, CDCl3) δ 7.42 (d, J = 8.4 Hz, 2H), 7.22 (d, J = 8.4 Hz, 2H), 3.50 (s, 2H), 2.50 (q, J = 7.1 Hz, 4H), 1.03 (t, J = 7.1 Hz, 6H). 13C{1H} NMR (125 MHz, CDCl3) δ 139.3, 131.3, 130.6, 120.5, 57.0, 46.9, 11.9. The spectroscopy data are consistent with previous literature.24 Br N S2 + NH2 • H2SO4 HN NHPMB CuOAc L-proline K3PO4 ACN, 100 °C, 4 h NH HN NHPMB N S3 1-(4-((diethylamino)methyl)phenyl)-3-(4-methoxybenzyl)guanidine (S3): Aryl halide S2 (0.24 g, 1 mmol), PMB-guanidine hemisulfate (0.23 g, 1 mmol), CuOAc (10 mol%, 0.10 mmol), l- proline (10 mol%, 0.20 mmol), and K3PO4 (1.27 g, 6.0 mmol) were mixed in a pressure tube with a magnetic stir bar and anhydrous acetonitrile (6 mL) was added. The reaction was heated to 100 °C for 4 h. After complete consumption of the starting material, ethyl acetate (30 mL) and H2O 226 (30 mL) were added. The separated aqueous layer was extracted with ethyl acetate (2 × 30 mL). The combined organic layers were washed with brine (40 mL), dried over sodium sulfate, and concentrated. The residue was purified with CombiFlash chromatography (silica gel) to give the desired product S3 (0.20 g, 58%). 1H NMR (500 MHz, DMSO-d6) δ 7.27 (d, J = 8.0 Hz, 2H), 7.09 (d, J = 7.8 Hz, 2H), 6.89 (d, J = 8.0 Hz, 3H), 6.71 (d, J = 6.6 Hz, 2H), 5.76 (s, 1H), 4.90 (s, 1H), 4.26 (s, 2H), 3.73 (s, 3H), 3.41 (s, 2H), 2.43 (q, J = 6.8 Hz, 4H), 0.96 (t, J = 6.8 Hz, 6H). 13C{1H} NMR (125 MHz, DMSO-d6) δ 158.1, 151.1, 132.7, 131.1, 129.1, 128.7, 122.5, 113.6, 56.6, 55.0, 45.8, 43.6, 11.7, (missing guanidine carbon). NH HN NHPMB NH • TFA HN NH2 TFA (excess) 100 °C, 20 mins N S3 N S4 1-(4-((diethylamino)methyl)phenyl)guanidine triflate salt (S4): A 15 mL pressured tube equipped with magnetic stirrer bar was charged with compound S3 (0.15 g, 0.44 mmol), then trifloroacetic acid (2 mL) was added. The reaction was sealed with teflon cap and placed in a preheated oil bath at 100 °C and then stirred for 20 minutes. The reaction was cooled to room temperature and the solvent removed under reduced pressure. The crude product was used directly without further purification. N N N NH • TFA HN NH2 + K2CO3 2-methoxyethanol 120 °C, 16 h N O 4-30 N S4 227 N N N N N N N H 4-27 Compound 4-30 (0.048 g, 0.22 mmol) was dissolved in 2-methoxyethanol (3 mL) and to the solution was added crude guanidine S4 (0.44 mmol) and potassium carbonate (0.091 g, 0.66 mmol). The solution was placed in a preheated oil bath and refluxed at 120 °C overnight. The reaction was then cooled down to room temperature, concentrated and product purified with CombiFlash chromatography (silica gel, 20-40 microns, gradient 0 – 5% methanol/dichloromethane) to give the desired product as a yellowish solid (0.041 g, 50%). mp = 124 – 125 °C. 1H NMR (500 MHz, CD3OD) δ 8.97 (d, J = 9.0 Hz, 1H), 8.56 (s, 1H), 8.42 – 8.38 (m, 1H), 8.29 (d, J = 5.3 Hz, 1H), 7.58 (d, J = 8.4 Hz, 2H), 7.27 (d, J = 8.4 Hz, 2H), 7.20 (dd, J = 9.0, 4.4 Hz, 1H), 7.11 (d, J = 5.3 Hz, 1H), 3.57 (s, 2H), 2.56 (q, J = 7.2 Hz, 4H), 1.09 (t, J = 7.2 Hz, 6H). 13C{1H} NMR (125 MHz, CD3OD) δ 160.4, 159.8, 157.6, 143.5, 139.2, 138.9, 132.8, 131.6, 129.7, 129.6, 119.9, 118.3, 110.6, 107.1, 56.3, 45.9, 9.9. FTIR (neat, cm-1): 3255, 3064, 2963,1609, 1570. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C21H24N7 +) 374.2088; found: 374.2091. NH2 NH2 + N conc. HNO3 EtOH, 100 °C, 16 h sealed teflon tube NH • HNO3 HN NH2 S5 1-phenylguanidine nitrate salt (S5): To a pressure tube equipped with stirrer bar was added aniline (0.91 mL, 10.0 mmol), ethanol (5 mL), cyanamide (50% in water, 1.20 mL, 15.0 mmol) and nitric acid (0.63 mL, 10.0 mmol). The tube was sealed and placed in a preheated oil bath at 100 °C and was allowed to stir for 16 h. The reaction was stopped, cooled to room temperature, and concentrated to give the crude product which was used directly for the next step without further purification. 228 N N N NH • HNO3 HN NH2 + K2CO3 2-methoxyethanol 120 °C, 16 h N O 4-30 S5 N N N N N N H 4-28 N-phenyl-4-(pyrazolo[1,5-b]pyridazin-3-yl)pyrimidin-2-amine (4-28): Compound 4-30 (0.10 g, 0.46 mmol) was dissolved in 2-methoxyethanol (4 mL) and to the solution was added crude phenylguanide S5 (0.18 g, 0.92 mmol) and potassium carbonate (0.19 g, 1.38 mmol). The solution was placed in a preheated oil bath and refluxed at 120 °C overnight. The reaction was then cooled down to room temperature, concentrated, and product purified with CombiFlash chromatography (silica gel, 20-40 microns, gradient 2% methanol/dichloromethane) to give the desired product as yellowish solid (0.073 g, 55%). 1H NMR (500 MHz, DMSO-d6) δ 9.57 (s, 1H), 9.16 (d, J = 8.7 Hz, 1H), 8.88 (s, 1H), 8.59 (dd, J = 4.4, 1.9 Hz, 1H), 8.45 (d, J = 5.2 Hz, 1H), 7.74 (d, J = 7.7 Hz, 2H), 7.44 (dd, J = 9.1, 4.4 Hz, 1H), 7.37 – 7.28 (m, 3H), 6.97 (t, J = 7.3 Hz, 1H). 13C{1H} NMR (125 MHz, DMSO-d6) δ 160.5, 159.8, 158.5, 144.5, 140.9, 140.3, 132.9, 130.0, 129.0, 122.0, 119.8, 119.2, 110.4, 108.1. The spectroscopy data are consistent with previous literature19 Cl N N Cl + N H NaH DMF, -5 to -10 °C, 0.5 h N N + N N N N Cl 4-31 Cl S6 1-(2-chloropyrimidin-4-yl)-1H-indole (4-31): To a solution of indole (1.17 g, 10.0 mmol) and 2,4-dichloropyrimidine (1.52 g, 10.20 mmol) in dimethylformamide (30 mL) between -5 to -10 °C was added a sodium hydride (60 wt% in mineral oil, 0.40 g, 10.0 mmol) slowly. The reaction was stirred for 30 minutes and then quenched with water (35 mL). The white solid was filtered and 229 purified CombiFlash chromatography (silica, 40-60 microns, gradient 5-30% ethyl acetate/hexane) to give 4-31 as white solid (0.53 g, 23%) and S6 as white solid (0.34 g, 15%). 4-31: mp = 128 – 129 °C. 1H NMR (500 MHz, CDCl3) δ 8.54 – 8.49 (m, 2H), 7.68 – 7.59 (m, 2H), 7.39 (ddd, J = 8.5, 7.1, 1.3 Hz, 1H), 7.32 – 7.23 (m, 2H), 6.78 (dd, J = 3.7, 0.9 Hz, 1H). 13C{1H} NMR (125 MHz, CDCl3) δ 161.0, 159.7, 159.3, 135.2, 131.2, 124.8, 124.1, 123.3, 121.4, 115.7, 109.7, 106.8. FTIR (neat, cm-1): 3142, 3114, 1574. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C12H9ClN3 +) 230.0480; found: 230.0484. S6: mp = 84 – 85 °C. 1H NMR (500 MHz, CDCl3) δ 8.79 – 8.74 (m, 1H), 8.54 (d, J = 5.2 Hz, 1H), 8.22 (d, J = 3.7 Hz, 1H), 7.63 (d, J = 7.7 Hz, 1H), 7.37 (ddd, J = 8.4, 7.3, 1.2 Hz, 1H), 7.30 – 7.24 (m, 1H), 7.05 (d, J = 5.2 Hz, 1H), 6.72 (dd, J = 3.7, 0.6 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ 161.6, 159.1, 157.4, 135.3, 131.4, 125.7, 124.0, 122.7, 120.9, 116.4, 115.9, 107.9. FTIR (neat, cm- 1): 3153, 3047, 1562, 1545. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C12H9ClN3 +) 230.0480; found: 230.0486. H2N N Cl N n-BuOH, 110 °C, 16 h N Cl 4-31 N N N N H 4-32 Cl Synthesized according to the general procedure for nucleophilic aromatic substitution with 4-31 (0.060 g, 0.26 mmol), 4-chlorobenzylamine (95 µL, 0.78 mmol) and n-butanol (3 mL) for 16 hours. Purified with CombiFlash chromatography (silica gel, 20-40 microns, gradient 0-100% hexane/ethyl acetate) to give a white solid (0.073 g, 84%). mp = 194 – 195 °C. 1H NMR (500 MHz, DMSO-d6) δ 8.30 (s, 1H), 8.11 – 7.90 (m, 2H), 7.58 (s, 1H), 7.38 (s, 4H), 7.26 – 7.07 (m, 2H), 6.96 (s, 1H), 6.74 (s, 1H), 4.57 (s, 2H). 13C{1H} NMR (125 MHz, DMSO-d6) δ 162.2, 159.8, 230 158.7, 139.4, 134.7, 131.1, 130.4, 129.0, 128.5, 128.2, 125.8, 123.3, 122.0, 120.8, 107.1, 97.9, 43.6. FTIR (neat, cm-1): 3222, 3142, 2990, 1611. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C19H16ClN4 +) 335.1058; found: 335.1064. 231 REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. Dunker, A. K.; Lawson, J. D.; Brown, C. J.; Williams, R. M.; Romero, P.; Oh, J. S.; Oldfield, C. J.; Campen, A. M.; Ratliff, C. M.; Hipps, K. W.; et al., J. Mol. Graphics Model. 2001, 19, 26-59. Dyson, H. J.; Wright, P. E., Nat. Rev. Mol. Cell Biol. 2005, 6, 197-208. DeForte, S.; Uversky, V. N., Molecules 2016, 21. Babu, M. 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Chem. 2019, 84, 6084- 6093. 233 Figure 4.7: 1H and 13C{1H} NMR spectra of 4-3 APPENDIX DG-3-66-product-cdcl3 _PROTON_01 TMS N N Cl 4 5 8 . 3 5 8 . 7 2 7 . l 3 c d c 6 2 7 . 1 9 0 . 4 9 0 . 0 2 0 . 5 1 9 . 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 DG-3-66-product-cdcl3 _ CARBON_01 TMS N N Cl . 4 1 6 1 . 5 9 5 1 . 6 2 5 1 . 9 1 2 1 . 4 3 0 1 . 0 0 0 1 l 3 c d c 4 7 7 . l 3 c d c 1 7 7 . l 3 c d c 9 6 7 . . 7 0 - 30000 25000 20000 15000 10000 5000 0 2100 2000 1900 1800 1700 1600 1500 1400 1300 1200 1100 1000 900 800 700 600 500 400 300 200 100 0 -100 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 234 Figure 4.8: 1H and 13C{1H} NMR spectra of 4-4 DG-3-68-tt10-15-cdcl3 _PROTON_01 H N N Cl 3 6 8 . 2 6 8 . 7 3 7 . 6 3 7 . l 3 c d c 6 2 7 . 7 4 3 . 6 0 0 . 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 0 0 1 . 7 9 0 . 1 9 0 . DG-3-68-tt10-15-cdcl3 _ CARBON_01 H N N Cl . 6 1 6 1 . 8 9 5 1 . 2 2 5 1 . 2 2 2 1 . 8 3 8 . 6 9 7 l 3 c d c 3 7 7 . l 3 c d c 0 7 7 . l 3 c d c 8 6 7 . 0 1 . 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 -10 235 75000 70000 65000 60000 55000 50000 45000 40000 35000 30000 25000 20000 15000 10000 5000 0 -5000 280 260 240 220 200 180 160 140 120 100 80 60 40 20 0 Figure 4.9: 1H and 13C{1H} NMR spectra of 4-6 DG-3-98-prod-DMSO _PROTON_01 0 8 9 . 5 2 9 . 4 2 9 . 4 2 9 . 4 2 9 . 4 2 9 . 3 2 9 . 3 2 9 . 0 1 9 . 9 0 9 . 9 0 9 . 8 0 9 . 8 0 9 . 8 0 9 . 8 4 8 . 8 4 8 . 7 4 8 . 6 4 8 . 6 4 8 . 5 4 8 . 5 4 8 . 2 1 8 . 1 1 8 . 2 1 8 . 1 1 8 . 0 1 8 . 0 1 8 . 9 0 8 . 9 0 8 . O 2 H 2 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 . I NH2 N N 4 1 2 . 7 8 0 . 5 0 1 . 7 9 0 . 8 9 0 . 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 DG-3-98-product_ CARBON_01 I NH2 N N . 3 3 5 1 . 4 7 3 1 . 3 5 3 1 . 4 9 2 1 o s m d 5 0 4 . o s m d 1 0 4 . o s m d 0 0 4 . o s m d 8 9 3 . o s m d 6 9 3 . o s m d 5 9 3 . 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 -10 236 90000 80000 70000 60000 50000 40000 30000 20000 10000 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 4.10: 1H and 13C{1H} NMR spectra of 4-7 DG-3-76-tt18-21_PROTON_01 N NN 2 0 9 . 9 8 8 . 9 8 8 . 7 8 8 . 7 8 8 . 2 7 8 . 1 7 8 . 8 6 8 . 8 6 8 . 7 6 8 . 7 6 8 . 6 0 8 . 5 0 8 . 8 5 7 . 7 5 7 . 6 5 7 . 5 5 7 . O 2 H 1 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 . N N Cl 13 12 11 10 9 DG-3-76-tt22-35 _ CARBON_01 3 9 0 . 9 9 0 . 4 9 0 . 4 9 0 . 3 0 1 . 3 0 1 . 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 N NN N N Cl . 2 0 6 1 . 6 4 4 1 . 7 0 4 1 . 9 8 2 1 . 3 0 2 1 . 4 5 1 1 . 2 0 6 1 . 6 4 4 1 . 7 0 4 1 . 9 8 2 1 . 3 0 2 1 . 4 5 1 1 160 150 140 130 120 f1 (ppm) o s m d 7 9 3 . o s m d 5 9 3 . o s m d 4 9 3 . o s m d 2 9 3 . o s m d 0 9 3 . 3 2 1 0 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 237 55000 50000 45000 40000 35000 30000 25000 20000 15000 10000 5000 0 -5000 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 Figure 4.11: 1H and 13C{1H} NMR spectra of 4-1 DG-3-78-tt20-22_PROTON_01 5 4 8 . 5 3 8 . 4 3 8 . 4 3 8 . 4 3 8 . 0 3 8 . 9 2 8 . 6 3 7 . 4 3 7 . 3 3 7 . 2 3 7 . 2 3 7 . 6 2 7 . 2 0 7 . 4 9 6 . 3 9 6 . 0 3 5 . 9 6 4 . 0 7 4 . 8 0 0 . 7 0 0 . 6 0 0 . N NN N N N H Cl 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 6 0 1 . 6 9 0 . 1 0 1 . 3 7 3 . 4 9 0 . 0 1 1 . 1 0 1 . 9 1 2 . . 2 2 6 1 . 0 0 6 1 . 1 8 5 1 . 9 2 4 1 . 3 9 3 1 . 8 7 3 1 . 0 3 3 1 . 9 2 3 1 . 5 9 2 1 . 7 8 2 1 . 5 8 2 1 . 6 7 1 1 . 8 0 1 1 . 7 6 0 1 DG-3-78-product-tt20-22_ CARBON_01 N NN N N N H Cl l 3 c d c 3 7 7 . l 3 c d c 0 7 7 . l 3 c d c 8 6 7 . . 1 5 4 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 238 21000 20000 19000 18000 17000 16000 15000 14000 13000 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 -1000 800 750 700 650 600 550 500 450 400 350 300 250 200 150 100 50 0 -50 Figure 4.12: 1H and 13C{1H} NMR spectra of 4-8 DG-4-68-tt13-22-DMSO _PROTON_01 6 1 9 . 6 1 9 . 4 1 9 . 4 1 9 . 9 7 8 . 8 5 8 . 8 5 8 . 7 5 8 . 7 5 8 . 4 2 8 . 3 2 8 . 2 4 7 . 1 4 7 . 0 4 7 . 9 3 7 . 0 1 7 . 9 0 7 . 7 6 6 . N NN N N NH2 O 2 H 4 3 3 . o s m d 9 4 2 . 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 2 0 1 . 9 9 0 . 8 9 0 . 1 0 1 . 9 0 1 . 8 0 1 . 4 8 1 . . 0 4 6 1 . 7 9 5 1 . 8 8 5 1 . 4 4 4 1 . 9 9 3 1 . 9 2 3 1 . 3 0 3 1 . 9 8 1 1 . 6 0 1 1 . 8 5 0 1 o s m d 5 0 4 . o s m d 3 0 4 . o s m d 1 0 4 . o s m d 0 0 4 . o s m d 8 9 3 . o s m d 6 9 3 . o s m d 5 9 3 . DG-4-68-tt13-22-DMSO _ CARBON_01 N NN N N NH2 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 -10 239 36000 34000 32000 30000 28000 26000 24000 22000 20000 18000 16000 14000 12000 10000 8000 6000 4000 2000 0 -2000 650 600 550 500 450 400 350 300 250 200 150 100 50 0 -50 Figure 4.13: 1H and 13C{1H} NMR spectra of 4-9 DG-5-48-DMSO _PROTON_01 1 9 8 . 9 8 8 . 0 8 8 . 7 5 8 . 6 5 8 . 6 5 8 . 1 3 8 . 0 3 8 . 3 4 7 . 2 4 7 . 1 4 7 . 0 4 7 . 0 1 7 . 9 0 7 . N NN N N N O 2 H 3 3 3 . 7 1 3 . o s m d 8 4 2 . 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 9 9 0 . 3 9 0 . 3 9 0 . 6 9 0 . 1 0 1 . 1 0 1 . 8 1 6 . . 3 2 6 1 . 3 9 5 1 . 4 8 5 1 . 3 4 4 1 . 1 0 4 1 . 7 2 3 1 . 6 9 2 1 . 4 9 1 1 . 9 0 1 1 . 0 5 0 1 o s m d 4 0 4 . o s m d 3 0 4 . o s m d 1 0 4 . o s m d 9 9 3 . o s m d 8 9 3 . o s m d 6 9 3 . o s m d 4 9 3 . . 4 7 3 DG-5-48-DMSO _ CARBON_01 N NN N N N 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 -10 240 24000 23000 22000 21000 20000 19000 18000 17000 16000 15000 14000 13000 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 -1000 -2000 1100 1000 900 800 700 600 500 400 300 200 100 0 -100 Figure 4.14: 1H and 13C{1H} NMR spectra of 4-11 DG-4-40-tt8-11_PROTON_01 N NN O O 3 5 8 . 3 5 8 . 1 5 8 . 1 5 8 . 5 4 8 . 2 4 8 . 2 4 8 . 1 4 8 . 1 4 8 . 7 2 7 . l 3 c d c 6 2 7 . 6 2 7 . 5 2 7 . 4 2 7 . 1 9 3 . 13 12 11 10 9 8 7 6 f1 (ppm) 5 DG-4-40-tt8-11_ CARBON_01 8 9 0 . 4 8 0 . 7 8 0 . 6 0 1 . . 1 3 6 1 . 2 3 4 1 . 4 2 4 1 . 0 5 3 1 . 1 8 2 1 . 2 9 1 1 . 2 4 0 1 N NN O O 5 2 3 . 4 3 2 1 0 -1 l 3 c d c 3 7 7 . l 3 c d c 1 7 7 . l 3 c d c 8 6 7 . . 6 1 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 -10 241 17000 16000 15000 14000 13000 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 -1000 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 4.15: 1H and 13C{1H} NMR spectra of 4-12 DG-4-41-DMSO _PROTON_01 5 8 2 1 . 4 6 8 . 4 6 8 . 3 6 8 . 3 6 8 . 4 5 8 . 3 5 8 . 2 5 8 . 1 5 8 . 9 4 8 . 1 5 7 . 0 5 7 . 9 4 7 . 9 4 7 . o s m d 0 5 2 . o s m d 9 4 2 . 5 3 3 . N NN O OH 5 0 1 . 8 9 0 . 5 0 1 . 3 8 0 . 9 0 1 . 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 DG-4-41-DMSO _ CARBON_01 . 0 4 6 1 . 8 4 4 1 . 5 2 4 1 . 0 5 3 1 . 3 8 2 1 . 7 0 2 1 . 9 4 0 1 o s m d 4 0 4 . o s m d 3 0 4 . o s m d 1 0 4 . o s m d 8 9 3 . o s m d 9 9 3 . o s m d 6 9 3 . o s m d 4 9 3 . N NN O OH 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 242 Figure 4.16: 1H and 13C{1H} NMR spectra of 4-13 DG-4-45-CDCl3 _PROTON_01 N NN Br 0 3 8 . 0 3 8 . 9 2 8 . 9 2 8 . 1 0 8 . 3 9 7 . 3 9 7 . 1 9 7 . 1 9 7 . l 3 c d c 6 2 7 . 7 0 7 . 6 0 7 . 5 0 7 . 4 0 7 . 13 12 11 10 9 8 DG-4-45-CDCl3 _ CARBON_01 8 9 0 . 3 9 0 . 2 0 1 . 7 0 1 . 7 6 f1 (ppm) N NN Br . 6 2 4 1 . 8 9 3 1 . 9 1 3 1 . 8 5 2 1 . 5 6 1 1 . 8 4 8 5 4 3 2 1 0 -1 l 3 c d c 3 7 7 . l 3 c d c 0 7 7 . l 3 c d c 8 6 7 . 0 1 . 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 -10 243 17000 16000 15000 14000 13000 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 -1000 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 -20 40000 38000 36000 34000 32000 30000 28000 26000 24000 22000 20000 18000 16000 14000 12000 10000 8000 6000 4000 2000 0 -2000 Figure 4.17: 1H NMR spectra of 4-10 DG-5-11-CDCl3 _PROTON_01 N NN 1 3 8 . 1 3 8 . 0 3 8 . 0 3 8 . 3 2 8 . 1 2 8 . 0 2 8 . 9 1 8 . 8 1 8 . 8 5 7 . 8 5 7 . 8 5 7 . 7 5 7 . 0 5 7 . 9 4 7 . 8 4 7 . 6 4 7 . 7 3 7 . 5 3 7 . 4 3 7 . l 3 c d c 7 2 7 . 5 0 7 . 4 0 7 . 3 0 7 . 2 0 7 . 6 2 1 . 8 0 0 . 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 2 8 0 . 9 8 0 . 8 1 1 . 3 9 1 . 7 0 2 . 9 9 0 . 2 1 1 . 244 Figure 4.18: 1H and 13C{1H} NMR spectra of 4-14 DG-5-96-tt21-CDCl3 _PROTON_01 4 6 8 . 4 6 8 . 3 6 8 . 0 1 8 . 0 1 8 . 0 1 8 . 9 0 8 . 8 0 8 . 8 0 8 . 6 6 7 . 4 6 7 . 7 5 7 . 7 5 7 . 6 5 7 . 6 5 7 . 5 5 7 . 5 5 7 . 4 5 7 . 4 5 7 . 3 5 7 . 3 5 7 . 3 5 7 . 2 5 7 . 2 5 7 . 1 5 7 . 0 5 7 . 0 5 7 . 0 5 7 . N N Cl l 3 c d c 7 2 7 . O 2 H 3 7 1 . 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 DG-5-96-tt21-CDCl3 _ CARBON_01 . 2 7 6 1 . 9 1 6 1 . 9 9 5 1 . 0 5 3 1 . 9 1 3 1 . 1 9 2 1 . 4 7 2 1 . 2 5 1 1 N N Cl l 3 c d c 3 7 7 . l 3 c d c 1 7 7 . l 3 c d c 8 6 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 -10 245 32000 30000 28000 26000 24000 22000 20000 18000 16000 14000 12000 10000 8000 6000 4000 2000 0 -2000 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 Figure 4.19: 1H and 13C{1H} NMR spectra of 4-15 l 3 c d c 6 2 7 . DG-5-101-CDCl3 _PROTON_01 N N N H Cl 7 2 8 . 2 0 8 . 1 0 8 . 1 0 8 . 0 0 8 . 7 4 7 . 7 4 7 . 6 4 7 . 5 4 7 . 5 4 7 . 4 4 7 . 4 4 7 . 2 3 7 . 0 3 7 . 9 2 7 . 8 2 7 . 7 2 7 . 13 12 11 10 9 8 7 DG-5-101-CDCl3 _ CARBON_01 9 9 0 . 6 9 1 . 8 9 2 . 0 9 3 . 3 0 1 . 0 0 7 . 9 9 6 . 7 0 6 . 8 6 4 . 7 6 4 . 4 0 1 . 6 f1 (ppm) 9 0 2 . 5 4 3 2 1 0 -1 l 3 c d c 3 7 7 . l 3 c d c 1 7 7 . l 3 c d c 8 6 7 . . 8 4 4 . 9 4 6 1 . 5 2 6 1 . 6 8 5 1 . 1 8 3 1 . 3 7 3 1 . 8 2 3 1 . 6 0 3 1 . 9 8 2 1 . 7 8 2 1 . 6 8 2 1 . 0 7 2 1 . 9 6 0 1 N N N H Cl 6500 6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 -500 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 246 Figure 4.20: 1H and 13C{1H} NMR spectra of 4-16 DG-4-47-tt31-35-DMSO _PROTON_01 2 5 8 . 1 5 8 . 3 4 8 . 3 4 8 . 2 4 8 . 2 4 8 . 2 4 8 . 1 4 8 . 1 4 8 . 1 9 7 . 0 9 7 . 1 5 7 . 1 5 7 . 0 5 7 . 0 5 7 . 0 5 7 . 9 4 7 . 9 4 7 . 9 4 7 . 5 2 7 . 4 2 7 . 4 2 7 . 3 2 7 . 2 2 7 . 2 2 7 . 1 2 7 . 9 0 2 1 . NH N N Cl O 2 H 6 3 3 . o s m d 0 5 2 . o s m d 0 5 2 . o s m d 9 4 2 . o s m d 9 4 2 . o s m d 9 4 2 . 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 . 5 5 6 1 . 7 0 6 1 . 0 9 5 1 . 7 7 3 1 . 6 1 3 1 . 4 5 2 1 . 2 3 2 1 . 1 2 2 1 . 8 1 2 1 . 0 5 1 1 . 8 2 1 1 . 3 2 1 1 o s m d 5 0 4 . o s m d 3 0 4 . o s m d 1 0 4 . o s m d 8 9 3 . o s m d 0 0 4 . o s m d 6 9 3 . o s m d 5 9 3 . DG-4-47-tt31-35-DMSO _ CARBON_02 NH N N Cl 55000 50000 45000 40000 35000 30000 25000 20000 15000 10000 5000 0 1900 1800 1700 1600 1500 1400 1300 1200 1100 1000 900 800 700 600 500 400 300 200 100 0 -100 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 247 Figure 4.21: 1H and 13C{1H} NMR spectra of 4-17 DG-4-57-DMSO _PROTON_01 7 6 1 1 . 9 1 8 . 3 1 8 . 2 1 8 . 9 5 7 . 8 5 7 . 7 5 7 . 2 4 7 . 0 4 7 . 8 3 7 . 6 3 7 . 5 3 7 . 4 3 7 . 4 3 7 . 5 1 7 . 3 1 7 . 2 1 7 . 3 0 7 . 2 0 7 . 8 5 4 . O 2 H 4 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 . NH N N N H Cl 1 0 1 . 1 0 1 . 2 0 1 . 9 9 0 . 6 8 4 . 4 0 1 . 2 9 1 . 4 1 2 . 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 DG-4-57-DMSO _ CARBON_01 NH N N N H Cl . 6 2 6 1 . 3 2 6 1 . 9 6 5 1 . 1 0 4 1 . 0 7 3 1 . 9 0 3 1 . 8 8 2 1 . 5 8 2 1 . 1 8 2 1 . 3 5 2 1 . 2 2 2 1 . 0 2 2 1 . 3 0 2 1 . 7 3 1 1 . 8 1 1 1 . 5 5 0 1 o s m d 0 0 4 . o s m d 9 9 3 . o s m d 7 9 3 . o s m d 4 9 3 . o s m d 5 9 3 . o s m d 2 9 3 . o s m d 0 9 3 . . 6 3 4 . 8 8 2 1 . 5 8 2 1 . 1 8 2 1 . 3 5 2 1 . 2 2 2 1 . 0 2 2 1 129 128 127 125 126 f1 (ppm) 124 123 122 . 6 2 6 1 . 3 2 6 1 . 9 6 5 1 160 f1 (ppm) 155 15 10 5 0 20 10 0 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 248 45000 40000 35000 30000 25000 20000 15000 10000 5000 0 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 4.22: 1H and 13C{1H} NMR spectra of 4-17 DG-5-89-DMSO _PROTON_01 NH2 N N N H Cl 4 6 7 . 3 6 7 . 2 3 7 . 2 3 7 . 1 3 7 . 0 3 7 . 0 3 7 . 8 2 7 . 7 2 7 . 1 9 6 . 0 3 6 . 9 6 5 . 8 6 5 . 9 3 4 . 8 3 4 . O 2 H 6 1 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 . 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 7 9 0 . 2 9 3 . 2 0 1 . 2 0 2 . 7 9 0 . 0 1 2 . . 4 4 6 1 . 0 2 6 1 . 1 5 5 1 . 6 0 4 1 . 3 1 3 1 . 3 9 2 1 . 4 8 2 1 . 9 5 9 o s m d 4 0 4 . o s m d 3 0 4 . o s m d 1 0 4 . o s m d 8 9 3 . o s m d 9 9 3 . o s m d 6 9 3 . o s m d 4 9 3 . . 5 3 4 DG-5-89-crashed-out-product-DMSO _ CARBON_01 NH2 N N N H Cl 8500 8000 7500 7000 6500 6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 -500 85 80 75 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 -10 249 Figure 4.23: 1H and 13C{1H} NMR spectra of 4-20 8 7 8 . 3 5 8 . 8 2 8 . 7 2 8 . 1 8 7 . 0 8 7 . 8 7 7 . 7 3 7 . 6 3 7 . 2 3 7 . 1 3 7 . 9 2 7 . 1 2 7 . 0 2 7 . 8 1 7 . 1 1 7 . 0 1 7 . 7 5 4 . 7 5 4 . 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 . DG-4-26-DMSO _PROTON_01 N N N N N N H 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 2 9 0 . 8 9 0 . 9 8 0 . 8 9 0 . 5 0 2 . 9 9 1 . 4 2 1 . 8 9 0 . 8 9 1 . . 3 2 6 1 . 1 9 5 1 . 3 8 5 1 . 9 3 4 1 . 4 0 4 1 . 5 9 3 1 . 4 2 3 1 . 3 8 2 1 . 3 7 2 1 . 9 6 2 1 . 6 6 2 1 . 5 8 1 1 . 2 0 1 1 . 6 5 0 1 o s m d 0 0 4 . o s m d 9 9 3 . o s m d 7 9 3 . o s m d 4 9 3 . o s m d 5 9 3 . o s m d 2 9 3 . o s m d 0 9 3 . . 3 4 4 DG-4-26-DMSO _ CARBON_02 N N N N N N H 80000 75000 70000 65000 60000 55000 50000 45000 40000 35000 30000 25000 20000 15000 10000 5000 0 -5000 32 30 28 26 24 22 20 18 16 14 12 10 8 6 4 2 0 -2 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 250 Figure 4.24: 1H and 13C{1H} NMR spectra of 4-21 8 7 8 . 4 5 8 . 7 2 8 . 6 2 8 . 3 7 7 . 2 7 7 . 0 7 7 . 9 2 7 . 8 2 7 . 0 1 7 . 9 0 7 . 8 8 6 . 6 8 6 . 9 4 4 . O 2 H 3 3 3 . 8 6 3 . 6 1 3 . 4 1 3 . o s m d 9 4 2 . o s m d 8 4 2 . o s m d 8 4 2 . DG-4-27-DMSO _PROTON_01 N N N N N N H OMe 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 5 9 0 . 8 2 1 . 6 8 0 . 6 9 0 . 4 9 2 . 4 9 0 . 6 8 1 . 1 1 2 . 9 0 3 . . 3 2 6 1 . 4 8 5 1 . 1 8 5 1 . 9 3 4 1 . 5 9 3 1 . 4 2 3 1 . 3 2 3 1 . 7 8 2 1 . 0 8 2 1 . 5 8 1 1 . 7 3 1 1 . 2 0 1 1 . 5 5 0 1 H O e M 6 8 4 . . 0 5 5 o s m d 0 0 4 . o s m d 9 9 3 . o s m d 7 9 3 . o s m d 5 9 3 . o s m d 4 9 3 . o s m d 2 9 3 . o s m d 0 9 3 . . 7 3 4 DG-4-27-DMSO _ CARBON_01 N N N N N N H OMe 85000 80000 75000 70000 65000 60000 55000 50000 45000 40000 35000 30000 25000 20000 15000 10000 5000 0 -5000 90 80 70 60 50 40 30 20 10 0 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 251 Figure 4.25: 1H and 13C{1H} NMR spectra of 4-22 DG-3-40-DMSO _PROTON_01 N N N 7 3 9 . 4 1 9 . 2 1 9 . 6 8 8 . 9 5 8 . 9 5 8 . 9 5 8 . 8 5 8 . 1 4 8 . 0 4 8 . 4 4 7 . 3 4 7 . 2 4 7 . 1 4 7 . 6 3 7 . 6 3 7 . 1 3 7 . 0 3 7 . 0 1 7 . 0 1 7 . 8 0 7 . 8 0 7 . 1 8 6 . 0 8 6 . 4 2 4 . 4 2 4 . 3 2 4 . 1 2 4 . 1 2 4 . 0 2 4 . 2 3 3 . o s m d 9 4 2 . o s m d 8 4 2 . o s m d 8 4 2 . N N N H O O 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 6 0 1 . 6 9 0 . 4 9 0 . 4 9 0 . 5 9 0 . 2 0 1 . 1 9 0 . 2 0 1 . 0 0 1 . 6 9 0 . 4 2 4 . DG-3-40-DMSO _ CARBON_01 N N N . 5 0 6 1 . 6 9 5 1 . 5 8 5 1 . 5 4 4 1 . 4 3 4 1 . 2 0 4 1 . 8 8 3 1 . 5 4 3 1 . 9 2 3 1 . 0 0 3 1 . 2 9 1 1 . 1 7 1 1 . 3 3 1 1 . 5 0 1 1 . 2 9 0 1 . 7 7 0 1 o s m d 5 0 4 . o s m d 3 0 4 . o s m d 1 0 4 . o s m d 8 9 3 . o s m d 9 9 3 . o s m d 6 9 3 . o s m d 4 9 3 . . 7 4 6 . 4 4 6 N N N H O O 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 -10 252 45000 40000 35000 30000 25000 20000 15000 10000 5000 0 110 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 4.26: 1H and 13C{1H} NMR spectra of 4-23 DG-3-85-DMSO _PROTON_01 N N N 3 5 9 . 2 2 9 . 0 2 9 . 9 8 8 . 1 6 8 . 1 6 8 . 0 6 8 . 0 6 8 . 8 4 8 . 7 4 8 . 5 4 7 . 4 4 7 . 3 4 7 . 2 4 7 . 8 3 7 . 7 3 7 . 4 0 7 . 4 0 7 . 3 0 7 . 6 1 6 . 5 1 6 . 5 1 6 . O 2 H 2 3 3 . 3 7 3 . o s m d 9 4 2 . o s m d 8 4 2 . o s m d 8 4 2 . N N N H OMe OMe 8 7 0 . 4 9 0 . 6 9 0 . 4 9 0 . 6 9 0 . 6 0 1 . 4 0 1 . 8 9 1 . 8 9 0 . 9 2 6 . 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 65000 60000 55000 50000 45000 40000 35000 30000 25000 20000 15000 10000 5000 0 -5000 253 Figure 4.27: 1H and 13C{1H} NMR spectra of 4-24 2 7 8 . 2 7 8 . 0 7 8 . 0 7 8 . 7 5 8 . 9 4 8 . 7 3 8 . 7 3 8 . 6 3 8 . 6 3 8 . 0 2 8 . 9 1 8 . 4 2 7 . 3 2 7 . 2 2 7 . 1 2 7 . 8 1 7 . 7 1 7 . 4 9 6 . 4 9 6 . 0 9 6 . 9 8 6 . 4 8 6 . 3 8 6 . 2 8 6 . 1 8 6 . O D H 8 8 4 . H O e M 4 3 3 . d o 3 d c 0 3 3 . 5 8 3 . 6 8 2 . 5 8 2 . 4 8 2 . DG-3-88-MeOH_PROTON_01 N N N N N N CF3 N NH2 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 3 0 1 . 4 1 0 . 7 9 0 . 7 0 1 . 4 9 0 . 1 0 1 . 1 0 1 . 3 9 0 . 2 0 1 . 5 9 0 . 7 7 3 . 8 1 4 . . 0 3 6 1 . 9 0 6 1 . 8 8 5 1 . 0 7 4 1 . 7 4 4 1 . 3 0 4 1 . 9 3 3 1 . 3 0 3 1 . 6 9 2 1 . 4 9 2 1 . 1 9 2 1 . 9 8 2 1 . 8 8 2 1 . 7 6 2 1 . 4 6 2 1 . 5 4 2 1 . 3 2 2 1 . 8 9 1 1 . 8 9 1 1 . 6 3 1 1 . 6 3 1 1 . 6 3 1 1 . 5 3 1 1 . 3 2 1 1 . 5 6 0 1 H O e M 8 9 4 . M C D 7 4 5 . d o 3 d c 5 9 4 . d o 3 d c 3 9 4 . d o 3 d c 2 9 4 . d o 3 d c 0 9 4 . d o 3 d c 8 8 4 . d o 3 d c 7 8 4 . d o 3 d c 5 8 4 . . 9 5 4 . 6 8 6 e n a x e H 1 2 3 . e n a x e h 3 0 2 . e n a x e h 2 4 1 . DG-3-88_ CARBON_01 N N N N N N CF3 N NH2 21000 20000 19000 18000 17000 16000 15000 14000 13000 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 -1000 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 -10 254 Figure 4.28: 1H and 13C{1H} NMR spectra of 4-25 DG-3-84-Methanol_PROTON_01 N N N 4 8 8 . 3 8 8 . 2 8 8 . 1 8 8 . 4 5 8 . 2 4 8 . 1 4 8 . 1 4 8 . 1 4 8 . 9 1 8 . 8 1 8 . 9 2 7 . 8 2 7 . 7 2 7 . 6 2 7 . 1 9 6 . 0 9 6 . O D H 8 8 4 . d o 3 d c 1 3 3 . d o 3 d c 1 3 3 . d o 3 d c 1 3 3 . d o 3 d c 0 3 3 . d o 3 d c 0 3 3 . 8 6 3 . 6 6 3 . 5 6 3 . 3 6 3 . 4 2 1 . 2 2 1 . 1 2 1 . N N N 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 8 9 0 . 1 9 0 . 0 9 0 . 1 9 0 . 9 9 0 . 1 0 1 . 8 0 4 . 1 2 6 . . 6 0 6 1 . 5 9 5 1 . 3 7 5 1 . 4 3 4 1 . 8 8 3 1 . 5 2 3 1 . 8 8 2 1 . 2 8 1 1 . 1 1 1 1 . 0 4 0 1 d o 3 d c 1 8 4 . d o 3 d c 9 7 4 . d o 3 d c 8 7 4 . d o 3 d c 6 7 4 . d o 3 d c 4 7 4 . d o 3 d c 2 7 4 . d o 3 d c 1 7 4 . . 0 2 4 . 1 2 1 DG-3-84-Methanol_ CARBON_01 N N N N N N 8500 8000 7500 7000 6500 6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 -500 300 250 200 150 100 50 0 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 255 Figure 4.29: 1H and 13C{1H} NMR spectra of 4-26 DG-3-92-tt9-13-DMSO _PROTON_01 5 9 9 . 4 1 9 . 2 1 9 . 1 9 8 . 3 6 8 . 2 6 8 . 4 5 8 . 3 5 8 . 7 3 8 . 4 9 7 . 2 9 7 . 4 5 7 . 3 5 7 . 1 5 7 . 9 4 7 . 8 4 7 . 7 4 7 . 6 4 7 . 2 4 7 . 0 4 7 . N N N N N N H CN O 2 H 2 3 3 . o s m d 8 4 2 . 5 9 0 . 5 9 0 . 5 9 0 . 8 9 0 . 4 9 0 . 2 0 1 . 1 1 1 . 3 1 2 . 8 9 0 . 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 . 0 0 6 1 . 0 0 6 1 . 6 8 5 1 . 6 4 4 1 . 9 1 4 1 . 5 0 4 1 . 0 3 3 1 . 5 0 3 1 . 8 9 2 1 . 2 5 2 1 . 9 3 2 1 . 7 1 2 1 . 6 9 1 1 . 5 9 1 1 . 8 1 1 1 . 3 0 1 1 . 1 9 0 1 o s m d 5 0 4 . o s m d 3 0 4 . o s m d 1 0 4 . o s m d 8 9 3 . o s m d 0 0 4 . o s m d 6 9 3 . o s m d 5 9 3 . 13 12 11 DG-3-92-tt9-13-DMSO _ CARBON_02 9 9 0 . 10 N N N N N N H CN 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 -10 256 20000 19000 18000 17000 16000 15000 14000 13000 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 -1000 65 60 55 50 45 40 35 30 25 20 15 10 5 0 -5 Figure 4.30: 1H and 13C{1H} NMR spectra of 4-29 DG-3-103-CDCl3 _PROTON_01 N NN O 4 7 8 . 4 7 8 . 2 7 8 . 2 7 8 . 7 4 8 . 6 4 8 . 6 4 8 . 6 4 8 . 2 4 8 . 2 3 7 . 1 3 7 . 0 3 7 . 9 2 7 . l 3 c d c 6 2 7 . 8 5 2 . 5 0 0 . 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 5 9 0 . 5 9 0 . 3 9 0 . 2 0 1 . 6 1 3 . . 3 2 9 1 . 8 3 4 1 . 3 2 4 1 . 4 4 3 1 . 0 9 2 1 . 0 0 2 1 . 1 3 1 1 DG-3-103-CDCl3 _ CARBON_01 N NN O l 3 c d c 3 7 7 . l 3 c d c 0 7 7 . l 3 c d c 8 6 7 . . 9 7 2 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 257 15000 14000 13000 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 -1000 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 -5 Figure 4.31: 1H and 13C{1H} NMR spectra of 4-30 DG-3-106-tt9-10-prod-cdcl3 _PROTON_01 N NN O N 9 7 8 . 9 7 8 . 7 7 8 . 7 7 8 . 3 3 8 . 3 3 8 . 2 3 8 . 2 3 8 . 6 7 7 . 3 7 7 . l 3 c d c 6 2 7 . 5 1 7 . 4 1 7 . 3 1 7 . 2 1 7 . 0 6 5 . 8 5 5 . 0 1 3 . 0 9 2 . 13 12 11 10 9 8 6 9 0 . 6 8 1 . 9 9 0 . 0 0 1 . 7 3 0 1 . 6 f1 (ppm) 3 0 3 . 4 1 3 . 5 4 3 2 1 0 -1 DG-3-106-prod-CDCL3 _ CARBON_01 N NN . 4 2 8 1 . 8 2 5 1 . 1 3 4 1 . 9 9 3 1 . 4 4 3 1 . 7 9 2 1 . 3 8 1 1 . 5 4 1 1 . 9 2 9 O N l 3 c d c 4 7 7 . l 3 c d c 1 7 7 . l 3 c d c 9 6 7 . . 0 5 4 . 3 7 3 22000 21000 20000 19000 18000 17000 16000 15000 14000 13000 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 -10 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 258 Figure 4.32: 1H and 13C{1H} NMR spectra of S2 DG-4-13-CDCl3 _PROTON_01 Br N 3 4 7 . 1 4 7 . l 3 c d c 6 2 7 . 3 2 7 . 1 2 7 . 0 5 3 . 2 5 2 . 1 5 2 . 9 4 2 . 8 4 2 . 4 0 1 . 3 0 1 . 1 0 1 . 7 0 0 . 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 8 8 0 . 3 9 0 . 3 0 1 . 9 0 2 . 7 0 3 . 1 0 -1 DG-4-13-CDCl3 _ CARBON_01 Br N . 3 9 3 1 . 3 1 3 1 . 6 0 3 1 . 5 0 2 1 l 3 c d c 4 7 7 . l 3 c d c 2 7 7 . l 3 c d c 9 6 7 . . 0 7 5 . 9 6 4 . 9 1 1 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 259 8000 7500 7000 6500 6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 -500 40 38 36 34 32 30 28 26 24 22 20 18 16 14 12 10 8 6 4 2 0 -2 -4 Figure 4.33: 1H and 13C{1H} NMR spectra of S3 8 2 7 . 7 2 7 . 0 1 7 . 8 0 7 . 9 8 6 . 0 9 6 . 1 7 6 . 0 7 6 . 6 7 5 . 0 9 4 . 6 2 4 . 3 7 3 . 1 4 3 . O 2 H 4 3 3 . o s m d 0 5 2 . 5 4 2 . 4 4 2 . 2 4 2 . 1 4 2 . 8 9 0 . 6 9 0 . 5 9 0 . DG-4-16-DMSO _PROTON_01 NH HN NHPMB N 13 12 11 10 9 8 7 6 f1 (ppm) 2 1 2 . 7 1 2 . 9 8 2 . 3 0 2 . 0 0 1 . 5 5 1 . 5 4 0 2 . 5 3 3 . 1 9 1 . 0 9 3 . 4 3 2 6 0 6 . 1 0 -1 . 1 8 5 1 . 1 1 5 1 . 7 2 3 1 . 1 1 3 1 . 1 9 2 1 . 7 8 2 1 . 5 2 2 1 . 6 3 1 1 o s m d 0 0 4 . o s m d 9 9 3 . o s m d 7 9 3 . o s m d 5 9 3 . o s m d 4 9 3 . o s m d 2 9 3 . o s m d 0 9 3 . . 6 6 5 . 0 5 5 . 8 5 4 . 6 3 4 . 7 1 1 DG-4-16-DMSO _ CARBON_01 NH HN NHPMB N 18000 17000 16000 15000 14000 13000 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 -1000 55 50 45 40 35 30 25 20 15 10 5 0 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 260 Figure 4.34: 1H and 13C{1H} NMR spectra of 4-27 DG-4-25-2nd-column-MeOD _PROTON_01 N N N 8 9 8 . 6 9 8 . 6 5 8 . 1 4 8 . 0 4 8 . 0 4 8 . 9 2 8 . 8 2 8 . 9 5 7 . 7 5 7 . 7 2 7 . 6 2 7 . 1 2 7 . 0 2 7 . 9 1 7 . 8 1 7 . 1 1 7 . 0 1 7 . 0 9 4 . 7 5 3 . 8 3 3 . 2 3 3 . d o 3 d c 2 3 3 . 2 3 3 . 1 3 3 . 1 3 3 . 8 5 2 . 7 5 2 . 5 5 2 . 4 5 2 . 1 1 1 . 9 0 1 . 8 0 1 . N N N N H 9 8 0 . 2 9 0 . 3 9 0 . 3 9 0 . 5 9 1 . 5 0 2 . 9 1 1 . 0 0 1 . 13 12 11 10 9 8 7 6 f1 (ppm) DG-4-25-2nd-column-MeOD _ CARBON_01 N N N . 4 0 6 1 . 8 9 5 1 . 6 7 5 1 . 5 3 4 1 . 2 9 3 1 . 9 8 3 1 . 8 2 3 1 . 6 1 3 1 . 7 9 2 1 . 6 9 2 1 . 9 9 1 1 . 3 8 1 1 . 6 0 1 1 . 1 7 0 1 7 6 1 . 5 6 9 1 . 8 8 0 . 8 2 4 . 4 3 2 5 3 6 . 1 0 -1 d o 3 d c 1 8 4 . d o 3 d c 9 7 4 . d o 3 d c 8 7 4 . d o 3 d c 6 7 4 . d o 3 d c 4 7 4 . d o 3 d c 3 7 4 . d o 3 d c 1 7 4 . . 3 6 5 . 9 5 4 9 9 . N N N N H 35000 30000 25000 20000 15000 10000 5000 0 420 400 380 360 340 320 300 280 260 240 220 200 180 160 140 120 100 80 60 40 20 0 -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 -10 261 Figure 4.35: 1H and 13C{1H} NMR spectra of 4-28 DG-3-130-DMSO _PROTON_01 7 5 9 . 6 1 9 . 5 1 9 . 8 8 8 . 0 6 8 . 0 6 8 . 9 5 8 . 9 5 8 . 6 4 8 . 5 4 8 . 5 7 7 . 3 7 7 . 5 4 7 . 4 4 7 . 3 4 7 . 2 4 7 . 5 3 7 . 4 3 7 . 3 3 7 . 2 3 7 . 0 3 7 . 9 9 6 . 7 9 6 . 6 9 6 . 4 7 5 . N N N N N N H o s m d 9 4 2 . o s m d 8 4 2 . o s m d 8 4 2 . 3 3 3 . 0 8 0 . 2 9 0 . 8 9 0 . 7 9 0 . 8 9 0 . 5 0 2 . 8 0 1 . 9 1 3 . 2 0 1 . 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 DG-3-130-DMSO _ CARBON_02 N N N N N N H . 5 0 6 1 . 8 9 5 1 . 5 8 5 1 . 5 4 4 1 . 9 0 4 1 . 3 0 4 1 . 9 2 3 1 . 0 0 3 1 . 0 9 2 1 . 0 2 2 1 . 8 9 1 1 . 2 9 1 1 . 4 0 1 1 . 1 8 0 1 o s m d 5 0 4 . o s m d 3 0 4 . o s m d 1 0 4 . o s m d 9 9 3 . o s m d 8 9 3 . o s m d 6 9 3 . o s m d 4 9 3 . 19000 18000 17000 16000 15000 14000 13000 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 -1000 300 250 200 150 100 50 0 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 262 Figure 4.36: 1H and 13C{1H} NMR spectra of 4-31 DG-4-62-tt18-26-CDCl3 _PROTON_01 2 5 8 . 2 5 8 . 2 5 8 . 1 5 8 . 0 5 8 . 6 6 7 . 5 6 7 . 3 6 7 . 3 6 7 . 3 6 7 . 2 6 7 . 1 6 7 . 1 6 7 . 0 4 7 . 0 4 7 . 9 3 7 . 9 3 7 . 8 3 7 . 7 3 7 . 7 3 7 . 0 3 7 . 0 3 7 . 9 2 7 . 9 2 7 . 7 2 7 . 7 2 7 . N N N Cl l 3 c d c 6 2 7 . 6 2 7 . 5 2 7 . 8 7 6 . 8 7 6 . 8 7 6 . 7 7 6 . 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 4 9 1 . 5 9 1 . 3 0 1 . 6 1 2 . 2 9 0 . DG-4-62-tt18-26-CDCl3_CARBON_02 . 0 1 6 1 . 7 9 5 1 . 3 9 5 1 . 2 5 3 1 . 2 1 3 1 . 8 4 2 1 . 1 4 2 1 . 3 3 2 1 . 4 1 2 1 . 7 5 1 1 . 7 9 0 1 . 8 6 0 1 N N N Cl l 3 c d c 3 7 7 . l 3 c d c 1 7 7 . l 3 c d c 8 6 7 . 8500 8000 7500 7000 6500 6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 -500 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 263 Figure 4.37: 1H and 13C{1H} NMR spectra of S6 DG-4-62-tt11-12-CDCl3 _PROTON_01 7 7 8 . 7 7 8 . 6 7 8 . 6 7 8 . 4 5 8 . 3 5 8 . 2 2 8 . 2 2 8 . 3 6 7 . 2 6 7 . 9 3 7 . 9 3 7 . 7 3 7 . 7 3 7 . 7 3 7 . 6 3 7 . 6 3 7 . 9 2 7 . 8 2 7 . N N N Cl l 3 c d c 7 2 7 . 6 2 7 . 6 2 7 . 5 2 7 . 6 0 7 . 5 0 7 . 2 7 6 . 2 7 6 . 2 7 6 . 1 7 6 . 0 3 5 . O 2 H 4 3 2 . 0 1 0 . 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 7 9 0 . 5 9 0 . 3 9 0 . 0 0 1 . 6 0 1 . 8 1 1 . 4 9 0 . 7 9 0 . DG-4-62-tt11-12-CDCl3 _ CARBON_01 N N N Cl . 6 1 6 1 . 1 9 5 1 . 4 7 5 1 . 3 5 3 1 . 4 1 3 1 . 7 5 2 1 . 0 4 2 1 . 7 2 2 1 . 9 0 2 1 . 4 6 1 1 . 9 5 1 1 . 9 7 0 1 l 3 c d c 3 7 7 . l 3 c d c 0 7 7 . l 3 c d c 8 6 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 -10 264 20000 19000 18000 17000 16000 15000 14000 13000 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 -1000 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 -5 Figure 4.38: 1H and 13C{1H} NMR spectra of 4-32 DG-4-63-DMSO _PROTON_01 0 3 8 . 6 0 8 . 5 0 8 . 4 0 8 . 1 0 8 . 8 5 7 . 8 3 7 . 6 2 7 . 1 2 7 . 4 1 7 . 7 0 7 . 6 9 6 . 4 7 6 . 7 5 4 . 3 3 3 . o s m d 9 4 2 . o s m d 8 4 2 . o s m d 8 4 2 . N N N N H Cl 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 5 9 0 . 3 0 2 . 7 9 0 . 1 0 4 . 1 0 2 . 8 0 1 . 3 9 0 . 2 0 2 . . 2 2 6 1 . 8 9 5 1 . 7 8 5 1 . 4 9 3 1 . 7 4 3 1 . 1 1 3 1 . 4 0 3 1 . 0 9 2 1 . 5 8 2 1 . 2 8 2 1 . 8 5 2 1 . 3 3 2 1 . 0 2 2 1 . 8 0 2 1 . 1 7 0 1 . 9 7 9 o s m d 9 9 3 . o s m d 7 9 3 . o s m d 5 9 3 . o s m d 4 9 3 . o s m d 2 9 3 . . 6 3 4 DG-4-63-DMSO _ CARBON_02 N N N N H Cl 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 -10 265 13000 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 -1000 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 Chapter 5: Progress towards the design and synthesis of molecular proteasome staples and probes 5.1 Introduction Proteasomes are intricate protein complexes responsible for the degradation and recycling of damaged or unwanted proteins within cells. These cellular "recycling centers" play a vital role in maintaining protein homeostasis and regulating various cellular processes, including cell cycle progression, DNA repair, and immune response.1 Among the various types of proteasomes, the human 20S proteasome has gained significant attention due to its central role in protein degradation. The 20S proteasome is composed of four stacked rings, each containing seven distinct subunits, forming a barrel-shaped structure (Figure 5.1). It possesses three types of proteolytic activities: caspase-like, trypsin-like, and chymotrypsin-like, enabling the proteasome to cleave proteins at different sites.2 The 20S proteasome can be divided into two distinct rings: the outer alpha rings and the inner beta rings (Figure 5.1a).3 The alpha ring of the 20S proteasome forms the outermost layer of the complex and provides structural stability. It consists of seven distinct alpha subunits (α1-α7) arranged in a circular manner (Figure 5.1c). These alpha subunits share a conserved proteasome-activating nucleotidase (PAN) domain, which contributes to the overall assembly and stability of the 20S proteasome complex. Additionally, specific alpha subunits within the alpha ring have specialized functions. For example, α1 and α2 subunits are involved in regulating the opening and closing of the proteasome gate for substrate entry and exit, while the α3 subunit contributes to the assembly and integrity of the complex, and the α4 subunit plays a role in substrate recognition and processing.4-5 266 Figure 5.1: Overview of the structure of 20S proteasome Dysregulation of proteasome activity has been implicated in several diseases, including cancer, neurodegenerative disorders, and autoimmune conditions (see Chapter 3).6-7 Consequently, the development of proteasome modulators has been proposed as a novel strategy to treat these neurodegenerative diseases.8-13 Our lab and others have identified various activators of the 20S proteasome that are hypothesized to interact directly the 𝛼-ring of the 20S proteasome to induce an open gate conformation.8,11-13 To understand the mechanism of activation of 20S proteasome by our small molecules, atomic force microscopy (AFM) imaging was used to study the gate opening dynamics 267 of 20S proteasome with TCH-165 (a known 20S proteasome activator).8 While AFM imaging showed that there was a significant increase in the population of open gate 20S proteasome particles in treated sample compared to control, binding mechanism could not be elucidated using this technique. In addition, previous computational studies from our lab showed that the small molecule activators of 20S proteasome interact with the inter-subunit pockets on the 𝛼-ring of the 20S proteasome to induce the open gate conformation, however, the binding mode of this small molecules still remain unclear via molecular docking.8,11-13 Identification of ligands binding site, especially the important subunits and residues needed to induce 20S proteasome gate opening would have a huge impact on the designing of future proteasome activators. Furthermore, if the small molecule binding site is determined to be the inter-subunit pockets on the 𝛼-rings, this would validate the molecular docking approach currently being used to predict the small molecule binding to the 20S proteasome. Several attempts have been made over the years in the group to identify the binding the binding site of small molecule activators of 20S proteasome using mass spectrometry and Cryo- EM. These attempts have failed partially due to the significant size difference between the 20S proteasome, which is approximately 750,000 Da, and the much smaller ligands used in the study, which are about 500 Da. In recent Cryo-EM attempts by Allison Vanecek, from the Tepe group, to obtain the structure of 20S proteasome bound to small molecule activator (SS-4-15, EC200 = 1.2 ± 0.6 µM) of the 20S proteasome, the structure of the ligand-bound 20S proteasome could not be obtained, likely due to the low proportion of ligand-bound 20S proteasomes complex compared to unbound ones. The 3D structure is reconstructed by averaging various 2D classes, so if only a small fraction of 20S proteasomes have a ligand bound, the electron density of the ligand may be 268 averaged out in the final 3D structure. Additional, high concentration of compound (∼200 µM) was used and resulted in denaturing of the 20S proteasome during sample preparation. We proposed we could overcome these challenges by utilizing more potent small molecule binders of the 20S proteasome. These compounds would have lower off rate and would increase the chance of obtaining a ligand-bound 20S proteasome. We hypothesized that we could design molecules that would target multiple binding pockets on the 20S proteasome since crystal structure and cryo-EM showed that the 20S proteasome have seven binding pockets on each 𝛼-ring. We called these molecules “proteasome staples”. The proteasome staples are also hypothesized to be better binders of the 20S proteasome and lock the 20S proteasome in a more rigid conformation that would enable image collection during cryo-EM studies better. The proteasome staples are also expected to have higher potency than traditional proteasome modulators, as they target multiple binding sites simultaneously. However, translating these molecules into therapeutics would be challenging due to their large size and their violation of Lipinski's Rule of Five for oral drugs. Nonetheless, they could serve as valuable tools for understanding how 20S proteasome activators bind to the protein. 5.2 Results and Discussion 5.2.1 Designing molecules i.e. “proteasome staples” that target multiple inter-subunit pockets on the 𝛼-rings of the 20S proteasome The proteasome staples are made of two main components: small molecule proteasome modulators and a linker (Figure 5.2). Proteasome modulators are compounds hypothesized to target the inter-subunit pockets of the 20S proteasome. Each proteasome modulator at each end of the linker is designed to target a different but adjacent inter-subunit pocket. When designing the staples, there were series of things that were considered. The first thing was identifying the 269 proteasome modulator of choice from the list of proteasome activators from our lab. A good choice of molecule would be one that have non-specific binding on the proteasome inter-subunit pocket i.e. the molecule should bind on multiple binding pockets on the 𝛼-ring, preferrable adjacent binding pockets. This would avoid having to use two different molecules to target different inter- subunit pocket on the 20S proteasome. Also, the molecule of choice should be easily functionalized with a handle that would allow easy coupling to any linker of choice. Figure 5.2: Generic structure for the 20S proteasome staples O N H H N O = Linker = Proteasome modulator Among all the known proteasome activators in our lab, we selected compound 5-1 (Figure 5.3). Compound 5-1 was selected as a molecule of choice because it has an indole moiety that could be easily functionalized at the C-3 position as a handle to incorporate a linker of choice. Although, docking studies predicted that 4 out of 9 preferred modes of 5-1 bind to the 𝛼1-2 inter- subunit pocket on the 20S proteasome and no other inter-subunit pocket. Interestingly, the derivatives of 5-1 such as the carboxylic acid 5-2 and amide derivatives 5-3 & 5-4 predominantly bind to both the 𝛼1-2 and 𝛼2-3 inter-subunits pockets making them a promising candidate for the proteasome staple design. Compounds 5-3 and 5-4 were used to mimic how one end of the proteasome staple would bind to the 20S proteasome. 270 Figure 5.3: Structure 5-1 and derivatives (a) Selected proteasome activator Cl N C-3 O N Cl (b) Derivatives of 5-1 O OH Cl 5-1 O NH Cl N O N Cl N O N Cl Cl O NH Cl N O N Cl Cl 5-2 Cl 5-3 Cl 5-4 Compound 5-1 was also selected because docking studies showed the C-3 position in the indole moiety to be exposed (Figure 5.4a), thereby indicating that there would be high possibility of maintaining similar binding pose even after functionalization with a linker. Similar result was seen with compound 5-3 in the 𝛼1-2 inter-subunit pocket of the 20S proteasome (Figure 5.4b). Both docking results further support the possibility of the compound maintaining its current binding pose in the proteasome after functionalizing with a linker and also allow the linker to extended outside of the binding pocket into the adjacent inter-subunit pocket for the other end of the staple to bind. 271 Figure 5.4: Docking pose of compounds 5-1 and 5-3 in 20S proteasome inter-subunit binding site Another important information to mention is that compound 5-3 and 5-4 maintained proteasome activity with EC200 of 3.1 µM and 2.4 µM respectively when compared to 5-1 with EC200 = 3.1 µM, indicating that the additional functionality at the C-3 position of the indole does not significantly impact the binding of the molecule to the 20S proteasome, which is desirable for eventual functionalization with a linker. Following the selection of the proteasome modulator, the next thing was selecting the linker of choice. Polyethylene glycol was selected as a linker of choice due to its water solubility and great biocompatibility.14 The biggest challenge after identifying the linker of choice was determining the suitable PEG length. To get an insight into the approximate number of PEG needed for compounds to reach both the 𝛼1-2 and 𝛼2-3 inter-subunit pockets, the distance between these two inter-subunit pockets was measured using Pymol and was determined to be between 28 Å – 30 Å (Figure 5.5) which is equivalent to about 5-6 PEG units. Although this length only indicates the straight distance between the two inter-subunit pockets, it does not account for folding or extra PEG units that would be needed within each of the inter-subunit pocket to extend to the outside of the proteasome. To account for folding and extra PEG units, more than 6 PEG units would be 272 needed to reach both the 𝛼1-2 and 𝛼2-3 inter-subunit pockets of the proteasome. Hence, several staples with varying PEG length were synthesized as discussed in section 5.2.3. Figure 5.5: Distance between the 𝛼1-2 and 𝛼2-3 inter-subunit pockets in Å 5.2.2 Synthesis of compound 5-1 and derivatives Compound 5-1 was synthesized in two steps starting from 5-5. Compound 5-5 was first acylated with chloroacetyl chloride to give amide 5-6 which was then alkylated with 6- chloroindole to give the desired compound 5-1 in 83% yield (Scheme 5.1). Vilsmeier-Haack reaction on 5-1 gave aldehyde 5-7 which was then oxidized with potassium permanganate to give carboxylic acid 5-2 in 55% yield. Alternatively, the carboxylic acid 5-2 can be synthesized from amide 5-6 by direct alkylation with 6-chloroindole-3-carboxylic acid in 87% yield, making compound 5-2 accessible in two steps from commercially available starting material 5-5 (Scheme 5.1). 273 Scheme 5.1: Synthesis of compound 5-1 and 5-2 Cl O + Cl Cl Et3N (2.0 equiv) DCM, 0 °C - rt, 16 h 78% Cl NH 5-5 Cl O N Cl 5-6 O H Cl N H (1.0 equiv) Cl NaH (1.02 equiv) DMF, 0 °C - rt, 16 h 83% 6-chloroindole-3-carboxylic acid NaH (2.02 equiv) DMF, rt, 16 h 87% O OH Cl O N N Cl 5-1 POCl3 (1.3 equiv) DMF, 0 °C – rt, 2 h then, NaOH workup 97% Cl Cl O N N KMnO4 (2.0 equiv) acetone, rt, 5 h 55% Cl O N N Cl Cl Cl 5-7 Cl 5-2 Following the synthesis of 5-2, compounds 5-3 and 5-4 were synthesized through HBTU coupling of the carboxylic with the corresponding amine. Compound 5-3 was synthesized in 61% yield and 5-4 in 81% yield. Scheme 5.2: Synthesis of amide 5-3 and 5-4 O OH Cl N O N Cl 5-2 O R NH R + H2N HBTU (1.2 equiv) DIPEA (2.4 equiv) DMF, rt, 12 h Cl N Cl O N Cl Cl 5-3: R = Me (61%) 5-4: R = n-Bu (81%) 5.2.3 Synthesis and evaluation of proteasome activity of the 20S proteasome staples From section 5.2.1, we determined that more than six PEG units is needed in the proteasome staple to be able to reach the 𝛼1-2 and 𝛼2-3 inter-subunit pockets. This number does not account for the folding and extra PEG units that would be needed within each of the inter- 274 subunit pocket to extend to the outside of the proteasome. I synthesized various staples with PEG units ranging from 5 to 21 to determine the optimal number of PEG units needed to maximize the activity of the staples, using compound 5-1 as the proteasome modulator. Carboxylic acid 5-2 was first coupled with PEG-5 (five PEG units) and PEG-8 (eight PEG units) amine via HBTU coupling, producing amides 5-8 and 5-9, respectively (Scheme 5.3). Boc deprotection of the protected end of the linkers in compound 5-8 with trifluoroacetic acid (TFA) yielded amine 5-10 in 87% over two steps. Similarly, 5-9 was deprotected to yield amine 5-11 in 67% over two steps. Compounds 5-10 and 5-11 were then reacted with carbonyl diimidazole (CDI) to give the proteasome staples 5-12 and 5-13 with 10 and 16 PEG units respectively. Scheme 5.3: Synthesis of proteasome staples 5-10 & 5-11 O OH NH2 O O n N H (1.0 equiv) HBTU (1.2 equiv) DIPEA (2.4 equiv) DMF, rt, 16 h Cl N O N Cl 5-2 CDI (1.0 equiv) THF, 70 °C, 12 h Cl Cl Cl R NH O n NH TFA (20 equiv) O DCM, rt, 16 h 5-10: n = 5, R= H, 87% 5-11: n = 8, R = H, 67% O Cl O N N Cl Cl 5-8: n = 5, R = Boc 5-9: n = 8, R = Boc O N H O N H N H O n O n N H N N O 5-12: n = 5, 76% 5-13: n = 8, 88% O Cl N N O Cl Cl Cl Additionally, the length of the PEG units could be increased to 21 PEG units by reacting compound 5-10 with dicarboxylic acid 5-14 to give staple 5-15 in 57% yield. 275 Scheme 5.4: Synthesis of proteasome staples 5-15 Cl O N NH O N NH2 O 8 HO O 5 OH O 5-14 (1.0 equiv) O HBTU (2.0 equiv) DIPEA (4.8 equiv) DMF, rt, 24 h 57% Cl Cl 2.0 equiv 5-11 H N O 5 O 8 O H N O O 8 HN O O NH 5-15 N O N Cl Cl Cl Cl N N O Cl Cl When evaluating the proteasome activity of these compounds, we envisioned that the staples could either bind to the 𝛼-ring of the 20S proteasome and activate the proteasome by inducing an open gate conformation or stabilize the closed conformation and therefore acting as a proteasome inhibitor. Following the synthesis of the three proteasome staples above, 5-12, 5-13, & 5-15, they were evaluated in both the 20S proteasome activation and inhibition assay by Allison Vanecek using fluorogenic 7-amino-methylcoumarin (AMC) conjugated small peptides assay. The experiment showed that compounds 5-12, 5-13, & 5-15 are inactive in the proteasome activation assay, even showing that there is a slight decrease in the activity of the 20S proteasome when compared to that of the vehicle. The result of this experiment is summarized in Figure 5.6A. However, when the compounds were tested in proteasome inhibition assay, they showed inhibition of the 20S proteasome at nanomolar concentration. Compound 5-12 with 10 PEG units showed the best activity with IC50 = 111 ± 2 nM. Compound 5-13 with 16 PEG units have an IC50 = 124 ± 2 nM and compound 5-15 with 21 PEG units have an IC50 196 ± 5 nM (Figure 5.6B). Also, when comparing the proteasome inhibition activity of 5-15 to 5-12, the enormous reduction in the activity could be the result of the length of the PEG linker which is significantly longer than needed. 276 Figure 5.6: Proteasome activation and inhibition assays with proteasome staples. Data obtained by Allison Vanecek To understand if the staples target two binding pockets on the 𝛼-ring has intended during the design of the compounds, compound 5-16 with 5 PEG units which would be too short to bridge the distance between the two inter-subunit pockets was also synthesized. Compound 5-16 should show little to no activity since more than 6 PEG units is needed for activity as determined in section 5.2.1. Compound 5-16 was synthesized from 5-10 through HBTU coupling with carboxylic 5-2 to yield the desired product in 78% yield (Scheme 5.5). As expected, 5-16 was inactive in the proteasome inhibition assay with IC50 > 20 µM further confirming the important of the right linker length for the staples. 277 Scheme 5.5: Synthesis of compound 5-16 NH2 O 5 Cl 5-2 (1.0 equiv) DIPEA (2.4 equiv) HBTU (1.0 equiv) DMF, rt, 12 h 78% O N NH O N 5-10 Cl Cl Cl N O N Cl Cl O NH O 5 5-16 Cl O N NH O N Cl Cl Following the synthesis of the three staples, I set out to investigate the effect of using a less active compound on both ends of the linker and how that would translate to the activity of the staples. Previous unpublished SAR studies on similar scaffolds showed that removing the chlorine on the parent molecule would lead to a significant loss in proteasome activity. I synthesized compound 5-17 which has no chlorine on either the phenyl rings or indole in two steps according to Scheme 5.6. As expected, compound 5-17 have an EC200 = 29 µM, which is about 10-fold less active than compound 5-1 with EC200 = 3.1 µM. Scheme 5.6: Synthesis of control compound 5-17 + NH Cl O Cl Et3N (2.0 equiv) DCM, 0 °C - rt, 16 h 96% O N Cl N H (1.0 equiv) NaH (1.02 equiv) DMF, 0 °C - rt, 16 h 86% O N N 5-19 5-17 After the identification of 5-17 as a less active proteasome enhancer (compared to 5.1) of the same class, proteasome staple 5-18 was synthesized using this compound as the proteasome modulator. The staple, 5-18 was synthesized similarly to the synthesis of 5-12 as shown in Scheme 5.7 and was evaluated in proteasome inhibition assay. Compound 5-18 have an IC50 = 1200 nM 278 which is also about 10-fold less active when compared to it chlorinated counterpart 5-12 with IC50 = 111 nM. This was an exciting result that showed that the activity of the proteasome modulator directly translates to the activity of the proteasome staple and also support that the staples most likely interact with the inter-subunit pockets on the 𝛼-rings of the 20S proteasome. Scheme 5.7: Synthesis of proteasome staple 5-18 O OH O OH 1. O O N Cl N H (1.0 equiv) NaH (2.05 equiv) DMF, 0 °C - rt, 16 h 36% O N N 5-19 5-20 NH2 2. O O 5 N H (1.0 equiv) HBTU (1.2 equiv) DIPEA (2.4 equiv) TFA (20 equiv) DCM, rt, 16 h quant. NH2 O 5 CDI (1.0 equiv) THF, 70 °C, 12 h 55% NH O N 5-21 O N O N H NH O 5-18 O 5 O N H N O N N O N NH 5 O To verify the direct interaction of the proteasome staples with the 20S proteasome, I used surface plasmon resonance (SPR) to investigate the direct binding of the staple with the proteasome. The SPR experiments were done in collaboration with Miracle Olatunde and Erica Lisabeth at the MSU drug discovery core. The Surface Plasmon Resonance (SPR) assay is a straightforward, label-free optical technique utilized for detecting biomolecular interactions and determining the dissociation constant (KD) of proteins and their ligands. It operates by detecting changes in mass on the chip surface. In this method, a metal surface, typically composed of gold or silver, is coated with carboxymethylated dextran or streptavidin. The protein or ligand of interest 279 is immobilized onto the metal surface, and a solution containing the analyte in an appropriate buffer is passed over it. Binding between the protein and analyte leads to alterations in the refractive index of light passing through the prism, resulting in a reduction in intensity. These changes are monitored in real-time, and the data is graphed as response units (RU) over time, known as a sensogram. Due to the enormous size of the proteasome, we choose to immobilize the staple on a streptavidin chip and flow the proteasome on the chip. The interaction between the staple and the proteasome should lead to a significant change in the mass which can be easily detected. Compound 5-24, an analogue of proteasome staple 5-12 that is functionalized with biotin, was first synthesized. Biotin is needed to immobilize the compound on the streptavidin chip for SPR experiment. HBTU coupling of 5-10 with dicarboxylic acid 5-22 followed by deprotection to give the desired compound 5-23 which was then used to synthesize the biotinylated compound 5- 24 (Scheme 5.8). SPR study of compound 5-24 after immobilization on streptavidin coated chip showed no interaction with the 20S proteasome when the proteasome was flowed over the chip at varying concentrations. I hypothesized that it could be due to the short linker length between the biotin-streptavidin on the surface of the chip and the proteasome staple, leading to the molecule being too close to the chip and not enough distance for the proteasome to interact with the molecule. 280 Scheme 5.8: Synthesis of biotinylated proteasome staple 5-24 NH2 O 5 + HO Cl O N NH O N Cl 5-10 2.0 equiv Cl O HN O O 5-22 O 1.0 equiv 1. HBTU (2.40 equiv) DIPEA (4.80 equiv) OH DMF, rt, 24 h 2. DCM/TFA (3:2) rt, 16 h 40% (2 steps) Cl Cl O N NH O N NH O 5 O NH2 H N O O O N HN 5 O N Cl Cl Cl 5-23 Cl O HN H NH H S O HO HN H S (1.5 equiv) O NH H DMAP (10 mol%) EDC • HCl (1.5 equiv) DMF, rt, 1 h 58% NH O O 5 O NH H N O O 5-24 Cl O N NH O N Cl Cl O N HN 5 O N Cl Cl Cl To test this hypothesis, I redesigned compound 5-24 to compound 5-27 which include 4 PEG units between the biotin and the rest of the staple to allow room for interaction with the proteasome. Compound 5-27 was synthesized following a similar approach for the synthesis of compound 5-24. It is important to note that, I switched from N-boc-dicarboxylic acid linker 5-22 ($312 / 0.10 g from A2B Chemical), to a cheaper linker 5-25 ($30 / 100 g) during the synthesis of compound 5-27 to allow access to more materials at a cheaper cost (Scheme 5.9). Compound 5- 31 was also synthesized according to scheme 5.10 as a less active control for the SPR experiment. 281 Scheme 5.9: Synthesis of compound 5-27 NH2 5 HO O O OH Cl O N NH O N Cl Cl 5-10 O NHBoc 5-25 (1.0 equiv) HBTU (2.4 equiv) DIPEA (4.8 equiv) DMF, rt, 24 h then, DCM/TFA (1:2) 67% (two steps) O NH NH 5 O NH2 O HN O N 5-26 Cl O N Cl Cl O 5 HN O N O N Cl HO O O O 4 N H O NH H HN H S (1.0 equiv) HBTU (1.2 equiv) DIPEA (2.4 equiv) DMF, rt, 16 h 82% Cl Cl N H O O N N Cl O 5 NH HN O O O HN 5-27 H N 4 O O O 5 HN O Cl Cl S H NH H N H O N O N Cl Cl Cl Following the synthesis of 5-27 and 5-31, the two compounds were evaluated in the proteasome inhibition assay. Compound 5-27 showed proteasome activity with IC50 = 254 nM and the 5-31 lost its proteasome activity as expected with IC50 > 20 µM. The SPR study was then conducted using 5-27 and 5-31. Compound 5-27 was immobilized on channel 1 and 2 while compound 5-31 was immobilized on channel 3 of the streptavidin coated chip (Figure 5.7a). A serial dilution of purified 20S proteasome in PBS buffer was then flowed over all three channels and a sensogram showing increase in response units (RUs) with increase in concentration of proteasome over time was generated for both compounds 5-27 (Figure 5.7b) and 5-31 (Figure 5.7c). The sensogram was then converted into a graph of RUs against log of concentration as shown in Figure 5.7d and Figure 5.7e respectively. From this study, the KD of compound 5-27 282 was determined to be between 0.031 nM – 0.80 nM and the control compound 5-31 ranged between 0.51 nM – 15000 nM. Scheme 5.10: Synthesis of less active compound 5-31 O OH + Cl O NaH (2.1 equiv) OMe DMF, 0 °C - rt, 16 h Cl Cl N H (1.0 equiv) 28% O OH H2N O 5 (1.0 equiv) NHBoc HBTU (1.20 equiv) DIPEA (2.4 equiv) N O OMe 5-28 DMF Cl then, DCM/TFA (5:3) NH2 O 5 O N NH O OMe 5-29 HO O OH O NHBoc 5-25 (1.0 equiv) HBTU (2.4 equiv) DIPEA (4.8 equiv) DMF, rt, 24 h then, DCM/TFA (1:2) 70% (two steps) HO O O O 4 N H O NH H HN H S (1.0 equiv) HBTU (1.2 equiv) DIPEA (2.4 equiv) DMF, rt, 16 h 79% Cl Cl O NH NH 5 O NH2 O HN O N O OMe 5-30 N H O O N MeO O 5 NH HN O O O 5-31 O 5 HN O N O MeO H N 4 O O Cl S H NH H N H O HN O 5 HN O O N MeO Cl From the SPR experiment, I was able to get binding with the proteasome with both compound 5-27 and the control 5-31 unlike compound 5-24 where proteasome binding was not observed. This supports that the short distance between the chip surface and the binding staple in compound 5-24 may be preventing the binding of the staple to the proteasome. In addition, the control compound 5-31 has a higher and wider range of KD when compared to the active compound 5-27 indicating that the compound have less binding to the proteasome. 283 Figure 5.7: (a) Sensogram showing the immobilization of compound 5-27 on channel 1 (red) and 2 (pink) and control compound 5-31 on channel 3 (blue). (b) Sensogram showing increase in RUs with increase in concentration of proteasome over time on channel 1 & 2 for compound 5-27 (DG- 6-136) (c) Sensogram showing increase in RUs with increase in concentration of proteasome over time channel 3 for compound 5-31 (DG-6-137). (d) A graph of RU against (log) concentration of proteasome for compound 5-27 (DG-6-136). (e) A graph of RU against (log) concentration of proteasome for compound 5-31 (DG-6-137) After verifying the interaction between proteasome staples and 20S proteasome using the in vitro inhibition assay and SPR, we decided to use photoaffinity labelling (PAL) to study the interaction between the proteasome staples and the proteasome with the goal of determining the 284 subunit(s) on the 𝛼-ring that the staples interact with. Photoaffinity labelling (PAL) is a sophisticated method for studying interaction between small molecules and protein. Usually, a molecule of interest is functionalized with photoreactive group and upon photolysis, the molecule traps its binding partner, usually a protein, covalently.15 When PAL is combined with analytical techniques such as mass spectrometry, a wide range of studies can be conducted. This enables the mapping of interactions between small molecules and proteins, as well as the determination of the small molecule's binding site on the protein.16 Among the photoreactive groups utilized in photoaffinity labeling, diazirines have attracted considerable attention. This is owing to their small size, long irradiation wavelength (350–355 nm), which minimizes damage to biological systems, and their short lifetime upon irradiation, thus diminishing nonspecific binding.17 Upon irradiation of diazirines with light, they generates carbene with then covalently bind with the nearest target molecule through C–C, O–H and X–H (X = N,S) insertions. Only few studies have been done in the literature on photoaffinity labelling of the proteasome. In 2010, Geurink et. al. used a combination of photoreactive group and a bio- orthogonal ligation handle incorporated into peptides to identify the β-subunits the peptide was binding to in the proteasome.18 Similarly, Zhu et al. employed PAL to identify the α-subunits to which peptides binding to the 20S proteasome, inducing an open gate conformation, were binding.19 While these two studies involved PAL and 20S proteasome, none of them used small molecule regulators of the 20S proteasome. I started by first synthesizing compound 5-33 by functionalizing the proteasome staple with amine handle 5-26 with probe 5-32 (Scheme 5.11). Probe 5-32 contains diazirine for photo crosslinking and an alkyne for click chemistry with reporter tags. Probe 5-32 has be used significantly in the literature for proteomic profiling,20-25 photoaffinity based fragment screening,26-28 and ligand mapping29-32. Probe 5-32 is also 285 commercially available but quite expensive ($436 for 100 mg from A2B) and published synthetic approach to access the compound requires significant synthetic effort to obtain the product in good yield.20,27 I have developed a novel approach to synthesize 5-32 in fewer steps and with a higher overall yield. The manuscript detailing this work is currently being prepared. Scheme 5.11: Syntheses of diazirine functionalized proteasome staples as probes for photoaffinity labelling of the 20S proteasome O NH NH 5 O NH2 O HN O N 5-26 Cl O N Cl Cl O 5 HN O N O N Cl Cl Cl NN HO O 5-32 (1.0 equiv) HBTU (1.3 equiv) DIPEA (2.4 equiv) DMF, rt, 24 h 75% 5.3 Future direction N N O NH 5 O NH O HN 5-33 Cl O NH O N O N Cl Cl O 5 HN O N O N Cl Cl Cl Following the synthesis of 5-33, Sydney Cobb is currently exploring its use in photoaffinity labeling studies with the 20S proteasome. We aim to covalently bind 5-33 to the 20S proteasome, then determine which subunit the compound interacts with by using SDS-PAGE after tagging the alkyne handle with a BODIPY probe. After successful result with SDS-PAGE, the subunit interacting with the staple can be purified by clicking the alkyne handle with a biotin and the subunit purified by a streptavidin column. The purified subunit(s) can then be characterized using mass spectrometry to determine their identities. Following the failed attempts in obtaining cryo-EM structure of small molecule bound 20S proteasome complex using previous small molecules from our lab, 5-33 could be the suitable compound needed for the cryo-EM experiments, considering its potent and unique binding interaction. Furthermore, 5-33 can covalently bind to the proteasome through the diazirine functionality and thereby eliminate the off rate of ligand when obtaining cryo-EM analysis. Since 286 another challenge that was faced while obtaining cryo-EM structure is sorting ligand bound 20S proteasome from unbound 20S proteasome, the alkyne handle in 5-33 can undergo a click reaction with an azide conjugated Au nanoparticle which would allow better sorting of ligand bound and unbound 20S proteasome. 5.4 Conclusion In this study, bivalent compounds called the “proteasome staples” that targets multiple inter-subunit pockets were developed. The proteasome staples are made up of a PEG linker and 20S proteasome modulators at each end of the linker. The effect of the linker length and the activity of the proteasome modulator were investigated on the 20S proteasome inhibition activity of the staples. The proteasome staples exhibit 20S proteasome inhibition activity at nM concentration and the binding activity of the most active proteasome staple was also verified using SPR studies. In addition, the most active staple was functionalized with a diazirine group which would allow future studies of the compound interaction with 20S proteasome using photoaffinity labelling studies and ultimately obtaining a cryo-EM structure of small molecule bound 20S proteasome. This would allow identification and characterization of the inter-subunit pocket(s) being targeted by small molecule modulators of the 20S proteasome. 5.5 Experimental General Information All flasks were oven-dried overnight and cooled under nitrogen. Reactions were carried out under a nitrogen atmosphere unless stated otherwise. All chemicals and solvents were purchased from commercial sources and used without further purification, unless otherwise mentioned. THF and DCM were dried by passing it through packed column of alumina or over activated 3Å molecular sieves. Infrared spectra were recorded on a Jasco Series 6600 FTIR spectrometer. The mass 287 spectrometer ionization method was ESI with a quadrupole detector. 1H and 13C NMR spectra were recorded on a 500 MHz spectrometer. Chemical shifts are reported relative to the residue peaks of the solvent: CDCl3: 7.26 ppm for 1H and 77.0 ppm for 13C, CD3OD: 3.31 ppm for 1H and 47.6 ppm for 13C, and DMSO-d6: 2.50 ppm for 1H and 39.5 ppm for 13C. Signal multiplicities are given as s (singlet), br (broad), d (doublet), t (triplet), dd (doublet of doublet), and m (multiplet). Reaction carried out at room temperature (rt) implies temperature range usually between 20 – 22 °C. Column chromatography was performed using a Teledyne ISCO CombiFlash® NextGen system with prepacked columns (RediSep® Normal-phase silica, 20-40 microns). TLCs were performed on pre-coated 0.25 mm thick silica gel 60 F254 plates and pre-coated 150 um thick, visualized using UV light and iodine staining. Cl NH O + Cl Cl Et3N DCM, 0 °C - rt, 16 h Cl O N Cl Cl 5-5 Cl 5-6 2-chloro-N,N-bis(4-chlorophenyl)acetamide (5-6): An 100 mL round bottom flask equipped with magnetic stirrer was charged with bis(4-chlorophenyl)amine (3.0 g, 12.60 mmol) and dichloromethane (50 mL). The resulting solution was cooled to 0 °C and then triethylamine (3.5 mL, 25.20 mmol) was added. A solution of chloroacetyl chloride (1.1 mL, 13.86 mmol) in dichloromethane (1.5 mL) was added slowly over 15 mins. After 4 hours, additional solution of chloroacetyl chloride (1.1 mL, 13.86 mmol) in dichloromethane (1.5 mL) was added. The reaction was then warmed up to room temperature and allowed to stirred overnight. The reaction was then diluted with sodium carbonate (50 mL) and the organic layer was collected. The aqueous phase was further extracted with dichloromethane (2 x 50 mL) and the organic phases were combined. The organics were dried over sodium sulfate, decanted and concentrated under reduced pressure 288 to give the crude product. The crude was then recrystallized in ethylacetate/hexane (4:1) to give the pure product has a pink solid (3.10 g, 78% yield). mp = 103 °C. 1H NMR (500 MHz, CDCl3) δ 7.39 (br, m, 4H), 7.23 (br, m, 4H), 4.01 (s, 2H). 13C{1H} NMR (125 MHz, CDCl3) δ 166.2, 140.2, 130.5, 129.7, 127.3, 42.5. FTIR (cm−1): 3094, 2989, 1670, 1486. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C14H11Cl3NO+), 313.9901; found: 313.9932 and 315.9904. Cl O N Cl Cl 5-6 + Cl N H DMF, 0 °C - rt, 16 h NaH Cl O N N Cl 5-1 Cl 2-(6-chloro-1H-indol-1-yl)-N,N-bis(4-chlorophenyl)acetamide (5-1): Sodium hydride (60% in mineral oil, 0.13 g, 3.24 mmol) was suspended in anhydrous N,N-dimethylformamide (5 mL) and cooled to 0 °C, then a solution of 6-chloroindole (0.48 g, 3.18 mmol) in N,N-dimethylformamide (5 mL) was added slowly via syringe. After 10 mins, a solution of 𝛼-chloroamide 5-6 (1.0 g, 3.18 mmol) in N,N-dimethylformamide (5 mL) was added in one-portion and the reaction was warmed up to room temperature and stirred overnight. Then, ethyl acetate (50 mL) and 1 M hydrochloric acid (30 mL) was added, and the organic layer was collected. The organic was further washed with 1 M hydrochloric acid (2 x 30 mL). The organic was dried over sodium sulfate, decanted and concentrated under reduced pressure to give the crude product which was used without further purification. mp = 174 °C. 1H NMR (500 MHz, CDCl3) δ 7.51(d, J = 8.7 Hz, 1H), 7.40 (s, 2H), 7.29 (s, 2H), 7.14 (d, J = 8.7 Hz, 4H), 7.08 (d, J = 7.7 Hz, 2H), 6.90 (d, J = 3.2 Hz, 1H), 6.47 (d, J = 3.2 Hz, 1H), 4.75 (s, 2H). 13C{1H} NMR (125 MHz, CDCl3) δ 167.1, 136.7, 130.7, 129.6, 129.2, 129.0, 127.9, 127.0, 126.8, 122.0, 120.6, 109.0, 102.7, 49.4. FTIR (neat, cm-1): 3062, 2939, 289 1668, 1490. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C22H16Cl3N2O+), 429.0323, 431.0294 & 433.0264; found: 429.0341, 431.0290, & 433.0287. Cl O N N Cl 5-1 POCl3 DMF, 0 °C – rt, 2 h then, NaOH workup Cl O H Cl O N N Cl 5-7 Cl 2-(6-chloro-3-formyl-1H-indol-1-yl)-N,N-bis(4-chlorophenyl)acetamide (5-7): A 100 mL round bottom flask equipped with magnetic stirrer was charged with N,N-dimethylformamide (5 mL) and cooled to 0 °C , then phosphorus oxychloride (0.39 mL, 4.13 mmol) was added slowly. The reaction was stirred for 15 mins, and a solution of the indole amide 5-1 (1.39 g, 3.18 mmol) in N,N-dimethylformamide (10 mL) was added. The reaction was stirred for 2 hours, diluted with water (30 mL) and basify with sodium hydroxide until pH ≈ 7 – 8. The reaction was extracted with ethyl acetate (3 x 30 mL) and the organics were combined and washed with lithium bromide (3 x 30 mL). The organic was dried over sodium sulfate, concentrated and recrystallized in ethyl acetate to give the desired product as a yellow foamy solid (1.45 g, quantitative). 1H NMR (500 MHz, DMSO-d6) δ 9.91 (s, 1H), 8.26 (s, 1H), 8.06 (d, J = 8.4 Hz, 1H), 7.83 (d, J = 1.8 Hz, 1H), 7.78 (s, 2H), 7.64 (s, 2H), 7.43 (s, 2H), 7.35 (s, 2H), 7.27 (dd, J = 8.4, 1.8 Hz, 1H), 5.08 (s, 2H). 13C{1H} NMR (125 MHz, DMSO-d6) δ 185.3, 166.6, 143.2, 139.0, 130.6, 129.4, 128.7 (overlaps with a broad aromatic carbon), 123.5, 123.1, 122.4, 117.7, 112.2, 49.8. FTIR (neat, cm−1): 3094, 2924, 1660, 1489. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C23H16Cl3N2O2 +), 457.0272; found, 457.0295 and 459.0269. 290 Cl O N Cl + Cl 5-6 O OH O OH Cl N H NaH Cl DMF, 0 °C - rt, 16 h O N N Cl Cl 5-2 1-(2-(bis(4-chlorophenyl)amino)-2-oxoethyl)-6-chloro-1H-indole-3-carboxylic acid (5-2): Sodium hydride (60% in mineral oil, 0.42 g, 10.49 mmol) was suspended in anhydrous N,N- dimethylformamide (5 mL) and cooled to 0 °C, then a solution of 6-chloroindole-3-carboxylic acid (0.93 g, 4.77 mmol) in N,N-dimethylformamide (10 mL) was added slowly via syringe. After 10 mins, a solution of 𝛼-chloroamide 5-6 (1.50 g, 4.77 mmol) in N,N-dimethylformamide (10 mL) was added in one-portion and the reaction was warmed up to room temperature and stirred overnight. Then, ethyl acetate (50 mL) and 1 M hydrochloric acid (30 mL) was added, and the organic layer was collected. The organic was further washed with 1 M hydrochloric acid (2 x 30 mL). The organic was dried over sodium sulfate, decanted and concentrated under reduced pressure. The crude was recrystallized in ethyl acetate to give the product as white solid (1.96 g, 87% yield). mp = 242 – 243 °C. 1H NMR (500 MHz, DMSO-d6) δ 12.15 (s, 1H), 8.04 (s, 1H), 7.95 (d, J = 8.5 Hz, 1H), 7.75 (d, J = 2.0 Hz, 3H), 7.62 (s, 2H), 7.42 (s, 2H), 7.32 (s, 2H), 7.18 (dd, J = 8.5, 1.9 Hz, 1H), 5.00 (s, 2H). 13C{1H} NMR (125 MHz, DMSO-d6) δ 166.8, 165.7, 138.4, 138.0, 131.6, 130.5, 129.4, 128.6, 127.6, 125.3, 122.3, 122.0, 111.7, 107.5, 49.5. FTIR (neat, cm−1): 3056, 2925, 1671, 1490. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C23H16Cl3N2O3 +), 473.0221; found: 473.0245 and 475.0219. 291 O OH Cl N O N Cl Cl O R NH + R H2N HBTU DIPEA DMF, rt, 12 h Cl N O N Cl Cl R = Me R = n-Bu In a 7mL vial, carboxylic acid (1.0 equiv, 0.127 mmol) was dissolved in N,N-dimethylformamide (1.5 mL) and HBTU (1.2 equiv, 0.152 mmol) and DIPEA (2.4 equiv, 0.305 mmol) were added. The reaction was stirred at room temperature for 10 mins, then amine (1.0 equiv, 0.127 mmol) was added and the reaction was stirred overnight for 12 hours. The solvent was removed under reduced pressure and the product was purified with CombiFlash chromatography (silica gel, 20−40 μm, gradient 20 – 70% ethyl acetate/hexane). O NH Cl N O N Cl 5-3 Cl 1-(2-(bis(4-chlorophenyl)amino)-2-oxoethyl)-6-chloro-N-methyl-1H-indole-3-carboxamide (5-3): Synthesized according to the procedure approach. Isolated as white solid (yield: 0.038 g, 61% yield). mp = 95 – 96 °C. 1H NMR (500 MHz, CD3OD) δ 8. 00 (d, J = 8.6 Hz, 1H), 7.57 (s, 1H), 7.43 (s, 4H), 7.33 (d, J = 1.8 Hz, 3H), 7.22 (s, 2H), 7.12 (dd, J = 8.6, 1.8 Hz, 1H), 4.91 (s, 2H), 2.89 (s, 3H). 13C{1H} NMR (125 MHz, CD3OD) δ 167.4, 166.4, 137.5, 132.0, 130.2, 129.9, 128.9, 128.4, 127.6, 124.7, 121.8, 121.4, 110.9, 109.8, 37.5, 25.0. FTIR (neat, cm−1): 3340, 3087, 292 2929, 1676, 1618, 1489. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C24H19Cl3N3O2 +), 486.0537; found: 486.0558 and 488.0532. O NH Cl N O N Cl 5-4 Cl 1-(2-(bis(4-chlorophenyl)amino)-2-oxoethyl)-N-butyl-6-chloro-1H-indole-3-carboxamide (5- 4): Synthesized according to the procedure approach. Isolated as off-white solid (yield: 0.054 mg, 81% yield). mp = 78 – 79 °C. 1H NMR (500 MHz, CD3OD) δ 8.01 (d, J = 8.6 Hz, 1H), 7.61 (s, 1H), 7.54 – 7.17 (m, 9H), 7.12 (dd, J = 8.6 1.8 Hz, 1H), 4.90 (s, 2H), 3.34 (t, J = 7.2 Hz, 2H), 1.57 (q, J = 7.6 Hz, 2H), 1.40 (h, J = 7.4 Hz, 2H), 0.96 (t, J = 7.4 Hz, 3H). 13C{1H} NMR (125 MHz, CD3OD) δ 167.4, 165.8, 137.5, 132.0, 130.2, 129.9, 128.8, 128.4, 127.6, 124.9, 121.9, 121.4, 111.1, 109.8, 48.9, 38.8, 31.5, 19.9, 12.8. FTIR (neat, cm−1): 3295, 2926, 1673, 1609, 1489. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C27H25Cl3N3O2 +), 528.1007; found, 528.1032 and 530.1007. O OH Cl N O N Cl 5-2 1. NH2 O 5 O N H O HBTU DIPEA DMF, rt, 16 h TFA DCM, rt, 16 h 2. Cl NH2 O 5 Cl NH O N O N 5-10 Cl Cl N-(17-amino-3,6,9,12,15-pentaoxaheptadecyl)-1-(2-(bis(4-chlorophenyl)amino)-2-oxoethyl)- 6-chloro-1H-indole-3-carboxamide (5-10): To a solution of indole carboxylic acid 5-2 (0.20 g, 293 0.422 mmol) in N,N-dimethylformamide (1.0 mL) at room temperature was added HBTU (0.19 g, 0.507 mmol) and DIPEA (0.18 mL, 1.01 mmol). The reaction was stirred for 15 mins and a solution of the PEG linker (0.16 g, 0.422 mmol) in N,N-dimethylformamide (0.5 mL) was added. The reaction was further stirred at room temperature overnight. Then, the solvent was removed under reduced pressure to give the crude N-boc product. The crude product was dissolved in dichloromethane (3 mL) and trifluoroacetic acid (0.65 mL, 8.44 mmol) and stirred overnight. The reaction was quenched with sodium carbonate (10 mL). The reaction was extracted with dichloromethane (3 x 10 mL) and the organics were combined and dried over sodium sulfate. The organic was then concentrated and purified with CombiFlash chromatography (silica gel, 20−40 μm, gradient 0−8% methanol (5% Et3N)/dichloromethane) to give the product as a white foamy solid (0.27 g, 87%). 1H NMR (500 MHz, CD3OD) δ 8.02 (d, J = 8.6 Hz, 1H), 7.73 (s, 1H), 7.48 (s, 4H), 7.35 (d, J = 1.8 Hz, 1H), 7.27 (s, 2H), 7.21 (s, 2H), 7.11 (dd, J = 8.6, 1.8 Hz, 1H), 4.90 (s, 2H), 3.65 – 3.45 (m, 22H), 2.80 (t, J = 5.2 Hz, 2H). 13C{1H} NMR (125 MHz, CD3OD) δ 167.2, 165.8, 137.6, 132.4, 130.3, 130.1, 128.9, 128.4, 127.7, 124.9, 121.9, 121.5, 110.8, 109.9, 70.2, 70.1, 70.1, 70.0, 70.0, 70.0, 69.9, 69.8, 69.7, 69.5, 65.5, 40.2, 38.9. FTIR (neat, cm−1): 3418, 2868, 1680, 1618, 1488. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C35H42Cl3N4O7 +), 735.2114; found, 735.2117, 736.2178, 737.2238, 738.2212, 739.2228, 740.2214, 741.2198. 294 Cl O N NH O N NH2 O 5 Cl CDI THF, 70 °C, 12 h Cl N N O O N H O 5 O N H N H 5-12 O Cl O N H 5 N N O Cl Cl 5-10 Cl Cl Cl N,N'-(19-oxo-3,6,9,12,15,23,26,29,32,35-decaoxa-18,20-diazaheptatriacontane-1,37- diyl)bis(1-(2-(bis(4-chlorophenyl)amino)-2-oxoethyl)-6-chloro-1H-indole-3-carboxamide) (5-12): Amine 5-10 (0.049 g, 0.067 mmol) was dissolved in tetrahydrofuran (1 mL) in a 15 mL pressure tube. Then carbonyldiimidazole CDI (5.4 mg, 0.034 mmol) was added. The reaction was placed in a preheated aluminum beads at 70 °C and stirred for 12 hours. The reaction was diluted with ethyl acetate (5 mL) and washed with 1 N hydrochloric acid (2 x 3 mL). The organic layer was collected, dried over sodium sulfate, decanted, and concentrated under reduced pressure. The reaction was then purified with CombiFlash chromatography (silica gel, 20−40 μm, gradient 0−8% methanol/dichloromethane) to give the product as a white foamy solid (0.039 g, 76%). 1H NMR (500 MHz, CDCl3) δ 8.03 (d, J = 8.5 Hz, 1H), 7.56 (s, 1H), 7.44 (s, 2H), 7.22 (s, 4H), 7.16 (dd, J = 8.5, 1.6 Hz, 1H), 7.11 (s, 2H), 7.06 (d, J = 1.6 Hz, 1H), 4.70 (d, J = 7.6 Hz, 1H), 3.67 – 3.50 (m, 20H), 3.45 (t, J = 5.2 Hz, 2H), 3.29 (t, J = 5.2 Hz, 2H). 13C{1H} NMR (125 MHz, CDCl3) δ 166.3, 164.5, 158.8, 137.1, 131.9, 130.8, 129.7, 129.2, 128.8, 126.9, 124.8, 122.4, 122.3, 111.9, 109.4, 70.6, 70.5, 70.4, 70.4, 70.4, 70.3, 70.3, 70.1, 70.0, 49.3, 45.7, 39.9, 39.1. FTIR (neat, cm−1): 3313, 3081, 2866, 1687, 1629. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C71H81Cl6N8O15 +), 1495.3948; found, 1495.3877, 1496.3909, 1497.3861, 1498.3882, 1499.3843, 1500.3857, 1501.3833, 1502.3842. 295 O OH Cl N O N Cl 5-2 1. NH2 O 8 O N H O HBTU DIPEA DMF, rt, 16 h TFA DCM, rt, 16 h 2. Cl NH2 O 8 Cl NH O N O N 5-11 Cl Cl N-(26-amino-3,6,9,12,15,18,21,24-octaoxahexacosyl)-1-(2-(bis(4-chlorophenyl)amino)-2- oxoethyl)-6-chloro-1H-indole-3-carboxamide (5-11): To a solution of the indole-carboxylic acid 5-2 (0.20 g, 0.422 mmol) in N,N-dimethylformamide (1.0 mL) at room temperature was added HBTU (0.19 g, 0.507 mmol) and DIPEA (0.18 mL, 1.01 mmol). The reaction was stirred for 15 mins and a solution of the PEG linker (0.22 g, 0.422 mmol) in N,N-dimethylformamide (0.5 mL) was added. The reaction was further stirred at room temperature overnight. Then, the solvent was removed under reduced pressure to give the crude product. The crude product was dissolved in dichloromethane (3 mL) and trifluoroacetic acid (0.65 mL, 8.44 mmol) and stirred overnight. The reaction was quenched with sodium carbonate (10 mL). The reaction was extracted with dichloromethane (3 x 10 mL) and the organics were combined and dried over sodium sulfate. The organic was then concentrated and purified with CombiFlash chromatography (silica gel, 20−40 μm, gradient 0−8% methanol (5% Et3N)/dichloromethane) to give the product as a clear oil (0.24 g, 67%). 1H NMR (500 MHz, CD3OD) δ 8.03 (d, J = 8.5 Hz, 1H), 7.72 (s, 1H), 7.49 (s, 4H), 7.36 (s, 1H), 7.30 (s, 4H), 7.13 (dd, J = 8.6, 1.8 Hz, 1H), 4.92 (d, J = 6.8 Hz, 1H), 4.85 (s, 2H, overlap with H2O), 3.67 – 3.43 (m, 36H), 2.74 (t, J = 5.1 Hz, 2H, NH2). 13C{1H} NMR (125 MHz, CD3OD) δ 168.5, 167.1, 138.9, 133.8, 131.6, 131.4, 130.2, 129.7, 129.0, 126.3, 123.3, 122.9, 112.2, 111.3, 73.2, 71.5, 71.5, 71.4, 71.4, 71.4, 71.4, 71.3, 71.2, 70.8, 296 66.9, 42.0, 40.3. FTIR (neat, cm−1): 3354, 3085, 2867, 1687, 1631. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C41H54Cl3N4O10 +), 867.2900; found, 867.2891 and 869.2870. Cl O N NH O N NH2 O 8 Cl CDI THF, 70 °C, 12 h Cl N N O O N H O 8 O N H N H 5-13 O Cl O N H 8 N N O Cl Cl 5-10 Cl Cl Cl N,N'-(28-oxo-3,6,9,12,15,18,21,24,32,35,38,41,44,47,50,53-hexadecaoxa-27,29- diazapentapentacontane-1,55-diyl)bis(1-(2-(bis(4-chlorophenyl)amino)-2-oxoethyl)-6- chloro-1H-indole-3-carboxamide) (5-13): Amine 5-10 (33 mg, 0.038 mmol) was dissolved in tetrahydrofuran (1 mL) in a 15 mL pressure tube. Then carbonyldiimidazole CDI (3.0 mg, 0.019 mmol) was added. The reaction was placed in a preheated aluminum beads at 70 °C and stirred for 12 hours. The reaction was diluted with ethyl acetate (5 mL) and washed with 1 N hydrochloric acid (2 x 3 mL). The organic layer was collected, dried over sodium sulfate, decanted, and concentrated under reduced pressure. The reaction was then purified with CombiFlash chromatography (silica gel, 20−40 μm, gradient 0−8% methanol/dichloromethane) to give the product as a white foamy solid (29 mg, 88%). 1H NMR (500 MHz, CD2Cl2) δ 7.91 (d, J = 8.4 Hz, 1H), 7.47 (s, 1H), 7.36 (s, 2H), 7.16 (s, 4H), 7.04 (d, J = 10.1 Hz, 4H), 6.62 (d, J = 5.9 Hz, 1H, NH), 4.62 (s, 2H), 3.65 – 3.34 (m, 36H). 13C{1H} NMR (126 MHz, cd2cl2) δ 166.1, 164.2, 158.5, 137.2, 132.0, 130.8, 130.0, 129.0, 128.6, 127.1, 124.8, 122.4, 121.9, 111.9, 109.6, 70.6, 70.4, 70.4, 70.4, 70.3, 70.3, 70.2, 70.1, 70.0, 49.3, 40.0, 39.1. FTIR (cm−1): 3338, 3089, 2868, 1685, 1633, 1488. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C83H105Cl6N8O21 +), 1759.5520; found, 1759.5155, 1760.5183, 1761.5134, 1762.5154, 1763.5120, 1764.5131, 1765.5116, 1766.5118. 297 NH2 O 8 HO NH O O N O N 5-11 Cl Cl Cl OH O O 5 5-14 HBTU DIPEA DMF, rt, 24 h H N O 5 O 8 O H N O O 8 HN O O NH 5-15 N O N Cl Cl Cl Cl N N O Cl Cl N1,N19-bis(1-(1-(2-(bis(4-chlorophenyl)amino)-2-oxoethyl)-6-chloro-1H-indol-3-yl)-1-oxo- 5,8,11,14,17,20,23,26-octaoxa-2-azaoctacosan-28-yl)-4,7,10,13,16- pentaoxanonadecanediamide (5-15): To a solution of the dicarboxylic acid 5-14 (8 mg, 0.023 mmol) in N,N-dimethylformamide (1.0 mL) at room temperature was added HBTU (17 mg, 0.046 mmol) and DIPEA (19 µL, 0.110 mmol). The reaction was stirred for 15 mins and a solution of the amine 5-11 (40 mg, 0.046 mmol) in N,N-dimethylformamide (1 mL) was added. The reaction was further stirred at room temperature overnight. Then, the solvent was removed under reduced pressure and purified with CombiFlash chromatography (silica gel, 20−40 μm, gradient 0−8% methanol/dichloromethane) to give the product as a clear oil (27 mg, 57%). 1H NMR (500 MHz, CD2Cl2) δ 8.03 (d, J = 8.7 Hz, 1H), 7.63 (s, 1H), 7.57 – 7.04 (m, 10H), 6.77 (s, 2H), 4.76 (s, 2H), 3.80 – 3.27 (m, 46H), 2.40 (t, J = 5.6 Hz, 2H). 13C{1H} NMR (125 MHz, CD2Cl2) δ 171.3, 166.1, 164.3, 137.3, 132.0, 130.8, 130.0, 129.0, 128.6, 127.1, 124.8, 122.3, 122.0, 111.9, 109.6, 70.4, 70.4, 70.3, 70.2, 70.2, 70.1, 70.0, 69.7, 67.1, 49.3, 39.1, 36.7. FTIR (neat, cm−1): 3322, 3092, 2866, 1689, 1539. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C96H129Cl6N8O27 +), 2038.7097; found, 2038.7106. 298 NH2 O 5 Cl 5-2 DIPEA HBTU DMF, rt, 12 h O N NH O N 5-10 Cl Cl O NH O 5 5-16 Cl O N NH O N Cl Cl Cl N O N Cl Cl N,N'-(3,6,9,12,15-pentaoxaheptadecane-1,17-diyl)bis(1-(2-(bis(4-chlorophenyl)amino)-2- oxoethyl)-6-chloro-1H-indole-3-carboxamide) (5-16): To a solution of the indole-carboxylic acid 5-2 (52 mg, 0.109 mmol) in N,N-dimethylformamide (1.5 mL) at room temperature was added HBTU (42 mg, 0.111 mmol) and DIPEA (46 µL, 0.262 mmol). The reaction was stirred for 15 mins and a solution of the amine 5-10 (80 mg, 0.109 mmol) in N,N-dimethylformamide (1.5 mL) was added. The reaction was further stirred at room temperature overnight. Then, the solvent was removed under reduced pressure and purified with CombiFlash chromatography (silica gel, 20−40 μm, gradient 0−8% methanol/dichloromethane) to give the product as a yellow foamy solid (101 mg, 78%). 1H NMR (500 MHz, CD2Cl2) δ 8.05 (d, J = 9.3 Hz, 1H), 7.65 (s, 1H), 7.50 (s, 2H), 7.40 – 7.11 (m, 8H), 4.74 (s, 2H), 3.76 – 3.43 (m, 13H). 13C{1H} NMR (125 MHz, CD2Cl2) δ 166.2, 164.2, 137.2, 132.0, 130.8, 129.9, 129.0, 128.5, 127.0, 124.8, 122.4, 121.9, 111.8, 109.5, 70.4, 70.4, 70.4, 70.1, 69.9, 49.2, 39.1. FTIR (neat, cm−1): 2875, 1676, 1631, 1549. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C58H55Cl6N6O9 +), 1189.2156; found,1189.2139, 1190.2173, 1191.2120, 1192.2152, 1193.2103, 1194.2125, 1195.2094, 1196.2094. 299 + NH O Cl Cl Et3N DCM, 0 °C - rt, 16 h O N Cl 5-19 2-chloro-N,N-diphenylacetamide (5-19): A 100 mL round bottom flask equipped with magnetic stirrer was charged with diphenylamine (2.0 g, 11.82 mmol) and dichloromethane (30 mL). The resulting solution was cooled to 0 °C and then triethylamine (3.29 mL, 23.64 mmol) was added. A solution of chloroacetyl chloride (1.03 mL, 13.00 mmol) in dichloromethane (1.5 mL) was added slowly over 15 mins. After 4 hours, additional solution of chloroacetyl chloride (1.03 mL, 13.00 mmol) in dichloromethane (1.5 mL) was added. The reaction was then warmed up to room temperature and allowed to stirred overnight. The reaction was then diluted with sodium carbonate (50 mL) and the organic layer was collected. The aqueous phase was further extracted with dichloromethane (2 x 50 mL) and the organic phases were combined. The organics were dried over sodium sulfate, decanted and concentrated under reduced pressure to give the crude product as yellow solid (2.78g, 96%). mp: 116 – 117 °C. 1H NMR (500 MHz, DMSO-d6) δ 7.60 – 7.28 (m, br, 10H), 4.19 (s, 2H). 13C{1H} NMR (125 MHz, DMSO-d6) δ 165.4, 129.8, 129.1, 128.6, 126.7, 43.2. FTIR (neat, cm−1): 3005, 2945, 1676, 1490. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C14H13ClNO+), 246.0680; found, 246.0712 and 248.0685. 300 Cl + O N 5-19 NaH N H DMF, 0 °C - rt, 16 h O N N 5-17 2-(1H-indol-1-yl)-N,N-diphenylacetamide (5-17): Sodium hydride (60% in mineral oil, 166 mg, 4.15 mmol) was suspended in anhydrous N,N-dimethylformamide (10 mL) and cooled to 0 °C, then a solution of indole (0.48 g, 4.07 mmol) in N,N-dimethylformamide (5 mL) was added slowly via syringe. After 10 mins, a solution of the 𝛼-chloroamide 5-19 (1.0 g, 4.07 mmol) in N,N- dimethylformamide (5 mL) was added in one-portion and the reaction was warmed up to room temperature and stirred overnight. Then, ethyl acetate (50 mL) and 1 M hydrochloric acid (30 mL) was added, and the organic layer was collected. The organic was further washed with 1 M hydrochloric acid (2 x 30 mL). The organic was dried over sodium sulfate, decanted, and concentrated under reduced pressure. The product was purified with CombiFlash chromatography (silica gel, 20−40 μm, gradient 5 – 15% ethyl acetate/hexane) to give a yellow foamy solid (1.15 g, 86% yield). 1H NMR (500 MHz, DMSO-d6) δ 7.83 – 7.16 (m, 13H), 7.13 (t, J = 7.5 Hz, 1H), 7.02 (t, J = 7.5 Hz, 1H), 6.40 (d, J = 3.2 Hz, 1H), 4.89 (s, 2H). 13C{1H} NMR (125 MHz, DMSO- d6) δ 167.5, 136.8, 130.6, 130.3, 129.4, 128.5, 127.0, 121.5, 120.7, 119.5, 110.1, 101.2, 48.9 (one of the broad phenyl carbon was not observed). FTIR (neat, cm−1): 3054, 2944, 1687, 1485. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C22H19N2O+), 327.1492; found, 327.1524. 301 O N Cl 5-19 O OH N H NaH DMF, 0 °C - rt, 16 h O O OH N N 5-20 1-(2-(diphenylamino)-2-oxoethyl)-1H-indole-3-carboxylic acid (5-20): Sodium hydride (60% in mineral oil, 163 mg, 4.07 mmol) was suspended in anhydrous N,N-dimethylformamide (5 mL) and cooled to 0 °C, then a solution of indole-3-carboxylic acid (327 mg, 2.03 mmol) in N,N- dimethylformamide (5 mL) was added slowly via syringe. After 10 mins, a solution of 𝛼- chloroamide (500 mg, 2.03 mmol) in N,N-dimethylformamide (5 mL) was added in one-portion and the reaction was warmed up to room temperature and stirred overnight. Then, ethyl acetate (30 mL) and 1 M hydrochloric acid (20 mL) was added and the organic layer was collected. The organic was further washed with 1 M hydrochloric acid (2 x 30 mL). The organic was dried over sodium sulfate, decanted and concentrated under reduced pressure. The product was purified with CombiFlash chromatography (silica gel, 20−40 μm, gradient 20 – 40% ethyl acetate/hexane) to give a yellow solid (267 mg, 36% yield). mp: 222 – 223 °C. 1H NMR (500 MHz, DMSO-d6) δ 11.99 (s, 1H), 7.99 (s, 1H), 7.83 – 7.26 (m, 11H), 7.27 – 7.12 (m, 3H), 4.98 (s, 2H). 13C{1H} NMR (125 MHz, DMSO-d6) δ 166.8, 166.1, 137.5, 137.2, 130.5, 129.4, 127.0, 126.7, 122.7, 121.7, 121.1, 111.2, 107.2, 49.3 (one of the broad phenyl carbon was not observed). FTIR (neat, cm−1): 3056, 2933, 1677, 1655, 1490. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C23H19N2O3 +), 371.1390; found, 371.1415. 302 O OH 1. NH2 O 5 O N H O O N N 5-20 HBTU DIPEA 2. TFA DCM, rt, 16 h NH2 O 5 NH O N O 5-21 N N-(17-amino-3,6,9,12,15-pentaoxaheptadecyl)-1-(2-(diphenylamino)-2-oxoethyl)-1H-indole- 3-carboxamide (5-21): To a solution of the indole-carboxylic acid (100 mg, 0.270 mmol) in N,N- dimethylformamide (1.0 mL) at room temperature was added HBTU (104 mg, 0.275 mmol) and DIPEA (112 µL, 0.650 mmol). The reaction was stirred for 10 mins and a solution of the PEG linker (103 mg, 0.270 mmol) in N,N-dimethylformamide (1.0 mL) was added. The reaction was further stirred at room temperature overnight. Then, the solvent was removed under reduced pressure to give the crude N-boc product. The crude product was dissolved in dichloromethane (3 mL) and trifluoroacetic acid (0.41 mL, 5.40 mmol) and stirred overnight. The reaction was quenched with sodium carbonate (10 mL). The reaction was extracted with dichloromethane (3 x 10 mL) and the organics were combined and dried over sodium sulfate. The organic was then concentrated and purified with CombiFlash chromatography (silica gel, 20−40 μm, gradient 0−8% methanol (5% Et3N)/dichloromethane) to give the product as a clear oil (193 mg, quantitative). 1H NMR (500 MHz, CD3OD) δ 8.12 (d, J = 7.9 Hz, 1H), 7.77 (s, 1H), 7.64 – 7.08 (m, 14H), 4.92 (s, 2H), 3.68 – 3.43 (m, 24H), 2.91 (t, J = 5.1 Hz, 2H). 13C{1H} NMR (125 MHz, CD3OD) δ 168.8, 167.7, 138.4, 133.5, 131.5, 130.1, 129.7, 127.7, 127.6, 123.9, 122.4, 122.2, 111.6, 110.9, 71.3, 71.2, 71.1, 71.1, 71.0, 70.9, 70.8, 68.9, 50.1, 303 40.7, 40.0. FTIR (neat, cm−1): 3343, 3050, 2872, 1682, 1626. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C35H45N4O7 +), 633.3283; found, 633.3313. NH O N 5-21 O N NH2 O 5 CDI THF, 70 °C, 12 h N O N H NH O O 5 5-18 O N H O N N O N NH 5 O N,N'-(19-oxo-3,6,9,12,15,23,26,29,32,35-decaoxa-18,20-diazaheptatriacontane-1,37- diyl)bis(1-(2-(diphenylamino)-2-oxoethyl)-1H-indole-3-carboxamide) (5-18): Amine 5-21 (50 mg, 0.079 mmol) was dissolved in tetrahydrofuran (1 mL) in a 15 mL pressure tube. Then carbonyldiimidazole CDI (6.4 mg, 0.040 mmol) was added. The reaction was placed in a preheated aluminum beads at 70 °C and stirred for 12 hours. The reaction was diluted with ethyl acetate (5 mL) and washed with 1 N hydrochloric acid (2 x 3 mL). The organic layer was collected, dried over sodium sulfate, decanted, and concentrated under reduced pressure. The reaction was then purified with CombiFlash chromatography (silica gel, 20−40 μm, gradient 0−8% methanol/dichloromethane) to give the product as a foamy white solid (29 mg, 55%). 1H NMR (500 MHz, CD3OD) δ 8.08 (d, J = 7.9 Hz, 1H), 7.69 (s, 1H), 7.62 – 7.12 (m, 13H), 4.93 (s, 2H), 3.66 – 3.47 (m, 20H), 3.40 (t, J = 5.4 Hz, 2H), 3.21 (t, J = 5.3 Hz, 2H). 13C{1H} NMR (125 MHz, CD3OD) δ 169.0, 167.7, 161.0, 138.4, 133.5, 131.5, 130.2, 129.8, 127.6, 127.6, 123.9, 122.5, 122.2, 111.8, 110.9, 71.5, 71.5, 71.4, 71.4, 71.4, 71.3, 71.2, 70.9, 50.3, 40.9, 40.3. 304 NH2 O 5 HO O 1. O HN O 5-22 HBTU DIPEA OH O Cl DMF, rt, 24 h 2. DCM/TFA rt, 16 h Cl Cl O N NH O N NH O N O N 5-10 Cl Cl NH O 5 O NH2 H N O O 5-23 Cl O N 5 HN O N Cl Cl Cl 4-amino-N1,N7-bis(1-(1-(2-(bis(4-chlorophenyl)amino)-2-oxoethyl)-6-chloro-1H-indol-3-yl)- 1-oxo-5,8,11,14,17-pentaoxa-2-azanonadecan-19-yl)heptanediamide (5-23): To a solution of the dicarboxylic acid 5-22 (19 mg, 0.068 mmol) in N,N-dimethylformamide (1.0 mL) at room temperature was added HBTU (62 mg, 0.163 mmol) and DIPEA (57 µL, 0.326 mmol). The reaction was stirred for 15 mins at room temperature and a solution of the amine 5-10 (100 mg, 0.136 mmol) in N,N-dimethylformamide (2.0 mL) was added. The reaction was further stirred at room temperature for 24 hours. Then, the solvent was removed under reduced pressure and the crude N-boc product was used directly for the next step. The crude N-boc product was dissolved in dichloromethane (1.5 mL) and trifluoroacetic acid (1.0 mL) and stirred overnight. The reaction was quenched with sodium carbonate (18 mL). The reaction was extracted with dichloromethane (3 x 15 mL) and the organics were combined and dried over sodium sulfate. The organic was then concentrated and purified with CombiFlash chromatography (silica gel, 20−40 μm, gradient 0−8% methanol (5% Et3N)/dichloromethane) to give the product as a foamy white solid (43.4 mg, 40% yield). 1H NMR (500 MHz, CD2Cl2) δ 8.08 (d, J = 8.4 Hz, 1H), 7.73 (s, 1H), 7.59 – 7.42 (m, 2H), 7.41 – 7.13 (m, 8H), 7.03 (t, J = 5.5 Hz, 1H, NH), 6.88 (t, J = 5.6 Hz, 1H, NH), 4.79 (s, 2H), 3.73 – 3.50 (m, 22H), 3.46 (q, J = 5.7 Hz, 2H), 3.32 (q, J = 5.4 Hz, 2H), 2.55 (q, J = 7.2 Hz, 1H), 2.21 (hept, J = 7.3 Hz, 2H), 1.70 (dq, J = 12.2, 7.4 Hz, 1H), 1.53 (dq, J = 12.2, 7.4 Hz, 1H). 13C{1H} NMR (125 MHz, CD2Cl2) δ 173.3, 166.6, 305 164.6, 137.6, 132.5, 131.1, 130.4, 129.4, 128.9, 127.5, 125.3, 122.8, 122.3, 112.2, 109.9, 70.8, 70.8, 70.7, 70.4, 70.4, 70.3, 70.0, 51.0, 49.7, 46.4, 39.5, 39.4, 33.2, 33.1. FTIR (neat, cm−1): 3287, 3063, 2868, 1685. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C77H92Cl6N9O16 +), 1608.4685; found, 1608.4606, 1609.4633, 1610.4592, 1611.4615, 1612.4583, 1613.4601, 1614.4575, 1615.4580, 1616.4565. O N NH O N Cl Cl O 5 NH O NH2 H N O O 5-23 Cl O N 5 HN O N O HO O NH H HN H S DMAP EDC • HCl DMF, rt, 16 h Cl Cl O N NH O N Cl Cl Cl Cl O HN H NH H S NH O O 5 O NH 5-24 H N O O O N 5 HN O N Cl Cl Cl N1,N7-bis(1-(1-(2-(bis(4-chlorophenyl)amino)-2-oxoethyl)-6-chloro-1H-indol-3-yl)-1-oxo- 5,8,11,14,17-pentaoxa-2-azanonadecan-19-yl)-4-(5-((3aS,4S,6aR)-2-oxohexahydro-1H- thieno[3,4-d]imidazol-4-yl)pentanamido)heptanediamide (5-24): A 15 mL round bottom flask equipped with magnetic stirrer bar was charged with amine 5-23 (31 mg, 0.019 mmol) and dimethylformamide (DMF) (2 mL). To the resulting solution was added biotin (7.1mg, 0.029 mmol), dimethylaminopyridine (DMAP) (10 mol%), and N-(3-Dimethylaminopropyl)-N′- ethylcarbodiimide hydrochloride (EDC•HCl) (6 mg, 0.029 mmol). The solution was further stirred at room temperature for 16 hours before the solvent was removed under reduced pressure. The crude was then purified with CombiFlash chromatography (silica gel, 20−40 μm, gradient 0−8% methanol/dichloromethane) to give the product as a white foamy solid (20.3 mg, 58%). 1H NMR (500 MHz, CD2Cl2) δ 8.08 (d, J = 8.5 Hz, 2H), 7.69 (d, J = 1.9 Hz, 2H), 7.58 – 7.06 (m, 20H), 7.03 – 6.87 (m, 4H), 6.80 (d, J = 8.5 Hz, 1H), 6.42 (s, 1H), 5.79 (s, 1H), 4.79 (s, 4H), 4.41 (dd, J = 8.0, 4.9 Hz, 1H), 4.21 (dd, J = 8.0, 4.5 Hz, 1H), 3.70 – 3.51 (m, 43H), 3.48 (t, J = 5.4 Hz, 4H), 306 3.42 – 3.25 (m, 5H), 2.19 – 2.08 (m, 4H), 2.10 – 2.03 (m, 2H), 1.75 – 1.60 (m, 6H), 1.55 (q, J = 7.3 Hz, 3H). 13C{1H} NMR (125 MHz, CD2Cl2) δ 173.4, 173.1, 173.0, 166.3, 164.3, 163.9, 137.2, 132.1, 130.8, 130.0, 129.1, 128.5, 127.1, 124.9, 122.5, 121.9, 111.9, 109.5, 70.4, 70.4, 70.4, 70.2, 70.1, 70.1, 70.0, 69.7, 69.7, 61.7, 60.1, 55.6, 49.3, 48.9, 40.5, 39.3, 39.2, 35.5, 32.9, 30.8, 27.9, 27.8, 25.3. FTIR (neat, cm−1): 3297, 2869, 1640, 1540. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C87H106Cl6N11O18S+), 1836.5535, 1837.5568, 1838.5505, 1839.5539; found 1836.5425, 1837.5443, 1838.5410, 1839.5427. NH2 5 HO O O OH Cl O N NH O N Cl Cl 5-10 O NHBoc 5-25 HBTU DIPEA DMF, rt, 24 h then, DCM/TFA O NH NH 5 O NH2 O HN O N 5-26 Cl O N Cl Cl O 5 HN O N O N Cl Cl Cl 2-amino-N1,N4-bis(1-(1-(2-(bis(4-chlorophenyl)amino)-2-oxoethyl)-6-chloro-1H-indol-3-yl)- 1-oxo-5,8,11,14,17-pentaoxa-2-azanonadecan-19-yl)succinimide (5-26): To a solution of amine 5-10 (100 mg, 0.136 mmol) in DMF (2 mL) was added dicarboxylic acid (14 mg, 0.062 mmol) followed by HBTU (56 mg, 0.149 mmol) and DIPEA (52 µL, 0.062 mmol). The solution was then stirred overnight and then concentrated under reduced pressure to give the crude N-boc protected product. The crude N-boc protected amine was redissolved in DCM (2 mL) and TFA (1 mL) and was stirred for another 16 hours. The reaction was diluted with DCM (5 mL) and quenched with sodium carbonate until pH ≈ 7. The DCM layer was collected, and the aqueous layer was further extracted with DCM (2 x 5 mL). The organics were combined, dried over sodium sulfate, decanted and then concentrated to give the crude amine. The crude was then purified with CombiFlash 307 chromatography (silica gel, 20−40 μm, gradient 0−10% methanol/dichloromethane) to give the product as off-white foamy solids (64 mg, 67%). 1H NMR (500 MHz, CD2Cl2) δ 8.12 – 8.06 (m, 2H), 7.71 (d, J = 8.2 Hz, 2H), 7.67 (t, J = 6.0 Hz, 1H), 7.53 – 7.15 (m, 20H), 7.07 (q, J = 5.5 Hz, 2H), 7.01 (t, J = 5.5 Hz, 1H), 4.78 (d, J = 2.6 Hz, 4H), 3.67 – 3.51 (m, 43H), 3.46 (dt, J = 8.4, 5.3 Hz, 4H), 3.34 (dh, J = 9.9, 5.2 Hz, 4H). 13C{1H} NMR (125 MHz, CD2Cl2) δ 174.2, 171.2, 166.3, 166.2, 164.3, 164.3, 137.2, 132.2, 132.1, 130.7, 130.0, 129.0, 128.5, 128.5, 127.1, 125.0, 125.0, 122.5, 122.5, 121.9, 111.8, 111.8, 109.5, 70.4, 70.4, 70.3, 70.3, 70.2, 70.2, 70.1, 70.1, 70.1, 69.6, 52.6, 49.3, 45.8, 40.9, 39.1, 39.1, 39.0. FTIR (neat, cm−1): 3302, 2922, 1670, 1544, 1275. HRMS (ESI-TOF) m/z: [M+2H]2+ calcd for (C74H87Cl6N9O16 2+), 784.7181, 785.2198, 785.7166, 786.2183; found 784.7188, 785.2202, 785.7183, 786.2192 O NH NH 5 O NH2 O HN O N 5-26 Cl O N Cl Cl O 5 HN O N O N Cl Cl O NH H HN H S O O 4 N H HBTU DIPEA DMF, rt, 16 h HO O Cl Cl Cl N H O O N N Cl O 5 NH HN O O O HN 5-27 S H NH H N H O H N 4 O O O 5 HN O N O N Cl Cl Cl To a solution of amine (64 mg, 0.0408 mmol) in DMF (3 mL) was added carboxylic acid (22 mg, 0.0449 mmol) followed by HBTU (19 mg, 0.0489 mmol) and DIPEA (17 µL, 0.0979 mmol). The solution was then stirred overnight and then concentrated under reduced pressure to give the crude product. The crude was then purified with CombiFlash chromatography (silica gel, 20−40 μm, gradient 0−10% methanol/dichloromethane) to give the product as a foamy white solid (68 mg, 82%). 1H NMR (500 MHz, CD2Cl2) δ 8.09 (d, J = 8.5 Hz, 2H), 7.76 (d, J = 7.8 Hz, 1H), 7.69 (d, J = 8.5 Hz, 2H), 7.61 – 7.09 (m, 20H), 7.02 (m, 2H), 6.89 (s, 1H), 6.24 (s, 1H), 5.62 (s, 1H), 5.35 (d, J = 6.0 Hz, 1H), 4.80 (s, 4H), 4.72 (q, J = 6.0 Hz, 1H), 4.50 – 4.38 (m, 1H), 4.37 – 4.17 (m, 308 1H), 4.00 – 3.21 (m, 66H), 3.11 (d, J = 4.5 Hz, 1H), 3.02 – 2.37 (m, 9H), 2.15 (t, J = 7.1 Hz, 2H), 1.61 (m, 4H), 1.39 (s, 2H). 13C{1H} NMR (125 MHz, CD2Cl2) δ 173.0, 171.4, 170.8, 166.4, 166.3, 164.3, 164.3, 163.8, 137.2, 132.2, 132.1, 130.7, 130.0, 129.0, 128.5, 127.2, 125.0, 122.5, 121.9, 111.8, 109.5, 70.4, 70.4, 70.4, 70.4, 70.3, 70.3, 70.2, 70.2, 70.1, 70.0, 69.9, 69.6, 69.5, 67.2, 61.8, 60.1, 55.7, 50.3, 49.3, 40.5, 39.3, 39.2, 39.2, 39.2, 37.6, 36.8, 35.5, 28.1, 28.0, 25.5. FTIR (neat, cm−1): 3296, 2864, 1642, 1538, 1278. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C95H121Cl6N12O23S+), 2041.6485, 2042.6518, 2043.6455, 2044.6489; found: 2041.6473, 2042.6494, 2043.6476, 2044.6476. O OH + Cl Cl N H O NaH OMe DMF, 0 °C - rt, 16 h Cl O OH N O OMe 5-28 6-chloro-1-(2-methoxy-2-oxoethyl)-1H-indole-3-carboxylic acid (5-28): To a suspension of NaH (60% in mineral oil, 2.1 equiv, 429 mg) in DMF (10 mL) at 0 °C was slowly added 6- chloroindole-3-carboxylic acid (1.0 equiv, 1.0 g). The solution was stirred for 15 minutes and methyl chloroacetate (1.0 equiv, 0.45 mL) was added. The reaction was then allowed to warm up to room temperature and stirred for 16 hours. Then, ethyl acetate (50 mL) and 1 M hydrochloric acid (30 mL) was added and the organic layer was collected. The organic was further washed with 1 M hydrochloric acid (2 x 30 mL). The organic was dried over sodium sulfate, decanted, and concentrated under reduced pressure to give the crude products. The crude was then purified with CombiFlash chromatography (silica gel, 20−40 μm, gradient 20−60% ethylacetate/hexane) to give the products as white solids (0.38 g, 28%). 1H NMR (500 MHz, acetone) δ 8.13 (d, J = 8.5 Hz, 1H), 8.12 (s, 1H), 7.63 (d, J = 1.8 Hz, 1H), 7.26 (dd, J = 8.5, 1.8 Hz, 1H), 5.27 (s, 2H), 3.75 (s, 309 3H). 13C{1H} NMR (125 MHz, acetone) δ 168.4, 164.8, 137.9, 136.9, 128.3, 125.6, 122.4, 122.1, 110.6, 107.8, 51.9, 47.4. FTIR (neat, cm-1): 3057, 2955, 1730, 1701. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C12H11ClNO4 +), 268.0372 & 270.0342; found: 268.0374 & 270.0359. OH H2N NHBoc O 5 HBTU DIPEA DMF then, DCM/TFA (5:3) Cl Cl O N O OMe 5-28 NH2 O 5 O N NH O OMe 5-29 methyl 2-(3-((17-amino-3,6,9,12,15-pentaoxaheptadecyl)carbamoyl)-6-chloro-1H-indol-1- yl)acetate (5-29): To a solution of indole carboxylic acid 5-28 (200 mg, 0.75 mmol) in N,N- dimethylformamide (2.0 mL) at room temperature was added HBTU (341 mg, 1.20 mmol) and DIPEA (0.31 mL, 1.8 mmol). The reaction was stirred for 15 mins and a solution of the PEG linker (284 mg, 0.75 mmol) in N,N-dimethylformamide (1.0 mL) was added. The reaction was further stirred at room temperature overnight. Then, the solvent was removed under reduced pressure to give the crude product. The crude product was dissolved in dichloromethane (5 mL) and trifluoroacetic acid (3 mL) and stirred overnight. The reaction was quenched with sodium carbonate (10 mL). The reaction was extracted with dichloromethane (3 x 10 mL) and the organics were combined and dried over sodium sulfate. The organic was then concentrated and purified with CombiFlash chromatography (silica gel, 20−40 μm, gradient 0−8% methanol (5% Et3N)/dichloromethane) to give the product as a white foamy solid (252 mg, 63%). 1H NMR (500 MHz, CD2Cl2) δ 8.22 (d, J = 8.5 Hz, 1H), 8.20 (s, 1H), 8.12 (t, J = 5.6 Hz, 1H), 7.88 (s, 2H), 7.26 (d, J = 1.8 Hz, 1H), 7.18 (dd, J = 8.5, 1.8 Hz, 1H), 4.87 (s, 2H), 3.75 (s, 3H), 3.72 – 3.47 (m, 24H). 13C{1H} NMR (125 MHz, CD2Cl2) δ 168.6, 164.6, 137.7, 132.1, 129.2, 125.5, 122.9, 122.5, 112.7, 110.1, 73.6, 70.9, 70.9, 70.9, 70.9, 310 70.8, 70.6, 70.6, 70.5, 48.2, 42.1, 39.6. FTIR (neat, cm−1): 3313, 2925, 1657, 1545, 1382. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C24H37ClN3O8 +), 530.2264 & 532.2235; found 530.2284 & 532.2267. HO NH2 O OH O 5 O NHBoc Cl O N NH O OMe 5-29 5-25 HBTU DIPEA DMF, rt, 24 h then, DCM/TFA (1:2) O N Cl O NH NH 5 O NH2 O HN O OMe 5-30 O 5 HN O N O MeO Cl dimethyl 2,2'-((22-amino-21,24-dioxo-5,8,11,14,17,28,31,34,37,40-decaoxa-2,20,25,43- tetraazatetratetracontanedioyl)bis(6-chloro-1H-indole-3,1-diyl))diacetate (5-30): To a solution of amine (195 mg, 0.368 mmol) in DMF (3 mL) was added dicarboxylic acid (39mg, 0.167 mmol) followed by HBTU (152 mg, 0.401 mmol) and DIPEA (140 µL, 0.802 mmol). The solution was then stirred overnight and then concentrated under reduced pressure to give the crude boc protected product. The crude boc protected amine was redissolved in DCM (5 mL) and TFA (3 mL) and was stirred for another 16 hours. The reaction was diluted with DCM (10 mL) and quenched with sodium carbonate until pH ≈ 7. The DCM layer was collected, and the aqueous layer was further extracted with DCM (2 x 10 mL). The organics were combined, dried over sodium sulfate, decanted and then concentrated to give the crude amine. The crude was then purified with CombiFlash chromatography (silica gel, 20−40 μm, gradient 0−8% methanol/dichloromethane) to give the product as a white foamy solid (135 mg, 70%). 1H NMR (500 MHz, CD2Cl2) δ 8.12 (dd, J = 8.5, 2.8 Hz, 2H), 7.80 (d, J = 7.6 Hz, 2H), 7.70 (t, J = 5.4 Hz, 1H), 7.28 (d, J = 1.6 Hz, 2H), 7.20 (dd, J = 8.5, 1.6 Hz, 2H), 7.11 (t, J = 5.2 Hz, 1H), 7.08 (t, J = 5.2 Hz, 1H), 7.02 (t, J = 5.2 Hz, 1H), 4.91 – 4.88 (m, 4H), 3.75 (s, 6H), 3.72 – 3.51 (m, 43H), 3.51 – 3.45 (m, 4H), 3.42 – 3.29 (m, 4H). 311 13C{1H} NMR (125 MHz, CD2Cl2) δ 174.1, 171.4, 168.6, 168.6, 164.6, 164.6, 137.6, 132.2, 132.2, 129.0, 125.4, 125.4, 122.9, 122.8, 122.3, 112.4, 112.3, 110.0, 70.7, 70.7, 70.7, 70.7, 70.7, 70.5, 70.5, 70.5, 70.4, 70.4, 70.3, 69.9, 69.9, 53.0, 52.9, 48.0, 40.9, 39.5, 39.4, 39.3. FTIR (neat, cm−1): 3311, 2922, 1653, 1543, 1384. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C52H76Cl2N7O18 +), 1156.4619, 1157.4652, 1158.4589; found: 1156.4667, 1157.4702, 1158.463. O N Cl O NH NH 5 O NH2 O HN O OMe 5-30 O 5 HN O N O MeO O O 4 N H HBTU DIPEA DMF, rt, 16 h HO O Cl O NH H HN H S Cl N H O O N MeO S H NH H N H O H N 4 O O O O O 5 NH HN O 5-31 HN O 5 HN O O N MeO Cl dimethyl 2,2'-((21,24-dioxo-22-(17-oxo-21-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4- d]imidazol-4-yl)-4,7,10,13-tetraoxa-16-azahenicosanamido)-5,8,11,14,17,28,31,34,37,40- decaoxa-2,20,25,43-tetraazatetratetracontanedioyl)bis(6-chloro-1H-indole-3,1- diyl))diacetate (5-31): To a solution of amine (135 mg, 0.117 mmol) in DMF (3 mL) was added carboxylic acid (63 mg, 0.129 mmol) followed by HBTU (53 mg, 0.140 mmol) and DIPEA (49 µL, 0.281 mmol). The solution was then stirred overnight and then concentrated under reduced pressure to give the crude product. The crude was then purified with CombiFlash chromatography (silica gel, 20−40 μm, gradient 0−8% methanol/dichloromethane) to give the product as a white foamy solid (151 mg, 79%). 1H NMR (500 MHz, CD2Cl2) δ 8.13 (d, J = 8.3 Hz, 2H), 7.79 (m, 3H), 7.43 (s, 1H), 7.30 (s, 3H), 7.23 – 7.11 (m, 4H), 6.98 (s, 1H), 6.40 (s, 1H), 5.88 (s, 1H), 5.35 (d, J = 5.3 Hz, 1H), 4.92 (s, 4H), 4.78 – 4.63 (m, 1H), 4.45 (s, 1H), 4.26 (s, 1H), 3.94 – 3.02 (m, 81H), 2.87 (d, J = 8.5 Hz, 1H), 2.74 (m, 2H), 2.66 – 2.54 (m, 1H), 2.48 (s, 2H), 2.17 (s, 2H), 1.64 (d, J = 35.0 Hz, 4H), 1.40 (s, 2H). 13C{1H} NMR (125 MHz, CD2Cl2) δ 173.1, 171.4, 170.8, 168.4, 164.3, 164.0, 137.3, 132.0, 132.0, 128.6, 125.1, 122.6, 122.0, 111.9, 109.7, 70.4, 70.3, 70.3, 70.2, 312 70.2, 70.1, 70.1, 70.0, 69.9, 69.6, 69.5, 67.2, 61.8, 60.2, 55.7, 52.7, 50.2, 47.7, 40.5, 39.3, 39.2, 39.2, 39.2, 37.6, 36.8, 35.6, 28.2, 28.1, 25.5. FTIR (neat, cm−1): 3308, 2864, 1749, 1639, 1544. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C73H112Cl2N10O25S2+), 815.3444, 815.8460, 816.3429, 816.8446; found: 815.3436, 815.8456, 816.3438, 816.8447. O NH NH 5 O NH2 O HN O N 5-26 Cl O N Cl Cl O 5 HN O N O N Cl Cl NN HO O 5-32 HBTU DIPEA Cl DMF, rt, 24 h N N O NH 5 O NH O HN O NH O N Cl O N Cl Cl O 5 5-33 HN O N Cl O N Cl Cl N1,N4-bis(1-(1-(2-(bis(4-chlorophenyl)amino)-2-oxoethyl)-6-chloro-1H-indol-3-yl)-1-oxo- 5,8,11,14,17-pentaoxa-2-azanonadecan-19-yl)-2-(3-(3-(but-3-yn-1-yl)-3H-diazirin-3- yl)propanamido)succinimide (5-33): Amine 5-26 (208 mg, 0.133 mmol) was dissolved in DMF (2 mL) and diazirine 5-32 (26.5 mg, 0.159 mmol) was added. Then, HBTU (66 mg, 0.173 mmol) and DIPEA (56 µL, 0.319 mmol) were added. The reaction was stirred in the dark and at room temperature for 24 hours. The reaction was diluted with DCM (20 mL) and 10% LiBr solution (10 mL). The DCM layer was collected and washed with 10% LiBr solution (3 x 10 mL). The organic layer was dried using sodium sulfate, concentrated, and purified with CombiFlash chromatography (silica gel, 20−40 μm, gradient 0−8% methanol/dichloromethane) to give the product as a white foamy solid (171 mg, 75%). 1H NMR (400 MHz, CD2Cl2) δ 8.14 – 8.04 (m, 2H), 7.71 (d, J = 10.4 Hz, 2H), 7.62 – 6.95 (m, 25H), 4.79 (d, J = 2.0 Hz, 4H), 3.57 (m, 41H), 3.42 (dd, J = 4.9, 2.8 Hz, 4H), 3.31 (dt, J = 5.6, 3.1 Hz, 4H), δ 2.06 (t, J = 2.6 Hz, 1H), 1.97 (m, 4H), 1.73 (t, J = 7.7 Hz, 2H), 1.57 (t, J = 7.5 Hz, 2H), 1.41 (d, J = 6.6 Hz, 2H). 13C{1H} NMR (101 MHz, CD2Cl2) δ 171.5, 171.2, 170.8, 166.4, 166.4, 164.5, 164.5, 137.2, 132.3, 132.3, 130.7, 130.0, 129.0, 128.5, 127.2, 313 125.0, 125.0, 122.5, 121.9, 111.7, 111.7, 109.5, 82.9, 70.3, 70.3, 70.3, 70.2, 70.2, 70.1, 70.1, 70.0, 70.0, 69.9, 69.5, 69.5, 69.1, 50.3, 49.3, 39.3, 39.2, 39.1, 37.6, 32.1, 29.9, 28.1, 28.0, 13.1. FTIR (neat, cm−1): 3262, 2895, 1639, 1538, 1268. HRMS (ESI-TOF) m/z: [M+H]+ calcd for (C82H95Cl6N11O17 2+), 858.7499, 859.2516, 859.7485, 860.2501; found: 858.7490, 859.2501, 859.7485, 860.2490. 314 REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. Hetz, C.; Glimcher, L. H., Current Opinion in Cell Biology 2011, 23, 123-125. Huber, E. M.; Heinemeyer, W.; Li, X.; Arendt, C. S.; Hochstrasser, M.; Groll, M., Nature Communications 2016, 7, 10900. Coux, O.; Tanaka, K.; Goldberg, A. L., Annu. Rev. Biochem 1996, 65, 801-847. Eisele, M. R.; Reed, R. G.; Rudack, T.; Schweitzer, A.; Beck, F.; Nagy, I.; Pfeifer, G.; Plitzko, J. M.; Baumeister, W.; Tomko, R. J., Jr.; et al., Cell Reports 2018, 24, 1301- 1315.e1305. Sahu, I.; Bajorek, M.; Tan, X.; Srividya, M.; Krutauz, D.; Reis, N.; Osmulski, P. A.; Gaczynska, M. E.; Glickman, M. H. A Role for the Proteasome Alpha2 Subunit N-Tail in Substrate Processing. In Biomolecules, 2023; Vol. 13. McNaught, K. S. P.; Olanow, C. 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DG-6-13-product-CDCl3 _PROTON_01 Cl O Cl N Cl 9 3 7 . 3 2 7 . 1 0 4 . 3 0 2 . 4 3 2 1 0 -1 l 3 c d c 4 7 7 . l 3 c d c 2 7 7 . l 3 c d c 9 6 7 . . 5 2 4 13 12 11 10 9 8 7 6 f1 (ppm) 5 DG-6-13-product-CDCl3 _ CARBON_01 3 9 3 . 0 0 4 . Cl O Cl N Cl . 2 6 6 1 . 2 0 4 1 . 5 0 3 1 . 7 9 2 1 . 3 7 2 1 . 2 0 4 1 . 5 0 3 1 . 7 9 2 1 . 3 7 2 1 8 6 4 2 0 142 140 138 136 134 f1 (ppm) 132 130 128 126 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 317 7000 6500 6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 -500 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 5.9: 1H and 13C{1H} NMR spectra of 5-1 DG-6-14-prod-CDCl3 _PROTON_01 1 5 7 . 1 5 7 . 9 4 7 . 9 4 7 . 0 4 7 . 9 2 7 . l 3 c d c 7 2 7 . 5 1 . 7 3 1 . 7 9 0 7 . 9 0 7 . 7 0 7 . 0 9 6 . 9 8 . 6 7 4 6 . 7 4 6 . 1 3 . 5 5 7 4 . O N N Cl Cl Cl 1 5 7 . 1 5 7 . 9 4 7 . 9 4 7 . 0 4 7 . 9 2 7 . l 3 c d c 7 2 7 . 5 1 . 7 3 1 . 7 9 0 7 . 9 0 7 . 7 0 7 . 0 9 6 . 9 8 . 6 800 600 400 200 0 2 0 . 1 2 9 . 1 9 1 . 2 2 9 3 . 1 9 . 1 8 9 0 . 7.6 7.5 7.4 7.3 7.2 f1 (ppm) 7.1 7.0 6.9 6. 8 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 2 0 . 1 2 9 . 1 9 1 . 2 2 9 3 . 1 9 . 1 8 9 0 . 3 9 0 . 6 1 . 2 l 3 c d c 3 7 7 . l 3 c d c 1 . 7 7 l 3 c d c 8 . 6 7 . 4 9 4 DG-6-14-prod-CDCl3 _ CARBON_02 1 . 7 6 1 . 7 6 3 1 . 7 0 3 1 . 6 9 2 1 . 2 9 2 1 . 0 9 2 1 . 9 7 2 1 . 0 7 2 1 8 . 6 2 1 . 0 2 2 1 . 6 0 2 1 . 0 9 0 1 . 7 2 0 1 O N N Cl Cl Cl . 7 0 3 1 . 6 9 2 1 . 2 9 2 1 . 0 9 2 1 . 9 7 2 1 . 0 7 2 1 8 . 6 2 1 80 60 40 20 0 132 131 130 129 f1 (ppm) 128 127 126 4000 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0 -200 380 360 340 320 300 280 260 240 220 200 180 160 140 120 100 80 60 40 20 0 -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 -10 318 Figure 5.10: 1H and 13C{1H} NMR spectra of 5-7 DG-6-15-DMSO _PROTON_01 O H 1 9 9 . 6 2 . 8 7 0 . 8 5 0 . 8 2 8 7 . 3 8 7 . 8 7 7 . 5 6 7 . 4 6 7 . 3 4 7 . 5 3 7 . 8 2 7 . 6 2 7 . 7 2 7 . 6 2 7 . 6 2 7 . M C D 6 7 5 . 8 0 5 . O 2 H 5 3 . 3 F M D 8 8 2 . F M D 3 7 2 . o s m d 1 5 2 . o s m d 0 5 2 . o s m d 0 5 2 . o s m d 0 5 2 . o s m d 9 4 2 . O N N Cl Cl Cl 2 8 7 . 3 8 7 . 8 7 7 . 5 6 7 . 4 6 7 . 3 4 7 . 5 3 7 . 8 2 7 . 6 2 7 . 7 2 7 . 6 2 7 . 6 2 7 . 1000 500 0 3 1 . 1 6 8 . 1 5 8 . 1 3 9 . 1 5 7 . 1 8 2 . 1 7. 8 7.7 7.6 7.5 f1 (ppm) 7.4 7.3 7.2 6 9 0 . 10 F M D 7 2 6 1 . . 6 6 6 1 6 0 . 1 4 0 . 1 3 1 . 1 6 8 . 1 5 8 . 1 3 9 . 1 5 7 . 1 8 2 . 1 9 8 7 6 f1 (ppm) 2 . 3 4 1 . 0 9 3 1 . 6 0 3 1 . 4 9 2 1 7 . 8 2 1 5 . 3 2 1 1 . 3 2 1 . 4 2 2 1 . 7 7 1 1 . 2 2 1 1 4 1 . 2 5 4 3 2 8 . 9 4 M C D 4 5 5 . 1 0 -1 F M D 2 6 3 . F M D 2 . 1 3 13 12 DG-6-15-DMSO _ CARBON_01 O H O N N Cl Cl 11 3 . 5 8 1 Cl 5 . 1 3 1 . 6 0 3 1 . 4 9 2 1 7 . 8 2 1 5 0 133 132 131 130 f1 (ppm) 129 128 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 319 4500 4000 3500 3000 2500 2000 1500 1000 500 0 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 -2 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 . Figure 5.11: 1H and 13C{1H} NMR spectra of 5-2 DG-6-31-DMSO _PROTON_01 5 1 2 1 . O OH O N N Cl Cl Cl 4 0 8 . 6 9 7 . 4 9 7 . 5 7 7 . 5 7 7 . 2 6 7 . 2 4 7 . 2 3 7 . 9 1 7 . 9 1 7 . 8 1 7 . 7 1 7 . 0 0 5 . 4 0 8 . 6 9 7 . 4 9 7 . 5 7 7 . 5 7 7 . 2 6 7 . 2 4 7 . 2 3 7 . 9 1 7 . 9 1 7 . 8 1 7 . 7 1 7 . 1 0 1 . 2 0 1 . 9 8 2 . 9 0 2 . 8 1 2 . 6 8 1 . 3 0 1 . 8 .4 8 .2 8 .0 7. 8 7.6 f1 (ppm) 7.4 7.2 7.0 6. 8 6000 5000 4000 3000 2000 1000 0 -1000 5 9 0 . 1 0 1 . 2 0 1 . 9 8 2 . 9 0 2 . 8 1 2 . 6 8 1 . 3 0 1 . 13 12 11 10 9 8 7 7 9 1 . 5 6 f1 (ppm) DG-6-31-DMSO _ CARBON_01 O OH . 8 6 6 1 . 7 5 6 1 . 4 8 3 1 . 0 8 3 1 . 6 1 3 1 . 5 0 3 1 . 4 9 2 1 . 6 8 2 1 . 6 7 2 1 . 3 5 2 1 . 3 2 2 1 . 0 2 2 1 . 7 1 1 1 . 5 7 0 1 4 3 2 1 0 -1 o s m d 4 0 4 . o s m d 3 0 4 . o s m d 1 0 4 . o s m d 8 9 3 . o s m d 9 9 3 . o s m d 6 9 3 . o s m d 4 9 3 . . 5 9 4 O N N Cl Cl Cl . 6 1 3 1 . 5 0 3 1 . 4 9 2 1 . 6 8 2 1 . 6 7 2 1 30 20 10 0 133 132 131 130 f1 (ppm) 129 128 127 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 320 16000 15000 14000 13000 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 -1000 -2000 -3000 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 5.12: 1H and 13C{1H} NMR spectra of 5-3 DG-6-49-MeOD _PROTON_01 O NH 0 0 8 . 9 9 7 . 7 5 7 . 3 4 7 . 3 3 7 . 2 3 7 . 9 2 7 . 2 2 7 . 3 1 7 . 2 1 7 . 1 1 7 . 1 1 7 . O D H 8 8 4 . 1 9 4 . d o 3 d c 1 3 3 . d o 3 d c 1 3 3 . d o 3 d c 1 3 3 . d o 3 d c 0 3 3 . d o 3 d c 0 3 3 . 9 8 2 . 9 7 2 . Cl N O N Cl Cl 7 5 7 . 3 4 7 . 3 3 7 . 2 3 7 . 9 2 7 . 2 2 7 . 3 1 7 . 2 1 7 . 1 1 7 . 1 1 7 . 0 0 1 . 5 8 3 . 4 9 2 . 2 0 2 . 4 0 1 . 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 f1 (ppm) 600 500 400 300 200 100 0 -100 6 9 0 . 0 0 1 . 5 8 3 . 4 9 2 . 2 0 2 . 4 0 1 . 13 12 11 10 9 8 7 6 f1 (ppm) DG-6-49-MeOD _ CARBON_01 O NH . 4 7 6 1 . 4 6 6 1 . 5 7 3 1 . 0 2 3 1 . 2 0 3 1 . 9 9 2 1 . 9 8 2 1 . 4 8 2 1 . 6 7 2 1 . 7 4 2 1 . 8 1 2 1 . 4 1 2 1 . 9 0 1 1 . 8 9 0 1 1 2 2 . 5 4 9 9 2 . 3 2 1 0 -1 d o 3 d c 1 8 4 . d o 3 d c 9 7 4 . d o 3 d c 8 7 4 . d o 3 d c 6 7 4 . d o 3 d c 4 7 4 . d o 3 d c 3 7 4 . d o 3 d c 1 7 4 . . 5 7 3 . 0 5 2 Cl N O N Cl Cl . 2 0 3 1 . 9 9 2 1 . 9 8 2 1 . 4 8 2 1 . 6 7 2 1 5 0 131 130 129 f1 (ppm) 128 127 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 321 9000 8500 8000 7500 7000 6500 6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 -500 90 80 70 60 50 40 30 20 10 0 Figure 5.13: 1H and 13C{1H} NMR spectra of 5-4 DG-6-47-MeOD _PROTON_01 O NH Cl N O N Cl 1 0 8 . 0 0 8 . 1 6 7 . 5 4 7 . 3 3 7 . 2 3 7 . 9 2 7 . 1 2 7 . 3 1 7 . 2 1 7 . 1 1 7 . 0 1 7 . 5 0 7 . O D H 8 8 4 . 0 9 4 . d o 3 d c 1 3 3 . d o 3 d c 1 3 3 . d o 3 d c 1 3 3 . d o 3 d c 0 3 3 . d o 3 d c 0 3 3 . 5 3 3 . 4 3 3 . 2 3 3 . 4 0 2 . 0 0 2 . 9 5 1 . 8 5 1 . 7 5 1 . 5 5 1 . 4 4 1 . 3 4 1 . 1 4 1 . 0 4 1 . 8 3 1 . 7 3 1 . 7 9 0 . 6 9 0 . 4 9 0 . 9 5 1 . 8 5 1 . 7 5 1 . 5 5 1 . 4 4 1 . 3 4 1 . 1 4 1 . 0 4 1 . 8 3 1 . 7 3 1 . 7 9 0 . 6 9 0 . 4 9 0 . 12000 11000 10000 9000 Cl 1 6 7 . 5 4 7 . 3 3 7 . 2 3 7 . 9 2 7 . 1 2 7 . 3 1 7 . 2 1 7 . 1 1 7 . 0 1 7 . 5 0 7 . 2000 8000 1000 500 1000 0 6 9 0 . 7.6 7.7 7 3 9 . 5 0 1 . 7.5 7.4 7.3 f1 (ppm) 7.2 7.1 7.0 0 1. 8 8 9 1 . 1.6 8 9 1 . 1.4 2 9 2 . 1.2 f1 (ppm) 1.0 0. 8 13 12 11 10 9 8 7 DG-6-47-MeOD _ CARBON_01 4 9 0 . 6 9 0 . 7 3 9 . 5 0 1 . 2 0 2 . 5 6 f1 (ppm) 6 9 1 . 8 9 1 . 8 9 1 . 2 9 2 . 4 3 2 1 0 -1 . 4 7 6 1 . 8 5 6 1 . 5 7 3 1 . 0 2 3 1 . 2 0 3 1 . 9 9 2 1 . 8 8 2 1 . 4 8 2 1 . 6 7 2 1 . 9 4 2 1 . 9 1 2 1 . 4 1 2 1 . 1 1 1 1 . 8 9 0 1 Cl . 2 0 3 1 . 9 9 2 1 . 8 8 2 1 . 4 8 2 1 . 6 7 2 1 O NH Cl N O N Cl 131 130 129 f1 (ppm) 128 127 d o 3 d c 1 7 4 . . 8 8 3 . 5 1 3 . 9 9 1 . 8 2 1 d o 3 d c 1 8 4 . d o 3 d c 0 8 4 . d o 3 d c 8 7 4 . d o 3 d c 6 7 4 . d o 3 d c 4 7 4 . d o 3 d c 3 7 4 . . 9 8 4 15 10 5 0 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 322 7000 6000 5000 4000 3000 2000 1000 0 -1000 110 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 5.14: 1H and 13C{1H} NMR spectra of 5-10 DG-6-37-MeOD _PROTON_01 NH2 O 5 Cl O N NH O N Cl Cl 10000 8000 6000 4000 2000 0 3 6 3 . 2 6 3 . 1 6 3 . 8 5 3 . 7 5 3 . 7 5 3 . 5 5 3 . 4 5 3 . 2 5 3 . 2 5 3 . 7 4 3 . 6 4 3 . d o 3 d c 2 3 3 . d o 3 d c 1 3 3 . d o 3 d c 1 3 3 . d o 3 d c 1 3 3 . d o 3 d c 0 3 3 . 5 1 2 2 . 3 .7 3 .6 3 .5 f1 (ppm) 3 .4 3 .3 O D H 7 8 4 . 0 9 4 . 4 6 3 . 3 6 3 . 2 6 3 . 1 6 3 . 9 5 3 . 8 5 3 . 7 5 3 . 7 5 3 . 5 5 3 . 4 5 3 . 2 5 3 . 2 5 3 . 8 4 3 . 7 4 3 . 6 4 3 . d o 3 d c 2 3 3 . d o 3 d c 1 3 3 . d o 3 d c 1 3 3 . d o 3 d c 1 3 3 . d o 3 d c 0 3 3 . 3 0 8 . 2 0 8 . 3 7 7 . 8 4 7 . 7 2 7 . 1 2 7 . 2 1 7 . 2 1 7 . 1 1 7 . 0 1 7 . 1 8 2 . 0 8 2 . 9 7 2 . 13 12 11 10 9 8 7 6 f1 (ppm) 1 0 1 . 2 0 1 . 8 8 3 . 0 1 1 . 0 7 3 . 7 0 1 . 6 9 1 . 5 5 1 2 2 . 4 8 8 1 . 3 2 1 0 -1 NH2 O 5 DG-6-37-MeOD _ CARBON_01 Cl O N NH O N Cl Cl . 2 7 6 1 . 8 5 6 1 . 6 7 3 1 . 4 2 3 1 . 3 0 3 1 . 1 0 3 1 . 9 8 2 1 . 4 8 2 1 . 7 7 2 1 . 9 4 2 1 . 9 1 2 1 . 5 1 2 1 . 8 0 1 1 . 9 9 0 1 . 2 0 7 . 1 0 7 . 1 0 7 . 0 0 7 . 0 0 7 . 0 0 7 . 8 9 6 . 9 9 6 . 7 9 6 . 5 9 6 . 5 5 6 . 2 0 4 . 9 8 3 . 4 2 3 1 . 3 0 3 1 . 1 0 3 1 . 9 8 2 1 . 4 8 2 1 . 7 7 2 1 . 2 0 7 . 1 0 7 . 1 0 7 . 0 0 7 . 0 0 7 . 0 0 7 . 9 9 6 . 8 9 6 . 7 9 6 . 5 9 6 60 40 20 0 200 150 100 50 0 133 132 131 130 f1 (ppm) 129 128 127 70.2 70.1 70.0 69.9 69. 8 69.7 69.6 69.5 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 -10 323 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 -1000 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 -5 Figure 5.15: 1H and 13C{1H} NMR spectra of 5-12 DG-6-39-CDCl3 _PROTON_02 O N H O O 5 N H N H O N H 5 Cl Cl N N O Cl l 3 c d c 7 2 7 . 4 0 8 . 2 0 8 . 6 5 7 . 4 4 7 . O Cl 2 2 7 . 6 1 7 . 6 1 7 . 5 1 7 . 4 1 7 . 1 1 7 . 6 0 7 . 6 0 7 . 0 7 4 . 9 6 4 . 5 6 3 . 4 6 3 . 2 6 3 . 2 6 3 . 0 6 3 . 9 5 3 . 9 5 3 . 6 5 3 . 5 5 3 . 2 5 3 . 6 4 3 . 5 4 3 . 4 4 3 . 0 3 3 . 9 2 3 . 8 2 3 . N N O Cl 5 6 3 . 4 6 3 . 2 6 3 . 2 6 3 . 0 6 3 . 9 5 3 . 9 5 3 . 6 5 3 . 5 5 3 . 2 5 3 . 6 4 3 . 5 4 3 . 4 4 3 . 0 3 3 . 9 2 3 . 8 2 3 . Cl 6 5 7 . 4 4 7 . l 3 c d c 7 2 7 . 2 2 7 . 6 1 7 . 6 1 7 . 5 1 7 . 4 1 7 . 1 1 7 . 6 0 7 . 6 0 7 . 2000 1500 1000 500 0 3 .7 0 5 0 2 . 3 .6 0 1 2 . 3 .5 f1 (ppm) 3 .4 1 0 2 . 3 .3 10000 5000 0 8 9 0 . 7.6 7.5 5 9 1 . 7.4 7 1 1 . 4 8 3 7.2 . 7.3 f1 (ppm) 5 2 1 . 2 9 1 7.1 . 7.0 6.9 0 0 1 . 8 9 0 . 5 9 1 . 4 8 3 . 7 1 1 . 2 9 1 . 5 2 1 . 9 9 0 . 0 5 0 2 . 0 1 2 . 1 0 2 . 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 DG-6-39-CDCl3 _ CARBON_01 . 3 6 6 1 . 5 4 6 1 . 8 8 5 1 . 1 7 3 1 . 9 1 3 1 . 8 0 3 1 . 7 9 2 1 . 2 9 2 1 . 8 8 2 1 . 9 6 2 1 . 8 4 2 1 . 4 2 2 1 . 3 2 2 1 . 9 1 1 1 . 4 9 0 1 . 6 0 7 . 5 0 7 . 4 0 7 . 4 0 7 . 4 0 7 . 3 0 7 . 3 0 7 . 1 0 7 . 0 0 7 M C D 5 3 5 . . 3 9 4 . 7 5 4 . 9 9 3 . 1 9 3 Cl Cl O N H O O 5 N H N H O N H 5 N N O Cl O Cl N N O Cl Cl . 9 1 3 1 . 8 0 3 1 . 7 9 2 1 . 2 9 2 1 . 8 8 2 1 . 9 6 2 1 . 6 0 7 . 5 0 7 . 4 0 7 . 4 0 7 . 4 0 7 . 3 0 7 . 3 0 7 . 1 0 7 . 0 0 7 100 50 0 133 132 131 130 f1 (ppm) 129 128 127 126 70.7 70.6 70.5 70.4 70.3 f1 (ppm) 70.2 70.1 70.0 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 -20 200 150 100 50 0 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 324 Figure 5.16: 1H and 13C{1H} NMR spectra of 5-11 DG-6-41-MeOD _PROTON_01 NH2 O 8 Cl O N NH O N Cl Cl 7.6 5 6 3 . 4 6 3 . 2 6 3 . 7 5 3 . 4 5 3 . 2 5 3 . 1 5 3 . 8 4 3 . 7 4 3 . 6 4 3 . 5 4 3 . 9 4 7 . 6 3 7 . 0 3 7 . 4 1 7 . 3 1 7 . 2 1 7 . 1 1 7 . 600 400 200 0 8000 6000 4000 2000 0 6 1 4 . 7.5 7.4 3 3 1 . 7.3 f1 (ppm) 5 5 3 . 7.2 3 0 1 . 7.1 3 .65 3 .60 . 9 3 6 3 3 .55 f1 (ppm) 3 .50 3 .45 O D H 5 8 4 . 2 9 4 . 1 9 4 . 5 6 3 . 4 6 3 . 2 6 3 . 7 5 3 . 4 5 3 . 2 5 3 . 1 5 3 . 8 4 3 . 7 4 3 . 6 4 3 . 5 4 3 . d o 3 d c 1 3 3 . d o 3 d c 1 3 3 . d o 3 d c 0 3 3 . d o 3 d c 0 3 3 . 3 0 8 . 2 0 8 . 2 7 7 . 9 4 7 . 6 3 7 . 0 3 7 . 4 1 7 . 3 1 7 . 2 1 7 . 1 1 7 . M C D 8 4 5 . 13 12 11 10 9 8 7 2 0 1 . 0 0 1 . 6 1 4 . 3 3 1 . 5 5 3 . 3 0 1 . 7 2 1 . 1 7 5 . 5 6 f1 (ppm) 5 7 2 . 4 7 2 . 3 7 2 . 9 3 6 3 . 4 8 1 . 4 3 2 1 0 -1 NH2 O 8 . 5 8 6 1 . 1 7 6 1 . 9 8 3 1 . 8 3 3 1 . 6 1 3 1 . 4 1 3 1 . 2 0 3 1 . 7 9 2 1 . 0 9 2 1 . 3 6 2 1 . 3 3 2 1 . 9 2 2 1 . 2 2 1 1 . 3 1 1 1 . 2 3 7 . 5 1 7 . 5 1 7 . 4 1 7 . 4 1 7 . 4 1 7 . 4 1 7 . 3 1 7 . 2 1 7 . 8 0 7 . 9 6 6 d o 3 d c 3 9 4 . d o 3 d c 2 9 4 . d o 3 d c 0 9 4 . d o 3 d c 8 8 4 . d o 3 d c 7 8 4 . M C D 9 4 5 . . 0 2 4 . 3 0 4 DG-6-41-MeOD _ CARBON_01 Cl O N NH O N Cl Cl . 6 1 3 1 . 4 1 3 1 . 2 0 3 1 . 7 9 2 1 . 0 9 2 1 . 5 1 7 . 5 1 7 . 4 1 7 . 4 1 7 . 4 1 7 . 4 1 7 . 3 1 7 . 2 1 7 20 10 0 300 200 100 0 132.5 132.0 131.5 131.0 130.5 130.0 129.5 129.0 f1 (ppm) 71.5 71.4 71.3 71.2 f1 (ppm) 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 250 200 150 100 50 0 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 325 Figure 5.17: 1H and 13C{1H} NMR spectra of 5-13 DG-6-72-CD2Cl2_PROTON_01 2 9 7 . 1 9 7 . 7 4 7 . 6 3 7 . 6 1 7 . 5 0 7 . 3 0 7 . 3 6 6 . 2 6 6 . 1 6 6 . l 2 c 2 d c 3 3 l 2 c 2 d c 2 3 l 2 c 2 d c 1 3 . . . 5 5 5 2 6 4 . 0 5 . 3 9 4 . 3 8 4 . 3 7 4 . 3 5 4 . 3 3 4 . 3 2 4 . 3 4 3 3 3 2 3 . . . 3 3 3 9 2 . 3 6 1 . 3 5 1 . 3 4 1 . 3 3 1 . 3 Cl Cl N N O O N H O N H N H O 8 O N H 8 O Cl N N O Cl Cl Cl 13 12 11 10 9 DG-6-72-CD2Cl2_ CARBON_01 5 9 0 . 8 6 9 0 . 3 9 . 1 2 0 4 . 3 0 4 . 8 0 . 1 1 1 . 2 . 2 0 6 3 0 9 . 1 7 6 f1 (ppm) 5 4 3 . 1 6 6 1 . 2 4 6 1 5 . 8 5 1 . 2 7 3 1 . 0 2 3 1 8 . 0 3 1 . 0 0 3 1 . 0 9 2 1 6 . 8 2 1 . 1 7 2 1 . 8 4 2 1 . 4 2 2 1 . 9 1 2 1 . 9 1 1 1 . 6 9 0 1 . 6 0 7 . 4 0 7 . 4 0 7 . 4 0 7 3 3 . . 0 7 0 7 . 2 0 7 . 1 0 7 . 0 0 7 l 2 c 2 d c 9 3 5 . 2 l 2 c 2 d c 3 . 3 5 1 0 -1 -2 3 . 9 4 . 0 0 4 . 1 9 3 l 2 c 2 d c 1 . 3 5 l 2 c 2 d c 7 . l 2 c 2 d c 5 . 3 5 3 5 . 0 2 3 1 8 . 0 3 1 . 0 0 3 1 . 0 9 2 1 6 . 8 2 1 . 1 7 2 1 . 6 0 7 . 4 0 7 . 4 0 7 . 4 0 7 3 3 . . 0 7 0 7 . 2 0 7 . 1 0 7 . 0 0 7 60 50 40 30 20 10 0 15 10 5 0 132 131 130 129 128 127 f1 (ppm) 70.7 70.6 70.5 70.4 70.3 70.2 70.1 70.0 69.9 69. 8 f1 (ppm) Cl Cl N N O O N H O N H N H O 8 O N H 8 O Cl N N O Cl Cl Cl 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 -10 326 2100 2000 1900 1800 1700 1600 1500 1400 1300 1200 1100 1000 900 800 700 600 500 400 300 200 100 0 -100 65 60 55 50 45 40 35 30 25 20 15 10 5 0 -5 Figure 5.18: 1H and 13C{1H} NMR spectra of 5-15 l 2 c 2 d c 1 3 5 . l 2 c 2 d c 2 3 5 . DG-6-73-CD2Cl2_PROTON_01 H N O 5 O 8 O H N O O 8 HN O 3 0 8 . 2 0 8 . 3 6 7 . 8 4 7 . 1 3 7 . 9 2 7 . 8 2 7 . 7 1 7 . 5 1 7 . 7 7 6 . O NH N O N Cl Cl Cl Cl N N O Cl Cl 6 7 4 . 7 6 3 . 6 6 3 . 5 6 3 . 1 6 3 . 5 5 3 . 0 6 3 . 4 5 3 . 9 4 3 . 7 4 3 . 8 4 3 . 6 3 3 . 5 3 3 . 1 4 2 . 0 4 2 . 9 3 2 . 7 0 0 . 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 -2 7 8 0 . 2 2 1 . 0 1 0 1 . 0 9 1 . 7 9 1 . 4 0 6 4 . 0 9 1 . l 2 c 2 d c 9 3 5 . l 2 c 2 d c 7 3 5 . l 2 c 2 d c 5 3 5 . l 2 c 2 d c 3 3 5 . l 2 c 2 d c 0 3 5 . . 3 9 4 . 1 9 3 . 7 6 3 DG-6-73-CD2Cl2_ CARBON_01 H N O 5 O 8 O . 3 1 7 1 . 1 6 6 1 . 3 4 6 1 H N O O 8 HN O O NH N O N Cl Cl Cl Cl N N O Cl . 8 0 3 1 Cl . 3 7 3 1 . 0 2 3 1 . 8 0 3 1 . 0 0 3 1 . 0 9 2 1 . 6 8 2 1 . 1 7 2 1 . 8 4 2 1 . 3 2 2 1 . 0 2 2 1 . 9 1 1 1 . 6 9 0 1 . 4 0 7 . 4 0 7 . 3 0 7 . 2 0 7 . 2 0 7 . 1 0 7 . 0 0 7 . 7 9 6 . 1 7 6 . 0 0 3 1 . 0 9 2 1 . 6 8 2 1 . 1 7 2 1 8 6 4 2 0 131 130 129 f1 (ppm) 128 127 1500 1400 1300 1200 1100 1000 900 800 700 600 500 400 300 200 100 0 -100 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 -10 327 Figure 5.19: 1H and 13C{1H} NMR spectra of 5-16 DG-6-51-CD2Cl2_PROTON_01 Cl 6 0 8 . 4 0 8 . 5 6 7 . 0 5 7 . 4 3 7 . 7 2 7 . 8 1 7 . 7 1 7 . 5 1 7 . 5 1 7 . 9 9 6 . 4 7 4 . 3 6 3 . 2 6 3 . 2 6 3 . 1 6 3 . 1 6 3 . 0 6 3 . 0 6 3 . 9 5 3 . 8 5 3 . 8 5 3 . 8 5 3 . 7 5 3 . 7 5 3 . 7 5 3 . 6 5 3 . 5 5 3 . 5 5 3 . 4 5 3 . 4 5 3 . A E T 7 0 3 . A E T 6 0 3 . A E T 4 0 3 . A E T 3 0 3 . F M D 3 9 2 . F M D 4 8 2 . A E T 0 3 1 . A E T 9 2 1 . A E T 7 2 1 . O NH O 5 Cl N O N Cl Cl O N NH O N Cl Cl 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 0 0 1 . 4 9 0 . 5 9 1 . 4 4 8 . 1 8 1 . 3 9 2 1 . l 2 c 2 d c 3 3 5 . l 2 c 2 d c 0 3 5 . . 2 9 4 . 1 9 3 l 2 c 2 d c 7 3 5 . l 2 c 2 d c 5 3 5 . l 2 c 2 d c 9 3 5 . DG-6-59-2nd-column-CD2Cl2_ CARBON_01 O NH O 5 Cl O N NH O N Cl Cl Cl . 2 6 6 1 . 2 4 6 1 . 2 7 3 1 . 0 2 3 1 . 8 0 3 1 . 9 9 2 1 . 0 9 2 1 . 5 8 2 1 . 0 7 2 1 . 8 4 2 1 . 4 2 2 1 . 9 1 2 1 . 8 1 1 1 . 5 9 0 1 . 4 0 7 . 4 0 7 . 4 0 7 . 1 0 7 . 9 9 6 Cl N O N Cl . 0 2 3 1 . 8 0 3 1 . 9 9 2 1 . 0 9 2 1 . 5 8 2 1 . 0 7 2 1 25 20 15 10 5 0 132 131 130 129 128 127 f1 (ppm) 7500 7000 6500 6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 -500 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 328 Figure 5.20: 1H and 13C{1H} NMR spectra of 5-19 DG-6-56-DMSO _PROTON_01 O N Cl 0 6 7 . 2 4 7 . 8 2 7 . o s m d 1 5 2 . o s m d 0 5 2 . o s m d 0 5 2 . o s m d 0 5 2 . o s m d 9 4 2 . 9 1 4 . 13 12 11 10 9 8 7 6 f1 (ppm) 5 DG-6-56-DMSO _ CARBON_01 8 1 0 1 . O N Cl . 6 3 5 6 1 . 3 8 9 2 1 . 4 1 9 2 1 . 1 6 8 2 1 . 0 7 6 2 1 0 0 2 . 4 3 2 1 0 -1 o s m d 2 0 0 4 . o s m d 6 8 9 3 . o s m d 9 6 9 3 . o s m d 2 5 9 3 . o s m d 5 3 9 3 . o s m d 9 1 9 3 . o s m d 2 0 9 3 . 4 2 3 4 . . 3 8 9 2 1 . 4 1 9 2 1 . 1 6 8 2 1 . 0 7 6 2 1 25 20 15 10 5 0 132 131 130 129 128 f1 (ppm) 127 126 125 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 329 9000 8500 8000 7500 7000 6500 6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 -500 65 60 55 50 45 40 35 30 25 20 15 10 5 0 -5 Figure 5.21: 1H and 13C{1H} NMR spectra of 5-17 DG-6-57-DMSO _PROTON_01 5 6 7 . 3 5 7 . 3 5 7 . 3 5 7 . 1 5 7 . 1 5 7 . 1 5 7 . 5 3 7 . 4 3 7 . 3 2 7 . 4 2 7 . 4 1 7 . 3 1 7 . 1 1 7 . 3 0 7 . 1 0 7 . 0 0 7 . 0 4 6 . 9 3 6 . 9 8 4 . O N N 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 . 5 6 7 . 3 5 7 . 3 5 7 . 3 5 7 . 1 5 7 . 1 5 7 . 1 5 7 . 5 3 7 . 4 3 7 . 3 2 7 . 4 2 7 . 4 1 7 . 3 1 7 . 1 1 7 . 3 0 7 . 1 0 7 . 1000 500 0 2 0 1 . 7. 8 7.7 7.6 . 0 0 3 1 7.5 7.4 f1 (ppm) 7.3 7.2 3 1 1 . 7.1 7 9 1 . 5 6 f1 (ppm) 4 3 2 1 0 -1 o s m d 3 0 4 . o s m d 1 0 4 . o s m d 0 0 4 . o s m d 8 9 3 . o s m d 6 9 3 . . 9 8 4 . 6 0 3 1 . 3 0 3 1 . 4 9 2 1 . 5 8 2 1 . 0 7 2 1 15 10 5 0 130 129 f1 (ppm) 128 127 13 12 11 10 9 8 7 DG-6-57-DMSO _ CARBON_01 0 0 3 1 . 3 1 1 . 2 0 1 . 9 8 0 . . 5 7 6 1 . 8 6 3 1 . 6 0 3 1 . 3 0 3 1 . 4 9 2 1 . 5 8 2 1 . 0 7 2 1 . 5 1 2 1 . 7 0 2 1 . 5 9 1 1 . 1 0 1 1 . 2 1 0 1 O N N 8500 8000 7500 7000 6500 6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 -500 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 330 Figure 5.22: 1H and 13C{1H} NMR spectra of 5-20 DG-6-58-DMSO _PROTON_01 9 9 7 . 9 6 7 . 4 5 7 . 7 4 7 . 6 4 7 . 6 4 7 . 3 3 7 . 5 2 7 . 4 2 7 . 3 2 7 . 3 2 7 . 2 2 7 . 1 2 7 . 0 2 7 . 9 1 7 . 8 1 7 . 7 1 7 . 6 1 7 . 8 9 4 . O 2 H 5 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 . 9 9 1 1 . O OH O N N 9 6 7 . 4 5 7 . 7 4 7 . 6 4 7 . 6 4 7 . 3 3 7 . 5 2 7 . 4 2 7 . 3 2 7 . 3 2 7 . 2 2 7 . 1 2 7 . 0 2 7 . 9 1 7 . 8 1 7 . 7 1 7 . 6 1 7 . 800 600 400 200 0 3 1 1 1 . 7.5 7.4 7.3 . 8 4 3 7.2 7.7 7.6 f1 (ppm) 4 9 0 . 12 13 DG-6-58-DMSO _ CARBON_01 11 10 9 8 7 3 1 1 1 . 8 4 3 . 7 3 1 . 0 0 2 . 5 6 f1 (ppm) . 8 6 6 1 . 1 6 6 1 . 5 7 3 1 . 2 7 3 1 . 5 0 3 1 . 4 9 2 1 . 0 7 2 1 . 7 6 2 1 . 7 2 2 1 . 7 1 2 1 . 1 1 2 1 . 2 1 1 1 . 2 7 0 1 4 3 2 1 0 -1 o s m d 3 0 4 . o s m d 1 0 4 . o s m d 9 9 3 . o s m d 8 9 3 . o s m d 6 9 3 . . 3 9 4 O OH O N N . 5 0 3 1 . 4 9 2 1 . 0 7 2 1 . 7 6 2 1 15 10 5 0 132 131 130 129 f1 (ppm) 128 127 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 331 7000 6500 6000 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 -500 65 60 55 50 45 40 35 30 25 20 15 10 5 0 -5 Figure 5.23: 1H and 13C{1H} NMR spectra of 5-21 DG-6-61-MeOD _PROTON_01 NH2 O 5 3 1 8 . 2 1 8 . 7 7 7 . 2 5 7 . 6 4 7 . 3 3 7 . 1 3 7 . 7 2 7 . 6 2 7 . 4 2 7 . 0 2 7 . 8 1 7 . 7 1 7 . O D H 6 9 4 . 2 9 4 . 4 6 3 . 3 6 3 . 2 6 3 . 1 6 3 . 1 6 3 . 0 6 3 . 0 6 3 . 9 5 3 . 8 5 3 . 7 5 3 . 6 5 3 . 7 5 3 . 6 5 3 . 5 5 3 . 4 5 3 . 3 5 3 . 2 5 3 . 2 5 3 . 1 5 3 . 0 5 3 . O N NH O N d o 3 d c 2 3 3 . d o 3 d c 1 3 3 . d o 3 d c 1 3 3 . d o 3 d c 1 3 3 . d o 3 d c 0 3 3 . 2 9 2 . 1 9 2 . 0 9 2 . 4500 13 12 11 10 9 8 7 6 f1 (ppm) DG-6-61-MeOD _ CARBON_01 6 9 0 . 5 9 0 . 8 1 3 1 . 3 0 2 . 5 1 9 2 2 . 4 6 9 1 . 3 O 5 NH2 . 8 8 6 1 . 7 7 6 1 . 4 8 3 1 . 5 3 3 1 . 5 1 3 1 . 1 0 3 1 . 7 9 2 1 . 7 7 2 1 . 6 7 2 1 . 9 3 2 1 . 4 2 2 1 . 2 2 2 1 . 6 1 1 1 . 9 0 1 1 . 3 1 7 . 2 1 7 . 1 1 7 . 1 1 7 . 0 1 7 . 9 0 7 . 8 0 7 . 9 8 6 . 1 0 5 2 1 0 -1 d o 3 d c 5 9 4 . d o 3 d c 3 9 4 . d o 3 d c 2 9 4 . d o 3 d c 0 9 4 . d o 3 d c 8 8 4 . d o 3 d c 7 8 4 . d o 3 d c 5 8 4 . A E T 3 7 4 . . 7 0 4 . 0 0 4 A E T 9 9 . O N NH O N . 3 1 7 . 2 1 7 . 1 1 7 . 1 1 7 . 0 1 7 . 9 0 7 . 8 0 7 300 200 100 0 71.5 71.4 71.3 71.2 71.1 71.0 f1 (ppm) 70.9 70. 8 70.7 70.6 60 50 40 30 20 10 0 . 5 1 3 1 . 1 0 3 1 . 7 9 2 1 131.5 131.0 130.5 f1 (ppm) 130.0 129.5 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 332 4000 3500 3000 2500 2000 1500 1000 500 0 900 850 800 750 700 650 600 550 500 450 400 350 300 250 200 150 100 50 0 -50 Figure 5.24: 1H and 13C{1H} NMR spectra of 5-18 DG-6-62-MeOD _PROTON_01 I D C 1 7 7 . 9 0 8 . 7 0 8 . 9 6 7 . 9 4 7 . 0 3 7 . 0 3 7 . 9 2 7 . 0 3 7 . 8 2 7 . 8 2 7 . 5 2 7 . 5 2 7 . 4 2 7 . 4 2 7 . 2 2 7 . 2 2 7 . 9 1 7 . 8 1 7 . 7 1 7 . 6 1 7 . 5 1 7 . 8 4 5 . I D C 6 0 7 . O D H 1 9 4 . 3 9 4 . 3 6 3 . 2 6 3 . 1 6 3 . 8 5 3 . 7 5 3 . 7 5 3 . 6 5 3 . 5 5 3 . 4 5 3 . 4 5 3 . 3 5 3 . 3 5 3 . 3 5 3 . 2 5 3 . 2 5 3 . 0 5 3 . 0 5 3 . 9 4 3 . 8 4 3 . 1 4 3 . 0 4 3 . 9 3 3 . d o 3 d c 1 3 3 . d o 3 d c 1 3 3 . d o 3 d c 0 3 3 . 12000 2 2 3 . 1 2 3 . 0 2 3 . 11000 O N H NH O O 5 O N H N O N N O N NH 5 O 9 4 7 . 0 3 7 . 0 3 7 . 9 2 7 . 0 3 7 . 8 2 7 . 8 2 7 . 5 2 7 . 5 2 7 . 4 2 7 . 4 2 7 . 2 2 7 . 2 2 7 . 9 1 7 . 8 1 7 . 7 1 7 . 6 1 7 . 5 1 7 . 7.6 7.5 7.4 8 3 3 1 . 7.3 7.2 f1 (ppm) 3 6 3 . 2 6 3 . 1 6 3 . 8 5 3 . 4 5 3 . 4 5 3 . 3 5 3 . 3 5 3 . 3 5 3 . 2 5 3 . 0 5 3 . 0 5 3 . 9 4 3 . 8 4 3 . 0 4 3 . d o 3 d c 1 3 3 . d o 3 d c 1 3 3 . d o 3 d c 0 3 3 . 1 2 3 . 3000 2000 1000 0 3 1 0 2 . . 0 0 2 3 .4 f1 (ppm) 3 .5 3 .7 3 .6 7 8 1 3 .2 . 3 .3 3 .1 1000 500 0 13 12 11 10 9 8 7 DG-6-62-MeOD _ CARBON_01 0 0 1 . 3 0 1 . 8 3 3 1 . 1 1 2 . 5 6 f1 (ppm) 3 1 0 2 . 0 0 2 . 7 8 1 . 4 3 2 1 0 -1 . 0 9 6 1 . 7 7 6 1 . 0 1 6 1 . 4 8 3 1 . 5 3 3 1 . 5 1 3 1 . 2 0 3 1 . 8 9 2 1 . 6 7 2 1 . 6 7 2 1 . 9 3 2 1 . 5 2 2 1 . 2 2 2 1 . 8 1 1 1 . 9 0 1 1 . 5 1 7 . 5 1 7 . 4 1 7 . 4 1 7 . 4 1 7 . 3 1 7 . 2 1 7 . 9 0 7 . 3 0 5 d o 3 d c 5 9 4 . d o 3 d c 3 9 4 . d o 3 d c 2 9 4 . d o 3 d c 0 9 4 . d o 3 d c 8 8 4 . d o 3 d c 7 8 4 . d o 3 d c 5 8 4 . . 9 0 4 . 3 0 4 O N H NH O O 5 O N H N O N N O N NH 5 O . 5 1 3 1 . 2 0 3 1 . 8 9 2 1 . 6 7 2 1 . 6 7 2 1 132 131 130 129 f1 (ppm) 128 127 . 5 1 7 . 5 1 7 . 4 1 7 . 4 1 7 . 4 1 7 . 3 1 7 . 2 1 7 . 9 0 7 10 71.6 5 0 71.5 71.4 71.3 71.2 71.1 71.0 70.9 f1 (ppm) 100 50 0 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 333 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 -1000 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 5.25: 1H and 13C{1H} NMR spectra of 5-23 DG-6-103-CD2Cl2_PROTON_01 9 0 8 . 7 0 8 . 3 7 7 . 0 5 7 . 0 5 7 . 6 3 7 . 9 2 7 . 9 1 7 . 9 1 7 . 7 1 7 . 4 0 7 . 3 0 7 . 2 0 7 . 9 8 6 . 8 8 6 . 7 8 6 . l 2 c 2 d c 4 3 5 . l 2 c 2 d c 3 3 5 . 1 8 4 . 9 7 4 . 6 6 3 . 5 6 3 . 4 6 3 . 3 6 3 . 2 6 3 . 1 6 3 . 0 6 3 . 0 6 3 . 9 5 3 . 9 5 3 . 8 5 3 . 6 5 3 . 5 5 3 . 4 5 3 . 3 5 3 . 2 5 3 . 8 4 3 . 7 4 3 . 6 4 3 . 5 4 3 . 3 3 3 . 2 3 3 . 1 3 3 . 0 3 3 . 5 9 2 . 7 8 2 . 7 5 2 . 6 5 2 . 4 5 2 . 3 5 2 . 4 2 2 . 2 2 2 . 1 2 2 . 9 1 2 . 8 1 2 . 6 1 2 . 0 9 1 . 1 7 1 . 0 7 1 . 9 6 1 . 8 6 1 . 7 6 1 . 5 5 1 . 4 5 1 . 3 5 1 . 1 5 1 . 0 5 1 . 3000 3200 Cl O N NH O N Cl Cl NH O 5 O NH2 H N O O 7 5 2 . 6 5 2 . 4 5 2 . 3 5 2 . 0 9 1 . 3 7 1 . 1 7 1 . 0 7 1 . 9 6 1 . 8 6 1 . 7 6 1 . 6 6 1 . 5 5 1 . 4 5 1 . 3 5 1 . 1 5 1 . 0 5 1 . O N 5 HN O N Cl Cl Cl 3 1 1 . 2.6 3 0 2 2.2 . 2.4 2.0 f1 (ppm) 1 1 1 . 7 0 1 . 1.6 1. 8 400 300 200 100 0 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 8 9 0 . 4 9 0 . 3 9 1 . 2 8 7 . 9 9 0 . 1 9 0 . 6 7 1 . 2 7 1 2 . 1 4 2 . 4 5 2 . 1 1 1 . 8 9 1 . 9 0 1 . 5 0 1 . DG-6-103-CD2Cl2_ CARBON_01 . 3 3 7 1 . 6 6 6 1 . 6 4 6 1 . 6 7 3 1 . 5 2 3 1 . 1 1 3 1 . 4 0 3 1 . 4 9 2 1 . 9 8 2 1 . 3 5 2 1 . 8 2 2 1 . 3 2 2 1 . 2 2 1 1 . 9 9 0 1 . 8 0 7 . 8 0 7 . 7 0 7 . 4 0 7 . 4 0 7 . 3 0 7 . 0 0 7 l 2 c 2 d c 3 4 5 . l 2 c 2 d c 1 4 5 . l 2 c 2 d c 8 3 5 . l 2 c 2 d c 6 3 5 . l 2 c 2 d c 4 3 5 . . 4 6 4 . 5 9 3 . 4 9 3 . 2 3 3 . 1 3 3 . 8 0 7 . 8 0 7 . 7 0 7 . 4 0 7 . 4 0 7 . 3 0 7 . 0 0 7 60 50 40 30 20 10 0 70. 8 70.6 70.4 70.2 70.0 69. 8 69.6 . 5 9 3 . 4 9 3 . 2 3 3 . 1 3 3 . 1 1 3 1 . 4 0 3 1 f1 (ppm) . 4 9 2 1 . 9 8 2 1 . 5 7 2 1 131 130 129 128 127 f1 (ppm) 15 10 5 0 41 40 39 38 37 f1 (ppm) 36 35 34 33 50 0 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 334 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0 -200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 5.26: 1H and 13C{1H} NMR spectra of 5-24 DG-6-111-2nd-column-DCM-d2_PROTON_01 9 6 7 9 0 7 0 8 8 . . . 9 6 7 . 0 5 7 . 6 3 7 . 9 2 7 . 9 1 . 7 9 1 . 7 8 1 . 7 7 1 . 7 7 1 . 7 6 1 . 7 0 0 7 . 0 0 7 . 9 9 6 . 9 9 6 . 8 9 6 . 8 9 6 . 6 9 6 . 5 9 6 . 4 9 6 . 1 9 6 . 0 9 6 . 9 8 . 6 1 8 . 6 9 7 6 . 2 4 6 . 9 7 5 . 5 3 4 3 3 3 . . . 5 5 5 9 7 4 . 6 6 . 3 5 6 . 3 4 6 . 3 3 6 . 3 3 6 . 3 2 6 . 3 9 5 . 3 9 5 . 3 8 5 . 3 6 5 . 3 5 5 . 3 9 4 . 3 8 4 . 3 7 4 . 3 6 3 5 3 3 3 2 3 2 3 1 3 . . . . . . 3 3 3 3 3 3 5 1 . 2 4 1 . 2 3 1 . 2 2 1 . 2 2 1 . 2 9 0 2 . 8 0 2 . 7 0 2 . 7 0 2 . 9 6 . 1 8 6 . 1 6 6 . 1 4 6 . 1 8 5 . 1 6 5 . 1 5 5 . 1 O HN H NH H S NH O O 5 O NH H N O O Cl O N NH O N Cl Cl O N 5 HN O N Cl Cl Cl 13 12 11 10 9 8 9 . 1 8 0 1 . 2 9 5 9 1 . 1 2 4 . 5 0 . 1 7 9 0 . 5 0 . 1 4 2 4 . 5 0 . 1 5 0 . 1 0 1 . 3 4 5 5 4 . 0 8 4 . 2 3 4 . 5 0 2 . 7 2 6 . 1 5 . 3 7 6 f1 (ppm) 5 4 3 2 1 0 -1 DG-6-111-2nd-column-DCM-d2_ CARBON_01 . 2 7 3 1 1 . 2 3 1 8 . 0 3 1 . 0 0 3 1 1 . 9 2 1 5 . 8 2 1 1 . 7 2 1 . 9 4 2 1 . 5 2 2 1 9 . 1 2 1 9 . 1 1 1 . 5 9 0 1 . 4 0 7 . 4 0 7 . 4 0 7 . 2 0 7 1 . 0 7 1 . 0 7 . 0 0 7 . 7 9 6 . 7 9 6 7 . 1 6 1 . 0 6 . 6 5 5 l 2 c 2 d c 9 3 5 . l 2 c 2 d c 7 . l 2 c 2 d c 4 . 3 5 3 5 l 2 c 2 d c 2 . 3 5 l 2 c 2 d c 0 . 3 5 3 . 9 4 . 9 8 4 . 5 0 4 3 . 9 3 . 2 9 3 . 5 5 3 . 9 2 3 8 . 0 3 . 9 7 2 . 8 7 2 3 . 5 2 O HN H 4 . 3 7 H 1 1 . 3 7 1 0 . 3 7 1 . 3 6 6 1 . 3 4 6 1 . 9 3 6 1 NH S NH O O 5 O NH H N O O Cl O N NH O N Cl Cl 5 HN O N O N . 0 1 2 3 1 Cl Cl . 7 7 0 3 1 Cl . 7 9 9 2 1 . 7 0 9 2 1 2 5 . 8 2 1 . 5 1 7 2 1 2 4 0 7 . 0 4 0 7 . 8 3 . 0 7 7 1 . 0 7 3 1 . 0 7 2 1 . 0 7 . 8 9 9 6 . 3 7 9 6 . 9 6 9 6 60 50 40 30 20 10 0 70.5 70.4 70.3 70.2 70.0 70.1 f1 (ppm) 69.9 69. 8 69.7 10 8 6 4 2 0 132 131 130 129 128 127 126 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 -10 335 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 45 40 35 30 25 20 15 10 5 0 Figure 5.27: 1H and 13C{1H} NMR spectra of 5-26 DG-6-129-CD2Cl2_PROTON_01 0 1 . 8 9 0 . 8 9 0 . 8 8 0 . 8 8 0 . 8 8 0 . 8 1 7 7 . 0 7 7 . 8 6 7 . 8 6 7 . 7 6 7 . 6 6 7 . 1 5 7 . 9 4 7 . 5 3 7 . 8 2 7 . 8 2 7 . 9 1 . 7 8 1 . 7 7 1 . 7 7 1 . 7 9 0 7 . 8 0 7 . 7 0 7 . 6 0 7 . 2 0 7 . 1 0 7 . 0 0 7 . 8 7 4 . 7 7 4 . 5 6 . 3 4 6 . 3 3 6 . 3 2 6 . 3 2 6 . 3 1 6 . 3 0 6 . 3 9 5 . 3 8 5 . 3 7 5 . 3 6 5 . 3 4 5 . 3 3 5 . 3 3 5 . 3 8 4 . 3 7 4 . 3 6 4 . 3 6 4 . 3 5 4 . 3 4 4 . 3 7 3 6 3 5 3 4 3 3 3 2 3 1 3 0 3 . . . . . . . . 3 3 3 3 3 3 3 3 O NH NH 5 O NH2 O HN O N Cl O N Cl Cl O 5 HN O N O N Cl Cl Cl 13 12 11 10 9 DG-6-129-CD2Cl2_ CARBON_01 5 9 . 1 8 3 9 . 1 6 0 . 1 0 9 9 1 . 7 9 . 1 2 0 . 1 8 8 . 3 0 0 . 3 4 9 5 4 . 9 3 4 . 7 6 f1 (ppm) 5 4 3 2 1 0 -1 l 2 c 2 d c 9 3 5 . l 2 c 2 d c 7 . l 2 c 2 d c 5 . 3 5 3 5 l 2 c 2 d c 3 . 3 5 l 2 c 2 d c 1 . 3 5 M C D 6 2 5 . 3 . 9 4 8 . 5 4 . 9 0 4 1 . 9 3 1 . 9 3 . 0 9 3 . 2 4 7 1 2 . 1 7 1 . 3 6 6 1 . 2 6 6 1 . 3 4 6 1 . 3 4 6 1 . 2 7 3 1 . 2 2 3 1 1 . 2 3 1 . 7 0 3 1 . 0 0 3 1 . 0 9 2 1 5 . 8 2 1 5 . 8 2 1 1 . 7 2 1 . 0 5 2 1 . 0 5 2 1 . 5 2 2 1 . 5 2 2 1 9 . 1 2 1 8 . 1 1 1 8 . 1 1 1 . 5 9 0 1 . 4 0 7 . 4 0 7 3 3 . . 0 7 0 7 . 2 0 7 . 2 0 7 1 . 0 7 1 . 0 7 1 . 0 7 . 6 9 6 O NH NH 5 O NH2 O HN O N Cl O N Cl Cl O 5 HN O N O N Cl Cl Cl 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0 -200 90 80 70 60 50 40 30 20 10 0 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 336 Figure 5.28: 1H and 13C{1H} NMR spectra of 5-27 DG-6-136-cd2cl2_PROTON_01 Cl Cl N H O O N N Cl S H NH H N H O H N 4 O O O 5 NH HN O O O HN O 5 HN O N O N Cl Cl Cl 4 6 3 . 4 6 3 . 3 6 3 . 9 5 3 . 6 5 3 . 6 4 3 . 5 4 3 . 4 4 3 . 7 3 3 . 6 3 3 . 5 3 3 . 4 3 3 . 3 3 3 . 2 3 3 . 1 3 3 . 2 1 3 . 1 1 3 . 2 9 2 . 0 9 2 . 7 8 2 . 5 8 2 . 4 8 2 . 3 8 2 . 7 7 2 . 6 7 2 . 4 7 2 . 3 7 2 . 9 6 2 . 6 6 2 . 3 6 2 . 2 6 2 . 0 6 2 . 9 5 2 . 7 4 2 . 6 4 2 . 6 1 2 . 5 1 2 . 3 1 2 . 0 1 8 . 8 0 8 . 7 7 7 . 5 7 7 . 0 7 7 . 8 6 7 . 1 5 7 . 0 4 7 . 9 3 7 . 6 3 7 . 0 3 7 . 3 2 7 . 2 2 7 . 9 1 7 . 7 1 7 . 4 0 7 . 0 0 7 . 9 8 6 . 0 8 4 . 4 7 4 . 2 7 4 . 1 7 4 . 0 7 4 . 4 4 4 . 3 4 4 . 2 4 4 . 5 2 4 . 5 2 4 . 3 2 4 . 2 6 5 . 5 3 5 . 4 3 5 . 4 2 6 . 4 6 1 . 2 6 1 . 1 6 1 . 0 6 1 . 8 5 1 . 9 3 1 . 8 2 1 . 3 0 1 . 1 0 1 . 0 0 1 . 1300 1200 1100 1000 900 800 700 600 500 400 300 200 100 0 -100 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 DG-6-136-cd2cl2_ CARBON_01 0 8 1 . 6 8 0 . 8 0 2 . 2 8 9 1 . 5 0 2 . 2 9 0 . 6 8 0 . 1 9 0 . 2 2 1 . 3 8 3 . 2 0 1 . 6 9 0 . 9 0 1 . 9 8 5 6 . 9 1 1 . 5 1 9 . 9 1 4 . 5 0 2 . 4 1 2 . 2 1 0 -1 . 0 3 7 1 . 4 1 7 1 . 8 0 7 1 . 4 6 6 1 . 3 6 6 1 . 3 4 6 1 . 3 4 6 1 . 8 3 6 1 . 2 7 3 1 . 2 2 3 1 . 1 2 3 1 . 7 0 3 1 . 0 0 3 1 . 0 9 2 1 . 5 8 2 1 . 2 7 2 1 . 0 5 2 1 . 5 2 2 1 . 9 1 2 1 . 8 1 1 1 . 5 9 0 1 . 4 0 7 . 4 0 7 . 4 0 7 . 4 0 7 . 3 0 7 . 3 0 7 . 2 0 7 . 2 0 7 . 1 0 7 . 0 0 7 . 9 9 6 . 6 9 6 . 5 9 6 . 2 7 6 . 8 1 6 . 1 0 6 . 7 5 5 l 2 c 2 d c 9 3 5 . l 2 c 2 d c 7 3 5 . l 2 c 2 d c 5 3 5 . l 2 c 2 d c 3 3 5 . l 2 c 2 d c 1 3 5 . . 3 0 5 . 3 9 4 . 3 9 3 . 2 9 3 . 2 9 3 . 2 9 3 . 8 6 3 . 5 5 3 . 1 8 2 . 0 8 2 . 5 5 2 90 Cl Cl N H O O N N Cl S H NH H N H O H N 4 O O O 5 NH HN O O O HN O 5 HN O O N N . 2 7 3 1 Cl Cl Cl . 2 2 3 1 . 1 2 3 1 . 7 0 3 1 . 0 0 3 1 . 0 9 2 1 . 5 8 2 1 . 2 7 2 1 . 0 5 2 1 . 5 2 2 1 . 9 1 2 1 135 130 f1 (ppm) 125 . 4 0 7 . 4 0 7 . 4 0 7 . 4 0 7 . 3 0 7 . 3 0 7 . 2 0 7 . 2 0 7 . 1 0 7 . 0 0 7 . 9 9 6 . 6 9 6 . 5 9 6 60 50 40 30 20 10 0 70.4 70.2 70.0 f1 (ppm) 69. 8 69.6 15 10 5 0 80 70 60 50 40 30 20 10 0 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 337 Figure 5.29: 1H and 13C{1H} NMR spectra of 5-28 DG-6-107-tt23-32-acetone_PROTON_01 O OH 4 1 . 8 2 1 . 8 2 1 . 8 3 6 7 . 3 6 7 . 7 2 7 . 7 2 7 . 5 2 7 . 5 2 7 . 7 2 . 5 5 7 . 3 e n o t e c a 0 1 . 2 e n o t e c a 7 0 2 . e n o t e c a 7 0 2 . e n o t e c a 6 0 2 . e n o t e c a 6 0 2 . e n o t e c a 5 0 2 . Cl N O OMe 13 12 11 10 9 8 7 6 f1 (ppm) 5 4 3 2 1 0 -1 1 0 . 1 5 9 0 . 9 8 . 0 0 0 . 1 9 8 . 1 4 9 2 . DG-6-107-tt23-32-acetone_ CARBON_01 e n o t e c a 5 5 0 2 . 4 . 8 6 1 . 8 4 6 1 . 9 7 3 1 . 9 6 3 1 3 . 8 2 1 . 6 5 2 1 . 4 2 2 1 1 . 2 2 1 . 6 0 1 1 . 8 7 0 1 e n o t e c a 4 9 2 . e n o t e c a 3 . 9 2 e n o t e c a 1 . 9 2 e n o t e c a 0 9 2 . e n o t e c a 8 . 8 2 e n o t e c a 7 . 8 2 e n o t e c a 5 . 8 2 9 . 1 5 . 4 7 4 O OH Cl N O OMe 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 -10 338 28000 26000 24000 22000 20000 18000 16000 14000 12000 10000 8000 6000 4000 2000 0 -2000 900 800 700 600 500 400 300 200 100 0 Figure 5.30: 1H and 13C{1H} NMR spectra of 5-29 DG-6-131-tt23-34-CD2Cl2_PROTON_01 3 2 . 8 2 2 . 8 0 2 . 8 3 1 . 8 2 1 . 8 1 1 . 8 7 8 7 . 6 2 7 . 6 2 7 . 9 1 7 . 8 1 7 . 7 1 7 . 7 1 7 . 7 8 4 . 5 7 . 3 1 7 . 3 0 7 . 3 9 6 . 3 6 6 . 3 5 6 . 3 4 6 . 3 3 6 . 3 3 6 . 3 2 6 . 3 1 6 . 3 0 6 . 3 9 5 . 3 9 5 . 3 8 5 . 3 7 5 . 3 6 5 . 3 5 5 . 3 4 5 . 3 3 5 . 3 2 5 . 3 2 5 . 3 1 5 . 3 1 5 . 3 1 5 . 3 NH2 O 5 Cl O N NH O OMe 5 7 . 3 0 7 . 3 9 6 . 3 6 6 . 3 5 6 . 3 4 6 . 3 3 6 . 3 3 6 . 3 2 6 . 3 1 6 . 3 0 6 . 3 9 5 . 3 9 5 . 3 8 5 . 3 7 5 . 3 6 5 . 3 5 5 . 3 4 5 . 3 3 5 . 3 2 5 . 3 2 5 . 3 1 5 . 3 1 5 . 3 1 5 . 3 2000 1000 0 1 1 . 3 3 .75 3 .70 3 .65 . 0 0 4 2 3 .60 f1 (ppm) 3 .55 3 .50 13 12 11 10 9 8 7 6 f1 (ppm) DG-6-115-DCM-d2_ CARBON_01 3 0 . 1 7 9 0 . 1 1 . 1 7 2 2 . 6 9 0 . 6 9 0 . 9 0 2 . 5 0 0 4 2 . 1 1 . 3 4 3 . 7 7 3 1 1 . 2 3 1 . 2 9 2 1 . 5 5 2 1 . 9 2 2 1 . 5 2 2 1 . 7 2 1 1 1 . 0 1 1 6 . 3 7 . 9 0 7 . 9 0 7 . 9 0 7 . 9 0 7 8 . 0 7 . 6 0 7 . 6 0 7 . 5 0 7 O 2 t E 1 . 6 6 6 . 8 6 1 . 6 4 6 1 NH2 O 5 Cl O N NH O OMe 2 M C D 2 . 3 5 2 . 8 4 1 . 2 4 . 6 9 3 1 0 -1 O 2 t E 5 5 1 . l 2 c 2 d c 3 4 5 . l 2 c 2 d c 1 . 4 5 l 2 c 2 d c 9 3 5 . l 2 c 2 d c 7 . l 2 c 2 d c 5 . 3 5 3 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 -10 339 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0 -200 -400 110 100 90 80 70 60 50 40 30 20 10 0 -10 Figure 5.31: 1H and 13C{1H} NMR spectra of 5-30 l 2 c 2 d c 4 3 5 . l 2 c 2 d c 4 3 5 . l 2 c 2 d c 4 3 5 . DG-6-134-CD2Cl2_PROTON_01 3 1 8 . 2 1 8 . 1 1 8 . 0 1 8 . 1 8 7 . 9 7 7 . 1 7 7 . 0 7 7 . 9 6 7 . 9 2 7 . 8 2 7 . 1 2 7 . 0 2 7 . 9 1 7 . 9 1 7 . 2 1 7 . 1 1 7 . 0 1 7 . 9 0 7 . 8 0 7 . 7 0 7 . 3 0 7 . 2 0 7 . 1 0 7 . 5 3 5 . O NH NH 5 O NH2 O HN O N Cl O OMe O 5 HN O N O MeO Cl 9 8 4 . 9 8 4 . 5 7 3 . 9 6 3 . 9 6 3 . 6 6 3 . 5 6 3 . 4 6 3 . 3 6 3 . 3 6 3 . 2 6 3 . 1 6 3 . 9 5 3 . 9 5 3 . 8 5 3 . 7 5 3 . 6 5 3 . 5 5 3 . 4 5 3 . 9 4 3 . 8 4 3 . 7 4 3 . 6 4 3 . 7 3 3 . 6 3 3 . 5 3 3 . 4 3 3 . 3 3 3 . 3 3 3 . 5 7 3 . 6 6 3 . 5 6 3 . 4 6 3 . 3 6 3 . 3 6 3 . 2 6 3 . 1 6 3 . 9 5 3 . 9 5 3 . 8 5 3 . 7 5 3 . 6 5 3 . 5 5 3 . 4 5 3 . 8 4 3 . 7 4 3 . 6 4 3 . 5 3 3 . 1500 1000 500 0 9 3 4 . 3 .4 3 .3 3 3 4 . 3 .5 6 1 6 . 3 .7 2 7 2 4 . 3 .6 f1 (ppm) 9 2 7 . 8 2 7 . 1 2 7 . 0 2 7 . 9 1 7 . 9 1 7 . 2 1 7 . 1 1 7 . 0 1 7 . 9 0 7 . 8 0 7 . 7 0 7 . 3 0 7 . 2 0 7 . 1 0 7 . 3 .9 3 . 8 200 100 0 7 8 1 7.2 . . 9 9 0 7.1 f1 (ppm) 8 9 0 . 1 0 1 . 7.0 6.9 9 7 1 . 7.3 7.4 13 12 11 10 9 8 7 0 9 1 . 3 9 1 . 4 9 0 . 9 7 1 . 7 8 1 . 9 9 0 . 8 9 0 . 1 0 1 . 7 9 3 . 5 6 f1 (ppm) 2 7 2 4 . 6 1 6 . 3 3 4 . 9 3 4 . 4 3 2 1 0 -1 l 2 c 2 d c 3 4 5 . DG-6-134-CD2Cl2_ CARBON_01 . 1 4 7 1 . 4 1 7 1 . 6 8 6 1 . 6 8 6 1 . 6 4 6 1 . 6 4 6 1 . 6 7 3 1 . 2 2 3 1 . 2 2 3 1 . 0 9 2 1 . 4 5 2 1 . 4 5 2 1 . 9 2 2 1 . 8 2 2 1 . 3 2 2 1 . 4 2 1 1 . 3 2 1 1 . 0 0 1 1 . 7 0 7 . 7 0 7 . 7 0 7 . 7 0 7 . 5 0 7 . 7 0 7 . 5 0 7 . 4 0 7 . 5 0 7 . 4 0 7 . 3 0 7 . 9 9 6 . 9 9 6 O NH NH 5 O NH2 O HN O N Cl O OMe O 5 HN O N O MeO Cl . 6 8 6 1 . 6 8 6 1 40 20 0 l 2 c 2 d c 1 4 5 . t N 3 E 8 8 . l 2 c 2 d c 8 3 5 . l 2 c 2 d c 6 3 5 . t N 3 E 0 6 4 . l 2 c 2 d c 4 3 5 . . 0 3 5 . 9 2 5 . 0 8 4 . 6 4 6 1 . 6 4 6 1 . 9 0 4 . 5 9 3 . 4 9 3 . 3 9 3 60 40 20 0 168 .7 168 .6 168 .5 f1 (ppm) 164. 8 164.6 f1 (ppm) 164.4 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 340 4000 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800 600 400 200 0 -200 280 260 240 220 200 180 160 140 120 100 80 60 40 20 0 -20 Figure 5.32: 1H and 13C{1H} NMR spectra of 5-31 DG-6-137-cd2cl2_PROTON_01 Cl N H O O N MeO O 5 NH HN O O O HN S H NH H N H O H N 4 O O O 5 HN O O N MeO Cl 5 7 3 . 9 6 3 . 9 5 3 . 8 5 3 . 7 5 3 . 6 4 3 . 7 3 3 . 3 3 3 . 3 2 3 . 2 1 3 . 2 1 3 . 8 8 2 . 6 8 2 . 8 7 2 . 7 7 2 . 5 7 2 . 4 7 2 . 2 7 2 . 9 6 2 . 3 6 2 . 2 6 2 . 0 6 2 . 9 5 2 . 8 4 2 . 7 1 2 . 6 3 5 . 5 3 5 . 2 9 4 . 2 7 4 . 1 7 4 . 5 4 4 . 6 2 4 . 4 1 8 . 2 1 8 . 2 8 7 . 1 8 7 . 8 7 7 . 6 7 7 . 3 4 7 . 0 3 7 . 1 2 7 . 9 1 7 . 5 1 7 . 8 9 6 . 0 4 6 . 8 8 5 . 8 6 1 . 7 6 1 . 1 6 1 . 0 4 1 . 8 2 1 . 6 0 1 . 4 0 1 . 3 0 1 . 8 8 0 . 4 0 2 . 5 2 3 . 9 1 1 . 5 9 2 . 2 8 3 . 4 0 1 . 2 0 1 . 2 0 1 . 6 9 0 . 7 2 4 . 6 0 1 . 6 0 1 . 5 2 1 . 4 2 1 8 . 7 1 1 . 5 1 2 . 4 5 1 . 8 0 2 . 7 1 2 . 7 4 4 . 6 2 2 . 6 f1 (ppm) S H NH H N H O 13 12 11 10 9 8 7 DG-6-137-cd2cl2_ CARBON_01 Cl N H O O N MeO H N 4 O O O 5 NH HN O O O HN O 5 HN O O N MeO Cl 5 4 3 2 1 0 -1 150 100 50 0 . 4 0 7 . 3 0 7 . 3 0 7 . 2 0 7 . 2 0 7 . 1 0 7 . 1 0 7 . 0 0 7 . 9 9 6 . 6 9 6 . 5 9 6 70.5 70.0 f1 (ppm) 69.5 . 4 0 7 . 3 0 7 . 3 0 7 . 2 0 7 . 2 0 7 . 1 0 7 . 1 0 7 . 0 0 7 . 9 9 6 . 6 9 6 . 5 9 6 . 2 7 6 . 8 1 6 . 2 0 6 . 7 5 5 . 1 3 7 1 . 4 1 7 1 . 8 0 7 1 . 4 8 6 1 . 3 4 6 1 . 0 4 6 1 . 3 7 3 1 . 0 2 3 1 . 0 2 3 1 . 6 8 2 1 . 1 5 2 1 . 6 2 2 1 . 0 2 2 1 . 9 1 1 1 . 7 9 0 1 l 2 c 2 d c 0 4 5 . l 2 c 2 d c 8 3 5 . l 2 c 2 d c 6 3 5 . l 2 c 2 d c 3 3 5 . l 2 c 2 d c 1 3 5 . . 7 2 5 . 2 0 5 . 7 7 4 . 5 0 4 . 3 9 3 . 2 9 3 . 2 9 3 . 2 9 3 . 6 7 3 . 8 6 3 . 6 5 3 . 2 8 2 . 1 8 2 . 5 5 2 850 800 750 700 650 600 550 500 450 400 350 300 250 200 150 100 50 0 -50 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10 230 220 210 200 190 180 170 160 150 140 130 120 110 f1 (ppm) 100 90 80 70 60 50 40 30 20 10 0 -10 341 Figure 5.33: 1H and 13C{1H} NMR spectra of 5-33 DG-7-42.10.fid 4 6 3 . 2 6 3 . 1 6 3 . 9 5 3 . 8 5 3 . 6 5 3 . 6 5 3 . 4 5 3 . 4 5 3 . 2 5 3 . 1 5 3 . 3 4 3 . 3 4 3 . 2 4 3 . 2 4 3 . 3 3 3 . 2 3 3 . 1 3 3 . 1 3 3 . 0 3 3 . 5.0×107 4.0×107 3 .0×107 2.0×107 1.0×107 0.0 N N NH O NH 5 O O O 3 . 8 3 .7 NH HN O N Cl O N Cl Cl . 9 6 0 4 3 .6 O N 1 4 4 . 3 .4 3 .5 f1 (ppm) 0 3 4 3 .3 . 3 .2 6 0 2 . 6 0 2 . 9 9 1 . 7 9 1 . 6 9 1 . 5 9 1 . 9 5 1 . 7 5 1 . 5 5 1 . 5 7 1 . 3 7 1 . 1 7 1 . 2 4 1 . 0 4 1 . Cl 6 8 0 . 2.1 8 9 3 . 2.0 1.9 1. 8 6 0 2 . 1.7 f1 (ppm) 5 0 2 1.6 . 1.5 7 2 2 1.4 . 1.3 1.2 4 6 3 . 2 6 3 . 1 6 3 . 9 5 3 . 8 5 3 . 6 5 3 . 6 5 3 . 4 5 3 . 4 5 3 . 2 5 3 . 1 5 3 . 3 4 3 . 3 4 3 . 2 4 3 . 2 4 3 . 3 3 3 . 2 3 3 . 1 3 3 . 1 3 3 . 0 3 3 . 7 9 2 . O 5 HN O N Cl Cl 1 1 8 . 0 1 8 . 9 0 8 . 8 0 8 . 8 0 8 . 3 7 7 . 0 7 7 . 7 4 7 . 5 4 7 . 1 4 7 . 0 4 7 . 8 3 7 . 6 3 7 . 5 3 7 . 4 3 7 . 9 2 7 . 2 2 7 . 1 2 7 . 9 1 7 . 8 1 7 . 8 1 7 . 6 1 7 . 6 1 7 . 4 1 7 . 9 7 4 . 9 7 4 . 7 0 2 . 6 0 2 . 6 0 2 . 9 9 1 . 8 9 1 . 7 9 1 . 6 9 1 . 5 9 1 . 4 9 1 . 5 7 1 . 3 7 1 . 1 7 1 . 9 5 1 . 7 5 1 . 5 5 1 . 2 4 1 . 0 4 1 . 14 13 12 11 10 9 8 7 f1 (ppm) 6 5 4 3 2 1 0 8 9 1 . 6 0 2 . 4 2 5 2 . 7 9 3 . 9 6 0 4 . 1 4 4 . 0 3 4 . 6 8 0 . 8 9 3 . 6 0 2 . 5 0 2 . 7 2 2 . DG-7-42.11.fid N N O NH 5 O NH O HN O NH O N Cl O N Cl Cl O 5 O HN O . 7 0 3 1 N N . 0 0 3 1 Cl . 3 0 7 . 3 0 7 . 3 0 7 . 2 0 7 . 2 0 7 . 1 0 7 . 1 0 7 . 0 0 7 . 0 0 7 . 9 9 6 . 5 9 6 . 5 9 6 0 69. 8 69.9 f1 (ppm) 69.7 69.6 69.5 69.4 Cl 150000 70.3 70.2 70.1 70.0 l 2 C 2 D C 0 4 5 . l 2 C 2 D C 8 3 5 . . 0 9 2 1 Cl . 2 7 2 1 M C D 2 4 5 . 100000 50000 0 l 2 C 2 D C 5 3 5 . l 2 C 2 D C 2 3 5 . l 2 C 2 D C 0 3 5 . . 3 0 5 . 3 9 4 131 130 129 f1 (ppm) 128 127 3 0 7 . . 3 0 7 . 3 0 7 . 2 0 7 . 2 0 7 . 1 0 7 . 1 0 7 . 0 0 7 . 0 0 7 . 9 9 6 . 5 9 6 . 5 9 6 . 1 9 6 . 5 1 7 1 . 2 1 7 1 . 8 0 7 1 . 4 6 6 1 . 4 6 6 1 . 5 4 6 1 . 5 4 6 1 . 2 7 3 1 . 3 2 3 1 . 3 2 3 1 . 7 0 3 1 . 5 8 2 1 . 0 5 2 1 . 0 5 2 1 . 5 2 2 1 . 9 1 2 1 . 7 1 1 1 . 7 1 1 1 . 5 9 0 1 . 9 2 8 . 3 9 3 . 2 9 3 . 1 9 3 . 6 7 3 . 1 2 3 . 9 9 2 . 1 8 2 . 0 8 2 . 1 3 1 210 200 190 180 170 160 150 140 130 120 110 100 f1 (ppm) 90 80 70 60 50 40 30 20 10 0 -10 342 3 .0×107 2.0×107 1.0×107 0.0 5.5×107 5.0×107 4.5×107 4.0×107 3 .5×107 3 .0×107 2.5×107 2.0×107 1.5×107 1.0×107 5.0×106 0.0 2000000 1800000 1600000 1400000 1200000 1000000 800000 600000 400000 200000 0 -200000 -5.0×106 3000000 3000000 2800000 2000000 2600000 2400000 1000000 2200000