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SYNTHESIS OF NOVEL QUATERNARY ALPHA-AMINO
ACIDS AND 2-IMIDAZOLINES DERIVED FROM
OXAZOL-5(4H)-ONES AND EVALUATION OF THEIR
PROTEASOMAL INHIBITION ACTIVITY

presented by

ROBERT ADAM MOSEY

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SYNTHESIS OF NOVEL QUATERNARY ALPHA-AMINO ACIDS AND
2-IMIDAZOLINES DERIVED FROM OXAZOL-5(4H)-ONES
AND EVALUATION OF THEIR PROTEASOMAL INHIBITION ACTIVITY

By

Robert Adam Mosey

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Chemistry
2010

ABSTRACT
SYNTHESIS OF NOVEL QUATERNARY ALPHA-AMINO ACIDS AND
2-IMIDAZOLINES DERIVED FROM OXAZOL-5(4H)-ONES
AND EVALUATION OF THEIR PROTEASOMAL INHIBITION ACTIVITY
By

Robert Adam Mosey

The research presented in this dissertation was focused towards the
synthesis of densely functionalized amino acid derivatives and heterocycles
derived from alkylation products of oxazoI-5(4H)-ones. The opening chapter
introduces oxazoI-5(4H)-ones and describes their reactivity and synthetic utility
for the construction of pharmaceutically relevant compound libraries. The
remaining chapters describe the development of an oxazol-5(4H)-one alkylation
and reduction protocol for the synthesis of quaternary a-amino acid derivatives
and the subsequent use of quaternary o-amino acid derivatives towards the
synthesis of proteasome inhibitors.

The first study in this dissertation describes the diastereoselective
synthesis of quaternary o-amino acid derivatives by means of a protocol involving
sequential oxazoI-5(4H)-one alkylation and hydride reduction. Previous studies
in our lab demonstrated that oxazol-5(4H)-ones participate in ene-type alkylation
reactions with enol ethers. The racemic reaction was revisited in our current
studies, wherein solvents were optimized and Bronsted acids were used to
promote diastereoselectivity. Hydride reduction of the oxazol-5(4H)-one
alkylation products was then performed, resulting in the stereoselective formation

of highly substituted tert-alkyl amino hydroxy carboxylic acids. Mechanistic

investigation also revealed that the ene—type oxazol-5(4H)-one alkylation reaction
likely occurs through a stepwise process.

The second study in this dissertation details the use of quaternary a-amino
acid derivatives prepared from oxazoI-5(4H)-one alkylation products towards the
synthesis of proteasome inhibitors and proteasome inhibitor precursors. A
quaternary o-amino acid derivative was first utilized to construct an advanced
intermediate used in the synthesis of salinosporamide A. A very similar
quaternary q-amino acid derivative was later used for the generation of a 2-
imidazoline scaffold. Further elaboration of the 2-imidazoline resulted in the
culmination of a 2-imidazoline bearing a B-lactone functionality. 2-lmidazolines
prepared in this chapter were then evaluated for their ability of inhibit proteolytic
activity of the mammalian 20$ proteasome.

The final study in this dissertation describes annulation reactions arising
from underutilized reactivity of 2-imidazolines. It was discovered that 2-
imidazolines underwent cyclization reactions with B-hydroxy carboxylic acids
when treated with BOP-Cl and triethylamine to afford molecules containing a
unique bicyclic oxazinone/imidazolidine ensemble. The reaction has been
explored and has revealed that N-Boc derivatives of the d-amino acids serine
and threonine may be used in the reaction to generate products
diastereoselectively. Muticyclic adducts prepared in this chapter were then
evaluated for their ability of inhibit proteolytic activity of the mammalian 20$

proteasome.

TABLE OF CONTENTS

LIST OF TABLES ................................................................................................ vii
LIST OF FIGURES ............................................................................................. viii
LIST OF SCHEMES ............................................................................................ xii
LIST OF SYMBOLS or ABBREVIATIONS .......................................................... xvi
CHAPTER I
OXAZOL-5(4H)-ONES AS PLURIPOTENT SCAFFOLDS FOR THE
SYNTHESES OF DIVERSE LIBRARIES OF COMPOUNDS ............................... 1
A. Introduction to drug discovery ..................................................................... 1
B. Introduction to oxazol-5(4H)-ones ............................................................... 7
C. Synthesis and general reactivity of oxazol-5(4H)-oneS ................................ 9
D. Nucleophilic reactions of oxazol-5(4H)-ones ............................................. 12
1. Acylation reactions ............................................................................... 13
2. Alkylation reactions .............................................................................. 14
3. Arylation reactions ................................................................................ 20
E. Electrophilic reactions of oxazol-5(4H)-ones ............................................. 21
F. Cycloaddition reactions of oxazol-5(4H)-ones ........................................... 23
1. [2+2] cycloaddition reactions ................................................................ 24
2. [3+2] cycloaddition reactions ................................................................ 26
G. Current studies .......................................................................................... 34
H. References ................................................................................................ 36
CHAPTER II

DIASTEREOSELECTIVE SYNTHESIS OF TERT-ALKYL AMINO HYDROXY
CARBOXYLIC ESTERS VIA AN INTERMOLECULAR ALKYLATION

REACTION OF OXAZOL-5(4H)-ONES AND ENOL ETHERS ........................... 47
A. Introduction to quaternary q-amino acids .................................................. 47
B. Syntheses of quaternary a-amino acids using oxazol-5(4H)-ones ............ 48
C. Introduction to tert-alkyl hydroxy carboxylic acids ..................................... 51
D. Synthesis of tert-alkyl hydroxy carboxylic acids from oxazol-5(4H)—ones .. 52
E. Syntheses of quaternary q-amino acid derivatives via nucleophilic

ring-opening reactions of quaternary oxazol-5(4H)-ones ........................... 56
F. Optimization of reaction diastereoselectivity .............................................. 58
G. Scope of alkylation/reduction reaction of oxazol-5(4H)-ones ..................... 62
H. Mechanistic investigation ........................................................................... 66
I. Enanioselective ene-type alkylation reaction of oxazoI-5-(4H)-ones ......... 68
J. Experimental ............................................................................................. 70

1. General information .............................................................................. 70

2. Materials .............................................................................................. 71
3. Synthesis and characterization of oxazoI-5-(4H)-one precursors ......... 71
4. General procedure for synthesis of oxazoI-5(4H)-ones ........................ 77
5. Synthesis of enol ethers and alkynyl ethers ......................................... 81
6. Synthesis of diamides .......................................................................... 82
7. General procedure for the synthesis of tert-alkyl amino
hydroxy carboxylic esters ..................................................................... 86
K. References .............................................................................................. 100
CHAPTER III
UTILIZATION OF OXAZOL-5(4H)-ONES TOWARDS THE SYNTHESIS OF
PROTEASOME INHIBITORS ........................................................................... 108
A. Introduction to the 26S proteasome ......................................................... 108
B. The 268 proteasome and the NF-KB pathway ......................................... 110
C. Proteasome inhibition by small molecules ............................................... 112
D. Introduction to salinosporamide A ........................................................... 118
E. Syntheses of salinosporamide A ............................................................. 120
F. Use of oxazol-5(4H)-ones towards the synthesis of salinosporamide A.. 128
G. Introduction to 2-imidazolines .................................................................. 133
H. Syntheses of 2-imidazolines .................................................................... 134
I. Synthesis of 2-imidazoline proteasome inhibitors .................................... 137
J. Evaluation of proteasome inhibition ......................................................... 142
K. Experimental ........................................................................................... 143
1. General lnforrnation ........................................................................... 143
2. Materials ............................................................................................ 144
3. Synthesis and Characterization .......................................................... 144
4. Procedure for proteasome inhibition assay ........................................ 159
L. References .............................................................................................. 161
CHAPTER IV
SYNTHESIS OF NOVEL MULTICYCLIC OXAZINONE/IMIDAZOLIDINE
ADDUCTS FROM AN ANNULATION REACTION OF 2-IMIDAZOLINES ........ 174
A. Reactivity of 2-imidazolines ..................................................................... 174
8. Synthesis of multicyclic imidazolidine/oxazinone adducts ....................... 177
C. Towards Chiral Resolution of Racemic 2-Imidazolines ............................ 187
D. Evaluation of proteasome inhibition ......................................................... 191
E. Experimental ........................................................................................... 193
1. General information ............................................................................ 193
2. Materials ............................................................................................ 194
3. Synthesis and Characterization .......................................................... 194
4. General procedure for the synthesis of multicyclic oxazinone
adducts .............................................................................................. 197
5. Decomposition of oxazinone/imidazolidine adducts ........................... 204
6. Procedure for proteasome inhibition assay ........................................ 204
F. References .............................................................................................. 206

CHAPTER V
CONCLUSION .................................................................................................. 212

vi

LIST OF TABLES

CHAPTER II

Table I I-1. Optimization of NaBH4 reduction of quaternary oxazoI-5(4H)-ones... 58
Table ”-2. Solvent screening to optimize selectivity in the alkylation reaction 59
Table "-3. Effect of Bronsted and Lewis acids on reaction diastereoselectivity.. 61

Table "-4. Alkylation/reduction reaction performed with various enol ethers ...... 64

Table “-5. Alkylation/reduction reaction with various oxazol-5(4H)-ones ........... 65
CHAPTER III

Table Ill-1. Inhibition of chymotrypsin-Iike activity of the 208 proteasome

by 2-imidazolines .............................................................................................. 143
CHAPTER IV

Table IV-1. Inhibition of chymotrypsin-Iike activity of the 208 proteasome by
oxazinone/imidazolidine adducts ...................................................................... 193

vii

LIST OF FIGURES

CHAPTER I

Figure l-1. Population of chemical space by chemical libraries prepared via
combinatorial synthesis and diversity-oriented synthesis ................... 5

Figure l-2. The five isomeric forms of oxazolones ................................................ 7

Figure I-3. Numbering of the oxazol-5(4H)-One system in accordance with the
Hantzsch-Widman rules ...................................................................... 9

CHAPTER II

Figure "-1. General structures of both a-amino and quaternary o-amino acids. 48

Figure "-2. Naturally occurring tert-alkyl amino hydroxy carboxylic acids ........... 52
Figure "-3. X-Ray crystal structure representation of ll-6A ................................. 62
Figure "-4. X-Ray crystal structure of "-268 ...................................................... 66
Figure "-5. Dimethyl 2-(4-methoxybenzamido)malonate (ll-31) ......................... 72

Figure "-6. 3-methoxy-2-(4-methoxybenzamido)-3-oxopropanoic acid (II-32).... 73

Figure "-7. Dimethyl 2-(4-(trifluoromethyl)benzamido)malonate (ll-33) .............. 74
Figure "-8. 3-methoxy-3-oxo-2-(4-(trifluoromethyl)benzamido)propanoic

acid (II-34) ......................................................................................... 75
Figure "-9. Dimethyl 2-(2-phenylacetamido)malonate (ll-35) ............................. 75

Figure "-10. 3-methoxy-3—oxo-2-(2-phenylacetamido)propanoic acid (II-36) ...... 75
Figure "-11. 2-(4-methoxyphenyl)-4-carbmethoxy-5(4H)-oxazolone (II-20) ........ 78

Figure "-12. 2-(4-trifluoromethylphenyI)-4-carbmethoxy-5(4H)-
oxazolone (II-21) ............................................................................. 79

Figure "-13. 2-ethyl-4-carbmethoxy-5(4H)-oxazolone (II-22) .............................. 80

Figure "-14. 2-benzyI-4-carbmethoxy-5(4H)-oxazolone (ll-23) ........................... 80

viii

Figure "-15.

Figure I I-16. Methyl 2-benzamidO-2-(benzylcarbamoyl)-3-

Benzyl vinyl ether (ll-2) ..................................................................

.82

(benzyloxy)butanoate (II-3) ............................................................. 82
Figure "-17. Methyl 2-benzamido-3-(benzyloxy)-2-carbamoylbutanoate (ll-4) 84
Figure "-18. Methyl-2-benzamido-3—tert-butoxy—2-

(hydroxymethyl)butanoate (II-6) ...................................................... 87
Figure ”-19. Methyl-2-benzamido—3-benzyloxy-2-

(hydroxymethyl)butanoate (ll-13) .................................................... 88
Figure "-20. Methyl-2-benzamido-3-ethoxy-2-

(hydroxymethyl)butanoate (II-14) .................................................... 90
Figure "-21. Methyl-2-(benzamido)-3-butoxy-2-(hydroxymethy|)but-3-

enoate (ll-18) .................................................................................. 91
Figure "-22. Methyl-2-(benzamido)—2-(hydroxymethyl)-3-

methoxy-3-methylbutanoate (ll-19) ................................................. 92
Figure "—23. Methyl-2-(4-methoxybenzamido)-3-tert-butoxy-2-

(hydroxymethyl)butanoate (II-24) .................................................... 93
Figure "-24. Methyl-2-(4-(trifluoromethyl)benzamido)-3-tert-butoxy-2-

(hydroxymethyl) butanoate (ll-25) ................................................... 95
Figure "-25. Methyl-3-tert-butoxy-2-(hydroxymethyl)-2-

(propionamido)butanoate (ll-26) ..................................................... 96
Figure ”-26. Methyl-2-(2-phenylacetamido)-3-tert-butoxy-2-

(hydroxymethyl)butanoate (ll-27) .................................................... 98

CHAPTER III

Figure III-1. Representation of the 208 and 26S mammalian proteasomes ..... 109

Figure III-2. The role of the proteasome in the NF-KB pathway ........................ 111
Figure Ill—3. Natural and synthetic proteasome inhibitors ................................. 114
Figure Ill-4. Natural pyrrolidinone proteasome inhibitors

and related congeners ................................................................... 120
Figure Ill-5. Structures of some biologically active 2-imidazolines .................... 134

Figure III-6. X-ray crystal structure of III-36 ...................................................... 140

Figure III-7. DL-(R)-Methyl 4-((R)-1-(tert-butoxy)ethyI)-2-(4-methoxyphenyl)-

4,5-dihydrooxazole-4-carboxylate (III-32) ...................................... 144
Figure III-8. DL-(2R, 3R)-Methyl 3-tert-butoxy-2-(hydroxymethyl)-2-
((4-methoxybenzyl)amino)butanoate (Ill-33) .................................. 145
Figure III-9. DL-(2R, 3R)-Methy| 2-((benzyloxy)methyl)-3-tert-butoxy-2-
((4-methoxybenzyl)amino)butanoate (Ill-34) .................................. 147
Figure III-10. DL-(2R, 3R)-Methyl 2-((benzyloxy)methyl)-3-tert-butoxy-2-
(N-(4-methoxybenzyl)acrylamido)butanoate (Ill-35) .................... 148
Figure Ill-11. DL-(2R, 3R)-Methyl 2-((benzyloxy)methyl)-3-hydroxy-2-
(N-(4-methoxybenzyl)acrylamido)butanoate (Ill-7) ...................... 149
Figure Ill-12. DL-(R)-Methyl 2-((benzyloxy)methyl)-2-
(N-(4-methoxybenzyl)acrylamido)-3-oxobutanoate (III-8) ............ 150
Figure Ill-13. DL—(2R, 3R)-Methyl 2-((benzyloxy)methyI)-3—hydroxy-2-
((4-methoxybenzyl)amino)butanoate (III-6) .................................. 151
Figure lll-14. DL-(R)-methyl 4-((R)-1 -tert-butoxyethyI)-2-phenyI-4, 5-
dihydrooxazole-4-carboxylate (Ill-38) ........... A ............................... 1 52
Figure III-15. DL-(ZS,3R)-methyl 2-benzamido-3-tert-butoxy-2-
formylbutanoate (III-39) ............................................................... 153
Figure Ill-16. DL-(R)-methyl 1-benzyI-4-((R)-1-(tert-butoxy)ethyI)-2-phenyl-
4,5-dihyd ro-1 H-imidazole-4-carboxylate (III-36) ........................... 154
Figure Ill-17. DL-(R)-methy| 1-benzyI-4-((R)-1-hydroxyethyl)-2-phenyl-
4,5-dihydro-1H-imidazole-4-carboxylate (Ill-41) ........................... 155
Figure III-18. DL-(R)—1-benzyl-4-((R)-1-hydroxyethyl)-2-phenyl-
4,5-dihydro-1H-imidazoIe-4-carboxylic acid (Ill-42) ..................... 157
Figure III-19. DL-(3R,4R)-7-benzyI-3-methyl-6-phenyl-2-oxa-
5,7—diazaspiro[3.4]oct-5-en-1-one (Ill-37) .................................... 158
CHAPTER IV
Figure lV-1. X-ray crystal structure of IV-1 ........................................................ 178
Figure lV-2. Major nOe correlations for IV-4A and lV-4B ................................. 184

Figure lV-3.
Figure IV-4.
Figure lV-5.

Figure lV—6.

Figure IV-7.

Figure IV-8.

Figure lV-9.

DL-(lV-1) ....................................................................................... 1 94
1-benzyl-2-phenyI-2-imidazoline (IV-3) ......................................... 196
lV-4A + IV-4B ............................................................................... 198
1-benzyl-1 0a-phenyl-2,3-dihydro-1 H-

benzo[e]imidazo[2,1-b][1 ,3]oxazin-5(10aH)-one (IV-5) ................. 199

tert-butyl ((6S,7R,8aS)-1-benzyl-7-methyl-5-oxo-83—phenyl
hexahydro-1 H-imidazo[2,1-b][1 ,3]oxazin-6-yl)carbamate (IV-6) 200

tert-butyl ((6S,8aS)-1-benzyl-5-oxo-8a-phenylhexahydro—1 H-
imidazo[2,1-b][1 ,3]oxazin-6-yl)carbamate (IV-7) ........................... 202

IV-9 ............................................................................................... 203

xi

LIST OF SCHEMES

CHAPTER I

Scheme l-1. Erlenmeyer and Mohr syntheses of oxazol-5(4H)-ones ................... 8
Scheme l-2. Cyclodehydrative formation of oxazoI-5(4H)-ones .......................... 10
Scheme l-3. Reactivity of oxazol-5(4H)-ones ..................................................... 11
Scheme l-4. Scaffolds available from oxazol-5(4H)-ones ................................... 12
Scheme l-5. Steglich rearrangment of O-acyl oxazoles ...................................... 13
Scheme l-6. Organocatalyzed asymmetric Steglich reactions ............................ 14
Scheme l-7. Alkylation of oxazoI-5(4H)-ones under basic conditions ................. 15

Scheme l-8. Selective oxazoI-5(4H)-one alkylation via phase transfer

catalysis ......................................................................................... 16
Scheme l-9. Selective Mannich alkylation reactions of oxazol-5(4H)-ones ........ 18
Scheme l-10. Organocatalytic Michael addition of oxazol-5(4H)-ones ............... 19

Scheme l-11. Pd-catalyzed asymmetric allylic alkylation of

oxazol-5(4H)-ones ....................................................................... 20
Scheme I-12. Arylation reaction of oxazoI-5(4H)-ones ....................................... 21
Scheme l-13. Synthesis of oxazoles from oxazol-5(4H)-ones ............................ 22

Scheme I-14. Use of an oxazoI-5(4H)-one in the synthesis of an indole

alkaloid isolated from Dendrodoa grossularia .............................. 23
Scheme l-15. Proposed mechanism of B-lactam formation ................................ 24
Scheme I-16. B-lactam formation from bicyclic mflnchnones ............................. 25
Scheme l-17. 1,3-dipolar cycloaddition reactions of oxazol-5(4H)-ones ............. 26
Scheme l-18. Pyrrole formation from oxazoI-5(4H)-ones and alkynes ............... 27

xii

Scheme I—19. Pyrroles derived from oxazol-5(4H)-ones and

alkyne equivalents ........................................................................ 28
Scheme l-20. lmidazole syntheses from oxazoI-5(4H)-ones .............................. 29
Scheme l-21. Solid-support synthesis of imidazoles .......................................... 29
Scheme l-22. Synthesis ofA1- and AZ-pyrrolines from oxazol-5(4H)-ones ......... 31

Scheme l-23. Exo selective synthesis of A1-pyrrolines from oxazol-5(4)-ones 32
Scheme I-24. Diasteroselective synthesis of AI-pyrrolines .............................. 33

Scheme I-25. Diastereoselective synthesis of 2-imidazolines via a cycloaddition
reaction of oxazoI-5(4H)-ones ....................................................... 34

Scheme l-26. Nonproteinogenic amino acids available from oxazoI-5(4H)-ones
for use towards the construction of proteasome inhibitors ........... 35

CHAPTER II

Scheme “-1. Synthesis of quaternary o-amino acids from oxazoI-5(4H)-ones... 48
Scheme ”-2. Resolution of racemic quaternary q-amino acid derivatives .......... 49
Scheme “-3. Kinetic resolution of racemic quaternary oxazol-5(4H)-ones ......... 51

Scheme "-4. Palladium-catalyzed alkylation of oxazol-5(4H)-ones with

allenes .......................................................................................... 54
Scheme "-5. Trost's synthesis of sphingofungin F ............................................. 55
Scheme "-6. lnterrnolecular alkylation reaction of oxazol-5(4H)-ones with

enol ethers and subsequent methanolysis .................................... 56
Scheme "-7. Synthesis of diamides from oxazol-5(4H)-ones ............................. 57

Scheme ”-8. Observation of O-alkylation intermediate in the alkyation
reaction ......................................................................................... 67

Scheme "-9. Oxazol-5(4H)-one alkylation using a deuterated alkoxy alkyne ..... 68

Scheme "-10. Terada's enantioselective quaternary q-amino acid synthesis 69

xiii

CHAPTER III

Scheme III-1. Mechanism of proteasomal inhibition by bortezomib and
salinosporamide A ..................................................................... 115

Scheme Ill-2. Strategy for the synthesis of salinosporamide A and a novel
B—lactone-containing 2-imidazoline ............................................ 1 18

Scheme III-3. Corey’s synthesis of the pyrrolidinone ring of
salinosporamide A ..................................................................... 122

Scheme III-4. Quaternary q-amino acid center preparation via an

oxazolidine ................................................................................. 123
Scheme Ill-5. The Danishefsky synthesis of salinosporamide A ...................... 124
Scheme III-6. Pyrrolidinone/B-lactone formation via bis-cyclization .................. 126
Scheme III-7. Pattenden synthesis of salinosporamide A ................................. 127
Scheme III-8. Hatakeyama synthesis of salinosporamide A ............................. 128

Scheme Ill-9. lnterrnolecular alkylation/reduction reaction of

oxazoI-5(4H)-ones ..................................................................... 129
Scheme Ill-10. Strategy for the synthesis of Corey intermediate Ill-8 ............... 130
Scheme Ill-11. Racemic synthesis of Corey intermediate III-8 ......................... 132
Scheme Ill-12. Synthesis of 2-imidazolines derived from diamines .................. 135

Scheme Ill-13. Synthesis of 2-imidazolines derived from B-hydroxy amides... 136
Scheme III-14. Synthesis of 2-imidazolines derived from oxazol-5(4H)-ones...137

Scheme Ill-15. Strategy for the synthesis of a C4-spiro- B-lactone
2-imidazoline ............................................................................ 138

Scheme IV-16. Synthesis of 2-imidazolines via an oxidation/reductive

amination protocol ................................................................... 139
Scheme lV-17. Synthesis of Ill-37 .................................................................... 141
CHAPTER IV
Scheme IV-1. Reactivity of 2-imidazolines and 2-imidazolinium salts .............. 175

xiv

Scheme lV-2.
Scheme lV-3.
Scheme IV-4.
Scheme lV-5.
Scheme lV-6.
Scheme IV-7.
Scheme lV-8.

Scheme lV-9.

Bicyclic adducts derived from 2-imidazolines and epoxides ....... 176
Reaction of azomethine ylides generated from 2-imidazolines..177
Synthesis of 2-imidazolines comprising a B-Iactone ................... 179
Oxazinone formation from stepwise amidation and annulation .. 180
Proposed mechanism of selective oxazinone ring formation ..... 182
Synthesis of oxazinone derivatives IV-4A and IV-4B ................. 184
Oxazinone/imidazolidine adducts derived from salicylic acid ..... 185

Bicyclic oxazinone adducts arising from amino acid
denvafives .................................................................................. 187

Scheme lV-10. Proposed origin of selectivity in the formation of lV-6

and lV-7 ................................................................................... 1 87
Scheme IV-11. Synthesis of IV-9 derived from IV-8 ......................................... 189
Scheme IV-12. Strategy for the resolution of 2-imidazoline enantiomers ......... 190
Scheme IV-13. TiCI4-mediated decomposition of lV-7 ...................................... 191

XV

LIST OF SYMBOLS or ABBREVIATIONS

4-PPY — 4-(1-Pyrrolidino)pyridine

BOP-Cl — Bis(2-oxo-3-oxazolidinyl)phosphonic chloride
CSA — Camphor Sulfonic Acid

DBU — 1,8-Diazabicyclo[5.4.0]undec-7-ene

DCE — 1,2-Dichloroethane

DCM — Dichloromethane

DEPT — Distortionless Enhancement by Polarization Transfer
DMAP — 4-Dimethylaminopyridine

DMF — N,N-Dimethylformamide

DMSO - Dimethylsulfoxide

DOS - Diversity Oriented Synthesis

EDCI - 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
EtOAC - Ethyl Acetate

EWG — Electron-withdrawing group

FDA — Food and Drug Administration

GCMS — Gas Chromatography - Mass Spectrometry
HPLC — High Performance Liquid Chromatography
HRMS — High Resolution Mass Spectrometry

HTS - High Throughput Screening

IBS - lmidazoline binding site

ICso - Half maximal inhibitory concentration

xvi

IKB - Inhibitor of NF-KB

IR — Infrared

LDA - Lithium diisopropylamide
LHMDS - Lithium bis(trimethylsilyl)amide
MS — Mass Spectrometry

MsCl — Methanesulfonyl chloride
nOe - Nuclear Overhauser effect
NF-KB - Nuclear Factor-KappaB
NMR — Nuclear Magnetic Resonance
Nu — Nucleophile

PMB -— para-Methoxybenzyl

rt — Room temperature

SAR — Structure Activity Relationship
SDS - Sodium dodecyl sulfate

TEA — Triethylamine

TFA — Trifluoroacetic acid

TFAA — Trifluoroacetic anhydride
THF — Tetrahydrofuran

TLC - Thin layer chromatography
TMS-Cl - Chlorotrimethylsilane

TOS — Target Oriented Synthesis
Tris - Tris(hydroxymethyl)aminomethane

UV — Ultraviolet

xvii

CHAPTER I
OXAZOL-5(4H)-ONES AS PLURIPOTENT SCAFFOLDS FOR THE
SYNTHESES OF DIVERSE LIBRARIES OF COMPOUNDS

A. Introduction to drug discovery.

Modern disease treatment has resulted in increases in both longevity and quality
of human life. During the course of human history, disease treatment has had a
synergistic relationship with drug discovery. Early drug discovery was honed in
early civilizations through crude experimentation, as components of plants, fungi,
minerals, and animals were administered to sick patients with varying degrees of
success. However, advancements in biological and physical sciences have
permitted the amendment of more sophisticated techniques to the sciences of
drug discovery and disease treatment. Future scientific developments are vital in
order to confront challenges associated with emerging and evolving diseases,
some of which undoubtedly will pose a legitimate threat to the stability and
livelihood of modern civilizations.

The goal of modern drug discovery is to study the pathology of diseases
and to apply appropriate methods for their prevention or treatment. Due to
recent scientific breakthroughs, such as the milestone completion of the human
genome project, many potential therapeutic targets have been identified which
were previously unknown. As such, researchers have become better enabled to
investigate and cure a host of diseases. However, it is noteworthy that while the
number of known drug targets has increased, the number of new drugs approved

by the FDA has steadily decreased.1

Pharmaceutical sciences have adopted the dogma of drug discovery and
actively pursue molecular candidates that modulate desired biological responses.
The process of drug discovery can be roughly divided into two parts: 1) the
identification of a suitable biological target for disease treatment and 2) the
identification and construction of a small molecule to act on that target. The
identification of a biological target is a challenging undertaking in itself as the
pathology of a given disease often encompasses numerous biological pathways,
some of which are important for additional biological functions. The search for an
appropriate small molecule to interact with the chosen biological target is an
equally daunting task, especially considering that the number of molecules
predicted to have drug-like properties is estimated to exceed the number of

1 Moreover, most

atoms occupying the planet (>1062 as compared to 1051).
molecules are biologically active in that they elicit some response when
introduced to a biological system.1 As such, the usual focus of pharmaceutical
chemistry is to pursue molecular candidates which elicit a selective response
when introduced to a biological system. Drugs with a selective mode of action or
a distinct biological target are valued in pharmaceutical research since unwanted
side effects often stem from undesirable and usually unknown biological
interactions. Recent studies have identified a significant number of undesirable
biological interactions of known drugs, bringing to light new opportunities for the
prediction and prevention of such interactions.2 Nonetheless, notable exceptions

exist and some nonselective drugs have proven to be of great therapeutic

importance. For example, many mood disorders including depression and

 

schizophrenia are considered to be polygenic in origin, and such disorders are
often treated successfully through the administration of drugs which affect
numerous biological targets.3

Screening large libraries of small molecules to identify selective
interactions with known receptors, enzymes, and other biological targets or
biological pathways of interest is a routine practice in modern drug discovery.4
Recent synthetic advances have allowed for rapid and efficient production of
compound libraries comprising a high degree of complexity. These collections of
compounds are populated in part by natural products and molecules that have
been inspired by nature.“ Such compound libraries are often screened against
numerous biological targets by a method known as high throughput screening
(HTS) to identify “hits”, or compounds that show some level of desired biological
activity.7 Once “hits” are identified, researchers often construct additional
compound libraries to identify structural features important for biological activity.
These structure-activity relationship (SAR) studies are useful for the development
of lead compounds and drug candidates. Unfortunately, this method may fail to
direct researchers towards identifying drug candidates if initial screens using
known compound libraries do not produce appropriate “hits”.

Fragment-based drug design is an emerging strategy used to identify
structural features within a small molecule which are required for biological
activitya'9 This approach utilizes a screen of low molecular weight compounds to
evaluate for weak binding (mM range) to biological targets of interest. The

screen often identifies numerous small molecules which interact with the target,

and these compounds are considered as fragments which may combined to
produce a potential drug candidate. This strategy is considered to be in its
infancy and has yet to produce any marketed drugs.10 However, it is viewed
favorably as a useful tool for enabling researchers with a reasonable starting
point in their search for drug candidates.

Once researchers have determined a course of focus for their drug
development studies, they often rely on common strategies for the rapid
production of compound libraries. Two frequently-used strategies are target-
Oriented synthesis (TOS) and combinatorial synthesis. TOS iS described as the
synthesis of a predetermined or predesigned molecular target. These target
molecules are often complex and may be synthesized from simpler fragments.
Researchers utilizing TOS routinely plan their syntheses via retrosynthetic
analyses to establish the building blocks, as well as the order of their
assemblage, required for the construction of molecular targets of interest.
Strategically planned synthetic routes are useful for the synthesis of desired
targets and often become amended for the construction of structurally related
analogs.

Combinatorial synthesis is often used in combination with TOS for the
construction of libraries comprised of structurally similar compounds.
Combinatorial synthesis is often described as parallel synthesis, Since numerous
compounds can be prepared in parallel from the same reaction through the
variation of reagents. This strategy is common especially in academic research

in order to investigate the scope of chemical reactions. However, while

 

combinatorial synthesis is useful for the rapid production of compound libraries,
the method is criticized by some for being too focused in terms of populating
chemical space. Chemical space is described as the theoretical landscape in
which all potential molecules exist, and molecules in this space are arranged
according to physical and electronic chemical descriptors. Compounds prepared
in parallel using combinatorial synthesis Often vary only slightly in terms of their
skeletal and stereochemical makeup and are considered to populate similar
chemical Space, as is depicted in Figure l-1. Needless to say, there is much to
learn about the relationship between chemical space and so-called biological
space. Therefore, accessing different regions of chemical space is considered to

be an advantageous strategy for the discovery of pharmaceutically relevant

 

molecules.
Descriptor 1 Descriptor 1
3..
Descriptor 2 Descriptor 2
Descriptor 3 Descriptor 3
Combinatorial Diversity-oriented
Synthesis Synthesis

Figure I-1. Population of chemical space by Chemical libraries prepared via

combinatorial synthesis and diversity-oriented synthesis.

One method used for the construction of compound collections in which

molecules differ greatly in their population of Chemical Space has been termed

11,12

diversity-oriented synthesis (DOS). DOS involves utilizing similar starting

scaffolds in orthogonal reactions to build a diverse set of compounds, which
undergo further derivatizations to generate molecules with increasing complexity
and diversity.12 DOS is undoubtedly more successful than TOS at populating a
variety of regions of Chemical space, as is depicted in Figure l-1. However, the
level of effectiveness by which an appropriate drug candidate can be discovered
by this approach is in question, as the new population of constructed compounds
is diluted into the large vastness of chemical space. Consequently, in
consideration of the time and resources needed for the construction of compound
libraries, there is arguably an equal, if not reduced, probability of producing a
compound suitable for drug development utilizing DOS when compared to TOS.

Drug discovery has played a Significant role in the development of modern
synthetic chemistry. The isolation and characterization of complex natural
products has often inspired researchers to pursue new chemical methods for
their preparation.13 Major synthetic advancements, notably in asymmetric
synthesis, have thus been required in order to emulate the amazing level of
molecular complexity found in natural products.“15 New methods of chemical
synthesis developed to construct products found in nature have in turn been
utilized in the construction of novel pharmaceuticals. The perpetual cycle of
discovery and advancement has had far reaching effects that have been felt
even in academia.

Discoveries involving synthetic reactions and methodologies have long
been and continue to be a hallmark of chemical education. Additionally,

synthetic and medicinal chemistry programs carried out at the academic level are

becoming increasingly more involved in drug discovery science. Therefore, it is
not uncommon for academic researchers to construct compound libraries
according to new methodologies with the explicit aim of obtaining drug-like
compounds. Such academic research currently ongoing in the Tepe lab involves
routine construction and screening of compound libraries derived from
oxazolones in order to elucidate molecular candidates which exhibit anti-cancer

properties.

B. Introduction to oxazol-5(4H)-ones.

Oxazolones are recognized for their utility as versatile scaffolds in organic
synthesis.16 Five isomeric forms of oxazolones exist, each of which exhibits
unique chemical reactivity (Figure I-2).17 Oxazol-5(4H)-ones, also called
“azlactones”, have been extensively utilized in the Tepe lab for the preparation of

diverse libraries of small molecules of heterocycles and amino acids.18

O R1 O R1 O 0 o 0 O
\Nr 0 1%)“) {fizz If“ :Q—RI
R2 0 R2 R2

R2
oxazol- oxazol- oxazol- oxazol- oxazol-
5(4H)-one or 5(2H)-one or 4(5H)-one or 2(3H)-one 2(5H)-one
azlactone psuedoxazolone isoxazolone

R1

Figure l-2. The five isomeric forms of oxazolones.

Oxazol-5(4H)-ones are cyclic esters of N-acyl a-amino acids and are
generally classified as being either saturated or unsaturated (Scheme M).

The first unsaturated oxazol-5(4H)-one was synthesized by PlOchl more than

a century ago in 1883 via condensation of benzaldehyde with hippuric acid in
the presence of acetic anhydride.19 Unsaturated oxazol-5(4H)-ones were
prepared in a similar fashion by Erlenmeyer, who established their correct
structure in 1900 and named them “azlactones” (Scheme l-1).20 Almost a
decade after Erlenmeyer’s discovery, Mohr and co-workers sucessfully
prepared the first saturated oxazol-5(4H)-ones by treating N-acyl amino acids
with acetic anhydride (Scheme l-1).2"23 The chemistry of oxazol-5(4H)-ones
then remained fairly unexplored until the 1940’s, at which time the structure of
penicillin was incorrectly thought to be an oxazol-5(4H)-one.24 Due to the high
interest of penicillin at the time, many research groups focused on the
reactivity of oxazol-5(4H)-ones. The structure of penicillin was eventually
corrected to contain a B-Iactam ring system instead of an oxazol-5(4H)-one
ring, but the information obtained from these studies was important for the

advancement of chemistry involving the oxazoI-5(4H)-one scaffold.

Erlenmeyer Synthesis

0 O R
JL ACZO WNI/O O unsaturated
ti
R

 

RI/ILfiAcozH + H R2 oxazol-5(4H)-one

 

Mohr Synthesis 2
0 R2 A 0 R1 0
R )LNACO H CZ Y Q saturated
1 H 2 N oxazol-5(4H)-one

R2

Scheme l-1. Erlenmeyer and Mohr syntheses of oxazol-5(4H)-ones.

A variety methods appear in the literature for naming oxazol-5(4H)-

ones.”25 The ring is generally numbered according to the Hantzsch-Widman

8

rules giving priority to the oxygen atom and numbering the ring in the direction
of the nitrogen atom as shown in Figure l-3.25 One method of naming refers
to the oxazol-5(4H)—one substrate as an amino acid derivative.20 For
example, the oxazol-5(4H)-one derived from N-benzoyl alanine would be
refered to as benzoyl alanine azlactone. A second method for naming oxazol-
5(4H)-ones describes the substrate as a dihydrooxazole.25 The oxazoI-5(4H)-
one derived from N-benzoyl alanine would then be referred to as 5-keto-2-
phenyI-4-methyI-4,5-dihydrooxazole. A third system, which will be used
throughout this disertation, assigns a parent name of oxazoI-5(4H)-one and
numbers substituents about the ring in accordance with the Hantzsch-Widman
rules. Using this system the oxazolone prepared from N-benzoyl alanine

would be described as 2-phenyl-4-methyI-5(4H)-oxazolone.

Y?
I 5 O

N34

R1

R2

Figure I-3. Numbering of the oxazol-5(4H)-one system in accordance with the

Hantzsch-Widman rules.

C. Synthesis and general reactivity of oxazol-5(4H)-ones.

Numerous examples exist for the preparation of oxazol-5(4H)-ones. Modern
syntheses of oxazol-5(4H)—ones often involve mild reaction conditions,
wherein N-acyI-q-amino acids are treated with dehydrating reagents (Scheme

l-2). N-acyl-o-amino acids used in these reactions are readily prepared under

9

Schotten-Baumann conditions using d-amino acids or from the N-acylation of
o-amino esters followed by hydrolysis.26 Since an abundance of natural and
unnatural q-amino acids are commerically available, researchers have easy
access to a wide variety of oxazol-5(4H)—ones. Traditionally, cyclodehydration
reactions of N-acyl-o-amino acids were performed in refluxing acetic acid,
conditions that often lead to reduced product purities and yields as well as
difficulties with product isolation. However, these issues are generally
avoided in modern preparations of oxazol-5(4H)-ones through the use of more

26.27

reactive dehydrating reagents, such as activated anhydrides and

carbodiimides.”

@Q‘m Dehydrating R1

0 O
“i Reagent : Ill/$0
R2

R1Iin2

 

Scheme l-2. Cyclodehydrative formation of oxazol-5(4H)-ones.

The oxazoI-5(4H)-one ring system contains numerous reactive sites
allowing for a wide variety of transformations (Scheme l-3). The acidic nature
of the proton(s) found at the C4 position of the oxazol-5(4H)-one scaffold

A3829 which can react with a

enables the formation of an oxazole enolate
range of electrophiles (Scheme l-3). Alternatively, the oxazoI-5(4H)-one ring
can be opened readily by a nucleophilic attack at the carbonyl carbon to
generate various protected amino acid derivatives (8, Scheme I-3).
Additionaly, treatment of oxazol-5(4H)-oneS with Lewis acids results in the

formation of a mesionic 1,3-dipole C (also known as a mt'mchnone) or of a

10

 

reactive ketene intermediate D (Scheme l-3), both of which undergo

cycloaddition reactions to yield novel heterocyclic compounds.”
R1 0
T 8
N? EIT'ZJNUC
R2
Ba s\ /(ucleophileB
i3;

Lewis acid
R1 0 9
v8 ‘——-- fit it
LA’% R1 N R2
R LA D

Scheme l-3. Reactivity of oxazol-5(4H)-ones.

The diverse reactivity of oxazol-5(4H)-ones makes them excellent
substrates for use in the synthesis of a variety of compounds (Scheme l-4).31
Highly substituted heterocyclic scaffolds can be directly accessed from oxazol-
5(4H)-ones with relative ease and in a stereoselective manner. Natural and
unnatural amino acids, as well as related derivatives, are also easily
constructed in enantiopure form by using oxazol-5(4H)-one intermediates.
The focus of the remainder of this chapter will be to describe some of the
synthetic applications of oxazol-5(4H)-ones used towards the constrCution of

biologically interesting scaffolds.

11

 

R
R1/LLN R3 R2 a
H R1 N s
O \ I / H R2 R3
( W
R4 R R4
R1YN H70 0 R
[J / R3 <——— N ———> NI “R3
R2 . R2 R3 002

 

 

 

Scheme l-4. Scaffolds available from oxazol-5(4H)-ones.

D. Nucleophilic reactions of oxazoI-5(4H)-ones.

Oxazol-5(4H)-ones are often recognized for the diverse transformations they
undergo due to their nucleophilic character. Oxazol-5(4H)-one a-protons, or
those at C4, have a relatively high acidity compared to q-amino acids, and
deprotonation results in the formation of an aromatic oxazole (A, Scheme 1-
3).”32 Oxazol-5(4H)-one deprotonation leads to cyclic enolates which are
less sterically encumbered than related acylic q-amino acids and display high
reactivity towards electrophiles. One consequence of facile enolate formation
is that optically active oxazol-5(4H)-ones undergo rapid racemization.28 Thus,

treatment of oxazol-5(4H)-ones with various electrophiles under basic

conditions is a practical method used for the preparation Of various racemic

12

quaternary a-amino acid progenitors. Advancements in recent years have
surfaced to address the lack of stereoselectivity in these reactions, often

through the employment of modern methods of organocatalysis.

1. Acylation reactions.

Early experiments by Steglich and coworkers demonstrated the utility of oxazol-
5(4H)-ones as nucleophiles. In 1970, Steglich and co-workers described the
synthesis of C4-acyl quaternary oxazol-5(4H)-ones by means of a rearrangement
of O-acylated oxazoles (Scheme l-5).33 The authors discovered that treatment of
the O-acylated oxazoles with nucleophilic organocatalysts such as 4-
dimethylaminopyridine (DMAP) readily promoted a rearrangement (now referred
to as the Steglich rearrangement) to afford quaternary oxazol-5(4H)-one
products. The generally accepted mechanism involves reversible DMAP-adduct
formation from the reaction of DMAP with O-acylated oxazole, followed by
acylation, which appears to be irreversible due to the stability of the oxazol-
5(4H)-one products (Scheme I-5).34

O DMAP

o O
R1O/U\O

2 _
N=< K — O 9 — Ar
ROD
N=<

Ar
0 Ar

 

 

L R1o’U‘8MAP

Scheme l-5. Steglich rearrangment of O-acyl oxazoles.

13

Since the initial discovery of the reaction, several chiral organocatalysts
have been used substoichiometrically in Steglich rearrangements to generate
enantiomerically enriched quaternary oxazol-5(4H)-ones.3““‘2 The first
asymmetric acyl migration of oxazol—5(4H)-ones was reported in 1998 by Fu and
co-workers, whose studies involved the use of the chiral DMAP derivative PPY*
(Scheme l-6).3“'35 Vedejs and co-workers demonstrated a similar asymmetric
acyl migration in which their DMAP derivative, TADMAP, was used as an
organocatalyst in the rearrangement (Scheme l-6).36'40 Both groups reported
obtaining quaternary oxazol-5(4H)-ones in good yields with a high degree of
enantioselectivity.

R1

0
O >TOR2 1-2 mol% catalyst I O
/ O * O

 

 

 

  

I r
N\2T tert-amyl alcohol NECO
R3 0 °C R23
E 13 C3R2
r M M g 0 up to 99% yield
e, e 2/ ‘Q °
N H ‘N | up to 95 /o ee
\ "'"CPh . Me Fe Me i
I , OAC . Me Me
N - ' Me
L (S)-TADMAP = . PPY* ,

 

 

 

 

Scheme l-6. Organocatalyzed asymmetric Steglich reactions.

2. Alkylation reactions.

Steglich and co-workers also investigated alkylation reactions of oxazol-5(4H)-
ones.43 In their studies, oxazol-5(4H)-ones were suspended in polar aprotic
solutions and treated with Ht‘Inig’S base in the presence of highly reactive

electrophiles to afford quaternary oxazol-5(4H)-ones in moderate to good yields

14

(Scheme l-7). The reaction yields both quaternary oxazol-5(4H)—ones and 0-
alkyl oxazoles, products which arise from respective C- and O-alkylation
reactions of the ambident enolate intermediate. The issue of chemoselectivity in
the alkylation of such an ambident nucleophile has Since been addressed by
utilizing the hard-soft properties of each of the two nucleophilic atoms. The
enolate oxygen is considered a hard base and it reacts with hard electrophiles,
whereas the softer carbon is expected to react with softer electrophiles.
Therefore, O-alkylation is expected to be more SN1-Iike when compared to C-
alkylation.44 Optimizations in reaction conditions, including the use of protic or
nonpolar solvents and the use of soft electrophiles, such as alkyl halides, have

thus been constructive for promoting the formation of C-alkylation products.

R1 0 R1 0 R1 0
118-=0 Ra-X . F7230 + \N'Z/g'OR3
Base R
R2 R2 3 R2
C-Alkylation O-Alkylation

Scheme l-7. Alkylation of oxazol-5(4H)-ones under basic conditions.

Recent developments have resulted in the preparation of enantiomerically
enriched quaternary oxazol-5(4H)—ones via alkylation reactions. Ooi and
coworkers have recently reported the use of a chiral tetraaminophosphonium
phase transfer catalyst M in biphasic mixtures to invoke enantioselectivity in

45,46

alkylation reactions of oxazol-5(4H)-ones (Scheme l-8). The authors

demonstrated the use of biphasic reaction mixtures that were either liquid-liquid

(cyclopentyl methyl ether - saturated K3PO4 aqueous solution)45 or liquid-solid

15

(tert-butyl methyl ether — ground solid K3PO4)46 in composition, both of which
were found to promote the generation of quaternary products in high yield and
enantiomeric excess. Furthermore, excellent diastereoselectivity was observed
in alkylation reactions of oxazoI-5(4H)-ones prepared from dipeptides,

tripeptides, and tetrapeptides (Scheme l-8).45

 

 

 

 

 

 

R1 0 R3-Br R
\NI/ O K3P04 (8 or aq) 1757(3)]:0 -
H (1 mol%) T_ .R A, Ole Ar
R2 R2 3 Ph W /’ Ph
up to 99% yield ”-3” ’
o . . \
up to 93 /o ee Ph““//N N Ph
NHBoc R343, NHBoc Ar \\Ar
RIWSTO O K3PO4 (3C1) WIRO O Ar = 3,5-(tBulhquSI)2-CSH3
Nf H (1 mol%) _ N72: . ' J
R2 7 R2 R3
up to 98% yield
up to 98% de

Scheme l-8. Selective oxazoI-5(4H)-one alkylation via phase transfer catalysis.

Studies aimed towards the development of new oxazol-5(4H)-one
alkylation reactions have also revealed methods for the stereoselective

47,48

generation of Mannich-type products. Such Mannich products are easily

converted into o-disubstituted a,B-diamino acids,49 which are valued as synthetic

t.50 All reported stereoselective

precursors to molecules of pharmaceutical interes
Mannich-type alkylation reactions of oxazol-5(4H)-ones have involved addition to
electron deficient imines in the presence of a chiral organocatalyst. Chiral

tetraaminophosphonium carboxylate catalysts (e.g. l-2) developed in Dr. Ooi’s

lab effectively promote this transformation to afford alkylation products in

16

excellent yields and stereoselectivities (Scheme l-9).47 An oxazol-5(4H)-one
enolate is initially generated in the reaction via deprotonation by the weakly basic
pivalate anion and becomes coordinated to the chiral phosphonium salt through
a defined hydrogen-bonding network. Stereoselective alkylation to the imine
electrophile then proceeds, followed by protonation of the resultant nitrogen
anion to regenerate catalytic pivalate.47 The use of small-molecule hydrogen-
bond donors, such as catalysts l-2, has become increasingly common in
reactions involving asymmetric organocatalysis.51 Cinchona alkaloids are one
such family of asymmetric H-bond donors that have shown utility in
stereoselective Mannich-type reactions of oxazoI-5(4H)--ones.52 Recently, Wang
and co-workers reported an asymmetric Cinchona alkaloid-catalyzed Mannich
reaction which allows access to enantiomers Opposite those prepared via Ooi’s
protocol (Scheme l-9).48 The reaction involves deprotonation of the oxazol-
5(4H)-one by the basic quinuclidine nitrogen of alkaloid I-3, and the resultant
enolate and ammonium species establish a chiral H-bonding complex, leading to

the observed stereoselective alkylation.

17

 

0 .280 Ar2 A” O
F0 251 -2 (2 mol%): TKO

 

 

 

 

 

 

 

 

N...
R1 R H THF, 40 °C 1 *NHSOZAIZ
:f R R2 up to 99% yield
/ r ,3 up to 93% ee
', Me MeH up to 7.821 dr
TMSO H N N ‘9 h’l‘fi
l3
MOe / \[Ph H eH Ph
\ l eOCOC(CH3)3
A” 0 Ar1
,Ts O
1‘7 0 N l-3 (20 mol%) Y O
1 +Ar2JLH Et O rt : N * ”HTS
R 2 ' R1 r2up to 97% yield
A up to 97% ee

up to >30:1 dr

Scheme l-9. Selective Mannich alkylation reactions of oxazol-5(4H)-ones.

Oxazol-5(4H)-one alkylation chemistry has recently been extended to
include stereoselective Michael addition reactions.53'54 In 2008, Jergensen and
co-workers demonstrated that the addition of racemic oxazol-5(4H)—ones to 0,8-
unsaturated aldehydes in the presence of chiral organocatalyst resulted in the
formation of quaternary substrates with two new stereogenic centers (Scheme l-
10).53 The authors reported complete control of the new Chiral center created
from the (LB-unsaturated aldehyde portion and good control of the stereocenter
formed on the oxazoI-5(4H)-one, with product diastereomeric ratios ranging from
2:1 to greater than 20:1 and enantiomeric excesses ranging between 83 and
96%. The reaction was found to tolerate various aldehydes as well as oxazol-

5(4H)-ones bearing a wide variety of substituents, leading to products containing

18

 

alkyl, benzyl, and aryl groups at all positions. In a similar manner,
organocatalyzed stereoselective additions of oxazol-5(4H)-ones to nitroalkenes
have been performed, and regioselectivity has been obtained in the reactions to

give rise to either C2 or C4 alkylation products.55'56

 

Ar
N AI'
H OTMS R1 _
1 R3\FN (10 mol%) R3 ,N‘ f 0
RW0 + OVRZ Ar= 3.5-(CFaizceHa> o R2
O toluene, rt 0
up to 91% yield
83-96% ee

up to >20:1 dr

Scheme l-10. Organocatalytic Michael addition of oxazol-5(4H)—ones.

Quaternary oxazol-5(4H)-ones have also been prepared through the
installation of allylic substituents. Trost and co-workers have developed several
methods to construct quaternary oxazol-5(4H)-ones via stereoselective allylic

alkylation reactions?“31

In one such preparation, the authors treated oxazol-
5(4H)-ones with allylic acetates under conditions of palladium catalysis with a
chiral diamine ligand I-4 (Scheme H1).58 The reaction was demonstrated using
racemic oxazol-5(4H)-ones bearing C4 alkyl substituents to give high yields of
prenylation and cinnamylation products (up to 95% yield) with excellent
enantiomeric excess (up to 99% ee).58 An alternative stereoselective palladium-
catalyzed allylic alkylation reaction protocol has since been reported by

Kawatsura and co-workers for the preparation of similar products in good

yields.‘52

19

 

 

O R2
N=<

l-4 (7.5 mol%)

 

 

+ P“ [n3-C3H5PdCl12 (2.5 mol %) R“ N=(O
R2 NEt3, toluene or CH3CN Ph
R1 \ OAc . up to 95% yield

up to 99% ee
R1 and R2=Me or O Q 0
R1=Ph and R2=H N‘H HN
cflenQD
l-4

 

 

 

Scheme I-11. Pd-catalyzed asymmetric allylic alkylation of oxazol-5(4H)-ones.

3. Arylation reactions.

The synthesis of quaternary oxazol-5(4H)-ones via the installation of aryl
substitutents has become a useful means for the preparation of unique
nonproteinogenic quaternary a-amino acids.”65 In 2003, Hartwig and co-
workers demonstrated the first palladium-catalyzed arylation reaction of
oxazol-5(4H)-ones (Scheme M2).64 The reaction involves the coupling of an
Sp2 carbon of an aryl bromides with the enolate of an oxazol-5(4H)-one. The
catalyst system for this reaction consists of Pd(OAc)2 and the sterically
hindered electron rich ligand Ad2P(t-Bu) (Scheme l-12). This protocol was
useful for the generation of quaternary substrates derived from various
oxazoI-5(4H)-ones and aryl bromides, with the highest yielding reactions
resulting from the use of electron rich or electron neutral aryl bromides (75-

85% vs ~60% for electron poor aryl bromides).

20

ArBr

 

Pd(OAc) 0
Ph 0 2 Ph O
“f0 Ad2P(‘Bu) _ \E f
R K2003 R Ar
Toluene

58-85% yield

Scheme l-12. Arylation reaction of oxazol-5(4H)-ones.

E. Electrophilic reactions of oxazoI-5(4H)-ones
The oxazol-5(4H)-one scaffold contains a highly electrophilic carbonyl center that
reacts with an assortment of nucleophiles. Exposure of oxazol-5(4H)-ones to
nucleophiles such as alcohols, amines, hydrides, and even arenes (under
FriedeI-Crafts conditions) has resulted in the construction of q-amino acid
derivatives and heterocyclic compounds (Scheme I-3, B).66'67 The electrophilic
Character of oxazoI-5(4H)-ones has also made them ideally suited for use in
medically-relevant biopolymers;68 such oxazol-5(4H)-one-containing polymers
have been shown to be useful for the prevention or promotion of mammalian cell
adhesion and bacterial biofilm formation.“"“:"70
A variety of heterocyclic scaffolds have been prepared from electrophilic
reactions of oxazol-5(4H)-ones. In an effort to develop a new route to highly
substituted oxazoles, Manasi Keni in the Tepe lab devised a one pot Friedel-
Crafts I Robinson-Gabriel synthesis for the production of 2,4,5-trisubstituted
oxazoles from oxazol-5(4H)-ones.71 Key to this protocol is the ability of oxazol-
5(4)-Ones to undergo Friedel-Crafts alkylation reactions with arenes to produce

7“" substrates which are known to undergo Robinson-

2-acylamino ketones,
Gabriel cyclodehydration reactions to afford oXazoles. The two sequential

21

reaction steps were combined in a one-pot synthesis using aluminum chloride
and trifluoromethanesulfonic acid, resulting in the culmination of oxazoles directly
from oxazoI-5(4H)-ones (Scheme M3).71 The reaction works well for a wide
variety of oxazol-5(4H)-one substrates containing both aromatic and alkyl
substituents. In agreement with known Friedel-Crafts reactions, the reaction
works best with either electron neutral or electron rich arenes, whereas electron
deficient arenes provide little or no product formation. It is believed that the
oxazol-5(4H)-one is first activated by the aluminum Chloride and a Friedel-Crafts
acylation ensues. The resulting 2-acylaminoketone becomes protonated by the
trifluoromethanesulfonic acid and then dehydrative cyclization proceeds to yield

the corresponding oxazole.

R3
R 0 O O 0 R2 R3
“(I e R )1."de
N AlCl3 1 H
R2

 

O
l TfOH
R3 — O R3 -
o O
MIG RN
N ,7 N
R2 H20 __ R2 _

 

 

Scheme I-13. Synthesis of oxazoles from oxazol—5(4H)-ones.

The electrophilic nature of oxazol-5(4H)-ones has also been utilized in
natural product synthesis. In 2008, Dr. Christopher Hupp in the Tepe lab
completed the first total synthesis of a marine alkaloid isolated from the marine

tunicate Dendrodoa grossularia.75 His synthesis featured a Claisen oxazole

22

rearrangement to first prepare a quaternary oxazol-5(4H)-one (Scheme l-14).
Methanolysis with NaOMe was used to open the electrophilic oxazol-5(4H)-one
ring, followed by subsequent rearrangement to furnish a hydantoin ring. Six
steps were then required to complete the synthesis of the natural product.75'76

The methodology developed by Dr. Hupp was also utilized to prepare analogs of

76,77

the natural product which were later evaluated for their biological activity.

  

   

O r H ' H
S N R’NYO r
T O EDCI N / O t
o NH/ /\H/ _. J
OEt N
TS NTS J

 

 

 

 

 

Ts TS

Scheme I-14. Use of an oxazol-5(4H)-one in the synthesis of an indole alkaloid
isolated from Dendmdoa grossulan’a.

F. Cycloaddition reactions of oxazol-5(4H)-ones

Cycloaddition reactions of oxazol-5(4H)-ones are useful in the generation of an
assortment of heterocycles?1 The ability of oxazol-5(4H)-ones to undergo
cycloaddition reactions arises from their reactive azomethine ylide and
amidoketene tautomers (C and D respectively, Scheme I-3), which are known to

react with dipolarophiles. Azomethine ylides are zwitterionic species comprising

23

a C-N-C fragment in which 4n electrons are delocalized across 3 p-orbitals.
These unstable dipolar species are generally prepared in situ by various means,
such as deprotonation of imine derivatives or therrnolysis of aziridines and 4-
oxazolines.78’80 Ketenes are also reactive species which generally need to be
generated in situ, often through treatment of an acid chloride with a tertiary amine
base. Ketenes react readily with nucleophiles to generate carboxylic acid

derivatives and are also known participants in polymerization reactions.“82

1. [2+2] cycloaddition reactions.

The cycloaddition reaction of ketenes with imines, known as the Staudinger
reaction,83 is a method used to prepare B-lactams, structural features common
to several families of antibiotics.84 Asymmetric variants of this reaction are
known, and B-lactams produced in the reactions are valued synthetically for
their utility as pharmacores.”87 Likewise, the B-lactam scaffold is accessible
via the Staudinger reaction of imines with oxazol-5(4H)-ones, as was first
demonstrated in 1971 by Huisgen and coworkers.88 In their studies, the
authors generated ketenes in situ by means of mesionic oxazoI-5(4H)-one
formation (Scheme MS). The ketenes were treated with imines and thermal

[2+2] cycloaddition reactions proceeded to afford B-lactams.

 

 

Ph ' 0 _ NR3 O R

0 e O ('5 ' IN 3

UNI/$0 /U\ JL R1/I\R2 )OL R2

HaC'O Ph N Ph —"’Ph N m,
P“ _ CH3 - CH3

 

 

Scheme I-15. Proposed mechanism of B-lactam formation.

24

Diastereoselective B-Iactam formation was later demonstrated by
Cremonesi and coworkers, who treated bicyclic oxazol-5(4H)-ones with imines
under basic conditions (Scheme M6).89 The authors illustrated good control
of diastereoselectivity through manipulation of the imine N-substituent. The
reaction provided mainly the cis-product (with respect to the sulfur and phenyl
groups) when performed with imines containing electron withdrawing moieties.
Conversely, high yields of trans B-lactams could be selectively formed when
imines contained electron donating groups.89 Both products are proposed to
arise from initial attack of the imine to the least-hindered side of the ketene, as
shown in Scheme l-16. Electron donating groups stabilize the initial iminium
intermediate to afford the trans products after the [2+2] cycloaddition.
However, the iminium intermediate is destabilized by electron withdrawing
groups, allowing the intermediate to undergo double bond isomerization
before ring formation occurs, thus giving rise to the thermodynamically favored

cis products.89

 

Ph
.COR \_ N—COR F‘x
[IL _NR1 :1 9 , Phsk N‘COR
S C=O NEt3,CHZCI{ P“ @9‘0 H"" N O
u reflux H R1 (521
20-91% yield trans
R 36-94% de 11
3%
[- 3 ( (\NHCOR F1
Se 0 se\( 9 H34. N‘COR
H (a 9-0 ——> Ph" 0
_N. '
Ph R1 R1
cis

Scheme I-16. B-lactam formation from bicyclic miJnchnones.

25

2. [3+2] cycloaddition reactions.

There are numerous literature examples wherein oxazol-5(4H)-ones participate
in [3+2] cycloaddition reactions with dipolarophiles to give rise to 5-membered
nitrogen heterocycles.‘°’1'78'90 Both aromatic and nonaromatic ring systems are
available via the reaction, and the product outcome is dependent upon the
dipolarophile used as well as reaction conditions. It is presumed that equilibrium
amounts of mesionic oxazol-5(4H)-ones react with dipolarophiles to initially form
bicyclic intermediates (Scheme l-17).91 Decarboxylation then proceeds and
aromatization ensues to afford the heterocyclic products. In this way, pyrroles

and imidazoles are prepared from alkynes and nitriles, respectively (Scheme I-

 

 

17).

R1YO 9 _R X\ R; R1 X
HNI / O X=CR3 W8 0 —> elf \ R3
9 x CR,N HN H C06
2 R R2 2

2
u r J l-co2
R1 R1

Scheme I-17. 1,3-dipolar cycloaddition reactions of oxazol-5(4H)-ones.

Pyrroles were first prepared via cycloadditions of oxazol-5(4H)-ones by
Huisgen, Gotthardt, and Bayer in 1964.92'93 The authors reportedly generated

oxazol-5(4H)-ones in situ and treated them with alkynes at various

26

temperatures to afford pyrroles in good yields. Using a variety of oxazol-
5(4H)-ones and alkynes, pyrroles were prepared with substituent diversity at
every atom of the heterocyclic scaffold (Scheme l-18).9""94 This procedure
has recently been amended to include in situ N-alkylation through the addition
of 2,6-di-tert-butylpyridine and a highly reactive alkylating reagent.95 In this
way, N-alkyl pyrroles are produced from nitrogen-unsubstituted N-acyl amino
acids.
R2
E9120 R4C: CR5 R3 \N/ R1

R5 R4
60-98% yield

Scheme l-18. Pyrrole formation from oxazol-5(4H)-ones and alkynes.

Similarly, pyrroles have been synthesized from mesionic oxazoI-5(4H)-
ones via reaction with alkyne equivalents. One such synthesis has been
reported using vinyl phosphonium salts as alkyne equivalents in cycloaddition
reactions with m'Linchnones.96 The reaction conditions involving heat
(refluxing THF/DMF solvent mixture) or sonication promote the cycloaddition,
after which decarboxylation and PPh3 elimination occur to afford the
anticipated products regioselectively in moderate yields (Scheme HQ). The
high regioselectivity in the reaction has been attributed to the strong
electrostatic forces between the phosphonium and carbonyl groups.96 The
preparation of pyrroles has also been reported via the reaction of miJnchnones

with vinyl-chlorinated alkenes (Scheme l-19).97 This reaction was performed

27

using a, B-unsaturated esters or ketones to regioselectively form the desired
pyrrole products in good yields. Placement of the chlorine substitutent on the
alkene is key to product formation, as its elimination after the decarboxylation

event gives rise to the aromatic products.97

 

 

Cl 0
32 R46.) 9 e FacMR‘: '32
R N R PPhBr O O R=CHOTOCH R N R
1W 3 4 3 R1~<\ I 4 6 5 4; 1M 3
®N R NEt3
R4 R2 3 CH3CN R400 CF3
35-68% yield 33 - 89 yield%

Scheme l-19. Pyrroles derived from oxazol-5(4H)-ones and alkyne

equivalents.

Oxazol-5(4H)-ones are also known to participate in [3+2] cycloaddition
reactions with nitriles and nitrile equivalents to generate imidazoles. Shortly
after reporting their pyrrole synthesis,94 Huisgen and coworkers reported a
similar decarboxylative cycloaddition reaction wherein imidazole formation
was observed when miinchnones were treated with electron-deficient nitriles
(Scheme l-20).98 The authors performed the reaction under a variety of
conditions to afford imidazoles with electron withdrawing substituents in
moderate yields. Consonni and coworkers prepared imidazoles through an
analagous reaction utilizing nitrile equivalents.99 The authors first generated
oxazol-5(4H)-ones in situ and treated them with N-
(phenylmethylene)benzenesulphonamides (Scheme l-20). The resulting
bicyclic intermediates consequently decomposed, releasing 002 and

benzenesulphinic acid, to form imidazole products. Pivotal to their approach

28

was the incorporation of the N-phenylsulphonyl moiety to act as a good

leaving group, and its expulsion permitted aromatization to yield the imidazole

 

 

products.
R2 R R2
1 O 9

SO Ph N

R1Wg/Z/R3 R4C-N all / O R4/\N 2 4 R1\« / R3
N R R2’ ' N
4 3 R4

41 - 71 yield% 20 - 65 yield%

Scheme I-20. lmidazole syntheses from oxazol-5(4H)-ones.

Formation of imidazole products by the aforementioned methods often
occur in low yields due to competing reactions of oxazoI-5(4H)-ones, such as
self-condensation, a process resulting in dimerized products.91 Bilodeau and
coworkers have demonstrated repression of self-condensation reactions of
oxazol-5(4H)-ones through the incorporation of solid-phase support.100 Their
studies involved the in situ formation of miinchnones through dehydration of
resin-bound amino acids followed by 1,3-dipolar cycloaddition reactions with
N-tosyl imines to give polymer-bound imidazoles (Scheme l-21). Liberation
from the resin by heating in acetic acid at 100 °C for 2 hours then afforded the

desired imidazoles in high yields.

Me i1 Me R1
:1 C02H___, R3/\ NTS \ R3
R2 0 EDCI RZBLN

O
é CHZCIZ
53- 99 yield%

Scheme I-21. Solid-support synthesis of imidazoles.

29

Preparations of nonaromatic pyrrolines via cycloaddition reactions
involving oxazol-5(4H)—ones have also been reported. Gotthardt, Huisgen,
and Schaefer investigated the formation of both A1-pyrrolines and A2-
pyrrolines from the reaction of mesionic oxazoI-5(4H)-ones with electron

”“103 For the construction of A1-pyrrolines.

deficient olefins (Scheme I-22).
the authors utilized N-unsubstituted oxazol-5(4H)-ones and gently heated
them in xylenes in the presence of alkenes (Scheme l-22).1°1'103 The
intermediate bicyclic adducts formed from the 1,3-dipolar cycloaddition then
underwent successive decarboxylation and protonation to afford A1-pyrrolines.
A1-pyrrolines contain an imine which is susceptible to imine-enamine
tautomerization, resulting in observed inversion of stereochemistry of the R5
substituent. (Scheme l-22).1°1'1°3 Exploitation of the equilibrium which exists
between the two tautomers enabled the authors to also explore the formation

2 The use of N-substituted oxazol-5(4H)—ones in similar

of Az-pyrrolines.10
cylcoaddions reaction with alkenes thus gave rise to Az-pyrrolines via
zwitterionic intermediates formed after decarboxylation (Scheme l-22). The
zwitterionic intermediates are proposed to undergo prototropic isomerization
to yield the more energetically favorable enamine tautomer.102 Such
zwitterionic intermediates are trapped as byclic adducts when an additional
alkene equivalent is added to the reaction mixture via a subsequent [3+2]
cycloaddition.102 The authors also demonstrated olefin regiocontrol through

quaternization of one of the pyrroline carbons.102 For example, use of 1,1-

diphenylethylene as a dipolarophile in the reaction generates a zwitterionic

30

intermediate that can only undergo the aforementioned isomerization via one

resonance contributer in order for product formation to occur (Scheme l-22).

—l

_ R5
R
R1\(0 0 [*4sz R1 ‘0‘ 4
Rl‘be $ (NR 0
R2 R3 R2”

l-co2

 

 

 

R5

R1 /
R s I

R,N 4 R2=Me,Ph 42E? HRTEER‘t

2 R

3
Az-pyrrolines A1-pyrrolines

‘i
R5

R1
/N / R4 RR2=Me :evm RLLiEm
R2 ’

R3

= (”02

 

Az-pyrrolines

Scheme l-22. Synthesis of A1- and Az-pyrrolines from oxazol-5(4H)—ones.

Diastereoselective preparations of A1-pyrrolines from oxazol-5(4H)—
ones have since been reported.‘°“'106 In 1989, Maryanoff and co-workers
demonstrated the exo-selective formation of a A1-pyrroline cycloadduct from
the exposure of an in situ-generated mesionic oxazol-5(4H)-one to an electron

1°“ The reaction, which was conducted in

deficient alkene (Scheme l-23).
refluxing acetic anhydride, selectively afforded the A1-pyrroline product in 80%
yield along with some dihydropyrazine (18%). The dihydopyrazine is derived

from the A1-pyrroline via successive decarboxylation and ring expansion, and

31

heating the A1-pyrroline in refluxing decalin affords the 7-membered

heterocycle in 70% yield.1°“'107

AcHN COZH AC2O
110 °C

 

Cl

  

 

 

 

decalin
reflux

NC \ / CN CI
N

Me H
70%

Scheme I-23. Exo selective synthesis of A1-pyrrolines from oxazol-5(4)-ones.

A more recent example of A1-pyrroline synthesis via an exo-selective
cycloaddition of oxazol-5(4H)-ones with alkenes was demonstrated by Dr.
Mahesh Peddibhotla in the Tepe group (Scheme l-24).106 The reaction
involved generation of mesionic oxazol-5(4H)-ones through exposure to Lewis
acids. In contrast to analogous cycloadditions which occurred under harsh
reaction conditions, the Lewis acid-mediated cycloadditions were carried out
at room temperature. Thus, treatment of oxazol-5(4H)-ones with 10 mol %
silver acetate and electron-deficient alkenes gave rise to A1-pyrrolines in good
yields. The exo-selective reaction provided pyrrolines with R2 and R3
substituents syn to each other. Moreover, the relationship between the R3

and R4 substituents was trans in the collected products, regardless of the

32

starting alkene geometry. This trans relationship is proposed to arise from
isomerization via imine-enamine tautomerization to give the more
thermodynamically stable product.106 Toste and co-workers have since
reported an analogous reaction which utilizes their chiral gold catalyst (S)-Cy-
SEGPHOS(AuOBz)2 to furnish A1-pyrrolines enantioselectively from oxazol-
5(4H)-ones.108

R

4 R3
0 O N R3 4'
R1‘<\ I R4 74 1352
N R1 N

R2 AgOAc (10 mol%) CO2H
THF, dark 59 - 95% yield

 

Scheme I-24. Diasteroselective synthesis of A1-pyrrolines.

Lewis acid-mediated cylcoadditions of oxazol-5(4H)-ones have also
been utilized for the preparation of 2-imidazolines.1°9'111 Dr. Mahesh
Peddibhotla of the Tepe lab demonstrated that treatment of oxazol-5(4H)-ones
with imines and TMS-Cl in refluxing dichloromethane diastereoselectively
afforded 2-imidazolines containing four point diversity (Scheme l-25).109
Various oxazol-5(4H)-ones and imines are tolerated in the reaction, giving rise
to 2-imidazolines containing alkyl, acyl, aryl, and heterocyclic substituents.110
The major products in this reaction were predominantly trans (with respect to
R2 and R3) when R1 was aryl, and it was steric interactions which were
postulated to direct the stereochemical outcome of the reaction”). The

reaction proceeds through a cycloaddition wherein the endo cycloadduct is

formed; it is proposed that steric interaction of the bulky silyl group with the

33

imine R3 substituent disfavors the exo approach.11o Additional studies by Dr.
Vasudah Sharma of the Tepe group later demonstrated complete reversal or
degradation of diastereoselectivity in the reaction through variation of

substituents about the oxazol-5(4H)-one ring.111

 

 

 

R R N TMS — R4
1Y0 0 3% R47 R\N'N£1 ____> N R3
M2: TMSCI H47VJ\(O R1‘<\N .R2
R2 CHzclz R3 R2 0 COZH

‘ ‘ 60 - 90% yield

Scheme l-25. Diastereoselective synthesis of 2-imidazolines via a cycloaddition

reaction of oxazol-5(4H)-ones.

G. Current studies.

The following chapters of this dissertation describe reactions which take
advantage of the nucleophilic and electrophilic properties of oxazoI-5(4H)-ones
for the preparation of nonproteinogenic amino acids (Scheme l-26). Such amino
acids prepared in these studies have proven to be valuable as building blocks
towards the construction of the natural product proteasome inhibitor
salinosporamide A.112 Furthermore, non-natural amino acids prepared from
oxazol-5(4H)-ones in the Tepe lab have been utilized to synthesize heterocycles
of interest, most notably 2-imidazolines, for use in our proteasome inhibition

studies.

34

.4
.4
. ?;
.tl
22%
i

 

 

N
COzMe
1 Ph
Me OlfTN/I
?H N g’Me
0 . 002Me /‘N i "
‘— JL" Me—.Ph ’N 3:0
R {j , th O o
Salinosporamide A O 3“ lmidazoline Proteasome
Inhibitors

Scheme I-26. Nonproteinogenic amino acids available from oxazol-5(4H)-ones

for use towards the construction of proteasome inhibitors.

35

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40

56.

57.

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59.

Cabrera, S.; Reyes, E.; Aleman, J.; Milelli, A.; Kobbelgaard, S.;
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Huang, Y.; Walji, A. M.; Larsen, C. H.; MacMillan, D. W. C.
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Trost, B. M.; Ariza, X. Enantioselective allylations of azlactones with
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Trost, B. M.; Dogra, K. Synthesis of novel quaternary amino acids using
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Trost, B. M.; Heinemann, C.; Ariza, X.; Weigand, S. Chiral recognition for
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Trost, B. M.; Jakel, C.; Plietker, B. Palladium-catalyzed asymmetric

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41

 

 

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Koen, M. J.; Morgan, J.; Pinhey, J. T.; Sherry, C. J. Reaction of 4-
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Liu, X. X.; Hartwig, J. F. Palladium-catalyzed alpha-arylation of azlactones
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Morgan, J.; Pinhey, J. T.; Sherry, C. J. Reaction of organolead triacetates
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Fernandez-Tejada, A.; Corzana, F.; Busto, J. H.; Jiménez-Osés, G.;
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Carter, H. E. Azlactones. Org. React. 1946, 3, 198-239.

Jewell, C. A.; Lynn, D. M. Multilayered polyelectrolyte assemblies as
platforms for the delivery of DNA and other nucleic acid-based
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Buck, M. E.; Breitbach, A. S.; Belgrade, S. K.; Blackwell, H. E.; Lynn, D.
M. Chemical Modification of Reactive Multilayered Films Fabricated from
Poly(2-alkenyl azlactone)s: Design of Surfaces that Prevent or Promote
Mammalian Cell Adhesion and Bacterial Biofilm Growth.
Biomacromolecules 2009, 10, 1564-1574.

Fredin, N. J.; Broderick, A. H.; Buck, M. E.; Lynn, D. M. Nanoimprinted
Thin Films of Reactive, Azlactone-Containing Polymers: Combining
Methods for the Topographic Patterning of Cell Substrates with
Opportunities for Facile Post-Fabrication Chemical Functionalization.
Biomacromolecules 2009, 10, 994-1003.

42

71.

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Keni, M.; Tepe, J. J. One-pot Friedel-Crafts/Robinson-Gabriel synthesis of
oxazoles using oxazolone templates. J. Org. Chem. 2005, 70, 4211-4213.

Mcclure, D. E.; Arison, B. H.; Jones, J. H.; Baldwin, J. J. Chiral Alpha-
Amino Ketones from the Friedel-Crafts Reaction of Protected Amino-
Acids. J. Org. Chem. 1981, 46, 2431-2432.

Rebek, J.; Tai, D. F.; Shue, Y. K. Synthesis of Ergot Alkaloids from
Tryptophan. J. Am. Chem. Soc. 1984, 106, 1813-1819.

Rebek, J.; Shue, Y. K. Total Synthesis of Rugulovasines. J. Am. Chem.
Soc. 1980, 102, 5426-5427.

Hupp. C. D.; Tepe, J. J. Total synthesis of a marine alkaloid from the
tunicate Dendrodoa grossularia. Org. Lett. 2008, 10, 3737-3739.

Hupp, C. D.; Tepe, J. J. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
Hydrochloride-Mediated Oxazole Rearrangement: Gaining Access to a
Unique Marine Alkaloid Scaffold. J. Org. Chem. 2009, 74, 3406-3413.

Please see the dissertation of Dr. Christopher Hupp for biological activity
data of the natural product and related analogs.

Coldham, |.; Hufton, R. Intramolecular dipolar cycloaddition reactions of
azomethine ylides. Chem. Rev. 2005, 105, 2765-2809.

Gothelf, K. V.; Jorgensen, K. A. Asymmetric 1,3-dipolar cycloaddition
reactions. Chem. Rev. 1998, 98, 863-909.

Pandey, G.; Banerjee, P.; Gadre, S. R. Construction of enantiopure
pyrrolidine ring system via asymmetric [3+2]-cycloaddition of azomethine

ylides. Chem. Rev. 2006, 106, 4484-4517.

Tidwell, T. T. Ketene Chemistry - the 2nd Golden-Age. Acc. Chem. Res.
1990, 23, 273-279. -

Orr, R. K.; Calter, M. A. Asymmetric synthesis using ketenes. Tetrahedron
2003, 59, 3545-3565.

43

 

luswlwq-gun,muq-Lfluw . . .1 ... . ,r .

 

83.

84.

85.

86.

87.

88.

89.

90.

91.

92.

Staudinger, H. Announcements from the chemical instiute at Strasbourg
University in Alsace, France - Ketene. Liebigs Ann. Chem. 1907, 356, 51-
123.

Durckheimer, W.; Blumbach, J.; Lattrell, R.; Scheunemann, K. H. Recent
Developments in the Field of Beta-Lactam Antibiotics. Angew. Chem. Int.
Ed. 1985, 24, 180-202.

France, 8.; Weatherwax, A.; Taggi, A. E.; Lectka, T. Advances in the
catalytic, asymmetric synthesis of beta-lactams. Acc. Chem. Res. 2004,
37, 592-600.

Lee, E. C.; Hodous, B. L.; Bergin, E.; Shih, C.; Fu, G. C. Catalytic
asymmetric Staudinger reactions to form beta-lactams: An unanticipated
dependence of diastereoselectivity on the choice of the nitrogen
substituent. J. Am. Chem. Soc. 2005, 127, 11586-11587.

Palomo, C.; Aizpurua, J. M.; Ganboa, l.; Oiarbide, M. Asymmetric
synthesis of beta-lactams by Staudinger ketene-imine cycloaddition
reaction. Eur. J. Org. Chem. 1999, 3223-3235.

Funke, E.; Huisgen, R. Ketenoid Reactivity of a Mesoionic Oxazol-5-One.
Chem. Ber. 1971, 104, 3222-3228.

Cremonesi, G.; Dalla Croce, P.; La Rosa, C. [2+2] Cycloaddition reactions
of imines with cyclic ketenes: Synthesis of 1,3-thiazolidine derived spiro-
beta-lactams and their transformations. Helv. Chim. Acta 2005, 88, 1580-
1588.

Padwa, A.; Pearson, W. H. Synthetic Applications of 1,3-Dipolar
Cycloaddition Chemistry Toward Heterocycles and Natural Products.

Gotthard, H.; Huisgen, R.; Bayer, H. O. 1,3-Dipolar Cycloaddition
Reactions .53. Question of 1,3-Dipolar Nature of Delta2-Oxazolin-5-Ones.
J. Am. Chem. Soc. 1970, 92, 4340-4344.

Huisgen, R.; Bayer, H. O.; Gotthardt, H. Azlactones as 1,3-Dipoles - New
Pyrrole Synthesis. Angew. Chem. Int. Ed. 1964, 3, 135-136.

44

93.

94.

95.

96.

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99.

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102.

Huisgen, R.; Bayer, H. 0.; Schaefer, F. C.; Gotthardt, H. New Type of
Mesoionic Aromatic Compound + Its 1,3-Dipolar Cycloaddition Reactions
with Acetylene Derivatives. Angew. Chem. Int. Ed. 1964, 3, 136-137.

Huisgen, R.; Gotthard.H; Bayer, H. O.; Schaefer, F. C. 1,3-Dipolar
Cycloadditions. 56. A Convenient Synthesis of N-Substituted Pyrroles
from Mesoionic Oxazolones and Alkynes. Chem. Ber. 1970, 103, 2611-
2624.

Hershenson, F. M.; Pavia, M. R. Synthesis of N-Substituted Pyrroles from
Azlactones Via 1,3-Oxazolium 5-Oxides. Synthesis 1988, 999-1001.

Clerici, F.; Gelmi, M. L.; Trimarco, P. 5(4H)-oxazolones. Part XI.
Cycloaddition reaction of oxazolones and munchnones to
triphenylvinylphosphonium salts as synthetic equivalents of alkynes.
Tetrahedron 1998, 54, 5763-5774.

Okano, T.; Uekawa, T.; Morishima, N.; Eguchi, 8. Synthesis of Beta-
(Trifluoromethyl)Pyrroles Via the Cycloaddition of Munchnones to
Electron-Deficient Trifluoromethylated Olefins. J. Org. Chem. 1991, 56,
5259-5262.

Brunn, E.; Funke, E.; Gotthard.H; Huisgen, R. 1.3-Dipolar Cycloadditions.
61. Cycloadditions of N-Substituted Oxazolium-5-Olates to Nitriles, Nitro,
Nitroso and A20 Compounds. Chem. Ber. 1971, 104, 1562-1572.

Consonni, R.; Croce, P. D.; Ferraccioli, R.; Larosa, C. A New Approach to
lmidazole Derivatives. J. Chem. Res., Synop. 1991, 188-189.

Bilodeau, M. T.; Cunningham, A. M. Solid-supported synthesis of
imidazoles: A strategy for direct resin-attachment to the imidazole core. J.
Org. Chem. 1998, 63, 2800-2801.

Gotthardt, H.; Huisgen, R.; Schaefer, F. C. Delta-2-Pyrroline Aus
Mesoionischen Oxazolen Und Olefinen. Tetrahedron Lett. 1964, 487-491.

Gotthard, H.; Huisgen, R. 1,3-Dipolar Cycloadditions .57. Preparation of

Delta2-Pyrrolines from N-Substituted Oxazolium 5-Oxides and Olefinic
Dipolarophiles. Chem. Ber. 1970, 103, 2625-2638.

45

 

103.

104.

105.

16.

107.

108.

109.

110.

111.

112.

Huisgen, R.; Gotthard.H; Bayer, H. O. 1,3-Dipolar Cycloadditions .55.
Delta1-Pyrrolines and 7-Azabicyclo 2,2,1!Heptane Derivatives from
Azlactones and Activated Alkenes. Chem. Ber. 1970, 103, 2368-2387.

Maryanoff, C. A.; Karash, C. B.; Turchi, l. J.; Corey, E. R.; Maryanoff, B. E.
Characterization of a Stable Carboxylic-Acid lnterrnediate from 1,3-Dipolar
Cyclo-Addition of a Munchnone with 1,2-Dicyanocyclobutene. J. Org.
Chem. 1989, 54, 3790-3792.

Melhado, A. D.; Luparia, M.; Toste, F. D. Au(l)-catalyzed enantioselective
1,3-dipolar cycloadditions of Munchnones with electron-deficient Alkenes.
J. Am. Chem. Soc. 2007, 129, 12638-12639.

Peddibhotla, S.; Tepe, J. J. Stereoselective synthesis of highly substituted
Delta(1)-pyrrolines: exo-selective 1,3-dipolar cycloaddition reactions with
azlactones. J. Am. Chem. Soc. 2004, 126, 12776-12777.

Maryanoff, C. A.; Turchi, I. J. Mechanism and Stereochemical Implications
of the Reaction of an Oxazolium-S-Oxide with 1,2-Dicyanocyclobutene -
an Am1 Study. Heterocycles 1993, 35, 649-657.

Melhado, A. D.; Luparia, M.; Toste, F. D. Au(l)-catalyzed enantioselective
1,3-dipolar cycloadditions of Munchnones with electron-deficient Alkenes.
J. Am. Chem. Soc. 2007, 129, 12638-12639.

Peddibhotla, S.; Jayakumar, S.; Tepe, J. J. Highly diastereoselective
multicomponent synthesis of unsymmetrical imidazolines. Org. Lett. 2002,
4, 533-3535.

Peddibhotla, S.; Tepe, J. J. Multicomponent synthesis of highly substituted
imidazolines via a silicon mediated 1,3-dipolar cycloaddition. Synthesis
2003, 1433-1440.

Sharma, V.; Tepe, J. l. Diastereochemical diversity of imidazoline
scaffolds via substrate controlled TMSCI mediated cycloaddition of
azlactones. Org. Lett. 2005, 7, 5091-5094.

Mosey, R. A.; Tepe, J. J. New synthetic route to access (+/-)

salinosporamide A via an oxazolone-mediated ene-type reaction.
Tetrahedron Lett. 2009, 50, 295-297.

46

CHAPTER II
DIASTEREOSELECTIVE SYNTHESIS OF TERT-ALKYL AMINO HYDROXY
CARBOXYLIC ESTERS VIA AN INTERMOLECULAR ALKYLATION
REACTION OF OXAZOL-5(4H)-ONES AND ENOL ETHERS

A. Introduction to quaternary a-amino acids.

The diversity of applications for which non-proteinogenic amino acids are used
make them valuable substrates within the fields of synthetic organic, bioorganic
and medicinal chemistry."2 One such group of amino acids that has received
considerable attention from the scientific community as of late are the quaternary
a-amino acids (or a,d-disubstituted d-amino acids) (Figure ll-1).3’1o The
presence of the additional alkyl substituent at the o-carbon of quaternary amino
acids generally results in changes in biophysical properties as compared to the
naturally occurring nonquaternary d-amino acid counterparts. When
incorporated into peptides, for example, the additional substituent often helps to
sterically constrain the free rotation of the residue’s side chain leading to unique
folding.“10 Peptides containing quaternary a-amino acids also generally exhibit
increased hydrophobicity, as well as an increased stability towards both
chemical”12 and metabolic”14 decomposition. These unique physical
properties make them intriguing tools for the design and study of peptides and
proteins. Additionally, naturally-occurring quaternary o-amino acids and
derivatives are found within the structures of many heterocyclic molecules that
exhibit interesting biological activities, making them attractive synthetic

targets.7'15

47

H N CO H H N CO H
2 Y 2 2 X 2
R R1 R2

d-amino acid quaternary

o-amino acid

Figure "-1. General structures of both d-amino and quaternary d-amino acids.

B. Syntheses of quaternary d-amino acids using oxazol-5(4H)-ones.

The importance of quaternary o-amino acids in synthetic and biological studies
has prompted interest in the development of new and efficient methods for their
construction.3*7'9'15 Classical methods for the synthesis of quaternary d-amino
acids include the alkylation of d-amino esters protected as imines16 and the
Strecker reaction17 of ketimines. An alternative approach is to prepare
quaternary d-amino acids from the oxazol-5(4H)-one scaffold.”21 Quaternary
substituted d-amino acid derivatives can be directly accessed from the
nucleophilic ring opening of quaternary substituted oxazol-5(4H)-ones, substrates

which have been generated using a variety of methods (Scheme "-1).19

R1 0
R1TNXCOZH :> RTgécoj Tit/{o
2

0R2 R3 R2 R3
Quaternary Quaternary OxazoI-5 4H -one
a-Amino Acid Oxazol-5(4H)-one ( )
Derivative

Scheme "-1. Synthesis of quaternary d-amino acids from oxazoI-5(4H)-ones.

OxazoI-5(4H)-one alkylation has been used strategically in the synthesis
of enantiomerically pure quaternary d-amino acid derivatives. Various methods

used for the generation of quaternary oxazoI-5(4H)-ones were described in

48

Chapter I, including alkylation reactions. Traditional alkylation reactions, like
those reported by Steglich, resulted in the formation of racemic quaternary
oxazol-5(4H)—ones. One method for the resolution of these racemic mixtures
involves nucleophilic ring opening of the oxazol-5(4H)-one scaffold by chiral
nucleophiles, thus producing separable diastereomeric mixtures (Scheme "-2).”
26 This technique has been successfully applied by Obrecht and co-workers for
the preparation of enantiomerically pure quaternary tyrosine derivatives.25 Their
strategy involved nucleophilic ring opening of racemic quaternary oxazol-5(4H)—
ones with chiral amino acid derivatives followed by separation of the resultant
diastereomers via silica gel chromatography (Scheme "-2). Selective cleavage
of the newly formed amide bond then afforded enantiomerically pure N-benzoyl

protected quaternary o-amino acids and/or esters.26

Ar Ar
N R-X R
)L 0 Base N 0
P“ O PhA
AF4-MeO-C6H4

° 0
H2N\_)LN

H

\Ph ,

 

Ar
° RH ° 0 ‘R H 0 O
P '.
Ph/lLNgYNJN PhJLN/QWNJN
O 3 H H O 5 H

Scheme "-2. Resolution of racemic quaternary a-amino acid derivatives.

49

The kinetic resolution of quaternary oxazol—5(4H)-ones is another means
for the preparation of chiral quaternary a-amino acid derivatives. Extensive
studies have demonstrated that nonquaternary oxazol-5(4H)—ones readily
undergo both small molecule- and enzyme-catalyzed dynamic kinetic resolutions
to afford high yields of enantiomerically pure d-amino acids and esters.”32
Recently, Tsuji and co-workers demonstrated the first transition-metal catalyzed
kinetic resolution of quaternary oxazol-5(4H)-ones (Scheme "-3).”34 Exposure
of quaternary oxazol-5(4H)-ones to a catalytic amount of copper in combination
with the chiral phosphine ligand (S)-DTBM-SEGPHOS activates quaternary
oxazol-5(4H)-ones towards methanolysis. Coordination of the oxazol-5(4H)-one
to the copper-ligand complex generates a chiral environment wherein
nucleophilic attack can occur at the reactive carbonyl carbon for only one of the
enantiomers. Thus, one oxazoI-5(4H)-one enantiomer undergoes nucleophilic

ring opening to yield the corresponding quaternary amino ester derivative,

whereas the opposing enantiomer remains unreacted.

50

 

2 mol% Cu(OAc)2 O O

 

 

R2 -0 (SI-DTBM-SEGPHOS sz“ N=/\O + R10R2"1"1O(C|-12)2C)MB
R3 2 eq M60<CH2)20H R3 LoiHRa
( toluene, 20 C , _<_ 99% ee 5 86% 66

IB

tBu 2
<2

(S)-DTBM-SEGPHOS

IWWHWHU ”In“ mum-4.“.

 

 

 

Scheme "-3. Kinetic resolution of racemic quaternary oxazol-5(4H)-ones.

C. Introduction to tert-alkyl hydroxy carboxylic acids.

Representative of quaternary a-amino acid derivatives are tart-alkyl amino
hydroxy carboxylic acids, densely functionalized quaternary carbon centers
surrounded by a combination of alkyl, amino, carboxylic, and hydroxyl
substituents.7'15 These structural motifs are present in numerous microorganism
metabolites including the sphingofungins, lactacystins, altemicidines,

7,15

oxazolomycins and many others (Figure "-2). The diverse and potent

biological activity of these metabolites has stimulated many researchers to

1 5. 35-39 40-45

pursue their total syntheses and in-depth biological evaluations.

51

 

9H 9H COZH

"C6H13WNH2 CI
0 OH \R

H Sphingofungin E R = OH
(+) Sphingofungin F R = H

[EL/so NH O

2 2

HHN NHQ
, JCOZH _

MeN = ”cow
r.

 

   
 

OH HO _:_ S
- \<‘0H NHAc
CONH2
(-) Altemicidin (+) Lactacystin
0 ,OH
N z:
<’ I \

 

Neooxazolomycin

Figure "-2. Naturally occurring tert-alkyl amino hydroxy carboxylic acids.

D. Synthesis of tert-alkyl hydroxy carboxylic acids from oxazol-5(4H)-ones.

Construction of the tert-alkyl amino hydroxy carboxylic acid moiety found within
the aforementioned biological targets of interest has proven to be a significant
synthetic challenge and has been the focus of many research groups.7'15
Approaches targeted towards the synthesis of the quaternary center in these
molecules have differed greatly due to the complexity of the target molecules and
a lack of general approaches available for the preparation of the core scaffold.
Some of these approaches will be discussed in Chapter III as methods for the

synthesis of such a quaternary center present in the marine metabolite

salinosporamide A.

52

Quaternary oxazol-5(4H)-ones have been prepared using a variety of
methods in which functionalities have been installed to make them suitable
precursors to tert-alkyl amino hydroxy carboxylic acids.19 Several of these
syntheses were described in Chapter I, including those in which the Steglich
rearrangement was used for the preparation of quaternary oxazol-5(4H)-ones
bearing C4 esters. Such quaternary oxazol-5(4H)-ones are susceptible to
hydride reduction, which results in ring decomposition to reveal amide and
alcohol functionalities. In this way, appropriately substituted quaternary oxazol-
5(4H)-ones are transformed into the corresponding tert-alkyl amino hydroxy
carboxylic acids.46

Quaternary oxazol-5(4H)—ones suitable for the construction of tert-alkyl
amino hydroxy carboxylic acids have also been generated through the
installation of allylic substituents. The Trost group has reported various
conditions used for this type of transformation. One such report describes the
regioselective and enantioselective preparation of quaternary oxazol-5(4H)-ones
under conditions of palladium catalysis using chiral diamine ligand ent-I-4
(Scheme "-4).47 The authors treated oxazol-5(4H)-ones with electron rich
alkoxy-substituted allenes to promote selective nucleophilic addition to the
substituted allene terminus, resulting in the culmination of quaternary products in

good yields and selectivity.48

53

II
o Pd(OCCF3)2 (2 mol%) 0

OBn RQLO ent-l-4 (6 mol%) > WC
+ _( KO‘Bu (2 mol%) R‘“

 

 

 

 

N— . . . H
Ph Hlppurlc Acrd (20 mol %) Ph
r CH20I2. rt . 67-87% yield
85-94% ee
Q Up to 20:1 dr
O . O
NH HN
PPHthP
ent-I-4

   

Scheme "-4. Palladium-catalyzed alkylation of oxazoI-5(4H)-ones with allenes.

In another report by the Trost group, an allylation reaction of oxazol-5(4H)-
ones was utilized to complete the total synthesis of the fungal metabolite
sphingofungin F.49'50 Using chemistry developed in their labs,51 the authors
treated 2-phenyl-4-methyl-5(4H)-oxazolone with a gem-diacetate-substituted
alkene under conditions of palladium catalysis with chiral diamine ligand I-4
(Scheme "-5). The resultant quaternary oxazoI-5(4H)-one then underwent
methanolysis to afford a quaternary o-amino acid derivative. The alkylation
reaction was reported to give the quaternary oxazoI-5(4H)-one in 70% yield with
89% ee. Several synthetic manipulations were then required to prepare the
natural product, which was formed in just 15 linear steps with an impressive 17%

overall yield.49'5°

54

O

 

\(KO NaH, M (1.5 mol%) OAcO
N ___< (na-C3H5PdCI)2 (0-5 "Ida/01 TBDPSOWO
Ph THF, -5 °C N=<
t OAc 70% yield Ph
TBDPSONOAC 89% ee 11

 

NH HN 9H 9H
P|P4 "C6H13WCOZH

II (+) Sphingofungin F
Scheme "-5. Trost's synthesis of sphingofungin F.

 

 

Recently, racemic quaternary oxazol-5(4H)-ones have been prepared via
an intermolecular ene-type alkylation reaction of oxazol-5(4H)ones and enol
ethers (Scheme "-6).52 Developed by Dr. Jason S. Fisk in the Tepe lab, this
reaction takes advantage of the strong enol character of the oxazol-5(4H)-one
ring. Oxazol-5(4H)-ones containing substituents that help stabilize the
substrate’s enol tautomer appear to best facilitate the reaction. For instance,
oxazoI-5(4H)-ones bearing acyl groups in the C4 position quantitatively provide
products in minutes at room temperature. However, the use of oxazol-5(4H)-
ones with C4 aryl substituents require higher temperatures for product formation.
Moreover, C4 alkyl-substituted oxazol-5(4H)-ones do not yield any alkylation
products under the reported reaction conditions. Methanolysis of the quaternary
oxazol-5(4H)-ones then affords quaternary malonate derivatives in excellent

yields.

55

 

 

th/O Z/OR th/O - H OR
N\8:O _1_-3_equ_iv, N? M N

,. Bz’
COzMe CHftC'Z MeOZC °' Meozc COzMe

0R NaOMe _<_99% yield

Scheme "-6. Intermolecular alkylation reaction of oxazol-5(4H)-ones with enol

ethers and subsequent methanolysis.

E. Syntheses of quaternary a-amino acid derivatives via nucleophilic ring-
opening reactions of quaternary oxazol-5(4H)-ones.
The oxazol-5(4H)—one ene-type alkylation reaction discovered by Dr. Fisk

enabled access to numerous quaternary amino acid derivatives which we
planned to use in the synthesis of natural products. As discussed in Chapter I,
oxazoI-5(4H)-ones react with a variety of nucleophiles, making them
appropriately suited for the synthesis of d-amino acid derivatives. In an effort to
desymmetrize the malonate products and effectively apply our alkylation
chemistry towards molecular targets of interest, we decided to exploit the
electrophilic nature of oxazoI-5(4H)-ones.

Initially, we investigated the formation of diamide products from ring-
opening reactions of quaternary oxazol-5(4H)-ones derived from our alkylation
reaction. We anticipated such products to be available from the addition of
amines to the same reaction flask containing quaternary oxazol-5(4H)-ones once
the alkylation reactions were complete, as could be monitored by TLC (Scheme
”-7). Indeed, treatment of 2-phenyI-4-carbmethoxy-5(4H)-oxazolone "-1 with
benzyl vinyl ether "-2 in refluxing CHZCIZ, followed by addition of either
benzylamine or ammonia to the reaction pot afford diamides "-3 and "-4 in 84%

and 92% yields respectively.

56

OBn

 

Ph 0 1 =/ OBn
Nfo ) "_2(1.5eq: n
CHZCIZ, reflux M26 Cit NHR
COzMe 2) NHZR 2 O

"'1 "-3 (R = Bn) 84% yield

"-4 (R = H) 92% yield

Scheme "-7. Synthesis of diamides from oxazoI-5(4H)-ones.

We were also interested in utilizing our alkylation chemistry in the
synthesis of tert-alkyl amino hydroxy carboxylic esters, which we anticipated
could be used to construct natural products and natural product analogs (Figure
“-2). We predicted that such compounds should be accessible via hydride

reduction‘“5'52‘55

of quaternary oxazol-5(4H)-ones obtained from our alkylation
reaction. To our delight, sodium borbhydride reduction of the quaternary adduct
prepared from the treatment of "-1 with tert-butyl vinyl ether "-5 proceeded
smoothly to give quaternary amino acid derivative "-6. Moreover, the mixture of
diastereomers generated in reaction were found to be separable via silica gel
chromatography (Table "-1). Optimal results were obtained in the reduction
when a solvent mixture composed of THF and water (2:1) was used (Table "-1,
condition 3). An organic solvent such as THF was required for solubility of the
oxazol-5(4H)-ones, and water as a co-solvent was found to increase the reaction
rate and yield, presumably by increasing the solubility of sodium borohydride

(Table "-1, conditions 13). Carboxylic acid products arising from hydrolysis of

quaternary oxazol-5(4H)-ones were observed in the reaction when water was

57

used as a co-solvent. However, these undesired products were minimized by

cooling the reaction to 0 °C.

O‘Bu

 

 

 

Ph 0 1 =/ O‘Bu
W0 ’ eke
CI"'20'2, rt MeOZC‘f OH
COzMe 2) NaBH4, solvent, ,,_6
"'1 temperature
Entry Solvent Temperature %Yield
(°C)
1 THF 25 65
2 H20 25 Low
3 THFzHZO (2:1) 0 91

 

Table "-1. Optimization of NaBH4 reduction of quaternary oxazoI-5(4H)-ones.

F. Optimization of reaction diastereoselectivity.
The aforementioned alkylation reaction developed in our lab was used to
generate quaternary oxazol-5(4H)-ones, albeit with little or no product
diastereoselectivity.52 In an effort to make this synthetic approach more broadly
applicable, we investigated several reaction conditions in an attempt to improve
the stereoselectivity of the reaction.“5

We initiated our optimization studies by first screening a variety of solvents
in the alkylation reaction of oxazol-5(4H)-one "-1 and tert-butyl vinyl ether "-5.
The majority of solvents used in the alkylation reaction provided little if any
diastereoselectivity, as was determined via 1H-NMR analysis of the reaction
mixtures after NaBH4 reduction. Reaction rates increased when polar solvents

were used, and products were completely formed in CH2CI2 in one hour (Table II-

58

2, entry 2). However, reaction times increased when solvents bearing lone pairs
were used, which can be attributed to hydrogen bond basicity (Table "-2, entries
1 and 3).“58 Finally, the use of a nonpolar solvent such as benzene greatly
decreased the reaction rate, yet an increase in stereoselectivity (d.r. = 67:33)
was observed (Table ”-2, entry 4). It is noteworthy that oxazol-5(4H)-one "-1
appears to be only sparingly soluble in benzene, and it is proposed that poor

solubility contributes to the increase in reaction time when using benzene as a

 

 

 

solvent.
O‘Bu
PhYo 1) =/ (1.5 eq.) H 9‘3” H 93“
NI 0 "'5 : 32’ \C + Z’N)-/\
CO M solvent, rt. time Meozc“ OH M6020 "’I_OH
"4 2 e 2) NaBH4 ll-6A "-68
Entry Solvent Time (h) 6A : SB % Yield
1 CH3CN 3 50: 50 90
2 CHZCIZ 1 55 :45 91
3 THF 19 52 :48 90
4 Benzene 36 67 : 33 90

 

Table "-2. Solvent screening to optimize selectivity in the alkylation reaction.

In an effort to increase the rate of the reaction while maintaining
selectivity, the use of protic catalysis was next investigated. Through screening a
variety of acids, we found that a substoichiometric amount of diphenyl phosphate
improved both the reaction rate and diastereoselectivity (Table "-3, entry 3).

Bronsted acids less acidic than diphenyl phosphate also improved the

59

diastereoselectivity of the reaction, but did little for increasing the reaction rate
(Table ”-3, entry 1). The use of more acidic Bronsted acids resulted in lower
yields of the desired product, presumably due to enol ether decomposition (Table
”-3, entries 4 and 5). Enol ether decomposition was notably problematic when
TFA was used in the reaction, as "-1 was not fully consumed even after 48
hours, as observed by TLC (Table "-3, entry 5). Chiral Bronsted acid (R)-(-)-1,1’-
binaphthyl-2,2’-diy| hydrogenphosphate provided the highest diastereoselectivity
(79:21) of the catalysts investigated, but provided little observable
enantioselectivity (2% ee) as determined by chiral HPLC (Table "-3, entry 3).59
Several other phosphoric acids prepared by Dr. Fisk were screened in the
reaction,59 and they were found to provide diastereomeric ratios similar to those
obtained through the use of commercially available diphenyl phosphate.
Additionally, several Lewis acids were screened resulting in little or no product
formation and low diastereoselectivity (Table "-3, entries 6-10). The significant
rate enhancement found when using Bronsted acids and not other Lewis acids is
suggestive of the catalyst protonating the enol ether to form an oxonium ions":61
The relative stereochemistry of the major diastereomer II-6A obtained using

conditions in Tables "-2 and "-3 has been established unambiguously via X-ray

crystal structure (Figure "-3).

60

t
1) OBu H O‘Bu H 9,3“

P“ O a
T ".5 (1-5 eq.)
Nfo : BZ’NFC + BZ,N>/\
Catalyst, Benzene, rt MeOZC OH MeOZC ”_OH
002““? 2) NaBH4
ll-1 lI-6A ll-GB

 

 

Entry Catalyst Time (hr) A:B %Yield

 

—‘L

3,5-Dinitrobenzoic acid 16 70 : 30 90

 

 

 

2 Diphenyl phosphate 3 75 : 25 90
3 (R)-(-)-1 ,1'-binaphthyl-2,2' 13 79 : 21 90
-din hydrogenphosphate
4 CSA 1 74 :26 83
5 TFA 48 67: 33 81
6 Ti(OiPr)4 48 67 : 33 61
7 Yb(OTf)3 48 - 3 -- 0
8 TMSOTf 48 " 3 " 0
9 Zn(OTf)2 48 52 : 48 24
10 Cu(OTf)2 48 " 3 " 0
CO Opgo QC. .0
CO 0 0H QOROH
(R)-(-)-1,1'-binaphthyI-2,2' Diphenyl
. -diyl hydrogenphosphate L Phosphate

 

 

 

 

 

 

Table "-3. Effect of Bronsted and Lewis acids on reaction diastereoselectivity.

61

 

0” (5%, C(17)
v’ cns) m;
9 f "‘ 0(5)
s O v
. ’9» we,
0(4)
_ Cg) 6(6)
0(5) =4) fr) 002)
xv 5‘. ’
91a. Nm 0(7) w) '
Si" , is? “'
U0(2) ~ “(1)
0(8)
g}\""2
I) W‘g‘éCIIOI
0(9) ¢

Figure "-3. X-Ray crystal structure representation of lI-6A.

G. Scope of alkylation/reduction reaction of oxazoI-5(4H)-ones.

Utilizing conditions optimized for diastereoselectivity, the scope of the reaction
was further explored. Exposure of 2-phenyl-4-carbmethoxy-5(4H)-oxazolone "-1
to various vinyl enol ethers in the presence of 10 mol % of diphenyl phosphate
resulted in high yields of the desired products after sodium borohydride reduction
(Table "-4). The tert-butyl and benzyl protecting groups yielded better
diastereoselectivity than the ethyl protecting group (Table “-4, entries 1-3),
indicating that enol ethers containing more sterically demanding oxygen
protecting groups generally produce better diastereoselectivities. The less

reactive higher substituted enol ethers gave lower yields and required the use of

62

heat (Table "-4, entries 4-6) to produce the desired products in good yields.
Both butoxyethyne "-11 and 2-methoxypropene "-12 (Table "-4, entries 7 and 8,
respectively) also provided reasonable yields of products when the reactions
were performed in dichloromethane. Dichloromethane was used as the solvent
for the formation of "-18 and "-19 to increase the rates of the reactions, being
that the products contained only one chiral center apiece. Furthermore,
reduction of the intermediate derived from the alkylation reaction of "-11 needed
to be performed in a 2:1 ratio of THF and EtOH at -41 °C. Hydrolysis or
esterification products were predominantly observed when water or ethanol,
respectively, were used as a cosolvent with THF at 0 °C. However, reducing the
temperature for the reduction reaction from 0 °C to -41 °C resulted in an increase

in product yield (67% yield at -41 °C versus 20% yield at 0 °C).

63

1) Enol Ether

 

 

 

 

P“ O Diphenyl- H
Y O ,N R + ,N R
hos hate 32 BZ
“Q: p p 1- MeOZCKOH MeOZCY”-OH
COzMe 2 fiegzfine: rt lI-6A and "-68 and
"-1 )leF/H‘lo ll-13A to ll-19A 11-133 to "-193
2 A B
Entry Substrate R Temp (°C) A:B %Yield
t
1 _/ot5u if“ 25 75:25 90
" a.
"-5 "(.35B
n
2 2703" EA 25 75:25 77
"-2 ll-13
3 _/OEt £3 25 57:33 85
_ ‘4
"-7 "-14
4 < :0 3:) 50 38:62 57
— 5.
"-8 11-15
OBn OBn
5 _ 5 50 59:31 48
8’ 12./V
(5:1 trans to cis)
"-9 (1-15
OMe
5 FUN“; H. i 50 50:40 33
"-10 "-1
n
7 : oneu O B“ 25 57
“‘5
"-11 "-18
OMe
OMe .
8 =§ X 25 52
“a.
(1-12 11-19

 

Table "-4. Alkylation/reduction reaction performed with various enol ethers.

64

The role of the 2-position of, the oxazol-5(4H)-one scaffold was also
investigated for its effect on reactivity and selectivity (Table "-5). Several oxazol-
5(4H)-ones with different substituents in the 2-position were prepared and were
reacted with tert-butyl vinyl ether "-5 in the presence of diphenyl phosphate.
Oxazol-5(4H)-ones with aryl substituents gave high yields and erosion of
diastereoselectivity was observed as electron deficiency increased (Table “-5,
entries 1-3). Selectivity was all but lost when oxazoI-5(4H)-ones containing alkyl
substituents were used (Table “-5, entries 4 and 5). The relative stereochemistry
of the diastereomer "-263 from the reaction of oxazol-5(4H)-one "-22 with "-5

was ascertained by X-ray analysis (Figure "-4).
t
1)_/__/O Bu

R "-5 R O t R o t
T0 O Diphenyl- \f OB” \f OB”

HN ‘ HN
Phosphate \<_\ + >/\

COzMe Benzene, rt "”902C 0” MeOZC "OH

 

 

 

"-1 and 2) NaBH4 Il-6A and "-63 and
"-20 to "-23 Il-24A to II-27A "-248 to "-278
A B
Entry R A:B % Yield
1 Ph 75 : 25 90
"-1 II-6A : "-68
2 4-MeO-Ph 74 : 26 88
“-20 ll-24A : "-243
3 4-CF3-Ph 67 : 33 81
"-21 II-25A : "-253
4 Et 43 : 57 50
"-22 II-26A : "-268
5 Bn 52 : 48 58
"-23 ll-27A : ll-27B

 

65

Table "-5. Alkylation/reduction reaction with various oxazol-5(4H)-ones.

0(1)
(ll/,5?
, 0(4)
$5 0(5)
, 0(2) ‘10),
(2;: 0(3)
"J 5\ 0(2)
4’1“ 0(11) 2“ ‘9“ \ («I/r)
‘ 0l1l ‘ «d zllr.‘ ‘.
~ - 0(4) x.-
/\\\ ”K
. (10) (443), 4:121! AIR Nlll cm ’5
(9)14. 510(5) . My 0(8)
'1“ ’ ~ .
0(13) 5‘5} 545' . , _ 0(9)
» 0(5) 1 “‘35" ’,
1; 0(3) Q
{$4 0(12) . .

Figure "-4. X-Ray crystal structure of "-268.

H. Mechanistic investigation.

The decrease in both stereoselectivity and yield in the reactions involving 2-alkyl-
oxazol-5(4H)-ones as compared to those involving 2-aryI-oxazol-5(4H)-ones may
potentially be explained by a difference in mechanism. To investigate this
phenomenon, we treated both 2-ethyl-4-carbmethoxy-5(4H)-oxazolone "-22 and
2-phenyI-4-carbmethoxy-5(4H)-oxazolone "-1 with tert-butyl vinyl ether "-5 in the
absence of catalyst (Scheme "-8). Analysis of the crude reaction mixtures
revealed an O-alkylated oxazole intermediate "-28 in the reaction involving "-22,
while the reaction of "-1 produced only C-alkylated product. Upon standing, we

observed the conversion of the O-alkylated intermediate to the C-alkylated

66

product along with some degradation of the enol ether component. This

observation infers that these ene-type reactions might proceed through an O to C

46.62.63

migration, although this was never observed for any of the 2-aryl

substituted oxazol-5(4H)-ones.

—

t _
EtYO =/O BU Et 0 >—OtBU Et 0
l o "-5 91’ o 91’ o
N -——- N / ——- N 013,,
COzMe CO Me MeOZC
"-23 "-28
_Observed IntermediateJ
O‘Bu ( ‘
Ph 0 =/ Ph t Ph
Y Q "-5 Y0 (>50 Bu Y0 O
COzMe cone MeOzC
"-1 _Not detected by NMR_

 

 

Scheme "-8. Observation of O-alkylation intermediate in the alkyation reaction.

To provide additional insight into the nature of the mechanism, we
conducted a deuterium study wherein oxazol-5(4H)-one "-1 was treated with
deuterium labeled alkoxy alkyne “-29 (Scheme "-9). The reaction resulted in a
1:1 mixture of diastereomers, indicating that these alkylation reactions may also
proceed via a step-wise mechanism. Furthermore, treatment of "-30 with excess
"-1 did not result in a loss of deuterium incorporation, demonstrating that the
acidic oxazol-5(4H)-one itself does not promote degradation of reaction
diastereoslectivity by isomerizing the enol ether generated in "-30. The results of
these deuterium-labeling studies are in agreement with studies previously
performed by Dr. Fisk, which also suggested the possibility of a stepwise
mechanism.

67

Ph D—E‘OB“ Ph

Y0 o "-29 _ g Y0 o
“If CHZCIZ, rt, 96% yield N OBu
COzMe Benzene, rt, 76% yield MeOzC l
"-1
"-30 D
1:1 mixture of

diastereomers

 

Scheme "-9. Oxazol-5(4H)-one alkylation using a deuterated alkoxy alkyne.

Supported by the significant rate enhancement found using only Bronsted
acids, we hypothesize that the acidic proton of the oxazol-5(4H)-one‘54 in the
absence of catalyst, or in this case the catalyst itself, protonates the enol ether,
forming an oxonium ion. However, coordination of the catalyst to the oxazolone
substrate, as typically seen with oxazol-5(4H)-ones, cannot be dismissed."“"65
Therefore, it is likely that the mechanistic nature of these alkylation reactions is
highly dependent on both the oxazol-5(4H)-one and enol ether being used in the

reaction.

I. Enantioselective ene-type alkylation reaction of oxazoI-5-(4H)-ones.

More recently, an asymmetric version of the intermolecular ene-type alkylation
reaction of oxazol-5(4H)-ones and enol ethers has been reported by Terada and
co-workers.66 Their work involves enol ether protonation by a chiral Bronsted
acid catalyst to generate a reactive oxocarbenium ion which is proposed to form
a hydrogen-bonding complex with the chiral conjugate base (Scheme ll-10).
Steric interactions between the resultant bulky chiral complex and the

nucleophilic oxazoI-5(4H)-one promote stereoselective alkylation that can only

68

occur when the reagents adopt appropriate orientations. Thus, treatment of

C2,C4-diaryl oxazol-5(4H)-ones with tert-butyl vinyl ether and a chiral phosphoric

acid followed by subsequent methanolysis afforded quaternary o-amino acid

derivatives in good yields with excellent diastereoselectivities and

enantioselectivities.6‘5

 

 

 

 

 

 

 

 

 

 

 

r _
58 9?,
R2’H\| catalyst > OR; 043'
0R1 H .... 1‘9 0 or G ’9
r 4? H H
CO R 3 R2 ' R2 '
‘ g e .
Opp l — 5) R1 5 R1 _
g g o‘OH : O O
i
R : j: />‘AH
R = 2,4,6—(i—Pr)3CGH2- ; A,2 N
catalyst l l)
OR1O OR1O 6:39
. :0
R2 OMe +R2 ., OMe MeONa ,o‘e A‘H
Arz NH Arz A MeOH H” "‘H 63 o
Ar1 o Ar1 o HA I N/>—Ar1
Major Minor _ R2 %’R1 Ar2 _
up to 99% yield

up to 99% ee, up to 96% de
Scheme "-10. Terada's enantioselective quaternary d-amino acid synthesis.

The enantioselective reaction reported by Terada and co-workers is an
excellent extension of work which was previously completed in the Tepe lab.
Undoubtedly, future studies involving alkylation reactions of oxazol-5(4H)-ones

performed in the Tepe lab will make use of chiral Bronsted acids, such as those

69

utilized by Terada, for the stereoselective preparation of densely functionalized

amino acid derivatives.

J. Experimental.

1. General information. Reactions were carried out in flame-dried glassware
under nitrogen atmosphere. All reactions were magnetically stirred and
monitored by TLC with 0.25 pm pre-coated silica gel plates using either UV light
or iodine to visualize the compounds. Column chromatography was carried out
on Silica Gel 60 (230-400 mesh) supplied by EM Science. Yields refer to
chromatographically and spectroscopically pure compounds unless otherwise
noted. Infrared spectra were recorded on a Nicolet lR/42 spectrometer. 1H and
13C NMR spectra were recorded on a Varian Unity Plus-500 spectrometer.
Chemical shifts are reported relative to the residual peaks of the solvent. The
following abbreviations are used to denote the multiplicities: s = singlet, d =
doublet, t = triplet, q = quartet, p = pentet, and m = multiplet. Diastereomeric
ratios were determined using the integration values obtained from 1H NMR. Gas
chromatography / low resolution mass spectra were recorded on a Hewlet-
Packard 5890 Series II gas chromatograph connected to a TRIO-1 EI mass
spectrometer. HRMS were obtained at the Mass Spectrometry Facility of
Michigan State University with a JEOL JMS HX-11O mass spectrometer.
Elemental analysis data were obtained on a Perkin Elmer 2400 Series ll CHNS/O
analyzer. Melting points were obtained using an Electrotherrnal® capillary

melting point apparatus and are uncorrected.

70

2. Materials. Reagents and solvents were purchased from commercial suppliers
and used without further purification. Anhydrous methylene chloride, benzene,
acetonitrile, and tetrahydrofuran were dispensed from a delivery system which
passes the solvents through a column packed with dry neutral alumina. Ethyl
vinyl ether "-7, teIt-butyl vinyl ether "-5, 2-methoxypropene Il-12, benzyl alcohol,
mercuric acetate, trifluoroacetic anhydride, sodium borohydride, benzylamine,
diphenyl phosphate, camphor sulfonic acid, trifluoroacetic acid, and 3,5-
dinitrobenzoic acid were all purchased from Sigma Aldrich, checked for purity,

and used without further purification.

3. Synthesis and characterization of oxazol-5-(4H)-one precursors. Oxazol-
5-(4H)-ones used in these studies were prepared in three steps starting from
commercially available dimethylaminomalonate HCI. The steps consist of
amidation of dimethylaminomalonate HCI, followed by monoester hydrolysis and
finally dehydration to give the desired oxazol-5-(4H)-one products. Additionally,
carboxylic acids . 2-benzamido-3-methoxy—3-oxopropanoic acid67 and 2-
(methoxycarbonyl)-2-(propionamido)acetic acid, the starting materials used to
synthesize "-1 and "-22, respectively, were prepared and characterized by Dr.
Jason S. Fisk. For further details regarding either the synthesis or
characterization of 2-benzamido-3-methoxy-3-oxopropanoic acid or 2-
(methoxycarbonyl)-2-(propionamido)acetic acid, please see the Ph.D.

dissertation of Jason S. Fisk.

71

o COzMe
N COzMe
\ H
O
"-31 '

Figure "-5. Dimethyl 2-(4-methoxybenzamido)malonate (II-31).

Dimethyl 2-(4-methoxybenzamido)ma|onate (ll-31).68 A suspension of
dimethylaminomalonate HCI (6.0435 g, 32.9 mmol) and TEA (4.60 mL, 32.9
mmol) in CH2C|2 (200 mL) stirred at room temperature for 45 minutes before
being cooled to 0 °C in an ice bath. The reaction was successively treated with
4-methoxybenzoic acid (5.0259 g, 33.1 mmol), DMAP (0.4060 g, 3.32 mmol),
and DCC (6.9428 g, 33.6 mmol), and the reaction stirred overnight while warming
to room temperature. The reaction was filtered, and the filtrate was concentrated
and subsequently diluted in THF to form a precipitate. The solid was filtered, and
the filtrate was again concentrated. This step was repeated until no additional
precipitate was formed upon dilution of the concentrated filtrate in THF. The
concentrated filtrate was then diluted in CH2C|2 and was washed successively
with 5% aqueous HCI solution, saturated NaHC03 solution, and brine before
being dried (Na2804) and concentrated. Purification via crystallization from
EtOAc and hexanes afforded 7.3 g of the title compound68 (79% yield) as a fluffy
crystalline solid (mp. = 100 — 101 °C). 1H NMR (500 MHz) (CDCI3): 6 3.827 and
3.831 (23, 9 H), 5.38 (d, J = 6.9 Hz, 1 H), 6.92 (d, J = 8.9 Hz, 2 H), 7.09 (d, J =
6.8 Hz, 1 H), 7.80 (d, J = 9.0 Hz, 2 H). 13C NMR (125 MHz) (CDCI3) 6: 53.4, 55.3,

56.4, 113.7, 125.0, 129.1, 162.7, 166.2, 166.9. IR (neat): 3327 cm", 1746 cm",

72

1610 cm", 1505 cm", 1254 cm", 1180 cm". HRMS (FAB): m/z calcd for
C13H15N05 [M+H], 282.0978; found, 282.0983.

0 COzMe
x
n COZH

\
O
"-32

Figure "-6. 3-methoxy-2-(4-methoxybenzamido)-3—oxopropanoic acid (II-32).

3-methoxy-2-(4-methoxybenzamido)-3-oxopropanoic acid (ll-32). To a
stirring solution of "-31 (2.0280 g, 7.21 mmol) in MeOH (60 mL) cooled to 0 °C in
an ice bath was added dropwise a solution of LiOH monohydrate (0.3034 g, 7.23
mmol) in H20 (60 mL). Once all LiOH had been added, the reaction warmed to
room temperature and stirred under N2 atmosphere for the requisite amount of
time, as monitored by TLC. The reaction was concentrated under vacuum to
remove most of the alcohol, and the remaining solution was acidified with
concentrated HCI. The aqueous layer was extracted with EtOAC (4 x 40 mL),
and the combined organic fractions were washed with brine before being dried
(NaZSO4) and concentrated under vacuum. Purification via crystallization from
EtOAc and hexanes afforded 1.6233 g of the title compound (84% yield) as a
crystalline solid (mp. = 106 - 107 °C). 1H NMR (500 MHz) ((CD3)ZSO): 6 3.73
(s, 3 H), 3.81 (s, 3 H), 5.27 (d, J = 7.6 Hz, 1 H), 7.01 (d, J = 8.7 Hz, 2 H), 7.92 (d,
J = 8.6 Hz, 2 H), 9.00 (d, J = 7.6 Hz, 1 H), 13.46 (broad s, 1 H). 13C NMR (125
MHz) ((CD3)2SO) 6: 52.6, 55.4, 56.6, 113.6, 125.3, 129.7, 162.1, 165.9, 167.8,
167.9. IR (neat): 3432 cm", 3347 cm", 1723 cm", 1608 cm", 1504 cm", 1250

cm". HRMS (FAB): m/z calcd for C12H14N06 [M+H], 268.0821; found, 268.0826.
73

o COzMe
N COzMe
H

"-33

Figure "-7. Dimethyl 2-(4-(trifluoromethyl)benzamido)malonate (ll-33).

Dimethyl 2-(4-(trifluoromethyl)benzamido)malonate (ll-33).68 Following the
procedure used for the synthesis of "-31, a 0 °C suspension of
dimethylaminomalonate HCI (2.0198 g, 11.0 mmol) and TEA (1.60 mL, 11.5
mmol) in CH2C|2 (140 mL) was successively treated with 4-
(trifluoromethyl)benzoic acid (2.0918 g, 11.0 mmol), DMAP (0.1371 g, 1.12
mmol), and DCC (2.2899 g, 11.1 mmol). Purification via column chromatography
on silica gel (6% ether / 94% CHZClz) afforded 3.0165 g of the title compound
(86% yield) as a white solid (mp. = 132 - 134 °C). 1H NMR (500 MHz) (CDCI3):
6 3.48 (s, 6 H), 5.02 (d, J = 6.9 Hz, 1 H), 6.88 (d, J = 6.7 Hz, 1 H), 7.34 (d, J = 8.2
Hz, 2 H), 7.58 (d, J = 8.1 Hz, 2 H). 13C NMR (125 MHz) (CDCI3) 6: 53.6, 56.5,
123.5 (q, J = 273 Hz), 125.7 (q, J = 4 Hz), 127.8, 133.8 (q, J = 33 Hz), 136.1,
165.6, 166.5. IR (neat): 3316 cm", 1743 cm", 1639 cm", 1530 cm", 1330 cm",
1165 cm", 1129 cm". HRMS (FAB): m/z calcd for cmHmr-gmo5 [M+H],

320.0746; found, 320.0751.

74

o cozrvre
J\
N COZH
H
F3C
"-34

Figure "-8. 3-methoxy-3-oxo-2-(4-(trifluoromethyl)benzamido)propanoic
acid (ll-34).

3-methoxy-3-oxo-2-(4-(trifluoromethyl)benzamido)propanoic acid (II-34).
Following the procedure used for the synthesis of “-32, a solution of "-33 (1.5862
g, 4.97 mmol) in MeOH (60 mL) was treated with a solution of LiOH monohydrate
(0.2084 g, 4.97 mmol) in H20 (60 mL). Purification via column chromatography
on silica gel (50% EtOAc / 50% hexanes —+ EtOAc —+ 10% MeOH / 90% EtOAc)
followed by recrystallization from hot benzene afforded 0.9357 g of the title
compound (62% yield) as a white solid (mp. = 145 — 146 °C). 1H NMR (500
MHz) ((CD3)ZSO): 6 3.73 (s, 3 H), 5.27 (d, J = 7.6 Hz, 1 H), 7.86 (d, J = 8.1 Hz, 2
H), 8.10 (d, J = 8.1 Hz, 2 H), 9.48 (d, J = 7.5 Hz, 1 H), 13.58 (broad s, 1 H). 13‘C
NMR (125 MHz) ((CD3)ZSO) 6: 52.7, 56.6, 123.9 (q, J = 273 Hz), 125.4 (q, J = 4
Hz), 127.1, 128.6, 131.6 (q, J = 32 Hz), 165.4, 167.36, 167.44. IR (neat): 3432
cm", 3347 cm", 3304 cm", 1718 cm’1, 1626 cm", 1530 cm", 1330 cm“, 1275
cm". HRMS (FAB): m/z calcd for C12H11F3N05 [M+H], 306.0589; found,
306.0601.

0 iOZMe
PMAn COZMe

"-35

Figure "-9. Dimethyl 2-(2-phenylacetamido)malonate (ll-35).

75

Dimethyl 2-(2-phenylacetamido)malonate (ll-35).67 To a stirring suspension of
dimethyl aminomalonate HCI (1.4085 g, 7.67 mmol) and TEA (2.15 mL, 15.4
mmol) in CH2CI2 (150 mL), cooled to 0 °C in an ice bath, was added phenylacetyl
chloride (1.65 mL, 12.5 mmol) and the reaction stirred overnight while warming to
room temperature. The reaction was washed successively with saturated
NaHC03 solution (x2), 5% HCI solution (x2), and brine, before being dried
(Na2804) and concentrated under vacuum. Purification via crystallization from
EtOAc and hexanes afforded 1.3628 g of the title compound (70% yield) as a
white fine crystalline solid (mp. = 85 - 86 °C). 1H NMR (500 MHz) (CDCI3): 6
3.66 (s, 2 H), 3.79 (s, 6 H), 5.19 (d, J = 6.9 Hz, 1 H), 6.45 (d, J = 6.0 Hz, 1 H),
7.26 — 7.40 (m, 5 H). 13c NMR (125 MHz) (CDCI3) 5: 43.1, 53.4, 56.2, 127.5,
129.0, 129.4, 134.0, 166.5, 170.6. IR (neat): 3316 cm", 1755 cm", 1743 cm“,
1645 cm", 1530 cm", 1183 cm". HRMS (FAB): m/z calcd for C13H16N05 [M+H],
266.1028; found, 266.1030.

0 002MB
PhJfiXCOZH

"-36

Figure "-10. 3-methoxy-3-oxo-2-(2-phenylacetamido)propanoic acid (II-36).

3-methoxy-3-oxo-2—(2-phenylacetamido)propanoic acid (ll-36).67 Following
the procedure used for the synthesis of "-32, a solution of “-35 (1.0080 g, 3.80
mmol) in MeOH (50 mL) was treated with a solution of Li0H monohydrate
(0.1602 g, 3.82 mmol) in H20 (50 mL). Purification via crystallization from hot

EtOAc and hexanes afforded 0.7843 g of the title compound (79% yield) as a

76

white crystalline solid (mp. = 132 — 133 °C). 1H NMR (500 MHz) ((CD3)2SO): 6
3.57 (s, 2 H), 3.68 (s, 3 H), 5.01 (d, J = 7.4 Hz, 1 H), 7.18 - 7.30 (m, 5 H), 8.87
(d, J = 7.4 Hz, 1 H), 13.54 (broad s, 1 H). 13c NMR (125 MHz) ((003)280) 5:
41.3, 52.6, 56.3, 126.4, 128.2, 129.0, 135.9, 167.47, 167.50, 170.4. IR (neat):
3341 cm", 3286 cm", 1755 cm", 1627 cm“, 1530 cm", 1232 cm". HRMS

(FAB): m/Z calcd for C12H14N05 [M+H], 252.0872; found, 252.0873.

4. General procedure for synthesis of oxazol-5(4H)-ones. 0xazol-5(4H)-ones

697° A suspension of

were synthesized using a known literature procedure.
carboxylic acid in anhydrous diethyl ether was treated with TFAA (2.2 eq)
dropwise and then stirred for 1.5 hours. The reaction was then cooled to 0°C and
water (1.1 eq) was added dropwise, after which the ice bath was removed and
the reaction stirred for an additional 1.5 hours. The reaction was cooled in a
freezer for 30 minutes causing the oxazol-5(4H)-one product to precipitate out of
solution which was collected via filtration and washed with cold diethyl ether.
The oxazoI-5(4H)-ones were then analyzed and used without further purification.
On occasion full characterization of oxazol-5(4H)-ones could not be completed
due to high moisture sensitivity of the compounds. Additionally for certain
oxazoI-5(4H)-ones carbon peaks may be hidden underneath the deuterated
solvent peaks. Using this general procedure "-1 was prepared and

characterized by Dr. Jason S. Fisk. For further details regarding either the

synthesis or characterization of "-1, please see the Ph.D. dissertation of Jason

77

S. Fisk or the supporting information of the publication in which this compound

was described .56

Figure "-11. 2-(4-methoxyphenyI)-4-carbmethoxy-5(4H)-oxazolone (II-20).

2-(4-methoxyphenyl)-4-carbmethoxy-5(4H)-oxazolone (II-20). Using the
general procedure, TFAA (0.68 g, 3.2 mmol) was added to a suspension of "-32
(0.40 g, 1.5 mmol) in diethyl ether (10 mL), and water (0.03 g, 2 mmol) was
added once the reaction had been cooled. Collection of the precipitate afforded
0.30 g of title compound (m.p. = 181 — 183 °C, 79% yield). The title compound
was used immediately without further purification in the preparation of "-24. 1H
NMR (500 MHz) (2:1 CDCI3:C505N): 6 3.74 (s, 3H), 3.90 (s, 3H), 6.98 - 7.02 (m,
2H), 8.08 - 8.12 (m, 2H), 9.26 (s, 1H). 13c NMR + DEPT (125 MHz) (2:1
CDCI3:C5D5N): 6 48.1 (—CH3), 53.1 (—CH3), 112.3 (aromatic -CH), 119.7
(quaternary —C), 124.9 (aromatic —CH), 144.4 (quaternary —C), 158.4 (quaternary
-C), 163.4 (quaternary —C), 166.2 (quaternary —C). IR (neat): 1779 cm", 1632
cm", 1514 cm", 1267 cm", 1113 cm". HRMS (FAB): m/z calcd for C12H12N05

[M+H], 250.0714; found, 250.0715.

78

002 Me
"-21

Figure "-12. 2-(4-trifluoromethylphenyl)-4-carbmethoxy-5(4H)-oxazolone (ll-21).

2-(4-trifluoromethylphenyl)-4-carbmethoxy-5(4H)-oxazolone (ll-21). Using the
general procedure, TFAA (0.71 g, 3.4 mmol) was added to a suspension of "-34
(0.47 g, 1.6 mmol) in diethyl ether (10 mL), and water (0.03 g, 2 mmol) was
added once the reaction had been cooled. Collection of the precipitate afforded
0.44 g of the title compound (solid, mp. = 192 - 194 °C, 97% yield). The title
compound was used immediately without further purification in the preparation of
"-25. 1H NMR (500 MHz) (2:1 CDCI3:C5D5N): 6 3.90 (s, 3H), 7.66 (d, J = 8.2 Hz,
2H), 8.19 (d, J = 8.1 Hz, 2H), 13.40 (broad s, 1H). 13C NMR + DEPT (125 MHz)
(2:1 CDCI3:C5D5N): 6 48.4 (—CH3), 98.5 (quaternary -C), 122.7 (quaternary —CF3,
q, J = 272 Hz), 123.1 (aromatic —CH), 123.6 (aromatic -CH, q, J = 4 Hz), 127.0
(quaternary -C, q, J = 32 Hz), 130.8 (quaternary —C), 142.5 (quaternary —C),
163.9 (quaternary —C), 167.1 (quaternary —C). IR (neat): 1796 cm", 1763 cm",
1644 cm", 1613 cm", 1337 cm", 1200 cm", 1121 cm“. HRMS (FAB): m/z

calcd for C12H9F3NO4 [M+H], 288.0485; found, 288.0484.

79

Et
r" o

002MG
"-22

Figure "-13. 2-ethyl-4-carbmethoxy-5(4H)-oxazolone (ll-22).

2-ethyl-4-carbmethoxy-5(4H)-oxazolone (ll-22). Using the general procedure,
TFAA (0.38 g, 1.80 mmol) was added to a suspension of 2-(methoxycarbonyl)-2-
(propionamido)acetic acid (0.15 g, 0.80 mmol) in diethyl ether (3 mL), and water
(16.5 mg, 0.92 mmol) was added once the reaction had been cooled. Collection
of the precipitate afforded 0.11 g of the title compound (80% yield). The title
compound is moisture sensitive and fairly unstable only allowing for partial
characterization. The title compound was used immediately without further
purification in the preparation of "-26. 1H NMR (500 MHz) (2:1 CDCI32C5D5N): 6
1.24 (t, J = 7.6 Hz, 3 H), 2.67 (q, J = 7.6 Hz, 2 H), 3.82 (s, 3 H), 15.83 (s, 1 H).
13C NMR + DEPT (125 MHz) (2:1 CDCI3:C5D5N): 6 8.5 (—CH3), 19.8 (—CH2), 48.0
(—CH3), 161.8 (quaternary —C). IR (KBr pellet): 3040 cm", 1789 cm", 1629 cm“,
1495 cm", 1269 cm“.

1° 0

COzMe
"-23

Figure "-14. 2-benzyl-4-carbmethoxy-5(4H)-oxazolone (ll-23).

2-benzyl-4-carbmethoxy-5(4H)-oxazolone (II-23). Using the general

procedure, TFAA (0.32 g, 3.45 mmol) was added to a suspension of "-36 (0.39
80

g, 1.6 mmol) in ether (6 mL), and water (0.03 g, 2 mmol) was added once the
reaction had been cooled. Collection of the precipitate afforded 0.28 g of the title
compound (75% yield). The title compound is moisture sensitive and fairly
unstable only allowing for partial characterization. The title compound was used
immediately without further purification in the preparation of "-27. 1H NMR (500
MHz) (2:1 CDCI32C5D5N): 6 3.82 (s, 3H), 4.00 (s, 2H), 7.18—7.22 (m, 1H), 7.24-
7.40 (m, 2H), 7.39-7.41 (m, 2H), 10.65 (broad s, 1H). 13C NMR (125 MHz)
((CD3)2SO): 6 32.8, 50.2, 127.6, 128.1, 128.8, 128.9, 129.0. IR (neat): 1784

cm", 1657 cm", 1501 cm“, 1456 cm", 1391 cm", 1206 cm".

5. Synthesis of enol ethers and alkynyl ethers. The following enol ethers
used in these studies were purchased and used as received: "-5 (ten-butyl vinyl
ether), "-7 (ethyl vinyl ether), "-8 (3,4-dihydro-(2H)-pyran), and "-12 (2-
methoxypropene). Vinyl ethers 1-((prop-1-enyloxy)methy|)benzene (ll-9)”72 and
1-methoxy-2-methylprop-1-ene (ll-10),73 as well as alkynyl ethers 1-butoxyethyne
(ll-11)”75 and 2-deutero-1-butoxyethyne (ll-30) were prepared by Dr. Jason S.
Fisk according to literature procedures. For further details regarding either the
synthesis or characterization of these compounds, please see the Ph.D.
dissertation of Jason S. Fisk or the supporting information of the publication in
which the compounds were described.56 The remaining enol ether used in these

studies, benzyl vinyl ether "-2, was prepared as follows:

81

OBn
=/

"-2

Figure "-15. Benzyl vinyl ether (ll-2).

Benzyl vinyl ether (ll-2). This compound was prepared according to a literature
procedure.76'77 A solution of benzyl alcohol (5.0 mL, 48 mmol) and Hg(0Ac)2
(0.557 g, 1.75 mmol) in ethyl vinyl ether (45.0 mL, 470 mmol) was prepared and
stirred under reflux for 16 hours before an additional portion of Hg(0Ac)2 (0.264
g, 0.828 mmol) was added. The reaction stirred under reflux for an additional 8
hours, after which it was concentrated in vacuo. The resulting crude oil was
eluted through a silica gel plug with hexanes, and the resultant eluent was
concentrated under vacuum to give the product as an oil. The spectral data
matches that previously reported in the literature.77 1H NMR (500 MHz) (CDCl3):
6 4.01 (dd, J1 = 2.1 Hz, J2 = 6.8 Hz, 1 H), 4.24 (dd, J1 = 2.1 Hz, J2 = 14.4 Hz, 1
H), 4.67 (s, 2 H), 6.49 (dd, J1 = 6.8 Hz, J2 = 14.3 Hz, 1 H), 7.19 — 7.33 (m, 5 H).

130 NMR (125 MHz) (c0013): 5 70.0, 87.3, 127.5, 127.9, 128.5, 136.8, 151.6.

6. Synthesis of diamides.

H QBn H OBn
Bz’N,.. + Bz’N,.
Me02C NHBn Me02C NHBn
0 0
ll-3A "-38

Figure "-16. Methyl 2-benzamido-2-(benzylcarbamoyl)-3-
(benzyloxy)butanoate (ll-3).

82

Methyl 2-benzamido-2-(benzylcarbamoyl)-3-(benzyloxy)butanoate (ll-3). A
suspension of "-2 (1.72 g, 9.10 mmol) and "-1 (1.0 g, 4.6 mmol) in CH2Cl2 (125
mL) stirred under reflux for 21 hours, after which the reaction was concentrated
to about half volume in vacuo. Benzylamine (1.80 mL, 16.5 mmol) was added
and the reaction stirred at room temperature under nitrogen atmosphere for 7
hours. The reaction was concentrated under vacuum and the resulting mixture
was purified via column chromatography on silica gel (nonlinear gradient from
20% EtOAc / 80% hexanes to 40% Et0Ac / 60% hexanes) to afford 1.76 g (84%
yield) of the title compound as a mixture of diastereomers. The relative
stereochemistry of each diastereomer was determined through analysis of the
1H-NMR spectrum obtained from aminolysis (with benzylamine) of a 3 : 1 mixture
of quaternary oxazol-5(4H)—one diastereomers obtained from the diphenyl
phospate-catalyzed reaction of "-1 with "-2. II-3A (solid, mp. = 98 — 100 °C): 1H
NMR (500 MHz) (CDCI3): 6 1.23 (d, J = 6.4 Hz, 3 H), 3.75 (s, 3 H), 4.41 (dd, J1 =
5.5 Hz, J2 = 14.9 Hz, 1 H), 4.52 (dd, J1 = 6.0 Hz, J2 = 14.9 Hz, 1 H), 4.69 (d, J =
10.8 Hz, 1 H), 4.87 — 4.92 (m, 2 H), 7.17 — 7.20 (m, 2 H), 7.25 — 7.33 (m, 8 H),
7.44 (t, J = 7.6 Hz, 2 H), 7.52 (t, J = 7.4 Hz, 1 H), 7.77 (t, J = 5.7 Hz, 1 H), 7.89
(d, J = 7.1 Hz, 2 H), 8.18 (s, 1 H). 13C NMR (125 MHz) (CDCI3) 6: 15.2, 43.9.
52.6, 67.4, 72.9, 74.9, 127.0, 127.1, 127.2, 127.5, 127.7, 128.2, 128.3, 131.7,
133.1, 137.1, 137.7, 165.3, 166.0, 168.4. IR (KBr pellet): 3358 cm", 3312 cm“,
1725 cm", 1649 cm", 1551 cm", 1532 cm", 1252 cm". MS (GCMS): m/z calcd
for C27H28N205 [M], 460.20; found, 460.3. Anal. Calcd. For C27H28N205: C,

70.42; H, 6.13; N, 6.08. Found: C, 70.40; H, 6.21; N, 6.06. "-33 (solid, mp. =

83

148 - 149 °C): 1H NMR (500 MHz) (coma): 5 1.43 (d, J = 6.3 Hz, 3 H), 3.74 (s, 3
H), 4.42 (d, J = 11.7 Hz, 1 H), 4.45 (q, J = 6.3 Hz, 1 H), 4.49 (dd, J1 = 5.6 Hz, J2 =
15.0 Hz, 1 H), 4.55 (dd, J1 = 5.8 Hz, J2 = 14.9 Hz, 1 H), 4.67 (d, J = 11.7 Hz, 1
H), 7.24 — 7.37 (m, 11 H), 7.44 (t, J = 7.6 Hz, 2 H), 7.53 (t, J = 7.4 Hz, 1 H), 7.81
- 7.84 (m, 2 H), 7.89 (t, J = 5.6 Hz, 1 H). 130 NMR (125 MHz) (coc13) 5: 15.2,
43.8, 53.2, 69.9, 71.5, 77.6, 127.22, 127.23,127.5, 127.6, 127.8, 128.40, 128.45,
128.49, 131.9, 133.4, 137.4, 137.9, 165.3, 167.5, 170.6. IR (KBr pellet): 3330
cm", 3276 cm", 1730 cm", 1661 cm", 1645 cm", 1532 cm", 1235 cm", 1132
cm". MS (GCMS): m/z calcd for C27H23N205 [M*], 460.20; found, 460.3. Anal.

Calcd. For C27H23N205: C, 70.42; H, 6.13; N, 6.08. Found: C, 70.13; H, 6.19; N,

6.06.
H QBn H OBn
Bz’N2 + Bz’N,..
MGOzc NH2 M6020 NH2
0 O
ll-4A "-43

Figure "-17. Methyl 2-benzamido-3-(benzyloxy)-2-carbamoylbutanoate (ll-4).

Methyl 2-benzamido-3-(benzyloxy)-2-carbamoylbutanoate (ll-4). A
suspension of "-2 (0.449 g, 2.38 mmol) and "-1 (0.400 g, 1.82 mmol) in CH2CI2
(100 mL) stirred under reflux for 24 hours, after which the reaction was
concentrated to approximately 25 mL in vacuo. The reaction was cooled to -78
°C and excess liquid NH3 was added. The reaction stirred overnight as it slowly
warmed to room temperature. The reaction was concentrated under vacuum and

the resulting mixture was purified via column chromatography on silica gel (55%

84

EtOAc / 45% hexanes) to afford 0.620 g (92% yield) of the title compound as a
mixture of diastereomers. The relative stereochemistry of each diastereomer
was determined through analysis of the 1H-NMR spectrum obtained from
aminolysis (with ammonia) of a 3 : 1 mixture of quaternary oxazol-5(4H)-one
diastereomers obtained from the diphenyl phosphate-catalyzed reaction of "-1
with "-2. ll-4A (solid, mp. = 64 - 66 °C): 1H NMR (500 MHz) (CDCI3): 6 1.24 (d,
J = 6.4 Hz, 3 H), 3.78 (s, 3 H), 4.71 (d, J = 10.7 Hz, 1 H), 4.84 (q, J = 6.4 Hz, 1
H), 4.89 (d, J = 10.7 Hz, 1 H), 5.72 (broad s, 1 H), 7.27 — 7.56 (m, 9 H), 7.82 —
7.85 (m, 2 H), 7.94 (s, 1 H). 13C NMR (125 MHz) (CDCI3) 6: 15.3, 53.0, 67.9,
73.3, 74.9, 127.2, 128.0, 128.2, 128.5, 128.6, 132.0, 133.3, 137.9, 166.4, 167.6,
168.4. IR (KBr pellet): 3357 cm“, 3295 cm", 3170 cm", 1722 cm", 1691 cm“,
1660 cm",1543 cm", 1250 cm", 1129 cm". MS (GCMS): m/z calcd for
C20H23N205 [M + H], 371.16; found: 371.2. Anal. Calcd. For C2oH22N205: C,
64.85; H, 5.99; N, 7.56; found: C, 64.56; H, 5.79; N, 7.67. "-43 (solid, mp. = 102
- 104 °C): 1H NMR (500 MHz) (CDCl3): 6 1.45 (d, J = 6.3 Hz, 3 H), 3.77 (s, 3 H),
4.40 (q, J = 6.3 Hz, 1 H), 4.47 (d, J = 11.7 Hz, 1 H), 4.70 (d, J = 11.7 Hz, 1 H),
5.58 (broad s, 1 H), 7.12 (s, 1 H), 7.30 - 7.56 (m, 8 H), 7.81 - 7.84 (m, 2 H). 13C
NMR (125 MHz) (CDCI3) 6: 15.1, 53.2, 69.8, 71.5, 77.4, 127.2, 127.6, 127.7,
128.3, 128.4, 131.8, 133.3, 137.4, 167.4, 167.6, 170.5. IR (KBr pellet): 3399 cm'
1, 3267 cm", 3214 cm", 1728 cm", 1677 cm", 1657 cm",1529 cm", 1292 cm".
MS (GCMS): m/z calcd for C2oH23N205 [M + H], 371.16; found, 371.4. Anal.
Calcd. For C2oH22N205: C, 64.85; H, 5.99; N, 7.56. Found: C, 64.47; H, 5.74; N,

7.72.

85

7. General procedure for the synthesis of ten-alkyl amino hydroxy
carboxylic esters. To a stirring suspension of oxazol-5(4H)-one (0.5 mmol) in
20 mL of solvent were successively added enol ether (0.75 mmol) and diphenyl
phosphate (0.05 mmol), and the reaction stirred at room temperature under
nitrogen atmosphere for the requisite amount of time as monitored by TLC. The
reaction was washed successively with saturated NaHC03 solution and brine
before being dried over M9804 and concentrated in vacuo. The crude reaction
was diluted in THF and cooled to 0 °C before NaBH4 (1 mmol) and cold H20
were added. The reaction stirred at 0 °C until complete by TLC. Saturated
NH4CI solution was added and the organic layer was extracted with CH2Cl2 (x3).
The combined organic extractions were dried (M9804) and concentrated. The
crude reaction mixtures were purified via column chromatography on silica gel
(diethyl ether / CH2CI2).

Using the general procedure, tert-alkyl amino hydroxy carboxylic esters ll-
15, "-16, and "-17 were all prepared and fully characterized by Jason S. Fisk.
Additionally, oxazol-5(4H)-one products "-28 and "-30 were prepared and
characterized by Jason S. Fisk. For further details regarding either the synthesis
or characterization of these compounds, please see the Ph.D. dissertation of
Jason S. Fisk or the supporting information of the publication these compounds

were described.56

86

H QtBU H OtBU
’ + N

Bz’N ,- Bz’ ,.
M602C OH M3020 OH
II-6A "-63

Figure "-18. Methyl-2-benzamido-3-tert-butoxy-2-
(hydroxymethyl)butanoate (ll-6).

Methyl-Z-benzamido-3-tert-butoxy-2-(hydroxymethyl)butanoate (ll-6). Using
the general procedure, a suspension of "-1 (0.10 g, 0.46 mmol), “-5 (69.1 mg,
0.69 mmol), and diphenyl phosphate (11.5 mg, 0.046 mmol) in anhydrous
benzene (20 mL) was stirred at room temperature for 3 hours. After washings,
the crude reaction intermediate was diluted in 3 mL THF and cooled to 0 °C
before NaBH4 (34.8 mg, 0.92 mmol) and water (1.5 mL) were added. Purification
via silica gel chromatography (7% ether / 93% CH2Cl2) afforded 0.14 g of the title
compound (90% yield) as a 3 : 1 ratio of diastereomers. Il-6A (solid, mp. = 83 —
85 °C.): 1H NMR (500 MHz) (CDCI3): 6 1.12 (s, 9H), 1.30 (d, J = 6.3 Hz, 3H),
3.79 (s, 3H), 3.95 (dd, J1 = 7.2 Hz, J2 = 11.6 Hz, 1H), 4.26 (dd, J1 = 6.3 Hz, J2 =
11.7 Hz, 1H), 4.37 (q, J = 6.3 Hz, 1H), 4.57 (t, J = 6.7 Hz, 1H (0-H)), 7.41-7.46
(m, 3H), 7.50 (t, J = 7.4 Hz, 1H), 7.79 (d, J = 7.7 Hz, 2H). 13c NMR + DEPT (125
MHz) (CDCI3) 6: 17.9 (-CH3), 28.6 (—CH3), 52.6 (-CH3), 64.1 (—CH2), 69.8 (-CH),
72.1 (quaternary -C), 75.2 (quaternary —C), 127.1 (aromatic -CH), 128.7
(aromatic -CH), 131.8 (aromatic —CH), 134.6 (aromatic quaternary —C), 169.4
(quaternary -C), 171.3 (quaternary -C). IR (neat): 3412 cm", 3320 cm'1, 1736
cm", 1653 cm", 1522 cm", 1487 cm“, 1234 cm". HRMS (FAB): m/z calcd for

C17H25N05 [M+H], 324.1811; found, 324.1823. Anal. Calcd. For C17H25N05: C,

87

63.14; H, 7.79; N, 4.33. Found: C, 63.04; H, 7.73; N, 4.55. "-63 (solid, mp. =
76 — 79 °C.): 1H NMR (500 MHz) (CDCI3): 6 1.13 (d, J = 6.2 Hz, 3H), 1.16 (s,
9H), 3.74 (s, 3H), 3.83 (t, J = 11.8 Hz, 1H), 3.95 (dd, J1 = 2.8 Hz, J2 = 11.8 Hz,
1H), 4.27 (q, J = 6.2 Hz, 1H), 5.71 (dd, J1 = 2.8 Hz, J2 = 11.6 Hz, 1H (0-H)), 7.26
(broad s, 1H), 7.42 - 7.54 (m, 3H), 7.79 (dd, J1 = 3.2 Hz, J2 = 5.3 Hz, 2H). 13‘C
NMR (125 MHz) + DEPT (CDCI3) 6: 19.1 (—CH3), 28.7 (—CH3), 52.7 (-CH3), 64.6
(—CH2), 68.6 (—CH), 70.0 (quaternary —C), 74.8 (quaternary -C), 127.0 (aromatic
—CH), 128.6 (aromatic —CH), 131.8 (aromatic -CH), 134.2 (aromatic quaternary -
C), 167.7 (quaternary —C), 171.2 (quaternary —C). IR (neat): 3405 cm", 3584 —
3156 cm", 1752 cm", 1669 cm", 1520 cm", 1487 cm", 1115 cm". HRMS

(FAB): m/z calcd for C17H25N05 [M+H], 324.1811; found, 324.1801.

H QB” H OBn
Bz’N g. + Bz’N \,
Me02C 0H Me02C 0H
lI-13A "-138

Figure "-19. Methyl-2-benzamido-3-benzyloxy-2-
(hydroxymethyl)butanoate (ll-13).

Methyl-2-benzamido-3-benzyloxy-2-(hydroxymethyl)butanoate (II-13): Using
the general procedure, a suspension of "-1 (0.21 g, 0.94 mmol), "-2 (0.19 g, 1.4
mmol), and diphenyl phosphate (24.1 mg, 0.096 mmol) in anhydrous benzene
(40 mL) was stirred at room temperature for 4 hours. After washings, the crude
reaction intermediate was diluted in 6 mL THF and cooled to 0 °C before NaBH4
(76.4 mg, 2.0 mmol) and water (3 mL) were added. Purification via silica gel

chromatography (17% ether I 83% CH2Cl2) afforded 0.26 g of the title compound

88

(77% yield) as a 3 : 1 ratio of diastereomers. ll-13A (solid, mp. = 96 - 98 °C.):
1H NMR (500 MHz) (CDCI3): 6 1.41 (d, J = 6.4 Hz, 3H), 3.84 (s, 3H), 4.05 (dd, J1
= 6.7 Hz, J2 = 11.8 Hz, 1H), 4.27 - 4.35 (m, 2H), 4.45 (d, J = 11.7 Hz, 1H), 4.49
(t, J = 6.9 Hz, 1H), 4.68 (d, J = 11.7 Hz, 1H), 7.26 — 7.29 (m, 5H), 7.39 (s, 1H),
7.41—7.45 (m, 2H), 7.51 - 7.55 (m, 1H), 7.72 — 7.76 (m, 2H). 13c NMR + DEPT
(125 MHz) (CDCl3) 6: 14.9 (—CH3), 52.8 (—CH3), 64.3 (—CH2), 69.3 (quaternary -
C), 71.7 (—CH2), 75.6 (—CH), 127.1 (aromatic —CH), 127.76 (aromatic -CH),
127.81 (aromatic —CH), 128.4 (aromatic —CH), 128.6 (aromatic —CH), 131.8
(aromatic -CH), 134.0 (aromatic quaternary —C), 137.6 (aromatic quaternary -C),
167.7 (quaternary —C), 170.7 (quaternary —C). IR (neat): 3415 cm", 1734 cm",
1653 cm", 1520 cm", 1485 cm", 1240 cm". MS (GCMS): m/z calcd for
C2oH23N05 [M], 357.16; found, 358.2. Anal. Calcd. For C2oH23N05: C, 67.21; H,
6.49; N, 3.92. Found: C, 66.96; H, 6.53; N, 4.09. "-138 (oil): 1H NMR (500 MHz)
(CDCI3): 6 1.22 (d, J = 6.3 Hz, 3H), 3.71 (s, 3H), 3.84 (dd, J1 = 7.8 Hz, J2 = 15.4
Hz, 1H), 4.04 (dd, J1 = 3.3 Hz, J2 = 11.9 Hz, 1H), 4.19 (q, J = 6.3 Hz, 1H), 4.42
(d, J = 11.8 Hz, 1H), 4.63 (d, J = 11.8 Hz, 1H), 5.42 (dd, J1 = 3.3 Hz, J2 = 11.3
Hz, 1H), 7.05 (broad s, 1H), 7.24—7.54 (m, 8H), 7.76 - 7.79 (m, 2H). 13c NMR +
DEPT (125 MHz) (CDCI3) 6: 13.9 (—CH3), 52.7 (—CH3), 63.9 (—CH2), 71.0 (-CH2),
71.4 (quaternary —C), 76.6 (-CH), 127.1 (aromatic -CH), 127.8 (aromatic -CH),
128.0 (aromatic —CH), 128.5 (aromatic —CH), 128.7 (aromatic -CH), 131.9
(aromatic —CH), 134.4 (aromatic quaternary —C), 137.3 (aromatic quaternary —C),

169.4 (quaternary —C), 171.1 (quaternary -C). IR (neat): 3412 cm", 3328 cm",

89

1749 cm", 1658 cm", 1520 cm", 1487 cm", 1069 cm". HRMS (FAB): m/z calcd

for C2oH24N05 [M+H], 358.1652; found, 358.1654.

H QEt H OEt
, N ' + , N
B2 2. 82 2.
MeOzC OH M602C OH
Il-14A "-148

Figure "-20. Methyl-2-benzamido-3-ethoxy-2-(hydroxymethyl)butanoate (ll-14).

Methyl-2-benzamido-3-ethoxy-2-(hydroxymethyl)butanoate (ll-14). Using the
general procedure, a suspension of "-1 (0.10 g, 0.46 mmol), "-7 (0.09 mL, 0.9
mmol), and diphenyl phosphate (11.3 mg, 0.045 mmol) in anhydrous benzene
(20 mL) was stirred at room temperature for 3 hours. After washings, the crude
reaction intermediate was diluted in 2.5 mL THF and cooled to 0 °C before
NaBH4 (34.7 mg, 0.92 mmol) and water (1.25 mL) were added. Purification via
silica gel chromatography (10% ether / 90% CH2Cl2) afforded 0.12 g of the title
compound (85% yield) as a 2 : 1 ratio of diastereomers. ll-14A (solid, mp. = 89 —
91 °C) 1H NMR (500 MHz) (CDCI3): 6 1.13 (t, J = 7.0 Hz, 3H), 1.30 (d, J = 6.4 Hz,
3H), 3.40 (dq, J1 = 7.0 Hz, J2 = 9.5 Hz, 1H), 3.65 (dq, J1 = 7.0 Hz, J2 = 9.5 Hz,
1H), 3.79 (s, 3H), 3.99 (dd, J1 = 7.2 Hz, J2 = 11.7 Hz, 1H), 4.14 (q, J = 6.4 Hz,
1H), 4.24 (dd, J1 = 6.0 Hz, J2 = 11.8 Hz, 1H), 4.49 (t, J = 6.8 Hz, 1H), 7.34 (s,
1H), 7.42 - 7.46 (m, 2H), 7.49 — 7.54 (m, 1H), 7.77 — 7.81 (m, 2H). 13C NMR +
DEPT (125 MHz) (CDCI3) 6: 15.1 (-CH3), 15.4 (-CH3), 52.8 (-CH3), 64.5 (-CH2),
65.4 (—CH2), 69.0 (quaternary —C), 75.7 (—CH), 127.1 (aromatic -CH), 128.7
(aromatic —CH), 131.9 (aromatic -CH), 134.1 (aromatic quaternary —C), 167.9

(quaternary —C), 170.9 (quaternary -C). IR (KBr pellet): 3413 cm“, 3352 cm",

90

1728 cm", 1644 cm", 1522 cm", 1489 cm", 1244 cm'1, 1067 cm". MS (GCMS):
m/z calcd for C15H21N05 [M"], 295.14; found, 295.9. Anal. Calcd. For C15H21N05:
c, 61.00; H, 7.17; N, 4.74. Found: c, 61.12; H, 7.19; N, 4.91. "-148 (oil): 1H
NMR (500 MHz) (CDCI3): 5 1.11 — 1.16 (m, 6H), 3.38 (dq, J1 = 7.0 Hz, J2 = 9.5
Hz, 1H), 3.63 (dq, J1 = 7.0, J2 = 9.5 Hz, 1H), 3.78 (s, 3H), 3.82 (dt, J; = 1.0 Hz, J2
= 11.8 Hz, 1H), 4.00 (dd, J1 = 3.1 Hz, J2 = 11.9 Hz, 1H), 4.07 (q, J = 6.3 Hz, 1H),
5.51 (dd, J1 = 3.1 Hz, J2 = 11.4 Hz, 1H), 7.02 (broad s, 1H), 7.45 (tt, J1 = 1.4 Hz,
J2 = 6.7 Hz, 2H), 7.50—7.55 (m, 1H), 7.78—7.81 (m, 2H). 130 NMR + DEPT (125
MHz) (CDCI3) 5: 14.01 (-CH3), 15.3 (-CH3), 52.8 (—CH3), 63.9 (—CH2), 64.7 (—
CH2), 71.4 (quaternary —C), 76.9 (—CH), 127.1 (aromatic -CH), 128.7 (aromatic —
CH), 131.9 (aromatic —CH), 134.5 (aromatic quaternary —C), 169.5 (quaternary —
C), 171.3 (quaternary —C). IR (neat): 3414 cm", 3327 cm", 1752 cm", 1661 cm’
1, 1522 cm", 1074 cm“. HRMS (FAB): m/z calcd for C15H22N05 [M+H],
296.1498; found, 296.1496.

H OBU

,N
82 2
Me02C 0H

"-18
Figure "-21. Methyl-2-(benzamido)-3-butoxy-2-(hydroxymethyl)but-3-
enoate (ll-18).

Methyl-2-(benzamido)-3-butoxy-2-(hydroxymethyl)but-3-enoate (II-18).
Using the general procedure, a suspension of "-1 (0.11 g, 0.49 mmol) and "-11
(72.7 mg, 0.74 mmol) in anhydrous dichloromethane (20 mL) was stirred at room

temperature overnight. After concentration, the crude reaction intermediate was

91

diluted in THF (3 mL) and cooled to -41 °C before anhydrous EtOH (1.5 mL) and
NaBH4 (92.7 mg, 2.45 mmol) were added. Purification via silica gel
chromatography (15% ether/ 85% CH2Cl2) afforded 0.11 g of the title compound
(67% yield) as an oil. 1H NMR (500 MHz) (CDCI3): 6 0.90 (t, J = 7.4 Hz, 3 H),
1.33 — 1.41 (m, 2 H), 1.60 — 1.68 (m, 2 H), 3.40 (dd, J1 = 5.1 Hz, J2 = 8.7 Hz, 1
H), 3.70 — 3.76 (m, 2 H), 3.80 (s, 3 H), 4.15 (dd, J1 = 8.7 Hz, J2 = 11.3 Hz, 1 H),
4.26 (d, J = 3.7 Hz, 1 H), 4.32 (d, J = 3.7 Hz, 1 H), 4.47 (dd, J1 = 4.9 Hz, J2 =
11.4 Hz, 1 H), 7.33 (broad s, 1 H), 7.41 — 7.46 (m, 2 H), 7.49 - 7.54 (m, 1 H),
7.77 — 7.81 (m, 2 H). 13c NMR + DEPT (125 MHz) (CDCI3): 5 13.7 (-CH3), 19.2
(—CH2), 30.6 (—CH2), 53.3 (—CH3), 64.1 (—CH2), 67.9 (-CH2), 84.6 (—CH2), 127.1
(aromatic —CH), 128.7 (aromatic —CH), 131.9 (aromatic —CH), 134.0 (aromatic
quaternary —C), 156.6 (quaternary —C), 167.2 (quaternary —C), 170.8 (quaternary
-C); IR (neat): 3575-3125 cm", 3416 cm", 1742 cm", 1651 cm", 1287 cm",

1225 cm"; HRMS (FAB): m/z calcd for C17H24N05 [M+H], 322.1656; found,

H
,N\{§0Me
32 2..
Me02C 0H
ll-19
Figure "-22. Methyl-2-(benzamido)-2-(hydroxymethyl)-3-methoxy-3-

322.1654.

methylbutanoate (ll-19).

Methyl-Z-(benzamido)-2—(hydroxymethyl)-3-methoxy-3-methylbutanoate (ll-
19). Using the general procedure, a suspension of "-1 (0.10 g, 0.46 mmol) and

"-12 (0.051 g, 0.70 mmol) in anhydrous benzene (20 mL) was stirred at room

92

temperature overnight. After washings, the crude reaction intermediate was
diluted in THF (3 mL) and cooled to 0 °C before NaBH4 (0.15 g, 4.1 mmol) and
water (0.1 mL) were added. Purification via silica gel chromatography (20%
ether / 80% CH2Cl2) afforded 0.084 g of the title compound (62% yield) as an oil.
1H NMR (500 MHz) (CDCI3) 6: 1.28 (s, 3 H), 1.41 (s, 3 H), 3.22 (s, 3 H), 3.76 (s,
3 H), 4.15 (dd, J1 = 9.9 Hz, J2 = 12.0 Hz, 1 H), 4.24 (dd, J1 = 4.3 Hz, J2 = 12.0 Hz,
1 H), 4.81 (dd, J1 = 4.3 Hz, J2 = 9.8 Hz, 1 H), 7.38 (broad s, 1 H), 7.41 — 7.46 (m,
2 H), 7.48 - 7.53 (m, 1 H), 7.77 — 7.80 (m, 2 H). 13c NMR + DEPT (125 MHz)
(CDCI3) 6: 20.3 (—CH3), 20.9 (-CH3), 49.2 (—CH3), 52.6 (-CH3), 62.9 (-CH2), 72.5
(quaternary —C), 79.6 (quaternary -C), 127.1 (aromatic -CH), 128.6 (aromatic —
CH), 131.7 (aromatic -CH), 134.7 (aromatic quaternary —C), 168.4 (quaternary —
C), 170.5 (quaternary —C). IR (neat): 3407 cm", 3299 cm", 1742 cm", 1659
cm", 1514 cm", 1485 cm", 1233 cm“, 1055 cm". HRMs (FAB): m/z calcd for

C15H22N05 [M+H], 296.1498; found, 296.1496.

Me0 Me0
QC gtBU Ofo OtBU

HN + HN

Meozc‘ CH20H Meozc" CH20H
II-24A "-248

Figure "-23. Methyl-2-(4-methoxybenzamido)-3-teIt-butoxy-2-
(hydroxymethyl)butanoate (ll-24).

Methyl-2-(4-methoxybenzamido)-3-tert-butoxy-2-(hydroxymethyl)butanoate
(ll-24). Using the general procedure, a suspension of "-20 (0.14 g, 0.55 mmol),

"-5 (0.084 g, 0.84 mmol), and diphenyl phosphate (14.3 mg, 0.057 mmol) in

93

anhydrous benzene (20 mL) was stirred at room temperature overnight. After
washings, the crude reaction intermediate was diluted in THF (3 mL) and cooled
to 0 °C before NaBH4 (46.0 mg, 1.2 mmol) and water (1.5 mL) were added.
Purification via silica gel chromatography (10% ether / 90% CH2Cl2) afforded
0.17 g of the title compound (88% yield) as a 2.9 : 1 ratio of diastereomers. ll-
24A (solid, mp. = 109 - 111): 1H NMR (500 MHz) (CDCI3): 6 1.11 (s, 9H), 1.30
(d, J = 6.3 Hz, 3 H), 3.79 (s, 3 H), 3.83 (s, 3 H), 3.94 (dd, J1 = 6.9 Hz, J2 = 11.7
Hz, 1 H), 4.25 (dd, J1 = 6.5 Hz, J2 = 11.7 Hz, 1 H), 4.36 (q, J = 6.3 Hz, 1 H), 4.72
(t, J = 6.7 Hz, 1 H), 6.90 — 6.94 (m, 2 H), 7.36 (broad s, 1 H), 7.73 — 7.78 (m, 2
H). 13c NMR + DEPT (125 MHz) (CDCI3) 5: 19.1 (—CH3), 28.7 (—CH3), 52.7 (—
CH3), 55.4 (—CH3), 64.8 (—CH2), 68.7 (-CH), 70.0 (quaternary -C), 74.8
(quaternary —C), 113.8 (aromatic -CH), 126.4 (aromatic quaternary —C), 128.9
(aromatic —CH), 162.4 (aromatic quaternary -C), 167.3 (quaternary —C), 171.3
(quaternary —C). IR (neat): 3600 — 3245 cm", 3416 cm", 1738 cm“, 1653 cm",
1609 cm", 1499 cm", 1258 cm". HRMs (FAB): m/z calcd for C18H23N05 [M +
H], 354.1917; found, 354.1914. "-248 (solid, mp. = 115 - 117): 1H NMR (500
MHz) (CDCI3): 6 1.13 (d, J = 6.2 Hz, 3 H), 1.16 (s, 9 H), 3.74 (s, 3 H), 3.80 — 3.86
(m, 4 H), 3.93 (dd, J1 = 2.7 Hz, J2 = 11.8 Hz, 1 H), 4.26 (q, J = 6.2 Hz, 1 H), 5.82
(dd, J1 = 2.7 Hz, J2 = 11.6 Hz, 1 H), 6.92 — 6.96 (m, 2 H), 7.18 (broad s, 1 H),
7.74 — 7.78 (m, 2 H). 13c NMR + DEPT (125 MHz) (CDCI3) 5: 17.9 (—CH3), 28.6
(—CH3), 52.6 (-CH3), 55.4 (—CH3), 64.2 (—CH2), 69.8 (—CH), 72.0 (quaternary —C),
75.1 (quaternary -C), 113.9 (aromatic —CH), 126.8 (aromatic quaternary —C),

128.9 (aromatic —CH), 162.5 (aromatic quaternary -C), 168.9 (quaternary -C),

94

171.5 (quaternary -C). IR (neat): 3411 cm", 3295 cm", 1752 cm", 1653 cm",
1607 cm“, 1499 cm", 1258 cm", 1115 cm". HRMS (FAB): m/z calcd for

C13H23N06 [M+H], 354.1917; found, 354.1915.

0Y0 QtBU 0Y0 OtBU

HNY\ + HN\/l\
Meozc“ CH20H Meozc" CH20H
lI-25A ll-25B

Figure "-24. Methyl-2-(4-(trifluoromethyl)benzamido)-3-tert-butoxy-2-
(hydroxymethyl) butanoate (ll-25).

Methyl-2—(4-(trifluoromethyl)benzamido)-3-tert-butoxy-2-(hydroxymethyl)

butanoate (ll-25). Using the general procedure, a suspension of "-21 (0.13 g,
0.45 mmol), "-5 (0.069 g, 0.68 mmol), and diphenyl phosphate (11.4 mg, 0.046
mmol) in anhydrous benzene (20 mL) was stirred at room temperature overnight.
After washings, the crude reaction intermediate was diluted in THF (2 mL) and
cooled to 0 °C before NaBH4 (38.4 mg, 1.0 mmol) and water (1 mL) were added.
Purification via silica gel chromatography (10% ether l 90% CH2Cl2) afforded
0.14 g of the title compound (81% yield), separated as a 2 : 1 ratio of
diastereomers. ll-25A (oil): 1H NMR (500 MHz) (CDCI3): 6 1.12 (s, 9 H), 1.29 (d,
J = 6.3 Hz, 3 H), 3.79 (s, 3 H), 3.92 - 3.99 (m, 1 H), 4.23 - 4.30 (m, 2 H), 4.37 (q,
J = 6.3 Hz, 1 H), 7.43 (s, 1 H), 7.67 — 7.71 (m, 2 H), 7.87 — 7.91 (m, 2 H). 13c
NMR + DEPT (125 MHz) (CDCI3) 6: 19.1 (—CH3), 28.7 (—CH3), 52.8 (-CH:,), 64.2
(—CH2), 68.6 (—CH), 70.0 (quaternary —C), 74.9 (quaternary —C), 123.6

(quaternary —CF3, q, J = 273 Hz), 125.7 (aromatic —CH, q, J = 4 Hz), 127.5

95

(aromatic -CH), 133.5 (aromatic quaternary —C, q, J = 33 Hz), 137.6 (aromatic
quaternary -C), 166.3 (quaternary —C), 171.1 (quaternary -C). IR (neat): 3447
cm", 3407 cm", 1740 cm", 1664 cm", 1653 cm", 1528 cm", 1327 cm", 1129
cm". HRMS (FAB): m/z calcd for C13H25F3N05 [M + H], 392.1685; found,
392.1687. "-258 (oil): 1H NMR (500 MHz) (CDCI3): 6 1.14 (d, J = 6.2 Hz, 3 H),
1.16 (s, 9 H), 3.75 (s, 3 H), 3.84 (dt, J1 = 1.0 Hz, J2 = 11.8 Hz, 1 H), 3.97 (dd, J1 =
2.9 Hz, J2 = 11.9 Hz, 1 H), 4.28 (q, J = 6.2 Hz, 1 H), 5.45 (dd, J1 = 2.9 Hz, J2 =
11.6 Hz, 1 H), 7.27 (s, 1 H), 7.71 — 7.74 (m, 2 H), 7.88 — 7.92 (m, 2 H). 13C NMR
+ DEPT (125 MHz) (CDCI3) 6: 17.9 (—CH3), 28.6 (—CH3), 52.7 (—CH3), 63.9 (-
CH2), 69.7 (—CH), 72.3 (quaternary —C), 75.4 (quaternary —C), 123.6 (quaternary
—CF3, q, J = 273 Hz), 125.8 (aromatic —CH, q, J = 4 Hz), 127.6 (aromatic -CH),
133.5 (aromatic quaternary -C, q, J = 33 Hz), 138.0 (aromatic quaternary —C),
168.1 (quaternary -C), 171.1 (quaternary —C). IR (neat): 3600 — 3250 cm", 3401
cm", 1754 cm", 1667 cm", 1530 cm", 1327 cm", 1121 cm". HRMS (FAB): m/z

calcd for C13H25F3N05 [M+H], 392.1685; found, 392.1687.

HN ’ + HN

Meozc“ CH20H Meozc’ CH20H
ll-26A "-268

Figure "-25. Methyl-3-tert-butoxy-2-(hydroxymethyl)-2-
(propionamido)butanoate (ll-26).

Methyl-3-tert-butoxy-2-(hydroxymethyl)-2-(propionamido)butanoate (ll-26).
Using the general procedure, a suspension of "-22 (0.17 g, 1.0 mmol), "-5 (0.15

g, 1.5 mmol), and diphenyl phosphate (23.8 mg, 0.095 mmol) in anhydrous

96

benzene (40 mL) was stirred at room temperature for 3 hours. After washings,
the crude reaction intermediate was diluted in THF (6 mL) and cooled to 0 °C
before NaBH4 (79.7 mg, 2.1 mmol) and water (3 mL) were added. Purification
via silica gel chromatography (10% ether / 90% CH2Cl2) afforded 0.14 g of the
title compound (50% yield) as a 1 : 1.3 ratio of diastereomers. lI-26A (oil): 1H
NMR (500 MHz) (CDCI3): 6 1.05 — 1.10 (m, 12 H), 1.17 (d, J = 6.3 Hz, 3 H), 2.19
- 2.30 (m, 2 H), 3.68 (s, 3H), 3.79 (dd, J1 = 6.8 Hz, J2 = 11.8 Hz, 1 H), 4.06 (dd,
J1 = 6.0 Hz, J2 = 11.6 Hz, 1 H), 4.15 (q, J = 6.3 Hz, 1 H), 4.67 (t, J = 6.5 Hz, 1 H),
6.60 (broad s, 1 H). 13C NMR (125 MHz) + DEPT (CDCl3) 6: 9.8 (-CH3), 17.7 (—
CH3), 28.6 (—CH3), 30.4 (—CH2), 52.5 (-CH3), 64.2 (—CH2), 69.6 (-CH), 71.8
(quaternary —C), 75.0 (quaternary -C), 171.3 (quaternary —C), 176.2 (quaternary
—C). IR (neat): 3413 cm", 3328 cm", 1742 cm", 1655 cm", 1516 cm", 1235 cm'
1. HRMS (FAB): m/z calcd for C13H26N05 [M+H], 276.1811; found, 276.1810. II-
263 (solid, mp. = 63 — 64 °C): 1H NMR (500 MHz) (CDCI3): 6 1.05 (d, J = 6.2
Hz, 3 H), 1.12 (s, 9 H), 1.19 (t, J = 7.6 Hz, 3 H), 2.32-2.41 (m, 2H), 3.67 — 3.75
(m, 4 H), 3.79 (dd, J1 = 2.4 Hz, J2 = 11.7 Hz, 1 H), 4.16 (q, J = 6.2 Hz, 1 H), 5.65
(d, J = 11.4 Hz, 1 H), 6.43 (s, 1 H). 13C NMR + DEPT (125 MHz) (CDCI3) 6: 9.4
(—CH3), 18.8 (-CH3), 28.5 (—CH3), 30.0 (-CH2), 52.5 (-CH3), 64.6 (—CH2), 68.7 (-
CH), 69.7 (quaternary —C), 74.4 (quaternary -C), 170.9 (quaternary —C), 174.4
(quaternary —C). IR (KBr pellet): 3360 cm", 3305 cm", 1755 cm", 1659 cm",
1541 cm", 1273 cm", 1115 cm". Ms (GCMS): m/z calcd for C11H22N03 [M -
C02Me], 216.16; found, 216.0. Anal. Calcd. For C13H25N05: C, 56.71; H, 9.15;

N, 5.09. Found: C, 56.96; H, 9.12; N, 5.06.

97

HN\(’\ + HNYK
Meozci CH20H Meozc" CH20H
lI-27A "-273

Figure "-26. Methyl-2-(2-phenylacetamido)-3-tert-butoxy-2-
(hydroxymethyl)butanoate (II-27).

Methyl-Z-(Z-phenylacetamido)-3-tert-butoxy-2-(hydroxymethyl)butanoate (ll-
27). Using the general procedure, a suspension of "-23 (0.24 g, 1.0 mmol), "-5
(0.16 g, 1.6 mmol), and diphenyl phosphate (27.0 mg, 0.11 mmol) in anhydrous
benzene (40 mL) was stirred at room temperature overnight. After washings, the
crude reaction intermediate was diluted in THF (6 mL) and cooled to 0 °C before
NaBH4 (84.8 mg, 2.2 mmol) and water (3 mL) were added. Purification via silica
gel chromatography (10% ether l 90% CH2Cl2) afforded 0.20 g of the title
compound (58% yield) as a 1.1 : 1 ratio of diastereomers. ll-27A (oil): 1H NMR
(500 MHz) (CDCI3): 6 1.02 (s, 9 H), 1.06 (d, J = 6.3 Hz, 3 H), 3.60 (d, J = 5.7 Hz,
2 H), 3.69 (s, 3 H), 3.82 (dd, J1 = 7.9 Hz, J2 = 11.7 Hz, 1 H), 4.03 -4.11 (m, 2 H),
4.43 (dd, J1 = 5.7 Hz, J2 = 7.7 Hz, 1 H), 6.53 (broad s, 1 H), 7.25 — 7.36 (m, 5 H).
13C NMR + DEPT (125 MHz) (CDCI3) 6: 18.8 (—CH3), 28.5 (-CH3), 44.2 (—CH2),
52.5 (-CH3), 64.8 (-CH2), 68.6 (—CH), 69.6 (quaternary —C), 74.5 (quaternary —
C), 127.4 (aromatic —CH), 128.9 (aromatic —CH), 129.5 (aromatic -CH), 134.4
(aromatic quaternary -C), 170.8 (quaternary —C), 172.0 (quaternary —C). IR
(neat): 3600 - 3470 cm", 3386 cm", 1740 cm", 1653 cm", 1509 cm", 1498 cm'
1, 1235 cm", 1078 cm". HRMS (FAB): m/z calcd for C18H28N05 [M + H],

338.1967; found, 338.1967. "-273 (oil): 1H NMR (500 MHz) (CDCI3): 5 0.87 (d, J

98

= 6.2 Hz, 3 H), 0.91 (s, 9 H), 3.63 — 3.71 (m, 6 H), 3.74 (dd, J1 = 2.9 Hz, J2 = 11.8
Hz, 1 H), 4.02 (q, J = 6.1 Hz, 1 H), 5.55 (dd, J1 = 2.9 Hz, J2 = 11.5 Hz, 1 H), 6.46
(broad s, 1 H), 7.24 — 7.36 (m, 5 H). 13C NMR + DEPT (125 MHz) (CDCI3) 6:
17.4 (-CH3), 28.3 (-CH3), 44.3 (—CH2), 52.4 (—CH3), 63.8 (—CH2), 69.4 (—CH),
71.8 (quaternary -C), 74.7 (quaternary —C), 127.4 (aromatic —CH), 129.0
(aromatic —CH), 129.7 (aromatic -CH), 134.4 (aromatic quaternary —C), 171.0
(quaternary —C), 173.5 (quaternary —C). IR (neat): 3366 cm", 3306 cm", 1754
cm", 1665 cm", 1522 cm", 1281 cm", 1063 cm". HRMs (FAB): m/z calcd for

C13H28NO5 [M+H], 338.1967; found, 338.1967.

99

K. References

1.

10.

Venkatraman, J.; Shankaramma, S. C.; Balaram, P. Design of folded
peptides. Chem. Rev. 2001, 101, 3131-3152.

Wang, L.; Schultz, P. G. Expanding the genetic code. Angew. Chem. Int.
Ed. 2005, 44, 34-66.

Cativiela, C.; Diaz-de-Villegas, M. D. Stereoselective synthesis of
quaternary alpha-amino acids. Part 1: Acyclic compounds. Tetrahedron:
Asymmetry 1998, 9, 3517-3599.

Cativiela, C.; Diaz-de-Villegas, M. D. Stereoselective synthesis of
quaternary alpha-amino acids. Part 2. Cyclic compounds. Tetrahedron:
Asymmetry 2000, 11, 645-732.

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Please see the dissertation of Jason S. Fisk.

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107

CHAPTER III
UTILIZATION OF OXAZOL-5(4H)-ONES TOWARDS THE SYNTHESIS
OF PROTEASOME INHIBITORS

A. Introduction to the 26S proteasome.
The synthesis and degradation of cellular proteins are integral processes
involved in maintaining biological homeostasis. Protein degradation, or
proteolysis, plays an essential role in many basic biological processes including
cell cycle control, cell differentiation, and apoptosis.1 Proteolysis is also a cellular
method used to regulate protein quality control, whereby misfolded, mutant, and
damaged proteins can be removed from the cellular environment.2 The sites
of proteolysis are compartmentalized within cells to prevent unregulated protein
hydrolysis, a process that would prove to be deleterious. In eukaryotic cells,
proteolysis occurs either inside the Iysosome, by action of the enclosed
proteases, or in the proteasome.3 However, it is commonly recognized that the
majority of intracellular proteolysis in all eukaryotic cells occurs within the
proteasome.4

The 268 proteasome is a large 2.5 MDa complex consisting of a core 208
particle, also known as the 208 proteasome, sandwiched between two 198
regulatory complexes, (Figure III-1).” Protein hydrolysis occurs within the cavity
of the 208 particle, a cylindrical structure comprised of four superimposed rings
(Figure III-1).9 The two outer a-rings are each made up of seven different 0-
subunits which surround a narrow entry channel into the 208 proteasome, and

the two inner B-rings each consist of seven B-subunits.7 Three of the seven [3-

108

subunits ((31, [32, and [35) in each of the two B-rings are catalytically active, and
proteins are hydrolyzed at these active sites via interaction with nucleophilic N-
terminal threonine residues.10 The three different active sites of protein
hydrolysis are often described based on their preference towards protein
substrates. The [31, I32, and [55 subunits exhibit specificity towards acidic, basic,
and hydrophobic residues respectively, such that they are said to have
respective caspase-like, trypsin-Iike, and chymotrypsin-Iike activities.6 However,
studies have demonstrated that the active sites within the proteasome are not

entirely specific with relation to the proteins they hydrolyze.11

   

20$ Proteasome 26$ Proteasome

Figure Ill-1. Representation of the 208 and 26S mammalian proteasomes.12

Protein degradation occurs selectively in the cytosol and nucleus of
eukaryotic cells by means of the ubiquitin-proteasome pathway. Proteins
become selected or “tagged” for degradation by the proteasome by means of
poly-ubiquitination, an enzymatic process by which a chain of ubiquitin molecules

becomes covalently bound to the substrate.”14 Poly-ubiquitinated peptides are

109

recognized by the 198 regulatory complex and are shuttled into the 208
proteasome. The 198 regulatory complex is critical for this process, and its
involvement includes binding poly-ubiquinated proteins, cleaving the poly-
ubiquitin chain, unfolding the substrate, and translocating it into the narrow entry
channel of the outer a ring of the 208 proteasome.6'15'16 Protein hydrolysis then
occurs with the assistance of water molecules at active sites within the [3 rings to
generate peptide fragments. The fragments may then undergo further proteolytic
events until oligopeptides are generated which are sufficiently small enough,
usually three to 22 amino acid residues in length, to diffuse through the

proteasomal entry channelsa'17

B. The 268 proteasome and the NF-kB pathway.
Multicellular organisms are routinely exposed to chemical, environmental,
mechanical, and microbiological stresses.13 Such stresses are managed in
organisms at the cellular level by inducible gene regulation. Inducible gene
regulators, such as the nucleic transcription factor NF-KB, are present within cells
and react to cellular stresses by transcribing a variety of genes as required.19
The NF-KB pathway has been a longstanding target of interest in the
scientific community. The NF-KB pathway is implicated in cellular responses

20'” and it is one of the many biological

such as inflammation and apoptosis,
pathways in eukaryotic cells regulated through the ubiquitin-proteasome
pathway.22 Under normal cellular conditions, NF-KB is located within the

cytoplasm and is complexed to the IKB protein (Figure III-2). However, cellular

110

stimulation by an agonist, such as chemotherapy, radiation, viruses, antigens, or
oxidants causes a cascade of events in which IKB undergoes successive
phosphorylation, poly-ubiquitination, and then proteolysis by the 26S
proteasome.12 Following the degradation of IKB, newly liberated NF-KB
translocates to the nucleus where a transcription event occurs, resulting in the
inhibition of the cellular event known as apoptosis, which is essentially
programmed cell death.

Extracellular Environment Cellular "Stress"

 

 

        

 

 

Ubiquitination Phosphorylation
le U u ‘ “IKB IKB
NF-KB U NF-KB NF-KB
Proteasome

 

NF'KB, I NF'KB Transcri tion ' " ‘.
. Inhlbltlon o
translocatlon '~ W ’ Apoptosis "

Nucleus _____ "

.9.
-- 0
. O
.---- ---.
-----..--—C’

Figure III-2. The role of the proteasome in the NF-KB pathway.

Recent discoveries concerning the NF-KB pathway have resulted in a
better understanding of diseases and their treatments, notably in the area of

anticancer research.""21'23"26 Treatments traditionally used in anticancer therapy,

111

such as chemotherapy and radiation, trigger the aforementioned cascade of
events that lead to release of NF-kB and subsequent transcription of genes

”'30 (Figure Ill-2). By this mechanism, cancerous

responsible for anti-apoptosis
cells survive anticancer treatments and are allowed to continue their rampant
replication. As noted above, NF-KB cannot translocate to the nucleus and trigger
the anti-apoptotic pathway until it is released from its complex with IKB. Since
the proteasome is responsible for the degradation of the NF-KB/IKB complex, it
has become an attractive target in anticancer therapy for sequestering NF-KB-
mediated anti-apoptosis. Indeed, proteasome inhibition has emerged as a useful
means of impeding cancerous cell survival, so much so that proteasome

L31 In

inhibitors have made their way into clinical use for anticancer treatmen
2003, the proteasome inhibitor bortezomib, also known as Velcade®, was
approved by the FDA for the treatment of multiple myeloma, and in 2005 it was
approved for the treatment of non-Hodgkin lymphoma.”34 Since then, other
small molecule proteasome inhibitors, such as carfilzomib and the natural marine
metabolite salinosporamide A, have since entered clinical studies for use as

anticancer agents.”40

C. Proteasome inhibition by small molecules.

Inhibition of the proteasome has been an intense focus of research in recent
years. A wealth of information regarding proteasomal structure and activity has
been attained from many of these studies through the use of small molecule

proteasome inhibitors.7'9"°"“‘16 Numerous proteasome inhibitors are currently

112

known, having been both isolated from natural sources and prepared
synthetically (Figure III-3). A number of these small molecules that inhibit the
proteasome also inhibit certain cellular proteases. Additionally, some
proteasome inhibitors exhibit little selectivity towards the different active sites of
the proteasome. As such, many synthetic proteasome inhibitors have been
designed to demonstrate excellent specificity towards the proteasome and even
to specific active sites.9’17'47 For example, vinyl sulfone Ac-APnLL-VS (Figure Ill-
3) exhibits potent and specific inhibition of the caspase-Iike active site of the 208
proteasome.9 Additionally, the demand for new clinical candidates for use in
disease treatment has required the preparation of small molecule proteasome

inhibitors which exhibit reduced toxicity.

113

LOH

O
C)Syringolin A HO

NH2 .
OH 0 “Yin/é
HO"

O H2N O
FellutamideB

  
  
   

 

OAc

H
30.1920“ng SallinosporamideA . OH .
(- )-Eplgallocatechln-3-gallate
(Velcade®)
i H O t O OH
O N “ks/[LN /O “J / U
H o = H k] N s

g
a)
A
u
N
0%
0;.
o
’ I
>
9
> o
3
,— o
'7
<
03

Figure Ill-3. Natural and synthetic proteasome inhibitors.

As was previously discussed, each protolytically active site within the 208
proteasome contains an N-terrninal threonine residue which acts to catalytically
cleave polypeptide amide bonds. Inhibitors of the 208 proteasome generally

contain electrophilic centers to which the N-terminal threonine residues

114

covalently bond (Scheme IlI-1).9"7'48 Thus, irreversible or slowly reversible bond
formation to small molecule inhibitors inactivates the proteasome towards further
proteolysis due to occupation of an active site. Proteolytic functionality of the
active site can only be reestablished once the covalent linkage as been
abolished. Several electrophilic functionalities exist within small molecules which
make them ideally suited for proteasome inhibition, including aldehydes, epoxy
ketones, B—lactones, esters, o,B-unsaturated amides, vinyl sulfones, and boronic
esters (Figure III—3). Of these functionalities, only boronic esters and vinyl
sulphones are representative of totally synthetic classes of proteasome

inhibitors.9'49

 

 

(\N H o ”Ill?“ /\N H o
0 HHo HO CH3 0 5 ”HO; (3
Bortezomib D I13N

N-terminal
threonine residue

 

 

  

0' 0 Km CH3C
Salinosporamide2 IL

 

N-terminal
threonine residue

Scheme III-1. Mechanism of proteasomal inhibition by bortezomib and

salinosporamide A.

115

However, a few compounds lacking the aforementioned electrophilic
functionalities are known to inhibit proteolytic activity.“7'5°'54 One such molecule,
TMC-95A, has been shown to competitively inhibit all active sites of the 208

5356 Disruption of

proteasome through the formation of H-bonding networks .
such noncovalent interactions and subsequent restoration of proteolytic activity
has been performed via dialysis, thereby demonstrating reversible inhibition by
TMC-95A.53'54 The discovery of this unique mode of proteasome inhibition has
energized researchers to prepare analogs of the natural product.50

Certain molecules can also inhibit proteolytic activity through allosteric
interactions. Inhibition of the 208 proteasome and the 26S proteasome has been
recently demonstrated through treatment with porcine bone-marrow derived 39-
amino acid peptide PR39 and related synthetic analogs.57 These polypeptides,
in which proline and arginine residues are abundant, cause gross changes in
proteasomal shape, as has been observed via atomic force microscopy.57 The
degree to which these peptides inhibit proteolysis has been shown to vary with
changes in pH, suggesting that the allosteric changes are promoted by strong
ionic and H-bonding interactions.”

Recently, studies in the Tepe lab have demonstrated inhibition of the
chymotrypsin-like activity of the 208 proteasome via treatment with appropriately
substituted 2-imidazolines. The evaluated 2-imidazolines do not appear to
covalently bind to the active site N-terminal threonine residues. Instead, it is

currently proposed that the 2-imidazolines inhibit the proteasome allosterically.

To the best of our knowledge, no small molecules have ever been reported to

116

inhibit the proteasome allosterically. This interesting discovery has prompted our
investigation into the synthesis of small libraries of novel 2-imidazolines to better
understand their mode of proteasome inhibition.

The 2-imidazolines prepared and evaluated in our lab have demonstrated
non-competitive inhibition of the chymotryspin-like active site of the 208
proteasome at low micromolar concentrations. Conversely, competitive inhibition
is observed when the proteasome is treated with small molecule inhibitors
comprised of pyrrolidinone and electrophilic B-lactone moieties, such as
salinosporamide A, omuralide, and a novel spiro-B-lactone compound prepared
in Jacobsen’s lab (Scheme Ill-2).58 Moreover, these small molecules inhibit the
chymotryspin-Iike active site at low nanomolar concentrations. All thus-far
evaluated 2-imidazolines prepared in our lab (Scheme Ill-2) have lacked an
electrophilic functionality, the likes of which are known to covalently adhere to
proteasomal active site threonine residues. Therefore, we became interested in
determining the effect that B-lactone incorporation into a 2-imidazoline might
have on the potency and mode of proteasome inhibition. We sought, then, to
synthesize a 2-imidazoline bearing a spiro-B—lactone, which we anticipated could
be derived from a tort-alkyl amino hydroxy carboxylic ester prepared through
oxazoI-5(4H)-one alkylation chemistry developed in the Tepe lab (Scheme Ill-
2).59 In addition, we envisioned that salinosporamide A could also be
constructed from a similar tert-alkyl amino hydroxy carboxylic ester starting
material (Scheme Ill-2). The remainder of Chapter III will describe our endeavors

towards 1) the racemic formal total synthesis of salinosporamide A and 2) the

117

synthesis and evaluation of the proposed B-lactone-containing 2-imidazoline

proteasome inhibitor.

 

Ph -
)zN

Bn’NwO

O .
Proposed 2-lmidazoline .

Proteasome Inhibitor ,

II

 

L

 

OH
R 0 o O Lcone
5f <—_— JL ArMe
N R N
COzMe H OtBU

 

 

 

 

 

o \/ W
O Bn’N "MR
HO O R CO2R
Jacobsen Tepe Lab
Proteasome -_ Proteasome
Inhibitor Inhibitors

Scheme Ill-2. Strategy for the synthesis of salinosporamide A and a novel
B-lactone-containing 2-imidazoline.

D. Introduction to salinosporamide A.

Numerous biologically active compounds have been isolated from marine
sources, resulting in the development of new therapeutic agents.60m In 2003,
efforts by Fenical and co-workers to discover new marine natural products

resulted in the cultivation and characterization of salinosporamide A from a strain

118

of seawater-requiring actinomycete bacteria (Figure lll-4).53'64 Salinosporamide
A has been shown to inhibit the 26S proteasome, and is currently being used in
clinical trials for anticancer therapy.6’3‘3'34 The structure of salinosporamide A
comprises a bicylic core, composed of a pyrrolidinone and a B-lactone. These
structural features are critical for the biological activity of natural product“65 and
are common to the biologically active terrestrial microbial products omuralide66
and cinnabaramide A67 (Figure Ill-4). Many of the structural features necessary

for activity in compounds of this class have been identified through structure-

8 58,68-

activity relationships,6 synthesis of congeners (e.g. Ill-1 - Ill-3, Figure Ill-4),

42'“ The complex molecular structure combined

78 and X-ray crystallography.
with the potent biological activity has made salinosporamide A an attractive
synthetic target; consequently, several groups have reported total syntheses of

79‘” Even though many elegant syntheses have been

the marine metabolite.
reported of this intriguing class of proteasome inhibitors, its potential clinical
relevance warrants the development of new synthetic approaches that may

provide access to additional candidates.

119

   

CH3(CH2)5 Més
Salinosporamide A Cinnabaramide A Omuralide
o H o

.«u OH
O

      

M5 M5 0
Ill-1 Ill-2 Ill-3

N
H

 

Figure Ill-4. Natural pyrrolidinone proteasome inhibitors and related congeners.

E. Syntheses of salinosporamide A.
Several structural features are present in salinosporamide A which have made it
an attractive and challenging synthetic target. The natural product contains five
contiguous stereogenic centers, two of which are quaternary, which bisect a
bicyclic pyrrolidinone/B-lactone ensemble and extend through a cyclohexenyl
substituent. Also featured is a chloroethyl substituent at C2 on the pyrrolidinone
ring (numbering shown in Figure III—4), which has been shown to contribute to the
potency of the marine metabolite as a proteasome inhibitor.68'86'87 The
quaternary stereocenter located at C4 of the pyrrolidinone ring is a tert-alkyl
hydroxy carboxylic ester, a quaternary q-amino acid subtype which was
described in Chapter II.

Salinosporamide A was first synthesized in 2004 in the labs of E. J.

Corey.84 The authors prepared the natural product in 17 linear steps beginning

120

from (S)-N-anisoyl threonine methyl ester Ill-4 (Scheme III-3). Key to their
synthetic strategy was early construction of the tert-alkyl amino hydroxy
carboxylic acid center, followed by successive formation of the pyrrolidinone and
B-lactone rings. The authors synthesized the tert-alkyl amino hydroxy carboxylic
acid center present in salinosporamide A from an 2-oxazoline intermediate.84 2-
oxazolines are known to undergo a variety of transformations and have been

“'90 Moreover, 2-

used to construct heterocycles and amino acid derivatives.
oxazolines have previously been used to construct the tart-alkyl amino hydroxy
carboxylic acid centers found in omuralide and lactacystin.“92 2-oxazoline Ill-5
was first prepared through cyclodehydration of Ill-4 with p-TsOH (Scheme Ill-3).84
Selective installation of the desired ether-functionalized methylene substituent
was then performed via treatment of Ill-5 with LDA and chloromethyl benzyl
ether, followed by acidic hydride reduction of the resultant quaternary 2-oxazoline
to afford amino alcohol ill-6. Successive N-acylation and alcohol oxidation
reactions gave rise to keto amide Ill-8, which was utilized to construct the desired
pyrrolidinone ring through a diastereoselective Baylis-Hillman-aldol reaction (d.r.
= 9:1) to yield Ill-9. The authors later reported the construction of a structurally

similar pyrrolidinone ring in high diastereoselectivity (d.r. = >9921) through an

unprecedented use of the Kulinkovich reagent.”74

121

HO 1. LDA

 

 

 

 

Me A PM
o r o CICHZOBn . %o Me
Arm/[L C02Me p-TsOH \n’ .....Me 69% = HN "”I—ZOBn
30% 2. NaCNBH
Ar=4H MeOPh COZMG ACQH 3 H0 Me
Ill-4 Ill-5 o Ill-6
90" 1.TMSCI
2. acrylyl chloride
IperEt
then H+
2%?) V 96%
PM PM
.. 2\Me Qurnuclldlneo N%02Me Bess-Martino N %02Me
,,,,,MeOB” ‘0 °C 7 d (3’0 Nl"—OBn¢ Periodinane "'11—an
OH d 90%;).196%/HO Me
III-9 ' ' Ill-8 Ill-7
l 10 steps

Salinosporamide A
Scheme III-3. Corey’s synthesis of the pyrrolidinone ring of salinosporamide A.

The tert-alkyl amino hydroxy carboxylic ester stereocenter present in
salinosporamide A has since been synthesized from oxazolidines, the saturated
analogs of 2-oxazolines. Total syntheses reported independently by the Potts
and Danishefsky groups described the use of oxazolidine ring systems as
templates for the asymmetric construction of the highly functionalized
stereocenter.”81 The total synthesis reported by Potts and co-workers involved
early preparation and use of N-acyl oxazolidine III-10. (Scheme Ill-4).81
Exposure of Ill-10 to basic conditions was performed to promote intramolecular
Aldol condensation, thereby establishing the C2, C3, and C4 stereocenters
present in pyrrolidinone intermediate Ill-11. In later synthetic steps,

decomposition of the oxazolidine ring was accomplished under acidic conditions,

122

and the resultant alcohol Ill-13 was oxidized to yield chiral quaternary amino

acid-containing III-14.81

?
NAG THF,I"(
\.—-’ 64%
ll/ C02Me 70% de
III-10

 

CF3CH20H, 60 °C

11,3-propanedithiol, HCI
94%

   

 

2. Dess—Martin periodinarg
3. N3H2PO4, NaC|02
2-methyl-2-butene BzO Ill-13
‘BuOH/HZO
63% for steps 2-3

 

l 5 steps

Salinosporamide A

Scheme Ill-4. Quaternary q-amino acid center preparation via an oxazolidine.

Danishefsky and co-workers prepared the quaternary amino acid center
by an alternate means in their total synthesis, instead utilizing bicyclic oxazolidine
Ill-15 in which the pyrrolidinone framework was already present (Scheme III-5).79
Similar to the Potts total synthesis, their synthesis also involved acidic
oxazolidine ring cleavage and subsequent oxidation to form the carbonyl
functionality present in Ill-18. However, this sequence was performed early and

prior to quaternization of the amino acid center. lmidate protection of the lactam

123

present in Ill-18 followed by cis-fused bicyclic ring formation via lactonization
under basic conditions furnished the anticipated quaternary center in bicyclic
adduct Ill-19. An additional 19 synthetic steps were then required to complete

the total synthesis.79

  

 

 

 

 

 

Ph‘
Ph (‘0
*/_O Q TfOH
0%)) 4 steps THF/H20
— ’l quant.
B O OCOZEt
"L15 " "L16
COztBU
EtO ,N ...,/ EtO
. , 19 steps ( LHMDS
SalinosporamideA /O <
THF, -20 °C
8 00
BnO III-19 BnO Ill-18

Scheme Ill-5. The Danishefsky synthesis of salinosporamide A.

Another common approach for the synthesis of the quaternary a-amino
acid center in salinosporamide A is the construction of the pyrrolidinone ring via
C3-C4 bond formation."2'83'85 The Romo group reported one such total synthesis
of the natural product wherein the fused pyrrolidinone/B-lactam ring system was
generated via bis-cyclization.82 The authors initially prepared B—ketoamide III-20
in 4 steps from commercially available O-benzyl-L-serine. Treatment of Ill-20
with modified Mukaiyama reagent Ill-21, organocatalyst 4-PPY, and HiJnig’s base
afforded bicyclic adduct Ill-22 as a mixture of diastereomers (Scheme Ill-6). The

authors propose that the bis-cyclization occurs in a stepwise fashion, whereby an

124

intramolecular aldol reaction first occurs through an ammonium enolate to form
the pyrrolidinone ring followed by lactonization of the resultant alkoxide
intermediate.82 In this way, the authors prepared an advanced racemic
intermediate containing the fused ring ensemble with an intact chloroethyl
substituent, all with the correct relative stereochemistry. The synthesis then
concluded through removal of the benzyl protecting groups and installation of the
cyclohexenyl substituent using Corey’s protocol.84 The Romo synthesis is quite
remarkable in that the labile B—lactone and halogen functionalities are installed in
early synthetic stages. In fact, Romo’s synthesis is the only reported total
synthesis of salinosporamide A which grossly strays from Corey’s original
protocol involving successive B-Iactone formation and halogen installation during

the final synthetic steps.84

125

o
PMB, )0H
0 N 9

OBn

O
PMB
N o
O —OBn
H Me

Cl Minor
Diastereomer

+ O _-
PMB
N
O 903" 4steps
H Me
CI Ill-22

Major
Diastereomer
25-35% yield

dr = 2-3 : 1

‘—

0
eaN/ Br
TfO n-Pr

Ill-21
CH2Cl2, -10 °C, 6 h

 

 

_CI

fp/

/ \
O "NG-JeBr
PMB o ‘n-Pr
o N

O
—OBn
Me

 

”(3'0

 

o . 4-PPY
n-Pr l iPr2NEt
l
O®_ _ eoe_
PMBN 03‘ / R PMBN_2/O—NO*R
_OBn ‘— 0 —osn
Me Me
Cl _ _Cl 4

 

 

 

 

Salinosporamide A

Scheme Ill-6. PyrrolidinonelB-lactone formation via bis-cyclization.

The synthesis of the C4 stereocenter of salinosporamide A has also been

performed through desymmetrization of malonate-containing pyrrolidinones.

The Pattenden group first utilized this approach in their total synthesis of the

marine metabolite (Scheme Ill-7).
malonate Ill-23 with acetic acid to perform an in situ deprotection of an acetal-

protected ketone and subsequent diastereoselective aldol cyclization to form

126

The authors prepared and treated amido

pyrrolidinone Ill-24. Next, protection of the alcohol and amide functionalities
present in Ill-24 preceded regioselective ester reduction to afford aldehyde Ill-25.
Installation of the cyclohexenyl substituent according to Corey’s protocol84 then

furnished Ill-26 with the desired functionalities positioned about the C4

 

stereocenter.
OBn
omo 4:1 HO 5 CB“
"160ch AcOHszg M602C '
MeOzC u o 65 °C, 4 d M9026“ N
71% H
III-23 lll-24
1. TMSOTf, 2,6—lutidine
~78 to 0 °C
2. PMB-Br, NaH, DMF
0 °C to rt
75% (2 steps)
3. Super-hydride (1.0 M in THF)
v CHZCIZ, -78 °C, 78%

 

OBn
TMSO? / ZnBr . OB"
MeOzC O TMSO;
@4 N O ¢THF 78 c Meozc‘f o
' .- ° OHC N
Ill-26 Ill-25
lSsteps

Salinosporamide A

 

Scheme Ill-7. Pattenden synthesis of salinosporamide A.

Desymmetrization of a malonate intermediate was also featured in the
total synthesis of salinosporamide A reported by Hatakeyama and coworkers.85
The authors utilized an intramolecular Conia-ene reaction to construct the
pyrrolidinone scaffold in III-28, and in later steps, substrate-controlled selective

127

 

 

ester reduction and subsequent oxidation afforded aldehydic ester III-30
(Scheme III-8). Only seven more steps, including cyclohexenyl installation via

Corey’s protocol,“ were then required to complete their total synthesis.

    

 

 

 

 

PMB In(OTf)3 PMB [MB
0
AcO O NYCOZMe (5 mol /o) O N "'fécgll'i‘lnee 4 steps 0 N -"%%hfiaee
\COzMe toluene 2 ...,, 2
\ 110 °C 0
Ill-27 95% OAc Ill-28 Ill-29
OBn
1.NaBH4
THF, EtOH
88%
2. Dess-Martin
Periodinane
CH2Cl2
ll 94%
_ , steps
Salinosporamide A ‘THF, -78 o C
88%

   

OBn III-31

Scheme Ill-8. Hatakeyama synthesis of salinosporamide A.

F. Use of oxazol-5(4H)-ones towards the synthesis of salinosporamide A.

In Chapter II the diastereoselective synthesis of tert-alkyl amino hydroxy
carboxylic esters by means of a sequential alkylation reaction of oxazol-5(4H)-
ones with enol ethers followed by hydride reduction was described (Scheme Ill-
9).59 The functionalities surrounding the quaternary stereocenter formed in the

reaction are represented at the C4 center in salinosporamide A. To the best of

128

 

our knowledge, this center had never been constructed via an oxazol-5(4H)-one

intermediate.

 

R1 0
T o
N R5 R
R2 — 1 0 OH
H R30 R4: 1,7 $30 NaBH4 o LR2R5
2 ——'
ene-type RR R )LN/QK‘R
. 5 1 4
R1 reaction R30 R H 0R3
4
I / OH
R2

Scheme Ill-9. Intermolecular alkylation/reduction reaction of oxazol-5(4H)—ones.

As previously discussed, the total synthesis of salinosporamide A by
Corey and co-workers involves the construction of pivotal intermediate Ill-8,
which undergoes cyclization via a Baylis-Hillman-aldol reaction to give the core
pyrrolidinone scaffold (Scheme Ill-10).“ Moreover, recent formal syntheses of
salinosporamide A have been reported in which molecules structurally analogous
to Ill-8 have been prepared for use in pyrrolidinone formation.”76 We
anticipated that this key intermediate in Corey’s synthesis should also be readily
accessible from our chemistry after a few functional group modifications. Due to
the skeletal diversity afforded by the use of various enol ethers in our oxazol-
5(4H)-one alkylation reaction (Table "-4), this could potentially serve as a
strategy for the synthesis of C3 analogues of salinosporamide A (Scheme Ill-10).

We sought to prepare Corey’s intermediate Ill-8,74“ then, in order to

129

demonstrate the utility of quaternary centers produced via the oxazol-5(4H)-one

template for the synthesis of salinosporamide A and related congeners.

 

Ill-80: R1=Me R0 R1

Scheme Ill-10. Strategy for the synthesis of Corey intermediate Ill-8.

The starting material for our synthesis of Ill-8 was obtained through our
alkylation/reduction reaction protocol to yield amino hydroxy carboxylic ester "-24
as a 3:1 mixture of diastereomers in 88% yield (Table "-5).59 For the synthesis of
Ill-8, only the major diastereomer Il-24A was used, as indicated in Scheme Ill-10.
However, it should be noted that the mixture of diastereomers could be used to
complete the racemic synthesis of salinosporamide A due to the destruction of
the second stereocenter during the oxidation of alcohols III-7 or Ill-6 (Scheme Ill-
11).“ The synthesis of Ill-8 initiated with the reduction of the amide functionality
present in tent-alkyl amino hydroxy carboxylic ester ll-24A. This reduction was
performed smoothly via cyclodehydration under basic conditions with MsCl
followed by oxazoline reduction with sodium cyanoborohydride in acetic acid to
afford amino-alcohol III-33 (Scheme Ill-11). This transformation accomplished
two goals in our synthesis: 1) the reduction of a stable amide in the presence of
an ester, a feat we found to be unsuccessful under a variety of attempted

conditions, and 2) the protection the amine as a PMB amine. The resultant

130

primary alcohol was treated under basic conditions with benzyl bromide to give
benzyl ether III-34 in 96% yield without any N-alkylation, an observation in
agreement with similar alkylation reactions of 1,2-amino alcohols.“ Acylation of
the secondary amine with acrylyl chloride under Corey’s conditions“ then gave
amide Ill-35 in 94% yield. Unfortunately, deprotection of the tert-butyl ether in Ill-
35 using acidic conditions proved to be problematic. The reaction was often
messy and low yielding, as treatment with TFA or aqueous phosphoric acid gave
25% and 27% yields respectively of alcohol Ill-7 as a single diastereomer.
Finally, oxidation of the free alcohol in Ill-7 was performed using the reported
procedure“ to cleanly afford racemic ketone Ill-8, the spectroscopic data of

which matches that of the known compound.

131

C|)H

 

 

 

 

0 ., C02Me MsCl Ar 0 NaCNBH3 pM
Ar/ILN/kae TEA F } AcOH HN %02Me
H O‘Bu DCE COzMe rt. 40 h t "“0“
Ar=4-MeOPh reflux; 7 h tBuO Me 73% BUD Me
Il-24A 92 /° III-32 III-33
BnBr, NaH
DMF, rt,
0.25 h
96%
pM PM 0 PM
‘ H P | I
O N %02Me 30:4C(|aq) O N %02Me VLCI HN %02Me
""'—OBn < 2 2 ""'—OBn < . :EW-OBn
I
/HO Me ”' 29 h /t Me PerEt tBuO Me
27 A BUG 0 °C 4 5 h
III-7 III-35 ’ ' III-34
94%
Dess-Martin Periodinane H3P04 (30)
CH2C|2, rt, 1.25 h CH20I2
98%
PM as described in 9'“
0 N %o23n§n t reference84 HNfgzg‘gn
/ 0 Me HO Me
III-3 Ill-6

Scheme Ill-11. Racemic synthesis of Corey intermediate Ill-8.

The disappointing yields obtained from the deprotection of the tert-butyl
ether prompted investigation into its removal at an earlier stage of the synthesis.

The original synthesis reported by Corey involved access to Ill-8 through

modification of alcohol Ill-6. We anticipated Ill-6 to also be accessible in our

synthesis through amendment of our synthetic route. Thus, deprotection of the
tert-butyl ether in compound Ill-34 was performed by treatment with aqueous
phosphoric acid95 to afford secondary alcohol Ill-6 in near quantitative yield,

thereby granting access to desired compound Ill-8.

132

This racemic synthesis of Ill-8 was completed in just 6 steps with a 15%
overall yield starting from lI-24A. However, racemic Ill-6 was derived from Ill-
24A in just 4 steps with an overall yield of 63%. In comparison, Corey’s original
route featured a six step synthesis starting from (S)-threonine methyl ester to
construct III-6 in 33% yield.“96 While our racemic synthesis of Ill-8 (and Ill-6)
appears no more efficient than Corey’s original protocol, it demonstrates a useful
synthetic application of highly functionalized molecules derived from oxazol-

5(4H)-one alkylation products.

G. Introduction to 2-imidazolines.

2-imidazolines have gained recognition for their ability to affect numerous
biological processes and for their utility as useful synthetic building blocks (Figure
lV-5). In biological systems, several 2-imidazolines have high affinity for
imidazoline binding sites (IBS), also known as imidazoline receptors. These IBS
are of interest to researchers for their role in hypertension, blood pressure
regulation, opioid dependence and tolerance, and neurological disorders.97
Studies have also indicated the possible application of 2-imidazolines as
antihyperglycemic,98 anti-inflammatory,99 antihypertensive,100 and

1 agents. Additionally, some highly substituted 2-

antihypercholesterolemic1o
imidazolines known as Nutlins have recently been found to bind to the MDM2
protein, thereby liberating p53 to suppress tumor growth in human cancer.102

Certain 2-imidazolines have also been found to sensitize leukemia T cells

towards chemotherapeutic agents.‘°3"“ Furthermore, 2-imidazolines have been

133

used as building blocks for the construction pharmaceutically relevant molecules

such as azapenams, dioxocyclams, and diazapinones."’5'106

0
”(if @226?
g; (3..

 

TCH-018 Nutlin 3 N3
Cl 0' HO fl
1 (KN Oxymetazoline
N N
N

H H
0' C|onidlne fiNUOH CIQ§.@

HN egg
0 i) Cl NJ

(:1: j/L Phentolamine
o ldazoxan TA'r-IsaanC}

Figure Ill-5. Structures of some biologically active 2-imidazolines.

H. Syntheses of 2-imidazolines.

A variety of methods appear in the literature for the preparation of 2-

107-109

imidazolines. Many early syntheses of the scaffold involve dehydrative

cyclization of monoacyl ethylenediamine derivatives. For example, Hill and
Aspinall demonstrated that 2-imidazolines were available from the treatment of
esters with ethylenediamine. The authors heated several esters with

ethylenediamine to afford 2-alkyl- and 2-aryl-2-imidazolines in good yields (2-

110,111

imidazoline numbering shown in Scheme lV-12). The reaction involves

initial monoamide formation followed by intramolecular cyclodehydrationm'113

134

Amides prepared from alkyl esters, such as acetate esters, could be isolated by
distillation and further converted to the desired 2-imidazolines by heating to high
temperatures in the presence of a dehydrating agent. However, the more
reactive amides prepared from aryl esters could not be isolated. Instead,
substrate dehydration occurred during attempts to purify the amino amides by

distillation, and the resultant 2-imidazolines were instead isolated.

 

 

 

 

 

H

R-CHO R N
H2N HNj
lR-COzEt 1x69

H

A k R N H
R/lLN/\/NHZ Dehydrating 53.] 5 CE)
Reagent 3 4 X)

Scheme III-12. Synthesis of 2-imidazolines derived from diamines.

2-imidazolines have also been conveniently generated from diamines and
aldehydes. Such reactions require the use of an oxidant, such as NBS,114
tBuOCl,115 orl2116'117 in order to generate desired 2-imidazolines (Scheme IV-12).
Initial intermolecular dehydration occurs to generate a 2-imidazolidine
intermediate114 followed by subsequent oxidation via treatment with an
electrophilic halogenating reagent to afford 2-imidazoline products. The protocol
involving the addition of an oxidant are attractive in that they are routinely
performed at room temperature, which allows for the preparation of heat-

sensitive compounds.

135

Alternatively, 2-imidazolines have been generated from hydroxyl-
substituted amides.118 Successive treatment of appropriately substituted [3-
hydroxy amides with SOClz and primary amines gives rise to 2-imidazolines,
which were observed to proceed via an imidoyl chloride intermediate (Scheme
lV-13).118 This reaction tolerates various aliphatic and aromatic amides, although
aryl amides exhibited greater reactivity. Consequently, less reactive aliphatic
amides required treatment with PCI5 in addition to SOClz to induce imidoyl

chloride formation and subsequent 2-imidazoline formation.

 

 

HO ,R Cl ..R SOC'Z I ,R
ij 3 SOCI2 i] 3 orPCl5 CF] 3
R1 N ‘R, R1 N *R, R1 ‘N ‘R,

H H

le-NHZ

1 3 \ w

\EI‘Z‘ ‘— R1 N6.) R4

R4 _ Cle .

 

 

Scheme Ill-13. Synthesis of 2-imidazolines derived from B—hydroxy amides.

The Tepe lab has also developed a method for the preparation of 2-
imidazolines, by which oxazol-5(4H)-ones undergo Lewis acid-mediated
cycloaddition reactions with imines, as was described in Chapter I (Scheme IV-
14). In this way, numerous 2-imidazolines have been prepared and evaluated for

their biological activity.22-‘°3'1°‘i-119

136

 

R
R1_<‘N R TMSCI R1T<‘N «£2
2 CHzclz COZH

60 - 90% yield

Scheme Ill-14. Synthesis of 2-imidazolines derived from oxazol-5(4H)-ones.

l. Synthesis of 2-imidazoline proteasome inhibitors.
Structure activity relationship studies are currently underway in our laboratories
to gain understanding into the mode of proteasome inhibition by 2-imidazolines.
We have become particularly interested in the synthesis of a 2-imidazoline
bearing a B-lactone functionality, such as Ill-37 (Scheme Ill-15). Unfortunately,
such a derivative was deemed to be unattainable by our current methodology
involving cycloaddition reactions of oxazol-5(4H)—ones (Scheme Ill-14). Thus, we
required a new strategy for the synthesis of 2-imidazolines comprising the carbon
framework present in desired 2-imidazoline Ill-37.

Apparent structural similarities are noticeable in proposed 2—imidazoline
III-37 and salinosporamide A. As such, it was envisioned that 2-imidazoline Ill-37
and salinosporamide A could be constructed from similar staring materials. Our
reported formal total synthesis of salinosporamide A involved modifications to
tert-alkyl amino hydroxy carboxylic ester ll-24A prepared from the alkylation
reaction of oxazol-5(4H)-ones and enol ethers (Scheme Ill-11).93 Therefore, we
anticipated preparation of 2-imidazoline Ill-37 to successfully arise through the

use of structurally similar starting material ll-6A (Scheme Ill-15).

137

Bn 8n

Ph Ph N
mt: \« OHCOzMe
:> N... jig/{rm
O O M6020 OtBU: OtBU
(+1-) Ill-37 (+1-) Ill-36 (+1-) ll-6A

Scheme Ill-15. Strategy for the synthesis of a C4-spiro- B—lactone 2-imidazoline.

Our proposed synthesis for the formation of Ill-37 involved initial
generation of the 2-imidazoline scaffold, followed by functional group modification
and subsequent B-lactone installation. Previous studies have demonstrated that
hydroxyl-substituted amides can be converted to the corresponding 2-
imidazolines by means of dehydration with SOClz or PCI5 (Scheme lV-12).118
However, various attempts to perform the cyclodehydration reaction according to
the published procedure were unsuccessful, and the only product obtained in the
reaction when II-6A was used was 2-oxazoline III-38 (Scheme lV-16).
Alternatively, we believed 2-imidazolines of interest to be attainable via the
corresponding amino amides, as was observed in the work of Hill and Aspinall
(Scheme lv-12).111 Thus, ll-6A was submitted to Swern or Dess-Martin oxidation
conditions immediately followed by reductive amination with NaBH(OAc)3 and
benzylamine to give rise to 2-imidazoline Ill-36 (Scheme lV-16). The structure of
Ill-36 has been confirmed via X-ray analysis, as is shown in Figure lV-6. Only a
minor amount of the intermediate amine III-40 was recovered from the reaction,
and it was observed to undergo cyclodehydration to form 2-imidazoline Ill-36
upon standing at room temperature for one week. A Dess-Martin
oxidation/reductive amination reaction sequence also generates the desired 2-

138

imidazoline when performed in a single pot, albeit in a lower yield (64% for the

synthesis of III-36).

 

 

 

 

o (RI-loo M (00002 OHC co2 Me NaBH<OA°)3 Bn—HN cone
)L ’ flee DMSO > HN Me BnNHz Me
Ph N CH2Cl2, r DCE, rt ' t
H O‘Bu -73 °C OJNPhO Bu 30% OiP hOBU
II-6A then TEA Ill-39 for 2 steps ‘ III-40 ‘
1. SOCIZ, reflux
2. BnNHz, TEA
>90%
Bn
PhYOé 3.3” ‘
N Ph N
NICeOZMe Pkg: E \N 00 Me
t ""' 2
BUO ‘ MeOzC O‘Bu tBuO:EMe
III-38 Ill-36 III-36

Scheme lV-16. Synthesis of 2-imidazolines via an oxidation/reductive amination

protocol.

139

 

 
 

- ‘ 0l2Bl
Cs” Nl2Bl
‘ ‘4 ‘ ‘
0(3Bl s): («v
' (\W 9
‘.\\\- , Q ,_
'1”! .3 ‘f? k.’
3“) (In, ‘ ///‘
;\"\\ ,,. . , v MB)»
5“ u ’4‘
DUB)

 

Figure Ill-6. X-ray crystal structure of Ill-36.

With 2-imidazoline Ill-36 in hand, we next focused our attention to the
synthesis of target 2-imidazoline "-37. Our synthetic strategy was to unveil the
alcohol and carboxylic acid functionalities present in Ill-36, available from the
ether and ester substituents respectively, and to use the reactive handles to
install the B—lactone funtionality (Scheme lV-17). Towards this end, Ill-36 was
modified through tert—butyl ether deprotection with aqueous phosphoric acid to
afford Ill-41 in 94% yield, followed by hydrolysis in 95% yield to give carboxylic
acid Ill-42 (Scheme IV-17). Treatment of III-42 with bis(2-oxo-3-

140

oxazolidinyl)phosphonic chloride (BOP-Cl) and triethylamine then resulted in the
generation of the desired B-lactone III-37. Purification of Ill-37 proved to be
problematic, though, due to its ease of hydrolysis, and only poor yields of impure-
product were ever recovered. The labile nature of Ill-37 appears to parallel the
reported instability of fl-lactone-containing salinosporamide A at physiological

H.657“120 However, from the reaction mixture containing Ill-37 was cleanly

p
isolated an unexpected product which was found to inhibit chymotrypsin-like
activity of the 208 proteasome at low micromolar concentrations. This
unexpected product and the chemistry associated with its synthesis will be

discussed in Chapter IV of this dissertation.

 

 

an H PO ( ) an
Ph N 3 4 aq N
\6 DCM ““6
N'” rt 6 h; N”
MeOzC O‘Bu 9’4% MeOzC OH
Ill-36 Ill-41
LiOH-HZO
THF, H20
95%
B” Bn
N I
th BOP-Cl (1.2 eq) PM“N
N""' A TEA (3 GQ) NMR
0 ‘ CHCI3 HOZC OH
Ill-37 rt, 1 hr III-42

<4% isolated

Scheme lV-17. Synthesis of Ill-37.

141

J. Evaluation of proteasome inhibition.

The 2-imidazolines prepared in this chapter were tested for their ability to inhibit
the chymotryptic-like activity of the 208 proteasome in an in vitro assay. The
results of the assay are shown in Table Ill-1. It was discovered that Ill-36, which
contains the bulky hydrophobic tart-butyl ether, inhibits chymotrypsin-Iike activity
at low micromolar levels (I050 = 7.5 pM). In comparison, the most potent 2-
imidazolines thus far evaluated in our lab have exhibited IC50 values in the range
of 1 — 2 pM. However, removal of the tert-butyl group appears to markedly
reduce potency, such that >20 uM of either Ill-41 or Ill-42 is necessary to inhibit
50% of proteolytic activity. Finally, a mixture containing Ill-37 was found to inhibit
chymotrypic activity at a low micromolar concentration (IC50 = 1.94 uM).
However, this mixture was comprised of III-37 and Vl-1 (1 : 3 ratio as determined
by 1H-NMR), and IV-1 alone was later found to be more potent than the mixture
(IC50 = 1.41 pM for IV-1). Therefore, the extent to which Ill-37 inhibits

chymotryptic—like activity of the proteasome is currently unknown.

142

Compound IC50 (uM)

 

Compound I050 (uM)

 

8n 8n
Ph N Ph N
Twp 7.45 T... >20
MeOzC O‘Bu HOZC OH
Ill-36 III-42
8n 8n
N N
th Ph\«
le- >20 N""' unknown
M6020 OH O O
Ill-41 Ill-37

 

Table Ill-1. Inhibition of chymotrypsin-like activity of the 208 proteasome by
2-imidazolines.

K. Experimental.

1. General Information. Reactions were carried out in flame-dried glassware
under nitrogen atmosphere. All reactions were magnetically stirred and
monitored by TLC with 0.25 pm pre-coated silica gel plates using either UV light
or iodine to visualize the compounds. Column chromatography was carried out
on Silica Gel 60 (230-400 mesh) supplied by EM Science. Yields refer to
chromatographically and spectroscopically pure compounds unless otherwise
noted. Infrared spectra were recorded on a Nicolet IR/42 spectrometer. 1H and
13C NMR spectra were recorded on a Varian Unity Plus-500 spectrometer.
Chemical shifts are reported relative to the residue peaks of the solvent (CDCI3:
7.27 ppm for 1H and 77.0 ppm for 13C). The following abbreviations are used to
denote the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, p =

pentet, and m = multiplet. HRMS were obtained at the Mass Spectrometry

143

Facility of Michigan State University with a JEOL JMS HX-110 mass
spectrometer. Melting points were obtained using an Electrothemlal® capillary

melting point apparatus and are uncorrected.

2. Materials. Reagents and solvents were purchased from commercial suppliers
and used without further purification. Anhydrous methylene chloride was
dispensed from a delivery system which passes the solvents through a column
packed with dry neutral alumina. Anhydrous chloroform and triethylamine were
distilled from calcium hydride and stored over activated molecular sieves prior to
use. The synthesis of ll-6A and II-24A were described in Chapter II of this

dissertation.

3. Synthesis and Characterization.

Q0.

N C02Me
tBuO Me
Ill-32

Figure Ill-7. DL-(R)-Methyl 4-((R)-1-(ten‘-butoxy)ethyl)-2-(4-methoxyphenyI)-4,5-
dihydrooxazole-4-carboxylate (Ill-32).

DL-(R)-Methyl 4-((R)-1-(tert-butoxy)ethyI)-2-(4-methoxyphenyl)-4,5-
dihydrooxazoIe-4-carboxylate (Ill-32). A 0 °C solution of II-24A (0.12 g, 0.34
mmol) and triethylamine (0.37 g, 3.6 mmol) in 1,2-dichloroethane (15 mL) was

treated with methanesulfonyl chloride (0.12 g, 1.0 mmol), and the reaction stirred

144

under nitrogen atmosphere while it warmed to room temperature. The reaction
was then refluxed until complete, as observed by TLC (about 7 hours). After
cooling to room temperature the reaction was diluted in CH2CI2 (15 mL) and was
made acidic by the addition of 30% aqueous HCI solution. The organic layer was
collected and the aqueous layer was extracted with CHZCIZ (2 x 30 mL). The
combined organic extracts were washed successively with saturated NaH003
solution and brine before being dried (M9804) and concentrated under vacuum.
Purification via silica gel chromatography (5% ether / 95% CH2Cl2) afforded 0.11
g of the title compound (96% yield) as an oil. 1H NMR (500 MHz) (CDCI3): 6 1.03
(d, J = 6.2 Hz, 3 H), 1.08 (s, 9 H), 3.73 (s, 3 H), 3.76 (s, 3 H), 4.43 (q, J = 6.2 Hz,
1 H), 4.53 (d, J = 9.5 Hz, 1 H), 4.90 (d, J = 9.5 Hz, 1 H), 6.83 (d, J = 8.9 Hz, 2 H),
7.86 (d, J = 8.9 Hz, 2 H). 13C NMR + DEPT (125 MHz) (CDCI3): 6 17.0 (—CH3),
28.5 (—CH3), 52.3 (—CH3), 55.1 (-CH3), 69.2 (-CH2), 70.0 (-CH), 73.9
(quaternary C), 82.2 (quaternary C), 113.4 (aromatic —CH), 119.4 (quaternary —
C), 130.2 (aromatic —CH), 162.2 (quaternary —C), 164.6 (quaternary -C), 172.4
(quaternary —C). IR (neat): 2978 cm", 1740 cm", 1638 cm", 1611 cm", 1514
cm'1, 1260 cm", 1082 cm". HRMS (FAB): m/z calcd for C13H25NO5 [M+H],
336.1805; found, 336.1820.
HZM%OZMe

"”'-OH

tBuO Me
III-33

Figure III-8. DL-(2R, 3R)-Methy| 3-tert-butoxy-2-(hydroxymethyl)-2-((4-
methoxybenzyl)amino)butanoate (III-33).

145

 

DL-(2R, 3R)-Methyl 3-tert-butoxy-2-(hydroxymethyl)-2-((4-
methoxybenzyl)amino)butanoate (Ill-33). A solution of III-32 (0.29 g, 0.86
mmol) in glacial acetic acid (4 mL) was treated with excess NaCNBH3 (0.21 g,
3.3 mmol) and the reaction stirred at room temperature for 2 days with more
NaCNBH3 (0.10 g, 1.6 mmol) added after 24 hours. The reaction was diluted
with H20 (10 mL) and the pH was adjusted to pH = 7 with the slow addition of
solid Na2C03. The reaction was extracted with EtOAc (3 x 25 mL) and the
combined organic extracts were dried (MgSO4) and concentrated under vacuum.
Purification via silica gel chromatography (9% ether / 91% CH2Cl2) afforded 0.21
g of the title compound (73% yield) as a white solid (mp. = 41 — 43 °C). 1H NMR
(500 MHz) (CDCI3): 6 1.19 (s, 9 H), 1.20 (d, J = 6.4 Hz, 3 H), 2.15 — 2.33 (broad
s, 1 H), 2.95 - 3.10 (broad s, 1 H), 3.58 (d, J = 12.4 Hz, 1 H), 3.65 (d, J = 12.3
Hz, 1 H), 3.77 (s, 3 H), 3.78 - 3.83 (m, 4 H), 3.86 (d, J = 11.6 Hz, 1 H), 4.01 (q, J
= 6.3 Hz, 1 H), 6.87 (d, J = 8.6 Hz, 2 H), 7.27 (d, J = 8.4 Hz, 2 H). 136 NMR +
DEPT (125 MHz) (CDCI3): 6 18.3 (—CH3), 28.7 (-CH3), 46.9 (—CH2), 51.6 (—CH3),
55.1 (—CH3), 60.6 (—CH2), 69.5 (quaternary -C), 69.8 (—CH), 74.5 (quaternary --
C), 113.8 (aromatic —CH), 129.0 (aromatic —CH), 132.4 (quaternary -C), 158.6
(quaternary -C), 173.4 (quaternary —C). IR (KBr pellet): 3391 cm", 3337 cm",
2973 cm", 1721 cm", 1617 cm", 1516 cm", 1240 cm", 1044 cm". HRMS

(FAB): m/z calcd for C18H30N05 [M+H], 340.2118; found, 340.2127.

146

EM?)
HN 02Me
""'-OBn

tBuO Me
III-34

Figure Ill-9. DL-(2R, 3R)-Methyl 2-((benzyloxy)methyl)-3—tert—butoxy-2-((4-
methoxybenzyl)amino)butanoate (Ill-34).

DL-(2R, 3R)-Methyl 2-((benzyloxy)methyl)-3-tert-butoxy-2-((4-
methoxybenzyl)amino)butanoate (Ill-34). A solution of Ill-33 (114.8 mg, 0.34
mmol) in DMF (2.5 mL) was treated successively with NaH (60% in mineral oil,
42.1 mg, 1.1 mmol) and benzyl bromide (64.8 mg, 0.38 mmol), and the reaction
stirred under nitrogen atmosphere until complete by TLC (about. 15 minutes).
The reaction was carefully quenched by the addition of saturated NH4CI solution
(5 mL) and then diluted in EtOAc (10 mL). The organic layer was collected and
the reaction was extracted with EtOAc (2 x 20 mL). The combined organic
extracts were washed with 10% LiCl solution (2 x 50 mL) before being dried
(NaZSO4) and concentrated under vacuum. Purification via silica gel
chromatography (2.5% ether / 97.5% CH2Cl2) afforded 0.14 g of the title
compound (96% yield) as an oil. 1H NMR (500 MHz) (CDCI3): 6 1.16 (s, 9 H),
1.20 (d, J = 6.2 Hz, 3 H), 2.11 - 2.21 (broad s, 1 H), 3.64 (d, J = 12.1 Hz, 1 H),
3.72 (s, 3H), 3.78 (d, J = 9.8 Hz, 1 H), 3.80 (s, 3 H), 3.87 (d, J = 12.1 Hz, 1 H),
3.93 (d, J = 9.7 Hz, 1 H), 4.04 (q, J = 6.2 Hz, 1 H), 4.56 (s, 2 H), 6.85 (d, J = 8.6
Hz, 2 H), 7.26 — 7.40 (m, 7 H). 13C NMR + DEPT (125 MHz) (CDCI3): 6 18.7 (-
CH3), 28.8 (—CH3), 47.6 (—CH2), 51.8 (—CH;.), 55.3 (-CH3), 69.7 (quaternary —C),

70.0 (—CH2), 71.3 (-CH), 73.4 (—CH2), 73.9 (quaternary —C), 113.6 (aromatic —

147

CH), 127.5 (aromatic —CH), 127.6 (aromatic -CH), 128.3 (aromatic -CH), 129.4
(aromatic -CH), 133.7 (aromatic quaternary —C), 138.4 (aromatic quaternary -—C),
158.4 (aromatic quaternary —C), 173.9 (quaternary —C). IR (neat): 3355 cm",
2977 cm", 1740 cm", 1513 cm“, 1456 cm", 1248 cm", 1192 cm", 1094 cm".
HRMS (FAB): m/z calcd for C25H35NO5 [M+H], 430.2588; found, 430.2569.

0 (M80...

""'—OBn
/
tBUO Me

Ill-35

Figure Ill-10. DL-(2R, 3R)-Methyl 2-((benzyloxy)methyl)-3-tert-butoxy-2-(N-(4-
methoxybenzyl)acrylamido)butanoate (Ill-35).

DL—(2R, 3R)-Methyl 2-((benzyloxy)methyl)-3-tert-butoxy-2-(N-(4-
methoxybenzyl)acrylamido)butanoate (III-35). A 0 °C solution of Ill-34 (112.9
mg, 0.26 mmol) and diisopropylethylamine (51.9 mg, 0.40 mmol) in CH2C|2 (1
mL) was treated with acryloyl chloride (35.8 mg, 0.40 mmol) and the reaction
stirred under nitrogen atmosphere at 0 °C for 4.5 hours. The reaction was diluted
in CHZClz and was washed successively with 5% HCl solution, saturated
NaHCOa solution, and brine before being dried (Na2804) and concentrated under
vacuum. Purification via silica gel chromatography (20% EtOAc / 80% hexanes)
afforded 119.0 mg of the title compound (94% yield) as an oil. 1H NMR (500
MHz) (CDCI3): 6 1.22 (s, 9 H), 1.34 (cl, J = 6.2 Hz, 3 H), 3.54 (d, J = 7.2 Hz, 1 H),
3.73 (s, 3 H), 3.84 (s, 3 H), 3.99 (d, J = 7.2 Hz, 1 H), 4.21 (d, J = 11.8 Hz, 1 H),
4.35 (d, J = 11.8 Hz, 1 H), 4.55 (q, J = 6.2 Hz, 1 H), 4.67 (d, J = 19.0 Hz, 1 H),

4.92 (d, J = 19.0 Hz, 1 H), 5.59 (dd, J. = 4.8 Hz, J2 = 7.6 Hz, 1 H), 6.33 - 6.37
148

 

(m, 2 H), 6.91 (d, J = 8.7 Hz, 2 H), 7.10 — 7.14 (m, 2 H), 7.22 — 7.29 (m, 5 H).
13C NMR + DEPT (125 MHz) (CDCI3): o 20.0 (-CH;.), 29.0 (-CH3), 48.8 (—CH2),
51.8 (—CH3), 55.3 (—CH;.), 69.5 (quaternary -C), 70.3 (—CH), 70.6 (-CH2), 73.0 (—
CH2), 74.2 (quaternary —C), 114.1 (aromatic —CH), 126.7 (—CH), 127.3 (—CH),
127.4 (—CH), 128.1 (—CH), 128.6 (-CH), 129.0 (—CH2), 131.0 (aromatic
quaternary -C), 137.5 (aromatic quaternary -C), 158.6 (aromatic quaternary —C),
167.6 (quaternary —C), 170.3 (quaternary —C). IR (neat): 2975 cm", 1750 cm",
1653 cm", 1512 cm", 1248 cm", 1188 cm". HRMS (FAB): m/z calcd for

023H33N05 [M+H], 484.2694; found, 484.2716.

0 (M60...

j I‘m-OBn

/HO Me
Ill-7

Figure Ill-11. DL-(2R, 3R)-Methyl 2-((benzyloxy)methyl)-3—hydroxy-2-(N-(4-
methoxybenzyl)acrylamido)butanoate (Ill-7).

DL-(2R, 3R)-Methyl 2-((benzyloxy)methyl)-3-hydroxy-2—(N-(4-
methoxybenzyl)acrylamido)butanoate (Ill-7). A solution of Ill-35 (21.6 mg,
0.045 mmol) in CH2Cl2 (0.5 mL) was treated with aqueous phosphoric acid (85%,
0.03 mL, 0.4 mmol) and the reaction stirred at room temperature for 20 hours.
The reaction was quenched with saturated NaHCO;. (5 mL) solution and diluted
in EtOAc (10 mL), and the organic layer was collected. The aqueous layer was
extracted with more EtOAc (2 x 15 mL) and the combined organic extracts were
dried (M9804) and concentrated under vacuum. Purification via silica gel
chromatography (40% EtOAc I 60% hexanes) afforded 5.2 mg of the title

149

 

compound“ (27% yield) as an oil. 1H NMR (500 MHz) (CDCI3): 6 1.23 (d, J = 6.5
Hz, 3 H), 3.16 (d, J = 7.3 Hz, 1 H), 3.80 (s, 3 H), 3.81 (s, 3 H), 3.83 (d, J = 10.3
Hz, 1 H), 4.00 (d, J = 10.3 Hz, 1 H), 4.38 (s, 2 H), 4.72 (p, J = 6.7 Hz, 1 H), 4.84
(s, 2 H), 5.60 (t, J = 6.2 Hz, 1 H), 6.35 (d, J = 6.0 Hz, 2 H), 6.87 (d, J = 8.6 Hz, 2
H), 7.18 (d, J = 6.6 Hz, 2 H), 7.24 - 7.33 (m, 5 H). 13C NMR (125 MHz) (CDCI3):
6 18.6, 48.7, 52.3, 55.3, 67.4, 69.5, 70.2, 73.5, 114.0, 126.9, 127.5, 127.7, 128.2,
128.4, 129.5, 130.8, 137.5, 158.6, 168.6, 171.7.
0 EM%OzMe
""'-OBn

/ 0 Me
III-8

Figure III-12. DL-(R)-Methyl 2-((benzyloxy)methyl)-2-(N-(4-
methoxybenzyl)acrylamido)-3-oxobutanoate (Ill-8).

DL-(R)-Methyl 2-((benzyloxy)methyl)-2-(N-(4-methoxybenzyl)acrylamido)-3-
oxobutanoate (Ill-8). This reaction was performed according to the known
procedure.“ To a stirring solution of Ill-7 (14.3 mg, 0.033 mmol) in CH2CI2 (0.5
mL) was added Dess-Martin periodinane (17.1 mg, 0.040 mmol) and the reaction
stirred at room temperature for 1.25 hours. The reaction was quenched with a
mixture of saturated aqueous NaHC03 and Na28203 solutions (1 : 1, 3 mL) and
extracted with EtOAC (3 x 5 mL). The combined organic extracts were dried
(M9804) and concentrated under vacuum. Purification via silica gel
chromatography (35% EtOAc / 65% hexanes) afforded 12.6 mg of the title
compound (89% yield) as an oil. The spectral data matches that previously

reported in the literature.“ 1H NMR (500 MHz) (CDCI3): 6 2.45 (s, 3 H), 3.78 —

150

3.82 (m, 5 H), 3.83 (s, 3 H), 4.31 (m, 2 H), 4.79 (d, J = 18.4 Hz, 1 H), 4.94 (d, J =
18.5 Hz, 1 H), 5.68 (dd, J. = 4.5 Hz, J2 = 7.7 Hz, 1 H), 6.38 - 6.42 (m, 2 H), 6.92
(d, J = 8.6 Hz, 2 H), 7.11 — 7.15 (m, 2 H), 7.26 — 7.31 (m, 3 H), 7.34 (d, J = 8.5
Hz, 2 H). 13C NMR + DEPT (125 MHz) (CDCI3): o 28.0 (—CH3), 48.8 (—CH2), 52.9
(-CH3), 55.3 (—CH3), 70.5 (—CH2), 73.7 (—CH2), 77.3 (quaternary —C), 114.1
(aromatic —CH), 127.1 (aromatic -CH), 127.5 (aromatic —CH), 127.9 (aromatic -
CH), 128.4 (aromatic —CH), 130.2 {—CHz), 130.4 (aromatic quaternary —C), 136.7
(aromatic quaternary —C), 158.8 (aromatic quaternary —C), 168.4 (quaternary -

C), 169.1 (quaternary —C), 198.0 (quaternary -C).

..W

IW—OBn

HO Me
Ill-6

Figure Ill-13. DL-(2R, 3R)-Methyl 2-((benzyloxy)methyl)—3-hydroxy-2-((4-
methoxybenzyl)amino)butanoate (III-6).

DL-(2R, 3R)-Methyl 2-((benzyloxy)methyI)-3-hydroxy-2—((4-
methoxybenzyl)amino)butanoate (Ill-6). A solution of III-34 (40.0 mg, 0.093
mmol) in CH2Cl2 (1 mL) was treated with aqueous phosphoric acid (85%, 0.04
mL, 0.6 mmol) and the reaction stirred at room temperature for 6 hours. The:
reaction was quenched with saturated NaHC03 (5 mL) solution and diluted in
EtOAc (10 mL), and the organic layer was collected. The aqueous layer was
extracted with more EtOAc (2 x 15 mL) and the combined organic extracts were
dried (M9804) and concentrated under vacuum to cleanly afford 34.0 mg of the

title compound (98% yield) as an oil without any further purification needed. The

151

 

spectral data matches that previously reported in the literature.“ 1H NMR (500
MHz) (CDCI3): 6 1.16 (d, J = 6.4 Hz, 3 H), 2.06 (broad s, 1 H), 3.37 (broad s, 1
H), 3.65 (d, J = 11.9 Hz, 1 H), 3.72 (d, J = 11.9 Hz, 1 H), 3.78 (s, 3 H), 3.82 (s,
3H), 3.83 (d, J = 9.8 Hz, 1 H), 3.88 (d, J = 9.8 Hz, 1 H), 4.00 (q, J = 6.4 Hz, 1 H),
4.56 (d, J = 2.3 Hz, 2 H), 6.88 (d, J = 8.5 Hz, 2 H), 7.24 — 7.40 (m, 7 H). 13C
NMR + DEPT (125 MHz) (CDCI3): 6 17.9 (—CH3), 47.4 (—CH2), 52.0 (-CH3), 55.3
(—CH;.), 69.3 (quaternary -C), 69.7 (—CH), 70.5 (—CH2), 73.5 (—CH2), 113.8
(aromatic —CH), 127.7 (aromatic -CH), 127.8 (aromatic —CH), 128.4 (aromatic —
CH), 129.4 (aromatic -CH), 132.4 (quaternary —C), 137.6 (quaternary -C), 158.7
(quaternary —C), 173.2 (quaternary —C). IR (neat): 3357 cm", 2926 cm", 1734
cm", 1514 cm", 1248 cm", 1100 cm". HRMS (FAB): m/z calcd for C21H28N05

[M+H], 374.1962; found, 374.1972.

Ph\«0\:
Nfcone
tBuO Me
III-38

Figure Ill-14. DL-(R)-methyl 4-((R)-1-tert-butoxyethyl)-2-
phenyl-4,5-dihydrooxazole-4-carboxylate (Ill-38).

DL—(R)-methyl 4-((R)-1-tert-butoxyethyl)-2-phenyI-4,5-dihydrooxazole-4-
carboxylate (Ill-38). Ill-38 has been prepared in our lab by various methods,
and the optimized procedure is as follows: A 0 °C solution of lI-6A (1.24 g, 3.85
mmol) and triethylamine (5.4 mL, 39 mmol) in anhydrous CH2CI2 (125 mL) was
treated with methanesulfonyl chloride (0.90 mL, 12 mmol), and the reaction

stirred under nitrogen atmosphere while it warmed to room temperature. The

152

reaction was then refluxed until complete, as observed by TLC (about 20 hours).
The reaction was neutralized with 30% aqueous HCI solution and was extracted
with CH2CI2 (3 x 150 mL). The combined organic extracts were dried (M9804)
and concentrated under vacuum. Purification via silica gel chromatography (7%
ether / 93% CH2Cl2) afforded 1.17 g of the title compound (solid, mp. = 68 — 69
°C, 99% yield). 1H NMR (500 MHz) (CDCl3): 6 1.04 (d, J = 6.2 Hz, 3 H), 1.09 (s,
9 H), 3.74 (s, 3 H), 4.45 (q, J = 6.2 Hz, 1 H), 4.57 (d, J = 9.5 Hz, 1 H), 4.93 (d, J =
9.5 Hz, 1 H), 7.33 (t, J = 7.6 Hz, 2 H), 7.42 (t, J = 7.4 Hz, 1 H), 7.90 - 7.94 (m, 2
H). 13C NMR (125 MHz) (CDCI3): 6 17.0, 28.5, 52.3, 69.4, 69.9, 73.9, 82.3,
127.0, 128.1, 128.4, 131.5, 164.8, 172.2. IR (neat): 2978 cm", 1742 cm", 1640
cm", 1264 cm", 1086 cm", 1069 cm". HRMS (FAB): m/z calcd for C17H24NO4
[M+H], 306.1705; found, 306.1703.

OHCQ 002MB

HN Me

t
0% PhO Bu

Ill-39

Figure III-15. DL-(28,3R)-methyl 2-benzamido-3-tert-butoxy-2-
formylbutanoate (Ill-39).

DL-(28,3R)-methyl 2-benzamido-3-tert-butoxy-2-formylbutanoate (Ill-39).
DMSO (0.38 mL, 5.3 mmol) was added dropwise to a stirring solution of oxalyl
chloride (0.23 mL, 2.6 mmol) in CH2Cl2 (4 mL), cooled to -78 °C, and the reaction
stirred under N2 atmosphere for 30 minutes. A solution of lI-6A (0.5619 g, 1.7
mmol) in CHZCIZ (3 mL) was added dropwise and the reaction stirred at -78 °C for

45 minutes. TEA (0.64 mL, 4.6 mmol) was then added and the reaction stirred at

153

-78 °C for 5 minutes before warming to room temperature. The reaction was
diluted with H20 (8 mL) and extracted with CH2CI2 (4 x 15 mL). The combined
organic extracts were dried (M9804) and concentrated to an oil, which was used
immediately in the next step. However, purification of the crude reaction via
silica gel chromatography (25% EtOAc / 75% hexanes) affords Ill-39 as an oil.
1H NMR (500 MHz) (CDCI3): 6 1.19 (s, 9 H), 1.28 (d, J = 6.2 Hz, 3 H), 3.79 (s, 3
H), 4.41 (q, J = 6.2 Hz, 1 H), 7.04 (s, 1 H), 7.43 (t, J = 7.5 Hz, 2 H), 7.51 (t, J =
7.5 Hz, 1 H), 7.80 (d, J = 7.1 Hz, 2 H), 9.89 (s, 1 H). 13C NMR + DEPT (125
MHz) (CDCI3): 6 19.2 (—CH;;), 28.6 (—CH3), 52.7 (—CH3), 68.9 (-CH), 72.5
(quaternary —C), 75.4 (quaternary —C), 127.1 (aromatic —CH), 128.5 (aromatic —
CH), 132.0 (aromatic -CH), 132.9 (aromatic quaternary —C), 167.5 (quaternary —
C), 167.9 (quaternary —C), 194.2 (quaternary —C). IR (neat): 3337 cm", 2880
cm", 1730 cm", 1653 cm", 1260 cm". HRMs (FAB): m/z calcd for C17H24N05
[M+H], 322.1654; found, 322.1663.

8n

'3th Me
N""'
MeOzC O‘Bu
Ill-36

Figure Ill-16. DL-(R)-methyl 1-benzyl-4-((R)-1-(tert-butoxy)ethyl)-2-phenyl-4,5-
dihydro-1H-imidazole-4-carboxylate (III-36).

DL-(R)-methyl 1-benzyl-4-((R)-1-(tart-butoxy)ethyI)-2-phenyl-4,5-dihydro-1H-
imidazole-4-carboxylate (III-36). To a stirring solution of crude Ill-39 in 1,2-

dichloroethane (12 mL) were added benzylamine (0.24 mL, 2.2 mmol) and

154

NaBH(OAc)3 (0.5102 g, 2.4 mmol), and the reaction stirred at room temperature
overnight. The reaction was quenched with NaH003 (25 mL) and extracted with
CH2Cl2 (3 x 30 mL). The combined organic extracts were dried (M9804) and
concentrated, and purification via silica gel chromatography (50% EtOAc / 50%
hexanes —> 80% EtOAc / 20% hexanes) was performed to give 0.5524 g of the
title compound (80% yield) as an off-white solid (mp. = 96 — 99 °C). 1H NMR
(500 MHz) (CDCI3): 6 1.11 — 1.15 (m, 12 H), 3.60 (d, J = 11.0 Hz, 1 H), 3.78 (s, 3
H), 4.09 (d, J = 11.0 Hz, 1 H), 4.30 (d, J = 15.8 Hz, 1 H), 4.40 (d, J = 15.8 Hz, 1
H), 4.47 (q, J = 6.1 Hz, 1 H), 7.25 — 7.32 (m, 3 H), 7.35 — 7.45 (m, 5 H), 7.56 -
7.60 (m, 2 H). 13'C NMR + DEPT (125 MHz) (CDCI3): 6 17.3 (—CH3), 28.8 (—CH;.),
51.6 (—CH2), 52.2 (—CH3), 52.3 (—CH2), 70.5 (—CH), 73.7 (quaternary —C), 80.4
(quaternary —C), 127.0 (aromatic -CH), 127.4 (aromatic —CH), 128.39 (aromatic
-CH), 128.40 (aromatic —CH), 128.7 (aromatic —CH), 130.2 (aromatic -CH),
130.4 (aromatic quaternary -C), 137.4 (aromatic quaternary -C), 166.1
(quaternary —C), 173.7 (quaternary -C). IR (KBr pellet): 2978 cm", 1728 cm",
1593 cm", 1260 cm". HRMs (FAB): m/z calcd for C24H30N203 [M+H],
395.2335; found, 395.2339. Anal. Calcd. For C24H31N203: C, 73.07; H, 7.66; N,
7.10. Found: C, 72.96; H, 7.77; N, 7.17.
8n
thl: Me

MGOZC OH
III-41

Figure Ill-1 7. DL-(R)-methyl 1-benzyl-4-((R)-1-hydroxyethyl)-2-phenyl-4,5-
dihyd ro-1 H-imidazoIe-4-carboxylate (III-41).

155

DL-(R)-methyl 1-benzyI-4-((R)-1-hydroxyethyI)-2-phenyl-4,5-dihydro-1H-
imidazoIe-4-carboxylate (Ill-41). A solution of Ill-36 (0.5524 g, 1.4 mmol) in
CHZCIZ (8.0 mL) was treated with aqueous phosphoric acid (85%, 0.49 mL, 8.4
mmol) and the reaction stirred at room temperature for 6 hours. The reaction
was quenched with saturated NaHCO;. solution (25 mL) and diluted in CH2Cl2 (15
mL), and the organic layer was collected. The aqueous layer was then extracted
with CH2C|2 (3 x 40 mL), and the combined organic extracts were dried (M9804)
and concentrated under vacuum to cleanly afford 0.4467 g of the title compound
(84% yield) as a white solid (mp. = 132 — 134 °C) without any further purification
needed. 1H NMR (500 MHz) (CDCI3): 6 1.18 (d, J = 6.4 Hz, 3 H), 2.64 (broad s,
1 H), 3.59 (d, J = 10.6 Hz, 1 H), 3.77 - 3.81 (m, 4 H), 4.23 (q, J = 6.4 Hz, 1 H),
4.35 (d, J = 2.7 Hz, 2 H), 7.21 — 7.48 (m, 8 H), 7.59 — 7.62 (m, 2 H). 13C NMR +
DEPT (125 MHz) (CDCI3): 6 17.0 (—CH;.), 52.0 (—CH2), 52.5 (—CH:.), 53.8 (—CH2),
70.8 (—CH), 79.1 (quaternary —C), 127.0 (aromatic —CH), 127.6 (aromatic -CH),
128.4 (aromatic —CH), 128.6 (aromatic —CH), 128.8 (aromatic -CH), 130.1
(aromatic quaternary —C), 130.4 (aromatic -CH), 136.9 (aromatic quaternary —C),
167.4 (quaternary -C), 174.3 (quaternary —C). IR (neat): 3202 cm", 2960 cm",
1735 cm", 1560 cm", 1257 cm". HRMS (FAB): m/z calcd for C20H23N203 [M+H],

339.1709; found, 339.1717.

156

 

Bn
N
PhW Me
NIH--
HOZC OH

III-42

Figure Ill-18. DL-(R)-1-benzyl-4-((R)-1-hydroxyethyl)-2-phenyl-4,5-dihydro-1 H-
imidazole-4-carboxylic acid (Ill-42).

DL-(R)-1-benzyl-4-((R)-1-hydroxyethyl)-2-phenyI-4,5-dihydro-1H-imidazole-

4-carboxylic acid (Ill-42). To a stirring solution of Ill-41 (27.5 mg, 0.081 mmol)
in THF (0.5 mL) and H20 (0.3 mL) was added LiOH'H20 (4.3 mg, 0.10 mmol) and
the reaction stirred at room temperature for 45 minutes. The reaction was made
acidic with concentrated HCI (about 3 drops) and concentrated under vacuum to
give a white residue, which was shown to be free of organic contaminants by 1H-
NMR. Removal of the inorganic salts was performed by dilution of the residue in
lPrOH and CHCI3 (1 : 9, 10 mL) followed by filtration of the resultant suspension
and concentration of the filtrate to give 25.0 mg of the product (95%) as a white
solid (mp. = 138 - 142 °C). 1H NMR (500 MHz) (CD300): 6 1.27 (d, J = 6.5 Hz,
3 H), 4.08 (q, J = 6.5 Hz, 1 H), 4.23 (dd, J. = 12.1 Hz, J2 = 15.4 Hz, 2 H), 4.64 (d,
J = 15.8 Hz, 1 H), 4.75 (d, J = 15.7 Hz, 1 H), 7.26 (d, J = 15.7 Hz, 2 H), 7.33 -
7.41 (m, 3 H), 7.67 (t, J = 7.8 Hz, 2 H), 7.75 — 7.79 (m, 3 H). 13C NMR + DEPT
(125 MHz) (CDgOD): 6 17.9 (-CH3), 51.8 (-CH2), 56.8 (—CH2), 70.4 (—CH), 73.7
(quaternary —C), 123.7 (aromatic quaternary -C), 128.7 (aromatic -CH), 129.8
(aromatic -CH), 129.8 (aromatic —CH), 130.3 (aromatic -CH), 130.8 (aromatic —

CH), 134.6 (aromatic quaternary -C), 134.9 (aromatic —CH), 168.4 (quaternary -

157

C), 172.2 (quaternary —C). IR (neat): 3218 cm", 2915 cm", 1732 cm", 1595 cm'
1, 1556 cm". HRMS (FAB): m/z calcd for C19H21N203 [M+H], 325.1552; found,

325.1555.

Figure Ill-19. DL-(3R,4R)-7-benzyl-3-methyl-6-phenyl-2-oxa-5,7-
diazaspiro[3.4]oct-5-en-1-one (Ill-37).

DL-(3R,4R)-7-benzyI-3-methyl-6-phenyl-2-oxa-5,7-diazaspiro[3.4]oct-5-en-1-

one (Ill-37). To a stirring suspension of Ill-42 (0.1185 g, 0.37 mmol) in CHCI3
(2.8 mL) were successively added TEA (0.15 mL, 1.1 mmol) and BOP-Cl (0.1167
g, 0.46 mmol), and the reaction mixture stirred vigorously at room temperature
under N2 atmosphere for 1.5 hours. The reaction was then diluted with CH2CI2 (7
mL) and washed with ice cold H20/brine mixture (5:1, 15 mL). The organic layer
was collected and the aqueous layer was extracted with CH2C|2 (2 x 15 mL). The
combined organic layers were dried (Na2804) and concentrated to a white
residue. Purification of the residue via silica gel chromatography (2% TEA I 35%
EtOAC / 63% hexanes on silica gel pretreated with TEA) afforded 30.1 mg of a 3 :
1 mixture containing the title compound (<4%) and lV-1. There appeared to be a
considerable amount of overlap between NMR peaks of III-37 and IV-1,
especially in the aromatic regions, such that only partial NMR data could be

extracted for Ill-37. 1H NMR (500 MHz) (CDCI3): 6 1.43 (d, J = 6.4 Hz, 3 H), 3.62

158

(d, J = 11.5 Hz, 1 H), 3.77 (d, J = 11.5 Hz, 1 H), 4.38 (d, J = 15.9 Hz, 1 H), 4.45
(d, J = 15.8 Hz, 1 H), 4.91 (q, J = 6.6 Hz, 1 H). 13C NMR (150 MHz) (CDCI3):
15.5, 50.7, 80.4, 84.5, 126.9, 127.9, 128.7, 129.0, 129.3, 130.9, 136.4, 168.8,

171.8.

4. Procedure for proteasome inhibition assay. The 2-imidazolines and
oxazinone/imidazolidine adducts prepared in this Chapter were tested for their
ability to inhibit the chymotryptic-like activity of the 208 proteasome in an in vitro
assay using purified human recombinant 20S proteasome (Boston Biochem,
Cambridge, Ma.) and the fluorogenic peptide substrate Suc—LLW-AMC (Boston
Biochem, Cambridge, Me.) All proteasome inhibition assays were performed by
Teri L. Lansdell according to a modification of known proceduresm'121
The modified procedure is as follows: An activated enzyme solution was first
prepared through addition of 208 proteasome to a reaction buffer (50 mM Tris-
HCI, pH 7.5, 0.03% SDS) to give a final concentration of 1 nM. To each well of a
black, clear bottom 96-well plate (Corning, Inc.) was added 100 uL of the
activated enzyme solution. Compounds being evaluated were diluted in neat
DMSO and 1 pL of test agent or vehicle (DMSO) was added to each well of the
96-well plate; compounds were evaluated at 20, 10, 5, 2.5, 1.3, and 0.6 uM final
concentrations. Enough chymotryptic substrate Suc-LLW-AMC was then added

to each well to give a final concentration of 100 (M. The rate of peptide

hydrolysis (Vmax) was determined using a Spectramax M5e (Molecular Devices,

159

Sunnyvale, Ca.) at 37° C over 30 minutes (A... 380, Am 440). The initial linear

portions of the curves were used to calculate the IC50 values.

160

 

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173

 

 

CHAPTER IV
SYNTHESIS OF NOVEL MULTICYCLIC OXAZINONEIIMIDAZOLIDINE
ADDUCTS FROM AN ANNULATION REACTION OF 2-IMIDAZOLINES

A. Reactivity of 2-imidazolines. .

The reactivity of 2-imidazolines arises from the basic amidine functionality
present within the heterocyclic scaffold."2 Both amidine nitrogen atoms react
with acids and electrophiles to generate a variety of substituted 2-imidazolines,
including those obtained from N-alkylation, N-acylation, and N-sulfonylation
reactions (Scheme lV-1).1 Substitution of both nitrogen atoms results in the
formation of 2-imidazolinium salts, many of which are isolable and stable. 2-
imidazolinium salts differ in reactivity from the precursor 2-imidazolines and are
susceptible to nucleophilic attack at the CZ or amidinium carbon (Scheme IV-1).
Thus exposure of 2-imidazolinium salts to nucleophiles such as alcohols, amines,
hydrides, malonate derivatives, and Grignard reagents results in the formation of
a wide variety of imidazolidine adducts?"28 Such imidazolidines have been used

2323 as well as synthetic precursors to N-heterocyclic

as organocatalysts
carbenes.23’25 Also characteristic of 2-imidazolines is their ability to undergo
hydrolysis when heated in basic or acidic aqueous solutions to give ring-opened

acyclic B-aminoamides (Scheme lV-1)."29

174

E E

 

Roll 69 RYN 56> M

l1 M $2
E
O
R/ILN/VNHR

t 9 R N
RYN Nu
(G) ———> X
N—7 NU N"7 NU = H20 é
R R

Scheme IV-1. Reactivity of 2-imidazolines and 2-imidazolinium salts.

Utilizing the combination of nucleophilic and electrophilic characteristics of
2-imidazolines in single-pot reactions has led to a number of interesting
transformations. Molecules bearing both nucleophilic and electrophilic properties
react with 2-imidazolines to generate novel annulation products. The earliest
such reaction was reported in 1966 by Dr. Feinauer and involved heating
epoxides in the presence of 2-imidazolines to form bicyclic products (Scheme IV-
2).4 The reaction likely proceeds through stepwise ring opening of the epoxide
by the amidine nitrogen followed by annulation via nucleophilic attack of the
resultant alkoxide to furnish the oxazolidine ring. Carmichael and co-workers
have recently synthesized a related imidazolidine-oxazolidine ring system
(Scheme lV-2). Their synthesis initiated with monoalkylation of ethylenediamine
with an epoxide, followed by imidazolinium ring formation. The resultant hydroxy
imidazolinium salt was then treated with BuLi to furnish the bicyclic adduct

through alkoxide attack onto the amidinium carbon (Scheme lV-2).25

175

 

 

 

Feinauer Synthesis

 

 

 

 

 

Ph EL FR 09 - R _‘Z_O
Et N ____R, 1 Et ___, tph XH R
\Elj A N/AN'Ph (:N \_,N
\_/
- - 18-92% 51-97%
BuLi
hexanes
Carm'chaelS th ' PhMe
I yn esus 1.HC|
2. HC(OMe)3 OHH
N PhMe, 90°C
H2N\_/NHR (OHNH NHR T NANR
3. Nal, MeZCO N69 9
44-67%|

Scheme lV-2. Bicyclic adducts derived from 2-imidazolines and epoxides.

2-imidazolines have also been shown to be progenitors of azomethine
ylides that can be used in cycloaddition reactions. Jones and co-workers have
demonstrated that N-alkylation with an appropriate alkyl halide generates 2-

imidazolinium salts.19'22

These particular 2-imidazolinium salts react as
azomethine ylides in cycloaddition reactions when treated with a base and
electron deficient alkenes, resulting in the formation of bicyclic adducts (Scheme
lV-3). The authors’ strategy relies on N-alkylation of 2-imidazolines with alkyl

halides bearing electron-withdrawing groups, such that azomethine ylides may

later be generated for the formation of annulation products.

176

 

 

 

 

 

 

(Ph l/Ph
N BrAEWG (N
> \
E.) EWG=cozR, 89%—7
COR, CN EWG
R
)\EWG
R = Me, Cl, H
v DBU
Ph — -
R EWGNr (Ph
‘ j R ,EWGN
N *—— f "(_7
ewe EWG‘i;g9 J
29-73% '

Scheme lV-3. Reaction of azomethine ylides generated from 2-imidazolines.

B. Synthesis of multicyclic imidazolidine/oxazinone adducts.

As previously described in Chapter III of this dissertation, the synthesis of 2-
imidazoline Ill-37 from lIl-42 proceeded in low yields, and the desired product
was isolated along with an unexpected product. X-ray crystal analysis revealed
the structure of this unexpected product to be tetracyclic imidazoline lV-1
(Scheme lV-4 and Figure lV-1). lV-1 was formed as the major product in the
reaction (in 44% yield), and interestingly it was found to inhibit the chymotrypsin-

like activity of the 208 proteasome at low micromolar concentrations.

177

 

Bn '3" Ph //Ph

 

ph N BOP-Cl(1.2 eq) Pkg” Me GYMN M
Tl».- TEA (3 eq) - fo + fiN N]: e
' Ph ’N
H020 OH CHC'a o O phf 0 o O
rt, 1 hr <4% isolated 44%
Ill-42 Ill-37 lV-1

Scheme lV-4. Synthesis of 2-imidazolines comprising a B-lactone.

The potency exhibited by IV-1 as an inhibitor of the chymotrypsin-like
activity of the 208 proteasome was found to rival that of most of the 2-
imidazolines thus far evaluated in our lab. Naturally, we were interested in
determining the extent to which certain structural components present in lV-1
contributed to its efficacy as a proteasome inhibitor. The three components
comprising IV-1 which were speculated to contribute most to the biological
activity were the amidine, the B-lactone, and the fused bicyclic
oxazinone/imidazolidine ensemble. Of these components, the unique fused
bicyclic oxazinone/imidazolidine ensemble is arguably the most visually striking.
Little is known about the reactivity or general biological profile of this scaffold
which has received scarce literature exposure.”31 We decided, then, to pursue
the construction of analogs of lV-1 as a means of investigating the novel scaffold
and its potential role in biological processes.

Initially, we focused our attention towards the formation of lV-1, as we
anticipated similar reactions would give rise to structurally related analogs. The
oxazinone ring in multicyclic adduct lV-1 is believed to arise from stepwise

intermolecular amide formation followed by annulation. Treatment of carboxylic

179

 

acid Ill-42 with TEA and BOPCI initially generates a mixed BOP-anhydride or
acid chloride intermediate (Scheme lV-5). lnterrnolecular amide formation is then
believed to occur through attack of the nucleophilic amidine onto the carboxylic
acid derivative to give rise to lV-2. Formation of the C-N bond would result in an
enhancement of electrophilicity at CZ, such that annulation should proceed to

afford the oxazinone ring present in lV-1.

 

 

Bn
Me 0“ BOP-Cl Me OH Ph)—N
TEA O-BOP NI \
anN 8"\N s V 7\
Ph Ph
Ill-42
Me 0 PhNBn — Me 0H3“ Bn—
+ § \.>@N
Bn~N N+R,‘—— 3an N ‘
FN 0 R2 FM 0 R>\R1
Ph Ph 2
lV-1 _ IV-2

 

 

Scheme lV-5. Oxazinone formation from stepwise amidation and annulation.

Novel heterocyclic adduct lV-1 was isolated as a single diastereomer, a
finding that warranted further investigation into the mechanism of its formation.
Interestingly, only heterodimer lV-1 was observed in the reaction (the relative
stereochemistry is depicted in Figure lV-1), which arises from the interaction of
one enantiomer of Ill-42 with the opposite enantiomer. One possibility for the
observed selectivity involves initial B-Iactone formation, followed by
intermolecular attack of the labile lactone by III-42 (or Ill-37) for the formation of

IV-1. However, when the reaction progress was monitored via kinetic 1H-NMR

180

studies it was observed that 2—imidazolines Ill-37 and lV-1 accumulated at a
similar rate and in overall equimolar amounts, suggesting that Ill-37 is likely not
consumed for the formation of lV-1. In light of similar rates of formation of Ill-37
and IV-1, it is envisioned that the 2—imidazoline approaches the activated
carbonyl from either face, leading to two interconvertible rotamer-like
intermediates (IV-2A and lV-ZB, Scheme lV-6). IV-2A is envisioned to undergo
facile ring formation via a chair transition state to generate the 6-membered
oxazinone ring. However, analogous oxazinone ring formation from intermediate
lV-ZB is less favorable, as ring formation would arise by means of a boat or twist-
boat transition state. Instead, it is proposed that the alcohol of lV-ZB intercepts
the imidazolinoyl amide carbonyl to form B-lactone Ill-37. However, B-lactone
formation from intermediate lV-2A is also plausible, and therefore cannot be
ruled out. Alternatively, it is possible that IV-2A and lV-ZB are preorganized into
chair conformations mediated by H-bonding between the activated carbonyl
oxygen and the nucleophilic alcohol.32 Such preorganized chair conformations
are predicted to be thermodynamically favorable, albeit oxazinone ring formation
from these conformations is anticipated to proceed through a twist-boat transition
state. Finally, the B-lactone ring present in lV-1 is believed to originate from
intramolecular attack of the alcohol onto the BOP-activated carbonyl, although it
has not been determined whether this lactone ring forms before or after

intermolecular adduct formation.

181

‘ A ’8" 0‘ :1 R2
011/ N 9%
Bn—N 0.99") Bn-N c. YNBn
/N R1 R2 / Ph
Ph Ph
IV-2A lV-ZB
Pth '3“
Me 0 N Ph N
NT
1 O 0
Ph
IV-1 Ill-37

Scheme lV-6. Proposed mechanism of selective oxazinone ring formation.

The mechanism proposed for the formation of lV-1 suggested that Ill-42
should react with various 2-imidazolines under similar reaction conditions.
Treatment of an in situ-generated mixed anhydride or acid chloride derived from
Ill-42 with different 2-imidazolines was anticipated to result in the formation of
adducts varying by substitution about the resultant imidazolidine ring. Our
hypothesis was tested by treating Ill-42 with lV-3 under basic conditions at room
temperature (Scheme lV-7). The desired product obtained from the use of 2-
imidazoline lV-3 was expected to contain all structural features present in lV-1
except the B—lactone moiety. Thus, the reaction would also to serve to probe the
extent to which the B-lactone contributed to the potency of IV-1 as a proteasome

inhibitor. Three equivalents of IV-3 were used in the annulation reaction to favor

182

 

 

 

the formation of the desired muticyclic adduct relative to Ill-37 and lV-1. To our
delight, the reaction afforded 69% yield of lV-4 as a 12 : 1 ratio of diastereomers,
along with minor amounts of Ill-37 and IV-1. The proposed relative
stereochemistry in each of the newly formed chiral centers in IV-4A and lV-4B
was determined via nOe analysis, as shown in Figure IV-2. Major diastereomer
lV-4A is proposed to arise from a transition state analogous to that for the
formation of lV-1. Additionally, diastereomer IV-4A was discovered to undergo
epimerization upon standing in CDCI3 for 2 weeks, thereby affording primarily
diastereomer IV-4B. The epimerization is believed to occur due to the trace acid
present in CDCI3, which promotes ring opening of the oxazinone ring via
protonation of the ether oxygen (Scheme lV-7). An similar observation has been
reported by Carmichael and coworkers, who treated their bicyclic ring system
(Scheme lV-2) with CDCI3, resulting in deuterium incorporation, presumably

through a ring-opened intermediate.25

183

 

 

 

 

Ph>_NBn
N1)
Bn (3 9%) Me 0+,“
Ph N BOP-C|(1.4 eq)
Tl TEA(3.5eq)_ Bn~N 451 NJ
HOZC OH CHCI3, rt, 2.5 hr pr?’ 0
III-42 69%, 12 : 1 d.r. IV-4A
(lV-4A:lV-4B)
1CDCl3
2weeks
Bn F Bn—
Me 0 EhN' ODPh N
U Me \er
BM .3 N ‘='— . fl
Ph 0 Bn’NxfN 0
lV-4B Ph

 

 

 

lV-4A IV-4B

Figure lV-2. Major nOe correlations for lV-4A and IV-4B.

The formation of lV-4 under our reaction conditions prompted our

investigation into the use of other reactive B—hydroxy carboxylic acid derivatives.

184

Exhaustive literature searches have revealed only one incidence of an annulation
reaction between a B-hydroxy carboxylic acid derivative and a 2-imidazoline.
The lone example was reported by Kollenz and co-workers, who demonstrated
that 2-thioethyl-2-imidazoline reacts with two equivalents of salicyloyl chloride
when heated in xylene to give a symmetrical multicyclic adduct (Scheme lV-8).30
In agreement with Kollenz’s results, we have observed that a bicyclic adduct lV-5
is available from the treatment of salicyloyl chloride, generated in situ from

salicylic acid and BOP-Cl, with lV-3 under basic conditions (Scheme lV-8).

Kollenz reaction

 

0
Cl 0 1‘1 0
EtS H N N
Yj OH > OX0
N Xylene
A
29%
Our Observation
Ph Bn
N
j: Ph
OH NJ Phr

 

o
CEKOH ”'3 - Elfin/fl)
BOPCIV
O TEA, CHCI3 0
rt, 3 h lV-5
63%

Scheme IV-8. Oxazinone/imidazolidine adducts derived from salicylic acid.

We proposed that oxazinone annulation products should also be available
from 2-imidazolines and simple B-hydroxy carboxylic acids. We decided to test

our hypothesis using the simple amino acid derivative N-Boc—L-threonine in the

185

reaction with 2-imidazoline lV-3. As anticipated, cyclization proceeded smoothly
to afford lV-6 in 94% yield as a single enantiomer (Scheme lV—9). The
stereochemistry of the new stereocenter was determined via nOe analysis and
was revealed be opposite of that observed in lV-1. It is believed that the
annulation proceeds through a chair transition state, the conformation of which
allows the sterically demanding methyl group to occupy an equatorial position.
Such a transition state favors the stereochemistry observed in IV-6 (Scheme IV-
10). The results obtained from the stereoselective formation of lV-6 encouraged
us to also investigate the use of N-Boc-L-serine in the annulation reaction with
lV-3. As anticipated, the annulation reaction proceeded smoothly to afford
bicyclic adduct lV-7 as a single enantiomer in 94% yield (Scheme lV-9).
Interestingly, nOe analysis revealed that the stereochemistry of the new
stereogenic was trans with respect to the carbamate. This annulation reaction is
proposed to proceed through a chair transition state, wherein the carbamate
occupies an equatorial position, to give the sterically strained cis disubstituted
oxazinone ring (Scheme lV-10). Subsequent rapid epimerization of the new
stereocenter, as was observed in the epimerization of lV-4, would then result in a
reduction of steric strain to give thermodynamically favored epimer lV-6.
Moreover, further epimerization of IV-6 or IV-‘I has not been observed, even after

allowing each compound to stand in CDCl3 at room temperature for two weeks.

186

 

OH OH
COzH KrCOZH 0th 3n

th
OP \FhN NHBoc Ph ,Bn NHBoc \FN
BocHN NJ BOP-Cl, TEA >rN BOP Cl TEA Bo GHNLWN J
o CHCl3, rt, 4 hr Nx) CHCI3, rt, 3 hr
l-6V 94% IV-3 94% lV-7

 

...-On.

Observed Observed
"De NKQ "De NKQ
Mb OTN M OTN
Boa-TIN Bocii-IN O

"mmmuuwww—uuwm—cww‘hflflmuu www—w-—-——uu———uu—un——m—-iunnmuu—mma—

m.m.m'=1 5.7—3:73;

 

 

 

 

 

 

 

NHBoc Bn
Me‘ OH PNh
NA>/\ _. 19:)
o 59me BocHN
Ph
lV-6
TN
/ ““OH ~—
®"\'\/N-Bn BocHN NJ BocHN’Eny J
o

IV-7

Scheme lV-10. Proposed origin of selectivity in the formation of lV-6 and lV-7.

C. Towards Chiral Resolution of Racemic 2-Imidazolines
Several studies in the Tepe lab have involved the synthesis of 2-imidazolines for
use in pharmaceutically relevant assays.33‘35 The majority of these compounds

have been prepared by means of a diastereoselective Lewis-acid catalyzed

187

dipolar cycloaddition reaction of oxazol-5(4H)-ones and imines, as was described

in Chapter I of this dissertation.”38

This chemistry allows for the rapid
production of a variety of racemic 2-imidazolines. However, ongoing studies in
our lab have required the use of enantiopure materials, and chiral HPLC has
been utilized to accommodate our needs. Unfortunately, our studies sometimes
require the use of multigram amounts of enantiopure 2-imidazolines, and such
large amounts have been difficult to obtain using our current methods. Our lab
has been interested, then, in developing chemical methods which might help us
to procure enantioenriched or enantiopure 2-imidazolines.

Recent developments in our lab have demonstrated stereoselective
adduct formation from an annulation reaction of 2-imidazolines. As was
previously discussed, only a single diastereomer was observed to arise from the
annulation reaction of either N-Boc—L-threonine or N-Boc-L-serine with lV-3.
Interestingly, preliminary studies indicate that the use of racemic cis-4,5-diphenyl
2-imidazoline IV-8 in the reaction with N-Boc—L-threonine results in the formation
of two products comprising a bicyclic oxazinone/imidazolidine ensemble
(Scheme IV-11). The two products were formed in nearly equal amounts in the
unoptimized reaction and are presumed to be diastereomers. Unfortunately, due
to difficulties encountered during purification, only one product was cleanly

isolated, the stereochemistry of which has not been unambiguously established

(IV-9, Scheme IV-11).

188

OH

 

COZH B
B n
ph N n NHBoc Me OQhN
E P“ BOP-CL TEA> N P“ + Diastereomer?
CHCI3, rt, 9 hr BocHN
Ph 21% yield 0 Ph
lV-8 of M9 IV-9

Scheme IV-11. Synthesis of IV-9 derived from IV-8.

We were encouraged by the results from the above reaction, and we
believed that such a reaction might be useful for the chiral resolution of racemic
2-imidazolines prepared in our lab. It is anticipated that racemic 2-imidazolines
of interest in our studies should undergo annulation reactions to afford separable
optically active oxazinone/imidazolidine adducts. Adducts prepared through
chemistry have been shown to be sensitive to acid and have been observed to
undergo epimerization, presumably through a ring-opened intermediate (Scheme
IV-7). This observation has prompted our investigation into acid-mediated
decomposition reactions of oxazinone/imidazolidine adducts as a means of
regenerating 2-imidazolines. It is envisioned that an annulation/decomposition
sequence would serve as a means of chiral resolution for racemic mixtures of 2-
imidazolines prepared in our lab, thereby obviating the need to separate

enantiomers via our current method involving chiral HPLC.

189

Me O N
E R1
Ph Bn BocHN éRz Ph Bn
T R1 0 3 T R1
”4.82 ::> + Ph B" :> ”£232
R3 Me Ong R3
optically pure N "R1 (+/-)
BocHN "R2

Mixture of Diastereomers

Scheme lV-12. Strategy for the resolution of 2-imidazoline enantiomers.

To this end, oxazinone/imidazolidine adduct decomposition has been
successfully demonstrated. Decomposition of IV-6 was initially attempted
through treatment with Bronsted acids in MeOH. Such reactions proceeded to
fully consume all starting material, but no 2-imidazoline was ever recovered in
the reactions, presumably due to 2-imidazoline hydrolysis."29 Oxazinone adduct
decomposition was then attempted via treatment with Lewis acids in a non-
nucleophilic solvent; similar conditions have been effective for the Lewis acid-
mediated cleavage of N-acyl hemiaminal ethers.”41 Thus, treatment of IV-6 with
one equivalent of TiCl4 in anhydrous CH2Cl2 followed by a water workup afforded
a 2.4 : 1 ratio of lV-6 to IV-3 with an unknown contaminant. Furthermore, the
analogous decomposition of lV-7 has been optimized to cleanly yield IV-3 in

greater than 90% yield (Scheme lV-13).

190

 

 

1. TiCI4 (1.5 eq)

 

Bn 0
o ghN CH2CI2,-41 Ctort 5n
\NrJ overnIght _ PhTN
BocHN 2. TEA (excess). NJ
0 then NaHCO3 (aq)
IV-7 90% IV-3

Scheme IV-13. TiCl4-mediated decomposition of IV-7.

Further studies involving the annulation reaction of 2-imidazolines with [3-
hydroxy carboxylic acid derivatives are underway in our labs. A major aim of
these studies is to utilize our newly discovered chemistry for the resolution of 2-
imidazolines prepared racemically by our chemistry. In addition, the annulation
reaction may be applied to the formation of other fused hetereocycles, such as
those derived from 2-oxazolines. We anticipate that the fused bicyclic
heterocycles prepared from our chemistry will be useful as building blocks in
synthesis. Moreover, compounds prepared from this chemistry may potentially
demonstrate activity against thus far unevaluated biological targets. However, in
order for anticipated applications to come to fruition, an increased knowledge
concerning the relationship between such compounds and biological activity is
necessary, and our continuing efforts in this area will hopefully bring such a

relationship to light.

D. Evaluation of proteasome inhibition.
The oxazinone/imidazolidine adducts prepared in this chapter were tested for

their ability to inhibit the chymotryptic-like activity of the 208 proteasome in an in

191

vitro assay. The results of the assay are shown in Table lV-1. B-Iactone-
containing multicyclic 2-imidazoline IV-1 was found to inhibit chymotrypsin-Iike
activity at low micromolar levels. Multicyclic 2-imidazolines lV-4A and IV-48 both
inhibited proteolytic activity at lower concentrations than lV-1, indicating that the
B-Iactone likely doesn't significantly contribute to the biological activity of lV-1.
Moreover, the parent 2-imidazolines from which lV-4A and lV-4B were derived
(Ill-42 and lV-3) were both found to be inactive as inhibitors (IC50 = >20 uM).42
Finally, >10 uM of fused heterocycle adducts IV-5 - lV-7, all of which lack 2-
imidazolines, was required to inhibit chymotrypsin-like activity. It is noteworthy
that multiheterocyclic adducts lV-1 and lV-4 inhibited proteolytic activity to a
greater extent than bicyclic adducts lV-5 — lV-7, as the results suggest that the
amidine portion of the 2-imidazoline may likely be important for inhibition of
proteolytic activity.

Multicyclic adducts lV-1 and IV-4 have demonstrated inhibition of the
chymotryptic-like activity of the 208 proteasome at low micromolar levels, yet it is
unlikely that either of these compounds will become lead compounds in our
studies due to their lengthy preparation. However, the synthesis and evaluation
of such compounds has given us insight into structural features which may be
important for inhibition of the 208 proteasome. Furthermore, the chemistry used
for the preparation of bicyclic oxazinone/imidazolidine adducts has demonstrated
to us the ease by which 2-imidazolines may undergo transformations stemming
from their underutilized reactivity. Such reactivity will be considered in the

planning and execution of reactions involving 2-imidazolines in our future studies.

192

 

Compound IC50 (pM) Compound IC50 (uM)

 

Bn
OPhN\ 13th
Bn\N>; 0:]: e 141 CEWNJ 15.9
o
o
>’Iv 1 lV-5
Bn
OPh. Eh Bn
B {V 0.74 Me O i N 15.9
"‘N = NJ
BocHN
0
MM lV-6
PhN.Bn Ph Bn
01”” 01*”
NJ 1.24 NJ 12.8
Br"NF BocHN
o
th lV-4B lV-7

Table lV-1. Inhibition of chymotrypsin-Iike activity of the 208 proteasome by

oxazinone/imidazolidine adducts.

E. Experimental.

1. General information. Reactions were carried out in flame-dried glassware
under nitrogen atmosphere. All reactions were magnetically stirred and
monitored by TLC with 0.25 pm pre-coated silica gel plates using either UV light
or iodine to visualize the compounds. Column chromatography was carried out
on Silica Gel 60 (230-400 mesh) supplied by EM Science. Yields refer to
chromatographically and spectroscopically pure compounds unless othenIvise
noted. Infrared spectra were recorded on a Nicolet lR/42 spectrometer. Optical
rotations were recorded on a Perkin Elmer 341 polarimeter. 1H and 13C NMR

spectra were recorded on either a Varian Unity Plus-500 spectrometer or a

193

 

Varian Inova-600 spectrometer. Chemical shifts are reported relative to the
residue peaks of the solvent. The following abbreviations are used to denote the
multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, and m =
multiplet. HRMS were obtained at the Mass Spectrometry Facility of Michigan
State University with a JEOL JMS HX-110 mass spectrometer. Melting points
were obtained using an Electrothermal® capillary melting point apparatus and are

uncorrected.

2. Materials. Reagents and solvents were purchased from commercial suppliers
and used without further purification. Anhydrous methylene chloride was
dispensed from a delivery system which passes the solvents through a column
packed with dry neutral alumina. Anhydrous chloroform, N,N-
dimethylforrnamide, and triethylamine were distilled from calcium hydride and
stored over activated molecular sieves prior to use. N-Boc—L-threonine,43 N-Boc—
L-serine,44 and IV-834 were synthesized according to literature preparations. The

synthesis of III-42 was described in Chapter III of this dissertation.

3. Synthesis and Characterization.

Figure IV-3. DL-(lV-1).

194

 

DL-(lV-1). To a stirring suspension of III-42 (0.1185 g, 0.37 mmol) in CHCI3 (2.8
mL) were successively added TEA (0.15 mL, 1.1 mmol) and BOP-Cl (0.1167 g,
0.46 mmol), and the reaction mixture stirred vigorously at room temperature
under N2 atmosphere for 1.5 hours. The reaction was then diluted with CH2C|2 (7
mL) and washed with ice cold H20/brine mixture (5:1, 15 mL). The organic layer
was collected and the aqueous layer was extracted with CH2CI2 (2 x 15 mL). The
organic layer was collected and the aqueous layer was extracted with CHzClz (2
x 15 mL). The combined organic layers were dried (N32504) and concentrated
to a white residue. Purification of the residue via silica gel chromatography (2%
TEA / 35% EtOAc / 63% hexanes on silica gel pretreated with TEA) afforded 48.8
mg of the title compound (44%) as a film. Recrystallization of the product from
benzene and petroleum ether afforded the product as clear crystals (m.p. = 204 -
206 °C). 1H NMR (500 MHz) (CDCI3): 6 1.24 (d, J = 6.3 Hz, 3H), 1.30 (d, J = 6.5
Hz, 3H), 2.50 (d, J = 12.0 Hz, 1H), 3.06 (d, J = 10.8 Hz, 1H), 3.19 (d, J = 12.0 Hz,
1H), 3.37 (d, J = 10.8 Hz, 1H), 3.55 (d, J = 12.9 Hz, 1H), 4.07 (d, J = 12.9 Hz,
1H), 4.17 (d, J = 16.0 Hz, 1H), 4.37 (d, J = 16.0 Hz, 1H), 4.75 (q, J = 6.3 Hz, 1H),
5.44 (q, J = 6.5 Hz, 1H), 7.11 (d, J = 7.3 Hz, 2H), 7.25 - 7.47 (m, 16 H), 7.65 —
7.69 (m, 2 H). 13C NMR + DEPT (125 MHz) (CDCI3): 5 14.0 (-CH3), 15.3 (—CH3),
47.1 (—CH2), 51.2 (-CH2), 52.1 (—CH2), 52.2 (—CH2), 72.0 (quaternary -C), 72.4
(—CH), 73.7 (quaternary —C), 75.8 (—CH), 103.2 (quaternary -C), 125.3 (aromatic
—CH), 126.9 (aromatic -CH), 127.4 (aromatic -CH), 127.4 (aromatic —CH), 128.3
(aromatic —CH), 128.5 (aromatic —CH), 128.5 (aromatic —CH), 128.6 (aromatic -

CH), 128.6 (aromatic -CH), 128.7 (aromatic —CH), 129.0 (aromatic —CH), 130.1

195

(aromatic quaternary —C), 130.5 (aromatic —CH), 136.9 (aromatic quaternary -C),
137.4 (aromatic quaternary -C), 139.6 (aromatic quaternary -C), 168.49
(quaternary -C), 168.50 (quaternary —C), 171.8 (quaternary —C). IR (neat): 2928
cm", 1834 cm“, 1694 cm", 1384 cm", 1087 cm". HRMS (FAB): m/z calcd for

C38H37N4O4 [M+H], 613.2815; found, 613.2816.

NFPh

\r
IIJ
v-3

Ph

Figure lV-4. 1-benzyl-2-phenyl-2-imidazoline (IV-3).

1-benzyl-2-phenyl-2-imidazoline (IV-3). According to modifications to a known
procedure,45 a stirring suspension of NaH (69.9 mg, 2.9 mmmol) in DMF (4 mL),
cooled to 0 °C in an ice bath, was treated dropwise with a solution of 2-phenyl-2-
imidazoline (0.3040 g, 2.1 mmol) in DMF (4 mL). After stirring at 0 °C for 25
minutes, benzyl bromide (0.30 mL, 2.5 mmol) was added and the reaction stirred
overnight under an atmosphere of N2 while warming to room temperature. The
reaction was poured over H20 (40 mL) and extracted with CHCI3 (4 x 30 mL).
The pooled organic extracts were washed successively with 10% aqueous LiCI
solution (3 x 100 mL) and brine before being dried (Na2804) and concentrated
under vacuum. Purification of the crude product via silica gel chromatography
(8% TEA / 92% EtOAc) was performed to give 0.3040 g of the title compound
(62% yield) as an off-white solid (mp. = 65 - 68 °C) The spectral data matches
that previously reported in the literature.46 1H NMR (500 MHz) (CDCI3): 5 3.36 (t,

J = 9.9 Hz, 2 H), 3.90 (t, J = 9.9 Hz, 2 H), 4.26 (s, 2 H), 7.23 — 7.27 (m, 3 H), 7.31
196

 

— 7.39 (m, 5 H), 7.59 — 7.63 (m, 2 H). 13c NMR + DEPT (125 MHz) (CDCI3): 5
50.7 (—CH2), 52.8 (-CH2), 53.2 (—CH2), 126.8 (aromatic —CH), 126.9 (aromatic —
CH), 127.8 (aromatic —CH), 128.1 (aromatic —CH), 128.3 (aromatic -CH), 129.5
(aromatic -CH), 131.1 (aromatic quaternary —C), 137.7 (aromatic quaternary —C),
167.0 (quaternary —C). HRMS (FAB): m/z calcd for C16H17N2 [M+H], 237.1392;

found, 237.1404.

4. General procedure for the synthesis of multicyclic oxazinone adducts.
The general procedure used for the formation of oxazinone adducts lV-4 — lV-7
and lV-9 is as follows: To a stirring 0.2 M solution of 2-imidazoline in CHCI3 were
added TEA (33 equivalents) and B—hydroxy carboxylic acid (1.1 equivalent). It
should be noted that an excess of 2-imidazoline (31.1 equivalents) relative to the
amount of B-hydroxy carboxylic acid can also be used to achieve similar product
yields. The reaction mixture was treated with BOP-Cl (1.4 equivalents) and
stirred at room temperature under nitrogen atmosphere for the requisite amount
of time, as monitored by TLC. The reaction was diluted in CH2CI2 (10X reaction
volume) and washed with saturated NaHCOalNaCI mixture (10:1 ratio of
saturated aqueous solutions, volume equal to organic layer). The organic layer
was collected and the aqueous layer was extracted with more CH2Cl2 (x2). The
pooled organic extracts were dried (Na2804) and concentrated. The crude

reaction mixtures were purified via column chromatography on silica gel.

197

 

Ph Ph
Me 0;:th Me 0%.:th
fiN/{KNJ + FN/ilrrNJ
Ph >41 0 Ph , 0
Ph

p
lV-4A IV-4B

Figure lV-5. IV-4A + IV-4B.

(IV-4A + lV-4B). Following the general procedure, BOP-Cl (75.2 mg, 0.30 mmol)
was added to a stirring solution of Ill-42 (67.7 mg, 0.21 mmol), lV-3 (149.9 mg,
0.63 mmol), and TEA (0.10 mL, 0.72 mmol) in CHC|3 (1.0 mL), and the reaction
stirred at room temperature for 2.5 hours. Purification via silica gel
chromatography (2% TEA/ 16% hexanes / 82% EtOAc on silica gel pretreated
with TEA) was performed to afford 78.2 mg of the title compound (oil, 69% yield)
as a 12 : 1 mixture of diastereomers. The mixture of diastereomers was allowed
to stand in CDCI3 for two weeks, after which it was observed that all of lV-4A had
epimerized to IV-4B. IV-4A: 1H NMR (500 MHz) (C0013): 5 1H NMR (500 MHz)
(CDCI3): 5 1.26 (d, J = 6.4 Hz, 3 H), 2.35 (dd, J1 = 8.8 Hz, J2 = 16.5 Hz, 1 H),
3.00 (d, J = 13.2 Hz, 1 H), 3.03 — 3.10 (m, 2 H), 3.52 (d, J = 10.7 Hz, 1 H), 3.57
(ddd,J1= 3.3 Hz, J2 = 8.7 Hz, J3 =11.7 Hz, 1 H), 4.10 (d, J = 13.2 Hz, 1 H), 4.13
— 4.19 (m, 2 H), 4.40 (d, J = 16.2 Hz, 1 H), 4.71 (q, J = 6.4 Hz, 1 H), 7.16 (d, J =
7.1 Hz, 2 H), 7.20 — 7.44 (m, 18 H), 7.67 - 7.70 (m, 2 H). 130 NMR + DEPT (125
MHz) (CDCI3): 5 14.0, 42.9, 44.8, 52.1, 52.3, 52.7, 69.9, 73.6, 101.1, 125.7,
126.7, 127.0, 127.3, 127.9, 128.1, 128.24, 128.25, 128.3, 128.5, 128.7, 130.3,
137.2, 138.4, 138.8, 187.9, 172.4. IR (neat): 1881 cm", 1592 cm", 1388 cm",

1232 cm". HRMS (FAB): m/z calcd for C35H35N402 [M+H], 543.2760; found,
198

 

543.2774. IV-4B: 1.23 (d, J = 6.4 Hz, 3 H), 2.34 (q, J = 9.2 Hz, 1 H), 2.93 — 3.01
(m, 2 H), 3.51 — 3.59 (m, 2 H), 3.84 (d, J = 10.2 Hz, 1 H), 3.90 (d, J = 12.9 Hz, 1
H), 4.01 (dt, J1 = 8.8 Hz, J2 = 11.8 Hz, 1 H), 4.17 (d, J = 15.7 Hz, 1 H), 4.22 (q, J
= 6.5 Hz, 1 H), 4.61 (d, J = 15.8 Hz, 1 H), 7.14 — 7.44 (m, 18 H), 7.62 (d, J = 6.8
Hz, 2 H). 130 NMR (150 MHz) (cosh): 5 14.3, 43.0, 44.6, 52.4, 52.8, 55.3, 71.8,
74.0, 103.2, 126.7, 127.0, 127.1, 127.5, 128.2, 128.25, 128.28, 128.5, 128.70,
128.73, 128.8, 130.3, 130.4, 137.39, 137.42, 137.8, 188.9, 172.2. IR (neat):
3080 cm", 2838 cm", 1888 cm", 1408 cm", 1218 cm", 1074 cm". HRMS

(FAB): m/z calcd for C35H35N402 [M+H], 543.2760; found, 543.2769.

Ph KP“

O+N
Cg?)

lV-5
Figure IV-6. 1-benzyI-10a-phenyI-2,3-dihydro-1 H-benzo[e]imidazo[2,1-
b][1 ,3]oxazin-5(10aH)-one (IV-5).

1-benzyl-10a-phenyl-2,3-dihydro-1H-benzo[e]imidazo[2,1-b][1,3]oxazin-

5(10aH)-one (IV-5). Following the general procedure, BOP-Cl (73.5 mg, 0.29
mmol) was added to a stirring solution of lV-3 (47.4 mg, 0.20 mmol), salicylic acid
(31.0 mg, 0.22 mmol), and TEA (0.090 mL, 0.64 mmol) in CHCI3 (1.0 mL), and
the reaction stirred at room temperature for 3 hours. Purification via silica gel
chromatography (50% EtOAc / 46% hexanes / 4% TEA) was performed to afford
45.3 mg of the title compound (63% yield) as a white solid (mp. = 141 - 144 °C).
1H NMR (500 MHz) (CDCI3): 5 2.93 — 2.99 (m, 1 H), 3.14 (dt, J1 = 6.7 Hz, J2 = 7.7

Hz, 1 H), 3.81 (d, J = 13.8 Hz, 1 H), 3.91 (dt, J1 = 7.0 Hz, J2 = 10.6 Hz, 1 H), 3.97

199

 

 

 

(d, J = 13.8 Hz, 1 H), 4.21 — 4.28 (m, 1 H), 6.99 (dt, J1 = 1.0 Hz, J2 = 7.6 Hz, 1
H), 7.12 (dd, J1 = 0.7 Hz, J2 = 8.2 Hz, 1 H), 7.15 - 7.20 (m, 2 H), 7.22 - 7.33 (m,
8 H), 7.40 — 7.50 (m, 3 H), 7.79 (dd, J1 = 1.7 Hz, J2 = 7.7 Hz, 1 H). 13c NMR +
DEPT (125 MHz) (CDCI3): 5 45.2 (—CH2), 47.0 (—CH2), 50.1 (-CH2), 104.2
(quaternary —C), 117.1 (aromatic -CH), 119.5 (aromatic quaternary —C), 122.3
(aromatic —CH), 127.27 (aromatic —CH), 127.32 (aromatic -CH), 128.0 (aromatic
—CH), 128.1 (aromatic —CH), 128.3 (aromatic —CH), 128.4 (aromatic —CH), 128.7
(aromatic —CH), 134.2 (aromatic —CH), 137.8 (aromatic quaternary —C), 138.9
(aromatic quaternary —C), 155.8 (aromatic quaternary —C), 161.4 (quaternary -
C). IR (neat): 3040 cm“, 2842 cm", 1871 cm", 1813 cm", 1471 cm", 1412 cm",
1247 cm". HRMS (FAB): m/z calcd for C23H21N2O2 [M+H], 357.1603; found,

357.1613.
Ph
Me O '3th
T3
BocHN
O
lV-6

Figure IV-7. tert—butyl ((68,7R,8aS)-1-benzyl-7-methyl-5-oxo—8a-
phenylhexahydro-1 H-imidazo[2,1-b][1 ,3]oxazin-6-yl)carbamate (IV-6).

tert-butyl ((68,7R,8aS)-1-benzyl-7-methyl-5-oxo-8a-phenylhexahydro-1 H-
imidazo[2,1-b][1,3]oxazin-6-yl)carbamate (IV-6). Following the general
procedure, BOP-Cl (0.2742 g, 1.08 mmol) was added to a stirring solution of lV-3
(0.1813 g, 0.77 mmol), N-Boc—L-threonine (0.1872 g, 0.85 mmol), and TEA (0.37
mL, 2.7 mmol) in CHCI3 (3.9 mL), and the reaction stirred at room temperature

for 4 hours. Purification via silica gel chromatography (60% EtOAc / 36%
200

 

 

hexanes /4% TEA) was performed to afford 0.3170 g of the title compound (94%
yield) as a fluffy white solid (mp. = 85 — 87 °C) , which appeared as a 4:1 ratio of
rotamers by 1H-NMR, [d]2°o = +270 (0 1.0, CH2Cl2). The rotamers were resolved
at 14 °C such that splitting was observed for individual proton signals of the
minor rotamer, whereas heating the sample to 41 °C resulted in a loss of splitting
and convergence of some signals. 1H NMR (500 MHz, 14 °C) (CDCI3): 5 1.11 (d,
J = 6.3 Hz, 3 H), 1.34 and 1.39 (2s, 9 H), 2.81 - 2.98 (m, 2 H), 3.52 - 3.63 (m, 2
H), 3.80 and 3.88 (2d, Jaw) = 14.0 Hz, J(3,33) = 14.4 Hz, 1 H), 4.07 - 4.17 (m, 1
H), 4.25 (ddd, J1 = 10.4 Hz, J2 = 6.1 Hz, J3 = 1.4 Hz, 1 H), 4.69 and 4.83 (2p,
J(4,69, = 6.5 Hz, J(4,83) = 6.4 Hz, 1 H), 5.35 and 5.64 (2d, J(5_35) = 4.8 Hz, J(5,54) =
5.4 Hz, 1 H), 7.06 (d, J = 6.8 Hz, 2 H), 7.16 — 7.25 (m, 3 H), 7.36 — 7.46 (m, 3 H),
7.57 (d, J = 7.3 Hz, 2 H). 13'C NMR + DEPT (125 MHz) (CDCl3): 5 17.0 (—CH3),
28.2 (—CH3), 45.3 (—CH2), 46.4 (—CH2), 49.3 (—CH2), 51.7 (-CH), 69.5 (—CH),
79.7 (quaternary -C), 101.9 (quaternary —C), 126.7 (aromatic -CH), 127.0
(aromatic —CH), 128.1 (aromatic —CH), 128.2 (aromatic -CH), 128.5 (aromatic —
CH), 129.0 (aromatic —CH), 138.4 (aromatic quaternary —C), 140.0 (aromatic
quaternary —C), 154.9 (quaternary -C), 166.8 (quaternary —C). IR (neat): 3413
cm", 3322 cm", 2976 cm", 1718 cm", 1686 cm", 1427 cm", 1185 cm". HRMs

(FAB): m/Z calcd for C25H32N304 [M+H], 438.2393; found, 438.2409.

201

 

Ph FF“
0 3 N
T)
BocHN
O
lV-7

Figure lV-8. tert-butyl ((6S,8aS)-1-benzyl-5-oxo-8a-phenylhexahydro-1H-
imidazo[2,1-b][1 ,3]oxazin-6-yl)carbamate (IV-7).

tert-butyl ((68,8aS)-1-benzyl-5-oxo-8a-phenylhexahydro-1H-imidazo[2,1-
b][1,3]oxazin-6-yl)carbamate (IV-7). Following the general procedure, BOP-Cl
(46.0 mg, 0.18 mmol) was added to a solution of N-Boc—L-serine (26.5 mg, 0.13
mmol), IV-3 (37.1 mg, 0.16 mmol), and TEA (0.060 mL, 0.43 mmol) in CHCI3
(0.60 mL). Purification via silica gel chromatography (50% EtOAc / 46% hexanes
/ 3% TEA) was performed to give 51.3 mg of the title compound (94% yield) as a
as an off-white solid (mp. = 134 — 136 °C), which appeared as a 5:1 ratio of
rotamers by 1H-NMR, [(11200 = +260 (0 1.0, CH2Cl2). The rotamers were resolved
at 5 °C such that splitting was observed for individual proton signals of the minor
rotamer, whereas heating the sample to 40 °C resulted in a loss of splitting and
convergence of some signals. 1H NMR at 5 °C (500 MHz, 5 °C) (CDCI3): 5 1.32
and 1.39 (2s, 9H), 2.82 — 2.88 and 2.91 - 2.98 (2m, 2H), 3.49 - 3.69 and 3.77 —
3.82 (2m, 4H), 3.87 — 3.93 and 3.95 — 4.02 (2m, 1 H), 4.19 - 4.29 (m, 1 H), 4.45
and 4.59 (2t, J(4,45) = 8.7 Hz, J(4,59) = 9.2 Hz, 1 H), 5.31 and 5.58 (s and d, J(5,58) =
5.4 Hz, 1 H), 7.05 — 7.09 (m, 2 H), 7.17 — 7.26 (m, 3 H), 7.38 - 7.47 (m, 3 H),
7.48 - 7.56 (m, 2 H). 13C NMR + DEPT (125 MHz) (CDCI3): 5 28.2 (—CH3), 44.8

(—CH2), 46.4 (—CH2), 48.2 (—CH), 49.8 (—CH2), 88.1 (—CH2), 79.9 (quaternary —C),

202

 

 

 

102.9 (quaternary -C), 126.6 (aromatic —CH), 127.1 (aromatic -CH), 128.19
(aromatic —CH), 128.23 (aromatic —CH), 128.7 (aromatic —CH), 129.2 (aromatic -
CH), 138.1 (aromatic quaternary -C), 138.5 (aromatic quaternary —C), 155.3
(quaternary —C), 167.3 (quaternary —C). IR (neat): 3331 cm", 2973 cm", 1711
cm", 1681 cm", 1428 cm", 1189 cm". HRMS (FAB): m/z calcd for C24H30N304

[M+H], 424.2238; found, 424.2237.
Bn
Me Ofizhhj
’VkKN Ph
BocHN
0 Ph
lV-9

Figure IV-9. IV-9.

(IV-9). Following the general procedure, BOP-CI (37.2 mg, 0.15 mmol) was
added to a stirring solution of lV-8 (39.8 mg, 0.10 mmol), N-Boc-L-threonine
(25.3 mg, 0.12 mmol), and TEA (0.050 mL, 0.36 mmol) in CHCI3 (3.9 mL), and
the reaction stirred at room temperature for 9 hours. Purification via silica gel
chromatography (10% EtOAc / 86% hexanes / 4% TEA) was performed to afford
12.5 mg of the title compound (21% yield) as a thin film, which appeared as a 3:1
ratio of rotamers by 1H-NMR, [812% = -0.5 (c 0.8, CH2Cl2). 1H NMR (600 MHz)
(CDCI3): 5 1.18 (d, J = 6.2 Hz, 3 H), 1.26 and 1.26 (2s, 9H), 3.66 (d, J = 15.8 Hz,
1 H), 4.26 (d, J = 15.9 Hz, 1 H), 4.28 -4.37 (m, 1 H), 4.67 — 4.73 and 4.81 -4.87
(2m, 1 H), 4.91 (d, J = 7.9 Hz, 1 H), 5.31 and 5.58 (s and d, J(5,58) = 5.6 Hz, 1 H),
5.50 (d, J = 7.9 Hz, 1 H), 6.65 (d, J = 7.3 Hz, 2 H), 6.86 — 7.00 (m, 8 H), 7.06 —

7.15 (m, 3 H), 7.20 - 7.23 (m, 2 H), 7.31 - 7.37 (3 H), 7.77 (d, J = 7.5 Hz, 2 H).

203

 

 

130 NMR (150 MHz) (CDCI3): o 17.1, 28.3, 48.5, 52.1, 85.8, 89.8, 70.0, 79.7,
103.9, 125.9, 127.1, 127.3, 127.39, 127.41, 127.45, 127.6, 128.2, 128.65,
128.72, 129.2, 129.5, 135.0, 137.4, 138.8, 139.7, 154.9, 189.2. IR (neat): 3415
cm", 3320 cm", 1718 cm", 1890 cm", 1404 cm", 1171 cm". HRMS (FAB): m/z

calcd for C37H40N304 [M+H], 590.3019; found, 590.3029.

5. Decomposition of oxazinone/imidazolidine adducts.

To a stirring solution of IV-7 (41.1 mg, 0.0970 mmol) in CH2CI2 (3.0 mL), cooled
to -41 °C in a CH3CN / CO2 (s) ice bath, was added dropwise a 1 M solution of
TiCl4 in CH2CI2 (0.15 mL, 0.15 mmol). The reaction stirred at -41 °C for 30
minutes and was then allowed to warm to room temperature while stirring
overnight. The reaction was then quenched through the successive addition of
TEA (0.5 mL) and aqueous NaHCOa solution (5 mL). The reaction was extracted
with EtOAc (3 x 15 mL) and the pooled organic extracts were dried (Na2SO4) and
concentrated to yield a crude product that contained only lV-3. Purification via
silica gel chromatography (8% TEA I 92% EtOAc) was performed to give 20.5 mg

of lV-3 (90% yield).

6. Procedure for proteasome inhibition assay. The 2-imidazolines and
oxazinone/imidazolidine adducts prepared in this chapter were tested for their
ability to inhibit the chymotryptic-like activity of the 208 proteasome in an in vitro
assay using purified human recombinant 20$ proteasome (Boston Biochem,

Cambridge, Ma.) and the fluorogenic peptide substrate Suc—LLW-AMC (Boston

204

 

 

Biochem, Cambridge, Ma.) All proteasome inhibition assays were performed by
Teri L. Lansdell according to a modification of known procedures.”48

The modified procedure is as follows: An activated enzyme solution was
first prepared through addition of 20S proteasome to a reaction buffer (50 mM
Tris-HCI, pH 7.5, 0.03% SDS) to give a final concentration of 1 nM. To each well
of a black, clear bottom 96-well plate (Corning, Inc.) was added 100 uL of the
activated enzyme solution. Compounds being evaluated were diluted in neat
DMSO and 1 pL of test agent or vehicle (DMSO) was added to each well of the
96-well plate; compounds were evaluated at 20, 10, 5, 2.5, 1.3, and 0.6 uM final
concentrations. Enough chymotryptic substrate Suc—LLW—AMC was then added
to each well to give a final concentration of 100 (TM. The rate of peptide
hydrolysis (Vmax) was determined using a Spectramax M5e (Molecular Devices,

Sunnyvale, Ca.) at 37° C over 30 minutes (lax 380, A...“ 440). The initial linear

portions of the curves were used to calculate the ICso values.

205

 

..l

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211

CHAPTER V
CONCLUSION

The studies described in Chapters ll - IV of this dissertation follow a linear
path that spans from oxazol-5(4H)-one alkylation to 2-imidazoline synthesis to 2-
imidazoline annulation, with each chapter building upon the results obtained from
the prior one. Such a progression in these studies may be viewed as a utilization
of DOS. As detailed in Chapter II, initial studies involved the development of a
protocol by which quaternary o-amino acid derivatives are diastereoselectively
synthesized from oxazol-5-(4H)-ones. This protocol allowed for the formation of
densely functionalized compounds containing reactive handles which were found
to be useful towards the synthesis of proteasome inhibitors, as was described in
Chapter III. In addition, the quaternary a-amino acid derivatives described in
Chapter II allowed access to 2-oxazoline and 2-imidazoline heterocyclic
scaffolds, the syntheses of which were described in Chapter III. Finally, the
surprising results observed during the synthesis of 2-imidazoline proteasome
inhibitors prompted our investigation into the development of 2-imidazoline
annulation chemistry, as was described in Chapter IV.

Future studies in the Tepe lab will undoubtedly utilize the oxazol-5(4H)-
one scaffold for the preparation of biologically active heterocyclic products.
Studies described in this dissertation in which oxazol-5(4H)-ones were used
primarily involved alkylation and ring-opening via hydride reduction. The
products derived from our alkylation/reduction protocol were of use towards the

synthesis of the marine metabolite salinosporamide A, and it is anticipated that

212

this chemistry may also be useful for the construction of other densely
functionalized natural products. Future SAR studies involving 2-imidazolines in
the Tepe lab are also anticipated and are expected to play a role in better
understanding proteasome inhibition as a means of anti-cancer treatment. The
chemistry developed for the construction of 2-imidazoline proteasome inhibitors,
as detailed in Chapter III, is expected to play a role in our future SAR studies by
granting access to 2-imidazolines not available by our previous chemical
methods. Moreover, it anticipated that the annulation reaction described in
Chapter IV may be utilized for the chiral resolution of 2-imidazolines. In
conclusion, it is envisioned that upcoming studies will prompt the development of
new chemistry in the Tepe lab, such that scores of compounds with desired

biological profiles may ultimately be prepared.

213

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