A Dissertation Entitled Synthesis and Biological Evaluation of Histone Deacetylase Inhibitor Largazole and Analogs by Pravin R

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A Dissertation

entitled

Synthesis and Biological Evaluation of Histone Deacetylase

Inhibitor Largazole and Analogs

Pravin R. Bhansali

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the

Doctor of Philosophy Degree in Medicinal Chemistry

______L. M. Viranga Tillekeratne, Ph. D., Committee Chair

______Amanda C. Bryant-Friedrich, Ph. D., Committee Member

______Paul W. Erhardt, Ph. D., Committee Member

______Kana Yamamoto, Ph. D., Committee Member

______Patricia Komuniecki, PhD,

Dean College of Graduate Studies

The University of Toledo

August 2011

An Abstract of

Synthesis and Biological Evaluation of Histone Deacetylase

Inhibitor Largazole and Analogs

Pravin R. Bhansali

Submitted to the Graduate Faculty in partial fulfillment of the requirements for the

Doctor of Philosophy Degree in Medicinal Chemistry

The University of Toledo

August 2011

Epigenetics, along with genetic mutations, are major etiological factors in carcinogenesis.

Epigenetics are heritable changes in gene expression that take place without modifying gene sequence. Histone acetylation modulated by two protein families, histone acetyltransferase (HAT) and histone deacetylase (HDAC) are the most extensively studied of these epigenetic processes. The fine balance of acetylation-deacetylation of histones in normal cells is disturbed in cancer cells. HDACs are a class of enzymes responsible for removal of acetyl groups from substrate proteins.

Recently isolated natural HDAC inhibitor largazole is very potent and selective in its activity for cancer cells. A molecule requires three structural elements to inhibit HDAC:

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1) a zinc-binding moiety, 2) a hydrophobic surface recognition group which occupies the lipophilic region of the enzyme and 3) a linker which interacts with hydrophobic channel.

The zinc-binding unit and surface recognition group are the potential targets for modification to achieve variations in activity and selectivity profiles of HDAC inhibitors.

We have successfully synthesized largazole and several largazole analogs with modified surface recognition groups and zinc-binding moieties to modulate potency and selectivity. Substitution of L-valine of largazole by hydrophobic amino acids such as D- naphthylalanine, L-naphthylalanine and L-allylglycine generated analogs with an altered hydrophobic core. Replacement of the thiol group of largazole with moieties containing multiple heteroatoms and hydroxamic acid gave analogs with modifications in the Zn 2+ binding moiety.

In our synthetic strategy, a stereoselective aldol condensation reaction set the stereochemistry at C-17 of the molecule. Synthesis of ( R)-α-methylcysteine as well as its enantiomer ( S)-α-methylcysteine provided access to the synthesis of largazole and its C-7 epimer for structure activity relationship (SAR) studies. Determination of the biological properties of these molecules has provided valuable SAR information that can direct the design and development of additional largazole analogs.

ACKNOWLEDGEMENTS

During the compilation and execution of my dissertation work, there were contributions from number of people. The main contribution came from Dr. Tillekeratne, my advisor. I am thankful for his valuable time, acumen, indispensable suggestions and constant support throughout my stay here to shape up my professional life.

I would like to extend my sincere gratitude to my committee members for their helpful suggestions and timely support. I am grateful to the department of medicinal and biological chemistry, UT for providing me an opportunity for dissertation work. I also appreciate efforts of journal club members to improve my synthetic ability. My heartfelt thanks to our collaborators Dr. Casero, Dr. Hanigan, Dr. Xie and Dr. Ahmad. It is great to have Dr. Kim, the NMR specialist; Steve, the glassblower; chemistry stockroom people and group members around to make the work atmosphere very conducive and professional. My sincere thanks to ACS Div. of Org. Chem., AAPS’ Drug Des. And Dis. section and Dr. Hoss for providing travel grants to attend three national conferences.

I am lucky to have caring family members. A special thanks to my soul mate, Meenakshi for her love, faith and unconditional support. I cannot forget my son, Tanishk who always took care of mine. In the end, I thank the almighty for his guidance.

TABLE OF CONTENTS

Abstract...………………………………………………………………………...... iii

Acknowledgements...... v

Table of contents...... vi

List of Tables...... viii

List of Figures...... ix

List of Schemes...... xi

List of spectra...... xiii

List of abbreviations...... xx

Chapter 1 Significance...... 01

1.1 Role of epigenetics in cancer...... 04

1.2 Crystal structure and mechanistic pathways...... 14

1.3 Innovation (Drug Design and Rationale)...... 25

Chapter 2 Results and Discussions...... 30

2.1 Synthesis of largazole...... 30

2.1.1 Synthesis of nitrile 7...... 31

2.1.2 Synthesis of ( R)- and ( S)-α-methylcysteine...... 32

2.1.3 Synthesis of acetyl Nagao auxillary ( 16 )...... 33

2.1.4 Synthesis of the t-butyl protected β-hydroxyamide 22 ...... 34

2.1.5 Attempts for synthesis of carboxylic acid 29 ...... 35

2.1.6 Synthesis of cyclic core 33 ...... 40

2.1.7 Failed attempts for conversion of cyclic core 33 to

largazole...... 42

2.1.8 Alternative Strategy to largazole with trityl as thiol protecting

group...... 42

2.2 Synthesis of analogs of T1 series...... 46

2.3 Unsuccessful attempt in the synthesis of analog T2 ...... 50

2.4 Synthesis of analog T3 ...... 53

2.5 Synthesis of analogs of T4 series...... 55

2.6 Synthesis of analog T5 ...... 57

2.7 Synthesis of analogs of T6 series...... 60

2.8 Attempts to synthesize 13-membered ring analog T7 ...... 61

2.9 Biological Studies...... 63

2.9.1 Results...... 63

2.9.2 Discussions...... 66

2.10 Future Directions...... 68

Chapter 3 Experimental Sections...... 74

3.1 Experimental Chemistry...... 74

3.2 Experimental Biology...... 131

References...... 132

Appendix...... 146

(1H and 13 C NMR Spectra)

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LIST OF TABLES

Table 1: Growth-inhibitory activity (GI 50 ) of natural product drugs...... 3

Table 2. A list of HDACis in clinical trials...... 9

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LIST OF FIGURES

Figure 1. Natural product anticancer drugs...... 2

Figure 2. Structure of largazole...... 3

Figure 3. Acetylation and deacetylation processes...... 4

Figure 4. HDACi binding elements...... 8

Figure 5. Structure of SAHA...... 8

Figure 6. Largazole and related natural products...... 11

Figure 7. Largazole analogs synthesized in other laboratories...... 13

Figure 8. Proposed mechanism of action of Zn 2+ -dependent HDACs...... 15

Figure 9. HDACis used in binding studies with HDAC4...... 16

Figure 10. Interaction of TFMK (yellow carbons) and HA (green carbons) with

HDAC4 catalytic domain...... 17

Figure 11. HDACis used in binding studies with HDAC7...... 17

Figure12. Serine phosphorylation site (red) in relation to the active site of HDAC8

where it shows interaction with two molecules of TSA...... 18

Figure 13. Some of the HDACis used in binding studies with HDAC8...... 19

Figure 14. Interaction of HDACis with flexible catalytic pockets of HDAC8...... 19

Figure 15. Linkerless selective HDAC8 inhibitors...... 20

Figure 16. Representative examples of selective HDAC1 and -2 inhibitors...... 21

Figure 17. Zinc binding site of HDAC8 showing the presence of Trp 141

at the bottom of the site...... 22

Figure 18. HDAC inhibitors designed for synthesis...... 26

Figure 19. Proposed binding of T4 series analogs with HDAC active site...... 28

Figure 20. Selective HDAC6 inhibitors...... 29

Figure 21. Growth inhibitory effects of largazole and analogs on HCT116 colon

carcinoma cells...... 64

Figure 22. Additional target molecules designed for future directions...... 69

Figure 23. Structure of phase I molecule CUDC-101...... 73

LIST OF SCHEMES

Scheme 1. Retrosynthetic analysis of largazole...... 31

Scheme 2. Synthesis of fragment 7...... 32

Scheme 3. Synthesis of ( R)- and ( S)-α-methylcysteine...... 33

Scheme 4. Synthesis of acetyl Nagao auxillary (16 )...... 33

Scheme 5. Synthesis of the t-butyl protected β-hydroxycarboxamide 22 ...... 34

Scheme 6. Mechanism for conversion of nitrile 7 to thiazole thiazoline derivative 29 ....36

Scheme 7. Failed attempt 1 for synthesis of 29 ...... 37

Scheme 8. Failed attempt 2 for synthesis of 29 ...... 38

Scheme 9. Failed Attempt 3 (model reaction 1)...... 38

Scheme 10. Reaction of benzoic acid with L-cysteine HCl...... 39

Scheme 11. Failed attempt 5 (model reaction 2)...... 40

Scheme 12. Synthesis of cyclic core 33 ...... 41

Scheme 13. Attempted conversion of 33 to largazole...... 42

Scheme 14. Attempts to synthesis of compound 34 ...... 43

Scheme 15. Synthesis of the β-hydroxycarboxamide 37 ...... 44

Scheme 16. Synthesis of largazole...... 45

Scheme 17. Synthesis of analog T1A ...... 46

Scheme 18. Synthesis of analog T1B ...... 47

Scheme 19. Attempted of Synthesis of analog T1C ...... 48

Scheme 20. Attempt for synthesis of analog T1D ...... 49

Scheme 21. Synthesis cyclic core 53 for analog T2 ...... 51

Scheme 22. Alternative route for analog T2 ...... 52

Scheme 23. Synthesis of analog T3 (C-7 epimer of largazole)...... 54

Scheme 24. Synthesis of analogs of type T4 with modified metal-binding domain...... 56

Scheme 25. Synthesis of alcohol 62 for the synthesis of cyclic core of T5 ...... 57

Scheme 26. Synthesis of cyclic core 66 ...... 58

Scheme 27. Synthetic pathway for side chain of target molecule T5 ...... 58

Scheme 28 . Synthesis of analog T5 ...... 59

Scheme 29. Synthesis of analog T6 ...... 60

Scheme 30. Attempt to the synthesis of 13 membered ring analog T7 ...... 61

Scheme 31. Alternative route of synthesis to cyclic core 74 ...... 62

Scheme 32: Synthesis of side chain fragments 83 of target molecule T8A ...... 71

Scheme 33. Alternative route to compound 83 ...... 71

Scheme 34. Synthesis of small ring analogs T9 ...... 72

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LIST OF SPECTRA

1H NMR of tert -butyl (4-carbamoylthiazol-2-yl)methylcarbamate ( 6) ...... 146

13 C NMR of tert -butyl (4-carbamoylthiazol-2-yl)methylcarbamate ( 6) ...... 147

1H NMR of tert -butyl (4-cyanothiazol-2-yl)methylcarbamate ( 7) ...... 147

13 C of tert -butyl (4-cyanothiazol-2-yl)methylcarbamate ( 7) ...... 148

1H NMR of 5-(tert -butylthiomethyl)-5-methylimidazolidine-2,4-dione ( 10 ) ...... 148

1H NMR of ( R)-α-methylcysteine HCl ( 13 ) ...... 149

13 C NMR of ( R)-α-methylcysteine HCl ( 13 ) ...... 149

1H NMR of ( R)-4-isopropylthiazolidine-2-thione ( 15 ) ...... 150

13 C NMR of ( R)-4-isopropylthiazolidine-2-thione ( 15 ) ...... 150

1H NMR of acetyl Nagao ( 16 ) ...... 151

13 C NMR of acetyl Nagao ( 16 ) ...... 151

1H NMR of 5-tosyloxy-1,3-dioxolane ...... 152

13 C NMR of 5-tosyloxy-1,3-dioxolane ...... 152

1H NMR of 1,3-dioxene ( 18 ) ...... 153

13 C NMR of 1,3-dioxene ( 18 ) ...... 153

1H NMR of 5-(tert -butylthio)pent-2E-enal ( 21 ) ...... 154

1H NMR of 7-(tert -butylthio)-3S-hydroxy-1-(4 R-isopropyl-2-thioxothiazolidin-3- yl)hept-4E-en-1-one ( 22 ) ...... 155

13 C NMR of 7-(tert -butylthio)-3S-hydroxy-1-(4 R-isopropyl-2-thioxothiazolidin-3- yl)hept-4E-en-1-one ( 22 ) ...... 157

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1H NMR of ( R)-methyl 2-(2-((( S,E)-7-(tert -butylthio)-3-hydroxyhept-4- enamido)methyl)thiazol-4-yl)-4-methyl-4,5-dihydrothiazole-4-carboxylate ( 31 ) ...... 158

13 C NMR of ( R)-methyl 2-(2-((( S,E)-7-(tert -butylthio)-3-hydroxyhept-4- enamido)methyl)thiazol-4-yl)-4-methyl-4,5-dihydrothiazole-4-carboxylate ( 31 ) ...... 159

1H NMR of ( R)-methyl 2-(2-((5 S,8 S)-8-(( E)-4-(tert -butylthio)but-1-enyl)-1-(9 H- fluoren-9-yl)-5-isopropyl-3,6,10-trioxo-2,7-dioxa-4,11-diazadodecan-12-yl)thiazol-4- yl)-4-methyl-4,5-dihydrothiazole-4-carboxylate ( 32 ) ...... 160

13 C NMR of ( R)-methyl 2-(2-((5 S,8 S)-8-(( E)-4-(tert -butylthio)but-1-enyl)-1-(9 H- fluoren-9-yl)-5-isopropyl-3,6,10-trioxo-2,7-dioxa-4,11-diazadodecan-12-yl)thiazol-4- yl)-4-methyl-4,5-dihydrothiazole-4-carboxylate ( 32 ) ...... 161

1H NMR of cyclized product 33 ...... 162

13 C NMR of cyclized product 33 ...... 163

1H NMR of (2 E)-5-[(triphenylmethyl)thio]-2-pentenal (36 ) ...... 164

13 C NMR of (2 E)-5-[(triphenylmethyl)thio]-2-pentenal ( 36 ) ...... 164

1H NMR of 3 S-hydroxy-1-(4 R-isopropyl-2-thioxo-thiazolidin-3-yl)-7-tritylsulfanyl- hept-4E-en-1-one ( 37 ) ...... 165

1H NMR of ( R)-methyl 2-(2-((3 S-hydroxy-7-(tritylthio)hept-4E- enamido)methyl)thiazol-4-yl)-4-methyl-4,5-dihydrothiazole-4-carboxylate ( 39 ) ...... 167

13 C NMR of ( R)-methyl 2-(2-((3 S-hydroxy-7-(tritylthio)hept-4E- enamido)methyl)thiazol-4-yl)-4-methyl-4,5-dihydrothiazole-4-carboxylate ( 39 ) ...... 168

1H NMR of (4 R)-methyl 2-(2-((8 S)-1-(9 H-fluoren-9-yl)-5-isopropyl-3,6,10-trioxo-8- (( E)-4-(tritylthio)-but-1-enyl)-2,7-dioxa-4,11-diazadodecan-12-yl)thiazol-4-yl)-4- methyl-4,5-dihydrothiazole-4-carboxylate ( 40 ) ...... 170

13 C NMR of (4 R)-methyl 2-(2-((8 S)-1-(9 H-fluoren-9-yl)-5-isopropyl-3,6,10-trioxo-8- (( E)-4-(tritylthio)-but-1-enyl)-2,7-dioxa-4,11-diazadodecan-12-yl)thiazol-4-yl)-4- methyl-4,5-dihydrothiazole-4-carboxylate ( 40 ) ...... 171

1H NMR of cyclic core 41 ...... 173

13 C of cyclic core 41 ...... 175

1H NMR of largazole ( 1)...... 176

13 C NMR of largazole ( 1) ...... 178

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13 C NMR of ( R)-methyl 2-(2-((5 R,8 S)-1-(9 H-fluoren-9-yl)-5-(naphthalen-1- ylmethyl)-3,6,10-trioxo-8-(( E)-4-(tritylthio)but-1-enyl)-2,7-dioxa-4,11-diazadodecan- 12-yl)thiazol-4-yl)-4-methyl-4,5-dihydrothiazole-4-carboxylate ( 42 ) ...... 181

1H NMR of cyclic core 43 ...... 182

13 C NMR of cyclic core 43 ...... 183

1H NMR of cyclic core 45 ...... 188

13 C NMR of cyclic core 45 ...... 189

1H NMR of analog T1B ...... 189

13 C NMR of T1B ...... 191

1H NMR of ( R)-methyl 2-(2-((5 S,8 S)-1-(9 H-fluoren-9-yl)-3,6,10-trioxo-5-(3-oxo-3- (tritylamino)propyl)-8-(( E)-4-(tritylthio)but-1-enyl)-2,7-dioxa-4,11-diazadodecan-12- yl)thiazol-4-yl)-4-methyl-4,5-dihydrothiazole-4-carboxylate ( 46 ) ...... 192

1H NMR of ( R)-methyl 2-(2-((5 S,8 S)-1-(9 H-fluoren-9-yl)-5-(naphthalen-1-ylmethyl)- 3,6,10-trioxo-8-(( E)-4-(tritylthio)but-1-enyl)-2,7-dioxa-4,11-diazadodecan-12- yl)thiazol-4-yl)-4-methyl-4,5-dihydrothiazole-4-carboxylate ( 48 ) ...... 195

13 C NMR of ( R)-methyl 2-(2-((5 S,8 S)-1-(9 H-fluoren-9-yl)-5-(naphthalen-1- ylmethyl)-3,6,10-trioxo-8-(( E)-4-(tritylthio)but-1-enyl)-2,7-dioxa-4,11-diazadodecan- 12-yl)thiazol-4-yl)-4-methyl-4,5-dihydrothiazole-4-carboxylate ( 48 ) ...... 196

1H NMR of ( R)-methyl 2-(2-((( S,E)-3-hydroxy-7-(tritylthio)hept-4- enamido)methyl)thiazol-4-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylate ( 51 ) ....197

13 C NMR of ( R)-methyl 2-(2-((( S,E)-3-hydroxy-7-(tritylthio)hept-4- enamido)methyl)thiazol-4-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylate ( 51 ) ....198

1H NMR of ( R)-methyl 2-(2-((5 S,8 S)-1-(9 H-fluoren-9-yl)-5-isopropyl-3,6,10-trioxo- 8-(( E)-4-(tritylthio)but-1-enyl)-2,7-dioxa-4,11-diazadodecan-12-yl)thiazol-4-yl)-5,5- dimethyl-4,5-dihydrothiazole-4-carboxylate ( 52 ) ...... 199

13 C NMR of ( R)-methyl 2-(2-((5 S,8 S)-1-(9 H-fluoren-9-yl)-5-isopropyl-3,6,10-trioxo- 8-(( E)-4-(tritylthio)but-1-enyl)-2,7-dioxa-4,11-diazadodecan-12-yl)thiazol-4-yl)-5,5- dimethyl-4,5-dihydrothiazole-4-carboxylate ( 52 ) ...... 200

1H NMR of ( R)-2,2,2-trichloroethyl 2-(2-(( tert -butoxycarbonylamino)methyl)thiazol- 4-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylate ( 54 ) ...... 201

13 C NMR of ( R)-2,2,2-trichloroethyl 2-(2-(( tert -butoxycarbonylamino)methyl)thiazol- 4-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylate ( 54 ) ...... 202

1H NMR of ( R)-2,2,2-trichloroethyl 2-(2-((( S,E)-3-hydroxy-7-(tritylthio)hept-4- enamido)methyl)thiazol-4-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylate ( 55 ) ....203

13 C NMR of ( R)-2,2,2-trichloroethyl 2-(2-((( S,E)-3-hydroxy-7-(tritylthio)hept-4- enamido)methyl)thiazol-4-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylate ( 55 ) ....204

1H NMR of ( R)-2,2,2-trichloroethyl 2-(2-((5 S,8 S)-1-(9 H-fluoren-9-yl)-5-isopropyl- 3,6,10-trioxo-8-(( E)-4-(tritylthio)but-1-enyl)-2,7-dioxa-4,11-diazadodecan-12- yl)thiazol-4-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylate ( 56 ) ...... 205

13 C NMR of ( R)-2,2,2-trichloroethyl 2-(2-((5 S,8 S)-1-(9 H-fluoren-9-yl)-5-isopropyl- 3,6,10-trioxo-8-(( E)-4-(tritylthio)but-1-enyl)-2,7-dioxa-4,11-diazadodecan-12- yl)thiazol-4-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylate ( 56 ) ...... 206

1H NMR of ( S)-methyl 2-(2-((( S,E)-3-hydroxy-7-(tritylthio)hept-4- enamido)methyl)thiazol-4-yl)-4-methyl-4,5-dihydrothiazole-4-carboxylate ( 59 ) ...... 207

13 C NMR of ( S)-methyl 2-(2-((( S,E)-3-hydroxy-7-(tritylthio)hept-4- enamido)methyl)thiazol-4-yl)-4-methyl-4,5-dihydrothiazole-4-carboxylate ( 59 ) ...... 208

1H NMR of ( S)-methyl 2-(2-((5 S,8 S)-1-(9 H-fluoren-9-yl)-5-isopropyl-3,6,10-trioxo- 8-(( E)-4-(tritylthio)but-1-enyl)-2,7-dioxa-4,11-diazadodecan-12-yl)thiazol-4-yl)-4- methyl-4,5-dihydrothiazole-4-carboxylate ( 60 ) ...... 210

13 C NMR of ( S)-methyl 2-(2-((5 S,8 S)-1-(9 H-fluoren-9-yl)-5-isopropyl-3,6,10-trioxo- 8-(( E)-4-(tritylthio)but-1-enyl)-2,7-dioxa-4,11-diazadodecan-12-yl)thiazol-4-yl)-4- methyl-4,5-dihydrothiazole-4-carboxylate ( 60 ) ...... 211

1H NMR of cyclic core 61 ...... 213

1H NMR of cyclic core 61 ...... 214

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1H NMR of analog T3 (C-7 largazole epimer) ...... 216

13 C NMR of analog T3 (C-7 largazole epimer) ...... 217

1H NMR of analog T4A ...... 219

13 C NMR of analog T4A ...... 220

1H NMR of analog T4B ...... 221

13 C of analog T4B ...... 222

1H NMR of analog T4C ...... 223

13 C NMR of analog T4C ...... 224

1H NMR of analog T4D ...... 225

1H NMR of ( S)-3-hydroxy-1-(( R)-4-isopropyl-2-thioxothiazolidin-3-yl)pent-4-en-1- one ( 62 ) ...... 226

13 C NMR of (S)-3-hydroxy-1-(( R)-4-isopropyl-2-thioxothiazolidin-3-yl)pent-4-en-1- one ( 62 ) ...... 227

1H NMR of ( R)-methyl 2-(2-((( S)-3-hydroxypent-4-enamido)methyl)thiazol-4-yl)-4- methyl-4,5-dihydrothiazole-4-carboxylate ( 64 ) ...... 227

13 C NMR of ( R)-methyl 2-(2-((( S)-3-hydroxypent-4-enamido)methyl)thiazol-4-yl)-4- methyl-4,5-dihydrothiazole-4-carboxylate ( 64 ) ...... 228

1H NMR of ( R)-methyl 2-(2-((5 S,8 S)-1-(9 H-fluoren-9-yl)-5-isopropyl-3,6,10-trioxo- 8-vinyl-2,7-dioxa-4,11-diazadodecan-12-yl)thiazol-4-yl)-4-methyl-4,5- dihydrothiazole-4-carboxylate ( 65 ) ...... 229

13 C NMR of ( R)-methyl 2-(2-((5 S,8S)-1-(9 H-fluoren-9-yl)-5-isopropyl-3,6,10-trioxo- 8-vinyl-2,7-dioxa-4,11-diazadodecan-12-yl)thiazol-4-yl)-4-methyl-4,5- dihydrothiazole-4-carboxylate ( 65 ) ...... 230

1H NMR of cyclic core 66 ...... 231

13 C NMR of cyclic core 66 ...... 232

1H NMR N-(benzyloxy)acrylamide ( 67 ) ...... 232

13 C NMR N-(benzyloxy)acrylamide ( 67 ) ...... 233

1H NMR of analog T5 ...... 234

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1H NMR of methyl 2-(( tert -butoxycarbonylamino)methyl)thiazole-4-carboxylate ( 69 ) 235

13 C NMR of methyl 2-(( tert -butoxycarbonylamino)methyl)thiazole-4-carboxylate (69 ) ...... 235

1H NMR of (R,E)-methyl 2-((3-hydroxy-7-(tritylthio)hept-4- enamido)methyl)thiazole-4-carboxylate ( 70 ) ...... 236

13 C NMR of (R,E)-methyl 2-((3-hydroxy-7-(tritylthio)hept-4- enamido)methyl)thiazole-4-carboxylate ( 70 ) ...... 237

1H NMR of methyl 2-((5 S,8 R)-8-(( E)-4-(acetylthio)but-1-enyl)-1-(9 H-fluoren-9-yl)- 5-isopropyl-3,6,10-trioxo-2,7-dioxa-4,11-diazadodecan-12-yl)thiazole-4-carboxylate (analog T6 )...... 238

13 C NMR of methyl 2-((5 S,8 R)-8-(( E)-4-(acetylthio)but-1-enyl)-1-(9 H-fluoren-9-yl)- 5-isopropyl-3,6,10-trioxo-2,7-dioxa-4,11-diazadodecan-12-yl)thiazole-4-carboxylate (analog T6 )...... 239

1H NMR of ( S, E)-methyl 2-((3-hydroxy-7-(tritylthio)hept-4enamido)methyl)thiazole- 4-carboxylate ( 72 ) ...... 240

13 C NMR of ( S, E)-methyl 2-((3-hydroxy-7-(tritylthio)hept- 4enamido)methyl)thiazole-4-carboxylate ( 72 ) ...... 241

1H NMR of methyl 2-((5 S,8 S)-1-(9 H-fluoren-9-yl)-5-isopropyl-3,6,10-trioxo-8-(( E)- 4-(tritylthio)but-1-enyl)-2,7-dioxa-4,11-diazadodecan-12-yl)thiazole-4-carboxylate (73 ) ...... 242

13 C NMR of methyl 2-((5 S,8 S)-1-(9 H-fluoren-9-yl)-5-isopropyl-3,6,10-trioxo-8-(( E)- 4-(tritylthio)but-1-enyl)-2,7-dioxa-4,11-diazadodecan-12-yl)thiazole-4-carboxylate (73 ) ...... 243

1H NMR of ( S,E)-2-(trimethylsilyl)ethyl 3-hydroxy-7-(tritylthio)hept-4-enoate ( 75 ) ....244

13 C NMR of ( S,E)-2-(trimethylsilyl)ethyl 3-hydroxy-7-(tritylthio)hept-4-enoate ( 75 ) ...245

1H NMR of ( S,E)-2-(trimethylsilyl)ethyl 3-(( S)-2-(((9 H-fluoren-9- yl)methoxy)carbonylamino)-3-methylbutanoyloxy)-7-(tritlthio)hept-4-enoate ( 76 ) ...... 246

13 C NMR of ( S,E)-2-(trimethylsilyl)ethyl 3-(( S)-2-(((9 H-fluoren-9- yl)methoxy)carbonylamino)-3-methylbutanoyloxy)-7-(tritlthio)hept-4-enoate ( 76 ) ...... 247

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1H NMR of ( S,E)-2-(trimethylsilyl)ethyl 3-(( S)-2-(2-(( tert - butoxycarbonylamino)methyl)thiazole-4carboxamido)-3-methylbutanoyloxy)-7- tritylthio)hept-4-enoate ( 77 ) ...... 248

13 C NMR of ( S,E)-2-(trimethylsilyl)ethyl 3-(( S)-2-(2-(( tert - butoxycarbonylamino)methyl)thiazole-4carboxamido)-3-methylbutanoyloxy)-7- tritylthio)hept-4-enoate ( 77 ) ...... 249

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LIST OF ABBREVIATIONS

AAPS American Association of Pharmaceutical Scientists ACS American Chemical society AML Acute myeloid leukemia AMP Adenosine 5'-monophosphate Boc tert -butyloxycarbonyl brsm Based on recovered starting material CDCl 3 Deuterated chloroform CDI carbonyl diimidazole CTCL Cutaneous T-cell lymphoma D2O Deuterated water DIBAL-H Diisobutylaluminium hydride DIPEA Diisopropylethylamine (Hunig's base) DMAP Dimethylaminopyridine DME Dimethoxyethane DMF Dimthylformamide DMSO Dimethyl sulphoxide DMSO-d6 Deuterated dimethyl sulphoxide DNA deoxyribonucleic acid EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide EGFR Epidermal growth factor receptor FK 228 Depsipeptide Romidepsin Fmoc 9H-Fluoren-9-ylmethoxycarbonyl HA Hydroxamic acid HAT Histoneacetyltransferase HATU O-(7-Azabenzotriazole-1-yl)-N, N,N’N’-tetramethyluronium hexafluorophosphate HDAC Histonedeacetylase HDACi Histonedeacetylase inhibitor HDACis Histonedeacetylase inhibitors HDACs Histone deacetylases HDLP histone deacetylase like protein HER Human epidermal growth factor receptor HOAt 1-Hydroxy-7-azabenzotriazole HPLC High performance liquid chromatography HRMS High resolution mass spectra HSP-90 Heat shock protein 90

Hunig's base Diisopropylethylamine, DIPEA MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4- sulfophenyl)-2H-tetrazolium NAD + Nicotinamide adenine dinucleotide NMR nuclear magnetic resonance PKA Protein kinase RNA Ribonucleic acid RT Room temperature SAHA Suberoylanilide hydroxamic acid SAR Structure activity relationship SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SM Starting Material SN1 First oder nucleophilic substitution SN2 Second order nucleophilic substitution TBAB tetrabutylamonnium bromide TEA Triethylamine TFA Trifluoroacetic acid TFMK Trifluoromethylketone THF Tetrahydrofuran TIPS Triisopropylsilane TLC Thin layer chromatography TRAIL Tumor necrosis factor related apoptosis inducing ligand TRAIL R1 Tumor necrosis factor related apoptosis inducing ligand receptor 1 TRAIL R2 Tumor necrosis factor related apoptosis inducing ligand receptor 2 TSA Trichostatin A USFDA United State Food and Drug Administration UT The University of Toledo

xxi

Chapter 1

1 Significance

A major challenge in cancer chemotherapy is the development of drugs which selectively kill cancer cells without affecting normal healthy cells. This is required to increase potency as well as to reduce toxic side effects. A cancer is a group of cells that undergo rapid multiplication due to disruption of the control mechanisms that regulate cell division. In most respects they are like normal cells and therefore, selectively targeting a drug molecule to cancer cells remains a challenging task. Natural products are a main source of lead molecules in the design and development of anticancer agents. 1 Many natural product based drugs such as vincristine, vinblastin, paclitaxel, docetaxel, etopside, teniposide, topotecan, irinotecan, actinomycin D, and doxorubicin with different mechanism of action have been used in cancer treatment (Figure 1). The blockbuster drug paclitaxel is an anti-mitotic agent and has limitations in clinical use due to development of multiple drug resistance. Actinomycin D is an antibiotic which inhibits RNA polymerase, while doxorubicin is a DNA intercalater. A common problem associated with all anticancer agents is lack of selectivity.

O OH O O OH O OH O O OH O HO O O OH O H O O CH Doxorubicin 3 O NH O OH OH O O H2N O Paclitaxel O N O O O N N N N O HN O N O O O HN O HN O O NH O O N NH2

O O Actinomycin D CH CH 3 3

Figure 1. Natural product anticancer drugs.

Recently, a new natural product largazole (Figure 2), isolated from a marine cynobacterium of the genus Symploca, was reported to possess surprisingly selective growth inhibitory activity on highly invasive transformed human mammary epithelial cells (MDA-MB-231) and transformed fibroblastic osteosarcoma (USOS) cells when compared to the corresponding normal cell lines NMuMG and NIH3T3, respectively

(Table 1).2

O S N Largazole NH N S O O O O

H3C(H2C)6 S HN

Figure 2. Structure of largazole

2 Table 1: Growth-inhibitory activity (GI 50 ) of natural product drugs

MDA-MB-231 NMuMG USOS NIH3T3

Largazole 7.7 nM 122 nM 55 nM 480 nM

Paclitaxel 7.0 nM 5.9 nM 12 nM 6.4 nM

Actinomycin D 0.5 nM 0.3 nM 0.8 nM 0.4 nM

Doxorubicin 310 nM 63 nM 220 nM 47 nM

Subsequent mechanistic studies showed that largazole is an inhibitor of histone

deacetylase (HDAC), an enzyme that plays a key role in cell division.3 Histone deacetylases (HDACs) are a class of enzymes responsible for removal of acetyl groups from substrate proteins, while the reverse reaction i.e. transfer of acetyl group from acetyl coenzyme Q to histone is catalyzed by histone acetyl transferase (HAT) 4 (Figure 3). The

normal cell’s finely balanced status of acetylation and deacetylation is modified in

tumorogenesis. 5 The deacetylated protein, positively charged at physiological pH,

interacts with the negatively charged DNA core to form transcriptionally inactive

condensed forms of genes. These epigenetic changes can result in silencing of tumor

suppressor genes leading to tumorogenesis. Inhibition of HDAC activates the

inappropriately silenced genes 6 and is thus an attractive target for anticancer drug

development.

H H HDAC H N N N NH2

O HAT O O

Figure 3. Acetylation and deacetylation processes.

1.1 Role of epigenetics in cancer

Apart from genetic mutations, epigenetics are a major player in carcinogenesis.

Epigenetics are heritable changes which take place without modifying gene sequence. 6

Chromatin is made up of many units of nucleosome which consists of 146 base pairs of

DNA wrapped around 8 histone molecules, 2 each of H 2A, H 2B, H 3, and H 4. Histones,

upon deacetylation, acquire positive charges at physiological pH and tightly interact with

the negatively charged DNA. In contrast, acetylated histones release DNA which is

transcriptionally active. 4, 6 In cancer, aberrant epigenetic silencing of several genes

including tumor suppressor genes is a common occurrence. 7 Aberrant epigenetic

silencing occurs as a result of abnormal promoter region CpG island DNA methylation in

concert with changes in covalent modification of histone proteins. 7g Enzymatic

modifications such as acetylation, methylation and phosphorylation of the lysine and

arginine residues of the N-terminus of histones regulate the access to DNA by

transcriptional factors thereby regulating gene expression. 8 Histone acetylation

modulated by the two protein families histone acetyltransferase and histone deacetylase is

the most extensively studied of these processes. 9 Epigenetic changes, unlike DNA

sequence changes, are reversible and thus targeting epigenetic gene regulation as an

anticancer strategy has attracted the attention of the scientific community.

HDAC proteins are a family of 18 enzymes and are classified into four classes based on

their size, cellular localization, number of catalytic active sites and homology to yeast

HDAC proteins: 10

Class I: HDAC1, -2, -3, and -8 resides in nucleus

Class IIa: HDAC4, -5, -7, and -9 shuttles between nucleus and cytoplasm

Class IIb: HDAC6 and -10 shuttles between nucleus and cytoplasm

Class III: Sirtuin proteins, consists of 7 types of proteins

Class IV: HDAC11 shuttles between nucleus and cytoplasm

Zn 2+ is predominantly required for activity of class I, II and IV HDACs. Class III HDACs do not require Zn 2+ but require NAD + for their catalytic activity. HDAC henceforth in following discussion refers to class I, -II and –IV HDACs only as HDAC inhibitors

(HDACis) inhibit all classes of HDAC other than class III. Association of class III Sirtuin proteins in offsetting age-related diseases such as type II diabetes, obesity and neurodegenerative diseases is known. 9b Although their full characterizations remains to

be accomplished, these HDAC isoforms have generally noticeable gene expression

patterns and may also differ in cellular localization and function. 10 HDAC proteins are involved in basic cellular events and disease states including cell growth, differentiation and cancer formation. 11 Distinctively over-expression of class I and class II HDAC proteins have been noticed in some cancers including ovarian (HDAC1-3), gastric

(HDAC2) and lung cancers (HDAC1 and -3) among others. 11 It has been proposed that

there is a possible correlation between HDAC8 and acute myeloid leukemia (AML).

Over-expression of HDAC6 was induced in some breast cancer cells. 11 Inhibition of

HDAC3 and -8 has been shown to be essential for inhibition of MCF-7 and KYO-1 cancer cell growth. 12 Targeting a single isoform/class can increase selectivity as well as

decrease toxicity.

All of the HDAC proteins except HDAC6 exist in multi-protein complexes with other

regulatory proteins and isolation of individual isoforms becomes difficult, which deters

the development of isoform selective HDAC inhibitors. 13 Class I enzymes consist of

around 500 amino acids while class II are made of about 1000 amino acids. 14 Class IIa

enzymes consist of a 600 amino acid residue extension at the N-terminus and they play a

role in regulatory and functional properties. 15 HDAC1 and -2 are highly related enzymes

with an overall sequence identity of about 82%. Phosporylation status of HDAC1 and -2

affects their activity. Hyperphosphorylation increases deacetylase activity while

hypophoshorylation has the opposite effect.16 HDAC3 has 34% overall sequence identity

with HDAC8. 16 HDAC4 and -5 are largely similar to each other (with overall sequence

identity 70%), and HDAC7 in turn is related to HDAC4 and -5 with ~ 58 and ~ 57%

overall similarity, respectively. 16 HDAC4, and to a lesser extent HDAC8 and -9, seem to

be expressed more in tumor tissues than in normal tissues. 16 HDAC5, -7 and -9 are

expressed in heart tissues and they can be correlated with their role in the development of

cardiac muscle tissue.16 Increased levels of HDAC5 mRNA in depressive patients have

been reported.17 HDAC6 has two independent catalytic domains- one at C-terminal and

the other at the N-terminal of the protein. C-terminal catalytic domain maintains its

activity even upon trimming of the N-terminal catalytic domain. Tubulin deacetylation activity is present in the C-terminal domain. 17 HDAC6 deacetylates tubulin and heat shock proteins apart from histones and is highly expressed in tumor cells. 16-17 It has been suggested that HDAC6 inhibitors are potential anticancer agents in the treatment of multiple myeloma 18 and they are also useful in inflammation, Huntington’s disease and various neurodegenerative disorders. 17, 19 Studies have shown that HDACs deacetylate many non-histone proteins as well. More than 1700 non-histone proteins have been detected as substrates for HDACs. 13 These non-histone proteins play a regulatory role in processes such as cell migration, cell proliferation and cell death. 20

X-ray crystallography and SAR studies of various HDAC inhibitors have shown that three key structural elements are required for a molecule to be effective as a HDAC inhibitor (Figure 4): 21 i) a metal binding domain which chelates with the Zn 2+ cation present at the active site ii) a linker, which occupies the hydrophobic channel iii) a surface recognition group, which interacts with hydrophobic residues on the rim of

the active site

Most HDAC inhibitors, including those in clinical trials, are reversible inhibitors, although irreversible inhibitors are known. 17 Irreversible inhibitors with a keto-epoxide covalently interacts with nucleophilic residues such as histidine, aspartate, and tyrosine of the active site. 17 The variety of functional groups used as zinc binding moieties include thiol, epoxy ketone, carboxylic acid, boronic acid, sulfoxide, sulfonamide,

phosphonamidate, phosphonate, phosphinate, trifluoroketone, hydroxamic acid, 2-amino benzamide, oxime amide, and α-thioamide.7e, 17

O S N Largazole NH N S O O O O

H3C(H2C)6 S HN

Linker Surface recognation cap group Zinc binding element

Figure 4. HDACi binding elements.

The selective activity of an HDAC inhibitor on a particular type of cancer cells may be attributed to variations in the composition of HDAC proteins. HDAC inhibitors have become a major area of research in the development of new drugs for the treatment of cancer. The HDAC inhibitors suberoylanilide hydroxamic acid (SAHA) (Figure 5), and depsipeptide FK 228 (Figure 6) were approved by the US FDA in October 2006 and

November 2009, respectively, for the treatment of cutaneous T-Cell lymphoma

(CTCL). 13

O H N OH N H O

Figure 5. Structure of SAHA.

A number of other HDAC inhibitors are in various phases of clinical trials (Table 2).

SAHA inhibits all isoforms of HDAC (thus called a pan-HDAC inhibitor) to varying degrees. The small cap group of SAHA interacts with the highly conserved region of the enzyme rim and is unable to make contact with the variable region which is further away from the opening of the channel of HDAC. The inability to interact with the variable region is responsible for its non-specificity. 22

Table 2. A list of HDACis in clinical trials 7b, 13, 23

Compound Chemical class Status Indication Butyrate Short chain Fatty acid Phase II Non-small cell lung cancer Valproic acid Carboxylic acid Phase I /II Non-small cell lung cancer Pivanex/ AN 9 Ester Phase I/II Chronic Lymphocytic Leukemia Ester Phase II Progressive or Recurrent Phenylbutyrare Brain Tumors

Belinostat Hydroxamate Phase II Refractory Peripheral T-Cell Lymphoma Panobinostat Hydroxamate Phase II Refractory Clear Cell Renal Carcinoma, Metastatic Thyroid Cancer, Refractory Colorectal Cancer Phase II/III Chronic Myeloid Leukemia CUDC-101 Hydroxamate. Phase I Advanced Solid Tumors Inhibits human epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2) and histone deacetylase (HDAC) JNJ-26481585 Hydroxamate Phase I Advanced Solid Malignancies and Lymphoma CHR-3996 Hydroxamate Phase I Advanced Solid Tumors

AR-42 Hydroxamate Phase I Advanced or Relapsed Multiple Myeloma, Chronic Lymphocytic Leukemia, or

Lymphoma

SB939 Hydroxamate Phase II Recurrent or Metastatic Prostate Cancer, Locally Advanced or Metastatic Solid Tumors PCI-24781 Hydroxamate Phase II Follicular Lymphoma Table 2 Continued from page 9 ITF2357/ Hydroxamate Phase II Refractory/Relapsed Givinostat Lymphocytic Leukemia 4SC-201/ Hydroxamate Phase II Relapsed or Refractory Resminostat Hodgkin's Lymphoma N-Acetyl Aminobenzamide Phase II Advanced Myeloma Dinaline (CI- 994)/ Tacedinaline MGCD0103 Aminobenzamide Phase II Refractory Chronic Lymphocytic Leukemia MS-275/ Aminobenzamide Phase II Metastatic Melanoma Entinostat

Phase I Advanced Solid Tumors or Lymphoma CG200745 Aminobenzamide Phase II Solid Tumors 4SC-202 2-Aminophenylcarbamoyl Phase I Advanced Hematologic Malignancies

Due to its highly selective activity on selected cancer cell lines and a strong

unprecedented picomolar inhibitory bias for class I HDACs (HDAC1, -2 and -3) over

class II HDAC6,3 largazole provides a new attractive lead molecule in the development of class and /isoform selective HDAC inhibitors. Interestingly, largazole shares a common 3-hydroxy-7-mercapto-4-heptenoic acid side chain with related natural product

HDAC inhibitors such as FK228 2, FR901375 3, and spiruchostatins 4 (Figure 6). All of

these agents are prodrugs which upon activation release the free thiol group necessary for

HDAC inhibitory activity. The thioester group present in largazole is hydrolyzed by

cellular esterases and/or lipases to release the free thiol function which constitutes the

domain that chelates Zn 2+ .3b In compounds 2, 3 and 4 the masked thiol is present as a disulfide bridge with a cysteine residue in the depsipeptide ring and is reduced to free thiol by glutathione mediated cellular reduction.24

9 8 O S O 6 7 10 N NH 3 12 HN NH O 2 3 S N O O O O O O NH 13 O O S H3C(H2C)6 S 17 15 HN 22 N H 1. Largazole S 2. FK228 O H N R N S O H O S OH O HN O N H H N NH O O O O HO N O O H S S 4. Spiruchostatin A R= Me 3. FR901375 Spiruchostatin B R= Et

Figure 6. Largazole and related natural products.

Total synthesis of largazole and several analogs along with some preliminary structure-

activity relationship (SAR) studies has been published. 3, 25 SAR studies (Figure 7) have

shown that variations in the length of the side-chain (A, B, C) resulted in decreased

activity. 25c, d Diminished activity resulted upon replacement of the side chain sulphur

atom with oxygen (D) or a methylene moiety (E).25a α-Aminoanilide group as a zinc

binding agent is known to produce HDAC1 and -2 selective activity 26 while α-

mercaptoacetamide compounds 27 showed preferential binding to HDAC6 over HDAC1, -

2, -8 and -10. However, thiol replacement with α-aminoanilide ( F, G) or α-

mercaptoacetamide ( H, I) did not produce noticeable selectivity. 25g The C-2 epimer J,

non-natural largazole enantiomer K, analog L with proline substituted for valine all had

reduced anticancer activity. 25g, 28 On the other hand, substitution of the thiazole-thiazoline moiety with an oxazole-oxazoline moiety (M) and cysteine substitution for α-methyl cysteine (N) gave equi-potent analogs. 25g Replacement of thiazole ring with pyridine (O) resulted in a 3-4 fold enhancement in HDAC inhibitory activity. In fact, the pyridine analog O is the most potent HDAC inhibitor known.25g The amide analog formed by substitution of ester oxygen atom at C-17 with an NH moiety (P) led to reduced activity.

This was attributed to a conformational change in the cap group of the molecule rather than to a change in hydrogen bonding ability as it occupies the hydrophobic region of the active site.25h

O S 7 O S 3 N NH 12 N 2 NH N S O OOO N S O OOO S N 6 n 17 15 H O N 6 H n = 1- Largazole D n =0, 2, 3-analogs A, B, C

O S O S N N NH NH N S N S NH2 O OOO O OOO N N n H 6 N H E H n = 2,3- analogs F,G

O S O S N NH N NH N S OOO N S H O OOO N HS N n H S N O 6 J H n = 1,2- analogs H, I O S O S N N N NH N S N S O OOO O OOO 6 S N S N L H 6 K H

H O S O O N N NH NH N S N O O OOO O OOO S N S N 6 6 N H M H

O O S S N N NH NH N N S O OOO O OONH

S N 6 S N 6 O H P H Figure 7. Largazole analogs synthesized in other laboratories.

1.2 Crystal structure and mechanistic pathways

Crystal structure information can dispense valuable directions for the design and

development of isoform and/ class-selective HDAC inhibitors. Currently, crystal

structures of HDAC2, -4, -7 and -8 are known. 13, 15, 17 It is assumed that the acetyl group of the lysine residue is held in the binding site by multiple hydrogen bonds during deacetylation by HDAC.22 A general catalytic mechanism for HDAC based on computational studies with histone deacetylase like protein (HDLP) by Vanommeslaeghe is shown in Figure 8.29 The Lewis acid-catalyzed carbonyl group activation by Zn 2+ facilitates the nucleophilic attack by a H2O molecule to generate a tetrahedral intermediate, which is stabilized by Tyr 297 and Zn 2+ . The stabilization of the intermediate transition state is mediated by Tyr 297 in HDLP, Tyr 303 in HDAC1, Tyr 304 in HDAC2,

306 976 709 Tyr in HDAC8, His and H 2O in HDAC4 and His in HDAC7. The tetrahedral intermediate breaks down to yield deacetylated lysine protein and acetate ion. 22

Class IIa HDAC enzymes have low activity as a result of low transition state stabilization due to the absence of Tyr 306 .15 A 320 amino acid residue structural homology between class I and –II HDACs at the catalytic site is known. 17 Class IIb enzymes are characterized by two catalytic domains. 15 Structures of HDAC4, -7 and -8 consist of two potassium ions near the catalytic site. 15 The four conserved zinc-binding ligands in class

IIa HDACs are His 665 , Cys 667 , His 678 , and Cys 751 which are missing in other HDACs. 15

Lack of transition state stabilizer Tyr 306 in HDAC4, and -7 (which is present in class I

HDACs) is responsible for their reduced enzymatic activity. 15 In HDAC4, Zn 2+ ion is

loosely bound to the protein. 15 In addition to inhibiting the enzyme, HDAC4 inhibitors

also hinder the role of class I HDAC3 as a promoter of HDAC4-

173 Asp173 Asp O HN O HN H N H N N 132 N His132 His H H substrate protein substrate protein 131 with acetyl lysine 131 His 166 His with acetyl lysine Asp in tetrahedral Asp166 N H intermediate N H NH N state NH N OH O H O H H O O O H Tyr297 H NH 2+ O 297 2+ O Zn NH Tyr Zn O NH N N NH N O H O N H O O O O O His170 His170 H H N O N O N N O H O H O O Asp168 Asp168 Asp258 Asp258

Asp173 O HN N H N His132 substrate protein with deacetylated lysine Asp166 N NH HN His131 OH 2 O H O Tyr297 NH 2+ O Zn NH O N O N H O O His170 H N O N O H O Asp168 Asp258

Figure 8. Proposed mechanism of action of Zn 2+ - dependent HDACs. (Adapted from Nat. Prod. Rep. , 2009 , 26 , 1293–1320).

regulated genes. 15 In a X-ray crystallographic study with HDAC4, it has been observed

that the trifluoromethyl ketone ( TFMK ) (Figure 9), upon hydration binds to HDAC4 in a

bidentate fashion as compared to the hydroxamate ( HA ) which interacts in a monodentate

manner. Unlike HA , TFMK fills the gap near Pro 800 and this leads to higher potency of

TFMK over HA (Figure 10).15 As this pocket is larger in HDAC1, -2 and -3 enzymes,

TFMK type compounds typically display 10-100 fold greater inhibition of class II

HDACs than class I HDACs. 15 Class IIa HDACs have a longer binding pocket near

His 976 due to the outward positioning of His 976 side chain. 15

O O N N N CF3 NH OH S N S N N HA O TFMK O

Figure 9. HDACis used in binding studies with HDAC4.

HDAC7 has a major role in cardiovascular development and disorders. 30 Inhibitor

binding studies with HDAC7 by X-ray crystallography (Figure 11) revealed that

benzamide inhibitors ( MS-275 , CI-994 , MGCD-0103 ), cyclic tetrapeptides (apicidin)

and short chain fatty acids (butyrate, and valproic acid) did not show binding abilities. 31

Binding of hydroxamate HDAC inhibitors (SAHA, TSA) to the active site Zn 2+ of

HDAC7 takes place in monodentate manner with participation of hydroxyl oxygen

only. 31 The carbonyl oxygen of hydroxamate is directed away from the zinc ion and is connected to a water molecule through hydrogen bonding. Amino acids present at the periphery of the pocket show conformational changes to the inhibitor upon its binding. 31

HDAC7 alone has low intrinsic catalytic activity while it is activated in the presence of

HDAC3. 31

Figure 10. Interaction of TFMK (yellow carbons) and HA (green carbons) with HDAC4 catalytic domain. Reprinted from Journal of Biological Chemistry 2008 , 283 (39), 26694- 26704.

O O O O N O N H O H N Trichostatin (TSA) HN NH O N O HN N N N NH H H 2 O N MGCD0103 Apicidin H O N NH H 2 O N OH CI-994 O O O Valproic acid O N NH H H 2 N O N MS-275 Butyrate O O

Figure 11. HDACis used in binding studies with HDAC7.

HDAC8 consists of 377 amino acids and is closely associated with phylogenetic border

between class I and class II HDACs. 32 HDAC8 activity is decreased upon cyclic AMP-

dependent protein kinase (PKA) phosphorylation of ser 39 which is responsible for the

hyperacetylation of histone H3 and H4. Serine is positioned at the surface of HDAC8,

approximately 20 Å from the opening to the active site and about 13 Å from the opening

to the second binding site (Figure 12). 32 A possible role of HDAC8 in acute myeloid

leukemia (AML) has been indicated. 32 Inversion, a chromosomal translocation, often

takes place in AML and produces a protein product which causes abnormal transcription

repression. HDACis reverse the repression produced by protein product association with

HDAC8. 32 HDAC8 consists of a wider active site pocket with broad surface opening. 32

The hydrophobic wall of the tunnel is lined with Phe 152 , Phe 208 , His 180 , Gly 151 , Met 274 and

Tyr 306 . These are preserved across the class I enzymes except that met 274 is replaced with

Leu in the other family members. 32

Figure12. Serine phosphorylation site (red) in relation to the active site of HDAC8 where it shows interaction with two molecules of TSA. Reprinted with permission from Structure 2004 , 12, 1325–1334.

In a HDACis binding study with HDAC8, it has been shown that hydroxamate inhibitors

bind in bidentate fashion. Somoza et al 33 observed that HDAC8 binding site is very flexible and can accommodate ligands with different structural motifs (Figure 13, 14) .

O HO NH HO O HN HN O CRA-A MS-344 NH O N N O

Figure 13. Some of the HDACis used in binding studies with HDAC8.

Figure 14. Interaction of HDACis with flexible catalytic pockets of HDAC8. TSA and CRA-A occupy two binding pockets while SAHA and MS-344 engage only one. Reprinted with permission from Structure 2004 , 12, 1325–1334.

This information was used by Ulrich et al 34 in the rational design of linkerless selective

HDAC8 inhibitors (Figure 15: compound R, S).

HN O OH O NH OH RS

HDAC1 IC50 (µM) >100.0 >100.0

HDAC6 IC50 (µM) 82.0 55.0

HDAC8 IC50 (µM) 0.7 0.3 Figure 15. Linkerless selective HDAC8 inhibitors.

It has been observed that proliferation of human lung, cervical and colon cancer cell lines were inhibited by knockdown of HDAC8 by RNA interference studies.35 HDAC1 and -3 have 43 % sequence identity with HDAC8. 35 In general, HDAC8 exists as a dimer of two molecules interacting head to head and each monomer binds with one Zn 2+ and two K + ions. 35 The presence of K + in the active site can affect the catalytic mechanism of the enzyme’s deacetylation process. 35 K+ can stabilize the transition state oxyanion and/ or the negatively charged acetate product. Also, the presence of K + ion may deliver the conformation required for catalytic activity. 35 Effective zinc binding of the large zinc interacting group, a 2-aminobenzanilide of MS-275 (Figure 16), is hindered because of the presence of Trp 141 at the bottom of the active site and leads to lack of inhibition of

HDAC8 by this compound as compared to HDAC1 and -3 which have Leu at 141 position (Figure 17).35

O N NH H H 2 N N MS-275; R = H T; R = Ph O

HDAC1IC50(nM) 125 10 R HDAC3IC50(nM) 370 6180

S S

O O O N N H N H H P NH2 MeO Q O N NH2 Me HDAC1 IC : 16 nM R 50 O HDAC1 IC50: 6 nM HCT-116 GI : 0.330 µM HCT-116 GI : 155 nM 50 50

Figure 16. Representative examples of selective HDAC1 and -2 inhibitors.

The presence of an aryl moiety para to amino group in aminobenzamide HDACis increases selectivity toward HDAC1 and -2 (Figure 16). This group is accommodated by

HDAC1 and -2 and not by other HDAC isoforms. This structural information was utilized by Miller et al of Merck research laboratories to develop several classes of selective HDAC1 and -2 inhibitors (Figure 16: compound Q, R).26

Figure 17. Zinc binding site of HDAC8 showing the presence of Trp 141 at the bottom of the site. Reprinted with permission from Proc. Natl. Acad. Sci. U. S. A. 2004, 101 (42), 15064-15069. "Copyright 2004 National Academy of Sciences, U.S.A."

α-Mercaptoacetamide compounds showed preferential binding to HDAC6 over HDAC1,

-2, -8 and -10. These compounds showed better neuroprotection. 27 As HDAC1 is required

for neuronal cell survival, selectivity of α-mercaptoacetamide compounds for HDAC6

over HDAC1 might be responsible for observed neuroprotection. 27

It has been suggested that HDACis arrest cell cycle at both the G1/S and G2/M phases. 36

As HDAC acts on histone as well as multiple non-histone proteins, many mechanistic

pathways have been suggested to include all possible events. 4 Acetylated histones were

the earliest substrates identified for HDACs. But a large number of other nuclear and

cytoplasmic proteins regulating different biological processes, whose activities are

modified through acetylation/ deacetylation, have been identified. Thus, this

posttranslational modification involving acetylation is emerging as a major mechanism of

regulating protein and cell function. 36

Proliferation arrest and TRAIL (Tumor Necrosis Factor Related Apoptosis Inducing

Ligand) mediated apoptosis are other outcomes of HDAC inhibition. HDACis induce p21

as well as TRAIL expression and thus cause tumor-selective death signaling in acute

myeloid leukemia (AML) cells and the blasts of individuals with AML. RNA

interference studies have shown that induction of p21, TRAIL and differentiation are

separate activities of HDACis.4 It has been observed that HDACis which act on HDAC1 and -2 cause TRAIL sensitization. These TRAIL proteins activate TRAIL receptors

TRAIL-R1 and TRAIL-R2 which commence activation of intrinsic and extrinsic death signaling cascades ultimately resulting in apoptosis. 17, 37 It has been suggested that

HDAC inhibitors are responsible for an increase in reactive oxygen species (ROS) in cancer cells preferentially over normal cells. 4 Reversible inhibition by HDAC inhibitors provide time for the normal cells to recover from the effect of the inhibitor, and thus survive in contrast to cancer cells. This could be another reason for selective inhibition of growth of cancer cells over normal cells. 20 Other proposed mechanisms include transformed cell cycle arrest, terminal cell differentiation, cell death by induction of intrinsic apoptotic pathway, activation of extrinsic apoptotic pathway, mitotic failure, autophasic cell death, polyploidy and senescence. 20

The effect of HDACis is not limited to cancer cells; they have also shown promising effects in a range of other disease states such as neurological disorders, arthritis,

inflammation, infectious diseases (such as HIV infections, malaria, fungal infections),

sickle cell anemia and cardiovascular diseases. 38 Toxicities associated with clinically

tested HDAC inhibitors include fatigue, vomiting, hypokalemia, diarrhea, and

thrombocytopenia. 5 Cardiac irregularities are also reported with many HDAC inhibitors. 5

HDAC isoform and/or class-selective inhibitors are likely to reduce side-effects and to have increased potency.

1.3 Innovation (Drug Design and Rationale)

In order to develop largazole analogs as potent isoform / class selective anticancer agents,

it was decided to:

1. Synthesize several largazole analogs of varying structures to determine the

structural elements required for improved activity with low toxicity. The designed

analogs incorporated changes in the surface recognition group as well as in the

zinc binding domain of largazole.

2. Determine HDAC inhibitory activity and HDAC isoform selectivity of these

synthesized analogs. This will also help to understand the role of individual

HDAC isoforms in certain disease conditions.

Less sequence homology between isoforms at the surface near the opening to the binding

pocket may be exploited to design isoform selective inhibitors. The area of the HDAC

enzyme bordering the binding pocket consists of many grooves which could be varyingly

occupied by multiple cap groups to impact selectivity as well as potency. 17 modifications of the zinc binding group and cap group has been extensively used to modulate selectivity of HDACi.11, 17 In order to increase isoform and/ or class selectivity for HDAC inhibitory activity, we propose to synthesize a number of largazole analogs T1- T7 (Figure 18) with modifications in the zinc binding group and cap group and to evaluate their HDAC inhibitory activity and selectivity profile. These compounds may help to define the role and possible pharmacological significance of isoform/class specific HDAC inhibitors in cancer treatment. 21, 39

H O S O S R N N NH NH S N N S O O O O O OOO HN H3C(H2C)6 S S N Amino acid analogs T1 6 H T2 R = R-1-naphthylmethyl, S-1-naphthylmethyl, S-Allyl, S-carbamoylethyl O S N O S NH N N S NH O O O N S O OOO S HN T4 H C(H C) S N R 3 2 6 H 1 C-7 epimer of largazoleT3 R1 = (CH3)2NCX, 2-hydroxybenzyl 2-pyridyl, 2-thenyl, O S NHFmoc R N 1 NH O N S O O O O O O H N S X R HO HN T6 O T5 O R 1 = S-isopropyl, R-isopropyl, R-1-naphthylmethyl, S-1-naphthylmethyl, S-Allyl, S-carbamoylethyl HN R = 4-methoxycarbonylthiazol-2-yl, cyclohexyl, 1-naphthyl

N S O O O O

H C(H C) S N 3 2 6 T7 H

Figure 18. HDAC inhibitors designed for synthesis

It has been shown that the replacement of L-valine (circled in structure of FK228 and largazole in Figure 5) with L-naphthylalanine and L-phenylalanine in FK228 resulted in enhancement of antitumor activity. 25l, 40 Interaction of the hydrophobic side chain of naphthylalanine with the hydrophobic elements of HDAC active site and changes in hydrogen bonding interactions are likely to be responsible for this enhanced activity profile. In order to determine the effect of such structural change in largazole, we designed several amino acid analogs of T1 series bearing hydrophobic as well as hydrophilic side chains such as R-1-naphthylmethyl, S-1-naphthylmethyl, S-Allyl, S- carbamoylethyl. 26

Analog T2 with a substituted thiazoline ring is designed to determine the effect of

increasing the steric bulk at C-8 on activity and selectivity. Also it will determine if (R)-

α-methyl cysteine HCl which is expensive and not readily available can be replaced with inexpensive and commercially available L-penicillamine.

Analog T3 is the C-7 epimer of largazole for structure activity relationship studies.

Changing configuration at C-7 changes the shape of the depsipeptide ring and will provide additional information about the SAR requirements of largazole.

Analogs T4 are designed such that sulphur and a second heteroatom present in the side- chain are 2/3 atoms away from each other can form a cyclic 5/6 membered transition state with Zn 2+ of HDAC enzyme (Figure 19). 41 As Zn (II) is a soft metal ion which can show different coordination numbers such as tetra-, penta and hexa-coordination, 42 binding of two heteroatoms to the Zn 2+ cation can increase its affinity for the enzyme and

may lead to enhancements in activity.

O S N NH N S OOO

S N 2 H X Largazole Zn

Figure 19. Proposed binding of T4 series analogs with HDAC active site.

The clinically approved HDAC inhibitor SAHA has a hydroxamic acid moiety as the zinc binding group. Although hydroxamic acid is rapidly hydrolyzed to the inactive carboxylic acid and is toxic, it is a widely studied zinc binding group because it is one of the most potent zinc binding agents and also it can form hydrogen bonds with various amino acids present in the enzyme. 22 Considering one order magnitude higher binding aptitude of hydroxamic acid over thiol, 42 we propose to replace the thiol group of largazole with a hydroxamic acid moiety to generate analog T5.

Molecules shown in Figure 19 are highly potent and selective HDAC6 inhibitors. 18b

Unique structural features of HDAC6 include a C-terminal zinc finger moiety and two catalytic domains. HDAC6 is also responsible for deacetylation of non-histone proteins such as α-tubulin and HSP-90. It is a much larger protein which does not complex with other isoforms and is widely present in the body. 17 It has been reported that HDAC6 inhibitors are Y-shaped molecules. 22 Molecules shown in Figure 20 are highly potent and selective HDAC6 inhibitors. 18b Target molecules T6 are designed to meet these structural requirements. It has been suggested that Boc cap group present in these molecules has

some favorable interactions with the HDAC6 enzyme. 17 Retaining the Fmoc group in the

designed analogs T6 will mimic steric hydrophobic bulk of Boc group to preserve its

complimentary interaction with the HDAC6 surface. 17 They will be synthesized and

tested against HDACs for HDAC6 isoform selectivity. HDAC6 inhibitors can be used in

the treatment of multiple myeloma and neurodegenerative Huntington’s disease. 17-19

O H C O R = , 3 O , , H3C H CH3 N SH R O Figure 20. Selective HDAC6 inhibitors.

Analog T7 is a smaller ring version of largazole designed to explore if the thiazoline ring is required for activity.

The synthesized compounds will be tested to determine GI 50 values by antiproliferative assay with cancer cell lines, HDAC inhibitory activity, effect on HDAC6 and HDAC isoform selectivity. Most of the biological testing would be carried out in the laboratory of Dr. Robert A. Casero, Professor of Oncology, the Sidney Kimmel Comprehensive

Cancer Center, the Johns Hopkins University, School of Medicine, Baltimore.

Chapter 2

2 Results and Discussions

2.1 Synthesis of largazole

The convergent synthetic approach adopted for synthesis of largazole is shown in retrosynthetic analysis (Scheme 1). It involves macrolactamization and side chain thioesterification of the intermediate 32 which could be derived by acyl transfer from fragment 22 to 30 followed by coupling with Fmoc-valine. Intermediate 30 can be synthesized from the thiazole nitrile 7 and ( R)-α-methylcysteine hydrochloride 13 .

Synthesis of 13 would also generate its enantiomer useful for making the C-7 epimer of largazole analogs. Fragment 22 is the product of acetate aldol reaction of the aldehyde 21 using acetyl Nagao 16 as the chiral auxiliary. 43

O S N NH N S O OOO

H3C(H2C)6 S N 1 Largazole H

MeOOC S N O S NHFmoc N OMe N S S O O O 30 N Cl- S NH 32 H3N

OH O S NC S N 22 S Cl- SH S OH N 7 H3N O S O 13 O Boc HN N S S H 21 SH 16 O Cl OO Acetyl Nagao NH t-Butyl mercaptan Chloroacetone Boc NH C 2 4 S OH Boc-thioglycinamide Glycerol formal

Scheme 1. Retrosynthetic analysis of largazole

2.1.1 Synthesis of nitrile 7

The synthesis of thiazole derivative 7 is shown in Scheme 2. Thiazole amide 6 was made in 60% overall yield from commercially available Boc-thioglycinamide and bromopyruvic acid in a one-pot reaction in which the initially formed carboxylic acid, after removal of water, was activated and treated with ammonia. This one pot protocol was found to be more efficient and gave the product in higher yield and in a much shorter

period of time than the previously reported methods. 25a Amide 6 was converted to the nitrile 7 using standard conditions in 99% yield. 25a We improved our own protocol by performing all reactions from Boc-thioglycinamide 4 to nitrile 7 in one pot with improved yield of 65% as compared to previous 59% overall yield. It also saved time and solvents required in the tedious purification of amide 6.

HOOC o Boc NH bromopyruvic acid S 1) Et3N, ClCOOEt, O C N 2 H N THF, 50 oC 2) NH OH,RT, 4 S 5 4 Boc HN 60% from 4

H2NOC NC S S N Et3N, TFAA, CH2Cl2, RT N 7 Boc NH 99% Boc NH 6 Scheme 2. Synthesis of fragment 7

2.1.2 Synthesis of ( R)- and ( S)-α-methylcysteine

The synthesis of ( R)-α-methylcysteine HCl 13 and its (S)-enantiomer 14 was achieved via the enzymatic resolution of racemic precursor 11 (Scheme 3). 44 Reaction of chloroacetone and t-butyl mercaptan under basic conditions gave 9, which was subjected to Bucherer-Berg conditions to produce hydantoin 10. This one-pot protocol gave 90% yield of 10 after two steps. Hydantoin 10 was converted to 11 in one-pot by hydrolysis with aqueous NaOH followed by KCNO treatment. Resolution of 11 with D- hydantoinase afforded 12 and the hydantoin enanantiomer 10a which were converted to

13 and 14, respectively, in quantitative yields.44

O O O 1) Aq.NaOH, reflux Aq NaOH S NaCN, HN NH Cl t-BuSH (NH )HCO , 2) KCNO, 70 oC 8 9 4 3 NH OH O 45% 4 S 90% f rom 8 10

D-hydantoinase O S Aq. NaCl, 40 oC O S SH OH 82% OH HCl, ref lux OH H NN H3N 2 H2NN H H quant. - O O Cl O 13 11 12 O i) LiOH, reflux Cl SH ii) HCl, reflux, OH HN NH H N quant. 3 O O S 14 10 a

Scheme 3. Synthesis of ( R)- and ( S)-α-methylcysteine

2.1.3 Synthesis of acetyl Nagao auxillary (16)

Nagao Auxillary was synthesized by refluxing D-valinol with CS2 in 1 N KOH (Scheme

4). 45 Acetyl Nagao 16 was synthesized by reacting Nagao auxillary with NaH and acetyl chloride in 92% yield.

S H2N NaH, acetyl chloride, S OH CS2, 1N KOH O ether, 0 oC then rt 120 oC HN S N S 92% 92% D-valinol 15

Nagao auxillary 16

Scheme 4. Synthesis of acetyl Nagao auxillary (16 )

2.1.4 Synthesis of the t-butyl protected β-hydroxyamide 22

Turning our attention to the synthesis of 22 (Scheme 5), commercially available glycerol formal 17 was converted to dioxene 18 in two steps. Glycerol formal was converted to the tosylate which was heated at 200 oC in the presence of KOH to generate the dioxene

18 . Alkylation of 18 with 2-[(2-bromoethyl)sulfanyl]-2-methylpropane (19 ) via the lithium derivative gave 20. Compound 20 underwent retro Diels-Alder reaction to give the aldehyde 2146 which was used in acetate aldol reaction to furnish the alcohol 22 in

81% diastereomeric excess. The two diastereomers were purified by flash column chromatography on silica gel in ethyl acetate: hexanes (10-30%). The S-configuration of the newly created chiral center of molecule 22 was established by modified Mosher ester

analysis. 47

1) tosyl chloride, pyridine, 0 oC OO 62% t-BuLi, THF, -78 oC OO OO o 2) Aq. KOH, 200 C S 44% Br S OH 18 19 85% 20 17

OH O o S toluene, 165 C O 16, TiCl4 S N microwave, 96% S H S 22 DIPEA, CH Cl , 21 2 2 -78 oC, 64% OH O dr 9.4: 1 (comp. 22:23) S S N S 23

Scheme 5. Synthesis of the t-butyl protected β-hydroxycarboxamide 22

2.1.5 Attempts for synthesis of carboxylic acid 29

The thiazole nitrile 7 was reacted with (R)-α-methyl cysteine HCl 13 to synthesize the thiazole-thiazoline derivative 29 as shown in Scheme 6 (vide infra). 25a, 25c Compound 7 consists of cyanide which acts as an electrophile and reacts with the nucleophilic thiol to give intermediate P1 . Another nucleophilic attack by amine leads to formation of intermediate P2 . Lone pair electron assistance for displacement of ammonia leads to the formation of product 29 . This reaction was initially performed using L-cysteine HCl instead of ( R)-α-methyl cysteine HCl 13 in a model reaction to obtain product 28 . As the reaction sequence requires conversion of the carboxylic acid 29 to its methyl ester, we attempted another model reaction (not shown) using the methyl ester of L-cysteine to see if the formation of the ester can be accomplished in one step. However, when ethyl or methyl ester of cysteine HCl was used under similar conditions, reaction was very

o sluggish even after 3 days of refluxing conditions with Et 3N in MeOH or stirring at 70 C with NaHCO 3 in MeOH and phosphate buffer pH 5.95 for 3 days.

O H S

HO N S N 28 H Boc HN S H HO NaHCO3, MeOH, phosphate NH3 buff er pH 5.95, 70 oC O Cl- or cysteine HCl Et3N, MeOH,reflux + N O Me H S S NaHCO3, 13, MeOH, N HO N o S phosphate buffer pH 5.95, 70 C N Boc HN 29 7 Boc HN H S Me HO Base NH 13 3 O Cl- H+ O NH H+ S NH2 HO N S S H S N N P1 P2 H N 2 COOH NHBoc Boc HN

Scheme 6. Mechanism for conversion of nitrile 7 to thiazole thiazoline derivative 29

A number of alternative methods were attempted by different routes before settling to the synthesis of 29 from compound 7 with NaHCO 3, MeOH and phosphate buffer pH 5.95 as in Scheme 6. Following paragraphs discuss each of the failed attempts.

Attempt 1

We initially used the more easily obtained ester 25 as the electrophile in expectation to obtain the same product 29 . Unfortunately reaction of ester 25 with compound 13 under similar conditions did not produce 29 (Scheme 7).

O O S HO N EtO S S NaHCO3, 13, MeOH, N N 25 X 29 phosphate buffer pH 5.9, 70 oC Boc HN Boc NH

Scheme 7. Failed attempt 1 for synthesis of 29

Attempt 2

Conversion of esters to amides by DIBAL-H is known. 48 If compound 25 is converted to intermediate 24 by DIBAL-H in the presence of 13 , the intermediate 24 may undergo cyclization to produce compound 29 ( Scheme 8). However when the ester 25 and 13 were treated with DIBAL-H (1.15 equiv), there was no disappearance of starting material. Increasing the DIBAL-H equivalence to 3.5 did not change reaction outcome, most probably due to the highly sterically hindered environment of the ester group or due to the presence of hetero atoms in compound 25 which can chelate with DIBAL-H.

HOOC SH DIBAL-H DIBAL-H HOOC NS 1.15 equiv, SH HN 3.5 equiv, COOH 13,THF COOEt 13, THF HN COOH NS O O NS XXNS NS NS NS 29 BocHN NH-Boc 29 NH-Boc NH-Boc BocHN 25 24 24 SH OH H3N 13 Cl- O

Scheme 8. Failed attempt 2 for synthesis of 29

Subsequent attempts were made using model reactions.

Attempt 3

Formation of amides from esters and amines using isopropyl magnesium chloride as

Lewis acid is known. 49 However, reaction of ethyl benzoate M1 and L- cysteine HCl in the presence of different Lewis acids including isopropyl magnesium chloride failed to give product M2 (Scheme 9). Increasing the amount of Lewis acid had no effect on the outcome of the reaction.

H H MgCl 6 equiv Me AlCl 10 equiv, HOOC COOEt 2 HOOC L-cysteine HCl 1.5 equiv, L-cysteine HCl 3 equiv, N S N S THF CH2Cl2 M1 M2 X X M2 ethyl benzoate

Scheme 9. Failed Attempt 3 (model reaction 1)

Attempt 4

In the next model reactions, amide coupling of benzoic acid M4 with L-cysteine HCl in the presence of CDI and triethylamine yielded compound M3 (Scheme 10). Although M3 was effectively cyclized by TiCl 4 to M2 , one pot-direct conversion of M4 to M2 was not successful.

COOH SH O CDI, TEA, CH2Cl2 M4 N COOH L-cysteine HCl H Benzoic acid X M3 TiCl4,CH2Cl2,THF

1)CDI, TEA,CH2Cl2 H 2) TiCl4 HOOC 3) THF N S M2

Scheme 10. Reaction of benzoic acid with L-cysteine HCl

The reaction was repeated with compound 5 in place of benzoic acid. Compound 5 was converted to amide 26 by reacting it with L-cysteine HCl, CDI and triethylamine

(Scheme 11). However, TiCl 4 mediated cyclization of intermediates 26 did not produce

50 28 . Attempted cyclization by an alternative reported method with Ph 3PO and Tf 2O was also unsuccessful.

HS COOH H OH HOOC CDI, TEA, THF, NH O L-cysteine HCl TiCl4,THF NS O X NS 4 attempts NS NS 5 26 NH-Boc 28 X NH-Boc NH-Boc Ph3PO, Tf 2O, CH3CN

H HOOC NS

NS 28 NH-Boc

Scheme 11. Failed attempt 5 (model reaction 2)

2.1.6 Synthesis of cyclic core 33

With the necessary building blocks in hand for largazole synthesis, the assembly of cyclic

core 33 was undertaken as shown in Scheme 12. The nitrile 7 was condensed with ( R)-α- methylcysteine HCl 13 to obtain the thiazole-thiazoline carboxylic acid 29 .25a, 25c The crude product 29, after simultaneous removal of Boc group and esterification of the free

carboxylic acid under acidic conditions in MeOH, gave 30. Acyl group was transferred 51

from 22 to 30 to obtain alcohol 31 in the presence of DMAP in dichloromethane. The

formation of 29 from nitrile 7 and the one pot conversion of 29 to alcohol 31 were carried

out efficiently with 78% yield for 3 steps. Yamaguchi esterification 3a, 52 was used to

couple Fmoc-L-valine to 31 to afford the acyclic precursor 32 in 95% yield. After

saponification and Fmoc group removal, macrocyclization with HOAt, HATU, and

Hunig’s base yielded the cyclized product 33 in 24% overall yield over the 3 steps.

O NC S S NaHCO3, 13, MeOH, N HO N S o phosphate buffer pH5.95, 70 C N Boc HN 29 7 Boc HN

MeOOC S HCl (g), MeOH N 31 O 22 S N S DMAP, ,CH2Cl2 OH O 78% f rom 7 MeO N S S NH N Fmoc-L-valine, 2,4,6-trichlorobenzoyl 30 - H N chloride, DIPEA, DMAP, THF, Cl 3 0 oC then RT 95%

o MeOOC S 1) 0.1 M LiOH,THF:H2O (4:1), 0 C O S N 2) Et2NH, acetonitrile N NHFmoc NH 3) HATU, HOAt, DIPEA, DMF S N S 24% for 3 steps N O O O OOO S N NH 33 H 32 S

Scheme 12. Synthesis of cyclic core 33

2.1.7 Failed attempts for conversion of cyclic core 33 to largazole

Having synthesized the advanced intermediate 33 , what remained to be done to prepare

largazole was to remove the t-butyl protecting group and to convert the resulting thiol to

the octanoate ester. Unfortunately, all efforts to remove the t-butyl group as illustrated in

53 54 Scheme 13 (with 1 M BBr 3; trifluoroacetic acid, anisole, mercuric acetate ) and

esterification with octanoyl chloride to form largazole failed. With BBr 3, mostly starting

material was recovered. Treatment with TFA/anisole/mercuric acetate indicated the

formation of a polar product (TLC) which was subjected to thioesterification. However

no largazole, largazole thiol or starting material was recovered.

attempt 1: 1 M BBr3, octanoyl chloride, CH2Cl2,RT attempt 2: TFA, anisole, mercuric acetate, o O O S 0 C; followed by DIPEA, octanoyl S chloride, DMAP, CH Cl ,RT N 2 2 N NH NH S N S N OOO X O OOO

H3C(H2C)6 S N S N H 33 H Largazole (1)

Scheme 13. Attempted conversion of 33 to largazole

2.1.8 Alternative Strategy to largazole with trityl as thiol protecting group

After unsuccessful attempts to remove t-butyl protecting group and to convert the resulting thiol to largazole in the final step, it was decided to change the protecting group from t-butyl to trityl group. For this purpose, synthesis of compound 34 was approached in a way similar to the synthesis of 20 (Scheme 14). Dioxene 18 was reacted with t-butyl lithium and reaction of the lithiated compound with 26 did not give product 34 . Changing

the order of addition i. e. slow addition of a solution of lithiated compound in THF to a

pre-cooled solution of 26 in THF at -78 oC did not help either. Attempts using the chloro compound 27 instead of the bromo compound 26 or changing reaction solvents too failed to yield 34 . After these unproductive efforts, it was decided to follow Janda’s protocol 55

for synthesis of aldehyde 36 (Scheme 15).

t-BuLi, THF or THF-ether or pentane-ether or ether, -78 oC OO Ph OO Ph S Ph Br S Ph Ph 34 18 26 Ph or S Ph Cl 27 Ph Ph Scheme 14. Attempts to synthesis of compound 34

Synthesis of alcohol 37 (Scheme 15) started with conjugate addition of triphenylmethanethiol to acrolein to give 35 which was used in a Wittig reaction with commercially available (triphenylphosphoranylidene)-acetaldehyde to yield aldehyde

36 .55 This one-pot protocol gave 65% overall yield for the two steps. Acetate aldol condensation of the aldehyde 36 with acetyl Nagao auxiliary ( 16 ) was used to synthesize alcohol 37 in 82% diastereomeric excess. 43 The two diastereomers were purified by flash column chromatography on silica gel in dichloromethane: hexanes (25-90%). The S- configuration of the newly created chiral center of molecule 37 was established by modified Mosher ester analysis. 47

(triphenylphosphoranylidene)- H Ph H triphenylmethanethiol, Ph acetaldehyde, benzene, 85 oC O Et3N, CH2Cl2,RT Ph S O 65% from acrolein Acrolein 35 Ph OH O S 16, TiCl , DIPEA, Ph Ph O 4 o Ph S N Ph CH2Cl2, -78 C S Ph S H 84%, dr 10:1 37 (yield 76.5% brsm) (comp. 37:38) + 36 Ph OH O S Ph Ph S N S 38 (diastereomer of 37) (yield 7.5% brsm)

Scheme 15. Synthesis of the β-hydroxycarboxamide 37

With the necessary building blocks in hand, the assembly of largazole was undertaken as shown in Scheme 16. The nitrile 7 was condensed with ( R)-α-methylcysteine HCl 13 to obtain the thiazole-thiazoline carboxylic acid 29 .25a After simultaneous removal of Boc group and esterification of the free carboxylic acid under acidic conditions in MeOH, acyl group was transferred 51 from 37 to 30 to obtain alcohol 39 . The formation of 29 from nitrile 7 and the one pot conversion of 29 to alcohol 39 proved very efficient with

78% yield for 3 steps. Yamaguchi esterification 3a, 52 was used to couple Fmoc-valine to alcohol 39 to afford the acyclic precursor 40 . After saponification and Fmoc group removal, macrocyclization with HOAt, HATU, and Hunig’s base yielded the cyclized product 41 in 56% overall yield over the 3 steps. Deprotection of trityl group gave largazole thiol which was esterified with octanoyl chloride using Hunig’s base to give largazole in 79% yield (based on recovered largazole thiol in esterification step) over two steps.

Final scheme for largazole synthesis

O NC S S NaHCO3, 13, MeOH, a)HCl (g), MeOH N HO N S b)i) EDC, MeOH, DIPEA phosphate buffer pH 5.95, 70 oC N ii) TFA, CH2Cl2 29 Boc HN 7 Boc HN

MeOOC S O S N 37 MeO N DMAP, ,CH2Cl2 S S 78% f rom 7 N N Ph OH O Ph 30 Ph - H N S NH Cl 3 39

Fmoc-valine, 2,4,6-trichloro S MeOOC 1) 0.1 M LiOH,THF:H O(4:1), 0 oC benzoyl chloride, DIPEA, 2 N 2) Et NH,CH Cl DMAP, THF, 0 oC then RT NHFmoc 2 2 2

97% N S 3) HATU, HOAt, DIPEA, CH2Cl2 Ph O O O 56% Ph Ph S NH 40

O S O N S NH 1) TFA, TIPS, CH Cl , N 2 2 NH N S Ph OOO 2) octanoyl chloride, DIPEA, CH2Cl2 N S Ph 79% brsm O OOO Ph S N 41 H H3C(H2C)6 S N Largazole (1) H

Scheme 16. Synthesis of largazole

2.2 Synthesis of analogs of T1 series

The three analogs of T1 series were synthesized in a similar manner to largazole except

using Fmoc-L-allylglycine, Fmoc-D-1-naphthylalanine and Fmoc-L-1-naphthylalanine

instead of Fmoc-L-valine in Yamaguchi condensation (conversion of compound 39 to

compound 40 for largazole).

Synthesis of analog T1A

Synthesis of analog T1A as shown in Scheme 17 started with the synthesis of compound

42 by Yamaguchi coupling of Fmoc-D-1-naphthylalanine with alcohol 39 . Cyclization of

compound 42 after hydrolysis of methyl ester and removal of Fmoc group gave cyclic core 43 in low yield of 14%. Gratifyingly, the final transformation of thioesterification after trityl group deprotection was more successful to yield the analog T1A in 50% overall yield for two steps.

Fmoc-D-1-naphthylalanine, MeOOC S MeOOC S 2,4,6-trichlorobenzoyl chloride, N DIPEA, DMAP, THF, 0 oC then RT NHFmoc N N S 55% Ph O O O N S Ph Ph OH O Ph NH Ph S 42 Ph S NH 39 o 1)0.1 M LiOH,THF:H20 (4:1), 0 C 2) Et2NH,CH2Cl2 3)HATU, HOAt, DIPEA, CH2Cl2 14%

O O S S 1) TFA, TIPS, CH2Cl2, N N NH NH 2) octanoyl chloride, N S N S DIPEA, CH2Cl2 Ph OOO O OOO 50% Ph Ph S N S N 6 43 H T1A H

Scheme 17. Synthesis of analog T1A

Synthesis of analog T1B

Synthesis of analog T1B is shown in Scheme 18. Yamaguchi coupling of alcohol 39 with

Fmoc-L-allylglycine produced compound 44. Macrocyclization of it after removal of protecting groups gave cyclic core 45 which was converted to final analog T1B as before in 35% overall yield for the two steps.

Fmoc-L-allylglycine, MeOOC S MeOOC S 2,4,6-trichlorobenzoyl chloride, N o NHFmoc N DIPEA, DMAP, THF, 0 C then RT N S 90% Ph O O O N S Ph Ph OH O Ph NH Ph S 44 Ph S NH 39 o 1) 0.1 M LiOH, THF:H2O (4:1), 0 C 2) Et2NH, CH2Cl2 3) HATU, HOAt, DIPEA, CH2Cl2 17%

O S O S 1) TFA, TIPS, CH Cl , 2 2 N N NH NH 2) octanoyl chloride, N S N S DIPEA, CH2Cl2 Ph OOO O OOO 35% Ph Ph S N S N H 6 H 45 T1B

Scheme 18. Synthesis of analog T1B

Unsuccessful attempt in the synthesis of analog T1C

Attempted synthesis of analog T1C as shown in Scheme 19 started with the synthesis of compound 46 by Yamaguchi coupling of Fmoc-Nδ-trityl-L-glutamine with alcohol 39 .

Cyclization of compound 46 after removal of protecting groups gave cyclic core 47 in

very low yield. Despite several attempts of purifications, it was not possible to obtain

cyclic core 47 in pure form. Therefore, it was decided to carry out next two

transformations without further purification. However, thioesterification after trityl group

deprotection was unsuccessful and no product of analog T1C was obtained.

Ph Fmoc-N(delta)-trityl-L-glutamine, Ph O MeOOC S Ph N MeOOC S 2,4,6-trichlorobenzoyl chloride, N o NHFmoc N DIPEA, DMAP, THF, 0 C then RT H N S 78% Ph O O O Ph N S Ph Ph Ph OH O Ph S NH 46 S NH o 39 1) 0.1 M LiOH, THF:H2O (4:1), 0 C or o 0.5 M LiOH, acetone:H2O (9:1), 0 C 2) Et2NH, CH2Cl2 3) HATU, HOAt, DIPEA, CH2Cl2

Ph Ph O O O S O S 1) TFA,TIPS,CH2Cl2, Ph H N N N N 2 NH H NH 2) octanoyl chloride,X N S N S DIPEA, CH2Cl2 Ph OOO O OOO Ph S N Ph S N 6 H H T1C 47

Scheme 19. Attempted of Synthesis of analog T1C

Synthesis of analog T1D

Attempted synthesis of analog T1D is shown in Scheme 20. Yamaguchi coupling of alcohol 39 with Fmoc-L-1-naphthylalanine produced compound 48 . Macrocyclization of it following removal of protecting groups gave cyclic core 49 in very low yield. Despite several attempts, it was not possible to obtain pure product of cyclic core 49. Therefore the last two transformations were carried out without further purification.

Thioesterification upon trityl deprotection followed by reverse phase HPLC purification on C18 column gave small amount of the partially purified product T1D . The synthesis

needs to be repeated to generate sufficient material for biological evaluations.

Fmoc-L-1-naphthylalanine, MeOOC S MeOOC S 2,4,6-trichlorobenzoyl chloride, N o NHFmoc N DIPEA, DMAP, THF, 0 C then RT N S 98% Ph O O O N S Ph Ph OH O Ph NH Ph S 48 Ph S NH 39 o 1) 0.1 M LiOH, THF:H2O (4:1), 0 C 2) Et2NH, CH2Cl2 3) HATU, HOAt, DIPEA, CH2Cl2

O S O S 1) TFA, TIPS, CH Cl , 2 2 N N NH NH 2) octanoyl chloride, N S N S DIPEA, CH2Cl2 Ph OOO O OOO Ph Ph S N S N H 6 H 49 T1D

Scheme 20. Attempt for synthesis of analog T1D

2.3 Unsuccessful attempt in the synthesis of analog T2

The assembly of analog T2 was undertaken as shown in Scheme 21. The nitrile 7 was condensed with commercially available L-penicillamine to obtain the thiazole-thiazoline carboxylic acid 50.25a After simultaneous removal of Boc group and esterification of the free carboxylic acid under acidic conditions in MeOH, acyl group was transferred 51 from

37 to obtain 51. The formation of 50 from nitrile 7 and the one pot conversion of 50 to alcohol 51 proved very efficient with 50% yield for the 3 steps. Yamaguchi esterification 3a, 52 was used to couple Fmoc-valine to alcohol 51 to afford the acyclic precursor 52. After saponification and Fmoc group removal, macrocyclization with

HOAt, HATU, and Hunig’s base yielded the cyclized product 53 in low yield over the 3 steps. As before, despite several attempts, pure product of cyclic core 53 could not be obtained. Incomplete saponification of 52 by LiOH in THF:H 2O was responsible for low yield of 53 .

H NaHCO , L-penicillamine, 3 HOOC S i) a) EDC, MeOH, DIPEA, NC Phosphate Buffer pH 5.95, b) HCl, MeOH o N MeOH, 70 C ii) DMAP, 37,CH2Cl2 N S 50 N S 50% from 7 7 BocHN BocHN H S H Fmoc-L-valine, MeOOC MeOOC S 2,4,6-trichlorobenzoyl chloride, N NHFmoc N DIPEA, DMAP, THF, 0 oC then RT N S 86% Ph O O O N S Ph OH O Ph NH S 52 S NH 51 o 1) 0.1 M LiOH, THF:H2O (4:1), 0 C Ph Ph 2) Et NH, CH Cl Ph 2 2 2 3) HATU,HOAt, DIPEA,CH2Cl2

H O S N NH N S Ph OOO Ph Ph S N H 53

Scheme 21. Synthesis cyclic core 53 for analog T2

As above mentioned route produced insufficient amount of cyclic core 53 , an alternative route to compound 53 was attempted. It started with esterification of carboxylic acid 50 with 2,2,2-trichloroethanol in presence of EDC and Hunig’s base to get 54 as shown in

Scheme 22. The Boc group of ester 54 was removed with acid and acyl group of 37 was transferred to obtain alcohol 55. The ester 56 was produced by Yamaguchi condensation of alcohol 55 with Fmoc-L-valine. The trichloroethyl ester group of 56 was cleaved with

43 Zn and NH 4OAc in THF. Usual Fmoc group removal followed by cyclization produced only 2 mg of impure cyclized core 53 (from 146 mg of compound 56 ). The impure 51

product, upon subjection to removal of trityl group and thioesterification did not produce analog T2 .

O H S H Cl3C HOOC S EDC, 2,2,2-trichloroethanol, O N i) HCl/Dioxane DIPEA, CH Cl N 2 2 ii) DMAP, 37,CH2Cl2 35% S 54 N 63% 50 N S Cl3C BocHN O H BocHN Fmoc-L-valine, S O H 2,4,6-trichlorobenzoyl chloride, O Cl C S N 3 o NHFmoc O N DIPEA, DMAP, THF, 0 C then RT N S 43% Ph O O O N S Ph Ph OH O Ph NH Ph S 56 Ph S NH 55 1) Zn, NH4OAc, THF 2) Et2NH,CH2Cl2 3)HATU, HOAt, DIPEA, CH2Cl2

H H O S O S 1) TFA, TIPS, CH Cl , 2 2 N N NH NH 2) octanoyl chloride,X N S N S DIPEA, CH2Cl2 Ph OOO O OOO Ph Ph S N S N H 6 H 53 T2 Scheme 22. Alternative route for analog T2

2.4 Synthesis of analog T3

Synthesis of target molecule T3 followed the same route as for largazole synthesis using

(S)-α-methylcysteine HCl (14 ) instead of ( R)-α-methylcysteine HCl to generate C-7 epimer of largazole (Scheme 23). The nitrile 7 was condensed with ( S)-α-methylcysteine to obtain the thiazole-thiazoline carboxylic acid 57.25a After simultaneous removal of Boc group and esterification of the free carboxylic acid under acidic conditions in MeOH, acyl group was transferred 51 from 37 to compound 58 to obtain alcohol 59. The formation of carboxylic acid 57 from nitrile 7 and the one pot conversion of 57 to alcohol 59 proved very efficient with 67% yield for 3 steps. Yamaguchi esterification 3a, 52 was used to couple Fmoc-valine to alcohol 59 to afford the acyclic precursor 60 in 93% yield. After

saponification and Fmoc group removal, macrocyclization with HOAt, HATU, and

Hunig’s base yielded the cyclized product 61 in 31% yield over the 3 steps.

Thioesterification with octanoyl chloride of cyclized core 61 upon removal of trityl group

produced analog T3 in 72% overall yield for two steps.

O NC S a) HCl (g), MeOH S NaHCO3, 14, MeOH, N HO N b) TFA, DCM o S phosphate bufferpH 5.95, 70 C N 57 Boc HN 7 Boc HN

O MeOOC S S N DMAP, 37,CH Cl MeO N S 2 2 N 67% from 7 N S Ph OH O 58 Ph - Cl H3N Ph S NH 59

Fmoc-valine, 2,4,6-trichloro S MeOOC 1) 0.1 M LiOH, THF:H O (4:1), 0 oC benzoyl chloride, DIPEA, 2 N 2) Et NH, CH Cl DMAP, THF, 0 oC then RT NHFmoc 2 2 2

93% N S 3) HATU, HOAt, DIPEA, CH2Cl2 Ph O O O 31% Ph Ph S NH 60 O S O S 1) TFA, TIPS, CH Cl , N 2 2 NH N NH 2) octanoyl chloride, DIPEA, CH2Cl2 N S 72% O OOO Ph N S Ph Ph OOO H C(H C) S N 3 2 6 H S N 61 H T3 (C-7 epimer of largazole)

Scheme 23. Synthesis of analog T3 (C-7 epimer of largazole)

2.5 Synthesis of analogs of T4 series

Scheme 24 shows the general method used for the synthesis of largazole analogs T4 ,

which incorporate modifications in the zinc binding motif. These analogs are designed

such that sulphur and a second heteroatom in the side chain are 2-3 atoms apart and are

thus capable of interacting with Zn 2+ via 5/6 membered cyclic transition states (Figure

19).

The analogs T4A , T4B , T4C and T4D were synthesized from the advanced

intermediate 41 after conversion to free thiol, by nucleophilic substitution of the

corresponding precursors by largazole thiol by either method A (basic) 56 or method B

(acidic) 57 as shown in Scheme 24. Method A comprises of removal of trityl group by

TFA to give thiol which reacted with alkyl halide precursor in the presence of CsCO 3 and tetrabutylammonium bromide (TBAB) to give S N2 displacement product. This two pot protocol gave an overall yield of 25%, and 19% of analogs T4A and T4D , respectively.

Analogs T4B and T4C were synthesized by method B. This one pot protocol consisted of

removal of trityl group of compound 41 by TFA followed by reaction of the resulting thiol with corresponding alcohol precursor in the same pot to give the SN1 substituted

product. This method gave 49% overall yield for both analogs T4B and T4C .

O S HBr O S N Br NH N N NH Ph N S Method A) i) TFA, TIPS, CH2Cl2 N S Ph Ph OOO OOO ii) Cs2CO3, TBAB, acetonitrile S N 41 H S N H N T4A Method A, 25%

N Cl O O S Method B) TFA, CH2Cl2 N NH N S CH OOO S 3 N OH OH H C S N 3 T4D H O OH Method A, 19%

O S O S N N NH NH N S N S OOO OOO S N S N T4C H T4B H OH Method B, 49% S Method B, 49%

Scheme 24. Synthesis of analogs of type T4 with modified metal-binding domain

2.6 Synthesis of analog T5

Synthesis of target molecule T5 began with synthesis of alcohol 62 by aldol reaction of

acrolein with acetyl Nagao auxillary ( 16 ) (Scheme 25). The two diastereomers were

separated by flash column chromatography on silica gel in ethyl acetate and hexanes (20-

90%) to get 62 in 77% diastereomeric excess. The absolute stereochemistry of newly

generated chiral center of 62 was determined by modified Mosher ester analysis. 47

OH O S 16,

H DIPEA, TiCl4, N S o CH2Cl2 , -78 C 62 O Acrolein 60%, dr: 7.6:1 (62:63) OH O S

N S 63

Scheme 25. Synthesis of alcohol 62 for the synthesis of cyclic core of T5

Alcohol 62 was used to synthesize the cyclic core 66 as in the synthesis of largazole

(Scheme 26). The assembly of cyclic core 66 began with simultaneous removal of Boc group and esterification of the free carboxylic acid of 29 under acidic conditions in

MeOH and acyl group transfer 51 from 62 to obtain alcohol 64 in one pot with 64% yield for 2 steps. Yamaguchi esterification 3a, 52 was used to couple Fmoc-L-valine with alcohol

64 to afford the acyclic precursor 65 in 83% yield. After saponification and Fmoc group removal, macrocyclization with HOAt, HATU and Hunig’s base yielded the cyclized product 66 in 35% yield over the 3 steps.

O S MeOOC S a)HCl (g), MeOH N HO N S b) i) EDC, MeOH,DIPEA N ii) TFA, CH2Cl2 29 N S c) DMAP, 62,CH Cl OH O Boc HN 2 2 64% NH 64

Fmoc-L-valine, 2,4,6-trichloro 83% benzoyl chloride, DIPEA, DMAP, THF, 0 oC then RT

MeOOC S O S o 1) 0.1 M LiOH, THF:H2O (4:1), 0 C N N 2) Et NH, CH Cl NHFmoc NH 2 2 2 3) HATU, HOAt, DIPEA, CH Cl N S 2 2 N S OOO 35% O O O

N NH 66 H 65

Scheme 26. Synthesis of cyclic core 66

Synthetic pathway to analog T5 side chain is depicted in Scheme 27. Reaction of acryloyl chloride with o-benzylhydroxylamine HCl in the presence of Hunig’s base gave product

67 in 62% yield.

DIPEA, Cl O CH2Cl2,RT O + O O Cl 62% N H3N acryloyl chloride H 67

Scheme 27. Synthetic pathway for side chain of target molecule T5

The cyclic core 66 was subjected to olefin metathesis with compound 67 using Grubbs

II nd generation or Nitro-Grela catalyst to afford precursor 68 of target molecule T5

(Scheme 28). Gratifyingly, olefin metathesis was accompanied by debenzylation and the

product T5 was isolated by reverse phase HPLC purification of the crude product on a

C18 column.

Grubbs IInd generation catalyst/ Grela catalyst, p-methoxyphenol O S O S o CH2Cl2, toluene, 110 C N N NH NH O N S O N S N OOO OOO 67 H H N O N N 68 H 66 H O

O S N NH N S OOO H N HO N H O T5

N N

Cl N N Ph Ru Cl Cl Ru P Cl O NO2

Grubbs IInd generation catalyst Grela catalyst

Scheme 28 . Synthesis of analog T5

2.7 Synthesis of analogs of T6 series

Synthesis of analog T6A as shown in Scheme 29 started with esterification of carboxylic acid 5 to get compound 69. Removal of Boc group of compound 69 by TFA in CH 2Cl 2 and acyl group transfer 51 from 38 gave the alcohol 70 in 81% yield. Yamaguchi esterification 3a, 52 was used to couple Fmoc-L-valine to alcohol 70 to afford the precursor

71. Compound 71 was taken to the next step without further purification. After removal of trityl group of compound 71 and thioesterification with acetyl chloride, it yielded analog T6 in 22% yield over the 2 steps.

MeOOC HOOC MeOOC EDC, MeOH, DIPEA 1) TFA, CH2Cl2 38 70 N S N S 2) , DMAP, CH2Cl2 5 68% N S 69 Ph 81% OH O Ph BocHN BocHN Ph S NH 2,4,6-trichlorobenzoyl chloride, Fmoc-L-valine, DMAP, DIPEA, THF, 0 oC then RT O MeO MeOOC NHFmoc 1) TFA, TIPS, CH2Cl2 NHFmoc N S 2) acetyl chloride, DIPEA, CH2Cl2 O O O O N S Ph O O O 22% Ph S NH Ph S NH T6 71

Scheme 29. Synthesis of analog T6

2.8 Attempts to synthesize 13-membered ring analog T7

Scheme 30 depicts attempts to synthesis of analog T7 . Removal of Boc group of

compound 69 with TFA, and transfer of acyl group from 37 with DMAP as acyl transfer

agent gave alcohol 72. Fmoc-L-valine coupling of alcohol 72 by Yamaguchi’s protocol yielded compound 73 in 92% yield. Macrocyclization after removal of protecting groups yielded partially purified 13-membered cyclic core 74. As each purification step resulted in loss of material, it was subjected to next set of reactions without further purification.

Usual protocol of trityl group removal with TFA and thioesterification with octanoic acid did not yield analog T7. As this approach gave low yields of the cyclic core 74 an alternative route to its synthesis was undertaken.

HOOC MeOOC MeOOC EDC, MeOH, DIPEA 1) TFA, CH2Cl2 N S N S 2) 37, DMAP, CH2Cl2 72 5 N S Ph OH O Ph BocHN BocHN 69 Ph S NH

2,4,6-trichlorobenzoyl chloride, Fmoc-L-valine, 92% DMAP,DIPEA, THF, 0 oC then RT O H N 1) 0.1 M LiOH, THF: H2O MeOOC 2) Et NH, CH Cl 74 S 2 2 2 NHFmoc N 3) HATU,HOAt, DIPEA,CH Cl Ph O O O 2 2 Ph N S Ph O O O Ph S NH Ph Ph S NH X 73 1) TFA, TIPS, CH2Cl2 2) Octanoyl Chloride, DIPEA, CH2Cl2 O H N

T7 N S O O O O

S NH

Scheme 30. Attempt to the synthesis of 13 membered ring analog T7

Acyl transfer from alcohol 37 to 2-trimethylsilylethanol yielded compound 75 which

upon coupling with Fmoc-L-valine gave compound 76 (Scheme 31). This was coupled

with carboxylic acid 5 to get precursor 77 for cyclic core synthesis. Although cyclization

after deprotection yielded compound 74, the amount obtained after purification was low.

Every purification step led to loss of material. Instability of cyclic core 74 on silica gel could be the reason for low yields obtained with its purification. Insufficient amount of cyclic core material 74 obtained deterred attempts to convert it to analog T7 .

Ph OH O S Ph 2-trimethylsilylethanol, Ph OH O Ph Ph S N DMAP, CH2Cl2 S Ph S O 37 75 Si

2,4,6-trichlorobenzoyl chloride, Fmoc-L-valine, DMAP, DIPEA, THF O

HN 1) Et NH, CH Cl NS 2 2 2 NHFmoc 2) HATU, HOAt, O 77 DIPEA, CH Cl , 5 Ph O O 2 2 O Ph NHBoc Ph O O Ph Ph S O Ph S O 1) TFA, CH Cl Si 2 2 Si 2) HATU, HOAt, 76 DIPEA, CH2Cl2

O H N 74 N S Ph O O O Ph Ph S NH

Scheme 31. Alternative route of synthesis to cyclic core 74

2.9 Biological Studies

2.9.1 Results

The Biological studies were carried out in the laboratory of Dr. Robert A. Casero,

Professor of Oncology, the Sidney Kimmel Comprehensive Cancer Center, the Johns

Hopkins University, School of Medicine, Baltimore. The antiproliferative activity of

analogs T1A , T1B , T3 , T4A , T4B, T4C and T6 were evaluated in the HCT116 colon

adenocarcinoma cell line by a standard 3-(4,5-dimethylthiazol-2-yl)-5-(3-

carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) reduction assay

(Promega) using largazole as the control as previously reported.58 Figure 21 show the results of the MTS assay after 96 h of treatment with 10 nM, 100 nM, 1 µM and 5 µM concentrations of the compounds. Largazole inhibited the growth of HCT116 cells with a

GI 50 of ~ 30 nM whereas the three side chain analogs T4A , T4B and T4C showed activity only at higher concentrations. Of the three, the pyridine analog T4A demonstrated the greatest effect on growth inhibition with a GI 50 of 1.0 µM. Of the analogs incorporating changes in the depsipeptide ring, the C-2 L-allyl substituted analog

T1B showed a GI 50 value of 10 nM. The C-7 epimer of largazole T3 is less active than largazole with a GI 50 value of 5 µM.

Figure 21. Growth inhibitory effects of largazole and analogs on HCT116 colon carcinoma cells.

To evaluate the effects of analogs T1B , T3 , T4A , T4B, and T4C on HDAC activity, the

downstream effects of HDAC inhibition on global histone H3 acetylation was evaluated

by Western blot analysis after 24 hours of cellular exposure to 10 nM, 100 nM, and 1 M

of each compound. Although no increase in global acetylation was observed in treated

cells exposed to analogs T3 , T4A , T4B and T4C at 100 nM, analog T1B showed

prominent increase in global acetylation at 100 nM. The effect of analog T1B on global

histone H3 acetylation is quite promising and is comparable to that of largazole at 100

nM. Analogs T4A and T4B showed a significant increase in global acetylation at 1 M,

though the effect is much lower than what was observed with largazole.

In order to ascertain the effect of analogs T4A , T4B and T4C on HDAC6 activity, the levels of α-tubulin acetylation after exposure to 10 nM, 100 nM and 1 µM were studied using Western blot analysis of whole cell lysates from cells treated for 24 hours. No

changes in α-tubulin acetylation were observed by exposure to any of the analogs, including largazole. This conforms to earlier findings that HDAC6 is not a target for largazole and T4 series analogs.10

2.9.2 Discussions

Largazole inhibited the growth of HCT116 cells with a GI 50 of ~30 nM. The three side

chain analogs T4A , T4B and T4C were less active, the pyridine analog T4A being the

most active of the three with a GI 50 of ~1.0 µM. The other two analogs were active at higher concentrations. This is consistent with significant increase of global H3

acetylation observed with largazole at 1 µM concentration and modest induction of

global H3 acetylation observed for analogs T4A and T4B . There was no change in acetylated α-tubulin levels with any of the compounds indicating they have no effect on

HDAC6. Thioethers bind weakly to Zn 2+ .59 These largazole analogs were designed to see whether the introduction of a second hetero atom would lead to stronger binding to the metal ion and also isoform/class selectivity. However, it is not possible to suggest if the diminished biological activity observed is due to poor affinity of thioether group for Zn 2+ ion or incompatibility of the modified metal binding moiety with the HDAC active site.

Despite the high sequence similarity within the active site, presence of discrete binding cavities in the vicinity of the metal ion may be taken advantage of to achieve isoform selectivity as exemplified by HDACis with substituted benzamide metal-binding domains which display class I selectivity. 26d, 41 The exploration of alternative metal-binding domains is one of the ways forward to the development of such isoform and class- selective HDACis.

Analog T1B with allyl group replacement of isopropyl group of valine was more potent than largazole. Substitution of the isopropyl group of valine at C-2 with other

hydrophobic and hydrophilic groups may lead to molecules with improved activity and selectivity.

Determination of HDAC isoform/class selectivity of all synthesized analogs is in progress.

2.10 Future Directions

The HDAC isoforms or subsets of isoforms pertinent to antitumor therapy are not clearly

understood. In addition, which HDAC substrates have the most critical roles in a

particular type of tumors is not known. 36 Although trial and error and serendipity have

played an important role in the development of HDAC inhibitors due to lack of

information about individual HDAC isoforms, rational drug design has led to a number of

clinically important HDACis.36 Some success has been achieved in the development of

isoform and/class selective HDACis based on binding studies of molecules to HDAC

proteins by X-ray crystallography. Apart from extending the synthetic efforts to generate

additional analogs of current series, HDAC4 selective compounds (analogs T8A and

T8B ), compounds (analogs T10 , T11 ,) with multiple sites of activity and HDAC6 selective analogs ( T12) as shown in Figure 22 are designed to increase the therapeutic efficacy of these molecules.

O S O S N N NH NH N S N S O O O O R1 O O O O HN F3C HN T8A T8B R2 R3

O Amino acid R NHFmoc Amino acid R3 O R1 NH O S O O O Me HN R2 O R / S O Small molecules T9 S R / S N N H R1 N Novel Multi-acting HDAC, EGFR and HER2 inhibitor T10

NHFmoc R3 Amino acid R1 O

HN O Me HN R2 R / S H2N N S N N H H X X = O, S N beta-homolysine Novel Multi-acting HDAC, EGFR and HER2 inhibitor T11

Amino acid NHFmoc O R O 1 N O O O

S O HDAC6 selective analogs T12

Figure 22. Additional target molecules designed for future directions.

Target molecules of T8 series

In target molecule T8 , the thiol group of largazole is replaced with a trifluoromethyl ketone moiety as HDACi with trifluoromethyl ketone functionality typically display a 10-

100 fold greater inhibition of class II than class I HDACs. 15 It has been shown that

HDAC4 (class II) inhibitors produce cellular sensitization to ionizing radiations. 39

Although the trifluoromethyl group fills the gap near Pro 800 , it is far away from His 976

which lies at the bottom of the biding pocket of HDAC4. Analogs of type T8B are designed to see if substituted phenyl ketone fills this gap and leads to additional HDAC4 selectivity. Electron withdrawing groups such as -NO 2, -CN, -SO 2NH 2, -COOR on phenyl ring serve three purposes: 1) they increase electropositivity of ketone; 2) they can form hydrogen bonds with His 976 , Pro 800 and other amino acids; 3) they fill the pocket around His 976 and Pro 800 . Additionally, presence of sterically hindered groups at 2,6- positions decreases chances of stabilization of hydrated ketone by Tyr 306 in class I HDAC

and thus increases their selectivity for class II HDAC.

Proposed synthesis of target molecule T8A can begin with Grignard reaction between 81

and 82 to give 83 as shown in Scheme 32. If Grignard reaction is complicated by formation of tertiary alcohol, Weinreb amide can be used instead of acid chloride to make

83 . An alternative route to 83 is depicted in scheme 33. 60 The cyclic core 66 (scheme 26)

can be subjected to olefin metathesis with 83 to afford the target molecule T8A.

O F3C + BrMg F C Cl 0 3 81 82 THF, -78 C O 83

Scheme 32: Synthesis of side chain fragments 83 of target molecule T8A

Target molecule T8B can be synthesized in a similar manner.

a) TMSCF3, CsF, DME, RT: b) Dess-Martin periodinane, O O CH2Cl2,RT H F C 84 3 83

Scheme 33. Alternative route to compound 83

Target molecules of T9 series:

Small molecules generally are preferred as drug candidates as their large-scale synthesis

for clinical use is more viable commercially. Seven-membered ring compounds T9 are

designed as small molecule analogs of largazole. Several analogs of T9 can be generated

using different amino acids. Also, stereochemical requirements can be deduced by

generating analogs using both D- and L-amino acids.

Synthesis of target molecule T9 is shown in Scheme 34. Acyl group transfer from 37 to

methanol gave 85 which can undergo Yamaguchi esterification with various Fmoc amino

acids to afford 86 . After saponification and Fmoc group removal, it can be subjected to

lactamization under specified conditions to give the cyclic intermediate 87 . Removal of

trityl group of 87 followed by acetylation will yield analogs of T9 series.

Ph OH O S Ph OH O Ph MeOH, DMAP, RT; Ph Ph S N S Ph S OMe 37 85 DIPEA, Fmoc-amino acid, THF, 0 oC then RT NH 2 NHFmoc i) 0.1 N LIOH, THF: H O , 0 oC; R O 2 ii) Diethylamine, DCM, RT R O 86a Ph O O Ph Ph O O Ph Ph S OH Ph S OMe 86 HOAt, HATU, DIPEA, CH Cl ,RT 2 2 O O R R O Ph O Ph O NH NH i) TFA, triisopropylsilane, CH Cl , S Ph 2 2 S 0 oC then RT; O 87 o small molecules T9 O ii) AcCl, DIPEA, CH2Cl2, 0 C then RT

Scheme 34. Synthesis of small ring analogs T9

Target molecules T10, T11 series

Involvement of human epidermal growth factor receptor (HER) family in tumorogenesis

is known. The four structurally linked tyrosine kinase receptor proteins of HER family

are the epidermal growth factor receptor (EGFR): (HER1), HER2, HER3 and HER4. 61

EGFR/HER2 inhibitor lapatinib and EGFR inhibitors erlotinib and gefitinib are FDA

approved drugs used against solid tumors. As these drugs suffer from limitations due to

development of drug resistance, Curis Inc. combined features of HDACi and

EGFR/HER2 inhibitors in CUDC-101 (Figure 23), which is in phase I clinical trials for

advanced head and neck, gastric, breast, liver and non-small cell lung cancer tumors.61

Based on the success of CUDC-101, 61 the proposed molecules T10, T11 are designed to have multiple sites of activity in a single molecule. The molecules combine HDAC,

EGFR, and HER2 inhibitory activity. In addition, analogs T11 are designed to include

two heteroatoms two atoms away from each other. The binding of these two heteroatoms

to Zn 2+ of HDAC enzymes would form a stable 5 membered transition state which may

potentiate the activity of these analogs.

HN C H CH N O HO N O CUDC-101 O N

Figure 23. Structure of phase I molecule CUDC-101.

Target molecules T12 series

HDAC inhibitors with phenylisoxazole as cap group are reported to have HDAC6 selectivity. 62 Few of these compounds have been shown to be 10-fold more potent than

SAHA. 62 Analogs T12 are designed to consist of phenylisoxazole along with Fmoc as cap groups and are Y shape molecules which retain their HDAC6 selectivity features.

Chapter 3

3 Experimental Sections

3.1 Experimental Chemistry

General:

THF was refluxed with Na and benzophenone and freshly distilled prior to use. NMR spectra were recorded on Varian INOVA 600 MHz and Varian VXRS 400 MHz instruments and calibrated using residual undeuterated solvent as internal reference

1 13 1 1 (CDCl 3: H NMR at δ 7.26, C NMR at δ 77.23; D 2O: H NMR at δ 4.8; DMSO-d6: H

NMR at δ 2.5, 13 C NMR at δ 39.51). Optical rotations were recorded on an AUTOPOL

III 589/546 polarimeter. High-resolution mass spectra (HRMS) were recorded on a

Micromass LCT Electrospray mass spectrometer at the Central Instrument Facility, the

Wayne State University, Detroit, Michigan and on a Micromass Q-Tof II mass spectrometer at Mass Spectrometry and Proteomics Facility, the Ohio State University,

Columbus, Ohio. Crude products were purified by flash column chromatography on silica gel (32-63 µ) purchased from Dynamic Adsorbents Inc. and by preparative thin layer chromatography on 1000 µ Uniplates purchased from Analtech Inc. using commercial solvents as specified. Microwave experiments were performed using Biotage Initiator.

Combiflash Companion by Teledyne ISCO Inc. was used for flash chromatographic 74

separations. HPLC analyses were performed on a Waters 1525 Binary Pump HPLC

system with Waters 2487 Dual Wavelength Absorbance Detector on a Symmetry C18

column (reverse phase, 5 , 4.6 mm x 150 mm) using a liner gradient of 10-100%

H2O:MeOH over 15-20 min; flow rate of 1 mL/min and UV detection at 254 nm. Luna 5

µ C 18 (2) 100A (size 250 X 10 mm 5 micron) column by Phenomenex Inc. was used for

HPLC separations. D-Hydantoinase, recombinant, immobilized from E. Coli (catalog no:

53765; CAS: [9030-74-4]) was purchased from Fluka Chemie AG.

tert -Butyl (4-carbamoylthiazol-2-yl)methylcarbamate (6).

A mixture of Boc-thioglycinamide 4 (0.95 g, 5 mmol, 1 equiv) and bromopyruvic acid

(0.835 g, 5 mmol, 1 equiv) in dry THF (20 mL) was stirred at 50 oC under nitrogen for 2

h. The reaction mixture was concentrated in vacuo and the residue was dried under

reduced pressure by repeated azeotropic removal of moisture with toluene. The crude

carboxylic acid residue was dissolved in dry THF (50 mL) and triethylamine (1.3 g,

12.94 mmol, 2.6 equiv) and ethyl chloroformate (0.681 g, 6.24 mmol, 1.25 equiv) were

added at 0 oC. The reaction mixture was stirred for 30 minutes at the same temperature.

Ammonium hydroxide (~ 1.1 g, 31.76 mmol, 5 equiv) was added and the reaction

mixture was stirred at room temperature for 2 h. It was concentrated in vacuo and

purified by flash column chromatography on silica gel in ethyl acetate/hexanes (33-

100%) to yield 6 (0.768 g, 60% over two steps from Boc-thioglycinamide); mp 153-154

o 25c o 1 C (lit. mp 153 C). H NMR (400 MHz, CDCl 3): δ 8.08 (s, 1H), 7.13 (br s, 1H), 5.94

(br s, 1H), 5.34 (br s, 1H), 4.59 (d, J = 5.6 Hz, 2H), 1.47 (s, 9H). 13 C NMR (100 MHz,

CDCl 3): δ 169.7, 163.1, 155.8, 149.3, 124.8, 80.8, 42.5, 28.5.

tert -Butyl-(4-cyanothiazol-2-yl)methylcarbamate (7).

To a solution of amide 6 (0.204 g, 0.797 mmol, 1 equiv) in dichloromethane (20 mL) at 0 oC was added triethylamine (0.174 g, 1.725 mmol, 2.16 equiv) followed by dropwise addition of trifluoroacetic anhydride (0.181 g, 0.863 mmol, 1.08 equiv). The reaction mixture was stirred at room temperature for 1 h, concentrated in vacuo and purified by flash chromatography on silica gel in ethyl acetate/hexanes (20-50%) to obtain the nitrile

o 25c o 1 7 (0.189 g, 99%). mp 84-85 C (lit. mp 84 C). H NMR (600 MHz, CDCl 3): δ 7.95

13 (s, 1H), 5.3 (s, 1H), 4.62 (d, J = 6 Hz, 2H), 1.47 (s, 9H). C NMR (100 MHz, CDCl 3): δ

171.7, 155.8, 131.1, 126.6, 114, 81, 42.5, 28.5.

5-(tert -Butylthiomethyl)-5-methylimidazolidine-2,4-dione (10).

To a 5% aqueous solution of NaOH (96 mL, 120 mmol, 1.2 equiv) was added t-butyl

mercaptan (9.02 g, 100 mmol, 1 equiv) at 0 oC. After 30 minutes, chloroacetone (9.25 g,

100 mmol, 1 equiv) was added at 0 oC and the mixture was stirred at room temperature

for 3 h. The two yellow layers of the reaction mixture became homogenous after addition

of NaCN (5.88 g, 120 mmol, 1.2 equiv), (NH 4)HCO 3 (27.7 g, 350 mmol, 3.5 equiv), and ammonium hydroxide solution (14.8 M, 31 ml, 459 mmol, 4.59 equiv). After stirring at

60 oC overnight, the reaction mixture was cooled and the pH was adjusted to 7-7.6 with

HCl. The precipitated product was filtered and dried in vacuo . It was recrystallized from

50% ethanol to get hydantoin 10 (19.44 g, 90%); mp 208-210 oC (lit. 63 mp 208-210 oC).

1 H NMR (400 MHz, DMSO-d6): δ 10.60 (s, 1H), 7.89 (s, 1H), 2.75 (s, 2H), 1.31 (s, 3H),

13 1.22 (s, 9H). C NMR (100 MHz, DMSO-d6): δ 177.2, 156.2, 62.1, 41.8, 35.1, 30.7,

23.6.

3-(tert -Butylthio)-2-methyl-2-ureidopropanoic acid (11).

A solution of hydantoin 10 (2.37 g, 10.97 mmol, 1 equiv) in aqueous NaOH (10% w/v,

35 mL, 87.5 mmol, 7.97 equiv) was refluxed at 120 oC for 60 h. After the completion of the reaction (TLC, 10% MeOH: CH 2Cl 2) the pH of the reaction mixture was adjusted to 8

with HCl when a thick precipitate was formed. The suspension was heated to 70 oC and a

solution of KCNO (3.1 g, 38.27 mmol, 3.49 equiv) in water (15 mL) was added drop

wise over 40 minutes along the sides of the flask. The reaction mixture was stirred at 70

oC for 7 h. After cooling, the pH of the reaction mixture was adjusted to 2 with HCl. The

product was filtered off, washed with water and dried to get compound 11 (1.15 g, 45%).

The crude product was taken to the next step without further purification.

(R)-3-(tert -Butylthio)-2-methyl-2-ureidopropanoic acid (12).

To an aq NaCl solution (3.2%, 7 mL) was added compound 11 (1.35 g, 5.77 mmol, 1

equiv) and pH of the mixture was adjusted to 6.5 with aq. NaOH (10%) solution.

Immobilized hydantoinase (0.45 g, 23.9 U) and magnesium sulphate monohydrate (8.0

mg) were added to it and the reaction mixture was stirred at 40 oC for 2 days while

maintaining the pH at 6.5 using 10% aq. H 2SO 4. The reaction mixture was cooled and the

precipitate of the enzyme and compound 10a was filtered off. The precipitate was washed

with water and extracted with ethyl acetate. The organic layer was washed with water and

concentrated to get 10a (0.553 g, 81.9%). The filtrate was washed with ethyl acetate (15

mL) twice. The pH of the aqueous layer was adjusted to 3 using HCl and the precipitate

of compound 12 was filtered off. It was washed with water and dried to get compound 12

(0.510 g, 81.9%).

(R)-α-Methylcysteine HCl (13).

A solution of compound 12 (1.5 g, 6.41 mmol, 1 equiv) in HCl (15 mL) was refluxed

under nitrogen for 3 days. The reaction mixture was azeotropped with isopropanol three

times. It was stirred with toluene at 60 oC for 1 hour and cooled. The crystals were filtered, washed with toluene and dried in vacuo to get compound 13 (1.1 g, quantitative).

20 64 1 [α]D + 7.88 ( c 0.33, H 2O) (lit. [α]D + 8.13 (( c 1.58, H 2O)). H NMR (400 MHz, D 2O):

δ 3.16 (d, J = 15.2 Hz, 1H), 2.86 (d, J = 15.2 Hz, 1H), 1.57 (s, 3H). 13 C NMR (100 MHz,

H2O): δ 173.2, 61.6, 30.3, 21.3.

(R)-4-isopropylthiazolidine-2-thione (15).45

A mixture of D-valinol (4 g, 38.83 mmol, 1 equiv), aqueous 1 N potassium hydroxide

(200 mL, 200 mmol, 5.15 equiv) and CS 2 (15.14 g, 199 mmol, 5.13 equiv) was refluxed

at 105 oC overnight. The reaction mixture was cooled to room temperature and extracted

with dichloromethane. The combined organic layers were dried over anhydrous sodium

sulphate and evaporated in vacuo . The crude product was purified by flash column

chromatography on silica gel in ethyl acetate: hexanes (5-20%) to obtain 15 (5.9 g, 92

20 65 20 1 %). [α]D + 34.0 ( c 1.0, CHCl 3). (lit. [α]D + 37 (c 1.0, CHCl 3)). H NMR (600 MHz,

CDCl 3): δ 7.43 (br s, 1H), 4.03 (dd, J = 8.4, 15.6 Hz, 1H), 3.52 (dd, J = 7.8, 10.8 Hz,

1H), 3.34 (dd, J = 9.0, 11.4 Hz, 1H), 1.93-1.99 (m, 1H), 1.04 (d, J = 6.6 Hz, 3H), 1.00 (d,

13 J = 6.6 Hz, 3H). C NMR (100 MHz, CDCl 3): δ 201.3, 70.2, 36.2, 32.3, 19.0, 18.5.

(R)-1-(4-isopropyl-2-thioxothiazolidin-3-yl)ethanone (16).

To a mixture of NaH (1.15g of 60% dispersion in oil, 28.65 mmol, 2.25 equiv) and

Nagao auxillary (15) (2.05 g, 12.7 mmol, 1 equiv) was added dry ether (100 mL) at 0 oC.

The reaction mixture was stirred for 10 minute at 0 oC and acetyl chloride (1.1 g, 14.01

mmol, 1.1 equiv) was added very slowly at 0 oC. It was stirred for 10 minutes at 0 oC and

then for 1 h at room temperature. The reaction mixture was treated with 1M HCl and

extracted with ethyl acetate (3 times). The organic extract was washed with brine, dried

over anhydrous sodium sulphate and concentrated in vacuo . The crude reaction product

was purified by flash column chromatography on silica gel in ethyl acetate: hexanes (5%)

1 to get compound 16 (2.378 g, 92%). H NMR (600 MHz, CDCl 3): δ 5.13-5.16 (m, 1H),

3.50 (dd, J = 7.8, 11.4 Hz, 1H), 3.02 (dd, J = 1.2, 11.4 Hz, 1H), 2.77 (s, 3H), 2.33-2.38

13 (m, 1H), 1.05 (d, J = 6.6 Hz, 3H), 0.97 (d, J = 7.2 Hz, 3H). C NMR (150 MHz, CDCl 3):

δ 203.4, 170.9, 71.4, 30.9, 30.6, 27.2, 19.3, 18.0.

1,3-Dioxene (18). 66

To a solution of glycerol formal (available as approx. 60: 40 isomeric mixture of of 5- hydroxy-1,3-dioxane: 4-hydroxymethyl-1,3-dioxolane) (26 g, 0.25 mol, 1 equiv) and toluenesulfonyl chloride (50 g, 0.2625 mol, 1.05 equiv) in dichloromethane (50 mL) was added anhydrous pyridine (25.5 mL, 0.316 mol, 1.26 equiv) through a dropping funnel at

0 °C over 2 h. 4-(Dimethylamino)pyridine (5.0 mg, 0.041 mmol, ) was added and the reaction mixture was stirred for 16 h at room temperature. It was partitioned between

H2O and dichloromethane and the aqueous phase was extracted with dichloromethane.

The combined organic extract was dried over anhydrous sodium sulphate and concentrated in vacuo . The residue was crystallized from Et 2O and hexanes to give 5- 79

1 tosyloxy-1,3-dioxane (27.34 g, 62%). H NMR (400 MHz, CDCl 3): δ 7.82 (d, J = 8.4 Hz,

2H), 7.36 (d, J = 8.0 Hz, 2H), 4.74-4.82 (m, 2H), 4.44-4.48 (m, 1H), 3.99 (d, J = 3.2 Hz,

1H), 3.96 (d, J = 3.6 Hz, 1H), 3.78 (dd, J = 6.0, 12.0 Hz, 2H), 2.46 (s, 3H). 13 C NMR

(100 MHz, CDCl 3): δ 145.5, 133.6, 130.2, 130.1, 128.2, 128.0, 93.7, 70.8, 68.8, 21.9.

To a homogeneous solution of KOH (9.8 g, 0.175 mol, 25 equiv) and triethylene glycol

(60 mL) at 50 °C was added 5-tosyloxy-1,3- dioxane (18 g, 0.07 mol, 1 equiv) in one portion. The resulting mixture was heated slowly to 200 °C under stirring, and H 2O and dioxene (18) formed were condensed in a Dean-Stark trap equipped with a dry ice condenser (-78 °C). n-Decane (100 mL) was added to the condensate, the phases were separated and the aqueous phase was extracted with n-decane. The combined organic extract was dried over sodium sulphate and the crude product was distilled to get 18 (2.32

1 g, 44%). H NMR (600 MHz, CDCl 3): δ 6.56 (dt, J = 2.4, 6.6 Hz, 1H), 5.06 (s, 2H), 4.92

13 (dt, J = 2.4, 6.6 Hz, 1H), 4.25 (t, J = 1.8 Hz, 2H). C NMR (100 MHz, CDCl 3): δ 143.8,

103.0, 90.7, 63.8.

6-(2-(tert -Butylthio)ethyl)-6H-1,3-diox-4-ene (20). 46

To a solution of dioxene 18 (0.516 g, 6 mmol, 1 equiv) in THF (8 mL) was added t-BuLi in pentane (1.6 M, 4 ml, 6.4 mmol, 1.07 equiv) with syringe pump over 25 minutes to give a light yellow solution. A precooled solution of 2-[(2-bromoethyl)sulfanyl]-2- methylpropane ( 19 ) (1.18 g, 6 mmol, 1 equiv) in THF (8 mL) at -78 oC was canulated slowly to the reaction mixture. After stirring for 4 hours at -78 oC it was allowed to warm to rt with stirring overnight. The reaction mixture was quenched with water and extracted with ether. The organic layer was washed with brine, dried over anhydride sodium 80

sulphate and concentrated in vacuo . It was partially purified on silica gel in ethyl acetate:

hexanes (0-2%) to afford 20 (1.03 g, ~ 85%). This was taken to the next step without further purification.

5-(tert -Butylthio)pent-2-E-enal (21). 46

Partially purified compound 20 (0.248 g, 0.0012 mmol) in anhydrous toluene (1.8 ml) was heated in a sealed vial in a microwave synthesizer at 165 oC for 110 min. The reaction mixture was concentrated and purified on silica gel in ethyl acetate: hexanes (1-

1 2%) to give 21 (0.211 g, 96%). H NMR (400 MHz, CDCl 3): δ 9.52 (d, 7.6, 1H), 6.87 (dt,

J = 6.8, 15.6 Hz, 1H), 6.16 (dd, J = 8.0, 16.0 Hz, 1H), 2.7 (t, J = 6.4 Hz, 2H), 2.61 (q, J =

13 6.8 Hz, 2H), 1.33 (s, 9H). C NMR (100 MHz, CDCl 3) δ 194.0, 156.3, 133.8, 42.7, 32.9,

+ 31.1, 26.6. HRMS-ESI ( m/z ): [M + H] calcd for C 9H17 OS, 173.1000; found, 173.0999.

7-(tert -Butylthio)-3S-hydroxy-1-(4 R-isopropyl-2-thioxothiazolidin-3-yl)hept-4E-en- 1-one (22). 43

To a stirred solution of acetyl Nagao chiral auxiliary 16 (0.318 g, 1.57 mmol, 1 equiv) in

o dichloromethane (15 mL) at 0 C was added TiCl 4 (0.327 g, 1.723 mmol, 1.1 equiv).

After stirring for 5 minutes, the reaction mixture was cooled to –78 oC and Hunig’s base

(0.222 g, 1.723 mmol, 1.1 equiv) was added. The reaction mixture was stirred for 2 h at the same temperature and the aldehyde 21 (0.27 g, 1.57 mmol, 1.0 equiv) in dichloromethane (4 mL) was added dropwise. The reaction mixture was stirred for 1 h at

-78 oC. It was removed from cooling bath, treated with water (15 mL), and diluted with dichloromethane (50 mL). The layers were separated and the aqueous layer was extracted with dichloromethane. The combined organic layer was washed with saturated NaCl (20

mL) and dried over anhydrous Na 2SO4. The solvent was removed in vacuo and the residue was purified by flash chromatography on silica gel in ethyl acetate/hexanes (5-

33%) to give the major isomer 22 as a thick yellow oil (0.216 g, 58% brsm), and the diastereomer 23 (0.023 g, 6%) with recovery of acetyl Nagao chiral auxiliary ( 16 ) (0.115

20 1 g). Data for major isomer 22 : [α]D - 277.6 ( c 0.135, CHCl 3). H NMR (400 MHz,

CDCl 3): δ 5.73-5.80 (m, 1H), 5.59 (dd, J = 6.0, 15.2 Hz, 1 H), 5.15 (t, J = 7.6 Hz, 1H),

4.63 (m, 1H), 3.61 (dd, J = 2.8, 17.6 Hz, 1H), 3.52 (dd, J = 8.0, 11.6 Hz, 1H), 3.30 (dd, J

= 9.2, 17.6 Hz, 1H), 3.03 (d, J = 11.6 Hz, 1H), 2.85 (s, 1H), 2.57 (t, J = 7.6 Hz, 2H),

2.27-2.39 (m, 3H), 1.30 (s, 9H), 1.05 (d, J = 6.8 Hz, 3H), 0.97 (d, J = 7.2 Hz, 3H). 13 C

(100 MHz, CDCl 3) δ 203.1, 172.7, 131.9, 130.7, 71.6, 68.7, 45.5, 42.23, 32.7, 31.2, 31.0,

+ 30.8, 28.0, 19.29, 18.0. HRMS-ESI ( m/z ): [M + Na] calcd for C 17 H29 NO 2S3Na,

398.1258; found, 398.1245.

(R)-2-(2-(( tert -Butoxycarbonylamino)methyl)thiazol-4-yl)-4,5-dihydrothiazole-4- carboxylic acid (29). 25a, 25c

To a well stirred mixture of the nitrile 7 (0.096 g, 0.4 mmol, 1 equiv) and NaHCO 3 (0.232

g, 2.76 mmol, 5.6 equiv) in methanol (5 mL) was added ( R)-α-methylcysteine

hydrochloride 13 (0.084 g, 0.491 mmol, 1.23 equiv) followed by phosphate buffer pH

5.95 (2.5 mL). The reaction mixture was degassed with nitrogen before stirring it under

nitrogen at 70 oC for 1 h. It was acidified with 1 M HCl and extracted with ethyl acetate

(15 mL) three times. The combined organic extract was washed with saturated NaCl

solution, dried over anhydrous sodium sulphate and concentrated to obtain the carboxylic

acid 29 (0.137 g) which was used in the next step without further purification.

(R)-Methyl 2-(2-((( S,E)-7-(tert -butylthio)-3-hydroxyhept-4-enamido)methyl)thiazol- 4-yl)-4-methyl-4,5-dihydrothiazole-4-carboxylate (31).

A solution of carboxylic acid 29 (0.121 g, 0.34 mmol, 1 equiv) in anhydrous methanol (5 mL) was bubbled with HCl gas for 5 minutes. The reaction mixture was stirred overnight at room temperature and concentrated in vacuo to give compound 30 which was

azeotropped using toluene before taking it to the next step. A mixture of above obtained

compound 30 and DMAP (0.108 g, 0.886 mmol, 2.61 equiv) in dichloromethane (2 mL)

was stirred for 5 minutes and a solution of aldol product 22 (0.124, 0.34 mmol, 1 equiv)

in dichloromethane (1 mL) was added. The reaction mixture was stirred for 2 h,

concentrated in vacuo and purified by flash chromatography on silica gel in ethyl

acetate/hexanes (20-100%) to afford the alcohol 31 (0.140 g, 78% over three steps from

20 1 nitrile 7). [ α]D – 15 ( c 0.1, CHCl 3). H NMR (400 MHz, CDCl 3): δ 7.90 (s, 1H), 7.32 (s,

1H), 5.66-5.72 (m, 1H), 5.52 (dd, J = 6.4, 15.2 Hz, 1H), 4.66-4.72 (m, 2H), 4.48 (s, 1H),

3.83 (d, J = 11.2 Hz, 1H), 3.76 (s, 3H), 3.24 (d, J =11.2 Hz, 1H), 2.52 (t, J = 7.6 Hz, 2H),

13 2.39-2.64 (m, 2H), 2.24 (q, J = 7.2 Hz, 2H). C NMR (100 MHz, CDCl 3): δ 173.8,

172.1, 168.2, 162.9, 148.3, 132.3, 130.6, 122.4, 84.6, 69.2, 53.1, 43.0, 42.2, 41.6, 40.9,

32.6, 31.1, 27.85, 24.1.

(R)-Methyl 2-(2-((5 S,8 S)-8-(( E)-4-(tert -butylthio)but-1-enyl)-1-(9 H-fluoren-9-yl)-5- isopropyl-3,6,10-trioxo-2,7-dioxa-4,11-diazadodecan-12-yl)thiazol-4-yl)-4-methyl- 4,5-dihydrothiazole-4-carboxylate (32)

To a solution of Fmoc-L-valine (0.013 g, 0.0383 mmol, 1.55 equiv) in THF (1 mL) at 0

oC were added Hunig’s base (0.007 g, 0.0575 mmol, 2.32 equiv) and 2,4,6-

trichlorobenzoyl chloride (0.0125 g, 0.0512 mmol, 2.07 equiv). The reaction mixture was

stirred at 0 oC for 1 h. When TLC indicated formation of the anhydride, alcohol 31

(0.012, 0.247 mmol, 1 equiv) in THF (1 mL) was added to the reaction mixture at 0 oC. It was stirred overnight at room temperature. The reaction mixture was concentrated in vacuo and flash chromatography purification on silica gel in ethyl acetate/hexanes (20-

20 1 100%) yielded the acyclic precursor 32 (0.019 g, 95%). [ α]D - 42 ( c 0.095, CHCl 3). H

NMR (400 MHz, CDCl 3): δ 7.90 (s, 1H), 7.75 (d, J = 7.6 Hz, 2H), 7.57 (d, J = 7.2 Hz,

2H), 7.39 (dt, J = 2.0, 7.6 Hz, 2H), 7.31 (t, J = 7.6 Hz, 2H), 6.86 (t, J = 6.0 Hz, 1H), 5.82-

5.90 (m, 1H), 5.65-5.70 (m, 1H), 5.56 (dd, J = 7.6, 15.6 Hz, 1H) 5.27 (d, J = 8.0 Hz, 1H),

4.69-4.79 (m, 2H), 4.32-4.41 (m, 2H), 4.19 (t, J = 6.8 Hz, 1H), 4.05-4.13 (m, 1H), 3.85

(d, J = 11.2 Hz, 1H), 3.78 (s, 3H), 3.24 (d, J = 11.6 Hz, 1H), 2.63 (d, J = 5.6 Hz, 2H),

2.53 (t, J = 7.2 Hz, 2H), 2.29 (q, J = 7.2 Hz, 2H), 2.06-2.13 (m, 1H), 1.76 (s, 1H), 1.62 (s,

3H), 1.29 (s, 9H), 0.95 (d, J = 6.8 Hz, 3H), 0.91 (d, J = 6.4 Hz, 3H). 13 C NMR (100 MHz,

CDCl 3): δ 173.8, 171.5, 169.2, 168.6, 162.9, 156.6, 148.5, 143.9, 143.9, 141.5, 141.5,

134.5, 127.9, 127.5, 127.3, 127.3, 125.3, 125.2, 122.3, 120.2, 120.2, 84.7, 72.5, 67.3,

59.7, 53.1, 47.3, 42.3, 41.7, 41.3, 32.7, 31.2, 31.1, 27.7, 24.2, 19.3, 18.2.

Cyclic core 33

To a stirred solution of 32 (0.029 g, 0.0351 mmol, 1 equiv) in THF/H 2O (4:1, 1.1 mL) at

0 oC was added 0.1 M LiOH (0.35 mL, 0.035 mmol, 1.0 equiv) dropwise over a period of

15 minutes. After stirring at 0 oC for 6 h it was acidified with 1 M HCl solution and extracted with EtOAc three times. The combined organic layer was washed with brine, dried over anhydrous sodium sulphate, and concentrated in vacuo . The product was purified by prep TLC to give the carboxylic acid. The carboxylic acid was dissolved in acetonitrile (3 mL) and treated with diethylamine (0.150 g, 1.46 mmol, 41.59 equiv).

After stirring at room temperature for 3 h, it was concentrated in vacuo to dryness to afford the free amino derivative. After drying azeotropically with toluene, it was treated with HATU (0.03 g, 0.0789 mmol, 2.25 equiv), HOAt (0.010 g, 0.0735 mmol, 2.09 equiv), dimethylformamide (30 mL, ~ 1 mM), and Hunig’s base (0.023 g, 0.173 mmol,

4.91 equiv) and the mixture was stirred for 30 h at room temperature. The reaction mixture was concentrated to dryness and was purified by flash chromatography on silica gel in ethyl acetate/hexanes (10-80%) to yield the cyclic core 33 (0.0048 g, 24% over 3

1 steps from 27 ). H NMR (400 MHz, CDCl 3): δ 7.76 (s, 1H), 7.17 (d, J = 9.6 Hz, 1H), 6.5

(dd, J = 3.0, 9.6 Hz, 1H), 5.87-5.92 (m, 1H), 5.68 (dt, J = 2.4, 7.2 Hz, 1H), 5.52 (dd, J =

7.2, 15.6 Hz, 1H), 5.29 (dd, J = 9.6, 17.4 Hz, 2H), 4.60 (dd, J = 3.0, 9.6 Hz, 1H), 4.26

(dd, J = 3.0, 17.4 Hz, 1H), 4.03 (d, J = 11.4 Hz, 1H), 3.28 (d, J = 11.4 Hz, 1H), 2.87 (dd,

J = 4.2, 16.2 Hz, 1H), 2.71 (dd, J = 3.0, 16.2 Hz, 1H), 2.56 (t, J = 7.2 Hz, 2H), 2.29-2.34

(m, 2H), 2.08-2.14 (m, 1H), 1.86 (s, 3H), 1.30 (s, 9H), 0.69 (d, J = 7.2 Hz, 3H), 0.50 (d, J

13 = 6.6 Hz, 3H). C NMR (100 MHz, CDCl 3): δ173.3, 169.7, 169.1, 168.2, 147.6, 133.9,

127.8, 124.4, 84.6, 72.3, 58.0, 43.5, 41.3, 40.8, 38.8, 34.4, 32.8, 31.2, 27.7, 24.4, 19.1,

16.8.

Attempted conversion of 33 to largazole:

attempts 1-3: 1 M BBr3, octanoyl chloride, CH2Cl2,RT attempt 4: TFA, anisole, mercuric acetate, 0 oC; followed by DIPEA, O S octanoyl chloride, DMAP, O S CH Cl ,RT N 2 2 N NH NH N S X N S OOO O OOO

S N H C(H C) S N 33 H 3 2 6 1 H Largazole

Attempt 1: 53 To a stirred solution of 33 (0.0032 g, 0.00579 mmol, 1 equiv) in dichloromethane (1 mL) were added octanoyl chloride (0.0019 g, 0.01166 mmol, 2 equiv) and BBr 3 in dichloromethane (1 M, 7 µL, 0.007 mmol, 1.21 equiv). The mixture was stirred overnight at room temperature. After TLC analysis, another portion of octanoyl chloride (0.0019 g, 0.01166 mmol, 2 equiv) was added and the mixture was stirred for another 5 h. Concentration of the reaction mixture in vacuo and purification by silica gel chromatography in ethyl acetate: hexanes gave the starting material 33 (0.002 g) and no largazole was isolated.

Attempt 2: To a stirred solution of 33 (0.002 g, 0.00362 mmol, 1 equiv) in dichloromethane (0.5 mL) were added octanoyl chloride (0.01435 g, 0.0876 mmol, 24 equiv) and BBr 3 in dichloromethane (1 M, 14 µL, 0.014 mmol, 3.87 equiv). The reaction

mixture was stirred for 2 days at room temperature. TLC analysis (4% MeOH: DCM) showed a new spot of low Rf (0.05), but chromatographic separation on silica gel in methanol: dichloromethane (4%) did not give any free thiol, largazole or the starting material.

Attempt 3: To a stirred solution of 33 (0.004 g, 0.00724 mmol, 1 equiv) in dichloromethane (0.5 mL) were added octanoyl chloride (0.0048 g, 0.0285 mmol, 3.93 equiv) and BBr 3 in dichloromethane (1 M, 14 µL, 0.014 mmol, 1.94 equiv). The reaction mixture was stirred at room temperature for 6 h. After monitoring by TLC more BBr 3 solution (2 equiv) and octanoyl chloride (8 equiv) were added and the reaction mixture was stirred overnight. Purification by column chromatography resulted in recovery of staring material (0.0025 g) but no thiol or largazole was isolated.

Attempt 4: 54 Trifluoroacetic acid (0.148 g, 1.30 mmol, 288 equiv) was added to the thioether 33 (0.0025 g, 0.0045 mmol, 1 equiv) at 0 oC. To the stirred reaction mixture were added anisole (0.001 g, 0.009 mmol, 2 equiv) and mercuric acetate (0.0015 g,

0.0047 mmol, 1.04 equiv) and the mixture was stirred for 30 min. at room temperature.

After the disappearance of the starting material (TLC) in favor of a more polar compound, reaction mixture was concentrated in vacuo . The residue was dissolved in acetonitrile (1 mL), and H 2S gas was bubbled through it to precipitate mercury as HgS.

The mixture was filtered and concentrated in vacuo .

The residue was dissolved in dry dichloromethane (0.5 mL) and treated with Hunig’s base (0.0059 g, 0.046 mmol, 10.23 equiv) and octanoyl chloride (0.0057 g, 0.035 mmol,

7.77 equiv). The reaction mixture was stirred at rt for 4 h. More Hunig’s base (0.0045 g,

0.035 mmol, 7.77 equiv) and DMAP (0.001 g, 0.0082 mmol, 1.82 equiv) were added and the mixture was stirred at room temperature. It failed to give any starting material or product.

(2 E)-5-[(Triphenylmethyl)thio]-2-pentenal (36). 55

To a solution of triphenylmethanethiol (6.91g , 25 mmol, 2.09 equiv) in dichloromethane

(100 mL) were added acrolein (1.965 g, 35 mmol, 2.9 equiv) and triethylamine (3.56 g,

35 mmol, 2.9 equiv). The reaction mixture was stirred for 1 h at room temperature and was concentrated to give the aldehyde 35 as a white solid, which was used in the next step without purification. A solution of the aldehyde 35 obtained above and

(triphenylphosphoranylidene)acetaldehyde (3.64 g, 11.96 mmol, 1 equiv) in dry benzene

(150 mL) was refluxed for 8 h. The reaction mixture was concentrated and purified by flash chromatography on silica gel in dichloromethane/hexanes (20-25%) to afford aldehyde 36 (5.83 g, 65% over the two steps); mp 140-141 oC. 1H NMR (400 MHz,

CDCl 3): δ 9.43 (d, J = 8.0 Hz, 1H), 7.42 (dd, J = 2.4, 7.6 Hz, 6H), 7.29 (dt, J = 2.0, 6.8

Hz, 6H), 7.22 (dt, J = 2.4, 7.2 Hz, 3H), 6.60-6.67 (m, 1H), 5.95-6.01 (dd, J = 8.0, 15.6

13 Hz, 1H), 2.29-2.37 (m, 4H). C NMR (100 MHz, CDCl 3): δ 194, 156, 144.7, 133.8,

129.7, 128.2, 127, 67.2, 31.9, 30.2.

3S-Hydroxy-1-(4 R-isopropyl-2-thioxo-thiazolidin-3-yl)-7-tritylsulfanyl-hept-4E-en- 1-one (37).

To a stirred solution of acetyl Nagao chiral auxiliary 16 (1.493 g, 7.355 mmol, 1 equiv) in

o dichloromethane (60 mL) at 0 C was added TiCl 4 (1.72 g, 9.05 mmol, 1.23equiv). After

stirring for 5 minutes, the reaction mixture was cooled to –78 oC and Hunig’s base (1.872

g, 9.2 mmol, 1.25 equiv) was added. The reaction mixture was stirred for 2 h at the same

temperature and the aldehyde 36 (2.6 g, 7.263 mmol, 0.987 equiv) in dichloromethane (8

mL) was added dropwise. The reaction mixture was stirred for 1 h at -78 oC. It was

removed from cooling bath, treated with water (15 mL), and diluted with

dichloromethane (50 mL). The layers were separated and the aqueous layer was extracted

with dichloromethane. The combined organic layer was washed with saturated NaCl (40

mL) and dried over anhydrous Na 2SO 4. The solvent was removed in vacuo and the

residue was purified by flash chromatography on silica gel in dichloromethane/hexanes

(25-90%) to give the major isomer 37 as a thick yellow oil (1.963 g, 76.5% brsm), and

the diastereomer 38 (0.193 g, 7.5%) with recovery of acetyl Nagao chiral auxiliary ( 16 )

20 1 (0.513 g). Data for major isomer 37 : [ α]D - 149 ( c 3.7, CHCl 3). H NMR (400 MHz,

CDCl 3): δ 7.41 (d, J = 7.2 Hz, 6H), 7.28 (t, J = 7.2 Hz, 6H), 7.21 (t, J = 7.2 Hz, 3H),

5.61-5.55 (m, 1H), 5.46 (dd, J = 6.0, 15.2 Hz, 1H), 5.12 (t, J = 6.8 Hz, 1H), 4.57 (t, J =

5.8 Hz, 1H), 3.56 (dd, J = 2.8, 17.6 Hz, 1H), 3.47 (dd, J = 7.6, 11.6 Hz, 1H), 3.28 (dd, J =

8.8, 17.6 Hz, 1H), 2.99 (d, J = 11.6 Hz, 1H), 2.82 (s, 1H), 2.37-2.32 (m, 1H), 2.21 (t, J =

7.2 Hz, 2H), 2.09 (q, J = 7.2 Hz, 2H), 1.05 (d, J = 6.8 Hz, 3H), 0.97 (d, J = 6.8 Hz, 3H).

13 C NMR (100 MHz, CDCl 3): δ 203.1, 172.7, 145, 132, 130.2, 129.7, 128, 126.8, 71.6,

68.6, 66.7, 45.4, 31.6, 31.6, 31, 30.8, 19.3, 18.0.

(R)-Methyl 2-(2-((3 S-hydroxy-7-(tritylthio)hept-4E-enamido)methyl)thiazol-4-yl)-4- methyl-4,5-dihydrothiazole-4-carboxylate (39).

A solution of carboxylic acid 29 (0.136 g, 0.38 mmol, 1 equiv) in anhydrous methanol (5 mL) was bubbled with HCl gas for 5 minutes. The reaction mixture was stirred overnight and concentrated in vacuo to give compound 30 which was azeotropped with toluene before taking it to the next step. A mixture of above obtained compound 30 and DMAP

(0.121 g, 0.992 mmol, 2.61 equiv) in dichloromethane (2 mL) was stirred for 5 minutes and a solution of aldol product 37 (0.214, 0.38 mmol, 1 equiv) in dichloromethane (1 mL) was added. The reaction mixture was stirred for 1 h, concentrated in vacuo and purified by flash chromatography on silica gel in ethyl acetate/hexanes (20-100%) to

20 1 afford the alcohol 39 (0.191 g, 78% over 3 steps a-c). [α]D - 11 ( c 3.55, CHCl 3). H

NMR (400 MHz, CDCl3): δ 7.90 (s, 1H), 7.38 (d, J = 8.4 Hz, 6H), 7.26 (t, J = 7.6 Hz,

6H), 7.19 (t, J = 7.6 Hz, 3H), 7.07 (t, J = 6.0 Hz, 1H), 5.50-5.58 (m, 1H), 5.37-5.43 (dd,

J = 6.0, 15.2 Hz, 1H), 4.63-4.74 (m, 2H), 4.43 (m, 1H), 3.86 (d, J = 11.6 Hz, 1H), 3.78 (s,

3H), 3.48 (s, 1H), 3.25 (d, J = 11.6 Hz, 1H), 2.34-2.46 (m, 2H), 2.18 (t, J = 7.2 Hz, 2H),

13 2.05 (q, J = 7.2, Hz, 2H), 1.63 (s, 3H). C NMR (100 MHz, CDCl 3): δ 173.8, 172.0,

168.1, 162.9, 148.4, 144.9, 132.4, 130.3, 129.7, 128, 126.8, 122.4, 84.6, 69.2, 66.7, 53.1,

+ 42.9, 41.6, 40.9, 31.6, 31.4, 24.1. HRMS-ESI ( m/z ): [M+H] calcd for C 36 H38 N3O4S3,

672.2019; found, 672.2024.

(4 R)-Methyl 2-(2-((8 S)-1-(9 H-fluoren-9-yl)-5-isopropyl-3,6,10-trioxo-8-(( E)-4- (tritylthio)-but-1-enyl)-2,7-dioxa-4,11-diazadodecan-12-yl)thiazol-4-yl)-4-methyl- 4,5-dihydrothiazole-4-carboxylate (40).

To a solution of Fmoc-L-valine (0.09 g, 0.266 mmol, 1 equiv) in THF (1 mL) at 0 oC were added Hunig’s base (0.45 g, 0.345 mmol, 1.29 equiv) and 2,4,6-trichlorobenzoyl chloride (0.78 g, 0.32 mmol, 1.2 equiv). The reaction mixture was stirred at 0 oC for 1 h.

When TLC indicated formation of the anhydride, alcohol 39 (0.07 g, 0.104 mmol, 0.39 equiv) in THF (1 mL) was added to the reaction mixture at 0 oC. It was stirred overnight at room temperature. The reaction mixture was concentrated in vacuo and flash chromatography purification on silica gel in ethyl acetate/hexanes (20-100%) yielded the

20 1 acyclic precursor 40 (0.100 g, 97%). [α]D -12 ( c 6.83, CHCl 3). H NMR (600 MHz,

CDCl 3): δ 7.89 (s, 1H), 7.76 (d, J = 7.2 Hz, 2H), 7.57 (d, J = 7.2 Hz, 2H), 7.41-7.37 (m, 8

H), 7.30 (t, J = 7.2 Hz, 2H), 7.26-7.29 (m, 6 H), 7.20 (t, J = 7.2 Hz, 3H), 6.74 (t, J = 8.4

Hz, 1H), 5.69-5.65 (m, 1H), 5.61 (dd, J = 6.0, 13.2 Hz, 1H), 5.42 (dd, J = 7.8, 15.0 Hz,

1H), 5.21 (d, J = 7.8 Hz, 1H), 4.7 (d, J = 6.0 Hz, 2H), 4.38 (dd, J = 7.2, 10.8 Hz, 1H),

4.33 (dd, J = 6.6, 10.8 Hz, 1H), 4.19 (t, J = 6.6 Hz, 1H), 4.05 (dd, J = 6.0, 8.4 Hz, 1H),

3.85 (d, J = 10.8 Hz, 1H), 3.78 (s, 3H), 3.24 (d, J = 10.4 Hz, 1H), 2.58 (d, J = 6.0 Hz,

2H), 2.2-2.12 (m, 2H), 2.07- 2.01 (m, 2H) 1.62 (s, 3H), 0.9 (d, J = 7.2 Hz, 3H), 0.85 (d, J

13 = 7.2 Hz, 3H). C NMR (100 MHz, CDCl 3): δ 173.8, 169.2, 168.6, 163, 156.6, 148.5,

144.9, 143.6, 141.5, 134.2, 129.7, 128.03, 127.9, 127.7, 127.3, 126.8, 125.2, 122.3,

120.15, 84.7, 72.42, 67.2, 66.8, 59.6, 53.1, 47.3, 41.7, 41.6, 41.3, 31.5, 31.3, 31, 29.9,

+ 24.1, 19.2, 18.1. HRMS-ESI ( m/z ): [M + H] calcd for C 56 H57 N4O7S3, 993.3397; found,

993.3389.

Cyclic core 41.3a

To a stirred solution of 40 (0.133 g, 0.134 mmol, 1 equiv) in THF/H 2O (4:1, 4 mL) at 0

oC was added 0.1 M LiOH (1.4 mL, 0.14 mmol, 1.045 equiv) dropwise over a period of

15 minutes. After stirring at 0 oC for 1 h, it was acidified with 1 M HCl solution and

extracted with EtOAc three times. The combined organic layer was washed with brine,

dried over anhydrous sodium sulphate, and concentrated and purified by prep TLC in

ethyl acetate to give the carboxylic acid which was carried to the next step. The

carboxylic acid was dissolved in dichloromethane (13 mL) and treated with diethylamine

(0.462 g, 6.32 mmol, 47.16 equiv). After stirring at room temperature for 3 h, it was

concentrated to dryness to afford the free amino derivative. After drying azeotropically

with toluene, it was treated with HATU (0.105 g, 0.276 mmol, 2.06 equiv), HOAt (0.038

g, 0.279 mmol, 2.08 equiv), dichloromethane (130 mL, ~ 1 mM), and Hunig’s base

(0.074 g, 0.575 mmol, 4.29 equiv) and the mixture was stirred for 30 h at room

temperature. The reaction mixture was concentrated to dryness and was purified by flash

chromatography on silica gel in ethyl acetate/hexanes (10-60%) to yield the cyclic core

20 1 41 (0.056 g, 57% over three steps from 2). [α]D + 2.5 ( c 0.95, CHCl 3). H NMR (400

MHz, CDCl 3): δ 7.73 (s, 1H), 7.37 (d, J = 8.4 Hz, 6H), 7.26 (t, J = 8 Hz, 6H), 7.20-7.15

(m, 4H), 6.49 (dd, J = 2.8, 9.2 Hz, 1H), 5.68-5.71 (m, 1H), 5.60 (m, 1H), 5.38 (dd, J =

6.8, 15.6 Hz, 1H), 5.19 (dd, J = 8.8, 17.6 Hz, 1H), 4.55 (dd, J = 3.2, 9.6 Hz, 1H), 4.11

(dd, J = 3.2, 17.6 Hz, 1H), 4.01 (d, J = 11.2Hz, 1H), 3.26 (d, J = 11.6 Hz, 1H), 2.77 (dd,

J = 9.6, 16.4 Hz, 1H), 2.64 (dd, J = 3.2, 16 Hz, 1H), 2.15-2.19 (m, 2H), 1.98-2.06 (m,

2H), 1.82 (s, 3H), 0.67 (d, J = 6.8 Hz, 3H), 0.50 (d, J = 6.8 Hz, 3H). 13 C NMR (100 MHz,

CDCl 3): δ 173.7, 169.4, 168.9, 168.1, 164.6, 147.6, 144.9, 133.3, 129.7, 128.1, 126.8,

124.3, 84.6, 72.0, 66.8, 58.0, 43.5, 41.2, 40.8, 34.2, 31.5, 31.4, 29.9, 24.4, 19.1, 16.9.

Largazole (1).

To a solution of 41 (0.033 g, 0.045 mmol, 1 equiv) in dichloromethane (5 mL) at 0 oC was added triisopropylsilane (0.013 g, 0.083 mmol, 1.85 equiv) followed by trifluoroacetic acid (0.307 g, 2.69 mmol, 59.85 equiv). After stirring for 3 h at room temperature, the reaction mixture was concentrated in vacuo . The crude product was purified by flash chromatography on silica gel in ethyl acetate/hexanes (20%) to first remove impurity followed by ethyl acetate to obtain the largazole thiol (0.022 g). To a stirred solution of largazole thiol in dichloromethane (7 mL) at 0 oC, were added, Hunig’s base (0.045 g, 0.35 mmol, 7.7 equiv) and octanoyl chloride (0.044 g, 0.27 mmol, 6 equiv). Catalytic DMAP (1.0 mg) in dichloromethane (0.1 mL) was added to the reaction mixture. After stirring for 4 h at room temperature, reaction was quenched with methanol and the mixture was concentrated in vacuo . Purification of the crude product by preparative thin layer chromatography on silica gel in ethyl acetate gave largazole (0.010

20 3a g, 79%, based on recovered largazole thiol 0.012 g). [α]D + 19.5 ( c 0.2, CHCl 3) (lit.

26.6 1 [α] D +38.9 ( c 0.027, MeOH)). H NMR (600 MHz, CDCl 3): δ 7.77 (s, 1H), 7.15 (d, J

= 9.6 Hz, 1H), 6.41 (dd, J = 2.4, 9.0 Hz, 1H), 5.79-5.84 (m, 1H), 5.66 (m, 1H), 5.50 (dd,

J = 6.6, 15.6 Hz, 1H), 5.29 (dd, J = 9.6, 18.0 Hz, 1H), 4.61 (dd, J = 3.0, 9.0 Hz, 1H), 4.27

(dd, J = 3.0, 17.4 Hz, 1H), 4.04 (d, J = 11.4 Hz, 1H), 3.28 (d, 11.4 Hz, 1H), 2.90 (t, J =

7.2 Hz, 2H), 2.85 (dd, J = 10.8, 16.2 Hz, 1H), 2.53 (t, J = 7.8 Hz, 2H), 2.31 (q, J = 7.2,

Hz, 2H), 2.10-2.12 (m, 1H), 1.87 (s, 3H), 1.62-1.67 (m, 2H), 1.26-1.31 (m, 8H), 0.87 (t, J

= 7.2 Hz, 3H), 0.68 (d, J = 7.2 Hz, 3H), 0.5 (d, 6.6 Hz, 3H). 13 C NMR (100 MHz, 93

CDCl 3): δ 199.6, 174.7, 169.6, 169.1, 168.1, 164.7, 147.7, 133.0, 128.6, 124.4, 84.7, 72.3,

57.9, 44.4, 43.6, 41.3, 40.7, 34.4, 32.5, 31.8, 29.1, 28.1, 25.9, 24.4, 22.8, 19.1, 16.8, 14.3.

+ HRMS- ESI ( m/z ): [M + Na] calcd for C 29 H42 N4O5S3Na, 645.2211 found, 645.2215. %

purity: 95.31% (HPLC)

(R)-Methyl 2-(2-((5 R,8 S)-1-(9 H-fluoren-9-yl)-5-(naphthalen-1-ylmethyl)-3,6,10- trioxo-8-(( E)-4-(tritylthio)but-1-enyl)-2,7-dioxa-4,11-diazadodecan-12-yl)thiazol-4- yl)-4-methyl-4,5-dihydrothiazole-4-carboxylate (42).

To a solution of Fmoc-D-1-naphthylalanine (0.130 g, 0.297 mmol, 3.06 equiv) in THF (1

mL) at 0 oC were added Hunig’s base (0.045 g, 0.345 mmol, 3.55 equiv) and 2,4,6- trichlorobenzoyl chloride (0.078 g, 0.32 mmol, 3.3 equiv). The reaction mixture was stirred at 0 oC for 1 h. When TLC indicated formation of the anhydride, alcohol 39 (0.065 g, 0.097, 1 equiv) in THF (1 mL) was added to the reaction mixture at 0 oC. It was stirred overnight at room temperature. The reaction mixture was concentrated in vacuo and flash chromatography purification on silica gel in ethyl acetate/hexanes (20-100%) yielded the

20 1 acyclic precursor 42 (0.058 g, 55%). [α]D - 1.38 ( c 0.65, CHCl 3). H NMR (400 MHz,

CDCl 3): δ 8.07 (d, J = 8.0 Hz, 1H), 7.80-7.85 (m, 2H), 7.75- 7.77 (m, 3H), 7.44-7.51(m,

4H), 7.34-7.41 (m, 9H), 7.23-7.29 (m, 9H), 7.15-7.19 (t, J = 7.2 Hz, 3H), 5.94 (t, J = 5.6

Hz, 1H), 5.55-5.62 (m, 1H), 5.39-5.46 (m, 2H), -5.32 (dd, J = 7.2, 15.6 Hz, 1H), 4.73 (q,

J = 7.2 Hz, 1H), 4.52 (dd, J = 6.0, 16.0 Hz, 1H), 4.42 (dd, J = 5.6, 16.0 Hz, 1H), 4.35 (dd,

J = 7.2, 10.4 Hz, 1H), 4.26 (dd, J = 7.2, 10.4 Hz, 1H), 4.13 (t, J = 7.2 Hz, 1H), 3.87 (d, J

= 11.2 Hz, 1H), 3.79 (s, 3H), 3.49 (d, J = 8.8 Hz, 1H), 3.24 (d, J = 11.2 Hz, 1H), 2.28

(dd, J = 6.0, 14.4 Hz, 1H), 2.15 (t, J = 7.2 Hz, 2H), 2.00-2.09 (m, 2H), 1.63 (s, 3H), 1.26

13 (t, J = 7.2 Hz, 1H). C (100 MHz, CDCl 3) δ 173.8, 170.9, 168.9, 167.9, 162.9, 155.8,

148.5, 144.9, 143.9, 143.8, 141.4, 133.9, 133.9, 132.5, 132.1, 129.7, 129.1, 128.2, 128.1,

127.9, 127.8, 127.3, 127.3, 127.2, 126.9, 126.8, 126.3, 125.6, 125.3, 125.2, 123.6, 122.1,

120.2, 84.7, 72.7, 67.3, 66.8, 55.0, 53.2, 47.2, 41.7, 41.3, 41.1, 36.2, 31.5, 31.3, 24.2.

Cyclic core 43.

To a stirred solution of 42 (0.058 g, 0.0532 mmol, 1 equiv) in THF/H 2O (4:1, 1.6 mL) at

0 oC was added 0.1 M LiOH (0.53 mL, 0.053 mmol, 1.00 equiv) dropwise over a period

of 15 minutes. After stirring at 0 oC for 1 h, it was acidified with 1 M HCl solution and

extracted with EtOAc three times. The combined organic layer was washed with brine,

dried over anhydrous sodium sulphate, and concentrated in vacuo . The residue was

purified by prep TLC in ethyl acetate to give the carboxylic acid. The carboxylic acid was

dissolved in dichloromethane (13 mL) and treated with diethylamine (0.1917 g, 2.70

mmol, 50.75 equiv). After stirring at room temperature for 3 h, it was concentrated in

vacuo to dryness to afford the free amino derivative. After drying azeotropically with toluene, it was treated with HATU (0.041 g, 0.108 mmol, 2.02 equiv), HOAt (0.015 g,

0.110 mmol, 2.07 equiv), dichloromethane (55 mL, ~ 1 mM), and Hunig’s base (0.030 g,

0.23 mmol, 4.32 equiv) and the mixture was stirred for 30 h at room temperature. The reaction mixture was concentrated to dryness and was purified by flash chromatography on silica gel in ethyl acetate/hexanes (10-60%) to yield the cyclic core 43 (0.0057 g,

20 1 14%). [α]D + 39.3 ( c 0.112, CHCl 3). H NMR (600 MHz, CDCl 3): δ 8.36 (d, J = 8.4 Hz,

1H), 7.80 (d, J = 8.4 Hz, 1H) 7.68 (d, J = 9.0 Hz, 1H), 7.67 (s, 1H), 7.55 (t, J = 7.8 Hz,

1H), 7.45 (t, J = 7.8 Hz, 1H), 7.39 (d, J = 8.4 Hz, 6H), 7.32 (d, J = 7.2 Hz, 1H), 7.26 (t, J

= 7.8 Hz, 6H), 7.19 (t, J = 7.2 Hz, 3H), 7.16 (d, J = 7.8 Hz, 1H), 7.06 (d, J = 6.6 Hz, 1H),

6.39 (s, 1H), 5.68 (t, J = 9.6 Hz, 1H), 5.61-5.66 (m, 1H), 5.21 (dd, J = 8.4, 15.6 Hz, 1H), 95

5.05 (dd, J = 8.4, 17.4 Hz, 1H), 4.83 (m, 1H), 4.20-4.24 (m, 2H), 3.68 (dd, J = 5.4, 13.8

Hz, 1H), 3.19-3.22 (m, 2H), 2.81 (dd, J = 9.8, 16.8 Hz, 1H), 2.55 (d, J = 16.2 Hz, 1H),

2.05-2.21 (m, 3H), 2.00-2.03 (m, 1H), 1.72 (s, 3H), 1.21 (dd, J = 6.6, 16.2 Hz, 1H), 0.92-

13 1.02 (m, 2H). C (150 MHz, CDCl 3) δ 174.2, 169.6, 167.9, 167.4, 162.9, 147.8, 145.0,

134.8, 134.0, 132.5, 132.1, 129.8, 128.8, 128.3, 128.2, 128.1, 126.9, 126.7, 125.9, 124.9,

124.5, 124.3, 84.9, 72.8, 66.9, 55.7, 41.8, 41.5, 40.3, 37.1, 31.6, 31.2, 26.4. HRMS- ESI

+ (m/z ): [M + H] calcd for C 48 H45 N4O4S3, 837.2603; found, 837.2599.

Analog T1A.

To a solution of 43 (0.005 g, 0.006 mmol, 1 equiv) in dichloromethane (1 mL) at 0 oC was added triisopropylsilane (0.0023 g, 0.0147 mmol, 2.44 equiv) followed by trifluoroacetic acid (0.046 g, 0.404 mmol, 67.25 equiv). After stirring for 3 h at room temperature, the reaction mixture was concentrated in vacuo . The crude product was purified by flash chromatography on silica gel in ethyl acetate/hexanes (20%) to first remove impurity followed by ethyl acetate to obtain the thiol. To a stirred solution of the thiol in dichloromethane (1 mL) at 0 oC were added Hunig’s base (0.006 g, 0.046 mmol,

7.7 equiv) and octanoyl chloride (0.006 g, 0.035 mmol, 5.83 equiv). After stirring for 4 h at room temperature, the reaction was quenched with methanol and the mixture was concentrated in vacuo . Purification of the crude product by preparative thin layer chromatography on silica gel in ethyl acetate and final purification by HPLC on C 18

20 column in methanol: water (20-100%) gave analog T1A (0.0022 g, 50%). [α]D + 45.0 ( c

1 0.1, CHCl 3). H NMR (600 MHz, CDCl 3): δ 8.40 (d, J = 8.4 Hz, 1H), 7.82 (d, J = 8.4 Hz,

1H), 7.72 (d, J = 7.8 Hz, 1H), 7.68 (s, 1H), 7.59 (t, J = 7.8 Hz, 1H), 7.47 (t, J = 7.8 Hz,

1H), 7.35 (d, J = 6.6 Hz, 1H), 7.26-7.29 (m, 1H), 7.15 (d, J = 7.2 Hz, 1H), 6.39-6.41 (m, 96

1H), 5.73-5.78 (m, 2H), 5.35 (dd, J = 8.4, 15.6 Hz, 1H), 5.07 (dd, J = 7.8, 16.8 Hz, 1H),

4.86 (q, J = 7.8 Hz, 1H), 4.25 (dd, J = 4.8, 16.8 Hz, 1H), 4.21 (d, J = 11.4 Hz, 1H), 3.72

(dd, J = 4.8, 13.8 Hz, 1H), 3.25 (dd, J = 9.6, 13.8 Hz, 1H), 3.21 (d, J = 11.4 Hz, 1H),

2.83-2.88 (m, 3H), 2.59 (d, J = 16.8 Hz, 1H), 2.51 (t, J = 7.2 Hz, 2H), 2.22-2.31 (m, 2H),

1.74 (s, 3H), 1.60-1.65 (m, 2H) 1.25 (m, 11H), 0.87 (t, J = 7.2 Hz, 3H). 13 C (150 MHz,

CDCl 3) δ 199.5, 174.2, 169.6, 167.9, 167.4, 162.9, 147.9, 134.2, 134.0, 132.5, 132.2,

128.9, 128.7, 128.3, 128.2, 126.7, 125.9, 124.9, 124.4, 124.3, 114.2, 85.0, 72.8, 55.7,

44.4, 41.8, 41.5, 40.4, 37.1, 32.4, 31.8, 29.9, 29.1, 28.0, 26.4, 25.8, 22.8, 14.2. HRMS-

+ ESI ( m/z ): [M + Na] calcd for C 32 H34 N4O5S3Na, 743.2372; found, 743.2372.

(R)-Methyl 2-(2-((5 S,8 S)-5-allyl-1-(9 H-fluoren-9-yl)-3,6,10-trioxo-8-(( E)-4- (tritylthio)but-1-enyl)-2,7-dioxa-4,11-diazadodecan-12-yl)thiazol-4-yl)-4-methyl-4,5- dihydrothiazole-4-carboxylate (44).

To a solution of Fmoc-L-allylglycine (0.046 g, 0.137 mmol, 2 equiv) in THF (1 mL) at 0 oC were added Hunig’s base (0.030 g, 0.230 mmol, 3.38 equiv) and 2,4,6- trichlorobenzoyl chloride (0.047 g, 0.192 mmol, 2.83 equiv). The reaction mixture was stirred at 0 oC for 1 h. When TLC indicated formation of the anhydride, alcohol 39 (0.046 g, 0.068 mmol, 1 equiv) in THF (1 mL) was added to the reaction mixture at 0 oC. It was stirred overnight at room temperature. The reaction mixture was concentrated in vacuo and flash chromatography purification on silica gel in ethyl acetate/hexanes (20-100%)

20 1 yielded the acyclic precursor 44 (0.061 g, 90%). [α]D - 12.53 ( c 1.7, CHCl 3). H NMR

(400 MHz, CDCl 3): δ 7.88 (s, 1H), 7.75 (d, J = 7.6 Hz, 2H), 7.56 (d, J = 7.2 Hz, 1H),

7.37-7.40 (m, 8H), 7.25-7.31 (m, 3H), 7.20 (t, J = 6.8 Hz, 3H), 6.84 (t, J = 6.0 Hz, 1H),

5.57-5.68 (m, 3H), 5.42 (dd, J = 7.2, 15.2, Hz, 1H), 5.32 (d, J = 7.6 Hz, 1H), 5.06-5.10

(m, 2H), 4.68 (d, J = 6.0 Hz, 2H), 4.31-4.38 (m, 2H), 4.26 (q, J = 6.4 Hz, 1H), 4.19 (t, J =

7.2 Hz, 1H), 3.85 (d, J = 11.2 Hz, 1H), 3.78 (s, 3H), 3.24 (d, J = 11.6 Hz, 1H), 2.58 (d, J

= 6.4 Hz, 2H), 2.49-2.54 (m, 1H), 2.38-2.44 (m, 1H), 2.19 (t, J = 6.8 Hz, 2H), 2.05 (q, J =

13 7.2 Hz, 2H), 1.85 (s, 1H), 1.62 (s, 3H). C NMR (100 MHz, CDCl 3) δ 173.8, 171.0,

169.2, 168.5, 163.0, 156.1, 148.5, 144.9, 143.9, 143.8, 141.5, 141.4, 134.0, 132.0, 129.7,

128.1, 127.9, 127.6, 127.3, 126.8, 125.3, 125.2, 122.3, 120.2, 119.8, 84.7, 72.7, 67.3,

66.8, 53.6, 53.1, 47.2, 41.7, 41.6, 41.2, 36.3, 31.5, 31.3, 24.1. HRMS-ESI (m/z ): [M +

+ Na] calcd for C 56 H54 N4O7S3Na, 1013.3052; found, 1013.3058.

Cyclic core 45.

To a stirred solution of 44 (0.061 g, 0.0615 mmol, 1 equiv) in THF/H 2O (4:1, 2 mL) at 0 oC was added 0.1 M LiOH (0.71 mL, 0.071 mmol, 1.15 equiv) dropwise over a period of

15 minutes. After stirring at 0 oC for 1 h, it was acidified with 1 M HCl solution and extracted with EtOAc three times. The combined organic layer was washed with brine, dried over anhydrous sodium sulphate, and concentrated in vacuo . The reaction mixture

was purified by preparative TLC in ethyl acetate: hexanes (70%) to give the carboxylic

acid (0.043 g, 0.044 mmol, 1 equiv) which was carried to the next step. During

purification, starting material 44 (0.01 g) was recovered. The carboxylic acid was

dissolved in dichloromethane (4.5 mL) and treated with diethylamine (0.156 g, 2.14

mmol, 48.4 equiv). After stirring at room temperature for 3 h it was concentrated to

dryness to afford the free amino derivative. After drying azeotropically with toluene it

was treated with HATU (0.041 g, 0.108 mmol, 2.45 equiv), HOAt (0.015 g, 0.110 mmol,

2.29 equiv), dichloromethane (60 mL, ~ 1 mM), and Hunig’s base (0.030 g, 0.23 mmol,

4.79 equiv) and the mixture was stirred for 30 h at room temperature. The reaction 98

mixture was concentrated to dryness and was purified by flash chromatography on silica

20 gel in ethyl acetate/hexanes (10-60%) to yield the cyclic core 45 (5.5 mg, 17%). [α]D

1 +20.0 ( c 0.28, CHCl 3). H NMR (600 MHz, CDCl 3): δ 7.69 (s, 1H), 7.37 (d, J = 7.8 Hz,

6H), 7.27 (t, J = 8.4 Hz, 6H), 7.19 (t, J = 8.4 Hz, 3H), 6.42 (dd, J = 3.6, 9.0 Hz, 1H),

5.64-5.70 (m, 2H), 5.36 (dd, J = 6.6, 15.0 Hz, 1H), 5.20 (dd, J = 9.6, 17.4 Hz, 1H), 4.69

(d, J = 16.8 Hz, 1H), 4.62-4.65 (m, 1H), 4.35 (d, J = 10.2 Hz, 1H), 4.16 (dd, J = 3.6, 18.0

Hz, 1H), 4.07 (d, J = 11.4 Hz, 1H), 4.21 (d, J = 11.4 Hz, 1H), 2.81 (dd, J = 10.2, 16.8 Hz,

1H), 2.63 (dd, J = 3.0, 16.8 Hz, 1H), 2.47-2.50 (m, 1H), 2.31-2.36 (m, 1H), 2.15-2.20 (m,

13 1H), 1.99-2.08 (m, 2H), 1.81 (s, 3H). C NMR (100 MHz, CDCl 3) δ 173.7, 169.5, 169.1,

167.8, 164.4, 147.9, 145.0, 133.7, 131.5, 129.8, 128.1, 128.0, 126.9, 119.5, 84.5, 72.5,

52.7, 42.7, 41.3, 40.6, 38.0, 31.6, 31.4, 24.8. HRMS-ESI ( m/z ):: [M + Na] + calcd for

C40 H40 N4O4S3Na, 759.2109; found, 759.2039.

Analog T1B.

To a solution of 45 (0.0055 g, 0.0075 mmol, 1 equiv) in dichloromethane (1 mL) at 0 oC was added triisopropylsilane (0.0023 g, 0.0147 mmol, 1.96 equiv) followed by trifluoroacetic acid (0.057 g, 0.498 mmol, 66.78 equiv). After stirring for 3 h at room temperature the reaction mixture was concentrated in vacuo . The crude product was

purified by flash chromatography on silica gel in ethyl acetate/hexanes (20%) to first

remove impurity followed by ethyl acetate to obtain the thiol. To a stirred solution of the

thiol in dichloromethane (1 mL) at 0 oC were added Hunig’s base (0.006 g, 0.046 mmol,

7.7 equiv) and octanoyl chloride (0.006 g, 0.035 mmol, 5.83 equiv). After stirring for 4 h

at room temperature reaction was quenched with methanol and the mixture was

concentrated in vacuo . Purification of the crude product by preparative thin layer 99

chromatography on silica gel in ethyl acetate as solvent gave analog T1B (0.0016 mg,

20 1 35%). [α]D + 25.56 ( c 0.08, CHCl 3). H NMR (600 MHz, CDCl 3): δ 7.71 (s, 1H), 7.19

(d, J = 8.4 Hz, 1H), 6.36 (dd, J = 3.0, 9.0 Hz, 1H), 4.79-4.83 (m, 1H), 5.73 (t, J = 8.4 Hz,

1H), 5.50 (dd, J = 7.2, 15.6 Hz, 1H) 5.26-5.32 (m, 3H), 4.67-4.72 (m 2H), 4.35 (d, J =

16.2 Hz, 1H), 4.27 (dd, J = 3.6, 18.0 Hz, 1H), 4.10 (d, J = 11.4 Hz, 1H), 3.22 (d, J = 11.4

Hz, 1H), 2.90 (t, J = 7.2 Hz, 2H), 2.87 (dd, J = 10.8, 16.8 Hz, 1H), 2.68 (dd, J = 2.4, 16.2

Hz, 1H), 2.53 (t, J = 7.2 Hz, 3H), 2.34-2.39 (m, 1H), 2.31 (q, J = 7.2 Hz, 2H), 1.85 (s,

3H), 1.63-1.67 (m, 2H), 1.25-1.32 (m, 13H), 0.88 (t, J = 7.2 Hz, 3H). 13 C NMR (150

MHz, CDCl 3) δ 199.7, 173.7, 169.5, 169.1, 167.8, 164.4, 147.9, 133.2, 131.5, 128.5,

124.3, 121.8, 119.5, 84.5, 72.6, 52.8, 44.4, 42.7, 41.3, 38.0, 32.5, 31.8, 29.9, 29.1, 28.1,

+ 25.9, 24.8, 22.8, 14.29. HRMS-ESI ( m/z ): [M + Na] calcd for C 29 H40 N4O5S3Na,

643.2059; found, 643.2046.

100

(R)-Methyl 2-(2-((5 S,8 S)-1-(9 H-fluoren-9-yl)-3,6,10-trioxo-5-(3-oxo-3- (tritylamino)propyl)-8-(( E)-4-(tritylthio)but-1-enyl)-2,7-dioxa-4,11-diazadodecan- 12-yl)thiazol-4-yl)-4-methyl-4,5-dihydrothiazole-4-carboxylate (46).

To a solution of Fmoc-L-Gln(Trt)-OH (0.096 g, 0.157 mmol, 2.00 equiv) in THF (1 mL)

at 0 oC were added Hunig’s base (0.028 g, 0.219 mmol, 2.78 equiv) and 2,4,6-

trichlorobenzoyl chloride (0.047 g, 0.192 mmol, 2.43 equiv). The reaction mixture was

stirred at 0 oC for 1 h. When TLC indicated formation of the anhydride, alcohol 39 (0.053 g, 0.079 mmol, 1 equiv) in THF (1 mL) was added to the reaction mixture at 0 oC. It was stirred overnight at room temperature. The reaction mixture was concentrated in vacuo and flash chromatography purification on silica gel in ethyl acetate/hexanes (20-60%)

20 1 yielded the acyclic precursor 46 (0.079, 78%). [α]D - 11.22 ( c 4.07, CHCl 3). H NMR

(400 MHz, CDCl 3): δ 7.82 (s, 1H), 7.73 (d, J = 7.6 Hz, 2H), 7.53 (d, J = 7.6 Hz, 2H),

7.37 (d, J = 7.6 Hz, 7H), 7.17- 7.28 (m, 27 H) 7.11 (t, J = 6.0 Hz, 1H), 6.86 (s, 1H), 5.76

(d, J = 7.2 Hz, 1H), 5.57-5.64 (m, 1H), 5.50 (q, J = 5.6 Hz, 1H), 5.40 (dd, J = 7.2, 15.2

Hz, 1H), 4.60 (dd, J = 6.4, 16.0 Hz, 1H), 4.47 (dd, J = 6.0, 16.0 Hz, 1H), 4.3-4.40 (m,

2H), 4.17 (t, J = 6.8, 1H), 3.82 (d, J = 11.2 Hz, 1H), 3.76 (s, 3H), 3.22 (d, J = 11.2 Hz,

1H), 2.42-2.52 (m, 2H), 2.26-2.34 (m, 2H), 2.14-2.21 (m, 2H), 2.00-2.08 (m,2H), 1.62

13 (s, 3H). C NMR (100 MHz, CDCl 3) δ 173.8, 171.0, 169.4, 168.9, 163.0, 156.5, 148.3,

144.9, 144.6, 143.9, 143.8, 141.4, 133.4, 129.7, 128.8, 128.1, 128.0, 127.9, 127.6, 127.3,

127.2, 126.8, 125.3, 122.1, 120.184.7, 72.7, 70.8, 67.1, 66.8, 54.1, 53.1, 47.3, 41.6, 41.4,

41.2, 33.0, 31.4, 31.3, 27.2, 24.1. HRMS-ESI ( m/z ): [M + Na] + calcd for

C36 H37 N3O4S3Na, 1286.4206; found, 1286.4214.

101

(R)-Methyl 2-(2-((5 S,8 S)-1-(9 H-fluoren-9-yl)-5-(naphthalen-1-ylmethyl)-3,6,10- trioxo-8-(( E)-4-(tritylthio)but-1-enyl)-2,7-dioxa-4,11-diazadodecan-12-yl)thiazol-4- yl)-4-methyl-4,5-dihydrothiazole-4-carboxylate (48).

To a solution of Fmoc-L-1-naphthylalanine (0.052 g, 0.119 mmol, 1.95 equiv) in THF (1

mL) at 0 oC were added Hunig’s base (0.028 g, 0.219 mmol, 3.59 equiv) and 2,4,6-

trichlorobenzoyl chloride (0.031 g, 0.128 mmol, 2.09 equiv). The reaction mixture was

stirred at 0 oC for 1 h. When TLC indicated formation of the anhydride, alcohol 39 (0.041 g, 0.061 mmol, 1 equiv) in THF (1 mL) was added to the reaction mixture at 0 oC. It was stirred overnight at room temperature. The reaction mixture was concentrated in vacuo and flash chromatography purification on silica gel in ethyl acetate/hexanes (20-60%)

20 1 yielded the acyclic precursor 48 (0.065 g, 98%). [α]D - 9.05 ( c 3.25, CHCl 3). H NMR

(600 MHz, CDCl 3): δ 8.02 (d, J = 8.4 Hz, 1H), 7.80 (m, 2H), 7.73 (dd, J = 2.4, 7.2 Hz,

2H), 7.68 (d, J = 8.4 Hz, 1H), 7.42-7.50 (m, 4H), 7.36-7.39 (m, 8H), 7.24-7.29 (m, 10H),

7.18 (t, J = 7.2 Hz, 4H), 6.61 (t, J = 6.0 Hz, 1H), 5.39-5.43 (m, 2H), 5.30-5.35 (m, 1H),

5.11 (dd, J = 7.2, 15.6 Hz, 1H), 4.53-4.60 (m, 2H), 4.52 (dd, J = 6.0, 16.2 Hz, 1H), 4.28-

4.35 (m, 2H), 4.09-4.11 (m, 1H), 3.82 (d, J = 11.4 Hz, 1H), 3.76 (s, 3H), 3.40-3.49 (m,

2H), 3.20 (d, J = 10.8 Hz, 1H), 2.41 (dd, J = 4.2, 14.4 Hz, 1H), 3.30 (dd, J = 6.6, 14.4 Hz

1H), 2.13 (t, J = 7.8 Hz, 2H) 1.97 (q, J = 7.2 Hz, 2H), 1.60 (s, 3H). 13 C NMR (100 MHz,

CDCl 3) δ 173.9, 171.2, 169.2, 168.8, 163.0, 156.1, 148.5, 145.0, 143.8, 143.8, 141.5,

134.0, 133.6, 132.1, 132.1, 84.7, 72.7, 71.6, 67.3, 66.8, 60.6, 55.1, 53.1, 47.2, 41.6, 41.5,

41.2, 35.5, 31.5, 31.3, 24.1, 21.3, 20.6, 14.4. HRMS-ESI ( m/z ): [M + Na] + calcd for

C64 H58 N4O7S3Na: 1113.3365; found: 1113.3384.

102

(R)-2-(2-(( tert -Butoxycarbonylamino)methyl)thiazol-4-yl)-5,5-dimethyl-4,5- dihydrothiazole-4-carboxylic acid (50).

To a well stirred mixture of the nitrile 7 (0.048 g, 0.2 mmol, 1 equiv) and NaHCO 3 (0.120

g, 1.43 mmol, 7.14 equiv) in methanol (2.5 mL) was added L-penicillamine (0.04 g,

0.268, 1.234 equiv) followed by phosphate buffer pH 5.95 (1.25 mL). The reaction

mixture was degassed with nitrogen before stirring it under nitrogen at 70 oC for 1 h. It was acidified with 1 M HCl and extracted with ethyl acetate (15 mL) three times. The combined organic extract was washed with saturated NaCl solution, dried over anhydrous sodium sulphate and concentrated to obtain the carboxylic acid 50 (0.07 g) which was

used in the next step without further purification.

(R)-Methyl 2-(2-((( S,E)-3-hydroxy-7-(tritylthio)hept-4-enamido)methyl)thiazol-4-yl)- 5,5-dimethyl-4,5-dihydrothiazole-4-carboxylate (51).

A solution of carboxylic acid 50 (0.07 g, 0.189 mmol, 1 equiv) in anhydrous methanol (5

mL) was bubbled with HCl gas for 5 minutes. The reaction mixture was stirred overnight

at room temperature and concentrated in vacuo and was azeotropped using toluene before taking it to the next step. A mixture of above obtained compound and DMAP (0.06 g,

0.492 mmol, 2.60 equiv) in dichloromethane (2 mL) was stirred for 5 minutes, and a solution of aldol product 37 (0.106, 0.189 mmol, 1 equiv) in dichloromethane (1 mL) was added. The reaction mixture was stirred for 1 h, concentrated in vacuo and purified by flash chromatography on silica gel in ethyl acetate/hexanes (20-100%) to afford the

20 1 alcohol 51 . [α]D - 5.28 ( c 3.6, CHCl 3). H NMR (400 MHz, CDCl 3): δ 7.88 (s, 1H), 7.38

(d, J = 7.6 Hz, 6H), 7.26 (t, J = 8.0 Hz, 6H), 7.19 (t, J = 7.2 Hz, 3H), 7.11 (q, J = 6.0 Hz,

1H), 5.50-5.57 (m, 1H), 5.39 (dd, J = 6.4, 15.6 Hz, 1H), 4.83 (s, 1H), 4.44-4.72 (m, 2H),

103

4.42 (s, 1H), 3.79 (s, 3H), 3.53 (s, 1H), 2.43 (dd, J = 3.6, 15.2 Hz, 1H), 2.38 (dd, J = 5.2,

13.2 Hz, 1H), 2.18 (t, J = 7.6 Hz, 2H), 2.03-2.08 (m, 2H), 1.75 (s, 3H), 1.43 (s, 3H). 13 C

NMR (100 MHz, CDCl 3) δ 172.1, 170.0, 168.1, 164.8, 148.8, 145.0, 132.4, 130.2, 129.7,

128.0, 126.8, 122.4, 86.0, 69.2, 66.7, 60.7, 60.6, 42.8, 40.9, 31.6, 31.4, 28.6, 26.1, 21.2,

14.4.

(R)-Methyl 2-(2-((5 S,8 S)-1-(9 H-fluoren-9-yl)-5-isopropyl-3,6,10-trioxo-8-(( E)-4- (tritylthio)but-1-enyl)-2,7-dioxa-4,11-diazadodecan-12-yl)thiazol-4-yl)-5,5-dimethyl- 4,5-dihydrothiazole-4-carboxylate (52).

To a solution of Fmoc-L-valine (0.080 g, 0.236 mmol, 2.48 equiv) in THF (1 mL) at 0 oC were added Hunig’s base (0.040 g, 0.310 mmol, 3.26 equiv) and 2,4,6-trichlorobenzoyl chloride (0.069 g, 0.281 mmol, 2.96 equiv). The reaction mixture was stirred at 0 oC for 1 h. When TLC indicated formation of the anhydride, alcohol 51 (0.065 g, 0.095 mmol, 1 equiv) in THF (1 mL) was added to the reaction mixture at 0 oC. It was stirred overnight at room temperature. The reaction mixture was concentrated in vacuo and flash chromatography purification on silica gel in ethyl acetate/hexanes (20-60%) yielded the

20 1 acyclic precursor 52. [α]D - 14.94 ( c 0.435, CHCl3). H NMR (400 MHz, CDCl 3): δ

7.89 (s, 1H), 7.76 (d, J = 7.2 Hz, 2H), 7.57 (d, J = 7.6 Hz, 2H), 7.35-7.41 (m, 8H), 7.25-

7.35 (m, 8H), 7.20 (t, J = 7.2 Hz, 3H), 6.77 (q, J = 8.4 Hz, 1H), 5.59-5.70 (m, 2H), 5.42

(dd, J = 7.6, 15.6 Hz, 1H), 5.24 (d, J = 8.4 Hz, 1H), 4.83 (s, 1H), 4.63-4.72 (m, 2H), 4.31-

4.39 (m, 2H), 4.19 (t, J = 6.8 Hz, 1h), 4.06 (t, J = 8.4 Hz, 1H), 3.80 (s, 3 H), 2.57 (d, J =

6.0 Hz, 2H), 2.12-2.20 (m, 2H), 1.99-2.08 (m, 3H), 1.75 (s, 3H), 1.43 (s, 3H), 0.89 (d, J =

13 6.8 Hz, 3H), 0.84 (d, J = 0.84 Hz, 3H). C NMR (100 MHz, CDCl 3) δ 169.9, 169.2,

168.7, 164.9, 156.5, 148.7, 144.9, 144.9, 143.9, 143.8, 141.4, 141.4, 134.1, 129.7, 128.0,

104

127.9, 127.7, 127.3, 127.2, 126.8, 125.2, 125.2, 120.1, 86.0, 72.4, 67.2, 66.7, 60.6, 60.5,

59.5, 52.4, 47.3, 41.5, 41.2, 31.4, 31.2, 31.0, 28.6, 26.8, 19.2, 18.0.

(R)-2,2,2-Trichloroethyl 2-(2-(( tert -butoxycarbonylamino)methyl)thiazol-4-yl)-5,5- dimethyl-4,5-dihydrothiazole-4-carboxylate (54).

To a solution of carboxylic acid 50 (0.410 g, 1.105 mmol, 1 equiv), EDC.HCl (0.380 g,

1.98 mmol, 1.79 equiv) and DMAP (0.146 g, 1.2 mmol, 1.09 equiv) in dichloromethane

(2 mL) were added Hunig’s base (0.519 g, 4.02 mmol, 3.63 equiv) and trichloroethanol

(1.55 g, 10 mmol, 9.05 equiv). The reaction mixture was stirred overnight at room temperature. The reaction mixture is partitioned between water and dichloromethane. The organic layer was washed with 10% HCl, 5% NaHCO 3, and brine, dried over anhydrous sodium sulphate and concentrated in vacuo . The crude reaction product was purified by flash column chromatography on silica gel in ethyl acetate: hexanes (9-25%) to get pure

20 1 ester 54 (0.195, 35%). [α]D - 5.77 ( c 0.26, CHCl 3). H NMR (600 MHz, CDCl 3): δ 7.95

(s, 1H), 5.23 (s, 1H), 4.98 (s, 1H), 4.92 (d, J = 12.0 Hz, 1H), 4.78 (d, J = 12.0 Hz, 1H),

4.62 (d, J = 6.6 Hz, 2H), 1.83 (s, 3H), 1.52 (s, 3H), 1.45 (s, 9H). 13 C NMR (100 MHz,

CDCl 3): δ 170.0, 168.1, 165.7, 155.9, 148.9, 122.1, 94.6, 85.8, 80.6, 74.9, 60.6, 42.5,

28.6, 28.3, 26.4.

105

(R)-2,2,2-Trichloroethyl 2-(2-((( S,E)-3-hydroxy-7-(tritylthio)hept-4- enamido)methyl)thiazol-4-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylate (55).

To a solution of ester 54 (0.111 g, 0.221 mmol, 1 equiv) in dichloromethane (3 mL) was added 4N HCl in 1,4-dioxane (4.5 mL) over 5 minutes. The reaction mixture was stirred for 1 h and concentrated in vacuo . The residue was azeotropped with toluene before

taking it to the next step. A mixture of above obtained compound and DMAP (0.07 g,

0.574 mmol, 2.60 equiv) in dichloromethane (2 mL) was stirred for 5 minutes and a

solution of aldol product 37 (0.124, 0.221 mmol, 1 equiv) in dichloromethane (1 mL) was

added. The reaction mixture was stirred for 1 h, concentrated in vacuo and purified by flash chromatography on silica gel in ethyl acetate/hexanes (20-100%) to afford the

20 1 alcohol 55 (0.111 g, 63%). [α]D - 5.6 ( c 1.25, CHCl 3). H NMR (400 MHz, CDCl 3): δ

7.9 (s, 1H), 7.37 (d, J = 7.6 Hz, 6H), 7.25 (t, J = 7.2 Hz, 6H), 7.18 (t, J = 6.8 Hz, 3H),

5.49-5.55 (m, 1H), 5.39 (dd, J = 6.0, 15.6 Hz, 1H), 5.00 (s, 1H), 4.92 (d, J = 12.0 Hz,

1H), 4.76 (d, J = 12.0 Hz, 1H), 4.30-4.70 (m, 2H), 4.41 (s, 1H), 3.58 (s, 1H), 2.35-2.44

(m, 2H), 2.18 (t, J = 7.2 Hz, 2H), 2.00-2.07 (m, 1H), 1.82 (s, 3H), 1.51 (s, 3H). 13 C NMR

(100 MHz, CDCl 3) δ 172.2, 168.4, 168.1, 165.8, 148.7, 145.1, 132.6, 130.3, 129.8, 128.1,

126.9, 122.6, 94.6, 85.7, 74.9, 69.3, 66.8, 60.8, 42.95, 41.0, 31.7, 31.5, 28.3, 26.4.

106

(R)-2,2,2-Trichloroethyl 2-(2-((5 S,8 S)-1-(9 H-fluoren-9-yl)-5-isopropyl-3,6,10-trioxo- 8-(( E)-4-(tritylthio)but-1-enyl)-2,7-dioxa-4,11-diazadodecan-12-yl)thiazol-4-yl)-5,5- dimethyl-4,5-dihydrothiazole-4-carboxylate (56).

To a solution of Fmoc-L-valine (0.146 g, 0.431 mmol, 1.98 equiv) in THF (1 mL) at 0 oC

were added Hunig’s base (0.074 g, 0.575 mmol, 2.64 equiv) and 2,4,6-trichlorobenzoyl

chloride (0.125 g, 0.512 mmol, 2.39 equiv). The reaction mixture was stirred at 0 oC for 1

h. When TLC indicated formation of the anhydride, alcohol 55 (0.175 g, 0.218 mmol, 1

equiv) in THF (1 mL) was added to the reaction mixture at 0 oC. It was stirred overnight

at room temperature. The reaction mixture was concentrated in vacuo and flash

chromatography purification on silica gel in ethyl acetate/hexanes (20-60%) yielded the

20 1 acyclic precursor 56 (0.106 g, 43%). [α]D - 7.14 ( c 0.28, CHCl 3). H NMR (600 MHz,

CDCl 3): δ 7.88 (s, 1H), 7.75 (d, J =10.8 Hz, 2H), 7.57 (d, J = 10.8 Hz, 2H), 7.37-7.39 (m,

8H), 7.26-7.32 (m, 8H), 7.20 (t, J = 11.4 Hz, 3H), 6.85 (q, J = 9.0 Hz, 1H), 5.60-5.69 (m,

2H), 5.42 (dd, J = 10.8, 15.2 Hz, 1H), 5.30 (d, J = 12.0 Hz, 1H), 4.95 (s, 1H), 4.92 (d, J =

18.0 Hz, 1H), 4.77 (d, J = 18.6 Hz, 1H), 4.79 (d, J = 8.4 Hz, 1H), 4.31-4.41 (m, 2H), 4.19

(t, J = 10.8 Hz, 1H), 4.06-4.12 (m, 1H), 2.58 (d, J = 9.0 Hz, 2H), 2.06-2.21 (m, 2H), 2.00-

2.08 (m, 1H), 1.82 (s, 3H), 1.51 (s, 3H), 0.90 (d, J = 10.2 Hz, 3H), 0.85 (d, J =10.2 Hz,

13 3H). C NMR (100 MHz, CDCl 3) δ 169.3, 168.8, 168.1, 165.6, 158.2, 156.6, 148.7,

145.0, 144.0, 143.9, 141.52, 134.3, 129.8, 128.1, 128.0, 127.8, 127.4, 126.9, 125.3, 125.3,

122.4, 120.2, 94.6, 85.8, 74.9, 72.5, 67.3, 66.9, 59.7, 47.4, 41.7, 41.4, 31.5, 31.4, 29.9,

28.3, 26.4, 21.3, 19.3, 18.1.

107

(S)-2-(2-(( tert -Butoxycarbonylamino)methyl)thiazol-4-yl)-4-methyl-4,5- dihydrothiazole-4-carboxylic acid (57).

To a well stirred mixture of the nitrile 7 (0.096 g, 0.4 mmol, 1 equiv) and NaHCO 3 (0.232

g, 2.76 mmol, 5.6 equiv) in methanol (5 mL) was added ( s)-α-methylcysteine hydrochloride 14 (0.084 g, 0.491, 1.23 equiv) followed by phosphate buffer pH 5.95 (2.5 mL). The reaction mixture was degassed with nitrogen before stirring it under nitrogen at

70 oC for 1 h. It was acidified with 1 M HCl and extracted with ethyl acetate (15 mL) three times. The combined organic extract was washed with saturated NaCl solution, dried over anhydrous sodium sulphate and concentrated to obtain the carboxylic acid 57

(0.112g) which was used in the next step without further purification.

(S)-Methyl 2-(2-((( S,E)-3-hydroxy-7-(tritylthio)hept-4-enamido)methyl)thiazol-4-yl)- 4-methyl-4,5-dihydrothiazole-4-carboxylate (59).

A solution of carboxylic acid 57 (0.056 g, 0.157 mmol, 1 equiv) in anhydrous methanol

(5 mL) was bubbled with HCl gas for 5 minutes. The reaction mixture was stirred overnight and concentrated in vacuo to give compound 58 which was azeotropped with toluene before taking it to the next step. A mixture of above obtained compound 58 and

DMAP (0.05 g, 0.410 mmol, 2.61 equiv) in dichloromethane (2 mL) was stirred for 5 minutes and was added a solution of aldol product 37 (0.088, 0.157 mmol, 1 equiv) in dichloromethane (1 mL). The reaction mixture was stirred for 1 h, concentrated in vacuo and purified by flash chromatography on silica gel in ethyl acetate/hexanes (20-100%) to

20 afford the alcohol 59 (0.07 g, 67% from nitrile 7 over 3 steps). [ α]D - 0.77 ( c 0.65,

1 CHCl 3). H NMR (600 MHz, CDCl 3): δ 7.91 (s, 1H), 7.37 (d, J = 7.2, Hz, 6H), 7.26 (t, J

= 6.6 Hz, 6H), 7.19 (t, J = 7.2 Hz, 3H), 6.89 (t, J = 6.0 Hz, 1H), 5.52-5.56 (m, 1H), 5.40

108

(dd, J = 6.6, 15.0 Hz, 1H), 4.71 (m, 2H), 4.43 (m, 1H), 3.86 (d, J = 11.4 Hz, 1H), 3.78 (s,

3H), 3.26 (d, J = 11.4 Hz, 1H), 2.44 (dd, J = 3.0, 15.6 Hz, 1H), 2.38 (dd, J = 8.4, 15.0 Hz,

1H), 2.18 (t, 7.8 Hz, 2H), 2.05 (q, J = 7.2 Hz, 2H), 1.62 (s, 3H). 13 C NMR (100 MHz,

CDCl 3): δ 173.8, 172.0, 167.9, 162.9, 148.5, 145.0, 132.4, 130.5, 129.7, 128.1, 126.8,

122.4, 84.7, 69.4, 66.8, 53.2, 42.9, 41.7, 41.0, 31.6, 31.5, 24.2. HRMS-ESI ( m/z ): [M +

+ Na] calcd for C 36 H37 N3O4S3Na, 694.1844; found, 694.1851.

(S)-Methyl 2-(2-((5 S,8 S)-1-(9 H-fluoren-9-yl)-5-isopropyl-3,6,10-trioxo-8-(( E)-4- (tritylthio)but-1-enyl)-2,7-dioxa-4,11-diazadodecan-12-yl)thiazol-4-yl)-4-methyl-4,5- dihydrothiazole-4-carboxylate (60).

To a solution of Fmoc-L-valine (0.071 g, 0.209 mmol, 2 equiv) in THF (1 mL) at 0 oC were added Hunig’s base (0.040 g, 0.31 mmol, 2.98 equiv) and 2,4,6-trichlorobenzoyl chloride (0.062 g, 0.255 mmol, 2.45 equiv). The reaction mixture was stirred at 0 oC for 1 h. When TLC indicated formation of the anhydride, alcohol 59 (0.07 g, 0.104 mmol, 1 equiv) in THF (1 mL) was added to the reaction mixture at 0 oC. It was stirred overnight at room temperature. The reaction mixture was concentrated in vacuo and flash chromatography purification on silica gel in ethyl acetate/hexanes (20-100%) yielded the

20 1 acyclic precursor 60 (0.095 g, 93%). [α]D - 4.18 ( c 2.75, CHCl 3). H NMR (400 MHz,

CDCl 3): δ 7.88 (s, 1H), 7.75 (d, J = 7.6 Hz, 2H), 7.57 (d, J = 7.6 Hz, 2H), 7.38 (d, J = 7.2

Hz, 8H), 7.25-7.30 (m, 8H), 7.20 (t, J = 7.2 Hz, 3H), 6.91 (t, J = 5.6 Hz, 1H), 5.59-5.71

(m, 2H), 5.39-5.46 (m, 1H), 5.30 ( d, J = 8.4 Hz, 1H), 4.69 (d, J = 6.0 Hz, 2H), 4.30-4.40

(m, 3H), 4.19 (q, J = 7.2 Hz, 1H), 4.07 (dd, J = 3.6, 6.0 Hz, 1H), 3.83 (d, J = 11.2 Hz,

1H), 3.77 (s, 3H), 3.24 (d, J = 11.6, 1H), 2.57 (d, J = 5.6 Hz, 2H), 2.14-2.20 (m, 2H),

2.00-2.07 (m, 2H), 1.62 (s, 3H), 0.89 (d, J = 6.8 Hz, 3H), 0.84 (d, J = 6.8 Hz, 3H). 13 C

109

NMR (100 MHz, CDCl 3): δ 173.8, 169.3, 168.6, 163.1, 156.6, 148.3, 144.9, 144.0,

143.9, 143.8, 141.4, 141.4, 134.2, 129.7, 127.9, 127.7, 127.3, 126.8, 125.2, 125.2, 84.6,

72.4, 67.2, 66.8, 59.6, 53.1, 47.3, 47.3, 41.6, 41.6, 41.2, 31.5, 31.3, 31.0, 24.1, 19.2, 18.1.

+ HRMS-ESI ( m/z): [M + Na] calcd for C 36 H37 N3O4S3Na, 1015.3209; found, 1015.3203.

Cyclic core 61.

To a stirred solution of 60 (0.095 mg, 0.096 mmol, 1 equiv) in THF/H 2O (4:1, 3 mL) at 0 oC was added 0.1 M LiOH (0.96 mL, 0.096 mmol, 1.0 equiv) dropwise over a period of

15 minutes. After stirring at 0 oC for 1 h it was acidified with 1 M HCl solution and was extracted with EtOAc three times. The combined organic layer was washed with brine, dried over anhydrous sodium sulphate and concentrated and purified by preparative TLC in ethyl acetate to give the carboxylic acid (0.052 g, 0.053 mmol, 1 equiv) which was carried to the next step. Also recovered starting ester 60 (0.039 g) during purification.

The carboxylic acid was dissolved in dichloromethane (4 mL) and treated with diethylamine (0.213 g, 2.92 mmol, 55.05 equiv). After stirring at room temperature for 3 h it was concentrated to dryness to afford the free amino derivative. After drying azeotropically with toluene it was treated with HATU (0.041 g, 0.108 mmol, 2.04 equiv),

HOAt (0.015 g, 0.110 mmol, 2.08 equiv), dichloromethane (60 mL, ~ 1 mM), and

Hunig’s base (0.030 g, 0.230 mmol, 4.33 equiv) and the mixture was stirred for 30 h at room temperature. The reaction mixture was concentrated to dryness and was purified by flash chromatography on silica gel in ethyl acetate/hexanes (10-60%) to yield the cyclic

20 1 core 61 (0.013 g, 31%). [α]D - 17.08 ( c 0.65, CHCl 3). H NMR (400 MHz, CDCl 3): δ

7.55 (s, 1H), 7.49 (d, J = 10.0 Hz, 1H), 7.34 (d, J = 8.0 Hz, 6H), 7.29 (t, J = 6.8 Hz, 6H),

7.23 (t, J = 7.2 Hz, 3H), 6.72 (t, J = 6.0 Hz, 1H), 5.56-5.57 (m, 1H), 5.14-5.20 (m, 1H), 110

5.03 (dd, J = 6.0, 15.6 Hz, 1H), 4.79 (dd, J = 7.2, 15.6 Hz, 1H), 4.57 (dd, J = 6.8, 14.4

Hz, 1H), 4.17 (d, J = 11.6 Hz, 1H), 3.94 (dd, J = 5.2, 15.6 Hz, 1H), 3.27 (d, J = 11.6 Hz,

1H), 2.64 (dd, J = 4.0, 10.4 Hz, 1H), 2.51 (dd, J = 4.0, 10.8 Hz, 1H), 2.07-2.25 (m, 3H),

1.80 (s, 3H), 1.52-1.58 (m, 1H), 1.00 (d, J = 6.8 Hz, 3H), 0.97 (d, J = 6.8 Hz, 3H). 13 C

NMR (100 MHz, CDCl 3): δ 174.1, 169.3, 167.5, 163.0, 148.6, 144.7, 130.9, 129.8, 129.7,

128.2, 128.1, 127.1, 121.8, 85.2, 71.5, 67.2, 58.3, 42.9, 42.3, 32.7, 31.1, 31.1, 26.6, 19.3,

+ 18.2. HRMS (ESI): m/z : [M + Na] calcd for C 40 H42 N4O4S3Na: 761.2266; found:

761.2266.

Analog T3 (C-7 epimer of largazole).

To a solution of 61 (0.013 g, 0.0176 mmol, 1 equiv) in dichloromethane (2.5 mL) at 0 oC was added triisopropylsilane (0.007 g, 0.044 mmol, 2.5 equiv) followed by trifluoroacetic acid (0.1535 g, 1.35 mmol, 76.5 equiv). After stirring for 3 h at room temperature the reaction mixture was concentrated in vacuo . The crude product was purified by flash chromatography on silica gel in ethyl acetate/hexanes (20%) to first remove impurity followed by methanol: ethyl acetate (1%) to obtain the thiol. To a stirred solution of the thiol in dichloromethane (1 mL) at 0 oC were added Hunig’s base (0.012 g, 0.092 mmol,

5.23 equiv) and octanoyl chloride (0.0114 g, 0.0699 mmol, 3.97 equiv). After stirring for

4 h at room temperature, reaction was quenched with methanol and the mixture was concentrated in vacuo . Purification of the crude product by preparative thin layer chromatography on silica gel in ethyl acetate: hexanes (30-60%) gave analog T3 (0.008

20 1 g, 72%). [α]D - 28.57 ( c 0.175, CHCl 3). H NMR (600 MHz, CDCl 3): δ 7.72 (s, 1H),

7.37 (d, J = 10.2 Hz, 1H), 6.65 (t, J = 6.0Hz, 1H), 5.62 (q, J = 4.2 Hz, 1H), 5.16-5.24 (m,

2H), 4.85 (dd, J = 7.2, 15.6 Hz, 1H), 4.62 (dd, J = 6.6, 10.2 Hz, 1H), 4.56 (dd, J = 4.8, 111

15.6 Hz, 1H), 4.18 (d, J = 11.4 Hz, 1H), 3.31 (d, J = 11.4 Hz, 1H), 2.51-2.65 (m, 4H),

2.51 (t, J = 7.8 Hz, 2H), 2.14-2.20 (m, 1H), 1.96 (q, J = 7.2 Hz, 2H), 1.84 (s, 3H), 1.62-

1.65 (m, 8H), 1.25-1.29 (m, 10 H), 1.03 (d, J = 7.2 Hz, 3H), 1.01 (d, J = 6.6 Hz, 3H),

13 0.88 (t, J = 7.2 Hz, 3H). C NMR (150 MHz, CDCl 3): δ 199.6, 174.2, 169.2, 169.1,

167.7, 163.9, 148.4, 130.6, 127.0, 122.7, 84.9, 71.4, 58.4, 44.4, 42.4, 42.2, 40.3, 32.9,

32.3, 31.8, 29.9, 29.1, 27.8, 26.4, 25.9, 22.8, 19.2, 18.3, 14.3. HRMS-ESI ( m/z ): [M +

+ Na] calcd for C 29 H42 N4O5S3Na, 645.2215; found, 645.2197.

Analog T4A.

To a solution of 41 (0.016 g, 0.0217 mmol, 1 equiv) in dichloromethane (2.5 mL) at 0 oC was added triisopropylsilane (0.007 g, 0.044 mmol, 2.03 equiv) followed by trifluoroacetic acid (0.1535 g, 1.35 mmol, 62.05 equiv). After stirring for 3 h at room temperature the reaction mixture was concentrated in vacuo . The crude product was purified by flash chromatography on silica gel in ethyl acetate/hexanes (20%) to first remove impurity followed by ethyl acetate to obtain the largazole thiol. To a stirred mixture of largazole thiol, 2-bromomethylpyridine HCl (0.012 g, 0.0472 mmol, 2.17 equiv), tetrabutylammonium bromide (0.007 g, 0.0217 mmol, 1 equiv), and cesium carbonate (0.033 g, 0.101 mmol, 4.66 equiv), was added acetonitrile (1.0 mL), and stirred for 20 h at room temperature. The reaction mixture was concentrated in vacuo and purified by preparative thin layer chromatography in methanol: dichloromethane (2.5%)

20 1 to obtain analog T4A (0.002 g, 25%). [α]D + 13.64 ( c 0.11, CHCl 3). H NMR (400

MHz, CDCl3): δ 8.52 (d, J = 4 Hz, 1H), 7.75 (s, 1H), 7.66 (dt, J = 1.6, 7.6 Hz, 1H), 7.36

(d, J = 8.0 Hz, 1H), 7.16 (m, 2H), 6.53 (dd, J = 3.2, 9.6 Hz, 1H), 5.82-5.89 (m, 1H), 5.63-

5.67 (m, 1H), 5.48 (dd, J = 6.8, 15.6 Hz, 1H), 5.28 (dd, J = 9.6, 17.6 Hz, 1H), 4.59 (dd, J 112

= 3.2, 9.2 Hz, 1H), 4.26 (dd, J = 3.2, 17.6 Hz), 4.03 (d, J = 11.2 Hz, 1H), 3.82 (s, 2H),

3.27 (d, J = 11.2 Hz, 1H), 2.85 (dd, J = 10.4, 16.4 Hz, 1H), 2.69 (dd, J = 3.2, 16.4 Hz,

1H), 2.53 (t, J = 7.6 Hz, 2H), 2.30 (q, J = 6.8 Hz, 2H), 2.05-2.12 (m, 1H), 1.85 (s, 3H),

0.69 (d, J = 6.8 Hz, 3H), 0.51 (d, J = 6.8 Hz, 3H). 13 C NMR (150 MHz, CDCl3) δ 173.8,

169.6, 169.1, 168.2, 164.8, 159.0, 149.4, 147.7, 137.0, 133.3, 128.2, 124.3, 123.3, 122.2,

84.7, 72.2, 58.0, 43.5, 41.3, 40.7, 38.3, 34.3, 32.2, 31.1, 24.5, 19.1, 16.9. HRMS-ESI

+ (m/z ): [M + Na] calcd for C 27 H33 N5O4S3Na, 610.1592; found, 610.1581.

Analog T4B.

To a solution of 41 (0.016 g, 0.0217 mmol, 1 equiv) in dichloromethane (2.5 mL) at 0 oC was added triisopropylsilane (0.007 g, 0.044 mmol, 2.03 equiv) followed by trifluoroacetic acid (0.1535 g, 1.35 mmol, 62.05 equiv). After stirring for 3 h at room temperature was added the 2-thiophene methanol (0.0126 g, 0.110 mmol, 5.06 equiv) and the reaction mixture was stirred for 50 minutes at room temperature. It was concentrated and purified by column chromatography on silica gel in ethyl acetate: hexanes (20-70%) which was repurified by prep TLC in methanol: dichloromethane (2.5%) to obtain analog

20 1 T4B (0.0063 g, 49%). [ α]D + 13.92 ( c 0.395, CHCl 3). H NMR (600 MHz, CDCl 3): δ

7.76 (s, 1H), 7.21 (t, J = 3.0 Hz, 1H), 7.17 (d, J = 9.6 Hz, 1H), 6.90-6.92 (m, 2H), 6.49

(dd, J = 3.0, 9.0 Hz, 1H), 5.84-5.89 (m, 1H), 5.64-5.68 (m, 1H), 5.49 (dd, J = 7.2, 15.6

Hz, 1H), 5.27 (dd, J = 9.0, 16.8 Hz, 1H), 4.60 (dd, J = 3.6, 9.6 Hz, 1H), 4.26 (dd, J = 3.0,

17.4 Hz, 1H), 4.03 ( d, J = 11.4 Hz, 1H), 3.90 (s, 3H), 3.27 (d, J = 11.4 Hz, 1H), 2.84 (dd,

J = 10.2, 16.2 Hz, 1H), 2.69 (dd, J = 3.6, 16.8 Hz, 1H), 2.53 (t, J = 7.2 Hz, 2H), 2.30 (q, J

= 7.2 Hz, 2H), 2.08-2.11 (m, 1H), 1.84 (s, 3H), 0.69 (d, J = 7.2 Hz, 3H), 0.50 (d, J = 6.6

13 Hz, 3H). C NMR (150 MHz, CDCl 3) δ 173.8, 169.6, 169.1, 168.1, 164.8, 147.7, 142.2, 113

133.3, 128.2, 126.9, 126.3, 125.1, 124.4, 84.6, 72.2, 58.0, 43.5, 41.3, 40.7, 34.3, 32.1,

+ 31.0, 30.8, 24.4, 19.1, 16.8. HRMS-ESI ( m/z ): [M + Na] calcd for C 26 H32 N4O4S4Na,

615.1204; found, 615.1184. % Purity: 95.7% (HPLC).

Analog T4C.

To a solution of 41 (0.0078 g, 0.0106 mmol, 1 equiv) in dichloromethane (1.25 mL) at 0 oC was added triisopropylsilane (0.00464 g, 0.029 mmol, 2.77 equiv) followed by trifluoroacetic acid (0.092 g, 0.808 mmol, 76.22 equiv). After stirring for 3 h at room temperature was added the 2-hydroxybenzyl alcohol (0.004 g, 0.0323 mmol, 3.04 equiv) and it was stirred for 2 h at room temperature. The reaction mixture was concentrated in vacuo and purified by column chromatography on silica gel in ethyl acetate: hexanes (20-

100%) followed by preparative TLC in ethyl acetate: hexanes (75%) to obtain analog

20 1 T4C (0.0031 g, 49%). [ α]D + 17.86 ( c 0.14, CHCl 3). H NMR (600 MHz, CDCl 3): δ

7.79 (s, 1H), 7.46 (s, 1H), 7.20 (d, J = 9.6 Hz, 1H), 7.14 (dd, J = 1.8, 7.2 Hz, 1H), 7.08

(dt, J = 1.8, 7.8 Hz, 1H), 6.82 (dt, J = 1.2, 7.8 Hz, 1H), 6.73 (dd, J = 1.2, 8.4 Hz), 6.70

(dd, J = 3.6, 8.4 Hz, 1H), 5.82-5.87 (m, 1H), 5.68-5.71 (m, 1H), 5.62 (dd, J = 6.0, 15.6

Hz, 1H), 5.19 (dd, J = 9.0, 17.4 Hz, 1H), 4.60 (dd, J = 3.6, 9.0 Hz, 1H), 4.20 (dd, J = 3.0,

17.4 Hz), 4.06 (d, J = 11.4 Hz, 1H), 3.80 (dd, J = 12.6, 27.6 Hz, 2H), 3.31 (d, J = 11.4

Hz, 1H), 2.88 (dd, J = 9.0, 16.2 Hz, 1H), 2.70 (dd, J = 3.0, 16.2 Hz, 1H), 2.58-2.62 (m,

1H), 2.46-2.51 (m, 1H), 2.28-2.33 (m, 2H), 2.06-2.12 (m, 1H), 1.85 (s, 3H), 0.71 (d, J =

7.2 Hz, 3H), 0.54 (d, J = 6.6 Hz, 3H). 13 C NMR (150 MHz, CDCl3) δ 173.6, 170.0,

168.9, 168.6, 165.8, 155.3, 147.4, 132.4, 130.9, 129.2, 128.9, 124.5, 123.6, 120.5, 116.7,

84.4, 71.7, 58.1, 43.6, 41.3, 40.9, 34.1, 32.2, 32.2, 31.1, 24.3, 19.1, 17.0. HRMS-ESI

+ (m/z ): [M + Na] calcd for C 28 H34 N4O5S3Na, 625.1589; found, 625.1562. 114

Analog T4D.

To a solution of 41 (0.0067 g, 0.0091 mmol, 1 equiv) in dichloromethane (1.25 mL) at 0 oC was added triisopropylsilane (0.004 g, 0.025 mmol, 2.69 equiv) followed by trifluoroacetic acid (0.077 g, 0.675 mmol, 74.22 equiv). After stirring for 3 h at room

temperature the reaction mixture was concentrated in vacuo . The crude product was

purified by flash chromatography on silica gel in ethyl acetate/hexanes (20%) to first

remove impurity followed by ethyl acetate to obtain the largazole thiol. To a stirred

mixture of largazole thiol, N,N-dimethyl-2-chloroacetamide (0.0142 g, 0.116 mmol,

12.77 equiv), tetrabutylammonium bromide (0.005 g, 0.0155 mmol, 1.7 equiv), and

cesium carbonate (0.025 g, 0.0767 mmol, 8.42 equiv), was added acetonitrile (0.4 mL),

and stirred for 20 h at room temperature. The reaction mixture was concentrated in vacuo

and the crude mixture was purified by preparative TLC in methanol:dichloromethane (2.5

%) followed by another preparative TLC in methanol:ethyl acetate (10%, 4 parts) and

1 hexane (1 part) to obtain analog T4D (0.001 g, 19%). H NMR (600 MHz, CDCl 3): δ

7.95 (s, 1H), 7.15 (d, J = 9.6 Hz, 1H), 6.47 (dd, J = 3.0, 9.0 Hz, 1H), 5.85-5.90 (m, 1H),

5.64-5.67 (m, 1H), 5.52 (dd, J = 6.6, 15.6 Hz, 1H), 5.27 (dd, J = 9.0, 17.4 Hz, 1H), 4.58

(dd, J = 3.6, 9.6 Hz, 1H), 4.26 (dd, J = 3.6, 18.0 Hz, 1H), 4.02 (d, J = 11.4 Hz, 1H), 3.26

(d, J = 10.8 Hz, 1H), 3.05 (s, 3H), 2.95 (s, 3H), 2.84 (dd, J = 10.2, 16.2 Hz, 2.66-2.70 (m,

3H), 2.36 (q, J = 7.2 Hz, 2H), 2.07-2.10 (m, 1H), 1.85 (s, 3H),0.68 (d, J = 7.2 Hz, 3H),

0.50 (d, J =7.2 Hz, 3H).

(S)-3-Hydroxy-1-((R)-4-isopropyl-2-thioxothiazolidin-3-yl)pent-4-en-1-one (62).

115

To a stirred solution of acetyl Nagao chiral auxiliary ( 16) (0.625 g, 3.079 mmol, 1 equiv)

o in dichloromethane (25 mL) at 0 C was added TiCl 4 (0.645 g, 3.39 mmol, 1.10 equiv).

After stirring for 5 minutes, the reaction mixture was cooled to –78 oC and Hunig’s base

(0.438 g, 3.39 mmol, 1.10 equiv) was added. The reaction mixture was stirred for 2 h at the same temperature. Acrolein (0.176 g, 3.146 mmol, 1.02 equiv) was added dropwise and the reaction mixture was stirred for 2.5 h at -78 oC. It was removed from cooling bath, treated with water (15 mL), and diluted with dichloromethane (50 mL). The layers were separated and the aqueous layer was extracted with dichloromethane. The combined organic layer was washed with saturated NaCl (40 mL) and dried over Na 2SO 4. The solvent was removed in vacuo and the residue was purified by flash chromatography on silica gel in dichloromethane/hexanes (25-90%) to give the major isomer 62 as a thick yellow oil (0.425 g, 53%), and the diastereomer 63 (0.053 g, 7.0%). Data for major

1 isomer 62 : H NMR (600 MHz, CDCl 3): δ 5.91-5.96 (m, 1H), 5.34 (dt, J = 1.2, 16.2 Hz,

1H), 5.15-5.19 (m, 2H), 4.66-4.68 (m, 1H), 3.67 (dd, J = 3.0, 18.0 Hz, 1H), 3.53 (dd, J =

7.8, 11.4 Hz, 1H), 3.31 (dd, J = 9.0, 17.4 Hz, 1H), 3.04 (dd, J = 1.2, 12.0 Hz, 1H), 2.87

(d, J = 4.8 Hz, 1H), 2.34-2.40 (m, 1H), 1.07 (d, J = 6.6 Hz, 3H), 0.99 (d, J = 7.2 Hz, 3H).

13 C NMR (100 MHz, CDCl 3): δ 203.2, 172.5, 138.9, 115.4, 71.5, 68.9, 45.2, 30.9, 30.8,

19.2, 17.9.

(R)-Methyl 2-(2-((( S)-3-hydroxypent-4-enamido)methyl)thiazol-4-yl)-4-methyl-4,5- dihydrothiazole-4-carboxylate (64).

A solution of carboxylic acid 29 (0.047 g, 0.131 mmol, 1 equiv) in anhydrous methanol

(5 mL) was bubbled with HCl gas for 5 minutes. The reaction mixture was stirred overnight and concentrated in vacuo which was azeotropped with toluene before taking it to the next step. A mixture of above obtained compound and DMAP (0.042 g, 0.344

116

mmol, 2.63 equiv) in dichloromethane (2 mL) was stirred for 5 minutes, and a solution of aldol product 62 (0.035, 0.135 mmol, 1.03 equiv) in dichloromethane (1 mL) was added.

The reaction mixture was stirred for 1 h, concentrated in vacuo and purified by flash chromatography on silica gel in ethyl acetate/hexanes (25-100%) to afford the alcohol 64

20 3a 26.3 1 (0.031 g, 64%). [ α]D - 23.04 ( c 1.15, CHCl 3) (lit. [α] D – 17.3 ( c 1.00, CHCl 3)). H

NMR (600 MHz, CDCl3): δ 7.95 (s, 1H), 6.76 (s, 1H), 5.87-5.92 (m, 1H), 5.33 (d, J =

16.8 Hz, 1H), 5.16 (d, J = 10.2 Hz, 1H), 4.74-4.82 (m, 2H), 4.57 (s, 1H), 3.89 (d, J = 11.4

Hz, 1H), 3.81 (s, 3H), 3.29 (d, J = 11.4 Hz, 1H), 2.55 (dd, J = 3.0, 15.6 Hz, 1H), 2.45 (dd,

13 J = 8.4, 15.0 Hz, 1H), 1.65 (s, 3H). C NMR (100 MHz, CDCl 3): δ 173.8, 172.1, 168.0,

162.9, 148.4, 139.2, 122.6, 115.6, 84.7, 69.6, 53.2, 42.7, 41.7, 40.9, 24.2.

(R)-Methyl 2-(2-((5 S,8 S)-1-(9 H-fluoren-9-yl)-5-isopropyl-3,6,10-trioxo-8-vinyl-2,7- dioxa-4,11-diazadodecan-12-yl)thiazol-4-yl)-4-methyl-4,5-dihydrothiazole-4- carboxylate (65).

To a solution of Fmoc-L-valine (0.110 g, 0.325 mmol, 2.26 equiv) in THF (1 mL) at 0 oC were added Hunig’s base (0.056 g, 0.431 mmol, 2.99 equiv) and 2,4,6-trichlorobenzoyl chloride (0.094 g, 0.384 mmol, 2.67 equiv). The reaction mixture was stirred at 0 oC for 1 h. When TLC indicated formation of the anhydride, alcohol 64 (0.053 g, 0.144 mmol, 1 equiv) in THF (1 mL) was added to the reaction mixture at 0 oC. It was stirred overnight at room temperature. The reaction mixture was concentrated in vacuo and flash chromatography purification on silica gel in ethyl acetate/hexanes (25-100%) yielded the

20 1 acyclic precursor 65 (0.082, 83%). [ α]D - 2.07 ( c 4.1, CHCl 3). H NMR (400 MHz,

CDCl 3): δ 7.88 (s, 1H), 7.73 (d, J = 7.6 Hz, 2H), 7.55 (d, J = 6.8 Hz, 2H), 7.37 (t, J = 8.0

Hz, 2H), 7.28 (t, J = 7.2 Hz, 2H), 7.19 (t, J = 6.0 Hz, 1H), 5.81-5.89 (m, 1H), 5.69 (q, J =

117

6.0 Hz, 1H), 5.45 (d, J = 8.4 Hz, 1H), 5.33 (d, J = 17.2 Hz, 1H), 5.22 (d, J = 10.8 Hz,

1H), 4.64-4.75 (m, 2H), 4.19-4.40 (m, 2H), 4.17 (t, J = 7.2 Hz, 1H), 4.11 (dd, J = 6.0, 8.0

Hz, 1H), 3.82 (d, J = 11.6 Hz, 1H), 3.75 (s, 3H), 3.22 (d, J = 11.6 Hz, 1H), 2.62 (d, J =

6.0 Hz, 2H), 2.03-2.11 (m, 1H), 1.6 (s, 3H), 0.94 (d, J = 6.8 Hz, 3H), 0.89 (d, J = 6.8 Hz,

13 3H). C NMR (100 MHz, CDCl 3): δ 173.7, 171.4, 169.2, 168.6, 162.9, 156.6, 148.3,

143.8, 143.8, 141.4, 134.4, 127.8, 127.2, 125.2, 122.3, 120.1, 118.6, 84.6, 72.5, 67.1,

59.7, 53.0, 47.2, 41.5, 41.2, 30.9, 24.0, 19.1, 18.0, 14.3.

Cyclic core 66.

To a stirred solution of 65 (0.0805 g, 0.117 mmol, 1 equiv) in THF/H 2O (4:1, 4 mL) at 0 oC was added 0.1 M LiOH (1.2 mL, 0.12 mmol, 1.03 equiv) dropwise over a period of 15 minutes. After being stirred at 0 oC for 1 h, it was acidified with 1 M HCl solution and extracted with EtOAc three times. The combined organic layer was washed with brine, dried over anhydrous sodium sulphate, and concentrated to give the carboxylic acid which was carried to the next step. The carboxylic acid was dissolved in dichloromethane

(10 mL) and treated with diethylamine (0.426 g, 5.84 mmol, 49.88 equiv). After stirring at room temperature for 3 h, it was concentrated in vacuo to dryness to afford the free amino derivative. After drying azeotropically with toluene, it was treated with HATU

(0.089 g, 0.234 mmol, 2.00 equiv), HOAt (0.032 g, 0.235 mmol, 2.00 equiv), dichloromethane (90 mL, ~ 1 mM), and Hunig’s base (0.061 g, 0.472 mmol, 4.034 equiv) and the mixture was stirred for 30 h at room temperature. The reaction mixture was concentrated to dryness and was purified by flash chromatography on silica gel in ethyl 118

20 acetate/hexanes (10-70%) to yield the cyclic core 66 (0.018 g, 35%). [ α]D + 36.82 ( c

3a 29.7 1 0.44, CHCl 3) (lit. [α]D + 22.8 ( c 0.33, MeOH)). H NMR (400 MHz, CDCl 3): δ 7.77

(s, 1H), 7.18 (d, J = 9.2 Hz, 1H), 6.47 (dd, J = 2.4, 9.2 Hz, 1H), 5.66-5.71 (m,1H), 5.24-

5.38 (m, 3H), 4.62 (dd, J = 3.2, 9.2 Hz, 1H), 4.26 (dd, J = 3.2, 17.6 Hz, 1H), 4.07 (d, J =

11.2 Hz, 1H), 3.27 (d, J = 11.2 Hz, 1H), 2.86 (dd, J = 10.4, 16.4 Hz, 1H), 2.72 (dd, J =

2.8, 16.4 Hz, 1H), 2.06-2.14 (m, 1H), 1.86 (s, 3H), 0.69 (d, J = 6.8 Hz, 3H), .51 (d, J =

13 6.8 Hz, 3H). C NMR (100 MHz, CDCl 3): δ 173.8, 169.5, 169.1, 168.1, 164.8, 147.6,

134.9, 124.5, 118.1, 84.6, 72.5, 57.9, 43.5, 41.3, 40.3, 34.4, 24.4, 19.1, 16.8.

N-(benzyloxy)acrylamide (67).

To a solution of o-benzyl hydroxylamine HCl (0.176 g, 1.1 mmol, 1.1 equiv) in

dichloromethane (2 mL) was added Hunig’s base (0.284 g, 2.2 mmol, 2.2 equiv). The

reaction mixture was cooled to 0 oC and was added acryloyl chloride (0.905 g, 1 mmol, 1

equiv) at 0 oC and the reaction mixture was stirred at room temperature for 2 h. The

reaction mixture was concentrated in vacuo and purified by flash column chromatography on silica gel in ethyl acetate: hexanes (10-20%) to get compound 67

1 (0.11 g, 62%). H NMR (600 MHz, DMSO-d6): δ 11.3(s, 1H), 7.35-7.39 (m, 5H), 6.16

(dd, J = 1.8, 17.4 Hz, 1H), 6.03 (dd, J = 10.2, 16.8 Hz, 1H), 5.65 (dd, J = 1.8, 10.2 Hz,

13 1H), 4.83 (s, 2H). C NMR (100 MHz, CDCl 3): δ 164.0, 135.3, 129.3, 128.7, 128.6,

127.6, 127.4, 78.3.

Analog T5.

A reaction mixture of cyclic core 66 (0.030 g, .0687 mmol, 1 equiv), compound 67

(0.015 g, 0.0847 mmol, 1.23 equiv), Grubb’s second generation catalyst (0.025 g, 0.0294 119

mmol, 0.428 equiv) and p-methoxyphenol (0.009 g, 0.0726 mmol, 1.06 equiv) in

dichloromethane (1.5 mL) was refluxed at 40 oC for 36 hours. As TLC analysis indicated

formation of a small amount of a new compound, additional Grubb’s catalyst (0.007 g,

0.008 mmol, 0.12 equiv) in toluene (1.2 mL) was added and the mixture was stirred at

110 oC for 7 hours. Following TLC analysis, another portion of compound 67 (0.015 g,

0.0847 mmol, 1.23 equiv) in toluene (0.5 mL) was added and the mixture was stirred at

110 oC for additional 7 hours. The reaction mixture was cooled, treated with DMSO (100

µl) and stirred overnight at room temperature. The reaction mixture was concentrated in

vacuo and purified by repeated flash chromatography on silica gel in ethyl acetate:hexanes (33-100%), and methanol:ethyl acetate (2%), followed by purification by successive preparative TLC in methanol:dichloromethane (4%), and acetone:hexane

(10%) and reverse phase HPLC on C18 column in water: methanol (10-100%) to obtain

1 T5 (0.0005 g, 1.5%). H NMR (400 MHz, CDCl 3): δ7.74 (s, 1H), 7.58 (d, J = 8.8 Hz,

1H), 7.11 (d, J = 6.0 Hz, 1H), 5.53 (q, J = 6.8 Hz, 1H), 5.26 (dd, J = 8.8, 16.8 Hz, 1H),

4.65 (dd, J = 6.6, 8.8 Hz, 1H), 4.25 (dd, J = 3.2, 17.2 Hz, 1H), 4.03 (d, J = 11.6 Hz, 1H),

3.49 (d, J = 4.8 Hz, 2H), 3.31-3.35 (m, 2H), 3.11 (dd, J = 5.2, 16.8 Hz, 1H), 2.05-2.15

(m, 1H), 1.86 (m, 3H), 0.74 (d, J = 2.0 Hz, 3H), 0.73 (d, J = 2.0 Hz, 3H).

Methyl 2-(( tert -butoxycarbonylamino)methyl)thiazole-4-carboxylate (69).

To a solution of carboxylic acid 5 (0.050 g, 0.195 mmol, 1 equiv), EDC.HCl (0.075 g,

0.39 mmol, 2 equiv) in MeOH (2 mL) was added with Hunig’s base (0.104 g, 0.8 mmol,

4 equiv). The reaction mixture was stirred overnight at room temperature and

concentrated in vacuo . The residue was partitioned between water and ethyl acetate. The

aqueous layer was extracted three times with ethyl acetate and the combined organic 120

extract was dried over anhydrous Na 2SO 4, filtered and concentrated in vacuo . The crude

reaction mixture was purified by flash column chromatography on silica gel in ethyl

1 acetate: hexanes (20-50%) to obtain 69 (0.036 g, 68%). H NMR (400 MHz, CDCl 3): δ

8.12 (s, 1H), 5.38 (s, 1H), 4.62 (d, J = 6.0 Hz, 2H), 3.92 (s, 3H), 1.44 (s, 9H). 13 C (100

MHz, CDCl 3): δ 170.4, 161.9, 155.8, 146.6, 128.3, 80.7, 52.7, 42.5, 28.5.

(R,E)-Methyl 2-((3-hydroxy-7-(tritylthio)hept-4-enamido)methyl)thiazole-4- carboxylate (70).

Ester 69 (0.086 g, 0.316 mmol, 1 equiv) was treated with dichloromethane: trifluoroacetic acid (2:1, 1.8 mL) at room temperature. The reaction mixture was stirred for 1 h at room temperature and concentrated in vacuo which was azeotropped with toluene before taking it to the next step. A mixture of above obtained compound and DMAP (0.039 g, 0.320 mmol, 1.01 equiv) in dichloromethane (2 mL) was treated with Hunig’s base (0.082 g,

0.636 mmol, 2.01 equiv) and stirred for 5 minutes. To the reaction mixture was added a solution of aldol diastereomer 38 (0.178, 0.316 mmol, 1 equiv) in dichloromethane (1

mL). The reaction mixture was stirred for 2 h, concentrated in vacuo and purified by flash

chromatography on silica gel in ethyl acetate/hexanes (25-80%) to afford the alcohol 70

20 1 (0.147 g, 81% over two steps from ester 69). [ α]D + 4.78 ( c 3.35, CHCl 3). H NMR (600

MHz, CDCl 3): δ 8.06 (s, 1H), 7.36 (d, J = 7.2 Hz, 6H), 7.25 (t, J = 7.2 Hz, 6H), 7.17 (t, J

= 7.2 Hz, 3H), 5.50-5.54 (m, 1H), 5.38 (dd, J = 6.0, 15.0 Hz, 1H), 4.64-4.73 (m, 2H),

4.41 (br s, 1H), 3.89 (s, 3H), 2.43 (dd, J = 3.0, 15.0, 2.37 (dd, J = 9.0, 15.0 Hz, 1H), 2.16

13 (t, J = 7.8 Hz, 2H), 2.03 (q, J = 7.2 Hz, 2H). C (150 MHz, CDCl 3): δ 172.3, 168.8,

161.8, 146.2, 144.9, 132.4, 130.2, 129.7, 128.7, 128.0, 126.8, 69.2, 66.7, 52.7, 42.9, 41.0,

+ 31.5, 31.4. HRMS-ESI ( m/z ): [M + Na] calcd for C 32 H32 N2O4S2Na, 595.1701; found,

595.1719.

121

Methyl 2-((5 S,8 R)-1-(9 H-fluoren-9-yl)-5-isopropyl-3,6,10-trioxo-8-(( E)-4- (tritylthio)but-1-enyl)-2,7-dioxa-4,11-diazadodecan-12-yl)thiazole-4-carboxylate (71).

To a solution of Fmoc-L-valine (0.018 g, 0.053 mmol, 2.0 equiv) in THF (1 mL) at 0 oC were added Hunig’s base (0.0104 g, 0.08 mmol, 3.07 equiv) and 2,4,6-trichlorobenzoyl chloride (0.0156 g, 0.064 mmol, 2.44 equiv). The reaction mixture was stirred at 0 oC for

1 h. When TLC indicated formation of the anhydride, alcohol 70 (0.015 g, 0.0262 mmol,

1 equiv) in THF (1 mL) was added to the reaction mixture at 0 oC. It was stirred overnight at room temperature. The reaction mixture was concentrated in vacuo and flash chromatography purification on silica gel in ethyl acetate/hexanes (33-800%) yielded partially pure 71. This was taken to the next step without further purification.

Methyl 2-((5 S,8 R)-8-(( E)-4-(acetylthio)but-1-enyl)-1-(9 H-fluoren-9-yl)-5-isopropyl- 3,6,10-trioxo-2,7-dioxa-4,11-diazadodecan-12-yl)thiazole-4-carboxylate (analog T6).

To a solution of 71 (0.035 g, 0.04 mmol, 1 equiv) in dichloromethane (5 mL) at 0 oC was added triisopropylsilane (0.013 g, 0.083 mmol, 2.08 equiv) followed by trifluoroacetic acid (0.307 g, 2.7 mmol, 67.5 equiv). After stirring for 3 h at room temperature, the reaction mixture was concentrated in vacuo . The crude product was purified by flash chromatography on silica gel in ethyl acetate/hexanes (20%) to first remove impurity followed by methanol: ethyl acetate (2%) to obtain the thiol. To a stirred solution of the thiol in dichloromethane (2 mL) at 0 oC were added Hunig’s base (0.0334 g, 0.2588 mmol, 6.47 equiv) and acetyl chloride (0.0132 g, 0.168 mmol, 4.2 equiv). After stirring for 4 h at room temperature, reaction was quenched with methanol and the mixture was concentrated in vacuo . Purification of the crude product by flash column chromatography

122

on silica gel in ethyl acetate: hexanes (40-100%) followed by preparative thin layer

chromatography purification in ethyl acetate: hexanes (75%) gave analog T6 (0.006 g,

20 1 22%). [ α]D + 10.71 ( c 0.28, CHCl 3). H NMR (600 MHz, CDCl 3): δ 8.11 (s, 1H), 7.74

(d, J = 7.8 Hz, 2H), 7.57 (d, J = 7.2 Hz, 2H), 7.38 (t, J = 7.8 Hz, 2H), 7.30 (dt, J = 4.2,

7.2 Hz, 2H), 6.61 (t, J = 6.0 Hz, 1H), 5.73-5.76 (m, 1H), 5.66 (dd, J = 4.8, 12.0 Hz, 1H),

5.51 (dd, J = 6.6, 15.6 Hz, 1H), 3.36 (d, J = 9.0, 1H), 4.76 (dd, J = 6.0, 16.2 Hz, 1H),

4.69 (dd, J = 6.0, 16.2 Hz, 1H), 4.39 (dd, J = 7.2, 10.2 Hz, 1H), 4.33 (dd, J = 6.6, 10.2

Hz, 1H), 4.19-4.23 (m, 2H), 2.86 (t, J = 7.2 Hz, 2H), 2.61 (dd, J = 7.8, 14.4 Hz, 1H), 2.55

(dd, J = 4.8, 15.0 Hz, 1H), 2.25-2.28 (m, 5H), 2.23-2.11 (m, 2H), 1.42 (d, J = 6.0 Hz,

13 1H), 0.92 (d, J = 6.6 Hz, 3H), 0.84 (d, J = 6.6 Hz, 3H). C (150 MHz, CDCl 3): δ 195.9,

171.1, 171.0, 169.2, 168.1, 161.8, 156.6, 156.6, 146.6, 144.0, 143.9, 141.5, 133.0, 128.6,

128.4, 127.9, 127.2, 125.3, 120.2, 72.4, 67.2, 59.3, 52.7, 47.3, 41.6, 41.2, 32.3, 31.2, 30.8,

+ 28.3, 19.2, 17.7, 17.6. HRMS-ESI ( m/z ): [M + Na] calcd for C 35 H39 N3O8S2Na,

716.2076; found, 716.2056.

(S, E)-Methyl 2-((3-hydroxy-7-(tritylthio)hept-4enamido)methyl)thiazole-4- carboxylate (72).

Compound 69 (0.036 g, 0.132 mmol, 1 equiv) was treated with TFA: DCM (1:5) (2 mL).

The reaction mixture was stirred for 2 h at room temperature. It was concentrated in

vacuo and azeotropped many times with toluene. The residue was treated with Hunig’s

base in MeOH, stirred for 1 h at room temperature and concentrated in vacuo . To a

mixture of the residue and DMAP (0.042 g, 0.344 mmol, 2.6 equiv) in dichloromethane

(1mL), was added aldol product 37 (0.074 g, 0.131 mmol, 1 equiv) in dichloromethane (1

mL). The reaction mixture was stirred for 2 hours at room temperature. The reaction

123

mixture was concentrated in vacuo and purified by flash column chromatography on

20 silica gel in ethyl acetate: hexanes (33-100%) to get 72 (0.07 g, 92%). [ α]D - 4.85 ( c

1 0.7, CHCl 3). H NMR (600 MHz, CDCl 3): δ 8.07 (s, 1H), 7.37 (dd, J = 1.8, 9.0 Hz, 6H),

7.25 (t, J = 9.0 Hz, 6H), 7.18 (t, J = 7.2 Hz, 3H), 5.50-5.54 (m, 1H), 5.39 (dd, J = 6.6,

15.6 Hz, 1H), 4.64-4.73 (m, 2H), 4.42 (s, 1H), 3.89 (s, 3H), 3.54 (s, 1H), 2.43 (dd, J =

3.0, 15.0 Hz, 1H), 2.37 (dd, J = 9.0, 15.6 Hz, 1H), 2.17 (t, J = 7.8 Hz, 2H), 2.00-2.05 (m,

13 2H). C (100 MHz, CDCl 3): δ 172.2, 168.7, 161.7, 146.2, 144.9, 132.4, 130.3, 129.7,

128.6, 128.0, 126.8, 69.2, 66.7, 52.7, 42.9, 41.0, 31.4.

Methyl 2-((5 S,8 S)-1-(9 H-fluoren-9-yl)-5-isopropyl-3,6,10-trioxo-8-(( E)-4- (tritylthio)but-1-enyl)-2,7-dioxa-4,11-diazadodecan-12-yl)thiazole-4-carboxylate (73).

To a solution of Fmoc-L-valine (0.107 g, 0.316 mmol, 1 equiv) in THF (1 mL) at 0 oC were added Hunig’s base (0.059 g, 0.46 mmol, 1.46 equiv) and 2,4,6-trichlorobenzoyl chloride (0.094 g, 0.38 mmol, 1.21 equiv). The reaction mixture was stirred at 0 oC for 1 h. When TLC indicated formation of the anhydride, alcohol 72 (0.09 g, 0.157 mmol, 0.5 equiv) in THF (1 mL) was added to the reaction mixture at 0 oC. It was stirred overnight at room temperature. The reaction mixture was concentrated in vacuo and flash chromatography purification of the residue on silica gel in ethyl acetate/hexanes (20-

20 1 80%) yielded the acyclic precursor 73 (0.13 g, 92%). [ α]D - 8.74 ( c 1.75, CHCl 3). H

NMR (400 MHz, CDCl 3): δ = 8.02 (s, 1H), 7.75 (d, J = 7.6 Hz, 2H), 7-37-7.40 (m, 8H),

7.25-7.31 (m, 8H), 7.20 (t, J = 7.2 Hz, 3H), 6.91 (t, J = 6.0 Hz, 1H), 5.6-5.7 (m, 2H), 5.41

(dd, J = 7.6, 15.6 Hz, 1 H), 5.31 (d, J = 8.4 Hz, 1H), 4.64-4.77 (m, 2H), 4.28-4.39 (m,

2H), 4.17 (t, J = 6.8 Hz, 1H), 4.06 (t, J = 6.8 Hz, 1H), 3.89 (s, 3H), 2.58 (d, J = 5.6 Hz,

124

2H), 2.13-2.19 (m, 2H), 1.99-2.07 (m, 3H), 0.89 (d, J = 6.8 Hz, 3H), 0.84 (d, J = 6.8 Hz,

13 3H). C (100 MHz, CDCl 3): δ 171.4, 169.7, 169.2, 161.8, 156.6, 146.4, 145.0, 143.9,

143.9, 141.5, 141.4, 134.2, 129.7, 128.6, 128.1, 127.9, 127.6, 127.3, 127.3, 126.8, 125.2,

125.2, 120.2, 120.2, 72.4, 67.3, 66.8, 59.6, 526, 47.3, 41.5, 41.4, 31.5, 31.3, 31.0, 19.2,

18.1.

125

Synthesis of cyclic core 74.

To a stirred solution of 73 (0.130 mg, 0.153 mmol, 1 equiv) in THF/H 2O (4:1, 5 mL) at 0 oC was added 0.1 M LiOH (1.6 mL, 0.16 mmol, 1.046 equiv) dropwise over a period of

15 minutes. After stirring at 0 oC for 4.5 h, the reaction mixture was acidified with 1 M

HCl solution and extracted with EtOAc three times. The combined organic layer was washed with brine, dried over anhydrous sodium sulphate, concentrated and purified by preparative TLC in ethyl acetate to give the carboxylic acid.

The carboxylic acid (0.097g, 0.118 mmol, 1 equiv) was dissolved in dichloromethane (10 mL) and treated with diethylamine (0.831 g, 11.38 mmol, 96.44 equiv). After stirring at room temperature for 3 h, it was concentrated in vacuo to dryness to afford the free amino derivative.

After drying azeotropically with toluene it was treated with HATU (0.084 g, 0.221 mmol,

2.0 equiv), HOAt (0.03 g, 0.221 mmol, 2.0 equiv), dichloromethane (110 mL, ~ 1 mM), and Hunig’s base (0.067 g, 0.517 mmol, 4.38 equiv) and the mixture was stirred for 30 h at room temperature. The reaction mixture was concentrated in vacuo to dryness and was purified by flash chromatography on silica gel in ethyl acetate/hexanes (10-100%) to yield the partially purified cyclic core 74 which was repurified by preparative TLC in ethyl acetate to obtain still partially pure cyclic core 74 (~ 0.012 g). This partially purified cyclic core was used in the next step without further purification.

126

Attempt to synthesize analog T7.

To a solution of 74 (0.012 g, 0.0188 mmol, 1 equiv) in dichloromethane (2.5 mL) at 0 oC was added triisopropylsilane (0.013 g, 0.083 mmol, 1.85 equiv) followed by trifluoroacetic acid (0.153 g, 1.35 mmol, 71.8 equiv). After stirring for 3 h at room temperature the reaction mixture was concentrated in vacuo . The crude product was

purified by flash chromatography on silica gel in ethyl acetate/hexanes (20%) to first

remove impurity followed by ethyl acetate and methanol: ethyl acetate (20%) to obtain

the thiol. To a stirred solution of thiol in dichloromethane (1 mL) at 0 oC were added

Hunig’s base (0.012 g, 0. 08 mmol, 4.28 equiv) and octanoyl chloride (0.0095 g, 0.058 mmol, 3.1 equiv). After stirring for 2.5 h at room temperature, reaction mixture was quenched with methanol and the mixture was concentrated in vacuo . Purification of the crude product by flash column chromatography on silica gel in ethyl acetate: hexanes

(10-100%) did not yield product.

(S,E)-2-(Trimethylsilyl)ethyl 3-hydroxy-7-(tritylthio)hept-4-enoate (75).

To a mixture of compound 37 (0.163 g, 0.29 mmol, 1 equiv) and DMAP (0.004 g, 0.033 mmol, 0.1 equiv) in dichloromethane (2 mL) was added 2-(trimethylsilyl)ethanol (0.043 g, 0.35 mmol, 1.2 equiv) and the mixture was stirred overnight at room temperature. It was concentrated in vacuo and purified by flash chromatography on silica gel in ethyl

20 acetate/hexanes (3-10%) to afford the alcohol 75 (0.05 g, 33%). [α]D - 1.27 ( c 0.315,

3b 24 1 CHCl 3) (lit. [α]D - 1.1 ( c = 2, CHCl3)). H NMR (600 MHz, CDCl 3): δ 7.39 (d, J = 7.2

Hz, 6H), 7.26 (t, J = 7.8 Hz, 6H), 7.19 (dt, J = 1.2, 7.2 Hz, 3H), 5.54-5.58 (m, 1H), 5.40

(dd, J = 6.6, 15.6 Hz, 1H), 4.42 (s, 1H), 4.17 (t, J = 9.0 Hz, 2H), 2.88 (s, 1H), 2.40-2.49

(m, 1H), 2.19 (t, J = 7.2 Hz, 2H), 2.03-2.08 (m, 2H), 0.97 (t, J = 9.0 Hz, 2H), 0.02 (s, 127

13 9H). C (100 MHz, CDCl 3): δ 172.7, 145.0, 132.1, 130.3, 129.7, 128.0, 126.8, 68.8,

63.3, 41.7, 31.6, 31.5, 17.5, -1.3.

(S,E)-2-(Trimethylsilyl)ethyl 3-(( S)-2-(((9 H-fluoren-9-yl)methoxy)carbonylamino)-3- methylbutanoyloxy)-7-(tritlthio)hept-4-enoate (76).

To a solution of Fmoc-L-valine (0.05 g, 0.147mmol, 1 equiv) in THF (1 mL) at 0 oC were added Hunig’s base (0.026 g, 0.196 mmol, 1.33 equiv) and 2,4,6-trichlorobenzoyl chloride (0.041 g, 0.166 mmol, 1.13 equiv). The reaction mixture was stirred at 0 oC for 1 h. When TLC indicated formation of the anhydride, alcohol 75 (0.05 g, 0.096 mmol, 0.66 equiv) in THF (1 mL) was added to the reaction mixture at 0 oC. It was stirred overnight at room temperature. The reaction mixture was concentrated in vacuo and flash chromatography purification on silica gel in ethyl acetate/hexanes (5-10%) yielded the

20 1 compound 76 (0.077 g, 99%). [α]D - 14.0 ( c 0.5, CHCl 3). H NMR (400 MHz, CDCl 3):

δ 7.78 (d, J = 7.6 Hz, 2H), 7.61 (dd, J = 3.0, 6.8 Hz, 2H), 7.40-7.42 (m, 8H), 7.26-7.35

(m, 8H), 7.21 (t, J = 7.2 Hz, 3H), 5.62-5.72 (m, 2H), 5.38 (dd, J = 7.6, 15.2 Hz, 1H), 5.31

(d, J = 8.8 Hz, 1H), 4.34-4.44 (m, 2H), 4.29 (dd, J = 4.4, 9.2 Hz, 1H), 4.26 (t, J = 7.2 Hz,

1H), 4.16 (t, J = 8.8, 2H), 2.69 (dd, J = 8.0, 15.6 Hz, 1H), 2.56 (dd, J = 5.6, 16.0 Hz, 1H),

2.11-2.21 (m, 3H), 2.05 (q, J = 6.8 Hz, 2H), 0.89-0.99 (m, 5H), 0.81 (d, J = 7.2 Hz, 3H),

13 0.03 (s, 9H). C (100 MHz, CDCl 3): δ = 171.1, 169.8, 156.3, 145.0, 144.1, 144.0, 141.5,

134.2, 129.7, 128.0, 127.9, 127.2, 126.8, 125.3, 120.2, 120.1, 72.0, 67.2, 66.8, 63.3, 58.9,

47.4, 39.9, 31.6, 31.5, 31.3, 19.2, 17.5, 17.5, -1.3.

128

(S,E)-2-(trimethylsilyl)ethyl 3-(( S)-2-(2-(( tert -butoxycarbonylamino)methyl)thiazole- 4-carboxamido)-3-methylbutanoyloxy)-7-tritylthio)hept-4-enoate (77)

The compound 76 (0.078g, 0.098 mmol, 1 equiv) was dissolved in dichloromethane (4.5 mL) and treated with diethylamine (0.355 g, 4.86 mmol, 49.6 equiv). After stirring at room temperature overnight, it was concentrated to dryness to afford the free amino derivative.

The free amine derivative along with HATU (0.074 g, 0.195 mmol, 2 equiv), HOAt

(0.027 g, 0.195 mmol, 2 equiv), compound 5 (0.038 g, 0.147 mmol, 1.52 equiv) were dissolved in dichloromethane (4 mL). To the above reaction mixture was added Hunig’s base (0.051 g, 0.39 mmol, 4 equiv) and the reaction mixture was stirred overnight at room temperature. It was concentrated in vacuo and purified by flash column chromatography on silica gel in ethyl acetate: hexanes (10-20%) to get 77 (0.064 g,

20 1 77%). [ α]D - 1.5 ( c 0.4, CHCl 3). H NMR (600 MHz, CDCl 3): δ 8.00 (s, 1H), 7.72 (d, J

= 9.0 Hz, 1H), 7.39 (dd, J = 1.8, 7.2 Hz, 6H), 7.28 (dt, J = 1.8, 7.2 Hz, 6H), 7.21 (dt, J =

1.2, 7.2 Hz, 3H), 5.67-5.71 (m, 1H), 5.63 (dd, J = 5.4, 7.8 Hz, 1H), 5.37 ( dd, J = 7.8,

15.6 Hz, 1H), 4.67 (dd, J = 4.8, 9.0 Hz, 1H), 4.54-5.64 (m, 2H), 4.13 (m, 2H), 2.68 (dd, J

= 7.8, 15.6 Hz, 1H), 2.54 (dd, J = 5.4, 15.6 Hz, 1H), 2.20-2.23 (m, 1H), 2.14-2.18 (m,

2H), 2.04 (q, J = 7.2 Hz, 2H), 1.47 (s, 9H), 0.94 (d, J = 7.2 Hz, 3H), 0.87 (d, J = 7.2 Hz,

13 3H), 0.00 (s, 9H). C (100 MHz, CDCl 3): δ = 170.8, 169.8, 169.4, 160.9, 155.8, 149.6,

145.0, 134.2, 129.7, 128.0, 127.9, 126.8, 124.1, 80.6, 72.0, 66.8, 63.3, 56.9, 42.5, 39.9,

31.8, 31.5, 31.2, 28.5, 19.3, 17.8, 17.4, -1.3.

129

Cyclic core 74.

To a solution of compound 77 (0.06 g, 0.07 mmol, 1 equiv) in dichloromethane was

added trifluoroacetic acid (1.075 g, 9.42 mmol, 134.6 equiv) very slowly. The reaction

mixture was stirred overnight at room temperature and concentrated in vacuo .

After drying azeotropically with toluene the residue was treated with HATU (0.054 g,

0.142 mmol, 2.0 equiv), HOAt (0.02 g, 0.147 mmol, 2.0 equiv), dichloromethane (70 mL,

~ 1 mM), and Hunig’s base (0.060 g, 0.46 mmol, 6.57 equiv) and the mixture was stirred

for 30 h at room temperature. The reaction mixture was concentrated in vacuo to dryness and was purified by flash chromatography on silica gel in ethyl acetate/hexanes (10-

100%) to yield the partially pure cyclic core 74 (~ 0.002 g).

130

3.2 Experimental Biology

Cytoproliferation assay:

Cell proliferation was measured using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-

carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) reduction assay with the CellTiter 96 One solution MTS assay as described by the manufacturer (Promega,

Madison, WI). Briefly, cells (3x10 4 cells/well) were seeded in quadruplicate in 96- well plates and allowed to attach overnight. Media was replaced with 100 uL of fresh medium containing the appropriate concentration of compounds T1B , T3 , T4A , T4B , T4C , or largazole. Following incubation at 37 °C, 5% CO , 20 µL of CellTiter 96 One Solution 2 was added per well and incubated 1.5 h at 37 °C and absorbance measured at 490 nm.

Evaluation of global histone acetylation levels:

HCT116 cells were treated for 24 hours with 10 nM, 100 nM, or 1 uM or DMSO. Cell lysates were extracted using RIPA buffer and equal amounts (30 ug/Lane) were loaded onto an SDS-PAGE gel. Standard Western blotting protocols were used using the

Invitrogen NuPAGE western blotting system. Primary antibodies used were AcH3

(Millipore), α-tubulin (Sigma), and β-actin (Sigma). Dye-conjugated secondary antibodies from Li-Cor Biosciences were used for detection and scanned using the

Odyssey Infrared Detection System (LI-COR Biosciences, Lincoln, NE).

131

References

1. (a) Cragg, G. M.; Grothaus, P. G.; Newman, D. J., Impact of natural products on

developing new anti-cancer agents. Chem Rev 2009, 109 (7), 3012-3043; (b) Sashidhara,

K. V.; White, K. N.; Crews, P., A selective account of effective paradigms and significant outcomes in the discovery of inspirational marine natural products. J Nat Prod 2009, 72

(3), 588-603; (c) Newman, D. J.; Cragg, G. M., Natural products as sources of new drugs over the last 25 years. J Nat Prod 2007, 70 (3), 461-477.

2. Taori, K.; Paul, V. J.; Luesch, H., Structure and Activity of Largazole, a Potent

Antiproliferative Agent from the Floridian Marine Cyanobacterium Symploca sp. J. Am.

Chem. Soc. 2008, 130 (6), 1806-1807.

3. (a) Ying, Y.; Taori, K.; Kim, H.; Hong, J.; Luesch, H., Total Synthesis and

Molecular Target of Largazole, a Histone Deacetylase Inhibitor. J. Am. Chem. Soc. 2008,

130 (26), 8455-8459; (b) Bowers, A.; West, N.; Taunton, J.; Schreiber, S. L.; Bradner, J.

E.; Williams, R. M., Total synthesis and biological mode of action of largazole: A potent

Class I histone deacetylase inhibitor. J. Am. Chem. Soc. 2008, 130 (33), 11219-11222.

4. Fouladi, M., Histone deacetylase inhibitors in cancer therapy. Cancer Invest 2006,

24 (5), 521-527.

5. Masetti, R.; Serravalle, S.; Biagi, C.; Pession, A., The role of HDACs inhibitors in childhood and adolescence acute leukemias. J Biomed Biotechnol 2011, 2011 , 148046.

132

6. Conley Barbara, A.; Wright John, J.; Kummar, S., Targeting epigenetic

abnormalities with histone deacetylase inhibitors. Cancer 2006, 107 (4), 832-840.

7. (a) Minucci, S.; Pelicci, P. G., Histone deacetylase inhibitors and the promise of

epigenetic (and more) treatments for cancer. Nat Rev Cancer 2006, 6 (1), 38-51; (b)

Bolden, J. E.; Peart, M. J.; Johnstone, R. W., Anticancer activities of histone deacetylase inhibitors. Nat Rev Drug Discov 2006, 5 (9), 769-784; (c) Smith, K. T.; Workman, J. L.,

Histone deacetylase inhibitors: anticancer compounds. Int J Biochem Cell Biol 2009, 41

(1), 21-25; (d) Mai, A.; Altucci, L., Epi-drugs to fight cancer: from chemistry to cancer

treatment, the road ahead. Int J Biochem Cell Biol 2009, 41 (1), 199-213; (e) Bertrand, P.,

Inside HDAC with HDAC inhibitors. Eur J Med Chem 2010, 45 (6), 2095-2116; (f)

Zhang, L.; Fang, H.; Xu, W., Strategies in developing promising histone deacetylase inhibitors. Med Res Rev 2010, 30 (4), 585-602; (g) Baylin, S. B.; Ohm, J. E., Epigenetic gene silencing in cancer - a mechanism for early oncogenic pathway addiction? Nat Rev

Cancer 2006, 6 (2), 107-116.

8. (a) Cloos, P. A. C.; Christensen, J.; Agger, K.; Helin, K., Erasing the methyl mark: histone demethylases at the center of cellular differentiation and disease. Genes &

Development 2008, 22 (9), 1115-1140; (b) Biel, M.; Wascholowski, V.; Giannis, A.,

Epigenetics--an epicenter of gene regulation: histones and histone-modifying enzymes.

Angew Chem Int Ed Engl 2005, 44 (21), 3186-3216.

9. (a) Mellert, H. S.; McMahon, S. B., Biochemical pathways that regulate acetyltransferase and deacetylase activity in mammalian cells. Trends Biochem Sci 2009,

34 (11), 571-578; (b) Hodawadekar, S. C.; Marmorstein, R., Chemistry of acetyl transfer

133

by histone modifying enzymes: structure, mechanism and implications for effector design. Oncogene 2007, 26 (37), 5528-5540.

10. Balasubramanian, S.; Verner, E.; Buggy, J. J., Isoform-specific histone deacetylase inhibitors: The next step? Cancer Lett. (Shannon, Irel.) 2009, 280 (2), 211-

221.

11. Bieliauskas, A. V.; Pflum, M. K. H., Isoform-selective histone deacetylase inhibitors. Chem. Soc. Rev. 2008, 37 (7), 1402-1413.

12. Montero, A.; Beierle, J. M.; Olsen, C. A.; Ghadiri, M. R., Design, synthesis, biological evaluation, and structural characterization of potent histone deacetylase inhibitors based on cyclic alpha /beta -tetrapeptide architectures. J. Am. Chem. Soc. 2009,

131 (8), 3033-3041.

13. Thaler, F.; Minucci, S., Next generation histone deacetylase inhibitors: the answer to the search for optimized epigenetic therapies? Expert Opin. Drug Discovery 2011, 6

(4), 393-404.

14. Hu, E.; Dul, E.; Sung, C. M.; Chen, Z.; Kirkpatrick, R.; Zhang, G. F.; Johanson,

K.; Liu, R.; Lago, A.; Hofmann, G.; Macarron, R.; de los Frailes, M.; Perez, P.; Krawiec,

J.; Winkler, J.; Jaye, M., Identification of novel isoform-selective inhibitors within class I histone deacetylases. J Pharmacol Exp Ther 2003, 307 (2), 720-728.

15. Bottomley, M. J.; Lo Surdo, P.; Di Giovine, P.; Cirillo, A.; Scarpelli, R.; Ferrigno,

F.; Jones, P.; Neddermann, P.; De Francesco, R.; Steinkuehler, C.; Gallinari, P.; Carfi, A.,

Structural and Functional Analysis of the Human HDAC4 Catalytic Domain Reveals a

Regulatory Structural Zinc-binding Domain. J. Biol. Chem. 2008, 283 (39), 26694-

26704.

134

16. de Ruijter, A. J.; van Gennip, A. H.; Caron, H. N.; Kemp, S.; van Kuilenburg, A.

B., Histone deacetylases (HDACs): characterization of the classical HDAC family.

Biochem J 2003, 370 (Pt 3), 737-749.

17. Butler, K. V.; Kozikowski, A. P., Chemical origins of isoform selectivity in

histone deacetylase inhibitors. Curr. Pharm. Des. 2008, 14 (6), 505-528.

18. (a) Hideshima, T.; Bradner, J. E.; Wong, J.; Chauhan, D.; Richardson, P.;

Schreiber, S. L.; Anderson, K. C., Small-molecule inhibition of proteasome and aggresome function induces synergistic antitumor activity in multiple myeloma. Proc

Natl Acad Sci U S A 2005, 102 (24), 8567-8572; (b) Suzuki, T.; Kouketsu, A.; Itoh, Y.;

Hisakawa, S.; Maeda, S.; Yoshida, M.; Nakagawa, H.; Miyata, N., Highly Potent and

Selective Histone Deacetylase 6 Inhibitors Designed Based on a Small-Molecular

Substrate. J. Med. Chem. 2006, 49 (16), 4809-4812.

19. (a) Langley, B.; Gensert, J. M.; Beal, M. F.; Ratan, R. R., Remodeling chromatin and stress resistance in the central nervous system: histone deacetylase inhibitors as novel and broadly effective neuroprotective agents. Curr Drug Targets CNS Neurol Disord

2005, 4 (1), 41-50; (b) Hoshino, M.; Tagawa, K.; Okuda, T.; Murata, M.; Oyanagi, K.;

Arai, N.; Mizutani, T.; Kanazawa, I.; Wanker, E. E.; Okazawa, H., Histone deacetylase activity is retained in primary neurons expressing mutant huntingtin protein. J

Neurochem 2003, 87 (1), 257-267; (c) Blanchard, F.; Chipoy, C., Histone deacetylase inhibitors: new drugs for the treatment of inflammatory diseases? Drug Discov Today

2005, 10 (3), 197-204; (d) Dompierre, J. P.; Godin, J. D.; Charrin, B. C.; Cordelieres, F.

P.; King, S. J.; Humbert, S.; Saudou, F., Histone deacetylase 6 inhibition compensates for

135

the transport deficit in Huntington's disease by increasing tubulin acetylation. J Neurosci

2007, 27 (13), 3571-3583.

20. Dokmanovic, M.; Clarke, C.; Marks, P. A., Histone Deacetylase Inhibitors:

Overview and Perspectives. Mol. Cancer Res. 2007, 5 (10), 981-989.

21. Miller, T. A.; Witter, D. J.; Belvedere, S., Histone deacetylase inhibitors. J. Med.

Chem. 2003, 46 (24), 5097-5116.

22. Newkirk, T. L.; Bowers, A. A.; Williams, R. M., Discovery, biological activity, synthesis and potential therapeutic utility of naturally occurring histone deacetylase inhibitors. Nat. Prod. Rep. 2009, 26 (10), 1293-1320.

23. Carafa, V.; Nebbioso, A.; Altucci, L., Histone deacetylase inhibitors: recent insights from basic to clinical knowledge & patenting of anti-cancer actions. Recent Pat

Anticancer Drug Discov 2011, 6 (1), 131-145.

24. Furumai, R.; Matsuyama, A.; Kobashi, N.; Lee, K. H.; Nishiyama, M.; Nakajima,

H.; Tanaka, A.; Komatsu, Y.; Nishino, N.; Yoshida, M.; Horinouchi, S., FK228

(depsipeptide) as a natural prodrug that inhibits class I histone deacetylases. Cancer Res

2002, 62 (17), 4916-4921.

25. (a) Nasveschuk, C. G.; Ungermannova, D.; Liu, X.; Phillips, A. J., A Concise

Total Synthesis of Largazole, Solution Structure, and Some Preliminary Structure

Activity Relationships. Org. Lett. 2008, 10 (16), 3595-3598; (b) Ghosh, A. K.; Kulkarni,

S., Enantioselective Total Synthesis of (+)-Largazole, a Potent Inhibitor of Histone

Deacetylase. Org. Lett. 2008, 10 (17), 3907-3909; (c) Seiser, T.; Kamena, F.; Cramer, N.,

Synthesis and biological activity of largazole and derivatives. Angew. Chem., Int. Ed.

2008, 47 (34), 6483-6485; (d) Ying, Y.; Liu, Y.; Byeon, S. R.; Kim, H.; Luesch, H.;

136

Hong, J., Synthesis and Activity of Largazole Analogues with Linker and Macrocycle

Modification. Org. Lett. 2008, 10 (18), 4021-4024; (e) Ren, Q.; Dai, L.; Zhang, H.; Tan,

W.; Xu, Z.; Ye, T., Total synthesis of largazole. Synlett 2008, (15), 2379-2383; (f)

Numajiri, Y.; Takahashi, T.; Takagi, M.; Shin-ya, K.; Doi, T., Total synthesis of

largazole and its biological evaluation. Pept. Sci. 2009, 45th , 41-44; (g) Bowers, A. A.;

West, N.; Newkirk, T. L.; Troutman-Youngman, A. E.; Schreiber, S. L.; Wiest, O.;

Bradner, J. E.; Williams, R. M., Synthesis and Histone Deacetylase Inhibitory Activity of

Largazole Analogs: Alteration of the Zinc-Binding Domain and Macrocyclic Scaffold.

Org. Lett. 2009, 11 (6), 1301-1304; (h) Bowers, A. A.; Greshock, T. J.; West, N.; Estiu,

G.; Schreiber, S. L.; Wiest, O.; Williams, R. M.; Bradner, J. E., Synthesis and

Conformation-Activity Relationships of the Peptide Isosteres of FK228 and Largazole. J.

Am. Chem. Soc. 2009, 131 (8), 2900-2905; (i) Chen, F.; Gao, A.-H.; Li, J.; Nan, F.-J.,

Synthesis and Biological Evaluation of C7-Demethyl Largazole Analogues.

ChemMedChem 2009, 4 (8), 1269-1272; (j) Wang, B.; Forsyth, C. J., Total synthesis of largazole - devolution of a novel synthetic strategy. Synthesis 2009, (17), 2873-2880; (k)

Souto, J. A.; Vaz, E.; Lepore, I.; Poppler, A.-C.; Franci, G.; Alvarez, R.; Altucci, L.; de

Lera, A. R., Synthesis and biological characterization of the histone deacetylase inhibitor largazole and C7-modified analogues. J. Med. Chem. 2010, 53 (12), 4654-4667; (l) Zeng,

X.; Yin, B.; Hu, Z.; Liao, C.; Liu, J.; Li, S.; Li, Z.; Nicklaus, M. C.; Zhou, G.; Jiang, S.,

Total synthesis and biological evaluation of Largazole and derivatives with promising selectivity for cancers cells. Org. Lett. 12 (6), 1368-1371.

26. (a) Heidebrecht, R. W., Jr.; Chenard, M.; Close, J.; Dahlberg, W. K.; Fleming, J.;

Grimm, J. B.; Hamill, J. E.; Harsch, A.; Haines, B. B.; Hughes, B.; Kral, A. M.;

137

Middleton, R. E.; Mushti, C.; Ozerova, N.; Szewczak, A. A.; Wang, H.; Wilson, K.;

Witter, D. J.; Secrist, J. P.; Miller, T. A., Exploring the pharmacokinetic properties of

phosphorus-containing selective HDAC 1 and 2 inhibitors (SHI-1:2). Bioorg Med Chem

Lett 2009, 19 (7), 2053-2058; (b) Kattar, S. D.; Surdi, L. M.; Zabierek, A.; Methot, J. L.;

Middleton, R. E.; Hughes, B.; Szewczak, A. A.; Dahlberg, W. K.; Kral, A. M.; Ozerova,

N.; Fleming, J. C.; Wang, H.; Secrist, P.; Harsch, A.; Hamill, J. E.; Cruz, J. C.; Kenific,

C. M.; Chenard, M.; Miller, T. A.; Berk, S. C.; Tempest, P., Parallel medicinal chemistry

approaches to selective HDAC1/HDAC2 inhibitor (SHI-1:2) optimization. Bioorg Med

Chem Lett 2009, 19 (4), 1168-1172; (c) Wilson, K. J.; Witter, D. J.; Grimm, J. B.;

Siliphaivanh, P.; Otte, K. M.; Kral, A. M.; Fleming, J. C.; Harsch, A.; Hamill, J. E.; Cruz,

J. C.; Chenard, M.; Szewczak, A. A.; Middleton, R. E.; Hughes, B. L.; Dahlberg, W. K.;

Secrist, J. P.; Miller, T. A., Phenylglycine and phenylalanine derivatives as potent and selective HDAC1 inhibitors (SHI-1). Bioorg Med Chem Lett 2008, 18 (6), 1859-1863; (d)

Witter, D. J.; Harrington, P.; Wilson, K. J.; Chenard, M.; Fleming, J. C.; Haines, B.; Kral,

A. M.; Secrist, J. P.; Miller, T. A., Optimization of biaryl Selective HDAC1&2 Inhibitors

(SHI-1:2). Bioorg Med Chem Lett 2008, 18 (2), 726-731.

27. Kozikowski, A. P.; Chen, Y.; Gaysin, A.; Chen, B.; D'Annibale, M. A.; Suto, C.

M.; Langley, B. C., Functional differences in epigenetic modulators-superiority of mercaptoacetamide-based histone deacetylase inhibitors relative to hydroxamates in cortical neuron neuroprotection studies. J Med Chem 2007, 50 (13), 3054-3061.

28. Wang, B.; Huang, P.-H.; Chen, C.-S.; Forsyth, C. J., Total Syntheses of the

Histone Deacetylase Inhibitors Largazole and 2-epi-Largazole: Application of N-

138

Heterocyclic Carbene Mediated Acylations in Complex Molecule Synthesis. J. Org.

Chem. 2011, 76 (4), 1140-1150.

29. Vanommeslaeghe, K.; De Proft, F.; Loverix, S.; Tourwe, D.; Geerlings, P.,

Theoretical study revealing the functioning of a novel combination of catalytic motifs in

histone deacetylase. Bioorg Med Chem 2005, 13 (12), 3987-3992.

30. Chang, S.; Young, B. D.; Li, S.; Qi, X.; Richardson, J. A.; Olson, E. N., Histone deacetylase 7 maintains vascular integrity by repressing matrix metalloproteinase 10. Cell

2006, 126 (2), 321-334.

31. Schuetz, A.; Min, J.; Allali-Hassani, A.; Schapira, M.; Shuen, M.; Loppnau, P.;

Mazitschek, R.; Kwiatkowski, N. P.; Lewis, T. A.; Maglathin, R. L.; McLean, T. H.;

Bochkarev, A.; Plotnikov, A. N.; Vedadi, M.; Arrowsmith, C. H., Human HDAC7 harbors a class IIa histone deacetylase-specific zinc binding motif and cryptic deacetylase activity. J Biol Chem 2008, 283 (17), 11355-11363.

32. Somoza, J. R.; Skene, R. J.; Katz, B. A.; Mol, C.; Ho, J. D.; Jennings, A. J.;

Luong, C.; Arvai, A.; Buggy, J. J.; Chi, E.; Tang, J.; Sang, B. C.; Verner, E.; Wynands,

R.; Leahy, E. M.; Dougan, D. R.; Snell, G.; Navre, M.; Knuth, M. W.; Swanson, R. V.;

McRee, D. E.; Tari, L. W., Structural snapshots of human HDAC8 provide insights into

the class I histone deacetylases. Structure 2004, 12 (7), 1325-1334.

33. Somoza, J. R.; Skene, R. J.; Katz, B. A.; Mol, C.; Ho, J. D.; Jennings, A. J.;

Luong, C.; Arvai, A.; Buggy, J. J.; Chi, E.; Tang, J.; Sang, B.-C.; Verner, E.; Wynands,

R.; Leahy, E. M.; Dougan, D. R.; Snell, G.; Navre, M.; Knuth, M. W.; Swanson, R. V.;

McRee, D. E.; Tari, L. W., Structural Snapshots of Human HDAC8 Provide Insights into

139

the Class I Histone Deacetylases. Structure (Cambridge, MA, U. S.) 2004, 12 (7), 1325-

1334.

34. Krennhrubec, K.; Marshall, B. L.; Hedglin, M.; Verdin, E.; Ulrich, S. M., Design and evaluation of 'Linkerless' hydroxamic acids as selective HDAC8 inhibitors. Bioorg

Med Chem Lett 2007, 17 (10), 2874-2878.

35. Vannini, A.; Volpari, C.; Filocamo, G.; Casavola, E. C.; Brunetti, M.; Renzoni,

D.; Chakravarty, P.; Paolini, C.; De Francesco, R.; Gallinari, P.; Steinkuehler, C.; Di

Marco, S., Crystal structure of a eukaryotic zinc-dependent histone deacetylase, human

HDAC8, complexed with a hydroxamic acid inhibitor. Proc. Natl. Acad. Sci. U. S. A.

2004, 101 (42), 15064-15069.

36. Atadja, P. W., HDAC inhibitors and cancer therapy. Prog Drug Res 2011, 67 ,

175-195.

37. Inoue, S.; Mai, A.; Dyer, M. J.; Cohen, G. M., Inhibition of histone deacetylase class I but not class II is critical for the sensitization of leukemic cells to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis. Cancer Res 2006, 66 (13),

6785-6792.

38. (a) Rotili, D.; Simonetti, G.; Savarino, A.; Palamara, A. T.; Migliaccio, A. R.;

Mai, A., Non-cancer uses of histone deacetylase inhibitors: effects on infectious diseases and beta-hemoglobinopathies. Curr Top Med Chem 2009, 9 (3), 272-291; (b) Wiech, N.

L.; Fisher, J. F.; Helquist, P.; Wiest, O., Inhibition of histone deacetylases: a pharmacological approach to the treatment of non-cancer disorders. Curr Top Med Chem

2009, 9 (3), 257-271.

140

39. Muraglia, E.; Altamura, S.; Branca, D.; Cecchetti, O.; Ferrigno, F.; Orsale, M. V.;

Palumbi, M. C.; Rowley, M.; Scarpelli, R.; Steinkuhler, C.; Jones, P., 2-

Trifluoroacetylthiophene oxadiazoles as potent and selective class II human histone

deacetylase inhibitors. Bioorg. Med. Chem. Lett. 2008, 18 (23), 6083-6087.

40. Di Maro, S.; Pong, R.-C.; Hsieh, J.-T.; Ahn, J.-M., Efficient solid-phase synthesis of FK228 analogues as potent antitumoral agents. J Med Chem 2008, 51 (21), 6639-6641.

41. (a) Methot, J. L.; Chakravarty, P. K.; Chenard, M.; Close, J.; Cruz, J. C.;

Dahlberg, W. K.; Fleming, J.; Hamblett, C. L.; Hamill, J. E.; Harrington, P.; Harsch, A.;

Heidebrecht, R.; Hughes, B.; Jung, J.; Kenific, C. M.; Kral, A. M.; Meinke, P. T.;

Middleton, R. E.; Ozerova, N.; Sloman, D. L.; Stanton, M. G.; Szewczak, A. A.;

Tyagarajan, S.; Witter, D. J.; Secrist, J. P.; Miller, T. A., Exploration of the internal cavity of histone deacetylase (HDAC) with selective HDAC1/HDAC2 inhibitors (SHI-

1:2). Bioorg Med Chem Lett 2008, 18 (3), 973-978; (b) Methot, J. L.; Hamblett, C. L.;

Mampreian, D. M.; Jung, J.; Harsch, A.; Szewczak, A. A.; Dahlberg, W. K.; Middleton,

R. E.; Hughes, B.; Fleming, J. C.; Wang, H.; Kral, A. M.; Ozerova, N.; Cruz, J. C.;

Haines, B.; Chenard, M.; Kenific, C. M.; Secrist, J. P.; Miller, T. A., SAR profiles of spirocyclic nicotinamide derived selective HDAC1/HDAC2 inhibitors (SHI-1:2). Bioorg

Med Chem Lett 2008, 18 (23), 6104-6109.

42. Wang, D.; Helquist, P.; Wiest, O., Zinc binding in HDAC inhibitors: a DFT study. J Org Chem 2007, 72 (14), 5446-5449.

43. Yurek-George, A.; Habens, F.; Brimmell, M.; Packham, G.; Ganesan, A., Total

Synthesis of Spiruchostatin A, a Potent Histone Deacetylase Inhibitor. J. Am. Chem. Soc.

2004, 126 (4), 1030-1031.

141

44. T. Ohishi, H. N., M. Sugawara, M. Izumida, T. Honda, K. Mori, N. Nogashima,

K. Inoue, Process for producing optically active alpha-methylcysteine derivative. US

patent 2006, US 2006/0105435 A1 .

45. Delaunay, D.; Toupet, L.; Lecorre, M., Reactivity of Beta-Amino Alcohols with

Carbon-Disulfide - Study on the Synthesis of 2-Oxazolidinethiones and 2-

Thiazolidinethiones. Journal of Organic Chemistry 1995, 60 (20), 6604-6607.

46. Funk, R. L.; Bolton, G. L., Metalation and alkylation of 4H-1,3-dioxin: a new

beta -acyl vinyl anion equivalent. J. Am. Chem. Soc. 1988, 110 (4), 1290-1292.

47. Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H., High-field FT NMR application of Mosher's method. The absolute configurations of marine terpenoids. J. Am.

Chem. Soc. 1991, 113 (11), 4092-4096.

48. Huang, P. Q.; Zheng, X.; Deng, X. M., DIBAL-H-H2NR and DIBAL-H-

(HNRR2)-R-1 center dot HCl complexes for efficient conversion of lactones and esters to amides. Tetrahedron Letters 2001, 42 (51), 9039-9041.

49. Paterson, I.; Florence, G. J.; Gerlach, K.; Scott, J. P.; Sereinig, N., A practical

synthesis of (+)-discodermolide and analogues: fragment union by complex aldol

reactions. J Am Chem Soc 2001, 123 (39), 9535-9544.

50. You, S. L.; Razavi, H.; Kelly, J. W., A biomimetic synthesis of thiazolines using hexaphenyloxodiphosphonium trifluoromethanesulfonate. Angew Chem Int Ed Engl

2003, 42 (1), 83-85.

51. Su, D.-W.; Wang, Y.-C.; Yan, T.-H., A novel DMAP-promoted oxazolidinethione deacylation. Application for the direct conversion of the initial chiral thioimide aldols to various ester protecting groups. Tetrahedron Lett. 1999, 40 (22), 4197-4198.

142

52. Inanaga, J.; Hirata, K.; Saeki, H.; Katsuki, T.; Yamaguchi, M., A rapid

esterification by mixed anhydride and its application to large-ring lactonization. Bull.

Chem. Soc. Jpn. 1979, 52 (7), 1989-1993.

53. Stuhr-Hansen, N., The tert-butyl moiety-a base resistant thiol protecting group smoothly replaced by the labile acetyl moiety. Synth. Commun. 2003, 33 (4), 641-646.

54. Clive, D. L. J.; Hisaindee, S.; Coltart, D. M., Derivatized Amino Acids Relevant to Native Peptide Synthesis by Chemical Ligation and Acyl Transfer. J. Org. Chem.

2003, 68 (24), 9247-9254.

55. Chen, Y.; Gambs, C.; Abe, Y.; Wentworth, P., Jr.; Janda, K. D., Total Synthesis of the Depsipeptide FR-901375. J. Org. Chem. 2003, 68 (23), 8902-8905.

56. Salvatore, R. N.; Smith, R. A.; Nischwitz, A. K.; Gavin, T., A mild and highly

convenient chemoselective alkylation of thiols using Cs2CO3-TBAI. Tetrahedron Lett.

2005, 46 (51), 8931-8935.

57. Richter, L. S.; Marsters, J. C., Jr.; Gadek, T. R., Two new procedures for the

introduction of benzyl-type protecting groups for thiols. Tetrahedron Lett. 1994, 35 (11),

1631-1634.

58. Sharma, S. K.; Wu, Y.; Steinbergs, N.; Crowley, M. L.; Hanson, A. S.; Casero, R.

A.; Woster, P. M., (Bis)urea and (bis)thiourea inhibitors of lysine-specific demethylase 1 as epigenetic modulators. J Med Chem 2010, 53 (14), 5197-5212.

59. Sigel, H.; Rheinberger, V. M.; Fischer, B. E., Stability of Metal Ion-Alkyl

Thioether Complexes in Solution - Ligating Properties of Isolated Sulfur-Atoms.

Inorganic Chemistry 1979, 18 (12), 3334-3339.

143

60. Ontoria, J. M.; Altamura, S.; Di Marco, A.; Ferrigno, F.; Laufer, R.; Muraglia, E.;

Palumbi, M. C.; Rowley, M.; Scarpelli, R.; Schultz-Fademrecht, C.; Serafini, S.;

Steinkuhler, C.; Jones, P., Identification of Novel, Selective, and Stable Inhibitors of

Class II Histone Deacetylases: Validation Studies of the Inhibition of the Enzymatic

Activity of HDAC4 by Small Molecules as a Novel Approach for Cancer Therapy. J.

Med. Chem. 2009, 52 (21), 6782-6789.

61. Cai, X.; Zhai, H. X.; Wang, J.; Forrester, J.; Qu, H.; Yin, L.; Lai, C. J.; Bao, R.;

Qian, C., Discovery of 7-(4-(3-ethynylphenylamino)-7-methoxyquinazolin-6-yloxy)-N- hydroxyheptanam ide (CUDc-101) as a potent multi-acting HDAC, EGFR, and HER2 inhibitor for the treatment of cancer. J Med Chem 2010, 53 (5), 2000-2009.

62. Kozikowski, A. P.; Tapadar, S.; Luchini, D. N.; Kim, K. H.; Billadeau, D. D., Use of the nitrile oxide cycloaddition (NOC) reaction for molecular probe generation: A new class of enzyme selective histone deacetylase inhibitors (HDACIs) showing picomolar activity at HDAC6. Journal of Medicinal Chemistry 2008, 51 (15), 4370-4373.

63. Oh, C. H.; Kang, Y. K.; Park, S. W.; Cho, J. H.; Kwon, S. K., Synthesis of New

Hydantoin-3-Acetic Acid-Derivatives. Bulletin of the Korean Chemical Society 1988, 9

(4), 231-235.

64. (a) Pattenden, G.; Thom, S. M.; Jones, M. F., Enantioselective Synthesis of 2-

Alkyl Substituted Cysteines. Tetrahedron 1993, 49 (10), 2131-2138; (b) Singh, S.,

Asymmetric synthesis of (R)- and (S)-α-methylcysteine. Recent Res. Dev. Org. Chem.

2004, 8 (Pt. 2), 323-339.

144

65. Li, Y.-Z.; Guo, C.-X.; Lu, X.-Y.; Zhao, X.-G.; Huang, H.-M., Synthesis and

quantum chemistry of (R) and (S)-N-cinnamyl-4-isopropylthiazolidine-2-thione. Youji

Huaxue 1998, 18 (6), 532-537.

66. Hoffmann, R. W.; Schafer, F.; Haeberlin, E.; Rohde, T.; Korber, K., Efficient homologation of aldehydes to 2,4,6-alkatrienals by means of an alpha -(1,3- dioxenylallyl)boronate. Synthesis 2000, (14), 2060-2068.

145

Appendix

1H and 13 C NMR Spectra

1H NMR of tert -butyl (4-carbamoylthiazol-2-yl)methylcarbamate (6) 1.47

H2NOC S 6 N

Boc NH 8.08 7.26 4.59 4.60 5.34 5.94 7.13

1.00 0.97 0.97 1.08 2.13 8.98

8 7 6 5 4 3 2 1 0 ppm 8.08 7.26

H2NOC S 6 N

Boc NH 4.59 4.60 5.34 5.94 7.13

1.00 0.97 0.97 1.08 2.13

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 ppm

146

13 C NMR of tert -butyl (4-carbamoylthiazol-2-yl)methylcarbamate (6)

77.54 77.23 76.91 163.11 149.33 42.52 169.65 80.78 155.79 124.80 H2NOC S 6 N

Boc NH 28.50

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

1H NMR of tert -butyl (4-cyanothiazol-2-yl)methylcarbamate (7)

1.47 1.59 NC S

7.26 N

Boc NH 7.95 4.62 4.61 5.30

1.00 0.59 1.89 12.06

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 ppm 4.62 4.61 5.30

NC S 7 N

Boc NH

0.59 1.89

5.35 5.30 5.25 5.20 5.15 5.10 5.05 5.00 4.95 4.90 4.85 4.80 4.75 4.70 4.65 4.60 4.55 4.50 ppm

147

13 C of tert -butyl (4-cyanothiazol-2-yl)methylcarbamate (7) 76.91 77.54 77.22

NC S N 7 Boc NH 42.53 114.00 126.61 81.03 155.80 171.65 131.08 2 8 .4 8

176 168 160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 ppm 1H NMR of 5-(tert -butylthiomethyl)-5-methylimidazolidine-2,4-dione (10)

H 1.22 O N 10 O N S H 1.31 2.75 7.89 10.60

1.00 1.11 2.14 9.05

13 12 11 10 9 8 7 6 5 4 3 2 1 0 -1 ppm 1.22

H O N 10 O

N 1.31 S H

3.03 9.05

1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 ppm 13 C NMR of 5-(tert -butylthiomethyl)-5-methylimidazolidine-2,4-dione (10)

35.13 156.20 62.09 177.22 41.84 39.51

H 23.64 O N 10 O N S H 30.69

180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm

148

1H NMR of ( R)-α-methylcysteine HCl (13) 4.80 1.57

SH OH H3N 2.89 3.14 Cl- O 2.85 3.18 13

1.00 0.97 2.80

4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

13 C NMR of ( R)-α-methylcysteine HCl (13) 30.33 61.60 173.23

13 SH OH H N 3 21.28 - Cl O 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

149

1H NMR of ( R)-4-isopropylthiazolidine-2-thione (15)

HN S

0.93 1.00 1.03 1.06 1.02 2.09 3.14

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm 13 C NMR of ( R)-4-isopropylthiazolidine-2-thione (15)

CDCl3 77.23 36.23 201.35 32.25 70.19 18.48 19.04

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

150

1H NMR of acetyl Nagao (16)

O S

N S CDCl3 7.26 16

1.00 1.051.06 3.12 1.01 3.15

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm

O S

N S

1.00 1.05 1.06 3.12 1.01 3.14 3.15 16 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

13 C NMR of acetyl Nagao (16)

CDCl3 77.23

O S

N S

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

151

1H NMR of 5-tosyloxy-1,3-dioxolane 2.46 O O S O O 7.81 7.83 7.377.35 7.26 O 4.794.76 3.963.95 3.81 3.79 1.59 4.80 4.74 4.463.993.98 3.78 4.82 4.47

1.93 2.09 1.10 0.93 2.38 3.22

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm

7.81 O 7.83 7.37 O 7.35 7.26 S O O O

1.93 2.09

7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 ppm

O 4.794.76 O 3.963.95 3.8 3.79 4.80 3.993.98 3.783.76 4.74 4.46 1 S O 4.82 4.474.47 4.454.44 4.48 4.44 O O

1.10 0.93 2.38 2.12

4.5 4.0 3.5 3.0 ppm

13 C NMR of 5-tosyloxy-1,3-dioxolane

O 93.70 68.82 77.5877.2776.95 O 145.51 133.56 95.83 66.95 S O O O

70.82 21.95 130.26

128.05

150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

152

1H NMR of 1,3-dioxene (18) 5.06

4.25 OO 4.25 7.26 6.5 6.56 4.924.91 2.17 1.25 4.90 0.87 6.577 4.92 18

1.00 2.11 2.09

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3. 3.0 2.5 2. 1.5 1. 0.5 0.0 ppm 5 0 0

4.25 4.25 4.24 OO 4.92 4.91 4.92 4.914.91 4.90 18

1.04 2.09

4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.0 ppm

OO 6.57 6.56 6.57 6.576.56 6.56

1.00

6.55 6.50 6.45 6.40 6.35 ppm

13 C NMR of 1,3-dioxene (18)

77.55 76.91 77.23 90.69 63.83

143.80 102.98

150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

153

1H NMR of 5-(tert -butylthio)pent-2E-enal (21) 1.33

S H 21

9.51 2.702.69 9.53 6.906.896.876.866.8 6.836.196.176.166.14 2.72 2.642.622.61 5

9 8 7 6 5 4 3 2 1 0 ppm

2.70 9.539.51 2.62 6.85 O 6.89 2.72 6.90 6.876.86 6.83 6.196.176.166.14 2.72 S H 21

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 ppm

13 C NMR of 5-(tert -butylthio)pent-2E-enal (21 ) 77.5477.23 76.91 32.94 26.64 O 42.65 S H 21

194.01 156.31 133.81

31.10

200 150 100 50 0 ppm

154

1H NMR of 7-(tert -butylthio)-3S-hydroxy-1-(4 R-isopropyl-2-thioxothiazolidin-3- yl)hept-4E-en-1-one (22)

1.31

1.07 0.98 1.05 0.96 OH O S

S N S 2.57 22

2.55 2.31 3.04 2.29 3.01 2.5 5.15 2.33 3.593.58 9 3.533.523.50 3.333.31 2.35 5.75 5.625.61 5.16 5.13 4.63 3.643.63 3.55 3.29 2.36 5.79 5.73 5.585.57 2.27 5.80 2.40

0.97 0.991.01 0.99 1.03 1.01 1.04 1.98 3.02 8.64 2.87

6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

5.15

5.75 5.625.61 5.16 5.13 4.63

5.79 OH O S 5.77 5.585.57 5.73 S N S 5.80 22

0.97 0.99 1.01 0.99

5.9 5.8 5.7 5.6 5.5 5.4 5.3 5.2 5.1 5.0 4.9 4.8 4.7 4.6 4.5 ppm

2.57

2.55 2.31 3.04 OH O S 2.29 3.01 2.59 2.33 3.59 S N 3.58 S 3.533.52 3.333.31 22 3.64 3.55 3.50 2.362.35 3.63 3.293.26 2.38 2.27 2.40

1.00 1.03 1.01 1.04 1.98 3.02 3.7 3.6 3.5 3.4 3.3 3.2 3.1 3.0 2.9 2.8 2.7 2.6 2.5 2.4 2.3 ppm

155

1.31

1.07 0.98 1.05 OH O S 0.96

S N S 22

8.64 2.87 2.72

1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 ppm

156

13 C NMR of 7-(tert -butylthio)-3S-hydroxy-1-(4 R-isopropyl-2-thioxothiazolidin-3- yl)hept-4E-en-1-one (22)

45.54 27.97 30.85 32.74 77.5477.2376.90 172.66 203.09 42.23

OH O S 18.01 S N 19.29 S 131.86130.67 68.72 22 71.57

31.17

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm

172.66 203.09

OH O S

S N S 22 130.6 131.8 7 6 205 200 195 190 185 180 175 170 165 160 155 150 145 140 135 130 ppm

45.54 27.97 30.85 32.74

42.23

OH O S

18.01 S N 19.29 68.72 S 71.57 22

31.17

70 65 60 55 50 45 40 35 30 25 20 ppm

157

1H NMR of ( R)-methyl 2-(2-((( S,E)-7-(tert -butylthio)-3-hydroxyhept-4- enamido)methyl)thiazol-4-yl)-4-methyl-4,5-dihydrothiazole-4-carboxylate (31)

1.26 MeOOC S N 3.76 31 N S 1.60 OH O 7.90 S NH 2.52 3.853.82 3.253.23 2.50 4.704.69 2.54 2.472.462.442.252.23 7.32 5.735.725.695.685.665.545.535.515.49 4.48

0.92 0.91 1.00 1.993.220.97 2.18 1.93 2.71 8.25

8.0 7.5 7.0 6.5 6.0 5.5 5. 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1. 0.5 0.0 0 ppm 0

3.76 1.60 1.26 MeOOC S N

31 2.52 N S OH O

S NH 3.82 3.25 2.5 3.85 3.23 0 2.25 2.54 2.23 2.472.462.44 2.42 4.48 2.27 2.22

0.98 1.14 3.22 0.97 2.181.79 1.93 2.71 8.25

4.5 4.0 3.5 3.0 2.5 2.0 1.5 ppm

158

13 C NMR of ( R)-methyl 2-(2-((( S,E)-7-(tert -butylthio)-3-hydroxyhept-4- enamido)methyl)thiazol-4-yl)-4-methyl-4,5-dihydrothiazole-4-carboxylate (31)

MeOOC S N 41.59 27.85 172.10 40.9132.55 173.68168.20162.88 148.27 31 84.57 42.96 N S 77.5477.2376.90 OH O

S NH

122.41 130.58 69.22 53.09 24.10 132.31

31.08 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 2 10 0 ppm 0

172.10 84.57 168.20 162.88 148.27 173.68

MeOOC S 122.41 N 31 130.58 N S OH O

S NH

132.31

175 170 165 160 155 150 145 140 135 130 125 120 115 110 105 100 95 90 85 ppm

41.59 27.85

40.91 32.55 MeOOC S 42.96 N 77.23 77.5476.90 31 N S 42.17 OH O

S NH

69.22 53.09 24.10

31.08

75 70 65 60 55 50 45 40 35 30 25 ppm

159

N S O O O 3.78 S NH 1.62 7.90 7.26 32 7.75 7.31 2.64 2.53 0.960.94 0.92 0.9 7.76 7.56 5.264.754.73 4.39 4.374.35 3.863.83 3.253.23 2.62 2.55 2.52 2.302.28 6.876.86 6.8 5.885.84 5.685.66 5.59 5.57 5.55 5.28 4.18 2.32 2.112.10 0 4

2.01 2.03 0.78 1.00 0.8 1.83 1.87 0.91 2.7 0.9 2.0 0.7 2.62 8.3 2.54 2 3 2 4 9 4 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm

7.90 7.31 7.26 7.75 7.76 7.56 7.33 7.58 7.39 7.40 MeOOC S N

7.29 NHFmoc 7.37 5.26 5.28 5.66 7.41 N S 6.86 5.68

O O O 5.84 5.59 5.88 5.55 5.57 6.87 5.65 6.84

NH 5.82 S 5.54 5.70 32 5.90

0.89 2.01 2.012.03 1.98 0.78 0.971.00 0.94 0.82

8.0 7.5 7.0 6.5 6.0 5.5 ppm 3.78 1.62 1.29

MeOOC S N NHFmoc N S O O O 0.96 0.92 S NH 0.94 32 2.53 2.64 2.62 4.75 3.25 3.83 4.73 2.52 3.86 4.37 3.23 2.30 2.28 4.35 2.55 4.39 4.21 4.09 4.10 4.11 4.08 2.32 2.27 2.10 2.08 4.18 4.32 4.41 2.07 2.11

1.83 1.87 0.99 0.91 2.73 0.92 1.74 2.04 1.94 0.79 2.628.34 2.54

4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

160

77.54 77.23 76.91 41.69 32.67 27.69 67.26 41.33 141.45 84.72 169.19 156.58 171.44 143.84 148.50 168.55 173.82 162.93 42.28

MeOOC S 18.16 19.25 53.13 59.70 31.06 134.52 47.32 N 127.51 72.52 24.17

NHFmoc 125.22

S 120.18

N 127.91 O O O

S NH 31.16 32

180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm 141.45 169.19 156.58 171.44 143.84 143.91 148.50 168.55 173.82 162.93

MeOOC S 122.28 N 134.52

NHFmoc 127.51

N S 125.22

O O O 120.15 120.18 S NH 32 127.91

175 170 165 160 155 150 145 140 135 130 125 120 ppm 77.54 77.23 76.91 41.69 32.67 27.69 67.26 41.33 84.72 42.28 18.16 19.25 53.13 59.70 31.06 47.32 24.17 72.52 31.16

90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 ppm

161

1H NMR of cyclized product 33

O S 5.30 1.30

7.26 N NH

N S2.80 OOO

S N 2.17 33 H 1.86 0.70 0.69 0.51 0.50 7.76 2.56 2.55 4.02 4.04 3.29 3.27 2.57 3.79 4.61 4.61 4.60 3.77 4.59 2.84 2.31 7.18 5.27 4.28 4.28 4.25 7.16 5.28 5.88 5.54 5.91 5.68 5.53 6.51 6.49 5.89 5.69 5.92

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm 7.26 5.30

O S N

7.76 NH N S OOO

S N 33 H 4.61 4.61 4.59 5.27 7.18 7.16 5.28 5.54 5.88 5.68 5.53 5.51 5.91 5.50 6.51 6.49 5.66 5.69 5.87 5.92

7.5 7.0 6.5 6.0 5.5 5.0 ppm 2.80 2.17 1.86 1.30

O 0.70 S 0.69 0.51 0.50

N 2.56 NH N S OOO 2.55 4.02 4.04 3.29 3.27 S 2.57 N 3.79 3.77 33 2.72 2.86 2.84

2.72 H 2.31 4.28 2.31 4.28 4.25 4.25 2.70 2.87 2.89 2.69

2.33

4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

162

13 C NMR of cyclized product 33

77.54 77.22 76.91 32.75 27.72 41.31 43.54 40.75 169.65 169.09 173.72 168.15 147.63 84.58 38.84 124.40 16.85 24.41 O S 19.14 57.97 72.26 34.37

133.86 N

127.80 NH N S OOO 31.19 S N 33 H

180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

169.65 169.09 173.72 168.15 84.58 147.63 124.40 O S N NH N S

OOO 133.86

S N 33 H 127.80

175 170 165 160 155 150 145 140 135 130 125 120 115 110 105 100 95 90 85 ppm 41.31 32.75 27.72 43.54 40.75 38.84

O S 16.85 24.41 19.14 57.97 72.26 N NH 34.37 N S OOO

S N 33 H 31.19

70 65 60 55 50 45 40 35 30 25 20 ppm

163

1H NMR of (2 E)-5-[(triphenylmethyl)thio]-2-pentenal (36) 7.26 7.41 7.29 7.43 7.22 7.31 9.44 9.42 7.20 6.61

36 6.65 5.99 6.01 5.97 5.95 6.63 6.67 6.60 6.64

0.89 6.07 6.43 1.00 0.88

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 ppm

Ph O Ph Ph S H 36 2.33 2.34 2.31 2.32 2.29 2.36 2.35 2.37

3.97

2.6 2.5 2.4 2.3 2.2 2.1 2.0 ppm

13 C NMR of (2 E)-5-[(triphenylmethyl)thio]-2-pentenal (36) 144.71 31.91 30.19 67.16

Ph O

193.95 Ph 156.00 133.77 Ph S H 36 129.68 128.15 126.96

192 184 176 168 160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16 8 0 ppm

164

1H NMR of 3 S-hydroxy-1-(4 R-isopropyl-2-thioxo-thiazolidin-3-yl)-7-tritylsulfanyl- hept-4E-en-1-one (37) 5.29 7.28 7.40

7.42 Ph OH O S Ph 1.06 7.26 0.98 1.04 Ph S N S 0.96

7.21 37 7.30 2.21 2.20 3.00 2.10 2.97 3.46 3.44 7.19 2.08 3.53 3.54 3.49 3.47 5.12 3.31 3.29 5.57 3.59 5.47 5.48 5.11 2.34 5.14 2.36 4.57 5.61 2.38 2.82 1.66

6.22 3.14 1.01 1.03 1.00 1.05 1.03 1.00 0.88 1.00 2.06 0.72 2.89

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 ppm

5.29 7.28 7.40 7.42 7.26 7.21 7.30 7.22

Ph HO O S Ph SN Ph S 7.19

37 5.12 5.57 5.47 5.48 5.11 5.14 5.61 5.43 5.55 5.45

6.22 6.53 3.14 1.00 1.01 1.03

7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3 6.2 6.1 6.0 5.9 5.8 5.7 5.6 5.5 5.4 5.3 5.2 5.1 ppm

165

1.06 0.98 1.04 Ph HO O S 0.96 Ph SN Ph S 37 2.21 2.20 3.00 2.10 2.97 3.46 3.44 2.08 3.53 3.54 3.49 3.47 3.31 3.29 3.59 3.58 2.34 2.23 2.36 2.12 3.27 3.24 2.33 2.38 2.82 1.66

0.97 1.05 1.03 1.00 0.88 1.00 2.00 2.06 0.72 2.85 2.89

3.5 3.0 2.5 2.0 1.5 1.0 ppm

13 C NMR of 3S-hydroxy-1-(4 R-isopropyl-2-thioxo-thiazolidin-3-yl)-7-tritylsulfanyl- hept-4E-en-1-one (37)

145.01 45.42 31.61 31.55 30.81 172.64 77.23 77.55 76.91 203.10 66.72 132.02 18.00 19.28 68.64 31.00 71.54 OH Ph O S Ph 126.76

Ph SN S 37 128.03 129.73

200 180 160 140 120 100 80 60 40 20 0 ppm

166

1H NMR of ( R)-methyl 2-(2-((3 S-hydroxy-7-(tritylthio)hept-4E- enamido)methyl)thiazol-4-yl)-4-methyl-4,5-dihydrothiazole-4-carboxylate (39)

MeOOC S

N 3.78

39 N S 1.63

7.26 Ph OH O 7.37 Ph 7.39 Ph S NH 7.24 7.90 7.19 7.21 2.18 3.27 3.84 3.24 3.87 4.68 2.17 2.06 4.70 4.67 7.17 2.42 2.40 7.08 5.52 5.43 5.41 3.48 4.43 7.06 5.56 5.37 5.50 5.39 2.45 2.34 2.46 5.57 4.63

0.85 5.95 0.93 1.02 1.01 2.87 1.00 1.00 2.07 2.70

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 ppm 7.26 7.37 7.39

MeOOC S 7.24 N 7.90

7.19 39 7.28 N S 7.21 Ph Ph OH O Ph S NH 7.17 7.08 5.52 5.43 5.41 7.06 7.09 5.56 5.37 5.39 5.50 5.54 5.57

0.85 5.95 6.67 3.01 0.93 1.00 1.02

8.0 7.5 7.0 6.5 6.0 5.5 ppm 3.78

MeOOC S N 39 N S Ph Ph OHO Ph S NH 3.27 3.84 3.24 3.87 4.68 4.70 4.67 3.48 4.43 4.72 4.64 4.74 4.63

2.00 1.01 0.97 2.87 0.92 1.00

4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1 ppm

167

1.63

MeOOC S N 39

N S Ph Ph OH O Ph S NH 2.18 2.17 2.06 2.42 2.04 2.40 2.41 2.37 2.20 2.07 2.45 2.34 2.02 2.36 2.46 2.47

0.97 1.00 1.91 2.07 2.70

2.9 2.8 2.7 2.6 2.5 2.4 2.3 2.2 2.1 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 ppm

13 C NMR of ( R)-methyl 2-(2-((3 S-hydroxy-7-(tritylthio)hept-4E- enamido)methyl)thiazol-4-yl)-4-methyl-4,5-dihydrothiazole-4-carboxylate (39)

144.92 41.64 31.56 31.43 40.89 172.02 42.88 168.04 77.23 77.54 76.91 84.63 148.35 162.87 173.73 66.70 122.39 53.13 69.19 24.14 132.39 MeOOC S

126.74 N 39 N S 128.00

129.66 Ph OHO Ph Ph S NH 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

144.92 172.02 168.04 148.35 162.87 173.73

MeOOC S 122.39 130.27 N 132.39

39 N S Ph Ph OH O

Ph S NH 126.74 128.00 129.66

170 165 160 155 150 145 140 135 130 125 ppm 168

41.64 31.56 31.43 40.89 42.88 77.23 76.91 77.54 84.63 66.70 53.13 69.19 24.14 MeOOC S N 39 N S Ph Ph OH O Ph S NH

85 80 75 70 65 60 55 50 45 40 35 30 25 ppm

169

MeOOC S

7.26 N 1.56 NHFmoc 40 N S Ph Ph OOO

Ph S NH 3.78 1.62 7.27 7.37 7.38 7.88 0.85 7.20 0.84 0.89 0.90 7.21 4.70 4.69 2.57 2.58 3.25 3.86 3.84 3.23 2.06 7.19 2.17 4.19 4.37 2.18 5.20 4.38 5.22 2.04 6.74 5.60 5.66 4.20 2.07 5.69 5.44 4.40

0.96 2.01 3.23 0.86 1.06 0.91 1.87 2.05 0.90 2.99 1.00 1.92 5.79 3.49 4.32

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 ppm 7.26

MeOOC S N NHFmoc 40 N S Ph Ph OOO 7.27 7.37 7.38 7.88 Ph S NH 7.20 7.21 7.31 7.56 7.75 7.57 7.32 7.76 7.40 7.19 7.40 7.41

0.96 2.26 2.01 9.06 2.57 5.48 3.23

7.90 7.85 7.80 7.75 7.70 7.65 7.60 7.55 7.50 7.45 7.40 7.35 7.30 7.25 7.20 7.15 ppm 3.78

MeOOC S N NHFmoc 4.70 4.69 40 N S Ph Ph OOO 3.84 3.86 Ph S NH 4.19 4.37 4.34 4.38 5.20 5.22 4.35 4.06 5.60 4.04 4.07 4.20 5.66 5.69 5.44 4.32 4.18 4.40 5.43 5.42 5.41 5.65 5.59

1.00 1.06 0.96 0.91 1.87 2.05 1.07 0.90 1.03 2.99

5.6 5.5 5.4 5.3 5.2 5.1 5.0 4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7 ppm

170

S MeOOC 1.62 1.56 0.90 0.89 0.85 0.84 N NHFmoc 40 N S Ph OOO

Ph 2.57 2.58 Ph S NH 3.25 3.23 2.06 2.17 2.18 2.04 2.07 2.03

1.00 1.92 2.28 5.79 3.49 4.32

3.3 3.2 3.1 3.0 2.9 2.8 2.7 2.6 2.5 2.4 2.3 2.2 2.1 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7 ppm 13 C NMR of (4 R)-methyl 2-(2-((8 S)-1-(9 H-fluoren-9-yl)-5-isopropyl-3,6,10-trioxo-8- (( E)-4-(tritylthio)-but-1-enyl)-2,7-dioxa-4,11-diazadodecan-12-yl)thiazol-4-yl)-4- methyl-4,5-dihydrothiazole-4-carboxylate (40) 145.01 41.73 77.62 76.99 77.31 31.55 67.29 31.37 141.51 41.34 169.23 84.77 156.63 143.91 171.44 143.98 168.62 148.53 66.86 29.96 173.88 162.99 60.66 14.47 18.16 53.16 19.26 59.69 31.10 134.23 125.30 72.50 47.38 24.22

120.22 MeOOC S N

126.86 NHFmoc N S Ph OOO 40 Ph 128.10 129.78 Ph S NH

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

171

145.01 141.51 169.23 156.63 143.91 171.44 143.98 148.53 168.62 173.88 162.99 122.35 127.76 134.23 MeOOC S 125.30 N NHFmoc 120.22 127.95 40 N S Ph Ph OOO Ph S NH 126.86 128.10 129.78

170 165 160 155 150 145 140 135 130 125 120 ppm 41.73 77.62 76.99 77.31 31.55 67.29 31.37 41.34 41.68 84.77 66.86 29.96 60.66 18.16 53.16 19.26 59.69 31.10 72.50 47.38 24.22 MeOOC S N NHFmoc

40 N S Ph Ph OOO

Ph S NH

85 80 75 70 65 60 55 50 45 40 35 30 25 20 ppm

172

1H NMR of cyclic core 41

7.36 O 7.26 S N 7.37 NH 1.82 S 41 N Ph Ph OOO 7.25 Ph S N H 0.67 7.19 0.50 0.66 0.49 1.24 7.73 7.18 2.18 2.19 2.06 3.27 2.65 4.00 4.02 3.25 4.56 7.15 4.12 5.60 4.54 4.09 6.50 6.48 2.01 5.40 2.76 5.68 2.75 5.39 5.18 5.37 5.71 5.20 5.70 5.21 0.85

0.97 3.93 0.96 1.00 1.02 1.00 1.04 1.03 1.02 2.00 3.20 2.97

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 ppm

7.36 7.26 7.37 7.25 7.27 7.19

O S 7.20 N NH 41 N S Ph OOO

7.73 Ph Ph S N 7.18 H 7.15 5.60 6.50 6.48 5.40 5.68 5.39 5.18 5.37 5.71 5.20 5.70 5.21 5.17 5.36

0.97 6.22 6.57 3.93 0.96 1.00 1.00 1.04 1.02

7.5 7.0 6.5 6.0 5.5 ppm

173

O S N NH 41 N S Ph OOO Ph Ph S N H 3.27 2.65 4.00 4.02 3.25 4.56 4.12 4.54 4.09 2.63 2.76 2.75 2.77 2.79

1.00 1.04 1.04 1.03 0.99 1.02

4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1 3.0 2.9 2.8 2.7 2.6 ppm 1.82 0.67 0.50 0.49 0.66

O S 1.24 N NH 41 N S Ph OOO Ph Ph S N H 2.18 2.19 2.06 2.05 2.04 2.00 2.01 2.17 2.03 1.99 0.85 2.15

2.00 3.43 3.20 2.93 2.97

2.3 2.2 2.1 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 ppm

174

13 C of cyclic core 41 144.90 76.92 77.55 77.23 31.39 31.54 169.43 41.19 168.94 40.76 43.49 168.06 173.65 29.90 84.57 66.76 147.60 164.62 124.32 24.42 19.09 34.25 16.92 71.98 O 57.98

133.27 S N NH 41 N S Ph Ph OOO 126.80

129.71 128.06 Ph S N H

180 160 140 120 100 80 60 40 20 0 ppm 144.90

O S N NH

169.43 41 N S Ph OOO 168.94 Ph Ph S N H 168.06 173.65 147.60 164.62 124.32 133.27 129.71 128.06 126.80

172 170 168 166 164 162 160 158 156 154 152 150 148 146 144 142 140 138 136 134 132 130 128 126 124 ppm

76.92 77.55 77.23 31.39 31.54 41.19 40.76 43.49 29.90 84.57 66.76 24.42 19.09 16.92 34.25 71.98 57.98

O S N NH 41 N S Ph Ph OOO Ph S N H

80 75 70 65 60 55 50 45 40 35 30 25 20 ppm

175

1H NMR of largazole (1)

O S 7.26 N Largazole NH N S 1.87 OOOO 1.62

H C(H C) S N 0.87

3 2 6 0.69 0.68 0.51 H 0.49 7.77 2.90 1.29 2.53 1.29 0.86 1.28 1.26 2.52 2.54 4.03 2.88 2.31 2.30 4.05 3.29 3.27 1.64 7.16 7.14 4.61 4.60 4.60 4.28 4.62 5.81 4.29 4.26 5.30 5.28 1.31 5.27 5.31 5.83 5.66 5.52 0.85 6.41 6.40 5.51 6.40 6.42

0.90 1.00 0.99 1.00 1.07 1.01 1.03 1.00 1.05 1.92 1.06 3.76 9.29 4.05 2.82

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 ppm 7.26

O S N Largazole NH N S OOOO 7.77

H3C(H 2C) 6 S N H 7.14 7.16 5.81 5.30 5.28 5.27 5.31 5.83 5.52 5.66 6.41 5.51 6.40 5.50 6.40 6.42

0.90 1.00 0.99 1.00 1.00 1.01 1.07

7.5 7.0 6.5 6.0 5.5 ppm

176

O S N Largazole NH N S OOOO 2.90 2.53 H3C(H 2C) 6 S N H 2.52 2.54 2.31 4.03 2.88 2.30 4.05 3.29 3.27 2.91 2.70 4.61 4.60 4.28 2.84 4.60 2.70 2.83 4.62 4.26 4.29 4.25 2.87 2.68 2.85 2.67 2.32 2.29 2.10 2.11

1.01 1.04 1.03 1.00 1.86 1.05 1.01 1.92 2.05 1.06

4.5 4.0 3.5 3.0 2.5 2.0 ppm 1.87

O S N 1.62 Largazole N N S OOOO 0.87 0.69 0.68

N 0.51 H3C(H 2C) 6 S 0.49 1.29 1.29 0.86 1.28 1.26 1.27 1.30 0.88 1.26 1.25 1.64 1.65 1.31 0.85 2.10 2.11

1.06 2.88 3.76 9.29 4.05 2.87 2.82

2.1 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 ppm

177

13 C NMR of largazole (1)

77.23 76.90 77.54

O S N NH Largazole N S OOOO 29.14 H3C(H 2C) 6 S N H 25.87 44.37 32.49 41.31 28.14 43.55 22.82 40.68 169.57 169.07 168.08 173.73 84.65 147.69 164.69 199.59 4 3014. 124.40 6 8516. 4 4334. 7 9357. 4 4324. 2 2672. 9 1119. 128.55 132.94

200 192 184 176 168 160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16 8 0 ppm

178

O S N Largazole NH N S OOOO

H3C(H 2C) 6 S N H 169.57 169.07 168.08 173.73 147.69 164.69 199.59 124.40 128.55 132.94

200 195 190 185 180 175 170 165 160 155 150 145 140 135 130 125 ppm 77.54 77.23 76.90 29.14

O S 25.87

N 44.37 Largazole NH 32.49 S 41.31

N 28.14 OOOO 31.84 43.55 22.82 40.68 H C(H C) S N

84.65 3 2 6 H 14.30 16.85 34.43 57.93 24.43 72.26 19.11

85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 ppm

179

1H NMR of ( R)-methyl 2-(2-((5 R,8 S)-1-(9 H-fluoren-9-yl)-5-(naphthalen-1-ylmethyl)- 3,6,10-trioxo-8-(( E)-4-(tritylthio)but-1-enyl)-2,7-dioxa-4,11-diazadodecan-12- yl)thiazol-4-yl)-4-methyl-4,5-dihydrothiazole-4-carboxylate (42) 3.79 1.63 7.25 7.35 7.37 7.23 7.17 7.19 7.75 7.80 3.26 3.84 7.77 7.49 3.23 3.87 2.05 2.15 3.51 3.49 1.26 7.15 7.51 4.13 2.13 0.87 7.83 2.02 5.39 0.88 5.41 0.88 4.51 5.43 5.94 4.33 5.45 5.34 4.35 1.28 4.74 4.72 4.44 2.27 2.17 2.08 1.24 0.85 2.29 5.95 5.92

0.88 2.77 9.41 0.81 1.820.84 1.162.82 1.77 0.96 2.38 2.87 1.20

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm 7.25 7.35 7.26 7.37 7.27 7.23 7.17 7.19 7.75 7.80 7.77 7.49 7.48 7.34 7.46 7.39 7.15 7.51 7.29 7.83 7.82 7.85 8.06 7.41 8.08

0.88 1.91 2.77 3.88 8.99 9.41 3.17

8.25 8.20 8.15 8.10 8.05 8.00 7.95 7.90 7.85 7.80 7.75 7.70 7.65 7.60 7.55 7.50 7.45 7.40 7.35 7.30 7.25 7.20 7.15 7.10 7.05 7.00 ppm 3.79 MeOOC S N NHFmoc

N S Ph O O O Ph Ph NH S 42 3.26 3.84 3.23 3.87 3.51 3.49 4.13 5.39 5.41 4.51 4.26 5.43 5.94 4.49 4.33 4.11 5.45 5.34 4.35 4.15 5.57 4.74 4.72 4.43 4.44 4.28 4.23 5.33 4.36 5.31 5.61 4.25 5.42 5.58 5.95 4.53 5.92 5.29 4.55 4.39 5.62 4.76

0.81 0.98 1.82 1.00 0.84 1.100.57 1.16 0.97 1.05 1.02 2.82 1.77 0.95

6.0 5.5 5.0 4.5 4.0 3.5 ppm

180

1.63 MeOOC S N NHFmoc

N S Ph O O O Ph Ph S NH 42 2.05 2.15 1.26 2.13 0.87 2.02 0.88 0.88 2.00 1.28 1.24 2.27 2.17 2.08 2.09 2.25 0.85 2.29 2.30

0.961.81 0.60 2.38 2.87 1.20

2.3 2.2 2.1 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7 ppm 13 C NMR of ( R)-methyl 2-(2-((5 R,8 S)-1-(9 H-fluoren-9-yl)-5-(naphthalen-1- ylmethyl)-3,6,10-trioxo-8-(( E)-4-(tritylthio)but-1-enyl)-2,7-dioxa-4,11-diazadodecan- 12-yl)thiazol-4-yl)-4-methyl-4,5-dihydrothiazole-4-carboxylate (42) 77.23 76.91 77.54 144.91 41.64 141.41 31.27 143.86 67.26 31.49 41.06 132.53 170.85 168.84 148.48 132.11 41.31 36.17 155.83 167.92 133.87 84.71 173.82 66.76 162.92 54.99 123.59 133.89 47.20 125.60 53.15 125.29 125.23 72.68 24.19 120.15 126.79 128.06 129.70

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm 144.91 141.41 143.86 132.53 148.48 132.11 168.84 170.85 155.83 167.92 133.87 173.82 162.92 133.89

MeOOC S N NHFmoc

N S Ph O O O Ph Ph S NH 42

175 170 165 160 155 150 145 140 135 ppm

181

122.05 123.59 127.76 126.24 125.60 129.06 125.29 125.23 128.19 127.32 126.85 127.21

127.24 120.15 127.89 MeOOC S N NHFmoc 126.79 N S Ph O O O Ph Ph S NH 42 128.06 129.70

130.0 129.5 129.0 128.5 128.0 127.5 127.0 126.5 126.0 125.5 125.0 124.5 124.0 123.5 123.0 122.5 122.0 121.5 121.0 120.5 120.0 ppm 1H NMR of cyclic core 43 7.26 7.38 1.72 7.40 7.19 7.24 2.05 7.67 7.20 7.17 1.26 3.21 1.59 3.19 7.68 7.80 4.22 7.81 7.45 0.97 7.06 1.25 4.20 2.56 0.93 2.10 8.35 1.27 0.95 2.16 3.00 1.19 5.30 4.11 5.68 4.21 4.13 3.20 4.83 4.82 4.24 3.22 2.02 5.62 4.24 2.18 6.39 5.23 5.22 5.21 5.04 2.11

0.99 1.94 8.91 0.92 1.971.00 1.95 0.98 1.98 1.02 3.28 2.921.36 2.00

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm

7.26

O S N NH N S 7.26 Ph OOO 7.38 Ph 7.40 Ph S N

43 H 7.27 7.19 7.24 7.67 7.20 7.17 7.68 7.80 7.81 7.45 7.06 7.55 7.32 7.31 8.37 8.35 7.04 7.47 7.16 7.53 7.44 7.56

0.99 1.03 1.94 1.09 1.13 6.041.16 8.91 3.96 1.00

8.4 8.3 8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 ppm

182

4.22 4.20 5.30 4.11 5.68 4.13 4.83 4.82 4.24 4.24 5.62 5.23 5.22 6.39 5.65 3.67 5.04 5.21 5.03 5.06 5.07 3.68 5.67 3.69 5.19 5.70 4.84 5.61 4.81 4.14 4.06 4.10 4.05

0.92 1.97 0.97 1.00 0.99 1.95 0.98

6.0 5.5 5.0 4.5 4.0 ppm 1.72

O S N NH

N S 2.05 Ph OOO Ph Ph S N 43 H 1.26 3.21 1.59 3.19 0.97 1.25 2.56 0.93 2.10 1.27 0.95 2.16 1.25 2.53 1.19 3.00 1.18 2.19 1.23 1.22 2.15 0.92 2.02 3.22 2.18 2.03 2.20 1.02 2.20 2.09 1.01 2.01 2.80 2.78 2.81 2.21 2.83 2.00 1.98

1.98 0.96 1.02 3.281.08 2.92 1.36 1.08 2.00

3.3 3.2 3.1 3.0 2.9 2.8 2.7 2.6 2.5 2.4 2.3 2.2 2.1 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 ppm 13 C NMR of cyclic core 43

128.09 129.76 126.85 77.22 77.01 77.43 31.59 126.69 55.70 125.88 37.14 72.80 31.24 124.91 124.29 144.98 40.30 134.81 41.80 26.41 41.44 132.49 167.41 169.60 66.88 174.14 167.91 162.89 147.80

180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

183

O S 144.98 N NH 134.81 N S Ph OOO Ph Ph S N 133.95 132.49 43 H 132.05 167.41 169.60 174.14 167.91 162.89 147.80

175 170 165 160 155 150 145 140 135 ppm 128.09 129.76 126.85 128.14 126.69 125.88 128.83 128.33 124.91 124.29 124.48

130.5 130.0 129.5 129.0 128.5 128.0 127.5 127.0 126.5 126.0 125.5 125.0 124.5 124.0 123.5 123.0 ppm 77.22 77.01 77.43 31.59 55.70 37.14 31.24 72.80 40.30 41.80 26.41 41.44 66.88 84.92

85 80 75 70 65 60 55 50 45 40 35 30 25 ppm

184

1H NMR of (R)-methyl 2-(2-((5 S,8 S)-5-allyl-1-(9 H-fluoren-9-yl)-3,6,10-trioxo-8-(( E)- 4-(tritylthio)but-1-enyl)-2,7-dioxa-4,11-diazadodecan-12-yl)thiazol-4-yl)-4-methyl- 4,5-dihydrothiazole-4-carboxylate (44) 3.78 7.38 1.62 7.27 7.39 7.25 7.26 7.20 7.88 7.22 7.74 7.76 2.05 1.85 4.69 4.67 2.58 4.35 5.05 2.19 2.57 3.83 3.25 3.86 3.22 2.17 7.18 2.06 4.19 5.59 4.37 5.64 5.60 5.62 5.08 5.31 5.33 1.26 6.84 4.20 2.43 6.83

0.78 2.15 8.730.85 3.012.10 1.832.00 2.900.94 1.76 2.09 2.77

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm 7.38 7.27 MeOOC S 7.39 N NHFmoc

7.25 N S 7.29

7.20 Ph O O O Ph 7.88 7.22

7.74 Ph 7.76 NH S 44 7.55 4.67 4.69 7.32 7.57 5.05 7.41 7.18 5.59 5.64 5.60 5.62 5.08 5.31 5.33 5.10 6.84 5.66 5.45 5.67 5.57 5.43 5.41 6.86 6.83 5.40 5.69 5.29

0.78 2.05 2.15 8.258.73 3.09 0.85 3.01 1.00 0.98 2.10 1.83

8.0 7.5 7.0 6.5 6.0 5.5 5.0 ppm 3.78 1.62 2.05 1.85 2.58 4.35 2.19 2.57 3.25 3.83 3.86 3.22 2.17 2.06 4.19 4.34 4.37 1.26 3.79 4.20 4.27 2.21 1.64 2.43 2.50 2.08 4.17 2.52 4.38 2.41 2.54 2.03 4.33 2.39 2.44 4.40

2.00 0.93 1.05 2.90 0.94 1.76 0.97 1.90 2.09 1.15 2.77

4.5 4.0 3.5 3.0 2.5 2.0 1.5 ppm

185

13 C NMR of (R)-methyl 2-(2-((5 S,8 S)-5-allyl-1-(9 H-fluoren-9-yl)-3,6,10-trioxo-8-(( E)- 4-(tritylthio)but-1-enyl)-2,7-dioxa-4,11-diazadodecan-12-yl)thiazol-4-yl)-4-methyl- 4,5-dihydrothiazole-4-carboxylate (44) 144.93 77.23 76.90 77.54 41.65 119.83 31.29 67.25 31.47 141.44 41.22 84.69 36.28 169.14 156.08 148.46 143.82 171.02 168.43 173.83 66.79 162.94 122.29 53.58 72.63 53.11 133.96 127.60 131.95 47.24 24.15 125.21 120.16 127.27 127.90 129.71 128.06

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm 144.93 141.44 169.14 148.46 143.82 156.08 143.89 168.43 171.02 173.83 162.94 133.96 131.95

175 170 165 160 155 150 145 140 135 ppm 119.83 122.29 127.60 125.21 120.16 127.27 127.90

MeOOC S N NHFmoc

129.71 N S Ph O O O Ph Ph S NH

128.06 44

130.5 130.0 129.5 129.0 128.5 128.0 127.5 127.0 126.5 126.0 125.5 125.0 124.5 124.0 123.5 123.0 122.5 122.0 121.5 121.0 120.5 120.0 119.5 119.0 118.5 ppm

186

MeOOC S N NHFmoc 77.23 76.90 77.54 41.65 N S Ph O O O 31.29 31.47 67.25 Ph 41.22 36.28 84.69 Ph S NH

66.79 44 53.58 72.63 53.11 47.24 24.15

85 80 75 70 65 60 55 50 45 40 35 30 25 ppm

187

1H NMR of cyclic core 45

Chloroform-d 1.63 7.26 1.83 7.38 2.05 7.39 5.30 7.21 1.26 1.25 7.70 7.22 1.27 7.20 4.09 4.08 3.23 4.12 4.11 3.21 2.18 2.19 5.68 5.67 4.72 4.37 2.66 4.35 4.69 2.80 5.26 4.66 4.65 2.82 5.23 2.63 2.03 5.71 5.40 2.35 6.44 6.43 5.70 5.66

0.88 6.55 0.91 1.88 1.09 0.950.930.94 0.92 0.95 1.08 3.93

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm

Chloroform-d 7.26 O S N NH N S 7.38 7.28 Ph OOO Ph 7.39 Ph S N 5.30 7.21 H 7.29 45 7.70 7.22 7.20 5.68 5.67 5.26 5.23 5.24 5.21 5.40 5.71 6.44 6.43 5.38 6.45 5.37 5.70 6.42 5.66 5.72

0.88 5.926.55 3.78 0.91 1.881.00 1.09

7.5 7.0 6.5 6.0 5.5 ppm

1.83 2.05 4.08 3.23 4.09 2.18 4.11 3.21 4.12 2.19 2.66 4.72 4.37 2.66 4.35 4.69 2.80 2.17 2.82 2.08 4.66 4.65 4.19 4.16 4.19 2.03 4.64 2.63 2.35 2.63 2.21 2.83 2.84 2.49 2.02 2.09 2.36 2.36 2.51 2.37 2.50 4.64 2.34 2.00 2.48 2.15

0.95 0.93 0.94 0.94 0.920.95 0.91 0.95 1.08 2.10 3.93 2.73

4.5 4.0 3.5 3.0 2.5 2.0 ppm

188

13 C NMR of cyclic core 45 76.93 77.57 77.25 144.99 119.47 31.39 41.29 31.56 42.66 40.58 169.07 37.97 169.46 173.67 167.81 84.50 147.87 29.94 164.37 124.23 24.78 52.86 72.45 133.67 126.86 128.13 129.79

180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm 144.99 119.47 169.07 169.46 173.67 167.81 147.87 164.37 124.23 131.50 127.96 133.67 O S N NH 126.86 N S Ph OOO Ph

Ph S N 128.13

45 H 129.79

175 170 165 160 155 150 145 140 135 130 125 120 ppm 77.57 77.25 76.93 31.39 41.29 31.56 42.66 40.58 37.97 84.50 29.94 24.78 52.86 72.45

85 80 75 70 65 60 55 50 45 40 35 30 25 ppm

1H NMR of analog T1B

189

7.26

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm 7.26 O S N NH N S O OOO

S N 6 H T1B

7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3 6.2 6.1 6.0 5.9 5.8 5.7 5.6 5.5 5.4 5.3 5.2 ppm

4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1 3.0 2.9 2.8 2.7 2.6 2.5 ppm

2.4 2.3 2.2 2.1 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 ppm

190

13 C NMR of T1B 77.23

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm

O S N NH N S O OOO

S N 6 H T1B

175 170 165 160 155 150 145 140 135 130 125 120 ppm 77.23

85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 ppm

191

1H NMR of ( R)-methyl 2-(2-((5 S,8 S)-1-(9 H-fluoren-9-yl)-3,6,10-trioxo-5-(3-oxo-3- (tritylamino)propyl)-8-(( E)-4-(tritylthio)but-1-enyl)-2,7-dioxa-4,11-diazadodecan- 12-yl)thiazol-4-yl)-4-methyl-4,5-dihydrothiazole-4-carboxylate (46) ethyl acetate 2.05

Ph Ph O MeOOC S Ph N N NHFmoc H N S ethyl acetate Ph O O O 1.26 Ph ethyl acetate Ph S NH

46 4.11

2.14 2.21 17.17 1.00 1.04 1.12 1.00 2.20 4.81 3.06 1.01 1.92 2.10 6.73 2.59 3.15 4.50

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 ppm

1.00 2.14 2.21 8.67 17.17 9.85 1.02 1.00

7.95 7.90 7.85 7.80 7.75 7.70 7.65 7.60 7.55 7.50 7.45 7.40 7.35 7.30 7.25 7.20 7.15 7.10 7.05 7.00 6.95 6.90 6.85 6.80 6.75 6.70 ppm

ethyl acetate 4.11

0.96 1.04 1.08 1.12 0.92 1.00 2.20 4.811.05 3.06

5.8 5.7 5.6 5.5 5.4 5.3 5.2 5.1 5.0 4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7 ppm

192

ethyl acetate 2.05

Ph ethyl acetate Ph O MeOOC S Ph N 1.26 N NHFmoc H N S Ph O O O Ph Ph NH S 46

1.01 1.92 2.10 2.10 6.73 2.59 3.15 4.50

3.3 3.2 3.1 3.0 2.9 2.8 2.7 2.6 2.5 2.4 2.3 2.2 2.1 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 ppm 13 C NMR of ( R)-methyl 2-(2-((5S,8S)-1-(9H-fluoren-9-yl)-3,6,10-trioxo-5-(3-oxo-3- (tritylamino)propyl)-8-((E)-4-(tritylthio)but-1-enyl)-2,7-dioxa-4,11-diazadodecan- 12-yl)thiazol-4-yl)-4-methyl-4,5-dihydrothiazole-4-carboxylate (46)

CDCl3 77.23

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm

175 170 165 160 155 150 145 140 135 130 125 120 ppm

193

CDCl3 77.23

85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 ppm

194

Ethyl acetate 2.05 MeOOC S N NHFmoc Ethyl acetate N S

Ph O O O 1.26 Ph Ethyl acetate Ph S NH 48 4.13

1.00 2.11 4.42 11.09 1.02 3.311.22 3.312.11 2.59 3.36 2.261.28 1.24 2.16 2.31 3.32 0.96 1.88

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

Ethyl acetate 4.13

1.00 2.11 4.42 9.03 11.09 1.02 3.311.22 3.312.11 2.59

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 ppm

Ethyl acetate 2.05

Ethyl acetate 1.26

1.13 3.36 2.26 1.28 0.99 1.24 2.16 1.94 2.31 1.41 3.32 0.96 1.88

3.5 3.0 2.5 2.0 1.5 1.0 ppm

195

13 C NMR of ( R)-methyl 2-(2-((5 S,8 S)-1-(9 H-fluoren-9-yl)-5-(naphthalen-1-ylmethyl)- 3,6,10-trioxo-8-(( E)-4-(tritylthio)but-1-enyl)-2,7-dioxa-4,11-diazadodecan-12- yl)thiazol-4-yl)-4-methyl-4,5-dihydrothiazole-4-carboxylate (48) CDCl3 77.23

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm

MeOOC S N NHFmoc

N S Ph O O O Ph Ph NH S 48

170 165 160 155 150 145 140 135 130 125 120 ppm

CDCl3 77.23

85 80 75 70 65 60 55 50 45 40 35 30 25 ppm

196

1H NMR of ( R)-methyl 2-(2-((( S,E)-3-hydroxy-7-(tritylthio)hept-4- enamido)methyl)thiazol-4-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylate (51)

Ethyl acetate

H 2.05 MeOOC S N

S N Ethyl acetate Ph OH O Ph Ph S NH 1.26 51 Ethyl acetate 4.11

1.00 8.03 1.15 1.291.09 2.41 1.29 1.67 3.41 1.20 1.32 5.39 3.17 3.07 2.47

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 ppm

1.00 7.638.03 3.99 1.15 1.29 1.28 1.09 2.41

7.5 7.0 6.5 6.0 5.5 5.0 ppm

Ethyl acetate 2.05

Ethyl acetate 1.26

Ethyl acetate 4.11

1.29 1.67 3.41 1.20 1.32 1.21 2.53 5.39 3.17 3.07 2.47

4.5 4.0 3.5 3.0 2.5 2.0 1.5 ppm

197

13 C NMR of ( R)-methyl 2-(2-((( S,E)-3-hydroxy-7-(tritylthio)hept-4- enamido)methyl)thiazol-4-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylate (51) CDCl3 77.23

H MeOOC S N

N S Ph OH O Ph Ph S NH 51

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm

175 170 165 160 155 150 145 140 135 130 125 ppm

CDCl3

77.23 Ethyl acetate 60.66 21.23 14.35

Ethyl acetate Ethyl acetate

85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 ppm

198

1H NMR of ( R)-methyl 2-(2-((5 S,8 S)-1-(9 H-fluoren-9-yl)-5-isopropyl-3,6,10-trioxo-8- (( E)-4-(tritylthio)but-1-enyl)-2,7-dioxa-4,11-diazadodecan-12-yl)thiazol-4-yl)-5,5- dimethyl-4,5-dihydrothiazole-4-carboxylate (52)

H MeOOC S N NHFmoc

N S Ph O O O Ph Ph NH S 52

3.18 3.27 17.47 1.22 3.031.26 1.26 2.81 3.02 1.62 4.31 2.81 4.51 1.20 4.27

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

1.00 3.18 3.27 17.47 1.22 3.03 1.26 1.26 2.81

8.0 7.5 7.0 6.5 6.0 5.5 5.0 ppm

3.02 1.62 1.44 4.31 2.81 2.38 4.51 1.20 4.27

4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

199

77.23 Ethyl acetate 60.53 21.23 14.35

Ethyl acetate Ethyl acetate

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm

H MeOOC S N NHFmoc

N S Ph O O O Ph Ph NH S 52

170 165 160 155 150 145 140 135 130 125 120 ppm

CDCl3

77.23 Ethyl acetate 60.53 21.23 14.35

Ethyl acetate Ethyl acetate

85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 ppm

200

1H NMR of ( R)-2,2,2-trichloroethyl 2-(2-(( tert -butoxycarbonylamino)methyl)thiazol- 4-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylate (54)

O H Cl3C S CDCl3 O N 7.26

54 N S H2O 1.57 BocHN

1.00 0.90 1.12 1.12 2.30 3.38 10.35

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 ppm

CDCl3 7.26

1.00 0.90 1.12 1.12 2.30

8.0 7.5 7.0 6.5 6.0 5.5 5.0 ppm

H2O 1.57

3.38 4.28 3.24 10.35

1.85 1.80 1.75 1.70 1.65 1.60 1.55 1.50 1.45 1.40 1.35 ppm

201

13 C NMR of ( R)-2,2,2-trichloroethyl 2-(2-(( tert - butoxycarbonylamino)methyl)thiazol-4-yl)-5,5-dimethyl-4,5-dihydrothiazole-4- carboxylate (54)

CDCl3 77.23

O H Cl3C S O N

54 N S

BocHN

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

170 165 160 155 150 145 140 135 130 125 120 115 110 105 100 95 ppm

202

1H NMR of ( R)-2,2,2-trichloroethyl 2-(2-((( S,E)-3-hydroxy-7-(tritylthio)hept-4- enamido)methyl)thiazol-4-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylate (55) Ethyl acetate 2.04 O H S Cl3C O N

N S Ethyl acetate

Ph OH O 1.26 Ph Ph S NH Ethyl acetate 55 4.11

0.77 6.60 1.030.972.07 1.01 1.94 0.91 2.25 4.882.56 2.52 3.13

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

0.77 6.386.60 4.13 1.001.03 0.97 0.92 2.07 1.01

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 ppm

Ethyl acetate 2.04

Ethyl acetate 1.26

Ethyl acetate 4.11

1.94 0.91 2.25 2.14 4.88 2.56 2.52 3.13

4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

203

13 C NMR of ( R)-2,2,2-trichloroethyl 2-(2-((( S,E)-3-hydroxy-7-(tritylthio)hept-4- enamido)methyl)thiazol-4-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylate (55)

CDCl3 77.23

O H S Cl3C O N

N S Ph OH O Ph Ph S NH 55

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

175 170 165 160 155 150 145 140 135 130 125 120 ppm

CDCl3 77.23

85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 ppm

204

2.26 2.27 8.95 0.81 2.00 1.00 0.87 1.69 2.23 1.81 4.662.43 2.66 3.94 8.48

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

0.69 2.26 2.27 8.448.95 3.13 0.81 2.00 1.00 0.98 0.87 0.83 1.69

8.0 7.5 7.0 6.5 6.0 5.5 5.0 ppm

Ethyl acetate 2.05 Cl3C O H S O N Ethyl acetate NHFmoc 1.26 N S Ph O O O Ethyl acetate Ph Ph NH 4.11 S 56

2.23 1.81 2.01 4.66 2.43 2.66 3.94 8.48

4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

205

13C NMR of ( R)-2,2,2-trichloroethyl 2-(2-((5 S,8 S)-1-(9 H-fluoren-9-yl)-5-isopropyl- 3,6,10-trioxo-8-(( E)-4-(tritylthio)but-1-enyl)-2,7-dioxa-4,11-diazadodecan-12- yl)thiazol-4-yl)-5,5-dimethyl-4,5-dihydrothiazole-4-carboxylate (56) CDCl3 77.23

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

Cl3C O H S O N NHFmoc N S Ph O O O Ph Ph NH S 56

170 165 160 155 150 145 140 135 130 125 120 ppm

CDCl3 77.23

Cl3C O H S O N NHFmoc

N S Ph O O O Ph Ph NH S 56

85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 ppm

206

1H NMR of (S)-methyl 2-(2-((( S,E)-3-hydroxy-7-(tritylthio)hept-4- enamido)methyl)thiazol-4-yl)-4-methyl-4,5-dihydrothiazole-4-carboxylate (59)

MeOOC S N

N S Ph OH O Ph Ph S NH 59

0.02 0.17 0.03 0.020.05 0.02 0.07 0.04 0.03 0.06 0.08

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 ppm

MeOOC S N

N S Ph OH O Ph Ph S NH 59

0.02 0.150.17 0.08 0.03 0.02 0.02

7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3 6.2 6.1 6.0 5.9 5.8 5.7 5.6 5.5 5.4 ppm

0.05 0.02 0.02 0.07 0.04 0.03 0.05 0.06 0.08

4.5 4.0 3.5 3.0 2.5 2.0 1.5 ppm

207

13 C NMR of ( S)-methyl 2-(2-((( S,E)-3-hydroxy-7-(tritylthio)hept-4- enamido)methyl)thiazol-4-yl)-4-methyl-4,5-dihydrothiazole-4-carboxylate (59)

MeOOC S N

N S Ph OH O Ph Ph S NH 59

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 ppm

175 170 165 160 155 150 145 140 135 130 125 120 ppm

208

MeOOC S N

N S Ph OH O Ph Ph S NH 59

85 80 75 70 65 60 55 50 45 40 35 30 25 ppm

209

1H NMR of ( S)-methyl 2-(2-((5 S,8 S)-1-(9 H-fluoren-9-yl)-5-isopropyl-3,6,10-trioxo-8- (( E)-4-(tritylthio)but-1-enyl)-2,7-dioxa-4,11-diazadodecan-12-yl)thiazol-4-yl)-4- methyl-4,5-dihydrothiazole-4-carboxylate (60)

MeOOC S N NHFmoc

N S Ph O O O Ph Ph S NH 60

2.37 2.52 12.98 0.97 2.241.181.02 2.00 2.19 1.163.25 1.08 1.98 3.883.65 4.24 3.77

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

0.97 2.37 2.52 9.3912.98 3.33 0.97 2.24 1.18 1.02 2.00

8.0 7.5 7.0 6.5 6.0 5.5 5.0 ppm

2.19 1.16 1.04 1.03 3.25 1.08 1.98 2.22 3.88 3.65 4.24 3.77

4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

210

MeOOC S N NHFmoc

N S Ph O O O Ph Ph S NH 60

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm

175 170 165 160 155 150 145 140 135 130 125 120 ppm

211

MeOOC S N NHFmoc

N S Ph O O O Ph Ph S NH 60

85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 ppm

212

1H NMR of cyclic core 61 Ethyl acetate 2.05

O S N NH Ethyl acetate Ph N S

Ph Ph OOO 1.26

S N Ethyl acetate 61 H 4.11

1.00 6.05 0.96 0.98 0.940.96 1.00 1.01 0.91 0.96 0.99 0.94 2.73 2.94 5.38

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

1.00 6.05 0.96 0.98 0.94 0.96 1.00 1.01

7.5 7.0 6.5 6.0 5.5 5.0 4.5 ppm

Ethyl acetate 2.05

Ethyl acetate 1.26

Ethyl acetate 4.11

0.91 0.96 0.99 0.90 0.94 2.73 2.94 5.38

4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

213

1H NMR of cyclic core 61

O S N NH Ph N S Ph Ph OOO

S N 61 H

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

175 170 165 160 155 150 145 140 135 130 125 120 ppm

214

O S N NH Ph N S Ph Ph OOO

S N 61 H

85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 ppm

215

1H NMR of analog T3 (C-7 largazole epimer) CDCl3 7.26

O S N NH N S O OOO

H C(H C) S N 3 2 6 H T3 (C-7 epimer of largazole)

0.88 1.00 1.06 1.26 1.97 1.01 1.29 1.05 1.18 3.881.46 3.552.57 10.63 2.89 3.37

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

CDCl3 7.26

0.88 1.00 1.06 1.26 1.971.01 1.29 1.02 1.05

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 ppm

1.18 3.88 2.23 1.46 2.15 3.55 2.79 2.57 10.63 2.99 3.37

3.4 3.3 3.2 3.1 3.0 2.9 2.8 2.7 2.6 2.5 2.4 2.3 2.2 2.1 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 ppm

216

13 C NMR of analog T3 (C-7 largazole epimer)

O S N NH N S O OOO

H C(H C) S N 3 2 6 H T3 (C-7 epimer of largazole)

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm

200 195 190 185 180 175 170 165 160 155 150 145 140 135 130 125 120 115 ppm

217

O S N NH N S O OOO

H C(H C) S N 3 2 6 H T3 (C-7 epimer of largazole)

70 65 60 55 50 45 40 35 30 25 20 15 ppm

218

1H NMR of analog T4A

7.26

O S N T4A NH N S OOO

S N H N

0.89 0.86 0.95 1.01 1.81 0.93 1.00 1.03 1.01 1.08

8.5 8.0 7.5 7.0 6.5 6.0 5.5 ppm

O S N NH T4A N S OOO

S N H N

1.00 1.00 1.07 1.95 1.01 0.99 1.04 1.932.01 1.23 2.97 2.77 2.62

4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

219

13 C NMR of analog T4A

O S 77.23 N NH T4A N S OOO

S N H N

180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

O S N T4A NH N S OOO

S N H N

170 165 160 155 150 145 140 135 130 125 120 ppm

O S N T4A NH N S OOO

S N H N

70 65 60 55 50 45 40 35 30 25 20 ppm

220

1H NMR of analog T4B

O S N T4B NH N S OOO

S N H S

7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3 6.2 6.1 6.0 5.9 5.8 5.7 5.6 5.5 5.4 5.3 5.2 ppm

O S N NH T4B N S OOO

S N H S

4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1 3.0 2.9 2.8 2.7 ppm

221

13 C of analog T4B O S N NH T4B

N S 77.23 OOO

S N H S

180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

O S N NH T4B N S OOO

S N H S

180 175 170 165 160 155 150 145 140 135 130 125 ppm

O S

77.23 N NH T4B N S OOO

S N H S

85 80 75 70 65 60 55 50 45 40 35 30 25 20 ppm

222

1H NMR of analog T4C

7.26

O S N T4C NH N S OOO

S N H OH

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm

O S N T4C NH N S OOO

S N H OH

2.7 2.6 2.5 2.4 2.3 2.2 2.1 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 ppm

223

13 C NMR of analog T4C

O S N 77.23 T4C NH N S OOO

S N H OH

180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

O S N T4C NH N S OOO

S N H OH

175 170 165 160 155 150 145 140 135 130 125 120 115 ppm 77.23

O S N T4C NH N S OOO

S N H OH

85 80 75 70 65 60 55 50 45 40 35 30 25 20 ppm

224

1H NMR of analog T4D CDCl3 7.26

dichloromethane 5.30

0.95 1.00 1.06 1.07 1.88 1.071.05 1.07 3.05 3.18 3.18 2.10 1.38 3.26 3.10 2.95

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

CDCl3 7.26

O S N NH dichloromethane N S 5.30 CH3 OOO N H3C S N T4D H O

0.95 1.00 1.061.07 1.05 1.88 1.071.05 1.07

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 ppm

3.05 3.01 3.181.08 3.18 2.10 1.38 3.26 3.10 2.95

3.0 2.5 2.0 1.5 1.0 0.5 ppm

225

1H NMR of ( S)-3-hydroxy-1-(( R)-4-isopropyl-2-thioxothiazolidin-3-yl)pent-4-en-1- one (62)

CDCl3 7.26

OH O S

N S

0.96 0.971.93 0.97 1.00 1.031.03 1.06 0.93 1.03 3.37 3.21

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

0.96 0.97 1.93 0.97 1.00 1.03 1.03 1.06 0.93

6.0 5.5 5.0 4.5 4.0 3.5 3.0 ppm

1.03 3.37 3.01 3.21

2.5 2.4 2.3 2.2 2.1 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 ppm

226

13 C NMR of (S)-3-hydroxy-1-(( R)-4-isopropyl-2-thioxothiazolidin-3-yl)pent-4-en-1- one (62)

CDCl3 77.23

OH O S

N S

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm 1H NMR of ( R)-methyl 2-(2-((( S)-3-hydroxypent-4-enamido)methyl)thiazol-4-yl)-4- methyl-4,5-dihydrothiazole-4-carboxylate (64) CDCl3 7.26 MeOOC S N

N S OH O

NH 64

1.00 1.02 1.081.15 1.082.15 1.07 3.36 1.76 1.10 3.40

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 ppm

CDCl3 7.26

MeOOC S N

N S OH O

NH 64

1.00 1.02 1.08 1.15 1.082.15 1.07

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 ppm

227

1.09 3.36 1.76 1.10 1.08 3.40

3.9 3.8 3.7 3.6 3.5 3.4 3.3 3.2 3.1 3.0 2.9 2.8 2.7 2.6 2.5 2.4 2.3 2.2 2.1 2.0 1.9 1.8 1.7 1.6 1.5 ppm 13 C NMR of ( R)-methyl 2-(2-((( S)-3-hydroxypent-4-enamido)methyl)thiazol-4-yl)-4- methyl-4,5-dihydrothiazole-4-carboxylate (64) 115.60 41.68 CDCl3 40.93 42.66 77.23 84.66 168.03 172.08 148.38 162.92 173.77 122.58 53.16 139.21 69.58 24.15

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 ppm

228

1H NMR of ( R)-methyl 2-(2-((5 S,8 S)-1-(9 H-fluoren-9-yl)-5-isopropyl-3,6,10-trioxo-8- vinyl-2,7-dioxa-4,11-diazadodecan-12-yl)thiazol-4-yl)-4-methyl-4,5-dihydrothiazole- 4-carboxylate (65)

MeOOC S N NHFmoc

N S O O O

NH 65

2.00 2.03 2.13 0.78 0.96 0.84 1.64 2.11 1.01 2.49 0.821.58 0.82 2.33 2.58

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

0.73 2.00 2.03 2.07 2.13 0.70 0.780.96 0.75 0.84 0.82 1.64

7.5 7.0 6.5 6.0 5.5 5.0 ppm

MeOOC S N NHFmoc

N S O O O

NH 65

2.11 1.010.80 0.862.49 0.82 1.58 0.82 2.33 2.58

4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

229

13 C NMR of ( R)-methyl 2-(2-((5 S,8 S)-1-(9 H-fluoren-9-yl)-5-isopropyl-3,6,10-trioxo- 8-vinyl-2,7-dioxa-4,11-diazadodecan-12-yl)thiazol-4-yl)-4-methyl-4,5- dihydrothiazole-4-carboxylate (65) 41.17

CDCl3 41.53 118.53 67.14 141.34 169.17 77.23 84.57 156.55 143.74 171.34 148.31 168.59 162.88 173.72 60.51 21.16 14.28 122.27 53.01 19.13 30.89 59.66 134.34 125.11 47.18 72.49 24.04 Ethyl acetate 127.19

120.07 Ethyl acetate 127.81

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm 118.53 141.34 169.17 156.55 143.74 171.34 148.31 168.59 162.88 173.72 122.27 134.34 125.11 127.19 120.07 127.81

175 170 165 160 155 150 145 140 135 130 125 120 ppm 41.17 41.53 CDCl3 67.14 77.23 84.57 60.51 21.16 14.28

MeOOC S 53.01 19.13 30.89 N 59.66 Ethyl acetate NHFmoc 47.18 72.49 24.04 N S Ethyl acetate O O O

NH 65

85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 ppm

230

1H NMR of cyclic core 66

O S N NH N S OOO Ethyl acetate

CdCl3 2.04 N Ethyl acetate 7.26 H Ethyl acetate 66 1.25 4.10

0.86 1.00 1.02 1.00 1.041.85 1.00 1.03 1.00 0.99 1.01 1.00 2.87 2.692.57

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

CdCl3 7.26

0.86 1.00 1.02 1.00 1.04 1.05 1.85 1.00

8.0 7.5 7.0 6.5 6.0 5.5 5.0 ppm

Ethyl acetate

2.04 Ethyl acetate

Ethyl acetate 1.25 4.10

1.03 1.00 0.99 1.01 1.01 1.00 2.87 2.69 2.57

4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

231

13 C NMR of cyclic core 66

CDCl3 118.14 41.27 40.33 77.23 43.51 169.52 168.08 84.56 169.07 173.77 147.63 164.80

O S N NH 124.45 16.80 N S 19.09

OOO 34.40 24.38 57.86 72.49 N 134.89 66 H

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm 1H NMR N-(benzyloxy)acrylamide (67) DMSO-d6 2.49

O O N H 67

0.84 5.07 1.21 1.13 2.00 3.77

11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 ppm

5.07 1.21 1.17 1.13

7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3 6.2 6.1 6.0 5.9 5.8 5.7 5.6 5.5 ppm

232

13 C NMR N-(benzyloxy)acrylamide (67) CDCl3 77.23 78.26 127.62 135.28 163.99 127.42

O O N H 67 128.58 129.26

170 165 160 155 150 145 140 135 130 125 120 115 110 105 100 95 90 85 80 75 70 65 60 ppm 127.62 127.42 128.72 128.58 129.26

131.0 130.5 130.0 129.5 129.0 128.5 128.0 127.5 127.0 126.5 126.0 125.5 125.0 124.5 124.0 123.5 123.0 ppm

233

1H NMR of analog T5 O S N CDCl3 NH

7.26 N S OOO H N HO N H O T5

1.101.26 1.00 1.18 1.32 1.29 1.36 1.36 3.401.52 1.61 3.54 4.28 6.48

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

CDCl3 7.26

1.101.26 1.00 1.18 1.32 1.29

7.5 7.0 6.5 6.0 5.5 5.0 4.5 ppm

1.36 3.40 3.15 1.52 1.61 3.54 4.28 6.48

4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

234

1H NMR of methyl 2-(( tert -butoxycarbonylamino)methyl)thiazole-4-carboxylate (69)

MeOOC

N S

CDCl3 BocHN 69 7.26

0.82 1.01 2.12 3.00 8.73

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 ppm 13 C NMR of methyl 2-(( tert -butoxycarbonylamino)methyl)thiazole-4-carboxylate (69) CDCl3 77.23 42.58 161.94 146.70 80.73 170.33 155.80 128.37 52.72 28.50

180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

235

1H NMR of (R,E)-methyl 2-((3-hydroxy-7-(tritylthio)hept-4- enamido)methyl)thiazole-4-carboxylate (70)

MeOOC

70 N S Ph OH O Ph Ph S NH

0.82 8.02 1.00 2.21 1.00 2.61 1.09 2.18

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 ppm

0.82 6.688.02 3.31 1.00 0.99

8.0 7.5 7.0 6.5 6.0 5.5 ppm

2.21 1.00 2.61 1.09 1.05 2.18 2.15

4.5 4.0 3.5 3.0 2.5 2.0 ppm

236

13 C NMR of (R,E)-methyl 2-((3-hydroxy-7-(tritylthio)hept-4- enamido)methyl)thiazole-4-carboxylate (70)

MeOOC 129.67 128.01 126.76 70 N S Ph OH O Ph Ph S NH 144.93 31.42

CDCl3 31.54 69.20 40.97 130.22 132.38 42.88 52.66 77.23 66.69 172.28 168.81 161.75 146.19 61.73

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 ppm 129.67 128.01 126.76 144.93 130.22 132.38 128.62 172.28 168.81 161.75 146.19

170 165 160 155 150 145 140 135 130 125 ppm 31.42 31.54 CDCl3 69.20 40.97 42.88 52.66 77.23 66.69 61.73

80 75 70 65 60 55 50 45 40 35 ppm

237

1H NMR of methyl 2-((5 S,8 R)-8-(( E)-4-(acetylthio)but-1-enyl)-1-(9 H-fluoren-9-yl)-5- isopropyl-3,6,10-trioxo-2,7-dioxa-4,11-diazadodecan-12-yl)thiazole-4-carboxylate (analog T6) CDCl3 7.26

MeOOC NHFmoc N S O O O O

S NH T6

0.87 1.97 2.01 2.02 0.77 0.91 0.93 1.00 0.98 1.802.87 1.67 1.05 4.562.24 2.97

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

CDCl3 7.26

0.87 1.97 2.01 2.02 2.01 0.77 0.91 0.920.93 0.91

8.0 7.5 7.0 6.5 6.0 5.5 ppm

1.000.92 0.980.93 1.80 2.87 1.670.91 1.05 4.56 2.24 2.97

4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

238

13 C NMR of methyl 2-((5 S,8 R)-8-(( E)-4-(acetylthio)but-1-enyl)-1-(9 H-fluoren-9-yl)- 5-isopropyl-3,6,10-trioxo-2,7-dioxa-4,11-diazadodecan-12-yl)thiazole-4-carboxylate (analog T6) 127.28 120.22 127.95 72.38 47.35 32.33 19.25 125.24 28.34 128.63 67.25 31.25 41.59 17.60 41.18 59.34 133.03 30.84 52.75 141.50 169.16 171.01 161.79 156.61 143.94 99.94 168.09 146.58 195.88

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm 127.28 120.22

MeOOC 127.95 NHFmoc N S O O O O 125.24 128.63 S NH

T6 133.03 141.50 169.16 171.01 161.79 156.61 143.94 168.09 146.58 195.88

200 195 190 185 180 175 170 165 160 155 150 145 140 135 130 125 120 ppm 72.38 47.35 32.33 19.25 28.34 67.25 31.25 41.59 17.60 41.18 59.34 30.84 52.75 29.92 99.94

100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 ppm

239

1H NMR of ( S, E)-methyl 2-((3-hydroxy-7-(tritylthio)hept- 4enamido)methyl)thiazole-4-carboxylate (72)

MeOOC 72 N S Ethyl acetate Ph OH O Ph 2.05 Ph S NH Ethyl acetate

Ethyl acetate 1.26 4.11

0.93 6.23 1.001.96 0.970.44 2.90 0.94 1.05 2.72 0.65

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 ppm

0.93 6.186.23 3.73 1.00 0.96

8.0 7.5 7.0 6.5 6.0 5.5 ppm

Ethyl acetate 2.05

Ethyl acetate 4.11

1.96 0.97 0.44 2.90 0.94 1.05 0.96 2.01 2.72

4.5 4.0 3.5 3.0 2.5 2.0 ppm

240

13 C NMR of ( S, E)-methyl 2-((3-hydroxy-7-(tritylthio)hept- 4enamido)methyl)thiazole-4-carboxylate (72)

144.93 CDCl3 31.44 172.20 41.00 42.92 146.23 168.73 77.23 161.74 66.70 52.67 69.21 132.38

126.75 MeOOC 72

128.01 N S Ph OH O Ph 129.67 Ph S NH

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 ppm 144.93 172.20 146.23 168.73 161.74 128.60 130.23 132.38 126.75 128.01 129.67

170 165 160 155 150 145 140 135 130 ppm

CDCl3 31.44 41.00 31.56 42.92 77.23 66.70 52.67 69.21

75 70 65 60 55 50 45 40 35 30 ppm

241

1H NMR of methyl 2-((5 S,8 S)-1-(9 H-fluoren-9-yl)-5-isopropyl-3,6,10-trioxo-8-(( E)-4- (tritylthio)but-1-enyl)-2,7-dioxa-4,11-diazadodecan-12-yl)thiazole-4-carboxylate (73)

MeOOC NHFmoc

N S Ph O O O Ph Ph S NH 73

0.94 2.21 2.30 9.32 0.98 2.172.27 2.09 2.16 1.12 3.31 1.97 3.54 4.31

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

0.94 2.21 2.30 8.729.32 3.34 0.98 2.17 1.00 2.27

8.0 7.5 7.0 6.5 6.0 5.5 ppm

2.09 2.16 1.12 1.10 3.31 1.97 2.17 3.54 4.31

4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

242

13 C NMR of methyl 2-((5 S,8 S)-1-(9 H-fluoren-9-yl)-5-isopropyl-3,6,10-trioxo-8-(( E)- 4-(tritylthio)but-1-enyl)-2,7-dioxa-4,11-diazadodecan-12-yl)thiazole-4-carboxylate (73)

CDCl3 144.94 77.23 67.25 31.28 31.47 41.37 141.45 41.54 143.88 156.59 169.19 146.33 161.82 66.78 18.05 19.18 31.02 52.59 59.60 47.26 134.19 72.42 125.18 120.18 126.81 129.72 128.05

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm 144.94 141.45 143.88 156.59 169.19 146.33 161.82 128.60 134.19 125.18 120.18

MeOOC 127.92 NHFmoc

N S 126.81 Ph O O O Ph Ph S NH 73 129.72 128.05

170 165 160 155 150 145 140 135 130 125 120 ppm

CDCl3 77.23 67.25 31.28 31.47 41.37 41.54 66.78 18.05 19.18 31.02 52.59 59.60 47.26 72.42

75 70 65 60 55 50 45 40 35 30 25 20 ppm

243

1H NMR of ( S,E)-2-(trimethylsilyl)ethyl 3-hydroxy-7-(tritylthio)hept-4-enoate (75)

Ph OH O Ph Ph S O 75 Si

12.63 1.521.00 0.47 1.05 0.44 0.48 0.96 2.22 0.99 3.92

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm

6.16 12.63 1.52 1.00 0.47 1.05

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 ppm

0.44 0.48 0.47 0.96 2.02 2.22 0.99 3.92

3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 ppm

244

13 C NMR of ( S,E)-2-(trimethylsilyl)ethyl 3-hydroxy-7-(tritylthio)hept-4-enoate (75)

145.02 41.67 31.61 63.24 17.49 172.69 -1.29 68.75 132.09 130.29 126.75 Ph OH O Ph Ph S O

128.01 75 Si 129.73

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

245

1H NMR of ( S,E)-2-(trimethylsilyl)ethyl 3-(( S)-2-(((9 H-fluoren-9- yl)methoxy)carbonylamino)-3-methylbutanoyloxy)-7-(tritlthio)hept-4-enoate (76)

NHFmoc

O Ph O O Ph Ph S O 76 Si

2.32 2.37 10.34 2.34 2.28 2.24 2.22 1.10 3.24 2.22 3.82 8.18

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm

NHFmoc

O Ph O O Ph Ph S O 76 Si

2.32 2.37 9.8810.34 3.86 2.34 2.28

7.5 7.0 6.5 6.0 5.5 ppm

2.240.98 2.22 1.00 1.10 3.24 2.41 3.28 3.82 8.18

4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm

246

13 C NMR of ( S,E)-2-(trimethylsilyl)ethyl 3-(( S)-2-(((9 H-fluoren-9- yl)methoxy)carbonylamino)-3-methylbutanoyloxy)-7-(tritlthio)hept-4-enoate (76)

144.96 31.25 141.44 31.52 63.33 39.82 67.18 156.29 143.93 169.80 17.45 171.05 19.16 -1.31 58.87 31.58 72.03 47.36 134.23 125.28 120.15 126.77 129.71 128.03

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm 144.96 141.44 156.29 143.93 169.80 171.05 134.23 125.28 120.15

NHFmoc 127.86 126.77 O Ph O O Ph Ph S O

76 Si 129.71 128.03

170 165 160 155 150 145 140 135 130 125 120 ppm 31.25 31.52 63.33 39.82 67.18 17.45 19.16 -1.31 58.87 31.58 72.03 47.36

80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 ppm

247

1H NMR of ( S,E)-2-(trimethylsilyl)ethyl 3-(( S)-2-(2-(( tert - butoxycarbonylamino)methyl)thiazole-4carboxamido)-3-methylbutanoyloxy)-7- tritylthio)hept-4-enoate (77)

HN NS O 77 Ph O O Ph NHBoc Ph S O Si

1.08 1.11 9.18 1.04 1.07 1.18 1.82 1.02 1.04 1.96 7.924.53 6.77

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm

HN NS O 77 Ph O O Ph NHBoc Ph S O Si

1.08 1.11 5.34 9.18 1.04 1.07 1.05 1.00 1.18 1.82

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 ppm

1.02 1.00 1.04 1.88 1.96 7.92 4.53 3.72 6.77

2.5 2.0 1.5 1.0 0.5 0.0 -0.5 ppm

248

13 C NMR of ( S,E)-2-(trimethylsilyl)ethyl 3-(( S)-2-(2-(( tert - butoxycarbonylamino)methyl)thiazole-4carboxamido)-3-methylbutanoyloxy)-7- tritylthio)hept-4-enoate (77)

CDCl3 144.94 31.25 63.31 77.23 39.86 31.52 169.80 160.85 170.80 17.42 149.54 66.77 42.47 -1.34 17.77 19.29 31.85 72.02 56.92 134.21 28.51 126.77 128.02 129.71

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

249