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Doctoral Dissertations Student Theses and Dissertations

Spring 2007

Synthetic methods for biologically relevant organofluorine compounds

Meher Perambuduru

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SYNTHETIC METHODS FOR BIOLOGICALLY RELEVANT ORGANOFLUORINE

COMPOUNDS

by

MEHERPERAMBUDURU

A DISSERTATION

Presented to the Faculty ofthe Graduate School of the

UNIVERSITY OF MISSOURI-ROLLA

In Partial Fulfillment of the Requirements for the Degree

DOCTOR OF PHILOSOPHY

m

CHEMISTRY

2007

V. PRAKASH REDDY EKKEHARD SINN ~~ NURANERCAL 111

ABSTRACT

This thesis describes novel synthetic methodologies for the synthesis of biologically relevant organofluorine compounds. Chapter 1 outlines the recent trends in this area and our synthetic procedures for the preparation of the gem-difluoromethylene analogues of the biologically active dipeptides, carnosine and carcinine. Synthesis of the latter compounds has been achieved using the fluorinated building block approach. Thus reaction of gem-difluoro-~­ alanine with histidine methyl ester dihydrochloride or histamine dihydrochloride using N­ benzyloxycarbonyl (N-Cbz) protecting strategy and EDCVHOBt peptide coupling protocol gave the gem-difluorinated version of the peptides carnosine and carcinine respectively. The gem­

Difluoro analogue of ~-alanine was prepared from a fluorine synthon, ethyl bromodifluoroacetate using Reforrnatsky reaction conditions.

Chapter 2 is focused on development of novel synthetic method for the preparation of gem-difluorodiarylmethanes from the corresponding carbonyl compounds. Thus, 2,2-diaryl-1,3- dithiolanes, readily obtainable from the corresponding diaryl ketones, were converted to the corresponding gem-difluorodiarylmethanes using a novel reagent combination SelectfluorR and

Olah's reagent, pyridinium polyhydrogen fluoride (PPHF). This reaction is convenient, effective, and requires simple workup and mild reaction conditions. The reaction, although broadly applicable for diaryl ketones, is not useful for the preparation of their aliphatic versions.

Chapter 3 delineates the preparation of trifluoromethylated arylsulfonamides by nucleophilic trifluoromethylation of aromatic N-tosylaldimines using trifluoromethyl trimethylsilane in the presence of N-heterocyclic carbene as a catalyst. N-heterocyclic carbene was produced in situ through the reaction from imidazolium chloride and potassium tert­ butoxide. Aromatic N-tosylaldimines with both electron-withdrawing and electron-donating groups underwent facile trifluoromethylation in the presence ofN-heterocyclic carbene to afford the corresponding trifluoromethylated sulfonamides in moderate to good yields. However, further optimizations are needed for the trifluoromethylation of aliphatic imines using this reagent. IV

ACKNOWLEDGMENTS

I am deeply grateful to my advisor, Dr. V. Prakash Reddy for his excellent guidance, encouragement and enthusiasm over the years he has provided. I appreciate his patience, insightful comments and for giving me the opportunity to think and learn independently and providing me the freedom in performing the research. Without his mentorship, constant support and caring this thesis would not have been possible.

I am thankful to Dr. Ekkehard Sinn, Dr. Yinfa Ma, Dr. Nuran Ercal and Dr.

Oliver C. Sitton for serving on my graduate committee and helping me throughout my

Ph.D. program.

I am particularly thankful to my beloved friend Mr. Ramesh Alleti for his friendship, guidance and support throughout my career and my life. I am greatly thankful to my friends Uday Kumar, Srinivas Reddy Dubbaka, Devender Pinnapareddy, Achanta

Srinivas, Vikram kanmukhla, and Srinivas Gorla for putting up with me and for their continuous support, encouragement and contribution.

I would like to express my deepest gratitude to my parents, brother and other family members for their abundant love, encouragement, and support in every decision I made and every step I took throughout my life. v

TABLE OF CONTENTS

ABSTRACT ...... iii

ACKNOWLEDGMENTS ...... iv

LIST OF FIGURES ...... viii

NOMENCLATURE ...... XV

l. SYNTHESIS OF GEM--DIFLUOROMETHYLENE ANALOGUES OF CARNOSINE AND GEM-DIFLUOROMETHYLENE CARCININE ...... 1

1. l INTRODUCTION ...... 1

1.1.1 Fluorinated versions of peptide analogues/mimetics ...... 1

1.1.2 Synthetic methods for selected fluorinated peptide analogues ...... !

1.1.2.1 Difluorostatone and difluorostatine peptide analogues ...... 2

1.1.2.2 Difluoroketomethylene peptide isostere ...... 6

1.1.2.3 Monofluoro ketomethylene peptide isostere ...... 9

1.1.2.4 Monofluoro hydroxyethylene peptide isosteres ...... 10

1.1.2.5 Fluoroalkene and (trifluoromethyl)alkene peptide isosteres ...... 14

1.1.2.6 Fluorinated amino acids and pep tides ...... 17

1.2 RESULTS AND DISCUSSIONS ...... 23

1.2.1 Synthesis of gem-difluoromethylene analogue of L-camosine ...... 27

1.2.2 Synthesis of gem-difluoromethylene carcinine ...... 36

1.3 CONCLUSIONS ...... 38

1.4 EXPERIMENTAL RESULTS ...... 39

1.4.1 General...... 39

1.4.2 Synthesis and properties of products ...... 40

1.4.3 NMR spectra of the products ...... 60 VI

1.5 REFERENCES ...... 82

2. A NOVEL AND CONVENIENT SYNTHETIC METHOD FOR THE PREPARATION OF GEM-DIFLUORODIARYLMETHYLENE COMPOUNDS ... 89

2.1 INTRODUCTION ...... 89

2.1.1 Aldehydes and ketones to gem-difluoromethylene compounds ...... 90

2.1.2 Hydrazones and Oximes to gem-difluoromethylene compounds...... 93

2.1.3 Dithioacetals and dithioketals to gem-difluoromethylene compounds ...... 95

2.1.4 Thiocarbonyls to gem-difluoromethylene compounds ...... 97

2.1.5 Azirines to gem-difluoromethylene compounds ...... 98

2.1.6 Olefinic and acetylenic compounds to gem-difluoromethylene compounds ...... 98

2.1.6.1 Alkenyl trifluoroborates ...... 98

2.1.6.2 Alkenes ...... 99

2.1.6.3 Allylamines ...... 100

2.1.6.4 Alkynes ...... 101

2.1. 7 Electrophilic difluorination ...... 102

2.2 RESULTS AND DISCUSSIONS ...... 104

2.3 CONCLUSIONS ...... 11 0

2.4 EXPERIMENTAL RESULTS ...... 110

241General. . ································· ...... 110 2.4.2 General procedure for the preparation of gem-difluoromethylene compounds ...... ················································· .110

2.4.3 Synthesis and properties of products ...... 111

2.4.5 NMR spectra ofproducts ...... ··· ...... ··· · ·· · ········· ·· ...... 116

2 .5 REFERENCES ······························ ...... 119 Vll

3. CARBENE CATALYZED TRIFLUOROMETHYLATIONS OF AROMATIC N- TOSYL-ALDIMINES ...... 124

3.1 INTRODUCTION ...... 124

3.1.1 Nucleophilic trifluoromethylation ofimines ...... l24

3.1.2 N-Hetereocyclic carbene as a catalyst...... 130

3.2 RESULTS AND DISCUSSIONS ...... 135

3.3 CONCLUSIONS ...... 139

3.4 EXPERIMENTAL RESULTS ...... 140

3.4.1 General...... : ...... 140

3.4.2 General procedure for the preparation of aromatic N-tosyl aldimines ..... 140

3.4.3 General procedure for the preparation of aliphatic N-tosyl aldimines ...... 141

3.4.4 General procedure for trifluoromethylation of aromatic N-tosyl aldimines ...... 141

3.4.5 Synthesis and properties ofproducts ...... : ...... 142

3.4.6 NMR spectra of products ...... 148

3.5 REFERENCES ...... 154

VITA ...... 158 Vlll

LIST OF FIGURES

Figure Page

1.1: Structural representations of slective fluorinated peptide analogues/mimetics/isosteres ...... 2

1.2: Structures of difluorostatine and difluorostatone analogues ...... 3

1.3: Hydration of difluorostatone peptide analogue ...... 3

1.4: Synthetic procedure for difluorostatine and difluorostatone analogues ...... 5

1.5: Hydration of peptide bond and difluoro ketone ...... 6

1.6: Synthetic procedure for difluoroketomethylene peptide isostere ...... 8

1. 7: Synthetic procedure for monofluoro ketomethylene peptide isostere ...... 10

1.8: Synthetic preocedure for monofluoro hydroxyethylene peptide isostere ...... 13

1.9: Synthetic procedure for fluoroalkene peptide isostere ...... 15

1.10: Synthetic procedure for (trifluoromethyl)alkene peptide isostere ...... 16

1.11: Synthetic procedure for chirally pure 4-trifluoro-B-lactams ...... 18

1.12: Alternative method for the preparation for 4-trifluoro-B-lactam ...... 18

1.13: Transformation of fluorinated B-lactams to fluorinated amino acids, peptides and taxoids ...... 19

1.14: Synthetic procedure for trifluoromethyl peptidomimetic hydroxamate ...... 21

1.15: Synthetic procedure for gem-difluoro analogue ofRhodopeptin...... 23

1.16: Structures of Carnosine, Carcinine, and Anserine ...... 24

1.17: Examples of reported carnosine analogues ...... 25

1.18: Structures of synthesized gem-difluoro analogues of carnosine and carcinine ...... 26

1.19: Retrosynthetic analysis of the gem-difluoromethylene analogue of L-carnosine .... 27 IX

1.20: Retrosynthetic analysis ofthe gem-difluoromethylene carcinine ...... 27

1.21: Preparation ofN-benzyl protectedgem-difluoro P-alanine ...... 28

1.22: Different methods for the peptide bond formation between histidine methyl ester hydrochloride and 3-(Dibenzylamino )-2,2-difluoropropanoic acid ...... 29

1.23: Coupling reaction between histamine hydrochloride and 3-(dibenzylamino )-2,2- difluoropropanoic acid ...... 30

1.24: Preparation ofN-PMP protected gem-difluoro P-alanine ...... 31

1.25: Synthesis of gem-difluoromethylene analogue of carnosine using PMP protecting method ...... 32

1.26: The preparation and reaction ofN-Cbz benzotriazolyl methylamine with ethyl bromodifluoroacetate...... 33

1.27: Preparation ofN-Cbz and N-Boc protected gem-difluoro P-alanine esters ...... 34

1.28: Preparation ofN-Cbz protected gem-difluoromethylene analogue of carnosine .... 35

1.29: Preparation ofN-Boc-gem-difluoromethylene analogue of carnosine ...... 35

1.30: Synthesis of gem-difluoromethylene analogue of carnosine from Cbz protecting method ...... 36

1.31: Synthesis of gem-difluoromethylene carcinine from PMP-protecting method ...... 3 7

1.32: Synthesis of gem-difluoromethylene carcinine from Cbz-protecting method ...... 38

1.33: 1H NMR spectra of compound 1.80 ...... 60

1.34: 13C NMR spectra of compound 1.80 ...... 60

1.35: 19F NMR spectra of compound 1.80 ...... 61

1.36: 1H NMR spectra of compound 1.81 ...... 61

1.37: 19FNMRspectraofcompound 1.81 ...... 62

1.38: 1H NMR spectra of compound 1.83 ...... 62

1.39: 13C NMR spectra of compound 1.83 ...... 63

1.40: 19F NMR spectra of compound 1.83 ...... 63 X

1.41: 1H NMR spectra of compound 1.85 ...... 64

1.42: 19F NMR spectra of compound 1.85 ...... 64

1.43: 13 C NMR spectra of compound 1.87 ...... 65

1.44: 19F NMR spectra of compound 1.87 ...... 65

1.45: 1H NMR spectra of compound 1.88 ...... 66

1.46: 13C NMR spectra of compound 1.88 ...... 66

1.47: 19F NMR spectra of compound 1.88 ...... 67

1.48: 1H NMR spectra of compound 1.89 ...... 67

1.49: 1H NMR spectra of compound 1.89 ...... 68

1.50: 19F NMR spectra of compound 1.89 ...... 68

1.51: 1H NMR spectra of compound 1.93 ...... 69

1.52: 1H NMR spectra of compound 1.92 ...... 69

1.53: 13C NMR spectra of compound 1.92 ...... 70

1.54: 19F NMR spectra of compound 1.92 ...... 70

1.55: 1H NMR spectra of compound 1.94 ...... 71

1.56: 1H NMR spectra of compound 1.95 ...... 71

1.57: 13C NMR spectra of compound 1.95 ...... 72

1.58: 19F NMR spectra of compound 1.95 ...... 72

1.59: 1H NMR spectra of compound 1.96 ...... 73

1.60: 13C NMR spectra of compound 1.96 ...... 73

1.61: 19F NMR spectra of compound 1.96 ...... 74

1.62: 1H NMR spectra of compound 1.98 ...... 74

1.63: 13C NMR spectra of compound 1.98 ...... 75 Xl

1.64: 19F NMR spectra of compound 1.98 ...... 75

1.65: 1H NMR spectra of compound 1.90 ...... 76

1.66: 13 C NMR spectra of compound 1.90 ...... 76

1.67: 19F NMR spectra of compound 1.90 ...... 77

1.68: 1H NMR spectra of compound 1.99 ...... 77

1.69: 13 C NMR spectra of compound 1.99 ...... 78

1.70: 19F NMR spectra of compound 1.99 ...... 78

1.71: 1HNMRspectraofcompound 1.101 ...... 79

1.72: 13 C NMR spectra of compound 1.101...... 79

1.73: 19F NMR spectra of compound 1.101 ...... 80

1.74: 1H NMR spectra of compound 1.100 ...... 80

1.75: 13 C NMR spectra of compound 1.100 ...... 81

1.76: 19F NMR spectra of compound 1.100 ...... 81

2.1. Structures of Gemicitabin and M3 antagonist...... 90

2.2: Conversion of aldehydes and ketones to gem-difluoromethylene compounds ...... 92

2.3: Mechanism of difluorination of carbonyl group with DAST ...... 93

2.4: Conversion ofhydrazones to gem-difluoromethylene compounds using IF ...... 93

2.5: Conversion ofhydrazones to gem-difluoromethylene compounds using NBS/PPHF ...... 94

2.6: Conversion of oximes to gem-difluoromethylene compounds ...... 94

2. 7: Conversion of dithiolanes to gem-difluoromethylene compounds using NBS/PPHF and DBH/PPHF ...... 95

2.8: Conversion of dithiolanes to gem-difluoromethylene compounds using Et2NCF2CHFCF3 and DBH/H20 ...... 96 xu

2.9: Conversion of diphenyl dithiolane to gem-difluoromethylene compound using p-Iodotoulenedifluoride ...... 96

2.I 0: Conversion of P-hydroxy orthothioesters to I, 1-difluoro-1-methylthio)ketones .... 97

2.II: Conversion of thiocarbonyls to gem-difluoromethylene compounds ...... 98

2.I2: Conversion azirines to 3,3-difluorophenylalanine ...... 98

2.I3: Conversion of alkenyl trifluoroborates to gem-difluoromethyl substituted alcohols ...... 99

2.14: Conversion of alkenes to gem-difluoromethylene compounds and the mechanism for the difluorination of alkenes ...... 100

2.15: Conversion of allyl amines toy- gem-difluoroamine ...... 10 1

2.16: Conversion of alkyne to gem-difluoromethylene compound and bromotluorination of alkene ...... I 0 I

2.17: Conversion of aryl substituted alkynes to a,a-difluoro-ketones ...... I 02

2.18: Electrophilic difluorination of P-ketoamides with Selectfluor...... I 02

2.19: Conversion of a,a-difluorolactams to (L)-4,4-difluoroglutamic acid ...... 103

2.20: Conversion of aryl ( a,a-difluoromethylenephosphonates) to gem-difluoromethylene compounds ...... I 04

2.21: Conversion of benzylic sulfonate esters to gem-dilfuoro-sulfonate esters ...... 104

2.22: Possible activation of thiolanes with the combination of Selecttluor and HF ...... 105

2.23: Conversion of 1,2-diphenyl-1 ,3-dithiolane to gem-dilfuorodiphenylmethane ...... 106

2.24: Conversion diarylsithiolanes to gem-difluorodiarylmethanes ...... 107

2.25: Reaction of dithiolanes from acetophenone, bromo-benzaldehyde and cyclododecanone with Selectfluor/PPHF ...... · .. · .. · .. · · · · .. · · · · .... I 08

2.26: Proposed mechanism for the formation of gem-difluoromethylene compounds from the carbonyl-dithiolanes and Selectfluor/PPHF ...... I 09

2.27: 19F NMR spectra of compound 2.10 ...... 116

2.28: 19F NMR spectra of compound 2.11 ...... ··········· ··· · .... 11 6 X Ill

2.29: 19F NMR spectra of compound 2.12 ...... 117

2.30: 19F NMR spectra of compound 2.13 ...... 117

2.31: 19F NMR spectra of compound 2.14 ...... 118

2.32: 19F NMR spectra of compound 2.15 ...... 118

3.1: Nucleophilic trifluoromethylation of azirines ...... 125

3.2: Nucleophilic trifluoromethylation of nitrones ...... 125

3.3: Nucleophilic trifluromethylation ofimines using TMSCF3 and TMS-Imidazole .... 126

3.4: Nucleophilic trifluoromethylation of N-aryl imines ofhexafluoroacetone ...... 126

3.5: Nucleophilic trifluoromethylation of N-tosylated amines using TBAT ...... 127

3.6: Nucleophilic trifluoromethylations of N-(tert-butylsufinyl)-imines, a,~­ unsaturated N-(tert-butylsufinyl)-imines and a-amino N-(tert-buty1sufiny1) imines ...... 128

3.7: Nucleophilic trifluoromethylation using CF3VTDAE ...... 128

3.8: Nucleophilic trifluoromethylation using Lewis base, AcOLi ...... 129

3.9: Nucleophilic trifluoromethylation using P(t-Bu)3-DMF system ...... 130

3.10: NHC catalyzed cyanosilylation of o-methoxybenzaldehyde ...... 131

3.11: Asymmetric cyanosilylation catalyzed by chiral NHC ...... 132

3.12: Possible reaction pathway for NHC catalyzed cyanosilylation ...... 132

3.13: Alternative NHC catalytic mechanism for cyanosilylation ...... 133

3.14: Cyanation of aldehydes, aldimines (Strecker reaction), ketones, and ketimines in the presence ofNHC ...... 134

3.15: Reaction of aziridines with silylated nucleophiles catalyzed by NHC ...... 134

3.16: NHC catalyzed trifluoromethylation of carbonyl compounds ...... 135

3.17: Preparation imidazolium chlorides ...... 136

3.18: Trifluromethylation of N-tosyl benzylaldimine in the presence ofNHCs ...... 137 XIV

3.19: Trifluoromethylation of aromatic N-tosyl aldimines in the presence ofNHC ...... 138

3.20: Trifluoromethylation of cyclohexyl-N-tosylaldimine in the presence of N-heterocyclic carbene ...... 139

3.21: 1H NMR spectra of compound 3.6 ...... 148

3.22: 1H NMR spectra of compound 3.7 ...... 148

3.23: 1H NMR spectra of compound 3.8 ...... 149

3.24: 1H NMR spectra of compound 3.9 ...... 149

3.25: 19F NMR spectra of compound 3.11 ...... 150

3.26: 1H NMR spectra of compound 3.16 ...... 150

3.27: 19F NMR spectra of compound 3.16 ...... 151

3.28: 1H NMR spectra of compound 3.17 ...... 151

3.29: 19F NMR spectra of compound 3.17 ...... 152

3.30: 1H NMR spectra of compound 3.18 ...... 152

3.31: 19F NMR spectra of compound 3.18 ...... 153

3.32: 1H NMR spectra of compound 3.19 ...... 153

3.33: 19F NMR spectra of compound 3.19 ...... 154 XV

NOMENCLATURE

Symbol Description gem geminal

alpha

beta

Boc tert-butoxy carbonyl

Cbz benzyloxy carbonyl lie isoleucine

AMP pyridylmethylamine

DCC N ,N -dicyclohexylcarbodiimide

HOBt 1-hydroxybenzotriazole

DEPC diethylphosphoryl cyanide

Ts tosyl n-BuLi n-butyllithium

LiN(TMS)z lithium bis(trimethylsilyl)amide

THF. tetrahydrofuran

HMPA hexamethylphosphoramide

TBDMSCl tert-butyldimethylsilyl chloride

TBP 4,4 '-thiobis(2-tert-butyl-6-methylphenol)

EDCI 1-[3-(dimethylamino )-propyl]-3-ethylcarbodiimide hydrochloride

DMSO dimethylsulfoxide

CDI carbonyldiimidazole

LDA lithium diisopropylamide XVl

NaHDMS sodium hexamethyldisilazide

TMSCl trimethylsilyl chloride

TBAF tetrabutylammonium fluoride

LHMDS lithium hexamethyldisilazide

Ac acetyl

DHP 3,4-2H-dihydropyran

KOt-Bu potassium tert-butoxide p-TsOH para-toulenesulfonic acid

PCC pyridinium chlorochromate

LiAlH4 lithium aluminium hydride

Bn benzyl

DIBAL-H diisobutylaluminium hydride

(DHQ)2PHAL 1,4-bis(dihydroquinine)phthalazine

DIAD diisopropyl azodicarboxylate

PMP para-methoxyphenyl

TIPSCl triisopropylsilyl chloride

CAN eerie ammonium nitrate

HOAt 1-hydroxy-7 -azabenzotriazole

HATU 0-(7 -azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate

TMP 1,1, 1-trimethylolpropane

DIPEA N,N-diisopropylethylamine

TF A trifluoroacetic acid

NMM N-methylmorpholine xvn

DMF dimethylformamide

BOP reagent benzotriazo1-1-yloxytris( dimethylamino )phosphonium hexafluorophosphate

DAST diethylaminosu1fur tetrafluoride

Deoxofluor bis(2-methoxyethy1)aminosu1fur trifluoride

NIS N-iodosuccinimide

NBS N -bromosuccinimide

PPHF pyridinium polyhydrogen fluoride

DBH 1,3-dihromo-5,5-dimethy1hydantoin

Selectfluor 1-ch1oromethyl-4-fluoro-1 ,4-diazoniahicyclo[2.2.2]octane his( tetrafluorohorate)

PVPHF 4-vinyl pyridinium poly(hydrogen fluoride)

Accufluor (N-fluoro-N-Hydroxy-1,4-diazobicyclo(2.2.2)octane his( tetrafluorohorate))

NFBS N-fluorohenzenesu1fonimide

TMSCF3 trimethy1si1yl trifluoromethane

TBAT tetrahuty1ammonium( triphenylsi1yl) di fluorosilicate

TMAF tetramethylammonium fluoride

TDAE tetrakis( dimethy1amino )-ethylene 1. SYNTHESIS OF gem-DIFLUOROMETHYLENE ANALOGUE OF CARNOSINE AND gem-DIFLUOROMETHYLENE CARCININE

1.1 INTRODUCTION:

1.1.1 Fluorinated versions of peptide analogues/mimetics: Peptide analogues are

widely used to elucidate structure - activity relationships, and replacement of the peptide

backbone with hydroxyrnethylene, ketomethylene, alkene, and other amino acid isosteres

has led to significant increases in bioavailability and oral activity. 1-3 The introduction of

fluorine into biologically active compounds can impart unique changes in physical,

chemical, and biological properties in those compounds.4'5 Fluorinated peptide analogues

are of particular interest in this regard.6-8 Various fluorine containing peptide analogues

have been reported in the literature. Among them, peptide mimetics containing fluorine

moieties such as fluorinated methyl ketones, difluorostatone, difluorostatine, difluoro

ketomethylene, monofluoro ketomethylene, monofluoro hydroxyethylene, fluoroalkene,

and (trifluoromethyl)alkene are well developed and used for biological activity studies. 9

1.1.2 Synthetic methods for selected fluorinated peptide analogues: Fluorinated

peptide analogues have been of particular importance in the development of inhibitors of

carboxylic ester and peptide hydrolases.8' 10-13 Brodbeck and co-workers first developed

the fluorinated aldehydes and ketones and described their use as inhibitors of

acetylcholinesterase. 14 Later, many different types of fluorinated peptide analogues were

synthesized and studied for their structure-activity relationships and inhibitory activities

towards aspartyl proteases, zinc metalloproteases, serine proteases, etc. General synthetic methods for selected fluorinated peptide analogues are discussed here. 2

Native peptide substrate

H 0 R2 H /yN0N~N'/ 0 R1 H 0

Fluorinated peptide mimetics/isosteres

H 0 H 0 0 I Y N0CFnH(3-n) /yNYxJlN..-\ 0 R1 0 R1 F F H

Fluorinated methyl ketones Difluorostatone

H 0 R2 H H OH 0 /yNY)clyNy /yNyl;(lN..-\ 0 R1 F F 0 0 R1 F F H

Difluoro ketomethylene Difluorostatine

H 0 R2 H R1 OH R2 /yN0ArN'f R1.N1~H N'/ 0 R1 F 0 F 0 Monofluoro ketomethylene Monofluoro hydroxyethylene

H R2 H H CF3 R2 H /yNyYyNy /yNyvyN'/ 0 R1 F 0 0 R1 0

Fluoroalkene (Trifluoromethyl)alkene

Figure 1.1: Structural representations of selective fluorinated peptide analogues/mimetics/isosteres

1.1.2.1 Difluorostatone and difluorostatine peptide analogues: Peptides which contain difluorostatin and difluorostatone residues have been shown to be potent inhibitors of the renin. 15-18 Renin is an aspartyl protease, 19-22 which cleaves the circulating a-globulin angiotensinogen to form the decapeptide angiotensin I. Angiotensin I is further converted to octapeptide angiotensin II by the angiotensin converting enzyme. Interest in the 3 blockade of the rennin has led to development of the highly potent inhibitors, which contain difluorostatin and difluorostatone moieties. The introduction of electron- withdrawing fluorine atoms adjacent to the carbonyl groups increases the electrophilicity of the carbonyl and thus facilitates the hydration of the ketone. The hydrated difluoro ketone was proposed to mimic the tetrahedral intermediate which forms during the enzyme-catalyzed hydrolysis of the peptide bond and thus acts as transition state analogue renin inhibitor. 16 The difluorostatone analogue has shown higher inhibitory activity than the corresponding nonfluorinated statone and difluorostatine analogues. 17

H OH 0 H 0 0 yN~~ YN~I y = F F y = F F Difluorostatine analogue Difluorostatone analogue

Figure 1.2: Structures of difluorostatine and difluorostatone analogues

H 0 0 YN~I + y = F F

Difluorostatone analogue Trasition state analogue inhibitor

Figure 1.3: Hydration of difluorostatone peptide analogue

Thaisrivongs and co-workers have reported the synthesis of the potent renin inhibitors with difluorostatine and difluorostatone residues usmg ethyl bromodifluoroacetate as fluorine synthon. 16' 18 As shown in Figure 1.4, N-Boc-leucine 1.1 4 was reacted with ethyl bromodifluoroacetate usmg activated zmc dust to afford diastereomeric adducts 1.2a and 1.2b. The ester 1.2b was hydrolyzed using sodium hydroxide and the resulting salt 1.3 was coupled to L-isoleucyl-2-pyridylmethylamine

(H-Ile-AMP) using N,N'-dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole

(HOBt) to obtain the adduct 1.4. Two more a~ino acids were sequentially added to the compound 1.4 using diethylphosphoryl cyanide (DEPC) with triethylamine as coupling reagent to obtain the intermediate 1.5. The peptide analogue with difluorostatine residue

1.6 was obtained by removal of tosyl group on histidine from the intermediate 1.5 using

1-hydroxybenzotriazole. The peptide analogue with difluorostatone residue 1. 7 was prepared by oxidation of hydroxy group from compound 1.5 using Swem oxidation conditions followed by removal of tosyl group on histidine. 5

0 OH BocHN~H __z_n_, B_r_c_F_ 2c_o_2_E_t_ + BocHN~C02 Et = F F Y1.1 y 1.2b

l~~~H. H20

OH OH 0 H-lle-AMP, DCC BocHN~C02Na BocHN~IIe-AMP = F F - F F HOBt Y 1.4 y 1.3

1. TFA, CH2CI2 Boc-His(Ts)-OH, DEPC, Et3N

2. TFA, CH2CI2 Boc-Phe-OH, DEPC, Et3N

OH 0 . fH Jl Boc-Phe-His-HN : J... Jl lie-AMP Boc-Phe-His(Ts)-HNY X ..,_lie-AMP __H_O_B_t__ YY ~ ..,_ - F F MeOH y 1.5 1.6 Difluorostatine analogue

1. Me2SO, (COCih CH2CI2, Et3N 2. HOBt, MeOH

0 0 Boc-Phe-His-HN- Jl Jl y X 'lie-AMP = F F y 1.7 Difluorostatone analogue

Figure 1.4: Synthetic procedure for difluorostatine and difluorostatone analogues 6

1.1.2.2 Difluoroketomethylene peptide isostere: The hydrated form of difluoroketomethylene motif resembles the putative tetrahedral intermediate I, m proteolytic cleavage of the peptide bond and differs only in the substitution of difluoromethylene for tetrahedral nitrogen.23

protease

Protease Substrate I. Tetrahedral Intermediate

R F F 0

"N~H HO OH R

II. Ketodifluoromethylene Dipeptide Isostere

Figure 1.5: Hydration of peptide bond and difluoro ketone

Hoover has reported the synthesis of ketodifluoromethylene dipeptide isostere usmg difluorodibromomethane (CF2Br2) as difluoromethylene source23 as shown in

Figure 1.6. The aldehyde 1.8 was difluoromethylenated using difluorodibromomethane followed by successive desilyalation using sodium hydroxide to obtain the compound

1.9. Compound 1.9 was esterified by sequential treatment with n-BuLi and 4- methylvaleryl chloride to give the compound 1.10. Enolisation of 1.10 with LiN(TMS)2 in THF, sequential addition of HMPA and tert-butyldimethylsilyl chloride in THF and hydrolysis gave the product 1.11. The imino ester 1.12 was prepared from the acid 1.11 by reacting with ammonia in the presence of oxalyl chloride, and successive 0-alkylation 7

with Meerwin salt (Et30BF4). The iodocyclization of imino ester 1.12 was done to obtain cyclic imidate 1.13 by the reaction of 1.12 with the mixture of iodine, sodium bicarbonate and catalytic amount of 4,4'-thiobis(2-tert-butyl-6-methylphenol) (TBP) in 1:1 dichloromethane-H20 in the dark at 25 °C for 24h. Hydrolysis of 1.13 with HCl in 2:1

THF-H20 produced acyclic hydrochloride, which was further converted to the benzyl carbamate 1.14 using Cbz-Cl. Iodide 1.14 was converted to the protected amino alcohol

1.15 by facile stereospecific hydrolysis under neutral conditions upon exposure to buffered peroxytrifluoroacetic acid in dichloromethane. The target difluoroketomethylene peptide mimic 1.16 was obtained by modified Pfitzner-Moffatt oxidation of amino alcohol 1.15 using 1-[3-(dimethylamino)-propyl]-3-ethylcarbodiimide hydrochloride and dichloroacetic acid in 1:1 toulene-DMSO at 25 °C. 8

1. CF 2Br2, THF HMPT

TMSO''. 2. NaOH H 1.9 1.10 1.8 1. LiN(TMSh , HMPA TBDMSCI 2. 2N HCI

r'J F.. F ~I 1. (COCih, NH3 ~COOH ~ . NH - 1.12 y 1.11 y

1. HCI OEt 2. CbzCI, NaHC03

1.14 y

OEt OEt y OH y 1.16 1.15

Figure 1.6: Synthetic procedure for difluoroketomethylene peptide isostere 9

1.1.2.3 Monofluoro ketomethylene peptide isostere: Monofluoroketomethylene peptide isosteres are very interesting peptide isosteres since the carbonyl group which is present adjacent to the monofluoromethylene group can be only partially hydrated in the presence of water so that interaction with several different types of protease is possible. There have been few reports on the synthesis of this type of peptide isosteres and peptide core units?4-26 In 1999, Hoffman has reported highly stereocontrolled synthesis of monofluoro ketomethylene dipeptide isosteres using Selectfluor as fluorinating reagent as shown in

Figure 1.7.27

According to Hoffinan's synthesis, tritylated amino acids 1.17 containing branched, unbranched, and heteroatom-containing side chains were converted to allyl ~­ ketoesters 1.18 in two steps using CDI, and LDA and allyl acetate. These ally keto esters

1.18 were alkylated with scalemic triflates 1.19, and decorboxylated to provide tritylated ketomethylene isosteres 1.20. The diastereoselectivity in the reaction, alkylation­ decarboxylation of allyl ~-ketoesters was quite good, ranging from 85-95% de. In this reaction, Palladium(O) not only cleaved the allyl group selectively but also catalyzed the decarboxylation to give tritylated ketomethylene isosteres as a mixture of diastereomers

1.20a and 1.20b. The syn isomer of 1.20a was converted to the corresponding (Z)-TMS enol ether 1.21 using NaHDMS-TMSCl. The bulky nitrogen substituent, trityl group on amino ketone caused the enolate formation to occur regiospecifically distal from the amino group by removal of a' -proton. And also the use of NaHDMS under kinetic conditions allowed the formation of only (Z) geometrical TMS enol ether 1.21. Later, the treatment of silyl enol ether 1.21 with Selectfluor in the presence of tetrabutylammonium 10 fluoride (TBAF) led to smooth a' -fluorination to produce monofluoro ketomethylene dipeptide isostere 1.22.

0 1. CDI 0 0 + R100H R 1 ~0allyl NHTr 2. 0 NHTr 1.17 8 )loallyl 1.18

1. NaH

2. Pd(PPh3)4

0 R2 0 R2 R 1 ~0Me + R 1 ~0Me NHTr 0 NHTr 0 1.20b 1.20a

1. NaHMDS 2. TMSCI

0 R2 TMSO R2 Selectfluor R10!YOMe R 1 ~0Me TBAF TrHN F 0 NHTr 0 1.22 1.21

Figure 1.7: Synthetic procedure for monofluoro ketomethylene peptide isostere

1.1.2.4 Monofluoro hydroxyethylene peptide isostere: HIV protease is one of the most significant protease targets in anti-infective therapy today. The inhibition of HIV protease has shown to extend the length and improve the quality of life of AIDS patients. The development and large-scale production of the stereochemically complex HIV protease 11

inhibitor indinavir by team of Merck scientists is one of the great recent achievements in

the pharmaceutical industry.28'29 Later, Myers and co-workers have synthesized all the

four diasteromers of stereospecific fluorine substituted hydroxyethylene dipeptide

isosteres of indinavir in search for higher potencies and properties. 30 Synthesis of

anti,anti isomer of fluorine substituted indinavir which was developed by Myers and co­

workers is presented here.

Nitroalkene 1.23 was reacted with the cold solution ( -78 °C) of the lithium enolate

derived from pseudoephedrine a-fluoroacetamide 1.24 to obtain the two distereomers

(ratio anti:syn = 1.7:1) of the conjugate addition product 1.25 which differs in only the~­

center. Diastereomerically pure anti isomer of 1.25 was obtained by recrystallization and

subjected to hydrolysis of the amide using 2N NaOH followed by diazomethylation using

oxalyl chloride and trimethylsilyldiazomethane to obtain the diazomethyl ketone 1.26.

Diazomethyl ketone was converted to tetrahydropyranyl ether 1.27 in three-step sequence

involving bromination using hydrobromic acid, reduction of fluoro ketone to form anti

configured fluorohydrin using sodium borohydride, and protection of alcohol using 3,4-

2H-dihydropyran (DHP). Nitroalkene 1.27 was converted to the corresponding

carboxylic acid upon sequential treatment with potassium tert-butoxide and potassium

permanganate in a buffered mixture of tert-butanol and water. The crude acid was

coupled with (1S,2R)-l-aminoindan-2-ol to afford amide and tetrahydropyranyl ether was cleaved to obtain hydroxyamide 1.28. The hydroxy amide within the resultant diol was

selectively protected using 2-methoxypropene and catalytic methanesulfonic acid. The resultant bromohydrin with protected hydroxy amide was reacted with potassium tert­ butoxide in THF at 0 °C to obtain the epoxide 1.29. Epoxide 1.29 was converted to 12 compound 1.31 by reacting with pyrazine 1.30 in 2-propanol followed by deprotection of benzyl carbamate by hydrogenolysis. Finally, alkylation of the secondary amine with 3- picolyl chloride hydrochloride and acidic methanolysis of the acetonide protecting group afforded the anti,anti isomer of fluorine substituted hydroxyethylene dipeptide isostere of indinavir 1.31. 13

CH3 0 LHMDS, LiCI + Syn isomer + Ph0N~F :: I OH CH3 1.24

1. 2N NaOH 2. (COCI)z, DMF TMSCHN2, THF

1. 48% aq HBr, AcOH THPQ (Ph 2. NaBH4, EtOH Br~N0 2 - 3. DHP, p-TsOH.H20 F F 1.27 1.26

1. t-BuOK, t-BuOH KMn04, Na2B40 7, H20 2. Amino alcohol, EDC.HCI HOSt, Et3N 3. p-Ts0H.H20

Ph ~H (' "H QH 1. 2-methoxypropene Br~N .,: CH3S03H F 0 8-..;: 2. t-BuOK 1.28 ~ #

Cbz, 1. ~) 2. 10% Pd/C, "'-NH EtOH O~NHt-Bu 1.30

Figure 1.8: Synthetic procedure for monofluoro hydroxyethylene peptide isostere 14

1.1.2.5 Fluoroalkene and (trifluoromethyl)alkene peptide isosteres: The fluoroalkene

group has been regarded as an isosteric replacement for the peptide bond on the basis of

1"t s p 1anar geometry an d d"trechon . o f po 1anzahon. . . 8 '31 ' 32 s evera1 groups have reported the

synthetic methods for these fluoroalkene peptide isosteres in the literature. 33 -40 In 1995,

Bartlett has synthesized tripeptide analogues with trans-fluoroalkene moiety to study

their structure-activity relationships and inhibitory properties towards zinc endopeptidase

thermolysin.41 The synthetic procedure developed by Bartlett is discussed here.

The mono protected diol 1.32 was oxidized to the aldehyde using Pyridinium

chlorochromate (PCC) and condensed with diethyl fluorooxalate to obtain fluoroacrylate

as a mixture of 1:2.3 mixture of E:Z isomers 1.33a and 1.33b. Diethyl fluorooxalate was

prepared in situ in the reaction using diethyl oxalate and ethyl fluoroacetate. The Z-

isomer of 1.33b was separated by column chromatography and converted to allylic

carbamate 1.34 by a three-step sequence involving amidation using ammonia in

methanol, reduction to amine using lithium aluminium hydride (LiAlH4), and protection of amine with benzyl chloroformate (Cbz-Cl). The silyl ether was deprotected and the homoallylic alcohol was oxidized to acid to obtain the compound 1.35. Finally, the coupling of acid 1.35 with phenyl alanine methyl ester using dimethylaminopropyl-3- ethyl carbodiimide (EDCI) and HOBt, and hydrolysis of the ester by lithium hydroxide afforded the fluoroalkene peptide isostere 1.36. 15

HO~OTBDMS------1.PCC F~OTBDMS Et0 2 C~OTBDMS + F - 2. NaH, (C02Eth, Et02C y y FCH2C02Et y 1.33b 1.32 1.33a

1. NH3, MeOH 2. LiAIH4 3. CbzCI, Et3N

TBAF CbzHN~C0 2 H CbzHN~OTBDMS F - F -y Jones reagent y 1.35 1.34

1. Phe-OMe, EDCI, HOBT iPr2NEt 2. LiOH

O (Ph CbzHN~Nl(OH F -y H 0

1.36

Figure 1.9: Synthetic procedure for fluoroalkene peptide isostere

Electrostatically, the (trifluoromethyl)alkene represents a better match of the amide bond than any other common alkene isostere. In 1998, Wipf and co-workers have reported the synthesis of (trifluoromethyl)alkene dipeptide isostere usmg trifluorotrichloroethane as trifluoromethyl group source.42 The synthetic procedure developed by Wipf and co-workers is discussed here.

Carboxylation of trifluorotrichloroethane 1.37, esterification with benzyl alcohol

(BnOH), and Reformatsky addition-elimination with acetaldehyde yielded the trifluoroalkene as a 2:3 mixture of (E)- and (Z)-isomers 1.38a and 1.38b. The (Z)-isomer 16

1.38b was isolated by column chromatography and subjected to Sharpless asymmetric dihydroxylation. The resulting diol obtained from Sharpless asymmetric dihydroxylation was converted to epoxide using Mitsunobu reaction conditions. Later, the reduction of benzyl ester to aldehyde using diisobutylaluminium hydride (DIBAL-H) and Witig chain extension of the resulting aldehyde provided the alkenyl oxirane 1.39. Akenyl oxirane

1.39 was converted to compound 1.40 by azide opening of epoxide, Straudinger reduction of azide to amine, conversion of amine to carbamate, and mesylation of alcohol. Finally, the allylic SN2' displacement of the mesylate and transamidation of the methyl ester provided the target (trifluoromethyl)alkene dipeptide isostere 1.41.

1. Zn, CuCI, C02 (650 psi) 2. BnOH, DCC, DMAP F3C - Cl3 ------~C02 Bn + 3. Zn, CuCI, MeCHO, TFAA 2 CF3 ~C0 Bn CF 1.37 1.38b 3 1.38a

1. Os04, K3FeCN03 (DHQhPHAL 2. Ph3P, DIAD 3. DIBAI-H 4. Ph3P=CHC02Me 1. NaN3, MeOH ~HBoc 2. Ph3P, H20 1 3. Boc 0, Et3N ~C02 Me 2 J , "" co,Me MsO CF3 0~ 4. MsCI, Et3N CF3 1.40 1.39

1. Me2Cu(CN)(ZnCih .2MgCI2.4LiCI

2. LiAI(NHMe)4

B l ~ ~CONHMe ocHW ):'( CF3 1.41

Figure 1.10: Synthetic procedure for (trifluoromethyl)alkene peptide isostere 17

1.1.2.6 Fluorinated amino acids and peptides: a-hydroxy-~-amino acids are well represented in a large number of biologically active compounds such as HN protease inhibitors, antitumor agents, and immunological response modifiers. Due to the enhancement of pharmacological properties by the incorporation of fluorine into the molecules, interest arose in the synthesis of fluorine containing a-hydroxy-~-amino acids which can serve as important bioactive compounds for medicinal chemistry and chemical biology. Kuznetsova and co-workers have reported a new synthetic approach to fluorine­ containing a-hydroxy-~-amino acids and their congeners from enantiopure trifluoromethyl containing ~-lactams using the ~-lactam synthon method.43 First, N-p­ methoxyphenyl trifluoroacetaldimine 1.42 was subjected to [2+2] ketene-imine cyclo addition using benzyloxyacetyl chloride as the ketene precursor to obtain racemic cis-3- benzyloxy-4-trifluoro-methyl- ~-lactam (±)-1.43. The benzyl protecting group was changed to acetyl group by hydrogenolysis followed by acetylation to obtain the racemic cis-3-acetoxy-4-trifluoromethyl- ~-lactam (±)-1.45. Racemic 1.45 was subjected to enzymatic optical resolution using PS-Amano lipase to obtain optically pure (3R,4R)-3- acetoxy-4-trifluoromethyl- ~-lactam (+)-1.45 and (3s, 4s)-3-hydroxy-4-trifluoromethyl­

~-lactam (-)-1.44. Hydroxy group was protected using triisopropylsilyl group in (-)-1.44 and (+ )-1.44 followed by the removal of N-p-methoxyphenyl group using eerie ammonium nitrate to obtain the precursors (+)-1.46 and (-)-1.46 for N-acetylation as shown in Figure 1.11. An alternative method was also developed for the preparation of

4-trifluoro-~-lactams involving the removal of N-protectingp-methoxyphenyl group from the racemic cis-3-acetoxy-4-trifluoromethyl- ~-lactam (±)-1.47 prior to enzymatic resolution as shown in Figure 1.12. 18

HO)={CF3 PhCH 2 OCH 2 COCI ' BnO)={CF3 Et3N N, 0 PMP 0 PMP (±) cis-1.44 (±) cis-1.43

A~O. DMAP, Py

AcO,, ,,CF HOr--ICF3 3 PS-Amano, buffer + o~'PMP 0}--N,PMP (+)-1.45 (-)-1.44 (±) cis-1.45

KOH 1. TIPSCI, Et3N

2. CAN

HO,,_ .,,CF3 ;=1, 0 PMP (+)-1.46

(-)-1.46 1. TIPSCI, Et3N

2. CAN

TIPSO,, ,,CF3

0 ~' H (+)-1.46 Figure 1.11: Synthetic procedure for chirally pure 4-trifluoro-~-lactams

AcO,, ,,CF3 HO~CF3 AcO;n:CF3 PS-Amano, buffer + o}--N,H 0 'H o~'H (±) cis-1.47 (+)-1.47 (-)-1.48

Figure 1.12: Alternative method for the preparation for 4-trifluoro-~-lactam 19

Later, the fluorinated P-lactam derivatives were transformed to fluorinated amino acids, dipeptide and taxoids. Fluorinated a-hydroxy-p-amino acids 1.50 were prepared by

N-acylation of the resulting P-lactams 1.49 followed by hydrolysis. Trifluoromethyl containing a-hydroxy-P-amino acid methyl esters 1.51 were prepared by methanolysis of

P-lactams. Trifluoromethyl containing dipeptides 1.52 and 1.53, and taxoids 1.54 were obtained by coupling P-lactams with amino acid esters and baccatins, respectively.

/ 1.51 PO/,;n, ,,CF3 0 Boc 1.49 / _\ 0 Rr 0 0 ~O)lN¥N~OEt H - H OP 1.52

1.54

Figure 1.13: Transformation of fluorinated P-lactams to fluorinated amino acids, peptides and taxoids 20

The trifluoromethyl group is frequently used as a mimic for various functional groups such as methyl, isopropyl and phenyl, and is found in a number of biologically active and therapeutic agents.44A5 The trifluoromethyl group is hydrophobic, bulky and electron-rich. It enhances the in vivo stability of a compound as the removal or substitution of a fluorine in the trifluoromethyl group (as opposed to its methyl isosteres) cannot be easily done by metabolic enzymes.46 Jacobson and co-workers have described a new family of potent peptidomimetic hydroxamate inhibitors against matrix metalloproteases.47,48 Later, Zanda and co-workers incorporated fluoroalkyl substituents at the quartemay position and synthesized molecule such as 1.62.49 The synthesis of fluoroalkyl peptidomimetic hydroxamate 1.62 reported by Zanda and co-workers is presented here.

The core malic unit with a-trifluoromethyl group was obtained by TiCl4 catalyzed

Aldol reaction of N-acyl-oxazolidine-2-thione 1.55 with ethyl trifluoropyruvate 1.56.

This gave 2.7:1 mixture of diastereomeric adducts 1.57a and 1.57b. The oxazolidin-2- thione was cleaved from 1.57a using potassium carbonate to obtain the carboxylic acid derivative and the resulting carboxylic acid was coupled with 1.60 using HOAt/HATU.

The coupling reaction generated the ~-lactam 1.59 in addition to the expected coupling product 1.61. The ~-lactam 1.59 was then separated and converted to coupling product

1.62. Later, the coupled product 1.61 was converted to the final trifluoromethyl peptidomimetic hydroxamate 1.62 by saponification followed by coupling with benzyl hydroxamate (BnONH2) and hydrogenolysis to cleave the benzyl group. 21

1.56 1.55

0 CF3 HOAt/HATU C02Et YcH TMP HO r:O,Et Ph 1.59 1.58

H,~NHCH 3 ,TMP 0 1.60

Figure 1.14: Synthetic procedure for trifluoromethyl peptidomimetic hydroxamate

Rhodopeptins, a new family of antifungal cyclic lipopeptides, were isolated from

Rhodococcus species Mer-N1033. 50-52 They are composed of three a-amino acids and one

~-amino acid with a lipophilic side chain. These Rhodopeptins have attractive fungicidal activities along with their unique structural characteristics. In 2000, Ohta and co-workers have reported their structure-activity relationship (SAR) studies on these molecules. 53 In their continuation work, they have reported the synthesis of difluoro54 analogues of

Rhodopeptin in the pursuit of identifying new Rhodopeptin analogues with decreased 22 toxicity and improved physiochemical properties.55 The synthesis of gem-difluoro analogues of Rhodopeptin which was reported by Ohta and co-workers is discussed here.

The aldehydes 1.63 was reacted with ethyl bromodifluoroacetate under

Reformatsky conditions and followed by hydrolysis of ester using sodium hydroxide, formation of p-methoxy phenyl amide (PMP) using p-anisidine and Bis-(2-oxo-3- oxazolidinyl)-phosphinyl chloride (Bop-Cl) to obtain a-gem-difluoro-~-hydroxy amide

1.64. Intramolecular Mitsunobu reaction of 1.64 using diethyl azodicarboxylate (DEAD) and triphenylphosphine (PPh3) afforded the racemic ~-lactam 1.65. The ring opening of

~-lactam 1.65 using sodium methoxide (MeONa) afforded the fully protected gem­ difluoro- ~-amino acid 1.66. The amino protecting conversion was accomplished by treatment with eerie (IV) ammonium nitrate (CAN) and di-tert-butyl carbonate ((Boc)20) which was necessary for the purification of the intermediate. After removal of the Boc group using trifluoro acetic acid (TFA) to obtain 1.67, conventional methods for peptide synthesis were used to obtain the gem-difluoro analogues of Rhodopeptin 1.68a and

1.68b. These gem-difluoro analogues retained the antifungal activity of the non­ derivatized Rhodopeptins and displayed improved physical properties such as less toxicity and more solubility. 55 23

1. Zn/THF F. F 2. NaOH/EtOH 0 BrXy 'Et 3. p-anisidine 0 BopCI, iPr2NEt 1.63

NH2 1. CAN NHPMP MeONa CH3(H2C)s~C02Me 2. (BochO CH3(H2C)s~C02Me F F 3. TFA F F MeOH 1.67 1.66.

O OH F N~N r H H + CH (H C)'' .. )\ 3 2 a HO~N \)_ ~ 0 NH2

1.68a 1.68b

Figure 1.15: Synthetic procedure for gem-difluoro analogue ofRhodopeptin

1.2 RESULTS AND DISCUSSIONS

Carnosine W-alanyl-L-histidine), a naturally occurring dipeptide, has unique role as antioxidant and antiglycating agent. 56-61 Based on these unique properties, it is able to attenuate cellular oxidative stress and can inhibit intracellular formation of ROS and

RNS. The attenuation of oxidative stress by carnosine can involve sequestering of ROS or chelation of metal ions. Structurally related compounds such as carcinine, and anserine also show antioxidant and neuronal protecting activities. 62 -64 24

H N~N~NH 2 tz.~ -A o N 0 OH H

Carnosine Carcinine Anserine

Figure 1.16: Structures of Carnosine, Carcinine, and Anserine

Although carnosine is abundantly present in excitable and nervous tissues (as high as 20 mM) in humans, its in vivo antioxidant properties are limited as it can be hydrolyzed to its constituent amino acids, histidine and P-alanine by serum carnosinase enzyme.65-67 Dietary supplementation of carnosine is shown to prevent the development of ischemic/reperfusion induced acute renal failure. 68 In light of its promising medicinal importance, there is lot of interest in developing analogues of carnosine with higher antioxidant, neuroprotective abilities and enzymatically stable analogues. Na- acetylcarnosine 1.69, for example, is somewhat resistant to hydrolysis by carnosinase and is used as a prodrug for opthamic use. 62 In 1999, Pinnen and co-workers reported the synthesis of sulfonamide peptide isosteres of carnosine, anserine, and isoanserine 1. 70-

1. 72 and studied their bioactivity. 69 These compounds have been reported to show higher stabilities towards carnosinase activity. In 2005, the same group have reported new synthetic carnosine analogues containing L(+)2,3-diaminopropanoic acid and studied the effect of N-acetylation on biochemical properties of carnosine. 70 Mono- and bis- acetylated compounds 1.73 and 1.74 are resistant to enzymatic hydrolysis by carnosinase and also act as competitive inhibitors of this enzyme. These compounds have shown identical hydroxy radical scavenging abilities to that of carnosine and also act as protective agents against peroxynitrite-dependent damage as determined by their ability 25

to prevent nitration of free tyrosine induced by this peroxynitrite radical. In 2005, Guiotto

and coworkers have reported the synthesis and neuroprotective abilities of histidyl

containing carnosine analogues 1.75-1.77 bearing hydrazide or 1,2-diol moieties. 71 These

carnosine analogues 1.75-1.77 have been demonstrated to show higher a,~-unsaturated

aldehydes-sequestering abilities than carnosine.

1.70 Ho \ -aN,11~NH 2 ~N ~ aA~ a* . N OH

1.72

1.73

N~NH2

lf..N) 0AN.-NH2 H H 1.77

Figure 1.17: Examples of reported carnosine analogues

Introduction of gem-difluoromethylene group into orgamc compounds dramatically alters biological activities and imparts increased lipophilicity to the molecules.72•73 The gem-Difluoromethylene group adjacent to the amide carbonyl group 26

can significantly increase the electophilicty of the carbonyl group, which would result in

more potent hydroxyl scavengers. In addition, these gem-difluoromethylene containing

carnosine and carcinine analogues could be useful as transition state analogues of enzyme

inhibitors.

H F. F N~N~NH2 ~ --A o tz N.. 0 OMe H

carnosine gem-difluoromethylene analogue of carnosine

carcinine gem-difluoromethylene carcinine

Figure 1.18: Structures of synthesized gem-difluoromethylene analogues of carnosine and carcinine

We envisioned that the preparation of gem-difluoro derivative of one of the

amino acids present in the dipeptide and coupling with another amino acid of the dipeptide would accomplish the synthesis of gem-difluoro analogue of dipeptide. Both carnosine and carcinine have P-alanine as one of the amino acids, and for this reason, the preparation of gem-difluoro- P-alanine has attracted our attention. Later, gem-difluoro- P- alanine was coupled with the respective amino acids to achieve the synthesis of gem- difluoro analogues of carnosine and carcinine. 27

H F F N--<'YN, I)(' -NH2 N~NH2 -.. FX ~H + HO lf ...._,. N,z !( . ."J "A r ~ > !( . ."J "A N 0 OMe N 0 OMe 0 H H Z = Protecting group li F F < + Br>

Figure 1.19: Retrosynthetic analysis ofthegem-difluoromethylene analogue ofL­ camosine

H,FX ~ N~Nlf ...._,NH2 -.. Fx ~H + HOlf ...._,N,z !( ~ 0 > N 0 H Z = Protecting group li

< +

Figure 1.20: Retrosynthetic analysis of the gem-difluoromethylene carcinine

1.2.1 Synthesis of gem-difluoromethylene analogue of L-carnosine: There have been several methods reported in the literature to prepare the fluoro-~-aminoacids. 74 Among them, Reformatsky-type of reactions using halodifluoroacetate as fluorine building block 28

are widely used to prepare ~-substituted a, a-difluoro-~-aminoacid derivatives.75-80

Protected verst on of gem-difluoro- ~-alanine, ethyl 3-(dibenylamino )-2,2-

difluoropropanoate 1.80 was prepared from N,N-(Bis-phenylmethyl)-benzotriazolyl-1-

methylamine81 '82 1.78 and ethyl bromodifluoroacetate 1.79 according literature

procedure80 using Reformtsky reaction conditions. Compound 1.78 was reacted with 1.79

in the presence of zinc dust activated with chlorotrimethylsilane at room temperature.

The reaction was completed in 3h and gave ethyl 3-(dibenylamino)-2,2-

difluoropropanoate 1.80 in 88% yield after column chromatography purification. Next,

the hydrolysis of ester group of 1.80 using IN LiOH afforded 3-(dibenzylamino)-2,2-

difluoropropanoic acid 1.81 in 74% yield.

F F ~'N Zn/TMSCI F F r-Ph , + EtO~Br EtO~N'-Ph N THF, RT, 3h l_ r-Ph 0 0 N'-Ph 1.79 1.80 1.78

MeOH 1N LiOH, 1RT, 1h CH3CN RT, 3h

r-Ph + HCHO HN'-Ph

1.81 Figure 1.21: Preparation ofN-benzyl protected gem-difluoro ~-alanine

After the preparation of free acid, 3-(dibenzylamino)-2,2-difluoropropanoic acid

1.81, different coupling methods were tested for the coupling with histidine methyl ester 29 hydrochloride to obtain the protected dipeptide in higher yields. First conventional coupling method, DCC/H0Bt83 was perfonned and obtained the product methyl 2-(3-

( dibenzylamino )-2,2-difluoropropanamido )-3-(lH-imidazol-4-yl)propanoate 1.83 in very

less yield (31 %) even after 36 h of reaction time. Later, peptide bond formation was performed using more advanced methods using EDCI!H0Bt84 and BOP reagent85

reagents. In both the cases, the product 1.83 was isolated in disappointing yields 42% and

36% respectively.

H~ FXF- r-Ph N~NH2 DCC, HOBT N~Nif '-/N'-Ph ~ -~ /!...... ) ~ 0 tz N oAoMe NMM. DMF H RT, 36h ~ O~oMe . 2 HCI 1.83 1.82 Yield= 31%

H~ FX ~ r-Ph N~NH2 E:OCI, HOBT N-0N If """N'-Ph - .... tz .~ -~ · NMM. DMF l!...) .A. o N oAoMe ~ 0 OMe H -2ooc - RT, 20h . 2 HCI 1.83 1.82 Yield= 42%

N~NH2 BOP reagent l( .~ -~ ---Et3N, CH3CN N oAoMe H RT. 10h .2 HCI 1.81 1.82 Yield= 36%

Figure 1.22: Different methods forth~ peptide ~ond form~tion between ~istid.ine methyl ester hydrochloride and 3-(Dtbenzylarnmo )-2,2-dtfluoropropanmc acid

Even the coupling reaction between 3-(dibenzylamino)-2,2-difluoropropanoic

acid 1.81 and histamine dihYdrochloride using EDCIIHOBt in the presence of N-methyl 30 morpholine in DMF was not efficient and gave the product N-(2-(lH-imidazol-4- yl)ethyl)-3-(dibenzylamino)-2,2-difluoropropanamide 1.85 in very low yield (26%).

N~NH2 EDCI, HOBT ( )\ + NMM, DMF ~ . 2 HCI -20°C - RT, 24h 1.84 1.81 Yield= 26% Figure1.23: Coupling reaction between histamine hydrochloride and 3-(dibenzylamino)- 2,2-difluoropropanoic acid

The lower yield in the case of coupling between the above compounds may be explained in terms of high steric hindrance caused by the amine protecting, dibenzyl group. Later we have turned our attention towards the preparation gem-difluoro-~- alanine derivative with less sterically demanding protecting group on amine.

According to that, ethyl 2,2-difluoro-3-( 4-methoxyphenylamino )propanoate 1.88, which has p-methoxy phenyl group (PMP)86 as protecting group on amine was prepared according to literature procedure. 87 Ethyl 2,2-difluoro-3-(4- methoxyphenylamino )propanoate 1.87 was prepared by reacting ethyl bromodifluoroacetate with N-( 4-methoxy phenyl)-benzotriazolyl-1-methylamine 1.86 in the presence of zinc dust/chlorotrimethyl silane at 50 °C for 3h. Later, hydrolysis of ester

1.87 using IN LiOH gave 2,2-difluoro-3-(4-methoxyphenylamino)propanoic acid 1.88 in

74% yield. 31

~'N F F F F CC , Zn/TMSCI N + EtO~Br EtO~NHPMP THF, 50°C, 3h 0 0 lNHPMP 1.87 1.86 1.79

MeOH 1RT, 2h 1N LiOH, CH3CN RT, 3h cc~N + HCHO + H2N-o-0Me H

F F HO~NHPMP

PMP= 0 ~OMe 1.88

Figure 1.24: Preparation ofN-PMP protected gem-difluoro ~-alanine

PMP-protected gem-difluoro- ~-alanine 1.88 was coupled with histidine

methylester using EDCI/HOBt to prepare PMP-protected gem-difluoromethylene

analogue of carnosine 1.89. In case of carnosine, the presence of free primary amine was responsible for its therapeutic activity. 71 Hence, we were interested in the deprotection of p-methoxy phenyl group from compound 1.89 to get the final compound gem- difluoromethylene analogue of carnosine 1.90 with free primary amine group.

Deprotection of p-methoxy phenyl group from 1.89 was attempted using eerie ammonium nitrate (CAN) mediated oxidative deprotection88 to get the final compound gem-difluoro analogue of carnosine 1.90. The reaction was not efficient and produced so many products corresponding to PMP deprotected and hydrolysis products of 1.89, and other uncharacterized products. Even after tedious work up and column chromatography 32

purification, the final compound gem-difluoro analogue of carnosine 1.90 was obtained in

impure form and in very low yield (14%).

F F N~NH2 EDCI, HOST !!___)_j_ + HO~NHPMP N 0 OMe 0 NMM, DMF H -20 °C- RT, 20h . 2 HCI 1.82 1.88 1.89

CAN, H20 CH3CN RT, 5h

1.90 Figure 1.25: Synthesis of gem-difluoromethylene analogue of carnosine using PMP protecting method

Because of the involvement of difficulties in the final deprotection step, the PMP protecting approach for the preparation of gem-difluoromethylene analogue of carnosine was not effective. Later, other protecting groups for amine such as carbonyl benzyloxy

(Cbz) and tert-butoxy carbonyl (Boc) were opted to get the higher overall yields.

According to that, N-( 4-benzyloxy carbonyl)-benzotriazolyl-1-methylamine 1.91 89 was prepared and reacted with ethyl bromodifluoroacetate in the presence of zinc dust and chlorotrimethylsilane to obtain gem-difluoro- ~-alanine ester with Cbz protecting group on amine 1.92. The reaction was not successful even at higher temperatures and longer reaction times, and all the starting material1.91 was recovered after the work up. 33

~~'N H2NI(O..__.. Ph __P_- T_s_O_H_._H_2o__ + HCHO + v--N' 0 Toulene, reflux, 15h 0 l N Ao/"'-. Ph H 1.91

~~'N F: F Zn/TMSCI F: F H v--N' 0 + EtO~Br X . EtO~NI(O..__.. Ph lNAO/"'-.Ph 0 THF, 50°C, 10h 0 0 H 1.79 1.92 1.91 Figure 1.26: The preparation and reaction ofN-Cbz benzotriazolyl methylamine with ethyl bromodifluoroacetate

After the unsuccessful attempt of Reformatsky reaction with 1.91, an indirect

method was followed to prepare Cbz protected gem-difluoro-~-alanine. Ethyl 3-

(dibenzylamino )-2,2-difluoropropanoate 1.80 from first step was taken and the dibenzyl

group was deprotected by hydrogenolysis in the presence of Pearlman's catalyst in dry

ethanol containing trifluoroacetic acid to obtain trifluoroacetate salt of free ~-amino ester

1.93. 8° Compound 1.93 was reacted with benzyl chloroformate in the presence of triethyl

amine to obtain the Cbz protected gem-difluoro-~-alanine 1.92 in 78% yield. t-Boc protected gem-difluoro-~-alanine 1.9490 was also prepared from 1.93 using di-tert-butyl- dicarbonate and pyridine . 34

H2/Pd(OHh EtO ~r-PhN...... _____ EtO ~ffiNH 3 Ph e EtOH, CF C02H 0 3 0 CF3C02 RT, 36 h 1.80 1.93

0

CIAO...... Ph F F EtO ~ffiNH3 EtO~NHCbz e Et3N, CH2CI2 0 CF3C02 0 0°C- RT, 4h 1.93 1.92

F F (f) (t-BochO F F EtO NH3 EtO~NHBoc e Py, CH2CI2 0 CF3C02 0 0°C- RT, 16h 1.93 1.94

Figure 1.27: Preparation ofN-Cbz and N-Boc protected gem-difluoro ~-alanine esters

N-Cbz protected ~-amino ester 1.92 was hydrolyzed to free acid 1.95 using 1 N

LiOH and coupled with histidine methylester hydrochloride using EDCIIHOBt in the presence of N-methyl morpholine in DMF to obtain protected version of gem-difluoro analogue of carnosine 1.96 in 78% yield. 35

F F F F NJ(XNH2 EtO~NHCbz HO~NHCbz + II ~ = RT, 3h "-N 0 A OMe 0 0 H 1.92 1.95 1_82 . 2 HCI

EDCI, HOST NMM, DMF -20 °C - RT, 20h

H F F N~N~NHCbz t!__) --A o N 0 OMe H 1.96

Figure 1.28: Preparation ofN-Cbz protected gem-difluoromethyelene analogue of carnosme

Simultaneously, Boc protected gem-difluoro analogue of carnosine 1.98 was prepared from N-Boc protected gem-difluoro ~-amino ester 1.94 by hydrolysis of ester group using LiOH91 in 2:2:1 ratio of THF/MeOH/H20 followed by coupling with histidine methylester hydrochloride using EDCIIHOBt in DMF.

F F F F N~NH2 EtO~NHBoc LiOH HO~NHBoc + {(_) -~ THF/MeOH/THF N oAoMe 0 RT, 12 h 0 H .2 HCI 1.94 1.97 1.82 EDCI, HOBT NMM, DMF -20 tO RT, 24h

H F F N~N~NHBoc {(_)-A o N 0 OMe H 1.98 Figure 1.29: Preparation ofN-Boc-gem-difluoromethylene analogue of carnosine 36

With the both Cbz and Boc protected gem-difluoro analogues of carnosine in hand, the first deprotection of Cbz group from 1. 75 was attempted in order to prepare final compound gem-difluoro analogue of carnosine. Compound 1.75 was subjected to hydrogenolysis in the presence catalytic amount of Pd in MeOH. The reaction mixture was stirred for 36h, Pd was removed by filtration, and the solvent was evaporated to get the final compound (S)-methyl 2-(3-amino-2,2-difluoropropanamido )-3-(1 H -imidazol-4- yl)propanoate 1.69 in very good yield (84%). The final Cbz deprotection reaction was simple, efficient and gave essentially pure final product the gem-difluoromethylene analogue of carnosine 1.69 in very good yield.

H F. F N----KYN~NHCbz Pd/C, H2 1(.~ -A o MeOH, 36h N 0 OMe H 1.96 1.90 Yield= 84%

Figure 1.30: Synthesis of gem-difluoromethylene analogue of carnosine from Cbz protecting method

Synthesis of gem-difluoromethylene analogue of carnosine 1.90 was achieved in seven steps starting from N,N-(bis-phenylmethyl)-benzotriazolyl-1-methylamine 1. 78 with an overall yield of23.2%.

1.2.2 Synthesis of gem-difluoromethylene carcinine: PMP protected gem-difluoro-P- alanine 1.88 was coupled with histamine hydrochloride using EDCVHOBt in DMF to prepare the protected version of gem-difluoromethylene-carcinine, N-(2-(lH-imidazol-4- yl)ethyl)-2,2-difluoro-3-( 4-methoxyphenylamino )propanamide 1.99. Later, deprotection of p-methoxy phenyl group from 1.99 was attempted using CAN mediated oxidative 37

deprotection method. Compound 1.99 was dissolved in acetonitrile, treated with aqueous

solution of CAN, and the solution was stirred for 3h. After work up and purification of

the residue, the final compound gem-difluoromethylene carcinine, N-(2-(lH-imidazol-4-

yl)cthyl)-3-amino-2,2-difluoropropanamide 1.100 was obtained in very low yield (22%)

accompanied by some other uncharacterized products.

H F F N~NH2 F F HO~NHPMP EDCI, HOBT N~N~NHPMP !!__ ~ + N . 2 HCI 0 NMM, DMF (( ~ 0 H N RT, 24h H 1.84 1.88 1.99

CAN, H20 CH3CN RT, 5h

Figure 1.31: Synthesis of gem-difluoromethylene carcinine using PMP-protecting method

Because of lower yields associated with PMP protecting grouping strategy, we

have turned our attention to Cbz protecting grqup strategy which was proved to be

effective in case of preparation of gem-difluoro analogue of carnosine. According to that,

Cbz protected difluoro P-alanine 1.95 coupled with histamine methylester hydrochloride using EDCVHOBt in the presence of N-methyl morpholine in DMF to obtain the protected version of gem-difluoro analogue of carcinine 1.101 in 72% yield. Later, deprotection of the Cbz group from 1.101 was attempted to prepare the final compound, 38 gem-difluoromethylene carcinine 1.100. Compound 1.101 was subjected to hydrogenolysis in the presence of a catalytic amount of Pd in MeOH. The reaction mixture was stirred for 36h, Pd was removed by filtration, and the solvent was evaporated to get the final compound N-(2-(lH-imidazol-4-yl)ethyl)-3-amino-2,2- difluoropropanamide 1.100 in very good yield (86%). The final Cbz deprotection reaction was simple, efficient and gave final product gem-difluoromethylene carcinine 1.100 in very good yield.

H F F F F N~N~NHCbz CbzHN~OH EDCI, HOBT + l( __ ~ 0 NMM, DMF N 0 -20 °C- RT, 16h H 1.101 1.84 1.95 Pd/C, H2 MeOH, 32h

1.100

Figure 1.32: Synthesis of gem-difluoromethylene carcinine using Cbz-protecting method

Synthesis of gem-difluoromethylene carcinine 1.100 was achieved in six steps starting from N,N-(bis-phenylmethyl)-benzotriazolyl-1-methylamine 1. 78 with an overall yield of 22%.

1.3 CONCLUSIONS

gem-Difluoromethylene analogues of carnosine and carcinine were synthesized in six steps starting from benzotriazolyl methylamines with an over all yields of23.2% and

22% respectively. The synthetic method for these compounds mainly consists of the 39

preparation of N-Cbz protected a,a-dif1uoro-~-alanine and its coupling with histidine

methylester dihydrochloride or histamine dihydrochloride followed by deprotection of

Cbz group by hydrogenolysis. N-Cbz protected a,a-difluoro-P-alanine was prepared by

deprotecting the dibenzyl group form N-dibenzyl protected a,a-difluoro-P-alanine ethyl

ester and reprotecting the resulting free amine with benzyl chloroformate foiiowed by

hydrolysis of ester. The reagent combination EDCUHOBT was found to be high yielding

for the coupling of N-dibenzyl-protected a,a-difluoro-P-alanine and histidine methyl ester

dihydrochloride and has been used for all the other coupling reactions. The final

deprotection of Cbz group by hydrogenolysis using Pd/C was simple and gave high

yields for the products. The developed synthetic procedure is simple, effective and can be

used to incorporate the gem-difluoromethylene group in other biologically interesting

peptides.

1.4 EXPERIMENTAL RESULTS

1.4.1 General: All reagents were purchased from commercial sources and used as

received. Column chromatography was carried out using Merck 60, 230-400 mesh. Thin

layer chromatography was carried out using silica gel coated polyester backed sheets.

GC/MS spectra were recorded on Hewlett-Packard 5989A spectrometer, equipped with a

Hewlett-Packard 5890 gas chromatograph. The 1H, 13C and 19F NMR spectra for CDCl3,

CD30CD3 and CD30D solutions were obtained on a INOV A-Varian 400 MHz spectrometer at 400, 100, and 386 MHz, respectively. The 1H NMR, 13 C NMR chemical shifts are referred to the residual solvents signals or the internal TMS (8 = 0.0). The 19F chemical shifts are referred to the internal CFCh (8 19F = 0.0). 40

1.4.2 Synthesis and properties of products

N,N-(Bis-phenylmethyl)-benzotriazolyl-1-methylamine (1. 78)

~'N, N l_ r-Ph N...... _ 1.78 Ph

Benzotriazole (5.97 g, 50 mmol) and dibenzyl amine (10.85 g, 55 mmol) were dissolved

in methanol (30 mL). To the resulting solution, 37% formaldehyde (4.5 mL, 60 mmol)

was added. Solid was formed instantaneously. The formed solid was filtered and

recrystallized from methanol to obtain the product N,N-(Bis-phenylmethyl)-

benzotriazolyl-1-methylamine 1. 78: yield 14.0 g (85%)

N-(4-methoxy phenyl)-benzotriazolyl-1-methylamine (1.86) CC} l_~-o-OMe 1.86

Benzotriazole (5.97 g, 50 mmol) and p-anisidine (10.85 g, 55 mmol) were dissolved in

methanol (30 mL). To the resulting solution, 37% formaldehyde (4.5 mL, 60 mmol) was

added. Solid was formed instantaneously. The formed solid was filtered and recrystallized from methanol to obtain the product N-(4-methoxy phenyl)-benzotriazolyl-

!-methylamine 1.86: yield 7.7 g (85%) 41

N-(4-benzyloxy carbonyl)-benzotriazolyl-1-methylamine (1.91)

1-(Hydroxylmethyl)-benzotriazole (2g, 13.41 mmol), benzyl carbamate (2.03 g, 13.41 mmol) and catalytic amount of toluene p-sulfinic acid monohydrate in 30 mL of toluene were refluxed for 15 h in an apparatus fitted with a Dean-Stark water separator. The mixture was cooled and the crystals formed were separated. Recrystallization from toluene gave the product 1.91: yield 2.6 g (70%)

Ethyl3-(dibenylamino)-2,2-difluoropropanoate (1.80)

~ FXF ~ r-Ph EtO If "'""N'-Ph 0 1.80

To the suspensiOn of zinc dust (0.927 g, 14.1 mmol) in dry THF (15 m L), chlorotrimethylsilane (1 mL, 7.9 mmol) was added under nitrogen atmosphere and stirred the mixture for 10 min. Ethyl bromodifluoroacetate (0.94 mL, 7.9 mmol) was added to the reaction mixture and stirred for 20 min, followed by the addition of N-(Bis- phenylmethyl)-benzotriazolyl-1-methylamine 1. 78 (2. 7 g, 8.13 mmol) in THF (20 mL) dropwise and allowed the reaction mixture to stir for 3h at room temperature. Reaction was quenched with saturated NaHC03 and solids were filtered on celite 545. The filtrate 42

was extracted with ethyl acetate (3 x 30 mL), washed with brine and dried over MgS04.

The solvent was evaporated and the residue was diluted with ether. The formed solid was

filtered and the ether was evaporated, and purified the residue by column

chromatography to give the product Ethyl 3-(dibenylamino)-2,2-difluoropropanoate 1.80

as a colorless oil: yield 2.26 g (88%)

1H NMR (400 MHz; CDCh) o 1.2 (t, J = 7.2 Hz, 3 H), 3.I4 (t, hH = I2.8 Hz, 2 H), 3.67

(s, 4 H), 4.I6 (q, J = 7. 2Hz, 2 H), 7.26-7.36 (m, IO H) ppm.

13 C NMR (IOO MHz; CDCh) o I4.0I, 55.49 (t, 2Jc-F = 26.6 Hz), 58.75, 62.96, I16.53 (t,

1Jc-F = 251.2 Hz), I27.57, I28.57, I29.42, I38.44, I64.09 (t, 2Jc-F = 31.9 Hz) ppm.

19F NMR (376 MHz; CDCh/CFCh) o-I06.83 (t, J = 13.9 Hz) ppm.

3-(Dibenzylamino)-2,2-difluoropropanoic acid (1.81)

To the solution of Ethyl 3-(dibenylamino )-2,2-difluoropropanoate 1.80 (760 mg, 2.28 mmol) in acetonitrile (10 mL), IN LiOH aqueous solution (10 mL) was added and the reaction mixture was stirred for 3h. The solvent was evaporated and diluted with water (5 mL) and washed with ether (10 mL). The aqueous phase was acidified with IN HCl (until pH I) and extracted with ethyl acetate (3 x 20 mL). The solvent was evaporated, and the residue was purified by column chromatography to give the product 3-( dibenzylamino )-

2,2-difluoropropanoic acid 1.81: yield 0.52 g (74%) 43

I H NMR (400 MHz; CDCh) o 3.46 {t, J = 12.8 Hz, 2 H), 4.18 (s, 4 H), 7.29-7.38 (m, 10

H), 10.98 (b, 1H)ppm.

13 C NMR (100 MHz; CD30CD3) o 55.74 (t, 2Jc-F = 26.6 Hz), 58.73 (t, 4Jc_F = 12.2 Hz),

116.88 (t, 1Jc-F = 249.7 Hz), 128.13, 129.07, 130.02, 138.53, 165.11 (t, 2Jc_F = 31.2 Hz) ppm.

19 F NMR (376 MHz; CD30CD3/CFCh) o -105.89 (t, J = 13.7 Hz) ppm.

Methyl 2-(3-( dibenzylamino )-2,2-difluoropropanamido)-3-(lH-imidazol-4- yl)propanoate (1.83)

Procedure 1: 3-(Dibenzylamino)-2,2-difluoropropanoic acid 1.81 (1 06 mg, 0.34 7 mmol), Histidine methylester hydrochloride (84 mg, 0.347 mmol) and 1-hydroxy benzotriazole (51.6 mg, 0.382 mmol) were taken in THF (10 mL), stirred under nitrogen atmosphere, and treated with 4-methylmorpholine (0.1 mL, 0. 7 mmol). The solution was cooled to 0 °C and added dicyclohexyl carbodiimide (71.5mg, 0.347 mmol) in dichloromethane (2.5 mL). The resulting solution was allowed to come slowly to room temperature and stirred for 24 h. The solvent was evaporated; saturated NaHC03 was added, and extracted with hot ethyl acetate (5 x 20 mL). The solvent was dried over

MgS04 and concentrated. The residue was purified by column chromatography to give the product methyl 2-(3-( dibenzylamino )-2,2-difluoropropanamido )-3-( I H-imidazol-4- yl)propanoate 1.83: yield 49 mg (31 %) 44

Procedure 2: 3-(Dibenzylamino )-2,2-difluoropropanoic acid 1.81 ( 106 mg, 0.34 7

mmol), histidine methylester hydrochloride 7 (84 mg, 0.347 mmol) and !-hydroxy

benzotriazole (51.6 mg, 0.382 mmol) were taken in dimethyl formamide (5 mL), stirred

under nitrogen atmosphere, and treated with 4-methylmorpholine (0.1 mL, 0. 7 mmol).

The solution was cooled to -20 °C and added dimethylaminopropyl-3-ethyl carbodiimide

(73.3 mg, 0.382 mmol). The resulting solution was allowed to come slowly to room temperature and stirred for 24 h. The solvent was evaporated, saturated NaHC03 was added, and extracted with hot ethyl acetate (5 x 20 mL). The solvent was dried over

MgS04 and concentrated. The residue was purified by column chromatography to give the product methyl 2-(3-( dibenzylamino )-2,2-difluoropropanamido )-3-( 1H-imidazol-4- yl)propanoate 1.83: yield 66 mg (42%)

Procedure 3: 3-(Dibenzylamino)-2,2-difluoropropanoic acid 1.81 (106 mg, 0.347 mmol), histidine methylester hydrochloride 7 (84 mg, 0.347 mmol) and BOP reagent

(154 mg, 0.347 mmol) were taken up in acetonitrile, stirred under nitrogen atmosphere, and treated with triethylamine (0.15 mL, 1.04 mmol). The mixture is stirred at room temperature for overnight. The precipitate was filtered off and washed with acetonitrile.

Acetonitrile was evaporated, the residue was dissolved in ethyl acetate, washed with 2N

HCl and saturated aqueous NaHC03. The organic layer was dried over MgS04, concentrated and purified by column chromatography to get the product methyl 2-(3-

( dibenzylamino )-2,2-difluoropropanamido )-3-( 1H-imidazol-4-yl)propanoate 1.83: yield

57 mg (36%)

1H NMR (400 MHz; CD30D) 8 3.12 (m, 2 H), 3.11 (m, 2 H), 3.63 (s, 3 H), 3.65 (s, 4 H),

4.74 (m, 1 H), 6.85 (s, 1 H), 7.26 (m, 10 H), 7.54 (s, 1 H) ppm. 45

13c NM ( R 100 MHz; CD30D) 8 29.79, 52.90, 54.04, 55.88 (t, 2Jc_F = 25.8 Hz), 59.14,

117.69, 118.41 (t, 1Jc-F = 252 Hz), 134.72, 136.48, 139.55, 166.05 (t, 2Jc-F = 28.9 Hz),

172.41 ppm.

19F NMR (376 MHz; CD30D/CFCb) 8-108.31 (m) ppm.

N-(2-(1H-imidazol-4-yl)ethyl)-3-(dibenzylamino )- 2,2-diflu orop ropanamide (1.85)

3-(Dibenzylamino)-2,2-difluoropropanoic acid 1.81 ( 200 mg, 0.656 mmol), histamine hydrochloride (121 mg, 0.656 mmol) and 1-hydroxy benzotriazole (97 .4 mg, 0. 721 mmol) were taken up in dimethyl formamide (5 mL), stirred under nitrogen atmosphere, and treated with 4-methylmorpholine (0.15 mL, 1.31 mmol). The solution was cooled to

-20 °C and added dimethylaminopropyl-3-ethyl carbodiimide (138 mg, 0. 721 mmol). The resulting solution was allowed to come slowly to room temperature and stirred for 24 h.

The solvent was evaporated, saturated NaHC03 was added, and extracted with hot ethyl acetate (5 x 20 mL). The solvent was dried over MgS04 and concentrated. The residue was purified by column chromatography to give the product N-(2-(1H-imidazol-4- yl)ethyl)-3-(dibenzylmino)-2,2-difluoropropanamide 1.85: yield 68 mg (26%)

1 . H NMR (400 MHz; CD30D) 8 2.81 (t, J = 7.2 Hz, 2 H), 3.11 (t, J = 13.6 Hz, 2 H), 3.47

(t, J = 7.2 Hz, 2 H), 3.63 (s, 4 H), 6.84 (s, 1 H), 7.27 (m, 10 H), 7.62 (s, 1 H) ppm. 46

13 C NMR (100 MHz; CD30D) 8 27.41, 40.39, 56.07 (t, 2Jc-r = 26.6 Hz), 59.30, 117.53,

118.60 (t, 1Jc-F = 251.2 Hz), 128.20, 129.25, 130.07, 136.07, 139.59, 166.18 (t, 2Jc-F =

28.8 Hz) ppm.

19 F NMR (376 MHz; CD30D/CFCh) 8-107.93 (m) ppm.

Ethyl 2,2-difluoro-3-(4-methoxyphenylamino )propanoate (1.87) FF Ho­ EtO~N ~ !J OMe 0 1.87

To the suspensiOn of zmc dust (647 mg, 9.9 mmol), in dry THF (1 0 mL), chlorotrimethylsilane (0.63 mL, 4.95 mmol) was added under nitrogen atmosphere and stirred the mixture for 10 min. Ethyl bromodifluoroacetate was added to the reaction mixture and stirred for 20 min, followed by the addition of N-( 4-methoxy phenyl)- benzotriazolyl-1-methylamine 1.86 (1.269 g, 4.95 mmol) in THF (15 mL) dropwise and the reaction mixture was stirred for 3h at 50 °C. Reaction was quenched with saturated

NaHC03 and solids were filtered on celite 545. The filtrate was extracted with ethyl acetate, washed with IN HCl and dried over MgS04. The solvent was evaporated and the residue was diluted with ether. The formed solid was filtered and the ether was evaporated of, and purified the residue by column chromatography to give the product ethyl 2,2-difluoro-3-( 4-methoxyphenylamino )propanoate 1.87 as a colorless oil: yield

0.97 (76%) 47

I H NMR (400 MHz; CDCh) 8 1.25 (t, J = 7.2 Hz, 3 H), 3.70 (s, 3 H), 3.71 (t, J11 _F = 12.8

Hz, 2 H), 4.22 (q, J = 7.2 Hz, 2 H), 6.62 (d, J = 8.8 Hz, 2 H), 6.75 (d, J = 9.2 Hz, 2 H)

ppm.

13C NMR (100 MHz; CDCh) 8 13.6, 48.15 (t, 2Jc-F = 27.3 Hz), 55.41, 62.90, 114.51 (t,

1Jc-F = 251.3 Hz), 114.61, 114.77, 140.77, 152.85, 163.46 (t, 2Jc-F = 31.8 Hz) ppm.

19 F NMR (376 MHz; CDCh/CFCh) 8-111.27 (t, J = 13.7 Hz) ppm.

2,2-Difluoro-3-(4-methoxyphenylamino )propanoic acid (1.88)

F. FHo­ HO~N ~ /; OMe 0 1.88

To the solution of Ethyl 2,2-difluoro-3-(4-methoxyphenylamino)propanoate 1.87 (1.2g,

4.63 mmol) in acetonitrile (15 mL), IN LiOH aqueous solution (15 mL) was added and the reaction mixture was stirred for 3h. The solvent was evaporated, diluted with water (5 mL) and washed with ether (10 mL). The aqueous phase was acidified with IN HCl (until pH l) and extracted with ethyl acetate (3 x 30 mL). The solvent was evaporated, and the residue was purified by column chromatography to give the product 2,2-difluoro-3-( 4- methoxyphenylamino)propanoic acid 1.88: yield 0.79 g (74%)

1H NMR (400 MHz; CD30CD3) 8 3.67 (s, 3 H), 3.77 (t, J = 14.4 Hz, 2 H), 6.73 (s, 4 H).

13C NMR (100 MHz; CD30CD3) 8 48.51 (t, 2Jc-F = 27.6 Hz), 55.68, II5.27, 116.18 (m),

142.68, 153.40, I65.I2 (m) ppm.

19F NMR (376 MHz; CD30CD3/CFCh) 8 -II0.67 (t, J = 13.7 Hz) ppm. 48

Methyl 2-(2,2-difluoro-3-(4-methoxyphenylamino )propanamido )-3-(1 H -imidazol-4- yl)propanoate (1.89)

2,2-Difluoro-3-(4-methoxyphenylamino)propanoic acid 1.88 (200 mg, 0.86 mmol),

histidine methylester hydrochloride (210 mg, 0.86 mmol) and 1-hydroxy benzotriazole

(129 mg, 0.95 mmol) were taken in dimethyl formamide (5 mL), stirred under nitrogen

atmosphere, and treated with 4-methylmorpholine (0.2 mL, 1.8 mmol). The solution was

cooled to -20 °C and added dimethylaminopropyl-3-ethyl carbodiimide (183 mg, 0.95 mmol). The resulting solution was allowed to come slowly to room temperature and

stirred for 24 h. The solvent was evaporated, saturated NaHC03 was added, and extracted with hot ethyl acetate (5 x 20 mL). The solvent was dried over MgS04 and concentrated.

The residue was purified by column chromatography to give the product Methyl 2-(2,2- difluoro-3-(4-methoxyphenylamino)propanamido)-3-(1H-imidazol-4-yl)propanoate 1.89: yield 235 mg (71 %)

1H NMR (400 MHz; CD30D) o 3.01 (dd, J = 8.8 Hz, 14.8 Hz, 1 H), 3.12 (dd, J = 5.2 Hz,

14.8 Hz, 1 H), 3.64 (m, 2 H), 3.66 (s, 3 H), 3.68 (s, 3 H), 4.68 (m,1 H), 6.63 (d, J = 8.8

Hz, 2 H), 6.72 (d, J = 9.2 Hz, 2 H), 6.79 (s, 1 H), 7.53 (s, 1 H) ppm. 49

13C NMR (100 MHz; CD30D) 8 29.54, 48.78 (t, 2Jc-F = 28.1 Hz), 52.95, 54.07, 56.11,

115.591, 115.67, 117.61 (t, 1Jc-F =252Hz), 117.78, 134.58, 136.43, 143.18, 153.88,

165.99 (t, 2Jc-F = 28.9 Hz), 172.41 ppm.

19 F NMR (376 MHz; CD30D/CFCb) 8-111.85 (m) ppm.

3-Ethoxy-2,2-difluoro-3-oxopropan-1-aminium 2,2,2-trifluoroacetate (1.93)

To a solution of Ethyl 3-(dibenylamino)-2,2-difluoropropanoate 1.80 (1 g, 3.0 mmol) in

EtOH (5 mL) and trifluoroacetic acid (0.28 mL, 3.6 mmol), Pd(OH)2 ( 20 wt.% on carbon) (100 mg) was added and the reaction mixture was stirred for 48 h under hydrogen atmosphere. The reaction mixture was filtered and evaporated the solvent to get the product 3-ethoxy-2,2-difluoro-3-oxopropan-1-aminium 2,2,2-trifluoroacetate 1.93: yield 0.66 g (82%)

1H NMR (400 MHz; DMSO-d6) 8 1.27 (t, J = 7.2 Hz, 3 H), 3.70 (t, J =16Hz, 2 H), 4.31

(q, J = 7.2 Hz, 2 H) ppm.

19F NMR (376 MHz; DMSO-dJCFCb) 8-74.03,-108.51 (t, J =17Hz) ppm. 50

E thy I 3-(benzyloxycarbonylamino)-2,2-difluoropropanoate (1.92)

F F H EtO~N[(O'-./Ph 0 0 1.92

3-Ethoxy-2,2-difluoro-3-oxopropan-1-aminium 2,2,2-trifluoroacetate 1.93 (1.22 g, 4.5 mmol) was taken up in dichloromethane (10 mL) and added triethylamine (1.4 mL, 10 mmol). The reaction mixture was cooled to 0 °C, benzyl chloroformate (0.7 mL, 4.95 mmol) was added and allowed to stir for 4h at room temperature. Water was added to the reaction mixture and extracted with dichloromethane (3 x 20 mL). The organic layer was washed with IN HCl, dried over MgS04, and evaporated. The obtained residue was purified by column chromatography to gtve the product Ethyl 3-

(benzyloxycarbonylamino )-2,2-difluoropropanoate 1.92: 1.02 g (78%)

1H NMR (400 MHz; CDCh) o 1.28 (t, J = 7.2 Hz, 3 H), 3.80 (m, 2 H), 4.26 (q, J = 7.2

Hz, 2 H), 5.08 (s, 2 H), 7.32 (m, 5 H) ppm.

13C NMR (100 MHz; CDCh) o 13.61, 43.59 (t, 2Jc-F = 28.1 Hz), 63.15, 67.13, 113.02 (t,

1Jc-F = 251.2 Hz), 127.97, 128.11, 128.37, 135.87, 156.13, 162.81 (t, 2Jc-F = 31.1 Hz) ppm.

19F NMR (376 MHz; CDCh/CFCh) o -112.22 (t, J = 13.7 Hz) ppm. 51

E thy I 3-(tert-butoxycarbonylamino)-2,2-difluoropropanoate (1.94)

F. F H EtO~NI(O~ 0 0 \- 1.94

3-Ethoxy-2,2-difluoro-3-oxopropan-1-aminium 2,2,2-trifluoroacetate 1.93 (0.42 g, 1.57 mmol) was taken up in dichloromethane (5 mL) and pyridine (0.5 mL). The reaction mixture was cooled to 0 °C, di-tert-butyl-dicarbonate (0.343 g, 1.57 mmol) in dichloromethane (1 mL) was added and the mixture was allowed to stir for overnight at room temperature. Water was added to the reaction mixture and extracted with dichloromethane (3 x 20 mL). The organic layers were pooled, dried over MgS04, and evaporated. The obtained residue was purified by column chromatography to give the product ethyl 3-(tert-butoxycarbonylamino)-2,2-difluoropropanoate 1.94: yield 0.31 g

(78%)

1H NMR (400 MHz; CDCh) () 1.11 (t, J = 6.8 Hz, 3 H), 1.19 (s, 9 H), 3.53 (m, 2 H), 4.08

(q, J = 7.2 Hz, 2 H) ppm.

3-(benzyloxycarbonylamino)-2,2-difluoropropanoic acid (1.95)

F. F H HO~NI(O'-./Ph 0 0 1.95

To the solution of Ethyl 3-(benzyloxycarbonylamino )-2,2-difluoropropanoate 1.92 (0.5 g,

1.74 mmol) in acetonitrile (13 mL), IN LiOH aqueous solution (13 mL) was added and 52

the reaction mixture was stirred for 3h. The solvent was evaporated and diluted with

water ( 5 mL) and washed with ether (1 0 mL ). The aqueous phase was acidified with 1N

HCl (until pH 1) and extracted with ethyl acetate (3 x 20 mL). The solvent was

evaporated, and the residue was purified by column chromatography to give the product

3-(benzyloxycarbonylamino)-2,2-difluoropropanoic acid 1.95: 0.33 g (74%)

I H NMR (400 MHz; CDCh) 8 3.72 (m, 2 H), 4.99 (s, 2 H), 7.23 (m, 5 H), 11.27 (s, 1 H)

ppm.

13 C NMR (100 MHz; CDCh) 8 43.56 (t, 2Jc-F = 27.3 Hz), 67.52, 113.08 (t, 1k-F = 250.4

Hz), 127.99, 128.26, 128.49, 135.64, 156.79, 165.44 (t, 2Jc-F = 31.1 Hz) ppm.

19 F NMR (376 MHz; CDCh/CFCh) 8 -112.78 (t, J = 13.7 Hz) ppm.

(S)-Methyl 2-(3-(benzyloxycarbonylamino)-2,2-difluoropropanamido )-3-( 1 H- imidazol-4-yl)propanoate (1.96)

1.96

3-(Benzyloxycarbonylamino)-2,2-difluoropropanoic acid 1.95 (147 mg, 0.567 mmol), histidine methylester hydrochloride (137 mg, 0.567 mmol) and 1-hydroxy benzotriazole

(85 mg, 0.624 mmol) were taken up in dimethyl formamide (5 mL), stirred under nitrogen atmosphere, and treated with 4-methylmorpholine (0.15 mL, 1.13 mmol). The solution was cooled to -20 °C and dimethylaminopropyl-3-ethyl carbodiimide (120 mg,

0.624 mmol) was added. The resulting solution was allowed to come slowly to room temperature and stirred for 24 h. The solvent was evaporated, saturated NaHC03 was 53

added, and the mixture was extracted with hot ethyl acetate (5 x 20 mL). The solvent was

dried over MgS04 and concentrated. The residue was purified by column

chromatography to give the product (S)-methyl 2-(3-(benzyloxycarbonylamino )-2,2-

difluoropropanamido)-3-(IH-imidazol-4-yl)propanoate 1.96: yield 182 mg (78%)

I H NMR (400 MHz; CD30D) 8 3.06 (dd, J = 8.8 Hz, 15 Hz, 1 H), 3.16 (dd, J = 5.6 Hz,

14.8 Hz, 1 H), 3.67 (s, 3 H), 4.74 (dd, J = 5.2 Hz, 8.4 Hz, 1 H), 5.07 (s, 2 H), 7.31 (m, 5

H), 6.87 (s, 1 H), 7.57 (s, 1 H) ppm.

uC NMR (100 MHz; CD30D) 8 29.43, 44.34 (t, 21c-F = 27.3 Hz), 52.99, 54.02, 67.82,

116.29 (t, 'lc-F = 252.7 Hz), 117.72, 128.82, 129.02, 129.43, 134.61, 136.49, 137.94,

158.67, 165.04 (t, 2Jc_F = 28.1 Hz), 172.31 ppm.

19 F NMR (376 MHz; CD30DICFCh) 8 -112.86 (m) ppm.

3-(tert-bu toxycarbonylamino)-2,2-difluoropropanoic acid (1.97)

F. F H HO~NI(O'(- o 0 1.97

Ethyl 3-(tert-butoxycarbonylamino )-2,2-difluoropropanoate 1.94 (280 mg, 1.11 mmol) was taken in 2:2:1 THF I MeOH I H20 (30 mL) and LiOH (28 mg, 1.162 mmol) was added. The reaction was allowed to stir at room temperature for 12 h. The volatiles were removed by rotary evaporation and the resulting aqueous solution was washed with dichloromethane (10 mL). The aqueous layer was then acidified with 10% citric acid solution and extracted with ethyl acetate (3 x 20 mL). The organic layers were combined, 54

dried over MgS04, and evaporated to get the product 3-(tert-butoxycarbonylamino)-2,2-

difluoropropanoic acid 1.97: yield 180 mg (72%)

I H NMR (400 MHz; CDCh) ~ 1.45 (s, 9 H), 3.66 (t, J =14Hz, 2 H) ppm.

I3c NMR (100 MHz; CDCh) ~ 30.76, 32.86, 45.95 (t, 2Jc-F = 26.5 Hz), 118.35 (t, 1Jc_F =

250.5 Hz), 160.74, 172.26 (m) ppm.

(S)-Methyl 2-(3-(tert-butoxycarbonylamino)-2,2-difluoropropanamido)-3-(1 H- imidazol-4-yl)propanoate (1.98)

3-(tert-Butoxycarbonylamino)-2,2-difluoropropanoic acid 1.97 (300 mg, 1.33 mmol), histidine methylester hydrochloride (323 mg, 1.33 mmol) and !-hydroxy benzotriazole

(199 mg, 1.47 mmol) were taken up in dimethyl formamide (5 mL), stirred under nitrogen atmosphere, and treated with 4-methylmorpholine (0.3 mL, 2.67 mmol). The solution was cooled to -20 °C and added dimethylaminopropyl-3-ethyl carbodiimide (281 mg, 1.47 mmol). The resulting solution was allowed to come slowly to room temperature and stirred for 24 h. The solvent was evaporated; saturated NaHC03 was added, and the solution was extracted with hot ethyl acetate (5 x 20 mL). The solvent was dried over

MgS04 and concentrated. The residue was purified by column chromatography to give the product (S)-methyl 2-(3-(tert-butoxycarbonylamino )-2,2-difluoropropanamido )-3-

(IH-imidazol-4-y1)propanoate 1.98: yield 0.32 g (65%) 55

I H NMR (400 MHz; CD30D) 8 1.42 (s, 9 H), 3.13 (m, 2 H), 3.63 (t, J = 14.8), 3.70 (s, 3

H), 4.72 (m, 1 H), 6.89 (s, 1 H), 7.61 (s, 1 H) ppm.

I3c NMR (100 MHz; CD30D) 8 28.62, 29.51, 43.89 (t, 2Jc-F = 27.4Hz), 52.99, 54.00,

116.27 (t, 1lc-F = 252.7 Hz), 117.86, 134.46, 136.50, 157.97, 165.15 (t, 2Jc-F = 28.1 Hz),

172.28 ppm.

19 F NMR (376 MHz; CD30D/CFCh) 8-113.13 (m) ppm.

(S )-Methyl 2-(3-amin o-2,2-difluoropropanamido)-3-(1 H -imidazol-4-yl)propanoate

(1.90)

Procedure 1: Solution of eerie ammonium nitrate (1.37 g, 2.5 mmol) in water (8 mL)

was added dropwise to the solution of PMP protected dipeptide 1.89 (191 mg, 0.5

mmo )in 5 mL of acetonitrile. The reaction mixture was stirred for 2 h and the solvent was

evaporated in vacuum. The residue was taken up in water and 5% NaHC03 was added

until pH 7 was reached, and then 20% aqueous solution of NazS03 was added and the

mixture was extracted with hot ethyl acetate (5 x 20 mL). The combined organic layers were washed with brine, and dried over MgS04. After filtration, the solvent was evaporated and the crude product was purified by column chromatography to obtain the product gem-difluoro analogue of camosme, (S)-methyl 2-(3-amino-2,2- difluoropropanamido)-3-(lH-imidazol-4-yl)propanoate 1.90: yield 20mg (14%) 56

Procedure 2: (S)-Methyl 2-(3-(benzyloxycarbonylamino )-2,2-difluoropropanamido )-3-

(1 H-imidazol-4-yl)propanoate 1.96 (170 mg, 0.415 mmol) was taken up in MeOH (5 mL)

and Pd ( 30 wt.% on carbon) (50 mg) was added. The reaction mixture was stirred for 36

h under hydrogen atmosphere, filtered, and evaporated to get the product (S)-Methyl 2-

(3-amino-2,2-difluoropropanamido)-3-(IH-imidazol-4-yl)propanoate 1.90: yield 96 mg

(84%)

I H NMR (400 MHz; CD30D) () 3.08 (t, JH-F = 14.8 Hz, 2 H), 3.09 (dd, J = 5.6 Hz, 14.6

Hz, I H), 3.20 (dd, J = 5.2 Hz, 15 Hz, 1 H), 3.72 (s, 3 H), 4.75 (m, 1 H), 6.89 (s, 1 H),

7.61 (s, 1 H) ppm.

13C NMR (100 MHz; CD30D/CDCh) o 27.57, 43.66 (t, 2Jc-F = 26.6 Hz), 51.48, 52.05,

115.33 (t, 1Jc-F = 251.2), 115.84, 132.43, 134.52, 163.66 (t, 2Jc-F = 28.9 Hz), 170.43 ppm.

19 F NMR (376 MHz; CD30D/CFCb) o -114.25 (t, J = 14.5 Hz) ppm.

N -(2-(1 H -imidazol-4-yl)ethyl)-2,2-difluoro-3-(4- methoxyphenylamino)propanamide

(1.99)

H F. FH-o­ N~N~N ~ !J OMe I(_~ o N H 1.99

2,2-Difluoro-3-( 4-methoxyphenylamino )propanoic acid 1.88 (300 mg, 1.3 mmol), histamine hydrochloride (239 mg, 1.3 mmol) and !-hydroxy benzotriazole ( 193 mg, 1.43 mmol) were taken up in dimethyl formamide (5 mL), stirred under nitrogen atmosphere, 57 and treated with 4-methylmorpholine (0.3 mL, 2.6 mmol). The solution was cooled to -20

°C and to it was added dimethylaminopropyl-3-ethyl carbodimide (274 mg, 1.43 mmol).

The resulting solution was allowed to come slowly to room temperature and stirred for 24 h. The solvent was evaporated, treated with NaHC03, and extracted with hot ethyl acetate

(5 x 20 mL). The solvent was dried over MgS04 and concentrated. The residue was purified by column chromatography to give the product N-(2-(lH-imidazol-4-yl)ethyl)-

2,2-difluoro-3-(4-methoxyphenylamino)propanamide 1.99: yield 280 mg (62%)

1H NMR (400 MHz; CD30D) () 2.70 {t, J = 7.2 Hz, 2 H), 3.41 (t, J = 7.2 Hz, 2 H), 3.66 (t,

JH-F = 13.6 Hz, 2 H), 3.67 (s, 1 H), 6.64 {d, J = 9.2 Hz, 2 H), 6.72 {d, J = 9.2 Hz, 2 H),

6.76 {s, 1 H), 7.62 (s, 1 H) ppm.

13C NMR (100 MHz; CD30D) () 25.54, 38.67, 46.96 {t, 2Jc-F = 28.1 Hz), 54.32, 113.37,

113.88, 115.86, 115.95 (t, 1Jc-F = 252 Hz), 134.04, 134.29, 141.41, 152.05, 164.29 (t,

2Jc-F = 28.1 Hz) ppm.

19F NMR (3 76 MHz; CD30D/CFCb) 8 -111.69 (t, h-H = 13.7 Hz) ppm.

Benzyl 3-(2-(lH-imidazol-4-yl)ethylamino)-2,2-difluoro-3-oxopropylcarbamate (1.101)

H F. F H N~N~NnO'-../Ph !!___ -~ 0 0 N H 1.101

3-(Benzyloxycarbonylamino)-2,2-difluoropropanoic acid 1.95 (178 mg, 0.687 mmol), histamine hydrochloride (126 mg, 0.687 mmol) and !-hydroxy benzotriazole (102 mg, 58

0.756 mmol) were taken up in dimethyl formamide (5 mL), stirred under nitrogen atmosphere, and treated with 4-methylmorpholine (0.15 mL, 1.374 mmol). The solution was cooled to -20 °C and dimethylaminopropyl-3-ethyl carbodiimidc ( 145 mg, 0. 756 mmol) was added. The resulting solution was allowed to come slowly to room temperature and stirred for 24 h. The solvent was evaporated, saturated NaHC03 was added, and the mixture was extracted with hot ethyl acetate (5 x 20 mL). The solvent was dried over MgS04 and concentrated. The residue was purified by column chromatography to give the product benzyl 3-(2-(lH-imidazol-4-yl)cthylamino)-2,2- difluoro-3-oxopropylcarbamate 1.101: yield 174 mg (72%)

1H NMR (400 MHz; CD30D) o 2.77 (t, J = 7.2 Hz, 2 H), 3.44 (t, J ~= 7.2 Hz, 2 H), 3.73 (t,

JH-F = 14Hz, 2 H), 5.06 (s, 1 H), 6.83 (s, 1 H), 7.25-7.31 (m, 5 H), 7.57 (s, 1 H) ppm.

2 13 C NMR (100 MHz; CD30D) o 27.37, 40.47, 44.38 (t, 1c-F = 28.1 Hz), 67.79, 116.42 (t,

1Jc-F =252Hz), 117.63, 128.82, 129.02, 129.44, 135.80, 136.12, 137.97, 158.71, 165.19

(t, 21c-F = 28.1 Hz) ppm.

19F NMR (376 MHz; CD30D/CFCh) o -112.56 (t, h--11 = 13.7 Hz) ppm.

N-(2-(1H-imidazol-4-yl)ethyl)-3-amino-2,2-difluoropropanamide (1.1 01)

H F F N~N~NH2 /(__ -~ 0 N H 1.101

Procedure 1: A solution ofceric ammonium nitrate (934 mg, 1.7 mmo1) in water (5 mL) was added dropwise to a solution of PMP-protected dipeptide 1.99 (120 mg, 0.3 7 mmol) 59 in 3 mL of acetonitrile. The reaction mixture was stirred for 2 h and the solvent was evaporated in vacuum. The residue was taken up in water treated with 5% NaHC03 until pH 7 was reached, and then 20% aqueous Na2S03 was added and the mixture was extracted with hot ethyl acetate (5 x 20 mL). The combined organic layers were washed with brine, and dried over MgS04. After filtration, the solvent was evaporated and the crude product was purified by column chromatography to obtain the product gem­ ditluoro analogue of carcinine, N-(2-(IH-imidazol-4-yl)ethyl)-3-amino-2,2- ditluoropropanamide 1.100: yield 18 mg (22%)

Procedure 2: Benzyl 3-(2-(1 H-imidazol-4-yl)ethylamino )-2,2-di fluoro-3- oxopropylcarbamate 1.101 (98 mg, 0.278 mmol) was taken in MeOH (5 mL) and added

Pd (30 wt.% on carbon) (50 mg). The reaction mixture was stirred for 36 h under hydrogen atmosphere, filtered, and evaporated to get the product N-(2-(1 H-imidazol-4- yl)ethyl)-3-amino-2,2-ditluoropropanamide 1.100: yield 52 mg (86%)

1H NMR (400 MHz; CD30D) a 2.82 (t, J = 7.2 Hz, 2 H), 3.11 (t, J11-F = 14.8 Hz, 2 H),

3.50 (t, J = 7.2 Hz, 2 H), 6.87 (s, 1 H), 7.62 (s, 1 H) ppm.

2 1 13C NMR (100 MHz; CD30D) a 27.32, 40.38, 45.40 (t, 1c-F = 26.5 Hz), 117.69 (t, k-F =

249.7 Hz), 117.95, 135.69, 136.21, 165.83 (t, 2Jc-F = 28.1 Hz) ppm.

19F NMR (376 MHz; CD30D/CFCh) 8-113.82 (t, J = 14.5 Hz) ppm. 60

1.4.3 NMR spectra of the products

IMPL! OI!C , • Yt' date Oct 17 1111 df'rq 3U . 7U IOIVII't COC1.t dn Hl ftlt tlllp dpwr :n ACOUISITION ctof 1 tt'rq .JII . 71t tn Hl d• """ • , .J ., •• d•f Ul np •·4132 ·-dteq . .., drtt .fb ...... , ... bl 11 .... PlOCUSINO tpwr 55 w-tfile pw 7.1 '~oc ..tof .1 aa'th "' ' aloe•" .n WtiCp t•tn not used .... ,LAOS 11 n .. n -· .. DUPlAV -~ - z.t ::.. as u.s.. V<.. . u .. .Je.. Sll.ll .. 1111. 1 ··-"•'" . .. 111. 000...... ph

. l l I ,..; ,I ...... -,- -~~~-.--.-- ..-.---..-., 8 3 2 PP•

Figure 1.33: 1H NMR spectra of compound 1.80

l I [

ZOI 180

Figure 1.34: 13C NMR spectra of compound 1.80 61

lAMPL! OfC • • VT d•U OCt 11 ZODii dfrq JU . 7U toh•nt COCU dn Hl fth •11p dpwr .u ACQUJSIHOH dot o srrq .J1i. Ut 1:n 'lt d•dllll """c •t o. uo dllf 2 10 np n ne d u q I W StOOO .I d ru f b UOII ... n bl u .... PRDC USINO tpwr ss ,. t .Jo pw S . l wtftl• : proc ..tof .o fn nt UU. ,. c;t au .. •1oclr n w•rr gatn not ut•d w•ltp rLAOS t1 n ...wnt tn n 1 .. .~ OISPU'I' tp -usn . .J ~: 54ii: we" 150. hz.. r.u ts su." rfl ISU7. 1 rfp 0 th to 1ns 111 . 100 n• no pill

. -.-.- ·- J--r-,-r-·-r-·~r , • r·-~~-~~-~ - 106 . 3 - 106 . 4 -1os .s -106 .6 - 106. 7 -1os.a -101 . 1 -101 .0 -101.1 -101 . 2 -107 . 3 -107.4 - 107 . 5 p~

Figure 1.35: 19F NMR spectra of compound 1.80

::;.

l ___!\..______J 11 10 a

Figure 1.36: 1H NMR spectra of compound 1.81 62

. .I 1

-101 . 2 -10&.3 ppll - lOS . S -115. 1 -115 . 7 -us·. a -105 . I -u&.o -101.1

Figure 1.37: 19F NMR spectra of compound 1.81

__ ..,JI r 7 . 5 7.1 •. 5 •••

Figure 1.38: 1H NMR spectra of compound 1.83 63

ll:alafl. delay t . ••• sac. o·~i::' li::~ ~!·~:::c J.lUI rapat"tttona . OISU'Yl Cll, lU, U1Ult MHt DlCOUP-Lf Hl, 3.U .71.JIS4'.) MH& cel\tti'MiouslvP.awar 4$ -· on 'WAt.Tl-11 •DIIIulriad DATA PII:OCEISlNCJ ltna. b'roa_dan1nl 1.1 Ha fT shr• 151.)1 l'ctt-al t I•• a hr , 54 •In, 41: Sei;

I ~I l I I dl ~~---~~~~-r~-,~~-.~-rro~~~~-~,--~~-.~-.-~-r~~~~~ -r-~~~~~~ 1 7 0 160 1 5 0 140 130 lZO 110 100 90 110 70 10 50 PPII

Figure 1.39: 13 C NMR spectra of compound 1.83

" ., {\I f,! • I \ J

-·~""j.,.... -111.1 - 1111 . 11 -111 . 0 -li! . Z -lit.• '' -107 . Z -1~1 . • -111.1 -tot .a -toa.o -toa.r -toe.• - 101 . 1 -····· ~

Figure 1.40: 19F NMR spectra of compound 1.83 64

ppa 8

Figure 1.41: 1H NMR spectra of compound 1.85

.

1 . ... "! ~ ~ .

1 1

~-./>~j~ -· ------~---~-- -~--~- --- ~- - --- ~.-..-~~~~..--,------, '9- ' -...... •107.4 •107.5 -107.1 •107.7 ·117.1 ·117.1 ·111.1 -101.1 -lii. Z •111.S •118. 4 ppe

Figure 1.42: 19F NMR spectra of compound 1.85 65

: ; ~ .

• N . ~ . ~ ~ . .N - ~"" . ... - -~=~ . 1 t;~ i pr 'f i 1 I i 1 I l I l I

180 140 120 100 ao 10 40 20 ppe

Figure 1.43: 13C NMR spectra of compound 1.87

IN ~ ~ 1 i

-111.3 -111.4 -111.5 -111.1 -111.1 -111.2

Figure 1.44: 19F NMR spectra of compound 1.87 66

.. 1

7.0 6 . 5 6 . 0 5. 5 5 . D 4 .5 4 . 0 3. 5 PPII

Figure 1.45: 1H NMR spectr~ of compound 1.88

! . .~ i

~ . i ~ i ~ 1 i I j L• J .! ..._. .. I I I I I I ' ' I I ' I ' ' - ' I I ' ' 111 Ul II 71 PPII 150 141 Ul Ul .. •• 110

Figure 1.46: 13C NMR spectra of compound 1.88 67

1

-109.8 -110 . 0 -uo . z -11o.4 -110.1 -110 .11 -111.1 -111. z -111.4 -111.1 -111.11 p ...

Figure 1.47: 19F NMR spectra of compound 1.88

I I I j 4 I I I I I ; J • I ' I I I I I I I I I I I ' ' ' ' ' I I I I 5.1 &.5 4.1 1.1 7.5 7.1 1.5 s.s

Figure 1.48: 1H NMR spectra of compound 1.89 68

~ I .1 ll

160 140 120 100 aa so 40 20 PPII

Figure 1.49: 1H spectra of compound 1.89

I I I I I I • I I I I ' I I I ; I I •• I I I I j I I I I I I I I I ' I ' I I I I ' • I I I I t I I -109.1 -111.5 -110.1 -111.5 -111.0 -111.5 -112.1 -11!.5 -113. 0 -111.5 -114.1 p~

Figure 1.50: 19F NMR spectra of compound 1.89 69

l

Figure 1.51: 1H NMR spectra of compound 1.93

t 8 5

Figure 1.52: 1H NMR spectra of compound 1.92 70

----~~~-----~~~---ull~------~·~~~J~--~1 --~~-- 160 140 120 100 ae .. 20 ppa

Figure 1.53: 13C NMR spectra of compound 1.92

SAMLI'LI or:c • .. , data Oct II ltll dfrq •olvant. COCll ""·'" ftla ••11 ...... "' ACQUl&ITIOII .. , ..• afrq 371.llt tfl 1"11 .. """ at t.tll .. , ...' ""IW 111111.1llltll ·-::= ...... 1'1t 11111" ••oc•nu• ...... '"'".. .II.. -vtflla•• '·.." ..tef .I r.;" ...... • aatlill ... "' &tl& ct Ill ' .. aloca • _, eatn .. _ftOt ••" 11 . ...--· ..'" .~" .. t:I8LI'L.-'f' ap -41UI. 7 .,.:: ····: ,_._ 1.11... ,.,,,..,,.. 11111...... 1 Ut 11. !:'..o,.. u•.••• !! = 1 ; I

-liZ .I - u:r.r - ur.a - ur. • - ur.s - ur.e -ur. 1 -uz.a - -111.7 -uz.a

Figure 1.54: 19F NMR spectra of compound 1.92 71

..__ ___l

.4. 5 4 . 0 3. 5 3.1 2. 5 r . a 1.5 1.1 a .s

Figure 1.55: 1H NMR spectra of compound 1.94

·~~·· ,.,, .. a.vtPLl otc. • vr •••• ,.,. I IU7 .-t"r~ Jtf, JII •otveAt CDI:1t f'lle ••P .It'" "'II ACQUI.ITJOfil ...." .. lfrtj 111 . 11t .. _. ..u, •...... , "'•• ...... , .... 1111 . 1 - ev ., .. ... ••,. "''n " ,...,.. II ..,.,- ... r.' , ' ·: r.•c ---...... ,.. nt.. .I ct I ....-·· olec:lr " ,.t• ,._,.., ulllll ...... " " -· •• ..•" ..•• ...... "' .,·' ...oc ...• ...u.tt... ·­rfl.. 1111 .1 •••••...... •, M CiiC ... 1 ii ~------J~~~ __)\______1l II • • • • - Figure 1.56: 1H NMR spectra of compound 1.95 72

~- - ~~ i i l 1 I 1 I. I .. l __,.-I ... ' ,.. "" - 1--.--..-• 170 160 151 uo 130 lZO 110 liD 10 •• 70 10 5I 40 p"'"

Figure 1.57: 13C NMR spectra of compound 1.95

1

Figure 1.58: 19F NMR spectra of compound 1.95 73

ore. • vr dfrq UI.7U lin Hl dpwt ,. dot 1 .. c .. , ...... ·­dral ... n P•otiSIJNO -wtftla .....'~oc not WIM" .... ' ...... ,

Q,IIPLAY .. u7a . .s- 1Ut .a ~ oc •• we • ... .,.1.11 ...... ,, U1a . 1 " Ull .3 11 ~~·... l.tlt n• cdc ph

7 . 5 1.5 1.0 5 . 5 ...... ___,___...... t-·. ll .... c. u •.n ....

Figure 1.59: 1H NMR spectra of compound 1.96

._... KC . a V1' ,. ""' •tr• 111.71& ..selve .. t ...... clll.., 111ft "' ftle IIIJ'W tl ACQUIIITION ••It efr• ltt . lal •• Wn'. .,'" J,.lll011 - ..- , ....w .... llret 1 . 1 ..IIV tllll. . t .... ft Ultt NDCIUt• ltl " ...... II.. -,. '·" r... , • "'''''r.oc " .. Hll• -· .. ., ..a1ec11 .. .. . -··...... , ,._"' ...... -·.. ••.. .• -· •• DIIPLAY .. 1111.1 .. 11UI,4 wo .. ..• ...... , ·-...'" ,...... 111 . 111 -••• ... ..

I ,L I j 1 I 1 1111 I - I I I I 1 I ' ' ' ' ' I I I ' I ' ' ' ' ' lll ' ' ' ' Ul Ill .. .. •• ... 111

Figure 1.60: 13C NMR spectra of compound 1.96 74

l AMIILt: O(C , a VT date Nov 4 t ttl dfr~ .Jti . 7U Solvent. cd.lod dn Ml ftle ••111 .,....,. u ACQUIBJTJON dof 1 s f'rt~ .J7t.ut tn I'll ... """ at 1 . 111 ••f' , ..' np UUal ·-...... lltttl . t .. ,. fb Slttt .. ... bl 11 ••oenatNO . 'tp..,r tl ,. ... ltv 1 . 1 -wtftlt . 111roc ..tot .t nt UU .. ct. tM .... alack 'Wtrr e•tn ttOt Utld _Ill, 11 HAOI ~ =:: ..'" .~ .. DIIIILAY I P - UIOU . .J ~: 1u:i: a< • owe: l S I 1'1&•• 5 . 1 5 h 101.01 rf'l 11411.1 rf~ 1 •• 1 t n• lU . tiG n• no ph

-111.5 -112 . a -112 . 5 .....

Figure 1.61: 19F NMR spectra of compound 1.96

.....,.,lf O(C . I YT dat.' O~t It '1111 tolvtnt cd3Dd dft Ml ffh .... """dpwr ""·'"II ACOUIIIfiDII ,,,., ....,.. ... '""'. til HI .. • , 3 . 741 ftlf Ill np UtM ...... ,,...... -...... •• II IJUCIIII- ,,.,.. II wtft1t- r."' , ..... ::tef '·:t .. ", at I .... ct I aloctt 11 e•t" not usM -· 11 ...-· •• "- . - :~ • DJIPLA't .. -t. o ...•• 1111 . 1 ...... I ,.,...... UIA.I.. ·­,,, liU . I II ••... lit ...... cdc ••

t • I I

Figure 1.62: 1H NMR spectra of compound 1.98 75

SAMIIll!: ore. • VT da'to Oct .u Ztol dfrq UI.IIZ sol>ttnt CDCl.J dn Ht dpwr 45 ftl :CQUlSlTlON UP dof 1 sfrq Ut .$31 d• "yy tn Cl.S •t l,lll d•f·- ....w np IIIII dttq sw 15111. I drtt fb 14111 n bt ...... '·' llllOCUIINO tJtWr SS lb 1.11 ,,pw 1 . .7 wtftle,,..c tof 1 nat uttd nt JUt •• u. ", c:t ... '" aloek 1 ....Wtl(p gafn '"'" uttd rLAGS 11 ... n -• tn n .. .~ -$5.4 11111 • • ...lla• Sll,tt..... 1101.1 Uts . t 1 ... 111.. ph

I J l J I 1 ~~~~~~~~~~~~~~~-~~~~~~~~~~~~~ r~ 180 1&0 140 1ZO lDI 80 II 20 - Figure 1.63: 13 C NMR spectra of compound 1.98

IAI'Ifi' Lt: Ot:C . & Vf data Oct u Utt .,, .. Jti . 7U solvent ct.Jod . Hl ft 1e IIIIP •pw.. r ACOUJ:aiTlON , .. tf'rq 3171.141 .. • tn ... .. < ....'" .., ... .. lUlU...... ;e 111111 . 1 .,·- .. 1.1 ,. 55111 . .. 11 ,AOCIISJNO ... .. ·-wtftle•• . ...''"'' ... r.oc ..... • not YIH 11141• •• u. " ..

·lU.:S 1li. 4- I I -ua.1 -u'a.t -u -112 . 8 -ur.t -u:11.1'

Figure 1.64: 19F NMR spectra of compound 1.98 76

I • ~~~~~~~~·~~~·~-~~~~~~~~~~· 5. D 4 . 5 4 . 1 3.5 3 . 0 2.5 PP'I 7 . 5 7.0 & . S &.D 5 . 5

Figure 1.65: 1H NMR spectra of compound 1.90

.

..::!:...... •.; : ..; ...... :.. : . . .. i.. ... ,r 1

4, • 1 • v : t I 4 I I 4 I' I 111 II II 171 110 151 Ul 111 121 111 " ..

Figure 1.66: 13C NMR spectra of compound 1.90 77

. N -

·1 1 ~II _ p.. --·---- - ______• .->' J \ -114.4 -114 . 1 -114 . 1 -115.0 -113. 8 -113 . 8 -114.8 -114 . 2

Figure 1.67: 19F NMR spectra of compound 1.90

j '------_) '-----J ""-...Ju___ __. --

I I • I f I I I I ' I ' I I I I ' I • 3.5 3.1 2.5 I ' 5.5 5.1 •. s ••• 7.5 7.1 1.5 •••

Figure 1.68: 1H NMR spectra of compound 1.99 78

l . I

:··· ~•ri•~·~•~·~·~•~•Ti~·~·~·~·•~·~·~·~•Ti~ ' ~;~,~·~~·~· .. ~: '~· ''l~\ . ~i.'~· r11~on•~·~••t~•~·~·~~·~·~·~·~ l~'~"~',,~,~·~·~·~lr~•~•,,~,· ~·~·~•r•~i ~~·••r•r•~,,,,~,~·~•rl~·~·~•i ppl 160 140 120 100 ao 10 40

Figure 1.69: 13C NMR spectra of compound 1.99

1

I I • I ( 4 I I I u I 1 -111.1 -'u'r.r ; I I ' I -u 1.~' -111.1 -ur.1 -111.2 -111.3 -1&&.5 -111-1

Figure 1.70: 19F NMR spectra of compound 1.99 79

,. ... , ....,

n ::" •n DUPt..A'I' .. 717 .... ::.. tna... 3. ,...... 11...... 11. rtl...... tH~:~ '"... ,h,, ...... n• cdc

Figure 1.71: 1H NMR spectra of compound 1.101

J.MPLl GIC. I 'IT ...~. ,. l 1117 dfr11 ut.7U •olvertt cdaod dn Hl 'f1h ••, .,_.,. .. ACQUIIITION ht I .,,.~ .... ~,J1 .. rn trt Cl.J .. , ..... np, ltttl'·'" . -.... 1.1 .... 11111.1 .,. .. fb ...... PltOCINl- "'',.,,..,. ..,.." - 1 . 11 Ol I ,., . M1 ...... , "'Ct .....lUI aloca 1 .... gatn ,._ wM not ...... -·· "in . •• .~ .. 018PlA'f '' -u.t :: 11111;: oc I we Ill ..... Jt . .. u -111.11 rfl JtN . I ,.,, 4trt.a '"..iltt .. .. Ut. lll..

I ,lL J.. ~ l I I I ,: I I' ' u ~ ~ Ill Ul .. •• Ul .. ': 111

Figure 1.72: 13C NMR spectra of compound 1.101 80

.... ,u OfC . 0 YT d•t• feb 1 1117 .. .,,.. Sti.1U colvant. cd.loct HI fil:COVnnrON .,.,...... •• s-rrq 3U.141 . tn fll ""' ... .. c •t ..... np 111131 ·-•••dseq ...... 111111 . 1 dras 1.1 fb ...... •• u 'lOCI MUG . tpwr II - pw ••• ...... 1:of .1 nt 111" ct ua alocll n wtrr gatn not u••' wa•p FLoloOS .•. 11 n wnt In . •• .~ .. OIS,LAY - uaat. 4 .. 1115.1 ~= ..0 ..we hf:•• ,.. •...... u ...'"" ...... lti . IU• ... •• 1 ! 11

-,-.~....,.,~....,.,~,-~,-~,-~...,-~...,.,~"'T'~.,..,.~-.,...-~-.,...-~TT'~,...... ,rr , .,-J· ..,.., ,, ~ rT~r_.....,._..,. -111.4 -111 .1 -111.& -ur.o -ur.z -112.4 -uz.a -111.a -111.1 -111. 1 - 111 . 4 -u1. 1 - 11a. a ~ I I I

Figure 1.73: 19F NMR spectra of compound 1.101

; I I . ' 4.1 ,-; I I 5 . 1 4.1 ' I I 5.5 a.o 7 . 5 7.1 1.5 •••

Figure 1.74: 1H NMR spectra of compound 1.100 81

----~--~1~~1· ~------~--~~J ~~----- ...-r~.,....,.-.-~.....,...... ,, ~.,...... ~,...... -..--.--, l 1 I t f f r·~~~.-.-.-~~--, '1 I I t l 0 0 ... t r '0 "t I 1· 1 1 • t 'f I I t I 1 ' 160 140 1ZO 100 80 10 •o ZO p ...

Figure 1.75: 13 C NMR spectra of compound 1.100

i 1

! i 1 1

-uJ.e -1n.1

Figure 1.76: 19F NMR spectra of compound 1.100 82

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2. A NOVEL AND CONVENIENT SYNTHETIC METHOD FOR THE PREPARATION OF gem-DIFLUORODIARYLMETHYLENE COMPOUNDS

2.1 INTRODUCTION

There has been considerable interest in the applications of gem-difluoro derivatives of organic compounds in biological and medicinal chemistry. Introduction of fluorine atom in the organic compounds changes their physical and chemical properties as well as biological activities. 1'2 The CF2 moiety has very similar van der Waals radius and electronic properties as those of oxygen atom, prompting the application of gem- difluoro compounds as isosteric and isopolar analogues of ethers, which can be potentially used as enzyme inhibitors.3 For example, alkoxy or aryloxy oxygen in alkyl- or aryl- phosphonates have been replaced with difluoromethylene group to increase their bioactivity.4 Such phosphonates with difluoromethylene group are hydrolytically stable and biologically more active than the corresponding non-fluorinated phosphonates.

The transformation of CH2 to CF2 in organic compounds has been used to develop stable and potent compounds with higher resistance toward metabolic degradation.

Recently, gem-difluoro-nucleosides have been developed to explore their medicinal applications.5•6 For example, 2' -deoxy-2', 2' -difluorocytidine (Gemcitabine) is currently one of the most effective and widely used anticancer drugs. 7•10 gem-Difluoro-avermectin derivatives have been used as potent anthelminitic and anticonvulsant agents. 11 (2R)-2-

[(lR)-3,3-difluorocyclopentyl]-2-hydroxy-2-phenylacetamides have been used as potent,

. . t 12,13 long-acting and orally active M3-antagoms s. 90

Gemicitabine

M3 antagonist

Figure 2.1: Structures of Gemicitabin and M3 antagonist

A variety of methods have been developed to prepare gem-difluoromethylene

compounds and these methods fall broadly in two classes. 4' 14-17 They are direct gem­

fluorination approach and the fluorinated building block approaches. Direct fluorination

is a powerful technique and particularly used at the final stages in synthetic schemes on

stable intermediates which can withstand the often forcing reaction conditions. The

building block approach is an effective method to incorporate CF2 synthon in organic

compounds using easily accessible fluorinated building blocks. Methods of introducing

fluorine directly are classified as nucleophilic and electrophilic fluorination methods

based on the source of fluorine from fluorinated reagents. Here in this introduction, a

short review of existing methods for direct fluorination is presented.

2.1.1 Aldehydes and ketones to gem-difluoromethylene compounds: The

transformation of aldehydes and ketones to difluoromethylene group is one of the most common and successful strategies to prepare difluoromethylene compounds. The 91 classical method to achieve this transformation uses sulfur tetrafluoride (SF4) with hydrogen fluoride as catalyst. 18-21 This method gives good yields for the desired products, but its use requires extremely cautious reaction conditions because of the toxicity, reactivity, and volatility ofSF4. In addition, special apparatus such as pressure equipment constructed of fluorine resistant material such as "Hastelloy-C" must be used and high temperatures are frequently necessary. Selenium tetrafluoride22 (SeF4) and moolybdenium tetrafluoride23•24 (MoF6) have been used for this transformation, but their application is limited because of their mildness in reactivity and involvement of hazards in their synthesis and use. These drawbacks have been circumvented to a greater extent by development of dialkylaminosulfur tetrafluoride. A series of these reagents have shown similar properties to SF4 in the transformation of carbonyl to gem-difluorination.25

The pioneering work of Middleton is responsible for the development of diethylaminosulfur tetrafluoride (DAST), a commercially available liquid that requires less forcing reaction conditions than SF4 and standard laboratory apparatus. 26 DAST is tolerable to a variety of other functional groups and has been used across many classes of natural products and other biologically interesting systems.27 As an alternative to DAST, bis(2-methoxyethyl)aminosulfur trifluoride (Deoxofluor) has been developed.

Deoxofluor is thermally more stable reagent and can transform aldehydes and ketones to

'ld . d' . 28 29 the corresponding gem-difluoromethylene compoun d s un d er mt reactton con thons. · 92

DAST or Deoxofluor or SF4 or SeF4

MeO~

Deoxofluor = N-SF3 MeO______.f

Figure 2.2: Conversion of aldehydes and ketones to gem-difluoromethylene compounds

Although the mechanism of DAST mediated gem-difluorination of carbonyl compounds has not been experimentally elucidated, the reaction may be initiated by the nucleophilic addition of HF (present in trace amounts in DAST) to the carbonyl group, followed by SN2-type reaction with DAST to give the a-fluoroalkoxydiethylaminosulfur difluoride and HF which further propagates the reaction. The a- fluoroalkoxydiethylaminosulfur difluoride intermediate may undergo intramolecular transfer of fluoride ion from sulfur to carbon with the elimination of diethylaminosulfinyl fluoride (Et2NSOF). Alternatively a carbocation intermediate may be formed which is trapped by the fluoride ion (intermolecular). The formation of the carbocation intermediate, in some cases, is evidenced by the formation of the corresponding fluoro- olefinic products. In polar solvents such as DMF, olefinic products are formed to a significant extent, while in nonpolar solvents such as CH2Ch, gem-difluoro-compounds are pred ommant . 1y 1ormec. d. 30-32 93

HF

R'~R F (favored in DMF)

(favored in CH2CI2)

Figure 2.3: Mechanism of difluorination of carbonyl group with DAST

A similar reaction mechanism has been suggested for the SF4, SeF4, MoF6, and deoxofluor mediated gem-difluorinations.

2.1.2 Hydrazones and Oximes to gem-difluoromethylene compounds: Hydrazone of aldehydes and ketones were transformed to the corresponding gem-difluoromethylene compounds using iodine monofluoride (IF) which is prepared from elemental 12, and F2.33

Side reactions were observed in this procedure when a-proton elimination competes with the second fluorination.

IF

IF

Figure 2.4: Conversion ofhydrazones to gem-difluoromethylene compounds using IF 94

Difluorination of hydrazones has also been effected by in situ generated 'BrF"

usmg the combination of N-bromosuccinimide (NBS) and pyridinium polyhydrogen

fluoride (PPHF).34 By using this procedure, the usage of hazardous elemental fluorine has

been avoided for these reactions.

NBS/PPHF

R =e.g, H, Ph; Ph, Ph; Ph, 4-MeO-Ph; H, PhCH2

Figure 2.5: Conversion ofhydrazones to gem-difluoromethylene compounds using NBS/PPHF

The combination of NIS/PPHF gave unsatisfactory results for difluorination of hydrazones. Unsubstituted hydrazones have severe drawbacks such as instability and propensity for the formation of azines, which are unreactive to IF. 34 However, azines react with NBS/PPHF and BrF3 to form gem-difluoromethylene compounds.

Oximes are unreactive with "BrF" but reacts with nitrosonium tetrafluoroborate

(NOBF4 ) to give good to excellent yields of the gem-difluoromethylene compounds. 35 A variety of oxime methyl ethers, 2,4-dinitrophenylhydrazones and azines react with BrF3 to give their correspondinggem-difluoro derivatives in good yields. 36 cS

Figure 2.6: Conversion of oximes to gem-difluoromethylene compounds 95

2.1.3 Dithioacetals and dithioketals to gem-difluoromethylene compounds:

Dithioacetals and dithioketals derived from aldehydes and ketones were converted to the corresponding gem-difluoro-compounds by using 1,3-dibromo-5,5-dimethylhydantoin

(DBH) or NBS with the presence of nucleophilic fluorine source PPHF in good yields. 37

But here in these procedures, electrophilic ring bromination has occurred in case of electron rich aromatic systems and gave side products.

n sxs NBS or DBH FXF R R PPHF R R

Br'N-P DBH= MejyN Me Br 0

Figure 2.7: Conversion of dithiolanes to gem-difluoromethylene compounds using NBS/PPHF and DBWPPHF

S02Ch and S02ClF have been used as an alternative to the NBS as a source of electrophilic species in this transformation to facilitate the work up of the reaction, as the only byproduets of the reaction are the volatile S02 and HCl. 311 Nitrosonium tetrafluoroborate in the combination with PPHF has also been used for this transformation?9 The desulfurative fluorination of dithiolanes was also done by using 1- diethylamino-1,1,2,2,3,3-hexafluoropropane in the presence of DBH as a source of electrophilic "Br+".40 1-Diethylamino-1,1,2,2,3,3-hexafluoropropane releases HF on reaction with water and thus acts as nucleophilic fluoride ion source in this reaction. BrF3 is also an effective reagent to bring about the transformation of dithiolanes to gem-

'fl 41-43 d1 uoro compounds. 96

Et2NCF2CHFCF3

DBH/H20

R =e.g., Ph, PhCH2, cycloalkyl 37-90%

Figure 2.8: Conversion of dithiolanes to gem-difluoromethylene compounds using EtzNCFzCHFCF3 and DBH/H20

Motherwell and coworkers have developed a method for the desulfurative gem­

difluorination of dithiolanes by using p-iodotoulenedifluoride.44.45 This reagent brings

about the gem-difluorination by supplying electrophilic F1 ion for the electrophilic

activation of dithiolanes, as well as nucleophilic fluoride ion for the fluorination of

carbonyl derivative center.

F F PhxPh 65%

Figure 2.9: Conversion of diphenyl dithiolane to gem-difluoromcthylene compound using p-Iodotoulenedi fluoride

This reaction is convenient for substrates involving stabilized carbocation as

intermediates. The variously substituted versions of p-iodotoulenedifluoride were also prepared and used for difluorination of dithioketals. Dithiolanes were also transfom1ed to the gem-difluoro compounds by anodic desulfurization of dithiolanes in Et 3N.3HF. The chemical oxidant can be replaced by an anode. 46 The ketone 1,3-dithiolanes and acyclic 97 dithioketals were converted to corresponding gem-difluoro compounds by anodic fluorodesulfurisation using triethylamine tris(hydrogen fluoride)47 as fluoride source. In contrast aryl dithioacetals gave gem-difluoroethters and aliphatic dithioacetals gave monofluoroethers under the same conditions. Kuruboshi has reported the conversion of P- hydroxy orthothioesters, derived from aldehydes, to ( 1, 1-difluoro-1-methylthio )ketones using oxidative desulfurisation conditions {TBATF/DBH)48 • The oxidation conditions used in this reaction were sufficient to oxidize the hydroxy group in the substrate to ketone simultaneously.

OH ~ /SMe TBATF/DBH R' '(-sMe SMe

25-95%

Figure 2.10: Conversion of P-hydroxy orthothioesters to 1, 1-difluoro-1- methylthio )ketones

2.1.4 Thiocarbonyls to gem-difluoromethylene compounds: The esters are relatively less electrophilic in nature compared to aldehydes and ketones and are unreactive in the presence of DAST. However, thioesters are more electrophilic than esters and are reactive toward DAST. Alkyl and aryl thioesters were easily prepared from esters using

Lawesson's reagent and reacted with DAST to obtain the gem-difluoroethers in good yields.49 And also fluorodesulfurisation ofthioesters using BrF3 and TBATFINBS or NIS were reported to prepare gem-difluoroethers from thioesters.50'51 Methyl xanthates,

ROCSSMe, underwent oxidative desulfurisation under similar conditions to afford difluoro(methylthio )methyl ethers, ROCF 2SMe. 52 98

DAST or BrF3

or TBATF/NBS

or TBATF/NIS

Figure 2.11: Conversion of thiocarbonyls to gem-difluoromethylene compounds

2.1.5 Azirines to gem-difluoromethylene compounds: 1-azirines were gem­

difluorinated using hydrogen fluoride as fluoride ion source. This is an useful method to

prepare p,p-di tluoroamines from 1-azirines. This method has been applied to synthesis of

3,3-di fluorophenylalanine. 53 • 54

PPHF

Figure 2.12: Conversion azirines to 3,3-difluorophenylalanine

2.1.6 Olefinic and acetylenic compounds to gem-difluoromethylene compounds

2.1.6.1 Alkenyl trifluoroborates: Alkenyl trifluoroborates were reacted with two mole equivalents of selectfluor in water or acetonitrile solvents to obtain the corresponding 99 difluoromethyl substituted alcohols and amides, respectively. 55 The intermediate, monofluoroalkene was isolated in these reactions. Alkenyl trifluoroborates were readily prepared from the corresponding alkenyl boronic acids by reacting with KHF2•

KHF2

R,IR2 =e.g., H!Ph; Ph/Ph; Me/Ph

Selectfluor, H20

Selectfluor, Selectfluor, CH3CN H20

Figure 2.13: Conversion of alkenyl trifluoroborates to gem-difluoromethyl substituted alcohols

2.1.6.2 Alkenes: Alkenes were converted to the corresponding gem-difluoro compounds by reacting with xenon difluorides in the presence of Iz. This reaction follows the mechanism involving electrophilic addition of IF to alkenes, fluorine exchange with xenon difluoride forming idosodifluoride, 1,2-alkyl shift accompanying the elimination of IF2, and fluoride ion attack on the carbocation center to give gem-difluoro compounds as shown in the Figure 2.14 .56 100

0°C, 0.5 h

0°C, 0.5 h

F F ~ XeF2 + 12 XeF2 [IF] ::y ~ I I~

F p- ~ ~ Figure 2.14: Conversion of alkenes to gem-difluoromethylene compounds and the mechanism for the difluorination of alkenes

2.1.6.3 Allylamines: Allylamines and diallylamines were reacted with HF-SbF5/NBS to

obtain y-gem-difluoroamines. 57 This reaction follows the mechanism involving the initial

formation ofthe cyclic bromonium ion which undergoes the 1,2-hydrideshift and fluoride

ion capture to give y-bromofluoro compound. Later, protolytic elimination of HBr

followed by fluoride ion attack on the resulting fluorocarbocation gives the the y-gem- difluoroamine. 101

HF-SbF5 +NBS

H H HF-SbF5 +NBS ~N~ F~N~F F F

Figure 2.15: Conversion of allyl amines toy- gem-difluoroamine

2.1.6.4 Alkynes: Hydrofluorination of alkynes were reported to occur with Markownikov addition using poly 4-vinyl pyridinium poly(hydrogen fluoride) (PVPHF)58 and with the combination of KHF2 and SiF4•5 9 Alkyl substituted alkynes were also converted to the-

CF2CX2 containing compounds by reacting with IF and BrF. Phenyl alkynes were prone to side reactions particularly with IF as the product undergoes further fluorination at the

CI2 center. Fluoroalkenes were likely the intermediates in these reactions and it has been reported separately that 2-fluoro-1-alkenes undergo bromofluorination regioselectively under mild conditions. 60 The regioselectivity in these reactions was attributed to the ability of fluorine to stabilize a-carbocation by electron donating resonance.

PVPHF

NBS

Figure 2.16: Conversion of alkyne to gem-difluoromethylene compound and bromo fluorination of alkene 102

Aryl substituted alkynes such as t-butyl phenyl acetylene, diphenyl acetylene

were reported to react with Accufluor, (N-fluoro-N-Hydroxy-1 ,4-

diazobicyclo(2.2.2)octane bis(tetrafluoroborate)) in aqueous acetonitrile to give their

corresponding a, a-difluoro-ketones. 61

0 Accufluor Ph-..::=--R Ph~R F F R =e.g., H, t-Bu, Ph, Me

Figure 2.17: Conversion of aryl substituted alkynes to a,a-difluoro-ketones

2.1.7 Electrophilic difluorination: Banks and Lawrence have reported the difluorination

of ~-ketoamides with Selectfluor under neutral conditions.62 The reaction times in these

reactions were significantly reduced by carrying out the two fluorinations sequentially and with a sodium enolate of the monofluoro intermediate for the second step. The relative ease of fluorination depends on the proportion of the enol tautomer and the usage of enolate in the reaction.

Selectfluor 40 °C, 647h

2. Selectfluor 27h,rt

Figure 2.18: Electrophilic difluorination of~-ketoamides with Selectfluor 103

Lactams were converted to a.,a.-difluorolactams by sequential reactions with lithium diisopropylamide (LDA) and Selectfluor.63 These difluorolactams were subsequently transformed to (L)-4,4-difluoroglutamic acid or its derivatives as shown in

Figure 2.19.

1. LDA, THF -78 °C 2. Selectfluor, THF, 0~ -78°C -to

1. LDA, THF -78 °C 2. Selectfluor, THF, -78°C

Figure 2.19: Conversion of a.,a.-difluorolactams to (L)-4,4-difluoroglutamic acid

Methyl- or ethyl- aryl (a.,a.-difluoromethylenephosphonates) were prepared from their corresponding phosphonates using an excess of sodium hexamethyldisilazide

(NaHMDS), and N-fluorobenzenesulfonimide (NFBS).64 This reaction tolerates a variety of functional groups in the aromatic ring, such as ester, ether, bromine, phenyl and nitro groups. 1,3-diaryl derived phosphonates were also converted to their corresponding benzylic gem-difluoro derivatives using the same conditions.64 However, this reaction was not successful for 1,4- diaryl derived phosphonates. 104

0 F. p;OEt

1. NaHMDS (2.2 eq.), THF, -78 °C F OEt O-...::::: 2. NFBS (2.5 eq.), THF, -78 °C "' X

X = e.g., H, ester, ether, Br, Ph, N02

1. NaHMDS, THF, -78 °C 0 2. NFBS, THF, -78 °C p_..OEt 'OEt F F

Figure 2.20: Conversion of aryl ( a,a-difluoromethylenephosphonates) to gem­ difluoromethylene compounds

Benzylic sulfonate esters were converted to their corresponding benzylic gem- difluoro-sulfonate esters by difluorination at a-center using t-butyllithium followed by reaction with NFBS.65

0 F. '~~OEt F 0 1. t-BuLi (2.2 eq.), THF, -78 °C 6-...::::: 2. NFBS (2.5 eq.), THF, -78 °C ...-: X

X= e.g., H, 4-N02, 4-Me

Fig 2.21: Conversion of benzylic sulfonate esters to gem-dilfuoro-sulfonate esters

2.2 RESULTS AND DISCUSSIONS

Chambers and coworkers have prepared gem-difluoromethylene compounds by reacting dithiolane derivatives of diaryketones with elemental fluorine-iodine 105

mixtures.66'67 This same reaction in aqueous medium, however, resulted in the hydrolysis of the substrates to the corresponding carbonyl compounds. We have reasoned that the reaction of Selectfluor with PPHF may involve the transiently formed F2 which can bring about the desulfurative fluorination in case of dithiolanes. Alternatively the Selectfluor can serve as p+ releasing agent, able to react with thio compounds to give fluorothionium ions, and the subsequent reaction with F should result in the desulfurative fluorination.

2BF4 HF + £6 /+ H

Or

Figure 2.22: Possible activation ofthiolanes with the combination ofSelectfluor and HF

2,2-diphenyl-1,3-dithiolane was prepared from 1,3-dithiolane and benzophenone

68 using BF3.Etz0 as catalyst, and reacted with solutions of PPHF and Selectfluor. The reaction was rapid even at 0 °C in methylene chloride (CHzCh), and in about 5 min complete conversion of 2,2-diphenyl-1,3-dithiolane 2.1 to the corresponding gem­ difluoro compound 2.2 was observed in very good yield. GC/MS of the reaction mixture 106

pnor to the workup has shown only one product corresponding to the gem­

difluoromethylene compound.

n Selectfluor/PPHF vv~ 2.1 2.2 Yiled = 86%

Figure 2.23: Conversion of 1,2-diphenyl-1 ,3-dithiolane to gem-dilfuorodiphenylmethane

With this promising result, several diaryl ketones were transformed into the dithiolanes using BF3.Et20 as catalyst, and the resulting dithiolanes were allowed to react with solutions of PPHF and Selectfluor. The reactions were rapid even at 0 °C, and in about 10 min complete conversion of the substrates to the gem-difluoro comounds was observed. Benzophenone dithiolanes, variously substituted with halogens or methyl groups, gave only the corresponding gem-difluoro compounds, as the single products, as shown by the GC/MS of the reaction mixture prior to the workup. 2-Adamantanone dithiolane 2.9 also gave quantitative conversion of the corresponding gem-difluoro compound 2.16 under these conditions. 107

Entry Dithiolane Product Yield(%) n

1 85 ~ ~ oxo2.3 Br oxo2.10 Br n

2 80 ~ ~ F 2.4 F oxo oxo 2.11 n

3 76 ~ ~ &02.12 Cl oxo2.5 Cl n 62 4 OXQ Cl Cl Cl JYO 2.6 Cl 2.13 n

81 5 OXQ F FJYO F 2.7 F 2.14 n 64 6 ~ OXo OMe oxo2•8 OMe 2.15 S) F 82 7 gts gtF 2.16 2.9

Figure 2.24: Conversion diaryldithiolanes to gem-difluorodiarylmethanes 108

However, acetophenone-dithiolane 2.17 gave the corresponding gem- difluoromethylene compound and the acetophenone in a ratio 6:4 under these reaction conditions. The dithiolanes derived from p-bromobenzaldehyde 2.20 and cyclododecanone 2.22 gave quantitative conversion to the corresponding carbonyl compounds. The lower yield observed for compound 2.17 and quantitative conversion of compounds 2.20 and 2.22 to the corresponding carbonyl compounds may be explained in terms of the relatively unstable nature of the incipient carbocation intermediates involved in these reactions.

0 Selectfluor/PPHF ~Me

2.18 2.19 2.17 (6 : 4)

0

Selecttluor/PPHF ~H Br

2.20 2.21

Selectfluor/PPHF

2.23 2.22

. · fd"tht"olanes from acetophenone, bromo-benzaldehyde and Ftgure 2.25: ReactiOn o 1 cyclododecanone with Selectfluor/PPHF

We have found that the reactions of dithiolanes with Selectfluor in aqueous

. . . PPHF ave the corresponding carbonyl compounds as the only acetomtnle .or m aqueous g 109 reaction products, in accordance with the observations of Wong and coworkers. 69 And also there was no noticeable reaction observed for dithiolanes with either HF or

Selectfluor alone in anhydrous CH2Ch under the reaction conditions used in this procedure.

This reaction may involve the fonnation of the intennediate fluoro-sulfonium ion by the attack of the "F+" from the Selectfluor or the transiently fonned fluorine molecule.

The fluoro-sulfonium ion then undergoes nucleophilic capture by the fluoride ion from the medium, and the process repeats to fonn the gem-difluoro compounds. If the intermediate carbocation fonned in the final desulfuration is not sufficiently long-lived, its reaction with the fluoride may be too slow so that it undergoes hydrolysis during the aqueous workup.

Selectfluor F or"F2"

0 -HF II .. RAR

· p d mechanism for the fonnation of gem-difluoromethylene F1gure 2 .26 : ropose compounds from the carbonyl-dithiolanes and Selectfluor/PPHF 110

2.3 CONCLUSIONS

The reaction between diaryldithiolanes with the combination of Selecttluor and

PPHF gave the corresponding gem-difluoro compounds in good to excellent yields. This

reaction is convenient, effective, and requires simple work up and mild reaction

conditions. The hydrolysis of the dithiolanes derived from aromatic aldehyde and

aliphatic ketone indicates that the reaction is only applicable to substrates which can

generate stabilized carbocation as reaction intermediates.

2.4 EXPERIMENTAL RESULTS

2.4.1 General: All reagents were purchased from commercial sources and used as

received. Colum chromatography was carried out using Merck 60, 230-400 mesh. Thin

layer chromatography was carried out using silica gel coated polyester backed sheets.

GC/MS spectra were recorded on Hewlett-Packard 5989A spectrometer, equipped with a

Hewlett-Packard 5890 gas chromatograph. The 1H and 19 F NMR spectra for CDCil

solutions were obtained on a !NOVA-Varian 400 MHz spectrometer at 400, and 386

MHz, respectively. The 1H NMR chemical shifts are referred to the internal TMS (8 =

0.0). The 19F chemical shifts are referred to the internal CFCh (8 19F == 0.0).

2.4.2 General procedure for the preparation of gem-difluoromethylene compounds:

Selectfluor (0. 71 g, 2 mmol) was dissolved in PPHF (wt % HF : Py = 61 : 39) in a 50 m L polyethylene bottle and cooled to 0 °C. The dithiolane (I mmol) in dichloromethane (1 0 mL) was added dropwise and allowed to stir for I 0 min at room temperature. The reaction mixture was then extracted with dichloromethane (3 X 30 mL), the combined organic layers were dried (MgS04), and the solvent was evaporated under reduced II I

pressure. The residue was purified by column chromatography (silica gel, 2.0% dicthyl

ether in hexane) to obtain gem-difluoromethylene product.

2.4.3 Synthesis and properties of products

Difluorodiphenylmethane (2.2)

2.2

Selectfluor (0.71 g, 2 mmol) was dissolved in PPHF (wt% HF: Py = 61 : 39) in a 50 mL

polyethylene bottle and cooled to 0 °C. The benzophenone dithiolanc, 2.1, (0.26g, 1

mmol) in dichloromethane (10 mL) was added dropwise and allowed to stir for 10 min at

room temperature. The reaction. mixture was then extracted with dichloromcthanc (3 X

30 mL), the combined organic layers were dried (MgS04), and the solvent was

evaporated under reduced pressure. The residue was purified by column chromatography

(silica gel, 2.0% diethyl ether in hexane) to give Difluorodiphenylmcthanc 2.2: yield

0.17g (86%)

MS: 204 (M+., 68), 183 (20), 127 (100), 107 (10), 77 (10)

1H NMR (CDCb): o 7.85 (dd, 4 H, J =7Hz, J =1.1 Hz), 7.64 (tt, 2 H, J = 7.4 Hz, 1.46

Hz), 7.54 (d, 4 H, J = 7.87 Hz) ppm.

19F NMR (CDCb): o-89.3 (s) ppm. 112

1-Bromo-4-(difluoro(phenyl)methyl)benzene (2.1 O)

~ u u Br 2.10

Prepared similarly to 2,2-phenyl-1,3-dithiolane 2.2. Yield 85%.

MS: 282 (M+., 41), 203 (60), 183 (84), 127 (93), 107 (30), 77 (100)

19 F NMR (CDCb): 8 -89.5 (s) ppm.

1-Fluoro-4-(difluoro(phenyl)methyl)benzene (2.11)

~ u UF 2.11

Prepared similarly to 2,2-phenyl-1 ,3-dithiolane 2.2. Yield 80%

MS: 222 (M\ 100), 203 (19), 145 (87}, 127 (77), 95 (18)

19F NMR (CDCb): 8 -88.1 (s), -111.5 (s) ppm. ll3

1-Chloro-4-(difluoro(phenyl)methyl)benzene (2.12)

~ v ~CI 2.12

Prepared similarly to 2,2-phenyl-1,3-dithiolane 2.2. Yield 76%

MS: 238(M+., 100), 219 (10), 203 (79), 161 (33), 127 (27)

19 F NMR (CDCb): o-89.2 (s) ppm.

bis(4-Chlorophenyl)difluoromethane (2.13)

Cl Cl 2.13

Prepared similarly to 2,2-phenyl-1 ,3-dithiolane 2.2. Yield 62%

MS: 272 (M+., 50), 237 (67), 201 (18), 161 (100), 111 (27), 75 (41)

19F NMR (CDCh): o-89.1 (s) ppm. 114

bis(4- Fluorophenyl)difluoromethane (2.14)

~ F~ llAF 2.14

Prepared similarly to 2,2-phenyl-1 ,3-dithiolane 2.2. Yield 81%

MS: 240 (M+., 100), 239 (29), 221 (17), 201 (7), 145 (78)

19 F NMR (CDCh): o -86.7 (s), -111.5 (s) ppm.

1-(Difluoro(phe nyl)methyl)-4-methoxybenzene (2.15)

~ V llAoMe 2.15

Prepared similarly to 2,2-phenyl-1,3-dithiolane 2.2. Yield 64%

MS: 234 (M+., 100), 203 (13), 157 (74), 127 (10), 77 (7)

19F NMR (CDCh): o -87.2 (s) ppm. 115

2,2-Difluorotricyclo[3.3.1.1 ]decane (2.16)

2.16

Prepared similarly to 2,2-phenyl-1,3-dithiolane 2.2. Yield 82%

MS: 172(M+., 14), 152(15), 109(25),93(41), 79(100),51 (71)

19 F NMR (CDCh): o-100.2 (s) ppm.

(1, 1-Difluoroethyl)benzene (2.18)

F. F if~ .0 2.18

Prepared similarly to 2,2-phenyl-1,3-dithiolane 2.2. Yield 48%

MS: 142 (M+., 34), 127 (57), 84 (100), 58 (74), 51 (44)

19F NMR (CDCh): o-87.9 (q, J =18Hz) ppm. 11 6 2.4.4 NMR spectra of products:

- 1

Figure 2.27: 19F NMR spectra of compound 2.10

J

l ! 1 1

Figure 2.28: 19F NMR spectra of compound 2.11 11 7

. 1

Figure 2.29: .19F NMR spectra of compound 2.12

·IH I I • -···

Figure 2.30: 19F NMR spectra of compound 2.13 118

I· '

.: 1

! .. I .I

I ...... - ..,.... u. 51 a -51 -110 -151 ., .. ~

Figure 2.31: 19F NMR spectra of compound 2.14

Figure 2.32: 19F NMR spectra of compound 2.15 119

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3. CARBENE CATALYZED TRIFLUOROMETHYLATIONS OF AROMATIC N-TOSYL-ALDIMINES

3.1 INTRODUCTION

3.1.1 Nucleophilic trifluoromethylation of imines: Trifluoromethyl-containing compounds have found a large number of applications ranging from biologically active drugs and agro-chemicals, to dyes and polymers. 1-3 In particular, the basicity of the amide bond is significantly reduced when the trifluoromethylated amines are incorporated into the peptides, due to the strongly electron withdrawing nature of the trifluoromethyl group. Thus, nonspecific proteolysis4 is attenuated when the latter amines are incorporated into the peptides. Further, these trifluoromethylated amines modify the solubility and desolvation properties5 of the derived peptides. A variety of methods have been reported in the literature for the preparation of trifluoromethylated amines6-10 and recently, the direct preparation of trifluoromethylated amines by nucleophilic trifluoromethylation has received much attention. A short survey of the existing methods for the preparation trifluoromethylated amines by nucleophilic trifluoromethylation using

TMSCF3 or CF3VTDAE is presented below.

Laurent and co-workers11 for the first time reported the trifluoromethylation of azirines using TMSCF3, and tetrabutylammonium fluoride (TBAF) as fluoride ion source.

The reaction is catalytic in fluoride ion initiators (Figure 3.1 ). The formation of thermodynamically unfavorable silicon nitrogen bond occurred probably due to the release ofhigh strain from the structure ofazirine upon addition oftrifluoromethyl group. 125

TBAF, THF

Figure 3.1: Nucleophilic trifluoromethylation of azirines

Nelson and co-workers 12 have reported the nucleophilic trifluoromethylation of nitrones with TMSCF3. Since nitrones are strongly electrophilic, they readily accept the trifluoromethide ion from TMSCF3 (Figure 3.2). The reactions proceeded smoothly at

-78 °C, but the limited solubility of nitrones necessitated the use of higher temperature.

The reactions were initiated by the addition of potassium tert-butoxide (t-BuOK). Other initiators such as KF and TBAF were ineffective as they yielded predominantly the addition-elimination products.

H~ A$A) + Ar N I THF, -78 °C 2

Figure 3.2: Nucleophilic trifluoromethylation of nitrones

Blazejewski and co-workers 13 have reported the nucleophilic trifluoromethylation of normal imines with TMSCF3. N-Trimethylsilylimidazole (TMS-Imidazole) was used in this reaction as an additional silylating reagent (Figure 3.3). The success in addition of trifluormethide ion on imine was based on preventing the decomposition of the intermediate which has a labile N-Si bond by trapping it with suitable electrophile, using

TMS-Imidazole. Lower yields for the desired trifluoromethylated amines were observed 126 due to the formation of byproducts, bistrifluoromethylated amme derivatives. The formation of bistrifluoromethylated amine derivative is thought to be due to the further attack of the trifluoromethide ion on the trifluoromethylated amine product.

i) CF3TMS I TMS-Imidazole

ii) CsF I THF iii) Si02 12M HCI 2-55% yield

R1 =Ph, 2-Naphthyl, 2-Furyl, 3-MeOC6H4

R2 = H, CF3, Me R3 = Ph, PhCH2, 3-CF3CsH4

Figure 3.3: Nucleophilic trifluromethylation ofimines using TMSCF3 and TMS­ Imidazole

Petrov14 have showed that N-aryl imines of hexafluoroacetone reacted with

TMSCF3 in the presence of cesium fluoride (CsF). The reactions were rapid in THF or monoglyme and required stoichiometric amount of fluoride ion initiator, CsF. (Figure

3.4)

TMSCF3, CsF

THF

48 - 84% yield

Figure 3.4: Nucleophilic trifluoromethylation of N-aryl imines ofhexafluoroacetone

1n 2001, Prakash and co-workers15 have reported their first success in nucleophilc trifluoromethylation of N-Tosyl aldimines by using non-metallic fluoride source 127 tetrabutylammonium(triphenylsilyl) difluorosilicate 16 (TBAT). N-Tosyl aldimines were used in these reactions to increase the electrophilicity of the imines and thus facilitating the attack of trifluoromethide ion on imines. When TBAF was used in these reactions, the products were obtained in lower yields with concomitant formation of trifluoromethane because of the strong hygroscopic nature of TBAF. The trifluromethylations of aromatic imines with electron withdrawing as well as electron donating group proceeded smoothly and gave the desired products in good yields. However, aliphatic sulfonamides and N-

Tosyl ketimines gave lower yields of the products. Subsequently, this methodology has been extended to N-(tert-butylsufinyl)-imines, 17 a,~-unsaturated N-(tert-butylsufinyl)- imines18 and a-amino N-(tert-butylsufinyl)- imines 19 to obtain the corresponding trifluoromethylated compounds in moderate to high yields. (Figure 3.6)

1. TMSCF3, TBAT

THF, 0- 5°C 2. Work up

Figure 3.5: Nucleophilic trifluoromethylation of N-tosylated amines using TBA T 128

9F3 9 TBAT R 1 ~N ... S'tBu H

1 R =p-CIC6H4, p-BrC6H4, 2-pyridyl, 2-furyl, Ph, 2-Naphthyl, tBu, PhCH2CH2

TMAF

Figure 3.6: Nucleophilic trifluoromethylations of N-(tert-butylsufinyl)- imines, a,p­ unsaturated N-(tert-butylsufinyl)- imines and a-amino N-(tert-butylsufinyl)- imines

Dolbier and co-workers20'21 have reported the nucleophilic trifluoromethylation of

N-tosyl aldimines and N-tolyl sulfinimines using the nucleophilic trifluoromethylation reagent derived from CF3I and tetrakis(dimethylamino)-ethylene (TDAE). This methodology was limited to nonenolizable aldimines. (Figure 3. 7)

DMF, -30 to 0 °C

DMF, -30 to 0 °C

Figure 3.7: Nucleophilic trifluoromethylation using CF3UTDAE 129

Mukaiyama and co-workers22 -24 have reported catalytic trifluoromethylation of various aldimines with (trifluoromethyl)trimethylsilane in the presence of Lewis bases.

Several lithium carboxylates such as lithium benzoate, acetate and pivalate were found to be effective bases in these reactions (Figure 3.8). However, weakly nucleophilic lithium trifluoroacetate did not promote this reaction. And also trifluoromethylation reactions did not proceed effectively in the presence of CsF and Lithium benzoate (BnOLi).

Nucleophilic trifluoromethylation of N-sulfonylimines, N-sulfinylimines and N- phosphonylimines proceeded smoothly in the presence of 10 mol% of lithium acetate. On the other hand, no trifluoromethylated products were observed when weak electrophiles such as N-phenylaldimine or N-benzylaldimine were used. Aromatic aldimincs with electron-withdrawing or electro-donating groups reacted smoothly to afford the trifluoromethylated products in good yields. Whereas aliphatic aldimines having a- protons adjacent to the imino group reacted smoothly to afford the trifluoromethylated products in high yields, those with a-protons did not undergo the trifluoromethylation.

AcOLi

DMF, -30 °C

AcOLi

DMF, -30 °C

AcOLi

DMF, -40 °C

Figure 3.8: Nucleophilic trifluoromethylation using Lewis base, AcOLi 130

25 Norio Shibata and co-workers have claimed that the P(t-Bu)3-DMF system

effectively promoted the nucleophilic trifluoromethylation reaction of N-tosylated imines

with TMSCF3. This reaction was limited to N-tosylbenzylaldimine and aromatic N-

tosylaldimines and required 100 mol% ofP(t-Bu)3. (Figure 3.9)

P(t-Bu)J TMSCF3 DMF, RT

Figure 3.9: Nucleophilic trifluoromethylation using P(t-Bu)3-DMF system

3.1.2 N-Hetereocyclic carbene as a catalyst: N-Heterocyclic carbenes (NHCs) have received considerable interest in recent years. Excellent a-donating properties of NHCs make them ligands of choice for transition metals and have been successfully employed as ligands in a wide range of transition metal-catalyzed reactions26-30, as well as substrates in multi-component reactions31 -34 • Further, they have been used as nucleophilic catalysts in various organic transformations such as transesterifications36-39, nucleophilic substitutions40-42, benzoin, and Stetter reactions.43-46

Suzuki and co-workers47 have reported that N-heterocyclic carbenes produced in situ from salts of imidazolium, benzimidazole, pyrido[1,2-c]imidazolium, imidazolium, thiazolium, and triazolium catalyze the addition of trimethylsilylcyanide to aldehydes to yield cyanohydrin trimethylsilyl ethers. (Figure 3.10) 131

0 OH 1. azolium salts t-BuOK, TMS-CN/THF, rt o::CN cc>OMe OMe

yields 85-96%

H Bn I@ e N(±) Br e N~N CC ') ~@ I \d N ~N-SN-Me I Ph pyrido[1,2-c]imidazolium benzimidazolium salt salt azolium salts = imidazolium salt

Bn ,Bn 11N@ e MexN@ e N _)) Br / ') Br 'N I Me S Me thiazolium salt triazolium salt imidazolinium salt

Figure 3.10: NHC catalyzed cyanosilylation of a-methoxybenzaldehyde

As suggested by the authors, the use of C2-symmetric imidazolidenyl carbene

derived from (R,R)-1 ,3-bis[(l-naphthyl)ethyl]imidazolium presumably led to

enantioselective cyanosilylation (Figure 3.11). They have also proposed a possible

reaction pathway for the NHC catalysis. Carbene attacks the electrophilic center of the

aldehyde and forms intermediate 3.2 which has been widely accepted in NHCs' catalysis.

Intermediate 3.2 reacts with TMS-CN to produce intermediate 3.3 and cyanide anion.

The cyanide anion nucleophilically attacks a carbon atom of 3.3. The imidazolium moiety of 3.3 acts as a leaving group, and nucleophilic substitution occurs to produce cyanohydrin silylether 3.4. In the case of the chiral NHC adds to aldehyde face selectively to produce diastereoisomeric intermediate 3.2 with an excess of a favored 132 isomer (Figure 3.12). The possible alternative catalytic mechanism was also discussed.

In particular, the NHC activates TMS-CN by coordination (Figure 3.13).

~OlH~OMe 1. imidazolium salt, t-BuOK, TMS-CN, -78 °C lXJ 2. w 22% ee

lmidazoilum salt=

Figure 3.11: Asymmetric cyanosilylation catalyzed by chiral NHC

· 3 12 p 'ble reaction pathway for NHC catalyzed cyanosilylation Ftgure • : osst 133

Figure 3~13: Alternative NHC catalytic mechanism for cyanosilylation

Aoyama, Kondo, and co-workers48-50 have described the cyanation reaction of aldehydes, aldimines (Strecker reaction), ketones, and ketimines with TMSCN in the presence of 1,3-bis(2,4,6-trimethylphenyl)imidazolium chloride and potassium tert- butoxide, as a nucleophilic organocatalyst. These cyanation reactions afforded the corresponding products in good yields under mild reaction conditions. 134

lmidazolium salt (10 mol%) KOt-Bu (9 mol%) TMSCN (1.2 equiv)

lmidazolium salt (5 mol%) KOt-Bu (4 mol%) TMSCN (1.2 equiv) THF, 0 °C

lmidazolium salt (5 mol%) KOt-Bu (4 mol%) TMSCN (1.2 equiv) DMF, r.t.

lmidazolium salt (5 mol%) KOt-Bu (4 mol%) TMSCN (1.5 equiv) DMF, r.t.; then H20 e HCI

lmidazolium sail= ---ct~)./~--9-

Figure 3.14: Cyanation of aldehydes, aldimines (strecker reaction), ketones, and ketimines in the presence ofNHC

N-Heterocylic carbenes have also reported to catalyze the ring opening of aziridines with silylated nucleophiles to afford the corresponding I ,2-difunctional

1 products in excellent yields under mild reaction conditions. 5

5mol%

Me3SiX, 40 °C X= N3, Cl, I

Figure 3.15: Reaction of aziridines with silylated nucleophiles catalyzed by NHC 135

3.2 RESULTS AND DISCUSSIONS

Recently, Song and co-workers52 have reported that both enolizable and nonenolizable aldehydes and a-ketoesters underwent facile tritluoromethylation at room temperature in the presence of only 0.5-1 mol% of the N-heterocyclic carbene 3.5 to afford the corresponding trifluoromethyl-containing alcohols in good yields. Under NHC catalysis, selective trifluoromethylation of aldehydes over ketones was achieved. This method offered much greater catalytic efficiency as well as broader substrate scope compared to other Lewis base catalyzed trifluoromethylation methods.

C)rN~NA ~ 3.5 \Y (0.5 mol%}

Figure 3.16: NHC catalyzed trifluoromethylation of carbonyl compounds

We envisioned that N-heterocyclic carbenes which have the reactivities similar to

Lewis bases such as phophines and amines can activate TMSCF3 and can catalyze the trifluoromethylation of aldimines. According to that, first we have prepared the precursors, imidazolium chlorides for the N-heterocyclic carbenes using the procedure reported by Arduengo.53 Glyoxal diimines 3.6 and 3.7 were prepared by condensation of glyoxal with two equivalents of 2,4,6-trimethylphenylamine and 2,6-diisopropylamine

. 1 L t glyoxal diimines were cyclised with chloromethylethyl ether to obtain respect tve y. a er, the desired imidiazolium chlorides 3.8 and 3·9 · 136

R I HXO n-PrOH + HlN H 0 H -...;:N I R 3.6, 3.7

3.6 and 3.8: R = y CICH20Et THF, 20- 40°C 1-(2 ,4, 6-trimethyl phenyl)

R e N Cl 3.7and3.9:R= V [+)>-H N I 1-(2,6-diisopropylphenyl) R 3.8, 3.9

Figure 3.17: Preparation imidazolium chlorides

N-tosyl benzylaldimine 3.10 was prepared according to reported procedurc54 and

reacted with TMSCF3 in the presence of N-heterocyclic carbene in THF solvent. N-

heterocyclic carbene was generated in situ through the reaction from 1,3-bis(2,4,6-

trimethylphenyl)imidazolium chloride 3.8 (1 0 mol%) and potassium tert-butoxide (9

mol%). The reaction was allowed to stir for 12 hand quenched with water and extracted

with ethyl acetate. The crude product has shown the peaks corresponding to the desired

trifluoromethylated product in NMR and the product was isolated in trace amounts by the

column chromatography. After this encouraging result, the reaction was conducted in a

more polar solvent DMF. To our delight, the reaction was completed in 5h and afforded

the product in 62% yield. Later, the reaction conditions were optimized and this led to

72% yield for the trifluoromethylated N-(Tosyl)benzylamine with 15 mol% of 1,3- bis(2,4,6-trimethylphenyl)imidazolium chloride and 13 mol% KOt-Bu. With the 137 optimized reaction conditions, the reaction was also conducted in the presence NHC generated from the reaction of 1,3-bis(2,6-diisopropylphenyl)imidazolium chloride 3.9 and potassium tert-butoxide. The reaction was not complete even after 1Oh and afforded the product in low yield (46%).

e Cl

N N ~ !J -41®-)J-\ I 3.8 /Ts N....-Ts (10 mol%) HN + TMSCF3 PhACF3 Ph)lH KOt-Bu (9 mol%) THF, RT, 12h 3.11 3.10 Trace amounts e Cl ,l$ - -4H-9-N, ~r ~ !J 3.8 /Ts HN N.,....Ts (15 mol%) + TMSCF3 PhACF3 KOt-Bu (13 mol%) Ph)lH DMF, RT,4h 3.11 3.10 yield= 72% e Cl ,l$H N ~N \ I

3.9 /Ts HN N.,....Ts (15 mol%) + TMSCF3 KOt-Bu (13 mol%) PhACF3 Ph)lH DMF, RT, 10h 3.11 3.10 yield= 46%

. h f of N-tosyl benzylaldimine in the presence ofNHCs Figure 3.18: Tnfluromet y1 a Ion 138

This methodology was extended to other aryl N-tosyl aldimines by reacting with

TMSCF3 to obtain the corresponding trifluoromethylated sulfonamides. Aromatic N-tosyl aldimines with both electron-withdrawing and electro-donating groups on phenyl ring reacted smoothly to afford the trifluoromethylated products in moderate to good yields.

Reactant Product Yield(%)

N,...Ts /Ts HN 82 dH ffcF 3 F F 3.12 3.16 /Ts N,...Ts HN 75 dH ffcF 3 Cl Cl 3.13 3.17 /Ts N,...Ts HN 78 dH ffcF 3 ON 02N 3.18 2 3.14

/Ts N,...Ts HN dH dcF, 70 H3C H3C 3.19 3.15

Figure J.19: Trifluoromethylation of aromatic N-tosyl aldimines in the presence of NHC 139

55 Reaction of aliphatic aldimine, cyclohexyl-N-tosyl aldimine with TMSCF3 in

the presence of excess catalyst has provided the trifluoromethylated product in trace

amounts, showing the peak corresponding to the product in 19F NMR.

/Ts HN + (i'cF, KOt-Bu (27 mol%) DMF, -10 °C, 1h 3.21

trace amounts

Figure 3.20: Trifluoromethylation of cyclohexyl-N-tosyl aldimine in the presence of N-heterocyclic carbene

3.3 CONCLUSIONS

N-Heterocyclic carbenes produced in situ from the imidazolium salts and potassium tert-butoxide were found to catalyze the nucleophilic trifluoromethylation of aromatic N-tosyl aldimines with TMSCF3. Aromatic N-tosyl aldimines with both electron-withdrawing and electron-donating groups underwent facile trifluoromethylation in the presence of the N-heterocyclic carbene (3.8) generated from 1,3-bis(2,4,6- trimethylphenyl)imidazolium chloride and potassium tert-butoxide to afford the corresponding trifluoromethylated sulfonamides in moderate to good yields. The NHC catalyzed reaction is broadly applicable for the trifluoromethylation of substituted benzaldehyde imines. However, further optimizations are needed for the trifluoromethylation of aliphatic imines using this reagent. 140

3.4 EXPERIMENTAL RESULTS

3.4.1 General: All reagents were purchased from commercial sources and used as

received. Column chromatography was carried out using Merck 60, 230-400 mesh. Thin

layer chromatography was carried out using silica gel coated polyester backed sheets.

GC/MS spectra were recorded on Hewlett-Packard 5989A spectrometer, equipped with a

1 Hewlett-Packard 5890 gas chromatograph. The H, 13C and 19F NMR spectra for CDC1 3

and dmso-d6 solutions were obtained on a INOV A-Varian 400 MHz spectrometer at 400,

100, and 386 MHz, respectively. The 1H NMR chemical shifts are referred to the residual

solvents signals or the internal TMS (8 = 0.0). The 19F chemical shifts are referred to the internal CFCh (8 19F = 0.0).

3.4.2 General procedure for the preparation of aromatic N-tosyl aldimines:

Method A: The respective aromatic aldehyde (5 mmol), p-toulenesulfonamide (5 mmol) and tetraethyl orthosilicate (5.5 mmol) were combined in a round bottomed flask equipped with a still head and heated at 160 °C under nitrogen for 5 h, during which time ethanol formed was collected in the receiving flask. On cooling, ethyl ether (50 mL) was added and the suspended solids were filtered and washed with ethyl ether. The crude product was recrystallised in ethyl acetate/hexane to obtain the aromatic N-tosyl aldimine.

Method B: The respective aromatic aldehyde ( 10 mmol) and p-toulenesulfonamide ( 10 mmol) were added to a round bottomed flash containing 50 mL of toluene. The reaction mixture was heated at reflux for 25 h, with azeotropic removal of water using Dean-Stark apparatus. The reaction mixture was allowed to cool to room temperature, followed by 141 concentration on the rotary evaporator. The crude product was recrystallised in diisopropylether to obtain the desired aromatic N-tosyl aldimine.

3.4.3 General procedure for the preparation of aliphatic N-tosyl aldimines: Aldehyde

(10 mmol), p-toulenesulfonamide (10 mmol)and sodium p-toulenesulfinate were added to a round bottomed flask containing 15 mL of formic acid and 15 mL of water. The solution was stirred for 12 hat room temperature and the resulting white precipitate was filtered off, washed with water (2 x 10 mL), then with pentane (10 mL). The crude solid was dissolved in dichloromethane (100 mL) and was added sat. aq NaHC03 (70 mL).

The solution was well stirred for 2 h at room temperature. The organic phase was decanted, the aqueous phase extracted with dichloromethane (70 mL) and the combined organic layers were dried (MgS04), filtered, and the solvent was removed under vacuum to obtain the aliphatic N-tosylaldimine.

3.4.4 General procedure for trifluoromethylation of aromatic N-tosyl aldimines:

Imidazolium chloride (15 mol%, 0.0724 mmol) was taken in 4 mL of DMF and was added potassium tert-butoxide (13 mol%, 0.063 mmol). After dissolution of imidazolium chloride (~20 min), aldimine (0.483 mmol) as solid and TMSCF3 (use either this or TMS­

CF3 consistently) (0.676 mmol) were added. The solution was stirred at room temperature for 4h, then quenched with water and extracted with ethyl acetate (3 x 15 mL). The organic layers were combined and successively washed with water and brine, dried over

Na2so4 and concentrated. The crude product was purified by silica gel chromatography

(10% EtOAc/Hexane) ~0 obtain the trifluoromethylated sulfonamide. 142

3.4.5 Synthesis and properties of products

Glyoxal-bis-(2,4,6-trimethylphenyl)imine (3.6)

3.6

To a solution of 2,4,6-trimethylphenylamine (13.52 g, 0.1 mol) in 70 ml of n-propanol was added at room temperature a mixture of 40% aqueous solution of glyoxal (7.26 g,

0.05 mol), 20 mL ofn-propanol and 10 ml ofwater. The mixture was stirred for 20h at r.t an then for 5h at 60 °C. Upon the addition of 40 mL of water, a yellow solid precipitated which was collected by filtration and dried in vacuum to obtain glyoxal-bis-(2,4,6- trimethylphenyl)imine 3.6: yield 14.6 g (75%)

1H NMR (400 MHz; CDCh) 8 2.15 (s, 12 H), 2.29 (s, 6 H), 6.90 (s, 4 H), 8.09 (s, 4 H),

8.13 (s, 2H) ppm.

Glyoxal-bis-(2,6-diisopropylphenyl)imine (3. 7)

3.7 143

To a solution of 2,6-diisopropylphenylamine (9.85 g, 0.056 mol) in 50 mL of n-propanol

was added at room temperature a mixture of 40% aqueous solution of glyoxal (3.63 g,

0.028 mol), 5 mL of n-propanol and 15 ml of water. The mixture was stirred for 2h at 70

°C. Upon the addition of 40 mL of water, a solid precipitated which was collected by

filtration and dried in vacuum to obtain glyoxal-bis-(2,6-diisopropylphenyl)imine 3.7:

yield 8.63 g (82%)

1H NMR (400 MHz; CDCb) 5 1.21 (d, J = 6.8 Hz, 12 H), 2.94 (septet, J = 6.8 H, 4H),

7.18 (m, 12H), 8.10 (s, 2H) ppm.

1,3-Bis-(2,4,6-trimethylphenyl)imidazolium chloride (3.8)

e Cl -q-N~~-9- 3.8

To a solution of chloromethylethyl ether (1.45 g, 15.38 mmol) in 9 ml ofTHF was added a so Iutwn · o f g 1yoxa 1- bt·s -(2 , 4 ,6-trimethylphenyl)imine 3.6 (4.5 g, 15.38 mmol) in 100 mL of THF. The flask was sealed under nitrogen with a septum and the mixtutre was stirred at room temperature. A so lI.d began to appear after 1hand the stirring was continued at r.t for 5 days. T h e 1orrnec. d solid precipitate was collected by filtration to obtain I ,3-bis-

(2,4,6-trimethylphenyl)imidazolium chloride 3.8: yield 1.68 g (32%) 144

I H NMR (400 MHz; dmso-d6) o2.12 (s, 12 H), 2.34 (s, 6 H), 7.19 (s, 4 H), 8.32 (s, 2H), 9.84 (s, IH) ppm.

1,3-Bis-(2,6-diisopropylphenyl)imidazolium chloride (3.9)

e Cl H 1$ N~N \ I

3.9

To a solution of chloromethylethyl ether (1.8 g, 18.30 mmol) in 3 mL of THF was added

glyoxal-bis-(2,6-diisopropylphenyl)imine 3. 7 (6.89 g, 18.30 mmol in 40 mL of TH F and

2 drops of water. The flask was sealed under nitrogen with a septum and the mixtutrc was

stirred at 40 °C. A solid began to appear after I h and the stirring was continued at the

same temperature for 16h and then the mixture was allowed to cool to room temperature

and precipitate was collected by filtration to obtain I ,3-bis-(2.6-

diisopropylphenyl)imidazolium chloride 3.9: yield 3.88 g (50%)

1H NMR (400 MHz; dmso-d6) o 1.14 (d, 1 = 6.8 Hz, 12 H), 1.24 (d. 1 :== 6.8 Hz, I 2 H).

2.33 (sept, J = 6.8 Hz, 4 H), 7.51 (d, J = 8.0 Hz, 4 H), 7.67 (t, 1 = 7.6 Hz, 2 H), 8.56 (s. 2

H), 10.20 (s, I H) ppm. 145

4-Methyl-N-(2,2,2-trifluoro-1-phenylethyl)benzenesulfonamide (3.11)

0 HN-~~CH3 d'CF 3 3.11

Imidazolium chloride 3.8 (25 mg, 0.0724 mmol) was taken in 4 mL of DMF and was added potassium tert-butoxide (7 mg, 0.063 mmol). After dissolution of the imidazolium chloride (~20 min), aldimine 3.10 (125 mg, 0.483 mmol) as solid and TMSCF, (0.1 mL,

0.676 mmol) were added. The solution was stirred for at room temperature tor 4h, then quenched with water and extracted with ethyl acetate (3 x 15 mL). The organic layers were combined and successively washed with water and brine, dried over Na2S04 and concentrated. The crude product was purified by silica gel chromatography (I 0% EtOAc) to obtain 4-Methyl-N-(2,2,2-trifluoro-1-phenylethyl)benzenesulfonamide 3.11: yield 114 mg (72%)

1H NMR (CDCh): 8 2.34 (s, 3H), 4.90 (q, J = 7.5 Hz, 1 H), 5.11 (b, I H), 7.12-7.33 (m, 7

H), 7.59 (d, J = 8.4 Hz, 2 H) Ppm.

19F NMR (CDCb): 8-74.36 (d, J = 7.6 Hz) ppm. 146

4-Methyl-N-(2,2,2-trifluoro-1-(4-fluorophenyl)ethyl)benzenesulfonamide (3.16)

0 HN-~D-CH 3 dCF3 F 3.16

Prepared similarly to 3.7. Yield 82%

1H NMR (CDCh): o 2.36 (s, 3 H), 4.84-4.92 (m, 1 H), 5.74 (d, J = 8.8 Hz, 1 H), 6.90-

6.95 (m, 2 H), 7.14-7.17 (m, 4 H), 7.58 (d, J = 8.4 Hz, 2 H) ppm.

19F NMR (CDCh): o-74.6 (d, J = 7.5 Hz), -112.0 (s) ppm.

N-(1-(4-Chlorophenyl)-2,2,2-trifluoroethyl)-4-methylbenzenesulfonamide (3.17)

II HN-~0~ ~ II CH3 ~CF~

CIN 3.17

Prepared similarly to 3.7. Yield 75%

IH NMR (CDCh): 0 2.32 (s, 3 H), 4.78-4.86 (m, 1 H), 5.61 (d, J = 9.2 Hz, 1 H), 7.04-

7.19 {m, 6H), 7.52 (d, J =8Hz, 2 H) ppm.

19F NMR (CDCh): o-74.4 (d, J == 7.6 Hz) ppm. 147

4-Methyl-N-(2,2,2-trifluoro-1-(4-nitrophenyl)ethyl)benzenesulfonamide (3.18)

Prepared similarly to 3.7. Yield 78%

1H NMR (CDCh): 8 2.35 (s, 3 H), 4.98-5.06 (m, 1 H), 6.19 (d, J = 9.2 Hz, 1 H), 7.16 (d, J

= 8.0 Hz, 2 H), 7.40 (d, J = 8.4 Hz, 2 H), 7.59 (d, J = 8.4 Hz, 2 H), 8.08 (d, J = 8.8 Hz, 2

H) ppm.

19F NMR (CDCh): 8 -74.0 (d, J = 7.6 Hz) ppm.

4-Methyl-N-(2,2,2-trifluoro-1-p-tolylethyl)benzenesulfonamide (3.19)

II HN-~0~ ~ !J CH3 ~CF~

HCN3 3.19

Prepared similarly to 3.7. Yield 70%

IH NMR (CDCh): 8 2.21 (s, 3 H), 2.28 (s, 3 H), 4.75-4.83 (m, 1 H), 5.73 (d, J = 9.2 Hz, 1

H), 6.95-7.00 (m, 4 H), 7.07 (d, J = 8.4 Hz, 2 H), 7.53 (d, J = 8.4 Hz, 2 H) ppm.

19p NMR (CDCh): 8-74.2 (d, J = 8.0 Hz) ppm. 148

3.4.6 NMR spectra of products:

~~~··· ....u ...c. , •• , ... ao"'''"' • cec11 ...... t t:t•ptrttur• lNOYA-411 ·-••r"

e~=~: t :::: ~:;!:·::c rib~i!:::l;:::.. ,,. .... , .... 'T ttae ltii.JI Total tt•• 1 hr, • ''"· 1 tee

------~------~Jl~~------_N~cll~------T" - J 1 a •

Figure 3.21: 1H NMR spectra of compound 3.6

.-u .,... lfC,. .V1'... ,.. ::1:.,.",.,. .lul 11 ~t:.. .. iiJr ; etr:OOU1• 1"1ft.ret ...... ::--· •.•:1 ·-r... •... ..t'lt ...... ::::MDCIMI... a.: •• It wtU1er.;oo .. ir... .~:...... , :: I .....,...... ,...... ••...... :.. E .. •.... ii ·-.....!J, dii, ...... "'·'"

-'

Figure 3.22: 1H NMR spectra of compound 3.7 149

U .M,lf DfC . I YT data Oct 1 Uti dfrq 3tf.114 IOh••nt DMSO Hl ..dp.,..r r t ':CouiSlT ION u p dof " sf'rq l tt . 7t& .... tn Hl .. A\ 3 , 74& , ' n p 441311: ...... 1\ol' ...... ·- dret fb .., ...... bl 11 NOCfiSINO . tpwf" II wtf1h pw 7 . 1 proc dl I fn tof t aath ..ct .• _, aloe: It .., •• p gatn ..... , " . ••h'"o .~ OIIPl4V .. ...,-l . .l ...I :~ ~ ... 17 . 13 su.u ··­.. lltl . 7 "' us.s ..... 111 . 111.. ,.. c llc ••

_,...... , -; · ----~------~ ppot & 3 t

Figure 3.23: 1H ,NMR spectra of compound 3.8

8AMPLf DEC. a \IT daU Aug I UU dfrq Stl . 7U t olven1. IJMIO dn Hl ,.,. .,., dpwr Sl ACQUISITION dof I tf'rq 5tt . 7U da nnn tn Hl at !.744 daf Ul' np UIJI ·-...... , ...... ,.. 1.1 ftl ...... ll P•OC::t:aiiND t,wr II wtflle :'r '·: ~:oc notvs" tot 1 nttl " n< I ' ct 1 drr eloc• " ._.., t•tn not 111aelll "*• HAGS . Witt tn" . •• ..• •• lltiiiLAV ., CJI.I ....,.. 4 ::.. • .. )1.74... .. 111··· ·­,,'". ""·'..... ••... J.lll• Ml CIIIC ,tw

J I - I 1 lO •

Figure 3.24: 1H NMR spectra of compound 3.9 150

I'll OIS("Vl ITANDAJtQ PAIU.t41TfRS

Pulse Sa ~u ance 1 tlpul lotv•nt ; COCl.J ..,.btent. 'Le.,.ereture INOVA-111 '"aaber ..

J I ------.---, I • - 1 -74 . .. -74 . 5 - 74 .• - 74.7 -a.a -73. 9 -74.0 -74.1 -74 . 2 -74.3

Figure 3.25: 19F NMR spectra of compound 3.11

ITANOAftD lH oeaU;Yl

1Mp1 lttlltl SAMPLl ore. 0 YT llatl Mlr 17 Zll1 tlfrq ....,., to1vent CDC1S ftll •• , .,_.. ..."' ACOUUlTtON .. , . tfrq 311.711 .. """ t" Ml . It .J , 7U , np ••••a ·-...... ,.IW 1111.1 ., ...... NOerattND " ... ..ll tpwr ss wtftll proc "' 7.1 - ttOt ... n" ..to'f .1 ....'" ' "'ct Jl " alocll" n .. gain ..ot utad ...... fUOO .... II . ••'" •" .. Ollf'LAY .. ..,... ."".. ::.. 11• .. u ...•. • ··­,,." ,.,...... ,, ..'"...... II.. .w cllc ..

~------_J ______A ____~~------I ·- 1 "

Figure 3.26: 1H NMR spectra of compound 3.16 151

r1t Otat:avf: t'f&N0.-.11.0 PAIIAMtllAt •••1 IZpul II(C • • YT dfr-. UI.7U Hl ..••wr ...... I ...... Ill' llr•• 1.1 Paocta•INO " ·-!:,.,..... t.JI ....'" ....warr 11 -·· ••.. . -· .I Dlt,LAY .. -11111 . 1 ::10 ...... I Ill .." 1U.N SU.II .. ttlll . l ··­ I '".."•... : n• ftO ph

-!0 -101 -110 -tn -uo ..... -10 -70 -ao

Figure 3.27: 19F NMR spectra of compound 3.16

a11p1 stilt" tMPll ore. • '

••• •• • •• • -

Figure 3.28: 1H NMR spectra of compound 3.17 152

Plt Otlllt.V~ STANOAit.D PARAN~Tf:RS ••P l •Zpu1

SAMPLI DEC • • YT lfat• Mar 11 1117 df'r lll 311,711 tol.,ent COC1.t lfn Hl dpwr 31 'ftl:cOUlllTlOM ••P Oof I •frq .1 71 . 1.11 fU """ tn .. , c np lltt.tl .. ... •t •·••• ·­...... •w 1011 11 . 1 ...... 'fb S Uit ho.o n b• 11 PROCIIIIND tpwr 55 1111 • • .,. rt ··: 'oftf'tle tof 1 not 111M nt 1 ft& "f C't .JSI •locw " l•tn not wtalf flAGS ••.." .• .. DISPLAY .. .. -:reut.l..... ~: II oc we .... ,., ,...... l ··­ 11151.1 1 •fp" ...... • M no P" •

------/ - 74 .11 _, ., -74.35 -74 ' 40 -74.45 - 74 .11 .... -74 . so .... - Figure 3.29: 19F NMR spectra of compound 3.17

e•ll lt'IUII .Mtf'LI DIC . a '

I I - Figure 3.30: tH NMR spectra of compound 3.18 153

t'U OHIUI.V( &TAHD .... O PAIIIAMITflltl u p1 stpul IAMPLt: ore. a. vT data Kllr U 1117 tolvant COC13 =~ ,. .. Jtt . 7:~ ''1:COUnntON .,.., dpvr, Jl tfrq 111 . 1.U .. . tn ,11 .. """ at 1 . 111 ..., . ,., 111131 ... ..dlllf • .., 111111 . 1 ,,... 1.1 t'b Ullt bl u NOCIUJNO " t pwr 55 -lb .... pv S . t wtft1a •• 0 proc t ot' 1 ,. not 11tH nt UZ4 ••th ", ct 11 alocr n warr gatn not u tad ...., flAOI ... " ~ wnt ••'" .~ .. OIIPUY lp -UUI.! ~: u:i~ .. 0 weh•••.. ,.. •Ill.••.. ,.,,rfl tll.Jt . .a th 11 tns U .I . IU n11 no ph

r-~~----.-~~~~-r~~~~-r-r~~-r~-r-r-~~~~----~------~-T~~---1 ~ -7&.00 -7&.05 -7& . 10 -7& . 11 - 7& . 11 - 7& .11 -73 . 85 -73.!10 -73 . !15

Figure 3.31: 19F NMR spectra of compound 3.18

ITANOUO IH GallflYf ...... ,. IAIM't.l ., ... DIC . OVT... .,.. ... u ...,. .. ''" . aohant CDCla .. 10 ftla ••P ..... "'0 ACQUIIJTIGN .. , srrq Jtt . 711 .. uo. at 1 . 744 , '""' "" .. - IW 1111...... 1 -:::.... a.: '~ bl ....11 HlOCUII­ vtftla tpvf'pw P.l" Mt 1111 .. •• 0 r." ", tor • ..... '"ct 111II ...... aloe • ,._-· .....n ...... " . .~ ••.. cnettu.v &81 • • ::.. 1111.1 :: on"0 ,,...... li .. ..U ...... 0 ,,...... ,..... c'c •• ..

l • • ' · --~----~· ------~------~----

Figure 3.32: 1H NMR spectra of compound 3.19 154

E j

______... j ~ ...... _~ -·-

~,-:•• ··~ ~ --.-,-~~....,-.--.-,...... ,.._ , l '" I I.,..... ~- T-..,...._,...._,__ .. -74 . 11 -74.11 -74.11 -74.21 -74.H •74.1a _14. 41 - F1gure. 3 .33: 19F NMR spectra of compound 3.19

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VITA

Meher Kumar Perambuduru was born in Mathala, INDIA, on August 291h, 1979.

He received his primary and secondary education from Z. P. P. H school, Nivagam, India.

He received his intermediate degree from Shanthinikethan junior college, Srikakulam,

India in 1996. He was awarded with Bachelor of Science degree from Andhra University,

Visakhapatnam, India in 1999. He received his Masters in Science from Hyderabad

Central University, Hyderabad, India in 2001. He continued his studies and was admitted as a graduate student for a Ph.D. in Department of Chemistry at University of Missouri

Rolla, Rolla, MO, USA, in 2002. He worked under the guidance of Dr. V. Prakash

Reddy to earn his Ph.D. in Chemistry in April, 2007