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THE SYNTaESIS OF NOVEL ORGANOFLUORINES BY ELECTROPHILIC FLUORINATION AND THEIR EVALUATION AS INHIBITORS OF PROTEIN TYROSINE PHOSPHATASE 1B

Christopher C. Kotoris

A thesis submitted in conformity with the requirements for the degree of Doctor of PhiIosophy Graduate Department of Chemistry

University of Toronto

O Copyright by Christopher C. Kotoris 2000

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Abstract

The Synthesis Of Novel Organofluorines By Electropbüic Fluorination And Their Evaluation As Inhibitors Of Protein Tyrosine Phosphatase lB,

Doctor of Philosophy, 2000, Christopher C. Kotoris, Department of Chemistry, University of Toronto.

Compounds b e h g non-hydrolyzable phosphate mimetics were prepared and

examined as inhibitors of protein tyrosine phosphatase 1 B (PTP 1 B).

These studies begin with a brief look at the importance of the phosphate group to

PTP1 B-peptide interactions. This was accomplished by examining peptides bearing unnatural,

non-phosphorylated tyrosine or phenylalanine derivatives as PTP 1 B inhibitors. These studies

indicate that the presence of a phosphate group or phosphate mimetic is crucial to peptide

binding.

We then examine non-peptidyl aryl denvatives bearing the most commonly used

phosphate niimetic, the difluoromethylenephosphonic acid (DFMP) group, as PTP 1 B

inhibitors. This involved developing a new approach, based on electrophilic fluorination of a-

phosphoryl carbanions, to the synthesis of aryl DFMP's. Factors affecting the electrophilic

fluorination yieIds, such as temperature. base and counterion were examined. Inhibition

studies reveaIed that these compounds were not significantly more potent inhibitors than the

parent compound, a,a-difluorobenzylphosphonic acid, with the exception k i n g the mefa-

phenyl substituted species which decreased the ICso by approximately 17-fold relative to a,a-

difluorobenzylphosphonic acid. However, certain compounds bearing two DFMP moieties

were very potent inhibitors. Possible reasons for this are discussed.

Further studies involved examining chiral a-monofluorophosphonates as PTPlB

inhibitors. This work includes the first description of a general method for preparing this cfass

of compounds. The key step in this procedure was the diastereosetective electrophilic

fluorination of a-carbanions of asymmetric phosphonamidates bearing (-)-ephedrine as a chiral

auxiliary. Removal of the ephedrine auxiliary afforded a-monofluorophosphoniç acids in

modest to good yields. Inhibition studies revealed that the R-enantiomers were approximately

10-fold more potent inhibitors than the corresponding S-enantiomers, but IO-fold less potent

than their a,a-difluoro analogues. Possible reasons for these differences are discussed.

Finally, we turned our attention to novel organofluorine functionalities that may be

either more amenable to cellular penetration or more readily converted to caged compounds

than DFMP-bearing compounds. Thus, compounds bearing monoanionic moieties. such as the

u-fluorotetrazole, carboxylate, malonyl and suifonate groups were prepared using

electrophilic fluorination and then examined as PTP 1 B inhibitors. Of these compounds, those

bearing the a-fluorosulfonate moiety were the most effective PTP I B inhibitors although these

were not as potent inhibitors as their DFMP analogues.

iii

This is dedicated to my fiancée

Faye

Acknowledeements

1 would like to express my deep gratitude to Professor Scott D. Taylor, my research

supervisor, for his guidance. patience and support, 1 thank the University of Toronto for

financial support through postgraduate research scholarships and Merck-Frosst Inc. for their

generous gifts of PTPIB.

I wish to thank the Taylor research group for their support, advice and fnendship' as

well as my fnends for their unique perspective on everything but chemistry.

Most irnportantly, 1 thank my family for their encouragement and support as well as my

fiancée Faye for her love, understanding, patience, and fiiendship.

Chapter 2 . Synthesis and Eviluatioa of Non-Phospbotyrosine Peptides as PTPlB Inhibitors

3 2.1 Introduction .................................................................................................................. -23

........................................................................................ 2.2 Experimental - 2 4

...............*.........**..*...... ......................................... 2.2.1 Materiais and Methods .... -24

................................................................................... 2.2.2 Syntheses -27

2.3 Results and Discussion ................................................................................................ 34

2.3.1 SynthesisofUnnaturalPhenylalanineDerivatives ...................................... 34

.................................................................... 2.3.2 Hexapeptide S ynthesis -37

.................................................................. 2.3.3 PTP 1 B Inhibition Studies 38

.......................................................................................... 2.1 Conclusions -43

Chapter 3 . Synthesis and Evaluation of Non-Peptidyl Inhibitors of PTPlB Bearing the Difluoromethylenephosphonic Acid Croup

........................................................................................... 3.1 Introduction 45

......................................................................... 3.1.1 The DFMP Moiety -45

3.1.2 SynthesisofArylDFMPs .................................................................. 47

....................................................................................... 3.2 Experimental -30

................................................... ....................*.........*. 3.2.1 Syntheses .. 51

.............................................................................. 3.3 Results and Discussion 73

............................. 3.3.1 Synthesis of Aryl DFMPs by Electrophilic Fluorination -73

.............................. 3.3.2 PTP Inhibition Studies with Difluorophosphonate Salts 79

................. 3.3.3 Difluorophosphonates as Potential Photoaffinity Labels for PTP's -93

......................................................................................... 3.4 Conclusions 101

Chapter 4 . Synthesis and Evaluation of Enantiomerically Pure a- Monofluoropbosphonic Acids

.......................................................................................... 4.1 Introduction 103

4.2 Experirnental ........................................................................................ 104

.................................................................................... 4.2.1 Syntheses 104

........................................................................... 4.3 Results and Discussion -122

.................................. 4.3.1 Chiral a-Monofluorophosphonic Acids: Overview -122

4.3.2 Diastereoselective Electrophilic Fluorination of an Asymmetric

Phosphonamide ............................................................................ 124

4.3.3 Diastereoseledve Electrophilic Fluorination of Asymmetrk

......................................................................... Phosphonamidates 126

......................................................................... 4.4 PTP 1 B Inhibition Studies 136

................................... 4.5 More About Diastereoselective Electrophilic Fluorination 140

......................................................................................... 4.6 Conclusions 143

Chapter 5 . Novel Phosphate Mimetics . ........................................................................................ 3.1 Introduction -145

...................................................................................... 5.2 Experimental -146

................................................................................... 5.2.1 Syntheses 146 - .......................................................................... 3.3 Results and Discussion -166

........................ 5.3.1 Synthesis of Benzylic a, a.Difluoronitriles and Tetrazoles -166

5.3.2 Synthesis of a, a.Difluorocarboxylates and a-Monofluoromalonyl . . ................................................................................. Denvatives 173

....................................................................... 5.4 PTPlB Inhibition Studies -176

....................................................................................... 5.5 Conclusions -178

Chapter 6 . Future Directions

................................................................................. 6.1 PTP 1 B Inhibitors -182

................................. 6.2 Other Applications of Chiral a~Monofluorophosphonates -184

.................................................................................................................. Appendix A 186

............................................................................................................................ References 193

............................................................................................ Publications -207

viii

Abbreviations

A la.. ................. Alanine

Arg ................. Arginine

Asn.. .............. .Asparagine

Asp ................. Aspartic acid

BSA.. ......... .Bovine serum albumin

n-BuLi.. ........ ..ri-Butyllithium

............ C ys.. Xysteine

DAST ............. Diethylarninosulfiir tri fluoride

DCC ............. ..Dicyclohexylcarbodiirnide

DCU.. ............. Dicyclohexylurea

DTT. ........... Dithiothreitol

D FMP ......... Di fluoromethy lenephosphonic acid

DIPEA. ....... .Diisopropylethylarnine

D MAP.. ......... -4-Dimethylaminopyridine

DMF.. ........... .Dimethylforrnamide

DM SO.. ........ .Dimethyl sulfoxide

DVB ......... ..Divinylbenzene

EDTA.. ....... Ethylenediaminetetraacetic acid

EGFR.. ....... .Epidemal growth factor receptor

FDP. . .......... Fluorescein diphosphate

F moc ............. -9-F luoreny lmethy loxycarbony l

FMP ........... .Fluorescein monophosphate

F2Pmp.. ....... Difluoromethylenephosphonyl phenylalanine

....... FOMT.. Fluoro-O-malonyltyroshe

................. Glu Glutamic acid

............... Gly.. Glycine

HATU.. ....... O-(7-azabenzoûiazol- 1 -yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate

................ His.. Histidine

HOB t ............. - 1 -Hydroxybenzotriazole

Ile.. ................ .Isoleucine

................ Irn.. .Imidazole

IRK. ............. insulin receptor kinase

IRS ............. .Insulin receptor substrate

............... Ka,. .Catalytic constant

.......... KDA.. Potassium diisopropylamide

KHMDS ....... .Potassium hexamethyldisilazide

............... K, .inhibition constant

................ Km.. .Michaelis constant

........... LD A. Lithium diisopropylamide

Leu.. ............... .Leucine

...... LiHMDS .Lithium hexamehyldisilazide

................ Lys.. Lysine

........... Met. .Methionine

.... NaHMDS.. Sodium hexamethyldisilazide

............ NBS N-bromosuccinirnide

............ NFS i N- fluorobenzenesul fonimide

NIDDM.. .... ..Non-insulin dependent diabetes mellitus

........ OMT.. .O-malonyltyrosine

.............. Pi. horganic phosphate

............ Pmp Phosphonomethylphenylalanine

....... pNPP.. ..p-Ntrophenyl phosphate

................ Pro .Proline

.......... PTKs.. .Protein tyrosine kinases

........... PTPs.. Protein tyrosine phosphatases

............ pTyr.. .PhosphoryIated tyrosine

................. R,. .Retention time (HPLC)

......... SDS.. Sodium dodecyl sulfate

.................. Ser Serine

TFA.. ............. .Triauoroacetic acid

THF ............... .TetrahydrofÙran

Thr.. ............... .Threonine

TMS ........,..... .Trirnethylsilane

TMSBr.. ...... .Trimethylsilyl brornide

Tyr.. ................. Tyrosine

Val.. ................. Valine

List of Tabies

- -- - -

Table Title Paee

K, values for pTyr and various peptides with rat PTP 1 . . . . . . . . . . . . . . . . . -24

Percent inhibition of PTP 1 B with 500 pM of hexapeptides 2.Sa-d.. . -3 8

Effect of base and counterion on the electrophilic fluorination of 3.5 with NFSi.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -74

Electrophilic fluorination of benzylic phosphonates with NFSi.. . . . . ..77

Percent inhibition of PTPlB and CD45 with 500 pM a,a-difluoromethy lenephosp honates 3.33-3.42. . . . . . . . . . . . . . . . . . . . . . . . . -80

Inhibition of PTP 1 B with 3.40,3.41 and 3.47,3.48,3.52 and 3.53.. .84

IC jo values for bis-phosphonates 3.56-3.59 with PTP I B . . . . . . . . . . . . . . .89

Effect of base and counterion on the electrophilic fluorination of phosphonamide 4.9 with WSi.. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. 125

Effect of base and counterion on the electrophilic fluorination of cis-isomer 4.13 with NFSi.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -129

Effect of base and counterion on the electrophilic fluorination of trans-isomer 4.16 with NFSi.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . .I29

Inhibition studies with phosphonic acids 1.19,3.40,4.1-4.4 and PTP I B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -136

Effect of base and counterion on the electrophilic fluorination of 4.A and 4.B .................... .................................. ....... ... ... 142

Ratio of "desired" to "undesired" isomer.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 143

Effect of base and counterion on the electrophilic fluorination of 5.7 with NFSi.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .I68

Electrophilic fluorination of benzylic nitriles with NFSi.. . . . . . . . . . . .. 170

xii

Effect of base and counterion on the electrophilic fluorinaion of 5.29 with NFSi ........................................................... 172

Percent inhibition of PTP 1 B with 500 p M non-fluorinated compounds ................................................................. -176

ICso values for fluorinated compounds 5.1-5.6,5.50,5.5 1. 1-19 and 3.40 ..................................................................... -177

xiii

List of Schemes

Scheme Title Page

The opposing reactions catalyzed by PTP's and PTK's ................. -2

................................... Common catalytic mechanism of PTP's -6

S ynthetic route for unnaturd L-phenylalanine derivatives 2.4a.d. .. -3 6

.......................... Solid-phase synthesis of hexapeptides 2.5a.d -38

Preparation of q l DFMPs via DAST fluorination of a.ketophosphonates ........................................................ -48

......... Preparation of aryl DFMPs via the cross-coupling approach -48

Electrophilic fluonnation of akyl phosphonates using NFSi ......... -49

Preparation of benzylic a, a.difluorophosphonate esters by electrophilic fluonnation ................................................... -50

Synthesis of substituted aryl(a, a~difluoromethylenephosphonates) .................................. by electrophilic f l u o ~ a t i o n using NFSi 76

Attempted fluorination of benzylic phosphonates 3.29 and 3.30 ..... -77

Possible route for decomposition products preventing formation ............................................................... of 3.31 and 3.32 78

Conversion of aryl difluorophosphonates into their ammonium salis ............................................................................ -79

Preparation of bis-DFMP biphenyl derivatives via electrophilic fluorination using NFSi ..................................................... -83

................................ Synthetic scheme for the synthesis of 3.52 86

Formation of a nitrene fiom aryl azides and its reaction with peptides ....................................................................... -94

............................................. First attempt to synthesize 3.61 95

xiv

Second attempt to synthesize 3.61 ....................................... -96

Thïrd attempt to synthesize 3.61 ......................................... -97

Synthesis of 3.61 ............................................................ 97

Reaction of benzophenone with light ................................... -99

Synthesis of benzophenone derivative 3.70 ........................... -100

Proposed synthetic route for the formation of enantiomerically Pure a-monofluorophosphonic acids using chiral auxiliaries ...... -123

Synthesis of a-monofluorophosphonamides 4.10 .................... -124

........................ Synthetic scheme for diastereomers 4.13.4.18 127

Synthetic scheme for fluorinated diastereomers 4.19430 .......... -128

Hydrolysis of 4.19.4.30 to give chiral acids 4.1.4.6 ................. -133

Synthesis of phosphonamidates 4.A and 4.B .......................... -141

Attempted synthesis of 5.23 via DAST fluonnation .................. 170

....................................... Synthesis of tetrazole derivatives -171

Attempted synthesis of unprotected and benzyl protected tetrazole . . ................................................................... denvatives 173

Synthesis of a. a.difluorocarboxylate derivatives ... .. ................ -174

Synthesis of malonyl and a-fluoromalonyl denvatives .............. -175

Reaction catalyzed by mandelate racemase ............................. 184

List of Figures

Figure Title Pape

Conserved structural segments required for catalysis by PTPI B. VHR, low M. phosphatase. YopS 1 and CD45 .............................. 5

"Movable" loop arnino acid sequence cornparison of a variety of PTP's ............................................................................. 5

Suggested transition state structures for the phosphorylation and . dephosphoryiation in PTP's. & the role of key conserved residues .. ... 8

Schematic of insulin-stirnulated receptor kinase autophosphorylation and the role of PTPI B in the enzymatic cascade ........................ -13

AnaIytical HPLC chromatograms of hexapeptides 2.5a and 2.5b .. - 3 9

...... Analytical HPLC chromatograms of hexapeptides 2 . 5 ~ and 2.5d 40

ESMS analysis of hexapeptides 2.5a and 2.5b .......................... -41

ESMS analysis of hexapeptides 2 . 5 ~ and 2.5d .......................... -42

................................... Inhibition of PTP 1 B by compound 3.48 85

Inhibition of PTP 1 B by compound 3.52 ................................... 87

Inhibition of PTP1 B by compound 3.59 .................................. -90

X-ray crystal structure of 4.19 ............................................. 131

X-ray crystal structure of 432. ............................................ 132

1 9 ~ - ~ ~ R ' s of 4.1.4.2 and a racemic mixture of 4.1 and 4.2 ........... 134

........................ .... Inhibition of PTPlB by compound 1.19 ...... 138

inhibition of PTP1 B by compound 4.1 .................................... 139

Inhibition of PTP 1 B by compound 5.4 .................................... 179

Inhibition of PTP 1 B by compound 5.5. ................................... 180

xvi

CHAPTER 1

PROTEIN TYROSINE PHOSPHATASES

1.1 OVERVIEW AND GLOBAL OBJECTIVES

A diverse array of stimuli, including hormones, cytokines and growth factors transmit

signals via pathways involving phosphorylation of specific cellular proteins.'.' The fate of a

cell. whether it will grow and divide, differentiate or die. is in turn dictated by these signal

transduction pathways. Thus, the regulation of protein phosphorylation and

dephosphorylation represents a fiindamental mechanism utilized by eukaryotic celts to govem

bioiogical processes and maintain homeostasis. Estimates suggest that one-third of cellular

proteins are phosphorylated. Protein phosphorylation in eukaryotic cells occurs mainly on

serine or threonine residues,' while tyrosine phosphorylation only accounts for 0.01 to 0.05%

of the total protein phosphorylation.' However. despite the small extent to which tyrosine

phosphoryIation occurs, this phenomenon has emerged as an extremely important form of

protein modification.

In vivo. tyrosine phosphorylation is dynamic and reversible. with protein tyrosine

kinases (PTK's) catalyzing the formation of phosphotyrosyl while protein tyrosine

phosphatases (PTP's) oppose this event by dephosphorylating tyrosine residues (Scheme 1.1 ).3

I t is now well-established that the PTKts and PTPts are key participants in signal transduction

pathways where they perform both positive and negative regdatory functions."or example.

they play important roles in the regulation of ce11 growth, mitogenesis, metabolism, gene

transcription and the immune respnse.' The imperfect operation of PTP's and PTK's.

integrated within the various signal transducing networks, has been implicated in a variety of

PTK's

Scheme 1.1. The opposing reactions catalyzed by PTP's and PTK's.

widespread diseases including diabete~.6-~ cancers 10-12 and immune dysfunctions. 1 3 . 1 4

Consequently, there has been much interest in the development of inhibitors of these enzymes

as they rnay have potentiai therapeutic properties. The focus of the work presented in this

thesis is on the PTP's- Over the last decade there has been an explosion of information about

PTP structure, regdation and fimction, resulting in the emergence of the study of PTP's as a

major field of research in both academia and industry. The development of new inhibitors of

these enzymes may not only provide a potential source of novel therapeutics but may also be a

source of chemical probes or tools for studying signal transduction pathways.

The global objective of the work presented in this thesis is the development of

inhibitors of a PTP known as PTPIB. There is now considerable evidence that PTPlB is

required for the down-regulation of the insuiin receptor kinase. Consequently. inhibitors of

this enzyme could be used as therapeutics for treating certain forms of diabetes. Towards this

objective, we present o u studies on the synthesis of compounds bearing non-hydrolyzable

phosphate mimetics and their evaluation as inhibitors of PTPI B.

1.2 OVERVIEW OF PTP1S

1.2.1 CLASSIFICATION OF PTP'S

To date, researchers have ascertained the identity of approximately 100 PTP1s. PTP's

have been categonzed into four different families. Two of these families are categorized

based on their subcellular localization. These are the receptor-like and cytoplasmic PTP's. As

far as peptide or protein substrates are concemed, the receptor-like and cytoplasmic PTP's

substrate specificity is strictly restricted to phosphotyrosyl peptides and proteins.'5.'6 The two

additional protein families are the dual specificity PTP's and low molecular weight PTP's.

These PTP's catalyze the dephosphorylation of phosphotyrosine. phosphothreonine and

phosphoserine residue~.".~~

The receptor-like PTP1s generally possess a variable extracellular domain. a single

transmembrane region and usually two tandemly repeated cytoplasmic PTP domains.

Leukocyte phosphatase ~ ~ 4 5 . ' ~ a major constituent of T- and B-cells involved in the immune

response of these cells. is regarded as the prototype for the general structure of the receptor

class of PTP1s. The significance of the two tandem homologous PTP domains is still unclear

as contradictory evidence to the fûnctionary or regulatory roles of the two domains remains

unresolved. Curreritly, al1 literature evidence illustrates that the first (membrane proximal)

PTP domain possesses phosphatase activity. However, it is unclear whether the second PTP

domain regulates the specificity andor activity of the first PTP domain or sirnply exhibits

independent substrate selectivity. 20-24

The cytoplasmic PTP's contain a single catalytic domain and various amino or

carboxyl terminal extensions. This group is exemplified by PTPIB?~ the first isolated PTP.

The intracellular PTP's dernonstrate a wide variety of protein modules flanking either side of

the conserved catalytic domain. These modules include (i) short hydrophobie segments that

can target the cytoplasmic face of intracellular membranes and (ii) S H 2 domains, which can

guide the PTP's to proteins/peptides containing phosphotyrosine residues. The dual specificity

phosphatases and the low molecutar weight phosphatases display minimal amino acid

sequence identity with the other two PTP families except for certain elements of a common

active site signature motif, which is discussed in more detail below.

Despite their primary structure variations and different active site substrate

specificities, al1 PTPs share similar tertiary structures, contain and require no metal cofactors.

hydrolyze p-nitrophenyl phosphate @NPP) and utilize a common mechanism of catalysis for

the hydrolysis of phosphate r n o n o e ~ t e r s . ~ ~ ~

1.2.2 STRUCTURE OF PTP'S

The PTP's are proteins whose tertiary structure is composed of highly twisted rnixed P-

sheets flanked by a-helices on either side. They can be proteins of greater than 400 amino

acids in which their diversity within the farnily originates from the non-catalytic segments

attached to the N- and C-termini of the PTP domains. Al1 PTP members, however. from

bacteria to mammals, have a catalytic domain of approximately 250 residues." There are two

major conserved sequence motifs within this 250 amino acid catalytic domain that are

cornmon to aH PTP's. These are the phosphate binding loop or PTP-loop and the "movable"

loop.

The PTP-loop contains an 1 1 -residue sequence. (1leNai)-His-Cys-X-Ala-Gly-X-Gly-

Arg-(Ser/Thr)-Gly (Figure 1 A), which is located within a crevice -9 angstrom deep2' This

important sequence foms the base of the active site pocket. Within this signature motif is a

segment that often contains several glycines (Gly-X-Gly-Arg-(Ser/Thr)-Gly) which allows

this section to form a loop that is important for binding substrates, especially that portion of

the substrate bearïng the phosphate moietym3

PHOSPHATASE PTP-LOOP MOVABLE LOOP

YOPS 1 I ~ G V G / @ A Wda PTP 1 B VH@AGIG~~SG W@l

CD45 V ~ S A G V - G w-@I VHR V-GY@SP

LOW M, PTP FI@$GNIC/EJSP

Figure 1.1. Conserved structural segments required for catalysis by PTP 1 B." the dual speci ficity PTP VHR,'~ low M, phosphatase.)O YopS 1 and ~ ~ 4 5 . l9

The "movable" loop is much smaller than the PTP-loop and consists of only three amino acids

(Figure 1 . 1 ): one of which is aiways aspartate. There is also often a tryptophan at the "hinge"

region of this loop (Figure 1.2).

Yersinia PTP

Yeast PTP 1 YFDLM@JM NKP

Yeast PTP2 Q ~ S C G V D

Human PTP 1 B HY- GVP

Human SHP- 1 QYL- GVP

~ u m a n Lar (D I ) QF- GVP

Figure 1.2. "Movable" loop amino acid sequence cornparison of a variety of PTP's.

1.2.3 MECHANISM OF CATALYSIS

It appears that al1 PTP's utilize a common mechanisrn. The mechanism that has been

proposed for PTP's is shown in Scheme 1.2. Substrate binding leads to the formation of an

O-H ' O', O : - - H ~ @ &P+ t/ -p;

O O-., Q-+<-w

Rotein Protein /- 6 H

- w o 7 OH

0-

Step 2

Phosphatase I

+ Pi

Scheme 1.2. Cornmon cataiytic mechanism of PTP's.

enzyme-substrate cornplex. This induces a confonnational change in the enzyme that brings

the conserved Asp residue of the "movable" loop close to the scissile oxygen of the substrate.

The phosphate group foms salt bridges with the conserved arginine of the PTP-loop. The

conserved cysteine of the PTP-loop attacks the phosphorus atom of the substrate. resulting in

cleavage of the O-P bond and formation of a covalent phosphoenzyme inter~nediate.~'

Phosphate ester hydrolysis is facilitated by the protonation of the ester oxygen by the

conserved Asp residue acting as a general acid. Once the E-P intermediate has been formed.

the same Asp residue (Glu in Yersinia PTP~') fûnctions as a general base by activating the

water molecule that approaches fiom the just-vacant leaving group side. This results in the

hydroiysis of the E-P intermediate and generates the non-covalent enzyme-phosphate

cornplex. The catalytic cycle of the enzyme is then completed with the dissociation of

The mechanism outlined in Scheme 1.2 is supported by crystallographic, kinetic and

mutagenesis studies. Crystal structures of PTP's, "fiee" and cornplexed with oxyanions such

as tungstate, or phosphotyrosine and phosphotyrosine-bearing peptides. show that binding of

these ligands induces a conformational change resulting in movement of the conserved Asp

residue into the active site. The "movable" loop covers the active site like a '.flap".33.34 This

positions the conserved Asp residue such that it foms a network of hydrogen bonds to the

oxyanion or phenolic oxygen of phosphotyrosine and a buried water rn~lecule.~' Thus, the

Asp residue is ideally positioned to donate a proton to the tyrosine-Ieaving group during the

first hydrolysis step and to act as a general base by activating a water rnolecule for the second

hydrolysis step (Scheme 1.2)." PTP hydrolysis of phosphotyrosine-containining peptides

exhibits a bell-shaped profile when vaiues of Lt are plotted vs. pH. This is indicative of

general acid and general base catalysis. Substitution of the invariant AS^^^^ in the Yersinia

PTP to Asn reduces catalytic activity 3-fold and causes the disappearance of the basic Iimb of

the pH profile indicating that this residue is acting as a general acid. The acidic limb is due to

protonation of Gluzw. Mutation of GluIw to a Gln resulted in significant loss of activity and

disappearance of the acidic limb of the pH profile indicating that it acts as a general base.'7

Site-directed mutagenesis of the invariant Arg residue results in almost complete loss

of enzyme a c t i ~ i t ~ . ' ~ ~ ' - ' ~ ~ ' These studies, however, have not illustrated whether the Arg

residue is required for PTP folding and structure, and/or catalysis. The Arg residue side chah

(guanidinium group) is planar in stmcture and possess the ability to form multiple hydrogen

bonds with phosphate rn~ieties.~' The three-dimensional structures of PTP-oxyanion and

PTP-pTyr complexes indicate that two of the oxygen atoms of the oxyanion and/or pTyr form

two hydrogen bonds with the positively charged guanidiniurn group of the invariant Arg

residue in the PTP-loop (Figure 1.3).

E-P FORMATION E-P BREAKDOWN

~ P ~ O Aspy0 Serfïhr general acid

SerIThr general base I O

1 I ?e O 1 O I

H O (i H O II I I I H

cys-sG FI O-R cys-sQ l? I

II i O- H

nucleophile -0 O' 1 1 -0 O- . . - salt bridges - ; I L 1 I

H H # H Fi lFn

Ars,,-/ 7 Arg- how y

Figure 1.3. Suggested transition state structures for the phosphorylation and dephosphorylation in PTP's, and the role of key conserved residues4

Hydrogen bonds are dso forged with the N-H amides of the peptide backbone making

up the PTP-loop. Consequently, one can deduce that the Arg side chah and the PTP-loop

peptide backbone amides collectively bind the phosphoryl group in the substrate. Kinetic

studies utiIizing the effect of altering the invariant Arg residue, by site-directed mutagenesis

within the active site, have depicted the invariant Arg residue as playing an important role in

substrate binding and. to an even greater extent, transition state s t ab i~ iza t ion .~ '~~ This is

esemplified by the fact that the Arg to Lys mutant showed minimal ha, improvement when

compared with Arg to Ala or Met mutant^.^' Although the Lys residue can partially replace

the Arg residue for substrate binding, it cannot form a coplanar bidentate complex with two of

the equatorial oxygen atoms present on the phosphate during catalysis. Thus, it cannot permit

the required stabilization of the trigond bipyrarnidd transition state provided by the

guanidinium group of arginine.

Site-directed mutagenesis studies have demonstrated that the invariant Cys residue is

absolutely essential for phosphatase activity. 15.18.24 Substituting the Cys residue with a Ser or

any other residue abolishes the PTP activity, yet, permits the protein to retain its ability to bind

phosphotyrosine-containg peptides/proteins.40 The pK, of the active site cysteine of the

Yersinia PTP has been estimated to be approximately 4.7 (the pK, for a normal cysteine is

8.3). Therefore, the cysteine residue exists as a thiolate anion at physiologicaI pH. The

crystal structures of PTP's complexed with oxyanions (such as tungstate, phosphate and

sulfate) and pTyr-containing peptides show that the cysteine sulfur atom is located at the

center of the PTP-loop within 3 to 4 angstroms of five backbone amide nitrogens. These

amides are electrostatically coupled via the carbonyl oxygens to hydrogen bonds radiating

away fiom the cysteine atom. This produces a network of microdipoles in which the positive

4 1.42 ends are pointed toward the cysteine thiol. This stabilizes the thiolate anion thus lowering

the pK, of the cysteine residue. It has been suggested that the sulfûr to oxygen substitution in

the Cys to Ser mutants leads to substantial strucWconfonnational and fiuictional alterations

which may result in the incomplete ionization of the hydroxyl group of the senne residuee4' In

fact, mutation of the Cys to a Ser eliminates the PTPts ability to form a phosphoenzyme

intemediate?' The higher pK. of serine compared to cysteine and the lower chemical

reactivity of the senne alkoxide, an inferior nucleophile compared to the cysteine thiolate, rnay

also contribute to the lack of catalytic activity. Direct evidence for the formation of the

phosphoenzyme intermediate has been obtained by trapping the covalent intermediate by

addition of SDS immediately after mixing the enzyme with a 32~-labelled phosphotyrosyl

peptide or with 3 2 ~ - p ~ ~ ~ . 40.4 Kinetic studies have shown hydrolysis of the covalent

intemediate is the rate-limiting step. 40.4446

It is believed that the hydroxyl group of the conserved Thr/Ser residue irnmediately

following the invariant Arg residue is important in the mediation of the breakdown of the E-P

intermediate and stabilizes the cysteine residue through hydrogen b ~ n d i n ~ ~ ~ (Figure 1.3). The

conserved histidine adjacent to the invariant cysteine, though not essentiai for PTP activity. is

believed to provide structura1 positioning of the cysteine residue and conformation of the

phosphate binding loop? Substitution of His with Asn or Ala altered the active site thiol pK,

to 5.99 and 7.35, r e ~ ~ e c t i v e l ~ . ~ ' Thus, the low pK, value of Cys (4.7 within the native PTP-

loop) suggests that ionic interactions are important in stabilizing the thiolate anion.

1.2.4 SUBSTRATE SPECLFICITY OF PTP's

A vast amount of time and effort has been placed on defining the substrate specificity

determinants for individual PTP's. Through the use of synthetic pTyr-containing peptides.

corresponding to natural phosphorylation sites in proteins, it was illustrated that PTP's display

ô range of k&K, values for these short peptide substrates indicating that PTP substrate

specificity is controlled, in part, at the primary structure level. 21.55.48-53 In particdar. it has

been shown that the undecapeptide DADEpTyrLIPQQG, modeled on an autophosphorylation

site ( T m 2 ) of the epidermal growth factor receptor (EGFR9g8-gg8) is an optimal peptide

substrate for Yersinio PTP and rat PTPI .'8.S1 Using various-sized synthetic pTyr-containing

peptides. corresponding to this and other autophosphorylation sites of the EGFR. provided a

pIatform fiom which the stnictural requirements of substrates for PTP's could be probed. For

exarnple, Zhang el al.'' utilized an Ala-scan in which each amino acid within the

phosphotyrosine-containing peptide substrate EGFR988-998 (DADEpTyrLIPQQG) was

sequentially replaced by Ala. Variations in the peptide length demonstrated that a minimum

requirement of six amino acid residues, four residues N-terminal to pTyr and one residue C-

terminal to pTyr, for efficient binding and catalysis. The Ala-scan demonstrated the

importance and preference for acidic residues at positions N-terminal to the pTyr for optimal

binding and turnover, especially at the pTyr-1 position.

1.3 PTPlB OVERVIEW

This thesis is concerned mainly with a PTP known as PTP 1 B- PTP 1 B, one of the most

studied of al1 the PTP's, is anchored by its C-terminus to the cytoplasmic face of the

endoplasmic reticulum and is the hurnan analogue of rat PTPI. It was isolated by Tonks et

al.'" and has been purified as a single polypeptide chain of 321 arnino acid residues with a

molecular weight of 37 kD. This, however, represents a truncated form of PTPIB, as the

cDNA indicates that this isolated enzyme corresponds to the NHz-terminal portion derived

from a full-length molecule of 435 amino acid residues. 25.56.57 To date, several crystal

structures of this enzyme complexed with various ligands have been reporteci. 35.41.58 The

truncated form of PTPl B is composed of 8 a-helices and 12 P-strands.

The conserved catalytic domain is approximately 250 amino acid residues (residues

30-278) which constitutes 57% of the protein sequence.ll This harbors the representative PTP

sequence motif (VV)HCXAGXXR(S/T)G. The 157 residue COOH-terminal non-catalytic

extension serves a regulatory function.'" The first 122 residues are predominantly hydrophilic

and contain several serine phosphorylation sites. Its final 35 residues target the enzyme to the

cytoplasmic face of membranes of the endoplasmic reticulum. Like most PTP's. PTPlB

displays a subtle preference for multiple acidic side chains within five amino acids amino-

terminal to the phosphotyrosine residue. In fact. the structure of PTPlB serves as the

prototype for comparing several structural characteristics of the PTP family of enzymes and

has provided the framework for understanding the mechanism of tyrosine dephosphorylation.

1.3.1 PTPlB AND DIABETES

Tyrosine phosphorylation is a crucial element for the initiation and propagation of

insulin action, which is mediated via the insulin receptor kinase (W), a transmembrane

59.60 glycoprotein with intrinsic PTK activity (Figure 1.4). Upon binding of insulin. a

conformational change in the receptor leads to a rapid autophosphorylation of multiple

tyrosine residues, including tyrosines 1 146, 1 150 and 1 15 1, on the intracellular portion of the

kinase regulatory domain. This subsequently enhances the intrinsic tyrosine kinase activity.

which in turn phosphorylates the various endogenous insulin receptor substrate proteins (Le.

IRS- 1) that propagate the insulin signaling e v e d 9

Insulin resistance is the inability of insulin to stimulate glucose uptake fiom blood to

muscle. Insulin-independent diabetes mellitus (also known as type II diabetes) is

c haracterized by insensitivity to insulin and resulting hypergl ycemia (high blood glucose

levels). Approximately 80% of people with diabetes suffer from the type II form. Insulin

resistance in NIDDM is believed to be caused by reduced insulin receptor kinase activity

rather than due to the deficiency of insulin.

IRK Tyr116 Insulin -

i

cascade of 2 O glycogen - phosphorylation - I

increase in receptor synthesis, etc. reactions tyrosine kinase activity

- plasma membrane

Figure 1.4. Schematic of insulin-stimulated receptor kinase autophosphorylation and the role of PTP 1 B in the enzymatic cascade.

PTP1 B 1

Although the precise in vivo function of PTPlB has not yet been established, there is

now considerable evidence that this enzyme may be involved in the down-regulation of insulin

signaling by dephosphorylating specific phosphotyrosine residues on the insulin receptor

kinase.8.6~-73 For exarnple, overexpression of PTPlB in ce11 cultures decreases the insulin-

stimulated receptor and/or IRS-1 phosphorylation, while reducing its level increases the

insulin initiated signaling pathways. 65.66.74 Moxharn et have shown that the G-protein

su bunit, Gia exhi bits insulin resistance characteristic of NIDDM. Gid deficiency was found

to increase PTP activity and attenuate insulin-stimulated tyrosine phosphorylation, in vivo. of

1 RS- 1. Most importantiy, drarnarically increased insulin sensirivity and dierary-induced

o besity resistance was prevalent in mice lacking PTPl B (P TPI B kiockour mouse) .72 This

suggests specific inhibitors of PTP 1 B may be useful as therapeutics for the treatment of type II

diabetes and obesity. For exarnple. vanadate, a phosphate analogue. has been shown to be an

insulin-mimetic and, in human clinical trials, has shown some potential in treating patients

with diabetes.'* It has k e n suggested that part of vanadate's insulin-mimetic effect is due to

its being a potent inhibitor of PTPIB. '678 However, vanadate is a non-specific phosphate

inhibitor and has a wide variety of effects on biological ~ ~ s t e r n s . ~ ~ M a t is really needed are

inhibitors that are specific for PTPI B.

1.1 PTP INHIBITOW

1 .I. 1 PEPTIDE-BASED INHIBITORS

The rnost logical approach towards the development of PTP-specific inhibitors is to

incorporate non-hydrolyzable phosphotyrosine mimetics in pIace of phosphotyrosine @Tyr.

1.1) in synthetic peptide substrates. This approach to PTP inhibition has been examined

e x t e n s i ~ e l y . ' ~ - ~ ~ ' ~ ~ inhibitors of this type were modeled after phosphonic acid derivatives of

pTyr. Phosphonic acids are phosphate isosteres in which the ester oxygen has been replaced

by a methylene unit and, therefore, are chemically and enzyrnatically resistant to P-C bond

cleavage. For example, phosphonornethylphenylalanine (Pmp, 1.2), utilized within the

peptide sequence D-A-D-E-X-L-NH2, has been examined as a PTPlB inhibitor. However,

this was a relatively poor PTP inhibitor exhibiting an ICso value of 200 p ~ . 8 ' It was

suggested that this was due to the higher pK, of the Pmp phosphonate group relative to the

pTyr phosphate moiety as well as the loss of the potential hydrogen bond associated with the

ester oxygen of pTyr. Thus, the difluoromethylene denvative 1.3 (FzPmp), was employed as a

pTyr mimetic." The difluoromethylenephosphonic acid (DFMP) group has been used

estensively as a phosphate biostere since the fluorines reduce the pKa of the phosphonate

group and are capable of acting as H-bond a~ce~ to r s . *~ The above mentioned hexamer

sequence bearing 1.3, was found to bind up to IO^ times better than the initial analogous

peptide bearing the non-fluoro analogue 1.2 (PTPIB ICso value of 0.1 p ~ ) . 8 ' Kinetic studies

suggest that the enhanced potency of the FzPmp-bearing peptides is attributed to specific

interactions of the fluorines with the PTP active site and not due to efTects on the phosphonate

pK, values."

COOH COOH

1.4 (OMT) 1.5 (FOMT) 1.6 (sTyr)

Albeit FzPmp-bearing peptide inhibitors display good potency, studies have suggested

that compounds bearing this moiety may not be able to cross the ce11 membrane. 82.87 This led

to the design of the dicarboxylic acid-containing malonate structures O-malony ltyrosine

(O MT 1 and fluoro-O-malonyltyrosine (FOMT 1 S). 82.88 The malonyl group has the

advantage in that is can be easily caged as an esterase labile or photolabile diester, transported

across the ce11 membrane, and once inside. regenerated as its di-acid form via cellular

esterases or light. However, peptides bearing OMT and FOMT were not as effective PTP

inhibitors as their F2Pmp-bearing peptide analogues. For example, the ICjo value for the

hexapeptide D-A-D-E-OMT-L with PTPlB was 10 pM while the FOMT analogue exhibited

an ICjo value of 1 PM. The increased potency of the FOMT analogue was attributed to the

introduction of new hydrogen-bonding interactions between the fluorine and the enzyme

active site and not to pK, effects as both OMT and FOMT are most likely diionized at

physiological Desrnarais ef al. examined a variety of peptides containing a sulfotyrosyl

residue (sTyr 1.6) as inhibitors of PTPl B and CD45. However. they were found to be

moderate inhibitors with ICso values in the low to mid-micromolar range.89 which is

considerabl y less than the analogous F2Pmp-bea~g peptides.

1.4.2 NON-PEPTIDYL INHIBITORS

Although peptide-based structures have produced potent and relatively selective PTP

inhibitors, there are a number of drawbacks to using peptidyl fhmeworks. Perhaps the most

serious of these shortcomings is their poor bioavailability: peptidyl inhibitors are usually

susceptible to proteolytic degradation and often exhibit poor cellular uptake. Consequently,

researchers have recently turned their efforts to developing non-peptidyl inhibitors of PTP's.

Indeed. most dmgs are not peptides but instead are small molecules, bearing functionalized

aromatic or heteroaromatic groups.

1.4.2.1 NON-SPECIFIC INORGANIC INHIBITORS

A number of metai-containing reversible inhibitors such as gallium nitrateg0 and

vanadate 76.77.9 1 have been reported. These species, which most likely fùnction as mimics of

inorganic phosphate or as transition state analogues. are also known to inhibit many other

enzymes involved in signal transduction pathways including ATPases and nucleases?'"'

Pervanadate, an irreversible PTP inhibitor, is speculated to inactivate PTP's by oxidizing the

critical Cys residuem7' However, the lack of selectivity and potentiai toxicity of these srnall

molecules limit their potential as therapeutically useh1 PTP inhibitors.

1.4.2.2 NATURAL PRODUCT-DERIWD INHIBITORS

Natural product screens have provided a wide variety of PTP inhibitors. The

aporphine aikaloids, nornucifenne (1.7), anonaine and roemerine (1.8) derived fiom the stem

and stem bark of Rollinia ulei exhibit potent inhibition of CD45 (IC jo = 5.3. 17, and 107 PM.

r e ~ ~ e c t i v e l ~ ) . ~ ~ Sulfircin (l.9), a sulfate-bearing marine natural product, was shown to be a

non-specific inhibitor of certain phosphatases such as cdc25A, VHR and PTP 1 B (IC jo = 7.8.

1.7 and 29.8 FM, r e ~ ~ e c t i v e l ~ ) . ~ ~ Several natural product derived inhibitors have been

discovered that are active against the VHR and cdc25 dual specificity phosphatases. Among

these are the Streptomycese-derived butyrolactone, RK-682 (1.10) and the stevastelins cyclic

depsipeptide irnmunosupressants (1.11), which exhibit non-cornpetitive inhibition of V H R . ~ ~ -

98 The benzoquinonoids, dnacin Al and B1 (1.12) inhibit the dual specificity phosphatase

cdc25B with ICso values of 14 1 and 64.4 pM, r e ~ ~ e c t i v e l ~ . ~ ~

In most cases, however, the structure-based design of new analogues of these compounds has

been hampered by a lack of understanding as to how these natural compounds interact with

PTP's.

R = H, anonaine R = Me, roemerine

stevastelin A, R = S03H stevastelin B, R = H

1.12

dnacin A l , R = CN dnacin 61. R = OH

1.4.2.3 MECHANISM-BASED IRREVERSIBLE INHIBITORS

Widlanski and e ers'^ first reported 4-fluoromethylphenylphosphate (1.13) as a

suicide inhibitor of human prostatic acid phosphatase. This enzyme, though not a tyrosine

phosphatase, is able to dephosphorylate a variety of phenyl phosphates. including

phosphotyrosyl residues. Withers et al.. 'O' however, have s h o w 4-

(difluoromethy1)phenylphosphate (1.14) to be a suicide inhibitor of SHP protein tyrosine

phosphatase. Enzyme-mediated phosphate ester cleavage is beiieved to forrn a reactive

quinone methide, which is subsequently attacked by nucleophilic residues at the active site

resulting in enzyme inactivation. 1.14 did not exhibit a high afEnity for PTPases and is most

likely a non-specific inhibitor. The a-halobenzylphosphonates (1.15) have been shown to

irreversibly inactivate the Yersinia PTP in a time- and concentrationdependent manner.'O'

These "quiescent affity labels" are reactive only when bound to an enzyme active site. The

order of inactivation is as follows: Br >> Cl > F. Recently, the menadione 1.16 was found to

inhibit cdc25 dual specificity phosphatases, while the suifone analogue of naphthoquinone

1.17, an antiturnor agent,lo3 was found to selectively inactivate P T P I B . ' ~ In spite of the

apparent selectivity of 1.17, mechanism-based irreversible inhibitors do not exhibit a high

affinity for PTP1s and typically undergo non-specific interactions with other cellular

nudeophiles.

1.4.2.4 STRUCTURED-BASED, NON-PEPTIDYL REVERSIBLE INHIBITORS

Much of the work on developing stmctured-based, non-peptidyl reversible inhibitors

has focussed on incorporating non-hydrolyzable phosphate mimetics into aromatic structures.

Frechette and coworkers reported that certain functionalized benzylic a-hydroxyphosphonic

acids are good inhibitors of ~ ~ 4 5 . " '

X = Br, CI, F 1.13, X = H 1.14, X = F

Researchers at Merck Frosst inc. have reported that

hydroxybutylidene-l,l -bisphosphonate), a potent inhibitor of bone resorption, is also an

inhibitor of PTPE."~

Burke and coworkers examined a series of phenyl derivatives bearing the

difluoromethylenephosphonic acid (DFMP) group as reversible PTP inhibitors. 'O7 From this

series, the naphthyl derivatives, 1.18 and 1.19, were moderate inhibitors of certain PTP's (Ki's

of 255 and 179 pM with PTPI B, respectively) in contrast to their non-fluorinated counterparts

which were very poor inhibitors. The addition of the hydroxyl group at the 4-position in 1.20.

yielded a slightly more potent inhibitor (Ki of 93 pM with PTPI B)." Surprisingly. the phenyl

derivatives 1.21a-d were poor inhibitors. The inhibition increases in the following order: X =

CH2 < CHOH < CHF < CF2. However, these denvatives were found to inhibit PP2A, a

serinehhreonine phosphatase, and are approximately 1000-fold less potent than the best

peptidyl inhibitors.

Recently, Taylor er a2.1°8 prepared the bis-DFMP compounds 1.22 and 1.23. These

naphthyi denvatives were even better inhibitors (Ki values of approximately 16 PM, with

PTP 1 B) and provided a small degree of selectivity between CD45 and PTP 1 B. Despite the

lack of absolute selectivity, these results demonstrate the potential of aryl DFMP derivatives

as non-peptidyl inhibitors of PTP's.

1.1 8 I

X 1.21a, X = CH2 1.22, 2,6 substitution 1.19, X = H b, X = CHOH 1.23. 2,7 substitution

1.20, X = OH c, X = CHF d, X = CF2

1.5 SPECIFIC OBJECTIVES

The specific objective of this thesis is to prepare non-peptidyl compounds bearing

current. as well as new, non-hydrolyzable phosphate mimetics and to examine these

compounds as inhibitors of PTPIB. Our studies begin with a brief look at the importance of

the phosphate group to PTP1 B-peptide interactions. This was accomplished by examining

peptides bearing unnatural, non-phosphorylated tyrosine or phenylalanine derivatives (Chapter

2) as PTPlB inhibitors. We then examine non-peptidyl compounds bearing the most

commonly used phosphate mimetic, the DFMP group, as PTPlB inhibitors. This work

involved developing a new approach, based on eiectrophilic fluorination. to the synthesis of

aryl DFMP's (Chapter 3). Further studies involved examinine chiral a-

monofluorophosphonates as PTPlB inhibitors. This work includes the first description of a

general method for preparing this class of compounds (Chapter 4). Finally, we tumed our

attention to novel organofluorine fiinctionalities that may be either more amenable to celtular

penetration or more readily converted to caged compounds than DFMP-bearing compounds

(Chapter 5) . Thus, a-fluorinated tetrazoles, carboxylates, malonyls and sulfonates were

prepared using electrophilic fluorhaion and then examined as PTP1 B inhibitors.

CHAPTER 2

SYNTHESIS AND EVALUATION OF NON-PHOSPHOTYROSINE PEPTIDES AS PTPlB INHIBITORS

2.1 INTRODUCTION

Before we began to pursue the design and synthesis of PTP inhibitors we wished to get

some idea as to just how important the presence of a phosphate mimetic was going to be, in

terms of conferring binding affinity, to PTPlB-inhibitor interactions. As far as substrate

binding to PTP 1 B is concemed, Dixon and coworkers have reported that PTP 1 B does not bind

to tyrosine-bearing peptides that lack a phosphate group. However, no experimental details

(Le. peptide sequence, concentration of peptide employed in the study) of these studies were

given.48 Nevertheless, this statement suggests that the presence of a phosphate group is

essential for substrate bïnding even though its precise contribution to binding, in terms of

kcaVmo1. was unknown.

Phosphotyrosine alone exhibits a low affinity for rat PTPl with a Km of approximately

5 rnM. However. certain peptides bearing pTyr have a much greater affinity for PTPI. For

example, the hexapeptide D-A-D-E-pTyr-M exhibits a Km for PTPl that is approximately 7 x

10)-fold less than that of pTyr (Table 2.1).'09 This difference in afinity is mainly due to the

enzymes preference for acidic side residues amino-termind to the phosphotyrosine residue.

The affinity of this peptide can be increased another 51-fold by substituting pTyr in the

peptide with FtPmp (see Table 2.1). Taken together, the incorporation of the additional arnino

acids to pTyr and the substitution of the bridging phosphate oxygen for a difluoromethylene

unit results in a 3.5 x 10'-fold increase in affinity compared to pTyr alone. Thus, we

hypothesized that peptides having the D-A-D-E-X-M sequence where X is a phenylalanine

Table 2.1. K, values for pTyr and various peptides with rat PTP1.

Substrate Km or Ki (PM)

Ptyr 4930a D-A-D-E-~T~~-M 0.7 1 ob D-A-D-E-F2Pmp-M 0.014~

'Value taken fiom reference 55. %ahes taken from reference 109.

derivative bearing a trifluoromethyl or difluoromethylene moiety at the para-position may

exhibit considerable inhibition of PTPlB, even though they would not contain a phosphate

group. By comparing the ICso's or Ki's of such peptides to that of D-A-D-E-F2Pmp-M, the

contribution of the phosphate group to PTP-peptide binding could be ascertained. Here we

present the synthesis of peptides bearing unnatural amino acids 2.la and 2.lb, as well as their

non-fluorinated analogues 2.lc and t.ld, and their evaluation as inhibitors of PTP 1 B.

2.2 EXPERIMENTAL

2.2.1 MATERIALS AND METHODS

C hemicals. Uniess otherwise noted, al1 reagents for syntheses were obtained fiom

commercial suppliers (Aldrich, Milwaukee. Wisconsin, USA or Lancaster Synthesis Inc.

Windham, New Hampshire, USA) and were used without fùrther purification.

Dic hloromethane was distilled fiom calcium hydride under argon. DMF was distilled under

reduced pressure fiom calcium hydride and stored over 4-A sieves under argon. Al1 glassware

was pre-dried prior to use and reactions involving moisture-sensitive reagents were executed

under an inert atmosphere of dry argon. Flash chromatography was performed using silica gel

60 (Toronto Research Chemicals, 230-400 mesh ASTM). Buffer chernicals and bovine serum

albumin (BSA) were obtained fiom Sigma Chemical Company. Enzyme assay solutions were

prepared with deionizeddistilled water. Fluorescein diphosphate (FDP) and human PTP 1 B

were a gift fiom Merck-Frosst Canada Inc. (Montreal, Canada).

Instrumentation. 'H- and ' 9 ~ - ~ ~ ~ spectra were recorded on a Varian 200-Gemini NMR.

Unless stated otherwise, al1 13c spectra were recorded on a Varian-400. For 'H-NMR spectra

run in CDCi3, chernical shifts (6) are reported in parts per million relative to the interna1

standard tetramethylsilane (TMS). For 'H spectra run in CD30D, chernical shifts (6) are

reported in parts per million relative to the residual CH3 peak at 6 3.30 ppm. For 'H spectra

run in D20, chemical shifls (6) are reported in parts per million relative to the residual HDO

peak at 6 4.68 ppm. For '-'c specha run in CDCl,, chemical shifts are reported in parts per

million relative to the CDCI, residual carbons (8 77.0 for the centrai peak). For I3c spectra

run in CD30D, chemical shifts are reported in parts per million relative to the CD30D carbon

(6 49.0 for the centrai peak). For "C spectra run in D20, chemical shifts (6) are reported in

parts per million relative to the CH3 peak of 3-(trimethylsi1yl)-1-propanesulfonic acid

(extemal). For I9~-NMR, chernical shifts (6) are reported in parts per million relative to

trifluoroacetic acid (extemal). Abbreviations s, d, t, q, qt, m and br are used for singlet,

doublet, triplet, quadruplet, quintuplet, multiplet and broad, respectively. Al1 NMR coupling

constants are given in Hz. Electron impact (EI) mass spectra were obtained on a Micromass

70-S-250 mass spectrometer. Electrospray mass spectra were obtained using a Micromass

Platform mass spectrometer. Al1 melting points were taken on a Fisher-Johns melting point

apparatus and are uncorrected. Analytical chiral HPLC was performed using a Waters LC

4000 System equipped with a Astec Chirobotic T #12024 chiral colurnn and a Waters 486

tunable absorbance detector set at 254 nm.

Solid-Phase Resins and Amino Acids for Peptide Synthesis. Unless otherwise stated,

Wang resin (1% DVB) and al1 amino acids used in solid-phase peptide syntheses were

purchased from Advanced Chemtech (Louisville, Kentucky, USA). Peptides were synthesized

rnanually, using Fmoc-amino acids, using a Memfield reaction vesse1 (30 ml, Cat. # SHG-

20330-PI) and a 180 degree variable rate fiask shaker (Cat. # SVR-22701-PI) obtained from

Peptides International Incorporated (Louisville, Kentucky, USA).

Peptide Purification and HPLC Analysis. Peptide purification and analyses were perfonned

on a Waters HPLC system using two model 600 pumps, a model 4000 gradient controller, a

mode1 70 10 Rheodyne injector and a model 486 UV detector. Irradiation was carried out with

a 250W GE heat lamp at 254 m. Al1 large scale purifications were performed on a

preparative scale Vydac #2 1 8TP 1 022 C 1 8 reverse-phase column ( 1 0-pn resin. 22 mm inside

diarneter by 250 mm length) using 0.1% TFA in waterlacetonit~ile. Al1 analytical peptide

analyses were performed on either a Vydac fC201HS54 Cl8 reverss-phase column (5-pm

resin, 4.6 mm inside diameter by 250 mm length) or an Astec Chirobiotic T #12024 chiral

column (5 -pn resin, 4.6 mm inside diameter by 250 mm length) using 0.1% TFA in

water1acetonitriIe and exhibited a single peak in the HPLC chromatogram.

Kinetic Studies with PTPIB. Unless othençrise noted, rates of PTPlB-catalyzed

dephosphorylation in the presence or absence of inhibitors were determined using FDP as

sub~trate"~ in assay buffer containhg 50 mM Bis-Tris, 2 mM EDTA, 5 mM DTT and 0.2

mg/cm3 BSA at 25 OC and pH 6.5. Stock solutions of inhibitors were prepared in assay b e e r

and found to be stable under these conditions indefinitely. Assays were carried out in 1 cm3

cuvettes with total volumes of 700 PL. Reactions were initiated by the addition of PTP t B

(final concentration 0.15 pg/cm3)- Rates were obtained by continuously monitoring the

production of FMP at 450 rn using a Varian Cary 1 spectrophotometer. The percentage

inhibition in the presence of inhibitors was performed in duplicate using FDP at Km

concentration (20 9M) with 500 pM inhibitor at pH 6.5.

2.2.2 SYNTHESES

General Procedure for the Preparation of 2,4-Disubstituted ~xazolones ."~

A mixture of the aldehyde, N-acetyl glycine (0.68 equiv.), anhydrous sodium acetate

(0.27 equiv.) and acetic anhydride (1.70 equiv.) was reflwed under argon for 2 h. The

mixture was then stirred at room temperature for an additional 12 h, at which time a yellow-

brown mass formed. The solidified mass was stirred with water (approximately 8-10 cm3

water/gram aldehyde) for 2 h. The resulting crude product was extracted with ethyl acetate,

washed thoroughly with water, dried (MgSOs) and concentrated in vacuo. Pure prodtict was

then obtained by recrystallization with ethyl acetatehexane.

2-Methyl-4-(4-trifluoromethylbenqlidene)~olone ( a ) . Yellow solid (41%). mp

96-98 OC; &(200 MHz; CDC13) 8.19 (2 H, d, J8.4, aryl), 7.70 (2 H, d, J9.2, aryl). 7.14 (1 H,

s, CH) and 2.44 (3 H, s, CH3)); 8~;(188 MHz; CDC13) (1 F, s, 50.0); 6,(100 MHz; CDC13) 167.6

(CO), 167.0, 142.7, 136.4, 132.1, 128.7, 125.7, 125.6, 125.3 (q, JCF 267.2, CF3), and 15.4

(CH3); d'z (EI) 255 (h.If, 100%), 227 (32), 185 (74), 166 ( 3 3 , 134 (36); Found: [Ml',

255.0508. CI 1 H8F3N02 requires m/r 255.0507.

2-Methyl4(4-trifluoromethoxybenzylidene)-5-osazolone (2.2b). Yellow solid (36%). rnp

101 -103 OC; &(200 MHz; CDC13) 8.14 (2 H, d, J 8.4, aryl), 7.28 (2 H, d, J 8.1, aryl), 7.1 1 (1

H, s, CH) and 2.42 (3 H, s, CH3)); &(188 MHz; CDC13) (1 F, s, 55.5); 6,(100 MHz; CDC13)

167.2 (CO), 166.8, 150.9, 133.7, 131.7, 129.0, 125.9 (q, J& 265.8, CF30), 120.8, and 15.5

(CH3); m/z (El) 271 (h.ir, 84%), 215 (15), 201 (100), 186 (17), 132 (30); Found: [Ml',

27 1 -0462. Cr iH8F3N03 requires m/z 27 1.0456.

2-Methyl-4-(4-methy~benzylidene)-5-0~azolone (2.2~). Yellow solid (38%). mp 125- 127

OC; &(200 MHz; CDCI3) 7.98 (2 H, d, J 8.1, aryl), 7.25 (2 H, d, J 8.0, aryl), 7.13 (1 H, s, CH)

and 2-41 (6 H, s, 2(CH3)); S,(100 MHz; CDC13) 167.7 (CO), 165.5. 141.8. 132.3, 131.9, 131.4.

130.7, l29.6,2 1.6 (CH3Ph), and 15.5 (CH,); m/r (EI) 20 1 (M', 49%), 173 (1 5), 13 1 (1 00)$ 103

(1 1); Found: FI]+, 201 .O797. Ci *Hi ,NO2 requires d z 201 .OWO.

2-MethyI-4-(4-methoxybenzylidene)-5arazolone (2.2d). Yellow solid (37%). mp 1 12- 1 14

OC: &(ZOO MHz; CDCI3) 8.07 (2 H, d, J9.1, aryl), 7.1 1 (1 H. s, CH). 6.96 (2 H, d, J 8.8. q l ) ,

3.87 (3 H, s, CH3) and 2.39 (3 H, s, CH3); S,(100 MHz; CDC13) 167.9 (CO), 164.9, 162.2,

134.2, 131.2, 129.7, 126.4, 114.5, 55.4 (CH30), and 15.4 (CH3); m/z (EI) 217 (M', 36%). 147

(100). 132 (36); Found: [Ml', 217.0746. CI IHI 1NO3 requires d z 217-0739.

Geaeral Procedure for the Reduction and Hydrolysis of ~xazolones."~

A mixture of PdClz (0.20 equiv.) and triethylarnine (1.16 equiv.) was stirred in

anhydrous ethanol (approximately 40-50 cm3/gram oxazolone), and a stream of hydrogen was

passed over for 5 min. Initially, the solution tunied black with the presence of white smoke

prior to becoming clear again. The desired oxazolone (1 equiv.) was then added and the

mixture was hydrogenated under an atmosphere of Hz (1 atm) ovemight. The solution turned

a pale yellow. The cataiyst was then filtered off and the filtrate concentrated in vacuo. The

resulting yellowish residue was then dissolved in CHC13 (approximately 25 ~ r n ~ / ~ r a r n of

oxazolone), washed with 10% HCI, water. dried (MgS04) and concentrated in vacuo. The

remaining yellow oil was dissolved in dioxane (approximately 5-10 ~ r n ~ / ~ r a r n oxazolone), an

equal volume amount of 10% KOH was added and the mixture was stirred at 30 OC for 5 h.

Upon evaporation, water (approximately 10 ~rn'/~ram oxazolone) was then added to the

resulting yellowish residue, which was then acidified to pH 2 with concentrated HCI. The

resulting precipitate was extracted with ethyl acetate, dried (MgS04) and concentrated in

vacuo to yield a pure white solid.

N-Acetyl-4-trifluoromethylphenylalanine (2.3a). White solid (50%). mp 183- 185 OC;

6"(200 MHz; CD30D) 7.59 (2 H, d, J 8.1, aryl), 7.43 (2 H, d, J 8.1, aryl), 4.7 1 (1 H, rn, CH),

3.04 (1 H, dd, J 5.1 & 13.9, PhCH), 2.94 (1 H, dd, J 5.2 & 14.0. PhCI-I), and 1.91 (3 H, s,

COCH3); &(188 MHz; CD30D) (1 F, s, 53.0); 8,(100 MHz; CD30D) 174.3 (COOH), 173.2

(COCH3), 143.2. 13 1.0, 126.3, 126.2,54.7 (CH), 38.3 (CHz), and 22.4 (COCH3); d z (ES) 274

(1 OO%), 2 15 (35%).

N-Acetyl-4-trifluorometho~phenylalanine (2.3b). White solid (42%). mp 146- 148 OC;

&(200 M W ; CD30D) 7.34 (2 H, d, J 8.8, aryl), 7.20 (2 H, d, J 8.0, aryl), 4.88 ( 1 H, m. CH),

3.24 (1 H, dd, J 5.2 & 14.0, PhCH), 2.99 (1 H, dd, J 5.1 & 14.0, PhCH), and 1.92 (3 H. s,

COCH3); 6,(188 MHz; CD30D) (1 F, s, 57.5); 6,(100 MHz; CD30D) 174.4 (COOH), 173.1

(COCH3), 149.6, 137. 8, 13 1.9, 121.8, 54.9 (CH), 37.9 (CH2), and 22.4 (COCH3); d' (ES)

29 1 (55%), 290 (100%)-

N-Acetyl-4-methylphenylalanine (2.3~). White solid (64%). mp 158-1 60 OC; aH(200 MHz;

CD30D) 7.10 (4 H, s, aryl), 4.62 (1 H, m, CH), 3.16 (1 H, dd, J 5.2 & 14.0, PhCH), 2.89 (1 H,

dd, J 9.2 & 14.0, PhCH), 2.30 (3 H, s, CH3Ph) and 1.91 (3 H, s, COCH3); 8,(100 MHz;

CDJOD) 174.8 (COOH), 173.1 (COCH3), 137.5, 135.3, 130.2, 130.1, 55.2 (CH), 38.2 (CH?),

22.5 (COCH3), and 21.1 (CH3Ph); m/z (ES) 220 (100%), 178 (35%).

N-Acetyl-4-methorypbenyIaIanine (23d). White solid (63%). mp 143- 145 OC; &(200

MHz; CD30D) 7.14 (2 H, d, J8.8, aryl), 6.84 (2 H, d, J8.8, aryl), 4.61 (1 H, m, CH), 3.77 (3

H, S. CH30Ph), 3.14 (1 H, dd, J5.2 & 13.9, PhCH), 2.88 (1 H, dd, J 5.2 & 14.1, PhCH), and

1-91 (3 H, s, COCH3); 8,(100 MHz; CD30D) 174.8 (COOH), 173.1 (COCH3), 160.2. 13 1.3.

130.5, 1 15.1, 55.9 (CH30Ph), 55.32 (CH), 37.79(CH2), and 22.43 (COCH3); d z (ES) 236

( 100%). 194 (30%).

General Procedure for L-Amino Acid ~eso lut ion . '~

The desired acetylated DL-phenylalanine derivative was suspended in distilled water

(approximately 1 cm3/0. 15 mrnol). Cobaltous acetate (0.0 1 equiv.) was added followed by the

dropwise addition of ammonium hydroxide (3 N), with stimng, until a pH of 7.5 was

achieved. Dry Acylase 1 powder (approximately 0.3 mg enzyrne/gram acetylated arnino acid)

was then added with gentle stimng to complete solution. The digest was then incubated in a

water bath (37 OC) for 18 h. The digest mixture, cooled to room temperature. was treated with

glacial acetic acid to pH 5 . A spatula tip of decolorizing charcoal was then added and the

mixture stirred for 1 h. Following suction filtration (Whatman #4 filter paper) and a water

wash of the residue, the colorless filtrate was treated with a few drops of I -hexanol and then

concentrated in vacuo to approxirnately I cm3. An 80% ethanol solution was then added,

enough to dissolve the white crystalline residue, and the solution was chilled for 18 h at 5 OC.

The resulting crystals were filtered and washed with cold ethanol to yield the desired pure L-

phenylalanine derivative.

(L)-4-Trinuorornethylpbeaylalanine (2.1a). White solid (50%). mp 2 10 OC (decomp.);

&(IO0 MHz; D20) 7.51 (2 H, d, 58.4, aryl), 7.26 (2 H, d, J8.4, aryl), 3.37 (1 H, m, CH), 2.89

(1 H. dd. J 5.5 & 13.5, PhCH) and 2.75 (1 H, dd, J 7.3 & 13.5, PhCH); 6 ~ ( l 8 8 MHz; DzO) (1

F. s. 52.9); d z (ES) 232 (100%); HPLC & = 5.19.

(L)-4-TrifluoromethoryphenylaIanine (2.1 b). White solid (5 1 %). mp 200 OC (decomp.);

6 ~ ( 2 0 0 MHz; D20) 7.14 (4 H, m, aryl), 3.33 (1 H, m, CH), 2.83 (1 H, dd, J5.5 & 13.6, PhCH)

and 2.68 (1 H. dd, J 7.3 & 13.6, PhCH); SF(188 MHz; Da) (1 F. s, 57.8); nv'z (ES) 248

( 100%); HPLC R, = 5.26.

(L)-4-Methylphenylalanine (2.1~). White solid (62%). mp 2 10 OC (decomp.); 6~(200 MHz;

DzO) 7.05 (4 H, m, aryl), 3.32 (1 H, m, CH), 2.81 (1 H, dd, J 5 . 5 & 13.5. PhCH). 2.63 (1 H,

dd, J 7.3 & 13.6, PhCm and 2.17 (3 H, s, CH3); m/z (ES) 178 (1 00%); HPLC R, = 6.1.

(L)-4-Methoxyphenylalanine (2.ld). White solid (67%). mp 190 OC (decomp.); 6&00

MHz; DzO) 7.05 (2 H, d, J8.4, q l ) , 6.81 (2 H, d, J8.4, aryl), 3.67 (3 H, s, CH30), 3.30 (1 H,

m, CH), 2.77 (1 H, dd, J5.5 & 13.7, PhCH) and 2.62 (1 H, dd, J7 .3 & 13.6, PhCH); m/,l (ES)

194 (1 00%); HPLC R, = 5.8.

General Procedure for the Formation of Fmoc-Protected Amino ~ c i d s . " ~

To a stirred solution of the appropriate L-amino acid (1 equiv.) and sodium

bicarbonate (1 equiv.), dissolved in a 5050 solution of water:acetone (1 cm3/10 mg of L-

arnino acid), was added 9-fluorenylmethyl succinimidyl carbonate (2 equiv.). ï h e reaction

was stirred ovemight. The mixture was then acidified to pH 2 with concentrated HCI and the

acetone removed under vacuo. The cmde product was dissolved in CHC13 and washed

subsequently with 0.1 N HCI and water. The organic phase was dried (MgS04) and

concentrated in vacuo. Silica gel chromatography (MeOH:CHCI3, I:99) yielded the pure

product as a white solid.

N-(9-Fluorenylmethyiorrycarbonyl)-(l)-4-trifluoromethylphenylalanine (2.4a). White

solid (79%). 6"(200 MHz; CDC13) 7.72 (2 H, d, J 7.7, q l ) , 7.55 (2 H, d. J 7.0, aryl), 7.46-

7.26 (4 H, m, aryl), 7.10 (2 H, br s, aryl), 5.24 (1 H, br d, NH), 4.66 (1 H, br m, CHCH20),

4.53-4.39 (2 H, r n CHzO), 4.19 (1 H, br t, CH) and 3.19 (2 H, m. CH2Ph); SF(188 MHz;

CDCI3) (1 F, s, 52.9).

N-(9-Fluorenylmethylory~arbonyl)-(L)-4-trifl~0r0meth0xyphenylaanine (2.4b). White

solid (88%). ZjH(200 MHz; CDCl3) 7.79 (2 H, d, J 7.7, aryl), 7.60-7.22 (10 H, m, aryl). 5.21 (1

H, br d, NH), 4.63 (1 H, br m, CHCH20), 4.58-4.39 (2 H, m CH20), 4.20 (1 H. br t, CH) and

3.24 (2 H, m, CH2Ph); 6~(188 MHz; CDC13) (1 F, s, 57.7).

N-(9-Fluorenylmethy~oxy~arbonyl)~)-4-methyIphenyIaIanine (2.4~). White solid (86%).

6H(200 MHz; CDC13) 7.77 (2 H, d, 5 7.0, aryl), 7.54 (2 H, bm, aryl), 7.44-7.27 (4 H. ml aryl).

7.13-7.02 (4 H, m, aryl), 5.24 ( I H, br d, NH), 4.69 (1 H, br m, CNCHtO), 4.50-4.36 (2 H, m

CH20), 4.21 (1 H, br t, CH), 3.15 (2 H, br t, CH2Ph) and 2.32 (3 H, s, CH3).

N-(9-Fluorenylmethyloxycarbonyl)-~)-4-methoxyphenyIaIanine (2.4d). White solid

(94%). &(200 MHz; CDC13) 7.76 (2 H, d, J 7.7, aryl), 7.53 (2 H, br s, q l ) , 7.43-7.25 (4 H,

m, aryl), 7.06 ( 2 H, d, J8.0, aryl), 6.82 (2 H, d,J8.0, aryl), 5.28 (1 H, br d, NH), 4.68 (1 H, br

d, J 2 1.6, CHCH20), 4.50-4.16 (3 H, m CHzO & CH), 3.75 (3 H, s, 0CH3) and 3.10 (2 H, br t,

CH2Ph).

Solid-Phase Peptide Syntheses.

The hexapeptides containing the sequence Asp-Ala-Asp-Glu-Xxx-Met were

synthesized via the Memfield solid phase where Xu = 2.1a-d with

appropriate N-termini protection.

Coupling of the First Amino Acid to the Resin.

Wang Resin 1% DVB (100 mg, 0.099 m o l ) was transferred to the Merrifield flask

containing DMF (4 cm3). The flask was s h a h for 5 min and the DMF removed. A mixture

of 15 mol % of the total molar quantity of amino acid of DMAP (7 mg, 0.0573 rnrnol) and 4

equiv. of DCC per equiv. of resin (82 mg, 0.396 mmol), in a 10 cm3 beaker. was dissolved in

DMF (1 cm3) and transferred to the Merrifield flask containing the resin. The beaker was

rinsed out with DMF (1 cm3) and added to the flask. In a 10 cm3 beaker. Fmoc-Met-OH (148

mg. 0.396 mmol) was dissolved in DMF (1 cm3) and added to the flask. The beaker was

rinsed out with DMF (1 cm3), added to the flask, and shaken for 4 h. The DMF was removed

and the resin washed successively with DMF (2 x 5 cm3) and CHzClt (5 x 5 cm3), shaking the

flask for 2 min during each wash.

Capping and Deprotection.

After removing any CH2C12 remaining from the washes DMF (1 cm3) was added. To

this acetic anhydride (37 a, 0.396 mol) and DMAP (7 mg, 0.0573 mmol) were added, and

the flask shaken for 1 h. The resin was then washed with DMF (5 x 5 cm3). shaking the flask

for 1 min during each wash. A 20% solution of piperidine in DMF (5 cm3) was added and the

flask shaken for 3 min. The piperidine solution was removed, another 5 cm3 was added, and

the flask shaken for an additionai 7 min. The piperidine solution was removed and the resin

washed with DMF (5 x 5 cm3) as before.

Subsequent Couplings.

In a 10 cm3 beaker, the desired Fmoc-Phe derivative (2 equiv.), HATü (75.3 mg,

0.198 mrnol) and DIPEA (68.5 PL, 0.396 mrnol) were dissolved in DMF (3 cm3). This was

transferred to the Merrifield flask. The beaker was ~ s e d out with DMF (1 cm3), added to the

flask and dlowed to shake for 1 h. The resin was then washed and deprotected as before.

Subsequent couplings of Fmoc-Glu(0tBu)-OH (85 mg, 0.1998 mmol), Fmoc-Asp(0tBu)-OH

(82 mg. 0.1993 rnmol), Fmoc-Ma-OH (62 mg, 0.1992 mmol) and Fmoc-Asp(0tBu)-OH (82

mg. 0.1 99 3 mmo 1), respective1 y, were performed as above.

CIeavage from the Resiir.

After the last deprotection and DMF wash, the resin was washed with CH2CIi (5 x 5

cm3). A solution of 50% TFA/CH2C12 (5 cm3) was then added to the Merrifield flask and

shaken for 4 h. The resin was pink in color. The clear filtrate was recovered and concentrated

under reduced pressure. The hexapeptides were then purified by preparative HPLC.

2.5a. D-A-D-E-(2.1a)-M. White solid. mp 185 OC (decomp.); SF(188 MHz; D20) 16.5: HPLC

R, = 8.1 ; m/z (ES) 795.01 (100%).

2.5b. D-A-D-E-(2.1 b)-M. White solid. mp 185 O C (decomp.); &(I 88 MHz D20) 20.7; HPLC

R, = 8.4; m/z (ES) 8 1 1 .O3 (1 00%).

2.5~. D-A-D-E-(2.1~)-M. White solid. mp 185 OC (decomp.); KPLC R, = 7.0; d z (ES) 741 -06

(1 00%).

2.5d. D-A-D-E-(2.ld)-M. White solid- mp 185 OC (decomp.); HPLC Rt = 6.1; m/z (ES)

757.08 (1 00%).

2.3 RESULTS AND DISCUSSION

2.3.1 SYNTHESIS OF UNNATURAL PHENYLANALINE DERIVATIVES

In addition to the fluoro derivatives 2.la and 2.lb we decided to prepare their non-

fluoro analogues 2.lc and 2.ld since this would allow us to detennine if the fluorines were

necessary for inhibition. We wished to prepare these amino acids in their enantiomerically

pure L-form since it is known that PTP 1 B has a distinct preference for peptides bearïng L-

amino a ~ i d s . * ~ Although there are many methods for preparing enantiomerically pure amino

acids."* we wanted to use a method that would enable us to start with readily available and

inexpensive starting materiais and reagents. Consequently, we chose the azlactone

mehod. 1 1 1.1 15.1 16 in this method, a 2,4-disubstituted 5-oxazolone (also known as an azlactone)

is prepared by condensation of 0x0 (aidehyde or ketone) compounds with N-benzoyl and/or

N-acyl derivatives of glycine. The oxazolones are then readily converted into the desired

amino acids by hydrogenation and hydrolysis to yieid racemic N-acyï amino acids.

Resolution of the racemic product yields the desired L- or D-amino acids. 1 1 1.1 12

The initial condensation of N-acetyl glycine with comrnercially available aldehydes

2.6a-d (Scheme 2.1) resulted in the formation of the desired oxazolones 2.2a-d in moderate

yields (3641%). Reduction of the oxazolones with hydrogen in ethano1 in the presence of a

palladium cataiyst and triethylamine followed by alkaline hydrolysis yielded the racemic N-

acyl amino acids 23a-d in moderate to good yields (42-64%). The enantiomerically pure

amino acids were obtained by enantioseiective hydrolysis of the N-acyl group using the

enzyme Aspergillus acylase I . " ~ This acylase is known to accept only L-amino acids as

substrates. The L-amino acids were obtained in good yields (50-67% based on the L-

enantiomer only). Amino acids 2.la-d were analyzed for enantiomeric purity using a chiral

HPLC column. Only a single peak was evident in ail of the chromatograrns indicating that the

amino acids were obtained in hi& enantiomeric purity. in preparation for their incorporation

into the hexapeptides, the L-amino acids were reacted with 9-fluorenylmethyl succinimidyl

carbonate yielding the desired Fmoc-protected phenylalanine derivatives 2.4a-d in exceIlent

yields (79-94%; Scheme 2.1).

N-acetyl glycine, sodium acetate,

sCHO acetic anhydride *

reflux 2h

R& "& acylase 1 (Aspergillus)

2.1 a-d

9-fluorenylrnethyl succinimidyl carbonate, sodium bicarbonate, H201acetone

1 . H2, PdCI2, NEt3, EtOH, 12 h

2. aq. KOH, dioxane

2.4a-d

Scheme 2.1. Synthetic route for unnaturd L-phenylalanine derivatives 2.4a-d.

2.3.2 HEXAPEPTIDE SYNTIIESIS

With the Fmoc-protected L-phenylalanine derivatives in hand, the final step in the

syntheses was to incorporate them into the desired hexapeptide. We chose the D-A-D-E-X-M

sequence since the analogous FzPrnp-bearing peptide, D-A-D-E-(F2Pmp)-M, is one of the best

PTP 1 B inhibitors ever made with a Ki of only 14 n~. ' ' ' Standard solid-phase peptide

synthesis techniques were employed using the Wang resin as the solid support. Thus, Fmoc-

protected methionine was attached to the Wang resin using DCC as coupling agent, in the

presence of catalytic amounts of DMAP. 114.1 17 Next, the resin was "capped to make sure that

there are no unfùnctionalized hydroxy groups on the resin. In other words, any hc t ional

groups on the resin that did not react with the first amino acid were acetylated with acetic

anhydride. The Fmoc-protecting group was removed by treatment with 20% piperidine in

DMF to give the resin-bound methionine with a fiee a-amino group. 1 15.1 18 After the resin was

washed, the next Fmoc-protected amino acid, in this case 2.4a-d (2 equiv.). was coupled to the

resin-bound methionine using O-(7-azabenzotriazol- 1 -y[)-N, N, N'. N'-tetramethyluronium

hexafluorophosphate (HATU, 2 equiv.) as coupling agent, in the presence of

diisopropylethylamine (4 equiv.) in DMF. Excess reagent was washed away from the resin

afier completion of coupling. Subsequent deprotections and couplings were performed in a

similar rnanner until the desired peptide was completely assembled. At this point, the peptide

was removed fiom the resin by treatment with 50% TFA in dichloromethane (Scheme 2.2).

Hexapeptides 2.5a-d were then purified by preparative reverse-phase HPLC. Reverse-phase

analytical HPLC analysis of the purified peptides reveaied only a single peak in the

chromatogram (Figures 2.1 and 2.2). Electrospray mass spectrometry was also used to

confirm the purity and identity of the hexapeptides (Figures 2.3 and 2.4).

Fmoc-Met-OH + Wang resin DCC DMAP

Fmoc-Met-COO-Wang resin

DMAP, 11:: anhydride

NH2-Met-COO-Wang resin .r 20% piperidine/DMF Frnoc-Met-COO-Wang resin

Coupling of Fmoc-amino acids with HATU, DIPEA, in DMF followed by deprotection with piperidine *

D-A-0-E-X-M-COO-Wang resin 50% TFA/CH2CI2 D-A-0-E-X-M

Scheme 2.2. Solid-phase synthesis of hexapeptides 2.5a-d.

2.3.3 PTPlB INHIBITION STUDIES

We initiated the inhibition studies using 500 pM of hexapeptides 2.5a-d. fluorescein

diphosphate (FDP) as s~bs t ra te"~ at Km concentration (20 pM) and PTPIB. The results are

given in Table 2.2. Al1 of the hexapeptides were found to be extremely poor inhibitors of

PTP 1 B. The fluorinated peptides exhibited oniy 10% inhibition while their non-fluorinated

Table 2.2. Percent inhibition of PTP 1 B with 500 pM of hexapeptides 2.5a-d.

Percent inhibition Peptide with 500 FM peptide

2.58 10+2 2 . 5 ~ 2 + 2 2.5b 10 I 2 2.5d 2 + 2

Figure 2.1. Analytical HPLC chromatograms of hexapeptides 2.5a and 2.5b.

Figure 2.2. AnaIytical HPLC chromatograrns of hexapeptides 2 . 5 ~ and 2.56.

Figure 2.3. ESMS analysis of hexapeptides 2.5a and 2.5b.

Figure 2.4. ESMS analysis of hexapeptides 2 . 5 ~ and 2.5d.

andogues displayed almost zero inhibition. Due to the low potency of these compounds it

was impractical to determine their ICso's as this would have required inordinate amounts of

peptide. We estimate that the IC50's of fluoro peptides 2.5a and 2.5b is probably in the 5-10

mM range and so is not that much different fiom pTyr, which is around 5 mM. It is

interesting to speculate why these peptides bind so poorly to PTPlB. It is not unrealistic to

expect that peptides 2.5a and/or 2.5b would have shown at least moderate inhibition of PTP 1 B

considering the tremendous contributions the D-A-D-E-X-M sequence and the fluorines make

to ligand binding as discussed earlier (see Table 2.1). It is possible that the presence of the

extra fluorine in these peptides affects binding. However, we believe that this is unlikely since

PTP 1 B can tolerate groups of varying size and shape at the para-position of tyrosine such as a

C F ~ P O ~ ' and a CF(COO-)z group. It is also possible that the first event in the binding of

peptidyl substrates to PTP 1 B is recognition of the phosphate group by the consewed arginine

of the PTP-loop. This may trigger a confortnational change that results in the proper

alignment of residues in the enzyme to form optimal interactions with other residues in the

peptide substrate. For example, salt bridges between Glu(P-1) and Asp(P-2) of the substrate

and Ara7 of the enzyme, and H-bonds between the N-H backbone of the peptide substrate and

Aspas of the enzyme.3s Although the crystal structures of PTPlB in the absence of bound

ligands have been obtained, data for only PTPlB-ligand complexes are available and so we

were unable to examine this hypothesis in more detail. 35.4 1.58

2.4 CONCLUSIONS

Although the amino acid sequence flanking pTyr and/or the introduction of fluorines at

the phosphonate a-methylene position are important structural features in determining PTP

substrate specificity, our studies dictate that without the presence of a phosphate moiety or

phosphate mimetic, the resulting peptide will not bind very well to the phosphatase. These

results are consistent with Dixon and coworkers' observation that peptides that do not bear a

phosphotyrosine residue do not bind to PTPIB.~* The precise contribution of the phosphate

group to substrate bindîng is still unknown. These results aiso suggest that the presence of a

phosphate mimetic was going to be an important component in the design of our PTPlB

inhibitors.

CHAPTER 3

SYNTHESIS AND EVALUATION OF NON-PEPTIDYL INHIBITORS OF PTP18 BEARING THE DlFLUOROMETHYLENEPHOSPHOMC ACID GROUP

3.1 iNTRODUCTION

3.1.1 THE= DFMP MOIETY

From our studies presented in Chapter 2, it was apparent that the presence of a

phosphate group is important for tight binding of substrates to PTP 1 B. Thus, it is likely that in

order to obtain potent inhibitors of PTPIB, whether based on peptidyl structures or small

molecules, a non-hydrolyzable phosphomimetic wodd be an important part of the inhibitor's

design.

As discussed in Chapter 1, a number of moieties have been examined as phosphate

rnimetics for obtaining PTP inhibitors such as methylenephosphonyl,8' sulfate.83

~ h i o ~ h o s ~ h a t e , ~ ~ r n a l ~ n ~ l , ~ ~ fluoromalonyl, '*-" a-hydroxyphosphonate~ ' OS and

81.107 difluoromethylphosphonyl groups. To date, the most effective of these groups. in terms

of obtaining PTP inhibitors, is the difluoromethylenephosphonic acid (DFMP) moiety (see

Chapter 1. section 1.4.2.4). Consequently, we decided to focus our initial efforts on

constnicting inhibitors bearing the DFMP moiety. The DFMP group (as well as the other

phosphate mimetics) has k e n used mainly for obtaining highly potent peptide-based

inhibitors (see Chapter 1, section 1 -4.1). However. since peptide-based inhibitors generall y

exhibit poor bioavailability, we wished to concentrate our efforts on non-peptidyl structures.

At the time our work began in this area, only a handful of reports had appeared describing

non-peptidyl inhibitors of PTP's and only two of these reports described non-peptidyl

inhibitors bearing the DFMP group (see Chapter 1, section 1.4.2.4). 58.1 O7 These compounds

were rdatively simple naphthyl DFMP derivatives (1.18-1.20) exhibiting modest affhities

(K,'s of 255, Z 79 and 93 PM, respectively) for PTPIB. Despite not k i n g highly selective and

approximately 1000-fold less potent than the best peptidyl inhibitors bearing the DFMP

gro~p,58.107 these results are significant in that they demonstrate the potential of non-peptidyl.

DFMP-bearing compounds as inhibitors of PTP's.

As a starting point for the development of non-peptidyl inhibitors of PTPlB. we

searched the existing literature regarding studies using non-peptidyl substrates. Montserat et

al. and Zhang have studied the active site specificity of rat PTPl (the rat homologue of human

PTf 1 B) with a number of non-peptidyl substrates. 119.120 The catalytic domain of this enzyme

has 97% sequence homology to the corresponding residues in hurnan PTPIB.~' Typically,

aromatic phosphates are found to be significantly better substrates (higher k&s and lower

Km's) than aliphatic phosphates. However, not al1 aromatic derivatives are created equal. P-

Naphthyl phosphate displays a lower Km (approximately 2 to 9-fold, depending on the pH)

than phenyl phosphate. Interestingly, the added presence of certain substituents on the phenyl

ring produced derivatives that displayed considerably lower Km levels than that of P-naphthyi

phosphate. 119.120 In general, substituents on the phenyl ring ortho and to a lesser extent meta to

the phosphate group have higher Km's compared to their para-substituted counterparts. This

has been attributed to stenc hindrance.'19 in addition, the substrate's af3nity towards PTP's

ofien significantly increased with the presence of negatively charged substituents remote from

the phosphate group.

In light of the above substrate and inhibitor studies, we decided to focus our efforts on

the synthesis of DFMP-bearing aryl derivatives. Our resolve was fürther strengthened by

reports describing small molecule aryl-bearing phosphonates as excellent inhibitors of

osteoclastic acid phosphatase ' 21 and human prostatic acid phosphatase. 122.1u These enzymes

are also known to have an affinity for phosphotyrosine. Consequently, we reasoned that an

appropriately functiondized aryl DFMP derivative could potentially exhibit potent inhibition

of PTP 1 B. Knowing that ortho and to a lesser extent meta substitution interferes with ligand

binding, 119.120 we focussed mainly upon para-substituted phenyl DFMP denvatives (3.1).

X = H, ester, ether, bromo, keto, phenyl, nitro

3.1.2 SYNTHESlS OF ARYL DFMPs

Central to Our approach to obtaining PTPlB inhibitors was an efficient method for

preparing the desired aryl DFMPs. There are currently three methods for preparing aryl

DFMPs: (1) diethylarninosulhu. trifluoride (DAST) fluorination of a -ke t~~hos~honates .~ '

(2) cross-couplings of aryl iodides to BrXCF2PO(OEt)2 where X is cdi" or ~n." ' in the

presence of stoichiometric quantities of C U C ~ ' ~ ~ or C U B ~ ' ~ ~ and (3) electrophilic fluorination

of benzylphosphonates.126 In al1 of these procedures, an a-difluorinated phosphonate ester is

initially prepared and then converted into the desired aryl DFMPs.

In the DAST procedure (Scheme 3.1),87 aryl DFMP compounds are prepared starting

from acid chlondes which are converted into a-ketophosphonates via an Arbuzov reaction

with a phosphite. To obtain the protected aryl DFMP compounds, the a-ketophosphonates

are reacted in a neat solution of excess DAST. Although this procedure has been used by

other groups for the preparation of aryl DFMPs, there are several serious drawbacks. First.

the reaction requires at least 5 equivalents of expensive DAST and thus is not very

econornical. Second, the reaction has been known to explode upon s c a l e - ~ ~ . " ~ Therefore.

we did not think that this would be a suitable method for prepax-ing this class of compounds.

Scheme 3.1. Preparation of aryl DFMPs via DAST fluorination of a-ketophosphonates.

In the cross-coupling approach (Scheme 3.2) zinc or cadmium dust is reacted with

diethyI bromodifluoromethylphosphonate, which is then transmetalated with CuBr or CuC1 to

form the organocopper species 3.4. Treatment with iodoarenes gives the desired aryl DFMPs

in modest to good yields. Although this procedure has been shown to be effective for

producing aryl DFMPs bearing a variety of substituents 124.'25 it requires the use of excess

diethyl bromodifluoromethylphosphonate, which is expensive and sometimes difficult to

remove after the synthesis.

1 . ZnJDMFirt - (Et0)2P(0)CF2CuZnBr2 Arllrt DMF

(Et0)2P(0)CF2Ar 2. CuBr

Scheme 3.2. Preparation of aryl DFMPs via the cross-coupling approach.

The third method for preparing aryl DFMPs, electrophilic fluorination of a-carbanions

of benzylic phosphonates, was developed in the Taylor gro~p.'26 This procedure was based

upon the work of Differding et al. who reported the preparation of mono- and

difluoroalkylphosphonates (non-benzylic) by electrophilic fluorination of a-carbanions with

N-fluorobenzenesulfonimide (NFS~).'~' This involved reacting alkyl phosphonates with one

equivalent of a strong base followed by reaction with one equivalent of NFSi to form the a-

monofluorophosphonate, which was isolated and purified. Repeating this procedure gave the

difluoro compounds in low to modest overall yields (1 1-36%; Scheme 3.3).

O 1. KDA (1 -3-2.0 eq.), THF, O -78 OC to -90 O C , 1 h repeat

O R,, P(OEt)2 P(oE~)~ - R P(oE~),

2. NFSi (1 -3-2.5 eq.), f HF: R~ F

x -78 OC to rt F F

Scheme 3.3. Electrophilic fluorination of alkyl phosphonates using NFSi (R= alkyl).

Taylor and coworkers found that benzylic a,a-difluorophosphonates could be

prepared in modest to good yields in a single step by reacting the phosphonates with 2.2

equivalents of NaHMDS at - 78 OC followed by the addition of 2.5 equivalents of NFSi

(Scheme 3.4).126 Although the scope of this procedure was not examined in detail. ive

reasoned that it may be used as a generai approach for the preparation of a wide variety of

aryl DFMPs. Benzylic phosphonates can be readily prepared by an Arbuzov reaction between

readily attainable benzylic haiides and phosphites. NFSi is less expensive than either DAST

or diethyl bromodifluoromethylphosphonate and purification should be readily achieved by

traditional silica gel chromatography.

-

i ; ( ~ ~ t h I - ~ . ~ ~ ~ . N ~ H M D s , P ( O E ~ ~

b THF, -78 OC

2. 2.5 eq. NFSÎ, THF,

X -78 OC to 20 OC X 3

X = H, OMe, NOz, Br

Scheme 3.4. Preparation of benzylic a,a-difluorophosphonate esters by electrophilic fluorination.

The objectives of the work presented in this chapter are two-fold. First, to examine the

scope of the electrophilic fluorination procedure for preparing aryl DFMPs. Second. to

examine the aryl DFMP compounds prepared using this procedure as inhibitors of PTP 1 B.

3.2 EXPERIMENTAL

For details conceming general materials and instrumentation used in the syntheses

discussed below see Chapter 2 (section 2.2.1) with the following additions and/or changes:

Chemicals. Tetrahydrofiuan (THF) and diethyl ether (ether) were distilled from

sodiumibenzophenone ketyl under argon. Dichioromethane, benzene and toluene were

distilled from calcium hyâride under argon. PP2A was purchased fiom Upstate

Biotechnology.

Instrumentation. "P-Nh4R spectra were recorded on a Varian 200-Gemini NMR. Al1 3 ' ~ -

NMR spectra were proton decoupled and chemical shifts (6) are reported in parts per niillion

relative to 85% phosphonc acid (extemal). Fast atom bombardment (FAB; negative ion) mass

spectra were obtained on a Micromass 70-S-20 m a s spectrometer.

3.2.1 SYNTHESES

Preparation of Betuyl Bromides.

If not cornmercially available, benzylic bromide precursors were prepared by literature

procedures, 128.129 using NBS/benzoyl peroxide in CC4 or N B S h in benzene. The benzyl

bron-iide precursors to compounds 3.11 and 3.29 were prepared as described below.

4-Bromomethylbenzoic acid benzyl ester (precursor to 3.11). Freshiy distilled benzyl

alco ho1 (0.50 cm3, 4.67 mmol), N,N-dimethyl-4-aminopyridine (0.057 g, 4.67 mmol) and

dicyclohexylcarbodiimide (DCC, 0.96 g, 4.67 mrnol) were added to a stirring solution of 4-

brornornethyl benzoic acid (1.0 g, 4.67 mmol) dissolved in anhydrous CH2C12 (25 cm3).

Formation of dicyclohexylurea as a precipitate was evident ahost immediately after the

addition of DCC. Reaction was stirred for 5 min, filtered and concentrated by rotary

evaporation. The crude product was purified by flash chromatography (silica, hexane:EtOAc.

5 2 . Rr = 0.6) to give the ester as a white solid (1.12 g, 74%). mp 60-62 OC; &(200 MHz;

CDC13) 8.06 (2 H, d, J8.1, aryl), 7.43 (7 H, m, aryl), 5.38 (2 H, s, 0CH2) and 4.50(2 H, S.

BrCH?); 6,(100 MHz; CDC13) 165.7 (CO). 142.6, 135.8, 130.1, 130.0, 128.9. 128.5, 128.2.

118.1, 66.7 (OCH*) and 32.1 (BrCH2); m / . (El) 306 (M'{8 '~r}, 19%), 199 (M'{"B~J -

0CH2Ph. 70), 91 (M+{*'B~} - 00CPhCH2Br, 100), 304 (M'{"B~), 19%), 197 ( M ' { 7 9 ~ r ) -

0CH2Ph. 69), 91 (M+{79~r} - 00CPhCH2Br, 100); Found [Ml'. 306.0074 & 304.0097.

C SH jBrOZ requires 306.0078 & 304.0099.

(4-Bromomethylpheny1)acetic acid benzyl ester (precursor to 3.29). This was prepared in

a manner similar to that described for 4-bromomethylbenzoic acid benzyl ester starting from

commercially available (4-bromomethylpheny1)acetic acid. Purification using column

chromatography (silica, hexane:EtOAc, 4: 1, Rf = 0.6) yielded pure 3.29 as a white soiid (88

%). mp 59-61 OC; 6"(200 MHz; CDCI3) 7.33 (9 H, m, aryl), 5.14 (2 H, s, 0CH2), 4.49 (2 H. s,

B K & ) and 3.67 (2 H, s, CH2): &(IO0 MHz; CDC13) 170.9 (CO)? 136.6, 135.7, 134.1. 129.7.

129.3. 128.5. 128.2, 128.1, 66.6 (OCH2), 40.9 (CH2) and 33.1 (BrCH2); d z (EI) 320

(wvffs '~r ) , 4%) 239 (w - 8'"9~r, 76), 185 (M+{81~r} - OOCCHzPh, 24), 91 (MT -

00CCH2PhCH2Br, IOO), 3 18 (h.if{79~r), 4%), 183 (M+{79~r) - OOCCHzPh. 26): Found

[MI-. 320.0247 & 3 18.0270. C16HL5Ba2 requires 320.0235 & 3 18.0275.

Preparation of Phosphonates.

Phosphonate 3.5 was purchased fiom Aldrich Chemical Co. and phosphonate 3.12 was

prepared by a literature procedure."O Unless otherwise stated. al1 other phosphonates were

prepared via an Arbuzov reaction with trimethyl or triethyl phosphite using the general

procedure described below.

General Procedure for the Preparation of Phosphonates.

The benzylic halide was 2dded to trimethyl or triethyl phosphite (10-15 equiv.) or to a

solution of trimethyl or triethyl phosphite (1 0-1 5 equiv.) in an equal volume of benzene and

heated to reflux for 6-18 hours. The reaction was cooled and then benzene andfor unreacted

phosphite and dirnethyl methylphosphonate or diethyl ethylphosphonate (formed during the

reaction) were removed by vacuum distillation. The phosphonates were obtained in pure forrn

by subjecting the residue to silica gel ~~omatography or by recrystallization.

Dimethyl benzylphosphonate (3.6).13' Column chromatography (silica. MeOH:CHC13, 298 .

Rf = 0.4) yielded pure 3.6 as a colorless oil (89%). &(200 MHz; CDC13) 7.30 (5 H, m, a*),

3 -69 (6 H, d, JHP 10.6, 2(CH3)) and 3.17 (2 H, d, J H ~ 2 1.6, CH2P); 6p(80 MHz; CDC13) 26.7 (1

P. s ) ; 6,(100 MHz; CDC13) 13 1.1 (d), 129.4 (d), 128.3 (d), 126.7 (d), 52.5 (d, JCp 6.6, 2(CH3))

and 32.6 (d, J'p 137.6, CH2); m/z (EI) 200 (w, 45%), 91 @If - PO(OMe)2, 100); Found

[Ml'. 200.0607. C9HI3O3P requires d z 200.0602.

Dimethyl 4-nitrobenzylphosphonate (3.7). Recrystallization fiom carbon tetrachloride

yielded pure 3.7 as a pale yellow solid (79%). mp 64-66 OC; 6"(200 MHz; CDC13) 8.14 (2 H.

d, J8.5, aryl), 7.44 (2 H, dd, J8 .8 & 2.2, aryl), 3.71 (6 H, d, JHp 11.0, 2(CH3)) and 3.25 (2 H,

d, JHP 22.3, CH2P); Sp(80 MHz; CDC13) 24.5 (1 P, s); 6,(100 MHz; CDC13) 147.1. 139.4 (d).

130.5 (d), 123.0, 52.8 (d, JCp 5.5. 2(CH3)) and 32.9 (d. JCp 137.2. CH2P); ndz (El) 245 (M-.

25%), 228 (M.+ - OH, 100); Found ml', 245.0449. C9H12NOjP requires d'.. 245.0453.

Diethyl 4-nitrobenzylphosphonate (3.8). Column chromatography (silica, EtOAc. Rf = 0.4)

yielded pure 3.8 as a yellow oil (94%). &(200 MHz; CDC13) 8.14 (2 H. d, J 8.4, aryl). 7.41 (2

H, dd, J 8 -6 & 2.4, aryl), 4.03 (4 H, m. 2(OCH2)), 3 -22 (2 H, d, J H ~ 22.3, CH2P) and 1 -23 (6 H.

t. J 7.2, 2(CH3)); 6p(80 MHz; CDC13) 21.9 (1 P, s); 6,(100 MHz; CDC13) 146.8, 139.7 (d).

130.5 (d), 123.5, 62.2 (d. J c p 7.3. 2(OCH2)), 33.7 (d, JCp 137.6, CH2P) and 16.1 (d. J C p 5.8.

2(CH;)); m/r (EI) 274 (MW. 100%), 137 (MW - PO(0Et)z. 54); Found [Ml-. 273.0766.

C H ioN05P requires m/z 273.0766.

Dimethyl 4-bromobenzylphosphonate (3.9).132 Column chromatography (silica. EtOAc. Rf

= 0.3) yielded pure 3.9 as a white solid (98%). mp 56-58 OC; 6~(200 MHz; CDC13) 7.43 (2 H.

d, J 8.8, q l ) , 7.16 (2 H, dd, J 8.5 & 2.6, q l ) , 3-67 (6 H, d, J H ~ 10.6. 2(CH3)) and 3.10 (2 H,

d, JHP 2 1.9, CH2P); 6p(80 MHz; CDC13) 25.7 (1 P, s); 6,(100 MHz; CDC13) 13 1.4. 13 1.1.

130.2, 120.8, 52.7 (d, JCP 6.6, 2(CH3)) and 32.1 (d, JCP 138.4. CHzP); m/l (EI) 280 (M-{"~r).

58%), 171 ( A f { " ~ r ) - PO(OMe)2, 94), 278 (I@{79~r), 58), 169 (M+{79~r) - PO(OMe)2.

100); Found [MI+, 277.9721 & 279.9702. C9HlzBr03P requires d z 277.9707 & 279.9687.

Diethyl 4-bromobeiizylpbospboaPte (3.10). 133 Colurnn chromatography (si 1 ica.

EtOAc:hexane, 1 : 1, Rr = 0.3) yielded pure 3.10 as a colorless oil(92%). &(200 MHz; CDC13)

7.44 (2 H, d, J 8.8, aryl), 7.18 (2 H, d, J 8.8, aryl), 4.02 (4 H, m, 2(OCHz)), 3.09 (2 H, d, JW

20.5, CH2P) and 1.25 (6 H, t, J 7.3. 2(CH3)); 6p(80 MHz; CDC13) 23.2 (1 P. s); 6,(100 MHz:

CDC13) 131.5 (d), 131.3 (d), 130.7 (d), 120.8 (d), 62.1 (d, JCp 6.6. 2(OCHz)), 33.2 (d. JCp

138.4, CHrP) and 16.2 (d, Jcp 5.8, 2(CH3)); mir (EI) 308 (Mf{8 i~r ) , 24%), 171 (M{"B~) -

PO(OEt)2, 1 OO), 306 ( M + { 7 9 ~ r ) , 24), 169 (M+{79~r) - PO(OEt)2, 97); Found [Ml', 306.00 15.

Cl 1 H 16Bf03P requires m/z 306.0020.

Dimethyl 4-carbobenzylory benzylphosphonate (3.1 1). Column chromatography (silica.

Et0Ac:hexane. 9:1, Rf = 0.3) yielded pure 3.11 as a white solid (86%). mp 66-68 OC: 6ci(200

MHz; CDC13) 8.02 (2 H. d, J 8.1, aryl), 7.38 (7 H, m, aryl), 5.35 (2 HI s, 0CH2), 3.66 (6 H. d,

JHP 10.6,2(CH3)) and 3.21 (2 H, d, JHP 22.4, CHiP); 6p(80 MHz; CDC13) 25.4 (1 P, s); iSc(lOO

MHz; CDC13) 165.9 (CO), 136.8 (d), 135.9, 129.8 (d), 129.6 (d). 128.8 (d)? 128.4, 128.1,

128.0. 66.5 (s, CH2), 52.8 (d, JCp 6.6, 2(CH3)) and 32.4 (d, JCp 137.7, CH2P); d' (EI) 334

(M', 6%), 227 (Mt - 0CH2Ph, 100), 91 (M'+ - 00CPhCH2PO(OMe)2, 59); Found [Mlr.

334.0970. C17H1905P requires m/r 334.0984.

Diethyl 4-benzoyibenzylphosphonate (3.13). 13' Column chromatography (silica.

Et0Ac:hexane. 7:3, Rf = 0.3) yielded pure 3.13 as a pale yellow oil (79%). &(200 MHz:

CDCI3) 7.74 (4 H, m, aryl), 7.43 (5 H, m, aryl), 4.02 (4 H, m, 2(OCH2)), 3.20 (2 H. d, J H ~

22.4, CH2P) and 1.24 (6 H, t, J 6.9, 2(CH3)); Sp(80 MHz; CDC13) 23.0 (1 P, s); 6,(100 MHz:

CDC13) 195.8 (CO), 137.3, 136.5 (d), 135.8 (d), 132.1, 130.0 (d), 129.6, 129.5 (d), 128.0. 62.0

(d, JiP 6-6, 2(OCH2)), 33.7 (d, J c p 137.7, CH2P) and 16.1 (d, Jip 6.6, 2(CH3)); m/z ( E I ) 332

(M', 57%)' 222 (Evl' - 1 10, 100), 195 (M' - PO(OEt12, 30); Found [MI+, 332.1 172. CisH2104P

requires mk 332.1 177.

Dimet hy 1 4-phenylbenzylphosphoarite (3.14). Column chromatography (silica,

MeOH:CHC13 1:99, Rf= 0.3) yielded pure 3.14 as a white solid (90%). mp 68-70 OC; 6@00

MHz; CDC13) 7.50 (9 H, m, aryl), 3.22 (2 H, d, J 2 1 -7, CHI) and 3 -7 1 (6 H. d, JHP 10.6.

2(CH3)); 6p(80 MHz; CDC13) 26.6 (1 P, s); 6,(100 MHz; CDC13) 140.5, 139.8 (d). 130.1 (d).

130.0 (d), 128.7, 127.3 (m), 126.9, 52.9 (d, JiP 6.9, 2(CH3)) and 32.4 (d, Jcp 138.6, CH2); rnk

(EI) 276 (M+, 44%), 167 (M' - PO(OMe)2, 100); Found [Ml', 276.0922. CicHi703P requires

d z 276.09 1 5 .

Dimethyl 3-phenylbenzylphosphonate (3.15). Colurnn chromatography (silica

Et0Ac:hexane 3: 1, Rf = 0.3) yielded pure 3.15 as a white solid (8 1 %). mp 46-48 OC; &&O0

MHz; CDC13) 7.44 (9 H, m, aryl), 3.70 (6 H, d, JHp 10.6. 2(CH3)) and 3.24 (2 H. d. JHp 21.6.

CH2); 6p(80 MHz; CDC13) 26.5 (1 P, s); 6,(100 MHz; CDC13) 141.5. 140.7, 131.7 (d). 128.9

(d). 128.6, 128.4 (t). 127.3, 127.0, 125.7 (d), 52.8 (d, JCP 6.6, 2(CH3)) and 32.8 (d. JCp 138.4,

CH2P): mk (EI) 276 (M. 100%), 167 (w - PO(OMe12, 87): Found [Ml'. 276.0918.

C l jHi703P requires d z 276.091 5.

Dimethyl 2-phenylbenzylphosphonate (3.16). Column chromatography (silica.

EtOAchexane 3:1, Rr = 0.3) yielded pure 3.16 as a pale yellow oil (86%). 6&00 MHz:

CDC13) 7.41 (9 H, m, aryl), 3.57 (6 H, d, Jf ip I l .O, 2(CH3)) and 3.21 (2 Hz d. JHP 22.0. CH?):

6p(80 MHz; CDC13) 27.1 (1 P, s); 8,(100 MHz; CDC13) 142.4 (d), 140.9, 130.3 (d), 129.3.

128.6 (d), 128.1, 127.3 (d): 127.0, 126.8 (d), 52.4 (d,JCp 6.6, z(CH3)) and 29.2 (d. JCP 138.4.

CH2P); m/z (EI) 276 (M', 99x1, 167 (M' - PO(OMe)2, 70). 165 (w - 11 1. 100); Found [Mld.

276.09 10. C isHi703P requires m/z 276.09 15.

Dimethyl 3,5-di-phenylbenzylphosphonate (3.17). Column chromatography (silica.

EtOAc:CH2C12, 2:3, Rf = 0.4) yielded pure 3.17 as a white solid (93%). mp 105-107 OC;

&(200 MHz; CDC13) 7.71-7.37 (13 H, m, aryi), 3.73 (6 H, d, JHp 1 1.7, 2(CH3)) and 3.3 1 (2 H,

d. J 21.9, CH2P); Sp(80 MHz; CDC13) 26.8 (1 P, s); 6,(100 MHz; CDC13) 142.2 (br d), 140.9.

132.6 (d). 128.8, 127.5 (br t), 127.3, 124.8 (d), 52.7 (d, JCp 6.4, Z(CH3)) and 33.1 (d, Jcp 138.2.

CH2P); d. (EI) 352 (hl', 100%), 256 (27), 243 (47), 165 (18); Found [M]', 352.1244.

CzoHzI03P requires m/z 352.1248.

33'-Bis[(dimethylphosphono)methyl]biphenyl (3.50a). Column chromatography

(MeOH:CHC13, 5:95, Rf = 0.3) yielded pure 3.50a as a colorless oil (85%). 6&00 MHz:

CDC13) 7.35 (8 H, m, aryl), 3.66 (1 2 H. d, JHp 1 1.7. 4(OCH3)) and 3.19 (4 H, d. J H ~ 20.5.

2(CH2P)); 6p(80 MHz; CDC13) 26.8 (1 P, s); 6,(lOO MHz; CDC13) 14 1 .O, 1 3 1 -9 (d), 138.6 (m).

125.6 (d). 52.6 (d, JCP 6.4, 4(OCH3)) and 32.8 (d. JCp 138.2. 2(CHzP)); d z (EI) 398 (MT.

74%), 289 (M+ - PO(OMe)2, 100); Found MI', 398.1044. C1&i2&P2 requires d . 3 9 8 . 1048.

4,l'-Bis[(dimetiiylphosphono)methylJbiphenyl (3.50b). Column chromatography

(MeOH:CHC13, 2:98, Rf = 0.3) yielded pure 3.50b as a white solid (87%). mp 128-1 30°C;

ZiH(200 MHz; CDC13) 7.54 (4 H, d. J7.7, aryl), 7.37 (4 H, dd, J8.0 & 2.4, aryl). 3.71 (12 H, d.

J1-IP 10.6, 4(OCH3)) and 3.21 (4 H, d, JHp 21.6. 2(CHz)); 6p(80 MHz; CDC13) 26.6 (1 P. s):

6,(100 MHz; CDC13) 139.23, 130.2 (d), 130.0 (d), 127.1, 52.9 (d, JCP 6.9, 4(OCH3)) and 32.5

(d, JCP 138.6, 2(CHzP)); d z (EI) 398 (w, 76%), 289 (M' - PO(OMe)l, 100); Found [Ml*.

398.1055. Ci8H2406P2 requires m/z 398.1048.

4,1'-Bis[(dimethylphosphono)methyl]benzophenone (3.71). Column chromatography

(silica. MeOH:CHCI3, 0.5:9.5, Rf = 0.2) yielded pure 3.71 as a white solid (61%). mp 91-93

OC; 6"(200 MHz; CDC13) 7.76 (4 H, d, J 8.5, aryl), 7.43 (4 H, d, J 2.2, aryl), 3.71 (1 2 H, d, JHp

11.0, 4(CH3)) and 3.25 (4 H, d, J H p 22.0, 2(CH2)); 6p(80 MHz; CDCt3) 25.5 (1 P. s); 6,(100

MHz; CDC13) 195.3 (CO), 136.1 (d), 135.9 (d), 130.1 (d), 129.4 (d), 52.7 (d, JCP 6.6, 4(CH3))

and 32.7 (d, Jcp 137.7,2(CH2)); d z (El) 426 (MC, 55%), 3 17 (M+ - PO(OMe)2, 21), 227 (M' -

PhcH~Po(oMe)~, 100); Found FI]+, 426.0986. C 19H2407P2 requires m/z 426.0997.

4-[(Dimethylphosphono)methyl]phenylacetic acid beozyl ester (3.29). Column

chrornatography (silica, EtOAc:hexane, 9:1, Rf = 0.3) yietded pure 3.29 as a colorless oil

(91%). 6~(200 MHz; CDCl3) 7.28 (4 H. s, aryl), 7.22 (5 H, s, aryl), 5.13 (2 H, s, 0CH2Ph),

3.66 (8 H, d, J 10.3, CH2C0 & 2(CH3)) and 3.1 1 (2 H, d, J H ~ 21.9, CHzP); 6p(80 MHz;

CDC13) 26.7 (1 P, s); 8,(100 MHz; CDC13) 170.9 (CO), 135.6, 132.4 (d), 129.9 (d), 129.7 (d).

129.3 (d), 128.3, 128.0, 127.9, 66.3 (s, BnCH2), 52.6 (d, J C p 6.6, 2(CH3))1 40.7 (s? CHr) and

32.2 (d. Jcp 138.4. CH2P); rn/r (El) 348 (MC, 19%), 2 13 (W - OOCCHzPh, 100); Found [Ml-.

348.1 128. CisH2i05P requires d z 348.1 127.

3-[(Dimethylphosphono)methyl]phenylacetic acid (3.30). A mixture of 3.29 (1 75 mg, 0.5

rnmol) and approximately 10 mg of 10% PdK, in ethyl acetate (10 cm3), was stirred for 15 h

under Hz (1 atm.). The mixture was filtered through Celite and concentrated by rotary

evaporation to give 101 mg (78%) of 3.30 as a white solid. No M e r purification was

necessary. mp 1 1 1 - 1 13 OC; ZiH(200 MHz; CDC13) 7.24 (4 H, s, aryl), 3 -67 (6 H, d, JHp 1 1 -7.

2(CH3)), 3.6 1 (2 H, s, CH2CO) and 3.16 (2 H, d, JHP 2 1 -9, CH2); 6p(80 MHz; CDC13) 27.1 ( 1

P, s); 6c(100 CDC13) 174.8 (CO), 132.9 (d), 129.7 (d). 129.5 (d), 129.4, 53.0 (d, JCP 6.6.

2(CH3)), 40.7 (s, CH2CO) and 32.1 (d, JCp 139.1, CHzP); nu" ( ~ 1 ) ~ ~ 258 (MI, 5%). 272 (W +

CHj. 9). 214 (W - CO2, 99), 149 (W - PO(OMe)2, 55); Found [Ml*. 258.0657. C i HisOjP

requires d z 258.0657.

Diethyl 4-azidobenzylphosphonate (3.63). 3.62 (OSOg, 0.002 1 mol) was dissolved in TFA

(7 cm3) and diazotized in the dark at O OC with NaN02 (0.28 g, 0.0041 mol). Afier stimng for

30 min, NaN3 (1 -34 g, 0.0206 mol) was added to the reaction mixture over a penod of severaI

minutes and the mixture was stirred for an additional 15 min. Ether (1 5 cm3) was then added

and the reaction stirred for 1 h. The crude product was then washed with water, dried

(MgSO4) and concentrated in v a m . Column chromatography (silica, EtOAc, Rf = 0.6)

yielded pure 3.63 as a pale yellow oil (0.46 g, 83%). 6"(200 MHz; CDC13) 7.27 (2 H. dd. J

2.6 & 8.8, aryl), 6.96 (2 H, d, J8.4, aryl), 4.00 (4 H, m, 2(OCHz)), 3.1 1 (2 H, d, J21.6. CH2P),

and 1.23 (6 H. t, J 7.2, 2(CH3)); &(80 MHz; CDC13) 23.71 (1 P, s); 6,(100 MHz: CDCl;)

1 3 8.8 (br d), 13 1.1 (d), 128.6 (d), 1 19.0 (d), 62.0 (d. J 6.4, 0CH2), 33.1 (d, JCp 139.1. CH2P)

and 16.2 (d, J 6.4, CH3); IR (cm-') 2 1 12.2; d z (EI) 269 (M', 10%). 24 1 (72). 185 (3)' 168

(38)' 109 (IOO), 81 (59); Found: M', 269.0939. Ci &i[6N303P requires m/r 269.0929.

4-Azidobenzoic acid"' (3.67). 4-Aminobenzoic acid (3.0 g, 0.02 19 mol) was dissolved in

TFA (45 cm3) and diazotized in the dark at O OC with sodium niaite (3.02 g, 0.0438 mol).

Afier stirring the solution for 30 min, sodium azide (14.2 g. 0.2186 mol) was slowly added IO

the reaction mixture over 10 min. AAer an additional 15 min, diethyl ether (30 cm3) was

added and the entire reaction mixture s h e d for 1 h. The product was isolated (water. ether.

Na2S04) and excess TFA removed as an azeotrope with benzene. The product was

recrystallized with ethedwater to yield 3.67 as a white solid (2.5 g' 7 1 %). mp 179 - 180 OC:

[lit.136 mp 185 OC]; 6"(200 MHz; CD30D) 7.97 (2 H, d, J 8.8, aryl) and 7.04 (2 H. d, J 8.8.

aryi); 6,(100 MHz; CD30D) 173.2 (COOH), 143.9, 133.9, 132.2 and 1 19.3; ~~(vlcm' l ; KBr)

2 107.4 cm-'; nt/r (EI) 163 OI.i', 27%), 135 (100), 107 (30), 89 (32), 79 (3 1 ), 64 (65); Found

[M]'. 163-0387. C7H5N302 requires m/s 163-0382.

4-[(DiethyIphosphono)oxomethyl]azidobeazene (3.68). Excess thionyl chloride was added

to 3.67 (0.7 g, 0.0043 mol) and the reaction mixture was stirred under reflux for 30 min. The

solution becarne clear. The excess thionyl chloide was distilled off ovemight, under reduced

pressure, leaving a yellowish solid. The product was recrystallized fiom petroleum ether and

concentrated under reduced pressure yielding an off-white precipitate (80%). mp 55 - 56 OC

[lit."' mp 57 - 58 OC]. Triethyl phosphite (584 pl, 1 eq.) was added dropwise to the acid

chlofide (0.6188 g, 0.0034 mol) at O OC. The resulting yellowish solution was stirred for 2 h.

Colurnn chromatography (silica, EtOAc, Rr = 0.35) yielded cmde 3.68 as a yellowish oïl

(21%). 6 ~ ( 2 0 0 MHz; CDC13) 7.92 (2 H, d, J8.7, aryl), 7.04 (2 H, d, J8.3, aryl). 4.08 (4 H. m.

(2)OCH2) and 1.33 (6 H, t, J 5.7, (2)CH3); 6p(80 MHz; CDC13) -3.24 & -15.48. Although the

'H-NMR indicates that the desired product was obtained, the 3 ' ~ - ~ ~ ~ displayed two peaks: 6

-3.24 (major) and 6 - 1 5.48 (minor, approx. 15%). We were unable to remove this impurity.

Sy o t bctic attempt: 4-[(~ieth~l~hos~hono)difluorometh~l] azidobeazenes7 (3.64). To 3.68

(0.2 g, 0.0007 mol), DAST (0.4 g, 0.33 ml, 3.5 eq.) was added dropwise at O OC. The reaction

was brought to room temperature and stirred overnight. The reaction mixture was diluted with

chlorofom (3 cm3). The solution was added dropwise to a well stirred suspension of NaHC03

in water (1.64 g in 7.5 cm3) at O OC. The suspension was stirred for an additional 15 min.

diluted with water (13 cm3) and extracted with chlorofom (3 x 40 cm3). The resulting

yellowish solution was dried (NarSOs) and concentrated under reduced pressure yielding a

yellowish oil. The 3 ' ~ - ~ ~ ~ indicated that the desired reaction did not occur.

Benzoyl-phosphonic acid dimethyl ester (3.2a). To a cooled solution (10 OC) of benzoyl

chloride (4.2 g, 0.0299 mol), P(OMe)3 (3.7 g, 0.0299 mol) was added dropwise with stirring

over 1 h, maintaining a temperature of 10 OC. The mixture was then stirred for an additional

hour at room temperature. Distillation yielded pure 3.2. as a paie yellow oil (84%).

MHz; CDCI3) 8.25 (2 H, d, J 7.3, aryl), 7.69-7.47 (3 H, m, aryl) and 3.92 (6 H. t, JHP 10.3,

2(CH3)); Sp(80 MHz; CDC13) - 1.6 (1 P, s); 6,(lOO MHz; CDCI3) 198.3 (d, CO), 134.7, 130.3,

1 29.6, 128.8 and 53.8 (br d, î(CH3)); d z (Er) 2 14 (w, 6%), 105 (1 OO), 77 (49); Found [Ml',

2 14.0393. C9Hi ,04P requires m/z 2 14.0395.

Benzoyl-phosphonic acid diethyl ester (3.2b). Same procedure as described for 3.2a using

P(OEt)3. Pale yellow oil (81%). 8"(200 MHz; CDCI3) 8.27 (2 H, d, J 7.4. aryl), 7.69-7.47 (3

H. m, aryl), 4.36-4-21 (4 H, m, 2(CH2)) and 1.39 (6 H, t, J 7.3, 2(CH3)); 6p(80 MHz; CDCI,) -

3.6 (1 P, s); 6,(100 MHz; CDC13) 199.0 (d, CO), 134.5, 130.3. 129.7. 128.7. 63.8 (br d.

?(CHz)) and 16.2 (br d, 2(CH3)); m/r (EI) 242 (h.I', 7%), 105 (100). 77 (36); Found [Ml'.

343.0772. CliHij04P requires m/z 243.0786.

Preparatiou of a,a-DiRuorornethylenephosphonates

Unless otherwise stated, al1 fluorination reactions were performed as descnbed below.

General Procedure for the Preparotion of a,a-Difluoromethylenepbosphonates.

To a solution of NaHMDS (Aldrich, 1.0 M in THF. 2.2 eq.) in dry THF

(approximately 0.4 cm3 N ~ H M D S / C ~ ' THF) at -78 OC was added a solution of the benzylic

phosphonate (1 .O eq.) in dry THF (approximately 15-20 cm3 THF/mmol phosphonate) over a

penod of 2 minutes. The resulting orange to dark red solution (sometimes a suspension

forms) was stirred for 1 h at -78 OC. A solution of NFSi (Aldrich. 2.5 eq.) in dry THF

(approxirnately 2-4 cm3 THF/rnmol NFSi) was added over a period of two minutes. during

which time the solution (suspension becomes a solution) turned fiom dark red or orange to

yeIIow-brown. Afier addition, the solution was stirred for 1-2 hours and then allowed to wann

to -30 OC during which time a precipitate formed. The reaction was quenched with 0.01 N

HCl and the resulting solution (precipitate dissolves) was extracted with EtOAc. The organics

were combined and washed with 5% NaHC03, bnne, dned (MgS04) and concentrated by

rotary evaporation to give a yellow residue, which was purified via flash chromatography. For

the formation of bis-difluoromethylenephosphonate derivatives, 5.5 equiv. NaHMDS and 7.3

equiv. NFSi were used.

[(Dimet hylphosphono)difluoromethyl] benzene (33a). Column chromatography (silica,

hexane:EtOAc, 73, Rr= 0.3) yielded pure 3.3a as a colorless oil(63%). &(200 MHz; CDC13)

7.50 (5 H, m, aryl) and 3.78 (6 H, d, JHP 10.6, 2(CH3)); Sp(80 MHz; CDC13) 6.5 (1 P. t, JPF

1 16.7); aF(l 88 MHz, CDC13) -3 1.6 (1 F, d, JFp 1 16.7); 6,(100 MHz; CDC13) 132.3 (id), 130.9

(d), 128.5 (bs), 136.1 (td), 1 18.3 (td, JCF 263.4 & JCp 21 8.8. CF2P) and 54.92 (m. OCW j): m k

(EI) 236 (M', 14%)' 127 (M' - PO(OMe)2, 100); Found [Ml', 236.0424. CsHl iF203P requires

d' 236.0414.

[(Diethylphosphono)difluoromethyl]beazeae (3.3b). Column chromatography (silica.

EtOAc:hexane, 3:2, Rf = 0.5) yielded pure 3.3b as a colorless oil(79%). &(200 MHz; CDC13)

7.64 (2 H, m, aryl), 7.48 (2 H, m, aryl), 4.18 (4 H, m, 2(CH2)) and 1.43 (6 H, t, J 7.1. 2(CH;));

Sp(80 MHz; CDC13) 4.4 (1 P, t, J ~ F 116.0); 6~(188 MHz; CDC13) -32.6 (1 F. d. JFp 116.0):

&(IO0 MHz; CDCI3) 132.5 (td), 130.6, 128.3, 126.1 (td), 117.9 (td, JCF 262.3 & JCp 21 8.2,

CF2), 64.7 (d, Jcp 7.4, 2(CH2)) and 16.2 (d, Jcp 5.8, 2(CH3)); m/z (El) 264 M. 20%), 127 (M

- PO(OEt)2, 100); Found [M]', 264.0726. CioHi3F20~P requires mk 264.0727.

4-[@imethylphosphono)dinuoromethyl~nitrobeene (3.18). Column chromatography

(silica. EtOAc:hexane, 1:1, Rf = 0.5) yielded pure 3.18 as a pale yellow solid (74%). &(ZOO

MHz; CDCI3) 8.05 (2 H, d, J 8-4, q l ) , 7.82 (2 H, d, J 7.7, aryl) and 3.89 (6 H, d, JHP 10.7.

S(CH3)); 6p(80 MHz; CDC13) 5.4 (1 P, t, J ~ F 110.0); 6~(188 MHz; CDCI3) -32.7 (1 F, d, J w

i 10.0); 6,(100 MHz; CDC13) 149.4, 138.4 (td), 127.5 (td), 1 17.3 (td, J& 265.0 & JCp 2 17.0,

CF2) and 55.2 (d, JCp 6.6, 2(CH3)); d z (EI) 28 1 (M+, 18%), 171 (bl+ - PO(OMe)2. 33) 109

(M' - 1 72, 100); Found [Ml', 28 1.0274. C I O H ~ ~ F ~ O ~ P requires nt/r 28 1.0264.

4-[(Diethylphosphono)difiuoromethylJnitrobeene (3.19). Column chromatography

(silica, EtOAc:hexane, 2:3, Rf = 0.4) yielded pure 3.19 as an oswhite solid (82%). mp 5 1-53

O C . 6~(200 MHz; CDCl3) 8.32 (2 H, cl. J 9.1, aryl), 7.82 (2 H, d, J 8.5, aryl), 4.25 (4 H. m.

l(OCH2)) and 1.35 (6 H, t, JHP 6.9, 2(CH3)); Q(80 MHz; CDC13) 3.1 (1 P, t, JpF 110.0);

s~(188 MHz; CDC13) -33.5 (1 F, d, J F ~ 110.0); &(100 MHz; CDC13) 149.2, 138.8 (td). 127.6

(td), 123.5. 1 17.1 (td, J& 264.2 & JCp 2 16.0, CF2), 65.1 (d. JCp 6.7, 2(CHz)) and 16.2 (d. JCp

4.8. ?(CH3)); r d z (EI) 310 (W, 7%), 173 (MH' - PO(OEt)2, IO), 109 - 201. 100):

Found [Ml', 309.0577. Cl 1 EiI4F2NO5P requires m/z 309.0578.

4-[(Dimethylphosphono)ditluoromethyl] b r o m o b e e n e (3.20). Column chromatography

(siiica. EtOAc:hexane, 3:7. Rf = 0.3) yielded pure 3.20 as a colorless oil (79%). 6&00 MHz;

CDClj) 7.60 (2 H, d, J8.8, iiryl), 7.48 (2 H, d, J8.5, aryl) and 3.83 (6 H, d, JHp 10.6, 2(CH3));

6p(80 MHz; CDC13) 6.0 (1 P, t, JpF 116.0); SF(188 MHz; CDC13) -32.1 (1 F. d. JFp 116.0):

6,(lOO MHz; CDC13) 13 1.7, 13 1.5, 127.7 (td), 125.5. 1 17.7 (td, JCF 263.4 & JCp 219.2. CF2)

and 54.8 (d, JCp 6.6, 2(CH3)); m/z (EI) 3 16 (M*{*'B~), 18%), 207 ( h f { 8 1 ~ r ) - PO(OMe)2. 99).

3 14 (l@{79~r). 19). 205 (h~l?{'~~r) - PO(OMe)2, 100); Found CM]', 3 15.95 1 1 & 3 13.9533.

C9HloBrFz03P requires m/z 3 15.9499 & 3 13.95 19.

4-[(Diethylphosphono)difluoromethyl] b r o m o b e e n e (3.21). Column chromatography

(silica, EtOAc:hexane, 3:7, Rf = 0.5) yielded pure 3.21 as a pale yellow oil (80%). 6&00

MHz; CDC13) 7.60 (2 H. d, J 8.8, q l ) , 7.49 (2 H, d, J 7.3, aryl), 4.21 (4 H, m. 2(CH2)) and

1 3 3 (6 H, t, JHp 7.4, 2(CH3)); +(80 MHz; CDCl3) 3.7 (1 P, t, J p ~ 1 14.5); &(188 MHz:

CDC13) -32.6 (1 F, d, JFp 1 14.5); 6,(lOO MHz; CDC13) 13 1 -6, 13 1.5 (m), 127.8 (td), 125.3 (d),

1 17.6 (td, JiF 263.5 & J C p 218.5. CF2), 64.7 (d, Jcp 6.6, 2(CHr)) and 16.2 (d, JCp 5.8. 2(CHj)):

m./' (EI) 344 ( M + { 8 1 ~ r ~ , 1 1%), 207 ( M + { ~ ' B ~ ) -PO(OEt)2, 93), 109 (W{"B~J - 235, 100),

342 (w { " ~ r ) , 1 1 ), 205 @f { 7 9 ~ r ) - PO(OEt)*, 96), 109 (M'{ 79 ~ r ) - 233, 1 00); Found [Mld,

34 1.98 19. C 11 Hr4BrF203P requises d z 34 1.9832.

Benzyl 4-((dimethylphosphono)difluoromethyl]benzoate (3.22). Column chromatography

(silica, EtOAc:hexane, 2:3, Rr = 0.6) yielded pure 3.22 as a colorless oil (72%). 6&00 MHz:

CDC13) 8.17 (2 H, d, J8.0, aryl), 7.70(2 H, d, J8.4, aryl), 7.40 (5 H, m, aryl), 5.39 (2 H. S.

OCH2) and 3.84 (6 H, d, J H p 10.6, 2(CH3)); 6p(80 MHz; CDC13) 6.0 (1 P, t, JpF 1 12.9); &(l88

MHz; CDC13) -32.5 (1 F, d, JFP 112.9); 6c(100 MHz; CDCl3) 165.4 (CO), 137.7 (m), 135.6.

132.4. 129.8, 128.6, 128.4, 128.2, 126.3 (t), 117.6 (td, JCF 264.0 & JCP 216.9, CF2), 67.1 (s,

CH2). 55.0 (d, JCp 5.9, 2(CH3)); m/l (EI) 370 (M', 5%), 263 (M' - 0CH2Ph 100), 761 (M' -

PO(OMe)2, 29); Found M', 370.0782. C17H17F205P requires d z 370.0782.

3-[@imethylphosphono)difluoromethylJmethoxybenzene (3.23). Column chromatography

(silica, EtOAc:hexane, 1 : 1, Rr = 0.4) yielded pure 3.23 as a colorless oil (80%). 6"(200 MHz:

CDC13) 7.51 (2 H, d, J8 .1 , aryl), 6.92 (2 H, d. J 8.7. aryl), 3.76 (9 W. d, OCH; and

2(POCH3)); 6p(80 MHz; CDC13) 6.78 (1 P, t, J ~ F 120.6); &(188 MHz; CDC13) -30.4 (1 F. d. J

120.6); &(IO0 MHz; CDC13) 161.5 (CO), 127.6 (td), 124.3 (td), 1 18.3 (td. J& 263.7 & Jcp

221.9. CF2), 55.3 (s, ArOCH3), 54.8 (d, JCp 7.4, 2(POCH3)); d~ (EI) 266 (W. 10%). 157

(Mt - PO(OMe)2, 100%); Found [w', 266.05 1 1. CioHi3F204P requires m/r 266.05 19.

4-[(Diethylphosphono)dinuoromethyl]benzophenone (3.24). Column chromatography

(silic- EtOAc:CH2C12, 0.5:9.5, Rf = 0.7) yielded pure 3.24 as a colorless oil (70%). 6~(200

MHz; CDC13) 7.81 (6 H, m, aryl), 7.53 (3 H, rn, aryl), 4.21 (4 H, m, 2(CH2)) and 1-31 (t. JHP

6.9, 2(CH3)); 6p(80 MHz; CDC13) 3.7 (1 P, t, J ~ F 113.7); &(Il38 MHz; CDC13) -32.9 (1 F. d)

JFP 1 13.7); 6,(lOO MHz; CDC13) 195.6 (CO), 139.6, 136.8, 136.2 (td), 132.7, 129.9, 129.7,

128.3, 126.2 (t), 117.6 (td, JCF 263.8 & JCp 217.3, CF2), 64.8 (d, J C p 7.3, 2(CHz)) and 16.1 (d.

JCP 5.9. 2(CH3)); m/r (EI) 368 @V, 100%), 231 (M' - PO(OEt)2, 96); Found [MI', 368.0978.

C 18H 19F209 requires ndz 368.0989.

4-14-((DimethyIphosphono)difluoromethyI)phenyI] benzene (3.25). Column

chromatography (silica, hexane:EtOAc 7:3, Rr= 0.3) yielded pure 3.25 as a white solid (59%)-

mp 75-78 OC; &(200 MHz; CDC13) 7.60 (9 H, m, aryl) and 3.87 (6 H, d, JHP 10.2, 2(CH3));

6p(80 MHz; CDC13) 6.6 (1 P, t, J ~ F 11 7.0); &(188 MHz; CDC13) -3 1.3 (1 FI d, J F ~ 1 17.0):

6,(100 MHz; CDC13) 143.8 (d), 140.0. 131.2 (td). 128.9, 128.0, 127.3 (br dd). 126.6 (td).

1 1 8.3 (td. J C ~ 263.4 & JCp 219.4. CFz), 54.9 (d, JCp 6.6. 2(CH3)); m/r (EI) 3 12 (W? 19%). 203

(M' - PO(OMe)2, 100); F o n d [MJ', 3 12.0730- CisHisFz03P requires d z 3 12.0727.

3-[4-((DimethyIphosphono)difluoromethyl)phenyI] benzene (3.26). Column

chromatography (silica, EtOAc:hexane, 1:1, Rr = 0.5) yielded pure 3.26 as a colorless oil

(60%). 6"(200 MHz; CDC13) 7.61 (9 H, m, aryl) and 3.86 (6 H, d, JHP 10.2 HL, 2(CHs)); 6p(80

MHz; CDC13) 6.6 (1 P, t, JpF 1 17.0); S~(188 MHz; CDC13) -3 1.4 (1 F, d, J F ~ 1 17.0); 6,(100

MHz; CDCI3) 141.7, 140.0, 133.0 (td), 129.6, 129.0, 128.9. 127.8, 127.2, 124.8 (m). 118.2 (td.

JcF 263.6 & JCp 21 8.2, CF2) and 54.8 (d, Jcp 6.6, 2(CH3)); d' (EI) 3 12 (W. 39%)_ 203 (W -

PO(OMe)2. 100), 183 (M' - 129. 20); Found [Ml'. 3 12.0744. CisHl jF203P requires m/l

3 12.0727.

2-(4-(@imethylphosphono)difluoromethyl)phenyl] benzene (3.27). Column

chromatography (silica, EtOAchexane, 1:1, Rf = 0.5) yielded pure 3.27 as a colorless oil

(46%). SH(200 MHz; CDC13) 7.50 (9 H, m, aryl) and 3.73 (6 H, d, J 10.2, 2(CH3)); 6p(80

MHz; CDCf3) 6.5 (1 P, t, JPF 1 17.5); S ~ ( l 8 8 MHz; CDC13) -22.0 (1 F, d, J F ~ 1 1 7.5); 6,(lOO

MHz; CDC13) 141.7 (m), 141.1, 132.7, 130.2, 129.4, 128.0 (br t), 127.3, 127.1, 127.0, 119.6

(rd. JcF 265.3 & JCp 218.5, CF2) and 54.8 (d, Jcp 6.6, 2(CH3)); mk (EI) 3 12 (MC, 37%), 203

(M' - PO(OMe)2, 76), 183 (M' - 129, 100); Found [Ml', 3 12.0740. CisHi5F203P requires d z

3 12.0727.

3,5-~4-(@imethylphosphono)difluoromethyl)di-phey1 benzene (3.28). Cotumn

chromatography (silica, CH2C12, Rf = 0.3) yielded pure 3.28 as a colorless oil (70%). 6 ~ ( 2 0 0

MHz; CDCl3) 7.93-7.40 (13 H, m, aryl) and 3.88 (6 H, d, JHP 11.7, 2(CH3)); 6p(80 MHz:

CDCI,) 6.7 (1 P, t, J ~ F 116.0); 6~(188 MHz; CDC13) -31.7 (1 F. d. JFP 116.0); 6c(100 MHz:

CDCI,) 142.1, 139.8, 133.4 (br td), 128.8, 128.3, 127.8, 127.1, 123.5 (br td), 118.1 (td? JCF

263.7 & JCP 218.3, CFz) and 54.8 (d, JCp 6.6, 2(CH3)); m/r (EI) 388 (W, 71%), 279 (100):

Found [MI', 388.1044. C Z I H ~ ~ F ~ O ~ P requires nt/r 388-1040.

3,3'-Bis[(dimethylpbospbono)düluoromethyl]biphenyl (3.51a). Column chromatography

(EtOAc:hexanes, 4:1, Rf = 0.3) yielded 3.51a as a paie yellow oil (29% yield). 6~(200 MHz:

CDC13) 7.30 (8 H. m, aryl), 3.87 (12 H, d, J 10.3, 4(OC&)); 6p(80 MHz; CDCl,) 6.6 (1 P, t,

JpF 1 16.0): 6F(l 88 MHz; CDCl3) -3 1.5 (1 F, d, JFp 1 16.0); SC(100 MHz; CDClj) 140.6. 136.0

(m). 129.7. 129.1, 125.5 (br td), 124.9 (br td), 118.1 (td, JCF 263.5 & Jcp 215.9, CFz), 51.6 (d,

JCP 7.3, 4(OCH3)); nc/z (ET) 470 (MC, 34%), 361 (M+ - PO(OMe)2, 100); Found [Ml'.

470.0671. C~8H20F40~2 requires d z 470.0671.

1,4'-Bis[(dimethylphosphono)dluoromethyI] b i p h e y (3.51 b). Column chromatography

(EtOAc:hexanes, 4:1, Rf = 0.4) yielded 3.51b as a white solid (34% yield). mp 92-94 OC;

6&00 MHz; CDCl3) 7.67 (8 H, rn, aryl). 3.85 (12 H, d, J 10.6, 4(OCH3)); 6p(80 MHz:

CDC13) 6.4 (1 P, t, J ~ F 1 16.7); 6 ~ ( l 8 8 MHz; CDCl3) -3 1.8 (1 F, d, J F ~ 1 16.7); 6c(100 MHz;

CDC13) 142.5, 132.0 (td), 127.4, 126.8 (td), 118.2 (td, k~ 262.8 & Jcp 218.9, CF2), 55.0 (d,

JCP 6.8, ~(OCHJ)); d z (EI) 470 (Mf, 14%), 361 (M+ - PO(OMe)2, 100), 252 (M' -

Z(PO(OMe)2), 39); Found [v', 470.0678. CifioF4OsPz requires nt/r 470.0671.

4,4'-Bis [(dimethyIphosphono)difhoromethyl~ benzophenone (3.72). Column

chromatography (silica, EtOAc, Rf = 0.6) yielded pure 3.72 as an off-white solid (28%). mp

1 14-1 16 O C ; &(200 MHz; CDCl3) 7.89 (4 H, d. J8.4, aryl), 7.76 (4 H, d, J8.0, aryl) and 3.89

(12 H, d, JHP 10.7, 4(CH3)); 8p(80 MHz; CDC13) 5.9 (1 P, t, J ~ F 113.0); &(188 MHz; CDC13) -

32.4 (1 F, d, JFP 113-0); Sc(lOO MHz; CDC13) 194.9 (CO), 139.0, 136.5 (td), 130.0, 126.4 (t),

1 17.7 (td, J C ~ 263.9 & Jcp 2 17.8, 2(CF2)) and 55.1 (d, Jcp 6.7, 4(CH3)); d z (EI) 498 (MyT

41%). 389 (M+ - PO(OMe)2, 100) 280 (W - 2(-PO(OMe)z), 6); Found [Ml+, 498.0601.

C 19H20F407P2 requires m/z 498.0620.

4-[(Dimethylphosphono)dinuoromethyl]benzoic acid (precursor to 3.34). A mixture of

3.22 (300 mg, 0.8 1 m o l ) and approximately 15 mg of 10% PdK, in methanol (5 cm3), was

stirred overnight under Hz (1 atm.). The mixture was filtered through Celite and concentrated

by rotary evaporation to give the acid as a white solid (90%). No M e r purification was

necessary. mp 124-126 OC; aH(200 MHz; CD30D) 8.18 (2 H, d, J 8.8, aryl). 7.74 (2 H. d, J

8.8, aryl) and 3.87 (6 H, d, J 10.3, 2(CH3)); 6p(80 MHz; CD3OD) 6.0 (1 P, t, JpF 1 14.5):

&(188 MHz; CD30D) -29.8 (1 F, d, JF;P 1 14.5); &(lOO MHz; CD30D) 167.6 (COOH). 136.8

(td), 133.9, 130.1, 126.5 (td), 118.3 (td. JCF 262.9 & J c p 219.7, CF2) a d 55.2 (d, JCp 7.3.

2(CH3)); miz (EI) 280 (MC, IO%), 171 (M? - PO(OMe)2, 100); Found [Ml', 280.03 12.

C F2O5P requires m/z 280.0302.

4-[4-(@iethylphosphono)difluorometbyl)phenyl]toluene (3.54). To 4-[(diethylphos-

phono)difluoromethyl]bromobenzene (410 mg, 1.2 1 rnmol) in deaerated ethanol (1 O cm'). was

added 4-methylphenylboronic acid (0.25 g, 1.5 eq.), Pd(0Ac)z (0.0 1 1 g, 0.042 eq.) and solid

Na2C03 (0.19 g, 1.5 eq.). The dark brown reaction mixture was s h e d at room temperature

for 36 h, under argon, diluted with ether (100 cm3) and filtered. The filtrate was washed with

1 N NaOH (1 x 100 cm3), brine (1 x 100 cm3), dried (MgS04) and concentrated by rotary

evaporation. Column chromatography (silica, EtOAc:hexane, 3:7, Rf = 0.3) yielded pure 3.54

as a white solid (71%). mp 42-44 OC; 6&00 MHz; CDC13) 7.67 (4 H, s, q l ) , 7.51 (2 H. d. J

7.3, aryl), 7.27 (2 H,d, J5.9, aryi),4.21 (4 H, rn, 2(CHz)), 2.41 (3 H, S. CH3Ar) and 1.34 (6 H.

t. J 6.6, 2(CH3)); 6p(80 MHz; CDC13) 4.4 (1 P, t, J ~ F 117.5); 6~(188 MHz; CDCl;) -32.2 (1 F,

d. JFP 117.5); 6,(100 MHz; CDC13) 143.7, 137.8, 137.2, 129.6, 127.0. 126.8, 126.7. 126.6,

11 8.3 (weak td, CFz), 64.6 (d, JCp 7.4, CH2), 20.93 (s, CH3&) and 16.2 (d, J C p 4.6, CH;); mk

(El) 354 (Mf, 23%), 2 17 (W - PO(OEt)*, 100); Found FI]', 354.1 196. CieH2i Fz03P requires

mk 354.1 194.

4-[4-(@iethylphosphooo)difluoromethyl)ben~e]diethyI benzylphosphonate (3.55). N-

Bromosuccinimide (0.14 g, 1 eq.) was added in several portions to a solution of 3.54 (275 mg.

0.78 m o l ) and benzoyl peroxide (0.0014 g, 0.0075 eq.) in anhydrous CC4 (25 cm3). Another

portion of benzoyl peroxide (0.0014 g) was added towards the final addition of NBS. The

mixture was refluxed for 2 h, cooled to room temperature, washed with water (2 x 15 cm3).

dried (MgS04) and concentrated by rotary evaporation. The crude reaction mixture was

passed through a s i k a gel column (hexaneEtOAc, 6:4, Rf = 0.4) yielding a partially purified

mixture of monobrominated and dibrominated products (- 5% of the total) as a colorless oil.

This crude mixture (170 mg) was then added to excess triethyl phosphite (10 cm3) and

refluxed for 10 h. The excess phosphite and diethyl ethylphosphonate (formed during the

reaction) were removed by vacuum distillation and the product was purified via silica gel

chromatography (EtOAc, Rr = 0.5) to give 3.55 as a yellow oil (73%). 6 ~ ( 2 0 0 MHz; CDC13)

7.68 (4 H. s, aryl), 7.56 (2H, d, J7.4, aryl), 7.39(2 H,d, J8.8,aryl),4.14(8 H, m, 4(Ci-i2)),

3.2 1 (2 H, d, JHp 22.0, CH2P) and 1.3 1 (12 H, m, 4(CH3)); 6p(80 MHz; CDC13) 23.9 (1 P, s);

4.30 (1 P, t, J~F 11 7.0); &(188 MHz; CDC13) -32.1 (1 F, d, JFP 117.0); SC(1 00 MHz; CDC13)

143.1, 138.5 (d), 131.6 (d), 131.4 (m), 130.3 (d), 127.3 (d), 126.9, 126.7 (td), 118.1 (weak td,

JCF 263.3 & JCp 218.9, CFz), 64.7 (d, JCp 7.4, CH2), 62.1 (d, JCp 6.6, CH2), 33.5 (d, JCp 138.4,

CHzP), 16.4 (d, JCp 5.1, 2(CH3)) and 16.3 (d, JCp 5.1, 2(CH3)); d z (EI) 490 (M+, 52%). 353

(AT+ - PO(OEI)~, 100); Found [Ml', 490.1486. CuH30F206P2 requires m/r 490.1472.

4-[@iethy~piiosphono)diîluoromethyl~azidobeene (3.61). A mixture of 3.19 (0.53 g, 1 -7 1

rnrnol) and zinc dust (5 eq.) in TFA (25 cm3) was stirred ovemight. The reaction was filtered.

cooled to O OC in an ice bath. NaNO2 (0.24 g, 2 eq.) was added and the mixture stirred in the

dark for 30 min. Nm (1.1 1 g, 10 eq.) was added over a period of 2 min and the mixture

stirred for an additional 15 min. Ether (50 cm3) was added and the mixture s h e d for 1 h.

The reaction mixture was then washed with water (1 x 50 cm3). The organic layer was dned

(Na2S04) and concentrated leaving an oily yellow residue. Column chromatography (silica,

EtOAc:CH2C12, 1 :9, Rf = 0.9) gave pure 3.64 as a yellow oil (76%). &(200 MHz: CDC13) 7.59

(2 H. d, J8.1, aryl), 7.09(2 H, d, J8.4, VI), 4.20 (4H, m. 2(CH2)) and 1.32 (6 H, t. J7.0.

2(CH3)); 6p(80 MHz; CDC13) 3.9 (1 P, t, JpF 117.0); &(188 MHz; CDC13) -31 -9 (1 F. d. JFP

1 17.0); 6,(lOO MHz; CDCl3) 142.7 (d), 129.0 (m), 127.9 (td), 1 18.9, 1 17.7 (td. JCF 262.9 &

J C p 219.7. CF2), 64.7 (d, J c p 6.6, 2(CH2)) and 16.3 (d, JCp 5.1, 2(CH3)); ~~(v lc rn- ' ; neat)

2 149.1 cm-'; rn/z (EI) 306 (MH', 5%), 169 (MW - PO(OEt)2, 25), 109 (MW -197. 100):

Found Ml', 305 .O74 1. Cl ,H i4FzN303P requires m l . 305.0747.

Preparation of Ammonium Salts of a,a-Difluoromethylenephospbonic Acids.

Deprotection of a,a-difluoromethylenephosphonate esters was accomplished using the

general procedure described below.

General Procedure for the Deprotection of a,a-Di2luoromethyIenephosphonate Esters.

To a solution of the a,a-difluoromethylenephosphonate ester in anhydrous CH2CI2

(approxirnately 1 cm3 CH2C12/0.1 mm01 phosphonate) was added TMSBr (approximately 1.5

equivalents of TMSBr per methyl or ethyl group). The solution was stirred at room

temperature for 12 hours (for methyl esters) or at reflux for 36 hours (for ethyl esters). The

solution was concentrated and the residue subjected to high vacuum for several hours. The

residue was dissolved in CHzClz (approximately 1 cm3 CHzC12/0.1 mm01 phosphonate) and a

solution of N&HC03 (2 equivalents per silyl ester moiety) in water (approxirnately 15 cm'

water!gram W H C 0 3 ) was added. The biphasic mixture was stirred vigorously for 30-60

minutes and the CH2C12 layer removed by rotary evaporation. The aqueous layer was then

lyophilized several times (3-4 tirnes) leaving the desired a,a-difluoromethylenephosphonic

acids as their ammonium salts (white flw powders) in near quantitative yields.

a,a-Difluorobenzylpbospboaic acid, ammonium salt (3.33). Prepared using the general

procedure from 3.3.. 6&00 MHz; DzO) 7.62 (2 H, m, aryi) and 7.49 (3 H, m, aryl); 6p(80

MHz: DzO) 5.8 (1 P, t, JPF 91.6); S~(188 MHz; D2O) -27.5 (1 F, d. J F ~ 91.6); &(100 MHz;

DzO) 137.2 (br td), 130.4, 129.0, 126.7 (br t), 122.8 (td, CF?); m/r (FAB) 207 ( M - ~ +- 1 H'?

100%).

4-[(Phosphono)difluoromethyl]benzoic acid, ammonium salt (3.34). Prepared using the

general procedure fiom 4-[@imethylphosphono)difluoromethyl] benzoic acid. 6"(200 MHz;

H 2 0 ) 7.89 (2 H, d, J 8.8, aryl) and 7.63 (2 H, d, J8.8, aryl); 6p(80 MHz; D20) 4.9 (1 P, t, JpF

97.7); 6~(188 MHz; D2O) 8.1 (1 F, d, J F ~ 97.7); &(100 MHz; D20) 175.7 (CO), 139.0 (td),

138.4, 129.6, 126.8 (t), 121 -9 (td, JcF 261.8 & JCp 189.2, CF2); m/z (FAB) 251 (M') + 2W,

100%).

4-[(Phosphono)difiuorometh~l]bromobenzeae, ammonium salt (3.35). Prepared using the

general procedure from 3.20.6~(200 MHz; D2O) 7.52 (2 H, d, J 8.4, aryl), 7.40 (2 H, d, J 8.4,

aryl); 6p(80 MHz; DzO) 5.4 (1 P, br t, JpF 91.5); 64188 MHz; D20) -28.0 (1 F, d, J F ~ 91.5);

6,(100 MHz; D20) 136.3 @r td), 13 1.9. 128.6 (t), 124.0, 122.0 (td. CF2); rdz (FAB) 285 ( M - ~

+ 1 W. 99%).

4-[(Phosphono)difiuoromethyl)nitrobenzene, ammonium salt (3.36). Prepared using the

general procedure from 3.18. 6~(200 MHz; D20) 8.19 (2 H, d, J 8.8, aryl) and 7.72 (2 H1 d, J

8.8, ~ 1 ) ; 6p(80 MHz; &O) 4.8 (1 P, t, JpF 89.0); ijF(188 MHz, D2O) -32.7 (1 F, d, JFP 89.0):

6,(100 MHz; DzO) 149.4, 149.7, 145.4 (br td), 128.7, 124.2, 122.9 (td, CF2); d z (FAB) 252

(M -2 + 1 H+, 99%).

Benzyl 4-[@hosphono)difiuoromethyl)be~oate, ammonium salt (3.37). Prepared using

the general procedure from 3.22. 6~(200 MHz; D20) 7.72 (2 H, br s, aryl), 7.52 (2 H. br S.

aryl), 7.03 (5 H, br s, aryl) and 4.94 (2 H, s, CH2); 6p(80 MHz; DzO) 4.7 (1 P, t, JpF 103.8):

ijF(188 MHz; DzO) -30.4 (1 F, d, JFp 103.8); 6,(100 M m , D20) 167.8 (CO), 139.7 (td), 136.4.

131.8, 130.2, 129.4, 129.1, 128.8, 127.1 (br t), 120.1 (td, JiF 260.4 & JCp 202.1, CF?) and 67.9

(S. CH2); m/i. (FAB) 341 (M2 + lHr, 100%).

4-[(Phosphono)difluoromethylJbenzophenone, ammonium salt (3.38). Prepared using the

general procedure fiom 3.24. 6~(200 MHz; DzO) 7.68 (7 H, br s, aryl) and 7.53 (2 H, br S.

aryl); 6p(80 MHz; DzO) 5.6 (1 P, t, J p F 92.0); 6~(188 MHz; DzO) -28.5 (1 FI d. JFp 92.0):

&(100 MHz; DzO) 201.5 (CO), 142.5 (td), 138.5, 137.7, 134.6, 131.3, 130.8, 129.6, 127.1 (t).

123.5 (td, JCF 262.2 & Jcp 180.2, CF2); ml' (Fm) 3 1 1 ( M - ~ + 1 H?, 100%).

2-[4-((Phosphono)düiuoromethyl)phenylJbenzene, ammonium salt (3.39). Prepared using

the general procedure fiom 3.27.8~(200 MHz; DzO) 7.87 (1 H, br s, aryl), 7.46 (7 H, rn, aryl)

and 7.24 (1 H, br s, aryl); 6p(80 MHz; D20) 5.4 (1 P, t, J ~ F 97.6); 6~(188 MHz; D20) -17.8 (1

F, d, JFP 97.6); Ôc(lOO MHz; D20) 143.4, 141.8, 133.2, 130.6, 130.4, 129.5 (t), 128.4, 128.2,

127.9, 123.1 (td, CF2); mir (FAB) 283 (M2 + 1H+, 100%).

3-(4-((Phosphono)dif luoromethyl)pheny~, ammonium salt (3.40). Prepared using

the general procedure fiom 3.26. SH(200 MHz; D20) 7.91 (1 H, s, aryi) and 7.57 (8 H, m.

aryl); tip(80 MHz; D2O) 5.9 (1 P, t, JpF 91 S); 6 ~ ( l 88 MHz; D20) -27.4 (1 F, d, JFP 9 1 -5):

6,(100 MHz; D20) 141.2 (d), 138.4 (td), 130.1, 129.8, 128.9, 128.1, 126.1 (t), 125.4 (t), 123.0

(td, CF2); m/z (FAB) 283 (M-2 + 1 W, 100%).

4-(4-((Phosphono)difhorometbyl)pbcayl]benzene, ammonium salt (3.41). Prepared using

the general procedure fiom 3.25. SH(200 MHz; D20) 7.72 (6 H, m, aryl) and 7.50 (3 H. m.

aryl); 6p(80 MHz; D20) 5.8 (1 P, br t, J p F 92.0); 6d 188 MHz; D20) -27.2 (1 F, d, JFp 92.0);

6c(100 MHz; DzO) 142.4, 141.0, 136.9 (br td), 130.1, 129.0, 128.1, 127.6 (br s), 127.5 (br s).

1 23. l (td, CF2); nt/r (FAB) 283 (M-~ + 1 Ht, 100%).

3,3-(4-((Phosphono)dinuoromethyl)di-phenbeee, ammonium salt (3.42). Prepared

using the general procedure fiom 3.28. 8~(200 MHz; D20) 7.77 (3 H, s, aryl), 7.42 (5 H, br d.

aryl) and 7.24-7.1 1 (5 H, m, aryl); 6p(80 MHz; DzO) 5.8 (1 P, t, JPF 88.5); ZiF(188 MHz; D20)

-27.6 (1 F, d, JFP 88.5); 6,(100 MHz; DzO) 141.7, 140.8, 139.0 (m), 129.9, 128.7. 127.9,

1 27.0, 124.6 (br t) and 1 19.8 (td, JcF 264.5 & Jcp 216.4, CF2); rn/z (Fm) 359 (c, 100%).

3,3'-Bis[(phosphono)difluorornethyl1biphenyl, ammonium salt (3.47). Prepared using the

general procedure fiom 3.51a. 6~(200 MHz; D20) 7.91 (2 H, s, aryl), 7.77 (2 H, s, aryl), 7.58

(4 H, m, aryl); 6p(80 MHz; &O) 6.90 (1 P, bs, J ~ F 94.6); S~(188 MHz; D20) -27.3 (1 F, d, J F ~

94.6); 6,(100 MHz; D20) 140.78, 137.40 (br td), 129.83, 129.23, 126.19 (br t), 125.33 (br t);

m/z (FAB) 413 ( M ~ + 3H+, 100%).

4,4'-Bis[(phosphono)difluoromethyllbiphenyl, ammonium salt (3.48). Prepared using the

general procedure from 3.Slb. 6~(200 MHz; DzO) 7.70 (4 H, d, J 8.8, aryl), 7.78 (4 H, d, J

8.4, aryl); Sp(80 MHz; D20) 6.18 (1 P, br t, J ~ F 93.9); &(188 MHz; DzO) -27.5 (1 F. d, JFP

93.9); 6,(100 MHz; DzO) 141 -8, 137.2 (br td), 127.6 (t), 123.1 (br td, CF2); m/z (FAB) 4 13 (M-

d + 3H+. 5 1%).

4-[4-((Phosphono)dinuoromethyl)benzeneJbe~lphosphoic acid, ammonium salt (3.52).

Prepared using the general procedure fiom 3.55. 6~(200 MHz; D20) 7.66 (6 H, br s, aryl).

7.39 (2 H, br S. aryl) and 3.03 (2 H, d, JHp 20.5, CH2P); 6p(80 MHz; D20) 21.21 (1 P. s): 6.2

(1 P, br s); &(Il38 MHz; D20) -37.1 (1 F, d, JFp 94.6); 6,( 100 MHz; DzO) 142.3, 138.5. 136.5

(d). 136.5 (m), 13 1.2 (d), 127.9. 127.6 (br t). 127.3, 123.0 (weak td, CF2) and 36.6 (d, JCp

126.7, CH2P); m/'(FAB) 377 ( M ~ + 3W, 100%).

1,4'-bis(phosphonomethyl)biphenyl, ammonium salt (3.53). Prepared using the general

procedure fiom 3.50b. 6~(200 MHz; D20) 7.69 (4 H, d, J 7.7, aryl), 7.45 (4 H. d, J 8.0. aryI).

3.10 (4 H, d, J20.8, CH2P); 6p(80 MHz; D20) 6.18 (1 P, s, 21.0); 6,(100 MHz; D20) 151.6

(d), 148.2 (d), 143.7 (d), 140.2,49.1 (d, J c p 128.6, CH2P); m/z (FAB) 341 (M-' + 3IT. 100°/a).

4-[(Pbospbono)difluoromethyl)azidobenzene, ammonium salt (3.61). Prepared using the

general procedure from 3.64. 6~(200 MHz; DzO) 7.60 (2 H, d, J 8.4. aryl), 7.18 (2 H. d. J 8.5.

aryl); 6p(80 MHz; D20) 4.6 (1 P, t, J ~ F 104.5); S~(188 MHz; D20) -29.2 (1 F, d, J F ~ 104.5);

&(100 MHz; DzO) 142.6, 131.6, 128.4, 119.7, 120.8 (td. JCF 256.6 & Jcp 199.2, CFZ): IR

( v l a n - ' ; i(Br disc) 2147.3 cm"; m/r (FAB) 248 (M-* + 1 H+, 100%).

1,4'-Bis[(phospbono)difluoromethyl J benzophenone, ammonium salt (3.70). Prepared using

the general procedure fiom 3.72. 6~(200 MHz; D2O) 7.87 (4 H, d, J 7.3, aryl) and 7.77 (4 H.

d. J 8.8, ~ 1 ) ; 6p(80 MHz; &O) 5.4 (1 P, t, JPF 92.0); 6~(188 MHz; D20) -28.6 (1 F, d. JFp

92.0); 6,(100 MHz; D2O) 201 .O (CO), 142.2 (td), 138.3, 130.9, 127.0 (t), 122.2 (td, JCF 257.8

& JCP 186.3, 2(CFz)); m./' (FAB) 441 (M4 + 3w, 26%).

Kinetic Studies with PTPlB. Kinetic studies with PTPlB were perfonned as described in

Chapter 2. section 2.2.1. ICso determinations were determined in duplicate at 9 or 10 different

inhibitor concentrations with FDP at Km concentration. Ki's were detennined by measuring

the initial rate (v) using various FDP concentrations (20, 25' 35, 50 and 100 ph4 FDP) at

various fixed concentrations of inhibitor. I/v vs l/[S] was plotted as shown in the following

equation:

1 = ((KmNmax)( 1 + [IlKi)) 1 Ils] + 1 N m a x

The slopes of these plots {(Km/Vmax)(l+ [T1Ki)) were determined using the program

Sigrnaplot or Excel. These slopes were replotted against the concentration of inhibitor and the

K,'s were obtained fiom the X-intercepts of these replots. AH Ki studies were determined

using Bis-Tris as buffer and were performed in duplicate.

Kinetic Studies with CD45. Kinetic studies with CD45 were performed as described for

PTP 1 B in Chapter 2, section 2.2.1 using a final concentration of 0.2 &cm3 of CD45.


3.3.1 SYNTHESIS OF ARYL DFMPs BY ELECTROPHILIC FLUORINATION

In Differding's original papa on the electrophilic fluorination of a-carbanions of non-

benzylic phosphonates, the reaction was carried out in two steps and the yield of fluorinated

product was found to be dependent on the nature of the cation in that KDA gave significantly

better yields than LDA.'~' NO explmations for this were given. Although Taylor and

coworkers found that benzylic a,a-difluorophosphonates could be prepared in modest to good

yields in a single step using NaHMDS as base,Iz6 the effect of base and counterion was not

examined. Thus, our first objective was to study the effect the base and counterion has on the

yield of benzylic a,a-difluorophosphonates using the one-step electrophilic fluorination

procedure. Utilizing commercially available diethyl benzylphosphonate 3.5 as a mode1

substrate, the fluorination reaction was performed using the one-step procedure with a variety

of bases and counterions. The results of these studies are shown in Table 3.1. In general.

hexamethyldisilazide bases gave equivalent or better yields than diisopropylamide bases.

Consistent with DiEerding's original report,I2' the yield depended strongly on the nature of the

cation. KDA gave a better yield than LDA although the yields using either base were Iow (33-

39%). Using a series of hexarnethyldisilazide bases, best yields were obtained using

Table 3.1. Effect of base and counterion on the electrophilic fluorination of 3.5 with NFSi.

-78 OC. 2. 2.5 e q NFSi, THF.

* O -78 OC

Base % Yield of 3.3b

KDA LDA

NaHMDS

KHMDS LiHMDS

sodium hexarnethyldisilazide (NaHMDS) followed by KHMDS and then LiHMDS. Thus it

t m e d out that the base (NaHMDS) utilized by Taylor and coworkers in their original report is

the best one. The. yield obtained here using NaHMDS as base was the same as that obtained

by Taylor and coworkers in their earlier studies. Performing the reaction with NaHMDS in

two steps a d o r at temperatures below -78 O C did not improve the yield. In alrnost al1 cases,

the product was readily obtained by performing a single purification by chromatography. The

effect of the counterion on the yield of the fluorination reaction is dificult to rationalize. We

are not aware of any studies on the differences in coordination of Na K or Li counterions with

phosphory 1-stabilized carbanions at low temperatures.

To compare our electrophilic fluorination procedure to other methods for preparing

aryl DFMPs, we synthesized the difluorophosphonates 3.3a and 3.3b via the DAST procedure

(Scheme 3. I).~' Employing dimethyl and diethyl benzoylphosphonates as mode1 substrates,

the fluorination reactions were performed using the procedure of Burke and cow~rkers.~' This

invoived reacting the ketophosphonates with neat DAST (5 equivalents) under argon at -78 OC

for several hours. This gave 3.3a and 3.3b in yields of 38% and 40%, respectively. which is

considerably less than the yield we obtained for 33b (79%) using our electrophilic

fluorination procedure. We d s o found that purification required several flash columns when

using the DAST procedure. Thus, the NFSi procedure is superior to the DAST procedure in

terrns of economy, yield and ease of purification. The yield of 3.3b obtained using our

electrophiiic fluorination method is alrnost identical to that obtained by Burton and Qiu via

CuCl-promoted coupling of (diethylphosphonyl)difluoromethylcadmium reagent with

iodobenzene (80%)'" and is superior to that obtained by Shibuya and coworken via CuBr-

promoted coupling of (diethy1phosphonyl)difluoromethylzinc reagent with iodobenzene.12'

To explore the scope of our electrophilic fluorination method a variety of substituted

benzyl phosphonates (3.6-3.17) were prepared and subjected to our electrophilic fluorination

conditions (2.2 equiv. NaHMDS, -78 OC in THF, followed by 2.5 equiv. of NFSi; Scheme

3.5). The benzylic phosphonates were prepared in two steps via an Arbuzov reaction between

trimethyl or triethyl phosphite and benzylic bromides, which were either commercially

benzene, reflux

Y p(oR)2 1. 2.2 ea. NaHMDS. THF, -78 OC t

V 2. 2.5 eq. NFSi, A THF, -78 O C

X = H, ester, ether, bromo, keto, phenyl, nitro

R = Me or Et

Scheme 3.5. Synthesis of substituted aryl(a,a-difluoromethylenephosphonates) by electrophilic fluorination using NFSi.

available or could be prepared in a single step by bromination of commercially available

toluene derivatives. In general, good yields were obtained for most of the fluorination

reactions (3.3a, 3.18-3.28; Table 3.2). Ethyl and methyl phosphonate esters could be used.

Although in most instances good yields were obtained using either ester, in general. slightly

better yields were obtained using the ethyl phosphonate esters. Both electron rich and electron

poor functional groups could be tolerated. The decrease in yield of the orrho-phenyl derivative

3.27 (46%) compared to the mefa-phenyl 3.26 and para-phenyl 3-25 derivatives (60% and

59'33, respectively) can be attributed to the increased steric demands of the orfho-substituted

phosphonate compared to the meta- and paru-substituted phosphonates. However, this effect

is relatively modest (13-14%), suggesting that the reaction is not overly sensitive to bulky

substituents at the ortho-position.

Fluonnation of benzylic phosphonates that contained an additional benzylic moiety at

the para-position using NFSi were unsuccessfu1. For example, selective fluorination of 3.29

(Scheme 3.6), which contains a benzyl carbon ester para to the phosphonate. was attempted

since the fluorination of benzylic carbon esters is known to proceed very poorly using

Table 3.2. Electrophilic fluorination of benzylic phosphonates with NFSi.

Substrate Product % Yield O O

II ~Z-f'<OR,,

X X

NaHMDS as base.13' However, neither the desired product nor any material arising from

mono- or difluorination of both the phosphonate and carbon ester methylene units could be

~7%: 1. NaHMDS, THF, -78 OC

2. NFSi X THF, -78 OC X

3.29, RI = OMe, X = CH2COOBn 3.31, RI = OMe. X = CH2COOBn 3.30, RI = OMe, X = CHzCOOH 3.32, RI = OMe, X = CH,COOH

Scheme 3.6. Attempted fluorination of benzylic phosphonates 3.29 and 3.30.

isolated and only unidentified decomposition products were obtained. Selective fluorinat ion

of the phosphonate 3.30 bearing the unprotected acid was also attempted. Reacting the

phosphonate 3.30 with 3.3 equiv. NaHMDS and 2.5 equiv. NFSi, we reasoned that initial

formation of the carboxylate anion would subsequentiy favor deprotonation of the methylene

unit next to the phosphonate. However, once again, only unidentified decomposition products

were obtained. The lack of formation of 3.31 and 3.32 may be due to the elimination of

fluorine fiom the initially formed monofluorinated products, as shown in Scheme 3.7, yielding

an intermediate, which may undergo m e r reaction.

____)

RI = COOBn

Scheme 3.7. Possible route for decomposition products preventing formation of 3.31 and 3.32.

In preparation for their evaluation as PTP inhibitors, al1 the a,a-

difluoromethylenephosphonates were converted to their corresponding salts via trimethylsilyl

bromide (TMSBr) in methylene chloride, followed by the addition of an aqueous solution of

ammonium bicarbonate. Removal of the organic layer in vacuo, followed by repeated

lyophilizations, yielded the a,a-difluoromethylenephosphonic acids as their ammonium salts

in near quantitative yields (Scheme 3.8). For compound 3.22, the benzyl group was first

removed by hydrogenation (Hz, P m , 90% yield) and then subjected to TMSBr/amrnonium

bicarbonate yielding the para-COOH compound (3.34; Table 3.3).

1. TMSBr (3 eq.), CH2CI2 Q 2- NHif~COi (3eq.) * Q X = Hl ester, ether,

bromo, keto, phenyl, nitro

R = Me or Et

Scheme 3.8. Conversion of aryl difluorophosphonates into their ammonium salts.

3.3.2 PTP INHIBITION STUDIES WITH DIFLUOROPHOSPHONATE SALTS

Salts 3.33-3.42 were examined for PTPIB inhibition. In addition, to see if any of these

compounds exhibited PTP selectivity, we a i s 0 examined their ability to inhibit CD45 CD45

is a transmembrane PTP expressed on the surface of hematopoietic cells. It is believed to be

responsibk for the dephosphorylation of Src PTK's, which results in the up-regulation of their

catalytic activity that eventually leads to ce11 a~t iva t i0n . l~~

An initial assessrnent of the inbibitory effects of these derivatives was performed

using 500 p M inhibitor with PTPlB or CD45 and FDP as substrate at a concentration equal to

its Km value (20 FM) at pH 6.5. The results, in terms of percent inhibition, are sumrnarized in

Table 3.3. Consistent with Burkes' studies,IO' the wubstituted compound, 3.33, is a very

poor inhibitor of PTPIB, resulting in only 41% inhibition at 500 FM. For PTPl B.

substitution at the para-position with non-aromatic moieties (3.34-3.36) did not significantly

improve the inhibition and, in one instance (3.34, the p-COOH compound) yielded a worse

inhibitor. Similarly, substitution at the para-position with aromatic groups (3.37, 3.38 and

3.41) did not significantiy improve the inhibition. However, addition of a phenyl group at the

Table 3.3. Percent inhibition of PTPI B and CD45 with 500 FM a,a-di fluoromethy lenephosphonates 3.33-3.42.

3.42, X = di-m-Ph 93 i 1 ND

'Errors are reported as * S.D.

mera-position (3.40) significantly improved the inhibitor potency. This appeared to be

entirely unique to the meta-substitution since the para-phenyl and ortho-phenyl substituted

derivatives (3.41 and 3.39) were moderate @ara-substituted) to very poor (ortho-substituted)

inhibitors. The ICso's of the unsubstituted compound 3.33 and the mefa-phenyl derivative

3.40 with PTP l B were detemined to be 6 10 p M and 35 FM, respectively. Thus, substitution

of benzyl difluoromethylenephosphonic acid with a mefa-phenyl group increases the

inhibition by a remarkable 17-fold. The meta-phenyl denvative is an even more potent

inhi bitor than the naphthyl derivatives 1-19 and 1.20. The lCIo for the para-phenyl derivative

(3.41) was only 2 10 FM. Since the percent inhibition of compounds 335-338 was v e y

similar to 3.41, it is lïkely that they would exhibit similar 1C5{s. Al1 compounds, including

the meta-phenyl denvative 3.40, were poor inhibitors of CD45 Thus, some inhibitory

selectivity c m be achieved using relatively simple aryl DFMP compounds.

We noted that the results in Table 3.3 followed the same trend as that observed by

Montserat et al. for the Km's of the andogous phosphate substrates with rat PTP 1. ' l 9 For

example, these workers have reported that para-carboxyphenyl phosphate exhibits a higher

Km (2.1 mM) than p-nitrophenyl phosphate (0.68 mM) with rat PTP1. Similarly, our studies

show that the para-carboxy-a,a-difluorobenzylphosphonic acid 3.34 is a poorer inhibitor of

human PTP 1 B than the para-nitro-a,a-difluorobenzyIphosphonic acid 3.36. Although meta-

substituted phenyl phosphates were usually poorer substrates than their para-substituted

analogues with rat PTP 1, l9 meta-phenyl phenylphosphate was an exception. The Km

reported for para-phenyl phenylphosphate, 2.4 mM, is approximately 7 times greater than that

reported for meta-phenyl phenylphosphate (0.32 mM).'19 Similady, the ICSo determined for

the para-phenyl derivative 3.41 (210 FM) is six times greater than the mefa-phenyl-a,a-

difluorobenzylphosphonic acid 3.40 (35 PM). However, non-peptidyl substrates meta-

substituted with aliphatic hydrocarbons (Le. ethyl, isopropyl) exhibited higher Km's than meta-

phenyl phenylphosphate with rat PTP1."9 This suggests that the effect observed with the

rneta-phenyl substituted compounds may involve more than just simple hydrophobic

interactions and may possibly involve pi-staclcing interactions with an aromatic residue

located close to the active site.

The above results suggest that studies using phenyl-based, non-peptidyl substrates

with rat PTPl could be used as a guide for the development of human PTPlB inhibitors

bearing the DFMP group. We therefore decided to examine additional DFMP-bearing

structures based on substrate studies with rat PTP1. An important observation made by

researchers at Merck-Frosst Inc. was that fluorescein diphosphate (FDP, 3.43) has a

significantly lower Km and hydrolysis of the first phosphate group occurs much faster than

fluorescein monophosphate (3.44) with PTPIB."* This is consistent with the studies by

Montserat er al. in that phenyl phosphate substrates bearing an additional charged moiety

(phosphate or carboxyiate) remote fiom the phosphate group exhibited very low Km's with rat

PTP1.1i9 This was especidly evident for certain symmetrical substrates bearing two

phosphate groups and one of these (3.45) is the most potent low molecular weight substrate

ever reported for a PTP. 3.45 exhibits a Km (16 PM) 19 times Iower than its mono-

phosphorylated analogue (3.46) and is almost as low as the best peptidyl substrates.

Consequently, we reasoned that an additional DFMP group on inhibitors such as 3.40 and

3.41 may yield very potent PTPI B inhibitors.

Taylor and coworkers have reported that naphthyl denvatives bearing two a,a-

difluorornethylenephosphonate esters could be prepared by reacting their non-fluoro analogues

with 5.5 equiv. NaHMDS followed by the addition of 7.3 equiv. N F S ~ . ' ~ ~ Consequently, we

reasoned that this approach could be used for the preparation of 3.47 and 3.48 (Scheme 3.9).

Thus. bis-benzyl bromides 3.49a and 3.49b were reacted with trimethyl phosphite to give the

bis-phosphonates 3.50s and 3.50b in near quantitative yields. Compounds 3.50~ and 3.50b

were reacted with 5.5 equiv. NaHMDS in TKF followed by the addition of 7.3 equiv. of NFSi

to give fluoro denvatives 3.51a and 3.51b in 29% and 34% yield, respectively. This is similar

to the yields obtaïned by Taylor and coworkers for the fluorination of bis-phosphonate

naphthyl c ~ r n ~ o u n d s . ' ~ ~ Reaction of 3.51. and 3.51b with TMSBrmHCO, gave the

desired DFMP compounds 3.47 and 3.48 in quantitative yields, respectively.

benzene, reflux

3.49a, meta-derivative 3.50a,b b, para-denvative

1. 5.5 eq. NaHMDS, THF, -78 OC

2. 7.3 eq. NFSi, THF, -78 OC

1.6 eq. TMSBr, (MO)2OPCFq CH&

3.47 or 3.48 4 2. NH4HC03 aq.

Scheme 3.9. Preparation of bis-DFMP biphenyl derivatives via electrophilic fluorination using NFSi.

Although, the meia bis-DFMP compound 3.47 was found to be a slightly poorer

inhibitor than the mono-DFMP compound 3.40, the para bis-DFMP analogue 3.48 completely

inhibited PTP 1 B at 500 pM and exhibited an ICso of 9.9 p M (Table 3.4). Compound 3.48 was

found to be a cornpetitive inhibitor exhibiting a Ki of 4.5 * I p M (Figure 3.1).

Table3.4. InhibitionofPTPlB with3.40,3.41,3.47,3.48,3.52 and 3.53.

% inhibition Phosphonate with 500 ph4 ICso (pM) Ki (CiM)

3.53 9* 1 2025 + 1 ND 'Errors rire reported as * S.D.

To ascertain if the fluorines are absolutely essential for potent inhibition. two

analogues of 3.48 were prepared in which al1 of the fluorines were removed fiom one (3.52) or

both (3.53) of the DFMP groups and these were examined for PTP 1 B inhibition. Cornpound

3.52 was prepared as outlined in Scheme 3.10. Suzuki coupling of 3.21 and para-

methylphenylboronic acid yielded the biphenyl derivative 3.54 in 71% yield. Free radical

bromination at the methyl group using M3S followed by an Arbuzov reaction with triethyl

phosphite yielded bis-phosphonate 3.55 in 73% yield. Removal of the ethyl groups in 3.55

using TMSBr, followed by treatment of the resulting TMS ester with ammonium bicarbonate,

gave the ammonium salt of 3.52 in 88% yield.

Figure 3.1. Inhibition of PTP 1 B by compound 3.48. (a) The activity of PTP 1 B (O. 15 pg/cm3) was measured at pH 6.5 as described under 'experimental procedures' in the presence of the following concentrations of 3.48: (a), O $M; (a), 5 FM; (A), 8 pM; (- ), 10 FM; (e), 12 FM; (r), 15 PM. (b) Replot of the slope fiom the double-reciprocal pIot versus concentrations of compound 3.48.

degassed ethanol 3.54

I P(OEt)3, reflux

Scheme 3.10. Synthetic scheme for the synthesis of 3.52.

The non-fluorinated analogue, 3.53, was a very poor inhibitor of PTP 1 B and exhibited

an IC so (2025 p.M; Table 3.4) approxirnately 200-fold greater than that of 3.48. Compound

3.52 exhibited an ICso of 51 FM (Table 3.4). More in-depth kinetic studies reveaied that this

compound is a cornpetitive inhibitor of PTPlB exhibiting a K i of 22 p M (Figure 3.2). Thus.

for this biphenyl series of bis-phosphonates, two fluorines alpha to just one of the phosphonate

groups enhance the enzymes afinity for the bis-phosphonate by approximately 40-fold.

However, the addition of two more a-fluorines on the second phosphonate group results in a

mere 5-fold afftnity enhancement.

Although there is no disputing the enhanced binding effect of the fluorines alpha to the

phosphonate group, their mode of action is controversial. There have k e n two mainstream

ideas regarding this phenornenon. One is that the fluorines may increase the by H-

Figure 3.2. Inhibition of PTPlB by compound 3.52. (a) The activity of PTP1 B (0.1 5 pg/cm3) was measured at pH 6.5 as described under 'experimentd procedures' in the presence of the following concentrations of 3.52: (e), O PM; ( i ), 1 O FM; (A ), 25 FM; (x), 40 PM; (+), 50 PM; (O), 75 pM. (b) Replot of the dope fkom the double-reciprocal plot versus concentrations of compound 3.52.

bonding with specific residues. The other focuses on pK, effects: at pH 6-7, the fluorinated

phosphonates are believed to bind tighter because they are completely ionized as compared to

the partially ionized non-fluorinated derivatives. To detennine whether the fluorines in 3.48

enhance binding via a pK, effect or some other type of interaction such as H-bonding, we

measured the inhibition constant of 3.48 as a function of pH. The second ionization constant

(pK,) of a,a-difluorobenzylphosphonic acid has been determinedB7 to be 5.71 and it is likely

that the second ionization constants of the individual DFMP groups on 3.48 will be similar.

Thus, we deterrnined the Ki of 3.48 with PTPlB at pH 5.5 and 7.2 (2.7 and 5.5 PM,

respectively; Lineweaver-Burk plots not show). Only a small difierence in K,'s over this pH

range (5.5, 6.5 and 7.2) was found, indicating that the dianionic and monoanionic forms bind

equally well and that inhibition is probably not due to pK, effects. This result is consistent

with a study by Chen et al. who found that F2Pmp-bearing peptides inhibit rat PTP 1 -catalyzed

dephosphorylation of pTyr-containing peptides in a pH-independent manner between 5.0 and

7 . 0 . ~ ~ This is also consistent with observations made by Burke and coworkers on the crystal

structure of 1.19 compiexed with PTPIB? It appears that the two fluorine atoms interact

with the phenyl ring of Pheigz via Van der Waal's contacts. Most irnportantly. the pro-R

fluorine appears to form an unusually strong H-bond with the amide group of Phelsz. This

phenomenon will be discussed in more detail in Chapter 4.

Based on the report that diphenylmethyl derived phosphates exhibited a greater af ini ty

for rat PTPl than their biphenyl phosphate derivati~es,"~ the bis-phosphonates 3.56-3.59

(Table 3.5) were synthesized" and examined for inhibition of PTPlB (Table 3.5). The 1,2-

diphenylethane derivative 3.57 was a better inhibitor than 3.48, yielding an ICso of 6.8 PM.

Any M e r increase in the length of the hydrocarbon Iinker ami resulted in only slightly better

inhibitors than 3.57 with the 1,4-biphenylbutane denvative, 3.59, exhibiting the best inhibition

with an ICso of 4.4 The inhibition of PTPl B with 3.59 was found to be competitive and a

K i of 1.5 pM (Figure 3.3) was detennined.

Table 3.5. ICso values for bis-phosphonates, 3.56- 3.59 with PTP 1 B.

3.59a, n = 4 4.4 k 0.4 'Prepared by Nicole Dinaut b Determined by author. 'Errors are reported as * S.D.

A number of compounds reported in this study (3.40, 3.42, 3.47, 3.48, 3.52 and 3.56-

3.59) are good inhibitors of PTPlB and are significantly better inhibitors of PTPlB than the

naphthyi DFMP inhibitors 1.19 and 1.20. Under our assay conditions, naphthyl derivative

1.19 exhibits an ICso of 95 * 10 pM with PTPIB. It should also be pointed out that Burke

and coworkers have reported that 1.19 is a good inhibitor of the serine/threonine phosphatase

PP~A."' We have also examined 1.19 wi?h PP2A and found that, under our assay conditions,

it has an ICso of 753 FM, which indicates that 1.19 is approximately 8 times less potent with

PP2A than with PTPIB.'~' We also examined 3.59 with PP2A and found that it has an ICso of

200 k 8 p M which is approximately 45 times greater than 3.59 with PTPIB.'~' Thus, 3.59 is

considerably more potent and selective than 1.19 in terms of selectivity between PP2A and

Figure 3.3. Inhibition of PTP 1 B by compound 3.59. (a) The activity of PTP 1 B (0.15 g/crn3) was measured at pH 6.5 as described under 'experirnental procedures' in the presence of the following concentrations of 3.59: (a), O pM; ( i ) , 1 PM; (A), 3 PM; ( v ), 5 FM; (+), 7 PM; (8), 9 FM. (b) Replot of the siope from the double-reciprocal plot versus concentrations of compound 3.59.

PTPlB. Unfortunately, inhibition studies with 1.20 and PP2A were not reported. It is also

important to note that the inhibitors obtained in this study are readily accessible, starting frorn

commerciaily available starting materials via synthetic routes that require only three to five-

steps.

It is remarkable that, although a,a-difluorobenzylphosphonic acid (3.33) is a very

poor inhibitor of PTPlB, simply by joining two such derivatives together much more potent

inhibitors of PTPlB can be obtained. Why does the presence of more than one phosphonate

or phosphate group enhance the binding of both substrates and inhibitors to PTP's? Fairly

recently. Zhang and coworkers reported the X-ray structure of PTPlB complexed with the

bis-phosphate compound 3.45 or saturathg pTyr. These studies revealed a second aryl

phosphate-binding site.'42 This is a low-affinity, non-catalytic binding site adjacent to the

active site. with Arg24 and kaj4 fonning important ionic interactions with the phosphate

group. These studies also reved that the high affrnity of 3.45 is not a result of one of the

phosphate groups occupying the cataiytic site and the other occupying the low afhity non-

catalytic site. Instead, 3.45 binds in two mutuaiiy exclusive modes. One of the phosphates of

3.45 binds in either the catalytic site or the non-catalytic site while the second phosphate

group is bound by a combination of electrostatic, hydrophobie, aromatic-aromatic and water-

mediated hydrogen bonds in an area slightly removed fiom both the catalytic and non-

catalytic sites. Zhang has suggested that a compound that could occupy both binding sites

simultaneously would be a very potent inhibitor of PTP1 B.

In collaboration with Zongchao Jia, a protein X-ray crystaitographer at Queen's

University, we very recently obtained the crystal structure of 3.59 complexed with PTP 1 B. '43

One of the DFMP groups in 3.59 occupies the high f inity catdytic site. However, the other

DFMP group does not occupy the low affinity site. The second DFMP group binds to PTP 1 B

via a sait bridge with Ar&,, not Are4 or Arg254. The crystal structure of PTP I B, in which the

conserved cysteine has been mutated to a serine (Cys2isSer mutant), complexed with

DADE@Tyr)L, a high affinity peptide substrate, reveals that the preference for acidic residues

at positions P-1 and P-2 in peptide substrates is mainly a result of salt bridges between Ara7

and the acidic side chains at the P-1 and P-2 positions in the peptide.35 It is also likely that

there are additional residues besides k g 4 , that are capable of interacting with anionic groups

since it is known that the presence of acidic residues at positions P-3 and P-4 in peptidyi

substrates aiso enhances their affinity for rat PTPl and PTPIB? Although it is possible that

one of the DFMP groups in the other bis-DFMP compounds (3.56-3.58) occupies the high

afinity catalytic site while the other DFMP group occupies the low affinity site. we think that

this is very unlikely, since changing the distance between the two DFMP groups does not

dramaticaliy increase or decrease their inhibitory potency relative to one another. It is most

likely that the second DFMP group in these compounds interacts with PTPlB via non-

specific, electrostatic interactions.

Very recently, Zhang and coworkers published a paper describing inhibitors of PTP 1 B

bearing two DFMP g-roups.lu The best inhibitor that was reported in this papa was

cornpound 3.60 which exhibited a Ki of 0.93 + 0.03 FM. These workers claimed that the most

likely explanation for the high affinity of this compound for PTPlB is that the two DFMP

groups occupy the two phosphate binding sites. However, this compound is only 1.5 times

more potent than our bis-DFMP inhibitor 3.59, which we know does not occupy both

phosphate binding sites. Thus, either the importance of this second phosphate binding site to

overall binding potency and inhibitor design has been overestimated or Zhang's inhibitor 3.60

does not simultaneously occupy both phosphate binding sites.

3.3.3 DIFLUOROPHOSPHONATES AS POTENTIAL PHOTOAFFINITY LABELS FOR PTP'S

Despite the growing number of potent PTP inhibitors, the precise in vivo fûnction of

the majority of them is still unknown. irreversible enzyme inhibitors, such as photoafinity

labels, are potential tools for elucidating the celluiar function of PTP's. Photoafinity labeling

is based on the modification of a n a d ligand via the incorporation of a chemically inert, yet,

photochemicaIly labile group. Once inadiated, the photolabile group forms a reactive species

that covalently modifies the ligand binding site. PhotoafEnity labeling of proteins has been

used for many years as a means of studying their in vivo fiction. 14'

Zhang and coworkers have used a peptide bearing a benzoy 1 phenylalanine residue as a

photoaffinity label for rat PTPl .146 This was used to identie amino acids involved in

recognition of protein substrates by PTP 1. However, this photoafiity label is peptide-based

and may be subject to proteolytic degradation and thus may not be suitable for ce11 studies.

Consequently, we wished to develop non-peptidyl photoafinity labels for PTP's.

One of the most common functionalities for photoaffinity labeling are aryl azides.

These species, when irradiated with light below 300 nm undergo photochernical

remangement to fonn a highly reactive Ntrene, which forms covalent bonds with the protein

(Scheme 3.1 1). Since PTPlB (and other PTP's) exhibit an f in i ty for aryl DFMPs, we

Scheme 3.1 1. Formation of a nitrene fiom aryl azides and its reaction with peptides.

hypothesized that such species bearing an azido group may be effective photoafinity labels

for PTP's. To test this hypothesis, we decided to prepare compound 3.61 as a mode1

photoaffinity label for PTPlB.

The synthesis of 3.61 himed out to be less than straighâonuard. Our fim attempt to

prepare 3.61 is outiined in Scheme 3.12. Phosphonate 3.8 was hydrogenated using Hz in the

presence of cataiytic 10Y0 Pd/C to form the aniline derivative 3.62 in 64% yield. Diazotization

of 3.62 followed by reaction with NaN3 af5orded aryl azide 3.63 in 83% yield. However,

attempts to fluorhate 3.63 using our electrophilic fluorination conditions were unsuccessful

and only unidentified decomposition products were obtained.

1. 2.2 eq. NaHMDS -78 O C , THF

2. 2.5 eq. NFSi, -78 O C , THF

N3 i Is 1. TMSBr/CH2C12 2. aq. NH4HC03

F 2 % ~ ~ % F2G

W O W 2

Scheme 3.12. First attempt to synthesize 3.61.

Pinhey and coworkers have described a method for preparing aryl azides by reacting

ary 1-lead tetraacetates, generated in situ from ary lboronic acids, with sodium azide. 147.138

Since the aryl bromide 3.20 could be readily prepared via our electrophilic bromination

methodology, we reasoned that this would be an expedient route to the aryl azide 3.65

(Scheme 3.1 3). Unfortunately, repeated attempts to form the arylboronic acid 3.66 from aryl

bromide 3.20 were unsuccesstùl. Performing the first step of the reaction (metallation with n-

BuLi) and then quenching the reaction with DzO followed by NMR analysis indicated that this

step proceeds fairly readily. It is possible that the second step does not proceed well due to the

highly electron withdrawing nature of the protected DFMP group.

1. n-BuLi, -78 OC 2. (iPrO)@ or (Me0)3B 3

3. HCI

1. TMSBr/CH2CI2 - 2. aq. NH4HC03 0

Scheme 3.13. Second attempt to synthesize 3.6 1.

For our third attempt, we reluctantly decided to attempt a synthesis of the aryl a i d e

photoactive probe by the DAST fluorination of the corresponding a-ketophosphonate 3.68

(Scheme 3.14). However, in this case, the formation of the a-ketophosphonate was hindered

by a cornpetitive reaction between the azide and the phosphite. The alternative route of

preparing the ketophosphonate first, followed by diazotization was not attempted since it is

unlikely that the unstable a-ketophosphonate would survive the diazotization conditions. .

TFA, NaN02, NaN3

COOH COOH Cocl

Scheme 3.14. Third attempt to synthesize 3.61.

Success was finaity obtained via the route outlined in Scheme 3.15. We reasoned that

the previously synthesized para-nitro derivative 3.19 could be converted to the a ide 3.64 by

reduction of the nitro group to the amine with Zdacid followed by diazotization and then

reaction of the diazonium salt with NaNj. By using Zdacid for reduction of the nitro group.

f . Zn dust, TFA 2. NaN02 3. NaN3 - $ 1. TMSBr/CH2CI2

2. aq. NH4HC03

Scheme 3.15. Synthesis of 3.61.

the resulting amine would be produced as the stable protonated sait 3.69 as opposed to the free

amine, which we knew was very unstable. Such reductions are normally carried out using

ZnIHCl. However, it was clear that HCl would not be a suitable acid for our reduction since

use of this acid would result in the hydrolysis of the phosphonate. We therefore attempted this

procedure using TFA as the acid and performing al1 three steps as a one-pot procedure without

any isolation of the ammonium intermediate.

Thus, 3.19 was s h e d ovemight in a solution of TFA in the presence of 5 equivalents

of zinc dust. Upon removal of the Zn dust (by filtration) the filtrate was cooled to O O C , 2

equivalents of NaN02 was added and the reaction was stined for 30 minutes. NaN3 was then

added and the reaction stirred at room temperature for an additional 15 minutes. Afier workup

and purification by flash chromatography the desired aryl azide 3.64 was obtained in a

remarkable 76% yield. Removal of the ethyl protecting groups on 3.64 using

TMSBr/N&HC03 gave the desired azide 3.61.

Unfortunately, inhibition studies with a i d e 3.61 revealed that this compound did not

have a very high af'fïnity for PTPlB, exhibiting an lCso of 95 W. For photoaffinity labeling

studies we wished to use a compound that exhibited a higher affinity for PTPlB so as to

rninimize non-specific (non-active site) labeling. By the time we had completed the synthesis

and inhibition studies with 3.61, we had d s o discovered that compounds bearing two aryl

DFMP groups (such as 3.57-3.59) were much better inhibitors of PTPlB than those bearing

one aryl DFMP group. Consequently, we designed benzophenone derivative 3.70 as a

potential photoaffinity label. Benzophenone derivatives have dso been used extensively as

photoaffinity labels. When irradiated with light at 350 nm, these species react preferentially

wi th unreactive C-H bonds (Scheme 3.1 6) .

i H-a bstraction

Scheme 3.16. Reaction of benzophenone with light.

Fortunately, compound 3.70 was considerably easier to prepare than azide 3.61

(Scheme 3.17). Thus, the bis-phosphonate 3.71 was prepared in an overall 61% yield fiorn

4,4'-bis(dimethy1)benzopheaone via an Arbuzov reaction on the bis-benzyl bromide.

Fluorination of 3.71 (5.5 equiv. NaHMDS, 7.3 equiv. NFSi) gave 3.72 in 28% yiefd. This is

approximately half the yield in which the mono(a,a-difluoromethylenephosphonyl)

I 1. 5.5 eq. NaHMDS, THF, -78 OC

2. 7.3 eq. NFSi, THF, -78 OC

Scheme 3.17. Synthesis of benzophenone denvative 3.70.

benzophenone denvative 3.24 was obtained. Reaction o f 3.72 with TMSBr/N&HC03 gave

3.70 in quantitative yield. Inhibition studies with 3.70 revealed that it has an lCso of 15 +_ 1

p M wîth PTPl B. Thus, 3.70 exhibits an affinity for PTPl B that is comparable to Zhang's

peptidyl photoafbity label for rat PTPl (K, = 9 C i ~ ) . ' 4 6 Photoaffinity labeling studies of

PTP I B with 3.70 are currentl y in progress in the Taylor lab.

3.4 CONCLUSIONS

In conclusion, we have demonstrated that a wide variety of aryl(a,a-

difluoromethylenephosphonates) can be synthesized in a one-pot procedure fiom the

corresponding phosphonates via electrophilic fluorination with NFSi. The fluorination

reaction proceeds well in the presence of an array of functional groups such as nitro, bromo.

ketone. ester, phenyl and ether groups. Phenyl and biphenyl derivatives containing two u,a-

difluoromethylenephosphonate groups can also be prepared. This procedure is compatible

with methyl or ethyl phosphonate esters but not with benzylic phosphonates containing an

additional benzylic moiety at the para-position. Conversion of the phosphonate esters to their

ammonium salts could be readily accomplished using TMSBr and aqueous ammonium

bicarbonate. This procedure was also used to prepare a potential photoaffinity label for

PTP 1 B.

The aryl DFMPs prepared using our electrophilic fluorination procedure were

exarnined for PTPl B inhibition. In generai, phenyl derivatives bearing a single DFMP group

were not significantly more potent inhibitors than the parent compound. a , a -

difluorobenzylphosphonic acid, with the exception being the meta-phenyl substituted species.

3.40, which decreased the IC50 by approximately 17-fold relative to a , a -

difluorobenzylphosphonic acid. This compound was not a good inhibitor of CD45 Modeling

studies with 3.40 and PTPlB are currently in progress in the Taylor group to ascertain the

reasons for the enhanced potency of 3.40 as compared to 3.41. Certain compounds bearing

two aryl DFMP moieties were very potent inhibitors with the best one (3.59) exhibiting a Ki of

1.5 pM. Compound 3.48 was exarnined in detail and it was found that the fluorines were

essential for potent inhibition. inhibition was pH-independent between pH 5.5-7.2 suggesting

that both the mono and dianionic forms of the individual DFMP groups bind equally well. X -

ray studies with 3.59 complexed with PTPIB revealed that one of the DFMP groups bound in

the active site while the other formed electrostatic interactions with Ar&,. Overall, this work

demonstrates that non-peptidyl compounds bearing the aryl DFMP group can be potent

inhibitors of PTPIB. To obtain more potent and selective inhibitors of PTPIB, studies are

underway in the Taylor group in which libraries of aryl DFMP derivatives are being prepared

using a combinatonal chemistry approach.

CHAPTER 4

SYNTHESIS AND EVALUATION OF ENANTIOMERICALLY PURE a- MONOFLUOROPHOSPHONIC ACIDS

4.1 INTRODUCTION

Despite the certainty that a-fluorophosphonates are potent PTP inhibitors, the question

of why a-fluorophosphonates are dramatically better inhibitors than their non-fluorinated

counterparts remaïns debatable. As discussed in Chapter 3, two possible explanations have

been put f o ~ a r d . * ~ One is that the PTP's require the dianionic fom of the phosphonates for

binding. At pH 6-7, the non-fluorinated phosphonates exist only partially as the dianions

while the fluorinated derivatives are almost completely ionizedg6 and therefore bind tighter.

However, it has been shown that both peptidyl and non-peptidyl compounds bearing the

DFMP group inhibit PTPlB in a pH-independent manner within the pH range spanning the

pKd of the phosphonate moiety. 86~'49 This indicates that the dianionic and monoanionic forms

of these inhibitors bind equally well and that the enhanced inhibition of the DFMP-bearing

inhibitors compared to their non-fluoro analogues is probably not due to pKa differences.

The other explanation is that the fluorines increase the &nity by H-bonding with

specific residues in the active site. This explanation is supported by data obtained fiom the

crystal structure of 1.19 complexed with PTPlB which was recently reported by Burke and

c o ~ o r k e r s . ~ ~ On the basis of this X-ray structure as well as molecular dynarnics calculations,

Burke has suggested that an unusually strong fluorine hydrogen bond exists between the pro-R

fluorine of 1.19 and the amido group of ~heigz? Burke has proposed, based on molecular

dynamics calculations, that the pro-R fluorine of 1.19 contributes an average of -4.6 kcaYmol

more interaction energy than the pro-S fluorine when binding to the A similar

interaction between PTP 1 B and the pro-R fluorine of a FzPmp-bearing peptide was noted in a

recent rnolecular dynamics study by Tracey and lover.'^' These results suggest that

enantiomericail y pure (R)-a-mono fluorophosphonic acids may be as effective inhibitors of

PTP 1 B as their difluoro analogues. To test this possibility and to gain further insight into the

role of the fluorines in PTP 1 B inhibition, we report here the synthesis of enantiomerically pure

a-monofluorophosphonic acids 4.1-4.6 and their evaluation as inhibitors of hurnan PTP 1 B.

4.3, R-enantiomer 4.4, S-enantiomer

4.2 EXPERIMENTAL

For details c o n c e h g general materiais and instrumentation see Chapter 2 (section

1.2.1 ) and Chapter 3 (section 3.2).

4.2.1 SYNTHESES

General Procedure for the Prepzrration of Phosphonic Acids 4.8,4.f 1 and 4.12.

Trimethylsilyl bromide (TMSBr, 5 equiv.) was added to a solution of either 2-

naphthylrnethylphosphonic acid methyl ester,lz6 biphenyl-3-ylmethylphosphonic acid methyl

esterlsl or benzylphosphonic acid diethyl ester (Aldrich, Milwaukee, Wisconsin, USA) in

anhydrous CHzC12 (approxirnately 5- 10 cm3 CHzClz/rnmol of ester). For the methyl esters, the

solution was stirred under an atmosphere of Nz at room temperature for 12 h. For the ethyl

ester, the solution was refluxed under an atmosphere of N2 for 24 h. The mixture was then

concentrated and placed under high vacuum for 2 h. The product was then redissolved in

CH2C12 and water (approximately 5-10 cm3 HzO/mmol of phosphonate) was added. The

mixture was s h e d vigorously for 30 min at which thne a white precipitate fonned. The

precipitate was then filtered, washed extensively with CHzC12 and recrystallized fiom water.

2-Naphtbylmethylphosphonic acid (4.8). White solid (97% yield); mp 225-227 OC (from

H20) (lit.'S2 mp 229-230 OC); &(200 MHz; CDIOD) 7.82-7.78 (4 H, br m), 7.46-7.44 (3 H. br

m) and 3.29 (2 H, d, JHp 20.5, CH2P); 6&30 MHz; CD30D) 26.3 (lP, s); 6,(100 MHz;

CD30D) 134.9 (d), 133.6 (d), 131.8 (d), 129.4 (d), 129.2 (d), 128.8 (d), 128.5 (d), 127.0, 126.6

and 36.0 (d, Jcp 134.8, CH2P); m/t (ES) 22 1 (1 00%).

Biphenyl-3-ylmethylphosphonie acid (4.1 1). White solid (79% yield); mp 1 72- 1 74 OC (fiom

HzO); 6~(200 MHz; CD30D) 7.61-7.30 (9 H, m), 3.18 (2 H, d, JHP 22.0. CH2P); Sp(80 MHz;

CD30D) 26.3 (lP, s); 6,(100 MHz; CD30D) 142.5 (d), 142.2, 134.8 (d), 129.9, 129.8. 129.6

(d). 128.3, 128.0, 127.8, 126.2 (d), and 35.8 (d, JCp 134.7, CHtP) ; d z (ES) 247 (1 00%).

Benzylphosphonic acid (4.12). White solid (99% yield); mp 17 1 - 1 72OC (from H20) (lit..'j3

1 69- 1 7 1 OC); 6#00 MHz; CD30D) 7.32-7.25 (5 H, m), 5.19 (2 H, s), 3.1 1 (2 H, d, JHP 22.0'

CH2P); &(80 MHz; CD30D) 26.2 (1 P, s); &(IO0 MHz; CD30D) 134.4 (d), 1 3 1 .O (d), 129.4,

127.6 and 36.0 (d, JCp 135.4, CH2P): m/z (ES) 171 (1000/0).

(3aR, 7aR)-(2-Naphthylmethyl)+a,4,5,6,7,7a-octahydro-1 $-dimethyl-1 JJ-benzodiaza-

phospholej-2-oxide (4.9). To a suspension of phosphonic acid 4.8 (2.8 g, 12.7 rnmol) in

anhydrous CH2C12 (25 cm3) and a catalytic quantity of DMF was added oxalyl chloride (4.83

g. 38.1 mmol) over a period of several minutes. The reaction was stirred for 4 h during which

time the reaction mixture became a clear pale yellow solution. The solution was concentrated

by rotary evaporation and placed under high vacuum ovemight during which tirne the solution

solidified yielding the crude phosphonic acid dichloride as a pale yellow solid. To a solution

of the cmde phosphonic acid dichlonde in anhydrous CH2C12 (30 cm3) was added a solution of

tron.s-(R,R)-N,N~-dimethyl-l,2-diaminocyclohexane, 4.7,'" (1.80 g, 12.7 m o l ) and Et3N

(3.57 cm3, 25.6 m o l ) in anhydrous CHzC12 (30 cm3) dropwise over a penod of 30 minutes

under an atmosphere of nitrogen. -Mer stirring for 12 h, the reaction mixture was filtered over

a short pad of Celite and washed with EtOAc (100 cm3). The crude residue was purified by

silica gel column chromatography using Me0H:EtOAc (2:98) as eluent to give pure 4.9 as a

white solid (2.58 g, 62%). mp 106-108 OC; 8~1200 MHz; CDC13) 7.83-7.71 (4 H, m), 7.50-

7.40 (3 H, m). 3.58-3.14 (2 H, m, CHzP), 2.72-2.66 (1 H, m, CHN), 2.60 (3 H. d, J 10.3.

NCH3). 2.42 (3 H, d, J 11.7, NCH,), 2.06-1.65 (5 H, m) and 1.29-0.89 (4 H, m); Sp(80 MHz;

CDC13) 75.5 (1 P1 s); Sc(100 MHz; CDC13) 133.4 (br s), 132.2 (br s), 130.7 (d), 128.5 (br m),

127.6 (br s), 127.4, 126.0, 125.4,64.6 (d, JCp 5.5),64.0 (d, J c ~ 7.3), 35.1 (ci, JCp 107.0, CH2P),

29.5. 28.5 (d, JCP 9.2), 28.1 (rn) and 24.2 (d. JCp 7.3); m/z (EI) 328 (w9 8%). 187 (33, 84

(1 00): Found: [m, 328-1702. CigHzsNzOP requires m/z 328-1705.

Generai Procedure for the Fluorinlition of 4.9 to gïve Diastereomeric (3aR,7aR)-2-il-

fluoronaphthylmethyl]-[3a,4,S,6,7,7a-octahydr0-1~-dimethyl-l~ J-beazodiazaphosphol-

el-2-oxide (4.10). To a solution of phosphonamide 4.9 in anhydrous THF (approximately 5-

10 cm3 THF/mmol of phosphonamide) at - 78 OC was added base (0.95 equiv.) over a period

of 2 minutes. The resulting orange solution was stirred for 2 h at - 78 OC. A solution of NFSi

(1.1 equiv.) in anhydrous THF (appmximately 2 4 cm3 THF/rnmol NFSi) was added over a

penod of 2 minutes, during which time the solution turned from orange to yellow-brown.

After addition, the solution was s h e d for 2 h at - 78 OC, during which time a precipitate

sometimes formed. The reaction was quenched with 10% N b C l and the resulting solution

(precipitate dissolves) was extracted with CHCI3. The combined organic layers were washed

with brine, dried (MgS04) and concentrated in vacuo to give a yellow residue. A reverse

addition procedure was also examined in which 1.1 equiv. of LiHMDS was added to 4.9 (1

equiv .) and NFSi ( 1.1 equiv.) over a penod of 1 h at -78 OC. After stimng an additional 2 h at

-78 OC the reaction was warmed to rt and quenched as described above. Column

chromatography of the crude residue gave 4.10 as a mixture of two diastereomers. For yields

and de's see Table 4.1. 6H(200 MHz; CD30D) 7.96-7.86 (4 H, m), 7.64-7.47 (3 H, m), 6.1 8 &

5.89 (1 H, dd, JHP 4.4, 3.7 and JHF 45.8, 45.4, CHF), 2.82-2.42 (5 H, m), 2.12-1.71 (7 H. m)

and 1.32-0.99 (4 H, m); Sp(80 MHz; CD30D) 37.7 (d, JpF 80.9) & 33.7 (d, JpF 82.4); 6F (188

MHz: CD30D) -1 13.4 (1 F, dd, JFH 45.8 and J F ~ 82.4) & -120.9 (1 F, dd, JW 45.4 and JFp

80.9); 6c(100 MHz: CD30D) 134.9 (d), 134.4, 132.9 (br dd), 129.1 (br m). 128.0 (br m),

126.6 (br dd), 126.0 (br t), 124.9 (br m), 98.0-87.5 (m CHF), 66.3 (d, JCp 6.4), 66.1 (d , JCp

1.6). 65.7 (br s), 65.5 (d, JCp 2.8), 64.9 (d, JCp 7.3), 30.8, 29.8-29.3 (m) and 25.3 (br m); nt'r

(Er) 546 (hr. 66%). 328 (IO), 279 (6), 187 (100), and 153 (12); Found: [M'], 346.1612.

C i9H2aFN20P requires m/i: 346.16 10.

General Procedure for the Preparation of Phosphonamidates 4.13-4.18.

A mixture of (1R,3S)-(-)-ephedrine (1 equiv.) and Et3N (2 equiv.) dissolved in

anhydrous toluene (approximately 1 -2 cm3 toluene/mmol (-)-ephedrine) was added dropwise

to a suspension of the crude phosphonic acid dichloride (1 equiv., prepared !Yom phosphonic

acids 4.8,4.11 and 4.12 using the procedure described above during the preparation of 4.9) in

anhydrous toluene (approximately 2 cm3 toluene/mmol phosphonic acid dichloride) at - 40 O

C, under an aûnosphere of nitrogen. After stirring for 1 h at - 40 OC, the reaction mixture was

stirred at room temperature overnight. The resulting yellow sluny (containing a white

precipitate) was diluted with EtOAc (100 cm3), washed with brine, dried (MgS04) and

concentrated in vacuo. Separation and purification of the tram- and cis-phosphonamidates

4.13-4.18 was achieved by silica gel column chromatography using Me0H:EtOAc (2:98) as

eluent for 4.13,4-14,4-16 and 4.17, and Me0H:EtOAc (3:97) as eluent for 4.15 and 4.18. The

cis-isomers 4.13-4.15 exhibited larger Rf values on silica TLC plates and eluted before the

crans-isomers 4.16-4.18 during column chromatography.

(2R, 4S, ~~)-(2-na~hth~lmeth~l)-3,4-dimeth~l-2~x0-5-~hen~1-2~~-l~~sxnu~hos~ho-

Iidine (4.13). White solid (24%); rnp 1 80- 1 82 OC (fiorn pentane/CHzClz); [alZzo -73 -2 (c

0.0284 in MeOH); 6~(200 MHz; CDC13) 7.82-7.74 (4 H, br m), 7.49-7.40 (3 H, br m), 7.30-

7-19 (5 H, brm), 4.74(1 H, brt, J5.9, HCO), 3.60 (2 H, d, JHp20.5, CHtP), 3.10-3.00 (1 H,

m, HCN), 2.67 (3 H, d, J 8.8, NCH3) and 0.64 (3 H, d, J 5.9, CCH3); 6,(80 MHz; CDC 13) 3 8 -4

(1 P, s); 6,(100 MHz; CDC13) 135.9 (d), 133.1 (d), 13 1.8 (d), 129.7 (d), 127.9 (br m), 127.8,

127.5 (ci), 127.4, 127.2, 126.0, 125.9, 125.6 (br d), 82.2 (PhCO), 58.1 (d. JCP 9.6, CH3CN).

34.4 (d. JCp 1 19.3, CHzP), 28.3 (d, JCp 5.8, NCH3) a d 14.2 (CH3C); nv'z (EI) 351 (M" 70%),

294 (2 l), 210 (46), 141 (64), 104 (100), 84 (85); Found: Ml', 35 1.1384. CzlHzzN02P

requires m/z 35 1.1388.

(ZS, 4S, 5~)-(2-na~hth~lmeth~l)-3,4-dimeth~~2-ox0-5-~hen~1-2h~-1~,2-0x~~hos~ho-

lidine (4.16). White solid (54%); mp 132-134 OC (hm pentane/CH2C12); [a]22D +I .1 (c

0.0239 in MeOH); 6~(200 MHz; CDC13) 7.82-7.78 (4 H, br m), 7.50-7.38 (3 H, br m), 7.22 (3

H, br s), 7.08 (2 H, brs), 5.70 (1 H, d, J5.9, HCO), 3.59 (2 H, d,JHp20.5, CH2P), 3.54-3.44(1

H, rn, HCN), 2.70 (3 H, d, J 8.8, NCH,) and 0.10 (3 H, d, J 7.3, CCH3); ap(80 MHz; CDC13)

36.8 (1 P, s); 6,(100 MHz; CDC13) 135.4 (d), 133.0 (d), 13 1.9 (d), 129.7 (d), 128.3 (d), 127.9

(br m), 127.8, 127.6, 127.3 (br d), 127.1, 126.0, 125.4, 125.3, 79.8 (PhCo), 60.6 (d, JCp 8.1,

CH3CN), 35.4 (d, JCP 122.3, CHzP), 29.8 (d, JCp 6.6, NCH3) and 13.3 (CH3C); m/z (EI) 351

(M', 50%). 294 (17), 210 (41), 141 (62), 104 (IOO), 84 (20); Found: [Mt, 351.1378.

CziH2NOzP requires d z 35 1.1388.

(2R 4S, 5~)-2-(meta-@heny1)be~1~3,4-dimeth~1-2-0x0-5-~hen~1-2~.~- 1,3,2-oraznphos-

pholidine (4.14). Colorless oil (24%); [alUD 47.1 (c 0.0133 in MeOH); bH(200 MHz;

CDCl3) 7.60-7.23 (9 H, m), 4.78 (1 H, t, J 5.9, HCO), 3.5 1 (2 H, d, JHp 20.5, CH2P), 3.32-3 -09

(1 H, m, HCN), 2.70 (3 H, d, J 10.3, NCH3) and 0.70 (3 H, d, J 7.4, CCH3); tip(80 MHz;

CDCI3) 38.2 (1 P. s); 6,(100 MHz; CDC13) 141 -4 (d). 140.5, 136.1 (d), 133.0 (d), 129.0 (d).

128.8, 128.5 (d), 128.3 (d), 128.2, 128.1, 127.4, 127.0, 126.1, 125.6 (d), 82.5 (PhCO), 58.4 (d,

JCP 9.5, CH&N), 34.6 (d, JCp 119.3, CHzP), 28.5 (d, JCp 5.9, NCH3) and 14.4 (CH3C): d z

(Er) 377 (w, 60%), 362 (19), 210 (28), 167 (17), 104 (100); Found: [Ml+, 377.1545.

CuH24N02P requires m/z 377.1545.

(2S, 4 S , 5~)-2-(rneta-(~ henyl) bewl)-3,4-dimethy1-2-~x~-5-Pheny~-2 hS- 1 ,3,2-oxazaP hos-

pholidine (4.17). White solid (56%); mp 120-1 22 OC (h pentanelCHzClt); [alXo -58.7 (c

0.0267 in MeOH); ijH(200 MHz; CDC13) 7.59-7.1 1 (9 H, m), 5.69 (1 H, d, J 5.9, HCO), 3.51

(2 H. d, JCP 19.0. CH2P), 3.55-3.45 (1 H, m, HCN), 2.74 (3 H. d, J 8.8, NCH3) and 0.08 (3 H,

d. J 7.3, CCH3); 6p(80 MHz; CDC13) 37.0 (1 P, s); 6,(100 MHz; CDC13) 141.2 (d). 140.3,

135.4 (d), 132.8 (d), 128.7 (m), 128.6, 127.9, 127.7, 127.2, 126.7, 125.4, 79.8 (PhCO), 60.9 (d.

JCP 7.4, CH&N), 35.0 (d, Jcp 122.3, CH2P), 29.7 (d, J'p 6.6, NCH3) and 13.2 (CH3C); d~

(EI) 377 (w, 100%), 362 (30), 210 (29), 167 (25), 104 (85); Found: [MI', 377.1549.

CuH24N02P requires m/r 377.1545.

(2~,4~,5~)-2-(be~1)-3,4~imeth~1-2-0~0-5-~ben~1-2~.~-1,3,2-0~aza~ho o i d i e (4.15).

White solid (20%); mp 133-134 OC; (fiom pentane/CHzC12); [a]22D -40.2 (c 0.0438 in MeOH);

6" (200 MHz; CDCl3) 7.36-7.23 (10 H, m), 4.67 (1 H, t. J 5.9, HCO), 3.44 (2 H, d, JHP 19.0,

CH2P), 3.17-3.03 (1 H, m, HCN), 2.68 (3 H, d, J 10.3, NCH3) and 0.68 (3 H, d, J 5.9, CCH3);

6 , (80 MHz; CDCI3) 38.4 (1 P, s); tic (100 MHz; CDCI3) 136.5 (br s), 132.8 (d), 129.7 (d),

128.6. 128.3, 128.1, 126.9 (br s), 126.3, 82.5 (PhCO). 58.6 (d, Jcp 9.1, CH3CN), 34.8 (d. JCp

119.8, CH2P), 28.5 (d, Jcp 5.5, NCH3) and 14.4 (CHIC); m/r (En 301 (M', 26%), 147 (24).

104 (100), 91 (39), 84 (47); Found: M', 301.1233. Ci~H2oN02P requires d z 301.1232.

(2~,1~,5~)-2-(be~l)-3,4-dimeth~l-2i)x0-5-~he11~1-2~.~-1,3,2-0xaza~h0~~h0iidine (4.18).

White solid (4 1 %): mp 169-1 71 OC (from pentane/CHzClz); [ a l U D -6.2 (c 0.0488 in MeOH);

&(zoo MHz; CDC13) 7.30-7.09 (10 H, m), 5.69 (1 H, d, J 5.8, HCO), 3.58-3.42 (1 H, m.

HCN), 3.42 (2 H, d, JHP 22.0, CH2P), 2.69 (3 H, d, J8 .7 , NCH3) and 0.15 (3 H, d, J7.3,

CCH3); &(80 MHz; CDC13) 37.0 (1 P, s); Zic(lOO MHz: CDC13) 136.1 (d), 132.7 (d), 130.1

(d). 128.6 (d), 128.3, 128.0, 126.9 (d), 125.8, 80.2 (PhCO), 61.3 (d, Jcp 7.4, CH3CN), 35.8 (d,

JCP 123.6. CH2P). 30.2 (d, JCp 5.4, NCH3) and 13.7 (CH3C); m/; (Ei) 301 (w, 19%). 147

(46). 104 (79), 97 (IOO), 91(46), 84 (93); Found: [Ml', 301.1239. C I ~ H ~ ~ N O ~ P requires d z

301.1232.

General Procedure for the Preparation of a-Fluoropbosphonamidates 4.19-4.30.

Same procedure as that described for the fluorination of 4.9 except phosphonamidates

4.13-4.18 were used as substrates. For the fluorination of phosphonamidates 4.14, 4.15, 4.17

and 4.18 NaHMDS was used as base. The fluorination of 4.13 and 4.16 was examined in

detail using a variety of bases, counterions and conditions. For the conditions and results of

these studies see Tables 4.2 and 4.3. Separation and purification of the fluorinated cis-isomers

4.19-4.24 was achieved by silica gel column chromatography using Et0Ac:hexane (1: 1 ) as

eluent. In the case of the cis-isomers 4.19-4.24, the S-enantiomers (stereochemistry at the a-

carbon) exhibited larger Rf values on silica TLC plates and eluted before the R-enantiomers

during column chromatography. Separation and purification of the fluorinated tram-isomers

4.25430 was achieved by silica gel coiumn chromatography using Et0Ac:hexane (7:3) as

eluent. In the case of the tram-isomers 4.25-4.30, the R-enantiomers (stereochemistry at the

a-carbon) exhibited larger Rf values on silica TLC plates and eluted before the S-enantiomers

during column chromatography.

(ZR, 4s. 5~)-2-((1~)-fluoro(na~hth~lmeth~l)~-3,4-dimeth~1-2-oxo-5-~hen~1-2~~-1,3,2-

oxazaphospholidine (4.19). White solid (see Table 4.2 for yields); mp 169-171 OC; (fiom

pentane/CHzClz); [alZo -102.5 (c 0.0268 in MeOH); &(ZOO MHz; CDCI,) 8.04 (1 H, s), 7.93-

7.84 (3 H, br m), 7.71 (1 H, d, J8.8), 7.52 (2 H, s), 7.37 (5 H, s), 6.21 (1 H, dd, J H p 8.8 and JHF

45.7. CHF), 5.72(1 H,d, J5.9, HCO), 3.77-3.69(1 H, m, HCN),2.23 (3 H, d, J8.8,NCH3)

and 0.78 (3 H. d, J 5.9, CCH3); 6p(80 MHz; Cm13) 28.9 (1 P, d, JpF 76.3); 6F(188 MHz;

CDC13) -125.1 (1 F, dd, J F ~ 45.7 and JFP 76.3); 6,(100 MHz; CDC13) 135.8 (d), 133.1. 132.8

(d), 130.9 (d). 128.3. 128.2, 128.2. 128.0, 127.7, 126.4, 125.8, 124.4 (dd), 123.0 (dd), 89.8

(dd, JCP 149.4 and JCF 188.9, CH);'), 82.5 (PhCO), 58.6 (d, JCp 9.5, CH3CN), 29.5 (d, JCp 4.3,

NCH3) and 14.0 (CH3C); m/; (EI) 369 (M', 66%), 210 (42), 159 (100), 104 (48); Found:

[M]', 369.1 298. C2, H2, FN02P requires m/r 369.1 294.

(2R, 4S, 5~)-2-[(1~)-luoro(na~hth~lmcth~l)]-3,44imeth~1-2-oxo-5-~hen~1-2~~-1,3,2-

oxazaphospholidine (4.22). White solid (see Table 4.2 for yields); mp 135-137 OC (fiom

pentane/CH2C12); [alao +40.8 (c 0.01 35 in MeOH); tjH(200 MHz; CDCl,) 7.99 (1 H, s)' 7.89-

7.81 (3 H, brm), 7.64(1 H,d, J7.3), 7.57-7.44(2 H, brm), 7.32(5 H, s),6.I5 (1 H, dd, JHP

4.4 and JHF 45.7, CHF), 5.45 (1 H, br t, J 6.6, HCO), 3.77-3.73 (1 H, m, HCN), 2.82 (3 H, d, J

10.2, NCH3) and 0.79 (3 H, d, J 5.8, CCH3); tip(80 MHz; CDCl3) 29.5 (1 P, d, JpF 85.5); 6F

(188 MHz; CDC13) -1 19.8 (1 F, dd, JFH 45.7 and JFp 85.5); 6,(100 MHz; CDC13) 135.8 (d),

133.4, 132.9 (d), 130.8 (dd), 128.4 (br d), 128.3, 128.3, 128.2, 127.7, 126.6, 126.4, 126.4,

126.1 (t), 123.8 (dd), 89.8 (dd. JCP 147.9 and JCF 186.0, CHF), 82.9 (PhCO), 58.5 (d, JCp 10.2,

CH3-), 28.5 (d. Jcp 6.6, NCH3) and 14.9 (d, J C p 3.6. CH3C); d' (EI) 369 (M. 31%). 210

(27), 159 (1 OO), 104 (60); Found: M', 369.129 1. Czi Hz 1 FN02P requires d z 369.1294.

(2~,4~,5~)-2-[(l~)-fluoro(meta-@hen~l)be~l)]-3,4-dimethy1-2-0~0-5-~hen~1-2h~-l~~-

oxazaphospholidine (4.20). White solid (43%); mp 1 26- 12% OC (fiom pentane/CHzClz);

[ a ] " D -85.3 (c 0.0252 in MeOH); aH(200 MHz; CDCI,) 7.83 (1 H, s). 7.66-7.30 (8 H, m). 6.12

( 1 H. dd, JHP 8.8 and J H ~ 45.8, CHF), 5.69 (1 H, d, J5.9. HCO), 3.80-3.66 (1 H, m, HCN),

2.3 1 (3 H. d, J 10.2. NCH3) and 0.8 1 (3 H, d, J 5.9, CCH3); &(80 MHz; CDC13) 28.9 (1 P. d,

JpF 76.3); S~(188 MHz; CDC13) -125.8 (1 F, dd, JFH 45.8 and J F p 76.3); Sc(lOO MHz; CDC13)

141.4, 140.4, 135.8 (d), 134.1 (d), 128.9, 128.8, 128.4, 128.2, 127.6, 127.2, 127.1. 125.8,

124.3 (dd), 124.0 (dd), 89.7 (dd, JCP 149.8 and JCF 189.3, CHF), 82.5 (PhCO), 58.7 (d, JCp 9.6,

CH3CN). 29.5 (d, Je 4.4, NCH3) and 14.1 (CH3C); d z (ET) 395 (M', 45%), 210 (71), 185

(60), 146 (42), 1 O4 (100); Found: pl', 395.1464. CuHuFNOzP requires m/z 395.1460.

( 2 ~ , 4 ~ , 5 ~ ) - 2 - ~ ( 1 ~ ) 4 u o r o ( m e t a ~ h e n ~ l ) b e ~ l ) ] - 3 , 4 - d i m e t h ~ ~ ~ - l , 3 , 2 -

oxazaphospholidine (4.23). White solid (25%); mp 147-149 OC ( h m pentane/CH2C12);

[ a j Z Z D +28.5 (c 0.0215 in MeOH); 6~(200 MHz; CDCI3) 7.75 (1 H, s), 7.66-7.34 (8 H, m),

6.06 (1 H, dd, JHp 4.4 and JHF 45.0, CHF), 5.44 (1 H, t, J6.6, HCO), 3.83-3.72 (1 H. m, HCN).

2.82 (3 H, d, J 10.3, NCH3) and 0.8 1 (3 H, d, J 7.3, CCH3); tip(80 MHz; CDC13) 29.6 (1 P, d,

JpF 86.2); SF(188 MHz; CDC13) -120.6 (1 F, dd, JFH 45.0 and J F p 86.2); S,(100 MHz; CDC13)

141.5 (br d), 140.5, 135.8 (d), 133.9 (dd), 129.0, 128.7, 128.4, 128.3, 127.7, 127.5, 127.2,

126.4, 125.4 (dd), 89.6 (dd, J C p 150.8 and JCF 186.0, C m ) , 83.0 (PhCO), 58.5 (d, JCp 10.2,

CHiCN), 28.4 (d, Jcp 6.6, NCH3) and 14.9 (CH3C); nu'z (El) 395 (M', 45%), 210 (73), 185

(63), 146 (42), 104 (100); Found: M', 395.1460. C23H23FNOzP requires nr/r 395.1450.

(2R, 4S, 5~)-2-[(l~)-fluoro(benz~l)]~dimeth~l-2-0x0-5-~hen~1-2~~-l~~-ox~zn~bos-

p holidine (4.2 1). White solid (29%); mp 129- 13 1 OC (fiom pentane/CH2Cl2); -93 -9 (c

0.041 1 in MeOH); 6~(200 MHz; CDC13) 7.53-7.26 (10 H, m), 5.98 (1 H, dd, JHP 4.4 and JHF

45.4, CHF), 5.38 (1 H, t, J6.6, HCO), 3.79-3.67 (1 H, m, HCN), 2.81 (3 H. d, J9.5. NCH3)

and 0.80 (3H, d, J 5.8, CCH3); 6p(80 MHz; CDC13) 29.5 (1 P, d, JpF 86.2); 6F (1 88 MHz;

CDC13) -120.4 (1 F, dd, JFH 45.4 and JFp 86.2); &(100 MHz; CDC13) 136.1 (d), 133.8 (d),

138.9 (br d), 128.5 (br s), 128.4, 126.8 (d), 126.6 (d), 126.5, 90.0 (dd, JCp 148.2 and JCF 185.8,

CHF), 82.9 (PhCO), 58.5 (d, JCP 9.2, CH&N), 28.5 (d, JCp 5.5, NCH3) and 14.9 (d, JCP 2.7.

CH3C); miz (EI) 319 (M', 18%), 210 (58), 192 (29)' 104 (LOO), 84 (98); Found: [Ml', -

319.1145. Ci7HI9FNOzP requkesdz319.1137.

(2R, 4S, SR)-2-((IR)-fluoro@eiuy 1 ) j - 3 , 4 - d i m e t h y l ~ x a z a p h o s -

pholidiae (4.24). White solid (1 5%), mp 1 14-1 16 OC (fiom pentane/CH2Cl2); [alUo +26.3 (c

0.0404 in MeOH); tiH(200 MHz; CDC13) 7.57-7.27 (10 H, m), 6.01 (1 H, dd, JHP 8.0 and JHF

45.8, CHF), 5.61 (1 H, br t, J 2.9, HCO), 3-74-3.60 (1 H, m, HCN), 2.25 (3 H, d, J 10.2,

NCH3) and 0.76 (3 H, d, J 5.9, CCH3); &(80 MHz; CDQ) 28.9 (1 P, d, JpF 79.4); & (1 88

MHz; CDC13) -125.8 (1 F, dd, JFH 45.8 and JFp 79.4); 6c(100 MHz; CDC13) 136.2 (br d).

133.9 (d), 128.5 @r s), 126.1 (br s), 125.6 (br s), 90.1 (dd, JCp 149.2 and JCF 188.5, CMF), 82.6

(PhCO), 58.9 (d, JCp 9.2, CH&N), 29.4 (d, JCp 4.6, NCH3) and 14.2 (d, JCp 2.7, CH3C); d z

(EI) 319 (M', 19%), 210 (59), 192 (27), 104 (100); Found: Fi]'. 319.1 126. Cl7HI9FNO2P

requires m/z 3 19.1 137.

(2S, 4S, SR)-2-[(l~)-fluoro(na~hth~lmeth~l) ] - 3 , 4 - d 5 - l $2-

oxazaphospholidine (4.25). White solid (see Table 4.3 for yields), mp 125-127 OC (fiorn

pentane/CHzC12); [ C Y ] ~ ~ +238.6 (c 0.0331 in MeOH); aH(200 MHz; CDC13) 7.99 (1 H, s),

7.93-7.84(3 H, bm), 7.66(1 H,d, J7.3), 7.55-7.47 (2 H, bm),7.39 (5 H, s), 6.22 (1 H,dd,

J H ~ 8.1 and J H ~ 45.8, CH"), 5.84 (1 H, d, J5.8, HCO), 3.74-3.61 (1 H, m, HCN), 2.33 (3 H, d.

J 8.8, NCH3) and 0.90 (3 H, d, J 7.3, CCH3); 6d80 MHz; CDC13) 28.0 (1 P, d, JpF 80.9); S

~(188 MHz; CDC13) -119.7 (1 F, dd, JFH 45.8 and JFp 80.9); S,(lOO MHz; CDC13) 135.4 (d),

133.1, 132.8 (d), 130.8 (d), 128.2, 128.2, 128.1, 128.0, 127.59, 126.4, 126.3, 135.7, 124.8

(dd), 123.1 (dd), 88.0 (dd, J C p 152.0 and JCF 192.3, CHF), 80.5 (PhCO), 61.1 (d, JCp 9.5,

CH3-), 30.8 (d, J& 5.1, NCH,) and 14.8 (d, JCp 3.7, m 3 C ) ; d z (EI) 369 (Mt, 61%), 210

(40), 1 59 (1 OO), 104 (43); Found: M+, 369.130 1. C21 H2iFN02P requires d. 369-1294.

(2S, 4S, 5 ~ ) - 2 - [ ( 1 ~ ) - f l u o r o ( n a ~ h t h ~ l m e t h ~ l ) ] - 3 , 4 - d i m e t h ~ l ~ ~ - l ~ ~ -

oxazaphospholidine (4.28). White solid (see Table 4.3 for yields). mp 18 1 - 1 83 OC (fiom

pentane/CHzC12); [a]"~ -21 5.3 (c 0.0273 in MeOH); 6~(200 MHz; CDC13) 8.06 (1 H, s), 7.95-

7.84(3 H, brm), 7.73 (1 H,d,J7.4),7.54-7.48(2H,brm), 7.42-7.27(5 H,m),6.04(1 H.dd,

JiIP 4.4 and JHF 45.8, CHF), 5.82 (1 H. d, J 5.8, HCO), 3.82-3.75 (1 H, m, HCN). 2.89 (3 H. d,

J 8.8, NCH3) and 0.79 (3 H, d, J 7.3, CCH3); 6480 MHz; CDC13) 28.5 (1 P, d, JpF 87.0); 6

F(l 88 MHz; CDC13) -1 14.9 (1 F, dd, JFH 45.8 and JFp 87.0); 6,(100 MHz; CDC13) 135.4 (d),

133.4, 132.9 (d), 130.9 (dd), 128.4 (br d), 128.3, 128.1, 128.1, 127.7, 126.5, 126.4, 126.3 (m).

125.6, 123.9 (dd), 87.9 (dd, J C p 151.6 and JCF 189.7, CHF), 80.8 (PhCO), 61.1 (d, JCp 8.8,

CH3CN), 29.5 (d, JCP 6.6. NCH3) and 14.1 (d, JCp 2.9, CH3C); mk (Ei) 369 (w, 66%), 2 10

(38). 159 (1 OO), 104 (42); Found: FI]', 369-1297. C2, H2iFN02P requires m/z 369.1294.

(ZS, 4S, 5 ~ ) - 2 - [ ( l ~ ) - f l u o r o ( l l t e t ~ - ( p h e n ~ l ) b e ~ l ) ~ - 3 , 4 - d i m e t h ~ l ~ -

oxazaphospboüdine (4.26). White hydroscopic solid (53%); [a lU~ +27.l (c 0.0240 in

MeOH); 6 ~ ( 2 0 0 MHz; CDC13) 7.85 (1 H, s), 7.71-7.34 (8 H, m), 6.19 (1 H, dd, J H ~ 8.1 and JHF

45.8. CHF), 5.93 (1 H, d, J 5.9, HCO), 3.80-3.74 (1 H, m, HCN), 2.50 (3 H, d, J 8.8, NCH3)

and 0.95 (3 H, d, J 5.9, CCH3); &(80 MHz; CDC13) 27.9 (1 P, d, J ~ F 82.4); &(l88 MHz;

CDC13) -120.3 (1 F, dd, JFH 45.8 and& 82.4); &(IO0 MHz; CDCI3) 141.3 (lx d), 140.3, 135.5

(d), 134.1 (d), 128.9, 128.7, 128.3, 128.2, 127.5, 127.3, 127.0, 125.8, 124.6 (dd). 124.3 (dd),

87.9 (dd, JCp 151.9 and JCF 195.2, CHF), 80.6 (PhCO), 61 -2 (d, JCp 9.5, CH3CN), 30.9 (d, JCp

5.9. NCH,) and 14.9 (CH3C); d z (ET) 395 (M', 49%), 210 (69), 185 (63), 146 (37), 104

(1 00); Found: [MI+, 395.1464. CnHzlFNOtP requires m/r 395.1454.

(2S, 4S, SR)-2-[(1 ~)-fluoro(me!u~hen~l)be~1)~-3,4-dimeth~1-2-0~0-5-~hen~1-2~~-1~~-

oxazaphosphoüdine (4.29). White solid (32%); mp 13 1 - 133 O C (from pentane/CH2Cl2):

[alZD -234.6 (c 0.0251 in MeOH); &(200 MHz; CDC13) 7.92 (1 H, s), 7.72-7.34 (8 H, m),

6.15 (1 H, dd, J H ~ 4.4 and JHF 45.6, CHF), 5.90 (1 H, d, J 5.8, HCO), 3.90-3.83 (1 H, m,

HCN), 2.98 (3 H, d, J 8.7, NCH3) and 0.84 (3 H, d, J 7.3, CCH3); 6p(80 MHz; CDC13) 28.3 (1

P. d, J p ~ 88.5); 6~(188 MHz; CDC13) -1 15.6 (1 F, dd, JFH 45.8 and JFP 88.5); Sc(lOO MHz;

CDC13) 141.4 (d), 140.3, 135.3 (d), 134.1 (dd), 128.9, 128.6, 128.2, 128.1, 127.5, 127.4,

127.0, 125.5. 125.4, 87.7 (dd, J C p 150.5 and J C ~ 187.8, CHF), 80.6 (PhCO), 61.0 (d, JCp 8.8.

CH3CN)' 29.4 (d. J& 6.6, NCH,) and 13.9 (CH3C); m/z (EI) 395 (M', 52%), 21 0 (73). 185

(65), 146 (40), 104 (100); Found: M', 395.1445. CuHuFNOzP requires d z 395.1450.

(2S, 4S, SR)-2-((1 R)-fluoro(benyl) J-3,4-dime thyl-2-~~~-5-Pheny1-2~5-1 ,3,2-oxazaPhos-

pholidine (4.27). Colorless oil (30%); [a ]"~ +26.3 (c 0.0476 in MeOH); 6~(200 MHz;

CDC13) 7.56-7.26 (10 H, m), 6.04 (1 H, dd, JHp 8.0 and JHF 45.8, CHF), 5.83 (1 H, d, J 5.9.

HCO), 3.74-3.61 (1 H, m, HCN), 2.38 (3 H, à, J 8.6, NCH3) and 0.86 (3H, d, J 6.6, CCH3); 6 p

(80 MHz; CDClj) 27.9 (1 P, d, JpF 79.3); &(188 MHz; CDC13) -120.3 (1 F, dd, JFH 45.8 and

JFp 79.3); 6c(100 MHz; CDC13) 135.8 (d), 133.8 (d), 128.4 (br s), 125.9 (br s), 88.3 (dd, JCP

151.9 and J C ~ 190.4, CHF), 80.7 (PhCO), 61.4 (d, Jcp 9.2, CH3CN), 30.9 (d, JCp 5.5, NCH3)

and 14.9 (d, JCp 3.7, CH3C); miz (EI) 3 19 (Mc, 3 1 %), 2 10 (69), 192 (33), 104 (1 00); Found:

[Ml*. 3 19.1 137. Cl7Hl9FNOZP requires m/z 3 19.1 137.

(2S1 43, SR)-2-((1 ~ ) - f l u o r o ( b e ~ l ) ] - 3 , 4 - d i m e t h ~ l ~ ~ hos-

pholidine (4.30). White solid (1 7%), mp 165- 167 OC (fiom pentane/CHzClz); [a lU~ - 1 08.3

(C 0.0263 in MeOH); &(200 MHz; CDC13) 7.62-7.1 1 (10 H, m), 5.87 (1 H, dd, JHP 4.4 and JCrF

45.5, CHF), 5.81 (1 H, d, J 5.8, HCO), 3.83-3.70 (1 H, m, HCN), 2.86 (3 H, d, J 8.8, NCH3)

and 0.76 (3 H, d, J 7.3, CCH3); &(80 MHz; CDC13) 28.3 (1 P, d, JpF 87.0); 6~(188 MHz;

CDC13) -1 15.2 (1 F, dd, JFH 45.5 and J F ~ 87.0); 6c(100 MHz; CDCI3) 135.7 (d), 133.7 (d).

129.9, 128.6, 128.4, 128.3, 126.9 (br t), 125.8, 88.2 (dd, JCp 151.9 and JCF 189.4, CHF), 80.9

(PhCO), 61.5 (d, JCP 8.3, CH3CN), 29.7 (d, JCp 6.4, NCH3) and 14.3 (d. JCp 2.7, CH3C) d z

(EI) 3 19 ( A f , 13%), 210 (43), 147 (100), 104 (72), 84 (38); Found: [Ml', 3 19.1 147.

C17Hr9FN02P requires m+ 3 19.1 137.

General Procedure for Synthesis of Chiral Phosphonic Acids 4.1-4.6.

A solution of the oxazaphospholone (4.19-4.30) in 10% TFlVMeOH (approximately 1

cm3 10% TFA-MeOWO.1 mm01 of 4.19-4.30) was stirred at 25 OC overnight. The solution

was then concentrated in vacuo, re-dissolved in benzene and re-concentrated a total of 3 times

prior to being placed under high vacuum for 2 h. The resulting product was dissolved in

anhydrous CHzCIz (approximately 3-5 cm3/1 mm01 of 4.19-4.30) and stirred at 25 OC for 24 h

in the presence of TMSBr (10 equiv.). The solution was concentrated in vacuo and placed

under high vacuum for several hours. Anhydrous ether was then added followed by filtration

and concentration of the filtrate (if a precipitate formed during the rotary evaporation, more

anhydrous ether was added and the suspension re-filtered). The filtrate was dissolved in

MeOH. containing 1 &op of water, and stirred for 15 min. The solution was then

concentrated in vacuo and the resulting product was washed extensively with benzene and

CHzCll to yield pure product. in addition to spectroscopie analysis of phosphonic acids 4.1-

4.6 (see below), their purity was also examined by analytical reverse phase HPLC (solvent A:

acetonitrile; solvent B: water with 0.1% TFA modifier) using the following gradient: O min:

25% A; 5 min: 25% A; 10 min: 100% A; 15 min: 100% A: 20 min: 75% A; 30 minutes: 25%

A. Al1 of the phosphonic acids were indicated to be pure by the critenon of analytical HPLC.

The ee's of the phosphonic acids were detemiined to be >97% using the "F-NMR method

described in section 4.2 (p.119).

(R)-Fluoro(2-naphthyI)niethylphosphonic acid (4.1). Obtained as a white solid in 57%

yield from 4.22 and 62% yield fiom 4.25; mp 180-1 8 1 OC (decornp.): HPLC retention time =

3-65 min; [ a ]22D + 45.7 (c 0.0433 in MeOH); 6"(200 MHz; CD30D) 7.97 (1 H, s), 7.89-7.85

(3 H. br m), 7.63 (1 H. d, J 8.8), 7.50-7.46 (2 H, br m) and 5.88 (1 H, dd, J H ~ 8.8 and JHF 45.8,

CHF); &(80 MHz; CD30D) 15.4 (1 P, d, JpF 83.9); S~(188 MHz; CD30D) -120.2 (1 F. dd,

JFH 45.8 and JFp 83.9); Sc(lOO MHz; CD30D) 134.9, 134.4 (d), 133.1 (dd), 129.1, 129.0 (br

d), 128.7, 127.6, 127.6 (m), 127.4, 125.5 (br dd) and 91.7 (dd, JCp 166.2 and J& 180.1. CHF);

d z (ES) 239 (100%).

(S)-FIuoro(2-naphthyI)methylphosphonic acid (4.2). Obtained as a white solid 58% yield

from 4.19 and 65% yield fiom 4.28. HPLC chromatograrn, rnp, and al1 spectral data were

identical to that reported for 4.1. Optical rotation was approximately the same as that obtained

for 4.1 but of opposite sign.

(R)-Fluoro(meto-(phenyl))ben.zylpbospho~ic acid (4.3). Obtained as a white soiid in 64%

yield from 4.23 and 70% yield fiom 4.26; HPLC retention time = 3.35 min; [alXD + 3 1.1 (c

0.02 1 1 in MeOH); mp 175 OC (decornp.); 6~(200 MHz; CD3OD) 7.76 (1 H, s). 7.63-7.30 (8 H.

br m) and 5.78 (1 H, dd, Jnp 8.8 and J H ~ 44.0, CHF); 6p(80 MHz; CD30D) 15.1 (1 P, d, JpF

85.4); 6 ~ ( l 8 8 MHz; CD30D) -120.6 (1 F, dd, JFH 44.0 and JFP 85.4); 6,(100 MHz. CD30D)

142.5, (d), 141.9, 136.4 (d), 129.9, 129.9, 128.6, 128.4, 128.0, 127.0 (t), 126.7 (t) and 91.5

(dd, JCP 165.5 and J& 180.1. CHF); m/r (ES) 265 (1 00%).

(S)-Fluoro(meta-(pbenyl))bewylphospbonic acid (4.4). Obtained as a white solid in 69%

yield from 4.20 and 67% yield from 4.29. HPLC chromatogram, mp and all spectral data were

identical to that reported for 43. Optical rotation was approximately the same as that obtained


(R)-Fluoro(phenyl)methylpbospbonic acid (4.5). Obtained as a white solid in 39Y0 yield

from 4.24 and 47% yield from 4.27; HPLC retention time = 3.57 min; [alZo + 36.9 (c 0.0489

in MeOH); rnp 114-1 15 OC; aH(200 MHz; CD30D) 7.51-7.37 (5 H, m) and 5.68 (1 H. dd, JHp

8.1 a d J H ~ 44.7. CHF); 6p(80 MHz; CD30D) 15.2 (1 P, d, JPF 85.5); &(188 MHz; CD3OD)

-120.5 (1 F, dd. JFH 44.7 and J F ~ 85.5); Sc(100 MHz; CDpOD) 135.8 (d), 129.9. 129.3. 128.2

(br t) and 9 1 -7 (dd, JCp 166.6 and JCF 179.4, CHF); m/r (ES) 189 ( 1 00%).

(S)-Fluoro(phenyl)methylphosphonic acid (4.6). Obtained as a white solid in 46% yield

from 4.21 and 41% yield fiom 4.30. HPLC chromatogram, mp and al1 spectral data were

identical to that reported for 4.5. Optical rotation was approximately the same as that obtained


Determination of Enantiomeric Exces of Phosphonic Acids 4.1-4.6. A mixture of the

phosphonic acid (4.1-4.6, 20-15 mg) and quinidine (1 equiv.) was dissolved in CDC13

(approximately 1 cm3), shaken until a homogeneous solution was obtained, and the 1 9 ~ - ~ M . R

was recorded. For each of the phosphonic acids 4.1-4.6, only a single set of doublet of

doublets was evident in the spectrograms indicating that 4.1-4.6 were obtained in >97% ee.

Dimethyl (2-naphthylmethyl)phosphonate. For a general procedure for the preparation of

phosphonates see Chapter 3, section 3.2.1. White solid (90%). mp 78-80 OC; 6~(200 MHz;

CDC13) 7.83-7.79 (4 H, br m, aryl), 7.49-7-46 (3 H, br m, aryl), 3.67 (6 H, d. JHP 10.2,

(OCH3)2) a d 3.34 (2 H, d, JHp 22.0, CHtP); &(80 MHz; CDC13) 26.7 (1 P, s): &(100 MHz:

CDCI3) 133.0 (d), 131.9 (d), 128.4 (d), 128.1 (d), 127.9 (d), 127.4 (d), 127.3 (d), 127.2. 125.8,

125.5, 52.5 (d, (OCHih) and 32.6 (d, JCp 137.7, CHzP); m/z (EI) 250 (h.I+, 100%), 154 (la),

141 (83), 1 15 (26); Found: M+, 250.0759. C13His03P requires m/z 250.0759.

Dimethyl difluoro(2-naphthyl)methylphosphonate. Same procedure as described for the

general procedure for the preparation of a,a-difluoromethylenephosphonates in Chapter 3.

section 3.2.1 using NaHMDS as base. Column chromatography (silica, EtOAc:hexane, 2 3 , Rr

= 0.5) yielded a white solid (78%); mp 59-61 OC; MHz; CDC13) 8.15 (1 H, s, aryl).

7.96-7.85 (3 H, br m, aryl), 7.69 (1 H, d, J7.4, aryl), 7.58-7.53 (2 H, br m, aryl) and 3.84 (6 H,

d, JHP 10.2, (OCH3)2); Sp(80 MHz; CDCl3) 6.4 (1 P, t, J ~ F 1 17.5); 6f(188 MHz; CDC13) -3 1.2

(2 F, d, J F ~ 117.5); 6,(100 MHz; CDCl3) 134.3, 132.6, 129.9 (br m), 128.8, 128.6, 127.8,

127.7, 126.9, 126.5 (td), 122.6 (t), 118.5 (td, JCP 218.7 & JCF 263.6, CF2) and 54.8 (d,

d z (EI) 286 (M+, 26%), 177 (100), 127 (14); Found: NI', 286.0567.

C 13H 13FZ03P requires m/z 286.0570.

Difluoro(2-nophthy1)methylpbosphonic acid. A solution of dimethyl difluoro(2-

naphthy1)methylphosphonate (500 mg, 1 -75 rnmol) in anhydrous CH2C12 (1 0 cm3) and TMSBr

(6.91 cm3, 5.24 mmol) was stirred at 25 OC for 12 h. The solution was concentrated in vacuo

and subjected to high vacuum for 2 h. Upon addition of water (5 cm3) a precipitate

immediately crashed out of solution. Filtration followed by a CH2C12 wash yielded a white

solid (563 mg, 90%). mp 122-124 OC; 6.4200 MHz; CD30D) 8.15 (1 H, s, aryl), 7.96-7.88 (3

H. br m, aryl), 7.70 (1 H, d, J 8.8, aryl) and 7.57-7.52 (2 H, br m. aryl); CD30D)

6.7 (1 P, t, J ~ F 114.4); 6d188 MHz; CD3OD) -30.4 (2 F, d, JFp 114.4); 6,(100 MHz; CD30D)

135.6, 133.9, 132.5 (br m), 129.7, 129.2, 128.8, 128.5, 127.8, 127.5 (br s), 124.1 (br t) and

1 19.9 (td, JCP 216.4 & JCF 264.6, CFt); m/z (ES) 257 (100%).

Ceneral Procedure for the Preparation of Phosphonamidates 4.A and 4.B.

To a solution of naphthylphosphonic acid (1.1 equiv.), derived fiom 4.8, and (S)-(-)-

a,a-diphenylpyrrolidine-2-methanol(1 equiv.) dissolved in anhydrous CH2C12 (approximately

8 cm3 CHrC12/mmol of (S)-diphenylprolinol) was added Et3N (2.2 equiv.) at O O C . The

reaction mixture was then allowed to wann to rt over 3 h and then stirred an additional 24 h.

The mixture was then filtered and concentrated in vacuo. Separation and purification of the

isorners, 4.A and 4.B, was achieved by silica gel column chromatography using

CH2C12:EtOAc (1 : 1) as eluent.

(2R, 5s)-[(2-Nap hthylmetbyl)]-4,4-diphenyl-3-oxa-1-azaphosp horylbicyclo(3.3.0 J octane

(l.A). White solid. mp 129-13 1 OC; MHz; CDC13) 7.73-7.17 (1 7 H, m, aryl), 3.84 (1

H, m), 3.70-3.55 (1 H, m), 3.36 (1 H, d, J4.4), 3.26 (1 H, d, J5.9), 3.01-2.82 (1 H, m) and

1.60-1.25 (4 H, m); 6p(80 MHz; CDC13) 42.3 (1 P, s); 6,(lOO MHz; CDC13) 143.4 (d), 141 -3

(d), 133.2 (d), 130.2 (br s), 129.6 (d), 128.4-127.2 (m), 126.6 (d), 125.2 (d), 88.2, 70.7 (d),

44.9, 37.3 (d, JCp 127.2, CbP) , 29.8 (d) and 26.0 (d); ml' (EI) 439 (W, 58%), 298 (14), 257

(25), 229 (37). 1 16 (100); Found: M+, 439.172 1. CZoH26N02P requires dz439.170 1 .

(2S, 5S)-[(2-Naphttiylmethyl)]-4,edipbenyl-3~xa-l-azciphosphorylbicyclo[3.3.0]octaae

(4.B). White solid. mp 106-108 OC; 6"(200 MHz; CDC13) 7.84-7.76 (5 H, br m, aryl), 7.58-

7.49(5 H,m,aryl), 7.42-7.22 (7 H, m,aryl)4.53 (1 H,m), 3.80-3.28 (4 H, m), 1.91-1.60(3 H.

m) and 1.00-0.89 (1 H, m); 6p(80 MHz; CDC13) 36.4 (1 Pl s); 6,(I00 MHz; CDC13) 144.5,

141.7 (d), 133.6 (br d), 132.5 (br s), 129.5 (d)? 128.5-127.4 (m), 126.2 (d), 125.7 (d), 89.4,

72.8 (d), 43.6 (d), 36.0 (ci, JCp 112.6, CHzP), 29.7 and 25.2; ntlr (EI) 439 (M', 27%), 298 (7),

257 (1 3), 229 (2 1 ), 1 16 (1 00); Found: M'. 439.1706. C28H26N02P requires d z 439.1 701.

Kinetic Studies with PTPIB. Rates of PTP 1 B-catalyzed dephosphorylation in the presence

or absence of inhibitors were determined using FDP as ~ubstrate"~ as descnbed in Chapter 2

(section 2.2.1) and Chapter 3 (section 3.2.1) with an assay b a e r containing lOOmM Bis-Tris,

4mM EDTA, 5 mM DTT and 0.2 Cig/cm3. at 25 O C and pH 6.5, in the presence of 5% DMSO.

X-ray Structural Characterization. X-ray structures of compounds 4.19-4.24 were

determined by Alan Lough in the Department of Chemistry at the University of Toronto. A

sunimary of selected crystallographic data is given in Appendix A, Tables 1 and 2. Data were

collected on a Nonius KappaCCD diffiactometer using graphite monochromated MoKa

radiation (A = 0.71073A). A combination of 1" phi and omega (with kappa offsets) scans were

used to collect sufficient data. The data fiames were integrated and scaled using the Denzo-

SMN package.'55 The structures were solved and refined using the SHELXTLWC V5.1 'j6

package. Refinement was by full-matrix least-squares on F~ using al1 data (negative

intensities included).


4.3.1 CHIRAL a-MONOFLUOROPHOSPHONIC ACIDS: O V E R W W

The a-monofluorophosphonic acids and phosphonate derivatives have received much

Iess attention than their difluoro counterparts as phosphate biosteres. This is in spite of the

fact that, unlike the difluoromethylene counterparts, their p h and electrostatic profiles are

almost identical to the phosphate group that they are designed to mimic. To our knowledge,

only two reports describing the synthesis of enantiomerically pure a-monofluorophosphonic

acids have appeared in the literature. "7~'58 in both instances, the a-monofluorophosphonic

acids were obtained via stereoselective hydrogenation of vinyl phosphonates

((E~O)~POCF=CRR').'~~~~~~ This procedure is not applicable to the preparation of chiral

benzylic a-monofluorophosphonates such as 4.1-4.6. Indeed, a general procedure for the

synthesis of enantiomerically pure a-monofluorophosphonic acids has never been reported.

Shibuya and coworkers have attempted to prepare enantiomerically enriched benzylic a-

rnonofluorophosphonates by DAST fluorination of opticaily active benzylic a-

hydroxyphosphonate denvatives. However these reactions proceeded with very Lou. ee (3-

j%).' 59

The general approach we have examined for the preparation of enantiomerically

enriched benzylic a-monofluorophosphonic acids is outlined in Scheme 4.2. Ln this approach,

fluorine is introduced in a diastereoselective manner by electrophilic fluorination of a-

carbanions of asymmecric phosphonamides or phosphonamidates using a diamine or chiral

amino alcohol as an auxiliary. Removd of the chiral auxiliary would yield the a-

monofluorophosphonic acids. This approach has been employed by a nurnber of other groups

for the preparation of enantiomerically enriched a-alkylphosphonic acids using alkyl halides

O. ' p chiral auxiliary

i. base 2. 'lF+l'

chiral auxitiary

F

I remove auxiliary

Scheme 4.1. Proposed synthetic route for the formation of enantiomericaily pure a- monofluorophosphonic acids using chiral auxiliarïes.

as electr~~hiles.'~~.~~~-'~~ Davis and coworkers have used a similar methodology for the

preparation of a wide variety of chiral a-monofluorocarbonyl compounds 163-167 usuig Evans'

oxazolidinone as a chiral au~iliar~.'~~ In addition, Differding and coworkers have shown that

racemic a-monofluoroalkylphosphonates can be prepared in modest yield by electrophilic

fluonnation of a-carbanions of achiral alkyl phosphonate esters with N-

fluorobenzenesulfonimide (NFS~).'~' in Chapter 3 we have shown that racemic benzylic a-

monofluorophosphonic acids can be readily prepared in good yield via electrophilic

fluorination using NFS~."' Consequently, we reasoned that the approach outlined in Scherne

4.1 may be a viable method for the preparation of chiral benzylic a-monofluorophosphonic

acids.

4.3.2 DIASTEREOSELECTIVE ELECTROPHlLIC FLUORINATION OF AN ASYMMETRIC PHOSPHONAMIDE

Initiaily, we examined tram-(R,R)-l,2-bis-N-methylafninocyclohexane (4.7) as a

chira1 auxiliary since it has been s h o w to be effective for the asymmetric synthesis of a-

alkylphosphonic acids and was readily obtainable.'" The naphthyl derivative 4.8 was used as

a mode1 system. Chiral phosphonamide 4.9 was prepared by reacting the phosphonic acid 4.8

with oxalyl chloridekat. DMF followed by reaction of the cmde phosphoryl chlonde with 4.7

in the presence of two equivaients of triethylamine (Scheme 4.2).

1. (C0C1)2, cat. DMF, 1. 0.95 eq. base, Me

O CH2CI2 ..ezb T ~ ~ , - ~ ~ ° C , 2 h - O.-~/K-(> r ~ ( ~ ~ 2 t

Ar 2. Et3N. CH2CI2 ArJ 2. 1.1 eq. NFSi. A~S/ ' 4.8 Y= b THF, -78 O C . 2 h F &

Scheme 4.2. Synthesis of a-monofluorophosphonamides 4.10.

Our previous studies (see Chapter 3, section 3.3.1) and those of ~ i f ferd in~" ' on the

electrophilic fluorination of benzyl phosphonates with NFSi indicated that the yields of

fluorinated product were dependent on the nature of the base and countenon. Consequently,

electrophilic fluorination of 4.9 with NFSi was examined with different bases and counterions.

The de of the reaction was detemined by examining the crude reaction mixture by ' 9 ~ - ~ ~ ~ .

Fluorination of 4.9 to give the fluorinated phosphonamides 4.10 was achieved by treating 4.9

with 0.95 equiv. of base at -78 OC for 2 h followed by the addition of 1 . l equiv. NFSi (Scheme

4.2). The yields and de's of these fluorination reactions are given in Table 4.1. The yield of

Table 4.1. Effect of base and counterion on the electrophilic fluorination of phosphonamide 4.9 with NFSi.

Base % yielda of 4.10 % dehc n-BuLi 68d 68d LiHMDS 8 1 7od, 16" NaHMDS 69d 1 KHMDS 71d 37d0

'Isolated yields. "De's were determined by obtaining the 19 F-NMR of the crude residue after aqueous workup of the reaction before chromatography. 'Absolute stereochernisûy not detemined. d~erforrned using 0.95 equiv. base and 1.1 equiv. NFSi as described in the experimentat section. 'Reverse addition: 1.1 equiv. of base was added to 4.9 (1 equiv.) and NFSi ( 1 . 1 equiv.) over a period of 1 h at -78 OC. Afler stimng an additional 2 h at -78 OC the reaction was warmed to rt and quenched as described in the experimental section. *Oppsite stereoisomer is favored.

the f l u o ~ a t i o n reaction was not highly dependent on the nature of the base or counterion with

good yields being obtained in al1 cases. However, the de of the reaction was highly dependent

on the nature of the cation but not the base itself. Bases with lithium counterions (n-BuLi and

LiHMDS) gave moderately good de's (68-70%) while NaHMDS and K H M D S gave low de's.

' 9 ~ - ~ ~ ~ analysis of the products obtained using KHMDS as base revealed preferential

formation of the stereoisomer opposite fiom that obtained when using the lithium and sodium

bases. Reverse addition of NFSi and LiHMDS (1.1 equiv. of LiHMDS added to a solution of

the phosphonamide and 1.1 equiv. NFSi at -78 O C over a period of 1 h) resulted in a large

decrease in the de (16%) and preferential formation of the stereoisomer opposite fiom that

obtained when adding the base fmt. The de's obtained with the lithium bases are comparable

to those obtained by Hanessian and coworkers (68-80%) on the asymmetric a-alkylation of

benzyl phosphonamides bearing 4.7 as auxiliary using aikyl halides as electrophiles and n-

BuLi as base.'" Unfortunately, separation of the diastereomeric products 4.10 proved to be

exceedingly difficuit. Even d e r several recrystaWations, diastereomerically pure 4.10 was

not obtained. Attempts to separate the two diastereomers by silica gel column

c hromatography were also unsuccessfid.

1.3.3 DIASTEREOSELECTIVE ELECTROPHILIC FLUORtNATION OF ASYMMETRIC PHOSPHONAMIDATES

Unable to utiIize the diamine auxiliary we next envisioned preparing aryl

phosphonamidates using (1 R,2S)-(-)-ephedrine as the chiral auxiliary as it was readily

available and had been used by Sting and Steglich for the asymmetric synthesis of a-

alkylphosphonic acids.I6' B-Naphthylphosphonic acid (4.8) was again used as the mode1

systern. Treating the arylphosphonic acid dichlonde, derived fiom 4.8, with (-)-ephedrine in

the presence of 2 equivalents of triethylamine yielded two diastereomeric phosphonamidates,

1.13 and 4.16, in 24% and 54% yield, respectively, which were readily separated by silica gel

chromatography (Scheme 4.3).

Diastereomer 4.13 was designated as the cis-isomer (phosphoryl and 5-methyl group

cis) while diastereomer 4.16 was designated as the tram-isomer (phosphoryl and 5-methyl

group ~ram) . The confi~gurations at phosphorus were assigned on the basis that in

oxazaphospholidines, such as 4.13 and 4.16, protons in a 13-cis-relationship to a P-O group

are deshielded. '69 Thus H-4 and H-5 in 4.16 appear m e r downfield than the corresponding

protons in 4.13. The configuration at phosphorus for al1 of the oxazaphospholidines prepared

in this work was determined in this manner and these configurations were later confirmed by

X-ray crystallography of some of the fluorinated derivatives (vide inpu).

1. (COC1)2, cat. DMF, CH2C12

2. (-)-ephedrine, Et3N,

1 toiuene. 4 0 OC - rt

cis-isomers trans-isomers v

separated by chromatography

Scheme 4.3. Synthetic scheme for diastereomers 4.13-4.18.

The fluorination of 4.13 and 4.16 was first attempted by subjecting them to the same

fluorination conditions as described above for 4.9 (Scheme 4.4). The results of these

fluorination reactions are shown in Tables 4.2 and 4.3. For both isomers, the yield and the de

of the reaction was highly dependent on the base and cation. NaHMDS gave the highest

yields (54% for cis-isomer 4.13 and 75% for tram-isomer 4.16). Lithium bases gave very low

de's (2-24%) for both 4.13 and 4.16. Modest de's were obtained with both 4.13 and

cis-isomers

= p-naphthyl = m-(Ph)C6H4 = Ph

base, NÇSi, THF, -78 OC

base, NFSi, THF, -78 OC

T

separated by chromatography T

separated by chromatograph y

4.19, Ar = p-naphthyl 4.22, Ar = p-naphthyl 4.25, Ar = P-naphthyl 4.28, Ar = P-naphthyl 4.20, Ar = rn-(Ph)C6H4 4.23, Ar = m-(?h)C6H4 4.26, Ar = m-(Ph)C6H4 4.29, Ar = m-(Ph)C6H4 4.21, Ar = Ph 4.24, Ar = Ph 4.27, Ar = Ph 4.30, Ar = Ph

Scheme 4.4. Synthetic scheme for fluorinated diastereomers 4.19-4.30.

1.16 using NaHMDS (58% and 54%) and KHMDS (36% and 40%). Although the de's of the

reactions were modest, the diastereomeric fluonnated phosphonamidates exhibited large

differences in mobility on silica gel (Rr differences ranged fiom 0.2-0.25) and could be readily

separated by silica gel flash chromatography. We also found that, after chomatographic

separation, the fluonnated cis-isomers 4.19 and 4.22 could be readily recrystallized and their

absotute stereochemistry determined by X-ray crystallography (Figures 4.1 and 4.2; see

Appendix A, Tables 1 and 2 for crystallographic data). Attempts to obtain X-ray quality

crystals of the tram-isomers 4.25 and 4.28 were unsuccessful. In an attempt to improve the de

Table 4.2. Effect of base and counterion on the electrophilic fluorination of cis-isomer 4.13 with NFSi.

Base % yielda of % deD 4.19 and 4.22

LiHMDS 29' 2 6) NaHMDS 54=, 47d 58', 46d (S) KHMDS 41' 36 ( 9 LDA 44' 8 ( 9 n-BuLi 33' 24 (s)

'Isolated yields. b ~ e ' s were determined by obtaining the ' 9 ~ - ~ ~ ~ of the cmde residue after aqueous workup of the resction before chromatography. '~erformed u& 0.95 equiv. base and 1.1 equiv. NFSi as described in the

d experimental section. Reverse addition: 1.1 equiv, of base was added to 4.13 (1 equiv.) and NFSi (1.1 equiv.) over a p e n d of 1 h at -78 OC. After stimng an

additional 2 h at -78 OC the reaction was warmed to rc and quenched as described in the experirnental section.

Table 4.3. Effect of base and counterion on the electrophilic fluorination of trans-isorner 4.16 with NFSi.

Base % yielda of % deb 4.25 and 4.28

LiHMDS 46' 2 (R) NaHMDS 79, 68d, 62' 54', 5od, 72' (R) KHMDS 62' 40 (R) LDA 66' 3 ( 9 n-BuLi 3 8" 18 (9

'Isolated yields. b ~ e ' s were determined by obtaining the 1 9 ~ -

NMR of the crude residue afier aqueous workup of the reaction before chromatography. 'Performed using 0.95 equiv. base and 1.1 equiv. NFSi as described in the experimental section. d~arne procedure as for (b) except 1.1 equiv. of base was used. 'Reverse addition: 1. I equiv. of base was added to 4.16 (1 equiv.) and NFSi (1. I equiv.) over a pend of I h at -78 OC. After stirring an additional 2 h at -78

OC the reaction was warmed to rt and quenched as described in the experimental section.

of the reactions, the reaction with NaHMDS was performed under a variety of different

conditions. Fluorination with the tram-isomer 4.16 using 1.1 equiv. of NaHMDS resulted in a

slight decrease in de and yield (Table 4.3). However, reverse addition of NFSi and NaHMDS

(1.1 equiv. of NaHMDS added to a solution of the phosphonamidate and 1.1 equiv. NFSi at -

78 OC over a period of 1 h) resuited in an increase in the de to 72% with 4.16 but a slight

decrease in the yield (Table 4.3); this was not the case with the cis-isomer 4.13 which resulted

in a decrease in de and yield (Table 4.2).

The mefa-@henyl)benzyl phosphonamidates, 4.14 and 4.17 (obtained in 24% and 56%.

respectively) and benzyl phosphonamidates, 4.15 and 4.18 (obtained in 20% and 41%.

respectively) were also prepared (Scheme 4.3). These phosphonamidates were fluorinated

using 0.95 equiv. NaHMDS and 1.1 equiv. NFSi to give the fluorinated cis-isomers 4.20,4.21.

1.23 and 4.24 in 43%, 29%, 25% and 15% yield, respectively, and the fluorinated trans-

isomers 4.26. 4.27, 4.29 and 4 3 0 in 53%, 30%, 32% and 17% yield, respectively (Scheme

4.4). For each fluonnation reaction, the fluorinated diastereomenc products could be readily

separated fiom one another by silica gel flash chromatography. The fluorination reaction of

the meta-@henyl)benzyt phosphonamidates (4.14 and 4.17) proceeded in overall good yields

(68%, cis-isomer; 85%, trans-isomer) but with low de's (25%, cis-isomer; 26%, frans-isomer).

The fluorination reaction of the ben@ phosphonamidates (4.15 and 4.18) also

proceeded in overall modest yields (44%, cis-isomer; 47%, tram-isomer) and Iow de's

(33% cis-isomer: 29% fians-isomer). Cis-isomers 4.20, 4.21,4.23 and 4.24 were also found

to be readily recrystallized and their structure and absolute stereochemistry was determined by

X-ray crystallography (Appendix A, Figures 1 -4).

Figure 4.1. X-ray crystai structure of 4.19.

Figure 4.2. X-ray crystal structure of 4.22.

The next step was to develop a racernization-fiee procedure for the removal of the

ephedrine auxiliary from f l u o ~ a t e d isomers 4.19-4.30 to obtain the desired enantiomerically

pure fiee acids 4.1-4.6. Since the absolute stereochemistry of al1 the fluorinated cis-isomers

4.19-4.24 was known, our initiai studies were performed using these phosphonamidates. Sting

and Steglich have reported that racemization-fiee hydrolysis of chiral ephedrine a-

alkylphosphonamidates can be accomplished by subjecting them to conc. HCI at 110 O C for 20

h.I6' However, applying these conditions to 4.19 and 4.22 resulted in a mixture of compounds

and we were unable to puri@ 4.1 and 4.2 fiom the mixture. Nevertheless, we found that the

auxiliary could be removed without racemization by modiming a procedure developed by

Calvo (Scheme 4.5).170 This involved treating 4.19-4.24 with 10% TFA/MeOH, followed by

reaction with TMSBr (1 0 equiv.). The contarninating ammonium salts were removed using

anhydrous ether followed by filtration. Hydrolysis of the TMS ester in methanoVHzO gave

the fiee acids 4.1-4.6 in modest to good yields (39-69%).

4.19, Ar = p-naphthyl 4.22, Ar = p-naphthyl 4.25, Ar = p-naphthyl 4.28, Ar = p-naphthyl 4.20, Ar = m-(Ph)C6H4 4.23, Ar = m-(Ph)C6H44.26, Ar = m-(Ph)C6H4 4.29, Ar = m-(Ph)C6H4 4.21, Ar = Ph 4.24, Ar = Ph 4.27, Ar = Ph 4.30, Ar = Ph

1.10% TFNMeOH 2. TMSBr, CH2C12 I I 1.10% TFAiMeOH

2. TMSBr, CH2CI2 I Scheme 4.5. Hydrolysis of 4.19-420 to give chirai acids 4.1-4.6.

We found that the enantiomeric purity of 4.1-4.6 could be readily detennined by

preparing solutions of 4.1-4.6 and 1 equiv. of the chirai base quinidine in CDC13 followed by

19 F-NMR analysis of the sait solutions. For example, the I9~-NMR spectra of the salts derived

from phosphonic acids 4.1 and 4.2 consist of a single set of doublet of doublets with each set

having a chernical shifi significantly different from the other set (Figure 4.3a and 4.3b). The

quinidine salts of a racemic mixture of 4.1 and 4.2 appear as two distinct sets of doublet of

doublets (Figure 4.3~). These results indicate that removal of the ephedrine auxiliary

Figure 4.3. (a) "F-NMR of 4.1 in the presence of 1 equiv. of quinidine. (b) ' 9 ~ - ~ ~ ~ of 4.2 in the presence of 1 equiv. of quinidine. (c) quinidine salts of a racemic mixture of 4.1 and 4.2.

proceeded without racemization and both 4.1 and 4.2 were obtained in high enantiorneric

purity (>97% ee). Similar resuits were obtained with acids 4.3-4.6. The epheârine auxiliary

was also removed fiom the fluorinated tram-isomers 4.25-4.30 to give acids 4.1-4.6 without

racemization and in modest to good yields using the procedwe described above (Scheme 4.5).

Since the absolute configuration of the phosphonic acids derived £tom cis-isomers

4.19-4.24 was known, it was then possible to determine the absolute configuration of the

phosphonic acids derived h m the pans-isomen 4.25-4.30 by analysis of their ' 9 ~ - ~ ~ ~

spectrum in the presence of one equiv. of quinidine. From the results of these studies, the

absolute stereochemistry of the trans-fluorophosphonamidates 4.25-4.30 was also determined.

The fluorination of the tram-isomers 4.16-4.18 using NaHMDS as base resulted in

preferential formation of the R-enantiomers (4.25-4.27). The fluorination studies with the

model tram-phosphonamidate 4.16 using a variety of different bases (Table 4.3)

demonstrated that the stereochemical outcome of the fluorination reaction is dependent upon

the nature of the base and counterion. When HMDS bases were used, the reaction proceeded

with preferential formation of the R-enantiomer 4.25; this preference was almost negligible

when lithium was the counterion. When other lithium bases were used, the S-enantiomer 4.28

was formed preferentially although the de's were very low (3-la%, Table 4.3). In contrast to

the fluonnation reactions with the tm-isomers, the fluorination reactions with the cis-

isomers 4.13-4.15 using NaHMDS proceeded with preferentiai formation of the S-

enantiomers. Studies with the model cis-isomer 4.13 showed that this was the case regardless

of the base and counterion, although with the lithium bases thïs preference was small to almost

negligible (Table 4.2).

The change or increase in stereoselectivity between lithium and sodium or potassium

bases may be due to a number of factors such as differences in the degree of complexation of

the cations with the phosphorus and other heteroatoms of the phosphonamidate, differences in

the degree of cornplexation of the cation with the solvent, differences in size of the cation, etc.

Further studies are necessary to ascertain the origin of the effect of cation on

diastereoselectivi ty.

1.4 PTPl B INHIBITION STUDIES

Inhibition studies with phosphonic acids 1.19, 3.40, 4.1-4.6 and PTPIB were

performed at pH 6.5 in bis-tris buffer-5% DMSO. The benzylphosphonic acids 4.5 and 4.6

were not inhibitors of the enzyme even at concentrations as high as 1.0 mM. The results of

the inhibition studies with 4.1-4.4 and their difluoro analogues are given in Table 4.4.

Table 4.4. Inhibition studies with phosphonic acids 1.19, 3.40, 4.1-4.4 and PTPIB. Phospho~c

4.4 3500 * 500 ND 'Errors are reported as standard deviations

Compounds 4.1-4.4 were found to be cornpetitive inhibitors of PTPlB. The R-

enantiomers 4.1 and 4.3 are not as effective inhibitors as their difluoro analogues 1.19 and

3.40 (Figures 4.4 and 4.5; compounds 1.19 and 4.1 shown), k ing 9.5-fold less potent

inhibitors than compounds 1.19 and 3.40. Thus, substitution of the pro-S fluorine with

hydrogen in 1.19 or 3.40 results in a 9.5-fold decrease in affmity for the enzyme. These

results indicate that the pro-S fluorine has some role in enhancing the affmity of the difluoro

86,149 inhibitors and support the argument that the fluorines do not enhance binding by reducing

the pK, of the phosphonic acid moiety. It is possible that this may be due to a direct

contribution of the pro-S fluorine to binding involving specific interactions of the pro-S

fluorine with residues in the active site. However, Burke's analysis of the PTPIB-1.19

complex did not uncover any significant role that the pro-S fluorine may have in binding

besides the formation of Van der Waal's interactions with the phenyl ring of Phelgz. 58

Nevertheless, it is possible that this interaction is necessary in order for the difluoro inhibitors

to be correctly positioned in the active site so that optimal interactions can be attained between

the enzyme and other functionalities on the inhibitor. This may include the formation of an

optimal fluorine H-bond between the pro-R fluorine and the N-H of Pheis2, hydrophobic

interactions between the aryl rings of the inhibitors and the side chains of certain m i n o acids

such as Tm6, Pheisz, Al-7 and Ilezig, and electrostatic binding of the phosphate group to the

positively charged phosphate binding site.18 It is ais0 possible that the strength of a FC-FH-

N hydrogen bond may be greater than a HC-FH-N hydrogen bond. However, we are not

aware of any theoretical or experimental studies supporting such an argument.

S-enantiomers 4.2 and 4.4 are approximately 105-fold poorer inhibitors than their

difluoro compounds 1.19 and 3.40. Thus, the substitution of the pro-R fluorine atoms in 1.19

and 3.40 with a hydrogen atom results in an approximately 105-fold decrease in affinity for

PTPIB. On the basis of the X-ray structure of the PTPIB-1.19 complex and molecular

dynarnics calculations, Burke and coworkers have suggested that the H-bond between the pro-

R fluorine of 1.19 and the backbone N-H of Phel82 may contribute as much -4.6 kcavmol to

Figure 4.1. Inhibition of P?P IB by compound 1.19. (a) The activity of PTP 1 B (0.15 @cm3) was measured at pH 6.5 as described under 'experimental procedures' in the presence of the following concentrations o f 1.19: (a), O pM; (i), 20 pM; (A), 40 PM; (X), 60 PM; (+), 80 FM; (O) , 100 PM. (b) Replot of the slope fiom the double-reciprocal plot versus concentrations of compound 1.19.

-0.06 -0.04 -0.02 O 0.02 0.04 0.06

[l IFDP] (p~")

Figure 4.5. Inhibition of PTPlB by compound 4.1. (a) The activity of PTPlB (O. 15 Cig/c/cm3) was measured at pH 6.5 as described under 'experimentai procedures' in the presence of the following concentrations of 4.1: (e), O FM; (m), 300 FM; ( b ) , 500 PM; (x), 750 PM; (O)'

1000 FM; (O), 1500 PM. (b) Replor of the slope fiom the double-reciprocal plot versus concentrations of compound 4.1.

the binding process.58 However, the 105-fold difference in *nity between 1.19 and 4.2

reported here corresponds to a difference of -2.75 kcdmol in binding energy which suggests

that -4.6 kcaVmol may be an overestimation of the strength of this H-bond. The subject of

fluorine hydrogen bonds involving C-F is a subject of much c o n t r o ~ e r s ~ . ' ~ ' ~ ' ~ ~ Nevertheless,

there is little doubt now that such H-bonds can form in certain instance^"^ although the

optimal strength of such bonds is still unknown. To our knowledge, studies estimating the

optimal strength of C-F"H-N hydrogen bonds have not been reported. However, a b iniiio

calculations by O'Hagan and coworkers predict that the strength of an optimal C-F"'H-O

hydrogen bond in a HO-H"'F-CH3 complex to be 2.4 kcal/rno~.'~' This value is close to the

value (-2.7 kcaVmol) that we have obtained for the difference in binding energy between 1.19

and 4.2.

1.5 MORE ABOUT DIASTEREOSELECTIVE ELECTROPHILIC FLUORINATION

Despite achieving our goal, which was the synthesis of enantiomerically pure a-

monofluorophosphonic acids, 4.1-4.6, the de's of the electrophilic fluorination reactions were

quite low. We wished to improve upon the de's by examining other chiral auxiliaries. The

auxiliary would have to meet certain requirements. First, it had to be readily available.

Second, it should promote the fluorination reaction to proceed in a high yield and de. Third.

it should be readily attached to the phosphonic acids in hi& yield and easily removed fiom the

fluorinated products in high yield. We envisioned that enantiopure (S)- or (R)-a,a-

di pheny l pyrrolidine-2-methanol, 4.3 1, would perhaps meet these requirements. This

compound, which is readily available, has been used very successfdly as a chiral ligand in

organic ~~nthesis ."~ In addition, we hypothesized that it could be readily removed fkom the

fluorinated product in a single step using mild acid and could therefore be recycled.

Phosphonic acid 4.8 was again used as our mode1 system. Treatment of the aryl

phosphonic dichloride, derived fiom 4.8, with 4.31 at O OC, in the presence of 2 equivalents of

triethylamine yielded two diastereomeric phosphonamidates, arbitrarily designated 4.A and

4.B (68% overall yield; 21% and 47%), which were readily separated by silica gel

chromatography (Scheme 4.6). We have not yet determined the absolute configuration of

either of these compounds. Both are crystalline compounds and so their absolute

configuration wilI be determined by X-ray crystallography .

1. Base 2. NFSi

Scheme 4.6. Synthesis of phosphonamidates 4.A and 4.8.

The fluorination of the isomers was attempted using the sarne fluorination conditions

as described for 4.9 (0.95 equiv. of base at -78 OC for 2 h followed by the addition of 1.1

equiv. of NFSi) using NaHMDS as base. We did not isolate the products. De's were

detemined by obfaining the l9~-FJMR of the cmde residue after aqueous workup of the

reaction and before chromatography. The results of these fluorination reactions using HMDS

bases are shown in Table 4.5. For both isomers, NaHMDS provides the best de's while

LiHMDS gives iow de's. With NaHMDS as base, the fluorination of one of the isomers

proceeded with a de of 33% while a highly respectable de of 89% was obtained with the other

diastereomer. Unfortunately, the 89% de was obtained with the diastereomer (4A or 4.B)

Table 4.5. Effect of base and counterion on the electrophilic fluorination of 4.A and 4.B.

Base % dea isomer- 1 % dea isomer-2 LiHMDS 16 2

KHMDS 69 28 'De's were determined by obtaining the ' 9 ~ - ~ ~ ~ of the cmde residue after aqueous workup of the reaction and before chromatography.

which was obtained as the minor isomer. We wished to increase the amount of the 'desired'

isomer by changing the reaction conditions. Results of these studies are shown in Table 4.6.

Ether was found to give the best ratio (2:l) in favor of the "desired" isomer. However, the

overall yield was low (20%). Increasing the temperature (gentle reflux) of the reaction

remarkably increased the ratio to 7: 1 in favor of the "desired" isomer. Although the ratio was

favorable, the overall yield of 32% (26% yield of "desired" isomer) was again low.

Nevertheless, these studies show that it is possible to alter the ratio and to obtain the 'desired'

isomer as the major product.

The above results suggest that it should be possible to develop a system that will allow

Table 4.6. Ratio of 'desired' to 'undesired' isomer. Solvent Temperature "desired i~orner"~

"undesired isomer" CH2Cl2 O OC - rt 1 /2

THF

Benzene O°C-rt 1/1

Ether O OC -rt 2/ 1

Ether O OC - reflux 7/ 1

'~etermined by obtaining the "P-NMR of the cmde midue afier aqueous workup of the reaction and before chrornatography.

us to perform diastereoselective electrophilic fluorhaion reactions on organophosphorus

substrates in high de. However, more work is necessary to achieve this goal. For example,

the X-ray crystal structures of the crystallïne isomers, 4.A and 4.B, must be obtained. Next,

the overall yield of the reaction to produce the "desired" isomer needs to be improved upon,

perhaps by longer reaction times. Different fluorination conditions and a racemization-fkee

hydrolysis procedure need to be investigated. Also, more chiral auxiliaries need to be

examined. Such studies are currently in progress in the Taylor group.

4.6 CONCLUSIONS

In conclusion, a-monofluorophosphonic acids 4.1-4.6 were synthesized in high

enantiomeric pwity. This was accomplished by the electrophilic fluorination of a-carbanions

of as ymmetric phosphonamidates bearing (-)-ephedrine as a chiral auxiliary, followeci by

chromatographie separation of the resulting diastereomers and removal of the auxiliary. The

synthetic methodology outlined here should be applicable to the synthesis of chiral a-

monofluorophosphonic acid inhibitors of other enzymes that bind or hydrolyze phosphate

esters. The Taylor group is currently investigating other chiral auciliaries in attempts to

improve the de's of the fluorination reactions. Inhibition studies with phosphonic acids 4.1-4.4

and PTP 1 B indicated that the pro-S fluorine in difluoro inhibitors 1.19 and 3.40 is essential for

good inhibition. Moreover, our inhibition studies demonstrate that the pro-R fluorine in the

di fluoro inhibitors contributes significantl y more than the pro4 fluorine towards PTP 1 B

affinity. however, the contribution of the pro-R fluorine to binding may have k e n

overestimated by previous workers?

CEIAPTER 5

NOVEL PHOSPHATE MIMETICS

5.1 INTRODUCTION

Although compounds bearing the DFMP group are good inhibitors of PTP's, studies by

Kole et al. suggest that inhibitors bearing this moiety may be incapable of penetrating cellular

membranes as a result of the dianionic nature of the DFMP group." Even though the

alternative of "caging" the DFMP derivatives with photolabile or enzyme labile groups existç,

forming ce11 permeable phosphonate esters, we opted for fmding a replacement for the DFMP

group as "caging" of these compounds can be problematic.176 Consequentiy, we wished to U

examine other functionalities that perfonn the same function as the DFMP group (in terms of

PTP inhibition) yet may allow for more facile cellular penetration.

Our studies in Chapter 2 indicate the importance of the phosphate group for ligand

binding to PTPIB. In addition, PTP's do not bind pTyr-containing peptides in which the

phosphate group is functionalized as a neutral ested7" Thus, it is not surprising that al1 of the

phosphomimetics that have been used for PTP 1 B inhibition are negativel y charged. We have

dso seen that the incorporation of fluorines into the phosphomimetics can significantly

enhance binding. Next to the DFMP group, perhaps the most effective phosphornimetic is the

dianionic fluoromaionyl (CF-malonyl) group (see Chapter 1, section 1.4.1 ). 82.88.177 Despite

being less effective PTP inhibitors than their DFMP-bearing a n a l ~ ~ u e s , ~ ~ " ~ compounds

bearing this phosphate mimetic are more readily converted into enzyme-labile diesters for

efficient delivery across ce11 membranes. However, we wished to develop phosphate

surrogates that are as effective or more effective than the CF-malonyl group, yet require no

further chernical modification for cellular studies. Anticipating that monoanionic

functionalities may be more amenable to cellular studies than dianionic species, we decided to

determine if monoanionic groups such as the a,a-difluorotetrazole (CFz-tetrazole), a,a-

difluorosulfonate (CF2-sulfonate) or a,a-difluorocarboxylate (CF2-carboxylate) moieties

could act as effective phosphate biosteres for PTP inhibition. Herein, we report the synthesis

of compounds 5.1, 53, 5.4 and 5.6 and the evaluation of these compounds, as well as

sulfonates 5.2 and 5.5, as inhibitors of PTP 1 B.

N-N N-N 5.1. X = CF~-~($N 5.4, X = c ~ ~ 4 . i ~ 5.2, X = CF2S03' 5.5, X = CF2S03-

5.3, X = CF2COO' 5.6, X = CF2COO'

5.2 EXPERIMENTAL

For detaifs concerning general matenals and instrumentation used in the syntheses

discussed below see Chapter 2 (section 2.2.1) and Chapter 3 (section 3.2).

5.2.1 SYNTHESES

Preparation of Non-Fluorinateâ Tetrazoles and Carboxylate Precursors.

5-(2-naphthylrnethy~)-2H-l,2,3,4-tetraz01e (5.33). A solution of 2-naphthylacetonitrile (2.5

g, 14.95 mrnol), sodium azide (0.88 g, 1.1 eq.) and ammonium chloride (1 -07 g, 1.1 eq.), in

anhydrous DMF (25 cm3), was heated at 80 OC under argon for 24 h (solution was yellow with

white precipitate deposited dong the walls of the reaction flask). The mixture was then

concentrated in vanro giving a yellow residue. Water (25 cm3) was added and the mixture

basified with 1 N NaOH followed by washes with ether (2 x 50 cm3). The aqueous layer was

then acidified to pH 3 with 1 N HCl and the resulting crude precipitate was filtered and

recrystallized fkom ethanol to give a white solid (67%). mp 152 - 154 OC; &(200 MHz;

CD30D) 7.76-7.87 (4 H, m, aryl), 7.35-7.50 (3 H, m, aryl) and 4.48 (2 H, s, CH2); Sc(lOO

MHz; CD30D) 157.1 (C-tetrazole), 134.9, 133.9, 133.9, 129.8, 128.7, 128.5, 127.5, 127.4.

127.1, 30.5 (s, CH2); m/z (EI) 210 (M', 65%), 182 (19), 166 (IO), 152 (21), 141 (100); Found

[M']. 210.0912. C12HION4 requires m/z 210.0905.

5-(1-naphthylmethy1)-2H-12 J,4-tetrnzole. Obtained using the general procedure as

descnbed for 5.33, starting fkom 1-naphthylacetonitrile. Recrystallized fiom ethanol as a

white solid (67%). mp 155 - 157 OC; SH(200 MHz; CD30D) 7.85-8.01 (3 H, m, aryl), 7.45-

7.54 (4 H, m, aryl) and 4.78 (2 H, s, CH2); 6c(100 MHz; CD30D) 157.2 (C-temole), 135.4,

132.8, 132.2, 129.9, 129.6, 128.5, 127.6, 127.0. 126.6, 124.2 and 28.1 (s, CH2); m/z (El) 210

(M'? 90%), 18 1 (1 7), 165 (53), 153 (66), 141 (100); Found [m, 210.0909. C 12HioN4 requires

w'z 2 lO.O9OS.

5-(2-naphthylmethy1)-2H-1 J J,4-tetrszole, sodium salt (5.27). Obtained using the general

procedure as descnbed for 5.33, except instead of acidification the crude product was HPLC

purified yielding 5.27 as a white solid (sodium sait) in quantitative yield- rnp 144-146 OC;

&(200 MHz; CD3OD) 7.84-7.73 (5 H, m, aryl), 7.47-7.37 (4 H, m, aryl) and 4.43 (2 H, s,

CH2); &(100 MHz; CD30D) 159.0 (C-tetrazole), 135.3, 134.9, 133.8, 129.4, 128.6, 128.2,

127.7, 127.2, 126.8 and 3 1.2 (s, CH2); m/r (FAB) 209 (1 00%).

5-(l-naphtbylmethyl)-2H-I,2,3,4-tetrrizoIe, sodium salt. Obtained using the general

procedure as described for 5.27, starting fkom 1 -naphthyIacetonitrile. HPLC purification

yielded a white solid (sodium salt) in quantitative yield. mp 129- 13 1 OC; &(200 MHz;

CD30D) 8.1 7 (1 H, d, J 8.8, aryl), 7.87-7.72 (3 H, m, aryl), 7.49-7.30 (5 H, m, aryl) and 4.62

(2 H, s, CH2); 6c(100 MHz; CD30D) 159.4 (C-tetrazole), 135.5, 134.0, 133.3, 129.8, 129.0,

128.4, 127.4, 127.0, 126.7, 124.7 and 28.9 (s, CH2); mit (FAB) 209 (1 00%).

5-(meta-(phenyi)phenyimetbyl~2H-1,2,3,4-tetrazoIe, sodium salt (5.28). Obtained using

the general procedure as described for 5.27, starting Eiom nitrile 5.16. HPLC purification

yielded a hygroscopic white solid (sodium salt) in quantitative yield. 6H(200 MHz; CD30D)

7.59-7.21 (9 H, m, aryl), and 4.25 (2 H, s, CH2); 6c(100 MHz; CD30D) 161.2 (C-tetrazole),

141.3, 140.8, 139.4, 129.7, 129.3, 128.3, 127.8, 127.7, 127.3, 125.5 and 31.4 (s, CH2); rn/z

(FAB) 235 (1 00%).

2 - ( te r t -Buty l ) -5 - (2 -n r iph thybe thy l ) -2H~o le (5.29). A solution of 5-(2-

naphthyhethyi)-2H-l,2,3,4-tetrazole (1 .O g, 4.76 mmol), tert-butyl alcohol (1 .O cm3, 2.17

equiv.) and concentrated HiS04 (0.12 cm3, 0.5 equiv.), in TFA (5 cm3, 12.98 equiv.), was

s~irred at room temperature overnight. The m i m e was diluted with ethyl acetate (20 cm3)'

washed subsequently with water (2 x 20 cm3), 10% NaOH (until the washings were basic),

water (2 x 20 cm3), dried (MgS04) and concentrated in vacuo. Column chromatography

(silica, CH2C12. Rf = 0.8) yielded pure 5.29 as a white solid (64%). mp 105 - 107 OC; &(200

MHz; CDC13) 7.77-7.82 (4 H, m, aryl), 7.42-7.47 (3 H, m, aryl), 4.40 (2 H, s, CH2) and 1.73 (9

H, s, C(CH3)3); 6c(100 MHz; CDC13) 164.7 (C-tetrazole), 134.5, 133.4, 132.2, 128.1, 127.6,

127.5, 127.2, 127.0, 125.9, 125.6, 63.5 (s, C(CH3))) , 32.0 (s, CH2) and 29.3 (s, C(CH3)3) ; mk

(Er) 266 (M', 78%), 223 (82), 166 (20), 153 (32), 141 (39), 56 (100); Found [m, 266.1540.

Ci 6H 18N4 requires d z 266-1532.

2-(tert-Bu~l)-5-(1-a~phthy~ethyl)-2W-l,4-tetole. Obtained using the general

procedure as dexribed for 5.29, starting fiom 5-(1-naphthylmethy1)-2H-1,2,3,4-tetrazole.

Column chromatography (silica, CH2C12, Rr = 0.8) yielded a white solid (61%). mp 64 - 66

OC; 6~(200 MHz; CDCI,) 8.22 (1 H, d, J 7.3, aryl), 7.86 (1 H, d, J 7.3, aryl), 7.75-7.83 (1 H,

m, aryl), 7.4 1-7.5 1 (4 H, m, aryl), 4.67 (2 H, s, CH2) and 1.70 (9 H, s, C(CH3)3); Sc(l 00 MHz;

CDCl3) 164.6 (C-tetrazole), 133.7, 133.1, 13 1.7, 128.6, 127.7, 127.0, 126.0, 125.6, 125.5,

123.9, 63.4 (s, C(CH3)3), 29.5 (s, CH*) and 29.2 (s, C(CH3)& m/z (Er) 266 (IM', 82%), 223

(38), 210 (92), 166 (43), 153 (100), 141 (54), 56 (69); Found [hf], 266.1533.

requires m/z 266.153 1.

2-Benql-5-(2-napbthylmethy1~2H-1~~,4-tet1*42ole (5.32). To a stirred mixture of 5.33

(200 mg, 0.95 mmol) and benzyl alcohol(0.1 cm3, 1 eq.) in anhydrous CHzClz (5 cm3), at 5 OC

under argon, was added triphenylphosphine (0.25 g, 1 eq.) followed by the dropwise addition

of neat diisopropyl azodicarboxylate (0.19 cm3, 1 eq.) over a penod of 10 minutes. The

resulting mixture was warmed to room temperature, stined for 2 days and then concentrated in

vacuo to give crude product. Column chromatography (silica, hexane:EtOAc. 7 3 , Rr = 0.6)

yielded pure 5.32 as a white solid (53%). mp 108 - 110 OC; 6~(200 MHz; CDC13) 7.81 (4 H,

brd. J8.8,aryl),7.48(3 H, brd, J4.4,aryl),7.45 (5 H, s,aryl),5.71 (2 H,s,CH2Ph)and4.43

(2 H, S. CH2); 8c(100 MHz; CDCL) 165.7 (C-tetrazole), 134.1, 133.4, 133.3, 132.3, 128.9.

128.8, 128.2, 127.6, 127.5, 127.3, 126.9, 126.0, 125.6, 56.5 (s, CH2Ph) and 31.9 (s, CH2); m/z

(Er) 300 (W, lOO%), 243 (28), 229 (39), 141 (58), 91 (71); FOU^ [m, 300.1386. Cl9HI6N4

requires d z 300.1 379.

Benzyl(2-naphthy1)acetate (5.37). 2-Naphthylacetic acid (2.0 g, 10.74 mmol) was dissolved

in rnethanol(45 cm3) and water (4.5 cm3). The resulting solution was titrated to pH 7.0 with a

20% aqueous solution of CszC03 (- 12 cm3). The mixture was then evaporated to dryness and

the resulting residue re-evaporated twice fiom DMF. The white cesium sait obtained was

s h e d with benzyl brornide (1.41 cm3, 1.1 eq.) in DMF (25 cm3) for 6 h. Upon evaporation to

dryness and treatment with water (100 cm3) the product solidified. The crude product was

taken into ethyl acetate (2 x 100 cm3), washed with brine, dried (Na2SO4) and concentrated in

vacuo to give crude product. Column chromatography (silica, hexane:EtOAc, 9: 1, Rf = 0.4)

yielded pure 5.37 as a white solid (2.57 g, 91%). mp 51 - 53 O C ; SH(200 MHz; CDC13) 7.75-

7.86 (4 H, m, aryl), 7.35-7.50 (3 H, mt aryl), 7.34 (5 H, s, aryl), 5.17 (2 H, S. 0CH2Ph) and

3.85 (2 H, s, CH2); 6,(100 MHz; CDC13) 171.2 (CO), 135.7, 133.3, 132.4. 131.3, 128.4, 128.1,

128.1, 127.9, 127.6, 127.5, 127.3, 126.0, 125.7, 66.6 (s, 0CH2Ph) and 41.4 (S. CH2). m/r (El)

276 (w, 49%), 141 (100), 1 15 (22), 9 1 (45); Found M', 276.1 153. C i8H1602 requires m/z

276.1 150.

Benyl (1-naphthy1)acetate. Obtained using the generai procedure as described for 5.37,

staring from 1 -naphthylacetic acid. CoIumn chromatography (silica, hexane:EtOAc. 9: 1, Rr =

0.4) yielded a colorless oil (93%). 6~(200 MHz; CDC13) 8.00 (1 H. br d' J 4.4, aryl), 7.79-

7.97 (2 H, m, aryl), 7.34-7.53 (4 H, m, aryl), 7.30 (5 H, s, aryl), 5.14 (2 H, s, 0CH2Ph) and

4.13 (2 H, s, CH& 8c(100 MHz; CDCl3) 171.2 (CO), 135.7, 133.7, 131.9, 130.3. 128.6, 128.3.

128.1, 128.0, 127.9, 127.8, 126.2, 125.6, 125.3, 123.7, 66.5 (s, 0CH2Ph) and 39.0 (s, CH2).

m/'. (EI) 276 (M+, 37%), 141 (100), 115 (18), 91 (39); Found M', 276.1 143. CigHi602

requires m/z 276.1 150.

Benzyl 2-(meta-bromophenyl)acetate (5.36). Obtained using the generai procedure as

described for 5.37, starting fiom 5.34. Column chromatography (silica, hexane:EtOAc, 4: 1, Rf

= 0.6) yielded pure 5.36 as a colorless oil (88%). &(ZOO MHz; CDC13) 7.19-7.47 (9 H, m,

aryl). 5.15 (2 H, s, OCH2Ph) and 3.64 (2 H, s, CH2); 6c(100 MHz; CDCb) 170.4 (CO), 136.3.

135.9, 132.5, 130.3, 130.1, 128.6, 128.3, 128.1, 127.9, 122.6, 66.8 (s, 0CH2Ph) and 40.9 (s,

CH2); (EI) L81"9'~r 306 (M*, 12%), 304 (12), 171 (16), 169 (16), 91 (100); Found [W],

304.0 1 18. C 15Hi30zBr requires m/z 304.0099.

Beny l 2-(3-biphenyiy1)acetate (5.38). To 536 (2.5 g, 0.0086 mmol) in deaerated ethanol

(50 cm3) was added phenylboronic acid (1.3 g, 1.2 eq.), Pd(0Ac)z (0.1 g, 0.05 eq.) and solid

Na2C03 (1 -4 g, 1.5 eq.). The dark brown reaction mixture was stirred at room temperature for

36 h, under argon. diluted with ether (100 cm3) and filtered. The filtrate was washed with 1 N

NaOH (1 x 100 cm3), brine (1 x 100 cm3), dned (MgS04) and concentrated down by rotary

evaporation. Column chromatography (silica, EtOAc:hexane, 1 :9, Rf = 0.3) yielded pure 5.38

as a colorless oil (70%). &(200 MHz; CDC13) 7.34-7.59 (14 H, m, aryl), 5.17 (2 H, s,

0CH2Ph) and 3.75 (2 H, s' CH2); 6~(100 MHz; CDC13) 171.1 (CO), 141.8, 141.2, 136.3,

134.7, 132.5, 130.4, 130.1, 129.1, 128.8, 128.6, 128.3, 128.2, 127.5, 127.3- 126.0, 66.7 (S.

0CH2Ph) and 41.6 (s, CH2); m/z (EI) 302 (M', 61%), 167 (100), 152 (23), 91 (99); Found

[m, 302.l298. CziH1802 requires dz302.1307.

General Procedure for the Preparation of a,a-Dauoro and a-Monofluoro Benzylic Nitriles, Tetrazoles and Carboxylic Esters.

To a solution of the benzylic nitrile, tetrazole or ester in anhydrous THF

(approximately 5- 10 cm3 THF/mmol of nitrile, tetrazole or ester) at -78 OC was added base

(2.2 equiv. for difluonnation, 1.1 equiv. for monofluonnation) over a period of 2 minutes.

The resulting orange to dark red solution was stirred for 1 h at -78 OC. A solution of NFSi

(2.5 equiv. for difluorination, 1.1 equiv. for rnonofiuorination) in anhydrous THF

(approximately 2-4 cm3 THF/mrnol NFSi) was added over a penod of 2 minutes, during which

time the solution tumed fiom dark red or orange to yellow-brown. mer addition, the solution

was stirred for 2-3 hours at -78 OC during which time a precipitate may form. The reaction

was quenched with 0.01 N HCI and the resulting solution (precipitate dissolves) was extracted

with CHCI,. The combined organic layen were washed with 5% NaHC03, brine, dried

(MgSO1) and concentrated in vucuo to give a yellow residue. Purification was achieved using

silica gel flash chromatography.

2J-Difluoro-2-(2-oaphthy1)acetonitriJe (5.8). Obtained using the general procedure

descnbed above fiom nitrile 5.7 using a variety of bases (see Table 5.1). Column

chromatography (silica, hexane:CHzC12, 8:2, RF = 0.5) yielded pure 5.8 as a white solid (50-

52%) using t-BuLi as base. mp 33-34 OC; 6~(200 MHz; CDC13) 8.22 (1 H. s, aryl), 7.91-8.04

(3 H. m, aryl) and 7.63-7.70 (3 H, m, aryl); 64188 MHz; CM31s) -6.8 (1 F, s); &(100 MHz;

CDCI;) 134.8, 132.1, 129.6, 128.9, 128.6, 128.2, 127.9, 127.5, 126.2 (t), 120.6 (t), 112.6 (t),

109.2 (t. J& 242.8, CF2); W" (EI) 203 (Mt, 100%), 184 (1 O), 177 (64), 153 (9), 127 ( 1 6):

Found [m, 203.0555. Ci2H7FzN requires m/r 203.0547.

2-Fluoro-2-(2-naphthyl)acetonitrile (5.9). Obtained using the general procedure described

above (monofluonnation conditions) fiom nitrile 5.7 using t-BuLi as base. Column

chromatography (silica, hexane:CHzCl~, 8:2, Rf = 0.4) yielded pure 5.9 as a white solid (60%).

mp 54-56 OC; 6~(200 MHz; CDCI3) 8.03 (1 H, d, J 1 1.7' aryl), 7.89-7.96 (3 H. m, aryl), 7.55-

7.63 (3 H. m, aryl) and 6.23 (1 H, d, JHF 46.9, C m ) ; &(l88 MHz; CDCI,) -91.1 (1 F. d, JFH

46.9); Sc(lOO MHz; CDC13) 134.2 (d), 132.6 (d), 129.5, 128.5 (d), 128.4 (d), 127.9' 127.8,

127.1, 123.5 (d), 115.3 (d, 533.7), 80.31 (d, JCF 181.6, CHF); 4. (EI) 185 (W, 100), 166

(1 j), 1 5 8 (29); Found m, 1 85.064 1. C i2HsFN requires ml. 185.064 1.

2,2-Difluoro-2-(1-aaphthyl)acetooitrile (5.25). Obtained using the general procedure

descnbed above fiom nitrile 5.24 using t-BuLi as base. Column chromatography (silica,

hexane:CH2C12, 8:2, Rf = 0.5) yielded pure 5.25 as a colorless oil (46%). bH(200 MHz;

CDC13) 8.28 (1 H, d, 58.8, aryl), 7.93-8.07 (3 H, m, aryl) and 7.48-7.73 (3 H, m, aryl); &(188

MHz; CDC13) -6.7 (1 F, s); &(100 MHz; CDC13) 133.9, 133.6, 129.1, 128.5, 128.1, 126.9,

126.0 (t), 125.3 (t), 124.2, 123.4 (t), 112.8 (t, J iF 48.0, CN) and 109.7 (t, J C ~ 243.5, CF2); m/z

(Er) 203 (MC, 100%), 184 (7), 177 (36), 153 (53), 127 (1 1); Found [M'], 203.0537. CizH7FzN

requires m/r 203.0546.

2-Fluoro-2-(1-naphthyl)acetonitrile. Obtained using the general procedure described above

(monofluonnation conditions) fiom nitrile 5.24 using t-BuLi as base. Column

chromatography (silica, hexane:CH2Clz, 8:2, Rf = 0.4) yielded a white solid (62%). mp 41 -

43 OC; &(200 MHz; CDC13) 7.5 1-7-71 (3 H, m, aryl), 7.83 (1 H, d, J 7.3, q l ) , 7.95-8.13 (3

H, m, aryl) and 6.68 (1 HT d, J"F 46.9, CHF); 8d188 MHz; CDCb) -93.7 (1 F, d, JFH 46.9);

&(IO0 MHz; CDC13) 133.8 (d), 132.3 (d), 129.9, 129.0, 127.8, 127.2 (d), 126.8, 126.6 (d),

124.8 (d), 122.6, 1 15.20 (d, JCF 33.7, CN) and 79.2 (d, JCF 180.8, CHF); d z (El) 185 (M?,

1 OO%), 166 (1 7), 158 (28); Found [m, 185.0645. Ci2H~FN requires m/s 185.0641.

2J-Difluoro-2-@ara-nitrophenyl)acetonitrile (5.17). Obtained using the general procedure

described above from nitrile 5.10 using NaHMDS as base. Column chromatography (silica,

hexane:CH2Cî2, 653.5, Rf = 0.5) yielded pure 5.17 as a colorless oil (34%). aH(200 MHz;

CDC13) 8.42 (2 H, d, J 8.8, aryl), 7.9 1 (2 H, d, J 8.8, aryl); 6~(188 MHz; CDC13) -9.1 (1 F, s);

6c(100 MHz; CDC13) 150.3, 136.6 (t), 126.8 (t), 124.5, 1 1 1.6 (t, JCF 47.0, CN), 107.45 (t, JiF

245.2, CF2); m/z (EI) 198 (M', 54%), 152 (100), 125 (39, 75 (1 8); Found [w], 198.024 1.

C8H4F2N2O2 requires d z 198.024 1.

2,t-Difluoro-2-@ara-bromophenyl)icetonitIe (5.18). Obtained using the general procedure

described above fiom nitrile 5.11 using LDA as base. Column chromatography (silica,

hexane:CH2C12, 6.5:3.5, Rf = 0.5) yielded pure 5.18 as a colorless oil (19%). &(200 MHz;

CDC13) 7.70 (2 H, d, J 8.8, aryl), 7.55 (2 Fi, d, J 7.3, aryl); 6~(188 MHz; CDC13) -7.8 (1 F, s);

6c(lOO MHz; CDC13) 132.6, 130.3 (t), 127.5 (t), 126.84 (t), 1 12.2 (t, JCF 48.0, CN) and 108.4

(t, J C ~ 243.5, CF2); m/r (ET) 233" (M', 99%), 231'~ (100), 207" (63), 205" (63), 152 (27);

Found [m, 230.9489. Cs&BrF2N requires m/z 230.9495.

2,2-Difluoro-2-(paru-methy1pbenyi)acetonite (5.19). Obtained using the general

procedure described above tiom nitrile 5.12 using t-BuLi as base. Column chromatography

(silica, hexane:CHzC12, 6.5:3.5, Rf = 0.5) yielded pure 5.19 as a colorless oil (44%). &(200

MHz; CDC13) 7.56 (2 H, d, J 8.8, aryl), 7.34 (2 H, d. J 7.4, aryl) and 2.44 (3 H, s, CH3):

sF(188 MHz; CDC13) -6.8 (1 F, s); &(100 MHz; CDC13) 143.2 (t), 129.8, 128.5 (t), 125.1 (t),

112.7 (t, JCF 48.4. CN), 109.1 (t, JCF 242.4, CF2) and 21.4 (s, CH3); m/z (EI) 167 (M', 99%),

152 (8), 141 (35),91 (100); Found m, 167.0546. C9H7FzN requires m/z 167-0547.

2,2-Difluoro-2-(ortko-methylphenyl)acetonte (5.20). Obtained using the general

procedure described above from nitrile 5.13 using t-BuLi as base. Colurnn chromatography

(silica, hexane:CHzC12, 6.5:3.5, Rf = 0.5) yielded pure 5.20 as a colorless oi1 (37%). &H(200

MHz; CDC13) 7.65 (1 H, d, J 7.3, aryl), 7.37-7.49 (1 H. m, aryl), 7.33 (2 H, br m, aryl) and

2.56 (3 H, s, CH3); 6~(188 MHz; CDCl3) -9.3 (1 F, s); &(100 MHz; CDC13) 136.7 (br t),

132.5, 132.3 (br t), 129.1 (t), 126.3, 125.8 (t), 112.5 (t,JCF 48.4, CN), 109.1 (t, JCF 243.9, CF2)

and 19.3 (s, CH3); mlz (EI) 167 (Mt, 77%), 140 (100), 91 (74); Found [Ml, 167.0345.

C9H7F2N requires d z 167.0547.

2,2-Difluoro-2-@ara-methoxypheny1)acetonitle (5.21). Obtained using the general

procedure described above from nitrile 5.14 using t-BuLi as base. Column chromatography

(silica, hexane:CH2C12, 653.5, Rr = 0.5) yielded pure 5.21 as a colorless oil (34%). aH(200

MHz; CDC13) 7.60 (2 H, d, J 8.8, aryl), 7.01 (2 H, d, J 8.8, aryi) and 3.88 (3 H, s, CH30);

&(188 MHz; CDC13) -5.3 (1 F, s); &(IO0 MHz; CDCb) 162.7, 126.9 (t), 123.3 (t), 1 14.5,

1 12.7 (t, JCF 49.1, CN), 109.03 (t, Jcr 241 -7, CF2) and 55.5 (s, CH30); m/z (EI) 259 (W.

29%), 234 (9), 183 (87), 164 (IS), 157 ( I O ) , 152 (12); Fomd Ml, 183.0493. CsH7F2N0

requires m/s 1 83 -0496.

2J-Difluoro-2-(meta-methoxypbenyl)acetonie (5.22). Obtained using the general

procedure described above fiom nitrile 5.15 using t-BuLi as base. Column chromatography

(silica, hexane:CH2C12, 653.5, Rf = 0.5) yielded pure 5.22 as a colorless oil (56%). &(200

MHz; CDC13) 7.12-7.49 (4 H, m, aryl) and 3.87 (3 H, s, CH30); &(188 MHz; CDCI3) -7.7 (1

F, s); 6c(100 MHz; CDC13) 160.1, 132.5 (t), 130.4, 118.3 (t), 117.3 (t), 112.5 (t, JiF 48.4,

CN), 1 10.47 (t), 108.65 (t, JCF 243.2, CF2) and 55.42 (s, CH30); m/z (EI) 183 M. 100%),

157 (1 5), 152 (1 8); Found m, 183.0487. C9H7FzN0 requires ni/r 183.0494.

2,2-Difluoro-2-(3-biphenyIyl)acetonitnIe (5.23). Obtained using the general procedure

described above fiom nitrile 5.16 using t-BuLi as base. Column chromatography (silica?

hexane:CH2Clz, 653.5, Rf = 0.5) yielded pure 5.23 as a colorless oil (47%). 6*(200 MHz:

CDC13) 7.81-7.88 (2 H, rn, q l ) , 7.60-7.65 (4 H. br m, aryl) and 7.43-7.54 (3 H, m. aryl);

&(188 MHz; CDCl3) -7.5 (1 F, s); &(100 MHz; CDC13) 6 142.7, 139.3, 13 1.8 (t), 13 1.2,

129.7, 129.1, 128.9 (t), 128.3, 127.2, 123.9 (t), 112.6 (t, JCF 48.0, CN), 108.9 (t, JCF 243.5,

CF2); m/z (EI) 229 (Mt, 100%), 203 (1 5 ) , 152 (1 5); Found [W], 229.0694. C14H9FZN

requires d z 229.0703.

2-(tert-Buty1)-5-[difluoro(2-napb t h y l ) m e t h 2 H - 4 t e t o e (530). Obtained using

the genera1 procedure described above fiom tetrazole 5.29 using a varïety of bases (see Table

5.3). Column chromatography (silica, CH2Clz:pentane, 64, Rf = 0.6) yielded pure 5.30 as a

white solid (5246%) using t-BuLi as base. mp 69-71 OC; 8"(200 MHz; CDCl3) 8.20 (1 H, s,

aryl), 7.86-7.96 (3 H, m, aryl), 7.73 (1 H, d, J 8.8, aryl), 7.52-7.60 (2 H, m, aryl) and 1 -77 (9

H, s, C(CH3),); 6d188 MHz; CDC13) -16.1 (1 F, s); &(IO0 MHz; CDC13) 162.5 (t, C-

tetrazole), 134.1, 132.4, 132.1 (t), 128.8, 128.6, 127.8, 127.5, 126.8, 125.6 (t), 122.4 (t), 115.6

(t. J C ~ 242.1, CF& 65.1 (s, C(CH3)3) and 29.3 (s, C(CH3)3); m/z (EI) 302 (M+, 60%). 246

(96), 218 (33), 177 (100), 170 (39), 57 (66); Found [m, 302.1347. C16H16F2N4 requires I)Uz

302.1343.

2-(tert-Butyl)-5-[difluoro(:l-naphthyl)methy-2H-,4-tetole. Obtained using the

general procedure described above fiom 2-(tert-butyl)-5-[(1-naphthyhethyl]-2H-l.2,3.4-

tetrazole using t-BuLi as base. Column chromatography (siIica, CH2C12:pentane, 6:4, Rr =

0.6) yielded a white solid (50%). mp 48 - 50 OC; &(200 MHz; CDCl3) 8.10-8.14 (1 H, br d,

aryl). 7.87-8.00 (3 H, m, aryl), 7.47-7.59 (3 H, m, aryl) and 1-74 (9 H, s, C(CH3)3); &(188

MHz; CDC13) -13.5 (1 F, s); ô~ (100 MHz; CDC13) 162.8 (t, C-tetrazole), 134.0, 13 1.9, 130.2

(t). 129.4, 128.8, 126.9, 126.0, 124.8 (t), 124.7, 124.5, 116.2 (t, JCF 241.7, CF2), 65.0 (s,

C(CH3),) and 29.2 (s, C(CH3)i); m/z (El) 302 (M', 62%), 190 (36), 177 (100), 57 (36); Found -

[Mt]. 302.1342. C 16H16FZN1 requires m/z 302.1343.

2 - ( t e r t - B u t y l ) - 5 - [ f l u o r o ( 2 - n a p h t h y l ) m e t ~ o e (5.31). Obtained using

the general procedure described above (monofluorination conditions) fiom tetrazole 5.29

using t-BuLi as base. Column chrornatography (silica, CH2C12:pentane, 6:4, Rr = 0.5) yielded

pure 5.31 as a white solid (61%). mp 75 - 77 OC; 8"(200 MHz; CDC13) 8.02 (1 H, s, aryl),

7.84-7.93 (3 H, m, aryl), 7.72 (1 H, d, J 1.5, q l ) , 7.49-7.54 (2 H, m, aryi), 6.93 (1 H, d, JHF

45.8, CHF) and 1 -76 (9 H, s, C(CH3)3); &(i 88 MHz; CDC13) -93.8 (1 F, d, JFH 45.8); Sc(l 00

MHz; CDCI3) 163.7 (d, C-tetrazole), 133.5 (d), 133.5, 132.8, 128.5, 128.2, 127.6, 126.7,

126.4, 126.4 (d), 123.9 (d), 86.9 (d, J C ~ 172.8, CHF), 64.4 (s, C(CH3)3) and 29.2 (s, C(CH3)3);

mk (El) 284 (1M: 50), 266 (43), 223 (41), 172 (44), 159 (98), 152 (27), 56 (100); Found

[m, 284.1439. Ci&Ii#N4 requires nr/s 284.1437.

2-(tert-Butyl)-5-[fluoro(l-nap~thyl)methyl-2-1,4-teole. Obtained using the

general procedure described above (monofluorination conditions) fiom 2-(tert-buty1)-5-[(l-

naphthylmethyl]-2H-l,2,3,4-tetrazole using t-BuLi as base. Colurnn chromatography (silica,

CHzClz:pentane, 6:4, Rf = 0.5) yielded a white solid (60%). mp 56 - 58 OC; &(200 MHz;

CDCI3) 8.07 (1 H, d, J 1.1, aryl), 7.79-7.92 (3 H, m, aryl), 7.49-7.59 (3 H, m, aryl). 7.36 (1 H.

S. CHF) and 1.73 (9 H, s, C(CH3)3); 6d188 MHz; CDC13) -98.1 (1 F, d, JFH 45.8); &(100

MHz; CDC13) 163.6 (d, C-tetrazole), 133.6, 131.8 (d), 129.9, 129.9, 128.8, 126.6. 125.9,

125.1. 124.9 (d). l23.2,85. 1 (d, JcF 172.8, CHF), 64.4 (s, C(CH3l3) and 29.2 (s, m/z

(Er) 284 (w, 49%), 266 (9), 228 (82), 171 (53), 159 (100), 152 (47), 57 (82); Found [MT],

284.144 1. C16H17FN4 requires m/z 284.1437.

Benzyl 2,2-difluoro-2-(2-naphthy1)acetate (5.40). Obtained using the general procedure

descnbed above fiom 5.37 using LDA as base. Column chromatography (silica?

hexane:EtOAc, 9.5:0.5, Rr = 0.4) yielded pure 5.40 as a white solid (62%). mp 65 - 67 OC;

&(200 MHz; CDC13) 8-10 (1 H, s, aryl), 7.86-7.94 (3 H, m, aryl), 7.55-7.68 (3 H, m, aryl),

7.32 (5 H, s, q l ) and 5.28 (2 H, s, 0CH2Ph); SF(188 MHz; CDC13) -26.07 (1 F, s); 6,(100

MHz; CDC13) 164.1 (t, CO), 134.2, 134.2, 132.3, 129.8 (t), 128.7, 128.7, 128.6, 128.2, 127.8,

127.7, 126.9, 125.8 (t), 121.8 (t), 113.6 (t, J C ~ 252.7, CF2) and 68.5 (s, 0CH2Ph); d z (EI)

3 12 (W, 34%), 1 77 (1 OO), 127 ( 17), 9 1 (63); Found NI', 3 12.0965. C 19H14F202 requires mir

3 12.0962.

Benzyi 2J-difluoro-2-(1-napbtbyl)acetate. Obtained using the general procedure described

above fkom benzyl2-(1 -naphthyl)acetate using LDA as base. Column chromatography (silica,

hexane:EtOAc, 950.5, Rf = 0.4) yielded a colorless oil (61%). &(200 MHz; CDC13) 8.15 (1

H, d, J 8.8, aryl), 7.85-8.00 (3 H, m, aryl), 7.48-7.57 (3 H, m, aryl), 7.20-7.32 (5 H, m. aryl)

and 5.25 (2 H, s, 0CH2Ph); SF(188 MHz; CDCI3) -24.5 (1 F, s); 8,(100 MHz, CDC13) 164.2 (t,

CO), 134.1. 133.7, 131.9, 129.2, 128.8, 128.6, 128.5, 128.0, 127.3, 126.2, 124.9 (t), 124.5,

124.1 (t), 1 14.3 (t, J& 252.3, CF2) and 68.4 (s, 0CH2Ph); m/z (El) 3 12 (M', 33%), 177 (100),

127 (1 3), 91 (60); Found M', 3 12.0959. C&&02 requires mk 3 12.0962.

Benzyl 2,Z-difluoro-2-(3-bipbeny1yl)acetate (5.39). Obtained using the general procedure

described above from 5.38 using LDA as base. Column chromatography (silica?

hexane:EtOAc, 9: 1, Rf = 0.4) yielded pure 539 as a colorless oil(40%). &(200 MHz; CDC13)

7 - 5 7 - 8 5 (14 H, m, aryl) and 5.31 (2 H, s, 0CH2Ph); &(188 MHz; CDC13) -28.0 (1 F, s);

&(100 MHz; CDC13) 163.9 (t, CO), 142.0, 140.1, 134.2, 134.2, 133.6 (t), 129.7, 129.1, 128.9,

138.7. 128.1 127.9, 127.2, 124.3 (m), 1 13.6 (t, JCF 252.6, CF2) and 68.3 (s, 0CH2Ph); m/z

(EI) 338 (M', 5 1 %), 302 (71, 203 (1 OO), 91 (68); Found m, 338.1 126. CziHi6Fz02 requires

m/' 338.1 1 18.

Benzyl 2-fluoro-2-(2-naphthy1)acetate. Obtained using the general procedure described

above (monofluorination conditions) fiom 5.37 using LDA as base. Column chromatography

(silica, hexane:EtOAc, 950.5, Rf = 0.3) yielded a white solid (64%). mp 66 - 68 OC; 6"(200

MHz; CDC13) 7.83-7.93 (4 H, rn, aryl), 7.51-7.58 (3 H, m, aryl), 7.3 1 (5 H, br s, aryl), 6.00 (1

H, d, J H ~ 47.6, CHF) and 5.29 & 5.18 (2 H, ABq, J 12.4, 0CH2Ph); &(188 MHz; CDCI3) -

103.55 (1 F, d, JFH 47.6); 6,(100 MHz; CDC13) 168.3 (d, CO), 134.8, 133.6 (d), 132.8, 131.3

(d), 128.7, 128.5, 128.4, 128.2, 128.1, 127.6, 126.9, 126.6 (d), 126.5, 123.5 (d), 89.4 (d, JCF

186.0, CHF) and 67.3 (s, 0CH2Ph); m/z (EI) 294 @A+, 29%), 159 (100), 133 (17), 91 (58);

Found [MI', 294.105 1. CipHlsF02 requires m/r 294.1056.

Benzyl 2-fluoro-2-(1-naphthy1)acetate. Obtained using the general procedure described

above (monofluorination conditions) fkom benzyl 2-(1-naphthy1)acetate using LDA as base.

Column chrornatography (silica, hexaneStOAc, 950.5, Rf = 0.3) yielded a white solid (65%).

rnp 73 - 75 OC; 6~(200 MHz; CDC13) 8.18 (1 H, br d, J 1.8, aryi), 7.90-7.95 (2 H. m, aryl),

7.49-7.63 (4 H, m, aryl), 7.19-7.31 (5 H, m, aryl), 6.43 (1 H, d, J H ~ 45.8, CHF) and 5.30 &

5.16 (2 H, ABq, J 12.4, OCHiPh); ad188 MHz; CDC13) -102.17 (1 F, d, JFH 45.8); &(IO0

MHz; CDC13) 168.7 (d, CO), 134.8, 133.8, 130.5, 130.5, 129.9 (d), 128.7, 128.4, 128.3, 128.0,

126.9, 126.9 (d), 126.1, 124.9, 123.6 (d), 88.6 (d, JiF 186.0, CHF) and 67.2 (s, 0CH2Ph); m/z

(EI) 294 (w, 3 1 %), 159 (100), 133 (20), 9 1 (55); Found FI]', 294.1052. Ci9Hi5F02 requires

d z 294-1054.

Preparation of Fluorinated Tetrazole Derivatives.

5-[Difluoro(2-napbthyl)methyl]-2H-1,2~,4-ietoIe (5.1). A suspension of 5.8 (100 mg,

0.49 rnrnol) and Na& (0.035 g, 1.1 equiv.) in anhydrous DMF (5 cm3) was stirred under argon

for 3 h at 65 OC. White still hot the solution was filtered and concentrated in vacuo. The

crude product was then HPLC purified yielding 5.1 as a white solid (sodium sait) in

quantitative yield. &(200 MHz; CD30D) 8.07 (1 H, s, aryl), 7.89-7.97 (3 H, br m, aryl) and

7.53-7.66 (3 H, m, aryl); 661 88 MHz; CD30D) -10.9 (1 F, s); &(100 MHz; CD30D) 1 6 1.2 (t.

C-tetrazote), 135.4, 135.2 (t), 133.9, 129.7, 129.5, 128.7, 128.4, 127.8, 126.4 (t), 123.6 (t) and

119.01 (t, JCF 238.4, CF2); m / . (FAB) 245 (100Yo).

5-[Difluoro(l-naphthyl)rnethyl]-2H-l,2,3,4-t01e. Obtained using the general procedure

described above fiom nitrile 5.25. HPLC purification yielded a white solid (sodium salt) in

quantitative yield. 6~(200 MHz; DzO) 8.09 (1 H, d, J 8.7, aryl), 7.93 (2 H, d, J 7.3, q l ) , 7.74

(1 H, d, J 8.8, aryl) and 7.42-7.65 (3 H, m, aryl); 64188 MHz; D20) -9.4 (1 F, s); &(IO0

MHz; DzO) 161.7 (t, C-tetrazole), 134.6, 132.8, 131.1 (t), 129.7, 127.9, 127.0, 125.5, 125.4 (t)

and 1 18.6 (t, JiF 238.4, CF& rn/z (FAB) 245 (100%).

5-[Difluoro(3-biphenyly1)methylJ-2H-l,2 J,4tetrazole (5.4). Obtained using the general

procedure described above fkom nitrile 5.23. HPLC purification yielded pure 5.4 as a white

solid (sodium salt) in quantitative yield. 6&00 MHz; DtO) 7.72 (1 H, s, aryl), 7.64 (1 H, d, J

7.3. aryl), 7.47-7.53 (4 H, br m, aryl) and 7.3 1-7.42 (3 H, m, aryl); &(188 MHz; DzO) -1 1.7 (1

F. s); &(IO0 MHz; DzO) 161.3 (t. C-tetrazole), 141 -9, 140.2, 136.9 (t), 130.2, 129.9, 129.7.

128.7, 127.7, 125.1 (t), 124.4 (t), 118.3 (î, JCF 239.1, CF2); mh (FAB) 271 (100%).

Deprotection of Carboxylic Ester Derivatives.

2,2-difluoro-2-(2-naphthy1)acetic acid (53). To 5.40 (250mg, 0.84 m o l ) dissolved in

EtOAc (-5 cm3) was added 10% Pd/C (15-20 mg) and the reaction mixture was stirred

overnight, under Ht (1 atm). The mixture was then fittered through Celite and concentrated in

vacuo. Column chromatography (silica, hexane:EtO~c, 4:1, Rr = 0.2) yielded pure 5.3 as a

white solid (71%). mp 180 OC (decomp.); 6~(200 MHz; CD30D) 8-15 (1 H. S. aryl), 7.92 ( 3

H, br d. aryl), 7.73 (1 H, d, J 8.8, aryl) and 7.54 (2 H, br s, aryl); 6~(188 MHz; CD30D) -22.5

(1 F. s); 6c(100 MHz; CD30D) 170.9 (t, CO), 135.2, 134.7 (t), 133.9. 129.5, 128.9, 128.7,

127.9, 127.5, 126.1 (t), 123.6 (t), 1 12.9 (t, JCF 252.2, CF2); m/z (FAB) 22 1 (1 00%).

2,2-difluoro-2-(1-naphthyl)acetic acid. Obtaining using the general procedure described

above, starting fiom benzyl 2,2difluoro-2-(1-naphthyl) acetate. Column chromatography

(silica hexane:EtOAc, 4:1, Rf = 0.2) yielded a white solid (73%). mp 180 OC (decomp.);

MHz; CD30D) 8.40 (1 H, br d, aryl), 7.96-7.83 (3 H, m, aryl) and 7.54 (3 H, m, aryl);

5~(188 MHz; CD30D) -18.5 (1 F, s); 6c(100 MHz; CD30D) 172.3 (t, CO), 135.3, 13 1.3.

129.3, 127.1, 126.6, 126.4, 125.5, 125.3, 125.2, 112.3 (t. JCF 254.4, CFt); m/z (FAB) 221

(80%). 177 (1 00).

2,2-difluoro-243-biphenyly1)acetic acid (5.6). Obtaining using the general procedure

described above, starting fiom 5.39. Column chromatography (silica, hexane:EtOAc. 4: 1, Rr

= 0.2) yielded pure 5.6 as a white solid (69%). mp 125 OC (decomp.); 6~(200 MHz; DzO)

7.67 (1 H, s, aryl), 7.42 (1 H, br s, aryl) and 7.01-7.27 (7 H, m, aryl); &(188 MHz; DtO) -22.4

(1 F, s); 6c(100 MHz; D20): 175.2 (CO), 156.3, 155.0, 150.7 (t), 144.6, 144.4, 144.2, 143.2,

142.2, 139.4 (br t), 138.8 (br t), 1 10.9 (t, JCF 250.8, CF2); m/z (FAB) 247 (1 00%).

2-(3-Biphenyly1)acetic acid (5.41). Obtaining using the general procedure described above,

starting fiom 5.40. Column chromatography (siiica, hexane:EtOAc, 4: 1. Rf = 0.1) yielded

pure 5.41 as a white solid (72%). mp 120 OC (decomp.); tiH(200 MHz; D20) 7.30-7.3s (3 H,

br m. q l ) , 7.08-7.16 (6 H, br m, aryl) and 3.43 (2 H, s, CH2); &(100 MHz; DzO): 183.5

(CO). 143.4, 143.2, 140.7, 131.9, 131.7, 131.1. 130.5, 130.3, 129.7, 127.6 and 47.4 (s, CH2);

m/z (FAB) 2 1 1 (45%).

Preparation of Malonyl and Fluoromalonyl Derivatives.

Di(tert-butyl) 2-(2-naphthyloxy) malonate (5.44). 2-Naphthol (650 mg, 4.5 m o l ) , Rh(1I)

acetate (87.7 mg, 0.044 eq.) and di-leri-butyl a-diaz~rnalonate,l'~ in anhydrous benzene. were

stirred ovemight under reflux. The mixture was cooled to roorn temperature. filtered through

Celite and concentrated in vacuo to give a crude yellow oil. Column chromatography (silica,

hexane:EtOAc, 9: 1, Rf = 0.4) yielded pure 5.44 as a white solid. mp 74-76 OC; 6~(200 MHz;

CDCI3) 7.68-7.80 (3 H, m, aryl), 7.30-7.44 (3 H, m, aryl), 7.15 (1 H, s, aryt), 5.15 (1 H, s, CH)

and 1-52 (1 8 H, s, î[C(CH&]; k(100 MHz; CDC13) 164.7 (CO), 155.3, 134.3, 129.6, 127.6,

126.9, 126.4, 124.2, 118.9, 109.1, 83.2 (s, C(CH3h), 78.3 (s, CH) and 27.9 (s, C(CH3)3); rn/z

(Er) 358 (hf, 60%), 246 (46), 202 (57), 157 (42), 127 (50), 57 (100); Found Ffl, 358.1780.

CziHz605 requires m/z 358.1 793.

Di(tert-butyl) 2-(1-naphtbylory) malonate. Obtained using the same procedure described

above. starting from 1 -naphthol. Colurnn chromatography (silica, hexane:EtOAc, 9: 1, Rr =

0.4) yielded a white solid (58%). mp 48-50 OC. MHz; CDC13) 8.48 (1 H, br d, aryl),

7.80 (1 H, br s, aryl), 7.53-7-48 (3 H, br m, aryi), 7.36-7.26 (1 H, m, aryl), 6.77 (1 H, d, J 7.3,

aryl), 5.16 (1 H, s, CH) and 1.51 (18 H, s, 2[C(CH3)3]; Sc(lOO MHz; CDC13) 164.8 (CO).

153.5, 134.8, 127.3, 126.6, 125.5, 125.3. 122.7, 122.1, 107.0, 83.1 (s, C(CH3)3), 78.8 (s, CH)

and 28.0 (s, C(CH3)3); m/z (Ei) 358 (M', 83%), 246 (94), 202 (56), 157 (65), 127 (53), 57

(100); Found [m, 358.1788. CziHz6Os requires m/r 358.1791.

Di(tert-butyl) 2-(3-biphenylol) malonate (5.45). Obtained using the same procedure

described above, starting from 3-biphenylol. Column chromatography (silica, hexane:EtOAc,

9: 1, Rf = 0.4) yielded a colorless oil (60%). &(200 MHz; CDC13) 7.3 1-7.59 (8 H, m, aryl),

6.92-7.06 (1 H, m, aryl), 5.06 (1 H, s, CH) and 1.51 (18 H, S. 2[C(CH;)3]; ac(lOO MHz:

CDC13) 164.7 (CO), 157.9, 142.9, 140.8, 129.7, 128.7, 127.4, 127.1, 121.1, 114.9, 114.5. 83.8

(s, ~ ( C H J ) ~ ) , 78.9 (s, CH) and 27.8 (s, C(CH3l3); m/z (EI) 384 (M?, 43%), 328 (4). 228 (60),

171 (35). 57 (100); Found m, 384.1942. CuHzsOs requires d z 384.1937.

Di(tert-butyl) 2-fïuoro-242-naphthyloxy) malonate (5.46). Obtained using the general

procedure described for the fluorinaion of benzylic nitriles, tetrazoles and carboxylic esters.

starting from 5.44 using 1.1 equiv. NaHMDS and 1.1 equiv. NFSi. Column chromatography

(silica, hexane:EtOAc, 9: 1, Rf = 0.5) yielded pure 5.46 as a colorless oil(74%). ijH(200 MHz;

CDCI3) 7.74-7.82 (3 H, rn, aryl), 7.60 (1 H, s, aryl), 7.27-7.5 1 (3 H, m, aryl) and 1.40 (1 8 H, s,

2[C(CH3)3]; 6F(188 MHz; CDC13) -36.3 (1 F, s); 6c(100 MHz; CDCI3) 161.4 (d, CO), 15 1.3,

133.9, 130.9, 129.3, 127.6 (d), 126.6, 125.3, 120.6 (br s), 116.1 (d), 104.4 (d, JCF 244.4, CHF),

84.4 (S. CHI)^) and 27.6 (S. C(CH3)3); d z (Eu 376 (M. 37%). 320 (19). 264 (84). 21 9

(26), 127 (46), 57 (1 00); Found FIç], 376.1682. C2iHuFOS requires d z 376-1686.

Di(tert-butyl) 2-fluoro-2-(1-naphthylory) malonate. Obtained using the general procedure

described for 5.46, starting fiom di(tert-butyl) 2-(1-naphthyloxy) malonate. Column

chromatography (silica, hexane:EtOAc, 9:1, Rr = 0.5) yielded a colorless oil (80%). 6~(200

MHz; CDC13) 8.36 ( I H, br d, aryl), 7.83-7.80 (1 H, m. aryl). 7.66-7.49 (3 H, m, aryl), 7.34 (2

H, br m. aryl) and 1.27 (18 H, s, 2[C(CH3)3]; ZiF(188 MHz; CDC13) -34.3 (1 F, s); 6c(100

MHz; CDC13) 161.2 (d, CO), 150.0, 134.6, 127.3, 126.6, 126.0, 125.2, 124.7, 122.9, 114.4,

104.8 (d, JCF 244.3, CHF), 84.3 (s, G(CH3)3) a d 27.4 (s, C(CH3);); m/z (EI) 376 (W. 41%).

320 (21), 264 (lOO), 127 (36), 57 (70); Found [m, 376.1688. C2iH25FOs requires d'

3 76.1686.

Di(tert-butyI) 2-fluoro-243-biphenylol) malonate (5.47). Obtained using the general

procedure descnbed for 5.46, starting fiom 5.45. Column chromatography (silica,

hexane:EtOAc, 9: 1, Rf = 0.5) yielded pure 5.47 as a colorless oil(68%). ZiH(200 MHz; CDCI;)

7.19-7.59 (9 H, m, aryl) and 1.41 (18 H, s, 2[C(CH3)3]; &(188 MHz; CDC1;) -35.4 (1 F, s);

&(100 MHz; CDCl3) 161.3 (d, CO), 154.2, 142.8, 140.3, 129.5, 128.8, 127.7, 127.0, 123.5,

120.6 (br d), 1 18.8 (br s), 104.3 (d, JCF 245.2, CHF), 84.4 (s, C(CI-13)3) and 27.6 (s, C(CH3)3);

d z (El) 402 (w, 27%), 290 (21), 246 (39, 201 (41), 153 (28), 57 (100); Found [M']?

402.1855. CuH27FOs requires d. 402.1843.

General Procedure for Malonyl and Fluoromalonyl Ester Hydrolysis.

The correspondhg mdonyl ester was dissolved in 90% TFA/CH2C12 and stirred at

room temperature for 1 hour. The solution was then concentrated in vacuo and placed under

hi& vacuum ovemight. The malonyl acids were then purified by preparative HPLC yielding

pure white solids in near quantitative yield.

2-(2-naphthyloxy)malonic acid (5.48). mp 136-1 38 OC; ZiH(200 MHz; CD30D) 7.77-7.89 (3

H, m. aryl), 7.18-7.52 (4 H, m, aryl) and 5.41 (1 H, s, CH); &-(IO0 MHz; CD30D) 169.2

(CO), 156.2, 135.6, 130.8, 128.6, 128.0, 127.6, 125.3, 124.9, 119.6 and 109.1 (S. CH); HPLC

R, 3.6; m/z (El) 246 (w, 2%), 202 (66), 144 (43), 127 (52), 115 (100); Found [M'], 246.0530.

Cl 3H 1 o05 requires m/z 246-0528.

2-(1-naphthylony)malonic acid. mp 146-148 OC; &(200 MHz; CD30D) 8.37-8.33 (1 H, br

m,aryl), 7.88(1 H, brs,aryl), 7.56(3 H, brd, J8.8,aryl), 7.38 (1 H, brt, J7.3,aryl),6.83 (1

H. d. J8.8. aryl) and 5.47 (1 H, s, CH); tic(lOO MHz; CD30D) 169.2 (CO), 154.1. 136.1,

128.4, 127.7, 126.9, 126.5, 126.4, 123.2, 122.9 and 107.2 (s, CH); HPLC & 3.3; m/z (EI) 246

(W. 4%), 202 (82), 143 (74), 127 (46), 115 (100); Found FIÇ], 246.0525. Ci3H1oO5 requires

d z 246.0528.

2-(3-Bipheny1ol)malonic acid (5.49). mp 118-120 OC; aH(200 MHz; CD30D) 6.97 (1 H, br

d, aryl), 6-71 -6.83 (7 H, m, aryl), 6.55 (1 H, br d) and 4.97 (1 H, s, CH); Zic(100 MHz;

CD30D) 169.2 (CO), 158.7, 144.2, 141.8, 131.0, 129.9, 129.8, 128.6, 128.0, 121.3, 114.9 and

1 14.3 (s, CH); HPLC R 3.8; m/z (EI) 272 (M', 3%), 228 (100), 202 (1 3), 183 (34). 153 (53).

1 1 S(24); Found Ffl, 272.0675. C 1sH12Os requires m/r 272.0684.

2-Fluoro-2-(2-naphthyloxy)malonic acid (5.50). mp 140 OC (decornp.); tjH(200 MHz;

CD30D) 7.79-7.88 (3 H, m, aryl), 7.60 (1 H, S. aryl) and 7.31-7.49 (3 H, m, aryl); 6d188

MHz; CD3OD) -34.1 (1 F, s); &(IO0 MHz; CD30D) 170.5 (d, CO), 152.8, 134.3, 130.4,

129.2, 127.5 (d), 126.5, 124.8, 120.5, 114.1 and 107.7 (d, JCF 244.8, CHF); HPLC & 3.3; m/z

(El) 264 (W, 6%), 155 (19), 144 (1 OO), 127 (30), 1 15 (71); FOU^ m, 264.0429. Ci3H9FOs

requires m/z 264.0434.

2-Fluoro-2-(1-naphthylory)malonic acid. mp 140 OC (decomp.); &(200 MW; CD30D)

8.27 (1 H, br d, aryl), 7.91-7.86 (1 H, br m, aryl), 7.69 (1 H, d, J8.8, aryl), 7.57-7.53 (2 H, br

m. ql) and 7.44-7.30 (2 H, m, aryl); &(188 MHz; CD30D) -32.5 (1 F, s); 6c(100 MHz;

CD;OD) 165.7 (d, CO), 150.9, 136.1, 128.5, 127.9 (d), 127.8, 127.1, 126.3, 125.7, 123.4,

1 1 4.1 (d). and 1 06.1 (d, JCF 245.4, CHF); HPLC RI 3.6; m/z (EI) 264 (MI, 4%), 202 (6), 1 44

( 100). 127 (25), 1 15 (98); Found FZC], 264.0422. Ci3H9FOs requires d z 264.0423.

2-Fluoro-2-(3-biphenylo1)malonic acid (5.51). mp 145 OC (decomp.); 6"(200 MHz;

CD;OD) 7.07 (1 H, br m, aryl) and 6.92-6.67 (8 H, m, aryl); 6~(188 MHz; CD30D) -33.2 (1

F. s): Sc(100 MHz; CD30D) 166.3 (d, CO), 154.9, 143.6, 141.4, 131.4, 130.2, 129.4, 128.1,

127.2. 120.7. 1 14.4 (d) and 1 13.1 (d, J C ~ 244.2, CHF); HPLC R 3.8; m/z (EI) 290 (Mt, 4%),

170 (100): 153 (19), 141 (39 , 1 15 (30); Found [M?, 290.0579. Ci5Hl lFOS requires d z

290.0590.

Kinetic Studies with PTPIB: Rates of PTP1B-catalyzed dephosphorylation in the presence

or absence of inhibitors were determined using FDP as substrate'1° as described in Chapter 2

(section 2.2.1) and Chapter 3 (section 3.2.1) with an assay buffer containhg 100mM Bis-Tris,

4mM EDTA, 5 mM DTT and 0.2 &cm3, at 25 OC and pH 6.5, in the presence of 10%

DMSO.


5.3.1 SYNTHESIS OF BENZYLIC a,a-DIFLUORONITRILES AND TETRAZOLES

The tetrazole hctionality plays an important role in medicinal chernistry as a result of

its ability to act as a biostere of other acidic m ~ i e t i e s . ' ~ ~ Numerous drugs bearing the tetrazole

moiety have been reported and therapeutics bearing this functionality usually exhibit good

cellular penetration.'79 Aithough we could not fhd any reports descnbing the tetrazole group

as a phosphate surrogate, its use as a highly effective sulfate surrogate with sulfotyrosine

binding proteins'7" suggested to us that the benzylic a,a-di fluorotetrazole (CFrtetrazole)

moiety may be an effective phosphate biostere for developing PTP inhibitors. Consequently,

we were interested in examining the benzylic a,a-difluorotetrazole moiety as a potential

phosphate biostere for developing PTP inhibitors.

Ideally, we wished to develop a methodology that requires a minimum nurnber of steps

using readily available and safe reagents. Perhaps, the most straightforward approach for

constructing tetrazoies is via reaction of nitriles with azide ion.180 Consequently, we

anticipated that a,a-difluorotetrazoles could be prepared by reacting a,a-difluoronitriles with

azide ion. Benzylic a,a-difluoronitriles have been prepared in a variety of ways.18' Perhaps

the most common method is that originally reported by Middleton and ~ i n ~ h a m . ' ~ ' ~ This

procedure involves DAST fluorination of a-ketoesters followed by reaction with amrnonia to

obtain the primary amide and subsequent dehydration of the amide with Pz05 to give a,a-

difluoronitriles in overall yields in the range of -50%. Although this approach produces the

desired fluoro compound, a rnulti-step synthesis is required. Another approach using

expensive DAST was reported by Bartmann and K r a ~ s e . " ~ ~ These workers reported the

synthesis of three benzylic a,a-difluoroacetoniûiles functionalized with hydrocarbon

substituents in overall low yields of 11-27%. This was accomplished by converthg benzoic

acid derivatives to the acid chlofides followed by reaction with CuCN or TMSCN to give the

a-ketonitriles followed by DAST fluorination. Recently, Hagele and Haas reported that this

procedure could be irnproved (a,a-difluorobenzeneacetonitrile obtained in 65%) by

performing the DAST fluorination in the presence of a catalytic amount of Zr&. l g L c Other

methods have been reported, however, these procedures generally proceed in poor yields and

require uncommon or highly toxic reagents and/or uncommon procedures. 181d-f

We envisioned preparing benzylic a,a-difluoronitriles via electrophilic fluorination of

a-carbanions of benzylic nitriles using the relatively cheap and commercially available

electrophilic fiuorinating agent, NFSi. We began o w studies using commercially available P-

naphthylacetonitrile 5.7 as a mode1 substrate for îhe fluorination reactions. AS mentioned in

Chapters 3 and 4, the yield of the fluorination product is often dependent upon the nature of

the base and counterion. Consequently, we first examined the fluorination of 5.7 using

different bases and countenons. The results of these studies are given in Table 5.1. The

reaction was initially performed using our one-step fluorinating procedure (2.2 equiv. of base

to a solution of the nitrile in THF at -78 C followed by the addition of 2.5 equiv. NFSi at -78

OC) that we had developed for the fluorination of benzylic phosphonates (Chapter 3). Aimost

al1 bases, irrespective of the cation, yielded the desired nitriIe 5.8 in low yields (9-16%; Table

5.1, entries 1-6). However, the utilization of t-BuLi proved tu be an exception, yielding 5.8 in

50% yield (Table 5.1, entry 7).

Interestingly, in cornparison to the less bulky n-BSi, t-BuLi displayed a 5-fold

increase in yield (Table 5.1, entries 6 and 7, respectively). This may be due to the n-BuLi

reacting with NFSi, a reaction that may not occur as readily with the more sterically hindered

Table 5.1. Effect of base and counterion on the electrophilic fluorination of 5.7 with NFSi.

1. Base, THF,

2. NFSi, THF,

Base E n t r ~ % Yielda of 5.8

LDA

KDA

NaHMDS

KHMDS

LiHMDS

n-BuLi

t-BuLi

t-BuLi

'lsolated yields. b~erformed in one-step (2.2 equiv. base, 2.5 equiv. NFSi) at -78 OC. 'Performed depwise by first adding 1 . 1 equiv. t-BuLi at -78 OC followed by 1.2 equiv. NFSi, stirred for 45 minutes and then this process was repeated. d~erformed in one-step (2.2 equiv. base, 2.5 equiv. NFSi) at -100 OC. 'Performed using 1 . 1 equiv. t-BuLi at -78 OC followed by 1.2 equiv. NFSi. gMonofluorinated product.

t-BuLi. It is also possible that the poor yields may be a result of attack of these bases on the

nitrile carbon of the a-fluonnated product, although this seems unlikely with the sterically

hindered bases. Due to the possibility that a reaction may be occurring between NFSi and t-

BuLi, when the fluorination was performed in a single step, we examined whether the yield

could be improved by performing the reaction in a stepwise fashion (1.1 equiv. t -BSi at -78

OC. stirred for 1 hour, followed by 1.2 equiv. NFSi, stirred for 2 houn and then repeating the

process). This did not result in a significant increase in yield. Performing the reaction at - 100

OC gave similar yields. Replacing the solvent, THF, with ether resulted in a lower yield.

Finally, the monofluorinated product 5.9 could be obtained (60% yield) using 1.1 equiv. t-

BuLi and 1.2 equiv. NFSi (Table 5.1, entry 8).

To examine the =ope of this reaction we attempted the fluorination of a variety of

substituted benzylnitriles. The results of these fluorinations are shown in Table 5.2. Yields of

the difluorinated products range fiom 3446% with the exception of the 4-bromo derivative.

AIthough t-BuLi was the best base for the naphthyl derivative 5.7, we found that with the 4-

nitro and 4-bromo derivatives (5.10 and 5.1 1 in Table 5.2), NaHMDS (for 4-nitro substituted)

and LDA (for 4-bromo substituted) worked k t . The low yield obtained with the bromo

derivative (1 9%) was due to significant loss of bromine fiom the aryl ring during the reaction.

To compare our methodology to the DAST approach reported by Hagele and ~aas, '* ' '

we also attempted to produce the biphenyl derivative 5.23 via DAST fluorination of the

corresponding a-ketonitrile starting with the cornmercially available acid 5.26 (Scheme 5.1).

However, we were unable to obtain any appreciable quantity of 5.23 in pure form using this

procedure. Thus, although the yields in Table 5.2 are modest, the fluorinated products are

obtained in a single step and, in our hands, overait higher yields than via DAST fluonnation of

ac y1 cyanides.

For inhibition studies, we converted only compounds 5.8 and 5.23 to the corresponding

tetrazoles since our previous studies indicated that the naphthyl and metu-biphenyl scaffolds

were most effective for PTP 1 B inhibition. Thus, the desired benzylic a,a-difluorotetrazoles

Table 5.2. Electrophilic fluorination of benzylic nitriles with NFSi.

Substrate Product Y ieldab

- -

'Isolated yields. b~erfomed in one-step (2.2 equiv. base, 2.5 equiv. NFSi) at -78 OC, 2 h. CPerformed using t-BuLi. d~erformed using NaHMDS. 'Performed using LDA.

cat. DMF 1. TMSCN a - Ph a t . Znl* Ph - --

Scheme 5.1. Attempted synthesis of 5.23 via DAST fluonnation.

5.1 and 5.4 were obtained in quantitative yields by reacting nitriles 5.8 and 5-23 with sodium

azide in DMF (Scheme 5.2). Unlike their non-fluonnated analogues 5.27 and 5.28 (derived

from 5.7 and 5.16, respectively) these reactions did not require any N-Cl as is usually the

case when converting nitriles to tetrazoles using N a 3 . 180

1. t-BuLi (2.2 eq.), THF,

r C ~ N -78 OC, 1 h w )Lc=N F

R 2. NFSi (2.5 eq.), THF, R -78 OC, 3 h

5.7, R = 9-naphthyl 5.16, R = m-(Ph)C6H4

I NaN3, NH4CI, DMF, 80 OC, 24 h

5.27, R = p-naphthyl 5.28, R = m-(Ph)CsH4

1 NaN3, DMF,

F F N-N w,.N R

5.1, R = p-naphthyl 5.4, R = m-(Ph)C6H4

Scheme 5.2. S ynthesis of tetrazole derivat ives.

In an attempt to increase the overall yields of the benzylic a,a-difluorotetrazoles, we

also examined whether it would be possible to obtain the fluorinated tetrazoles by direct

fluorination of protected tetrazoles. We first attempted using a t-butyl-protected tetrazole

since the t-butyl group is a common protecting group for tetrazoles. '79c Thus, t-butyl-protected

tetrazole 5.29 was constructed by reacting 5.27 with t-BuOH/H2S04 in 'T'FA. Compound 5.29

was then used as a mode1 substrate for the fluorination reaction. EIectrophilic fluorination was

performed via our typical procedure using a variety o f bases and cations (entries 1-8, Table

Table 53. Effect of base and counterion on the electrophilic fluorination of 5.29 with NFSi.

1. Base, THF, -78 OC *

2. NFSi, THF, \ \

Base EnW % Yielda of 5.30

LDA

KDA

NaHMDS

KHMDS

LHMDS

n-BuLi

t-BuLi

t-BuLi

'Isofated yields. b~erformed in one-step (2.2 equiv. base, 2.5 equiv. NFSi) at -78 OC. 'Performed stepwise by first adding 1.1 equiv. t-BuLi at -78 OC followed by 1.2 equiv. NFSi, stirred for 45 minutes and then this process was repeated. d~erfonned using 1 . I equiv. t-BuLi at -78 OC followed by 1.2 equiv. NFSi. 'Monofluorinated product.

As with the nitriles, t-BuLi was the most effective base with the one-step and two-step

procedures giving similar yields of fluorinated product 5.30 (52-56%, entry 7, Table 5.3).lg2

The monofluorinated tetrazole 531 was also obtained (61% yield) using 1 . 1 equiv. t-BuLi and

1.2 equiv. NFSi (Table 5.3, entry 8). We d s o attempted the fluonnation reaction using a

benzyl protecting group instead of a t-butyl protecting group but this was unsuccessfÙ1 as was

the fluorination of unprotected 5.33 using three equivdents of base and NFSi as fluorinating

agent (Scheme 5.3). The yield for the fluorination of tetrazole 5.29 was similar to that

obtained for the fluorination of nitrile 5.7. Thus, since this approach also requires a protection

and eventuai deprotection of the tetrazole, it appears that no advantage can be gained (in ternis

of yield) by fluorinating the protected tetrazoles rather than the nitriles.

"'=N 1. 2.2 eq. t-BuLi, -78 OC, THF

Ph \ N 2. 2.5 eq. NFSi,

5.32 -78 OC, THF

N=v 1. 3.2 eq. t-BuLi. -78 OC, THF

2. 2.5 eq. NFSi,

5.33 -78 OC, THF

Scheme 5.3. Attempted synthesis of unprotected and benzyl protected tetrazole denvatives.

5.3.2 SYNTHESIS OF a,a-DIFLUOROCARBOXYLATES GND a-MONOFLUORO- MALONYL DEWATIVES

Electrophilic fluorination has been used extensively for the preparation of a-

fluorinated esters. 183v'" Consequently, we chose this approach for the synthesis of the CF2-

carboxylates 5.3 and 5.6 (Scheme 5.4). However, we used LDA instead of NaHMDS as it

appears to be the base of choice (in terms of yield) for the fluonnation of ester

(for 5.36) 20% aq. CsC03, P~IB(OH)~ (1.2 eq.),

r ~ m ~ benzyl bromide. Y=-

Na2C03 (1 -5 eq.), /-COOR' R MeOH/H20 R 5 mol % ~ d ( 0 ~ c ) z ) R

5.34, R = m-(Br)C6H4 DMF, rt

5.36, R = m-(&)C6H4 H2 (1 a m l ~ 5-38. R = m-(Ph)C&

5.35, R = P-naphthyl 5.37, R = pnaphthyl 5% Pd/C R' = Bn

5.41, R = rn-(Ph)C6H4

(for 5.37 8 6.38) R '=H L

f /

I 1. LDA (2.2 eq.), THF, -78 OC, 1 h

2. NFSi (2.5 eq.), THF,

HZ (1 atm), -78 OC, 3 h

F 5% PdlC,

)LCOOH EtOAc, 12 h 4

R F - c m , R

Scheme 5.4. Synthesis of a,a-difluorocarboxylate derivatives.

deri~atives. '~~*'" To begin, the commercially available acids 5.34 and 5.35 were converted to

their corresponding benzyl esters 5.36 and 5.37 in excellent yields (89-91%) using Cs2C03

and benzyl bromide. A Suzuki reaction with ester 5.36 and phenylboronic acid yielded the

biphenyl ester 5.38 (70%). Next, esters 5.37 and 5.38 were treated with 2.2 equiv. LDA at -78

OC followed by the addition of 2.5 equiv. NFSi to give difluoro esters 5.39 and 5.40 in modest

to good yield (40% and 60%, respectively). Hydrogenolysis of 5.39 and 5.40 yielded the CF2-

carboxylates 5.3 and 5.6 in good yields (80% and 93%, respectively).

Since the malonyl and CF-malonyl groups have been championed as phosphate

mimetics, we prepared malonyl derivatives 5.48-5.51 so that we could compare the

effectiveness of the CF2-tetrazole, CF2-sulfonate and CF2-carboxylate moieties as phosphate

mimetics to the malonyl and CF-malonyl groups. Compounds 5.50 and 5.51 were prepared

using the procedure developed by Burke et al. (Scheme 5.5).8z~v'85 The phenol derivatives

5.42 and 5.43 were refluxed in benzene in the presence of previously prepared di-tert-butyl a-

dia~omalonate"~ (5.52) and rhodium diacetate to give r-butyl malonate esters 5.44 and 5.45

(63% and 60% respectively). The corresponding fluorinated derivatives 5.46 and 5.47 were

prepared in good yields (74% and 68%, respectively) via our electrophilic fluorination

conditions (1.1 equiv. NaHMDS at -78 OC in THF, 1.2 equiv. NFSi at -78 OC in THF). The

desired malonyl denvatives 5.48-5.51 were obtained via hydrolysis of the corresponding t-

butyl esters with TFA/CH2C12 (85-96% yields).

1. NaHMDS (1 -1 eq.), Rh(ll)acetate (4.4 mol%), THF, -78 OC, 1 h 5.52, benzene, 2. NFSi (1.1 eq.), THF, reflux, 18 h -78 O C 3 h

R-OH - R-O-CH(COO~BU)~ R-O-CF(COO~BU)~

5.42, R = p-naphthyl 5.44, R = p-naphthyl 5.46, R = P-naphthyl 5.43, R = m(Ph)C6H4 5.45, R = m(Ph)C6H4 5.47, R = m(Ph)C6H4

90% TFA/CH2CI2, rt, 1 h

5.18, R = p-naphthyl 5.50, R = p-naphthyl 5.49, R = m(Ph)C6H4 5.54, R = m(Ph)C6H4

Scheme 5.5. Synthesis of malonyl and a-fluoromalonyl derivatives.

The CFrsulfonate derivatives, 5.2 and 5.5, as well as their non-fluorinated analogues.

5.53 and 5.54, were prepared by Dr. Mei-Jin Chen, a pst-doctoral fellow in the Taylor group,

via electrophilic fluorination of sulfonate esters. Ia6

5.4 PTPIB CNHIBITION STUDIES

We initiated the inhibition studies by first exarnining the non-fluorinated compounds

5.27, 5.28, 5.35,5.41, 5.48, 5.49,5.53, 5.54,4.8 and 4.11 using 500 pM inhibitor with PTPIB

and fluorescein diphosphate as substrate at Km concentration (20 pM), at pH 6.5 and 10%

DMSO. The results are given in Table 5.4. Not surpnsingly, al1 of these compounds are poor

inhibiton of PTPl B. In general, the non-fluorinated phosphate counterparts 4.8 and 4.11

appear to be slightly better inhibitors with the exception of the naphthyl tetrazole derivative

5.27 and the malonyl denvatives 5.48 and 5.49.

Anticipating better inhibitory potency, ICsols were determined for the fluorinated

compounds. The results are given in Table 5.5. These compounds were better inhibitors than

their non-fluorinated analogues, yet still not as effective as their DFMP analogues. The CF2-

carboxylates 5.3 and 5.6 displayed the worst inhibition. Their ICso's were 18 and 29 times

higher than their DFMP-bearing analogues, respectively.

Table 5.4. Percent inhibition of PTPI B with 500 FM non-fluorinated compounds.

Compound Percent Inhibition8 Compound

Percent Inhibition8

5.53 CH2S03' 5 k 2 5.54 CH2S03' 10*2

5.27 CH2-tetrazole 12*2 5.28 CH2-tetrazole 1 5 & 2

5.35 CH2COO- 4 * 2 5.41 CH2COO' 10 * 2

4.8 ~ ~ 2 ~ 0 3 ' ~ 9 * 2 4.11 C H ~ P O ~ - ~ 1 7 k 2

5.48 OCH(COO> 3 7 + 2 5.49 OCF(C00?2 39 + 2

%rot-s are reported as * the standard deviation of two determinations.

Table 5.5. ICso values for fluorinated compounds 5.1-5.6,5.50,5.51, 1.19 and 3.40.

Compound X Y ICa(@+f)' Compouiid X Y I G o Owa

5.1 CF2 tetrazole 230 * 12 5.4 CF2 tetrazole 195 * 10

5.3 CF2 COO' 640*18 5.6 CF2 coo- 435*12

5.50 OCF (C0032 320 k 1 1 5.5 1 OCF ( ~ 0 0 3 ~ 250 * 10

1.19 CF2 PO^-^ 35 sb 3.40 CF2 PO^-^ 1 5 1 3 ~

"IC5,'s were detennined using eight different inhibitor concentrations. Enors are reported as * the standard deviation of two determinations. bThe presence of 10% DMSO in the assay mixture results in lower ICS,,'s than previously reported values (see Chapter 4, section 4.4) which were obtained in 5% DMSO solution.

The CF2-sulfonates 5.2 and 5.5 were the most effective phosphate surrogates. They

exhibited ICso's that were ody 5 and 7.6 times higher than that of DFMP inhibitors 1.19 and

3.40, respectively. Although sulfate-bearing compounds inhibit PTPs, 74.83.89 the well-known

labili ty of phenolic sulfateslg7 lhits their use as potential therapeutics. The sulfonates

studied here are highly stable compounds. Sulfonates, in general, are highly acidic

compounds and therefore are most Iikely to be completely ionized at the pH under which these

studies were performed (pH 6.5). Thus, the enhanced inhibitory effect of the CF2-sulfonates

compared to their non-fluorinated counterparts is most likely a result of a direct interaction of

the fluorines with residues in the enzyme active site and is not due to pK, effects. The CF2-

tetrazole derivatives 5.1 and 5.4 were 6.5 and 13 times less effective inhibitors than their

DFMP analogues. The pK,'s of the conjugate acids of 5.27 and 5.1 were determined by

potentiornetric titration in 10% DMSO/H20 to be 5.3 and 3.9, respectively. Thus, both exist

mainly in the anionic form at pH 6.5. Again, this suggests that the enhanced inhibition found

with the fluorinated derivatives as compared to the non-fluo~ated denvatives is due to

interaction of the fluorines with residues in the enzyme active site.

The Kits of compounds 5.4 and 5.5 were detennined (Figures 5.1 and 5.2). Both were

found to be cornpetitive inhibitors with Kits of 98 * 9 pM and 49 + 7 pM, respectively.

Despite the CFz-sulfonate and CFz-tetrazole compounds k ing Iess effective inhibitors than

their DFMP analogues, these compounds were better inhibitors than the dianionic CF-malonyl

compounds 5.50 and 5.51. Thus, it appears that the CFz-sulfonate and CF2-tetrazole groups

are more effective phosphate biosteres than the CF-malonate group. Although it is possible

that the CFz-sulfonate compounds, as is the case with the CF-malonyl derivatives, may require

caging for cellular studies, it is very possible that this will not be the case for the CF2-tetrazole

compounds since tetrazole-bearing compounds usually exhibit good cellular penetration. 179b

5.5 CONCLUSIONS

We have demonstrated that benzylic a,a-difluoro nitriles and tetrazoles can be

prepared by electrophilic fluorination of a-carbanions of their non-fluorinated analogues.

These functionalities, as well as the CF2-sulfonate and CFz-carboxylate groups, were

examined as phosphate mimetics for obtaining PTP inhibitors. Of these four moieties. the

CF2-sulfonate group appears to be the most effective phosphate mimetic, although it is not as

effective as the DFMP group. Studies are in progress in the Taylor group, in collaboration

with Merck-Frosst Inc., to determine the ce11 permeability of compounds bearing the CF2-

sulfonate and CFz-tetrazole groups. It should also be noted that this procedure has the

potential to provide novel classes of organofluonnes with applications beyond the scope of

PTP inhibitors. The benzylic tetrazole group is an important biostere in medicinai chemistry

and the a-difluorination of this moiety will provide an additional potential means for

-0.09 -0.06 -0.03 O 0.03 0.06

[1 IFDP] (j .M1)

Figure 5.1. inhibition of PTP 1 B by compound 5.4. (a) The activity of PTP 1 B (0.1 5 j.@cm3) was measured at pH 6.5 as described under 'experirnental procedures' in the presence of the following concentrations of 5.4: (O), O pM; (i), 100 pM; (A), 150 pM; (X), 200 pM; (e), 250 ph4; (O), 300 pM. (b) Replot of the slope fiom the double-reciprocal plot versus concentrations of compound 5.4.

Figure 5.2. Inhibition of PTP 1 B by compound 5 5 . (a) The activity of PTPI B (0.15 Fig/crn3) was measured at pH 6.5 as descnbed under 'experimental procedures' in the presence of the following concentrations of 5.5: (a), O pM; (i), 50 pM; (A), 100 pM; (x), 150 pM; (e), 200 FM; (O), 250 pM. (b) Replot of the slope fiom the double-reciprocal plot versus concentrations of compound 5.5.

increasing the bioactivity of compounds bearing the tetrazole moiety. Finally, this tactic can

now be extended to the development of inhibitors of enzymes that hydrolyze phenyl sulfates

such as aryl sulfatases and steroid suifatases and proteins that bind tyrosyl sulfates. Recently,

there has been considerable interest in the development of steroid sulfatase inhibitors,ls8 some

of which are estrone suifonates in which the labile S-O bond in the substrate is replaced by a

methylene unit.lsad Conversion of these compounds to the a,a-difluorosulfonate denvatives

may provide a means of increasing the potency of these compounds. The CF*-sulfonate and

CF2-tetrazoie groups may also be useful in the development of inhibitors of other

therapeutically important proteins that recognize pTyr (such as SH2 domains).

CHAPTER 6

FUTURE DIRECTIONS

6.1 PTPlB INHIBITORS

We believe that the studies presented here on the development of PTPlB inhibitors

provide insight into the stnrctural requirements for the fihue development of potent small

molecule inhibiton of PTPlB. We envision that the following features will be important for

the future development of PTPlB inhibitors: (1) a phosphate mimetic attached to an aromatic

or heteroaromatic scaffold; (2) functiondization of the basic aromatic or heteroaromatic

scaffold with other aromatic or heteroaromatic fûnctionalities and (3) an additional moiety that

can either bind to the second aryl phosphate binding site andior forge strong non-specific

interactions with positively charged Arg residues beyond the binding pocket. Studies are

currently undenvay in the Taylor group to construct compounds having the above features

using a combinatorid chemistry approach. For exarnple, studies are in progress to construct

libraries of compounds having general structure 6 . 1 . ' ~ ~ Here, a phenyl scaffold is

functionalized with (1) the CF2-sulfonate or DFMP group; (2) an aromatic or heteroaromatic

moiety at the meta-position and (3) an aikyl chah which has a polar or anionic group.

heteroaromatic

6.1, X = ~ 0 ~ - ~ or SOg

Until quite recently, some of the compowids in this thesis were among the most potent

small molecule inhibitors of PTPlB rq>orted in the literature. Not surprisingly, a nurnber of

pharmaceutical companies have begun programs to develop inhibitors of PTPlB. Very

6.4 6.5 (Roaiglitazone)

recently, researchers at Wyeth-Ayerst reported that compounds 6.2 and 6.3 are very potent and

moderately selective inhibitors of PTP 1 B. Compound 6.4 was arrîved at by random

screening of the Wyeth-Ayerst compound collection which resulted in the identification of

cornpound 6.4 as a moderate inhibitor of PTPl B.'% More or less random modifications of 6.4

led to compound 6.2. This compound was show to be an orally active antidiabetic agent in

mouse models. Compound 6 3 was based on the Smith-Kline Beecham's (SKB) antidiabetic

agent Rosiglitazone 6.5, which has recently been approved for marketing in the United States.

Although there is no published evidence to suggest that 6.5 acts by inhibition of PTPIB,

Wyeth-Ayent chose this compound as a starting point for 6.X19' In addition to king a potent

inhibitor of PTP1 B, cornpound 6.3 was found to normalize plasma glucose in mouse models.

Neither compound 6.2 nor 6.3 appear to have al1 of the components that we have listed above

for obtaining potent PTPlB inhibitors. However, how compounds 6.2 and 6.3 interact with

PTPl B is unknown and it is possible that the above-mentioned criteria are indeed met with

these compounds but in ways that are not immediately evident.

6.2 OTHER APf LICATIONS OF CHIRAL a-MONOFLUOROPHOSPHONATES

Our studies on the preparation of chiral a-monofluorophosphonic acids presented in

Chapter 4 represent the fust general approach for the preparation of this class of compounds.

In addition to king useful probes for examining PTPIB-inhibitor interactions, we anticipate

that chiral a-monofluorophosphonic acids will f h d broad applications as inhibitors or probes

of other enzymes. For example, in collaboration with Professor Steven Bearne, in the

Biochemistry Department at Dalhousie University, we are exarnining a-fluorinated phosphonic

acids as inhibitors of mandelate racemase. Mandelate racemase catalyzes the racernization of

R- and S-enantiomers of mandelate via abstraction of the a-hydrogen (Scheme 6.1 ). ' 92 The

mandelate racemase ,

Rsnantiomer S-enantiomer

Scheme 6.1. Reaction catalyzed by mandelate racemase.

significance of this seemingly simple reaction, r e q u i ~ g only a divalent cation (ie. M ~ ' ~ ) , is

its application as a mode1 for the comprehension of enzyme-catalyzed reaction hdamentals.

That is. this single substrate/product enzyme provides the basis for the formatiodbreakage of

carbon-hydrogen bonds. In addition, the ability of mandelate racemase to bind to and catalyze

both substrate enantiomers with essentially equal afinity and kinetic syrnmetry provides an

intriguing conceptualization of how a chiral enzyme (inherentiy asymmetric active si te) c m

catalyze a syrnrnetrical reaction. Also, it raises questions as to how the enzymes can catalyze

rapid carbon-hydrogen bond cleavage of carbon acids with relatively high pK, values. The

reaction is believed to proceed via a two-base mechanism, LysLb6 and Hisz97 abstracting the a-

proton fiom (9-mandelate and (R)-mandelate respectively. 193 Glu3 l, is believed to function as

a general a ~ i d . ' ~ ~ In collaboration with the Bearne group, we have shown that a-

hydroxybenzylphosphonic acid 6.6 and a,a-difluorobenzylphosphonic acid (6.7) are potent

inhibitors of this enyme.lg5 Recent studies have shown that the enzyme preferably binds the

R-enantiomer of 6.6.'" We have recently sent chiral compounds 4.5 and 4.6 to the Bearne

group and studies are undenvay to see if the enzyme discriminates, in ternis of binding

affinity, between these two enantiomers. We anticipate that these studies will enhance our

understanding of the mechanism of this interesting enzyme.

Appendh A

Figure 1. X-ray crystal structure of 4.20.

Table 1. Crystallographic data for compounds 4.19-4.21 4.19 4.20 4.2 1

C21 H2lFN02P CuHvFNOzP C 1 7H19FN02P C hemical formula Formula weight Crystal system Space group Absorption coefficient Unit ce11 dimensions

369.36 orthorhombic p2(1)2(1)2(1) O. 172 mm-' a = 5.7212(4) A

a = 90° b = 1 1 .S757(l6) A

p = 900 c = 28.177(4) A

y = 90" 1866.1(4) A' 100.0(1) K 4 6987 1586 [P(int) = O. 1431 R1 = 0.0887 wR2 = 0.1 171 RI = 0.0521 wR2 = 0.1049

395.39 orthorhornbic P2(1)2(1)2 O. 167 mm-' a = 1 l.8451(5) A

a = 90" b = 27.381 S(l9) A

P = 90" c = 6.0985(8) A

y = 90" 1978.0(3) A' 100.0(1) K 4 9348 3440 [li(int) = 0.0901 R1 =O.1102 wR2 = O. 1080 R1= 0.0552 wR2 = 0.0949

3 19-30 orthorhombic PX 1 )2( 1 )2( 1) 0.191 mm-' a = 5.7208(2) A

a = 90" b = 11.2412(8) A

p = 90° c = 24.5839(15) A

y = 90' 1 58O.96(16) A3 100.0(1) K 4 7844 3556 [R(int) = 0.0701 R1 = 0.0717 wR2 = O. 1 O00 R1 = 0.0452 wR2 = 0.0924

Unit ce11 volume Temperature z Reflections collected Independent reflections R indices (ail data)

Final R indices

Table 2. Crystailographic data for compounds 4.22-4.24 4.22 4.23 4.24

C hemical formula CZIHZIFNOZP CuHuFN02P C I iH19FNozp Formula weight 369.36 395.39 3 19.30 Crystal system monoclinic orthorhom bic monoclinic Space group pz( 1 ) P2(1)2(1)2 p2( 1 Absorption coefficient 0.1 7 1 mm-' 0.162 mm" O. 1 89 mm" Unit ce11 dimensions a = 6.6666(4) A a = 12.272(2)(5) A a = 9.9593(7) A

a = 90' a = 90' a = 90" b = 12.1266(5) A b = 24.9928(3) A b = 9.9593(7) A

p = 96.743(3)' P = 90" f3 = 92.346(3)' c = 11.6697 A c=6.6447(7)A c=9.9593(7)A

y = 90" y = 90" y = 90' Unit ceII volurne 936.89(8) A3 2038.1(2) A3 798.51(9) A3 Temperature 200.0(1) K 100.0(1) K 100.0(1) K z 2 4 2 Reflections collected 6432 1242 1 10240 Independent 3690 4608 3541 reflections m(int) = 0.0321 [R(int) = 0.05 11 p(int) = 0.0551 R indices (al1 data) R1 = 0.0393 R1 = 0.0473 RI = 0,0416

wR.2 = 0.0800 wR2 = 0.0796 wR2 = 0.0937 Final R indices RI = 0.0328 RI = 0.0354 RI = 0.0366

wR2 = 0.0771 wR2 = 0.0766 wR2 = 0.0915 Goodness-of-fit on F~ 1 .O50 1 .O52 1 .O34

1.

2.

3.

4.

5.

6 .

7.

8.

9.

1 O.

I I .

12.

13.

14.

15.

16,

17.

18.

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PUBLICATIONS

Kotoris, C. C.; Wen, W .; Taylor, S. D., "Preparation of Chiral a-Monofluorophosphonic Acids and Their Evaluation as Inhibi tors of Protein Tyrosine Phosphatase 1 B" J. Chem. Soc., Perkins Tram 1, in press.

Taylor, S. D.; Kotoris, C. C.; Hum, G., "Recent Advances in Electrophilic Fluorination" Tetrahedron, 1999, 55, 1 243 1 (Review Article).

Kotoris, C. C.; Chen, Md.; Taylor, S. D., "Preparation of Benzylic a,a-Difluoronitriles, - tetrazoles, and -sulfonates via Electrophilic Fluorination" J. Org. Chem., 1998,63,8052.

Kotoris, C. C.; Chen, M-J.; Taylor, S. D., "Novel Phosphate Mimetics for the Design of Non- Peptidyt Inhibitors of Protein Tyrosine Phosphatases" Bioorg. Med Chem. Lett-, 1998, 8, 3375.

Taylor, S. D.; Kotoris, C, C.; Dinaut, A. N.; Chen, M-J.; Rarnachandran, C.; Huang, Z., "Potent Non-Peptidyl Inhibitors of Protein Tyrosine Phosphatase 1 B" Bioorg. Med. Chem., 1998, 6, 1457.

Taylor, S. D.; Kotoris, C. C.; Dinaut, A. N.; Chen, M-J., "Synthesis of Aryl (Difluoromethylenephosphonates) via Electrophilic Fluorination of a-Carbanions of Benzylic Phosphonates with N-Fluorobenzenesulfonimide" Tetrahedron, 1998,54, 169 1.

Kotoris, C. C. "Studies on intramolecular Peptide Bond Cleavage and Design and Synthesis of Protein Tyrosine Phosphatase inhibitors" Masters of Science, 1996.

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