A re-examination of the difluoromethylenesulfonic acid group as a phosphotyrosine mimic for PTP1B inhibition

Bioorganic & Medicinal Chemistry 16 (2008) 6764–6777

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Bioorganic & Medicinal Chemistry

journal homepage: w ww.elsevier.com/locate/bmc

A re-examination of the diﬂuoromethylenesulfonic acid group

as a phosphotyrosine mimic for PTP1B inhibition

Munawar Hussain ^a,b, Vanessa Ahmed ^a, Bryan Hill ^a,, Zaheer Ahmed ^b, Scott D. Taylor ^a,

*

^aDepartment of Chemistry, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada N2L 3G1

^bInternational Center for Chemical and Biological Sciences, H.E.J. Research Institute of Chemistry, University of Karachi, Karachi 75270, Pakistan

a r t i c l e i n f o

a b s t r a c t

Article history:

Protein tyrosine phosphatase 1B (PTP1B) is involved in the down-regulation of insulin signaling and is a

well-validated therapeutic target for the treatment of diabetes and obesity. Key to the design of potent

inhibitors of PTP1B is a moiety that effectively mimics the phosphate group of the natural phosphotyro-

sine substrate. Diﬂuoromethylsulfonomethylphenylalanine (F₂Smp) is one of the best monoanionic pTyr

mimics reported to date. However, the diﬂuoromethylenesulfonic acid (DFMS) group as a phosphate

mimic has not been carefully evaluated in the context of a non-peptidyl platform. Here we present a care-

ful examination of the DFMS group as a phosphate mimic. This was achieved by ﬁrst constructing an ana-

log of a previously reported high afﬁnity, non-peptidyl PTP1B inhibitor (compound 2, IC₅₀= 8 nM) in

which a diﬂuoromethylenephosphonic acid group is replaced with the DFMS moiety (compound 6).

We also report the synthesis of its non-ﬂuorinated methylenesulfonic analog (compound 7), as well as

two other derivatives in which a distal sulfonamide moiety is replaced with a diﬂuoromethylenesulfona-

mide group (compounds 8 and 9). Compounds 2 and 6–9 were examined as PTP1B inhibitors. Replacing

the distal sulfonamide moiety with a diﬂuoromethylenesulfonamide group had only a modest effect on

inhibitor potency. However, compound 6 was approximately a 1000-fold poorer inhibitor than com-

pound 2. Most signiﬁcantly, inhibition studies with compound 7 and a peptide bearing sulfonomethyl-

phenylalanine revealed that the ﬂuorines have little effect on the potency of the DFMS-bearing

inhibitors. This is in contrast to a previous assumption that the ﬂuorines in DFMS-bearing inhibitors con-

tributed signiﬁcantly to their potency. This may in part explain the large difference in potency between

the DFMS and DFMP-bearing compounds. These results also demonstrate that sulfonomethylphenylala-

nine, a pTyr mimic that is readily constructed, is a relatively good pTyr mimic in comparison to most oth-

ers that have been reported when examined in the context of the DADE-X-LNH₂peptide platform.

Received 24 March 2008

Revised 16 May 2008

Accepted 28 May 2008

Available online 12 June 2008

Keywords:

PTP1B

Non-peptidyl inhibitors

Phosphotyrosine mimics

1. Introduction

been reported.⁴One of the earliest, and still one of the most effec-

tive mimics, is the diﬂuoromethylenephosphonic acid (DFMP,

Protein tyrosine phosphatases (PTPs) catalyze the dephospho-

rylation of phosphotyrosine residues in peptides and proteins.

Among the approximately 100 human tyrosine phosphatases that

have been identiﬁed so far,¹protein tyrosine phosphatase 1B

(PTP1B) has garnered the most attention as far as inhibitor design

is concerned.²PTP1B is involved in the down-regulation of insulin

signaling and is considered to be a well-validated therapeutic tar-

get for the treatment of diabetes and obesity.³A key component in

the design of PTP1B inhibitors and indeed, almost all phosphatase

inhibitors, is the presence of a hydrolytically stable phosphotyro-

sine (pTyr) or phosphate mimic and dozens of such mimics have

ꢀCF₂PO^ꢀ2) group.⁵A wide variety of highly potent PTP1B inhibi-

3

tors, such as peptide 1^5a–c(K_i= 24 nM)⁶and small molecule 2

(IC₅₀= 8 nM),⁷have been prepared bearing this mimic (Fig. 1).

However, the potential problems that are associated with develop-

ing highly polar and charged phosphonate compounds into thera-

peutics have led to an intense search for phosphate mimics that are

less highly charged yet still contribute signiﬁcantly to inhibitor po-

tency when incorporated into a small molecule platform. Several

years ago we examined the diﬂuoromethylene sulfonic acid (DFMS,

ꢀCF₂SO^ꢀ₃) group as a phosphate mimic for PTP1B inhibition.^8a,bWe

reasoned that the DFMS group would be an effective monoanionic

phosphate mimic for several reasons. First, Desmarais and cowork-

ers examined a series of peptides bearing sulfotyrosine (sTyr or sY)

as PTP1B inhibitors and found that even relatively simple peptides,

such as AcDE(sY)L, are good reversible competitive inhibitors with

* Corresponding author. Tel.: +1 519 888 4567x3325; fax: +1 519 746 0435.

E-mail address: s5taylor@sciborg.uwaterloo.ca (S.D. Taylor).

Present address: Department of Chemistry, Brandon University, Fourth Floor, John

R. Brodie Science Centre, 270 18th Street, Brandon, Manitoba, Canada R7A 6A9.

IC₅₀’s in the low

lM region which indicates that these peptides

doi:10.1016/j.bmc.2008.05.062

M. Hussain et al. / Bioorg. Med. Chem. 16 (2008) 6764–6777

6765

bind almost as well as the best peptide substrates.⁹These results

S

Br

R

suggested to us that the substitution of the phosphorus in pTyr

for a sulfur (to sTyr) does not have a serious deleterious effect on

ligand binding. Second, it has been shown that peptide 1 is almost

1000 times more effective as a pTyr mimic than the corresponding

peptide bearing phosphonomethylphenylalanine (the non-ﬂuori-

nated analog of peptide 1).^5a,cMoreover, pH and crystallographic

studies suggest that the inhibitory-enhancing effect of the ﬂuo-

rines in DFMP-bearing inhibitors is due to H-bonding of the ﬂuo-

rines with residues in the active site and not due to a pK_a

effect.^5c,10Although the DFMS-bearing peptide 3 was found to be

Br

R'

-1

6, R = CF SO , R' = SO NH

2

3

2

-1

7, R = CH SO , R' = SO NH

2

3

2

-2

8, R = CF PO , R' =CF SO NH

2

4

2

-1

9, R = CF SO , R' = CF SO NH

2

3

2

Figure 2. Structures of compounds 6–9.

over 100-fold (IC₅₀= 10 lM) less potent a competitive PTP1B

inhibitor than peptide 1, the DFMS group is still a much more effec-

tive phosphate mimic than other monoanionic mimics that have

been evaluated in this hexapeptide platform.^8b,11This 100-fold dif-

ference in potency was in stark contrast to the only 6-fold differ-

Although Romsicki et al. reported⁷that the synthesis of inhibi-

tor 2 is described in a patent by Sing et al., we were unable to ﬁnd

any mention of 2 in this document.^12aThe preparation of the phos-

phonate portion of compound 2, phosphonate 10, was describe-

d,^12ahowever, the synthesis of compound 2 itself as well as the

biphenyl sulfonamide portion, compound 13, was not. Compound

2 is just one of a series of similar compounds reported by MerckF-

rosst,^7,12a–cseveral of which are potent PTP1B inhibitors and some

of these compounds have proven to be useful as tools for elucidat-

ing the role of PTP1B in signaling pathways.^7,13a,bThe ortho-bromo

phosphonate portion is a common feature of this series of inhibi-

tors and so an efﬁcient synthesis of phosphonate 10 as well as its

precursors is of considerable interest. Sing et al. prepared phospho-

nate 10 in seven steps in an overall 17% yield starting from readily

prepared ethyl 4-amino-3-bromobenzoate (Scheme 2).^12aIn this

ence in potency between naphthyl sulfonate compound

4

(IC₅₀= 175 M) and its phosphonate analog 5 (IC₅₀= 35 M), two

l

relatively simple non-peptidyl competitive inhibitors.^8aOne possi-

ble reason for the 100-fold versus 6-fold difference is that the pep-

tide scaffold may be preventing the F₂Smp residue from forming

optimal interactions in the active site as suggested by Gao et al.

for other monoanionic pTyr mimics.¹¹However, neither compound

4 nor 5 was q highly potent inhibitor and, consequently, this sim-

ple naphthyl platform may not be the most effective platform for

assessing the DFMS group as a phosphate mimic in a non-peptidyl

platform. In order to make a more complete assessment of the

DFMS group as a phosphate mimic for PTP1B inhibition, we wished

to replace the DFMP group in a potent a non-peptidyl competitive

inhibitor, such as compound 2,⁷with a DFMS group and then com-

pare their inhibitory potencies. In this report, we describe an

effective synthesis of inhibitor 2, its DFMS analog 6, and its

non-ﬂuorinated methylenesulfonyl analog 7 (Figs. 1 and 2). The

synthesis, the ﬂuorines were introduced by subjecting

a-keto-

phosphonate 19 to DAST, which gave the corresponding

a,a-di-

ﬂuoro derivative 20 in a 46% yield. Although this is a viable

synthesis of 10 we wished to avoid using DAST for safety reasons

and also reduce the number of steps. We reasoned that a more efﬁ-

cient and DAST-free synthesis could be achieved by introducing the

ﬂuorines by electrophilic ﬂuorination of a benzylic phospho-

nate.^14a–cThus, phosphonate 22 was prepared in 94% yield from

benzyl bromide 21¹⁵via a Michaelis–Arbuzov reaction using tri-

ethylphosphite (Scheme 3). Electrophilic ﬂuorination of phospho-

nate 22 using NaHMDS and N-ﬂuorobenzenesulfonimide

(NFSi)^14a–cgave ﬂuorophosphonate 23 in 57% yield. Reduction of

the ester using NaBH₄gave alcohol 24 in 72% yield which was con-

verted into bromide 20 in 83% yield using CBr₄/PPh₃. Finally, reac-

tion of bromide 20 with potassium thioacetate gave phosphonate

10 in 92% yield. Thus, phosphonate 10 was prepared in just ﬁve

steps with an overall yield of 29%, a signiﬁcant improvement over

the previously reported procedure.

synthesis of two other derivatives,

8 and 9, in which the

sulfonamide moiety in compounds 2 and 6 is replaced with a diﬂu-

oromethylenesulfonamide group is also described. Inhibition stud-

ies with these compounds and PTP1B reveal surprising results

concerning the use of benzylicsulfonates as PTP1B inhibitors.

2. Results and discussion

2.1. Syntheses

The general approach to the synthesis of compounds 2 and 6–9

is outlined in Scheme 1.^12aThe protected aryl phosphonate or sul-

fonate portions (10–12) would be prepared bearing a thioacetate

which, upon thiol deprotection, would be reacted in situ with the

biphenyl sulfonamide portions, 13 and 14, bearing a benzylic bro-

mide moiety. Deprotection of the phosphonate and sulfonate

would yield the ﬁnal compounds.

The sulfonate analog of 10, compound 11, was prepared using a

strategy similar to that used for phosphonate 10 in that a benzylic

sulfonate was prepared and then subjected to electrophilic ﬂuori-

nation (Scheme 4). Reaction of benzyl bromide 21 with potassium

thioacetate gave thioester 25 in 99% yield. Oxidative chlorination

R

O^-

P

O

S

O

O^-

F

O^-

F

O

P

^-O

S

D-A-D-E

LNH₂

N

H

Br

F

O

F

Br

-2

SO₂NH₂

1, R = CF₂PO₃

3, R = CF₂SO₃

-

2

4

5

Figure 1. PTP1B inhibitors 1–5.

6766

M. Hussain et al. / Bioorg. Med. Chem. 16 (2008) 6764–6777

-2

S

2, R = CF PO , R' = SO NH

2

4

2

Br

R

-1

6, R = CF SO , R' = SO NH

2

3

2

-1

7, R = CH SO , R' = SO NH

2

3

2

Br

R'

-2

8, R = CF PO , R' =CF SO NH

2

4

2

-1

9, R = CF SO , R' = CF SO NH

2

3

2

SAc

Br

+

R

Br

R'

13, R' = SO NH

10, R = CF PO(OEt)

2

14, R' =CF SO NH

11, R = CF SO nPt

2

3

12, R = CH SO nPt

2

3

nPt = neopentyl

Scheme 1.

POBr₃, DMF

CH₂Cl₂

Br

NaNO₂, HCl

DIBAL

OHC

CH₂OH

OHC

H₂N

CO₂Et

NC

CO₂Et

then

CuCN, NaCN

Br

17 (100%)

16 (83%)

15 (58%)

LIHMDS

HPO(OEt)₂

O

P

O

P

O

P

Br

SAc

Br

EtO

HO

P

EtO

KSAc

DAST

MnO₂

F

Br

F

Br

O

Br

10 (86%)

19 (97%)

18 (91%)

20 (46%)

Scheme 2.

O

P

O

P

EtO

Br

2.5 equiv NaHMDS

2.5 equiv NFSi,

-78 ºC, 2 h then rt, 16 h

EtO

2.3 equiv P(OEt)₃,

reflux 3 h

CO₂Me

F

Br

21

23 (57%)

22 (94%)

6 equiv NaBH₄THF,

MeOH, 65-75 ºC, 2 hrs

O

P

O

P

O

P

EtO

1.1. equiv KSAc,

DMF, 30 min, rt

EtO

1.1equiv CBr₄,

CH₂SAc

CH₂OH

CH₂Br

F

1.1 equiv PPh₃

F

Br

CH₂Cl₂, 0 ºC, 2 hrs

24 (72%)

10 (92%)

20 (83%)

Scheme 3.

of 25 gave chlorosulfonate 26, which was isolated in a 98% yield.

Chlorosulfonate 26 was converted into neopentyl (nPt) ester 27

in 81% yield using neopentyl alcohol and triethylamine. Electro-

philic ﬂuorination^8b,16a,bof benzylic sulfonate 27 proceeded well

giving diﬂuoromethylenesulfonate 28 in 81% yield. Reduction of

the ester moiety in 28, bromination of the resulting alcohol 29,

and conversion of benzylbromide 30 to sulfonate 11 were per-

formed using the same procedures described above for the synthe-

sis of 10 and all three reactions proceeded in excellent yields. The

overall yield of sulfonate 11 from benzyl bromide 21 was 54%.

The non-ﬂuorinated sulfonate 12 was prepared from ester 27 using

the same reduction, bromination, and thioacetylation procedure

and all three of these reactions proceeded in excellent yield

(Scheme 4, compounds 31 and 32).

As mentioned previously, we were unable to ﬁnd any descrip-

tion in the primary or patent literature of the biphenyl sulfonamide

M. Hussain et al. / Bioorg. Med. Chem. 16 (2008) 6764–6777

6767

O

S

O

Cl

AcS

Br

Cl , CHCl ,

1.03 equiv KSAc

CO₂Me

2

3

CO₂Me

HOAc, H O

DMF, 30 min, rt

Br

2

Br

26 (98%)

21

25 (99%)

1.5 equiv

neopentyl (nPt)

alcohol,

1.3 equiv Et₃N,

CH₂Cl₂, O/N

6 equiv NaBH₄,THF, MeOH, 65 ºC, 2 h

O

S

O

nPtO

S

F

2.5 equiv NaHMDS,

3.0 equiv NFSi

nPtO

S

X

CH₂OH

CO₂Me

6 equiv NaBH₄,THF,

MeOH, 65 ºC 2 h

CO₂Me

O

F

Br

THF, -78 ºC, 2 h

then at rt O/N

Br

28 (81%)

29, X = CF₂(99%)

31, X = CH₂(98%)

27 (81%)

1.1 equiv PPh₃,

1.1 equiv CBr₄,

0 ºC, 2 h

O

S

O

1.8 equiv KSAc,

DMF, rt, 30 min

nPtO

S

X

CH₂SAc

X

CH₂Br

O

Br

30, X = CF₂(89%)

32, X = CH₂(88%)

11, X = CF₂(99%)

12, X = CH₂(92%)

Scheme 4.

portion of inhibitor 2, compound 13. Our synthesis of compound

13 is shown in Scheme 5. Nitration of commercially available 2-

bromobenzenesulfonyl chloride gave compound 33 in an isolated

96% yield.¹⁷Reaction of the sulfonyl chloride with ammonia in eth-

anol gave sulfonamide 34 in 87% yield.¹⁷The nitro group in 34 was

readily reduced to the amine 35 in 96% yield using Zn/HCl. Diazo-

tization of 35 followed by reaction of the diazonium salt with KI

gave compound 36 in 78% yield. A tolyl group was installed using

a Suzuki reaction which gave biphenyl derivative 37 in 64% yield.

Finally, sulfonamide 13 was prepared in 89% yield by irradiating

a reﬂuxing solution (oil bath) of 37 and NBS in the presence of a

catalytic amount of benzoylperoxide with a 250 W lamp. The over-

all yield of 13 was 36%.

Our synthesis of biphenyl a,a-diﬂuorosulfonamide 14 is out-

lined in Scheme 6. 4-Bromo-3-methylaniline was converted to

iodobenzene derivative 38 in 81% yield by diazotization and

then reaction of the diazonium salt with KI (Scheme 6).¹⁸Bro-

mination of 38 using the same procedure as for the bromina-

tion of 37 (NBS, benzoylperoxide, hm), where the reaction was

heated to reﬂux using an oil bath, resulted in the formation

of a signiﬁcant quantity of deiodinated material. However, it

was found that by heating the reaction to gentle reﬂux with

the heat generated from the lamp (no oil bath), deiodination

could be kept to a minimum and the desired benzyl bromide

derivative 39 was obtained in a 65% yield after careful chroma-

tography. Compound 39 was reacted with KSAc and the

2 equiv HNO

NH /EtOH, THF,

rt, 30 min

6 equiv Zn, HCl

3

O N

Br

O

Br

O

O N

2

Br

O

H N

2

Br

O

2

THF, rt 2.5 hrs

H SO

2

4

S

O

Cl

O

Cl

O

NH

2

O

NH

2

33 (92%)

35 (96%)

34 (87%)

1. 1.02 equiv NaNO , HCl

2

2. 1.02 equiv KI, 0 ºC 5 min

then 90 ºC,10 min

1.1 equiv NBS,

0.2 equiv (PhCOO)

Br

2.2 equiv tolylboronic acid

8 mol% Pd(PPh ) ,

2

Br

O

I

Br

O

H C

3

Br

O

3 4

hν, reflux (oil bath),

benzene, 6 h

S

K CO , THF,100 ºC O/N

2

3

O

NH

O

NH

2

O

NH

2

37 (64%)

36 (78%)

13 (89%)

Scheme 5.

6768

M. Hussain et al. / Bioorg. Med. Chem. 16 (2008) 6764–6777

cat. (PhCOO)

1.1 equiv NBS, hν

benzene, reflux 6 h

1. 1.02 equiv NaNO , HCl

2. 3 equiv KI, 0 ºC 5 min

then , 100 ºC, 10 min

2

I

Br

I

Br

H N

2

Br

CH

CH Br

CH

3

2

3

38 (81%)

39 (65%)

1.02 equiv KSAc

DMF, rt, 30 min

2 equiv

2.4 equiv NaHMDS

2.1 equiv NFSi,

-78 ºC, 2 h then

at rt O/N

2

Cl , AcOH/

diallylamine,

CH Cl , O/N

2

H O,/CH Cl

I

Br

2

I

Br

SO N(allyl)

I

Br

I

Br

SO Cl

2

SAc

2

SO N(allyl)

2

F

41

40 (99%)

42 (84%over two steps)

43 (94%)

1.02 equiv tolyl-boronic acid

20 mol% Pd(PPh ) K CO ,

3 4

2

3

THF,100 ºC O/N

20 equiv

Barbituric acid

1.1 equiv NBS,

cat. (PhCOO)

2

H C

Br

H C

3

Br

20 mol % Pd(PPh )

3

BrCH

Br

hν, benzene

reflux 6 h

2

3 4

2

CH CN, reflux 18 h

3

SO NH

SO N(allyl)

2

SO NH

2

F

14 (88%)

44 (73%)

45 (86%)

Scheme 6.

resulting thioacetate 40 (99% yield) was subjected to oxidative

chlorination to give crude sulfonyl chloride 41. We were unable

to isolate 41 in pure form and so crude 41 was reacted with

diallyl amine to give protected sulfonamide 42 in an 84% yield

(two steps). Once again, we found that the ﬂuorines could be

readily introduced by electrophilic ﬂuorination of benzylic sul-

fonamide 42 using NaHMDS/NFSi.^19a,bThis gave the desired

diﬂuoromethylenesulfonamide 43 in an outstanding 94% yield.

A Suzuki reaction between 43 and tolylboronic acid gave biaryl

species 44 in 73% yield. The temperature of this reaction was

crucial to its success as temperatures over 100 °C resulted in

a signiﬁcant amount of reaction occurring at the bromine while

temperatures less than 100 °C resulted in a slow and often

incomplete reaction even after 24 h. The allyl groups in 44 were

removed in 86% yield using 20 mol% Pd(PPh₃)₄and barbituric

acid in reﬂuxing acetonitrile.^19aBromination of the resulting

primary sulfonamide 45 using the same procedure as described

for the bromination of 38 gave compound 14 in 88% yield. The

overall yield of 14 was 23%.

Phosphonate 10 was coupled to sulfonamides 13 and 14 to give

compounds 46 and 49 in 95% and 72% yields, respectively, by stir-

ring a cold aqueous ethanolic solution of the sulfonamides and the

phosphonate containing excess NaOH for 30 min. (Scheme 7). The

phosphonate groups were deprotected by treatment with TMSBr

then with methanol which gave phosphonate compounds 2 and

8 in 98% and 92% yield as their free acids which could be converted

to their sodium salts using a Dowex Na⁺form ion exchange

column.

Compounds 47, 48 and 50 were prepared in good yield by cou-

pling sulfonates 11 and 12 to sulfonamides 13 and 14 using the

same procedure as that described for 46 and 49 (Scheme 7) The

neopentyl groups from compounds 47, 48, and 50 were removed

using reﬂuxing 1 N HCl. Compounds 6 and 7 were obtained in pure

form as their ammonium salts in 96% and 76% yield, respectively,

by subjecting the crude material to silica gel ﬂash chromatography

using MeOH/NH₄OH/CH₂Cl₂as eluent. This puriﬁcation procedure

was not successful for compound 9 and RP-HPLC was required to

obtain pure 9 and the deprotection yield was 43%.

SAc

R

Br

excess NaOH,

EtOH,

0 ºC 30 min

10, R = CF PO(OEt)

2

S

For phosphonates 46 and 49:

11, R = CF SO nPt

Br

R

2

3

3-4 equiv TMSBr, CH Cl

2

12, R = CH SO nPt

2 (98%), 6 (96%),

7 (76%), 8 (92%),

9 (43%)

2

+

3

then MeOH

R'

46, R = CF PO(OEt) , R' = SO NH (95%)

Br

For sulfonates 47, 48 and 50:

1 N HCl, reflux

Br

2

Br

47, R = CF SO nPt, R' = SO NH (78%)

2

3

2

48, R = CH SO nPt, R' = SO NH (76%)

R'

2

3

2

13, R' = SO NH

2

49, R = R = CF PO(OEt) , R' =CF SO NH (72%)

2

14, R' =CF SO NH

2

50, R = CF SO nPt, R' = CF SO NH (83%)

2

3

2

Scheme 7.

M. Hussain et al. / Bioorg. Med. Chem. 16 (2008) 6764–6777

6769

2.2. Inhibition studies

ined as a PTP1B inhibitor. However, the non-ﬂuorinated analog of

inhibitor 4 exhibited only 10% inhibition at 500

M^8awhich indi-

l

IC₅₀determinations were carried out under conditions similar

to those reported by MerckFrosst for compound 2^7,20(50 mM

Bis–Tris HCl buffer, pH 6.3 containing 2% glycerol, 0.1% Triton

X-100, 4% DMSO, 4.5 mM DTT and 1.8 mM EDTA) with the only sig-

niﬁcant difference being the presence of 4% DMSO.²¹6,8-Diﬂuoro-

4-methylumbelliferyl phosphate (DiFMUP) was used as substrate

cates that the ﬂuorines in inhibitor 4 contribute signiﬁcantly to

the binding of compound 4 to PTP1B. Therefore, we had assumed

that the ﬂuorines in peptide 3 were also making a signiﬁcant con-

tribution to the potency of this peptide. To determine if this is in-

deed the case, we prepared peptide 51²²and examined it, as well

as peptide 3, as PTP1B inhibitors under the conditions described

above. Under these conditions, peptide 3 exhibited an IC₅₀of

at K_mconcentration (5 l

M).^20,21Although none of the new com-

pounds (6–9) were as potent as the parent compound 2, some sur-

prising and informative results were obtained (Table 1). Compound

6 is a 1000-fold less potent inhibitor than its phosphonate analog,

compound 2. This large difference in potency prompted us to

examine whether the modality of inhibition of sulfonate 6 was dif-

ferent from that of competitive inhibitors 2–5. However, sulfonate

6 also behaved as a purely competitive inhibitor with a K_iof

24 lM. Most signiﬁcantly, the IC₅₀of peptide 51 was 44 lM, which

is less than two-fold higher than that of peptide 3. Thus, our previ-

ous assumption that the ﬂuorines in peptide 3 contributed signiﬁ-

cantly to its potency was incorrect. The inability of the ﬂuorines in

inhibitors 3 and 6 to make beneﬁcial contacts with active site res-

idues may partly explain the very large (100- to 1000-fold) differ-

ence in potency between these compounds and their DFMP

analogs.²³On the other hand, compound 4 is much smaller than

peptide 3 and compound 6, and so compound 4 may have greater

freedom of motion in the active site. If so, then this may allow the

ﬂuorines in inhibitor 4 to make some beneﬁcial contacts with ac-

tive site residues while residues in peptide 3 and moieties in com-

pound 6 outside the active site may restrict the mobility of the

arylDFMS group in the active site which could limit the ability of

the ﬂuorines to make beneﬁcial contacts. It is worth mentioning

that, with the exception of sulfonodiﬂuoromethylphenylalanine

(in peptide 3), sulfonomethylphenylalanine (in peptide 51) is a

considerably more effective pTyr mimic than other monoanionic

pTyr mimics that have been examined in this hexapeptide

(DADE-X-LNH₂, X = pTyr mimic) platform.¹¹

In compounds 8 and 9 the sulfonamide moiety in compounds 2

and 6 is replaced with a diﬂuoromethylenesulfonamide group. This

modiﬁcation was pursued since this allowed us to increase the

hydrophobicity of these compounds while at the same time lower-

ing the pK_aof the sulfonamide by about three orders of magnitude

(from 10.5 to 7.5) by making just a minor structural change.^24a,b

Phosphonate inhibitor 8 exhibited a 5-fold increase in IC₅₀com-

pared to compound 2 (Table 1). Further studies revealed that 8 is

a competitive inhibitor with a K_iof 24 nM, still a very potent inhib-

itor. However, sulfonate 9 exhibited a slight decrease in IC₅₀com-

pared to sulfonate 6 (Table 1). Moreover, inhibitor 9 exhibited

10.6 lM and inhibition was reversible (Fig. 3). Surprisingly, inhibi-

tion studies with the nonﬂuorinated analog of inhibitor 6, com-

pound 7, revealed that it has an IC₅₀that is almost the same as

that of compound 6. More detailed studies with inhibitor 7 re-

vealed that it too is a competitive inhibitor with a K_iof 10.3 lM

which is essentially the same as that of compound 6 The non-ﬂuo-

rinated analog of peptide 3, peptide 51 (Fig. 4), was never exam-

Table 1

Inhibition of PTP1B with compounds 2, and 6–9

Compound

IC₅₀

(l

M)

K_i(lM)

2

6

7

8

9

0.0060 0.0004

ND

13

19

3

10.6 0.4

10.3 1.8

0.024 0.003

3.3 0.5

0.030 0.002

6.0 0.4

6

4

2

reversible mixed inhibition (Fig. 5) with a K_iof 3.3

lM and an

a

K_iof 12.5 M indicating that this more hydrophobic compound

l

is probably binding somewhat differently to PTP1B compared to

compound 8 and this may account for their different trends in K_i’s.

Although monoanionic inhibitors 6, 7, and 9 are considerably

less potent than their phosphonate counterparts, they compare

favorably, in terms of their afﬁnity for PTP1B, to the majority of

0

1

2

1/[diFMUP] (1/μM)

8

6

4

2

Figure 3. Lineweaver–Burk plot for the inhibition of PTP1B with inhibitor 6 in

50 mM Bis–Tris HCl, pH 6.3, 20% glycerol, 5 mM DTT, 2 mM EDTA, 0.1% Triton

X-100. (

ꢁ

) 0

l

M, (j) 5

lM, (

N) 10

lM, (.) 20 lM.

O

S

OH

O

D-A-D-E

LNH

2

0

1

2

N

1/[diFMUP] (1/μM)

H

O

51

Figure 5. Lineweaver–Burk plot for the inhibition of PTP1B with inhibitor 9 in

50 mM Bis–Tris HCl, pH 6.3, 20% glycerol, 5 mM DTT, 2 mM EDTA, 0.1% Triton

Figure 4. Structure of peptide 51.

X-100. (

ꢁ

) 0

l

M, (j) 2.5

l

M, ( ) 5 lM, (.) 10 lM.

N

6770

M. Hussain et al. / Bioorg. Med. Chem. 16 (2008) 6764–6777

the best non-peptidyl monoanionic PTP1B inhibitors that have

been reported in the literature. Amarasinghe et al. reported a

to tetramethylsilane (d 0.0, internal standard). For ¹H NMR in

CD₃OD, chemical shifts are reported in ppm relative to the solvent

residual peak (d 3.31, central peak). For ¹H NMR in D₂O, chemical

shifts are reported in ppm relative to the solvent residual peak (d

4.79). For ¹H NMR in DMSO-d₆, chemical shifts are reported in

ppm relative to the solvent residual peak (d 2.49). For ¹H NMR

in acetone-d₆, chemical shifts (d) are reported in ppm relative to

the solvent residual peak (d 2.09). For ¹³C NMR in CDCl₃, chemical

shifts are reported in ppm relative to CDCl₃(d 77.0 for the central

peak). For ¹³C NMR spectra run in CD₃OD, chemical shifts are re-

ported in ppm relative to the solvent residual peak (d 49.00, cen-

tral peak). For ¹³C NMR in DMSO-d₆, chemical shifts are reported

in ppm relative to DMSO-d₆, (d 39.5 for the central peak). For ¹³C

NMR spectra run in D₂O, chemical shifts are reported in ppm rel-

ative to CH₃OH in D₂O (d 49.5, external standard). For ¹³C NMR

run in acetone-d₆, chemical shifts (d) are reported in ppm relative

to the solvent residual peak (d 30.6). All ³¹P NMR spectra chemi-

cals shifts are reported in ppm relative to 85% H₃PO₄(d 0.0, exter-

nal standard). All ¹⁹F spectra chemical shifts are reported in ppm

relative to an external CFCl₃standard (d 0.0, CFCl₃). CIMS were run

in the positive mode using ammonia as ionizing. Melting points

were determined on a Fisher–Johns melting point apparatus and

are uncorrected.

monoanionic sulfamide PTP1B inhibitor with an IC₅₀of 2.3 lM

though the mode of inhibition was not reported.^25aResearchers

at Abbott have reported monoanionic inhibitors bearing either a

2-(hydroxyphenoxy)phenyl acetic acid moiety or a isoxazole car-

boxylic acid group with K_i’s of 6–9

have reported a monoanionic pyrazine carboxylic acid-based

PTP1B competitive inhibitor with a K_iof 4

M.^25dHowever, it

l

M.^25b,cResearchers at Sunesis

l

should be noted that neither our sulfonate inhibitors nor any of

the other above mentioned monoanionic inhibitors are as potent

as the monoanionic inhibitors bearing the (S)-isothiazolidinone

phosphate mimic recently reported by workers at Incyte.^25e

Although the (S)-isothiazolidinone phosphate mimic has never

been examined in the DADE-X-LNH₂platform, studies with other

smaller peptides as well as with non-peptidyl compounds bearing

this moiety suggest that it is the most effective monoanionic phos-

phate reported to date and appears to be even more effective than

the DFMP group.

3. Conclusions

An efﬁcient synthesis of protected phosphonate 10, an impor-

tant biophore for obtaining potent non-peptidyl PTP1B inhibitors,

as well as an effective synthesis of known DFMP-bearing inhibitor

2, one of the most potent non-peptidyl PTP1B inhibitors reported

to date, was achieved. The synthesis of the DFMS analog of com-

pound 2 (compound 6), and that of its non-ﬂuorinated methyl-

enesulfonic analog (7), as well as two other derivatives in

which a distal sulfonamide moiety is replaced with a diﬂuorom-

ethylenesulfonamide group, were also described. DFMS-bearing

inhibitor 6 was a much less potent PTP1B inhibitor than phospho-

nate 2. Replacing the distal sulfonamide moiety with a diﬂuorom-

ethylenesulfonamide group in compound 2 did not yield a more

potent inhibitor though this substitution in compound 6 did yield

a slightly better inhibitor. Surprisingly, inhibition studies with the

non-ﬂuorinated sulfonates, compound 7 and peptide 51 bearing

sulfonomethylphenylalanine, revealed that the ﬂuorines had little

effect on the potency of the DFMS-bearing inhibitors which was

in contrast to our previously held assumption that the ﬂuorines

in DFMS-bearing inhibitors contributed signiﬁcantly to their po-

tency. This may in part explain the large difference in potency be-

tween the DFMS and DFMP bearing compounds. These results

also show that sulfonomethylphenylalanine, a pTyr mimic that

is more easily prepared than sulfonodiﬂuoromethylphenylala-

nine,^16b,22is one of the best monoanionic pTyr mimics reported

so far when examined in the context of the DADE-X-LNH₂

platform.

4.1.2. {[2-Bromo-4-(4⁰-bromo-3⁰-sulfamoylbiphenyl-4-ylmethyl-

sulfanylmethyl)-phenyl]diﬂuoromethyl}phosphonic acid (2)

To a solution of 46 (0.113 g, 0.159 mmol) in dry CH₂Cl₂(10 mL)

under Ar was added trimethylsilyl bromide (0.97 g, 4.0 equiv,

0.63 mmol) and the mixture was stirred at rt for 20 h. The mixture

was concentrated and then the residue was dissolved in methanol

and stirred for 30 min. The solution was concentrated and the res-

idue was dissolved in methanol and concentrated again. This gave

pure 2 as a colorless oil (0.103 g, 98%). For inhibition studies, com-

pound 2 was dissolved in water and passed through a Dowex Na⁺

form ion exchange column to give 2 as a white disodium salt. The

spectral data given below is for the free acid. ¹H NMR (300 MHz,

acetone-d₆) d 3.73 (2H, s), 3.75 (2H, s), 7.40–7.43 (3H, d, J = 8 Hz),

7.58–7.64 (4H, m), 7.75 (1H, dd, J = 8.4 Hz), 7.86 (1H, d,

J = 8.3 Hz), 8.32 (1H, s); ¹³C NMR (75 MHz, acetone-d₆) d 34.2,

35.2, 118.0, 118.3 (dt, J = 264.0, 216.0 Hz), 119.7, 126.9, 127.6,

127.8, 129.9, 130.1 (t, J = 7.7 Hz), 131.2, 135.5, 135.6, 137.1,

138.8, 140.3, 143.2, 143.6, 205.5; ³¹P NMR (121 MHz, acetone-d₆)

d 5.4 (t, J = 111.0 Hz); ¹⁹F NMR (282 MHz, acetone-d₆) d ꢀ105.0

(d, J = 112.7 Hz). LR^ꢀESIMS m/z (relative intensity) 653 ([MꢀH]^ꢀ,

100); HR^ꢀESIMS m/z calcd for C₂₁H₁₈Br₂F₂NO₅PS₂([M-H]^ꢀ),

653.8620, found 653.8614.

4.1.3. [2-Bromo-4-(4⁰-bromo-3⁰-sulfamoylbiphenyl-4-ylmethyl-

sulfanyl methyl)phenyl]diﬂuoromethanesulfonate, ammonium

salt (6)

4. Experimental

4.1. Chemistry

A solution of compound 47 (0.0112 g, 0.0160 mmol) in 1 N HCl

(10 mL) was reﬂuxed for 4 h. The solution was concentrated by

high-vacuum rotary evaporation and the resulting residue was

subjected to ﬂash chromatography (2:0.5:10 MeOH/NH₄OH/

CH₂Cl₂) which gave compound 6 as a white ammonium salt

(0.010 g, 95%). ¹H NMR (500 MHz, CD₃OD) d 3.64 (2H, s), 3.68

(2H, s), 7.32 (1H, d, J = 8.1 Hz), 7.38 (2H, d, J = 8.2 Hz), 7.55 (1H,

s), 7.59 (2H, d, J = 8.2 Hz), 7.67 (1H, d, J = 8.2 Hz), 7.70 (1H, dd,

J = 8.3 Hz), 7.83 (1H, d, J = 8.3 Hz), 8.33 (1H, d, J = 2.3 Hz); ¹⁹F

NMR (282 MHz, CD₃OD) d ꢀ99.1; ¹³C NMR (75 MHz, CD₃OD) d

34.0, 34.8, 117.7, 119.7 (t, J = 279.6 Hz), 120.1, 126.5, 127.0,

127.3, 129.5, 129.9, 130.5 (t, J = 7.7 Hz), 131.0, 135.1, 135.3,

137.1, 138.5, 140.4, 142.7, 143.0; LR^ꢀESIMS m/z (relative intensity)

4.1.1. General

All starting materials and reagents were obtained commer-

cially and used without further puriﬁcation with the exception

of TMSBr, which was distilled under Ar and stored under Ar in a

Schlenk tube. Tetrahydrofuran (THF) was distilled from sodium

metal in the presence of benzophenone under argon. CH₂Cl₂was

distilled from calcium hydride under nitrogen. DMF was distilled

under aspirator pressure from CaH and stored under Ar. Methanol

was dried by reﬂuxing over magnesium metal and distilled under

Ar and stored over 4 Å molecular sieves. Flash chromatography

was performed using silica gel (60 Å, 230–400 mesh). For ¹H

NMR in CDCl₃, chemical shifts (d) are reported in ppm relative

+

653 ([Mꢀ NH₄]^ꢀ, 100); HR^ꢀESMS m/z calcd for C₂₁H₁₇Br₂F₂NO₅S₃

+

([Mꢀ NH₄]^ꢀ), 653.8525, found 653.8525.

M. Hussain et al. / Bioorg. Med. Chem. 16 (2008) 6764–6777

6771

4.1.4. [2-Bromo-4-(4⁰-bromo-3⁰-sulfamoylbiphenyl-4-ylmethyl-

sulfanylmethyl)phenyl]-methanesulfonate, ammonium salt (7)

A suspension of compound 48 (0.160 g, 0.232 mmol) in 1 N

HCl (15 mL) was reﬂuxed for 12 h. The solution was concentrated

by high-vacuum rotary evaporation and the resulting residue was

subjected to ﬂash chromatography (2:0.5:10 MeOH/NH₄OH/

CH₂Cl₂) which gave pure compound 7 as a white ammonium salt

(0.100 g, 76%). ¹H NMR (300 MHz, CD₃OD) d 3.63 (2H, s), 3.60 (2H,

s), 4.25 (2H, s) 7.21 (1H, d, J = 7.9 Hz), 7.37 (2H, d, J = 8.2 Hz), 7.45

(1H), 7.53 (1H, d, J = 7.9 Hz), 7.58 (2H, d, J = 8.2 Hz), 7.70 (1H, dd,

J = 8.3 Hz), 7.83 (1H, d, J = 8.3 Hz), 8.32 (1H, d, J = 2.2 Hz) ; ¹³C

NMR (75 MHz, CD₃OD) d 34.3, 34.7, 55.7, 117.8, 124.9, 120.1,

126.5, 127.3, 127.6, 129.6, 130.9, 131.5, 132.0, 132.8, 135.4,

137.1, 138.7, 140.5, 142.8; LR^ꢀESIMS m/z (relative intensity) 618

(½M ꢀ NH^þ₄ꢂꢀ, 100); HR^ꢀESIMS m/z calcd for C₂₁H₁₈Br₂NO₅S₃

at rt for 30 min. The DMF was removed by high-vacuum rotary

evaporation and the residue dissolved in CH₂Cl₂. The resulting

solution was washed with water, then dried (Na₂SO₄), and concen-

trated. The residue was subjected to ﬂash chromatography (1:1

EtOAc/hexane) which gave pure 10 as a colorless oil (0.711 g,

92%). ¹H NMR (300 MHz, CDCl₃) d 1.20 (6H, t, J = 7.1 Hz), 2.21

(3H, s), 3.93 (2H, s), 4.03–4.16 (4H, m), 7.19 (1H, d, J = 8.1 Hz),

7.40 (1H, d, J = 8.1 Hz), 7.47 (1H, s); ³¹P NMR (121 MHz, CDCl₃) d

6.51 (t, J = 114.4 Hz); ¹⁹F NMR (282 MHz, CDCl₃) d ꢀ104.0 (d,

J = 114.0 Hz); ¹³C NMR (75 MHz, CDCl₃) d 16.3 (d, J = 5.4), 30.1,

32.0, 64.9 (d, J = 6.9 Hz), 117.9 (dt, J = 265.9, 219.1 Hz), 120.0 (t,

J_CP= 3.0), 127.6, 129.8 (dt, J = 8.7, 1.8 Hz), 130.5 (J = 21.3, 14.3),

135.4, 142.5 (d, J = 1.41), 194.1; LREIMS m/z (%) 430 (M⁺, 23), 389

(100); HREIMS m/z calcd for C₁₄H₁₈B_rF₂O₄PS (M⁺) 429.9815, found

429.9819.

+

([Mꢀ NH₄]^ꢀ), m/z 617.8714, found 617.8714.

4.1.8. Thioacetic acid S{3bromo-4-[(2,2-dimethylpropoxysulfonyl)

diﬂuoromethyl]-benzyl}ester (11)

4.1.5. ({2-Bromo-4-[4⁰-bromo-3⁰-(diﬂuorosulfamoylmethyl)

biphenyl-4-ylmethylsulfanylmethyl]phenyl}diﬂuoromethyl)

phosphonic acid (8)

Prepared in a 96% yield from compound 30 using the same pro-

cedure as that described for compound 10. The following quanti-

ties were used: 30 (0.100 g, 0.22 mmol), potassium thioacetate

(0.047 g, 1.8 equiv, 0.40 mmol), DMF (5 mL). The residue was sub-

jected to ﬂash chromatography (0.5:9.5 EtOAc/hexane) which gave

11 as a colorless oil. ¹H NMR (300 MHz, CDCl₃) d 0.97 (9H, s), 2.32

(3H, s), 4.03 (2H, s), 4.12 (2H, s), 7.33 (1H, d, J = 8.1 Hz), 7.57 (1H, d,

J = 8.2 Hz), 7.62 (1H, s); ¹⁹F NMR (285 MHz, CDCl₃) d ꢀ94.8; ¹³C

NMR (75 MHz, CDCl₃) d 25.7, 29.6, 30.1, 31.9, 84.6, 120.5 (t,

J = 286.7 Hz), 120.9, 126.0 (t, J = 21.1 Hz), 127.8, 130.8 (t,

J = 7.9 Hz), 135.8, 144.3, 194.0; LR-ESIMS m/z (relative intensity)

462 (M⁺, 100); HR-ESIMS m/z calcd for C₁₅H₁₉BrF₂O₄S₂(M⁺)

462.0220, found 462.0224.

Prepared in a 92% yield from compound 49 using the same

procedure as that described for compound 2. The following quan-

tities were used: 49 (0.026 g, 0.034 mmol), dry CH₂Cl₂(4 mL), and

TMSBr (0.016 g, 2.9 equiv, 0.10 mmol). For inhibition studies,

compound 8 was dissolved in water and passed through a Dowex

Na⁺form ion exchange column to give 8 as a white disodium salt.

The spectral data given below are for the free acid. ¹H NMR

(300 MHz, CD₃OD) d 3.64 (2H, s), 3.68 (2H, s) 7.32–7.38 (3H, m),

7.56 (4H, m), 7.67 (1H, d, J = 8.2 Hz), 7.79 (1H, d, J = 8.4 Hz), 7.90

(1H, s); ³¹P NMR (121 MHz, CD₃OD) d 5.5 (t, J = 110.5 Hz); ¹⁹F

NMR (282 MHz, CD₃OD) d ꢀ99.2, ꢀ106.0 (d, J = 110.1 Hz); ¹³C

NMR (125.8 MHz, CD₃OD) d 34.0, 38.5, 119.2 (bs), 119.7 (bs),

120.1 (t, J = 283.2 Hz), 126.6, 127.3, 128.87 (t, J = 8.0 Hz), 129.5,

129.7 (t, J = 8.1 Hz) 130.9, 131.5, 135.2, 135.8, 137.3, 138.4,

139.9, 142.9 LR^ꢀESIMS m/z (relative intensity) 703 ([Mꢀ1]^ꢀ,

100); HR^ꢀESIMS m/z calcd for C₂₂H₁₇Br₂F₄NO₅PS₂(MꢀH)^ꢀm/z

703.8589, found 703.8597.

4.1.9. Thioacetic acid S-(3-bromo-4-isobutoxysulfonylmethyl-

benzyl) ester (12)

Prepared using the same procedure as that used for com-

pound 10. The following quantities were used: compound 32

(0.555 g, 1.34 mmol), potassium thioacetate (0.179 g, 1.80 equiv,

1.57 mmol), DMF (5 mL). The residue was subjected to ﬂash

chromatography (1:4 EtOAc/ hexane) which gave pure 12 as a

white solid (92%). Mp 108–110 °C; ¹H NMR (300 MHz, CDCl₃) d

0.88 (9H, s), 2.31 (3H, s), 3.76 (2H, sj), 4.02 (2H, s), 4.54 (2H,

s), 7.23 (1H, d, J = 7.8 Hz), 7.43 (1H, d, J = 7.9 Hz), 7.52 (1H, s);

¹³C NMR (75 MHz, CDCl₃) d 26.0, 30.3, 31.7, 32.2, 55.4, 79.5,

125.4, 127.0, 128.3, 132.6, 133.4, 140.9, 194.4; LRCIMS m/z (rel-

ative intensity) 426 ([M+NH₄]⁺, 100), 258 (10), 214 (35); HREIMS

4.1.6. {2-Bromo-4-[4⁰-bromo-3⁰-(diﬂuorosulfamoylmethyl)

biphenyl-4-ylmethylsulfanylmethyl]-phenyl}diﬂuorometha-

nesulfonic acid (9)

A suspension of 50, (0.090 g, 0.116 mmol), in 1 N HCl (20 mL)

was reﬂuxed for 4 h. The solution was concentrated by high-vac-

uum rotary evaporation which gave 75 mg of crude 9 as a light

brown oil. Silica gel ﬂash chromatography (2:0.5:10 MeOH/

NH₄OH/CH₂Cl₂) of crude 9 failed to give pure product. To obtain

pure 9 the impure material from the silica ﬂash column was sub-

jected to reverse-phase semi-preparative HPLC (C-18 column,

55% 0.1% TFA in H₂O/45% CH₃CN to 20% 0.1% TFA in H₂O/80%

CH₃CN over 60 minutes, ﬂow rate = 8 mL/min, R_t= 39 min) which

gave pure 9 as its free acid (0.035 g, white powder, 43%). ¹H NMR

(300 MHz, acetone-d₆) d 3.71 (2H, s), 3.74 (2H, s), 7.35 (1H, d,

J = 8.0 Hz), 7.42 (2H, d, J = 8.1 Hz) 7.57–7.63 (3H, m), 7.70–7.78

(2H, s) 7.84 (1H, d, J = 8.4 Hz), 7.95 (1H, d, J = 1.8 Hz); ¹⁹F NMR

(285 MHz, acetone-d₆) d ꢀ99.1, ꢀ99.0; ¹³C NMR (75 MHz, CD₃OD)

d 34.1, 35.0, 119.2, 119.8 (t, J = 280.5 Hz), 120.1 (t, J = 284.2 Hz),

126.6, 127.2, 128.9 (t, J = 21.5 Hz), 128.9 (t, J = 7.8 Hz), 129.6,

129.9 (t, J = 22.5 Hz), 130.6 (t, J = 7.7 Hz), 131.0, 135.2, 135.9,

137.2, 138.4, 139.9, 143.1; LR^ꢀESIMS m/z (relative intensity) 704

([MꢀH]^ꢀ, 100); HR^ꢀESIMS m/z calcd for C₂₂H₁₆Br₂F₄NO₅S₃

([MꢀH]^ꢀ) 703.8493, found 703.8483.

m/z calcd for

C

₁₃H₁₈BrO₃S₂([M-(CH₃CO)]⁺) 364.9886, found

364.9876.

4.1.10. 4-Bromo-4⁰-bromomethylbiphenyl-3-sulfonic acid

amide (13)

A solution of compound 37 (0.300 g, 0.92 mmol), N-bromosuc-

cinimide (0.181 g, 1.1 equiv, 1.02 mmol) and benzoylperoxide

(0.048 g, 0.20 equiv, 0.18 mmol) in benzene (25 mL) was reﬂuxed

(oil bath) under Ar for 6 h during which a 250 W lamp was shone

on the reaction. The reaction was quenched with water then ex-

tracted with EtOAc. The combined organics were dried (Na₂SO₄)

and concentrated and the resulting residue subjected to ﬂash

chromatography (2:8 EtOAc/hexane) to give 13 as a white solid

(0.333 g, 89%). Mp 151–153 °C; ¹H NMR (300 MHz, acetone-d₆) d

4.68 (2H, s), 6.76 (2H, br s), 7.58 (2H, d, J = 8.3 Hz), 7.68 (2H, d,

J = 8.3 Hz), 7.76 (1H, d, J = 8.2 Hz), 7.87 (1H, d, J = 8.2 Hz), 8.33

(1H, d, J = 2.3 Hz); ¹³C NMR (75 MHz, acetone-d₆) d 32.9, 118.3,

127.2, 127.6, 130.0, 131.3, 135.7, 138.4, 138.7, 140.0, 143.3; LRE-

IMS m/z (relative intensity) 403 (M⁺, 11), 326 (100), 165 (22);

4.1.7. Thioacetic acid S-{3-bromo-4-[(diethoxyphosphoryl)di-

ﬂuoromethyl]benzyl}ester (10)

A solution of 20 (0. 777 g, 1.79 mmol) and potassium thioace-

tate (0.228 g, 1.12 equiv, 1.57 mmol) in DMF (15 mL) was stirred

HREIMS m/z calcd for C

₁₃H₁₁Br₂NO₂S (M⁺) 402.8877, found

402.8885.

6772

M. Hussain et al. / Bioorg. Med. Chem. 16 (2008) 6764–6777

4.1.11. (4-Bromo-4⁰-bromomethylbiphenyl-3-yl)diﬂuoro-metha-

nesulfonamide (14)

centrated. The residue was subjected to ﬂash chromatography

(1:1 EtOAc/hexane) which gave 23 as a white solid (1.25 g, 57%).

Mp 35 °C; ¹H NMR (300 MHz, CDCl₃) d 1.09 (6H, t, J = 7.2 Hz),

3.67 (3H, s), 3.93–4.07 (4H, m), 7.45 (1H, d, J = 8.3 Hz), 7.76 (1H,

d, J = 8.2 Hz), 8.03 (1H, s); ³¹P NMR (121 MHz, CDCl₃) d 4.9 (t,

To a solution of 45 (0.100 g, 0.266 mmol) in benzene (7 mL)

were added N-bromosuccinimide (0.052 g, 1.1 equiv, 0.29 mmol)

and benzoylperoxide (0.008 g, 0.1 equiv, 0.03 mmol). The mixture

was gently reﬂuxed under Ar by placing a 250 W lamp close to

the reaction vessel. After 3 h, water was added and the mixture

was extracted with EtOAc. The combined organics were concen-

trated and the resulting residue was subjected to ﬂash chromatog-

raphy twice (3.5:6.5 EtOAc/hexane) which gave pure 14 as a white

solid (0.105 g, 88%). Mp 152–154 °C; ¹H NMR (300 MHz, acetone-

d₆) d 4.68 (2H, s), 7.39 (2H, br s), 7.57 (2H, d, J = 7.6 Hz), 7.66

(2H, d, J = 7.5 Hz), 7.75 (1H, d, J = 10.5 Hz), 7.84 (1H, d, J = 8.0 Hz),

7.94 (1H, s); ¹⁹F NMR (285 MHz, acetone-d₆) d ꢀ98.6; ¹³C NMR

(75 MHz, acetone-d₆) d 32.9, 119.7, 120.2 (t, J = 284.5 Hz), 127.2,

129.2 (t, J = 7.6 Hz), 130.0, 131.34, 136.2, 138.3, 138.6, 139.6,

142.5; LREIMS m/z (relative intensity) 453 (M⁺, 12), 395 (100),

296 (68); HREIMS m/z calcd for C₁₄H₁₁Br₂F₂NO₂S (M⁺) 452.8845,

found 452.8838.

J = 111.0 Hz); ¹⁹F NMR (282 MHz, CDCl₃)

d

ꢀ105.0 (d,

J = 110.8 Hz); ¹³C NMR (75 MHz, CDCl₃) d 16.1 (d, J = 5.4), 52.34,

64.8 (d, J = 6.9 Hz,), 117.5 (dt, J = 266.6, 217.0 Hz), 119.9 (app. dt,

J = 2.9 Hz, J = 2.9 Hz), 127.8, 129.7 (dt, J = 8.7, 1.7 Hz), 133.2, 135.6

(dt, J = 21.1, 14.1 Hz), 135.9, 164.4; LREIMS m/z (%) 400 (M⁺, 2),

321 (100), 265 (48); HREIMS m/z calcd for C₁₃H₁₆BrF₂O₅P (M⁺)

399.9887, found 399.9876.

4.1.15. [(2-Bromo-4-hydroxymethylphenyl)diﬂuoromethyl]-

phosphonic acid diethyl ester (24)

A suspension of 23 (1.20 g, 3.00 mmol) and NaBH₄(0.67 g, 6.0

equiv, 18 mmol) in dry THF (8 mL) under Ar was stirred at 65 °C

for 15 min. MeOH (8 mL) was added dropwise over 20 min and

the mixture was stirred for 2 h at 65 °C. The reaction was quenched

with a satd NH₄Cl solution, water was added and the mixture was

extracted several times with CH₂Cl₂. The combined organisc were

dried (Na₂SO₄) and concentrated. The residue was subjected to

ﬂash chromatography (4:1 EtOAc/hexane) which gave pure 24 as

a colorless oil (0.80 g, 72%). ¹H NMR (300 MHz, CDCl₃) d 1.24 (6H,

t, J = 7.1 Hz), 4.02–4.21 (5H, m, OH peak merged), 4.53 (2H, s),

7.23 (1H, d, J = 8.2 Hz), 7.44 (1H, d, J = 8.2 Hz), 7.58 (1H, s); ³¹P

NMR (121 MHz, CDCl₃) d 5.5 (t, J = 114.6 Hz); ¹⁹F NMR (282 MHz,

CDCl₃) d 104.0 (d, J = 115.9 Hz); ¹³C NMR (75 MHz, CDCl₃) d 16.3

(d, J = 5.5), 62.9, 65.2 (d, J = 7.0 Hz), 117.5 (dt, J = 265.7, 220.7 Hz),

119.9, 125.1, 129.6 (dt, J = 8.7, 1.9 Hz), 129.8 (dt, J = 21.3, 14.3

Hz), 133.1, 146.4; LRCIMS m/z (%) 390 ([M + NH₄]⁺, 100), 312

(20); HREIMS m/z calcd for C₁₂H₁₆F₂O₄P ([MꢀBr]⁺) 293.0754, found

293.0760.

4.1.12. [(2-Bromo-4-bromomethylphenyl)diﬂuoromethyl]phos-

phonic acid, diethyl ester (20)

To a solution of 24 (0.80 g, 2.2 mmol) in CH₂Cl₂(20 mL) at 0 °C

were added carbon tetrabromide (0.78 g, 1.1 equiv, 2.4 mmol) and

triphenylphosphine (0.62 g, 1.1 equiv, 2.4 mmol), and the mixture

was stirred for 2 h. The reaction was quenched with water, ex-

tracted with CH₂Cl₂, and the combined organics were dried

(Na₂SO₄) and concentrated. The resulting residue was subjected

to ﬂash chromatography (1:1 EtOAc/hexane) which gave pure 20

as a colorless oil (0.78 g, 83%). ¹H NMR (300 MHz, CDCl₃) d 1.22

(6H, t, J = 7.1 Hz), 4.06–4.19 (4H, m), 4.30 (2H, s), 7.30 (1H, d,

J = 8.2 Hz), 7.47 (1H, d, J = 8.2 Hz), 7.58 (1H, s); ³¹P NMR

(121 MHz, CDCl₃) d 5.30 (t, J = 111.0 Hz); ¹⁹F NMR (282 MHz,

CDCl₃) d ꢀ104.0 (d, J = 112.9 Hz); ¹³C NMR (75 MHz, CDCl₃) d

16.3 (d, J = 5.5 Hz), 30.7, 65.0 (d, J = 6.9 Hz), 117.8 (dt, J = 266.2,

218.7 Hz), 120.1 (app. dt, J_CP= 3.1 Hz, J = 3.1 Hz), 127.7, 130.1 (dt,

J = 8.7, 1.9 Hz), 131.55 (dt, J = 21.4, 14.2 Hz), 135.6, 141.9 (d,

J = 1.4 Hz). LRCIMS m/z (%) 454 ([M + NH₄]⁺, 100), 355 (11); HRE-

IMS m/z calcd for C₁₂H₁₅B_rF₂O₃P ([M – Br]⁺) 356.9889, found

356.9881.

4.1.16. 4-Acetylsulfanylmethyl-3-bromo-benzoic acid methyl

ester (25)

A solution of compound 21 (21.3 g, 69.0 mmol) and potassium

thioacetate (8.07 g, 1.03 equiv, 70.8 mmol) in DMF (60 mL) was

stirred at rt for 30 min. The reaction was diluted with water and

extracted with CH₂Cl₂. The combined organics were dried

(Na₂SO₄), then concentrated, and the resulting solid residue re-

crystallized in CH₂Cl₂/hexane which gave compound 25 as a crys-

talline white solid (20.65 g, 99%). Mp 62–63 °C; ¹H NMR (300 MHz,

CDCl₃) d 2.30 (3H, s), 3.86 (3H, s), 4.19 (2H, s), 7.47 (1H, d,

J = 8.0 Hz), 7.84 (1H, dd, J = 8.1 Hz), 8.15 (1H, s); ¹³C NMR

(75 MHz, CDCl₃) d 30.2, 33.7, 52.4, 124.3, 128.6, 130.7, 131.0,

133.8, 142.2, 165.4, 194.4; LRCIMS m/z (relative intensity) 320

([M+NH₄]⁺, 100), 223 (42); HREIMS m/z calcd for C₁₀H₈O₂BrS

([MꢀOMe]⁺) 270.9423, found 270.9431.

4.1.13. 3-Bromo-4-(diethoxyphosphorylmethyl)benzoic acid

methyl ester (22)

This was prepared according to the procedure of Gilbert et

al.²⁶A mixture of 3-bromo-4-(bromomethyl)benzoic acid methyl

ester (21)¹⁵(4.05 g, 13.3 mmol) and triethyl phosphite (5.33 g,

2.40 equiv, 32.0 mmol) was reﬂuxed for 3h. The excess triethyl

phosphite was removed by distillation and the residue was sub-

jected to ﬂash chromatography (1:1 EtOAc/Hexanes) which gave

pure 22 as a colorless oil (4.55 g, 94%). ¹H NMR (300 MHz, CDCl₃)

d 1.22 (6H, t, J = 7.1 Hz), 3.41 (2H, d, J = 22.5 Hz), 3.88 (3H, s),

3.97–4.10 (4H, m), 7.49 (1H, dd, J = 8.1 Hz), 7.89 (1H, d,

J = 7.9 Hz), 8.20 (1H, s); ³¹P NMR (121 MHz, CDCl₃) d 24.1. LRE-

IMS m/z (%) 333 ([MꢀOMe]⁺, 4) 285 (100) 229 (85); HREIMS

4.1.17. 3-Bromo-4-chlorosulfonylmethylbenzoic acid methyl

ester (26)

To a suspension of thioacetate 25 (5.03 g, 16.7 mmol) in glacial

acetic acid (32 mL) and ice water (16 mL) was added CHCl₃drop-

wise until the mixture become clear. The reaction was placed in

an ice bath and Cl₂gas was bubbled through the mixture for 2 h.

Nitrogen was then bubbled through the solution to remove excess

Cl₂and then mixture was diluted with water and extracted with

CH₂Cl₂. The combined organics were dried (Na₂SO₄) and concen-

trated to give pure 26 as colorless ﬁne crystals (5.32 g, 98%). Mp

99–101 °C; ¹H NMR (300 MHz, CDCl₃) d 3.93 (3H, s), 5.16 (2H, s),

7.68 (1H, d, J = 8.1 Hz), 8.04 (1H, dd, J = 8.0 Hz), 8.33 (1H, d,

J = 1.4 Hz); ¹³C NMR (75 MHz, CDCl₃) d 52.7, 69.4, 126.2, 128.8,

130.8, 133.2, 133.3, 134.6, 164.8; LREIMS m/z (relative intensity)

325 (M⁺, 6) 227 (100); HREIMS m/z calcd for C₉H₈O₄BrClS (M⁺)

325.9015, found 325.9020.

m/z calcd for

332.9884.

C

₁₂H₁₅BrO₄P ([MꢀOMe]⁺), 332.9891, found

4.1.14. 3-Bromo-4-[(diethoxyphosphoryl)diﬂuoromethyl]benzoic

acid, methyl ester (23)

To a solution of phosphonate 22 (2.00 g, 5.47 mmol) and N-ﬂu-

orobenzenesulfonimide (4.31 g, 2.50 equiv, 13.7 mmol) in dry THF

(50 mL) at ꢀ78 °C was added NaHMDS (1.0 M in THF, 13.7 mL, 2.50

equiv, 13.7 mmol) over 10 min. The mixture was stirred for 2 h at

ꢀ78 °C, then allowed to warm to rt, and stirred for 16 h. The reac-

tion was quenched with a satd NH₄Cl solution, extracted with

CH₂Cl₂, and the combined organic layers dried (Na₂SO₄) and con-

M. Hussain et al. / Bioorg. Med. Chem. 16 (2008) 6764–6777

6773

4.1.18. 3-Bromo-4-(2,2-dimethylpropoxysulfonylmethyl)benzoic

acid methyl ester (27)

7.73 (1H, s); ¹⁹F NMR (285 MHz, CDCl₃) d ꢀ95.0; ¹³C NMR

(75 MHz, CDCl₃) d 25.8, 30.3, 32.1, 84.9, 120.4 (t, J = 286.9 Hz),

121, 127.1 (t, J = 21.2 Hz), 128.0, 131.1 (t, J = 8.8 Hz), 136.1,

143.6; LRCIMS m/z (relative intensity) 466 ([M + NH₄]⁺, 100); HRE-

To a solution of compound 26 (5.06 g, 15.5 mmol) and neopen-

tyl alcohol (2.04 g, 1.5 equiv, 23.3 mmol), in dry CH₂Cl₂(50 mL) at

0 °C under Ar was added triethylamine (2.03 mL, 1.3 equiv,

20.2 mmol) dropwise over 10 min. The mixture was stirred for

16 h at rt, quenched with H₂O, and extracted with CH₂Cl₂. The

combined organics were dried (Na₂SO₄) and concentrated and

the resulting residue subjected to ﬂash chromatography which

gave pure 27 as a colorless crystalline solid (4.72 g, 81%). Mp 51–

53 °C; ¹H NMR (300 MHz, CDCl₃) d 0.84 (9H, s), 3.75 (2H, s), 3.84

(3H, s), 4.59 (2H, s), 7.56 (1H, d, J = 8.1 Hz), 7.90 (1H, dd,

J = 7.9 Hz), 8.2 (1H, d, J = 1.5 Hz); ¹³C NMR (75 MHz, CDCl₃) d

25.9, 31.7, 52.5, 55.5, 79.6, 125.4, 128.6, 132.1, 132.6, 132.9,

134.1, 165.0; LREIMS m/z (relative intensity) 378 (M⁺, 6), 229

(100); HREIMS m/z calcd for C₁₄H₁₉BrO₅S (M⁺) 378.0137, found

378.0142.

IMS m/z calcd for

C

₁₃H₁₆BrF₂O₃S ([MꢀBr]⁺) 370.9946, found

370.9948.

4.1.22. (2-Bromo-4-hydroxymethylphenyl)methanesulfonic

acid, 2,2-dimethylpropyl ester (31)

A suspension of compound 27 (0.570 g, 1.51 mmol) and NaBH₄

(0.335 g, 6.0 equiv, 9.05 mmol) in dry THF (8 mL) was gently re-

ﬂuxed under Ar for 15 min. MeOH (8 mL) was added, dropwise

over 20 min and gentle reﬂuxing was continued for an additional

2 h. The reaction was quenched with a satd aq NH₄Cl solution,

H₂O was added and the mixture was extracted with CH₂Cl₂. The

combined organics were dried (Na₂SO₄) then concentrated, and

the resulting residue subjected to ﬂash chromatography which

gave pure 31 as a colorless oil (0.525 g, 98%). ¹H NMR (300 MHz,

CDCl₃) d 0.88 (9H, s), 3.05 (1H, br s), 3.77 (2H, s), 4.54 (4H, s),

7.22 (1H, d, J = 7.9), 7.43 (1H, d, J = 7.8 Hz), 7.55 (1H, s); ¹³C NMR

(75 MHz, CDCl₃) d 26.0, 31.7, 55.4, 63.4, 79.6, 125.5, 125.9, 126.7,

131.1, 132.5, 144.1; LREIMS m/z (relative intensity) 350 (M⁺, 10),

199 (100); HREIMS m/z calcd for C₁₃H₁₉BrO₄S (M⁺) 350.0187, found

350.0190.

4.1.19. 3-Bromo-4-(2,2-dimethylpropoxysulfonylmethyl)benzoic

acid methyl ester (28)

To a solution of compound 27 (2.00 g, 5.28 mmol) and N-ﬂuoro-

benzenesulfonimide (5.00 g, 3.0 equiv, 15.9 mmol) in dry THF

(100 mL) at ꢀ78 °C was added NaHMDS (1.0 M in THF, 13.3 mL,

2.5 equiv, 13.3 mmol) over 30 min. The mixture was stirred for

2 h at ꢀ78 °C then allowed to warm to rt, and stirred for 16 h.

The reaction was quenched with aq satd NH₄Cl solution and ex-

tracted with diethyl ether. The combined organics were dried

(Na₂SO₄) and then concentrated. The resulting residue was sub-

jected to ﬂash chromatography which gave pure 28 as a white solid

(1.78 g, 81%). Mp 61–63 °C; ¹H NMR (300 MHz, CDCl₃) d 0.97 (9H,

s), 3.90 (3H, s), 4.15 (2H, s), 7.74 (1H, d, J = 8.3 Hz), 8.02 (1H, d,

J = 8.3 Hz), 8.31 (1H, s); ¹⁹F NMR (285 MHz, CDCl₃) d ꢀ95.5; ¹³C

4.1.23. (2-Bromo-4-bromomethylphenyl)methanesulfonic acid,

2,2-dimethylpropyl ester (32)

To a solution of compound 31 (0.580 g, 1.65 mmol) in CH₂Cl₂

(25 mL) at 0 °C were added carbon tetrabromide (0.606 g, 1.10

equiv, 1.83 mmol) and triphenylphosphine 0.480 g, 1.10 equiv,

1.83 mmol) and the mixture was stirred for 1 h. The mixture was

concentrated by rotary evaporation at rt and the resulting residue

was subjected to ﬂash chromatography which gave pure 32 as a

white solid (0.593 g, 86%). Mp 81–83 °C; ¹H NMR (300 MHz, CDCl₃)

d 0.87 (9H, s), 3.77 (2H, s), 4.36 (2H, s), 4.55 (2H, s), 7.32 (1H, d,

J = 7.9 Hz), 7.48 (1H, d, J = 7.9 Hz), 7.61 (1H, s); ¹³C NMR (75 MHz,

CDCl₃) d 26.0, 31.3, 31.7, 55.3, 79.6, 125.5, 128.2, 128.5, 132.9,

133.6, 140.5; LREIMS m/z (relative intensity) 411 (M⁺, 4) 333

NMR (75 MHz, CDCl₃)

d 25.8, 32.1, 52.8, 85.0, 120.3 (t,

J = 287.4 Hz), 121.08 (t, J = 2.5 Hz), 128.1, 130.9 (t, J = 13.9 Hz),

131.32 (t, J = 21.0 Hz), 134.7, 136.6, 164.4; LR⁺ESIMS m/z (relative

intensity) 415 ([M+1]⁺, 100); HR⁺ESIMS m/z calcd for

C

₁₄H₁₈BrF₂O₅S ([M+1]⁺) 415.0026, found 415.0034.

4.1.20. (2-Bromo-4-hydroxymethyl-phenyl)diﬂuoromethanesul-

fonic acid, 2,2-dimethyl-propyl ester (29)

(20), 262 (100); HREIMS m/z calcd for C

₁₃H₁₈Br₂O₃S (M⁺)

411.9343, found 411.9337.

A suspension of compound 28 (1.00 g, 2.41 mmol) and NaBH₄

(6.0 equiv, 14.5 mmol) in dry THF (8 mL) was gently reﬂuxed under

Ar for 15 min. MeOH (8 mL) was added dropwise over 20 min and

the mixture was stirred under gentle reﬂux for a further 2 h. The

reaction was quenched with satd aq NH₄Cl solution, water was

then added and the mixture was extracted with diethyl ether.

The combined organics were dried (Na₂SO₄) and then concen-

trated. The resulting residue was subjected to ﬂash chromatogra-

phy which gave pure 29 as light pinkish oil (0.92 g, 99%). ¹H

NMR (300 MHz, CDCl₃) d 0.95 (9H, s), 4.12 (2H, s), 4.55 (2H, s),

7.29 (1H, d, J = 8.1 Hz), 7.58 (1H, d, J = 8.1 Hz), 7.63 (1H, s); ¹⁹F

NMR (285 MHz, CDCl₃) d ꢀ94.7; ¹³C NMR (75 MHz, CDCl₃) d 25.8,

32.1, 63.0, 84.8, 120.7 (t, J = 286.8 Hz), 120.9 (t, J = 2.7 Hz), 125.3,

125.9 (t, J = 21.1 Hz), 130.7 (t, J = 7.8 Hz), 133.4, 147.4; LRCIMS

m/z (relative intensity) 404 (M+NH₄)⁺, 100).

4.1.24. 2-Bromo-5-nitrobenzenesulfonyl chloride (33)

To a suspension of 2-bromobenzenesulfonyl chloride (5.00 g,

19.6 mmol) in conc. H₂SO₄(80 mL) was added a solution of conc.

H₂SO₄(3.6 mL)/69% HNO₃(3.6 mL, 39.2 mmol, 2 equiv) dropwise

over a period of 30 min. The mixture becomes a clear yellow solu-

tion. The mixture was stirred for 4 h, then added slowly to ice

water (800 mL) with vigorous stirring. After the ice melted the sus-

pension was extracted with CHCl₃. The combined organics were

dried (MgSO₄) and concentrated to give 33 as a crude pale yellow

solid. ¹H NMR of the crude solid revealed that it consisted of 95% of

the desired product 33 and 5% of the isomer resulting from nitra-

tion at the 3-position. Re-crystallization from hexane gave pure

33 (5.40 g, 92%). Mp 91–92 °C (lit.¹⁷mp 92 °C); ¹H NMR

(300 MHz, CDCl₃) d 8.09 (1H, d, J = 8.5 Hz), 8.37 (1H, d, J = 8.4 Hz),

9.00 (1H, s).

4.1.21. (2-Bromo-4-bromomethylphenyl)diﬂuoromethanesul-

fonic acid, 2,2-dimethylpropyl ester (30)

4.1.25. 2-Bromo-5-nitrobenzenesulfonamide (34)

To a solution of 29 (0.900 g, 2.30 mmol) in dry CH₂Cl₂(50 mL) at

0 °C were added carbon tetrabromide (0.85 g, 1.10 equiv,

2.56 mmol) and triphenylphosphine (0.610 g, 1.1 equiv,

2.56 mmol) and stirred for 2 h. The mixture was concentrated at

rt by rotary evaporation and the resulting residue subjected to

ﬂash chromatography which gave pure 30 as a colorless oil

(0.93 g, 89%). ¹H NMR (300 MHz, CDCl₃) d 0.99 (9H, s), 4.15 (2H,

s), 4.38 (2H, s), 7.43 (1H, d, J = 8.1 Hz), 7.65 (1H, d, J = 8.2 Hz),

To a solution of 33 (5.60 g, 18.8 mmol) in dry THF (80 mL) at

0 °C was added slowly a solution of NH₃in EtOH (2 M, 80 mL).

The mixture was allowed to warm to rt and then stirred for

20 min. Water was added and the mixture was extracted with

CH₂Cl₂. The combined organics were dried (Na₂SO₄) and concen-

trated and the resulting residue solid was washed with cold CH₂Cl₂

which gave compound 34 as a white solid (4.6 g, 87%). Mp 197–

199 °C (lit.¹⁷mp 204–205 °C); ¹H NMR (300 MHz, DMSO-d₆) d

6774

M. Hussain et al. / Bioorg. Med. Chem. 16 (2008) 6764–6777

7.95 (2H, br s), 8.11 (1H, d, J = 8.7 Hz), 8.27 (1H, dd, J = 8.6 Hz), 8.65

(1H, d, J = 2.5 Hz).

(300 MHz, acetone-d₆) d 2.33 (3H, s), 7.22 (1H, dd, J = 10.0 Hz),

7.33 (1H, d, J = 8.3 Hz), 7.55 (1H, dd, J = 1.4 Hz).

4.1.30. 1-Bromo-2-bromomethyl-4-iodobenzene (39)

4.1.26. 5-Amino-2-bromobenzenesulfonamide (35)

The following procedure is based on that of Kiang et al.¹⁸To a

solution of 38 (1.43 g, 4.80 mmol) in benzene (20 mL) were added

N-bromosuccinimide (0.974 g, 1.1 equiv, 5.32 mmol) and benzoyl-

peroxide (0.234 g, 0.20 equiv, 0.964 mmol). The mixture was

gently reﬂuxed under Ar by placing a 250W lamp close to the reac-

tion vessel. After 6 h, water was added and the mixture was ex-

tracted with CH₂Cl₂. The combined organics were washed with

satd aq NaHCO₃, then dried (Na₂SO₄), and concentrated. The result-

ing residue was subjected to ﬂash chromatography three times

(hexane) which gave pure 39 as a white solid (1.18 g, 65%). The

To a solution of 34 (0.140 g, 0.500 mmol) in THF/conc. HCl

(6.0 mL, 1:1) was added Zn powder (0.195 g, 6.0 equiv, 3.00 mmol)

portion-wise. The mixture was stirred at rt for 2.5 h and then neu-

tralized with satd aq NaHCO₃. The mixture was diluted with water

and extracted with EtOAc. The combined organics were concen-

trated and the residue subjected to ﬂash chromatography which

gave compound 35 as a white solid (0.120 g, 96%). Mp 148–

150 °C; ¹H NMR (300 MHz, acetone-d₆) d 5.22 (2H, br s), 6.47

(2H, br s), 6.73 (1H, dd, J = 8.5 Hz), 7.37 (1H, d, J = 8.6 Hz), 7.4

(1H, d, J = 2.7 Hz); ¹³C NMR (300 MHz, acetone-d₆) d 103.2, 114.9,

118;.2, 135.1, 142.6, 148.3; LREIMS m/z (relative intensity) 250

(M⁺, 100) 170 (17); HREIMS m/z calcd for C₆H₇BrN₂O₂S (M⁺),

249.9412, found 249.9409.

¹H NMR was consistent with that reported in the literature.^{18 1}

H

NMR (300 MHz, CDCl₃) d 4.48 (2H, s), 7.27 (1H, d, J = 8.4 Hz), 7.45

(1H, dd, J = 8.4 Hz), 7.75 (1H, d, J = 2.0 Hz).

4.1.31. Thioacetic acid S(2-bromo-5-iodobenzyl) ester (40)

Prepared using the same procedure as that described for com-

pound 10. The following quantities were used: 39 (6.56 g,

17.5 mmol), potassium thioacetate (2.19 g, 1.10 equiv, 0.40 mmol),

DMF (50 mL). Pure 40 (6.49 g, 99%) was obtained as a crystalline

white solid after aqueous workup. Mp 70–72 °C; ¹H NMR

(300 MHz, CDCl₃) d 2.30 (3H, s), 4.09 (2H, s), 7.18 (1H, d,

J = 8.4 Hz), 7.35 (1H, dd, J = 8.3 Hz), 7.71 (1H, d, J = 1.8 Hz), ¹³C

NMR (75 MHz, CDCl₃) d 30.4, 33.5, 92.7, 124.4, 134.4, 137.9,

139.5, 139.7, 194.3; LREIMS m/z (relative intensity) 370 (M⁺, 3),

291 (100); HREIMS m/z calcd for C₉H₈BrIOS (M⁺) 369.8524, found

369.8527.

4.1.27. 2-Bromo-5-iodobenzenesulfonamide (36)

To a solution of compound 35 (0.250 g, 1.00 mmol) dissolved in

H₂O/conc. HCl (4 mL, 3:1) at 0 °C was added a solution of NaNO₂

(0.070 g, 1.02 equiv, 1.02 mmol) in H₂O (3 mL) and stirred for 5

min. To this was added a solution of KI (0.498 g, 3.0 equiv,

3.00 mmol) in H₂O (3 mL) and the mixture was heated at 90 °C

for 10 min. Water was added and the mixture was extracted with

EtOAc. The combined organics were washed with an aq solution of

Na₂S₂O₃then dried (Na₂SO₄) and concentrated. The resulting resi-

due was subjected to ﬂash chromatography (3:7 EtOAc/hexane)

which gave 36 as a white solid (0.283 g, 78%). Mp: 179–181 °C;

¹H NMR (300 MHz, acetone-d₆) d 6.86 (2H, br s), 7.60 (1H, dd,

J = 8.3 Hz), 7.85 (1H, d, J = 8.2 Hz), 8.35 (1H, s); ¹³C NMR (75 MHz,

acetone-d₆) d 91.7, 119.2, 136.9, 137.9, 142.1, 144.2; LREIMS m/z

(relative intensity) 361 (M⁺, 100) 280 (19), 154 (12); HREIMS m/z

calcd for C₆H₅BrINO₂S (M)⁺360.8269, found 360.8269.

4.1.32. (2-Bromo-5-iodophenyl)methanesulfonyl chloride (41)

Chlorine gas was bubbled through a solution of 40 (0.370 g,

1.00 mmol) in acetic acid/water (16 mL, 3:1) at 0 °C for 1 h. The

mixture was purged with N₂and air to remove excessive Cl₂, water

was added, and the mixture was extracted with CH₂Cl₂. The com-

bined organics were dried (Na₂SO₄) and concentrated. The result-

ing sulfonyl chloride was unstable and so the crude product was

used immediately for the next step.

4.1.28. 4-Bromo-4⁰-methylbiphenyl-3-sulfonic acid amide (37)

To a solution of 36 (0.181 g, 0.5 mmol), tetrakis(triphenylphos-

phine)palladium(0) (0.058 g, 8 mol%) and 4-tolylboronic acid

(0.15 g, 2.2 equiv, 1.1 mmol) in dry THF (20 mL) was added a 2M

solution of K₂CO₃(2 mL) and the mixture was reﬂuxed under Ar

for 16 h. The reaction was diluted with water and extracted with

EtOAc. The combined organics were dried (Na₂SO₄) and concen-

trated and the resulting residue subjected to ﬂash chromatography

(2:8 EtOAc/hexane) which gave 37 as a white solid (0.105 g, 64%).

Mp 155–157 °C; ¹H NMR (300 MHz, acetone-d₆) d 2.38 (3H, s), 5.27

(2H, br s), 7.24 (2H, d, J = 7.7 Hz), 7.44 (2H, d, J = 7.7 Hz), 7.55 (1H,

d, J = 8.2 Hz), 7.72 (1H, d, J = 8.2 Hz), 8.30 (1H, s); ¹³C NMR

(75 MHz, acetone-d₆) d 20.2, 117.6, 126.6, 127.4, 129.8, 131.0,

135.5, 135.6, 138.3, 140.6, 143.1; LREIMS m/z (relative intensity)

325 (M⁺, 100), 245 (71), 165 (30); HRMS m/z calcd for

4.1.33. N,N-Diallyl-(2-bromo-5-iodo-phenyl)methane

sulfonamide (42)

To a solution of crude 41 in CH₂Cl₂(15 mL) was added diallyl-

amine (0.31 mL, 2.5 equiv, 2.5 mmol) and the mixture was stirred

for 1 h. Water was added and the mixture was extracted with

CH₂Cl₂and the combined organics were dried (Na₂SO₄) and con-

centrated. The resulting residue was subjected to ﬂash chromatog-

raphy (0.5:9.5 EtOAc/hexane) which gave pure 42 as a white solid

(0.383 g, 84% over two steps). Mp 60–62 °C; ¹H NMR (300 MHz,

CDCl₃)

d 3.74 (4H, d, J = 6.4 Hz), 4.33 (2H, s), 5.17 (4H, d,

J = 11.7 Hz), 5.65 (2H, m), 7.27 (1H, d, J = 8.4 Hz), 7.46 (1H, d,

J = 8.4 Hz), 7.82 (1H, s). ¹³C NMR (75 MHz, CDCl₃) d 49.7, 57.3,

92.4, 119.5, 125.5, 131.5, 132.7, 134.6, 139.1, 141.1; LREIMS m/z

(relative intensity) 455 (M⁺, 3) 399 (12), 331 (100); HREIMS m/z

calcd for C₁₃H₁₅BrINO₂S (M⁺) 454.9052, found 454.9055.

C

₁₃H₁₂BrNO₂S (M⁺) 324.9772, found 324.9775.

4.1.29. 1-Bromo-4-iodo-2-methylbenzene (38)

The following procedure is based on that of Kiang et al.¹⁸To a

solution of 4-bromo-3-methylaniline (2.06 g, 11.0 mmol) in H₂O/

conc.HCl (60 mL, 3:1) at 0 °C was added a solution of NaNO₂

(0.774 g, 1.02 equiv, 11.2 mmol) in H₂O (8 mL) and stirred for 5

min. A solution of KI (5.48 g, 3.00 equiv, 33.0 mmol) in H₂O

(8 mL) was added and the reaction was heated to 100 °C for

10 min. After cooling to rt, water was added and the mixture was

extracted with EtOAc. The combined organics were washed with

an aq solution of Na₂S₂O₃, then dried (Na₂SO₄), and concentrated.

The resulting residue was subjected to ﬂash chromatography

(100% hexane) to give 38 as white solid (2.63 g, 81%). ¹H NMR

was consistent with that reported in the literature.¹⁸¹H NMR

4.1.34. N,N-Diallyl-C-(2-bromo-5-iodophenyl)-C,C-diﬂuorometha-

nesulfonamide (43)

To a solution of 42 (1.46 g, 3.21 mmol) and N-ﬂuorobenzene-

sulfonimide (2.09 g, 2.10 equiv, 6.64 mmol) in dry THF (50 mL) at

ꢀ78 °C was added NaHMDS (1.0 M in THF, 7.54 mL, 2.40 equiv,

7.54 mmol) over 10 min. The mixture was stirred at ꢀ78 °C for

2 h, then allowed to warm to rt, and then stirred for 16 h. The reac-

tion was quenched with satd aq NH₄Cl and then extracted with

CH₂Cl₂. The combined organics were dried (Na₂SO₄), then concen-

trated, and the resulting residue was subjected to ﬂash chromatog-

M. Hussain et al. / Bioorg. Med. Chem. 16 (2008) 6764–6777

6775

raphy (0.5:9.5 EtOAc/hexane) which gave pure 43 as a white solid

(1.44 g, 94%). Mp 89–91 °C; ¹H NMR (300 MHz, CDCl₃) d 3.98 (4H,

d, J = 6.5 Hz), 5.22–5.29 (4H, m), 5.72–5.86 (2H, m), 7.37 (1H, d,

J = 8.4 Hz), 7.61 (1H, d, J = 8.4 Hz), 7.92 (1H, d, J = 1.9 Hz); ¹⁹F

NMR (285 MHz, CDCl₃) d ꢀ97.0; ¹³C NMR (75 MHz, CDCl₃) d 50.1,

91.8, 120.2, 120.6 (t, J = 285 Hz), 120.8, 130.21 (t, J = 21.7 Hz),

132.2, 137.1, 139.1 (t, J = 8.2 Hz), 141.8; LREIMS m/z (relative

intensity) 491 (M⁺, 3), 331 (100), 204 (10); HREIMS m/z calcd for

br s), 4.13–4.24 (4H, m), 6.80 (2H), 7.40–7.45 (3H), 7.57–7.63

(4H, m), 7.74 (1H, dd, J = 8.2 Hz), 7.85 (1H, d, J = 8.2 Hz), 8.30 (1H,

d, J = 1.7 Hz); ³¹P NMR (121 MHz, acetone-d₆)

d 6.2 (t, J =

110.9 Hz); ¹⁹F NMR (282 MHz, acetone-d₆) d ꢀ104.0 (d, J =

ꢀ111.6 Hz); ¹³C NMR (75 MHz, acetone-d₆) d 16.7 (d, J = 5.2),

35.2, 36.1, 65.5 (d, J = 6.8 Hz), 118.8, 119.1 (dt, J = 265.1,

218.4 Hz), 120.3, 127.7, 128.4, 128.8, 130.7, 130.8 (t, J = 7.3 Hz),

132.0, 136.4, 136.5, 137.9, 139.5, 141.1, 144.0, 144.8; LR⁺ESIMS

m/z (relative intensity) 712 ([M+1]⁺, 100); HR⁺ESIMS m/z calcd

for C₂₅H₂₆Br₂F₂NO₅PS₂([M+1]⁺) 711.9403, found 711.9418.

C

₁₃H₁₃BrF₂INO₂S (M⁺) 490.88631 found 490.8871.

4.1.35. N,N-Diallyl(4-bromo-4⁰-methyl-biphenyl-3-yl)diﬂuoro-

methanesulfonamide (44)

4.1.38. [2-Bromo-4-(4⁰-bromo-3⁰-sulfamoylbiphenyl-4-ylme-

thylsulfanylmethyl)phenyl]-diﬂuoromethanesulfonic acid, 2,

2-dimethylpropyl ester (47)

To a solution of 43 (0.461 g, 0.940 mmol), tetrakis(triphenyl-

phosphine)palladium(0) (0.231 g, 20 mol%) and 4-tolylboronic acid

(0.150 g, 1.10 equiv, 1.02 mmol) in dry THF (25 mL) was added and

a 2 M aq K₂CO₃solution (2.0 mL). The mixture was reﬂuxed under

Ar for 16 h. After cooling to rt, water was added and the mixture

was extracted with CH₂Cl₂. The combined organics were dried

(Na₂SO₄) and concentrated and the resulting residue subjected to

ﬂash chromatography (1:4 EtOAc/hexane) which gave pure 44 as

a white solid (0.313 g, 73%). Mp 80–82 °C; ¹H NMR (300 MHz,

CDCl₃) d 2.39 (3H, s), 4.05 (4H, d, J = 7.5 Hz), 5.25–5.32 (4H, m),

5.86 (2H, m), 7.25 (2H, d, J = 8.0 Hz), 7.47 (2H, d, J = 8.1 Hz) 7.52

(1H, d, J = 8.4 Hz), 7.73 (1H, d, J = 8.3 Hz), 7.89 (1H, d, J = 2.1 Hz);

¹⁹F NMR (285 MHz, CDCl₃) d ꢀ96.3; ¹³C NMR (75 MHz, CDCl₃) d

21.2, 50.2, 119.3, 120.0, 121.8 (t, J = 284 Hz), 126.9, 128.6 (t,

J = 9.1 Hz), 129.2 (t, J = 8.0 Hz), 129.8, 131.1, 132.5, 135.8, 136.0,

138.2, 140.5; LREIMS m/z (relative intensity) 455 (M⁺, 9), 295

(100); HREIMS m/z calcd for C₂₀H₂₀BrF₂NO₂S (M⁺) 455.0366, found

455.0357.

Prepared using the same procedure as that described for 46. The

following quantities were used: compound 11 (0.033 g, 1.0 equiv,

0.074 mmol), compound 13 (0.030 g, 1.0 equiv, 0.074 mmol), EtOH

(5 mL), and 2 N NaOH (2.0 mL, 54 equiv, 4.0 mmol). Flash chroma-

tography (3:7 EtOAc/hexane) of the crude residue gave pure 47 as a

semisolid (0.054 g, 78%). ¹H NMR (300 MHz, acetone-d₆) d 0.98

(9H, s), 3.77 (2H, s), 3.79 (2H, s), 4.20 (s, 2H), 6.8 (2H, s), 7.42

(2H, d, J = 8.1 Hz), 7.55 (1H, d, J = 8.1 Hz), 7.63 (2H, d, J = 8.1 Hz),

7.69–7.77 (3H), 7.86 (1H, d, J = 8.3 Hz), 8.33 (1H, d, J = 2.1 Hz);

¹⁹F NMR (285 MHz, acetone-d₆) d ꢀ95.3; ¹³C NMR (75 MHz, ace-

tone-d₆) d 25.1, 31.8, 34.3, 35.3, 84.6, 118.0, 120.3 (t, J = 2.6 Hz),

120.7 (t, J = 285.3 Hz), 125.5 (t, J = 21.2 Hz), 126.9, 127.6, 128.4,

129.9, 130.8 (t, J = 7.9 Hz), 131.2, 135.6, 136.1, 137.2, 138.6,

140.3, 143.2, 146.1; LR⁺ESIMS m/z (relative intensity) 742

([M+NH₄]⁺, 100); HR⁺ESIMS m/z calcd for C₂₆H₃₁Br₂F₂N₂O₅S₃

([M+NH₄]⁺) 742.9730, found 742.9739.

4.1.36. (4-Bromo-4⁰-methylbiphenyl-3-yl)diﬂuoromethanesul-

fonamide (45)

4.1.39. [2-Bromo-4-(4⁰-bromo-3⁰-sulfamoylbiphenyl-4-ylme-

thylsulfanylmethyl)phenyl]-methanesulfonic acid, 2,2-dime-

thylpropyl ester (48)

A solution of 44 (0.320 g, 0.70 mmol), tetrakis(triphenylphos-

phine)palladium(0) (0.161 g, 20 mol%), and 1,3-dimethylbarbituric

acid (2.18 g, 20.0 equiv, 14.0 mmol) in dry CH₃CN (30 mL) was re-

ﬂuxed vigorously for 18 h. Water was added and the mixture was

extracted with EtOAc and the combined organics concentrated. The

residue was dissolved in THF/6N HCl (120 mL, 3:1), stirred for 1h,

diluted with H₂O, and extracted with EtOAc. The combined organ-

ics were dried (Na₂SO₄), then concentrated, and the resulting resi-

due subjected to ﬂash silica chromatography (3:7 EtOAc/hexane)

which gave pure 45 as a white solid (0.225 g, 86%). Mp 149–

151 °C; ¹H NMR (300 MHz, a acetone-d₆) d 2.38 (3H, s), 5.08 (2H,

br, s), 7.24 (2H, d, J = 6.4 Hz), 7.44 (2H, d, J = 7.8 Hz) 7.55 (1H, d,

J = 8.4 Hz), 7.74 (1H, d, J = 8.3 Hz), 7.87 (1H, s,); ¹⁹F NMR

(285 MHz, acetone-d₆) d ꢀ98.6; ¹³C NMR (75 MHz, acetone-d₆) d

20.2, 119.0 (t, J = 2.5 Hz), 120.3 (t, J = 283.3 Hz), 126.7, 128.4 (t,

J = 16.4 Hz), 129.0 (t, J = 7.8 Hz), 129.8, 131.1, 135.6, 136.1, 138.3,

140.3; LREIMS m/z (relative intensity) 375 (M⁺, 20), 295 (100);

HREIMS m/z calcd for C₁₄H₁₂BrF₂NO₂S (M⁺) 374.9740, found

374.9737.

Prepared using the same procedure as that used for compound

46. The following quantities were used: compound13 (0.101 g, 1.0

equiv, 0.250 mmol), 12 (0.102 g, 1.0 equiv, 0.250 mmol), EtOH (5

mL), and 2 N NaOH (2.0 mL, 16 equiv, 4.0 mmol). The residue

was subjected to ﬂash chromatography (3:7 EtOAc/hexane) which

gave pure 48 as semisolid (0.130 g, 76%). ¹H NMR (300 MHz, ace-

tone-d₆) d 0.92 (9H, s), 3.72 (2H, s), 3.74 (2H, s), 3.88 (s, 2H), 4.72

(2H), 6.81 (2H, s), 7.36–7.43 (3H, m), 7.54–7.64 (4H, m), 7.74

(1H, d, J = 8.3 Hz), 7.85 (1H, d, J = 8.3 Hz), 8.32 (1H, s); ¹³C NMR

(75 MHz, acetone-d₆) d 25.4, 31.4, 34.5, 35.2, 54.7, 79.3, 118.0,

125.1, 126.9, 127.3, 127.5, 128.4, 129.8, 131.2, 132.9, 133.3,

135.6, 137.0, 138.8, 140.3, 141.9, 143.1; LR⁺ESIMS m/z (relative

intensity) 707 ([M + NH₄]⁺, 100); HR⁺ESIMS m/z calcd for

C

₂₆H₃₃Br₂N₂O₅S₃([M+NH₄]⁺) m/z 706.9918, found 706.9924.

4.1.40. ([2-Bromo-4-[4⁰-bromo-3⁰-(diﬂuorosulfamoylmethyl)

biphenyl-4-ylmethylsulfanyl-methyl)phenyl]diﬂuoromethyl)

phosphonic acid diethyl ester (49)

Prepared from compounds 10 and 14 in 72% yield using the

same procedure as that described for 46. The following quantities

were used: 14 (0.030 g, 1.0 equiv, 0.066 mmol), 10 (0.029 g, 1.0

equiv, 0.066 mmol), EtOH (5 mL), and 2 N NaOH (1.0 mL, 30 equiv,

2.0 mmol). Flash chromatography (2:3 EtOAc/hexane) of the crude

residue gave pure 49 as a semisolid. ¹H NMR (300 MHz, acetone-

d₆) d 1.28 (6H, t, J = 6.3 Hz), 3.76 (4H, br s), 4.13–4.24 (4H, m),

7.41–7.47 (5H, NH₂peak merged), 7.57–7.65 (4H, m), 7.76 (1H, d,

J = 8.2 Hz), 7.85 (1H, d, J = 8.2 Hz), 7.94 (1H, d, J = 1.7 Hz); ³¹P

NMR (121 MHz, acetone-d₆) d 5.1 (t, J = 110.9 Hz); ¹⁹F NMR (282

MHz, acetone-d₆) d ꢀ98.6, ꢀ104.0 (d, J = 111.8 Hz); ¹³C NMR

(125.75 MHz, acetone-d₆) d 14.3, 35.2, 36.1, 65.5 (d, J = 6.7 Hz),

119.1 (dt, J = 265.0, 218.2 Hz), 120.2, 120.4 (t, J = 3.6 Hz), 121.1 (t,

J = 283.3 Hz), 127.8, 128.8, 129.8 (t, J = 22.7 Hz), 130.0 (t,

4.1.37. {[2-Bromo-4-(4⁰-bromo-3⁰-sulfamoylbiphenyl-4-ylmethy

lsulfanylmethyl)phenyl]diﬂuoro-methyl}phosphonic acid diethyl

ester (46)

To a suspension of 10 (0. 100 g, 1 equiv, 0.233 mmol) and 13

(0.094 g, 1 equiv, 0.233 mmol) in EtOH (5 mL) was added CHCl₃

(3 mL) to make the solution clear. The solution was cooled to

0 °C (ice bath) and an aqueous solution of NaOH (2.0 mL, 2 N, 17

equiv, 4.0 mmol) was added and the mixture stirred for 30 min.

The mixture was diluted with water and extracted with EtOAc.

The combined organics were dried (Na₂SO₄) and concentrated.

The resulting residue was subjected to ﬂash chromatography (3:7

EtOAc/hexane) which gave pure 46 as a semisolid (0.158 g, 96%).

¹H NMR (300 MHz, acetone-d₆) d 1.27 (6H, t, J = 7.0 Hz), 3.75 (4H,

6776

M. Hussain et al. / Bioorg. Med. Chem. 16 (2008) 6764–6777

J = 7.6 Hz), 130.7, 130.9 (t, J = 7.9 Hz), 131.1 (dt, J = 21.5, 15.0 Hz),

132.1, 136.4, 137.0, 138.0, 139.6, 140.8, 144.9. LR⁺ESIMS m/z (rela-

tive intensity) 761 ([M+1]⁺, 100); HR⁺ESIMS m/z calcd for

HR^ꢀESMS m/z calculated for C₃₂H₄₇N₇O₁₅S (MꢀH)^ꢀ800.2773,

found 800.2764.

C

₂₆H₂₇Br₂F₄NO₅PS₂(M+1)⁺m/z 761.9388 found 761.9371.

4.2. Inhibition studies

4.1.41. [2-Bromo-4-[4⁰-bromo-3⁰-(diﬂuorosulfamoylmethyl)

biphenyl-4-ylmethylsulfanylmethyl]-phenyl}diﬂuoromethane-

sulfonic acid 2,2-dimethyl-propyl ester (50)

4.2.1. IC₅₀and K_ideterminations

Stock solutions of the inhibitors were prepared in 40% DMSO/

60% buffer containing 50 mM Bis–Tris HCl, pH 6.3. 10

inhibitor stock solution was added to the wells of a 96-well

microtiter plate containing 90 L of 5.5 M diFMUP in 50 mM

Bis–Tris HCl, pH 6.3, 5 mM DTT, and 2 mM EDTA. The reactions

were initiated at 25 °C with 10

L of a 30 nM solution of PTP1B²⁷

lL of each

Prepared using the same procedure as that described for 46. The

following quantities were used: 11 (0.038 g, 1.0 equiv,

0.085 mmol), 14 (0.039 g, 1.0 equiv, 0.085 mmol), EtOH (10 mL),

2 N NaOH (1.0 mL, 23.5 equiv, 2.0 mmol). Flash chromatography

(3:7 EtOAc/hexane) of the crude residue gave pure 50 as a semi-

solid (0.055 g, 83%). ¹H NMR (300 MHz, CDCl₃) d 1.00 (9H, s),

3.58 (2H, s), 3.64 (2H, s), 4.16 (2H, s), 5.16 (2H, s), 7.33 (3H, s),

7.47–7.62 (5H, m), 7.76 (1H, d, J = 8.2 Hz), 7.89 (1H, s); ¹⁹F NMR

(285 MHz, CDCl₃) d ꢀ96.9, ꢀ94.7; ¹³C NMR (75 MHz, CDCl₃) d

25.9, 32.2, 34.6, 35.6, 84.8, 119.7, 120.1 (t, J = 284.4 Hz), 120.7 (t,

J = 286.3 Hz), 120.96, 125.9 (t, J = 21.1 Hz), 127.2, 127.7 (t,

J = 21.3 Hz), 127.9, 129.5 (t, J = 7.8 Hz), 129.7, 130.8 (t, J = 7.8 Hz),

131.6, 136.1, 136.2, 137.5, 137.8, 140.2, 144.7; LR⁺ESIMS m/z (rel-

ative intensity) 704, ([M-C₅H₁₁]⁺, 100); HR⁺ESIMS m/z calcd for

C₂₂H₁₆Br₂F₄NO₅S₃([MꢀC₅H₁₁]⁺), 703.8493, found 703.8507.

l

in a buffer containing 50 mM Bis–Tris HCl, pH 6.3, 20% glycerol,

5 mM DTT, 2 mM EDTA, and 0.1% Triton X-100. The production

of ﬂuorescent product diFMU was monitored for 10 min using

a spectroﬂuorimeter platereader with excitation and emission

at 360 nM and 460 nM, respectively. The initial rates of enzyme

activity in relative ﬂuorescence units per second (RFU/s) were

used to determine the IC₅₀’s and K_i’s. For IC₅₀’s, the ratio of the

initial rate in the presence of inhibitor (V_i) to that in the absence

of inhibitor (V_o) was calculated and plotted as a semi-log curve

in Graﬁt, from which the IC₅₀value was calculated based on the

following equation: V_i= V_o/[1 + ([I]/IC₅₀)^S] + B, where V_iis the

initial rate of reaction at an inhibitor concentration of [I]; V_ois

the velocity in the absence of inhibitor; B is background and s

is the slope factor equal to V_oꢀ B. For K_i’s, data were plotted

as Lineweaver–Burk graphs and K_ivalues were calculated from

replots of the slopes or intercepts of the Lineweaver–Burk

graphs according to the equations for mixed or competitive

inhibition.

4.1.42. Synthesis of peptide 51

Fmoc-L-Phe(p-CH₂SO₃Na)OH was prepared according to the

procedure of Miranda et al.²²Solid-phase peptide synthesis was

carried out manually using Fmoc strategy using the Rink amide

AMS resin (0.71 mmol/g). The resin was preswollen in CH₂Cl₂for

2 h. Coupling of Fmoc-

orophenyl ester of Fmoc-

L

-LeuOH was carried out with the pentaﬂu-

-leucine (4.0 equiv), HOBt (4.0 equiv) in

L

Acknowledgments

DMF (stirred 10 min at room temperature before being added to

the resin) and then shaken with resin (2ꢃ 60 min). The resin

was then rinsed with DMF (6ꢃ 3 min) and CH₂Cl₂(2ꢃ 3 min).

All remaining coupling reactions were carried out as follows: A

solution of Fmoc-amino acid (4.0 equiv), HATU or HBTU (4.0

equiv), HABt or HOBt (4.0 equiv), DIEA (4.0 equiv), and DMF

(3 mL) was stirred at room temperature for 10 min. This solution

was then added to the resin and shaken for 60 min. Each coupling

was carried out twice. The resin was washed after each coupling

with DMF (3 mL, 6ꢃ 3 min) and CH₂Cl₂(3 mL, 2ꢃ 3 min). The

Fmoc group was removed using 20% piperidine/DMF (2ꢃ 15

min), and then the resin was washed with DMF (3 mL, 4ꢃ 3

min) 1:1 DMF:CH₂Cl₂(3 mL, 1ꢃ 3 min) and CH₂Cl₂(3 mL, 1ꢃ 3

min). The side chains of both the Asp and Glu were protected as

t-butyl esters. Once the peptide was assembled the ﬁnal Fmoc

group was removed and the resin dried overnight. The resin was

placed in a small round-bottomed ﬂask and Reagent K (82.5%

TFA, 2.5% ethanedithiol, 5% water, 5% thioanisole, and 5% phenol)

was added and stirred for 2.5 hours at room temperature. The mix-

ture was ﬁltered and the residue evaporated. To this mixture was

added cold methyl t-butylether (0 °C) and the suspension trans-

ferred to a 35 mL centrifuge tube. The mixture was centrifuged at

3500 rpm at 0 °C for 10 min and then the solvent was decanted

off. This process was repeated two more times affording a white

crude peptide. The peptide was dried overnight in the fumehood,

then placed in water and cooled, and lyophilized which gave crude

peptide 51 as a ﬂuffy white powder. Pure peptide 51 was obtained

by semi-preparative RP-HPLC using a C-18 column (250 ꢃ 21.2

mm, 10 micron) and was eluted using a linear gradient of 95%

H₂O containing 0.1%TFA/5% CH₃CN to 60% H₂O containing

0.1%TFA/40% CH₃CN over 70 minutes at a ﬂow rate of 8 mL/min.

The retention time was 32 min and the peptide was detected using

a detector set at 220 nM. Analytical RP-HPLC (C-18 column,

This work was supported by a Discovery Grant from the Natural

Sciences and Engineering Research Council of Canada (NSERC).

M.H. thanks the Higher Education Commission of Pakistan for

ﬁnancial support.

Supplementary data

Supplementary data (Lineweaver–Burk plots for compound 7

and 8, IC₅₀plot for peptides 3 and 51 and data from reversibility

experiments) associated with this article can be found in the online

version at doi:10.1016/j.bmc.2008.05.062.

References and notes

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between the IC₅₀’s could be made. The IC₅₀for compound

2 under our

conditions (6 nM) is similar to that reported by MerckFrosst for this compound

(8 nM) in the absence of DMSO which indicates that the presence of 4% DMSO

in the assay mixture does not signiﬁcantly affect ligand binding. Moreover, the

Article Doi

A re-examination of the difluoromethylenesulfonic acid group as a phosphotyrosine mimic for PTP1B inhibition

DOI: 10.1016/j.bmc.2008.05.062

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