Potent benzimidazole sulfonamide protein tyrosine phosphatase 1B inhibitors containing the heterocyclic (S)-isothiazolidinone phosphotyrosine mimetic

Article abstract of DOI:10.1021/jm0600904

Potent nonpeptidic benzimidazole sulfonamide inhibitors of protein tyrosine phosphatase 1B (PTP1B) were derived from the optimization of a tripeptide containing the novel (S)-isothiazolidinone ((S)-IZD) phosphotyrosine (pTyr) mimetic. An X-ray cocrystal s

Full text of DOI:10.1021/jm0600904

3774
J. Med. Chem. 2006, 49, 3774-3789
Articles
Potent Benzimidazole Sulfonamide Protein Tyrosine Phosphatase 1B Inhibitors Containing the
Heterocyclic (S)-Isothiazolidinone Phosphotyrosine Mimetic
Andrew P. Combs,*,† Wenyu Zhu,† Matthew L. Crawley,† Brian Glass,† Padmaja Polam,† Richard B. Sparks,† Dilip Modi,†
Amy Takvorian,† Erin McLaughlin,† Eddy W. Yue,† Zelda Wasserman,† Michael Bower,† Min Wei,‡ Mark Rupar,‡ Paul J. Ala,‡
Brian M. Reid,‡ Dawn Ellis,‡ Lucie Gonneville,‡ Thomas Emm,§ Nancy Taylor,§ Swamy Yeleswaram,§ Yanlong Li,‡
Richard Wynn,‡ Timothy C. Burn,‡ Gregory Hollis,‡ Phillip C. C. Liu,‡ and Brian Metcalf†
Incyte Corporation, DiscoVery Chemistry, Applied Technology, and Drug Metabolism, Experimental Station, Route 141 and Henry Clay Road,
Wilmington, Delaware 19880
ReceiVed January 26, 2006
Potent nonpeptidic benzimidazole sulfonamide inhibitors of protein tyrosine phosphatase 1B (PTP1B) were
derived from the optimization of a tripeptide containing the novel (S)-isothiazolidinone ((S)-IZD)
phosphotyrosine (pTyr) mimetic. An X-ray cocrystal structure of inhibitor 46/PTP1B at 1.8 Å resolution
demonstrated that the benzimidazole sulfonamides form a bidentate H bond to Asp48 as designed, although
the aryl group of the sulfonamide unexpectedly interacts intramolecularly in a pi-stacking manner with the
benzimidazole. The ortho substitution to the (S)-IZD on the aryl ring afforded low nanomolar enzyme
inhibitors of PTP1B that also displayed low caco-2 permeability and cellular activity in an insulin receptor
(IR) phosphorylation assay and an Akt phosphorylation assay. The design, synthesis, and SAR of this novel
series of benzimidazole sulfonamide containing (S)-IZD inhibitors of PTP1B are presented herein.
Introduction
described elsewhere.6 In summary, we determined that the (S)-
IZD heterocycle is the most potent pTyr mimetic known to date,
being ∼10-fold more potent than the difluoromethylphosphonate
(DFMP) when incorporated into dipeptide inhibitors and 5-fold
more potent than the analogous thiadiazolidinone (TDZ) het-
erocyclic pTyr mimetic. However, despite the significant
improvement in the enzyme potency of these peptidic (S)-IZD
inhibitors, they also lacked cellular activity because of poor
permeability.
Protein tyrosine phosphatase 1B (PTP1Ba), the prototypical
protein tyrosine phosphatase (PTP) from a family of ∼112
intracellular PTPs, has been strongly implicated by in vitro and
in vivo experiments as a negative regulator of both insulin and
leptin signal transduction pathways.1 The observation that
PTP1B knockout mice exhibit increased insulin sensitivity and
are highly resistant to weight gain upon high fat feeding strongly
suggests that PTP1B is a viable drug target for the treatment of
diabetes and obesity.2,3 Thus, a potent cell permeable inhibitor
of PTP1B has been sought by nearly all pharmaceutical
companies to test this hypothesis.4
Medicinal chemistry efforts to date have focused on the
identification of inhibitors that contain strong binding, nonhy-
drolysable tyrosine phosphonate (pTyr) mimetics to affect potent
binding to PTP1B. A variety of pTyr mimetics have been
discovered and incorporated into potent small molecule inhibi-
tors of PTP1B. Unfortunately, they lack cell permeability and
oral bioavailability because of the strong negative charge carried
by the pTyr mimetics as well as their high molecular weight
and/or peptidic nature.
We rationalized that the diffusely monoanionic (S)-IZD pTyr
mimetic was not solely responsible for the lack of permeability
and that the reduction in the peptidic nature of the inhibitors
might cause a net improvment in permeability. The C-terminal
primary amide was replaced with a benzimidazole with a 20-
fold improvement in potency. The N-terminal truncation of the
peptide followed by parallel synthesis and purification of
N-acylated libraries identified the sulfonamide as a potent
replacement for the two terminal peptides. The introduction of
ortho substituents on the aromatic ring to the (S)-IZD provided
relatively low molecular weight (MW ∼450-650) PTP1B
inhibitors with low nanomolar enzyme potency. The ortho-
methyl benzimidazole sulfonamide derivative 79a has excep-
tional enzyme potency and was shown to be cell active in an
IR phosphorylation assay as well as in an Akt phosphorylation
assay. Details of the synthesis and SAR are presented herein.
Chemistry. The synthesis of peptides and benzimidazole
sulfonamide derivatives incorporating the (S)-IZD pTyr mimetic
followed our route previously reported to derive peptide
derivative 1 starting with commercially available phenylalanine
boronic acid derivative 3 (Scheme 1).5 Acid 3 was coupled with
chloroisothiazolinone 4 in a Suzuki7 cross-coupling to generate
oxidized core 5 in moderate yield. The reduction-deprotection
of 5 using palladium on carbon under 50 psi of hydrogen gas
gave 6 in high yield. The coupling of acid 6 with benzenedi-
We recently disclosed the structure-based design of novel
isothiazolidinone (IZD) pTyr mimetics and their incorporation
into the peptidic inhibitors of PTP1B.5 A thorough evaluation
of the structure activity relationships of various pTyr hetero-
cyclic mimetics and the peptidic portion of these inhibitors is
* To whom correspondence should be addressed. Tel: 302-498-6832.
Fax: 302-425-2750. E-mail: acombs@incyte.com.
† Discovery Chemistry.
‡ Applied Technology.
§ Drug Metabolism.
a Abbreviations: PTP1B, protein tyrosine phosphatase 1B; IR, insulin
receptor; pTyr, phosphotyrosine; (S)-IZD, (S)-isothiazolidinone; TZD,
thiadiazolidinone; DFMP, difluoromethylphosphonate.
10.1021/jm0600904 CCC: $33.50 © 2006 American Chemical Society
Published on Web 06/08/2006

Protein Tyrosine Phosphatase 1B Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3775
Scheme 1. Synthesis of Benzimidazole-Containing (S)-IZD Analoguesa
ï
a (a) 16 mol % Pd(dppf)Cl2, 3 equiv Et3N, DCM, 90 C, 16 h, 68%; (b) 10 wt % Pd/C, EtOH, 93%; (c) 7, HATU, DIEA, rt, 16 h, 48%; For compds 9,
10, 12, 14, and 16, chiral separation of (R/S)-IZD diastereomers was performed; (d) AcOH, 40 ïC, 2 h; (e) TFA, 30 min, rt, (83% for 2 steps); (f) Boc-Phe,
HATU, DIEA, rt, 2 h; (g) TFA, 30 min, rt, (h) Ac2O, DIEA, DCM, 2 h, rt (80% for 2 steps); (i) MsOH, ACN, rt, 60% (j) R3Cl, Et3N, DCM, rt, 5 h; (k)
TFA, µW, 1 min (20-80% for 2 steps).
Scheme 2. Synthesis of ortho-Fluoro Substituted (S)-IZD-Containing Analoguesa
a (a) 10 mol % Pd(dppf)Cl2, 4, 3 equiv Et3N, toluene, 90 ïC, 16 h, 61%; (b) 10% Pd/C, EtOH, rt, 16 h, 82%; (c) NBS, benzoyl peroxide, CCl4, 80 ïC,
4 h, 67%; (d) AgNO3, EtOH, H2O, reflux, 1 h, 85%; (e) DBU, 61, CH2Cl2, 74%; (f) 0.5 mol % R,R-(-)-1,2-bis(o-methoxyphenyl)(phenyl)phosphinoethan(1,5-
cyclooctadiene rhodium; (I), EtOH, 50 psi H2, 12 h, 93%, 97% ee, 1:1 dr; (g) Chiralcel AD column, 47%, 97% ee, single diastereomer; (h) TFA, 130 ïC,
µW, 6 min, 74%; (i) Boc2O, Et3N, CH2Cl2, 97%; (j) LiOH, H2O, MeOH, 99%; (k) BOPCl, 7, iPr2NEt, DMF, rt, 1 h; (l) AcOH, 40 ïC, 1.5 h; (m) TFA/
CH2Cl2 ) 1:5; (n) Et3N, 66, CH2Cl2.
amine 7 followed by cyclization under acidic conditions at room
temperature for 24 h afforded the desired benzimidazole. Careful
control of the reaction temperature was critical because ring
closure at higher temperatures proceeded more rapidly but

3776 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
Scheme 3. Synthesis of ortho-Methyl Substituted Derivativesa
Combs et al.
a (a) LiBH4, THF, rt, 2 days, 82%; (b) DMSO, oxalyl chloride, CH2Cl2, -78 ïC, 96%; (c) DBU, 70, CH2Cl2, 85%; (d) 0.5 mol % R,R-(-)-1,2-bis(o-
methoxyphenyl)(phenyl)phosphinoethan(1,5-cyclooctadiene rhodium (I), EtOH, 50 psi H2, 12 h, 83%, 98% ee; (e) Pd(OAc)2, Et3N, 4,4,5,5-tetramethyl-
1,3,2-dioxaborolane, 91% (f) NaIO4, NH4OAc, THF, H2O, rt, 18 h, 80%; (g) 15 mol % Pd(dppf)Cl2, 4, 3 equiv Et3N, toluene, 80 ïC, 24 h, 62%; (h)
ï
ï
L-selectride, THF, -78 C, 88%; (i) TFA, 130 C, µW, 6 min, 74%; (j) BnOC(O)Cl, Et3N, CH2Cl2; (k) LiOH, H2O, MeOH; (l) BOPCl, iPr2NEt, 7, DMF,
rt, 1 h, 82%; (m) AcOH, 40 ïC, 1.5 h; (n) 2.2 equiv SEMCl, iPr2NEt, CH2Cl2; (o) Chiralcel AD column, 98% ee, single diastereomer; (p) 10 wt % Pd/C,
MeOH, H2; (q) Et3N, 66, CH2Cl2; (r) TFA, 130 ïC, µW, 6 min;
returned mixtures of diastereomers at the alpha center of the
amino acid. At this point in the synthesis, the (R/S)-IZD
diastereomers were easily separated by chiral HPLC to afford
two discrete isomers, (S)-IZD and diastereomer 8 is shown in
Scheme 1. Both diastereomers were further elaborated to the
final compounds, providing an active and inactive pair. The
active isomer was assigned to the (S)-IZD on the basis that all
X-ray cocrystal structures solved to date have the (S)-IZD isomer
bound, such as 46/PTP1B. All other analogues were derived
from diastereomer 8. The removal of the Boc group from 8
with TFA furnished the free amine, which could be acylated
with a variety of reagents under mild conditions to give amides,
ureas, sulfonamides, and carbamates such as 10-56.
The synthesis of each desired analogue ortho substituted on
the aryl ring to the (S)-IZD necessitated the optimization of a
unique sequence of chemical reactions. The ortho-fluoro and
ortho-methyl derivatives were synthesized via a similar Suzuki
cross-coupling reaction of chloroisothiazolinone 4 and a properly
substituted arylboronic acid. The construction of the ortho-
bromo and ortho-chloro derivatives relied on a chemoselective
and regioselective Heck coupling of an ortho substituted
arylidodide with isothiazolidinone 82. The separation of (R/S)-
IZD isomers was also necessary in these routes, and the (S)-
IZD isomer was again assigned by synthesis of the two (R/S)-
IZD diastereomers and the determination of the active compound.
The details of each sequence of reactions are described below.
The ortho-fluoro derivatives were synthesized via a similar
Suzuki cross-coupling approach, though accessing the key amino
acid-containing (S)-IZD intermediate 64 was more challenging.
Starting with commercially available fluoroboronic acid 57 and
chloro-isothiazolinone heterocycle 4, palladium catalyzed cross-
coupling employing dppf as the ligand gave a 61% yield of
core 58 (Scheme 2). The reduction of heterocyclic olefin
followed by the formation of the dibromo acetal equivalent 59
proceeded in 51% yield for two steps. Aldehyde 60 was
unmasked using silver nitrite in refluxing aqueous ethanol in
85% yield. A Horner-Wadsworth-Emmons type olefination
with phosphonate 61 afforded unsaturated amino acid 62. The
reduction of 62 utilizing R,R-(-)-1,2-bis(o-methoxyphenyl)-
(phenyl)phosphinoethan(1,5-cyclooctadiene) rhodium8 pro-
ceeded with >97% ee to give a 1:1 mixture of diastereomers at
the heterocycle, which were separated on a Chiral AD HPLC
column. Single isomer 63 was then globally deprotected by
microwave irradiation in TFA to afford the free amine. Classical
thermal conditions gave significantly lower yields in this
deprotection sequence for most derivatives. Reprotection of the
amine as the Boc carbamate and saponification of the ester
generated the key amino acid containing (S)-IZD intermediate

Protein Tyrosine Phosphatase 1B Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3777
Scheme 4. Synthesis of ortho-Chloro Substituted (S)-IZD-Containing Analoguesa
a (a) Zn0, NH4Cl, MeOH, H2O; (b) NCS, DMF; (c) NaNO2, 1 N aq HCl, KI, 51%) (3 steps); (d) Pd(OAc)2, Bu4NCl, 81, Et3N, DMF, 100 ïC, 64%; (e)
ï
ï
LiBH4, MeOH, 0 C, 72%; (f) Chiralcel AD column, 48%, 97% ee, single diastereomer; (g) TFA, 130 C, µW, 0.25 h, 88%; (h) Boc2O, Et3N, CH2Cl2; (i)
LiOH, H2O, MeOH, 81% (2 steps); (j) BOPCl, 7, iPr2NEt, DMF, rt, 1 h (k) AcOH, 40 ïC, 1.5 h, 72% (2 steps); (l) TFA/CH2Cl2 ) 1:5; (m) Et3N, 66,
CH2Cl2, 76% (2 steps).
Scheme 5. Synthesis of ortho-Bromo Substituted (S)-IZD-Containing Analoguesa
a (a) Zn0, NH4Cl, MeOH, H2O; (b) NBS, DMF; (c) NaNO2, 1 N aq. HCl, KI 64% (3 steps); (d) Pd(OAc)2, Bu4NCl, 82, Et3N, DMF, 100 ïC, 52%; (e)
LiBH4, MeOH, 0 ïC, 82%; (f) TFA, 130 ïC, µW, 0.25 h, 99%; (g) CbzCl, Et3N, CH2Cl2; (h) LiOH, H2O, MeOH, 81% (2 steps); (i) BOPCl, iPr2NEt, DMF,
rt, 1 h (j) AcOH, 40 ïC, 1.5 h, 72% (2 steps); (k)TMSI, CH3CN; (l) Et3N, 66, CH2Cl2, 68% (2 steps).
64. Peptide coupling with diamine 7 followed by in situ
cyclization in acetic acid afforded benzimidazole 65. The
N-terminal amine of the benzimidazole derivatives were depro-
tected with TFA and reacted with various arylsulfonyl chlorides
66 to furnish benzimidazole sulfonamide derivatives 67 (a-e).
Synthesis of the ortho-methyl scaffold also employed a
Suzuki cross-coupling to attach the IZD to the scaffold (Scheme
3). However, it was again necessary to reconstruct the overall
synthetic route compared to those of the unsubstituted and ortho-
fluoro syntheses. Commercial bromide 68 was converted in 80%
yield over two steps to aldehyde 69 via a reduction-oxidation
sequence. Olefination of the aldehyde with phosphonate ester
70 afforded unsaturated amino acid 71 in 85% yield. The
reduction of 71 utilizing R,R-(-)-1,2-bis(o-methoxyphenyl)-

3778 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
Combs et al.
Figure 2. Peptidomimetic approach to optimizing peptide-(S)-IZD
inhibitors.
Table 1. SAR of the N-terminal Truncation of Peptide Benzimidazoles
Figure 1. X-ray crystal structure of compound 1 bound to PTP1B.
The molecular surface of the protein is shown with the (S)-IZD
heterocycle binding in the phosphotyrosine binding pocket. The
bidentate hydrogen bond of the inhibitor’s peptide-like backbone with
Asp48, also shown, is indicated with dotted lines.
(phenyl)phosphinoethan(1,5-cyclooctadiene) rhodium proceeded
with >97% ee to give 72. Bromide 72 was converted to boronic
acid 73 via coupling to the borate ester followed by oxidative
cleavage. Boronic acid 73 was cross-coupled with chloro-
isothiazolinone heterocycle 4 employing the same conditions
utilized for the ortho-fluoro derivative to give IZD scaffold 74.
The reduction of the heterocycle with sodium borohydride and
subsequent global deprotection furnished amine salt 75 in 66%
yield. Reprotection of the amine as the Cbz carbamate followed
by saponification afforded acid 76 in quantitative yield. The
coupling of acid 76 with diamine 7 and then in situ cyclization
with acetic acid provide the desired benzimidazoles 77. At this
point, the 1:1 diastereomeric mixture at the heterocycle of 77
was separated utilizing a Chiralcel AD HPLC column. The
single diastereomer was then bis-SEM protected to supply
intermediate 78. The removal of the Cbz group using hydro-
genation followed by coupling with arylsulfonyl chlorides 66
and subsequent deprotection with TFA afforded final products
79.
The synthesis of the ortho halogenated (S)-IZD derivatives
(chloro and bromo) could not be affected by a Suzuki cross-
coupling because of chemoselectivity issues. A novel synthetic
strategy was devised to couple the IZD to ortho-halo iodophenyl
derivatives 81 and 88 through chemoselective and regioselective
Heck reactions (Schemes 4 and 5). The synthesis of ortho-chloro
derivative 87 is outlined below. The synthesis of ortho-bromo
derivative 93 was performed as shown in Scheme 5 with only
a slight modification in the protecting group strategy.
Key chloro-iodo-derivative 81 was readily obtained from
commercial nitro-phenylalanine 80. The nitro group of 80 was
reduced to an amino group using zinc and ammonium chloride
in methanol, followed by ortho chlorination to give an inter-
mediate 2-chloroaniline. The aniline was diazatized using
sodium nitrite under acidic conditions and the resulting inter-
mediate trapped with potassium iodide to afford 81 in 51% yield
over three steps. This intermediate was used in the key Heck9
reaction with heterocycle 82. The halogenated core 81 was
coupled with 82 in 64% yield with classical ligandless conditions
utilizing palladium acetate, tetra-n-butylammonium chloride, and
triethylamine in DMF.10 The reduction of 83 using lithium
borohydride followed by the separation of the diastereomers
utilizing chiral HPLC afforded single enantiomer 84. Global
deprotection using trifluoroacetic acid in the microwave at 130
°C, followed by Boc reprotection of the amine with di-tert-
butyl dicarbonate and then saponification of the ester supplied
a The pNPP assay. b The mixture of (R/S)-IZD diastereomers.
acid 85 in 71% yield over three steps. The coupling of
benzenediamine 7 to scaffold 85 and cyclization under acidic
conditions gave benzimidazole 86 in 72% yield. The removal
of the carbamate with TFA followed by sulfonylation afforded
the desired ortho-chloro benzimidazole sulfonamide 87 in 76%
yield.
Results and Discussion
In our effort to improve the cell permeability and the overall
pharmacokinetic properties of our peptidic (S)-IZD-containing
inhibitors of PTP1B, we endeavored to eliminate the peptidic
nature of lead compound 1. We initially focused on replacing
the C-terminal amides with less polar, more hydrophobic groups.
An inspection of the X-ray structures of several inhibitors,
including 1, revealed a conserved bidentate hydrogen bond
between the C-terminal amide NH and the adjacent amide NH
with Asp48 of PTP1B (Figure 1). Previous analogs from our
lab had also shown that N-terminal tertiary amides were
completely inactive against PTP1B, corroborating that this is
an important interaction. Several heterocycles 2 were thus

Protein Tyrosine Phosphatase 1B Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3779
Table 3. SAR of Benzimidazole Substituents
Table 2. SAR of Arylsulfonamide Substituents
PTP1B
pNPP
R2
PTP1B
pNPP
IC50
IC50
compdb
2-
3-
4-
5-
6-
(nM)a
compdb
R1
(nM)a
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
100 (32)c
120
66 (43)c
80
160
60
110
170
77
110
300
85
80
130
67
51
45
63
48
94
80
240
280
160
460
79
34
47
48
49
50
51
52
53
54
55
56c
H
80
190
140
120
53
140
150
160
130
240
460
F
4-Me
5-Me
F
F
5-F
Cl
5-CN
Cl
5-Cl
Cl
5-CO2Me
5-CF3
5-OMe
4-OH
Br
Br
Br
Me
Me
CF3
CN
4-tert-butyl
Me
CF3
a The pNPP assay. b The compounds were synthesized in parallel as a
mixture of 4 diastereomers (two major: (R/S)-IZD and two minor: alpha-
center of amino acid) and purified by preparative LCMS. c The compound
was submitted as a mixture of 2 diastereomers ((R/S)-IZD).
CF3
CN
ureas, carbamates, sulfonamides) was synthesized using scaffold
8, as a mixture of diastereomers, in an effort to identify
replacements for the N-terminal peptide portion of the inhibitor.
Gratifyingly, arylsulfonamides were rapidly identified as potent
PTP1B inhibitors. The new benzimidazole sulfonamide lead 14
is nonpeptidic and lower in molecular weight than parent 10
yet has comparable potency (IC50 ) 32 nM). The aryl
functionality was important for potency since the simple
methylsulfonamide 13 was 5-fold less active. A focused
compound library of substituted arylsulfonamides was synthe-
sized in parallel and purified by automated LCMS11,12 to
optimize this functionality. A partial, yet representative set of
compounds is shown Table 2. The SAR from this study revealed
that a variety of meta or para substituted derivatives were more
potent than the ortho substituted compounds, although they are
only 2-3-fold better than the unsubstituted parent 14.
OMe
Cl
OMe
Ph
Cl
Cl
Cl
Cl
Cl
Cl
Cl
F
Cl
Cl
Cl
F
Cl
F
51
F
F
F
110
140
160
110
72
F
F
F
F
a A pNPP assay. b The compounds were synthesized in parallel as a
mixture of 4 diastereomers (two major: (R/S)-IZD and two minor: alpha-
center of amino acid) and purified by preparative LCMS. c The pNPP data
for the separated single diastereomer.
A small library of substituted benzimidazoles was synthesized
and showed that a wide variety of functional groups could also
be appended to the benzimidazole (Table 3). All 3-substituted
derivatives were approximately equipotent against PTP1B,
whereas the large tert-butyl substituent 56 at the 2-position was
less active. Modeling predicted that the benzimidazole would
bind in a solvent exposed region on the surface of PTP1B, which
we have termed the E-site. The expected orientation of the
benzimidazole effectively explains the lack of SAR in the
5-position because these groups would project into the solvent
and would not interact with the protein. The loss of activity
with large substituents 56 at the 4-position could also be
rationalized on the basis of a steric clash with Asp48 or Phe182.
The relatively flat SAR for the sulfonamide was more difficult
to rationalize on the basis of our design and modeling, which
predicted that the arylsulfonamide NH and benzimidazole NH
bidentate H-bond interaction with Asp48 would necessitate that
the aryl group of the sulfonamide bind in an extended
conformation against the protein in the so-called C-site. An
X-ray cocrystal structure of benzimidazole sulfonamide 46/
PTP1B solved to 1.8 Å resolution confirmed the presence of
the bidentate H-bonding pattern and the position of the
targeted, which could maintain the bidentate hydrogen bonding
interaction with Asp48 (Figure 2). The benzimidazole moiety
proved to be a very effective replacement for the amide.
Benzimidazole peptide 10 was ∼10-fold more potent than the
primary amide 1 with an IC50 ) 35 nM (Table 1). The mode of
inhibition experiments unequivocally demonstrated that 10 was
a competitive and reversible inhibitor of PTP1B.
The truncation of the N-terminus of peptide inhibitor 10
demonstrated that the peptide portion of the inhibitor signifi-
cantly contributed to the binding of this molecule to PTP1B
(Table 1). The elimination of the terminal acetamide in
compound 11 gave a surprising >20-fold loss in activity. It is
clear that the hydrogen bond between the amide carbonyl and
Arg54 provides a significant amount of the binding energy
observed with these peptidic ligands. Truncating further to the
simple acylbenzimidazole derivative 12 resulted in an additional
2-fold loss in activity, suggesting that the hydrophobic phenyl
substituent of the peptide plays only a minor role in binding to
PTP1B. The desolvation of the hydrophobic side chain in this
solvent exposed region is likely a contributing factor.
A diverse compound library of different acyl groups (amides,

3780 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
Combs et al.
Figure 4. Modeling of ortho-methyl (S)-IZD. (A) Molecular surface
of 46 (orange mesh) shown with the molecular surface of the
phosphotyrosine binding site of PTP1B (transparent gray), illustrating
the complementarity of the ligand in the enzyme active site. (B)
Molecular surface of a model of ortho-methyl derivative 79a with the
unchanged enzyme surface showing that ortho-methyl occupies the
entire available volume of the D-site, a small preformed pocket adjacent
to the catalytic site of PTP1B.
Figure 3. The X-ray cocrystal structure of compound 46/PTP1B at
1.8 Å resolution. (A) Structure of compound 46; (B) bidentate hydrogen
bond with Asp48 shown between the benzimidazole and sulfonamide
NHs; (C) top view showing the intramolecular pi-stacking of the
ligands’ arylsulfonamide and benzimidazole groups.
improving the binding (5-20-fold) of nearly all PTP1B inhibitor
chemotypes by binding into the relatively small D-site imbedded
deep into the protein adjacent to the catalytic site. Thus, a series
of ortho substituents to the (S)-IZD, including halo and alkyl
groups, was targeted. The synthesis of each different ortho
substituted scaffold required the development of a unique
synthesis of 14-18 linear steps. The significant synthetic effort
was rewarded with the identification of the most potent enzyme
inhibitors of this series, and the first compounds to show cellular
activites. The ortho-F derivatives 67 were the most potent,
giving 2-4-fold increases in enzyme potency compared to that
of the unsubstituted parent, whereas the ortho-chloro 87 and
ortho-methyl 79 congeners were approximately equipotent
(Table 4). The sterically larger ortho-Br derivative 93 was much
less active. Although it is known that the D-site accommodates
halogens and small hydrophobic substituents, it was surprising
that the ortho-bromo derivative was not active because ortho-
bromo difluoromethylphosphonate (DFMP) derivatives have
been reported to increase potency as much as 20-fold over the
unsubstituted DFMP derivatives.13 One possible explanation is
that the steric bulk of the bromide adjacent to the (S)-IZD causes
the optimal binding conformation of the heterocycle to be of
much higher energy, thus resulting in a net loss in binding
affinity. For the ortho-chloro and ortho-methyl derivatives, we
propose that this negative interaction with the heterocycle is
less severe for these smaller substituents and that they are nearly
optimal for filling the available space in the D-site (Figure 4).
benzimidazole in the E-site, but revealed an unexpected
conformation for the arylsulfonamide (Figure 3). One observes
a 180° rotation of the sulfonamide apparently driven by the
intramolecular pi-stacking of the aryl ring of the sulfonamide
and benzimidazole while maintaining the H bond to Asp48. The
X-ray structure indicates that the aryl of the sulfonamide does
not bind with the protein in any site but extends into the solvent
with only intramolecular interactions. The failure to significantly
improve the binding of these ligands by substitution on the
arylsulfonamide is consistent with the observed binding mode
in the solid state because the aryl ring does not interact with
the protein, although the observed high affinity of these
benzimidazole sulfonamide derivatives attests to the strength
of this intramolecular interaction. This unique conformation of
the ligand has been observed in all X-ray cocrystal structures
of benzimidazole sulfonamide inhibitors solved to date. A
detailed analysis of these cocrystal structures will be reported
elsewhere.
Further optimization of the benzimidazolesulfonamide lead
focused on substitutions ortho to the (S)-IZD heterocyclic pTyr
mimetic on the aryl ring. Substituting ortho to pTyr mimetics
has been shown to be an effective strategy for significantly

Protein Tyrosine Phosphatase 1B Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3781
Table 4. SAR of ortho-Substituted (S)-IZD Benzimidazole
Sulfonamides
that the heterologous overexpression of an IR leads to constitu-
tive autophosphorylation of the receptor in the absence of the
ligand14,15 presumably because of overwhelming endogenous
negative regulation by PTP1B. Indeed this was the case. As
shown in Figure 5A, the overexpression of IR leads to
detectable, constitutive autophosphorylation (lane 1) that is
reduced upon the coexpression of PTP1B (lane 2). This assay
format proved more robust than the potentiation of ligand
stimulated IR phosphorylation, which is a transient phenomenon
that is difficult to control during tests of large numbers of
compounds in a miniaturized format. As validation of this assay,
a specific shRNA that reduces PTP1B protein levels by
approximately 50% reverses the suppression of IR phosphory-
lation levels by PTP1B (compare lanes 1 vs 4 and 2 vs 3).
Therefore, in this cellular assay, there is direct and dynamic
regulation of IR phosphorylation by PTP1B. Treatment of cells
with the nonselective tyrosine phosphatase inhibitor bpV also
reverses the effect of PTP1B on pIR levels.
PTP1B
pNPP
IC50
IR phos
assay fold
shift at
caco-2
Pm
compd
X
R2
R1
(nM)a
80 µMb
(×10-6 cm/s)
14
H
F
F
F
H
H
3-F
3-CF3
H
H
H
H
H
H
H
5-Cl
H
H
H
H
H
32
18
15
10
50
1.0 ( 0.3c
1.6 ( 0.6
1.1
2.3
1.0
0.4
0.2
<0.1
0.3
<0.1
<0.1c
0.4
n.t.
<0.1
67a
67b
67c
87
Cl
Br
Me
Me
Me 3-CF3,
4-Br
93
340c 0.7c
The ortho-methyl (S)-IZD compound 79a significantly in-
creased the levels of IR phosphorylation in a dose-dependent
manner (up to 4-fold at 400 µM) (Figure 5B). As a control,
inactive (R)-IZD diastereomer 79b was tested and found not to
affect pIR levels up to 80 µM, providing further confirmation
that the observed cellular activity of inhibitor 79a was mediated
through intracellular inhibition of PTP1B. To determine whether
the inhibition of PTP1B impacted signaling downstream of IR,
a phospho-Akt (Ser473) ELISA was performed using human
hepatoma HepG2 cells. The treatment with 80 µM of compound
79a stimulated Akt phosphorylation by 55% and 217% in the
absence or presence of 2 nM insulin compared with that of either
DMSO control, demonstrating that inhibition of PTP1B in these
cells potentiated insulin signaling.
79a
79bd
79c
35
>10000
16
2.8 ( 0.8
1.0
2.0
79d
79e
Me 2-CN
Me 3-Cl
H
H
18
76
1.4
1.9
<0.1
0.2
a The pNPP assay. b The IRp assay results are given as averages of
triplicates taken on the same day. The standard deviations are calculated
for compounds 14 and 67a on the basis of triplicate assays performed on
three different days. SD for 79a was calculated on the basis of 10 different
day triplicate runs. c The assay result is from a mixture of (R/S)-IZD isomers.
d 79d is a single diastereomer with (R)-IZD heterocycle configuration.
The activity of the ortho-fluoro derivative is likely optimal
because of its ability to partially fill the D-site while maintaining
the optimal conformation of the (S)-IZD heteocycle in the
catalytic site. The increased activity of the ortho-fluoro deriva-
tives compared to that of the ortho-chloro and ortho-methyl
derivatives can also be attributed to their intrinsic ability to
hydrogen bond to Lys120 in the D-site. The ortho-fluoro
derivatives may also pull the density from the pi-cloud of the
phenyl ring and, thus, increase the ring-stacking interaction with
the Phe182 that caps the ligand in the catalytic site.
A comparison of analogous benzimidazole sulfonamides with
different ortho substituents showed that ortho-fluoro derivatives
67 also exhibited modest cellular activity, whereas unsubstituted
14, ortho-chloro 87, and ortho-bromo 93 derivatives were all
inactive. The observed small increases in caco-2 permeability
and correlated cellular activities for ortho-methyl 79 and ortho-
fluoro 67 derivatives suggests that hydrophobic shielding of the
(S)-IZD by the ortho-methyl group in particular may play a role
in increasing the cell permeability of these (S)-IZD-containing
PTP1B inhibitors.
Cellular Activity. Cellular activities for our most potent
PTP1B inhibitors were assessed using an insulin receptor (IR)
phosphorylation assay. We took advantage of the observation
Figure 5. ortho-Methyl (S)-IZD derivative 79a enhances cellular insulin receptor phosphorylation. (A) Insulin receptor (IR) phosphorylation assay.
The transfection of HEK293 cells with plasmids encoding hIR leads to the autophosphorylation of the receptor on tyrosines 1162 and 1163 (IRpYpY),
which can be quantitated by a specific ELISA. Cotransfection of PTP1B increases the PTP1B protein with concomitant down modulation of
IRpYpY levels. shRNA against PTP1B suppresses PTP1B protein accumulation and restores IRpYpY levels. (B) Cells treated with compound 79a,
the enzyme active (S)-IZD isomer, demonstrate a dose-dependent increase in IRpYpY levels, whereas the enzyme inactive compound 79b fails to
modulate IR phosphorylation. Additional controls include the incubation of cells with 10 µM bpV(pic) or PTP1B shRNA to block PTP1B activity
or the protein and two doses of insulin 10 and 100 nM. The inset shows the relative stimulation compared with the DMSO control for compounds
79a and its (R)-IZD diastereomer 79b.

3782 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
Combs et al.
(d, J ) 8.0 Hz, 1H), 5.22 (t, J ) 9.6 Hz, 1H), 4.35 (m, 1H), 3.40
(m, 1H), 3.15 (dd, J ) 17.2, 8.4 Hz, 1H), 3.03 (dd, J ) 13.6, 4.4
Hz, 1H), 2.81 (m, 1H), 1.55 (s, 9H), 1.25 (s, 9H). LCMS found for
C21H31N2O7S (M + Na)+: m/z ) 477.0.
Conclusion
The structure-based design and synthesis of nonpeptidic
ligands of PTP1B led to the identification of potent, cell
permeable benzimidazole sulfonamide inhibitors. The X-ray
cocrystal structure of benzimidazole sulfonamide 46 revealed
an unusual bound conformation that effectively explains the lack
of SAR for substitution on either the sulfonamide or benzimi-
dazole aryl rings. An ortho substitution to the (S)-IZD hetero-
cyclic pTyr mimetic resulted in the discovery of potent ortho-
fluoro and ortho-methyl enzyme inhibitors, such as 79a, with
cellular activity in an IR phosphorylation assay. These results
provide further evidence that nonpeptidic compounds incorpo-
rating the (S)-IZD heterocyclic pTyr mimetic can be potent, cell
permeable inhibitors of phosphatases, such as PTP1B. Further
optimization of these nonpeptidic ligands to improve their cell
permeability and, thus, cellular activity will be necessary to
provide PTP1B inhibitors suitable for in vivo proof of principle
animal studies in diabetes and obesity.
(5S)-5-4-[(2S)-2-Amino-2-(1H-benzimidazol-2-yl)ethyl]phenyl-
2-tert-butylisothiazolidin-3-one 1,1-dioxide Bis(trifluoroacetate)
(8). (2S)-2-[(tert-butoxycarbonyl)amino]-3-[4-(2-tert-butyl-1,1-di-
oxido-3-oxoisothiazolidin-5-yl)phenyl]propanoic acid (6) (160 mg,
0.35 mmol), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uro-
nium hexafluorophosphate (160 mg, 0.42 mmol), DMF (1.1 mL),
and N,N-diisopropylethylamine (0.30 mL, 1.8 mmol) were premixed
for 5 min and then added to 1,2-benzenediamine (7) (57 mg, 0.53
mmol) and stirred at room temperature overnight. The preformed
ester was yellow in color and, upon addition to the diamine,
continued to darken over several minutes. Purification by prepara-
tive LCMS and lyophillization gave the diastereomeric mixture (95
mg, 48%). The mixture (60 mg) was treated with Si-dipiperidine
(Silicycle, 1 mmol/g) in MeOH (3 mL) for 1 h and filtered.
Separation on Chiralcel OD using 60% EtOH/hex and injecting in
MeOH gave tert-butyl ((1S)-2-[(2-aminophenyl)amino]-1-4-[(5S)-
2-tert-butyl-1,1-dioxido-3-oxoisothiazolidin-5-yl]benzyl-2-oxoeth-
yl)carbamate (12 mg, 20%) (Peak2). 1H NMR (400 MHz, d6-
DMSO): δ 9.55 (s, 1H), 7.40 (dd, J ) 18.4, 8.0 Hz, 4H), 7.20 (d,
J ) 7.6 Hz, 1H), 7.00 (m, 2H), 6.90 (d, J ) 7.6 Hz, 1H), 6.75 (m,
1H), 5.23 (t, J ) 9.6 Hz, 1H), 42.5 (m, 1H), 3.40 (dd, J ) 17.6,
10.0 Hz, 1H), 3.18 (dd, J ) 17.6, 8.4 Hz, 1H), 3.08 (dd, J ) 13.6,
4.8 Hz, 1H), 2.88 (m, 1H), 1.48 (s, 9H), 1.30 (s, 9H). LCMS found
for C27H37N4O6S (M + H)+: m/z ) 545.2. tert-Butyl ((1S)-2-[(2-
aminophenyl)amino]-1-4-[(5S)-2-tert-butyl-1,1-dioxido-3-oxoisothi-
azolidin-5-yl]benzyl-2-oxoethyl)carbamate (12 mg, 0.022 mmol)
was dissolved in acetic acid (2 mL) and stirred at 40 °C for 2 h.
LCMS indicated complete cyclization. Evaporation and Boc
removal with TFA at room temperature for 30 min before
re-evaporation gave the desired single diastereomer amine 8 as the
Experimental Section
Chemistry. All reactions were run under an atmosphere of dry
nitrogen. All solvents were used without further purification as
acquired from commercial sources. NMR spectra were obtained
using a Varian Mercury-300, a Mercury-400, or an Inova-500
spectrometer. The chemical shifts are reported in parts per million
relative to tetramethylsilane (TMS) as the internal standard. All
final products important to SAR and discussed in the text were
characterized by H NMR, 13C NMR, HRMS, LCMS, and two
HPLC methods.
1
Purifications by flash chromatography were performed on
RediSep columns using an Isco CombiFlash SG100c. Preparative
LCMS purifications were performed on a Waters FractionLynx
system using mass directed fractionation and compound-specific
method optimization (J. Comb. Chem. 2004, 6, 874-883). The LC
method utilized a Waters SunFire column (19 × 100 mm, 5 µM
particle size), with a water/0.1% TFA and acetonitrile/0.1% TFA
gradient at a flow rate of 30 mL/min over a total run time of 5
min.
1
bis TFA salt (12 mg, 83%). H NMR (400 MHz, d6-DMSO): δ
8.70 (s, 3H), 7.60 (dd, J ) 6.0, 3.2 Hz, 2H), 7.38 (d, J ) 8.4 Hz,
2H), 7.20 (m, 4H), 5.22 (dd, J ) 11.4, 8.8 Hz, 1H), 4.83 (m, 1H),
3.42 (dd, J ) 14.0, 8.4 Hz, 1H), 3.35 (dd, J ) 16.8, 10.0 Hz, 2H),
3.08 (dd, J ) 17.2, 8.4 Hz, 1H), 1.52 (s, 9H). LCMS found for
C26H29F6N4O7S (M + H)+: m/z ) 427.2.
(2S)-2-(Acetylamino)-N-((1S)-1-(1H-benzimidazol-2-yl)-2-4-
[(5S)-2-tert-butyl-1,1-dioxido-3-oxoisothiazolidin-5-yl]phenylethyl)-
3-phenylpropanamide Trifluoroacetate (9). (5S)-5-4-[(2S)-2-
amino-2-(1H-benzimidazol-2-yl)ethyl]phenyl-2-tert-butylisothiazolidin-
3-one 1,1-dioxide bis(trifluoroacetate) (8) (10.0 mg, 0.015 mmol)
was stirred in DMSO (0.5 mL), and N,N-diisopropylethylamine (7
µL, 0.04 mmol) was added. In a separate vial, N-(tert-butoxycar-
bonyl)-L-phenylalanine (6.1 mg, 0.023 mmol), N,N,N′,N′-tetra-
methyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (8.7
mg, 0.023 mmol) and N,N-diisopropylethylamine (13 µL, 0.075
mol) were stirred in NMP (0.5 mL) for 5 min before adding to the
above prepared amine solution. The mixture was stirred at room
temperature overnight. The mixture was diluted with methanol and
purified by preparative LCMS to give (12 mg, 100%) the BocPhe
intermediate. LCMS found for C36H43N5O6S (M + H)+: m/z )
674.3. This intermediate, tert-butyl (1S)-2-[((1S)-1-(1H-benzimi-
dazol-2-yl)-2-4-[(5S)-2-tert-butyl-1,1-dioxido-3-oxoisothiazolidin-
5-yl]phenylethyl)amino]-1-benzyl-2-oxoethylcarbamate, (12 mg,
0.018 mmol) was stirred in TFA (1 mL) for 30 min. This amine
was stirred in acetonitrile (2 mL) and treated with Si-dipiperidine
(Silicycle, 1 mmol/g, 100 mg) for 30 min. Filtration and evaporation
gave the free base, which was stirred in dioxane (1 mL) and treated
with acetic anhydride (5 µL, 0.05 mmol) for 1 h. Purification by
preparative LCMS gave the desired amide (10 mg, 80%). 1H NMR
(400 MHz, CD3OD): δ 7.75 (m, 2H), 7.61 (m, 2H), 7.41 (d, J )
8.4 Hz, 2H), 7.30 (d, J ) 8.4 Hz, 2H), 7.06 (m, 2H), 7.04 (m, 3H),
5.48 (dd, J ) 8.0, 8.0 Hz, 1H), 5.05 (dd, J ) 9.2, 9.2 Hz, 1H),
4.55 (dd, J ) 7.2, 7.2 Hz, 1H), 3.47 (m, 2H), 3.19 (m, 2H), 2.96
(dd, J ) 7.2, 6.4 Hz, 1H), 2.78 (dd, J ) 9.2, 9.2 Hz, 1H), 1.89 (s,
3H), 1.60 (s, 9H). LCMS found for C33H37N5O5S (M + H)+: m/z
) 616.2.
Benzyl N-(tert-butoxycarbonyl)-4-(2-tert-butyl-1,1-dioxido-3-
oxo-2,3-dihydroisothiazol-5-yl)-L-phenylalaninate (5). (4-(2S)-
3-(benzyloxy)-2-[(tert-butoxycarbonyl)amino]-3-oxopropylphenyl)-
boronic acid (3) (250 mg, 0.63 mmol), 2-tert-butyl-5-chloro-1,1-
dioxo-1,2-dihydro-1λ6-isothiazol-3-one (4) (330 mg, 1.5 mmol),
potassium carbonate (433 mg, 3.13 mmol), and 1,4-dioxane (2 mL)
were combined and degassed with nitrogen gas for 10 min before
[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) com-
plexed with dichloromethane (1:1) (80 mg, 0.10 mmol) was added,
and degassing continued for another 10 min. The orange suspension
was heated to 80 °C overnight. The mixture was filtered through a
plug of silica with 25% ethyl acetate/hexanes, and concentration
of the filtrates gave the crude material, which was purified on silica
1
gel to give 5 (230 mg, 68% yield). H NMR (400 MHz, CDCl3):
δ 7.65(d, J ) 8.8 Hz, 2H), 7.35 (d, J ) 8.8 Hz, 2H), 7.30 (m, 4H),
7.00 (s, 1H), 5.12 (dd, J ) 23.6, 12.0 Hz, 2H), 4.43 (dd, J ) 9.2,
6.8 Hz, 1H), 3.20 (dd, J ) 14.0, 6.4 Hz, 1H), 3.00 (dd, J ) 13.6,
9.2 Hz, 1H), 1.70 (s, 9H), 1.40 (s, 9H). LCMS found for
C28H35N2O7S (M + Na)+: m/z ) 565.0.
(2S)-2-[(tert-Butoxycarbonyl)amino]-3-[4-(2-tert-butyl-1,1-di-
oxido-3-oxoisothiazolidin-5-yl)phenyl]propanoic acid (6). Benzyl
N-(tert-butoxycarbonyl)-4-(2-tert-butyl-1,1-dioxido-3-oxo-2,3-dihy-
droisothiazol-5-yl)-L-phenylalaninate (5) (230 mg, 0.42 mmol) and
10% palladium on carbon (115 mg) in ethanol (3 mL) were shaken
overnight on a Parr hydrogenation apparatus. LCMS indicated a
fully reduced heterocycle, but only half of the benzyl ester was
cleaved. The mixture was resubmitted to the same conditions
overnight after filtration to remove the catalyst. LCMS indicated
complete reaction. Filtration and concentration gave 6 (180 mg,
93%). NMR (400 MHz, CDCl3): δ 7.40 (d, J ) 8.0 Hz, 2H), 7.31

Protein Tyrosine Phosphatase 1B Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3783
(2S)-2-(Acetylamino)-N-((1S)-1-(1H-benzimidazol-2-yl)-2-4-
[(5S)-1,1-dioxido-3-oxoisothiazolidin-5-yl]phenylethyl)-3-phenyl-
propanamide Trifluoroacetate (10). (2S)-2-(acetylamino)-N-((1S)-
1-(1H-benzimidazol-2-yl)-2-4-[(5S)-2-tert-butyl-1,1-dioxido-3-oxo-
isothiazolidin-5-yl]phenylethyl)-3-phenylpropanamide trifluoro-
acetate (9) (10 mg, 0.01 mmol) was stirred in acetonitrile (1 mL),
and methanesulfonic acid (150 µL) was added. The clear solution
was evaporated under a nitrogen stream for 30 min to remove most
of the acetonitrile. The solution was diluted with acetonitrile and
purified by preparative LCMS to give the desired product as a
2H), 5.06 (t, J ) 8.6 Hz, 1H), 5.01 (dd, J ) 8.7, 6.7 Hz, 1H), 3.29
(m, 2H), 3.28 (m, 2H). 13C NMR (125 MHz, CD3OD): δ 170.9,
163.7 (J ) 257.3 Hz), 154.3, 142.6, 137.5, 132.7, 132.7, 130.9,
130.8, 130.2, 127.9, 124.0, 121.3, 115.3, 115.1, 66.3, 53.9, 40.1,
39.0. HRMS calcd for C24H21FN4O5S2 (M + H)+: 529.1017; found,
529.1014.
2-tert-Butyl-5-(2-fluoro-4-methylphenyl)isothiazol-3(2H)-
one 1,1-dioxide (58). (2-Fluoro-4-methylphenyl)boronic acid (57)
(19.5 g, 0.127 mol), 2-tert-butyl-5-chloro-1,1-dioxo-1,2-dihydro-
1λ6-isothiazol-3-one (4) (30.5 g, 0.136 mol), and [1,1′-bis(diphen-
ylphosphino)ferrocene]-dichloropalladium(II) complexed with dichlo-
romethane (1:1) (10.4 g, 12.7 mmol) were dissolved in toluene (500
mL) and treated with triethylamine (53 mL, 0.38 mol). The reaction
was degassed and placed under an atmosphere of nitrogen. The
reaction was then heated to 90 °C for 18 h. The reaction was diluted
with ethyl acetate (500 mL) and washed with 1 N aqueous
hydrochloric acid (500 mL). The ethyl acetate was dried over
sodium sulfate, filtered, and concentrated in vacuo. The crude
material was filtered through Celite and then silica gel. The crude
material was then purified by silica gel chromatography (5-10%
ethyl acetate/hexanes) to afford the product as a white solid (24.5
g, 65%). 1H NMR (400 MHz, CDCl3): δ 7.85 (t, J ) 7.8 Hz, 1H),
7.11-7.03 (m, 2H), 6.86 (d, J ) 1.8 Hz, 1H), 2.42 (s, 3H), 1.73
(s, 9H).
1
colorless glass, (6 mg, 60%). H NMR (400 MHz, CD3OD): δ
7.74 (m, 2H), 7.61 (m, 2H), 7.42 (d, J ) 7.9 Hz, 2H), 7.28 (d, J )
7.9 Hz, 2H), 7.07 (m, 2H), 7.02 (m, 2H), 7.02 (m, 1H), 5.48 (dd,
J ) 7.5, 7.5 Hz, 1H), 5.12 (dd, J ) 8.6, 8.6 Hz, 1H), 4.56 (dd, J
) 7.5, 7.5 Hz, 1H), 3.46 (m, 2H), 3.31 (m, 2H), 2.98 (dd, J )
13.8, 6.9 Hz, 1H), 2.89 (dd, J ) 13.7, 8.6 Hz, 1H), 1.89 (s, 3H).
13C NMR (125 MHz, DMSO-d6(+TFA)): δ 174.2, 173.8, 170.2,
154.1, 138.3, 138.1, 132.3, 131.0, 130.9, 130.2, 130.2, 129.4, 128.0,
128.0, 115.3, 66.4, 56.3, 50.0, 39.1, 38.4, 38.2, 22.6. HRMS calcd
for C29H29N5O5S (M + H)+: 560.1889; found, 560.1963.
N-((1S)-1-(1H-Benzimidazol-2-yl)-2-4-[(5S)-1,1-dioxido-3-
oxoisothiazolidin-5-yl]phenylethyl)acetamide Trifluoroacetate
(12). (5S)-5-4-[(2S)-2-amino-2-(1H-benzimidazol-2-yl)ethyl]phenyl-
2-tert-butylisothiazolidin-3-one 1,1-dioxide bis(trifluoroacetate) (30
mg, 0.046 mmol) was stirred in N,N-dimethylformamide (0.5 mL),
pyridine (0.5 mL), and acetic anhydride (0.5 mL) for 2 h at room
temperature. The reaction was quenched with methanol and purified
by preparative LCMS to give the tert-butyl intermediate. TFA
cleavage of tert-butyl in a microwave at 130 °C for 3 min followed
by evaporation and preparative LCMS gave the desired product
2-tert-Butyl-5-[4-(dibromomethyl)-2-fluorophenyl]isothiazo-
lidin-3-one 1,1-dioxide (59). 2-tert-Butyl-5-(2-fluoro-4-methyl-
phenyl)isothiazol-3(2H)-one 1,1-dioxide (58) (4.5 g) in ethanol (120
mL) was treated with palladium (900 mg, 8.5 mmol) (10%
palladium on carbon) and placed on a Par hydrogenator under a
50 psi atmosphere of hydrogen for 24 h. The reaction solution was
filtered though Celite. After concentration, the crude material was
purified by silica gel chromatography (3-10% ethyl acetate/
1
(16 mg, 66%). H NMR (400 MHz, DMSO-d6): δ 8.54 (d, J )
8.1 Hz, 1H), 7.54 (m, 2H), 7.36 (d, J ) 8.4 Hz, 2H), 7.29 (d, J )
8.4 Hz, 2H), 7.20 (m, 2H), 5.31 (dt, J ) 7.9, 6.1 Hz, 1H), 5.15 (t,
J ) 9.0 Hz, 1H), 3.42 (dd, J ) 14.1,5.6 Hz, 1H), 3.32 (dd, J )
17.1, 9.8 Hz, 1H), 3.17 (dd, J ) 17.0, 8.4 Hz, 1H), 3.14 (dd, J )
14.3, 9.2 Hz, 1H), 1.82 (s, 3H). 13C NMR (125 MHz, DMSO-d6):
δ 169.2, 169.1, 154.5, 138.8, 137.1, 129.3, 128.9, 127.6, 122.1,
114.1, 64.1, 48.5, 38.3, 37.4, 22.3. HRMS calcd for C20H20N4O4S
(M + H)+: 413.1285; found, 413.1285.
1
hexanes) to afford the product as a white solid (3.6 g, 79%). H
NMR (400 MHz, CDCl3): δ7.21-7.17 (m, 1H), 7.02-6.96 (m,
2H), 5.04 (dd, J ) 9.0, 7.0 Hz, 1H), 3.26 (dd, J ) 17.2, 9.0 Hz,
1H), 3.08 (dd, J ) 17.2, 7.0 Hz, 1H), 2.37 (s, 3H), 1.66 (s, 9H).
2-tert-Butyl-5-(2-fluoro-4-methylphenyl)isothiazolidin-3-one 1,1-
dioxide (6.2 g, 21 mmol), N-bromosuccinimide (11.0 g, 62 mmol),
and benzoyl peroxide (1.2 g, 5.2 mmol) in carbon tetrachloride (100
mL) were heated at reflux for 3.5 h. The solution was filtered to
remove the solid, washed with satd. aqueous sodium bicarbonate
solution (200 mL), dried over sodium sulfate, and concentrated.
Purification by silica gel chromatography (10-30% ethyl acetate/
N-((1S)-1-(1H-Benzimidazol-2-yl)-2-4-[(5S)-1,1-dioxido-3-ox-
oisothiazolidin-5-yl]phenylethyl)benzenesulfonamide Trifluo-
roacetate (14). (5S)-5-4-[(2S)-2-amino-2-(1H-benzimidazol-2-yl)-
ethyl]phenyl-2-tert-butylisothiazolidin-3-one 1,1-dioxide bis(trifluoro-
acetate) (30 mg, 0.04 mmol) was stirred in methylene chloride (3.0
mL), and benzenesulfonyl chloride (16.2 mg, 0.0917 mmol) was
added. The resulting solution was stirred for 5 h before evaporation
and purification by preparative LCMS to yield the desired product
1
hexanes) afforded the product as a white solid (5.8 g, 61%). H
NMR (400 MHz, CDCl3): δ 7.45-7.33 (m, 3H), 6.59 (s, 1H), 5.07
(dd, J ) 8.8, 7.2 Hz, 1H), 3.31 (dd, J ) 17.2, 8.8 Hz, 1H), 3.12
(dd, J ) 17.1, 7.2 Hz, 1H), 1.65 (s, 9H). LCMS found for C14H17-
Br2FNNaO3S (M + Na)+: m/z ) 480.
1
as the trifluoroacetate salt (12 mg, 42%). H NMR (400 MHz,
DMSO-d6 (+TFA)): δ 9.02 (d, J ) 5.8 Hz, 1H), 7.74 (m, 2H),
7.52 (d, J ) 7.7 Hz, 2H), 7.49 (m, 2H), 7.44 (t, J ) 7.5 Hz, 1H),
7.32 (t, J ) 7.5 Hz, 1H), 7.25 (d, J ) 8.0 Hz, 2H), 7.14 (d, J ) 8.0
Hz, 2H), 5.23 (dd, J ) 9.4, 8.6 Hz, 1H), 4.89 (m, 1H), 3.37 (dd, J
) 17.3, 9.9 Hz, 1H), 3.30 (dd, J ) 13.9, 6.0 Hz, 1H), 3.23 (dd, J
) 17.1, 7.9 Hz, 1H), 3.18 (dd, J ) 13.8, 8.5 Hz, 1H). 13C NMR
(125 MHz, DMSO-d6(+TFA)): δ 168.5, 152.7, 136.5, 138.8, 132.5,
131.5, 129.3, 129.2, 128.9, 127.7, 126.2, 125.5, 114.4, 64.1, 51.7,
38.3, 37.1. HRMS calcd for C24H22N4O5S2 (M + H)+: 511.1112;
found, 511.1122.
4-(2-tert-Butyl-1,1-dioxido-3-oxoisothiazolidin-5-yl)-3-fluo-
robenzaldehyde (60). 2-tert-Butyl-5-[4-(dibromomethyl)-2-fluo-
rophenyl]isothiazolidin-3-one 1,1-dioxide (59) (4.2 g, 9.2 mmol)
and silver nitrate (3.12 g, 18.4 mmol) in ethanol (200 mL) and
water (50 mL) were heated at reflux for 0.5 h. The solution was
cooled to room temperature, filtered to remove the precipitate, and
concentrated to remove most of the ethanol. The residue was diluted
with ethyl acetate (200 mL). The organic layer was washed with
water (200 mL) and then dried over sodium sulfate. Concentration
in vacuo followed by purification by silica gel chromatography
(30% ethyl acetate/hexanes) afforded the product as a white solid
(2.4 g, 83%). 1H NMR (400 MHz, CDCl3): δ 10.0 (d, J ) 2.0 Hz,
1H), 7.78-7.75 (m, 1H), 7.70-7.65 (m, 1H), 7.56-7.52 (m, 1H),
5.13 (dd, J ) 8.9, 6.6 Hz, 1H), 3.35 (dd, J ) 17.2, 8.9 Hz, 1H),
3.15 (dd, J ) 17.2, 6.6 Hz, 1H), 1.66 (s, 9H). LCMS found for
C14H17FNNaO4S (M + Na)+: m/z ) 336.
Methyl (2Z)-2-[(tert-butoxycarbonyl)amino]-3-[4-(2-tert-butyl-
1,1-dioxido-3-oxoisothiazolidin-5-yl)-3-fluorophenyl]acrylate (62).
Methyl [(tert-butoxycarbonyl)amino](dimethoxyphosphoryl)acetate
(61) (3.53 g, 11.9 mmol) in methylene chloride (180 mL) was
treated with 1,8-diazabicyclo[5.4.0]undec-7-ene (1.92 mL, 12.9
mmol). After five minutes, 4-(2-tert-butyl-1,1-dioxido-3-oxoisothia-
zolidin-5-yl)-3-fluorobenzaldehyde (60) (3.1 g, 9.9 mmol) was
N-((1S)-1-(1H-Benzimidazol-2-yl)-2-4-[(5S)-1,1-dioxido-3-ox-
oisothiazolidin-5-yl]phenylethyl)-3-fluorobenzenesulfonamide Tri-
fluoroacetate (16). (5S)-5-4-[(2S)-2-amino-2-(1H-benzimidazol-2-
yl)ethyl]phenyl-2-tert-butylisothiazolidin-3-one 1,1-dioxide bis(tri-
fluoroacetate) (45 mg, 0.069 mmol) was stirred in methylene
chloride (3.0 mL) with N,N-diisopropylethylamine (48 µL, 0.27
mmol). 3-Fluorobenzenesulfonyl chloride (27 mg, 0.14 mmol) was
added, and the resulting solution was stirred for 5 h before
evaporation and purification by preparative LCMS to yield the
1
desired product (14 mg, 32%). H NMR (400 MHz, CD3OD): δ
7.70 (m, 2H), 7.57 (m, 2H), 7.42 (dt, J ) 7.9, 1.4 Hz, 1H), 7.37
(dd, J ) 8.2, 5.2 Hz, 1H), 7.34 (dt, J ) 8.2, 1.9 Hz, 1H), 7.27 (d,
8.1 Hz, 2H), 7.22 (td, J ) 7.8, 2.7 Hz, 1H), 7.14 (d, J ) 8.0 Hz,

3784 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
Combs et al.
added and the solution stirred at room temperature for 1 h. The
solution was diluted with methylene chloride (50 mL), washed with
1 N aqueous hydrochloric acid solution (250 mL), and the organic
phase dried over sodium sulfate. Purification by silica gel chro-
matography (5-25% ethyl acetate/hexanes) afforded the product
5.43-5.39 (m, 2H), 3.44-3.21 (m, 4H), 1.42 (s, 9H). LCMS found
for C23H26FN4O5S (M + H)+: m/z ) 489.
N-{(1S)-1-(1H-Benzimidazol-2-yl)-2-[4-(1,1-dioxido-3-oxoiso-
thiazolidin-5-yl)-3-fluorophenyl]ethyl}benzenesulfonamide
Trifluoroacetate (67a). tert-Butyl {(1S)-1-(5-chloro-1H-benzimi-
dazol-2-yl)-2-[4-(1,1-dioxido-3-oxoisthiazolidin-5-yl)-3-fluoro-
phenyl]-ethyl}carbamate trifluoroacetate (12.4 mg, 2.06 µmol) in
methylene chloride (1 mL) was treated with trifluoroacetic acid
(0.5 mL). The solution stirred at room temperature for 1 h and was
then concentrated in vacuo. The residue was dissolved in methanol
(1 mL) and treated with triethylamine (20 µL, 14.4 µmol) and
benzensulfonyl chloride (10.5 µL, 8.2 µmol). The solution was
stirred 4 h at room temperature and then purified by preparative
1
as a white foam (3.7 g, 77%). H NMR (400 MHz, CDCl3): δ
7.35-7.29 (m, 3H), 7.17 (s, 1H), 5.04 (dd, J ) 9.0, 7.1 Hz, 1H),
3.87 (s, 3H), 3.29 (dd, J ) 17.2, 9.0 Hz, 1H), 3.17 (dd, J ) 17.2,
7.0 Hz, 1H), 1.65 (s, 9H), 1.39 (s, 9H). LCMS found for C22H30-
FN2NaO7S (M + Na)+: m/z ) 507.
Methyl (2S)-2-[(tert-butoxycarbonyl)amino]-3-{4-[(5S)-2-tert-
butyl-1,1-dioxido-3-oxoisothiazolidin-5-yl]-3-fluorophenyl}-
propanoate (63). Methyl (2Z)-2-[(tert-butoxycarbonyl)amino]-3-
[4-(2-tert-butyl-1,1-dioxido-3-oxoisothiazolidin-5-yl)-3-fluoro-
phenyl]acrylate (62) (1.24 g, 2.56 mmol) in ethanol (100 mL) was
degassed, and (R,R)-(-)-1,2-bis[(o-methoxyphenyl)(phenyl)phos-
phino]ethane(1,5-cyclooctadiene) rhodium (I) tetrafluroborate (58
mg, 0.077 mmol) was added under nitrogen. The solution was
placed under a hydrogen atmosphere (50 psi) and shaken for 16 h.
The solution was concentrated in vacuo and purified by silica gel
chromatography (10-40% ethyl acetate/hexanes) to afford the
product as a white foam (1.14 g, 92%). The crude residue was
purified by normal phase chiral HPLC (ChiralCel OD-H [20 ×
250 mm, 5 µm], 30% EtOH/70% hexanes, 15 mL/min, 30 °C) to
yield isomer 1 (peak 1) (531 mg, active isomer) and isomer 2 (peak
2) (493 mg, inactive isomer). 1H NMR (400 MHz, CDCl3): δ 7.29-
7.27 (m, 1H), 7.03-6.95 (m, 2H), 5.06-5.02 (m, 2H), 4.61-4.57
(brs, 1H), 3.73 (s, 3H), 3.31-3.27 (m, 1H), 3.15-3.06 (m, 3H),
1.66 (s, 9H), 1.42 (s, 9H). LCMS found for C22H32FN2NaO7S (M
+ Na)+: m/z ) 509.
1
HPLC to afford the product as a white solid (7.5 mg, 57%). H
NMR (400 MHz, d6-DMSO): δ 9.07 (d, J ) 5.9 Hz, 1H), 7.78
(m, 2H), 7.53 (m, 2H), 7.50 (d, J ) 7.3 Hz, 2H), 7.45 (t, J ) 7.4
Hz, 1H), 7.35 (m, 1H), 7.33 (m, 2H), 7.04 (d, J ) 11.3 Hz, 1H),
6.99 (dd, J ) 8.1, 1.6 Hz, 1H), 5.38 (t, J ) 8.5 Hz, 1H), 4.96 (m,
1H), 3.36 (dd, J ) 8.5, 2.1 Hz, 2H), 3.31 (dd, J ) 13.6, 4.4 Hz,
1H), 3.16 (dd, J ) 13.9, 9.9 Hz, 1H). 13C NMR (125 MHz, d6-
DMSO, 30 °C): δ 168.7, 160.2, 152.7, 139.2, 138.6, 132.6, 131.0,
130.2, 128.9, 126.1, 125.8, 125.4, 116.3, 115.7, 114.3, 57.9, 51.4,
37.5, 36.3. HRMS calcd for C24H22FN4O5S2 (M + H)+: 529.1025;
found, 529.0981.
N-(1S)-1-(1H-Benzimidazol-2-yl)-2-{4-[(5S)-1,1-dioxido-3-ox-
oisothiazolidin-5-yl]-3-fluorophenyl}ethyl-3-fluorobenzenesulfona-
mide Trifluoroacetate (67b). 5-4-[(2S)-2-Amino-2-(1H-benzim-
idazol-2-yl)ethyl]-2-fluorophenylisothiazolidin-3-one 1,1-dioxide
bis(trifluoroacetate) (12 mg, 0.019 mmol) in methylene chloride
(2 mL) was treated with triethylamine (16 µL, 0.12 mmol) and
3-fluorobenzenesulfonyl chloride (10 µL, 0.078 mmol). After 3 h
at room temperature, the solution was purified by preparative LCMS
(2S)-2-[(tert-Butoxycarbonyl)amino]-3-{4-[(5S)-1,1-dioxido-3-
oxoisothiazolidin-5-yl]-3-fluorophenyl}propanoic Acid (64). Meth-
yl (2S)-2-[(tert-Butoxycarbonyl)amino]-3-{4-[(5S)-2-tert-butyl-1,1-
dioxido-3-oxo-isothiazolidin-5-yl]-3-fluorophenyl}propanoate (63)
(1.10 g, 2.26 mmol) in trifluoroacetic acid (10 mL) was heated for
2 min at 130 °C in the microwave. The solution was concentrated
in vacuo and purified by preparative LCMS to afford the product
as a white solid (614 mg, 61%). 1H NMR (400 MHz, CD3OD): δ
7.53 (m, 1H), 7.18-7.14 (m, 2H), 5.33-5.30 (m, 1H), 4.39 (m,
1H), 3.82 (s, 3H), 3.43-3.12 (m, 4H). LCMS found for C13H16-
FN2O5S (M + H)+: m/z ) 331. Methyl (2S)-2-amino-3-{4-[(5S)-
1,1-dioxido-3-oxoisothiazolidin-5-yl]-3-fluorophenyl}-propanoate
trifluoroacetate (558 mg, 1.26 mmol) in methylene chloride (50
mL) was treated with di-tert-butyl dicarbonate (548 mg, 2.51 mmol)
and triethylamine (0.88 mL, 6.3 mmol). The solution was stirred
at room temperature for 2 h. The solution was concentrated in
vacuo, diluted with methanol (8 mL), and then treated with 2 M
lithium hydroxide in water (1.5 mL). After 2 h, the solution was
acidified using a 1 N aqueous hydrochloric acid solution (2 mL),
and the solution was directly purified by preparative LCMS to afford
1
to afford the product as a white solid (9.2 mg, 72%). H NMR
(400 MHz, d6-DMSO): δ 9.20 (d, J ) 6.3 Hz, 1H), 7.78 (dd, J )
6.9, 3.4 Hz, 2H), 7.53 (dd, J ) 6.1, 2.1 Hz, 2H), 7.38 (m, 1H),
7.37 (m, 1H), 7.34 (m, 1H), 7.33 (m, 1H), 7.30 (m, 1H), 7.10 (d,
J ) 11.2 Hz, 1H), 7.05 (d, J ) 8.0 Hz, 1H), 5.40 (t, J ) 7.8 Hz,
1H), 5.04 (m, 1H), 3.38 (m, 2H), 3.36 (m, 1H), 3.16 (dd, J ) 13.6,
9.5 Hz, 1H). 13C NMR (125 MHz, d6-DMSO, 30 °C): δ 168.6,
161.2, 160.1, 150.2, 140.9, 139.4, 131.5, 131.3, 130.1, 125.6, 125.5,
122.3, 119.7, 116.5, 115.7, 114.4, 113.4, 57.9, 51.8, 37.8, 36.3.
HRMS calcd for C24H21F2N4O5S2 (M + H)+: 547.0931; found,
547.0941.
5-{4-[(2S)-2-(5-Chloro-1H-benzimidazol-2-yl)-2({[3-(tri-
fluoromethyl)phenyl]sulfonyl}amino)ethyl]-2-fluorophenyl}-3-
oxoisothiazolidin-2-ide 1,1-dioxide Trifluoroacetate (67c). tert-
Butyl {(1S)-1-(5-chloro-1H-benzimidazol-2-yl)-2-[4-(1,1-dioxido-
3-oxoisthiazolidin-5-yl)-3-fluorophenyl]-ethyl}carbamate trifluo-
roacetate (236 mg, 0.370 mmol) in methylene chloride (15 mL)
was treated with trifluoroacetic acid (5 mL). The solution was stirred
at room temperature for 1 h and then concentrated in vacuo. The
residue was dissolved in methanol (8 mL) and treated with
triethylamine (0.51 mL, 3.70 mmol) and m-trifluoromethyl benze-
nesulfonyl chloride (0.36 mL, 2.2 mmol). The solution was stirred
4 h at room temperature and then purified by preparative HPLC to
afford the product as a white solid (131 mg, 54%). 1H NMR (400
MHz, CD3OD): δ 7.94 (s, 1H), 7.78 (d, J ) 7.8 Hz, 1H), 7.75 (d,
J ) 2.0 Hz, 1H), 7.73 (d, J ) 7.6 Hz, 1H), 7.68 (d, J ) 8.6 Hz,
1H), 7.53 (dd, J ) 8.8, 2.0 Hz, 1H), 7.52 (dd, J ) 7.5, 7.5 Hz,
1H), 7.31 (dd, J ) 7.8, 7.8 Hz, 1H), 6.96 (dd, J ) 11.0, 1.5 Hz,
1H), 5.28 (dd, J ) 8.1, 8.1 Hz, 1H), 5.13 (dd, J ) 7.6, 5.7 Hz,
1H), 3.35 (m, 1H), 3.33 (m, 1H), 3.27 (m, 1H), 3.26 (m, 1H). 13C
1
the product as a white solid (507 mg, 97%). H NMR (400 MHz,
CD3OD): δ 7.47-7.42 (m, 1H), 7.18-7.11 (m, 2H), 5.39-5.35
(m, 1H), 4.39-4.35 (m, 1H), 3.35-3.28 (m, 2H), 3.25-3.20 (m,
1H), 2.97-2.91 (m, 1H), 1.35 (s, 9H). LCMS found for C17H22-
FN2NaO7S (M + Na)+: m/z ) 439.
tert-Butyl (1S)-1-(1H-benzimidazol-2-yl)-2-{4-[(5S)-1,1-dioxido-
3-oxoisothiazolidin-5-yl]-3-fluorophenyl}ethylcarbamate Tri-
fluoroacetate (65). (2S)-2-[(tert-Butoxycarbonyl)amino]-3-{4-[(5S)-
1,1-dioxido-3-oxoisothiazolidin-5-yl]-3-fluorophenyl}propanoic acid
(64) (402 mg, 0.965 mmol) in DMF (30 mL) was treated with
benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluoro-
phosphate (512 mg, 1.16 mmol). After 5 min at room temperature,
N,N-diisopropylethylamine (0.50 mL, 2.9 mmol) and 1,2-benzene-
diamine (7) (156 mg, 1.45 mmol) were added and the solution
stirred for 2 h. The solution was concentrated in vacuo and the
residue dissolved in acetic acid (5 mL) and heated at 40 °C for 2
h. The acetic acid was removed in vacuo, and the residue was
purified by preparative LCMS to afford the product as a white solid
NMR (125 MHz, d6-DMSO, 30 °C): δ 170.5, 162.5, 155.1, 142.1,
140.5 134.0, 133.0, 132.0, 131.9, 131.7, 131.4, 131.4, 130.7, 128.3,
126.8, 124.7, 124.6, 118.0, 117.6, 116.7, 115.3, 59.9, 53.3, 39.5,
38.0. HRMS calcd for C24H22FN4O5S2 (M + H)+: 631.0508; found,
631.0633.
4-Bromo-3-methylbenzaldehyde (69). A solution of methyl
4-bromo-3-methylbenzoate (68) (50.0 g, 218 mmol), lithium
tetrahydroborate (5.70 g, 262 mmol), and tetrahydrofuran (500 mL)
was stirred at 25 °C for 2 days. The reaction was cooled to 0 °C,
1
(373 mg, 64%). H NMR (400 MHz, CD3OD): δ 7.75-7.71 (m,
2H), 7.59-7.56 (m, 2H), 7.47-7.43 (m, 1H), 7.15-7.09 (m, 2H),

Protein Tyrosine Phosphatase 1B Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3785
1
quenched with saturated NH4Cl solution, and diluted with ethyl
acetate. The aqueous layers were extracted with ethyl acetate three
times, dried with sodium sulfate, filtered, and concentrated in vacuo.
The crude residue was purified by flash column chromatography
desired product (1.25 g, 91%). H NMR (400 MHz, CDCl3): δ
7.67 (d, J ) 8.0 Hz, 1Η), 7.37-7.25 (m, 5Η), 6.90 (m, 2Η), 5.22
(d, J ) 8.4 Hz, 1Η), 5.10 (m, 2Η), 4.64 (m, 1Η), 3.70 (s, 3Η),
3.06 (m, 2Η), 2.48 (s, 3Η), 1.33 (s, 12Η). LCMS found for C25H33-
BNO6 (M + H)+: m/z ) 454. A solution of methyl (2S)-2-
[(benzyloxy)carbonyl]amino-3-[3-methyl-4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)phenyl]propanoate (6.90 g, 15.2 mmol),
sodium periodate (16.3 g, 76.1 mmol), THF (150 mL), ammonium
acetate (4.69 g, 60.9 mmol), and water (150 mL) was stirred at 25
°C for 24 h. The reaction was diluted with ethyl acetate and 1 N
HCl solution. The aqueous solution was extracted once with ethyl
acetate. The combine organic solutions were washed with brine,
dried with sodium sulfate, filtered, and concentrated in vacuo. The
crude residue was crystallized in ethyl acetate to yield the desired
1
to yield the desired product (36.0 g, 82%). H NMR (400 MHz,
CDCl3): δ 7.49 (d, J ) 8.2 Hz, 1Η), 7.21 (d, J ) 1.3 Hz, 1Η),
7.02 (m, 1Η), 4.59 (s, 2Η), 2.39 (s, 3Η). LCMS found for C8H10-
BrO (M + H-H2O)+: m/z ) 183, 185. To a solution of dimethyl
sulfoxide (21.2 mL, 0.298 mol) in methylene chloride (150 mL)
was added oxalyl chloride (5.0 mL, 0.060 mol) at -78 °C under
an atmosphere of nitrogen. The resulting mixture was stirred at
the same temperature for 20 min. A solution of (4-bromo-3-
methylphenyl)methanol (6.0 g, 30 mmol) in methylene chloride
(50 mL) was cannulated into the reaction flask. After stirring for
1.0 h, triethylamine (21 mL, 0.15 mol) was added. The reaction
mixture was stirred at -78 °C for 1 h and warmed to room
temperature for 1 h. The reaction was quenched with 1 N HCl
solution, and the aqueous phase was separated and extracted once
with methylene chloride. The combined organic solutions were
washed with brine, dried over sodium sulfate, filtered, and
concentrated in vacuo. The crude residue was purified by flash
column chromatography to yield the desired product (5.7 g, 96%).
1H NMR (400 MHz, CDCl3): δ 9.96 (s, 1Η), 7.73 (m, 1Η), 7.71
(s, 1Η), 7.55 (m, 1Η), 2.49 (s, 3Η).
1
product (5.65 g, 80%). H NMR (400 MHz, CD3OD): δ 7.3 (m,
5Η), 7.16 (d, J ) 7.5 Hz, 1Η), 7.0 (m, 2Η), 5.02 (m, 2Η), 4.42
(dd, J ) 9.4, 5.3 Hz, 1Η), 3.68 (s, 3Η), 3.10 (dd, J ) 13.8, 5.3
Hz, 1Η), 2.88 (dd, J ) 13.6, 9.4 Hz, 1Η), 2.28 (s, 3Η). LCMS
found for C19H23BNO6 (M + H)+: m/z ) 372.
Methyl (2S)-2-[(benzyloxy)carbonyl]amino-3-[4-(2-tert-butyl-
1,1-dioxido-3-oxo-2,3-dihydroisothiazol-5-yl)-3-methylphenyl]-
propanoate (74). A solution of 2-tert-butyl-5-chloro-1,1-dioxo-
1,2-dihydro-1λ6-isothiazol-3-one (4) (1.86 g, 8.30 mmol), [4-((2S)-
2-[(benzyloxy)carbonyl]amino-3-methoxy-3-oxopropyl)-2-meth-
ylphenyl]boronic acid (73) (2.80 g, 7.54 mmol), [1,1′-bis(diphen-
ylphosphino)ferrocene]dichloropalladium(II) complexed with dichlo-
romethane (1:1) (924 mg, 1.13 mmol), potassium carbonate (5.21
g, 37.7 mmol), and 1,4-dioxane (38 mL) was degassed with nitrogen
and stirred at 80 °C for 24 h. The reaction was diluted with water
and extracted three times with ethyl acetate, dried with sodium
sulfate, filtered, and concentrated in vacuo. The crude residue was
purified by flash column chromatography to yield the desired
product (2.4 g, 62%). 1H NMR (400 MHz, CDCl3): δ 7.61 (d, J )
8.0 Hz, 1Η), 7.35 (m, 5Η), 7.03 (m, 2Η), 6.46 (s, 1Η), 5.27 (d, J
) 8.0 Hz, 1Η), 5.11 (m, 2Η), 4.67 (m, 1Η), 3.74 (s, 3Η), 3.14
(dd, J ) 11.9, 5.9 Hz, 1H), 3.07 (dd, J ) 11.9, 6.1 Hz, 1H), 2.36
(s, 3 Η), 1.73 (s, 9 Η). LCMS found for C26H31N2O7S (M + H)+:
m/z ) 515.
Methyl (2Z)-2-[(benzyloxy)carbonyl]amino-3-(4-bromo-3-
methylphenyl)acrylate (71). To a solution of N-(benzyloxycar-
bonyl)phosphonoglycine trimethyl ester (70) (10.0 g, 30.2 mmol)
in methylene chloride (200 mL) was added 1,8-diazabicyclo[5.4.0]-
undec-7-ene (4.96 mL, 33.2 mmol) at room temperature under an
atmosphere of nitrogen. After stirring for 10 min, a solution of
4-bromo-3-methylbenzaldehyde (6.01 g, 30.2 mmol) in methylene
chloride (50 mL) was cannulated into the reaction solution. The
resulting solution was stirred at room temperature for 1.5 h. The
reaction was diluted with ethyl acetate and quenched with 1.0 N
HCl solution. The aqueous layer was extracted twice with ethyl
acetate. The combined organic solutions were washed with brine,
dried over sodium sulfate, filtered, and concentrated in vacuo. The
crude residue was purified by flash column chromatography to yield
1
the desired product (10.4 g, 85%). H NMR (400 MHz, CDCl3):
Methyl (2S)-2-[(benzyloxy)carbonyl]amino-3-[4-(2-tert-bu-
tyl-1,1-dioxido-3-oxoisothiazolidin-5-yl)-3-methylphenyl]pro-
panoate (75). To a solution of methyl (2S)-2-[(benzyloxy)carbonyl]-
amino-3-[4-(2-tert-butyl-1,1-dioxido-3-oxo-2,3-dihydroisothiazol-
5-yl)-3-methylphenyl]propanoate (74) (570 mg, 1.11 mmol) in THF
(11 mL) was added 1 M l-selectride in THF (2.1 mL) at -78 °C.
After stirring for 10 min, the reaction was quenched with acetic
acid (1.0 mL) and diluted with water. The aqueous phase was
separated and extracted three times with ethyl acetate. The combined
organic solutions were dried with sodium sulfate, filtered, and
concentrated in vacuo. The crude residue was purified by flash
column chromatography to yield the desired product (501 mg, 88%).
1H NMR (400 MHz, CDCl3): δ 7.36-7.31 (m, 5Η), 7.20 (dd, J )
8.0, 3.1 Hz, 2Η), 7.00 (m, 2Η), 5.21 (d, J ) 8.6 Hz, 1Η), 5.10 (m,
2Η), 5.04 (t, J ) 8.3 Hz, 1H), 4.65 (m, 1Η), 3.72 (s, 3Η), 3.23
(dd, J ) 17.0, 8.4 Hz, 1Η), 3.1 (m, 3Η), 2.43 (s, 3Η), 1.65 (s,
9Η). LCMS found for C26H33N2O7S (M + H)+: m/z ) 517.
(2S)-2-[(Benzyloxy)carbonyl]amino-3-[4-(1,1-dioxido-3-ox-
oisothiazolidin-5-yl)-3-methylphenyl]propanoic Acid (76). A
solution of methyl (2S)-2-[(benzyloxy)carbonyl]amino-3-[4-(2-tert-
butyl-1,1-dioxido-3-oxoisothiazolidin-5-yl)-3-methylphenyl]pro-
panoate (75) (450 mg, 0.871 mmol) in trifluoroacetic acid (3 mL)
was heated in a microwave reactor at 130 °C for 5 min, and the
solvent was removed in vacuo. The residue was purified with
δ 7.44 (d, J ) 8.5 Hz, 1Η), 7.3 (m, 7Η), 7.17 (m, 1Η), 5.11 (s,
2Η), 3.82 (s, 3Η), 2.33 (s, 3Η). LCMS found for C19H18BrNO4Na
(M + Na)+: m/z ) 426.
Methyl (2S)-2-[(benzyloxy)carbonyl]amino-3-(4-bromo-3-
methylphenyl)propanoate (72). A solution of methyl-2-[(benzyl-
oxy)carbonyl]amino-3-(4-bromo-3-methylphenyl)acrylate (71) (10.4
g, 25.7 mmol) in ethanol (200 mL) was degassed with nitrogen.
(R,R)-(-)-1,2-Bis[(o-methoxyphenyl)(phenyl)phosphino]ethane(1,5-
cyclooctadiene) rhodium (I) tetrafluroborate (194 mg, 257 µmol)
was added under nitrogen. The reaction was placed under a
hydrogen atmosphere (50 psi) and shaken for 24 h. The solvent
was removed under reduced pressure. The crude residue was
purified by flash column chromatography to yield the desired
product (8.7 g, 83%). 1H NMR (400 MHz, CD3OD): δ 7.40 (d, J
) 8.2 Hz, 1Η), 7.33-7.25 (m, 5Η), 7.13 (s, 1Η), 6.91 (dd, J )
8.2, 1.9 Hz, 1Η), 5.00 (m, 2 Η), 4.42 (dd, J ) 9.6, 5.3 Hz, 1Η),
3.70 (s, 3Η), 3.09 (dd, J ) 13.9, 5.3 Hz, 1Η), 2.84 (dd, J ) 13.9,
9.8 Hz, 1Η), 2.32 (s, 3Η). LCMS found for C19H20BrNO4Na (M
+ Na)+: m/z ) 428.
[4-((2S)-2-[(Benzyloxy)carbonyl]amino-3-methoxy-3-oxopro-
pyl)-2-methylphenyl]boronic Acid (73). To a mixture of methyl
(2S)-2-[(benzyloxy)carbonyl]amino-3-(4-bromo-3-methylphenyl)-
propanoate (72) (1.23 g, 3.03 mmol), 1,4-dioxane (8 mL), triethyl-
amine (1.7 mL, 12 mmol), palladium acetate (17 mg, 0.076 mmol),
and O-(dicyclohexylphosphino)biphenyl (106 mg, 0.303 mmol) was
added dropwise 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.10 mL,
7.57 mmol). The greenish reaction mixture was stirred at 80 °C
for 30 min. The reaction was quenched with saturated NH4Cl
solution. The aqueous solution was extracted twice with ethyl
acetate. The organic solutions were washed with brine, dried with
sodium sulfate, filtered, and concentrated in vacuo. The crude
residue was purified by flash column chromatography to yield the
1
preparative LCMS to give the desired product (210 mg, 74%). H
NMR (400 MHz, CD3OD): δ 7.45 (dd, J ) 7.6, 6.0 Hz, 1Η), 7.23-
7.17 (m, 2Η), 5.47 (t, J ) 8.1 Hz, 1Η), 4.34 (m, 1Η), 3.81 (s,
3Η), 3.37-3.30 (m, 2Η), 3.25 (d, J ) 5.9 Hz, 1Η), 3.13 (dd, J )
14.4, 7.8 Hz, 1Η), 2.49 (s, 3Η). LCMS found for C14H20N2O5S
(M + H)+: m/z ) 327. Methyl (2S)-2-amino-3-[4-(1,1-dioxido-3-
oxoisothiazolidin-5-yl)-3-methylphenyl]propanoate trifluoroacetate
(286 mg, 0.649 mmol) in methanol (7 mL) was treated with
triethylamine (362 µL, 2.60 mmol) and cooled to 0 °C. Benzyl

3786 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
Combs et al.
chloroformate (116 µL, 0.812 mmol) was added and the solution
stirred for 2 h. Lithium hydroxide (4.0 M) in water (0.81 mL) was
added and the solution stirred for additional 1 h. The reaction
soluiton was acidified with 1 N HCl and extracted with ethyl acetate.
The crude product was used in the next step without purification.
LCMS found for C21H23N2O7S (M + H)+: m/z ) 447.
) 8.1 Hz, 1H), 7.00 (d, J ) 7.7 Hz, 1H), 6.82 (s, 1H), 5.38 (t, J
) 8.4 Hz, 1H), 4.91 (t, J ) 7.9 Hz, 1H), 3.27 (m, 2H), 3.22 (m,
2H), 2.24 (s, 3H); 13C NMR (125 MHz, CD3OD): δ 170.9, 155.0,
140.9, 140.6, 137.5, 134.2, 132.8, 132.5, 130.3, 129.4, 129.2, 128.4,
128.0, 127.4, 115.4, 62.9, 53.9, 40.5, 40.0, 19.9; HRMS calculated
for C25H25N4O5S2 (M + H)+: m/z ) 525.1245.
N-((1S)-1-(1H-Benzimidazol-2-yl)-2-{4-[(5R)-1,1-dioxido-3-
oxoisothiazolidin-5-yl]-3-methylphenyl}ethyl)benzene-
sulfonamide (79b). The title compound was prepared using a
procedure similar to that used to prepare 79a using a single
enantiomer with (R)-IZD of 78. 1H NMR (500 MHz, CD3OD): δ
7.68 (m, 2H), 7.64 (dt, J ) 7.2, 1.4 Hz, 2H), 7.53 (m, 2H), 7.50
(tt, J ) 7.5, 1.2 Hz, 1H), 7.37 (tt, J ) 7.5, 1.2 Hz, 2H), 7.19 (d, J
) 8.1 Hz, 1H), 6.93 (d, J ) 1.4 Hz, 1H), 6.86 (dd, J ) 8.0, 1.6
Hz, 1H), 5.32 (t, J ) 8.1 Hz, 1H), 4.90 (dd, J ) 8.4, 7.2 Hz, 1H),
3.26 (dd, J ) 11.6, 8.1, 2H), 3.21 (t, J ) 7.2 Hz, 2H), 2.31 (s,
3H); 13C NMR (125 MHz, CD3OD): δ 172.3, 154.7, 140.6, 140.4,
137.2, 134.5, 133.3, 132.6, 130.4, 129.7, 129.3, 128.3, 128.1, 127.6,
115.4, 62.6, 54.1, 40.4, 39.5, 20.2; HRMS calculated for
C25H24N4O5S2Na (M + Na)+: 547.1075; found, 547.1075.
N-((1S)-1-(1H-Benzimidazol-2-yl)-2-{4-[(5S)-1,1-dioxido-3-
oxoisothiazolidin-5-yl]-3-methylphenyl}ethyl)-4-bromo-3-(tri-
fluoromethyl)benzenesulfonamide (79c). Compound 79c was
prepared using a procedure similar to that used to prepare compound
79a. 1H NMR (500 MHz, CD3OD, 30 °C): δ 7.97 (d, J ) 2.3 Hz,
1H), 7.76 (d, J ) 8.4 Hz, 1H), 7.70 (m, 2H), 7.60 (dd, J ) 8.5, 2.3
Hz, 1H), 7.57 (m, 2H), 7.29 (d, J ) 8.1 Hz, 1H), 7.08 (dd, J )
8.0, 1.3 Hz, 1H), 6.93 (d, J ) 1.3 Hz, 1H), 5.38 (t, J ) 8.1 Hz,
1H), 5.05 (dd, J ) 8.6, 7.4 Hz, 1H), 3.33 (m, 2H), 3.28 (m, 2H),
2.28 (s, 3H); 13C NMR (125 MHz, CD3OD, 30 °C): δ 170.9, 153.9,
141.1, 140.6, 137.7, 132.8, 132.7, 132.5, 131.8, 129.4, 129.1, 128.5,
128.0, 127.4, 123.7, 120.3, 115.3, 115.2, 62.6, 53.8, 40.0, 38.9,
20.2; HRMS calculated for C26H23BrF3N4O5S2 (M + H)+: 671.0245;
found, 671.0218.
N-((1S)-1-(1H-Benzimidazol-2-yl)-2-{4-[(5S)-1,1-dioxido-3-
oxoisothiazolidin-5-yl]-3-methylphenyl}ethyl)-2-cyanoben-
zenesulfonamide (79d). Compound 79d was prepared using a
procedure similar to that used to prepare compound 79a. 1H NMR
(500 MHz, CD3OD, 30 °C): δ 7.90 (d, J ) 7.9 Hz, 1H), 7.80 (m,
2H), 7.69 (d, J ) 7.4 Hz, 1H), 7.66 (t, J ) 7.7 Hz, 1H), 7.62 (d,
J ) 7.4 Hz, 1H), 7.61 (m, 2H), 7.25 (d, J ) 7.9 Hz, 1H), 7.08 (dd,
J ) 7.8, 1.5 Hz, 1H), 7.01 (d, J ) 1.1 Hz, 1H), 5.39 (t, J ) 8.5
Hz, 1H), 5.18 (dd, J ) 10.2, 4.5 Hz, 1H), 3.34 (dd, J ) 14.3, 4.8
Hz, 1H), 3.31 (m, 2H), 3.21 (dd, J ) 14.4, 10.6 Hz, 1H), 2.31 (s,
3H); 13C NMR (125 MHz, CD3OD, 30 °C): δ 171.1, 154.7, 142.9,
140.9, 137.6, 137.5, 134.9, 134.7, 133.2, 133.0, 131.2, 130.1, 129.2,
129.0, 128.5, 117.3, 115.8, 111.4, 62.7, 54.3, 40.2, 39.4, 20.8;
HRMS calculated for C26H24N5O5S2 (M + H)+: 550.1219; found,
550.1194.
Benzyl (1S)-1-(1H-benzimidazol-2-yl)-2-[4-(1,1-dioxido-3-ox-
oisothiazolidin-5-yl)-3-methylphenyl]ethylcarbamate (77). A so-
lution of (2S)-2-[(benzyloxy)carbonyl]amino-3-[4-(1,1-dioxido-3-
oxoisothiazolidin-5-yl)-3-methylphenyl]propanoic acid (76) (200
mg, 0.448 mmol) in DMF (2 mL) was treated with benzotriazol-
1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (0.218
g, 0.493 mmol). After stirring for 10 min at 0 °C, a solution of
1,2-benzenediamine (7) (72.7 mg, 0.672 mmol) and N,N-diisopro-
pylethylamine (0.39 mL, 2.24 mmol) in DMF (1 mL) was
cannulated into the reaction flask. The solution was stirred at 25
°C for 2 h. The solution was diluted with ethyl acetate and washed
with satd. aqueous sodium bicarbonate solution and 1.0 N hydro-
chloric acid solution. The organic phase was dried over sodium
sulfate, filtered, and concentrated in vacuo. The crude residue was
purified by preparative LCMS to yield the desired product (196
1
mg, 82%). H NMR (400 MHz, CD3OD): δ 7.47 (d, J ) 8.0 Hz,
0.5Η), 7.39-7.22 (m, 9Η), 7.12 (d, J ) 7.8 Hz, 0.5Η), 7.07 (s,
1Η), 6.82 (d, J ) 7.6 Hz, 0.5Η), 6.71 (d, J ) 7.4 Hz, 0.5Η), 5.50
(t, J ) 8.2 Hz, 1Η), 5.10 (s, 2Η), 4.47 (m, 1Η), 3.3 (m, 2Η), 3.07
(m, 2Η), 2.48 (s, 1.5Η), 2.38 (s, 1.5Η). LCMS found for
C27H29N4O6S (M + H)+: m/z ) 537. A solution of benzyl (1S)-
2-[(2-aminophenyl)amino]-1-[4-(1,1-dioxido-3-oxoisothiazolidin-5-
yl)-3-methylbenzyl]-2-oxoethylcarbamate trifluoroacetate (1.20 g,
1.84 mmol) and acetic acid (40 mL) was stirred at 40 °C for 2 h.
The solvent was removed in vacuo. The crude product was used in
the next step without purification. LCMS found for C27H27N4O5S
(M + H)+: m/z ) 519.
Benzyl [(1S)-2-[4-((5S)-1,1-dioxido-3-oxo-2-{[2-(trimethylsilyl)-
ethoxy]methyl}isothiazolidin-5-yl)-3-methylphenyl]-1-(1-{[2-(tri-
methylsilyl)propoxy]methyl}-1H-benzimidazol-2-yl)ethyl]car-
bamate (78). A solution of benzyl (1S)-1-(1H-benzimidazol-2-yl)-
2-[4-(1,1-dioxido-3-oxoisothiazolidin-5-yl)-3-methylphenyl]-
ethylcarbamate acetate (77) (956 mg, 1.65 mmol), 2-trimethylsilyl)-
ethoxy methyl chloride (0.73 mL, 4.1 mmol), N,N-diisopropyl-
ethylamine (1.73 mL, 9.91 mmol), and methylene chloride (30 mL)
was stirred at 25 °C for 3 h. The reaction was diluted with 1 N
HCl solution and extracted three times with ethyl acetate, dried
with sodium sulfate, filtered, and concentrated in vacuo. The crude
residue was purified by normal phase chiral HPLC (ChiralCel OD-H
(20 × 250 mm, 5 µm), 30% EtOH/70% hexanes, 15 mL/min, 30
1
°C) to yield 78 (390 mg, 30%). H NMR (400 MHz, CD3OD): δ
7.79 (m, 1Η), 7.64 (m, 1Η), 7.42-7.30 (m, 9Η), 7.16 (s, 1Η),
5.64 (d, J ) 11.5 Hz, 1Η), 5.52-5.45 (m, 3H), 5.17-5.00 (m,
4Η), 3.76 (t, J ) 8.2 Hz, 2Η), 3.58-3.37 (m, 6Η), 2.42 (s, 3Η),
1.02 (t, J ) 8.0 Hz, 2Η), 0.96-0.86 (m, 2Η), 0.11 (s, 9Η), 0.00
(s, 9Η). LCMS found for C39H55N4O7SSi2 (M + H)+: m/z ) 779.
N-((1S)-1-(1H-Benzimidazol-2-yl)-2-{4-[(5S)-1,1-dioxido-3-
oxoisothiazolidin-5-yl]-3-methylphenyl}ethyl)benzene-
sulfonamide (79a). A solution of benzyl [(1S)-2-[4-((5S)-1,1-
dioxido-3-oxo-2-{[2-(trimethylsilyl)ethoxy]methyl}isothiazolidin-
5-yl)-3-methylphenyl]-1-(1-{[2-(trimethylsilyl)propoxy]methyl}-
1H-benzimidazol-2-yl)ethyl]carbamate (78) (50.0 mg, 64.2 µmol),
methanol (3.5 mL), and 10% palladium on carbon (15 mg, 14 µmol)
was degassed and placed under a hydrogen balloon at 25 °C for
1.5 h. The reaction was diluted with methanol, filtered through
Celite, and concentrated in vacuo. The crude product (28.0 mg,
34.7 µmol), methylene chloride (1.7 mL), N,N-diisopropylethyl-
amine (30 µL, 0.17 mmol), and benzenesulfonyl chloride (66) (8.9
µL, 70 µmol) were stirred at 25 °C for 5 h. The reaction was
concentrated and treated with trifluoroacetic acid (1.3 mL). The
reaction was heated at 130 °C for 2 min in the microwave. The
reaction was concentrated and purified by preparative LCMS to
yield desired product 79a (6.5 mg, 29%). 1H NMR (500 MHz, CD3-
OD): δ 7.69 (m, 2H), 7.63 (d, J ) 7.6 Hz, 2H), 7.53 (m, 2H),
7.50 (tt, J ) 7.4, 1.2 Hz, 1H), 7.39 (t, J ) 7.7 Hz, 2H), 7.27 (d, J
N-((1S)-1-(1H-Benzimidazol-2-yl)-2-{4-[(5S)-1,1-dioxido-3-
oxoisothiazolidin-5-yl]-3-methylphenyl}ethyl)-3-chloroben-
zenesulfonamide (79e). Compound 79e was prepared in a manner
1
similar to that used for compound 79a. H NMR (500 MHz, d6-
DMSO +10 µL TFA, 30 °C): δ 9.26 (d, J ) 6.1 Hz, 1H), 7.80
(m, 2H), 7.56 (m, 2H), 7.53 (s, 1H), 7.46 (d, J ) 8.1 Hz, 1H), 7.41
(d, J ) 8.0 Hz, 1H), 7.32 (t, J ) 8.0 Hz, 1H), 7.26 (d, J ) 8.0 Hz,
1H), 7.03 (s, 1H), 7.02 (d, J ) 8.1 Hz, 1H), 5.49 (t, J ) 8.6 Hz,
1H), 5.03 (dd, J ) 9.2, 4.5 Hz, 1H), 3.39 (dd, J ) 17.1, 9.2 Hz,
1H), 3.28 (dd, J ) 14.1, 4.7 Hz, 1H), 3.22 (dd, J ) 17.3, 8.6 Hz,
1H), 3.12 (dd, J ) 13.9, 9.6 Hz, 1H), 2.27 (s, 3H); 13C NMR (125
MHz, d6-DMSO +10 µL of TFA, 30 °C): δ 168.7, 152.6, 140.5,
138.3, 136.0, 133.7, 132.5, 131.3, 130.8, 130.6, 128.1, 126.8, 126.4,
126.2, 126.0, 124.7, 114.3, 60.6, 51.6, 37.7, 37.3, 19.4; HRMS
calculated for C25H24ClN4O5S2 (M + H)+: 559.0881; found,
559.0881.
Methyl (2S)-2-[(tert-butoxycarbonyl)amino]-3-(3-chloro-4-
iodophenyl)propanoate (81). Methyl (2S)-3-(4-aminophenyl)-2-
[(tert-butoxycarbonyl)amino]propanoate (80) (1.0 g, 3.22 mmol)
and N-chlorosuccinimide (474 mg, 3.55 mmol) were dissolved in
DMF (20 mL) and allowed to stir under an atmosphere of nitrogen
for 24 h. The reaction was quenched with water and diluted with

Protein Tyrosine Phosphatase 1B Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3787
ethyl acetate (100 mL). The organic phase was separated, dried
over sodium sulfate, filtered, and concentrated in vacuo. The crude
material was purified by silica gel column (5-20% ethyl acetate/
was dissolved in trifluoroacetic acid (3 mL) and heated in the
microwave at 130 °C for 5 min. The reaction was concentrated in
vacuo and then dissolved in methylene chloride (5 mL). The
solution was treated with triethylamine (364 µL, 2.61 mmol) and
di-tert-butyl-dicarbonate (228 mg, 0.152 mmol). The reaction was
stirred at room temperature for 2 h and then treated with an aqueous
lithium hydroxide solution (4.0 M, 0.52 mL) and allowed to stir
for 2 h. The reaction was quenched with aqueous hydrochloric acid
solution (1.0 N, 5 mL) and purified by reverse phase HPLC to
afford the product as a white solid (184 mg, 81%). 1H NMR (400
MHz, CD3OD): δ 7.44-7.41 (m, 1H), 7.18-7.13 (m, 1H), 5.37
(brs, 1H), 4.36-4.33 (m, 1H), 3.38-3.32 (m, 3H), 2.98-2.95 (m,
1H), 1.37 (s, 9H). LCMS found for C17H21ClN2O7S (M + H)+:
m/z ) 433.
tert-Butyl (1S)-1-(1H-benzimidazol-2-yl)-2-{3-chloro-4-[(5S)-
1,1-dioxido-3-oxoisothiazolidin-5-yl]phenyl}ethylcarbamate Tri-
fluoroacetate (86). tert-Butyl (1S)-2-[(2-aminophenyl)amino]-1-
{3-chloro-4-[(5S)-1,1-dioxido-3-oxo-isothiazolidin-5-yl]benzyl}-2-
oxoethylcarbamate (85) (75 mg, 0.17 mmol) in DMF (2 mL) was
treated with benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate (91.8 mg, 0.207 mmol). After 5 min at room
temperature, N,N-diisopropylethylamine (0.090 mL, 0.519 mmol)
and 1,2-benzendiamine 7 (28 mg, 0.26 mmol) were added and the
solution stirred for 1 h at room temperature. The solution was
concentrated and then heated in acetic acid (5 mL) at 40 °C for 1
h. The solution was concentrated in vacuo. Purification by prepara-
tive LCMS afforded the product as a white solid (77 mg, 72%). 1H
NMR (400 MHz, CD3OD): δ 7.76-7.72 (m, 2H), 7.59-7.55 (m,
2H), 7.43-7.40 (m, 1H), 7.17-7.12 (m, 2H), 5.44-5.40 (m, 2H),
3.40-3.23 (m, 3H), 3.13-3.11 (m, 1H), 1.37 (s, 9H). LCMS found
for C24H26ClN4O5S (M + H)+: m/z ) 506.
N-(1S)-1-(1H-Benzimidazol-2-yl)-2-{3-chloro-4-[(5S)-1,1-di-
oxido-3-oxoisothiazolidin-5-yl]phenyl}ethylbenzenesul-
fonamide Trifluoroacetate (87). (2S)-N-(2-Aminophenyl)-3-{3-
chloro-4-[(5S)-1,1-dioxido-3-oxoisothiazolidin-5-yl]phenyl}-2-[(phen-
ylsulfonyl)-amino]propanamide trifluoroacetate (86) (22.9 mg, 0.037
mmol) was treated with trifluoroacetic acid (1 mL) in methylene
chloride (5 mL). After 1 h at room temperature, the solution was
concentrated in vacuo, and the residue was dissolved in methanol
(2.0 mL). Triethylamine (0.050 mL, 0.36 mmol) and benzenesulfo-
nyl chloride (66) (0.028 mL, 0.022 mmol) were added. After 3 h
at room temperature, the solution was concentrated. Purification
by preparative LCMS afforded the product as a white solid (18.5
mg, 76%). 1H NMR (400 MHz, d6-DMSO, 30 °C): δ 9.02 (d, J )
6.2 Hz, 1H), 7.76 (m, 2H), 7.50 (m, 2H), 7.49 (m, 2H), 7.43 (m,
2H), 7.40 (d, J ) 8.2 Hz, 1H), 7.34 (d, J ) 1.5 Hz, 1H), 7.31 (m,
2H), 7.16 (dd, J ) 8.1, 1.3 Hz, 1H), 5.56 (t, J ) 8.2 Hz, 1H), 4.91
(m, 1H), 3.42 (dd, J ) 17.2, 8.5 Hz, 1H), 3.36 (dd, J ) 17.0, 7.9
Hz, 1H), 3.29 (dd, J ) 13.9, 3.8 Hz, 1H), 3.11 (dd, J ) 13.6, 10.0
Hz, 1H) 13C NMR (125 MHz, d6-DMSO, 30 °C): δ 168.9, 152.9,
138.8, 138.7, 134.3, 132.5, 132.0, 130.3, 129.8, 128.8, 128.2, 126.5,
125.8, 125.4, 114.5, 60.7, 51.7, 37.4. HRMS calcd for C24H22-
ClN4O5S2 (M + H)+: 545.0721; found, 545.0729.
1
hexanes) to afford the product as a clear oil (645 mg, 61%). H
NMR (400 MHz, CD3OD): δ 7.05 (s, 1H), 6.90-6.87 (m, 1H),
6.76-6.74 (m, 1H), 4.27-4.23 (m, 1H), 3.68 (s, 3H), 2.97-2.92
(m, 1H), 2.78-2.72 (m, 1H), 1.39 (s, 9H); LCMS found for C10H14-
ClN2O2 (M + H)+: m/z ) 229. Methyl (2S)-3-(4-amino-3-
chlorophenyl)-2-[(tert-butoxycarbonyl)amino]propanoate (3.00 g,
9.12 mmol) in aqueous hydrochloric acid solution (1.0 N, 100 mL)
at 0 °C was treated with a solution of sodium nitrite (692 mg, 10.0
mmol) in water (10 mL). The solution was stirred at 0 °C for 30
min. A solution of potassium iodide (1.89 g, 11.4 mmol) in water
(10 mL) was added and the solution stirred 30 min at room
temperature and then 10 min at 35 °C. The solution was diluted
with ethyl acetate (200 mL) and quenched with an aqueous sodium
thiosulfate solution (1.0 N, 500 mL), and the organic phase was
separated. The organic layer was washed with aqueous hydrochloric
acid solution (1.0 N, 100 mL), brine (100 mL), dried over sodium
sulfate, and concentrated in vacuo. Purification by silica gel
chromatography (10-40% ethyl acetate/hexanes) afforded the
product as a yellow solid (2.1 g, 53%). 1H NMR (400 MHz, CD3-
OD): δ 7.80 (d, J ) 8.0 Hz, 1H), 7.37 (s, 1H), 6.90 (d, J ) 8.2
Hz, 1H), 4.36-4.33 (m, 1H), 3.70 (s, 3H), 3.12-3.07 (m, 1H),
2.86-2.80 (m, 1H), 1.37 (s, 9H). LCMS found for C10H12ClINO2
(M + H)+: m/z ) 340.
Methyl (2S)-2-[(tert-butoxycarbonyl)amino]-3-[4-(2-tert-butyl-
1,1-dioxido-3-oxo-2,3-dihydroisothiazol-5-yl)-3-chlorophenyl]-
propanoate (83). 2-tert-Butylisothiazol-3(2H)-one 1,1-dioxide (82)
(284 mg, 1.50 mmol), palladium acetate (48 mg, 0.21 mmol), and
tetra-N-butylammonium chloride (238 mg, 0.858 mmol) were
combined. To this mixture was added methyl (2S)-2-[(tert-butoxy-
carbonyl)amino]-3-(3-chloro-4-iodophenyl)propanoate (377 mg,
0.858 mmol) in DMF (4 mL). The reaction was treated with
triethylamine (0.60 mL, 4.3 mmol). The reaction was degassed and
then allowed to stir under an atmosphere of nitrogen at 65 °C for
2 h. The reaction was quenched with water (50 mL), diluted with
ethyl acetate (100 mL), and washed with an aqueous hydrochloric
acid solution (1.0 N, 100 mL). The organic solution was dried over
sodium sulfate, filtered. and concentrated in vacuo. The crude
material was purified by silica gel chromatography (5-25% ethyl
acetate/hexanes) to afford the product as a white solid (480 mg,
64%). 1H NMR (400 MHz, CD3OD): δ 7.80 (d, J ) 8.2 Hz, 1H),
7.54 (s, 1H), 7.35 (d, J ) 8.0 Hz, 1H), 7.03 (s, 1H), 4.44-4.40
(m, 1H), 3.73 (s, 3H), 3.25-3.20 (m, 1H), 2.95 (dd, J ) 10.0,
13.7 Hz, 1H), 1.70 (s, 9H), 1.37 (s, 9H). LCMS found for C17H22-
ClN2O5S (M + H)+: m/z ) 401.
Methyl (2S)-2-[(tert-butoxycarbonyl)amino]-3-{4-[(5S)-(2-
tert-butyl-1,1-dioxido-3-oxoisothiazolidin-5-yl]-3-chlorophen-
yl}propanoate (84). Methyl (2S)-2-[(tert-butoxycarbonyl)amino]-
3-[4-(2-tert-butyl-1,1-dioxido-3-oxo-2,3-dihydroisothiazol-5-yl)-3-
chlorophenyl]propanoate (83) (300 mg, 0.598 mmol) in THF (6
mL) was cooled to 0 °C and treated with a lithium tetrahydroborate
solution in THF (2.0 M, 0.33 mL). The reaction was allowed to
stir for 0.5 h at room temperature. After the addition of acetic acid
(0.5 mL), the solution was diluted with ethyl acetate (100 mL),
washed with water (100 mL), separated, dried over sodium sulfate,
filtered, and concentrated in vacuo. The crude material was purified
by silica gel chromatography (5-30% ethyl acetate/hexanes) to
afford the product as a white solid (162 mg, 54%). The two
diastereomers were separated by normal phase chiral HPLC
(ChiralCel OD-H (20 × 250 mm, 5 µm), 30% EtOH/70% hexanes,
15 mL/min, 30 °C) to yield 84 (103 mg, 48%). 1H NMR (400 MHz,
CD3OD): δ 7.45-7.42 (m, 2H), 7.31-7.27 (m, 1H), 5.59 (t, J )
8.0 Hz, 1H), 4.39-4.36 (m, 1H), 3.70 (s, 3H), 3.38-3.33 (m, 1H),
3.25-3.14 (m, 2H), 2.95-2.92 (m, 1H), 1.67 (s, 9H), 1.37 (s, 9H).
LCMS found for C17H24ClN2O5S (M + H)+: m/z ) 403.
Methyl (2S)-3-(3-bromo-4-iodophenyl)-2-[(tert-butoxycarbo-
nyl)amino-]propanoate (88). Methyl (2S)-2-[(tert-butoxycarbonyl)-
amino]-3-(4-nitrophenyl)propanoate (80) (44.0 g, 136 mmol) in
methanol (750 mL) and water (75 mL) was treated with ammonium
chloride (10.9 g, 203 mmol) and then zinc (71 g, 1.1 mol). The
solution was stirred at reflux for 1 h. The solution was filtered
through Celite, the residue was concentrated under vacuum to
remove the methanol, and the water solution was extracted with
ethyl acetate (500 mL) and dried over sodium sulfate. The product
1
was a yellow foam (38.2 g, 96%). H NMR (400 MHz, CD3OD):
δ 6.91 (d, J ) 8.4 Hz, 2H), 6.65 (d, J ) 8.3 Hz, 2H), 4.28-4.22
(m, 1H), 3.66 (s, 3H), 2.98-2.90 (m, 1H), 2.86-2.78 (m, 1H),
1.39 (s, 9H). LCMS found for C15H22N2O4Na (M + Na)+: m/z )
317.
(2S)-2-(tert-Butoxycarbonyl)amino-3-{3-chloro-4-[(5S)-1,1-di-
oxido-3-oxoisothiazolidin-5-yl]phenyl}propanoic Acid (85). Meth-
yl (2S)-2-amino-3-{3-chloro-4-[(5S)-1,1-dioxido-3-oxoisothiazolidin-
5-yl]phenyl}-propanoate trifluoroacetate (84) (350 mg, 0.70 mmol)
Methyl (2S)-3-(4-aminophenyl)-2-[(tert-butoxycarbonyl)amino]-
propanoate (17.2 g, 58.4 mmol) in DMF (400 mL) was treated with
N-bromosuccinimide (11.4 g, 64.3 mmol). The solution was stirred
at room temperature overnight. The solution was diluted with ethyl

3788 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13
Combs et al.
acetate (500 mL), washed with water (500 mL), a satd. aqueous
sodium bicarbonate solution (500 mL), and an aqueous hydrochloric
acid solution (1.0 N, 500 mL). The organic phase was dried over
sodium sulfate and concentrated in vacuo. Silica gel chromatography
(10-50% ethyl acetate/hexanes) afforded the product as a viscous,
concentrated in vacuo to give a white foam (420 mg, 99%). The
product was used in subsequent steps without purification. 1H NMR
(500 MHz, CDCl3): δ 7.68 (dd, J ) 14.2, 1.6 Hz, 1H), 7.56-7.51
(m, 1H), 7.40-7.36 (m, 1H), 5.65 (t, J ) 7.4 Hz, 1H), 4.39 (t, J )
6.8 Hz, 1H), 3.82 (s, 3H), 3.47-3.42 (m, 1H), 3.36-3.25 (m, 2H),
2.18-3.11 (m, 1H). LCMS found for C13H16BrN2O5S (M + Na)+:
m/z ) 391.
1
slightly yellow gel (20.1 g, 92%). H NMR (400 MHz, CD3OD):
δ 7.21 (d, J ) 1.7 Hz, 1H), 6.92 (dd, J ) 8.2, 1.8 Hz), 6.75 (d, J
) 8.2 Hz, 1H), 4.27-4.23 (m, 1H), 3.68 (s, 3H), 2.97-2.92 (m,
1H), 2.77-2.72 (m, 1H), 1.39 (s, 9H). LCMS found for C15H21-
BrN2O4Na (M + Na)+: m/z ) 396.
Methyl (2S)-2-amino-3-[3-bromo-4-(1,1-dioxido-3-oxoisothia-
zolidin-5-yl)phenyl]-propanoate trifluoroacetate (83.8 mg, 0.166
mmol) in methanol (2 mL) was treated with triethylamine (92 µL,
0.66 mmol) and chilled to 0 °C. Benzyl chloroformate (20.8 µL,
0.146 mmol) was added and the solution stirred 2 h at 0 °C. An
aqueous lithium hydroxide solution (4.0 M, 0.21 mL) was added
and the solution stirred 2 h. Purification by preparative LCMS afford
Methyl (2S)-3-(4-amino-3-bromophenyl)-2-[(tert-butoxycarbo-
nyl)amino]-propanoate (19.8 g, 53.0 mmol) suspended in 1 N
aqueous hydrochloric acid (500 mL) was treated dropwise at 0 °C
with sodium nitrite (3.66 g, 53.0 mmol) in water (75 mL). After
15 min at 0 °C, potassium iodide (8.81 g, 53.1 mmol) in water (75
mL) was added, and the solution was heated at 40 °C for 15 min.
The solution was quenched with a satd. aqueous sodium thiosulfate
solution (100 mL) and extracted with ethyl acetate (500 mL). The
organic phase was washed with a 0.1 N hydrochloric acid solution
(500 mL) and a satd. aqueous sodium bicarbonate solution (500
mL) and dried over sodium sulfate. Purification by silica gel
chromatography (10-40% ethyl acetate/hexanes) afforded the
product as slightly yellow solid (18.7 g, 73%). 1H NMR (400 MHz,
CD3OD): δ 7.80 (d, J ) 8.0 Hz, 1H), 7.55 (d, J ) 1.8 Hz, 1H),
6.92 (dd, J ) 8.0, 1.9 Hz), 4.36-4.29 (m, 1H), 3.71 (s, 3H), 3.10-
3.04 (m, 1H), 2.85-2.79 (m, 1H), 1.37 (s, 9H). LCMS found for
C15H19BrINO4Na (M + Na)+: m/z ) 506.
Methyl (2S)-3-[3-bromo-4-(2-tert-butyl-1,1-dioxido-3-oxo-2,3-
dihydroisothiazol-5-yl)phenyl]-2-[(tert-butoxycarbonyl)amino]-
propanoate (89). Methyl (2S)-3-(3-bromo-4-iodophenyl)-2-[(tert-
butoxycarbonyl)amino]-propanoate (2.35 g, 4.85 mmol), 2-tert-
butylisothiazol-3(2H)-one 1,1-dioxide (82) (1.61 g, 8.49 mmol),
palladium acetate (218 mg, 0.971 mmol), tetra-N-butylammonium
chloride (1.35 g, 4.85 mmol), and then triethylamine (2.03 mL,
14.6 mmol) were dissolved in DMF (40 mL). The solution was
degassed and then stirred with heating at 70 °C under nitrogen for
120 min. LCMS indicated an absence of starting material. The
solution was diluted with ethyl acetate (150 mL) and washed with
water (150 mL) and a 1.0 N aqueous hydrochloric acid solution
(150 mL). The organic phase was filtered through Celite with ethyl
acetate washing. The organic solution was dried over sodium sulfate,
concentrated in vacuo, and then purified by silica gel chromatog-
raphy (10-25% ethyl acetate/hexanes) to afford the product as a
yellow solid (1.38 g, 52%). 1H NMR (500 MHz, CD3OD): δ 7.80
(d, J ) 8.2 Hz, 1H), 7.55 (s, 1H), 7.23 (d, J ) 8.2 Hz, 1H), 6.89
(s, 1H), 5.08-5.05 (m, 1H), 4.62-4.59 (m, 1H), 3.76 (s, 3H), 3.23-
3.20 (m, 1H), 3.06-3.02 (m, 1H), 1.79 (s, 9H), 1.43 (s, 9H). LCMS
found for C22H29BrN2O7SNa (M + Na)+: m/z ) 567.
1
the product as a white solid (69 mg, 81%). H NMR (400 MHz,
CDCl3): δ 7.48 (brs, 1H), 7.46-7.43 (m, 1H), 7.31-0.7.26 (m,
6H), 5.65 (t, J ) 8.2 Hz, 1H), 5.03 (s, 2H), 4.45-4.41 (m, 1H),
3.45 (dd, J ) 17.5, 8.5 Hz, 1H), 3.36-3.19 (m, 2H), (2.94 dd, J )
14.2, 9.7 Hz, 1H). LCMS found for C20H20BrN2O7S (M + H)+:
m/z ) 511.
Benzyl (1S)-1-(1H-benzimidazol-2-yl)-2-[3-bromo-4-(1,1-di-
oxido-3-oxoisothiazolidin-5-yl)phenyl]ethylcarbamate Trifluo-
roacetate (92). (2S)-2-[(Benzyloxy)-carbonyl]amino-3-[3-bromo-
4-(1,1-dioxido-3-oxoisothiazolidin-5-yl)phenyl]propanoic acid (91)
(65.3 mg, 0.128 mmol) in DMF (4 mL) was treated with
benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluoro-
phosphate (67.8 mg, 0.153 mmol). After 5 min, 1,2-benzenediamine
7 (20.7 mg, 0.192 mmol) and N,N-diisopropylethylamine (111 µL,
0.638 mmol) were added and the solution stirred at room temper-
ature for 2 h. Purification by preparative LCMS afforded the product
1
as a white solid (58 mg, 76%). H NMR (400 MHz, CD3OD): δ
7.72 (s, 1H), 7.56-7.23 (m, 10H), 6.91-6.85 (m, 1H), 5.67 (t, J
) 7.7 Hz, 1H), 5.09 (s, 2H), 4.50-4.47 (m, 1H), 3.46 (dd, J )
16.5, 8.8 Hz, 1H), 3.34-3.24 (m, 1H), 3.26-3.08 (m, 2H). LCMS
found for C26H26BrN4O6S (M + H)+: m/z ) 601.
Benzyl (1S)-2-[(2-aminophenyl)amino]-1-[3-bromo-4-(1,1-dioxido-
3-oxoisothiazolidin-5-yl)benzyl]-2-oxoethylcarbamate (28.2 mg,
0.0469 mmol) was dissolved in acetic acid (2.76 mL) and stirred
at 60 °C for 1 h. The solution was concentrated and purified by
preparative LCMS to afford the product as a white solid (26.1 mg,
95%). 1H NMR (400 MHz, CD3OD): δ 7.79-7.56 (m, 5H), 7.42-
7.12 (m, 7H), 5.61-5.57 (m, 1H), 5.35-5.31 (m, 1H), 5.03 (s,
2H), 3.47-3.28 (m, 3H), 3.22-3.14 (m, 1H). LCMS found for
C26H24BrN4O5S (M + H)+: m/z ) 583.
N-{(1S)-1-(1H-Benzimidazol-2-yl)-2-[3-bromo-4-(1,1-dioxido-
3-oxoisothiazolidin-5-yl)phenyl]ethyl}benzenesulfonamide (93).
Benzyl (1S)-1-(1H-benzimidazol-2-yl)-2-[3-bromo-4-(1,1-dioxido-
3-oxoisothiazolidin-5-yl)phenyl]ethylcarbamate (92) (15.8 mg, 2.71
mmol) in acetonitrile (0.5 mL) was treated with iodotrimethylsilane
(11.6 µL, 8.12 mmol) at 25 °C and stirred for 30 min. The solution
was quenched with a 1 N aqueous HCl solution (0.5 mL), diluted
with acetonitrile (1 mL), and directly purified by preparative LCMS
to afford the product as a clear solid (15.1 mg, 99%). To a solution
of 5-4-[(2S)-2-amino-2-(1H-benzimidazol-2-yl)ethyl]-2-bromophe-
nylisothiazolidin-3-one 1,1-dioxide trifluoroacetate (11.3 mg, 20.0
mmol) and triethylamine (8.4 mL, 60 mmol) in methanol (0.9 mL)
was added a solution of benzenesulfonyl chloride (66) (3.0 µL, 24
mmol) and methanol (0.1 mL), and the resulting mixture was stirred
at 25 °C for 2h. The reaction solution was diluted with water and
methanol and purified by preparative LCMS to yield the desired
Methyl (2S)-3-[3-bromo-4-(2-tert-butyl-1,1-dioxido-3-oxoiso-
thiazolidin-5-yl)phenyl]-2-[(tert-butoxycarbonyl)amino]-
propanoate (90). Methyl (2S)-3-[3-bromo-4-(2-tert-butyl-1,1-di-
oxido-3-oxo-2,3-dihydroisothiazol-5-yl)phenyl]-2-[(tert-butoxycar-
bonyl)-amino]propanoate (89) (834 mg, 1.53 mmol) in THF (20
mL,) was chilled to 0 °C and treated dropwise with 2 M lithium
tetrahydroborate in tetrahydrofuran (0.80 mL). The solution was
stirred at 0 °C for 30 min. After 10 drops of acetic acid was added,
the solution was diluted with ethyl acetate (150 mL), washed with
water (150 mL), and dried over sodium sulfate. Silica gel chro-
matography (10-40% ethyl acetate/hexanes) afforded the product
1
as a slightly yellow foam (682 mg, 82%). H NMR (400 MHz,
1
CDCl3): δ 7.49-7.45 (m, 1H), 7.32-7.29 (m, 1H), 7.24-7.18 (m,
1H), 5.46-5.42 (m, 1H), 5.05-5.00 (m, 1H), 4.60-4.54 (m, 1H),
3.35-3.30 (m, 1H), 3.20-3.16 (m, 1H), 3.08-3.00 (m, 2H), 1.68
(s, 9H), 1.43 (s, 9H). LCMS found for C22H31BrN2O7S (M +
Na)+: m/z ) 569.
product (8.1 mg, 68%). H NMR (400 MHz, CD3OD): δ 7.77-
7.74 (m, 2H), 7.64-7.52 (m, 5H), 7.45-7.25 (m, 5H), 5.56-5.52
(m, 1H), 4.98-5.90 (m, 1H), 3.46-3.40 (m, 2H), 3.26-3.15 (m,
2H); HRMS calcd for C21H24BrN4O5S2 (M + Na)+: 611.0034;
found, 611.0016.
(2S)-2-[(Benzyloxy)carbonyl]amino-3-[3-bromo-4-(1,1-dioxido-
3-oxoisothiazolidin-5-yl)phenyl]propanoic Acid (91). Methyl
(2S)-3-[3-bromo-4-(2-tert-butyl-1,1-dioxido-3-oxoisothiazolidin-5-
yl)phenyl]-2-[(tert-butoxycarbonyl)amino]propanoate (90) (458 mg,
0.654 mmol) was dissolved in trifluoroacetic acid (10 mL) and
heated at 130 °C in the microwave for 2 min. The solution was
PTP1B Expression and Biochemical Assays. Human PTP1B,
SHP1, SHP2, and TC-PTP were expressed in E. coli, essentially
as described in the literature (J. Biol. Chem. 2000, 275, 10300-
10307; J. Struct. Biol. 1997 120, 201-203; Cell 1998 92, 441-
450; and J. Biol. Chem. 2002 277, 19982-19990). PTP enzymatic
assays were performed as described in the literature (J. Am. Chem.

Protein Tyrosine Phosphatase 1B Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 13 3789
Soc. 2003, 125, 4087-4096). The pNPP assay was performed as
described in the literature with the following modifications. The
reactions were carried out in clear bottom 384-well plates containing
80 µL per well. Dilutions of compounds were made in DMSO,
and PTP1B in the assay buffer (25 mM bis-tris-propane at pH 7.0,
1 mM EDTA, 0.1 mg/mL of BSA) was added (final [DMSO] of
2%). To 60 µL of this mixture, 20 µL of 4-nitrophenyl phosphate
(pNPP) was added per well, for a final pNPP concentration of 1
mM and a final PTP1B concentration of 5 nM. The rate of formation
of the phenolate ion was monitored at 410 nm on a Spectramax
384 plate reader. The slopes of initial reaction rates (15 min
reactions) were plotted and fitted to the sigmoidal dose-response
equation to obtain IC50 values of the compounds (GraphPad
Software Prism 3.0). For the mode of inhibition analysis, final pNPP
concentrations in the reactions varied between 0.46 and 30 mM.
The initial reaction rates of these reactions were fitted to the
Michaelis-Mention equation by nonlinear regression analysis
(Graphpad Prism). A competitive Ki value was determined by linear
regression of a plot of Km vs [inhibitor] such that Ki ) -(x-
intercept).
Insulin Receptor (IR) and Akt Phosphorylation Cellular
Assays. HEK293-MSR cells were transfected with plasmids encod-
ing full length human PTP1B and the insulin receptor using Fugene
6. In some experiments, a plasmid encoding a short hairpin RNA
(shRNA) against PTP1B (CGGATTAAACTACATCAAGAc-
gaaTCTTGATGTAGTTTAATCC, corresponding to nucleotides
165 to184) was cotransfected as a control. The cells were dispensed
into 96-well plates and allowed to attach for 24 h. Compounds were
diluted from DMSO stocks (0.4% final v/v) into DMEM without
the serum, and the cells were treated for 16 h. The nonselective
tyrosine phosphatase inhibitor bpV (pic) and insulin were added
for 30 min prior to harvesting. The cells were lysed in a buffer (25
mM Tris-HCl at pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5%
Deoxycholate; 1 mM EDTA supplemented with a protease inhibitor
cocktail) on ice, the cell extracts were clarified by centrifugation,
and the supernatants were used for the detection of phospho-IR
using an ELISA (Biosource), which specifically detects the activated
IR on the critical regulatory phospho-tyrosine residues (Y1162 and
Y1163) in the beta subunit (Ellis et al., 1986). Data were normalized
to total protein content as determined by the bicinchoninic acid
assay. In preliminary experiments, the data was normalized to total
IR using an anti-IR ELISA (Biosource) and total protein content
with good correlation between the two methods. For the measure-
ment of Akt phosphorylation, HepG2 cells were seeded into
collagen-coated 96-well plates. Compound treatment and cell lysis
were performed exactly as described above, and detection was
achieved using a phospho-Akt ELISA (Ser473, Biosource) and
normalized to total protein content. For immunoblotting experi-
ments, cell lysates were separated by SDS-PAGE, transferred to
nitrocellulose filters, and probed with antibodies to PTP1B (On-
cogene Research Products, Ab-1) or phospho-Insulin Receptor
(pY1162pY1163, Biosource) and appropriate horseradish peroxidase
conjugated secondary antibodies and developed with enhanced
chemiluminescence reagents.
Supporting Information Available: HPLC purity analysis. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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Acknowledgment. We thank Laurine Galya, Mei Li, and
Karl Blom for NMR spectroscopic, analytical, and mass
spectroscopic assistance, respectively. We thank Mary Becker-
Pasha and Milton Hillman for the expression and purification
of enzymes, respectively. We thank Rakesh Kohli at the
University of Pennsylvania for providing HRMS data.
JM0600904