Sulfonylated aminothiazoles as new small molecule inhibitors of protein phosphatases

Article abstract of DOI:10.1016/S0960-894X(00)00658-2

Based on a previously identified lead structure, SC-ααδ9, we have developed a versatile new chemical scaffold that can be readily modified to generate libraries of both Tyr and dual specificity phosphatase inhibitors with reduced molecular weight and lipophilicity. The most potent analogue identified to date, aminothiazole 8z, inhibits the dual specificity phosphatase Cdc25B with a K_i of 4.6 ± 0.4 μM and a Hill coefficient of 2.

Full text of DOI:10.1016/S0960-894X(00)00658-2

Bioorganic & Medicinal Chemistry Letters 11 (2001) 313±317
Sulfonylated Aminothiazoles as New Small Molecule Inhibitors of
Protein Phosphatases
Peter Wipf,a,* Diana C. Aslan,a Eileen C. Southwickb and John S. Lazob
aDepartment of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, USA
bDepartment of Pharmacology, University of Pittsburgh, Pittsburgh, PA 15261, USA
Received 29 September 2000; accepted 11 November 2000
AbstractÐBased on a previously identi®ed lead structure, SC-ꢀꢀꢁ9, we have developed a versatile new chemical sca€old that can
be readily modi®ed to generate libraries of both Tyr and dual speci®city phosphatase inhibitors with reduced molecular weight and
lipophilicity. The most potent analogue identi®ed to date, aminothiazole 8z, inhibits the dual speci®city phosphatase Cdc25B with a
Ki of 4.6 Æ 0.4 mM and a Hill coecient of 2. # 2001 Elsevier Science Ltd. All rights reserved.
The control of biological pathways exerted by reversible
phosphorylation of enzymes and receptors extends from
cell division, cell growth, and cellular transport to
apoptosis and muscle contraction.1 As part of a pro-
gram to develop phosphatase inhibitors as mechanistic
probes in cell biology and as potential antiproliferative
agents,2 4 we have used combinatorial chemistry strate-
gies to develop pharmacophores 1 and 2 as inhibitors of
Ser/Thr protein phosphatases (PP1 and PP2A), Tyr
protein phosphatases (PTP1B), and, especially, Ser/Thr/
Tyr dual speci®city phosphatases (Cdc25 and VHR).5
While the Tyr protein phosphatases and dual speci®city
phosphatases share a common HC(X)5R catalytic site
and mechanism of catalysis, they have unique biological
roles. Thus, PTP1B is believed to regulate intracellular
insulin signaling6a while VHR functions to dephos-
phorylate the mitogen activated protein kinase sub-
strates Erk1 and Erk2.6b Cdc25A and its isoforms B and
C play a crucial role in the regulation of the cell cycle.7
Each Cdc25 isoform is involved in a distinct cell cycle
event: Cdc25A expressed early in the G1 phase controls
entry into S phase and appears to be a crucial player in
the DNA damage checkpoint mechanism.1a,8 Cdc25B is
expressed in G1 and G2 and is essential for preinitiation
of G2/M transition and possibly S phase progression.9
Cdc25C activates the mitotic kinase Cdk1 at the G2/M
checkpoint.10 Overexpression of Cdc25 has been impli-
cated in cancerogenesis,7 and given the importance of
cell cycle checkpoints in maintaining genetic integrity
and immune function, the identi®cation of phosphatase-
selective inhibitors should be potentially useful for the
development of novel chemotherapeutic agents.
Our prior lead structure, the combinatorial library-
derived SC-ꢀꢀꢁ9, displayed low-micromolar activity
against Cdc25B and sub-micromolar inhibition of
PTP1B as well as moderate cancer cell-speci®c in vitro
toxicity,3 but in our SAR studies with sca€olds 1 and 2,
we were unable to reduce the lipophilicity and molecular
weight of this compound without ablating enzyme inhi-
bitory activity.4 An intriguing aspect of the structure±
activity pro®le of SC-ꢀꢀꢁ9, however, was the pro-
nounced in¯uence of substituents at the oxazole moiety
on the biological e€ects of this compound.2,3 Accord-
ingly, we decided to study a new heterocyclic library
developed around the oxazole segment of SC-ꢀꢀꢁ9.
Aminothiazoles have almost ubiquitous presence in
pharmaceutical test samples and have been used as
building blocks in dopamine and ®brinogen (GpIIb-
IIIa) receptor antagonists,11,12 DNA gyrase inhibitors,13
antibiotics14 and inhibitors of kynurenine 3-hydroxylase
cyclin dependent kinases,15,16 among others. N-Sulfon-
amide derivatives of aminothiazoles o€er many possibi-
lities for substituent modi®cations without being
subjected to the ready metabolism of amide derivatives.
a,b-Unsaturated sulfonamide derivatives retain con-
siderable electrophilic character,17 and since DSPases
contain an active-site cysteine residue,18 we investigated
sulfonamides of type 8 as a new class of potentially
irreversible Cdc25 inhibitors. The preparation of the
aminothiazole component was readily accomplished by
*Corresponding author. Tel.: +1-412-624-8606; fax: +1-412-624-
0787; e-mail: pwipf@pitt.edu
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00658-2

314
P. Wipf et al. / Bioorg. Med. Chem. Lett. 11 (2001) 313±317
a Hantzsch synthesis (Scheme 1).19 According to the
method of King and Hlavacek,20 aldehydes were con-
verted to the a-methylene ketones that were not com-
mercially available and exposed to a mixture of thiourea
and iodine at 100 ꢀC. a,b-Unsaturated sulfonylchlorides
were obtained according to the protocols of Gennari
and Roush.17,21 Sulfonates 6 were isolated exclusively as
trans-isomers after condensations of commercially
available aldehydes with phosphonate 5 and were con-
verted to the desired sulfonylchlorides 7 by cleavage of
the ethyl group and treatment with triphenylphosphine
and sulfuryl chloride.22 A small library of N-sulfonated
aminothiazoles was synthesized by treatment of 4 with 7
in the presence of pyridine as an acid scavenger.23
Yields for the coupling reaction varied from 10% to
73% depending on the reactivity of the sulfonyl chlor-
ides. In accordance with the SAR derived from the
oxazole segment of SC-ꢀꢀꢁ9, most of the substituents
were aliphatic or aromatic groups.
was interesting that all of the most reactive compounds
except 8e had n-propyl moieties in R1. A more detailed
analysis of the modi®cation on these domains was
informative. For example, some variation in the aro-
matic halogens was tolerated on the R domain for
Cdc25B inhibition but steric limitations existed. Thus,
the 4-Cl phenyl containing thiazole 8dd and the 4-CF3
phenyl containing 8ii had IC50 values of 21 and 25 mM,
respectively, while the biphenyl containing 8cc had an
IC50 value of 40 mM. The 4-CF3 phenyl containing 8y
(IC50=20 mM) and 4-chlorophenyl containing 8aa
(IC50=34 mM) were more potent Cdc25B inhibitors
compared to the biphenyl containing 8t (IC50=46 mM).
Similarly, both 8w and 8z showed low micromolar IC50
values while 8ee showed an IC50 for Cdc25B of >100
mM. The R1 position had a considerable role in de®ning
the potency of the sulfonylated aminothiazoles with
phenyl substitutions being more potent than the ethyl or
n-decyl substitutions. For example, 8d had an IC50 of 26
mM while both 8o and 8r were essentially inactive at 100
mM. Similarly, 8a and 8e were more active than their
congeners 8n or 8q. The R2 domain had considerable
¯exibility in accepting substitutions. Generally, an aro-
matic residue containing an electron-withdrawing group
performed well. Thus, 8cc was superior to congeners
lacking the di¯uorophenyl substitution, such as 8x, 8u,
or 8v, but was equivalent to 8bb. Kinetic analyses of 8z
were most compatible with competitive inhibition of
Cdc25B with a Ki of 4.6 Æ 0.4 mM and a Hill coecient
of 2. For 8e a Ki of 36 Æ 6 mM was found when the best
®t model, a competitive S-parabolic model, was used,
which is consistent with two molecules of the inhibitor
binding with the enzyme. It is interesting that the crystal
structure of the Cdc25B catalytic domain revealed two
independent potential binding sites for substrates.24 Our
Sulfonylated aminothiazoles 8 were evaluated for their
ability to inhibit the in vitro enzyme activity of full
length, recombinant, human Cdc25B, VHR, and PTP1B
by our previously described methods3 using O-methyl-
¯uorescein monophosphate as a substrate. Many ami-
nothiazoles were e€ective in the low micromolar range,
although none had submicromolar median inhibitory
concentration (IC50) values. As illustrated in Table 1, 15
of the 35 newly synthesized compounds had IC50 values
for Cdc25B <50 mM and ®ve had IC50 values ꢁ25 mM,
indicating the rich potential of this minimal pharmaco-
phore. Among the best inhibitors, namely 8w, 8y, 8dd,
8e, 8ii, and 8z, except for 8e all were substituted with
halogenated aromatics in R and R2. Because the parent
SC-aad9 pharmacophore favored diphenyl oxazoles,3 it
Scheme 1.

P. Wipf et al. / Bioorg. Med. Chem. Lett. 11 (2001) 313±317
315
Table 1. Structures of thiazoles 8 and IC50 values (mM Æ SEM) for Cdc25B, VHR, and PTP1Ba
Thiazole
R
R1
R2
Cdc25B
VHR
PTP1B
8a
8b
8c
8d
8e
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Et
Et
Et
(4-NO2)Ph
(4-Me)Ph
2-Furyl
54 Æ 4
>100
>100
26 Æ 2
22 Æ 2
>100
34 Æ 3
33 Æ 1
72 Æ 12
26 Æ 4
58 Æ 1
76 Æ 3
70 Æ 20
88 Æ 10
>100
47 Æ 3
>100
>100
40 Æ 2
30 Æ 1
>100
25 Æ 1
24 Æ 2
33 Æ 7
20 Æ 1
39 Æ 3
32 Æ 1
34 Æ 1
50 Æ 2
>100
58 Æ 4
>100
>100
38 Æ 3
45 Æ 2
>100
36 Æ 1
49 Æ 2
85 Æ 4
59 Æ 20
>100
58 Æ 3
90 Æ 7
83 Æ 4
>100
>100
>100
>100
98 Æ 5
58 Æ 12
>100
>100
10 Æ 5
>100
20 Æ 3
24 Æ 4
32 Æ 6
35 Æ 5
77 Æ 8
20 Æ 8
>100
51 Æ 2
>100
>100
29 Æ 2
(2-Cl)Ph
2-Naphthyl
(3,4-diOMe)Ph
(4-CF3)Ph
n-Nonyl
(3,4-diF)Ph
(4-MeCO2)Ph
(4-n-Bu)Ph
(4-CN)Ph
(4-Ph)Ph
(4-NO2)Ph
(2-Cl)Ph
(4-MeSO2)Ph
2-Naphthyl
(2-Cl)Ph
2-Naphthyl
(4-Me)Ph
(4-CF3)Ph
2-Naphthyl
(2-Cl)Ph
(4-n-Bu)Ph
(4-Me)Ph
(2-Cl)Ph
(4-Me)Ph
(4-Ph)Ph
(3,4-diF)Ph
(3,4-diF)Ph
(2-Cl)Ph
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
8q
8r
>100
89 Æ 11
>100
32 Æ 3
Ph
Ph
Et
n-Decyl
n-Propyl
n-Propyl
n-Propyl
n-Propyl
n-Propyl
n-Propyl
n-Propyl
n-Propyl
n-Propyl
n-Propyl
n-Propyl
n-Propyl
n-Propyl
Me
>100
>100
8s
8t
2-Naphthyl
(4-Ph)Ph
(4-Ph)Ph
(4-Ph)Ph
(4-CF3)Ph
(4-Ph)Ph
(4-CF3)Ph
(4-Cl)Ph
(4-Cl)Ph
(4-Cl)Ph
(4-Ph)Ph
(4-Cl)Ph
(4-Ph)Ph
(4-Cl)Ph
(4-Cl)Ph
(4-Cl)Ph
(4-CF3)Ph
69 Æ 2
46 Æ 2
92 Æ 0
>100
14 Æ 4
80 Æ 9
20 Æ 4
26 Æ 4
34 Æ 8
38 Æ 8
40 Æ 12
21 Æ 4
>100
34 Æ 5
21 Æ 1
42 Æ 1
57 Æ 1
25 Æ 1
54 Æ 12
31 Æ 16
32 Æ 9
48 Æ 20
27 Æ 8
22 Æ 6
20 Æ 5
82 Æ 7
43 Æ 1
>100
8u
8v
8w
8x
8y
8z
8aa
8bb
8cc
8dd
8ee
8€
8gg
8hh
8ii
(3,4-diF)Ph
(2-Cl)Ph
(2-Cl)Ph
33 Æ 3
89 Æ 3
>100
25 Æ 1
H
Me
n-Propyl
>100
31 Æ 1
(3,4-diF)Ph
aIC50 values were generally calculated with a minimum of ®ve concentrations (0.1 to 100 mM) for the Cdc25B assays and three concentrations (1, 10,
and 100 mM) for VHR and PTP1B assays.
analysis did not reveal any evidence for covalent
interactions of aminothiazoles with Cdc25B.
compared to our prior lead structure. Of particular
interest is the lack of a requirement for an obvious sur-
rogate phosphate, such as the carboxylate in SC-ꢀꢀꢁ9.
The parallel inhibitor pro®les of most of the compounds
in this limited library con®rm the general similarity in
the architecture of the active site of Cdc25, VHR, and
PTP1B. The considerable activity di€erences among the
members of the library, however, also reinforce the
possibility that selective small molecule inhibitors
should be attainable. Among the ca. 40 aminothiazoles
of type 8 tested to date, we have already identi®ed a
compound (8z) with a Ki for Cdc25B below that of SC-
ꢀꢀꢁ9. Thus, we expect that the sulfonylated ami-
nothiazole sca€old will continue to be useful in the
pursuit of potent and selective phosphatase inhibitors.
Consistent with the conserved enzymology and sig-
nature motif of the active site of Cdc25, VHR, and
PTP1B, almost all of the compounds that were inactive
against Cdc25B were also inactive against VHR and
PTP1B (Table 1). Nevertheless, exceptions were noted.
Thus, while 8dd and 8cc exhibited di€erence in their
inhibitory activity against Cdc25B and PTP1B, they
were equally e€ective against VHR. Thiazoles 8i, 8q, 8u,
and 8v retained anti-VHR activity even though they
lacked signi®cant inhibitory activity against Cdc25B or
PTP1B. This may re¯ect di€erences in the variable
amino acids juxtaposed to the catalytic cysteine and
essential arginine in the catalytic site. As was previously
noted,3 the regions surrounding the active sites of Cdc25
and PTP1B are hydrophobic, which may explain the
enhanced inhibition seen with aminothiazoles containing
aromatic moieties.
Acknowledgements
This work was supported in part by the US Department
of Defense, the Fiske Drug Discovery Fund and Merck
& Co., and by the National Institutes of Health (CA
78039). We thank Kathleen Cooley and Diane Luci for
their assistance with enzyme inhibition assays and
aminothiazole preparation.
In summary, we have identi®ed a versatile new chemical
sca€old that can be readily modi®ed to generate librar-
ies of both Tyr and dual speci®city phosphatase inhibi-
tors with reduced molecular weight and lipophilicity

316
P. Wipf et al. / Bioorg. Med. Chem. Lett. 11 (2001) 313±317
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23. All new products were characterized by 1H NMR, 13C
NMR, and HRMS. A typical procedure: 2-(2-Chlorophenyl)-
ethenesulfonic acid ethyl ester (6, R2=2-ClPh). To a solution
of triethyl a-phosphonomethane sulfonate (5, 1.0 g, 3.8 mmol)
in THF (50 mL) was added at 78 ꢀC under N2 n-BuLi (1.6 M
in hexanes, 2.4 mL, 3.8 mmol). The reaction mixture was stir-
red for 15 min at 78 ꢀC, then 2-chlorobenzaldehyde (0.39
mL, 3.5 mmol) was added dropwise as a solution in THF (5
mL). After the addition of aldehyde was complete, the reac-
tion mixture was warmed up to 22 ꢀC, stirred for 12 h, and
quenched with satd aq NaCl. The organic layer was separated,
the aqueous layer was extracted with Et2O, the combined
organic layers were dried (MgSO4) and the solvent evaporated
under reduced pressure. The solid precipitated from the resi-
due was recrystallized from CH2Cl2/hexane to give pure 6
(0.57 g, 66%) as a colorless powder. Alternatively, the crude
mixture can be puri®ed by chromatography on SiO2 (hex-
anes:Et2O, 6:1): mp 69±70 ꢀC (hexanes); H NMR (CDCl3) d
1
7.95 (d, J=15.6 Hz, 1H), 7.57 (d, J=6.9 Hz, 1H), 7.42±7.31
(m, 3H), 6.79 (d, J=15.3 Hz, 1H), 4.27±4.20 (m, 2H), 1.38 (t,
J=6.9 Hz, 3H); 13C NMR (CDCl3) d 140.4, 135.1, 132.4,
130.4, 130.3, 128.3, 127.5, 124.0, 67.4, 15.0; HRMS (EI) calcd
for C10H11ClO3S 246.0117, found 246.0122.
2-(2-Chlorophenyl)-ethenesulfonyl chloride (7, R2=2-
ClPh). To a solution of 6 (0.44 g, 1.8 mmol) in acetone (50
mL) was added tetrabutylammonium iodide (0.99 g, 2.7
mmol). The reaction mixture was heated at re¯ux overnight,
cooled to 22 ꢀC, and concentrated under reduced pressure, and
the residue was used without puri®cation. To a solution of
triphenylphosphine (0.94 g, 3.6 mmol) in CH2Cl2 (50 mL) was
added at 0 ꢀC under N2 sulfuryl chloride (0.32 mL, 3.9 mmol)
and after 15 min the crude ammonium salt obtained in the
previous step as a solution in CH2Cl2 (10 mL). After stirring
overnight, the solution was concentrated under reduced pres-
sure and the crude residue was puri®ed by chromatography
on SiO2 (Et2O:hexanes, 1:4) to give sulfonyl chloride 7 (0.33
g, 78%) as a gray solid: mp 59±60 ꢀC (hexanes); IR (neat)
1
12. Badorc, A.; Bordes, M.-F.; de Cointet, P.; Savi, P.; Ber-
nat, A.; Lale, A.; Petitou, M.; Ma€rand, J.-P.; Herbert, J.-M.
J. Med. Chem. 1997, 40, 3393.
13. Boehm, H.-J.; Boehringer, M.; Bur, D.; Gmuender, H.;
Huber, W.; Klaus, W.; Kostrewa, D.; Kuehne, H.; Luebbers,
T.; Meunier-Keller, N.; Mueller, F. J. Med. Chem. 2000, 43,
2664.
14. Heinze-Krauss, I.; Angehrn, P.; Guerry, P.; Hebeisen, P.;
Hubschwerlen, C.; Kompis, I.; Page, M. G. P.; Richter,
H. G. F.; Runtz, V.; Stalder, H.; Weiss, U.; Wei, C.-C. J. Med.
Chem. 1996, 39, 1864.
3074, 1602, 1587, 1431, 1166 cm
;
1H NMR (CDCl3) d
8.15 (d, J=15.3 Hz, 1H), 7.63±7.27 (m, 5H); 13C NMR
(CDCl3) d 141.3, 136.4, 133.6, 132.3, 131.0, 129.0, 128.9,
127.8; HRMS (EI) calcd for C8H6Cl2O2S 235.9466, found
235.9473.
5-Propyl-4-(4-tri¯uoromethyl-phenyl)-thiazol-2-ylamine (4,
R=4-F3CPh, R1=n-Pr). A mixture of thiourea (0.17 g, 2.2
mmol), I2 (0.14 g, 1.1 mmol) and 1-(4-tri¯uoromethylphenyl)-
pentan-1-one (0.25 g, 1.1 mmol) was stirred at 100 ꢀC for 14 h.
The cooled reaction mixture was washed onto a fritted ®lter
with Et2O and CH2Cl2. The ®ltrate was concentrated under

P. Wipf et al. / Bioorg. Med. Chem. Lett. 11 (2001) 313±317
317
reduced pressure and the residue was dissolved in a minimum
amount of boiling water, cooled, made basic with NH4OH
solution and extracted with ethyl acetate. The organic layer
was dried (MgSO4) and concentrated to give an oil from which
4 (0.23 g, 74%) crystallized as a yellow solid: mp 47±49 ꢀC
(ethyl acetate); IR (neat) 3402, 3098, 2967, 1618, 1529, 1332,
CH2Cl2 (1 mL). The reaction mixture was stirred at 22 ꢀC for 2
days, and concentrated under reduced pressure. The crude
residue was puri®ed by chromatography on SiO2
(CH2Cl2:Et2O, 1:1) to give 8w (72 mg, 64%) as a colorless
solid: mp 162±163 ꢀC (CH2Cl2); IR (neat) 3441, 2919, 1478,
1
1224, 1169, 1130 cm 1; H NMR (CDCl3) d 7.92 (d, J=15.6
1
1167 cm 1; H NMR (CDCl3) d 7.64 (s, 4H), 5.47 (bs, 2H),
Hz, 1H), 7.65±7.23 (m, 8H), 6.91 (d, J=15.3 Hz, 1H), 2.61 (t,
J=7.5 Hz, 2H), 1.70±1.60 (m, 2H), 0.93 (t, J=7.4 Hz, 3H);
13C NMR (CDCl3) d 166.8, 135.8, 134.8, 132.7, 132.0, 131.3,
130.7, 130.1, 128.8, 128.1, 127.9, 127.2, 125.9, 124.1, 121.9,
28.5, 24.2, 13.6; HRMS (EI) calcd for C21H18ClF3N2O2S2
486.0450, found 486.0460.
24. Reynolds, R. A.; Yem, A. W.; Wolfe, C. L.; Deibel, M. R.,
Jr.; Chidester, C. G.; Watenpaugh, K. D. J. Mol. Biol. 1999,
293, 559.
2.73 (t, J=7.7 Hz, 2H), 1.70±1.61 (m, 2H), 0.96 (t, J=7.4 Hz,
3H); 13C NMR (CDCl3) d 165.4, 144.1, 138.8, 128.5, 125.1,
28.9, 25.2, 13.5; HRMS (EI) calcd for C13H13F3N2S 286.0752,
found 286.0752.
2-(2-Chlorophenyl)-ethenesulfonic acid [5-propyl-4-(4-tri-
¯uoromethylphenyl)-thiazol-2-yl]-amide (8w). A solution of 4
(0.079 g, 0.28 mmol) in pyridine (2 mL) was treated at 22 ꢀC
with a solution of sulfonyl chloride 7 (0.055 g, 0.23 mmol) in