Discovery of a novel submicromolar inhibitor of the lymphoid specific tyrosine phosphatase

Article abstract of DOI:10.1016/j.bmcl.2008.03.079

We report here a class of thiazolidine-2,4-diones and 2-thioxothiazolidin-4-ones as potent inhibitors of the lymphoid specific tyrosine phosphatase (Lyp) identified from high throughput screens. Chemical modification by incorporating the known phosphotyro

Full text of DOI:10.1016/j.bmcl.2008.03.079

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2840–2844
Discovery of a novel submicromolar inhibitor
of the lymphoid specific tyrosine phosphatase
Yuli Xie,a Yidong Liu,a Gangli Gong,a Alison Rinderspacher,a Shi-Xian Deng,a
b
b
b
b
c
ˇ
´
Deborah H. Smith, Udo Toebben, Effie Tzilianos, Lars Branden, Dusica Vidovic,
Caty Chung,c Stephan Schurer,c Lutz Tautzd and Donald W. Landrya,*
¨
aDepartment of Medicine, Columbia University, 650 W 168th Street, BB 1029, New York, NY 10032, USA
bDepartment of Physiology and Cellular Biophysics, Columbia University, New York, NY 10032, USA
cScientific Computing, The Scripps Research Institute, Jupiter, FL 33458, USA
dInfectious and Inflammatory Disease Center, Burnham Institute for Medical Research, La Jolla, CA 92037, USA
Received 3 March 2008; revised 24 March 2008; accepted 31 March 2008
Available online 8 April 2008
Abstract—We report here a class of thiazolidine-2,4-diones and 2-thioxothiazolidin-4-ones as potent inhibitors of the lymphoid spe-
cific tyrosine phosphatase (Lyp) identified from high throughput screens. Chemical modification by incorporating the known phos-
photyrosine (pTyr) mimics led to the discovery of a salicylate-based inhibitor with submicromolar potency.
Ó 2008 Elsevier Ltd. All rights reserved.
The lymphoid specific phosphatase (Lyp), encoded by
the PTPN22 gene, plays an important role in T cell sig-
naling. A single-nucleotide polymorphism in PTPN22 is
associated with autoimmune disorders such as type 1
diabetes, rheumatoid arthritis, systemic lupus erythe-
matosus, and Grave’s disease. The autoimmunity-pre-
disposing allele corresponds to a gain-of-function
mutation, producing a variant phosphatase that is more
active than the wild type enzyme in inhibiting T cell sig-
naling.1,2 Inhibition of Lyp therefore provides a novel
approach to the treatment of a broad spectrum of auto-
immune diseases.
at 360 nm and emission at 465 nm was recorded at the
endpoint of the reaction. From the compound libraries
provided by NIH, a variety of active hits were identified
through the high throughput screens and were con-
firmed manually by the dose–response assays. Intrigu-
ingly,
a
number of these compounds share
a
thiazolidine core structure and thus form an active clus-
ter, providing a starting point for the assessment of
structure–activity relationships and medicinal chemistry
efforts (Fig. 1).
Protein tyrosine phosphatases (PTPases) share a highly
conserved active site, the phosphotyrosine (pTyr) bind-
ing pocket that is the main target for PTPase inhibitor
design. Thus, most inhibitors share a pharmacophore
structurally similar to the pTyr substrate. Effective pTyr
mimics are often charged bidentate anions that compet-
itively bind to the highly polarized pTyr pocket. Several
classes of mimics have been reported,4 including the dif-
luoromethylenephosphonates (DFMP) and benzoic
acids such as 2-(oxalylamino)-benzoic acids (OBA), sal-
icylic acids (SA), and its derivatives (Fig. 2).
To identify small molecule inhibitors of Lyp, high
throughput screens were performed at Columbia Uni-
versity as part of the Molecular Library Screening Cen-
ter Network (MLSCN) of the NIH Roadmap for
Medical Research. The Lyp assay3 was established using
a bacterially expressed fully active 62 kDa N-terminal
catalytic domain of Lyp. Compounds were assayed for
their capacity to inhibit Lyp-catalyzed conversion of
DiFMUP(6,8-difluoro-4-methylumbeliferyl phosphate)
to a fluorescent product. Fluorescence with excitation
We noticed that several of the thiazolidine hits con-
tained benzoic acid moiety, and thus could be replaced
with a pTyr-mimicking fragment. Fragment-based drug
design is a new approach that has been successfully ap-
plied to challenging targets.5 This strategy allows hits to
Keywords: Thiazolidine; Lyp; pTyr mimics; Salicylate.
*
Corresponding author. Tel.: +1 212 3055839; fax: +1 212 3053475;
e-mail: dwl1@columbia.edu
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.03.079

Y. Xie et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2840–2844
COOH
2841
COOH
COOH
N
N
O
N
O
S
S
S
O
O
O
O
O
O
N
O
O
O
N
N
S
S
S
O
S
O
N
N
NH
HN
NH
O
O
OH
OH
CU-01000104193 CU-01000104171
CU-01000095611
CU-01000089901
CU-01000095625
IC50=1.0uM
IC50=2.5uM
IC50=4.8uM
IC50=1.9uM
IC50=1.9uM
COOH
COOH
S
N
S
N
O
O
S
S
O
O
N
S
S
O
COOH
O
O
O
N
O
O
N
O
S
N
S
S
N
S
O
O
HN
HN
COOH
HOOC
CU-01000066973
CU-01000092109
CU-01000066932
CU-01000104073
CU-01000088853
IC50=0.98uM
IC50=3.3uM
IC50=14uM
IC50=3.5uM
IC50=1.3uM
Figure 1. An active cluster of Lyp inhibitors.
othiazolidinyl esters 3a–3d were synthesized from thia-
zolidine-2, 4-dione 1, which was first converted to
potassium salt 2 by potassium hydroxide and subse-
quently alkylated with methyl (tert-butyl) bromoacetate
in acetone at 50 °C, or methyl (tert-butyl) bromopropi-
onate in dimethylformamide at 90 °C in the presence
of potassium iodide. Acids 4a–4b were obtained by the
treatment of the tert-butyl esters 3c–3d with trifluoroace-
tic acid. Suzuki coupling of boronic acid 5a–5b with 5-
iodo-2-hydroxybenzoate 6a– 6b conveniently yielded
salicylic acid-derived aldehydes 7a–7d, which were con-
densed with thiazolidine-diones 3a, 3c, 4a, 4b, and com-
mercially available 2-thioxothiazolidin-4-ones 4c–4d in
toluene to afford the final product 8a– 8s in yields of
72–85%.
O
O
P
O
P
O
O
O
N
H
O
O
O
F
F
O
O
O
pTyr
DFMP
OBA
O
O
O
O
O
O
O
O
CMBA
SA
Figure 2. The phosphotyrosine (pTyr) mimic.
be optimized by combining and linking different frag-
ments. We reasoned that combining a known pTyr sur-
rogate with the thiazolidine core structure we identified
from high throughput screens might result in synergisti-
cally improved potency. Salicylic acid and its deriva-
tives, which are potent pTyr surrogates, were chosen
as our initial building blocks for novel Lyp inhibitors.
A triacid analog 11 was synthesized according to
Scheme 2.6 Alkylation of compound 7a with 2-bromo-
acetate gave aldehyde 9, followed by deprotection of
methyl ester with lithium hydroxide to yield aldehyde
10. Similar condensation of aldehydes 10 and 4d readily
afforded the desired product 11 in a total yield of 62%
over three steps.
As shown in Scheme 1, a series of thiazolidine-dione and
2-thioxothiazolidin-4-one compounds with an appended
salicylic moiety were designed and synthesized.6 Diox-
To further modify the salicylic compounds, Schiff base
analogs with the thiazolidine-dione head group replaced
with the hydantoin ring were synthesized.6 As outlined

2842
Y. Xie et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2840–2844
O
K
H
N
O
O
Br
CO2R
n
KOH
N
CO2R
n
N
O
O
S
Acetone,
50 ºC, 5 h
EtOH,70 ºC, 5 h
S
1
S
2
O
or
3a, R=CH3, n=1
KI, DMF,
3b
3c
90 ºC, 12 h
, R=CH3, n=2
t
, R= Bu, n=1
3d, R=tBu, n=2
O
O
t
TFA, CH2Cl2
rt, 2h
N
CO2 Bu
N
CO2H
n
S
S
n
O
O
4a,n=1; 4b,n=2
3c, n=1; 3d, n=2
CO2R'
OH
(Ph3P)2PdCl2,
K2CO3
HO
OHC
+
X
X
B(OH)2
R'O2C
I
OHC
THF,reflux,3 h
5a
5b
,X=S
,X=O;
6a
6b
,R'=H
,R'=CH3;
7a
7b
, X=S,R'=CH3
7d, X=S,R'=H
,X=O,R'=CH3;
7c,X=O,R'=H;
HO2C
H
CO2R'
OH
O
N
O
O
N
X
3a 3c 4a 4d
,
,
-
S
S
S
RO2C
N
S
S
piperazine acetate,
toluene,reflux, 3-5h
n
Y
8a 8s
-
4c
4d
Scheme 1. The synthesis of Lyp inhibitors 8a–8s.
O
LiOH
Br
CO2CH3
HO
O
H3CO2C
CHO
O
CHO
H3CO2C
EtOH, rt, 2 h
H3CO2C
K2CO3,DMF
rt, 24 h
9
7a
CO2H
O
O
HO2C
O
O
S
4d
HO2C
N
O
CHO
HO2C
piperazine acetate
toluene, reflux, 5 h
S
HO2C
11
10
Scheme 2. The synthesis of the triacid analog 11.
in Scheme 3, simply mixing aldehydes with the hydro-
chloride salt of 1-aminohydantoin in ethanol readily
gave the corresponding Schiff bases 12a–12d in quantita-
tive yields. The synthesis of 17a and 17b started from the
protection of 1-aminohydantoin with benzaldehyde by
forming imine 13. Deprotonation of 13 followed by
alkylation with bromoacetate provided the imidyl esters
15a and 15b. Simultaneous deprotection of ester and
imine by refluxing in hydrochloric acid afforded the cor-
responding amino acid 16. Condensation between the
hydantoin-derived amino acid and aldehydes 7c and 7d
furnished the Schiff base analogs 17a and 17b in good
yields.
A total of 25 salicylic compounds were synthesized, and
their ability to inhibit Lyp was tested in vitro with IC50s
determined from the dose–response assays. Results for
thiazolidine compounds are summarized in Table 1,
while the results for Schiff base analogs are shown in Ta-
ble 2. Seventeen compounds showed reasonable activi-
ties against Lyp. Among them, compound 8p was
shown to be a submicromolar inhibitor with an impres-
sive IC50 of 0.39 lM. Several structure–activity relation-
ships could be observed from these data. The presence
of a free carboxyl group in the molecule is essential
for the activity. For example, diester compounds 8b,
8c, 8d, and 8k are basically inactive, consistent with

Y. Xie et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2840–2844
2843
O
CO2R'
HN
H
N
N
N
O
EtOH, rt, 24 h
X
OH
O
O
+
N
X
OHC
HCl•H2N
OH
CO2R'
7a 7d
-
12a
12b
12d
, X=O, R'=CH3;
, X=S, R'=CH3;
, X=O, R'=H
benzaldehyde
EtOH, rt, 24 h
12c
, X=S, R'=H
O
O
O
Br
CO2R
KOH
N N
O
N N
N
N
N
HN
RO2C
KN
EtOH, 70 ºC, 2 h
acetone, 50 ºC, 24 h
O
O
14
13
15a
15b
, R = Me,
, R=tBu
O
N
O
HO2C
7c 7d
,
N
N
1N HCl
N NH2•HCl
N
17a
,X=O
X
HO2C
O
17b,X=S
EtOH
reflux,12 h
O
rt, 24 h
OH
CO2H
16
Scheme 3. The synthesis of Schiff base analogs.
Table 1. The Lyp inhibitory activities of compounds 8a–8s and 11
CO2R'
OH
O
CO2H
O
X
O
O
S
S
RO2C
N
HO2C
N
n
Y
11
8a-8s
HO2C
S
a
Compound
R
R0
n
X
Y
IC50 (lM)
8a
8b
8c
8d
8e
8f
H
CH3
CH3
CH3
CH3
H
1
2
2
1
1
2
2
1
1
1
1
1
1
1
2
2
1
1
0
O
O
S
S
21.0
44.0
>44
>44
8.7
CH3
CH3
CH3
H
O
O
O
S
S
O
O
S
H
H
O
O
O
S
5.1
5.9
8g
8h
8i
H
H
H
H
S
2.8
5.4
H
H
S
8j
H
CH3
CH3
CH3
CH3
H
S
S
4.3
8k
8l
CH3
H
O
S
O
O
O
O
O
O
O
O
S
>44
18
8m
8n
8o
8p
8q
8r
8s
11
H
O
O
O
S
>44
5.6
H
H
CH3
CH3
H
4.3
H
0.39
4.8
CH3
CH3
O
S
H
1.8
1.4
H
O
>44
a IC50 values are determined from a single experiment performed in triplicate.
the expectation that negative charge is important for the
activity of phosphatase inhibitors. As demonstrated by
compounds 8j, 8r, 8m, and 8q, the thiophene ring is gen-
erally more potent than the furan ring. Interestingly, 2-
thione also seems preferable to 2-one in the thiozolidine
ring that is best illustrated by increased potency of com-
pounds 8a and 8j. The length of the side chain (n = 0, 1,
and 2), depending on the overall structure of the com-
pound, has a varying impact on the activities. 2-(carb-
oxymethoxy) benzoic acid (CMBA), a derivative of
salicylic acid, is also a potent pTyr mimic. However,
our attempt to replace salicylic acid with CMBA re-

2844
Y. Xie et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2840–2844
Table 2. The inhibitory activities of the Schiff base analogs
ities via high throughput screening. Improved potency
(<1 lM) was achieved by rational design and chemical
synthesis. PTPase selectivity and cell studies of the
promising compounds are currently being pursued.
O
R
N
N
N
X
O
References and notes
OH
CO2R'
12a-12d; 17a-17b
1. Bottini, N.; Musumeci, L.; Alonso, A.; Rahmouni, S.;
Nika, K.; Rostamkhani, M.; MacMurray, J.; Meloni, G.
F.; Lucarelli, P.; Pellecchia, M., et al. Nat. Genet. 2004, 36,
337.
2. Vang, T.; Congia, M.; Macis, M. D.; Musumeci, L.; Orru,
V.; Zavattari, P.; Nika, K.; Tautz, L.; Tasken, K.; Cucca,
F., et al. Nat. Genet. 2005, 37, 1317.
a
Compound
R
R0
X
IC50 (lM)
12a
12b
12c
12d
17a
17b
H
CH3
H
O
O
S
>44
>44
>44
34.0
36.0
>44
H
H
CH3
H
H
S
3. See the assay protocol published in PubChem (AID: 606).
4. Burke, T. R.; Lee, K. Acc. Chem. Res. 2003, 36, 426.
5. Hajduk, P. J.; Greer, J. Nat. Rev. Drug Disc. 2007, 6, 211.
6. Analytical data for representative compounds: 8p, 1H
NMR (300 MHz, CD3OD) d 10.65 (br, 1H), 8.12 (s, 1H),
7.99 (s, 1H), 7.91 (d, 1H), 7.68 (d, 1H), 7.62 (d, 1H), 7.06 (d,
CH2CO2H
CH2CO2H
H
O
S
H
a IC50 values are determined from a single experiment performed in
triplicate.
1H), 3.90 (s, 3H), 3.83–3.80 (m, 2H), 2.60–2.55 (m, 2H); 13
C
sulted in a great loss of activity of compound 11 com-
pared with 8e, indicating that the free hydroxyl group
is necessary for retaining the activity. Coincidently,
when this manuscript was being prepared, the crystal
structure of Lyp in complex with a salicylic acid-based
inhibitor was published.7 It was shown that the hydroxyl
group of salicylic acid makes critical polar interactions
in the active site of Lyp.
NMR (75 MHz, CD3OD) d 172.5, 168.8, 166.9, 165.7,
160.6, 149.8, 137.5, 136.2, 133.2, 127.7, 126.8, 125.5, 124.8,
119.3, 118.6, 115.1, 53.4, 38.4, 32.4; ESI-MS(M++H): 434.3.
Compound 11, 1H NMR (300 MHz, CD3OD) 8.11 (s, 1H),
7.93 (d, 1H), 7.73 (s, 1H), 7.39 (d, 1H), 7.31 (d, 1H), 7.22(d,
1H), 4.86 (s, 2H), 4.72 (s, 2H); 13C NMR (75 MHz,
CD3OD) d 194.2, 170.3, 167.9, 167.3, 166.6, 158.2, 157.7,
149.5, 129.4, 127.6, 124.7, 123.3, 122.0, 120.0, 118.0, 115.2,
110.4, 66.0, 45.8; ESI-MS(M++H): 464.0. Compound 12d,
1H NMR (300 MHz, CD3OD) d 11.2 (s, 1H), 8.01 (s, 1H),
7.98 (s, 1H), 7.85 (d, 1H), 7.43 (d, 1H), 7.35 (d, 1H), 7.01 (d,
1H), 4.32 (s, 2H); 13C NMR (75 MHz, CD3OD) d 171.9,
169.5, 161.5, 153.8, 144.6, 138.8, 138.2, 133.4, 132.4, 127.5,
125.3, 124.2, 118.9, 114.3, 49.9; ESI-MS(M++H): 346.3.
7. Yu, Xiao; Sun, J. P.; He, Y.; Guo, X.; Liu, S.; Zhou, B.;
Hudmon, A.; Zhang, Z. Y. Proc. Natl. Acad. Sci. U.S.A.
2007, 104, 19767.
As can be seen from Table 2, the Schiff base analogs are
poor inhibitors in general. Only compounds 12d and 17a
showed moderate activities with the IC50 values of 34.0
and 36.0 lM, respectively.
In summary, we have successfully identified a cluster of
thiozolidine compounds with good Lyp inhibitory activ-