Synthesis and characterization of a potent and selective protein tyrosine phosphatase inhibitor, 2-[(4-methylthiopyridin-2-yl)methylsulfinyl]benzimidazole

Article abstract of DOI:10.1016/S0960-894X(00)00539-4

The synthesis and biological activity of a series of 2-[(4-methylthiopyridin-2-yl)methylsulfinyl]benzimidazoles are described. These compounds have potent inhibitory effects against the protein tyrosine phosphatase activity of CD45. Enzymatic analysis with several phosphatases revealed that compound 5a had high specificity for CD45 compared with serine/threonine phosphatases (PP1, PP2A), tyrosine phosphatases (LAR, PTP1B and PTP-S2) and dual phosphatase (VHR). (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00539-4

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2657±2660
Synthesis and Characterization of a Potent and Selective Protein
Tyrosine Phosphatase Inhibitor,
2-[(4-Methylthiopyridin-2-yl)methylsul®nyl]benzimidazole
Takuya Hamaguchi,a,* Akiko Takahashi,a Terumi Kagamizono,a Akira Manaka,a
Masakazu Satoa and Hiroyuki Osadab
aMedical Research Laboratories, Taisho Pharmaceutical Co., Ltd, Yoshino-cho 1-403, Omiya-shi, Saitama 330-8530, Japan
bAntibiotics Laboratory, The Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako-shi, Saitama 351-01, Japan
Received 24 July 2000; accepted 16 September 2000
AbstractÐThe synthesis and biological activity of a series of 2-[(4-methylthiopyridin-2-yl)methylsul®nyl]benzimidazoles are descri-
bed. These compounds have potent inhibitory e€ects against the protein tyrosine phosphatase activity of CD45. Enzymatic analysis
with several phosphatases revealed that compound 5a had high speci®city for CD45 compared with serine/threonine phosphatases
(PP1, PP2A), tyrosine phosphatases (LAR, PTP1B and PTP-S2) and dual phosphatase (VHR). # 2000 Elsevier Science Ltd. All
rights reserved.
Introduction
hampered by the absence of PTPase-speci®c agents.
Sodium orthovanadate6 and phenylarsine oxide (PAO)7
are known to be PTPase inhibitors. However, both
compounds have no speci®city for PTPases. Therefore,
more selective PTPase inhibitors are needed.
Tyrosine phosphorylation of receptors is a feature of
cell activation in a variety of biological systems. Tyr-
osine phosphorylation level is determined by the bal-
ances of activities of protein tyrosine kinases (PTKs)
and protein tyrosine phosphatases (PTPases). While
PTKs have been extensively studied, the characteriza-
tion of the equally important PTPases has recently been
undertaken. For example, CD45 is a typical receptor-
type PTPase, which consists of a putative ligand-bind-
ing extracellular domain, a transmembrane segment,
and an intracellular catalytic region. The intracellular
catalytic regions of receptor-type PTPases are highly
homologous both to each other and to the non-recep-
tor-type PTPases. A large number of studies have
shown that CD45 dephosphorylates the phosphotyr-
osine of Src-family kinases, permitting these kinases to
become activated in T and B cells.1 5 CD45 is a possible
target for drugs in the treatment of several immune dis-
eases.
To discover new immunosuppressive reagents, we have
been screening new PTPase inhibitors from among both
synthetic compounds and microbial metabolites. Pre-
viously, we found potent and speci®c PTPase inhibitors,
such as RK-6828 and the stevastelins.9 Using them, we
evaluated the roles of PTPases in cellular signal trans-
duction. In subsequent screening for PTPase inhibitors,
we found benzimidazole derivatives, which exhibited
95% inhibition of human CD45 PTPase activity at 5 mg/
mL concentration. A variety of benzimidazole com-
pounds are known, but their PTPase inhibitory e€ects
have not been reported. In addition, these compounds
had potent and speci®c inhibitory e€ects on the PTPase
activity of CD45. In this study, 2-[(4-methylthiopyridin-
2-yl)methylsul®nyl]benzimidazoles were synthesized and
evaluated for their ability to inhibit the several PTPase
activities. Speci®c inhibitors against CD45 are useful for
the treatment of several immune diseases.
PTPase-speci®c inhibitors could potentially serve as
useful tools in elucidating the physiological signi®cance
of protein tyrosine dephosphorylation in cellular signal
transduction pathways. In contrast to tyrosine kinases,
elucidation in detail of the roles of PTPase has been
Chemistry
For structure±activity relationship studies, we pre-
pared benzimidazole derivatives. The syntheses of the
*Corresponding author. Tel.: +81-48-663-1111; fax: +81-48-652-
7254; e-mail: s14795@ccm.taisho.co.jp
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00539-4

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T. Hamaguchi et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2657±2660
Scheme 1. (a) (i) concd HCl/AcOH, re¯ux; (ii) H2, 10% Pd/C/EtOH, rt; (iii) EtOCS2K/H2O, re¯ux; (b) 2 N NaOH aq/EtOH, 50±90 ꢀC; (c) m-
chloroperbenzoic acid/CHCl3, 50 ꢀC.
compounds (4a, 5a±d) are outlined in Scheme 1. 5-Sub-
stituted 2-mercapto-1H-benzimidazoles (2a, b) were
synthesized from corresponding acetanilide derivatives
(1a, b) in the previously described manner.10 4-Sub-
stituted 2-(chloromethyl)pyridines (3a±c) were pur-
chased or derived from corresponding picoline-1-oxides
via Pummerer-like rearrangement and chlorination with
thionyl chloride in chloroform. Coupling reactions of
3a±c with 2a or 2b were performed in aqueous basic
conditions to obtain the sulfenyl derivatives (4a±d), and
continuous selective oxidation of the benzimidazolyl
sulfur atom using m-chloroperbenzoic acid were
subjected to obtain sulfoxides 5a±d.
thiopyridyl moiety to 4-methylpyridyl 5c or pyridyl
moiety 5d, inhibitory e€ects were decreased in a graded
fashion. The 4-methylthiopyridyl moiety was required
for inhibitory activity.
Protein phosphatases are categorized into three
groups:12 the protein serine/threonine phosphatases
(PPases), PTPases and dual-speci®city phosphatases
(DSPases). These protein phosphatases play important
roles in regulating a variety of fundamental cellular
processes. Since 5a was most potent against CD45, we
investigated the inhibitory speci®city of 5a against PP-
ases (PP1 and PP2A), PTPases (CD45, LAR, PTP1B
and PTP-S2) and DSPase (VHR).13 As shown in Table
2, 5a had no inhibitory e€ect on either PP1 or PP2A
activities at a concentration of 1000 mM. On the other
hand, it exhibited inhibitory e€ects on CD45, LAR,
PTP1B and PTP-S2 with IC50 values of 0.28, 18.2, 55.4
and 55.2 mM, respectively. Sodium orthovanadate, a
non-speci®c PTPase inhibitor, also inhibited only
PTPases activities. These ®ndings indicated that 5a was
an inhibitor of PTPases and had potent inhibitory
e€ects on receptor-type PTPases (CD45 and LAR)
compared with non-receptor-type PTPase (PTP1B and
PTP-S2). Furthermore, 5a also exhibited a slight inhi-
bition of VHR, with an IC50 of 50.2 mM. Since vanadate
inhibited CD45 and LAR with IC50 values of 34.2 and
30.8 mM, respectively, 5a had some degree of enzyme
speci®city for CD45.
Biological Studies
The e€ects of compounds on CD45 were examined
using p-nitrophenyl phosphate as a substrate at 37 ꢀC
and pH 6.5.11 IC50 values are summarized in Table 1.
Compound 5a had a potent inhibitory e€ect on CD45
with an IC50 value of 0.28 mM. On the other hand,
4a had no inhibitory e€ect on CD45 at a concentra-
tion of 1000 mM. The substituents at the 5-position and
2-sul®nyl moiety of benzimidazole were required for
this inhibitory e€ect. With a change from the 4-methyl-
Table 1. Inhibitory e€ects on PTPase activity of CD45
Table 2. Speci®city of inhibition by 5a of several phosphatases
Activity IC50 (mM)
Compound
R1
R2
A
IC50 (mM)
PPases
PTPases
DSPase
4a
5a
5b
5c
5d
i-Propyl
i-Propyl
Cyclohexyl
i-Propyl
i-Propyl
SCH3
SCH3
SCH3
CH3
H
S
>1000
0.28
80.5
380
SˆO
SˆO
SˆO
SˆO
Compound PP1 PP2A CD45 LAR PTP1B PTP-S2 VHR
5a >1000 >1000 0.28 18.2
Vanadate >1000 >1000 34.2 30.8
55.4
30.2
55.2
38.1
50.2
105.8
120

T. Hamaguchi et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2657±2660
2659
The 2-[(2-pyridylmethyl)sul®nyl]benzimidazoles, such as
omeprazole, have attracted considerable attention as
potential therapeutics for the treatment of peptic
ulcer.14 Some evidence suggests that omeprazole oxi-
dizes SH groups of the (H+ K+)-ATPase resulting in
enzyme inhibition after omeprazole is transformed into
the active form by acid.15,16 This mechanism of action is
supported by chemical evidence in that 2-[(2-pyr-
idylmethyl)sul®nyl]benzimidazole is transformed to
intermediate cyclic sulfenamides followed by coupling
with adequate thiol to form disul®de17 (Scheme 2).
Without exception, all PTPases contain an active site
signature motif, (I/V)HCXAGXGR(S/T)G, which
enfolds the catalytic cysteinyl residue.18 To investigate
whether 5a must be transformed to the active form for
showing an inhibitory e€ect on PTPase, we evaluated
the in¯uence of glutathion (GSH) on the inhibitory
e€ect of 5a against CD45. GSH is an SH-containing
compound, which disrupts the binding of omeprazole to
(H+ K+)-ATPase.19,20 Compound 5a preincubated in
acidic conditions (in 0.1 M HCl) was added to PTPase
assay medium with or without 10 mM GSH at 37 ꢀC for
30 min.11 As shown in Fig. 1a, 5a inhibited the PTPase
activity of CD45 without a€ecting GSH. In addition,
the inhibitory e€ects of 5a were the same with or with-
out treatment in acidic conditions (Fig. 1b). We investi-
gated the inhibitory e€ect of omeprazole on PTPase
activity, but found it had no e€ect up to 1 mM (data not
shown). The di€erence in structures between omepra-
zole and 5a is the absence of a 4-methylthiopyridyl
group in the former. These ®ndings indicated that the
mechanism of inhibition by 5a did not involve form-
ation of a disul®de bond between the intermediate
cyclic sulfenamide and the catalytic cysteinyl residue
of PTPase.
Scheme 2. Possible mechanism for acid-transformation of 5a. 5a is
transformed into the intermediate cyclic sulfenamide (A), followed by
coupling with adequate thiols (RSH) to form the ultimate disul®des (B).
CD45 is a transmembrane PTPase expressed on most
hematopoietic cells. The exact function of CD45 in
lymphocyte activation is currently under intense inves-
tigation in many laboratories. Accumulating evidence
has shown that CD45 is an essential component of
receptor signaling pathways, since it dephosphorylates
the negative regulatory tyrosine residues of Src-family
kinases. In T cell activation, the PTPase activity of
CD45 activates Lck by dephosphorylation of a C-term-
inal tyrosine residue.5 In immature B cells, another Src
family PTK, Fyn, appears to be a selective substrate for
CD45, compared with Lck and Syk.21 Some studies
have reported that CD45 is necessary for activation of
FceRI-associated Lyn,22 the dephosphorylation of
receptor subunits,23 and the initiation of calcium in¯ux
in mast cell activation.24 Since 5a suppressed histamine
release from rat peritoneal mast cells and mouse pas-
sive-sensitized anaphylaxis reaction induced by mono-
clonal anti-DNP IgE and DNP-BSA (Hamaguchi et al.,
unpublished data), CD45 may be involved in these pro-
cesses. These results indicate that PTPase inhibitors are
useful in the treatment of allergies.
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