Discovery and SAR of 2-amino-5-[(thiomethyl)aryl]thiazoles as potent and selective Itk inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.01.115

A series of structurally novel aminothiazole based small molecule inhibitors of Itk were prepared to elucidate their structure-activity relationships (SARs), selectivity and cell activity in inhibiting IL-2 secretion in a Jurkat T-cell assay. Compound 2 i

Full text of DOI:10.1016/j.bmcl.2006.01.115

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2411–2415
Discovery and SAR of 2-amino-5-[(thiomethyl)aryl]thiazoles
as potent and selective Itk inhibitors
Jagabandhu Das,* Chunjian Liu,* Robert V. Moquin, James Lin, Joseph A. Furch,
Steven H. Spergel, Kim W. McIntyre, David J. Shuster, Kathleen D. O’Day,
Becky Penhallow, Chen-Yi Hung, Steven B. Kanner, Tai-An Lin, John H. Dodd,
Joel C. Barrish and John Wityak
Bristol-Myers Squibb Pharmaceutical Research Institute, PO Box 4000, Princeton, NJ 08543-4000, USA
Received 3 January 2006; revised 24 January 2006; accepted 26 January 2006
Available online 14 February 2006
Abstract—A series of structurally novel aminothiazole based small molecule inhibitors of Itk were prepared to elucidate their struc-
ture–activity relationships (SARs), selectivity and cell activity in inhibiting IL-2 secretion in a Jurkat T-cell assay. Compound 2 is
identified as a potent and selective Itk inhibitor which inhibits anti-TCR antibody induced IL-2 production in mice in vivo.
Ó 2006 Elsevier Ltd. All rights reserved.
Mammalian immunity relies on the activation of T
cells upon antigen presentation. Following the engage-
ment of the T-cell antigen receptor (TCR), T-cell acti-
vation is initiated by sequential activation of three
distinct classes of non-receptor protein tyrosine ki-
nases, namely the Src family kinases (Lck and Lyn),
the Syk family kinases (ZAP-70 and Syk), and the
Tec family kinases (Itk, Txk, and Tec).1 Inhibition
of any of these kinases will impede the initiation sig-
nals and block the T-cell activation following antigen
presentation.
secretion, and proliferation.3 In addition, Itk-deficient
mice are unable to establish functional Th2 cells result-
ing in their inability to clear parasitic infections depen-
dent upon a Th2 response.4 These studies suggest that
a selective Itk inhibitor should be useful as an immuno-
suppressive and/or anti-inflammatory agent and may be
an attractive modulator of dysregulated allergic path-
ways mediated by Th2 cells.5
Directed screening of our internal compound libraries6,8
using an HTRF assay identified the aminothiazole 1 as a
selective ATP-competitive Itk inhibitor.7 In this report,
we outline our initial efforts in optimizing this series
leading to compound 2 as a potent and selective Itk
inhibitor in vitro with excellent activity in reducing
IL-2 production, and T cell proliferation in both human
and mouse cells (Figure 1). In addition, 2 is shown to
suppress anti-TCR antibody induced IL-2 production
in mice in vivo.
Itk (interleukin-2-inducible T-cell kinases), also known
as Emt or Tsk, is expressed mainly in T-, natural killer,
and mast cells. Itk is tyrosine phosphorylated and acti-
vated in response to cross linking of TCR, CD28 or
CD2, and has been implicated in thymocyte develop-
ment and activation of T cells through TCR and
CD28 engagement. Within the cell, Itk is regulated by
membrane recruitment, followed by Lck phosphoryla-
tion, and then autophosphorylation.2 Gene knockout
studies have revealed that mice lacking Itk have fewer
number of T cells, especially CD4+ T cells, and matured
T cells isolated from these mice exhibit defective TCR
mediated responses such as calcium mobilization, IL-2
The synthesis of these compounds follows a general syn-
thetic route that is illustrated with the preparation of 2
in Scheme 1. Accordingly, 2-amino-4-thiocyanatothiaz-
ole8 was treated with 2 equiv of sodium borohydride in
ethanol at 0–5 °C for 2 h and then reacted with methyl
5-chloromethyl-2,3-dimethylbenzoate9 at rt for 16 h to
form a methyl ester, which was saponified with aq. sodi-
um hydroxide solution in methanol under reflux to form
acid 3 in 72% overall yield.
Keywords: Selective Itk inhibitors; Thiazoles.
*
Corresponding authors. Tel.: +1 609 252 5068; fax: +1 609 252
6804; e-mail: jagabandhu.das@bms.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.01.115

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J. Das et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2411–2415
O
N
S
O
O
N
S
O
N
H
N
H
Me
S
N
H
N
N
Me
Me
S
Me
N
Me
N
Me
N
Me
Me
1
Me
O
2
=
O
Me
Itk IC50 = 1 μM
Jurkat IL-2 I50 = 22 μM
Itk IC50
Jurkat IL-2 I50 = 700 nM
96 nM
Figure 1. Itk inhibitory activity of aminothiazoles 1 and 2.
N
S
N
S
a, b
O
N
H2N
H2N
c
O
H2N
S
OH
SCN
S
S
N
N
Me
Me
3
4
Me
Me
Me
O
O
N
O
N
S
O
O
N
H
e
d
N
H
S
H
S
N
Me
Me
Me
S
N
N
Cl
N
Me
N
Me
Me
Me
Me
2
Me
O
5
Me
O
Scheme 1. Reagents and conditions: (a) NaBH4, EtOH, methyl 5-chloromethyl-2,3-dimethylbenzoate, 87%; (b) MeOH, 1 N aq. NaOH, D, 86%; (c)
N-acetylpiperazine, BOP reagent, 4-methylmorpholine, DMF, 60 °C, 88%; (d) 4-chloromethylbenzoyl chloride, Py, CH2Cl2; (e) 3,3-dimethyl-2-
butanamine, DMF, 55 °C, 30% overall yield in two steps.
Reaction of acid 3 with N-acetylpiperazine in DMF in
the presence of BOP reagent and 4-methylmorpholine
at 50 °C for 5 h afforded compound 4 (88% yield), which
was treated with 4-chloromethylbenzoyl chloride in
dichloromethane in the presence of pyridine at rt for
16 h to form benzamide 5. Benzamide 5 was heated with
6 equiv. of 3,3-dimethyl-2-butanamine in DMF at 55 °C
for 16 h to form 2 (BMS-488516) in 30% overall yield in
two steps.
Aminothiazole 1 and its analogs were tested for their
ability to inhibit the phosphorylation of an exogenous
Table 1. SAR for thiazole ring modification
O
O
Table 2. SAR for linker modification
Z
N
S
N
O
N
S
H
O
Me
N
Me
N
N
Y
N
X
H
Me
O
Me
N
Me
N
6,a
Itk inhibition IC50 (lM)
Me
Compound
Z
O
N
S
6
1
1.0
Compound
X–Y
Itk inhibition IC50 (lM)
S
1
1.0
Me
N
4
1a
1b
1c
1d
1e
1f
1g
>50
39
S
S
1h
1i
>50
5.0
4.6
1.7
1.6
1.6
0.6
Ph
N
S
O
N
S
N
>50
>50
>50
>50
>50
1j
S
S
N
N
N
H
N
1k
1l
O
N
N
N
N
O
S
S
S
S
S
N
N
1m
1n
N
S
a Values throughout are means of three independent experiments,
standard deviation 10%.

J. Das et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2411–2415
Table 5. SAR for amide modification
2413
Table 3. SAR for amide modification
O
O
N
S
N
O
O
2
R
N
N
S
S
N
S
Z
H
H
Me
N
Me
N
Me
Me
Me
O
6
Compound
Z
Itk inhibition IC50 (lM)
Compound
R
Itk inhibition Jurkat IL-2
C50, lM,6
O
inhibition
6,a
C50
(lM)
1
1.0
N
Me
N
1al
0.3
2.0
Me
N
1o
1p
1q
1r
1s
1t
NHPh
>50
11.2
>50
2.2
Me
Me
1am
1an
1ao
0.04
0.32
0.096
13.5
5.7
N
HN
HO
HO
N
NH(CH2)2NHAc
H
OH
N
N
2.2
N
Me
Me
O
H
N
2.1
Me
Me
N
Me
N
2
0.09
0.7
Me
Me
O
2.6
N
H
N
1ap
1aq
0.14
2.4
0.7
N
1u
1v
1w
0.9
Me
N
N
H
Me
Me
N
N
0.035
N
H
Me
NMe2
14.8
1.44
a Values are means of three independent experiments, standard devi-
ation 20%.
NH
N
N
N
N
substrate (GST-fused SLP-76)6 using a recombinant Itk
kinase domain as source of the enzyme (IC50, Tables 1–5).
N
Table 1 outlines the SAR observed with the thiazole ring
modification. Introduction of a substituent in the 4-po-
sition of the thiazole (1a and 1b) resulted in a significant
loss of potency. Replacement of the thiazole ring with
isosteric five-membered ring heterocycles (1c and 1d)
or six-membered ring systems (1e and 1f) or a benzo-
thiazole ring (1g) was equally unsuccessful thereby
indicating that the thiazole ring system was optimal
for Itk inhibitory activity.
Table 4. SAR for thioaryl modification
O
N
O
N
S
S
N
H
2
Me
4
6
N
N
Me
R
Me
O
6
Compound
R
H
Itk inhibition IC50 (lM)
1
1x
1.0
37
2-Me
4-Me
5-Me
6-Me
5-NO2
5-NH2
Table 2 summarizes relevant SAR for the thioalkylaryl
linker. Oxidation of the thioether linkage to its sulfoxide
(1h) resulted in complete loss of potency. Replacement
of the thioether moiety with an olefin (1i) or substitution
of the aryl ring with an olefin (1j) led to an approximate-
ly 5-fold loss in activity. The phenyl ring can be replaced
with several 5-membered heterocycles (1k–1n) without
any loss of Itk inhibitory potency.
1y
1z
0.38
0.26
15
1aa
1ab
1ac
1ad
1ae
1af
1ag
1ah
1ai
1aj
1ak
1al
13
0.54
14
5-NHAc
4-NH2
4-NHAc
4-NO2
4-Cl
0.73
2.42
1.09
1.03
0.35
0.35
0.72
0.30
To optimize the potency, we next turned to the modifi-
cation of the N-acetyl-piperazine amide moiety (Table
3). Replacement with benzamide (1o) or N,N-dimethyl
amide (1v) groups significantly attenuated the potency.
Substitution of the piperazine ring with an 2-acetami-
4-OMe
5-OMe
5-Cl
4,5-Di-Me

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J. Das et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2411–2415
Table 6. Enzyme selectivity of selected Itk inhibitors
6
Itk IC50 (nM)
a
Compound
Bmax
Btka
>500
>1400
Teca
Txka
Lcka
Syka
>500
>1400
Cdk2a
2
1aq
96
35
>500
1000
>500
325
>500
90
270
90
33
300
a Selectivity ratio: IC50/IC50 (Itk).
doethylenediamine side chain (1q) was also not tolerat-
ed. Replacement of the N-acetylpiperazine with a piper-
idine group (1p) also significantly reduced the potency.
However, the piperazine ring can be replaced with a
homopiperazine (1s) or a morpholine moiety (1t) with-
out any loss of potency. Finally substitution of the ace-
tyl group attached to the piperazine ring with either a
tetrazole (1w), a pyrimidine ring (1u) or hydroxyethyl
group (1r) was tolerated.
Introduction of substituents on the central phenyl ring
had a significant effect on Itk inhibitory activity of this
series of analogs (Table 4). A methyl group scan demon-
strated that such substitution at either C4- or C5-posi-
tion (1y and 1z) led to a 3- and 4-fold increase in
potency, respectively. However, such substitution at
C2 (1x) or C6 (1aa) was detrimental to activity. A gen-
eral SAR trend was observed regarding substituents at
the C4- and C5-positions. The nature and size of the
substituents at C4 had a minor effect on potency. In con-
trast, Itk inhibitory activity was more sensitive with re-
gard to substituents at C5. An electron-deficient
function (1ab) or a large group (1ad) was not tolerated.
A small alkyl substituent (1y and 1z), or electron-rich
groups (1ac, 1ae, 1ai, and 1aj) at C4 and C5 led to the
more potent analogs in this series. 4,5-Dimethyl-substi-
tuted analog (1al) was also identified as a potent Itk
inhibitor.
Figure 2. Dose-dependent inhibition of anti-CD3 antibody induced
serum IL-2 production in mice (data in black). Serum concentrations
of 2 (data in red).
Compounds 1aq and 2 were tested for their selectivity
over a panel of kinases (Table 6). Compound 2 showed
a better selectivity profile than 1aq with respect to other
Tec family kinases. It was >500-fold selective against all
Tec family kinases and showed at least 30-fold selectiv-
ity against all other protein kinases tested. Furthermore,
2 also exhibited competitive kinetics with respect to ATP
in its inhibition of Itk.6
Based on its potent enzyme activity and selectivity, 2
was selected for further evaluation. Compound 2 inhib-
ited anti-CD3-antibody induced IL-2 secretion both in
human Jurkat T-cells (IC50 = 0.70 0.18 lM), PBMC
(IC50 = 0.65 0.14 lM), and in murine EL4 cells
(IC50 = 0.25 0.07 lM) and splenocytes (IC50 = 1.36
0.40 lM). Compound 2 was also tested for its ability
to inhibit IL-2 production in vivo in mice following
intravenous injection of anti-CD3 antibody. Analog 2
dose dependently suppressed IL-2 production in serum
(50 and 100 mpk, sc) in line with its serum drug concen-
trations (Figure 2).
Because of its excellent potency, further optimization
was focused on the 4,5-it di-Me analog (1al). We con-
centrated on SAR studies at the thiazole 2-carboxamide
functionality (Table 5). Replacement of the N,N-di-Me
group at the para position of the benzamide with a
piperazine ring (1am) resulted in a 7-fold increase in en-
zyme activity in vitro. Despite its excellent in vitro
potency compound 1am was 7-fold less potent than
compound 1al in a Jurkat cell-based IL-2 production
inhibition assay. Similarly the corresponding benzyl
alcohol analog (1an) was 3-fold less potent in the Jurkat
cell assay, despite being equipotent to compound 1an
against Itk. A pronounced increase in biochemical and
cellular potency was observed on introduction of a large
hydrophobic aminomethyl moiety at the para position
of the benzamide group. Compound 2 was identified
as a potent Itk inhibitor (IC50 = 90 nM) with a Jurkat
T-cell IL-2 IC50 value of 700 nM. Furthermore, replace-
ment of the benzamide group in 1al with a pyrrolo-2-
carboxamide group (1ap) retained both biochemical
and cell potencies of the parent analog. Introduction
of a large hydrophobic aminomethyl group at the C5-
position of the pyrrole ring led to analog 1aq, which
was equipotent with 2 in both biochemical and cell-
based assays. Compounds 1aq and 2 were thus selected
for further characterization.
In summary, starting from a screening lead 1 with mod-
est biochemical and cell potency we identified a series of
potent and selective Itk inhibitors as exemplified by 2
through a systematic SAR approach. We also demon-
strated the potency of this analog in an in vivo mouse
model. To our knowledge, 2 is one of the first examples
of a potent and selective small molecule Itk inhibitor
reported to date and should serve as a useful tool in
investigating the role of Itk in T cell signaling.
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