Discovery and SAR of 2-amino-5-(thioaryl)thiazoles as potent and selective Itk inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.04.060

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 3 is identified as a potent and selective Itk inhibitor which inhibits anti-TCR antibody induced IL-2 production in mice in vivo and was previously reported to reduce lung inflammation in a mouse model of ovalbumin induced allergy/asthma.

Full text of DOI:10.1016/j.bmcl.2006.04.060

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3706–3712
Discovery and SAR of 2-amino-5-(thioaryl)thiazoles
as potent and selective Itk inhibitors
Jagabandhu Das,* Joseph A. Furch, Chunjian Liu, Robert V. Moquin, James Lin,
Steven H. Spergel, Kim W. McIntyre, David J. Shuster, Kathleen D. O’Day,
Becky Penhallow, Chen-Yi Hung, Arthur M. Doweyko, Amrita Kamath,
Hongjian Zhang, Punit Marathe, 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 10 March 2006; accepted 20 April 2006
Available online 6 May 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 3 is
identified as a potent and selective Itk inhibitor which inhibits anti-TCR antibody induced IL-2 production in mice in vivo and was
previously reported to reduce lung inflammation in a mouse model of ovalbumin induced allergy/asthma.
Ó 2006 Elsevier Ltd. All rights reserved.
Itk (Interleukin-2-inducible T cell kinases), also known
as Emt or Tsk, is expressed mainly in T, natural killer,
and mast cells.1 Itk is tyrosine phosphorylated and acti-
vated in response to cross linking of TCR, CD28 or
CD2 and has been implicated in thymocyte development
and activation of T cells through TCR and CD28
engagement.2 Gene knockout studies have revealed that
In an earlier report we described SAR optimization of
the directed screening hit 1 that led to the discovery of
2 as a potent and selective Itk inhibitor.7 In this commu-
nication, we outline SAR studies in a related and novel
2-amino-5-thioaryl-thiazole series that culminated in the
identification of 3 as a highly potent and selective small
molecule Itk inhibitor with excellent activity in reducing
T cell proliferation in vitro and Il-2 production in mice
in vivo (Fig. 1). As reported earlier,8 compound 3 reduc-
es lung inflammation in a mouse model of ovalbumin
induced allergy/asthma.
mice lacking Itk have fewer T cells, especially CD4+
T
cells, and mature T cells isolated from these mice exhibit
defective TCR mediated responses such as calcium
mobilization, IL-2 secretion, and proliferation.3 In addi-
tion, Itk deficient mice are unable to establish functional
Th2 cells resulting in their inability to clear parasitic
infections dependent upon a Th2 response.4 Recent
studies have shown that Itk deficient mice have drasti-
cally reduced lung inflammation, eosinophil infiltration,
and mucous production in response to OVA induced
induction of allergic asthma.5 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.6
The synthesis of compounds related to 3 is shown in
Schemes 1–3. Scheme 1 outlines a general route to the
carboxamide analogs 9. Reaction of commercially avail-
able 2-amino-5-bromothiazole 4 with the appropriately
substituted 3-carboxythiophenol 5 in the presence of sodi-
um methoxide in methanol followed by addition of anhy-
drous hydrogen chloride in dioxane formed the thioether
6. Protection of the amino group as a tert-butylcarbamate
and subsequent base hydrolysis of the methyl ester affor-
ded acid 7. Coupling of acid 7 with the appropriate amines
under standard conditions followed by BOC-deprotec-
tion with TFA formed amine 8 which was further coupled
with an acid chloride in the presence of pyridine to form 9.
Keywords: Amino-(thioaryl)thiazoles; Selective Itk inhibitor.
*
A similar but more concise route was used for the syn-
thesis of 3. Reaction of 2-amino-5-bromothiazole 4
Corresponding author. 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.2006.04.060

J. Das et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3706–3712
3707
O
N
S
O
O
N
S
O
Me
N
H
N
H
H
N
N
Me
Me
S
N
S
Me
N
Me
N
Me
N
Me
Me
Me
2
Me
O
1
O
Itk IC50 = 96 nM
Jurkat IL-2 I50 = 700 nM
Itk IC50 = 1 μM
Jurkat IL-2 I50 = 22 μM
O
Me
S
OMe
N
O
N
S
Me
N
Me
N
H
N
Me
Me
H
O
Me
3
Itk IC50 = 19 nM
Jurkat IL-2 I50 = 250 nM
Figure 1. Itk inhibitory activity of aminothiazoles 1–3.
R1
R1
N
S
N
S
a
H2N
+
H2N
CO2H
Br
CO2Me
S
HS
4
5
6
R1
Me
O
N
S
b, c
d, e
Me
O
N
Me
CO2H
S
H
7
R1
R1
O
R2
N
R2
N
N
N
f
H2N
R4
N
R3
R3
S
S
S
S
H
O
O
9
8
Scheme 1. Reagents: (a) i—NaOMe, MeOH; ii—4 M HCl, dioxane; (b) di-tert-butylcarbonate, DMAP, THF; (c) aq NaOH, MeOH; (d) EDAC,
HOAt, i-Pr2NEt, R2R3NH, THF; (e) TFA; (f) CH2Cl2, Py, R4COCl.
Me
Me
S
OMe
OMe
N
S
N
S
a
b
H2N
+
H2N
CO2H
CO2H
Br
HS
4
11
10
O
O
Me
OMe
N
O
N
S
N
Me
OMe
Me
N
S
c
N
Me
N
H2N
S
H
Cl
N
S
O
13
O
12
O
Me
OMe
N
O
N
N
Me
Me
N
d
H
Me
Me
S
S
H
N
O
3
Me
Scheme 2. Reagents: (a) NaOMe, MeOH, 87%; (b) EDAC, HOAt, i-Pr2NEt, N-acetylpiperazine, THF, 78%; (c) CH2Cl2, i-Pr2NEt,
4-chloromethylbenzoyl chloride; (d) MeOH, 2-amino-3,3-dimethylbutane, 36% overall yield in two steps.
and 3-carboxy-4-methoxy-6-methylthiophenol 10 with
methanolic sodium methoxide at 0 °C to rt formed acid
11 which was then treated with N-acetylpiperazine to
form aminothiazole 12. Subsequent reaction of 12 with
4-chloromethylbenzoyl chloride and diisopropylethyl
amine at ambient temperature formed 13 which upon
treatment with 2-amino-3,3-dimethylbutane in refluxing
methanol afforded 3. This synthetic route eliminated the
protection/deprotection steps outlined in Scheme 1.
Preparation of 2-heteroarylamine analogs required a
different approach as illustrated in Scheme 3 with the
synthesis of 2-aminopyridine 18. Reaction of 2-amino-
pyridine 14 with benzoylisothiocyanate in acetone

3708
J. Das et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3706–3712
a, b, c
N
S
N
S
d
NH2
N
N
N
H
N
N
Br
H
14
15
16
O
e
N
S
N
f
N
Me
N
N
N
N
N
S
CO2H
S
H
S
H
O
17
18
Scheme 3. Reagents: (a) acetone, benzoylisothiocyanate, 100%; (b) aq NaOH, 73%; (c) aq EtOH, chloroacetaldehyde, 79%; (d) bromine, AcOH,
86%; (e) 3-carboxythiophenol, NaOMe, MeOH, 86%; (f) EDAC, HOAt, i-Pr2NEt, N-acetylpiperazine, THF, 100%.
followed by basic hydrolysis of the benzoyl protecting
group afforded a thiourea intermediate9 which was con-
densed with chloroacetaldehyde10 in aq ethanol to form
thiazole 15. Bromination of 15 with bromine and acetic
acid at 40 °C afforded 5-bromothiazole 16 which was
then treated with 3-carboxythiophenol to form the acid
17. Reaction of 17 with N-acetylpiperazine under stan-
dard conditions afforded 2-pyridylamino derivative 18.
This synthetic route is quite general and was used for
the synthesis of other heteroaryl amino derivatives.
Table 2. SAR for thioaryl linker modification
O
Me
R1
N
O
N
S
4
6
N
2
N
S
H
O
Me
N
Me
Compound
R1
Itk inhibition
IC50 (lM)8
Aminothiazole 1 and its analogs were tested for their
ability to inhibit the phosphorylation of an exogenous
substrate (GST-fused SLP-76)8 using a recombinant
Itk kinase domain as source of the enzyme (IC50,
Tables 1–6).
9a
9b
9c
9d
9e
9f
H
2-Me
0.97
>25
4-Me
6-Me
4-Cl
0.29
0.94
0.6
4-NH2
4-OH
4-OMe
4-NHAc
5-NH2
5-CF3
0.09
0.03
0.034
0.25
1.13
19.24
0.21
3.9
Table 1 outlines the SAR observed with the thiomethyl
ether linkage modification of the initial screening hit 1.
Truncation of the thiomethyl linker by deletion of either
the methylene group (9a) or the sulfur atom (1a) retains
the biochemical potency of the parent 1. In the 4-tert-
butyl benzamide series the thiomethyl linker can be re-
placed with an extended ether (1b) or N-methylated
amine (1c) without any loss of potency. The N-desmeth-
yl analog (1d) corresponding to 1c is at least an order of
magnitude less potent. Based on its biochemical potency
and selectivity over other kinases (data not shown) 9a
was selected for further optimization.
9g
9h
9i
9j
9k
9l
4,5-di-Me
5,6-di-Me
4,6-di-Me
4-OH,6-Me
9m
9n
9o
9p
0.06
0.012
0.018
4-OMe,6-Me
A profound increase in biochemical potency was
observed upon introduction of substituents on the
central thiophenyl ring (Table 2). A methyl group scan
revealed the importance of such substitution at the
C4- or C6-position of the phenyl ring (9d–e). A wide
variety of small substituents (9e–i) is tolerated at the
C4-position. In general electron donating groups led
to more potent analogs (9f–h). A similar trend is
observed with respect to substitution at the C5-position
of the phenyl ring (9j–k). Finally disubstitution in the
phenyl ring led to some of the more potent analogs.
4-Hydroxy-6-methyl and 4-methoxy-6-methyl analogs
9o and 9p are identified as the two most active
compounds in this series.
Table 1. SAR for thioether linker modification
O
Me
N
O
N
S
N
N
X
H
O
R
Compound
X
R
Itk inhibition
IC50 (lM)8
1
SCH2
S
CH2
Me2N
Me2N
Me2N
t-Bu
1.0
0.97
0.82
0.93
2.19
22.34
9a
1a
1b
1c
1d
To optimize the potency further, we next turned to the
modification of the C20-benzamide moiety of compound
9p (Table 3). Replacement of the N,N-dimethyl group
on the benzamide with an ether (9q–r) has little impact
on the biochemical and cell potency of these analogs.
CH2OCH2
CH2N(Me) CH2
CH2NH CH2
t-Bu
t-Bu

J. Das et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3706–3712
3709
Table 3. SAR for C2-amide modification
O
Me
N
OMe
Me
O
N
N
2
R
N
S
S
H
O
Compound
R
Itk inhibition IC50 (lM)8
Jurkat Il-2 inhibition IC50 (lM)8
9p
0.018
0.28
0.42
0.36
3.4
Me
N
Me
9q
9r
9s
0.02
MeO
Me
0.047
0.029
Me
Me
O
H
N
H
N
Me
Me
3
0.015
0.014
0.009
0.25
0.25
0.69
Me
Me
H
N
9t
9u
Table 4. SAR for C2 amine modification
O
Me
R1
N
N
2
4
6
N
R
2
N
S
S
H
O
Compound
R
R1
Itk inhibition
IC50 (lM)8
Jurkat IL-2 inhibition
IC50 (lM)8
18a
18
Ph
2-Pyridyl
3-Pyridyl
H
34.2
0.69
NDa
24
H
18b
18c
18d
18e
18e
18f
18g
18h
H
>50
NDa
NDa
NDa
NDa
2.18
0.37
0.46
0.23
2-Pyrimidinyl
2-Pyrazinyl
2-Thiazolyl
2-Pyridyl
H
46.3
H
6.06
7.07
H
4-OH
0.03
6-Br-2-pyridyl
4,6-di-Me-2-pyridyl
4,6-di-Me-2-pyridyl
4-OH
4-OH
4-OMe, 6-Me
0.007
0.007
0.006
a ND, not determined.
Furthermore, the benzylamine analog 9s is an order of
magnitude less potent than the parent 9p in the cell
based assay despite being biochemically equipotent.
Introduction of a large hydrophobic group on the ben-
zyl amine led to analog 3 as a highly potent Itk inhibitor
both biochemically and in the cell. The benzamide can
be replaced with a pyrrolo amide (9t) or cyclopropyl
amide (9u) with no loss of potency in vitro. However,
the cyclopropyl amide 9u is roughly 2-fold less potent
in the Jurkat cell based assay.
We also investigated the effect of replacing the C20-
carboxamide with an aryl/heteroaryl amine (Table 4).
Replacement with aniline (18a) resulted in substantial

3710
J. Das et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3706–3712
Table 5. SAR for C2 and C3 carboxamide modification
OMe
R1
Me
O
N
2
S
3
R
N
S
H
O
Compound
R
R1
Itk inhibition
IC50 (lM)8
Jurkat Il-2 inhibition
IC50 (lM)8
MLM ratea
O
9u
9v
0.009
0.26
0.69
1.0
High
Me
N
N
High
Me
O
O
N
9w
9x
9y
0.019
0.32
0.21
25.32
0.26
Moderate
Low
Me
N
N
N
N
HO2C
Me
H
Me
N
N
0.007
Moderate
H
a MLM (mouse liver microsome) rate (nmol/min/mg of protein): high >0.3; moderate 0.1–0.29; low <0.1.
Table 6. SAR for C3 carboxamide modification
OH
R1
N
3
Br
N
N
S
S
H
O
Compound
18f
R1
Itk inhibition
IC50 (lM)8
Jurkat Il-2 inhibition
IC50 (lM)8
MLM ratea
High
O
0.007
0.034
0.015
0.37
0.37
>50
N
N
Me
N
18j
Low
CO2H
18k
>50
Low
N
CO2H
18l
N(Me)CH2CO2H
>10
Low
N
18m
0.083
>10
Low
NH
SO2Me
O
a MLM (mouse liver microsome) rate (nmol/min/mg of protein): high >0.3; moderate 0.1–0.29; low <0.1.
loss of potency. Heteroaryl amine substitution revealed
that only the 2-pyridyl amine analog 18 retained the
biochemical potency, while the isomeric pyridine deriva-
tive 18b or other heteroaryl amines (18c–e) were signifi-
cantly less potent. In the 2-pyridyl series an increase in
potency was achieved by introduction of substituent in
the central thiophenyl ring (18e). Substitution on the
pyridine ring further increased both biochemical and cell
potency. Compounds 18f–h were identified as some of
the most potent analogs in this series.
Our SAR studies so far identified several extremely
potent Itk inhibitors (3, 9p, 9t, and 18f–h) in this novel
2-amino-5-arylthio thiazole series. However, in vitro
metabolism studies with both mouse and human liver
enzyme homogenates revealed that this class of analogs

J. Das et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3706–3712
3711
in general suffered from a rapid rate (>0.3 nmol/min/mg
of protein) of degradation. To address this issue, we
investigated the effect of incorporating an acidic or basic
functionality in the molecule that could reduce its
affinity for the cytochrome P450 isozymes. Table 5 out-
lines these results in the C20-carboxamide series. For
example, replacement of the N-acetylpiperazine group
in the cyclopropyl amide analog 9u with basic sidechains
Prior to assessing activity in vivo, the pharmacokinetic
profile of 3 was determined in mouse (Table 8). Single
iv and oral doses of 3 were administered. Concentra-
tions of 3 were determined in plasma using an LC/MS
assay. Plasma concentrations declined with a mean elim-
ination half-life of 1.4 h upon oral dosing. Analog 3 suf-
fered from a high rate of clearance compared to the
mouse hepatic blood flow. The oral bioavailability of 3
based on the dose-normalized AUCs after oral and iv
dosing was 6.4% in mice.
(9v–w) or introduction of
a methylaminomethyl
functionality in the pyrroloamide analog (9y) had little
impact on the metabolic degradation rates of these
compounds. The metabolically stable carboxylic acid
analog 9x was significantly less potent against the
enzyme, and more importantly, in the cellular assay.
Compound 3 was tested for its ability to inhibit IL-2
production in vivo in mice following intravenous injec-
tion of anti-CD3 antibody (Fig. 2). A 50% inhibition
of serum IL-2 production was observed at a dose of
50 mg/kg, sc, irrespective of the amount of the antibody
used. In a separate experiment, the serum concentration
of 3 was determined to be 1.89 0.34 lM (n = 5), 1.75 h
after administration at 50 mg/kg, sc. In addition we
reported earlier8 that compound 3 significantly reduced
lung inflammation at a dose of 25 mg/kg, sc in a mouse
model of ovalbumin induced allergy/asthma.
A similar trend was observed with the 2-pyridylamino
analogs (Table 6). Several metabolically stable analogs
were identified through the introduction of an acid
(18j–l) or an acid surrogate group (18m). Some of these
analogs (18j–k) retained the intrinsic biochemical poten-
cy of the parent compound 18f but lacked any cellular
activity in the Jurkat Il-2 inhibition assay. To summa-
rize, the rapid rate of metabolic degradation of this class
of compounds could not be addressed without subse-
quent loss of cellular potency.
In summary, starting from a screening lead 1 with
modest biochemical and cell potency we identified a
novel series of potent and selective Itk inhibitors as
exemplified by 3 through systematic SAR optimiza-
tion. The problem of the metabolic instability of this
series could not be solved without concomitant loss
of cell potency. However, we demonstrated the poten-
cy of compound 3 in an in vivo mouse model. To our
knowledge, 3 is the first example of a potent and
selective small molecule Itk inhibitor to show efficacy
in a mouse model of lung inflammation. This analog
Based on its potent enzyme and cellular activity,
compound 3 was characterized for enzyme selectivity
in vitro. As shown in Table 7, analog 3 exhibited exqui-
site selectivity over other kinases. It was at least 200-fold
selective over Tec family kinases and over 55-fold selec-
tive against other kinases tested. In addition 3 showed
ATP competitive kinetics8.
Based on potency and selectivity, 3 was analyzed further
in cells. Compound 3 inhibited anti-CD3 antibody
induced IL-2 secretion both in human Jurkat T-cells
Table 8. Single dose pharmacokinetic parameters of compound 3 in
mice
(IC50 = 0.25 0.11 lM), PBMCs (IC50 = 0.39
and in murine EL4 cells (IC50 = 0.07 0.04 lM) and
splenocytes (IC50 = 0.38 0.18 lM). The murine EL4
0.16 lM)
iv route
Oral route
Dose
10 mg/kg
Dose
20 mg/kg
cell line appears to be the most sensitive to Itk inhibition.
Half-life (h)
Clearance
(mL/min/kg)
MRT (h)
1.3
Half-life (h)
Cmax (lM)
1.4
0.069
116
Table 7. Enzyme selectivity of compound 3
1
MRT (h)
Tmax (h)
F (%)
3.1
1
IC50 (lM)8 (selectivity ratio)a
Tmax (h)
Vss (L/kg)
0.05
7
Enzyme
6.4
Itk
Txk
0.019 (1)
11 (550)
Tec
Btk
17 (850)
4.1 (200)
7000
6000
5000
4000
3000
2000
1000
0
Vehicle
Bmax
Lck
Fyn
>50 (>2500)
2.4 (120)
1.1 (55)
Compound 3
Syk
ZAP-70
IR
>50 (>2500)
>50 (>2500)
1.1 (55)
*
*
EGFR
Cdk2
ERK-1
PKA
PKC
Akt1
IKKb
GSK-3b
>50 (>2500)
29 (1450)
>50 (>2500)
>50 (>2500)
24 (1200)
2ug
10ug
Anti-CD3 Antibody
>50 (>2500)
>50 (>2500)
36 (1800)
Figure 2. Inhibition of anti-CD3 antibody induced serum IL-2
production in mice in vivo by 50 mg/kg of compound 3. *p < 0.05
(Student’s t test).
a Selectivity ratio, IC50/IC50 (Itk).

3712
J. Das et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3706–3712
5. Mueller, C.; August, A. J. Immunol. 2003, 170, 5056.
6. Kanner, S. B.; Perez-Villar, J. J. Trends Immunol. 2003, 24,
249.
should serve as a useful tool in investigating the role
of Itk in T cell signaling.
7. Das, J.; Liu, C.; Moquin, R. V.; Lin, J.; Furch, J. A.;
Spergel, S. H.; McIntyre, K. W.; Shuster, D. J.; O’Day, K.
D.; Penhallow, B.; Hung, C.-Y.; Kanner, S. B.; Lin, T.-A.;
Dodd, J. H.; Barrish, J. C.; Wityak, J. Bioorg. Med. Chem.
Lett. 2006, 16, 2411.
8. Lin, T.-A.; McIntyre, K. W.; Das, J.; Liu, C.; O’Day,
K. D.; Penhallow, B.; Hung, C.-Y.; Whitney, G. S.;
Shuster, D. J.; Yang, X.; Townsend, R.; Postelnek, J.;
Spergel, S. H.; Lin, J.; Moquin, R. V.; Furch, J. A.;
Kamath, A. V.; Zhang, H.; Marathe, P. H.; Perez-Villar,
J. J.; Doweyko, A.; Killar, L.; Dodd, J. H.; Barrish, J.
C.; Wityak, J.; Kanner, S. B. Biochemistry 2004, 43,
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