Discovery and initial SAR of 2-amino-5-carboxamidothiazoles as inhibitors of the Src-family kinase p56Lck

Article abstract of DOI:10.1016/j.bmcl.2003.08.054

A novel series of 2-amino-5-carboxamidothiazoles were identified as inhibitors of Lck. Structure-activity studies demonstrate the structural requirements for potent Lck activity. Cyclopropylamide 11d is a potent Lck inhibitor having sub-micromolar activity in a PBL proliferation assay.

Full text of DOI:10.1016/j.bmcl.2003.08.054

Bioorganic & Medicinal Chemistry Letters 13 (2003) 4007–4010
Discovery and Initial SAR of 2-Amino-5-carboxamidothiazoles as
Inhibitors of the Src-family Kinase p56Lck
John Wityak,a,* Jagabandhu Das,a,* Robert V. Moquin,a Zhongqi Shen,a James Lin,a
Ping Chen,a Arthur M. Doweyko,b Sidney Pitt,c Suhong Pang,c Ding Ren Shen,c
Qiong Fang,c Henry F. de Fex,c Gary L. Schieven,c Steven B. Kannerc
and Joel C. Barrisha
aDepartment of Discovery Chemistry, Bristol-Myers Squibb Pharmaceutical Research Institute, PO Box 4000,
Princeton, NJ 08543-4000, USA
bDepartment of Macromolecular Structure, Bristol-Myers Squibb Pharmaceutical Research Institute, PO Box 4000,
Princeton, NJ 08543-4000, USA
cDepartment of Immunology, Inflammation, and Pulmonary Drug Discovery,
Bristol-Myers Squibb Pharmaceutical Research Institute, PO Box 4000, Princeton, NJ 08543-4000, USA
Received 29 May 2003; revised 25 August 2003; accepted 27 August 2003
Abstract—Anovel series of 2-amino-5-carboxamidothiazoles were identified as inhibitors of Lck. Structure–activity studies
demonstrate the structural requirements for potent Lck activity. Cyclopropylamide 11d is a potent Lck inhibitor having sub-
micromolar activity in a PBL proliferation assay.
# 2003 Elsevier Ltd. All rights reserved.
The tyrosine kinase p56Lck is a member of the Src
family of kinases expressed predominantly in T cells and
NK cells. It plays essential roles in T cell development,1
activation,2 and T cell antigen receptor (TCR) signaling.
Lck phosphorylates the immunoreceptor tyrosine acti-
vation motif (ITAM) sequences of the invariant chains
(including the z chain) within the TCR. Once phos-
phorylated, the z chain binds ZAP-70 via its SH2
domains.2c Phosphorylation of ZAP-70 by Lck, and the
combined action of ZAP-70, Lck, and the related Src
family kinase Fyn drives downstream signaling events
and leads ultimately to calcium flux, and T cell activa-
tion and proliferation. Genetics experiments using
Lckꢀ/ꢀ mice have validated Lck as an immunosup-
pressive molecular target.1a These mice display a SCID-
like syndrome and are unable to reject skin grafts
despite the presence of peripheral T cells.3 In addition,
over expression of a dominant negative form of Lck
leads to early arrest of thymocyte development prior to
expression of CD4, CD8, and the TCR.1b Aselective
Lck inhibitor should inhibit T cell activation and have
potential utility in the treatment of acute and chronic T
cell mediated autoimmune and inflammatory disorders
such as solid organ transplant graft rejection, multiple
sclerosis, rheumatoid arthritis, psoriasis, and delayed
type hypersensitivity reactions. Recent reviews describe
inhibitors and highlight the therapeutic potential of Src
family and Lck inhibition.4
We recently reported the discovery of a novel series of
imidazoquinoxalines as Lck kinase domain inhibitors.5
In addition, high throughput screening of our com-
pound collection led to the identification of 2-amino-
thiazole 1 as a weakly active Lck inhibitor (Fig. 1). In
this report we describe our initial efforts toward opti-
mizing the Lck activity of this lead.
*Corresponding author. Tel.:+1-858-332-4783; fax:+1-858-812-1648;
e-mail: jwityak@gnf.org
Figure 1. Activity for 2-aminothiazole 1.
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.08.054

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J. Wityak et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4007–4010
Screening was initiated using mouse Lck (mLck), and
subsequently continued using human Lck (hLck). Since
Lck from these two species has a sequence homology of
84.4%,6 it was expected that this change would have
little bearing on the resulting SAR. Indeed, the bridging
assay data revealed a good correlation between the
mouse and human assay results.7
activity. Our SAR studies continued with a round of
parallel synthesis in which the 2,4,6-trimethylaniline
moiety of 7a was replaced with a diverse set of amines.
As summarized in Table 1, this survey revealed a nar-
row SAR that favored certain secondary arylamides
(7a–e) over analogues having a secondary or tertiary
alkylamide (7f–j). Furthermore, the importance of small
substituents on the 2- and 6-positions of the arylamide
is clearly evident as 7a–e have superior Lck activity
versus those analogues lacking one or both substituents
(7k–o) or having sterically demanding groups at these
positions (7p–s). In this respect, the SAR parallels that
observed with other series of inhibitors.5a,9 These sub-
stituents likely serve to orient the aniline ring in a
conformation orthogonal to the thiazole (vide infra).
The thiazoles studied were synthesized as shown in
Schemes 1 and 2. In Scheme 1, the commercially avail-
able 4-methyl- (2), 4-ethyl- (3), 4-phenyl- (4), and 4-tri-
fluoromethyl- (5) ethyl 2-aminothiazole-5-carboxylates
were converted to their corresponding Boc-derivatives
followed by ester saponification. Activation of the
resulting thiazole-5-carboxylates 6 through formation of
the acyl chloride followed by reaction with various
amines then afforded the desired thiazole derivatives
7–10.
We next focussed on developing the SAR with respect
to the thiazole 2-amino group (Table 2). Alarge number
of amides, carbamates, and ureas were prepared using a
parallel synthesis strategy. This effort resulted in Lck
inhibitors having greatly improved activity. Areduction
in steric bulk appeared to be beneficial (7a vs 7t). Cer-
tain aryl and heteroaryl amides also showed good
activity (7u, 7v), and alkylureas 7w and 7x were partic-
ularly active. In a direct comparison of 2,4,6-trimethyl-
anilines (7u, 7v, 7x) with their 2-chloro-6-methyl aniline
counterparts (7y, 7z, 7aa), the former were 2–20-fold
more active than the latter against Lck.
The 4-des(alkyl)thiazoles 11 were prepared according to
Scheme 2. Starting from ethyl 2-aminothiazole-5-car-
boxylate (12),8 protection of the amine as the Boc-deri-
vative followed by saponification afforded acid 13. This
material was activated through formation of the acyl
chloride, coupled with an appropriate aniline derivative,
and deprotected to give amine 14. Amine 14 was then
acylated to afford the corresponding urea, carbamate,
or amide derivatives 11.
Simply protecting the 2-amino group of 1 as the Boc-
derivative 7a resulted in a 2-fold improvement in Lck
Compounds 7bb and 7cc (Fig. 2) serve to demonstrate
the importance of the amide NH moieties to Lck activ-
ity. Each compound was inactive when tested at a con-
centration of 50 mM. As a comparison, 7a had an IC50
of 1.5–3 mM. From analysis of the docking of 11e into
Table 1. 2-t-Butyloxycarbonylamino-4-methylthiazole 5-carboxamide
SAR
Compd
R2
R3
mLck
hLck
IC50, mMa
IC50, mMa
Scheme 1. Synthesis of 4-substituted thiazole derivatives: (a) Boc2O,
DMAP, THF; (b) NaOH, MeOH (aq); (c) (COCl)2, DMF, THF; (d)
iPr2Net, THF; (e) TFA, CH2Cl2; (f) R0OCOCl, DMAP, THF or
PhOCOCl, THF followed by NHR01R02 or RCOCl, DMAP, THF.
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
7s
H
H
H
H
H
H
H
H
—
—
H
H
H
H
H
H
H
H
H
2,4,6-Me3 aniline
2-Cl, 6-Me aniline
2,6-Me2 aniline
4-Br, 2,6-Me2 aniline
2,6-Cl2 aniline
2,2-Me2 propylamine
1,2-Me2 propylamine
2,5-Me2 3-pyrroline
4-Formylpiperazine
Cyclohexylmethylamine
Aniline
2,4-Cl2 aniline
2-NO2 aniline
3-OMe, 5-CF3 aniline
2-Me aniline
2-Me, 6-iPr aniline
2,6-iPr2 aniline
2-OMe, 6-Me aniline
2-tBu, 6-Me aniline
3
9.6
7.5
2.4
1.5
28
4.4
1.4
12.5
—
>50
—
—
—
—
—
—
—
—
—
—
—
—
>50
>50
>50
>50
>50
>50
>50
>50
>50
45.7
40.3
>50
24.3
>50
Scheme 2. Synthesis of 4-hydridothiazole derivatives: (a) Boc2O,
DMAP, THF; (b) NaOH, MeOH (aq); (c) (COCl)2, DMF, THF; (d)
—
R2R3NH, iPr2NEt, THF; (e) TFA, CH2Cl2; (f) R1OCOCl, DMAP,
0
aValues throughout are mean of three experiments, standard deviation
ꢁ10%.
THF or PhOCOCl, THF followed by NHR1R1 or R1COCl, DMAP,
THF.

J. Wityak et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4007–4010
Table 2. 4-Methylthiazole 2-amino substituent SAR
4009
Table 3 were inactive when tested at a concentration of
30 mM. In an apparent SAR paradox, while pursuing
the 4-des(alkyl) series further it was found that when
Boc was replaced by certain amides (11b,c), these com-
pounds had roughly similar activities to their 4-methyl
counterparts 7y and 7z. Furthermore, the combination
of a cyclopropylamide with the 4-des(alkyl) core as in
11d and 11e resulted in the discovery of the most highly
active Lck inhibitors from this series. In a direct com-
parison of 11d with the corresponding 4-methylthiazole
7dd, 11d was found to have a 40-fold potency advan-
tage. In a PBL proliferation assay using costimulation
with anti-CD3 and anti-CD28, 11d had an IC50 of 0.88
mM, while 11e had an IC50 of 1.8 mM.
Compd
R1
R2
R3
mLck
hLck
IC50, mM IC50, mM
7t
7u
7v
7w
7x
7y
7z
MeO–
2-Furyl–
Phenyl–
MeNH–
n-BuNH–
2-Furyl–
Phenyl–
n-BuNH–
H
H
H
H
H
H
H
H
(2,4,6-Me3)Ph
(2,4,6-Me3)Ph
(2,4,6-Me3 Ph
(2,4,6-Me3)Ph
(2,4,6-Me3)Ph
(2-Cl, 6-Me)Ph
(2-Cl, 6-Me)Ph
(2-Cl, 6-Me)Ph
0.72
—
0.32
0.17
0.070
—
0.7
0.36
0.80
0.24
0.030
0.71
4.5
—
—
7aa
0.62
Table 4 shows representative selectivity data for 11d
from a panel of potential kinase targets. Excellent
selectivity was observed for the receptor tyrosine and
serine/threonine kinases from this panel. However, sig-
nificant selectivity was not observed for the other seven
Src family members (Src, Fyn, and Hck data shown)
nor for Jak3.
In summary, we identified a novel thiazole lead as a
weak Lck inhibitor through high-throughput screening
of our in-house compound collection. Through parallel
synthesis and SAR studies, the Lck activity of this lead
Figure 2. N-Methyl analogues 7bb and 7cc.
the ATP binding site of the published X-ray structure,10
we surmise that these amide NH groups may be
engaged in significant hydrogen bonding interactions
(Fig. 3); the aniline NH appears to be favorably posi-
tioned for a hydrogen bonding interaction with the
Thr316 side-chain hydroxyl, while the 2-aminothiazole
moiety appears in close contact for hydrogen bonding
with the protein backbone carbonyl and NH of Met319.
This binding mode is also consistent with the observed
preference for 2,6-disubstitution of the aniline. Such
substitution ensures that the aniline adopts a con-
formation permitting it to occupy an angular hydro-
phobic pocket common to tyrosine kinases.
Table 3. Thiazole 4-position SAR
Compd
R1
R2
R3
R4
hLck
IC50 (mM)
7a
8
9
10
11a
11b
11c
11d
11e
7dd
(CH3)3CO–
(CH3)3CO–
(CH3)3CO–
(CH3)3CO–
(CH3)3CO–
2-Furyl–
Phenyl–
H
H
H
H
H
H
H
H
H
H
(2,4,6-Me3)Ph
(2,4,6-Me3)Ph
(2,4,6-Me3)Ph
(2,4,6-Me3)Ph
(2,4,6-Me3)Ph
(2-Cl, 6-Me)Ph
(2-Cl, 6-Me)Ph
(2-Cl, 6-Me)Ph
(2,4,6-Me3)Ph
(2-Cl, 6-Me)Ph
Me
Et
Ph
CF3
H
H
H
H
H
Me
1.5
>30
>30
>30
>30
3.1
0.89
0.035
0.018
1.4
Turning our attention to the 4-position of the thiazole,
it was found in a series of compounds in which the 2-
amino substituent was Boc that groups larger than
methyl were not well tolerated. Analogues 8–11a in
c-Pr–
c-Pr–
c-Pr–
Table 4. Kinase activity of 11d
Kinase IC50 (mM)
Fold
selectivity
Kinase IC50 (mM)
Fold
selectivity
Lck
Fyn
Src
Hck
Jak3
FAK
0.035
0.071
0.010
0.31
0.044
>25
—
—
—
10
—
>7000
Cdk2
p38
FGF
KDR
HER1
HER2
>25
25
>25
>25
10
>700
700
>700
>700
300
Figure 3. Proposed binding interactions of 11e with Lck kinase
domain.
>25
>700

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J. Wityak et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4007–4010
was optimized and a set of structural features associated
with potent activity established. We identified 11d as a
potent Lck inhibitor having sub-micromolar activity in
a PBL proliferation assay. When tested against a panel
of potential kinase targets, 11d was determined to be a
potent pan-Src family/Jak3 kinase inhibitor. However,
excellent selectivity was observed against kinases from
several other kinase families. Further development of
the SAR, and optimization of the cell and in vivo
activity of this series will be reported in due course.
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´
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Acknowledgements
We would like to thank the Discovery Analytical Sci-
ences group for analytical support.
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