Discovery of novel 2-(aminoheteroaryl)-thiazole-5-carboxamides as potent and orally active Src-family kinase p56Lck inhibitors

Article abstract of DOI:10.1016/j.bmcl.2004.09.093

A series of substituted 2-(aminoheteroaryl)-thiazole-5-carboxamide analogs have been synthesized as novel, potent inhibitors of the Src-family kinase p56^Lck. Among them, compound 2 displayed superior in vitro potency and excellent in vivo efficacy. A series of substituted 2-(aminoheteroaryl)- thiazole-5-carboxamide analogs have been synthesized as novel, potent inhibitors of the Src-family kinase p56^Lck. Among them, compound 2 displayed superior in vitro potency and excellent in vivo efficacy.

Full text of DOI:10.1016/j.bmcl.2004.09.093

Bioorganic & Medicinal Chemistry Letters 14 (2004) 6061–6066
Discovery of novel 2-(aminoheteroaryl)-thiazole-5-carboxamides
as potent and orally active Src-family kinase p56Lck inhibitors
Ping Chen,a,* Derek Norris,a Jagabandhu Das,a Steven H. Spergel,a John Wityak,a
Leslie Leith,a Rulin Zhao,a Bang-Chi Chen,a Sidney Pitt,b Suhong Pang,b Ding Ren Shen,b
Rosemary Zhang,b Henry F. De Fex,b Arthur M. Doweyko,c Kim W. McIntyre,b
David J. Shuster,b Kamelia Behnia,d Gary L. Schievenb and Joel C. Barrisha
aDepartment of Discovery Chemistry, Bristol-Myers Squibb Pharmaceutical Research Institute,
PO Box 4000, Princeton, NJ 08543-4000, USA
bDepartment of Immunology, Inflammation and Pulmonary Drug Discovery,
Bristol-Myers Squibb Pharmaceutical Research Institute, PO Box 4000, Princeton, NJ 08543-4000, USA
cDepartment of Macromolecular Structure, Bristol-Myers Squibb Pharmaceutical Research Institute,
PO Box 4000, Princeton, NJ 08543-4000, USA
dDepartment of Metabolism and Pharmacokinetics, Bristol-Myers Squibb Pharmaceutical Research Institute,
PO Box 4000, Princeton, NJ 08543-4000, USA
Received 1 September 2004; revised 22 September 2004; accepted 22 September 2004
Available online 22 October 2004
Abstract—A series of substituted 2-(aminoheteroaryl)-thiazole-5-carboxamide analogs have been synthesized as novel, potent inhib-
itors of the Src-family kinase p56Lck. Among them, compound 2 displayed superior in vitro potency and excellent in vivo efficacy.
Ó 2004 Elsevier Ltd. All rights reserved.
The tyrosine kinase p56Lck has been shown to play a piv-
otal role in T cell development1 and activation.2 T cells
that lack Lck or contain mutant Lck are unable to re-
spond to stimulation via the TCR.2a,3 Lck knockout
mice or those with over expression of an Lck dominant
negative show early arrest in thymocyte development.1
Inhibition of Lck may be useful for the treatment of
both chronic and acute T cell-mediated autoimmune
and inflammatory disorders such as rheumatoid arthri-
tis, multiple sclerosis, transplant rejection and delayed
hypersensitivity (DTH) reactions. Recent reviews have
described several classes of Lck inhibitors and high-
lighted their therapeutic potential.4
pound 2 as a highly potent (Ki = 130pM) and orally ac-
tive Lck inhibitor (Fig. 1). In addition, compound 2
displays excellent activity against T cell proliferation
(IC50 = 80nM) and in vivo efficacy in a rodent rheuma-
toid arthritis model (rat adjuvant arthritis).
Our initial effort in the modification of 1 to improve its
enzyme activity was focused on: (1) replacement of the
cyclopropyl group with alkyl, aryl, and heterocyclic
(including heteroaryl) groups of different sizes; and (2)
replacement of the amide linker with other linkers such
Me
We recently reported the discovery of 2-(carboxamido)-
thiazole-5-carboxamides, represented by 1, as potent
Lck inhibitors with modest cellular activity.5 This paper
describes our effort in the optimization of the in vitro
potency of 1 which has led to the identification of com-
O
N
N
N
N
Me
Me
H
N
H
N
N
H
Me
N
H
S
S
O
O
Cl
Cl
1
2
IC
IC
= 35 nM
50 (T cell prolif.)
IC
= 1.2 nM
50 (Lck)
50 (Lck)
= 884 nM
Ki
= 130 pM
(Lck)
IC
= 80 nM
Keywords: Lck inhibitor; pan-Src-family inhibitor.
*
50 (T cell prolif.)
Corresponding author. Tel.: +1 6092525809; fax: +1 6092526601;
e-mail: ping.chen@bms.com
Figure 1. Activity for 1 and 2.
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.09.093

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P. Chen et al. / Bioorg. Med. Chem. Lett. 14(2004) 6061–6066
as carbamate or sulfonamide. Unfortunately, this ap-
proach led to little or no success. In most cases, subtle
changes resulted in a >1000-fold drop in enzyme inhib-
itory activity.6 While a few analogs displayed enzyme
activity comparable to 1 (IC50 = 30–50nM), none of
them showed the desired level of cellular potency
(<0.5lM). Using a homology model of the p56Lck active
site coupled with the observed SAR, we rationalized that
while the carbonyl of the C2-amide of 1 may not be in-
volved in any active H-bonding interactions with the en-
zyme, its conformational rigidity directs the small
cyclopropyl group into an optimal hydrophobic binding
pocket. This optimal binding interaction derived from
the small and rigid cyclopropyl amide was most likely
disrupted when a larger alkyl or aryl group, a less con-
formational constrained group, or a sterically bulkier
linker (such as a carbamate or a sulfonamide) were used.
Accordingly, we turned our attention to a conforma-
tionally rigid Ôamide mimicÕ as a possible replacement
for the cyclopropyl amide of 1 and a heteroaryl amine
appeared to be a suitable starting point to serve this
purpose.
4,6-dichloro-2-methylpyrimidine was used in Scheme 1,
where R1 = Me and X = Cl) to afford the corresponding
analog 5a. Alternatively, the conversion of amine 4a to
bromide 4b followed by coupling with an aminohetero-
cycle (in this case, 2-methyl-4-chloropyrimidine was
used) in the presence of a base afforded 5b (R1 = Me
and X = H). If desired, 5a (Y = Cl) could be further
homologated to give 6 (R2 = amines). For illustration,
2-aminoethylpyrrolidine was used in this coupling reac-
tion as shown in Scheme 1 (step f).
Alternatively, deprotonation of 2-chlorothiazole 77
using n-butyl lithium followed by quenching with com-
mercially available arylisocyanates at ꢀ78°C gave rise
to 2-chloro-5-carboxamidothiazoles 8 in excellent yield
(>90%). Protection of the amide 8 with the p-meth-
oxybenzyl (PMB) bromide and reaction with an
appropriate aminoheteroaryl, such as 4-amino-2,6-
dimethylpyrimidine in the presence of a base, followed
by the removal of the PMB protecting group similarly
gave analog 5c (where R1 = Y = Me) in excellent overall
yield (>95%), as shown in Scheme 2. Since the C2-chlo-
rothiazole 8 is significantly less reactive than the C2-
bromothiazole 4b, protection of the amide in 8 prior
to the coupling with aminoheterocycles is necessary to
minimize the undesired cross coupling side reaction. In
general, this route is complementary, and sometimes
superior to the first one described in Scheme 1, especially
when a properly activated heterocycle cannot be ob-
tained readily.
The heteroaryl aminothiazole-5-carboxamides described
in this report were synthesized as depicted in Schemes 1
and 2. In Scheme 1, the acid 35 was coupled to an appro-
priate aniline via the acid chloride intermediate.
Removal of the Boc protecting group provided the
intermediate 4a. Amine 4a was then coupled to an
appropriately activated heteroaryl core (for illustration,
Since our previous SAR5 in this series of inhibitors dem-
onstrated that 2-chloro-6-methyl aniline was an optimal
pharmacophore for excellent Lck binding affinity, this
particular aniline was used throughout our SAR studies.
For simplicity, data on only a limited set of analogs are
presented in Tables 1–3 to describe the SAR.
N
a-c
e (or e')
N
H
N
X
BocHN
S
OH
S
R
O
O
3
4a (X= NH )
2
d
f
4b (X = Br)
R1
R1
N
N
N
N
N
N
H
N
H
N
Table 1. 2-Arylaminothiazole-5-carboxamide SAR (compounds 1,
9–14)
Y
N
R2
N
S
S
H
H
R
R
O
O
N
Me
H
Ar
5a (R = H or Me, Y = Cl)
N
N
N
1
1
6 (R = H, R =
)
N
H
1
2
S
5b (R = H or Me, Y= H)
H
O
Cl
Scheme 1. Synthesis of C2-heteroaromatic aminothiazole derivatives:
(a) (COCl)2, cat. DMF, THF; (b) ArNH2, i-Pr2NEt, THF; (c) TFA,
CH2Cl2; (d) NaNO2, CuBr; (e) 4,6-dichloro-2-methylpyrimidine, NaH,
THF; (e0) 4-amino-6-chloro-2-methylpyrimidine, NaH (or NaHMDS);
(f) 1-(2-hydroxyethyl)piperazine, neat, 80°C.
b
Compds
Ar
na
Lck (enzyme)b
IC50,nMa
T Cell Prolif.
IC50,nMa
1
9
35
884
210
0.6
N
N
10
11
12
13
14
1.2
6.7
10
140
870
270
350
–
a
b-d
N
H
N
Cl
Cl
S
S
N
N
R
O
8
7
R1
N
N
N
H
N
N
N
Y
N
H
4
S
R
O
5c (R1 = Me, Y = Me)
374
Scheme 2. Synthesis of C2-heteroaromatic aminothiazole derivatives:
(a) n-BuLi, ArN@C@O, ꢀ78°C; (b) p-MeO-C6H4CH2Cl, NaH, THF;
(c) 4-amino-2-methylpyrimidine, NaH, THF; (d) TFA, anisole, TfOH.
a Values throughout are mean of three experiments, standard deviation
10%.
b For detailed assay conditions, see Ref. 17.

P. Chen et al. / Bioorg. Med. Chem. Lett. 14(2004) 6061–6066
6063
Table 2. 2-Arylaminothiazole-5-carboxamide SAR (compounds 15–
21)
assay. Compound 9 showed a 4-fold improvement of
IC50 against T cell proliferation over 1. Exchange of ani-
line in 9 with 2-aminopyridine essentially retained in vi-
tro potency (10). However, similar substitution using the
regio-isomeric 3- and 4-aminopyridines led to the loss of
intrinsic activities (11 and 12). To investigate if basicity
played a significant role in the enhancement of intrinsic
activity, the 3-aminopyridazine analog 13 was prepared.
The activity of 13 is between that of 10 and 11, a result
consistent with the observation that the 2-pyridyl sub-
stituent is more potent than the 3-pyridyl substituent
and that basicity does not play a significant role in the
binding affinity.8 Finally, replacing the aniline of 9 with
a cyclohexyl amine (14) is detrimental to the activity.
The more than 400-fold loss of enzyme activity clearly
indicates a steric restriction in this region, consistent
with the observation that the C2-cyclobutyl amide, a
very close analog of 1, is less active against Lck.6
N
Me
H
Ar
N
N
H
S
O
Cl
Compds
Ar
Lck (enzyme)b
IC50,nMa
T cell prolif.b
IC50,nMa
Me
15
<1
106
N
Me
Me
N
16
17
1.1
0.5
156
85
N
N
N
18
19
7.3
4
365
140
Me
Me
Our next effort focused on developing the SAR with
respect to the substitution pattern on the 2-amino-
heteroaromatic ring system of 10. While 2- or 3-methyl-
aminopyridyl analogs (15 and 16) were equipotent to
10, the 4- and 5-methyl substitution (17 and 18) resulted
in reduction of activity (5- to 7-fold). In addition, appro-
priate disubstitution was tolerated (19 and 2 vs 10).
Switching pyridine to pyrimidine led to a dramatic re-
sult: while the 4,6-dimethyl-2-aminopyrimidine analog
2 displayed excellent intrinsic potency, its regio-isomer
20 was substantially less potent, indicating the geometric
preference of polar heteroatoms in the small hydropho-
bic pocket (vide infra). Interestingly, the corresponding
di-substituted aniline was tolerated with respect to en-
zyme binding but a considerable drop in cellular activity
was observed (21 vs 9) (Table 2).
N
Me
Me
Me
2
1 (Ki = 130pM)
80
—
N
Me
20
21
50
2
N
Me
Me
N
Me
883
a Values throughout are mean of three experiments, standard deviation
10%.
b For detailed assay conditions, see Ref. 17.
Replacement of the 2-cyclopropylamido functionality of
16 with a simple aniline resulted in a significant gain
(>30-fold) of enzyme potency, as demonstrated by com-
pound 9 in Table 1. This dramatic enhancement of en-
zyme activity was also reflected in the cellular based
Further optimization was focused on the enhancement
of the cellular activity of 2. This was achieved via
the introduction of solubility-enhancing side chains9
containing polar and weakly basic groups onto the
Table 3. 2-Arylaminothiazole-5-carboxamide SAR (compounds 2, 22–25)
R1
2
1X
6
N3
N
Me
H
N
R2
4
N
S
5
H
O
Cl
Compds
X
R1
R2
Lck (enzyme)b
IC50,nMa
T cell prolif.b
IC50,nMa
2
N
N
Me
H
Me
1 (Ki = 130pM)
80
N
22
0.4 (Ki = 60 pM)
19
N
H
O
O
N
N
23
24
25
N
H
H
HN
O
4
17
3
N
H
N
<1
<1
CH
H
H
6
N
N
26
CsA
CH
<1
NA
11
50
N
H
a Values throughout are mean of three experiments, standard deviation 10%.
b For detailed assay conditions, see Ref. 17.

6064
P. Chen et al. / Bioorg. Med. Chem. Lett. 14(2004) 6061–6066
2-amino-heteroaromatic rings of 19 and/or 2. Using a
parallel synthetic approach outlined in Scheme1 (step
f), analogs with sub-nanomolar activities against Lck
and single digit nanomolar inhibitory activities against
T cell proliferation were identified (Table 3). With the
exception of 23, incorporation of side chains bearing
polar groups at the C6 position of 2 in general led to
compounds with better Lck affinity relative to the
corresponding parent and, more significantly, a substan-
tial boost (4- to 25-fold) in cellular potency (22 and 24).
A similar result was also observed in the 2-aminopyr-
idine series. Analogs containing (S)-methyl-piperazine
(25) and morpholino-2-ethylamine (26) side chains at
the C2 position (numbering according to the structure
shown in Table 3) also displayed superior cell potency
in comparison to their parent compound 10. The in-
creased in vitro potency of 22 and 24–26 as compared
to 2 and 10 can be attributed, at least in part, to the en-
hanced aqueous solubility since our modeling studies
indicate that these side chains are pointed toward the
solvent front.10
Figure 2. Proposed binding interactions of 2 with Lck kinase domain.
Van der Waals surfaces are shown (inset) illustrating the fit of the
pendant pyrimidine ring in the hydrophobic cleft of the binding site.
Me
Me
In contrast to the in vitro assays, the physiological level
of ATP in intracellular settings is thought significantly
higher and may approach 500lM or above. Such a
change in ATP concentration can potentially generate
a drastic reduction in binding affinity for any ATP-com-
petitive inhibitors. To gain insight into the change of
magnitude in binding affinity against Lck, compound 2
was further tested at a higher ATP concentration. Not
surprisingly, in the presence of 1 mM ATP, compound
2 displayed a significant loss in binding affinity toward
the enzyme, with an IC50 of 5.5 0.1lM and a Ki value
of 247 94nM (n = 2).
N
N
N
N
N
N
Me
Me Me
N
H
N
Me
N
Me
Me
N
H
S
S
O
O
Cl
Cl
27
IC50 (Lck) = 277 nM
28
IC50 (Lck) = 2,345 nM
Figure 3. Enzyme activity of 27 and 28.
The proposed binding mode for 2 is consistent with that
previously published for 1 in the ATP binding pocket.5
This is illustrated in Figure 2 using a model of the
p56Lck ATP binding site based on the published struc-
ture.11 The H-bonding donor/acceptor system at
Met319/aminothiazole appears to play a central role in
orientation of the ligand, with the deep hydrophobic
pocket occupied by the angular 2-chloro-6-methylani-
line ring. In addition, there is a likelihood that some
interaction occurs between the Thr316 hydroxyl acting
as a H-bond acceptor and the carboxamide NH as these
groups are modeled to about 2.8 Angstroms of each
other. N-substitution of 2 with a methyl functionality
led to dramatic loss of enzyme binding affinity (27 and
28, Fig. 3) relative to its parent. In addition, the pendant
2,6-dimethyl-2-amino pyrimidine ring occupies a rela-
tively narrow hydrophobic cleft created by Leu251,
Tyr318 and Gly322 (Fig. 2, inset). This feature may ex-
plain the added affinity of the pendant ring series as such
burial of hydrophobic surfaces for both the ligand and
protein is energetically favored in aqueous solution.
The improvement of physicochemical properties derived
from the incorporation of polar side chains and/or the
enhancement of cell permeability are unlikely to be the
sole contributing factors for the superior cellular po-
tency observed for compounds 22–26. To ensure that
this extraordinary level of cellular potency was not asso-
ciated with a cytotoxic effect, analogs within this series,
including 22, were routinely assayed for cytotoxicity in
an Alamar Blue Assay. In general, compounds such as
22 and closely relate related analogs showed no sign of
cytotoxicity to Jurkat (T) and Ramos (B) cells. To put
the superior T cell potency of these analogs into perspec-
tive, cyclosporine A (CsA), a potent anti-inflammatory
drug on the market, was used as a comparator
(IC50(T
prolif.) = 50nM, Table 3). Cyclosporin A
cell
suppresses the T cell proliferation by inhibiting the
phosphatase activity of calcineurin on NFAT, thereby
preventing its translocation to the nucleus and suppress-
ing IL-2 mRNA transcription. On the other hand, inhi-
bition of Lck by compounds such as 22 blocks the signal
transduction cascade considerably upstream of calcineu-
rin, presumably suppressing the activities of signaling
molecules from multiple pathways. In addition, com-
pounds in this series potently inhibit several other kin-
ases (vide infra), thus profound suppression of T cell
proliferation may also be due to an additive or synergis-
tic effect of inhibiting multiple kinase activities and/or
signaling pathways.
Selectivity over other kinases is an important factor for
consideration when developing a chronically adminis-
tered therapeutic agent. As a representative analog in
this series, compound 2 was screened against in-house
kinase panels and the relevant selectivity data is shown
in Table 4. In general, excellent selectivity is observed
for 2 against the receptor tyrosine and serine/threonine
kinases whereas no selectivity against the Src-family
kinases (Fyn, Src, Hck, Blk, and Fgr) or JAK3

P. Chen et al. / Bioorg. Med. Chem. Lett. 14(2004) 6061–6066
Table 4. Kinase selectivity profile for compound 2
6065
Kinase
Enzyme
IC50, lMa
Kinase
Enzyme
IC50, lMa
Lck
Fyn
Src
0.001
0.001
JAK3
0.006
>25
>25
CDK2
CDK4
FGFR1
KDR
<0.001
0.002
0.001
Hck
Blk
Fgr
>10
>10
<0.001
HER2
5.2
a Values throughout are mean of three experiments, standard deviation
10%.
was observed, presumably due to the high sequence
homology within their ATP binding sites.
Figure 4. Rat adjuvant arthritis studies using 2.
Compound 2 was selected as a prototype from this series
of Lck inhibitors for further evaluation. The in vivo
pharmacokinetic profile of 2 was determined in rats.
Upon single doses of 10mpk iv and po, 2 showed a Tmax
at 1h with peak plasma exposure of 2lM (Cmax). Both
plasma half life (T1/2) and the mean residence time
(MRT) of 2 are approximately 4h. The volume of distri-
bution (Vss) is 12L/kg, indicating that significant parti-
tioning of 2 into the tissues can be expected. The rate
of plasma clearance (Cl) is moderate at 29mL/min/kg,
giving the oral bioavailability of 2 as 65% in rats. In
addition, compound 2 displayed a 93.6% plasma
protein binding in mouse.
(Ugo Basile, Italy). Fourteen days after adjuvant immu-
nization, rats with clinical signs of disease were random-
ized into treatment groups (n = 8/group). Compound 2
was administered orally twice daily at 1.0 or 3.0mg/kg
and compared to the once daily methotrexate (MTX)
treated group at 0.1mg/kg. As shown in Figure 2, com-
pound 2 showed a dose-dependent inhibition of paw
swelling in this animal model of rheumatoid arthritis.
At 3.0mg/kg, treatment with 2 slowed disease progres-
sion significantly (p > 0.05; StudentÕs test) when com-
pared to the vehicle treatment group within the first
24h after administration was initiated. The 1.0mg/kg
dose level was less efficacious, but still demonstrated
significant inhibition of paw swelling after one week of
treatment. In contrast to the vehicle control rats which
showed loss of body weight due to disease progression,
rats that received 2 appeared healthy and actually
gained weight over the course of treatment.
Studies have shown that T cells plays an important role
in the pathogenesis of rheumatoid arthritis (RA).12 Re-
cent clinical investigation in RA patients using a modu-
lator of T cell co-stimulation (CTLA4-Ig) demonstrated
significant improvement of signs and symptoms of rheu-
matoid arthritis as well as the health-related quality
of life,13 suggesting that a strategy of targeting T cell
responses could lead to therapeutic benefits for
RA patients. pan-Src-family kinase inhibitors could
potentially provide benefit in the rat adjuvant arthritis
model by action on Src-family kinases in multiple cell
types. The inhibition of Lck and Fyn would be expected
to block T cell activation. In addition, macrophages are
believed to play an important role in the rat adjuvant
arthritis model. Many Fc-c receptor-induced functional
responses and signaling events have been reported to be
diminished or delayed in macrophages from mice defi-
cient in the Src-family kinases Hck, Fgr, and Lyn,
including immunoglobulin (Ig)G-coated erythrocyte
phagocytosis and the respiratory burst.14 The same
macrophages have also been reported to have impaired
integrin-mediated signal transduction, altered cytoskele-
tal structure and reduced motility.15 Taken together,
pan-Src-family kinase inhibition might therefore con-
tribute to efficacy in an in vivo model by inhibiting
responses of T cells and macrophages beyond the inhibi-
tion of T cell responses that would be expected from an
Lck-specific inhibitor. Based on its encouraging in vitro
potency and PK profile in rats, we evaluated 2 in a rat
adjuvant arthritis model (Fig. 4). In this model, male Le-
wis rats were immunized with complete FreundÕs adju-
vant at the base of the tail. Hind paw volumes were
measured by volume displacement plethysmometry
In summary, we have identified a series of novel 2-(ami-
noheteroaryl)thiazole-5-carboxamide analogs, repre-
sented by compound 2, as potent Lck inhibitors with
superior T cell activity over CsA. Compound 2 dis-
played a pharmacokinetic profile suitable for oral
administration and demonstrated excellent in vivo effi-
cacy comparable to the known anti-inflammatory drug
methotrexate in a rodent inflammation model. Addi-
tional characterization of 2 and related analogs from
this series will be reported in due course.16
Acknowledgements
We would like to thank the Discovery Analytical
Sciences group for analytical support.
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