Discovery of 2-amino-heteroaryl-benzothiazole-6-anilides as potent p56lck inhibitors

Article abstract of DOI:10.1016/S0960-894X(03)00511-0

A series of structurally novel benzothiazole based small molecule inhibitors of p56^lck was prepared to elucidate their structure-activity relationships (SAR), selectivity and cell activity in the T-cell proliferation assay. BMS-350751 (2) and BMS-358233 (3) are identified as potent Lck inhibitors with excellent cellular activities against T-cell proliferation.

Full text of DOI:10.1016/S0960-894X(03)00511-0

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2587–2590
Discovery of 2-Amino-heteroaryl-benzothiazole-6-anilides as
Potent p56lck Inhibitors
Jagabandhu Das,* Robert V. Moquin, James Lin, Chunjian Liu, Arthur M. Doweyko,
Henry F. DeFex, Qiong Fang, Suhong Pang, Sidney Pitt, Ding Ren Shen,
Gary L. Schieven, Joel C. Barrish and John Wityak
Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543-4000, USA
Received 24 March 2003; accepted 17 April 2003
Abstract—A series of structurally novel benzothiazole based small molecule inhibitors of p56lck was prepared to elucidate their
structure–activity relationships (SAR), selectivity and cell activity in the T-cell proliferation assay. BMS-350751 (2) and BMS-
358233 (3) are identified as potent Lck inhibitors with excellent cellular activities against T-cell proliferation.
# 2003 Elsevier Ltd. All rights reserved.
p56lck (Lck), a member of the Src family of non-recep-
tor protein tyrosine kinases is expressed primarily in
T-lymphocytes and natural killer cells. In T cells, Lck is
absolutely required for T-cell antigen receptor (TCR)
signaling, culminating in IL-2 gene expression and
effector functions.1 T-cells lacking Lck are shown to be
severely impaired in TCR tyrosine phosphorylation and
are unable to undergo activation via the TCR.2 Selective
inhibitors of Lck may have potential therapeutic utility
in the treatment of T cell mediated disorders such as
autoimmune and inflammatory diseases and in the
prevention of solid organ transplant rejection.3
Accordingly, 2-aminobenzothiazole-6-anilide4 4 was
treated with 2 equiv each of copper(II) bromide and
tert-butyl nitrite in acetonitrile at 0 ꢀC to room tem-
perature to form the corresponding 2-bromo-benzo-
thiazole-6-anilides 5. Compound
5 served as an
advanced intermediate for the synthesis of the 2-amino-
benzothiazole analogues. Reaction of 5 with a primary
amine (R1NH2) at an elevated temperature (105–110 ꢀC,
In a previous communication4 we described structure–
activity relationship (SAR) studies leading to a novel
benzothiazole template, that culminated in the dis-
covery of BMS-243117 (1) as a potent, and selective Lck
inhibitor. We outline here cognate studies with the goal
of discovering suitable replacements for the urea moiety
of 1, leading to 2-aminopyridyl analogue 2 (BMS-
350751) and 2-aminopyrimidinyl analogue 3 (BMS-
358233) as highly potent Lck inhibitors in vitro with
excellent activity in a T-cell proliferation assay (Fig. 1).
The synthesis, and SAR studies leading to these analo-
gues are described.
Figure 1. Lck inhibitory activity of benzothiazoles 1, 2, and 3.
The synthesis of these compounds follows a general
synthetic route that is illustrated in Scheme 1.
*Corresponding author. Tel.: +1-609-252-5068; fax: +1-609-252-
6804; e-mail: jagabandhu.das@bms.com
Scheme 1. (a) CuBr2, t-BuNO2, CH3CN, 78–95%; (b) R1NH2,
105–110 ꢀC or R1NH2, THF, NaH, 60 ꢀC, 30–75%.
0960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00511-0

2588
J. Das et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2587–2590
neat) afforded the secondary amine 6. Alternatively, 5
can be treated with the amine (R1NH2, 4ꢀequiv) in pre-
sence of sodium hydride (8 equiv) at 60 C for several
hours to form 6. The products were purified either by
trituration or automated preparative HPLC on a
and ready availability of 2-aminopyridines and 4-ami-
nopyrimidines, further optimization was carried out on
both of these series. Table 2 outlines the effect of sub-
stitution in the pyridine ring. In the 2,4,6-trimethylani-
line series, a methyl substitution at the C4, C5, or C6
position was well tolerated while such substitution at C3
was quite detrimental for potency. The 3,5-dimethyl
analogue 6z was an approximately 8-fold more potent
inhibitor than the parent compound 6h. A somewhat
different SAR trend was observed with the 2-chloro-6-
methylaniline analogues. Unlike in the 2,4,6-trimethy-
laniline series, the 6-Me analogue 6ad was significantly
more potent than the 4-Me analogue 6ab and the 5-Me
analogue 6ac. Compounds 6ad, 6ae, and 6af were iden-
tified as some of the more potent Lck inhibitors. A fur-
ther increase in Lck inhibition was observed by
introduction of a polar functionality at either C4 or C6-
position of the pyridine ring. Compounds 2, and 6ah–
6ao were identified as highly potent Lck inhibitors.
However several of these analogues (6ah–6ak) were
observed to be cytotoxic towards T-cells in an Alamar
Blue based assay.5 Compound 2 was identified as the
most potent and non-cytotoxic Lck inhibitor (IC50=500
pM) in this series.
reversed phase column.
A parallel solution-phase
approach was used to prepare a wide variety of analo-
gues using these conditions.
Compound 2 (BMS-350751) and its analogues were
tested for their ability to inhibit the phosphorylation of
an exogenous substrate5 by human Lck enzyme (IC50,
Tables 1–3).
Table 1 outlines the SAR observed with the C2 amine
modification. In our prior communication4 we demon-
strated the importance of the 2,6-substitution in the
aniline ring. Therefore, the present SAR studies were
carried out using the 2,4,6-trimethylaniline and
2-chloro-6-methylaniline series. In the 2,4,6-trimethyl-
aniline series introduction of an aniline moiety (6b)
resulted in a 9-fold increase in Lck inhibitory potency
compared to the 2-aminoanalogue 6a. In contrast,
cyclopropylmethyl (6c) or 5-membered heterocyclic
substituents (6d–g and 6m) were not tolerated. A more
significant increase in Lck inhibitory activity was
observed with the 2-pyridyl analogue 6h while the iso-
meric 3-pyridyl and 4-pyridyl derivatives (6i and 6j) were
less potent. More potent Lck inhibitors were however
identified in the 2-chloro-6-methylaniline series (6r–u).
Table 3 summarizes similar studies in the 4-pyrimidine
series. Substitution in the pyrimidine ring has led to
some very potent Lck inhibitors in this series. A wide
variety of substitution at C2, C6 or at both positions
was well tolerated. A significant boost in activity was
observed with a polar hydroxyethyl amine substituent
either at C2 (6at), or C6 (6ba). Similarly, an ethyl sub-
stituent (6av), and an amino substituent (6ay) or a
Because of excellent potency of the 2-pyridyl analogues
(6h and 6r) and the 4-pyrimidinyl derivatives (6l and 6s),
Table 2. SAR for C2-amino-pyridine modification
Table 1. SAR for C2-amine modification
Compd
R
R1
Lck inhibition
IC50, mM5
Compd
R
R1
Lck inhibition
IC50, mM5
6h
6v
6w
6x
6y
6z
6aa
6ab
6ac
6ad
6ae
6af
6ag
6ah
6ai
6aj
6ak
6al
6am
2
2,4,6-Me3
2,4,6-Me3
2,4,6-Me3
2,4,6-Me3
2,4,6-Me3
2,4,6-Me3
2-Cl,6-Me
2-Cl,6-Me
2-Cl,6-Me
2-Cl,6-Me
2-Cl,6-Me
2-Cl,6-Me
2-Cl,6-Me
2-Cl,6-Me
2-Cl,6-Me
2-Cl,6-Me
2-Cl,6-Me
2-Cl,6-Me
2-Cl,6-Me
2-Cl,6-Me
2-Cl,6-Me
2-Cl,6-Me
H
3-Me
4-Me
5-Me
6-Me
3,5-di-Me
H
4-Me
5-Me
6-Me
0.138
>3.1
0.032
0.072
0.18
0.017
0.084
0.02
0.035
0.009
0.008
0.005
0.403
0.009
0.001
0.001
0.002
0.010
0.013
0.0005
0.002
0.006
6a
6b
6c
6d
6e
6f
6g
6h
6i
2,4,6-Me3
2,4,6-Me3
2,4,6-Me3
2,4,6-Me3
2,4,6-Me3
2,4,6-Me3
2,4,6-Me3
2,4,6-Me3
2,4,6-Me3
2,4,6-Me3
2,4,6-Me3
2,4,6-Me3
2,4,6-Me3
2,4,6-Me3
2-Cl,6-Me
2-Cl,6-Me
2-Cl,6-Me
2-Cl,6-Me
2-Cl,6-Me
2-Cl,6-Me
2-Cl,6-Me
H
Ph
15.07
1.74
Cyclopropyl-CH2
2-Thiazolyl
2-Imidazolyl
4-Imidazolyl
5-(1,2,4-Triazolyl)
2-Pyridyl
>3.1
>3.1
>3.1
>3.1
>3.1
0.138
>3.1
>3.1
>3.1
0.403
1.89
0.235
0.327
>3.1
1.59
0.084
0.027
0.041
0.015
3-Pyridyl
4-Pyridyl
6j
6k
6l
4,6-di-Me
4-Et
4-NH2
2-Pyrimidinyl
4-Pyrimidinyl
3-(1,2,4-Triazinyl)
2-Pyrazinyl
Ph
2-Imidazolyl
3-Pyrazolyl
2-Pyridyl
4-Me2NCH2
4-NMe-PiperazinoCH2
4-MorpholinoCH2
6-NH2
6-Me2NCH2
6-MorpholinoCH2
6-HOCH2CH2NH
6-AcNHCH2CH2NH
6-H2NCMe2CH2NH
6m
6n
6o
6p
6q
6r
6s
6t
4-Pyrimidinyl
2-Pyridazinyl
3-Pyrazinyl
6an
6ao
6u

J. Das et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2587–2590
2589
methylamino substituent (6az) resulted in Lck inhibitors
with improved activity. Some of the most potent inhib-
itors were the 2,6-disubstituted pyrimidine analogues (3,
6bd–6bi). Compounds 3, 6az, and 6bg were selected for
further characterization.
phoaminophosphonic acid-adenylate ester,
a
non-
hydrolyzable ATP mimic). As illustrated in Figure 2
both BMS-350751 (2) and BMS-358233 (3) are thought
to bind in an extended conformation to the ATP-bind-
ing site of Lck. The 2,6-disubstituted aniline of 2 and 3
occupies a clearly defined hydrophobic pocket (common
to all Src family kinases) which is not occupied by ATP.
The 2,6-disubstitution skews the aniline ring out of
plane with respect to the benzothiazole core and this in
turn allows the phenyl ring to fit into the narrow and
angular hydrophobic pocket. In addition, both of these
analogues are likely to make several productive hydro-
gen bond interactions within the active site: the NH of
the aniline is hydrogen bonded to Thr316 side chain
hydroxyl, the benzothiazole nitrogen is in H-bond con-
tact with Tyr318, and the two NHs’ of the amino-pyri-
dine or the amino-pyrimidine moiety are in contact with
the backbone carbonyls of Met319, and Glu320,
respectively. The lack of selectivity exhibited by this
series of benzothiazoles compared to the urea analogue
1 is not clearly understood but it may be due to differ-
ences in the amino acid sidechains found at the edge of
the ATP binding pocket in other kinases. For example,
Lck residues Glu320 and Asn321 are replaced in Src by
Ser 320 and Lys321, respectively, and in Fyn by Asn320
and Lys321, respectively. These differences represent
significant alterations in the polarity of the binding site
surface features at and near the region surrounding the
pendant heterocyclic-substituted benzothiazoles.
Pyridine 2 and the pyrimidine analogues 3, 6az, and 6bg
were tested for their activity against other Src family
kinases and for cell activity in a T-cell proliferation
assay (Table 4). These compounds were potent inhibi-
tors of Src and Fyn kinases, in addition to Lck. Fur-
thermore, these analogues were shown to be potent
inhibitors of anti-CD3/anti-CD28 induced PBL pro-
liferation.5 Compound 3 was found to be the most
potent inhibitor (IC50=262 nM) in this assay.
A binding model for these benzothiazole Lck inhibitors
was developed based on the published coordinates6 of
the activated Lck kinase domain bound to ANP (phos-
Table 3. SAR for C2-amino-pyrimidine modification
Compd
R
R1
Lck inhibition
IC50, mM5
In summary, we have described the design and optimi-
zation of a novel series of 2-heteroarylamino-benzo-
thiazole Lck inhibitors. SAR studies identified several
substituted pyridyl and pyrimidinyl analogues as highly
potent inhibitors. BMS-350751 (2) and BMS-358233 (3)
were identified as some of the most highly active Lck
inhibitors in this series and displayed excellent potency
in a T-cell proliferation assay.
6l
2,4,6-Me3
2,4,6-Me3
2,4,6-Me3
2-Cl,6-Me
2-Cl,6-Me
2-Cl,6-Me
2-Cl,6-Me
2-Cl,6-Me
2-Cl,6-Me
2-Cl,6-Me
2-Cl,6-Me
2-Cl,6-Me
2-Cl,6-Me
2-Cl,6-Me
2-Cl,6-Me
2-Cl,6-Me
2-Cl,6-Me
2-Cl,6-Me
2-Cl,6-Me
2-Cl,6-Me
2-Cl,6-Me
H
2,6-di-Me
6-Et
H
2-Cl
0.403
0.053
0.077
0.027
0.031
0.020
0.003
0.021
0.005
0.020
0.013
0.008
0.002
0.008
0.006
0.020
0.007
0.006
0.007
0.002
0.006
0.001
0.010
6ap
6aq
6s
6ar
6as
6at
6au
6av
6aw
6ax
6ay
6az
6ba
6bb
6bc
6bd
6be
6bf
6bg
6bh
3
2-MeNH
2-HOCH2CH2NH
6-Me
6-Et
6-Cl
6-OMe
6-NH2
6-MeNH
6-HOCH2CH2NH
6-AcNHCH2CH2NH
2,6-di-Me
2-Me,6-MeNHCH2
2-Me,6-MorpholinoCH2
2-Me,6-MeNHCH2
2-HOCH2CH2NH,6-Me
2-HOCMe2CH2NH,6-Me
2-Cl,6-Me 2-(2-tetrahydrofurylCH2NH),6-Me
2-Cl,6-Me 2-MorpholinoCH2,6-Me
6bi
Table 4. Enzyme selectivity of selected Lck inhibitors
Compd
Lck
Ki, nM5
Src
Ki, nM5
Fyn
Ki, nM5
T-Cell Prolif.
IC50, nM5
1
2
3
6az
6bg
5.5
0.9
0.54
6.6
1.1
333
13
3.2
17
100
2.4
1.1
8.8
4
1100
475
262
690
329
Figure 2. Proposed superimposed conformations of 2 (cyan) and 3
(magenta) bound to the ATP binding site of Lck.
10

2590
J. Das et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2587–2590
reader was used for fluorescence measurements using an exci-
References and Notes
tation wavelength of 360 nm and an emission wavelength of
460 nm. Readings were taken immediately following ATP
addition and every 10 to 30 seconds thereafter depending on
the reaction rate. Fluorescence measeurements were recorded
until the signal reached 90% of the initial signal. At least 50
data points were collected in order to calculate the slope, or
rate of the reaction. The rate was then converted to mM pro-
duct/min and plotted against inhibitor concentration. Ki was
determined by nonlinear least squares regression with the
Kaleidagraph1 software (Synergy Software) according to
established procedure (see: Bieth, J. G. Meth. Enzymol. 1995,
248, 59). Assays for Src, and Fyn were run under the same
conditions as for Lck. (c) PBL Proliferation Assay: For a
description of this assay, see ref 4. (d) Alamar Blue Assay:
Normal human peripheral blood mononuclear cells (PBMC)
were isolated from human heparinized whole blood, and were
grown in the presence of 5 mg/mL PHA (phytohemaglutinin)
for 2–4 days in RPMI 1640 media with 10% fetal bovine
serum to generate PHA blasts. Inhibitor compounds (or
DMSO as a control) were diluted to a final concentration of
30 mM in 96-well Costar tissue culture plate. PHA blasts were
washed two times, resuspended in RPMI 1640 containing 10%
fetal bovine serum, and then were added 3Â105/well to the
plate containing test compounds, in a total volume of 200 mL.
The plate was incubated at 37 ꢀC for 24 h. Alamar Blue solu-
tion (Biosource catalog # DAL 1100) was then added at 20
mL/well to each well and incubated another 4 h at 37 ꢀC. The
plate was read in a CytoFluor Multi-well Plate Reader-4000
(PerSeptive Biosystems) using an excitation wavelength of 530
nm and an emission wavelength of 590 nm.
1. (a) Molina, T. J.; Kishihara, K.; Siderovski, D. P.; van
Ewijk, W.; Narendran, A.; Timms, E.; Wakeham, A.; Paige,
C. J.; Hartman, K.-U.; Veillette, A.; Davison, D.; Mak, T. W.
Nature 1992, 357, 161. (b) Levin, D. S.; Anderson, S. J.; For-
bush, K. A.; Perlmutter, R. M. EMBO J. 1993, 12, 1671. (c)
Levitzki, A. Top. Curr. Chem. 2001, 211, 5. (d) Dowden, J.;
Ward, S. G. Exp. Opin. Ther. Patents 2001, 11, 295.
2. (a) Straus, D. B.; Weiss, A. Cell 1992, 70, 585. (b) Chan,
A. C.; Desai, D. M.; Weiss, A. C. Ann. Rev. Immunol. 1994,
12, 555. (c) Weiss, A.; Littman, D. Cell 1994, 76, 263. (d)
Hanke, J. H.; Gardner, J. P.; Dow, R. L.; Changelian, P. S.;
Brissette, W. H.; Weringer, E. J.; Pollok, B. A.; Connelly, P. A. J.
Biol. Chem. 1996, 271, 695. (e) Van Oers, N. S. C.; Lowin-Kropt,
B.; Finlay, D.; Connolly, K.; Weiss, A. Immunity 1996, 5, 429.
3. Hanke, J. H.; Pollok, B. A.; Changelian, P. S. Inflammation
Res. 1995, 44, 357.
4. Das, J.; Lin, J.; Moquin, R. V.; Shen, Z.; Spergel, S. H.;
Wityak, J.; Doweyko, A. M.; De Fex, H.; Fang, Q.; Pang, S.;
Pitt, S.; Shen, D. R.; Schieven, G. L.; Barrish, J. C. Bioorg.
Med. Chem. Lett. 2003, 13, 2145.
5. (a) Lck Enzyme Assay: For a description of the assay, see
ref 4. Lck enzyme assays were run in triplicate and the values
are mean of these experiments, standard deviation Æ10%. (b)
Ki determination assay: A coupled assay (see: Cole, P. A.;
Grace, M. R.; Phillips, R. S.; Burn, P.; Walsh, C. T. J. Biol.
Chem. 1995, 270, 22105) was used to monitor the tyrosine
phosphorylation of the Lck specific peptide AEEEIYGVL-
FAKKKK by GST-Lck. The following reaction conditions
were used: 50 mM Tris–HCl pH 8.0, 5 mM MgCl2, 0.15 M
NaCl, 200 mM substrate peptide, 1 mM PEP, 200 mM NADH,
1 unit of LDH, 0.6 unit of PK, 9 nM GST-Lck, and 1–2 mM
ATP. The assay was carried out in a 96 well plate at 26 ꢀC. For
each reaction 78 mL of reaction mixture (all reaction compo-
nents except ATP) and 2 mL of inhibitor solution in DMSO
was added. The reaction was initiated by addition of 20 mL of
5 mM ATP solution. The Cytofluor (Molecular Devices) plate
6. Two crystal structures of the catalytic domain of Lck
(Tyr394 phosphorylated) were published. See: (a) Yamaguchi,
H.; Hendrickson, W. A. Nature 1996, 384, 484. (b) Zhu, X.;
Kim, J. L.; Newcomb, J. R.; Rose, P. E.; Stover, D. R.;
Toledo, L. M.; Zhao, H.; Morgenstern, K. A. Structure 1999,
7, 651 Protein Data Bank (1QPC pdb, Research Collaboratory
for Structural Bioinformatics.