Discovery of 2-amino-6-carboxamidobenzothiazoles as potent Lck inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.02.079

A novel series of 2-amino-6-carboxamidobenzothiazole was discovered to have potent Lck inhibitory properties. A highly efficient chemistry was developed. Also described are the detailed SAR study and the BaF3 cell line profiling for this series.

Full text of DOI:10.1016/j.bmcl.2008.02.079

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2324–2328
Discovery of 2-amino-6-carboxamidobenzothiazoles
as potent Lck inhibitors
Shenlin Huang,* Zuosheng Liu, Shin-Shay Tian, Mark Sandberg, Tracy A. Spalding,
Russell Romeo, Maya Iskandar, Zhiliang Wang, Donald Karanewsky and Yun He*,
The Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121, USA
Received 13 January 2008; revised 28 February 2008; accepted 29 February 2008
Available online 6 March 2008
Abstract—A novel series of 2-amino-6-carboxamidobenzothiazole was discovered to have potent Lck inhibitory properties. A highly
efficient chemistry was developed. Also described are the detailed SAR study and the BaF3 cell line profiling for this series.
Ó 2008 Elsevier Ltd. All rights reserved.
Lymphocyte specific protein tyrosine kinase (Lck), a
member of the Src family of non-receptor protein tyro-
sine kinases, is predominantly expressed in T-lympho-
cytes and natural killer cells.1,2 Lck plays an essential
role in the T cell receptor (TCR) signal transduction
pathway.3 It phosphorylates the f chain of the TCR
complex on specific tyrosine residue located within a
motif termed the immunoreceptor tyrosine activation
motif (ITAM), resulting in coupling of TCR with pro-
tein tyrosine kinase ZAP-70 via its Src homology-2
(SH2) domains.1 Subsequent phosphorylation of ZAP-
70 by Lck4 triggers a series of downstream cascade
events that ultimately leads to cytokine release, T cell
activation and proliferation.5 Genetically modified mice
with Lck mutations exhibit defects in T cell maturation
and signalling.6 These findings indicate that Lck inhibi-
tors should inhibit T cell activation and therefore be use-
ful therapies for T cell-mediated autoimmune diseases
and graft rejection. Additionally, a selective Lck inhibi-
tor will offer a more desirable safety profile compared to
other immuno-suppressive agents (e.g., cyclosporine or
steroids currently used for treating T cell-mediated auto-
immune and inflammatory diseases) since Lck is exclu-
sively expressed in lymphoid cells.2
Developing small molecule Lck inhibitors has been the
focus of major pharmaceutical research in recent years.7
Potent and bioavailable Lck inhibitors have been identi-
fied and demonstrated in vivo efficacy in several models
of T cell dependent immune responses.8 Our research ef-
forts towards modifying the 2-amino-6-carbo-
xamidobenzothiazole series previously reported by
Bristol-Myers Squibb9 resulted in a novel scaffold with
potent Lck inhibitory properties. Described here are
the chemistry for analogue synthesis, structure–activity
relationship (SAR) study and kinase profiling for this
series.
As shown in Scheme 1, the chemistry started by coupling
of 2-tert-butoxycarbonylaminobenzothiazole-6-carbox-
ylic acid (1a) with N-(4-amino-3-methylphenyl)-3-trifluo-
romethylbenzamide (1b) in the presence of HATU to
provide compound 2. After removing the Boc group with
TFA, the amino intermediate 3 was treated with different
isocyanates to generate the urea analogues 4a–e. Numer-
ous carbamates 4f–j were also generated by reacting com-
pound
3
with different chloroformates. Several
aminoalkyl and heteroaryl groups were attached to the
–NH at the C-2 position via chemistry described in
Scheme 2. First, mixing 2-aminobenzothiazole-6-carbox-
ylic acid methyl ester (5) with tert-butyl nitrite and CuBr2
provided bromo ester 6, which was saponified to carbox-
ylic acid 7. Treating this intermediate with oxalyl chloride
and compound 1b in sequence resulted in bisamide 8.
Then, the 2-bromide was displaced with various amines
to generate analogues 9a–h. For the synthesis of com-
pounds 9d–h, the addition of NaH was necessary in order
to generate a more active N anion.
Keywords: Lck inhibitor; 2-Amino-6-carboxamidobenzothiazole.
*
Corresponding authors. Tel.: +1 858 332 4684; fax: +1 858 812 1684
(S.H.); tel.: +86 21 3895 4910x2060; fax: +86 21 5079 0292 (Y.H.);
e-mail addresses: shuang2@gnf.org; yun.he@Roche.com
Present address: Roche R&D Center (China) Ltd, 720 Cai Lun
Road, Building 5, Pudong, Shanghai 211203, China.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.02.079

S. Huang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2324–2328
2325
H2N
O
CF3
N
H
1b
N
H
N
N
S
Boc
H
N
Boc
N
H
b
N
H
CF3
S
OH
O
a
O
O
1a
2
O
N
S
N
S
H
N
H
N
H
N
H
c
H2N
R
N
H
N
CF3
CF3
O
O
O
O
4a-j
3
Scheme 1. Synthesis of analogues 4a–j. Reagents: (a) N,N-diisopropylethylamine, HATU, DMF, 100%; (b) TFA, 85%; (c) RNCO for 4a–e, 80–90%
or RO(CO)Cl for 4f–j, 40–60%.
N
S
N
S
N
S
b
a
c, d
H2N
Br
Br
O
O
OH
O
O
5
O
6
7
N
S
N
S
H
R
H
H
N
H
N
Br
N
e
N
H
N
CF3
CF3
O
O
O
O
8
9a-h
Scheme 2. Synthesis of analogues 9a–h. Reagents: (a) tert-BuONO, CuBr2, CH3CN, 88%; (b) LiOH, THF/H2O (1:1), 85%; (c) (COCl)2, CH2Cl2; (d)
1b, iso-Pr2NEt, CH2Cl2, 97%; (e) RNH2, DMF, 60–90%.
Table 1. Cellular activity of compounds 3, 4a–j, 9a–h
All analogues were tested for their cellular activity using
a TEL-Lck transformed BaF3 cell line that was stably
transfected with a Cytomegalovirus immediate early
promoter upstream of a luciferase gene. Briefly, the 30
portion of Lck was fused to the 50 region of TEL, a gene
encoding a member of the ETS transcription factor fam-
ily. The TEL-Lck fusion protein includes the catalytic
domain of Lck and the TEL-specific oligomerization do-
main. TEL-induced oligomerization of TEL-Lck re-
sulted in the constitutive activation of its tyrosine
kinase activity and conferred cytokine-independent pro-
liferation to the interleukin-3-dependent BaF3 hemato-
poietic cell line. Transformed BaF3 cells were plated in
384-well plates (25,000 cells per well) and incubated with
serial dilutions of inhibitor or DMSO for 48 h. Lucifer-
ase expression was used as a measure of cell prolifera-
tion/survival and was evaluated with the Bright-Glo
Luciferase Assay System (Promega, Madison, WI).
N
S
H
N
R
H
N
N
H
CF3
O
O
Compound
R
H
Cellular activity
IC50 (lM)
3
0.170
0.021
0.016
0.016
0.094
0.125
0.030
0.030
0.008
0.112
0.131
0.081
4a
4b
4c
4d
4e
4f
CH3NHC(O)
CH3CH2NHC(O)
CyclopentylNHC(O)
PhNHC(O)
Pyrid-3-ylNHC(O)
CH3OC(O)
4g
4h
4i
IsopropylOC(O)
CH3OCH2CH2OC(O)
PhOC(O)
4j
4-MethoxyphenylOC(O)
N,N-(CH3)2NCH2CH2CH2
9a
9b
9c
9d
9e
9f
The inhibition of cell proliferation results is shown in Ta-
ble 1. Compound 3 with an unsubstituted amino group
only showed moderate cellular potency with an IC50 of
170 nM. Converting the amine to urea resulted in more
potent compounds. A 10-fold increase in potency was ob-
served with simple alkyl and carbocyclic ureas (4a, 4b, 4c),
though the potency improvement for aryl and heteroaryl
ureas (4d, 4e) was marginal. Transforming the amine to
carbamate was also beneficial to cellular potency. As seen
with urea analogues, alkyl carbamates (4f, 4g, 4h) were
more anti-proliferative than aryl carbamates (4i, 4j). In
1-Methylpiperazin-4-ylCH2CH2 0.058
Morpholin-4-ylCH2CH2CH2
4-Methylpyridin-2-yl
4,6-Dimehylpyrimidin-2-yl
2,4-Dimethylpyrimidin-6-yl
Pyrazin-2-yl
0.078
0.016
0.053
0.0006
0.004
0.008
9g
9h
5-Methylisoxazol-3-yl
particular, compound 4h had a single-digit nM IC50, pos-
sibly due to its high cellular permeability. Adding amino
groups (9a–c) not only improved solubility, but also in-

2326
S. Huang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2324–2328
O
O
N
S
N
S
N
S
a
b
H2N
N
H
N
H
O
N
H
N
H
O
OH
O
O
O
O
10
12
11
O
N
S
N
S
H
N
c
H
N
H
N
H
d
NHBoc
N
H
N
H
N
NH2
O
O
O
13
14
O
N
e
H
H
N
N
N
N
R
S
H
H
O
15a-i
Scheme 3. Synthesis of analogues 15a–i. Reagents: (a) EtNCO, THF, 86%; (b) LiOH, 1,4-dioxane/H2O (1:1), 92%; (c) 5-tert-butoxycarbonylamino-
2-methylaniline, HATU, Et3N, DMF, 82%; (d) TFA, 92%; (e) RC(O)OH, HATU, Et3N, DMF, 82%.
creased cellular potency. Attaching heteroaryls (9d–h)
generated the most potent analogues, for example, com-
pounds 9d and 9e had single-digit nM IC50 and the IC50
for compound 9f was <1 nM, indicating there might be
additional interactions between the Lck ATP binding
pocket and these heteroaryls.
barely changed the potency. On the other hand, 4-Cl
on the 5-CF3-phenyl ring (15d) seemed to be harmful,
resulting in a threefold loss in potency. The negative im-
pact by substituting the CF3 with CH3O (15e), Me (15f)
or Cl (15g) was even more severe. Replacing 3-CF3-phe-
nyl group with 2-tert-butylpyridin-4-yl group (15h) or 2-
tert-butylthien-5-yl (15i) had very little effect in terms of
cellular inhibition.
The second stage of our SAR study was focused on the
right side benzoyl functionality while fixing the left side
as an ethyl urea group. Using the chemistry shown in
Scheme 3, 2-aminobenzothiazole-6-carboxylic acid ethyl
ester (10) was reacted with ethyl isocyanate to generate
compound 11, which was saponified to carboxylic acid
12. It was then coupled with compound 5-tert-butoxy-
carbonylamino-2-methyl-aniline to provide amide 13.
After removing the Boc group with TFA, the amino
intermediate 14 was coupled with various carboxylic
acids to provide analogues 15a–i. The cellular data are
displayed in Table 2. Adding F to the C-3 position of
the 5-CF3-phenyl ring (15a) increased potency slightly
while the effect of 4-methylimidizol-1-yl (15b) at the
same position was minimal. Attaching solubilizing
group 4-methylpiperizin-1-yl (15c) to the C-3 position
Using chemistry in Scheme 4, compounds 16 and 18
were synthesized to explore whether the 2-NH is essen-
tial for cellular activity. Methylation of the 2-NH of
compound 2 under Mitsunobu conditions afforded ana-
logue 16. Next, the Boc was removed with TFA to gen-
erate intermediate 17, which was reacted with ethyl
isocyanate to afford analogue 18. Methylation of the
2-NH resulted in a dramatic loss in cellular potency
for both carbamate 16 and urea 18 (data shown in Table
3), demonstrating this NH is crucial for Lck potency. A
synthesis was also developed to examine the effect of
bromination of the middle ring. As shown in Scheme
5, intermediate 3 was reacted with tert-butyl nitrite
and CuBr2 to generate dibromo compound 19. Next,
displacement of the 2-Br with 2-(morpholin-4-yl)ethyla-
mine provided analogue 20, which was nearly inactive in
BaF3 cells (see Table 3), showing that adding Br to the
middle ring was detrimental to Lck potency. Finally, we
examined the effect of reversing the amide group at the
right side. By similar chemistry in Scheme 1, compounds
21 and 22 were synthesized, both of which exhibited sev-
eral fold lower cellular potency relative to their corre-
sponding analogues 4b and 4c.
Table 2. Cellular activity for compounds 15a–i
O
N
H
N
H
N
N
H
N
H
R
S
O
O
Compound
R
Cellular
activity
Compounds 4b, 4c, 4f and 4h were also tested in two dif-
ferent Jurkat cell lines that have the interleukin 2 (IL-2)
promoter driving luciferase. In the Jurkat IL-2 anti-
CD3+28 assay, the T cell receptor is stimulated by incu-
bating cells with plate bound anti-CD3+CD28 antibod-
ies, leading to an increase in IL-2 promoter activity.
Therefore, Lck inhibitors would block this activation.
On the other hand, a Jurkat IL-2 PMA/Iono assay is
used to measure general off target effects, as the IL-2
promoter is stimulated by PMA and ionomycin in a T cell
IC50 (lM)
15a
15b
15c
15d
15e
15f
15g
15h
15i
3-F-5-CF3-phenyl
0.007
3-(4-Methylimidazol-1-yl)-5-CF3-phenyl 0.015
3-(4-Methylpiperizin-1-yl)-5-CF3-phenyl 0.019
4-Cl-3-CF3-phenyl
3-Methoxyphenyl
3-Methylphenyl
0.050
0.158
0.112
0.104
0.009
0.012
3-Chlorophenyl
2-tert-Butylpyridin-4-yl
2-tert-Butylthien-5-yl

S. Huang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2324–2328
2327
O
N
S
O
H
N
H
N
a
N
S
O
N
H
H
N
H
N
CF3
O
N
O
CF3
O
O
O
O
2
16
N
H
N
S
H
H
N
b
c
H
N
N
N
N
N
H
N
CF3
S
H
CF3
O
O
O
O
17
18
Scheme 4. Synthesis of analogues 16 and 18. Reagents: (a) Ph3P, DIAD, MeOH, THF, 53%; (b) TFA, 99%; (c) EtNCO, Et3N, THF, 71%.
Table 3. Cellular activity for compounds 16, 18, 20–22
N
S
CF3
R1
H
N
X
N
R2
R3
O
Compound
R1
R2
R3
–X–
Cellular activity IC50 (lM)
2
tert-ButylOC(O)
tert-ButylOC(O)
CH3CH2NHC(O)
H
H
H
H
Br
H
H
–NH(CO)–
–NH(CO)–
–NH(CO)–
–NH(CO)–
–(CO)NH–
–(CO)NH–
0.036
0.955
>10
16
18
20
21
22
CH3
CH3
H
2-(Morpholin-4-yl)CH3CH2
CH3CH2NHC(O)
CyclopentylNHC(O)
2.099
0.118
0.139
H
H
N
H
N
S
H
N
H
N
a
H
N
H2N
N
Br
CF3
S
CF3
O
O
O
O
Br
3
19
O
N
S
N
H
N
b
H
N
N
H
CF3
O
O
Br
20
Scheme 5. Synthesis of analogue 20. Reagents: (a) tert-BuONO, CuBr2, CH3CN, 72%; (b) 2-(morpholin-4-yl)ethylamine, DMF, 70%.
Table 4. Cellular activity of compounds 4b, 4c, 4f and 4h
receptor independent manner. For the anti-CD3+28 as-
say, anti-mouse IgG was bound to the plate using a so-
dium bicarbonate buffer followed by incubation with
OKT3 (mouse anti-human CD3) and mouse anti-human
CD28 antibodies. Plates were then washed and 100,000
Jurkat IL-2-luc cells were plated out in 0.05 mL for 5 h.
For the PMA/Iono assay, 50,000 cells were plated out in
0.025 mL followed by the addition of 0.025 mL of
40 ng/mL PMA and 1.5 lg/mL ionomycin. Luciferase
activity was evaluated with the Bright-Glo Luciferase As-
say System. As expected, these potent Lck inhibitors
exhibited high potency in the Jurkat IL-2 anti-
CD3+CD28 assay and low off target activity as deter-
mined in the Jurkat IL-2 PMA/Iono assay (see Table 4).
Compound
Jurkat cell/anti-CD3+
CD28 IC50 (lM)
Jurkat cell/PMA/Iono
IC50 (lM)
4b
4c
4f
0.044
0.062
0.036
0.012
0.373
0.812
0.851
0.438
4h
lent selectivity was achieved against a number of
structurally diverse kinases including JAK3, KDR,
InsR, Alk, FGFR-3 and Flt3. Both compounds also
showed good selectivity against Sky though their po-
tency for two other Src family members Lyn and Src re-
mains relatively high.
Compounds 4a and 4g were selected for screening
against a panel of BaF3 cell lines transformed with dif-
ferent kinases. The results are shown in Table 5. Excel-
In summary, a novel series of 2-amino-6-carbo-
xamidobenzothiazole was discovered to have potent

2328
S. Huang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2324–2328
Table 5. BaF3 cell line profiling of compounds 4a and 4g
Compound
IC50 (lM)
BaF3/Lck BaF3/Lyn BaF3/Src BaF3/Sky BaF3/JAK3 BaF3/KDR BaF3/InsR BaF3/Alk BaF3/FGFR3 BaF3/FLT3
4a
4g
0.021
0.030
0.094
0.268
0.301
0.517
1.105
2.436
5.01
>10
1.608
3.283
1.276
13.5
1.633
6.109
1.984
12.603
3.141
>10
6. Molina, T. J.; Kishihara, K.; Siderovskid, D. P.; van Ewijk,
W.; Narendran, A.; Timms, E.; Wakeham, A.; Paige, C. J.;
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McRae, B.; Wallace, C.; Leach, M.; Ratnofsky, S.; Belan-
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Lck inhibitory properties. A highly efficient chemistry
was developed. Many potent analogues were synthesized
with urea, carbamate, heteroarylamine or alkylamine at
the C-2 position. The SAR for the middle ring, the right
side aryl group was also explored. The selectivity profile
was herein included. Future studies will be directed to-
wards determination of PK properties and in vivo
efficacy.
References and notes
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