2-Aminobenzimidazoles as potent ITK antagonists: trans-stilbene-like moieties targeting the kinase specificity pocket

Article abstract of DOI:10.1016/j.bmcl.2008.09.098

Based on the information from molecular modeling and X-ray crystal structures, the kinase specificity pocket of ITK could be occupied upon extension of the right-hand-side of the 2-benzimidazole core of the inhibitors. 2-Aminobenzimidazoles with a trans-stilbene-like extension were designed and synthesized as novel ITK antagonists. Significant improvement on binding affinity and cellular activity were obtained through the trans-stilbene-like antagonists. Several compounds showed inhibitory activity in an IL-2 functional assay.

Full text of DOI:10.1016/j.bmcl.2008.09.098

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6218–6221
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2-Aminobenzimidazoles as potent ITK antagonists: trans-stilbene-like
moieties targeting the kinase specificity pocket
a
a
b
b
b
Ho Yin Lo a, , Jörg Bentzien , Roman W. Fleck , Steven S. Pullen , Hnin Hnin Khine , Joseph R. Woska Jr. ,
*
Stanley Z. Kugler c, Mohammed A. Kashem c, Hidenori Takahashi a
a Medicinal Chemistry, Boehringer Ingelheim Pharmaceuticals Inc., 900 Ridgebury Road, P.O. Box 368, Ridgefield, CT 06877, USA
b Immunology and Inflammation, Boehringer Ingelheim Pharmaceuticals Inc., 900 Ridgebury Road, P.O. Box 368, Ridgefield, CT 06877, USA
c Biomolecular Screening, Boehringer Ingelheim Pharmaceuticals Inc., 900 Ridgebury Road, P.O. Box 368, Ridgefield, CT 06877, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on the information from molecular modeling and X-ray crystal structures, the kinase specificity
pocket of ITK could be occupied upon extension of the right-hand-side of the 2-benzimidazole core of
the inhibitors. 2-Aminobenzimidazoles with a trans-stilbene-like extension were designed and synthe-
sized as novel ITK antagonists. Significant improvement on binding affinity and cellular activity were
obtained through the trans-stilbene-like antagonists. Several compounds showed inhibitory activity in
an IL-2 functional assay.
Received 5 September 2008
Revised 25 September 2008
Accepted 29 September 2008
Available online 2 October 2008
Keywords:
ITK
Ó 2008 Elsevier Ltd. All rights reserved.
T-cell kinase
Aminobenzimidazoles
Inflammation
Kinase specificity pocket
Interleukin-2-inducible T-cell kinase (ITK) is a tyrosine kinase
from the Tec kinase family.1 ITK is expressed in T-cells, NK cells
and mast cells.2 Studies using ITK deficient murine CD4+ T-cells
showed reduced IL-2, IL-4, IL-5, and IL-13 production upon T-cell
receptor stimulation.3–5 The hypothesis that a selective ITK antag-
onist would reduce the production of T-cell cytokines, which con-
tribute to inflammation, is attractive for the treatment of
inflammatory diseases such as rheumatoid arthritis and allergic
asthma.6
Val419
Phe435
Lys391
Met438
H
N
O
N
S
KSP
Met410
Glu406
Asp500
N
O
N
Leu489
NH2
1
O
Recently, we have discovered that 2-aminobenzimidazoles are
potent ITK inhibitors.7 Lead compounds such as 1 showed signifi-
cant inhibitory activity in an ITK DELFIA screening assay
(IC50 = 25 nM). However, their potency was severely reduced in a
Figure 1. The structure and binding mode of 1 in ITK.
kinase specificity pocket (KSP). The KSP spans a 7.3-Å space
from the ‘gate-keeper’ Phe435 to Met410. It contains hydrophobic
cell assay measuring Ca+ flux using DT40/ITK cells (IC50 = 2.4
lM).
(Leu418 Leu489 and Val419
, , ) as well as hydrophilic residues
Furthermore, compounds such as 1 had a low selectivity over insu-
lin receptor kinase (IRK) (IC50 = 145 nM). Therefore, we embarked
in a synthetic effort to improve potency and selectivity.
The binding mode of compound 1 was predicted by molecular
modeling and confirmed by X-ray crystallography of the en-
zyme–inhibitor complex. (Fig. 1).8 Interestingly, the aminobenzim-
idazole 1 adopts a tautomeric form that allows the establishment
of a key hydrogen bonding with the backbone Met438. On the
right-hand side, the thiophene group points toward an unoccupied
(Lys391, Glu406, Met410, and Asp500). It was envisioned that appro-
priate substitutions of the thiophene ring could occupy the KSP,
and hence improve binding affinity for ITK while enhancing selec-
tivity over IRK. To extend into the KSP, the ‘gate-keeper’ residue
Phe435, which resides at the entrance of the KSP, had to be circum-
vented. We designed a trans-stilbene-like extension, predicted by
molecular modeling to present the correct orientation necessary
for occupying the KSP, while avoiding Phe435 (Fig. 2).
The preparation of various trans-stilbene-like analogs is illus-
trated in Schemes 1 and 2. In order to synthesize a number of ana-
logs efficiently, a parallel synthesis approach was used. Common
precursors such as 5, 6, and 7 were prepared as the divergent
* Corresponding author. Tel.: +1 203 798 4923.
E-mail address: ho-yin.lo@boehringer-ingelheim.com (H.Y. Lo).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.098

H. Y. Lo et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6218–6221
6219
pared efficiently by a standard amide coupling reaction with the
corresponding acid partners, (5-bromothiophene-2-carboxylic
acid, 5-formyl-2-thiophenecarboxylic acid, and 5-acetylthio-
phene-2-carboxylic acid), respectively.
Starting from intermediates 5, 6, and 7, various types of deriva-
tives featuring different olefinic linkers could be prepared. The syn-
theses of some representative examples are described in Scheme 2.
For the analogs carrying a di-substituted olefinic linker and
aromatic end-moieties such as pyridine 8a, a Heck-type coupling
protocol was adopted, using precursor 5 as the starting material,
and the corresponding vinyl aromatic system as the coupling part-
ners.9 For mono-substituted olefin analogs such as 8b, a Stille-type
coupling was performed with precursor 5 and tributylvinyl tin as
the starting materials. A Wittig reaction was used for the efficient
preparation of nitrile 8c from precursor 6. Compounds bearing a
tri-substituted olefin linker, such as 9a and 9b, were synthesized
from precursor 7, starting with a Wittig protocol to yield 9a. Bro-
mide 9a could then become the precursor for the preparation of
several compounds featuring aromatic moieties, such as 9b, In this
case, palladium-catalyzed coupling reactions with the correspond-
ing boronic acids were carried out.
F435
O
N
H
N
N
R1
R2
S
O
N
The analogs were tested in the ITK DELFIA assay10a to determine
the intrinsic binding affinity, a Ca+ flux assay with the DT40/ITK
cell line10b to determine the cell activity, and also in an IRK DELFIA
assay for selectivity. The results are summarized in Tables 1 and 2.
For di-substituted olefinic type of inhibitors (Table 1), introduc-
tion of the olefinic linker with small substitutions such as 8b, 8c,
and 8e, showed more than 5-fold improvement on molecular and
cellular activity of ITK and superior selectivity over IRK compared
with the lead compound 1. Phenyl substitution analog 8f however
only maintained potency and selectivity profile as 8b, though we
had hypothesized that the aromatic system should had occupied
the KSP and further enhanced the compound profile. Extensive
studies on the substitution pattern on the aromatic ring were ini-
tiated. Electronic effect on the binding was the first factor to be
investigated. Compounds with an electron donating group (8h,
8i, and 8j) or an electron withdrawing group (8k, 8l, 8m, and 8r)
on all positions on the aromatic ring were synthesized. All of these
NH2
O
General formula
Figure 2. Molecular modeling of 8d (R1 = H, R2 = 4-pyridine). The 4-pyridine ring
resides in the KSP may provides hydrogen bonding interactions with amino acid
residues such as Lys391
.
points for the analogs’ syntheses (Scheme 1). Simple acylation of
commercially available fluoronitroaniline with benzoyl chloride,
followed by methylation of the corresponding amide, gave tri-
substituted amide 2 in good yield. SNAr displacement of the fluo-
rine in 2 with b-alanine amide, followed by reduction of nitro
group led to aniline 3. Formation of benzimidazole 4 from amide
3 was accomplished with cyanogen bromide without incident.
With benzimidazole 4 in hand, precursors 5, 6, and 7 were pre-
H2N
NO2
F
N
NH2
N
NO2
F
b
a
O
O
O
N
H
NH2
2
3
4
c
Br
O
N
S
H
N
d
N
N
N
N
NH2
O
O
N
5
NH2
NH2
O
O
e
f
O
O
O
N
S
O
N
S
H
H
N
H
N
N
N
O
O
N
N
NH2
NH2
O
O
6
7
Scheme 1. General synthetic route for common precursors. Reagents and conditions: (a) i—benzoyl chloride, K2CO3, EtOAc, rt, 98%; ii—NaH, MeI, THF, 0 °C to rt, 80%; (b) i—b-
alanine amide, i-Pr2NEt, DMF, 80 °C, 75%; ii—10% Pd/C, H2, EtOH, 100%; (c) cyanogen bromide, EtOH, rt, 88%; (d) 5-bromothiophene-2-carboxylic acid, EDCI, HOBt, i-Pr2NEt,
DMF, rt, 93%; (e) 5-acetylthiophene-2-carboxylic acid, EDCI, HOBt, i-Pr2NEt, DMF, rt, 90%; (f) 5-formyl-2-thiophenecarboxylic acid, EDCI, HOBt, i-Pr2NEt, DMF, rt, 83%.

6220
H. Y. Lo et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6218–6221
N
O
H
S
N
a
N
N
N
+
5
5
6
N
O
NH2
O
8a
O
N
S
b
H
N
N
N
O
NH2
O
8b
N
N
c
O
S
H
N
N
+
(Ph)3P
N
O
N
NH2
O
8c
Br
O
S
H
N
N
Br
(Ph)3P
d
N
+
O
7
N
Br
NH2
O
9a
N
e
O
N
S
H
N
N
O
N
NH2
O
9b
Scheme 2. Synthetic routes for representative targets. Reagents and conditions: (a) Pd2(dba)3, (tBu)3P, N-methyl-dicyclohexylamine, Et4NCl, DMF, 100 °C, 12 h, 30%; (b)
Tributylvinyl tin, Pd(PPh3)4, 100 °C microwave, 1 h, 54%; (c) THF, rt, 12 h, 50%; (d) nBuLi, THF, 0 °C, 60%; (e) pyridine-3-boronic acid, Cs2CO3, 120 °C, 12 h, 54%.
compounds showed inferior molecular potency compared with
parent phenyl analog 8f, and there was no significant difference
between compounds with electron-donating groups and elec-
tron-withdrawing groups. We hypothesized that the loss of activ-
ity was mainly due to steric hindrance imposed by the
substitutions. The next modification evaluated was the effect of
introducing hydrogen bond donors or acceptors separately at each
position on the phenyl ring. A hydrogen bonding acceptor such as
pyridine (8g, 8a, and 8d) offered no advantage over phenyl analog
8f, and the 4-pyridine analog 8d showed a 10-fold decrease in
molecular potency compared with 8g and 8a. A hydrogen bonding
donor such as amine (8n, 8p, and 8q) showed a similar profile as
the pyridine analogs. There is no obvious impact on the binding
affinity with either hydrogen bonding donors or acceptors in that
region of the molecule.
most potent compound in the series in terms of cell activity;
though with only moderate selectivity over IRK.
The idea of a tri-substituted olefinic linker was based on the
hypothesis that the presence of an extra methyl group could
provide additional steric hindrance between the olefinic linker
and the thiophene ring, which in turn would generate a different
turning angle toward the KSP. Representative compounds of this
type were synthesized and tested. The results are summarized in
Table 2. To our disappointment, the ‘tri-substituted’ analogs
were generally weaker in terms of binding affinity toward ITK
when compared with to their ‘di-substituted’ partners. The rea-
son behind the loss of binding affinity might be due to the steric
hindrance between the newly introduced methyl group and the
gatekeeper Phe435
.
Some of the of potent compounds in the ‘di-substituted’ series
were tested in a functional assay 11, in which IL-2 production
was measured using human CD4+ T-cells stimulated with anti-
CD3 and anti-CD28 mAbs. These compounds (8v and 8x) had IC50
values in the low-micromolar range. Compared with the lead com-
Different heterocycles bearing hydrogen bond donors were also
introduced to fine tune the affinity of the inhibitors. Compounds
such as aminopyridine 8w and aminopyrimidine 8x offered no
improvement over the parent aminophenyl analogs 8f. However,
introduction of smaller heterocycles such as imidazole and pyra-
zole provided an advantage. Significant improvement in cell activ-
pound 1, which had no inhibitory activity (IC50 > 5 lM) when
tested, the improvement of molecular activity by the series did
ity was achieved with pyrazole 8v (0.28 lM), which was also the
translate to the cellular functional assay.

H. Y. Lo et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6218–6221
6221
Table 1
Results of di-substituted inhibitors’ activities with different R2 (refer to general formula in Fig. 2)
a
a
a
Compound
R1
R2
ITK IC50
(lM)
IRK IC50
(lM)
Ca+ flux IC50
(lM)
IL-2 inhibition IC50
>5
(lM)
1
—
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
—
H
0.025
0.007
0.005
0.010
0.011
0.006
0.005
0.065
0.067
0.013
0.058
0.270
0.030
0.130
0.006
0.009
0.006
0.042
0.009
0.008
0.012
0.009
0.007
0.005
0.145
0.48
3.25
>5
0.97
4.00
>5
>5
>5
>5
>5
2.4
8b
8c
8e
8f
8g
8a
8d
8h
8i
0.38
0.39
0.54
0.62
0.76
0.69
0.99
>5
1.90
>5
>5
>5
>5
0.62
1.40
0.57
>5
0.74
0.81
2.2
0.28
2
0.41
CN
COOMe
Ph
2-Pyridine
3-Pyridine
4-Pyridine
2-MeO-Ph
3-MeO-Ph
4-MeO-Ph
2-CF3-Ph
8j
8k
8l
>5
>5
>5
3-CF3-Ph
4-CF3-Ph
8m
8n
8p
8q
8r
8s
8t
8u
8v
8w
8x
2-NH2-Ph
3-NH2-Ph
4-NH2-Ph
3-NO2-Ph
4-OH-Ph
2-Pyrazine
1-Imidazole
4-(1H)-Pyrazole
6-Pyridine-2-ylamine
5-Pyrimidin-2-ylamine
1.95
4.90
3.30
4.30
2.20
>5
>5
0.33
3.8
3
1.5
1.6
a
Values are means of three experiments.
Table 2
5. Fowell, D. J.; Shinkai, K.; Liao, X. C.; Beebe, A. M.; Coffman, R. L.; Littman, D. R.;
Locksley, R. M. Immunity 1999, 11, 399.
6. Kanner, S. B.; Perez-Villar, J. J. Trends Immunol. 2003, 24, 249.
Results of tri-substituted inhibitors’ activities with different R2 (refer to general
formula in Fig. 2)
7. (a) Snow, R. J.; Abeywardane, A.; Cywin, C. L.; Lord, J.; Kashem, M. A.; Khine,
H. H.; Kowalski, J. A.; Pullen, S. S.; Roth, G. P.; Sarko, C. R.; Wilson, N. S.;
Winters, M.; Wolak, J. P. Bioorg. Med. Chem. Lett. 2007, 17, 3660; (b) Winters,
M. P.; Robinson, D. J.; Khine, H. H.; Pullen, S. S.; Woska, J. R., Jr.; Raymond, E.
L.; Sellati, R.; Cywin, C. L.; Snow, R. J.; Kashem, M. A.; Wolak, J. P.; King, J.;
Kaplita, P. V.; Liu, L. H.; Farrell, T. M.; DesJarlais, R.; Roth, G. P.; Takahashi, H.;
Moriaty, K. J. Bioorg. Med. Chem. Lett. 2008, 18, 5541; (c) Moriarty, K. J.;
Takahashi, H.; Pullen, S. S.; Khine, H. H.; Sallati, R. H.; Raymond, E. L.; Woska,
J. R., Jr.; Jeanfavre, D. D.; Roth, G. P.; Winters, M. P.; Qiao, L.; Ryan, D.;
DesJarlais, R.; Robinson, D.; Wilson, M.; Bobko, M.; Cook, B. N.; Lo, H. Y.;
Nemoto, P. A.; Kashem, M. A., et al Bioorg. Med. Chem. Lett. 2008, 18, 5545;
(d) Moriarty, K. J.; Winters, M.; Qiao, L.; Ryan, D.; DesJarlis, R.; Robinson, D.;
Cook, B. N.; Kashem, M. A.; Kaplita, P. V.; Liu, L. H.; Farrell, T. M.; Khine, H.
H.; King, J.; Pullen, S. S.; Roth, G. P.; Magolda, R.; Takahashi, H. Bioorg. Med.
Chem. Lett. 2008, 18, 5537.
R1
R2
ITK IC50
M)
IRK IC50
M)
Ca+ flux IC50
(lM)
a
a
a
Compound
(l
(l
9c
9d
9e
9f
9b
9h
Me
Me
Me
Me
Me
Me
H
CN
COOMe
Ph
3-Pyridine
4-Pyridine
0.037
0.063
0.039
0.075
0.039
0.059
>5
0.29
0.29
1.10
3
1.6
0.83
4.50
3.60
>5
>5
>5
a
Values are means of three experiments.
Concerning the selectivity over other Tec kinase family, com-
pound 8x showed high activity on BTK (5 nM) and moderate selec-
tivity over TXK (700 nM). However the implication of the
selectivity over other Tec kinase on the indication (inflammation)
is unclear at this point.
In conclusion, a series of trans-stilbene-like 2-aminobenzimid-
azole inhibitors were designed and synthesized. The intrinsic
potencies, cellular activities, and selectivity of the compounds
were improved when compared with the lead compound. Further-
more, some of the top compounds showed activity in the IL-2 func-
tional assay. It is predicted that the improvement in activity is due
to partial occupancy of the KSP. Further studies in this area are
underway.
8. White, A.; Abeywardane, A.; Cook, B. N.; Fuschetto, N.; Gautschi, E.; John, A.;
Kroe, R. R.; Kronkaitis, A.; Li, Xiang; Pullen, S. S.; Roma, T.; Moriarty, K. J.; Roth,
G. P.; Snow, R. J.; Studts, J. M.; Takahashi, H.; Farmer, B.T. II.; J. Biol. Chem,
submitted for publication.
9. Representative procedure for the Heck type coupling reactions: To a solution of
bromothiophene 5 (50 mg, 0.095 mmol) in DMF (5 mL) were added 2-vinyl
pyridine (0.02 mL, 0.19 mmol), Pd2(dba)3 (9 mg, 0.01 mmol), tri-tert-
butylphosphine (21 mg, 0.095 mmol), N-methyl-dicyclohexylamine (0.02 mL,
0.095 mmol) and Et4NCl (16 mg, 0.095 mmol). The solution was heated to
100 °C in a sealed tube for 12 h. The reddish-brown solution was cooled down
and 3-mercaptopropyl-functionalized silica gel (100 mg) was added. The
mixture was stirred for 10 min and was filtered. The filtrate was washed
with water and extracted with EtOAc. The organic layer was concentrated and
the residue was purified by column chromatography with 5% MeOH in CH2Cl2
as the eluent to afford vinyl pyridine 8 g (39 mg, 75%) as colorless oil.
10. (a) Kashem, M. A.; Nelson, R. M.; Yingling, J. D.; Pullen, S. S.; Prokopowicz, A. S.,
III; Jones, J. W.; Wolak, J. P.; Rogers, G. R.; Morelock, M. M.; Snow, R. J.; Homon,
C. A.; Jakes, S. J. Biomol. Screening 2007, 12, 70; (b) Takata, R.; Kurosaki, T. J. Exp.
Med. 1996, 184, 31.
References and notes
11. CD4+ T-cells are isolated from whole blood by positive selection. Purified T-
cells are activated through the TCR and CD28 via anti-CD3 and anti-CD28
mAbs. Compounds are diluted in 10% DMSO to a final concentration of .25%
1. Smith, C. I.; Islam, T. C.; Mattsson, P. T.; Mohamed, A. J.; Nore, B. F.; Vihinen, M.
Bioessays 2001, 23, 436.
2. Forssell, J.; Sideras, P.; Eriksson, C.; Malm-Erjefält, M.; Rydell-Törmänen, K.;
Ericsson, P.; Erjefält, J. S. Am. J. Respir. Cell. Mol. Biol 2005, 32, 511.
3. Schaeffer, E. M.; Yap, G. S.; Lewis, C. M.; Czar, M. J.; McVicar, D. W.; Cheever, A.
W.; Sher, A.; Schwartzberg, P. L. Nat. Immune 2001, 2, 1183.
4. Schaeffer, E. M.; Debnath, J.; Yap, G.; McVicar, D.; Liao, X. C.; Littman, D. R.;
Sher, A.; Varmus, H. E.; Lenardo, M. J.; Schwartzberg, P. L. Science 1999, 284,
638.
DMSO at every dose. Fifty thousand cells are added in 100
lL of media/well
followed by 100 L of Compound or DMSO alone. Cells incubate overnight at
l
37 °C and then the supernatants are analyzed for IL-2 with the R&D Systems IL-
2 ELISA kit (Cat. No. D2050) following a 1:10 dilution. IC50s are calculated by
SAS.