2-Aminobenzimidazoles as potent ITK antagonists: de novo design of a pyrrole system targeting additional hydrogen bonding interaction

Article abstract of DOI:10.1016/j.tetlet.2008.10.057

Based on information from molecular modeling, a series of 2-aminobenzimidazoles with pyrrole moieties were designed and synthesized as ITK antagonists. Results showed that a significant improvement of intrinsic and cell-based potency was achieved. X-ray c

Full text of DOI:10.1016/j.tetlet.2008.10.057

Tetrahedron Letters 49 (2008) 7337–7340
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
2-Aminobenzimidazoles as potent ITK antagonists: de novo design
of a pyrrole system targeting additional hydrogen bonding interaction
a
a
a
a
b
Ho Yin Lo a, , Jörg Bentzien , Andre White , Chuk C. Man , Roman W. Fleck , Steven S. Pullen ,
Hnin Hnin Khine b, Josephine King b, Joseph R. Woska Jr. b, John P. Wolak c, Mohammed A. Kashem c,
Gregory P. Roth d, Hidenori Takahashi a
*
a Boehringer Ingelheim Pharmaceuticals Inc., Medicinal Chemistry, 900 Ridgebury Rd./PO Box 368, Ridgefield, CT 06877, USA
b Boehringer Ingelheim Pharmaceuticals Inc., Immunology and Inflammation, 900 Ridgebury Rd./PO Box 368, Ridgefield, CT 06877, USA
c Boehringer Ingelheim Pharmaceuticals Inc., Biomolecular screening, 900 Ridgebury Rd./PO Box 368, Ridgefield, CT 06877, USA
d Burnham Institute for Medical Research at Lake Nona, Orlando, FL 32819, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on information from molecular modeling, a series of 2-aminobenzimidazoles with pyrrole moieties
were designed and synthesized as ITK antagonists. Results showed that a significant improvement of
intrinsic and cell-based potency was achieved. X-ray crystallographic analysis of an inhibitor complex
with ITK confirmed the prediction from the de novo design that the pyrrole moiety of the inhibitor would
form an additional hydrogen bonding interaction with Glu436 in the catalytic domain, and hence
improve overall binding affinity of the inhibitor.
Received 27 September 2008
Revised 10 October 2008
Accepted 13 October 2008
Available online 17 October 2008
Ó 2008 Elsevier Ltd. All rights reserved.
During the quest for novel anti-inflammatory agents and mech-
anisms which could be used on the treatment of inflammatory dis-
ease, such as allergic asthma, rheumatoid arthritis, and multiple
sclerosis, we pursued Interleukin-2 inducible T-cell kinase (ITK)1
as a promising target.
Val419
Glu436
S
Lys391
Met438
Phe435
H
N
O
N
KSP
Met410
Glu406
Asp500
N
O
In our recent search for novel small molecule inhibitors for ITK,2
we discovered a series of potent, selective ITK antagonists featur-
ing 2-aminobenzimidazole scaffold as a key pharmacophore. Lead
compound such as 1 (Fig. 1) exhibited a good activity profile:
IC50’s were determined to be 140 nM in an ITK DELFIA assay3a for
the intrinsic binding affinity and 780 nM in an Insulin Receptor Ki-
nase (IRK) DELFIA assay for the selectivity. However, compound 1
N
Leu489
O
N
1
Figure 1. Illustration of the binding mode of 1 in ITK.
was not active (IC50 > 3 l
M) in a DT40/ITK cell assay,3b which
might be due to the relatively low intrinsic potency of the
Leu,489 and Val419
) , ,
and hydrophilic residues (Lys391 Glu406
Met,410 and Asp500). In order to access the KSP from the thiophene
side of the inhibitor effectively, the design had to take into
consideration structural modifications that would avoid creating
steric interactions with Phe435. Molecular modeling suggested that
a compound such as 2 which features a pyrrole moiety off the
2-amide position could potentially act as a hydrogen bonding
donor through interactions with the carbonyl group of Glu436 as
shown in Figure 2. Furthermore, an additional hydrogen bond
could also be achieved through interactions with Lys391 and the
carbonyl group off the 3-position of the pyrrole ring. Thirdly, the
‘pyrrole-carbonyl-aromatic’ substitution pattern might provide
the correct angle necessary to avoid Phe435 and occupy the KSP.
This would provide an opportunity for additional interactions with
various amino acids (Ser499, Asp,500 and Glu406) within the pocket.
compound.
Based on an X-ray co-crystal structure of a structurally related
ITK inhibitor bound to the human ITK kinase domain,4 we realized
that beside the double ‘backbone’ hydrogen bonding interactions
between the amide carbonyl group of Met438 and the N–H of the
benzimidazole core, Glu436 might be able to provide an additional
hydrogen bonding interaction by its amide moiety.
Next to Glu436, there was an unoccupied kinase specificity
pocket (KSP). The KSP is about 7.3 Å deep (measuring from the
‘gate-keeper’ Phe435 to Met410) and contains hydrophobic (Leu418
,
* Corresponding author. Tel.: +1 203 798 4923.
E-mail address: ho-yin.lo@boehringer-ingelheim.com (H. Y. Lo).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.10.057

7338
H. Y. Lo et al. / Tetrahedron Letters 49 (2008) 7337–7340
H
O
H
N
H
N
O
O
O
O
a
b
N
O
O
H
HO
9
8
O
H
O
N
N
H
N
N
c
O
N
N
O
10
Scheme 2. Synthetic route for the preparation of 2-substituted pyrrole type of
compound. Reagents and conditions: (a) POCl3, DMF, DCE, 70%; (b) (i) MeMgBr, THF,
91%; (ii) Dess–Martin periodinane, 96%; (iii) 1 N NaOH, MeOH, 74%; (c) 5, EDCI,
HOBt, iPr2NEt, DMF, 60%.
ate with benzoyl chloride, followed by acid hydrolysis of the corre-
sponding ester. With both 2-aminobenzimidazole 5 and pyrrole 6
in hands, the amide coupling reaction gave final target 7 in modest
yield.
For 2-substituted pyrrole analogs, such as 10 (Scheme 2), the
preparation of the pyrrole moiety started from
a-formylation of
Figure 2. Molecular modeling and diagram show the binding mode of 2 in ITK
(orange: ITK; grey: 2).
ethyl pyrrole-2-carboxylate to give aldehyde 8. The addition of a
methyl group with the use of Grignard reagent followed by oxida-
tion and acid hydrolysis gave 3-acetylpyrrole-carboxylic acid 9 in
excellent yield. Simple amide coupling reaction between 9 and 2-
aminobenzimidazole 5 provided the target 2-substituted acetyl
pyrrole 10.
The analogs were tested in an ITK DELFIA assay to determine
the intrinsic binding affinities; an IRK DELFIA assay to
determine the selectivity; and a DT40/ITK Ca+ flux cell assay to
determine the cell activities. Selected compounds from the series
were also tested in a functional assay6 measuring IL-2 inhibition
using human CD4+ T-cells stimulated with anti-CD-3 and anti-
CD-28 mAbs. The results were shown in Table 1.
Successful design of this pyrrole type of compounds was antici-
pated to improve binding affinity for ITK and selectivity over IRK.
The synthetic routes toward the 2-aminobenzimidazole-pyrrole
type of compounds were shown in Schemes 1 and 2. For the syn-
thesis of 3-substituted pyrrole analogs, such as 7 (Scheme 1), com-
mercially available 4-fluoro-3-nitroaniline was used as the starting
material. Acylation of 4-fluoro-3-nitroaniline with benzoyl chlo-
ride followed by methylation of the resulting amide gave amide
3 in good yield. SNAr displacement of the fluorine in 3 with 1-(3-
aminopropyl)-2-pyrrolidinone followed by transition metal
reduction of the nitro group provided diamine 4. The formation
of the 2-aminobenzimidazole core 5 was carried out with cyano-
gen bromide in good yield. Pyrrole moiety 6 was prepared by firstly
performing a Friedel–Craft acylation5 of ethyl pyrrole-2-carboxyl-
As predicted from the modeling study, the introduction of a pyr-
role moiety proved to be beneficial in terms of binding affinity.
3-Nitropyrrole 11 and bromopyrrole 13 exhibited significant
improvements in both molecular and cellular activities compared
H2N
NO2
F
N
NH2
N
NO2
F
b
a
O
O
O
N
H
N
3
4
5
c
H
N
O
N
d
H
N
N
N
N
N
NH2
O
O
O
H
N
N
O
HO
N
N
O
6
O
O
e
7
H
N
O
O
Scheme 1. Synthetic route for 3-substituted pyrrole analogs. Reagents and conditions: (a) (i) benzoyl chloride, K2CO3, EtOAc, rt, 98%; (ii) NaH, MeI, THF, 0 °C to rt, 80%; (b) (i)
1-(3-aminopropyl)-2-pyrrolidinone, iPr2NEt, DMF, 80 °C, 70%; (ii) Pd/C, H2, EtOH, 100%; (c) cyanogen bromide, EtOH, rt, 80%; (d) 6, EDCI, HOBt, iPr2NEt, DMF, rt, 60%; (e) (i)
benzoyl chloride, AlCl3, CH2Cl2, 0 °C, 76%; (ii) NaOH, MeOH, 100%.

H. Y. Lo et al. / Tetrahedron Letters 49 (2008) 7337–7340
7339
Table 1
Results of selected 2-aminobenzimidazole-pyrrole analogs
R1
R2
R3
O
N
N
H
N
N
O
N
N
O
R1
R2
R3
ITK IC50
(
lM)
IRK IC50
(lM)
Ca+flux IC50
(lM)
IL-2 inhibition IC50 (lM)
a
a
a
a
Compounds
1
—
H
CH3
H
CH3
H
H
H
H
H
—
H
H
H
H
H
Acetyl
H
H
H
—
NO2
NO2
Br
H
Acetyl
H
Benzoyl
2-Fluorobenzoyl
2-Methylbenzoyl
0.140
0.024
3.9
0.018
>5
0.026
0.077
0.021
0.025
0.1
0.78
0.89
>5
0.28
>5
>5
>5
4.1
4
>5
>3
0.77
>5
0.89
>5
1.6
>5
0.19
0.19
>5
11
12
13
14
15
10
7
0.97
1.5
16
17
a
Values are means of three experiments.
to the lead compound 1. Unfortunately, direct comparison with 1
could not be accomplished due to the chemical instability of the
corresponding unsubstituted pyrrole analog. In general, electron-
withdrawing group had to be incorporated into the pyrrole ring
in order to increase the overall chemical stability of the inhibitor.
To test our hypothesis that the pyrrole N–H could indeed act as
a donor for the hydrogen bonding, N-methyl pyrrole analogs such
as 12 and 14 were prepared as a counter proof. As the modeling
predicted, both compounds showed a significant loss in binding
affinity compared to 11 and 1, respectively. Additionally, the
methyl group might induce an unfavorable steric hindrance, which
would be anticipated to lead to a loss of activity.
Next, the opportunity for additional hydrogen bonding by the
carbonyl substitution on either 2- or 3-position of the pyrrole ring
was investigated. Introduction of an acetyl group in the 2- and
3-position of the pyrrole ring gave acetylpyrrole compounds 10
and 15, respectively. Both compounds showed comparable mole-
cular potency with 3-substitution leading to slightly more potent
inhibitors (26 nM vs 77 nM). However, there was no significant
improvement of potency by the introduction of this additional
carbonyl group compared to the bromo-substituted pyrrole 13.
Incorporation of benzoyl group in the 3-position of the pyrrole ring
(7) retained molecular potency and a 4-fold improvement in the
cell activity compared to bromopyrrole 13.
To investigate the possibility of occupying the KSP by the
phenyl group of compound 7 (IC50 = 21 nM), substitution patterns
on the benzene ring were explored. Selected from all of the analogs
that were synthesized for this purpose, the o-substituted phenyl
analogs provided the most interesting results. First of all, an elec-
tron-withdrawing group such as fluoro in 16 did not alter the bind-
ing affinity of the inhibitor. However, with the introduction of
steric hindrance, such as methyl group in 17, the binding affinity
of the inhibitor diminished significantly. This result pointed to
the direction that the benzene ring in 7 was positioned in a very
confined environment that even a small change in the torsional an-
gle between the carbonyl and the benzene ring would affect the
binding affinity of the inhibitor. With the promising result of ben-
Figure 3. Crystal structure of 7 in ITK: (green: 7; grey: ITK; only in the key amino
acid moieties in the KSP were shown).
imidazole core resided near Met438 and formed two backbone
hydrogen bonds with the kinase as predicted. Encouragingly, the
crystal structure showed that the carbonyl group in Glu436 was
within hydrogen bonding distance (3.1 Å) to the pyrrole nitrogen,
indicating that an additional hydrogen bond had indeed been
established. This accounts for the improvements in the potencies
and IRK selectivity over lead compound 1. However, to our disap-
pointment, the benzoyl group in the crystal structure did not occu-
py the KSP (region around Leu418, Met410, Phe,501 and Asp500 in the
figure). The benzene ring rotated away from the KSP. Also, LYS391
was disordered and could not possibly form extra hydrogen bond
with the carbonyl group on the inhibitor. This result explained
the lack of further improvement in binding affinity of the inhibitors
upon incorporation of carbonyl substitutions.
zoyl pyrrole 7 in a functional assay (IC50 = 0.97
lM), compared to
the lack of activity (>5 M) of lead compound 1, along with an
improved IRK selectivity, the occupation of the KSP seemed to be
a very possible scenario.
To verify the binding mode of benzoyl pyrrole 7, an X-ray co-
crystal structure was generated. As shown in Figure 3, the benz-
l
In conclusion, a series of 2-benzimidazole-pyrrole type ITK
inhibitors were developed by de novo design aiming for additional
hydrogen bonding interactions. Experimental results demon-
strated a significant improvement of binding affinity (7-fold in-
crease compared to lead compound 1. Finally, an X-ray co-crystal

7340
H. Y. Lo et al. / Tetrahedron Letters 49 (2008) 7337–7340
J. R.; 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. doi:10.1016/j.bmcl.2008.09.015, in
press; (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. doi:10.1016/j.bmcl.2008.09.017, in press.
structure of one of the most potent compounds in the series con-
firmed the predicted binding mode, revealing that an additional
hydrogen bonding interaction was indeed established with the
introduction of pyrrole moiety. However, the KSP was not occupied
by the design. Further refinement of the modeling and synthetic
efforts in the program will allow better design for the occupation
of the KSP and hence will provide superior ITK inhibitors in the
future.
3. (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, 70–83; (b) Takata, R.; Kurosaki, T. J. Exp.
Med. 1996, 184, 31.
4. 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.
Acknowledgment
The authors would like to thank Dr. Stéphane De Lombaert for
helpful advice on the preparation of this Letter.
5. Representative procedure for Friedel–Craft acylation: To a solution of ethyl
pyrrole-2-carboxylate (200 mg, 1.4 mmol) in CH2Cl2 (10 mL), were added
benzoyl chloride (0.3 mL, 2.8 mmol) and AlCl3 (375 mg, 2.8 mmol) at 0 °C
under nitrogen atmosphere. The solution was warmed to room temperature for
12 h. The solution was cooled back to 4 °C and saturated NaHCO3 solution
(10 mL) was added. The solution was extracted with CH2Cl2, and the organic
layer was collected and dried with MgSO4. The solution was filtered
and concentrated. The residue was then dissolved in MeOH/H2O (10 mL/2 mL).
1 N NaOH (2 mL) was added, and the solution was stirred at room temperature
for 12 h. The pH of the solution was adjusted to 6 by the addition of 0.1 N
HCl. The solution was then concentrated, and the residue was purified by
column chromatography to afford benzoyl pyrrole 6 (230 mg, 76%) as a white
solid.
References and notes
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M. Bioessays 2001, 23, 436–446; (b) Forssel, J.; Sideras, P.; Eriksson, C.; Malm-
Erjefält, M.; Rydell-Törmänen, K.; Ericsson, P.; Erjefält, J. S. Am. J. Respir. Cell. Mol.
Biol. 2005, 32, 511–520; (c) Schaeffer, E. M.; Yap, G. S.; Lewis, C. M.; Czar, M. J.;
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2. (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, 3660–3665; (b) Winters, M. P.;
Robinson, D. J.; Khine, H. H.; Pullen, S. S.; Woska, Jr., J. R.; Raymond, E. L.; Sellati,
R.; Cywin, C. L.; Snow, R. J.; Kashem, M. A.; Wolak, J. P.; King, J.; Kaplita, P. V.; Liu,
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6. 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% DMSO at
every dose. 50,000 cells are added in 100 lL of media/well followed by 100 lL of
compound or DMSO alone. Cells are incubated overnight at 37 °C, and then the
supernatants are analyzed for IL-2 with the R&D Systems IL-2 ELISA kit
(Cat.#D2050) following a 1:10 dilution. IC50’s are calculated by SAS.