5-Aminomethyl-1H-benzimidazoles as orally active inhibitors of inducible T-cell kinase (Itk)

Article abstract of DOI:10.1016/j.bmcl.2008.09.016

A series of novel 5-aminomethyl-1H-benzimidazole based inhibitors of Itk were prepared. Structure-activity relationships, selectivity and cell activity are reported for this series. Compound 2, a potent and selective antagonist of Itk, inhibited anti-CD3 antibody induced IL-2 production in vivo in mice.

Full text of DOI:10.1016/j.bmcl.2008.09.016

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5541–5544
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
5-Aminomethyl-1H-benzimidazoles as orally active inhibitors of inducible
T-cell kinase (Itk)
Michael P. Winters b, Darius J. Robinson b, Hnin Hnin Khine a, Steven S. Pullen a, Joseph R. Woska Jr. a,
Ernest L. Raymond a, Rosemarie Sellati a, Charles L. Cywin a, Roger J. Snow a, Mohammed A. Kashem a,
John P. Wolak a, Josephine King a, Paul V. Kaplita a, Lisa H. Liu a, Thomas M. Farrell a, Renee DesJarlais b,
b
Gregory P. Roth c, Hidenori Takahashi a, , Kevin J. Moriarty
*
a Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, PO Box 368, Ridgefield, CT 06877, USA
b Johnson & Johnson Pharmaceutical Research and Development, 665 Stockton Drive, Exton, PA 19341, USA
c 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:
Received 15 July 2008
Revised 29 August 2008
Accepted 3 September 2008
Available online 7 September 2008
A series of novel 5-aminomethyl-1H-benzimidazole based inhibitors of Itk were prepared. Structure–
activity relationships, selectivity and cell activity are reported for this series. Compound 2, a potent
and selective antagonist of Itk, inhibited anti-CD3 antibody induced IL-2 production in vivo in mice.
Ó 2008 Elsevier Ltd. All rights reserved.
This paper is dedicated to the memory of Dr.
Ronald L. Magolda.
Keywords:
SAR
Itk inhibitor
Benzimidazole
In vivo mouse model
Antigen recognition by T-cells is the initiating stimulus for T-
cell activation. T-cell activation can lead to proliferation, secretion
of cytokines, and initiation of regulatory and cytolytic effector
functions. Engagement of the T-cell antigen receptor (TCR) results
in the rapid recruitment and activation of three classes of non-
receptor tyrosine kinases, the Src family (Lck and Lyn), the Syk
family (Zap-70 and Syk) and the Tec family (Itk, Txk, and Tec).1–3
Inhibiting any of these tyrosine kinases would potentially impede
T-cell activation following antigen presentation by blocking the
signaling cascade.4
peripheral blood T-cells from patients with moderate or severe
atopic dermatitis than in controls or patients with mild atopic der-
matitis.9,10 These studies suggest that a selective Itk inhibitor may
be useful as an anti-inflammatory or immunosuppressive agent for
the treatment of dysregulated pathways mediated by Th2 cells.
Thiazole inhibitors of Itk were disclosed recently,11,12 as were
the hit to lead studies that eventually led to the benzimidazole ser-
ies of compound 1 which we previously identified.13 Compound 1
had an IC50 value of 12 nM in the Itk DELFIA-based enzyme assay,
IC50 of 0. 3343
ited good selectivity against a panel of protein kinases.14 Com-
pound 1 was not toxic (TC50 > 10 M; Jurkat) and showed good
l
M in the DT40 Ca2+ flux cellular assay and exhib-
Interleukin-2 (IL-2)-inducible T-cell kinase (Itk) is a key mem-
ber of the Tec kinase family and a number of factors point to the
importance of this kinase in immune disease. Deletion of Itk in
mice results in reduced TCR-induced proliferation and secretion
l
stability against both human and rat liver microsomes. In the
course of exploring the functionality necessary for Itk activity,
the 5-amide was converted to a methylamino moiety 2 (Fig. 1).
Although less potent than 1 in the DELFIA assay (46 nM), it had
of the cytokines IL-2, IL-4, IL-5, IL-10, and IFN-c 5–7
In a mouse
.
model of allergic asthma, lung inflammation, eosinophil infiltra-
tion, and mucous production are drastically reduced in response
to challenge with the allergen OVA in the absence of Itk.8 Further-
more, the Itk gene is reported to be more highly expressed in
similar potency (0.41 lM) in the DT40 cellular assay. Compound
2 became the starting point for a new lead series, and this report
details the SAR of this novel series.15
The general synthetic route to this novel benzimidazole series is
illustrated by the preparation of 2 (Scheme 1). Accordingly, 4-flu-
oro-3-nitrobenzylalcohol 3 was converted to 4 by treatment with
* Corresponding author. Tel.: +1 203 798 5049; fax: +1 203 798 5297.
E-mail address: hidenori.takahashi@boehringer-ingelheim.com (H. Takahashi).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.016

5542
M. P. Winters et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5541–5544
Table 1
O
O
Modifications of the N-methylcyclohexyl group
N
CN
CN
N
N
N
N
N
NH
NH
O
O
R1
CN
N
N
N
R
NH
NH2
O
MeO
1
2
MeO
Itk IC50 = 0.046 μM
DT40 IC50 = 0.41 μM
Itk IC50 = 0.012 μM
DT40 IC50 = 0.34 μM
Compound
R1
R
Itk IC50 (lM)
2
7
8
Cyclohexyl
H
Cyclohexyl
Phenyl
Cyclopentylmethyl
2-Methoxyethyl
CH3
CH3
H
H
CH3
H
0.046
0.17
Figure 1. Itk inhibitory activity for compounds 1 and 2.
0.045
0.048
0.034
0.081
0.11
9
10
11
12
13
NO2
a, b
TIPSO
c, d
NO2
F
HO
4-(N-Methylpiperidine)methyl
H
R1 and R = morpholine
0.069
N
OMe
H
3
4
in place of cyclohexyl. Changing R1 to less hydrophobic groups, 2-
methoxyethyl (11) and 4-(N-methylpiperidine)methyl (12), re-
sulted in a 2- and 3-fold loss in potency, respectively. Replacement
of the N-methylcyclohexyl group with a morpholine (13) resulted
in a slight loss in potency compared to 2. This SAR data is consis-
tent with the binding mode of compounds similar to 1 that were
crystallized with Itk.16 In the crystal structure, the equivalent
hydrophobic group of 1 (the left side phenyl) interacts with mostly
hydrophobic residues on the surface of the protein.
O
N
TIPSO
CN
N
N
NH2
HO
NH
N
e, f
MeO
MeO
5
6
O
CN
N
N
N
NH
g, h
Having established the requirement for the N-methylcyclohexyl
position of the benzimidazole, the SAR around the R2 substituent
was explored (Table 2). In another series (1), this position was
found to have a profound influence on the selectivity against insu-
lin receptor kinase (IRK). Substitution of p-cyano with p-CF3 (14)
resulted in a complete loss of kinase activity while replacement
with p-chloro (15) had no effect demonstrating that there is a size
limitation at this position. In addition, both 2 and 15 had excellent
selectivity against IRK. Replacement of the p-cyano with the m-cy-
ano (16) resulted in a 2-fold increase in potency while the m-bro-
mo (17) gave a 26-fold loss in potency compared to 2. It is
interesting to note that the IRK selectivity decreased substantially
with meta-substitution. Heterocycles 5-isoxazole (18) and 4-pyri-
dyl (19) were both well tolerated and had good selectivity versus
IRK, and 3-pyridyl substitution (20) resulted in a 6 nM compound
with excellent selectivity against IRK.
MeO
2
Scheme 1. General synthetic scheme for the synthesis of 5-aminomethylbenzim-
idazoles. Reagents and conditions: (a) TIPSOTf, 2,6-lutidine, CH2Cl2, rt, 24 h;
(b) 3-methoxypropan-1-amine, DIEA, CH3CN, rt to 50 °C, 24 h, 61% over two steps;
(c) NH4HCO2, 10% Pd/C, ethanol, rt, 3 h, 91%; (d) BrCN, ethanol, rt, 24 h, 88%;
(e) 4-cyanobenzoyl chloride, DIEA, DMAP, CH2Cl2, rt, 24 h, 77%; (f) 0.01 N HCl,
dioxane, 95 °C, 3 h, 92%; (g) MnO2, acetone, rt, 24 h, 84%; (h) N-methylcyclohex-
anamine, NaBH(OAc)3, (EtO)3CH, CH2Cl2, rt, 24 h, 42%.
TIPSOTf and 2,6-lutidine followed by displacement of the fluorine
with 3-methoxypropylamine. The nitro group was reduced with
ammonium formate and 10% Pd/C, and the resulting diamine cy-
clized to the 2-aminobenzimidazole 5 with cyanogen bromide.
Acylation of the 2-amino with 4-cyanobenzoylchloride followed
by TIPS deprotection with dilute HCl at 95 °C gave 6. The resulting
benzyl alcohol 6 was oxidized to the aldehyde with MnO2, and
lastly reductive amination with cyclohexylmethylamine in the
presence of sodium triacetoxyborohydride provided the desired
compound 2.
All compounds were screened for inhibitory activity against Itk
in a DELFIA assay. For the purposes of determining selectivity, test-
ing of selected compounds was conducted against related kinases
Tec, Bmx, Txk, and Btk as well as IRK (insulin receptor kinase),
Syk and Lyn. Selected compounds were tested for cellular activity
by measuring inhibition of Ca2+ flux in DT40 cells13 and the inhibi-
tion of T-cell receptor induced IL-2 production in human T-cells.
In an effort to identify pharmacophores that would give im-
proved potency against Itk we focused our initial explorations on
the N-methylcyclohexyl group (Table 1). Removal of the cyclohexyl
group (7) resulted in a 4-fold loss in potency while removal of the
methyl (8) showed no effect, as did replacement of the cyclohexyl
with phenyl (9). Confirming that large hydrophobic groups are pre-
ferred at this position, the only replacement that afforded a slight
increase in potency was the introduction of cyclopentylmethyl (10)
Table 2
SAR of C-2 amide linkage
O
R3
N
N
N
NH
MeO
Compound
R2
Itk IC50
(lM)
Selectivity versus IRKa
2
14
15
16
17
18
19
20
p-CN-Phenyl
p-CF3-Phenyl
p-Cl-Phenyl
m-CN-Phenyl
m-Br-Phenyl
5-Isoxazole
4-Pyridyl
0.046
>10
0.046
0.020
1.2
0.047
0.036
0.006
>217
1
>217
17
6.6
115
64
3-Pyridyl
163
a
Selectivity ratio IC50 IRK/IC50 Itk.

M. P. Winters et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5541–5544
5543
Table 6
Microsome stability study
Having identified a set of optimum pharmacophores for the C-2
amide linkage (R2) of the benzimidazole as well as the most favor-
able changes at the C-5 position (R and R1), we next undertook a
rapid evaluation of the N-1 (R3) site in-conjunction with changes
to R, R1, and R2 (Table 3). First, keeping R, R1, and R2 constant as
CH3, cyclohexyl and p-CN-phenyl, respectively, changing R3 from
3-methoxypropyl (2) to b-alanine amide (21) gave an almost equi-
potent compound, while changing to 3-(N1-imidazo)propyl (22) or
3-hydroxypropyl (23) gave a slight increase in potency. Another
example, 3-(N1-imidazo)propyl (24) for 3-methoxypropyl (18)
when R2 is 5-isoxazole, also gave a negligible change in potency.
These results confirm that for the conservative changes to R3
shown, there is an almost flat SAR. This is consistent with the crys-
tal structure of compounds such as 1 wherein this group does not
interact with the protein and extends toward solvent.15 Interest-
ingly, two changes that by themselves did not give an increase in
potency alone, R1 to phenyl and R2 to 5-isoxazole (26), gave a
15-fold potency increase when combined, 3 nM versus Itk.
Compound
RLM CLH (%QH) t1/2 (min)
HLM CLH (%QH) t1/2 (min)
2
8
26
3435
1789
669
5433
27,107
6422
120
100
80
60
40
20
0
Mean +/- STDEV
*** p value > 0.001 vs Vehicle
***
***
***
Five compounds were selected for further evaluation in the
DT40 cell-based Ca2+ flux inhibition assay (Table 4). Compounds
2, 8, 15, and 24 showed good activity in the cell-based assay, all
***
50 mg/kg
100mg/kg
30mg/kg
10mg/kg
Vechicle Cyclosporin A
with IC50s of approximately 0.5 lM. Of these, 2 and 8 were selected
compound 2, p.o.
for further study. Compound 26 showed outstanding potency in
the DT40 cell assay, 75 nM, mimicking its performance in the en-
zyme assay, and was also selected for further study.
Figure 2. Effects of Itk inhibition on in vivo IL-2 production in anti-CD3 stimulated
Balb/c mice.
Compounds 2, 8, and 26 were tested for selectivity against a pa-
nel of kinases (Table 5). All showed >50-fold selectivity against the
kinases screened with the exception of Txk. Compounds 2 and 8
showed selectivity of about 20-fold against Txk while 26 showed
much improved selectivity against Txk.
All three compounds were incubated with human liver micro-
somes; 8 had excellent microsomal stability while 2 was good
and 26 fair (Table 6). The stability in rat liver microsomes mim-
icked the human, although the t1/2 for 26 was very short, only
9 min. For this reason, inhibitors 2 and 8 were chosen for evalua-
tion in the functional assays.
Table 3
SAR of N-1 position
O
R2
R
N
N
N
Compounds 2 and 8 were evaluated in a human CD4+ T-cell co-
stimulation assay. Human CD4+ T-cells were co-stimulated with
immobilized anti-CD3 and anti-CD28 antibodies in the presence
NH
R1
R3
of compound or DMSO for 20 h. IL-2 and IFN-
measured by ELISA. Compound 2 inhibited the anti-CD3 and
CD28 antibody induced production of IL-2 (IC50 0.6 M) and
IFN- (IC50 1.1 M) while 8 inhibited IL-2 and IFN- at IC50s of
1.0 and 2.3 M, respectively. Inhibitor 2 showed excellent selectiv-
ity against five isoforms of cytochrome P450, 1A2, 2C9, 2C19, 2D6,
and 3A4 with IC50s > 14 M. A pharmacokinetic study of 2 in mice
c levels were then
Compound
R
R1
R2
R3
Itk IC50 (lM)
2
21
22
23
18
24
25
26
CH3 C6H11 p-CN-Phenyl 3-Methoxypropyl
CH3 C6H11 p-CN-Phenyl –CH2CH2CONH2
CH3 C6H11 p-CN-Phenyl 3-(N1-Imidazo) propyl 0.030
0.046
0.041
l
c
l
c
CH3 C6H11 p-CN-Phenyl –CH2CH2CH2OH
0.027
0.047
0.046
0.053
0.003
l
CH3 C6H11 5-Isoxazole
CH3 C6H11 5-Isoxazole
3-Methoxypropyl
3-(N1-Imidazo)propyl
3-Methoxypropyl
3-Methoxypropyl
l
H
H
C6H11 5-Isoxazole
C6H5 5-Isoxazole
revealed an iv t1/2 of 5 h, clearance of 43 mL/min/kg, and oral bio-
availability of 17%. Compound 2 was also tested for its ability to in-
hibit IL-2 production in vivo in mice following iv injection of anti-
CD3 antibody (Fig. 2). Inhibitor 2 dose-dependently inhibited IL-2
production in anti-CD3 stimulated Balb/c mice at oral doses of
10, 30, and 100 mg/kg.
Table 4
DT40 cell assay
Compound
Itk IC50
(l
M)
DT40 IC50 (lM)
In conclusion we identified a series of potent and selective
inhibitors of Itk as exemplified by compound 2. We have also
shown that 2 demonstrated good activity in vivo in mice following
oral administration. Continuing efforts focused on further optimiz-
ing the benzimidazole series are currently underway and will be
reported at a later date.
2
8
15
24
26
0.046
0.045
0.046
0.046
0.003
0.41
0.65
0.60
0.54
0.075
References and notes
Table 5
Enzyme selectivity of selected Itk inhibitors
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Itk IC50
(
lM)
IRKa
Btka
Syka
Bmxa
Txka
Teca
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2
8
26
0.046
0.045
0.003
>217
>222
>3333
61
70
1658
>217
>222
>3333
>217
>222
>3333
24
22
149
>217
>222
68
a
Selectivity ratio IC50/IC50 Itk.

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