5-Aminomethylbenzimidazoles as potent ITK antagonists

Article abstract of DOI:10.1016/j.bmcl.2009.02.012

Benzamide 1 demonstrated good potency as a selective ITK inhibitor, however the amide moiety was found to be hydrolytically labile in vivo, resulting in low oral exposure and the generation of mutagenic aromatic amine metabolites. Replacing the benzamide with a benzylamine linker not only addressed the toxicity issue, but also improved the cellular and functional potency as well as the drug-like properties. SAR studies around the benzylamines and the identification of 10n and 10o as excellent tools for proof-of-concept studies are described.

Full text of DOI:10.1016/j.bmcl.2009.02.012

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1588–1591
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
5-Aminomethylbenzimdazoles as potent ITK antagonists
a
a
a
a
Doris Riether a, , Renée Zindell , Jennifer A. Kowalski , Brian N. Cook , Jörg Bentzien ,
Stéphane De Lombaert a, David Thomson a, Stanley Z. Kugler Jr. a, Donna Skow a, Leslie S. Martin a,
Ernest L. Raymond b, Hnin Hnin Khine c, Kathy O’Shea b, Joseph R. Woska Jr. b, Deborah Jeanfavre b,
Rosemarie Sellati b, Kerry L. M. Ralph b, Jennifer Ahlberg d, Gabriel Labissiere d, Mohammed A. Kashem a,
Steven S. Pullen c, Hidenori Takahashi a
*
a Medicinal Chemistry Department, Boehringer Ingelheim Pharmaceuticals Inc., 900 Ridgebury Rd./PO Box 368, Ridgefield, CT 06877, USA
b Inflammation and Immunology Departments, Boehringer Ingelheim Pharmaceuticals Inc., 900 Ridgebury Rd./PO Box 368, Ridgefield, CT 06877, USA
c Cardiovascular Disease Department, Boehringer Ingelheim Pharmaceuticals Inc., 900 Ridgebury Rd./PO Box 368, Ridgefield, CT 06877, USA
d Department of Drug Discovery Support, Boehringer Ingelheim Pharmaceuticals Inc., 900 Ridgebury Rd./PO 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:
Benzamide 1 demonstrated good potency as a selective ITK inhibitor, however the amide moiety was
found to be hydrolytically labile in vivo, resulting in low oral exposure and the generation of mutagenic
aromatic amine metabolites. Replacing the benzamide with a benzylamine linker not only addressed the
toxicity issue, but also improved the cellular and functional potency as well as the drug-like properties.
SAR studies around the benzylamines and the identification of 10n and 10o as excellent tools for proof-
of-concept studies are described.
Received 6 January 2009
Revised 3 February 2009
Accepted 4 February 2009
Available online 8 February 2009
Keywords:
ITK
Ó 2009 Elsevier Ltd. All rights reserved.
Interleukin-2 inducible T-cell kinase
5-Aminobenzimidazole
Interleukin-2 inducible T-cell kinase (ITK) is a tyrosine kinase
from the Tec kinase family1 that is expressed in T-cells, NK cells
and mast cells. Itk deficient murine CD4+ T-cells demonstrate re-
duced IL-2, IL-4, IL-5 and IL-13 production upon T-cell receptor
stimulation.2–4 This suggests that a selective ITK inhibitor would
reduce the production of these pro-inflammatory cytokines and
hence ITK is considered an attractive target for the potential treat-
ment of inflammatory diseases such as psoriasis and allergic
asthma.5
Recently, we have reported the discovery of 5-aminobenzimi-
dazoles as potent inhibitors of ITK.6 Compounds such as 1 (Scheme
1) showed significant inhibitory activity in an ITK DELFIA-based
molecular assay7 (IC50 = 3 nM) and good cellular potency
(IC50 = 110 nM), as measured in a stably transfected, ITK-express-
ing DT40 cell line assay.8 However, in human hepatocytes com-
pounds such as 1 suffered from amide bond cleavage, generating
reactive aniline metabolites 2a and 2b. Upon subsequent testing,
it was observed that, upon further metabolic activation in vitro,
these metabolites formed adducts with glutathione (GSH) and
demonstrated mutagenicity in an Ames test. These results were
indicative of the formation of one or more reactive metabolites.
Metabolites 2a and 2b were also generated in vivo in rat and the
amide bond hydrolysis contributed to the very low plasma levels
observed in mice after oral administration. Therefore, we em-
barked in a synthetic effort to identify potent, orally bioavailable
ITK inhibitors that are not metabolized to a toxicophoric aniline.
The X-ray crystal structure of ITK9 with a related compound
bearing a cyclohexyl amide demonstrated that the cyclohexyl
group forms a lipophilic interaction with a shallow pocket on the
enzyme surface. Assuming that 1 binds in a similar fashion, a
molecular modeling study suggested a number of opportunities
for replacing the benzamide linker. Previously, we have reported
a few examples of linkers such as benzylamine, ketone and ether
linkers that were tolerated in this position.10 These alternative
linkers had been assessed at an early stage of the lead optimization
effort, prior to the identification of the more preferred biaryl right
hand side groups, and so their impact had not been fully deter-
mined. Therefore, upon achieving a significant increase in intrinsic
potency with the biaryl right hand piece of the aminobenzimidaz-
ole based ITK inhibitors,6 we decided to re-visit the more polar lin-
ker systems, such as benzylamines, to improve the overall physical
properties as well as the potency in the functional assay of our ITK
inhibitor series. In this Letter, we discuss an SAR of a new class of
very potent ITK inhibitors having a benzylamine linker between
the core and the left hand side functional groups.
To systematically explore the SAR, a variety of benzylamine
analogs were prepared, as illustrated in Scheme 2. Compound
* Corresponding author. Tel.: +1 203 791 6203.
E-mail address: doris.riether@boehringer-ingelheim.com (D. Riether).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.012

D. Riether et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1588–1591
1589
R1
N
O
N
O
N
human hepatocytes
H
N
H
N
N
& rat in vivo
N
N
S
S
H
O
O
O
N
N
OH
OH
1
2a: R1 = Me
2b: R2 = H
Scheme 1. Benzamide 1: Cleavage to aniline metabolites 2a and b.
O
HO
N
S
6
c
O
N
H
N
O
N
N
N
NO2
F
NO2
TIPSO
S
TIPSO
HO
TIPSO
NH2
O
b
a
N
OH
N
H
3
4
5
7
OH
OH
O
N
O
N
H
N
H
N
N
N
N
S
S
N
H
O
O
e
O
d
N
9b
8
OH
OH
Scheme 2. Reagents and conditions: (a) (i) TIPS triflate, 2,6-lutidine, DCM; (ii) 2,2-dimethyl ethanolamine, CH3CN, 61% over 2 steps; (b) (i) 10% Pd/C, NH þHCOOꢀ, EtOH, 91%;
4
(ii) cyanogen bromide, EtOH, rt, 88%; (c) EDC, HOBt, DIEA, 75%; (e) (i) 0.01 N HCl, dioxane, 95 °C, 92%; (ii) MnO2, acetone, 84%; (f) amine, NaBH(OAc)3, DCM, 75–95%.
9b was used as the starting point. In order to rapidly explore the
SAR, we resorted to a parallel synthesis approach. Aldehyde pre-
cursor 8 was prepared as the divergent point for analog synthe-
ses (Scheme 2).
tration used, we developed a complementary assay using a higher
concentration of ATP (1 mM) in order to extend the dynamic range
of the assay, thereby allowing comparisons between the most po-
tent inhibitors.7 The compounds were further tested for selectivity
against insulin receptor kinase (IRK) due to their previously ob-
served occasional cross-reactivity with this enzyme. The cellular
potency of the compounds was determined first in a stably trans-
fected ITK-expressing DT40 cell line assay.8 The most potent com-
pounds from the series were tested thereafter in a functional assay
measuring inhibition of IL-2 secretion using human CD4+ T-cells
stimulated with anti-CD3 and anti-CD28 mAbs. The results are
shown in Table 1.
Benzylamine inhibitor 9a showed a significant loss in ITK bind-
ing affinity and a complete loss in cellular potency when compared
to benzamide 1. In contrast to the tertiary amine 9a, secondary
amine 9b was fourfold less potent than 1 in the ITK binding assay,
twofold less potent in the cellular assay, and equipotent to 1 in the
functional assay. A similar trend was observed for cyclohexyl ana-
logs 9c and 9d which were potent inhibitors in the functional assay
Protection of commercially available benzyl alcohol 3 as the
TIPS ether, followed by displacement of the fluorine with 2,2-di-
methyl ethanolamine (SnAr), gave amine 4 in good yields. After
reduction of the nitro group of 4, the formation of benzimidazole
5 was accomplished uneventfully with cyanogen bromide. Stan-
dard amide coupling with thiophene acid 611 yielded 7. Acidic
deprotection of the benzyl alcohol, followed by MnO2 oxidation,
gave aldehyde intermediate 8. The reductive aminations were per-
formed using polymer-supported reagents such as MP–BH(OAc)3
or MP–BH3CN. Scavenging of the remaining starting materials with
PS-Benzaldehyde, PS-Isocyanate and PS-Trisamine, allowed effi-
cient access to a wide variety of benzylamines such as 9b. All of
the analogs prepared were tested in an ITK DELFIA assay to deter-
mine their intrinsic binding affinities. Since the predicted assay
range does not extend below 2 nM, based on the enzyme concen-
Table 1
Results of selected benzylamine analogs 9 with different R1 and R2 in comparison with the corresponding benzamide 1
O
H
R1
N
N
N
S
N
N
O
R2
OH
9
R1
R2
ITK IC50 (nM)
ITKb IC50 (nM)
IRK IC50 (nM)
DT40 Cell IC50 (nM)
IL-2 inhibition IC50 (nM)
a
a
a
a
a
Compds
1
–
Ph
Ph
–
Me
H
Me
H
3
200
na
na
670
940
940
na
na
350
700
110
na
180
180
210
440
9a
9b
9c
9d
100
11
8
460
230
180
Cyclohexyl
Cyclohexyl
8
a
Values are means of at least three separate experiments, (na = not active: IC50 > 5
High ATP (1 mM) concentration ITK molecular assay.
lM).
b

1590
D. Riether et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1588–1591
Table 2
Results of selected lipophilic benzylamine analogs 10
O
H
R1
N
N
N
NH
S
N
N
R2
OH
10
R1
R2
ITK IC50 (nM)
ITKb IC50 (nM)
IRK IC50 (nM)
DT40 Cell IC50 (nM)
IL-2 inhibition IC50 (nM)
a
a
a
a
a
Compds
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
10o
10p
Ph
Ph
Me
H
Me
H
H
H
Me
H
Me
H
H
H
8
2
2
1
4
2
3
1
1
2
1
1
1
1
1
2
780
75
160
99
900
420
490
140
100
390
140
20
5500
1700
480
400
1200
500
570
480
310
600
360
93
120
21
13
20
na
na
26
19
6
25
12
3
2
4
150
59
93
Cyclohexyl
Cyclohexyl
H
Me
Me
Benzyl
3-Pyridyl-methyl
1-Methyl-propyl
2-Methyl-propyl
(1R)-1,2,2-Trimethylpropyl
(1R)-1,2,2-Trimethylpropyl
(1S)-1,2,2-Trimethylpropyl
(1S)-1,2,2-Trimethylpropyl
2-N,N-Dimethylaminoethyl
130
37
91
23
86
50
64
20
18
150
Me
H
Me
Me
20
13
8
180
230
160
400
390
3
62
a
Values are means of at least three separate experiments, (na = not active: IC50 > 5
High ATP (1 mM) concentration ITK molecular assay.
lM).
b
Table 3
Results of selected cyclic benzylamine analogs 11
O
H
N
N
NH
S
R
N
N
OH
11
ITKb IC50 (nM)
IRK IC50 (nM)
DT40 Cell IC50 (nM)
IL-2 inhibition IC50 (nM)
a
a
a
a
a
Compds
R
ITK IC50 (nM)
11b
11c
11d
11e
11f
Piperidine
2-Hydroxymethylpiperidine
Morpholine
3,5-Dimethylmorpholine
3-Hydroxypyrrolidine
3
3
2
1
2
250
660
220
98
550
1100
320
320
420
11
58
13
3
120
230
46
310
130
a
Values are means of at least three separate experiments, (na = not active: IC50 > 5
High ATP (1 mM) concentration ITK molecular assay.
lM).
b
with IC50s of 230 nM and 180 nM, respectively. This data suggested
that with the benzylamine class of inhibitors, the loss of potency
observed between the assays (ITK binding vs cellular assay using
DT40/ITK cells vs functional assay) is significantly smaller than
with the benzamide series. This phenomenon can possibly be ex-
plained by differences in aqueous solubility and protein plasma
binding between benzamides and benzylamines. While benzamide
amine 10e led to an inactive compound in the cellular assay, de-
spite good binding potency. Introduction of one methyl group as
in 10f was not sufficient to introduce any cellular potency. In con-
trast the N,N-dimethyl amine 10g demonstrated moderate potency
in the cellular assay, as well as in the functional assay despite sim-
Table 4
Pharmacokinetic parameters of 10n in Sprague–Dawley ratsa administered in PEG/
1 has a low solubility of 2
bound in a dialysis plasma protein binding assay, benzylamine 9d
displays moderate solubility of 27 g/mL and a substantially high-
lg/mL in pH 7 buffer and is 98.7% protein
H2O = 70:30
Administered iv (1 mg/kg)
l
b
t1/2
2.5 h
er unbound fraction (93.3% protein bound).
Vdss
CL
AUC0-1
11.6 L/kg
Further optimization and SAR exploration was performed with-
in the pyrazole RHS class of inhibitors 10 which we had found to be
more potent when compared to the methyl-oxazole class 9.6 To ac-
cess the pyrazole derivatives 10, the same synthetic route as de-
scribed in Scheme 2 was applied using the appropriate thiophene
carboxylic acid in the amide coupling step.
Pyrazoles 10a–d (Table 2) were prepared for direct comparison
to the corresponding methyl-oxazoles 9a–d and displayed compa-
rable SAR trends. Removing all substituents to yield unsubstituted
53.1 mL minꢀ1 kgꢀ1
314 ng h/mL
Administered po (10 mg/kg)
Tmax
1.2 h
Cmax
AUC0-1
F (%)
92 ng/mL
659 ng h/mL
21%
a
Values represent the means only for n = 3.
Terminal half-life.
b

D. Riether et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1588–1591
1591
Figure 1. Effect of ITK inhibitor 10n on in vivo cytokine production in anti-CD3 stimulated Balb/c mice.
ilar binding potency as 10f. Increasing the lipophilicity of R1 gener-
ally led to an increase in potency in both the cellular and functional
assays. Both aromatic substituents such as benzyl 10h and 3-pyri-
dylmethyl 10i, as well as aliphatic substituents were well toler-
ated. Among the most potent substituents within the
benzylamine series we identified 1,2,2-trimethylpropyl as in 10l–
o. The stereochemistry of the 1,2,2-trimethylpropyl group had only
a marginal effect on the functional potency. In the functional assay
the (S)-enantiomers 10n and 10o achieved potencies of 20 nM and
18 nM, respectively, while the (R)-enantiomers 10l and 10m dem-
onstrated potencies of 50 nM and 64 nM, respectively. Compounds
10n and 10o were profiled further and the additional data is dis-
cussed below.
injection) (Fig. 1). The terminal plasma concentrations (8 h post
dose) of 10n were 1.3 M, 0.4 M and 0.1 M, respectively, dem-
onstrating that the in vivo EC50 was comparable to the HWB IC50
In summary, we have demonstrated that the amide moiety of
benzamide ITK inhibitors such as 1 can be replaced with a benzyl-
amine linker to address the liability of forming potentially toxic
metabolites. The new benzylamines generally demonstrated in-
creased potency in assays using primary human cells and are
among the most potent orally bioavailable ITK inhibitors to date.
l
l
l
.
References and notes
1. (a) Smith, C. I.; Islam, T. C.; Mattsson, P. T.; Mohamed, A. J.; Nore, B. F.; Vihinen,
M. Bioassays 2001, 23, 436–446; (b) 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.
2. Schaeffer, E. M.; Yap, G. S.; Lewis, C. M.; Czar, M. J.; McVicar, D. W.; Cheever, A.
W.; Sher, A.; Schwartzberg, P. L. Nat. Immunol. 2001, 2, 1183.
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Sher, A.; Varmus, H. E.; Lenardo, M. J.; Schwartzberg, P. L. Science 1999, 284,
638.
The SAR further demonstrated that secondary (R2 = H) and ter-
tiary (R2 = Me) amines are generally very comparable. Further
exploration of R1 showed that polar substituents such as hydroxyl
and methoxy (not shown), or basic amines such as dimethylamino
substituents as in 10p led to a loss in potency. In comparison to
acyclic amines, cyclic amines, as shown in Table 3, are slightly less
potent and polar substituents such as in 11c and 11f again de-
creased potency.
4. Fowell, D. J.; Shinkai, K.; Liao, X. C.; Beebe, A. M.; Coffman, R. L.; Littman, D. R.;
Locksley, R. M. Immunity 1999, 11, 399.
5. Kanner, S. B.; Perez-Villar, J. J. Trends Immunol. 2003, 24, 249.
6. Cook, B. N.; Bentzien, J.; White, A.; Nemoto, P. A.; Wang, J.; Man, C. C.;
Soleymanzadeh, F.; Khine, H. H.; Kashem, M. A.; Kugler, S. Z., Jr.; Wolak, J. P.;
Roth, G. P.; De Lombaert, S.; Pullen, S. S.; Takahashi, H. Bioorg. Med. Chem. Lett.
2009, 19, 773.
7. 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. Screen. 2007, 12, 70.
8. Snow, R. J.; Abeywardane, A.; Campbell, S.; Lord, J.; Kashem, M. A.; Khine, H. H.;
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S.; Winters, M. P.; Wolak, J. P.; Cywin, C. L. Bioorg. Med. Chem. Lett. 2007, 17(13),
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Selected compounds were further profiled in a human whole
blood assay (HWB) in which IL-2 secretion is measured after stim-
ulation using staphylococcal entertoxin B. While benzamide 1
demonstrated an inhibitory potency of only 10
say, benzylamines 10n and 10o were found to be much more po-
tent, with IC50 of 240 nM and 690 nM, respectively. The
lM in the HWB as-
s
pharmacokinetic profile of 10n in Sprague–Dawley rats is shown
in Table 4. The compound demonstrated a high volume of distribu-
tion at steady-state (11.6 L/kg) and a rapid plasma clearance
(53.1 mL min/kg), resulting in a half-life of 2.5 h and a 21% bio-
availability at 10 mg/kg.
Compound 10n was also tested for its in vivo activity in a mouse
model of T-cell activation. Female Balb/c mice were dosed orally
with 10n, followed 5 h later with an i.v. injection of anti-CD3.
The ITK inhibitor 10n dose-dependently (15, 5, and 1.5 mg/kg)
inhibited IL-2 and IL-4 production (measured 3 h post anti-CD3
10. 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.; Moriarty, K. J. Bioorg. Med. Chem. Lett. 2008, 18(20), 5541–5544.
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