Itk kinase inhibitors: Initial efforts to improve the metabolical stability and the cell activity of the benzimidazole lead

Article abstract of DOI:10.1016/j.bmcl.2008.09.017

Previously, we reported a series of novel benzimidazole based Itk inhibitors that exhibited excellent enzymatic potency and selectivity but low microsomal stability. Employing a structure based approach a new series of inhibitors with comparable potency a

Full text of DOI:10.1016/j.bmcl.2008.09.017

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5537–5540
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Itk kinase inhibitors: Initial efforts to improve the metabolical stability
and the cell activity of the benzimidazole lead
Kevin J. Moriarty c, Michael Winters c, Lei Qiao c, Declan Ryan c, Renee DesJarlis c, Darius Robinson c,
Brian N. Cook a, Mohammed A. Kashem a, Paul V. Kaplita b, Lisa H. Liu b, Thomas M. Farrell b,
Hnin Hnin Khine b, Josephine King b, Steven S. Pullen b, Gregory P. Roth d, Ronald Magolda a, Hidenori Takahashi a,
*
a Department of Medicinal Chemistry, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, PO Box 368, Ridgefield, CT 06877, USA
b Department of Immunology and Inflammation, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, PO Box 368, Ridgefield, CT 06877, USA
c Johnson & Johnson, PRDUS, Springhouse, PA 19477, 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:
Received 15 July 2008
Revised 29 August 2008
Accepted 3 September 2008
Available online 7 September 2008
Previously, we reported a series of novel benzimidazole based Itk inhibitors that exhibited excellent
enzymatic potency and selectivity but low microsomal stability. Employing a structure based approach
a new series of inhibitors with comparable potency and selectivity to the original series and with a poten-
tial for improved microsome stability was identified.
Ó 2008 Elsevier Ltd. All rights reserved.
This paper is dedicated to the memory of Dr.
Ronald L. Magolda.
Keywords:
SAR
Itk inhibitor
Metabolic stability
Liver microsomes
The Tec-family of tyrosine kinases (Bmx, Btk, Itk, Rlk/Txk, and
Tec) comprises five highly homologous proteins that are primarily
expressed in T-cells and other hemopoietic cell types, three of
which, Itk, Rlk/Txk, and Tec, are expressed in T-cells. Itk plays a
critical function during the initial steps of T-cell-receptor signaling
resulting in a cascade of downstream signaling pathways leading
to T-cell activation, differentiation and the production of cytokines
O
O
N
H
N
Me
N
N
S
S
H
N
Me
Me
Me
Me
Me
Me
O
1
Figure 1. Structure of ITK inhibitor reported by Bristol Myers Squibb.
such as IL-2 and IFN-c.
1–5 A selective inhibitor of Itk should prevent
T-cell activation and thus have broad application for the treatment
of T-cell-dependent processes such as autoimmune and inflamma-
tory diseases as well as organ transplant rejection.6–13
Bristol Myers Squibb disclosed a series of 2-amino-5-[(thio-
methyl)aryl]thiazoles, exemplified by compound 1, which were
potent and selective Itk inhibitors.14,15 Compound 1 was shown
to be effective at inhibiting IL-2 production in mice in a dose
dependent manner (50 and 100 mpk, sc) following an intravenous
injection of anti-CD3 antibody (Fig. 1).
Several of these compounds displayed excellent selectivity for
inhibition of Itk over other kinases. The benzimidazole class Itk
inhibitors were however rapidly metabolized in human liver
microsomal condition. In vitro metabolite identification studies
revealed the cyclohexyl C4 as the primary metabolic position,
and the key pathway for metabolism was shown to be hydroxyl-
ation at the C4 site. Introduction of gem difluorides (3) at C4
dramatically improved the metabolic stability and eliminated the
lability of a short in vitro half-life. However, the addition of the
fluorines resulted in a loss of cellular potency presumably due to
a decrease in cellular penetration since there was no observed loss
in enzymatic activity. Encouraged by these preliminary results, we
focused our efforts on identifying a replacement for the cyclohexyl
group that maintained metabolic stability coupled with good
We recently reported the discovery of a series of benzimidaz-
oles (2 and 3) which are potent Itk kinase inhibitors (Fig. 2).16
* 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.2008.09.017

5538
K. J. Moriarty et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5537–5540
F
CH3
N
O
CH3
N
F
O
CN
N
N
CN
N
N
NH
NH
O
O
O
NH2
H2N
O
2
3
Itk IC50 = 0.004μM
Itk IC50 = 0.008μM
DT40 IC50 = 2.3μ
HLM CLH = 19 %QH
DT40 IC50 = 0.30μM
HLM CLH = 93 %QH
Figure 2. Structures of benzimidazole lead ITK inhibitors.
enzyme and cellular activity. In this paper, we present our initial
efforts in our efforts to discovery novel, selective and improved
metabolically stable inhibitors of Itk.
H
N
NO2
F
H2N
NO2
F
b
a
O
The crystal structure of the compound 4/Itk complex revealed
that the cyclohexyl ring is situated in a pocket located on the sur-
face of the protein adjacent to the ATP binding site.17 The cyclo-
hexyl group binds in a hydrophobic subsite consisting of Phe437,
Leu379, Ile369 and the methylene chains of Lys387 and Gln367.
The C-5 anilide linkage adopts a cis-orientation with the carbonyl
pointing toward the solvent and the N-methyl group is situated to-
ward the exposed cleft region (Fig. 3).
5
6
NH2
NH
CH3
H3C
N
NO2
F
N
c,d
O
O
NH2
7
8
O
Docking studies using the above crystal structure indicated
that a phenyl ring would be readily accommodated in the hydro-
phobic pocket and offered the possibility of replacing the cyclo-
hexyl ring with a potentially less susceptible pharmacophore.
Our efforts in the modification of the benzimidazole series, to im-
prove its metabolic stability focused on replacing the cyclohexyl
ring with aryl rings.
CH3
N
N
N
NH2
O
O
f
e
9
NH2
O
CH3
N
CN
N
N
NH
O
10
NH2
O
Scheme 1. Representative synthetic scheme of 2-amino-1H-benzimidazole class
Itk inhibitors. Reagents and conditions: (a) C6H5COCl, pyridine, CH2Cl2, 0 °C to rt,
99%; (b) excess CH3I, NaH, THF, rt, 24 h, 96%; (c) H2NCH2CH2CONH2, DIEA, CH3CN,
50 °C, 24 h, 92%; (d) NiCl2Á6H2O, NaBH4, CH3OH, rt, 24 h, 86%; (e) BrCN, EtOH; rt,
24 h, 83%; (e) (p-CN)C6H4CO2H, PyBOP, DIEA, DMF, rt, 24 h, 93%.
The synthesis of these compounds follows a general synthetic
route that is illustrated with the preparation of 10 in Scheme 1.
The commercially available 4-fluoro-3-nitro-phenylamine (5) was
acylated with benzoyl chloride in the presence of pyridine to give
compound 6. The resulting anilide was then reacted with excess
methyl iodide in the presence of NaH at room temperature to give
compound 7. The fluoride was displaced with b-alanine amide at
50 °C for 12 h, and the nitro group was then reduced quantitatively
18
with NiCl2 6H2O/NaBH4 in methanol to give the phenylenedi-
amine compound 8. Compound 8 was then treated with cyanogen
bromide in ethanol at room temperature overnight to form com-
pound 9. The 2-amino group was acylated with 4-cyanobenzoic
acid using PyBOP/DIEA in DMF afforded 10 in 60% overall yield.
The benzimidazole 10 and its analogs were tested for their abil-
ity to inhibit the phosphorylation of substrate using a recombinant
Itk kinase domain as the source of enzyme.19
CH3
O
S
N
N
N
NH
O
Replacement of the cyclohexyl with a phenyl ring (2 vs 10) re-
sulted in no loss in potency against Itk in the enzyme assay but
lead to an approximately 2-fold improvement in the DT40 cellular
assay19 (Table 1). Substitution in the para position (11–15) was
well tolerated in the Itk enzyme assay yielding analogs with com-
NH2
O
4
Figure 3. X-ray structure of compound 4 bound in Itk.

K. J. Moriarty et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5537–5540
5539
Table 1
Table 3
SAR of substitutions on C-5 anilide position
SAR of aromatic amide on C-2 position
F
CH3
N
O
CH3
F
O
Ar
N
N
N
CN
N
NH
O
NH
O
N
NH2
O
NH2
O
Compound
Ar
Itk IC50
(
lM)
IRK IC50 (lM)
3
18
10
19
20
21
22
23
24
25
C6H5
C6H4(p-CN)
C6H4(p-Cl)
C6H3(3-CH3,4-CN)
5-Picolinonitrile
5-Cyanothiophene
2-Thiophene
5-Isoxazole
3-Furyl
0.003
0.003
0.006
1.9
0.023
0.008
0.014
0.010
0.010
0.12
>10
>10
>10
>10
>10
0.11
>10
0.32
CH3
N
O
R
CN
N
N
NH
O
NH2
O
10-17
Compound
R
Itk IC50
(l
M)
DT40 IC50 (lM)
3
—
H
0.008
0.003
0.002
0.012
0.010
0.007
0.035
0.004
0.027
2.3
observed that modifications of the Ar position had a significant
influence on the selectivity profile versus the insulin receptor ki-
nase (IRK). Conscious of this, we profiled the 5-aryl analogs against
both Itk and IRK. A similar selectivity profile was found for these
derivatives. para-Substitution (10, 19, and 21) yielded inhibitors
with both excellent Itk inhibitory potency and improved selectivity
against IRK while the 2-thiophene (23) and the 3-furyl (25) showed
diminished selectivity. The 5-cyanothiophene (22) and the 5-isox-
azole (24) were also tolerated providing analogs with improved
selectivity compared to compound 18 while di-substitution of
the aryl ring (20) was found to be detrimental to activity. These re-
sults were consistent with our previously proposed rationale for
the improved selectivity of the para-substitutents. The improved
selectivity results from a steric interaction with the gatekeeper res-
idue (Met) at the opening of the Selectivity Pocket of the insulin
receptor kinase.16
We next focused our explorations on the N–CH3 position of the
5-anilinde (Table 4). As a possible site of metabolism we focused
on identifying potential replacements for the methyl group. The
ethyl and propyl groups were all tolerated at this position. There
was however a slight preference for the ethyl over the propyl indi-
cating the previous size requirement at this position.16
Based on the enzyme potency, three compounds (10, 19, and
27) were selected for further evaluation. The compounds were
screened for cellular activity, selectivity, microsome stability, and
cytotoxicity activity (Tables 5 and 6). The inhibitors all exhibited
10
11
12
13
14
15
16
17
0.34
0.33
0.26
0.37
1.1
0.50
0.72
7.2
p-F
p-Cl
p-CH3
p-OCH3
p-OCF3
3-Pyridyl
4-Pyridyl
parable activity to 10. Replacement of the phenyl ring with either a
3- or 4-pyridyl (16 and 17) was also well tolerated in the enzyme
assay. Introduction of substitutions on the phenyl ring, however,
had a significant effect on the in vitro cellular activity for this ser-
ies. The p-fluoro (11), p-chloro (12), p-methyl (13), p-OCF3 (15),
and 3-pyridyl (16) groups gave compounds that had improved cel-
lular activity compared to 10 while the, p-OCH3 (14) and 4-pyridyl
analogs (17) resulted in a loss of potency compared to 10.
Generally, analogs 10–13 and 15 displayed the best balance of
enzyme and cellular potencies. In the series, compounds with
low ALogP (<2.8) and high TPSA > 100 Å2 coupled with molecular
weights >500 displayed a more pronounced biochemical to cellular
assay shift in IC50 values (Table 2).20 These results are suggestive of
poor cellular permeability consistent with compounds in the liter-
ature with similar characteristics.20 Interestingly, the region-iso-
mers of 3- and 4-pyridyl analogs (compound 16 and 17,
respectively) showed a 10-fold difference in cell potency even
though both have similar physicochemical properties.
Table 3 summarizes the relevant SAR observed for the Ar substi-
tutions at C-2 position. From our previous SAR studies of the cyclo-
hexyl on the left-hand side of the molecule derivatives, we
Table 4
SAR of N-substitutions at C-5 position
R
O
NH
N
Ar
N
N
O
Table 2
Predicted physicochemical properties of C-5 anilide analogs
Compound
R
AlogP
TPSA (Å2)
MW
NH2
O
3
—
H
p-F
p-Cl
p-CH3
p-OCH3
p-OCF3
3-Pyridyl
4-Pyridyl
2.6
2.9
3.1
3.5
3.3
2.8
5.0
1.7
1.7
134
134
134
134
134
143
143
147
147
508.5
466.5
484.5
501.0
480.5
496.5
550.5
467.5
467.5
Compound
R
Ar
Itk IC50 (lM)
10
11
12
13
14
15
16
17
10
19
26
27
28
29
30
CH3
CH3
CH3
Et
Et
Pr
C6H4(p-CN)
C6H4(p-Cl)
5-Isoxazole
C6H4(p-CN)
5-Isoxazole
C6H4(p-CN)
C6H4(p-Cl)
0.003
0.006
0.010
0.007
0.027
0.018
0.054
Pr

5540
K. J. Moriarty et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5537–5540
Table 5
References and notes
Cellular activity, cytotoxicity and microsome stability of selected compounds
1. Kane, L. P.; Lin, J. Curr. Opin. Immunol. 2000, 12, 242.
2. Samelson, L. E. Annu. Rev. Immunol. 2002, 20, 371.
3. Ohashi, P. S. Nat. Rev. Immunol. 2002, 2, 427.
Compound Itk IC50
M)
DT40 IC50
M)
Jurkat TC50
(lM)
HLM CLH
(%QH)
RLM CLH
(%QH)
(
l
(
l
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.
5. Tomlinson, M. G.; Kane, L. P.; Su, J.; Kadlecek, T. A.; Mollenauer, M. N.; Weiss, A.
Mol. Cell. Biol. 2004, 24, 2455.
10
19
27
0.003
0.006
0.007
0.34
0.34
0.16
>10
5.8
>10
32
48
57
35
46
nd
nd, not determined.
6. Liao, X. C.; Littman, D. R. Immunity 1995, 3, 757.
7. Miller, A. T.; Wilcox, H. M.; Lai, Z.; Berg, L. J. J. Exp. Med. 1998, 187, 1721.
9. Fowell, D. J.; Shinkai, K.; Liao, X. C.; Beebe, A. M.; Coffman, R. L.; Littman, D. R.;
Locksley, R. M. Immunity 1999, 11, 399.
Table 6
Selectivity results of selected compounds
10. Forssell, J.; Sideras, P.; Eriksson, C.; Malm-Erjefalt, M.; Rydell-Tormanen, K.;
Ericsson, P. O.; Erjefalt, J. S. Am. J. Respir. Cell. Mol. Biol. 2005, 32, 511.
11. Graves, P. E.; Siroux, V.; Guerra, S.; Klimecki, W. T.; Martinez, F. D. J. Allergy Clin.
Immunol. 2005, 116, 650.
Compound IRK IC50 Lyn IC50 Tec IC50
Txk IC50
M)
Cyp 2C9
M)
Cyp 3A4
(lM)
(l
M) M) M)
(
l
(
l
(
l
(
l
10
19
27
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.56
0.50
0.31
>30
>30
>30
>30
>30
>30
12. Matsumoto, Y.; Oshida, T.; Obayashi, I.; Imai, Y.; Matsui, K.; Yoshida, N. L.;
Nagata, N.; Ogawa, K.; Obayashi, M.; Kashiwabara, T.; Gunji, S.; Nagasu, T.;
Sugita, Y.; Tanaka, T.; Tsujimoto, G.; Katsunuma, T.; Akasawa, A.; Saito, H. Int.
Arch. Allergy Immunol. 2002, 129, 327.
13. Liu, K. Q.; Bunnell, S. C.; Gurniak, C. B.; Berg, L. J. J. Exp. Med. 1998, 187, 1721.
14. Lin, T. A.; McIntyre, K. W.; Das, J.; Liu, C.; O’Day, K. D.; Penhallow, B.; Hung, C.
Y.; Whitney, G. S.; Shuster, D. J.; Yang, X.; Townsend, R.; Postelnek, J.; Spergel, S.
H.; Lin, J.; Moquin, R. V.; Furch, J. A.; Doweyko, A.; Killar, L.; Dodd, J. H.; Barrish,
J. C.; Wityak, J.; Kanner, S. B. Biochemistry 2004, 43, 11056.
15. Das, J.; Liu, C.; Moquin, R. V.; Lin, J.; Furch, J. A.; Spergel, S. H.; Doweyko, A. M.;
McIntyre, K. W.; Shuster, D. J.; O’Day, K. D.; Penhallow, B.; Hung, C. Y.; Kanner,
S. B.; Lin, T. A.; Dodd, J. H.; Barrish, J. C.; Wityak, J. Bioorg. Med. Chem. Lett. 2006,
16, 4148.
16. 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.; Wilson, M.; Bobko, M.; Robinson, D.; Cook,
B. N.; Lo, H. Y.; Nemoto, P. A.; Kashem, M. A.; Wolak, J. P.; White, A.; Kaplita,
P. V.; Liu, L. H.; Magolda, R.; Tomczuk, B. Bioorg. Med. Chem. Lett. 2008, 18,
5545.
17. White, A.; Abeywardane, A.; Cook, B. N.; Fuschetto, N.; Gautschi, E.; John, A.;
Kroe, R. R.; Kronkaitis, A.; Li, X.; 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.
good cellular potency in the DT40 cellular assay. All the com-
pounds exhibited improved stability compared to 2 against human
microsomes, and greater than 500-fold separations between the
DT40 IC50 and TC50 values. The compounds also showed excellent
selectivity against IRK, Lyn, Tec and a 44- to 188-fold selectivity
against Txk. Unfortunately although the compounds did show an
improvement there still was a high degree of metabolism indicat-
ing additional susceptible sites on the compounds. There was how-
ever an observed 2-fold improvement when the N–CH3 was
replaced with a N–Et which did point to one of the possible sites
of metabolism for the series. Additional profiling is currently
underway to identify the other sites so additional corrective mod-
ifications can be carried out to improve the stability of the series.
In summary, starting from a series that showed excellent enzy-
matic potency and selectivity but poor microsomal stability, we
identified a new series that retained excellent potency, selectivity
and cellular activity and with the potential for improved micro-
some stability. Further improvements and modifications of the ser-
ies focusing on improving the stability and leading to an orally
active Itk inhibitor will be presented in forthcoming publications.
18. Nose, A.; Kudo, T. Chem. Pharm. Bull. 1981, 29, 1159.
19. (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. Screen. 2007, 12, 70; (b) Snow, R. J.; Abeywardane, A.;
Campbell, S.; Lord, J.; Kashem, M. A.; Khine, H. H.; King, J.; Kowalski, J. A.;
Pullen, S. S.; Roma, T.; Roth, G. P.; Sarko, C. R.; Wilson, N. S.; Winters, M. P.;
Wolak, J. P.; Cywin, C. L. Bioorg. Med. Chem. Lett. 2007, 17, 3660.
20. (a) Ertl, P.; Rohde, B.; Selzer, P. J. Med. Chem. 2000, 43, 3714; (b) Ghose, A. K.;
Viswanadhan, V. N.; Wendoloski, J. J. J. Phys. Chem. A 1998, 102, 3762.