Benzimidazole- and benzoxazole-based inhibitors of Rho kinase

Article abstract of DOI:10.1016/j.bmcl.2008.10.095

Inhibitors of Rho kinase have been developed based on two distinct scaffolds, benzimidazoles, and benzoxazoles. SAR studies and efforts to optimize the initial lead compounds are described. Novel selective inhibitors of ROCK-II with excellent potency in b

Full text of DOI:10.1016/j.bmcl.2008.10.095

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6390–6393
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Benzimidazole- and benzoxazole-based inhibitors of Rho kinase
E. Hampton Sessions, Yan Yin, Thomas D. Bannister, Amiee Weiser, Evelyn Griffin,
Jennifer Pocas, Michael D. Cameron, Claudia Ruiz, Li Lin, Stephan C. Schürer,
*
Thomas Schröter, Philip LoGrasso, Yangbo Feng
Department of Molecular Therapeutics, and Translational Research Institute, The Scripps Research Institute Florida, 130 Scripps Way, #2A1, Jupiter, FL 33458, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Inhibitors of Rho kinase have been developed based on two distinct scaffolds, benzimidazoles, and benz-
oxazoles. SAR studies and efforts to optimize the initial lead compounds are described. Novel selective
inhibitors of ROCK-II with excellent potency in both enzyme and cell-based assays were obtained. These
inhibitors possess good microsomal stability, low cytochrome P-450 inhibitions and good oral
bioavailability.
Received 8 September 2008
Revised 16 October 2008
Accepted 17 October 2008
Available online 25 October 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Rock-II
Rho kinase
Glaucoma
Hypertension
Benzimidazole
Benzoxazole
Chroman
Aminopyrimidine
Pyrazole
Rho kinase (ROCK) is a serine/threonine kinase that is an impor-
tant regulator in smooth cell contraction. Two isoforms of Rho
groups working toward the development of novel Rho kinase
inhibitors.13
kinase are known, ROCK-I (or ROKb) and ROCK-II (or ROK
a
), which
A focused medicinal chemistry effort based on literature ROCK
inhibitors and leads from an in-house HTS campaign14 led to amide
1 (Fig. 1) as an early lead compound12 with high affinity for ROCK-
II in enzyme assays15 (IC50 < 2 nM), good selectivity over the
related kinase PKA16 (IC50 = 127 nM), and high potency in a cell-
based myosin light chain bis-phosphorylation (ppMLC) assay17
(IC50 = 25 nM). While these initial data were promising, we sought
to address the potential lability of the central amide bond, which
upon hydrolysis would lead to primary anilines, a group often
associated with potential toxicity. One strategy for amide replace-
ment involved the synthesis of the benzoxazole and benzimidazole
ring-fused analogs 2 and 3.
share 92% sequence identity in the ATP binding pocket.1 ROCK is
activated via the binding of the GTP-bound G protein Rho. Once
activated, ROCK phosphorylates numerous substrates affecting cel-
lular contraction, cytoskeleton regulation, and microtubule regula-
tion. One example of these effects is the phosphorylation of myosin
light chain (MLC) which, when phosphorylated, leads to cell con-
traction. ROCK is closely related to other members of the AGC fam-
ily of kinases such as CDC42-binding kinase (MRCK), protein kinase
A (PKA) and protein kinase B (Akt).2
There are many potential therapeutic applications for an inhib-
itor of ROCK, including hypertension,3 inflammation,4 ischemic
stroke,5 cancer,6 multiple sclerosis,7 erectile dysfunction,8 and
glaucoma.9 So far, this potential has only been realized by one clin-
ically approved inhibitor (fasudil, marketed in Japan for cerebral
vasospasm10). Our goal was to identify and develop potent and
selective inhibitors of ROCK with acceptable pharmacokinetic
and safety profiles. Our initial progress toward this goal has been
previously reported.11,12 Additionally, there are several other
We found that both benzoxazole 2 and benzimidazole 3 main-
tain many of the desirable properties of amide 1. In particular,
* Corresponding author. Tel.: +1 561 228 2201.
E-mail address: yfeng@scripps.edu (Y. Feng).
Figure 1. Structures of lead compounds 1–3.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.095

E. H. Sessions et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6390–6393
6391
benzoxazole 2 has high affinity for ROCK-II (IC50 = 21 nM) with
moderate PKA selectivity (IC50 = 1206 nM). The potency in the
cell-based ppMLC assay was, however, reduced (IC50 = 330 nM).
Benzimidazole 3 has high ROCK-II affinity (IC50 = 27 nM) and cell-
based potency (IC50 = 86 nM), but with reduced selectivity for
PKA (IC50 = 168 nM). With these promising initial results, we
sought to optimize both scaffolds as novel ROCK inhibitors.
Analogs 7 of benzoxazole 2 were readily synthesized by a short
sequence (Scheme 1) that began with amide coupling of commer-
cially available aniline 4 and a chroman carboxylic acid to yield
amide 5. The nitrogen heterocycle was then incorporated through
standard palladium coupling chemistry, giving intermediate 6. Clo-
sure of the benzoxazole ring was lastly accomplished under acidic
conditions, giving the desired product 7.
Scheme 2. Synthesis of benzimidazole 11.
Similarly, a short sequence (Scheme 2) involving sequential
amide coupling, dehydration, and Suzuki coupling with an appro-
priate aryl boronic acid (or alternatively a two-step palladium cat-
alyzed borylation/Suzuki coupling sequence with an aryl halide)
gave the targeted benzimidazoles 11.
The necessary chromanylcarboxylic acids were synthesized as
previously described,12 or according to Scheme 3. The isopropoxy
chroman 13 was derived from the corresponding methoxy sub-
strate via BBr3 mediated demethylation followed by alkylation.
The carboxamide 15 was synthesized from the methyl ester via a
two-step hydrolysis/peptide coupling sequence.
Scheme 3. Synthesis of chroman derivatives.
We studied the effect of various nitrogen-containing heterocy-
cles upon ROCK-II potency and selectivity. Representative analogs
are shown in Table 1. Addition of a methyl group on the 3-position
of the pyrazole modestly improved the ROCK-II potency, PKA selec-
tivity and stability to human liver microsomes, though potency in
the cell-based assays (ppMLC) was reduced. More promising was
compound 18. The pyridine substitution in this compound im-
proved the enzyme potency, significantly raised PKA selectivity
(158-fold vs 6-fold), and improved cell-based potency (IC50
< 6 nM). Unfortunately, 18 was found to be a potent inhibitor of
several cytochrome p450 (CYP) metabolizing enzymes, as were
lead compounds 2 and 3.18 The CYP inhibition was effectively re-
duced by using 2-aminopyrimidine as the heterocycle. Thus benz-
imidazole 19 and benzoxazole 20 had minimal inhibition of the
CYP enzymes studied, with high stability to human liver micro-
somes, and with high potency in ROCK-II enzyme and cell-based
assays.
Next we explored the effect of various substituents on the chro-
man ring, hoping that improved interactions within the ROCK-II
binding pocket would lead to improvements in ROCK-II affinity
and PKA selectivity. To this end, several 6-substituted chromans
were incorporated into the scaffolds as shown in Table 2.19 The
addition of a methoxy or an amide group improved both potency
and PKA selectivity. Compound 21 had high affinity for ROCK-II
(2 nM) with enhanced selectivity versus PKA (240-fold). Benzoxaz-
Table 1
Varying the nitrogen heterocycle of benzimidazoles and benzoxazoles.
b
Compound
R
X
Enzyme assays
IC50 (nM)
Cell assay
ppMLC
IC50 (nM)
t1/2 (min) HLM
a
a
ROCK-II
PKA
2
O
N
21
1206
330
86
40
20
3
27
9
168
122
778
317
16
17
18
N
O
N
ndc
533
<6
30
70
29
9
2
19
N
O
8
198
66
nd
44
57
20
27
1336
a
Values are means of 2 or more experiments with errors within 15% of the mean.
2 mg/mL human liver microsomes were used in stability studies.
nd, not determined.
b
c
ole 22 was similarly potent and significantly more selective
(ROCK-II IC50 = 2 nM, ꢀ950-fold selectivity for PKA). Larger alkoxy
groups were not well-tolerated (e.g., isopropoxy derivatives 23 and
24). Fluoro substitution (e.g., 25 and 26), a methyl group (27) or a
Scheme 1. Synthesis of benzoxazole 7.

6392
E. H. Sessions et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6390–6393
Table 2
Table 3
Evaluation of 6-substituted chromans.
In vivo (rat) pharmacokinetic data for inhibitors 21, 22, and 32.a
Compound Cl (mL/
min/kg)
Vss (L/kg)
t1/2 (h)
AUC po
M h)
Cmax po
M)
Oral F
(%)
(
l
(
l
21
22
32
17 2.6 0.89 0.11 2.3 0.2 1.9 0.7 0.90 0.58 35 13
8.4 0.3 1.6 0.1 2.6 0.2 6.6 1.3 0.97 0.20 63 12
12 4.4 0.63 0.20 1.4 0.1 2.2 0.3 0.74 0.03 29 4.4
b
Compound
R
X
Enzyme assays
IC50 (nM)
Cell assay
ppMLC IC50
(nM)
t1/2 (min)
a
a
HLM
aData was generated from three determinations. Dosed at 1.0 mg/kg (iv) or 2.0 mg/
kg (po).
ROCK-II
PKA
21
22
23
24
25
26
27
28
29
30
31
–OMe
–OMe
–OiPr
–OiPr
–F
–F
–Me
–CO2Me
–CO2Me
N
O
N
O
N
O
O
N
O
N
N
2
2
602
99
46
nd
nd
nd
28
nd
28
34
nd
nd
45
91
25
18
nd
79
25
1911
6382
nd
270
2912
6389
10,190 271
nd
669
75
296
294
6
15
78
6
24
<1
<1
nd
212
23
–CONHBn
nd, not determined.
a
Values are means of 2 or more experiments with errors within 80% of the mean.
2 mg/mL human liver microsomes were used in stability studies.
b
Figure 3. Optimized conformation of 22 docked into the ROCK-II structure.
carbomethoxy group (28, 29) were all tolerated, though with
somewhat reduced affinity for ROCK-II. Replacement of the ester
with amides not only improved microsomal stability, as expected,
but also resulted in higher potency (benzimidazole 30, 31). Inter-
estingly, even larger C-6 amide substituents were tolerated (e.g.,
benzimidazole 31) whereas larger alkoxy groups (e.g., benzimid-
azole 23) were not. This survey of 6-substituted chroman deriva-
tives led us to conclude that methoxy and carboxamide
substituents are particularly helpful in enhancing affinity for
ROCK-II.
A limited SAR study on the effects of substituents on the benz-
imidazole ring system (not shown) revealed that in general only
small groups were well-tolerated. Benzimidazole 32, with a fluo-
rine atom incorporated (Fig. 2),20 had excellent affinity for ROCK-
II, selectivity against PKA, and high potency in the cell-based
ppMLC assay (IC50 < 6 nM). Additionally, this compound was
screened against several related kinases, and proved to have excel-
lent selectivity against all kinases tested. Screening against ROCK-I
for compound 32 and those listed in Tables 1 and 2 is undergoing
in our labs. Preliminary results (data not shown) demonstrated
that these compounds were typically pan-ROCK inhibitors.
The most promising derivatives in both the benzimidazole and
benzoxazole scaffolds were evaluated for their in vivo pharmacoki-
netic properties (Table 3). All of the compounds shown had both
high oral bioavailability and systemic exposure. The high bioavail-
ability of benzoxazole 22 (F = 63%) was especially noteworthy.
To identify likely binding modes of our inhibitors, we docked
benzimidazole 21 and benzoxazole 22 into a model of ROCK-II as
previously described.12,21 The initial docking results failed to show
any direct interaction between the enzyme and the benzimidazole
(or benzoxazole) nitrogen, an interaction anticipated based upon
the binding mode of related ROCK-II inhibitors.12,21 Inclusion of a
water molecule, which is frequently observed in the phosphate
binding region in ROCK crystal structures,22,23 could act as a bridge
between the benzimidazole nitrogen and the enzyme. Thus, we
optimized the enzyme–ligand complex with a water molecule by
molecular dynamics and minimization and created a docking
model from the minimized complex. Into this enzyme structure
21 and 22 were docked and the best conformation obtained for
22 is shown in Figure 3 (21 has essentially the same conformation).
Figure 3 shows the S enantiomer of 22 and our modeling indicated
that the opposite enantiomer showed a similar binding mode.21
In this model, the 2-aminopyrimidine moiety forms a typical
two hydrogen bond pattern with the Glu170 and Met172 residues
of the hinge region. An important hydrophobic interaction is
formed by the chroman moiety with the hydrophobic binding
pocket under the P-loop which is characterized by Phe103,
Ala102, Leu123, and the carbon chain of Lys121. The benzoxazole
or benzimidazole groups potentially interact with the Lys121 and
Asp232 side chains via a water bridge. Docking experiments start-
ing from an alternate ROCK-II structure suggested that an alternate
orientation of the central benzimidazole/benzoxazole ring may
also be possible, however, the essential hydrogen bonding with
the hinge region and the hydrophobic interactions of the chroman
are maintained in that structure as well.
In conclusion, a potent series of benzimidazole- and benzoxaz-
ole-based ROCK inhibitors have been developed from the initial
amide lead 1. Optimization of the nitrogen heterocycle led to com-
pounds with improved microsomal stability and reduced CYP inhi-
bition, while substitution of the chroman ring produced
compounds with improved potency and selectivity against PKA.
The benzoxazole series have good ROCK-II potency and selectivity
against PKA with excellent pharmacokinetic properties. The benz-
imidazole series possess excellent biochemical and cell-based po-
tency, with ꢀ100-fold selectivity over PKA and many also have
good pharmacokinetic properties. Future efforts to optimize these
compounds will focus on the enantioselective synthesis and evalu-
ation of both enantiomers, the development of isoform selective
ROCK (I and II) inhibitors and the achievement of pharmacokinetic
properties tailored to specific therapeutic applications. These stud-
ies will be reported in due course.
Figure 2. Structure and IC50 values of fluorinated derivative 32.

E. H. Sessions et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6390–6393
6393
S. F.; Elkins, P. A.; Kirkpatrick, R. B.; Dul, E.; Khandekar, S. S.; Yi, T.; Jung, D. K.;
Wright, L. L.; Smith, G. K.; Behm, D. J.; Doe, C. P.; Bentley, R.; Chen, Z. X.; Hu, E.;
Lee, D. J. Med. Chem. 2007, 50, 2; (b) Goodman, K. B.; Cui, H.; Dowdell, S. E.;
Gaitanopoulos, D. E.; Ivy, R. L.; Sehon, C. A.; Stavenger, R. A.; Wang, G. Z.; Viet,
A. Q.; Xu, W.; Ye, G.; Semus, S. F.; Evans, C.; Fries, H. E.; Jolivette, L. J.;
Kirkpatrick, R. B.; Dul, E.; Khandekar, S. S.; Yi, T.; Jung, D. K.; Wright, L. L.;
Smith, G. K.; Behm, D. J.; Bentley, R.; Doe, C. P.; Hu, E.; Lee, D. J. Med. Chem.
2007, 50, 6; (c) Iwakubo, M.; Takami, A.; Okada, Y.; Kawata, T.; Tagami, Y.; Sato,
M.; Sugiyama, T.; Fukushima, K.; Taya, S.; Amano, M.; Kaibuchi, K.; Iijima, H.
Bioorg. Med. Chem. 2007, 15, 1022.
Acknowledgments
We thank Prof. Patrick Griffin and Prof. William Roush for their
support and Dr. Derek Duckett and Ms. Weimin Chen for the coun-
ter screening against p38 and JNK kinases.
References and notes
14. Schröter, T.; Minond, D.; Weiser, A.; Dao, C.; Habel, J.; Spicer, T.; Chase,
P.; Baillargeon, P.; Scampavia, L.; Schurer, S.; Chung, C.; Mader, C.;
Southern, M.; Tsinoremas, N.; LoGrasso, P.; Hodder, P. J. Biomol. Screen
2008, 13, 40.
1. Nakagawa, O.; Fujisawa, K.; Ishizaki, T.; Saito, Y.; Nakao, K.; Narumiya, S. FEBS
Lett. 1996, 392, 189.
2. Riento, K.; Ridley, A. J. Nat. Rev. Mol. Cell. Biol. 2003, 4, 446.
3. (a) Uehata, M.; Ishizaki, T.; Sato, H.; Ono, T.; Kawahara, T.; Morishita, T.;
Tamakawa, H.; Yamagami, K.; Inui, J.; Maekawa, M.; Narumiya, S. Nature 1997,
389, 990; (b) Doe, C.; Bentley, R.; Behm, D. J.; Lafferty, R.; Stavenger, R.; Jung, D.;
Bamford, M.; Panchal, T.; Grygielko, E.; Wright, L. L.; Smith, G. K.; Chen, Z.;
Webb, C.; Khandekar, S.; Yi, T.; Kirkpatrick, R.; Dul, E.; Jolivette, L.; Marino, J. P.,
Jr.; Willette, R.; Lee, D.; Hu, E. J. Pharmacol. Exp. Ther. 2007, 320, 89.
4. Kataoka, C.; Egashira, K.; Inoue, S.; Takemoto, M.; Ni, W.; Koyanagi, M.;
Kitamoto, S.; Usui Hypertension 2002, 39, 245.
15. Assays were performed using the STK2 kinase system from Cisbio.
5 lL
mixture of a 1-lM STK2 substrate and ATP (20 lM) in STK-buffer was added to
the wells using a BioRAPTR FRDTM Workstation (Aurora Discovery). 20 nL of test
compounds was dispensed. Reaction was started by addition 0.5 nM ROCK-II in
STK-buffer. After 4 h at rt the reaction was stopped by addition of 10
antibody and 62.5 nM Sa-XL in detection buffer. After 1 h at rt the plates were
read on the Viewlux in HTRF mode.
lL of 1Â
16. PKA was chosen as a routine preliminary kinase counterscreen because it is
among the kinases most closely related to ROCK, along with MRCK and Akt.
Promising compounds will be more thoroughly profiled, particularly against a
much larger panel of kinases and also assessed for affinity to other relevant
receptors, enzymes, and ion channels.
5. Satoh, S.; Toshima, Y.; Ikegaki, I.; Iwasaki, M.; Asano, T. Brain Res. 2007, 1128,
175.
6. Ying, H.; Biroc, S. L.; Li, W. W., et al Mol. Cancer Ther. 2006, 5, 2158.
7. Sun, X.; Minohara, M.; Kikuchi, H.; Ishizu, T.; Tanaka, M.; Piao, H.; Osoegawa,
M.; Ohyagi, Y.; Shimokawa, H.; Kira, J. I. J. Neuroimmunol. 2006, 180, 126.
8. Bivalacqua, T. J.; Champion, H. C.; Usta, M. F.; Cellek, S.; Chitaley, K.; Webb, R.
C.; Lewis, R. L.; Mills, T. M.; Hellstrom, W. J. G.; Kadowitz, P. J. Proc. Natl. Acad.
Sci. U.S.A. 2004, 101, 9121.
9. (a) Tanihara, H.; Inatani, M.; Honjo, M.; Tokushige, H.; Azuma, J.; Araie, M. Arch.
Ophthalmol. 2008, 3, 309; (b) Waki, M.; Yoshida, Y.; Oka, T.; Azuma, M. Curr. Eye
Res. 2001, 22, 470.
10. Shibuya, M.; Suzuki, Y.; Sugita, K.; Saito, I.; Sasaki, T.; Takakura, K.; Nagata, I.;
Kikuchi, H.; Takemae, T.; Hidaka, H., et al J. Neurosurg. 1992, 76, 571.
11. Feng, Y.; Cameron, M. D.; Frackowiak, B.; Griffin, E.; Lin, L.; Ruiz, C.; Schröter, T.;
LoGrasso, P. Bioorg. Med. Chem. Lett. 2007, 17, 2355.
12. Chen, Y. T.; Bannister, T.; Weiser, A.; Griffin, E.; Lin, L.; Ruiz, C.; Cameron, M.;
Duckett, D.; Schurer, S.; Schröter, T.; LoGrasso, P.; Feng, Y. Bioorg. Med. Chem.
Lett. 2008, 18, 6406.
17. Schröter, T.; Griffin, E.; Weiser, A.; Feng, Y.; LoGrasso, P. Biochem. Biophys. Res.
Commun. 2008, 374(2), 356.
18. % Inhibition of CYP450 isoforms 2C9/2D6/3A4/1A2 at 10
lM were: 2: 80/41/
15/27; 3: 84/84/47/51; 18: 95/92/96/90; 19: 13/15/-3/31; 20: -8/-4/-6/0.
19. Preliminary SAR, not shown, indicated that substitution at the 6-position is
preferred.
20. The synthesis of 32 follows the Scheme
2 procedures, starting with the
fluorinated diamine, which is available by SnCl2 reduction of the nitro group of
commercially available 2-amino-4-bromo-6-fluoro-nitrobenzene.
21. Feng, Y.; Yin, Y.; Weiser, A.; Griffin, E.; Cameron, M.; Lin, L.; Ruiz, C.; Schurer, S.;
Inoue, T.; Rao, V.; Schroter, T.; LoGrasso, P. J. Med. Chem. 2008. doi:10.1021/
jm800986w.
22. Yamaguchi, H.; Kasa, M.; Amano, M.; Kaibuchi, K.; Hakoshima, T. Structure
2006, 14, 589.
23. Yamaguchi, H.; Miwa, Y.; Kasa, M.; Kitano, K.; Amano, M.; Kaibuchi, K.;
Hakoshima, T. J. Biochem. 2006, 140, 305.
13. (a) Stavenger, R. A.; Cui, H.; Dowdell, S. E.; Franz, R. G.; Gaitanopoulos, D. E.;
Goodman, K. B.; Hilfiker, M. A.; Ivy, R. L.; Leber, J. D.; Marino, J. P., Jr.; Oh, H.-Y.;
Viet, A. Q.; Xu, W.; Ye, G.; Zhang, D.; Zhao, Y.; Jolivette, L. J.; Head, M. S.; Semus,