Chroman-3-amides as potent Rho kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.10.080

Inhibition of Rho kinase (ROCK) is an attractive strategy for the treatment of diseases such as hypertension, glaucoma, and cancer. Here we report chroman-3-amides as highly potent ROCK inhibitors with sufficient kinase selectivity, excellent cell activit

Full text of DOI:10.1016/j.bmcl.2008.10.080

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6406–6409
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Chroman-3-amides as potent Rho kinase inhibitors
Yen Ting Chen, Thomas D. Bannister, Amiee Weiser, Evelyn Griffin, Li Lin, Claudia Ruiz, Michael D. Cameron,
*
Stephan Schürer, Derek Duckett, 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:
Inhibition of Rho kinase (ROCK) is an attractive strategy for the treatment of diseases such as hyperten-
sion, glaucoma, and cancer. Here we report chroman-3-amides as highly potent ROCK inhibitors with suf-
ficient kinase selectivity, excellent cell activity, good microsomal stability, and desirable pharmacokinetic
properties for study as potential therapeutic agents.
Received 18 August 2008
Revised 16 October 2008
Accepted 17 October 2008
Available online 22 October 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Rho kinase
ROCK
ROCK-II
Glaucoma
Kinase
Chromans
ROCK-I and ROCK-II are highly homologous isoforms1 of Rho ki-
nase (ROCK), a downstream effector of the small GTPase Rho. These
serine-threonine kinases have numerous cellular functions,2
including regulation of the formation of stress fibers and focal
adhesions via the phosphorylation of myosin light chain (MLC).3
Inhibition of ROCK promotes effects such as relaxation of vascular
smooth muscle fibers,4 alteration of intercellular junctions in the
trabecular meshwork of the eye,5 and promotion of neuronal out-
growth.6 ROCK inhibitors have been proposed for treating hyper-
tension,4 ischemic stroke,7 cancer,8 erectile dysfunction,9
glaucoma,10 multiple sclerosis,11 and spinal cord injury.12 Fasudil,
the only marketed ROCK inhibitor, is used in Japan for cerebral
vasospasm. In recent years, a variety of other ROCK inhibitors have
been reported,13 including aminofurazans,14 indazoles,15 and
isoquinolines.16
We began a program to identify potent and selective ROCK
inhibitors with pharmacokinetic (PK) profiles favorable for ther-
apy. The analysis of known ROCK ligands, paired with the study
of hits from a high-throughput screen, led us to identify benzodi-
oxane 1 as a promising lead (Fig. 1). This compound was a potent
inhibitor of ROCK-II (IC50 = 2 nM) with fair selectivity (ꢀ100-fold)
over the highly homologous protein kinase A (PKA)17 and was po-
tent in a cell-based myosin light chain bis-phosphorylation assay
(ppMLC)18 (IC50 = 72 nM). In PK studies, benzodioxane 1 had mod-
est stability in vitro upon exposure to the human liver microsome
Figure 1. Structures of benzodioxane 1 and chroman 2.
(HLM, t1/2 = 20 min) and had essentially no oral bioavailability in
rats (F < 1%).
We reasoned that the instability of benzodioxane 1 toward liver
microsomes might correlate with electron deficiency of the amide.
Chroman 2, in which the oxygen atom
a- to the amide in com-
pound 1 was replaced with a methylene group was prepared and
indeed had improved human microsomal stability (t1/2 = 65 min)
with similar affinity for ROCK-II (IC50 < 2 nM) and selectivity over
PKA (Table 1). Moreover, compound 2 showed enhanced potency
in the cell-based ppMLC assay (IC50 = 25 nM) and slightly better
oral bioavailability (F = 6%).
The straightforward synthesis of compound 2 and its close ana-
logs is outlined in Scheme 1. Coupling of acid 3 with bromoaniline
4 followed by Suzuki heteroarylation with 1H-4-pyrazoleboronic
acid pinacol ester under microwave irradiation gave compounds
with the general structure 5.
Dual routes to the preparation of the substituted chroman-3-
carboxylic acid are shown in Scheme 2. Ethoxycarbonylation of
4-chromanone 6 gave b-ketoester 7.19 Cationic hydrogenation of
the ketone to chroman 8, followed by saponification afforded acid
9. Alternatively, substituted salicylaldehyde 10 and benzyl acrylate
* Corresponding author. Tel.: +1 561 228 2201.
E-mail address: yfeng@scripps.edu (Y. Feng).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.080

Y. T. Chen et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6406–6409
6407
procedure.20 Coupling of functionalized acid 9 and the substituted
anilines and Suzuki coupling was accomplished by the two-step
sequence illustrated in Scheme 1 to give the desired inhibitors.
Inhibitors containing an amide group at C6 of the chroman ring
were prepared as described in Scheme 4. In this route, acid 9a was
coupled to bromoaniline (4, R2 = H) to give bromide 14, which was
saponified to acid 15. Coupling of acid 15 with available amines
and microwave-assisted Suzuki heteroarylation gave chroman
amides with the general structure 16.
Table 1
Kinase inhibition and stability of chroman 2 and analogs.
R2
Enzyme assays
IC50 (nM)
ppMLC cell
assay IC50 (nM)
t1/2 (min)
b
Compound
a
a
HLM
ROCK-II
PKA
Table 1 summarizes the effects of substitution in the central
phenyl ring upon affinity for ROCK-II and PKA, activity in the
cell-based ppMLC assay, and stability in vitro to a preparation of
human liver microsomes (HLM).21 Compounds with R2 = methoxy
in the 20 or 30 position (inhibitors 17 and 18) retained ROCK-II po-
tency relative to 2 with enhanced cell-based activity (3- to 4-fold)
but were less stable to HLM. Several fluorinated analogs (com-
pounds 19–22) have similar potency and cell-based activity yet
have neither enhanced PKA selectivity nor increased stability. Most
encouraging, however, was the finding that inhibitor 23, with a 2-
dimethylaminoethoxy substituent, maintained the potency and
stability of the lead with a marked improvement in PKA selectivity
(PKA/ROCK-II ratio = 470, vs 60 for inhibitor 2) and with slightly
enhanced activity (fourfold) in the cell-based ppMLC assay. Regio-
isomer 24 was curiously a far less effective ROCK-II inhibitor.
We next investigated the effects of substituent R3 on the chro-
man ring (Table 2). Compound 25, with a C8 methoxy group, had
less affinity for ROCK-II than chroman 2, whereas 6-methoxy
substituted analog 26 retained ROCK-II potency with enhanced
selectivity (140-fold) over PKA, although microsomal stability
was reduced. Other small groups at the C6 position were well-tol-
erated with respect to ROCK-II inhibition, with compounds 27 and
31 displaying high potency in the cell-based ppMLC assay. Replac-
ing the metabolically labile ester with an amide led to compound
33, a potent ROCK-II inhibitor with over 200-fold selectivity for
PKA. In brief, chromans containing 6-methoxy, 6-fluoro, and 6-pri-
mary amide substitutions (inhibitors 26, 27, and 33, respectively)
possessed the best balance of properties.
2
H
2
4
3
4
4
5
17
3
127
290
109
87
82
117
531
25
7
6
80
15
6
65
8
17
18
19
20
21
22
23
30-OMe
20-OMe
14
40
37
25
13
69
20-F
30,50-F
20-OMe,50-F
20-F,50-OMe
20-Dimethyl-
aminoethoxy
30-Dimethyl-
aminoethoxy
8
6
1416
24
640
>20,000
ndc
ndc
a
Values are means of two or more experiments. The error in these values is
within 30% of the mean.
b
2 mg/mL human liver microsomes were used in stability studies.
Not determined.
c
Scheme 1. Reagents and conditions: (i) HATU, N-methylmorpholine, DMF, rt, 14 h;
(ii) 1H-4-pyrazoleboronic acid pinacol ester, Pd(PPh3)4, Na2CO3, toluene, ethanol,
H2O, 1 h, 140 °C, lW.
We sought to determine if the effects of substituents R2 and R3
in tuning ROCK-II inhibition, selectivity, stability, and especially
potency in the cell-based assay could be combined. Hence, we pre-
pared a series of chromans with the 20-methoxy, 30-methoxy, or 20-
dimethylaminoethoxy group and these compounds were more
effective in the cell-based assay relative to their unsubstituted
counterparts (Table 3). The PKA selectivity trend was mixed for
the 20- and 30-methoxy compounds. However, 20-dimethylamino-
ethoxy analogs 38 and 47 were remarkably selective (>10,000-fold)
for ROCK-II over PKA.
Scheme 2. (a) Reagents and conditions: (i) LiHMDS, THF, ꢁ78 °C, 30 min; (ii)
ethylchloro-formate, THF, ꢁ78 °C, 1 h; (b) Et3SiH, TFA, rt, 14 h; (c) LiOH, methanol,
THF, H2O, rt, 2 h; (d) benzyl acrylate, DABCO, 0.5 h, 160 °C,
lW; (e) H2, 10% Pd/C,
methanol, rt, 14 h.
condensed to give chromene 11, which upon hydrogenolysis also
provided acid 9.
We explored the effects of structural variation in the central
ring using commercially available 4-bromoaryl amines and also
dimethylaminoethoxy analogs 4a and 4b. Amine 4a was prepared
from 4-bromo-2-fluoro-1-nitrobenzene (12) via nucleophilic
substitution followed by stannous chloride reduction (Scheme 3).
3-Dimethylaminoethoxy analog 4b was prepared by a literature
Scheme 4. Reagents and conditions: (a) 4-bromoaniline, HATU, N-methylmorpho-
line, DMF, rt, 14 h; (b) LiOH, methanol, THF, rt, 2 h, 90%; (c) (i) NHR1R2, HATU,
N-methylmorpholine, DMF, rt, 14 h; (ii) 1H-4-pyrazoleboronic acid pinacol ester,
Scheme 3. Reagents and conditions: (a) NaH, 2-(dimethylamino)ethanol, THF, rt,
7 h, 92%; (b) SnCl2, ethanol, 70 °C, 1.5 h, 71%.
Pd(PPh3)4, Na2CO3, toluene, ethanol, H2O, 1 h, 140 °C, lW.

6408
Y. T. Chen et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6406–6409
Table 2
Glu170 and Met172 in the kinase hinge region, a third between the
Kinase inhibition and stability of substituted chromans.
chroman amide carbonyl and Lys121 in the phosphate binding re-
gion, and a fourth between the dimethylaminoethoxy moiety and
Asp176. Co-crystal structures of ROCK with ligands Fasudil and
Y-27632 have been previously reported.23 The docking pose shown
in Figure 2 suggests that our ROCK-II inhibitors have combined the
ligand–enzyme interactions from both Fasudil and Y-27632.
In this model the chroman group also interacts in a hydrophobic
binding region composed of Phe103 of the flexible P loop, Leu123,
the carbon chain of Lys121, and Phe136.23b The 6-methoxy group
on the chroman may maximize this interaction and explain the
high ROCK-II affinity seen in this series. Exceptional PKA selectivity
may be due to three factors: (i) increased steric hindrance for bind-
ing to PKA due to Thr184 versus Ala231 in ROCK-II (ii) poor inter-
action of the dimethylaminoethoxy moiety with Glu128 in PKA, in
contrast to its favorable interaction with the corresponding Asp176
in ROCK-II, and (iii) optimized interaction of the chroman ring with
the hydrophobic binding region and the flexible and distinct P loop
of ROCK-II.
In summary, we have synthesized a series of chroman-3-amides
with remarkable inhibition of ROCK-II and other favorable proper-
ties for further study. Assessment of the in vivo efficacy of these
inhibitors in therapeutically relevant animal models, SAR studies
in the pyrrole region, the enantioselective synthesis and evaluation
of both enantiomers, and a detailed analysis of factors affecting
affinity for the enzyme isoforms, ROCK-I and ROCK-II, are under-
way and will be reported in due course.
Compound R3
Enzyme assays
IC50 (nM)
ppMLC cell t1/2 (min)
assay IC50
(nM)
b
a
a
HLM
ROCK –II PKA
25
26
27
28
29
30
31
32
33
34
35
8-OMe
6-OMe
6-F
6-Cl
6-Me
6-CO2H
6-CO2Me
6-CONMe2
6-CONH(CH2)2NMe2
6-CON(Me)(CH2)2NMe2
6-CON(CH2CH2)2NMe
285
3
3734
437
ndc
38
71
nd
29
105
138
34
nd
20
70
35
4
270
5
>20,000 3487
10
30
10
86
<2
93
78
2000
16850
>20,000 22
1644
206
4000
751
500
47
>20,000 1500
3062
51
41
ndc
a
Average of two or more measurements. The error in these values is within 30%
of the average.
b
2 mg/mL human liver microsomes were used in stability studies.
Not determined.
c
Compounds 23, 38, and 41 were selected for further selectivity
screening and were essentially inactive against several other ki-
nases except for MRCK
a
(Table 4),22 the selectivity for which was
moderate (ꢀ100-fold). Pharmacokinetic evaluation in rats showed
that these compounds had improved oral bioavailability (27–35%)
relative to lead compounds 1 and 2 (Table 5). Inhibitor 38 dis-
played an especially intriguing pharmacokinetic profile, with high
systemic exposure upon oral delivery.
Inhibitor 38 was docked into the catalytic domain of ROCK-II
(gray, modeled from PDB entry 2ETR, chain A) by methods de-
scribed previously22a and the obtained binding mode is illustrated
in Figure 2. This picture shows the S enantiomer of 38 and our
docking suggested that the opposite enantiomer had a similar
binding motif.22a Compound 38 (orange) forms four hydrogen
bonds with ROCK-II: two between the pyrazole group and residues
Table 4
Kinase inhibition of selected chromans.
a
Compound
Enzyme assay IC50 (nM)
ROCK-II
PKA
1416
>20,000
7316
AKT1
MRCK
a
JNK3
p38
23
38
41
3
<2
<2
>20,000
>20,000
>20,000
210
150
79
>20,000
>20,000
>20,000
>20,000
>20,000
>20,000
a
Average of two or more measurements with error in these values within 60%
of the average.
Table 3
Kinase inhibition and stability of substituted chromans.
a
a
b
Compound
R3
R2
Enzyme assay IC50 (nM)
ROCK-II PKA
1348
ppMLC cell assay IC50 (nM)
t1/2 (min) HLM
36
37
6-OMe
6-OMe
30-OMe
20-OMe
3
3
<6
<6
6
12
327
38
(SR3850)
39
40
41
42
43
44
45
6-OMe
6-F
6-F
20-Dimethyl-aminoethoxy
30-OMe
<2
7
3
<2
2
3
5
12
4
>20,000
1518
705
7316
955
1259
2390
1628
1764
>20,000
<6
<6
<6
<6
38
66
< 6
52
35
16
46
28
23
56
36
36
62
6
20-OMe
6-F
20-Dimethyl-aminoethoxy
30-OMe
6-Cl
6-Cl
6-Cl
6-Me
6-Me
6-Me
20-OMe
20-Dimethyl-aminoethoxy
30-OMe
46
47
20-OMe
13
62
20-Dimethyl-aminoethoxy
<2
a
Average of two or more measurements with error in these values within 30% of the average.
2 mg/mL human liver microsomes were used in stability studies.
b

Y. T. Chen et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6406–6409
6409
Table 5
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; (c) Hirooka,
Y.; Shimokawa, H.; Takeshita, A. Drug News Perspect. 2004, 17, 523.
5. Thieme, H.; Nuskovski, M.; Nass, J. U.; Pleyer, U.; Strauss, O.; Wiederholt, M.
Invest. Ophthalmol. Vis. Sci. 2000, 13, 4240.
6. Amano, M.; Chihara, K.; Nakamura, N.; Fukata, Y.; Yano, T.; Shibata, M.; Ikebe,
M.; Kaibuchi, K. Genes Cells 1998, 3, 177.
7. Satoh, S.; Toshima, Y.; Ikegaki, I.; Iwasaki, M.; Asano, T. Brain Res. 2007, 1128,
175.
8. (a) Nakajima, M.; Hayashi, K.; Egi, Y.; Katayama, K.; Amano, Y.; Uehata, M.;
Ohtsuki, M.; Fujii, A.; Oshita, K.; Kataoka, H. Cancer Chemother. Pharmacol. 2003,
52, 319; (b) Nakajima, M.; Hayashi, K.; Katayama, K.; Amano, Y.; Egi, Y.; Uehata,
M.; Goto, N.; Kondo, T. Eur. J. Pharmacol. 2003, 459, 113; (c) Gaggioli, C.; Hooper,
S.; Hidalgo-Carcedo, C.; Grosse, R.; Marshall, J. F.; Harrington, K.; Sahai, E. Nat.
Cell Biol. 2007, 12, 1392.
Rat ADME data for selected ROCK-II inhibitorsa.
Compound
38
23
41
Dose iv/po (mg/kg)
Cl (mL/min/kg)
Vss (L/kg)
t1/2 (h)
Cmax po (
1.0/2.0
67 11
1.0/2.0
1.0/2.0
61 11
5.9 0.9
1.5 0.2
0.10 0.01
23
2
4.6 0.9
0.9 0.4
0.17 0.04
33 11
1.7 0.1
1.0 0.1
0.32 0.07
l
M)
Oral F (%)
35
2
27
5
a
Data was generated from three determinations.
9. 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. USA 2004, 101, 9121.
10. Waki, M.; Yoshida, Y.; Oka, T.; Azuma, M. Curr. Eye Res. 2001, 22, 470.
11. 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.
12. Chan, C. C.; Khodarahmi, K.; Liu, J.; Sutherland, D.; Oschipok, L. W.; Steeves, J.
D.; Tetzlaff, W. Exp. Neurol. 2005, 196, 352.
13. Liao, J. K.; Seto, M.; Noma, K. J. Cardiovasc. Pharmacol. 2007, 50, 17.
14. 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,
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.
15. (a) Feng, Y.; Cameron, M. D.; Frackowiak, B.; Griffin, E.; Lin, L.; Ruiz, C.;
Schröter, T.; LoGrasso, P. Bioorg. Med. Chem. Lett. 2007, 17, 2355; (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.
16. (a) 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. Lett. 2007, 15, 1022; (b) Tamura, M.; Nakao, H.; Yoshizaki, H.;
Shiratsuchi, M.; Shigyo, H.; Yamada, H.; Ozawa, T.; Totsuka, J.; Hidaka, H.
Biochim. Biophys. Acta 2005, 245, 1754.
Figure 2. Docking of compound 38 into the ROCK-II kinase domain.
Acknowledgments
17. Jacobs, M.; Hayakawa, K.; Swenson, L.; Bellorn, S.; Fleming, M.; Taslimi, P.;
Doran, J. J. Biol. Chem. 2006, 281, 260.
We thank Prof. William Roush and Prof. Patrick Griffin for their
support, Ms. Weimin Chen for her assistance with JNK3 and p38
assays, and Dr. Hampton Sessions for helpful discussions.
18. Schröter, T.; Griffin, E.; Weiser, A.; Feng, Y.; LoGrasso, P. Biochem. Biophys. Res.
Comm. 2008, 374, 356–360.
19. Castonguay, L. A.; Hagmann, W. K.; Lin, L. S.; Shah, S. K. WO 03086288, 2003.
20. Gaster, L. M.; Wyman, P. A. WO 9850346, 1998.
21. ROCK and PKA assays were performed as described in: Schröter, T.; Minond, D.;
Weiser, A.; Dao, C.; Habel, J.; Spicer, T.; Chase, P.; Baillargeon, P.; Scampavia, L.;
Schürer, S. C.; Chung, C.; Mader, C.; Southern, M.; Tsinoremas, N.; LoGrasso, P.;
Hodder, P. J. Biomol. Screen. 2008, 13, 17.
22. (a) AKT1 and MRCK (assays were performed as described in: Feng, Y.; Yin, Y.;
Weiser, A.; Griffin, E.; Cameron, M. D.; Lin, L.; Ruiz, C.; Schürer, S. C.; Inoue, T.;
Rao, P. V.; Schröter, T.; LoGrasso, P. J. Med. Chem. 2008, DOI: 10.1021/
jm800986w.; (b) Jiang, R.; Duckett, D.; Chen, W.; Habel, J.; Ling, Y. Y.; LoGrasso,
P.; Kamenecka, T. M. Bioorg. Med. Chem. Lett. 2007, 17, 6378.
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