Substituted 2H-isoquinolin-1-one as potent Rho-Kinase inhibitors. Part 1: Hit-to-lead account

Article abstract of DOI:10.1016/j.bmcl.2010.04.070

Two closely related scaffolds were identified through an uHTS campaign as desirable starting points for the development of Rho-Kinase (ROCK) inhibitors. Here, we describe our hit-to-lead evaluation process which culminated in the rapid discovery of potent leads such as 22 which successfully demonstrated an early in vivo proof of concept for anti-hypertensive activity.

Full text of DOI:10.1016/j.bmcl.2010.04.070

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3235–3239
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Substituted 2H-isoquinolin-1-one as potent Rho-Kinase inhibitors. Part 1:
Hit-to-lead account
d
b
a
b
c
Frank Wu a, , Frank H. Büttner , Rhonda Chen , Eugene Hickey , Scott Jakes , Paul Kaplita ,
Mohammed A. Kashem a, Steven Kerr b, Stanley Kugler a, Zofia Paw b, Anthony Prokopowicz a,
Cheng-Kon Shih b, Roger Snow a, Erick Young a, Charles L. Cywin a
*
a Department of Medicinal Chemistry, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Rd., Ridgefield, CT 06877, USA
b Department of Cardiometabolic Diseases, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Rd., Ridgefield, CT 06877, USA
c Department of Drug Discovery Support, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Rd., Ridgefield, CT 06877, USA
d Boehringer Ingelheim Pharma GmbH and Co. KG, Birkendorfer Strasse 65, D-88397 Biberach an der Riss, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Two closely related scaffolds were identified through an uHTS campaign as desirable starting points for
the development of Rho-Kinase (ROCK) inhibitors. Here, we describe our hit-to-lead evaluation process
which culminated in the rapid discovery of potent leads such as 22 which successfully demonstrated
an early in vivo proof of concept for anti-hypertensive activity.
Received 22 February 2010
Revised 12 April 2010
Accepted 16 April 2010
Available online 22 April 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keyword:
Rho-Kinase inhibitors
Rho-Kinase (ROCK) is a member of the serine–threonine protein
kinase family. ROCK exists in two isoforms, ROCK 1 and ROCK 2.1
ROCK has been identified as an effector molecule of RhoA, a small
GTP-binding protein (G protein) that is upregulated in a variety of
pathological conditions. RhoA/ROCK signaling pathway plays an
important role in signal transduction initiated by several vasoac-
tive factors, for example angiotensin II,2a urotension II,2b endothe-
lin-1,2c serotonin,2d norepinephrine2e and platelet-derived growth
factor (PDGF).2f Many of these factors are implicated in the patho-
genesis of cardiovascular disease. One well established contribut-
ing role ROCK plays in cardiovascular disease is hypertension via
increased smooth muscle contraction.3 Mechanistically ROCK cata-
lyzes an inactivating phosphorylation of the myosin-binding sub-
unit of myosin light chain phosphatase, which leads to prolonged
smooth muscle contraction and increased vascular tone.1,3 There-
fore, ROCK inhibition represents a new approach to treat the car-
diovascular diseases such as hypertension.
and reasonable kinase selectivity profiles to serve as tools for
further therapeutic target validation and a starting point for lead
optimization.
Boehringer Ingelheim’s compound collection was screened in a
homogenous luciferase assay using ROCK 2, substrate AKRRRLSSL-
RA, and a luciferin–luciferase detection reagent to quantify resid-
ual ATP.4 Included in the screening collection was a library
designed as potential ROCK inhibitors based in part on the work
of Takami et al.5 This consisted of bicyclic cores with hydrogen
bonding moieties that could potentially bind to the hinge region
of the kinase. The cores were attached through an amide linker
to a set of hydrophobic fragments. Two members of this library
were the quinazolinone inhibitor 1 and the isoquinolone 2 with
modest potency of 2.9 and 1.7 lM, respectively. Several related
heterocyclic cores containing potential H-bond donor and acceptor
H
N
A number of ROCK inhibitors have been reported in recent
years,4 the most cited being the isoquinoline derivative Fasudil
and the pyridine-based Y-27632 (Fig. 1). Here, we wish to report
our discovery and hit-to-lead evaluation of a novel series of substi-
tuted 2H-isoquinolin-1-one and 3H-quinazolin-4-one ROCK inhib-
itors (Fig. 1). These compounds provide desirable levels of potency
NH2
H
N
S
X
N
R
H
O
O
H
N
H
HN
O
X = C, N
N
O
O
N
1 (X = N, R = benzyl)
2 (X = C, R = benzyl)
Y-27632
Fasudil
* Corresponding author. Tel.: +1 203 791 6317.
Figure 1. Structures of Fasudil, Y-27632 and compound 1 and 2.
E-mail address: frank.wu@boehringer-ingelheim.com (F. Wu).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.070

3236
F. Wu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3235–3239
Table 3
SAR studies of linker
motifs were not tolerated (Table 1). The phthalazindione 3, phthal-
imide 4 and benzotriazole 5 showed no activity up to 10 M. Our
l
initial goal was to demonstrate tractable SAR and evaluate the
opportunity to improve potency. To this end, results of exploration
of the SAR of 1 are summarized in Tables 2–4.
Table 2 shows the results of phenyl ring amide regioisomer SAR
using both quinazolinone isoquinolone cores as templates. These
studies showed an absolute requirement for attachment at the b
position suggesting a strong spatial requirement of the hinge bind-
ing moiety with regard to a proper orientation of the benzyl amide
moiety.
Y
HN
O
Compound
Y
ROCK 2 IC50 (nM)
1700
H
N
2
We next explored replacements of the exocyclic amide linker.
Observed in Table 3, carbamate 12 and urea 13 were three- to four-
O
O
H
N
O
12
13
14
360
520
Table 1
SAR of heterocycle core
H
N
H
N
H
N
O
S
heterocycle
O
H
N
O
>10,000
Compound
Heterocycle
ROCK 2 IC50 (nM)
2900
O
N
HN
Cambrex PKLight ATP Detection Reagent using luciferin–luciferase to quantify
residual ATP. Assay values reported as the mean of n P 2 independent replicates,
and standard error (SE) typically <20%.
1
O
HN
2
3
1700
Table 4
SAR studies of R2 amide of molecule
O
O
H
R2
N
HN
HN
>10,000
O
HN
1
R
O
O
O
Compound
R1
R2
ROCK 2 IC50 (nM)
HN
2
H
PhCH2–
PhCH2–
CH3–
1700
320
1300
>10,000
280
430
57
12
19
26
5100
210
110
48
4
5
>10,000
>10,000
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
O
Ph
N
N
PhCH2CH2–
Cyclohexylmethyl–
NH2(Ph)CH–
MeNH(Ph)CH–
Cyclopropylamino(Ph)CH–
Me2N(Ph)CH–
PhCH2NH(Ph)CH–
PhCH2NH(Me)CH–
PhCH2NHCH2–
(S)-NH2(PhCH2)CH–
(R)-NH2(PhCH2)CH–
N
H
Cambrex PKLight ATP Detection Reagent using luciferin–luciferase to quantify
residual ATP. Assay values reported as the mean of n P 2 independent replicates,
and standard error (SE) typically <20%.
160
Table 2
SAR studies of regioisomers
Cambrex PKLight ATP Detection Reagent using luciferin–luciferase to quantify
residual ATP. Assay values reported as the mean of n P 2 independent replicates,
and standard error (SE) typically <20%.
H
N
a
b
X
O
O
HN
c
d
fold more potent than 2; however, sulfonamide 14 did not show
significant activity up to 10 lM. The loss of activity of 14 may be
Compound
X
Position
ROCK 2 IC50 (nM)
due to the fact that, unlike amide bonds, sulfonamides adopt pre-
dominantly a cis-bond rotamer, resulting in an unfavorable orien-
tation of the molecule.
A summary of amide substituent R2 SAR is presented in Table 4.
We used 7-chloro substituted isoquinolone 15 as a starting refer-
ence as it provides a fivefold improvement in potency than non-
substituted core (15 vs 2).
1
6
7
8
2
9
10
11
N
N
N
N
C
C
C
C
b
a
c
d
b
a
c
2900
>10,000
>10,000
>10,000
1700
>10,000
>10,000
>10,000
d
Replacing the benzyl group with a methyl group resulted in a
fourfold loss of potency (15 vs 16), while replacement of the benzyl
group with phenyl was detrimental (15 vs 17). These data implied
Cambrex PKLight ATP Detection Reagent using luciferin–luciferase to quantify
residual ATP. Assay values reported as the mean of n P 2 independent replicates,
and standard error (SE) typically <20%.

F. Wu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3235–3239
3237
that there exists an important hydrophobic interaction in the re-
gion, which requires a proper orientation of the phenyl group.
Compound 17 failed to achieve this conformation. Increasing linker
length (phenethyl 18 vs 15) had no significant effect on the activ-
ity, and a cyclohexylmethyl replacement of the benzyl group (19 vs
15) was tolerated. More interestingly a significant increase of
activity was observed in 20 with the incorporation of an alpha-
amino group. Modulation with N-substitution with methyl and
cyclopropyl groups further improved the potency to 12 and
19 nM, respectively (21–22). Compound 23 with a N,N-dimethyl
group had a slight decrease of activity relative to 21. These data
suggested that the basic amino group, most likely protonated at
physiological pH, participates in a specific interaction with the pro-
tein as a salt bridge. A significant loss of activity (>200-fold) was
observed when further increasing size of N-substitution group
(e.g., with benzyl group 24). It should be noted that this loss of po-
tency was able to be mostly restored by decreasing the size of the
group substituted on the alpha carbon as was seen in 25–26. These
results hinted that this portion of molecule cannot tolerate the two
bulky/hydrophobic moieties simultaneously. Compound 20 was
originally synthesized as a racemic mixture as phenylglycine 20
was known to racemize readily. In order to examine the chiral pref-
binding region pocket, interacting with acid residue of Asp232
and the side chain carbonyl of Asn219. This model was instrumen-
tal in guiding our early SAR work, and proved to be consistent with
X-ray crystallography work, which became available later.8
The ATP binding sites of kinase proteins are relatively conserved
which poses a significant challenge discovering of highly selective
inhibitors. In this program a two-stage kinase selectivity testing
strategy was implemented. Compounds were first tested against
a panel of counter screen kinases Prkce, DMPK, cdc42, Prkcl2,
which were chosen as representatives based on their similarity
to Rho-Kinase in terms of sequence homology, phylogenetic rela-
tionship, biological relationship and chemical susceptibility,
respectively. The most promising compounds were then profiled
against an extended panel of about 50 kinases. Several representa-
tive inhibitors are shown in Table 5 which demonstrate improved
selectivity relative to Fasudil and Y-27632. The SAR trends indi-
cated a balance between potency and selectivity could be achieved
by modulating the amino substitution of molecule (e.g., compound
22). These compounds were found to be dual inhibitors of ROCK 1
and ROCK 2.
Table 5
erence, we synthesized the respective L- and D-phenylanine ana-
Selectivity profile in counter screening kinases
logues (27 and 28) as surrogates, and noticed that there was a
moderate stereochemical preference of (S)-configuration for the
biological activity.
Compound ROCK 2
ROCK 1
IC50
Prkce
IC50
DMPK
IC50
Cdc42
IC50
Prkcl2
IC50
IC50
(nM)
(nM)
(nM)
(nM)
(nM)
(nM)
The series was confirmed being ATP competitive in biochemical
experiments. The crystal structure of ROCK was not available when
this work was started. Among the kinases whose three-dimen-
sional structures were known, PKA was chosen as a template for
homology modeling based on its reasonable homology (56%, 39%
identity) throughout the kinase domain to Rho-kinase.6 Addition-
ally, co-crystal structures of ROCK inhibitors in PKA had been pub-
lished.6 We used Schrödinger’s PRIME software to build the
homology model and GLIDE for the docking studies.7 Shown in
Figure 2 is a docking model of compound 23 (preferred enantio-
mer) in the ATP binding site. In the modeling the isoquinolinone
moiety has specific interactions with the protein, forming two
essential H-bonds in the hinge region, one with the backbone NH
of Met172 and the other being to the backbone carbonyl of
15
20
21
22
23
26
Fasudil
Y-27632
320
57
12
19
26
110
400
61
180
59
14
39
19
53
660
46
>8000
>8000
>8000
>8000
>8000
>8000
3000
>8000
>8000
>8000
>8000
>8000
>8000
>8000
>8000
5400
1700
1300
>8000
>8000
>8000
>8000
>8000
1400
510
110
>8000
2300
3000
780
335
70
Cambrex PKLight ATP Detection Reagent using luciferin–luciferase to quantify
residual ATP. Assay values reported as the mean of n P 2 independent replicates,
and standard error (SE) typically <20%.
Table 6
An extended kinase panel profiling of 22
Glu170. The isoquinolinone ring appears to enjoy favorable
p inter-
actions with the protein, and the isoquinolone-substituted chlorine
interacts with Phe384 from the C-terminal loop. The exocyclic
amide linker appears to serve as a spacer group without direct
interaction with the backbone of the protein. The phenyl group,
which orients itself nearly perpendicular to the isoquinolinone
ring, sits nicely in a hydrophobic groove underneath the glycine-
rich loop. The basic amino group inserts into the ATP phosphate
Kinase
Percent of control
@3
Kinase
Percent of control
@3
l
M
lM
ROCK 2
PKBdeltaPH
S6K1
PKB-beta
PRK2
0.55
17
15
39
16
31
JNK3 alpha1
GSK3B
CAMK-1
PBK
MAPKAPK2
MST2
84
103
100
100
95
PKA (rec)
86
MAPKAPK1A 31
MAPKAP-K3
CDK2/
cyclinA
MAPK2/
ERK2
94
89
MSK1
ERK8
35
54
74
103
111
MAPKAP-
K1b
SGK
MKK1
smMLCK
PKCA
MARK3
AuroraB
CHK2
SRPK1
NEK2A
CSK
PRAK
PDK1
CK1
DYRK1A
SAPK2AP38
70
76
85
68
41
76
82
91
94
63
94
90
69
95
PKD1
NEK7
MNK2 alpha
NEK6
CK2
AMPK
CHK1
JNK1A1
EF2K
Src
PIM2
PLK1
MNK1
73
90
90
104
98
86
90
93
105
88
87
93
89
Figure 2. A docking pose of compound 23 in the ATP pocket.

3238
F. Wu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3235–3239
Table 7
bolic stability as well as in vivo rat pk studies. This exercise
Results of functional activity and profiling
highlighted an overall promising profile as starting point for opti-
mization and focused our attention on metabolic stability as a
key parameter. In spite of low in vitro metabolic stability, 22 was
found to have sufficient oral exposure in rat pk studies to be ad-
vanced as an early proof of concept tool compound in a disease-re-
lated spontaneously hypertensive rat (SHR) in vivo model.10
Compound 22 demonstrated a moderate but statistically signifi-
cant activity in attenuating hypertension at the highest dose
tested, 40 mg/kg (shown in Fig. 3).
Albeit with marginal effect, compound 22 dose dependently de-
creased mean arterial pressure (MAP). The maximum response was
14% decrease in blood pressure at 4 h with a 40 mg/kg p.o. dose
and the duration of the response was approximately 8 h.
The general synthetic procedure for our compounds is illus-
trated in Schemes 1–3. Schemes 1 and 2 depict the synthesis of
the amino isoquinolinone and quinazolinone intermediates.10 Syn-
thesis of 22 is provided as representative example in Scheme 3.
A nitro substituted cinnamic acid 29 was reduced to the corre-
sponding amine using tin II chloride in hydrochloric acid and was
subsequently acetylated The resulting cinnamic acid 30 was then
converted to the corresponding acyl azide 31, which was converted
in a Curtius rearrangement to isoquinolinone by heating in dieth-
ylene glycol dibutyl ether at a temperature of 230–260 °C. In the
case where R1 is H, a mixture of regioisomeric products was
formed (32a or 32b), which was separated by chromatography.
The acetyl group was removed by hydrolysis under acidic condi-
tions using concentrated hydrochloric acid, generating the desired
amino isoquinolinone intermediate 32 (Scheme 1).
Heating the nitro anthranilic acid 33 with formamidine at a
temperature of 200 °C provided nitro 34, which was reduced to
amino quinazolinone intermediate 35 by catalytic hydrogenation
over palladium (Scheme 2).
Shown in Scheme 3, intermediate 32 was treated with 2-chloro-
phenylacetyl chloride in DMF in the presence of triethylamine, and
the resulting chloro 36 was converted to the final compound 22 by
reacting with cyclopropyl amine.
In summary, a novel series of potent ROCK inhibitors with an
improved kinase selectivity profile were discovered. These inhibi-
tors have demonstrated potent functional tissue activity and
in vivo activity, suggesting their potential therapeutic utility in car-
diovascular diseases such as hypertension. Further drug-like prop-
Compound
22
23
Fasudil
Y-27632
ROCK 2 IC50 (nM)
Aortic ring IC50 (nM)
Cytotoxic (
Solubility pH 7
19
26
90
>10
6
440
7500
>10
>60
10/10
73/nd
nd
nd
nd
nd
260
4750
>10
>32
nd
>90/nd
nd
nd
570
>10
16
lM)
l
g/ml
Caco-2 (10 cm/s) (A to B/B to A) 20/23 32/20
HLM/RLMa t1/2 min
Cl (ml/min/kg)
AUC p.o. (ng h/mL)
T1/2 p.o. (h)
21/7
82
1293
2.7
64
>10
>30
15/3
123
30
2.5
2
nd
nd
% Fb
Cyp inh. (
l
M) 1A2/2D6/3A4
M)
>10
nd
>10/3.6/>10 >10/3.2/>10
nd nd
hERG inhibition (
l
a
Compounds were incubated with liver microsomes at a concentration of 1 mg
protein/ml.
b
In vivo rat pk data was generated at dose of 1.0 mg/kg iv and 10 mg/kg po
Illustrated as an example in Table 6, compound 22 showed no
significant inhibitory activity at 3 M against other kinases in the
l
panel except PKB delta, S6K1, PRK2 (less than 30% of control).
In addition to kinase selectivity screening, representative com-
pounds were further profiled in a general selectivity testing. Com-
pound 22 demonstrated a good selectivity profile against a panel of
53 receptors, ion channels, and enzymes and exhibited <40% inhi-
bition of all of them at 10 lM (data not shown).
Inhibition of ROCK has been reported to relax blood vessels
ex vivo.9 Shown in Table 7, these compounds produce a dose
dependent relaxation effect in phenylephrine pre-constricted rat
aortic rings. The relaxation effect of these compounds was revers-
ible, as contractility could be restored by washing the treated
aortic rings. The difference between enzymatic and tissue activities
may partially be due to the higher ATP concentration in the cellular
level as well as compound permeability. In comparison with
Fasudil and Y-27632, compound 22 demonstrated greater than
10-fold improvement in potency in both the enzymatic and
functional tissue assays. Furthermore, compounds did not induce
cytotoxic effects in HeLa cells up to 10 lM concentration.
In order to assess the key areas for further optimization of this
series, compounds were profiled in a battery of target independent
drug-like properties assays. These assays include solubility, perme-
ability, CYP450 inhibition, hERG channel inhibition, in vitro meta-
Blood Pressure Response (MAP) in conscious SHR (Telemetry)
CMC-T; P.O. (n = 5)
C M C -T; P .O . (n=5)
22; 40 mg/kg; P.O. (n = 5)
170
160
150
140
130
120
110
100
90
BI 633,276; 40m g/kg; P.O . (n=5)
22; 20 mg/kg; P.O. (n = 5)
BI 633,276; 20m g/kg; P.O . (n=5)
BI 633,276; 5m g/kg; P .O . (n=5)
22; 5 mg/kg; P.O. (n = 5)
*
*
** **
(-14%)
Dosing Time
9AM
(* p < .05 vs Veh.)
(** p < .01 vs Veh.)
-5
0
5
10
15
20
Time (Hours)
Figure 3. Effect of 22 on the mean arterial pressure (MAP) in conscious SHR. Compound was administered as a single dose oral gavage. Reported results represent the mean
arterial pressure for three treated animals.

F. Wu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3235–3239
3239
a) EtOCOCl,
Et3N, acetone
b) NaN3
O
1. SnCl2, conc HCl
2. Ac2O, AcOH
O
H
N
NO2
R2
HO
O
HO
O
R2
30
29
H
N
(BuO(CH2)2)2O,
Bu3N, 240oC
H
N
HN
R2
O
N3
HN
+
R2
O
HN
O
R2
O
Vb
O
31
31b
31a
conc. HCl
reflux, 1h
NH2
R2
HN
O
32
Scheme 1. Synthesis of intermediate 32.
formamidine
hydrochloride
200oC
NO2
R2
NO2
R2
H2N
HO
N
O
NH2
R2
N
H2, Pd/C
DMF/MeOH
HN
HN
O
O
33
35
34
Scheme 2. Synthesis of intermediate 35.
Cl
H
N
H
N
Cl
NH2
Cl
NH2
Cl
N
O
H
HN
O
HN
O
HN
Cl
Cl
TEA/DMF
DMF
O
O
O
32
36
22
Scheme 3. Synthesis of 22 as an example.
26157; (c) Takami, A.; Iwakubo, M.; Okada, Y.; Kawata, T.; Odai, H.;
Takahashi, N.; Shindo, K.; Kimura, K.; Tagami, Y.; Miyake, M.; Fukushima,
K.; Inagaki, M.; Amano, M.; Kaibuchi, K.; Iijima, H. Bioorg. Med. Chem. 2004,
12, 2115.
erty profiling qualified this series of compounds, represented by
22, as a starting point for lead optimization.
6. Breitenlechner, C.; Gassel, M.; Hidaka, H.; Kinzel, V.; Huber, R.; Engh, R.;
Bossemeyer, D. Structure 2003, 11, 1595. Since our initial work, the co-
structures of Fasudil, hydroxyfasudil, H-1152P, and Y-27632 have been
reported within ROCK I and II (see Ref. 8).
7. Maestro 8.0.314, GLIDE 4.5213, and PRIME 1.6313 were utilized from within
Schrödinger Suite 2007 (Schrödinger, LLC, Portland, OR).
8. (a) Jacobs, M.; Hayakawa, K.; Swenson, L.; Bellon, S.; Fleming, M.; Taslimi, P.;
Doran, J. J. Biol. Chem. 2006, 281, 260; (b) Yamaguchi, H.; Kasa, M.; Amano, M.;
Kaibuchi, K.; Hakoshima, T. Structure 2006, 14, 589.
9. 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.
References and notes
1. Ishizaki, T.; Ishizaki, T.; Maekawa, M.; Fujisawa, K.; Okawa, K.; Iwamatsu, A.;
Fujita, A.; Watanabe, N.; Saito, Y.; Kakizuka, A.; Morii, N.; Narumiya, S. EMBO J.
1996, 15, 1885.
2. (a) Yamakawa, T.; Tanaka, S.; Numaguchi, K.; Yamakawa, Y.; Motley, E.; Ichihara,
S.; Inagami, T. Hypertension 2000, 35, 313; (b) Sauzeau, V.; Mellionnec, E.;
Bertoglio, J.; Scalbert, E.; Pacaud, P.; Loirand, G. Circ. Res. 2001, 88, 1102; (c)
Tangkijvanich, P.; Tam, S.; Yee, H. Hepatology 2001, 33, 74; (d) Shimokawa, H. Jpn.
Circ. J. 2000, 64, 1; (e) Martinez, M.; Randriamboavonjy, V.; Ohlmann, P.; Komas,
N.; Duarte, J.; Schneider, F.; Stoclet, J. C.; Andriantsitohaina, R. Am. J. Physiol. 2000,
279, H1228; (f) Kishi, H.; Bao, J.; Kohama, K. J. Biochem. 2000, 128, 719.
3. Somlyo, A. P. Nature 1997, 389, 908.
4. Morwick, T.; Büttner, F.; Cywin, C.; Dahmann, G.; Hickey, E.; Jakes, S.; Kaplita,
P.; Kashem, M.; Kerr, S.; Kugler, S.; Mao, W.; Marshall, D.; Paw, Z.; Shih, C.; Wu,
F.; Young, E. J. Med. Chem. 2010, 53, 759.
5. (a) Hu, E.; Lee, D. Expert Opin. Ther. Targets 2005, 9, 715; (b) Engh, R.;
Girod, A.; Kinzel, V.; Huber, R.; Bossemeyer, D. J. Biol. Chem. 1996, 271,
10. (a) Eloy, F.; Deryckere, A. J. Heterocyclic Chem. 1971, 8, 57; (b) Briet, N.; Brookes,
M.; Davenport, R.; Galvin, F.; Gilbert, P.; Mack, S.; Sabin, V. Tetrahedron 2002,
58, 5761; (c) Chao, Q.; Deng, L.; Shih, H.; Leoni, L.; Genini, D.; Carson, D.;
Cottam, H. J. Med. Chem. 1999, 42, 3860.