Discovery of substituted 4-(pyrazol-4-yl)-phenylbenzodioxane-2-carboxamides as potent and highly selective Rho kinase (ROCK-II) inhibitors

Article abstract of DOI:10.1021/jm800986w

The identification of a new class of potent and selective ROCK-II inhibitors is presented. Compound 5 (SR-3677) had an IC₅₀ of ～3 nM in enzyme and cell based assays and had an off-target hit rate of 1.4% against 353 kinases, and inhibited only 3 out of 70 nonkinase enzymes and receptors. Pharmacology studies showed that 5 was efficacious in both, increasing ex vivo aqueous humor outflow in porcine eyes and inhibiting myosin light chain phosphorylation.

Full text of DOI:10.1021/jm800986w

6642
J. Med. Chem. 2008, 51, 6642–6645
Chart 1. SAR Evolution
Discovery of Substituted 4-(Pyrazol-4-yl)-
phenylbenzodioxane-2-carboxamides as
Potent and Highly Selective Rho Kinase
(ROCK-II) Inhibitors
Yangbo Feng,† Yan Yin,† Amiee Weiser,† Evelyn Griffin,†
Michael D. Cameron,† Li Lin,† Claudia Ruiz,†
Stephan C. Schu¨rer,† Toshihiro Inoue,‡ P. Vasanth Rao,‡
Thomas Schro¨ter,† and Philip LoGrasso*,†
Department of Molecular Therapeutics, and Translational Research
Institute, The Scripps Research Institute, Florida, 5353 Parkside
DriVe, Jupiter, Florida 33458, and Department of Ophthalmology,
Pharmacology and Cancer Biology, Duke UniVersity School of
Medicine, Durham, North Carolina 27710
highly selective against other kinases and nonkinase enzymes
and receptors (ideally with IC50 values >1 µM). An in-house
HTS campaign24 led to the discovery of compound 1 (Chart
1), a pyridine-thiazole based amide compound. Although 1 is a
potent ROCK-II inhibitor (Table 1, IC50 ) 7.2 nM) with good
selectivity against a few other kinases, it has a relatively large
shift in cell-based potency as assessed by myosin light chain
bisphosphorylation (ppMLC) (IC50 ) 137 nM).25 The optimiza-
tion of 1 started by replacing the central thiazole group with a
phenyl ring (Chart 1). As shown in Table 1, the potency of the
resulting compound 2 was reduced in both enzyme and cell-
based assays compared to that of 1. We reasoned that the
pyridine-phenyl system in 2 might have perturbed the favorable
geometry that was present in the pyridine-thiazole structure of
1. A molecule with a 5-membered nitrogen-containing hetero-
cycle linked to the central phenyl ring might mimic the original
pyridine-thiazole system. Thus, we prepared compound 3, which
contained a pyrazole to replace the pyridine in 1 as the hinge
binding element.
The new pyrazole-phenyl scaffold of 3 was even more potent
(Table 1) than compound 1. Introduction of additional func-
tionality on the central aryl ring was used to improve the
inhibitor’s overall pharmaceutical properties and/or to enhance
the selectivity. We thus prepared compound 4. This methoxy
substituted ROCK inhibitor showed good selectivity against
PKA (∼300-fold) and Akt1 (∼2700-fold), and its cell potency
was also improved (IC50 ) 30 nM vs IC50 ) 72 nM in 3).
However, the selectivity against MRCK (IC50 ) 367 nM), the
most closely related kinase to ROCK that we tested, decreased
(110-fold vs 790-fold in 3). Finally, a dimethylaminoethoxy
moiety was introduced to replace the methoxy group to give
compound 5. This novel ROCK-II inhibitor showed high
potency in biochemical and cell-based assays as well as high
selectivity against all kinases tested (Table 1 and Supporting
Information). The IC50 value of 5 for ROCK-I was 56 ( 12
nM (n ) 4). The equal potency between biochemical and cell-
based assays for 5 could be due to cell accumulation, differences
in cell permeability, or enzyme kinetics. It should be pointed
out that compounds 1-5 are all racemates.
ReceiVed August 4, 2008
Abstract: The identification of a new class of potent and selective
ROCK-II inhibitors is presented. Compound 5 (SR-3677) had an IC50
of ∼3 nM in enzyme and cell based assays and had an off-target hit
rate of 1.4% against 353 kinases, and inhibited only 3 out of 70
nonkinase enzymes and receptors. Pharmacology studies showed that
5 was efficacious in both, increasing ex ViVo aqueous humor outflow
in porcine eyes and inhibiting myosin light chain phosphorylation.
RhoA and its downstream kinase (ROCK) play an important
role in the regulation of smooth muscle contraction1 and neurite
growth retraction.2 The abnormal activation of the ROCK
pathway has also been observed in many disorders of the central
nervous system.3 Therefore, the inhibition of ROCK is a
promising strategy for the treatment of various diseases such
as hypertension,4 coronary and cerebral vasospasm,5 erectile
dysfunction,6 glaucoma,7,8 asthma,9 multiple sclerosis (MS),3
atherosclerosis,10 stroke,11 and cancer.12
Most ROCK inhibitors reported in the literature contain
isoquinoline (e.g., Fasudila and H-1152P),13,14 pyridine (such
as Y-2763215 and Y-39983,8,16 which is in clinical trials for
the treatment of glaucoma17), or indazole18-22 moieties. These
compounds are either moderately potent in ROCK biochemical
assays (IC50 > 100 nM) or have poor cell activities (IC50 values
in cell-based assays normally in the range of 0.1 µM-10 µM).
A promising new class of ROCK inhibitors (ROCK-I) based
on aminofurazan-azabenzimidazoles was recently reported.23
Some compounds in this series were shown to have lower than
100 nM IC50 values in cell-based assays.
Our goal was to discover potent ROCK-II inhibitors with IC50
values less than 10 nM in biochemical assays and less than 100
nM in cell-based assays. In addition, these inhibitors should be
* Corresponding author. Phone: 561-799-8881. Fax: 561-799-8958.
E-mail: lograsso@scripps.edu.
†
The synthesis of 5 (Scheme 1) began from 2-fluoro-4-bromo-
nitrobenzene (6). Nucleophilic substitution was used to introduce
the side chain dimethylaminoethoxy group to provide the
nitrobenzene derivative (7). This intermediate was then reduced
with SnCl2 to aryl amine (8). Amide formation on the newly
formed amino group was accomplished in DMF using HATU
as the coupling reagent in the presence of DIEA to give the
bromide derivative (9). A Suzuki coupling reaction using
Pd[P(Ph)3]4 as the catalyst in the presence of K2CO3 was then
The Scripps Research Institute, Florida.
Duke University School of Medicine.
‡
a Abbreviations: Fasudil, 5-(1,4-diazepan-1-ylsulfonyl)isoquinoline;
H-1152P, (S)-4-methyl-5-(2-methyl-1,4-diazepan-1-ylsulfonyl)isoquinoline;
Y-27632, (R)-4-(1-aminoethyl)-N-(pyridin-4-yl)cyclohexanecarboxamide;
Y-39983, (R)-4-(1-aminoethyl)-N-(1H-pyrrolo[2,3-b]pyridin-4-yl)benza-
mide; AUC, pharmacokinetic area under curve; Cl, pharmacokinetic
clearance; Cmax, pharmacokinetic maximum concentration; F, oral bio-
availability; HLMS, human liver microsomal stability; hERG, human ether-
a-go-go; HTS, high-throughput screening; ppMLC, bis-phospho myosin light
chain; TM, trabecular meshwork; Vd, volume of distribution.
10.1021/jm800986w CCC: $40.75
2008 American Chemical Society
Published on Web 10/04/2008

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6643
kinase IC50 (µM)
Table 1. Data for Biochemical and Cell-Based Assays
ROCK-II IC50
ppMLC IC50
(µM)
cmpd
(µM)
PKA
MRCK
Akt1
1
0.0072 ( 0.0023
(n ) 2)
0.046 ( 0.032
(n ) 2)
0.0015 ( 0.0007
(n ) 2)
0.0033 ( 0.002
(n ) 2)
0.0032 ( 0.0023
(n ) 9)
0.137
0.637 ( 0.119
(n ) 2)
0.692 ( 0.021
(n ) 2)
0.186 ( 0.016
(n ) 2)
0.940 ( 0.019
(n ) 2)
4.850 ( 0.212
(n ) 2)
7.050 ( 2.900
(n ) 2)
1.190 ( 0.057
(n ) 2)
0.367 ( 0.188
(n ) 2)
>20
(n ) 1)a
0.657 ( 0.119
(n ) 2)
(n ) 2)
2
3
4
5
6.436 ( 2.834
(n ) 4)
1.410 ( 0.646
(n ) 4)
8.819 ( 2.000
(n ) 2)
7.491 ( 1.402
(n ) 2)
0.072 ( 0.057
(n ) 4)
0.030
(n ) 1)a
0.0035
3.968 ( 1.680
(n ) 5)
1.190 ( 0.509
(n ) 2)
(n ) 1)a
a The compound was tested as replicates on the plate, and the IC50 data was calculated by drawing a curve from both data sets.
Scheme 1. Synthesis of 5a
a panel of 70 nonkinase enzymes and receptors, 5 inhibited only
three adrenergic receptors: R1a, R1b, and R2a with 53%, 68%,
and 76% inhibition, respectively, at 3 µM inhibitor concentra-
tion, which indicates IC50 values in the 1 µM range or ∼300
times higher than the IC50 against ROCK-II in cell-based assays.
Furthermore, inhibition of the human-ether-a-go-go (hERG) was
only 31% at 3 µM (Supporting Information). All these counter
screening results demonstrated that 5 is not only a potent
compound but also a highly selective ROCK-II inhibitor.
Cocrystal structures of ROCK with ligands Fasudil and
Y-27632 have previously been reported.27,28 Our docking results
of 5 to ROCK-II (Supporting Information) explain well the
observed SAR. There are several key binding elements that
contribute to the high potency and selectivity of 5. The best
scoring pose (Figure 1) indicated that the pyrazole headgroup
binds to the hinge region of the enzyme through two H-bonds:
one between the NH of Met-172 and the N of the pyrazole ring
and another between the backbone CdO of Glu-170 and the
NH of the pyrazole ring. In the phosphate binding region, the
amide CdO of 5 forms a hydrogen bond with the side chain
NH of Lys-121. A very important H-bonding interaction occurs
in the ribose binding region between the protonated tertiary
amine of the dimethylaminoethoxy group of 5 and the Asp-
176 carboxylate side chain. This interaction is probably the
reason for the higher selectivity of 5 compared to that of
compounds 1-4 (Table 1) in which this H-bond is absent. This
specific interaction was observed in the crystal structure of the
ROCK-II-Fasudil complex but was not seen in the cocrystal
structure of Y-27632 with ROCK-II (bovine) and ROCK-I
(human). Thus, we have combined the binding motifs of both
Fasudil and Y-27632 into 5.
It is important to point out that there is a hydrophobic pocket
under the P-loop in this enzyme-ligand complex (Supporting
Information), and the benzodioxane phenyl ring of 5 is immersed
inside this pocket. It is believed that the hydrophobic interaction
of the benzodioxane phenyl ring with the hydrophobic surface
of this pocket is the dominating factor that contributes to the
high potency of 5. To demonstrate the significance of this
interaction, two compounds (10 and 11) without the benzo-
dioxane moiety (Figure 2) were synthesized and evaluated. The
binding of 10 and 11 to ROCK-II is much weaker (IC50 ) 920
nM and 760 nM, respectively, compared to 3 nM in 5). This
hydrophobic interaction further explains why the very simple
structures 1, 3, and 4 have high affinity for ROCK-II.
a Reagents and conditions: (a) NaH, THF, 2-(dimethyl-amino)ethanol,
rt, 92%; (b) SnCl2, ethanol, 70 °C, 91%; (c) benzodioxane-2-carboxylic
acid, HATU/DIEA, DMF, rt, 75%; (d) 4-1H-pyrazoleboronic acid, pinacol
ester, Pd[P(Ph)3]4, K2CO3, dioxane/H2O, 95 °C, 35%.
applied to provide 5. Similar procedures were also used for the
preparation of compounds 2, 3, and 4.
The in Vitro drug metabolism and in ViVo pharmaco-kinetic
properties of these ROCK-II inhibitors were evaluated (Table
2). Four human CYP-450 enzymes (2C9, 2D6, 3A4, and 1A2)
were selected for routine screening. The HTS hit compound 1
strongly inhibited all four enzymes. Replacement of the thiazole
group with an unsubstituted phenyl ring (compounds 2 and 3)
significantly reduced CYP inhibitions. Addition of a dimethy-
laminoethoxy group (5) to the central phenyl ring eliminated
the 3A4 inhibition seen in the original hit 1. Overall, none of
these compounds possessed good in Vitro drug metabolism and
in ViVo pharmacokinetic properties desired for systemic ap-
plications. The inhibitors had either low human liver microsomal
stability (HLMS), high clearance (Cl), or low AUC (i.v.) and
Cmax (p.o.). In addition, all of the compounds had none or very
low oral bioavailability (%F). However, as required in a soft
drug approach, these properties are desirable for the topical
application of a drug for treating glaucoma, which is one of
the promising applications for ROCK inhibitors.
In addition to selectivity studies against the kinases listed in
Table 1, 5 was also profiled against a large panel of kinases
and other nonkinase but therapeutically relevant enzymes and
receptors. In the profiling versus 353 kinases (Ambit screen26)
in single-point inhibition using 3 µM of 5 (Supporting Informa-
tion), 5 was discovered to hit only 5 other kinases with
percentage inhibition greater than 50%: Akt3, Clk1, Clk2, Clk4,
and Lats2. All other kinases showed either no or very low
(<10%) inhibition. An off-target hit rate of only 1.4% (5 out
of 352) is believed to be one among the few best known ATP-
competitive kinase inhibitors.26 In the profiling studies against
The effect of 5 on ex ViVo aqueous humor outflow facility
from the trabecular meshwork (TM) of porcine eyes is shown
in Figure 3. The results show that continuous exposure of 25
µM 5 increases the outflow facility by 60% at 1 h perfusion,
increasing to 70-80% for the 2-5 h time points (p < 0.01).
This 70-80% increase in outflow is the maximal response that

6644 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Table 2. Data for Pharmacokinetics in Vitro and in ViVo
Letters
in ViVo rat PKc (i.v., 1 mg/kg; p.o., 2 mg/kg)
CYP-450sa
% inh. at
10 µM
HLMS at 2 mg/mL
Cl (i.v.)
mL/min/kg
T1/2
Vd (i.v.)
AUC (i.v.)
µM-h
Cmax (p.o.)
µM
cmpd
F (%)
T1/2 (min)b
(iv) h
L/kg
1
2
3
4
5
96/94/80/90
64/33/51/23
87/40/1/20
93/79/36/92
80/75/0/46
18
6
20
15
35
181
161
18
11
61
0.4
0.5
2.2
2.6
0.8
3.7
5.3
1.9
1.0
3.0
0.3
0.3
2.8
4.3
0.7
0.0
0.0
0.02
0.12
0.05
0
0
0.3
0.4
7.0
a CYP-450s: 2C9/2D6/3A4/1A2. b HLMS: human liver microsomal stability. c Data reported is the mean of 3 determinations, and the standard error is
within 30% of the mean except for Cmax, which is within 50%.
Figure 4. Western blot analysis of p-MLC in the TM tissue derived
from the porcine eyes untreated (control) and perfused with 25 µM 5.
Figure 1. Structure of compound 5 (green) docked in the catalytic
domain of the ROCK-II homology model (gray).
not tested, it may be that lower doses of 5 would also be
maximally effective in increasing aqueous humor outflow and
thus be more reflective of the intrinsic potency differences
between 5 and Y-27632. These studies are currently ongoing
in our laboratories. These results underscore the important role
ROCK plays in aqueous humor outflow and illustrate the
promise of this class of compounds for potential intraocular
pressure lowering in glaucoma.
In an effort to ensure that 5 acted through inhibition of ROCK
ex ViVo, we monitored the phosphorylation state of myosin light
chain in trabecular meshwork from porcine eyes. Figure 4
presents the Western blot analysis for p-MLC in TM tissue
isolated from porcine eyes that were perfused with 25 µM 5 at
15 mmHg for 5 h. The results show a strong cross-reactive band
for p-MLC in the control lane indicating large amounts of
p-MLC. TM tissue derived from the eyes perfused with 25 µM
5 shows a very faint band for p-MLC suggesting greater than
90% inhibition of Rho kinase in the TM tissue at this dose.
Actin immuno-cross-reactivity for both samples showed equal
loading of protein between wells. These data strongly support
the notion that selective ROCK inhibitors from this scaffold
are potent modulators of myosin light chain phosphorylation
in TM tissue and correlate nicely with the increased outflow
facility seen in these very same porcine eyes (Figure 3).
In summary, we have identified a new class of potent and
highly selective ROCK-II inhibitors. These novel compounds
possess a pyrazole group that can function as the hinge binding
moiety and a side chain with H-bonding capability that can
enhance the inhibitor’s selectivity. Compound 5 is very potent
in both enzyme and cell-based assays with single digit IC50
values. Pharmacology studies showed that 5 was efficacious in
both increasing aqueous humor outflow and in target modula-
tion-reduction of p-MLC. Further optimizations of this scaffold,
including the synthesis and evaluation of each enantiomer of
5, and more pharmacology studies are underway in our
laboratories and will be reported in due course.
Figure 2. Compounds 10 and 11 and their ROCK-II potency.
Figure 3. Time course for the aqueous humor outflow facility by 25
µM 5 perfused in enucleated porcine eyes (N ) 3; average ( SE; *p
< 0.05; **p < 0.01).
can be attained and is achieved at doses 2-4-fold lower than
needed for Y-27632,29,30 a well studied ROCK inhibitor. The
2-4-fold lower dose of 5 utilized to increase aqueous humor
outflow compared to Y-27632 is more modest than one might
expect given the difference in biochemical- and cell-based
potency between the two compounds and may be due to
compound accessibility to the different regions (trabecular
meshwork, juxtacanilicular (JCT) area, and Schlemm’s canal)
of the aqueous outflow pathway. Since lower doses of 5 were
Acknowledgment. We thank Dr. Michael Chalmers for the
high resolution mass spectra, Ambit Bioscience for the kinase

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6645
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panel screening, and MDS for the nonkinase enzyme and
receptor screening. We are also grateful to Professor Patrick
Griffin and Professor William Roush for their support. Y. Feng
thanks Dr. Yen Ting Chen for reading the manuscript and for
the helpful discussion. This work was supported in part by NIH
grant R01-EY018590 (P.V.R.).
Supporting Information Available: More docking information
for 5; procedures for biologic testing; and experimental procedures
and spectra data for compounds 2, 3, 4, 5, 10, and 11. This material
is available free of charge via the Internet at http://pubs.acs.org/
instruct/jmcmar.pdf.
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