Development of dihydropyridone indazole amides as selective rho-kinase inhibitors

Article abstract of DOI:10.1021/jm0609014

Rho kinase (ROCK1) mediates vascular smooth muscle contraction and is a potential target for the treatment of hypertension and related disorders. Indazole amide 3 was identified as a potent and selective ROCK1 inhibitor but possessed poor oral bioavailabi

Full text of DOI:10.1021/jm0609014

6
J. Med. Chem. 2007, 50, 6-9
functions have been identified to date, and undesired kinase
Development of Dihydropyridone Indazole
Amides as Selective Rho-Kinase Inhibitors
activity could negatively impact the long-term safety and
tolerability of a ROCK1 inhibitor.7 To assess potential kinase
selectivity issues, compounds were screened against a panel of
33 kinases including rat RSK18 and p70S6K. RSK1 and p70S6K
were of particular concern: both are involved in cell prolifera-
tion and their active sites share a high degree of sequence
homology with ROCK1.9
Krista B. Goodman,* Haifeng Cui, Sarah E. Dowdell,
Dimitri E. Gaitanopoulos, Robert L. Ivy, Clark A. Sehon,
Robert A. Stavenger, Gren Z. Wang, Andrew Q. Viet,
Weiwei Xu, Guosen Ye, Simon F. Semus,
Christopher Evans, Harvey E. Fries, Larry J. Jolivette,
Robert B. Kirkpatrick, Edward Dul, Sanjay S. Khandekar,
Tracey Yi, David K. Jung, Lois L. Wright, Gary K. Smith,
David J. Behm, Ross Bentley, Christopher P. Doe,
Erding Hu, and Dennis Lee
Departments of Medicinal Chemistry, Biology, and Drug
Metabolism and Pharmacokinetics, CardioVascular and Urogential
Center of Excellence in Drug DiscoVery, Departments of
Computational, Analytical, and Structural Sciences, Gene
Expression and Protein Biochemistry, High Throughput Chemistry,
and Screening and Compound Profiling, DiscoVery Research,
GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, King of
Prussia, PennsylVania 19406
Figure 1. Activity and kinase selectivity of the initial dihydropyri-
midinone lead. Data represent the average value for two or more
measurements. Standard error is typically within 2-fold of the reported
mean.
ReceiVed July 28, 2006
Screening identified dihydropyrimidinyl indazole amide 3 as
promising lead. Not only was 3 a potent ROCK1 inhibitor, but
it also possessed exciting selectivity (>30-fold) against a panel
of 33 kinases, including RSK1 and p70S6K (Figure 1).
Compound 3 was further evaluated in the rat aortic ring
dilation assay to establish functional activity.10 Although
compound 3 displayed cellular activity, a significant shift (>50-
fold) was observed relative to in vitro potency. In addition, the
pharmacokinetic profile of 3 was characterized by low oral
bioavailability and high clearance (Oral F ) not quantifiable,
Cl ) 50 mL/min/kg) in rats. Based on these observations, the
primary goal for lead optimization was to identify modifications
that would improve the pharmacokinetic profile for this chemical
series.
Abstract: Rho kinase (ROCK1) mediates vascular smooth muscle
contraction and is a potential target for the treatment of hypertension
and related disorders. Indazole amide 3 was identified as a potent and
selective ROCK1 inhibitor but possessed poor oral bioavailability.
Optimization of this lead resulted in the discovery of a series of
dihydropyridones, exemplified by 13, with improved pharmacokinetic
parameters relative to the initial lead. Indazole substitution played a
critical role in decreasing clearance and improving oral bioavailability.
Rho-associated kinase (ROCK1)1 is a serine/threonine kinase
that has been implicated in a variety of cellular processes
including vascular smooth muscle contraction, stress-fiber
formation, cell migration, and gene expression.2 In vascular
smooth muscle contraction, ROCK1 plays a central role in the
calcium-sensitization pathway.3 Activation of ROCK1 indirectly
regulates the phosphorylation state of myosin light chain, leading
to increased vascular smooth muscle contraction. Inhibition of
this pathway represents a promising strategy for the treatment
of a variety of cardiovascular diseases, including hypertension.4
Several ROCK1 inhibitors have been reported in the literature,5
including 1 (Y-27632)3 and 2 (Fasudil).6 These compounds have
been used extensively to elucidate the role of Rho-kinase in
vivo. Importantly, studies have shown that both compounds
lower blood pressure in preclinical models of hypertension.3,6
Given these encouraging observations, we sought to develop a
potent, selective ROCK1 inhibitor for use as an antihypertensive
agent.
Figure 2. Structure of 3 docked in the active site of the ROCK1
homology model. Key hydrogen bonds to the protein backbone are
highlighted in green.
To provide insight into its binding mode, compound 3 was
docked into the active site of our ROCK1 homology model
(Figure 2).11 The lowest energy structure oriented the indazole
toward the hinge region of the kinase, making two key hydrogen
bonds with the protein backbone. This binding mode is
consistent with previously described kinase inhibitors bearing
an indazole ring.4 Indeed, methylation of compound 3 at N1 of
the indazole resulted in a dramatic loss of activity (ROCK1 IC50
From the outset, achieving kinase selectivity was a high
priority. Over 500 protein kinases with a range of biological
* To whom correspondence should be addressed. Phone: 610-270-6635.
Fax: 610-270-4490. E-mail: krista.b.goodman@gsk.com.
10.1021/jm0609014 CCC: $37.00 © 2007 American Chemical Society
Published on Web 12/15/2006

Letters
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 1
Scheme 1. Synthesis of Dihydropyridonesa
7
Table 1. Profiles of Initial Screening Hit and Pyridone Analog
cmpd
3
4
IC50a (nM)
a Reagents: (a) Meldrum’s acid, R1CHO, NH4OAc, HOAc; (b) NaOH,
MeOH, H2O; (c) 5-aminoindazole, EDC, Et3N, DMF; (d) Meldrum’s acid,
R1CHO, NH4OAc, AcOH.
ROCK1
rat aorta
RSK1
14
760
3100
2850
51
200
1410
1100
p70S6K
rat PK parametersb
dosec (mg/kg)
1.3
1.6
Cmaxc (ng/mL)
567 ( 112
668 ( 121
1.0 ( 0.5
47 ( 10
2.1 ( 0.3
14 ( 5
T1/2 (h)
0.7 ( 0.2
Cl (mL/min/kg)
Vdss (L/kg)
F (%)
49 ( 5
2.2 ( 0.2
not quantifiable
a Data represent the average value for two or more measurements.
Standard error is typically within 2-fold of the reported mean. b PK data
were determined from an iv/po crossover study. Pharmacokinetic parameters
represent average values from three tested animals. c Values are reported
for the iv leg of the study.
Figure 3. LC/MS/MS analysis of parent 11 and its M+16 metabolite
generated by incubation with rat hepatocytes.
> 2500 nM). In the model, the aryl group occupies a
hydrophobic region beneath the glycine-rich loop. However, no
significant interactions between the dihydropyrimidinone core
and the protein were identified, suggesting that this heterocycle
may serve primarily as a scaffold to appropriately orient the
indazole and the aryl group for optimal binding.
We then investigated whether metabolism could be limiting oral
bioavailability. Compound 11 was incubated with rat hepato-
cytes, and incubates were subsequently analyzed by LC/MS/
MS for the presence of metabolites (Figure 3). Three chromato-
graphically resolved M+16 metabolites were observed. Subse-
quent fragmentation of one of these metabolites identified a
fragment (m/z 150.0596), which suggested that oxidation was
occurring on the indazole. Concurrently, the dihydropyridone
fragment was recovered unchanged (m/z 282.0144), supporting
the conclusion that oxidation had occurred on the indazole.
Based on these observations, we directed our chemical strategy
toward compounds with diverse indazole substitution patterns
in an attempt to block sites of metabolism and potentially
improve oral bioavailability and half-life by reducing clearance.
A variety of indazole substitution patterns were explored
(Table 3). Substitution at C3 and C6 had little effect on ROCK1
inhibition (13 and 15) or kinase selectivity. In contrast, addition
of a fluorine substituent at C4 dramatically reduced potency
(14). Interestingly, while substitution at C7 maintained ROCK1
potency, selectivity against key kinases suffered (16). In contrast
to its modest effects on ROCK1 activity, indazole substitution
Because our binding hypothesis suggested that modification
of the dihydropyrimidinone core could be tolerated, our initial
strategy focused on identifying core replacements with improved
cellular activity and oral bioavailability. Mindful of previous
work that drew a correlation between hydrogen bond count and
oral bioavailability in rats,12 compounds with a decreased
hydrogen bond count in the central ring were targeted. Indeed,
simple modification of the core to a pyridone 4 was well
tolerated (Table 1) and selectivity against the kinase panel was
maintained. Importantly, rat pharmacokinetic studies demon-
strated that 4 had enhanced oral bioavailability (14%) relative
to 3. Furthermore, these modifications decreased the shift
between the in vitro enzyme assay and the rat aortic ring dilation
assay from greater than 50-fold for compound 3 to 4-fold for
compound 4. Therefore, optimization of 4 became the new focus
of our lead optimization efforts.
Dihydropyridone analogs were rapidly synthesized by two
routes outlined in Scheme 1. Methylacetoacetate was condensed
with Meldrum’s acid, an aryl aldehyde, and ammonium acetate
to afford the substituted dihydropyridone 6.13 Saponification of
the ester provided acid 7, which smoothly underwent coupling
with an indazole to provide indazole amide 9. Alternatively,
ketoamide 8 could be treated with Meldrum’s acid, an aryl
aldehyde, and ammonium acetate to provide indazole amide 9
in a single step.
Pyridone derivatives with varying aryl groups (R1) were
prepared and evaluated for ROCK1 inhibition. Close analogs
of 4 incorporating a variety of aryl substituents had similar
activity and selectivity (Table 2). Replacement of fluorine with
trifluoromethyl resulted in a 10-fold increase in ROCK1 potency
and maintained the desired selectivity profile. However, these
modifications did not significantly improve oral bioavailability.
Figure 4. Effect of 15 on mean arterial pressure in spontaneously
hypertensive rats. Compound was administered by single dose oral
gavage. Reported data represent the average decrease in mean arterial
pressure for five treated animals.

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Journal of Medicinal Chemistry, 2007, Vol. 50, No. 1
Letters
Table 2. Profile of Dihydropyridones with Varying Aryl Rings
kinase IC50a (nM)
rat PK parametersb
c
rat aortaa
IC50 (nM)
dosec
(mg/kg)
Cmax
T1/2
(h)
Cl
Vdss
(L/kg)
Oral F
(%)
compd
R
ROCK1
RSK1
p70S6K
(ng/mL)
(mL/min/kg)
4
4-FPh
51
5
8
1410
740
620
1150
nd
560
1550
200
39
nd
1.6
0.9
0.9
1.7
668 ( 121
315 ( 43
334 ( 29
700 ( 35
1.0 ( 0.5
0.7 ( 0.1
0.8 ( 0.01
0.9 ( 0.1
47 ( 10
51 ( 4
57 ( 1
45 ( 8
2.1 ( 0.3
2.3 ( 0.2
2.4 ( 0.3
1.8 ( 0.3
14 ( 5
22 ( 4
1 ( 1
10
11
12
4-CF3Ph
3,4-diClPh
2-naphthyl
9
1100
76
13 ( 2
a Data represent the average value for two or more measurements. Standard error is typically within 2-fold of the reported mean. b PK data were determined
from an iv/po crossover study. Pharmacokinetic parameters represent average values from three tested animals. c Values are reported for the iv leg of the
study.
Table 3. Effect of Indazole Substitution on Kinase Selectivity and Rat DMPK Parameters
a Data represent the average value for two or more measurements. Standard error is typically within 2-fold of the reported mean. b PK data were determined
from an iv/po crossover study. Pharmacokinetic parameters represent average values from three tested animals. c Values are reported for the iv leg of the
study.
at C3, C6, and C7 had a pronounced effect on the rat PK profile.
While substitution at C3 and C7 had only modest impact on
oral bioavailability (13 and 17), incorporation of fluorine at C6
dramatically increased oral bioavailability (15). This mode of
substitution provided a general handle for improving pharma-
cokinetics in this series. Importantly, the improved profile of
15 provided an ideal tool compound to explore the effects of
indazole-derived ROCK1 inhibitors in vivo.
Spontaneously hypertensive rats (SHRs) were treated with
compound 15 to establish its effect on mean arterial pressure
(Figure 4). Indeed, treatment of animals with 15 resulted in a
dose-dependent decrease in mean arterial pressure.14 A maxi-
mum decrease of 50 mmHg was observed approximately 2 h
after oral administration at 30 mg/kg. These data represent a
significant increase in efficacy compared to 2 (∼15 mmHg
decrease at 30 mg/kg, data not shown).
with 61% oral bioavailability. Compound 15 dramatically
reduced mean arterial pressure in spontaneously hypertensive
rats after oral administration. Further studies around the inda-
zole-dihydropyridones will be reported in due course.
Supporting Information Available: Synthetic procedures and
characterization data for all compounds. Background information
on the ROCK homology model. In vitro incubation and analytical
methodologies for metabolite ID experiments. Procedures for
enzyme assays and acute SHR studies. This information is available
free of charge via the Internet at http://pubs.acs.org.
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(h)
plasma concn
(ng/mL)
0.5
1
2
4
8
193
192
190
110
74
24
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JM0609014