Potent, selective and orally bioavailable dihydropyrimidine inhibitors of Rho kinase (ROCK1) as potential therapeutic agents for cardiovascular diseases

Article abstract of DOI:10.1021/jm8005096

Recent studies using known Rho-associated kinase isoform 1 (ROCK1) inhibitors along with cellular and molecular biology data have revealed a pivotal role of this enzyme in many aspects of cardiovascular function. Here we report a series of ROCK1 inhibitors which were originally derived from a dihydropyrimidinone core 1. Our efforts focused on the optimization of dihydropyrimidine 2, which resulted in the identification of a series of dihydropyrimidines with improved pharmacokinetics and P450 properties.

Full text of DOI:10.1021/jm8005096

J. Med. Chem. 2008, 51, 6631–6634
6631
Table 1. Activity, Kinase Selectivity, and Pharmacokinetics of the
Initial Dihydropyrimidinone and Dihydropyrimidine Leads
Potent, Selective and Orally Bioavailable
Dihydropyrimidine Inhibitors of Rho Kinase
(ROCK1) as Potential Therapeutic Agents for
Cardiovascular Diseases
Clark A. Sehon,*,† Gren Z. Wang,† Andrew Q. Viet,†
Krista B. Goodman,† Sarah E. Dowdell,† Patricia A. Elkins,§
Simon F. Semus,§ Christopher Evans,‡ Larry J. Jolivette,‡
Robert B. Kirkpatrick,| Edward Dul,| Sanjay S. Khandekar,|
Tracey Yi,| Lois L. Wright, Gary K. Smith,
IC509 (nM)
14
ROCK1
RSK1
p70S6K
rat aorta
105
3100
2850
760
5300
4500
1300
David J. Behm,# Ross Bentley, Christopher P. Doe,
Erding Hu,# and Dennis Lee†
pharmacokinetics (rat)10
1.3
dose11 (mg/kg)
1.8
T
1/2 (h)
CL (mL/min/kg)
dss (L/kg)
oral F (%)
0.7 ( 0.2
1.0 ( 0.7
15 ( 2.8
0.8 ( 0.2
16 ( 7.5
Departments of Medicinal Chemistry, InVestigatiVe Biology,
Vascular Biology, and Drug Metabolism and Pharmacokinetics,
CVU CEDD, Department of Computation and Structural Sciences
and Department of Gene Expression and Protein Biochemistry,
GlaxoSmithKline, 709 Swedeland Road, King of Prussia,
PennsylVania 19406, and Department of Assay DeVelopment and
Compound Profiling, GlaxoSmithKline, Research Triangle Park,
North Carolina 27709
49 ( 5
V
2.2 ( 0.2
not detectable
of dihydropyrimidinone derived amides (1) (Table 1).5 After
extensive lead optimization, the dihydropyrimidinone series was
found to have limited oral bioavailability and cellular activity.
During the course of the chemistry effort, an alternative
scaffold containing a dihydropyrimidine core (2) was identified.
In comparison to the dihydropyrimidinone 1, the dihydropyri-
midine 2 exhibited improved pharmacokinetics (PK) and
diminished ROCK1 activity. In addition, compound 2 had
significant P450 liabilities (2C9, 0.64 µM; 2D6, 0.09 µM; 3A4,
0.35 µM). Overall, compound 2 maintained good selectivity
(>30 fold) against a panel of 33 kinases, including RSK16 and
p70S6K6 (Table 1) and had reasonable activity in the phenyle-
pherine induced rat aortic tissue functional assay. Our initial
goal was to survey substitution around the dihydropyrimidine
core and to determine if the improved PK was general to the
series and if the undesired P450 inhibition could be overcome.
Preparation of dihydropyrimidines such as 7 was carried out
in a two-step process utilizing the modified Biginelli cyclization
as the key ring forming reaction.7 Condensation of 5-aminoin-
dazole with diketene afforded ketoamide 4, which underwent
ReceiVed May 2, 2008
Abstract: Recent studies using known Rho-associated kinase isoform
1 (ROCK1) inhibitors along with cellular and molecular biology data
have revealed a pivotal role of this enzyme in many aspects of
cardiovascular function. Here we report a series of ROCK1 inhibitors
which were originally derived from a dihydropyrimidinone core 1. Our
efforts focused on the optimization of dihydropyrimidine 2, which
resulted in the identification of a series of dihydropyrimidines with
improved pharmacokinetics and P450 properties.
Rho-associated kinase isoform 1 (ROCK1a)1 is an enzyme
involved in diverse cellular signaling functions such as smooth
muscle contraction, cytoskeleton rearrangement, cell migration,
and proliferation. Recent studies using ROCK1 inhibitors such
as 30 (Fausidil)2,3 and 31 (Y-27632)3 along with cellular and
molecular biology data have revealed a pivotal role of this
serine-threonine kinase in many aspects of cardiovascular
function. ROCK1 is a potential therapeutic target in the
treatment of cardiovascular diseases such as hypertension.4
Scheme 1. Synthesis of Dihydropyrimidinesa
Recently, we and others described the discovery of 5-sub-
stituted indazoles as ROCK1 inhibitors, including a new class
* To whom correspondence should be addressed. Phone: 610-270-6031.
Fax: 610-270-4490. E-mail: clark.a.sehon@gsk.com.
†
Department of Medicinal Chemistry.
Department of Drug Metabolism and Pharmacokinetics.
Department of Computational and Structural Sciences.
Department of Gene Expression and Protein Biochemistry.
‡
§
|
Department of Assay Development and Compound Profiling.
Department of Vascular Biology.
#
Department of Investigative and Cardiac Biology.
a Abbreviations: ATP, adenosine triphosphate; ROCK1, Rho-associated
kinase isoform 1; RSK1, ribosomal S6 kinase 1; SAR, structure activity
relationship; SHR, spontaneously hypertensive rat.
a (a) Diketene, CH3CN, 84%; (b) Al(CH3)3, NH4Cl, toluene, 95%; KOAc
(2 equiv), DMSO, 100 °C, 3 h, 32%.
10.1021/jm8005096 CCC: $40.75
2008 American Chemical Society
Published on Web 10/09/2008

6632 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Table 2. SAR of 2-Phenyl Substituents
Letters
Table 3. Screening of a Series of Substituted Aldehydes
Table 4. Activity, Kinase Selectivity, and Pharmacokinetics of
Compound 21
Biginelli condensation in the presence of an appropriately
substituted amidine8 6 and an aldehyde to afford 7 (Scheme 1).
Optimal conditions were determined to be excess KOAc in
DMSO at 100 °C for ∼3 h. The overall yields were modest but
sufficient to complete the preparation of a large number of
compounds to determine initial SAR.
Our initial array focused on simple substitution on the aryl
ring introduced from the benzamidine 6 (Table 2). In general,
a wide variety of substitutions were tolerated. The 4-MeO
compound 7 had the most promising rat PK (oral bioavailability
of 35% vs 0% for cmpd 12) and was selected for further
exploration.
SAR around the 4-position was well established and appeared
to parallel that of the dihydropyrimidinone and dihydropyridone
series reported recently.5 From this small array (Table 3), the
2-F,4-Cl substitution found in compound 21 was optimal for
activity against ROCK1.
Because of the P450 issues associated with the 4-MeO series
(compound 21), we then focused our efforts on substitution of
pyridyl ring found in compound 12. In comparison with
compound 21 (Table 5), the ROCK1 enzyme activity was
maintained and CYP2D6 inhibition was substantially dimin-
Compound 21 had >100-fold selectivity over selected kinases
and good oral bioavailability at 58% (Table 4). In addition, it
had encouraging functional activity in the rat aortic contraction
assay (IC50 ) 200 nM). Unfortunately, there was significant
P450 inhibition against CYP2D6 (Table 5). This undesired
CYP2D6 activity appeared to be general for the 4-MeO series
and proved to be insurmountable.
Concurrently, the crystal structure of the complex of com-
pound 12 with ROCK1 was solved. As expected, the indazole
binds with the ROCK1 hinge region by forming two key
hydrogen bonding interactions (Figure 1). In addition, there is
a potential interaction between the 4-pyridine nitrogen and
Lys200. This interaction may be responsible for the increased
binding affinity associated with compounds containing
hydrogen bond acceptors in the para position (e.g., MeO in
compound 7).
Figure 1. Crystal structure of compound 12 bound to the ATP site of
ROCK1. An unexpected hydrogen bond interaction was observed with
the unsubstituted 4-pyridyl nitrogen and Lys200. The blue sphere
represents a water molecule.

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6633
Table 5. Optimizing P450 of Compound 21
Table 7. P450 Inhibition of Compounds 25 and 27
P450 IC50 (µM)9
compd
CYP2C9
CYP2D6
CYP3A4
25
27
2.5
8.4
5.2
2.1
2.5
5.3
Table 8. Comparison of Indazole and 6-F-Indazole Analogues10
Table 6. Substituted Indazole Analogues
compd
R1
Cl
Cl
Cl
MeO
MeO
EtO
Me
R2
R3
ROCK1 IC50 (nM)9
22
25
26
23
27
28
29
H
F
H
H
F
H
H
Me
H
H
4
6
15
2
6
11
10
F
F
H
H
ished. However, the oral bioavailability was decreased. Interest-
ingly, ortho substitution on the pyridine does not affect the
activity, suggesting the hydrogen bond interaction with Lys200
may be weak.
On the basis of the related dihydropyridone series,5 it was
determined that C6 substitution on the indazole was crucial for
improving oral bioavailability. With this in mind, a small series
of dihydropyrimidines with a variety of substituted pyridines
and incorporation of the key C6 fluoroindazole substitution was
prepared.
Indazole substitution resulted in only minimal loss of binding
activity when compared to unsubstituted compound 22 (Table
6). Two compounds, 25 and 27, were then advanced to P450
(Table 7) and rat PK studies. Both compounds had dramatically
improved oral bioavailability (49% and 53%), and the chloro
analogue 25 showed an improvement in half-life in rats (Table
8) and was selected for in vivo efficacy studies.
Figure 2. Effect of 25 on mean arterial pressure in conscious SHR.
Compound was administered by single dose oral gavage. Reported data
represent the average decrease in mean arterial pressure for five treated
animals.
At 30 mg/kg (po), compound 25 induced a 25 mmHg (t ) 3 h)
drop in blood pressure, thus demonstrating potent in vivo activity
for the series (Figure 2).
Conclusion
A series of potent and selective dihydropyrimidine inhibitors
of ROCK1 have been described. From the initial lead 2,
significant improvements were made in the overall profile for
this series of ROCK1 inhibitors. Incorporation of substitution
next to the 4-pyridyl nitrogen provided compounds with
Compound 25 (Rat aorta IC50 ) 256 nM) was profiled in a
spontaneously hypertensive rat (SHR) model of hypertension.

6634 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Letters
therapeutic agents for cardiovascular diseases. Curr. Opin. InVest.
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improved P450 profiles. In addition, indazole substitution
provided very encouraging DMPK profiles. Final optimization
led to compound 25, which has promising oral bioavailability
(49% in rat and 19% in monkey), good half-life (1.8 h in rat
and 2.2 h in monkey), maintained good selectivity against a
panel of 31 kinases (>100 fold), as well as RSK16 and p70S6K6
(RSK1 IC50 ) 398 nM, p70S6K IC50 ) 1000 nM), and a
dramatically improved P450 profile (>2.2 µM at all isozymes
tested). Finally, oral administration of compound 25 gave a
robust 25 mmHg reduction mean arterial pressure in spontane-
ously hypertensive rats at a single dose of 30 mg/kg.
(5) (a) 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.; Yi, T.; Jung, D. K.; Wright,
L. L.; Smith, G. K.; Behm, D. J.; Doe, C. P.; Bentley, R.; Hu, E.;
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Selective Rho-Kinase Inhibitors. J. Med. Chem. 2007, 50 (1), 6–9.
(b) Feng, Y.; Cameron, M. D.; Frackowiak, B.; Griffin, E.; Lin, L.;
Ruiz, C.; Schroeter, T.; LoGrasso, P. Structure-activity relationships,
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(6) (a) Inhibition of RSK1 or p70S6K has been implicated in a number
of cellular functions. Although the specific consequence of inhibition
is not clearly understood, knowing the cross-activity of this series,
we sought to remove the activities against these targets. For a
leading reference on RSK1, see: Hauge, C.; Fro¨din, M. RSK and
MSK in MAP kinase signalling. J. Cell Sci. 2006, 119, 3021–3033.
(b) RAJIV6For a leading reference on p70S6K, see: Berven, L. A.;
Crouch, M. F. Cellular function of p70S6K: A role in regulating cell
motility. Immunol. Cell Biol. 2000, 78, 447–451.
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K.; Baader, E.; Bartman, W.; Berrgmann, A.; Granzer, B.; Jendralla,
H.; Kerekjarto, B. V.; Krause, R.; Paulas, E.; Schubert, W.; Wess, G.
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(9) Data represent the average value for two or more measurements.
Standard error is typically within 2-fold of the reported mean.
(10) PK data were determined from an iv/po crossover study. Pharmaco-
kinetic parameters represent average values from three tested animals.
(11) Values are reported for the iv leg of the study.
Note Added after ASAP Publication. This paper published
ASAP on October 9, 2008 with an incorrect presentation of
compounds 30 and 31. The correct version was published on
October 15, 2008.
Supporting Information Available: Synthetic procedures and
characterization data for all compounds. Procedures for ROCK
assays and acute SHR studies. This material is available free of
charge via the Internet at http://pubs.acs.org.
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JM8005096