Discovery of a phenylpyrazole amide ROCK inhibitor as a tool molecule for in vivo studies

Article abstract of DOI:10.1016/j.bmcl.2020.127495

Structure-activity relationship optimization on a series of phenylpyrazole amides led to the identification of a dual ROCK1 and ROCK2 inhibitor (25) which demonstrated good potency, kinome selectivity and favorable pharmacokinetic profiles. Compound 25 was selected as a tool molecule for in vivo studies including evaluating hemodynamic effects in telemeterized mice, from which moderate decreases in blood pressure were observed.

Full text of DOI:10.1016/j.bmcl.2020.127495

Journal Pre-proofs
Discovery of a Phenylpyrazole Amide ROCK Inhibitor as a Tool Molecule
for in vivo Studies
Zilun Hu, Cailan Wang, Peter W. Glunz, Julia Li, Nathan L. Cheadle, Alice
Y. Chen, Xue-Qing Chen, Joseph E. Myers, Victor R. Guarino, Anne Rose,
John S. Sack, Doree Sitkoff, David S. Taylor, Songmei Xu, Chunhong Yan,
Hongwei Zhang, Lisa Zhang, James Hennan, Leonard P. Adam, Ruth R.
Wexler, Mimi L. Quan
PII:
S0960-894X(20)30606-5
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127495
BMCL 127495
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
11 July 2020
8 August 2020
10 August 2020
Please cite this article as: Hu, Z., Wang, C., Glunz, P.W., Li, J., Cheadle, N.L., Chen, A.Y., Chen, X-Q., Myers,
J.E., Guarino, V.R., Rose, A., Sack, J.S., Sitkoff, D., Taylor, D.S., Xu, S., Yan, C., Zhang, H., Zhang, L.,
Hennan, J., Adam, L.P., Wexler, R.R., Quan, M.L., Discovery of a Phenylpyrazole Amide ROCK Inhibitor as a
Tool Molecule for in vivo Studies, Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/
10.1016/j.bmcl.2020.127495
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier Ltd.

Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
Discovery of a Phenylpyrazole Amide ROCK
Inhibitor as a Tool Molecule for in vivo
Studies
Leave this area blank for abstract info.
Zilun Hu,* Cailan Wang, Peter W. Glunz, Julia Li, Nathan L. Cheadle, Alice Y. Chen, Xue-Qing Chen, Joseph
E. Myers, Victor R. Guarino, Anne Rose, John S. Sack, Doree Sitkoff, David S. Taylor, Songmei Xu, Chunhong
Yan, Hongwei Zhang, Lisa Zhang, James Hennan, Leonard P. Adam, Ruth R. Wexler, and Mimi L. Quan
MeO
O
25
HN
N
ROCK1 IC50: 5.6 nM
ROCK2 IC50: 7.2 nM
NH NH2

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Discovery of a Phenylpyrazole Amide ROCK Inhibitor as a Tool Molecule for in
vivo Studies
Zilun Hu,* Cailan Wang, Peter W. Glunz, Julia Li, Nathan L. Cheadle, Alice Y. Chen, Xue-Qing Chen, Joseph E.
Myers, Victor R. Guarino, Anne Rose, John S. Sack, Doree Sitkoff, David S. Taylor, Songmei Xu, Chunhong Yan,
Hongwei Zhang, Lisa Zhang, James Hennan, Leonard P. Adam, Ruth R. Wexler, and Mimi L. Quan
Research & Early Development, Bristol Myers Squibb, P.O. Box 5400, Princeton, New Jersey 08543-5400, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Structure-activity relationship optimization on a series of phenylpyrazole amides led to the
identification of a dual ROCK1 and ROCK2 inhibitor (25) which demonstrated good potency,
kinome selectivity and favorable pharmacokinetic profiles. Compound 25 was selected as a tool
molecule for in vivo studies including evaluating hemodynamic effects in telemeterized mice,
from which moderate decreases in blood pressure were observed.
2009 Elsevier Ltd. All rights reserved.
Keywords:
ROCK
Rho kinase inhibitor
Dual inhibitor
Phenylpyrazole
Telemetry study
As members of the serine-threonine protein kinase family, Rho
associated protein kinases (ROCK) are involved in signaling
pathways of many cellular functions, such as actin organization,
cell adhesion, cell migration, and cytokinesis.1 Signal transduction
involving the ROCK pathway is initiated by several vasoactive
factors, many of which are implicated in the pathogenesis of
cardiovascular disease.2 Activated ROCK phosphorylates the
myosin-binding subunit of myosin light chain phosphatase, which
inhibits activity of the phosphatase and leads to muscle contraction
by regulating the shape and function of the actomyosin complex.3-4
Contraction of smooth muscle in the vasculature increases blood
pressure. Therefore, inhibition of ROCK activity can lower blood
pressure and has the potential to positively affect cardiovascular
diseases.5-7 There are two known human ROCK isoforms, ROCK1
and ROCK2, which have highly similar kinase domains of 92%
sequence identity overall, and 100% identity within the ATP
binding pocket.8
studies of Y-27632 have illustrated the link between ROCK and
cardiovascular disease in animal models.17-19 These compounds
demonstrated either moderate ROCK inhibitory activities or
kinome selectivity. The pursuit of ROCK inhibitors with improved
potency and kinome selectivity remains an active research area.
Cl
Cl
H
H
MeO
N
MeO
O
N
hinge modification
O
O
HN
N
N
2
1
ROCK1 IC50: 6.4 nM
ROCK2 IC50: 3.3 nM
ROCK1 IC50: 15 nM
ROCK2 IC50: 7.0 nM
reverse amide
O
Cl
MeO
N
H
HN
Preclinical and clinical stage ROCK inhibitors have been
reported, and dual ROCK1/2 inhibitors have been marketed.
Among them, Fasudil,9-10 Ripasudil11-12 and Netarsudil13 are
marketed for cerebral vasospasm and glaucoma/ocular
hypertension. ROCK2 selective inhibitor KD025 (SLx-2119) is
being investigated in clinical trials for the treatment of
chronic graft versus host disease (cGvHD), idiopathic pulmonary
fibrosis (IPF), and moderate to severe psoriasis.14-16 Pre-clinical
N
3
ROCK1 IC50: 5.0 nM
ROCK2 IC50: 2.3 nM
Figure 1. Lead identification.

Our initial lead compound 1 (Figure 1) was identified from
in-house kinase data mining. Compound 1 was a dual ROCK1/2
inhibitor with ROCK1 and ROCK2 IC50 values of 15 nM and 7.0
nM, respectively. However, compound 1 showed unwanted off-
target kinase activity. The oxazole moiety present in the molecule
was presumed to be the hinge binder. Replacing the oxazole with
a pyrazole, which was successfully used as hinge binders in
reported literature,20 resulted in aniline amide 2 with ROCK1 and
ROCK2 activities of single digit nanomolar IC50 values. The
corresponding benzamide analog 3 demonstrated similar ROCK
inhibitory potencies.
selectivity index is the ratio of active kinases with IC50<100-fold of ROCK2
out of tested kinases (>130 kinases).
Structure-activity relationship (SAR) of benzamides based on 3
was investigated, and the results are listed in Table 1. It was
observed that replacing the ortho-chlorobenzylamine with ortho-
methoxybenzylamine resulted in a dramatic loss in activity (4 vs.
3). However meta-methoxybenzylamine amide retained ROCK1/2
inhibitory activities (5 vs. 4). Introducing a benzylic methyl
substitution showed a 4- to 7-fold improvement of ROCK1/2
potencies, and compound 6 demonstrated ROCK1 and ROCK2
IC50 values of 0.9 nM and 0.3 nM, respectively. The kinome
selectivity index, FSI100, of 6, which is the ratio of active kinases
with IC50<100-fold of ROCK2 out of kinases tested was 2.2,
indicating it was selective. However, the metabolic stability of 6
in human liver microsomes (HLM) was poor. The fluoro and
difluoro analogs (7, 8) retained potency with improved HLM
stability, and compound 8 showed improved kinome selectivity
with a FSI100 value of 0.7. Amino substitution (9) enhanced
ROCK1 and ROCK2 potencies with IC50 values of 0.8 nM and 0.3
nM, respectively, but was less selective against other kinases.
Addition of a hydroxyl moiety improved metabolic stability (10),
but decreased ROCK1 activity when combined with a fluoro
substituted phenyl group (11, 12).
Figure 2. X-ray co-crystal structure of 10 with ROCK2 (A) and comparison of
co-crystals in ROCK1 and ROCK2 (B)
X-ray crystal structures of 10 in ROCK1 (3.3 Å) and ROCK2
(3.0 Å) were obtained (Figure 2).22 Compound 10 binds in the
DFG-in conformation with pyrazole as the hinge binder, and the
terminal phenyl ring is located under the p-loop and against the c-
helix. The amide carbonyl group interacts with conserved lysine
121 (ROCK2 residue numbering), and the hydroxy group binds to
the aspartic acid of each enzyme’s DFG motif. As illustrated in
Figure 2B, compound 10 binds very similarly in the active sites of
ROCK1 and ROCK2. These co-crystals bear a strong similarity to
the ROCK2-bound crystal structure of a chromenopyridine
chemotype recently reported by our group.23 In both cases, the
ligands in general, form an obtuse angle between the planar
hinge/middle section and the terminal phenyl ring tucked under the
p-loop. This overall binding mode is also seen in other recently
published ROCK1 and ROCK2 co-crystal structures.24-26
Table 1. Benzamide SAR.
O
HN
N
HN R
MeO
IC50 (nM)a,21
HLM
Cpd#
R
MetStabb
(Rem%)
FSI100c
ROCK1
ROCK2
66

4
5
6
182
6.7
0.9
-
-
OMe
1.2
0.3
49
32
4.5
2.2


OMe
OMe

7
8
3.1
5.4
1.3
1.6
72
79
3.0
0.7
Table 2. Central phenyl substitution.
F
F
F
O
OMe


HN
N
HN
2
3
R
Me
9
0.8
7.9
0.3
2.3
93
87
5.5
3.1
NH2
OH
IC50 (nM)
HLM MetStab
(Rem%)
32
Cpd#
R
FSI100
ROCK1
ROCK2
0.3


10
6
3-OMe
3-Et
0.9
0.8
8.7
10
2.2
1.0
1.5
1.5
2.2
1.4
0.8
-
F
F
13
14
15
16
17
18
19
0.4
1.0
0.7
6.7
1.4
1.1
110
38
74
59
67
44
49
-
11
12
24
16
2.1
2.1
90
92
2.8
1.4
3-Me
3-Cl
F
OH
OH
3-CN
3-CF3
2-OMe
2-Me
56

7.1
5.4
1170
aCaliper assay IC50 data listed in Table 1-3. See reference 21 for details.
bHuman liver microsome (HML) metabolic stability remaining percentage
(Rem%) after incubated for 10 min in 0.5 uM. concentration. cFSI100: fraction

20
2-F,3-OMe
6.0
1.0
57
0.7
metabolic stability. Methyl, chloro and cyano substituted analogs
(14 - 16) were more stable in human liver microsomes than 6, but
had reduced affinity for ROCK1. While 2-OMe and 2-F/3-OMe
substitutions maintained potency (18, 20) and good kinome
selectivity, significantly weaker ROCK binding was observed for
the 2-Me analog (19). This is most likely due to disruption of the
nearly planar phenyl-amide dihedral angle seen in the X-ray co-
crystal structures of 10 in the ROCK enzymes while the planar
arrangement is likely maintained in 18 through the intramolecular
H-bond between OMe and the amide NH.27
Effects of substitution on the central phenyl ring were
investigated. As shown in Table 2, electron donating and
withdrawing groups at the 3-position were generally tolerated with
good kinome selectivity (FSI100), and the resulting impact on
potency, metabolic stability and selectivity were subtle. Analogs
with 3-methoxy and ethyl substitutions (6, 13) were potent with
sub-nanomolar IC50 values. However, both showed poor HLM
Table 3. Aniline amides
HN
N
NH
R
MeO
O
IC50 (nM)
HLM MetStab
Kinome Selectivityb
(T1/2, min)a
Cpd#
R
ROCK1
6.4
ROCK2
3.3



2
36
15
2/35
0/22
0/31
1/22
1/19
Cl
21
22
23
24
21
14
4.3
10
7.0
4.0
5.0
8.0
OH Cl
NH2 Cl
20

Cl
Cl
31
NH2

NTc
NH2
6/261 (FSI100 = 2.0)
(none < 100 nM)

25
26
7.2
48
5.6
11
41
62
NH2
NH2

0/23
aHuman liver microsome (HML) metabolic stability half-life (T1/2 in min).
bKinome selectivity is illustrated as the number of kinases with IC50 <1 M out of the panel of total tested kinases in this set of compounds.
cNot tested.
Aniline amide SAR was further explored based on compound
2. Benzylic substitution with polar groups was introduced aiming
to improve metabolic stability and solubility while maintaining
potency. Compound 21 with the hydroxyl substitution retained
ROCK2 potency and was slightly less potent for ROCK1
compared to 2. Analogs with an amine substitution derived from
(R)-phenylglycine and (R)-phenylalanine (22 - 24) showed
comparable activities to 2. Further homologation resulted in
analog 25, which maintained both ROCK1 and ROCK2 potencies
with a balanced profile of kinome selectivity and metabolic
stability. The corresponding enantiomer (26) was less potent.
The synthesis of the benzamides in Table 1 is shown in Scheme
1. Suzuki coupling between boronate 27 and bromide 28 followed
by the removal of Boc protection gave ester 29. Hydrolysis of ester
29 afforded acid 30, which was coupled with different amines to
provide analogs 3 -12. Following similar procedures outlined in
Scheme 1, compounds in Table 2 were prepared from the
appropriately substituted starting materials.
MeO
Br
MeO
a
b
O
O
BocN
N
+
B
BocN
N
NH2
NH2
27
31
32
MeO
O
MeO
a
b
OMe
O
OMe
O
B
+
BocN
HN
N
Br
O
c
N
MeO
O
MeO
O
28
27
29
BocN
N
HN
N
NH NHBoc
NH NH2
MeO
MeO
34
25
OH
O
HN
O
R
c
HN
N
HN
Scheme 2. Reagents and conditions: (a) PdCl2 (dppf)-DCM adduct, K3PO4,
toluene/H2O, 100 °C, 83% (b) (R)-2-((tert-butoxycarbonyl)amino)-4-
phenylbutanoic acid (33), HATU, DIEA, DMF, 0 °C, 100% (c) TFA, DCM, rt,
97%
N
30
3
12
-
Scheme 1. Reagents and conditions: (a) (i) PdCl2(dppf)-DCM adduct, K3PO4,
dioxane/H2O, 100 °C, 3 h (ii) TFA, DCM, rt, 30 min, 70% for two steps (b)
LiOH, THF/H2O, rt, 5 h, 100% (c) RNH2, HATU, DIEA, DMF, 0°C to rt, 30 –
95%.
Compounds in Table 3 were synthesized as exemplified by the
synthesis of compound 25 described in Scheme 2. Suzuki coupling
between 27 and bromide 31 afforded aniline 32. Amide formation

Figure 3. Effects of 25 on MAP in mice: A single oral dose administration of
1 and 10 mg/kg formulated in 2% CMC/0.5% Tween 80/97.5% water vehicle
was given at time 0 (arrow). Arterial blood pressure was recorded continuously
throughout the study period. Data were averaged for 1 hour intervals
throughout the 48 hour continuous data recordings (24 hours pre-dose and 24
hours after dose) for data display.
between 32 and amino acid 33, followed by the removal of Boc
protection, provided 25 in high yield.
Selected compounds with good overall profiles including
potent ROCK inhibition, kinome selectivity, metabolic stability,
and PAMPA permeability at both pH 5.5 and 7.4, were evaluated
in mouse pharmacokinetic (PK) studies, and the results are shown
in Table 4. These compounds exhibited moderate to good
In conclusion, a series of phenyl pyrazole amides was identified
and optimized for dual ROCK1/2 potencies and kinome
selectivity. Compound 25 was discovered to be a potent dual
ROCK1/2 inhibitor with good kinome selectivity and a favorable
pharmacokinetic profile. The hemodynamic effects of 25 were
investigated in mouse telemetry studies. Moderate decreases in
blood pressure were observed. Further characterization of 25 as a
dual ROCK1/2 inhibitor in additional animal models will be
reported in the future.
exposures after oral dosing. Both compound
8 and 25
demonstrated good PK profiles with Cmax of 23 M and 28 M,
respectively, and an AUC of 140 M*h. Compound 25 was further
studied as a tool molecule for in vivo characterization.
Dual ROCK inhibitors have been reported to have potential
applications for the treatment of hypertension as well as other
cardiovascular diseases.17-19 Thus, we determined the
hemodynamic effects of compound 25. Compound 25 was
investigated in conscious telemeterized mice at doses of 1 and 10
mpk, PO. As shown in Figure 3, decreases in mean arterial blood
pressure (MAP) were observed at both doses. The decreases were
moderate with maximum effects of -9 mmHg (-8%) and -11
mmHg (-10%), respectively. The duration of effects lasted 6 - 8
hours post dosing with the effects appearing related to the dose.
Acknowledgments
The authors would like thank Karen Rossi for the X-ray crystal
structure pictures, and BMS Lead Discovery and Optimization for
generating kinome selectivity, PAMPA and HLM metabolic
stability data. The X-ray co-crystal structure of compound 10 with
ROCK2 was obtained at Proteros. We also thank Drs. Heather
Finlay, Ellen Kick, and Joseph Luettgen for reviewing this
manuscript.
Table 4. Mouse PK profiles of selected compounds
References and notes
Mouse PO PK
PAMPA
Dose*
(mpk)
Cpd#
permeability
(Pc, nm/sec, pH 5.5/7.4)
Cmax
(M)
AUC
(M*h)
1.
2.
Riento, K.; Ridley, A. J. Nat. Rev. Mol. Cell. Biol. 2003, 4, 446-
456.
Shimokawa, H.; Rashid, M. TRENDS Pharmacol. Sci. 2007, 28,
296-302.
Somlyo, A.P. Nature, 1997, 389, 909-911.
Schofield A. V.; Bernard, O. Crit. Rev. Biochem. Mol. Biol. 2013,
48, 301-316.
Knipe, R. S.; Tager, A. M.; Liao, J. K. Pharmacol. Rev. 2015, 67,
103-117.
Loirand, G. Pharmacol. Rev. 2015, 67, 1074-1095.
Feng, Y.; LoGrasso, P. V.; Defert, O.; Li, R. J. Med. Chem. 2016,
59, 2269-2300.
Matsui, T.; Amano, M.; Yamamoto, T.; Chihara, K.; Nakafuku, M.;
Ito, M.; Nakano, T.; Okawa, K.; Iwamatsu, A.; Kaibuchi, K. EMBO
J. 1996, 15, 2208-2216.
8
610/610
105/110
520/440
290/200
450/740
10a
10a
2b
23
21
1.3
12
28
140
10
12
14
25
90
3.
4.
5.0
35
10b
10c
5.
140
6.
7.
*Formulation vehicles: (a) 10% Cremaphor EL/15% NMP/75% water (b) 10%
EtOH/10% DMAC/10% Cremaphor EL/70% water (c) 10% EtOH/10%
TPGS/10% water/70% PEG400
8.
9.
Nagumo, H.; Sasaki, Y.; Ono, Y.; Okamoto, H.; Seto, M.; Takuwa,
Y. Am. J. Phys.- Cell Phys. 2000, 278, C57-65.
10. Naraoka, M., Munakata, A.; Matsuda, N.; Shimamura, N.; Ohkuma,
H. Transl. Stroke Res. 2013, 4, 368-374.
11. Garnock-Jones, K. P. Drugs 2014, 74, 2211-2215.
12. Inoue, T.; Tanihara, H. Expert. Opin. Pharmacther. 2017, 18, 1669-
1673.
13. Lin, C.-W.; Sherman, B.; Moore, L. A.; Laethem, C. L.; Lu, D.-
W.; Pattabiraman, P. P.; Rao, P. V.; DeLong, M. A.; Casey C.
Kopczynski, C. C. J. Ocul. Pharmacol. Ther. 2018, 34, 40-51.
14. Zanin-Zhorov, A.; Weiss, J. M.; Nyuydzefe, M. S.; Chen, W.;
Scher, J. U.; Mo, R.; Depoil, D.; Rao, N.; Liu, B.; Wei, J.; Lucas,
S.; Koslow, M.; Roche, Maria; Schueller, O.; Weiss, S.;
Poyurovsky, M. V.; Tonra, J.; Hippen, K. L.; Dustin, M. L.; Blazar,
B. R.; Liu, C.-J; Waksal, S. D. Proc. Nat. Acad. Sc. USA
2014, 111, 16814-16819.
15. Park, J.; Chun, K.-H. Gene, 2020, 144474.
16. KD025 - Clinical Trials, ClinicalTrials.gov, NCT03919799, April,
2020.
17. Cantoni, S.; Pastore, F.; Cavalli, S.; Marchini, G.; Villetti,
G.; Nassini, R.; De Logu, F.; Facchinetti, F. Eur. Respiratory
J. 2016, 48, PA5100.
18. Uehata, M.; Ishizaki, T.;Satoh, H.; Ono, T.; Kawahara, T.;
Morishita, T.; Tamakawa, H.; Yamagami, K.; Inui, J.; Maekawa,
M.; Narumiya, S. Nature, 1997, 389, 990-994.
19. Kobayashi, N.; Horinaka, S.; Mita, S.; Nakano, S.; Honda, T.;
Yoshida, K.; Kobayashi, T.; Matsuoka, H.; Cardiovascular Res.,
2002, 55, 757-767.
20. (a) Chen, Y. T.; Bannister, T. D.; Weiser, A.; Griffin, E.; Lin, L.;
Ruiz, C.; Cameron, M. D.; Schurer, S.; Duckett, D.; Schroter, T.;
LoGrasso, P.; Feng, Y. Bioorg. Med. Chem. Lett. 2008, 18, 6406-

6409. (b) Feng,Y.; Yin, Y.; Weiser, A.; Griffin, E.; Cameron, M.
D.; Lin, L.; Ruiz, C.; Schurer, S. C.; Inoue, T.; Rao, P. V.; Schröter,
T.; LoGrasso, P. J. Med. Chem. 2008, 51, 6642–6645. (c) Fang, X.;
Yin, Y.; Chen, Y. T.; Yao, L.; Wang, B.; Cameron, M. D.; Lin, L.;
Khan, S.; Ruiz, C.; Schroter, T.; Grant, W.; Weiser, A.; Pocas, J.;
Pachori, A.; Schurer, S.; LoGrasso, P.; Feng, Y. J. Med. Chem.
2010, 53, 5727–5737. (d) Fang, X.; Chen, Y. T.; Sessions, E. H.;
Chowdhury, S.; Vojkovsky, T.; Yin, Y.; Pocas, J. R.; Grant, W.;
Schröter, T.; Lin, L.; Ruiz, C.; Cameron, M. D.; LoGrasso, P.;
Bannister, T. D.; Feng, Y. Bioorg. Med. Chem. Lett. 2011, 21, 1844-
1848.
Freiberg, G. M.; Gopalakrishnan, S. M.; Harris, C. M.; Honore, M.
P.; Kage, K. L.; Kapecki, N. J.; Ling, C.; Ma, J.; Mack, H.; Mamo,
M.; Maurus, S.; McRae, B.; Moore, N. S.; Mueller, B. K.; Mueller,
R.; Namovic, M. T.; Patel, K.; Pratt, S. D.; Putman, C. B.; Queeney,
K. L.; Sarris, K. K.; Schaffter, L. M.; Stoll, V.; Vasudevan, A.;
Wang, L.; Wang, L.; Wirthl, W.; Yach, K. J Med. Chem. 2018, 61,
11074-11100.
27. A search of the Cambridge Crystallographic Data Centre’s small
molecule crystallographic database (Conquest version 20.0.5;
Bruno, I. J.; Cole, J. C.; Edgington, P. R.; Kessler, M.; Macrae, C.
F.; McCabe, P.; Pearson, J.; Taylor, R. Acta Cryst., 2002, B58, 389-
397) using the query 2-methyl, 6-H-benzamide yields 50 hits; the
smallest phenyl-amide dihedral angle (C(Me)CC(O)N) observed is
50 degrees. In contrast, the query 2-fluoro, 6-H-benzamide
produces 73 hits; of these, 26 possess a phenyl-amide dihedral angle
(C(F)CC(O)N below 20 degrees. The corresponding dihedral
angles in the co-crystal structures of 10 in ROCK1 and ROCK2 are
18 degrees and 10 degrees, respectively.
21. Details of kinase inhibitors in vitro potency (IC50) determination
were described in reference 23.
22. The PDB IDs are 7JOU (10/ROCK1) and 7JOV (10/ROCK2).
23. Hu, Z.; Wang, C.; Sitkoff, D.; Cheadle, N.; Xu, S.; Muckelbauer, J.
K.; Adam, L. P.; Wexler, R. R.; Quan, M. L. Bioorg. Med. Chem.
Lett. Accepted.
24. Gao, H.; Marhefka, C; Jacobs, M. D.; Cao, J.; Bandarage, U.
K.; Green, J. Bioorg. Med. Chem. Lett. 2018, 28, 2616-2621.
25. Bandarage, U. K.; Cao, J.; Come, J. H.; Court, J. J.; Gao, H.; Jacobs,
M. D.; Marhefka, C.; Nanthakumar, S.; Green, J. Bioorg. Med.
Chem. Lett. 2018, 28, 2622-2626.
Click here to remove instruction text...
26. Hobson, A. D.; Judge, R. A.; Aguirre, A. L.; Brown, B. S.; Cui, Y.;
Ding, P.; Dominguez, E.; DiGiammarino, E.; Egan, D. A.;

Highlights:
.
SAR optimization is explored on a series of
phenylpyrazole amides in the discovery of dual
ROCK1 and ROCK2 inhibitors.
.
.
ROCK1 and ROCK2 co-crystal structures of a
dual inhibitor are described.
An in vivo tool molecule which demonstrates
good potency, kinome selectivity, and favorable
pharmacokinetic profile is identified.
The tool molecule is evaluated for
.
hemodynamic effects in telemeterized mice,
from which moderate decreases in blood
pressure are observed.