ROCK inhibitors 4: Structure-activity relationship studies of 7-azaindole-based rho kinase (ROCK) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2020.127721

Rho kinase (ROCK) inhibitors are of therapeutic value for the treatment of disorders such as hypertension and glaucoma, and potentially of wider use against diseases such as cancer and multiple sclerosis. We previously reported a series of potent and selective ROCK inhibitors based on a substituted 7-azaindole scaffold. Here we extend the SAR exploration of the 7-azaindole series to identify leads for further evaluation. New compounds such as 16, 17, 19, 21 and 22 showed excellent ROCK potency and protein kinase A (PKA) selectivity, combined with microsome and hepatocyte stability.

Full text of DOI:10.1016/j.bmcl.2020.127721

Bioorg. Med. Chem. Lett. 33 (2021) 127721
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
ROCK inhibitors 4: Structure-activity relationship studies of 7-azaindole--
based rho kinase (ROCK) inhibitors
Upul K. Bandarage*, John Court, Huai Gao, Suganthini Nanthakumar, Jon H. Come,
Simon Giroux, Jeremy Green
Vertex Pharmaceuticals Incorporated, 50 Northern Avenue, Boston, MA 02210, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
Rho kinase (ROCK) inhibitors are of therapeutic value for the treatment of disorders such as hypertension and
glaucoma, and potentially of wider use against diseases such as cancer and multiple sclerosis. We previously
reported a series of potent and selective ROCK inhibitors based on a substituted 7-azaindole scaffold. Here we
extend the SAR exploration of the 7-azaindole series to identify leads for further evaluation. New compounds
such as 16, 17, 19, 21 and 22 showed excellent ROCK potency and protein kinase A (PKA) selectivity, combined
with microsome and hepatocyte stability.
Rho kinase (ROCK)
protein kinase A (PKA)
7-Azaindole
Thiazole
Introduction
Results and discussion
The Rho-associated protein kinases (ROCK) are serine-threonine-
kinases of the AGC-family and are involved in the regulation of vital
cellular processes including motility, division, differentiation and
contraction.1 The two isoforms, ROCK I and ROCK II, are highly ho-
mologous (92% sequence identity) in their ATP-binding domain and
share a 65% overall sequence identity.2 Upon activation by GTP-bound
Rho, ROCK phosphorylates various protein targets, such as myosin light
chain3 and myosin light chain phosphatase,4 LIM kinase,5 and zipper-
interacting kinase (ZIPK)6 which lead to cell signaling that promotes
changes in cell motility, adhesion,7 stress fiber formation,7 and force
generation through smooth muscle contraction8. ROCK inhibitors are of
potential therapeutic value for the treatment of glaucoma9 and other
diseases.10–13
In our previous reports we described a pyridine series of ROCK in-
hibitors18 and the development of a related 7-azaindole-derived series.17
During the course of this work we explored the SAR of the central het-
erocycle ring in the context of the pyridine series. In this work we re-
evaluated this ring in the context of the 7-azaindole series, as well as
explored the effects of substituents on the 7-azaindole group.
We first explored the replacement of the thiazole ring of compound 1
with five- and six-membered heterocyclic ring systems including
regioisomeric thiazoles, thiophenes and pyrimidines, as well as thia-
diazole (Fig. 2). Based on our previous studies,17,18 we incorporated the
m-methanesulfonamide phenylacetyl group to interact with the glycine-
rich P-loop region of the ATP-binding pocket.
Enzyme inhibition data for the replacement of the thiazole ring of
compound 1 with various five- and six-membered heterocycles is sum-
marized in Table 1. Most of these new analogs showed strong inhibition
of ROCK-1with Ki values typically below 100 nM and good selectivity
against PKA. In particular, regioisomeric thiazole 3, thiophene
regioisomer 4, and thiadiazole 6 all displayed >30-fold selectivity
against PKA while thiophene regioisomer 5 showed the weakest selec-
tivity against PKA. Both pyrimidine regioisomers 7 and 8 showed good
ROCK potency and somewhat reduced PKA selectivity with respect to
the other 5-membered heterocycles in the series. Overall, the replace-
ment of the thiazole moiety of compound 1 with various five- and six-
membered heterocycles did not further improve either ROCK potency
To date, three ROCK inhibitors have been approved for clinical use:
fasudil is approved for the treatment of cerebral vasospasm14 in Japan
and China, and ripasudil and netarsudil are approved for the treatment
of glaucoma in Japan and the U.S. respectively.15,16
We previously reported a new series of very potent ROCK inhibitors
derived from a 3-substituted-7-azaindole scaffold exemplified by com-
pounds 1 and 2 which exhibited excellent potency against ROCK, good
selectivity against the closely related AGC family kinase PKA, and good
CYP inhibition profiles (Fig. 1).17 In our continued efforts to identify
potent and selective ROCK inhibitors for clinical development, we
further explored the SAR of the 3-substituted-7-azaindole series.
* Corresponding author.
E-mail address: upul_bandarage@vrtx.com (U.K. Bandarage).
https://doi.org/10.1016/j.bmcl.2020.127721
Received 15 October 2020; Received in revised form 17 November 2020; Accepted 23 November 2020
Available online 28 November 2020
0960-894X/© 2020 Elsevier Ltd. All rights reserved.

U.K. Bandarage et al.
Bioorganic & Medicinal Chemistry Letters 33 (2021) 127721
Fig. 1. Structures of ROCK I inhibitors 1 and 2.17
Table 2. Most of the compounds showed excellent ROCK enzyme po-
tency and PKA selectivity except the 4-chloro analog 13, which showed
very poor selectivity against PKA. Fluorine-substituted analogs 10 and
11 showed improved THP-1 migration cellular potencies compared with
other substituted azaindoles. This observation prompted us to synthesize
additional fluorine-substituted analogs at the 4- and 5- positions of the 7-
azaindole ring.
In previous studies17, we reported that 7-azaindole ROCK inhibitors
containing m-substituted phenylacetamides bearing solubilizing groups
such as piperazines and piperidines, attached via two- and three-carbon
linkers, can increase both ROCK potency and PKA selectively due to an
additional binding interaction with the acidic side chain of Asp177 in
ROCK. The corresponding residue in PKA (Gln84) is both neutral and
larger, thus conferring desirable potency, selectivity and solubility
properties17,18Therefore, we synthesized compounds containing solu-
bilizing groups bearing the beneficial 4 - and 5-fluorine substituted 7-
azaindoles with the aim of further enhancing cellular potency and
PKA selectivity. The results are shown in Table 3. The 4- and 5-fluorine
Fig. 2. Replacement of compound 1 thiazole with 5- and 6-membered
heterocycles.
or PKA selectivity. Methyl-substituted thiazole 9 showed reduced ROCK
potency and PKA selectivity. Furthermore, most of these new analogs
showed reduced cellular activity in the THP-1 cell migration assay.
Thiadiazole 6 showed decreased cell potencies possibly due to poor cell
permeability and/or low compound solubility.
We next explored 7-azaindole substituents at the 4- and 5-positions
with groups including F, Cl, Br and OH and the results are outlined in
Table 1
ROCK inhibition and PKA selectivity data for ring A replacements.
Compound
A
ROCK I Ki (
μ
M)a
PKA Selectivity
PKA Ki /ROCK I Ki
11
THP-1 migration
CYP 3A4
LLEb
(ROCK I)
6
IC50
0.02
(μM)
IC50
8.3
(μM)
1
0.006
3
4
5
0.01
0.008
0.1
31
39
1
0.37
0.34
ND
12
6
6.1
5.8
4.7
5
6
7
8
0.007
0.048
0.007
34
23
20
1.52
8
100
48
7
5.9
6.7
0.82
100
9
0.052
31
ND
5
4.6
a
b
Minimum Significant Ratio (MSR)19 = 3.6; i.e., compounds that have a difference in Ki of at least a factor of 3.6 are considered significantly different19
LLE: Lipophilic ligand efficiency (LLE = pKi - clogP)20
2

U.K. Bandarage et al.
Bioorganic & Medicinal Chemistry Letters 33 (2021) 127721
Table 2
Table 4
In vitro hepatic stability and hERG profiles for selected compounds.
ROCK inhibition and PKA selectivity data for substituted 7-azaindoles.
Compound
1
2
11
18
21
22
Rat Clint (µL/min/106 cells)
6.7
4
26.8
7.5
11.7
4.3
32
11.2
5.7
20.5
6.3
Human Clint
3.3
(µL/min/106 cells)
hERG IC50
(μ
M)
>30
>30
>30
>30
>30
>30
Table 5
Rat IV (1 mpk) and PO (3 mpk) PK profiles of selected compounds.
Compound
X
ROCK
PKA
THP-1
CYP
3A4
LLE
Compound
1
2
18
20
21
22
I Ki
Selectivity
migration
(μ
M)
IC50 (μM)
Rat IV PK (1 mg/kg)
CL (mL/min/kg)
PKA Ki
IC50
(ROCK I)
7
114
0.71
10
71
128
5.4
68
66
/ROCK I Ki
(μM)
T
1/2 (hr)
2
3.2
14
5.5
19
3.8
8.7
0.25
1
H
0.006
0.004
0.003
0.007
0.092
0.011
0.022
11
31
53
107
2
0.02
0.08
0.07
1.3
8
6
Vss (L/kg)
0.6
2.4
24
10
11
12
13
14
15
4-F
36
6
6.3
6.3
6.4
4.4
5.3
4.8
AUC0-inf (µg.h/mL)
0.15
0.24
0.13
0.25
5-F
Rat PO PK (3 mg/kg)
5-OH
4-Cl
5-Cl
5-Br
5
AUC0-inf (µg.h/mL)
9.2
0.9
38
0.6
0.06
46
ND
8
C
max
(µg/mL)
46
32
0.31
10
8
%F
35
substituted 7-azaindoles containing 2 and 3 carbon linked N-methyl
piperazine (19, 16, 17) and three-carbon linked piperidine (21 and 22)
showed >140-fold PKA selectivity. Compound 22 showed the highest
PKA selectivity (>480-fold) while maintaining excellent ROCK potency.
However, these new analogs displayed reduced cellular potency
compared with respect to unsubstituted 7-azaindoles (cf 2, 18 and 20).
The metabolic stability of selected compounds was next evaluated in
human and rat hepatocytes (Table 4). Generally, these compounds dis-
played moderate stability in rat hepatocyte and good stability in human
hepatocytes. In addition, these compounds show no inhibition of the
hERG channel (all are >30 µM).
oral dosing and showed good oral bioavailability.
Chemistry
The syntheses of the described inhibitors are illustrated in Schemes
1–5. Regioisomeric thiazole 3 was synthesized in six steps as illustrated
in Scheme 1. 3-Cyano-7-azaindole 23 was first converted to carbox-
amide 24 by treatment with concentrated sulfuric acid. Reaction of 24
with Lawesson’s reagent afforded thiocarboxamide 25 which was
converted to thiazolocarboxylic acid 26 by heating with bromopyruvic
acid in ethanol. Curtius rearrangement of 26 with DPPA and t-BuOH
and followed by removed of Boc group with TFA afforded amino-
thiazole 27. The coupling of 27 with m-methylsulfonamidophenyl-
In vivo rat PK profiling of select compounds is summarized in Table 5.
All compounds bearing a basic solubilizing chain showed high plasma
clearance (>65), high volumes of distribution (>10) and longer T1/2
(>3 h) except compound 2. Compounds 1 and 18 were also evaluated by
21
acetic acid in the presence of BtSO2CH3 afforded desired target
compound 3.
Table 3
Selected data for 4- and 5-fluoro-7-azaindoles with solubilizing groups.
Compound
R
X
ROCK I Ki (
μ
M)
PKA Selectivity
PKA Ki /ROCK I Ki
293
THP-1 migration IC50
0.02
(μM)
CYP 3A4
LLE
IC50
19
(μ
M)
(ROCK I)
4.9
2
H
0.006
16
17
18
19
20
21
4-F
5-F
H
0.01
180
240
50
0.4
ND
ND
18
4.4
4.7
4.9
5
0.005
0.006
0.003
0.005
0.003
0.26
0.02
0.4
5-F
H
320
17
27
0.02
0.05
21
4.4
4.7
4-F
143
100
22
5-F
0.001
480
0.14
ND
4.9
3

U.K. Bandarage et al.
Bioorganic & Medicinal Chemistry Letters 33 (2021) 127721
Scheme 1. Synthesis of 3-thiazolo-7-azaindole 3. Reagents and conditions: (a) c.H2SO4, RT, 18 h; (b) Lawesson’s reagent, THF, 70 ◦C; (c) bromopyruvic acid, EtOH,
reflux, 2 h; (d) DPPA, t-BuOH, DIPEA, 90 ◦C, 2 h; (e) TFA, DCM, RT, 3 h; (f) m-methylsulfonamidophenylacetic acid, BtSO2CH3, Et3N, THF, DMF, 180 ◦C,
W, 15 min.
μ
Scheme 2. Synthesis of thiopheno-7-azaindole 4. Reagents and conditions: (a) DMF, POCl3, 60 ◦C, 4 h; (b) ethyl 2-mercaptoacetate, NaOEt, EtOH, reflux, 90 min; (c)
1 M NaOH, dioxane, 80 ◦C, 90 min; (d) DPPA, t-BuOH, DIPEA, 90 ◦C, 2 h; (e) TFA, DCM, RT, 3 h (f) m-methylsulfonamidophenylacetic acid, BtSO2CH3, Et3N, THF,
DMF, 180 ◦C, µW, 15 min.
Scheme 3. Synthesis of thiophene-7-azaindole 5. Reagents and conditions: (a) t-butyl-4-bromothiophen-2-ylcarbamate, K2CO3, Pd(PPh3)4, K2CO3, DME, 160 ◦C,
μW,
30 min; (b) TFA, DCM, RT, 1 h; (c) m-methylsulfonamidophenylacetic acid, BtSO2CH3, Et3N, THF, DMF, 180 ◦C,
μW, 15 min; (d) LiOH, THF, water.
4

U.K. Bandarage et al.
Bioorganic & Medicinal Chemistry Letters 33 (2021) 127721
Scheme 4. Synthesis of thiadiazolo-7-azindole 6. Reagents and conditions: (a) thiosemicarbazide, TFA, 100 ◦C, 2 h; (b) m-methylsulfonamidophenylacetic acid,
BtSO2CH3, Et3N, THF, DMF, 180 ◦C,
W, 15 min.
μ
Scheme 5. Synthesis of 3-pyrimidine-7-azaindoles 7 and 8. Reagents and conditions: (a) 4-bromopyrimidine 2-amine, Pd(dppf)2.Cl2. DCM, dioxane; (b) m-meth-
ylsulfonamidophenylacetic acid, BtSO2CH3, Et3N, THF, DMF, 180 ◦C,
(dppf)2.Cl2. DCM, dioxane.
μ
W, 15 min; (c) 4 N HCl, dioxane, CH3CN, 80 ◦C, 3 h; (d) 6-bromopyrimidine-4-amine, Pd
Scheme 6. Synthesis of substituted 3-thiazolo-7-azaindoles 9–15. Reagents and conditions: (a) bromoacetyl bromides, AlCl3, CH2Cl2, 50 ◦C, 1 h; (b) thiourea,
ethanol, 70 ◦C, 1 h; (c) m-methylsulfonamidophenylacetic acid, BtSO2CH3, Et3N, THF, DMF, 180 ◦C,
W, 15 min.
μ
Compound 4 was synthesized in six steps as outlined in Scheme 2.
The reaction of 3-acetyl-7-azaindole 28 with POCl3 afforded com-
pound 29, which was reacted with ethyl 2-mercaptoacetate and so-
dium ethoxide in EtOH to afford thiophene ethylester 30. The
hydrolysis of ester 30, followed by Curtius rearrangement and removal
of the Boc group afforded compound 32. Coupling of 32 with m-
methylsulfonamidophenylacetic acid in the presence of MeSO2Bt
afforded target compound 4.
5

U.K. Bandarage et al.
Bioorganic & Medicinal Chemistry Letters 33 (2021) 127721
Regioisomeric thiophene 5 was synthesized in four steps as outlined
in Scheme 3. Suzuki coupling of boronate ester 33 with tert-butyl-4-
bromothiophen-2-ylcarbamate afforded compound 34. Coupling of 34
with m-methylsulfonamidophenylacetic acid in the presence of MeS-
O2Bt, followed by removal of the tosyl group afforded target compound
5.
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
The authors thank Greg May for analytical chemistry support, Dr
Caroline Chandra Tjin for useful comments and suggestions and Dr
Reiko Arimoto for DMPK assistance.
Thiadiazole 6 was synthesized in two steps as outlined in Scheme 4.
3-Cyano-7-azaindole, 23 was reacted with thiosemicarbazide in TFA to
afford aminothiadiazole 35 which was coupled with m-methyl-
sulfonamidophenylacetic acid to afford target compound 6.
The syntheses of pyrimidine containing compounds 7 and 8 are
outlined in Scheme 5. Suzuki coupling of boronate ester 33 with 4-bro-
mopyrimidine-2-amine or 6-bromopyrimidine-4-amine afforded ami-
nopyrimidines 36 or 37 respectively. Amide coupling, followed by
removal of the tosyl group with HCl, afforded target compounds 7 and 8.
3-Thiazolo-7-azaindoles 9–15 were prepared in three steps as shown
in Scheme 6. Friedel-Crafts acylation of commercially available azain-
doles 38a-g with bromoacetyl bromide in DCM afforded 3-(2-bromoa-
cetyl)-7-azaindole 39a-g in good yields. Hantzsch thiazole synthesis of
azaindoles 39a-g and thiourea afforded aminothiazoles 40a-g. Amida-
tions of compounds 40a-g with m-methylsulfonamidophenylacetic acid
were performed as previously described to afford final products 9–15 in
good yields.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2020.127721.
References
1 Godoy MAF, de Rattan S. Trends Pharmacol Sci. 2011;32:384–393.
2 Nakagawa O, Fujisawa K, Ishizaki T, Saito Y, Nakao K, Narumiya S. FEBS Lett. 1996;
392:189.
3 Riento K, Ridley AJ. J Nat Rev Mol Cell Biol. 2003;4:446.
4 Muranyi A, Derkach D, Erdodi F, Kiss A, Ito M, Hartshorne DJ. FEBS Lett. 2005;579:
6611.
5 Scott RW, Olson MF. J Mol Med. 2007;85:555.
6 Hagerty L, Weitzel DH, Chambers J, et al. J Biol Chem. 2007;282:4884.
7 Ishizaki T, Naito M, Fujisawa K, Maekawa M, Watanabe S. FEBS Lett. 1997;404:118.
8 Kureishi Y, Kobayashi S, Amano M, Kimura K, Kanaide HJ. Biol Chem. 1997;272:
12257.
Conclusion
9 Waki M, Yoshida Y, Oka T, Azuma M. Curr Eye Res. 2001;2:470.
10 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.
11 (a) Hirooka Y, Shimokawa H, Takeshita A. Drug News Perspect. 2004;17:523.(b)
Doggrell SA. Opin Invest Drug. 2005;14:1157.
We synthesized a series of 7-azaindole-based ROCK inhibitors and
evaluated their biological activity. Many of these compounds exhibited
excellent ROCK potency and good selectivity against the closely related
AGC kinase, PKA. In addition, top compounds showed good microsomal
and hepatocyte stability, lacked hERG inhibition and good overall CYP
3A4 inhibition profiles. In vivo PK in rats indicated that compounds with
solubilizing chains are rapidly cleared from plasma and highly distrib-
uted, consistent with other kinase inhibitors bearing solubilizing groups.
Replacement of the central thiazole or thiophene ring with other het-
erocycles did not enhance compound profiles. Azaindole substituents,
most notably 4-F and 5-F, may offer an improved overall profile and
warrant further investigation.
12 (a) Nakajima M, Hayashi K, Egi Y, et al. Cancer Chemother Pharmacol. 2003;52:319.
(b) Nakajima M, Hayashi K, Katayama K, et al. Eur J Pharmacol. 2003;459:113.
13 Sun XJ, Minohara M, Kikuchi H, et al. J Neuroimmunol. 2006;180:126.
14 Shibuya M, Satoh S, Suzuki Y, Mcdonald R, ed. Cerebral asospasm: advances in
research and treatment. New York: Thieme, 2004, 284-287.
15 Garnock-Jones KP. Drugs. 2014;74:2211–2215.
16 Sturdivant JM, Royalty SM, Lin C-W, et al. Bioorg Med Chem Lett. 2016;26:
2475–2480.
17 Bandarage UK, Cao J, Come JH, et al. Bioorg Med Chem Lett. 2018;28:2622–2626.
18 Green J, Cao J, Bandarage UK, et al. J Med Chem. 2015;58:5028–5037.
19 Haas JV, Eastwood BJ, Iversen PW, Weidner JR. Minimum Significant Ratio-A
Statistic to Assess Assay Variability. In Sittampalam GS (Ed.) Assay Guidance
Manual, Eli Lilly & Company and the National Center for Advancing Translational
Sciences:Bethesda (MD), 2004.
20 Leeson PD, Springthorpe B. Nat Rev Drug Discov. 2007;6:881–890.
21 Katritzky AR, He H-Y, Suzuki K. J Org Chem. 2000;65:8210–8213.
Declaration of Competing Interest
The authors declare that they have no known competing financial
6