Fragment-based discovery of 6-substituted isoquinolin-1-amine based ROCK-I inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.11.060

Fragment-based NMR screening of a small literature focused library led to identification of a historical thrombin/FactorXa building block, 17A, that was found to be a ROCK-I inhibitor. In the absence of an X-ray structure, fragment growth afforded 6-substituted isoquinolin-1-amine derivatives which were profiled in the primary ROCK-I IMAP assay. Compounds 23A and 23E were selected as fragment optimized hits for further profiling. Compound 23A has similar ROCK-1 affinity, potency and cell based efficacy to the first generation ROCK inhibitors, however, it has a superior PK profile in C57 mouse. Compound 23E demonstrates the feasibility of improving ROCK-1 affinity, potency and cell based efficacy for the series, however, it has a poor PK profile relative to 23A.

Full text of DOI:10.1016/j.bmcl.2010.11.060

Bioorganic & Medicinal Chemistry Letters 21 (2011) 97–101
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Fragment-based discovery of 6-substituted isoquinolin-1-amine
based ROCK-I inhibitors
⇑
Peter Ray , Jane Wright, Julia Adam, Johnathan Bennett, Sylviane Boucharens, Darcey Black, Andrew Cook,
Angus R. Brown, Ola Epemolu, Dan Fletcher, Anders Haunso, Margaret Huggett, Phil Jones, Steven Laats,
Amanda Lyons, Jordi Mestres, Jos de Man, Richard Morphy, Zoran Rankovic, Brad Sherborne,
Lorcan Sherry, Nicole van Straten, Paul Westwood, Guido Z. R. Zaman
Discovery Research, MSD, Newhouse, Lanarkshire ML1 5SH, Scotland, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Fragment-based NMR screening of a small literature focused library led to identification of a historical
thrombin/FactorXa building block, 17A, that was found to be a ROCK-I inhibitor. In the absence of an
X-ray structure, fragment growth afforded 6-substituted isoquinolin-1-amine derivatives which were
profiled in the primary ROCK-I IMAP assay. Compounds 23A and 23E were selected as fragment opti-
mized hits for further profiling. Compound 23A has similar ROCK-1 affinity, potency and cell based effi-
cacy to the first generation ROCK inhibitors, however, it has a superior PK profile in C57 mouse.
Compound 23E demonstrates the feasibility of improving ROCK-1 affinity, potency and cell based efficacy
for the series, however, it has a poor PK profile relative to 23A.
Received 22 October 2010
Revised 12 November 2010
Accepted 15 November 2010
Available online 19 November 2010
Keywords:
Rho kinase
ROCK-I
ROCK inhibitors
Fragment-based drug discovery
FBDD
Ó 2010 Published by Elsevier Ltd.
STD NMR
Rho kinase (ROCK) belongs to the AGC family of serine/threo-
nine kinases that are activated via interaction with the small
GTP-binding protein RhoA. Two isoforms have been described,
ROCK-I and ROCK-II, which are highly homologous (65% overall
homology; 92% identity in kinase domains) and exhibit absolute
identity in their ATP-binding site.1 ROCK is ubiquitously expressed
across a range of cell types within various tissues and is implicated
in a number of important physiological functions including regula-
tion of smooth muscle contraction, cytoskeleton re-arrangement,
cell migration and proliferation and inflammatory responses.2
Accumulating clinical and pre-clinical evidence indicates that
ROCK is an important target for several cardiovascular diseases
including angina pectoris, hypertension, coronary vasospasm,
restenosis after percutaneous coronary intervention and arterio-
sclerosis.3 Clinical studies using the ROCK inhibitor Fasudil have
demonstrated efficacy and safety in treating cerebral vasospasm,
ischemic stroke and stable angina. Furthermore, pre-clinical stud-
ies suggest additional therapeutic potential for ROCK inhibitors
in a wide range of diseases including cancer, glaucoma and CNS
diseases such as Alzheimer’s disease and neuropathic pain.4
First generation ROCK inhibitors, including hydroxyl-Fasudil
(the active metabolite of Fasudil) and Y-27632, have been known
for a number of years (Fig. 1).5
More recently, there have been extensive research efforts
towards identifying novel classes of potent and selective ROCK
inhibitors.6 To this end, specialized screening techniques including
fragment-based hit discovery are being utilized to identify novel,
tractable low-MW hit compounds.7 Herein we describe a frag-
ment-based approach which afforded a novel low molecular
weight ROCK inhibitor with improved pharmacokinetics relative
to the first generation compounds, for the potential treatment of
atherosclerosis.
A small library of 69 compounds was created by fragmenting a
database of kinase inhibitors that included existing ROCK
H
N
N
N
O
O
O S
N
O
NH
O
O S
N
H
N
N
H2N
H
H
Hydroxy-Fasudil
Y-27632
Fasudil
⇑
Corresponding author. Tel.: +44 (0)1698 736127.
E-mail address: peter.ray@merck.com (P. Ray).
Figure 1. First generation ROCK inhibitors.
0960-894X/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2010.11.060

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P. Ray et al. / Bioorg. Med. Chem. Lett. 21 (2011) 97–101
inhibitors such as Y-27632 and Fasudil. Stock solutions were pre-
pared (500 M or 100 M) and saturation transfer NMR used to
NH2
l
l
N
screen for binders.7 Forty-one fragments bound to ROCK-I and
NMR competition binding experiments with Y-27632 showed that
16 bound in a similar manner to the ATP-binding site (see Fig. 2).
The NMR competition experiments suggested that fragments
10–16, which are structurally related to both Fasudil and
Y-27632, were the most potent ATP binders (Table 1). Fragments
10, 13 and 14 were prioritised based on analysis of the literature,
which indicated their potential to deliver a novel series of
ROCK-I inhibitors.9
NH2
17A
Figure 3. Thrombin 6-(aminomethyl)isoquinolin-1-amine fragment 17A.
Since the 6-substituted isoquinolin-1-amine core was relatively
unexplored in the kinase arena at the time, it was felt that elabo-
rating this fused bicyclic ring system would offer good scope for
generating a novel series. Fragment-based approaches benefit
enormously from biostructural information but at that time no
X-ray structure for ROCK was available. However, extensive use
had been made of PKA for co-crystallization of ROCK-I ligands, so
to guide fragment growth efforts, compound 17A was docked into
a PKA-based homology model of ROCK-I (Fig. 4). The preferred
binding mode indicated the isoquinolin-1-amine core co-ordinat-
ing with the protein backbone in the ATP-binding site in a
conformation identical to that observed for the adenine ring from
the X-ray structure of a non-hydrolysable ATP mimetic, rather than
that adopted by the isoquinoline ring of Fasudil.13
Fragments 10, 13 and 14 were used as templates for similarity
and sub-structure searches of the corporate building block collec-
tion, with a focus on identifying bulk quantities of non-commer-
cially available cores.
One such building block, 17A (Fig. 3), was derived from a histor-
ical thrombin inhibitor project.10 With a pKa of ꢀ7.6, the amino iso-
quinoline moiety exists, at physiological pH, in equilibrium
between the neutral and protonated forms.11 It was envisaged that
the neutral form would be able to bind to the kinase’s hinge region
and when tested in the ROCK-I IMAPÒ,12 assay 17A exhibited a
pIC50 of 5.11 0.03.
In this orientation the pendant aminomethyl group is placed in
the ribose binding region14 normally exploited by hydrophobic
substituents in other inhibitor series. So we designed one library
in which hydrophobic substituents were appended to the amino-
methyl group (Table 2). Additionally the docking model suggested
moving the basic amino group further from the bicyclic core, in
order to provide better access to the phosphate binding region of
the ATP-binding site. So plans for a second library were drawn
up with positively charged substituents to explore this hypothesis
(Table 4). The first fragment growth library from 17A was achieved
via reductive amination of N-(6-formylisoquinolin-1-yl)benzamide
18 with a variety of amines, followed by deprotection as described
in Scheme 1.
The N-(6-formylisoquinolin-1-yl)benzamide 18 was prepared
by Knoevenagel condensation of 3-bromobenzaldehyde 19 with
malonic acid in refluxing ethanol in the presence of pyridine to af-
ford 3-bromocinnamic acid, which was treated with thionyl chlo-
ride in refluxing toluene to give the acid chloride 20. Acid
chloride 20 was dissolved in acetone and the resulting solution
added to a mixture of sodium azide in water/acetone at 0 °C. After
stirring vigorously for 1 h, the reaction mixture was poured onto
ice-water and the 3-bromocinnamoyl azide precipitated as a white
solid. A solution of the azide in dichloromethane was then added
dropwise to preheated (140 °C) diphenyl ether to give the pre-
sumed Curtius-re-arrangement product, with evolution of nitrogen
N
O
N
N
NH2
N
N
H
1
2
3
O
H
N
O
N
N
H2N
N
N
HO
O
4
5
6
7
8
OH
N
H2N
N
O
H
S
N
N
N
N
N
N
N
N
N
H2N
H
N
H
9
10
11
12
13
NH2
N
N
N
N
O
O
N
16
14
15
Figure 2. Fragments 1–16, identified from ROCK-I NMR competition binding
experiments with Y-27632.
Table 1
A
comparison of the results from the ROCK-1 NMR
competition experiments
Fragment
% drop in intensity of
binding signals on
addition of Y-27632
1
2
20
25
3
30
4
34
5
38
6
40
7
60
8
64
9
68
10
11
12
13
14
15
16
70
>80
>80
>80
>80
>80
>80
Figure 4. Docking of fragment 17A (orange) into the ROCK-I homology model
alongside modeled Fasudil (cyan) and ATP mimetic (pink).

P. Ray et al. / Bioorg. Med. Chem. Lett. 21 (2011) 97–101
99
Table 2
Table 3
ROCK-I IMAP SAR for R amines, 17A–M
Further in vitro profiling of 17G
NH2
NH2
N
N
H
R
N
Ph
a
17G
Compounds
R
ROCK-I (IMAP) pIC50
5.11 0.03
17A
NH2
17Ga
N
17B
17C
6.37 0.07
6.23 0.3
ROCK-I pKi
7.2
5.89 0.05
6.5
0.48/3.0
<4.5
5.5
ROCK-I IMAP pIC50
ROCK-II IMAP pIC50
LE/LipE
THP migration pIC50
hERG (Dofetilide) pKi
H
N
O
H
N
Cl
Cl
17D
17E
17F
6.11 0.04
6.09 0.04
6.00 0.03
a
pIC50 values are shown as mean standard deviation from
n = 2–5 separate experiments (except where n = 1).
H
N
H
N
A diverse array of reductive aminations were investigated and a
number of 6-(aminomethyl)isoquinolin-1-amine derivatives pre-
pared for SAR evaluation in the ROCK-I IMAP assay. Compounds
which afforded a pIC50 >5 in the ROCK-I IMAP assay, 17B–O, are
shown in Table 2. Compounds with significantly improved potency
compared to 17A were identified such as 17B, 17C, 17G and 17K.
These compounds were further profiled in vitro in order to evalu-
ate the scope for further optimization.
The data for compound 17G is shown in Table 3. As expected
from the close ATP-site homology, a similar ROCK-II IMAP potency
was obtained. However, 17G was poorly active in the THP cell
based (MCP-1) migration assay, which provides an in vitro under-
standing of MCP-1-induced transendothelial migration of mono-
cytes in unstable atherosclerotic lesions. The compound also
possessed significant hERG binding.
As a key element of the design of our second fragment-growing
library based on extending the linker to the basic amine, ether
linked cyclic amines were prioritised. Cyclic amines are highly rep-
resented within the ROCK literature and it was rationalized that
linking readily available piperidines to a 6-hydroxy-isoquinolin-
1-amine core would provide compounds with reduced rotational
bonds and possibly improved ligand-lipophilicity efficiency values
relative to compounds 17A–M. Piperidine derivatives of 6-
(oxy)isoquinolin-1-amine, 23A–F, were readily accessible from
the route described in Scheme 2. The 6-hydroxy-isoquinolin-1-
amine core 28 was prepared from commercially available 24.
Oxidation of 24 with mCPBA, followed by treatment with POCl3
afforded 25, which was converted to 26 following reaction with
phenol and potassium hydroxide. Phenoxy derivative 26 was then
treated withammonium acetateto afford 27, which was deprotected
OMe
H
N
17G
17H
17I
5.89 0.05
5.83 0.09
5.77 0.05
H
N
N
H
H
N
CF3
H
N
17J
5.73 0.04
6.24 0.09
N
O
H
N
17K
H
N
17L
5.46 0.04
5.17
H
N
17M
a
pIC50 values are shown as mean standard deviation from n = 2–5 separate
experiments (except where n = 1).
gas. The dichloromethane was removed under reduced pressure
and the resulting diphenyl ether isocyanate solution heated
(250–260 °C) to afford 6-bromoisoquinolinone 21. Conversion of
21 to 1-amino-6-bromoisoquinoline 22 involved treatment with
POCl3, followed by reaction with phenol and potassium hydroxide
and then ammonium acetate. The aminoisoquinoline 22 was
N-benzoyl protected, the bromine lithiated and then quenched
with N,N-dimethylformamide to afford aldehyde 18.
Cl
OPh
O
N
a, b
c
N
N
Cl
HN
a, b
h, i
c, d
j, k
e, f, g
Br
O
N
OMe
Br
Br
OMe
N
OMe
O
Br
24
25
26
19
20
21
NH2
Ph
O
NH2
NH2
d
e
f
NH2
HN
N
N
N
OMs
OMe
g, h
OH
R
R
27
28
( )m
O
N
Boc
22
29
18
17A-M
NH2
NH2
NBoc
( )m
N
N
N
Scheme 1. Synthesis of N-(6-formylisoquinolin-1-yl)benzamide 18 and reductive
amination, followed by deprotection to afford 6-(aminomethyl)isoquinolin-1-
amine based ROCK-I inhibitors 17A–O. Reagents and conditions: (a) CH2(CO2H)2,
EtOH, pyridine, 16 h, reflux; (b) SOCl2, toluene, 1 h, reflux; (c) NaN3, H2O, acetone,
1.5 h, 0 °C; (d) Ph2O, 2 h, 140 °C then 2 h, 250–260 °C; (e) POCl3, CHCl3, 2 h, reflux;
(f) PhOH, KOH, xylene, 4 h, reflux; (g) NH4OAc, 160 °C; (h) (PhCO)2O, pyridine, 1 h,
125 °C; (i) n-BuLi, THF, À78 °C, DMF added and allowed to warm to 0 °C; (j) THF,
amine, MP-cyanoborohydride resin; (k) AcOH, 6 N HCl, 24 h at 104 °C.
( )m
O
O
30
23
Scheme 2. Synthesis of 6-(oxy)isoquinolin-1-amine 25A–F based ROCK inhibitors.
Reagents and conditions: (a) mCPBA, DCM; (b) POCl3, 6 h, 90 °C; (c) PhOH, KOH, 3 h,
140 °C; (d) NH4OAc, 16 h, 150 °C; (e) BBr3, DCM; (f) PS-BEMP, MeCN, 160 °C; (g)
TFA, DCM; (h) HOAc, CH3CN, RCHO, NaBH(OAc)3.

100
P. Ray et al. / Bioorg. Med. Chem. Lett. 21 (2011) 97–101
Table 5
with boron tribromide to afford 28. PS-BEMP mediated SN2 displace-
ment of the readily prepared mesylates 29 afforded ether 30. N-Boc
deprotection of 30 afforded compounds23A–C. Reductive amination
with the appropriate amine 23A–C (R = H) provided compounds
23D–F.
Profiling of compound 23A and the N-benzylated derivative 23E selected optimized
fragments
23Aa
23Ea
ROCK-I pKi
7.4
n.d.
ROCK-I IMAP pIC50
ROCK-II IMAP pIC50
THP migration pIC50
LE/LipE
5.67 0.16
5.95 0.04
5.3
0.58/3.9
All >120
70
6.3/3.6
Clean
243
6.62 0.01
7.04 0.09
6.11 0.21
—/2.4
12/22/>120
27
The SAR for 23A–F, in the ROCK-I IMAP assay is shown in
Table 4. The potencies obtained for piperidines 23A and 23B were
encouraging and improved following N-benzylation to afford 23D
and 23E. Compounds 23A, 23B, 23D and 23E represented a signif-
icant improvement over the original fragment hit 17A illustrating
the benefit of employing an extended rigidified linker between
the isoquinolin-1-amine and the basic amine. As predicted by the
homology model, the importance of the basic nitrogen on the
piperidine ring is illustrated by the inactivity of compound 23G rel-
ative to 23A.
HLM/RLM/MLM (min, T1/2
)
Rat Heps (min, T1/2
)
Caco-2(>5 Â 10À6 cm/s) A–B/B–A
Cyp inhibition (2C19, 3A4, 1A2, 2D6, 2C9)
MW
2.2/1.5
All >3
333
lM
c log P/c log D
hERG (Dofetilide) pKi
2/À1
4.2/3.2
6.1
Wistar rat
4.7
C57 mouse
>75
9.5
11
5.8
Bioavailability (F%)
Clp (ml/min/kg)
Vss (L/kg)
7
In order to evaluate the scope for further optimization, com-
pound 23A and its N-benzylated derivative 23E were selected for
further in vitro and in vivo pharmacokinetic profiling (Table 5).
Compound 23A had comparable binding affinity, potency and cell
based efficacy to the first generation ROCK inhibitors hydroxyl-
Fasudil and Y-27632 (Table 6), which were benchmarked in the
same assay formats. The LiPE and selectivity over hERG for 23A
was improved relative to 17G. Also unlike 17G, 23A showed activ-
ity in the THP migration assay. Compound 23E exhibited higher
activity than 23A in both the IMAP and THP assays, but its hERG
activity was also high. In the C57 mouse, 23A had an excellent
in vivo PK profile relative to the first generation ROCK-I inhibitors
(Tables 5 and 6). In contrast, the more lipophilic N-benzyl deriva-
tive 23E, had poor in vivo bioavailability with high clearance and
volume in the Wistar rat. Compound 23A was screened in dupli-
295
8.2
8.2
T1/2 (h)
a
pIC50 values are shown as mean standard deviation from n = 2–5 separate
experiments (except where n = 1).
Table 6
Benchmarking of first generation ROCK inhibitors
Fasudila
HO-Fasudila
Y-27632a
ROCK-I pKi
7.0
7.5
7.6
ROCK-I IMAP pIC50
ROCK-II IMAP pIC50
THP (MCP1) pIC50
hERG (Dofetilide) pKi
C57 mouse
Bioavailability (F%)
Clp (ml/min/kg)
Vss (L/kg)
5.5 0.1
5.87 0.02
5.16 0.09
4.6
6.12 0.02
6.24 0.06
5.16 0.31
5.1
6.06 0.02
6.61 0.09
5.58 0.26
4.1
cate at 10 l
M in Invitrogen’s SelectScreenÒ profiling service. Out
of 245 kinases, 23A inhibited 25 with >80% inhibition. These ki-
nases included PKB, CK1, DYRK3, EPHA8, FES, CRK4, HGK, PDGFRA,
3.6
318
1.8
57
34
2.4
0.75
52
50.9
3.8
T1/2 (h)
0.02
1.67
a
pIC50 values are shown as mean standard deviation from n = 2–5 separate
Table 4
experiments (except where n = 1).
ROCK-I IMAP SAR for R2 amines, 23A–F
NH2
PLK3, PKA, PKD, PKC, PRKG, PRKX, RET, ROCK-I, ROCK-II, SGK2,
SNFILK2, Tie2 and RSE.
N
R
In conclusion, a historical thrombin building block 17A, was
identified by first conducting a focused NMR screen of fragments
from known ROCK and other kinase inhibitors, and then mining
the corporate building block collection for novel cores. Then by
employing a fragment growth and linker modification strategy, a
ROCK-I inhibitor 23A was discovered with favorable properties rel-
ative to known ROCK-I inhibitors. This approach highlights the
utility of focused literature-based fragment screening, followed
by substructure and similarity searches to identify related cores
suitable for fragment growth to afford novel compounds. In partic-
ular, 23A has superior in vivo pharmacokinetics in C57 mouse
compared to the first generation ROCK inhibitors, as well as similar
ROCK-I affinity and cell based efficacy. Although 23E demonstrated
improved affinity and cell based efficacy, it suffered from poor
in vivo bioavailability with a high clearance and volume in the
rat. Compounds 23A and 23E were considered suitable starting
points for further optimization into a lead candidate, with the goal
of improving ROCK-I potency and selectivity, whilst maintaining
good in vivo PK. This hit to lead candidate optimization program
will be described in a subsequent communication.
a
Compounds
R
ROCK-I (IMAP) pIC50
O
23A
5.67 0.16
N
H
O
23B
23C
5.71 0.02
5.44 0.18
N
H
O
O
N
H
23D
6.62 0.09
N
N
Ph
O
23E
23F
23G
6.62 0.01
5.37 0.01
<4
Ph
O
N
Ph
O
N
Supplementary data
SO2Me
a
pIC50 values are shown as mean standard deviation from n = 2–5 separate
experiments (except where n = 1).
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.11.060.

P. Ray et al. / Bioorg. Med. Chem. Lett. 21 (2011) 97–101
101
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