Optimisation of 6-substituted isoquinolin-1-amine based ROCK-I inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.12.104

Rho kinase is an important target implicated in a variety of cardiovascular diseases. Herein, we report the optimisation of the fragment derived ATP-competitive ROCK inhibitors 1 and 2 into lead compound 14A. The initial goal of improving ROCK-I potency relative to 1, whilst maintaining a good PK profile, was achieved through removal of the aminoisoquinoline basic centre. Lead 14A was equipotent against both ROCK-I and ROCK-II, showed good in vivo efficacy in the spontaneous hypertensive rat model, and was further optimised to demonstrate the scope for improving selectivity over PKA versus hydroxy Fasudil 3.

Full text of DOI:10.1016/j.bmcl.2010.12.104

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1084–1088
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
BMCL Digest
Optimisation of 6-substituted isoquinolin-1-amine based ROCK-I inhibitors
⇑
Peter Ray , Jane Wright, Julia Adam, Sylviane Boucharens, Darcey Black, Angus R. Brown, Ola Epemolu,
Dan Fletcher, Margaret Huggett, Phil Jones, Steven Laats, Amanda Lyons, Jos de Man, Richard Morphy,
Brad Sherborne, Lorcan Sherry, Nicole van Straten, Paul Westwood, Mark York
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:
Rho kinase is an important target implicated in a variety of cardiovascular diseases. Herein, we report the
optimisation of the fragment derived ATP-competitive ROCK inhibitors 1 and 2 into lead compound 14A.
The initial goal of improving ROCK-I potency relative to 1, whilst maintaining a good PK profile, was
achieved through removal of the aminoisoquinoline basic centre. Lead 14A was equipotent against both
ROCK-I and ROCK-II, showed good in vivo efficacy in the spontaneous hypertensive rat model, and was
further optimised to demonstrate the scope for improving selectivity over PKA versus hydroxy Fasudil 3.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 12 November 2010
Revised 21 December 2010
Accepted 22 December 2010
Available online 28 December 2010
Keywords:
Rho kinase
ROCK-I
ROCK-II
Serine-threonine kinase
Inhibitor
Optimisation
Rho kinase (ROCK) belongs to the AGC family of serine / threonine
kinases and interacts with the GTP-binding protein RhoA to afford
active protein. Two iso-forms of Rho kinase have been described
(ROCK-I and ROCK-II) and they share high homology in the ATP bind-
ing site. RhoA and its downstream kinase (ROCK) have been impli-
cated in a number of important roles such as regulation of smooth
muscle contraction, cytoskeleton rearrangement, cell migration
and proliferation. Furthermore, a large amount of pre-clinical re-
search effort with various inhibitors has demonstrated that both
RhoA and ROCK are important targets for several cardiovascular
diseases.1
Acute clinical studies with Fasudil, which is presumed to act via
its active metabolite hydroxyl Fasudil 3, have demonstrated effi-
cacy and safety in treating cerebral vasospasm, ischemic stroke
and stable angina. Fasudil has also shown beneficial effects in a
number of other cardiovascular diseases, including angina pectoris,
hypertension, coronary vasospasm, restenosis after percutaneous
coronary intervention and arteriosclerosis.2 Recently, there has
been a surge in the research effort towards identifying potent
and selective ROCK inhibitors.3
However, it has superior in vivo pharmacokinetics in the C57
mouse. Although derivative 2 demonstrated the scope to improve
affinity, potency and cell based efficacy, it suffered for poor
in vivo bioavailability and also had a high clearance and volume
in the rat (Table 1).4
Herein, we report on the optimisation of compounds 1 and 2.
The optimisation goal was to increase the potency of 1, whilst
maintaining the good pharmacokinetic properties. Compound 1
has poor Caco-2 permeability, however, it has excellent bioavail-
ability and it was rationalized that this small dibasic compound
was absorbed predominantly via the para-cellular route. The
Caco-2 permeability for 2 was worse, despite the increased
c log P/log D. Furthermore, 2 suffered from poor bioavailability
and it was rationalized that the dibasic nature of this larger com-
pound was problematic for absorption,5 as it could not be absorbed
via the para-cellular route.
An initial effort was made to reduce the pKa of the piperidine
amine 1 through alkylations with suitable electron withdrawing
derivatives. However, the resultant compounds had both poor
ROCK-1 potency and microsomal stability. A further attempt was
also made to lower the lipophilicity of the benzyl derivative in 2,
by changing the phenyl to various heterocycles. However, Caco-2
permeability remained an issue and only moderate to poor liver
microsomal stabilities were obtained (data not shown).
Since the aminoisoquinoline core is reported to have a pKa of
ꢀ7.5,6 subsequent optimisation focused on analogues of isoquino-
line with differing substituents at the 1-position that would signif-
icantly alter the pKa. Isoquinoline itself was a very interesting
Previously, we reported the ROCK-I inhibitor 1, which was ob-
tained from fragment based approach. Compound 1 has similar
ROCK-I affinity, potency and cell based efficacy (which provides
an in vitro understanding of MCP-1-induced migration of mono-
cytes in unstable atherosclerotic lesions) to hydroxy Fasudil 3.
⇑
Corresponding author. Tel.: +44 (0)1698 736127.
E-mail address: p.ray@btinternet.com (P. Ray).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.104

P. Ray et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1084–1088
1085
Table 1
Table 2
ROCK-I hit compounds 1, 2 and hydroxy Fasudil 3
ROCK-I IMAP SAR for isoquinolines 8A–C
NH2
NH2
O
N
N
N
HN
N
NH
N
Ph
O
O
O
R
S
O
O
N
NH
a
Compounds
R
ROCK-I (IMAP) pIC50
1a
2a
nd
3a
8A
8B
8C
rac-H
S-Bn
R-Bn
5.75 0.02
6.77 0.04
7.14 0.02
ROCK-I pKi
7.43 0.08
5.67 0.16
5.95 0.04
5.30 0.28
6.3
7.46 0.13
6.12 0.02
6.24 0.06
5.16 0.31
nd
ROCK-I IMAP pIC50
ROCK-II IMAP pIC50
THP migration pIC50
6.62 0.01
7.04 0.09
6.11 0.21
2.2
a
pIC50 values are shown as mean standard deviation from n = 2–5 separate
experiments (except where n = 1).
Caco-2 (5 Â 10À6 cm/s)
A–B/B–A
3.6
1.5
C57 Mouse
>75
9.5
11
5.8
Wistar Rat
7
295
8.2
8.2
C57 Mouse
57
34
2.4
0.75
O
O
Bioavailability (F%)
Clp (mL/min/kg)
Vss (L/kg)
a,b
c,d
g
e
HN
HN
O
HO
OMe
OH
OMe
OMe
H
O
H
10
9
11
12
14
T1/2 (h)
a
O
pIC50 values are shown as mean standard deviation from n = 2–5 separate
experiments (except where n = 1).
O
f
n( )
HN
n( )
O
NBoc
( )m
NH
( )m
HN
O
m = 0,1,2
n = 0,1
13
analogue and this was obtained by demethylation of 6 to afford the
phenol, followed by a Mitsunobu coupling with N-boc-3-hydroxy-
piperidine to give 7. TFA deprotection afforded isoquinoline 8A,
which could be converted to the N-benzyl derivative by reductive
alkylation with benzaldehyde, and then resolved to afford the sin-
gle enantiomers 8B and 8C (Scheme 1).
Scheme 2. Reagents and conditions: (a) Ph3PCHCO2Me, toluene; (b) NaOH; (c)
DPPA, NEt3, toluene; (d) diphenylmethane, >250 °C; (e) BBr3, DCM or HCl; (f) PS-
BEMP, MeCN, 160 °C; (g) TFA, DCM.
Table 3
Isoquinoline 8A showed ROCK-1 activity comparable to 1 and
enantiomers 8B and 8C showed improved potency (Table 2). Com-
pound 8B was profiled in vivo and showed improved bioavailabil-
ity with respect to 2. However, the pharmacokinetic profile of 8B
was still not optimal, as it was associated with a high clearance
and high volume of distribution in the rat. Furthermore, 8B showed
potent 2D6 and 3A4 cytochrome P450 inhibition (Table 4).
The above data supported the rationale for further modification
of the pKa in order to improve the pharmacokinetics. Hence, our
attention shifted onto the isoquinolin-1-one building block 9,
which was synthesized as shown in Scheme 2. Homologation of
3-methoxybenzaldehyde 10 afforded acid 11, which was reacted
with diphenylphosphoryl azide and then heated to temperatures
in excess of 250 °C to afford isoquinolin-1-one 12. Deprotection
of 12 with BBr3 afforded phenol 9. With 9 in hand, SN2 PS-BEMP
mediated displacement of the readily prepared mesylates of
N-Boc-protected aminoalcohols afforded ethers 13A–G. N-Boc
deprotection of 13A–G afforded compounds 14A–G.
Gratifyingly, S-isomer 14A had an ROCK-I pIC50 of 7.21 0.22
(Table 3). The R-isomer 14B and the 4-substituted piperidine 14C
were both less potent. However, the azepine 14D also showed good
ROCK-I potency with its enantiomer 14E being less potent. The
pyrrolidines 14F and 14G showed moderate potencies.
Compound 14A was selected for further profiling and was found
to have potent ROCK-I affinity as well as similar potency in the
ROCK-II IMAP assay as ROCK-I (Table 4). It showed improved po-
tency in the THP migration assay relative to hydroxyl Fasudil 3
and no major inhibition of any of the CYP isoforms studied. Due
to the slow rate of metabolism, the enzymes responsible for any
metabolism of 14A could not be identified. Rat and human plasma
ROCK-I IMAP SAR for R2 amines, 14A–G
O
HN
R
a
Compounds
R
ROCK-I (IMAP) pIC50
O
14A
7.21 0.22
N
H
O
14B
14C
14D
6.17 0.04
6.73 0.10
7.24 0.07
N
H
O
O
NH
N
H
O
14E
6.48 0.11
N
H
O
O
14F
14G
6.28 0.10
6.06 0.16
N
H
N
H
a
pIC50 values are shown as mean standard deviation from n = 2–5 separate
experiments (except where n = 1).
protein binding for 14A were 20% and 6%, respectively. This low le-
vel of binding across species indicates that most of the drug in the
plasma will be available for effect. The pharmacokinetic studies for
14A in rat show suitable oral bioavailability. Furthermore, both
distribution volume and clearance are moderate, resulting in a
suitable half life. The estimated fraction of the dose absorbed
was 62% which is consistent with its high solubility, low molecular
a,b
N
N
c
N
NBoc
N R
OMe
O
O
7
R=H
8A
8B
8C
6
d
R=CH2Ph (Enantiomer 1)
R=CH2Ph (Enantiomer 2)
Scheme 1. Reagents and conditions: (a) BBr3, DCM, À78 °C to 20 °C; (b) N-boc-3-
hydroxypiperidine, DEAD, PPh3, THF, 20 °C; (c) TFA, DCM.

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P. Ray et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1084–1088
Table 4
potent ROCK-I inhibitor with improved permeability and a suitable
pharmacokinetic profile.
Profiling of compounds 8B and 14A
8Ba
14Aa
Compound 14A was evaluated in the NovaScreen general side
effect panel of 64 assays at 10 lM and only showed limited binding
ROCK-I pKi
nd
8.6
to the serotonin and dopamine transporters (56% and 43%, respec-
tively). Compound 14A showed limited hERG binding affinity in
the [3H]-dofetilide binding assay, with a pKi <4, and produced
<20% inhibition of hERG tail current in a high throughput electro-
ROCK-I IMAP pIC50
ROCK-II IMAP pIC50
THP migration pIC50
6.77 0.04
7.10 0.08
6.48 0.01
nd
21/nd
11.6/17.6
3A4, 2D6 <1
7.21 0.22
7.41 0.07
6.18 0.16
<12, 24, 17
<6/2
H/R/M liver micr: Clint, lL/mL/mg
R/H Heps:Clint, mL/mL/1 Â 106 cells
Caco-2 (Â10À6 cm/s) A–B/B–A
2C19, 3A4, 1A2, 2D6, 2C9
Cyp inhibition
physiology assay at 10 lM. Compound 14A showed no activity in
14.3/11.2
All
the Vitotox assay in the presence or absence of S9 liver extract
and did not activate the human pregnane X receptor (PXR) or the
aryl hydrocarbon receptor (AhR).
In vivo, 14A reduced blood pressure (BP) in the spontaneous
hypertensive rat model by 34% at 10 mg/kg and 15% at 3 mg/kg
po (Fig. 1). The effect was greater than that observed for the refer-
ence standard Y-27632.7
l
M
M
& rest >5
5.7
l
>5
<4
rat
31
42
8.2
lM
hERG (Dofetilide) pKi
Wistar rat
Bioavailability (F%)
Clp (mL/min/kg)
Vss (L/kg)
T1/2 (h)
rat
37
184
8.9
0.85
1.75
a
The full kinase selectivity profiles for hydroxy Fasudil 3 (at
pIC50 values are shown as mean standard deviation from n = 2–5 separate
experiments (except where n = 1).
10 lM) and 14A (at 1 lM) were obtained from Millipore. The pro-
file for 14A compares well with that of hydroxy Fasudil 3. Com-
pound 14A appeared to be less selective than hydroxy Fasudil 3
for the AGC-related kinases [in particular PKA, PKC (d,
g, l) and
110
100
90
PrkX]. Overall, 14A was highly selective over the panel of kinases
tested, with selectivity of >25-fold over >92% of the non-AGC fam-
ily kinases tested. It is known that Fasudil, and its more potent, ac-
tive metabolite hydroxy Fasudil 3, are efficacious in man with no
apparent side effects up to 80 mg/kg t.i.d (po) for 8 weeks.8 Thus,
a selectivity profile which is equivalent or better than that of hy-
droxy Fasudil 3 is believed to be a desirable goal. Further full dose
response profiling confirmed that 14A was less selective for PKC &
PKA isoforms compared to hydroxy Fasudil 3 (Table 5).
80
Vehicle
1mg/kg
3mg/kg
10mg/kg
70
Using a homology model for ROCK-I, developed initially using
PKA as a template and then modified according to the published
ROCK-I crystal structures, docking studies with known ligands
and lead 14A provided suggestions for isoquinolone series modifi-
cations.9 Compound 14A appears tightly bound in the ATP binding
pocket making interactions through the adenosine and ribose
binding regions with limited exploration of space not occupied
by ATP. The model was then used to identify residues available
for interactions with 14A. The sequence differences between
ROCK-I, PKA and the PKC isoforms were defined (Table 6) and
mapped onto the ROCK-I homology model (Fig. 2). The computa-
tional modeling data suggested a focus on interactions with resi-
dues I82, M128 and A215. The appropriate areas of space relative
to the proposed binding mode for 14A are highlighted in magenta
in the Figure 2. Additional areas highlighted are those in: Green,
which might afford PKA and some PKC subtype selectivity (F368,
L205 and M153). Orange, those which would only afford selectivity
over PKA (D160, D202).
60
0
2
4
6
8
10
Time
12
14
16
18
20
22
Figure 1. The effect of 14A on mean arterial pressure (MAP) in the SHR model.
Table 5
Full dose response data for 14A and hydroxy Fasudil 3
Kinase
Assay format
pIC50
14A
3
PKCd (h)
RFBA
RFBA
RFBA
RFBA
RFBA
RFBA
IMAPa
6.5
6.4
6.3
7.1
6.5
6.6
7.21 0.22
4.6
4.7
5.0
5.2
5.0
5.5
6.12 0.02
PKC
PKC
g
l
(h)
(h)
PrkX (h)
PKA
ROCK-I
ROCK-I
a
pIC50 values are shown as mean standard deviation from n = 2–5 separate
From analysis of the selectivity data from the literature, it was
clear that selectivity over PKA was a key issue for optimisation. It
was hypothesized that focusing on improving selectivity over
PKA would afford compounds with an overall improved selectivity
profile. Initial full dose response PKA profiling of 14A, 14C and 14D
experiments (except where n = 1).
weight and permeability in the Caco-2 assay. Hence, the strategy to
modulate one of the basic centers was successful in affording a
Table 6
ATP site residue for closely related AGC kinase
Res. No.
Rock
PKCdelta
PKCeta
PKCmu
PKCepsilon
PKCiota
PKCbeta
PKCgamma
PKCzeta
PKA (h)
82
128
153
160
202
205
215
368
I
L
L
L
L
L
L
L
L
I
F
I
D
D
L
T
F
L
L
L
L
I
F
I
D
D
L
T
F
L
L
M
M
D
D
L
M
D
D
L
A
F
M
D
D
L
A
F
M
D
E
L
C
Y
M
D
D
L
A
F
M
D
D
M
A
F
M
D
D
M
T
M
E
E
L
T
F
A
F
F

P. Ray et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1084–1088
1087
O
O
HN
( )n
( )m
O
NH2
( )n
( )m
R2
R2
O
a,b
R2
O
c
HN
HN
HN
OH
NHBoc
R1
R1
R1
( )n
( )m
MsO
Scheme 3. Reagents and conditions: (a) PS-BEMP, MeCN, 160 °C; (b) TFA, DCM;
(c) acetaldehyde, MeCN, AcOH, NaBH(OAc)3.
ROCK-1 and PKA potency. However, 15B maintained a similar
ROCK-I potency and afforded a slightly improved selectivity over
PKA (Table 7).
The next optimisation cycle involved making changes to the
amine scaffold within the orange residue differences for PKA with-
in Figure 2. Amines, 16A–E, were readily prepared according to
Scheme 3. Compound 16A lost potency at both ROCK-1 and PKA,
however, improved selectivity over PKA was obtained. The cyclo-
hexyl derivative 16B retained ROCK-1 potency and the selectivity
over PKA was improved. Furthermore, reductive amination of
16B afforded 16C, which had suitable ROCK-I potency and good
selectivity over PKA. Further homologated amines such as 16D
and 16E were also prepared and both showed promising ROCK-I
potencies and improved selectivity over PKA versus the lead 14A
(Table 7).
In conclusion, the fragment derived aminoisoquinolines 1 and 2,
were optimised into isoquinolone lead 14A through a strategy of
removing the aminoisoquinoline basic centre. Compound 14A is
ꢀ10-fold more potent than hydroxy Fasudil 3 and is highly selec-
tive in a large panel of receptor and enzyme targets. In vitro, 14A
demonstrated concentration-dependent inhibition of MCP-1 stim-
ulated chemotaxis. In vivo, 14A lowered blood pressure in the
spontaneous hypertensive rat model of arterial hypertension and
the antihypertensive effect was superior to that of the first gener-
ation reference compound Y-27632. Full dose response kinase pro-
filing revealed that selectivity over PKA was an issue and further
optimisation provided compounds which demonstrate the scope
for improving selectivity over PKA.
Figure 2. Sequence differences between ROCK-I, PKA and the PKC isoforms mapped
onto the ROCK-I homology model. Magenta areas might afford PKA and PKC
subtype selectivity. Green areas might afford PKA and some PKC subtype selectivity.
Orange area might afford selectivity over PKA.
suggested that changing the ring size had little effect on selectivity
over PKA (Table 7). In order to probe the green and magenta resi-
due differences for PKA within Figure 2, derivatives 15A and 15B
were prepared using equivalent synthetic methodology to that
described in Scheme 2. Compound 15A showed a decrease in both
Table 7
ROCK-I IMAP and PKA RFBA pIC50 data, 14A,C,D, 15A,B and 16A–E
O
2
R
HN
R3
R1
Compd R1
R2
R3
PKA
RFBA
pIC50
ROCK-I
IMAP pIC50
ROCK-I
minus PKA
a
O
14A
14C
14D
H
H
H
H
H
H
6.5
5.8
6.4
7.21 0.22
6.73 0.10
7.24 0.07
0.7
0.9
0.8
N
H
References and notes
O
O
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NH
N
H
O
O
15A
15B
16A
Me
H
H
5.6
6.3
5.2
6.23 0.05
7.32 0.04
6.64 0.06
0.6
1
N
H
Me
H
N
H
O
O
H
1.4
NH2
16B
16C
H
H
H
H
6.2
5.1
7.50 0.14
6.92 0.09
1.3
1.8
NH2
O
O
NHEt
NH2
16D
16E
H
H
H
H
5.5
5.2
7.09 0.06
6.78 0.03
1.6
1.6
O
NH2
a
pIC50 values are shown as mean standard deviation from n = 2–5 separate
experiments (except where n = 1).

1088
P. Ray et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1084–1088
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