2
K. Sumi et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
including hypertension,19 cerebral infraction,20 cancer,21 cardio-
vascular disease,22 neurodegenerative disease,23 and glaucoma.24
At present, fasudil (1, HA-1077; Fig. 1) is the only successfully
developed ROCK inhibitor, as it has been approved in Japan to treat
cerebral vasospasm after hemorrhage in the subarachnoid space.25
ROCK inhibitors are expected to serve as a new class of antiglauco-
ma agents because of their IOP-lowering effect, which is conferred
through the relaxation of trabecular meshwork cells, leading to a
decrease in the elevated resistance of aqueous humor outflow.26–
a, b
c, d, e
OH
OMs
H2N
H2N
CbzHN
H2N
OH
n
3
4
5a: n = 1
5b: n = 2
Boc
N
f, g
OTBS
n
Cl
SO2
X
7a: X = Br
6
7b: X = Cl
7c: X = F
n = 1 or 2
N
33
Furthermore, the development of several experimental ROCK
inhibitors as antiglaucoma agents is advancing as companies
Boc
N
compete to reach the market first.34–36
OH
n
n
HN
BocN
Previously, Hidaka, who discovered fasudil and pioneered re-
search into protein kinase inhibitors represented by the H-series
compounds,25,37 and co-workers succeeded in developing the
powerful ROCK inhibitor H-1152 (2) and its derivatives.38–40 In
addition, our research group recently demonstrated the significant
IOP-lowering effect of H-1152 in ocular normo- and hypertensive
rabbits.41 In the present study, we synthesized H-1152 analogs,
including some compounds previously reported as selective ROCK
inhibitors,40 and examined their inhibitory effects on ROCK under
the same conditions. Furthermore, their IOP-lowering effects were
evaluated in ocular normotensive monkey models. In this study,
we reveal that isoquinoline-5-sulfonamides are promising
compounds for treating glaucoma and discuss the correlation
between ROCK inhibition and the IOP-reducing activities of these
compounds.
We designed and synthesized various isoquinoline-5-
sulfonamide compounds (H-0103–H-0107, H-1001, H-1005)
based on the structure of H-1152. The structural aspects of these
compounds are as follows. (1) The methyl group at the 4-position
of the isoquinoline ring of H-1152 was replaced with a fluoro,
chloro, or bromo group. (2) A methyl group was introduced at
either methylene near the sulfonylated amino group of the cyclic
diamine. (3) Hexahydro-1H-1,4-diazepine or the larger hexahy-
dro-1H-1,4-diazocane was used as a cyclic diamine. The synthetic
route of H-0103–0107 is shown in Scheme 1. Starting from
N
SO2
X
h
SO2
X
N
N
8
9
n = 1 or 2
n = 1 or 2
n
HN
i
H-0103: = 1, X = Cl
N
n
SO2
X
H-0104: n = 1, X = Br
H-0105: n = 2, X = F
H-0106: n = 2, X = Cl
H-0107
: n = 2, X = Br
N 2HCl
Scheme 1. Synthesis of H-0103–H-0107. Reagents and conditions: (a) CbzCl,
iPr2NEt, CH2Cl2, room temperature; (b) CH3SO2Cl, Et3N, CH2Cl2, room temperature;
(c) 5a–b, THF, reflux, then Boc2O, Et3N, CH2Cl2, room temperature, 66% in three
steps (n = 1), 42% in three steps (n = 2); (d) TBSCl, imidazole, CH2Cl2, room
temperature, 95% (n = 1), 70% (n = 2); (e) H2, Pd–C, MeOH, room temperature, 90%
(n = 1), 87% (n = 2); (f) 7a–c, Et3N, CH2Cl2, room temperature, 60% (n = 1, X = Cl), 71%
(n = 1, X = Br), 66% (n = 2, X = F), 79% (n = 2, X = Cl), 70% (n = 2, X = Br); (g) Bu4NF,
THF, room temperature, 86% (n = 1, X = Cl), 86% (n = 1, X = Br), 92% (n = 2, X = F), 89%
(n = 2, X = Cl), 63% (n = 2, X = Br); (h) PPh3, diisopropyl azodicarboxylate, THF, room
temperature, 96% (n = 1, X = Cl), 90% (n = 1, X = Br), 97% (n = 2, X = F); (i) 4 M HCl/
1,4-dioxane–ethyl acetate, room temperature, 54% (n = 1, X = Cl), 96% (n = 1, X = Br),
72% (n = 2, X = F), 65% in two steps (n = 2, X = Cl), 81% in two steps (n = 2, X = Br).
L
-alaninol (3), N-Cbz protection followed by O-mesylation affor-
ded 4. After the alkylation of 4 with 3-amino-1-propanol (5a)
or 4-amino-1-butanol (5b) in refluxing THF, the resultant second-
ary amino and hydroxy groups were successively protected by
Boc and TBS groups, respectively, and then the Cbz group was re-
moved by catalytic hydrogenolysis to give 6. Condensation of
amine 6 with 4-haloisoquinoline-5-sulfonyl chloride 7a–c42 in
dichloromethane followed by deprotection of TBS group by using
tetrabutylammonium fluoride gave 8. Intramolecular cyclization
of 8 under Mitsunobu’s condition gave 9. Finally, deprotection
of the Boc group and simultaneous salt formation by adding the
1,4-dioxane solution of HCl to 9 dissolved in ethyl acetate fur-
nished the desired H-0103–H-0107. H-1001 and H-1005 were
prepared in a similar way, starting from racemic 3-aminobutyric
acid or 4-aminopentanoic acid and using 2-aminoethanol for
alkylation (Scheme 2).
for H-0106 (IC50 = 21 nM). Furthermore, neither the substituted
position of the methyl group on the cyclic diamine nor the
stereochemistry of this carbon center affected the inhibitory activ-
ity, as illustrated by the results of racemic H-1001 (IC50 = 18 nM)
and H-1005 (IC50 = 29 nM).
To evaluate the IOP-lowering effects of these seven compounds,
a 1% phosphate buffer solution of each compound was topically
administered into the eyes of cynomolgus monkeys. As shown in
Figure 2, H-0106 exhibited the most potent IOP-lowering effect
among the tested compounds (maximum reduction, À4.9 mmHg;
duration, 10 h). H-0104 also strongly decreased IOP (maximum
reduction, À4.5 mmHg; duration, 10 h). The IOP-lowering effect
of H-0103, which displayed potent ROCK inhibitory activity almost
equal to that of H-0104 or H-0106, was obviously weaker than
those of both compounds (maximum reduction, À4 mmHg; dura-
tion, 7–8 h). Although H-0105 and H-0107 inhibited ROCK in a
similar manner, with IC50 values of 39 and 48 nM, respectively,
their IOP-lowering effects greatly differed. In cases of H-0105 and
H-0107, the maximum reductions of IOP were À2.8 and
À4.6 mmHg, respectively, and the durations of reduction were 4
and 9 h, respectively. Furthermore, although H-1001 and H-1005,
which are the isomers of H-0104 and H-0106, respectively,
inhibited ROCK to a similar extent as their isomers, they displayed
considerably weak IOP-lowering activity with short durations
(maximum reduction, À2.3 mmHg; duration, 3–4 h) (Fig. 2B).
The discrepancy of the ROCK inhibitory and IOP-reducing activ-
ities of some compounds can be attributed to differences in their
ocular penetration ability and/or the metabolic stability. Another
Using the seven synthetic isoquinoline-5-sulfonamide com-
pounds, we examined their inhibitory effects on ROCK2. As shown
in Table 1, all of the tested compounds were found to potently
inhibit ROCK2 similar to H-1152 (IC50 = 18 nM). H-0103 (IC50
=
25 nM) or H-0104 (IC50 = 28 nM), in which the 4-methyl group of
the isoquinoline ring of H-1152 was replaced with a halogen, such
as Cl or Br, did not display significantly decreased inhibitory activ-
ity. Ring expansion from hexahydro-1H-1,4-diazepine (n = 1,
7-membered ring) to hexahydro-1H-1,4-diazocane (n = 2, 8-mem-
bered ring) was also an acceptable modification in terms of ROCK
inhibition (IC50 = 21–48 nM). In this case, in particular, substitution
of the chloro group at the 4-position of the isoquinoline ring was
most preferable for ROCK inhibition, as illustrated by the result