Discovery of 3,4-dihydrobenzo[f][1,4]oxazepin-5(2H)-one derivatives as a new class of ROCK inhibitors for the treatment of glaucoma

Article abstract of DOI:10.1016/j.bmcl.2021.128138

The Rho-associated protein kinases (ROCKs) are associated with the pathology of glaucoma and discovery of ROCK inhibitors has attracted much attention in recent years. Herein, we report a series of 3,4-dihydrobenzo[f][1,4]oxazepin-5(2H)-one derivatives as a new class of ROCK inhibitors. Structure-activity relationship studies led to the discovery of compound 12b, which showed potent activities against ROCK I and ROCK Ⅱ with IC₅₀ values of 93 nM and 3 nM, respectively. 12b also displayed considerable selectivity for ROCKs. The mean IOP-lowering effect of 12b in an ocular normotensive model was 34.3%, and no obvious hyperemia was observed. Overall, this study provides a good starting point for ROCK-targeting drug discovery against glaucoma.

Full text of DOI:10.1016/j.bmcl.2021.128138

Bioorg. Med. Chem. Lett. 45 (2021) 128138
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 3,4-dihydrobenzo[f][1,4]oxazepin-5(2H)-one derivatives as a
new class of ROCK inhibitors for the treatment of glaucoma
a
,
1
b
,
1
b
b
b,
c
b
Yumeng Sun , Yueshan Li , Zhuang Miao , Ruicheng Yang , Yun Zhang , Ming Wu ,
b
a,*
Guifeng Lin , Linli Li
a
Key Laboratory of Drug Targeting and Drug Delivery System of Ministry of Education, West China School of Pharmacy, Sichuan University, Sichuan 610041, China
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China
Macular Disease Research Laboratory, Department of Ophthalmology, West China Hospital, Sichuan University, Sichuan 610041, China
b
c
A R T I C L E I N F O
A B S T R A C T
Keywords:
The Rho-associated protein kinases (ROCKs) are associated with the pathology of glaucoma and discovery of
ROCK inhibitors
Structure–activity relationship
Glaucoma
ROCK inhibitors has attracted much attention in recent years. Herein, we report a series of 3,4-dihydrobenzo[f]
[
1,4]oxazepin-5(2H)-one derivatives as a new class of ROCK inhibitors. Structure-activity relationship studies led
to the discovery of compound 12b, which showed potent activities against ROCK I and ROCK II with IC50 values
of 93 nM and 3 nM, respectively. 12b also displayed considerable selectivity for ROCKs. The mean IOP-lowering
effect of 12b in an ocular normotensive model was 34.3%, and no obvious hyperemia was observed. Overall, this
study provides a good starting point for ROCK-targeting drug discovery against glaucoma.
IOP-lowering effect
7
,15,17
Among them, ripasudil18 and
Glaucoma is one of the primary causes of blindness and the global
number of patients with glaucoma is estimated to be more than 111.8
million in 2040.1 Glaucoma patients often bear a combination of
symptoms including elevated intraocular pressure (IOP), irreversible
optic neuropathy and different degrees of corneal endothelial cell
39983, SR-3677, INS117548.
netarsudil,1
9,20
have been approved to use clinically as anti-glaucoma
,2
drugs. Despite a good therapeutic effect, these two drugs show an
adverse effect of conjunctival hyperemia, which could be due to the poor
kinase selectivity.1
,21–24
Therefore, discovering more potent and selec-
3
–5
dysfunction until loss of visual field. Lowing IOP is the most efficient
strategy for the treatment of glaucoma.6 Notably, each millimeter of
mercury (mmHg) reduction in IOP, the risk of the glaucomatous field
impairment progression can be decreased by 10–19% for glaucoma
patients.7
tive ROCK inhibitors is necessary at present.
To identify new ROCK inhibitors, we screened our in-house chemical
library containing about 1000 compounds synthesized by our group,
which led to the retrieving of a ROCK inhibitor with a new scaffold 3,4-
dihydroisoquinolin-1(2H)-one (Hit 1, Fig. 2). Hit 1 is a dual ROCK I/II
Rho-associated protein kinases (ROCKs), which are widely expressed
in the trabecular meshwork (TM), have been demonstrated to play an
inhibitor with IC50 values of 2.062 ± 0.103
μ
M and 0.632 ± 0.059
μ
M,
respectively. A further structural optimization was carried out in this
investigation.
8
important role in the pathology of glaucoma. After activation by Rho-
GTPase, ROCK phosphorylate the intracellular downstream sub-
strates,9 such as myosin phosphatase targeting subunit 1 (MYPT1),
The structural optimization and SAR analyses were focused on three
regions: 3,4-dihydroisoquinolin-1(2H)-one (region I), 1H-indazole (re-
gion II) and anisole (region III).
myosin light chain (MLC), and LIM kinases (LIMK).1
0–13
ROCK inhibitors
have been demonstrated to be able to efficiently decrease IOP by acting
on TM and altering the cytoskeleton.14 Meanwhile, it also plays a role in
In the first step, we fixed region II and III, and varied region I. A total
of four compounds (6, 10a-c) were synthesized. As shown in Scheme 1,
3-methoxybenzylamine reacted with 4-bromo-2-hydroxybenzoyl chlo-
ride (2), which was prepared by refluxing reaction of 4-bromo-2-hydrox-
optic nerve protection through increasing retinal vascular perfusion and
promoting optic nerve regeneration.1
5,16
Currently, a number of ROCK
inhibitors have been reported to anti-glaucoma. Fig. 1 shows several
representative examples, including ripasudil, netarsudil, Y-27632, Y-
ybenzoic acid (1) in SOCl
2
, to generate intermediate 3. Compound 4 was
then obtained by cyclization from intermediate 3. Suzuki-Miyaura
*
Corresponding author.
E-mail address: lilinli@scu.edu.cn (L. Li).
These authors contributed equally to this work.
1
https://doi.org/10.1016/j.bmcl.2021.128138
Received 5 February 2021; Received in revised form 16 May 2021; Accepted 20 May 2021
Available online 24 May 2021
0
960-894X/© 2021 Elsevier Ltd. All rights reserved.

Y. Sun et al.
Bioorganic & Medicinal Chemistry Letters 45 (2021) 128138
Fig. 1. Structures of representative ROCK inhibitors for anti-glaucoma.
Fig. 2. The chemical structure of Hit 1 together with regions (I, II and III) that are the focuses for the structure–activity relationship analysis.
coupling of 4 with commercially available reagent 5 produced final
product 6. Schmidt rearrangements were carried out on commercially
available reagents 7a-c to give intermediates 8a-c. Suzuki-Miyaura
coupling of intermediates 9a-c obtained from nucleophilic substitution
reaction of 8a-c provided final compounds 10a-c.
optimal fragment 3,4-dihydrobenzo[f][1,4]oxazepin-5(2H)-one and re-
gion III as its original subgroup. A total of eight compounds were pre-
pared. Scheme 2 shows the synthesis routes of compounds 12a-g and 16.
Intermediate 9b was coupled with different boronic acids or pinacol
boronate esters through Suzuki-Miyaura coupling to give desired com-
pounds 12a-g. Intermediate 14 was obtained from 6-bromochroman-4-
one (13) through Schmidt rearrangement. 14 was then treated with 3-
methoxybenzyl bromide and sodium hydride in dry THF to give 15.
Finally, the desired product 16 was obtained through Suzuki-Miyaura
coupling of 15 with pyridin-4-ylboronic acid.
The kinase inhibitory activities of these compounds are given in
Table 1. Replacement of the 3,4-dihydroisoquinolin-1(2H)-one moiety
with benzo[e][1,3]oxazin moiety (6) improved the potency slightly.
Expansion of the ring of Hit 1 (10a) by inserting a methylene led to a
decrease in potency for both ROCK I and II inhibition, while the same
ring expansion of 6 (10b) resulted in slight and 3-fold increase in the
ROCK I and ROCK II inhibitions, respectively. Introduction of two
methyl groups to the 2-position of 10b caused a decrease in ROCK
inhibitory activity.
Table 2 summarizes the chemical structures and ROCK inhibitory
activities of these compounds. Replacement of 1H-indazole in 10b with
1,3-benzodioxol (12a) led to the loss of activity. While 4-pyridine
replacement (12b) made a significant increase to the ROCK inhibitory
activity. However, compound 16 with 7-position pyridine substitution
In the second step, we optimized region II with region I fixed as the
2

Y. Sun et al.
Bioorganic & Medicinal Chemistry Letters 45 (2021) 128138
◦
◦
Scheme 1. Synthesis routes of compounds 6 and 10a-c. Reagents and conditions: (i) SOCl
2
◦
, DMF, 85 C, 5 h; (ii) TEA, THF, 25 C, 8 h, 80%; (iii) Dibromomethane,
◦
, Methanesulfonic acid, DCM, 25 ◦C, 70–85%. (vi)
Cs
2
CO
3
, MeCN, 80 C, 24 h, 40%; (iv) Pd(PPh
3
)
4
, K
2
CO
3
, 1,4-dioxane/H
2
O (10/1), N
2
, 100 C,10 h, 50%. (v) NaN
, 100 ◦C, 12 h, 55–58%.
3
NaH, dry THF, 0 to 50 ◦C, 4 h, 75–90%; (vii) Pd(PPh
)
4
, K CO , 1,4-dioxane/H
3
2
3
2
O (10/1), N
2
◦
3 4 2 3
) , Cs CO , 100 C, 12 h; (ii) DCM,
Scheme 2. Synthesis routes of compounds 12a-g and 16. Reagents and conditions: (i) 1,4-dioxane/water (V/V = 5/1), Pd(PPh
◦
◦
◦
◦
NaN
3
, CH
3
SO
3
H, 0 C to 25 C, 12 h; (iii) dry THF, 1-(bromomethyl)-3-methoxybenzene, NaH, 0 C to 25 C, 12 h; (iv) 1,4-dioxane/water (V/V = 5/1), pyridin-4-
◦
ylboronic acid, PdCl
2
(dppf)⋅CH
2
Cl
2
adduct, Cs
2
CO
3
, 100 C, 12 h.
decreased ROCK inhibitory activity. An amino-substitution at 2-position
12c) resulted in a decrease in potency compared with 12b, and further
In the third step, region III was optimized with region I and II fixed as
their optimal groups. We synthesized six compounds (19a-f). The syn-
thesis of these compounds is shown in Scheme 3. Suzuki-Miyaura
coupling of pyridin-4-ylboronic acid (11b) with various 4-benzyl-8-
bromo-3,4-dihydrobenzo[f][1,4]oxazepin-5(2H)-one derivatives (18a-
f), which were obtain from substitution reactions of 8b with various
commercially available benzyl bromide (17a-f), afforded final
(
position adjustment of nitrogen and amino led to a loss of activity (12d).
Pyrimidine replacement in region II also resulted in a loss of potency
(
12e). Finally, we replaced the 4-pyridine with azaindole, and the po-
tencies of the resulting compounds (12f and 12 g) did not exceed that of
2b.
1
3

Y. Sun et al.
Bioorganic & Medicinal Chemistry Letters 45 (2021) 128138
Scheme 3. Synthesis routes of compounds 19a-f. Reagents and conditions: (i) DMF, NaH, 0 C to 25 ◦C, 12 h; (ii) 1,4-dioxane/water (V/V = 5/1), pyridin-4-
◦
◦
ylboronic acid, Pd(PPh
3
)
4
or PdCl
2
(dppf)⋅CH
2
Cl
2
adduct, Cs
2
CO
3
or K
2
CO
3
, 100 C, 12 h.
compounds 19a-f.
Table 1
Table 3 shows chemical structures and ROCK inhibitory activities of
compounds 19a-f. Compared with the 3-methoxy substitution of phenyl
in 12b, other substituents at 3-position (19a-e) did not improve the
potency against ROCK I and II. Adding another methoxy moiety at 5-po-
sition also decreased the activity.
Structures and in vitro biological activities of compounds Hit 1, 6 and 10a-c.
The above structural optimization and SAR analyses led to the dis-
covery of a number of compounds that showed potent activity against
both ROCK I and ROCK II. 12b is the most active compound. Further
studies to this compound including kinase selectivity, binding mode
prediction, and anti-glaucoma activity, will be carried out in the follows.
To examine the kinase selectivity, we tested the kinase inhibition
profile of 12b at a fixed concentration of 10 µM. Here 100 representative
kinases were selected. The inhibition rates of 12b against the selected
kinases are shown in Table S1. Fig. 3 displays the human kinome
dendrogram based on the measured inhibition rates. 12b exhibited a
high inhibition rate against only one kinase LIMK2 except ROCK I and II;
LIMK2 is the direct downstream kinase of the ROCKs. 11 The calculated
selectivity scores, S (5), S (10) and S (20), are 0.029,0.049 and 0.069,
respectively. All of these data indicate that 12b is a good selective ROCK
inhibitor.
a
Compound
Hit 1
Region I
IC50 (µM)
ROCK I
ROCK II
2.062 ± 0.103
0.632 ± 0.059
6
1.462 ± 0.099
0.434 ± 0.012
2.115 ± 0.087
0.182 ± 0.002
0.749 ± 0.023
1
1
1
0a
0b
0c
> 10
Molecular docking was then used to predict the binding modes of
1
2b with ROCK I and II (Fig. 4). The structures of ROCK I and II were
taken from the RCSB Protein Data Bank (PDB entries: 6E9W for ROCK I,
and 6ED6 for ROCK II). The two ROCK isoforms share 92% similarity in
their amino acid sequence,23 and their active pockets (the ATP-binding
site) are very similar. As predicted by molecular docking, 12b occupies
the active pockets of ROCK I and II with the same pose and orientation.
In both ROCK I and II, the nitrogen of pyridine of 12b forms a hydrogen
bonding interaction with the NH backbone of Met (Met156 in ROCK I,
and Met172 in ROCK II) in the hinge region. In addition, the amide
carbonyl of 12b forms another hydrogen bonding interaction with
Lys105 in ROCK I, and Lys121 in ROCK II. The only difference is that the
oxygen atom of 3-methoxy group of 12b forms the third hydrogen
bonding interaction with Lys216 in ROCK II, but the same interaction
does not be formed in ROCK I due to the unfavorable distance between
the oxygen atom of 3-methoxy group of 12b and Lys200 (corresponding
to Lys216 in ROCK II). This could be used to interpret that 12b has a
higher inhibitory activity against ROCK II than ROCK I.
1.080 ± 0.150
6.012 ± 0.260
a
IC50 values were determined from Kinase Profiler of Eurofins. The data are
averages of two independent experiments reported as the mean ± SD.
4

Y. Sun et al.
Bioorganic & Medicinal Chemistry Letters 45 (2021) 128138
Table 2
Table 3
Chemical structures and in vitro biological activities of compounds 10b, 12a-g
Chemical structures and in vitro biological activities of compounds 12b, 19a-f.
and 16.
a
Compound
n
8
Region II
IC50 (µM)
ROCK I
a
Compound
Region III
IC50 (µM)
ROCK I
ROCK II
ROCK II
1
0b
1.080 ± 0.150
0.182 ± 0.002
1
1
2b
9a
0.093 ± 0.004
0.003 ± 0.001
1.964 ± 0.621
9.242 ± 1.209
0.133 ± 0.014
0.495 ± 0.316
1
2a
8
> 10
> 10
1
9b
1
1
2b
2c
8
8
0.093 ± 0.004
1.145 ± 0.107
0.003 ± 0.001
0.015 ± 0.003
1
1
1
1
9c
9d
9e
9f
3.099 ± 0.202
0.208 ± 0.004
0.668 ± 0.125
4.493 ± 0.039
0.050 ± 0.009
0.009 ± 0.001
0.029 ± 0.027
0.217 ± 0.062
1
2d
8
> 10
> 10
1
1
2e
2f
8
8
> 10
> 10
0.103 ± 0.026
0.015 ± 0.007
1
1
2g
6
8
7
> 10
> 10
5.535 ± 1.102
0.761 ± 0.096
a
IC50 values were determined from Kinase Profiler of Eurofins. The data are
averages of two independent experiments reported as the mean ± SD.
Normotensive NZW rabbits were adopted to evaluate the IOP-
lowering effect of 12b in vivo. As shown in Fig. 6, comparing with
vehicle, 12b rapidly and significantly lowered IOP. The prominent IOP
reduction was observed at 0.5 h after dose administration and was
sustained for 2–3 h. The highest average IOP reduction was achieved at
a
IC50 values were determined from Kinase Profiler of Eurofins. The data are
averages of two independent experiments reported as the mean ± SD.
1
h after instillation, and was 34.3% from baseline (4 mmHg). No
Western blot was then performed to determine the effect of 12b on
the ROCK signaling in intact cells. Here we detected the expression
levels of MYPT1 and its phosphorylated form (p-MYPT1) in SH-SY5Y cell
line ; MYPT1 is a direct downstream signal protein of ROCKs.12 As
shown in Fig 5, 12b treatment dose-dependently inhibited the phos-
phorylation of MYPT1, but did not impact the expression of MYPT1.
These results indicated that 12b could efficiently suppress the ROCK
signaling in intact cells.
obvious hyperemia was noticed during the course of the experiment,
suggesting that 12b could potentially separate the desired IOP-lowering
effect of ROCK inhibitors from commonly observed side effect.
In summary, 12b is a potent and selective ROCK inhibitor with a new
chemical scaffold. It could efficiently lower IOP in normotensive NZW
rabbits and did not show obvious hyperemia. Overall, 12b is a good lead
compound of anti-glaucoma and deserves further investigations.
13
5

Y. Sun et al.
Bioorganic & Medicinal Chemistry Letters 45 (2021) 128138
Fig. 3. The kinase selectivity of 12b shown on the human kinome dendrogram.
Fig. 4. Predicted binding modes of 12b with ROCK I (A) and ROCK II (B). Dashed red lines indicate the hydrogen bonding interactions. The crystal structures of
ROCK I (PDB entry: 6E9W) and ROCK II (PDB entry: 6ED6) were taken from the RCSB Protein Data Bank. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
Fig. 5. 12b reduced the level of p-MYPT1. SH-SY5Y cells were treated with various concentrations of 12b (0.1, 0.3 and 1 M) for 12 h. Quantification of immu-
μ
**
***
noblots is presented in the right panel (* P < 0.05, P < 0.01,
P < 0.001 vs. control, t test).
6

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Bioorganic & Medicinal Chemistry Letters 45 (2021) 128138
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