Discovery of the ROCK inhibitor netarsudil for the treatment of open-angle glaucoma

Article abstract of DOI:10.1016/j.bmcl.2016.03.104

Inhibition of Rho kinase (ROCK) to improve fluid outflow through the trabecular meshwork and lower intraocular pressure is a strategy for the development of new anti-glaucoma agents. Alpha-aryl-beta-amino isoquinoline analogs were identified as potent ROCK inhibitors. Compounds that provided a longer duration of intraocular pressure reduction in Dutch Belted rabbits also inhibited norepinephrine transporter. Ester 60 improved bioavailability of its parent ROCK inhibitor, 29 (K_i = 0.2 nM) and demonstrated an effective and sustained IOP reduction for 24 h after dosing. From these studies, netarsudil (a.k.a. AR-13324) was discovered and is currently in clinical trials for the treatment of glaucoma and ocular hypertension.

Full text of DOI:10.1016/j.bmcl.2016.03.104

Accepted Manuscript
Discovery of the ROCK inhibitor netarsudil for the treatment of open-angle
glaucoma
Jill M. Sturdivant, Susan M. Royalty, Cheng-Wen Lin, Lori A. Moore, Jeffrey
D. Yingling, Carmen L. Laethem, Bryan Sherman, Geoffrey R. Heintzelman,
Casey C. Kopczynski, Mitchell A. deLong
PII:
S0960-894X(16)30337-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.03.104
BMCL 23747
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 February 2016
28 March 2016
29 March 2016
Please cite this article as: Sturdivant, J.M., Royalty, S.M., Lin, C-W., Moore, L.A., Yingling, J.D., Laethem, C.L.,
Sherman, B., Heintzelman, G.R., Kopczynski, C.C., deLong, M.A., Discovery of the ROCK inhibitor netarsudil for
the treatment of open-angle glaucoma, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/
10.1016/j.bmcl.2016.03.104
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Discovery of ROCK inhibitor netarsudil for
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Jill M. Sturdivant,* Susan M. Royalty, Cheng-Wen Lin, Lori A. Moore, Jeffrey D. Yingling, Carmen L.
Laethem, Bryan Sherman, Geoffrey R. Heintzelman, Casey C. Kopczynski, Mitchell A. deLong

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Discovery of the ROCK inhibitor netarsudil for the treatment of open-angle
glaucoma.
Jill M. Sturdivant,* Susan M. Royalty, Cheng-Wen Lin, Lori A. Moore, Jeffrey D.
Yingling, Carmen L. Laethem, Bryan Sherman, Geoffrey R. Heintzelman, Casey C.
Kopczynski, Mitchell A. deLong
a Aerie Pharmaceuticals, Inc. 4301 Emperor Boulevard, Suite 400 Durham, NC 27703
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Inhibition of Rho kinase (ROCK) to improve fluid outflow through the trabecular meshwork and
lower intraocular pressure is a strategy for the development of new anti-glaucoma agents. Αlpha-
aryl-beta-amino isoquinoline analogs were identified as potent ROCK inhibitors. Compounds
that provided a longer duration of intraocular pressure reduction in Dutch Belted rabbits also
inhibited norepinephrine transporter. Ester 60 improved bioavailability of its parent ROCK
inhibitor, 29 (Ki = 0.2 nM) and demonstrated an effective and sustained IOP reduction for 24 h
after dosing. From these studies, netarsudil (a.k.a.AR-13324) was discovered and is currently in
clinical trials for the treatment of glaucoma and ocular hypertension.
Keywords:
Rho kinase (ROCK)
Glaucoma
Trabecular meshwork
Intraocular pressure (IOP)
AR-13324
2009 Elsevier Ltd. All rights reserved.
One of the most common causes of blindness worldwide is
glaucoma, a group of diseases characterized by progressive optic
nerve damage. Elevated intraocular pressure (IOP) is a major
risk factor for glaucoma and lowering of IOP is currently the
only available treatment.1 Common IOP-lowering therapies
include beta-blockers (e.g. timolol)2 and carbonic anhydrase
inhibitors (e.g. dorzolamide)3 which lower IOP by decreasing the
production of aqueous humor by the ciliary body. Alpha agonists
(e.g. brimonidine)4 reduce IOP by decreasing aqueous humor
production and increasing uveoscleral outflow. The most
commonly prescribed medications, the prostaglandin F2α
analogues (e.g. latanoprost)5 reduce IOP by increasing aqueous
humor outflow through the uveoscleral pathway. There are
currently no medications that target the diseased trabecular
outflow pathway in the eye, which is the underlying cause of
elevated IOP in glaucoma.6
including rabbit and monkey.9 SNJ-1656 (also known as Y-
39983) was the first ROCK inhibitor to demonstrate an IOP-
lowering effect in human subjects.10 Its IOP-lowering effect
peaked at 4 hours after dosing and was no longer effective 24
hours after dosing. Recently, the ROCK inhibitor ripasudil
(previously K-115), a fluorinated analog of fasudil, was approved
in Japan as an adjunctive therapy for the treatment of glaucoma
®
and ocular hypertension (Glanatek 0.4%).11 In a Phase 2
clinical study, ripasudil 0.4% demonstrated peak IOP reduction at
two hours after dosing and required twice-daily dosing to
maintain efficacy throughout the day.12
Over the last several years, Aerie has developed inhibitors of
Rho kinase (ROCK) as a way to directly treat the diseased tissue
of the trabecular meshwork and to lower IOP in patients with
glaucoma.7 The Rho kinases are serine/threonine protein kinases
that exist as 2 isoforms, ROCK1 and ROCK2, 8 which are widely
expressed in many tissues including the trabecular meshwork.6-8
ROCK promotes the assembly of actin stress fibers and focal
adhesions and regulates cell contraction and motility.6
Figure 1. Structure of AR-12286
Aerie’s Rho kinase program previously identified the amino
isoquinoline amide AR-12286 as a potent ROCK inhibitor (Ki =
2.0 nM) that effectively lowered IOP in animal models and
human subjects (Figure 1).13 The goal of the present study was to
discover ROCK inhibitors with a more durable IOP-lowering
effect to allow once-daily dosing in patients. We describe the
discovery of novel amino-isoquinoline amide ROCK inhibitors
Inhibition of ROCK by various inhibitors has demonstrated
increased aqueous humor outflow through the trabecular
meshwork with concomitant reduction of IOP in animal models

that provide a longer duration of IOP-lowering effect in animal
models than previous ROCK inhibitors. The most effective
compounds were also found to have inhibitory activity against
the norepinephrine transporter.
The long duration of IOP lowering for 3 was a unique feature
relative to the ROCK inhibitors AR-12286 and SNJ-1656, as well
as other published ROCK inhibitors.9-13 To screen for potential
activity against other targets, 3 was tested against a panel of 79
human proteins including transmembrane receptors, transporters,
channels, and CYTP450s (Eurofins PanLabs, Taipei, Taiwan).
For comparison, AR-12286 was screened against the same
panel. When assayed at a concentration of 10 μM, 3 produced
≥70% inhibition against 5 proteins: norepinephrine transporter
(NET), serotonin reuptake transporter (SERT), and CYP450
2C19, 2D6, and 3A4. AR-12286 had no activity against these
proteins with the exception of CYP450 2D6 (67% inhibition).
The inhibitory activity of 3 against NET and SERT was
considered of interest since both adrenergic and serotonergic
signaling are involved in IOP regulation,17 and the adrenergic
agonists epinephrine and brimonidine are in clinical use as IOP-
lowering drugs for glaucoma.18Because of the potent and
sustained efficacy of the α-aryl β-amino isoquinoline analog 3, a
continued SAR effort was further explored (Table 2).
Substitution at the 4-position of the α-aryl ring with both electron
withdrawing and donating substituents resulted in better ROCK2,
HTM and PTM activity compared with substitution at the 2 or 3
positions.
Table 1 summarizes the SAR derived from extending the
amino alkyl arm of α-aryl amino isoquinoline analogs. The α-aryl
α-amino isoquinoline analog
2 displayed potent ROCK2
inhibition (Ki = 1.5 nM) but was less effective at disrupting focal
adhesions and actin stress fibers in cell-based assays conducted
with SV-40 transformed human trabecular meshwork cells14
(HTM) and primary porcine trabecular meshwork cells15 (PTM),
respectively. Analogs including α-aryl-β-amino isoquinoline 3
and α-aryl-γ-amino isoquinoline 6, with extended alkyl-amino
arms, displayed a significant improvement in both HTM and
PTM assays. The β-amino analog 3 was twice as potent against
ROCK2 (Ki = 0.8 nM) relative to the α- and γamino analogs 2 and
6, respectively (Ki = 1.5 nM). Furthermore, the S-enantiomer 4
of the β-amino analog 3 was 42 times more potent against
ROCK2 than the R-enantiomer 5.
Table 1.
Extension of the amino alkyl arm
Table 2.
SAR of the α-aryl β-amino isoquinoline
ROCK2a
Ki nM
2.3
HTMb
IC50 nM
278
PTMc
IC50 nM
243
Cpd
1/SNJd
R
n.a.
2
3
4
5
6
NH2
1.5
0.8
0.4
16
5609
123
47
1216
47
2196
137
179
1816
97
-CH2NH2
(S)-CH2NH2
(R)-CH2NH2
-CH2CH2NH2
ROCK2a
Ki nM
2.2
1.0
1.0
1.0
2.0
1.2
1.3
0.9
0.4
0.8
1.0
3.3
0.4
1.3
0.6
5.5
1.0
10.3
2.0
1.3
2.0
3.0
0.2
2.7
HTMb
IC50 nM
266
139
201
102
219
70
192
383
64
208
90
PTMc
IC50 nM
456
260
298
175
247
200
245
455
129
414
200
511
84
459
1015
5147
796
2954
448
670
448
1376
485
201
Cpd
X1,X2
Y
1.5
7
8
9
H
2-fluoro
3-fluoro
4-fluoro
4-fluoro
3,4-difluoro
2-chloro
3-chloro
4-chloro
2,4-dichloro
2-methyl
3-methyl
4-methyl
3-OH
OH
H
H
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
a ROCK2 enzyme inhibition assay.
b,c human (HTM) and porcine (PTM) trabecular meshwork cell
assays; data represent average of at least duplicate runs.
d Reference ROCK inhibitor SNJ-1656
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
n.a. not applicable
Analogs 2, 3 and 6 were then compared to AR-12286
and SNJ-1656 (1) for their ability to lower IOP in Dutch Belted
rabbits.16 The compounds were dissolved in aqueous, buffered
solutions (0.1% - 0.3%, pH-5-6.8) and the rabbits were dosed (1
drop, 30 μL) by topical ocular administration after the baseline
(time 0) measurement of IOP and a second dose (0.1% - 0.3%, 1
drop, 30 μL) was given immediately after the 24 hour IOP
measurement. IOP measurements were taken at 1, 2, 4, 8, and 24
hours after dosing each day. Each of the treated eyes was
compared with its untreated contralateral control eye to
determine the change in IOP (ΔIOP). On day 2, the maximum
ΔIOP produced by AR-12286, 0.3% was -6.8 mmHg, p<0.01 at 2
hours post-dose and ΔIOP at 8 hours post-dose was -2.2 mmHg,
p<0.01. SNJ-1656 (1), 0.3% produced a maximum ΔIOP of -5.5
mmHg, p<0.05, at 2 hours post-dose on day 2 and a ΔIOP at 8
hours post dose of -0.5 mmHg, p=0.13. In comparison, the α-aryl
β-amino isoquinoline amide 3 at 0.2% produced a maximum ΔIOP
of -8.5 mmHg, p< 0.01 at 4 h post-dose on Day 2 and a sustained
ΔIOP at 8 h post-dose of -8.0 mmHg, p<0.01. Less effective
reductions in IOP were observed for 2 (0.3%, max ΔIOP = -2.3
mmHg, p<0.05, 2 h; ΔIOP = 0 mmHg, 8 h) and 6 (0.3%, max ΔIOP
324
39
1721
193
11002
672
4239
214
111
214
248
48
4-OH
3,4-OH
3-CH3,4-OH
3-OCH3, 4-OH
3-OCH3
4-OCH3
4-OCH2C6H5
3-CH2OH
4-CH2OH
3-CF3
4-CF3
α-naphthyl
298
493
4239
1.8
1.0
0.7
152
2954
1015
33
β-naphthyl
H
193
a enzyme inhibition data are an average of at least duplicate runs
b,c
porcine and human trabecular meshwork cell assays; data
represent average of at least duplicate runs
= -4.8 mmHg, p<0.001, 4 h; ΔIOP = -3.1 mmHg p<0.05, 8h).
Moderate conjunctival hyperemia and mild chemosis (swelling)
were the only observed side effects of compound 3.
The most potent ROCK2 inhibitors (Ki <1nM) that were also
effective in the HTM and PTM cells included 4-chlorophenyl 15

(ROCK2 Ki = 0.4 nM), 4-methyl-phenyl 19 (ROCK2 Ki = 0.4
nM), 4-hydroxyl phenyl 21 (ROCK2 Ki = 0.6 nM), 4-
hydroxylmethyl phenyl, 29 (ROCK2 Ki = 0.2 nM), and the β-
naphthyl analog, 33 (ROCK2 Ki = 0.7 nM). Also tested and
compared with the 6-aminoisoquinoline (6-AIQ) parents 3 and 10
were the 1-hydroxy-6-aminoisoquinoline analogs 7 and 11 which
were 2 times less potent in the ROCK2, PTM and HTM assays
(Table 2).
6-aminoisoquinoline; (e) NH2NH2, MeOH, reflux (f) Boc2O,
NEt3, CH2Cl2; (g) TBAF, THF; (h) 4N HCl-dioxane, CH2Cl2, rt.
Both 21 and 29 as well as other analogs in Table 2 were
synthesized following Scheme 1. Alternatively, some were
prepared from commercially available N-Boc- α- substituted
phenyl amino acids which were coupled with 6-AIQ and
deprotected.
hydroxyphenyl)
The methyl esters of the methyl 2-(4-
acetate (34) and methyl 2-(4-
These compounds were tested in Dutch Belted rabbits
for their ability to lower IOP. Both 4-chloro and 4-methyl
analogs 15 and 19 were dosed topically at 0.1% on day 1 and
0.3% on day 2. The β-naphthyl analog 33 was dosed at 0.1%. The
4-chloro analog 15 (max. ΔIOP = -8.1 mmHg, p<0.01, 8 h, day 2),
4-methylated compound 19 (max. ΔIOP = -9.6 mmHg, p<0.01,
8h, day 1) and α-β-naphthyl analog 33 (max. ΔIOP = -7.2 mmHg,
p<0.05, 24h, day 1) produced significant maximum IOP
reductions with moderate hyperemia and mild chemosis.
(hydroxymethyl)phenyl) acetate (35) were TIPS protected using
TIPS-OTf to give 36 (91%) and 37 (78%). Alkylation with N-
(bromomethyl)phthalimide20 gave 38 (68%) and 39 (82%).
Hydrolysis of these methyl esters with lithium hydroxide also
cleaved the imide ring to give the 2-carboxylbenzamides 40 and
41. Using excess EDC, and coupling with 6-aminoisoquinoline
provided isoquinoline amide analogs 42 (85% over 2 steps) and
43 (82% over 2 steps) with the cyclic imide intact. Aminolysis
with hydrazine and Boc-protection gave the N-Boc amino
isoquinoline amide analogs 44 (70% over 2 steps) and 45 (74%
over 2 steps). Deprotection of the silyl protecting groups with
TBAF gave N-Boc-protected phenol (46, 93%) and N-Boc
protected benzyl alcohol (47, 92%). Final N-Boc deprotection
with 4-N HCl- dioxane in CH2Cl2 gave 21 (96%) and 29 (89%).
In an attempt to reduce the hyperemia and improve
corneal penetration, esters were prepared of the most potent
ROCK2 inhibitors, 4-hydroxyl phenyl 21 and 4-hydroxymethyl
phenyl 29. Topical ocular ester prodrugs with improved corneal
penetration have been successfully developed previously for
adrenergic agonists and prostaglandin analogues.5a,17 The
hydroxylated compounds 21 and 29 demonstrated sub-nanomolar
potency against ROCK2 in vitro (Table 2) but did not lower IOP
as well in Dutch Belted rabbits, with maximum ΔIOPs of -1.5
mmHg, p<0.01 and -2.1 mmHg, p<0.05, respectively. This may
be explained by their inability to penetrate the cornea. With three
primary layers, consisting of the outer lipophilic epithelium, the
thick hydrophilic stroma, and the inner endothelium, the cornea
presents a strong resistance barrier which makes it a challenge to
develop topical ophthalmic drugs.19
Table 3.
Esters of the α-4-hydroxy phenyl β-amino isoquinoline amide.
ROCK2a HTMb
Ave
ΔIOPc
mmHg
Cpd
48
49
50
51
52
53
54
R
Ki nM
XC50
nM
Irritat.
Scored
-6.5, 4 h
-4.8, 8 h
CH(CH3)2
-C(CH3)3
C6H5
42
14
330
167
74
0.8
c 0.8
-5.9, 4 h
-7.1, 8 h
1.8
c 1.5
359
6.8
-7.8, 4 h
-3.7, 8 h
0.9
0.4
-6.0, 4 h
-6.0, 8 h
CH2C6H5
1.8
2,4-
dimethyl-
C6H5
3,5-
dimethyl
C6H5
-6.8, 4 h
-6.5, 8 h
821
275
2459
1250
0.5
0.5
-7.3, 4 h,
-5.5, 8 h
-2.2, 4 h
-0.3, 8 h
3-pyridyl
-CH2NH2
4.8
1
832
156
0.2
0
-1.1, 4 h
-0.1, 8 h
55
a
enzyme inhibition data are an average of at least duplicate runs.
b human trabecular meshwork cell assay; data represent average of at
least duplicate runs.
c
Scheme 1. Reagents and conditions: (a) TIPS-OTf, 2,6-
lutidine, CH2Cl2; (b) LiHMDS, N-bromomethylphthalimide,
THF, -78°C- 0°C; (c) LiOH*H2O, THF-H2O; (d) EDC, DMAP,
0.1% compound dosed on day 1 and 0.3% dosed on day 2; IOP
numbers from day 2;p =0.1-0.001.d average irritation score on Day
2, based on Draize method,
c = chemosis.

p =0.1-0.001.
d average irritation score on Day 2, based on Draize method,
c = chemosis.
dosed 0.08% on days 1 and 2.
Esters of the 4-hydroxyl phenyl parent 46 and the 4-
hydroxymethyl phenyl parent 47 were prepared with both
hydrophilic and hydrophobic side chains following Scheme 2.
e
The esters in both classes maintained ROCK2 and HTM
activity, though the activity was less that of the respective parent
compounds (Tables 3 and 4). These compounds were tested for
IOP lowering in Dutch Belted rabbits at 0.1% on day one (1 drop,
30 μL) and the dose was increased to 0.3% on day 2. IOP
measurements were taken at 1, 2, 4, 8, 24 hours after dosing each
day. Irritation was scored according to the Draize scale21 at each
time point. To identify the optimal ester to carry forward, several
criteria had to be met. The compound had to be dissolved and
stable in a topical formulation. It also had to demonstrate a
significant and sustained reduction in IOP with minimal
hyperemia and no chemosis. The isopropyl and tert-butyl esters
48 and 49 in the 4-hydroxy phenyl class met the criteria for IOP
lowering (48, Day 2 ΔIOP = -6.5 mmHg, p<0.01, 4 h, 49, ΔIOP =
-7.1 mmHg, p<0.001, 8 h), but moderate hyperemia and mild
chemosis were observed at the higher dose level (0.3%) on day 2
(Table 3).
Scheme 2. Reagents and conditions: (a) EDC, DMAP,
RCO2H, pyridine; (b) 4N HCl-dioxane, CH2Cl2, rt.
Table 4.
Esters of the α-4-hydroxymethyl phenyl β-amino isoquinoline
amide.
Improved irritation scores and effective IOP lowering were
observed for the benzoate 50 (max ΔIOP = -7.8 mmHg, p<0.01, 4
h), phenylacetate 51 (max ΔIOP = -6.0 mmHg, p<0.001, 4 h) and
the 2, 4-dimethylbenzoate 52 (max ΔIOP = -6.8 mmHg, p<0.001,
4 h), on day 2, producing mild to moderate hyperemia and
sustained reductions in IOP at 8 hours post-dose (Table 3). The
more hydrophilic esters (54, 55) did not lower IOP as well and
appeared to have a similar efficacy profile as the 4-hydroxyl
phenyl parent 21.
ROCK
2a
Ki nM
HTMb
XC50
nM
Ave
Irritat
Scored
ΔIOPc
Cpd
R
mmHg
-7.3, 4 h
-7.1, 8 h
1.0
c 0.5
56
CH(CH3)2
3.2
21.8
1.7
0.8
4.2
0.9
1.6
0.7
ND
83
424
72
The esters in the 4-hydroxymethyl phenyl class typically
exhibited more pronounced reductions in IOP than the 4-hydroxy
phenyl esters (Table 4). The hydroxymethyl isopropyl 56 (max
ΔIOP = -7.3 mmHg, p<0.01, 4 h) and tert-butyl ester 57 (max
ΔIOP = -8.1 mmHg, p<0.01, 8 h) effectively reduced IOP at 0.3%
on day 2, but also produced moderate hyperemia and mild
chemosis. Benzoate ester 58 showed an impressive max IOP
reduction of -9.7 mmHg, p<0.01, with no chemosis, but produced
a relatively high hyperemia score. The 2,4-dimethyl benzoate
analog 60 displayed a significant IOP reduction on day 2 of -6.3
mmHg, p<0.05, at 4 hours post-dose and a sustained IOP
reduction of -5.5 mmHg, p<0.05, at 8 hours post-dose, and
produced only mild hyperemia with no chemosis. Interestingly,
the 3,5-dimethyl benzoate 61 also showed potent and sustained
IOP lowering, but the hyperemia score was more than double the
2,4-dimethyl analog 60 at 0.3% on day 2. The more hydrophilic
esters (63, 64) did not produce the large reductions in IOP that
were observed for the hydrophobicesters.
2.0
c 0.5
-3.5, 4 h
-8.1, 8 h
57
58
59
60
61
62
63
C (CH3)3
C6H5
-6.8, 4 h
-9.7, 8 h
1.0
0.7
0.4
-6.0, 4 h
-6.0, 8 h
CH2C6H5
118
250
65
2,4-
dimethyl
C6H5
-6.3, 4 h
-5.5, 8 h
3,5-
dimethyl
C6H5
-7.7, 4 h
-9.7, 8 h
1.1
0.4
0.5
-8.9, 4 h
-5.4, 8 h
44
(CH2)3CH3
3-pyridyle
-4.3, 4 h
-1.8, 8 h
65
-2.1 4 h
-1.3, 8 h
0
0
64
CH2NMee
38
a
enzyme inhibition data are an average of at least duplicate runs.
b human trabecular meshwork cell assay; data represent average of at
least duplicate runs.
c
0.1% compound dosed on day 1 and 0.3% dosed on day 2;

IOPs were measured in Dutch Belted rabbits. The rabbits were dosed (1
drop, 30 μL) after the baseline (time 0) measurements. Mean baseline IOP
(on day 1, prior to dosing) in the study eye ranges from 24.2-25.5 mmHg.
IOP measurements were taken at 1, 2, 4, 8 and 24 hours after time 0.
Each of the treated eyes was compared with its contralateral control eye to
determine the change in (Δ)IOP.
1
0
-1
-2
-3
-4
-5
In summary, compounds representing a new class of potent
ROCK inhibitors have been developed that can be formulated
and dosed as a topical eye drop, penetrate the cornea, and
significantly lower IOP in a sustained manner. 2,4-Dimethyl
benzoate 60 displayed improved bioavailability with minimum
irritation in Dutch Belted rabbits. A sustained IOP reduction was
observed for 60, which appears to be a unique trait in this class,
as compared to previously described classes of ROCK inhibitors.
This sustained IOP reduction may be related to factors including
corneal penetration, rate of hydrolysis by the corneal esterases,
and the inhibition of norepinephrine transporter. Continued
studies on 60 identified the S-enantiomer, netarsudil (a.k.a. AR-
0
5
10
Time (hr)
Figure 2. ΔIOP with 60 (▲ 0.1%) and parent alcohol 29 (•
0.1%). IOPs were measured in Dutch Belted rabbits. The rabbits were
dosed (1 drop, 30 μL) after the baseline (time 0) measurements. Mean
baseline IOP (on day 1, prior to dosing) in the study eye ranged from
21.1-24.4 mmHg. IOP measuremts were taken at 1, 2, 4, 8 and 24 hours
after time 0. Each of the treated eyes was compared with its contralateral
control eye to determine the change in IOP (ΔIOP).
15
20
25
13324), as the active antipode (ROCK2 Ki=
2
nM).22
Subsequently, netarsudil 0.02% was shown in two Phase II
clinical trials to produce significant ΔIOPs that ranged from -5.7
to -6.3 mmHg after four weeks of dosing in patients with
glaucoma and ocular hypertension.23 Netarsudil was well
tolerated, with trace to mild hyperemia being the most frequently
reported adverse event.23 Currently, netarsudil is in Phase III
clinical trials for the treatment of open-angle glaucoma and
ocular hypertension.
The 2,4-dimethyl benzoate 60 (0.1%) was compared directly
with both the 4-hydroxymethyl phenyl parent 29 (0.1%) and
ROCK inhibitor SNJ-1656 1 (0.1%). As expected, 60 displayed
improvement in IOP reduction compared to the 4-hydroxymethyl
phenyl parent 29 (Figure 1). At the 2, 4, 8 and 24 h time points,
60 was 2, 1.6, 1.5, and 0.7 mmHg better than its parent alcohol
29. Given that the ROCK2 inhibitory activity of 60 was 20-fold
lower than the parent 29, the superior IOP-lowering activity of 60
is likely due to improved corneal penetration and bioavailability.
Acknowledgments
We thank John Arnold (Samford University McWhorter
School of Pharmacy, Birmingham, Alabama) for characterization
of AR-12286 activity in rabbits.
60 (0.1%) also displayed much larger reductions in IOP at 8
hours (-3.2 mmHg) and 24 hours (-1.5 mmHg) post-dose than
SNJ-1656 (1) 0.1% (8 h, -0.1 mmHg; 24 h, 0 mmHg, Figure 2).
Given the similarity of this sustained IOP reduction to that
observed for 3, 60 was tested for inhibitory activity against NET
and SERT (Eurofins PanLabs, Taipei, Taiwan). At 10 μM, the
2,4-dimethyl benzoate 60 demonstrated 96% inhibition of NET
and 94% inhibition of SERT. In comparison, the 4-
hydroxymethyl phenyl parent 29 demonstrated 48% and 39%
inhibition of NET and SERT, respectively. 60 (500 nM) was also
tested against a panel of 442 human protein kinases (DiscoverX,
Fremont, CA). 11 Kinases were inhibited >90%, but only
ROCK1 and ROCK2 (each 93% inhibition) and PKC (delta,
91%, and eta, 93% inhibition) have been identified as potential
targets for lowering IOP. Similarly 29 (500 nM) inhibited 12
kinases >90 including ROCK1 and ROCK2 (100% inhibition
each) and PKC (delta, 98%, epsilon, 93%, eta, 98% inhibition).
References and notes
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Figure 3. ΔIOP with 60 (▲ 0.1%) and SNJ-1656 (•0.1%).
15
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