Novel 1H-imidazol-2-amine derivatives as potent and orally active vascular adhesion protein-1 (VAP-1) inhibitors for diabetic macular edema treatment

Article abstract of DOI:10.1016/j.bmc.2013.04.011

Novel thiazole derivatives were synthesized and evaluated as vascular adhesion protein-1 (VAP-1) inhibitors. Although we previously identified a compound (2) with potent VAP-1 inhibitory activity in rats, the human activity was relatively weak. Here, to improve the human VAP-1 inhibitory activity of compound 2, we first evaluated the structure-activity relationships of guanidine bioisosteres as simple small molecules and identified a 1H-benzimidazol-2-amine (5) with potent activity compared to phenylguanidine (1). Based on the structure of compound 5, we synthesized a highly potent VAP-1 inhibitor (37b; human IC₅₀ = 0.019 μM, rat IC₅₀ = 0.0051 μM). Orally administered compound 37b also markedly inhibited ocular permeability in streptozotocin-induced diabetic rats after oral administration, suggesting it is a promising compound for the treatment of diabetic macular edema.

Full text of DOI:10.1016/j.bmc.2013.04.011

Bioorganic & Medicinal Chemistry 21 (2013) 3873–3881
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel 1H-imidazol-2-amine derivatives as potent and orally active
vascular adhesion protein-1 (VAP-1) inhibitors for diabetic macular
edema treatment q
a
a
a
a
Takayuki Inoue a, , Masataka Morita , Takashi Tojo , Akira Nagashima , Ayako Moritomo ,
⇑
Hiroshi Miyake b
a Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
b Astellas Research Technology Co., Ltd, 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel thiazole derivatives were synthesized and evaluated as vascular adhesion protein-1 (VAP-1) inhib-
itors. Although we previously identified a compound (2) with potent VAP-1 inhibitory activity in rats, the
human activity was relatively weak. Here, to improve the human VAP-1 inhibitory activity of compound
2, we first evaluated the structure–activity relationships of guanidine bioisosteres as simple small
molecules and identified a 1H-benzimidazol-2-amine (5) with potent activity compared to phenylguani-
dine (1). Based on the structure of compound 5, we synthesized a highly potent VAP-1 inhibitor (37b;
Received 14 March 2013
Revised 2 April 2013
Accepted 4 April 2013
Available online 19 April 2013
Keywords:
Vascular adhesion protein-1
Inhibitor
Macular edema
Guanidine bioisostere
1H-Imidazol-2-amine derivative
human IC50 = 0.019 lM, rat IC50 = 0.0051 lM). Orally administered compound 37b also markedly
inhibited ocular permeability in streptozotocin-induced diabetic rats after oral administration,
suggesting it is a promising compound for the treatment of diabetic macular edema.
Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
1. Introduction
tissue has been observed in many inflammation-associated dis-
eases.7 VAP-1 was recently reported to be expressed on retinal vas-
Vascular adhesion protein-1 (VAP-1) is an endothelial surface
glycoprotein with homology to semicarbazide-sensitive amine
oxidases. Two types of VAP-1 have been identified: a membrane-
binding type present in vascular endothelium and a soluble type
found in serum. Membrane-bound VAP-1 adheres to white blood
cells and lymphocytes and is involved in inflammation,1 whereas
the soluble form displays amine oxidase activity that is involved
in amine detoxification.
VAP-1 is highly expressed in vascular endothelial cells within
inflamed areas and is responsible for the metabolism of primary
amines, such as methylamine and aminoacetone,2,3 and leukocyte
trafficking. VAP-1 catalyzes the oxidative deamination of primary
amines to produce the corresponding aldehydes, in addition to
the cytotoxic reaction products ammonia and hydrogen peroxide,4
which may be associated with a number of vasculopathies.5,6 Con-
sistent with these properties, an increase in plasma and
membrane-associated VAP-1 derived from adipocytes and vascular
cular endothelial cells, where it functions in leukocyte recruitment
on retinal vessels.8 The accumulation of toxic by-products and in-
creased leukocyte adhesion activity are indicative of the break-
down of the blood–retinal barrier and are thought to lead to the
severe macular edema that is often seen in diabetic retinopathy,
which is the leading cause of blindness in humans. Even minimal
disruption of the macular region due to edema can cause severe vi-
sual loss, and if left untreated, can lead to irreversible changes in
the macular region and the exacerbation of retinopathy.
We recently described a novel VAP-1 inhibitor, compound 2,
which was synthesized from a high-throughput screening (HTS)
hit compound.9 However, compound 2 showed approximately
16-fold lower VAP-1 inhibitory activity in humans than in rats (hu-
man IC50, 0.23 lM; rat IC50, 0.014 lM). Therefore, to improve the
human VAP-1 inhibitory activity of compound 2, here, we at-
tempted to further modify the phenylguanidine moiety, which
was previously identified as being critical for human VAP-1 inhib-
itory activity based on the results of a docking study between com-
pound 2 and human VAP-1 enzyme.9 We initially attempted to
identify bioisosteres of phenylguanidine (1) with human VAP-1
inhibitory activity using the design strategy shown in Figure 1.
Additionally, we conducted optimization of the phenylguanidine
bioisostere, 1H-benzimidazol-2-amine (5), and introduced the
q
This is an open-access article distributed under the terms of the Creative
Commons Attribution-NonCommercial-No Derivative Works License, which per-
mits non-commercial use, distribution, and reproduction in any medium, provided
the original author and source are credited.
⇑
Corresponding author. Tel.: +81 29 829 6227; fax: +81 29 854 1519.
E-mail address: takayuki.inoue@astellas.com (T. Inoue).
0968-0896/$ - see front matter Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.04.011

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T. Inoue et al. / Bioorg. Med. Chem. 21 (2013) 3873–3881
S
NH
NH2
HN
N
NH
NH2
N
H
O
N
H
2
1
IC50 h:0.23µM, r:0.014µM
IC50 h:>100µM, r:78µM
Evaluation of bioisosteres
S
H
N
H
N
N
HN
N
NH2
NH2
NH2
N
H
N
O
N
Introduction
of thiazole moiety
Optimization
37b
19
5
IC50 h:0.019µM, r:0.0051µM
IC50 h:4.1µM, r:1.0µM
IC50 h:32µM, r:0.42µM
Figure 1. Summary for synthesizing bioisosteres of phenylguanidine.
NH
O
O
H
N
H
N
H2N
N
O
H
16
a
Br
O
b
N
H
NH2
O
N
17
N
18
15
H
N
N
O
c
b
NH2
N
H
Br
a
N
N
22
H
20
21
23
Scheme 1. Reagents and conditions: (a) compound 16, DMF; (b) 6 M HCl, MeOH, reflux; (c) (1) Br2, HBr, AcOH, (2) acetone.
–
Br
MeO C
2
+
PPh
3
S
25
O
S
H
N
b
a
HN
N
CO Me
2
N
CO Me
2
27
O
26
O
–
c
Br
+
HO
NH
O
PPh
3
H2N
N
O
35
O
S
28
S
O
S
H
d
H
N
b
OH
HN
CHO
a
HN
OH
N
N
N
n
O
O
29
24
O
34a : n=0
34b : n=1
34c : n=2
–
S
Cl
+
S
PPh
3
H N
c
N
31
O
S
H
N
b
OHC
a
H
N
CO Me
2
CO Me
2
CO Me
N
2
N
O
33
30
S
O
32
O
O
H
H
N
N
NH2
NH
S
e
H
N
N
N
HN
HCl
N
N
n
n
37a : n=0
37b : n=1
37c : n=2
36a : n=0
36b : n=1
36c : n=2
O
O
Scheme 2. Reagents and conditions: (a) compounds 25 or 28 or 31, tBuOK, DMF; (b) H2 (3 atm), 10% Pd–C, AcOH, MeOH, THF; (c) 1 M NaOH, EtOH, reflux; (d) (1) (COCl)2,
CH2Cl2, DMF, (2) TMSCH2N2, CH2Cl2, (3) 4 M HCl/AcOEt, CH2Cl2/DMF, (4) compound 35, DMF; (e) 4 M HCl/AcOEt, MeOH.

T. Inoue et al. / Bioorg. Med. Chem. 21 (2013) 3873–3881
3875
thiazole pharmacophore of compound 2 to the obtained 4-benzyl-
Table 1
1H-imidazol-2-amine (19). Consequently, we succeeded in
synthesizing compound 37b, which displayed strong inhibitory
activities against both human and rat VAP-1.
VAP-1 inhibitory activities of phenylguanidine bioisosteres
Compd
Structure
VAP-1 Humana
VAP-1 Ratb
In this paper, we discuss the synthesis and structure–activity
relationships (SARs) of this series of novel 1H-imidazol-2-amine
derivatives as candidate human VAP-1 inhibitors.
NH
NH2
1c
>100
78
N
H
N
3d,c
>100
>100
>100
>100
2. Chemistry
N
H
N
H
NH2
N
The synthesis of 1H-imidazol-2-amine derivatives 18 and 23 is
shown in Scheme 1. Cyclization of 2-bromo-1-phenylethanone
(15) with N-carbamimidoylacetamide (16) gave N-(4-phenyl-1H-
imidazol-2-yl)acetamide (17). Deprotection of the acetyl group in
compound 17 with 6 M hydrogen chloride in refluxing methanol
gave 4-phenyl-1H-imidazol-2-amine (18). Bromination of 4-phe-
nylbutan-2-one (20) with bromine gave 1-bromo-4-phenylbutan-
2-one (21). 4-(2-Phenylethyl)-1H-imidazol-2-amine (23) was
obtained from 21 by the same procedure as that used for
compound 18.
Scheme 2 shows the syntheses of compounds 37a–c. The Wittig
reaction of aldehyde derivatives 24 and 30 with the corresponding
phosphonium salts 25, 28, and 31 and subsequent hydrogenation
in the presence of palladium on carbon gave intermediates 27,
33, and 34b. Hydrolysis of ester derivatives 27 and 33 with 1 M so-
dium hydroxide in refluxing ethanol gave the corresponding
carboxylic acid derivatives 34a,c.
4e,c
N
N
NH2
5c
6c
4.1
1.0
N
H
N
>100
>100
N
NH2
a
b
c
IC50 data for human VAP-1 in
l
M (n = 2).
IC50 data for rat VAP-1 in
Commercially available.
Hydrochloride salt.
lM (n = 2).
d
e
Hydrobromide salt.
Table 2
VAP-1 inhibitory activities of 1H-benzimidazol-2-amine derivatives
Treatment of 34a–c with oxalyl chloride followed by ketoniza-
tion with (trimethylsilyl)diazomethane, substitution of the
trimethylsilyl group with chloride, and cyclization with tert-butyl
carbamimidoylcarbamate (35) provided 36a–c, respectively. The
deprotection of the tert-butoxycarbonyl (Boc) group of 36a–c
afforded the desired compounds 37a–c.
Compd
Structure
VAP-1 Humana
VAP-1 Ratb
1.0
N
NH2
5c
4.1
N
H
N
7c
>100
74
>100
63
NH2
NH2
S
3. Results and discussion
8d,c
N
H
The compounds were evaluated for in vitro VAP-1 inhibitory
activity against human and rat VAP-1. VAP-1 activities were deter-
mined using a radiochemical enzyme assay with 14C-benzylamine
as an artificial substrate. The VAP-1 enzymes used in the assays
were prepared from Chinese hamster ovary (CHO) cells stably
expressing human or rat VAP-1.
N
9c
>100
>100
>100
>100
N
H
N
NH2
10e,c
N
H
We previously demonstrated that the guanidine moiety of com-
pound 2 forms tight hydrogen bonds network with Asp386, but can
also bind covalently to the trihydroxyphenylalanine quinone (TPQ)
471, indicating that the guanidine moiety is important for the VAP-
1 inhibitor activity.9 Here, we therefore focused on the synthesis of
compounds with close structural similarity to phenylguanidine,
such as imidazoline derivatives (3, 4), 1H-benzimidazol-2-amine
(5), and quinazolin-2-amine (6). The VAP-1 inhibitory activities
of compounds 3–6 are shown in Table 1. Imidazoline derivatives
3 and 4, and quinazolin-2-amine (6) showed no inhibitory activity,
whereas 1H-benzimidazol-2-amine (5) exhibited strong VAP-1
inhibitory activity.
We next attempted to optimize the inhibitory activity of com-
pound 5 (Table 2). Thiazole derivative 7 had no detectable VAP-1
inhibitory activity, suggesting that the NH of the imidazole moiety
is required for the activity. 1H-Indol-2-amine (8) also exhibited a
marked reduction in inhibitory activity compared to 5, implying
that the guanidine moiety is essential for VAP-1 inhibition. Deam-
ination of 5 was also deleterious to the inhibitory activity (9), a re-
sult that is consistent with the fact that a primary amine-forming
Schiff base is essential for the VAP-1 inhibitory activity of 5.9,13,14
Aminomethyl analogue 10 also lost the inhibitory activity, suggest-
ing that the introduction of a methylene group increased the size of
N
NH2
11f,c
>100
90
N
H
N
N
12c
13c
>100
>100
80
NH2
N
NH2
9.3
N
H
N
14c
>100
1.0
NH2
N
H
a
b
c
d
e
f
IC50 data for human VAP-1 in
l
M (n = 2).
IC50 data for rat VAP-1 in
Commercially available.
Hydrochloride salt.
Dihydrochloride salts.
Hemisulfate salt.
lM (n = 2).
the guanidine moiety, thereby preventing the interaction of 10
with VAP-1. As the deletion of the benzene ring (11) also resulted
in a significant reduction in VAP-1 inhibition, the benzene ring ap-

3876
T. Inoue et al. / Bioorg. Med. Chem. 21 (2013) 3873–3881
Figure 2. Computational docking results for compound 5 with human VAP-1. (A) Best docking solution (lowest binding energy) calculated by GOLD ver.5.1. for compound 5
(ball and stick representation; compound is colored blue for nitrogen and green for carbon) surrounded by the human VAP-1 active site. Key receptor residues are indicated.
(B) Results of the docking analysis represented as an Active LP image (green, hydrophobic regions of the surface; blue, mildly polar regions; and purple, hydrogen-bonding
regions).
the thiazole moiety of the other main pharmacophore found in
compound 2. Unfortunately, all of the generated compounds (12–
14) had no inhibitory activity against human VAP-1.
Table 3
VAP-1 inhibitory activities of 1H-imidazol-2-amine derivatives
To understand the SAR of 1H-benzimidazole-2-amine, we
examined compound 5 using a human VAP-1 docking model9–14
with the GOLD program (version 5.1; Fig. 2). As an initial docking
structure, the amine group at the 2-position of 5 formed a Schiff
base intermediate with the TPQ moiety of VAP-1. In addition to
the covalent interaction of the primary amine, the modeling also
suggested that the NH of the imidazole ring of 5 formed a hydrogen
Compd
Structure
VAP-1
VAP-1
Ratb
Humana
N
NH2
5c
4.1
1.0
44
N
H
H
N
NH2
bond with Asp386, the imidazole ring formed a
tion with TPQ471 (–OH), the benzene ring formed a
tion with Tyr384, and the CH of the benzene ring formed a
proton– interaction with Phe389. We considered that even com-
p–proton interac-
18
>100
32
N
p–p
interac-
H
N
p
19c
23
0.42
4.8
NH2
pact molecules such as 5 could exert potent inhibitory activity due
to these five types of intermolecular interactions. In addition, as
compound 5 just fit in the active center pocket of VAP-1, the intro-
duction of additional substituents would likely block entry of the
compound into the active site. These results were in approximate
agreement with the SAR findings. Consequently, we considered
that it was impossible to add any substituents into the benzimid-
azole moiety. Therefore, we searched for other bioisosteres of
1H-benzimidazol-2-amine with human VAP-1 inhibitory activity.
Based on the high potency of 1H-benzimidazol-2-amine (5), we
examined whether spatial room could be generated by introducing
flexibility between the phenyl and imidazole rings. First, optimiza-
tion of the distance between the phenyl and imidazole rings of 5
was conducted by examining compounds with different linkers
(Table 3). Among the examined compounds (18, 19, and 23), com-
pound 19, which contained a methylene linker between the phenyl
and imidazole rings, showed the strongest inhibitory activity
against human VAP-1. Compound 18, which did not have a linker,
and 23, which contained an ethylene linker, had no detectable hu-
man VAP-1 inhibitory activity.
N
H
N
NH2
>100
N
S
H
N
37ad
37bd
13
0.15
NH
N
N
O
NH2
H
N
S
N
O
0.019
0.0051
H
N
NH2
N
S
H
N
N
37cd
2.2
0.018
NH
O
N
NH2
a
b
c
The results of the docking analysis of 19 with human VAP-1 are
shown in Figure 3. The analysis indicated that the NH of the imid-
azole ring formed a hydrogen bond with Asn470, the imidazole
IC50 data for human VAP-1 in
l
M (n = 2).
IC50 data for rat VAP-1 in
Commercially available.
Hydrochloride salt.
lM (n = 2).
d
ring formed a
p–p interaction with Tyr384, and the benzene ring
formed a –proton interaction with Leu469. Compound 19 showed
p
approximately 8-fold less potent human VAP-1 inhibitory activity
compared to 5, suggesting that the benzene ring of compound 19
is slightly exposed from the VAP-1 binding pocket due to the pres-
ence of a methylene moiety between the phenyl and imidazole
rings, resulting in reduced affinity for VAP-1. However, we pre-
dicted that introducing a substituent at the 4-position of the ben-
zene ring would be possible because this position faces toward the
pears necessary for activity. Among the examined derivatives,
1H-benzimidazol-2-amine (5) showed the most potent inhibitory
activity against human and rat VAP-1. Subsequently,
we introduced a methyl group at the 1, 4, or 5 position of 1H-benz-
imidazole-2-amine to determine the appropriate position to attach

T. Inoue et al. / Bioorg. Med. Chem. 21 (2013) 3873–3881
3877
Figure 3. Computational docking results for compounds 5 and 19 with human VAP-1. (A) Best docking solution (lowest binding energy) calculated by GOLD ver.5.1. for
compounds 5 (stick representation; compound is colored blue for nitrogen and green for carbon) and 19 (ball and stick representation; compound is colored blue for nitrogen
and pink for carbon) surrounded by the human VAP-1 active site. Key receptor residues are indicated. (B) Results of the docking analysis represented as an Active LP image
(green, hydrophobic surface regions; blue, mildly polar regions; and purple, hydrogen-bonding regions).
solvent side. Compound 19 showed greater than 3- and 186-fold
improvements in human and rat VAP-1 inhibitory activities,
respectively, compared to 1. Therefore, we decided to introduce a
thiazole moiety into compound 19, generating compound 37b.
Compound 37b showed satisfactory potency against both human
and rat VAP-1 (Table 3), and had 12-fold higher inhibitory activity
against human VAP-1 than 2. Although compounds 18 and 23 had
no detectable human VAP-1 inhibitory activity, we synthesized
their thiazole derivatives (37a and 37c) for confirmation. Consis-
tent with the SAR results, both compounds exhibited lower po-
tency compared to 37b.
following two reasons: first, the anchor-part substructure of 37b
(19: IC50 = 32 M) is more potent than that of 2 (1: IC50 = >100 M)
and second, the thiazole moiety of 37b is positioned optimally in
the active site of VAP-1, resulting three times as many interactions
with the enzyme as 2 (Fig. 4).
The inhibitory properties of 37b were further compared against
other amine oxidases (AOs), including human monoamine oxidase
(MAO)-A and -B, using a fluorometric enzymatic assay (Table 4).
Compound 37b displayed greater than 580-fold selectivity for
VAP-1 inhibitory activity over MAO-A/B. We also examined the
inhibitory effect of 37b on plasma VAP-1 activity in streptozotocin
(STZ)-induced diabetic rats (Fig. 5A).9 Significant inhibitory activity
was observed at a dose of 10 mg/kg after oral (po) administration.
l
l
The results of the docking analysis of 37b with human VAP-1
are shown in Figure 4. As expected, the amidothiazole moiety
interacted with the active site of human VAP-1 enzyme. Specifi-
cally, the thiazole ring of 37b formed
p–proton interactions with
Table 4
Thr212 and Leu447, the sulfur atom in the thiazole ring made an
Selectivity of compounds 2 and 37b for VAP-1 and MAO-A,B
S–O interaction9,15 with the Thr210 backbone carbonyl oxygen,
Compd
VAP-1
Human
VAP-1
Rat
MAOa-A
Human
MAOa-B
Human
the amide moiety formed a proton–
p interaction with Tyr176,
and the acetyl moiety formed a CH–O interaction with Asp180. To-
gether, these analysis results suggest that strong interactions be-
tween the amidothiazole moiety and human VAP-1 enzyme led
to the increased inhibitory activity of 37b compared to 2. In partic-
ular, we speculate that 37b is more potent than 2 due to the
IC50
(l
M)b
IC50
(l
M)b
IC50
(l
M)b
IC50 (l
M)b
2
37b
0.23
0.019
0.014
0.0051
>100
>100
60
11
a
MAO, monoamine oxidase.
n = 2.
b
Figure 4. Computational docking results for compounds 2 and 37b with human VAP-1. Best docking solution (lowest binding energy) calculated by GOLD ver.5.1. for
compounds 2 (stick representation; compound is colored blue for nitrogen, red for oxygen, yellow for sulfur, and cyan for carbon) and 37b (ball and stick representation;
compound is colored blue for nitrogen, red for oxygen, yellow for sulfur, and orange for carbon) surrounded by the human VAP-1 active site. Key receptor residues are
indicated.

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T. Inoue et al. / Bioorg. Med. Chem. 21 (2013) 3873–3881
Ocular permeability
Plasma VAP-1
A
B
20
15
10
5
sham (n=10)
sham (n=10)
3000
2000
1000
0
STZ control (n=12)
37b 10mg/kg,po (n=12)
STZ control (n=12)
37b 10mg/kg,po (n=12)
***
***
ns
###
###
0
Figure 5. Pharmacodynamic profile and pharmacology of compound 37b in rats. (A) Inhibitory effect on plasma VAP-1 activity in streptozotocin (STZ)-induced diabetic rats
(n = 10–12, 10 mg/kg, po) at 2 weeks after treatment with compound 37b. The plasma VAP-1 activity was measured using a radiolabeled enzyme assay with 14C-benzylamine
as the substrate. (B) Effect of compound 37b (n = 10–12, 10 mg/kg, po) on ocular permeability in STZ-induced diabetic rats. ⁄⁄⁄P <0.001 and ###P <0.001, Student’s t-test versus
sham and STZ control groups, respectively. Error bars indicate standard error.
Finally, we evaluated the effect of 37b on ocular permeability in
STZ-induced diabetic rats as a pathological model of macular ede-
ma (Fig. 5B). The oral administration of 37b at 10 mg/kg had an
inhibitory effect on ocular permeability, suggesting that 37b is a
promising compound for the treatment of diabetic macular edema.
202; HRMS (ESI) Calcd for C11H12N3O (M+H)+: 202.0980, found:
202.0986.
5.1.2. 4-Phenyl-1H-imidazol-2-amine (18)
A mixture of 17 (500 mg), 6 M HCl (2.2 mL), and MeOH (2.5 mL)
was refluxed for 3 h. After cooling the mixture to room tempera-
ture, the precipitate was filtered off and the filtrate was then con-
centrated in vacuo. The obtained residue was dissolved in H2O and
the resulting solution was neutralized with 1 M NaOH. The precip-
itate was collected in vacuo and then washed with H2O and Et2O to
give 18 (131 mg, 33%) as a pale brown solid. 1H NMR (DMSO-d6) d
5.25 (2H, s), 6.96 (1H, s), 7.07 (1H, t, J = 7.5 Hz), 7.26 (2H, t,
J = 7.5 Hz), 7.57 (2H, d, J = 7.5 Hz), 10.53 (1H, br s); FAB MS m/e
(M+H)+ 160; HRMS (ESI) Calcd for C9H10N3 (M+H)+: 160.0875,
found: 160.0871; Anal. Calcd for C9H9N3Á0.3H2O: C, 65.68; H,
5.88; N, 25.53. Found: C, 65.81; H, 5.49; N, 25.19.
4. Conclusion
We have developed a 1H-imidazol-2-amine derivative with an
acetamidothiazole moiety that functions as a novel orally active
VAP-1 inhibitor. The compound, 37b, which was synthesized based
on the evaluation and optimization of guanidine bioisosteres, is the
most potent VAP-1 inhibitor reported to date and has excellent
selectivity for VAP-1 compared to other AOs. Furthermore, orally
administered 37b was effective in a pathological model of macular
edema, suggesting it is a promising compound for the treatment of
diabetic macular edema.
5.1.3. 1-Bromo-4-phenylbutan-2-one (21)
To a solution of 4-phenylbutan-2-one (20) (3.0 g) in AcOH
(7 mL) and 48% aqueous HBr (3 mL) was added a solution of Br2
(6.5 g) in AcOH (5 mL) at 0 °C. After stirring the reaction mixture
at room temperature for 4 h, acetone (30 mL) was added to the
solution, and the reaction mixture was further stirred at room tem-
perature for 14 h. The mixture was then concentrated in vacuo, di-
luted with brine, and extracted twice with CH2Cl2. The combined
organic layer was dried over anhydrous MgSO4, and then evapo-
rated in vacuo. The obtained residue was purified by flash column
chromatography over silica gel with hexane/AcOEt (40:1–10:1) as
an eluent to give 21 (1.5 g, 34%) as a pale brown oil. CI MS m/e (M)+
227.
5. Experimental
5.1. Chemistry
1H NMR spectra were measured with a JEOL EX400 or GX500
spectrometer. Chemical shifts are expressed in d units using tetra-
methylsilane as the standard (in the NMR description, s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet; and br, broad peak).
Mass spectra were recorded with Hitachi M-80 or JEOL JMS-
DX300 spectrometers. Silica gel column chromatography was per-
formed using Wakogel C-200 or Merck Silica Gel 60. Unless other-
wise noted, all commercial reagents and solvents were used
without further purification.
5.1.4. N-[4-(2-Phenylethyl)-1H-imidazol-2-yl]acetamide (22)
Compound 22 was prepared from 21 and N-carbamimidoylace-
tamide (16) according to the same procedure used for compound
17 and was obtained as an off-white solid (10% yield). 1H NMR
(DMSO-d6) d 2.02 (3H, s), 2.67–2.74 (2H, m), 2.81–2.88 (2H, m),
6.39 (1H, s), 7.13–7.29 (5H, m), 10.97 (2H, br s); FAB MS m/e
(M+H)+ 230; HRMS (ESI) Calcd for C13H16N3O (M+H)+: 230.1293,
found: 230.1293.
The following compounds were purchased from commercial
sources and used without further purification: 1 (2002-16-6), 3
(1848-75-5), 4 (41213-54-1), 5 (934-32-7), 6 (1687-51-0), 7
(136-95-8), 8 (27878-37-1), 9 (51-17-2), 10 (5993-91-9), 11
(1450-93-7), 12 (1622-57-7), 13 (6285-68-3), 14 (171082-91-0),
and 19 (862281-44-5).
5.1.1. N-(4-Phenyl-1H-imidazol-2-yl)acetamide (17)
A solution of 2-bromo-1-phenylethanone (15) (1.0 g) in DMF
(20 mL) was added to a solution of N-carbamimidoylacetamide
(16) (1.0 g) at 0 °C. The reaction mixture was stirred at room
temperature for 15 h and then concentrated in vacuo. The obtained
solid was washed with CH3CN to give 17 (546 mg, 54%) as an off-
white solid. 1H NMR (DMSO-d6) d 2.07 (3H, s), 7.16 (1H, t,
J = 7.5 Hz), 7.25 (1H, s), 7.32 (2H, t, J = 7.5 Hz), 7.71 (2H, d,
J = 7.5 Hz), 11.22 (1H, s), 11.61 (1H, br s); FAB MS m/e (M+H)+
5.1.5. 4-(2-Phenylethyl)-1H-imidazol-2-amine (23)
Compound 23 was prepared from 22 according to the same
procedure used for compound 18 and was obtained as a pale yel-
low solid (93% yield). 1H NMR (DMSO-d6) d 2.55–2.61 (2H, m),
2.77–2.83 (2H, m), 5.05 (2H, br s), 6.12 (1H, s), 7.13–7.29 (5H,
m), 10.11 (1H, br s); FAB MS m/e (M+H)+ 188; HRMS (ESI) Calcd
for C11H14N3 (M+H)+: 188.1188, found: 188.1190.

T. Inoue et al. / Bioorg. Med. Chem. 21 (2013) 3873–3881
3879
5.1.6. A mixture of methyl 4-[(Z)-2-(2-acetamido-1,3-thiazol-4-
yl)vinyl]benzoate and methyl 4-[(E)-2-(2-acetamido-1,3-
thiazol-4-yl)vinyl]benzoate (26)
J = 7.5 Hz), 2.80 (2H, t, J = 7.5 Hz), 2.89 (4H, m), 3.57 (3H, s), 6.72
(1H, s), 7.11 (4H, s), 12.06 (1H, br s); FAB MS m/e (M+H)+ 333;
HRMS (ESI) Calcd for C17H21N2O3S (M+H)+: 333.1273, found:
333.1274.
Potassium tert-butoxide (3.9 g) was added to a mixture of [4-
(methoxycarbonyl)benzyl](triphenyl)phosphonium bromide (25)
(14.3 g) and DMF (75 mL) at 0 °C under a nitrogen atmosphere.
The reaction mixture was stirred at 0 °C for 15 min, and N-(4-form-
ylthiazol-2-yl)acetamide (24)9 (5.0 g) was then added to the mix-
ture at 0 °C. The reaction mixture was further stirred at room
temperature for 13 h and then poured into ice–H2O. The precipi-
tate was collected in vacuo and washed with Et2O to give 26
(Z:E = 2:1) (7.8 g, 88%) as an off-white solid. Mp 175–177 °C; 1H
NMR (DMSO-d6) d 2.13 (3H Â 2/3, s), 2.16 (3H Â 1/3, s), 3.85 (3H,
s), 6.61 (2H Â 2/3, s), 7.05 (1H Â 2/3, s), 7.26 (1H Â 1/3, d,
J = 15.5 Hz), 7.26 (1H Â 1/3, s), 7.37 (1H Â 1/3, d, J = 15.5 Hz), 7.64
(2H Â 2/3, d, J = 8.5 Hz), 7.69 (2H Â 1/3, d, J = 8.5 Hz), 7.90
(2H Â 2/3, d, J = 8.5 Hz), 7.94 (2H Â 1/3, d, J = 8.5 Hz), 12.05 (1H,
br s); FAB MS m/e (M+H)+ 303.
5.1.11. 4-[2-(2-Acetamido-1,3-thiazol-4-yl)ethyl]benzoic acid
(34a)
To a solution of 27 (330 mg) in dioxane (3.3 mL) was added 1 M
NaOH (0.43 mL) at 0 °C and then the mixture was refluxed for 2 h.
Volatiles were evaporated in vacuo and the obtained residue was
dissolved in H2O and washed once with AcOEt. The aqueous layer
was adjusted to pH 2 using 1 M HCl, and the resulting precipitate
was collected by filtration to give 34a (218 mg, 69%) as a colorless
solid. 1H NMR (DMSO-d6) d 2.11 (3H, s), 2.87–3.05 (4H, m), 6.73
(1H, s), 7.32 (2H, d, J = 8.0 Hz), 7.84 (2H, d, J = 8.0 Hz), 12.06 (1H,
br s), 12.78 (1H, br s); FAB MS m/e (M+H)+ 291; HRMS (ESI) Calcd
for C14H15N2O3S (M+H)+: 291.0803, found: 291.0808; Anal. Calcd
for C14H14N2O3SÁ0.08C2H4O2: C, 57.62; H, 4.89; N, 9.49; S, 10.86.
Found: C, 58.00; H, 4.80; N, 9.23; S, 10.38.
5.1.7. Methyl 4-[2-(2-acetamido-1,3-thiazol-4-yl)ethyl]benzoate
(27)
5.1.12. {4-[2-(2-Acetamido-1,3-thiazol-4-yl)ethyl]phenyl}acetic
acid (34b)
A mixture of 26 (119 g), 10% palladium on carbon (118 g), AcOH
(300 mL), and DMF (2000 mL) was stirred under a hydrogen atmo-
sphere (3 atm) at room temperature for 6 h, and then filtered
through a CeliteÒ pad. The filtrate was concentrated in vacuo and
the residual solid was suspended in AcOEt, and then filtered in va-
cuo. The filtrate was concentrated in vacuo and the obtained resi-
due was purified by flash column chromatography over silica gel
with hexane/AcOEt (1:2) to CHCl3/MeOH (10:1) as an eluent and
triturated with Et2O to give 27 (69 g, 57%) as a pale yellow solid.
Mp 170–171 °C; 1H NMR (DMSO-d6) d 2.11 (3H, s), 2.86–2.95
(2H, m), 2.97–3.05 (2H, m), 3.83 (3H, s), 6.72 (1H, s), 7.35 (2H, d,
J = 8.5 Hz), 7.87 (2H, d, J = 8.5 Hz), 12.08 (1H, br s); FAB MS m/e
(M+H)+ 305; HRMS (ESI) Calcd for C15H17N2O3S (M+H)+:
305.0960, found: 305.0966.
Compound 34b was prepared from 29 according to the same
procedure used for compound 27 and was obtained as an off-white
solid (29% yield). 1H NMR (DMSO-d6) d 2.11 (3H, s), 2.86–2.93 (4H,
m), 3.50 (2H, s), 6.74 (1H, s), 7.14 (4H, s), 12.07 (1H, s), 12.24 (1H,
br s); FAB MS m/e (M+H)+ 305; HRMS (ESI) Calcd for C15H17N2O3S
(M+H)+: 305.0960, found: 305.0962.
5.1.13. 3-{4-[2-(2-Acetamido-1,3-thiazol-4-
yl)ethyl]phenyl}propanoic acid (34c)
Compound 34c was prepared from 33 according to the same
procedure used for compound 34a and was obtained as a colorless
solid (88% yield). 1H NMR (DMSO-d6) d 2.11 (3H, s), 2.52 (2H, t,
J = 7.5 Hz), 2.77 (2H, t, J = 7.5 Hz), 2.89 (4H, m), 6.73 (1H, s), 7.11
(4H, s), 12.10 (1H, br s); FAB MS m/e (M+H)+ 319; HRMS (ESI) Calcd
for C16H19N2O3S (M+H)+: 319.1116, found: 319.1122.
5.1.8. A mixture of {4-[(Z)-2-(2-acetamido-1,3-thiazol-4-
yl)vinyl]phenyl}acetic acid and {4-[(E)-2-(2-acetamido-1,3-
thiazol-4-yl)vinyl]phenyl}acetic acid (29)
Compound 29 was prepared from 24 and [4-(carboxy-
methyl)benzyl](triphenyl) phosphonium bromide (28) according
to the same procedure used for compound 26 and was obtained
as a white powder (Z:E = 5:1) (94% yield). 1H NMR (DMSO-d6) d
2.12 (3H Â 5/6, s), 2.15 (3H Â 1/6, s), 3.52 (2H Â 5/6, s), 3.54
(2H Â 1/6, s), 6.46 (1H Â 5/6, d, J = 12.5 Hz), 6.54 (1H Â 5/6, d,
J = 12.5 Hz), 6.95 (1H, s), 7.11–7.49 (6H Â 1/6, m), 7.20 (2H Â 5/6,
d, J = 8.0 Hz), 7.41 (2H Â 5/6, d, J = 8.0 Hz), 12.10 (1H, br s), 12.42
(1H, br s); FAB MS m/e (M+H)+ 303.
5.1.14. tert-Butyl (4-{4-[2-(2-acetamido-1,3-thiazol-4-
yl)ethyl]phenyl}-1H-imidazol-2-yl)carbamate (36a)
To a solution of 34a (300 mg) in CH2Cl2 (4.5 mL) was added
dropwise oxalyl chloride (0.16 mL) at 0 °C under a nitrogen atmo-
sphere. After stirring for 5 min, two drops of DMF were added to
the reaction mixture, which was then stirred under ice-cooling
for 1 h. The solvent was then evaporated to give acid chloride as
a darkish yellow powder, which was then dissolved in CH2Cl2
(4.5 mL) and the resulting solution was cooled in an ice-bath. To
the solution was added dropwise 2 M (trimethylsilyl)diazometh-
ane/hexane (1.0 mL) at 0 °C under a nitrogen atmosphere. The
reaction mixture was stirred at room temperature for 2 h, and then
4 M HCl/AcOEt (1.0 mL) and DMF (2 mL) were added at 0 °C under
a nitrogen atmosphere. After stirring the solution at room temper-
ature for 12 h, the organic solvent was removed in vacuo. The
obtained halo-ketone was dissolved in DMF (6 mL) and then
tert-butyl carbamimidoylcarbamate (35) (493 mg) was added to
the solution at 0 °C under a nitrogen atmosphere. The reaction
mixture was stirred at 65 °C for 7 h and then concentrated in va-
cuo. The obtained residue was purified by flash column chromatog-
raphy over silica gel with CHCl3/MeOH (50:1–20:1) as an eluent to
give 36a (101 mg, 23%) as an off-white solid. 1H NMR (DMSO-d6) d
1.58 (9H, s), 2.12 (3H, s), 2.85–2.98 (4H, m), 6.55 (1H, s), 6.72 (1H,
s), 7.16 (2H, d, J = 8.0 Hz), 7.62 (2H, d, J = 8.0 Hz), 8.25 (1H, br s),
12.08 (1H, br s); FAB MS m/e (M+H)+ 428; HRMS (ESI) Calcd for
C21H26N5O3S (M+H)+: 428.1756, found: 428.1760.
5.1.9. Methyl (2E)-3-{4-[(E)-2-(2-acetamido-1,3-thiazol-4-
yl)vinyl]phenyl}acrylate (32)
Compound 32 was prepared from methyl (2E)-3-(4-formylphe-
nyl)acrylate (30) and [(2-acetamido-1,3-thiazol-4-yl)methyl](tri-
phenyl)phosphonium chloride (31)9 according to the same
procedure used for compound 26 and was obtained as a pale yel-
low wax (70% yield). 1H NMR (DMSO-d6) d 2.15 (3H, s), 3.73 (3H,
s), 6.66 (1H, d, J = 16.0 Hz), 7.24 (1H, d, J = 14.5 Hz), 7.55–7.78
(2H, m), 7.95 (4H, s), 12.20 (1H, br s); FAB MS m/e (M+H)+ 329;
HRMS (ESI) Calcd for C17H17N2O3S (M+H)+: 329.0960, found:
329.0960.
5.1.10. Methyl 3-{4-[2-(2-acetamido-1,3-thiazol-4-
yl)ethyl]phenyl}propanoate (33)
Compound 33 was prepared from 32 according to the same pro-
cedure used for compound 27 and was obtained as a colorless wax
(76% yield). 1H NMR (DMSO-d6) d 2.11 (3H, s), 2.60 (2H, t,

3880
T. Inoue et al. / Bioorg. Med. Chem. 21 (2013) 3873–3881
5.1.15. tert-Butyl (4-{4-[2-(2-acetamido-1,3-thiazol-4-
yl)ethyl]benzyl}-1H-imidazol-2-yl)carbamate (36b)
was defined using CbH from Leu468 as the central atom with a ra-
dius of 20 Å. The ligand was docked covalently to nitrogen atom N1
of PAQ1729 (PAQ is used PDB entry 2C1111) to assign TPQ and the
ligand. Each ligand was docked 10 times.
Compound 36b was prepared from 34b and 35 according to the
same procedure used for compound 36a and was obtained as a yel-
low amorphous solid (22% yield). 1H NMR (DMSO-d6) d 1.52 (9H, s),
2.11 (3H, s), 2.79–2.99 (4H, m), 3.52–3.64 (2H, m), 5.15 (1H, br s),
6.36 (1H, s), 6.49–6.59 (1H, m), 6.72 (1H, s), 7.06–7.18 (4H, m),
12.05 (1H, br s); FAB MS m/e (M+H)+ 442; HRMS (ESI) Calcd for
5.3. Biology
5.3.1. Inhibitory effect on human and rat VAP-1 enzyme
activities
C
22H28N5O3S (M+H)+: 442.1913, found: 442.1913.
Human and rat VAP-1 enzyme activities were measured using a
radiochemistry enzymatic assay with 14C-benzylamine (American
Radiolabeled Chemicals, St. Louis, MO, USA) as a substrate.21 An en-
zyme suspension prepared from Chinese Hamster Ovary (CHO)
cells stably expressing human or rat VAP-1 enzyme was preincu-
bated with the test compound in a 96-well microplate at room
temperature for 30 min. The enzyme suspension was then incu-
bated with 14C-benzylamine (final concentration of 1 Â 10À5 mol/
5.1.16. tert-Butyl (4-{4-[2-(2-acetamido-1,3-thiazol-4-
yl)ethyl]phenethyl}-1H-imidazol-2-yl)carbamate (36c)
Compound 36c was prepared from 34c and 35 according to the
same procedure used for compound 36a and was obtained as an
off-white amorphous solid (26% yield). 1H NMR (DMSO-d6) d 1.53
(9H, s), 2.11 (3H, s), 2.70–2.93 (8H, m), 6.38 (1H, s), 6.72 (1H, s),
7.10 (4H, s), 12.06 (1H, br s); FAB MS m/e (M+H)+ 456; HRMS
(ESI) Calcd for C23H30N5O3S (M+H)+: 456.2069, found: 456.2074.
L) in a final volume of 50
was stopped by the addition of 2 mol/L (50
dation products were extracted directly with a toluene scintillator
(200 L), and the radioactivity was measured with a scintillation
l
L at 37 °C for 1 h. The enzymatic reaction
lL) citric acid. The oxi-
5.1.17. N-(4-{2-[4-(2-Amino-1H-imidazol-4-
yl)phenyl]ethyl}thiazol-2-yl)acetamide hydrochloride (37a)
A mixture of 36a (32 mg), 4 M HCl/AcOEt (0.4 mL), and MeOH
(1 mL) was stirred at room temperature for 10 h under a nitrogen
atmosphere. The solvent was removed in vacuo and the residual
amorphous substance was solidified with AcOEt to give 37a
(21 mg, 76%) as an off-white solid. 1H NMR (DMSO-d6) d 2.11
(3H, s), 2.88–2.96 (4H, m), 6.50 (1H, s), 6.71 (1H, s), 7.25 (2H, d,
J = 8.0 Hz), 7.55 (2H, d, J = 8.0 Hz), 9.14 (1H, br s), 12.01 (1H, br
s); FAB MS m/e (M+H)+ 328; HRMS (ESI) Calcd for C16H18N5OS
(M+H)+: 328.1232, found: 328.1231.
l
spectrometer.
5.3.2. Inhibitory effect on MAO-A and MAO-B activities
MAO-A and MAO-B enzyme activities were measured using a
fluorometric enzymatic assay with 5-hydroxytriptamine and ben-
zylamine as enzyme substrates, respectively. An enzyme suspen-
sion prepared with recombinant monoamine oxidase (MAO)-A
and B (Supersomes MAO A/BÒ, BD Gentest, MA, USA) was used in
the human MAO-A and MAO-B assays. The enzymatic reaction
was performed using the AmplexÒ Red Oxidase Assay kit (Molecu-
lar Probes). Briefly, compound 37b and a substrate mixture con-
taining AmplexÒ Red reagent were reacted at 37 °C for 1 h with
the MAO-A and MAO-B enzyme suspensions. The fluorescence of
the resulting mixture was measured using a fluorescent plate read-
er (SPECTRA MaxÒ M2; Molecular Devices, Osaka, Japan).
5.1.18. N-[4-(2-{4-[(2-Amino-1H-imidazol-4-
yl)methyl]phenyl}ethyl)thiazol-2-yl]acetamide hydrochloride
(37b)
Compound 37b was prepared from 36b according to the same
procedure used for compound 37a and was obtained as a pale yel-
low solid (74% yield). 1H NMR (DMSO-d6) d 2.12 (3H, s), 2.77–2.96
(4H, m), 3.71–3.85 (2H, m), 6.57 (1H, s), 6.72 (1H, s), 7.078–7.21
(4H, m), 11.64 (1H, s), 12.05 (1H, s); FAB MS m/e (M+H)+ 342;
5.3.3. Measurement of ocular permeability in the diabetic rats
Male Sprague–Dawley (SD) rats (6 weeks old) were purchased
from Japan Charles River Laboratories (Yokohama, Japan). Follow-
ing 1 week of acclimation, one group of animals was weighed
and injected intraperitoneally with 65 mg/kg STZ (Sigma–Aldrich,
St. Louis, MO, USA) in citrate-buffered saline. A second group
underwent sham treatments with citrate-buffered saline. Two
weeks after STZ injection, plasma glucose was measured from col-
lected plasma samples. The blood glucose value was measured
using a colorimetric method, and rats that showed 350 mg/dL
blood glucose levels 2 weeks after STZ treatment were diagnosed
with diabetes.
Compound 37b was given daily for 2 weeks starting 2 weeks
after the completion of STZ treatment. Ocular vascular permeabil-
ity was examined 24 h after the date of the final administration of
37b. Ocular permeability was evaluated based on fluorescein leak-
age into the vitreous 30 min after tail vein administration of
40 mg/mL/kg sodium fluorescein solution. Permeability was ex-
pressed as the intravitreal concentration/plasma concentration ra-
tio of fluorescein. Measurement of the fluorescein was performed
using a SpectraMaxÒ fluorescent plate reader (Molecular Devices,
Osaka, Japan).
HRMS (ESI) Calcd for
342.1388.
C
17H20N5OS (M+H)+: 342.1389, found:
5.1.19. N-[4-(2-{4-[(2-Amino-1H-imidazol-4-
yl)ethyl]phenyl}ethyl)thiazol-2-yl]acetamide hydrochloride
(37c)
Compound 37c was prepared from 36c according to the same
procedure used for compound 37a and was obtained as an off-
white solid (96% yield). 1H NMR (DMSO-d6) d 2.12 (3H, s), 2.65–
2.95 (8H, m), 6.51 (1H, br s), 6.71 (1H, s), 7.12 (4H, s), 11.58 (1H,
br s), 12.07 (1H, br s); FAB MS m/e (M+H)+ 356; HRMS (ESI) Calcd
for C18H22N5OS (M+H)+: 356.1545, found: 356.1540; Solubility pH
5 = 197
lg/mL, pH 7 = 28 lg/mL.
5.2. Molecular modeling
5.2.1. Human VAP-1 model
side-chain conformational search and minimization for
A
Leu469 of the three-dimensional (3D) structure of VAP-1 with
TPQ in an active conformation (PDB-code: 2C11,11 resolution:
2.90 Å) was performed using the LowMode MD16 function in the
Molecular Operating Environment (MOE) program17 with the
MMFF94x forcefield. The bound 2-hydrazinopyridine ligand was
then deleted.
5.3.4. Measurement of plasma VAP-1 activity
On the day after the last dosing with compound 37b in STZ dia-
betic rats, VAP-1 activities in plasma samples collected from each
group were measured using a radio-enzyme assay with 14C-ben-
zylamine as the substrate.21 Each plasma sample was incubated
with 14C-benzylamine (final concentration of 1 Â 10À5 mol/L) to a
5.2.2. Docking study
Compounds were docked to the human VAP-1 model using the
docking program GOLD version 5.1.18–20 The ligand-binding pocket

T. Inoue et al. / Bioorg. Med. Chem. 21 (2013) 3873–3881
3881
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final volume of 100
stopped by the addition of 2 mol/L (100
dation products were then extracted with toluene/AcOEt (500
The radioactivity of the supernatant (200 L) was then measured
l
L at 37 °C for 1 h. The enzymatic reaction was
L) citric acid, and the oxi-
L).
l
l
l
with 5 mL scintillation cocktail using a scintillation spectrometer.
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Acknowledgements
The authors are grateful to the staff of the Division of Analytical
Science Laboratories for performing elemental analysis and
spectral measurements. We thank Drs. Shinya Nagashima and
Shimpei Kawakami for their valuable comments and assistance
in the preparation of this manuscript.
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