Synthesis and antitumor activity of 1,2,4-triazoles having 1,4-benzodioxan fragment as a novel class of potent methionine aminopeptidase type II inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.08.063

A series of 1,2,4-triazole derivatives containing 1,4-benzodioxan (5a-5q) have been designed, synthesized, structurally determined, and their biological activities were evaluated as potential MetAP2 inhibitors. All the synthesized compounds were first reported. Among the compounds, compound 5k showed the most potent biological activity against HEPG2 cancer cell line (IC₅₀ = 0.81 μM for HEPG2 and IC₅₀ = 0.93 μM for MetAP2), which was comparable to the positive control. Docking simulation by positioning compound 5k into the MetAP2 structure active site was performed to explore the possible binding model. The results of apoptosis and Western-blot assay demonstrated that compound 5k possessed good antitumor activity against HEPG2 cancer cell line. Therefore, compound 5k with potent inhibitory activity in tumor growth inhibition may be a potential antitumor agent against HEPG2 cancer cell.

Full text of DOI:10.1016/j.bmc.2011.08.063

Bioorganic & Medicinal Chemistry 19 (2011) 5948–5954
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and antitumor activity of 1,2,4-triazoles having 1,4-benzodioxan
fragment as a novel class of potent methionine aminopeptidase type II
inhibitors
Ya-Ping Hou , Juan Sun , Zhong-Hua Pang, Peng-Cheng Lv, Dong-Dong Li, Li Yan, Hong-Jia Zhang,
⇑
⇑
⇑
Emily Xi Zheng , Jing Zhao , Hai-Liang Zhu
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of China
Nassau University Medical Center, 2201 Hempstead Turnpike, East Meadow, NY 11554, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 1,2,4-triazole derivatives containing 1,4-benzodioxan (5a–5q) have been designed, synthe-
sized, structurally determined, and their biological activities were evaluated as potential MetAP2 inhib-
itors. All the synthesized compounds were first reported. Among the compounds, compound 5k showed
Received 24 June 2011
Revised 26 August 2011
Accepted 27 August 2011
Available online 1 September 2011
the most potent biological activity against HEPG2 cancer cell line (IC50 = 0.81
lM for HEPG2 and
IC50 = 0.93 M for MetAP2), which was comparable to the positive control. Docking simulation by posi-
l
tioning compound 5k into the MetAP2 structure active site was performed to explore the possible bind-
ing model. The results of apoptosis and Western-blot assay demonstrated that compound 5k possessed
good antitumor activity against HEPG2 cancer cell line. Therefore, compound 5k with potent inhibitory
activity in tumor growth inhibition may be a potential antitumor agent against HEPG2 cancer cell.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Triazole
Benzodioxan
Antitumor activity
Auto Dock
MetAP2
1. Introduction
The derivatives of 1,2,4-triazole have a high potential for biolog-
ical activity. The following 1,2,4-triazole derivatives are applied in
Cancer is one of the degenerative diseases of old age and in-
creases with age in both rodents and humans.1 Cancer continues
to be a worldwide killer, despite the enormous amount of research
and rapid developments during the past decade.2 According to re-
cent statistics, cancer accounts for about 23% of the total deaths in
the USA and is the second most common cause of death after heart
disease.3 Therefore, there is an increasing need for new therapies,
especially those based on current knowledge of cancer biology as
well as those taking advantage of the cancer cells phenotype, de-
scribed by Hanahan and Weinberg.4
Methionine aminopeptidases (MetAPs) play an important role
in removing the initiator methionine residue from nascent poly-
peptide chains and are believed to be one of the essential enzymes
involved in protein maturation.5 In yeasts and humans, two pro-
teins are known to possess MetAP activity, MetAP1 and MetAP2.6
MetAP2 appears to play a critical role in cell proliferation and tu-
mor growth. It is expressed at higher concentrations in tumors as
compared to normal cells.7 Available reports also suggested that
MetAP2 plays an important role in the growth of different types
of tumors.6
medicine: alprazolam (tranquilizer), estazolam (hypnotic, sedative,
tranquilizer), rilmazafon (hypnotic, anxiolytic, used in the case of
neurotic insomnia), benatradin (diuretic), trapidil (hypotensive),
trazodon (antidepressant, anxiolytic), and so on.8 The derivatives
of 1,2,4-triazole possess a wide range of antimicrobial8–11 and
antitumour8,12,13 activities. Recently, the compounds containing
1,2,4-triazole were discovered as a novel class of potent tubulin
polymerization inhibitors.14,15
By examining the MetAP2 pocket, we hypothesized that an ex-
tra group (1,4-benzodioxan) could form an additional hydrogen
bonding between oxygen atoms from 1,4-benzodioxan and the
hydrogen of some amino in the active site of MetAP2, such as
ARG 337, ILE 338, PHE 387, and so on. The additional binding
may lead to increased activities. The 1,4-benzodioxan (14BZD)
structure is an important pharmacophore and is found widely in
nature.16 1,4-benzodioxan structure was expected to show strong
biological activities.17 Some 1,4-benzodioxan derivatives are anti-
psychotic agents and others are used by living species in their
chemical communication systems. For example, 9-methoxystro-
bilurin L. having a 1,4-benzodioxan structure was suggested to
have strong antifungal activity.17 Phendioxan and mephendioxan
are recognized as a selective a
-adrenoceptor antagonists.18–22
⇑
Corresponding authors. Tel.: +86 25 8359 2572; fax: +86 25 8359 2672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
To the best of our knowledge, few reports have been dedicated
to the synthesis and MetAP2 inhibitory activity of 1,2,4-triazole
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.08.063

Y.-P. Hou et al. / Bioorg. Med. Chem. 19 (2011) 5948–5954
5949
derivatives containing 1,4-benzodioxan fragment. Herein, in con-
tinuation to extend our research on antitumor compounds with
MetAP2 structure inhibitory activity, we report in the present work
the synthesis and structure–activity relationships of a series of
1,2,4-triazole derivatives containing 1,4-benzodioxan as antitumor
agents. Biological evaluation indicated that some of the synthe-
sized compounds were potent inhibitors of MetAP2.
As illustrated in Table 1, the active analogs showed a distinctive
potential pattern of selectivity as well as broad-spectrum antitu-
mor activity. With regard to selectivity against individual cell lines,
most of the compounds showed effectiveness against cell line Hu-
man hepatocellular liver carcinoma HEPG2 with IC50 values range
of 0.81–60.01
compounds, compound 5k showed the most potent biological
activity (IC50 = 0.81 M). HELA human cervical cancer cell line
proved to be sensitive toward compounds 5f and 5m with IC50 con-
centration range of 15.03–18.60 g/mL comparative to TNP-470
(2.02 M). Regarding SW1116 human colorectal carcinoma cell
line, higher sensitivity was observed with compounds 5o and 5q
with IC50 values range of 5.81–7.35 M, comparable to TNP-470
(5.54 M). BGC823 human gastric cancer cell line was proved to
be a little sensitive toward compounds 5b, 5e and 5k with IC50 con-
centration range of 17.43–26.45 M comparative to TNP-470
(1.48 M). Compounds 5i and 5j were proved to be ineffective
against the four cell lines.
lM comparative to TNP-470 (0.86 lM). Among these
l
2. Results and discussion
2.1. Chemistry
l
l
The synthesis of 5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4-
phenyl-4H-1,2,4-tria zole-3-thiol 4 containing 1,4-benzodioxan
was prepared as shown in Scheme 1. It was synthesized for the first
time and prepared in five steps. Firstly, 2,3-dihydrobenzo[b][1,4]
dioxine-6-carboxylic acids gave 2,3-dihydrobenzo[b][1,4]dioxine-
6-carboxylates 1, catalyzed by concentrated sulfuric acid in metha-
nol. Secondly, 2,3-dihydrobenzo[b][1,4]dioxine-6-carbohydrazides
were prepared by treatment of 1 with hydrazine hydrate (85%) in
ethanol. Then, on treatment with phenyl isothiocyanate in presence
of ethanol, 2 yielded 2-(2,3-dihydrobenzo[b][1,4]dioxine-6-car-
bonyl)-N-phenylhydrazine carbothioamides 3. Finally, a solution
of NaOH (2 N) containing 3 was stirred under refluxing for 30 min,
yielding the desired compounds 4 according to the reported
procedure.11
l
l
l
l
The activity of the tested compounds could be correlated to
structure variations. Through investigating on the selectivity of
the tested compounds over the four cell lines, it was clear that
the tested compounds showed stronger activities against HEPG2
than other three cancer cell lines. Among the tested compounds,
compound 5k showed the most potent biological activity against
HEPG2 cancer cell line with IC50 value of 0.81 lM. Structure–activ-
ity relationship (SAR) analysis indicated that compounds with elec-
Seventeen 1,2,4-triazole derivatives (5a–5q) were prepared by
refluxing in anhydrous acetonitrile of 4 with different benzyl bro-
mide compoundsas shownin Scheme 2 according tothe literature.11
tron-withdrawing group showed stronger activity than that with
electron-donating group, with all the IC50 values below 50 lM
against HEPG2. In further study of compounds with electron-with-
drawing group, different group led to different antitumor activity,
and the potency order was F (fluorine) > Cl (chlorine) > Br (bro-
mine) > NO2 (nitro-group). With regard to the F-substituted com-
pounds, di-substituted compounds 5m and 5n have lower
activity comparing with the mono-substituted compounds 5k
and 5l. Substituents at different positions led to different antitu-
mor activities and the potency order was ortho- > meta- > para-.
2.2. Biological activity
2.2.1. Antiproliferation assay
All the synthesized derivatives (5a–5q) were evaluated for their
ability to antiproliferative activity against HEPG2, HELA, SW1116
and BGC823. The results were summarized in Table 1.
O
O
O
O
O
a
b
OCH3
OH
O
1
O
O
H
N
H
N
O
NH2
O
O
N
H
c
N
H
S
O
3
2
SH
N
d
N
O
O
N
4
Scheme 1. Synthesis of compound 4. Reagents and conditions: (a) methanol, concentrated sulfuric acid, reflux, 12 h; (b) hydrazine hydrate (85%), ethanol, reflux, 5 d; (c)
phenyl isothiocyanate, ethanol, reflux, 30 min; (d) NaOH (2 N), reflux, 30 min.

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Y.-P. Hou et al. / Bioorg. Med. Chem. 19 (2011) 5948–5954
O
O
O
O
N
N
N
R3
R2
R4
N
e
N
N
+
Br
HS
S
R1
R1
R4
R2
R3
4
5a-q
5j R1 = R2 = R4 = H, R3 = CH3
5k R1 = F, R2 = R3 = R4 = H
5a R1 = R2 = R3 = R4 = H
5b R1 = NO2; R2 = R3 = R4 =H
5l
R1 = R2 = R4 = H, R3 = F
5c
R1 = R3 = R4 = H, R2 = NO2
5m R1 = R3 = F, R2 = R4 = H
5n R1 = R4 = F, R2 = R3 = H
5d R1 = R2 = R4 =H, R3=NO2
5e R1 = Cl, R2 = R3 = R4 = H
5o
R1 = Br, R2 = R3 = R4 = H
5f
5g R1 = R2 = R4 = H, R3 = Cl;
5h R1 = R3 = R4 = H, R2 = OCH3;
5i
R1 = R3 = R4 = H, R2 = Cl
5p R1 = R3 = R4 = H, R2 = Br
5q R1 = R2 = R4 = H, R3 = Br
R1 = CH3; R2 = R3 = R4 = H
Scheme 2. General synthesis of compounds (5a–q). Reagents and conditions: (e) reflux, acetonitrile.
Table 1
Antiproliferative activity of the synthesized compounds (5a–5q)
Table 2
MetAP2 inhibitory activity of the selected compounds
IC50
(
l
M)
Compound
MetAP2 (IC50, lM)
Compound
HEPG2
HELA
SW1116
BGC823
5a
5b
5d
5e
5f
5g
5h
5i
5k
6.01
11.78
12.39
1.84
1.92
3.45
30.71
17.66
0.93
2.24
3.95
4.80
5.37
5.91
7.29
1.05
4
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
60.01
5.96
12.02
49.28
13.77
0.97
2.39
3.53
35.12
20.49
56.76
0.81
1.02
1.96
2.07
4.73
5.21
7.94
0.86
51.92
32.75
69.78
45.24
36.18
28.96
15.03
79.18
>100
36.01
49.74
24.25
46.92
18.60
35.17
>100
44.20
54.63
2.02
38.36
7.93
8.30
7.59
13.84
9.67
47.04
73.42
26.45
30.07
48.59
17.43
51.24
79.32
>100
>100
44.28
18.57
>100
>100
84.16
>100
65. 64
33.76
1.48
10.64
8.30
10.08
12.70
16.16
13.57
20.79
7.83
9.01
7.35
26.17
5.81
5.54
5l
5m
5n
5o
5p
5q
5k
5l
5m
5n
5o
5p
5q
TNP-470
TNP-470
2.2.3. Apoptosis assay
Apoptosis is an essential mechanism used to eliminate activated
HEPG2 cells during the shut-down process of excessive immune re-
sponses and maintain proper immune homeostasis, while deficient
apoptosis of activated HEPG2 cell is associated with a wide variety
of immune disorders. As a representative of these triazole deriva-
tives, compound 5k was studied in vitro. We detected the mecha-
nism of compound 5k inhibition activity by flow cytometry (FCM)
(Fig. 3) and found that the compound could induce the apoptosis
of activated HEPG2 cells in a dose-dependent manner. As shown
2.2.2. MetAP2 inhibitory assay
The MetAP2 inhibitory potency of the triazole derivatives con-
taining 1,4-benzodioxan was examined and the results were sum-
marized in Table 2. Most of the tested compounds displayed potent
MetAP2 inhibiting activity. Among them, compound 5k showed
the most potent inhibitory with IC50 of 0.93 lM. The results of Me-
tAP2 inhibitory activity of the tested compounds were in agree-
ment to the structure relationships (SAR) of their antitumor
activities. This agreement suggested that the potent antitumor
activities of the synthetsized compounds were likely related to
their MetAP2 inhibitory activities.
in Figure 3, HEPG2 cells were treated with 0.5, 1.0, 2.0 and 4.0 lM
of compound 5k for 24 h. The compound increased the percentage
of apoptosis by Annexin V-FITC/PI staining in a dose-dependent

Y.-P. Hou et al. / Bioorg. Med. Chem. 19 (2011) 5948–5954
5951
Figure 1. Molecular docking modeling of compound 5k with MetAP2: compound 5k is nicely bound to the MetAP2 with its oxygen atom of triazole ring project toward the
amino hydrogen of ARG 337, forming two H-bond interactions, H–OÁ Á ÁH: 2.100 Å, 148.9o and H–OÁ Á ÁH: 2.139 Å, 145.7o, respectively.
manner. The result indicated that compound 5k induced apoptosis
of HEPG2 cells.
2.2.4. Western-blot assay
In an effort to study the preliminary mechanism of the com-
pound with potent inhibitory activity, the western-blot experi-
ment was performed to explore the effect of compound 5k. The
western-blot results were summarized in Figure 4, confirming
compound 5k’s inhibitory activity.
2.3. Binding model of compound 5k into MetAP2
In an effort to elucidate the possible mechanism by which the
title compounds can induce anticancer activity in the four cells
and guide further SAR studies, molecular docking of the potent
inhibitor 5k into ATP binding site of MetAP2 was performed on
the binding model based on the MetAP2 (2EA4.pdb). The binding
Figure 2. 3D model of the interaction between compound 5k and MetAP2 binding
site. MetAP2 is represented by molecular surface. Compound 5k is depicted by
models of compound 5k and MetAP2 were depicted in Figures 1
sticks and balls.
Figure 3. HEPG2 cells isolated from naïve mice were cultured with anticancer and various concentrations of 5k for 24 h. Cells were stained by Annexin VeFITC/PI and
apoptosis was analyzed by flow cytometry. Inhibition including early and late apoptosis.

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Y.-P. Hou et al. / Bioorg. Med. Chem. 19 (2011) 5948–5954
5 d The sovlent was removed leaving oil which was dissovled in
chloroform (20 ml) and extracted with water (40 ml). After drying
the organic layer with anhydrous Na2SO4 and evaporating the sol-
vent under reduced pressure and a solid appeared. The solid was
recyrstallized from ethanol to obtain the compound 2. (Scheme 1)
4.1.3. Synthesis of 2-(2,3-dihydrobenzo[b][1,4]dioxine-6-car-
bonyl)-N-phenylhydrazinecarbothioamide (3)
Figure 4. Compound 5k was examined by western blotting. Data are representative
To a solution of compound 2 (1 mmol) in ethanol (50 mL), phe-
nyl isothiocyanate (1.5 mmol) was added and the solution was stir-
red under reflux for 30 min. The sovlent was removed under
reduced pressure leaving a residue which was washed with water
(60 ml) and a solid appeared. The solid was recyrstallized from eth-
anol to obtain the compound 3. (Scheme 1)
of three independent experiments.
and 2. In the binding model, compound 5k was nicely bound to the
MetAP2 with its oxygen atom of triazole ring project toward the
amino hydrogen of ARG 337, forming two H-bond interactions,
H–OÁ Á ÁH: 2.100 Å, 148.9o and H–OÁ Á ÁH: 2.139 Å, 145.7o, respec-
tively. The molecular docking results suggested that compound
5k was a potential inhibitor of MetAP2.
4.1.4. Synthesis of 5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4-
phenyl-4H-1,2,4- triazole-3-thiol (4)
The compound 3 (1 mmol) was added in NaOH (2 N, 50 mL),
and the solution was stirred under reflux for 30 min. The resulting
solution was cooled to room temperature and acidified to pH 3–4
with 37% HCl. The precipitate formed was filtered, washed with
water to afford the desired compound 4. (Scheme 1)
3. Conclusions
A series of 1,2,4-triazole derivatives containing 1,4-benzodi-
oxan have been synthesized and evaluated for their antitumor
activities. Compound 5k demonstrated the most potent inhibitory
activity that inhibited the growth of HEPG2 cells with IC50 of
White powder, yield 96%, mp: 281–284 °C. 1H NMR (500 MHz,
CDCl3): 10.65 (s, 1H), 7.49–7.58 (m, 3H), 7.32–7.35 (m, 2H), 6.89
(s, 1H), 6.72 (d, J = 8.0 Hz, 2H), 4.22–4.28 (m, 4H); MS (ESI):
312.36. (C16H14N3O2S, [M+H]+). Anal. Calcd for C16H13N3O2S: C,
61.72; H, 4.21; N, 13.50. Found: C, 61.49; H, 4.04; N, 13.88.
0.81
0.93
l
l
M
and inhibited the activity of MetAP2 with IC50 of
M, which was comparable to the positive control TNP-470.
In order to gain deeper understanding of the structure–activity
relationships observed at the MetAP2, molecular docking of the
most potent inhibitor 5k into the binding site of MetAP2 was per-
formed on the binding model based on the MetAP2 complex struc-
ture. Analysis of the compound 5k’s binding conformation
demonstrated that compound 5k was stabilized by hydrogen
bonding interaction with ARG 337. Apoptosis assay and Western-
blot results showed the compound 5k was a potential antitumor
agent.
4.1.5. Synthesis of 3-(benzylthio)-5-(2,3-dihydrobenzo[b][1,4]
dioxin-6-yl)-4-phenyl-4H-1,2,4-triazole-substituted-benzyl
bromide (5a–q)
To a solution of compound 4 (1 mmol) in acetonitrile, the corre-
sponding benzyl bromide compounds (1 mmol) were added and
the mixture was stirred under reflux for 4–8 h in the presence of
NaOH (2 mmol). Then, the solvent was removed under reduced
pressure and a solid obtained. The solid was recrystallized from
acetonitrile to afford compounds 5a–q. (Scheme 2)
4. Experimental section
4.1. Methods of synthesis
4.1.5.1. 3-(Benzylthio)-5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-
4-phenyl-4H-1,2,4-triazole (5a).
White powder, yield 87%,
mp: 181–184 °C. 1H NMR (500 MHz, CDCl3): 7.45–7.50 (m, 3H),
7.35–7.36 (m, 2H), 7.30–7.32 (m, 1H), 7.27–7.28 (m, 2H), 7.10–
7.11 (m, 2H), 6.99 (s, 1H), 6.89 (d, J = 8.5 Hz, 1H), 6.75 (d,
J = 8.5 Hz, 1H), 4.50 (s, 2H), 4.21–4.26 (m, 4H). MS (ESI): 402.48
(C23H20N3O2S, [M+H]+). Anal. Calcd for C23H19N3O2S: C, 68.81; H,
4.77; N, 10.47. Found: C, 68.43; H, 4.94; N, 10.81.
All chemicals used were purchased from Aldrich (USA). The elu-
ates were monitored using TLC. Melting points (uncorrected) were
determined on a XT4MP apparatus (Taike Corp., Beijing, China). ESI
mass spectra were obtained on a Mariner System 5304 mass spec-
trometer, and 1H NMR spectra were recorded on a DPX500 spec-
trometer at 25 °C with TMS and solvent signals allotted as
internal standards, Chemical shifts are reported in ppm (d). Ele-
mental analyses were performed on a CHN-O-Rapid instrument
and were within 0.4% of the theoretical values.
4.1.5.2. 3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-5-(2-nitroben-
zylthio)-4-phenyl-4H-1,2,4-trazole (5b).
Wheat power, yield
82%, mp: 179–182 °C. 1H NMR (500 MHz, CDCl3): 8.08 (d,
J = 8.0 Hz, 1H), 7.95 (d, J = 8.0 Hz, 1H), 7.58–7.61 (m, 1H), 7.48–
7.51 (m, 1H), 7.45–7.46 (m, 3H), 7.11 (d, J = 7.5 Hz, 2H), 6.96 (s,
1H), 6.84 (d, J = 8.5 Hz, 1H), 6.73 (d, J = 9.0 Hz, 1H), 4.86 (s, 2H),
4.20–4.25 (m, 4H). MS (ESI): 447.48 (C23H19N4O4S, [M+H]+). Anal.
Calcd for C23H18N4O4S: C, 61.87; H, 4.06; N, 12.55. Found: C,
61.50; H, 4.33; N, 12.29.
4.1.1. Synthesis of methyl 2,3-dihydrobenzo[b][1,4]dioxine-6-
carboxylate (1)
2,3-Dihydrobenzo[b][1,4]dioxine-6-carboxylic acid (1 mmol) in
methanol (30 ml) was treated with concentrated sulfuric acid
(0.5 ml) under reflux overnight. The sovlent was removed in vacuo
leaving oil which was dissovled in ethyl acetate (20 ml) and ex-
tracted with water (40 ml). After drying the organic layer with
anhydrous Na2SO4 and evaporating the solvent under reduced
pressure and a solid appeared. The solid was recyrstallized from
ethanol to obtain the compound 1. (Scheme 1)
4.1.5.3. 3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-5-(3-nitroben-
zylthio)-4-phenyl-4H-1,2,4-triazole (5c).
Brown power,
yield 84%, mp: 164–167 °C. 1H NMR (500 MHz, CDCl3): 8.23 (s,
1H), 8.12 (d, J = 7.5 Hz, 1H), 7.81 (d, J = 7.5 Hz, 1H), 7.50–7.52 (m,
1H), 7.46–7.49 (m, 3H), 7.15 (d, J = 7.0 Hz, 2H), 6.97 (s, 1H), 6.84
(d, J = 8.5 Hz, 1H), 6.74 (d, J = 8.5 Hz, 1H), 4.55 (s, 2H), 4.21–4.25
(m, 4H). MS (ESI): 447.48 (C23H19N4O4S, [M+H]+). Anal. Calcd for
(C23H18N4O4S: C, 61.87; H, 4.06; N, 12.55. Found: C, 61.50; H,
4.39; N, 12.22.
4.1.2. Synthesis of 2,3-dihydrobenzo[b][1,4]dioxine-6-carbo-
hydrazide (2)
A stirred solution of compound 1 (0.1 mol) in ethanol (50 mL)
was treated with the hydrazine hydrate (85%), under reflux for

Y.-P. Hou et al. / Bioorg. Med. Chem. 19 (2011) 5948–5954
5953
4.1.5.4. 3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-5-(4-nitroben-
zylthio)-4-phenyl-4H-1,2,4-triazole (5d). Yellow power,
4.1.5.11. 3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-5-(2-fluorob-
enzylthio)-4-phenyl-4H-1,2,4-triazole (5k). White powder,
yield 85%, mp: 161–164 °C. 1H NMR (500 MHz, CDCl3): 8.16 (d,
J = 8.5 Hz, 2H), 7.61 (d, J = 8.5 Hz, 2H), 7.47–7.53 (m, 3H), 7.14
(d, J = 8.5 Hz, 2H), 6.97 (s, 1H), 6.85 (d, J = 8.5 Hz, 1H), 6.74 (d,
J = 8.5 Hz, 1H), 4.55 (s, 2H), 4.21–4.26 (m, 4H). MS (ESI): 447.48
(C23H19N4O4S, [M+H]+). Anal. Calcd for (C23H18N4O4S: C, 61.87;
H, 4.06; N, 12.55. Found: C, 61.50; H, 4.43; N, 12.32.
yield 85%, mp: 173–175 °C. 1H NMR (500 MHz, CDCl3): 7.48–7.51
(m, 3H), 7.46 (d, J = 6.0 Hz, 1H), 7.26 (d, J = 7.0 Hz, 1H), 7.12 (d,
J = 7.5 Hz, 2H), 7.06–7.09 (m, 1H), 7.01–7.05 (m, 1H), 6.98 (s, 1H),
6.87 (d, J = 8.5 Hz, 1H), 6.77 (d, J = 8.0 Hz, 1H), 4.52 (s, 2H), 4.21–
4.26 (m, 4H). MS (ESI): 420.47 (C23H19FN3O2S, [M+H]+). Anal. Calcd
for C23H18FN3O2S: C, 65.86; H, 4.33; N, 10.02. Found: C, 65.53; H,
4.50; N, 10.25.
4.1.5.5.
3-(2-Chlorobenzylthio)-5-(2,3-dihydrobenzo[b][1,4]
White pow-
dioxin-6-yl)-4-phenyl-4H-1,2,4-triazole (5e).
4.1.5.12. 3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-5-(4-fluorob-
der, yield 94%, mp: 201–203 °C. 1H NMR (500 MHz, CDCl3):
7.57–7.58 (m, 1H), 7.46–7.49 (m, 3H), 7.35–7.36 (m, 1H), 7.20–
7.23 (m, 2H), 7.09 (d, J = 7.5 Hz, 2H), 6.98 (s, 1H), 6.86 (d,
J = 8 Hz, 1H), 6.75 (d, J = 8.5 Hz, 1H), 4.60 (s, 2H), 4.22–4.24 (m,
4H). MS (ESI): 436.93 (C23H19ClN3O2S, [M+H]+). Anal. Calcd for
C23H18ClN3O2S: C, 63.37; H, 4.16; N, 9.64. Found: C, 63.08; H,
4.32; N, 9.30.
enzylthio)-4-phenyl-4H-1,2,4-triazole (5l).
White powder,
yield 85%, mp: 157–159 °C. 1H NMR (500 MHz, CDCl3): 7.46–7.52
(m, 3H), 7.33–7.36 (m, 2H), 7.12 (d, J = 7.5 Hz, 2H), 6.99 (d,
J = 8.0 Hz, 1H), 6.98 (s, 1H), 6.97 (d, J = 9.0 Hz, 1H), 6.87 (d,
J = 9.0 Hz, 1H), 6.75 (d, J = 8.0 Hz, 1H), 4.46 (s, 2H), 4.22–4.25 (m,
4H). MS (ESI): 420.47 (C23H19FN3O2S, [M+H]+). Anal. Calcd for
C23H18FN3O2S: C, 65.86; H, 4.33; N, 10.02. Found: C, 65.58; H,
4.06; N, 9.79.
4.1.5.6.
3-(3-Chlorobenzylthio)-5-(2,3-dihydrobenzo[b][1,4]
White powder,
4.1.5.13.
3-(2,4-Difluorobenzylthio)-5-(2,3-dihydrobenzo[b]
(5m). White
dioxin-6-yl)-4-phenyl-4H-1,2,4-triazole(5f).
yield 91%, mp: 123–125 °C. 1H NMR (500 MHz, CDCl3): 7.46–7.51
(m, 3H), 7.34 (s, 1H), 7.22–7.27 (m, 3H), 7.11 (d, J = 7.5 Hz, 2H),
6.98 (s, 1H), 6.86 (d, J = 8.5 Hz, 1H), 6.44 (d, J = 8.5 Hz, 1H), 4.44
(s, 2H), 4.22–4.25 (m, 4H). MS (ESI): 436.93 (C23H19ClN3O2S,
[M+H]+). Anal. Calcd for C23H18ClN3O2S: C, 63.37; H, 4.16; N,
9.64. Found: C, 63.54; H, 4.53; N, 9.27.
[1,4]dioxin-6-yl)-4-phenyl-4H-1,2,4-triazole
powder, yield 87%, mp: 133–135 °C. 1H NMR (500 MHz, CDCl3):
7.47–7.54 (m, 4H), 7.15 (d, J = 6.5 Hz,2H), 6.99 (s, 1H), 6.87 (d,
J = 8.5 Hz, 1H), 6.79–6.82 (m, 2H), 6.75 (d, J = 8.5 Hz, 1H), 4.48 (s,
2H), 4.21–4.26 (m, 4H). MS (ESI): 438.46 (C23H18F2N3O2S,
[M+H]+). Anal. Calcd for C23H17F2N3O2S: C, 63.15; H, 3.92; N,
9.61. Found: C, 63.42; H, 3.69; N, 9.38.
4.1.5.7.
3-(4-Chlorobenzylthio)-5-(2,3-dihydrobenzo[b][1,4]
White powder,
4.1.5.14.
3-(2,6-Difluorobenzylthio)-5-(2,3-dihydrobenzo[b]
(5n). White
dioxin-6-yl)-4-phenyl-4H-1,2,4-triazole(5g).
yield 92%, mp: 174–177 °C. 1H NMR (500 MHz, CDCl3): 7.47–7.48
(m, 3H), 7.31 (d, J = 7.5 Hz, 2H), 7.26 (d, J = 7.5 Hz, 2H), 7.11 (d,
J = 6.5 Hz, 2H), 6.98 (s, 1H), 6.85 (d, J = 8.5 Hz, 1H), 6.74 (d,
J = 8.5 Hz, 1H), 4.44 (s, 2H), 4.22–4.24 (m, 4H); MS (ESI): 436.93
(C23H19ClN3O2S, [M+H]+). Anal. Calcd for C23H18ClN3O2S: C,
63.37; H, 4.16; N, 9.64. Found: C, 63.09; H, 4.44; N, 9.28.
[1,4]dioxin-6-yl)-4-phenyl-4H-1,2,4-triazole
powder, yield 89%, mp: 194–196 °C. 1H NMR (500 MHz, CDCl3):
7.49–7.50 (m, 3H), 7.23–7.24 (m, 1H), 7.21 (d, J = 8.5 Hz,2H), 7.01
(s, 1H), 6.86–6.89 (m, 3H), 6.76 (d, J = 8.5 Hz, 1H), 4.51 (s, 2H),
4.22–4.27 (m, 4H). MS (ESI): 438.46 (C23H18F2N3O2S, [M+H]+).
Anal. Calcd for C23H17F2N3O2S: C, 63.15; H, 3.92; N, 9.61. Found:
C, 63.42; H, 3.59; N, 9.24.
4.1.5.8.
3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-5-(3-meth-
(5h). White
4.1.5.15.
3-(2-Bromobenzylthio)-5-(2,3-dihydrobenzo[b][1,4]
White pow-
oxybenzylthio)-4-phenyl-4H-1,2,4-triazole
dioxin-6-yl)-4-phenyl-4H-1,2,4-triazole (5o).
powder, yield 93%, mp: 115–118 °C. 1H NMR (500 MHz, CDCl3):
7.43–7.49 (m, 3H), 7.19–7.22 (m, 1H), 7.08 (d, J = 8.0 Hz, 2H),
6.97 (s, 1H), 6.92 (d, J = 7.5 Hz, 1H), 6.88 (s, 1H), 6.85 (d,
J = 8.0 Hz, 1H), 6.80–6.82 (m, 1H), 6.74 (d, J = 8.5 Hz, 1H), 4.45 (s,
2H), 4.21–4.26 (m, 4H), 3.78 (s, 3H). MS (ESI): 432.51
(C24H22N3O3S, [M+H]+). Anal. Calcd for C24H21N3O3S: C, 66.80; H,
4.91; N, 9.74. Found: C, 66.53; H, 4.63; N, 9.91.
der, yield 89%, mp: 192–194 °C. 1H NMR (500 MHz, CDCl3): 7.60
(d, J = 7.0 Hz, 1H), 7.55 (d, J = 7.5 Hz, 1H), 7.45–7.50 (m, 3H),
7.24–7.27 (m, 1H), 7.13–7.16 (m, 1H), 7.09 (d, J = 7.5 Hz, 2H),
6.98 (s, 1H), 6.87 (d, J = 8.5 Hz, 1H), 6.75 (d, J = 8.5 Hz, 1H), 4.62
(s, 2H), 4.21–4.26 (m, 4H). MS (ESI): 481.38 (C23H19BrN3O2S,
[M+H]+) Calcd for C23H18BrN3O2S: C, 57.51; H, 3.78; N, 8.75. Found:
C, 57.24; H, 3.95; N, 8.48.
4.1.5.9. 3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-5-(2-methylb-
4.1.5.16.
3-(3-Bromobenzylthio)-5-(2,3-dihydrobenzo[b][1,4]
White pow-
enzylthio)-4-phenyl-4H-1,2,4-triazole (5i).
White powder,
dioxin-6-yl)-4-phenyl-4H-1,2,4-triazole (5p).
yield 90%, mp: 166–168 °C. 1H NMR (500 MHz, CDCl3): 7.42–7.49
(m, 3H), 7.27 (d, J = 7.5 Hz, 1H), 7.17 (m, 1H), 7.14 (d, J = 8 Hz,
1H), 7.10 (m, 1H), 7.06 (d, J = 7.5 Hz, 2H), 6.98 (s, 1H), 6.86 (d,
J = 8 Hz, 1H), 6.74 (d, J = 8.5 Hz, 1H), 4.49 (s, 2H), 4.21–4.25 (m,
4H), 2.33 (s, 3H). MS (ESI): 416.51 (C24H22N3O2S, [M+H]+). Anal.
Calcd for C24H21N3O2S: C, 69.37; H, 5.09; N, 10.11. Found: C,
69.75; H, 5.32; N, 10.44.
der, yield 92%, mp: 121–124 °C. 1H NMR (500 MHz, CDCl3): 7.46–
7.51 (m, 4H), 7.39 (d, J = 7.5 Hz, 1H), 7.30 (d, J = 7.5 Hz, 1H),
7.14–7.17 (m, 1H), 7.09 (d, J = 8.5 Hz, 2H), 6.97 (s, 1H), 6.85 (d,
J = 8.5 Hz, 1H), 6.741 (d, J = 8 Hz, 1H), 4.42 (s, 2H), 4.21–4.25 (m,
4H). MS (ESI): 481.38 (C23H19BrN3O2S, [M+H]+) Calcd for
C23H18BrN3O2S: C, 57.51; H, 3.78; N, 8.75. Found: C, 57.24; H,
3.43; N, 8.42.
4.1.5.10. 3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-5-(4-methylb-
4.1.5.17.
3-(4-Bromobenzylthio)-5-(2,3-dihydrobenzo[b][1,4]
enzylthio)-4-phenyl-4H-1,2,4-triazole (5j).
White powder,
dioxin-6-yl)-4-phenyl-4H-1,2,4-triazole (5q).
White pow-
yield 91%, mp: 175–178 °C. 1H NMR (500 MHz, CDCl3): 7.44–7.50
(m, 3H), 7.24–7.25 (m, 2H), 7.10–7.13 (m, 4H), 6.99 (s, 1H), 6.87
(d, J = 8.5 Hz, 1H), 6.75 (d, J = 8.5 Hz, 1H), 4.47 (s, 2H), 4.22–4.25
(m, 4H), 2.33 (s, 3H). MS (ESI): 416.51 (C24H22N3O2S, [M+H]+).
Anal. Calcd for C24H21N3O2S: C, 69.37; H, 5.09; N, 10.11. Found:
C, 69.05; H, 5.45; N, 10.43.
der, yield 92%, mp: 189–191 °C. 1H NMR (500 MHz, CDCl3): 7.46–
7.51 (m, 3H), 7.42 (d, J = 8.5 Hz, 2H), 7.26 (d, J = 8.0 Hz, 2H), 7.12
(d, J = 7.0 Hz, 2H), 6.98 (s, 1H), 6.87 (d, J = 8.5 Hz, 1H), 6.75 (d,
J = 8.5 Hz, 1H), 4.43 (s, 2H), 4.21–4.26 (m, 4H). MS (ESI): 481.38
(C23H19BrN3O2S, [M+H]+) Calcd for C23H18BrN3O2S: C, 57.51; H,
3.78; N, 8.75. Found: C, 57.79; H, 3.42; N, 8.47.

5954
Y.-P. Hou et al. / Bioorg. Med. Chem. 19 (2011) 5948–5954
4.2. Cell proliferation assay
4.6. Experimental protocol of docking study
The antitumor activities of compounds 5a–5q were determined
using a standard (MTT)-based colorimetric assay (Sigma). Seed 104
cells per well into 96-well plates, incubate at 37 °C, 5% CO2 for 24 h.
The automated docking studies were carried out using Auto
Dock version 4.0. First, AutoGrid component of the program precal-
culates a three-dimensional grid of interaction energies based on
the macromolecular target using the AMBER force field. Then auto-
mated docking studies were carried out to evaluate the binding
free energy of the inhibitors within the macromolecules.
Then add 100
into wells to maintain the final concentration of drug as 40, 20,
6.67, 2.22, 0.74, 0.25 and 0.082 g/mL. One concentration should
lL a series concentration of drug-containing medium
l
be triplicated. And TNP-470 was used for the positive control. After
48 h, cell survival was determined by the addition of an MTT solu-
The three-dimensional structures of the aforementioned com-
pounds were constructed using Chem. 3D ultra 11.0 software
[Chemical Structure Drawing Standard; Cambridge Soft corpora-
tion, USA (2009)], then they were energetically minimized by using
MOPAC with 100 iterations and minimum RMS gradient of 0.10.
The Gasteiger-Hückel charges of ligands were assigned. The crystal
structures of MetAP2 (PDB code: 2EA4) complex were retrieved
from the RCSB Protein Data Bank (http://www.rcsb.org/pdb/
home/home.do). All bound waters and ligands were eliminated
from the protein and the polar hydrogens and the Kollman-united
charges were added to the proteins.
tion (25
lL of 5 mg/mL MTT in PBS). After 4 h, discard the medium
and add 100
lL DMSO; the plates were votexed for 10 min to make
completely dissolution. Optical absorbance was measured at
490 nm.
4.3. MetAP2. inhibitory assay
Seventeen 1,2,4-triazole derivatives containing 1,4-benzodioxan
were tested in a search for small molecule inhibitors of MetAP2,
which was purchased from R&D Systems (Minneapolis, MN). In a
typical study, MetAP2 was incubated for 4 h at room temperature
with or without the presence of the triazole derivatives, the final
concentration of drug as 40, 20, 6.67, 2.22, 0.74, 0.25 and
Acknowledgments
This work was supported by Jiangsu National Science Founda-
tion (No. BK2009239), the Fundamental Research Funds for the
Central Universities (No. 1092020804, 1106020824 and
1096020802), and the Six Kinds of Professional Elite Foundation
of Jiangsu Province (J.Z.).
0.082 lM. The results were reported in Table 2.
4.4. Apoptosis assay
HEPG2 cells were treated with various concentrations of com-
pound 5k for 24 h and then stained with both Annexin V-FITC
(fluorescein isothiocyanate) and propidium iodide (PI). Then sam-
ples were analyzed by FACSCalibur flow cytometer (Becton Dickin-
son, SanJose, CA).
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