Synthesis, structure and hypoxic cytotoxicity of 3-amino-1,2,4- benzotriazine-1,4-dioxide derivatives

Article abstract of DOI:10.1002/ardp.200600201

A series of novel 3-amino-1,2,4-benzotriazine-1,4-dioxide derivatives were synthesized and screened for their in vitro cytotoxicity against promyelocyte leukemia HL-60, androgen-independent prostate tumor PC3, hepatocellular carcinoma Bel-7402, human esop

Full text of DOI:10.1002/ardp.200600201

258
Arch. Pharm. Chem. Life Sci. 2007, 340, 258–263
Full Paper
Synthesis, Structure and Hypoxic Cytotoxicity of
3-Amino-1,2,4-benzotriazine-1,4-dioxide Derivatives
Faqin Jiang1, Qinjie Weng2, Rong Sheng1, Qing Xia1, Qiaojun He2, Bo Yang2, and Yongzhou Hu1
1 ZJU-ENS Joint Laboratory of Medicinal Chemistry, College of Pharmaceutical Sciences, Zhejiang University,
Hangzhou, China
2 Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University,
Hangzhou, China
A series of novel 3-amino-1,2,4-benzotriazine-1,4-dioxide derivatives were synthesized and scree-
ned for their in vitro cytotoxicity against promyelocytic leukemia HL-60, androgen-independent
prostate tumor PC3, hepatocellular carcinoma Bel-7402, human esophagus tumor ECA-109, and
human breast tumor MCF-7 cell lines in hypoxia and in normoxia. Most compounds showed hig-
her cytotoxic activity both in hypoxia and in normoxia. Among them, compounds 61 and 62
showed more potent cytotoxic activity and hypoxic selectivity when compared to tirapazamine.
Keywords: Cytotoxicity / Hypoxia / Tirapazamine /
Received: November 30, 2006; accepted: February 2, 2007
DOI 10.1002/ardp.200600201
sent work is an extension of our ongoing efforts towards
Introduction
the development and identification of new molecules
possessing cytotoxic activity and hypoxic selectivity.
Accordingly, we have designed and synthesized a series
of 3-amino-1,2,4-benzotriazine-1,4-dioxide derivatives by
adding small lipophilic groups to the C-3 amino group
and electron-adopting substituents or electron-donating
substituents to the benzene ring of tirapazamine. The
synthesized compounds were evaluated for their cyto-
toxicity and hypoxia selectivity in vitro.
Solid tumors in the hypoxic areas are more resistant to
chemotherapy and radiotherapy than aerobic ones. How-
ever, hypoxia also promotes the production of a class of
important and specific antitumor prodrugs, namely bior-
eductive agents, including quinones, nitro derivatives,
and N-oxides [1–4].
Among a large number of N-oxides, a benzotriazine
dioxide named tirapazamine, (3-amino-1,2,4-benzotria-
zine-1,4-dioxide, SR4233, WIN 59075, TPZ), is in phase II
and III of clinical trials, alone or in combination with cis-
platin-based chemotherapy and radiotherapy [5, 6]. How-
ever, some reports showed the antitumor efficacy of tira-
pazamine is reduced by its limited diffusion to reach all
hypoxic tumor cells [7, 8]. Therefore, the discovery of new
prodrugs with properly optimized biological properties
is a challenge in this research area. In the previous work
of our team, we found that introduction of some lipophi-
lic groups at the C-3 amino group could improve the
activity and hypoxic selectivity of N-oxides [9]. The pre-
Results and discussion
3-Amino-7-substituted-1,2,4-benzotriazine-1,4-dioxide
derivatives 46–68 were prepared according to the known
methods with minor modification as shown in Scheme 1
[9–11]. Treatment of o-nitroaniline 1–4 with triphosgene
in toluene under reflux for 3 h resulted in 2-nitrophenyl
isocyanates 5–8. Reacting 5–8 with anhydrous ammonia
gas provided 2-nitrophenylureas 9–12, followed by cycli-
zation of 9–12 in 30% aq-NaOH afforded the 3-hydroxy-
1,2,4- benzotriazine-1-oxides 13–16. 3-Hydroxy-1,2,4-ben-
zotriazine-1-oxide 13 was nitrified by a mixture of nitric
acid and vitriol to yield 3-hydroxy-7-nitro-1,2,4-benzotria-
zine-1-oxide 17.
Correspondence: Yongzhou Hu, Zhejiang University, Hangzhou
310058, China.
E-mail 1: huyz@zju.edu.cn (Y.Z. Hu)
E-mail 2: yang924@zju.edu.cn (B. Yang)
Fax: +86 571 88208460
i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2007, 340, 258–263
Hypoxic Cytotoxicity
259
mary amines to yield the 3-amino-1,2,4-benzotriazine-1-
oxides 23–45, which were oxidized with 30% hydrogen
peroxide to afford the desired 3-amino-1,2,4-benzotria-
zine-1,4-dioxides 46–68. The structures of the synthe-
sized compounds were determined by IR, MS, and NMR.
A preliminary evaluation of the cytotoxic activity of all
the target compounds was performed by MTT assays in
vitro. Five cancer cell lines (HL-60: promyelocytic leuke-
mia, BEL-7402: hepatocellular carcinoma, ECA-109:
human esophagus tumor, MCF-7: human breast cancer,
PC-3: androgen-independent prostate tumor) were
employed in the present study. Cancer cells were seeded
in 96-well microtiter plates (4000 cells/well) and main-
tained in hypoxia and in normoxia, respectively. TPZ was
considered as the reference drug according to the litera-
ture method [12]. The IC50 values and the hypoxic cyto-
toxicity ratio (HCR, the ratio of IC50 values in normoxia /
in hypoxia) are shown in Table 1 and Table 2, respec-
tively.
Scheme 1. Design and synthesis of the TPZ derivatives.
As shown in Table 1, most tested compounds exhibited
higher cytotoxic activities than TPZ in hypoxia toward
some tested cancer cell lines. The substituents on the ben-
Chlorination of 13–17 with phosphorus oxychloride
produced 3-chloro-1,2,4-benzotriazine-1-oxides 18–22.
Compounds 18–22 were substituted with various pri-
Table 1. Cytotoxicity of TPZ derivatives (46–68) against five human cancer cell lines in hypoxia and in normoxia.
Comp.
Cytotoxicity (IC50, lM)a)
HL-60
Nc)
BEL-7402
N
ECA-109
MCF-7
N
PC-3
N
Hb)
H
H
N
H
H
TPZ
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
1.6 l 0.8
1.8 l 0.6
0.6 l 0.2
0.4 l 0.1
0.4 l 0.2
0.4 l 0.2
1.3 l 0.4
0.8 l 0.3
0.4 l 0.2
3.0 l 0.8
2.1 l 0.5
2.3 l 0.4
0.9 l 0.4
0.8 l 0.3
0.3 l 0.2
0.2 l 0.1
0.2 l 0.1
0.2 l 0.1
0.7 l 0.2
0.6 l 0.3
0.7 l 0.2
1.4 l 0.8
1.2 l 0.5
0.6 l 0.2
1.8 l 0.6
1.7 l 0.3
1.9 l 0.6
3.2 l 0.7
2.2 l 0.2
1.0 l 0.1
1.2 l 0.4
2.3 l 0.9
6.4 l 1.0
3.2 l 0.8
1.6 l 0.3
1.0 l 0.3
1.0 l 0.2
3.7 l 1.0
1.8 l 0.7
0.5 l 0.2
0.3 l 0.1
0.4 l 0.1
1.2 l 0.4
0.6 l 0.2
0.9 l 0.2
1.0 l 0.4
0.3 l 0.4
1.3 l 0.3
1.6 l 0.2
3.0 l 0.8
4.1 l 0.3
2.6 l 0.2
2.5 l 0.5
2.0 l 0.7
3.5 l 0.8
4.0 l 0.9
3.5 l 0.7
3.6 l 0.2
3.3 l 0.6
2.5 l 0.4
1.1 l 0.2
6.0 l 1.0
3.6 l 0.8
1.3 l 0.9
0.5 l 0.1
0.5 l 0.2
1.2 l 0.3
1.1 l 0.2
1.4 l 0.6
1.6 l 0.2
0.6 l 0.2
1.9 l 0.7
1.9 l 0.6
0.7 l 0.1
0.8 l 0.2
1.7 l 0.5
1.1 l 0.2
0.7 l 0.3
0.5 l 0.1
3.3 l 0.8
3.2 l 0.8
1.7 l 0.6
1.6 l 0.3
0.7 l 0.2
2.9 l 0.5
1.7 l 0.6
1.5 l 0.3
0.4 l 0.1
0.7 l 0.2
1.2 l 0.5
0.5 l 0.1
0.8 l 0.4
0.3 l 0.1
0.7 l 0.5
0.7 l 0.2
0.9 l 0.3
0.6 l 0.2
4.7 l 0.8 2.6 l 0.6
4.5 l 0.4 2.7 l 0.5
2.9 l 0.9 2.4 l 0.5
3.1 l 0.6 1.2 l 0.2
3.0 l 0.5 1.1 l 0.6
2.7 l 0.1 1.1 l 0.2
4.7 l 0.8 2.0 l 0.4
15.1 l 2.1 3.2 l 0.8
4.2 l 0.8 2.3 l 0.4
8.5 l 1.5 2.4 l 0.9
6.1 l 1.2 1.3 l 0.3
3.2 l 0.2 3.6 l 0.8
3.5 l 1.0 1.7 l 0.6
1.9 l 0.6 1.7 l 0.5
1.1 l 0.2 0.4 l 0.1
1.7 l 0.3 0.4 l 0.1
3.5 l 0.8 0.6 l 0.3
1.4 l 0.4 0.6 l 0.2
2.6 l 0.3 0.8 l 0.5
0.7 l 0.1 0.5 l 0.1
1.1 l 0.5 0.3 l 0.1
1.1 l 0.3 0.5 l 0.1
1.3 l 0.3 0.6 l 0.1
0.9 l 0.2 0.7 l 0.2
4.5 l 0.4
1.2 l 0.3
2.0 l 0.4
2.7 l 0.6
2.5 l 0.5
1.8 l 0.5
1.5 l 0.3
3.4 l 0.6
3.3 l 0.5
6.0 l 0.7
6.1 l 0.9
1.6 l 0.6
4.8 l 0.8
2.8 l 0.1
1.8 l 0.9
1.2 l 0.5
0.7 l 0.2
1.7 l 0.3
3.0 l 0.8
1.2 l 0.6
2.1 l 0.3
2.2 l 0.4
8.5 l 1.0
3.9 l 0.3
1.8 l 0.6
4.5 l 0.1
2.9 l 0.9
1.8 l 0.6
1.8 l 0.7
1.1 l 0.3
1.9 l 0.6
1.3 l 0.5
2.4 l 0.5
2.9 l 0.5
3.1 l 0.3
2.6 l 0.9
1.7 l 0.2
1.9 l 0.1
2.0 l 0.5
1.6 l 0.7
0.7 l 0.2
1.6 l 0.8
1.5 l 0.6
0.7 l 0.2
0.8 l 0.4
0.6 l 0.2
1.1 l 0.2
1.0 l 0.3
0.6 l 0.1
5.7 l 0.5
3.0 l 0.7
2.2 l 0.2
2.4 l 0.6
2.1 l 0.5
5.3 l 0.9
4.2 l 0.7
4.6 l 0.7
5.2 l 0.9
5.0 l 0.8
3.2 l 0.5
4.8 l 0.8
4.0 l 0.8
1.6 l 0.5
0.8 l 0.1
1.6 l 0.2
1.9 l 0.6
0.9 l 0.3
0.4 l 0.2
0.2 l 0.1
0.4 l 0.1
1.0 l 0.2
0.8 l 0.2
8.6 l 0.4
5.0 l 0.9
3.8 l 0.8
4.1 l 0.3
3.4 l 0.2
7.0 l 0.4
4.3 l 0.6
7.4 l 0.5
7.4 l 0.3
3.8 l 0.7
5.8 l 0.9
2.1 l 0.3
1.8 l 0.5
0.7 l 0.2
1.0 l 0.2
3.4 l 0.6
4.0 l 0.8
1.6 l 0.5
4.0 l 0.6
3.6 l 0.3
9.1 l 0.9
2.4 l 0.9
3.4 l 0.5
a)
Each experiment was independently performed three times.
H = Hypoxia: the percentage of oxygen is 3%.
N = Normoxia: the percentage of oxygen is 20%.
b)
c)
i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

260
F. Jiang et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 258–263
Table 2. The hypoxic cytotoxicity ratio (normoxia/hypoxia) of TPZ derivatives 46–68.
Comp.
HCRa, b)
Comp.
HCRa, b)
HL-60
BEL-7402 ECA-109 MCF-7
PC-3
HL-60
BEL-7402 ECA-109 MCF-7
PC-3
TPZ
46
47
48
49
50
51
52
53
54
55
56
1.1
1.6
2.8
4.1
2.5
4.2
1.0
2.9
7.9
1.01
1.21
no
1.8
2.2
no
1.1
2.0
2.8
1.74
no
1.1
6.6
5.4
1.7
2.8
4.1
5.1
1.4
4.7
1.7
2.1
1.3
1.9
2.1
1.9
2.7
1.3
2.0
2.19
3.79
no
3.8
4.4
1.9
1.5
2.3
2.3
2.1
1.3
57
58
59
60
61
62
63
64
65
66
67
68
2.2
2.4
5.1
4.0
6.4
6.9
1.0
1.4
no
1.6
2.0
2.4
1.6
1.0
no
1.8
1.6
1.5
1.68
1.44
1.17
2.0
1.3
2.7
2.6
2.9
2.8
3.2
2.2
2.8
2.4
3.84
2.1
2.7
3.0
1.1
no
no
no
1.61
1.20
no
1.0
no
1.4
2.0
1.3
1.4
1.9
1.6
1.07
no
2.5
1.2
1.5
2.07
2.54
1.07
5.31
8.77
1.12
1.21
2.46
1.21
no
no
1.09
1.60
1.47
1.45
1.89
a)
The HCR was the ratio of IC50 values in normoxia and in hypoxia.
Each value was the mean of three times of independent experiment.
b)
zene ring or at the 3-position of TPZ could affect the cyto-
toxic activity by changing the electronic properties and
the lipophilic properties of the entire molecule. When
the group was an electron-adopting substituent on the
benzene ring such as chlorine or a nitro group (e.g. 61,
62, and 64), it showed lower IC50 values than that of an
electron-donating substituent such as a methyl or a
methyloxy group. The side chains at the 3-position of TPZ
could intensively influence the cytotoxic activity. When
the side chain was an alkyl or an aromatic group, the
compounds showed lower IC50 values than TPZ (e.g. 48–
50). At the same time, all the tested compounds showed
different activity toward various kinds of cancer cells and
exhibited higher activity toward HL-60 cells and ECA-109
cells, and lower activity toward PC-3 cells.
Experimental
Melting points were recorded on a Bꢀchi B-540 melting point
apparatus and are uncorrected (Bꢀchi Labortechnik, Flawil,
Switzerland). The H-NMR spectra were recorded on a Bruker
1
Avance DMX-400 NMR-spectrometer (400 MHz, 298C; Bruker
Bioscience, Billerica, MA, USA). Chemical shift are given in ppm
relative to tetramethylsilane (TMS) as internal standard (0 ppm).
Infrared (IR) spectra were performed on a Bruker VECTOR 22
spectrophotometer (cm– 1). Mass spectra were recorded on an
Esquire-LC-00075 mass spectrometer (Bruker). Purifications were
performed by chromatography over silica gel. All reagents were
purchased from commercial suppliers and dried and purified
when necessary.
Chemistry
The compounds 13–16 were synthesized according to the
known method [9].
In conclusion, all compounds showed higher activity
in hypoxia than in normoxia against all the tested cancer
cells as shown in Table 2. Compounds with electron-
adopting substituents on the benzene ring showed
higher cytotoxic activity but lower hypoxia selectivity.
The compounds with a nitro group showed excellent
activity but were avoid of hypoxia selectivity (e.g. 64–68).
The chloro compounds (e.g. 61, 62) showed exciting
results in the cytotoxic activity and hypoxia selectivity
and would be likely candidates to be further modified for
obtaining second-generation prodrugs with optimized
micropharmacokinetic properties. The pharmacokinetic
experiment on these compounds is currently under
investigation in our laboratory.
3-Hydroxy-7-nitro-1,2,4-benzotriazine-1-oxide 17
3-Hydroxy-1,2,4-benzotriazine-1-oxide 13 was nitrified by the
mixture of nitric acid and vitriol at 608C for 2 h, and then the
mixture was poured into ice water (200 mL), the precipitate was
filtrated and dried to get a yellow solid 17; yield 92.3%; m.p.
211–2138C; MS (ESI): 209 [M+H]; 1H-NMR (DMSO, 400 MHz) d:
11.34 (s, 1H, OH), 8.69 (s, 1H, CH), 8.51 (d, 1H, CH, J = 8.8 Hz), 7.45
(d, 1H, CH, J = 9.2 Hz).
General procedure for the synthesis of 3-Chloro-7-
substituted-1,2,4-benzotriazine-1-oxides 18–22
3-Hydroxy-1,2,4-benzotriazine-1-oxide 13 (0.1 mol) and phos-
phorus oxychloride (70 mL) were refluxed for 30 min. The mix-
ture was evaporated under vacuum and the residue was poured
on ice. The product was crystallized from methanol to yield the
compound 18. Compounds 19–22 were also prepared by the pro-
cedure described above.
This work was financial supported by the National Natural
Science Foundation of China (20602030), the Science and Tech-
nology Department of Zhejiang province of China (2006c23002)
and the Zhejiang Natural Science Foundation (No. R205120).
i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2007, 340, 258–263
Hypoxic Cytotoxicity
261
3-Chloro-1,2,4-benzotriazine-1-oxide 18
3-(n-Hexylamino)-1,2,4-benzotriazine-1,4-dioxide 47
Yield 86.0%; m.p. 119–1208C (lit. [11]: m.p. 117–1188C); MS (ESI):
Yield 21.9%; m.p. 153–1558C; MS (ESI): 263 [M+H]; 1H-NMR
(CDCl3, 400 MHz) d: 8.26 (t, 1H, CH, J = 6.4 Hz), 8.15 (d, 2H, CH, J =
8.4 Hz), 8.07 (t, 1H, CH, J = 8.8 Hz), 7.87 (t, 1H, NH, J = 8.0 Hz),
1.18–1.22 (m, 10H, CH2), 0.77~0.80 (m, 3H, CH3); IR (KBr): 3255,
3108, 2918, 2849, 1597, 1493, 1412, 1358, 1176, 1108, 1078, 860,
1
182 [M+H]; H-NMR (CDCl3, 400 MHz) d: 8.45 (d, 1H, CH, J = 8.8
Hz), 8.02 (d, 2H, CH, J = 4.0 Hz), 7.78–7.81 (m, 1H, CH); IR (KBr):
3079, 3038, 2218, 1979, 1859, 1664, 1608, 1569, 1488, 1451,
1412, 1357, 1323, 1267, 1210, 1180, 1124, 1085, 967, 767 cm– 1
.
763, 721 cm– 1
.
3-Chloro-7-methyl-1,2,4-benzotriazine-1-oxide 19
3-Cyclohexylamino-1,2,4-benzotriazine-1,4-dioxide 48
Yield 15.3%; m.p. 190–1968C; MS (ESI): 261 [M+H]; 1H-NMR
(DMSO, 400 MHz) d: 8.17 (d, 1H, CH, J = 8.0 Hz), 8.10 (d, 1H, CH, J =
8.0 Hz), 7.88–7.94 (m, 2H, CH), 7.53 (t, 1H, NH, J = 7.6 Hz), 3.69–
3.75 (m, 1H, CH), 1.87 (d, 2H, CH2, J = 12.8 Hz), 1.73 (d, 2H, CH2, J =
11.8 Hz), 1.60 (d, 1H, CH2, J = 13.2 Hz), 1.43~ 1.53 (m, 2H, CH2),
1.26–1.37 (m, 2H, CH2), 1.11–1.17 (m, 1H, CH2); IR (KBr): 3246,
1739, 1619, 1493, 1414, 1357, 1227, 1184, 1089, 970, 865, 771,
Yield 80%; m.p. 180–1818C (lit. [13]: m.p. 177–1798C); MS (ESI):
1
196 [M+H]; H-NMR (CDCl3, 400 MHz) d: 8.20 (s, 1H, CH), 7.89 (d,
1H, CH, J = 8.4 Hz), 7.82 (d, 1H, CH, J = 8.4 Hz), 2.612 (s, 3H, CH3).
3-Chloro-7-methoxyl-1,2,4-benzotriazine-1-oxide 20
Yield 76%; m.p. 184–1868C (lit. [14]: m.p. 188.5–190.58C); MS
(ESI): 212 [M+H]; 1H-NMR (CDCl3, 400 MHz) d: 7.89 (d, 1H, CH, J =
8.8 Hz), 7.66 (s, 1H, CH), 7.62 (d, 1H, CH, J = 8.8 Hz), 4.01 (s, 3H,
OCH3).
725 cm– 1
.
3-Phenylamino-1,2,4-benzotriazine-1,4-dioxide 49
Yield 17.6%; m.p. 203–2048C; MS (ESI): 255 [M+H]; 1H-NMR
(CDCl3, 400 MHz) d: 8.15 (d, 2H, CH, J = 8.4 Hz), 8.09 (d, 2H, CH, J =
8.4 Hz), 7.92 (d, 2H, CH, J = 8.0 Hz), 7.89 (t, 2H, CH, J = 6.8 Hz), 7.52
(t, 2H, CH, NH, J = 7.2 Hz); IR (KBr): 3309, 2928, 1646, 1590, 1491,
1366, 1174, 1105, 1025, 998, 839, 771, 725 cm-1.
3-Chloro-7-chloro-1,2,4-benzotriazine-1-oxide 21
Yield 89%; m.p. 154–1568C (lit. [14]: m.p. 157–158.58C); MS (ESI):
216 [M+H]; 1H-NMR (CDCl3, 400 MHz) d: 8.40 (s, 1H, CH), 7.92–
7.98 (m, 2H, CH).
3-Chloro-7-nitro-1,2,4-benzotriazine-1-oxide 22
Yield 84.4%; m.p. 163–1688C; MS (ESI): 227 [M+H]; 1H-NMR
(CDCl3, 400 MHz) d: 8.98 (s, 1H), 8.45 (d, 1H, CH, J = 9.2 Hz), 8.13
(d, 1H, CH, J = 9.2 Hz).
3-Benzylamino-1,2,4-benzotriazine-1,4-dioxide 50
Yield 33.2%; m.p. 206–2078C; MS (ESI): 269 [M+H]; 1H-NMR
(CDCl3, 400 MHz) d: 8.84 (t, 1H, CH, J = 6.4 Hz), 8.01–8.15 (m, 2H,
CH), 7.89 (t, 1H, CH, J = 7.2 Hz), 7.51 (t, 1H, NH, J = 8.0 Hz), 7.34 (d,
2H, CH2, J = 8.8 Hz), 7.27 (t, 2H, CH, J = 7.2 Hz), 7.20 (t, 1H, CH, J =
5.6 Hz), 4.58 (d, 2H, CH, J = 6.8 Hz); IR (KBr): 3387, 3012, 1603,
TPZ was prepared by the known method as a red solid [11] m.
p. 222–2248C (lit. [11]: m.p. 2208C); MS (ESI): 179 [M+1]; 1H-NMR
(CDCl3, 400 MHz) d: 8.31 (d, 2H, CH, J = 8.8 Hz), 7.86 (t, 1H, CH, J =
7.6 Hz), 7.53 (t, 1H, CH, J = 7.6 Hz), 6.15 (s, 2H, NH2); IR (KBr):
3413, 3263, 3084, 1626, 1596, 1517, 1487, 1416, 1356, 1165,
1574, 1498, 1417, 1356, 1206, 1188, 1103, 1085, 844, 726 cm– 1
.
1099, 858, 833, 777, 767, 728 cm– 1
.
3-Ethylamino-7-methyl-1,2,4-benzotriazine-1,4-dioxide
51
Yield 35.9%; m.p. 201–2038C; MS(ESI): 221 [M+H];1H-NMR (CDCl3,
400 MHz): d 8.19 (d, 1H, CH, J = 8.8 Hz), 8.13 (s, CH, 1H), 7.70 (d,
1H, CH, J = 8.8 Hz), 7.01 (s, 1H, NH), 3.63~3.66 (m, 2H, CH2), 2.527
(s, 3H, CH3), 1.340–1.377 (t, 3H, CH3, J = 7.2 Hz); IR (KBr): 3254,
1608, 1507, 1414, 1379, 1354, 1330, 1160, 1091, 996, 894, 814,
General procedure for the synthesis of 3-Alkylamino-7-
substituted-1,2,4-benzotriazine-1,4-dioxides 46–68
A mixture of 3-chloro-1,2,4-benzotriazine-1-oxide 18 (2.75 mmol)
and ethylamine (2.75 mmol) in ethanol (10 mL) was stirred at
room temperature for 12 h and then the precipitate was fil-
trated to yield the 3-ethylamino-1,2,4-benzotriazine-1-oxide 23.
Compound 23 (2.00 mmol) was oxidized by 30% hydrogen perox-
ide (10 mL) and acetic acid (20 mL) at 608C for 24 h. The mixture
was neutralized by saturated aqueous sodium bicarbonate solu-
tion and extracted with dichloromethane (36100 mL), and then
the combined organic fractions were dried over anhydrous
sodium sulfate and evaporated under vacuum. The residue was
chromatographed over silica gel and eluted with a gradient of
(25%–100%) EtOAc/petroleum to get 3-ethylamino-1,2,4-benzo-
triazine-1,4-dioxide 46. Compounds 47–68 were also prepared
by the procedure described above.
777, 725 cm– 1
.
3-(n-Hexylamino)-7-methyl-1,2,4-benzotriazine-1,4-
dioxide 52
Yield 28.7%; m.p. 162–1708C; MS (ESI): 277 [M+H]; 1H-NMR
(CDCl3, 400 MHz) d: 8.18 (d, 1H, CH, J = 8.8 Hz), 8.12 (s, 1H, CH),
7.69 (d, 1H, CH, J = 8.8 Hz), 7.04 (s, 1H, NH), 3.55–3.61(m, 2H,
CH2), 2.52 (s, 3H, CH3), 1.31–1.44 (m, 8H, CH2), 0.84–0.91 (m, 3H,
CH3); IR (KBr): 3264, 2923, 2854, 1604, 1504, 1464, 1415, 1379,
1344, 1314, 1286, 1230, 1157, 1135,1110, 1083, 1014, 984, 897,
815, 786 cm– 1
.
3-Cyclohexylamino-7-methyl-1,2,4-benzotriazine-1,4-
3-Ethylamino-1,2,4-benzotriazine-1,4-dioxide 46
Yield 32.7%; m.p. 183–1848C; MS (ESI): 207 [M+H]; 1H-NMR
(CDCl3, 400 MHz) d: 8.25 (d, 2H, CH, J = 8.8 Hz), 7.80 (t, 1H, CH, J =
7.6 Hz), 7.43 (t, 1H, CH, J = 8.0 Hz), 7.04 (s, 1H, NH), 3.55–3.62 (m,
2H, CH2), 1.29 (t, 3H, CH3, J = 7.6 Hz); IR (KBr): 3263, 2982, 1618,
1594, 1491, 1411, 1359, 1323, 1181, 1158, 1107, 1085, 765, 720
dioxide 53
Yield 38.6%; m.p. 201–2038C; MS (ESI): 275 [M+H]; 1H-NMR
(CDCl3, 400 MHz) d: 8.17 (d, 1H, CH, J = 8.4 Hz), 8.11 (s, 1H, CH),
7.68 (d, 1H, CH, J = 8.4 Hz), 6.96 (s, 1H, NH), 3.94–3.97 (m, 1H,
CH), 2.51 (s, 3H, CH3), 2.07–2.10 (d, 2H, CH2, J = 10.0 Hz), 1.65–
1.68 (d, 2H, CH2, J = 13.2 Hz), 1.36~1.45 (m, 4H, CH2), 1.23–1.28
cm– 1
.
i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

262
F. Jiang et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 258–263
(m, 2H, CH2); IR (KBr): 3250, 2936, 2855, 1661, 1584, 1501, 1414,
1375, 1341, 1291, 1232, 1254, 1112, 1087,1019, 960, 925, 892,
3-(n-Hexylamino)-7-chloro-1,2,4-benzotriazine-1,4-
dioxide 60
870, 847, 809, 774 cm– 1
.
Yield 31.3%; m.p. 181–1838C; MS (ESI): 297 [M+H]; 1H-NMR
(CDCl3,, 400 MHz) d: 8.34 (d, 1H, CH, J = 8.8 Hz), 8.26 (s, 1H, CH),
7.78 (d, 1H, CH, J = 8.8 Hz), 7.09 (s, 1H, NH), 3.56–3.61 (m, 2H,
CH2), 1.68l1.73 (m, 2H, CH2), 1.40–1.44 (m, 2H, CH2), 1.30–1.35
(m, 4H, CH2), 0.68l0.91 (m, 3H, CH3); IR (KBr): 3408, 3294, 3070,
1610, 1490, 1407, 1380, 1313, 1159, 1120, 1087, 913, 815, 755
3-Phenylamino-7-methyl-1,2,4-benzotriazine-1,4-dioxide
54
Yield 15.3%; m.p. 221–2238C; MS (ESI): 269 [M+H]; 1H-NMR
(CDCl3, 400 MHz) d: 8.04 (d, 1H, CH, J = 8.8 Hz), 7.99 (s, 1H, CH),
7.92 (m, 5H, CH), 7.75 (d, 1H, CH, J = 8.8 Hz), 7.00 (s, 1H, NH), 3.31
(s, 3H, CH3); IR (KBr): 3408, 3265, 1604, 1516, 1416, 1353, 1160,
cm– 1
.
1100, 1024, 900, 812, 770 cm– 1
.
3-Cyclohexylamino-7-chloro-1,2,4-benzotriazine-1,4-
dioxide 61
Yield 45.8%; m.p. 191–1928C; MS (ESI): 295 [M+H]; 1H-NMR
(CDCl3, 400 MHz) d: 8.33 (s, 1H, CH), 8.24 (d, 1H, CH, J = 8.8 Hz),
7.78 (d, 1H, CH, J = 8.8 Hz), 7.01 (s, 1H, NH), 3.94–3.98 (m, 1H,
CH), 2.05–2.10 (m, 2H, CH2), 1.79l1.83 (m, 2H, CH2), 1.86–1.90
(m, 1H, CH2), 1.35~1.49 (m, 4H, CH2), 1.23–1.30 (m, 1H, CH2); IR
(KBr): 3290, 3090, 3068, 2927, 1592, 1489, 1462, 1445, 1405,
1382, 1371, 1334, 1298, 1272, 1193, 1162, 1120, 1107, 1082, 961,
3-Benzylamino-7-methyl-1,2,4-benzotriazine-1,4-dioxide
55
Yield 37.6%; m.p. 222–2238C; MS (ESI): 283 [M+H]; 1H-NMR
(DMSO, 400 MHz) d: 8.26 (d, 1H, CH, J = 8.8 Hz), 8.15 (s, 1H, NH),
7.75 (d, 2H, CH, J = 8.8 Hz), 7.25–7.26 (m, 5H, CH), 6.26 (s, 2H,
CH2), 2.55 (s, 3H, CH3); IR (KBr): 3408, 3266, 1604, 1516, 1353,
1334, 1160, 1100, 1024, 900,812, 770 cm– 1
.
912, 884, 845, 817, 756 cm– 1
.
3-Ethylamino-7-methyloxy-1,2,4-benzotriazine-1,4-
3-Phenylamino-7-chloro-1,2,4-benzotriazine-1,4-dioxide
dioxide 56
Yield 23.5%; m.p. 205–2078C; MS (ESI): 237 [M+H]; 1H-NMR
(CDCl3, 400 MHz) d: 8.19 (d, 1H, CH, J = 9.2 Hz), 7.57 (s, 1H, CH),
7.49 (d, 1H, CH, J = 9.2 Hz), 6.90 (s, 1H, NH), 3.94 (s, 3H, OCH3),
3.57~3.64 (m, 2H, CH2), 1.30 (t, 3H, CH3, J = 6.8 Hz); IR (KBr): 3245,
1605, 1504, 1472, 1386, 1358, 1310, 1257, 1198, 1155,
62
1
Yield 27.3%; m.p. 2958C; MS (ESI): 289 [M+H]; H-NMR (CDCl3,
400 MHz) d: 8.36 (s, 1H, CH), 8.35 (d, 1H, CH, J = 9.0 Hz), 8.33 (d,
1H, CH, J = 9.0 Hz), 7.83–7.86 (m, 5H, CH), 7.01 (s, 1H, NH); IR
(KBr): 3408, 3276, 3064, 1621, 1600, 1523, 1487, 1403, 1381,
1358, 1164, 1097, 1024, 915, 876, 815, 751 cm– 1
.
1112,1081, 1008, 898,857, 826, 787 cm– 1
.
3-Cyclohexylamino-7-methyloxy-1,2,4-benzotriazine-1,4-
3-Benzylamino-7-chloro-1,2,4-benzotriazine-1,4-dioxide
dioxide 57
63
Yield 26.1%; m.p. 208–2098C; MS (ESI): 291 [M+H]; 1H-NMR
(CDCl3, 400 MHz) d: 8.18 (d, 1H, CH, J = 9.2 Hz), 7.56 (s, 1H, CH),
7.47 (d, 1H, CH, J = 9.2 Hz), 6.82 (s, 1H, NH), 3.92 (s, 4H, CH, OCH3),
2.08 (d, 2H, CH2, J = 9.6 Hz), 1.77–1.80 (m, 2H, CH2), 1.65–1.67 (d,
1H, CH2, J = 9.6 Hz), 1.35–1.47 (m, 4H, CH2), 1.24–1.32 (m, 1H,
CH2); IR (KBr): 3247, 2939, 1591, 1506, 1453, 1421, 1389, 1332,
1292, 1257, 1183, 1147, 1116, 1094, 1018, 964, 891,842, 816, 779
Yield 42.2%; m.p. 275–2768C; MS (ESI): 303 [M+H];1H-NMR(CDCl3,
400 MHz) d: 8.34 (s, 1H, CH), 8.26 (d, 1H, CH, J = 9.2 Hz), 7.94 (d,
1H, CH, J = 9.2 Hz), 7.31–7.44 (m, 5H, CH), 5.30 (s, 1H, NH), 7.77
(s, 2H, CH2); IR (KBr): 3411, 3283, 3091, 3068, 1600, 1526, 1489,
1407, 1379, 1358, 1325, 1261, 1163, 1123, 1095, 1024, 950, 915,
877, 816, 753 cm– 1
.
cm– 1
.
3-Ethylamino-7-nitro-1,2,4-benzotriazine-1,4-dioxide 64
Yield 33.6%; m.p. 203–2058C; MS (ESI): 252 [M+H]; 1H-NMR
(CDCl3, 400 MHz) d: 8.93 (s, 1H, CH), 8.60 (d, 1H, CH, J = 9.6 Hz),
8.42 (d, 1H, CH, J = 9.6 Hz), 7.32 (s, 1H, NH), 3.70–3.70 (m, 2H,
CH2), 1.40 (t, 3H, CH3, J = 6.8 Hz); IR (KBr): 3233, 3101, 1634, 1601,
1530, 1497, 1428, 1392, 1331, 1236, 1161, 1132, 1093, 995, 920,
3-Benzylamino-7-methyloxy-1,2,4-benzotriazine-1,4-
dioxide 58
Yield 24.8%; m.p. 210–2148C; MS (ESI): 299 [M+H]; 1H-NMR
(DMSO, 400 MHz) d: 8.11 (d, 1H, CH, J = 8.4 Hz), 8.04 (s, 1H, CH),
7.45 (m, 5H, CH), 7.37 (d, 1H, CH, J = 8.4 Hz), 6.78 (s, 1H, NH), 4.87
(s, 2H, CH2), 3.78 (s, 3H, OCH3); IR (KBr): 3423, 3352, 1646, 1535,
1473, 1415, 1397, 1358, 1275, 1204, 1168, 1121,1037, 845, 789
cm–1.
894, 830, 802, 745 cm– 1
.
3-(n-Hexylamino)-7-nitro-1,2,4-benzotriazine-1,4-dioxide
65
Yield 25.3%; m.p. 201–2048C; MS (ESI): 308 [M+H]; 1H-NMR
(CDCl3,, 400 MHz) d: 8.91 (s, 1H, CH), 8.86 (d, 1H, CH, J = 10.0 Hz),
8.55 (d, 1H, CH, J = 11.6 Hz), 8.24 (s, 1H, NH), 3.30l3.42 (m, 2H,
CH2), 3.31–3.38 (m, 4H, CH2), 1.62–1.67 (m, 4H, CH2), 0.92 (t, 3H,
CH3, J = 7.2 Hz); IR (KBr): 3251, 3080, 1633, 1602, 1529, 1498,
1471, 1432, 1392, 1330, 1296, 1157, 1134, 1107, 1082, 1056, 981,
3-Ethylamino-7-chloro-1,2,4-benzotriazine-1,4-dioxide 59
Yield 39.5%; m.p. 205–2098C; MS (ESI): 241 [M+H]; 1H-NMR
(CDCl3, 400 MHz) d: 8.33 (s, 1H, CH), 8.24 (d, 1H, CH, J = 9.2 Hz),
7.78 (d, 1H, CH, J = 9.2 Hz), 7.09 (s, 1H, NH), 3.60–3.67 (m, 2H,
CH2), 1.36 (t, 3H, CH3, J = 7.2 Hz); IR (KBr): 3243, 2971, 2361, 2338,
1607, 1489, 1442, 1413, 1386, 1359, 1332, 1290, 1204, 1169,
1127, 1091, 1059, 993, 975, 873, 823, 806, 756 cm– 1
.
920, 831, 750 cm– 1
.
i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2007, 340, 258–263
Hypoxic Cytotoxicity
263
(final: 0.25 mg/mL) for another 4 h of incubation. After these
4 h, 100 lL of DMSO was added to each well and optical density
(OD) was read at 570 nm. The IC50 values were calculated using
the PrismPad computer program (GraphPad Software, Inc., San
Diego, CA, USA) and were defined as concentration of drug caus-
ing 50% inhibition in absorbance compared with control (vehi-
cle) cells. All of the experiments were performed in triplicate
and the IC50 values were derived from the mean OD values of the
triplicate tests.
3-Cyclohexylamino-7-nitro-1,2,4-benzotriazine-1,4-
dioxide 66
Yield 39.4%; m.p. 178–1838C; MS (ESI): 306 [M+H]; 1H-NMR
(DMSO, 400 MHz) d: 8.88 (s, 1H, CH), 8.54 (d, 1H, CH, J = 9.8 Hz),
8.25 (d, 1H, CH, J = 9.8 Hz), 6.89 (s, 1H, NH), 3.81 (s, 1H, CH), 3.31l
3.48 (m, 2H, CH2), 1.85–1.88 (m, 2H, CH2), 1.74–1.78 (m, 2H,
CH2), 1.30–1.36 (m, 4H, CH2); IR (KBr): 3311, 3277, 3058, 2940,
2856, 1592, 1530, 1494, 1465, 1418, 1393, 1372, 1336, 1292,
1159, 1135, 1104, 1084, 1022, 962, 942, 924, 891, 841, 749 cm– 1
.
3-Phenylamino-7-nitro-1,2,4-benzotriazine-1,4-dioxide 67
Yield 19.1%; m.p. 257–2618C; MS (ESI): 300 [M+H]; 1H-
NMR(DMSO, 400 MHz) d: 8.85 (s, 1H, CH), 8.57 (d, 1H, CH, J =
11.6 Hz), 8.30 (d, 1H, CH, J = 11.6 Hz), 7.59–7.87 (m, 5H, CH), 7.22
(s, 1H, NH); IR (KBr): 3411, 3278, 3106, 3062, 1866, 1744, 1632,
1604, 1535, 1510, 1489, 1472, 1413, 1394, 1361, 1341, 1265,
References
[1] M. H. Torre, D. Gambino, J. Araujo, H. Cerecetto, et al.,
Eur. J. Med. Chem. 2005, 40, 473–480.
1166, 1099, 938, 913, 854, 815, 747 cm– 1
.
[2] M. P. Hay, S. A. Gamage, M. S. Kovacs, F. B. Pruijn, et al., J.
Med. Chem. 2003, 46, 169–182.
[3] M. P. Hay, F. B. Pruijn, S. A. Gamage, H. D. S. Liyanage, et
al., J. Med. Chem. 2004, 47, 475–488.
3-Benzylamino-7-nitro-1,2,4-benzotriazine-1,4-dioxide 68
Yield 31.9%; m.p. 276–2818C; MS (ESI): 314 [M+H]; 1H-NMR
(DMSO, 400 MHz) d: 8.83 (s, 1H, CH), 8.64 (d, 1H, CH, J = 11.6 Hz),
8.27 (d, 1H, CH, J = 11.6 Hz), 7.43–7.75 (m, 5H, CH), 7.10 (s, 1H,
NH), 4.69 (s, 2H, CH2); IR (KBr): 3404, 3274, 3110, 3055, 2921,
2851, 1632, 1602, 1536, 1489, 1471, 1412, 1385, 1361, 1338,
[4] W. A. Denny, Eur. J. Med. Chem. 2001, 36, 577–595.
[5] J. M. Brown, L. H. Wang, Anti–Cancer Drug Des. 1998, 13,
529–539.
[6] S. K. Williamson, J. J. Crowley, P. N. Lara Jr, J. McCoy, et
al., J. Clin. Oncol. 2005, 23, 9097–9104.
1261, 1165, 1099, 1026, 941, 912, 840, 813, 747 cm– 1
.
[7] K. O. Hicks, F. B. Pruijn, J. R. Sturman, W. A. Denny, W. R.
Cell culture and cytotoxicity assay
Wilson, Cancer Res. 2003, 63, 5970–5977.
All cancer cells, promyelocytic leukemia HL-60, androgen-inde-
pendent prostate tumor PC-3, hepatocellular carcinoma BEL-
7402, human esophagus tumor ECA-109, and human breast can-
cer MCF-7, were cultured in RPMI-1640 medium with heat-inacti-
vated 10% fetal bovine serum in hypoxic atmosphere with 3%
O2, 5% CO2 and in normoxic atmosphere with 20% O2, 5% CO2 at
378C. All the compounds were dissolved in DMSO at the concen-
trations 1.0 mg/mL and were then diluted to the appropriate
concentrations. Cells were seeded in 96-well microtiter plates
(4000 cells/well). After 24-hour incubation in appropriate med-
ium, cells were treated with various concentrations (0, 0.2, 1.0,
5.0, 25.0, 50.0 lM) of all tested compounds, and incubated in
normoxia and in hypoxia for 72 h, respectively. Afterwards,
10 lL of stock 3-[4,5-dimethylthia-zol-2-yl]-2,5-diphenyltetrazo-
lium bromide (MTT, Sigma) solution was added to each well
[8] J. M. Brown, W. R. Wilson, Nat. Rev. Cancer 2004, 4, 437–
447.
[9] F. Q. Jiang, B. Yang, L. L. Fan, Q. J. He, Y. Z. Hu, Bioorg.
Med. Chem. Lett. 2006, 16, 4209–4213.
[10] F. J. Wolf, R. M. Wilson, K. Pfister, M. Tishler, J. Am. Chem.
Soc. 1954, 76, 4611–4613.
[11] R. F. Robbins, K. Schofield, J. Chem. Soc. 1957, 3186–3194.
[12] B. Yang, R. C. Patrick, Clin. Cancer Res. 2005, 11, 2774–
2780.
[13] W. O. Foye, J. M. Kauffman, Y.-H. Kim, J. Heterocyclic Chem.
1982, 19, 497–501.
[14] J. Jiu, G. P. Mueller, J. Org. Chem. 1959, 24, 813–318.
i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com