Synthesis, hypoxia-selective cytotoxicity of new 3-amino-1,2,4- benzotriazine-1,4-dioxide derivatives

Article abstract of DOI:10.1016/j.ejmech.2011.01.007

We reported the synthesis, hypoxic cytotoxic activities and selectivities of 18 new 3-(alkoxymethylamino)-1,2,4-benzotriazine 1,4-dioxides. The synthesized compounds were screened in vitro against 5 cell lines: K562, SMMC-7721, A549, PC-3 and KB in hypoxia and in normoxia. Some of them showed higher or similar cytotoxic activity when compared to tirapazamine. Physico-chemical study showed the positive correlation between hypoxic activity and lipophilicity within a certain range. Preliminary mechanism study on the potent derivatives 4b, 4l and 4m indicated that the cytotoxic activities of these compounds might be mediated by inducing apoptosis.

Full text of DOI:10.1016/j.ejmech.2011.01.007

European Journal of Medicinal Chemistry 46 (2011) 919e926
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, hypoxia-selective cytotoxicity of new 3-amino-1,2,4-benzotriazine-1,
4-dioxide derivatives
,
Qing Xia a, Ling Zhang a, Jun Zhang b, Rong Sheng a, Bo Yang b, Qiaojun He b, Yongzhou Hu a
*
a ZJU-ENS Joint Laboratory of Medicinal Chemistry, College of Pharmaceutical Sciences, Zhejiang University, Zijingang Campus, Hangzhou 310058, China
b Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
a r t i c l e i n f o
a b s t r a c t
Article history:
We reported the synthesis, hypoxic cytotoxic activities and selectivities of 18 new 3-(alkoxymethyl-
amino)-1,2,4-benzotriazine 1,4-dioxides. The synthesized compounds were screened in vitro against 5
cell lines: K562, SMMC-7721, A549, PC-3 and KB in hypoxia and in normoxia. Some of them showed
higher or similar cytotoxic activity when compared to tirapazamine. Physico-chemical study showed the
positive correlation between hypoxic activity and lipophilicity within a certain range. Preliminary
mechanism study on the potent derivatives 4b, 4l and 4m indicated that the cytotoxic activities of these
compounds might be mediated by inducing apoptosis.
Received 19 October 2010
Received in revised form
2 December 2010
Accepted 7 January 2011
Available online 15 January 2011
Keywords:
Ó 2011 Elsevier Masson SAS. All rights reserved.
3-Amino-1,2,4-benzotriazine-1,4-dioxide
derivatives
Synthesis
Hypoxia
Antitumor activity
1. Introduction
molecules that targeting the hypoxic fraction of a solid tumor for
hypoxic regions are far away from blood vasculatures. Although
Hypoxia, formed by chaotic growth and an inefficient micro-
vasculature system within the tumor [1], not only drives tumor
metabolism, progression, invasion, and metastasis [2e6], but also
plays a negative role in the treatment of human tumors because of
their resistance to radiotherapy and chemotherapy through various
mechanisms [7e10]. Thus, tumor hypoxia gives an opportunity for
the development of bioreductive agents, which can be selectively
activated within hypoxic tissues [11e13].
A range of chemical classes has been explored since this concept
was first proposed: N-oxides, quinones, nitro-aromatics, and metal
complexes [14]. Tirapazamine (TPZ), a benzotriazine 1,4-dioxide, is
one of such tumor-selective prodrug which undergoes enzymic
one-electron reduction to form a DNA damaging species, which
may affect DNA strand breaks [15e17]. TPZ has been studied
extensively in vitro and in vivo and has already demonstrated
significant activity in a wide range of clinical trials in combination
with radiotherapies and cisplatin-based chemotherapies [18e20].
Physico-chemical property, associating closely with the pene-
tration of anticancer drugs [21], is one of the primary concerns for
being a potent hypoxic antitumor compound, the ability of TPZ to
kill hypoxic cells in tumors is generally limited by its poor extra-
vascular transport [22,23]. Moreover, TPZ displays significant
toxicities in the clinical setting [18,19]. Therefore, many efforts are
currently involved in developing new analogues with higher bio-
logical activity and/or lower systemic toxicity. Hay’s group had
made a lot of contribution in this field. They applied the spatially
resolved pharmacokinetic/pharmacodynamic (PK/PD) model to
guide drug synthesis and identified several benzotriazine 1,4-
dioxides with improved extravascular transport (EVT) and hypoxic
activity [24,25].
In the previous work of our team, some lipophilic groups were
introduced into the C-3 amino group to improve the activity of
benzotriazine 1,4-dioxides. Most of the compounds showed more
potent activity than TPZ [26,27]. The structureeactivity relation-
ships demonstrated that when the compound had an electron-
donating substituent such as methoxy group on the 7-position of
benzotriazine ring, it showed higher hypoxic selectivity than that of
an electron-withdrawing substituent [27]. In order to find new
molecules possessing cytotoxic activity and hypoxic selectivity,
a series of 3-(alkoxymethylamino)-1,2,4-benzotriazine 1,4-dioxides
were synthesized and evaluated for their cytotoxicity and selec-
tivity in vitro.
* Corresponding author: Tel./fax: þ86 571 88208460.
E-mail address: huyz@zju.edu.cn (Y. Hu).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.01.007

920
Q. Xia et al. / European Journal of Medicinal Chemistry 46 (2011) 919e926
cell line SMMC-7721, lung cancer cell line A549, human prostate
cancer cell line PC-3 and human epidermoid tumor cell line KB
under normoxic and hypoxic conditions. TPZ was employed as
a positive control. The results are summarized in Table 1.
As shown in Table 1, some tested benzotriazine 1,4-dioxide
derivatives showed higher or similar cytotoxic activity and selec-
tivity in comparison with TPZ against the tested cancer cell lines
except KB cell line. The substituent on the 3-position or 7-position
of benzotriazine ring had significant impact on activity because of
the disparity on the electronic and lipophilic properties of the
molecule. Obviously, compounds (e.g. 4ie4n) with an aromatic
group at 3-position side chain terminal displayed more potent
cytotoxic activity than those with a 3-alkyl side chain (e.g. 4ae4h)
against most tested cell lines. On the other hand, introduction of
methoxy group into 7-position of benzotriazine ring improved both
cytotoxicity and selectivity. For example, the cytotoxic activity and
selectivity in hypoxia against K562 cell line of 4q was 7.5 fold and
114 fold better than those of TPZ, respectively.
Comparing the cytotoxic activity of 4i and 4k with that of 4j, 4m
and 4n suggested that an electron-withdrawing group on benzene
ring of 3-position side chain increased cytotoxicity against most
tested cell lines, particularly for SMMC-7721 and K562. These 3-
(alkoxymethylamino)-1,2,4- benzotriazine 1,4-dioxide compounds
with oxygen atom at the 3-position side chain shown more favor-
able hypoxic selectivity compared with previous reported 3-amino-
1,2,4-benzotriazine 1,4-dioxide compounds [26,27].
Scheme 1. The synthetic route of compounds 4aer. Reagents and conditions:
(a) NH2CN, cHCl, 100 ꢀC; (b) 30% aq. NaOH, 100 ꢀC; (c) R2OH, (HCHO)n, CH3COOH,
85 ꢀC; (d) MCPBA, NaHCO3, DCM, 20 ꢀC.
2. Results and discussion
2.1. Chemistry
The synthetic route of benzotriazine 1,4-dioxide analogues 4aer
is shown in Scheme 1. Substituted nitroanilines 1aeb were con-
verted to 3-amino-1,2,4-benzotriazine 1-oxides 2aeb using the
method of Mason and Tennant [28]. Treatment of 2aeb with
paraformaldehyde and alcohol derivatives in the presence of cata-
lytic amount of acetic acid at 85 ꢀC for 3 h resulted in 3-(alkox-
ymethylamino)-1,2,4 -benzotriazine 1-oxides 3aer in high yields
(70e90%). Oxidation of 3aer with MCPBA at room temperature in
dichloromethane gave target compounds 4aer. The structures of all
the newly synthesized compounds were determined by IR, 1H NMR,
13C NMR and mass spectrum.
2.2.2. Mechanism studies
To gain preliminary insight into the mechanisms of action of
these compounds, 4b, 4l and 4m were further assayed for their
effect on cell cycle progression and proteins expression. The results
are presented as follows.
2.2. Pharmacology
2.2.1. In vitro cytotoxic activities
DNA fragmentation is a common feature of apoptotic cell death.
Propidium iodide staining for Sub-G1 content analysis is used to
characterize the apoptosis process. As shown in Fig. 1(A), after
All the synthesized compounds were evaluated for their cyto-
toxic activities in vitro against human cancer cell lines including
chronic myeloid leukemia cell line K562, hepatocellular carcinoma
Table 1
Cytotoxicity of TPZ derivatives against five cancer cell lines in hypoxia and in normoxia.
O
R1
N
N
R2
O
N
O
N
H
Compound
R1
R2
Cytotoxicity (IC50, mM)
SMMC-7721
K562
H
KB
H
A549
H
PC3
H
Ha
Nb
N
N
N
N
TPZ
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
e
4.75
2.48
4.23
5.53
3.99
3.05
2.86
4.17
3.09
4.79
0.81
2.47
1.15
0.78
0.52
1.07
0.55
1.19
0.74
32.79
6.67
1.81
3.79
4.99
3.89
3.24
3.3
5.85
4.88
6.17
4.69
1.85
2.24
0.66
1.24
0.65
3.52
0.45
0.24
0.25
19.41
7.8
18.71
9.07
6.29
1.58
2.80
1.95
1.75
1.22
3.04
4.39
2.52
5.72
1.30
2.51
4.06
0.39
0.58
3.20
2.81
2.40
0.22
1.93
2.77
4.09
3.74
2.32
2.90
2.11
1.20
3.49
4.14
2.19
2.81
3.32
4.27
3.44
2.54
2.51
4.47
0.22
7.43
1.17
4.27
7.4
20.97
7.51
15.9
14.2
14.67
9.2
11.06
3.25
1.21
13.41
5.76
6.08
14.49
7.65
3.07
6.57
3.63
8.58
4.14
CH3
C2H5
n-C3H7
n-C4H9
7.45
10.52
7.46
6.31
6.46
7.27
5.18
2.61
10.75
1.47
2.71
7.64
8.42
2.89
2.81
5.04
2.50
3.13
27.12
19.52
21.09
16.98
15.87
28.15
7.03
20.12
14.5
12.9
15.42
17.61
17.63
14.53
13.11
10.78
2.97
16.11
3.26
8.72
20.1
18.3
5.65
4.28
7.11
27.52
6.69
22.54
10.21
15.24
39.54
5.21
22.08
13.99
12.26
8.26
7.4
6.01
8.51
7.52
6.67
0.39
11.86
5.1
10.67
52.76
9.31
>100
10.61
30.9
88.52
>100
n-C5H11
iso-C5H11
Cyclohexyl
n-C7H15
CH2-C6H5
CH2-C6H4-4-Cl
CH(CH3)-C6H5
CH2-C6H4-4-OMe
CH2-C6H4-4-CF3
CH2-C6H4-3-NO2
n-C3H7
14.01
14.21
19.8
21.96
9.45
8.54
16.6
OMe
OMe
OMe
OMe
2.88
11.99
16.95
18.12
12.41
n-C5H11
CH2-C6H5
CH(CH3)-C6H5
12.86
21.39
8.04
a
H ¼ hypoxia: the percentage of oxygen is 3%.
N ¼ normoxia: the percentage of oxygen is 20%.
b

Q. Xia et al. / European Journal of Medicinal Chemistry 46 (2011) 919e926
921
Fig. 1. (A) K562 cells were incubated in normoxia and in hypoxia, and were treated with 4m, 4b, 4l (10 mM) for 48 h. After treatment, cells were harvested and detected of apoptosis
by flow cytometry using PI apoptosis detection kit. (B) K562 cells were harvested after the same treatment as A, cell extract were collected and immunoblotted with procaspase-3,
cleaved caspase-3, PARP and XIAP antibodies.
treatment with 4b, 4l, 4m (10.0
m
M) for 48 h in normoxia and in
retention behavior of chemical substances in HPLC could be used in
QSAR studies to replace the hydrophobicity parameter of drugs [29].
In order to investigate the relationship between lipophilicity
and hypoxic activity, retention times of the 3-amino-1,2,4-benzo-
triazine-1,4-dioxide derivatives was measured by an RP-HPLC. And
RI values of tested compounds were calculated by an equation
hypoxia, a characteristic cleavage of DNA was observed in K562
cells. The percentages of apoptotic cells induced by 4m, 4b and 4l
were 9.50%, 14.13% and 14.95% in normoxia, respectively, while that
in hypoxia were 32.11%, 33.07% and 54.24%, respectively. Sponta-
neous apoptosis of the control was seen in 5.27% of cells in nor-
moxia and 13.03% in hypoxia. These results suggested that these
compounds serve as potential hypoxic-selective compounds,
mainly owing to the undergoing of apoptosis.
Table 2
Retention index (RI) of tirapazamine derivatives and their cytotoxicity against K562
cell line.
In most cases, the activation of caspase cascade accompanies
with the apoptosis. The expression of procaspase-3, cleaved cas-
pase-3, XIAP and PARP were measured in K562 cells treated with
Compound RI
IC50
HCR Compound. RI
IC50
HCR
(K562)
(K562)
compound 4m, 4b, 4l (10.0
and 4l decrease the protein levels of procaspase-3, XIAP and PARP,
and induce the cleavage of caspase-3 in hypoxia.
m
M, 48 h). As shown in Fig. 1(B), 4m, 4b
4a
4b
4c
4d
4e
4f
4g
4h
4i
392.4 3.79
432.4 4.99
2.06 4j
3.09 4k
4.53 4l
5.44 4m
4.40 4n
2.24 4o
2.21 4p
0.48 4q
3.43 4r
737.8 1.85
720.5 2.24
1.76
3.89
30.45
16.21
8.69
440.8 3.89
481.2 3.24
690.5 3.3
740.6 5.85
738.3 4.88
760.1 6.17
716.9 4.69
603.7 0.66
632.3 1.24
658.1 0.65
430.2 3.52
647.9 0.45
585.2 0.24
628.4 0.25
2.3. Physico-chemical characteristics
1.22
15.80
114.67
26.76
Lipophilicity of the drug molecules plays an important role in
relation to their biological activity. Baker et al. reported that the

922
Q. Xia et al. / European Journal of Medicinal Chemistry 46 (2011) 919e926
Fig. 2. The correlation between cytotoxicity or HCR of tirapazamine derivatives and their RI values.
based on the retention times of the alkan-2-ones used. The results
are summarized in Table 2 and Fig. 2.
H-7), 6.08 (br s, 1H, NeH), 5.03 (d, 2H, J ¼ 7.6 Hz, CH2), 3.44 (s, 3H,
CH3); MS (ESI): m/s ¼ 207 [M þ 1]þ.
As shown in Fig. 2, hypoxic activities increased with the lip-
ophilicity of compounds in the range from 380 to 580 in terms of RI
values while negative correlation was shown when the RI values exc-
eeded 580, indicating that introducing appropriate lipophilic groups
enhanced cytotoxicity. On the other hand, compounds with the RI
values in the range from 580 to 650 showed better hypoxic selectivity.
4.1.3. 3-(Ethoxymethylamino)-1,2,4-benzotriazine 1-oxide (3b)
Yellow solid (74.4% yield); IR (KBr, cmꢁ1) 3309, 3059, 2970, 2915,
1567, 1495, 1406, 1316, 1064, 1006, 763, 645; 1H NMR (400 MHz,
CDCl3)
d
8.28 (d, 1H, J ¼ 8.0 Hz, H-8), 7.74 (td, 1H, J ¼ 8.0, 1.6 Hz, H-
6), 7.67 (d, 1H, J ¼ 8.0 Hz, H-5), 7.35 (td, 1H, J ¼ 8.0, 1.6 Hz, H-7), 6.17
(br s, 1H, NeH), 5.07 (d, 2H, J ¼ 7.2 Hz, CH2), 3.65 (q, 2H, J ¼ 6.8 Hz,
CH2), 1.24 (t, 3H, J ¼ 6.8 Hz, CH3); MS (ESI): m/s ¼ 221 [M þ 1]þ.
3. Conclusions
In summary, a new series of 3-(alkoxymethylamino)-1,2,4-ben-
zotriazine 1,4-dioxides 4aer have been synthesized and evaluated
for their hypoxic cytotoxicity and selectivity. Some compounds
showed higher or similar cytotoxic activity compared to TPZ in
hypoxia, particularly for SMMC-7721 and K562. Substituents on the
benzotriazine ring or at the 3-position affected the activity, as
introduction of a methoxy group at 7-position improved both
cytotoxicity and selectivity. And compounds with aromatic group at
3-position side chain terminal were more active than corresponding
compounds with alkyl groups. Physico-chemical study showed the
positive correlation between hypoxic activity and lipophilicity
within a certain range. Mechanism study using the potent deriva-
tives 4b, 4l and 4m revealed that the cytotoxic activities of these
compounds might be mediated by inducing apoptosis.
4.1.4. 3-(Propoxymethylamino)-1,2,4-benzotriazine 1-oxide (3c)
Yellow solid (73.8% yield); IR (KBr, cmꢁ1) 3314, 3060, 2928, 2871,
1697, 1565, 1493, 1411, 1317, 1069, 941, 758, 658, 591; 1H NMR
(400 MHz, CDCl3)
d
8.28 (d, 1H, J ¼ 8.8 Hz, H-8), 7.74 (t, 1H,
J ¼ 7.6 Hz, H-6), 7.67 (d, 1H, J ¼ 8.0 Hz, H-5), 7.36 (t, 1H, J ¼ 7.8 Hz, H-
7), 6.36 (br s, 1H, NeH), 5.07 (d, 2H, J ¼ 6.8 Hz, CH2), 3.54 (t, 2H,
J ¼ 6.6 Hz, CH2), 1.63 (m, 2H, CH2), 0.91 (t, 3H, J ¼ 7.4 Hz, CH3); MS
(ESI): m/s ¼ 235 [M þ 1]þ.
4.1.5. 3-(Butoxymethylamino)-1,2,4-benzotriazine 1-oxide (3d)
Yellow solid (88.0% yield); IR (KBr, cmꢁ1) 3311, 3063, 2945, 2865,
1564, 1491, 1415, 1357, 1237, 1074, 757, 644; 1H NMR (400 MHz,
CDCl3)
d
8.28 (d,1H, J ¼ 8.0 Hz, H-8), 7.73 (t,1H, J ¼ 8.0 Hz, H-6), 7.67
(d, 1H, J ¼ 8.0 Hz, H-5), 7.35 (t, 1H, J ¼ 8.0 Hz, H-7), 6.38 (br s, 1H,
NeH), 5.07 (d, 2H, J ¼ 7.2 Hz, CH2), 3.58 (t, 2H, J ¼ 6.8 Hz, CH2), 1.57
(m, 2H, CH2), 1.35 (m, 2H, CH2), 0.89 (t, 3H, J ¼ 7.2 Hz, CH3); MS
(ESI): m/s ¼ 249 [M þ 1]þ.
4. Experimental protocols
4.1. Synthesis
4.1.6. 3-(Pentyloxymethylamino)-1,2,4-benzotriazine 1-oxide (3e)
Yellow solid (79.8% yield); IR (KBr, cmꢁ1) 3304, 3060, 2925,
2860, 1563, 1492, 1413, 1319, 1236, 1075, 760, 647; 1H NMR
General: melting points were obtained on a B-540 Büchi melting-
point apparatus and are uncorrected. IR spectra were performed on
a Brüker VECTOR 22 FTIR spectrophotometer in KBr pellets
(400e4000 cmꢁ1). 1H NMR spectra were recorded on a Brüker AM
400 instrument at 400 MHz (chemical shifts are expressed as
(400 MHz, CDCl3)
d
8.27 (d, 1H, J ¼ 8.0 Hz, H-8), 7.72 (t, 1H,
J ¼ 7.6 Hz, H-6), 7.66 (d, 1H, J ¼ 8.0 Hz, H-5), 7.34 (t,1H, J ¼ 7.8 Hz, H-
7), 6.29 (t, 1H, J ¼ 6.8 Hz, NeH), 5.07 (d, 2H, J ¼ 6.8 Hz, CH2), 3.57 (t,
2H, J ¼ 6.6 Hz, CH2), 1.59 (m, 2H, CH2), 1.29 (m, 4H, CH2 and CH2),
0.86 (t, 3H, J ¼ 6.8 Hz, CH3); MS (ESI): m/s ¼ 263 [M þ 1]þ.
d
values relative toTMS as internal standard). Mass spectra (MS), ESI
(positive) were recorded on an Esquire-LC-00075 spectrometer.
4.1.1. General procedure for the synthesis of 3-
(Alkoxymethylamino)-1,2,4-benzotriazine 1-oxides 3aer
4.1.7. 3-(iso-Pentyloxymethylamino)-1,2,4-benzotriazine 1-oxide
(3f)
A mixture of substituted 3-amino-1,2,4-benzotriazine 1-oxides
2aeb (1.1 mmol), paraformaldehyde (4.7 mmol) and corresponding
alcohols (4 mL) was stirred at 85 ꢀC for 3 h in the presence of
catalytic amount of acetic acid. Then the solvent was removed under
vacuum and the residue was purified by silica gel column chroma-
tography (EtOAc/petrolemnether,1:2) to give pure compounds3aer.
Yellow solid (83.3% yield); IR (KBr, cmꢁ1) 3307, 3060, 2917, 2866,
1565, 1492, 1413, 1358, 1319, 1237, 1076, 763, 659; 1H NMR
(400 MHz, CDCl3)
d
8.29 (d, 1H, J ¼ 8.0 Hz, H-8), 7.74 (t, 1H,
J ¼ 7.6 Hz, H-6), 7.69 (d, 1H, J ¼ 8.0 Hz, H-5), 7.36 (t,1H, J ¼ 7.8 Hz, H-
7), 5.93 (t, 1H, J ¼ 6.8 Hz, NeH), 5.06 (d, 2H, J ¼ 7.2 Hz, CH2), 3.61 (t,
2H, J ¼ 6.8 Hz, CH2), 1.69 (m, 1H, CH), 1.49 (m, 2H, CH2), 0.88 (d, 6H,
J ¼ 6.8 Hz, CH3 and CH3); MS (ESI): m/s ¼ 263 [M þ 1]þ.
4.1.2. 3-(Methoxymethylamino)-1,2,4-benzotriazine 1-oxide (3a)
Yellow solid (70.6% yield); IR (KBr, cmꢁ1) 3323, 3059, 2938,
2827, 1562, 1497, 1420, 1112, 1060, 902, 761, 643, 578; 1H NMR
4.1.8. 3-(Cyclohexyloxymethylamino)-1,2,4-benzotriazine 1-oxide (3g)
Yellow solid (76.3% yield); IR (KBr, cmꢁ1) 3308, 3056, 2926, 2851,
1564, 1496, 1410, 1313, 1240, 1061, 763, 661; 1H NMR (400 MHz,
(400 MHz, CDCl3)
1.6 Hz, H-6), 7.69 (d, 1H, J ¼ 8.0 Hz, H-5), 7.37 (td, 1H, J ¼ 8.0, 1.6 Hz,
d
8.30 (d, 1H, J ¼ 8.0 Hz, H-8), 7.75 (td, 1H, J ¼ 8.0,
CDCl3)
d
8.28 (d, 1H, J ¼ 8.8 Hz, H-8), 7.74 (t, 1H, J ¼ 7.6 Hz, H-6), 7.66

Q. Xia et al. / European Journal of Medicinal Chemistry 46 (2011) 919e926
923
(d, 1H, J ¼ 8.0 Hz, H-5), 7.36 (t, 1H, J ¼ 8.0 Hz, H-7), 6.02 (t, 1H,
J ¼ 6.6 Hz, NeH), 5.11 (d, 2H, J ¼ 7.2 Hz, CH2), 3.56 (m, 1H, CH), 1.95
(m, 2H, CH2), 1.74 (m, 2H, CH2), 1.35 (m, 6H, CH2, CH2 and CH2); MS
(ESI): m/s ¼ 275 [M þ 1]þ.
(400 MHz, DMSO-d6)
d
8.75 (t,1H, J ¼ 6.4 Hz, NeH), 8.17 (m, 2H, Ar),
8.10 (dd, 1H, J ¼ 8.0, 1.2 Hz, H-8), 7.84 (td, 1H, J ¼ 8.0, 1.2 Hz, H-6),
7.79 (d, 1H, J ¼ 8.0 Hz, H-5), 7.62 (m, 2H, Ar), 7.43 (td, 1H,
J ¼ 8.0,1.6 Hz, H-7), 4.98 (d, 2H, J ¼ 6.8 Hz, CH2), 4.73 (s, 2H, CH2);
MS (ESI): m/s ¼ 328 [M þ 1]þ.
4.1.9. 3-(Heptyloxymethylamino)-1,2,4-benzotriazine 1-oxide (3h)
Yellow solid (76.2% yield); IR (KBr, cmꢁ1) 3304, 3060, 2923,
2854, 1563, 1493, 1414, 1360, 1320, 1238, 1098, 761, 657; 1H NMR
4.1.16. 3-(Propoxymethylamino)-7-methoxy-1,2,4-benzotriazine 1-
oxide (3o)
(400 MHz, CDCl3)
d
8.28 (dd, 1H, J ¼ 8.6, 1.2 Hz, H-8), 7.73 (td, 1H,
Yellow solid (89.7% yield); IR (KBr, cmꢁ1) 3330, 3104, 2945, 2864,
J ¼ 7.6, 1.6 Hz, H-6), 7.66 (d, 1H, J ¼ 7.6 Hz, H-5), 7.35 (td, 1H, J ¼ 7.6,
1.2 Hz, H-7), 6.11 (t, 1H, J ¼ 6.8 Hz, NeH), 5.05 (d, 2H, J ¼ 7.2 Hz,
CH2), 3.57 (t, 2H, J ¼ 6.4 Hz, CH2), 1.58 (m, 2H, CH2), 1.29 (m, 8H,
4CH2), 0.86 (m, 3H, CH3); MS (ESI): m/s ¼ 291 [M þ 1]þ.
1564,1507, 1395,1253,1171,1069, 1023, 844, 759, 646, 580; 1H NMR
(400 MHz, CDCl3)
d
7.63 (m, 2H, H-8 and H-5), 7.45 (dd, 1H, J ¼ 8.8,
2.4 Hz, H-6), 5.88 (t,1H, J ¼ 6.8 Hz, NeH), 5.07 (d, 2H, J ¼ 7.2 Hz, CH2),
3.95 (s, 3H, CH3), 3.56 (t, 2H, J ¼ 6.8 Hz, CH2), 1.65 (m, 2H, CH2), 0.94
(t, 3H, J ¼ 7.4 Hz, CH3); MS (ESI): m/s ¼ 265 [M þ 1]þ.
4.1.10. 3-(Benzyloxymethylamino)-1,2,4-benzotriazine 1-oxide (3i)
Yellow solid (90.3% yield); IR (KBr, cmꢁ1) 3298, 3057, 2931, 2856,
1561, 1494, 1413, 1320, 1073, 955, 766, 725, 659, 653, 460; 1H NMR
4.1.17. 3-(Pentyloxymethylamino)-7-methoxy-1,2,4-benzotriazine
1-oxide (3p)
(400 MHz, CDCl3)
d
8.29 (d, 1H, J ¼ 8.0 Hz, H-8), 7.75 (td, 1H, J ¼ 8.0,
Yellow solid (84.1% yield); IR (KBr, cmꢁ1) 3338, 2926, 2858,1565,
1.6 Hz, H-6), 7.69 (d, 1H, J ¼ 8.0 Hz, H-5), 7.32 (m, 6H, H-7 and Ar),
6.19 (t, 1H, J ¼ 6.4 Hz, NeH), 5.14 (d, 2H, J ¼ 7.6 Hz, CH2), 4.69 (d, 2H,
J ¼ 8.8 Hz, CH2); MS (ESI): m/s ¼ 283 [M þ 1]þ.
1507, 1394, 1256, 1175, 1076, 1032, 942, 844, 758, 644, 568; 1H NMR
(400 MHz, CDCl3)
d
7.60 (m, 2H, H-8 and H-5), 7.42 (dd, 1H, J ¼ 8.8,
2.8 Hz, H-6), 6.02 (t, 1H, J ¼ 6.4 Hz, NeH), 5.07 (d, 2H, J ¼ 6.8 Hz,
CH2), 3.92 (s, 3H, CH3), 3.56 (t, 2H, J ¼ 6.8 Hz, CH2), 1.59 (m, 2H,
CH2), 1.31 (m, 4H, CH2 and CH2), 0.86 (t, 3H, J ¼ 7.0 Hz, CH3); MS
(ESI): m/s ¼ 293 [M þ 1]þ.
4.1.11. 3-((4-Chlorobenzyloxy)methylamino)-1,2,4-benzotriazine 1-
oxide (3j)
Yellow solid (77.7% yield); IR (KBr, cmꢁ1) 3320, 3053, 2926, 2857,
1696, 1564, 1493, 1413, 1318, 1075, 950, 794, 640, 586; 1H NMR
4.1.18. 3-(Benzyloxymethylamino)-7-methoxy-1,2,4-benzotriazine
1-oxide (3q)
(400 MHz, CDCl3)
d
8.29 (dd, 1H, J ¼ 8.0, 0.8 Hz, H-8), 7.76 (td, 1H,
J ¼ 8.0, 1.6 Hz, H-6), 7.67 (d, 1H, J ¼ 8.0 Hz, H-5), 7.38 (td, 1H, J ¼ 8.0,
1.6 Hz, H-7), 7.31 (m, 4H, Ar), 6.14 (t, 1H, J ¼ 7.2 Hz, NeH), 5.13 (d,
2H, J ¼ 7.6 Hz, CH2), 4.64 (s, 2H, CH2); MS (ESI): m/s ¼ 317 [M þ 1]þ.
Yellow solid (84.5% yield); IR (KBr, cmꢁ1) 3311, 3054, 2935, 2848,
1568,1504,1393,1310,1264,1124,1058, 843, 735, 688, 575; 1H NMR
(400 MHz, CDCl3)
d
7.60 (m, 2H, H-8 and H-5), 7.43 (dd, 1H, J ¼ 9.6,
2.4 Hz, H-6), 7.32 (m, 5H, AreH), 5.88 (t, 1H, J ¼ 7.2 Hz, NeH), 5.11
4.1.12. 3-((1-Phenylethoxy)methylamino)-1,2,4-benzotriazine 1-
oxide (3k)
(d, 2H, J ¼ 7.2 Hz, CH2), 4.67(s, 2H, CH2), 3.92 (s, 3H, CH3); 13C NMR
(125 MHz, CDCl3)
d 158.35, 158.04, 149.56, 145.06, 131.85, 129.40,
Yellow solid (79.8% yield); IR (KBr, cmꢁ1) 3319, 3063, 2985, 2929,
128.69, 128.45, 128.12, 120.10, 98.22, 71.99, 70.50, 56.38; MS (ESI):
2865, 1567, 1492, 1419, 1375, 1318, 1238, 1041, 760, 685; 1H NMR
m/s ¼ 313 [M þ 1]þ.
(400 MHz, CDCl3)
d
8.26 (d, 1H, J ¼ 8.8 Hz, H-8), 7.73 (td, 1H, J ¼ 7.6,
0.8 Hz, H-6), 7.63 (d, 1H, J ¼ 8.8 Hz, H-5), 7.30 (m, 6H, H-7 and Ar),
6.25 (t,1H, J ¼ 7.0 Hz, NeH), 4.99 (m, 2H, CH2), 4.74 (q,1H, J ¼ 6.5 Hz,
CH), 1.45 (d, 3H, J ¼ 6.8 Hz, CH3); MS (ESI): m/s ¼ 297 [M þ 1]þ.
4.1.19. 3-((1-Phenylethoxy)methylamino)-7-methoxy-1,2,4-
benzotriazine 1-oxide (3r)
Yellow solid (83.7% yield); IR (KBr, cmꢁ1) 3308, 3050, 2933,
2846, 1571, 1502, 1379, 1305, 1257, 1121, 1053, 837, 729, 686, 561; 1H
4.1.13. 3-((4-Methoxybenzyloxy)methylamino)-1,2,4-benzotriazine
1-oxide (3l)
NMR (400 MHz, CDCl3) d 7.58 (m, 2H, H-8 and H-5), 7.43 (dd, 1H,
J ¼ 9.2, 2.8 Hz, H-7), 7.31 (m, 5H, Ar), 5.81 (t, 1H, J ¼ 6.8 Hz, NeH),
4.98 (m, 2H, CH2), 4.73 (q, 1H, J ¼ 6.4 Hz, CH), 3.93 (s, 3H, CH3), 1.46
(d, 3H, J ¼ 6.4 Hz, CH3); MS (ESI): m/s ¼ 327 [M þ 1]þ.
Yellow solid (81.6% yield); IR (KBr, cmꢁ1) 3332, 3027, 2930, 2868,
1561, 1505, 1413, 1317, 1245, 1071, 954, 814, 764, 644; 1H NMR
(400 MHz, CDCl3)
d
8.29 (dd, 1H, J ¼ 8.0, 0.8 Hz, H-8), 7.74 (td, 1H,
J ¼ 8.0, 1.6 Hz, H-6), 7.69 (d, 1H, J ¼ 8.0 Hz, H-5), 7.37 (td, 1H, J ¼ 8.0,
1.2 Hz, H-7), 7.29 (m, 2H, Ar), 6.88 (m, 2H, Ar), 6.06 (t, 1H, J ¼ 6.6 Hz,
NeH), 5.11 (d, 2H, J ¼ 6.8 Hz, CH2), 4.61 (d, 2H, J ¼ 5.6 Hz, CH2), 3.80
4.1.20. General procedure for the synthesis of 3-
(Alkoxymethylamino)-1,2,4-benzotriazine 1,4-dioxides 4aer
A solution of MCPBA (0.30 mmol) in DCM (2 mL) was added
dropwise to a stirred solution of 3-(alkoxymethylamino)-1,2,4-
benzotriazine 1-oxides 3aer (0.22 mmol) in DCM (4 mL) and
NaHCO3 (0.44 mmol), and the mixture was stirred at room
temperature for 6 h. The suspension was filtered through Celite, the
solvent was evaporated, and the residue was purified by chroma-
tography (EtOAc/DCM, 3:1), to give 3-(alkoxymethylamino)-1,2,4-
benzotriazine 1,4-dioxides 4aer (about 50%) as a red solid, and the
starting material 3aer (about 40%) which could be recycled.
(d, 3H, J ¼ 6.8 Hz, CH3); 13C NMR (125 MHz, CDCl3)
d 159.50, 158.97,
148.77, 135.97, 131.63, 130.16, 129.76, 127.17, 126.07, 120.58, 114.02,
71.60, 70.22, 55.52; MS (ESI): m/s ¼ 313 [M þ 1]þ.
4.1.14. 3-((4-(Trifluoromethyl)benzyloxy)methylamino)-1,2,4-
benzotriazine 1-oxide (3m)
Yellow solid (81.6% yield); IR (KBr, cmꢁ1) 3297, 3065, 2930, 2858,
1564, 1497, 1416, 1327, 1064, 1105, 1072, 953, 821, 763, 659; 1H NMR
(400 MHz, DMSO-d6)
d
8.75 (t, 1H, J ¼ 5.8 Hz, NeH), 8.18 (d, 1H,
J ¼ 8.4 Hz, H-8), 7.85 (t,1H, J ¼ 7.6 Hz, H-5), 7.66 (m, 3H, H-6 and Ar),
7.56 (d, 2H, J ¼ 8.0 Hz, Ar), 7.43 (t, 1H, J ¼ 7.8 Hz, H-7), 4.96 (d, 2H,
J ¼ 6.4 Hz, CH2), 4.69 (s, 2H, CH2); MS (ESI): m/s ¼ 351 [M þ 1]þ.
4.1.21. 3-(Methoxymethylamino)-1,2,4-benzotriazine 1,4-dioxide
(4a)
Red solid (50.1% yield), mp 177e180 ꢀC; IR (KBr, cmꢁ1) 3248,
3075, 2946, 2837, 1598, 1491, 1403, 1353, 1116, 1080, 951, 902, 857,
4.1.15. 3-((3-Nitrobenzyloxy)methylamino)-1,2,4-benzotriazine 1-
oxide (3n)
767, 660, 558; 1H NMR (400 MHz, CDCl3)
d 8.38 (m, 2H, H-8 and H-
5), 7.93 (t, 1H, J ¼ 7.4 Hz, H-6), 7.82 (br s, 1H, NeH), 7.60 (t, 1H,
J ¼ 7.8 Hz, H-7), 5.08 (d, 2H, J ¼ 5.6 Hz, CH2), 3.44 (s, 3H, CH3); MS
(ESI): m/s ¼ 223 [M þ 1]þ.
Yellow solid (81.6% yield); IR (KBr, cmꢁ1) 3324, 3091, 2941, 2835,
1564, 1527, 1412, 1347, 1121, 1065, 943, 809, 761, 664; 1H NMR

924
Q. Xia et al. / European Journal of Medicinal Chemistry 46 (2011) 919e926
4.1.22. 3-(Ethoxymethylamino)-1,2,4-benzotriazine 1,4-dioxide (4b)
Red solid (59.7% yield), mp 162e166 ꢀC; IR (KBr, cmꢁ1) 3231,
3094, 2974, 2859, 1591, 1492, 1417, 1339, 1177, 1110, 1061, 948, 767,
1165, 1110, 1091, 1063, 947, 863, 761, 720, 645; 1H NMR (400 MHz,
CDCl3)
d
8.35 (m, 2H, H-8 and H-5), 7.92 (t, 1H, J ¼ 7.6 Hz, H-6), 7.88
(br s, 1H, NeH), 7.56 (t, 1H, J ¼ 8.0 Hz, H-7), 5.09 (d, 2H, J ¼ 6.4 Hz,
CH2), 3.56 (t, 2H, J ¼ 6.4 Hz, CH2), 1.56 (m, 2H, CH2), 1.28 (m, 8H,
653, 510; 1H NMR (500 MHz, CDCl3)
d 8.34 (m, 2H, H-8 and H-5),
7.91 (t, 1H, J ¼ 7.6 Hz, H-6), 7.84 (br s, 1H, NeH), 7.57 (t, 1H,
J ¼ 7.8 Hz, H-7), 5.09 (d, 2H, J ¼ 6.0 Hz, CH2), 3.63 (q, 2H, J ¼ 7.2 Hz,
CH2), 1.20 (t, 3H, J ¼ 7.2 Hz, CH3); 13C NMR (125 MHz, CDCl3)
4CH2), 0.85 (m, 3H, CH3); MS (ESI): m/s ¼ 307 [M þ 1] þ
.
4.1.29. 3-(Benzyloxymethylamino)-1,2,4-benzotriazine 1,4-dioxide
(4i)
d
149.92, 138.59, 136.17, 131.52, 128.17, 121.96, 117.99, 71.99, 64.51,
15.28; MS (ESI): m/s ¼ 237 [M þ 1]þ.
Red solid (53.4% yield), mp 158e162 ꢀC; IR (KBr, cmꢁ1) 3342,
2925, 2857, 1601, 1491, 1409, 1347, 1172, 1079, 962, 854, 723; 1H
4.1.23. 3-(Propoxymethylamino)-1,2,4-benzotriazine 1,4-dioxide
(4c)
NMR (400 MHz, DMSO-d6)
d
9.20 (t, 1H, J ¼ 6.8 Hz, NeH), 8.24 (d,
1H, J ¼ 9.2 Hz, H-8), 8.19 (d, 1H, J ¼ 9.2 Hz, H-5), 7.97 (td, 1H, J ¼ 8.0,
1.2 Hz, H-6), 7.63 (td, 1H, J ¼ 8.0, 0.8 Hz, H-7), 7.32 (m, 5H, Ar), 4.95
(d, 2H, J ¼ 6.4 Hz, CH2), 4.59 (s, 2H, CH2); MS (ESI): m/s ¼ 299
[M þ 1]þ.
Red solid (58.2% yield), mp 160e163 ꢀC; IR (KBr, cmꢁ1) 3243,
3100, 2926, 2862, 1595, 1492, 1416, 1175, 1076, 946, 765, 653, 510;
1H NMR (400 MHz, CDCl3)
d 8.34 (m, 2H, H-8 and H-5), 7.89 (m, 2H,
H-6 and NeH), 7.55 (t, 1H, J ¼ 8.0 Hz, H-7), 5.08 (d, 2H, J ¼ 5.6 Hz,
CH2), 3.52 (t, 2H, J ¼ 6.8 Hz, CH2), 1.58 (m, 2H, CH2), 0.89 (t, 3H,
J ¼ 7.4 Hz, CH3); MS (ESI): m/s ¼ 251 [M þ 1]þ.
4.1.30. 3-((4-Chlorobenzyloxy)methylamino)-1,2,4-benzotriazine
1,4-dioxide (4j)
Red solid (49.3% yield), mp 196e198 ꢀC; IR (KBr, cmꢁ1) 3234,
3100, 2932, 2821, 1595, 1490, 1413, 1341, 1175, 1077, 953, 859, 767,
4.1.24. 3-(Butoxymethylamino)-1,2,4-benzotriazine 1,4-dioxide
(4d)
654, 560; 1H NMR (400 MHz, CDCl3)
d 8.36 (m, 2H, H-8 and H-5),
Red solid (58.2% yield), mp 158e160 ꢀC; IR (KBr, cmꢁ1) 3254,
3027, 2949, 2860, 1598, 1492, 1413, 1350, 1175, 1079, 944, 860, 766,
7.93 (t, 1H, J ¼ 7.6 Hz, H-6), 7.88 (br s, 1H, NeH), 7.59 (t, 1H,
J ¼ 8.0 Hz, H-7), 7.27 (s, 4H, Ar), 5.17 (d, 2H, J ¼ 6.4 Hz, CH2), 4.64 (s,
2H, CH2); MS (ESI): m/s ¼ 333 [M þ 1]þ.
721, 652; 1H NMR (400 MHz, CDCl3)
d 8.36 (m, 2H, H-8 and H-5),
7.91 (t, 1H, J ¼ 7.2 Hz, H-6), 7.72 (br s, 1H, NeH), 7.58 (t, 1H,
J ¼ 8.0 Hz, H-7), 5.10 (d, 2H, J ¼ 5.6 Hz, CH2), 3.58 (t, 2H, J ¼ 6.6 Hz,
CH2), 1.57 (m, 2H, CH2), 1.36 (m, 2H, CH2), 0.90 (t, 3H, J ¼ 7.4 Hz,
4.1.31. 3-((1-Phenylethoxy)methylamino)-1,2,4-benzotriazine 1,4-
dioxide (4k)
CH3); 13C NMR (125 MHz, CDCl3)
d
149.93, 138.59, 136.19, 131.50,
Red solid (55.4% yield), mp 149e152 ꢀC; IR (KBr, cmꢁ1) 3258,
3084, 2891,1590,1492,1418,1362,1332,1183,1166,1113,1069,1041,
128.14, 121.96, 117.98, 72.22, 68.91, 31.82, 19.47, 14.09; MS (ESI): m/
s ¼ 265 [M þ 1]þ.
952, 861, 764, 721, 700, 646; 1H NMR (400 MHz, CDCl3)
d 8.32 (m,
2H, H-8 and H-5), 7.89 (t, 1H, J ¼ 7.8 Hz, H-6), 7.67 (br s, 1H, NeH),
7.56 (t, 1H, J ¼ 7.8 Hz, H-7), 7.26 (m, 5H, Ar), 4.52 (m, 2H, CH2), 4.72
(q, 1H, J ¼ 6.8 Hz, CH), 1.45 (d, 3H, J ¼ 6.8 Hz, CH3); MS (ESI): m/
s ¼ 313 [M þ 1]þ.
4.1.25. 3-(Pentyloxymethylamino)-1,2,4-benzotriazine 1,4-dioxide
(4e)
Red solid (62.2% yield), mp 133e135 ꢀC; IR (KBr, cmꢁ1) 3254,
2955, 2932, 2857, 1594, 1493, 1418, 1361, 1181, 1111, 1081, 948, 864,
762, 721, 646; 1H NMR (400 MHz, CDCl3)
d
8.38 (m, 2H, H-8 and H-
4.1.32. 3-((4-Methoxybenzyloxy)methylamino)-1,2,4-benzotriazine
1-oxide 1,4-dioxide (4l)
5), 7.93 (t, 1H, J ¼ 7.6 Hz, H-6), 7.87 (br s, 1H, NeH), 7.59 (t, 1H,
J ¼ 8.0 Hz, H-7), 5.11 (d, 2H, J ¼ 5.6 Hz, CH2), 3.59 (t, 2H, J ¼ 6.4 Hz,
CH2), 1.60 (m, 2H, CH2), 1.32 (m, 4H, CH2 and CH2), 0.88 (t, 3H,
J ¼ 7.4 Hz, CH3); MS (ESI): m/s ¼ 279 [M þ 1]þ.
Red solid (52.8% yield), mp 174e177 ꢀC; IR (KBr, cmꢁ1) 3236,
3103, 2924, 2876, 1597, 1504, 1414, 1344, 1245, 1175, 1115, 1064, 953,
859, 766, 720, 654, 527; 1H NMR (500 MHz, CDCl3)
d 8.34 (m, 2H, H-
8 and H-5), 7.90 (m, 2H, H-6 and NeH), 7.57 (t, 1H, J ¼ 8.0 Hz, H-7),
7.26 (d, 2H, J ¼ 8.0 Hz, Ar), 6.83 (d, 2H, J ¼ 8.4 Hz, Ar), 5.14 (d, 2H,
J ¼ 7.0 Hz, CH2), 4.59 (s, 2H, CH2), 3.75 (s, 3H, CH3); 13C NMR
4.1.26. 3-(iso-Pentyloxymethylamino)-1,2,4-benzotriazine 1,4-
dioxide (4f)
Red solid (63.9% yield), mp 153e156 ꢀC; IR (KBr, cmꢁ1) 3250,
2950, 2933, 2864, 1593, 1493, 1417, 1360, 1335, 1181, 1166, 1109,
(125 MHz, CDCl3) d 159.59, 149.87, 138.56, 136.16, 131.54, 129.68,
129.45, 128.20, 121.93, 117.98, 114.08, 71.41, 70.65, 55.49; MS (ESI):
1077, 948, 764, 721, 659; 1H NMR (400 MHz, CDCl3)
d
8.37 (m, 2H,
m/s ¼ 329 [M þ 1]þ.
H-8 and H-5), 7.92 (t, 1H, J ¼ 7.2 Hz, H-6), 7.79 (br s, 1H, NeH), 7.58
(t, 1H, J ¼ 8.0 Hz, H-7), 5.10 (d, 2H, J ¼ 5.6 Hz, CH2), 3.61 (t, 2H,
J ¼ 6.8 Hz, CH2), 1.67 (m, 2H, CH2), 1.49 (m, 1H, CH), 0.89 (d, 6H,
J ¼ 6.4 Hz, CH3 and CH3); MS (ESI): m/s ¼ 279 [M þ 1]þ.
4.1.33. 3-((4-(Trifluoromethyl)benzyloxy)methylamino)-1,2,4-
benzotriazine 1,4-dioxide (4m)
Red solid (45.9% yield), mp 190e193 ꢀC; IR (KBr, cmꢁ1) 3257,
3107, 2931, 1594, 1494, 1416, 1359, 1333, 1184, 1163, 1112, 1067, 1018,
4.1.27. 3-(Cyclohexyloxymethylamino)-1,2,4-benzotriazine 1,4-
dioxide (4g)
952, 866, 824, 765, 722, 660; 1H NMR (400 MHz, DMSO-d6)
d 9.19 (t,
1H, J ¼ 7.0 Hz, NeH), 8.24 (d, 1H, J ¼ 8.4 Hz, H-8), 8.18 (d, 1H,
J ¼ 8.4 Hz, H-6), 7.98 (t, 1H, J ¼ 8.0 Hz, H-5), 7.65 (m, 3H, H-7 and
Ar), 7.55 (d, 2H, J ¼ 8.4 Hz, Ar), 4.98 (d, 2H, J ¼ 6.8 Hz, CH2), 4.70 (s,
Red solid (51.7% yield), mp 154e158 ꢀC; IR (KBr, cmꢁ1) 3258,
2928, 2849, 1592, 1493, 1418, 1362, 1335, 1182, 1166, 1110, 1065, 949,
862, 763, 720, 646; 1H NMR (400 MHz, CDCl3)
d
8.34 (m, 2H, H-8
2H, CH2); 13C NMR (125 MHz, DMSO-d6)
d 150.27, 143.99, 143.46,
and H-5), 7.90 (t,1H, J ¼ 7.8 Hz, H-6), 7.80 (br s,1H, NeH), 7.55 (t,1H,
J ¼ 8.0 Hz, H-7), 5.13 (d, 2H, J ¼ 6.0 Hz, CH2), 3.54 (m, 1H, CH), 1.91
(m, 2H, CH2), 1.72 (m, 2H, CH2), 1.30 (m, 6H, CH2, CH2 and CH2); MS
(ESI): m/s ¼ 291 [M þ 1]þ.
139.15, 136.30, 131.63, 128.64, 128.54, 125.71, 125.69, 121.84, 117.96,
71.84, 68.96; MS (ESI): m/s ¼ 367 [M þ 1] þ
.
4.1.34. 3-((3-Nitrobenzyloxy)methylamino)-1,2,4-benzotriazine
1,4-dioxide (4n)
4.1.28. 3-(Heptyloxymethylamino)-1,2,4-benzotriazine 1,4-dioxide
(4h)
Red solid (35.8% yield), mp 186e189 ꢀC; IR (KBr, cmꢁ1) 3241,
3101, 2932, 2825, 1593, 1527, 1415, 1350, 1176, 1077, 950, 732, 654,
Red solid (56.7% yield), mp 156e159 ꢀC; IR (KBr, cmꢁ1) 3256,
2956, 2926, 2853, 1593, 1546, 1492, 1460, 1418, 1360, 1335, 1181,
555; 1H NMR (400 MHz, DMSO-d6)
8.24 (d, 1H, J ¼ 8.4 Hz, H-8), 8.18 (m, 2H, H-5 and Ar), 8.09 (d, 1H,
d
9.22 (t, 1H, J ¼ 6.4 Hz, NeH),

Q. Xia et al. / European Journal of Medicinal Chemistry 46 (2011) 919e926
925
J ¼ 8.4 Hz, Ar), 7.98 (t, 1H, J ¼ 7.8 Hz, H-6), 7.80 (d, 1H, J ¼ 7.2 Hz, H-
7), 7.63 (m, 2H, Ar), 5.00 (d, 2H, J ¼ 6.4 Hz, CH2), 4.74(s, 2H, CH2); MS
(ESI): m/s ¼ 344 [M þ 1]þ.
well) for 24 h and subsequently treated with different concentra-
tions of all tested compounds under normoxia or hypoxia for 72 h,
respectively. Viable cells were determined using 3-(4,5-dime-
thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay kit (MTT,
Sigma) according to the manufacturer’s instructions. The concen-
tration of drug causing 50% inhibition in absorbance compared
with control cells (IC50) was calculated using the software of dos-
eeeffect analysis with microcomputers.
4.1.35. 3-(Propoxymethylamino)-7-methoxy-1,2,4-benzotriazine
1,4-dioxide (4o)
Red solid (53.6% yield), mp 149e153 ꢀC; IR (KBr, cmꢁ1) 3253,
3077, 2930, 2867, 1592, 1507, 1388, 1335, 1250, 1119, 1084, 969, 731,
1
633, 550; H NMR (400 MHz, CDCl3)
d
8.23 (d, 1H, J ¼ 9.6 Hz, H-8),
7.73 (t, 1H, J ¼ 6.8 Hz, NeH), 7.58 (d, 1H, J ¼ 2.4 Hz, H-5), 7.51 (dd,
1H, J ¼ 9.6, 2.8 Hz, H-6), 5.06 (d, 2H, J ¼ 6.8 Hz, CH2), 3.95 (s, 3H,
CH3), 3.52 (t, 2H, J ¼ 6.8 Hz, CH2), 1.58 (m, 2H, CH2), 0.88 (t, 3H,
4.2.2. Flow cytometry analysis [30]
DNA content was monitored using flow cytometry. After drug
exposure, K562 cells were harvested, washed with PBS, centrifuged,
and fixed with 75% ethanol at ꢁ20 ꢀC overnight. The fixed cells
J ¼ 7.2 Hz, CH3); 13C NMR (125 MHz, CDCl3)
d 159.71, 149.07, 134.62,
132.13, 129.49, 119.33, 99.31, 72.24, 70.69, 56.66, 23.04, 10.75; MS
were resuspended in 500.0 ml of PBS containing 100 mg/ml RNase
(ESI): m/s ¼ 281 [M þ 1]þ.
and incubated at 37 ꢀC for 30 min. After incubation, the cells were
stained with 10 g/ml propidium iodide (PI, Sigma, St. Louis, MO)
m
4.1.36. 3-(Pentyloxymethylamino)-7-methoxy-1,2,4-benzotriazine
1,4-dioxide (4p)
for 30 min at room temperature in the dark. Flow cytometry was
analyzed with a FACSCalibur flow cytometer (BD Biosciences, San
Jose, CA).
Red solid (62.0% yield), mp 133e135 ꢀC; IR (KBr, cmꢁ1) 3260,
3074, 2932, 2857, 1591, 1508, 1420, 1383, 1336, 1258, 1244, 1193,
1153, 1115, 1085, 1021, 973, 817, 797, 731, 629; 1H NMR (400 MHz,
4.2.3. Western blot analysis [30]
CDCl3)
d
8.24 (d, 1H, J ¼ 9.2 Hz, H-8), 7.69 (t, 1H, J ¼ 6.6 Hz, NeH),
After treatment, cells were collected and lysed in a lysis buffer
[150 mM NaCl, 50 mM TriseHCl pH 8.0, 2 mM ethylene glycol-bis
(b-aminoethyl ether), 2 mM EDTA, 25 mM NaF, 25 mM b-glycer-
ophos-phate, 0.2% Triton X-100, 0.3% Nonidet P-40, and 0.1 mM
phenylmethyl-sulfonyl fluoride]. Protein extracts were resolved by
8e15% SDS-PAGE and transferred to PVDF membranes, and
western blot analysis was carried out using specific primary anti-
bodies and then HRP-labeled secondary antibodies. The signals
were visualized using enhanced chemiluminescent (ECL) detection
reagents.
7.60 (d, 1H, J ¼ 2.8 Hz, H-5), 7.52 (dd, 1H, J ¼ 9.6, 2.4 Hz, H-6), 5.06
(d, 2H, J ¼ 7.2 Hz, CH2), 3.95 (s, 3H, CH3), 3.55 (t, 2H, J ¼ 6.6 Hz, CH2),
1.57 (m, 2H, CH2), 1.29 (m, 4H, CH2 and CH2), 0.86 (t, 3H, J ¼ 7.0 Hz,
CH3); MS (ESI): m/s ¼ 309 [M þ 1]þ.
4.1.37. 3-(Benzyloxymethylamino)-7-methoxy-1,2,4-benzotriazine
1,4-dioxide (4q)
Red solid (48.6% yield), mp 148e152 ꢀC; IR (KBr, cmꢁ1) 3256,
2939, 2821, 1604, 1504, 1383, 1328, 1259, 1112, 1015, 970, 835, 788,
691, 554; 1H NMR (400 MHz, CDCl3)
d
8.22 (d, 1H, J ¼ 9.2 Hz, H-8),
7.76 (t, 1H, J ¼ 6.8 Hz, NeH), 7.59 (d, 1H, J ¼ 2.4 Hz, H-5), 7.52 (dd,
1H, J ¼ 9.2, 2.4 Hz, H-6), 7.28 (m, 5H, Ar), 5.14 (d, 2H, J ¼ 6.8 Hz,
CH2), 4.66 (s, 2H, CH2), 3.95 (s, 3H, CH3); 13C NMR (125 MHz, CDCl3)
4.3. Retention index
4.3.1. Chromatographic conditions
d
159.82, 148.95, 137.50, 134.62, 132.26, 129.46, 128.70, 128.12,
The analysis was performed on a Diamonsil C18 column (4.6 mm
128.02, 119.38, 99.30, 71.74, 70.92, 56.67; MS (ESI): m/s ¼ 329
200 mm, 5
mm). The optimum separation in HPLC was carried out
[M þ 1]þ.
with a mobile phase composed of methanol:water (60:40, v/v) at
a flow-rate of 1.0 ml/min. The volume of sample injected was 20 ml,
the column temperature was set at 20 ꢀC, and the isocratic elution
condition was monitored at 280 or 350 nm, according to the UV
absorption of the sample.
4.1.38. 3-((1-Phenylethoxy)methylamino)-7-methoxy-1,2,4-
benzotriazine 1,4-dioxide (4r)
Red solid (46.7% yield), mp 156e159 ꢀC; IR (KBr, cmꢁ1) 3354,
3111, 2967, 2826, 1591, 1509, 1454, 1418, 1384, 1329, 1265, 1160,
1120, 1079, 1032, 1014, 956, 835, 793, 762, 728, 701, 614, 552; 1H
4.3.2. RI evaluation
NMR (400 MHz, CDCl3)
d
8.19 (d, J ¼ 9.6 Hz, 1H, H-8), 7.59 (d,
Each of the 3-amino-1,2,4-benzotriazine-1,4-dioxide derivatives
and alkan-2-ones was dissolved in methanol (0.1 mg/ml) and then
injected into the HPLC apparatus. The retention times of the 3-
amino-1,2,4-benzotriazine-1,4-dioxide derivatives and alkan-2-
ones were recorded. The capacity factors (Kx ) and RI values of the
samples were calculated using the equations as follows:
J ¼ 2.4 Hz,1H, H-5), 7.51 (m, 2H, H-6 and NeH), 7.30 (m, 4H, Ar), 7.18
(t, 1H, J ¼ 7.2 Hz, Ar), 4.99 (m, 2H, CH2), 4.71 (q, 1H, J ¼ 6.4 Hz, CH),
3.96 (s, 3H, CH3), 1.44 (d, 3H, J ¼ 6.4 Hz, CH3); MS (ESI): m/s ¼ 343
[M þ 1]þ.
0
4.2. Pharmacology
Kx0 ¼ ðtx ꢁ t0Þ=t0
Five human cancer cell lines (K562, SMMC-7721, A549, PC-3,
and KB) were purchased from Cell Bank of China Science Academy
(Shanghai, China). The above cells were cultured in RPMI-1640
(Invitrogen Corp., Carlsbad, CA) medium with heat-inactivated 10%
fetal bovine serum, penicillin (100 units/mL) and streptomycin
À
ÁꢀÀ
Á
RI ¼ 100N þ 100 logKx0 ꢁ logKn0
logKn0 þ1 ꢁ logKn0
where x ¼ 3-amino-1,2,4-benzotriazine-1,4-dioxide derivatives
measured, n ¼ alkan-2-one eluting immediately before x,
n þ 1 ¼ alkan-2-one eluting immediately after x and N ¼ carbon
number of alkan-2-one. The K0 values were calculated using the
retention time of NaNO2 as a dead time.
(100 mg/mL) and incubated in hypoxic atmosphere with 3% O2, 5%
CO2 and in normoxic atmosphere with 20% O2, 5% CO2 at 37 ꢀC.
4.2.1. Cytotoxicity assay [30]
The cytotoxic activity in vitro was measured using the MTT
assay. All the compounds were dissolved in DMSO at the concen-
trations 10.0 mg/mL and were then diluted to the appropriate
concentrations. Cells were plated in 96-well plates (5 ꢂ103 per
Acknowledgements
This study was financially supported by the National Key Tech
Project for Major Creation of New Drugs (NO. 2009ZX09501-003),

926
Q. Xia et al. / European Journal of Medicinal Chemistry 46 (2011) 919e926
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