Synthesis and hypoxic-cytotoxic activity of some 3-amino-1,2,4-benzotriazine-1,4-dioxide derivatives

Article abstract of DOI:10.1016/j.bmcl.2006.05.095

A series of 3-amino-1,2,4-benzotriazine-1,4-dioxide derivatives 1 have been synthesized and evaluated for their cytotoxic activity in vitro against human leukemia cell lines: Molt-4, K562, HL60, human liver cancer cell Hep-G2, human prostate cancer cell PC-3 in hypoxia. Most of the compounds showed more potent activity than TPZ. Compounds 1i and 1m displayed encouraging superior activity against Molt-4 and HL-60 cell lines. Three potential derivatives received the test of the activity in hypoxia and in normoxia against Molt-4 and HL-60 cell lines and showed obvious hypoxia selectivity. Further mechanism study revealed that the cytotoxic activities of compounds 1i and 1k in Molt-4 cells might be mediated by modulation of p53 protein expression and mitochondrial membrane potential (ΔΨ_m).

Full text of DOI:10.1016/j.bmcl.2006.05.095

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4209–4213
Synthesis and hypoxic–cytotoxic activity of some
3-amino-1,2,4-benzotriazine-1,4-dioxide derivatives
Faqin Jiang,a Bo Yang,b Lingling Fan,b Qiaojun Heb and Yongzhou Hua,*
aZJU-ENS Joint Laboratory of Medicinal Chemistry, School of Pharmaceutical Sciences, Zhejiang University,
Hubin campus, Hangzhou 310031, China
bDepartment of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Zhejiang University,
Hubin campus, Hangzhou 310031, China
Received 23 January 2006; revised 11 May 2006; accepted 29 May 2006
Available online 13 June 2006
Abstract—A series of 3-amino-1,2,4-benzotriazine-1,4-dioxide derivatives 1 have been synthesized and evaluated for their cytotoxic
activity in vitro against human leukemia cell lines: Molt-4, K562, HL60, human liver cancer cell Hep-G2, human prostate cancer cell
PC-3 in hypoxia. Most of the compounds showed more potent activity than TPZ. Compounds 1i and 1m displayed encouraging
superior activity against Molt-4 and HL-60 cell lines. Three potential derivatives received the test of the activity in hypoxia and
in normoxia against Molt-4 and HL-60 cell lines and showed obvious hypoxia selectivity. Further mechanism study revealed that
the cytotoxic activities of compounds 1i and 1k in Molt-4 cells might be mediated by modulation of p53 protein expression and
mitochondrial membrane potential (DWm).
Ó 2006 Elsevier Ltd. All rights reserved.
The hypoxic cells in solid tumor lead to their resistance
to radiotherapy and chemotherapy, as well as increasing
the probability of tumor metastases.1–6 However, tumor
hypoxia also provides a target for the therapeutic strat-
egies. One such strategy is to use bioreductive prodrugs
that are transformed after administration under hypoxic
conditions, either by metabolism or by spontaneous
chemical breakdown, to form pharmacologically active
species. There are four main classes of bioreductive
cells). And one of the most reliable methods for improv-
ing their diffusion ability is to increase its lipophilicity by
incorporating with a properly lipophilic group.17–20
In addition, many previous studies reported, for the
acidic atmosphere of cancer cells, the derivatives of
TPZ with basic group in 3-position such as amino group
and substituted amino group showed more activity and
hypoxia selectivity.18–20
prodrugs:
N-oxides,8–11 and metal complexes.12–15
nitroaromatics/heterocyclics7,
quinones,
In order to improve the lipophilicity of TPZ and find
some compounds with higher activity against tumor in
hypoxia, in this article, some alkyl and aromatic groups
were introduced into the amino group of 3-position in
TPZ. We synthesized 15 known and new compounds,21
and evaluated for their cytotoxic activity in vitro. Fur-
ther study on the mechanisms of cytotoxic activity in
hypoxia was also performed with two potent com-
pounds in Molt-4 cells.
Among the N-oxides, tirapazamine (3-amine-1,2,4-ben-
zotriazine-1,4-dioxide, SR4233, WIN 59075, TPZ) as
one of the most promising prodrugs is in phases II
and III clinical trials.16
Some studies indicate the diffusion ability of TPZ deriv-
atives can affect the cytotoxic activity (those with low
diffusion ability cannot diffuse relatively large distance
from functional blood vessels to reach hypoxic tumor
3-Amino-1,2,4-benzotriazine-1,4-dioxide derivatives 1
were prepared as shown in Scheme 1. Treatment of
o-nitroaniline 2 with triphosgene in toluene under reflux
for 3 h resulted in 2-nitrophenyl isocyanate 3. Reacting 3
with anhydrous ammonia gas provided 2-nitrophenyl-
urea 4. Cyclization of 4 in 30% aq NaOH afforded the
3-hydroxy-1,2,4-benzotriazine-1-oxide 5. Chlorination
Keywords: 3-Amino-1,2,4-benzotriazine-1,4-dioxide derivatives; Syn-
thesis; Cytotoxicity; Hypoxia.
*
Corresponding author. Tel.: +86 571 87217210; fax: +86 571
87217412; e-mail: huyz@zjuem.zju.edu.cn
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.05.095

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F. Jiang et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4209–4213
lines (Molt-4, K562, and HL60), human liver cancer cell
line (Hep-G2), and human prostate cancer cell line (PC-
3) in hypoxia, using TPZ as the reference drug accord-
ing to the reported methods.24 After 48 h treatment,
the cell growth rates were determined by MTT assay
and the IC50 values were calculated with LOGIT meth-
od. The results are summarized in Table 1.
Three potential compounds 1i, 1k, 1m and TPZ were
selected to test the cytotoxicity against Molt-4 and
HL-60 cell lines in normoxia or in hypoxia for 48 h.
The hypoxic cytotoxicity ratio (HCR), and the IC50 val-
ues in normoxia and in hypoxia, was calculated. The
results are presented in Table 2.
Scheme 1. The synthetic route of compounds 1a–o. Reagents and
conditions: (a) (COCl2)3, toluene, reflux, 3 h; (b) NH3; (c) NaOH; (d)
AcOH; (e) POCl3, reflux, 3 h; (f) R1–NH–R2, ethanol, reflux, 12 h; (g)
AcOH/H2O2, 50 °C, 24 h.
As shown in Table 1, most tested compounds exhibited
higher cytotoxic activities than TPZ in hypoxia, in par-
ticular for leukemia cell lines. Obviously, the cytotoxic
potency of 1a–o against these five cancer cell lines was
highly dependent on structures of the 3-alkylamino side
chains. When R1 was alkyl group or aromatic group and
R2 was hydrogen (e.g., 1a–1m), they showed lower IC50
values than TPZ for most of the tested cancer cell lines.
Among them, compound 1i, when R1 was substituted
aromatic group, showed 0.6 lM and 0.9 lM IC50 values
at the tested Molt-4 and HL-60 cell lines. Compound 1m
was the most potent cytotoxic compound against all of
the tested cancer cell lines, with IC50 values in the range
of 0.8–7.7 lM. It can be assumed that the nitro group on
the benzene ring was important for the cytotoxic activi-
ties. On the other hand, compounds 1a–1d, in particular
1c, with alkylamino in 3-position were less active than
of 5 with phosphorus oxychloride produced 3-chloro-
1,2,4-benzotriazine-1-oxide 6 according to the known
methods with minor modifications.22,23 Compound 6
was substituted with various primary or secondary
amines to yield the 3-amino-1,2,4-benzotriazine-1-oxide
7, which was oxidized by 30% hydrogen peroxide to af-
ford the desired 3-amino-1,2,4-benzotriazine-1,4-dioxide
1. The structures of compounds obtained were deter-
mined by IR, MS, NMR, and elemental analysis.
All the prepared compounds were evaluated for their
cytotoxic activity in vitro against human leukemia cell
Table 1. Cytotoxicity of the target compounds against five human cancer cell lines in vitro
Compound
R1
R2
Cytotoxicity (IC50, lM)a
Molt-4
HL-60
K562
Hep-G2
PC-3
TPZ
1a
H
–CH2CH3
H
H
H
H
H
H
4.6 0.3
3.3 0.8
4.9 1.5
3.0 1.0
2.6 1.7
1.9 1.0
7.0 2.6
9.0 4.2
7.2 2.8
25.8 6.0
10.7 1.3
3.6 1.2
5.2 0.8
4.5 2.3
4.7 0.5
11.2 3.4
3.7 0.6
6.6 1.0
19.1 2.2
13.5 0.7
17.4 8.8
>56
22.3 4.7
13.3 2.6
18.2 1.7
>56
1b
1c
–(CH2)7CH3–n
–CH2CH@CH2
–CH2CH2OH
1d
1e
23.2 3.1
13.3 0.7
22.4 5.3
15.9 4.8
1f
H
1.3 0.7
1.4 1.1
1.1 0.2
0.6 0.4
2.3 1.8
2.2 0.6
2.3 0.7
0.8 0.3
2.5 1.1
2.9 0.9
1.5 0.8
0.9 0.2
8.3 2.8
2.5 0.4
5.8 1.6
1.4 0.8
2.6 1.1
2.4 0.7
2.5 0.9
2.9 0.2
10.6 1.1
1.9 0.1
2.2 1.0
3.1 0.8
9.5 2.1
15.0 6.4
10.6 0.5
10.1 0.5
17.4 7.4
8.2 1.3
9.7 1.5
7.7 0.8
10.5 3.6
17.0 3.2
10.3 2.0
12.0 10.6
16.1 2.4
10.7 0.9
10.6 0.9
7.4 2.5
1g
1h
1i
H
H
H
1j
H
1k
1l
H
H
1m
H
1n
1o
–CH2CH3
–CH2CH3
>17.6
>16.1
>49
>60
>55
>47
>107
>95
>107
>95
a Each experiment was independently performed three times and expressed as means SD.

F. Jiang et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4209–4213
4211
Table 2. Cytotoxicity data of derivatives against Molt-4 and HL-60 cells in normoxia and in hypoxia
d
d
Compound
IC50 (Molt-4, lM)
IC50 (HL-60, lM)
Normoxiaa
15.8
Hypoxiab
HCRc
Normoxiaa
Hypoxiab
HCRc
TPZ
1i
3
4.6 0.3
0.6 0.4
2.2 0.6
0.8 0.3
3.43
8.67
3.68
6.25
16.1 3.4
6.8 1.3
6.3 2.4
6.0 2.1
7.0 2.6
0.9 0.2
2.5 0.4
1.4 0.8
2.31
7.56
2.53
4.29
5.2 1.4
8.1 3.0
5.0 1.7
1k
1m
a IC50 values in normoxia.
b IC50 values in hypoxia (3% O2).
c Hypoxic cytotoxicity ratio = IC50 (normoxia)/IC50 (hypoxia).
d Each experiment was independently performed three times and expressed as means SD.
corresponding 3-substituted phenylamino analogues 1e–
1m. When both of R1 and R2 were alkyl group, such as
1n and 1o it did lead to a loss of inhibitory activity.
These results suggest that 1i and 1m might be promising
cytotoxins in hypoxia and be worthy to be evaluated
intensively.
(1i, 1k) and TPZ as the positive drug in Molt-4 cells.
Molt-4 cells were treated with tested compounds for
12 h or 24 h, respectively, then the apoptosis, mitochon-
drial membrane potential (DWm), and p53 expression
were determined according to the reported methods.24
The results are shown in Figures 1–3. All experiments
were repeated three times.
Three potential compounds were more cytotoxic in
hypoxia than in normoxia against Molt-4 and HL-60
cells. The HCR of these compounds against Molt-4
and HL-60 cell lines ranged from 3 to 9 (Table 2).
Molt-4 cells were cultured in complete medium with
2.0 lM TPZ, 1i, and 1k for 24 h in 3% O2 condition.
As shown in Figure 1, after 24 h, TPZ, 1i, and 1k in-
duced apoptosis in 11%, 45% and 33%, respectively,
while spontaneous apoptosis of the control (without
drug) was 3%. With the same concentration, apoptotic
Further study on the mechanisms of cytotoxic activity
in hypoxia was performed with two potent compounds
Figure 1. TPZ, 1k and 1i induced apoptosis in Molt-4 cells. Molt-4 cells were treated with 2.0 lM TPZ, 1k, and 1i in hypoxia. Without (A)/with 1i
(B), 1k (C) or TPZ (D) for 24 h. Apoptosis was assessed by propidium iodide staining of lysed cell nuclei. The DNA content of 20,000 events was
analyzed per test by flow cytometry. Apo, apoptosis. Two additional experiments yielded equivalent results.

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F. Jiang et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4209–4213
treated with 2.0 lM TPZ, 1i, and 1k for 12 h in hypoxia
were determined. Loss of DWm could cause the release of
cytochrome C and other factors that trigger apoptosis.25
With JC-1 staining, mitochondria depolarization is spe-
cifically indicated by a fluorescence emission shift from
red to green (and the yellow fluorescence is intermediate
state). DWm in Molt-4 cells was reduced by TPZ, 1i, and
1k after 12 h treatment and the quantitated data from
flow cytometry after JC-1 staining are shown in Figure
2, comparing with control, Molt-4 cells treated with 1i,
1k, and TPZ exhibited a mass of yellow and green fluo-
rescence, and 1i and 1k showed the stronger ability in
DWm decline than TPZ at the same concentration level
(P < 0.05–0.01), suggesting that 1i and 1k might possess
more potent cytotoxic activity through mitochondrial
pathway.
P53 is involved in cell apoptosis and DNA damage. The
expression of p53 in Molt-4 cells treated with TPZ, li, or
1k was performed by Western blot analysis. As shown in
Figure 3, 2.0 lM TPZ, 1i, and 1k increased p53 level in
Molt-4 cells after 24 h of exposure in hypoxia. Accord-
ing to the data we have obtained, we confirmed that 1i
and 1k exerted higher cytotoxicity, and the activity
was related to the p53-mediated apoptosis pathway.
In summary, a series of 3-amino-1,2,4-benzotriazine-1,4-
dioxide derivatives, 1a–o, have been synthesized and
evaluated for their cytotoxic activities in hypoxia. Most
tested compounds had higher cytotoxic activities than
TPZ in vitro, and their cytotoxic potency was highly
dependent on structures of the 3-alkylamino side chains.
Three tested compounds showed higher hypoxic selec-
tivity against Molt-4 and HL-60 cell lines. Mechanism
study revealed that the cytotoxic activities of these com-
pounds might be mediated by modulation of p53 protein
expression and mitochondrial membrane potential
(DWm).
Figure 2. TPZ, 1i, and 1k induced DWm loss in Molt-4 cells treated
with 2.0 lM TPZ, 1i, and 1k for 12 h in hypoxia. Without (A)/with 1i
(B), 1k (C) or TPZ (D). DWm loss was assessed by JC-1 staining in
which mitochondria depolarization is indicated by an increase in the
green to red fluorescence intensity ratio. The quantitated data from
flow cytometry are represented as a histogram (E). Values represent
means SD for three separate experiments performed in triplicate.
**, P < 0.01, *, P < 0.05 versus TPZ group.
Acknowledgment
Financial support of this research by Science and Tech-
nology Department of Zhejiang province (2006c23002)
is gratefully acknowledged by the authors.
P53
Supplementary data
-Tubulin
Supplementary data associated with this article can be
found in the online version at doi:10.1016/j.bmcl.
2006.05.095.
Control
TPZ
1k
1i
Figure 3. Induction of p53 overexpression in Molt-4 cells treated with
2.0 lM TPZ, 1i, and 1k for 24 h in hypoxia was observed. a-Tublin
was shown as a loading control. Two additional experiments yielded
equivalent results.
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