Design, synthesis, and antitumor activities of some novel substituted 1,2,3-benzotriazines

Article abstract of DOI:10.3390/molecules13061427

A series of novel substituted 1,2,3-benzotriazines based on the structures of vatalanib succinate (PTK787) and vandetanib (ZD6474) were designed and synthesized. The antiproliferative effects of these compounds were tested on microvascular endothelial cel

Full text of DOI:10.3390/molecules13061427

Molecules 2008, 13, 1427-1440; DOI: 10.3390/molecules13061427
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.org/molecules
Article
Design, Synthesis, and Antitumor Activities of Some Novel
Substituted 1,2,3-Benzotriazines
Jin-Ling Lv 1, Rui Wang 1, Dan Liu 1, Gang Guo 1, Yong-Kui Jing 2 and Lin-Xiang Zhao 1,*
1
Shenyang Pharmaceutical University, Shenyang, 110016, P.R. China; E-mails:
jinlinglv12@yahoo.com.cn (Lv); ruiwang36@yahoo.com.cn (Wang); sammyld@163.com(Liu);
guogang19761126@sina.com (Guo)
2
Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA;
E-mail: yongkui.jing@mssm.edu
* Author to whom correspondence should be addressed. E-mail: zhaolinxiang@syphu.edu.cn; Tel.:
(+86)24-23986420; Fax: (+86)24-23986420.
Received: 5 June 2008; in revised form: 22 June 2008 / Accepted: 23 June 2008 / Published: 24 June
2008
Abstract: A series of novel substituted 1,2,3-benzotriazines based on the structures of
vatalanib succinate (PTK787) and vandetanib (ZD6474) were designed and synthesized.
The antiproliferative effects of these compounds were tested on microvascular endothelial
cells (MVECs) using the MTT assay. Introduction of a methoxy and a 3-chloropropoxy
group into the 1,2,3-benzotriazines increased the antiproliferative effects. 4-(3-Chloro-4-
fluoroanilino)-7-(3-chloropropoxy)-6-methoxy-1,2,3-benzotriazine (8m) was the most
effective compound. It was 4-10 fold more potent than PTK787 in inhibiting the growth of
T47D breast cancer cells, DU145 and PC-3 prostate cancer cells, LL/2 murine Lewis lung
cancer cells and B16F0 melanoma cells.
Keywords: 1,2,3-Benzotriazine, synthesis, antiproliferative activity, vatalanib succinate.

Molecules 2008, 13
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Introduction
Vascular endothelial growth factor (VEGF) plays an important role in both physiological and
pathological angiogenesis by binding to its receptor, VEGFR [1]. The expression of VEGFR is
increased in the majority of cancers and is associated with survival, migration and invasion of solid
tumor cells [1]. Elevated VEGF levels have been found to increase resistance to chemotherapy [2, 3]
and VEGF signaling has been used as a therapeutic target for the treatment of cancer. VEGFR
inhibitors such as vatalanib succinate (PTK787/ZK222584) and vandetanib (ZD6474) have been used
in clinical trials in several types of cancer [4-7]. Although both agents showed antitumor activities
alone, combinations with chemotherapeutic agents seem to be required for reaching therapeutic effects
[8-12]. To improve the antiproliferative abilities of PTK787 and ZD6474 (Figure 1), we have analyzed
the structures of PTK787 and ZD6474. PTK787 contains 2,3-benzodizine and ZD6474 contains 1,3-
benzodizine. We thought that the replacement of the benzodizine with 1,2,3-benzotriazine might have
increased activities compared to PTK787. Thus, we designed and synthesized eighteen novel
compounds (8a-m) with a basic 1,2,3-benzotriazine scaffold and the introduction of a methoxy group
or a short alkoxy group. The antiproliferative effects of these compounds were determined in
microvascular endothelial cells (MVECs) using MTT assay and the antiproliferative effects of
compound 8m were found to be the most profound in inhibiting growth of MVECs. Therefore, it was
further investigated in T47D breast cancer cells, DU145 and PC-3 prostate cancer cells, LL/2 murine
Lewis lung cancer cells and B16F0 melanoma cells.
Figure 1. Structures of vatalanib succinate (PTK787) and vandetanib (ZD6474).
Cl
F
Br
HN
COOH
COOH
HN
N
N
O
O
N
N
N
N
PTK787
ZD6474
Results and Discussion
Design and synthesis of novel 1,2,3-benzotriazines
Recently, it was reported that 1,2,4-benzotriazines were inhibitors of Src and the binding model of
1,2,4-benzotriazines with Src showed the importance of the N1 and N2 atoms [13, 14]. We considered
that VEGFR and Src might react with substrates in a similar fashion as both of them are tyrosine
kinases. Both PTK787 and ZD6474 contain a substituted anilino group. ZD6474 contains a methoxy
group at the C6 position and a short side chain at the C7 position. We thought that these substituents

Molecules 2008, 13
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are required for the antitumor activity. Thus, we decided using 1,2,3-benzotriazine to replace the
phthalazine in PTK787 or the quinazoline in ZD6474 and to add a methoxy group at the C6 position
and a short alkoxy group at the C7 position of 1,2,3-benzotriazine. A series of target compounds, 8a-r,
were obtained and their structures are listed in Table 1.
Compounds 8a-r were prepared according to the synthetic route outlined in Scheme 1. 4-Cyano-2-
methoxyphenol (1) reacted with various alkyl halides to afford intermediates 2a-c in satisfactory
o
yields. Compounds 2a-c were selectively nitrated at 30 C to give nitro compounds 3a-c in 87-95%
yields. The reduction of the nitro compounds 3a-c to the corresponding amines 4a-c was catalyzed by
o
Pd/C [15]. Compounds 4a-c were diazotized and then coupled with substituted anilines at 0 C,
followed by chromatographic purification to afford triazenes 6b-r. Compound 6a was prepared by the
coupling of diazotized 2-aminobenzonitrile (5) and 4-chloroaniline. The cyclization of compounds 6a-
r in 70% ethanol formed intermediates 7a-r, which then rearranged to compounds 8a-r after refluxing
in acetic acid [16]. As intermediates 7a-r were unstable, they were used directly in the next step
without further purification.
Scheme 1. Synthetic route to the target compounds.
O
CN
O
CN
O
CN
O
CN
a
c
b
R1
NO2
HO
R1
R1
NH2
2a R1= CH3CH2O
2b R1=CH3(CH2)4O
2c R1=Cl(CH2)3O
3a R1= CH3CH2O
3b R1=CH3(CH2)4O
3c R1=Cl(CH2)3O
4a R1= CH3CH2O
4b R1=CH3(CH2)4O
4c R1=Cl(CH2)3O
1
R3
NH
N
HN
R3
R2
R1
CN
R2
R1
R2
R1
d
e
f
N
4a-c or 5
R3
N
N
N
N
N
N
N
H
5: 2-aminobenzonitrile
6a-r
7a-r
8a-r
Reagents and conditions: (a) alkyl halides, DMF, K2CO3, 37 oC; (b) HNO3, 30 oC; (c) Pd/C,
cyclohexene, ethanol, reflux; (d) NaNO2/HCl, 0 oC; substituted aniline, 0 oC; (e) 70% ethanol,
reflux; (f) AcOH, reflux.
Antiproliferative activities in MVECs
The antiproliferative effects of compounds 8a-r in MVECs were determined (Table 1) and the
structure-activity relationships were analyzed. The results revealed that compounds with a methoxy
group (R2) at the C6 position and an alkoxy group (R1) at the C7 position (8b-r) had an increased
activity compared to that of compound 8a which did not have substitutions at the C6 and C7 positions.
The compounds with a 3-chloropropoxy group (R1) at the C7 position (8h-r) were more active than
compounds with an ethoxy group (8b-d) or a pentyloxy group (8e-g).

Molecules 2008, 13
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By comparing the activities of compounds with a methoxy group at the C6 position and a 3-
chloropropoxy group at the C7 position, but with a different substituted group (R3) at the C4 anilino
group (8h-r), it revealed that a substitution at the C4 anilino increased the antiproliferative activity in
MVECs compared to compound 8r, which did not have a substitution at the C4 anilino group. The
electronic effect of a substituent on the anilino group did not influence the antiproliferative activities
(comparing compounds 8k-o). Compounds with two substituents at the C3’ and C4’ positions of the
C4 anilino group (8i, 8m, and 8p) were more active than compounds with two substituents at the C3’
and C5’ positions (8h) or compounds with one substituent (8j, 8k, 8l, 8n, 8o, 8q). Compound 8m was
the most potent one in inhibiting proliferation of MVECs with a GI50 value of 7.98 μM. The
compounds with substituents at the anilino group (compounds 8h-q) were more effective than PTK787
in inhibiting growth of MVECs after the introduction of a methoxy group at the C6 position and a 3-
chloropropoxy group at the C7 position.
Table 1. The structures of target compounds and their antiproliferative effects in MVECs.
Compounds
R1
R2
R3
4-Cl
GI50 (μM) a
>80
8a
H
H
8b
CH3CH2O
CH3CH2O
CH3CH2O
CH3(CH2)4O
CH3(CH2)4O
CH3(CH2)4O
Cl(CH2)3O
Cl(CH2)3O
Cl(CH2)3O
Cl(CH2)3O
Cl(CH2)3O
Cl(CH2)3O
Cl(CH2)3O
Cl(CH2)3O
Cl(CH2)3O
Cl(CH2)3O
Cl(CH2)3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
4-Cl
26.54±1.43
42.56±2.79
38.81±1.86
25.35±1.02
42.64±2.27
37.98±1.98
28.84±1.40
11.02±0.49
18.59±1.31
17.08±0.65
23.05±0.92
7.98±0.35
24.86±0.86
23.19±1.12
15.22±0.51
21.38±1.54
>80
8c
4-CH3
3-Cl, 4-F
4-Cl
8d
8e
8f
3-OCF3
4-OCF3
3,5-di-Cl
3,4-di-Cl
4-Cl
8g
8h
8i
8j
8k
4-CH3
4-F
8l
8m
3-Cl, 4-F
3-OCF3
4-OCF3
3-CF3, 4-F
3-CF3
8n
8o
8p
8q
8r
H
vatalanib succinate
38.15±2.07
(PTK787)
a. GI50 is the concentration that inhibits 50% of cell growth. The cells were treated with various
concentrations of the tested compounds for 4 days and cell growth inhibition was determined
using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data
shown are means ± SD of three independent experiments.

Molecules 2008, 13
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The antiproliferative effects of compound 8m were then tested in tumor cell lines. The
antiproliferative effects of compound 8m were determined in human T47 breast cancer cells, DU-145
and PC-3 prostate cancer cells, murine LL/2 Lewis lung cancer cells and B16F0 melanoma cells using
MTT assay (Table 2). Compound 8m was more effective than PTK787 to inhibit cell growth in all the
tested cell lines.
Table 2. Antiproliferative effects of compound 8m and PTK787 in several tumor cell lines.
GI50 (μM) a
Compounds
T47D
DU-145
4.96±0.43
41.28±2.47
PC-3
LL/2
B16F0
8m
5.04±0.32
36.32±1.88
6.91±0.30
3.79±0.19
5.19±0.22
18.71±1.67
PTK787
63.68±3.55 31.15±2.01
a. Cells were treated with various concentrations of the tested compounds for 4 days and the cell
growth inhibition was determined using the MTT assay. Data shown are means ± SD of three
independent experiments.
Conclusions
In summary, our data indicate that 1,2,3-benzotriazines substituted with a methoxy group at the C6
position and a 3-chloropropoxy group at the C7 position exhibit antiproliferative activities. Most of
these compounds are more effective than PTK787 in inhibiting proliferation of MVECs. Compound
8m is the most potent and exhibits greater anti-tumor cell growth activity activities than PTK787.
Since we did not stimulate the cell growth of MVECs and tumor cells with VEGF, the observed
antiproliferative effects of these compounds, including PTK787, may be through a pathway
independent of VEGFR inhibition. Because of the high similarity of 1,2,3-benzotriazine and 1,2,4-
benzotriazine, the increased antiproliferative effects of the designed compounds in tumor cells may be
due to the inhibition of Src, or other tyrosine kinases which is not inhibited by PTK787 [17]. The
effects of our novel 1,2,3-benzotriazines on the activities of VEGFRs, Src and other kinases are under
investigation.
Experimental
General
Reagents (analytical grade) were obtained from commercial suppliers and used without further
1
13
purification unless otherwise noted. H- and C-NMR spectra were recorded on a Bruker ARX-300
instrument with tetramethylsilane as the internal standard. IR spectra were recorded on a Bruker IR-
27G spectrometer. MS were determined on either Finnigan MAT/USA spectrometer (LC-MS).
Elemental analysis was determined on a Carlo-Erba 1106 Elemental analysis instrument (Carlo Erba,
Milan, Italy). The melting points were determined on an electrically heated X4 digital visual melting
point apparatus and were uncorrected. The structures of the synthesized compounds were characterized

Molecules 2008, 13
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by melting point (mp), infrared (IR) spectra, nuclear magnetic resonance (NMR) spectra and mass
spectral (MS) data.
General procedure for the synthesis of 4-substituted-3-methoxybenzonitriles 2a-c
A solution of 4-cyano-2-methoxyphenol (1, 1.00 g, 6.70 mmol) in anhydrous DMF (4.00 mL) was
stirred and cooled with a water bath. K2CO3 (1.39 g, 10.1 mmol) was added and the mixture was
stirred at 20 oC for 1 h. The corresponding alkyl halide (8.13 mmol) was added dropwise, the mixture
o
was stirred at room temperature overnight, then heated at 37 C for 6 h and finally poured into a
mixture of ice/H2O (100 mL). After stirring for 10 min, a precipitate was formed. It was filtered off,
washed with H2O, and air-dried to yield the 4-substituted-3-methoxybenzonitriles 2a-c as white solids.
o
1
4-Ethoxy-3-methoxybenzonitrile (2a): Yield: 91.8%; mp: 102-103 C. H-NMR (DMSO-d6) δ: 7.36
(2H, m, H-2, H-6), 7.04 (1H, s, H-5), 4.11 (2H, q, J = 6.9 Hz, CH3CH2O-), 3.81 (3H, s, -OCH3), 1.35
(3H, t, J = 6.9 Hz, CH3CH2O-); LC-MS: 178.1 (M+H)+; Anal. Calcd. for C10H11NO2: C 67.78, H 6.26,
N 7.90; Found: C 67.79, H 6.24, N 7.91.
o
1
3-Methoxy-4-pentyloxybenzonitrile (2b): Yield: 89.0%; mp: 55-56 C. H-NMR (DMSO-d6) δ: 7.37
(2H, m, H-2, H-6), 7.09 (1H, d, J = 8.1 Hz, H-5), 4.02 (2H, t, CH3(CH2)3CH2O-), 3.79 (3H, s, -OCH3),
1.71 (2H, m, CH3CH2CH2CH2CH2O-), 1.34 (4H, m, CH3CH2CH2CH2CH2O-), 0.88 (3H, t, J = 6.9 Hz,
CH3CH2CH2CH2CH2O-); LC-MS: 220.1 (M+H)+; Anal. Calcd. for C13H17NO2: C 71.21, H 7.81, N
6.39; Found: C 71.20, H 7.80, N 6.39.
1
4-(3-Chloropropoxy)-3-methoxybenzonitrile (2c): Yield: 87.2%. H-NMR (DMSO-d6) δ: 7.41 (2H, m,
H-2, H-6), 7.15 (1H, d, J = 8.8 Hz, H-5), 4.16 (2H, t, J = 6.0 Hz, ClCH2CH2CH2O-), 3.79 (5H, m, -
OCH3, ClCH2CH2CH2O-), 2.20 (2H, m, ClCH2CH2CH2O-); LC-MS: 226.1 (M+H)+; Anal. Calcd. for
C11H12ClNO2: C 58.54, H 5.36, N 6.21; Found: C 58.52, H 5.37, N 6.20.
General procedure for the synthesis of 4-substituted-5-methoxy-2-nitrobenzonitriles, 3a-c
A solution of 4-substituted-3-methoxybenzonitriles 2 (9.10 mmol) in nitric acid (10 mL) was heated
to 30 oC for 2 h, poured into ice-water (100 mL), filtered, and washed with water to afford compounds
3 as light yellow solids.
o
1
4-Ethoxy-5-methoxy-2-nitrobenzonitrile (3a): Yield: 88.1%; mp: 198-199 C. H-NMR (DMSO-d6) δ:
7.85 (1H, s, H-3), 7.70 (1H, s, H-6), 4.25 (2H, q, J = 6.9 Hz, CH3CH2O-), 3.97 (3H, s, -OCH3), 1.37
(3H, t, J = 6.9 Hz, CH3CH2O-); LC-MS: 223.1 (M+H)+; Anal. Calcd. for C10H10N2O4: C 54.05, H
4.54, N 12.61; Found: C 54.03, H 4.55, N 12.60.
5-Methoxy-2-nitro-4-pentyloxybenzonitrile (3b): Yield: 92.9%; mp: 136-137 oC. 1H-NMR (DMSO-d6)
δ: 7.89 (1H, s, H-3), 7.80 (1H, s, H-6), 4.16 (2H, t, J = 6.3 Hz, CH3CH2CH2CH2CH2O-), 3.95 (3H, s, -
OCH3), 1.72 (2H, m, CH3CH2CH2CH2CH2O-), 1.35 (4H, m, CH3CH2CH2CH2CH2O-), 0.89 (3H, t, J =

Molecules 2008, 13
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6.9 Hz, CH3CH2CH2CH2CH2O-); LC-MS: 265.1 (M+H)+; Anal. Calcd. for C13H16N2O4: C 59.08, H
6.10, N 10.60; Found: C 59.10, H 6.11, N 10.59.
o
1
4-(3-Chloropropoxy)-5-methoxy-2-nitrobenzonitrile (3c): Yield: 95.3%; mp: 133-134 C. H-NMR
(DMSO-d6) δ: 7.91 (1H, s, H-3), 7.71 (1H, s, H-6), 4.32 (2H, t, J = 6.0 Hz, ClCH2CH2CH2O-), 4.00
(3H, s, -OCH3), 3.78 (2H, t, J = 6.3 Hz, ClCH2CH2CH2O-), 2.23 (2H, m, ClCH2CH2CH2O-); LC-MS:
271.0 (M+H)+; Anal. Calcd. for C11H11ClN2O4: C 48.81, H 4.10, N 10.35; Found: C 48.82, H 4.11, N
10.34.
General procedure for the synthesis of 4-substituted-2-amino-5-methoxybenzonitriles, 4a-c
A mixture of compound 3 (3.69 mmol) and Pd/C (10%, 7.00 g) in anhydrous ethanol (10.0 mL) was
stirred and heated under reflux. Cyclohexene (2.30 mL, 22.6 mmol) was added dropwise. The mixture
was refluxed overnight, then cooled to 40 °C, filtered, and washed with ethanol. The filtrate was
concentrated to yield a solid. The crude product was suspended in ethanol, stirred at 40 °C for 30 min,
cooled to room temperature, filtered to produce pure compound 4 as a yellow solid.
1
2-Amino-4-ethoxy-5-methoxybenzonitrile (4a): Yield: 49.1%; mp: 135-136 °C. H-NMR (DMSO-d6)
δ: 6.87 (1H, s, H-6), 6.39 (1H, s, H-3), 5.58 (2H, s, NH2), 3.96 (2H, q, J = 6.9 Hz, CH3CH2O-), 3.65
(3H, s, -OCH3), 1.33 (3H, t, J = 6.9 Hz, CH3CH2O-); LC-MS: 193.1 (M+H)+; Anal. Calcd. for
C10H12N2O2: C 62.49, H 6.29, N 14.57; Found: C 62.50, H 6.30, N 14.58.
1
2-Amino-5-methoxy-4-pentyloxybenzonitrile (4b): Yield: 50.1%; mp: 136-137 °C. H-NMR (DMSO-
d6) δ: 6.85 (1H, s, H-6), 6.38 (1H, s, H-3), 5.57 (2H, s, NH2), 3.88 (2H, t, J = 6.3 Hz,
CH3CH2CH2CH2CH2O-), 3.63 (3H, s, -OCH3), 1.70 (2H, m, CH3CH2CH2CH2CH2O-), 1.34 (4H, m,
CH3CH2CH2CH2CH2O-), 0.88 (3H, t, J = 6.9 Hz, CH3CH2CH2CH2CH2O-); LC-MS: 235.1 (M+H)+;
Anal. Calcd. for C13H18N2O2: C 66.64, H 7.74, N 11.96; Found: C 66.62, H 7.73, N 11.97.
o
1
2-Amino-4-(3-chloropropoxy)-5-methoxybenzonitrile (4c): Yield: 47.2%; mp: 118-119 C. H-NMR
(DMSO-d6) δ: 6.89 (1H, s, H-6), 6.43 (1H, s, H-3), 5.62 (2H, s, NH2), 4.03 (2H, t, J = 6.1 Hz,
ClCH2CH2CH2O-), 3.77 (2H, t, J = 6.4 Hz, ClCH2CH2CH2O-), 3.65 (3H, s, -OCH3), 2.18 (2H, m,
ClCH2CH2CH2O-); LC-MS: 241.1 (M+H)+; Anal. Calcd. for C11H13ClN2O2: C 54.89, H 5.44, N 11.64;
Found: C 54.88, H 5.45, N 11.63.
General procedure for the synthesis of 7-alkoxy-6-methoxy-4-substituted-1,2,3-benzotriazines 8a-r
Compound 4 (or compound 5 when preparing 8a, 10.0 mmol) in 10N-hydrochloric acid (30.0 mL)
o
was cooled to 0 C and diazotized with sodium nitrite (0.71 g) in water (10.0 mL). The diazonium
o
solution was neutralized with excess of sodium acetate trihydrate and stirred for 2 h at 0 C with a
o
corresponding substituted aniline (10.0 mmol). The solution was kept overnight at 4 C, filtered, and
washed with water. The crude material was purified on a silica gel column with petroleum ether : ethyl
acetate (v/v) = 10:1 to yield a yellow solid 6. Compound 6 was boiled in 70% ethanol (25.0 mL) for 1

Molecules 2008, 13
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h, then the solution was evaporated under reduced pressure to dryness. Acetic acid (10.0 mL) was
added and the solution was refluxed for 2 h, cooled, poured into water (100 mL), filtered, and dried to
afford 7-alkoxyl-6-methoxy-4-substituted-1,2,3-benzotriazines 8.
o
4-(4-chloroanilino)-1,2,3-benzotriazine (8a): Yield: 68.9%; mp: 230 C. IR (KBr): 3266, 3086, 1621,
1
1559, 1493, 1424, 1147, 766 cm-1; H-NMR (DMSO-d6) δ: 10.02 (1H, s, NH), 8.62 (1H, d, J = 8.1
Hz), 8.23 (1H, d, J = 8.1 Hz), 8.13 (1H, t, J = 8.2 Hz), 8.05 (1H, m), 7.90 (2H, m), 7.52 (2H, m); LC-
MS: 257.1 (M+H)+; 13C-NMR (DMSO-d6) δ : 151.02, 143.53, 137.58, 134.73, 132.38, 128.66, 128.06,
127.47, 124.28, 121.87, 109.04; Anal. Calcd. for C13H9ClN4: C 60.83, H 3.53, N 21.83; Found: C
60.79, H 3.51, N 21.85.
o
4-(4-Chloroanilino)-7-ethoxy-6-methoxy-1,2,3-benzotriazine (8b): Yield: 72.1%; mp: 230 C. IR
(KBr): 3296, 2928, 1612, 1560, 1494, 1423, 1291, 818 cm-1; 1H-NMR (DMSO-d6) δ: 9.68 (1H, s, NH),
7.92 (3H, m, H-8, H-2’, H-6’), 7.51 (3H, m, H-8, H-3’, H-5’), 4.29 (2H, q, J = 6.6 Hz, CH3CH2O-),
4.03 (3H, s, -OCH3), 1.44 (3H, t, J = 6.9 Hz, CH3CH2O-); LC-MS: 331.1 (M+H)+; 13C-NMR (DMSO-
d6) δ: 154.22 (C-7), 153.27 (C-6), 150.12 (C-10), 141.65 (C-9), 137.95 (C-1’), 128.57, 127.48, 123.95,
106.78 (C-5), 103.84 (C-4), 100.09 (C-8), 64.74 (CH3CH2O-), 56.73 (-OCH3), 14.42 (CH3CH2O-);
Anal. Calcd. for C16H15ClN4O2: C 58.10, H 4.57, N 16.94; Found: C 58.01, H 4.56, N 16.96.
o
7-Ethoxy-6-methoxy-4-(4-methylanilino)-1,2,3-benzotriazine (8c): Yield: 73.2%; mp: 249-251 C. IR
(KBr): 33381, 2980, 1614, 1512, 1426, 1283, 1244, 1103, 856, 816 cm-1; 1H-NMR (DMSO-d6) δ: 9.50
(s, 1H, NH), 7.90 (s, 1H, H-8), 7.70 (d, 2H, J = 8.2 Hz, ArH-2’H, 6’H), 7.53 (s, 1H, H-5), 7.25 (d, 2H,
J = 8.2 Hz, ArH-3’H, 5’H), 4.29 (q, 2H, J = 6.9 Hz, CH3CH2O-), 4.02 (s, 3H, CH3O-), 2.34 (s, 3H,
13
ArH-4’CH3), 1.44 (t, 3H, J = 6.8 Hz, CH3CH2O-); LC-MS: 311.2 (M+H)+; C-NMR (DMSO-d6) δ:
154.16, 153.19, 150.30, 141.62, 138.86, 128.72, 124.04, 122.70, 106.77, 103.82, 100.12, 64.73, 56.72,
20.89, 14.46; Anal. Calcd. for C17H18N4O2: C 65.79, H 5.85, N 18.05; Found: C 65.68, H 5.81, N
18.07.
4-(3-Chloro-4-fluoroanilino)-7-ethoxy-6-methoxy-1,2,3-benzotriazine (8d): Yield: 64.1%; mp: 248-
1
250 oC. IR (KBr): 3288, 1609, 1554, 1500, 1417, 1292, 1237, 1222, 1106 cm-1; H-NMR (DMSO-d6)
δ: 9.66 (s, 1H, NH), 8.17 (m, 1H, ArH-2’H), 7.87 (s, 1H, H-8), 7.82 (m, 1H, ArH-5’H), 7.58 (s, 1H, H-
5), 7.51 (d, 1H, J = 9 Hz, ArH-6’H), 4.31 (q, 2H, J = 6.9 Hz, CH3CH2O-), 4.03 (s, 3H, CH3O-), 1.44 (t,
3H, J = 6.9 Hz, CH3CH2O-); LC-MS: 349.2 (M+H)+; 13C-NMR (DMSO-d6) δ: 154.43, 154.29, 153.62,
152.10, 150.35, 141.80, 136.65, 124.10, 123.00, 122.90, 119.55, 117.10, 116.80, 107.15, 104.13,
100.28, 64.69, 56.65, 14.46; Anal. Calcd. for C16H14ClFN4O2: C 55.10, H 4.05, N 16.06; Found: C
55.00, H 4.07, N 16.04.
o
4-(4-Chloroanilino)-6-methoxy-7-pentyloxy-1,2,3-benzotriazine (8e): Yield: 69.3%; mp: 213 C. IR
(KBr): 3378, 2954, 1612, 1562, 1507, 1426, 1245, 817 cm-1; 1H-NMR (DMSO-d6) δ: 9.63 (1H, s, NH),
7.91 (3H, m, H-8, H-2’, H-6’), 7.53 (3H, m, H-8, H-3’, H-5’), 4.24 (2H, t, J = 6.0 Hz,
CH3CH2CH2CH2CH2O-), 4.03 (3H, s, -OCH3), 1.82 (2H, m, CH3CH2CH2CH2CH2O-), 1.40 (4H, m,
CH3CH2CH2CH2CH2O-), 0.92 (3H, t, J = 6.0 Hz, CH3CH2CH2CH2CH2O); LC-MS: 373.2 (M+H)+;

Molecules 2008, 13
1435
13C-NMR (DMSO-d6) δ: 154.31 (C-7), 153.27 (C-6), 150.30 (C-10), 141.63 (C-9), 138.88 (C-1’),
128.73, 127.40, 123.91, 106.72 (C-5), 103.82 (C-4), 100.05 (C-8), 68.98 (CH3CH2CH2CH2CH2O-),
56.79 (-OCH3), 28.17 (CH3CH2CH2CH2CH2O-), 27.78 (CH3CH2CH2CH2CH2O-), 21.98
(CH3CH2CH2CH2CH2O-), 14.04 (CH3CH2CH2CH2CH2O-); Anal. Calcd. for C19H21ClN4O2: C 61.21,
H 5.68, N 15.03; Found: C 61.11, H 5.64, N 14.99.
6-Methoxy-7-pentyloxy-4-(3-trifluoromethoxyanilino)-1,2,3-benzotriazine (8f): Yield: 73.1%; mp:
o
1
181-183 C. IR (KBr): 3421, 2931, 1613, 1508, 1446, 1258, 1216, 1163, 1105, 845, 784 cm-1; H-
NMR (DMSO-d6) δ: 9.72 (s, 1H, NH), 8.04 (s, 1H, ArH-2’H), 7.92 (d, 1H, J = 8.9 Hz, ArH-4’H), 7.91
(s, 1H, H-8), 7.60 (s, 1H, H-5), 7.56 (t, 2H, J = 8.6 Hz, ArH-5’H), 7.14 (d, 1H, J = 8.7 Hz, ArH-6’H),
4.24 (t, 2H, J = 6.3 Hz, CH3CH2CH2CH2CH2O-), 4.05 (s, 3H, CH3O-), 1.83 (m, 2H,
CH3CH2CH2CH2CH2O-), 1.42 (m, 4H, CH3CH2CH2CH2CH2O-), 0.92 (t, 3H, J = 6.9 Hz,
CH3CH2CH2CH2CH2O-); LC-MS: 423.2 (M+H)+; 13C-NMR (DMSO-d6) δ: 154.23, 153.52, 150.28,
148.76, 141.90, 141.06, 130.41, 124.25, 121.80, 118.10, 115.20, 114.04, 106.92, 104.11, 100.19, 68.98,
56.79, 28.17, 27.78, 21.98, 14.04; Anal. Calcd. for C20H21F3N4O3: C 56.87, H 5.01, N 13.26; Found: C
56.81, H 5.00, N 13.23.
6-Methoxy-7-pentyloxy-4-(4-trifluoromethoxyanilino)-1,2,3-benzotriazine (8g): Yield: 70.2%; mp:
o
1
230-232 C. IR (KBr): 3426, 2959, 1615, 1510, 1428, 1247, 1200, 1165, 1102, 850, 786 cm-1; H-
NMR (DMSO-d6) δ: 9.69 (s, 1H, NH), 7.98 (d, 2H, J = 9.0 Hz, ArH-3’H, 5’H), 7.90 (s, 1H, H-8), 7.58
(s, 1H, H-5), 7.46 (d, 2H, J = 8.7 Hz, ArH-2’H, 6’H), 4.24 (t, 2H, J = 6.3 Hz, CH3CH2CH2CH2CH2O-),
4.04 (s, 3H, CH3O-), 1.83 (m, 2H, CH3CH2CH2CH2CH2O-), 1.42 (m, 4H, CH3CH2CH2CH2CH2O-),
0.92 (t, 3H, J = 6.9 Hz, CH3CH2CH2CH2CH2O-); LC-MS: 423.2 (M+H)+; 13C-NMR (DMSO-d6) δ:
154.31, 153.27, 150.30, 144.05, 141.54, 137.91, 125.24, 123.93, 122.14, 121.45, 106.86, 103.83,
100.08, 68.97, 56.76, 28.17, 27.76, 21.97, 14.04; Anal. Calcd. for C20H21F3N4O3: C 56.87, H 5.01, N
13.26; Found: C 56.88, H 5.03, N 13.27.
7-(3-Chloropropoxy)-4-(3,5-dichloroanilino)-6-methoxy-1,2,3-benzotriazine (8h): Yield: 72.9%; mp:
o
1
110-112 C. IR (KBr): 3430, 2919, 1620, 1508, 1423, 1289, 849 cm-1; H-NMR (DMSO-d6) δ: 9.74
(1H, s, NH), 8.10 (2H, d, J = 1.77 Hz, H-2’, H-6’), 7.91 (1H, s, H-8), 7.68 (1H, s, H-5), 7.37 (1H, s, H-
4’), 4.39 (2H, t, J = 6.15 Hz, ClCH2CH2CH2O-), 4.06 (3H, s, -OCH3), 3.84 (2H, t, J = 6.5 Hz,
ClCH2CH2CH2O-), 2.30 (2H, m, ClCH2CH2CH2O-); LC-MS: 413.0 (M+H)+; 13C-NMR (DMSO-d6) δ:
154.57 (C-7), 153.92 (C-6), 150.31 (C-10), 142.11 (C-9), 141.94, 134.28, 122.82, 120.06, 107.51 (C-
5), 104.54 (C-4), 100.44 (C-8), 66.29 (ClCH2CH2CH2O-), 57.26 (-OCH3), 42.19 (ClCH2CH2CH2O-),
31.84 (ClCH2CH2CH2O-); Anal. Calcd. for C17H15Cl3N4O2: C 49.36, H 3.65, N 13.54; Found: C 49.28,
H 3.66, N 13.56.
7-(3-Chloropropoxy)-4-(3,4-dichlorophenyl)-6-methoxy-1,2,3-benzotriazine (8i): Yield: 72.4%; mp:
o
1
120-122 C. IR (KBr): 3424, 1616, 1510, 1426, 1281, 848 cm-1; H-NMR (DMSO-d6) δ: 9.74 (1H, s,
NH), 8.31 (1H, d, J = 2.31 Hz, H-2’), 7.93 (2H, m, H-8, H-6’), 7.71 (1H, m, H-5’), 7.67 (1H, s, H-5),
4.38 (2H, t, J = 6.0 Hz, ClCH2CH2CH2O-), 4.05 (3H, s, -OCH3), 3.84 (2H, t, J = 6.4 Hz,

Molecules 2008, 13
1436
ClCH2CH2CH2O-), 2.30 (2H, m, ClCH2CH2CH2O-); LC-MS: 413.0 (M+H)+; 13C-NMR (DMSO-d6) δ:
154.11 (C-7), 153.43 (C-6), 149.95 (C-10), 141.66 (C-9), 139.23, 130.84, 130.51, 125.02, 123.14,
121.90, 107.10 (C-5), 104.11 (C-4), 100.08 (C-8), 65.86 (ClCH2CH2CH2O-), 56.85 (-OCH3), 41.87
(ClCH2CH2CH2O-), 31.46 (ClCH2CH2CH2O-); Anal. Calcd. for C17H15Cl3N4O2: C 49.36, H 3.65, N
13.54; Found: C 49.31, H 3.66, N 13.56.
4-(4-Chloroanilino)-7-(3-chloropropoxy)-6-methoxy-1,2,3-benzotriazine (8j): Yield: 67.9%; mp: 253
oC. IR (KBr): 3425, 2925, 1616, 1561, 1495, 1424, 1245, 839 cm-1; 1H-NMR (DMSO-d6) δ: 9.66 (1H,
s, NH), 7.91 (3H, m, H-8, H-2’, H-6’), 7.62 (1H, s, H-5), 7.50 (2H, d, J = 8.8 Hz, H-3’, H-5’), 4.37
(2H, t, J = 6.0 Hz, ClCH2CH2CH2O-), 4.04 (3H, s, -OCH3), 3.84 (2H, t, J = 6.4 Hz, ClCH2CH2CH2O-),
2.29 (2H, m, ClCH2CH2CH2O-); LC-MS: 379.0 (M+H)+; 13C-NMR (DMSO-d6) δ: 153.99 (C-7),
153.29 (C-6), 150.12 (C-10), 141.57 (C-9), 137.91 (C-1’), 128.59, 127.54, 123.99, 107.07 (C-5),
104.07 (C-4), 100.23 (C-8), 65.82 (ClCH2CH2CH2O-), 56.82 (-OCH3), 41.87 (ClCH2CH2CH2O-),
31.47 (ClCH2CH2CH2O-); Anal. Calcd. for C17H16Cl2N4O2: C 53.84, H 4.25, N 14.77; Found: C 53.79,
H 4.24, N 14.71.
7-(3-Chloropropoxy)-6-methoxy-4-(4-methylanilino)-1,2,3-benzotriazine (8k): Yield: 70.1%; mp: 247
oC. IR (KBr): 3420, 2924, 1615, 1513, 1425, 1282, 1242, 859 cm-1; 1H-NMR (DMSO-d6) δ: 9.55 (1H,
s, NH), 7.92 (1H, s, H-8), 7.69 (2H, d, J = 8.4 Hz, H-2’, H-6’), 7.59 (1H, s, H-5), 7.25 (2H, d, J = 8.4
Hz, H-3’, H-5’), 4.37 (2H, t, J = 6.0 Hz, ClCH2CH2CH2O-), 4.03 (3H, s, -OCH3), 3.84 (2H, t, J = 6.3
Hz, ClCH2CH2CH2O-), 2.34 (3H, s, -CH3), 2.29 (2H, m, ClCH2CH2CH2O-); LC-MS: 359.1 (M+H)+;
13C-NMR (DMSO-d6) δ: 154.23 (C-7), 153.51 (C-6), 150.62 (C-10), 141.59 (C-9), 136.43 (C-1’),
133.64, 129.48, 123.22, 107.28 (C-5), 104.42 (C-4), 100.86 (C-8), 66.14 (ClCH2CH2CH2O-), 57.22 (-
OCH3), 42.24 (ClCH2CH2CH2O-), 31.84 (ClCH2CH2CH2O-), 20.99(Ph-CH3); Anal. Calcd. for
C18H19ClN4O2: C 60.25, H 5.34, N 15.61; Found: C 60.28, H 5.33, N 15.56.
7-(3-Chloropropoxy)-4-(4-fluoroanilino)-6-methoxy-1,2,3-benzotriazine (8l): Yield: 71.2%; mp: 166
oC. IR (KBr): 3437, 1620, 1510, 1428, 1284, 842 cm-1; 1H-NMR (DMSO-d6) δ: 9.63 (1H, s, NH), 7.91
(1H, s, H-8), 7.84 (2H, m, H-2’, H-6’), 7.61 (1H, s, H-5), 7.29 (2H, t, J = 8.7 Hz, H-3’, H-5’), 4.37
(2H, t, J = 6.0 Hz, ClCH2CH2CH2O-), 4.03 (3H, s, -OCH3), 3.84 (2H, t, J = 6.4 Hz, ClCH2CH2CH2O-),
2.29 (2H, m, ClCH2CH2CH2O-); LC-MS: 363.1 (M+H)+; 13C-NMR (DMSO-d6) δ: 160.67, 157.48,
154.28 (C-7), 153.56 (C-6), 150.64 (C-10), 141.79 (C-9), 135.46 (C-1’), 125.15, 125.04, 115.79,
115.50, 107.40 (C-5), 104.29 (C-4), 100.67 (C-8), 66.18 (ClCH2CH2CH2O-), 57.17 (-OCH3), 42.22
(ClCH2CH2CH2O-), 31.86 (ClCH2CH2CH2O-); Anal. Calcd. for C17H16ClFN4O2: C 56.28, H 4.45, N
15.44; Found: C 56.20, H 4.43, N 15.56.
4-(3-Chloro-4-fluoroanilino)-7-(3-chloropropoxy)-6-methoxy-1,2,3-benzotriazine (8m): Yield: 77.1%;
mp: 138-139 oC. IR (KBr): 3314, 2923, 1616, 1571, 1511, 1427, 1285, 849 cm-1; 1H-NMR (DMSO-d6)
δ: 9.70 (1H, s, NH), 8.18 (1H, d, J = 4.4 Hz, H-2’), 7.90 (1H, s, H-8), 7.82 (1H, m, H-6’), 7.64 (1H, s,
H-5), 7.51 (1H, t, J = 9.1 Hz, H-5’), 4.38 (2H, t, J = 5.9 Hz, ClCH2CH2CH2O-), 4.04 (3H, s, -OCH3),
3.84 (2H, t, J = 6.4 Hz, ClCH2CH2CH2O-), 2.29 (2H, m, ClCH2CH2CH2O-); LC-MS: 397.0 (M+H)+;
13C-NMR (DMSO-d6) δ: 155.47 (C-4’), 154.32 (C-7), 153.64 (C-6), 152.25 (C-4’), 150.32 (C-10),

Molecules 2008, 13
1437
141.81 (C-9), 136.57 (C-1’), 124.15 (C-2’), 122.93 (C-6’), 122.84 (C-6’), 119.47 (C-6’), 117.14 (C-
5’), 116.85 (C-5’), 107.33 (C-5), 104.28 (C-4), 100.56 (C-8), 66.19 (ClCH2CH2CH2O-), 57.21 (-
OCH3), 42.18 (ClCH2CH2CH2O-), 31.86 (ClCH2CH2CH2O-); Anal. Calcd. for C17H15Cl2FN4O2: C
51.40, H 3.81, N 14.10; Found: C 51.41, H 3.85, N 14.08.
7-(3-Chloropropoxy)-6-methoxy-4-(3-trifluoromethoxyanilino)-1,2,3-benzotriazine
(8n):
Yield:
65.3%; mp: 154-155 oC. IR (KBr): 3427, 1615, 1572, 1511, 1448, 1256, 854 cm-1; 1H-NMR (DMSO-
d6) δ: 9.75 (1H, s, NH), 8.05 (1H, s, H-2’), 7.93 (2H, s, H-8, H-6’), 7.65 (1H, s, H-5), 7.59 (1H, t, J =
8.2 Hz, H-5’), 7.15 (1H, d, J = 7.9 Hz, H-4’), 4.38 (2H, t, J = 5.9 Hz, ClCH2CH2CH2O-), 4.06 (3H, s, -
OCH3), 3.84 (2H, t, J = 6.4 Hz, ClCH2CH2CH2O-), 2.29 (2H, m, ClCH2CH2CH2O-); LC-MS: 429.1
13
(M+H)+; C-NMR (DMSO-d6) δ: 154.34 (C-7), 153.65 (C-6), 150.40 (C-10), 148.82, 141.96 (C-9),
141.12 (C-1’), 130.54, 124.05, 122.30, 118.90, 115.91, 114.64, 107.32 (C-5), 104.41 (C-4),100.46 (C-
8), 66.15 (ClCH2CH2CH2O-), 57.17 (-OCH3), 42.17 (ClCH2CH2CH2O-), 31.82 (ClCH2CH2CH2O-);
Anal. Calcd. for C18H16ClF3N4O3: C 50.42, H 3.76, N 13.07; Found: C 50.37, H 3.78, N 13.06.
7-(3-Chloropropoxy)-6-methoxy-4-(4-trifluoromethoxyanilino)-1,2,3-benzotriazine (8o): Yield: 63.0%;
o
1
mp: 150 C. IR (KBr): 3444, 1618, 1574, 1510, 1429, 1247, 852 cm-1; H-NMR (DMSO-d6) δ: 9.68
(1H, s, NH), 7.93 (2H, d, J = 9.0 Hz, H-2’, H-6’), 7.88 (1H, s, H-8), 7.59 (1H, s, H-5), 7.40 (2H, d, J =
8.7 Hz, H-3’, H-5’), 4.33 (2H, t, J = 5.9 Hz, ClCH2CH2CH2O-), 4.00 (3H, s, -OCH3), 3.79 (2H, t, J =
6.6 Hz, ClCH2CH2CH2O-), 2.25 (2H, m, ClCH2CH2CH2O-); LC-MS: 429.1 (M+H)+; 13C-NMR
(DMSO-d6) δ: 154.41 (C-7), 153.70 (C-6), 150.54 (C-10), 144.48, 141.97 (C-9), 138.51 (C-1’),
125.64, 124.23, 122.34, 121.79, 107.46 (C-5), 104.43 (C-4), 100.68 (C-8), 66.23 (ClCH2CH2CH2O-),
57.22 (-OCH3), 42.21 (ClCH2CH2CH2O-), 31.85 (ClCH2CH2CH2O-); Anal. Calcd. for
C18H16ClF3N4O3: C 50.42, H 3.76, N 13.07; Found: C 50.39, H 3.75, N 13.04.
7-(3-Chloropropoxy)-4-(4-fluoro-3-trifluoromethylanilino)-6-methoxy-1,2,3-benzotriazine (8p): Yield:
o
1
71.1%; mp: 150 C. IR (KBr): 3452, 2920, 1614, 1508, 1432, 1290, 1129 cm-1; H-NMR (DMSO-d6)
δ: 9.82 (1H, s, NH), 8.18 (2H, m, H-6’, H-2’), 7.91 (1H, s, H-8), 7.65 (1H, s, H-5), 7.60 (1H, m, H-5’),
4.38 (2H, t, J = 6.0 Hz, ClCH2CH2CH2O-), 4.05 (3H, s, -OCH3), 3.84 (2H, t, J = 6.3 Hz,
ClCH2CH2CH2O-), 2.30 (2H, m, ClCH2CH2CH2O-); LC-MS: 431.1 (M+H)+; 13C-NMR (DMSO-d6) δ:
156.36, 154.04 (C-7), 153.35 (C-6), 150.01 (C-10), 141.53 (C-9), 135.74 (C-1’), 128.37, 128.26,
124.46, 120.85, 120.26, 117.61, 117.33, 107.05 (C-5), 103.94 (C-4), 100.10 (C-8), 65.83
(ClCH2CH2CH2O-), 56.80 (-OCH3), 41.78 (ClCH2CH2CH2O-), 31.44 (ClCH2CH2CH2O-); Anal.
Calcd. for C18H15ClF4N4O2: C 50.19, H 3.51, N 13.01; Found: C 50.17, H 3.53, N 13.00.
7-(3-Chloropropoxy)-6-methoxy-4-(3-trifluoromethylanilino)-1,2,3-benzotriazine (8q): Yield: 75.2%;
mp: 180-181 oC. IR (KBr): 3438, 2925, 1623, 1576, 1510, 1448, 1244, 799 cm-1; 1H-NMR (DMSO-d6)
δ: 9.81 (1H, s, NH), 8.30 (1H, s, H-2’), 8.25 (1H, d, J = 8.4 Hz, H-6’), 7.94 (1H, s, H-8), 7.70 (1H, d, J
= 7.8 Hz, H-5’), 7.66 (1H, s, H-5), 7.51 (1H, d, J = 8.1 Hz, H-4’), 4.38 (2H, t, J = 6.0 Hz,
ClCH2CH2CH2O-), 4.06 (3H, s, -OCH3), 3.84 (2H, t, J = 6.4 Hz, ClCH2CH2CH2O-), 2.29 (2H, m,
ClCH2CH2CH2O-); LC-MS: 413.1 (M+H)+; 13C-NMR (DMSO-d6) δ: 154.10 (C-7), 153.41 (C-6),
150.14 (C-10), 141.66 (C-9), 139.87 (C-1’), 129.88, 129.53, 129.32, 125.71, 119.93, 118.17, 107.09
(C-5), 104.11 (C-4), 100.14 (C-8), 65.85 (ClCH2CH2CH2O-), 56.83 (-OCH3), 41.84

Molecules 2008, 13
1438
(ClCH2CH2CH2O), 31.45 (ClCH2CH2CH2O-); Anal. Calcd. for C18H16ClF3N4O2: C 52.37, H 3.91, N
13.57; Found: C 52.32, H 3.94, N 13.56.
o
4-Anilino-7-(3-chloropropoxy)-6-methoxy-1,2,3-benzotriazine (8r): Yield: 70.1%; mp: 243 C. IR
1
(KBr): 3435, 2922, 1614, 1566, 1502, 1447, 1421, 1285, 1241, 1108, 850, 744 cm-1; H-NMR
(DMSO-d6) δ: 9.59 (1H, s, NH), 7.94 (1H, s, H-8), 7.84 (2H, d, J = 7.8 Hz, ArH-2’H, 6’H), 7.61 (1H,
s, H-5), 7.45 (2H, t, J = 7.8 Hz, ArH-3’H, 5’H), 7.18 (1H, t, J = 7.5 Hz, ArH-4’H), 4.37 (2H, t, J = 6.0
Hz, ClCH2CH2CH2O-), 4.04 (3H, s, -OCH3), 3.84 (2H, t, J = 6.3 Hz, ClCH2CH2CH2O-), 2.29 (2H, m,
13
ClCH2CH2CH2O-); LC-MS: 345.2 (M+H)+; C-NMR (DMSO-d6) δ: 153.88 (C-7), 153.17 (C-6),
150.28 (C-10), 141.50 (C-9), 138.83 (C-1’), 128.68 (C-3’, C-5’), 124.03 (C-4’), 122.88 (C-2’, C-6’),
107.01 (C-5), 104.02 (C-4), 100.31 (C-8), 65.78 (ClCH2CH2CH2O-), 56.81 (-OCH3), 41.89
(ClCH2CH2CH2O-), 31.48 (ClCH2CH2CH2O-); Anal. Calcd. for C17H17ClN4O2: C 59.22, H 4.97, N
16.25; Found: C 59.28, H 4.96, N 16.17.
Antiproliferative activity assay
Cell lines: The cell lines used were obtained from ATCC (Rockville, MD, USA). T47D human breast
cancer cells, DU145 and PC-3 human prostate cancer cells were cultured in RPMI1640 medium
(Gibco; New York, NY, USA) supplemented with 100 units/mL penicillin, 100 µg/mL streptomycin, 1
mM L-glutamine, 5 µg/mL insulin and 10% (v/v) heat-inactivated fetal bovine serum (FBS). MVECs,
LL/2 murine lewis lung carcinoma cells and B16F0 melanoma cells were cultured in DMEM with 4
mM L-glutamine adjusted to contain 4.50 g/L glucose and 10% (v/v) heat-inactivated FBS.
Cell growth inhibition assay: The antiproliferative activities of these compounds were determined by
the MTT assay. Cells (2×103 cells/well in 96-well plates) were incubated for 24 h, then various
concentrations of each compound were added to each well and the cells were cultured for another 4
days at 37 oC. MTT solution (50 μL of 2 mg/mL) was added per well and the culture continued for an
additional 4 h. The medium was removed by aspiration and the cells were dissolved in 200 μL DMSO.
The absorbance at 570 nm was measured in the 96-well plate reader. Growth inhibition is reported as
compared to untreated cells (%) and GI50 concentration was calculated [18].
Acknowledgements
This work is supported by the Natural Science Foundation of Liaoning Province of China (No.
20062060).
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