Synthesis and biological evaluation of substituted 1,2,3-benzotriazines and pyrido[3,2-d]-1,2,3-triazines as inhibitors of vascular endothelial growth factor receptor-2

Article abstract of DOI:10.1016/j.bmc.2013.10.009

A novel series of substituted 1,2,3-benzotriazines and pyrido[3,2-d]-1,2,3- triazines were synthesized. The abilities of these compounds to inhibit the VEGFR-2 kinase activity and the proliferation of human microvascular endothelial cells (MVECs) were det

Full text of DOI:10.1016/j.bmc.2013.10.009

Bioorganic & Medicinal Chemistry 21 (2013) 7807–7815
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of substituted
1,2,3-benzotriazines and pyrido[3,2-d]-1,2,3-triazines as inhibitors
of vascular endothelial growth factor receptor-2
Xing-Wang Zhao a, Dan Liu a, Sheng-Lin Luan a, Guo-Dong Hu a, Jin-Ling Lv a, Yong-Kui Jing b,
Lin-Xiang Zhao a,
⇑
a Key Laboratory of Structure-Based Drugs Design & Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China
b Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of substituted 1,2,3-benzotriazines and pyrido[3,2-d]-1,2,3-triazines were synthesized. The
abilities of these compounds to inhibit the VEGFR-2 kinase activity and the proliferation of human micro-
vascular endothelial cells (MVECs) were determined. 6-Methoxy-4-substituted-1,2,3-benzotriazines and
4-substituted-6-chloro-pyrido[3,2-d]-1,2,3-triazines have the abilities of inhibiting the VEGFR-2 kinase
activity, but only the 4-substituted-6-chloro-pyrido[3,2-d]-1,2,3-triazines exhibit good growth inhibitory
effects on MVECs. Compound 6-chloro-4-(3-trifluoromethylanilino)-pyrido[3,2-d][1,2,3]triazin (11d) is
less half active than PTK787 to inhibit the VEGFR-2 kinase activity, but is more active than PTK787 to
inhibit the growth of MVECs. The potential binding modes of 6d, 11d, and CTZ12 in complex with their
putative intracellular target, VEGFR-2, were predicted using Surflex-Dock.
Received 1 September 2013
Revised 9 October 2013
Accepted 10 October 2013
Available online 21 October 2013
Keywords:
1,2,3-Benzotriazines
Pyrido[3,2-d]-1,2,3-triazines
VEGFR-2 kinase
MVECs
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
Vascular endothelial growth factor receptor-2 (VEGFR-2) is a
receptor tyrosine kinase expressed on the endothelial cells, which
belongs to the immunoglobulin subclass of the receptor tyrosine
kinase superfamily and has seven Ig-like extracellular domains
with a single transmembrane helix with an intracellular kinase
region.1,2 Following binding of vascular endothelial growth factor
A (VEGFA), VEGFR-2 has the potential to form either a homodimer
or a heterodimer complex with VEGFR-1, resulting in intracellular
tyrosine phosphorylation.3 The downstream effects of VEGFR-2
signaling which culminate in angiogenesis include a potent in-
crease in vascular permeability and promotion of endothelial cell
migration, proliferation, and survival.4–9 Disruption of VEGF signal-
ing pathway by either specific binding of circulating VEGF or inhib-
iting receptor tyrosine kinases with small molecules has been
found to inhibit angiogenesis, tumor progression and dissemina-
tion.10–13 The recombinant humanized anti-VEGF monoclonal anti-
body Avastin has been approved for the treatment of metastatic
colorectal cancer.14 The small molecule VEGFR-2 kinase inhibitor
sunitinib (SU11248) was approved by the FDA for the treatment
Figure 1. Structures of PTK787 and CTZ12.
molecules have been developed as potent VEGFR-2 inhibitors for
the treatment of various cancers and some are currently undergo-
ing phase III clinical studies, including PTK787 (vatalanib,
ZK222584). PTK787 inhibited VEGF-induced autophosphorylation
of VEGFR-2 and endothelial cell proliferation.16,17
Previously we have synthesized a series of novel substituted
1,2,3-benzotriazines with a similar backbone structure and found
that they could inhibit the proliferation of human microvascular
endothelial cells (MVECs). The most effective compound 4-(3-
chloro-4-fluoroanilino-)-7-(3-chloropropoxy)-6-methoxy-1,2,3-
benzotriazine (CTZ12) was 4–10 fold more potent than PTK787 in
inhibiting the growth of MVECs (Fig. 1).18 To improve the ability of
of advanced renal cell carcinoma.15
A number of synthetic
⇑
Corresponding author. Tel./fax: +86 024 23986420.
E-mail addresses: linxiang.zhao@vip.sina.com, zhaolinxiang@syphu.edu.cn (L.-X.
Zhao).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.10.009

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X.-W. Zhao et al. / Bioorg. Med. Chem. 21 (2013) 7807–7815
Scheme 1. Synthetic route to the target compounds 6a–6z. Reagents and conditions: (a) alkyl halide, K2CO3, DMF, 37 °C, 6 h; (b) HNO3, 30 °C, 2 h; (c) Pd/C, cyclohexene, EtOH,
reflux, overnight; (d) NaNO2/10 N HCl, 0 °C; substituted aniline, 4 °C, 2 h; (e) 70% EtOH, reflux, 1 h; (f) AcOH, reflux, 2 h.
CTZ12 to inhibit the proliferation of MVECs, a new series of substi-
tuted 1,2,3-benzotriazines as well as the pyrido[3,2-d]-1,2,3-tri-
azine derivatives were synthesized. The anti-proliferative effects
of these compounds on MVECs as well as their abilities of inhibit-
ing VEGFR-2 kinase activity were tested and analyzed.
acetate, compounds (10a–10j) were boiled in 70% ethanol, which
were then directly rearranged to the final compounds (11a–11j)
in refluxed acetic acid after evaporating the ethanol.
3. Biological evaluation and discussion
2. Chemistry
Although CTZ12 was more effective than PTK787 in inhibiting
the proliferation of MVECs, it has minimal ability of inhibiting
the VEGFR-2 kinase activity (Table 1). Replacement of 3-chloro-
4-fluoro at the C4 anilino group (CTZ12) with 4-trifluoromethyl
(6y) or 4-methyl (6z) did not improve the ability of inhibiting VEG-
FR-2 activity, but decreased the ability of inhibiting proliferation of
MVECs. Replacement of 7-chloropropoxy at the C7 position of
1,2,3-benzotriazine with an ethoxy group (6a–6i), but not with a
n-butoxy (6j–6r) or a pentlyoxy (6s–6x), improved the ability of
inhibiting VEGFR-2 activity. However, all these compounds de-
creased the ability of inhibiting cell growth of MVECs. Also replace-
ment of 3-chloro-4-fluoro at the C4 anilino group with a different
substituted group (X) (6a–6i) did not significantly influence the
ability of inhibiting VEGFR-2 activity. Replacing 7-(3-chloroprop-
oxy)-6-methoxy-1,2,3-benzotriazine with 6-chloro-pyrido[3,2-d]-
1,2,3-triazin evidently improved the ability of inhibiting VEGFR-2
activity (11a–11j) with similar or improved ability of inhibiting
growth of MVECs comparing to CTZ12 (Table 2). Although these
4-substituted-6-chloro-pyrido[3,2-d]-1,2,3-triazines showed less
activities than PTK787 to inhibit VEGFR-2 activity, they were more
effective than PTK787 to inhibit growth of MVECs. Based on these
data we found that: (a) Introduction of a N atom at 5-position of
1,2,3-benzotriazines (6-chloro-pyrido[3,2-d]-1,2,3-triazine ring
system) derivatives improved the ability of inhibiting VEGFR-2
activity (11a–11j) with similar or better ability of inhibiting cell
growth of MVECs comparing to CTZ12; (b) 7-site alkoxyl group
Compounds 7-substituted-6-methoxy-4-substituted-1,2,3-ben-
zotriazines (6a–6z) were synthesized according to the synthetic
route as described in Scheme 1. The intermediates 4-substituted-
3-methoxybenzonitriles (2a–2d) were prepared by the reaction
of different alkyl halides with 4-cyano-2-methoxyphenol (1) in
the presence of potassium carbonate in DMF. The selectively nitra-
tion of compounds (2a–2d) in HNO3 gave the nitro compounds
(3a–3d) in good yields, following with the reduction reaction by
Pd/C as catalyst to obtain the corresponding amines (4a–4d). Com-
pounds (4a–4z) were diazotized and then coupled with substituted
anilines at 0 °C, which were purified by recrystallization with ethyl
acetate to afford triazenes (5a–5z). Compounds (5a–5z) were
boiled in 70% ethanol, then directly rearranged to the final com-
pounds (6a–6z) in refluxed acetic acid after evaporating the
ethanol.
The preparation of target compounds 4-substituted-6-chloro-
pyrido[3,2-d]-1,2,3-triazines (11a–11j) were described in
Scheme 2. The intermediate 3-amino-6-chloropicolinonitrile (9)
was synthesized by the reaction of 2,6-dichloro-3-nitropyridine
with cuprous cyanide in N-methyl-pyrrolidone, followed by the
reduction with sodium hyposulfite in dichloromethane/aqua solu-
tion using TBAB as a phase-transfer catalyst. Subsequently, the
intermediate 9 was diazotized and then coupled with substituted
anilines (7a–7j). After purification by recrystallization with ethyl
Scheme 2. Synthetic route to the target compounds 11a–11j. Reagents and conditions: (a) CuCN, NMP, DMF, 180 °C, 15 min; (b) Na2S2O4, TBAB, CH2Cl2, H2O, 30 °C, 4 h; (c)
NaNO2/10 N HCl, 0 °C; substituted aniline, 0 °C, 2 h; (d) 70% EtOH, reflux, 1 h; (e) AcOH, reflux, 2 h.

X.-W. Zhao et al. / Bioorg. Med. Chem. 21 (2013) 7807–7815
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Table 1
The structures of target compounds 6a–6z and their abilities of inhibiting VEGFR-2 activity and the proliferation of MVECs
R2
HN
O
R1
N
N
N
a
b
Compd R1
R2
VEGFR-2 inhibition (%) MVECs GI50
(l
M) Compd R1
R2
VEGFR-2 inhibition (%) MVECs GI50 (lM)
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
Ethoxy
3-F-4-Br
3-CF3
4-CF3
3-OCF3
4-OCF3
3-Cl
3-CF3-4-F 46.78 0.22
2-F
3-Br
35.77 0.56
37.34 1.43
24.66 0.66
49.20 2.46
38.44 1.48
45.50 0.19
73.30 0.06
>80
>80
23.28 1.50
>80
59.99 1.13
31.65 0.57
>80
39.79 0.79
>80
15.50 0.66
>80
30.04 0.47
16.06 1.48
6o
6p
6q
6r
6s
6t
6u
6v
n-Butoxy
2-F
3-Br
4-F
12.03 1.65
<10
<10
12.03 0.62
18.98 2.51
>80
16.80 0.68
>80
>80
25.34 0.94
>80
27.93 1.33
42.64 1.18
37.98 1.14
>80
44.38 1.37
17.08 0.65
7.98 0.35
38.15 2.07
Ethoxy
Ethoxy
Ethoxy
Ethoxy
Ethoxy
Ethoxy
Ethoxy
Ethoxy
n-Butoxy 3-F-4-Br
n-Butoxy 3,4-diCl
n-Butoxy 4-CF3
n-Butoxy 4-OCF3
n-Butoxy
n-Butoxy
n-Butoxy
Pentlyoxy
Pentlyoxy
Pentlyoxy
Pentlyoxy
Pentlyoxy
Pentlyoxy
4-CH3
4-Cl
3-F-4-Br 19.78 0.28
3,5-DiF
3-OCF3
4-OCF3
4-F
10.70 1.17
<10
11.22 0.38
11.50 1.57
<10
14.20 1.99
<10
100
48.87 1.15
44.10 1.26
<10
10.70 2.49
<10
6w
6x
6y
Chloropropoxy 4-CF3
Chloropropoxy 4-CH3
6z
10.96 1.74
CTZ12
n-Butoxy 3-CF3-4-F 12.57 2.23
PTK787c
a
VEGFR-2 inhibition: the inhibition rate of the compounds against VEGFR-2 kinase at the concentration of 10
l
g/mL.
b
GI50: the concentration of the compounds (
lM) producing 50% cell growth inhibition after 4 days of drug exposure, as determined by the MTT assay. Data shown are
means SD of three independent experiments.
c
Used as positive control.
Table 2
The structures of target compounds 11a–11j and their abilities of inhibiting VEGFR-2 activity and the proliferation of MVECs
R3
HN
Cl
N
N
N
N
VEGFR-2 inhibitiona (%)
MVECs GI50
(
lM)
Compd
R3
VEGFR-2 inhibition (%)
MVECs GI50 (lM)
b
Compd
R3
11a
11b
11c
11d
11e
11f
4-Cl
29.83 2.21
30.75 1.16
30.08 0.99
40.00 1.12
17.24 1.20
29.93 2.21
10.50 0.55
9.56 1.17
7.93 1.42
5.25 0.09
8.06 0.10
4.28 0.94
11g
11h
11i
11j
4-OCF3
3-CF3-4-F
2-F
29.67 2.23
12.08 2.99
31.56 0.54
30.80 0.98
<10
11.49 0.99
10.78 1.40
10.83 0.14
7.24 0.09
7.98 0.35
38.15 2.07
3-F-4-Br
3,5-DiCl
3-CF3
4-CF3
3-OCF3
3-Br
CTZ12
PTK787c
100
a
VEGFR-2 inhibition: the inhibition rate of the compounds against VEGFR-2 kinase at the concentration of 10
l
g/mL.
b
GI50: the concentration of the compounds (
lM) producing 50% cell growth inhibition after 4 days of drug exposure, as determined by the MTT assay. Data shown are
means SD of three independent experiments.
c
Used as positive control.
of the 1,2,3-benzotriazines derivatives is an important moiety to
inhibit VEGFR-2 kinase activity. Replacement of 7-chloropropoxy
at the C7 position of 1,2,3-benzotriazine with an ethoxy group
(6a–6i), but not with a n-butoxy (6j–6r) or a pentlyoxy (6s–6x),
improved the ability of inhibiting VEGFR-2 activity.
A docking analysis was carried out to compare the potential
binding modes of some compounds with VEGFR-2. To illustrate
the docking protocol, the native ligand 4-amino-furo[2,3-d]pyrim-
idine was removed from the Protein Data Bank structure (PDB en-
try 1YWN)19 and redocked into the ATP-binding site of VEGFR-2.
The docked result was compared to the crystal structure of the
bound ligand–protein complex and illustrated the post-docked li-
gand binds well to the cleft of the VEGFR-2. The RMSD value be-
tween the original ligand and post-docked ligand is 0.329. These
docking results better reconstruct the crystal structure and the
parameters are suitable for studying.
Using this model, PTK787 was also docked into the ATP-binding
site of VEGFR-2 kinase and the binding mode was almost consis-
tent to the data reported previously.20 The phthalazine bicycle
makes hydrophobic contacts with residues Leu 1033, Gly 920,
Cys917, and Leu 838, the aniline moiety occupies the hydrophobic
pocket formed by residues Val 914, Val 912, Glu915, Val 897, Leu
887, Glu883, Lys 866, and Cys1043, and the pyridyl ring engages
in
a hydrophobic region containing the residues Gly920,
Leu1033, Cys1043, and Asn1031 (Fig. 2).
Compounds 6d, 11d with medium inhibiting ability and CTZ12
with minimal inhibiting ability on VEGFR-2 activity were selected
for docking evaluation as representative examples. The predicted
binding modes of 6d, 11d, and CTZ12 are shown in Figure 3. The
anilino moiety of 6d, 11d, and CTZ12 is located in the hydrophobic
pocket formed by residues Lys 866, Val 897, Leu 887, Val 912, Val
914, Cys 1043, and Phe 1045, while the 1,2,3-benzotriazine and

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X.-W. Zhao et al. / Bioorg. Med. Chem. 21 (2013) 7807–7815
Figure 2. Binding model of compound PTK787 in complex with VEGFR-2 kinase.
PTK787 is shown in stick model colored by green and the receptor is shown in lines
model colored by atom type.
pyrido[3,2-d]-1,2,3-triazine bicycle make hydrophobic contacts
with residues Leu 838, Gly 920, and Leu 1033. Unlike compounds
6d and CTZ12, the anilino NH group of compound 11d forms direct
hydrogen bonds with the backbone of Cys 917. This may explain
why compound 11d shows more potent inhibitory activity against
VEGFR-2 than 6d and CTZ12. Glu 915 and Cys 917 are the residues
of the hinge region in the ATP-binding site of VEGFR-2 which may
be involved in hydrogen contracts with the adenine ring of ATP and
maybe allow water-mediated hydrogen bonds with the anilino NH
group of the inhibitor PTK787, 6d, and CTZ12).20 The reason that
CTZ12 is less effective to inhibit the VEGFR-2 activity than 6d
might be due to the steric hindrance of 3-chloropropoxy group,
which is too large to enter into the hydrophobic region formed
by the residues of Gly920, Leu1033, Cys1043, and Asn1031.
Based on the predicted binding modes in Figure 3, it seems that
introducing a small substituted group at 7- or 8-position of 1,2,3-
benzotriazines or pyrido[3,2-d]-1,2,3-triazines forming hydrogen
bonds with CYS917, GLY920 would improve the activity. Also, in
the cavity formed by VAL912, GLU883, and LEU887, adding hydro-
phobic groups with ability to form hydrogen bonds will elevate the
activity.
4. Conclusion
A series of substituted 1,2,3-benzotriazines and pyrido[3,2-d]-
1,2,3-triazines were synthesized and evaluated for their abilities
to inhibit the VEGFR-2 kinase activity and the proliferation
of MVECs. 6-methoxy-4-substituted-1,2,3-benzotriazines and 4-
substituted-6-chloro-pyrido[3,2-d]-1,2,3-triazines have improved
ability of inhibiting VEGFR-2 activity, but only 4-substituted-
6-chloro-pyrido [3,2-d]-1,2,3-triazines exhibit good growth
inhibitory effects on MVECs. 4-substituted-6-chloro-pyrido
[3,2-d]-1,2,3-triazines are worthy of further studies.
Figure 3. Binding models of compounds 6d, 11d, and CTZ12 in complex with
VEGFR-2 kinase. The hydrogen bonds are labeled as yellow broken lines.
Compounds 6d, 11d, and CTZ12 are shown in stick model colored by magenta
and the receptor is shown in lines model colored by atom type.
recorded on a Bruker ARX-300 instrument with tetramethylsilane
(TMS) as the internal standard. IR spectra (KBr disks) were re-
corded on a Bruker IFS55 instrument. Elemental analysis was
determined on a Carlo-Erba 1106 Elemental analysis instrument
(Carlo Erba, Milan, Italy). All chemicals were obtained from com-
mercial suppliers and used without purification. TLC analysis was
carried out on silica gel plates GF254 (Qindao Haiyang Chemical,
China). 2-cyano-3-nitro-6-chloropyridine is synthesized in accor-
dance with literature procedure.21
5. Experimental protocols
5.1. Chemistry
The melting points were determined on an electrically heated
X4 digital visual melting point apparatus and were uncorrected.
Mass spectra (MS) were determined on either Finnigan MAT/USA
spectrometer (LC–MS). 1H NMR and 13C NMR spectrum were

X.-W. Zhao et al. / Bioorg. Med. Chem. 21 (2013) 7807–7815
7811
5.2. General procedure for the synthesis of 2-amino-5-methoxy-
4-substituted benzonitriles (4a–4d)
5.3.3. 7-Ethoxy-6-methoxy-4-(4-trifluoromethylanilino)-1,2,3-
benzotriazine (6c)
Yield: 71%. Mp: 242–244 °C. ESI-MS: m/z, 365.1 [M+H]+. IR
(KBr): 3439, 1614, 1508, 1427, 1326, 1103, 847 cmꢀ1 1H NMR
A solution of 4-cyano-2-methoxyphenol (1) (6.70 mmol) in
anhydrous DMF (4.00 mL) was stirred and cooled with an ice bath.
K2CO3 (10.1 mmol) was added and the mixture was stirred at 20 °C
for 1 h. The corresponding alkyl halide (8.13 mmol) was added
dropwisely. The mixture was stirred at room temperature over-
night, then heated at 37 °C for 6 h and finally poured into ice-water
(100 mL). After stirring for 10 min, a precipitate was formed, then
filtered off, washed with H2O, and air-dried. Without any additional
purification, the resulting 3-methoxy-4-substituted-benzonitriles
(2a–2d) (9.10 mmol) in nitric acid (10 mL) was heated at 30 °C for
2 h, poured into ice-water (100 mL), filtered, and washed with
H2O to afford 5-methoxy-2-nitro-4-substituted-benzonitrile (3a–
3d). Then a mixture of the intermediate (3a–3d) (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
dropwisely. The mixture was refluxed overnight, then cooled to
40 °C, filtered, and washed with ethanol. The filtrate was concen-
trated 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 compounds (4a–4d) as yellow solids.
;
(DMSO-d6) d: 9.81 (s, 1H, NH), 8.16 (d, 2H, J = 8.4 Hz, arom), 7.94
(s, 1H, arom), 7.81 (d, 2H, J = 8.7 Hz, arom), 7.60 (s, 1H, arom),
4.31 (q, 2H, J = 6.9 Hz, CH2), 4.05 (s, 3H, CH3), 1.45 (t, 3H,
J = 6.9 Hz, CH3). Anal. Calcd for C17H15F3N4O2: C, 56.04; H, 4.15;
N, 15.38; Found: C, 56.07; H, 4.18; N, 15.41.
5.3.4. 7-Ethoxy-6-methoxy-4-(3-trifluoromethoxyanilino)-
1,2,3-benzotriazine (6d)
Yield:70%. Mp: 207–209 °C. ESI-MS: m/z, 381.2 [M+H]+. IR
(KBr): 3426, 1616, 1510, 1444, 1423, 1255, 1166, 1105,
869 cmꢀ1 1H NMR (DMSO-d6) d: 9.73 (s, 1H, NH), 8.04 (s, 1H,
;
arom), 7.92 (m, 2H, arom), 7.58 (m, 2H, arom), 7.15 (d, 2H,
J = 7.8 Hz, arom), 4.31 (q, 2H, J = 6.9 Hz, CH2), 4.04 (s, 3H, CH3),
1.45 (t, 3H, J = 6.9 Hz, CH3). Anal. Calcd for C17H15F3N4O3: C,
53.69; H, 3.98; N, 14.73; Found: C, 53.65; H, 3.96; N, 14.69.
5.3.5. 7-Ethoxy-6-methoxy-4-(4-trifluoromethoxyanilino)-
1,2,3-benzotriazine (6e)
Yield: 69%. Mp: 224–226 °C. ESI-MS: m/z, 381.2 [M+H]+. IR
(KBr): 3429, 1615, 1509, 1428, 1246, 1164, 1102, 1015,
5.3. General procedure for the synthesis of final compounds 6a–
6z
851 cmꢀ1 1H NMR (DMSO-d6) d: 9.70 (s, 1H, NH), 7.98 (d, 2H,
;
J = 9 Hz, arom), 7.90 (s, 1H, arom), 7.57 (s, 1H, arom), 7.46 (d, 2H,
J = 8.7 Hz, arom), 4.30 (q, 2H, J = 6.9 Hz, CH2), 4.04 (s, 3H, CH3),
1.45 (t, 3H, J = 6.9 Hz, CH3). Anal. Calcd for C17H15F3N4O3: C,
53.69; H, 3.98; N, 14.73; Found: C, 53.72; H, 3.99; N, 14.76.
Compounds
2-amino-5-methoxy-4-substitutedbenzonitriles
(4a–4d) (10.0 mmol) in 10 N hydrochloric acid (30.0 mL) were
cooled to 0 °C and diazotized with sodium nitrite (0.71 g) in water
(10.0 mL). The diazonium solution was neutralized with excess of
sodium acetate trihydrate and stirred for 2 h at 0 °C with the cor-
responding substituted anilines (10.0 mmol). The solution was
kept overnight at 4 °C, filtered, and washed with water. The crude
products were then purified by recrystallization with ethyl acetate
to obtain intermediate (5a–5z). Compounds (5a–5z) were boiled in
70% ethanol (25.0 mL) for 1 h, and then evaporated under reduced
pressure to dryness. Acetic acid (10.0 mL) was added and the solu-
tion was refluxed for 2 h, cooled, poured into water (100 mL), fil-
tered and dried. The crude products thus obtained were
recrystallized from ethanol to give the final compounds 7-alk-
oxyl-6-methoxy-4-substituted-1,2,3-benzotriazines (6a–6z).
5.3.6. 4-(3-Chloroanilino)-7-ethoxy-6-methoxy-1,2,3-
benzotriazine (6f)
Yield: 66%. Mp: 237–239 °C. ESI-MS: m/z, 331.1 [M+H]+. IR
(KBr): 3377, 3296, 2924, 1612, 1510, 1430, 1292, 1248, 1106,
1101, 839 cmꢀ1 1H NMR (DMSO-d6) d: 9.65 (s, 1H, NH), 8.09 (s,
;
1H, arom), 7.90 (s, 1H, arom), 7.84 (d, 1H, J = 8.1 Hz, arom), 7.58
(s, 1H, arom), 7.48 (t, 1H, J = 8.1 Hz, arom), 7.22 (d, 1H, J = 8.1 Hz,
arom), 4.31 (q, 2H, J = 6.9 Hz, CH2), 4.03 (s, 3H, CH3), 1.44 (t, 3H,
J = 6.9 Hz, CH3). 13C-NMR (DMSO-d6) d: 154.22, 153.27, 150.12,
141.65, 137.94, 126.57, 127.48, 123.97, 64.74, 56.73, 14.42. Anal.
Calcd for C16H15ClN4O2: C, 58.10; H, 4.57; N, 16.94; Found: C,
58.14; H, 4.60; N, 16.96.
5.3.1. 7-Ethoxy-4-(3-fluoro-4-bromoanilino)-6-methoxy-1,2,3-
benzotriazine (6a)
5.3.7. 7-Ethoxy-4-(4-fluoro-3-trifluoromethylanilino)-6-
methoxy-1,2,3-benzotriazine (6g)
Yield: 68%. Mp: 238–240 °C. ESI-MS: m/z, 393.0 [M+H]+. IR
(KBr): 3326, 2918, 1682, 1623, 1509, 1432, 1289, 1243, 1106,
Yield: 70%. Mp: 216–218 °C. ESI-MS: m/z, 383.2 [M+H]+. IR
(KBr): 3451, 1615, 1508, 1433, 1326, 1326, 1291, 1236, 1131,
843 cmꢀ1
;
1H NMR (DMSO-d6) d: 9.74 (s, 1H, NH), 8.13 (dd, 1H,
842 cmꢀ1 1H NMR (DMSO-d6) d: 9.79 (s, 1H, NH), 8.28 (m, 2H,
;
J = 2.1 Hz, 11.7 Hz, arom), 7.89 (s, 1H, H-8), 7.72 (m, 2H, arom),
7.59 (s, 1H, arom), 4.31 (q, 2H, J = 6.9 Hz, CH2), 4.04 (s, 3H,
CH3),1.44 (t, 3H, J = 6.9 Hz,CH3). Anal. Calcd for C16H14BrFN4O2: C,
48.87; H, 3.59; N, 14.25; Found: C, 48.88; H, 3.61; N, 14.26.
arom), 7.88 (s, 1H, arom), 7.61 (m, 2H, arom), 4.31 (q, 2H,
J = 6.9 Hz, CH2), 4.02 (s, 3H, CH3), 1.45 (t, 3H, J = 6.9 Hz, CH3). Anal.
Calcd for C17H14F4N4O2: C, 53.41; H, 3.69; N, 14.65; Found: C,
53.36; H, 3.65; N, 14.60.
5.3.2. 7-Ethoxy-6-methoxy-4-(3-trifluoromethylanilino)-1,2,3-
benzotriazine (6b)
5.3.8. 7-Ethoxy-4-(2-fluoroanilino)-6-methoxy-1,2,3-
benzotriazine (6h)
Yield: 70%. Mp: 228–230 °C. ESI-MS: m/z, 365.1 [M+H]+. IR
(KBr): 3344, 3132, 2936, 1702, 1619, 1572, 1509, 1447, 1332,
Yield: 66%. Mp: 224–226 °C. ESI-MS: m/z, 315.2 [M+H]+. IR
(KBr): 3273, 2919, 2849, 1609, 1567, 1505, 1455, 1422, 1286,
1290, 1243, 1163, 1112, 840, 797, 699.2 cmꢀ1
;
1H NMR (DMSO-
1237, 1105, 763 cmꢀ1 1H NMR (DMSO-d6) d: 9.67 (s, 1H, NH),
;
d6) d: 9.78 (s, 1H, NH), 8.30 (s, 1H, arom), 8.24 (d, 1H, arom),
7.92 (s, 1H, arom), 7.69 (t, 1H, arom), 7.60 (s, 1H, H-5), 7.52 (d,
1H, arom), 4.31 (q, 2H, J = 6.9 Hz, CH2), 4.05 (s, 3H, CH3), 1.45 (t,
3H, J = 6.9 Hz, CH3). Anal. Calcd for C17H15F3N4O2: C, 56.04; H,
4.15; N, 15.38; Found: C, 56.02; H, 4.13; N, 15.35.
7.88 (s, 1H, arom), 7.59 (m, 2H, arom), 7.36 (m, 3H, arom), 4.31
(q, 2H, J = 6.9 Hz, CH2), 4.02 (s, 3H, CH3), 1.45 (t, 3H, J = 6.9 Hz,
CH3). Anal. Calcd for C16H15FN4O2: C, 61.14; H, 4.81; N, 17.83;
Found: C, 61.11; H, 4.79; N, 17.81.

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X.-W. Zhao et al. / Bioorg. Med. Chem. 21 (2013) 7807–7815
5.3.9. 4-(3-Bromoanilino)-7-ethoxy-6-methoxy-1,2,3-
benzotriazine (6i)
H18F4N4O2: C, 55.61; H, 4.42; N, 13.65; Found: C, 55.58; H, 4.40;
N, 13.61.
Yield: 68%. Mp: 232–234 °C. ESI-MS: m/z, 375.0 [M+H]+. IR
(KBr): 3429, 2925, 1613, 1506, 1428, 1291, 1243, 1105, 859,
5.3.15. 7-n-Butoxy-4-(2-fluoroanilino)-6-methoxy-1,2,3-
benzotriazine (6o)
781.9 cmꢀ1 1H NMR (DMSO-d6) d: 9.64 (s, 1H, NH), 8.21 (s, 1H,
;
arom), 7.91 (d,1H, J = 7.8 Hz, arom), 7.89 (s, 1H, arom), 7.58 (s,
1H, arom), 7.39 (m, 2H, arom), 4.30 (q, 2H, J = 6.9 Hz, CH2), 4.04
(s, 3H, CH3), 1.44 (t, 3H, J = 6.9 Hz, CH3). Anal. Calcd for C16H15BrN4-
O2: C, 51.22; H, 4.03; N, 14.93; Found: C, 51.23; H, 4.06; N, 14.96.
Yield: 61%. Mp: 225–227 °C. ESI-MS: m/z, 343.2 [M+H]+. IR
(KBr): 3357, 2958, 1611, 1503, 1453, 1112, 840, 758 cmꢀ1 1H
;
NMR (DMSO-d6) d: 9.66 (s, 1H, NH), 7.87 (s, 1H, arom), 7.59 (m,
2H, arom), 7.34 (m, 3H, arom), 4.24 (t, 2H, J = 6.4 Hz, CH2), 4.01
(s, 3H, CH3), 1.81 (m, 2H, CH2), 1.49 (m, 2H, CH2), 0.97 (t, 3H,
J = 7.3 Hz, CH3). Anal. Calcd for C18H19FN4O2: C, 63.15; H, 5.59; N,
16.36; Found: C, 63.16; H, 5.61; N, 16.38.
5.3.10. 7-n-Butoxy-4-(3-fluoro-4-bromoanilino)-6-methoxy-
1,2,3-benzotriazine (6j)
Yield: 64%.Mp: 227–229 °C. ESI-MS: m/z, 421.2 [M+H]+. IR
(KBr): 3423, 2959, 1614, 1511, 1429, 1289, 1240, 1104,
5.3.16. 4-(4-Bromoanilino)-7-n-butoxy-6-methoxy-1,2,3-
benzotriazine (6p)
846 cmꢀ1 1H NMR (DMSO-d6) d: 9.75 (s, 1H, NH), 8.13 (d, 1H,
;
J = 11.6 Hz, arom), 7.90 (s, 1H, arom), 7.72 (m, 2H, arom), 7.61 (s,
1H, arom), 4.25 (t, 2H, J = 6.4 Hz, CH2), 4.04 (s, 3H, CH3), 1.81 (m,
2H, CH2), 1.51 (m, 2H, CH2), 0.97 (t, 3H, J = 7.3 Hz, CH3). Anal. Calcd
for C18H18BrFN4O2: C, 51.32; H, 4.31; N, 13.30; Found: C, 51.30; H,
4.28; N, 13.29.
Yield: 64%. Mp: 231–232 °C. ESI-MS: m/z, 403.7 [M+H]+. IR
(KBr): 3426, 2930, 1613, 1508, 1428, 1291, 1106, 855, 780 cmꢀ1
;
1H NMR (DMSO-d6) d: 9.66 (s, 1H, NH), 8.22 (s, 1H, arom), 7.91
(m, 2H, H-8, arom), 7.59 (s, 1H, arom), 7.36 (m, 2H, arom), 4.25
(t, 2H, J = 6.4 Hz, CH2), 4.04 (s, 3H, CH3), 1.81 (m, 2H, CH2), 1.49
(m, 2H, CH2), 0.97 (t, 3H, J = 7.3 Hz, CH3). Anal. Calcd for C18H19-
BrN4O2: C, 53.61; H, 4.75; N, 13.89; Found: C, 53.54; H, 4.68; N,
13.82.
5.3.11. 7-n-Butoxy-4-(3,4-dichloroanilino)-6-methoxy-1,2,3-
benzotriazine (6k)
Yield: 55%. Mp: 213–215 °C. ESI-MS: m/z, 393.2 [M+H]+. IR
(KBr): 3424, 2959, 1614, 1508, 1425, 1239, 1104, 854 cmꢀ1
;
1H
5.3.17. 7-n-Butoxy-4-(4-fluoroanilino)-6-methoxy-1,2,3-
benzotriazine (6q)
NMR (DMSO-d6) d: 9.71 (s, 1H, NH), 8.31 (d, 1H, J = 2.1 Hz, arom),
7.92 (d, 1H, J = 6.9 Hz, arom), 7.88 (s, 1H, arom), 7.70 (d, 1H,
J = 8.7 Hz, arom), 7.61 (s, 1H, arom), 4.25 (t, 2H, CH2), 4.04 (s, 3H,
CH3), 1.81 (m, 2H, CH2), 1.49 (m, 2H, CH2), 0.97 (t, 3H, J = 7.3 Hz,
CH3). Anal. Calcd for C18H18Cl2N4O2: C, 54.97; H, 4.61; N, 14.25;
Found: C, 54.99; H, 4.64; N, 14.28.
Yield: 57%. Mp: 220–222 °C. ESI-MS: m/z, 343.1 [M+H]+. IR
(KBr): 3430, 2960, 1614, 1572, 1509, 1428, 1222, 1107, 1014,
830 cmꢀ1 1H NMR (DMSO-d6) d: 9.59 (s, 1H, NH), 7.88 (s, 1H,
;
arom), 7.83 (m, 2H, arom), 7.71 (s, 1H, arom), 7.29 (m, 2H, arom),
4.26 (t, 2H, J = 6.4 Hz, CH2), 4.03 (s, 3H, CH3), 1.81 (m, 2H, CH2),
1.49 (m, 2H, CH2), 0.97 (t, 3H, J = 7.3 Hz, CH3). Anal. Calcd for C18-
H19FN4O2: C, 63.15; H, 5.59; N, 16.36; Found: C, 63.20; H, 5.63;
N, 16.41.
5.3.12. 7-n-Butoxy-6-methoxy-4-(4-trifluoromethylanilino)-
1,2,3-benzotriazine (6l)
Yield: 50%. Mp: 238–240 °C. ESI-MS: m/z, 393.2 [M+H]+. IR
(KBr): 3333, 3133, 2964, 2937, 1608, 1569, 1508, 1446, 1336,
5.3.18. 7-n-Butoxy-4-(4-methylanilino)-6-methoxy-1,2,3-
benzotriazine (6r)
1292, 1244, 1162, 1127, 800 cmꢀ1 1H NMR (DMSO-d6) d: 9.77 (s,
;
1H, NH), 8.31 (s, 1H, arom), 8.25 (d, 1H, arom), 7.90 (s, 1H, arom),
7.69 (t, 1H, arom), 7.60 (s, 1H, arom), 7.51 (d, 1H, arom), 4.25 (t, 2H,
J = 6.4 Hz, CH2), 4.05 (s, 3H, CH3), 1.81 (m, 2H, CH2), 1.49 (m, 2H,
CH2), 0.98 (t, 3H, J = 7.3 Hz, CH3). 13C NMR (DMSO-d6) d: 154.93,
153.91, 150.46, 143.25, 142.28, 126.29, 125.86, 124.06, 123.87,
123.65, 122.14, 107.22, 104.39, 100.37, 69.13, 57.18, 30.85, 19.20,
14.12. Anal. Calcd for C19H19F3N4O2: C, 58.16; H, 4.88; N, 14.28;
Found: C, 58.20; H, 4.92; N, 14.32.
Yield:56%. Mp: 214–215 °C. ESI-MS: m/z, 339.2 [M+H]+. IR
(KBr): 3335, 2958, 2927, 2872, 2851, 1613, 1569, 1512, 1428,
1286, 1236, 1102, 852 cmꢀ1 1H NMR (DMSO-d6) d: 9.51 (s, 1H,
;
NH), 7.90 (s, 1H, arom), 7.70 (d, 2H, J = 8.2 Hz, arom), 7.54 (s, 1H,
arom), 7.25 (d, 2H, J = 8.2 Hz, arom), 4.24 (t, 2H, J = 6.4 Hz, CH2),
4.02 (s, 3H, CH3), 2.34 (s, 3H, CH3), 1.81 (m, 2H, CH2), 1.49 (m,
2H, CH2), 0.97 (t, 3H, J = 7.3 Hz, CH3). Anal. Calcd for C19H22N4O2:
C, 67.44; H, 6.55; N, 16.56; Found: C, 67.42; H, 6.53; N, 16.55.
5.3.13. 7-n-Butoxy-4-(4-trifluoromethoxyanilino)-6-methoxy-
1,2,3-benzotriazine (6m)
5.3.19. 4-(4-Chloroanilino)-6-methoxy-7-pentyloxy-1,2,3-
benzotriazine (6s)
Yield: 63%. Mp: 232–234 °C. ESI-MS: m/z, 409.2 [M+H]+. IR
(KBr): 3298, 2963, 1615, 1509, 1425, 1249, 1200, 1166, 1100,
Yield: 72%. Mp: 212–214 °C. ESI-MS: m/z, 373.2 [M+H]+. IR
(KBr): 3378, 2954, 1612, 1507, 1425, 1245, 1101, 1014,
855 cmꢀ1
;
1H-NMR (DMSO-d6) d: 9.69 (s, 1H, NH), 7.97 (d, 2H,
817 cmꢀ1 1H NMR (DMSO-d6) d: 9.63 (s, 1H, NH), 7.91 (m, 3H,
;
J = 9 Hz, arom), 7.90 (s, 1H, H-8), 7.58 (s, 1H, arom), 7.46 (d, 2H,
J = 8.7 Hz, arom), 4.25 (t, 2H, J = 6.4 Hz, CH2), 4.04 (s, 3H, CH3),
1.81 (m, 2H, CH2), 1.49 (m, 2H, CH2), 0.97 (t, 3H, J = 7.3 Hz, CH3).
Anal. Calcd for C19H19F3N4O3: C, 55.88; H, 4.69; N, 13.72; Found:
C, 55.91; H, 4.72; N, 13.74.
H-8, arom), 7.57 (s, 1H, arom), 7.50 (d, 2H, J = 8.4 Hz, arom), 4.24
(t, 2H, J = 6.4 Hz, CH2), 4.03 (s, 3H, CH3), 1.81 (m, 2H, CH2), 1.40
(m, 4H, 2ꢁCH2), 0.92 (t, 3H, J = 7.0 Hz, CH3). 13C NMR (DMSO-d6)
d: 154.31, 153.27, 150.30, 141.63, 138.88, 128.73, 128.73, 124.04,
122.69, 122.69, 106.81, 103.82, 100.15, 68.98, 56.79, 28.17, 27.78,
21.98, 14.04. Anal. Calcd for C19H21ClN4O2: C, 61.21; H, 5.68; N,
15.03; Found: C, 61.25; H, 5.71; N, 15.06.
5.3.14. 7-n-Butoxy-4-(4-fluoro-3-trifluoromethylanilino)-6-
methoxy-1,2,3-benzotriazine (6n)
Yield: 62%. Mp: 233–235 °C. ESI-MS: m/z, 411.2 [M+H]+. IR
(KBr): 3360, 2962, 1612, 1508, 1432, 1290, 1234, 1137,
5.3.20. 4-(4-Bromo-3-fluoroanilino)-6-methoxy-7-pentyloxy-
1,2,3-benzotriazine (6t)
830 cmꢀ1
;
1H NMR (DMSO-d6) d: 9.78 (s, 1H, NH), 8.29 (m, 2H,
Yield: 75%. Mp: 220–222 °C. ESI-MS: m/z, 435.0 [M+H]+. IR
(KBr): 3265, 2956, 1613, 1510, 1429, 1288, 1239, 1177, 1104,
arom), 7.87 (s, 1H, arom), 7.63 (d, 1H, J = 9.6 Hz, arom), 7.61 (s,
1H, arom), 4.25 (t, 2H, CH2), 4.04 (s, 3H, CH3), 1.81 (m, 2H, CH2),
1.49 (m, 2H, CH2), 0.97 (t, 3H, J = 7.3 Hz, CH3). Anal. Calcd for C19-
841, 819, 786 cmꢀ1 1H NMR (DMSO-d6) d: 9.75 (s, 1H, NH), 8.16
;
(m, 1H, arom), 7.90 (s, 1H, arom), 7.73 (m, 2H, arom), 7.61 (s, 1H,

X.-W. Zhao et al. / Bioorg. Med. Chem. 21 (2013) 7807–7815
7813
arom), 4.25 (t, 2H, CH2), 4.04 (s, 3H, CH3), 1.83 (m, 2H, CH2), 1.42
(m, 4H, 2ꢁCH2), 0.93 (t, 3H, CH3). Anal. Calcd for C19H20BrFN4O2:
C, 52.43; H, 4.63; N, 12.87; Found: C, 52.41; H, 4.60; N, 12.84.
d, J = 8.4 Hz, arom), 7.59 (1H, s, arom), 7.25 (2H, d, J = 8.4 Hz, arom),
4.37 (2H, t, J = 6.0 Hz, CH2), 4.03 (3H, s, CH3), 3.84 (2H, t, J = 6.3 Hz,
CH2), 2.34 (3H, s, CH3), 2.29 (2H, m, CH2). 13C NMR (DMSO-d6) d:
154.23, 153.52, 150.62, 141.59, 136.43, 133.64, 129.48, 123.22,
107.28, 104.42, 100.86, 66.14, 57.22, 42.24, 31.84, 20.99. Anal.
Calcd for C18H19ClN4O2: C, 60.25; H, 5.34; N, 15.61; Found: C,
60.28; H, 5.33; N, 15.56.
5.3.21. 4-(3,5-Difluoroanilino)-6-methoxy-7-pentyloxy-1,2,3-
benzotriazine (6u)
Yield: 68%. Mp: 192–194 °C. ESI-MS: m/z, 375.1 [M+H]+. IR
(KBr): 3345, 2936, 1620, 1512, 1449, 1291, 1235, 1114, 992, 846,
784 cmꢀ1
;
1H NMR (DMSO-d6) d: 9.77 (s, 1H, NH), 7.89 (s, 1H,
5.4. General procedure for the synthesis of final compounds 4-
arom), 7.75 (d, 2H, J = 8.7 Hz, arom), 7.62 (s, 1H, arom), 7.00 (t,
1H, J = 9.3 Hz, arom), 4.24 (t, 2H, J = 6.4 Hz, CH2), 4.05 (s, 3H,
CH3), 1.83 (m, 2H, CH2), 1.42 (m, 4H, 2ꢁCH2), 0.92 (t, 3H,
J = 7.2 Hz, CH3). Anal. Calcd for C19H20F2N4O2: C, 60.95; H, 5.38;
N, 14.97; Found: C, 60.99; H, 5.41; N, 15.02.
substituted-6-chloro-pyrido-[3,2-d]-1,2,3-triazine (11a–11j)
A mixture of 2,6-dichloro-3-nitropyridine (7) (26 mmol), cu-
prous cyanide (0.59 mml) and appropriate amount of N-methyl
pyrrolidone was heated at 180 °C for 15 min, then cooled to 10 °C
and poured into ice-water (200 mL) and stirred for 30 min. The
solution was then filtered, washed with water and dried. The result-
ing 2-cyano-3-nitro-6-chloropyridine (8) was extracted with boil-
ing toluene (50 mL) for 3 times, combined the organic phases and
washed with water and saturated sodium chloride solution. Then
the organic phase was dried, filtered, evaporated under reduced
pressure to dryness, and purified by recrystallization with a mix-
ture of petroleum ether/ethyl acetate (4/1). The reduction of the
intermediate 8 (10 mmol) was reacted with sodium hyposulfite
(80 mmol, and 40 mmol 2 h later) in dichloromethane/aqua
(20 mL/20 mL) solution, and added TBAB (1 mmol) as a phase-
transfer catalyst. The mixture was stirred at room temperature for
3 h, and then extracted with dichloromethane (40 mL) for 3 times.
The combined organic phase was dried with anhydrous magnesium
sulfate, filtered and evaporated under reduced pressure to yield 2-
cyano-6-chloro-3-pyridinylamine (9). Compound 9 was added into
3 mL of hydrochloric acid (10 mol/L), and the aqueous solution of
sodium nitrite (0.072 g/1.0 mL) was added dropwise under stirring
for 20 min in an ice-salt bath. The diazonium solution was reacted
with the corresponding substituted anilines (10.0 mmol) and then
neutralized with excess of sodium acetate trihydrate, stirred for
2 h at 0 °C. The solution was kept overnight, filtered, and washed
with water to yield the crude products. The crude products were
then purified by recrystallization with ethyl acetate to obtain inter-
5.3.22. 6-Methoxy-7-pentyloxy-4-(3-trifluoromethoxyanilino)-
1,2,3-benzotriazine (6v)
Yield: 73%. Mp: 181–183 °C. ESI-MS: m/z, 423.2 [M+H]+. IR (KBr):
3421, 2931, 1613, 1508, 1446, 1258, 1216, 1163, 1105, 845,
784 cmꢀ1; 1H NMR (DMSO-d6) d: 9.72 (s, 1H, NH), 8.04 (s, 1H, arom),
7.92 (d, 1H, J = 8.9 Hz, arom), 7.91 (s, 1H, arom), 7.60 (s, 1H, arom),
7.56 (t, 2H, J = 8.6 Hz, arom), 7.14 (d, 1H, J = 8.7 Hz, arom), 4.24 (t,
2H, J = 6.3 Hz, CH2), 4.05 (s, 3H, CH3), 1.83 (m, 2H, CH2), 1.42 (m,
4H, 2ꢁCH2), 0.92 (t, 3H, J = 6.9 Hz, CH3). Anal. Calcd for C20H21F3N4-
O3: C, 56.87; H, 5.01; N, 13.26; Found: C, 56.92; H, 5.08; N, 13.31.
5.3.23. 6-Methoxy-7-pentyloxy-4-(4-trifluoromethoxyanilino)-
1,2,3-benzotriazine (6w)
Yield: 70%. Mp: 230–232 °C. ESI-MS: m/z, 423.2 [M+H]+. IR
(KBr): 3426, 2959, 1615, 1510, 1428, 1247, 1200, 1165, 1102,
850, 786 cmꢀ1 1H NMR (DMSO-d6) d: 9.69 (s, 1H, NH), 7.98 (d,
;
2H, J = 9.0 Hz, arom), 7.90 (s, 1H, arom), 7.58 (s, 1H, arom), 7.46
(d, 2H, J = 8.7 Hz, arom), 4.24 (t, 2H, J = 6.3 Hz, CH2), 4.04 (s, 3H,
CH3), 1.83 (m, 2H, CH2), 1.42 (m, 4H, 2ꢁCH2), 0.92 (t, 3H,
J = 6.9 Hz, CH3). Anal. Calcd for C20H21F3N4O3: C, 56.87; H, 5.01;
N, 13.26; Found: C, 56.82; H, 4.97; N, 13.22.
5.3.24. 6-Methoxy-4-(4-fluoroanilino)-7-pentyloxy-1,2,3-
benzotriazine (6x)
mediates
1-substituted-3-(2-cyano-6-chloro-3-pyridyl)triazene
Yield: 64%. Mp: 213–215 °C. ESI-MS: m/z, 357.2 [M+H]+. IR
(KBr): 3369, 2953, 1616, 1509, 1428, 1283, 1222, 1158, 1107,
(10a–10j). The intermediates 10a–10j were boiled in 70% ethanol
(25.0 mL) for 1 h, and then evaporated under reduced pressure to
dryness. Acetic acid (10.0 mL) was added and the solution was re-
fluxed for 2 h, cooled, poured into water (100 mL), filtered and
dried. The crude products thus obtained were recrystallized from
ethanol to give the final compounds 4-substituted-6-chloro-pyr-
ido[3,2-d][1,2,3]triazines (11a–11j).
832, 786 cmꢀ1 1H NMR (DMSO-d6) d: 9.60 (s, 1H, NH), 7.88 (s,
;
1H, arom), 7.82 (m, 2H, arom), 7.55 (s, 1H, arom), 7.29 (m, 2H,
arom), 4.23 (t, 2H, J = 6.3 Hz, CH2), 4.03 (s, 3H, CH3), 1.83 (m, 2H,
CH2), 1.42 (m, 4H, 2ꢁCH2), 0.92 (t, 3H, J = 7.0 Hz, CH3). Anal. Calcd
for C19H21FN4O2: C, 64.03; H, 5.94; N, 15.72; Found: C, 64.06; H,
5.96; N, 15.77.
5.4.1. 6-Chloro-4-(4-chloroanilino)-pyrido[3,2-d]-1,2,3-triazine
(11a)
5.3.25. 7-(3-Chloropropoxy)-6-methoxy-4-(4-
trifluoromethylanilino)-1,2,3-benzotriazine (6y)
Yield: 43%. Mp: 207–209 °C. ESI-MS: m/z, 314.0 [M+H]+. IR
Yield: 70%. Mp: 179–180 °C. ESI-MS: m/z, 413.1 [M+H]+. IR
(KBr): 3387, 2933, 1616, 1567, 1509, 1425, 1321, 1249, 1121,
(KBr): 3272, 3145, 1609, 1558, 1505, 1140, 827 cmꢀ1 1H NMR
;
(DMSO-d6) d: 10.49 (s, 1H, NH), 8.68 (d, 1H, J = 8.7 Hz, H-8), 8.20
(d, 1H, J = 8.7 Hz, H-7), 8.07 (d, 2H, J = 8.7 Hz, arom-20 H, 60 H),
7.51 (d, 2H, J = 8.7 Hz, arom-30 H, 50 H). 13C NMR (DMSO-d6) d:
154.24, 150.66, 139.14, 138.28, 136.91, 131.71, 131.71, 128.55,
128.55, 126.01, 124.50, 124.50. Anal. Calcd for C12H7Cl2N5: C,
49.34; H, 2.42; N, 23.97 Found: C, 49.33; H, 2.41; N, 23.95.
848 cmꢀ1 1H NMR (DMSO-d6) d: 9.83 (s, 1H, NH), 8.16 (d, 2H,
;
J = 8.5 Hz, arom), 7.96 (s, 1H, arom), 7.82 (d, 2H, J = 8.6 Hz, arom),
7.66 (s, 1H, arom), 4.37 (t, 2H, J = 6.0 Hz, CH2), 4.06 (s, 3H, CH3),
3.84 (t, 2H, J = 6.4 Hz, CH2), 2.29 (m, 2H, CH2). 13C NMR (DMSO-
d6) d: 154.50, 153.81, 150.45, 143.20, 142.16, 126.25, 125.85,
124.05, 123.90, 123.68, 122.16, 107.42, 104.60, 100.56, 66.25,
57.25, 42.23, 31.86. Anal. Calcd for C18H16ClF3N4O2: C, 52.37; H,
3.91; N, 13.57; Found: C, 52.40; H, 3.98; N, 14.61.
5.4.2. 4-(4-Bromo-3-fluoroanilino)-6-chloro-pyrido[3,2-d]-
1,2,3-triazine (11b)
Yield: 45%. Mp: 200–202 °C. ESI-MS: m/z, 354.0 [M+H]+. IR
5.3.26. 7-(3-Chloropropoxy)-6-methoxy-4-(4-methylanilino)-
1,2,3-benzotriazine (6z)
(KBr): 3441, 3290, 2920, 1606, 1505, 1447, 1136, 840, 642 cmꢀ1
;
1H NMR (DMSO-d6) d: 10.63 (s, 1H, NH), 8.72 (d, 1H, J = 8.7 Hz,
H-8),8.23 (m, 2H, H-7, arom), 7.96 (d, 1H, J = 8.7 Hz, arom), 7.77
(t, 1H, J = 8.4 Hz, arom). Anal. Calcd for C12H6BrClFN5: C, 40.65; H,
1.71; N, 19.75, Found: C, 40.71; H, 1.78; N, 19.79.
Yield: 70.1%; Mp: 245–247 °C. ESI-MS: m/z, 359.1 [M+H]+. IR
(KBr): 3420, 2924, 1615, 1513, 1425, 1282, 1242, 859 cmꢀ1 1H
;
NMR (DMSO-d6) d: 9.55 (1H, s, NH), 7.92 (1H, s, arom), 7.69 (2H,

7814
X.-W. Zhao et al. / Bioorg. Med. Chem. 21 (2013) 7807–7815
5.4.3. 6-Chloro-4-(3,4-dichloroanilino)-pyrido[3,2-d]-1,2,3-
triazine (11c)
760 cmꢀ1 1H NMR (DMSO-d6) d: 10.36 (s, 1H, NH), 8.69 (d, 1H,
;
J = 8.7 Hz, H-8), 8.21 (d, 1H, J = 8.7 Hz, H-7), 7.71(t, 1H, J = 8.4 Hz,
arom-60 H), 7.36 (m, 3H, arom-30 H, 40 H, 50 H). Calcd for C12H7-
ClFN5: C, 52.28; H, 2.56; N, 25.40 Found: C, 52.35; H, 2.58; N, 25.44.
Yield: 48%. Mp: 190–192 °C. ESI-MS: m/z, 326.0 [M+H]+. IR
(KBr): 3357, 1600, 1556, 1505, 1450, 1145, 843 cm-1 1H NMR
;
(DMSO-d6) d: 10.64 (s, 1H, NH), 8.74 (d, 1H, J = 8.7 Hz, H-8), 8.28
(s, 2H, arom-30 H, 50 H), 8.24 (d, 1H, J = 8.7 Hz, H-7), 7.43 (s, 1H,
arom-40 H). Calcd for C12H6Cl3N5: C, 44.13; H, 1.85; Cl, 32.57; N,
21.45, Found: C, 44.16; H, 1.86; N, 21.47.
5.4.10. 4-(4-Bromoanilino)-6-chloro-pyrido[3,2-d]-1,2,3-
triazine (11j)
Yield: 49%. Mp: 205–207 °C. ESI-MS: m/z, 336.0 [M+H]+. IR
(KBr): 3345, 1605, 1505, 1477, 1144, 843, 777 cmꢀ1 1H NMR
;
5.4.4. 6-Chloro-4-(3-trifluoromethylanilino)-pyrido[3,2-d]-
1,2,3-triazine (11d)
(DMSO-d6) d: 10.50 (s, 1H, NH), 8.70 (d, 1H, J = 8.7 Hz, H-8), 8.38
(s, 1H, arom-20 H), 8.21 (d, 1H, J = 9.0 Hz, H-7), 8.05 (d, 1H,
J = 6.3 Hz, arom-60 H), 7.43 (m, 2H, arom-40 H, 50 H). Calcd for C12-
H7BrClN5: C, 42.82; H, 2.10; N, 20.81; Found: C, 42.88; H, 2.17; N,
20.87.
Yield: 47%. Mp: 190–192 °C. ESI-MS: m/z, 326.0 [M+H]+. IR
(KBr): 3303, 1606, 1548, 1509, 1465, 1335, 1105, 843, 802 cmꢀ1
;
1H NMR (DMSO-d6) d: 10.66 (s, 1H, NH), 8.72 (d, 1H, J = 8.7 Hz,
H-8), 8.52 (s, 1H, arom-20 H), 8.38 (d, 1H, J = 8.4 Hz, arom-40 H),
8.25 (d, 1H, J = 8.7 Hz, H-7), 7.70 (t, 1H, J = 8.1 Hz, arom-50 H),
7.56 (d, 1H, J = 7.8 Hz, arom-60 H). Calcd for C13H7ClF3N5: C,
47.94; H, 2.17; N, 21.50 Found: C, 47.92; H, 2.14; N, 21.48.
5.5. VEGFR-2 kinase inhibition assay
VEGFR-2 Kinase Assay Kit, purchased from CST (Cell Signaling
Technology) with a serial number of 7788, was used to evaluate
the inhibitory effects of target compounds on the VEGFR-2 kinase
activity. PTK787 was used as the positive control for VEGFR-2 ki-
nase, and 0.1% (v/v) DMSO was the negative control. The assay
was performed in 96-well polystyrene round-bottomed plates
5.4.5. 6-Chloro-4-(4-trifluoromethylanilino)-pyrido[3,2-d]-
1,2,3-triazine (11e)
Yield: 46%. Mp: 206–208 °C. ESI-MS: m/z, 326.0 [M+H]+. IR
(KBr): 3250, 3051, 1614, 1499, 1454, 1321, 1110, 1067,
846 cmꢀ1
;
1H NMR (DMSO-d6) d: 10.66 (s, 1H, NH), 8.72 (d, 1H,
pre-coated with 3
final volume of 100
10 g/mL of the selected compound were added in each well.
The reaction was initiated by adding 8 ng/ L of VEGFR-2 kinase
lM poly (Glu, Tyr)4:1 as a substrate and with a
J = 8.7 Hz, H-8), 8.32 (d, 2H, J = 8.4 Hz, arom-30 H, 50 H), 8.23 (d,
1H, J = 8.7 Hz, H-7), 7.82 (d, 2H, J = 8.4 Hz, arom-20 H, 60 H). 13C
NMR (DMSO-d6) d: 154.88, 151.12, 142.04, 139.57, 138.77,
132.21, 126.61 (q, J = 3.75 Hz), 126.29, 126.18 (q, J = 3.6 Hz),
124.80 (q, J = 270 Hz, –CF3), 124.76 (q, J = 31.8 Hz, –CCF3), 122.98,
120.86. Calcd for C13H7ClF3N5: C, 47.94; H, 2.17; N, 21.50 Found:
C, 47.98; H, 2.20; N, 21.56.
l
L in each well. A solution of 40 M ATP and
l
l
l
and incubated for 5 min at room temperature. After adding ATP
solution and incubating for 30 min at room temperature, the reac-
tion was stopped by adding 50 mM EDTA. Next, 100
tyrosine mAb antibody was added. After 1 h of incubation at room
temperature, the plate was washed three times with 200 L PBS-T.
Then 100 L of anti-rat IgG (1:500 dilution) and anti-rabbit IgG
lL of Phospho-
l
5.4.6. 6-Chloro-4-(3-trifluoromethoxyanilino)-pyrido[3,2-d]-
1,2,3-triazine (11f)
l
(1:1000 dilution) diluted with PBS-T containing 1% BSA was added.
The plate was reincubated at room temperature for 30 min, and
Yield: 40%. Mp: 147–149 °C. ESI-MS: m/z, 342.0 [M+H]+. IR
(KBr): 3292, 1613, 1505, 1464, 1286, 1133, 882, 841 cmꢀ1
;
1H
washed five times as before. Finally, 100
and incubated at room temperature for 15 min. The reaction was
terminated by the addition of 100 L of 2 N H2SO4, and A450 was
lL of TMB was added
NMR (DMSO-d6) d: 10.61 (s, 1H, NH), 8.71 (d, 1H, J = 8.7 Hz, H-8),
8.18 (m, 3H, H-7, arom-20 H, 40 H), 7.58 (t, 1H, J = 8.1 Hz, arom-50
H), 7.19 (d, 1H, J = 8.1 Hz, arom-60 H). 13C NMR (DMSO-d6) d:
154.78, 151.12, 148.76, 140.08, 139.56, 138.70, 132.15, 130.64,
126.32, 121.47 (q, J = 255 Hz, –CF3), 118.07, 116.92, 115.26. Calcd
for C13H7ClF3N5O: C, 45.70; H, 2.06; N, 20.50; Found: C, 45.74; H,
2.09; N, 20.56.
l
measured on a microplate plate reader (Bio-Rad™). The enzyme
inhibition rate (%) was calculated using the equation:Inhibition
rate (%) = [1ꢀ(A450/A450 control)] ꢁ 100%
5.5.1. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay
5.4.7. 6-Chloro-4-(4-trifluoromethoxyanilino)-pyrido[3,2-d]-
1,2,3-triazine (11g)
The anti-proliferative activities of target compounds on MVECs
were evaluated by the MTT assay with PTK787 as the positive con-
trol. MVECs cells were cultured in DMEM with 4 mM L-glutamine
and adjusted to contain 4.50 g/L glucose and 10% (v/v) heat-inacti-
vated fetal bovine serum. Cells (2 ꢁ 103 cells/well in 100 ml med-
ium) were incubated for 24 h, then various concentrations of
each compound mixed in 100 ml medium were added to each well.
The cells were cultured for another 4 days at 37 °C. MTT solution
Yield: 45%. Mp: 165–167 °C. ESI-MS: m/z, 342.0 [M+H]+. IR
(KBr): 3281, 1611, 1508, 1266, 1157, 1031, 847 cmꢀ1
;
1H NMR
(DMSO-d6) d: 10.56 (s, 1H, NH), 8.69 (d, 1H, J = 8.7 Hz, H-8), 8.21
(d, 1H, J = 8.7 Hz, H-7), 8.13 (d, 2H, J = 9.0 Hz, arom-30 H, 50 H),
7.47 (d, 2H, J = 8.4 Hz, arom-20 H, 60 H). Calcd for C13H7ClF3N5O:
C, 45.70; H, 2.06; N, 20.50; Found: C, 45.77; H, 2.12; N, 20.53.
(50
tinued for an additional 4 h. The medium was removed by aspira-
tion and the cells were dissolved in 200 L DMSO. The absorbance
at 570 nm was measured in the 96-well plate reader. All of the
compounds were tested three times. Growth inhibition is reported
as compared to untreated cells (%) and GI50 concentration was
calculated.
lL of 2 mg/mL) was added per well and the cultures were con-
5.4.8. 6-Chloro-4-(4-fluoro-3-trifluoromethylanilino)-
pyrido[3,2-d]-1,2,3-triazine (11h)
l
Yield: 43%.Mp: 234–236 °C. ESI-MS: m/z, 344.0 [M+H]+. IR (KBr):
3307, 2924, 1593, 1510, 1457, 1133, 837 cmꢀ1; 1H NMR (DMSO-d6)
d: 10.70 (s, 1H, NH), 8.71 (d, 1H, J = 8.7 Hz, H-8), 8.51 (m, 1H, arom-
60 H), 8.42 (m, 1H, arom-20 H), 8.22 (d, 1H, J = 8.7 Hz, H-7), 7.62 (t,
1H, J = 9.6 Hz, arom-50 H). Calcd for C13H6ClF4N5: C, 45.43; H,
1.76; N, 20.38; Found: C, 45.41; H, 1.75; N, 20.36.
5.6. Molecular modeling
5.4.9. 6-Chloro-4-(2-fluoroanilino)-pyrido[3,2-d][3,2-d]-1,2,3-
triazine (11i)
The crystal structure of the VEGFR-2 kinase in complex with a
4-amino-furo[2,3-d]pyrimidine inhibitor was downloaded from
the Protein Data Bank (PDB entry 1YWN).19 All calculations and
manipulations were performed using the Surflex-Dock in the Sybyl
Yield: 41%. Mp: 195–197 °C. ESI-MS: m/z, 276.1 [M+H]+. IR
(KBr): 3356, 3065, 1620, 1601, 1514, 1465, 1139, 1010, 841,

X.-W. Zhao et al. / Bioorg. Med. Chem. 21 (2013) 7807–7815
7815
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structure were removed and hydrogen atoms were added.
Compounds PTK787, 6d,11d, and CTZ12 were built using the
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parameters were used except for max conformation per fragment
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and the maximum number of pulses per legend was 50. The dock-
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the same conformation and the rationality of the conformation.
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Acknowledgments
This work is supported by the National Major Scientific and
Technological Special Project for ‘Significant New Drugs Develop-
ment’ during the Eleven Five-year Plan Period (grant no.
2009ZX09103-080) and the authors want to thank Tripos for offer-
ing us free evaluation using their software.
17. Scott, E. N.; Meinhardt, G.; Jacques, C.; Laurent, D.; Thomas, A. L. Expert Opin.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.10.009.
20. Bold, G.; Altmann, K. H.; Frei, J.; Lang, M.; Manley, P. W.; Traxler, P.; Wietfeld,
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