Synthesis and Docking Studies of Some 1,2,3-Benzotriazine-4-one Derivatives as Potential Anticancer Agents

Article abstract of DOI:10.1002/jhet.3452

Newly synthesized 1,2,3-benzotriazine-4-one derivatives substituted at position-3 were characterized by various analytical and spectral techniques. The in vitro antitumor activity was evaluated against three different cell lines (liver cells cancer, colorectal cancer, and breast cancer), where compounds 7b, 15, and 25 showed strong antitumor activity with IC₅₀ ranging from 5.54 to 16.26?μM. In addition, molecular modeling studies using MOE were performed to investigate their binding modes to the C-Met kinase active site. Docking results demonstrated that all new compounds recognized the active sites of C-Met kinase and form different types of bonding interactions with key active site amino acid residues.

Full text of DOI:10.1002/jhet.3452

Month 2019 Synthesis and Docking Studies of Some 1,2,3-Benzotriazine-4-one Deriv-
atives as Potential Anticancer Agents
*
Ahmed A. Fadda, Ehab Abdel-Latif,
Ahmed Fekri,
and Amal R. Mostafa
Department of Chemistry, Faculty of Science, Mansoura University, Mansoura 35516, Egypt
*E-mail: ehabattia00@gmx.net
Received August 24, 2018
DOI 10.1002/jhet.3452
Published online 00 Month 2019 in Wiley Online Library (wileyonlinelibrary.com).
Newly synthesized 1,2,3-benzotriazine-4-one derivatives substituted at position-3 were characterized by
various analytical and spectral techniques. The in vitro antitumor activity was evaluated against three differ-
ent cell lines (liver cells cancer, colorectal cancer, and breast cancer), where compounds 7b, 15, and 25
showed strong antitumor activity with IC50 ranging from 5.54 to 16.26 μM. In addition, molecular modeling
studies using MOE were performed to investigate their binding modes to the C-Met kinase active site.
Docking results demonstrated that all new compounds recognized the active sites of C-Met kinase and form
different types of bonding interactions with key active site amino acid residues.
J. Heterocyclic Chem., 00, 00 (2019).
INTRODUCTION
RESULTS AND DISCUSSION
The three possible isomers of triazine (1,2,3-triazines,
1,2,4-triazines, and 1,3,5-triazines) have great interest in
medicinal chemistry due to their wide range of
activities such as antimicrobial [1,2], anti-inflammatory
[3,4], analgesic [5], antihistaminic [6], anticancer [7],
antiviral [8], and antiangiogenic [9]. The benzo-fused
derivatives of 1,2,3-triazines have attracted much
attention in both the medicinal and agrochemical fields
[10–12]. Many pharmacological properties for 1,2,3-
benzotriazine-4-ones have been reported, including
drugs with sedative, anesthetic, antitumor, antiarthritic,
diuretic, and antitubercular activities [13–18]. In this
paper, the key 2-cyano-N-(4-oxobenzo [d][1,2,3]triazin-
3(4H)-yl) acetamide (5) has been synthesized and
utilized as a precursor for various 1,2,3-benzotriazine-4-
ones containing various heterocycles (e.g., pyridine,
chromene, thiazole, and pyrazole) at position-3. The
resulting series of 3-substituted 1,2,3-benzotriazine-4-
ones was evaluated for their anticancer activities against
liver cells cancer, colorectal cancer, and breast cancer.
Chemistry. The reaction of cyanoacetic hydrazide (1)
with isatoic anhydride (2) has been achieved by refluxing
in ethanol to furnish 2-amino-N0-(2-cyanoacetyl)
benzohydrazide (3) in 67% yield. The reaction
mechanism involves nucleophilic attack of the amino part
from cyanoacetic hydrazide on the carbonyl group of
isatoic anhydride, which leads to ring opening and
decarboxylation to produce our target benzohydrazide 3
(Scheme 1). The chemical structure of this
benzohydrazide was proved based on both elemental and
spectral data. The IR absorption bands clearly indicated
the presence of NH2 and NH functions at 3459, 3363,
and 3250 cmꢀ1, nitrile function (C ≡ N) at 2261 cmꢀ1
,
and two carbonyl functions at 1687 and 1654 cmꢀ1. The
1H NMR spectrum revealed singlet for two protons at
3.78 ppm (CH2), singlet at 6.40 ppm for two protons
(NH2), two triplet signals at 6.50 and 7.21, and two
doublet signals at 6.74 and 7.53 ppm (aromatic protons).
Finally, singlet at 10.16 ppm for the two protons of imine
functions (NH).
© 2019 Wiley Periodicals, Inc.

A. A. Fadda, E. Abdel-Latif, A. Fekri, and A. R. Mostafa
Vol 000
Scheme 1. Synthetic pathway to 2-cyano-N-(4-oxobenzotriazin-3-yl)
acetamides 5 and 7.
and p-hydroxybenzaldehyde) furnished the corresponding
α,β-unsaturated nitrile derivatives 9a–d (Scheme 2). The
IR spectrum of 9a was characterized by the presence of
absorptions at 3287, 2123, and 1682 cmꢀ1 corresponding
to NH, nitrile, and carbonyl functions, respectively. The
1H NMR of 9b displayed singlet at 3.87 ppm for three
protons (OCH3), doublet at 7.14 ppm, and multiplet in
the region 7.95–8.35 for the aromatic protons in addition
to singlet at 9.16 ppm for the olefinic proton (CH=C).
The reaction of α,β-unsaturated nitrile derivatives 9a–d
with malononitrile (10) has been achieved by heating the
two reactants under reflux in ethyl alcohol and drops of
piperidine to produce the corresponding N-benzotriazinyl-
pyridone derivatives 12a–d. The reaction proceeds via a
nucleophilic attack of active methylene (from
malononitrile) on the synthesized α,β-unsaturated nitrile 9
to form the Michael adduct 11 which underwent
intramolecular cyclization and auto-oxidation achieve the
desired product 12. The chemical structures of 12a–d
have been secured by their IR, 1H NMR, and their
alternative synthesis from the reaction of the
When 2-amino-N0-(2-cyanoacetyl) benzohydrazide (3)
treated with nitrous acid (NaNO2/HCl) at 0–5°C, it readily
diazotized to form the intermediate diazonium chloride 4,
which underwent in situ intramolecular cyclization to
afford 2-cyano-N-(4-oxobenzo [d][1,2,3]triazin-3(4H)-yl)
acetamide (5) in 77% yield. The chemical structure of 5
was verified on the basis of spectral data, the IR exhibited
absorptions at 3174 cmꢀ1 corresponding to NH group,
2260 cmꢀ1 for the nitrile function (C ≡ N) while
absorptions at 1723 and 1693 cmꢀ1 was attributed to the
carbonyl functions (C=O). The protons of methylene
cyanoacetamide
(namely, 4-methylbenzylidene-malononitrile,
5
with arylidene-malononitriles 13
4-
methoxybenzylidene-malononitrile, 4-chlorobenzylidene-
malononitrile, and 4-hydroxybenzylidene-malononitrile)
in boiling ethyl alcohol and few drops of piperidine. The
spectroscopic data of the obtained N-benzotriazinyl-
pyridones 12a–d were in complete agreement with the
assigned structures. IR spectrum of 12a showed
absorptions at 3424 and 3365 cmꢀ1 for the amino group
(NH2), 2205 cmꢀ1 for the nitrile function (C ≡ N) in
addition to 1676 and 1625 cmꢀ1 for carbonyl groups
(C=O). 1H NMR spectrum of the same compound
exhibited singlet at 3.82 pm corresponding to three
protons of (OCH3) function, singlet at 7.22 ppm for the
protons of amino group (NH2) in addition to doublet,
triplet and multiplet signals in the region 6.94–8.28 for
the aromatic protons.
1
group (CH2) appeared as singlet at 4.17 ppm in the H
NMR spectrum. The methylene group of cyanoacetamide
derivative 5 proved to be reactive toward electrophilic
coupling reaction with aryl diazonium chlorides. Thus, it
coupled with various diazotized anilines 6 (namely,
aniline, 4-toluidine, and 4-anisidine) in ethyl alcohol
containing sodium acetate at 0–5°C to produce the
corresponding 2-oxo-2-((4-oxobenzotriazin-3-yl)amino)-
N-aryl-acetohydrazonoyl cyanide derivatives 7a–c. The
structures of these synthesized hydrazones were
characterized by spectral data. For example, the IR
spectrum of 7b showed absorption bands at 3327 and
3230 cmꢀ1 referring to imino groups (NH), and nitrile
group (C ≡ N) was detected at 2214 cmꢀ1 in addition to
two peaks that appeared at 1704 and 1682 cmꢀ1 for the
carbonyl groups (C=O). The 1H NMR spectrum of 7c
revealed singlet at 3.77 ppm for three protons of methoxy
group (OCH3). The signals of aromatic protons resonated
as two doublet at 6.99 and 7.72 ppm, two triplet at 8.03
and 8.20 ppm, and multiplet at 8.28–8.36 ppm. The
protons of two NH groups were characterized as two
singlet signals at 12.06 and 12.33 ppm.
Scheme 2. Synthesis of N-benzotriazinyl-pyridones 12a–d.
Knoevenagel condensation of cyanoacetamide 5 with
various para substituted benzaldehydes (namely,
p-tolualdehyde, p-anisaldehyde, p-chlorobenzaldehyde,
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Benzotriazine-4-ones as Anticancer Agents
Scheme 4. Synthesis of 2-cyano-2-(thiazol-2-ylidene)-N-(4-oxo-
benzotriazin-3-yl) acetamides 21, 23, and 25.
When cyanoacetamide 5 heated with salicylaldehyde
(14) in ethanol and drops of piperidine for approximate
time, 2-imino-N-(4-oxobenzo [d][1,2,3]triazin-3(4H)-yl)-
2H-chromene-3-carboxamide (15) has been separated in
moderate yield (Scheme 3). The infrared spectrum of 15
was characterized by absorptions at 3442, 1709, and
1650 cmꢀ1 attributable to the NH, carbonyl (C=O)
1
functions, respectively. The H NMR spectrum revealed
multiplet bands at 6.99–8.59 ppm assigned for the
aromatic protons and singlet signal at 9.48 ppm
corresponding to the proton of chromene C4.
Furthermore, condensation of cyanoacetamide 5 with
acetyl acetone (16) in refluxed ethanol and few drops
of
triethylamine
furnished
the
corresponding
N-benzotriazinyl-2-pyridinone scaffold 17. In the 1H
NMR spectrum of 17, the protons of two methyl groups
(2CH3) appeared as two singlet signals at 2.26 and
2.54 ppm and the proton at C-5 of the pyridine ring
appeared as singlet signal at 6.25 ppm, while the aromatic
protons appeared as two triplet signals at 7.49 and
8.11 ppm and two doublet signals at 8.29 and 8.43 ppm.
The basic catalyzed addition of benzotriazinyl
cyanoacetamide 5 to phenyl isothiocyanate (18) was
achieved by stirring the reactants in DMF containing
multiplet in the region 7.04–8.38 ppm for the aromatic
protons, and a further singlet signal deshielded at
10.87 ppm corresponding to one proton (NH). In addition,
in situ addition of ethyl bromoacetate to 19 prompted the
construction of 2-cyano-2-(4-oxo-thiazolidin-2-ylidene)-
N-(4-oxobenzotriazin-3-yl) acetamide scaffold 23 with
molecular formula C19H12N6O3S. The IR spectrum
solid potassium hydroxide for
6 h to yield the
corresponding non-isolable potassium sulfide salt 19,
which underwent further in situ heterocyclization
reactions with α-halogenated reagents 20, 22, and 24 to
furnish the corresponding 3-phenylthiazole derivatives
(Scheme 4). First, in situ treatment of 19 with
chloroacetone and/or phenacyl chloride at room
temperature prompted the formation of 2-cyano-2-(4-
substituted-thiazol-2-ylidene)-N-(4-oxo-benzotriazin-3-yl)
acetamides 21a and 21b, respectively. The IR spectrum of
21a revealed absorptions at 3211 cmꢀ1 referring to the NH
function, 2194 cmꢀ1 due to the nitrile function (C ≡ N),
while the absorptions at 1712 and 1693 cmꢀ1 were
displayed the absorption of NH group at 3320 cmꢀ1
,
nitrile group (C ≡ N) at 2195 cmꢀ1, and three carbonyl
groups at 1736, 1712, and 1677 cmꢀ1. Finally, in situ
cyclization of 19 using chloroacetonitrile was responsible
for the production of 2-cyano-2-(4-imino-thiazolidin-2-
ylidene)-N-(4-oxobenzotriazin-3-yl) acetamide scaffold 25
in moderate yield. 1H NMR spectrum was utilized to
confirm the structure of 25, the protons of methylene
group (CH2) appeared as singlet at 3.89 ppm, the aromatic
protons resonated as multiplet at 7.32–8.31 ppm, and the
protons of two NH groups appeared as two singlet signals
at 10.24 and 11.13 ppm.
1
attributed to the carbonyl groups. The H NMR spectrum
of the same compound exhibited singlet signal at
2.50 ppm referring to three protons (CH3), singlet at
5.20 ppm for the proton at C-5 of the thiazole ring,
Enaminonitrile derivative 27 was easily synthesized
through the reaction of cyanoacetamide
5
with
dimethylformamide-dimethylacetal (26) by heating in dry
xylene (Scheme 5). The chemical structure of 27 has been
secured because of its spectroscopic analyses. The IR
spectrum exhibited absorptions at 3342 cmꢀ1 for the NH
function, 2195 cmꢀ1 for the nitrile function (C ≡ N), and
Scheme 3. Synthesis of benzotriazine derivatives 15 and 17.
1
1688, 1668 cmꢀ1 for the carbonyl groups (C=O). The H
NMR spectrum of 27 revealed two singlet signals at 3.25
and 3.36 ppm for the protons of two methyl groups N
(CH3)2 and singlet signal at 7.87 ppm for the olefinic
proton (CH=C). The aromatic protons resonated as two
triplet and multiplet signals the region 7.98–8.30 ppm,
while the proton of NH function was singlet at
11.15 ppm. The behavior of the enaminonitrile 27 toward
hydrazine hydrate (an example of nitrogen nucleophiles)
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A. A. Fadda, E. Abdel-Latif, A. Fekri, and A. R. Mostafa
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Scheme 5. Synthesis of 5-amino-N-(4-oxobenzotriazin-3-yl)-1H-
pyrazole-4-carboxamides 29 and 33.
benzotriazine-4-one scaffolds were performed against
three types of human cancer cell lines: HepG2
(hepatocellular
carcinoma),
HTC-116
(colorectal
carcinoma), and MCF-7 (mammary gland breast cancer).
Cytotoxic potential of these 1,2,3-benzotriazine-4-ones on
carcinogenic cells was tested by the MTT method [19,20].
The cytotoxicity evaluation involved the use of
doxorubicin as antitumor drug reference standard. From
the obtained results in Table 1, we observe that the tested
1,2,3-benzotriazine-4-one compounds displayed important
differences in the cytotoxic activity. Thus, it can be
mentioned that varying the heterocyclic moiety at
position-3 of the benzotriazine-4-one nucleus causes
significant changes in cytotoxicity. Among the tested
compounds, compounds 7b (bearing tolyl-hydrazonoyl
cyanide group) and 25 (4-imino-thiazolidin-2-ylidene
group) were found to be the most potent scaffolds against
all three cell lines with IC50 values from 5.54 to
11.84 μM. Luckily, compounds 15 (having 2-oxo-2H-
chromene moiety) and 21b (4-phenyl-thiazol-2-ylidene
moiety) displayed strong activities toward HCT-116 and
MCF-7 and moderate activities toward HepG2. Moreover,
compounds 7a, 9b, 12d, 21a, and 23 possessed moderate
anti-proliferative activities against HepG2, HCT-116, and
MCF-7 cell lines. Unfortunately, some of the synthesized
1,2,3-benzotriazine-4-one derivatives 5, 7c, 9c, 17, 27,
to attain polyfunctionally substituted azole was studied. Thus,
5-amino-N-(4-oxobenzotriazin-3-yl)-1H-pyrazole-4-
carboxamide (29) was picked up as a sole product by boiling
enaminonitrile 27 with hydrazine hydrate (28) in ethyl
alcohol. The IR spectrum clearly indicated the lack of any
absorption due to cyano function. In the 1H NMR
spectrum, the absence of singlet signals for the protons of
methyl groups clearly indicated the loss of N (CH3)2
group. The reaction of cyanoacetamide 5 with carbon
disulfide (30) in DMSO containing potassium hydroxide
proceeded by stirring for 6 h at room temperature,
followed by in situ addition of 2 mol of methyl iodide
(31) gave the corresponding ketene dithioacetal derivative
32. The presence of absorptions at 3245 cmꢀ1 (NH),
2201 cmꢀ1 (C ≡ N), and 1708 and 1683 cmꢀ1 (C=O) in
Table 1
Cytotoxic activity of the synthesized 1,2,3-benzotriazine-4-one scaffolds
against HepG2, HCT-116, and MCF-6 cancer cell lines.
In vitro Cytotoxicity IC50 (μM)
Compound
HepG2
HCT-116
MCF-7
1
DOX
5
7a
7b
7c
9a
9b
9c
9d
12a
12b
12c
12d
15
4.50 0.2
78.36 3.9
68.15 3.7
7.67 0.9
85.57 4.0
57.36 2.2
52.40 2.8
86.17 4.2
60.52 2.8
41.78 1.4
47.36 1.6
55.03 1.9
69.91 3.7
45.54 2.5
>100
32.85 2.1
25.74 1.8
16.26 1.3
5.54 0.6
58.75 3.4
54.68 1.6
93.62 4.5
64.12 3.8
5.23 0.3
55.62 3.3
30.64 2.3
6.59 0.5
4.17 0.2
52.60 3.5
23.09 1.9
8.28 0.7
88.93 4.8
64.38 3.1
34.09 2.9
78.19 3.9
56.72 2.4
40.11 1.7
50.11 2.3
50.22 1.6
37.21 3.0
15.39 1.6
95.47 5.1
28.23 2.4
13.77 1.2
9.15 0.9
11.84 1.1
53.15 3.6
49.26 1.4
>100
the IR spectrum secured the structure of 32. The N MR
spectrum displayed two singlet signals at 2.66 and
2.73 ppm for the protons of two methyl groups, triplet
and multiplet signals in the region 8.01–8.34 for the
aromatic protons, in addition to singlet at 12.33 for the
proton of NH group. The aminopyrazole derivative 33
was synthesized in 46% yield by boiling a suspension of
ketene dithioacetal 32 with hydrazine hydrate (28) in
ethyl alcohol for 4 h as shown in Scheme 5. The IR
spectrum of 33 exhibited absorptions at 3443 and
3324 cmꢀ1 for the (NH2 and NH) function, in addition to
broad absorption near 1620 cmꢀ1 for the carbonyl
79.34 4.1
61.20 2.8
35.89 2.6
67.71 3.8
58.22 2.6
38.24 1.8
45.38 1.8
49.58 1.2
46.69 3.1
20.35 1.7
75.37 3.9
27.80 2.0
17.36 1.4
13.92 1.2
10.53 0.9
64.18 3.8
58.33 1.8
95.55 4.7
68.24 3.1
17
21a
21b
23
25
27
29
32
33
1
function (C=O). The H NMR spectrum displayed singlet
at 2.61 ppm for three protons (CH3), singlet at 7.33 ppm
for two protons (NH2), triplet and multiplet signals in the
region 7.52–8.24 ppm for the aromatic protons, and two
singlet signals at 10.51 and 11.87 ppm for the protons of
two NH groups.
56.44 2.2
Anticancer activity (in vitro cytotoxicity activity). The
pharmacological activities of the synthesized 1,2,3-
IC50 values are the mean SD of three separate experiments. DOX,
doxorubicin.
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Benzotriazine-4-ones as Anticancer Agents
and 32 did not manifest significant anti-proliferative
activities against the tested cell lines.
factor/scatter factor (HGF/SF). Simulation of both
possible binding modes and affinities of this series of
new ligands was performed using MOE v10.2009
software and c-Met kinase active site amino acids (PDB.
I.D.3ce3). The native cocrystallized ligand was redocked
in the active site after preparation of the protein using
standard procedure to initially validate docking process.
The results of validation showed the program docked the
ligand in the active site using the same binding pose as
the X-ray crystal structure, as reflected thorough a low
relative mean standard deviation (1.48 A ̊) (Fig. 1). The
Molecular docking study. Motivated by the promising
results of the antitumor screening data of our newly
synthesized benzotriazine-4-one scaffolds and the
grounds of having two FDA approved c-Met kinase
inhibitors (Crizotinib and Cabozantinib) for cancer
treatment, we performed molecular docking studies to
investigate the underlying mechanism of possible
molecular interaction of these compounds with c-Met
tyrosine kinase and the receptor of hepatocyte growth
Figure 1. (A) Docking validations 2D ligand interaction and (B) 3D superposition of native ligand and redocked native ligand at the binding site of C-Met
kinase [PDB ID: 3ce3; relative mean standard deviation (RMSD) = 1.4855; S = ꢀ8.2936 kcal/mol]. [Color figure can be viewed at wileyonlinelibrary.com]
Figure 2. (A) 2D and (B) 3D ligand interaction of 7b with the active sides amino acid of C-Met kinase (S = ꢀ5.9088) in comparison with native ligands.
[Color figure can be viewed at wileyonlinelibrary.com]
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A. A. Fadda, E. Abdel-Latif, A. Fekri, and A. R. Mostafa
Vol 000
native ligand was bound to the active site’s amino acids
with a network of hydrogen bonds, thus resulting in a
binding energy score of S = ꢀ8.2936 kcal/mol (Fig. 1).
The benzotriazine-4-one scaffolds have been prepared
for docking simulation as described in the experimental
(docking studies); results show that all members of this
series of compounds were able to recognize the active
site of the c-Met kinase and perform different types of
bonding interactions with the key active site amino acid
residues. As representatives, 2D and 3D binding
interactions of the candidates 7b, 15, and 25 are
displayed in the top and bottom panels of Figures 2, 3,
and 4, respectively. Benzotriazine-4-one compound 15
accommodated favorably in the active pocket of the
receptor and made binding interactions with the key
active site amino acid residues.
Compound 7b formed four hydrogen bonds, one
between the C=O of the triazine ring and GLY 1224
(3.0A ̊) and the other between the C=O of the amidic
group and MET1131 (3.18 A ̊). The third bond is between
the NH of the amidic group and ASP1222 (2.62 A ̊),
while the last hydrogen bond is between C=N─NH and
Figure 3. (A) 2D and (B) 3D ligand interaction of 15 with the active sides amino acid of C-Met kinase (S = ꢀ22.1657) in comparison with native ligands.
[Color figure can be viewed at wileyonlinelibrary.com]
Figure 4. (A) 2D and (B) 3D ligand interaction of 25 with the active sides amino acid of C-Met kinase (S = ꢀ1.9895) in comparison with native ligands.
[Color figure can be viewed at wileyonlinelibrary.com]
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Benzotriazine-4-ones as Anticancer Agents
MET1131 (3.77 A ̊). It is noteworthy that 7b revealed a
good binding energy score better than the native ligand
(S = ꢀ22.1657 kcal/mol) (Fig. 2).
Thermo Scientific Nicolet iS10 FTIR spectrometer
(Waltham, MA). 1H NMR spectra were recorded in
DMSO-d6 on Bruker WP spectrometer (Rheinstetten,
Germany) at 400 MHz using TMS as an internal standard.
The mass spectra were recorded on a Quadrupole GC/MS
Thermo Scientific Focus/DSQII (Waltham, MA) at
70 eV. Elemental analyses (C, H, and N) were determined
on Perkin-Elmer 2400 analyzer (PerkinElmer Instruments,
Shelton, CT).
Synthesis of 2-amino-N0-(2-cyanoacetyl) benzohydrazide
(3). To a suspension of isatoic anhydride (2) (0.05 mol,
8.15 g) in absolute ethanol (50 mL), cyanoacetic
hydrazide (1) (0.05 mol, 5.0 g) was added and then
refluxed for 4 h. The precipitate that formed on cooling
was filtered to separate the desired product 3.
Compound 15 formed two hydrogen bonds, one
between the N=N group on the triazine ring and
GLY1224 (2.98A ̊) and the other between the C=O of the
amidic group and ASP1222 (1.7A ̊). It is noteworthy that
15 revealed a good binding energy score compared with
the native ligand (S = ꢀ22.1657 kcal/mol) (Fig. 3).
Furthermore, compound 25 showed good interactions
with the active site amino acids of C-Met kinase
(S = ꢀ1.9895 kcal/mol) (Fig. 4). The observed binding
was mediated by a network of interactions between 25
and C-Met kinase where the NH of the amidic group
formed hydrogen bond with MET1131 (3.37 A ̊).
Besides, the imine group (C=N) of the thiazole ring
displayed hydrogen bond with HIS1202 (3.40 A ̊).
Finally, a hydrogen bond was observed between the CN
group and ASP1222 (3.31 A ̊). Comparing 15 with 25
could lead to a conclusion that the introduction of the
thiazole ring to the backbone of the benzotriazine ring
system in case of 25 demonstrated additional advantages
by acting as a platform for hydrogen bond. In conclusion,
the molecular docking was used to give a preliminary
impression of the possible underlying mechanism of
action of some promising newly synthesized compounds.
White crystals, yield = 67%, mp = 173–175°C. IR (v/
cmꢀ1): 3459, 3363, 3250 (NH2 and NH), 2261 (C ≡ N),
1
1687, 1654 (C=O). H NMR (δ/ppm): 3.78 (s, 2H, CH2),
6.40 (s, 2H, NH2), 6.50 (t, 1H, Ar-H), 6.74 (d, 1H, Ar-
H), 7.21 (t, 1H, Ar-H), 7.53 (d, 1H, Ar-H), 10.16 (s, 2H,
2NH). Analysis for C10H10N4O2 (218.08): Calcd: C,
55.04; H, 4.62; N, 25.68%. Found: C, 55.22; H, 4.70; N,
25.77%.
Synthesis of 3-(2-cyanoacetamido)-4-oxo-3H-benzo [d]
[1,2,3]-triazine (5).
Sodium nitrite solution (0.7 g in
15 mL H2O) was added drop by drop to a stirred
suspension of benzohydrazide 3 (0.01 mol, 2.18 g) in
5 mL conc. HCl at 0–5°C. The reaction mixture was
stirred for an hour at 0–5°C, and the solid product that
was obtained by filtration was recrystallized by heating in
ethyl alcohol.
CONCLUSION
This paper reports on the synthesis of various
heterocycles incorporating 1,2,3-benzotriazine-4-one
moiety in order to evaluate their anticancer activity. The
key 2-cyano-N-(4-oxobenzo [d][1,2,3]triazin-3(4H)-yl)
acetamide (5) (which prepared in 77% yield via
Buff crystals, yield = 77%, mp = 195–197°C. IR (v/
1
cmꢀ1): 3174 (NH), 2260 (C ≡ N), 1723, 1693 (C=O). H
NMR (δ/ppm): 4.17 (s, 2H, CH2), 8.02–8.33 (m, 4H,
Ar-H), 12.32 (s, 1H, NH). MS (EI): m/z (%) = 229
(molecular ion, 12), 201 (14), 185 (32), 147 (27), 134
(base peak, 100.0), 105 (26), 76 (47), 51 (12). Analysis
for C10H7N5O2 (229.06): Calcd: C, 52.40; H, 3.08; N,
30.56%. Found: C, 52.52; H, 3.03; N, 30.64%.
intramolecular
diazo-coupling
of
2-amino-N0-(2-
cyanoacetyl)benzohydrazide) was utilized as a precursor
for the synthesis of N-benzotriazinyl-pyridones 12a–d, 2-
imino-N-(benzotriazin-3-yl)-2H-chromene derivative 15,
2-cyano-2-(thiazol-2-ylidene)-N-(benzotriazinyl)
Synthesis of 3-(2-cyano-2-arylhydrazono-acetamido)-4-
acetamides 21, 23, and 25, and 5-amino-N-
(benzotriazinyl)-pyrazoles 29 and 33. The in vitro
evaluation of their antitumor activity against liver cells
cancer, colorectal cancer, and breast cancer revealed that
the benzotriazine-4-one scaffolds 7b, 15, and 25 showed
the highest antitumor activity with IC50 ranging from
5.54 to 16.26 μM.
oxo-4H-benzo [d][1–3] triazines 7a–c.
To a cold
suspension (0–5°C) of p-substituted aniline (0.005 mol)
in 1.5 mL conc. HCl NaNO2 solution (0.35 g in 5 mL
H2O) was added dropwise with stirring. This freshly
obtained diazonium chloride solution was added
dropwise to a cold suspension of the 3-cyanoacetamido-
benzotriazine derivative 3 (0.005 mol, 1.14 g) in 25 mL
ethanol and 1.5 g of sodium acetate. The stirring time
was continued at 0–5°C for additional an hour, and the
solid product that collected by filtration was recrystallized
EXPERIMENTAL
by heating in ethyl alcohol.
All melting points (uncorrected) were determined on an
electrothermal Gallenkamp apparatus (Weiss-Gallenkamp,
Loughborough, UK). The IR spectra were measured on a
3-(2-Cyano-2-(phenylhydrazono)-acetamido)-4-oxo-4H-
benzo [d][1,2,3] triazine (7a).
Yellow crystals,
yield = 56%, mp = 238–240°C. IR (v/cmꢀ1): 3339, 3231
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

A. A. Fadda, E. Abdel-Latif, A. Fekri, and A. R. Mostafa
Vol 000
1
1
(NH), 2216 (C ≡ N), 1692 (broad, C=O). H NMR (δ/
ppm): 7.18–7.62 (m, 5H, Ar-H), 8.05–8.32 (m, 4H, Ar-
H), 11.48 (s, 1H, NH), 12.26 (s, 1H, NH). MS (EI): m/z
(%) = 333 (molecular ion, 31), 208 (base peak, 100.0),
180 (44), 152 (74), 119 (21), 91 (17), 76 (33), 51 (16).
Analysis for C16H11N7O2 (333.10): Calcd: C, 57.66; H,
2123 (C ≡ N), 1692 (C=O). H NMR (δ/ppm): 7.35–8.18
(m, 8H, Ar-H), 8.86 (s, 1H, olefinic CH=C), 11.46 (s,
1H, NH). Analysis for C17H10N5O2Cl (351.05): Calcd: C,
58.05; H, 2.87; N, 19.91%. Found: C, 58.17; H, 2.95; N,
19.98%.
2-Cyano-3-(4-hydroxyphenyl)-N-(4-oxobenzo
[d][1,2,3]
triazin-3(4H)-yl) acrylamide (9d).
Yellow crystals,
3.33; N, 29.42%. Found: C, 57.78; H, 3.41; N, 29.55%.
3-(2-Cyano-2-(4-tolylhydrazono)-acetamido)-4-oxo-4H-
yield = 57%, mp = 293–295°C. IR (v/cmꢀ1): 3221 (NH),
1
benzo [d][1,2,3] triazine (7b).
Yellow crystals,
2208 (C ≡ N), 1690 (C=O). H NMR (δ/ppm): 6.88 (d,
yield = 77%, mp = 258–260°C. IR (v/cmꢀ1): 3327, 3230
2H, Ar-H), 7.55–8.25 (m, 6H, Ar-H), 8.71 (s, 1H,
olefinic CH=C), 9.26 (s, 1H, OH), 11.78 (s, 1H, NH).
Analysis for C17H11N5O3 (333.09): Calcd: C, 61.26; H,
3.33; N, 21.01%. Found: C, 61.17; H, 3.24; N, 20.91%.
1
(NH), 2214 (C ≡ N), 1704, 1682 (C=O). H NMR (δ/
ppm): 2.31 (s, 3H, CH3), 7.24 (d, 2H, Ar-H), 7.69 (d,
2H, Ar-H), 8.02–8.37 (m, 4H, Ar-H), 12.04 (s, 1H, NH),
12.31 (s, 1H, NH). Analysis for C17H13N7O2 (347.11):
Calcd: C, 58.79; H, 3.77; N, 28.23%. Found: C, 58.63;
Synthesis of 6-amino-4-aryl-2-oxo-1-(4-oxobenzo [d][1,2,3]
triazin-3(4H)-yl)-1,2-dihydropyridine-3,5-dicarbonitrile
derivatives 12a–d.
Method A. A mixture of 9
H, 3.69; N, 28.11%.
3-(2-Cyano-2-(4-anisylhydrazono)-acetamido)-4-oxo-4H-
benzo [d][1,2,3] triazine (7c). Brown crystals, yield = 65%,
(0.002 mol) and malononitrile (10) (0.002 mol, 0.13 g)
has been refluxed for 2 h in 20 mL ethanol containing
five drops of piperidine. The precipitate that separated on
cooling to room temperature was filtered and dried.
mp = 230–232°C. IR (v/cmꢀ1): 3328, 3230 (NH), 2210
1
(C ≡ N), 1683 (broad, C=O). H NMR (δ/ppm): 3.77 (s,
3H, OCH3), 6.99 (d, 2H, Ar-H), 7.72 (d, 2H, Ar-H), 8.03
(t, 1H, Ar-H), 8.20 (t, 1H, Ar-H) 8.28–8.36 (m, 2H, Ar-
H), 12.06 (s, 1H, NH), 12.33 (s, 1H, NH). Analysis for
C17H13N7O3 (363.11): Calcd: C, 56.20; H, 3.61; N,
26.99%. Found: C, 56.35; H, 3.69; N, 26.93%.
Method B. To a solution of 3-cyanoacetamido-
benzotriazine derivative 5 (0.002 mol, 0.46 g) in 15 mL
ethanol, different 2-arylidene-malononitrile derivatives 13
(0.002 mol) and five drops piperidine were added. The
reaction mixture was boiled under reflux for 2 h, and the
precipitate that formed was filtered to give the desired
Synthesis of 2-cyano-N-(4-oxobenzo [d][1,2,3]triazin-3(4H)-
yl)-3-phenylacrylamide derivatives 9a–d. To a solution of
3-cyanoacetamido-benzotriazine derivative 5 (0.005 mol,
1.14 g) in 15 mL ethanol, 0.005 mol of different
substituted benzaldehyde 8 (namely, p-tolualdehyde,
pyridine derivatives 12.
6-Amino-2-oxo-1-(4-oxobenzo [d][1,2,3]triazin-3(4H)-yl)-4-
(4-tolyl)-1,2-dihydropyridine-3,5-dicarbonitrile (12a).
Pale
yellow crystals, yield = 48%, mp = 278–280°C. IR (v/
cmꢀ1): 3424, 3365 (NH2), 2205 (C ≡ N), 1676, 1625
p-anisaldehyde,
p-chlorobenzaldehyde,
and
p-
1
(C=O). H NMR (δ/ppm): 2.40 (s, 3H, CH3), 7.23–7.38
hydroxybenzaldehyde), and five drops of piperidine were
added. The reaction mixture was refluxed for 2 h, and the
solid that formed on cooling was filtered and dried.
(m, 6H, Ar-H, and NH2), 7.89 (t, 1H, Ar-H), 8.05 (t, 1H,
Ar-H), 8.19–8.26 (m, 2H, Ar-H). Analysis for
C21H13N7O2 (395.11): Calcd: C, 63.79; H, 3.31; N,
24.80%. Found: C, 63.66; H, 3.25; N, 24.71%.
6-Amino-4-(4-anisyl)-2-oxo-1-(4-oxobenzo [d][1,2,3]triazin-
3(4H)-yl)-1,2-dihydropyridine-3,5-dicarbonitrile (12b).
2-Cyano-N-(4-oxobenzo
[d][1,2,3]triazin-3(4H)-yl)-3-(4-
tolyl)-acrylamide (9a).
Beige crystals, yield = 62%,
mp = 290–292°C. IR ( v /cmꢀ1): 3287 (NH), 2123
1
(C ≡ N), 1682 (C=O). H NMR (δ/ppm): 2.35 (s, 3H,
Greenish yellow crystals, yield = 50%, mp = 283–285°C.
CH3), 7.31–8.26 (m, 8H, Ar-H), 9.04 (s, 1H, olefinic
CH=C), 11.12 (s, 1H, NH). MS (EI): m/z (%) = 331
(molecular ion, 18), 208 (base peak, 100), 180 (88), 152
(72), 150 (17), 121 (45), 76 (31), 51 (6). Analysis for
C18H13N5O2 (331.11): Calcd: C, 65.25; H, 3.95; N,
IR ( v /cmꢀ1): 3431, 3366 (NH2), 2204 (C ≡ N), 1676,
1
1627 (C=O). H NMR (δ/ppm): 3.82 (s, 3H, OCH3), 6.94
(d, 2H, Ar-H), 7.22 (s, 2H, NH2), 7.54 (d, 2H, Ar-H),
7.91 (t, 1H, Ar-H), 8.04 (t, 1H, Ar-H), 8.21–8.28 (m, 2H,
Ar-H). Analysis for C21H13N7O3 (411.11): Calcd: C,
61.31; H, 3.19; N, 23.83%. Found: C, 61.44; H, 3.30; N,
21.14%. Found: C, 65.42; H, 3.99; N, 21.23%.
3-(4-Anisyl)-2-cyano-N-(4-oxobenzo [d][1,2,3]triazin-3(4H)-
yl)-acrylamide (9b).
Yellow crystals, yield = 55%,
23.97%.
mp = 290–292°C. IR ( v /cmꢀ1): 3274 (NH), 2205
6-Amino-4-(4-chlorophenyl)-2-oxo-1-(4-oxobenzo [d][1,2,3]
triazin-3(4H)-yl)-1,2-dihydropyridine-3,5-dicarbonitrile
(12c). Brown powder, yield = 45%, mp = 288–290°C. IR
1
(C ≡ N), 1661 (C=O). H NMR (δ/ppm): 3.87 (s, 3H,
OCH3), 7.14 (d, 2H, Ar-H), 7.95–8.35 (m, 6H, Ar-H),
9.16 (s, 1H, olefinic CH=C), 11.68 (s, 1H, NH). Analysis
for C18H13N5O3 (347.10): Calcd: C, 62.24; H, 3.77; N,
20.16%. Found: C, 62.35; H, 3.79; N, 20.22%.
3-(4-Chlorophenyl)-2-cyano-N-(4-oxobenzo [d][1,2,3]triazin-
(v/cmꢀ1): 3335, 3196 (NH2), 2206 (C ≡ N), 1674 (C=O).
1H NMR (δ/ppm): 7.22–7.42 (m, 6H, Ar-H and NH2),
7.87 (t, 1H, Ar-H), 8.06 (t, 1H, Ar-H), 8.18–8.30 (m, 2H,
Ar-H). Analysis for C20H10N7O2Cl (415.06): Calcd: C,
57.77; H, 2.42; N, 23.58%. Found: C, 57.89; H, 2.51; N,
23.67%.
3(4H)-yl) acrylamide (9c).
Pale brown crystals,
yield = 65%, mp = 283–285°C. IR (v/cmꢀ1): 3265 (NH),
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2019
Benzotriazine-4-ones as Anticancer Agents
6-Amino-4-(4-hydroxyphenyl)-2-oxo-1-(4-oxobenzo
[1,2,3]triazin-3(4H)-yl)-1,2-dihydropyridine-3,5-dicarbonitrile
[d]
Analysis for C20H14N6O2S (402.09): Calcd: C, 59.69; H,
3.51; N, 20.88%. Found: C, 59.77; H, 3.54; N, 20.95%.
2-Cyano-2-(3,4-diphenylthiazol-2(3H)-ylidene)-N-(4-
oxobenzo [d][1,2,3]triazin-3(4H)-yl) acetamide (21b).
Yellow crystals, yield = 62%, mp = 198–200°C. IR (v/
cmꢀ1): IR ( v /cmꢀ1): 3246 (NH), 2194 (C ≡ N), 1692,
1643 (C=O). 1H NMR (δ/ppm): 6.88 (s, 1H, thiazole
H5), 7.16–8.36 (m, 14H, Ar-H), 10.97 (s, 1H, NH).
Analysis for C25H16N6O2S (464.11): Calcd: C, 64.64; H,
(12d). Beige crystals, yield = 43%, mp > 300°C. IR (v/
cmꢀ1): 3424, 3365, 3152 (NH2 and OH), 2205, 2182
1
(C ≡ N), 1677, 1625 (C=O). H NMR (δ/ppm): 6.86 (d,
2H, Ar-H), 7.16 (s, 2H, NH2), 7.44 (d, 2H, Ar-H), 7.86
(t, 1H, Ar-H), 8.02 (t, 1H, Ar-H), 8.18–8.26 (m, 2H, Ar-
H), 9.32 (s, 1H, OH). Analysis for C20H11N7O2 (397.09):
Calcd: C, 60.45; H, 2.79; N, 24.68%. Found: C, 60.57;
H, 2.91; N, 24.75%.
3.47; N, 18.09%. Found: C, 64.51; H, 3.41; N, 17.98%.
2-Cyano-2-(4-oxo-3-phenylthiazolidin-2-ylidene)-N-(4-
oxobenzo [d][1,2,3]triazin-3(4H)-yl) acetamide (23).
crystals, yield = 66%, mp = 277–280°C. IR ( v /cmꢀ1):
Synthesis of 2-imino-N-(4-oxobenzo [d][1,2,3]triazin-3(4H)-
yl)-2H-chromene-3-carboxamide (15). To a suspension of
Buff
3-cyanoacetamido-benzotriazine derivative 5 (0.002 mol,
0.45 g) in 15 mL ethyl alcohol, salicylaldehyde (14)
(0.002 mol, 0.24 mL) and five drops of piperidine were
added and refluxed for 2 h. The solid that was obtained
on cooling was picked up by filtration.
1
3320 (NH), 2195 (C ≡ N), 1736, 1712, 1677 (C=O). H
NMR (δ/ppm): 4.08 (s, 2H, CH2), 7.18–8.31 (m, 9H, Ar-
H), 11.69 (s, 1H, NH). Analysis for C19H12N6O3S
(404.07): Calcd: C, 56.43; H, 2.99; N, 20.78%. Found:
Beige crystals, yield = 53%, mp = 210–212°C. IR (v/
cmꢀ1): 3442 (NH), 1709, 1650 (C=O). 1H NMR
(δ/ppm): 6.99–8.59 (m, 8H, Ar-H), 9.48 (s, 1H,
chromene H4). Analysis for C17H11N5O3 (333.09):
Calcd: C, 61.26; H, 3.33; N, 21.01%. Found: C, 61.37;
C, 56.31; H, 2.91; N, 20.69%.
2-Cyano-2-(4-imino-3-phenylthiazolidin-2-ylidene)-N-(4-
oxobenzo [d][1,2,3]triazin-3(4H)-yl) acetamide (25). Brown
crystals, yield = 46%, mp = 230–231°C. IR ( v /cmꢀ1):
1
3434, 3277 (NH), 2189 (C ≡ N), 1699, 1650 (C=O). H
NMR (δ/ppm): 3.89 (s, 2H, CH2), 7.32–8.31 (m, 9H, Ar-
H), 10.24 (s, 1H, NH), 11.13 (s, 1H, NH). Analysis for
C19H13N7O2S (403.09): Calcd: C, 56.57; H, 3.25; N,
24.30%. Found: C, 56.66; H, 3.34; N, 24.39%.
H, 3.42; N, 21.12%.
Synthesis of 4,6-dimethyl-2-oxo-1-(4-oxobenzo [d][1,2,3]
triazin-3(4H)-yl)-1,2-dihydropyridine-3-carbonitrile (17).
A
mixture of 3-cyanoacetamido-benzotriazine derivative 5
(0.002 mol, 0.46 g) and acetyl acetone (16) (0.002 mol,
0.2 mL) was refluxed in 15 mL ethyl alcohol and drops of
triethylamine for 4 h. The mixture was cooled to 25°C and
the precipitate that formed was filtered and dried.
Orange crystals, yield = 52%, mp = 250–252°C. IR (v/
cmꢀ1): 2223 (C ≡ N), 1719, 1681 (C=O). 1H NMR
(δ/ppm): 2.26 (s, 3H, CH3), 2.54 (s, 3H, CH3), 6.25 (s,
1H, pyridine H5), 7.94 (t, 1H, Ar-H), 8.11 (t, 1H, Ar-H),
8.29 (d, 1H, Ar-H), 8.43 (d, 1H, Ar-H). Analysis for
C15H11N5O2 (293.09): Calcd: C, 61.43; H, 3.78; N,
23.88%. Found: C, 61.30; H, 3.70; N, 23.75%.
Synthesis of 2-cyano-N-(4-oxobenzo [d][1,2,3]triazin-3(4H)-
yl)-2-(3-phenylthiazolidin-2-ylidene) acetamide derivatives 21,
23, and 25. To a stirred suspension of KOH (0.01 mol,
0.56 g) in 20 mL DMF, the 3-cyanoacetamido-
benzotriazine derivative 5 (0.01 mol, 2.30 g) and phenyl
isothiocyanate (18) (0.01 mol, 1.2 mL) were added. The
mixture was allowed to stir for 6 h. Chloroacetone,
Synthesis of 2-cyano-3-(dimethylamino)-N-(4-oxobenzo [d]
[1,2,3]-triazin-3(4H)-yl) acrylamide (27). To a solution of
3-cyanoacetamido-benzotriazine derivative 5 (0.005 mol,
1.15 g) in boiling dry xylene (15 mL), dimethylformamide
dimethylacetal (DMF-DMA) (0.005 mol, 0.56 mL) was
added. The reaction mixture was refluxed for 4 h, cooled
and diluted with water, and the solid product was filtered
and recrystallized by heating in ethyl alcohol.
Pale yellow crystals, yield = 64%, mp = 198–200°C. IR
(v/cmꢀ1): 3342 (NH), 2195 (C ≡ N), 1688, 1668 (C=O). 1H
NMR (δ/ppm): 3.25 (s, 3H, CH3), 3.36 (s, 3H, CH3), 7.87
(s, 1H, olefinic CH=C), 7.98 (t, 1H, Ar-H), 8.15 (t, 1H, Ar-
H), 8.28–8.30 (m, 2H, Ar-H), 11.15 (s, 1H, NH). Analysis
for C13H12N6O2 (284.10): Calcd: C, 54.93; H, 4.25; N,
29.56%. Found: C, 54.80; H, 4.19; N, 29.47%.
Synthesis of 5-amino-N-(4-oxobenzo [d][1,2,3]triazin-3(4H)-
yl)-1H-pyrazole-4-carboxamide (29).
To a suspension of
enaminonitrile 27 (0.005 mol, 1.42 g) in 20 mL ethyl
alcohol, hydrazine hydrate (0.01 mol, 0.50 mL) was added
and then boiled under reflux for 2 h. After cooling, the
solid product that formed was filtered and dried.
phenacyl
chloride,
ethyl
bromoacetate,
and/or
chloroacetonitrile (0.01 mol) were added, and the stirring
time was continued for further 4 h. The precipitate that
formed on dilution with ice-cold water was collected by
filtration and recrystallized by heating in ethyl alcohol.
2-Cyano-2-(4-methyl-3-phenylthiazol-2(3H)-ylidene)-N-(4-
oxo-benzo [d][1,2,3]triazin-3(4H)-yl) acetamide (21a). Pale
Pale brown crystals, yield = 52%, mp > 300°C. IR (v/
cmꢀ1): 3437, 3362, 3206 (NH2 and NH), 1648 (broad,
1
C=O). H NMR (δ/ppm): 7.05 (s, 2H, NH2), 7.84 (t, 1H,
Ar-H), 8.06 (t, 1H, Ar-H), 8.17–8.26 (m, 2H, Ar-H), 8.62
(s, 1H, pyrazole H3), 11.24 (s, 1H, NH), 12.92 (s, 1H,
NH). Analysis for C11H9N7O2 (271.08): Calcd: C, 48.71;
H, 3.34; N, 36.15%. Found: C, 48.88; H, 3.41; N, 36.23%.
yellow crystals, yield = 45%, mp = 260–262°C. IR (v/
1
cmꢀ1): 3211 (NH), 2194 (C ≡ N), 1712, 1693 (C=O). H
NMR (δ/ppm): 2.50 (s, 3H, CH3), 5.20 (s, 1H, thiazole
H5), 7.04–8.38 (m, 9H, Ar-H), 10.87 (s, 1H, NH).
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

A. A. Fadda, E. Abdel-Latif, A. Fekri, and A. R. Mostafa
Vol 000
Synthesis of 2-cyano-3,3-bis (methylthio)-N-(4 oxobenzo [d]
[1,2,3]-triazin-3(4H)-yl) acrylamide (32). To a suspension
interest. In general, the scope of docking studies is thus
concerned with the generation and evaluation of plausible
structures of intermolecular complexes.
of solid KOH (0.02 mol, 1.12 g) in DMSO (20 mL), the
3-cyanoacetamido-benzotriazine derivative 5 (0.01 mol,
2.29 g) was added followed by the addition of carbon
disulfide (0.02 mol, 1.5 mL). The mixture was stirred
overnight, then methyl iodide (0.02 mol, 1.30 mL) was
added, and stirring time was continued for additional 4 h.
The precipitate that formed, on dilution with ice-cold
water, was filtered and recrystallized by heating in ethyl
alcohol.
This part of the study was aiming to perform virtual
screening through molecular docking of the selected
compounds in the active site of tyrosine kinase receptor
domain of the hepatocyte growth factor receptor C-MET
(PDB I.D.:3ce3). Data obtained for the most two active
compounds in this study, namely, 8 and 11b, are described
herein. During the docking procedure, the default settings
were applied with the aid of MOE v10.2009.
Yellow crystals, yield = 55%, mp = 185–187°C. IR (v/
The C-Met kinase protein structure was first repaired
and then appropriately protonated in the presence of
ligand using the protonate 3D process in MOE and then
was used directly for docking. Docking validation was
first performed to confirm the appropriateness of the
chosen docking parameters, which were found to be the
default parameters, through determining the ability of the
cocrystallized ligand to accurately identify the binding
pocket and also to form similar binding interactions like
the ones originally attained in its crystal structure. The
completeness of the validation procedure was assessed
based on the value of the relative mean standard
deviation obtained for the redocked native ligand.
Thereafter, the candidate ligands were prepared by
performing a systematic conformational search associated
with energy minimization. The lowest energy conformer
for each candidate was utilized for performing the
docking study. Docking of the investigated new
compounds was performed using the default parameters
with the aid of triangle matcher placement method with
London dG scoring, which was used for the docking runs,
and the 10 docking poses retained by the software were in-
dividually examined for final choice of the best docking
pose. The best docking conformer for each candidate li-
gand was selected based on a set of parameters, namely,
the S score reflecting the ligand’s binding energy, energy
of placement, energy of conformation, and ligand–receptor
binding interactions.
1
cmꢀ1): 3245 (NH), 2201 (C ≡ N), 1708, 1683 (C=O). H
NMR (δ/ppm): 2.66 (s, 3H, CH3), 2.73 (s, 3H, CH3),
8.01 (t, 1H, Ar-H), 8.18 (t, 1H, Ar-H), 8.30–8.34 (m, 2H,
Ar-H), 12.33 (s, 1H, NH). Analysis for C13H11N5O2S2
(333.04): Calcd: C, 46.84; H, 3.33; N, 21.01%. Found:
C, 46.98; H, 3.35; N, 21.13%.
Synthesis of 3-amino-5-(methylthio)-N-(4-oxobenzo [d]
[1,2,3]-triazin-3(4H)-yl)-1H-pyrazole-4-carboxamide (33).
A mixture of ketene dithioacetal 32 (0.002 mol, 0.66 g)
and hydrazine hydrate (0.002 mol, 0.1 mL) was boiled
under reflux for 4 h in 15 mL ethyl alcohol. After
cooling, the solid that formed was filtered and dried.
Off-white needles, yield = 46%, mp = 290–292°C. IR (v/
cmꢀ1): 3443, 3324 (NH2 and NH), 1620 (broad, C=O). 1H
NMR (δ/ppm): 2.61 (s, 3H, CH3), 7.33 (s, 2H, NH2), 7.52
(t, 1H, Ar-H), 7.70 (t, 1H, Ar-H), 8.15–8.24 (m, 2H, Ar-H),
10.51 (s, 1H, NH), 11.87 (s, 1H, NH). Analysis for
C12H11N7O2S (317.07): Calcd: C, 45.42; H, 3.49; N,
30.90%. Found: C, 45.30; H, 3.43; N, 30.82%.
Anticancer screening.
The evaluation of in vitro
cytotoxicity effects of the synthesized 1,2,3-
benzotriazine-4-one scaffolds was carried out against
three human cancer cell lines (namely, hepatocellular
carcinoma HepG-2, colorectal carcinoma HCT-116, and
mammary gland breast cancer MCF-7). These cell lines
were obtained from ATCC via Holding company for
biological products and vaccines (VACSERA), Cairo,
Egypt. Cytotoxicity determinations are based on the trans-
formation of the yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide (MTT) to a purple formazan
derivative by mitochondrial succinate dehydrogenase in
practical cells. The method of this MTT test was performed
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Docking studies. Molecular docking software attempts
to simulate the structure (or structures) of the
intermolecular complex formed between two or more
molecules, usually a large structure of a protein complexing
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to suggest the binding modes of protein inhibitors [22–25]
to the protein active site. Most docking algorithms generate
a large number of possible structures, so they require a
means to score each structure to identify those of most
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