Design, Synthesis, and Anticancer Evaluation of Tetrazole-Fused Benzoxazole Derivatives as Tubulin Binding Agents

Article abstract of DOI:10.1134/S1070363218100250

A novel series of tetrazole fused benzoxazole derivatives 9a–9j are synthesized, and their structures are characterized by ¹H and ¹³C NMR, and mass spectra. The compounds 9a, 9b, 9g, 9h, and 9j demonstrate the highest activity. The compounds 9b and 9g exhibit good anticancer activity, in particular against MCF7, Hop62, and A-549 cell lines with the range of GI₅₀ values from <0.1 to 4.56 μM. Molecular docking study is carried out for the compounds 9a–9j, according to which the compound 9b forms one hydrogen bond with THR766 with the highest docking score (–7.33). This indicates that 9b is effeciently binging to tubulin site.

Full text of DOI:10.1134/S1070363218100250

ISSN 1070-3632, Russian Journal of General Chemistry, 2018, Vol. 88, No. 10, pp. 2183–2189. © Pleiades Publishing, Ltd., 2018.
Design, Synthesis, and Anticancer Evaluation
of Tetrazole-Fused Benzoxazole Derivatives
as Tubulin Binding Agents1
P. Ravikumara,b*, G. S. B. Raoljib, K. Venkata Sastryc, S. Kalidasua,b, and T. Balaarajud
a
Department of Chemistry, JNT University, Hyderabad, Telangana, 500082 India
*e-mail: ravipolothi@gmail.com
b GVK Biosciences Private Limited, Nacharam, IDA Mallapur, Hyderabad, Telangana, 500076 India
c CMR College of Pharmacy, Medchal, Hyderabad, Telangana 501401, India
d
Department of Chemistry, School of Science, Gitam University, Hyderabad, Telangana, 502329 India
Received August 10, 2018
Abstract―A novel series of tetrazole fused benzoxazole derivatives 9a–9j are synthesized, and their structures
1
are characterized by H and 13C NMR, and mass spectra. The compounds 9a, 9b, 9g, 9h, and 9j demonstrate
the highest activity. The compounds 9b and 9g exhibit good anticancer activity, in particular against MCF7,
Hop62, and A-549 cell lines with the range of GI50 values from <0.1 to 4.56 µM. Molecular docking study is
carried out for the compounds 9a–9j, according to which the compound 9b forms one hydrogen bond with
THR766 with the highest docking score (–7.33). This indicates that 9b is effeciently binging to tubulin site.
Keywords: tetraxole, benzoxazole, cephalosporin, anticancer activity, molecular docking
DOI: 10.1134/S1070363218100250
INTRODUCTION
by 1H and 13C NMR and mass spectra. The synthesized
compounds were evaluated for their anticancer
activity.
Benzoxazole demonstrates interesting pharmacological
activities such as antibacterial [1], antituberculosis [2],
antitumor [3], antibiotic [4], antihelmintic [5], anti-
microbial [6, 7], antifungal [6], and COX-2 inhibitory
[8]. The compounds MUK-1 (1) and DMUK-1 (2)
(Fig. 1) demonstrated potent antibacterial activity
against Gram positive and Gram-negative bacteria
[9, 10].
RESULTS AND DISCUSSION
Synthesis. The synthetic approach compounds
9a–9j is outlined in Scheme 1. The key intermediate
for preparation of the new analogues was 2-(4-azido-
phenyl)-1,3-benzoxazole (7). The starting compound
2-amino-phenol (4) was condensed with 4-amino-
benzaldehyde (5) in ethanol. The semi-product reacted
with Pb(OAc)4 in acetic acid to afford pure cyclized
benzoxazole 6. The compound 6 underwent diazotiza-
tion under the action of 10% HCl and NaNO2. The
following addition of NaN3 gave pure azide key
intermediate 7. Finally, azide 7 reacted with different
substituted aromatic nitrile compounds 8a–8j in
isopropanol in the presence of ZnBr2 to give pure
target compounds 9a–9j.
Tetrazoles are nitrogen containing heterocyclic
compounds that demonstrate a broad range of
biological activities include antibacterial [11], anti-
inflammatory [12], antiviral [13], antitumor [14],
antiproliferative [15] herbicidal [16], analgesic [17],
and antifungal activity [18]. Molecules of highly
efficient antibacterial drug Cephalosporin (3) (Fig. 1)
consist of the tetrazole nucleus to which β-lactam is
attached [19].
In view of the above, we have synthesized a series
of benzoxazole fused tetrazole derivatives. The struc-
tures of the synthesized compounds were characterized
Biological evaluation. In vitro cytotoxicity. The
newly synthesized compounds 9a–9j were screened for
their anticancer activity against four different human
cancer cell lines MCF-7 (breast cancer), KB (oral
cancer), Hop62 and A-549 (lung cancer) (Table 1).
1 The text was submitted by the authors in English.
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2184
RAVIKUMAR et al.
(b)
(a)
(c)
O
O
OMe
MeO
OH
N
S
N
O
N
O
N
N
CH3
N
OH
HO
H
S
N
N
N
N
O
O
1
O
2
O
CO2Na
3
Fig. 1. Structure of (a) MUK-1 (1), (b) DMUK-1 (2), and (c) cephalosporin (3).
Adriamycin was used as a positive control. The
compounds demonstrated good anticancer activity.
Among those, the compounds 9a, 9b, 9g, 9h, and 9j
demonstrated more potent activity. The compounds 9b
and 9g exhibited high anticancer activity (MCF7,
Hop62, and A-549 cell lines) in the range of GI50 from
<0.1 to 4.56 µM. Preliminary structure activity
relationship (SAR) for these compounds 9a–9j
indicated that compound 9a without a substituent on
the phenyl ring exhibited moderate activity against two
cancer cell lines (MCF-7 = 2.89±0.37 µM) and (A549 =
2.11±0.24 µM). Introduction of electron donating
groups, 3,4,5-trimethoxyphenyl derivative 9b, induced
more potent anticancer activity towards MCF-7,
Hop62, and A-549 cell lines, with GI50 values of
1.45±0.30, 1.67±0.28 and 0.1±0.002 µM, respectively.
The electrons withdrawing 4-nitro group on the phenyl
ring in compound 9g resulted in its most potent
activity against three cell lines (MCF-7
=
1.34±0.27µM), (Hop62 = 1.22±0.23 µM) and (A549 =
0.12±0.01µM). The 3-nitrophenyl containing compound
9h was of lower activity (MCF-7= 1.19±0.51µM),
(KB = 2.55±0.16 µM), (Hop62 = 2.90±0.31µM), and
(A549 = 1.30±0.26 µM) than 9g. Another interesting
phenomena was observed with the compound (9j) with
the 4-trifluoromethyl group on the phenyl ring. It
demonstrated relatively low activity (MCF-7=
2.15±0.90 µM) and (KB = 2.09±0.86 µM).
Molecular docking. PDB id 4hj0 had been
obtained from protein data bank and used for docking
and in silico analysis. Molecular docking results
Scheme 1. Synthetic approach to the title compounds 9a–9j.
CHO
NH2
OH
N
(1) EtOH, reflux
NH2
(2) Pb(OAc)4,
AcOH
O
NH2
4
5
6
CN
R
N
O
N
N
O
N
N
NaNO2, HCl
8a−8j
N
N3
NaN3, room temperature,
4 h
IPA, ZnBr2
reflux, 12 h
7
R
9a−9j
R = H (8a, 9a), 3,4,5-trimethoxy (8b, 9b), 4-methoxy (8c, 9c), 4-chloro (8d, 9d), 4-bromo (8e, 9e), 4-fluoro (8f, 9f), 4-nitro
(8g, 9g), 3-nitro (8h, 9h), 4-trifluoromethyl (8i, 9i), 4-methyl (8j, 9j).
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 88 No. 10 2018

DESIGN, SYNTHESIS, AND ANTICANCER EVALUATION OF TETRAZOLE-FUSED
Table 1. Cytotoxic activity (GI50 µM)a of compounds 9a–9j
2185
Compound
9a
MCF-7b
2.89±0.37
1.45±0.30
34.6±1.38
2.77±0.38
5.67±0.23
14.6±1.67
1.34±0.27
1.19±0.51
23.9±1.23
2.15±0.90
0.17±0.018
KBc
Hop62d
3.67±0.21
1.67±0.28
No active
8.34±0.25
9.34±0.33
No active
1.22±0.23
2.90±0.31
No active
No active
0.14±0.022
A-549d
9.79±0.24
2.78±0.32
7.28±0.32
12.5±0.68
No active
7.40±0.29
4.56±0.35
2.55±0.16
No active
2.09±0.86
0.17±0.025
2.11±0.24
0.1±0.002
9.24±0.12
13.2±1.17
17.9±1.02
24.8±3.20
0.12±0.01
1.30±0.26
47.2±2.67
17.4±1.0
9b
9c
9d
9e
9f
9g
9h
9i
9j
Adriamycin
7.25±0.56
a 50% growth inhibition and the values are mean of three determinations. b Breast cancer. c Oral cancer. d Lung cancer.
Table 2. Binding energies, number of hydrogen bonds and residues involved in H-bonding for compounds 9a–9j against
EGFR (PDB ID: 4hjo)
Comp. no. Binding energy, kcal/mol [4]
Docking Score Number of H-bonds Residues involved in H-bonding
9b
9g
9c
9i
–62.072
–59.840
–58.521
–57.965
–61.082
–56.487
–55.126
–61.639
–62.139
–61.510
–7.338
–7.087
–6.927
–6.871
–6.807
–6.792
–6.727
–6.327
–5.773
–5.063
1
1
2
2
1
2
2
1
0
1
THR766
THR766
THR766, MET769
THR766, MET769
ASP831
9a
9j
THR766, MET769
THR766, MET769
ASP831
9f
9e
9d
9h
0
ASP831
revealed that compound 9b was the most preferred
one, based on the analysis, followed by compounds 9g
and 9c and so on (Table 2). Molecules of compound
9b could form one hydrogen bond with THR766 with
highest docking score (–7.33) among the designed
molecules and the glide energy also lower than the
EXPERIMENTAL
All chemicals and reagents were obtained from
Aldrich (Sigma–Aldrich, St. Louis, MO, USA),
Lancaster (Alfa Aesar, Johnson Matthey Company,
Ward Hill, MA, USA) and used without further
purification. Reactions were monitored by TLC,
performed on silica gel glass plates containing 60 F-
254, and visualized by UV light or iodine indicator. 1H
others (Figs.
2 and 3). It demonstrated few
hydrophobic interactions with specific residues, like
LEU694, LUE768, ALA719, and LEU834.
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RAVIKUMAR et al.
Fig. 2. Binding poses and interactions of compound 9b
with the binding sites of the target protein EGFR (PDB ID:
4hjo).
Fig. 3. (Ribbon Model) The detailed binding pose and
interactions of compound 9b with the binding sites of the
target protein EGFR (PDB ID: 4hjo).
and 13C NMR spectra were measured on a Bruker
UXNMR/XWIN-NMR (400 MHz, 300 MHz) spectro-
meter using DMSO-d6 as a solvent and TMS as an
internal standard. ESI spectra were measured on a
Micro mass, Quattro LC using ESI+ software with
capillary voltage 3.98 kV and ESI mode positive ion
trap detector. Melting points were determined on an
electrothermal melting point apparatus, and are
uncorrected.
product was purified by column chromatography with
ethyl acetate/hexane (1 : 9) to afford pure compound 7.
Yield 95%, mp 298–300°C. 1H NMR spectrum, δ, ppm:
7.21 d (1H, J = 8.02 Hz), 7.29 t (1H J = 7.61 Hz), 7.32
d (2H, J = 8.05 Hz), 7.39 t (1H, J = 7.49 Hz), 7.43 d
(1H, J = 8.23 Hz), 7.54 d (2H, J = 8.05 Hz). MS (ESI):
237 [M + H]+.
Synthesis of tetrazole-fused benzoxazole derivatives
9a–9j. A mixture of a benzonitrile 8a–8j (1 mmol)
with azide 7 (1.1 mmol), ZnBr2 (1 mmol), water
(40 mL), and isopropylalcohol (10 mL) was refluxed
for 24 h upon vigorous stirring. HCl (3 N, 30 mL) And
ethyl acetate (100 mL) were added and the process was
continued until the solid in the flask disappeared and
pH of the aqueous layer became 1. The organic layer
was separated and aqueous layer was treated with
2×100 mL of ethyl acetate. The combined organic
layers were evaporated. 200 mL of 0.25 N NaOH were
added, and the mixture was stirred for 30 min until the
original precipitate was dissolved and the suspension
of zinc hydroxide was formed. The suspension was
filtered off, and the solid washed with 20 mL of 1 N
NaOH. 40 mL of 3 N HCl were added to the filtrate
upon vigorous stirring causing tetrazole to precipitate.
Tetrazole was filtered off and washed with 2×20 mL of
3 N HCl and dried in a drying oven to afford the
corresponding pure compound 9a–9j.
4-(1,3-Benzoxazol-2-yl)aniline (6). The mixture of
2-aminophenol 4 (10 g, 91.63 mmol) with 4-amino-
benzaldehyde 5 (11.3 g, 91.63 mmol) was refluxed in
10 mL of ethanol for 3 h. After cooling down the
reaction mixture, ethanol was removed in vacuum. The
resulting Schiff base was dissolved in 12 mL of acetic
acid, and lead tetraacetate (40 g, 91.63 mmol) was
added. The mixture was stirred at room temperature
for 1 h, then diluted with 30 mL H2O, extracted with
ethyl acetate, and dried over anhydrous Na2SO4. The
solvent was removed in vacuum, and the crude product
was purified by column chromatography with ethyl
acetate/hexane (3 : 7) to afford pure compound 6.
1
Yield 92%, mp 359–361°C. H NMR spectrum, δ,
ppm: 5.97 s (2H), 6.57– 6.88 m (2H), 7.30 d.d.d (2H,
J = 6.4, 5.4, 3.7 Hz), 7.65 d.d (2H, J = 6.4, 2.6 Hz),
7.76 –7.98 m (2H). MS (ESI): 211 [M + H]+.
2-(4-Azidophenyl)-1,3-benzoxazole (7). The
amine derivative 6 (12 g, 57.14 mmol) was dissolved
in 10% aqueous HCl and cooled down to 0°C. Upon
addition of a solution of NaNO2 (3.9 g, 57.14 mmol),
the reaction mixture was stirred for 10 min at 0–5°C.
Sodium azide (3.7 g, 57.14 mmol) was added and the
mixture was stirred at room temperature for 4 h. The
reaction mixture was diluted with ethyl acetate. The
organic layer was washed with brine and dried over
Na2SO4. After evaporation of the solvent, the crude
2-[4-(5-Phenyl-1H-tetrazol-1-yl)phenyl]benzo[d]-
1
oxazole (9a). Yield 70%, mp 310–312°C. H NMR
spectrum, δ, ppm: 7.13–7.19 m (3H), 7.44–7.54 m
(3H), 7.61 t (1H J = 7.4 Hz ), 7.66–7.75 m (4H), 7.85 d
(2H, J = 8.34 Hz). 13C NMR spectrum, δ, ppm: 111.4,
119.6, 124.7, 125.4, 126.7, 128.4, 129.5, 130.5, 132.4,
133.4, 134.8, 143.5, 143.8, 149.6, 150.7, 160.4. Found,
%: C 70.71; H 3.82; N 20.59. C20H13N5O. Calculated,
%: C 70.79; H 3.86; N 20.64. MS (ESI): 340 [M + H]+.
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DESIGN, SYNTHESIS, AND ANTICANCER EVALUATION OF TETRAZOLE-FUSED
2187
2-{4-[5-(3,4,5-Trimethoxyphenyl)-1H-tetrazol-1-
yl]phenyl}benzo[d]oxazole (9b). Yield 58%, mp 320–
322°C. 1H NMR spectrum, δ, ppm: 3.86 s (3H), 3.90 s
(6H), 7.14–7.20 m (3H), 7.43–7.55 m (3H), 7.61 s
(2H), 7.84 d (2H, J = 8.33 Hz). 13C NMR spectrum, δ,
ppm: 57.7, 61.5, 106.5, 111.5, 119.7, 123.5, 124.6,
125.6, 126.6, 128.4, 134.5, 141.4, 142.5, 143.6, 148.4,
150.4, 155.4, 160.6. Found, %: C 64.27; H 4.49; N
16.36. C23H19N5O4. Calculated, %: C 64.33; H 4.46; N
16.31. MS (ESI): 430 [M + H]+.
2-{4-[5-(4-Nitrophenyl)-1H-tetrazol-1-yl]phenyl}-
benzo[d]oxazole (9g). Yield 71%, mp 336–338°C. H
1
NMR spectrum, δ, ppm: 7.16–7.23 m (3H), 7.46–7.55
m (3H), 7.64 d (2H, J = 8.37 Hz), 7.69 d (2H, J = 8.37
Hz), 7.86 d (2H, J = 8.38 Hz). 13C NMR spectrum, δ,
ppm: 111.8, 119.8, 124.5, 125.7, 126.6, 128.7, 131.4,
134.6, 135.7, 143.6, 143.8, 146.7, 149.7, 150.7, 161.6.
Found, %: C 62.47; H 3.18; N 21.91. C20H12N6O3.
Calculated, %: C 62.50; H 3.15; N 21.87. MS (ESI):
385 [M + H]+.
2-{4-[5-(4-Methoxyphenyl)-1H-tetrazol-1-yl]-
phenyl}benzo[d]oxazole (9c). Yield 69%, mp 317–
319°C. H NMR spectrum, δ, ppm: 3.86 s (3H), 7.14–
7.21 m (3H), 7.27 d (2H, J = 8.23 Hz), 7.44–7.55 m
(3H), 7.62 d (2H, J = 8.23 Hz), 7.85 d (2H, J = 8.33 Hz).
13C NMR spectrum, δ, ppm: 57.6, 111.6, 118.6, 119.6,
123.4, 124.5, 125.6, 126.4, 128.6, 130.5, 134.5, 143.4,
143.9, 149.4, 150.5, 159.5, 160.5. Found, %: C 68.24;
H 4.12; N 18.99. C21H15N5O2. Calculated, %: C 68.28;
H 4.09; N 18.96. MS (ESI): 370 [M + H]+.
2-{4-[5-(3-Nitrophenyl)-1H-tetrazol-1-yl]phenyl}-
benzo[d]oxazole (9h). Yield 68%, mp 339–341°C. 1H
NMR spectrum, δ, ppm: 7.16–7.22 m (3H), 7.46–7.54
m (3H), 7.61 d (1H, J = 8.35 Hz), 7.65 s (1H), 7.70–
7.75 m (2H), 7.85 d (2H, J = 8.37 Hz). 13C NMR
spectrum, δ, ppm: 111.7, 119.8, 122.5, 124.6, 125.6,
126.5, 126.8, 128.6, 130.6, 132.4, 134.6, 135.5, 139.6,
143.5, 150.5, 150.7, 151.4, 161.8. Found, %: C 62.44;
H 3.11; N 21.90. C20H12N6O3. Calculated, %: C 62.50;
H 3.15; N 21.87. MS (ESI): 385 [M + H]+.
1
2-{4-[5-(4-Chlorophenyl)-1H-tetrazol-1-yl]-
phenyl}benzo[d]oxazole (9d). Yield 70%, mp 327–
329°C. H NMR spectrum, δ, ppm: 7.15–7.22 m (3H),
7.45–7.54 m (3H), 7.63 d (2H, J = 8.33 Hz), 7.69 d
(2H, J = 8.33 Hz), 7.86 d (2H, J = 8.41 Hz). 13C NMR
spectrum, δ, ppm: 111.7, 119.7, 124.5, 125.6, 126.6,
127.5, 127.9, 131.4, 132.6, 134.5, 136.6, 143.5, 143.8,
149.5, 150.5, 160.7. Found, %: C 64.21; H 3.22; N
18.77. C20H12ClN5O. Calculated, %: C 64.26; H 3.24;
N 18.74. MS (ESI): 374 [M + H]+.
2-[4-(5-p-Tolyl-1H-tetrazol-1-yl)phenyl]benzo[d]-
oxazole (9i). Yield 71%, mp 308–310°C. H NMR
1
1
spectrum, δ, ppm: 2.45 s (3H), 7.14–7.53 m (3H), 7.45–7.58
m (5H), 7.60 d (2H, J = 8.32 Hz), 7.83 d (2H, J =
8.34 Hz). 13C NMR spectrum, δ, ppm: 31.6, 111.6,
119.6, 124.6, 125.6, 126.5, 127.4, 128.5, 129.7, 132.5,
134.5, 141.4, 143.6, 143.9, 149.7, 150.7, 160.5. Found,
%: C 71.33; H 4.17; N 19.88. C21H15N5O. Calculated,
%: C 71.38; H 4.28; N 19.82. MS (ESI): 354 [M + H]+.
2-{4-[5-(4-(Trifluoromethyl)phenyl)-1H-tetrazol-
1-yl]phenyl}benzo[d]oxazole (9j). Yield 61%, mp 315–
2-{4-[5-(4-Bromophenyl)-1H-tetrazol-1-yl]-
phenyl}benzo[d]oxazole (9e). Yield 66%, mp 331–
1
317°C. H NMR spectrum, δ, ppm: 7.15–7.22 m (3H),
1
333°C. H NMR spectrum, δ, ppm: 7.15–7.23 m (3H),
7.44–7.55 m (3H), 7.63 d (2H, J = 8.32 Hz), 7.67 d
(2H, J = 8.32 Hz), 7.82 d (2H, J = 8.36 Hz). 13C NMR
spectrum, δ, ppm: 111.7, 114.6, 119.6, 124.6, 125.6,
126.7, 127.6, 128.7, 130.6, 131.6, 134.6, 135.6, 143,5,
143.8, 149.7, 150.6, 160.6. Found, %: C 61.92; H 2.97;
N 17.19. C21H12F3N5O. Calculated, %: C 61.92; H
2.97; N 17.19. MS (ESI): 408 [M + H]+.
7.37 d (2H, J = 8.34 Hz), 7.46–7.55 m (3H), 7.64 d
(2H, J = 8.34 Hz), 7.86 d (2H, J = 8.38 Hz). 13C NMR
spectrum, δ, ppm: 111.7, 119.7, 124.5, 125.6, 126.7,
126.9, 128.5, 128.8, 132.5, 133.6, 134.6, 143.5, 143.8,
149.7, 150.6, 161.6. Found, %: C 57.39; H 2.91; N 16.77.
C20H12BrN5O. Calculated, %: C 57.43; H 2.89; N 16.74.
MS (ESI): 418 [M + H]+.
Procedure for MTT-assay. The synthesized
compounds 9a–9j have been evaluated for their in
vitro cytotoxicity in human cancer cell lines. A
protocol of 48 h continuous drug exposure has been
used and a sulforhodamine B (SRB) protein assay has
been used to estimate cell viability or growth. The cell
lines were grown in DMEM medium containing 10%
fetal bovine serum and 2 mM L-glutamine and were
inoculated into 96 well microtiter plates in 90 mL at
plating densities depending on the doubling time of
2-{4-[5-(4-Fluorophenyl)-1H-tetrazol-1-yl]-
phenyl}benzo[d]oxazole (9f). Yield 69%, mp 314–
316°C. H NMR spectrum, δ, ppm: 7.14–7.22 m (3H),
7.44–7.60 m (5H), 7.63 d (2H, J = 8.32 Hz), 7.84 d
(2H, J = 8.37 Hz). 13C NMR spectrum, δ, ppm: 111.7,
116.5, 119.6, 124.5, 125.6, 125.9, 126.4, 128.7, 132.7,
134.5, 143.4, 143.7, 149.7, 150.7, 151.3, 160.5. Found,
%: C 67.17; H 3.33; N 19.64. C20H12FN5O. Calculated,
%: C 67.22; H 3.38; N 19.60. MS (ESI): 358 [M + H]+.
1
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RAVIKUMAR et al.
individual cell lines. The microtiter plates were
incubated at 37°C, 5%CO2, 95% air, and 100% relative
humidity for 24 h prior to addition of experimental
drugs. Aliquots of 10 mL of the drug dilutions were
added to the appropriate microtiter wells already
containing 90 mL of cells. For each compound four
concentrations (0.1, 1, 10, and 100 μM) were
evaluated and each was carried out in triplicate wells.
Plates were incubated further for 48 h and the assay
was terminated by addition of 50 mL of cold
trichloroacetic acid (10%TCA, final concentration) and
incubated for 60 min at 4°C. The plates were washed
five times with tap water and air dried. Sulforhod-
amine B (SRB) solution (50 mL, 0.4% w/v) in 1%
acetic acid was added to each cell, and plates were
incubated for 20 min at room temperature. The
residual dye was removed by washing five times with
1% acetic acid. The plates were air dried. Bound stain
was subsequently eluted with trizma base (10 mM),
and the absorbance was read on an ELISA plate reader
at 540 nm with 690 nm reference wavelength. Percent
growth was calculated on a plate by plate basis for test
wells relative to control wells. The above deter-
minations were repeated three times. Percent growth
was expressed as the ratio of average absorbance of the
test well to the average absorbance of the control
wells.
(Schrodinger, LLC, New York, NY, 2017) with the
following steps:
(1) The missing side chains were added to the
crystal structure by Schrodinger’s Prime 3.0.
(2) Hydrogen bond atoms were added and water
molecules beyond 0 Å of the co-crystallized ligand
were removed.
(3) Protonation states of entire systems were
adjusted to pH range of 7.0±–4.0 using Epik.
(4) Hydrogen bond networks and flip orientations/
tautomeric states of Gln, Asn, and His residues were
optimized.
(5) The geometry optimization was performed to a
maximum root mean square deviation (RMSD) of
0.3 Å with the OPLS2005 force field.
Receptor grid generation. Protein grid was generated
using receptor grid generation module in Glide program.
The binding region was defined using a co-crystal ligand.
Docking studies. Docking experiment was
performed with the solved 3D-structure of EGFR
tyrosine kinase domain (PDB id 4hjo) using Glide
module in Schrodinger. Authenticity of this docking
method to predict the bioactive conformation was
validated using the X-ray structure of tyrosine kinase
in a complex with a co-crystal erlotinib. Co-crystal
erlotinib was redocked into the active site of 4hjo and
the best predicted bound conformation having lowest
docking energy was selected. Glide uses an explicit
water model for modelling salvation effects. The
demonstrated research output and results are the
structural model exploration and involved
methodology is very effective, which was analyzed in
few of research papers [20–22]. In this methodology,
we took G-score, which is a modified and extended
empirical scoring function based on Chem-Score the
docking method that is XP descriptors been used to
score (G-score) for ligand includes drug like properties
such as Lipo-EvdW, H-bond, and RotPenal. Lipo-
EvdW is the Chem-Score lipophilic pair term and
fraction of the complete receptor-ligand VdW’s
energy. H-bond is the Chem-Score H-bonding pair term.
Molecular modelling and drug design. The
binding mode of the compounds at the active site of
proposed inhibitors was explored using Glide In-silico
protocol (Schrödinger software package Maestro 2017-2
(Glide, Schrödinger, LLC, New York, NY, 2017).
Search of an in-house database was utilized to
prioritize molecules for the synthesis of few designed
core structures. The PDB ID 4hjo was used for
docking study which was downloaded from protein
data bank (www.rcsb.org)
Ligand preparation. All ligands were drawn using
maestro software and prepared for docking by ligprep
module (LigPrep, Schrödinger, LLC, New York, NY,
2017). The docking in-silico experiments were
performed through Glide (Grid based Ligand Docking
with Energetics) program in Schrodinger Software
(maestro) Suite.
Binding energy calculations. The binding free
energies were gauged by using Prime MMGBSA
method. The Molecular Mechanics Generalized Born
Surface Area [23, 24]. (MM-GBSA) method was used
to calculate binding free energies for the designed
molecules by combining molecular mechanics calcula-
Preparation of protein structures. The crystal
structure of tubulin (PDB id 4hjo) was considered to
get insights into the binding modes of ligands. The
protein X-ray structure was prepared using the Protein
Preparation Wizard implemented in Maestro 11.4
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 88 No. 10 2018

DESIGN, SYNTHESIS, AND ANTICANCER EVALUATION OF TETRAZOLE-FUSED
2189
Eur. J. Med. Chem., 2004, vol. 39, p. 291. doi 10.1016/
j.ejmech.2003.11.014
tions and continuum (implicit) solvation models. These
models (Implicit solvent) are often used to compute
free energies of solute solvent interactions and signi-
ficantly improve the computational speed and reduce
errors in statistical averaging that result from
incomplete sampling of solvent conformations.
7. Turan-Zitoni, G., Demirayak, S., Ozdemir, A.,
Kaplancıklı, Z.A., and Yildiz, M.T., Eur. J. Med Chem.,
2004, vol. 39, p. 267. doi 10.1016/j.ejmech.2003.11.001
8. Srinivas, A., Vidyasagar, J., Swathi, K., and
Sarangapani, M., J. Chem. Pharm. Res., 2010, vol. 2,
p. 213.
CONCLUSIONS
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A novel series of tetrazole fused benzoxazole
derivatives 9a–9j is synthesized. These compounds are
evaluated for their anticancer activity against four
human cancer cell lines such as MCF-7 (Beast cancer),
KB (Oral cancer), Hop62 and A-549 (Lung cancer).
The compounds 9a, 9b, 9g, 9h, and 9j demonstrate
more potent activity. The compounds 9b and 9g
exhibit good anticancer activity, in particular MCF7,
Hop62, and A-549 cell lines, and the range of GI50
value is <0.1 to 4.56 µM. Molecular docking study is
carried out for the compounds 9a–9j. Among these,
compound 9b forms one hydrogen bond with THR766
with the highest docking score (–7.33), which indicates
its effective binging to tubulin site.
p. 788
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Gill, C.H., Bioorg. Med. Chem. Lett., 2008, vol. 18,
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ACKNOWLEDGEMENTS
We would like to express our gratitude and thanks
to Department of Chemistry, Jawaharlal Nehru
Technological University, Hyderabad for constant
encouragement during this research program, and
thanks to GVK Bio, Hyderabad for providing basic
research facility.
18. Scheffler, R.J., Colmer, S., Tynan, H., Demain, A.L.,
and Gullo, V.P., Appl. Microbiol. Biotechnol., 2013,
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CONFLICT OF INTERESTS
No conflict of interests was declared by the authors.
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