Synthesis and selective cytotoxicity of novel biphenyl-based tetrazole derivatives

Article abstract of DOI:10.1007/s00044-014-1010-4

Cancer today represents a significant public health problem worldwide, and the challenge is to produce cost-effective drugs. Recently, biphenyl compounds as well as tetrazole derivatives is known for their potential nonselective anticancer activities. In search of novel selective anticancer agents, a series of newly hybrid molecules was designed and synthesized by combining the structural features of biphenyl and tetrazole moieties. The structures of newly synthesized compounds were characterized using spectroscopic techniques (FT-IR, ¹H NMR, ¹³C NMR, and HMBC). Cytotoxic evaluations of these novels compound on human cancer cell lines showed a significant anticancer activity against more than one tested cell lines. Compounds 5n, 5j, and 5o proved to exhibit the strongest and selective cytotoxic effect on HepG2 and MCF-7 lines. Taken together, this study has led to the development of promising leads for cancer therapy.

Full text of DOI:10.1007/s00044-014-1010-4

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-014-1010-4
ORIGINAL RESEARCH
Synthesis and selective cytotoxicity of novel biphenyl-based
tetrazole derivatives
Mahesh H. Malani
Ahmed S. Ibrahim
•
•
Bharatkumar Z. Dholakiya
Farid A. Badria
•
Received: 14 August 2013 / Accepted: 14 April 2014
Ó Springer Science+Business Media New York 2014
Abstract Cancer today represents a significant public
health problem worldwide, and the challenge is to produce
cost-effective drugs. Recently, biphenyl compounds as well
as tetrazole derivatives is known for their potential non-
selective anticancer activities. In search of novel selective
anticancer agents, a series of newly hybrid molecules was
designed and synthesized by combining the structural
features of biphenyl and tetrazole moieties. The structures
of newly synthesized compounds were characterized using
Keywords Tetrazole ꢀ Cancer ꢀ Chemotherapy ꢀ
Cytotoxity ꢀ Antiproliferative
Introduction
Cancer (a term comprising over 100 types of malignancy)
is one of the major burdens of chronic disease in the world.
According to Cancer Society statistics, more than one-
quarter of all death rates is attributed to cancer. Never-
theless, it is very difficult to cure this disease and to clarify
its pathogenesis due to the intricate etiology of cancer.
1
13
spectroscopic techniques (FT-IR, H NMR, C NMR, and
HMBC). Cytotoxic evaluations of these novels compound
on human cancer cell lines showed a significant anticancer
activity against more than one tested cell lines. Compounds
nd
(Cancer Facts & Figs. 2 Ed., 2011; Colorectal Cancer
5
n, 5j, and 5o proved to exhibit the strongest and selective
Facts & Fig., 2011; Canadian Cancer Society., 2012).
Chemotherapy is the last chance for many cancer survivors
and perhaps the only alternative for patients who have had
multiple resections and maximum irradiation. After four
decades of the cancer chemotherapy era, research has
achieved a great success, and a large number of chemo-
therapeutic agents have been discovered since that time.
However, only a few have earned a solid position in the list
of useful drugs due to the associated side effects to varying
degrees, and the emergence of drug resistance. Therefore,
developing new anticancer drugs with a better potency and
specificity against cancer cells has become the hotspot of
global media attention.(Choi et al., 2008; Rapp et al., 2003;
Yuancai and Si-shen, 2005).
cytotoxic effect on HepG2 and MCF-7 lines. Taken toge-
ther, this study has led to the development of promising
leads for cancer therapy.
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-014-1010-4) contains supplementary
material, which is available to authorized users.
M. H. Malani ꢀ B. Z. Dholakiya (&)
Department of Applied Chemistry, S.V. National Institute of
Technology, Ichchhanath, Surat 395007, Gujarat, India
e-mail: bharat281173@gmail.com
M. H. Malani
e-mail: maheshmalani.svnit@gmail.com
Nowadays, biphenyl compounds as well as tetrazole
derivatives are known for their varied biological activities,
such as antibacterial, anti-inflammatory, and recently
anticancer activities. Accordingly, a suitable combination
of these structural features is a rational strategy that may
result in the design of new molecules in which the char-
acteristics of various components are modulated, amplified,
or give rise to entirely new properties. Although syntheses
A. S. Ibrahim
Department of Biochemistry, Faculty of Pharmacy, Mansoura
University, Mansoura 35516, Egypt
F. A. Badria
Department of Pharmacognosy, Faculty of Pharmacy, Mansoura
University, Mansoura 35516, Egypt
123

Med Chem Res
of tetrazole derivatives have been reported since the mid-
century, there is still a dearth of efficient processes. The
well-known and the most efficient route for such prepara-
tion is the [3?2] cycloaddition between hydrazoic acid and
cyanide derivatives. (Kiyoto et al., 1998) However,
hydrazoic acid is highly explosive. As such, the use of
sodium azide as a substrate in place of the hydrazoic acid
would be practically convenient. Unfortunately, the [3?2]
cycloaddition energy barrier would significantly lower with
hydrazoic acid than the azide ion. To overcome this energy
limitation, synthesis has been designed either to control the
hydrazoic acid formation (Sauer et al., 1960) or to use a
large excess of azide ions in the presence of metal catalysts
Lower aqueous layer was collected, washed with 25 ml
xylene, and the pH was adjusted to 4–5 by 50 % HCl. The
aqueous layer was stirred for 60 min, and solid product was
separated, filtered and finally washed with water. Drying
was carried out at 60–70°c for 12 h, and white-colored
powder (2) (18.8 gm, 87 % yield) was obtained (Murli
Krishna et al., 2011).
0
Synthesis of trityl protected 5-(4 -methylbiphenyl-2-
yl)-2H-tetrazole (3) compound
0
Trityl protected 5-(4 -methylbiphenyl-2-yl)-2H-tetrazole
compound (3) (Scheme 1) was prepared at present work
using the reported technique. (Grigor et al., 2001) Mixture
of compound (2) 15.0 gm (0.0634 mol), triphenyl methanol
(
Steven and Wittenberger, 1993) or strong Lewis Acids.
Bret and Michael, 1993) Overall, these procedures are
(
undesirable due to the longer reaction times, high tem-
perature, low yields, or non-recoverable catalysts. In the
present study, we have described the synthesis of 20 novel
hybrid tetrazole and biphenyl derivatives, and their evo-
lution toward tumor growth inhibitory activities over a
panel of human cancer cell lines to develop novel, selec-
tive, and active anticancer agents.
1
7.35 gm (0.066 mol), and 70 ml toluene was charged in
three-neck flat-bottom reaction flask. Reaction mass was
stirred at 28–30 °C for 15 min, and 2–3 drops of concen-
trated sulphuric acid were added. Reaction mass was
continuously stirred, and the temperature was maintained at
110–112 °C for 2.0 h. Simultaneously, water was removed
by azeotropic distillation using toluene as distillation sol-
vent. After completion of reaction, reaction mass was
cooled to 28–30 °C, filtered, and dried at 65–70 °C. White
solid powder (3) (27.3 gm 90 % yield) was obtained.
Experimental
Materials and methods
0
Synthesis of brominated trityl protected 5-(4 -
All reagents were of analytical grade and were used
without further purification. Solvents employed were
purified by standard procedure before use. Brominated
trityl group protected biphenyl tetrazol was prepared as per
reported process (Somisetti and Sudhaker, 2011), and
methylbiphenyl-2-yl)-2H-tetrazole compound
0
Brominated trityl protected 5-(4 -methylbiphenyl-2-yl)-2H-
tetrazole compound (4) was prepared using the reported
process.(Somisetti and Sudhaker, 2011) Mixture of com-
pound (3) 15.0 gm (0.0313 mol), DBDMH 6.0 gm
0
sodium azide was purchased from Aldrich, and 4 -methyl-
biphenyl-2-carbonitrile was purchased from Acros Organ-
ics-India.
(
0.020 mol), AIBN 1.02 gm (0.0062 mol), and dichloro-
methane (75 ml) as a solvent was charged in three-neck flat-
bottom reaction flask. Reaction mass was continuously
stirred, and initially temperature was raised to 28–30 °C and
then maintain to reflux for 8–9 h. After completion of the
reaction, reaction mass was cooled to 20–22 °C, and 10 ml
water was added, stirred and transfer in separating funnel.
Lower organic layer from separating funnel was separated
and washed with sodium bicarbonate solution followed by
washing with water, and treated with sodium sulfate. The
organic layer was evaporated to dryness, and white-colored
solid product (4) (14.79 gm 85 % yield) was obtained.
0
Synthesis of 5-(4 -methylbiphenyl-2-yl)-2H-tetrazole
0
Mixture of 19.3 gm (0.1 mol) 4 -methyl-biphenyl-2-car-
bonitrile (1) and 18.8 gm (0.137 mol) triethylamine HCl
and 8.9 gm (0.137 mol) sodium azide in 100 ml xylene
was charged in a reaction kettle equipment with stirrer,
thermometer, and water condenser. Reaction mass was
stirred at 28–30 °C for 1.0 h, and then temperature was
raised to 138–142 °C (reflux) and maintained for 28 h.
Progress of the reaction was monitored on TLC, and after
completion of the reaction, reaction mass was cooled to
room temperature and 50 ml water was added. pH of
reaction mass containing water was adjusted to 10–11 by
the addition of 10 % NaOH solution, stirred for 30 min,
settled in separating funnel, and the layer was separated.
Synthesis of Amino based tetrazole compounds (5a–5t)
Compounds (5a–5t) were prepared by addition of compound
(4) (1.0 mol), substituted aromatic’s amine (1.1 mol), and
1
23

Med Chem Res
Scheme 1 Reaction scheme:
reaction scheme of synthesized
compounds
acetone as a solvent in three-neck flat-bottom reaction flask.
Reaction mass was stirred at 28–30 °C for 15–20 min, and tri
ethyl amine (1.1 mol) as an acid scavenger was added.
Reaction mass was continuously stirred, and the temperature
was maintained 55–60 °C for 3.0 h. Progress of the reaction
was monitored by TLC. After completion of the reaction,
reaction mass was cooled to 28–30 °C, filtered and washed
with acetone. De-protection of trityl group was carried out by
charging wet cake with methanol in three-neck flat-bottom
flask and was stirred at 28–30 °C with addition of 4 N HCl to
adjust pH 5–6, and temperature was raised and maintain at
40–45 °C for 1.0 h. The solvent was distilled under vacuum
at 40–45 °C temperature, and the solvent was added (water
and ethyl acetate). Again, reaction mass was stirred for
10 min and transferred to separating funnel and settled. Top
organic layer was separated and treated with sodium sulfate
and evaporated to dryness. Solid product (5a–5t) having
yield 75–95 % was obtained.
123

Med Chem Res
Table 1 Physico chemical parameter of synthesized compounds
Sr.
no.
Compounds
Code
%
Yield
Color (solid powder) Melting
Point
Compounds name
0
1
2
5a
5b
77 %
95 %
Off White
198–200 °C 1-[4-({[2 -(2H-tetrazol-5-yl)biphenyl-4-
yl]methyl}amino)phenyl]ethanone
0
Dirty White to
Creamish
167–168 °C 4-({[2 -(2H-tetrazol-5-yl)biphenyl-4-
yl]methyl}amino)benzenesulfonic acid
0
3
4
5
6
7
5c
5d
5e
5f
81 %
75 %
83 %
78 %
79 %
Light Brown
White
194–196 °C 4-({[2 -(2H-tetrazol-5-yl)biphenyl-4-yl]methyl}amino)phenol
0
193–194 °C N-{[2 -(2H-tetrazol-5-yl)biphenyl-4-yl]methyl}aniline
0
White to Creamish
White
210–212 °C 2-({[2 -(2H-tetrazol-5-yl)biphenyl-4-yl]methyl}amino)benzoic acid
0
165–168 °C N-{[2 -(2H-tetrazol-5-yl)biphenyl-4-yl]methyl}naphthalen-1-amine
0
5g
White
172–174 °C 1-phenyl-N-{[2 -(2H-tetrazol-5-yl)biphenyl-4-
yl]methyl}methanamine
0
8
9
5h
5i
82 %
84 %
85 %
88 %
90 %
92 %
83 %
82 %
79 %
77 %
White
178–180 °C 2-methyl-N-{[2 -(2H-tetrazol-5-yl)biphenyl-4-yl]methyl}aniline
0
White
184–185 °C 3-methyl-N-{[2 -(2H-tetrazol-5-yl)biphenyl-4-yl]methyl}aniline
0
10
11
12
13
14
15
16
17
5j
White
188–190 °C 4-methyl-N-{[2 -(2H-tetrazol-5-yl)biphenyl-4-yl]methyl}aniline
0
5k
5l
Light Yellow
Off White
Creamish Powder
White
177–179 °C 2-nitro-N-{[2 -(2H-tetrazol-5-yl)biphenyl-4-yl]methyl}aniline
187–189 °C 3-nitro-N-{[2 -(2H-tetrazol-5-yl)biphenyl-4-yl]methyl}aniline
192–193 °C 4-nitro-N-{[2 -(2H-tetrazol-5-yl)biphenyl-4-yl]methyl}aniline
194–195 °C 2-chloro-N-{[2 -(2H-tetrazol-5-yl)biphenyl-4-yl]methyl}aniline
0
0
5m
5n
5o
5p
5q
0
0
White
191–193 °C 4-chloro-N-{[2 -(2H-tetrazol-5-yl)biphenyl-4-yl]methyl}aniline
0
White
197–199 °C 4-fluoro-N-{[2 -(2H-tetrazol-5-yl)biphenyl-4-yl]methyl}aniline
0
White
201–203 °C 5-methyl-N-{[2 -(2H-tetrazol-5-yl)biphenyl-4-yl]methyl}-1,3-thiazol-
2
-amine
0
1
8
5r
91 %
Yellow Colored
117–122 °C 4-methyl-N-{[2 -(2H-tetrazol-5-yl)biphenyl-4-yl]methyl}piperazin-1-
amine
0
1
9
0
5s
5t
86 %
88 %
Off White
White
182–183 °C N-{[2 -(2H-tetrazol-5-yl)biphenyl-4-yl]methyl}pyridin-2-amine
0
2
197–198 °C N-{[2 -(2H-tetrazol-5-yl)biphenyl-4-yl]methyl}pyridin-3-amine
4
Biological evaluation of the isolated compounds
diluting in medium containing 10 % calf serum to 2 9 10
cells per 20 ml culture, aliquoting into each well of a tray,
and culturing until confluent. Microtiter trays with con-
fluent monolayer cultures of cells were inverted, the
medium shaken out, and replaced with serial dilutions of
sterile compounds in triplicate. In each tray, the last row of
wells was reserved for controls that were not treated with
compounds. Trays were cultured for 96 h. Trays were
inverted onto a paper towel pad, and the remaining cells
rinsed carefully with medium and fixed with 3.7 % (v/v)
formaldehyde in saline for at least 20 min. The fixed cells
were rinsed with water, and examined visually. The cyto-
toxic activity is identified as confluent, relatively unaltered
monolayers of stained cells treated with the investigated
compounds. Cytotoxicity was estimated as the concentra-
tion that caused approximately 50 % loss of the monolayer.
5-fluorouracil was used as a positive control.
Cytotoxicity and antitumor assays
Samples were prepared for assay by dissolving in 50 ml of
dimethyl sulfoxide (DMSO), and diluting aliquots into a
sterile culture medium at 0.4 mg/ml. These solutions were
sub-diluted to 0.02 mg/ml in sterile medium, and the two
solutions were used as stocks to test samples at 100, 50, 20,
1
0, 5, 2, and 1 mg/ml in triplicate in the wells of microtiter
plates. The compounds were assayed in triplicate on
monolayers grown in Dulbecco’s modified Eagle’s medium
supplemented with 10 % (v/v) fetal calf serum (HyClone
Laboratories, Ogden, UT), 60 mg/ml Penicillin G, and
1
00 mg/ml streptomycin sulfate maintained at 37 °C in a
humidified atmosphere containing about 5 % (v/v) CO in
2
air. All medium components were obtained from Sigma
Chemical Co., St. Louis, MO, unless otherwise indicated.
Subcultures of cells for screening were grown in the wells
of microtiter trays (Falcon Microtest III 96-wells trays,
Becton–Dickinson Labware, Lincolin Park, NJ) by sus-
pending cells in medium following trypsin–EDTA treat-
ment, counting the suspension with a hemocytometer,
Results and discussion
Physicochemical parameters of the compounds are pre-
sented in Table 1. All the compounds were colored and
1
23

Med Chem Res
stable in air. They were insoluble in water, but soluble in
organic solvents like ethylacetae, ethanol, DMF and
DMSO.
3229 (–NH), 2480 (–C=N), 851 (p-disubstituted
Ar). MW:-368; ESIMS; M/Z 368.
0
4
-({[2 -(2H-tetrazol-5-yl)biphenyl-4-
yl]methyl}amino)benzenesulfonic acid (5b)
FTIR & NMR spectral studies
1
Yield 95 %; m.p. 167–168 °C; H NMR (400 MHz,
The important infrared spectral bands and their tentative
assignments for the highly yielded biphenyl-based tetrazole
compound, 5b, (Supplementary Fig. 1), and its derivatives
were recorded as KBr disks, IR spectrum of the compounds
DMSO), 1.25 d ppm (2H, d, J = 14.6 Hz, –CH –NH), 3.34
2
d ppm (3H, s, -OCH ), 4.48 d ppm (1H, s, –NH, tetrazole),
3
6
.58–7.94 d ppm (8H, m, –Ar–H), 7.98 d ppm (1H, t,
1
J = 24.56 Hz, –NHCH2); C NMR (100 MHz, DMSO-
3
-
1
5
b showed a characteristic band between 3,417 cm
d6): d 46.58 (C-18), 113.5 (C-21,C-25), 128.55 (C-13,C-
confirming the presence of -OH groups of tetrazole com-
pounds and a characteristic band around at 3,336 and
1
1
7), 128.89 (C-8,C-10), 129.06 (C-14,C-16), 131.58 (C-9),
31.96 (C-22,C-24), 136.42 (C-6), 141.59 (C-15), 149.65
-
1
3
,227 cm
confirming the presence of -NH groups of
-
C-20); FTIR (KBr) tmax cm : 3417 (-OH), 3336 (–NH
1
(
tetrazole and secondary amine respectively. NMR spectral
tetrazole), 3227 (–NH), 2490 (–C=N), 1028(-S=O), 851 (p-
data of compound 5b revealed signals 1.25 d ppm for
disubstituted Ar). MW:-421; ESIMS; M/Z 421.
-
CH NH group, singlet at 4.48 d ppm for proton of –NH
2
tertazole group, and signals at 7.98 d ppm for –NH-CH
(
2
0
1
Supplementary Fig. 2). The C NMR data of compounds
3
4
-({[2 -(2H-tetrazol-5-yl)biphenyl-4-
yl]methyl}amino)phenol (5c)
5
1
b revealed signals between 163 d ppm for –C=N group, at
48 d ppm for Ar–C–SO group, and at 129, 127, 112, d
2
1
Yield 81 %; m.p. 194–196 °C; H NMR (400 MHz,
ppm for aromatic carbon, ESIMS; M/Z (Supplementary
DMSO), 1.27 d ppm (2H, d, J = 14.5 Hz, –CH –NH), 4.50
Figs. 3 and 4) (Silverstein and Webster, 1997).
2
d ppm (1H, s, –NH, tetrazole), 6.60–7.96 d ppm (8H, m,
–
Ar–H), 8.1 d ppm (1H, t, J = 24.46 Hz, –NHCH ), 9.1 d
Characterization data of synthesized compounds
2
1
3
ppm (1H, s, -OH); C NMR (100 MHz, DMSO-d ): d
6
4
5.88 (C-18), 112.8 (C-21,C-25), 127.55 (C-13,C-17),
127.92 (C-8,C-10), 128.26 (C-14,C-16), 129.77 (C-9),
29.96 (C-22,C-24), 135.482 (C-6), 140.79 (C-15), 148.68
2
2
3
0
2
2
1
24
25
R
1
2
-
1
(C-20); FTIR (KBr) tmax cm : 3420 (-OH), 3330 (–NH
tetrazole), 3222 (–NH), 2482 (–C=N), 851 (p-disubstituted
NH
1
8
5
19
Ar). MW:-343; ESIMS; M/Z 343.
1
1
1
6
7
14
13
0
N-{[2 -(2H-tetrazol-5-yl)biphenyl-4-yl]methyl}aniline (5d)
1
2
0
N
N
3
4
2
1
NH
Yield 75 %; m.p. 193–194 °C; H NMR (400 MHz,
DMSO), 1.30 d ppm (2H, d, J = 14.4 Hz, –CH –NH), 4.52
d ppm (1H, s, –NH, tetrazole), 6.62–7.98 d ppm (9H, m,
Ar–H), 8.2 d ppm (1H, t, J = 24.52 Hz, –NHCH2);
7
1
8
9
6
N
2
5
11
1
13
–
C
NMR (100 MHz, DMSO-d ): d 44.68 (C-18), 111.4 (C-
6
2
1,C-25), 126.35 (C-13,C-17), 126.19 (C-8,C-10), 127.16
0
1
-[4-({[2 -(2H-tetrazol-5-yl)biphenyl-4-yl] methyl}amino)
(C-14,C-16), 128.57 (C-9), 128.86 (C-22,C-24), 134.22 (C-
-
6), 139.69 (C-15), 147.45 (C-20); FTIR (KBr) tmax cm
1
phenyl]ethanone (5a)
:
3
340 (–NHtetrazole), 3230 (–NH), 2480 (–C=N), 848 (p-
1
Yield 77 %; m.p. 198–200 °C; H NMR (400 MHz,
disubstituted Ar).MW:-327; ESIMS; M/Z 327.
DMSO), 1.27 d ppm (2H, d, J = 14.7 Hz, –CH –NH), 3.4
2
0
d ppm (3H, s, –CH ), 4.46 d ppm (1H, s, –NH, tetrazole),
2-({[2 -(2H-tetrazol-5-yl)biphenyl-4-
3
6
.60–7.94 d ppm (8H, m, –Ar–H), 7.99 d ppm (1H, t,
J = 24.6 Hz, –NHCH2); C NMR (100 MHz, DMSO-d6):
yl]methyl}amino)benzoic acid (5e)
1
3
1
d 44.68 (C-18), 111.5 (C-21,C-25), 126.33 (C-13,C-17),
Yield 83 %; m.p. 210–212 °C; H NMR (400 MHz,
1
1
26.89 (C-8,C-10), 127.06 (C-14,C-16), 128.77 (C-9),
29.15 (C-22,C-24), 134.42 (C-6), 139.69 (C-15), 147.25
DMSO), 1.28 d ppm (2H, d, J = 14.58 Hz, –CH –NH),
4.52 d ppm (1H, s, –NH, tetrazole), 6.64–8.0 d ppm (9H, m,
2
-
1
(
C-20); FTIR (KBr) tmax cm : 3338 (–NH tetrazole),
–Ar–H), 8.2 d ppm (1H, t, J = 24.6 Hz, –NHCH ), 10.2 d
2
123

Med Chem Res
1
3
0
ppm (1H, s, –COOH); C NMR (100 MHz, DMSO-d ): d
3-methyl-N-{[2 -(2H-tetrazol-5-yl)biphenyl-4-
6
4
1
1
5.88 (C-18), 112.95 (C-21,C-25), 127.85 (C-13,C-17),
28.11 (C-8,C-10), 128.56 (C-14,C-16), 129.97 (C-9),
30.16 (C-22,C-24), 135.82 (C-6), 140.89 (C-15), 148.75
yl]methyl}aniline (5i)
1
Yield 84 %; m.p. 184–185 °C; H NMR (400 MHz,
-
1
(C-20); FTIR (KBr) tmax cm : 3417 (-OH), 3332 (–NH
tetrazole), 3220 (–NH), 2482 (–C=N), 1710 (-CO), 846 (p-
DMSO), 1.24 d ppm (2H, d, J = 14.3 Hz, –CH
d ppm (3H, s, –CH ), 4.44 d ppm (1H, s, –NH, tetrazole),
6.51–7.81 d ppm (10H, m, –Ar–H), 7.90 d ppm (1H, t,
2
–NH), 3.13
3
disubstituted Ar). MW:-370; ESIMS; M/Z 370.
1
3
J = 24.26 Hz, –NHCH2); C NMR (100 MHz, DMSO-
d6): d 44.28 (C-18), 111.45 (C-21,C-25), 126.55 (C-13,
C-17), 126.89 (C-8,C-10), 127.16 (C-14,C-16), 128.77
(C-9), 129.06 (C-22,C-24), 134.22 (C-6), 139.99 (C-15),
0
N-{[2 -(2H-tetrazol-5-yl)biphenyl-4-yl]methyl}naphthalen-
-amine (5f)
1
-
1
147.35 (C-20); FTIR (KBr) tmax cm : 3334 (–NH tetra-
1
Yield 78 %; m.p. 165–168 °C; H NMR (400 MHz,
DMSO), 1.23 d ppm (2H, d, J = 14.7 Hz, –CH –NH), 4.46
zole), 3228 (-NH), 2488 (–C=N), 852 (p-disubstituted Ar).
MW:-342; ESIMS; M/Z 342.
2
d ppm (1H, s, –NH, tetrazole), 6.56–7.90 d ppm (11H, m,
1
3
0
–
Ar–H), 7.96 d ppm (1H, t, J = 24.3 Hz, –NHCH2);
C
4-methyl-N-{[2 -(2H-tetrazol-5-yl)biphenyl-4-
NMR (100 MHz, DMSO-d ): d 46.78 (C-18), 113.1 (C-
yl]methyl}aniline (5j)
6
2
1,C-25), 128.15 (C-13,C-17), 128.59 (C-8,C-10), 128.96
C-14,C-16), 129.17 (C-9), 130.16 (C-22,C-24), 134.12 (C-
), 139.19 (C-15), 147.25 (C-20); FTIR (KBr) t
1
(
Yield 85 %; m.p. 188–190 °C; H NMR (400 MHz,
6
DMSO), 1.26 d ppm (2H, d, J = 14.5 Hz, –CH
d ppm (3H, s, –CH ), 4.46 d ppm (1H, s, –NH, tetrazole),
6.51–7.85 d ppm (8H, m, –Ar–H), 7.94 d ppm (1H, t,
2
–NH), 3.18
max
-
1
cm : 3338 (–NH tetrazole), 3225 (–NH), 2478 (–C=N),
3
8
45 (p-disubstituted Ar). MW:-377; ESIMS; M/Z 377.
1
3
J = 24.15 Hz, –NHCH2); C NMR (100 MHz, DMSO-
0
d
1
1
6
): d 45.98 (C-18), 112.85 (C-21,C-25), 128.15 (C-13,C-
7), 128.59 (C-8,C-10), 128.96 (C-14,C-16), 129.97 (C-9),
1
-phenyl-N-{[2 -(2H-tetrazol-5-yl)biphenyl-4-
yl]methyl}methanamine (5g)
29.86 (C-22,C-24), 135.82 (C-6), 141.69 (C-15), 149.35
-
1
1
(C-20); FTIR (KBr) tmax cm : 3334 (–NH tetrazole),
3
Yield 79 %; m.p. 172–174 °C; H NMR (400 MHz,
DMSO), 1.29 d ppm (2H, d, J = 14.65 Hz, –CH –NH),
.52 d ppm (1H, s, –NH, tetrazole), 6.5 d ppm (1H, –NH–
228 (–NH), 2488 (–C=N), 851 (p-disubstituted Ar). MW:-
2
3
42; ESIMS; M/Z 342.
4
CH ), 6.61–7.98 d ppm (9H, m, –Ar–H), 8.1 d ppm (1H, t,
2
0
1
J = 23.9 Hz, –NHCH2); C NMR (100 MHz, DMSO-d6):
3
2-nitro-N-{[2 -(2H-tetrazol-5-yl)biphenyl-4-
yl]methyl}aniline (5k)
d 45.18 (C-18), 112.15 (C-21,C-25), 127.25 (C-13,C-17),
1
1
27.19 (C-8,C-10), 128.36 (C-14,C-16), 129.27 (C-9),
29.66 (C-22,C-24), 135.12 (C-6), 140.33 (C-15), 148.45
1
Yield 88 %; m.p. 177–179 °C; H NMR (400 MHz,
-
1
DMSO), 1.25 d ppm (2H, d, J = 14.6 Hz, –CH
d ppm (1H, s, –NH, tetrazole), 6.58–7.96 d ppm (10H, m,
Ar–H), 7.94 d ppm (1H, t, J = 24.74 Hz, –NHCH2);
NMR (100 MHz, DMSO-d ): d 46.68 (C-18), 112.5 (C-
2
–NH), 4.48
(
C-20); FTIR (KBr) tmax cm : 3340 (–NH tetrazole),
3
230 (–NH), 2472 (–C=N), 848 (p-disubstituted Ar). MW:-
1
3
–
C
3
41; ESIMS; M/Z 341.
6
2
1,C-25), 127.95 (C-13,C-17), 128.19 (C-8,C-10), 128.56
0
2
-methyl-N-{[2 -(2H-tetrazol-5-yl)biphenyl-4-
(C-14,C-16), 130.17 (C-9), 130.66 (C-22,C-24), 136.22 (C-
-
6), 141.19 (C-15), 149.25 (C-20); FTIR (KBr) tmax cm
1
yl]methyl}aniline (5h)
:
3
336 (–NH tetrazole), 3227 (–NH), 2485 (–C=N), 1550
1
Yield 82 %; m.p. 178–180 °C; H NMR (400 MHz,
(-NO ), 852 (p-disubstituted Ar). MW:-372; ESIMS; M/
2
DMSO), 1.22 d ppm (2H, d, J = 14.56 Hz, –CH –NH), 3.1
Z 372.
2
d ppm (3H, s, –CH ), 4.42 d ppm (1H, s, –NH, tetrazole),
3
0
6
.49–7.80 d ppm (10H, m, –Ar–H), 7.92 d ppm (1H, t,
J = 24.1 Hz, –NHCH2); C NMR (100 MHz, DMSO-d6):
3-nitro-N-{[2 -(2H-tetrazol-5-yl)biphenyl-4-
1
3
yl]methyl}aniline (5l)
d 46.25 (C-18), 113.5 (C-21,C-25), 128.15 (C-13,C-17),
1
1
1
28.59 (C-8,C-10), 128.86 (C-14,C-16), 129.97 (C-9),
30.46 (C-22,C-24), 136.52 (C-6), 141.69 (C-15), 149.35
Yield 90 %; m.p. 187–189 °C; H NMR (400 MHz,
DMSO), 1.27 d ppm (2H, d, J = 14.2 Hz, –CH –NH), 4.50
d ppm (1H, s, –NH, tetrazole), 6.60–7.98 d ppm (10H, m,
–Ar–H), 7.96 d ppm (1H, t, J = 24.36 Hz, –NHCH2);
2
-
1
(
C-20); FTIR (KBr) tmax cm : 3332 (–NH tetrazole),
1
3
3
225 (–NH), 2485 (–C=N), 848 (p-disubstituted Ar). MW:-
C
3
42; ESIMS; M/Z 342.
NMR (100 MHz, DMSO-d ): d 43.88 (C-18), 111.95
6
1
23

Med Chem Res
0
Table 2 Antiproliferative activities of target compounds on different
cancer cell lines
2-chloro-N-{[2 -(2H-tetrazol-5-yl)biphenyl-4-
yl]methyl}aniline (5n)
a
Compounds IC50 (lM)
1
Yield 83 %; m.p. 194–195 °C; H NMR (400 MHz,
WI-38
VERO
MCF-7
HEPG-2
DMSO), 1.24 d ppm (2H, d, J = 14.55 Hz, –CH –NH),
2
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
a
a
b
c
92 ± 0.08
85 ± 0.17
92 ± 0.05
88 ± 0.12
97 ± 0.13
82 ± 0.12
95 ± 0.19
40 ± 0.14
110 ± 0.05
115 ± 0.34
105 ± 0.12
80 ± 0.25
75 ± 0.09
80 ± 0.14 70 ± 1.34 67 ± 0.29
88 ± 0.12 71 ± 1.32 60 ± 0.29
89 ± 0.13 55 ± 1.30 67 ± 0.15
84 ± 0.15 86 ± 1.74 66 ± 0.23
90 ± 0.12 60 ± 1.32 56 ± 0.21
70 ± 0.15 64 ± 1.25 70 ± 0.23
88 ± 0.15 78 ± 1.35 63 ± 0.21
39 ± 0.18 58 ± 1.24 52 ± 0.21
95 ± 0.05 30 ± 0.05 25 ± 0.32
95 ± 0.11 20 ± 0.35 15 ± 0.35
85 ± 0.04 30 ± 0.01 25 ± 0.05
90 ± 0.22 35 ± 0.22 30 ± 3.01
85 ± 0.24 40 ± 0.05 30 ± 0.05
4.50 d ppm (1H, s, –NH, tetrazole), 6.61–7.99 d ppm (10H,
1
3
m, –Ar–H), 8.1 d ppm (1H, t, J = 24.16 Hz, –NHCH2);
C
NMR (100 MHz, DMSO-d ): d 45.28 (C-18), 112.5 (C-
6
d
e
21,C-25), 127.55 (C-13,C-17), 127.79 (C-8,C-10), 128.26
(C-14,C-16), 129.37 (C-9), 129.66 (C-22,C-24), 135.82 (C-
-
1
6), 140.89 (C-15), 148.75 (C-20); FTIR (KBr) tmax cm
:
f
3338 (–NH tetrazole), 3229 (–NH), 2483 (–C=N), 710 (-C–
g
h
i
Cl) 856 (p-disubstituted Ar). MW:-362; ESIMS; M/Z 362.
0
4
-chloro-N-{[2 -(2H-tetrazol-5-yl)biphenyl-4-
j
yl]methyl}aniline (5o)
k
l
1
Yield 82 %; m.p. 191–193 °C; H NMR (400 MHz,
m
n
o
p
q
r
DMSO), 1.26 d ppm (2H, d, J = 14.52 Hz, –CH -NH),
2
105 ± 0.07 125 ± 0.36 25 ± 1.21 10 ± 0.14
100 ± 0.04 105 ± 0.03 25 ± 0.25 20 ± 0.32
4.52 d ppm (1H, s, –NH, tetrazole), 6.63–7.97 d ppm (8H,
m, –Ar–H), 8.12 d ppm (1H, t, J = 24.52 Hz, –NHCH );
2
90 ± 0.17
60 ± 0.14
23 ± 0.13
80 ± 0.10
78 ± 0.17
10 ± 0.05
90 ± 0.15 50 ± 1.34 40 ± 0.29
56 ± 0.14 50 ± 1.34 47 ± 0.29
19 ± 0.15 30 ± 1.30 41 ± 0.26
85 ± 0.12 70 ± 1.29 50 ± 0.27
69 ± 0.17 80 ± 0.11 94 ± 0.23
08 ± 1.02 10 ± 0.48 05 ± 1.36
1
3
C NMR (100 MHz, DMSO-d ): d 46.88 (C-18), 113.45
6
(C-21,C-25), 128.45 (C-13,C-17), 128.89 (C-8,C-10),
1
1
29.06 (C-14,C-16), 129.57 (C-9), 130.06 (C-22,C-24),
36.62 (C-6), 141.49 (C-15), 148.55 (C-20); FTIR (KBr)
s
t
-
1
tmax cm : 3336 (–NH tetrazole), 3227 (–NH), 2481
–C=N), 725 (-C–Cl) 852 (p-disubstituted Ar). MW:-
-FU
(
(
1–100 (weak), 100–200 (very weak), Above 200 (non-cytotoxic)
IC50, lM): 1–10 (very strong), 11–25 (strong), 26–50 (moderate),
362; ESIMS; M/Z 362.
5
0
4
-fluoro-N-{[2 -(2H-tetrazol-5-yl)biphenyl-4-
yl]methyl}aniline (5p)
(
C-21,C-25), 126.85 (C-13,C-17), 127.09 (C-8,C-10),
1
Yield 79 %; m.p. 197–199 °C; H NMR (400 MHz,
1
1
27.66 (C-14,C-16), 128.37 (C-9), 129.06 (C-22,C-24),
33.72 (C-6), 138.89 (C-15), 146.95 (C-20); FTIR (KBr)
DMSO), 1.27 d ppm (2H, d, J = 14.33 Hz, –CH –NH), 4.55
d ppm (1H, s, –NH, tetrazole), 6.64–8.1 d ppm (8H, m, –Ar–
H), 8.3 d ppm (1H, t, J = 24.1 Hz, –NHCH2); C NMR
100 MHz, DMSO-d ): d 45.73 (C-18), 112.58 (C-21,C-25),
2
-
1
tmax cm : 3332 (–NH tetrazole), 3225 (–NH), 2488
–C=N), 1555 (-NO ), 855 (p-disubstituted Ar). MW:-
1
3
(
2
(
6
3
72; ESIMS; M/Z 372.
1
27.35 (C-13,C-17), 127.79 (C-8,C-10), 127.96 (C-14,C-
1
6), 129.77 (C-9), 130.08 (C-22,C-24), 135.62 (C-6), 140.89
0
-1
4
-nitro-N-{[2 -(2H-tetrazol-5-yl)biphenyl-4-
(C-15), 148.75 (C-20); FTIR (KBr) tmax cm : 3340 (–NH
tetrazole), 3233 (–NH), 2486 (–C=N), 1110 (-C–F) 857 (p-
disubstituted Ar). MW:-345; ESIMS; M/Z 345.
yl]methyl}aniline (5m)
1
Yield 92 %; m.p. 192–193 °C; H NMR (400 MHz,
DMSO), 1.22 d ppm (2H, d, J = 14.4 Hz, –CH –NH), 4.46
0
2
5-methyl-N-{[2 -(2H-tetrazol-5-yl)biphenyl-4-yl]methyl}-
d ppm (1H, s, –NH, tetrazole), 6.56–7.94 d ppm (8H, m,
1,3-thiazol-2-amine (5q)
1
3
–
Ar–H), 7.92 d ppm (1H, t, J = 24.28 Hz, –NHCH2);
C
1
NMR (100 MHz, DMSO-d ): d 46.38 (C-18), 113.15 (C-
Yield 77 %; m.p. 201–203 °C; H NMR (400 MHz,
6
2
1,C-25), 128.05 (C-13,C-17), 128.69 (C-8,C-10), 129.06
DMSO), 1.25 d ppm (2H, d, J = 14.45 Hz, –CH –NH),
2
(
C-14,C-16), 130.07 (C-9), 130.86 (C-22,C-24), 136.42 (C-
-
2.98 d ppm (3H, s, –CH ), 4.46 d ppm (1H, s, –NH, tet-
3
1
6
), 141.69 (C-15), 149.45 (C-20); FTIR (KBr) tmax cm
:
razole), 6.54–7.92 d ppm (7H, m, –Ar–H), 7.96 d ppm (1H,
1
3
3
334 (–NH tetrazole), 3229 (–NH), 2488 (–C=N), 1558
t, J = 24.22 Hz, –NHCH2); C NMR (100 MHz, DMSO-
d6): d 44.75 (C-18), 111.3 (C-21,C-25), 126.55 (C-13,
C-17), 126.89 (C-8,C-10), 127.26 (C-14,C-16), 128.47
(
-NO ), 854 (p-disubstituted Ar). MW:-372; ESIMS; M/
2
Z 372.
123

Med Chem Res
Fig. 1 Comparison of cytotoxic
activities of target compounds
on different cancer cell lines
(
C-9), 128.96 (C-22,C-24), 134.52 (C-6), 139.79 (C-15),
-
ppm (1H, t, J = 24.52 Hz, –NHCH ), 8.3 d ppm (4H,m,
2
1
13
1
47.65 (C-20); FTIR (KBr) tmax cm : 3419 (-OH), 3339
–NH tetrazole), 3229 (–NH), 2480 (–C=N), 735(-C–S)
pyridine ring); C NMR (100 MHz, DMSO-d ): d 45.58 (C-
6
(
18), 112.82 (C-21,C-25), 127.35 (C-13,C-17), 127.89 (C-8,C-
8
48 (p-disubstituted Ar). MW:-347; ESIMS; M/Z 347.
10), 128.36 (C-14,C-16), 129.27 (C-9), 129.96 (C-22,C-24),
1
35.82 (C-6), 140.89 (C-15), 148.95 (C-20); FTIR (KBr) t
max
-1
cm : 3342 (–NH tetrazole), 3228 (–NH), 2468 (–C=N), 848
0
4
-methyl-N-{[2 -(2H-tetrazol-5-yl)biphenyl-4-
(p-disubstituted Ar). MW:-328; ESIMS; M/Z 328.
yl]methyl}piperazin-1-amine (5r)
0
N-{[2 -(2H-tetrazol-5-yl)biphenyl-4-yl]methyl}pyridin-3-
1
Yield 91 %; m.p. 117–122 °C; H NMR (400 MHz, DMSO),
amine (5t)
1
.22 d ppm (2H, d, J = 14.1 Hz, –CH –NH), 3.15 d ppm (3H,
2
1
s, –CH ), 4.42 d ppm (1H, s, –NH, tetrazole), 6.48–7.82 d ppm
Yield 88 %; m.p. 197–198 °C; H NMR (400 MHz,
DMSO), 1.28 d ppm (2H, d, J = 14.36 Hz, –CH –NH),
4.49 d ppm (1H, s, –NH, tetrazole), 6.55–7.89 d ppm (6H,
3
(
6H, m, –Ar–H), 7.92 d ppm (1H, t, J = 24.46 Hz, –NHCH ),
2
2
1
3
8
.11 d ppm (4H,m, piperazine ring); C NMR (100 MHz,
DMSO-d ): d 46.88 (C-18), 114.5 (C-21,C-25), 128.45 (C-
m, –Ar–H), 7.98 d ppm (1H, t, J = 24.68 Hz, –NHCH ),
8.44 d ppm (4H,m, pyridine ring); C NMR (100 MHz,
6
2
1
3
1
3,C-17), 128.89 (C-8,C-10), 129.08 (C-14,C-16), 130.17 (C-
9
), 130.86 (C-22,C-24), 136.42 (C-6), 141.39 (C-15), 149.25
DMSO-d ): d 46.68 (C-18), 113.53 (C-21,C-25), 128.45
(C-13,C-17), 128.86 (C-8,C-10), 129.08 (C-14,C-16),
6
-1
(
C-20); FTIR (KBr) tmax cm : 3340 (–NH tetrazole), 3230
–NH), 2470 (–C=N), 855 (p-disubstituted Ar). MW:-
(
130.17 (C-9), 130.86 (C-22,C-24), 136.52 (C-6), 141.79
-
1
3
49; ESIMS; M/Z 349.
(C-15), 149.35 (C-20); FTIR (KBr) t max cm : 3340 (–NH
tetrazole), 3225 (–NH), 2462 (–C=N), 845 (p-disubsti-
tuted Ar). MW:-328; ESIMS; M/Z 328.
0
N-{[2 -(2H-tetrazol-5-yl)biphenyl-4-yl]methyl}pyridin-2-
amine (5s)
Cytotoxicity and antitumor assays
1
Yield 86 %; m.p. 182–183 °C; H NMR (400 MHz, DMSO),
1
.25 d ppm (2H, d, J = 14.2 Hz, –CH –NH), 4.46 d ppm (1H,
s, –NH, tetrazole), 6.52–7.85 d ppm (6H, m, –Ar–H), 7.95 d
Cytotoxicity was expressed as the concentration that
caused a 50 % loss of the cell monolayer (IC ). The
2
5
0
1
23

Med Chem Res
results of our preliminary screening indicated that com-
pounds 5n, 5j, and 5o exhibited a very strong and a
selective cytotoxic activity against both HEPG-2 and
MCF-7, whereas compounds 5l, 5m, 5p, 5q, and 5r showed
a moderate cytotoxic activity against the aforementioned
cell lines. Other compounds 5a-h and 5s-t showed a weak
cytotoxicity activity (Table 2). Interestingly, compounds 5i
and 5k showed a moderate cytotoxic activity against MCF-
tetrazole moieties and evaluated their anticancer activities.
Out of a set of 20 molecules, three compounds exhibited a
significant and a selective anticancer activity against
HEPG-2 as well as MCF-7 that could be used as leads for
further investigations in the area of liver and breast cancer
therapies.
Acknowledgments We wish to thank Department of Applied
Chemistry, SVNIT, Surat for providing laboratory facilities and
financial support. We are grateful to Mr. Avtar Singh SAIF Punjab
University for spectral analysis.
7
while they had a strong activity against HEPG-2. On the
other hand, all tested compounds showed a weak cytotoxic
activity against both WI-38 and VERO cell lines (Fig. 1).
Subsequently, we may conclude the following structure
activity relationships for the promo sing biphenyl-based
tetrazole derivatives. The presence of the basic skeleton
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cell lines. Particularly, CL at ortho position gives a very
strong cytotoxicity compared to para position. F substitu-
tion had a moderate cytotoxic activity compared to chlo-
rine. The presence of NO , an electron withdrawing group
2
(
strongly deactivating group), in position 2, 3, or 4 of the
benzene ring showed a moderate cytotoxicity toward
HEPG-2 and MCF-7 cell lines (e.g., Compounds 5k, 5l, or
5
m). However, the presence of the electron releasing
group, CH , in position 2, 3, or 4 showed a strong to
3
moderate cytotoxicity activity toward HEPG-2 and MCF-7
(
compounds 5h, 5i, and 5j). Following this further, the
presence of electron withdrawing or releasing groups at
any subsequent position of the benzene ring in tetrazole
motif showed a weak cytotoxicity against WI-38 and
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4
:105–111
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Conclusion
In conclusion, we have designed hybrid molecules on the
basis of the biological significance of biphenyl and
123