Facile synthesis of novel quinoline derivatives as anticancer agents

Article abstract of DOI:10.1007/s00044-013-0855-2

Convenient and efficient synthesis of novel N-(4-Acetyl-4,5-dihydro-5-(7,8, 9-substituted-tetrazolo[1,5-A]-quinolin-4-yl)-1,3,4-thiadiazol-2-yl)acetamides 4a-j and their in vitro anticancer activity against two cell lines viz., human breast cancer cell line MCF7 and human cervix cancer cell line HeLa were carried out. GI50, LC50, TGI values were evaluated. Two of the compounds 4e and 4i with halogen substituent at 7th position of the target molecules have shown potent activity against human cervix cancer cell line HeLa. DNA cleavage studies revealed that most of these compounds show partial cleavage and few of them show complete cleavage of DNA.

Full text of DOI:10.1007/s00044-013-0855-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0855-2
ORIGINAL RESEARCH
Facile synthesis of novel quinoline derivatives as anticancer agents
•
Sheetal Babu Marganakop Ravindra Ramappa Kamble
•
•
Joy Hoskeri D. Jagadish Prasad Gangadhar Yamanappa Meti
•
Received: 13 June 2013 / Accepted: 17 October 2013
Ó Springer Science+Business Media New York 2013
Abstract Convenient and efficient synthesis of novel N-
(4-acetyl-4,5-dihydro-5-(7,8,9-substituted-tetrazolo[1,5-a]-
quinolin-4-yl)-1,3,4-thiadiazol-2-yl)acetamides 4a–j and
their in vitro anticancer activity against two cell lines viz.,
human breast cancer cell line MCF7 and human cervix
cancer cell line HeLa were carried out. GI50, LC50, TGI
values were evaluated. Two of the compounds 4e and 4i
with halogen substituent at 7th position of the target mol-
ecules have shown potent activity against human cervix
cancer cell line HeLa. DNA cleavage studies revealed that
most of these compounds show partial cleavage and few of
them show complete cleavage of DNA.
Introduction
Quinoline, thiadiazole, and tetrazole derivatives are known
to possess wide range of pharmacological activities (Brian
et al., 2010; Adnan et al., 2004; Zarubaev et al., 2010;
Chebolu et al., 2011; Malleshappa et al., 2011). Many of
the derivatives of these compounds have been used as
active drugs and thorough literature review (Abeer and
Daneshtalab, 2011) has also revealed that more efficient
lead molecules are designed by joining two or more
pharmacologically active heterocyclic systems together in
a single molecular framework (Fig. 1). Therefore, synthe-
sis of such molecules is challenging and may lead to
invention of new molecules of remedial importance.
Cancer is due to the result of uncontrolled growth of the
cells and hence it is one of the major diseases causing death
worldwide. Despite of extensive investigation in case of
breast cancer there is still no known cure. Cervical cancer
which mainly occurs in the uterus has emerged as a killer
disease globally. It is mostly seen in women above
35 years of age. World health organization has predicted
that the number of people affected by cervical cancer in
developing countries would triple in another two decades.
Cancer chemotherapy is highly selective and not associated
with the serious toxicities. Tetrazole and quinoline deriv-
atives have been reported as promising candidates for
anticancer activity.
Keywords Anticancer activity Á Breast cancer cell line
MCF7 Á Cervix cancer cell line HeLa Á DNA cleavage Á
Quinoline
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-013-0855-2) contains supplementary
material, which is available to authorized users.
S. B. Marganakop Á R. R. Kamble (&) Á G. Y. Meti
Department of Studies in Chemistry, Karnatak University,
Dharwad 580003, India
Understanding these facts and in continuation of our
research exploration (Sheetal et al., 2012) of novel quinoline
derivatives, we have carried out structural modifications to
N-[4-acetyl-5-(6, 7, 8-substituted-2-chloroquinolin-3-yl)-
4,5-dihydro-1,3,4-thiadiazol-2-yl]-acetamides by simple
convenient methods. Hence, we have made an attempt to
fuse tetrazole moiety with quinoline derivatized with 1,3,4-
thiadiazolidine ring so as to get three heterocyclic rings in a
e-mail: kamchem9@gmail.com
J. Hoskeri
Department of Studies and Research in Biotechnology,
The Oxford College of Science, Bangalore, India
D. J. Prasad
Department of Studies in Chemistry, Mangalore University,
Mangalgangotri, Konaje 574199, India
123

Med Chem Res
Fig. 1 Molecules with
anticancer activity
O
H
O
O
O
OH
O
O
OH
O
OH
OH
OH
N
N
O
O
O
O
H
O
OH
O
HO
O
OH
O
NH2
H2N
OH
H3C
OH
Camptothecin
Adriamycin
Doxorubcin
Scheme 1 Synthesis of
compounds 4a–j
a R1=R2=R3=H, b R1=R3=H,
R2=CH3, c R1=R3=H,
R2=OCH3, d R1=CH3,
R2=R3=H, e R1=R3=H, R2=Br,
f R1=OCH3, R2=R3=H,
g R1=R2=H, R3=OCH3,
h R1=H, R2=R3=OCH3,
i R1=R3=H, R2=Cl, j R1=R2=H,
R3=CH3. Reaction conditions:
(a) Ac2O, 90 °C; (b) NaN3,
EtOH, AcOH, reflux; (c) Ac2O,
90 °C; (d) NaN3, H2O, EtOH,
reflux
single molecular frame work. DNA cleavage study and
in vitro anticancer activity of these compounds were carried
out against human breast cancer cell line MCF7 and human
cervix cancer HeLa cell line. GI50, LC50, TGI values were
evaluated.
Table 1 Comparison of % yield for the preparation of compounds
4a–j by conventional and by one-pot reaction
Entry no.
Conventional method
One-pot method
4a
4b
4c
4d
4e
4f
78.0
82.0
75.0
70.0
65.0
75.8
71.2
65.8
78.0
70.0
85.5
87.0
78.1
72.2
66.0
85.0
76.6
68.9
84.5
74.3
Results and discussion
Chemistry
4g
4h
4i
The title compounds 4a–j were prepared by two methods
as depicted in Scheme 1, % yield from direct conversion
from 2a–j is better than the conventional method (Table 1).
The proposed mechanism of the reaction is depicted in
Scheme 2. The structures of the compounds 4a–j were
4j
1
confirmed by their elemental analysis, IR, H NMR, and
derivative 3a–j appeared around d 8.1 ppm. This proton
was shifted to d 6.2–7.3 ppm in the title compounds 4a–
j and hence confirmed the cyclization of thiosemicarbazone
to 1,3,4-thiadiazole 4a–j. The protons of the substituent
groups and the aromatic protons appeared in their respec-
tive regions. 13C NMR spectral studies indicated the
number of signals in consistent with the number of mag-
netically non-equivalent carbon atoms. Mass spectral ana-
lysis has shown molecular ion peak for compound 4a at
355, and a peak corresponding to loss of N3 at 313. Loss of
COCH3 at 271 and a base peak at 43 corresponding to
mass spectral data. The IR spectra of the title compounds
4a–j have shown mainly a sharp strong absorption band
around 1,650–1,680 cm-1 which corresponds to amide
carbonyl group. Another medium absorption band was
observed at around 1,602–1,642 cm-1 due to C=N
stretching.
1
The H NMR spectral analysis of the title compounds
4a–j revealed a characteristic singlet in the range d
6.2–7.3 ppm responsible for C5-H of the 1,3,4-thiadiazole
ring. The imine proton (-CH=N) in the thiosemicarbazone
123

Med Chem Res
Scheme 2 Proposed
mechanism for the synthesis of
4a–j
O
O
O
O
H
N
NH2
H
N
NH2
N
S
N+
H
S
H
Cl
N
N
Cl
2a
O
N
S
NH2
N
Cl
N
Ac2O
O
N
NHCOCH3
N
S
3a
N
Cl
-
N-
N
+
N
O
O
N
S
N
S
NHCOCH3
NHCOCH3
N
N
N
N
N-
N
N
N
+
N
4a
COCH3 (Scheme 3) other compounds also have shown
corresponding peaks at their respective positions.
compounds 4e and 4i may be attributed to the halogen
substituent (Yang et al., 2010; Tashfeen et al., 2008) on the
quinoline ring and partial activity of the compounds 4b, 4d,
and 4j due to methyl substituents. Electron withdrawing
atoms such as Cl, Br on the quinoline ring enhanced the
activity of the molecules, whereas electron donating groups
like CH3, OCH3 retarded the anticancer activity. This
observation reveals that electron withdrawing substituent
in quinoline nucleus improves the anticancer activity of
these molecules.
Anti cancer activity
The anticancer activity studies reveal that two compounds
4e and 4i exhibited good activity against human cervix
cancer cell line HeLa. None of the compounds have shown
good activity against human breast cancer cell line MCF7.
From the LC50, TGI, GI50 values, and cell growth curves
(Tables 2, 3; Figs. 2, 3) it is clear that compounds 4e and 4i
with bromo and chloro group at 7th position of target
molecule exhibited good activity. Compounds 4b, 4d, and
4j with methyl groups (at 7, 8, and 9th position, respec-
tively) have exhibited partial activity against human cervix
cancer cell line HeLa. Unsubstituted and methoxy substi-
tuted compounds 4a, 4c, 4f, 4g, and 4h have not shown any
reasonable activity against both the tested cell lines.
However, the compound 4d has shown partial anticancer
activity against both the cell lines. Potent activity of the
DNA cleavage activity
A number of studies have shown that the clinical efficacies
of many drugs have correlation with their abilities to
induce enzyme-mediated DNA cleavage. The inhibitory
potency of the test compounds was assessed by comparing
the cleavage of DNA by control and the title compounds.
The gel electrophoresis was used for the analysis of DNA
cleavage activity of title compounds 4a–j. It was observed
123

Med Chem Res
Scheme 3 Mass spectral
fragmentation pattern for
compound 4a
+
H3COC
N
N
NHCOCH3
S
( M+
)
N
m/z 355
N
m/z =43
Base peak
N
N
N3
COCH3
+
H3COC
+
N
N
H3COC
NHCOCH3
S
N
N
NH2
S
N
m/z 314
N
N
m/z =313
N
N
COCH3
N3
+
+
H3COC
H3COC
N
N
N
NH
S
S
N2
N
N
m/z = 271
m/z = 243
COCH3
N
+
NH
N
S
N
m/z = 214
Table 2 LC50, TGI and GI50 values at lM drug concentration
against human breast cancer cell line MCF7
Table 3 LC50, TGI, and GI50 values at lM drug concentration
against human cervix cancer cell line HeLa
Entry no.
LC50
TGI
GI50a
Entry no.
LC50
TGI
GI50a
4a
4b
4c
4d
4e
4f
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[0.1
[100
[100
[100
95.33
[100
[100
[100
[100
[100
98.93
\0.1
4a
4b
4c
4d
4e
4f
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[100
[0.1
[100
81.29
[100
85.08
\0.1
[100
[100
[100
\0.1
83.26
\0.1
4g
4h
4i
4g
4h
4i
4j
4j
ADR
ADR
a
a
GI50 B 1 lM is considered to be active
GI50 B 1 lM is considered to be active
123

Med Chem Res
from the gel photograph that compounds 4a–c, e, g, h, and
i (with 100 lg concentration) have shown partial cleavage
of DNA, whereas compounds 4d and 4f showed significant
diminishing of band with a prominent streak indicating
complete DNA cleavage (Fig. 4).
Experimental
Chemistry
Melting points were measured using open capillary in a
melting point apparatus. FT-IR spectra were recorded on
Nicolet 5700 spectrophotometer using KBr pellets. The 1H
NMR and 13C NMR spectra were recorded on a Varian
(300 MHz) FT-NMR spectrometer in DMSO-D6 with TMS
as an internal standard. Mass spectra were recorded on a
GCMS-SC\AD\17-004 Mass spectrometer. All the reagents
and solvents were of analytical grade, and used as supplied
unless otherwise stated. TLC was performed on silica gel
coated plates for monitoring the reactions. Elemental
analyses were performed on Heraus CHN analyzer. The
anticancer activities, DNA cleavage evaluations were car-
ried out at ACTREC, Tata Memorial Centre, Mumbai,
Maharashtra, India and Biogenics, Research and training
centre in Biotechnology, Hubballi, Karnataka, India.
The thiosemicarbazones 2a–j and corresponding N-[4-
acetyl-5-(6,7,8-substituted-2-chloroquinolin-3-yl)-4,5-
dihydro-1,3,4-thiadiazol-2-yl]-acetamides 3a–j were pre-
pared by literature methods (Sheetal et al., 2012; Meth-Cohn
et al., 1981; Wiles et al., 1907; El-Sayed et al., 2004). Fur-
ther, these were converted to title compounds N-(4-acetyl-
4,5-dihydro-5-(7,8,9-substituted-tetrazolo[1,5-a]-quinolin-
4-yl)-1,3,4-thiadiazol-2-yl)acetamide (4a–j) (Scheme 1).
Fig. 2 Cell growth curve for human cervix HeLa cell lines
Synthesis
General procedure for the preparation of N-(4-acetyl-4,5-
dihydro-5-(7,8,9-substituted-tetrazolo[1,5-a]quinolin-4-yl)-
1,3,4-thiadiazol-2-yl)acetamide (4a–j).
Compound (3a–j, 0.005 mol), sodium azide (0.01 mol),
acetic acid (1 ml), and ethanol (10 ml) were charged in
round bottom flask fitted with condenser and stirred. The
reaction mixture was slowly heated and refluxed for 3–4 h.
After completion of the reaction (monitored by TLC), the
product was filtered and washed with ethanol. The crude
product was purified by crystallization from ethanol: DMF
Fig. 3 Cell growth curve for human breast cancer MCF7 cell lines
Fig. 4 DNA cleavage studies
for compounds 4a–j
123

Med Chem Res
(60:40) to get colorless to pale brown needles of the
NHCOCH3); 24.12 (Ar–CH3); MS m/z (%): 369 (M?,
14.0), 327 (12.0), 285 (4), 43 (100); CHN analysis; cal-
culated for C16H15N7O2S: C, 52.02; H, 4.09; N, 26.54.
Found: C, 52.04; H, 4.12; N, 26.52.
compounds (4a–j).
One-pot synthesis of compounds (4a–j)
N-(4-Acetyl-4,5-dihydro-5-(7-methoxy-tetrazolo[1,5-a]
A solution of compound (2a–j) (0.005 mol) in acetic
anhydride (4.0 ml) was heated at 80–90 °C for 1 h. The
reaction mixture was cooled to room temperature and acetic
anhydride was removed under reduced pressure. To the
reaction mixture, sodium azide (0.01 mol) in water (2.0 ml)
and ethanol (10.0 ml) were added and the reaction mixture
was heated gently initially for 30 min and then refluxed for
3–4 h. After completion of the reaction (monitored by
TLC), the product was filtered and washed with ethanol and
then poured into ice-cold water. The precipitate thus
obtained was filtered off, washed with water, dried, and
crystallized from Ethanol:DMF (60:40) to get colorless to
pale brown needles of the compounds (4a–j).
quinolin-4-yl)-1,3,4-thiadiazol-2-yl)acetamide (4c)
Yellow crystals, m.p. 240–242 °C; IR (cm-1): 3221, 3164,
2950, 1653, 1606, 1411; 1H NMR (300 MHz, d ppm,
DMSO-D6); 2.02 (s, 3H, CH3 of NHCOCH3), 2.35 (s, 3H,
CH3 of N4–COCH3), 3.90 (s, 3H, OCH3), 6.97 (s, 1H, C5-H
of thiadiazole), 7.48–7.98 (m, 4H, Ar–H, C5-H,C6-H, C8-H,
C9-H), 11.85 (s, 1H, NHCO); 13C NMR (300 MHz, d ppm,
DMSO-D6);169.51 (C=O of NHCOCH3), 168.32 (C=O of
NCOCH3), 150.07, 144.31 (C2 of thiadiazole ring), 107.40,
156.91, 122.97, 129.79, 140.26, 129.22, 133.62, 130.91
(quinoline), 68.50 (C5 of thiadiazole ring), 54.71 (CH3 of
OCH3), 21.40 (CH3 of NCOCH3), 21.82 (CH3 of NHC-
OCH3); MS m/z (%): 386 (M?, 18.0), 375 (55), 359 (42),
343 (12), 331 (30), 43 (100). CHN analysis: calculated for
C16H15N7O3S: C, 49.86; H, 3.92; N, 25.44. Found: C,
49.90; H, 3.91; N, 26.52.
N-(4-Acetyl-4,5-dihydro-5-(tetrazolo[1,5-a]quinolin-4-yl)-
1,3,4-thiadiazol-2-yl)acetamide (4a)
Colorless crystals, m.p. 235–237 °C; IR (cm-1): 3452,
3261, 2984, 1668, 1610; 1H NMR (300 MHz, d ppm,
DMSO-D6); 2.03 (s, 3H, CH3 of NHCOCH3), 2.31 (s, 3H,
CH3 of N4–COCH3), 7.01 (s, 1H, C5-H of thiadiazole),
7.69–8.31 (m, 4H, Ar–H, C6-H, C7-H, C8-H, C9-H), 8.64
(s, 1H, C5-H), 11.72 (s, 1H, NHCO); 13C NMR (300 MHz,
d ppm, DMSO-D6); 169.72 (C=O of NHCOCH3), 168.22
(C=O of NCOCH3), 144.4 (C2 of thiadiazole ring), 151.22,
127.61, 126.51, 128.78, 128.41, 146.21, 128.34, 133.22,
130.23 (quinoline), 68.61 (C5 of thiadiazole ring), 21.32
(CH3 of NCOCH3), 21.91 (CH3 of NHCOCH3); MS m/
z (%): 355 (M? 16.0), 313 (12), 271 (5), 43 (100); CHN
analysis; calculated for C15H13N7O2S: C, 50.70; H, 3.69;
N, 27.59. Found: C, 50.72; H, 3.70; N, 27.60.
N-(4-Acetyl-4,5-dihydro-5-(9-methyl-tetrazolo[1,5-a]
quinolin-4-yl)-1,3,4-thiadiazol-2-yl)acetamide (4d)
Yellow crystals, m.p. 210–212 °C; IR (cm-1): 3411, 3252,
2934, 1666, 1633, 1422; 1H NMR (300 MHz, d ppm,
DMSO-D6); 2.08 (s, 3H, CH3 of NH COCH3), 2.15 (s, 3H,
CH3 of N4–COCH3), 2.42 (s, 3H, Ar–CH3) 7.02 (s, 1H, C5-
H of thiadiazole), 7.30–8.01 (m, 3H, Ar–H, C6-H, C7-H,
C8-H), 8.61 (s, 1H, C5-H), 11.12 (s, 1H, NHCO); 13C NMR
(300 MHz, d ppm, DMSO-D6); 169.49 (C=O of NHC-
OCH3), 167.67 (C=O of NCOCH3), 144.41 (C2 of thiadi-
azole ring), 151.11, 126.81, 128.31, 137.65, 129.60,
127.03, 143.22, 126.82, 133.93, 129.42 (quinoline), 68.60
(C5 of thiadiazole ring), 21.50 (CH3 of NCOCH3), 21.72
(CH3 of NHCOCH3), 24.06 (Ar–CH3); MS m/z (%): 369
(M?, 13.0), 327 (11), 285 (6), 43 (100); CHN analysis;
calculated for C16H15N7O2S: C,52.02; H, 4.09; N, 26.54.
Found: C, 52.04; H, 4.10; N, 26.50.
N-(4-Acetyl-4,5-dihydro-5-(7-methyl-tetrazolo[1,5-a]
quinolin-4-yl)-1,3,4-thiadiazol-2-yl)acetamide (4b)
Pale yellow crystals, m.p. 226–228 °C; IR (cm-1): 3442,
3247, 2924, 1663, 1623, 1400; 1H NMR (300 MHz, d ppm,
DMSO-D6); 2.12 (s, 3H, CH3 of NHCOCH3), 2.22 (s, 3H,
CH3 of N4-COCH3), 2.30 (s, 3H, CH3 of Ar–CH3) 7.21 (s,
1H, C5-H of thiadiazole), 7.59–8.23 (m, 3H, Ar–H, C6-H,
N-(4-Acetyl-4,5-dihydro-5-(7-bromo-tetrazolo[1,5-a]
quinolin-4-yl)-1,3,4-thiadiazol-2-yl)acetamide (4e)
Yellow crystals, m.p. 228–230 °C; IR (cm-1): 3435, 2921,
2854, 1705, 1658, 1611, 1486; 1H NMR (300 MHz, d ppm,
DMSO-D6); 2.02 (s, 3H, CH3 of NHCOCH3), 2.31 (s, 3H,
CH3 of N4–COCH3), 7.24 (s, 1H, C5-H of thiadiazole),
7.99 (s, 1H, Ar H, C5-H), 8.11–8.61 (m, 3H, Ar–H, C6-H,
C8-H,C9-H),11.46 (s, 1H, NHCO); 13C NMR (300 MHz, d
ppm, DMSO-D6); 169.62 (C=O of NHCOCH3), 168.30
C8-H, C9-H), 6.75 (s, 1H, C5-H), 11.12 (s, 1H, NHCO); 13
C
NMR (300 MHz, d ppm, DMSO-D6); 169.61 (C=O of
NHCOCH3), 167.82 (C=O of NCOCH3), 144.22 (C2 of
thiadiazole ring), 150.41 126.60, 131.61, 130.72, 127.43,
142.65, 128.13, 134.22, 129.12 (quinoline), 68.60 (C5 of
thiadiazole ring), 21.41 (CH3 of NCOCH3), 21.29 (CH3 of
123

Med Chem Res
(C=O of NCOCH3), 144.10 (C2 of thiadiazole ring), 152.21,
128.20, 120.90, 131.84, 128.56, 144.22, 126.31, 133.41,
130.79 (quinoline), 68.41 (C5 of thiadiazole ring), 21.31
(CH3 of NCOCH3), 21.89 (CH3 of NHCOCH3); MS m/z (%):
435 (M?2, 22), 433 (M?, 19), 391 (22), 349 (34), 43 (100):
CHN analysis; calculated for C15H12BrN7O2S : C, 41.49; H,
2.79; N, 22.58. Found: C, 41.48; H, 2.77; N, 22.61.
H), 11.42 (s, 1H, NHCO); 13C NMR (300 MHz, d ppm,
DMSO-D6); 169.40 (C=O of NHCOCH3), 168.62 (C=O of
NCOCH3), 144.22 (C2 of thiadiazole ring), 107.42, 152.55,
150.10, 108.20, 140.11, 124.90, 133.50, 128.11 (quino-
line), 68.43 (C5 of thiadiazole ring), 56.20 (OCH3), 21.51
(CH3 of NCOCH3), 21.90 (CH3 of NHCOCH3); MS m/
z (%): 415 (M?, 25), 372 (54), 329 (50), 271 (49), 43 (100);
CHN analysis: calculated for C17H17N7O4S: C, 50.70; H,
3.69; N, 27.59. Found: C, 50.72; H, 3.66; N, 25.58.
N-(4-Acetyl-4,5-dihydro-5-(9-methoxy-tetrazolo[1,5-a]
quinolin-4-yl)-1,3,4-thiadiazol-2-yl)acetamide (4f)
N-(4-Acetyl-4,5-dihydro-5-(7-chloro-tetrazolo[1,5-a]
Yellow crystals, m.p. 215–217 °C; IR (cm-1): 3231, 3166,
2953, 1663, 1606, 1414; 1H NMR (300 MHz, d ppm,
DMSO-D6); 2.03 (s, 3H, CH3 of NHCOCH3), 2.55 (s, 3H,
CH3 of N4–COCH3), 3.96 (s, 3H, OCH3), 6.99 (s, 1H, C5-H
of thiadiazole), 7.45–8.01 (m, 4H, Ar–H, quin C5-H, C6-H,
C7-H, C8-H), 11.32 (s, 1H, NHCO); 13C NMR (300 MHz,
d ppm, DMSO-D6); 169.51 (C=O of NHCOCH3), 168.31
(C=O of NCOCH3), 144.30 (C2 of thiadiazole ring),
151.20, 117.91, 126.54, 105.9, 155.04, 138.56, 129.14,
134.33, 130.91, (quinoline), 68.52 (C5 of thiadiazole ring),
55.11 (CH3 of OCH3), 21.50 (CH3 of NCOCH3), 21.91
(CH3 of NHCOCH3); MS m/z (%): 387 (M?, 20), 375 (63),
359 (46), 343 (14),331 (31), 43 (100); CHN analysis: cal-
culated for C16H15N7O3S: C, 49.86; H, 3.92; N, 25.44.
Found: C, 49.90; H, 3.90; N, 25.42.
quinolin-4-yl)-1,3,4-thiadiazol-2-yl)acetamide (4i)
Yellow crystals, m.p. 231–233 °C; IR (cm-1): 3445, 2932,
2856, 1702, 1661, 1621, 1486; 1H NMR (300 MHz, d ppm,
DMSO-D6); 2.22 (s, 3H, CH3 of NHCOCH3), 2.35 (s, 3H,
CH3 of N4–COCH3), 7.14 (s, 1H, C5-H of thiadiazole),
7.89 (s, 1H, C5-H), 8.48–8.89 (m, 3H, C6-H, C8-H,C9-H),
11.47 (s, 1H, NHCO); 13C NMR (300 MHz, d ppm,
DMSO-D6); 169.71 (C=O of NHCOCH3), 168.31 (C=O of
NCOCH3), 152.23, 144.15 (C2 of thiadiazole ring), 125.10,
132.23, 130.20, 130.51, 141.23, 129.10, 133.50, 130.61
(quinoline), 68.90 (C5 of thiadiazole ring), 21.33 (CH3 of
NCOCH3), 21.90 (CH3 of NHCOCH3); MS m/z (%): 391
(M?2, 27.0), 389 (M?, 9.0), 347 (22.3), 305 (40), 43 (100);
CHN analysis; calculated for C15H12ClN7O2S: C, 46.22; H,
3.10; N, 25.15. Found: C, 46.19; H, 3.11; N, 25.16.
N-(4-Acetyl-4,5-dihydro-5-(8-methoxy-tetrazolo[1,5-a]
quinolin-4-yl)-1,3,4-thiadiazol-2-yl)acetamide (4g)
N-(4-Acetyl-4,5-dihydro-5-(8-methyl-tetrazolo[1,5-a]
quinolin-4-yl)-1,3,4-thiadiazol-2-yl)acetamide (4j)
Yellow crystals, m.p. 225–226 °C; IR (cm-1): 3235, 3150,
2973, 1661, 1600, 1411; 1H NMR (300 MHz, d ppm, DMSO-
D6); 2.02 (s, 3H, CH3 of NHCOCH3), 2.67 (s, 3H, CH3 of N4–
COCH3), 3.87 (s, 3H, OCH3), 6.80 (s, 1H, C5-H of thiadia-
zole), 7.45–7.97 (m, 4H, Ar–H, C5-H,C6-H, C7-H, C9-H),
11.55 (s, 1H, NHCO); 13C NMR (300 MHz, d ppm, DMSO-
D6);169.51(C=OofNHCOCH3), 168.30(C=OofNCOCH3),
144.39 (C2 of thiadiazole ring), 152.30, 129.70, 119.51,
150.09, 106.21, 144.20, 124.12, 134.20, 127.91 (quinoline),
68.64 (C5 of thiadiazole ring), 55.81 (CH3 of OCH3), 21.40
(CH3 of NCOCH3), 21.81 (CH3 of NHCOCH3); MS m/z (%):
387 (M?, 25), 375 (62), 359 (56), 343 (20), 331 (41), 43 (100);
CHN analysis: calculated for C16H15N7O3S: C, 49.86; H,
3.82; N, 25.49. Found: C, 49.91; H, 3.95; N, 25.46.
Yellow crystals, m.p. 210–212 °C; IR (cm-1): 3415, 3242,
2912, 1658, 1622, 1427; 1H NMR (300 MHz, d ppm,
DMSO-D6): 2.04 (s, 3H, CH3 of NH COCH3), 2.12 (s, 3H,
CH3 of N4–COCH3), 2.52 (s, 3H, Ar–CH3) 7.15 (s, 1H, C5-
H of thiadiazole),7.40–8.08 (m, 3H, C6-H, C7-H, C9-H),
8.51 (s, 1H, C5-H of thiadiazole), 11.22 (s, 1H, NHCO);
13C NMR (300 MHz, d ppm, DMSO-D6); 169.50 (C=O of
NHCOCH3), 167.71 (C=O of NCOCH3), 144.40 (C2 of
thiadiazole ring), 151.20, 126.51, 128.20, 129.51, 137.22,
143.12, 126.94, 133.84, 129.34 (quinoline), 68.62 (C5 of
thiadiazole ring), 21.52 (CH3 of NCOCH3), 21.71 (CH3 of
NHCOCH3), 24.40 (Ar–CH3); MS m/z (%): 369 (M?, 15),
327 (11), 285 (6), 43 (100); CHN analysis; calculated for
C16H15N7O2S: C, 52.02; H, 4.09; N, 26.54. Found: C,
52.04; H, 4.12; N, 26.52.
N-(4-Acetyl-4,5-dihydro-5-(7,8-dimethoxy-tetrazolo[1,5-a]
quinolin-4-yl)-1,3,4-thiadiazol-2-yl)acetamide (4h)
Anticancer activity assay
Yellow crystals, m.p. 222–223 °C; IR (cm-1): 3235 3120,
2978, 1662, 1620, 1422; 1H NMR (300 MHz, d ppm,
DMSO-D6); 2.01 (s, 3H, CH3 of NHCOCH3), 2.65 (s, 3H,
CH3 of N4–COCH3), 3.91 (s, 3H, OCH3), 6.52 (s, 1H, C5-H
of thiadiazole), 7.01–7.81 (m, 3H, Ar–H, C5-H, C6-H, C9-
Colorimetric cytotoxicity assay for in vitro anticancer
screening is a rapid, sensitive, and inexpensive method
(Skehan et al., 1990) for measuring the cellular protein
content of adherent and suspension cultures in 96-well
123

Med Chem Res
Isolation of DNA
microtitre plates. Cultures fixed with trichloroacetic acid
were stained for 30 min with sulforhodamine B [SRB,
0.4 % (w/v)] dissolved in acetic acid (1 %). Unbound dye
was removed by four washes with acetic acid (1 %), and
protein-bound dye was extracted with 10 mM unbuffered
tris base [tris (hydroxymethyl) aminomethane] for deter-
mination of optical density in a computer-interfaced,
96-well micro titer plate reader. The SRB assay results
were linear with the number of cells and with values for
cellular protein measured by both the Lowry and Bradford
assays at densities ranging from sparse sub confluence to
multilayered supraconfluence. The signal-to-noise ratio at
564 nm was approximately 1.5 with 1,000 cells per well.
The sensitivity of the SRB assay compared favorably with
sensitivities of several fluorescence assays and was supe-
rior to those of both the Lowry and Bradford assays and to
those of 20 other visible dyes. The SRB assay provided a
colorimetric end point that is nondestructive, indefinitely
stable, and visible to the naked eye. It provided a sensitive
measure of drug-induced cytotoxicity, and was useful in
quantitating clonogenicity, and is well suited to high-vol-
ume, automated drug screening. SRB fluoresces strongly
with laser excitation at 488 nm and can be measured
quantitatively at the single-cell level by static fluorescence
cytometry.
DNA of E. coli was isolated using the literature procedure
(Sambrook et al., 1989). The bacterial culture (1.5 ml) was
centrifuged to obtain the pellet. The pellet was then dis-
solved in lysis buffer (0.5 ml, 100 mM tris pH 8.0, 50 mM
EDTA, 50 mM lysozyme). Saturated phenol (0.5 ml) was
added and incubated at 55 °C for 10 min. The mixture was
centrifuged at 10,000 rpm for 10 min and to the superna-
tant liquid equal volume of chloroform:isoamyl alcohol
(24:1) and 1/20th volume of 3 M sodium acetate (pH 4.8)
were added. Further, the mixture was centrifuged at
10,000 rpm for 10 min and to the supernatant liquid chilled
absolute alcohol (three volumes) was added. The precipi-
tated DNA was separated by centrifugation. The pellet was
dried and dissolved in Tris buffer (10 mM tris pH 8.0) and
stored under cold condition. The final compounds 4a–
j (100 lg) were added separately to the DNA sample. The
sample mixtures were incubated at 37 °C for 2 h. The
electrophoresis of the samples was done.
Agarose (200 mg) was dissolved in TAE buffer (25 ml)
(4.84 g Tris base, pH 8.0, 0.5 M EDTA/1 l) by boiling. When
the gel attained &55 °C, it was poured into the gel cassette fitted
with comb. The gel was then allowed to solidify. The comb was
carefully removed and the gel was placed in the electrophore-
sis chamber flooded with TAE buffer. DNA sample (20 ll,
mixed with bromophenol blue dye at 1:1 ratio), was loaded
carefully into the wells, along with standard DNA marker (DNA
molecular weight marker (k DNA Hind III digest, Banglore
Genei, Bangalore) and constant 50 V of electricity was passed
for around 45 min. The gel was removed and carefully stained
with ETBR solution (10 lg/ml) for 10–15 min and the bands
were observed under UV transilluminator.
All the title compounds 4a–j were evaluated for in vitro
activity against two cell lines viz, human breast cancer cell
line MCF7 and human cervix cancer cell line HeLa. For
each compound dose–response curves against each cell line
were measured. SRB protein assay has been used to esti-
mate cell viability or growth. Three experiments were
carried out for each cell line at 10-fold dilutions of four
concentrations ranging from 10-4 to 10-7 M. The per-
centage growth was evaluated spectrophotometrically ver-
sus negative control not treated with test agents and
positive control, treated with Adriamycin, a proven anti-
cancer agent. The average of the three values was indicated
in the (Fig. 2, 3). The cytotoxic effects of each compound
can be expressed as the LC50 (concentration of drug
causing 50 % cell kill), TGI (concentration of drug causing
total inhibition of cell growth), and GI50 (concentration of
drug causing 50 % inhibition of cell growth). These values
were calculated and are depicted in the (Tables 2, 3).
Conclusion
Compounds containing quinoline-1,3,4-thiadiazole and
tetrazole nucleus in a single molecular frame work were
efficiently synthesized and in vitro anticancer activity and
DNA cleavage studies were carried out. In vitro anticancer
activity revealed that these compounds are selective in their
action because only two of our synthesized compounds 4e
and 4i have exhibited good activity against Hela cell line.
Hence, compounds 4e and 4i may be considered as new
leads for the development of antitumoural agents. DNA
cleavage study indicated that most of these compounds
have DNA cleavage properties.
DNA cleavage study
Culture media
Nutrient broth was used for the growth of the organism.
The media (50 ml) was prepared and autoclaved for
15 min at 121 °C, 15 lb pressure. The autoclaved media
were inoculated with the seed culture and incubated at
37 °C for 24 h.
Acknowledgments The authors thank ACTREC, Mumbai and
Biogenics, Hubballi, for assistance in conducting pharmacological
assay and USIC, Karnatak University Dharwad—580003 for spectral
characterization.
123

Med Chem Res
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, a
laboratory manual, 2nd edn. Cold Spring Harbor Laboratory,
New York
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