MEDICINAL CHEMISTRY RESEARCH
Med Chem Res (2014) 23:2727–2735 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 / Published online: 5 November 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 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. Keywords Anticancer activity Breast cancer cell line MCF7 Cervix cancer cell line HeLa DNA cleavage 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 R. R. Kamble (&) G. Y. Meti Department of Studies in Chemistry, Karnatak University, Dharwad 580003, India e-mail:
[email protected] 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
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, synthesis 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 derivatives have been reported as promising candidates for anticancer activity. 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 1,3,4thiadiazolidine ring so as to get three heterocyclic rings in a
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Fig. 1 Molecules with anticancer activity
O
OH
O
O
O
OH
O OH
OH
N
OH
N O
O
OH O
O
H O
H2N
O
OH O
OH
O
O
H
HO O
NH2
H3C Camptothecin
Doxorubcin
OH Adriamycin
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.
Results and discussion Chemistry 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 confirmed by their elemental analysis, IR, 1H NMR, and 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. The 1H 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
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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
78.0
85.5
4b
82.0
87.0
4c
75.0
78.1
4d
70.0
72.2
4e
65.0
66.0
4f
75.8
85.0
4g 4h
71.2 65.8
76.6 68.9
4i
78.0
84.5
4j
70.0
74.3
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 respective regions. 13C NMR spectral studies indicated the number of signals in consistent with the number of magnetically non-equivalent carbon atoms. Mass spectral analysis 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
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Scheme 2 Proposed mechanism for the synthesis of 4a–j
O O O
N
O
H N
NH2
H
S
N+
NH2
N
H
S H
N
Cl Cl
N
2a O N
N
NH2
S Cl
N
A c2O O N N
NHCOCH 3
S
-
O N
NHCOCH 3
N
S N
N
N
N +
N
-
O
N
N
3a
Cl
N
NHCOCH 3
S N
N
N
N
N
N-
+
4a COCH3 (Scheme 3) other compounds also have shown corresponding peaks at their respective positions. 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, respectively) have exhibited partial activity against human cervix cancer cell line HeLa. Unsubstituted and methoxy substituted 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
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. 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
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Scheme 3 Mass spectral fragmentation pattern for compound 4a
+ H3COC
N
N
NHCOCH3 S N m/z =43 Base peak
N
N
( M+ )
m/z 355
N N3
COCH3
+ H3COC
+ H3COC
N N
S
NH2 S N
N
N
N N NHCOCH3
m/z 314
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 + N NH 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
[100 [100
[100 [100
[100 [100
4a 4b
[100 [100
[100 [100
[100 81.29
4c
[100
[100
[100
4c
[100
[100
[100
4d
[100
[100
95.33
4d
[100
[100
85.08
4e
[100
[100
[100
4e
[100
[100
\0.1
4f
[100
[100
[100
4f
[100
[100
[100
4g
[100
[100
[100
4g
[100
[100
[100
4h
[100
[100
[100
4h
[100
[100
[100
4i
[100
[100
[100
4i
[100
[100
\0.1
4j
[100
[100
98.93
4j
[100
[100
83.26
ADR
[100
[0.1
\0.1
ADR
[100
[0.1
\0.1
a
GI50 B 1 lM is considered to be active
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a
GI50 B 1 lM is considered to be active
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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).
Fig. 2 Cell growth curve for human cervix HeLa cell lines
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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 carried 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-[4acetyl-5-(6,7,8-substituted-2-chloroquinolin-3-yl)-4,5dihydro-1,3,4-thiadiazol-2-yl]-acetamides 3a–j were prepared by literature methods (Sheetal et al., 2012; Meth-Cohn et al., 1981; Wiles et al., 1907; El-Sayed et al., 2004). Further, these were converted to title compounds N-(4-acetyl4,5-dihydro-5-(7,8,9-substituted-tetrazolo[1,5-a]-quinolin4-yl)-1,3,4-thiadiazol-2-yl)acetamide (4a–j) (Scheme 1).
Synthesis
Fig. 3 Cell growth curve for human breast cancer MCF7 cell lines
General procedure for the preparation of N-(4-acetyl-4,5dihydro-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. 4 DNA cleavage studies for compounds 4a–j
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(60:40) to get colorless to pale brown needles of the compounds (4a–j).
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NHCOCH3); 24.12 (Ar–CH3); MS m/z (%): 369 (M?, 14.0), 327 (12.0), 285 (4), 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.
One-pot synthesis of compounds (4a–j) 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). 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-(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, C8-H, C9-H), 6.75 (s, 1H, C5-H), 11.12 (s, 1H, NHCO); 13C 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
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N-(4-Acetyl-4,5-dihydro-5-(7-methoxy-tetrazolo[1,5-a] 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 NHCOCH3); 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-(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, C5H 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 NHCOCH3), 167.67 (C=O of NCOCH3), 144.41 (C2 of thiadiazole 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-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
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(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. N-(4-Acetyl-4,5-dihydro-5-(9-methoxy-tetrazolo[1,5-a] quinolin-4-yl)-1,3,4-thiadiazol-2-yl)acetamide (4f) 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: calculated for C16H15N7O3S: C, 49.86; H, 3.92; N, 25.44. Found: C, 49.90; H, 3.90; N, 25.42. N-(4-Acetyl-4,5-dihydro-5-(8-methoxy-tetrazolo[1,5-a] quinolin-4-yl)-1,3,4-thiadiazol-2-yl)acetamide (4g) Yellow crystals, m.p. 225–226 °C; IR (cm-1): 3235, 3150, 2973, 1661, 1600, 1411; 1H NMR (300 MHz, d ppm, DMSOD6); 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 thiadiazole), 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, DMSOD6); 169.51 (C=O of NHCOCH3), 168.30 (C=O of NCOCH3), 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. 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)
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 (quinoline), 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-(7-chloro-tetrazolo[1,5-a] 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-methyl-tetrazolo[1,5-a] quinolin-4-yl)-1,3,4-thiadiazol-2-yl)acetamide (4j) 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, C5H 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); 13 C 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. Anticancer activity assay
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Yellow crystals, m.p. 222–223 °C; IR (cm ): 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
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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 determination 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 superior 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-volume, 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. 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 estimate 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 percentage growth was evaluated spectrophotometrically versus negative control not treated with test agents and positive control, treated with Adriamycin, a proven anticancer 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). 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.
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Isolation of DNA 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 dissolved 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 supernatant 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 precipitated 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 electrophoresis 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.
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. Acknowledgments The authors thank ACTREC, Mumbai and Biogenics, Hubballi, for assistance in conducting pharmacological assay and USIC, Karnatak University Dharwad—580003 for spectral characterization.
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