Synthesis and preliminary anticancer activity of new 1H-4,5-dihydro-3-(3- hydroxy-2-naphthyl)-4-substituted-1,2,4-triazoline-5-thiones.

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1 Indian Journal of Chemistry Vol. 44B, ovember 2005, pp Synthesis and preliminary anticancer activity of new 1H-4,5-dihydro-3-(3- hydroxy-2-naphthyl)-4-substituted-1,2,4-triazoline-5-thiones. Part II H ese Dogan, Arzu Duran & Sevim Rollas* Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Marmara University, Haydarpasa, Istanbul, Turkey sevim@sevimrollas.com Received 18 June 2004; accepted (revised) 9 March 2005 The compounds 1H-4,5-dihydro-3-(3-hydroxy-2-naphthyl)-4-substituted-1,2,4-triazoline-5-thiones have been synthesised from 3-hydroxy-2-naphthoic acid hydrazide in two steps. The structural elucidation of the synthesised compounds has been performed by 1 H MR and mass spectroscopic data besides elemental analyses. Some of the compounds are tested in vitro for their ability to inhibit growth of 60 different human cancer cell lines by the ational Cancer Institute and notable cytotoxic effect on some cancer cell lines is observed from some of them as expected in comparison with the control agent 5-fluorouracil. Keywords: 1H-4,5-dihydro-3-(3-hydroxy-2-naphthyl)-4-substituted-1,2,4-triazoline-5-thiones, anticancer activity IPC: Int.Cl. 7 C 07 D // A 61 P 35/02 Cancer chemotheraphy has become increasingly important in recent years. It is important to develop new anticancer drugs, because most antineoplastic drugs are highly toxic. The newer improved potential anticancer agents are usually superior to older drugs. This fact prompted us to synthesize new compounds. Although some work has been reported on the anticancer activity of thiadiazole derivatives 1-8, but no work on the anticancer activity of triazole derivatives has been reported so far, but our earlier studies and the present study showed that 1,2,4-triazoline-5- thiones also have cytotoxicity on some human tumor cell lines in case of 1,3,4-thiadiazole derivatives 8. This led us to investigate anticancer activity of 1,2,4- triazoline-5-thione derivatives. In a previous study, we had synthesized a series of 3,4-disubstituted-1,2,4-triazoline-5-thiones 10 and one of them, 1H-4,5-dihydro-3-(3-acetyloxy-2-naphthyl)- 4-ethyl-1,2,4-triazoline-5-thione 3a was evaluated for its in vitro primary cytotoxicity in the ational Cancer Institute 9. This compound was highly active against leukemia cell line, HL-60 (TB) in the in vitro screen and its value was (Table I). When this value was compared with the values of two anticancer drugs, thioguanine (-5.96) and 5- fluorouracil (5-FU,-4.81) on same cancer cell line 11, it appeared that there was an enormous difference between the values of of the anticancer drugs and the synthesized compound. Thus, the cytotoxicity of the synthesized compound was better than those of other two drugs. In the present study, five new analogs of 1,2,4-triazoline-5-thione were synthesized, determined structurally and two of them were screened on 60 human tumor cell lines associated with two previously synthesized and reported compounds 9 namely, 1H-4,5-dihydro-3-(3-benzoyloxy-2-naphthyl)-4-ethyl-1,2,4-triazoline-5-thione 3a 1 and 1H-4,5-dihydro-1-(2,6-dichlorobenzoyl)-3-(3- hydroxy-2-naphthyl)-4-ethyl-1,2,4-triazoline-5-thione 3a 2 (Scheme I). The compounds were synthesised in two steps (Scheme II). In the first step, 3-hydroxy-2-naphthoic acid hydrazide was added to 4-(bromo/chloro/ methoxy)phenyl, 3-(trifluoromethyl)-phenyl and phenethyl isothiocyanates for the synthesis of 1-(3- hydroxy-2-naphthoyl)-4-substituted thiosemicarbazides 12 1g-k. In the second step, 1H-4,5-dihydro-3-(3- hydroxy-2-naphthyl)-4-substituted-1,2,4-triazoline-5- thiones 2g-k were obtained by the cyclization of the thiosemicarbazides in alkaline medium 9,10, followed by acidification with hydrochloric acid. Elemental analysis and spectroscopic methods confirmed the structures of the compounds. The most characteristic proton which proves the structure of 1,2,4-triazoline-5-thione moiety is the H proton. In the 1 H MR spectrum, the most important evidence

2 2302 IDIA J. CHEM., SEC B, OVEMBER 2005 Table I In vitro tumour cell growth inhibition of the compounds Panel/Cell Line 2h 2k 3a 1 3a 2 3a 9 5-FU 11 Leukemia CCRF-CEM > > HL-60 (TB) > > > K-562 >-4.00 > > > > MOLT >-4.00 >-4.00 > > > RPMI SR >-4.00 >-4.00 > > > on-small cell lung cancer A549/ATCC > >-4.00 >-4.30 >-4.30 >-4.30 > EKVX > >-4.30 >-4.30 > HOP >-4.30 > > > HOP > >-4.00 >-4.30 >-4.30 >-4.30 > > CI-H >-4.30 > > CI-H > > CI-H322M > >-4.00 >-4.30 >-4.30 >-4.30 > > CI-H > CI-H >-4.30 > Colon cancer COLO > >-4.00 >-4.30 > > HCT > > > HCT > >-4.30 > > HT >-4.30 >-4.30 > > KM > > >-4.30 >-4.30 > > SW > >-4.00 >-4.30 > > > Central nervous system cancer SF >-4.30 >-4.30 >-4.30 > > SF > > > > > SF >-4.30 >-4.30 >-4.30 > > SB > >-4.00 >-4.30 >-4.30 >-4.30 > SB > >-4.00 >-4.30 > > U >-4.30 > > Melanoma LOX IMVI >-4.30 >-4.30 > > MALME-3M > >-4.30 > > M > >-4.30 > > SK-MEL > SK-MEL > >-4.00 >-4.30 >-4.30 >-4.30 > > SK-MEL > UACC >-4.30 >-4.30 >-4.30 > UACC > > Contd

3 DOGA et al.: SYTHESIS OF SUBSTITUTED THIOES 2303 Table I In vitro tumour cell growth inhibition of the compounds Contd Panel/Cell Line 2h 2k 3a 1 3a 2 3a 9 5-FU 11 Leukemia Ovarian cancer IGROVI > OVCAR > > > OVCAR > >-4.00 >-4.30 >-4.30 >-4.30 > > OVCAR >-4.30 >-4.30 >-4.30 > > OVCAR > >-4.00 >-4.30 > > SK-OV > >-4.00 >-4.30 > > Renal cancer >-4.30 > > ACH > >-4.30 > > > CAKI > > > RXF >-4.30 > > S12C > > TK >-4.30 >-4.30 > UO > > Prostate cancer PC > >-4.30 >-4.30 > DU > >-4.00 >-4.30 > > Breast cancer MCF >-4.30 >-4.30 >-4.30 > > CI/ADR-RES >-4.30 >-4.30 > MDA-MB-231/ATCC > > HS 578T MDA-MB > >-4.30 >-4.30 >-4.30 > > BT > > T-47D > > > > Meangraph midpoint MG-MID Cl 3a 1 H S C 2 H 5 OCOC 6 H 5 Scheme I C O Cl S C 2 H 5 3a 2

4 2304 IDIA J. CHEM., SEC B, OVEMBER 2005 CH 2 CHCSHR + RCS 1g-k Sa H S a R HCl g h a R Oa f e d 2g-k R: d* CF 3 Br Cl c* OCH 3 CH 2 CH 2 c* g h i j k Scheme II e* d* for differentiating thiosemicarbazide and 1,2,4-triazoline-5-thione is the difference of the number of H protons and the frequency which these protons undergo to resonance. The initial compound thiosemicarbazide shows three H peaks at ppm 12. When thiosemicarbazide is converted to 1,2,4- triazoline-5-thione, these peaks are removed from spectrum and 1,2,4-triazoline-5-thione ring shows only one H peak at a higher frequency. In the 1 H MR spectra, H peak of triazoline ring was observed at ppm as a singlet. All the other protons characterising the compounds were obtained as expected and are marked with some letters on the rings (Scheme II). The mass spectrum of 2j which was selected as prototype, showed molecular ion (M + ) peak, its relative intensity is 100%. The mass fragmentation pattern was in accordance with those reported in the literature 9. Results and Discussion 1,2,4-Triazoline-5-thione derivatives 2h and 2k and previously reported compounds 9 3a 1 and 3a 2 were tested in vitro for their ability to inhibit growth of 60 different human cancer cell lines including leukemias, melanomas, and cancers of lung, colon, ovary, prostate, breast, kidney and central nervous system origin (Table I) at tenfold dilutions of five concentrations ranging from 10-4 to 10-8 M. Primary anticancer assay was performed in accordance with the protocol of the Drug Evaluation Branch, ational Cancer Institute, Bethesda The 48 hour continuous drug exposure protocol was used, and a sulforhodamine B (SRB) protein assay was used to estimate cell viability or growth. The percentage growth was obtained spectrophotometrically. For all the cell lines, the 50% growth inhibition ( ) and total growth inhibition () were determined and expressed as the logarithmic form ( and ). The means of these values (MG-MID) were calculated and the deviations of individual values from the means were found. egative values from the mean indicated more sensitive cell lines. The compounds having the individual values which were equal or smaller than-4, were declared to be active 8,11,13. When the compounds were compared with thioguanine, the values of 2h (-5.88) and 3a 1 (-5.82) were found to be lower than that of thioguanine (-5.32) 11 on ovarian cancer cell line (IGROVI). Similarly, the values of 2h (-4.60) and 2k (-4.44) were lower than that of thioguanine (-4.10) 11 on central nervous system cancer cell line (SB-19). In view of this evidence, the cytotoxicity of 2h, 2k and 3a 1 on the above two cell lines was comparable to those of thioguanine. For breast cancer, these values of 2h (-5.05, on MDA- MB-231/ATCC) and 3a 2 (-5.06, on HS 578T) were

5 DOGA et al.: SYTHESIS OF SUBSTITUTED THIOES 2305 also comparable to those of thioguanine (-5.71 and -5.13, respectively) 11. The values of the compounds on other cell lines were higher than those of thioguanine, but a lot of them were lower than those of 5-FU (Table I). The values of 5- FU were-4.77 and-3.91, for ovarian cancer cell line (IGROVI) and central nervous system cancer cell line (SB-19) respectively, therefore 2h, 2k and 3a 1 are also much more active than 5-FU against above cell lines. When the compounds were compared with 5-FU, it was seen that they have lower values than that of 5-FU on following cancer cell lines. All of the tested compounds were highly active on breast cancer cell lines (MDA-MB-231/ATCC, HS 578T, BT-549 and T-47D), melanoma cell line (SK-MEL-2), because their values were lower than those of 5-FU. For the non-small cell lung cancer cell lines (EKVX and CI-H 522), the values of 2h, 2k and 3a 1 were-4.54,-4.47,-4.97 and-5.25,-4.82, -4.96, respectively and all of them were lower than those of 5-FU (-3.27 and-4.44). These values of 2h, 2k and 3a 2 on same cancer for CI-H226 (-4.63, -4.70,-4.85) were also lower than that of 5-FU (-3.56). The cytotoxicities on the central nervous system cancer cell lines were as follows: SF-268: 2h(-5.19); SF-295: 2h(-4.88), 2k(-4.37), 3a 1 (-4.57), 3a 2 (-4.46); SB-19: 2h(-4.60), 2k(-4.44); SB-75: 2h(-4.54), 2k(-4.14), 3a 2 (-4.37); U251:2h:(-4.89), 2k:(-4.69), 3a 2 (-4.71), and all of them were lower than 5-FU (-4.28,-4.28,-3.91,-3.78 and-4.37, respectively). 3a 1 (-5.50) exhibited higher cytotoxicity than the others comparable cytotoxicity on overian cancer cell line (OVCAR-3) in comparison to 5-FU (-4.53). Furthermore, the values of 2h (-5.88), 2k (-4.83), 3a 1 (-5.82) and 3a 2 (-5.18) were higher than that of 5-FU (-4.77) on cell line IGROVI, especially 2h and 3a 1 were the most active componds. The most active among the tested compounds was 2h bearing p-chlorophenyl linked to 4. It showed the most marked effects in vitro screening on non-small cell lung cancer cell lines (EKVX, CI-H226, CI- H522), central nervous system cancer cell lines (SF-268, SF-295, SB-19, SB-75, U251), melanoma cell line (SK-MEL-2), ovarian cancer cell lines (IGROVI, OVCAR-5, SK-OV-3), renal cancer cell line (TK-10) and breast cancer cell lines (MDA- MB-231/ATCC, HS 578T, BT-549). On same cancer cell lines, the values of 5-FU were lower than those of 2h. When the compounds were screened in vitro against central nervous system cancer, 2h showed more cytotoxicity than other three compounds on all of the cell lines. However, the values of 2h were comparable to those of 5-FU for leukemia cell lines (CCRF-CEM, SR), colon cancer cell lines (KM12, SW-620), melanoma cell lines (MALME- 3M, M14, SK-MEL-5, UACC-257, UACC-62), ovarian cancer cell lines (OVCAR-3, OVCAR-4), renal cancer cell line (S12C) and breast cancer cell lines (CI/ADR-RES, MDA-MB-435, T-47D). These preliminary results show that most of the compounds are much more active than 5-fluorouracil against most cancer cell lines, and may be considered promising for the development of new anticancer agents. Experimental Section Chemicals used in the experiments were commercially available and were used without further purification. Melting points were determined using a Büchi 530 melting point apparatus in open glass capillaries and are uncorrected. Elemental analyses were performed with Leco CHS-932 elemental analyzer. UV spectra were recorded on a Shimadzu UV 2100S spectrometer; (1 mg/100 ml in ethanol), 1 H MR spectra on a Bruker AVAC DPX 400 spectrometer in DMSO-d 6 using TMS as internal standard (chemical shifts in δ, ppm); and mass spectra on a Fisons Instruments VG Platform II LS-MS spectrometer. General procedure for the preparation of 1H-4, 5-dihydro-3-(3-hydroxy-2-naphthyl)-4-substituted- 1, 2, 4-triazoline-5-thiones 2g-k. Equimolar amounts of 3-hydroxy-2-naphthoic acid hydrazide and appropriate isothiocyanate were refluxed in ethanol for 3 hr. The precipitated thiosemicarbazides 1g-k were recrystallised from ethanol 12, dissolved in 2 sodium hydroxide and heated under reflux for 2 hr. After cooling, the solution was acidified with HCl. The precipitate was filtered, washed with water and crystallised from ethanol. 2g: yield 68.4%, m.p. 290 o C; UV (λ max, nm): (ε 48212); 1 H MR: δ (1H, s, H), (1H, s, ), 8.06 (1H, s, CH a ), 7.82 (1H, d, CH h ), 7.68 (1H, d, CH e ), 7.46 (1H, t, CH f ), 7.33 (1H, t, CH g ), 7.07 (1H, s, CH d ), 7.56 (2H, d, CH b* ), 7.26 (2H, d, Ch a* ). Anal. Calcd for C 18 H 12 Br 3 OS: C, 54.28; H, 3.04;, Found: C, 55.08; H, 2.93;, %. 2h: yield 93.0%, m.p. 294 o C; UV (λ max, nm): (ε 19849); 1 H MR: δ (1H, s, H), (1H,

6 2306 IDIA J. CHEM., SEC B, OVEMBER 2005 s, ), 8.07 (1H, s, CH a ), 7.83 (1H, d, CH h ), 7.68 (1H, d, CH e ), (3H, m, CH f and CH b* ), (3H, m, CH g and CH a* ), 7.07 (1H, s, Ch d ). Anal. Calcd. for C 18 H 12 Cl 3 OS: C, 61.10; H, 3.42;, Found: C, 62.05; H, 3.28;, %. 2i: yield 67.4%, m.p. 270 o C; UV (λ max, nm): (ε 25645); 1 H MR: δ (1H, s, H), (1H, s, ), 8.10 (1H, s, CH a ), 7.83 (1H, d, CH h ), 7.67 (1H, d, CH e ), 7.46 (1H, t, CH f ), 7.33 (1H, t, CH g ), 7.06 (1H, s, CH d ), 7.77 (1H, s, CH d* ), 7.71 (1H, t, CH b* ), 7.58 (2H, d, CH a* and Ch c* ), protons of ethanol were at 1.07 (t), 3.43 (m), 4.30(t) ppm and these peaks which have very low intensities were proportional to the number of protons. Anal. Calcd. for C 19 H 12 F 3 3 OS 1/5 C 2 H 5 : C, 58.75; H, 3.35;, Found: C, 59.02; H, 3.30;, %. 2j: yield 90.5%, m.p. 278 o C; UV (λ max, nm): (ε 35849); 1 H MR: δ (1H, s, H), (1H, s, ), 8.01 (1H, s, CH a ), 7.80 (1H, d, CH h ), 7.67 (1H, d, CH e ), 7.45 (1H, t, CH f ), 7.31 (1H, t, CH g ), 7.08 (1H, s, CH d ), 7.21 (2H, d, CH a* ), 6.87 (2H, d, CH b* ), 3.70 (3H, s, CH 3 ); EI-MS (m/z, %): 349 (M +, 100), 348 (43.04), 169 (11.00), 141 (11.31), 140 (11.08), 122 (6.88), 114 (10.05), 113 (5.14). Anal. Calcd. for C 19 H 15 3 O 2 S: C, 65.31; H, 4.33;, Found: C, 65.49; H, 4.04;, 11.96%. 2k: yield 90.6%, m.p. 202 o C; UV (λ max, nm): (ε 68583); 1 H MR: δ (1H, s, H), (1H, s, ), 7.78 (1H, d, CH h ), 7.73 (1H, d, CH e ), 7.50 (1H, t, CH f ), (2H, m, CH a and CH g ), 7.31 (1H, s, CH d ), (2H, t, CH b* ), 7.09 (1H, t, CH c* ), 6.81 (2H, d, CH a* ), 4.07 (2H, t, CH 2e* ), 2.96 (2H, t, CH 2d* ). Anal. Calcd. for C 20 H 17 3 OS: C, 69.14; H, 4.93;, Found: C, 70.21; H, 5.03;, %. In vitro cytotoxicity screening 8,11,13 The human tumor cell lines of the cancer screening panel were grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mm l-glutamine. For a typical screening experiment, cells were inoculated into 96-well microtiter plates in 100 μl at plating densities ranging from 5000 to cells/well depending on the doubling time of individual cell lines. After cell inoculation, the microtiter plates were incubated at 37 o C, 5% CO 2, 95% air and 100% relative humidity for 24 hr prior to addition of experimental drugs. After 24 hr, two plates of each cell line were fixed in situ with trichloroacetic acid (TCA), to represent a measurement of the cell population for each cell line at the time of drug addition (T z ). Experimental drugs were solubilized in dimethyl sulfoxide at 400-fold the desired final maximum test concentration and stored frozen prior to use. At the time of drug addition, an aliquot of frozen concentrate was thawed and diluted to twice the desired final maximum test concentration with complete medium containing 50 μg.ml -1 gentamicin. Additional four, 10-fold or ½-log serial dilutions were made to provide a total of five drug concentrations plus control. Aliquots of 100 μl of these different drug dilutions were added to the appropriate microtiter wells already containing 100 μl of medium, resulting in the required final drug concentrations. Following drug addition, the plates were incubated for an additional 48 hr at 37 C, 5% CO 2, 95% air, and 100% relative humidity. For adherent cells, the assay was terminated by the addition of cold TCA. Cells were fixed in situ by the gentle addition of 50 μl of cold 50% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 min at 4 o C. The supernatant was discarded, and the plates were washed five times with tap water and air dried. SRB solution (100 μl) at 0.4% (w/v) in 1% acetic acid was added to each well, and plates were incubated for 10 min at room temperature. After staining, unbound dye was removed by washing five times with 1% acetic acid and the plates were air dried. Bound stain was subsequently solubilized with 10 mm trizma base, and the absorbance was read on an automated plate reader at a wavelength of 515 nm. For suspension cells, the methodology was the same except that the assay was terminated by fixing settled cells at the bottom of the wells by gently adding 50 μl of 80% TCA (final concentration, 16%TCA). Using the seven absorbance measurements [time zero (T z ), control growth (C), and test growth in the presence of drug at the five concentration levels (T i )], the percentage growth was calculated at each of the drug concentration levels. Percentage growth inhibition (GI) was calculated as: T T /( C T ) for concentrations for [( i z ) z ] 100 which T i T z [( T )/ T ] 100 T for concentrations i z z for which T i < T z. Three dose response parameters were calculated for each experimental agent. was calculated from [( T i Tz )/( C Tz )] 100 = 50, which was the drug concentration resulting in a 50% reduction in the net protein increase (as measured by SRB staining) in control cells during the drug incubation. The drug

7 DOGA et al.: SYTHESIS OF SUBSTITUTED THIOES 2307 concentration resulting in was calculated from T i = T z. Values were calculated for each of these parameters if the level of activity was reached; however, if the effect was not reached or was exceeded, the value for that parameter was expressed as greater or less than the maximum or minimum concentration tested. Acknowledgements Authors thank the members of Drug Research and Development, Division of Cancer Research, ational Cancer Institute, Bethesda, Maryland, for the in vitro anticancer screening of the compounds. This research was supported by The Research Fund of Marmara University, project number SA/ References 1 Oleson J J, Sloboda A, Troy W P, Halliday S L, Landes M J, Angier R B, Semb J, Cyr K & Williams J H, J Am Chem Soc,77(2), 1955, Gagiu F, Binder U & Gyorfi Z, J Prakt Chem, 36(1-2), 1967,108; Chem Abstr, 67, 1967, 82169g. 3 Tutoveanu M, Comanita E, Andrei A & Mikle I, Bul Inst Politeh Iasi, 18(1-2), 1972, 95; Chem Abstr, 77, 1972, h. 4 Miyamoto K, Koshiura R, Mori M, Yokoi H, Mori C, Hasegawa T & Takatori K, Chem Pharm Bull, 33(11), 1985, Gadad A K, Karki S S, Rajurkar V G & Bhongade B A, Arzneim-Forsch / Drug Res, 49, 1999, Andreani A, Leoni A, Locatelli A, Morigi R, Rambaldi M, Recanatini M & Garaliene V, Bioorg Med Chem, 8, 2000, Chou J Y, Lai S Y, Pan S L, Jow G M, Chern J W & Guh J H, Biochem-Pharmacol, 66(1), 2003, Terzioglu & Gürsoy A, Eur J Med Chem, 38, 2003, Duran A, Dogan H & Rollas S, Il Farmaco, 57, 2002, Dogan H, Rollas S & Erdeniz H, Il Farmaco, 53, 1998, Gürsoy A & Karali, Eur J Med Chem, 38, 2003, Dogan H, Duran A & Yemni E, Drug Metabol Drug Interact, 15, 1999, Karali, Eur J Med Chem, 37, 2002, Monks A, Scudiero D, Skehan P, Shoemaker R, Paull K, Vistica D, Hose C, Langley J, Cronise P, Vaigro-Wolff A, Gray-Goodrich M, Campbell H, Mayo J & Boyd M, J atl Cancer Inst, 83(11), 1991, Kuo S, Lee H, Juang J, Lin Y, Wu T, Chang J, Lednicer D, Paull K D, Lin C M, Hamel E & Lee K, J Med Chem, 36, 1993, Leteurtre F, Kohlhagen G, Paull K D & Pommier Y J, J atl Cancer Inst, 86, 1994, 1239.