Synthesis, characterization and anticancer studies of ...

Journal of Saudi Chemical Society (2012) xxx, xxx–xxx

King Saud University

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ORIGINAL ARTICLE

Synthesis, characterization and anticancer studies of new steroidal oxadiazole, pyrrole and pyrazole derivatives Shamsuzzaman *, Tabassum Siddiqui, Mohd Gulfam Alam, Ayaz Mahmood Dar Department of Chemistry, Aligarh Muslim University, Aligarh 202 002, India Received 2 February 2012; accepted 27 April 2012

KEYWORDS Hydrazide; Oxadiazole; Pyrrole; Pyrazole; Anticancer; MTT assay

Abstract In the present study steroidal derivatives, 3b-[50 -mercapto-10 ,30 ,40 -oxadiazole-2-yl]methoxy cholest-5-ene 2, 3b-[20 ,50 -dimethylpyrrole-1-yl]aminocarbonylmethoxycholest-5-ene 3 and 3b-[30 ,50 dimethyl pyrazole-1-yl]carbonylmethoxycholest-5-ene 4 have been synthesized from cholest-5-en3b-O-acetyl hydrazide 1 using CS2/KOH, acetonyl acetone and acetyl acetone, respectively as reagents and are characterized by IR, 1H NMR,13C NMR, MS and elemental analysis. Compounds 2–4 were also evaluated for anticancer activity against human leukemia cell line (HL-60) by MTT assay and compound 4 displayed the promising behavior by showing better anticancer activity. ª 2012 King Saud University. Production and hosting by Elsevier B.V. All rights reserved.

1. Introduction 1,3,4-Oxadiazoles, pyrroles, pyrazoles and their derivatives represent an important class of heterocyclic compounds with broad spectrum of biological activity. 1,3,4-Oxadiazoles have been reported to possess insecticidal (Zheng et al., 2003), herbicidal (Chavan et al., 2006), antibacterial (Shivarama Hollaa et al., 2000), antifungal (Liu et al., 2008), analgesic (Narayana et al., 2005), anti-inflammatory (Koksal et al., 2008), antimalarial (Zareef et al., 2007a), antiviral (Farghaly and El-Kashef, 2006), anti-HBV (El-Essawy et al., 2007), antianxiety (Amr et al., 2008), anticancer (Kumar et al., 2008), anti-HIV (Zareef et al., 2007b), antitubercular (Macaev et al., 2005) and anticonvulsant activities (Zarghi et al., 2005). Substituted pyrroles have been used as intermediates in the synthesis of mitomycin anti* Corresponding author. Tel.: +91 9411003465. E-mail address: [email protected] (Shamsuzzaman). Peer review under responsibility of King Saud University.

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tumor antibiotics possessing antibacterial activity (De Leon and Ganem, 1997; Gilchrist, 1998; Luly and Rapoport, 1983). Functionalized pyrroles are building blocks of natural tetrapyrrole pigments (Dutton et al., 1983) such as porphobilinogen or bilirubin and various other natural products and their analogs (Shen et al., 1993). Pyrazoles and their derivatives are of much importance on account of their use in therapy in different diseases (El-Hawash et al., 2006; Salgin-Goksen et al., 2007; Savelli and Alessandro, 1996). They are also reported to possess antibacterial (Abdallah et al., 2005), fungicidal (Devasia et al., 2006), antidiuretic (Vicini et al., 2002), anticancer (Rostom, 2006; Ibrahim et al., 2007), anti-HIV (Rida et al., 2005; Salih, 2008), antitumor (Salgin-Goksen et al., 2007), analgesic-anti-inflammatory (Gulcan et al., 2003; Onkol et al., 2008) and anticonvulsant (Onkol et al., 2004) properties. This gave a great impetus to the search for potential pharmacologically active drugs carrying such moieties. Taking into consideration the existing cancer therapies, chemotherapy has turned out to be one of the most significant treatments in cancer management (Harrison et al., 2009). The natural product based drugs, Paclitaxel and Docetaxel, are extensively used in the treatment of a wide variety of cancers

1319-6103 ª 2012 King Saud University. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jscs.2012.04.009

Please cite this article in press as: Shamsuzzaman, et al., Synthesis, characterization and anticancer studies of new steroidal oxadiazole, pyrrole and pyrazole derivatives. Journal of Saudi Chemical Society (2012), http://dx.doi.org/10.1016/j.jscs.2012.04.009

2 because of their efficacy (Carlson, 2008). Researchers are directed toward the search for anticancer drugs with limited harmful side effects particularly with respect to the immune system. Prompted by these observations and in continuation of our researches (Shamsuzzaman et al., 2010) on biologically active heterocycles we hereby report the synthesis of some new substituted steroidal pyrazole, 1,3,4-oxadiazole and pyrrole derivatives which have also been evaluated for their anticancer activity against human leukemia cell line. 2. Experimental 2.1. Chemistry All melting points were recorded in open capillary tube and are uncorrected. Infrared spectra were recorded in KBr pellets with spectrolab interspec 2020 FT-IR spectrometer and values are given in cm1. 1H and 13C NMR spectra were run in CDCl3 at 300 and 100 MHz, respectively using TMS as an internal standard and values are given in ppm (d). TLC plates were coated with silica gel G, light petroleum ether refers to a fraction of bp. 60–80 C. Anhydrous sodium sulfate was used as drying agent. The abbreviations s, d, dd, m and br denote ‘‘singlet, doublet, double-doublet, multiplet and broad’’, respectively. 2.2. 3b-[30 ,50 -Mercapto -10 ,30 ,40 -oxadiazole-2-yl]methoxycho lest-5-ene (2) To a solution of compound 1 (Satyanarayana and Siddiqui, 1994) (1.08 mmol) in absolute ethanol (40 ml), KOH and CS2 dissolved in absolute ethanol (10 ml) in equimolar amounts were added and the reaction mixture was heated under reflux till the evolution of H2S ceased (21 h.). The reaction mixture was cooled to room temperature, poured in ice cold water and neutralized with dil. HCl. The precipitated solid was filtered, dissolved in diethyl ether, washed with water and dried over anhydrous Na2SO4. Evaporation of solvent and recrystallization from ethanol provided compound 2. Solid; Yield: 67.5%; mp 125–126 C; Anal. Calcd for C30H48N2O2S: C, 71.92, H, 9.68, N, 5.55. Found C, 71.95, H, 9.66, N, 5.59; IR (KBr) t cm1: 1625 (C‚N), 1385 (C– N), 1220 (C–O), 1195 (C‚S); 1H NMR (CDCl3, 300 MHz, ppm): d 9.06 (br s, 1H, NH, exchangeable with D2O), 5.38 (dd, J = 8 Hz, 4 Hz, 1H, C6-H), 4.6 (m, W1/2, 15 Hz, 1H, axial, C3a-H), 3.54 (s, 2H, O-CH2), 1.15 (s, 3H, C10-CH3), 0.74 (s, 3H, C13-CH3), 0.90, 0.85 (other steroidal side-chain methyl protons). 13 C NMR (CDCl3, 100 MHz, ppm) d 156.5, 155.1, 148.9, 122.9, 75.9, 73.7, 48.3, 46.3, 42.78, 40.5, 39.8, 39.40, 35.8, 33.49, 33.2, 32.7, 31.9, 30.3, 29.5, 29.3, 28.7, 25.4, 22.4, 22.2, 21.7, 21.1, 20.8, 20.4, 20.3, 18.6; MS: m/z [M+] 500.

Shamsuzzaman et al. was filtered, washed with water, air dried and then recrystallized from ethanol to get compound 3. Solid; yield 69%; mp 131–133 C; Anal. Calcd for C35H56N2O2: C, 78.31, H, 10.51, N, 5.22. Found C,78.29, H, 10.55, N, 5.20; IR (KBr) t cm1: 1753 (C‚O), 1615 (C‚C), 1340 (C–N), 1196 cm1 (C–O); 1H NMR (CDCl3, 300 MHz, ppm): d 8.3(s, 1H, NH, exchangeable with D2O), 5.71 (s, 2H, 2 · CH of pyrrole), 5.28 (dd 1H, J = 9 Hz, 5 Hz, C6-H), 2.34 (s, 6H, 2 · CH3), 4.58 (m, 1H, W1/2, 16 Hz, axial, C3a-H), 4.86 (s, 2H, O-CH2), 1.17 (s, 3H, C10-CH3), 0.74 (s, 3H, C13CH3), 0.93, 0.84 (other steroidal side-chain methyl protons). 13 C NMR (CDCl3, 100 MHz, ppm) d 173.2, 149.1, 130.3, 130.1, 122.9, 108.2, 108.1, 75.6, 74.5, 48.5, 48.3, 46.5, 42.9, 40.4, 40.3, 39.8, 39.6, 35.8, 33.4, 32.4, 31.7, 30.3, 29.7, 29.6, 28.5, 25.4, 22.5, 22.3, 21.5, 21.3, 20.4, 20.3, 20.2, 18.6, 5.3, 5.1; ESI MS: m/z [M+] 536. 2.4. 3b-[30 ,50 -Dimethyl pyrazole-1-yl]carbonylmethoxycholest5-ene (4) To a solution of compound 1 (1.08 mmol) in glacial acetic acid (50 ml), a solution of acetyl acetone (1.08 mmol) and 1 N glacial acetic acid (5 ml), in absolute ethanol (40 ml) was slowly added. The mixture was refluxed at 100–125 C in an oil bath for 8 h. After the completion (TLC) the reaction mixture was poured into ice cold water (80–100 ml) and allowed to stand overnight. The precipitated solid was filtered, taken in diethyl ether, washed with water, dried over anhydrous sodium sulfate and recrystallized from ethanol to afford compound 4. Solid; Yield: 71%; mp 138–141 C; Anal. Calcd for C34H54N2O2: C, 78.11, H, 10.41, N, 5.36. Found; C, 78.09, H, 10.33, N, 5.35; IR (KBr) t cm1; 1716 (C‚O), 1624 (C‚N), 1620 (C‚C), 1397 (C–N), 1215, 1089 (C–O); 1H NMR (CDCl3, 300 MHz, ppm): d 5.6 (dd, J = 7 Hz, 3 Hz, 1H, C6-H), 5.49 (s, 1H, pyrazole ring), 4.82 (m, W1/2, 14 Hz, axial, 1H, C3a-H), 4.27 (s, 2H, O-CH2-), 1.56 (s, 6H, 2 · CH3 of pyrazole ring), 1.17 (s, 3H, C10-CH3), 0.72 (s, 3H, C13-CH3), 0.91, 0.83 (other steroidal side-chain methyl protons). 13 C NMR (CDCl3, 100 MHz, ppm) d 200.3, 149.1, 147.3, 147.2, 122.9, 106.1, 74.8, 67.8, 48.5, 46.4, 42.9, 40.6, 39.8, 39.7, 39.4, 35.8, 32.7, 32.5, 31.9, 30.3, 29.6, 29.5, 28.6, 25.4, 22.5, 22.4, 21.7, 21.3, 20.9, 20.5, 20.3, 18.6, 13.7, 7.3; ESI MS: m/z [M+] 522. 2.5. Anticancer activity

2.3. 3b-[20 ,50 -Dimethylpyrrole-1-yl]aminocarbonylmethoxy cholest-5-ene (3)

2.5.1. Cell lines and culture conditions Human cancer cell line HL-60 cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 10U penicillin and 100 lg/ml streptomycin at 37 C with 5% CO2 in a humidified atmosphere. Fresh medium was given every second day and on the day before the experiments were done. Cells were passaged at preconfluent densities, using a solution containing 0.05% trypsin and 0.5 mM EDTA.

A solution of compound 1 (1.08 mmol) in absolute ethanol (25 ml) was added to a solution of acetonyl acetone (1.08 mmol) and 1 N glacial acetic acid (5 ml). The mixture was heated on a boiling water bath for 7 h. The progress of reaction was monitored by TLC. After completion, the reaction mixture was concentrated to half of its original volume and poured in ice cold water (100–150 ml). The separated solid

2.5.2. Cell viability assay (MTT) The anticancer activity in vitro was measured using the MTT assay. The assay was carried out according to known protocol (Slater et al., 1963; Van de Loosdrecht et al., 1994). Exponentially growing cells were harvested and plated in 96-well plates at a concentration of 1 · 104 cells/well. After 24 h incubation at 37 C under humidified 5% CO2 to allow cell attachment,


Synthesis, characterization and anticancer studies of new steroidal oxadiazole, pyrrole and pyrazole derivatives the cells in the wells were, respectively, treated with target compounds at various concentrations for 48 h. The concentration of DMSO was always kept below 1.25%, which was found to be non-toxic to the cells. A solution of 3-(4,5-dimethylthizao1-2-y1)-2,5-diphenyltetrazolium bromide (MTT), was prepared at 5 mg/ml in phosphate buffered saline (PBS; 1.5 mM KH2PO4, 6.5 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl; pH 7.4). Of this solution 20 ll was added to each well. After incubation for 4 h at 37 C in a humidified incubator with 5% CO2, the medium/MTT mixtures were removed, and the formazan crystals formed by the mitochondrial dehydrogenase activity of vital cells were dissolved in 100 ll of DMSO per well. The absorbance of the wells was read with a microplate reader (Bio-Rad Instruments) at 570 nm. Effects of the drug cell viability were calculated using cells treated with DMSO as control. 2.5.3. Data analysis Cell survival was calculated using the formula: Survival (%) = [(absorbance of treated cellsabsorbance of culture medium)/(absorbance of untreated cellsabsorbance of culture medium)] · 100 (Woerdenbag et al., 1993). The experiment was done in triplicate and the inhibitory concentration (IC) values were calculated from a dose response curve. IC50 is the concentration in ‘lM’ required for 50% inhibition of cell growth as compared to that of untreated control. IC50 values were determined from the linear portion of the curve by calculating the concentration of agent that reduced absorbance in treated cells, compared to control cells, by 50%. Evaluation is based on mean values from three independent experiments, each comprising at least six microcultures per concentration level. 3. Results and discussion 3.1. Chemistry Newly synthesized compounds 3b-[50 -mercapto-10 ,30 ,40 -oxadiazole-2-yl] methoxycholest-5-ene 2, 3b-[20 ,50 -dimethyl pyrrole1-yl] aminocarbonyl methoxy cholest-5-ene 3 and 3b-[30 , 50 -dimethylpyrazole-1-yl]carbonylmethoxycholest-5-ene 4 were synthesized from cholesten-5-en-3b-O-acetylhydrazide 1. Compound 1 in turn was synthesized from ethyl-5-cholesten3b-O-acetate B (Satyanarayana and Siddiqui, 1994). The targeted steroidal derivatives 2–4 were obtained with CS2/KOH, acetonyl acetone and acetyl acetone, respectively. The reaction pathway has been summarized in Scheme 1. The synthesized compounds 2–4 were characterized by IR, NMR, MS and elemental analyses. The selected indicative bands in IR spectra of targeted compounds 2–4 provide useful information for determining their structures. The IR spectrum of steroidal 1,3,4-oxadiazole 2 exhibited characteristic absorption bands at 1625 (C‚N), 1385 (C–N), 1220 (C–O) and 1195 cm1 (C‚S). In the IR spectrum of steroidal pyrrole 3 characteristic absorption bands at 1753, 1615, 1340 and 1196 cm1 were due to C‚O, C‚C, C–N and C–O groups, respectively. Similarly the IR spectrum of steroidal pyrazole 4 showed absorption bands at 1716 (C‚O), 1624 (C‚N), 1620 (C‚C), 1397 (C–N), 1215 and 1089 cm1 (C–O). The structures of compounds 2–4 were also supported by the study of their 1H NMR spectra. The 1H NMR spectrum of compound 2 displayed a single proton singlet at d 9.06

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(exchangeable with D2O) for NH of oxadiazole ring, a twoproton singlet at d 3.54 (O-CH2–), a double-doublet for a single proton at d 5.38 (J = 8 Hz, 4 Hz) for C6-H, and a one-proton broad multiplet centered at d 4.6 (W1/2, 15 Hz, axial) for C3a-H. Steroidal pyrrole 3 in its 1H NMR spectrum showed a singlet for one-proton at d 8.3 (–NH, exchangeable with D2O), two olefinic protons of pyrrole ring at d 5.71, a double-doublet for a single proton at d 5.28 (J = 9 Hz, 5 Hz, C6-H), a oneproton broad multiplet centered at d 4.58 (W1/2, 16 Hz, axial) for C3a-H and another singlet for two-protons at d 4.86 (OCH2). Six methyl protons of pyrrole ring appeared as singlet at d 2.34. In the 1H NMR spectrum of compound 4, two olefinic protons appeared separately at d 5.6 (J = 7 Hz, 3 Hz, C6H) and 5.49 (pyrazole ring) as a double-doublet and a singlet, respectively. It also showed a two-proton singlet at d 4.72 for OCH2, another singlet for six protons (2 · CH3) at 1.56 and a broad multiplet integrating for one-proton at d 4.82 (W1/2, 14 Hz, axial) for C3a-H. Other prominent peaks of steroidal nucleus in the compounds 2–4 were observed at their characteristic positions and are given in experimental section. 13 C NMR spectra of compounds 2–4 were in good agreement with the proposed structures. The presence of the 1,3, 4-oxadiazole unit in compound 2 was supported by the appearance of two quaternary signals (C20 and C50 ) in the range d 156.5–155.1 in the 13C NMR spectrum. The methylene carbon attached to oxadiazole moiety appeared at d 73.7. Similarly the structure of pyrrole 3 was also confirmed by the 13C NMR which displayed signals at d 130.1–130.3 for C20 and C50 of pyrrole ring and d 108.2–108.1 for C30 and C40 of pyrrole ring. The two methyl carbons attached at C20 and C50 of pyrrole ring are also found to appear at d 5.1–5.3. Other important signals appeared at d 173.2 and 74.5 for –NHCO and for –OCH2, respectively. In the 13C NMR spectrum of compound 4 the carbonyl and methylene carbons appeared at d 200.3 and 67.8, respectively. The carbons of pyrazole moiety appeared at d 149.1 (C30 ), 106.1 (C40 ), 147.3 (C50 ) and 7.3–13.7 (2 · CH3). Other characteristic peaks of 13C NMR for compounds 2–4 are given in the experimental section. In ESI mass analysis the distinctive signals were observed in the mass spectra of compounds 2–4 which followed the definite fragmentation pattern. The molecular ion peaks (M+) for compounds 2–4 were observed at m/z 500, 536 and 522, respectively. 3.2. Anticancer activity In vitro evaluation of anticancer activity of the synthesized compounds was carried out using the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Slater et al., 1963). The conversion of the soluble yellowish MTT to the insoluble purple formazan by active mitochondrial lactate dehydrogenase of living cells has been used to develop an assay system for measurement of cell proliferation. Cell viability was measured with the purple formazan that was metabolized from MTT mitochondrial lactate dehydrogenase, which is active only in live cells. The screening of compounds 2–4 was done using the human leukemia cell line. Anticancer potency of the compounds was indicated by IC50 values that were calculated by linear regression analysis of the concentration-response curves obtained for each compound. Anticancer study showed that compound 4 was most active toward HL-60 cells


4

Shamsuzzaman et al. C8H17

C8H17

Hydrazine hydrate

Cl-CH2-CO-OEt

HO

O EtO

A

C C O H2

B

C8H17

O

H2 N

NH

C

O

CS2 / KOH

O

CH2

S

C O H2

N

N

H

1 Acetonyl acetone

A

2 ce

ty la

ce

to ne O O

C

H2 C

CH3 N

N H

CH3

H3C O

C

CH2

N

N

H

4

O

CH3

3 Scheme 1

Formation of steroidal oxadiazole, pyrrole and pyrazole.

among the three compounds with IC50 = 14.32 while the IC50 for compounds 2 and 3 were 17.33 and 18.57, respectively. It may be inferred that compound 4 contains ‘[30 ,50 -dimethylpyrazole-1-yl] carbonylmethoxy’ moiety attached at 3b position which may be responsible for this enhanced anticancer activity. 4. Conclusion The present work involves the simple and convenient synthesis of steroidal 1,3,4-oxadiazole, pyrrole and pyrazole 2–4 in reasonably good yields. This strategy offered a very straightforward and efficient method to access steroidal 1,3,4oxadiazole, pyrrole and pyrazole. The products 2–4 after being characterized by IR, 1H NMR, 13C NMR, MS and elemental analysis, were screened for anticancer study against HL-60 cell line during which compound 4 has been identified more promising by showing better anticancer behavior (IC50 = 14.32). Compounds 2 and 3 also showed moderate to good anticancer activity by showing IC50 = 17.33 and 18.57, respectively. This study also supports that pyrazole moiety after being attached with steroidal nucleus may be the factor responsible for enhanced anticancer behavior as pyrazoles are being considered better anticancer agents (Rostom, 2006; Ibrahim et al., 2007). In conclusion the present study showed that the synthesized compounds can be used as templates for future development through modification and derivatization to design more potent and selective anticancer agents.

Acknowledgments The authors thank the Chairman, Department of Chemistry, A.M.U., Aligarh, for providing necessary research facilities. Facilities provided by SAP (DRS-1) for their generous research support are also gratefully acknowledged. Authors also thank the Indian Institute of Integral Medicine (IIIM), Jammu for biological studies of the compounds and the UGC, New Delhi for financial support in the form of Major Research Project [UGC-Scheme-F. No.33-263/2007 (SR)]. References Abdallah, M.A., Riyadh, S.M., Abbas, I.M., Gomha, S.M., 2005. J. Chin. Chem. Soc. 52, 987–994. Amr, A.E.E., Mohamed, S.F., Abdel-Hafez, N.A., Abdalla, M.M., 2008. Monatsh Chem. 139, 1491–1498. Carlson, R., 2008. Exp. Opin. Invest. Drugs 17, 707–722. Chavan, V.P., Sonawane, S.A., Shingare, M.S., Karale, B.K., 2006. Chem. Heterocycl. Compd. 42, 625–630. De Leon, C.Y., Ganem, B., 1997. Tetrahedron 53, 7731–7752. Devasia, R.A., Jones, T.F., Ward, J., Stafford, L., Hardin, H., Bopp, C.M., Beatty, M., Mintz, E., Schaffner, W., 2006. Am. J. Med. 119, 168–176. Dutton, C.J., Fookes, C.J.R., Battersby, A.R., 1983. J. Chem. Soc., Chem. Commun., 1237–1238. El-Essawy, F.A., Khattab, A.F., Abdel-Rahman, A.A.H., 2007. Monatsh Chem. 138, 777–785.


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