- Email: [email protected]
Design, synthesis, molecular docking and biological evaluation of new dithiocarbamates substituted benzimidazole and chalcones as possible chemotherapeutic agents
Bioorganic & Medicinal Chemistry Letters 22 (2012) 3274–3277
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, molecular docking and biological evaluation of new dithiocarbamates substituted benzimidazole and chalcones as possible chemotherapeutic agents Keerthana Bacharaju a, Swathi Reddy Jambula a, Sreekanth Sivan b, Saritha JyostnaTangeda a, Vijjulatha Manga b,⇑ a b
Sarojini Naidu Vanitha Pharmacy Maha Vidyalaya, Osmania University, Hyderabad 500001, Andhra Pradesh, India Molecular Modeling and Medicinal Chemistry Group, Department of Chemistry, Nizam College, Osmania University, Hyderabad 500001, Andhra Pradesh, India
a r t i c l e
i n f o
Article history: Received 29 December 2011 Revised 3 March 2012 Accepted 6 March 2012 Available online 11 March 2012
a b s t r a c t A series of novel dithiocarbamates with benzimidazole and chalcone scaffold have been designed synthesised and evaluated for their antimitotic activity. Compounds 4c and 9d display the most promising antimitotic activity with IC50 of 1.66 lM and 1.52 lM respectively. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Benzimidazole Chalcone Dithiocarbamate Antimitotic Molecular docking
Benzimidazoles are one of the most extensively studied classes of heterocyclic compounds known for their wide range of biological activities.1,2 Extensive studies have been carried out on benzimidazoles and their chemotherapeutic activity.3,4 On the other side, dithiocarbamates are a common class of organic molecules, that form mono and bidentate coordination with transition metals. Transition metal complexes of dithiocarbamate present a wide range of biological activities5 and are recently applied in the treatment of cancer.6,7 Since brassinin (Fig. 1), a phytoalexin first isolated from cabbage had cancer preventive activity, structural modification on this compound led to the synthesis of isobrassinin (Fig. 1)8 and a series of dithiocarbamates, some of these were found to have antitumor activity.9 Beside the compounds mentioned above chalcones are the biogenetic precursors of all known flavonoids and isoflavanoids and are abundant in edible plants.10 They exhibit a broad spectrum of pharmacological activities such as anticancer,11 antiinflammatary,12 antimalarial,13 antifungal,14 antilipidemic,15 antiviral,16 antileshmanial,17 antiulcer18 and antioxidant activities.19 Recently Yong Qian and coworkers reported a series of chalcone derivatives (Fig. 1), with dithiocarbamate moieties which possessed potential antiproliferative and anti-tubulin properties.20 Microtubules are among the most important molecular targets for cancer chemo⇑ Corresponding author. Tel.: +91 040 23234321; fax: +91 040 23240806. E-mail address: [email protected] (V. Manga). 0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2012.03.018
therapeutic agents. These small molecules bind to the tubulin, interfering with the polymerisation or depolymerisation of microtubules and there by inducing cell cycle arrest, resulting in cell death or apoptosis. Although many new chalcone derivatives have been synthesized as potential antitumor agents, there is very scarce recent literature data on antitumor potentials of dithiocarbamate-substituted chalcones. This combination should provide favourable structural properties of both dithiocarbamate and chalcone moieties. It improves their binding affinities with the protein via hydrogen bonding, hydrophobic contact and to further explore their chemotherapeutic properties. New molecules were designed in which indole moiety, is isosterically replaced with benzimidazole. A series of novel derivatives containing both benzimidazole nucleus and dithiocarbamate as side chain possessing different substituents on nitrogen linked through methylene group at second position of benzimidazole were synthesized. Route applied for the synthesis of a various dithiocarbamate analogues is summarized in Schemes 1 and 2. Key intermediate 2-chloromethylbenzimidazole (2) (Scheme 1) was prepared by the addition of chloroaceticacid to o-phenylenediamine in 4 N hydrochloric acid as reported earlier.21 Dithiocarbamates were synthesized from various amines and carbon disulfide, using dimethylformamide as solvent and anhydrous potassium phosphate as base, followed by treatment with 2-chloromethylbenzimidazoles.22 Chalconedithiocarbamates were obtained by
3275
K. Bacharaju et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3274–3277
H N
SCH3
S
S
N H
N H Brassinin
N H
SR
Isobrassinin O
S
Cl N
H3C
S
H3C
3-(4-chlorophenyl)-3-oxo-1-phenylpropyl diethylcarbamodithioate Figure 1. Structure of known chalcone derivatives.
one pot reaction of various amines, carbondisulfide and intermediate chalcone23 (Scheme 2). Synthesized compounds were evaluated for their biological activity using chick pea seeds (Cicer arietinum)24 for their potential antimitotic activity, it is a rapid and inexpensive cytotoxicity test for the preliminary screening of new drugs.25,26 Synthesized compounds were found to inhibit the growth of the germinating roots from the seeds. Inhibitory activity values (IC50) are listed in Table 1. NH2
Among the series compound 4c (piperidine) showed highest potency with IC50 of 1.66 lM. Compounds 4a (pyrrolidine) and 4b (morpholine) were found to be equipotent with IC50 1.8 lM and 1.89 lM respectively. In case of the compounds with alkyl substituted aminodithiocarbamates, compound with diethyl (4e) substitution showed greater potency than the corresponding dimethyl amine (4d) with IC50 values 1.86 and 2.12 lM respectively. However compound 4f and 4g with propyl amine and butyl amine exhibited similar activity like 4d as evidenced from the IC50 values 2.29 and 2.11 lM respectively. Overall results indicate that the cyclic amines have higher activity when compared to aliphatic amines. Antimitotic activity of 9a–9f series, compound 9d (2-amino benzothiazole side chain) showed highest potency of 1.52 lM. Compound 9a (pyrrolidine) and 9c (morpholine) were equipotent with IC50 1.67 and 1.65 lM respectively. On the contrary compound 9b (piperidine) showed slight decrease in potency (1.85 lM). In case of the compounds with dialkyl amine side chain the compound 9f (diethyl amine) showed greater potency than the corresponding compound 9e (dimethyl amine) with IC50 1.84 lM and 2.43 lM, respectively. In order to gain more insight into the interaction between these new series of dithiocarbamate derivatives and b-tubulin that is involved in assembly of microtubules. Crystal structure of b-tubulin of bovine (pdb id: 1SA0) was download from the protein data bank (Since the plant b-tubulin experimental structure is not available and it has 94% sequence similarity with bovine). GLIDE 5.6 was
ClCH2COOH 4N HCl, 100 ºC
NH2
Cl
N
R2
N H
1
3
2
CS2, Anhydrous K3PO4
1 2
4a: 4b: 4c: 4d: 4e: 4f: 4g:
+
R1 HN
NR R = Pyrrolidinyl NR1R2 = morpholinyl NR1R2 = piperdinyl NR1R2 = N,N-dimethyl NR1R2 = N,N-diethyl NR1= H, R2 = N-propyl NR1= H, R2 = N-butyl
DMF, RT 2Hrs R1 N
H N
S
R2
S N
4a - g Scheme 1. Synthetic route to the benzimidazole dithiocarbamates (4a–4g).
O
CH3
O KOH, MeOH
+ 5
+
RT
6
O
R1 HN
R2
3
7
CS2, CH2Cl2
9a: 9b: 9c: 9d: 9e: 9f:
NR1R2 = pyrrolidinyl NR1R2 = piperidinyl NR1R2 = morpholinyl NR1= H, R2 = benzothiazolyl NR1R2 = N,N-dimethyl NR1R2 = N,N-diethyl
O
S
R1
9a-f Scheme 2. Synthetic route to the chalcone dithiocarbamates (9a–9f).
S N
R2
3276
K. Bacharaju et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3274–3277
Table 1 The antimitotic activity and GlideScore of title compounds 4a–g and 9a–f
R
1
N
H N
R
S
2
O
S
S
S N
R
4a - 4g
*
1
N
R
2
9a-f
Compound
R1R2
Antimitotic activity IC50 ±SDa (lM)
GlideScore (SP docking) Kcal/mol
GlideScore (XP docking) Kcal/mol
Cisplatin* 4a 4b 4c 4d 4e 4f 4g 9a 9b 9c 9d 9e 9f
— Pyrrolidinyl Morpholinyl Piperdinyl N,N-Dimethyl N,N-Diethyl R1 = H, R2 = N-Propyl R1 = H, R2 = N-butyl Pyrrolidinyl Piperdinyl Morpholinyl R1 = H, R2 = Benzothiazolyl N,N-Dimethyl N,N-Diethyl
1.21 ± 0.21 1.8 ± 0.45 1.89 ± 0.47 1.66 ± 0.44 2.12 ± 0.50 1.86 ± 0.51 2.29 ± 0.47 2.11 ± 0.49 1.67 ± 0.42 1.85 ± 0.44 1.65 ± 0.39 1.52 ± 0.40 2.43 ± 0.48 1.84 ± 0.43
— 6.656 6.726 6.546 5.814 6.060 5.764 5.914 6.506 6.504 6.999 7.774 5.587 6.107
— 7.090 7.119 6.354 6.594 6.756 7.085 7.787 7.192 7.945 6.983 8.637 7.246 8.004
Cisplatin was taken as a standard, as it inhibits tubulin assembly into microtubules. IC50 values are average of three replicate assays.
a
Figure 2. Dock pose of 9d in the active site of bovine b-tubulin showing a hydrogen bond interaction with Val 315. All the aromatic rings are deeply embedded into the hydrophobic pockets.
used for molecular docking. Protein was prepared using protein preparation wizard in Maestro 9.0 applying default parameters; a grid was generated around the active site by selecting the cocrystalized ligand. Receptor van der Waals scaling for non polar atoms was kept at 0.9. Molecules were built using Maestro build panel
and prepared by LigPrep 2.0 application. Low energy conformation of the ligands were selected and docked into the grid generated for the protein using both standard precision (SP) and extra precision (XP) docking modes. Dock pose of each ligand was analysed for interactions with the receptor. Molecules showed purely
K. Bacharaju et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3274–3277
hydrophobic interactions with the protein active site. Molecules were deeply embedded into the hydrophobic pocket formed by Cys241, Leu245 and Leu255 amino acids in the active site (Fig. 2). Most active molecule in the series (9d) showed high dockscore of 7.774 and 8.637 kcal/mol in SP and XP docking protocol respectively, this can be explained in terms of hydrophobic interaction of the phenyl rings, specifically benzothiazole ring that is occupying the extended hydrophobic cavity, it also showed one hydrogen bond interaction with Val315 ( Fig. 2). Molecules 4a–c and 9a–c showed moderate dockscore ranging from 6.504 to 6.999 kcal/mol, this is due to the hydrophobic interaction of cyclic systems, but lack of hydrogen bond interaction has reduced the dockscore compared to 9d this is also evident from the IC50 values. Molecules 4d and 9e having a dimethyl group instead of cyclic system has least activity and dockscores due to reduced hydrophobic characteristics. Molecule 4e and 9f having diethyl substitution that increases hydrophobic nature showed improved dockscore than dimethyl substituted 4d and 9e molecules similar to the experimental IC50 values. In molecules 4f and 4g only single substitution of N-propyl and N-butyl groups is present that resulted in considerable decrease in dockscore in accordance to the IC50 values. A regression analysis between the SP dockscore and IC50 values gave a regression coefficient (r2) value of 0.761 representing the significant correlation between the biological activities and docking analysis. In conclusion, a novel class of benzimidazoledithiocarbamate and chalcone dithiocarbamate derivatives were synthesized by introducing various amines on dithiocarbamate side chain. Acyclic amines showed less potency compared to cyclic groups. The main aim of the study was to evaluate these compounds for their antimitotic activity and screen them for further invitro studies. Although these molecules showed less potency compared to Cisplatin, toxicity wise these molecules can be considered as potent antimitotic derivatives. Acknowledgments We gratefully acknowledge support for this research from Council of Scientific and Industrial Research (Project No. 01/ (2436)/10/EMR-II), Department of Science and Technology, New Delhi, India, University Grants Commission, New Delhi, India. We also acknowledge Sarojini Naidu Vanitha Pharmacy Maha Vidyalaya and Department of chemistry, Nizam College, Hyderabad, India. We also acknowledge Schrödinger Inc. for GLIDE software, Tripos Inc. for SYBYLX-1.2. References and notes 1. 2. 3. 4. 5.
6. 7. 8. 9. 10. 11.
Horton, D. A.; Bourne, G. T.; Smythe, M. L. Chem. Rev. 2003, 103, 893. Rastogi, S.; Sharma, S. Synthesis 1983, 11, 861. Ozden, S.; Atabey, D.; Yildiz, S.; Goker, H. Bioorg. Med. Chem. 2005, 13, 1587. Boiani, M.; Gonazalez, M. Mini. Rev. Med. Chem. 2005, 5, 409. (a) Caladas, E. D.; Hosana Conceicua, M.; Miranda, M. C. C.; Souzana, L.; Lima, J. F. J. Agric. Food. Chem. 2001, 49, 4521; (b) Goal, A.; Mazur, S. J.; Fattah, R. J.; Hartman, T. L.; Turpin, J. A.; Huang, M.; Rice, W. G.; Appella, E.; Inman, J. K. Bioorg. Med. Chem. Lett. 2002, 12, 767. Schonenbreger, V. H.; Lippert, P. Pharmazie 1972, 72, 139. Scozzafava, A.; Mastrolorenzo, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 1887, 2000, 10. Csomos, P.; Zupko, I.; Rethy, B.; Fodor, L.; Falkay, G.; Bernath, G. Bioorg. Med. Chem. Lett. 2008, 16, 6273. Cao, S. L.; Feng, Y. P.; Jiang, Y. Y.; Liu, S. Y.; Ding, D. Y.; Li, R. T. Bioorg. Med. Chem. Lett. 1915, 2005, 15. Chetana, B. P.; Mahajan, S. K.; Suvarna, A. K. J. Pharm. Sci. Res. 2009, 1(3), 11. Subhashish, M.; Vijayendra, K.; Ashok, K. Prasad; Hanumanth, R.; Raj, G.; Marc, E. Bracke; Carl, E. O.; Subash, C. J.; Virender, S. P. Bioorg. Med. Chem. 2001, 9, 337.
3277
12. Javier, R.; Jose, N. D.; Jaime, E. C.; Gricela, L.; Miguel, P. M.; Luisa, F. Eur. J. Med. Chem. 2002, 37, 699. 13. Satish, K. A.; Nidhi, M.; Rajesh, K. B.; Manish, S.; Amit, B.; Lokesh, C. M.; Virendra, K. B. Med. Chem. Res. 2009, 18, 407. 14. Lathchev, K. L.; Batovska, D. J.; Parushev, St. P.; Ubiyvovk, V. M.; Sibirny, A. A. Eur. J. Med. Chem. 2008, 43, 2220. 15. Santos, L.; Curi, P. R.; Correa, R.; Cechinel, F. V.; Nunes, R. J.; Yunes, R. A. Arch. Pharm. 2006, 339, 541. 16. Mallikarjuni, K. G. E-J. Chem. 2005, 2, 58. 17. Simon, F. N.; Saren, B. C.; Gabriele, C.; Arsalan, K.; Tommy, L. J. Med. Chem. 1998, 41, 4819. 18. Kyogoku, K.; Hatayama, K.; Yokomri, S.; Saziki, R.; Nakane, S.; Sasajima, M.; Sawada, J.; Ohzeki, M.; Tanaka, J. Chem. Pharm Bull. 1979, 27, 294. 19. Belsare, D. P.; Pal, S. C.; Kazi, A. A.; Kankate, R. S.; Vanjari, S. S. Int. J. ChemTech Res. 2010, 2, 1080. 20. Yong, Q.; Gao, Y. M.; Ying, Y.; Kui, C.; Qing, Z. Z.; Wen, J. M.; Lei, S.; Jing, Z. Bioorg. Med. Chem. 2010, 18, 4310. 21. U.S. Patent 4714764, 1987; Chem. Abstr. 1983, 98, 46775h. 22. General procedure for synthesis of benzimadazoledithiocarbmates 4a–g: Equimolar mixture of amine 3 and anhydrous potassium phosphate in dimethyl formamide was stirred at room temperature for 5 min, and then carbondisulfide (2 equiv) was added. The reaction mixture was stirred for additional 20 min, and then appropriate 2-chloromethylbenzimidazole 2, (1 equiv) was added. Stirring was continued at room temperature until the reaction was completed as monitored by TLC. The mixture was poured into cold water then extracted with ethylacetate (3 30 mL), organic phase was washed once with water and dried over sodium sulfate and filtered. Solvent was evaporated under reduced pressure and the resultant residue was chromatographed over silica gel using mixture of petroleum ether and ethylacetate as eluent. Compound (4a): Yield: 92%; Melting point:160–162 °C; IR (KBr) cm 1: 3394(NH), 2921(CH); 1H NMR [CDCl3,400 MHz]: d (ppm) 2.0–2.11(m, 4H, (CH2)2), 3.62–3.65(t, 2H, NCH2), 3.97–4.01(t, 2H, NCH2), 4.88(s, 2H, CH2S), 7.21–7.26 (m, 4H, Ar-H); EI-MS: 278(M+1)+. Compound (4b): Yield: 96%; Melting point:186–188 °C; IR (KBr) cm 1: 3221(NH), 2921(CH); 1H NMR [DMSO, 200 MHz]: d (ppm) 3.66 (m, 4H, O(CH2)2), 3.98–4.18 (br s, 4H, N(CH2)2), 4.76 (s, 2H, CH2S), 7.12–7.47 (m, 4H, Ar-H), 12.37 (br s, 1H, NH); EI-MS: 294[M+1]+. Compound (4c): Yield: 91%; Melting point:168–170 °C; IR (KBr) cm 1: 3451 (NH), 2936 (CH); 1H NMR (CDCl3, 400 MHz): d (ppm) 1.67–1.73 (d, 6H, piperidine (CH2)3), 3.86 (s, 2H, NCH2) 4.34 (s, 2H, NCH2), 4.91 (s, 2H, CH2S), 7.22–7.26 (m, 4H, Ar-H). 23. General procedure for synthesis of chalconedithiocarbamates 9a–f: Carbondisulfide (0.15 mL, 2.5 mM) and chalcone 7 (0.42 g, 2 mM) was dissolved in dichloromethane (10 mL) and the solution was cooled to 0 °C in an ice bath. Amine 3 (2.25 mM) was slowly added and the reaction mixture was stirred at 0 °C for 30 min. Then, the solution was warmed to room temperature and stirred for another 24 h, and the reaction was monitored by TLC. After the end of the reaction, solvents were removed in vaccum and the residue was purified by column chromatography on silica gel (ethyl acetate– petroleum ether) affording compound dithiocarbamates. Compound (9a): Yield: 92%; Melting point:116–118 °C; IR (KBr) cm 1: 1679 (C@O), 2869 (aliphatic); 1H NMR [CDCl3, 400 MHz]: 1.94–2.06 (m, 4H, pyrrolidine (CH2)2), 3.57–3.63 (m, 2H, NCH2), 3.71–3.78 (m, 1H, CH2 of CO gp), 3.90–3.94 (t, 2H, NCH2), 4.10–4.16 (m, 1H, CH2 of CO gp), 5.74–5.77 (dd, 1H, SCH), 7.25–7.96 (m, 10H, Ar-H); MASS (ESI): 378 [M+Na]+. Compound (9b): Yield: 82%; Melting point:118–120 °C; IR (KBr) cm 1: 1681 (C@O), 2939 (aliphatic); 1H NMR [CDCl3, 400 MHz]: 1.69 (s, 6H, piperidine (CH2)3), 3.72–3.78 (m, 1H, CH2 of CO gp), 3.83 (br s, 2H, NCH2), 4.12–4.17 (m, 1H, CH2 of CO gp), 4.27 (br s, 2H, NCH2), 5.70–5.74 (dd, 1H, SCH), 7.22–7.53 (m, 10H, Ar-H); MASS (ESI):392 [M+Na]+ Compound (9d): Yield: 85%; Melting point:148–150 °C; IR (KBr) cm 1:1667 (C@O), 3341 (N–H), 2856 (aliphatic); 1H NMR [CDCl3, 300 MHz]: 3.55 (s, 1H, CH2), 3.76 (s, 1H, CH2), 5.44 (s, 1H, SCH), 6.79 (br s, 1H, NH), 7.05-7.85 (m, 14H, Ar-H). 24. Murthy, G.S.; Francis T.P.; Rajendra, C. Singh.; Nagendra, H.G.; Naik, C. Current Sci. 2010, 100, 1399. Procedure: Chick pea seeds (Cicer arietinum) of good quality were soaked overnight with water to hasten the germinating process. These were distributed in a group of 10 each in Petri dishes on moistened filter paper. Drug solutions were prepared 1% DMSO at concentrations ranging from 1 mL and added to the filter paper in the Petri dishes. One Petri dish served as control (DMSO) and one served as standard (cisplatin). The seeds were allowed to germinate for 7 days and care was taken to moisten the filter paper with control and drug solutions every 24 h. The length of the radicals was measured in cm at the end of 7th day and percent mean values of the DMSO (control) treated and 1% growth inhibition is calculated. 25. Vijay, L. K.; Singhal, A. Biocell 2009, 33, 19. 26. Sharma, C. B. S. R. Current Sci. 1983, 52, 1000.
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, molecular docking and biological evaluation of new dithiocarbamates substituted benzimidazole and chalcones as possible chemotherapeutic agents Keerthana Bacharaju a, Swathi Reddy Jambula a, Sreekanth Sivan b, Saritha JyostnaTangeda a, Vijjulatha Manga b,⇑ a b
Sarojini Naidu Vanitha Pharmacy Maha Vidyalaya, Osmania University, Hyderabad 500001, Andhra Pradesh, India Molecular Modeling and Medicinal Chemistry Group, Department of Chemistry, Nizam College, Osmania University, Hyderabad 500001, Andhra Pradesh, India
a r t i c l e
i n f o
Article history: Received 29 December 2011 Revised 3 March 2012 Accepted 6 March 2012 Available online 11 March 2012
a b s t r a c t A series of novel dithiocarbamates with benzimidazole and chalcone scaffold have been designed synthesised and evaluated for their antimitotic activity. Compounds 4c and 9d display the most promising antimitotic activity with IC50 of 1.66 lM and 1.52 lM respectively. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Benzimidazole Chalcone Dithiocarbamate Antimitotic Molecular docking
Benzimidazoles are one of the most extensively studied classes of heterocyclic compounds known for their wide range of biological activities.1,2 Extensive studies have been carried out on benzimidazoles and their chemotherapeutic activity.3,4 On the other side, dithiocarbamates are a common class of organic molecules, that form mono and bidentate coordination with transition metals. Transition metal complexes of dithiocarbamate present a wide range of biological activities5 and are recently applied in the treatment of cancer.6,7 Since brassinin (Fig. 1), a phytoalexin first isolated from cabbage had cancer preventive activity, structural modification on this compound led to the synthesis of isobrassinin (Fig. 1)8 and a series of dithiocarbamates, some of these were found to have antitumor activity.9 Beside the compounds mentioned above chalcones are the biogenetic precursors of all known flavonoids and isoflavanoids and are abundant in edible plants.10 They exhibit a broad spectrum of pharmacological activities such as anticancer,11 antiinflammatary,12 antimalarial,13 antifungal,14 antilipidemic,15 antiviral,16 antileshmanial,17 antiulcer18 and antioxidant activities.19 Recently Yong Qian and coworkers reported a series of chalcone derivatives (Fig. 1), with dithiocarbamate moieties which possessed potential antiproliferative and anti-tubulin properties.20 Microtubules are among the most important molecular targets for cancer chemo⇑ Corresponding author. Tel.: +91 040 23234321; fax: +91 040 23240806. E-mail address: [email protected] (V. Manga). 0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2012.03.018
therapeutic agents. These small molecules bind to the tubulin, interfering with the polymerisation or depolymerisation of microtubules and there by inducing cell cycle arrest, resulting in cell death or apoptosis. Although many new chalcone derivatives have been synthesized as potential antitumor agents, there is very scarce recent literature data on antitumor potentials of dithiocarbamate-substituted chalcones. This combination should provide favourable structural properties of both dithiocarbamate and chalcone moieties. It improves their binding affinities with the protein via hydrogen bonding, hydrophobic contact and to further explore their chemotherapeutic properties. New molecules were designed in which indole moiety, is isosterically replaced with benzimidazole. A series of novel derivatives containing both benzimidazole nucleus and dithiocarbamate as side chain possessing different substituents on nitrogen linked through methylene group at second position of benzimidazole were synthesized. Route applied for the synthesis of a various dithiocarbamate analogues is summarized in Schemes 1 and 2. Key intermediate 2-chloromethylbenzimidazole (2) (Scheme 1) was prepared by the addition of chloroaceticacid to o-phenylenediamine in 4 N hydrochloric acid as reported earlier.21 Dithiocarbamates were synthesized from various amines and carbon disulfide, using dimethylformamide as solvent and anhydrous potassium phosphate as base, followed by treatment with 2-chloromethylbenzimidazoles.22 Chalconedithiocarbamates were obtained by
3275
K. Bacharaju et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3274–3277
H N
SCH3
S
S
N H
N H Brassinin
N H
SR
Isobrassinin O
S
Cl N
H3C
S
H3C
3-(4-chlorophenyl)-3-oxo-1-phenylpropyl diethylcarbamodithioate Figure 1. Structure of known chalcone derivatives.
one pot reaction of various amines, carbondisulfide and intermediate chalcone23 (Scheme 2). Synthesized compounds were evaluated for their biological activity using chick pea seeds (Cicer arietinum)24 for their potential antimitotic activity, it is a rapid and inexpensive cytotoxicity test for the preliminary screening of new drugs.25,26 Synthesized compounds were found to inhibit the growth of the germinating roots from the seeds. Inhibitory activity values (IC50) are listed in Table 1. NH2
Among the series compound 4c (piperidine) showed highest potency with IC50 of 1.66 lM. Compounds 4a (pyrrolidine) and 4b (morpholine) were found to be equipotent with IC50 1.8 lM and 1.89 lM respectively. In case of the compounds with alkyl substituted aminodithiocarbamates, compound with diethyl (4e) substitution showed greater potency than the corresponding dimethyl amine (4d) with IC50 values 1.86 and 2.12 lM respectively. However compound 4f and 4g with propyl amine and butyl amine exhibited similar activity like 4d as evidenced from the IC50 values 2.29 and 2.11 lM respectively. Overall results indicate that the cyclic amines have higher activity when compared to aliphatic amines. Antimitotic activity of 9a–9f series, compound 9d (2-amino benzothiazole side chain) showed highest potency of 1.52 lM. Compound 9a (pyrrolidine) and 9c (morpholine) were equipotent with IC50 1.67 and 1.65 lM respectively. On the contrary compound 9b (piperidine) showed slight decrease in potency (1.85 lM). In case of the compounds with dialkyl amine side chain the compound 9f (diethyl amine) showed greater potency than the corresponding compound 9e (dimethyl amine) with IC50 1.84 lM and 2.43 lM, respectively. In order to gain more insight into the interaction between these new series of dithiocarbamate derivatives and b-tubulin that is involved in assembly of microtubules. Crystal structure of b-tubulin of bovine (pdb id: 1SA0) was download from the protein data bank (Since the plant b-tubulin experimental structure is not available and it has 94% sequence similarity with bovine). GLIDE 5.6 was
ClCH2COOH 4N HCl, 100 ºC
NH2
Cl
N
R2
N H
1
3
2
CS2, Anhydrous K3PO4
1 2
4a: 4b: 4c: 4d: 4e: 4f: 4g:
+
R1 HN
NR R = Pyrrolidinyl NR1R2 = morpholinyl NR1R2 = piperdinyl NR1R2 = N,N-dimethyl NR1R2 = N,N-diethyl NR1= H, R2 = N-propyl NR1= H, R2 = N-butyl
DMF, RT 2Hrs R1 N
H N
S
R2
S N
4a - g Scheme 1. Synthetic route to the benzimidazole dithiocarbamates (4a–4g).
O
CH3
O KOH, MeOH
+ 5
+
RT
6
O
R1 HN
R2
3
7
CS2, CH2Cl2
9a: 9b: 9c: 9d: 9e: 9f:
NR1R2 = pyrrolidinyl NR1R2 = piperidinyl NR1R2 = morpholinyl NR1= H, R2 = benzothiazolyl NR1R2 = N,N-dimethyl NR1R2 = N,N-diethyl
O
S
R1
9a-f Scheme 2. Synthetic route to the chalcone dithiocarbamates (9a–9f).
S N
R2
3276
K. Bacharaju et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3274–3277
Table 1 The antimitotic activity and GlideScore of title compounds 4a–g and 9a–f
R
1
N
H N
R
S
2
O
S
S
S N
R
4a - 4g
*
1
N
R
2
9a-f
Compound
R1R2
Antimitotic activity IC50 ±SDa (lM)
GlideScore (SP docking) Kcal/mol
GlideScore (XP docking) Kcal/mol
Cisplatin* 4a 4b 4c 4d 4e 4f 4g 9a 9b 9c 9d 9e 9f
— Pyrrolidinyl Morpholinyl Piperdinyl N,N-Dimethyl N,N-Diethyl R1 = H, R2 = N-Propyl R1 = H, R2 = N-butyl Pyrrolidinyl Piperdinyl Morpholinyl R1 = H, R2 = Benzothiazolyl N,N-Dimethyl N,N-Diethyl
1.21 ± 0.21 1.8 ± 0.45 1.89 ± 0.47 1.66 ± 0.44 2.12 ± 0.50 1.86 ± 0.51 2.29 ± 0.47 2.11 ± 0.49 1.67 ± 0.42 1.85 ± 0.44 1.65 ± 0.39 1.52 ± 0.40 2.43 ± 0.48 1.84 ± 0.43
— 6.656 6.726 6.546 5.814 6.060 5.764 5.914 6.506 6.504 6.999 7.774 5.587 6.107
— 7.090 7.119 6.354 6.594 6.756 7.085 7.787 7.192 7.945 6.983 8.637 7.246 8.004
Cisplatin was taken as a standard, as it inhibits tubulin assembly into microtubules. IC50 values are average of three replicate assays.
a
Figure 2. Dock pose of 9d in the active site of bovine b-tubulin showing a hydrogen bond interaction with Val 315. All the aromatic rings are deeply embedded into the hydrophobic pockets.
used for molecular docking. Protein was prepared using protein preparation wizard in Maestro 9.0 applying default parameters; a grid was generated around the active site by selecting the cocrystalized ligand. Receptor van der Waals scaling for non polar atoms was kept at 0.9. Molecules were built using Maestro build panel
and prepared by LigPrep 2.0 application. Low energy conformation of the ligands were selected and docked into the grid generated for the protein using both standard precision (SP) and extra precision (XP) docking modes. Dock pose of each ligand was analysed for interactions with the receptor. Molecules showed purely
K. Bacharaju et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3274–3277
hydrophobic interactions with the protein active site. Molecules were deeply embedded into the hydrophobic pocket formed by Cys241, Leu245 and Leu255 amino acids in the active site (Fig. 2). Most active molecule in the series (9d) showed high dockscore of 7.774 and 8.637 kcal/mol in SP and XP docking protocol respectively, this can be explained in terms of hydrophobic interaction of the phenyl rings, specifically benzothiazole ring that is occupying the extended hydrophobic cavity, it also showed one hydrogen bond interaction with Val315 ( Fig. 2). Molecules 4a–c and 9a–c showed moderate dockscore ranging from 6.504 to 6.999 kcal/mol, this is due to the hydrophobic interaction of cyclic systems, but lack of hydrogen bond interaction has reduced the dockscore compared to 9d this is also evident from the IC50 values. Molecules 4d and 9e having a dimethyl group instead of cyclic system has least activity and dockscores due to reduced hydrophobic characteristics. Molecule 4e and 9f having diethyl substitution that increases hydrophobic nature showed improved dockscore than dimethyl substituted 4d and 9e molecules similar to the experimental IC50 values. In molecules 4f and 4g only single substitution of N-propyl and N-butyl groups is present that resulted in considerable decrease in dockscore in accordance to the IC50 values. A regression analysis between the SP dockscore and IC50 values gave a regression coefficient (r2) value of 0.761 representing the significant correlation between the biological activities and docking analysis. In conclusion, a novel class of benzimidazoledithiocarbamate and chalcone dithiocarbamate derivatives were synthesized by introducing various amines on dithiocarbamate side chain. Acyclic amines showed less potency compared to cyclic groups. The main aim of the study was to evaluate these compounds for their antimitotic activity and screen them for further invitro studies. Although these molecules showed less potency compared to Cisplatin, toxicity wise these molecules can be considered as potent antimitotic derivatives. Acknowledgments We gratefully acknowledge support for this research from Council of Scientific and Industrial Research (Project No. 01/ (2436)/10/EMR-II), Department of Science and Technology, New Delhi, India, University Grants Commission, New Delhi, India. We also acknowledge Sarojini Naidu Vanitha Pharmacy Maha Vidyalaya and Department of chemistry, Nizam College, Hyderabad, India. We also acknowledge Schrödinger Inc. for GLIDE software, Tripos Inc. for SYBYLX-1.2. References and notes 1. 2. 3. 4. 5.
6. 7. 8. 9. 10. 11.
Horton, D. A.; Bourne, G. T.; Smythe, M. L. Chem. Rev. 2003, 103, 893. Rastogi, S.; Sharma, S. Synthesis 1983, 11, 861. Ozden, S.; Atabey, D.; Yildiz, S.; Goker, H. Bioorg. Med. Chem. 2005, 13, 1587. Boiani, M.; Gonazalez, M. Mini. Rev. Med. Chem. 2005, 5, 409. (a) Caladas, E. D.; Hosana Conceicua, M.; Miranda, M. C. C.; Souzana, L.; Lima, J. F. J. Agric. Food. Chem. 2001, 49, 4521; (b) Goal, A.; Mazur, S. J.; Fattah, R. J.; Hartman, T. L.; Turpin, J. A.; Huang, M.; Rice, W. G.; Appella, E.; Inman, J. K. Bioorg. Med. Chem. Lett. 2002, 12, 767. Schonenbreger, V. H.; Lippert, P. Pharmazie 1972, 72, 139. Scozzafava, A.; Mastrolorenzo, A.; Supuran, C. T. Bioorg. Med. Chem. Lett. 1887, 2000, 10. Csomos, P.; Zupko, I.; Rethy, B.; Fodor, L.; Falkay, G.; Bernath, G. Bioorg. Med. Chem. Lett. 2008, 16, 6273. Cao, S. L.; Feng, Y. P.; Jiang, Y. Y.; Liu, S. Y.; Ding, D. Y.; Li, R. T. Bioorg. Med. Chem. Lett. 1915, 2005, 15. Chetana, B. P.; Mahajan, S. K.; Suvarna, A. K. J. Pharm. Sci. Res. 2009, 1(3), 11. Subhashish, M.; Vijayendra, K.; Ashok, K. Prasad; Hanumanth, R.; Raj, G.; Marc, E. Bracke; Carl, E. O.; Subash, C. J.; Virender, S. P. Bioorg. Med. Chem. 2001, 9, 337.
3277
12. Javier, R.; Jose, N. D.; Jaime, E. C.; Gricela, L.; Miguel, P. M.; Luisa, F. Eur. J. Med. Chem. 2002, 37, 699. 13. Satish, K. A.; Nidhi, M.; Rajesh, K. B.; Manish, S.; Amit, B.; Lokesh, C. M.; Virendra, K. B. Med. Chem. Res. 2009, 18, 407. 14. Lathchev, K. L.; Batovska, D. J.; Parushev, St. P.; Ubiyvovk, V. M.; Sibirny, A. A. Eur. J. Med. Chem. 2008, 43, 2220. 15. Santos, L.; Curi, P. R.; Correa, R.; Cechinel, F. V.; Nunes, R. J.; Yunes, R. A. Arch. Pharm. 2006, 339, 541. 16. Mallikarjuni, K. G. E-J. Chem. 2005, 2, 58. 17. Simon, F. N.; Saren, B. C.; Gabriele, C.; Arsalan, K.; Tommy, L. J. Med. Chem. 1998, 41, 4819. 18. Kyogoku, K.; Hatayama, K.; Yokomri, S.; Saziki, R.; Nakane, S.; Sasajima, M.; Sawada, J.; Ohzeki, M.; Tanaka, J. Chem. Pharm Bull. 1979, 27, 294. 19. Belsare, D. P.; Pal, S. C.; Kazi, A. A.; Kankate, R. S.; Vanjari, S. S. Int. J. ChemTech Res. 2010, 2, 1080. 20. Yong, Q.; Gao, Y. M.; Ying, Y.; Kui, C.; Qing, Z. Z.; Wen, J. M.; Lei, S.; Jing, Z. Bioorg. Med. Chem. 2010, 18, 4310. 21. U.S. Patent 4714764, 1987; Chem. Abstr. 1983, 98, 46775h. 22. General procedure for synthesis of benzimadazoledithiocarbmates 4a–g: Equimolar mixture of amine 3 and anhydrous potassium phosphate in dimethyl formamide was stirred at room temperature for 5 min, and then carbondisulfide (2 equiv) was added. The reaction mixture was stirred for additional 20 min, and then appropriate 2-chloromethylbenzimidazole 2, (1 equiv) was added. Stirring was continued at room temperature until the reaction was completed as monitored by TLC. The mixture was poured into cold water then extracted with ethylacetate (3 30 mL), organic phase was washed once with water and dried over sodium sulfate and filtered. Solvent was evaporated under reduced pressure and the resultant residue was chromatographed over silica gel using mixture of petroleum ether and ethylacetate as eluent. Compound (4a): Yield: 92%; Melting point:160–162 °C; IR (KBr) cm 1: 3394(NH), 2921(CH); 1H NMR [CDCl3,400 MHz]: d (ppm) 2.0–2.11(m, 4H, (CH2)2), 3.62–3.65(t, 2H, NCH2), 3.97–4.01(t, 2H, NCH2), 4.88(s, 2H, CH2S), 7.21–7.26 (m, 4H, Ar-H); EI-MS: 278(M+1)+. Compound (4b): Yield: 96%; Melting point:186–188 °C; IR (KBr) cm 1: 3221(NH), 2921(CH); 1H NMR [DMSO, 200 MHz]: d (ppm) 3.66 (m, 4H, O(CH2)2), 3.98–4.18 (br s, 4H, N(CH2)2), 4.76 (s, 2H, CH2S), 7.12–7.47 (m, 4H, Ar-H), 12.37 (br s, 1H, NH); EI-MS: 294[M+1]+. Compound (4c): Yield: 91%; Melting point:168–170 °C; IR (KBr) cm 1: 3451 (NH), 2936 (CH); 1H NMR (CDCl3, 400 MHz): d (ppm) 1.67–1.73 (d, 6H, piperidine (CH2)3), 3.86 (s, 2H, NCH2) 4.34 (s, 2H, NCH2), 4.91 (s, 2H, CH2S), 7.22–7.26 (m, 4H, Ar-H). 23. General procedure for synthesis of chalconedithiocarbamates 9a–f: Carbondisulfide (0.15 mL, 2.5 mM) and chalcone 7 (0.42 g, 2 mM) was dissolved in dichloromethane (10 mL) and the solution was cooled to 0 °C in an ice bath. Amine 3 (2.25 mM) was slowly added and the reaction mixture was stirred at 0 °C for 30 min. Then, the solution was warmed to room temperature and stirred for another 24 h, and the reaction was monitored by TLC. After the end of the reaction, solvents were removed in vaccum and the residue was purified by column chromatography on silica gel (ethyl acetate– petroleum ether) affording compound dithiocarbamates. Compound (9a): Yield: 92%; Melting point:116–118 °C; IR (KBr) cm 1: 1679 (C@O), 2869 (aliphatic); 1H NMR [CDCl3, 400 MHz]: 1.94–2.06 (m, 4H, pyrrolidine (CH2)2), 3.57–3.63 (m, 2H, NCH2), 3.71–3.78 (m, 1H, CH2 of CO gp), 3.90–3.94 (t, 2H, NCH2), 4.10–4.16 (m, 1H, CH2 of CO gp), 5.74–5.77 (dd, 1H, SCH), 7.25–7.96 (m, 10H, Ar-H); MASS (ESI): 378 [M+Na]+. Compound (9b): Yield: 82%; Melting point:118–120 °C; IR (KBr) cm 1: 1681 (C@O), 2939 (aliphatic); 1H NMR [CDCl3, 400 MHz]: 1.69 (s, 6H, piperidine (CH2)3), 3.72–3.78 (m, 1H, CH2 of CO gp), 3.83 (br s, 2H, NCH2), 4.12–4.17 (m, 1H, CH2 of CO gp), 4.27 (br s, 2H, NCH2), 5.70–5.74 (dd, 1H, SCH), 7.22–7.53 (m, 10H, Ar-H); MASS (ESI):392 [M+Na]+ Compound (9d): Yield: 85%; Melting point:148–150 °C; IR (KBr) cm 1:1667 (C@O), 3341 (N–H), 2856 (aliphatic); 1H NMR [CDCl3, 300 MHz]: 3.55 (s, 1H, CH2), 3.76 (s, 1H, CH2), 5.44 (s, 1H, SCH), 6.79 (br s, 1H, NH), 7.05-7.85 (m, 14H, Ar-H). 24. Murthy, G.S.; Francis T.P.; Rajendra, C. Singh.; Nagendra, H.G.; Naik, C. Current Sci. 2010, 100, 1399. Procedure: Chick pea seeds (Cicer arietinum) of good quality were soaked overnight with water to hasten the germinating process. These were distributed in a group of 10 each in Petri dishes on moistened filter paper. Drug solutions were prepared 1% DMSO at concentrations ranging from 1 mL and added to the filter paper in the Petri dishes. One Petri dish served as control (DMSO) and one served as standard (cisplatin). The seeds were allowed to germinate for 7 days and care was taken to moisten the filter paper with control and drug solutions every 24 h. The length of the radicals was measured in cm at the end of 7th day and percent mean values of the DMSO (control) treated and 1% growth inhibition is calculated. 25. Vijay, L. K.; Singhal, A. Biocell 2009, 33, 19. 26. Sharma, C. B. S. R. Current Sci. 1983, 52, 1000.