A parent benzothiazole molecule was synthesized by Jacobson synthesis, then it is subjected to treatment with various aromatic aldehydes to get the corresponding Schiff bases followed by esterification of carboxyl group by using various alcohols. The structures of synthesized compounds were confirmed by various spectroscopic methods such as IR, NMR and mass spectroscopy. The products were evaluated for their antimicrobial activity. Some of the compounds exhibited potent activity when compared with the standards

Sushant Sudhir Pande, T. Panneer Selvam, Padmavathi P. Prabhu, C. S. Shastry

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 Research in Pharmacy 1(2) : 6-12, 2011                                                                 ISSN : 2231-539X www.researchinpharmacy.com   Regular Article Design, synthesis, characterization and biological evaluation of Benzothiazole-6-carboxylate derivatives  Padmavathi P. Prabhu*a, C. S. Shastryb, Sushant Sudhir Pandea, T. Panneer Selvama  aDepartment of Pharmaceutical chemistry,  Srinivas College of Pharmacy, Valachil,  Mangalore- 574 143,  Karnataka, India. bDepartment of Pharmacology, NGSMIPS, Paneer, Deralakatte, Mangalore. Corresponding Author email: padmapprabhu@gmail.com    A parent benzothiazole molecule was synthesized by Jacobson synthesis, then it is subjected to treatment with various aromatic aldehydes to get the corresponding Schiff bases followed by esterification of carboxyl group by using various alcohols. The structures of synthesized compounds were confirmed by various spectroscopic methods such as IR, NMR and mass spectroscopy. The products were evaluated for their antimicrobial activity. Some of the compounds exhibited potent activity when compared with the standards.   Key words: Benzothiazole; Schiff base; Esterification; Antimicrobial activity.   Benzothiazole nucleus is present in various compounds and it is responsible for various biological activities. The present work mainly focuses on the benzothiazoles with potential activities that are now in development.  Benzothiazoles are bicyclic ring system with multiple applications. Amino-benzothiazoles constitute an important class of compounds.  In recent year heterocyclic compounds analogues and derivatives have attracted strong interest due to their useful biological and pharmacological properties.  Proposed work is based upon the development of some newer structurally related compounds of benzothiazoles and evaluation of biological activity. Benzothiazoles shows diverse biological activities ranging from antiparasitic (Alaimo, 1974), anti-inflammatory (Singh, 1986), antitumor (Schnur, 1991 and  Hutchinson, 2003),   immunosuppressive (Paget, 1969 and Hugershoff, 1901),  anti- tubercular activity (Leon, 1951). Such wide range of activities motivated us to develop newer analogues of the benzothiazole expecting better biological activity. Synthesis of the basic nucleus is well established and the proposed derivatives can be synthesized based upon the literature available about the reaction involved. Although they have been known from long ago to be biologically active (Caleta, 2004, Bhusari, 2000 and Nargund, 1998), their varied biological features are still of great scientific interest. Benzothiazoles show antitumor activity, especially the phenyl-substituted benzothiazoles (Leong, 2004 and Bradshaw, 2002). Substituted 6-nitro- and 6-aminobenzothiazoles (Hutchinson, 2001) show antimicrobial activity. In connection with our earlier studies on the synthesis and biologic properties of substituted benzothiazoles (Prabhu, 2011 and Prabhu, 2010) we now focused our attention on the synthesis and biological evaluation of novel alkyl-2-(4-(benzylideneamino) phenyl) benzo thiazole-6-carboxylate as analogous of 2-(4-(benzylideneamino) phenyl) benzo  Padmavathi et al. /Research in Pharmacy 1(2) : 6-12, 2011  7  thiazole-6-carboxylic acid. We have given below a brief account of various alterations conducted on benzothiazole ring and their antimicrobial activity.  Material and methods Melting points of synthesized compounds were determined by melting point apparatus and uncorrected. NMR spectra were recorded on BRUKER-spectrospin 400MHz spectrometer in CDCl3, Tetra methyl silane (TMS; δ = 0.00 ppm) served as internal standards for 1H NMR. The corresponding residual non-deuterated solvent signal (CDCl3: δ = 77.00 ppm) was used as internal standards for 13C NMR. IR spectra were measured using a JASCO FT/IR-410 spectrometer, and Perkin-Elmer FT/IR Spectrum BX, GX. Mass spectra were measured with Micromass Q-Tof (ESI-HRMS). Column chromatography was conducted on Silica gel 230-400 mesh (Merck) and preparative thin-layer chromatography was carried out using SILICA GEL GF-254. Spectras were obtained from IISC Bangalore. The physical data of the derivatives are listed in table I. The scheme of synthesis is given in schemes 1 and 2.  In scheme 1 parent benzothiazole molecule was synthesized by Jacobson method using Lawsson’s reagent, the product obtained was used for the synthesis of various benzothiazole-6-carboxylate derivatives as shown in the scheme 2.  Scheme 1: Synthesis of parent benzothiazole molecule   Scheme 2: Synthesis of benzothiazole-6-carboxylate derivatives Padmavathi et al. /Research in Pharmacy 1(2) : 6-12, 2011  8  Procedure Synthesis of N-(4-Carboxy phenyl)-4- nitro phenyl benzamide (1a) : To a solution of p-amino benzoic acid (11.5 g, 62.6 mmol) in dichloro methane (100 ml) was added pyridine (40 ml) followed by addition of mixture of p-nitro benzoyl chloride (8 ml, 68.9 mmol) in toluene (30 ml). Then, the mixture was heated at reflux for 5 hrs., after this, toluene was removed under vacuum, 100 ml water was added then product was extracted with dichloromethane (3×100ml) and dichloromethane layer was rinsed with 1M HCl (200 ml) followed by washing with 20% 100ml aqueous solution of sodium carbonate. The organic layer was then dried over sodium sulphate (Na2SO4) and concentrated to produce benzamide derivative 1a as a purple crystalline solid. The compound was recrystallised from dichloromethane and hexane. Yield = 86 %. Synthesis of N-(4-Carboxy phenyl)-4- nitrophenyl thio benzamide (2a): To a solution compound 1a (1.0 g) in 40 ml dry toluene was added lawesson’s reagent (0.6 molar eq). The mixture was heated under an atmosphere of nitrogen at reflux for 2 hrs. after which it was concentrated and purified by column chromatography to give yellow crystals. Yield 85 % Synthesis of 6-carboxy-2-(P-nitrophenyl) benzothiazole (3a): To the compound 2a (0.1g) in 0.5 ml ethanol was added 1.5 M 7 ml NaOH. The solution was cooled in an ice-bath and freshly prepared aqueous potassium ferricyanide (2-3 molar equivalents) was added. The reaction mixture was stirred at room temperature for 24 hr.; then, the mixture was neutralized with 1M HCl and extracted with ethyl acetate (3×75ml). The organic layer was dried over Na2SO4. Then organic layer was removed in vacuum and the residue is purified by recrystallisation from ethanol or ethyl acetate: hexanes to give white needles. Yield 79% Synthesis of 6-carboxy-2-(p-aminophenyl) benzothiazole (4a): To 6-carboxy-2-(p-nitrophenyl)-benzothiazole 3a (5g, 25 mmol) in 20ml ethanol: 10ml water was added iron powder (4.1g, 75 mmol) (325 mesh) and ammonium chloride (0.7g, 12.5 mmol).The reaction was stirred at 85oC for one hr., cooled to room temperature, and filtered through celite. The filter cake was then washed with 100ml toluene and the filtrate and filtrate was washed with water (2 ×100 ml). The toluene layer was dried over MgSO4 or Na2SO4 and concentrated to a solid that was triturated with hexane (25 ml). The solid was separated by filtration. Yield = 86% General procedure for the synthesis of Schiff bases of benzothiazole (5a-5c):  6-carboxy-2-(P-aminophenyl) benzothiazole 4a (0.025 moles) was dissolved in 20 ml ethanol, followed by drop wise addition of substituted aromatic aldehyde (0.030 moles) dissolved in 10ml ethanol at room temperature. The reaction mixture was stirred for 24 hr at room temperature. Then, ethanol was evaporated and product was recrystallized from ethyl acetate: hexane mixture. General Procedure for the synthesis of corresponding esters of benzothiazoles (P5a-P5h): To the corresponding compound (5a-5c) (5.58 mmoles) added corresponding alcohol (27.9 mmoles). Followed by the addition of thionyl chloride (6.69 mmoles). Then reaction mixture was refluxed for 5 hrs. The excess of thionyl chloride and the alcohol was removed under reduced pressure. The product washed with sufficient cold Petroleum ether, dried and weighted. Table I indicates yield and M.P. of various corresponding esters synthesized Spectral data for various synthesized compounds  Methyl 2-(4-(3-fluorobenzylideneamino) phenyl) benzo [d] thiazole-6-carboxylate (5a): IR (neat) cm-1: 1742(C=O), 1630(C=N), 1714(C=C); 1HNMR (CDCl3): δ 3.9 (s, 3H, -CH3), δ 6.9-8.7 (m, 12H, aromatic); 13CNMR δ 55.06, 119.7, 120.0, 120.1, 124.2, 127.6, 127.7, 130.3,  131.4, 134.0, 136.4, 141.1 , 141.7, 141.8, 157.2, 164.1, 165.4, 168, 169.8. HR-ESIMS: Calculated for C22H15FN2O2S (390.43010): found 390.42020.  Padmavathi et al. /Research in Pharmacy 1(2) : 6-12, 2011  9  OOSNNRR'= -CH3, -CH2CH3, -CH2C6H5 etcSr No. -RCode % Yield M.P.1 m-FPhysical naturelight yellow crystals 722 light Yellow crystals 753 Light yellow crystals 764 yellow crystals 715 yellow crystals 726 757 colourless crystals 74Table I270-272280-282290-292261-264228-231272-274246-248OOHSNNR R'-OHSOCl2R'-R'-CH2C6H5-C2H5-CH3p-NO2 -CH3-C2H5-CH2C6H5colourless crystals-C2H58yellow crystals76 249-251-CH2C6H5R= -F,-NO2, -OCH3,P5aP5bP5cP5dP5eP5fP5gP5hp-OCH3m-Fm-Fp-NO2p-NO2p-OCH3  Ethyl-2-(4-(3-fluorobenzylideneamino) phenyl) benzo[d] thiazole-6-carboxylate (5b):IR (neat) cm-1: 1746(C=O), 1634(C=N), 1710(C=C) HNMR (CDCl3): δ 1.37-1.40 (t, 3H, -CH3, J=5.6), δ 4.3-4.36 (q, 2H, -CH2, J=5.5), δ 7.0-9.0 (m, 12H, aromatic; 13CNMR δ 14.8, 61.4, 116.9, 117.1, 118.1, 118.9, 121.4, 124.7, 124.8, 127.9, 127.7, 128.5, 129.9, 130.0,  130.1, 133.5, 138.4, 139.0, 154.3, 161.2, 162.5, 165.1, 166.7. HR-ESIMS: Calculated for C23H17FN2O2S (404.45668): found 404.44433.   Benzyl 2-(4-(3-fluorobenzylideneamino) phenyl)benzo[d]thiazole-6-carboxylate (5c):IR (neat) cm-1: 1736(C=O), 1710(C=C), 1622(C=N); 1HNMR (CDCl3): δ 5.4 (s, 2H, -CH2), δ 7.0-8.8 (m, 17H, aromatic); 13CNMR δ 66.8, 117.9, 118.5, 118.7, 121.3, 124.6, 127.8, 128.1, 128.3, 130.0, 133.4, 137.0, 138.2, 138.7, 154.1, 161.4, 162.4, 165.0, 166.9. HR-ESIMS: Calculated for C28H19FN2O2S (466.52606): found 466.51334.  Methyl 2-(4-(4-nitrobenzylideneamino) phenyl)benzo[d]thiazole-6-carboxylate (5d):IR (neat) cm-1: 1754(C=O), 1710(C=C), 1622(C=N); 1HNMR (CDCl3): δ 3.9 (s, 3H, -CH3), δ 7.4-8.8 (m, 12H, aromatic); 13CNMR δ 124.6, 128.7, 132.0, 132.2, 134.7, 135.9, 138.5, 138.9, 140.9, 145.9, 147.4, 159.2, 161.7, 168.6, 169.8, 170.5, 174.3, 187.5. HR-ESIMS: Calculated for C22H15N3O4S (417.43720): found 417.42620.  Ethyl 2-(4-(4-nitrobenzylideneamino) phenyl)benzo[d]thiazole-6-carboxylate (5e):IR (neat) cm-1: 1742(C=O), 1710(C=C), 1620(C=N); 1HNMR (CDCl3): δ 1.36-1.39 (t, 3H, -CH3, J=5.7), δ 4.31-4.35 (q, 2H, -CH2, J=5.6), δ 7.4-8.8 (m, 12H, aromatic); 13CNMR δ 14.4, 61.1, 117.8, 121.1, 124.4, 124.4, 127.6, 128.2, 129.8, 129.8, 131.3, 133.8, 139.8, 151.5, 154.0, 161.0, 162.2, 162.9, 166.4. HR-ESIMS: Calculated for C23H17N3O4S (431.46378): found 431.45541.  Benzyl 2-(4-(4-nitrobenzylideneamino) phenyl)benzo[d]thiazole-6-carboxylate (5f):IR (neat) cm-1: 1741(C=O), 1710(C=C), 626(C=N); 1HNMR (CDCl3): δ 5.4 (s, 2H, -CH2), δ 7.1-8.8 (m, 17H, aromatic); 13CNMR δ 61.1, 117.2, 120.5, 123.8, 127.0, 127.4, 127.5,  Padmavathi et al. /Research in Pharmacy 1(2) : 6-12, 2011  10  127.6, 129.2, 130.7, 1323.6, 136.3, 137.5, 139.2, 150.9, 153.4, 160.4, 161.6, 162.2, 166.1. HR-ESIMS: Calculated for C28H19N3O4S (493.53316): found 493.52206.  Ethyl 2-(4-(4-methoxy benzylidene amino) phenyl) benzo[d] thiazole-6-carboxylate (5g): IR (neat) cm-1: 1745(C=O), 1623(C=N), 1710(C=C); 1HNMR (CDCl3): δ 1.37-1.40 (t, 3H, -CH3, J=5.6), δ 3.8 (s, 3H, -OCH3), δ 4.32-4.36 (q, 2H, -CH2, J=5.5), δ 6.9-8.9 (m, 12H, aromatic); 13CNMR δ 13.5, 54.9, 60.1, 113.8, 116.8, 120.2, 123.5, 126.7, 127.2, 127.3, 128.9, 131.2, 132.1, 137.1, 153.0, 160.0, 161.3, 161.9, 162.9, 165.5. HR-ESIMS: Calculated for C24H20N2O3S (416.49220): found 416.48210.  Benzyl 2-(4-(4-methoxybenzylideneamino) phenyl)benzo[d] thiazole-6-carboxylate (P5h): IR (neat) cm-1: 1741(C=O), 1622(C=N), 1710(C=C) 1HNMR (CDCl3): δ 3.80 (s, 3H, -OCH3,), δ 5.4 (s, 2H, -CH2), δ 6.8-8.9 (m, 17H, aromatic).13CNMR δ 40.5, 51.3, 98.9, 102.4, 105.8, 109.1, 112.3, 112.6, 112.8, 112.9, 114.5, 116.8, 117.9, 121.5, 122.7, 145.6, 146.9, 147.5, 148.5, 151.4. HR-ESIMS: Calculated for C29H22N2O3S (478.56158): found 478.55238.  Biological evaluation In vitro antimicrobial study Benzothiazoles show a wide spectrum of chemotherapeutic activity and a considerable amount of work has been done on the synthesis of new potent antibacterial and antifungal benzothiazoles. Synthesized compounds were screened for their in-vitro antibacterial activity against P.aeruginosa, E.coli, S.aureus, B.subtilis at 100 µg/ml and in-vitro antifungal activity against Candida albicans and Aspergillus Niger activities at 100 µg/ml concentration. Standard antibacterial ampicillin and standard antifungal ketoconazole were also screened under similar conditions for comparison. DMSO was used as a solvent control. The culture media was nutrient agar and method employed was cup plate method (Cruichshank, 1975). All the compounds showed varying degree of antibacterial activity. All the compounds were far less active than the standard drug taken.   Results and discussion  Amongst the synthesized compounds several compounds exhibit antifungal and anti-bacterial activity. Compounds P5d, P5e, P5f, P5g have shown significant antibacterial activity Compound P5e& P5f was most significant We were pleased to observe significant activity of compound P5e, P5f &P5g against Candida albicans and Aspergillus niger. While other compound shown less significant activity against bacteria and fungi. The antimicrobial activity and antifungal activity studies have shown in table II and table III respectively. Table II: Results of anti-bacterial activity Sl.No code Diameter of zone of inhibition ( in mm)   P.aeruginosa E.coli S.aureus B.subtilis 01 P5a 15 13 14 16 02 P5b 10 10 13 12 03 P5c 12 10 12 13 04 P5d 16 16 15 16 05 P5e 18 16 17 16 06 P5f 17 16 18 16 07 P5g 16 15 16 16 08 P5h 15 12 11 14 09 Ampicillin 21 20 22 20 10 DMSO - - - -   Padmavathi et al. /Research in Pharmacy 1(2) : 6-12, 2011  11  Table III: Results of Anti-Fungal Activity   Conclusion Various benzothiazole derivatives having 4-(benzylideneamino) phenyl substituents at position 2 and different carboxylate substituents at position 6 were synthesized with a view of enhancing the biological activity. The structure of newly synthesized compounds was confirmed by IR, 1H NMR, Mass spectra. Further evaluation of antimicrobial activity was carried out. The synthesis of various 2-(4-(benzylideneamino) phenyl) benzothiazole-6-carboxylate derivatives by the described method resulted in products with good yield. Micro b2iological evaluation of the synthesized compounds showed good to moderate activity.   Acknowledgement   The authors are thankful to the authorities of A. ShamaRao Foundation Mangalore, Karnataka, India, Nitte Education Trust Mangalore, and Karnataka, India for providing necessary facilities and Indian Institute of Sciences, Bangalore for carrying out IR, NMR and mass spectra.   References  Alaimo RJ and Pelosi SS. 1974. Antiparasitic thiocyanatobenzothiazoles. J. Med. Chem. 17: 775-776.  Bhusari KP.  2000. Synthesis and antitubercular activity of some substituted 2-(4-aminophenylsulphon amido) benzothiazoles. Indian. Heterocycl. 9: 213-216.  Bradshaw TD, Chua MS, Browne HL, Trapani V, Sausville EA and Stevens MF. 2002. In vitro evaluation of amino acid prodrugs of novel antitumour 2-(4-amino-3-methyl phenyl) benzothia-zoles.  Brit. J. Cancer. 86: 1348. Caleta I, Rdisa GM, Mrvos-Sermek D, Cetina M, Tralic-Kulenovic V, Pavelic K and Karminski-Zamola. 2004. 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Design, synthesis, characterization and biological evaluation of Benzothiazole-6-carboxylate derivatives

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Design, synthesis, characterization and biological evaluation of Benzothiazole-6-carboxylate derivatives

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