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Letters in Drug Design & Discovery, 2009, 6, 502-507
Microwave-Assisted Solvent-Free Synthesis of N-alkyl Benzotriazole Derivatives: Antimicrobial Studies Raghu Ningegowda1, C.V. Kavitha2, B.S. Priya2, S.L. Gaonkar2, M.V. Tejesvi3, K.S. Rangappa*,2 and S. Nanjunda Swamy*,3 1
Syngene International Ltd, Biocon Park, Bangalore 560 099, Karnataka, India
2
Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore-570006, Karnataka, India
3
Department of Studies in Biotechnology, University of Mysore, Manasagangotri, Mysore-570 006, Karnataka, India
4
Department of Biotechnology, Sri Jayachamarajendra College of Engineering, Mysore-570006, Karnataka, India Received March 19, 2009: Revised June 02, 2009: Accepted June 03, 2009
Abstract: To elucidate further our structural activity relation on the chemistry and antimicrobial activity, a series of novel N-alkylated benzotriazole derivatives bearing pharmaceutically important substituted biphenyl and benzyl halides were synthesized. The synthesized compounds were characterized and tested for in vitro antimicrobial activities by MIC determination against a panel of susceptible and resistant Gram-positive and Gram-negative microorganisms. The synthesis has been carried out using microwave irradiation method with solvent and without solvent. The obtained results showed high yields. Interestingly, compounds 2a and 2b showed a two fold increased antibacterial activity than the standard drug tested and the compounds 2d and 2e showed potent antifungal activity than the standard drug tested.
Keywords: Benzotriazole, N-alkylation, Solvent-free, Microwave irradiation, Antimicrobials. INTRODUCTION Because of the increasing bacterial resistance to antibiotics, the discovery and development of novel antimicrobials is one of the most important fields of research today. Derivatization of heterocyclic pharmacophores represents a versatile approach to generate chemical diversity for lead identification and optimization. Benzotriazoles have found wide applications in organic synthesis, as dyestuffs and fluorescent compounds, corrosion inhibitors, photo stabilizers and agrochemicals [1]. Benzotriazole is a good leaving group and it has been widely used as synthetic auxiliary in organic synthesis [2]. The synthesis of furthermore functionalized benzotriazoles is considered to become important subjects of synthetic and biological researches since they are expected to exhibit novel properties applicable to such fields. On the other hand, benzotriazole derivatives have shown potential antimycobacterial [4], antitubercular [5], antitumor [6], antiproliferative [7, 8], anesthetic [9], and anti-inflammatory activities [10, 11]. Microwave heating has emerged as a powerful technique to promote a variety of chemical reactions [12-16]. Microwave reactions under solvent-free conditions are attractive in offering reduced pollution and offer low cost together with simplicity in processing and handling [17-20]. Nalkylation of aromatic compounds involving nitrogen heterocycles are important reactions in organic synthesis. Our previous report on the synthesis and antimicrobial activities of *Address correspondence to these authors at the Department of Biotechnology, Sri Jayachamarajendra College of Engineering, Mysore-570006, Karnataka, India; Tel: +91-821-2515770; Mob: +91-9844646879; Fax: +91821-2548290; E-mail:
[email protected] Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore-570006, Karnataka, India; Tel: +91-821-2419661; Fax: +91821-2412191; E-mail:
[email protected] 1570-1808/09 $55.00+.00
N-alkylated benzotriazole unraveled that N-substituted groups attributed their role in enhancing the activity [21]. Inspired by these results, in the present investigation we designed and synthesized a novel benzotriazole derivatives having heteroaromatic moieties. The focus of the investigation was to probe the optimal structural requirement of these compounds with regard to antimicrobial activities and further elucidate the SAR’s. We report here the synthesis of Nalkylated benzotriazole and their antimicrobial activity. MATERIALS AND METHODS Melting points were recorded on a SEALCO-605 melting point apparatus and were uncorrected. IR spectra were recorded on a Jasco FT-IR 8000 spectrometer. Spectra were recorded on Bruker AMX-400 MHz using CDCl3 as solvent with TMS as internal standard. Elemental analysis was obtained on a Vario-EL instrument. Silica gel GF-254 was used for thin layer chromatography. All of the reagents and chemicals were purchased from Sigma Aldrich Chemicals Pvt Ltd and used without further purifications. EXPERIMENTAL General Procedure for the Synthesis of N-Alkylated Benzotriazole Derivatives Microwave with Solvent The compound benzotriazole 1 and aralkyl halides were dissolved in N,N-dimethylformamide. The solution was introduced into microwave oven and irradiated for 40-50 s at 60% power. After completion of the reaction, the dark red mass was poured on ice-cold water. The product was extracted in ethyl acetate (6 volumes x 3), and the combined © 2009 Bentham Science Publishers Ltd.
Microwave-Assisted Solvent-Free Synthesis of N-alkyl Benzotriazole
Table 1.
Letters in Drug Design & Discovery, 2009, Vol. 6, No. 7
503
Reaction Condition and Physical Data of Bioactive Benzotriazole Derivatives
Compound
Time taken for the reaction MW irr (s)
R
With solvent
Without solvent
40
30
40
2a
Yield Eluent x
Mp (C) With solvent
Without solvent
9:1
88
97
110-112
20
9:1
86
94
90-92
50
30
9:1
85
90
98-99
50
30
9:1
86
94
102-104
40
20
9:1
88
95
89-90
O O
2b O O
N
N 2c N
O N
2d O
2e
Br X=Benzene: ethyl acetate.
organic layer was washed with water, dried, and distilled completely. The resultant oil was crystallized from ethanoln-hexane (1:1) mixture to give pure product 2 (a-e) (Table 1). Microwave without Solvent A conical flask charged with grounded equimolar mixtures of benzotriazole and aralkyl halides is kept at 20-30 s in a microwave oven at 60% power. After completion of the reaction, which was monitored by TLC, the residue was
poured into ice-cold water and worked up as described earlier. Compound 2a: 4 -benzotriazol-1-yl-methyl-biphenyl-2carboxylic acid tert-butyl ester was obtained by using benzotriazole (1g, 8.39 mmol), and 4 -bromomethyl-biphenyl-2carboxylic acid tert-butyl ester (2.91g). IR max (KBr): 3091, 2912, 2899, 1749, 1410, 1329, 1159 cm-1. 1H NMR (CDCl3, 400MHz, ): 1.04 (s, 9H, -C(CH3)3), 6.1 (s, 2H, -CH2), 7.2-
504 Letters in Drug Design & Discovery, 2009, Vol. 6, No. 7
Ningegowda et al.
8.1 (m, 12 H, Ar-H). Anal. Calcd for C24H23N3O2: C, 74.78; H, 6.01; N, 10.90; Found: C, 74.76; H, 6.00; N, 10.91. Compound 2b: 4 -benzotriazol-1-yl-methyl-biphenyl-2carboxylic acid methyl ester was obtained by using benzotriazole (1g, 8.39 mmol) and 4 -bromomethyl-biphenyl-2carboxylic acid methyl ester(2.56g). IR max (KBr): 3091, 2902, 2899, 1736, 1412, 1235, 1192 cm-1. 1H NMR (CDCl3, 400MHz, ): 3.42 (s, 3H, -OCH3), 5.9 (s, 2H, -CH2), 7.528.12 (m, 12H, Ar-H). Anal. Calcd for C21H17N3O2: C, 73.45; H, 4.99; N, 12.24; Found: C, 73.46; H, 5.00; N, 12.22. Compound 2c: 1-[2 -(1-trityl-1H-tetrazol-5-yl)-biphenyl4-yl-methyl]-1H- benzotriazole was obtained by using benzotriazole (1g, 8.39 mmol), and 5-(4 -bromomethylbiphenyl-2-yl)-1-trityl-1H-tetrazole (4.67g). IR max (KBr): 3032, 2961, 1621, 1414, 1309 cm-1. 1H NMR (CDCl3, 400MHz, ): 5.8 (s, 2H, -CH2), 6.9-8.1 (m, 29H, Ar-H). Anal. Calcd for C39H29N7: C, 78.63; H, 4.91; N, 16.46; Found: C, 78.64; H, 4.90; N, 16.48. Compound 2d: 2-(3-benzotriazol-1-yl-propyl)-isoindole1,3-dione was obtained by using benzotriazole (1g, 8.39 mmol), and 2-(2-Chloro-ethyl)-isoindole-1,3-dione (1.24g). IR max (KBr): 3021, 2890, 1691, 1400, 1312 cm-1. 1H NMR (CDCl3, 400MHz, ): 4.9 (t, 2H, -N-CH2), 3.9 (t, 2H, -NCH2), 2.8 (quintet, 2H, -CH2-). Anal. Calcd for C17H14N4O2: C, 66.66; H, 4.61; N, 18.29; Found: C, 66.67; H, 4.65; N, 18.30. Compound 2e: 1-(4-bromo-benzyl)-1H-benzotriazole was obtained by using benzotriazole (1g, 8.39 mmol), and 1bromo-4-bromomethyl-benzene (2.10g). IR max (KBr): 3020, 2902, 1410, 1310 cm-1. 1H NMR (CDCl3, 400MHz, ): 5.3 (s, 2H, -CH2), 7.02 (d, 2H, Ar-H)7.35 (d, 2H, Ar-H), 7.6-8.0 (m, 4H, Ar-H). Anal. Calcd for C13H10N3Br: C, 54.19; H, 3.50; N, 14.58; Found: C, 54.17; H, 3.52; N, 14.60. Microbiology: In Vitro Evaluation of Antimicrobial Activity Bacteria and fungal species used in this study were obtained from the Department of Studies in Biotechnology, N N
University of Mysore, India, namely, Bacillus substilis, Escherichia coli, Pseudomonas fluorescens, Xanthomonas campestris pvs, Xanthomonas oryzae, Aspergillus niger, Aspergillus flavus, Fusarium oxysporum, Trichoderma species, and Fusarium monaliforme. The bacterial strains were maintained on LB agar medium and the filamentous fungi were maintained on potato dextrose agar (PDA) medium at 28 C. The disk diffusion method [22] was used to determine antibacterial and antifungal activity of synthesized compounds. Paper discs with DMSO were used as negative controls. The bacteria were grown in LB broth overnight, centrifuged at 5,000 rpm for 5 mins, pellet was suspended in double distilled water and was used to inoculate the plates. The filamentous fungal inoculum was prepared with the spores derived from 5 to 15 days culture on PDA medium. The mycelia were covered with 10ml of distilled water and the conidia were scraped using a sterile pipette. The spores were recovered after filtration on sterile absorbent cotton and were resuspended in sterile distilled water. The cell density of each inoculum was adjusted with hemocytometer in order to obtain a final concentration of approximately 10 4 CFU/ml and 106 spores/ml for the bacteria and filamentous fungi, respectively. Nystatin (Himedia) was used as a positive control for fungi, and streptomycin and tetracycline for bacteria. Each disk contained 10 g of standard drugs and 25 g synthesized compounds. Plates were first kept at 4°C for at least 2 hours to allow the diffusion of chemicals and then incubated at 28°C. Inhibition zones were measured after 24 hours of incubation for bacteria and after 48 hours of incubation for fungi. RESULTS AND DISCUSSION Chemistry The synthesis of the title compounds was carried out by N-alkylation of benzotriazole with different bioactive aralkyl halides, such as 4 -bromomethyl-biphenyl-2-carboxylic acid tert-butyl ester 2a, 4 -bromomethyl-biphenyl-2-carboxylic acid methyl ester 2b, 5-(4 -bromomethyl-biphenyl-2-yl)-1trityl-1H-tetrazole 2c, 2-(2-Chloro-ethyl)-isoindole-1,3-dione 2d and 1-bromo-4-bromomethyl-benzene 2e, under microN
R-X
N
N
Solvent-free
H
MW.Irr
N R 2 (a-e)
1 Br
Br
Br N
N
Br
O
N N O O
Where R-X =
2a
N
O O
2b
O
2c
Scheme 1. Schematic representation of synthesis of benzotriazole derivatives 2(a-e).
2d
Cl
Br 2e
Microwave-Assisted Solvent-Free Synthesis of N-alkyl Benzotriazole
Letters in Drug Design & Discovery, 2009, Vol. 6, No. 7
wave irradiation with solvent and without solvent as shown in Scheme 1. In comparison to our earlier conventional (thermal) heating method, microwave irradiation with solvent, solvent-free microwave heating offers more advantages, such as reduced reaction time (20-30s), low cost, simplicity in processing, reduced pollution, and high yield. The results are depicted in Table 1. The N-alkylated benzotriazole derivatives 2(a-e) were characterized by IR, 1H NMR, and C H N analysis. The 1H NMR spectrum of all the synthesized compounds 2 (a-e) showed the peak at 4.9-6.1ppm due to N-CH2 group indicates the formation of the product and the peak at 11ppm due to –NH group of benzotriazole was disappeared. The formation of the product was further confirmed by correct elemental analyses. Biology In view of synthesizing new antimicrobials, newer benzotriazole derivatives 2(a-e) have been synthesized and evaluated their efficacy as antimicrobials in vitro by the disk diffusion method [12,14] against different strains. Tests were performed in triplicate and the results are reported as means of at least three determinations. The results showed that all the compounds were effective compared to standard drugs against the bacterial strains Table 2.
Minimal Inhibitory Concentration (MIC) in Method
a
tested as shown in Tables 2 and 3. Antibacterial activities shown by the compounds were in the following order 2b 2a 2c 2d 2e. Compounds 2b and 2a bearing biphenyl carboxylic methyl ester and biphenyl carboxylic acid tertbutyl ester groups, respectively, showed a two fold increased inhibitory activity than the standard drug tested. Difference in the inhibitory activity can be attributed for the presence of biphenyl having tetrazole moiety with three phenyl groups and isoindole 1,3 dione moiety in compounds 2c and 2d, respectively. Compound 2e bearing 4-bromo methyl phenyl exhibited similar potency as that of standard. Antifungal activities shown by the compounds were as shown in Table 4 and Table 5. Antifungal activities shown were in the following order 2e2d2a2b. The compounds 2e, 2d, 2a and 2b showed more potency than the standard nystatin at tested concentration. Compounds 2c did not show any significant inhibitory activity against any of the fungal strains tested. CONCLUSION In summary, we have synthesized novel derivatives of Nalkylated benzotriazoles bearing pharmaceutically important bioactive substituents 2(a-e) under both conventional and microwave irradiation technique. It is thus concluded that
g/ml of Compounds Against Tested Bacterial Strains by Microdilution
Minimal inhibitory concentration (MIC) in g/ml a
Compound Bacillus-substilis
Escherichia – -coli
Pseudomonas -fluorescens
Xanthomonas -campestris pvs.
Xanthomonas -oryzae
2a
90.35
100.42
90.36
80.32
120.53
2b
80.31
90.36
70.29
60.28
110.49
2c
100.42
120.53
100.42
90.41
130.50
2d
150.66
140.64
130.55
100.43
150.67
2e
240.96
190.89
180.86
120.53
190.89
Streptomycin
250.98
190.89
170.84
-
-
Tetracycline
-
-
-
130.56
200.94
Values are means of three determinations, the ranges of which are less than 5% of the mean in all cases.
Table 3.
Inhibitory Zone (Diameter) mm of Compounds Against Tested Bacterial Strains by Disk Diffusion Method Inhibitory Zone (diameter) mm a
Compound
505
Bacillus-substilis
Escherichia -coli
Pseudomonas -fluorescens
Xanthomonas -campestris pvs.
Xanthomonas -oryzae
2a
260.99
371.55
381.56
331.25
291.10
2b
281.0
391.62
411.76
351.34
311.05
2c
24.0.89
321.0
351.35
301.42
271.02
2d
210.84
29 1.0
311.05
271.11
240.98 160.45
2e
140.59
190.75
220.83
180.65
Streptomycin
14 0.60
200.76
210.81
-
-
Tetracycline
-
-
-
170.58
160.49
Streptomycin sulphate (10 g/disc); Tetracycline (10 g/disc) were used as positive reference and Compounds (25 g/disc). a Values are means of three determinations, the ranges of which are less than 5% of the mean in all cases.
506 Letters in Drug Design & Discovery, 2009, Vol. 6, No. 7
Table 4.
Ningegowda et al.
Minimal Inhibitory Concentration (MIC) in M of Compounds Against Tested Fungal Strains by Turbidometric Method Inhibitory Zone (diameter) mm a
Compound
a
Aspergillus-niger
Aspergillus – -flavus
Fusarium -oxysporum
Trichoderma-species
Fusarium-monaliforme
2a
140.45
160.57
130.38
150.55
170.57
2b
160.56
180.65
150.58
170.58
190.67
2c
411.76
481.96
461.94
391.56
401.86
2d
120.45
140.45
150.46
140.47
110.35
2e
90.23
100.25
110.27
100.24
120.38
Nystatin
311.21
341.34
381.55
301.02
321.26
Values are means of three determinations, the ranges of which are less than 5% of the mean in all cases.
Table 5.
Inhibitory Zone (Diameter) mm of Compounds Against Tested Fungal Strains by Disk Diffusion Method Inhibitory Zone (diameter) mm a
Compound Aspergillus-niger
Aspergillus – -flavus
Fusarium -oxysporum
Trichoderma-species
Fusarium-monaliforme
2a
220.87
230.86
260.91
270.95
220.72
2b
200.79
210.78
240.85
250.87
190.63
2c
50.11
70.17
40.10
60.13
80.16
2d
240.89
260.94
280.93
301.24
250.87
2e
270.92
301.18
321.17
371.75
341.46
Nystatin
140.45
160.56
180.68
200.75
190.57
Nystatin (10 g/disc) was used as positive reference and compounds (25 g/disc). a Values are means of three determinations, the ranges of which are less than 5% of the mean in all cases.
under microwave heating in the absence of solvent, the products 2(a-e) were conveniently and efficiently prepared in good yields, typically in the range of 80-85%. The pharmacological evaluation indicated that the activity of the benzotriazole ring with biphenyl moiety having different substituents may serve as a new class of antimicrobials and found to be non-strain dependent. Further, the research on modification of the title compounds to understand the structure activity relationship and the mechanism of inhibition is underway ACKNOWLEDGEMENTS We thank NMR Research Center, IISC, Bangalore for the NMR spectral analysis. One of the authors SNS thanks JSS Mahavidyapeetha and Dr. B.G. Sangameshwara Principal, SJCE, Mysore for constant encouragement and support. REFERENCES [1] [2] [3] [4]
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