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Synthesis and evaluation of antimicrobial activity of novel 1,3,4-oxadiazole derivatives.

Introduction

The earliest evidence of successful chemotherapy is from ancient Peru, where the Indians used bark from the cinchona tree to treat malaria. Modern chemotherapy has been dated to the work of Paul Ehrlich in Germany, who sought systematically to discover effective agents to treat trypanosomiasis and syphilis. Ehrlich postulated that it would be possible to find chemicals that were selectively toxic for parasites but not toxic to humans. Progress in the development of novel antibacterial agents has been great, but the development of effective, nontoxic antifungal and antiviral agents has been slow. Amphotericin B, isolated in the 1950s, remains an effective antifungal agent, although newer agents such as Fluconazole are now widely used. An antimicrobial is a substance that kills or inhibits the growth of microbes such as bacteria (antibacterial activity), fungi (antifungal activity) and viruses (antiviral activity). Any attempt to discuss the chemotherapeutic properties of heterocyclic compounds must, of necessity, be confined to a limited aspect of the subject. Therefore, the present discussion will be limited to monocyclic compounds with 5-membered ring. By definition, this includes not only compounds with a single 5-membered ring but also substances with two or more rings, one of which must be six membered. The polyene antibiotics, which apparently act by binding to membrane sterols, contain a rigid hydrophobic center and a flexible hydrophilic section. Structurally, polyenes are tightly packed rod sheld in rigid extension by the polyene portion. They interact with fungal cells to produce a membrane-polyene complex that alters the membrane permeability, resulting in internal acidification of the fungus with exchange of [K.sup.+] and sugars;

loss of phosphate esters, organic acids, nucleotides; and eventual leakage of cell protein. In effect, the polyene makes a pore in the fungal membrane and the contents of the fungus leak out. Although numerous polyene antibiotics have been isolated, only amphotericin B is used systemically. Nystatin is used as a topical agent and primaricin as an ophthalmic preparation. A number of other agents interfere with the synthesis of fungal lipid membranes. These agents belong to a class of compounds referred to as imidazoles: miconazole, ketoconazole, clotrimazole, and fluconazole. These compounds inhibit the incorporation of subunits into ergosterol and may also directly damage the membrane. The development of antifungal agents has lagged behind that of antibacterial agents. This is a predictable consequence of the cellular structure of the organisms involved. This difficulty complicates experiments designed to evaluate the in vitro or in vivo properties of a potential antifungal agent [1-5]. Tetrazole derivatives possess broad spectrum of biological activity in both medicinal and pharmaceutical such as antibacterial, antifungal, antiviral, analgesic anti-inflammatory, antiulcer [6-13]. 1,3,4-Oxadiazoles show various biological activities and have been synthesized from different compounds. Many reaction schemes were followed for the synthesis of the ring and 1, 3,4-oxadiazole ring showed diversity in biological activity. 1,3,4-Oxadiazole is popularly known for its antimicrobial, anti-inflammatory, pesticidal, antihypertensive activities etc [14-22]. It is well known that the synthesis of heterocyclic compounds tends to contain multi-structure in a molecule. The ring formation involves the condensation reaction. In each step, a water molecule is formed.

The challenge is to develop the ring system by incorporating the tetrazole nucleus into it through the proposed reaction scheme. In this study, it was planned to incorporate the oxadiazole ring system into tetrazole ring, as it has not been reported earlier. Synthesis of derivatives of 1,3,4-oxadiazoles from different benzaldehydes and Schiff bases. Characterization of the synthesized compounds along with their antimicrobial activity on different strains of bacteria and fungi has been performed.

Material and Methods

General

All chemicals and solvents were purchased from Qualigens and were of AR-grade purity. All reactions are carried out at laboratory condition. Melting points were determined with open capillary and were uncorrected. FT-IR spectra were recorded on a Shimadzu FT-IR model 8010 spectrophotometer, [sup.1]H NMR spectra were recorded in DMSO on a Varian Mercury FT-NMR model YH-300 instrument using TMS as internal standard. Mass spectra were recorded on GC-MS auto tune EI instrument.

General procedure for synthesis of compound 1: A mixture of phenyl tetrazole 5 g (0.03 mol) in methanol is prepared, stirred well to dissolve compound 1.To this add 3.67 mL (0.03 mol) of ethyl chloroacetate drop wise with continuous stirring to get clear solution. Reflux the reaction mixture on water bath for about 2 hours. A solid residue was obtained by cooling at room temperature. The product was filtered, dried, recrystalised from warm ethanol.

General procedure for synthesis of compound 2: Ethyl (5-phenyl-1H-tetrazol-1-yl) acetate 9 g (0.03 mol) was condensed with 1.95 mL (0.03 mol) 99% hydrazine hydrate. Reflux the reaction mixture on water bath for about 5 hours. The solid residue of acetohydrazide was obtained by cooling. The product was filtered, dried, recrystalised from warm ethanol.

General procedure the synthesis of Schiff's bases (3a-h) [23]: 2-(5-phenyl-1H-tetrazol-1-yl) acetohydrazide (1) 2 g (0.009 mol) was refluxed with various aromatic aldehydes (0.009 mol) in the presence of sulphuric acid for 6 h. The reaction mixture was then poured into the crushed ice. The resultant solid was washed with distilled water, dried and recrystalised from ethanol.

General procedure for synthesis of 1,3,4-oxadiazole (4a-h): A mixture of compounds Schiff bases 3a-h (0.01 mol) and acetic anhydride (5 mL) was refluxed for 2 h. The mixture was cooled, poured onto crushed ice and allowed to stand at room temperature overnight. The separated solid was washed with water, dried and recrystalised from ethanol.

Biological evaluation

Antimicrobial activity by minimum inhibitory concentration (MIC) method

The minimum inhibitory concentration (MIC) of the test substances against Staphylococcus aureus, Escheshia coli, Candida albicans and Aspergillus niger was determined by liquid broth method of two fold serial dilution technique [24-25]. In this assay, the minimum concentration of each test substance required to inhibit the growth of microorganism was determined.

For this assay, a series of assay tubes were prepared containing uniform volume (1 mL) of sterile SD broth and equal volume of known concentration of test substance was added. The test substance in the first tube was serially diluted in twofold decreasing concentrations through the sixth tube and seventh tube was left without test substance as positive control. The tubes with the test substance i.e. from one to seventh were inoculated with 1 mL of inoculums (1 x [10.sup.6] CFU per mL). The final concentration of test substance ranged from 1000 to 31.25 [micro]g/mL Solvent control and sterility controls were maintained in the experiment. The tubes were incubated at 28[degrees]C for 48 h. Standard antibiotic, ampicillin and fluconazole were tested as standard drug at concentrations ranging from 100 to 3.12 [micro]g/mL.

The tubes were inspected visually to determine the growth of the organism as indicated by turbidity (In fact, turbidity of the culture medium is indicative of the presence of a large number of cells), the tubes in which the antibiotic is present in concentration sufficient to inhibit fungal growth remain clear. In experimental terms the MIC is the concentration of the drug present in the last clear tube, i.e. in the tube having the lowest concentration in which growth is not observed.

Results and Discussion

Herein we have described the synthesis (Scheme 1), characterization and biological evaluation of some 1,3,4-oxadiazole containing 5-phenyltetrazole. All compounds were analyzed satisfactorily by CHN elemental analysis. All the IR and NMR spectral characteristics of different 1,3,4-oxadiazole are in good agreement with proposed structure and are shown in experimental section. The IR spectra of compounds 4a-h shows absorption bands at 3057 due to (Ar-H str.), 1656 due to C=N ring stretch and 2376, 2247 due -NCH2. Similarly absorption also occurs at 1285(N-N=N-), 1108 and 1138 (tetrazole ring). The 1H NMR spectra shows chemical shift at 7.10-8.10 due to aromatic protons, 6.30 (s, [sup.1]H CH of 1,3,4-oxadiazole), 3.98 (d, 2H CH2), 2.15(s, 3H,CH3).The results of spectral data are in good agreement with the structure of synthesized compounds. The physicochemical and spectral data of the compounds 4a-h are described in tables 1 and 2.

The antimicrobial activity of all the synthesized compounds is shown in Table 3. All the compounds exhibited significant antibacterial and antifungal activities. Good antibacterial activity was observed in 4d, 4b, 4g against S. aureus. Whereas compounds 4f, 4g, 4d and 4e showed noticeable activity against E. coli. Compound 4d, 4g, 4f showed marked activity against A. niger and C. albicans. The compound 4f bearing methoxy group shows MIC at 62.5 [micro]g/mL against E. coli and C. albicans and 125 [micro]g/mL against S. aureus and A. niger respectively.

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Conclusion

Conclusively, a variety of 1,3,4-oxadiazole derivatives containing 5phenyltetrazole have been successfully synthesized in appreciable yields by simple synthetic route from 5-phenyltetrazole and screened in vitro for their antimicrobial activities against both strains of Gram-positive and Gram-negative bacteria. Most of the compounds found to possess good antimicrobial activity at minimal inhibitory concentration.

Form the above evidence; it is clear that 1,3,4-oxadiazole derivatives can be used to discover bioactive synthetic products that may serve as leads for the development of new pharmaceuticals that address hither to unmet therapeutic needs. It is hoped that this study would lead to the establishment of some compounds that could be used to formulate new and more potent antimicrobial drugs of synthetic origin.

Acknowledgments

The authors are thankful to University of Pune, India, for providing spectral data, and Principal, MES College of Pharmacy, Sonai, for providing necessary facilities.

References and Notes

[1] Arthur, M.; Courvalin, P. Antimicrob Agents Chemother. 1993, 37, 1563.

[2] Murray, B. J. Infect. Dis. 1991, 163, 1185.

[3] Gale, E. F.; Cundliffe, E.; Reynolds, P. E.; Richmond, M. H.; Waring, M. J. The Molecular Basis of Antibiotic Action, 2nd Ed. New York: John Wiley & Sons, 1981, 473.

[4] Foye, O. W. Principles of Medicinal Chemistry, 3rd Ed. Varghese Publishing House (P) Ltd, 1974, 676-736.

[5] Tripathi, K.; D. Essential of Medical Pharmacology, 5th Ed. Jaypee Brothers Medical Publishers (P) Ltda. 2003, 627-629.

[6] Silverstein, R. M.; Webster, X. F. Spectrometric Identification of Organic Compounds, 6th Ed. John Wiley & Sons (Asia) Pvt. Ltd, Singapore, 2005, 81-109.

[7] Mulwad, V. V.; Pawar, R. B.; Chaskar, A. C. J. Korean Chem. Soc.2008, 52, 249.

[8] Rajasekaran, A.; Sankar, N.; Murugesh, A.; Kalasalingam, R. A.; Archives of Pharmacal Research 2006, 29, 535.

[9] Upadhayaya, R. S.; Jain, S.; Sinha, N.; Kishore, N.; Chandra, R.; Arora, S. K. Eur. J. Med. Chem. 2004, 39, 579.

[10] Bachar, S. C.; Lahiri, S. C. Pharmazie 2004, 59, 435.

[11] Ray, S. M.; Lahiri, S. C. J. Ind. Chem. Soc. 1990, 67, 324.

[12] Aliasghar, J.; Khalili D.; Clercq, E. D.; Salmi, C.; Brunel, J. M. Molecules 2007, 12, 1720.

[13] Adamec, J.; Waisser, K.; Kunes, J.; Kaustova, J. Arch. Pharm. 2005, 338, 385.

[14] Zhang, Z. Y.; Chu, C. H.; Hui, X. P. Ind. J. Chem. 2002, 416, 2176.

[15] Singh, H.; Srivastava, M. K.; Singh, B. K.; Singh, S. K.; Dubey, G. Ind. J. Chem. 2001, 406, 159.

[16] Mogilaiah, K.; Sakram, S.; Ind. J. Chem. 2004, 436, 2014.

[17] Ajitha, M.; Rajnarayana, K.; Sangarapani, K. Pharmazie 2002, 57, 796.

[18] Bhat, K. S.; Karthikeyan, M. S.; Holla, B. S.; Shetty, N. S. Ind. J. Chem. 2004, 436, 1765.

[19] Aboraia, A. S.; Rahman, H. M. R.; Mahfouz, N. M.; Gendy, A. E. L. 6ioorg. Med. Chem. 2006, 14, 1236.

[20] Amir, M.; Shahani, S.; Ind. J. Heterocycl. Chem. 1998, 8, 107.

[21] Axena, S.; Verma, M. Ind. J. Pharm. Sci. 1992, 54, 1.

[22] Mullican, D.; Wilson, M. W.; Cannor, D. T.; Kostlan, C. R.; Schirier, D. J.; Dyer R. D. J. Med. Chem. 1993, 36, 1090.

[23] Bhaskar, V. H.; Mohite, P. B. Digest J. Nanomaterials and 6iostructures 2010, 5, 177.

[24] Indian Pharmacopoeia; Vol. II (P-Z); Published by the Controller of Publications, Delhi, 1996, A-100.

[25] Gibbons, S.; Birgit, O.; Jhonsen, I. Fitoterapia 2002, 73, 300.

P. B. Mohite (a) and V. H. Bhaskar * (b)

(a) Department of Pharmaceutical Chemistry, MES College of Pharmacy, Sonai, Ahmednagar, India, 414 105

(b) MP Patel College of Pharmacy, Kapadwanj, Gujrat, India-687 320

Received: 19 January 2011; revised: 18 June 2011; accepted: 20 June 2011. Available online: 28 September 2011.

* Corresponding authors: mohitepb@rediffmail.com
Table 1. Physicochemical characterization of titled compounds

Sr.         R                         Mol. Form                   Mol.
No                                                                 Wt

1           H          [C.sub.18][H.sub.16][N.sub.6][O.sub.2]     348

2          2-Cl        [C.sub.18][H.sub.15]Cl[N.sub.6][O.sub.2]   382

3          4-Cl        [C.sub.18][H.sub.15]Cl[N.sub.6][O.sub.2]   382

4          4-Br        [C.sub.18][H.sub.15]Br[N.sub.6][O.sub.2]   427

5      4-C[H.sub.3]    [C.sub.19][H.sub.18][N.sub.6][O.sub.2]     362

6     4-OC[H.sub.3]    [C.sub.19][H.sub.18][N.sub.6][O.sub.3]     378

7      3-N[O.sub.2]    [C.sub.18][H.sub.15][N.sub.7][O.sub.4]     393

8     [(C[H.sub.3]).   [C.sub.20][H.sub.21][N.sub.7][O.sub.3]     391
        sub.2]-N-

Sr.   M.P. ([degrees]C)   Yield   C, H, N Calculated (found)
No                         (%)

                                    C %      H %       N %

1     128-130[degrees]C    62%     62.06     4.63     24.12
                                  (60.00)   (4.60)   (24.08)
2     136-138[degrees]C    70%     56.48     3.95     21.95
                                  (56.22)   (3.90)   (21.90)
3     138-140[degrees]C    72%     56.48     3.95     21.95
                                  (56.20)   (3.90)   (21.88)
4     144-146[degrees]C    68%     50.60     3.54     19.67
                                  (50.10)   (3.48)   (19.58)
5     180-182[degrees]C    69%     62.97     5.01     23.19
                                  (62.75)   (4.96)   (23.05)
6     155-157[degrees]C    74%     60.31     4.79     22.21
                                  (60.15)   (4.60)   (22.10)
7     188-190[degrees]C    68%     54.96     3.84     24.93
                                  (54.80)   (3.75)   (24.78)
8     180-182[degrees]C    55%     61.37     5.41     25.05
                                  (61.20)   (5.30)   (24.98)

Table 2. Spectral Characterization of titled compounds

Sr.         R         IR (KBr) [cm.sup.-1]
No.

4a          H         3057(Aromatic C-H), 2376,2247(-
                        NC[H.sub.2]),1656 (-C=N of
                        1,3,4oxadiazole),832(C-H def
                        disubstituted benzene)
4b        2-Cl        30570(Aromatic C-H), 2376,2247(-
                        NC[H.sub.2]),1652 (-C=N of
                        1,3,4oxadiazole),828(C-H def
                        disubstituted benzene),776(C-Cl).
4c        4-Cl        3048(Aromatic C-H), 2376,2247(-
                        NC[H.sub.2]),1658 (-C=N of
                        1,3,4oxadiazole),842(C-H def
                        disubstituted benzene),776(C-Cl).
4d        4-Br        3040(Aromatic C-H), 2376,2247(-
                        NC[H.sub.2]),1651 (-C=N of
                        1,3,4oxadiazole),838(C-H def
                        disubstituted benzene),697(C-Br).
4e    4-C[H.sub.3]    3060(Aromatic C-H), 2376,2247(-
                        NC[H.sub.2]),1656 (-C=N of 1,3,4
                        oxadiazole),840(C-H def
                        disubstituted benzene)
4f    3-OC[H.sub.3]   3045(Aromatic C-H), 2376,2247(-
                        NC[H.sub.2]),1648 (-C=N of 1,3,4
                        oxadiazole),832(C-H def
                        disubstituted benzene),1165(-
                        OC[H.sub.3]).
4g    3-N[O.sub.2]    3048(Aromatic C-H), 2376,2247(-
                        NC[H.sub.2]),1645 (-C=N of 1,3,4
                        oxadiazole),835(C-H def
                        disubstituted benzene),1564
                        (N[O.sub.2]).
4h    (C[H.sub.3])    3042(Aromatic C-H), 2376,2247(-
           2-N          NC[H.sub.2]),1642 (-C=N of 1,3,4
                        oxadiazole),828(C-H def
                        disubstituted benzene)

Sr.   1H NMR(300.00 MHz)              m/e
No.   [delta] ppm                    ratio

4a    7.10-8.10 (m, 10H, Ar),         348
        6.30(s,1H,CH)3.98 (s, 2H,
        -C[H.sub.2]),2.15(s,3H,
        C[H.sub.3])
4b    7.10-8.10 (m, 9H, Ar),          383
        6.28(s,1H,CH)3.88 (s, 2H,   [M + 1]
        -C[H.sub.2]),2.15(s,3H,
        C[H.sub.3])
4c    7.10-8.10 (m, 9H, Ar),          383
        6.28(s,1H,CH)3.90 (s, 2H,   [M + 1]
        -C[H.sub.2]),2.15(s,3H,
        C[H.sub.3])
4d    7.10-8.10 (m, 9H, Ar),          427
        6.24(s,1H,CH)3.85 (s, 2H,
        -C[H.sub.2]),2.15(s,3H,
        C[H.sub.3])
4e    7.10-8.10 (m, 9H, Ar),          362
        6.42(s,1H,CH)3.80 (s, 2H,
       -C[H.sub.2]),2.15(s,3H,
        C[H.sub.3])
4f    7.10-8.10 (m, 9H, Ar),          380
        6.15(s,1H,CH)3.75 (s, 2H,   [M + 2]
        -C[H.sub.2]),2.15(s,3H,
        C[H.sub.3])

4g    7.10-8.10 (m, 9H, Ar),          393
        6.24(s,1H,CH)3.70 (s, 2H,
        -C[H.sub.2]),2.15(s,3H,
        C[H.sub.3])

4h    7.10-8.10 (m, 9H, Ar),          392
        6.26(s,1H,CH)3.94(s, 2H,    [M + 1]
        -C[H.sub.2]),2.15(s,3H,
        C[H.sub.3])

Table 3. In vitro antimicrobial activity of
1,3,4-oxadiazole derivatives

Compound      Minimum Inhibitory Conc.(MIC) in ug/ml

                S.     E. Coli   A. niger      C.
              aureus                        albicans

4a             250       250       500        500
4b             125       250       250        125
4c             500       250       250        250
4d             62.5      125       125        250
4e             250       125       250        125
4f             125      62.5       125        62.5
4g             125      62.5       125        500
4h             500       250       250        125
Ampicillin     6.25     6.25        --         --
Fluconazole     --       --        6.25       6.25
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Author:Mohite, P.B.; Bhaskar, V.H.
Publication:Orbital: The Electronic Journal of Chemistry
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Date:Apr 1, 2011
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