SYNTHESIS AND BIOEVALUATION OF KETOCONAZOLE THIOSEMICARBAZONE ANALOGUES

SYNTHESIS AND BIOEVALUATION OF KETOCONAZOLE THIOSEMICARBAZONE ANALOGUES

1. K. Agrawal ¹ and Y. Murti*²

Rajiv Academy for Pharmacy, Mathura-281001, Uttar Pradesh, India

Institute of Pharmaceutical Research, GLA University, Mathura-281406, Uttar Pradesh, India

ABSTRACT

Ketoconazole (KTZ) is a synthetic antifungal drug used to prevent and treat fungal infections, especially in immunocompromised patients such as those with AIDS. Resistance to ketoconazole has been observed in a number of clinical fungal isolates, including C. albicans. Thus new effective agents with less toxicity against fungal infection are urgently required. With this view, ketoconazole thiosemicarbazone analogues (Compounds 1-10) were synthesized wherein condensation of different thiosemicarbazides substituted by different cyclic and aromatic amines with the KTZ was done. Investigation of in-vitro antifungal activity of compounds was done by broth microdilution assay method against five pathogenic fungi Aspergillus niger, Candida albicans, Candida krusei, Candida glabrata and Candida tropicalis. Ketoconazole was used as reference for inhibitory activity against fungi. All the compounds were found potent antifungal agents, while compounds 8, 9 and 10 exhibited excellent in-vitro antifungal activity showing importance of halogenated compounds.

Antifungal activity

INTRODUCTION: Ketoconazole (KTZ) ^1-5 is a potent, orally active, broad-spectrum antifungal agent, which was recently developed from imidazole. KTZ derivatives is most often used to treat fungal infections that can spread to different parts of the body through the bloodstream such as yeast infections of the mouth, skin, urinary tract, and blood, and certain fungal infections that begin on the skin or in the lungs and can spread through the body. However, resistances to KTZ in many pathogenic fungi as well as several side effects are also well documented ⁶.

Thiosemicarbazones are a class of small molecules that have been evaluated for antifungal activity ^7-8. In addition various thiosemicarbazone derivatives have been shown to possess anticancer, anti-proliferative, antioxidant and many other biological properties ^9-13.

In view of the antifungal activity of the above pharmacophores, it was envisaged that the combined effect of these entities will result in increased antifungal activity. Therefore, it was planned to synthesize ketoconazole thiosemicarbazone analogues (Compounds 1-10) with a hope to obtained potent antifungal agents.

Experimental:

Instrumentation: Melting points were taken in open capillary tube and were uncorrected. The reaction was monitored by thin layer chromatography on silica gel G plates (Merck Silica-60F₂₅₄) and the final products were purified by recrystallization from absolute methanol. All the newly synthesized compounds were confirmed by FTIR (Recorded on a FTIR-8400S spectro- photometer, SHIMADZU), ¹H NMR (Recorded on Bruker NMR spectrophotometer in deuterium-substituted DMSO form using TMS as internal standard) and elemental analysis.

Synthetic procedure: Cyclic or aromatic amine on reaction with carbon disulfide in aqueous sodium hydroxide solution and dimethyl sulfate afforded substituted methyl carbamodithioates which on treatment with hydrazine hydrate yielded substituted thiosemicarbazides. Condensation of different substituted thiosemicarbazides with the KTZ was done in methanol (Scheme 1).

SCHEME 1

Where R = cyclohexyl (1), cyclooctyl (2), phenyl (3), o-methyl phenyl (4), p-methyl phenyl (5), o-nitro phenyl (6), p-nitro phenyl (7), o-chloro phenyl (8), p-chloro phenyl (9) and 2,4-dichloro phenyl (10).

The reaction mixture was refluxed for 12 hrs and left overnight at room temperature. After cooling, the solid was filtered and recrystallized from appropriate solvent to give desired ketoconazole thiosemicarbazone analogues 1-10.

The structures of the synthesized compounds have been determined by their elemental analysis, FTIR, ¹H NMR spectra. Physicochemical, analytical and spectral data of synthesized compounds are given in table 1 & 2.

TABLE 1: PHYSICOCHEMICAL AND ANALYTICAL DATA OF SYNTHESIZED COMPOUNDS (1-10)

TABLE 2: SPECTRAL DATA OF SYNTHESIZED COMPOUNDS (1-10)

Determination of in vitro Antifungal Activity: All the compounds were evaluated for their in-vitro antifungal activity against Aspergillus niger, Candida albicans, Candida krusei, Candida glabrata and Candida tropicalis using broth microdilution assay method ¹⁴. Ketoconazole was used as reference for inhibitory activity against fungi and was also screened under similar conditions for comparison. Serial dilutions of the test compounds and KTZ were prepared in Mueller-Hinton agar.

Suspensions of each microorganism were prepared to contain 10⁵ cfu/mL (colony forming unit/mL) and the plates were incubated at 35^oC for 24 h and 48 h for all fungal strains tested and recorded as the MIC expressed in (µg/mL).

These experiments were duplicated to define the MIC values. The MIC was defined as at least 80 % inhibition of the growth of control. The data of activity is summarized in table 3.

TABLE 3: MICS VALUES (µg/ml) FOR FUNGI OF THE SYNTHESIZED COMPOUNDS (1-10)

 RESULT AND DISCUSSION: In the present study, ketoconazole thiosemicarbazone derivatives have been synthesized by using different substituted thiosemicarbazides with KTZ. TLC confirmed the purity of the synthesized compounds. The structural elucidation was done by using spectral and elemental analysis data. The data given in table 3 reveal good in-vitro antifungal activity of synthesized compounds (1-10) against the tested fungal strains. When compared to KTZ, compounds 8, 9 and 10 are more active with less MIC value showing importance of halogenated compounds.

CONCLUSION: The study on ketoconazole thiosemi- carbazones as candidate antifungals suggests the beneficial potential of these leads that need to be further explored in order to discover and develop better and yet safer therapeutic agents for fungal infections.

ACKNOWLEDGEMENT: The authors are thankful to the Head, SAIF, CDRI, Lucknow, for providing spectral facilities.

REFERENCES:

1. Heel RC, Brogden RN, Carmine A, Morley PA, Speight TM and Avery GS: Ketoconazole: A review of its therapeutic efficacy in superficial and systemic fungal infections. Drugs 1982; 23:1-36.
2. Bossche HV, Willemsens G, Cools W, Cornelissen F, Lauwers W and Cutsem V: In vitro and in vivo effects of the antimycotic drug ketoconazole on sterol synthesis. Antimicrobial Agents and Chemotherapy 1980; 17:922-928.
3. Kraemer FB and Pont A: Inhibition of cholesterol synthesis by ketoconazole. American Journal of Medicine 1986; 80:616-622.
4. Ballard SA, Lodola A and Tarbit MH: A comparative study of 1-substituted imidazole and 1, 2, 4-triazole antifungal compounds as inhibitors of testosterone hydroxylations catalysed by mouse hepatic microsomal cytochrome P-450. Biochemical Pharmacology 1988; 37:4643-4651.
5. Shepherd FA, Hoffert B, Evans WK, Emery G and Trachtenberg J: Ketoconazole. Use in the treatment of ectopic adrenocorticotropic hormone production and Cushing's syndrome in small-cell lung cancer. Archives of Internal Medicine 1985; 145:863-864.
6. Richard F, Michelle AC and Pamela CC. Lippincotts Illustrated Reviews Pharmacology, Edition 4; CBS Publishers & Distributors, 2008.
7. Chandra R, Pandey OP and Sengupta SK: Organophosphorus derivatives containing piperazine dithiosemicarbazones as chemotherapeutants against fungal pathogens of sugarcane. Journal of Agricultural and Food Chemistry 2005; 53:2181-2184.
8. Pandeya SN, Sriram D, Nath G and Clercq E: Synthesis, antibacterial, antifungal and anti HIV activities of Schiff and Mannich bases derived from isatin derivatives and N-[4(4- chlorophenyl) thiazol-2-yl] thiosemicarbazide. European Journal of Pharmaceutical Sciences 1999; 9:25-31.
9. Richardson DR, Sharpe PC, Lovejoy DB, Senaratne D, Kalinowski DS, Islam M and Bernhardt PV: Dipyridyl thiosemicarbazone chelators with potent and selective antitumor activity form iron complexes with redox activity. Journal of Medicinal Chemistry 2006; 49:6510-6521.
10. Kovala-Demertzi D, Yadav PN, Wiecek SJ, Skoulika TV and Demertzis MA: Zinc(II) complexes derived from pyridine-2-carbaldehyde thiosemicarbazone and (1E)-1-pyridin-2-ylethan-1-one thiosemicarbazone. Synthesis, crystal structures and antiproliferative activity of zinc(II) complexes. Journal of Inorganic Biochemistry 2006; 100(9):1558-1567.
11. Karatepe M and Karatas F: Antioxidant, pro-oxidant effect of the thiosemicarbazone derivative Schiff base (4-(1-phenylmethylcyclobutane-3-yl)-2-(2-hydroxybenzylidene hydrazino) thiazole) and its metal complexes on rats. Cell Biochemistry and Function 2006; 24(6):547-554.
12. Afrasiabi ES, Lin W, Ma Y, Campana C and Padhye S: Nickel (II) complexes of naphthaquinone thiosemicarbazone and semicarbazone: synthesis, structure, spectroscopy, and biological activity. Journal of Inorganic Biochemistry 2005; 99(7):1526-1531.
13. Belicchi-Ferrari M, Bisceglie F, Casoli C, Durot S, Morgenstern-Badarau I, Pelosi G, Pilotti E, Pinelli S and Tarasconi P: Copper(II) and cobalt(III) pyridoxal thiosemicarbazone complexes with nitroprusside as counterion: syntheses, electronic properties, and antileukemic activity. Journal of Medicinal Chemistry 2005; 48(5):1671-1675.
14. Goto S, Jo K, Kawakita T, Mitsuhashi S, Nishino T, Ohsawa N and Tanami H: Determi- nation method of minimum inhibitory concentrations. Chemotherapy 1981; 29:76-79.
    

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