Synthesis, Antifungal and Toxicity Screening of Newer Isoniazid Derivatives

A series of Isoninicotinic acid hydrazide (INH) incorporated derivatives of thiazolidin-4-one (2a-h, 3a-h), azetidin-2one (4a-h) and 1,3,4-oxadiazole (5a-h) were synthesized in satisfactory yield and pharmacologically evaluated for their in vitro antifungal activity. All the synthesized compounds were in good agreement with elemental and spectral data. A majority of the tested compounds showed good to moderate antifungal activity against all tested pathogenic fungal strains. To evaluate the toxicity of the compounds on liver, estimation of enzymes was also carried out. *Corresponding author: Sadaf Jamal Gilani, Department of Pharmaceutical Chemistry, KIET School of Pharmacy, Ghaziabad, UP, India, Tel: 919891128162; E-mail: gilanisadaf@gmail.com Received March 04, 2014; Accepted April 15, 2014; Published April 17, 2014 Citation: Gilani SJ, Maurya DP, Katiyar D, Goel R, Nagarajan K, et al. (2014) Synthesis, Antifungal and Toxicity Screening of Newer Isoniazid Derivatives. Med chem 4: 428-434. doi:10.4172/2161-0444.1000174 Copyright: © 2014 Gilani SJ, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Introduction
Antimicrobial agents are those inhibitory chemicals which are employed to kill microorganisms or prevent their growth. Infectious diseases account for approximately one-half of all deaths in tropical countries. Although deaths from bacterial and fungal infections have dropped in the developed world, these are still major causes of death in the developing world [1]. In addition, primary and opportunistic fungal infections continue to increase rapidly because of the increased number of immunocompromised patients (AIDS, cancer and transplants) [2]. Antimicrobials reduce or completely block the growth and multiplication of bacteria. This has made them unique for the control of deadly infectious diseases caused by a variety of pathogens. They have transformed our ability to treat infectious diseases such as pneumonia, meningitis, tuberculosis, malaria and AIDS [3]. Literature survey revealed that thiazolidin-4-ones are a class of heterocycles which have attracted significant interest in medicinal chemistry and they have a wide range of pharmaceutical and biological activities including antimicrobial [4], anti-inflammatory, analgesic, antitubercular and antidiabetic [5][6][7][8]. Similarly, The azetidin-2-one derivatives have been reported to possess a wide range of biological activities like antibacterial, antifungal, anti-inflammatory, anticonvulsant, anticancer and antitubercular [9][10][11][12][13][14]. In addition, 1,3,4-oxadiazoles are a class of heterocycles which have attracted significant interest in medicinal chemistry and they have a wide range of pharmaceutical and biological activities including antimicrobial, anti-inflammatory and analgesic [15][16][17]. In the design of new compounds, development of hybrid molecules through the combination of different pharmacophores in one structure may lead to compounds with increased antifungal activity. In view of the above mentioned facts and in continuation of our interest in the synthesis of heterocycles containing isoniazid moiety, to identify new candidates that may be of value in designing new, potent, selective and less toxic antifungal agents, we report herein the synthesis and antifungal evaluation of some novel structural hybrids incorporating both the isoniazid moiety with thiazolidin-4-one, azetidin-2-one and 1,3,4-oxadiazole ring systems through different linkages. Further, Enzyme estimation was also carried out to assess the toxicity effects of the compounds on liver.

Experimental
All the solvents were of AR grade and were obtained from Merck, CDH and S.D. Fine chemicals. Melting points were determined in open capillary tubes and are uncorrected. All the compounds were subjected to elemental analysis (CHN) and the measured values agreed within ± 0.4% with the calculated ones. Thin layer chromatography was performed on silica gel G (Merck). The spots were developed in an iodine chamber and visualized with an ultraviolet lamp. The solvent systems used were benzene:acetone (8:2, v/v) and toluene:ethyl acetate:formic acid (5:4:1, v/v). Ashless Whatman No. 1 filter paper was used for vacuum filtration. The IR spectra were recorded in KBr pellets on a (BIO-RAD FTS 135) WIN-IR spectrophotometer. The FAB mass spectra of all the compounds were recorded on a JEOL SX102/DA-600 mass spectrometer using argon/xenon (6 kV, 10 mA) as the FAB gas. The 1 H-NMR spectra were recorded on a Bruker model DPX 300 FT-NMR spectrometer in CDCl 3 using tetramethylsilane (Me 4 Si, TMS) as an internal standard. The chemical shifts are reported in the δ ppm scale [18]

General procedure for the synthesis of N-(3-chloro-2-(2substitutedphenyl)-4-oxaazetidin-1-yl) isonicotinamide (4ah)
A solution of 1 (0.01 mol) in dioxane (20 mL) was added to a well stirred mixture of chloroacetylchloride (0.012 mol) and triethylamine (Et 3 N) (0.012 mol) in dioxane (10 mL) at 0-5ºC. The reaction mixture was then stirred for 8h, kept for 2days at room temperature and then treated with cold water. The solid thus obtained was filtered, washed with water and recrystallized from methanol to yield 4a-h.

Antifungal activity
Antifungal activity of the synthesized compounds were determined in vitro by using serial plate dilution method [19,20] against C. albicans (ATCC 2091), A. niger (MTCC 281), A. flavus (MTCC 277), M. purpureous (MTCC 369) and P. citrinum (NCIM 768) at 100 μg/ mL, 50 μg/mL, 25 μg/mL, 12.5 μg/mL and 6.25 μg/mL concentrations, respectively, in the nutrient agar media. Standard antibiotic ketoconazole was used as reference drug at 25 μg/mL, 12.5 μg/mL and 6.25 μg/mL. Solutions of required concentrations of test compounds were prepared by dissolving the compounds in DMSO. The minimum inhibitory concentration (MIC) obtained for the test compounds and standard drug are reported in Table 1. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of the compounds that inhibited visible growth of microorganisms on the plate.

Animals
Male albino mice (Swiss, 18-25 gm) were used as experimental animals. The test compounds were suspended in polyethylene glycol (PEG). The animals were maintained on an adequate diet and allowed free access to food and water except during the short time they were removed from cages for testing. The animals were maintained at room temperature (25-30°C). All the experimental protocols were carried out with the permission from Institutional Animal Ethics Committee (IAEC). Animals were obtained from Central Animal House Facility, Hamdard University, New Delhi-110062, India. Registration number and date of registration of Animal House Facility (173/CPCSEA, 28, JAN-2000).

Assessment of liver function
Liver functions such as serum glutamate oxaloacetate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT) were assessed by a reported method [21]. The alkaline phosphatase was also measured according to the reported procedures [22,23]. All data are recorded in Table 2.
The antifungal activity study revealed that all the compounds tested showed good to moderate antifungal activity against all pathogenic strains (MIC 6.25 μg/mL, 12.5 μg/mL, 25 μg/mL and 50 μg/mL). Structure and biological activity relationship of title compounds showed that the presence of 4-chloro phenyl, 4-methoxy phenyl groups (MIC 6.25 and12.5 μg/mL) are responsible for good antifungal activity.
Thus, various thiazolidine (2a-h, 3a-h), azetidine (4a-h) and oxadiazole (5a-h) derivatives of isoniazid were prepared with the objective of developing better antifungal agents. The derivatives of the aforementioned rings were found to have a promising class of compounds with an interesting pharmacological profile. Further, it is clear from structure activity relationship (SAR), that the thiazolidine derivatives (2b, 2e, 3b, 3e) were found to be more active than azetidine (4b, 4e) and oxadiazole (5b, 5e) derivatives.

Assessment of liver function
The most active compounds (2b, 4b and 5b) of the series were evaluated further for their hepatotoxic effects by assessing the liver enzymes. Any significant changes in the level of enzymes are indicative of liver disorders. Levels of alkaline phosphatase, SGOT and SGPT enzymes were measured and the results are expressed as mean ± SEM. Compound 4b showed significant rise in the alkaline phosphatase and SGPT level with P<0.01 when compared to control. The rise in SGOT level was also found to be significant with P<0.05. Compound 5b was also found to increase the alkaline phosphatase and SGPT levels significantly with P<0.05. The rise in SGOT level was not significant   with compound 5b. Compound 2b showed no significant change in all the three enzymes and can be considered to have no hepatotoxicity.

Conclusion
Thus, various thiazolidin-4-ones, (2a-h, 3a-h) azetidin-2-ones (4ah) and 1,3,4-oxadiazole (5a-h) derivatives of isoniazid were prepared with the objective of developing better antifungal agents. All the derivatives were found to have a promising class of compounds with an interesting pharmacological profile. Among these the compound N-(2-(4-Chlorophenyl)-4-oxothiazolidin-3-yl) isonicotinamide (2b) showed maximum antifungal activity with no hepatotoxicity effect. Hence, it is clear from structure activity relationship (SAR), that thiazolidin-4-ones derivatives were more active than azetidin-2-ones and 1,3,4-oxadiazole derivatives. Also a common result was obtained for parent drug isoniazid, which showed moderate activity against all pathogenic fungal strains. In conclusion, the isoniazid incorporated hydrazone derivatives can be regarded as a newer class of antifungal agents. They were also found to be less toxic which indicates better tolerability of the compounds having strong future prospects.