Synthesis, in vitro and in vivo antifungal activity of 5-phenylthio-2,4-bisbenzyloxypyrimidine: a novel nucleobase.

A pyrimidne nucleobase, 5-phenylthio-2,4-bisbenzyloxypyrimidine and its analogs were synthesized and scanned for in vitro antifungal activity using cup-plate and macrobroth dilution method against Candida albicans, Aspergillus niger, Aspergillus flavus and Aspergllus fumigatus. In the cup-plate method, 5-phenylthio-2,4-bisbenzyloxypyrimidine showed very good antifungal activity compared to clotrimazole at the concentrations of 100 and 1000 μg/ml and in the macrobroth dilution method, it showed comparable activity with respect to standard drugs fluconazole and itraconaole. In vivo antifungal activity of 5-phenylthio-2,4-bisbenzyloxypyrimidine at the dose levels of 10 and 30 mg/kg was carried by causing systemic infection of mice using the same fungi used in in vitro testing. The results from in vivo studies with 5-phenylthio-2,4-bisbenzyloxypyrimidine and fluconazole indicated that 5-phenylthio-2,4-bisbenzyloxypyrimidine had similar potency as fluconazole at both dose levels.

There is a continuous need for developing potent and safe antifungal agents against fungal pathogens due to rapid emergence of drug resistant mutant fungi, increasing risk of opportunistic infections in immunocompromised patients such as those on cancer chemotherapy, organ transplantation and/or suffering with human immunodeficiency virus infection[1]. The clinical utility of triazole agents such as fluconazole and itraconazole is quite limited, especially against drug resistant Candida species[2], many of which are also have been reported to be resistant to amphotericin B[3], ketoconazole[4], or flucytosine[5]. In view of this, here we present the synthesis, in vitro and in vivo antifungal study of a 5-phenylthio-2,4-bisbenzyloxypyrimidine (PTBP) as preliminary trails. Synthesis of PTBP (Scheme 1, compound 6) and its analogs were carried out as reported earlier by our group[6].

Sabouraud agar media (SDA) was used for the antifungal screening and was followed as described in the literature[7]. This evaluation was carried out against Candida albians, Aspergillus niger, Aspergillus flavus, and Aspergillus fumigatus. Test solution and standard drug clotrimazole were prepared at the concentration of 1000 and 100 μg/ml in DMF. Diameter of zone of inhibition was measured in mm after 24 h of incubation at 37°. Sabouraud dextrose broth was used for the evaluation of the synthesized compound against A. niger, A. flavus, A. fumigatus and RPMI-1640 with glutamine and without bicarbonate broth (pH 7.0) was used for C. albicans[8]. Minimum inhibitory concentration (MIC) of the compound was estimated according to literature and compared to that obtained for itraconazole, fluconazole and clotrimazole[2-910]. Test solution and all standard drugs were prepared at the concentration of 512 μg/ml in distilled dimethylsulphoxide (DMSO) and treated as stock solutions. MICs were scanned ranging the concentration from 256 μg/ml to 0.031 μg/ml. Haemocytometer was used to quantify the inoculum size or spore load per ml[11]. MICs were determined using Elico SL 171 spectrophotometer after 48 h of incubation at 37°. Amount of growth of fungal culture was determined by measuring the turbidity at 630 nm (the more turbid the suspension, the less light will be transmitted through. Since, turbidity is directly proportional to number of cells). MICs were defined as the concentration of the compound that inhibited the complete growth.

In vivo antifungal activity[12] was carried using Swiss albino mice of equal sex, weighing between 18-20 g (4-6 week old) were fed standard pellet diet and given tap water ad libitum. The experiment was approved by the Institutional Animal Ethics Committee. Mice were divided into five groups of 8 mice each for evaluating against C. albicans and A. niger and five groups of 6 mice each were used in case of A. flavus and A. fumigatus. First, second, third, fourth and fifth group were injected with quantified number of fungal spores via tail intravenous route (0.2 ml). Fungal suspension was prepared in sterile saline. Haemocytometer was used to quantify the inoculum size and 6.25×106 cells of C. albicans, 1.13×104cells of A. niger, 5.23×104cells of A. flavus and 1.03×105 cells of A. fumigatus were used. PTBP solution and standard drug, fluconazole were prepared in Tween-80 and suspended in distilled water and sterilized before injection. After 24 h of injection with fungal spores, first and second group of animals were treated with PTBP solution of 10 mg/kg and 30 mg/kg, respectively. Third and fourth groups received fluconazole at the dose of 10 mg/kg and 30 mg/kg, respectively. Fifth group received saline and served as control. Injections used for all groups were intraperitoneal for 5 days. Mice were kept under observation for 15 days and deaths occurred during the experiment were recorded during this period. On day 16 survived mice were sacrificed by cervical dislocation and both survived and moribund mice were dissected, liver and lung were removed and triturated in 2 ml of sterile saline and poured on Sabouraud agar media, incubated for 48 h for the growth of fungal burden. Data were analysed by one-way ANOVA followed by Duncan multiple range test (DMRT) and the level of significance was set at P<0.05.

Synthetic route for the preparation of nucleobases (3-6). 3.R = CH3, 4.R = CH2CH3, 5.R = CH2(CH)3, 6.R = CH2C6H5

In the present study, PTBP showed very good in vitro antifungal activity against all the tested fungi, A. niger, A. flavus, A. fumigatus and C. albicans compared to standard drug, clotrimazole at both the concentration level of 100 μg/ml and 1000 μg/ml. Hence, we decided to study further in vivo antifungal activity of PTBP (Table 1). MIC of PTBP was carried against four fungi, C. albicans, A. niger, A. flavus and A. fumigatus and was compared with fluconazole, itraconazole and clotrimazole (Table 2). MIC of PTBP, fluconazole, itraconazole and clotrimazole against C. albicans remained same i.e., 128 μg/ml. Clotrimazole has the lowest MIC (32 μg/ml) against A. niger followed by fluconazole (64 μg/ml), PTBP (128 μg/ml) and itraconazole (128 μg/ml). Similarly, clotrimazole exhibited lowest MIC against A. flavus (16 μg/ml) and A. fumigatus (32 μg/ml). However, PTBP has significant less MIC (64 μg/ml) compared to fluconazole (128 μg/ml) and itraconazole (128 μg/ml) against A. flavus and has the same MIC of fluconazole and itraconazole against A. fumigatus (128 μg/ml). Results of the study demonstrated that, PTBP had comparable MIC against all the four fungi compared to fluconazole and itraconazole except clotrimazole which showed superior activity against aspergillus sps.

TABLE 1

IN VITRO ANTIFUNGAL ACTIVITY USING CUP-PLATE METHOD

TABLE 2

MIC OF PTBP AND STANDARD DRUGS (μg/ml)

The in vivo antifungal activity of PTBP was compared with standard drug, fluconazole at two dose levels, 10 mg/kg and 30 mg/kg (Table 3). No deaths were occurred during the study period against C. albicans and A. niger but fungal burden studies showed that there was a significant difference between treated and control groups at the level of P<0.05. PTBP showed more efficiency in reducing the C. albicans spores in both liver and lung at the dose of 30 mg/kg compared to fluconazole at the same dose level. However, at the dose of 10 mg/kg, PTBP and fluconazole showed similar effect in fungal spore reduction (in lung as well as liver). In case of antifungal activity against A. niger there was no significant difference between PTBP and fluconazole treated mice at the dose level of 10 mg/kg and 30 mg/kg. This suggested that PTBP had the same efficiency as that of standard drug. Deaths were observed in all treated (except 30 mg/kg fluconazole group) and control groups of A. fumigatus infected mice before the completion of course of experiment. Two female mice and one female mouse were dead in 10 mg/kg fluconazole and 10 mg/kg PTBP groups respectively. One female mouse was dead in PTBP treated at the dose 30 mg/kg and in control group. However, fungal burden study showed that there was no significant difference between PTBP and fluconazole. In control group of A. flavus, five of six mice were dead and only one mouse was alive during the course of experiment. Deaths were also occurred in all the treated groups but numbers of deaths were less compared to control group. Three deaths were occurred in 30 mg/kg fluconazole and 10 mg/kg PTBP groups. Finding of fungal burden of A. flavus in control group showed the uncountable number of spores in lungs of three male mice and liver of one male mouse. Similarly, uncountable numbers of A. flavus spores were also found in one male mouse of 10 mg/kg fluconazole group. DMRT pos-test showed that PTBP was comparable with fluconazole at both the dose levels at P<0.05. However, it should be noted that in control group all the mice were dead except one female mouse. This indicated that fluconazole as well PTBP prolonged the survival rate of the mice. Therefore, in vivo antifungal activity of PTBP and fluconazole indicated that PTBP had similar potency as fluconazole at the dose of 10 mg/kg and 30 mg/kg against all the tested fungi.

TABLE 3

RECOVERY OF FUNGAL BURDEN FROM ORGANS OF MICE