In vitro antifungal activity of 2-(3,4-dimethyl-2,5-dihydro-1H-pyrrol-2-yl)-1-methylethyl pentanoate, a dihydropyrrole derivative

Pathogens belonging to fungal genera Aspergillus and Candida are ubiquitous and global in distribution (Randhawa & Khan, 1987). These infections are increasingly recognized as an emerging threat to public health. In immunocompromised hosts, infections by Aspergillus, Candida, Histoplasma, etc., become invasive and disseminate from the primary site of infection to other parts of the body including the gut, kidney, spleen and brain.

Invasive aspergillosis is reported to be associated with a mortality rate of 55 % (Denning & Stevens, 1990). Mortality due to Aspergillus infection in bone marrow transplant recipients was observed to be as high as 80 % despite appropriate chemotherapy (Meyer, 1990). Cerebral aspergillosis presents the symptoms of acute meningitis and is always fatal.

Candida species have been found to be the fourth most prevalent group of pathogens and have been isolated from 8 % of patients with nosocomial bloodstream infections (Pfaller, 1994). They are also identified as critical pathogens in infections of wounds and other body fluids (Powderly et al., 1988). The change in mortality rate associated with disseminated candidiasis has been insignificant even after treatment with effective antifungal drugs (Pfaller et al., 1999).

The drugs currently available for treatment of various fungal infections are primarily polyenes and the azole class of compounds. Amphotericin B, which is considered to be the drug of choice, has been found to be highly nephrotoxic and less effective in invasive aspergillosis (Powderly et al., 1988; Rex et al., 1995). Further, the development of fungal resistance against most of the available drugs has been observed (Pfaller et al., 1998a, b; Powderly, 1994). The increased occurrence of mycoses in immunocompromised patients and the development of resistance in fungi to current drugs has emphasized the need for developing new antifungal compounds with minimal adverse effects in humans.

Several bioactive molecules, including antifungals, from plants have been reported. 2-(3,4-dimethyl-2,5-dihydro-1H-pyrrol-2-yl)-1-methylethyl pentanoate, a novel antifungal molecule, was isolated from the plant Datura metel L (Rajesh et al., 2001; Dabur et al., 2004). The results of preliminary experiments showed this molecule to have anti-Aspergillus properties. The present study deals with detailed investigations on the antifungal spectrum and potency of 2-(3,4-dimethyl-2,5-dihydro-1H-pyrrol-2-yl)-1-methylethyl pentanoate against 10 clinical isolates of Candida and 19 clinical isolates of Aspergillus.

Strains.
Clinical isolates of Candida and Aspergillus, namely Candida albicans, Candida tropicalis, Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger, were obtained from the Microbiology Department, Vallabhbhai Patel Chest Institute, Delhi. These were used along with the standard strains procured from the Indian Type Culture Collection, IARI, Delhi. Quality control strains of C. albicans (ITCC 4718), C. tropicalis (ITCC 1634), A. fumigatus (ITCC 4517), A. flavus (ITCC 5192) and A. niger (ITCC 5405) were included in each test as recommended by the National Committee for Clinical Laboratory Standards (NCCLS).

Antifungal agents.
The compound 2-(3,4-dimethyl-2,5-dihydro-1H-pyrrol-2-yl)-1-methylethyl pentanoate was isolated from D. metel as described previously (Rajesh et al., 2001; Dabur et al., 2004) and characterized. The compound was dissolved in dimethyl sulphoxide (DMSO) and diluted with distilled water. Amphotericin B was used as the standard drug. Freshly prepared solutions of the test compound and the standard drug were used in the study.

Antifungal susceptibility tests.
Evaluations of the susceptibility of Candida were made by the microbroth dilution method as per NCCLS document M27-A (NCCLS, 1997). The fungi used as inocula were grown overnight on Sabouraud dextrose agar (E. Merck) at 35 °C. Tests were performed in RPMI 1640 (Gibco-BRL) buffered to pH 7.0 with 0.165 M morpholinepropanesulphonic acid (MOPS; Sigma). The MIC₉₀ was considered to be the lowest concentration of the compound that inhibited the visible growth of fungi. Effects of different media were determined by using buffered RPMI 1640 supplemented with 20.0 g glucose l¹, yeast nitrogen broth (pH 7.0; Difco laboratories) supplemented with 5.0 g glucose l¹, antibiotic medium 3 (Becton Dickson Microbioogy systems) and Sabouraud dextrose broth (E. Merck). These media were substituted for buffered RPMI 1640 as recommended by the NCCLS (1997). Inoculum effects were determined as per NCCLS (1997), except that strains were suspended to a turbidity equivalent to that of a 0.5 McFarland standard in 0.9 % (w/v) NaCl and were further diluted in 0.9 % NaCl to achieve the desired inoculum levels. Inoculum densities were verified by determining the number of viable colonies per millilitre on Sabouraud dextrose agar after serial dilutions in 0.9 % NaCl.

The activity of 2-(3,4-dimethyl-2,5-dihydro-1H-pyrrol-2-yl)-1- methylethyl pentanoate against Aspergillus isolates was also determined by the microbroth dilution method. Cultures were grown on Sabouraud dextrose agar at 37 °C until sporulation. Stock spore suspensions were prepared with yeast nitrogen broth (pH 7.0) supplemented with 5.0 g glucose l¹ and 25 % (v/v) glycerol, and were stored at 4 °C until use. The c.f.u. ml¹ was determined by plating serial dilutions on Sabouraud dextrose agar plates.

Before inoculation for susceptibility tests, the spore suspensions were diluted to achieve 2 x 10⁴ to 1 x 10⁵ c.f.u. ml¹ in yeast nitrogen broth with 0.5 % glucose (pH 7.0) and were incubated for 24 h at 37 °C to germinate the spores. Serial twofold dilutions of the test compound were made in yeast nitrogen broth plus 0.5 % glucose (pH 7.0) in 100 µl volumes and were inoculated with 100 µl of the germinated spore suspensions. The cultures were incubated for 72 h at 37 °C. The MIC₉₀ was determined as the lowest concentration that inhibited visible fungal growth.

Time-kill analysis.
C. albicans ITCC 4718 was grown on Sabouraud dextrose agar at 35 °C for 24 h. Isolated colonies were selected and suspended in 0.9 % NaCl to a turbidity equivalent to that of 0.5 McFarland standard. Flasks were prepared that contained RPMI 1640 buffered with 0.165 M MOPS to pH 7.0 and four times the MIC₉₀ of the test compound or no compound (growth control). The flasks were inoculated with yeast suspension to a final concentration of approximately 10⁵ c.f.u. ml¹. The cultures were incubated at 35 °C with shaking for up to 24 h. At the defined time intervals, aliquots were removed and the number of viable colonies per millilitre was determined on Sabouraud dextrose agar after serial dilution in 0.9 % NaCl.

In vitro activity of the dihydropyrrole derivative
Nineteen strains of Aspergillus and 10 strains of Candida were employed to evaluate the antifungal potential of the novel compound, 2-(3,4-dimethyl-2,5-dihydro-1H-pyrrol-2-yl)-1-methylethyl pentanoate. Its MIC₉₀ was found to be in the concentration range of 21.87 to 43.75 µg ml¹ against all 29 strains of fungi. The growth of all the pathogenic fungal strains, i.e. C. albicans, C. tropicalis, A. fumigatus, A. flavus and A. niger, was inhibited by the compound. Antibiotic medium 3 was used to identify resistance, if any, in yeasts against the compound and amphotericin B as described by Rex et al. (1995). The MIC₉₀ against the yeasts was found to be 10.93 µg ml¹ in antibiotic medium 3 (Table 1). Variations in susceptibility in the group of fungal strains tested with 2-(3,4-dimethyl-2,5-dihydro-1H-pyrrol-2-yl)-1-methylethyl pentanoate were observed; however, resistance against the compound was not seen. It was observed that 2-(3,4-dimethyl-2,5-dihydro-1H-pyrrol-2-yl)-1-methylethyl pentanoate had broad-spectrum and potent antifungal activity against pathogenic fungi.

Table 1. Effect of different test media on in vitro activity of 2-(3,4-dimethyl-2,5-dihydro-1H-pyrrol-2-yl)-1-methylethyl pentanoate ND, Not determined; YNB, yeast nitrogen broth.

The fungicidal activity of 2-(3,4-dimethyl-2,5-dihydro-1H-pyrrol-2-yl)-1-methylethyl pentanoate against clinical isolates of C. albicans was analysed by time-kill analysis (Fig. 1). When the compound was tested at four times its MIC₉₀, it caused 99.299.8 % loss of yeast viability after 8 h of incubation, while amphotericin B caused a 99.9 % loss of viability after 4 h of incubation.

(15K): Fig. 1. Time-kill analysis of 2-(3,4-dimethyl-2,5-dihydro-1H-pyrrol-2-yl)-1-methylethyl pentanoate and amphotericin B at antibiotic concentrations equal to four times the MIC90 for C. albicans. , Drug-free control; ", 2-(3,4-dimethyl-2,5-dihydro-1H-pyrrol-2-yl)-1-methylethyl pentanoate; , amphotericin B, 1 µg ml1.

The MIC₉₀ of the compound against all the 19 strains of Aspergillus was found to be 21.8743.75 µg ml¹ while the MIC₉₀ of amphotericin B was 0.51.0 µg ml¹. Substantially inhibited growth at sub-MIC₉₀ concentrations of the compound was also detected visually by light microscope (Nikon). Significant growth inhibition up to a concentration of 21.87 µg ml¹ of 2-(3,4-dimethyl-2,5-dihydro-1H- pyrrol-2-yl)-1-methylethyl pentanoate was detected microscopically (Fig. 2). Significant growth inhibition is reported for some antibiotics at sub-MIC₉₀ concentrations by other test methods and is suggested to be therapeutically relevant on the basis of data obtained with animal models (Bartizal et al., 1997; Kurtz et al., 1994; Zhanel et al., 1997). Therefore, the results of in vivo efficacy tests in animal models are required to resolve this point.

(63K): Fig. 2. Inhibition of A. fumigatus growth after 12 h of incubation by 2-(3,4-dimethyl-2,5- dihydro-1H-pyrrol-2-yl)-1-methylethyl pentanoate. A, normal germinating spores; B, spores treated with 10.93 µg ml1 of compound, abnormal growth; C, spores treated with 21.87 µg ml1 of compound, no growth.

Effect of test method on in vitro activity of dihydropyrrole derivative
The test method can affect the level of antifungal activity determined (Rex et al., 1993, 1995). The NCCLS recommend the use of RPMI 1640 buffered to pH 7.0 with MOPS and supplemented with 20.0 g glucose l¹ as the test medium. We compared the in vitro activity of 2-(3,4-dimethyl-2,5- dihydro-1H-pyrrol-2-yl)-1-methylethyl pentanoate against 10 strains of Candida in five test media, namely RPMI 1640, RPMI 1640 supplemented with glucose, yeast nitrogen broth supplemented with glucose, antibiotic medium 3 and Sabouraud dextrose broth (Table 1). The addition of glucose to RPMI 1640 did not alter the activity of 2-(3,4-dimethyl-2,5-dihydro-1H-pyrrol-2-yl)-1-methylethyl pentanoate. The MIC₉₀ of the compound was approximately twofold higher in yeast nitrogen broth than in RPMI 1640. In contrast, the MIC₉₀ of 2-(3,4-dimethyl-2,5-dihydro-1H-pyrrol-2-yl)-1-methylethyl pentanoate was about twofold lower in antibiotic medium 3 and Sabouraud dextrose broth than in RPMI 1640. Rex et al. (1995) reported similar observations. Amphotericin B was found to be most active in antibiotic medium 3 and Sabouraud dextrose broth.

Conclusion
The novel compound 2-(3,4-dimethyl-2,5-dihydro-1H- pyrrol-2-yl)-1-methylethyl pentanoate isolated from D. metel was found to have potent activity against all of the 29 pathogenic strains of fungi, i.e. isolates of C. albicans, C. tropicalis, A. fumigatus, A. niger and A. flavus, tested in the present study, and the size of the inocula did not significantly affect the in vitro activity of the compound. The activity of the compound in antibiotic medium 3 against C. albicans and C. tropicalis was highest, the MIC₉₀ being 10.93 µg ml¹. It was observed that 2-(3,4-dimethyl-2,5-dihydro-1H-pyrrol-2-yl)-1-methylethyl pentanoate is fungicidal and resistance against this compound was not found in any of the strains tested in the present study.

The authors are thankful to Bundelkhand University, Jhansi, and the Institute of Genomics and Integrative Biology, Delhi, for providing necessary facilities.