Naftifine, a new antimycotic drug of the allylamine class, is a potent inhibitor of ergosterol biosynthesis in Candida albicans. Treated cells showed a dose-dependent drop in ergosterol content; the level was reduced by 60% at concentrations of >50 mg/liter, causing total inhibition of growth. This inhibition coincided with a heavy accumulation of the sterol precursor squalene. Radiolabeling experiments showed that the inhibition of sterol synthesis was complete within 10 min of exposure of cells to the compound. Control cells incorporated [14C]acetate into nonsaponifiable lipids composed primarily of ergosterol, whereas naftifinetreated cells accumulated only labeled squalene. When the drug was removed by washing cells thoroughly in 1% Tween 80, the accumulated squalene was further metabolized to ergosterol. A similar pattern of inhibition was observed in sterol biosynthesis from [14C]mevalonate in a cell-free system. At 50 mg/liter, naftifine gave >99% inhibition of sterol biosynthesis both in whole cells and in cell extracts of C. albicans. The primary action of naftifine appears to be the blocking of fungal squalene epoxidation.Naftifine is an allylamine antimycotic drug with activity against a wide range of pathogenic fungi, particularly dermatophytes both in vitro (4) and in vivo (12). In clinical trials, the compound proved very effective as a topical treatment for dermatomycoses (3). At higher concentrations, naftifine also has in vitro activity against Candida albicans (4) and may be clinically useful against this organism. As naftifine represents an entirely new chemical class of antifungal agents, it is important to establish the mode of action in a range of target organisms. Earlier work with the dermatophyte Trichophyton mentagrophytes and the yeast Candida parapsilosis (11) indicated that naftifine interferes with ergosterol biosynthesis by blocking squalene metabolism. In this report, we describe the activity of naftifine against C. albicans, using several experimental models designed to quantify the effects on ergosterol biosynthesis in comparison with the inhibition of cell growth.( Extraction of lipids. Cells were harvested by centrifugation and treated by saponification in 50% ethanol containing 15% KOH and 0.1% pyrogallol for 2 h under reflux. After the addition of 2 volumes of water, the nonsaponifiable lipid (NSL) was extracted three times with petroleum ether (40 to 60°C). The petrol extract was washed with water, dried over anhydrous sodium sulfate, filtered, and brought to dryness in a rotary evaporator. NSL was dissolved in cyclohexane and stored at -20°C in the dark. For quantitative analysis, a known amount of cholesterol was added as an internal standard before the extraction.Gas chromatography. Components of NSL were separated by using a Siemens gas chromatograph (Sichromat 1) with a glass column (110 by 0.6 cm) with 1% OV3 silicone on Chromosorb G (Supelco, Inc., Bellefonte, Pa.) in the stationary phase. The carrier gas was helium (23 ml/min), and the temperature range program was ...

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Assays based on reporter gene technology represent today an important tool in the pharmaceutical industry for discovering novel compound classes interfering with the activation and signaling of target cells after stimulation. Here we describe a reporter gene assay targeting mast cell activation of IgE plus antigen, established in an attempt to identify substances preventing type I allergy (allergic rhinitis, allergic conjunctivitis, allergic asthma, and acute and chronic urticaria). The assay is based on a murine mast cell line designated CPII, stimulation by IgE plus antigen, and a reporter gene construct with the TNF alpha promoter linked to luciferase as a read-out system. Via screening about 50,000 substances, compound 2 was found to inhibit the reporter gene induction in the submicromolar range in this assay. Analogues of compound 2 of the 2,3,4-trihydropyrimidino[2,1-a]isoquinoline type were synthesized starting from 2-alkyl-substituted benzonitriles via aminolysis with 1,3-diaminopropane, dimetalation of 2-substituted 2-phenyl-1,4,5,6-tetrahydropyrimidines with n- and sec-butylithium, reaction with carboxylic acid methyl esters, and finally acidic dehydration. From about 50 derivatives, compound 41 was selected as a lead structure with an IC50 of 0.2 microM and a TC50 of 2.7 microM. In a first profiling in secondary assays, it effectively interfered with the production of mediators such as TNF alpha, IL-4, IL-6, IL-13, and leukotriene synthesis as measured by the corresponding ELISAs. In addition, a passive cutaneous anaphylaxis in mice (a typical type I reaction) is inhibited to more than 90% by compound 41, when administered intradermally 90 min before challenge.