Phthalides are bioactive compounds that naturally occur in the family Apiaceae. Considering their potentially versatile applications, it is desirable to determine their physical properties, activity and metabolic pathways. This study aimed to examine the utility of whole-cell biocatalysts for obtaining 3-butyl-3-hydroxyphthalide, which is the metabolite formulated during mammalian metabolism of 3-n-butylidenephthalide. We performed transformations using 10 strains of fungi, five of which efficiently produced 3-butyl-3-hydroxyphthalide. The product yield, determined by high-performance liquid chromatography, reached 97.6% when Aspergillus candidus AM 386 was used as the biocatalyst. Increasing the scale of the process resulted in isolation yields of 29–45% after purification via reversed-phase thin layer chromatography, depending on the strain of the microorganism used. We proposed different mechanisms for product formation; however, hydration of 3-n-butylidenephthalide seems to be the most probable. Additionally, all phthalides were tested against clinical strains of Candida albicans using the microdilution method. Two phthalides showed a minimum inhibitory concentration, required to inhibit the growth of 50% of organisms, below 50 µg/mL. The 3-n-butylidenephthalide metabolite was generally inactive, and this feature in combination with its low lipophilicity suggests its involvement in the detoxification pathway. The log P value of tested compounds was in the range of 2.09–3.38.

The resistance of Candida albicans and other pathogenic yeasts to azole antifungal drugs has increased rapidly in recent years and is a significant problem in clinical therapy. The current state of pharmacological knowledge precludes the withdrawal of azole drugs, as no other active substances have yet been developed that could effectively replace them. Therefore, one of the anti-yeast strategies may be therapies that can rely on the synergistic action of natural compounds and azoles, limiting the use of azole drugs against candidiasis. Synergy assays performed in vitro were used to assess drug interactions Fractional Inhibitory Concentration Index. The synergistic effect of fluconazole (1) and three synthetic lactones identical to those naturally occurring in celery plants—3-n-butylphthalide (2), 3-n-butylidenephthalide (3), 3-n-butyl-4,5,6,7-tetrahydrophthalide (4)—against Candida albicans ATCC 10231, C. albicans ATCC 2091, and C. guilliermondii KKP 3390 was compared with the performance of the individual compounds separately. MIC90 (the amount of fungistatic substance (in µg/mL) inhibiting yeast growth by 90%) was determined as 5.96–6.25 µg/mL for fluconazole (1) and 92–150 µg/mL for lactones 2–4. With the simultaneous administration of fluconazole (1) and one of the lactones 2–4, it was found that they act synergistically, and to achieve the same effect it is sufficient to use 0.58–6.73 µg/mL fluconazole (1) and 1.26–20.18 µg/mL of lactones 2–4. As fluconazole and phthalide lactones show synergy, 11 new fluconazole analogues with lower toxicity and lower inhibitory activity for CYP2C19, CYP1A2, and CYP2C9, were designed after in silico testing. The lipophilicity was also analyzed. A three-carbon alcohol with two rings was preserved. In all compounds 5–15, the 1,2,4-triazole rings were replaced with 1,2,3-triazole or tetrazole rings. The hydroxyl group was free or esterified with phenylacetic acid or thiophene-2-carboxylic acid chlorides or with adipic acid. In structures 11 and 12 the hydroxyl group was replaced with the fragment -CH2Cl or = CH2. Additionally, the difluorophenyl ring was replaced with unsubstituted phenyl. The structures of the obtained compounds were determined by 1H NMR, and 13C NMR spectroscopy. Molecular masses were established by GC-MS or elemental analysis. The MIC50 and MIC90 of all compounds 1–15 were determined against Candida albicans ATCC 10231, C. albicans ATCC 2091, AM 38/20, C. guilliermondii KKP 3390, and C. zeylanoides KKP 3528. The MIC50 values for the newly prepared compounds ranged from 38.45 to 260.81 µg/mL. The 90% inhibitory dose was at least twice as high. Large differences in the effect of fluconazole analogues 5–15 on individual strains were observed. A synergistic effect on three strains—Candida albicans ATCC 10231, C. albicans ATCC 2091, C. guilliermondii KKP 339—was observed. Fractional inhibitory concentrations FIC50 and FIC90 were tested for the most active lactone, 3-n-butylphthalide, and seven fluconazole analogues. The strongest synergistic effect was observed for the strain C. albicans ATCC 10231, FIC 0.04–0.48. The growth inhibitory amount of azole is from 25 to 55 µg/mL and from 3.13 to 25.3 µg/mL for 3-n-butylphthalide. Based on biological research, the influence of the structure on the fungistatic activity and the synergistic effect were determined.