The equimolar mixture of 3-isochromanone (3.38 mmol) and the corresponding aromatic aldehyde (3.38 mmol) was heated (140°C) in the presence of catalytic amount of piperidine (four drops) in argon atmosphere under stirring for 1 h. Then, the reaction mixture was taken up in ethanol and cooled down. The crystals separated were filtered off and washed with cold ethanol. The products were purified by means of column chromatography. (See details at the compounds.) The conversion was always 100%.

Obtained from a mixture of 3-isochromanone (0.50 g; 3.38 mmol) and 4-nitrobenzaldehyde (0.51 g, 3.38 mmol) with the above method. The reaction mixture was separated by column chromatography (silica gel, CH₂Cl₂/CH₃OH = 10:0.01) to afford 17E (85%) and 17Z (15%) based on separation (0.69 g, 72%). 17E R_f = 0.37 (silica gel, dichloromethane/methanol = 10:0.05), yellow crystalline solid from methanol. m.p. 145–146°C. IR ν_max (cm^–1) (KBr) 1726 (st, C = O). ¹H NMR (500 MHz, dmso-d₆) δ (ppm) 5.47 (s, 2H), 7.13 (d, J = 7.6 Hz, 1H), 7.20 (t, J = 7.2 Hz, 1H), 7.38 (dt, J = 7.6, 0.7 Hz, 1H), 7.47 (d, J = 7.6 Hz, 1H), 7.72 (d, J = 8.7 Hz, 2H), 7.79 (s, 1H), 8.21 (d, J = 8.7 Hz, 2H).¹³C NMR. (125 MHz, dmso-d₆) δ (ppm) 68.8, 123.7, 125.8, 127.0, 128.1, 128.2, 129.0, 129.1, 130.4, 133.5, 135.0, 141.3, 147.2, 167.1, ³J(Hα-C3) = 7.1 Hz. Anal Calcd for C₁₆H₁₁NO₄: C, 68.33; H, 3.94; Found: C, 68.22; H, 4.11.

Prepared from a mixture of 3-isochromanone (0.50 g; 3.38 mmol) and mesitaldehyde (98%) (0.51 g, 3.37 mmol) with the above method (0.82 g, 89%). R_f = 0.52 (silica gel, dichloromethane/methanol = 10:0.01), m.p.155°C (dec.) yellow crystalline solid from methanol. IR ν_max (cm^–1) (KBr) 1726 (st, C = O). Anal Calcd for C₁₉H₁₈O₂: C, 81.99; H, 6.52; Found: C, 82.18; H, 6.65.

Prepared from a mixture of 3-isochromanone (0.50 g; 3.38 mmol) and p-tolualdehyde (0.41 g, 3.38 mmol) with the above method. The reaction mixture was purified via column chromatography (silica gel, toluene/ethyl acetate = 95.5:4.5) to afford 19E (0.49 g, 58%). R_f = 0.49 (silica gel, toluene/ethyl acetate = 10:1), m.p.125–26°C, yellow crystalline solid from methanol. IR ν_max (cm^–1) (KBr) 1716 (st, C = O). Anal Calcd for C₁₇H₁₄O₂: C, 81.58; H, 5.64; Found: C, 80.69; H, 5.70.

Prepared from a mixture of 3-isochromanone (0.50 g; 3.38 mmol) and o-tolualdehyde (0.41 g, 3.38 mmol) with the above method. The reaction mixture was purified via column chromatography (silica gel, toluene/ethyl acetate = 95.5:4.5) to afford 20E (0.42 g, 50%). R_f = 0.49 (silica gel, toluene/ethyl acetate = 10:1), m.p.78°C (dec.), yellow crystalline solid from methanol. IR ν_max (cm^–1) (KBr) 1722 (st, C = O). Anal Calcd for C₁₇H₁₄O₂: C, 81.58; H, 5.64; Found: C, 81.70; H, 5.79.

Prepared from a mixture of 3-isochromanone (0.50 g; 3.38 mmol) and 4-anisaldehyde (0.47 g, 3.34 mmol) with the above method. The reaction mixture was purified via column chromatography (silica gel, toluene/ethyl acetate = 95.5:4.5) to afford 21E (0.73 g, 81%). R_f = 0.43 (silica gel, toluene/ethyl acetate = 10:1), m.p.129–131°C, yellow crystalline solid from methanol. IR ν_max (cm^–1) (KBr) 1707 (st, C = O). Anal Calcd for C₁₇H₁₄O₃: C, 76.68; H, 5.30; Found: C, 76.79; H, 5.49.

Prepared from a mixture of 3-isochromanone (0.50 g; 3.38 mmol) and 3-anisaldehyde (0.47 g, 3.34 mmol) with the above method. The reaction mixture was purified via column chromatography (silica gel, toluene/ethyl acetate = 95.5:4.5) to afford 22E (0.59g, 66%), R_f = 0.43 (silica gel, toluene/ethyl acetate = 10:1), m.p.102–103°C, yellow crystalline solid from methanol. IR ν_max (cm^–1) (KBr) 1719 (st, C = O). Anal Calcd for C₁₇H₁₄O₃: C, 76.68; H, 5.30; Found: C, 76.79; H, 5.48.

Prepared from a mixture of 3-isochromanone (0.50 g; 3.38 mmol) and 4-chlorobenzaldehyde (0.48 g, 3.34 mmol) with the above method. The reaction mixture was purified via column chromatography (silica gel, toluene/ethyl acetate = 95.5:4.5) to afford 23E (0.68 g, 74%), R_f = 0.44 (silica gel, toluene/ethyl acetate = 10:1), m.p.163–165°C, yellow crystalline solid from methanol. IR ν_max (cm^–1) (KBr) 1716 (st, C = O). Anal Calcd for C₁₆H₁₁ClO₂: C, 70.99; H, 4.10; Found: C, 71.15; H, 4.22.

Obtained from a mixture of 3-isochromanone (0.50 g; 3.38 mmol) and 3-nitrobenzaldehyde (0.51 g, 3.38 mmol) with the above method. The reaction mixture was separated by column chromatography (silica gel, dichloromethane/ methanol = 10:0.05) to afford 65% 24E and 35% 24Z stereoisomers (0.56 g, 58%). 24E: R_f = 0.55 (silica gel, dichloromethane/methanol = 10:0.1), m.p. 183°C, yellow crystalline solid from methanol. IR ν_max (cm^–1) (KBr) 1720 (st, C = O). Anal Calcd for C₁₆H₁₁N₁O₄: C, 68.33; H, 3.94; Found: C, 68.22; H, 4.19.

Prepared from a mixture of 3-isochromanone (0.50 g; 3.38 mmol) and 3,4,5-trimethoxybenzaldehyde (95%) (0.70 g, 3.38 mmol) with the above method. The reaction mixture was purified via column chromatography (silica gel, toluene/ethyl acetate = 95.5:4.5) to give 25E (0.75 g, 68%), R_f = 0.34 (silica gel, toluene/ethyl acetate = 8:2), m.p.165–167°C, yellow crystalline solid from methanol. IR ν_max (cm^–1) (KBr) 1723 (st, C = O). Anal Calcd for C₁₉H₁₈O₅: C, 69.93; H, 5.56; Found: C, 70.11; H, 5.64.

Prepared from a mixture of 3-isochromanone (0.50 g; 3.38 mmol) and veratraldehyde (95%) (0.60 g, 3.43 mmol) with the above method. The reaction mixture was purified via column chromatography (silica gel, toluene/ethyl acetate = 95.5:4.5) to give 26E as a faint yellow crystalline solid from methanol (0.71g, 64%), R_f = 0.36 (silica gel, toluene/ethyl acetate = 8:2), m.p.129–131°C. IR ν_max (cm^–1) (KBr) 1717 (st, C = O). Anal Calcd for C₁₈H₁₆O₄: C, 72.96; H, 5.44; Found: C, 73.09; H, 5.58.

Obtained from a mixture of 3-isochromanone (0.50 g; 3.38 mmol) and piperonal (0.51 g, 3.37 mmol) with the method above. The reaction mixture was separated by column chromatography (silica gel, toluene/ethyl acetate = 95.5:4.5) to afford 40% 27E and 60 % 27Z stereoisomers (0.63 g, 66%). 27E R_f = 0.42 (silica gel, toluene/ethyl acetate = 10:1), m.p.153–155°C, yellow crystalline solid from methanol. IR ν_max (cm^–1) (KBr) 1716 (st, C = O). Anal Calcd for C₁₇H₁₂O₄: C, 72.85; H, 4.32; Found: C, 72.95; H, 4.50.

Obtained from a mixture of 3-isochromanone (0.50 g; 3.38 mmol) and 3-chlorobenzaldehyde (95%) (0.50 g, 3.38 mmol) with the above method. The reaction mixture was separated by column chromatography (silica gel, dichloromethane) to afford 80% 28E and 20% 28Z (0.61 g, 67%). 28E R_f = 0.37 (silica gel, dichloromethane), m.p.132–133°C, yellow crystalline solid from methanol. IR ν_max (cm^–1) (KBr) 1724 (st, C = O). Anal Calcd for C₁₆H₁₁ClO₂: C, 70.99; H, 4.10; Found: C, 71.18; H, 4.23.

Prepared from a mixture of 3-isochromanone (0.50 g; 3.38 mmol) and 4-hydroxybenzaldehyde (0.41 g, 3.38 mmol) with the above method. The reaction mixture was purified via column chromatography (silica gel, dichloromethane/methanol = 10:0.05) to give 29E as a faint yellow crystalline solid from methanol (0.62 g, 73%). R_f = 0.14 (silica gel, dichloromethane/methanol = 10:0.15), m.p. 212–213°C. IR ν_max (cm^–1) (KBr) 3444 (st, OH), 1703 (st, C = O). Anal Calcd for C₁₆H₁₂O₃: C, 76.18; H, 4.79; Found: C, 76.30; H, 4.91.

Prepared from a mixture of 3-isochromanone (0.50 g; 3.38 mmol) and vanillin (0.51 g, 3.38 mmol) with the above method. The reaction mixture was purified via column chromatography (silica gel, dichloromethane/methanol = 10:0.05) to give 30E as a faint yellow crystalline solid from methanol (0.60 g, 63%), R_f = 0.24 (silica gel, dichloromethane/methanol = 10:0.1), m.p. 140–141°C. IR ν_max (cm^–1) (KBr) 3346 (st, OH), 1696 (st C = O). Anal Calcd for C₁₇H₁₄O₄: C, 72.33; H, 5.00; Found: C, 72.41; H, 5.12.

Strains used in these experiments: C. albicans wild-type ATCC 26555, C. albicans wild-type SC5314 (ATCC MYA-2876), C. albicans wild-type ATCC 90028, S. cerevisiae wild-type CVX12-3A (MATα ura3-373-251-328 his4-34 leu2-3-112), S. cerevisiae MATα pbr1-1 leu1 ura3-373-251-328, S. pombe wild-type 972 h^–, S. pombe pbr1-8 leu1-32 h^–, S. pombe pbr1-6 leu1-32 h^–.

The antifungal activity of isochromanones was determined with agar dilution method by using Sabouraud-chloramphenicol agar as previously published (Zacchino et al., 1997, 1999). Stock solutions of tested compounds (10 mg/mL in DMSO) were diluted to afford serial 2-fold dilutions that were added to each medium yielding in concentrations ranging from 0.10 to 250 μg/ mL. The final concentration of DMSO in the assay was not higher than 2.5%.

As standard amphotericin B was applied (Inj. FUNGIZONE 50mg Bristol-Myers Squibb Hungary Kft.). The antifungal caspofungin was a generous gift from Merck Sharp & Dohme.

Standard YEPD (Yeast Extract, Peptone, Dextrose) (Sherman, 1990), YEPD+S (YEPD with 1.2M sorbitol), YES (Yeast Extract, Dextrose and Supplements of Adenine, Histidine, Leucine, Uracil and Lysine) (Alfa et al., 1993) and YES+S (YES with 1.2 M sorbitol) (Ribas et al., 1991; Ishiguro et al., 1997), media were used for determination of MIC values of the tested compounds. A stock solution of 10 mg/ml of each tested compounds was prepared in DMSO and serial dilutions in DMSO were prepared. The final concentration of DMSO was 2% in the YEPD medium. This concentration of DMSO does not influence the viability and sensitivity of the tested yeast strains. The serial dilutions of compounds were dispensed into test tubes with 1 ml of YEPD or YEPD+S to make final concentrations of 200, 100, 50, 25, 12.5, 6.26, 3.125, 1.6, 0.8 and 0.4 μg/ml. The tested yeast strains were previously grown in YEPD, YEPD+S, YES or YES+S agar plates at 30°C for 48 h.

For inoculation of YEPD or YEPD+S macro test tubes a suspension of tested fungus was produced with 10⁵ cfu/ml (cfu - colony forming unit). 10 μl of suspension was used for inoculation of each tube. After 48 h incubation at 30°C the tubes were checked. The MIC value is the lowest concentration of the tested compound that could inhibit the fungal growth and the medium remained clear. From each tube 10 μl culture was plated on YEPD agar medium to check the contamination. The MIC tests were carried out in three parallels.

Micro-culture assays of large numbers of samples were analyzed as described) (Martins et al., 2011). Essentially early log-phase cells were grown in YEPD, YEPD+S, YES or YES+S (1.2 M sorbitol) medium at 30°C, and diluted to cell density of 4 X 10⁶ cells/ml in YEPD, YEPD+S, YES or YES+S medium and aliquots of 100 μl were dispensed into each small test tube (3 ml test tube size). Then, another 100 μl of YEPD, YEPD+S, YES or YES+S with the corresponding dilution of each compound at 2X concentration (4% DMSO) was added. The final volume was 200 μl, with the cells diluted to of 2 X 10⁶ cells/ml and each compound diluted to 1X final concentration, containing increasing concentrations of antifungal (0, 1, 2, 5, 10, 20, 50, 100 and 200 μg/ml) or an equivalent volume of solvent (2% DMSO). The cells were grown at 30°C in an orbital roller and turbidity was analyzed after 24 and 48 h of incubation. The MIC was determined as the minimal concentration of tested compound that produced complete cell growth inhibition and kept the medium clear. The MIC values were calculated from at least three independent experiments.

According to the previous experiments of the authors the GS-specific antifungals showed the following in vivo MIC values – in brackets – for the different S. pombe strains given in μg/ml: S. pombe wild-type: papulacandin (5), enfumafungin (10), pneumocandin (5), caspofungin (10); S. pombe pbr1-8 mutant: papulacandin (>100), enfumafungin (>100), pneumocandin (>100), caspofungin (50); S. pombe pbr1-6 mutant: papulacandin (>100), enfumafungin (>100), pneumocandin (50), caspofungin (30) (Martins et al., 2011). In this study we have used caspofungin as a standard control of the in vivo MIC values in S. pombe, S. cerevisiae and C. albicans cells.

β(1,3)-D-glucan synthase assay was executed as published previously) (Ishiguro et al., 1997; Martins et al., 2011). Cell extracts were obtained from early log-phase cells grown in YES medium at 28°C. Membrane enzyme extracts were resuspended in buffer A (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 1 mM β-mercaptoethanol) containing 33% glycerol and 50 μM GTPγS and stored at −80°C. The standard assay mixture contained 5 μl of enzyme (15–25 μg protein), 150 μM GTPγS, and 2 μl of increasing concentrations of tested compound (0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10 and 20 μg in 2 μl of DMSO, from dilutions of a stock solution of 10 mg/ml in DMSO and kept at −20°C) or an equivalent volume of 2 μl of solvent DMSO, in a total volume of 40 μl. The reaction was incubated for 30 min at 30°C and stopped by addition of 1 ml 10% trichloroacetic acid. The IC50 was determined as the concentration of tested compound that produced half-maximal inhibitory concentration of the in vitro GS activity and the IC30 was the concentration of tested compound that produced 30% inhibition of the maximal activity. All reactions were carried out in duplicate, and the values were calculated from three independent cell cultures.

According to the previous experiments of the authors the control antifungal caspofungin used in this study showed the following in vitro IC50 values – in brackets - of the enzymatic GS activity for the different S. pombe strains given in μg/ml: S. pombe wild-type: caspofungin (0.3); S. pombe pbr1-8 mutant: caspofungin (250); S. pombe pbr1-6 mutant: caspofungin (150) (Martins et al., 2011). In this study we have used caspofungin as a standard control of the IC50 and IC30 values for in vitro assays of enzymatic GS activity.

To study the structure-antifungal activity in the two molecular library of isochromanones (1E-16E and 17E-30E) different type of structures were studied. The effect of the structural changes on the antifungal effect was investigated. Thus the aromatic groups were either homo- or heteroaromatic, the substituents of the aromatic ring were selected as both electron withdrawing and electron donating ones. As for the influence of the stereochemical factor, the tested compounds are of E- or Z-configuration around the exocyclic double bond.

A series of yeasts was chosen for the antifungal screening as wild-type and mutants resistant to the specific fungal cell wall GS inhibitors papulacandins and echinocandins (Cortés et al., 2019): fission yeast S. pombe wild-type (972 h^–) and pbr1-8 and pbr1-6 resistant mutants, budding yeast S. cerevisiae wild-type and pbr1-1 resistant mutant, and the pathogenic dimorphic fungus C. albicans wild-type (ATCC 26555, SC5314 and ATCC 90028) (Castro et al., 1995; Martins et al., 2011). Four types of complete media were used for the fungal culture in the screening: standard YEPD, YES, YEPD+S (1.2 M sorbitol) and YES+S media were applied for the vegetative growth of yeast cells. Sorbitol is an osmotic stabilizer, and YEPD+S or YES+S media were used for the analysis of osmotic protection, either in cell wall deficient mutants or against the lethal antifungal effects (Ribas et al., 1991; Castro et al., 1995; Frost et al., 1995; Cortés et al., 2012; Muñoz et al., 2013).

The screening of the first generation of isochromanones was carried out in YEPD and YEPD+S media (1E-16E). The fungal strains analyzed are: C. albicans wild-type ATCC 26555, SC5314 and ATCC 90028, S. cerevisiae wild-type CVX12-3A and pbr1-1 mutant, and S. pombe wild-type (972h^–), pbr1-8 mutant and pbr1-6 mutant. The results are summarized in Table 4. The bold numbers in Tables 4–6 indicate the more efficient compounds. In general, under these conditions the tested compounds (1E-16E) showed a high activity. The weak or moderate efficacy against the C. albicans wild-type strains was observed by some substances (3E, 4Z, 5Z, 10E, 15, and 16E). The efficiency toward the S. pombe pbr1-6 mutant strain was the highest inhibitory activity, except 7E, 11E, 12 and 14E. The highest activity was observed at the 1E- phenyl derivative, 3E –pyridyl derivative, 4Z- methylpyrrolyl compound, 5Z- pyrrolyl derivative, 8Z-2-chloro derivative. As regards the other S. pombe pbr1-8 mutant strain, the antifungal activity of the tested compounds was slightly lower, but the substances with the best activity are partly identical to the best compounds mentioned with pbr1-6, (i.e., 1E, 3E, 4Z, 5Z).

This family of isochromanones showed weak or moderate antifungal activity toward the C. albicans wild-type strains (3E, 4Z, 5Z, 9E and 16E), and 4Z- methylpyrrolyl compound proved to be the most active one

Next, the first generation of isochromanones (1E-16E) was also screened in YES medium (Table 5) and exerted a weaker antifungal potency than in YEPD medium and also than the second family of isochromanones in YES medium (17E-30E) (Table 6). The strains are partly different from the previously mentioned ones (Tables 5, 6) analyzing two wild type C. albicans strains because they behaved similarly in YEPD. It is remarkable that there was no effect against either the C. albicans, the S. cerevisiae, or the S. pombe wild-type strains, except with 30E. Interestingly, the S. cerevisiae and more specially the S. pombe pbr1-6 and pbr1-8 mutants resistant to specific fungal cell wall inhibitors showed different degrees of sensitivity. The results of these antifungal assays in YES medium are displayed in Tables 5, 6.

The S. pombe pbr1-6 mutant strain was the more vulnerable one, as several isochromanones of the first generation exerted a medium or good antifungal effect (e.g., 1E-1Z- phenyl derivatives, 3E –pyridyl derivative, 4Z –methylpyrrolyl, 5Z - pyrrolyl compound and 13E furyl derivative). The S. cerevisiae strains were less sensitive toward the isochromanones, from the first generation of the isochromanones almost only one substance, the 13E furyl derivative - the most efficient antifungal from this series- proved to be efficient and 1E and 11E to be slightly efficient against the S. cerevisiae pbr1-1 mutant strains.

We wanted to see the bioactivity of the second family of isochromanones to see if they had a better antifungal activity in YES with respect to WT and pbr mutants. This family of compounds (17E-30E), generally showed higher antifungal activity than the first generation (Tables 5, 6). With the exception of 30E- a vanillin derivative – all of the compounds were inefficient against the wild-type strains of S. pombe, S. cerevisiae and C. albicans. Several substances proved to be very active specially against the S. pombe pbr1-6 mutant strain, some compounds with electron withdrawing groups as 17Z -4-nitrophenyl derivative, 23E -4-chlorophenyl derivative, 28E-28Z -3-chlorophenyl derivatives, but some substances with electron donating groups also exerted an excellent antifungal potency as 19E -4-methylphenyl derivative, 21E 4-methoxyphenyl derivative. Some compounds showed a medium antifungal potency (as 20E, 26E). As regards the antifungal activity of the E-Z stereoisomers, there was a difference in the potency of 17E and 17Z, the latter was ten times more efficient.

Most of the previously mentioned compounds showed antifungal activity also against the S. pombe pbr1-8 mutant strain, although in some cases it was weaker being the most efficient: 17Z -4-nitrophenyl derivative, 20E -2-methylphenyl derivative, 22E- 3-methoxyphenyl derivative. Out of the second generation of isochromanones the 30E – vanillin derivative - had the broadest spectrum showing antifungal activity against all of the strains except the C. albicans wild-type strains.

The type of medium i. e. composition, incubation times and temperature can influence the growth and detectability of fungi from the clinical samples and the MIC values of the tested strains (Als et al., 2010; Cruz et al., 2013; Balouiri et al., 2016). The Sabouraud and YEPD media are effective and most useful as media for subculture (Washington et al., 2006). Modified RPMI medium is proposed as a standard medium for determination of MIC values of fungi (Washington et al., 2006; Espinel-Ingroff and Pfaller, 2007; Clinical and Laboratory Standard Institute, 2008). In our experiments we used the standard YES and YEPD media and they were optimal for our experimental strains and assays.

Summarizing the results of the antifungal screenings, the most efficient compounds with activity and the lowest MIC values against several strains are the following compounds ordered by their antifungal activity: 1E- phenyl derivative, 3E –pyridyl derivative, 4Z- methylpyrrolyl compound, 5Z- pyrrolyl derivative, 8Z-2-chloro derivative, 9E – 2,3-dihydroxy derivative and the 15 coumarine from the first family, from the second family the 17Z -4-nitrophenyl, 19E -4-methylphenyl, 23E -4-chlorophenyl and 28E-28Z -3-chlorophenyl derivatives.

In order to explore the mechanism of action of the tested compounds, the osmotic protection against the antifungal effect was analyzed. The inhibitory effect of the compounds in YEPD and YES media were compared with their inhibitory effect in YEPD or YES medium containing 1.2 M sorbitol (YEPD+S or YES+S). These media have been used to detect osmotically fragile mutants that can be rescued in the presence of sorbitol. These mutants present defects in their cell wall synthesis and composition (Ribas et al., 1991; Cortés et al., 2012; Muñoz et al., 2013). Similarly, the osmotic protection can partially protect the cells against the specific cell wall synthesis antifungals (Frost et al., 1995). Therefore, we tested in YEPD+S and YES+S the inhibitory effect of the compounds that showed some in vivo inhibition in YES medium (Tables 4–6). In all of the cases sorbitol protected the cells increasing the in vivo MIC of the tested compound (Tables 4–6), suggesting that the mode of action of the compounds could be inhibiting the synthesis of essential cell wall polymers, as it has been described for the specific GS inhibitors papulacandins and echinocandins (Martins et al., 2011; Brown et al., 2012; Kathiravan et al., 2012; Cortés et al., 2019). To explore this possibility of the mode of action of the tested drugs, their putative inhibitory capacity of the enzymatic GS activity was also examined in vitro (Tables 7, 8). According to these tests, the first family of isochromanones acted as poor inhibitor of this enzyme, compounds 1E – 1Z showed weak inhibitory effect against the in vitro GS of S. pombe pbr1-6 mutant strain (Table 7). Other compounds that had no in vivo effect showed some weak in vitro inhibitory effect detected as IC30 (30% of the maximal inhibitory concentration) but not as IC50 (half-maximal inhibitory concentration), indicating that some in vitro inhibition was present but it never or hardly reached the 50% of inhibition. Interestingly, the in vitro inhibition was higher in the GS enzyme of pbr1-6 mutant, as observed in vivo in living cells. All these data are in accordance with their weak antifungal effect.

Regarding the second generation of isochromanones, they produced a much higher inhibitory effect against the GS of S. pombe pbr1-6 mutant strain (as 17E–17Z – 4-nitrophenyl, 18E – 2,4,6-trimethylphenyl, 19E – 4-methylphenyl, 22E – 3-methoxyphenyl and the 30E - vanillin derivative, Table 8). The inhibition of pbr1-6 GS was even stronger considering the IC30 value compared to that of wild-type GS. Concerning the influence of the aromatic moieties there are both electron withdrawing and electron donating ones among these substituents. The inhibition of these compounds on the other S. pombe pbr1-8 mutant strain was slight (17E, 23E, 25E, 26E, 28E, 28Z, and 30E). As before, the effect was more pronounced considering the IC30. Finally, the wild-type GS also showed a noticeable sensitivity to drug 30E. These promising results open new possibilities of antifungal strategies that will require, however, to be explored in deep with echinocandin resistant mutants of pathogenic fungi in future studies.