Compounds S and C were obtained by isolation from the entire plant of Macleaya microcarpa (Maxim) Fedde according to the method reported by us [26] and used as the starting materials to synthesize S₁–S₈ and C₁–C₈ (Figure 2), respectively, using the synthetic route outlined in Scheme 1.

Structural modifications of S and C included introduction of alkoxyl, cyano and acetonyl at C-6 by nucleophilic addition, and the reduction and oxidation of the C=N double bond at the C-6 position. Compounds S₁–S₅/C₁–C₅ and S₆–S₈/C₆–C₈ were prepared according to the methods recently reported by us [19,26].

The structures of all compounds were elucidated by spectroscopic analyses including ESI-MS, ¹H- and ¹³C-NMR spectra. The spectral data of all the compounds were in agreement with that previously reported by us [19,26]. It should be noted that S₁–S₆ and C₁–C₆ were racemates and used as such for the bioactivity assays. We tried to resolve the racemates to their enantiomers but were unsuccessful. The main reason is that these compounds very easily convert back to their corresponding parent compounds S or C under acidic or even weakly acidic conditions.

Unlike the parent compounds S or C, all the derivatives of S or C lack the C=N⁺ moiety, and their only structural difference lies in their different substituents at the 6 position. S₇ and C₇ were the reduction products of S and C, respectively. The significant difference of the activities between S and S₇ or C and C₇ showed that the iminium moiety in the molecule S or C is the determinant for their antifungal activity.

S₁–S₄ or C₁–C₄ have N,O-acetal structures and both S₅ and C₅ are structurally α-hydroxynitrile derivatives. All these compounds are able to easily convert back to the corresponding parent compound S or C under acidic conditions [27,28], including the slightly acidic environment of the lysosome of cells [29]. Unlike S₁–S₅ and C₁–C₅, compounds S₆–S₈ and C₆–C₈ could not undergo a similar transformation under the same conditions. The significant difference in activities between S₁–S₅ and S₆–S₈ or between C₁–C₅ and C₆–C₈ as well as the lack of a significant difference between the of S₁–S₅ or C₁–C₅ strongly suggest that the activities of S₁–S₅ and C₁–C₅ might well come from their corresponding hydrolytic products, i.e., S or C. In other words, S and C might be the real active compounds of S₁–S₅ and C₁–C₅, respectively, while S₁–S₅ and C₁–C₅ might be only the precursors of S and C. In addition, comparing the activities of C with that of S or C₁–C₅ with S₁–S₅, we could find that for most of the fungi, a 7,8-methoxy group, relative to a 7,8-methylenedioxy group, could enhance the activity to a certain degree. It was worth noting that the structure and antifungal activity relationships were very similar to the case of their reported antibacterial activities [26].

Sanguinarine iodide (S) and chelerythrine iodide (C) were obtained in our laboratory by isolation from the whole plant of M. microcarpa (Maxim) Fedde [24]. Melting points (m.p.) were determined on XT-4 micro-melting point apparatus and are uncorrected. ¹H-NMR and ¹³C-NMR spectra were recorded with a Bruker AVANCE Ⅲ instrument operating at 500 and 125 MHz, respectively, using TMS as an internal standard. ESI-MS was measured on a Trace mass spectrometer. Thiabendazole (TBZ, ≥99.1%), a commercial fungicide standard, was purchased from Sigma-Aldrich Trading Co. Ltd. (Shanghai, China). Dimethyl sulfoxide (DMSO) was purchased from J&K China Chemical Ltd. (Beijing, China). Other reagents were obtained locally and of analytical grade. The water used was redistilled and ion-free. Curvularia lunata, Alternaria alternate, Fusarium solani, Fusarium oxysporum sp. vasinfectum, Valsa mali, Fusarium oxysporum sp. niveum and Pyricularia oryzae were isolated, identified and provided by the Center of Pesticide Research, Northwest A&F University, (Shaanxi, China). These fungi were grown on PDA plates at 28 °C and maintained by periodic subculturing at 4 °C.

Compounds S₁–S₅ and C₁–C₅ were prepared according to the methods and the procedure recently reported by us [19]. Compounds S₆–S₈ and C₆–C₈ were prepared the procedure according to the methods previously reported by us [26]. The spectral data of S₁–S₈ and C₁–C₈ are consistent with our previous papers [19,26].

The antifungal activity in vitro was assayed by the growth rate method [30] with slight modifications. The tested fungi maintained on potato-dextrose-agar (PDA) medium slants were subcultured for 48 h in Petri dishes prior to testing and used for inoculation of fungal strains on PDA plates. The tested compounds were completely dissolved in DMSO, and then diluted by water to provide the stock solution (1.0 µg/mL) in 5% DMSO aqueous solution. The stock solution was completely mixed with the autoclaved PDA medium to provide a medium containing 100 µg/mL of sample and then poured into the Petri dishes in a laminar flow chamber. Thiabendazole solution (1.0 µg/mL) in 5% DMSO aqueous solution and 5% DMSO aqueous solution were used as the treated control and the untreated control, respectively. When the medium in the plates was partially solidified, a 5-mm thick and 4-mm diameter disc of fungus cut from earlier subcultured Petri dishes was placed at the centre of the semi-solid medium. The dishes were kept in an incubator at 28 °C for 72 h. Each experiment was carried out in triplicates. The diameters (in mm) of inhibition zones were measured in three different directions and the growth inhibition rates were calculated according to the following formula and expressed as means ± S.D.: Growth inhibition rate (%) = [(d_c − d₀) − (d_s − d₀)]/(d_c − d₀) × 100 where d₀: Diameter of the fungus cut, d_c: Diameter of the untreated control fungus, d_s: Diameter of the sample-treated fungus.

Based on in vitro antifungal activity screening results, S, S₁, C and C₁ were selected to determine their toxicity. According to the method described above, a stock solution containing 800 μg/mL samples was prepared in 5% DMSO aqueous solution. Then the stock solution was completely mixed with the autoclaved PDA medium to prepare a set of media containing 120, 90, 80, 40, 20, 10, 5 and 2.5 μg/mL samples, respectively. 5% DMSO aqueous solution was used as the untreated control. According to the method described above, the antifungal activity for each concentration was determined. Each experiment was carried out in triplicate. The average inhibition rate for each test was calculated. The concentration of the compound was transformed to the corresponding logarithm value (log₁₀C). A linear regression, namely toxicity regression equation, was established by using the least square method. EC₅₀ values were calculated from the toxicity regression equation.

SPSS statistics V17.0 software was used to analyze the data and establish toxicity regression equationa. Duncan multiple comparison test was performed on the data to determine significant differences between the inhibition rates of different compounds at the same concentration.