Melting points (m.p.) were obtained using a microscope apparatus (XT-4, Beijing Tech Instrument Co., China). Nuclear magnetic resonance (¹H NMR and ¹³C NMR) was determined on a Bruker NMR spectrometer (Bruker, Germany). High-resolution mass spectrometry (HRMS) was performed on a Thermo Scientific Q Exactive Plus instrument (Thermo Fisher Scientific, United States).

Intermediates 1 and 2 were obtained by referring to the previously reported methods (Wu W. et al., 2020).

To a 100-ml three round-bottom flask, intermediate 2 (0.01 mol), ethyl 4-hydroxybenzoate (0.012 mol), Cs₂CO₃ (0.02 mol), and acetone (50 ml) were added. After reacting for 2–4 h at room temperature, the solvent was vacuum evaporated. The residues were recrystallized from ethanol to give pure intermediate 3.

To a solution of intermediate 3 (20 mmol) in 40 ml absolute methanol, 80% hydrazine hydrate (60 mmol) was added dropwise. After reacting for 5–7 h under reflux conditions, the reaction was quenched to room temperature. The white solids precipitated from the reaction solution were filtrated and recrystallized from ethanol to give pure intermediate 4.

To a mixture of intermediate 4 (30 mmol), KOH (45 mmol), and ethanol (500 ml), carbon disulfide (36 mmol) was added dropwise. The white precipitates were filtered, dried under vacuum, and then added to 30 ml precooled concentrated H₂SO₄. After stirring for 2 h at 0°C, the mixture was poured into 1,000 ml ice water and neutralized with sodium bicarbonate saturated solution (Wu et al., 2016a; Wu et al., 2016b). The filtrate was acidified with 5% hydrochloric acid, and the produced solid was filtered and recrystallized from ethanol to give the key intermediate 5.

Intermediate 5 (2 mmol), NaOH (2.2 mmol) dissolved in 15 ml water, and substituted benzyl chloride (2.1 mmol) were added in a 100-ml three round-bottom flask and stirred at room temperature for 2–4 h (Scheme 1). Upon completion of reaction, the residues were filtered and recrystallized from ethanol to produce the pure target compounds 6a–6t. The physical properties, NMR, and HRMS for title compounds are reported in Supplementary Data S1, and the spectral data of 6a are shown below. 2-((2-methylbenzyl)thio)-5-(4-((6-(trifluoromethyl)pyrimidin-4-yl)oxy)phenyl)-1,3,4-thiadiazole (6a). White solid; yield 65.24%; m. p. 104–107°C; ¹H NMR (600 MHz, DMSO-d ₆ , ppm) δ: 8.99 (s, 1H, pyrimidine-H), 8.04–8.02 (m, 2H, phenyl-H), 7.86 (s, 1H, pyrimidine-H), 7.50–7.48 (m, 4H, phenyl-H), 7.42 (d, 1H, J = 5.4 Hz, phenyl-H), 7.23–7.17 (m, 3H, phenyl-H), 4.65 (s, 2H, -SCH₂-), 2.41 (s, 3H, pyrimidine-CH₃); ¹³C NMR (150 MHz, DMSO-d ₆ , ppm) δ: 170.32, 167.66, 165.34, 159.73, 156.22 (q, J = 35.1 Hz), 154.29, 137.37, 134.12, 130.98, 130.59, 129.74, 128.65, 127.66, 126.62, 123.27, 121.80 (q, J = 272.7 Hz), 116.13, 107.07, 36.66, 19.26; HRMS (ESI) calcd for C₂₁H₁₅ON₄S₂F₃ [M+Na]⁺: 483.05249, found: 483.05316.

The in vitro antifungal activity was determined according to the mycelial growth rate method (Zhang et al., 2018; Wang et al., 2019; Wu Q. et al., 2020). Each target compound (5 mg) was dissolved in DMSO (1 ml) and added to 9 ml H₂O and 90 ml potato dextrose agar (PDA) medium to prepare 9 dishes of mixed PDA plates with a concentration of 50 μg/ml. After that, a 0.4-cm diameter of each test fungus was put onto the middle of mixed PDA plates and fostered in an incubator at 28°C for 3–4 days. After the mycelia diameter of the untreated PDA plate reached 5–6 cm, the inhibition rates I (%) are calculated using the following formula, where C (cm) and T (cm) represent the fungi diameters of the untreated and treated PDA plates, respectively. Inhibition rate  I   ( % ) = ( C − T ) / ( C − 0 . 4 ) × 100

In the ¹H NMR data of compound 6a, a singlet appears at 4.65 ppm and indicates the presence of the -SCH₂- group. The CH proton of the 6-trifluoromethylpyrimidine ring appeared as two singlets at 8.99 and 7.86 ppm. Meanwhile, in the ¹³C NMR data of compound 6a, two signals at 170.32 and 167.66 ppm indicated the presence of C proton in the 1,3,4-thiadiazole group. One quartet at 156.22 ppm indicated the presence of -CF₃ in the pyrimidine fragment. In addition, compound 6a was confirmed correctly by combining HRMS data with the [M + Na]⁺ peaks.

As shown in Table 1, compounds 6c, 6g, and 6h exhibited higher in vitro antifungal activity against Phomopsis sp., and the inhibition rates were 89.6%, 88.7%, and 89.2%, respectively, compared to that of pyrimethanil (85.1%). Meanwhile, Table 1 shows that the inhibitory activity values of compounds 6g, 6h, and 6q against B. cinerea were 86.1%, 90.7%, and 88.3%, respectively, which were superior to that of pyrimethanil (82.8%). In addition, compound 6h possessed similar bioactivity against B. dothidea (82.6%) to that of pyrimethanil (84.4%).

Table 2 shows that compounds 6c, 6g, and 6h had the EC₅₀ values of 25.4, 28.8, and 25.9 μg/ml, respectively, which were better than that of pyrimethanil (32.1 μg/ml). Meanwhile, compounds 6g (EC₅₀ = 57.5 μg/ml) and 6h (EC₅₀ = 50.8 μg/ml) exhibited better in vitro bioactivity on B. cinerea than pyrimethanil (62.8 μg/ml). Meanwhile, compounds 6g (EC₅₀ = 67.8 μg/ml) and 6h (EC₅₀ = 63.6 μg/ml) exhibited lower in vitro bioactivity against B. dothidea than pyrimethanil (57.6 μg/ml).

Further structure–activity relationship analysis indicated that more than 80% of the title compounds showed excellent antifungal activity against Phomopsis sp. and B. cinerea. Meanwhile, changing R₁ (H or CH₃) did not significantly improve the antifungal activity of the compound. Only against Phomopsis sp., the number of compounds (R₁ = H) with activity higher than 80% is twice that of compounds (R₁ = CH₃). In addition, the introduction of strong electron withdraw groups (CN and CF₃) into R₂ was able to enhance the activity of the compounds, while the introduction of an alkyl group (CH₃) cannot obviously improve the antifungal activity of the compounds.