Design, Synthesis, In Vitro Antifungal Activity and Mechanism Study of the Novel 4-Substituted Mandelic Acid Derivatives

Plant diseases caused by phytopathogenic fungi are a serious threat in the process of crop production and cause large economic losses to global agriculture. To obtain high-antifungal-activity compounds with novel action mechanisms, a series of 4-substituted mandelic acid derivatives containing a 1,3,4-oxadiazole moiety were designed and synthesized. In vitro bioassay results revealed that some compounds exhibited excellent activity against the tested fungi. Among them, the EC50 values of E13 against Gibberella saubinetii (G. saubinetii), E6 against Verticillium dahlia (V. dahlia), and E18 against Sclerotinia sclerotiorum (S. sclerotiorum) were 20.4, 12.7, and 8.0 mg/L, respectively, which were highly superior to that of the commercialized fungicide mandipropamid. The morphological studies of G. saubinetii with a fluorescence microscope (FM) and scanning electron microscope (SEM) indicated that E13 broke the surface of the hyphae and destroyed cell membrane integrity with increased concentration, thereby inhibiting fungal reproduction. Further cytoplasmic content leakage determination results showed a dramatic increase of the nucleic acid and protein concentrations in mycelia with E13 treatment, which also indicated that the title compound E13 could destroy cell membrane integrity and affect the growth of fungi. These results provide important information for further study of the mechanism of action of mandelic acid derivatives and their structural derivatization.


Introduction
Plant diseases caused by phytopathogenic fungi are a serious threat to the process of crop production and cause large economic losses to global agriculture [1,2]. More than 8000 species of fungi are known to cause plant diseases, such as Fusarium graminearum (F. graminearum), Gibberella saubinetii (G. saubinetii), Sclerotinia sclerotiorum (S. sclerotiorum), Botrytis cinerea (B. cinerea), etc., and these fungi cause 85% of plant diseases and have caused huge economic crop losses [3]. Moreover, some fungi can also produce toxins that are harmful to animal and human health, such as aflatoxins, fumonisins, and trichocenes, which will last for many years [4,5]. Therefore, the prevention and control of fungal diseases have become an urgent problem in agricultural production. Currently, chemical control is still an effective method to prevent plant fungal diseases. However, the frequent use and abuse of chemical pesticides not only bring about environmental pollution and pesticide residue problems but also accelerate the emergence of resistance in pathogenic fungi [6,7]. At present, it is urgent to develop new antifungal pesticides with high efficiency, novel action mechanism, no cross-resistance, and environmental friendliness [8].
To obtain high antifungal activity compounds with a novel action mechanism, a series of 4-substituted mandelic acid derivatives containing a "1,3,4-oxadiazole thioether" were designed and synthesized ( Figure 2). In vitro antifngal bioassays indicated that some compounds exhibited excellent antifungal activities against three tested fungi (G. saubinetii, V. dahlia, and S. sclerotiorum). In order to investigate the antifungal mechanism of the highly active compounds, we conducted morphological studies using the compound E13. The morphological study of G. saubinetii hyphae with the treatment of title compound E13 by FM and SEM revealed that it could remarkably break the surface morphology of mycelia. Furthermore, the results of intracellular nucleic acid and protein leakage experiments further demonstrated that title compound E13 could destroy the integrity of the cell membrane of G. saubinetii, thus inhibiting the growth of mycelium. These results provide important clues for the further mechanism study and derivatization of mandelic acid derivatives used for plant disease control.  [14]. Compound 2 is from Wang et al., 2019 [15]. Compound 3 is from Xiang et al., 2020 [16]. Compound 4 is from Wu et al., 2019 [17]. Compound 5 is from Gan et al., 2015 [18] and Gan et al. [19], 2017. Compound 6 is from Yang et al., 2020 [20]. Compound 7 is from Chen et al., 2020 [21].
To obtain high antifungal activity compounds with a novel action mechanism, a series of 4-substituted mandelic acid derivatives containing a "1,3,4-oxadiazole thioether" were designed and synthesized ( Figure 2). In vitro antifngal bioassays indicated that some compounds exhibited excellent antifungal activities against three tested fungi (G. saubinetii, V. dahlia, and S. sclerotiorum). In order to investigate the antifungal mechanism of the highly active compounds, we conducted morphological studies using the compound E 13 . The morphological study of G. saubinetii hyphae with the treatment of title compound E 13 by FM and SEM revealed that it could remarkably break the surface morphology of mycelia. Furthermore, the results of intracellular nucleic acid and protein leakage experiments further demonstrated that title compound E 13 could destroy the integrity of the cell membrane of G. saubinetii, thus inhibiting the growth of mycelium. These results provide important clues for the further mechanism study and derivatization of mandelic acid derivatives used for plant disease control.

Chemistry
The synthetic route is depicted in Figure 3. As initial materials, 4-substituted mandelic acids were used to synthesize key intermediate ester B. Then, C was obtained from B through hydrazinolysis in the presence of 80% hydrazine hydrate and reacted with CS2

Chemistry
The synthetic route is depicted in Figure 3. As initial materials, 4-substituted mandelic acids were used to synthesize key intermediate ester B. Then, C was obtained from B through hydrazinolysis in the presence of 80% hydrazine hydrate and reacted with CS 2 under base conditions to form intermediate thiol D. Title compounds E 1 -E 28 were synthesized from D with different substituted benzyl halides with K 2 CO 3 in cetonitrile and water. The title compounds were characterized by 1 H nuclear magnetic resonance (NMR), 13 C NMR, 19 F NMR spectroscopy, and high-resolution mass spectrometry (HRMS).

Chemistry
The synthetic route is depicted in Figure 3. As initial materials, 4-substituted mandelic acids were used to synthesize key intermediate ester B. Then, C was obtained from B through hydrazinolysis in the presence of 80% hydrazine hydrate and reacted with CS2 under base conditions to form intermediate thiol D. Title compounds E1-E28 were synthesized from D with different substituted benzyl halides with K2CO3 in cetonitrile and water. The title compounds were characterized by 1 H nuclear magnetic resonance (NMR), 13 C NMR, 19 F NMR spectroscopy, and high-resolution mass spectrometry (HRMS).

Crystal Structure
The crystal structure of title compound E9 was confirmed by X-ray single-crystal diffraction analysis ( Figure 4). A combination of absorption correction and Lorentz polarization was adopted. Structural solutions and F2-based full-matrix least-squares refinements were conducted using SHELXT-14 software. An isotropic refinement of the nonhydrogen atoms was then carried out. Subsequently, the scattering factors of the neutral atoms were analyzed by incorporating an anomalous dispersion correction. The majority of H2O molecules in the crystalline structure were removed using the SQUEEZE option in PLATON.

Crystal Structure
The crystal structure of title compound E 9 was confirmed by X-ray single-crystal diffraction analysis ( Figure 4). A combination of absorption correction and Lorentz polarization was adopted. Structural solutions and F 2 -based full-matrix least-squares refinements were conducted using SHELXT-14 software. An isotropic refinement of the nonhydrogen atoms was then carried out. Subsequently, the scattering factors of the neutral atoms were analyzed by incorporating an anomalous dispersion correction. The majority of H 2 O molecules in the crystalline structure were removed using the SQUEEZE option in PLATON.

In Vitro Antifungal Activity Analysis
The preliminary antifungal activity results of the title compounds against seven plant phytogenic fungi at 100 mg/L are shown in Table 1. As indicated, some title compounds exhibited excellent inhibitory activities against the seven tested fungi at a concentration of 100 mg/L. In detail, compounds E 10 , E 13, and E 14 showed good antifungal activity against G. saubinetii, with inhibition rates of 78.5%, 78.3%, and 80.2%, respectively, which were higher than mandipropamid (19.1%). The inhibition rates of E 6 , E 10 , E 13 , E 14, and E 20 against V. dahliae were 93.8%, 91.8%, 100%, 95.9%, and 94.4%, respectively, which were much higher than mandipropamid (27.2%). Compounds E 9 , E 13, and E 17 exhibited 92.8%, 92.5%, and 89.7% inhibition of S. sclerotiorum, respectively, which was much better than that of the commercialized fungicide mandipropamid (14.0%). Compounds E 10 , E 13, and E 14 showed good antifungal activity against Fusarium oxysporum, with inhibition rates of 69.4%, 70.3%, and 68.8%, respectively, which were higher than mandipropamid (26.5%). Compounds E 5 , E 7 , E 10, and E 13 showed remarkable antifungal activity against T. cucumeris, with inhibition rates of 84.9%, 81.4%, 81.1%, and 87.5%, respectively, which were higher than hymexazol (79.3%). These results indicate the broad-spectrum inhibitory activities of 4-substituted mandelic acid derivatives. As shown in Table 2, some title compounds showed obviously superior antifungal activity than commercial pesticides. For example, the EC 50 values of E 13 , E 14, and E 17 against G. saubinetii were 20.4, 21.5, and 22.0 mg/L, respectively; the EC 50 values of E 6 , E 13, and E 18 against V. dahlia were 12.7, 18.5, and 15.7 mg/L, respectively; the EC 50 values of E 8 , E 9 , and E 18 against S. sclerotiorum were 13.8, 10.3, and 8.0 mg/L, respectively; the EC 50 values of E 7 and E 13 against T. cucumeris were 5.7 and 7.1 mg/L, respectively; which were much higher than corresponding control fungicides. The preliminary structure-activity relationship analysis showed that when R 2 were the same substituted phenyl groups and R 1 were electron-withdrawing groups, the anti-G. saubinetii and anti-S. sclerotiorum activities were higher than electron-donating groups, such as E 9 > E 2 , E 13 /E 20 > E 6 , E 14 /E 21 > E 7 . When R 1 was the same group and R 2 was substituted phenyl groups, the anti-G. saubinetii and anti-S. sclerotiorum activities were higher than that of non-substituted phenyl groups, such as E 2 /E 7 > E 1 , E 9 /E 10 /E 13 /E 14 > E 8 . In addition, when R 1 is a halogen group and the para-substituent group or orthosubstituted group of R 2 are halogen groups, the anti-G. saubinetii and anti-S. sclerotiorum activities were higher than electron-donating groups, such as E 13 /E 14 > E 9 , E 24 /E 25 > E 22 . Table 1. Inhibition effect of title compounds against seven pathogenic fungi at 100 mg/L a .

Morphological Study of G. saubinetii Fungus with SEM
To investigate the antifungal mechanism, we conducted morphological studies using the compound E 13 . The morphology of G. saubineti treated with title compound E 13 was observed by SEM ( Figure 5). As shown in Figure 5A, hyphae grew normally with the treatment of 1% DMSO (CK group), and the morphology was relatively complete. When treated with the compound E 13 at the concentration of 2 eq. EC 50 , the surface of the mycelia began to shrink obviously, as shown in the red circle in Figure 5B. With the concentration of E 13 increased to 4 eq. EC 50 , the change in the mycelia morphology intensified, and some holes appeared on the surface of the mycelia, which were marked by red arrows in Figure 5C. When the concentration reached 8 eq. EC 50 , all the mycelia were contracted, and some surfaces were seriously damaged, as shown in the red circle in Figure 5D. We speculated that compound E 13 could destroy the cell membrane and wall of G. saubineti, culminating in the death of hyphae.
the compound E13. The morphology of G. saubineti treated with title compound E13 was observed by SEM ( Figure 5). As shown in Figure 5A, hyphae grew normally with the treatment of 1% DMSO (CK group), and the morphology was relatively complete. When treated with the compound E13 at the concentration of 2 eq. EC50, the surface of the mycelia began to shrink obviously, as shown in the red circle in Figure 5B. With the concentration of E13 increased to 4 eq. EC50, the change in the mycelia morphology intensified, and some holes appeared on the surface of the mycelia, which were marked by red arrows in Figure  5C. When the concentration reached 8 eq. EC50, all the mycelia were contracted, and some surfaces were seriously damaged, as shown in the red circle in Figure 5D. We speculated that compound E13 could destroy the cell membrane and wall of G. saubineti, culminating in the death of hyphae.

Morphological Study of G. saubinetii Fungus with FM
To further determine whether the membrane of mycelium was damaged, propidium iodide (PI) dye was used to stain the DNA, and fluorescence microscopy (FM) was used to observe its fluorescence. PI can enter the cell across the damaged cellular membrane and specifically bind with DNA to emit red fluorescence [22]. As shown in panels A, B, and C of Figure 6, the cellular membrane of the hyphal cells without staining was colorless. However, when compared with the blank control ( Figure 6D), strong fluorescence intensity was observed by FM in the G. saubinetii hyphae treated with E13 at a concentration of 2 eq. and 4 eq. EC50 ( Figure 6E,F). Moreover, as shown in Figure 6E,F, the higher the concentration of E13, the more red-colored hyphae appear. Therefore, the FM observation further proved that target compound E13 could destroy the membrane integrity of G. saubinetii, and the effect was dose-dependent

Morphological Study of G. saubinetii Fungus with FM
To further determine whether the membrane of mycelium was damaged, propidium iodide (PI) dye was used to stain the DNA, and fluorescence microscopy (FM) was used to observe its fluorescence. PI can enter the cell across the damaged cellular membrane and specifically bind with DNA to emit red fluorescence [22]. As shown in panels A, B, and C of Figure 6, the cellular membrane of the hyphal cells without staining was colorless. However, when compared with the blank control ( Figure 6D), strong fluorescence intensity was observed by FM in the G. saubinetii hyphae treated with E 13 at a concentration of 2 eq. and 4 eq. EC 50 (Figure 6E,F). Moreover, as shown in Figure 6E,F, the higher the concentration of E 13 , the more red-colored hyphae appear. Therefore, the FM observation further proved that target compound E 13 could destroy the membrane integrity of G. saubinetii, and the effect was dose-dependent.

Mechanistic Study of the Antifungal Activity of E13
Further cytoplasmic content leakage assay confirmed the membrane of mycelia was damaged. Nucleic acid and protein concentrations in mycelial suspensions can be evaluated by measuring the changes in absorbance at 260 and 280 nm [23]. As shown in Figure   Figure 6.

Mechanistic Study of the Antifungal Activity of E 13
Further cytoplasmic content leakage assay confirmed the membrane of mycelia was damaged. Nucleic acid and protein concentrations in mycelial suspensions can be evaluated by measuring the changes in absorbance at 260 and 280 nm [23]. As shown in Figure 7, compared with the blank control (CK) group, the absorbance values at 260 and 280 nm were significantly increased within 2 h after treatment with 2 eq. and 4 eq. EC 50 of compound E 13 and stabilized at approximately 8 h. Therefore, it can be inferred that the nucleic acids and proteins of G. saubinetii mycelia were significantly released after treatment with E 13 , and the effect was dose-dependent. This result was consistent with the morphological observation by SEM and FM experiments. When treated with the title compound E 13, the cell membrane integrity was damaged, and the nucleic acids and proteins were released, thereby inhibiting the growth of fungi.

Mechanistic Study of the Antifungal Activity of E13
Further cytoplasmic content leakage assay confirmed the membrane of mycelia was damaged. Nucleic acid and protein concentrations in mycelial suspensions can be evaluated by measuring the changes in absorbance at 260 and 280 nm [23]. As shown in Figure  7, compared with the blank control (CK) group, the absorbance values at 260 and 280 nm were significantly increased within 2 h after treatment with 2 eq. and 4 eq. EC50 of compound E13 and stabilized at approximately 8 h. Therefore, it can be inferred that the nucleic acids and proteins of G. saubinetii mycelia were significantly released after treatment with E13, and the effect was dose-dependent. This result was consistent with the morphological observation by SEM and FM experiments. When treated with the title compound E13, the cell membrane integrity was damaged, and the nucleic acids and proteins were released, thereby inhibiting the growth of fungi.

Instruments and Chemicals
1 H nuclear magnetic resonance (NMR), 13 C NMR, and 19 F NMR spectra were acquired on a Bruker 400 NMR spectrometer (Bruker Corporation, Karlsruhe, Germany) with tet-

Instruments and Chemicals
1 H nuclear magnetic resonance (NMR), 13 C NMR, and 19 F NMR spectra were acquired on a Bruker 400 NMR spectrometer (Bruker Corporation, Karlsruhe, Germany) with tetramethylsilane (TMS) as the internal standard and DMSO-d 6 as the solvent. High-resolution mass spectrometry (HRMS) data were obtained on a Thermo Scientific Q Exactive (Thermo Scientific, Waltham, MA, USA). X-ray crystallographic data were collected by a D8 QUEST (Bruker Corporation, Karlsruhe, Germany). The morphology of the fungus was observed by a Nova Nano SEM450 scanning electron microscope (SEM) instrument (Thermo Fisher Scientific, Waltham, MA, USA) and an Olympus-BX53F fluorescence microscope (FM) (Olympus Ltd., Tokyo, Japan). The cytoplasmic content leakage assay was performed by Cytation™5 multimode readers (BioTek Instruments, Inc., Winooski, VT, USA). HPLC analysis was performed on LC-2030C (Shimadzu, Kyoto, Japan). Melting points were measured with SGW X-4B binocular microscope melting point apparatus (Inesa, Shanghai, China). All reagents and solvents are commercially available with chemical or analytical purity. Benzyl halides were purchased from Shanghai Haohong Scientific Co., Ltd. (Shanghai, China).

Crystallographic Analysis
A single crystal of title compound E 9 suitable for X-ray diffraction analysis was obtained by slow evaporation of DMF solution at room temperature. Crystallographic data of compound E 9 : colorless crystal, C 17 Table S1 in the Supporting Information. The supplementary data for title compound E 9 have been deposited in the Cambridge Crystallographic Data Centre (accessed on 18 September 2020, http://www.ccdc.cam.ac.uk/conts/retrieving.html) under deposition number 2050255.

General Procedure for the Synthesis of Intermediate B
Intermediate B was synthesized, as reported previously [24]. A mixture of 4-substituted mandelic acid (0.1 mol), 98% sulfuric acid (0.05 mol), and methanol (50 mL) was stirred at 80 • C for 3 h, and the reaction was monitored by TLC. Then, the solvent was concentrated under reduced pressure to obtain B, which was used in the next reaction without further purification.

General Synthesis Procedure for Intermediates C and D
Intermediate B (0.1 mol) and 80% hydrazine hydrate (0.2 mol) were dissolved in methanol (20 mL) and reacted at 100 • C for 30 min. Then, the solvent was concentrated in vacuo to obtain C. To a solution of KOH (0.15 mol) in 20 mL ethanol, C (0.1 mol) was added, and CS 2 (0.2 mol) was slowly added dropwise into the solution, reacted at r.t. for 12 h and then reacted at 80 • C for 10 h. The solution was concentrated in vacuo. The pH value was adjusted to 3 to 4 with 2 M hydrochloric acid solution, and the mixture was extracted with ethyl acetate (50 mL × 3) and dried with anhydrous sodium sulfate. The ethyl acetate was removed under reduced pressure to obtain D, which was used in subsequent reactions without further purification.

General Synthesis Procedure for Target Compounds E 1 -E 28
To a solution of D (4.0 mmol) in CH 3 CN/H 2 O (5.0 mL/5.0 mL), K 2 CO 3 (8.0 mmol) and different benzyl halides (4.0 mmol) were added and reacted at r.t. for 24 h and then concentrated in vacuo. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate = 20/1~10/1) to obtain the title compounds E 1 -E 28 , and the physical and spectral data are provided in the Supporting Information Figures S1-S28.

In Vitro Antifungal Activity
The fungicidal activities of E 1 -E 28 were tested in vitro against seven plant pathogenic fungi (G. saubinetii, V. dahlia, S. sclerotiorum, F. oxysporum, F. proliferatum, T. cucumeris, and P. capsici) using a mycelial growth inhibition method [25,26]. The preliminary activity screening concentration of the title compounds was 100 mg/L. The mycelial dishes of fungi that were used for testing were cut from the PDA medium cultivated at 25 ± 1 • C and were approximately 4 mm in diameter; the disks were inoculated in the middle of a PDA plate with a germ-free inoculation needle and then incubated for 3 to 5 days at the same temperature. DMSO (1%) in sterile distilled water served as a blank control (CK), whereas the commercialized fungicides mandipropamid and hymexazol served as the positive controls. Each treatment was conducted in triplicate. When the mycelia of the blank control grew to 6 cm, the diameters of the mycelia treated with the title compounds were recorded. Inhibitory effects on these fungi were calculated using the formula I (%) = [(C − T)/(C − 0.4)] × 100, where C represents the diameter of fungal growth of the blank control, T represents the diameter of the fungi with the treated compound, and I represents the inhibition rate. Standard deviation (SD) values were calculated based on the inhibition data of three repetitions for each test compound. Based on the in vitro antifungal activity results, the median effective concentrations (EC 50 ) values of the highly active compounds were further determined according to the method described above. A series of activity screening concentrations of the title compounds and positive control consisting of 100, 50, 25, 12.5 or 6.25 mg/L were prepared. EC 50 values were calculated by linear regression analysis with SPSS software 20.0. The regression equations of the title compounds are provided in Tables S2-S6 in the Supporting Information.

Morphological Observation of G. saubinetii by Scanning Electron Microscopy
To observe the effects of the high-antifungal-activity title compounds on the morphology of fungi, the morphology of the hyphae after treatment with the compound E 13 was observed by SEM on the basis of previous methods [27][28][29]. For the SEM experiments, six fungal cakes (4.0 mm) of the G. saubinetii were taken and incubated in PDB (potato dextrose broth) medium at 28 • C and 180 rpm for 24 h, and then compound E 13 was added to each group at different concentrations (0, 2 eq., 4 eq., and 8 eq. EC 50 ), and incubated under the same conditions for 24 h. The mycelia were centrifuged (6000 rpm, 5 min, 4 • C) and washed with PBS buffer (pH 7.2) three times. Then, 2.5% glutaraldehyde was added, and the samples were placed in a refrigerator at 4 • C overnight and dehydrated with gradient ethanol (30%, 50%, 70%, 90%, and 100%) and t-butanol for 15 min. The dehydrated mycelia were dried in a vacuum and sprayed with gold. The mycelia were observed and photographed on a Nova Nano SEM scanning electron microscopy.

Morphological Observation of G. saubinetii by Fluorescence Microscope
According to previously reported methods [30,31], G. saubinetii hyphae were cultured in potato dextrose broth (PDB) medium at 28 • C and 180 rpm for 24 h and then treated with 0, 2, and 4 eq. EC 50 of compound E 13 for 24 h. The PDB medium was removed by centrifuging at 4 • C and 6000 rpm for 5 min, and the hyphae were stained with 10 µL of propidium iodide (PI) solution (20 mg/L). The hyphae were incubated at 37 • C for 15 min in the dark and then washed with PBS three times. A coverslip was placed on the hyphae, and the samples were observed and photographed using an Olympus-BX53F fluorescence microscopy.

Determination of Cytoplasmic Content Leakage
The leakage of cytoplasmic contents from G. saubinetii mycelia treated with E 13 was measured according to the methods described previously with minor modifications [32,33]. Six cakes (4.0 mm) of G. saubinetii fungus were placed in 100 mL of PDB at 28 • C for 3 days at 180 rpm. The hyphae were harvested and washed three times with sterile distilled water. The mycelia (4.0 g) were suspended in 20 mL of distilled water containing compound E 13 at various concentrations (0, 2 eq., and 4 eq. EC 50 ) and incubated on a rotary shaker at 28 • C for 8 h. The absorbance values of the supernatant at 260 nm and 280 nm were determined at 0 h, 2 h, 4 h, 6 h, and 8 h, respectively. Each experiment was conducted in triplicate.

Statistical Analysis
Each experiment was performed in triplicate. The results are expressed as the means ± SD. The EC 50 values were evaluated with the regression equation and r value. All the data in the same group were evaluated by the Q-test. Differences between groups were compared by one-way analysis of variance (ANOVA; Duncan's multiple range test) with p < 0.05. Statistical tests were performed using the SPSS software package (version 20.0, IBM).

Conclusions
A series of 4-substituted mandelic acid derivatives containing a 1,3,4-oxadiazole moiety were designed and synthesized. In vitro bioassay results revealed that some title compounds exhibited excellent activity against seven kinds of pathogenic fungi. Among them, the EC 50 values of E 13 against G. saubinetii, E 6 against V. dahlia, and E 18 against S. sclerotiorum were 20.4, 12.7, and 8.0 mg/L, respectively, which were highly superior to that of the commercialized fungicide mandipropamid. A morphological study of G. saubinetii with FM and SEM indicated that E 13 broke the surface of the hyphae and destroyed cell membrane integrity with the increased concentration, thereby inhibiting fungal reproduction. Further cytoplasmic content leakage determination results showed a dramatic increase of the nucleic acid and protein concentrations in mycelia with E 13 treatment, which also indicated that the title compound E 13 could destroy the cell membrane integrity and affect the growth of fungi. Further mechanistic research is planned for future completion.