ResearchonCrystal StructureandFungicidalActivityof theAmide Derivatives Based on the Natural Products Sinapic Acid and Mycophenolic Acid

Structural optimization based on natural products is an important and effective way to discover new green pesticides. Here, two series of amide derivatives based on sinapic acid and mycophenolic acid were designed in combination with the fungicidal natural product piperlongumine and synthesized by preparing the carboxylic acid into acyl chloride and then reacting with the corresponding aromatic amines, respectively. .e resulting structures were successively characterized by H NMR, 13 C NMR, and HRMS. .e crystal structures of molecules I-4 and II-5 were analyzed for structure validation. .e in vitro inhibitory activity indicated that most of the target products exhibited fungicidal activity equivalent to or even better than fluopyram against Physalospora piricola. .e in vivo fungicidal activity demonstrated that the compounds I-5 and II-4 displayed almost the same preventative activity as carbendazim and fluopyram at 200 μgmL. .e TEM observation revealed that the fungicidal activity of the target molecules against Physalospora piricola may be due to the influence on the mitochondria in the cell structure. .ese results will provide valuable theoretical guidance for developing the new green fungicides.


Introduction
Agrochemicals are important production materials for agricultural production, and their development plays an important role in ensuring national food security, agricultural product quality, ecological environment safety, and public health [1]. However, the continuous application of traditional chemical fungicides has produced many negative effects including ecological environment pollution, pathogen resistance, and poison for beneficial insects and microorganisms [2,3]. erefore, the development of efficient, safe, low residual, and environmentally friendly green fungicides has become the inevitable trend for pesticide innovation [4,5]. e discovery of lead compounds and the exploration of mechanism of action are the key to the development and innovation of fungicides. Structural optimization based on natural products has become an effective way to develop new green fungicides, which has important guiding significance for practicing new development concepts and promoting green development of agrochemicals [6][7][8][9][10][11]. For example, coumoxystrobin was successfully developed based on natural products strobilurin and coumarin ( Figure 1) [12]. Moreover, natural product strobilurin A-derived methoxyacrylate fungicides have occupied the top position in the market sales of fungicides [6].
Mycophenolic acid (MPA) was discovered by Gosio in 1893 in a strain of Penicillium fungus and was found to possess broad biological activity such as antifungal, antiviral, anticancer, and antipsoriasis properties [13,14]. Sinapic acid is widely distributed in the plants and belongs to hydroxycinnamic acid. Furthermore, commercial fungicides dimethomorph, pyrimorph, and flumorph were successively developed based on natural product cinnamic acid (Figure 2) [15][16][17][18]. In this project, combined with the structure of fungicidal natural product piperlongumine [19,20], two series of amide compounds based on natural products sinapic acid and mycophenolic acid were designed, synthesized, and evaluated their fungicidal activity against the common agricultural pathogens (Figure 3).

Materials and Equipment.
e materials and reagents used in the organic synthesis reactions were of analytical grade and purchased from Energy Chemical and Bide Pharmatech Ltd. Melting points were measured on a X-5 binocular microscope (Yuhua Co., Ltd., China). 1 H NMR and 13 C NMR were provided on a AVANCE NEO-500 MHz spectrometer (Bruker, Germany). HRMS was recorded on a Xevo G2-XS QTof spectrometer (Waters, USA). X-ray crystal structure was determined on a D8 Venture diffractometer (Bruker, Germany). e purification of target compounds was performed by the column chromatography on silica gel (200-300 mesh).

X-Ray Crystal Structure Determination.
e crystals of the target compounds I-4 and II-5 were cultivated from a mixed solvent of methanol, ethyl acetate, and n-hexane, respectively. All measurements were made on a Bruker D8 Venture diffractometer with Mo-Kα radiation (λ � 0.71073Å). e crystal data of the compound I-4 were collected at 298 K, and the colorless crystal is of monoclinic system, space group C 2/c, with a � 28.208 (3)

Fungicidal Activity Measurement.
With fluopyram and carbendazim as positive controls, the mycelial growth inhibition method was used to determine the in vitro inhibitory activities of the target compounds against common agricultural pathogens according to the previously reported procedures [21,22], and each treatment was repeated at least three times. e tested pathogens include Rhizoctonia solani (RS), Gibberella zeae (GZ), Botrytis cinerea (BC), Physalospora piricola (PP), Cercospora circumscissa Sacc. (CS), Colletotrichum capsici (CC), Alternaria kikuchiana Tanaka (AK), and Alternaria sp. (AS). e in vivo fungicidal activity of compounds I-5 and II-4 against Physalospora piricola was performed on apples referring to literature methods [23]. e target molecule (5.0 mg) was dissolved in dimethyl sulfoxide (30 μL) and diluted with 0.1% Tween-80 aqueous solution to provide the test stock solution (200 μg·mL −1 ), which was sprayed with the same volume on healthy apples. Subsequently, the fungi cake containing Physalospora piricola with a diameter of 7 mm was inoculated. After cultivation at 25°C for 5 days, the average lesion area was measured to calculate the preventative activity. Each in vivo fungicidal activity screening was carried out for at least five repeats.

Transmission Electron Microscope (TEM) Investigation.
Physalospora piricola hyphae were obtained by incubation in PDB medium at 25°C for 72 h and centrifugation at 7000 rpm for 3 min, which were then resuspended in PDB medium to treat with compounds I-5 and II-4 (200 μg ·mL −1 ) for 24 h, respectively. Subsequently, the treated hyphae were provided by centrifugation and fixed with 2.5% glutaraldehyde. e ultrastructure observation of the hyphae treated with compounds I-5 and II-4 (200 μg·mL −1 ) was performed by Shiyanjia Lab on a TEM according to the standard procedures.

Organic Synthesis.
Herein, the important intermediates and target molecules I-1-I-5 and II-1-II-5 were provided referring to the reported procedures (Scheme 1). In the preparation of target compound I-1-I-5, the phenolic hydroxyl group in sinapic acid was firstly reacted with acetic anhydride to produce (E)-3-(4-acetoxy-3,5-dimethoxyphenyl)acrylic acid [24], which was further reacted with thionyl chloride under the catalyzed condition of DMF (3 drops) to provide (E)-3-(4-acetoxy-3,5-dimethoxyphenyl) acrylic chloride. Finally, the target compound I-1-I-5 was synthesized by reacting the acyl chloride 2 with the corresponding aromatic amines, respectively. In addition, the condensing reagents such as EDCI-HOBt, HATU-DIEA, or TBTU-DIEA were also taken to explore the condensation of the carboxylic acid 1 and substituted aromatic amines; however, the yields of the products were low. e target compound II-1-II-5 was obtained according to the same steps described above. Subsequently, the obtained structures were identified and characterized by 1 H NMR, 13 C NMR, and HRMS.

Crystal Structure Analysis.
e crystal structure analysis is beneficial in investigating the physical and chemical properties of the molecules. In this study, several crystal structure characteristics were also illustrated through the crystal structures and packing of molecules I-4 and II-5 ( Figure 4, CCDC numbers 2095769 and 2095768). e selected bond lengths and angles are presented in Table 1, and the selected dihedral angles are shown in Table 2.    From the crystal packing, the π-π interactions occurred between the benzene rings of the adjacent molecules, which strengthen the integration of the crystal molecules (Figures 4(c) and 4(d)).

Fungicidal Inhibitory Activity.
e in vitro inhibitory activities of the target compounds against the common agricultural pathogens were investigated, and the results are shown in Table 3. From the data, most of the target compounds exhibited weak-to-moderate fungicidal activity against Gibberella zeae, Rhizoctonia solani, Botrytis cinerea, Cercospora circumscissa Sacc, Alternaria kikuchiana Tanaka, Colletotrichum capsici, and Alternaria sp. However, all compounds showed moderate-to-good fungicidal activity against Physalospora piricola, even better than fluopyram. For example, compounds I-1, I-4, and I-5 exhibited higher inhibitory activity than fluopyram, with the inhibitory rates of 76.2%, 73.3%, and 73.5%, respectively. It can be concluded that the compounds I-1-I-5 and II-1-II-5 displayed high selectivity for the fungicidal activity against Physalospora piricola. In terms of the relationship between the structures and the initial inhibitory activity, the structural modification had different effects on the inhibitory activities of target compounds against the different pathogens. For example, compared with the electron-withdrawing trifluoromethyl and chlorine groups, the introduction of the electron-donating methyl, methoxy, or isopropoxy group at the benzene ring was beneficial to improving the fungicidal activity of I-4 and I-5 against Physalospora piricola. For instance, the inhibition rates of compounds I-4 and I-5 were 73.3% and 73.5%, respectively, which were apparently higher than those of compounds I-2 and I-3. However, this structural modification had no significant effects on the inhibitory activity of the compounds II-2-II-5 against Physalospora piricola. To further investigate the fungicidal activity of the target compounds against Physalospora piricola, the EC 50 values were measured and the results are exhibited in Table 4. It could be found that most of the target compounds exhibited fungicidal activity equivalent to or even better than

TEM Observation.
To further explore the effects of molecules I-5 and II-4 on the hyphae, the ultrastructure of Physalospora piricola hyphae treated with distilled water, compounds I-5 and II-4 (200 μg·mL −1 ), was observed on a TEM, and the results are illustrated in Figure 6. From the data, the cell wall and plasma membrane of the cell structures in the control and tested groups were normal, and mitochondria could be clearly observed in the control group. However, the mitochondria in the cell structure treated with I-5 and II-4 were blurred or even disappeared. Based on this, it could be speculated that the fungicidal activity of the target compounds against Physalospora piricola may be due to the influence of I-1-I-5 and II-1-II-5 on the mitochondria in the cell structure.

Conclusion
In summary, two series of sinapic acid-derived and mycophenolic acid-derived amide derivatives were designed and synthesized. e obtained structures were characterized by 1 H NMR, 13 C NMR, HRMS, and X-ray crystal diffraction. e in vitro and in vivo fungicidal activity screening indicated that compared with other tested pathogens, most of the target compounds exhibited excellent fungicidal activity against Physalospora piricola, of which the compounds I-5 and II-4 displayed almost the same preventative activity as carbendazim and fluopyram at 200 μg·mL −1 . e TEM observation further revealed that the fungicidal activity of the target compounds against Physalospora piricola may be due to the influence on the mitochondria in the cell structure.

Data Availability
e data used to support the findings of this study are included within the article and the supplementary information file(s). Crystallographic data for the structures reported in this manuscript have been deposited with the Cambridge Crystallographic Data Centre under the CCDC numbers: 2095769 (compound I-4) and 2095768 (compound II-5). Copies of these data can be obtained free of charge from http://www.ccdc.cam.ac.uk/ data_request/cif.

Conflicts of Interest
ere are no conflicts of interest to declare.

Supplementary Materials
e supporting information contained X-ray crystal data of the compounds I-4 and II-5, and 1 H NMR, 13 C NMR, and