Design, Synthesis and Bioactivity Evaluation of Novel 2-(pyrazol-4-yl)-1,3,4-oxadiazoles Containing an Imidazole Fragment as Antibacterial Agents

Imidazole alkaloids, a common class of five-membered aromatic heterocyclic compounds, exist widely in plants, animals and marine organisms. Because of imidazole’s extensive and excellent biological and pharmacological activities, it has always been a topic of major interest for researchers and has been widely used as an active moiety in search of bioactive molecules. To find more efficient antibacterial compounds, a series of novel imidazole-fragment-decorated 2-(pyrazol-4-yl)-1,3,4-oxadiazoles were designed and synthesized based on our previous works via the active substructure splicing principle, and their bioactivities were systematically evaluated both in vitro and in vivo. The bioassays showed that some of the target compounds displayed excellent in vitro antibacterial activity toward three virulent phytopathogenic bacteria, including Xanthomonas oryzae pv. oryzae (Xoo), Xanthomonas axonopodis pv. citri (Xac) and Pseudomonas syringae pv. actinidiae (Psa), affording the lowest EC50 values of 7.40 (7c), 5.44 (9a) and 12.85 (9a) μg/mL, respectively. Meanwhile, compound 7c possessed good in vivo protective and curative activities to manage rice bacterial leaf blight at 200 μg/mL, with control efficacies of 47.34% and 41.18%, respectively. Furthermore, compound 9a showed commendable in vivo protective and curative activities to manage kiwifruit bacterial canker at 200 μg/mL, with control efficacies of 46.05% and 32.89%, respectively, which were much better than those of the commercial bactericide TC (31.58% and 17.11%, respectively). In addition, the antibacterial mechanism suggested that these new types of title compounds could negatively impact the cell membranes of phytopathogenic bacteria cells and cause the leakage of the intracellular component, thereby leading to the killing of bacteria. All these findings confirm that novel 2-(pyrazol-4-yl)-1,3,4-oxadiazoles containing an imidazole fragment are promising lead compounds for discovering new bactericidal agents.


Introduction
Imidazole alkaloids, a common class of five-membered aromatic heterocyclic compounds containing two intersite nitrogen atoms, exist widely in plants, animals, microorganisms and marine organisms [1]. Imidazole is a dominant skeleton in drug development that exists in the core structure of many medicines and pesticides and has been extensively explored by scientific researchers [2,3]. As a good pharmacodynamic group, imidazole can synergically enhance drug efficacy, improve in vitro and in vivo bioactivity, and reduce biotoxicity, and it also possesses excellent bioavailability, good tissue penetration and relatively low adverse reactions [1][2][3]. To date, imidazoles have become one of the most important skeletons in medicine and pesticide discovery, and many novel bioactive molecules have been reported based on this advantageous framework, such as antibacterial [4,5], antifungal [6], antiviral [7], anti-inflammatory [8], anticancer [9], antioxidant [10]
Furthermore, all the structures of the title compounds were identified by NMR and HRMS. In the 1 H NMR spectra of the synthesized title compounds, three single peaks (s) stably occurred in the range of δ 7.51-7.40, 7.06-6.98 and 6.91-6.88 ppm, which belonged to protons of the imidazole ring. Other peaks in the range of δ 6.90-8.20 ppm were contributed by the pyrazol group and phenyl group, respectively. The triplet peak (t) between 4.00 ppm and 3.90 ppm was the CH 2 group, which connected with the imidazole moiety. Meanwhile, other triplet peaks in the ranges of δ 3.82-3.72 and 3.30-3.20 ppm were the signals of the O-CH 2 -(compounds 7a-7f and 8a-8d) and S-CH 2 -protons (compounds 9a-9d), respectively. Specifically, the singlet peaks at δ 4.09-4.07 ppm on the spectra of compounds 8a-8d were the protons of the CH 3 group. The peaks in the range from δ 2.00-1.00 ppm, were for the protons of alkyl linker. In the 13 C NMR spectra, the quartet (q) peaks at δ 130.9-129.6 and 119.5-119.0 ppm showed the carbon of CF 3 and CCF 3 , respectively. Moreover, the characteristic peaks of pyrazol-C 4 and C 5 of compounds 8a-8d and pyrazol-C 3 and C 4 in target compounds 7a-7f and 9a-d appeared at δ 139.0-138. 8 and 109.2-108.2 ppm, respectively. The characteristic peaks of the imidazole moiety were signaled at δ 137.3-137.0, 129.5-129.2 and 118.9-118.0 ppm, respectively. Notably, two high peaks at δ~130.0 and~125.5 ppm in the spectra of target compounds 7a-7f and 9a-d, respectively, were validated as being the carbons of phenyl. The peaks that occurred at δ 48.0-26.0 ppm were mainly contributed by the signals of alkyl linker; distinctively, the CH 3 of target compounds 8a-8d were also signaled at this region ranging from δ 39.9 ppm to 39.8 ppm, and showed as a quartet. In 19 F NMR spectra, the CF 3 signals of compounds 7a-7f and 9a-9d mainly occurred at δ −55.8 or −55.7 ppm, whereas these CF 3 signal appeared in the range from δ −58.0 ppm to −57.8 ppm for compounds 8a-8d. Moreover, in HR(ESI)MS spectra, the exact molecular weights of all the synthesized target compounds were found according to the theoretical values, which stated that the designed molecules had been successfully synthesized. In addition, the purities of the active compounds were >97% by HPLC verification. The above-mentioned analysis declared that the title compounds were successful prepared, and characterization details can be found in the Supplementary Materials.

The 50% Effective Concentration (EC 50 ) Test
Furthermore, to assess the specific antibacterial activity, active compounds with inhibition rates over 50% at 50 µg/mL and the positive controls BT and TC were chosen to determine the EC 50 via our previously reported method [39,40]. The bioassay ( Table 2) results showed that most of the target compounds exhibited excellent antibacterial activity and high selectivity toward Xoo, affording active EC 50   The EC 50 values were also transformed from mass concentrations to molar concentrations, and a similar regularity in the bioactivity of the title compounds was performed. For instance, compound 7c exhibited the lowest EC 50 value of 16.55 µM toward Xoo, which was significantly better than that of BT (97.85 µM) and TC (234.16 µM). Furthermore, compound 9a had outstanding bactericidal effects toward Xac and Psa, giving EC 50 values of 11.76 and 27.75 µM, respectively, which were superior to BT (154.73 and 351.56 µM, respectively) and TC (204.21 and 228.59 µM, respectively).
In addition, according to the statistical analysis, the in vitro EC 50 values showed significant differences toward all the tested pathogens between the active compounds and the positive controls (BT and TC). These outstanding results declared that the title compounds had great potential for controlling intractable phytopathogenic bacteria.

Structure-Activity Relationship (SAR) Analysis
The preliminary SAR analysis showed that the antibacterial activity of 5-(1-phenyl-5-(trifluoromethyl)-1H-pyrazol-4-yl)-1,3,4-oxadiazole-2-ol derivatives decorated with an imidazole moiety 7a~7f presented a fluctuation trend with the increase in the length of the alkyl linker from 4 to 12, affording the best EC 50 values (7.40 and 11.22 µg/mL) toward Xoo and Xac when the carbon number of the alkyl linker was 6 (7c), respectively. For anti-Psa, compounds 7a (n = 6) and 7b (n = 8) showed moderate potencies, with EC 50 values of 40.71 and 28.40 µg/mL, respectively, whereas the ability was sharply reduced when the alkyl linker was over 6 (compounds 7c~7f) with EC 50 values over 50 µg/mL. Thus, appropriate alkyl chain length was beneficial for enhancing antibacterial potency due to the title compounds possessing suitable hydrophobicity. All these results demonstrated that a short alkyl linker is more beneficial for bioactivity than a long alkyl linker, which suggested that the hydrophilicity of the compound had a significant effect on the bioactivity. These molecules might interact with the cell membrane via electrostatic interactions and enter inside the bacterial cell via the endocytosis. Then hydrophobic fragments (alkyl chain) would penetrate the bacterial membrane, disrupt the function of the cell membranes (such as the permeability, etc.) and cause the leakage of intracellular material, thereby leading to the death of bacterial cells. This possible hypothesis is proposed referring to the previous study [45].
To investigate the effect of the position (R 1 ) and variety (R 2 ) of the pyrazole ring on the antibacterial ability, four (1-methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-ol derivatives decorated with an imidazole moiety 8a~8d were synthesized based on target compounds 7c~7f. As shown in Tables 1 and 2, the antibacterial competence on anti-Xac was sharply decreased when the site of the -CF 3 group was transferred from 5-to 3-and the phenyl was replaced by methyl, such as compound 7c (EC 50 = 11.22 µg/mL, n = 6) > compound 8a (EC 50 > 50 µg/mL, n = 6). Similarly, the same results were summarized for compounds 8b~8d against Xoo when the length of the alkyl linker was increased from 8 to 12, which indicated that the 5-(1-phenyl-5-(trifluoromethyl)-1H-pyrazol-4-yl)-1,3,4-oxadiazole-2-ol moiety was more favorable for enhancing the anti-Xoo ability than the 1-methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)-1,3,4-oxadiazol-2-ol moiety. The above findings stated that the increased lipophilic property of R 2 was of more benefit for the bioactivity than that of a low lipophilic group. Meanwhile, when the alkyl length was n = 8, 10 and 12 (compounds 8b~8d), a partly enhanced antibacterial potency appeared toward Xac and Psa, compared to that of the relative compounds 7d~7f, with improved EC 50 values ranging from 11.22 and > 50 µg/mL to 8.72 and 35.24 µg/mL, respectively. These results suggest that the position (R 1 ) and variety (R 2 ) of the substituent on the pyrazole ring have an indeterminate influence on the bactericidal activity, which also relates to the carbon number of the alkyl linker.
Furthermore, these EC 50 values were transformed from mass concentrations to molar concentrations. The antibacterial activities of compounds 7a~7f also presented an undulant trend when the length of the alkyl linker increased from 4 to 12, affording their active EC 50 values that ranged from 16.55 to >100 µM toward Xoo, 25.09 to >100 µM toward Xac and 65.56 to >100 µM toward Psa, respectively. This indicates that the too-long length of alkyl linkers was unfavorable to increase the antibacterial competence, e.g., the compound 7f (n =12) provided EC 50 values over 100 µM toward all the tested bacteria. In addition, the antibacterial competence on anti-Xac and Xoo was sharply decreased when the position of the -CF 3 group was transferred from 5-to 3-and the phenyl was replaced by methyl, such as compound 7c (EC 50 = 16.55 and 25.09 µM, respectively, n = 6) >compound 8a (EC 50 were over 100 µM, n = 6). However, the significantly improved potency toward Xac was observed when the alkyl linker was n = 8 and 10, e.g., 7d (EC 50 > 100 µM, n = 8) < 8b (EC 50 = 26.40 µM, n = 8), and 7e (EC 50 = 72.79 µM, n = 10) < 8c (EC 50 = 19.76 µM, n = 10). As for anti-Psa, compound 8c showed the enhanced power with the moderate EC 50 value of 79.87 µM. The results suggested that the position (R 1 ) and variety (R 2 ) of the substituent on the pyrazole ring and the length of alkyl linker possessed an uncertain influence on the bactericidal activity. Finally, a globally reduced tendency against Xoo was observed when the O atom was replaced by the S atom, especially the compounds with the alkyl chain of n equals 6 and 10 where the activity decreased significantly, e.g., 7c (EC 50 = 16.55 µM, n = 6) < 9a (EC 50 = 26.77 µM, n = 6), and 7e (EC 50 = 17.46 µM, n = 10) < 9c (EC 50 = 52.51 µM, n = 10). In addition, the compounds possessed an equivalent activity in the alkyl chain of 8 (7d and 9b). For anti-Xac and anti-Psa activities, the S-atom-containing compound 9a (EC 50 values were 11.76 and 27.75 µM, respectively) displayed enhanced antibacterial power to the relative O-atom-containing compound 7c (EC 50 values were 25.09 against Xoo and >100 µM toward Psa, respectively) with the alkyl length of n = 6, but the bioactivities were sharply weakened with the increased alkyl lengths, which declared that the decreased water solubility of the molecule was unfavorable to bioactivity, and the S and O atom possessed an uncertainly effect on the bioactivities.
The ADME properties of compounds 7c and 9a were assessed by using the ADMETlab 2.0 software [46]. The predicted results are displayed in Table S2; these two compounds had acceptable physicochemical properties, ADMET, and drug-like properties. For instance, compounds 7c and 9a had better safety in some aspects (such as AMES toxicity, eye corrosion, eye irritation, etc.). Interestingly, compounds 7c and 9a were shown to meet the Lipinski rule and Golden Triangle. These results suggest that these two compounds possess good pharmacokinetic characteristics for new agrochemical discovery.

In Vivo Bioassays of Compound 7c against Rice Bacterial Blight
Based on the in vitro bioassays, the active compound 7c (the lowest EC 50 value of 7.40 µg/mL toward Xoo) was chosen to evaluate the in vivo antibacterial effects against rice bacterial blight via the pot experiment [39]. The results (Figure 3 and Table 3) showed that compound 7c possessed good in vivo protective and curative activities to manage rice bacterial leaf blight at 200 µg/mL, with control efficacies of 47.34% and 41.18%, respectively, which were much better than those of the commercial bactericide TC (35.12% and 37.50%, respectively) and partly superior to BT (48.28% and 31.37%, respectively). In particular, the designed compounds showed low phytotoxicity toward plants due to the compounds not causing any lesions or necrosis on the rice leaves and stem. These results indicate that compound 7c possesses promising applications for controlling rice bacterial blight and could be considered as a lead molecule to develop novel agricultural bactericides.

In Vivo Bioassays of Compound 9a against Kiwifruit Bacterial Canker
Based on the in vitro bioassays, the active compound 9a (afforded with the lowest EC 50 value of 12.85 µg/mL toward Psa) was chosen to assess the in vivo anti-Psa activity via the pot experiment [47][48][49]. The results (Table 4) showed that compound 9a presented commendable protective and curative activities against kiwifruit bacterial canker at 200 µg/mL with control efficiencies of 46.05% and 32.89%, respectively, which were much better than those of the commercial bactericide TC (31.58% and 17.11%, respectively). For the observations at 14 days after inoculation (Figure 4), severe blackening with pyogenic exudate was observed on the wounds of negative controls (red circle). In contrast, only a little white exudate was discovered around the wounds without signs of obvious deterioration after treatment with compounds 9a and TC, which indicated that our designed molecular skeleton was a promising core to develop a novel bactericidal agent for controlling kiwifruit bacterial canker.

Growth Effect of Compound 7c toward Xoo
To investigate the probable action mechanism of the designed molecules, the growth effect assay was carried out according to the previously reported method with some modifications [50]. The results ( Figure 5) displayed that the bacterial growth was slightly restrained after treatment with 1 EC 50 and 2 EC 50 of compound 7c at an early stage (0-12 h), whereas a rapid increase growth rate was observed after 12 h and kept similar OD 595 values with the CK after 24 h. However, after incubating with the 4 EC 50 compounds, the growth curve showed a significant downward trend. All these findings suggest that compound 7c showed a bacteriostatic effect toward Xoo at the low concentrations (<4 EC 50 ), whereas a bactericidal effect was displayed at the high dosages (>4 EC 50 ).

Morphological Observation of Xoo Cells by Scanning Electron Microscopy (SEM)
To observe the morphological changes and cell membrane integrity of phytopathogens after treatment with our synthesized compounds, SEM technology was employed according to our reported method [40]. As displayed in Figure 6, the morphology and cell membrane integrity of Xoo cells were obviously affected after incubation with compound 7c for 15 h. Clearly, the cells in the negative controls had a full surface with a uniform, complete and regular shape. Comparatively, partial collapse and shrinking occurred in small amounts of Xoo cells when treated with a low concentration of compound 7c at 7.40 µg/mL (1 × EC 50 ) (Figure 6b), and more serious damage was observed at the increased concentrations (Figure 6c-e), with most of the cells collapsing, shrinking, distorting and flattening at 14.80 µg/mL (2 × EC 50 ), 29.60 µg/mL (4 × EC 50 ) and 59.2 µg/mL (8 × EC 50 ). In particular, almost all Xoo cells were severely damaged at the drug dose of 118.2 µg/mL (16 × EC 50 ) (Figure 6f), with the morphology changing to one of shrinking, collapsing and breaking, and leakage holes appearing on the surface of the majority of the Xoo cell membrane. All these observations indicate that compound 7c has a strong impact on the morphology and cell membrane of Xoo cells.

Membrane Permeability Changes by Propidium Iodide (PI) Staining Experiment
Permeability of the cell membrane has important physiological functions for the movement of water inside and outside the cell, the exchange of various substances, and the maintenance of pH and osmotic pressure [51,52]. To study the membrane permeability of the tested phytopathogenic bacteria affected by our designed compounds, the Xoo cells were detected by using a typical PI staining assay [53], in which the nonfluorescent dye PI can bind to DNA and RNA in cells with a damaged cell membrane and produce strong red fluorescence, but it cannot pass through cells with intact cell membranes, so the intensity and distribution of red fluorescence indirectly reflects the membrane permeability [54]. Clearly, the progressively elevated red fluorescence intensity and increased number of fluorescent cells (Figure 7b-f) showed that enhanced membrane permeability of Xoo cells occurred after incubation with compound 7c for 15 h when compared with the cells in the negative control (Figure 7a). All these findings demonstrate that our designed 2-(pyrazol-4-yl)-1,3,4-oxadiazoles could change the cell membrane permeability of Xoo and cause the dysfunction of cell metabolism, which might be a key factor in revealing outstanding antibacterial potency. All these findings are in accordance with the outcomes of SEM observation.

General Synthetic Protocols for Target Compounds 7a~7f, 8a~8d and 9a~9d
As depicted in Scheme 1, the intermediates 2-6 were obtained according to our previously reported methods [37,38]. For the details, see Supplementary Materials. Then, the corresponding dibromo alkane (1.41 mM) was slowly dropped into the reaction system and continually reacted at room temperature for another 2 h. After that, the reaction was diluted with ethyl acetate, washed by saturated ammonium chloride solution, dried using anhydrous sodium sulfate and evaporated under vacuum. Subsequently, the crude product was added into a mixture of imidazole (1.00 mM), NaH (1.20 mM) and DMF (2 mL) under ice bath condition, and reacted at room temperature for 4 h. After that, the reaction was diluted with ethyl acetate, washed by saturated ammonium chloride solution, dried by anhydrous sodium sulfate and evaporated under vacuum. Finally, the target compounds were purified by column chromatography on a silica gel using CH 2 Cl 2 and CH 3 OH (30:1) as the eluant to afford the desired products 7a~7f, 8a~8d and 9a~9d, respectively. All the NMR and HRMS spectra for target compounds are displayed in the Supplementary Materials.

Methods for General Bioassays
The in vitro bioassays against three phytopathogenic bacteria Xoo, Xac and Psa, in vivo pot experiment for managing rice bacterial leaf blight and SEM imaging experiment were according to our reported methods [39][40][41][42]. All the detailed descriptions are displayed in Supplementary Materials.

In Vivo Antibacterial Bioassay of Compound 9a against Kiwifruit Bacterial Canker
The in vitro bioassays of the active compound 9a against kiwifruit bacterial canker were carried out according to our reported method with some modifications [45][46][47]. The commercial bactericide thiodiazole copper (TC, 20% suspending agent) and an equivalent DMSO were used as the positive and negative (CK) controls, respectively. Briefly, the healthy kiwifruit pot with smooth surface were cleaned up using a degreasing cotton soaked with water. Then, three wounds were made with 1 mm width and down to xylem using a sterilized knife on each plant. For the protective assay, 10 µL of drug solution (200 µg/mL) or DMSO solution were added into the corresponding wounds, then 10 µL of Psa bacterial suspension (OD 595 = 0.1) was inoculated at 24 h after addition. For the curative assay, there was only a time change on adding drug solution and Psa bacterial suspension. After 30 min for each operation, all the treatments were cultured in a climate chamber (95% RH) under 14 h lighting at 14 • C and 10 h dark at 10 • C. The length of lesion was measured 14 days after inoculation. All treatments were carried out in triplicate. The control efficiencies (I) were calculated by the following equation: Corrected lesion length (cm) = measured lesion length − 1.0 Control efficiency I (%) = (C − T)/C × 100 In the equation, C and T are the average corrected length of lesion of the negative control and the treatment group, respectively.

Growth Effect Assay of Compound 7c against Xoo
To further know the probable action of the designed molecules, we performed an in vitro growth curve assay on Xoo via the previously reported method with some modifications [50]. Firstly, some bacterial colony was incubated into fresh NB broth at 28 • C. After overnight growth, the cultures were adjusted to the OD 595 value of~0.5 by sterile NB broth, then 200 µL of the adjusted Xoo solutions was added into a 96-well plate and supplemented with compound 7c at concentrations of 1 EC 50 , 2 EC 50 , 4 EC 50 , 8 EC 50 and 16 EC 50 , respectively. The plates only containing the adjusted Xoo solutions were used as the blank controls. After that, the samples were incubated in a Cytation™ 5 multi-mode readers at 28 • C for 27 h, and the OD 595 values were detected every 3 h. Finally, the growth curve was drawn by using origin 8.

Membrane Permeability Changes by Propidium Iodide (PI) Staining Experiment
In this assay, the method was according to the guidebook of a commercial PI staining kit. Briefly, compound 7c was added into 2 mL Xoo solution with OD 595 of 0.2 to give the final concentrations of 1 × EC 50 (7.40 µg/mL), 2 × EC 50 (14.80 µg/mL), 4 × EC 50 (29.60 µg/mL), 8 × EC 50 (59.2 µg/mL) and 16 × EC 50 (118.2 µg/mL), respectively, and the equivalent volume of DMSO was used as blank control. Then, all the treatments were incubated in a constant temperature shaker under the conditions of 28 • C and 180 rpm for 14 h. After that, all the samples were washed with PBS (10 mM, pH = 7.2) 3 times and resuspended in 100 µL PBS. Subsequently, the bacterial cells were stained with 10 µL PI stain (20 µg/mL) for 30 min, and then washed by PBS 2 times to remove the extracellular stain. Finally, all the treated Xoo cells were visualized on an Olympus BX53 microscope under red fluorescent channel with exposure time of 2 s.

Data and Statistical Analysis
The data of in vitro antibacterial assay were analyzed by using excel 2013 (Microsoft Corporation, Washington, DC, USA) and shown as the average mean ± standard deviation (SD) of three replicate data [55]; the detailed processes are described in Supplementary Materials. In addition, Duncan's multiple range test of one-way analysis of variance (ANOVA, p = 0.05) was conducted in SPSS ver. 25.0 statistical software (SPSS Inc., Chicago, IL, USA) for statistical analysis, and the EC 50 values and in vivo bioassay results of different treatments were considered statistically significant when p-value was < 0.05 [56].

Conclusions
In summary, fourteen novel 2-(pyrazol-4-yl)-1,3,4-oxadiazoles decorated with an imidazole moiety were designed and synthesized via a four-step reaction, and their structures were identified by 1 H NMR, 13 C NMR, 19 F NMR and HRMS. The in vitro bioassays revealed that most of the target compounds showed moderate to good antibacterial activity against three intractable phytopathogenic bacteria, Xoo, Xac and Psa, with the lowest EC 50 values of 7.40 (7c), 5.44 (9a) and 12.85 (9a) µg/mL, respectively, which were more powerful than those of the commercial bactericides BT (31.94, 50.51 and 114.76 µg/mL, respectively) and TC (76.81, 66.98 and 74.98 µg/mL, respectively). The structure-activity relationship (SAR) analysis showed that the alkyl chain, the position (R 1 ) and variety (R 2 ) of the substituent on the pyrazole ring, and the S and O atom on 1,3,4-oxadiazole ring presented crucial effects on the bioactivities. Furthermore, the pot experiments showed that compound 7c possessed good in vivo protective and curative activities to manage rice bacterial leaf blight at 200 µg/mL, with control efficacies of 47.34% and 41.18%, respectively, which were much better than those of the commercial bactericide TC (35.12% and 37.50%, respectively) and partly superior to BT (48.28% and 31.37%, respectively). Meanwhile, compound 9a presented commendable in vivo protective and curative activities against kiwifruit bacterial canker at 200 µg/mL, with control efficiencies of 46.05% and 32.89%, respectively, which were much better than those of the commercial bactericide TC (31.58% and 17.11%, respectively). Finally, the growth effect assay, SEM observations and PI staining experiment mutually verified that these designed molecules could negatively impact the cell membrane of phytopathogenic bacteria cells and cause the leakage of the intracellular component, thereby leading to the killing of bacteria. All these findings indicate that the imidazole-decorated 2-(pyrazol-4-yl)-1,3,4-oxadiazoles 7c and 9a can be considered as lead molecules to develop novel agricultural bactericides.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules28062442/s1. Figures S1-S56 show the 1 H NMR, 13 C NMR, 19 F NMR and HRMS spectra of target compounds 7a~7f, 8a~8d and 9a~9d. Figures S57-S58 and Table S1 show the HPLC spectra of target compounds 7c and 9a, respectively. Table S2 shows the results of ADME properties' prediction of compounds 7c and 9a. Table S3 shows the toxic regression equation and correlation coefficient (R 2 ) of active compounds against Xoo, Xac and Psa.