Benzamides Substituted with Quinoline-Linked 1,2,4-Oxadiazole: Synthesis, Biological Activity and Toxicity to Zebrafish Embryo

To develop new compounds with high activity, broad spectrum and low-toxicity, 17 benzamides substituted with quinoline-linked 1,2,4-oxadiazole were designed using the splicing principle of active substructures and were synthesized. The biological activities were evaluated against 10 fungi, indicating that some of the synthetic compounds showed excellent fungicidal activities. For example, at 50 mg/L, the inhibitory activity of 13p (3-Cl-4-Cl substituted, 86.1%) against Sclerotinia sclerotiorum was superior to that of quinoxyfen (77.8%), and the inhibitory activity of 13f (3-CF3 substituted, 77.8%) was comparable to that of quinoxyfen. The fungicidal activities of 13f and 13p to Sclerotinia sclerotiorum were better than that of quinoxyfen (14.19 mg/L), with EC50 of 6.67 mg/L and 5.17 mg/L, respectively. Furthermore, the acute toxicity of 13p was 19.42 mg/L, classifying it as a low-toxic compound.

In our efforts to develop potent fungicides, we have previously reported the synthesis and biological activity studies of 1,2,4-oxadiazole-substituted benzamide derivatives [22,23]. Some of them exhibited good fungicidal activities. In view of the facts mentioned above, to further improve the fungicidal activities of these compounds, we designed ( Figure 2) a series of novel 1,2,4-oxadiazole-substituted benzamides using the splicing principle of active substructures and synthesized them by introducing a quinoline scaffold at the 5-position of 1,2,4-oxadiazole. The chemical structures of these new compounds were confirmed by 1 H-NMR, 13 C-NMR, and HRMS, their fungicidal activities were studied and a toxicity test with zebrafish embryo was performed.

Synthesis of Target Compounds
The synthetic pathway to target compounds 13a-13q is shown in Scheme 1. The starting material 3,5-dichloroaniline 1 underwent addition, hydrolysis, cyclization, and oxidation reaction to give 5,7-dichloro-4-hydroxyquinoline 5. During the addition reaction (the first reaction in Scheme 1), methanesulfonic acid (MSA) was selected as the acid catalyst and the optimum molar ratio of 1:0.2 was determined, which greatly improved the yield of the reaction. In step 4, we performed a preliminary screening of the oxidant, and finally determined to take the K2S2O8/H2SO4 system as the oxidant to afford compound 5 in the best yield. The influence of different reaction conditions on the yield of the compound 5 is shown in Table 1. Moreover, acetonitrile was chosen as the solvent in which the product had low solubility so that it could precipitate to obtain the solid easily.

Synthesis of Target Compounds
The synthetic pathway to target compounds 13a-13q is shown in Scheme 1. The starting material 3,5-dichloroaniline 1 underwent addition, hydrolysis, cyclization, and oxidation reaction to give 5,7-dichloro-4-hydroxyquinoline 5. During the addition reaction (the first reaction in Scheme 1), methanesulfonic acid (MSA) was selected as the acid catalyst and the optimum molar ratio of 1:0.2 was determined, which greatly improved the yield of the reaction. In step 4, we performed a preliminary screening of the oxidant, and finally determined to take the K2S2O8/H2SO4 system as the oxidant to afford compound 5 in the best yield. The influence of different reaction conditions on the yield of the compound 5 is shown in Table 1. Moreover, acetonitrile was chosen as the solvent in which the product had low solubility so that it could precipitate to obtain the solid easily.

Synthesis of Target Compounds
The synthetic pathway to target compounds 13a-13q is shown in Scheme 1. The starting material 3,5-dichloroaniline 1 underwent addition, hydrolysis, cyclization, and oxidation reaction to give 5,7-dichloro-4-hydroxyquinoline 5. During the addition reaction (the first reaction in Scheme 1), methanesulfonic acid (MSA) was selected as the acid catalyst and the optimum molar ratio of 1:0.2 was determined, which greatly improved the yield of the reaction. In step 4, we performed a preliminary screening of the oxidant, and finally determined to take the K 2 S 2 O 8 /H 2 SO 4 system as the oxidant to afford compound 5 in the best yield. The influence of different reaction conditions on the yield of the compound 5 is shown in Table 1. Moreover, acetonitrile was chosen as the solvent in which the product had low solubility so that it could precipitate to obtain the solid easily. The synthesis of amine oxime 9 was similar to our previous procedures [23]. It should be noted that this reaction could not be carried out for a long time to avoid the form of amide by-products. Afterwards, intermediate 12 was prepared from compound 9 via cyclization, hydrolysis, and condensation reaction. In addition, the hydrolysis reaction was carried out under acidic condition to avoid by-products as chlorine-substituted alkanes would hydrolyzed readily than ester groups under alkaline condition (Scheme 2).  The synthesis of amine oxime 9 was similar to our previous procedures [23]. It should be noted that this reaction could not be carried out for a long time to avoid the form of amide by-products. Afterwards, intermediate 12 was prepared from compound 9 via cyclization, hydrolysis, and condensation reaction. In addition, the hydrolysis reaction was carried out under acidic condition to avoid by-products as chlorine-substituted alkanes would hydrolyzed readily than ester groups under alkaline condition (Scheme 2).  The synthesis of amine oxime 9 was similar to our previous procedures [23]. It should be noted that this reaction could not be carried out for a long time to avoid the form of amide by-products. Afterwards, intermediate 12 was prepared from compound 9 via cyclization, hydrolysis, and condensation reaction. In addition, the hydrolysis reaction was carried out under acidic condition to avoid by-products as chlorine-substituted alkanes would hydrolyzed readily than ester groups under alkaline condition (Scheme 2). Finally, Williamson ether synthesis of compound 5 with 12 formed the target compounds 13.

Spectrum Analysis of Target Compounds
All the target compounds were confirmed by 1 H-NMR, 13 C-NMR, and HRMS. The target compound 13f was taken as an example to conduct spectrum analysis. In the 1 H-NMR spectra of 13f, the -NH-proton signal was found at δ 10.75 ppm. In addition, the single peak at 6.04 ppm was the peak of -CH 2 -between ether bond and 1,2,4-oxadiazoles.
In the 13 C-NMR spectra of compound 13f, the appearances of signals at 167.83 ppm and 165.40 ppm were assigned to the carbons of the 1,2,4-oxadiazole ring. In the HRMS spectrogram, the calculated value of the ion peak of this compound was [M + Na] + 559.0546, and the measured value was [M + Na] + 559.0549. The absolute error was within 0.003.

Biological Activities of Target Compounds
The results of the fungicidal activities test of the target compounds against 10 fungi are shown in Table 2. At 50 mg/L, all the target compounds 13a-13q were found to exhibit certain inhibitory activity against the 10 fungi tested. Overall, the target compounds showed better inhibitory activity against Sclerotinia sclerotiorum, ranging from 47.2% to 86.1%. Among them, the inhibitory rate of compound 13p (86.1%) was superior to the control drug quinoxyfen (77.8%), and the inhibitory rate of compound 13f was 77.8%, which was similar to quinoxyfen. In addition, the inhibition rates of compounds 13a, 13b, 13d and 13o against Sclerotinia sclerotiorum were 75.0%, 72.2%, 75.0% and 75.0%, respectively, which are slightly lower than that of quinoxyfen. Other compounds also exhibited moderate inhibitory activity (47.2-69.4%). For Alternaria solani, Gibberella zeae, Phytophthora capsica and Physalospora piricola, some compounds possessed better inhibitory activities than quinoxyfen, but their inhibition rates were less than 50%. As can be seen from Table 3, the EC 50 of compounds 13f and 13p against Sclerotinia sclerotiorum were 6.67 mg/L and 5.17 mg/L, respectively, which were significantly superior to quinoxyfen (14.19 mg/L). Structure-activity relationship (SAR) results for these target compounds showed that when the substituent of the benzene ring was 3-CF 3 or 3,4-(Cl) 2 , their inhibitory activities were obviously superior to others. Overall, electron withdrawing groups are beneficial to inhibitory activity.

Toxicity to Zebrafish Embryo
According to the fungicidal activity results (Figure 3), we selected compound 13p with better activity to study the lethal and teratogenic effects exposure on zebrafish embryos from 6 to 96 hpf (hours post fertilization). When the concentration of 13p was below 40 mg/L, the mortality rate increased sharply as the concentration increased. Afterwards, the mortality rate exceeded 90% at 40 mg/L. The resulting LC 50 value for compound 13p was 19.42 mg/L, and it was classified as a low-toxic compound [24].

Toxicity to Zebrafish Embryo
According to the fungicidal activity results (Figure 3), we selected compound 13p with better activity to study the lethal and teratogenic effects exposure on zebrafish embryos from 6 to 96 hpf (hours post fertilization). When the concentration of 13p was below 40 mg/L, the mortality rate increased sharply as the concentration increased. Afterwards, the mortality rate exceeded 90% at 40 mg/L. The resulting LC50 value for compound 13p was 19.42 mg/L, and it was classified as a low-toxic compound [24]. As the time and concentration increased, zebrafish embryos showed obvious developmental delay (Figure 4), such as bent spine, pericardial cyst, yolk cyst and even malformation. At 72 hpf, compared to the control group, the zebrafish embryo exposed at 10 mg/L and 20 mg/L showed obvious yolk cyst. At 96 hpf, pericardial cyst and bent spine appeared on the zebrafish embryo exposed at 10 mg/L and 20 mg/L.  As the time and concentration increased, zebrafish embryos showed obvious developmental delay (Figure 4), such as bent spine, pericardial cyst, yolk cyst and even malformation. At 72 hpf, compared to the control group, the zebrafish embryo exposed at 10 mg/L and 20 mg/L showed obvious yolk cyst. At 96 hpf, pericardial cyst and bent spine appeared on the zebrafish embryo exposed at 10 mg/L and 20 mg/L. with better activity to study the lethal and teratogenic effects exposure on zebrafish embryos from 6 to 96 hpf (hours post fertilization). When the concentration of 13p was below 40 mg/L, the mortality rate increased sharply as the concentration increased. Afterwards, the mortality rate exceeded 90% at 40 mg/L. The resulting LC50 value for compound 13p was 19.42 mg/L, and it was classified as a low-toxic compound [24]. As the time and concentration increased, zebrafish embryos showed obvious developmental delay (Figure 4), such as bent spine, pericardial cyst, yolk cyst and even malformation. At 72 hpf, compared to the control group, the zebrafish embryo exposed at 10 mg/L and 20 mg/L showed obvious yolk cyst. At 96 hpf, pericardial cyst and bent spine appeared on the zebrafish embryo exposed at 10 mg/L and 20 mg/L.

General Information
Melting points were determined using an X-4 digital microscopic melting point detector (Taike, Beijing, China) and the thermometer was uncorrected. 1 H-NMR and 13 C-NMR spectra were measured on BRUKER Avance 500 MHz spectrometer (Bruker 500 MHz, Fallanden, Switzerland) using CDCl 3 or DMSO as the solvent. High-resolution electrospray mass spectra (HR-ESI-MS) were determined using an UPLC H CLASS/QTOF G2 XS mass spectrometer (Waters, Milford, CT, USA). All the reagents were analytical grade or synthesized in our laboratory. The characterization data for all synthetic compounds are provided in the Supplementary Materials.
Ethics statement: The Institutional Animal Care and Use Committee (IACUC) at Wenzhou Medical University (SYXK 2019-0009, 4 April 2019 to 4 April 2024) approved our study plan for proper use of zebrafish. All studies were carried out in strict accordance with the guidelines of the IACUC. All dissections were performed on ice, and all efforts were made to minimize suffering. (2) 3,5-dichloroaniline 1 (16.20 g, 0.10 mol) and ethyl acrylate (30.00 g, 0.30 mol) were sequentially added to a three-necked flask, heated, and stirred until dissolved completely. The mixture of MSA (1.44 g) and water (2.70 g) was added dropwise, then reacted at 60 • C for 16 h. After the reaction was completed, the mixture was cooled to room temperature, unreacted ethyl acrylate was removed under reduced pressure. The remnant was dissolved in toluene (300 mL) and washed with HCl. Finally, the organic layer was dried with anhydrous MgSO 4

5,7-Dichloro-4-hydroxyquinoline (5)
Conc. H 2 SO 4 (2.00 g) was added slowly to a solution of compound 4 (5.00 g, 23.00 mmol) in acetonitrile (35.00 g). Afterwards, K 2 S 2 O 8 (8.00 g) was added when the temperature reached 50 • C. The mixture was then reflux for 4 h. TLC was used to track the reaction progress. After the reaction was completed, the mixture was cooled to room temperature to precipitate solid. The solid was filtered, washed with water, and dried to obtain product 5 (4.60 g). Yield: 93.4%.

Methyl-3-(N-hydroxycarbamimidoyl)benzoate (9)
The synthesis of intermediate 9 was performed with reference to our previous work.

Methyl 3-(5-(chloromethyl)-1,2,4-oxadiazol-3-yl)benzoate (10)
To a three-necked flask, we added intermediate 9 (0.97 g, 5.00 mmol), triethylamine (1.20 g, 12.00 mmol) and dry toluene (100 mL). Stirring was started at 0 • C for 2 h followed by the dropwise addition of chloroacetyl chloride (0.58 g, 5.20 mmol). This was then reacted at 0 • C for another 3 h. The mixture was further heated to reflux for about 2 h. The mixture was then cooled to room temperature and sequentially washed with water and saturated sodium chloride solution. The organic layer was dried with Na 2 SO 4 and the solvent was removed to give 0.93 g yellow solid. Yield: 73.8%; 1 H-NMR (500 MHz, Chloroform-d) 3.2.7. 3-(5-(Chloromethyl)-1,2,4-oxadiazol-3-yl)benzoic Acid (11) Compound 10 (5.00 g, 0.02 mol), CH 3 COOH (30 mL), and HCl (30 mL) were added to a three-necked flask and reacted at 70 • C for 3 h. After the reaction was completed, the mixture was cooled to room temperature to precipitate white solid. The white solid was filtered, washed with water, and dried to give compound 11 (4.45 (12) The solution of compound 11 (0.24 g, 1.00 mmol) in SOCl 2 (5 mL) was reacted at reflux for 3 h. The SOCl 2 was removed and THF (30 mL) was added subsequently. Then, the mixture of substituted aniline (1.20 mmol), triethylamine (2.5 mmol) and THF (1 mL) was added dropwise under ice bath. Stirred overnight, separated by column chromatography to give intermediate 12.      CO 3 (0.35 g) and DMF (10 mL) were added to a round bottom flask. The mixture was reacted at 60 • C for 5 h. Afterwards, the mixture was cooled to room temperature and poured into water (100 mL) then extracted with ethyl acetate. The extraction was dried over anhydrous MgSO 4 and filtered. After that the filtration was concentrated and separated by column chromatography to give target compounds 13.

Conclusions
In conclusion, a total of 17 novel benzamides containing quinoline-linked 1,2,4oxadiazole moiety were designed using splicing principle of active substructures and synthesized via Williamson ether synthesis. The structures of target compounds were confirmed by 1 H NMR, 13 C NMR, and HRMS. The bioassay results showed that 13a-13q displayed certain inhibitory activity against 10 fungi tested, especially 13f and 13p. It is worth mentioning that the fungicidal activities of 13f and 13p to Sclerotinia sclerotiorum were better than quinoxyfen (14.19 mg/L) with EC 50 of 6.67 mg/L and 5.17 mg/L, and their inhibition rates were equal (77.8%) or higher (86.1%) than quinoxyfen (77.8%) at 50 mg/L. Moreover, the acute toxicity of 13p was 19.42 mg/L, which was classified as a low-toxic compound. Hence, these compounds could potentially be lead compounds for further study.