Synthesis and Biological Activity of Benzamides Substituted with Pyridine-Linked 1,2,4-Oxadiazole

To find pesticidal lead compounds with high activity, a series of novel benzamides substituted with pyridine-linked 1,2,4-oxadiazole were designed by bioisosterism, and synthesized easily via esterification, cyanation, cyclization and aminolysis reactions. The structures of the target compounds were confirmed by 1H-NMR, 13C-NMR and HRMS. The preliminary bioassay showed that most compounds had good larvicidal activities against mosquito larvae at 10 mg/L, especially compound 7a, with a larvicidal activity as high as 100%, and even at 1 mg/L was still 40%; at 50 mg/L, all the target compounds showed good fungicidal activities against the eight tested fungi. Moreover, compound 7h exhibited better inhibitory activity (90.5%) than fluxapyroxad (63.6%) against Botrytis cinereal. Therefore, this type of compound can be further studied.

Amide compounds could be roughly classified as carboxyamides, mandelic acid amides and phenylamides according to their chemical structures. Through analyzing the reported amide compounds, it was found that when the carboxylic acid parts of the compounds include the structural units of pyridine-linked heterocycle, amide compounds usually have better insecticidal or fungicidal activities. Most of the compounds that have been reported include the structural units of pyridine-linked pyrazole, such as the commercial insecticide chlorantraniliprole (Figure 1) and the compound A (Figure 1, at 50 mg/L, its fungicidal activity against Botrytis cinerea was 80%) synthesized by Xu et al. [21]. However, there are few reports about pyridine-linked 1,2,4-oxadiazole. Moreover, Syngenta has reported a lot of 1,2,4-oxadiazole substituted benzamides such as compounds B (Figure 1) and C (Figure 1) that had good fungicidal activities [22,23]. In view of these facts mentioned above, to find pesticides with high biological activity, using A and B as lead compounds and replacing the structure of trifluoromethyl with the pyridine ring, a series of novel benzamides substituted with pyridine-linked 1,2,4-oxadiazole have been designed and synthesized according to bioisosterism (Figure 2). Target compounds were confirmed by 1 H-NMR, 13 C-NMR and HRMS. Their insecticidal and fungicidal activities were studied and the result showed that the target compounds had good insecticidal and fungicidal activities. The synthetic route of the target compounds is shown in Scheme 1.
Molecules 2020, 25, x 2 of 12 ( Figure 1) that had good fungicidal activities [22,23]. In view of these facts mentioned above, to find pesticides with high biological activity, using A and B as lead compounds and replacing the structure of trifluoromethyl with the pyridine ring, a series of novel benzamides substituted with pyridinelinked 1,2,4-oxadiazole have been designed and synthesized according to bioisosterism (Figure 2). Target compounds were confirmed by 1 H-NMR, 13 C-NMR and HRMS. Their insecticidal and fungicidal activities were studied and the result showed that the target compounds had good insecticidal and fungicidal activities. The synthetic route of the target compounds is shown in Scheme 1.   Molecules 2020, 25, x 2 of 12 ( Figure 1) that had good fungicidal activities [22,23]. In view of these facts mentioned above, to find pesticides with high biological activity, using A and B as lead compounds and replacing the structure of trifluoromethyl with the pyridine ring, a series of novel benzamides substituted with pyridinelinked 1,2,4-oxadiazole have been designed and synthesized according to bioisosterism (Figure 2). Target compounds were confirmed by 1 H-NMR, 13 C-NMR and HRMS. Their insecticidal and fungicidal activities were studied and the result showed that the target compounds had good insecticidal and fungicidal activities. The synthetic route of the target compounds is shown in Scheme 1.
In step 2, the iodine atom on the benzene ring being replaced by a cyano group is a typical nucleophilic substitution reaction. NaCN and KCN are two common nucleophilic reagents. In fact, these two reagents are highly toxic, so we chose CuCN, which has relatively low toxicity, as the cyanidation reagent to reduce the risk in the experiment and the harm to the environment. In addition, the process of our experiment was different from that reported in former papers [24,25]. Using small natural organic molecule L-proline as the catalyst, DMF as the solvent and under the condition where temperature gradually increased, the amount of by-product decreased and the yield of product 3 was the highest [26]. The influence of different experimental conditions on the yield of product 3 is shown in Table 1.
In step 2, the iodine atom on the benzene ring being replaced by a cyano group is a typical nucleophilic substitution reaction. NaCN and KCN are two common nucleophilic reagents. In fact, these two reagents are highly toxic, so we chose CuCN, which has relatively low toxicity, as the cyanidation reagent to reduce the risk in the experiment and the harm to the environment. In addition, the process of our experiment was different from that reported in former papers [24,25]. Using small natural organic molecule l-proline as the catalyst, DMF as the solvent and under the condition where temperature gradually increased, the amount of by-product decreased and the yield of product 3 was the highest [26]. The influence of different experimental conditions on the yield of product 3 is shown in Table 1. In step 4, the formation of 1,2,4-oxadiazole was achieved in a one-pot reaction. 3,6-dichloropicolinoyl chloride that was freshly prepared was dropped into the solution of methyl-2-chloro-5-(N -hydroxycarbamimidoyl) benzoate 4 and triethylamine in toluene at 0 • C, to give the intermediate of methyl-2-chloro-5-(N-(3,6-dichloropicolinoyl)-N -hydroxycarbamimidoyl)benzoate. Next, the intermediate was cyclized to produce 1,2,4-oxadiazole at reflux (Scheme 2). In this way, the self-cyclization of compound 4 was avoided because of the higher reactivity of acid chloride.

Spectrum Analysis of Target Compounds
The target compound 7a was taken as an example to conduct spectrum analysis. In the 1 H-NMR spectra of compound 7a, the singlet at δ 10.09 ppm was the NH peak. The signals of benzene and pyridine rings were assigned at 8.37-7.04 ppm. In the 13 C-NMR spectra of compound 7a, the C=O signal could be found at 171.75 ppm. The appearance of signals at 167.27 and 163.91 ppm was 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] + 466.9840, and the measured value was [M + Na] + 466.9840. The absolute error was within 0.003.

Biological Activities of Target Compounds
The results of the insecticidal activity test of the target compounds are shown in Tables 2 and 3. In Table 2, the death rates of compound 7 are all below 50% against mythimna sepatara, helicoverpa armigera and pyrausta nubilalis at 500 mg/L. Therefore, the insecticidal activities of this series of compounds were not good enough against the three targets. Nevertheless, we can see that compound 7 exhibited good larvicidal activity against mosquito larvae from Table 3. The larvicidal activities of compounds 7a and 7f were 100% at 10 mg/L. Furthermore, it was found that the larvicidal activity of compound 7a was 100% at 2 mg/L. Even at 1 mg/L, the larvicidal activity was still 40%. It exhibited better larvicidal potency than etoxazole against mosquito larvae. It can be seen from the compound 7h, 7i and 7j that the position of the substituent has little effect on the larvicidal activity of the target compound. Furthermore, as for compounds 7a to 7n, from the general trend in the larvicidal activity, it can be concluded that the less steric substitution attached to aniline may reduce the obstacles of the target compound binding to the target receptor and help to bring about an increase in activity.

Spectrum Analysis of Target Compounds
The target compound 7a was taken as an example to conduct spectrum analysis. In the 1 H-NMR spectra of compound 7a, the singlet at δ 10.09 ppm was the NH peak. The signals of benzene and pyridine rings were assigned at 8.37-7.04 ppm. In the 13 C-NMR spectra of compound 7a, the C=O signal could be found at 171.75 ppm. The appearance of signals at 167.27 and 163.91 ppm was 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] + 466.9840, and the measured value was [M + Na] + 466.9840. The absolute error was within 0.003.

Biological Activities of Target Compounds
The results of the insecticidal activity test of the target compounds are shown in Tables 2 and 3. In Table 2, the death rates of compound 7 are all below 50% against mythimna sepatara, helicoverpa armigera and pyrausta nubilalis at 500 mg/L. Therefore, the insecticidal activities of this series of compounds were not good enough against the three targets. Nevertheless, we can see that compound 7 exhibited good larvicidal activity against mosquito larvae from Table 3. The larvicidal activities of compounds 7a and 7f were 100% at 10 mg/L. Furthermore, it was found that the larvicidal activity of compound 7a was 100% at 2 mg/L. Even at 1 mg/L, the larvicidal activity was still 40%. It exhibited better larvicidal potency than etoxazole against mosquito larvae. It can be seen from the compound 7h, 7i and 7j that the position of the substituent has little effect on the larvicidal activity of the target compound. Furthermore, as for compounds 7a to 7n, from the general trend in the larvicidal activity, it can be concluded that the less steric substitution attached to aniline may reduce the obstacles of the target compound binding to the target receptor and help to bring about an increase in activity.

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 NMR spectrometer (Bruker 500 MHz, Fallanden, Switzerland); High-resolution electrospray mass spectra (HR-ESI-MS) were determined using an UPLC H-CLASS/QTOF G2-XS mass spectrometer (Waters, Milford, MA, USA). All the reagents and solvents were in analytical purity. The characterisation data for all synthesised compounds are provided in the Supporting Information file.

Methyl 2-chloro-5-iodobenzoate 2
2-chloro-5-iodobenzoic acid 1 (2.8 g, 0.01 mol), methanol (50 mL) and H 2 SO4 (0.5 mL) were added to a three-necked flask and reacted at reflux for about 8 h. After the mixture was cooled to room temperature, methanol was removed under reduced pressure. Then, EtOAc (50 mL) was added and the pH adjusted to 7-8 by using NaHCO 3 . The organic layer was dried with Na 2 SO 4 . The solvent was removed to give 2.71 g creamy white solid. Yield: 92%, m.p.

Methyl 2-chloro-5-(N -hydroxycarbamimidoyl)benzoate 4
Methyl 2-chloro-5-cyanobenzoate 3 (1.4 g, 7.2 mmol) was added to a three-necked flask and dissolved by ethanol (45 mL). Stirring was started at room temperature, and then hydroxylamine hydrochloride (0.75 g) and triethylamine (1.1 g) were gradually added. The mixture was stirred for 3 h. Then the solvent was removed under reduced pressure and the remnant was dissolved in EtOAc (50 mL) and saturated saline (50 mL). The organic layer was dried with Na 2 SO 4 and evaporated, to give 1. 3,6-dichloropicolinic acid (1.1 g, 5 mmol) and SOCl 2 (20 mL) were added to a round bottom flask. The mixture was stirred and refluxed for 2 h. Then, SOCl 2 was removed under reduced pressure to give 3,6-dichloropicolinoyl chloride.
Intermediate 4 (1.1 g, 5 mmol), triethylamine (1.2 g, 5 mmol) and dry toluene (100 mL) were added to a three-necked flask. The mixture was stirred at 0 • C for 2 h. After that, the prepared 3,6-dichloropicolinoyl chloride (dissolved by 30 mL dry toluene) was dropped into the flask. The mixture continued to be stirred for 1 h at 0 • C. Then, the temperature was increased to reflux for 2 h. When the mixture was cooled to room temperature, it was washed by saturated sodium chloride solution (150 mL × 3). The organic layer was dried by Na 2 SO 4 and removed under reduced pressure to give 1. Intermediate 5 (0.8 g, 2.0 mmol) and THF (40 mL) were added to a three-necked flask. After being dissolved, 30% NaOH (5 mL) was also added to the flask and refluxed for 2 h. After the mixture was cooled to room temperature, the solvent was removed. Then, the pH was adjusted to 2-3 with HCl and 0.7g white solid precipitate was obtained. Yield: 94%, m.p. 203-204 • C; 1 H-NMR (500 MHz, DMSO-d 6

Preparation of Target Compound 7
Intermediate 6 (4.1 g, 11 mmol), triethylamine (0.2 g), DCM (30 mL) and EDCI (0.3 g) were added to a three-necked flask. Substituted aniline (12 mmol) was stirred and dropped into the flask at 0 • C. TLC was used to track reaction progress. At last, target compound 7 was obtained by using the method of column chromatography separation. temperature gradually in the cyanation reaction, we got the best yield (79%) and reduced the risk of this experiment. The structures of the target compounds were confirmed by 1 H-NMR, 13 C-NMR and HRMS. The preliminary bioassay results showed that most compounds had good larvicidal activity against mosquito larvae at 10 mg/L, especially compound 7a with excellent larvicidal activity (100%); even at 1 mg/L, the larvicidal actiity was still 40%; at 50 mg/L, all the target compounds showed good fungicidal activity against the eight tested fungi. Compound 7h exhibited good inhibitory activity (90.5%) against Botrytis cinereal, which was better than fluxapyroxad (63.6%). In addition, it had moderate inhibitory activities against Alternaria solani (50.0%), Sclerotinia sclerotiorum (80.8%) and Thanatephorus cucumeris (84.8%). Therefore, these compounds could potentially be the lead compounds for further optimisation.