Synthesis, Larvicidal Activities and Antifungal Activities of Novel Chlorantraniliprole Derivatives and Their Target in the Ryanodine Receptor

In order to identify novel chlorantraniliprole derivatives as potential insecticides or fungicides, 25 analogues of chlorantraniliprole were synthesized. The insecticidal activities against oriental armyworm and the antifungal activities against five typical fungi of these derivatives were tested. Compounds 2u, 2x and 2y exhibited good activities against oriental armyworm, especially compounds 2u and 2x which showed higher larvicidal activities than indoxacarb. Moreover, all of the tested compounds exhibited activities against five typical fungi. The Ki values of all synthesized compounds were calculated using AutoDock4. The relationship between the Ki values and the results of insecticidal activities against oriental armyworm further indicated that the membrane-spanning domain protein of the ryanodine receptor might contain chlorantraniliprole binding sites.


Introduction
Lepidopteran pests such as the oriental armyworm (Mythimna separata) have become difficult to control because of their emerging resistance to various types of traditional insecticides [1,2]. In order to control the oriental armyworm effectively, there were currently two new types of diamide insecticides with exceptional insecticidal activities on a range of lepidopteran pests, the phthalic diamides, such as flubendiamide [3,4] and the anthranilic diamides, such as chlorantraniliprole [5] and cyantraniliprole ( Figure 1) [6].
Chlorantraniliprole, discovered by DuPont, has excellent control of lepidopteran pests, low mammalian toxicity and a favorable environmental profile. The widespread use of chlorantraniliprole on lepidopteran pests in the future may result in pest resistance and residues in the field [7]. Thus, designing novel chlorantraniliprole derivatives as new insecticides has attracted considerable research attention. However, a reasonable design strategy could not be proposed because of the lack of a clear target for chlorantraniliprol. In our previous work [8], we proposed the ryanodine receptor [9] as a possible binding target for chlorantraniliprole and its derivatives using an AutoDock4 analysis, and we initially confirmed that the membrane-spanning domain protein of the ryanodine receptor might have specific binding site(s) for chlorantraniliprole derivatives. Figure 2a shows the relative position relationship between the proposed ryanodine receptor (in white color) and chlorantraniliprole (in blue color). Diamide insecticides activate insect ryanodine (Ry)-sensitive intracellular Ca 2+ channels by affecting calcium release [10], as seen in Figure 2b. We initially identified the target by comparing the relationship between the insecticidal activities against diamondback moth and the molecular docking results. Both diamondback moth and oriental armyworm are lepidopteran pests [11]. Therefore, in this study, oriental armyworm was selected for the first time for a bioactivity study, which examines the relationship with the Ki values of molecular docking. On the other hand, it was reported [12] that chlorantraniliprole derivatives might also show antifungal effects. Therefore, in this work the in vitro antifungal activity of some chlorantraniliprole derivatives was tested too. The chlorantraniliprole derivatives obtained herein were also used as new probes to bind with the proposed acceptor to further confirm that the membrane-spanning domain protein of ryanodine receptor was the special target binding with chlorantraniliprole and its derivatives.

Chemistry
Compound 1 was obtained by literature methods (Scheme 1) [7,[17][18][19]. The nineteen compounds 2b-t were synthesized according to our previous work [8]. Among the new compounds 2a, 2u-y, compound 2u was conveniently obtained by treatment of compound 1 with methyl 2-aminoacetate without any other catalyst or base at room temperature. Due to the insolubility of glycine in the reaction solvents (tetrahydrofuran or dichloromethane), a mixture of pyridine and water was selected as solvent in the preparation of compound 2v. In the case of compounds 2w and 2x, a strong base (NaH) was needed, while triethylamine was employed for compound 2w.

Larvicidal Activities and Structure-Activity Relationships (SARs)
The results in Table 1 indicate that all of the tested compounds 2a-y, except for four compounds (2a, 2q, 2r and 2w) showed the same larvicidal activities (100%) as chlorantraniliprole, indoxacarb and avermectins at the concentrations of 100 and 200 mg/L. These results reveal that most of the chlorantraniliprole derivatives in our study exhibited considerable insecticidal activities against oriental armyworm.
The larvicidal activities of all compounds except for three compounds (2u, 2x and 2y) were lower than those of chlorantraniliprole and avermectins when the concentration was under 50 mg/L, however, compounds 2u and 2x showed obviously better activities (both 100%) than indoxacarb (40%), even when the concentration was lowered to 5 mg/L, while, compound 2y displayed the same larvicidal activity (40%) as indoxacarb at this concentration. Notably, these two compounds 2u and 2x displayed the same activity (100%) as chlorantraniliprole and avermectins at the concentration of 5 mg/L. In Table 2, the preliminary structure-activity relationship (SAR) data is summarized. All compounds with cyano groups (R2 = CN) instead of a chloride group commonly had low insecticidal activities against oriental armyworm. For example, compounds 2o, 2p, 2q, 2r, 2s and 2t in Table 1 had no insecticidal activities when the concentration was less than 25 mg/L.
Most of the researchers in previous work [20][21][22] preserved the anthranilic amide moiety, which suggested that this structure was an important pharmacophore in those compounds [23]. The number of methylene groups in the amide moiety was also an important element for the insecticidal activity and two methylenes was the most favorable number for high activity. For example, compounds 2c, 2d, 2j, 2l and 2n (n = 2) had high insecticidal activities (100%), while compounds 2g and 2h (n = 3 or 4) showed lower activities (60%). Obviously, when R4 and R5 had the same substituent groups ( Table 2) as in compounds 2d, 2g and 2h (R4 = R5 = ethyl group), the insecticidal activities were dramatically reduced.
* The insecticidal activities against oriental armyworm at a concentration of 50 mg/L.
In addition, the substitution of R4 or R5 at the tail of the amide moiety was a significant factor affecting the insecticidal activities. When either one of R4 or R5 was substituted by hydrogen, most compounds (2j, 2l and 2n) displayed relatively high activities (100%), except compound 2k (40%), while, when both R4 and R5 were substituted by alkyl groups, most of compounds (2g, 2h, and 2i) displayed relatively lower activities (60%), except compound 2d (100%), which indicated that the secondary amine at the tail of the amide moiety was an necessary pharmacophore. In conclusion, the substitution at the terminal positions of the amide moiety and the number of methylene groups might be important factors that influence the insecticidal activities, which could be verified when more chlorantraniliprole derivatives are designed to increase the insecticidal activities against oriental armyworm.
The activities on FO and CA for all compounds, except 2t and 2f (both were 0%) showed varying inhibitory activities (ranging from 4.2% to 41.7%). Regarding the antifungal activities on PP, all tested compounds showed low to good inhibitory activities (from 5.3% to 78.9%) and two compounds (2h and 2j) showed higher activities (78.9%, 63%, respectively) than other compounds. The activities on AS for all compounds except for three (2k, 2l and 2t, no activity) showed inhibitory activities ranging from 11.1% to 50.0%. The activities of all tested compounds on FG were commonly higher than those on FO and AS. All compounds showed low to average inhibitory activities (from 14.8% to 51.9%) against FG and the compound 2b showed better activity (51.9%) than the other compounds.

Molecular Docking Results
The results in Table 4 reveal that ten compounds (2e, 2l, 2m, 2o, 2q, 2r, 2s, 2t, 2w and 2y) with unavailable Ki values except for four of them (2e, 2l, 2m and 2y) displayed low biological activities (≤60%) against oriental armyworm, while the other 15 compounds (2a, 2b, 2c, 2d, 2f, 2g, 2h, 2i, 2j,  2k, 2n, 2p, 2u, 2v and 2x) with available Ki value, except one compound (2a), generally displayed medium to high biological activities (40%-100%). These results further confirm that the membrane-spanning domain protein (GenBank accession no. JF927788) of the ryanodine receptor selected in our previous work [8] might have specific binding site(s) for chlorantraniliprole and its derivatives, and identification of the possible binding site is worthy of further study from a protein molecular level point of view. However, the docking software program might affect the accuracy of the Ki values and the biological diversity of pests plays an important role in the experimental activity tests. These reasons could explain why compounds 2e, 2l, 2m and 2y with unavailable Ki values showed high activities (100%) and compound 2a with available Ki values showed relatively no activity.

General
Melting points (mp) of the products were determined in open capillary tubes and are uncorrected. The products were purified by column chromatography by using silica gel (200-300 mesh). 1 H-NMR and 13 C-NMR spectra were recorded on a Varian-400 instrument at room temperature with TMS as an internal standard and CDCl3 or DMSO-d6 as solvents. Mass spectra were recorded with a JEOL MS-D 300 mass spectrometer. The reactions were monitored by analytical thin-layer chromatography TLC with ultraviolet (UV) light and the TLC was carried out on silica gel GF254. All reagents were purchased from Acros (Geel, Belgium) or Alfa Aesar (Deisenhofen, Germany). The anhydrous solvents were dried and purified according to standard techniques before use.

Syntheses
The intermediate compound 1 was obtained in six steps by literature methods [7,17,18]. The nineteen compounds 2b-t were synthesized according to our previous methods [8]. The other six new title compounds 2a, 2u-y were prepared as follows:

Biological Assay Methods
All of the tested compounds had the purity of more than 95%, and all bioassays were performed on representative test organisms reared in the laboratory. Evaluations were based on a percentage scale of 0-100, in which 0 = no activity and 100 = total kill. The standard deviations of the tested biological values were ±5%.

Larvicidal Activities against Oriental Armyworm (Mythimna separata Walker)
The larvicidal activities against oriental armyworm were tested by foliar application [7]. The test was repeated at 25 °C ± 1 °C according to statistical requirements. The tested compounds were dissolved in acetone and diluted with water to the required concentrations from 200 to 5 mg/L for bioassay. Individual corn leaves were placed on moistened pieces of filter paper in Petri dishes. The leaves were then dipped in the test solution and allowed to dry. The dishes were infested with 10 fourth-instar oriental armyworm larvae. Percentage mortalities were evaluated 24 h after treatment. Each treatment was replicated for three times. For comparative purposes, chlorantraniliprole, indoxacarb and avermectins were selected as the controls under the same conditions.

Antifungal Bioassay: Inhibitory Effects on Phytopathogenic Fungi
The five phytopathogenic fungi chosen included Fusarium oxysporum (FO), Cercospora arachidicola (CA), Physalospora piricola (PP), Alternaria solani (AS), and Fusarium graminearum (FG). All the fungi are typical and often occur in the Chinese agro-ecosystem. The antifungal activities of nineteen chlorantraniliprole derivatives (2b-l, 2o-u and 2x) were tested in vitro by the poisoned food technique at the concentration of 50 µg/mL by dissolving the compounds in DMSO and sterilized water (containing 1% Tween) and diluting with PDA in a Petri dish [24][25][26].

Molecular Docking Methods
In our study, the membrane-spanning domain protein of the ryanodine receptor was selected from GenBank (accession No. JF927788) [8] as the possible specific receptor, which was proposed to bind with small molecules to give the Ki values using AutoDock4. For the AutoDock4 docking methods readers can refer to our previous work [8]. The Ki values were used to evaluate the binding energy between the small molecules and their possible receptor. The analysis of the relationship between the Ki values and the insecticidal activities against oriental armyworm could thus afford further information about the specific receptor.

Conclusions
A series of novel chlorantraniliprole derivatives containing different amide groups and anthraniloyl moieties were designed and synthesized. Their insecticidal activities against oriental armyworm and the antifungal activities against five typical fungi were evaluated. The results indicated that all tested compounds except three (2a, 2r and 2w) exhibited favorable insecticidal activities against oriental armyworm. In particular, compounds 2u and 2x showed obviously better activities than indoxacarb even when the concentration was lowered to 5 mg/L. The preliminary structure-activity relationship of the title compounds indicated that compounds with a cyano group (R2 = CN) had low insecticidal activities. Two methylene groups and a secondary amine in the amide moiety were necessary factors for increasing the insecticidal activities. Moreover, all of the tested compounds exhibited activities against five typical fungi. The molecular docking results revealed that most of the compounds with available Ki values exhibited moderate to high activities against oriental armyworm. The relationship between the Ki values and the insecticidal activities suggested that the proposed membrane-spanning domain protein (GenBank accession no. JF927788) of the ryanodine receptor has special binding site(s) and might be the receptor of chlorantraniliprole and its analogs. Confirmation of this is a worthy topic for further study.