Design, Synthesis, and SAR of Novel 2-Glycinamide Cyclohexyl Sulfonamide Derivatives against Botrytis cinerea

N-(2-trifluoromethyl-4-chlorophenyl)-2-oxocyclohexyl sulfonamide (chesulfamide) is in the limelight as a novel fungicide, and has fungicidal activity against Botrytis cinerea. For exploring more novel structures, 33 new compounds were synthesized by N-alkylation and acid–amine coupling reactions with chesulfamide as the core moiety, and their structures were characterized and established by 1H-NMR, 13C-NMR, MS, and elemental analysis. The structure of (1R,2S)-2-(2-(N-(4-chloro-2-trifluoromethylphenyl)sulfamoyl)-cyclohexylamino)-N-(2-trifluoromethylphenyl) acetamide (II-19) was defined by X-ray single crystal diffraction. The in vivo and in vitro fungicidal activities against B. cinerea were evaluated. The bioassay results of mycelial growth demonstrated that most compounds exhibited excellent inhibitory activity against B. cinerea at 50 μg mL−1, and 7 compounds showed lower EC50 values than boscalid (EC50 = 4.46 μg mL−1) against B. cinerea (CY-09). In cucumber pot experiment, the inhibitory rates of four compounds (II-4, II-5, II-12, and II-13) against B. cinerea were 90.48, 93.45, 92.86, and 91.07, which were better than cyprodinil (88.69%), the best performing of all controls. In tomato pot experiment, the control efficacy of two analogs (II-8 and II-15) were 87.98 and 87.97% at 200 μg mL−1, which were significantly higher than boscalid (78.10%). Most compounds have an excellent fungicidal effect on B. cinerea, with potential as a lead compound for developing new pesticides.


Introduction
Plant-pathogenic fungi, with the characteristics of diversity and wide distribution, are causing a destructive economic and humanitarian problem, which threatens food security and biodiversity [1][2][3][4][5]. B. cinerea, one of the important representative fungi, has a necrotic lifestyle, and attacks over 200 major dicotyledonous plants in temperate and subtropical regions, causing soft rotting of all aerial plant parts, as well as decaying vegetables, fruits, and flowers after harvesting [6,7]. Despite the availability of many fungicides to control it, many of them have failed due to the genetic plasticity [6]. The reality alkyl, sulfonyl, benzene, piperidine, and acylated tetrazole, in order to get more diverse products ( Figure  3), but the cyclohexylsulfonamide structure that we linked in target compounds had not yet been done before. Here, we designed and synthesized a series of 2-glycinamide cyclohexyl sulfonamide derivatives. Their comprehensive fungicidal activity against B. cinerea were evaluated, and the structure-activity relationship was analyzed. The synthetic routes of intermediates and target compounds were shown in Schemes 1 and 2, respectively.

Chemistry
The synthetic routes for the target compounds are shown in Scheme 2. Substituted chloroacetamide (Intermediate I) was synthesized by the reaction of amines and chloroacetyl chloride in dichloromethane (CH2Cl2) as solvent in the presence of triethylamine (Et3N). The target compounds II-1 to II-28 were obtained by the nitrogen alkylation reaction of N- (2-
By comparing the two methods used to synthesize the target compounds, we found the later method resulted in higher yields and easier purification. Prior to this, we also tried to use potassium tert-butoxide [32], magnesium methoxide, and calcium chloride [33] as catalysts, trying to promote the amidation reaction of L-4 with amines, but we abandoned this method finally, because of its low yield.
The structures of the target compounds were characterized by 1 H-NMR, 13 C-NMR, MS and elemental analysis. Twenty two of them were published in a Chinese patent [34]. The 1 H-NMR and 13 C-NMR spectra are available in Supplementary Data. Additionally, the structure of II-19 was confirmed by X-ray single crystal diffraction (Figure 4a). The molecular structure of II-19 existed as a chair conformation, the sulfonamide moiety on the equatorial bond position and the glycinamide moiety was linked to cyclohexane by an axial bond. The first chiral carbon atom (C11) had the S configuration and the second chiral carbon atom (C10) had the R configuration. Based on the structural similarities of all the target compounds, it was supposed that all the compounds had the same configuration, the same as II-19. Interestingly, a reported compound (Figure 4b) with similar structure showed proton transfer [25], which was not observed in this study. We found that the H atom on SO 2 -NH (N2) was trapped by the N atom (N1) attached to the cyclohexane, whereas compound II-19 did not present this behavior. Since the amino group (N2) linked to the acetamido structure (which probably exerts an electron withdrawing effect) may acquire a weaker basicity, the lack of proton migration phenomenon appears to be justified. same configuration, the same as II-19. Interestingly, a reported compound (Figure 4b) with similar structure showed proton transfer [25], which was not observed in this study. We found that the H atom on SO2-NH (N2) was trapped by the N atom (N1) attached to the cyclohexane, whereas compound II-19 did not present this behavior. Since the amino group (N2) linked to the acetamido structure (which probably exerts an electron withdrawing effect) may acquire a weaker basicity, the lack of proton migration phenomenon appears to be justified.
(a) Target compound II- 19 (b) Structure in our previous study Figure 4. X-ray crystal structures (red represents oxygen, green is fluorin, light blue stands for hydrogen, deep blue symbolizes nitrogen).

Biological Activity and Structure-Activity Relationship Study
The mycelium inhibition of all target compounds against B. cinerea was initially tested at 50 μg mL −1 , and most of them exhibited significant fungicidal activity similar or superior to control (Tables  1 and 2). The inhibitory rate of II-4, II-12, and II-13 (90.66%, 90.41%, and 90.90%) was higher than boscalid (89.18%) against CY-09. Next, we further carried out a concentration gradient test on CY-09 and calculated the EC50 values (Table 1). The result showed that the EC50 values of 14 compounds were less than procymidone (EC50 = 10.31 μg/mL). II-5, II-6, II-19, II-26, and II-28 had higher fungicidal activity than all positive control, with the EC50 values of 3.38, 3.38, 3.26, 3.40, and 3.57 μg mL −1 . II-4, II-11, II-18, II-30, II-31, and II-33 also showed outstanding fungicidal activity, which were significantly better than carbendazim, and substantially the same or better than the other four. Based on the results, 9 compounds were tested on HLD-15 and DL-11 in the same manner, and further confirmed the high fungicidal activity again ( Table 2).

Biological Activity and Structure-Activity Relationship Study
The mycelium inhibition of all target compounds against B. cinerea was initially tested at 50 µg mL −1 , and most of them exhibited significant fungicidal activity similar or superior to control (Tables 1 and 2). The inhibitory rate of II-4, II-12, and II-13 (90.66%, 90.41%, and 90.90%) was higher than boscalid (89.18%) against CY-09. Next, we further carried out a concentration gradient test on CY-09 and calculated the EC 50 values ( Table 1). The result showed that the EC 50 values of 14 compounds were less than procymidone (EC 50 = 10.31 µg/mL). II-5, II-6, II-19, II-26, and II-28 had higher fungicidal activity than all positive control, with the EC 50 values of 3.38, 3.38, 3.26, 3.40, and 3.57 µg mL −1 . II-4, II-11, II-18, II-30, II-31, and II-33 also showed outstanding fungicidal activity, which were significantly better than carbendazim, and substantially the same or better than the other four. Based on the results, 9 compounds were tested on HLD-15 and DL-11 in the same manner, and further confirmed the high fungicidal activity again ( Table 2). The compounds with outstanding fungicidal activity in vitro tests also demonstrated excellent control effect and stable performance in cucumber and tomato pot experiments (Tables 3 and 4). Meanwhile, there was no phytotoxicity occurred during the tests. II-4, II-5, II-12 and II-13, which showed superb activity in vitro tests, had higher inhibition rates (90.48%, 93.45%, 92.86%, and 91.07%) in cucumber pot test than carbendazim (59.52%), procymidone (83.33%), boscalid (88.10%), pyrimethanil (82.14%), and cyprodinil (88.69%). In tomato pot test, the control efficacy of II-4, II-5, II-7, II-8, II-15, and II-33 were 67.19%, 68.43%, 73.83%, 80.14%, 76.96%, and 72.02% at the concentration of 500 µg mL −1 , which were similar to boscalid (77.05%), the best performing amongst the controls. Subsequently, we selected 9 compounds to test, and reduced the concentration to 200 µg mL −1 . The results showed that the test compounds basically maintained high control effect, and the control efficacy of II-4, II-5, II-8, and II-15 (78.51%, 78.91%, 87.98%, and 87.97%) were better than all control agents. Due to the growth status of tomato seedlings and the pathogenicity of spores at different stages, the two batches of results did not show a directly proportional to the concentration, but overall, results showed the same trend, that specific compounds showed higher effect than control.  Accordingly, by analyzing the experimental results, we determined the relationship between chemical structures and fungicidal activity as (1) the compounds with substituents on benzene ring were better than that without substituents (II-1), and fluorine and bromine atoms on benzene ring showed better activity, while fluorine was the best; (2) Substituents on phenyl ring were more active at paraand ortho-position than at meta-position. Activity was generally low with substituents only at meta-position (II-2, II-16, II-20, II-22) and with little activity at both 3-and 5-position (II-21); (3) Compared II-3, II-4, II-5, II-13 and II-14, we found that there was no significant difference between di-and multi-substituted compounds, but it was obviously better than the mono-substituted; (4) The halogen atoms on the benzene ring were superior to the electron-donating groups (CH 3 O-); (5) Comparison of II-18 and II-27 or comparison among II-15, II-26, and II-28 showed that extension of the carbon chain between the amine group and benzene ring increased in vitro fungicidal activity; (6) By comparing II-29 to II-33, it can be concluded that increasing the length of the alkyl carbon chain enhanced fungicidal activity, and the cyclic structure (II-31) had a higher activity than the chain structure (II-30) of the same carbon number. In total, compounds with substituted benzene ring showed more stable and highly fungicidal activity than compounds with alkyl substituents.
Based on overall bioassay results, the compounds had a stronger fungicidal effect on in vitro assays and cucumber pot experiments (infected with mycelium) compared to tomato pot experiments (infected with spraying spores), that is to say, the compounds had slightly better inhibitory effects on mycelium than spores.

Materials and Instrumentation
All solvents and reagents were commercially available for analytical reagent (AR) grade and dried prior to use. Column chromatography (Silica gel: 200-300 mesh) was used to purify target compounds. The melting points were determined by the X-5 binocular microscope melting point apparatus (Beijing Tech Instrument Co. Ltd., Beijing, China). 1 H-NMR and 13 C-NMR spectra were recorded on a Bruker 600 MHz and 101 MHz spectrometer (Bruker, Karlsruhe, Germany), using dimethyl sulfoxide (DMSO-d 6 ) as solvent and tetramethylsilane (TMS) as the internal standard. MS data were obtained on the 7000C Triple Quad GC/MS and 6460 Triple Quad LC/MS Mass Spectrometers (Agilent Technologies, Santa Clara, CA, USA).

Synthesis of Substituted Chloroacetamide I
Under nitrogen atmosphere, dry CH 2 Cl 2 (30 mL), amine (0.02 mol), and Et 3 N (0.05 mol) were added to a three-necked round bottom flask and stirred for 0.5 h, then chloroacetyl chloride dropped slowly and reacted for 3 h at room temperature. Then, the solution was washed with 2 mol L −1 hydrochloric acid (30 mL), saturated aq NaHCO 3 solution (30 mL), and brine (40 mL), successively, then dried over anhydrous Na 2 SO 4 and filtered. After evaporating CH 2 Cl 2 in vacuum, the obtained crude product was refined by recrystallization using ethyl acetate/petroleum ether.

Fungal Bioassay
In vitro and in vivo fungicidal activities of all target compounds against B. cinerea were tested by mycelium growth inhibition tests and greenhouse pot experiments. Three strains of B. cinerea (CY-09, DL-11, HLD-15) were isolated from naturally infected tomato leaves in three different areas (CY-09, DL-11, and HLD-15 came from Chaoyang, Dalian, and Huludao, respectively) in Liaoning Province, China. Carbendazim, procymidone, boscalid, pyrimethamine, and cyprodinil were provided by Shenyang Research Institute of the Chemical Industry, National Pesticides Engineering Research Centre and used as positive control.

Evaluation of Target Compounds II on the Inhibition of B. cinerea
Preliminary tests verified the in vitro fungicidal effect of B. cinerea on plates by the mycelium growth inhibition method. Commercial fungicides were used as positive control, while acetone was the blank. Compounds to be tested were dissolved in acetone and diluted with sterilized molten potato-dextrose agar (PDA) medium to obtain the drug-containing medium at the final concentration of 50 µg mL −1 , and poured it into sterile 90 mm Petri dishes (15 mL per dish). The medium solidified and reduced to room temperature, and 5 mm plugs of B. cinerea culture were placed in the center of the PDA plates. Then, the plates were sealed with parafilm and incubated under a regular 12:12 h light/dark regimen at 26 • C for 96 h. The radial growth diameters were measured, and relative inhibition rate of treatment compared to blank assay was calculated by the following equation: where d d was the diameter of the original disk (5 mm), d c and d t represented the average diameter of 3 replicates of the control and treatment plates, respectively.

Evaluation of the Fungicidal Activity on B. cinerea by Concentration Gradient Test
Based on the result of multiple preliminary tests, the EC 50 values of specific compounds were evaluated to verify further fungicidal effect on B. cinerea by the method as previously described.
The four concentrations of the tested compounds in PDA were 50, 12.5, 3.125 and 0.78125 µg mL −1 . The EC 50 values were calculated using log-probit analysis.

In Vivo Fungicidal Activity against B. cinerea (CY-09) by Greenhouse Pot Experiments
(1). Evaluation of Fungicidal Activity on Cucumber Leaves The compounds to be tested were processed to 5% emulsifiable concentrate (EC) formulations and diluted with water to 500 µg mL −1 . The rest of the procedures were given in ref [17]. The seedlings were maintained in a greenhouse pot at 24 ± 1 • C and relative humidity above 90%. When the blank control was infected completely, the lesion diameters of all cotyledons were measured. The calculation method was referred to the formula given in Section 3.3.1.
(2). Evaluation of Fungicidal Activity on Tomato Leaves The conidia of B. cinerea were obtained from a 4-week-old PDA medium, which was cultured at 25 • C. The mixtures of 10 mL sterile distilled water and 0.05 mL Tween 80 were poured into culture dishes to let the spores fall off. The spore-rich suspension was filtered through four layers of sterilized gauze to remove mycelium. Finally, the conidia concentration was measured using a hemocytometer and diluted to 10 6 -10 7 spores per mL. The processing method of the compound-containing EC formulation at the concentration of 500 µg mL −1 and 200 µg mL −1 was identical to Section 3.3.3 (1). When the number of tomato leaves was about 30, the solution of the compound to be tested were sprayed evenly onto tomato leaves and dried naturally. Subsequently, the spore suspension was sprayed on seedlings and plants kept in the same condition as test Section 3.3.3 (1). When the blank assay was infected completely, the Disease Index of all leaves were investigated and the Control Efficiency was calculated [37].

Conclusions
In summary, 33 novel 2-glycinamide cyclohexyl sulfonamide derivatives were designed and synthesized. II-4, II-5, and II-15 were found as the best active compounds in inhibiting B. cinerea in this series of compounds, which were superior to the commercial fungicides carbendazim, procymidone, boscalid, pyrimethanil, and cyprodinil in both in vitro and in vivo tests. The structure-activity relationship showed that docking glycinamide on the lead compound and introducing a benzene ring structure with two fluorine atoms at 2,4-or 2,5-positions could greatly enhance activity. Studies on the structural optimization and mechanism of action are in progress. We believe these compounds will become promising candidates for pesticides.
Supplementary Materials: Supplementary materials are available online. The 1 H-NMR and 13 C-NMR spectra of target compounds II and detailed description of the crystal structure of II-19.