Synthesis, Fungicidal Activity and SAR of 2-Thiazolamide/Pyrazolamide-Cyclohexylsulfonamides against Botrytis cinerea

In order to explore more efficient sulfonamides against Botrytis cinereal, 36 novel cyclohexylsulfonamides were synthesized by N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDCI) and 1-hydroxybenzotriazole (HOBt) condensation reaction using chesulfamide as a lead compound, introducing thiazole and pyrazole active groups. Their structures were characterized by 1H-NMR, 13C-NMR, mass spectrum (MS), and elemental analysis. Compound III -31 was further confirmed by X-ray single crystal diffraction. The in vitro and in vivo fungicidal activities against B. cinerea were evaluated by three bioassay methods. The results of mycelial growth demonstrated that median effective concentration (EC50) values of nine compounds were close to boscalid (EC50 = 1.72 µg/mL) and procymidone (EC50 = 1.79 µg/mL) against B. cinerea (KZ-9). In the spore germination experiment, it was found that compounds III-19 and III-31 inhibited germination 93.89 and 98.00%, respectively; at 10 µg/mL, they approached boscalid (95.97%). In the tomato pot experiment, the control effects of two compounds (III-21 and III-27) were 89.80 and 87.90%, respectively, at 200 µg/mL which were significantly higher than boscalid (81.99%). The structure–activity relationship (SAR) was also discussed, which provided a valuable idea for developing new fungicides.


Introduction
Botrytis cinerea is one of the top 10 fungal pathogens that causes grey mould or Botrytis blight. It mainly affects vegetables and fruits, as well as a large number of flowers, shrubs, weeds and trees worldwide [1][2][3][4][5]. Many fungicides have failed to control B. cinerea because of their high risk for fungicide resistance development [6]. Therefore, it is worth developing and synthesizing new safe products that can effectively control B. cinerea.
It is a common familiarity that sulfonamide compounds have excellent biological activities not only in the field of pharmaceuticals [7,8] but also as agrochemicals [9][10][11][12], such as herbicides and fungicides. A novel sulfonamide fungicide chesulfamide [13,14] is different from other commercial fungicides; it showed excellent fungicidal activity against B. cinerea due to its unique mechanism of action, including (1) acting on mycelium cell membrane, (2) disturbing DNA, and (3) inducing disease resistance of plants [13,15]. Therefore, chesulfamide was used as a lead compound.
In the current situation, thiazolamide and pyrazolamide fungicides have become the dominant fungicide varieties, such as Ethaboxam, Thfluzamide [16], Bixafen [17], Sedaxane [18],      In this study, we used chesulfamide as a lead compound, reducing the carbonyl group to an amino group [26,27], and the EDCI/HOBt condensation method [24,25], interfacing with the pyrazole carboxylic acid and thiazole carboxylic acid. In order to improve the fungicidal activities against B. cinerea, the benzene ring (linked to NH) was optimized, which was different from the structure of 2-trifluoromethyl-4-chlorobenzene before. Moreover, 18 novel 2-thiazolamide cyclohexylsulfonamides and 18 novel 2-pyrazolamide cyclohexylsulfonamides were designed and synthesized (Scheme 1b). The in vivo and in vitro fungicidal activities against B. cinerea were evaluated and the structure-activity relationship was analyzed. The synthetic route of the target compounds was shown in Scheme 2. Molecules 2019, 24,  O Ⅰ Ⅱ Ⅲ Figure 2. X-ray crystal structures.

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Scheme 2. Synthetic route of the target compounds III.

Chemistry
The corresponding aminocyclohexylsulfonamide (I), HOBt and EDCI in the presence of triethylamine (Et 3 N) in dichloromethane (CH 2 Cl 2 ) as solvent were allowed to react with different carboxylic acids (II) at 0 • C. Finally, 36 novel cyclohexylsulfonamides compounds III were synthesized.
The structure of compound III-31 was analyzed by X-ray single crystal diffraction. The structure (CCDC: 1917351) was shown in Figure 2a and available in Supplementary Data. X-ray diffraction provided a relative configuration of chiral centers, hence it proved the cis-configuration at the cyclohexane ring. Probably, the compound III-31 was racemates, which mixed both enantiomers ("C 7 "R, "C 12 "S and "C 7 "S, "C 12 "R). A compound reported (Figure 2b) [26] with a similar structure showed that there were two intramolecular hydrogen bonds in the structure, N 1 -H . . . O 3 and N 2 -H . . . O 2 . On the contrary, in our study, compound III-31 had no intramolecular hydrogen bonds, but had intermolecular hydrogen bonds, with N 1 in one molecule being linked to O 3 in another molecule, eventually forming the chain structure ( Figure 2c). O Ⅰ Ⅱ Ⅲ Figure 2. X-ray crystal structures. Additionally, in this work, the chemical structures of all the synthesized compounds were characterized by nuclear magnetic resonance (NMR), mass spectrum (MS) and elemental analysis. The 1 H NMR and 13 C NMR spectra are available in Supplementary Data. In the 1 H NMR spectra (600 MHz, DMSO-d 6 ) of compound III-28 (Figure 3a), the protons on the benzene ring were revealed in a low field in the range of δ H 7.4 to 7.6 ppm, while cyclohexyl and methyl group gave signals in the range of δ H 1.0 to 5.0 ppm. However, pyrazole-H and SO 2 NH appeared at δ H 8.36 ppm and 10.13 ppm, respectively. The carboxamide proton of NH-C=O showed doublet (d) at δ H 7.67 ppm, J = 8. 4 Hz. The signal of proton of CH-SO 2 at δ H 4.64 ppm revealed only coupling constants less than 9 Hz, indicating the equatorial position of this proton. Analogously, the axial orientation of the proton on CH-N was revealed by its splitting to doublet of triplets (dt) at δ H 3.52 ppm with one diaxial coupling of J = 11.4 Hz and two axial equatorial couplings of J = 3.6 Hz. The splitting pattern of protons on CHF 2 is very characteristic. Generally, the proton of CHF 2 showed triplet (t) at δ H 7.26ppm, J = 54 Hz.
In the 13 C-NMR spectra (151 MHz, DMSO-d 6 ) of compound III-28 (Figure 3b), the signals in the range of δ C 0 to 70 ppm were assigned to methyl and cyclohexyl groups. Benzene ring carbons and most of the pyrazole carbons resonated in the range of δ C 112 to 140 ppm and the signal at δ C 161 ppm belonged to C=O. The difluoromethyl group caused the triplet at δ C 110.16 ppm with 1 J FC = 234.05 Hz, while the adjacent carbon signal at δ C 144.78 ppm is split into a triplet due to 2 J FC = 22.80 Hz.
In the 13 C-NMR spectra (151 MHz, DMSO-d 6 ) of compound III-25 ( Figure 3c), there were very interesting phenomena due to seven fluorine atoms. Carbon signals of C-F in the F-Ph showed a doublet at δ C 162.10 ppm, 1 J FC = 246.43 Hz, while carbon signals of C-F in 2,4,5-trifluorobenzene, which appeared in the range of δ C 145 to 151 ppm, showed 1 J FC couplings of 245.38, 244.77, 239.03 Hz, respectively, and further long-range C-F coupling. The carbon signals of CF 3 appeared as a quartet at δ C 119.61 ppm with 1 J FC = 270.74 Hz, while the quartet of the adjacent carbon at δ C 129.96 ppm showed the 2 J FC coupling of 39. 26 Hz. In order to screen out active compounds quickly and correctly, the target compounds were 121 tested by an in vitro mycelium growth inhibition assay and the spore germination method.

Biological Assay and Structure-Activity Relationship Study
In order to screen out active compounds quickly and correctly, the target compounds were tested by an in vitro mycelium growth inhibition assay and the spore germination method. Then, active compounds were tested in tomato pot experiments.

In Vitro Fungicidal Activity against B. cinerea
The germicidal virulence test of 36 target compounds was tested against the B. cinerea strain (KZ-9), with boscalid and procymidone as standard fungicides. The EC 50 values of 17 compounds were lower than 10 µg/mL. Compounds III-19 and III-27 showed better in vitro fungicidal activity against KZ-9, with the EC 50 values of 1.99 and 2.04 µg/mL (Table 1). Some compounds showed excellent fungicidal activity against KZ-9 and were rescreened out by another B. cinerea strain (CY-09). The EC 50 value of compound III-21 against CY-09 was 2.33 µg/mL, which showed its excellent fungicidal activity ( Table 2). The results represented in Table 3  The results represented in Table 3  The results represented in Table 3 indicated that several compounds can inhibit the spore The results represented in Table 3 indicated that several compounds can inhibit the spore germination of B. cinerea at the concentration of 10 µg/mL. Among these, compounds III-19 and III-31 showed superb activity on spore germination, which had higher inhibition rates (93.89 and 98.0%) were comparable to boscalid (95.97%).

In Vivo Fungicidal Activity against B. cinerea
After that, the in vivo control effect of active compounds against B. cinerea on tomato leaves were tested at 200 µg/mL. The results showed that the control effects of compounds III-19, III-21 and III-27 were 78.00, 89.80 and 87.90%, respectively, which were similar to boscalid (81.99%). The three compounds showed excellent activities in in vivo tests (Table 4). Accordingly, by analyzing the experimental results, the structure-activity relationship (SAR) was summarized as follows: (1) Compounds III-10-III-18 had the same alkyl-thiazole structure as that of Thifluzamide while compounds III-1-III-9 had phenylthiazole structure. It was noted that compounds III-1-III-9 showed significantly improved fungicidal activities. In addition, compounds containing pyrazoles revealed the same results; it showed that fungicidal activities of compounds III-19-III-27 were better than compounds III-28-III-36. In total, compounds with benzene ring-substituted thiazole or pyrazole active groups showed more highly fungicidal activities. (2) For 2-thiazolamide-cyclohexylsulfonamides (III-1-III-18), the activity was significantly increased when the benzene ring (linked to NH) contained fluorine-containing groups (2-F, 3-F and 2,4,5-F), such as compounds III-1, III-2, III-9 and III-11. When the benzene ring (linked to NH) contained chlorine, the phenylthiazole compounds III-3 and III-4 showed excellent fungicidal activity. However, fungicidal activity was mediocre on the benzene ring containing bromine. (3) For 2-pyrazolamide-cyclohexylsulfonamides (III-19-III-36), the activity was higher when the benzene ring (linked to NH) contained fluorine-containing groups (2-F, 3-F), such as phenylpyrazole compounds III-19 and III-27, but, if the substituent was a 2,4,5-fluorine, the fungicidal activity slightly decreased. Additionally, when the benzene ring (linked to NH) contained 4-chlorine, the compounds III-21 and III-36 showed excellent activity. When the benzene ring (linked to NH) contained bromine, the phenylpyrazole compounds III-23 and III-24 showed excellent activity. 4) It could be seen that the compounds III containing pyrazole substituent showed higher fungicidal activity than thiazole substituent.

Materials and Instrumentation
The reagents and solvents were commercially available for analytical reagent (AR) grade and were used as received or were dried prior to use, as needed. The melting point was measured by X-5 binocular microscope melting point apparatus (Beijing Tech Instruments Co. Ltd., Beijing, China). Using dimethyl sulfoxide (DMSO-d 6 ) as solvent and tetramethylsilane (TMS) as the internal standard, 1 H-NMR spectra were recorded on 300 MHz and 600 MHz spectrometers and 13 C-NMR spectra were recorded on 75 MHz and 151 MHz spectrometers (Bruker, Karlsruhe, Germany). 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). Elemental analyses were determined on a Vario EL III elemental analyser (Elementar Analysensysteme GmbH, Frankfurt, Germany).

Synthesis of N-Substituted Phenyl-2-Aminocyclohexylsulfonamides I
The synthesis of N-substituted phenyl-2-aminocyclohexylsulfonamide I was done according to the reported method [10,21,27]. In this paper, 2-trifluoromethyl-4-chloroaniline in the reference was replaced by different substituted aniline, but the experimental methods were consistent. The yield of the compounds I was 40-75%.

Fungicidal Activity Bioassays
The in vitro and in vivo fungicidal activities of all the target compounds against B. cinerea were tested by mycelium growth inhibition assay, spore germination experiment and tomato pot experiment, respectively. The B. cinerea strains (KZ-9, CY-09) were collected from different regions of Liaoning, China, and cultured on potato dextrose agar (PDA) for several generations. Boscalid and procymidone (with the purity more than 95%) are provided by the Shenyang Research Institute of the Chemical Industry.

Evaluation of the Fungicidal Activity on B. cinerea by Mycelium Growth Experiments
Using the mycelium growth rate method, the compounds were dissolved in acetone and mixed with sterile molten PDA to obtain four concentrations of 50, 12.50, 3.125 and 0.78 µg/mL, poured them into sterile 90 mm diameter Petri dishes under aseptic conditions, respectively. A 5 mm B. cinerea plug was inoculated in the center of 90 mm PDA petri dish. Boscalid and procymidone were used as positive control agents while acetone was used as blank control. The EC 50 value was calculated according to mycelium growth. [25] 3.3.2. Evaluation of the Fungicidal Activity on B. cinerea by Spore Germination Experiments The effect of target compounds III on spore germination against B. cinerea was determined by the concave slide method. The method was given in reference [25]. The commercial fungicide boscalid was used as the positive control, and the results were given in Table 3. 3.3.3. In Vivo Fungicidal Activity against B. cinerea by Tomato Pot Experiments The compound was first prepared to 5% emulsifiable concentrate (EC) and diluted with water into an aqueous solution of 200 µg/mL. Tomato seedlings at the 6-8 leaves stage on the main stem were selected as objects. Spraying the solution evenly on the tomato plants and upon naturally drying, a spore suspension of B. cinerea with 5 × 10 5 spores per mL was evenly sprayed on the tomato seedlings. It was placed in a greenhouse at 20-25 • C, with humidity above 80%. Waiting for observation, using boscalid as control agents, and an emulsifiable concentrate without agent was the blank control and then disease incidence and index on tomato leaves were calculated. [25]

Conclusions
In conclusion, 36 novel sulfonamide compounds were designed and synthesized in this paper. The thiazole and pyrazole active groups were introduced by the method of active substructure splicing, and benzene ring (linked to NH) was optimized. In vitro and in vivo fungicidal activities against B.cinerea were evaluated, and some target compounds showed excellent activity. Moreover, the structure-activity relationship revealed that the compounds containing phenylpyrazole amide structure showed better activity effects, while fungicidal activity was highest at the meta-substituted fluorine atom on the benzene ring (linked to NH), such as compound III-19 showing notable in vitro fungicidal activity. Thus, the present results laid the foundation for further structural design and fungicide screening.

Conflicts of Interest:
The authors declare no conflict of interest.