Synthesis of 2-Acyloxycyclohexylsulfonamides and Evaluation on Their Fungicidal Activity

Eighteen N-substituted phenyl-2-acyloxycyclohexylsulfonamides (III) were designed and synthesized by the reaction of N-substituted phenyl-2-hydroxyl-cycloalkylsulfonamides (I, R1) with acyl chloride (II, R2) in dichloromethane under the catalysis of TMEDA and molecular sieve. High fungicidal active compound N-(2,4,5-trichlorophenyl)-2-(2-ethoxyacetoxy) cyclohexylsulfonamide (III-18) was screened out. Mycelia growth assay against the Botrytis cinerea exhibited that EC50 and EC80 of compound III-18 were 4.17 and 17.15 μg mL−1 respectively, which was better than the commercial fungicide procymidone (EC50 = 4.46 μg mL−1 and EC80 = 35.02 μg mL−1). For in vivo activity against B. cinerea in living leaf of cucumber, the control effect of compound III-18 was better than the fungicide cyprodinil. In addition, this new compound had broader fungicidal spectra than chlorothalonil.

The ester group is the common bioactive moiety and 2-hydroxycycloalkylsulfonamides possess promising fungicidal activity. In continuation of our research on the synthesis of biological cycloalkylsulfonamide compounds, esterification was applied to the N-substituted phenyl-2hydroxycycloalkylsulfonamides (I), which was as the lead compounds to get the title compounds III (Scheme 1). Their fungicidal activity against B. cinerea and other 10 kinds of pathogenic fungi were evaluated to obtain the optimized compounds with significantly improved fungicidal activity and broad antifungal spectra.

Synthesis and Structure Elucidation
Oxalyl chloride was used as the chlorinating agent in this experiment, which was reacted rapidly under the catalysis of DMF. The reaction made a very high yield of the acyl chloride, which was reacted with the compounds containing hydroxy group under the catalysis of TMDEA and molecular sieves to obtain a very high yield of target compounds.
In the 1 H NMR spectra of the compound III-5, 2-H α was split by 1-H α , 3-H e and 3-H α in the cyclohexane, forming the splitting ddd peak ( Figure 2). It may be the trans chair-conformation in Figure 3. Due to the directly induced effect of the sulfonyl group and the ortho-position effect of the acyloxy to the 1-H α , which made the CH signal slightly wide single peak in the spectra, the peak type was similar to that of NH in sulfonamide structure. In the IR spectrum, the very obvious ester carbonyl stretching vibration appeared around 1700 cm −1 . NH stretching vibration was around 3200 cm −1 to 3500 cm −1 .    From the results of mycelial growth rate (Table 1) and structure-activity relationship (SAR), it could be seen that acyloxy substituent showed higher antifungal activity when it was a small group. Among them, 2-substituted benzoyloxy compounds showed generally lower activity, while 2-(2-alkoxyacetyloxy) cycloalkylsulfonamides showed good activity. The same structure-activity relationship appeared in the test of detached leaves of cucumber (Table 1). It showed obvious bioactivity when the R 2 was ethoxymethyl and methoxymethyl. The bioactivity of the N-(2,4,5-trichlorophenyl)-2-acyloxycyclohexylsulfonamides were better than that of the N-(2-trifluoromethyl-4-chlorinephenyl)-2-acyloxycyclohexyl-sulfonamides. The fungicidal activity of III-18 was a little higher than that of the procymidone in vitro tests. While the detached leaves test showed that the control efficiency of III-18 was significantly higher than that of the procymidone. According to Regulation (EC) No 1107/2009, procymidone is no longer approved in the European Union. Because of the great fungicidal activity, III-18 could be a potential substitute of this fungicide. The letters a-l denoted the results of difference significance analysis. Means followed by the same letter within the same column are not significantly different (p ≥ 0.05, Fisher's LSD multiple comparison test).

Fungicidal Activity of Compounds III-9 and III-18 against Ten Pathogenic Fungi
The screening data in Table 2 indicated that the two compounds III-9 and III-18 had broad fungicidal spectra. III-9 exhibited moderate activity against ten pathogenic fungi. Compound III-18 exhibited excellent bioactivity against the most of the selected fungus species (except for the R. solani) and the inhibitory rate was similar to or better than the commercialized fungicide chlorothalonil. In addition, the concentration gradient experiment was evaluated by the method of cucumber leaf to check the fungicidal activity of compounds III-18, N-(2,4,5-trichlorophenyl)-2hydroxycycloalkylsulfonamide (19) and N-(2,4,5-trichlorophenyl)-2-oxycycloalkylsulfonamide (20). The results in Table 3 showed that the activity of the compounds was gradually improved after continuous structural optimization. From the results, it could be seen that the bioactivity was not obviously different between III-18 and 19, which was much better than the activity of 20, and a little better than that of the control cyprodinil. The pathogen infection symptoms ( Figure 4) showed that the leaves treated by the compound III-18 were still green and the disease spots were wet blotch plaque, which was better than the leaves treated by other compounds. The leaves treated by 20 and blank control were black and rotten.

General Information
Infrared (IR) spectra were recorded in potassium bromide disks on a Perkin Elmer Spectrum 65 spectrophotometer. Nuclear magnetic resonance (NMR) spectra were recorded in CDCl 3 unless indicated otherwise with Bruker 600 MHz or 300 MHz spectrometers, using tetramethylsilane (TMS) as the internal standard. Elemental analysis was performed by the analytical center at the Institute of Chemistry, Chinese Academy of Sciences, Beijing, China. Melting points were measured on an X-5 melting-point apparatus, and the thermometer was uncorrected. The solvents and reagents were used as received or were dried prior to use as needed.

Synthesis of Compounds I
Compounds I were synthesized according to the method given in the references [25] and [27].

Synthesis of Compounds III-1-III-18
According to the method given in the reference [28], to the solution of I (0.01 mol), N,N,N',N'-tetramethylethylenediamine (TMEDA, 0.006 mol) and 3A molecular sieves (2 g) in dry dichloromethane (30 mL), acyl chloride II (0.011 mol) was added dropwise at room temperature. The mixture was stirred at room temperature for 2 h. And the reaction was quenched by ice water (20 mL × 2), filtered and dried with anhydrous magnesium sulfate. After evaporating the solvent under vacuum, the crude product was purified by silica gel chromatography using petroleum/ethyl acetate (10/1, v/v) as eluant to obtain III (Scheme 1).

Effect of the Title Compounds on the Mycelial Growth
Fungicidal activities of the compounds against B. cinerea were evaluated using the method given in reference [27]. The title compounds III were dissolved in acetone and mixed with sterile molten potato dextrose agar (PDA) to obtain the gradient concentrations of 50, 25, 12.5, 6.25, 3.13, 1.56 and 0.78 μg mL −1 . The commercial fungicide procymidone (with a percentage composition of 96%) was used as the positive control. The inhibition rate was calculated according to Equation (1). The EC 50 and EC 80 values of compounds III were estimated using logit analysis, and the results are given in Table 1.
where I 1 is the inhibition rate, D 1 is the average diameter of mycelia in the blank test, and D 0 is the average diameter of mycelia in the presence of compounds.

Effect of Compounds III-9 and III-18 against Ten Major Crop Diseases
The fungicidal activity of compounds III-9 and III-18 against ten kinds of fungi was assessed using the mycelium growth test on PDA. The compounds were dissolved in acetone and mixed PDA to obtain final concentration of 50 μg mL −1 . Chlorothalonil was used as the positive control. Other test conditions were the same with the method given in the mycelial growth, and the results were shown in Table 2. 3.3.3. Effect on the Ability of B. cinerea to Colonize Detached Leaves of Cucumber The compounds III-1-III-18 were confected to 2.5% EC formulations, which were diluted to concentration of 500 μg mL −1 with water to obtain the solutions that were spread on the surface of the cucumber leaves. After air drying for 2 h at 23 °C , the upper sides of the leaves were inoculated with 5 mm plugs of B. cinerea, which was maintained on PDA [25]. The cucumber leaves were maintained at 24 ± 1 °C in culture dishes. The inhibition rate was calculated and the results were shown in Table 1.

In Vivo Fungicidal Activity against B. cinerea by Pot Culture Test Method in Greenhouse
Using pot culture test method according to the reference [25], the in vivo fungicidal activity of the title compounds against B. cinerea was evaluated in greenhouse. B. cinerea was maintained on potato dextrose agar (PDA) medium at 4 °C . The culture plates were cultivated at 24 ± 1 °C . Germination was conducted by soaking cucumber seeds in water for 2 h at 50 °C and then keeping the seeds moist for 24 h at 28 °C in an incubator. When the radicles were 0.5 cm, the seeds were grown in plastic pots containing a 1:1 (v/v) mixture of vermiculite and peat. Cucumber plants used for inoculations were at the stage of two seed leaves. The compounds III-18, 19 and 20 were confected to 2.5% EC formulations, which were diluted to concentration of 500, 125 and 31.25 μg mL −1 with water to obtain the solutions. Tested compounds and commercial fungicides were sprayed with a hand spray on the surface of the seed leaves. Water sprayed seed leaves were set as the CK (Figure 4). After drying, the upper sides of the leaves were inoculated with 5 mm plugs of B. cinerea, which was maintained on PDA [25]. The plants were maintained at 24 ± 1 °C and above 80% relative humidity in greenhouse. The fungicidal activity was evaluated and the results were shown in Table 3 and

Conclusions
N-substituted phenyl-2-acyloxycyclohexylsulfonamides were designed and synthesized. Their structures were confirmed by 1 H NMR, IR and elementary analysis. Their fungicidal activity was better than the leading compounds N-substituted phenyl-2-hydroxycycloalkylsulfonamides. Among them, compound III-18 showed better in vitro and in vivo fungicidal activity than the fungicides procymidone and cyprodinil. In addition, this new compound had broader fungicidal spectra than chlorothalonil. The preliminary structure and activity relationship showed that the 2-alkoxyacetoxy groups were considerably more likely to improve the activity of these compounds.