Synthesis, Fungicidal Activity, and Structure Activity Relationship of β-Acylaminocycloalkylsulfonamides against Botrytis cinerea

Abstract

In order to discover new antifungal agrochemicals that could have highly active and novel motifs, thirty-six new 2-acylaminocycloalkylsulfonamides (IV) were synthesized. Their structures were characterized and confirmed by ¹H NMR, ¹³C NMR, IR, MS, elemental analysis and X-ray single crystal diffraction. In vitro and in vivo activities against various Botrytis cinerea strains were evaluated. Bioassay results revealed that most of the title compounds exhibited excellent in vitro fungicidal activity, in which compound IV-26 showed the highest activity against sensitive, low-resistant, moderate-resistant and high-resistant strains of B. cinerea compared with the positive fungicide procymidone. Meanwhile in vivo fungicidal activity of compound IV-31 was better than the commercial fungicides procymidone and chesulfamide in greenhouse trial. The structure activity relationship (SAR) was also discussed and the results were of importance to the structural optimization and development of more potent sulfonamides antifungal agents.

Botrytis cinerea (teleomorph: Botryotinia fuckeliana) is an airborne plant pathogen with a necrotrophic lifestyle attacking over 200 crop hosts worldwide. Many kinds of fungicides have been failed to control this plant disease due to its genetic plasticity¹. Moreover, the continuous use of fungicides, such as carbendazim, diethofencarb, procymidone, and pyrimethanil etc, has led to the growing resistance of this plant pathogen to fungicides². Thus, phytofungal disease control is urgently necessitated the discovery and development of new antifungal agents with highly active, low resistance and novel motifs for plant protection.

As very important sulfur-containing analogs of amino carboxylic acids, 2-aminoethanesulfonic acid was first isolated from ox bile in 19^th century by Tiedemann and Gmelin, which name ‘taurine’ was attributed by Gmelin³,⁴. In addition, to be an essential amino acid of human body, taurine has also shown a variety of biological functions⁵,⁶,⁷,⁸,⁹,¹⁰,¹¹,¹²,¹³. Its derivatives had been received much more attention around the world. For example, ASPA (3-amino-2-sulfopropanoic acid, Fig. 1) and CA (2-amino-3-sulfopropanoic acid, Fig. 1), the simple substituted taurines, showed some anti-inflammatory activities¹⁴. As representatives of cyclic taurine derivatives, TAPS ((1S,2S)-2-aminocyclopentane-1-sulfonic acid, Fig. 1) and PSA (piperidi-3-sulfonic acid, Fig. 1) gave different effects on ATP-dependent calcium ion uptake¹⁵, while CAHS ((1R,2S)-2-aminocyclohexane-1-sulfonic acid, Fig. 1) and TAHS ((1S,2S)-2-aminocyclohexane-1-sulfonic acid, Fig. 1) had the thermoregulation ability via interaction with the central serotonergic system¹⁶.

Figure 1. Taurine derivatives.

Besides, 2-aminoethanesulfonic acid had been found as key structural moieties in some natural products, such as dimethyl arsenic aminosulfonate (A, Fig. 1), which was isolated from Sargassum lacerifolium¹⁷. Flavocristamides (B, Fig. 1), isolated from a marine bacterium Flavobacterium sp., was able to inhibit the enzyme DNA polymerase α¹⁸. 5-Taurinomethyluridine (C, Fig. 1) was discovered in mammalian mitochondrial tRNAs¹⁹, which was considered to be responsible for precise codon recognition and the absence of these derivatives led to mitochondrial encephalomyopathic diseases. In addition, taurine deoxyadenosine monophosphates (Tau-dAMP, D, Fig. 1) were recently developed as potential substrates for the HIV-1 reverse transcriptase²⁰.

Studies on the synthesis and biological activity of taurine analogues 2-acylaminoethylsulfonamides have been reported frequently. For instance, β-aminoethanesulfonyl azides E (Fig. 1)²¹ and taurine-containing peptidomimetics F (Fig. 1)²² were synthesized, and 2-indole-acylsulfonamides G (Fig. 1)²³ was used as myeloid cell leukemia-1 inhibitors.

While 2-aminocycloalkylsulfonic acid and its derivatives were rarely reported so far²⁴,²⁵, and there are no reports on the synthesis of 2-acylaminocycloalkyl-sulfonamides. Although its application in field of medicine was primarily reported, in agricultural research was still poorly applied. Recently, our group reported a series of 2-oxycycloalkylsulfonamides (H-N, Figs 2 and 3), which possessed highly fungicidal activity²⁶,²⁷,²⁸,²⁹, of which compound L (chesulfamide, Fig. 3) could be great promise and a lead compound in fungicide research and development. Based on the lead structure of compound L, compounds M and N (Fig. 3) were designed and synthesized with much higher fungicidal activity³⁰,³¹,³².

Figure 2. 2-Oxycycloalkylsulfonamides derivatives.

Figure 3. The designed strategy for the key intermediates II and title compounds IV.

These findings encouraged us to further extend the structural modification of compound L with the aim to find more potent antifungal agents. In this paper, 2-acylaminocycloalkylsulfonamides (IV, Fig. 3) were constructed by reaction of reductive amination and acylation (Fig. 4). The single-crystal structure of the title compounds IV-3 and IV-31 were analyzed. The fungicidal activity of the title compounds against various B. cinerea strains was evaluated. According to their fungicidal activities, structure-activity relationship (SAR) was also discussed.

Figure 4. Synthetic route for the key intermediates II and the title compounds IV-1 to IV-36.

Results and Discussion

Synthesis and Structure Elucidation

The synthetic route of title compounds IV-1 to IV-36 was outlined in Fig. 4 using 2-oxycycloalkylsulfonamides as a starting material. Reductive amination method in ref. 30 was applied to the treatment of ketones with ammonia in ethanol and titanium (IV) isopropoxide, followed by in situ sodium borohydride reduction. In our experiments, however, the method is improved. For the synthesis of compounds II from compounds I, the ethanol solution of ammonia was replaced directly by continuous passing of ammonia gas. The reaction was completed in a short time by monitoring ammonia gas pressure upto 20 mmHg. It was easy to operate and, and the yield of compounds II were from 42% to 96%. In addition, the title compounds IV were easily obtained by the reaction of compounds II with acyl chloride. Yields of title compounds IV were generally high (over 90%).

Crystal structures of compounds IV-3 and IV-31 were analyzed by X-ray single crystal diffraction. Their structures were shown in Fig. 5, and their crystal data were shown in Table S1 to Table S3. Compound IV-3 was typical chair conformation, in which the chiralities of the 9^th and the 14^th carbon atoms on cyclohexane were R and S respectively. In addition, the bulky sulfonamide group was on equatorial bond and the smaller amide group was on axial bond. The spatial configuration was presented as cis-1, 2-disubstituent. Two benzene rings were far apart, which avoided the steric hindrance effect. For compound IV-31, the chiralities of the 3^th and the 9^th carbon atoms on cycloheptane were S and R respectively. Similarly, that of the two groups was also on the same side of the ring plane and its space conformation was cis-1, 2-disubstituent. Specific optical rotation of compounds IV-3 and IV-31 were tested as −39.2° and −0.67° respectively. Compound IV-3 possessed specific optical rotation value of −39.2° due to the better stability of cyclohexane, while compound IV-31 was unstable in methanol solution, of which two conformations were mutually transformed to a raceme, resulting in the optical activity disappeared.

Figure 5. Crystal structures of IV-3 and IV-31.

The structures of the synthesized compounds were confirmed by ¹H NMR, ¹³C NMR, IR, LC-MS and elemental analysis. Due to the structural similarity, all the compounds showed similar spectroscopic characteristics. In ¹H NMR spectra of compounds II and IV, the protons on the benzene ring appeared in low field in the range of δ_H 7.0 to 8.0 ppm, while cycloalkyl group gave signals in the range of δ_H 0 to 5.0 ppm appointed to the protons of CH₃-, CH₂- and CH-. In addition, active hydrogen atoms of -NH₂ and SO₂NH- in compounds II appeared around 8.3 ppm, and these two types of hydrogen signals were combined together to represent a broad singlet. The reason may be that active hydrogen of SO₂NH- is transferred to -NH₂, forming a structure of -NH³⁺. While that of O = C-NH and SO₂NH- in compound IV appeared around 8.3 ppm and 9.3 ppm respectively.

Coupling splitting of protons on CH-SO₂, is very characteristic. Generally, the proton of CH-SO₂ showed doublet of doublet of doublets (ddd), such as compounds IV-5, IV-7, IV-14, IV-21 and IV-23, and corresponding splitting of proton on CH-N was triplet of doublets (td) (Fig. 6). While in the spectra of some compounds, such as compound IV-3, the proton of CH-SO₂ showed doublet of doublets (dd), instead of ddd, and corresponding splitting of proton on CH-N was doublet of triplets (dt) (Fig. 7). However, there were some compounds which coupling splitting of protons on CH-SO₂ were special because the conformation was dynamic, such as compounds IV-13, IV-15, IV-16, IV-30 and IV-32 and so on, the protons of CH-SO₂ and CH-N showed different signal peak type. This phenomenon was very interesting, but the reason was still unknown. We choose the dominant conformation to explain the normal splitting characteristic of CH-SO₂.

Figure 6. ¹H NMR spectrum of compound IV-5.

Figure 7. ¹H NMR spectrum of compound IV-3.

Taking the ¹H NMR spectra of compound IV-3 as an example (Fig. 7), according to the crystal structure, the conformation diagram (Fig. 8) of compound IV-3 was drawn to explain the reasons for this splitting characteristic. As shown in Fig. 8, the proton of C₁₄ linked -SO₂ was located in axial bond (C₁₄-H_a) due to coupling splitting effects of equatorial bond (C9-H_e) on C₉, equatorial bond (C₁₃-H_e) and axial bond (C₁₃-H_a) on C₁₃. Normally the proton of C₁₄ linked -SO₂ showed ddd signal due to the magnetic non-equivalence of these three protons (C₉-H_e, C₁₃-H_e and C₁₃-H_a), appeared as dd signal. Its spatial conformation was shown in Fig. 8(a), which can be determined from the single crystal structure in Fig. 5. It showed a strong coupling splitting effect due to C₁₄-H_a, C₉-H_e and C₁₃-H_e lying on one side of cyclohexane plane and at a close distance, while it showed a weak coupling splitting effect due to C₁₃-H_a and C₁₄-Ha lying on both sides of cyclohexane plane and at a distant position, which led to its split signal invisible in the spectrum. Therefore, in ¹H NMR spectra C₁₄-H_a showed double doublets.

Figure 8. Conformation of compound IV-3 according to the crystal structure.

Correspondingly, the proton of C₉-H_e linked -NH showed double triplets. The reason can be explained by its spatial conformation. As shown in Fig. 8(b), the difference of magnetic non-equivalence of two protons between C₁₀-H_e and C₁₀-H_a is small due to the protons adjacent C₁₀-H_e and C₁₀-H_a close to proton of C₉-H_e. So proton of C₉-H_e affected by protons of C₁₀-H_e and C₁₀-H_a, which signal showed coupling splitting of triplets, and protons of C₉-H_e affected by protons of C₁₄-H_e, which signal showed coupling splitting of doublets. Therefore, in ¹H NMR spectra double triplets were assigned to the proton of C9-H_e.

In ¹³C NMR spectra (see supplementary information), compounds I, II and IV revealed signals of carbon in the range of δ_C 0 to 70 ppm assigned to methyl, methylene and methane on naphthene, and carbon signals of benzene ring and trifluoromethyl in the range of δ_C 115 to 140 ppm in low field. Compounds I and IV gave carbon signals around 202 ppm and 166 ppm respectively assigned to C = O.

In IR spectra of compounds I and IV, the absorption peak of carbonyl stretching vibration appeared around 1700 cm⁻¹ and 1650 cm⁻¹, respectively. While the absorption peak of imino group stretching vibration appeared around 3300 cm⁻¹. In addition, the stretching vibration absorption (-NH₂ and -SO₂NH) of compounds II appeared around 3500 cm⁻¹ and 3150 cm⁻¹.

In LC-MS (ES⁺ mode) spectrum of IV-1 (Fig. 9), the quasi-molecular ion peak was 491 [M + H]⁺, which accorded with the nitrogen rule. Firstly, sulfonamide bond was broken into a characteristic ion peak at m/z 296, and then fragment ion peak of m/z 135 was obtained by amide bond fracture. Finally, fragment ion peaks of m/z 92 and m/z 77 were obtained via McLafferty rearrangement on the benzene ring and after losing a methylene, respectively. According to the above analysis, fragment missing was reasonable.

Figure 9. MS (ES⁺ mode) analysis of IV-1 with the fragmentation patterns.

Bioassay of Fungicidal Activities

B. cinerea strains showed multiple physiological characteristics because of the different living environment and fungicide application level. As a result, sensitivities of strains from different areas are also disparate to new compounds.

Fungicidal activity and structure-activity relationship of compounds IV-1~IV-29

In order to screen out active compounds correctly and quickly, firstly the title compounds (IV-1~IV-29) were tested against two B cinerea strains (Dd-15 and Sy-10), which inhibition rates were shown in Fig. 10. Two-factor analysis of variance between strains and compounds was conducted by SPSS20.0. Analytical results showed that there were sensitivity differences in the twenty-nine new compounds against the two strains. For example, the activity of the title compounds against Dd-15 was generally high, and average inhibition rate was about 76.0%. While the activity was relatively low against Sy-10, which average inhibition rate was about 58.4%. According to the analysis of bioactivity against two strains, there were twenty-one compounds, which fungicidal activities were higher than that of the positive control procymidone.

Figure 10. Fungicidal activity of compounds IV-1~IV-29 against two B. cinerea strains (Sy-10 and Dd-15, 50 mg/L).

The preliminary structure-activity relationship can be summarized in four points. First, for substituent benzoyl chloride (IV-1~IV-17), fungicidal activity was mediocre on the benzene ring containing two substituents, and substituted phenyl groups at ortho- and para-position with methoxyl group and fluorine atom showed excellent activity. However, fungicidal activity was higher at meta-substituted methyl group and meta-substituted chlorine atom. When trifluoromethyl group was on the benzene ring such as compounds IV-15 and IV-16, fungicidal activity was the highest. Second, for alkylacyl chloride (IV-18~IV-23), fungicidal activity of compounds showed a rising trend with the increase of carbon number in the alkyl group, for example, that of which containing n-hexanoyl chloride (IV-22) or n-heptanoyl chloride (IV-23) was the highest. Third, for halogenated acetyl chloride (IV-24~IV-27), the bioactivity increased with the increase of chlorine atom number, and activity of chloro-substituted compounds was higher than that of the bromo-substituted ones. Finally, for 2-alkoxyl acetyl chloride (IV-28 and IV-29), the activity of 2-ethoxyl acetyl chloride was higher than that of the 2-methoxyl acetyl chloride. As a result, eleven highly active compounds were chosen as candidates in the second round screening.

As shown in Fig. 11, eleven compounds were screened out to determine fungicidal activity against other six different B. cinerea strains. Two-factor analysis of variance results showed that there remained significant differences in sensitivities of the six B. cinerea strains to the title compounds. For example, the average inhibition rates of eleven compounds against As-12, Cy-07, Dd-04, Dl-17, Fs-06 and Hld-16 were 51.97%, 29.17%, 62.82%, 55.84%, 35.70% and 47.74% respectively. The activities of eleven compounds against the six B. cinerea strains could be divided into eight subsets (a–h), in which those of compounds IV-23, IV-24, IV-26 and IV-29 were higher than the positive control procymidone. These four compounds were selected to do the further study and their EC₅₀ values were evaluated and shown in Table 1.

Figure 11. Fungicidal activity of title compounds IV against six B. cinerea strains (50 mg/L).

Table 1. EC₅₀ values of title compounds IV against six B. cinerea strains.

Compound Number	R2	EC50 (mg/L) (95% confidence limits of EC50)
		As-12	Cy-07	Dd-04	Dl-17	Fs-06	Hld-16
IV-23 ab	CH3(CH2)5―	13.87 (9.25–20.81)	18.13 (9.92–33.14)	9.14 (6.34–13.18)	15.19 (11.63–19.85)	13.63 (10.12–18.36)	19.40 (13.67–27.53)
IV-24 ab	ClCH2―	16.14 (11.94–21.81)	12.34 (8.44–18.05)	8.89 (5.94–13.30)	13.69 (10.06–18.62)	25.81 (14.13–47.16)	25.44 (19.80–32.68)
IV-26 a	Cl3C―	4.79 (3.37–6.81)	1.60 (0.65–3.91)	1.97 (1.18–3.28)	0.37 (0.07–2.08)	7.56 (4.57–12.51)	5.88 (3.36–10.30)
IV-29 ab	C2H5OCH2―	13.27 (10.57–16.67)	10.37 (6.78–15.88)	5.29 (3.51–7.99)	11.81 (8.50–16.39)	8.94 (7.20–11.09)	31.90 (19.81–51.36)
procymidone bc	—	16.93 (10.28–27.88)	21.36 (14.18–32.18)	8.17 (5.80–11.49)	2.49 (1.33–4.66)	75.84 (36.17–159.00)	12.91 (9.15–18.20)

The letters a–c denoted the difference significance analysis results of the same compound against six different strains. Means followed by the same letter within the same column are not significantly different (p > 0.05, Fisher1s LSD multiple comparison test).

Fungicidal activities of all the four title compounds were higher than that of the positive fungicide procymidone. Overall, the fungicidal activities of compound IV-26 against six strains (As-12, Cy-07, Dd-04, Dl-17, Fs-06, and Hld-16), IV-29 against four strains (As-12, Cy-07, Dd-04, and Fs-06), IV-23 and IV-24 against three strains (As-12, Cy-07, and Fs-06) were higher than those of procymidone. Activities of the four title compounds against the six B. cinerea strains were different, for example, EC₅₀ values of compound IV-26 against the six strains were 0.37~7.56 mg/L, while those of procymidone were 2.49~75.84 mg/L. Referring to resistant grading standards to procymidone³³,³⁴, Dd-04 and Dl-17 were low-resistant strains; As-12, Cy-07 and Hld-16 were moderate-resistant strains; Fs-06 was high-resistant strain. The results displayed that the in vitro activities of compound IV-26 against all the resistant strains were excellent.

After the above test, structure-activity relationship between acyl chloride and fungicidal activity was confirmed. It was to be sure that trichloroacetyl chloride had the greatest contribution to the fungicidal activity for the twenty-nine acyl chlorides. Therefore, trichloroacetyl chloride was marked as the active group in the later structural modification of cycloalkyl group (IV-30~IV-36). Moreover, compared to the fungicidal activity screened for one strain, it was more reliable for different strains from different areas to choose as the test targets.

Fungicidal activity and structure-activity relationship of compounds IV-26 and IV-30~IV-36

As shown in Table 2, after the structural modification of cycloalkyl group, compounds IV-30~IV-36 also had very high fungicidal activity against five other B. cinerea strains. Referring to resistant grading standards to procymidone³³,³⁴, As-11 was sensitive strain; Dl-11 was low-resistant strain; Cy-09 was moderate-resistant strain; Fs-11 and Hld-15 were high-resistant strains. The statistical results of SPSS showed that compounds IV-26, IV-30, IV-31, IV-32, IV-33 and IV-34 exhibited excellent activity. It was found that the size of cycloalkyl group was important factor to determine the fungicidal activity compared with that of IV-26. For example, the EC₅₀ values of compounds IV-26, IV-30 and IV-31 were 0.15~3.64 mg/L, 0.66~11.68 mg/L and 0.82~9.49 mg/L, respectively. The activities of compound IV-26 containing 6-membered ring were better than those of compounds IV-30 and IV-31 respectively containing 5- and 7-membered ring. In addition, it was found that the types of substituent alkyl group on the cyclohexane had a significant effect on the fungicidal activity by comparing activities of compounds IV-32~IV-36. The activity decreased with the increase of alkyl carbon number. Moreover, the position of alkyl group also had effect on the activity. For example, compared with compounds IV-32, IV-33 and IV-34, their fungicidal activity was para-methyl > ortho-methyl > meta-methyl in the order.

Table 2. EC₅₀ values of title compounds IV-26 and IV-30~IV-36 against five B. cinerea strains.

Compound number	n	R1	EC50 (mg/L) (95% confidence limits of EC50)
			As-11	Cy-09	Dl-11	Fs-11	Hld-15
IV-26 a	2	H	0.41 (0.08–2.01)	1.13 (0.46–2.75)	0.15 (0.02–1.26)	3.64 (1.72–7.72)	1.87 (1.06–3.31)
IV-30 a	1	H	0.66 (0.17–2.48)	2.28 (1.54–3.38)	0.77 (0.32–1.87)	11.68 (9.08–15.03)	0.85 (0.39–1.86)
IV-31 a	3	H	4.59 (2.87–7.33)	1.36 (0.58–3.15)	0.96 (0.47–1.99)	9.49 (2.22–40.56)	0.82 (0.20–3.26)
IV-32 a	2	3-CH3	14.76 (2.92–74.55)	10.71 (0.71–160.53)	0.01 (0.00–25.75)	7.93 (2.60–24.18)	0.96 (0.13–7.13)
IV-33 a	2	4-CH3	0.18 (0.01–2.95)	2.23 (0.37–13.43)	0.15 (0.02–1.32)	15.56 (5.34–45.32)	0.15 (0.01–2.67)
IV-34 a	2	5-CH3	0.56 (0.06–4.85)	6.19 (2.94–13.04)	2.22 (1.37–3.59)	16.75 (7.52–37.27)	1.19 (0.47–2.98)
IV-35 ab	2	5-C2H5	51.4 (2.52–1049.18)	19.87 (1.42–277.31)	0.22 (0.01–4.01)	16.42 (5.30–50.84)	31.99 (3.84–266.66)
IV-36 c	2	5-C(CH3)3	>100	44.12 (18.73–103.92)	9.49 (6.30–14.28)	>100	30.76 (18.10–52.25)
procymidone bc	—	—	0.22 (0.07–0.65)	20.00 (14.52–27.55)	4.40 (3.43–5.65)	>100	>100

The letters a–d denoted the difference significance analysis results of the same compound against five different strains. Means followed by the same letter within the same column are not significantly different (p > 0.05, Fisher1s LSD multiple comparison test).

In vivo fungicidal activity against B. cinerea on leaves of cucumber (mycelium inoculation method)

Six compounds (IV-25, IV-26, IV-30, IV-31, IV-32, and IV-33) were tested for their in vivo fungicidal activity on leaves of cucumber, and the leading compound chesulfamide (L, Fig. 3) was used as the positive control. The bioassay results in Table 3 showed that the control efficiency of compound IV-31 was significantly higher than that of the positive control chesulfamide. Fungicidal activity of compounds IV-26, IV-30, IV-32 and IV-33 was equivalent to the chesulfamide.

Table 3. Control efficiency of compounds against B. cinerea on leaves of cucumber.

Compd.	Inhibition rate (%) ± SEM
IV-25	24.54 ± 11.43 b
IV-26	37.58 ± 32.58 ab
IV-30	35.78 ± 16.95 ab
IV-31	64.30 ± 15.57 a
IV-32	39.37 ± 22.08 ab
IV-33	36.68 ± 34.42 ab
chesulfamide	32.11 ± 23.32 ab

The letters a–b denoted the results of difference significance analysis. Means followed by the same letter within the same column are not significantly different (p > 0.05, Fisher1s LSD multiple comparison test).

Compared with the previous work, the structure of the title compounds was modified and new. Meanwhile, their fungicidal activity had greater improvement than that of the lead compound. From the point of view of chemical synthesis, novel key intermediates 2-aminocycloalkylsulfonamides (II-1~II-8) were obtained, which had the vital significance for obtaining the title molecules with structural diversity. In addition, the effective improvements of synthesis method for compounds II were made, which greatly increased the yield and the reaction progress. On the other hand, structural characterization of title compounds IV was described in detail. In particular, the NMR spectra are very characteristic. The single crystal structure was obtained, which provided the basis for accurately structural analysis. According to the single crystal structure, the computer-aided design could be simulated, which is helpful to further molecular design and structural optimization. Preliminary mechnism study indicated that cyclohexyl alkyl sulfonamides might inhibit the growth of gray mould by affecting the synthesis of the internal substance³⁵. The elucidation of the mode of action of these new compound is worth research, which will be studied in detail in the future.

Conclusion

In conclusion, we reported the synthesis of a new series of 2-acylaminocycloalkylsulfonamides and their in vitro and in vivo fungicidal activities against various B. cinerea strains were evaluated. Some title compounds showed notable activity, especially compound IV-31 was of great potential to be developed as new antifungal agents for plant protection. Moreover, single crystal structure of compound IV-31 was determined to assist the further molecular design and structural modification. In addition, the SAR results indicated that structure of acylchloride and naphthenic scaffold had significant effects on the activity. Thus, the present results were of great promise for the design and development of novel sulfonamides antifungal agents. Further research was necessary on the more extensive structural modification and the broad determination of the fungicidal spectra.

Materials and Methods

General

Nuclear magnetic resonance (NMR) spectra were recorded in CDCl₃ and DMSO-d₆ unless indicated otherwise with a Bruker Avance III 600 MHz spectrometer (Bruker, Fallanden, Switzerland), using tetramethylsilane (TMS) as an internal standard. Infrared (IR) spectra were recorded on a Shimadzu IR Affinity-1 spectrophotometer (Shimadzu, Kyoto, Japan) with KBr disks. UPLC-MS/MS (Agilent, Palo Alto, CA. USA): ACQUITY UPLC BEH C₁₈ chromatographic column (2.1 mm × 100 mm, 1.7 μm); column temperature: 40 °C; mobile phase: solvent A for acetonitrile, solvent B for 0.1% formic acid-water solution; gradient elution program: 10% A at the initial time of 0 min, and then 90% A~10% B in the range of 0 to 2.0 min, 50% A in the range of 2.0 to 4.0 min, 10% A~90% B in the range of 4.0 to 4.2 min, 10% A in the range of 4.2 to 5.2 min; velocity of flow: 0.2 mL/min; sampling volume: 3 μL. Ion source: ESI; acquisition methods: using multiple reaction monitoring and electrospray ionization in positive mode. Melting points were determined on an X-5 melting-point apparatus (Beijing Tech Instrument Co., Ltd., Beijing, China), and the thermometer was uncorrected. Optical rotation was measured on an automatic polarimeter (ATOGO AP-300; condition: λ = 589 nm, L = 100 mm, Temp. = 22.0 °C). The solvents and reagents were used as received or were dried prior to use, as needed. High resolution mass spectra for new compounds were recorded on a G2-XS QTof Mass Spectrometry Facility (Waters, Milford, MA, USA). Elemental analysis was carried out with a Flash EA 1112 elemantal analyzer (Thermo Finnigan, Bremen, Germany).

Botrytis cinerea strains

Thirteen different B. cinerea strains, Sy-10, Dd-04, Dd-15, Hld-15, Hld-16, Fs-06, Fs-11 Dl-11, Dl-17, Cy-07, Cy-09, As-11 and As-12, were isolated from damaged parts of tomato in a greenhouse in Shenyang, Dandong, Huludao, Fushun, Dalian, Chaoyang and Anshan respectively, Liaoning Province, China, in April 2014, and cultured on potato dextrose agar (PDA) at 28 °C and maintained at 4 °C with periodic subculturing.

Synthesis

The synthetic routes of the key intermediates II and title compounds IV were outlined in Fig. 4.

Synthesis of N-(2-trifluoromethyl-4-chlorophenyl)-2-oxocyclohexylsulfonamides I-1~I-8

Compounds I were synthesized according to the method given in the ref. 26. The synthetic route of compounds I-1 to I-8 was outlined in Fig. 4. I-1 (n = 1, R¹ = H), I-2 (n = 0, R¹ = H), I-3 (n = 2, R¹ = H) were already known³⁰ and I-4~I-8 are new compounds. Their physical data and spectra data were shown as follows:

N-(2-trifluoromethyl-4-chlorophenyl)-3-methyl-2-oxocyclohexylsulfonamide (I-4)

(n = 1, R¹ = 3-Me) Colorless crystal; yield, 71%; mp 108–109 °C; ¹H NMR (CDCl₃) δ: 1.11 (d, J = 6.4 Hz, 3H, CH₃), 1.47–2.64 (m, 7H, C₄H₇), 3.97 (dd, J = 13.4, 5.3 Hz, 1H, CH-SO₂), 7.37 (s, 1H, SO₂-NH), 7.51–7.71 (m, 3H, Ph-H); ¹³C NMR (DMSO-d₆) δ: 14.41, 23.59, 30.20, 35.94, 45.28, 70.74, 118.99, 121.97, 123.79, 126.94, 131.63, 132.20, 133.43, 204.54; IR (ν, cm⁻¹): 3344, 1708; MS (z/e): 369(M⁺), 195, 175, 111, 83, 55; Anal. Calcd for C₁₄H₁₅ClF₃NO₃S: C, 45.47; H, 4.09; N, 3.79; found: C, 45.31; H, 3.94; N, 3.92.

N-(2-trifluoromethyl-4-chlorophenyl)-4-methyl-2-oxocyclohexylsulfonamide (I-5)

(n = 1, R¹ = 4-Me) Colorless crystal; yield, 91%; mp 97–99 °C; ¹H NMR (CDCl₃) δ: 1.34–2.62 (m, 10H, C₅H₁₀), 3.90 (dd, J = 13.0, 5.7 Hz, 1H, CH-SO₂), 7.35 (s, 1H, SO₂-NH), 7.51–7.71 (m, 3H, Ph-H); ¹³C NMR (DMSO-d₆) δ: 21.84, 26.24, 28.86, 34.55, 48.07, 69.24, 118.64, 118.99, 125.61, 126.98, 132.23, 133.28, 145.66, 202.41; IR (ν, cm⁻¹): 3365, 1708; MS (z/e): 369(M⁺), 148, 131, 126, 120, 91; Anal. Calcd for C₁₄H₁₅ClF₃NO₃S: C, 45.47; H, 4.09; N, 3.79; found: C, 45.63; H, 3.98; N, 3.57.

N-(2-trifluoromethyl-4-chlorophenyl)-5-methyl-2-oxocyclohexylsulfonamide(I-6)

(n = 1, R¹ = 5-Me) Colorless crystal; yield, 94%; mp 104–105 °C; ¹H NMR (CDCl₃) δ: 1.07–2.63 (m, 10H, C₅H₁₀), 3.99 (dd, J = 13.3, 5.4 Hz, 1H, CH-SO₂), 7.37 (s, 1H, SO₂-NH), 7.51–7.69 (m, 3H, Ph-H); ¹³C NMR (DMSO-d₆) δ: 21.16, 26.44, 30.32, 34.52, 36.63, 69.63, 119.00, 121.97, 123.78, 126.91, 131.69, 132.19, 133.44, 203.09; IR (ν, cm⁻¹): 3367, 1710; MS (z/e): 369(M⁺), 352, 306, 195, 175, 55; Anal. Calcd for C₁₄H₁₅ClF₃NO₃S: C, 45.47; H, 4.09; N, 3.79; found: C, 45.66; H, 4.31; N, 3.59.

N-(2-trifluoromethyl-4-chlorophenyl)-5-ethyl-2-oxocyclohexylsulfonamide (I-7)

(n = 1, R¹ = 5-Et) Colorless crystal; yield, 99%; mp 90~93 °C; ¹H NMR (CDCl₃) δ: 0.96–2.66 (m, 12H, C₆H₁₂), 3.98 (dd, J = 12.5, 5.4 Hz, 1H, CH-SO₂), 7.38 (s, 1H, SO₂-NH), 7.52–7.70 (m, 3H, Ph-H); ¹³C NMR (DMSO-d₆) δ: 11.80, 28.21, 32.23, 34.41, 36.63, 41.27, 69.70, 121.97, 123.79, 126.91, 131.70, 132.21, 133.46, 133.82, 203.20; IR (ν, cm⁻¹): 3375, 1714; MS (z/e): 383(M⁺), 366, 320, 195, 175, 55; MS (z/e): 383(M⁺), 366, 320, 195, 175, 55; Anal. Calcd for C₁₅H₁₇ClF₃NO₃S: C, 46.94; H, 4.46; N, 3.65; found: C, 47.11; H, 4.37; N, 3.78.

N-(2-trifluoromethyl-4-chlorophenyl)-5-tertiarybutyl-2-oxocyclohexylsulfonamide (I-8)

(n = 1, R¹ = 5-t-Bu) Colorless crystal; yield, 93%; mp 86–89 °C; ¹H NMR (CDCl₃) δ: 0.93–2.70 (m, 16H, C₈H₁₆), 3.95 (dd, J = 13.3, 5.3 Hz, 1H, CH-SO₂), 7.38 (s, 1H, SO₂-NH), 7.52–7.70 (m, 3H, Ph-H); ¹³C NMR (DMSO-d₆) δ: 8.97, 27.63, 30.23, 32.64, 41.31, 44.82, 69.96, 119.01, 123.78, 126.91, 131.76, 132.28, 133.49, 133.99, 203.08; IR (ν, cm⁻¹): 3329, 1714; MS (z/e): 280, 194, 175, 154, 69, 57; Anal. Calcd for C₁₇H₂₁ClF₃NO₃S; C, 49.57; H, 5.14; N, 3.40; found; C, 49.68; H, 4.95; N, 3.61.

Synthesis of the key intermediates N-(2-trifluoromethyl-4-chlorophenyl)-2-aminocycloalkylsulfonamides II-1~II-8

The synthetic route of compounds II-1 to II-8 was outlined in Fig. 4, according to the method given in the ref. 36, under a nitrogen atmosphere, compounds I (30 mmol) and titanium (IV) isopropoxide (17 mL, 60 mmol) in dry ethyl alcohol (150 mL) were stirred, while the ammonia gas passed through the reaction mixture and maintained the pressure of ammonia upto 20 mmHg at room temperature for 6 h, which was monitored by TLC analysis. Then sodium borohydride (1.7 g, 45 mmol) was added slowly to the resulting mixture at room temperature and stirred for 3 h. The reaction was quenched by addition of ammonium hydroxide solution (2 M, 120 mL). The resulting inorganic precipitate was filtered off, and washed with ethyl acetate (150 mL). The filtrate was concentrated under reduced pressure to remove ethyl acetate, and then extracted with ethyl acetate (200 mL). The combined organic extracts were washed with brine (300 mL), dried over anhydrous Na₂SO₄, evaporated under reduced pressure, and recrystallized from methanol to afford pure key intermediates II. Their physical and spectra data were shown as follows.

N-(2-trifluoromethyl-4-chlorophenyl)-2-aminocyclohexylsulfonamide (II-1)

(n = 1, R¹ = H) Colorless crystal, yield, 73%; mp 252–254 °C; ¹H NMR (DMSO-d₆) δ: 1.32–2.00 (m, 8H, 4CH₂), 2.89 (dt, J = 12.4, 3.1 Hz, 1H, CH-N), 3.79 (d, J = 2.1 Hz, 1H, CH-SO₂), 7.27–7.42 (m, 3H, Ph-H), 8.21 (s, 3H, NH₂ + NH); ¹³C NMR (DMSO-d₆) δ: 24.07, 24.17, 25.09, 30.36, 50.21, 60.99, 118.65, 122.25, 123.87, 125.68, 125.84, 132.23, 147.54; IR (ν, cm⁻¹): 3516, 3078; MS (z/e): 357[M + H]⁺, 175, 162, 98, 81; Anal. Calcd for C₁₇H₂₁ClF₃NO₃S: C, 43.76; H, 4.52; N, 7.85. found: C, 43.88; H, 4.69; N, 7.61.

N-(2-trifluoromethyl-4-chlorophenyl)-2-aminocyclopentylsulfonamide (II-2)

(n = 0, R¹ = H) White powder; yield, 95%; mp 183–186 °C; ¹H NMR (DMSO-d₆) δ: 1.52–2.05 (m, 6H, 3CH₂), 3.40–3.44(m, 1H, CH-N), 3.64 (dd, J = 11.6, 6.6 Hz, 1H, CH-SO₂), 7.27–7.47 (m, 3H, Ph-H), 8.14 (s, 3H, NH₂ + NH); ¹³C NMR (DMSO-d₆) δ: 21.72, 26.00, 30.71, 51.90, 61.45, 118.71, 121.93, 123.88, 125.68, 125.86, 132.24, 147.70; IR (ν, cm⁻¹): 3614, 3198; MS (z/e): 342(M⁺), 196, 176, 148, 84, 67; Anal. Calcd for C₁₂H₁₄ClF₃N₂O₂S: C, 42.05; H, 4.12; N, 8.17; found: C, 41.92; H, 3.98; N, 8.35.

N-(2-trifluoromethyl-4-chlorophenyl)-2-aminocycloheptyl sulfonamide (II-3)

(n = 2, R¹ = H) White powder; yield, 91%; mp 230–232 °C; ¹H NMR (DMSO-d₆) δ: 1.41–2.28 (m, 10H, 5CH₂), 2.92 (dd, J = 10.0, 2.2 Hz, 1H, CH-N), 3.99 (td, J = 5.5, 2.4 Hz, 1H, CH-SO₂), 7.27–7.39 (m, 3H, Ph-H), 8.27 (s, 3H, NH₂ + NH); ¹³C NMR (DMSO-d₆) δ: 21.81, 22.09, 25.95, 27.41, 32.06, 49.81, 61.71, 118.64, 122.28, 123.92, 125.73, 125.80, 132.23, 147.29; IR (ν, cm⁻¹): 3523, 3095; MS (z/e): 370, 194, 174; Anal. Calcd for C₁₄H₁₈ClF₃N₂O₂S; C, 45.35; H, 4.89; N, 7.55; found; C, 45.21; H, 5.02; N, 7.63.

N-(2-trifluoromethyl-4-chlorophenyl)-3-methyl-2-aminocyclohexylsulfonamide (II-4)

(n = 1, R¹ = 3-Me) White powder; yield, 79%; mp 213–216 °C; ¹H NMR (DMSO-d₆) δ: 0.85–2.11 (m, 10H, C₅H₁₀), 2.83 (td, J = 11.5, 3.5 Hz, 1H, CH-N), 3.18 (td, J = 11.4, 4.4 Hz, 1H, CH-SO₂), 7.27–7.42 (m, 3H, Ph-H), 8.34 (s, 3H, NH₂ + NH); ¹³C NMR (DMSO-d₆) δ: 17.05, 19.17, 21.96, 24.89, 31.34, 52.13, 56.10, 118.64, 122.26, 123.87, 125.68, 125.79, 132.20, 147.29; IR (ν, cm⁻¹): 3599, 3140; MS (z/e): 370(M⁺), 176, 112, 95, 67; Anal. Calcd for C₁₄H₁₈ClF₃N₂O₂S: C, 45.35; H, 4.89; N, 7.55; found: C, 45.18; H, 4.62; N, 7.69.

N-(2-trifluoromethyl-4-chlorophenyl)-4-methyl-2-aminocyclohexylsulfonamide (II-5)

(n = 1, R¹ = 4-Me) White powder; yield, 86%; mp 230–233 °C; ¹H NMR (DMSO-d₆) δ: 0.74–2.08 (m, 10H, C₅H₁₀), 3.11 (s, 1H, CH-N), 3.29 (s, 1H, CH-SO₂), 7.22–7.51 (m, 3H, Ph-H), 8.25 (s, 3H, NH₂ + NH); ¹³C NMR (DMSO-d₆) δ: 22.27, 25.40, 28.00, 31.26, 33.51, 50.43, 55.57, 118.60, 122.07, 123.86, 125.66, 125.83, 132.15, 147.74; IR (ν, cm⁻¹): 3523, 3072; MS (z/e): 370(M⁺), 176, 112, 95, 67, 55; Anal. Calcd for C₁₄H₁₈ClF₃N₂O₂S: C, 45.35; H, 4.89; N, 7.55; found: C, 45.56; H, 4.69; N, 7.41.

N-(2-trifluoromethyl-4-chlorophenyl)-5-methyl-2-aminocyclohexylsulfonamide (II-6)

(n = 1, R¹ = 5-Me) White powder; yield, 85%; mp 250–252 °C; ¹H NMR (DMSO-d₆) δ: 0.90–2.12 (m, 10H, C₅H₁₀), 2.83 (td, J = 11.5, 3.5 Hz, 1H, CH-N), 3.18 (td, J = 11.4, 4.4 Hz, 1H, CH-SO₂), 7.28–7.42 (m, 3H, Ph-H),8.35 (s, 3H, NH₂ + NH); ¹³C NMR (DMSO-d₆) δ: 22.14, 25.68, 30.73, 32.35, 50.00, 55.70, 60.67, 118.52, 118.67, 122.14, 122.28, 125.85, 132.23, 147.44; IR (ν, cm⁻¹): 3523, 3170; MS (z/e): 370(M⁺), 278, 250, 197; Anal. Calcd for C₁₄H₁₈ClF₃N₂O₂S: C, 45.35; H, 4.89; N, 7.55; found: C, 45.57; H, 4.98; N, 7.38.

N-(2-trifluoromethyl-4-chlorophenyl)-5-ethyl-2-aminocyclohexylsulfonamide (II-7)

(n = 1, R¹ = 5-Et) White powder; yield, 54%; mp 227–230 °C; ¹H NMR (DMSO-d₆) δ: 0.81–2.04 (m, 12H, C₆H₁₂), 3.05–3.06 (m, 1H, CH-N), 3.55–3.56 (m, 1H, CH-SO₂), 7.26–7.46 (m, 3H, Ph-H), 8.19 (s, 3H, NH₂ + NH); ¹³C NMR (DMSO-d₆) δ: 12.02, 24.62, 27.03, 29.29, 32.53, 37.50, 46.60, 55.72, 118.55, 122.07, 122.27, 125.84, 132.15, 132.23, 147.67; IR (ν, cm⁻¹): 3523, 3277; MS (z/e): 384(M⁺), 194, 174, 95, 67, 56; Anal. Calcd for C₁₅H₂₀ClF₃N₂O₂S: C, 46.81; H, 5.24; N, 7.28; found: C, 47.02; H, 5.06; N, 7.51.

N-(2-trifluoromethyl-4-chlorophenyl)-5-tertiarybutyl-2-aminocyclohexylsulfonamide (II-8)

(n = 1, R¹ = 5-t-Bu) White powder; yield, 42%; mp 230–233 °C; ¹H NMR (DMSO-d₆) δ: 0.77–2.45 (m, 16H, C₈H₁₆), 2.87 (d, J = 12.2 Hz, 1H, CH-N), 3.76 (s, 1H, CH-SO₂), 7.27–7.41 (m, 3H, Ph-H), 8.20 (s, 3H, NH₂ + NH); ¹³C NMR (DMSO-d₆) δ: 19.56, 22.51, 27.66, 28.86, 32.74, 46.15, 46.23, 60.46, 118.64, 122.32, 123.90, 125.70, 125.82, 132.18, 147.31; IR (ν, cm⁻¹): 3502, 3109; MS (z/e): 412(M⁺), 397, 355, 194, 154; Anal. Calcd for C₁₇H₂₄ClF₃N₂O₂S: C, 49.45; H, 5.86; N, 6.78; found: C, 49.25; H, 6.04; N, 6.66.

Synthesis of acyl chlorides III

Substituent benzoyl chlorides (III-1~III-17), acetyl chlorides (III-18~III-23), halogenated acetyl chlorides (III-24~III-27), alkoxylacetyl chlorides (III-28~III-29) were synthesized according to the given method in the ref. 32.

Synthesis of title compounds 2-acylaminocycloalkylsulfonamides IV-1~IV-29 and IV-30~IV-36

Under nitrogen, acyl chlorides III (3 mmol) were dropwise added to the solution of II (3 mmol) and triethylamine (Et₃N, 3.9 mmol) in dry dichloromethane (40 mL). (Fig. 4) The solution was stirred at room temperature for 2 h. The mixture was filtered and washed with 3 M HCl (30 mL), saturated NaHCO₃ (30 mL), and brine (40 mL). After dried by anhydrous Na₂SO₄ and concentrated in vacuo, the crude product was recrystallized with the acetone/petroleum ether to afford pure IV. Their physical data and spectra data were shown as follows.

N-(2-trifluoromethyl-4-chlorophenyl)-2-(2-methoxybenzoylamino) cyclohexylsulfonamide (IV-1)

(R² = 2-CH₃OC₆H₄) White solid; yield, 95%; mp 129–130 °C; ¹H NMR (DMSO-d₆) δ: 1.48–2.14 (m, 8H, 4CH₂), 3.55 (dt, J = 11.9, 3.3 Hz, 1H, CH-N), 3.94 (s, 3H, OCH₃), 4.64 (dd, J = 6.9, 3.4 Hz, 1H, CH-SO₂), 7.05–7.89 (m, 7H, Ph-H), 8.60 (d, J = 7.3 Hz, 1H, CO-NH), 9.57 (s, 1H, SO₂-NH); ¹³C NMR (DMSO-d₆) δ: 20.07, 22.53, 24.01, 29.88, 45.54, 56.60, 62.79, 112.70, 121.11, 121.96, 122.02, 126.85, 127.07, 131.24, 131.29, 131.49, 133.21, 133.64, 133.84, 157.79, 164.40; IR (ν, cm⁻¹): 3370, 3121, 1649; MS (z/e): 491[M + H]⁺, 296, 135, 92, 77; Anal. Calcd for C₂₁H₂₂ClF₃N₂O₄S: C,51.38; H, 4.52; N, 5.71; found: C, 51.62; H, 4.69; N, 5.52.

N-(2-trifluoromethyl-4-chlorophenyl)-2-(4-methoxybenzoylamino) cyclohexylsulfonamide (IV-2)

(R² = 4-CH₃OC₆H₄) White solid; yield, 86%; mp 139–141 °C; ¹H NMR (DMSO-d₆) δ: 1.37–2.17 (m, 8H, 4CH₂), 3.51 (dt, J = 11.8, 3.4 Hz, 1H, CH-N), 3.81 (s, 3H, OCH₃), 4.72 (dd, J = 7.9, 3.6 Hz, 1H, CH-SO₂), 6.97–7.79 (m, 7H, Ph-H), 7.91 (d, J = 8.5 Hz, 1H, CO-NH), 9.43 (s, 1H, SO₂-NH); ¹³C NMR (DMSO-d₆) δ: 19.71, 21.72, 24.15, 30.65, 45.26, 55.74, 63.08, 113.62, 113.62, 122.05, 123.87, 126.55, 127.07, 127.41, 129.98, 131.01, 131.26, 133.62, 134.04, 161.95, 166.86; IR (ν, cm⁻¹): 3390, 3080, 1631; HRMS-ESI, m/z calcd for C₂₁H₂₃ClF₃N₂O₄S, [M + H]⁺491.1019; found, 491.1022.

N-(2-trifluoromethyl-4-chlorophenyl)-2-(2-methylbenzoylamino) cyclohexylsulfonamide (IV-3)

(R² = 2-CH₃C₆H₄) White crystal; yield, 96%; mp 192–194 °C; ¹H NMR (DMSO-d₆) δ: 1.36–1.96 (m, 8H, 4CH₂), 2.33 (s, 3H, CH₃), 3.50 (dt, J = 11.9, 3.6 Hz, 1H, CH-N), 4.81 (dd, J = 8.9, 3.1 Hz, 1H, CH-SO₂), 7.20–7.81 (m, 7H, Ph-H), 8.38 (d, J = 9.2 Hz, 1H, CO-NH), 9.36 (s, 1H, SO₂-NH); ¹³C NMR (DMSO-d₆) δ: 19.40, 19.55, 21.36, 24.39, 31.18, 44.43, 63.36, 122.08, 123.89, 125.57, 126.97, 128.00, 129.56, 130.46, 130.68, 131.03, 133.62, 134.15, 135.86, 137.37, 169.89; IR (ν, cm⁻¹): 3388, 3074, 1645; MS (z/e): 475[M + H]⁺, 280, 216, 119, 91; Anal. Calcd for C₂₁H₂₂ClF₃N₂O₃S: C,53.11; H, 4.67; N, 5.90; found: C, 53.35; H, 4.50; N, 6.09.

N-(2-trifluoromethyl-4-chlorophenyl)-2-(3-methylbenzoylamino) cyclohexylsulfonamide (IV-4)

(R² = 3-CH₃C₆H₄) White crystal; yield, 43%; mp 176–177 °C; ¹H NMR (DMSO-d₆) δ: 1.32–2.12 (m, 8H, 4CH₂), 2.36 (s, 3H, CH₃), 3.54–3.49 (m, 1H, CH-N), 4.74 (d, J = 4.3 Hz, 1H, CH-SO₂), 7.34–7.80 (m, 7H, Ph-H), 8.04 (d, J = 8.6 Hz, 1H, CO-NH), 9.42 (s, 1H, SO₂-NH); ¹³C NMR (DMSO-d₆) δ: 19.71, 21.32, 21.71, 24.15, 30.61, 45.27, 63.04, 122.05, 123.87, 125.31, 127.02, 128.30, 128.60, 130.95, 131.23, 132.01, 133.62, 134.03, 135.29, 137.60, 167.60; IR (ν, cm⁻¹): 3385, 3046, 1629; MS (z/e): 475[M + H]⁺, 280, 216, 119, 91; Anal. Calcd for C₂₁H₂₂ClF₃N₂O₃S: C, 53.11; H, 4.67; N, 5.90; found: C, 52.94; H, 4.57; N, 5.71.

N-(2-trifluoromethyl-4-chlorophenyl)-2-(4-methylbenzoylamino) cyclohexylsulfonamide (IV-5)

(R² = 4-CH₃C₆H₄) White solid; yield, 98%; mp 217–219 °C; ¹H NMR (DMSO-d₆) δ: 1.32–2.37 (m, 8H, 4CH₂), 2.33 (s, 3H, CH₃), 3.41 (td, J = 11.1, 2.9 Hz, 1H, CH-N), 4.23 (ddd, J = 19.2, 10.6, 4.0 Hz, 1H, CH-SO₂), 7.23–7.80 (m, 7H, 7H, Ph-H), 8.36 (d, J = 8.5 Hz, 1H, CO-NH), 9.36 (s, 1H, SO₂-NH); ¹³C NMR (DMSO-d₆) δ: 21.32, 24.36, 24.45, 27.08, 32.95, 48.53, 65.11, 122.03, 123.84, 126.78, 126.97, 127.69, 128.91, 128.98, 131.28, 131.33, 132.37, 133.58, 134.25, 141.24, 165.99; IR (ν, cm⁻¹): 3346, 3045, 1630; MS (z/e): 475[M + H]⁺, 280, 216, 119, 91; Anal. Calcd for C₂₁H₂₂ClF₃N₂O₃S: C, 53.11; H, 4.67; N, 5.90; found: C, 53.31; H, 4.88; N, 6.12.

N-(2-trifluoromethyl-4-chlorophenyl)-2-(2,4-dimethylbenzoylamino) cyclohexylsulfonamide (IV-6)

(R² = 2,4-(CH₃)₂C₆H₃) White solid; yield, 95%; mp 179–181 °C; ¹H NMR (DMSO-d₆) δ: 1.35–2.01 (m, 8H, 4CH₂), 2.30 (d, J = 5.4 Hz, 6H, CH₃ + CH₃), 3.50 (dt, J = 11.6, 3.6 Hz, 1H, CH-N), 4.78 (dd, J = 8.8, 3.1 Hz, 1H, CH-SO₂), 7.03–7.81 (m, 6H, Ph-H), 8.27 (d, J = 9.1 Hz, 1H, CO-NH), 9.37 (s, 1H, SO₂-NH); ¹³C NMR (DMSO-d₆) δ: 19.41, 19.59, 21.16, 21.36, 24.37, 31.13, 44.46, 63.39, 122.08, 123.89, 126.02, 126.96, 128.20, 130.59, 130.97, 131.14, 133.62, 134.18, 134.40, 136.01, 139.11, 170.00; IR (ν, cm⁻¹): 3392, 3053, 1645; MS (z/e): 489[M + H]⁺, 294, 133, 105, 79; Anal. Calcd for C₂₂H₂₄ClF₃N₂O₃S: C, 54.04; H, 4.95; N, 5.73; found: C, 53.89; H, 5.08; N, 5.54.

N-(2-trifluoromethyl-4-chlorophenyl)-2-(3,5-dimethylbenzoylamino) cyclohexylsulfonamide (IV-7)

(R² = 3,5-(CH₃)₂C₆H₃) White solid; yield, 93%; mp 193–195 °C; ¹H NMR (DMSO-d₆) δ: 1.33–2.31 (m, 8H, 4CH₂), 2.28 (s, 6H, CH₃ + CH₃), 3.41 (td, J = 11.0, 2.8 Hz, 1H, CH-N), 4.23 (ddd, J = 19.2, 10.5, 4.0 Hz, 1H, CH-SO₂), 7.12–7.79 (m, 6H, 6H, Ph-H), 8.35 (d, J = 8.5 Hz, 1H, CO-NH), 9.33 (s, 1H, SO₂-NH); ¹³C NMR (DMSO-d₆) δ: 21.21, 24.34, 24.45, 27.04, 32.87, 48.47, 63.01, 65.13, 122.04, 123.85, 125.37, 125.82, 126.99, 131.07, 131.16, 132.65, 133.58, 134.28, 135.17, 137.46, 137.52, 166.38; IR (ν, cm⁻¹): 3324, 2977, 1626; MS (z/e): 489[M + H]⁺, 294, 230, 133, 105; Anal. Calcd for C₂₂H₂₄ClF₃N₂O₃S: C, 54.04; H, 4.95; N, 5.73; found: C, 54.27; H, 4.77; N, 5.93.

N-(2-trifluoromethyl-4-chlorophenyl)-2-(2-fluorobenzoylamino) cyclohexylsulfonamide (IV-8)

(R² = 2-FC₆H₄) White solid; yield, 96%; mp 167–169 °C; ¹H NMR (DMSO-d₆) δ: 1.38–2.11 (m, 8H, 4CH₂), 3.54 (m, 1H, CH-N), 4.76 (s, 1H, CH-SO₂), 7.26–7.83 (m, 7H, Ph-H), 8.29 (dd, J = 7.2, 65.3 Hz, 1H, CO-NH), 9.41 (s, 1H, SO₂-NH); ¹³C NMR (DMSO-d₆) δ: 19.65, 21.76, 24.12, 30.55, 45.14, 62.95, 116.41, 122.04, 123.86, 124.63, 127.02, 130.71, 130.98, 131.28, 132.74, 133.63, 133.97, 158.90, 160.55, 164.18; IR (ν, cm⁻¹): 3308, 3077, 1635; MS (z/e): 479[M + H]⁺, 284, 220, 123, 95; Anal. Calcd for C₂₀H₁₉ClF₄N₂O₃S: C, 50.16; H, 4.00; N, 5.85; found: C, 50.38; H, 4.14; N, 5.77.

N-(2-trifluoromethyl-4-chlorophenyl)-2-(3-fluorobenzoylamino) cyclohexylsulfonamide (IV-9)

(R² = 3-FC₆H₄) White solid; yield, 94%; mp 192–194 °C; ¹H NMR (DMSO-d₆) δ: 1.31–2.20 (m, 8H, 4CH₂), 3.52 (d, J = 11.8 Hz, 1H, CH-N), 4.76 (s, 1 H, CH-SO₂), 7.38–7.80 (m, 7H, Ph-H), 8.21 (d, J = 8.5 Hz, 1H, CO-NH), 9.41 (s, 1H, SO₂-NH); ¹³C NMR (DMSO-d₆) δ: 19.65, 21.65, 24.13, 30.61, 45.33, 62.96, 115.08, 118.36, 122.04, 123.85, 124.39, 127.09, 130.54, 130.60, 131.19, 131.36, 133.62, 133.96, 137.66, 166.04; IR (ν, cm⁻¹): 3378, 3050, 1630; MS (z/e): 479[M + H]⁺, 284, 220, 123, 95; Anal. Calcd for C₂₀H₁₉ClF₄N₂O₃S: C, 50.16; H, 4.00; N, 5.85; found: C, 50.31; H, 3.83; N, 6.02.

N-(2-trifluoromethyl-4-chlorophenyl)-2-(2-chlorobenzoylamino) cyclohexylsulfonamide (IV-10)

(R² = 2-ClC₆H₄) White solid; yield, 97%; mp 199–201 °C; ¹H NMR (DMSO-d₆) δ: 1.33–2.20 (m, 8H, 4CH₂), 3.52 (m, 1H, CH-N), 4.78 (dd, J = 3.0, 8.9 Hz, 1H, CH-SO₂), 7.46–7.81 (m, 7H, Ph-H), 8.58 (d, J = 9.2 Hz, 1H, CO-NH), 9.37 (s, 1H, SO₂-NH); ¹³C NMR (DMSO-d₆) δ: 19.43, 21.41, 24.31, 30.94, 44.55, 63.14, 122.06, 123.88, 127.02, 127.20, 129.59, 129.74, 130.53, 130.92, 130.99, 131.18, 133.64, 134.07, 137.17, 166.65; IR (ν, cm⁻¹): 3386, 3083, 1655; MS (z/e): 495[M + H]⁺, 300, 236, 139, 111; Anal. Calcd for C₂₀H₁₉Cl₂F₃N₂O₃S: C, 48.49; H, 3.87; N, 5.66; found: C, 48.62; H, 4.01; N, 5.39.

N-(2-trifluoromethyl-4-chlorophenyl)-2-(3-chlorobenzoylamino) cyclohexylsulfonamide (IV-11)

(R² = 3-ClC₆H₄) White solid; yield, 95%; mp 184–186 °C; ¹H NMR (DMSO-d₆) δ: 1.33–2.07 (m, 8H, 4CH₂), 3.51–3.53 (m, 1H, CH-N), 4.76 (s, 1H, CH-SO₂), 7.46–7.81 (m, 7H, Ph-H), 8.58 (d, J = 8.6 Hz, 1H, CO-NH), 9.37 (s, 1H, SO₂-NH); ¹³C NMR (DMSO-d₆) δ: 19.66, 21.65, 24.13, 30.62, 45.32, 62.96, 122.04, 123.86, 126.98, 127.08, 128.02, 130.41, 131.12, 131.25, 131.33, 133.15, 133.62, 133.97, 137.34, 166.04; IR (ν, cm⁻¹): 3353, 3069, 1638; MS (z/e): 495[M + H]⁺, 300, 236, 139, 111; Anal. Calcd for C₂₀H₁₉Cl₂F₃N₂O₃S: C, 48.49; H, 3.87; N, 5.66; found: C, 48.70; H, 3.66; N, 5.87.

N-(2-trifluoromethyl-4-chlorophenyl)-2-(4-chlorobenzoylamino) cyclohexylsulfonamide (IV-12)

(R² = 4-ClC₆H₄) White solid; yield, 67%; mp 188–190 °C; ¹H NMR (DMSO-d₆) δ: 1.29–2.17 (m, 8H, 4CH₂), 3.51 (dt, J = 11.9, 3.5 Hz, 1H, CH-N), 4.74 (dd, J = 8.1, 3.6 Hz, 1H, CH-SO₂), 7.50–7.86 (m, 7H, Ph-H), 8.19 (d, J = 8.7 Hz, 1H, CO-NH), 9.40 (s, 1H, SO₂-NH); ¹³C NMR (DMSO-d₆) δ: 19.67, 21.66, 24.13, 30.62, 45.32, 62.99, 122.04, 123.86, 126.61, 126.81, 127.04, 128.45, 130.12, 131.09, 131.31, 133.62, 133.99, 134.06, 136.25, 166.41; IR (ν, cm⁻¹): 3386, 3081, 1634; MS (z/e): 495[M + H]⁺, 300, 236, 139, 111; Anal. Calcd for C₂₀H₁₉Cl₂F₃N₂O₃S: C, 48.49; H, 3.87; N, 5.66; found: C, 48.33; H, 4.10; N, 5.81.

N-(2-trifluoromethyl-4-chlorophenyl)-2-(2,6-dichlorobenzoylamino) cyclohexylsulfonamide (IV-13)

(R² = 2,6-Cl₂C₆H₃) White solid; yield, 90%; mp 216–218 °C; ¹H NMR (DMSO-d₆) δ: 1.34–2.19 (m, 8H, 4CH₂), 3.35–3.38 (m, 1H, CH-N), 4.45–4.49 (m, 1H, CH-SO₂), 7.41–7.81 (m, 6H, Ph-H), 8.86 (d, J = 7.8 Hz, 1H, CO-NH), 9.46 (s, 1H, SO₂-NH); ¹³C NMR (DMSO-d₆) δ: 21.81, 22.31, 24.27, 29.38, 46.63, 62.44, 121.98, 123.79, 127.17, 127.73, 128.18, 128.40, 131.33, 131.69, 131.80, 131.85, 133.63, 134.03, 136.63, 163.20; IR (ν, cm⁻¹): 3309, 3110, 1645; HRMS-ESI, m/z calcd for C₂₀H₁₉Cl₃F₃N₂O₃S [M + H]⁺529.0134; found, 529.0128.

N-(2-trifluoromethyl-4-chlorophenyl)-2-(3,5-dichlorobenzoylamino) cyclohexylsulfonamide (IV-14)

(R² = 3,5-Cl₂C₆H₃) White solid; yield, 98%; mp 236–238 °C; ¹H NMR (DMSO-d₆) δ: 1.33–2.38 (m, 8H, 4CH₂), 3.39 (td, J = 11.2, 3.0 Hz, 1H, CH-N), 4.21 (ddd, J = 19.3, 10.7, 4.0 Hz, 1H, CH-SO₂), 7.56–7.80 (m, 6H, Ph-H), 8.70 (d, J = 8.5 Hz, 1H, CO-NH), 9.39 (s, 1H, SO₂-NH); ¹³C NMR (DMSO-d₆) δ: 24.30, 24.38, 27.03, 32.83, 48.76, 64.92, 120.19, 122.01, 123.83, 126.45, 126.95, 127.03, 127.15, 130.84, 131.50, 133.60, 134.13, 134.51, 138.39, 163.14; IR (ν, cm⁻¹): 3268, 3086, 1644; MS (z/e): 529[M + H]⁺, 334, 270, 173, 145; Anal. Calcd for C₂₀H₁₈Cl₃F₃N₂O₃S: C, 45.34; H, 3.42; N, 5.29; found: C, 45.27; H, 3.67; N, 5.04.

N-(2-trifluoromethyl-4-chlorophenyl)-2-(2-trifluoromethylbenzoylamino) cyclohexylsulfonamide (IV-15)

(R² = 2-CF₃C₆H₄) Colorless crystal; yield, 90%; mp 88–89 °C; ¹H NMR (DMSO-d₆) δ: 1.34–2.03 (m, 8H, 4CH₂), 3.49–3.51 (m, 1H, CH-N), 4.80 (s, 1H, CH-SO₂), 7.56–7.81 (m, 7H, Ph-H), 8.67 (d, J = 5.5 Hz, 1H, CO-NH), 9.33 (s, 1H, SO₂-NH); ¹³C NMR (DMSO-d₆) δ: 19.25, 21.37, 24.33, 30.78, 44.42, 63.10, 122.06, 123.29, 123.87, 125.11, 126.31, 127.06, 129.18, 129.86, 131.21, 131.33, 132.52, 133.63, 134.03, 136.78, 167.22; IR (ν, cm⁻¹): 3338, 3195, 1657; MS (z/e): 529[M + H]⁺, 334, 270, 173, 145; Anal. Calcd for C₂₁H₁₉ClF₆N₂O₃S: C, 47.69; H, 3.62; N, 5.30; found: C, 47.89; H, 3.52; N, 5.21.

N-(2-trifluoromethyl-4-chlorophenyl)-2-(3-trifluoromethylbenzoylamino) cyclohexylsulfonamide (IV-16)

(R² = 3-CF₃C₆H₄) White solid; yield, 89%; mp 178–179 °C; ¹H NMR (DMSO-d₆) δ: 1.42–2.19 (m, 8H, 4CH₂), 3.52 (d, J = 11.3 Hz, 1H, CH-N), 4.77 (s, 1H, CH-SO₂), 7.60–8.11 (m, 7H, Ph-H), 8.41 (d, J = 8.7 Hz, 1H, CO-NH), 9.41 (s, 1H, SO₂-NH); ¹³C NMR (DMSO-d₆) δ: 19.75, 21.74, 24.06, 30.57, 45.42, 62.88, 122.04, 123.51, 123.85, 124.86, 125.31, 127.06, 128.03, 129.05, 129.26, 129.69, 131.13, 132.32, 133.63, 136.29, 166.09; IR (ν, cm⁻¹): 3381, 3092, 1636; MS (z/e): 529[M + H]⁺, 334, 270, 173, 145; Anal. Calcd for C₂₁H₁₉ClF₆N₂O₃S: C, 47.69; H, 3.62; N, 5.30; found: C, 47.55; H, 3.76; N, 5.13.

N-(2-trifluoromethyl-4-chlorophenyl)-2-(2-methoxy-5-chlorobenzoylamino) cyclohexylsulfonamide (IV-17)

(R² = 2-CH₃O-5-ClC₆H₃) White solid; yield, 97%; mp 140–141 °C; ¹H NMR (DMSO-d₆) δ: 1.44–2.15 (m, 8H, 4CH₂), 3.53–3.56 (m, 1H, CH-N), 3.93 (s, 3H, OCH₃), 4.63 (dd, J = 7.0, 3.4 Hz, 1H, CH-SO₂), 7.22–7.80 (m, 6H, Ph-H), 8.58 (d, J = 7.4 Hz, 1H, CO-NH), 9.54 (s, 1H, SO₂-NH); ¹³C NMR (DMSO-d₆) δ: 20.01, 22.44, 23.96, 29.84, 45.62, 57.02, 62.71, 114.86, 122.01, 123.83, 124.09, 124.98, 127.08, 130.23, 131.29, 131.53, 132.40, 133.64, 133.79, 156.49, 163.29; IR (ν, cm⁻¹): 3380, 3125, 1653; MS (z/e): 525[M + H]⁺, 266, 169, 126, 111; Anal. Calcd for C₂₁H₂₁Cl₂F₃N₂O₄S: C, 48.01; H, 4.03; N, 5.33; found: C, 48.26; H, 3.90; N, 5.62.

N-(2-trifluoromethyl-4-chlorophenyl)-2-(acetylamino) cyclohexylsulfonamide (IV-18)

(R² = Me) Colorless crystal; yield, 99%; mp 145–146 °C; ¹H NMR (DMSO-d₆) δ: 1.34–2.03 (m, 8H, 4CH₂), 1.87 (s, 3H, CH₃), 3.39 (dt, J = 12.1, 3.2 Hz, 1H, CH-N), 4.55 (dd, J = 8.8, 2.9 Hz, 1H, CH-SO₂), 7.60–7.78 (m, 3H, Ph-H), 7.95 (d, J = 9.2 Hz, 1H, CO-NH), 9.26 (s, 1H, SO₂-NH); ¹³C NMR (DMSO-d₆) δ: 19.49, 21.40, 23.08, 24.24, 30.87, 44.07, 63.19, 122.06, 123.87, 126.91, 129.99, 130.72, 133.59, 134.22, 170.44; IR (ν, cm⁻¹): 3390, 3037, 1657; MS (z/e): 398[M]⁺, 194, 159, 140; Anal. Calcd for C₁₅H₁₈ClF₃N₂O₃S: C, 45.17; H, 4.55; N, 7.02; found: C, 44.95; H, 4.21; N, 7.26.

N-(2-trifluoromethyl-4-chlorophenyl)-2-(propionylamino) cyclohexylsulfonamide (IV-19)

(R² = Et) Colorless crystal; yield, 98%; mp 165–167 °C; ¹H NMR (DMSO-d₆) δ: 0.98 (t, J = 7.6 Hz, 3H, CH₃), 1.29–2.00 (m, 8H, 4CH₂), 2.15 (q, J = 7.6 Hz, 2H, CH₂), 3.39 (dt, J = 12.0, 3.3 Hz, 1H, CH-N), 4.56 (dd, J = 8.8, 3.1 Hz, 1H, CH-SO₂), 7.59–7.79 (m, 3H, Ph-H), 7.84 (d, J = 9.2 Hz, 1H, CO-NH), 9.25 (s, 1H, SO₂-NH); ¹³C NMR (DMSO-d₆) δ: 10.19, 19.52, 21.45, 24.24, 28.76, 30.87, 43.94, 63.27, 122.05, 123.87, 126.94, 130.24, 130.80, 133.56, 134.19, 174.04; IR (ν, cm⁻¹): 3375, 3095, 1643; MS (z/e): 412[M]⁺, 218, 154, 69, 57; Anal. Calcd for C₁₆H₂₀ClF₃N₂O₃S: C, 46.55; H, 4.88; N, 6.79; found: C, 46.27; H, 5.01; N, 6.58.

N-(2-trifluoromethyl-4-chlorophenyl)-2-(n-butyrylamino) cyclohexylsulfonamide (IV-20)

(R² = n-propyl) Colorless crystal; yield, 93%; mp 119–121 °C; ¹H NMR (DMSO-d₆) δ: 0.86 (t, J = 4.8 Hz, 3H, CH₃), 1.32–2.14 (m, 12H, 6CH₂), 3.35–3.40 (m, 1H, CH-N), 4.56 (d, J = 4.0 Hz, 1H, CH-SO₂), 7.59–7.80 (m, 3H, Ph-H), 7.87 (d, J = 6.4 Hz, 1H, CO-NH), 9.29 (s, 1H, SO₂-NH); ¹³C NMR (DMSO-d₆) δ: 13.98, 19.10, 19.49, 21.46, 24.26, 30.98, 37.56, 43.96, 63.26, 122.06, 123.87, 126.98, 130.35, 130.85, 133.59, 134.19, 173.11; IR (ν, cm⁻¹): 3392, 3101, 1658; HRMS-ESI, m/z calcd for C₁₇H₂₃ClF₃N₂O₃S [M + H]⁺427.1070; found, 427.1076.

N-(2-trifluoromethyl-4-chlorophenyl)-2-(n-valerylamino) cyclohexylsulfonamide (IV-21)

(R² = n-butyl) White crystal; yield, 92%; mp 175–177 °C; ¹H NMR (DMSO-d₆) δ: 0.84 (t, J = 6.9 Hz, 3H, CH₃), 1.23–2.24 (m, 14H, 7CH₂), 3.20 (td, J = 10.1, 3.4 Hz, 1H, CH-N), 4.06 (ddd, J = 18.7, 9.3, 4.0 Hz, 1H, CH-SO₂), 7.57–7.80 (m, 3H, Ph-H), 7.93 (d, J = 8.5 Hz, 1H, CO-NH), 9.31 (s, 1H, SO₂-NH); ¹³C NMR (DMSO-d₆) δ: 14.26, 22.34, 23.81, 25.31, 26.31, 28.64, 31.41, 35.89, 47.42, 65.02, 122.02, 123.83, 126.92, 130.97, 131.09, 133.55, 134.25, 172.43; IR (ν, cm⁻¹): 3360, 1647; MS (z/e): 440[M]⁺, 246, 195, 57; Anal. Calcd for C₁₈H₂₄ClF₃N₂O₃S: C, 49.03; H, 5.49; N, 6.35; found: C, 49.25; H, 5.30; N, 6.28.

N-(2-trifluoromethyl-4-chlorophenyl)-2-(n-hexanoylamino) cyclohexylsulfonamide (IV-22)

(R² = n-pentyl) White crystal; yield, 87%; mp 119–121 °C; ¹H NMR (DMSO-d₆) δ: 0.86 (t, J = 7.3 Hz, 3H, CH₃), 1.21–2.19 (m, 16H, 8CH₂), 3.40 (d, J = 11.9 Hz, 1H, CH-N), 4.57 (d, J = 5.4 Hz, 1H, CH-SO₂), 7.60–7.86 (m, 3H, Ph-H), 7.79 (s, 1H, CO-NH), 9.26 (s, 1H, SO₂-NH); ¹³C NMR (DMSO-d₆) δ: 14.28, 22.34, 23.91, 25.31, 26.31, 28.64, 31.41, 32.30, 35.88, 47.41, 64.99, 109.90, 122.02, 123.84, 126.99, 131.01, 133.57, 134.24, 172.41; IR (ν, cm⁻¹): 3376, 3029, 1646; HRMS-ESI, m/z calcd for C₁₉H₂₇ClF₃N₂O₃S [M + H]⁺455.1383; found, 455.1389.

N-(2-trifluoromethyl-4-chlorophenyl)-2-(n-heptanoylamino) cyclohexylsulfonamide (IV-23)

(R² = n-hexyl) White crystal; yield, 95%; mp 105–106 °C; ¹H NMR (DMSO-d₆) δ: 0.84 (t, J = 7.4 Hz, 3H, CH₃), 1.20–2.24 (m, 18H, 9CH₂), 3.20 (td, J = 10.1, 3.4 Hz, 1H, CH-N), 4.06 (ddd, J = 18.7, 9.4, 4.0 Hz, 1H, CH-SO₂), 7.57–7.80 (m, 3H, Ph-H), 7.93 (d, J = 8.5 Hz, 1H, CO-NH), 9.32 (s, 1H, SO₂-NH); ¹³C NMR (DMSO-d₆) δ: 14.11, 22.14, 23.82, 23.93, 26.34, 27.51, 32.33, 35.61, 40.44, 47.42, 65.03, 122.03, 123.84, 126.98, 130.99, 131.09, 133.57, 134.25, 172.41; IR (ν, cm⁻¹): 3325, 3110, 1643; MS (z/e): 468[M]⁺, 274, 195, 113; Anal. Calcd for C₂₀H₂₈ClF₃N₂O₃S: C, 51.22; H, 6.02; N, 5.97; found: C, 51.42; H, 5.90; N, 6.15.

N-(2-trifluoromethyl-4-chlorophenyl)-2-(2-chloroacetylamino) cyclohexylsulfonamide (IV-24)

(R² = ClCH₂) Gray solid; yield, 94%; mp 121–124 °C; ¹H NMR (DMSO-d₆) δ: 1.28–2.03 (m, 8H, 4CH₂), 3.42 (dt, J = 12.1, 3.4 Hz, 1H, CH-N), 4.10 (dd, J = 53.0, 12.9 Hz, 2H, CH₂), 4.52 (dd, J = 8.3, 3.3 Hz, 1H, CH-SO₂), 7.59–7.80 (m, 3H, Ph-H), 8.19 (d, J = 8.8 Hz, 1H, CO-NH), 9.30 (s, 1H, SO₂-NH); ¹³C NMR (DMSO-d₆) δ: 19.63, 21.70, 24.05, 30.49, 43.00, 44.89, 62.73, 122.02, 123.84, 127.04, 131.06, 131.33, 133.64, 133.92, 166.10; IR (ν, cm⁻¹): 3383, 3180, 1678; HRMS-ESI, m/z calcd for C₁₅H₁₈Cl₂F₃N₂O₃S [M + H]⁺433.0367; found, 433.0371.

N-(2-trifluoromethyl-4-chlorophenyl)-2-(2,2-dichloroacetylamino) cyclohexylsulfonamide (IV-25)

(R² = Cl₂CH) Colorless crystal; yield, 82%; mp 179–180 °C; ¹H NMR (DMSO-d₆) δ: 1.44–1.95 (m, 8H, 4 CH₂), 3.44 (dt, J = 3.3, 12.2 Hz, 1H, CH-N), 4.49 (dd, J = 8.2, 3.5 Hz, 1H, CH-SO₂),6.54 (s, 1H, CH-Cl₂), 7.50–7.81 (m, 3H, Ph-H), 8.47 (d, J = 8.6 Hz, 1H, CO-NH), 9.41 (s, 1H, SO₂-NH); ¹³C NMR (DMSO-d₆) δ: 14.47, 19.61, 23.98, 30.25, 45.35, 62.37, 66.82, 122.01, 123.83, 127.12, 131.58, 133.65, 133.80, 162.80, 163.24; IR (ν, cm⁻¹): 3367, 3273, 1672; MS (z/e): 468(M⁺), 274, 210, 130, 81, 64; Anal. Calcd for C₁₅H₁₆ClF₃N₂O₃S: C, 38.52; H, 3.45; N, 5.99; found: C, 38.66; H, 3.59; N, 5.73.

N-(2-trifluoromethyl-4-chlorophenyl)-2-(2,2,2-chloroacetylamino) cyclohexylsulfonamide (IV-26)

(R² = Cl₃C) Colorless crystal; yield, 88%; mp 151–154 °C; ¹H NMR (DMSO-d₆) δ: 1.28–2.37 (m, 8H, 4CH₂), 3.51–3.58 (m, 1H, CH-N), 3.95–4.04 (m, 1H, CH-SO₂), 7.55–7.81 (m, 3H, Ph-H), 8.85 (d, J = 8.4 Hz, 1H, CO-NH), 9.46 (s, 1H, SO₂-NH); ¹³C NMR (DMSO-d₆) δ: 24.27, 27.11, 28.68, 31.88, 50.33, 63.74, 93.26, 121.99, 123.81, 127.05, 131.72, 132.05, 133.64, 134.09, 160.27; IR (ν, cm⁻¹): 3421, 3311, 1708; MS (z/e): 306, 242, 161; Anal. Calcd for C₁₅H₁₅Cl₄F₃N₂O₃S: C, 35.88; H, 3.01; N, 5.58; found: C, 36.01; H, 3.22; N, 5.47.

N-(2-trifluoromethyl-4-chlorophenyl)-2-(2-bromoacetylamino) cyclohexylsulfonamide (IV-27)

(R² = BrCH₂) Colorless crystal; yield, 90%; mp 131–132 °C; ¹H NMR (DMSO-d₆) δ: 1.33–2.08 (m, 8H, 4CH₂), 3.41 (m, 1H, CH-N), 3.88–4.16 (m, 2H, CH₂-Br), 4.52 (s, 1H, CH-SO₂), 7.59–7.81 (m, 3H, Ph-H), 8.33 (dd, J = 9.0, 61.8 Hz, 1H, CO-NH), 9.32 (d, J = 18.6 Hz, 1H, SO₂-NH); ¹³C NMR (DMSO-d₆) δ: 19.63, 21.70, 24.06, 30.50, 43.00, 44.89, 62.74, 122.02, 123.84, 127.06, 131.05, 131.32, 133.62, 133.92, 166.11; IR (ν, cm⁻¹): 3383, 3090, 1678; MS (z/e): 477[M + H]⁺, 80; Anal. Calcd for C₁₅H₁₇BrClF₃N₂O₃S: C, 37.71; H, 3.59; N, 5.86; found: C, 37.94; H, 3.47; N, 5.62.

N-(2-trifluoromethyl-4-chlorophenyl)-2-(2-methoxyacetylamino) cyclohexylsulfonamide (IV-28)

(R² = CH₃OCH₂) White solid; yield, 79%; mp 125–127 °C; ¹H NMR (DMSO-d₆) δ: 1.38–2.10 (m, 8 H, 4CH₂), 3.31 (s, 3H, OCH₃), 3.47 (dt, J = 11.5, 3.3 Hz, 1H, CH-N), 3.79–3.86 (m, 2H, OCH₂), 4.51 (dd, J = 7.4, 3.5 Hz, 1H, CH-SO₂), 7.55 (d, J = 8.0 Hz, 1H, CO-NH), 7.60–7.81 (m, 3H, Ph-H), 9.42 (s, 1H, SO₂-NH); ¹³C NMR (DMSO-d₆) δ: 19.80, 21.98, 23.87, 30.17, 44.51, 58.98, 62.62, 71.52, 122.02, 123.83, 127.10, 127.15, 131.05, 131.36, 133.66, 169.47; IR (ν, cm⁻¹): 3394, 3099, 1647; MS (z/e): 428[M]⁺, 234, 195, 170; Anal. Calcd for C₁₆H₂₀ClF₃N₂O₄S: C, 44.81; H, 4.70; N, 6.53; found: C, 45.03; H, 4.52; N, 6.77.

N-(2-trifluoromethyl-4-chlorophenyl)-2-(2-ethoxyacetylamino) cyclohexylsulfonamide (IV-29)

(R² = C₂H₅OCH₂) Colorless crystal; yield, 32%; mp 125–127 °C; ¹H NMR (DMSO-d₆) δ: 1.14 (t, J = 7.0 Hz, 3H, CH₃) 1.30–2.07 (m, 8H, 4CH₂), 3.46–3.51 (m, 3H, OCH₂-CO, CH-N), 3.86 (q, J = 15.3 Hz, 2H, OCH₂), 4.47 (dd, J = 7.3, 3.6 Hz, 1H, CH-SO₂), 7.53 (d, J = 7.8 Hz, 1H, CO-NH), 7.60–7.81 (m, 3H, Ph-H), 9.48 (s, 1H, SO₂-NH); ¹³C NMR (DMSO-d₆) δ: 15.32, 19.88, 22.11, 23.82, 29.99, 44.66, 62.51, 66.62, 69.67, 109.90, 115.73, 122.01, 123.82, 127.07, 131.18, 133.66, 169.71; IR (ν, cm⁻¹): 3412, 3070, 1681; HRMS-ESI, m/z calcd for C₁₇H₂₃ClF₃N₂O₄S [M + H]⁺443.1019; found, 443.1024.

N-(2-trifluoromethyl-4-chlorophenyl)-2-(2,2,2-trichloroacetylamino) cyclopentylsulfonamide (IV-30)

(n = 0, R¹ = H) Colorless crystal; yield, 98%; mp 86–88 °C; ¹H NMR (DMSO-d₆) δ: 1.63–2.28 (m, 6H, 3CH₂), 3.94 (q, J = 7.5 Hz, 1H, CH-N), 4.40 (p, J = 7.1 Hz, 1H, CH-SO₂), 7.59–7.82 (m, 3H, Ph-H), 8.65 (d, J = 6.9 Hz, 1H, CO-NH), 9.72 (s, 1H, SO₂-NH); ¹³C NMR (DMSO-d₆) δ: 21.44, 26.54, 30.73, 53.99, 63.48, 92.81, 121.93, 123.75, 127.25, 131.84, 132.01, 133.56, 133.73, 161.25; IR (ν, cm⁻¹): 3363, 3190, 1726; MS (z/e): 488(M)⁺, 294, 230, 164, 67; Anal. Calcd for C₁₄H₁₃Cl₄F₃N₂O₃S: C, 34.45; H, 2.68; N, 5.74; found: C, 34.56; H, 2.87; N, 5.49.

N-(2-trifluoromethyl-4-chlorophenyl)-2-(2,2,2-trichloroacetylamino) cycloheptylsulfonamide (IV-31)

(n = 2, R¹ = H) Colorless crystal; yield, 97%; mp 114–115 °C; ¹H NMR (DMSO-d₆) δ: 1.17–2.17 (m, 10H, 5CH₂), 3.57–3.59 (m, 1H, CH-N), 4.57 (s, 1H, CH-SO₂), 7.60–7.78 (m, 3H, Ph-H), 8.34 (d, J = 6.5 Hz, 1H, CO-NH), 9.58 (s, 1H, SO₂-NH); ¹³H NMR (DMSO-d₆) δ: 8.96, 22.60, 23.79, 25.95, 27.86, 31.06, 46.04, 50.26, 65.05, 93.06, 122.09, 123.90, 125.72, 127.12, 130.96, 133.59, 160.72; IR (ν, cm⁻¹): 3381, 3242, 1710; MS (z/e): 530(M)⁺, 320, 256, 162, 95, 67; Anal. Calcd for C₁₆H₁₇Cl₄F₃N₂O₃S: C, 37.23; H, 3.32; N, 5.43; found: C, 37.55; H, 3.21; N, 5.62.

N-(2-trifluoromethyl-4-chlorophenyl)-3-methyl-2-(2,2,2-trichloroacetylamino) cyclohexylsulfonamide (IV-32)

(n = 1, R¹ = 3-Me) Colorless crystal; yield, 96%; mp 137~139 °C; ¹H NMR (DMSO-d₆) δ: 0.93–2.35 (m, 10H, 5CH₂), 3.75 (dd, J = 9.4, 4.6 Hz, 1H, CH-N), 3.97 (td, J = 8.6, 4.2 Hz, 1H, CH-SO₂), 7.54–7.81 (m, 3H, Ph-H), 8.55 (d, J = 8.2 Hz, 1H, CO-NH), 9.66 (s, 1H, SO₂-NH); ¹³C NMR (DMSO-d₆) δ: 18.24, 19.65, 24.93, 31.96, 50.48, 55.50, 60.87, 92.94, 121.97, 123.79, 127.19, 130.90, 131.59, 133.66, 133.73, 161.15; IR (ν, cm⁻¹): 3398, 3250, 1707; MS (z/e): 516(M)⁺, 322, 258, 164, 95, 67; Anal. Calcd for C₁₆H₁₇Cl₄F₃N₂O₃S: C, 37.23; H, 3.32; N, 5.43; found: C, 37.42; H, 3.12; N, 5.60.

N-(2-trifluoromethyl-4-chlorophenyl)-4-methyl-2-(2,2,2-trichloroacetylamino) cyclohexylsulfonamide (IV-33)

(n = 1, R¹ = 4-Me) Colorless crystal; yield, 93%; mp 123–124 °C; ¹H NMR (DMSO-d₆) δ: 0.93 (d, J = 6.0 Hz, 3H, CH₃), 1.40–2.40 (m, 7H, C₄H₇), 3.82 (s, 1H, CH-N), 4.10–4.20 (m, 1H, CH-SO₂), 7.53–7.82 (m, 3H, Ph-H), 8.81 (d, J = 7.2 Hz, 1H, CO-NH), 9.85 (s, 1H, SO₂-NH); ¹³C NMR (DMSO-d₆) δ: 22.18, 25.42, 28.17, 31.15, 34.89, 51.80, 59.87, 92.66, 121.92, 123.74, 127.29, 131.20, 131.89, 133.53, 133.80, 160.81; IR (ν, cm⁻¹): 3360, 3226, 1693; MS (z/e): 516(M)⁺, 259, 224, 202, 112, 81; Anal. Calcd for C₁₆H₁₇Cl₄F₃N₂O₃S: C, 37.23; H, 3.32; N, 5.43; found: C,36.99; H, 3.21; N, 5.60.

N-(2-trifluoromethyl-4-chlorophenyl)-5-methyl-2-(2,2,2-trichloroacetylamino) cyclohexylsulfonamide (IV-34)

(n = 1, R¹ = 5-Me) White crystal; yield, 98%; mp 144–145 °C; ¹H NMR (DMSO-d₆) δ: 0.95–2.33 (m, 10H, 5CH₂), 3.64 (td, J = 11.6, 2.9 Hz, 1H, CH-N), 3.90–4.03 (m, 1H, CH-SO₂), 7.55–7.81 (m, 3H, Ph-H), 8.83 (d, J = 8.4 Hz, 1H, CO-NH), 9.45 (s, 1H, SO₂-NH); ¹³C NMR (DMSO-d₆) δ: 22.11, 31.03, 31.71, 32.72, 35.13, 50.22, 63.45, 93.26, 122.00, 123.81, 127.07, 131.67, 132.05, 133.61, 134.11, 160.32; IR (ν, cm⁻¹): 3431, 3336, 1687; MS (z/e): 516(M)⁺, 322, 258, 95, 67, 55; Anal. Calcd for C₁₆H₁₇Cl₄F₃N₂O₃S: C, 37.23; H, 3.32; N, 5.43; found: C, 37.08; H, 3.53; N, 5.27.

N-(2-trifluoromethyl-4-chlorophenyl)-5-ethyl-2-(2,2,2-trichloroacetylamino) cyclohexylsulfonamide (IV-35)

(n = 1, R¹ = 5-Et) White crystal; yield, 93%; mp 122–125 °C; ¹H NMR (DMSO-d₆) δ: 0.84(t, J = 7.4 Hz, 3H, CH₃), 1.15–2.35 (m, 9H, C₅H₉), 3.83 (d, J = 4.4 Hz, 1H, CH-N), 4.17 (dd, J = 10.4, 6.7 Hz, 1H, CH-SO₂), 7.55–7.84 (m, 3H, Ph-H), 8.67 (d, J = 6.6 Hz, 1H, CO-NH), 9.81 (s, 1H, SO₂-NH); ¹³C NMR (DMSO-d₆) δ: 11.75, 26.04, 27.69, 28.54, 30.29, 32.20, 51.19, 60.10, 92.73, 121.94, 123.76, 125.58, 127.26, 131.13, 131.84, 133.76, 160.91; IR (ν, cm⁻¹): 3394, 3261, 1705; MS (z/e): 530(M)⁺, 320, 256, 162, 95, 67; Anal. Calcd for C₁₇H₁₉Cl₄F₃N₂O₃S: C, 38.51; H, 3.61; N, 5.28; found: C, 38.69; H, 3.50; N, 5.04.

N-(2-trifluoromethyl-4-chlorophenyl)-5-tertiarybutyl-2-(2,2,2-trichloroacetylamino) cyclohexylsulfonamide (IV-36)

(n = 1, R¹ = 5-t-Bu) Colorless crystal; yield, 90%; mp 124–126 °C; ¹H NMR (DMSO-d₆) δ: 0.87–2.21 (m, 16H, C₈H₁₆), 3.57 (dt, J = 12.9, 3.3 Hz, 1H, CH-N), 4.38 (dd, J = 5.9, 3.1 Hz, 1H, CH-SO₂), 7.61–7.83 (m, 3H, Ph-H), 7.97 (d, J = 6.0 Hz, 1H, CO-NH), 9.85 (s, 1H, SO₂-NH); ¹³C NMR (DMSO-d₆) δ: 20.55, 22.95, 27.55, 32.69, 46.21, 47.02, 62.47, 92.92, 121.95, 123.77, 127.12, 132.13, 132.36, 133.42, 133.72, 160.95; IR (ν, cm⁻¹): 3400, 3284, 1708; MS (z/e): 530(M)⁺, 109, 67; Anal. Calcd for C₁₉H₂₃Cl₄F₃N₂O₃S: C, 40.88; H, 4.15; N, 5.02; found: C, 41.02; H, 3.98; N, 5.21.

Bioassays

In vitro fungicidal activity

In vitro effects of compounds against B. cinerea were evaluated by mycelium growth rate method³⁰,³¹,³². The tested compounds were dissolved in DMSO (dimethyl sulfoxide) and mixed with sterile molten potato dextrose agar (PDA) to a final concentration of 50 mg/L. EC₅₀ values were estimated using logit analysis. The concentration gradients were 50, 12.5, 3.13, 0.78 mg/L on PDA and a commercial fungicide procymidone was used as the positive control. EXCEL 2010 was used to analyze bioassay data. The variance analysis was carried out by using SPSS 20.0 software for the inhibition rate, EC₅₀ and control efficiency.

The relative inhibition rate of the synthetic compounds compared to blank control was calculated via the following equation (1):

In which, I stands for the rate of inhibition (%), C is the diameter of mycelia in the blank control test (in mm), and T is the diameter of mycelia in the presence of tested compounds (in mm).

In vivo antifungal activity

In vivo effects were checked on leaves of cucumber (Cucumis sarivus L.) by mycelium inoculation method with pot cultural test in greenhouse³⁷,³⁸,³⁹,⁴⁰. The cucumber seedlings at 2–3 leaf stages were used to assay the fungicidal activity against B. cinerea. The compounds were confected to 2.5% EC (emulsifiable concentrate) formulation. The formulation was diluted to 500 mg/L with water and sprayed on the surface of the cucumber leaves. After air drying, the surface of the leaves was inoculated with 6 mm plugs of B. cinerea, which was maintained on potato dextrose agar (PDA). This procedure was repeated three times, and nine replicates were performed per treatment. The chesulfamide (L, Fig. 3) was used as the positive control.

The fungicidal activity was assessed when the untreated cucumber plant (blank control) fully developed symptoms. The area of inoculated leaves covered by disease symptoms was evaluated and compared to that of untreated ones to determine the average disease index. The relative control efficacy of compounds compared to the blank assay was calculated via the following equation (2):

where I is relative control efficacy, CK is the average disease index during the blank assay and PT is the average disease index after treatment during testing.

Additional Information

How to cite this article: Liu, C.-H. et al. Synthesis, Fungicidal Activity, and Structure Activity Relationship of β-Acylaminocycloalkylsulfonamides against Botrytis cinerea. Sci. Rep. 7, 42096; doi: 10.1038/srep42096 (2017).

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