ZONG Gung-NingLI Feng-YunFAN Zhi-Jin②MAO Wu-ToSONG Hi-BinCHEN LiZHU Yu-JieXU Jing-HuSONG Yin-QiWANG Ji-Rn(Stte Key Lortory of Elemento-orgnic Chemistry, Collortive Innovtion Center of Chemicl Science nd Engineering (Tinjin), Nnki University, Tinjin 300071, Chin)(College of Chemistry nd Phrmcy Engineering, Nnyng Norml University, Nnyng, Henn 473061, Chin)

Synthesis, Crystal Structure and Biological Activity of 2-(3,4-Dichloroisothiazol-5-yl)-4-(trifluoromethyl)-4,5-dihydrothiazol-4-yl-3-methylbenzoate①

ZONG Guang-NingaLI Feng-YunaFAN Zhi-Jina②MAO Wu-TaobSONG Hai-BinaCHEN LaiaZHU Yu-JieaXU Jing-HuaaSONG Yin-QiaWANG Jia-Ranaa(State Key Laboratory of Elemento-organic Chemistry, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin 300071, China)b(College of Chemistry and Pharmacy Engineering, Nanyang Normal University, Nanyang, Henan 473061, China)

The title compound diethyl 2-(3,4-dichloroisothiazol-5-yl)-4-(trifluoromethyl)-4,5-dihydrothiazol-4-yl-3-methylbenzoate (C15H9Cl2F3N2O2S2, Mr= 441.26) was prepared from methyl 3,4-dichloroisothiazole-5-carboxylate as the starting material by four steps of reaction.Its structure was characterized by IR,1H-NMR,13C-NMR, EA and single-crystal X-ray diffraction.The crystal of the title compound belongs to the monoclinic system, space group P21/c with a = 8.8437(18), b = 16.128(3), c = 12.305(3) ?, β = 91.68(3)o, V = 1754.4(6) ?3, Z = 4, Dc= 1.671 g/cm3, μ(MoKa) = 0.71073 mm-1, F(000) = 888, R = 0.0384 and wR = 0.0778.Weak π-π interactions occur between the isothiazole rings and phenyl rings of adjacent molecules to form a one-dimensional chain and stabilize the crystal structure.Bioassay indicates that the title compound has good activity against the fungi and TMV tested.

isothiazole, 4,5-dihydrothiazole, synthesis, crystal structure, biological activity;

1 INTRODUCTION

Aggressive pathogens cause disease and great losses in agriculture all over the world[1].A great amount of fungicides are used to control plant disease every year.In contrast to traditional fungicide, plant elicitors induce systemic acquired resistance of host plant against abroad spectrum of disease without direct activities against pathogens itself[2-4].Heterocyclic compounds have various biological activities[5,6].Isotianil, a derivative of isothiazole-5-carboxylic acid, is a novel plant elicitor with broad spectrum of diseases controlling effects by cooperating with other fungicides or insecticides[7-9].Isothiazole and their derivatives present a wide range of biological activities including insecticidal activity[10,11], fungicidal activity[7,12], herbicidal activity[13], and systemic acquired resistance[3].Some isothiazoles also present pharmaceutical activity as of acyl guanidine inhibitors[14],antiglycation reagents[15]and dual functional inhibitors[16].

Phytoalexins are antimicrobial substances produced by plants in response to infection or stress with low molecular weight[17].Types of phytoalexins produced by crucifers have good fungicidal activity, which have unique structures containing isothiazole or thiazole related rings and at least one sulfur atom, for example spirobrassinin[18].Research revealed that the heterocyclic ring with 4,5-dihydrothiazole of spirobrassinin and its analogs played a key role in keeping the antifungal activity[19,20].Some compounds bearing the 4,5-dihydrothiazole ring showed broad-spectrum biological activity[21].A series of substituted 2-(pyridin-3-yl)-4-(trifluoromethyl)-4,5-dihydrothiazol-4-yl benzoates has been found as potential fungicidal chemicals with good activity[22].

Another type of heterocyclic compounds 2-(3,4-dichloroisothiazol-5-yl)-4-(trifluoromethyl)-4,5-dihydrothiazol-4-yl-3-methylbenzoate with active substructures of 3,4-dichloroisothiazole and 4,5-dihydrothiazole moieties were designed and synthesized here according to the principle of pesticide designation and the description in Scheme 1[2], and their crystal structure and activity were also evaluated.

Scheme 1.Schematic structure and synthesis of the target compound

2 EXPERIMENTAL

All reagents and solvents for synthesis and analyses were of analytical grade and used without further purification.Column chromatography purification was carried out by using silica gel (200～300) with ethyl acetate and petroleum ether as eluent.The melting point was measured on an X-4 binocular microscope (Gongyi Tech.Instrument Co., Henan, China), and the temperature was not corrected.Infrared (IR) spectra were recorded on a Bruker Vector 22 Fourier transform infrared (FTIR) spectrometer using KBr pellets.Hydrogen Nuclear Magnetic Resonance (1H NMR) spectra were measured at 400 MHz using a Bruker AV-400 spectrometer with deutero-chloroform (CDCl3) as the solvent and tetramethylsilane (TMS) as the internal standard.Elemental analyses (EA) data were obtained on a Vario EL CUBE instrument made by German.The single-crystal structure was determined on a Rigaku Saturn 724 CCD diffractometer.The equipment was operated using Mo-Kα radiation (λ = 0.71073 ?).

2.1 Synthesis

Intermediate 4 was synthesized by a three-step process (Scheme 1).White solid, 3,4-dichloroisothiazole- 5-carboxamide, 2 was synthesized from 2.12 g (10 mmol) ethyl methyl 3,4-dichloroisothiazole-5-carboxylate 1 via an aminolysis reaction at room temperature in a good yield (96%);1H NMR(400 MHz, DMSO-d6): δ 8.35 (s, br, 1H), 8.08 (s, br, 1H).Lawson’s reagent (2.42 g, 6 mmol) was added to a solution of compound 2 (1.97 g, 10 mmol) in anhydrous toluene (50 mL).After refluxing for 3 hours and concentrated under reduced pressure, the obtained residue was purified by chromatography on silica gel (petroleum ether : ethyl acetate, 3:1, v/v) to give a white solid, 3,4-dichloroisothiazole-5-carbothioamide 3;1H NMR(400 MHz, CDCl3): δ 6.80 (s, br, 1H), 6.24 (s, br, 1H).A solution of compound 3 (1.0 g, 5.0 mmol) and 3-bromo-1,1,1-trifluoropropan-2-one (1.0 g, 5.0 mmol) in anhydrousethanol (20 mL) was refluxed and stirred for 16 h.After concentration under reduced pressure, the residue was purified by chromatography on silica gel (petroleum ether : ethyl acetate, 10:1, v/v) to give a yellow solid, 2-(3,4-dichloroisothiazol-5-yl)-4-(trifluoromethyl)-4,5-dihydrothiazol-4-ol 4;1H NMR (400 MHz, CDCl3): δ 3.76 (dd, J=54.5 Hz, 10.2 Hz, 2H), 3.65(s, 1H).

To a stirred solution of compound 4 (0.32 g, 1.0 mmol) and 3-methylbenzoyl chloride in anhydrous CH2Cl2(15 mL) at 0 ℃ was added triethylamine (0.15 g, 1.5 mmol) dropwise.After being wormed to ambient temperature gradually, the resulting mixture was stirred for about 10 h.Then, the reaction mixture was diluted with CH2Cl2(20 mL) and washed with aqueous HCl (2 mol/L), saturated aqueous NaHCO3and brine, and finally dried over anhydrous Na2SO4.After concentration, the crude product was purified by chromatography on silica gel using petroleum ether (60～90 ℃) and ethyl acetate (v/v = 10:1) as the eluent.The pure title compound was obtained as a white solid with the yield of 65%; m.p.: 100～102 ℃.IR (KBr pellet press, ν, cm-1): 2921 (CH3), 1741 (C=O), 1573(Ar, C=C), 1506(Ar, C=C), 1425(Ar, C=C).1H NMR (400 MHz, CDCl3) δ 7.87 (d, J = 5.7 Hz, 2H), 7.46 (d, J = 7.5 Hz, 1H), 7.40 (d, J = 7.9 Hz, 1H), 3.99 (s, 2H), 2.44 (s, 3H).13C NMR (101 MHz, CDCl3) δ 168.29 (s), 163.80 (s), 152.32 (s), 138.58 (s), 134.97 (s), 130.58 (s), 128.58 (s), 127.29 (s), 124.24 (s), 123.77 (s), 120.94 (s), 108.44～107.50 (q), 36.69 (s), 21.27 (s).EA clacd.for C15H9Cl2F3N2O2S2: C, 40.83; H, 2.06; N, 6.35%.Found: C, 41.14; H, 2.08; N, 6.37%.

2.2 Crystal data and structure determination

The crystal of the target compound was cultivated from the mixture of ethyl acetate and dichloromethane with 1:1 (v/v).The colorless crystal of the title compound with dimensions of 0.20mm × 0.18mm × 0.12mm was selected and mounted on a glass fiber for X-ray diffraction analysis.All measurements were made on a Rigaku Saturn 724 CCD diffractometer MoKα radiation (λ = 0.71073 ?).The data were collected at 113(2) K and the crystal is of monoclinic system, space group P21/c, with a = 8.8437(18), b = 16.128(3), c = 12.305(3) ?, β = 91.68(3)o, V = 1754.4(6) ?3, Z = 4, density (calculated) = 1.671 g/cm3, and linear absorption coefficient 0.200 mm-1.In the range of 2.08≤θ≤27.90°, 17654 integrated reflections were collected, reduced to a data set of 4182 unique with Rint= 0.0384, and completeness of data (to theta = 25.02°) of 99.9%.Data were collected and processed using Crystal Clear (Rigaku).An empirical absorption correction was applied using Crystal Clear (Rigaku).The structure was solved by direct methods with the SHELXS-97 program[23].Refinements were done by the full-matrix least-squares on F2with SHELXL-97[24].All of the non-H atoms were refined anisotropically by full-matrix least-squares to give the final R = 0.0384 and wR = 0.0778 ((0.0310P)2+ 0.6562P], where P =with (Δ/σ)max= 0.004 and S = 1.036 by using the SHELXL program.The hydrogen atoms were located from a difference Fourier map and refined isotropically.The corrections for absorption was multi-scan, Tmin= 0.8808 and Tmax= 0.9259.

2.3 Biological screening Fungicide screening

Preliminary screening was conducted by fungi growth inhibition method according to the reference using potato dextrose agar (PDA) as cultivation medium[25].A stock solution of the target compound was prepared at 500 μg/mL using sterilized water containing 2 drops of N,N-dimethylformamide (DMF) as a solvent, then 1 mL of the stock solution was transferred into a 10 cm diameter of Petri dish.9 mL of PDA was then added to prepare the plate containing 50 μg/mL of the test compound.Before the plate solidification, the PDA was thoroughly mixed by turning around the Petri dish in the sterilized hood 5 times to scatter the compound in PDA evenly.Then, a fungi cake in 4 mm diameter was inoculated on the plate and cultured in the culture tank at 24～26 ℃.The diameter of fungi spread was measured 2 days later.Growth inhibitionwas then calculated using the corresponding control.Representative fungi used in this study included Alternaria solani (AS), Botrytis cinerea (BC), Cercospora arachidicola (CA), Gibberella zeae (GZ), Phytophthora infestans (Mont) de Bary (PI), Physalospora piricola (PP), Pellicularia sasakii (PS), Sclerotinia sclerotiorum (SS), and Rhizoctonia cerealis (RC).

Insecticide activity of the target compound against Mythimna separata

Insecticidal activity of the target compounds against M.separata was tested using the leaf-disk method[26,27].Fresh corn leaves were dipped into the 200 μg/mL test water solution for 10 s which was prepared with a 5% of acetone to help the compound dissolve.After air-drying for evaporating off the acetone and water, the treated leaves were cut into small pieces and placed in Petri dishes with a 10 cm diameter.Thirty individuals of M.separata were transferred into each Petri dish.The Petri dishes were finally fastened with rubber bands and placed in a standard cultivation room for 72 h at 25 ℃ with 80% humidity.The percentage of mortalities was evaluated according to the corresponding CK which uses water to dispose only.The insects having no reaction by touching with a brush pen were regarded as a death.

Curative effect of the target compounds on TMV in vivo

Healthy fresh tobacco plants at six-leaf stage were selected for the tests.TMV at a concentration of 5.88 × 10-2μg/mL was inoculated on the whole leaves using the conventional juice robbing method.After the leaves were dried in greenhouse, the compound solution (100 μg/mL) was smeared on the upper three leaves, and the solvent was smeared on the lower three leaves as control.The local lesion numbers were then recorded 2～3 days after inoculation.Three replicates were performed for the target compound, respectively.

The activities of protection, inactivation, and curative effects against TMV were calculated by the average number of viral inflammations on the inoculated leaves with the corresponding control according to equation (1):

Where Y is the antivirus inhibition ratio (protection, inactivation, and curative effects in vivo) (%), CK is the average number of viral inflammations on the control leaves in vivo, and A is the average number of viral inflammations on the target compound treated leaves in vivo.

Protective effect of the target compound against TMV in vivo

Healthy fresh tobacco plants at six-leaf stage were selected for the tests.The target compound solution (100 μg/mL) was smeared on the whole leaves, and then the leaves were dried in the greenhouse.After 12 h, TMV at a concentration of 5.88×10-2μg/mL was inoculated on the upper three leaves using the conventional juice robbing method, and the solvent was smeared on the lower three leaves as a control.The local lesion numbers were then recorded 2～3 days after inoculation.Three replicates were performed for the target compound, respectively.

Inactivation effect of the target compounds against TMV in vivo

Healthy fresh tobacco plants at six-leaf stage were selected for the tests.The TMV virus at a concentration of 5.88×10-2μg/mL was inhibited by mixing with the target compound solution (100 μg/mL) at the same volume for 30 min.Then the mixture was inoculated on the upper three leaves using the conventional juice robbing method, and the solvent was smeared on the lower three leaves as a control.The local lesion numbers were then recorded 2～3 days after inoculation.Three replicates were performed for the target compound, respectively.

Screening for systemic acquired resistance

Systemic acquired resistance of the target compound was detected using tobacco against the tobacco mosaic virus (TMV) system as described in Ref.2.The induction activity was evaluated using the antivirus inhibition ratio, which was calculatedby the average number of viral inflammations on the inoculated leaves with the corresponding control accordingly.Tiadinil, ribavirin and ningnanmycin were used as positive controls, respectively, and the target compound was tested at the concentration of 100 μg/mL.

3 RESULTS AND DISCUSSION

The molecular structure is shown in Fig.1.The selected bond lengths, bond angles and torsion angles are listed in Table 1.

Fig.1.Molecular structure of the title compound shown as thermal probability

Table 1.Selected Bond Lengths (?), Bond Angles (°) and Torsion Angles (°) for the Title Compound

As shown in Table 1, bond lengths and bond angles within the isothiazole ring agree well with the values reported[28].The sum of N(2)–C(4)–C(3), N(2)–C(4)–S(2) and C(3)–C(4)–S(2) angles is 360°, indicating the sp2hybridization state of C(4) atom.The dihedral angle between the isothiazole ring and the plane formed by N(2), C(4) and S(2) atoms is 3.318(12)°, which indicates the existence of strong conjugative effect between the imine group including S(2) atom and isothiazole ring; due to this strong conjugative effect, the bond length of C(3)–C(4) is 1.467(3) ?, which is slightly shorter than that of a typical C–C bond (1.53 ?)[29].The torsion angles of C(6)–N(2)–C(4)–S(2) and C(5)–S(2)–C(4)–N(2) are 1.4(2) and 1.02(17)°, respectively, indicating the obvious distortion of the non-aromatic 4,5-dihydrothiazole ring; it is very interesting that, all five atoms of the 4,5-dihydrothiazole ring almost exist coplanar.In the crystal structure, because of sp3hybridization state of the C(6) atom, the stable conformation of the molecule looks like a “L” in Fig.1.Owing to the p-πconjugate effect, the bond lengths of S(1)–C(3) and O(1)–C(8) are 1.7138(19) and 1.370(3) ?, respectively, which are much shorter than that of the S(2)–C(5) (1.810(2) ?) and O(1)–C(6) (1.437(2) ?) bonds.Due to the π-π conjugation of phenyl ring and the carbonyl group at C(9)–C(8)–O(2), the bond length of C(8)=O(2) (1.204(2) ?) is slightly shorter than that of the normal C=O bond (1.22 ?)[30].Not only weak π-π interactions occur between the isothiazole and phenyl rings of the adjacent molecules, but also exist between isothiazole and isothiazole rings, phenyl and phenyl rings of the adjacent molecules, which form a one-dimensional chain structure (Fig.2).

Fig.2.Crystal packing of the title compound

Table 2.Fungicidal Activity of the Title Compound (Inhibition Rate, %, 50 μg/mL)

Table 3.Antiviral Activity of the Title Compound against Tobacco Mosaic Virus (%, 100 μg/mL)±SD

4 BIOLOGICAL ACTIVITY

The inhibition effects of the title compound against nine typical fungi were tested.The results as compared with commercialized azoxystrobin are shown in Table 2.The preliminary screening results indicated that the title compound presents good fungicidal activity against GZ, BC and PS with the inhibition rates of 82.35%, 87.18% and 87.95%, respectively, which is higher than that of the azoxystrobin.The title compound also possesses good fungicidal activity against PP, SS and RC with the inhibition rates of 91.03%, 95.77% and 88.10%, respectively, equal to that of azoxystrobin.Furthermore, the title compound showed a potential fungicidal activity with broad-spectrum as above 48% of the inhibition rate against nine fungi.Screening against TMV of the title compound was conducted for protection, inactivation, and curative effect and induction activities in vivo (Table 3).The induction of systemic acquired resistance for tobacco against tobacco mosaic virus (TMV) determination was also detected under the concentration of 100 μg/mL according to the reported reference[2].The result indicated that the title compound had a good antiviral activity as comparedwith the positive controls tiadinil, ribavirin and ningnanmycin, especially it has moderate degree of induction effect with 39.64% of the activity.This is higher than that of ribavirin (23.87%) and ningnanmycin (17.57 %), which is almost equal to that of TDL at 100 μg/mL; moreover, the curative effect is higher than that of ningnanmycin (28.89%) and tiadinil (14.67%); however, inactivation and protection effects were lower than ningnanmycin.The larvicidal activity of the target compound against M.separata was tested by leaf disk method[26,27], and the insect mortality was 35% at 200 μg/mL.The result demonstrated that the title compound had certain extent of insecticidal activity.

As discussed above, the isothiazole title compound was designed by the principle of combination of bioactive substructure with four steps.The X-ray diffraction confirmed its structure.Bioassay results indicated that the title compound was a good pesticide lead with various biological activities.Dihydrothiazole group will be derived with other sulfur containing heterocycles to enhance its biological activity.

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10.14102/j.cnki.0254-5861.2011-0598

8 December 2014; accepted 31 March 2015 (CCDC 1028315)

① This study was funded in part by the Tianjin Natural Science Foundation (No.14JCYBJC20400), the "111" Project of Ministry of Education of China (No.B06005) and NFFTBS (No.J1103306)

② Corresponding author.Fan Zhi-Jin, born in 1968, professor.E-mail: fanzj@nankai.edu.cn