Synthesis, Crystal Structure and Fungicidal Activity of 3-Chloro-4-(3,4-dichloroisothiazol-5-yl)-5-hydroxy-7-methyl-2H-chromen-2-ones①

LV You LI Kun HAO Ze-Sheng KALININA Ttin A. GLUKHAREVA Ttin V.② FAN Zhi-Jin②

a (State Key Laboratory of Elemento-organic Chemistry,College of Chemistry, Nankai University, Tianjin300071, China)

b (The Ural Federal University Named after the First President of Russia B. N. Yeltsin,Yeltsin UrFU620002, Ekaterinburg, Russia)

ABSTRACT 3-Chloro-4-(3,4-dichloroisothiazol-5-yl)-5-hydroxy-7-methyl-2H-chromen-2-ones, a kind of coumarin derivatives, were synthesized by β-ketoester formation and cyclization. Target compound 5e was crystallized from methanol for structural identification as monoclinic crystal system, space group C2/c with a =16.2700(6), b = 7.1801(5), c = 23.4861(10) Å, V = 2742.6(2) Å3, Z = 8, Dc = 1.756 g/cm3, F(000) = 1456 and μ =0.827 mm-1. 8308 Reflections were collected (6.01≤2θ≤50.05°), of which 2428 were unique (Rint = 0.0432) and used in all calculations. The final R = 0.0408 (I > 2σ(I)) and wR = 0.1056 (reflections). In vitro bioassay indicated that compounds 5d & 5e possessed good activity against Botrytis cinerea, Physalospora piricola, Rhizoctonia solani, and Sclerotinia sclerotiorum with lower EC50 valuesfalling between 0.50 and 4.85 µg/mL than that of positive control osthole with its EC50 values between 7.38 and 74.59 µg/mL. In vivo screening showed that 5e exhibited 98% and 95% efficacy against Pseudoperonospora cubensis (Berk. & Curt.) Rostov.at 100 and 50 µg/mL,respectively. Our studies discovered that the combination of bioactive substructures of isothiazole with coumarin was an effective way to novel fungicide development.

Keywords: coumarin, synthesis, crystal structure, fungicidal activity;

1 INTRODUCTION

Coumarin (2H-1-benzopyran-2-one) and its derivatives are an important heterocyclic structure in natural products[1],which can be found in many synthetic and naturally occurring drugs and agrochemicals[2,3]. They have received widespread consideration in drug and pesticide discovery due to their impressive pharmacological and physiological activities, such as anticoagulant[4], antibacterial[5,6], antiviral[7], antitumor[8],fungicidal[2,3], anti-inflammatory agents[6,9]and anti-HIV activity[9,10]. Introducing an aromatic ring into the 4-position of coumarin would benefit the biological performance of target compounds[8]. As an active substructure, 3,4-dichloroisothiazole showed good fungicidal activity in pesticide lead discovery[11,12].

Target identification and molecular design are important bases for novel pesticide development[13-16]. Our group focused on agrichemical lead discovery and their mode of action, different pyrazole-thiazoles[17], pyrazole-aromatics[18],and thiadizole derivatives[19]were discovered with various kinds of fungicidal activities. Here, by combining the bioactive substructures of coumarin and isothiazole,3,4-chloroisothiazole-containing coumarins were designed(Fig.1) and synthesized (Scheme 1) for fungicidal activity determination, one of which was chosen for its crystal structure identification.

Scheme 1. Synthetic route of the target compounds 5a～5e

Fig.1. Design of the target compounds

2 EXPERIMENTAL

2. 1 Instruments and reagents

Melting point was measured on an X-4 Digital Type Melting Point Tester (Gongyi, China) and uncorrected.1H NMR spectra were recorded on a Bruker AV400 spectrometer (400 MHz) (Wisconsin, United States of America) and chemical shifts were reported in ppm.13C NMR and19F NMR spectra were recorded on a Bruker AV400 spectrometer (101 and 376 MHz) (Wisconsin, United States of America) with complete proton decoupling. High- resolution mass spectra (HRMS) were recorded with an Agilent 6520 Q-TOF LC/MS instrument (Agilent Techno- logies Inc. State of California, United States of America). Elemental analyses were performed with a Vario EL CUBE elemental analyzer(Elementar Analysensysteme GMBH, Germany). Crystal structure was determined on a SuperNova, Single source at offset/far, Eos diffractometer. All of the solvents and materials were of reagent grade and purified as required.

2. 2 Synthetic procedure for the target compounds

The general procedure for the synthesis of compounds 5 is shown in Scheme 1. The key intermediates,β-ketoesters 3a～3d were obtained by Blaise reaction from compounds 1 and 2.Meanwhile,β-ketoester 3e was prepared by the chlorination of compound 3a with sulfuryl chloride.

A solution of compounds 3 (1.0 mmol) and 5-methylresorcin (1.05 mmol) in trifluoroacetic acid (5.0 mL) was heated under refluxing for 12～24 h. The mixture was quenched with water (10 mL) and extracted with ethyl acetate (3 × 10 mL). The combined organic solution was washed with water(2 × 15 mL), saturated aqueous NaHCO3(15 mL) and brine(15 mL) respectively, dried over anhydrous Na2SO4, and then concentrated under reduced pressure. After that, the residue was purified by column chromatography on a silica gel(100～200 mesh) with a mixture of ethyl acetate/petroleum ether (60～90 °C fraction) (1:10～1:3, v/v) to give compounds 5a～5e (yields between 6% and 91%).

Analytical data for compound 5aPale yellow powder;yield: 91%; m.p.: over 220oC.1H NMR (400 MHz, DMSOd6)δ10.61 (s, 1H), 6.75 (s, 1H), 6.50 (s, 1H), 6.39 (s, 1H),2.30 (s, 3H);13C NMR (101 MHz, DMSO-d6)δ159.6, 158.7,155.0, 154.5, 145.6, 144.4, 141.5, 120.1, 115.7, 111.9, 107.8,104.0, 21.3. Anal. Calcd. (%) for C13H7Cl2NO3S: C, 47.58; H,2.15; N, 4.27. Found (%): C, 47.70; H, 2.51; N, 4.27.

Analytical data for compound 5bPale yellow powder;yield: 21%; m.p.: over 220oC.1H NMR (400 MHz,DMSO-d6)δ10.37 (s, 1H), 6.72 (d,J= 1.5 Hz, 1H), 6.45 (d,J= 1.6 Hz, 1H), 2.27 (s, 3H), 1.83 (s, 3H);13C NMR (101 MHz, DMSO-d6)δ160.6, 160.2, 154.7, 153.4, 146.5, 143.2,136.9, 123.1, 120.0, 112.5, 108.0, 105.1, 21.6, 14.4. Anal.calcd (%) for C14H9Cl2NO3S: C, 49.14; H, 2.65; N, 4.09.Found (%): C, 49.12; H, 2.92; N, 4.05.

Analytical data for compound 5cPale yellow powder;yield: 6%; m.p.: over 220oC.1H NMR (400 MHz, DMSO-d6)δ10.38 (s, 1H), 6.73 (d,J= 1.5 Hz, 1H), 6.44 (d,J= 1.5 Hz,1H), 2.27 (s, 3H), 2.28～2.01 (m, 2H), 0.99 (t,J= 7.4 Hz,3H);13C NMR (101 MHz, DMSO-d6)δ159.5, 159.4, 154.5,153.1, 146.0, 142.9, 136.1, 128.0, 120.0, 112.0, 107.6, 104.5,21.7, 21.2, 13.2; HRMS (m/z) calcd. for C15H11Cl2NO3S(M+H)+: 355.9915, found: 355.9907.

Analytical data for compound 5dPale yellow solid;yield: 51%; m.p.: 209～211oC.1H NMR (400 MHz,DMSO-d6)δ10.58 (s, 1H), 6.84～6.80 (m, 1H), 6.57～6.51(m, 1H), 2.30 (s, 3H);13C NMR (101 MHz, DMSO-d6)δ155.1 (d,J= 6.2 Hz), 153.8 (d,J= 29.9 Hz), 153.2, 151.1 (d,J= 2.7 Hz), 146.0, 142.9 (d,J= 2.6 Hz), 141.6 (d,J= 253.5 Hz), 121.8 (d,J= 13.6 Hz), 121.0, 112.4, 107.8, 102.9, 21.1;19F NMR (376 MHz, CD3OD)δ–132.5; HRMS (m/z) calcd.for C13H6Cl2FNO3S (M+H)+: 345.9507, found: 345.9506.

Analytical data for compound 5eColorless crystal;yield: 56%; m.p.: over 220oC.1H NMR (400 MHz,DMSO-d6)δ10.69 (s, 1H), 6.81～6.80 (m, 1H), 6.54～6.49(m, 1H), 2.30 (s, 3H);13C NMR (101 MHz, DMSO-d6)δ158.2, 155.9, 154.6, 152.5, 145.9, 144.5, 138.6, 119.9, 119.1,112.5, 107.8, 104.8, 21.3; HRMS (m/z) calcd. for C13H6Cl3NO3S (M+H)+: 361.9212, found: 361.9208.

2. 3 Structure determination

The colorless crystal of the title compound 5e with dimensions of 0.80mm × 0.50mm × 0.20mm was cultured from methanol and selected for X-ray diffraction analysis.The data were collected on a SuperNova Single Crystal diffractometer equipped with mirror-monochromatic MoKαradiation (λ= 0.71073 Å) with anωscan mode at 130.5(4) K.In the range of 3.01°≤θ≤25.02°, a total of 8308 reflections were collected with 2428 unique ones (Rint= 0.0432), of which 2258 were observed withI> 2σ(I) for refinements.Using Olex2[20], the structure was solved with the XS[21]structure solution program by direct methods and refined with the ShelXL[22]refinement package using least-squares minimization. All of the non-hydrogen atoms were located with successive difference Fourier syntheses. The hydrogen atoms were added according to the theoretical models. The final full-matrix least-squares refinement converged atR=0.0408,wR= 0.1056 (w= 1/[σ2(Fo)2+ (0.0483P)2+ 9.6548P],whereP= (Fo2+ 2Fc2)/3),S= 1.074, (Δρ)max= 1.64, (Δρ)min= –0.56 e/Å3and (Δ/σ)max= 0.001.

2. 4 In vitro fungicidal activity determination

Fungicidal activities of the target compounds 5 were evaluated at 25 µg/mL according to the previously established procedures[23]. Nine representative fungi,A. s:Alternaria solani;B. c:Botrytis cinerea;C. a:Cercospora arachidicola;G. z:Gibberella zeae;P. i:Phytophthora infestans(Mont)de Bary;P. p:Physalospora piricola;P. s:Pellicularia sasakii,R. s:Rhizoctonia solani;andS. s:Sclerotinia sclerotiorum, were chosen. The commercial coumarin fungicide osthole was chosen as positive control.Any compounds with inhibition over 85% were chosen for further median effective concentration (EC50) determination according to reported procedures[23].

2. 5 In vivo fungicidal activity test

The protective activity of compounds 5 against cucumber downy mildew (Pseudoperonospora cubensis(Berk. & Curt.)Rostov.), cucumber gray mold (B. cinereaPers. ex Fr.),cucumber powdery mildew (Sphaerotheca fuliginea) and rice sheath blight (R. solaniKuhn) were determined by pot bioassay according to reported procedures[24]in Shenyang Research Institute of Chemical Industry (Shenyang, China) at a concentration of 200 µg/mL respectively. Any compounds with inhibition over 60% were further tested at lower concentration.

3 RESULTS AND DISCUSSION

As shown in Scheme 1, the target compound 5 was synthesized by a Pechmann condensation reaction with acceptable yields. Their structures were characterized by1H NMR,13C NMR,19F NMR, HRMS and/or elemental analyses.The crystal structure of compound 5e cultured from methanol and shown in Fig.2 was confirmed by X-ray diffraction.

The selected bond lengths, bond angels and torsional angels are shown in Tables 1 and 2. All bond lengths and bond angles within coumarinyl substructure appear to be normally relative to the closely related compounds in literature[25]. Meanwhile, bond lengths and bond angles within the isothiazole ring agree well with the values reported[26]. The torsional angles (all close to 180° or 0°) and measured dihedral angles between benzene and pyranone(2.9°) indicated the coplanar whole coumarinyl. Each molecule has a coumarinyl and an isothiazole moiety with a measured dihedral angle of 92.2° between these two moieties.However, when the isothiazole was substituted by benzene,the corresponding dihedral angle became 126.4° with the fungicidal activity decreasing[8,25]. In this case, we believed that increasing the dihedral angle between coumarinyl and isothiazole substructures could improve its fungicidal activity.Classical intermolecular O–H· O hydrogen bond with bond length of 1.96 Å and bond angle of 174.88° was observed between the hydroxyl and carbonyl groups in the target molecule. There is noπ-πinteraction due to the large distance between adjacent coumarin or its adjacent isothiazole.However, the molecules are linked together by a hydrogen bond nearly parallel to theabplane to form infinite ribbons along theaaxis, which were stacked alternately along thecaxis (Fig.3).

Table 1. Selected Bond Lengths (Å) and Bond Angles (°) for Compound 5e

Table 2. Selected Torsional Angles (°) for Compound 5e

Fig.2. X-ray crystal structure of compound 5e

Fig.3. One-dimensional chain structure of 5e. The hydrogen bond distance (Å) and angle (°): H(3)···O(2A) 1.96 Å;O(3)–H(3)···O(2A) 175°. Symmetry operation: A =1/2+x, –1/2+y, z

The target compound 5 was evaluated forin vitrofungicidal activities against nine phytopathogenic fungi at a concentration of 25 µg/mL as compared with the positive control osthole. As shown in Table 3, all compounds displayed various degrees of fungicidal activities against the fungi tested. Particularly, compounds 5d & 5e exhibited good to excellentin vitroactivities againstB. cinerea,P. piricola,R.solaniandS. sclerotiorumat a concentration of 25 µg/mL with inhibition over 85%. To better compare the fungicidal potency of 5d & 5e, their EC50values againstB. cinerea,P.piricola,R. solaniandS. sclerotiorumwere determined. As shown in Table 4, 5d and 5e showed lower EC50values falling between 0.50 and 4.85 µg/mL against the fungi tested.They were more active than osthole (EC50values between 7.38 and 74.59 µg/mL).

Table 3. In Vitro Fungicidal Activities (Inhibition Rate /%) of Compound 5a

Table 4. EC50 Values of Compound 5 with Inhibition over 85% at 25 µg/mL In Vitro

In vivoprotective activities of compounds 5d & 5e againstP. cubensis(Berk. & Curt.) Rostov.,B. cinereaPers. ex Fr.,S.fuliginea, andR. solaniKuhnat a concentration of 200µg/mL were tested with the data listed in Table 5. Both of them showed no activity againstB. cinereaandS. fuliginea.Compound 5d exhibited good activity againstR. solaniwith 70% of inhibition. Meanwhile, 5e showed good activity againstP. cubensiswith 75% of efficacy. Even when the concentration lowered to 100 and 50 µg/mL, 5e still had excellent activity againstP. cubensiswith the inhibition of 98%and 95%, respectively.

Table 5. In Vivo Fungicidal Activity of Compound 5 at Different Concentrations

The results of this study (Tables 3～5) suggested that 3,4-dichloroisothiazole-containing coumarins were good fungicidal leads with better activity than that of the positive control ostholein vitroandin vivo, deserving for further study.

Dedicated to the 100th Anniversary of Chemistry at Nankai University.