Synthesis, Antifungal Activity and 3D-QSAR Study of Novel 1,3,4-Thiadiazole-Thiourea Compounds Containing gem-Dimethylcyclopropane Ring

ZOU Renxuan, DUAN Wengui, LIN Guishan, LI Baoyu, CUI Yucheng

(School of Chemistry and Chemical Engineering,Guangxi University,Nanning 530004,China)

Abstract：A series of novel 1,3,4-thiadiazole-thiourea compounds containing gem-dimethylcyclopropane ring 8a-8t were designed and synthesized by multi-step reactions in search of novel antifungal molecules.Structures of all target compounds were characterized by FT-IR, 1H NMR, 13C NMR, ESI-MS and elemental analysis.The antifungal activities of the target compounds were preliminarily evaluated.The bioassay results revealed that, at 50 mg/L, the inhibitory rate of compound 8l(R=m-BrC6H4)against P.piricola was 79.5%, which was better than that of the positive control chlorothalonil.In order to design more effective antifungal compounds against P.piricola, analysis of the three-dimensional quantitative structure-activity relationship(3D-QSAR)was performed using the comparative molecular field analysis(CoMFA)method.The reasonable and effective 3D-QSAR model(r2=0.991, q2=0.514)was established.

Key word：3-carene;gem-dimethylcyclopropane ring;1,3,4-thiadiazole-thiourea;antifungal activity;3D-QSAR

3-Carene, a natural bicyclic monoterpene mainly found in various essential oils and turpentine oils[1-3], is one of the rare compounds containing a three-membered gem-dimethylcyclopropane ring in the nature[4-5].3-Carene exhibited various biological activities, such as anti-inflammatory[1], anticancer[6], antimicrobial[7-8], fumigant[9], antioxidant[10-11], and semiochemical properties[12].It can serve as starting material to synthesize bioactive compounds containing gem-dimethylcyclopropane ring[13].Cyclopropane derivatives play important roles in the fields of medicine and pesticides.They have received considerable attention because of their broad-spectrum bioactivities, such as antimicrobial[14], antitumor[15], insecticidal[16], anti-inflammatory[17], and herbicidal[18]activities.On the other hand, compounds including 1,3,4-thiadiazole moiety, display insecticidal[19], antiviral[20], antifungal[21], and antitumor activities[22].Besides, compounds with thiourea scaffold hold diverse pharmacological properties such as insecticidal[23], herbicidal[24-25], antifungal[26], antitubercular[27], and antimicrobial[28]activities.

Herein, taking the above-mentioned clues into consideration along with in continuation of our interest in the works on natural product-based bioactive compounds[29-31], a series of 1,3,4-thiadiazole-thiourea compounds containing gem-dimethylcyclopropane ring were designed and synthesized by integrating bioactive 1,3,4-thiadiazole and thiourea moieties into the skeleton of gem-dimethylcyclopropane ring converted from 3-carene.All target compounds were characterized by means of FT-IR,1H NMR,13C NMR, ESI-MS and elemental analysis.Their antifungal activities were evaluated as well.Furthermore, the quantitative structure-activity relationship was found out through 3D-QSAR analysis of the antifungal activity of the target compounds againstP.piricola.The reasonable and effective 3D-QSAR model provided a theoretical basis for the subsequent optimization of the series of compounds and the discovery of new potential antifungal agents with higher activity.

1 Materials and Methods

1.1 Materials and instruments

3-Carene(GC purity 98%, racemate)was purchased from Wuzhou Pine Chemicals Co., Ltd..Other reagents were purchased from commercial suppliers and used as received.1H NMR and13C NMR spectra were carried out in CDCl3on Avance III HD 500 and 600 MHz spectrometer with TMS as the internal standard(Bruker Co., Ltd., Zurich Switzerland).The HPLC analysis were performed on a Waters 1525 instrument equipped with chromatographic column SunFire C18(4.6 mm×150 mm)and 2998PDA detector.IR spectra were recorded on a Nicolet iS50 FT-IR spectrometer(Thermo Scientific Co., Ltd., Madison, WI, USA)by means of KBr pelleting.MS spectra were measured by means of the electrospray ionization(ESI)method on TSQ Quantum Access MAX HPLC/MS instrument(Thermo Scientific Co., Ltd., Waltham, MA, USA).Melting points were detected on a MP420 automatic melting point apparatus(Hanon Instruments Co., Ltd., Jinan, China).They were not corrected.The elemental analysis was measured by 2400 Series II CHNS/O Elemental Analyzer(PerkinElmer Co., Ltd., Waltham, MA, USA).

1.2 Synthesis of 2-(2-(2,2-dimethyl-3-propylcyclopropyl)acetyl)hydrazine-1-carbothioamide(5)

According to our previous report[4]compounds2,3and4were prepared.As illustrated in Fig.1, under anhydrous atmosphere, compound4(7.3 g, 35.6 mmol)was dissolved in THF(75 mL).Thiosemicarbazide(7.1 g, 77.9 mol)was then added and the reaction mixture was aged at room temperature for several hours.After the reaction was complete, the solution was quenched with aqueous NaHCO3(50 mL)and extracted with EtOAc(100 mL).The organic phases were washed with aqueous NaHCO3(50 mL), then brine, dried over anhydrous sodium sulfate, and evaporated in vacuo.Then, the crude product was purified by silica gel column chromatography(V(ethyl acetate)∶V(petroleum ether)=1∶2)to obtain intermediate5.

Fig.1 Synthesis of 1, 3, 4-thiadiazole-thiourea compounds containing gem-dimethylcyclopropane ring 8a-8t

1.3 Synthesis of N-(5-((2,2-dimethyl-3-propylcyclopropyl)methyl)-1,3,4-thiadiazol-2-yl)acetamide(6)

According to the report[32], under anhydrous atmosphere, acetyl chloride(17 mL, 239 mmol)was added dropwise to compound5(4.1 g, 17.0 mmol)under stirring.Then the reaction mixture was stirred at room temperature for 48 h.After completion of the reaction, deionized water(5 mL)was added to damage the unreacted acetyl chloride and acidified with aqueous NaHCO3to pH 7.5.Afterwards, the mixture was extracted with 50 mL EtOAc several times.The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, and evaporated in vacuo to obtain the intermediate6.

1.4 Synthesis of 5-((2,2-dimethyl-3-propylcyclopropyl)methyl)-1,3,4-thiadiazol-2-amine(7)

To a mixture of intermediate6(3.2 g, 12 mmol)and anhydrous ethanol(20 mL), a solution of NaOH(0.80 g, 20 mmol)in water(10.0 mL)was added slowly at room temperature under stirring.Then the reaction mixture was stirred at 110 ℃ for 24 h.TLC was used to monitor the reaction process.After completion of the reaction, the solution was extracted with EtOAc several times.The organic phases were dried over anhydrous sodium sulfate, and evaporated in vacuo to obtain the intermediate7.

1.5 General procedure for synthesis of compounds 8a-8t

To a mixture of compound7(0.23 g, 1 mmol)and sodium hydroxide(0.04 g, 1 mmol)in acetonitrile(20 mL), substituted isothiocyanate(1.2 mmol)was added and stirred vigorously at room temperature.Then, the reaction mixture was heated to 90 ℃ and stirred for 1 h.After the reaction was finished, the residual sodium hydroxide was removed through filtration.The filtrate was concentrated in vacuo and the residue was purified by silica gel chromatography to afford the target compounds8a-8t.

1.6 Antifungal activity test

Theinvitroantifungal activities of all the target compounds8a-8tagainst eight fungi including cucumber fusarium wilt(Fusariumoxysporumf.sp.cucumerinum), apple ring rot(Physalosporapiricola), peanut speckle(Cercosporaarachidicola), watermelon anthracnose(Colleterichumorbiculare), wheat scab(Gibberellazeae), tomato early blight(Alternariasolani), rice sheath blight(Rhzioeotniasolani), and corn southern leaf blight(Bipolarismaydis)were performed at the concentration of 50 mg/L[33].Commercially available microbicide chlorothalonil was used as a positive control.The relative inhibition of the circle of mycelium compared with the blank assay was calculated by use of the equation:

I=(DCK-DT)/DCK×100%

Where:DCK—the extended diameter of the circle of mycelium during the blank assay, mm;DT—the extended diameter of the circle of mycelium during testing, mm.

1.7 3D-QSAR analysis

Molecular modeling was performed using SYBYL-X 2.1.1 software(Tripos, Inc., St.Louis, MO, USA).Complete conformational optimization of each structure was performed using a conjugate gradient procedure based on the Tripos force field and Gasteiger-Hückel charges.Compound8l(R=m-BrC6H4)was used as a template to build the other molecular structures.According to the common skeleton marked with an asterisk shown in Fig.2, sixteen optimized molecules containing benzene ring were superimposed(Fig.3).The values of the CoMFA field were automatically calculated by the SYBYL/CoMFA routine.A predictive 3D-QSAR model was established using CoMFA descriptors as independent variables and ED values as dependent variables.The cross-validation with the leave-one-out mothed was carried out to obtain the cross-validatedq2and the optimal number of components.Then, a non-cross-validation analysis under the optimal number of components was performed.The modeling capability was indicated by the correlation coefficient squaredr2, and the prediction capability was judged by ther2andq2.

Fig.2 The asterisk skeleton of title compounds

Fig.3 Superposition modes of compounds

2 Results and Discussion

2.1 Synthesis and characterization

The synthetic route of target compounds8a-8tis illustrated in Fig.1, the intermediates2,3and4were prepared by our previously reported method[4].Intermediate5was prepared by the N-acylation reaction of intermediate4with thiosemicarbazide.In the conversion process of intermediates5to7by cyclization, excessive acetyl chloride was used to produce intermediate6first.According to the report[32], the existence of acetyl chloride could assist the formation of 1, 3, 4-thiadiazole, and retain the three-membered ring, with the proposed reason that an almost anhydrous environment could avoid the generation of proton.However, the excessive acetyl chloride would cause N-acylation reaction of the amino group, resulting in intermediate6.Then, intermediate6was hydrolyzed under strong alkaline condition to give intermediate7.Finally, twenty target compounds8a-8twere synthesized by the nucleophilic addition of intermediate7with a series of substituted isothiocyanate compounds.

The intermediates5-7and the target compounds8a,8c,8e,8g,8j,8l,8m,8r, and8twere characterized as examples, and the characterization data of the other target compounds were shown in the auxiliary materials.

2.2 Antifungal activity

Theinvitroantifungal activities of the target compounds8a-8tagainstF.oxysporumf.sp.cucumerinum,C.arachidicola,C.orbiculare,P.piricola,G.zeae,A.solani,R.solani, andB.maydiswere preliminarily assayed at the concentration of 50 mg/L, with the commercial antifungal agent chlorothalonil used as the positive control.The results are listed in Table 1.

Table 1 Antifungal activity of 1, 3, 4-thiadiazole-thiourea compounds containing gem-dimethylcyclopropane ring 8a-8t(50 mg/L)

It was found that, all target compounds exhibited certain antifungal activity against the eight tested fungi at the specified concentration.In general, all target compounds exhibited the best activity againstP.piricolawith an average inhibition rate of 30.8% andF.oxysporumf.sp.cucumerinumcame second with an average inhibition activity of 28.7%.Compounds8a(R=C6H5)and8c(R=m-CH3C6H4)showed the same antifungal activity of 71.8% againstP.piricola.Besides, compound8l(R=m-BrC6H4)displayed antifungal activity of 79.5%, better than that of the positive control(chlorothalonil)with inhibition rate of 75.0%.In order to investigate the structure-activity relationship, 3D-QSAR study was subsequently carried out.

2.3 CoMFA analysis

The 3D-QSAR analysis of the antifungal activity againstP.piricolaof the target compounds was carried out by the CoMFA method.According to a report in the literature[34], the antifungal activity againstP.piricolawas expressed in terms of activity factor(ED)by the formula:

ED=lg{I/[(100-I)×MW]}

Sixteen target compounds in the training set and two compounds in the test set are presented in Table 2.

Table 2 ED values of experimental and predicted activities

A predictive 3D-QSAR model with the value of non-validated correlational coefficient(r2=0.991), the Fischer ratio(F=171.581), standard error of estimate(S=0.067)and the cross-validated correlational coefficient(q2=0.514)was established.As shown in Fig.4, the Scatter plot of predictedEDvaluesvsexperimentalEDvalues was presented.All data were concentrated near theX=Yline.It indicated that the 3D-QSAR model was reasonable and effective.

Fig.4 Predicted ED value of CoMFA model vs experimental ED value

The electrostatic and steric contribution maps of CoMFA are shown in Fig.5.The contribution rate of the steric field was 75.3% while the electrostatic field was 24.7%.This revealed that the steric field was the major contributor to the increase in the antifungal activity againstP.piricola.In Fig.5(a), there were some green regions located around the 2, 3, or 4-position of the benzene ring.This indicated that the bulky groups at these positions were favorable to enhance the antifungal activity.For example, compounds8c(R=m-CH3C6H4),8g(R=m-FC6H4)and8l(R=m-BrC6H4)displayed higher antifungal activity.

a.steric; b.electrostatic

In Fig.5(b), the electrostatic field contours were displayed in two distinguishing colors.The blue area indicated that the introduction of electron-donating groups was beneficial to improve activity.The red area indicated that the introduction of electron-withdrawing groups was beneficial to improve activity.Therefore, the introduction of electron-donating groups at the 2-position and electron-withdrawing groups at the 3-position of benzene rings were both favorable for antifungal activity.For instance, compounds8g(R=m-FC6H4),8l(R=m-BrC6H4)and8n(R=m-NO2C6H4)exhibited better antifungal activity than that of8e(R=m-OCH3C6H4).

Based on 3D-QSAR analysis above, a novel compound(Fig.6)was designed and the predictedEDvalue was calculated by the established CoMFA model.As a result, the designed compound(ED=-1.677)showed potential excellent antifungal activity with inhibition rates of 89.8%, which deserved further study.

Fig.6 Newly designed molecule of 1-(2-(tert-butyl)phenyl)-3-(5-((2,2-dimethyl-3-propylcyclopropyl)methyl)-1,3,4-thiadiazol-2-yl)thiourea

3 Conclusions

Twenty novel 1, 3, 4-thiadiazole-thiourea compounds containing gem-dimethylcyclopropane ring were designed, synthesized, characterized, and evaluated for their antifungal activities by using the natural product 3-carene as starting material.The bioassay result revealed that, at 50 mg/L, compounds8l(R=m-BrC6H4)exhibited better antifungal activities of 79.5% againstP.piricola.This showed better antifungal activity than that of the commercial fungicide chlorothalonil.Compound8lwas worthy of further investigation.In order to design more effective antifungal compounds againstP.piricola, the 3D-QSAR analysis was carried out using the CoMFA method.A reasonable and effective 3D-QSAR model(r2=0.991,q2=0.514)was established, and a new molecule with modification on phenyl group was designed.

Acknowledgments

The authors are grateful to the State Key Laboratory of Element-Organic Chemistry, Nankai University, China, for the bioassay test.