Facile synthesis and antifungal activity of dithiocarbamate derivatives bearing an amide moiety J. Serb. Chem. Soc. 80 (11) 1367–1374 (2015) UDC 547.496.2–327:542.913:615.282–188 JSCS–4803 Original scientific paper 1367 Facile synthesis and antifungal activity of dithiocarbamate derivatives bearing an amide moiety YU-WEN LI* and SHU-TAO LI School of Chemistry and Pharmacy, Qingdao Agricultural University, Qingdao, P. R. China (Received 14 January, accepted 24 May 2015) Abstract: Two series of novel dithiocarbamate derivatives bearing an amide moiety, 3a–i and 4a–i, were synthesized by a facile method, and the structures of the derivatives were confirmed by elemental analysis and 1H-NMR, 13C- -NMR and high-resolution mass spectrometry (HRMS). Their antifungal acti- vity against five phytopathogenic fungi were evaluated, and the results showed that most of the target compounds displayed low antifungal activity in vitro against Gibberella zeae, Cytospora sp., Colletotrichum gloeosporioides, Alt- ernaria solani, and Fusarium solani at a concentration of 100 mg L-1. How- ever, two compounds, 4f and 4g, exhibited significant activity against A. solani and C. gloeosporioides, respectively. Keywords: antifungal activity; dithiocarbamate derivatives; amide moiety; syn- thesis. INTRODUCTION Plant disease arising from phytopathogenic fungi is one of the major causes of severe losses in agriculture and horticulture crop production worldwide, and poses a threat to global food security.1 In the past years, fungicides have contri- buted enormously to reduce crop loss caused by phytopathogenic fungi. How- ever, the main problem associated with the application of fungicides is the emer- gence of fungicide resistance. Therefore, it is necessary to develop efficient fun- gicides with novel structures to obviate this resistance. Due to their multiple biological profiles, carboxylic acid amide compounds have found application not only in medicinal chemistry but also in pesticide che- mistry, i.e., as insectides,2–4 fungicides,5 herbicides6 and plant growth regul- ators.7 Dithiocarbamate derivatives, on the other hand, have played important roles in medicinal and pesticide chemistry because of their diverse activities.8–11 In pesticide chemistry, dithiocarbamate derivatives have also served as fungi- cides.12 An important strategy for drug discovery has emerged that consists of * Corresponding author. E-mail: ywli@qau.edu.cn doi: 10.2298/JSC150114047L 1368 LI and LI hybridizing two bioactive molecules or pharmacophores to generate a novel class of molecules with a potentially stronger bioactivity profile.13,14 Thus, inspired by the biological importance of carboxylic acid amides and dithiocarbamates as fun- gicides in the pesticide field, herein, the synthesis of novel carboxylic acid amide–dithiocarbamate hybrids 3a–i and 4a–i and their antifungal activities are reported. RESULTS AND DISCUSSION Chemistry In this study, the starting materials, 2-chloroacetamides 2a–i, were prepared according to the literature15 with some modifications (Scheme 1). The inter- mediates 2a–g were synthesized by treatment of 2-chloroacetyl chloride with 1a–g, respectively, in a mixture of acetic acid and a saturated solution of sodium ace- tate. However, this procedure was unsuitable for the synthesis of intermediates 2h and 2i due to difficulties encountered in the separation and purification of the liquid 2h and 2i from the liquid mixture of HOAc and NaOAc. Thus, inter- mediates 2h and 2i were synthesized by reaction of 2-chloroacetyl chloride with 1h and 1i, respectively, in the presence of triethylamine as acid-scavenger and dichloromethane as solvent. With intermediates 2a–i available, the target com- pounds 3a–i and 4a–i were synthesized. However, conventional synthesis of dithiocarbamates involves costly and toxic chemical reagents, such as thiophos- gene.16 Current strategies could partially alleviate the expense and toxicity for the synthesis by several one-pot syntheses via the reaction of carbon disulfide with amine and alkyl halides or acrylates.17 Still, several drawbacks of these one- -pot procedures remained inevitable, such as the use of strong bases, high reac- ClCH2COCl + H2NR NaOAc HOAc ClCH2CONHR ClCH2COCl + YHN CH2Cl2 (C2H5)3N N Y O Cl 1a-1g 2a-2g 1h-1i 2h-2i 2h: Y = CH2 2i: Y = O NO2 F Cl Cl OH a b c d e f g R = Scheme 1. Synthesis of intermediates 2a–i. SYNTHESIS AND ANTIFUNGAL ACTIVITY OF DITHIOCARBBAMATES 1369 tion temperatures, long reaction time, and harmful organic solvents.18 To circum- vent the drawbacks associated with the previous procedures for the preparation of dithiocarbamates, the target compounds 3a–i and 4a–i were prepared according to Scheme 2. Scheme 2. Synthesis of target compounds 3a–i and 4a–i. As illustrated in Scheme 2, the synthesis method was improved such that the reactions of carbon disulfide, triethylamine and piperidine or morpholine were realized at 0 °C to obtain intermediates 3 and 4, respectively, as white solids. To the thus-obtained intermediates 3 and 4, absolute ethanol was added in situ, lead- ing to the respective suspensions of 3 and 4 in absolute ethanol. Subsequently, reaction of the suspended 3 and 4 in ethanol with the intermediates 2a–i at 50 °C led to the generation of the target compounds 3a–i and 4a–i, respectively. Upon completion of the reaction, 3a–i and 4a–i were in situ precipitated by cooling the corresponding reaction mixture down to 0 °C and collected by filtration. Since more or less 3a–i or 4a–i remained in the mother liquor, more sequential steps were indispensable to recover the residual 3a–i and 4a–i from their correspond- ing mother liquor. Firstly, evaporation of the mother liquor to dryness afforded a solid mixture containing the side-product triethylamine hydrochloride and resi- dual 3a–i and 4a–i, respectively. Furthermore, given that triethylamine hydro- chloride is not soluble in ether solvents while 3a–i and 4a–i are, the addition of 2-methyltetrahydrofuran to the thus-obtained solid mixtures left a white preci- pitate of triethylamine hydrochloride, a useful reagent in drug synthesis,19 that was removed by filtration. The filtrates were then concentrated under reduced pressure to give the respective residuals 3a–i and 4a–i. Finally, recrystallization of the residuals 3a–i and 4a–i from 95 % ethanol led to the desired target com- pounds. The notable advantages of the present procedure over previous ones are that it is easy to conduct in terms of generation, separation, and purification of the target compounds 3a–i and 4a–i and is readily performed in one pot with only 1370 LI and LI one solvent by modulating the temperature in the range from 50 to 0 °C. In addition, taking the recovered 3a–i and 4a–i into account, the overall yields of the products were almost quantitative. Antifungal activity: in vitro screening of compounds 3a–3i and 4a–4i All the newly synthesized target compounds 3a–i and 4a–i were evaluated in vitro for their antifungal activity against five phytopathogenic fungi, i.e., Gibber- ella zeae, Cytospora sp., Colletotrichum gloeosporioides, Alternaria solani and Fusarium solani at concentration of 100 mg L–1. As summarized in Table I, most target compounds displayed low antifungal activities against these five phyto- pathogenic fungi at the indicated concentration with exception of compounds 4f and 4g. Compound 4f displayed 77.26 % inhibition of A. solani, while compound 4g exhibited 74.87 % inhibition of C. gloeosporioides at a concentration of 100 mg L–1. To understand further the role of different groups of the compounds in conferring the antifungal activity, it is necessary to compare their structures. Structurally, compounds 3a–i and 4a–i are derived from same scaffold but with different substituents, leading to the difference in the antifungal activity. Gen- erally, compounds 4a–4i were superior to the corresponding compounds 3a–i in terms of their antifungal activity, suggesting that the presence of morpholinyl group in 4a–i conferred better antifungal activity than the corresponding piper- TABLE I. Fungicidal activities of the target compounds 3a–i and 4a–i at a concentration of 100 mg L-1; Hy – hymexazol Compound Antifungal activity (Inhibition rate, %) G. zeae A. solani C. gloeosporioides Cytospora sp. F. solani 3a 3b 33.41 30.32 24.52 23.48 20.25 24.97 5 42.13 26.75 24.46 3c 34.42 21.47 14.81 36.29 34.79 3d 39.52 41.23 38.65 39.49 36.13 3e 21.78 30.14 23.41 31.73 35.47 3f 43.97 46.13 40.94 49.78 41.64 3g 40.27 43.94 39.08 46.66 39.35 3h 19.24 23.56 34.75 28.91 14.34 3i 27.88 29.76 30.51 32.14 30.09 4a 41.77 36.72 38.13 32.43 38.19 4b 46.18 39.76 30.67 51.09 32.08 4c 40.07 28.94 34.96 46.81 40.11 4d 47.14 45.97 47.08 52.13 43.93 4e 23.93 35.46 26.43 33.19 39.86 4f 58.11 77.26 59.04 57.89 53.98 4g 62.93 51.67 74.87 60.09 56.89 4h 30.08 32.77 47.69 38.06 45.77 4i 31.18 35.26 43.22 39.18 34.17 Hy 90.56 82.15 53.78 46.69 79.23 SYNTHESIS AND ANTIFUNGAL ACTIVITY OF DITHIOCARBBAMATES 1371 idinyl group in compounds 3a–i. More interestingly, compounds 4f and 4g, bearing two substituents on the benzene ring, displayed significantly higher anti- fungal activities relative to the compounds with a single substituent on the ben- zene ring. In addition, the compounds with fluorine substituent on the benzene ring regardless of the substituent on the carbamic moiety, such as 3d and 4d, showed higher antifungal activities (although not dramatic) compared to other corresponding compounds without fluorine substituent on the benzene ring. Although most of the target compounds display low inhibition rate against myc- elia growth of these five tested fungi at concentration of 100 mg/L, target com- pounds 4f–g could be potential lead structures for further discovery of novel antifungal agrochemicals. EXPERIMENTAL Chemistry All the employed chemicals were obtained from Qingdao Justness Reagent Company (China) and used without further purification. The melting points were measured using a WRS-1B digital melting point apparatus. The 1H-NMR spectra were recorded on a Bruker DRX-400 Advance spectrometer at 400 MHz using TMS as an internal standard. The physical, analytical and spectral data of the synthesized compounds are given in the Supplementary material to this paper. General procedure for the synthesis of compounds 2a–g A substituted aniline (34.3 mmol) was added to 12.5 mL of a saturated solution of sodium acetate, followed by 12.5 mL acetic acid. The suspension was cooled to 0 °C, and 2- chloroacetyl chloride (34.4 mmol, 2.75 ml) was added dropwise to the suspension at ≤5 °C. During the addition of 2-chloroacetyl chloride, the suspension dissipated and the mixture clarified. Before the addition of 2-chloroacetyl chloride was complete, a white precipitate began to form. Upon completion of the addition, the heterogeneous mixture was brought to 25 °C and stirred at room temperature for 2 h. The white precipitate was filtered, washed with distilled water (2×5 mL) and dried under vacuum to afford the crude product. Recrystal- lization of crude 2a–g from absolute ethanol gave the desired 2a–g, respectively. The phys- ical, analytic and spectral data of 2a–g are summarized in the Supplementary material to this paper. General procedure for synthesis of compounds 2h and 2i To a stirred solution of triethylamine (17.2 mmol, 2.5 mL) and 17.2 mmol piperidine 1h (or morpholine 1i) in 10 mL CH2Cl2, 2-chloroacetyl chloride (17.2 mmol, 1.38 mL) was added dropwise at 0 °C. Upon completion of the dropwise addition, the solution was brought to room temperature and stirred for 2 h. Subsequently, the resulting solution was diluted with 20 mL CH2Cl2 and successively washed with 20 mL water and 20mL brine. The CH2Cl2 layer was separated and concentrated under reduced pressure to give crude 1h (or 1i) which was further purified by chromatography to afford the desired 1h (or 1i) as a yellowish oil. The physical, analytical and spectral data of 2h and 2i are summarized in the Supplementary material to this paper. 1372 LI and LI General procedure for the synthesis of compounds 3a–i and 4a–i To a solution of triethylamine (17.2 mmol, 1.5 mL) and 17.2 mmol piperidine 1h (or morpholine 1i), carbon disulfide (18.9 m mol, 1.2 mL) was added dropwise at 0 °C to form a white solid 3 (or 4). Upon completion of the addition, 15 mL absolute ethanol was added to form a slurry of the white solid 3 (or 4), to which 17.2 mmol respective intermediate 2a–i was added. The mixture was heated to 50 °C to afford a clear solution. The clear solution was kept stirring at 50 °C for 3 h and then cooled to 0 °C. The cooled solution was kept for 2 h at 0 °C and the precipitate of 3a–i (or 4a–i) was collected by filtration and the corresponding filtrate was evaporated to dryness leading to a solid mixture of triethylamine hydrochloride and residual 3a–i (or 4a–i). Then, the addition of 2-methyltetrahydrofuran to the thus obtained solid mixture led to triethylamine hydrochloride by filtration. The corresponding filtrate was concentrated under vacuum to give the residual 3a–i (or 4a–i). Recrystallization of thus obtained residual 3a–i (or 4a–i) from 95 % ethanol led to the desired 3a–i (or 4a–i). Taking the recovered 3a–i and 4a–i into account, the overall yield of 3a–i and 4a–i was almost quantitative. The physical, analytical and spectral data for 3a–i and 4a–i are summarized in the Supplementary material to this paper. Antifungal activity Antifungal activities of target compounds 3a–i and 4a–i were evaluated in vitro against five phytopathogenic fungi (Gibberella zeae, Cytospora sp., Colletotrichum gloeosporioides, Alternaria solani and Fusarium solani) using the mycelium growth rate method.20,21 All the fungi were provided by the Qingdao Agricultural University. The strains were retrieved from the storage tube and cultured for 2 weeks at 25 °C on potato dextrose agar (PDA). The antifungal activity was assessed as follows: PDA medium was prepared in flasks and sterilized. The target compounds 3a–i and 4a–i were dissolved in acetone prior to mixing with molten agar at 55 °C, and the concentration of the target compounds 3a–i and 4a–i were 100 mg L-1. The PDA medium was then poured into sterilized Petri dishes. The five fungi were incubated in PDA at 25 °C for 7 days to obtain new mycelium for the fungicidal assays, and a mycelia disk of 4 mm in diameter cut from culture medium was picked up with a sterilized inoculation needle and inoculated in the centre of the PDA Petri dishes. The inoculated Petri dishes were incubated at 25 °C ℃ for 3–4 days. Acetone was used as the control, and the commercially available agricultural fungicide hymexazol served as the positive control. Each compound was measured in three replicates, and each colony diameter of the three replicates was measured 4 times by the cross bracketing method. The inhibition rate (IR) was calculated according to the following formula: ( )% 100 4 − = − C T IR C where C is the average diameter of mycelia in the blank test and T is the average diameter of mycelia on PDA treated with the target compounds. CONCLUSIONS In summary, a series of dithiocarbamates bearing an amide moiety 3a–i and 4a–i were synthesized in almost quantitative yield by a facile and convenient pro- cedure. Especially, the synthesis, separation, and purification by recrystallization could be conducted in one-pot and in same medium by regulating the temperature from 50 to 0 °C. The results of the bioassay indicated that most target compounds SYNTHESIS AND ANTIFUNGAL ACTIVITY OF DITHIOCARBBAMATES 1373 displayed low activities against G. zeae, Cytospora sp., C. gloeosporioides, A. solani and F. solani at a concentration of 100 mg L–1. However, the compounds 4f and 4g gave significant inhibition rates against A. solani and C. gloeospo- rioides, respectively, at a concentration of 100 mg L–1. Generally, the antifungal activities of compounds 4a–i, which have a morpholinyl substituent, are superior to the corresponding compounds 3a–i with the piperidinyl substituent. Addition- ally, the number of substituents on benzene ring influenced the antifungal activity as evidenced by the fact that the compounds bearing two substituents on the ben- zene ring, such as 3f and 3g and 4f and 4g, displayed better antifungal activities than the compounds with a single substituent on the benzene ring. Although most of the target compounds displayed low inhibition rates against mycelia growth of the five tested fungi at concentrations of 100 mg L–1, the target compounds 4f and 4g could be potentially leading structures for further discovery of novel anti- fungal agrochemicals. SUPPLEMENTARY MATERIAL Physical, analytical and spectral data of the synthesized compounds are available electro- nically from http://www.shd.org.rs/JSCS/, or from the corresponding author on request. Acknowledgement. This work was financially supported by the Natural Science Found- ation of Shandong Province (ZR2011BL003). И З В О Д ЈЕДНОСТАВНА СИНТЕЗА АМИДНИХ ДЕРИВАТА ДИТИОКАРБАМАТА И ЊИХОВА АНТИФУНГАЛНА АКТИВНОСТ YU-WEN LI и SHU-TAO LI School of Chemistry and Pharmacy, Qingdao Agricultural University, Qingdao, P. R. China Синтетисана је серија нових деривата дитиокарбамата који садрже амидну групу, 3a–i и 4a–i, применом олакшаног поступка и добијеним дериватима је структура потвр- ђена 1H-NMR, 13C-NMR спектроскопијом, елементалном анализом и масеном спектро- метријом високе резолуције (HRMS). Испитана је антифунгална активност према пет фитопатогених гљива. Резултати су показали да већина деривата показује in vitro актив- ност према Gibberella zeae, Cytospora sp., Colletotrichum gloeosporioides, Alternaria solani и Fusarium solani при концентрацији 100 mg L-1. Једињења 4f и 4g показују значајну актив- ност према A. solani и C. gloeosporioides, редом. (Примљено 14. јануара, прихваћено 24. маја 2015) REFERENCES 1. S. Savary, P. S. Teng, L. Willocquet, F. W. Nutter, Annu. Rev. Phytopathol. 44 (2006) 89 2. J. C. Yang, J. B. Zhang, B. S. Chai, C. L. Liu, Chin. J. Pestic. Sci. 47 (2008) 6 3. X. T. Xu, F. Leng, W. G. Duan, G. S. Lin, Q. J. Mo, W. K. Wang, Chem. Bulletin 75 (2012) 463 (in Chinese) 4. G. 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