Fungal diseases of agricultural crops pose a serious threat to high yields and product quality, which leads to significant economic losses. Consumption of food and feed contaminated with fungi and soiled with mycotoxins has a detrimental effect on the health of humans and animals and can even lead to the development of leukemia and the formation of malignant tumors [1]. Fungicidal preparations can reduce the spread of fungal diseases, increase agricultural productivity, and are used in medicine to treat various fungal infections.

Widely used fungicides with systemic effects are azoles. The action mechanism of these drugs is due to a violation of the biosynthesis of ergosterol, the main component of the cell membrane of phytopathogen fungi, which leads to death due to the impossibility of osmotic nutrition. Azole drugs inhibit the stage of C14 demethylation of lanosterol by the CYP51 enzyme of the cytochrome P450 family. 1,2,4-Triazole or imidazole fragments are a pharmacophore group that binds to the target – the iron atom of the heme porphyrin ring [2, 3]. Long-term use of azole drugs leads to the emergence of resistance [4]. As a result, there is a need to search and create new highly active fungicides.

One of the trends in modifying azole drugs is the inclusion of two or more heterocyclic fragments into the structure [3], which makes it possible to increase their activity, reduce toxicity and persistence.

In our previously proposed model [5], azole is a pharmacophore group responsible for binding to the active site of the enzyme (Scheme 1). The second heterocycle can also bind to the heme iron atom, which is typical for sulfur-containing heterocycles, for example, thiazole, which is part of the structure of ravuconazole and isavuconazole [6]. The lipophilic fragment associated with the linker ensures close interaction of the molecule with the apoenzyme.

Scheme
scheme 1

1.

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Many sulfur-containing heterocyclic compounds, including 1,2,3-dithiazole derivatives [7–12], demonstrate a wide range of biological activity. The biological properties of 4-chloro-N-aryl-1,2,3-dithiazol-5-imines, which have antibacterial, herbicidal, antiviral, and antitumor activity, have been most fully studied [13–17]. We have proposed a new model of compounds capable of inhibiting the CYP51 enzyme [6] (Scheme 2). The structure of the compounds includes two heterocycles connected by a linker: an azole, 1,2,3-dithiazol-5-imine, and a lipophilic sterol-emulating fragment. The terminal part of the molecule, represented by 1,2,4-triazole or imidazole rings, is responsible for binding to heme in the active site of cytochrome P450. The linker, consisting of a piperazinylethyl group, allows more precise binding within C14-alpha demethylase, due to the formation of new hydrogen bonds with the amino acids of the enzyme, and provides a certain lability of the molecular skeleton of the target compounds. The 1,2,3-Dithiazole-5-iminium fragment can provide additional binding in the active site, as well as independently bind to heme through the interaction of the electron pair of the sulfur atom in position 2 of the 1,2,3-dithiazole ring.

Scheme
scheme 2

2.

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The azoles model proposed in this study differs in the linearity of the structure (azole–linker–heterocycle–lipophilic group system) from the classical model of azole drugs, where the structure is branched, in particular, an additional heterocycle and a lipophilic group are attached to the linker. Azole drugs with a relatively long piperazine linker and additional heterocycles, for example, itraconazole and posaconazole, have been used in medicine since the 90s as systemic antimycotic drugs [18].

To obtain the target compounds, a three-step synthesis scheme was developed from Appel’s salt (4,5-dichloro-1,2,3-dithiazol-1-ium chloride) [19] (Scheme 3, Table 1).

Scheme
scheme 3

3.

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Table 1. Reaction conditions and yield of compounds 1–4
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A series of 4-chloro-N-aryl-1,2,3-dithiazol-5-imines 1 was prepared by reacting Appel’s salt with various substituted anilines. In the case of p-phenyl-substituted imines, the yield of reaction products was 47–90%; for sterically more hindered di- and trichlorophenyl derivatives, the yield of reaction products significantly decreased (26–66%) (Table 1).

Next, imines 1 were reacted with 1,4-diazabicyclo[2.2.2]octane (DABCO) similarly to the methods described in the literature [20–23]. Presumably, the nucleophilic attack on 4-chloro-1,2,3-dithiazol-5-imines occurs nonselectively, this reaction proceeds via the ANRORC mechanism, which explains the formation of many by-products. As suggested by the authors of the researches [20, 24], the interaction of 4-chloro-N-aryl-1,2,3-dithiazol-5-imines with various amines occurs at the S3 position of the dithiazole ring with ring opening and chlorine elimination. Further, depending on the amine structure, a repeated nucleophilic attack can occur either at the carbon atom of the imino group (path a) or at the carbon atom of the cyano group (path b) with subsequent closure of the 1,2,3-dithiazole ring (Scheme 4).

Scheme
scheme 4

4.

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Intermediate product 2a was obtained in the highest yield of 48%, which is slightly lower than the yield obtained in the synthesis of a similar compound when reacting with unsubstituted aniline [20]. It was revealed that reactions of this type lead to the formation of by-product dithiazole-5-thione (according to the results of high-resolution mass spectrometry), degradation products of the dithiazole ring and tarring, which is accompanied by a significant decrease in yield to 12%.

The resulting 4-[4-(2-chloroethyl)piperazin-1-yl]N-aryl-1,2,3-dithiazol-5-imines 2a–2d were treated with sodium 1,2,4-triazolate or imidazolate to obtain the target compounds 3a–3c and 4a. The reaction with the sodium salt of 1,2,4-triazole proceeds easily, compounds 3a–3c were obtained in 54–86% yield (Table 1). The reaction with sodium imidazolate occurs with a lower yield (30%) and is accompanied by the formation of by-products. This may likely be due to the higher basicity of imidazole compared to 1,2,4-triazole and, therefore, the possibility of opening the 1,2,3-dithiazole moiety by reacting the imidazole with the electrophilic ring sulfur atom.

The resulting compounds 1a–1e, 3a–3c, and 4a were researched in vitro for fungicidal activity against 6 types of phytopathogenic fungi. The tests were carried out according to a well-known method [25]: the effect of compounds on the radial growth of phytopathogens was studied at a concentration of 30 mg/L on potato sucrose agar; the fungicide triadimefon was used as a standard for comparison (Table 2).

Table 2. Fungicidal activity of compounds 1, 3, 4
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According to studies results, some compounds exhibit high fungicidal activity against a number of phytopathogen fungi. It is worth noting that the most active compounds are intermediate 4-chloro-N-aryl-5H-1,2,3-dithiazol-5-imines 1a–1d. We associate the high activity of these compounds with the possible formation of active metabolites – cyanothioanilides, the formation of which is responsible for the antiviral activity of similar compounds [15]. Cyanothioanilides are known to be active against Phytophthora infestans [26].

Thus, a series of 4-[4-(2-azolylethyl)piperazin-1-yl]-N-aryl-5H-1,2,3-dithiazol-5-imines was obtained. The fungicidal activity of both these compounds and the intermediate 4-chloro-N-aryl-5H-1,2,3-dithiazol-5-imines was studied in vitro against 6 species of phytopathogenic fungi. The greatest activity was demonstrated by 4-chloro-N-aryl-5H-1,2,3-dithiazol-5-imines, which are superior in fungitoxicity to the standard triadimefon against 4 types of phytopagens, and 4-chloro-N-(4-chlorophenyl)-5H-1,2,3-dithiazol-5-imine and 4-chloro-N-(4-methoxyphenyl)-5H-1,2,3-dithiazol-5-imine completely inhibit the mycelial growth of Rhizoctonia solani and Sclerotinia sclerotiorum.

EXPERIMENTAL

1H and 13C NMR spectra were recorded on a Bruker 300 MHz pulsed broadband magnetic resonance spectrometer in CDCl3 and DMSO-d6, internal standard—TMS. Mass spectra were obtained on a high-performance liquid chromatograph with a QExactive ThermoScientific high-resolution mass spectrometer in electrospray ionization mode at atmospheric pressure when recording positive ions in the range of 80–750 Da with a resolution of 35 000 (HYPERSILGoldaQ column with a length of 150 mm and an internal diameter of 2.1 mm, mobile phase—acetonitrile–water–formic acid, capillary voltage—4000 V).

Appel’s salt (4,5-dichloro-1,2,3-dithiazol-1-ium chloride) was synthesized similarly to the method described in [18].

General procedure for synthesis of 4-chloro-N-aryl-5H-1,2,3-dithiazol-5-imines 1a–1e. To a mixture of 1.44 mmol of 4,5-dichloro-1,2,3-dithiazol-1-ium chloride (Appel’s salt) and 1.44 mmol of the corresponding aniline in 3 mL of anhydrous methylene chloride was added 2.88 mmol of pyridine in 1 mL of methylene chloride. The reaction mixture was stirred at room temperature in an argon atmosphere for 8 h. The solvent was removed in vacuum, and the residue was separated by flash chromatography (petroleum ether–ethyl acetate, 2 : 1).

4-Chloro-N-(4-chlorophenyl)-5H-1,2,3-ditiazol-5-imine (1a). Yield 47%, mp 105–107°С. 1Н NMR spectrum (CDCl3), δ, ppm: 7.18 d (2H, CHAr, 3J 8.6 Hz), 7.42 d (2H, CHAr, 3J 8.6 Hz).

4-Chloro-N-(4-bromophenyl)-5H-1,2,3-ditiazol-5-imine (1b). Yield 81%, mp 118–120°С. 1Н NMR spectrum (CDCl3), δ, ppm: 7.09 d (2H, CHAr, 3J 8.5 Hz), 7.56 d (2H, CHAr, 3J 8.5 Hz).

4-Chloro-N-(4-methoxyphenyl)-5H-1,2,3-ditiazol-5-imine (1c). Yield 90%, mp 89–90°С. 1Н NMR spectrum (CDCl3), δ, ppm: 3.84 s (3Н, ОСН3), 6.98 d (2H, CHAr, 3J 8.8 Hz), 7.27 d (2H, CHAr, 3J 8.9 Hz).

4-Chloro-N-(2,6-dichlorophenyl)-5H-1,2,3-ditiazol-5-imine (1d). Yield 66%, mp 150–152°С. 1Н NMR spectrum (DMSO-d6), δ, ppm: 7.26 t (1H, CHAr, 3J 7.9 Hz), 7.60 m (2H, CHAr).

4-Chloro-N-(2,4,6-trichlorophenyl)-5H-1,2,3-ditiazol-5-imine (1e). Yield 26%, mp 98–100°С. 1Н NMR spectrum (DMSO-d6), δ, ppm: 7.84 s (2H, CHAr).

General procedure for obtaining of 4-[4-(2chloroethyl)piperazin-1-yl]-N-aryl-1,2,3-ditiazol-5-imines 2a–d. A mixture of 0.2 mmol 4-chloro-N-aryl-5H-1,2,3-dithiazol-5-imine and 0.4 mmol DABCO in 8 mL of chlorobenzene was refluxed for 5–8 h. The reaction mixture was cooled, the solvent was removed in vacuum, the residue separated by column chromatography.

4-[4-(2-Chloroethyl)piperazin-1-yl]-N-(4-chlorophenyl)-1,2,3-ditiazol-5-imine (2a). Yield 48% (eluent—petroleum ether–ethyl acetate, 2 : 1). 1Н NMR spectrum (CDCl3), δ, ppm: 2.68 t (4H, CH2, 3J 4.2 Hz), 2.80 t (2H, CH2,3J 4.2 Hz), 3.63 m (2H, CH2), 3.78 m (4H, CH2), 7.08 d (2H, CHAr, 3J 8.8 Hz), 7.40 d (2H, CHAr, 3J 8.7 Hz). 13С NMR spectrum (CDCl3), δC, ppm: 41.54, 49.06, 53.48, 60.51, 121.66, 130.54, 131.44, 151.55, 158.96, 161.87.

4-[4-(2-Chloroethyl)piperazin-1-yl]-N-(4-bromophenyl)-1,2,3-ditiazol-5-imine (2b). Yield 22% (eluent—petroleum ether–ethyl acetate, 5 : 2). 1Н NMR spectrum (CDCl3), δ, ppm: 2.65 t (4H, CH2, 3J 4.3 Hz), 2.78 t (2H, CH2, 3J 4.3 Hz), 3.60 m (2H, CH2), 3.76 m (4H, CH2), 7.00 d (2H, CHAr, 3J 8.7 Hz), 7.53 d (2H, CHAr, 3J 8.7 Hz). 13С NMR spectrum (CDCl3), δС, ppm: 41.56, 49.10, 53.51, 60.52, 121.93, 132.54, 133.73, 149.61, 158.99, 161.85.

4-[4-(2-Chloroethyl)piperazin-1-yl]-N-(4-methoxyphenyl)-1,2,3-ditiazol-5-imine (2c). Yield 37% (eluent—petroleum ether–ethyl acetate, 3 : 1). 1Н NMR spectrum (CDCl3), δ, ppm: 2.69 t (4H, CH2, 3J 4.2 Hz), 2.81 t (2H, CH2, 3J 4.2 Hz), 3.64 t (2H, CH2, 3J 4.2 Hz), 3.80 m (4H, CH2), 3.85 s (3H, CH3), 7.13 m (4H, CHAr). 13С NMR spectrum (CDCl3), δС, ppm: 41.49, 48.97, 53.47, 55.63, 60.36, 115.44, 121.70, 151.55, 158.92, 159.52, 161.87.

4-[4-(2-Chloroethyl)piperazin-1-yl]-N-(2,6-dichlorophenyl)-1,2,3-ditiazol-5-imine (2d). Yield 12% (eluent—petroleum ether–ethyl acetate, 3 : 1). 1Н NMR spectrum (CDCl3), δ, ppm: 2.71 t (4H, CH2, 3J 4.3 Hz), 2.92 t (2H, CH2, 3J 4.3 Hz), 3.49 m (2H, CH2), 3.84 m (4H, CH2), 7.33 m (3H, CHAr). 13С NMR spectrum (CDCl3), δС, ppm: 41.53, 49.06, 53.51, 60.54, 123.75, 128.69, 131.58, 151.53, 158.92, 161.83.

General procedure for obtaining of 4-[4-(2-azolylethyl)piperazin-1-yl]-N-aryl-5H-1,2,3-ditiazol-5-imines 3a–3c and 4a. To a solution of 0.038 mmol corresponding 4-[4-(2-chloroethyl)piperazin-1-yl]-N-aryl-1,2,3-dithiazol-5-imine was added 0.038 mmol of sodium 1,2,4- triazolate or 0.038 mmol of sodium imidazolate in 3 mL of anhydrous acetonitrile. The reaction mixture was refluxed for 8–10 h and then cooled. The precipitate was filtered off, and the filtrate was evaporated in vacuum. The residue was separated by column chromatography.

4-{4-[2-(1H-1,2,4-Triazol-1-yl)ethyl]piperazin-1-yl}-N-(4-chlorophenyl)-5H-1,2,3-ditiazol-5-imine (3a). Yield 86% (eluent—dichloromethane–methanol, 4 : 1). 1Н NMR spectrum (CDCl3), δ, ppm: 2.56 t (4H, CH2, 3J 4.3 Hz), 2.80 t (2H, CH2, 3J 4.3 Hz), 3.66 m (2H, CH2), 4.25 m (4H, CH2), 6.99 d (2H, CHAr, 3J 8.5 Hz), 7.32 d (2H, CHAr, 3J 8.5 Hz), 7.88 s (С3HTrz), 8.14 s (С5HTrz). Mass spectrum (HRMS), m/z: 408.0832 [M + H]+ (calculated for C16H19ClN7S2+: 408.0826).

4-{4-[2-(1H-1,2,4-Triazol-1-yl)ethyl]piperazin-1-yl}-N-(4-bromophenyl)-5H-1,2,3-ditiazol-5-imine (3b). Yiled 65% (eluent—dichloromethane–methanol, 2 : 1). 1Н NMR spectrum (CDCl3), δ, ppm: 2.63 t (4H, CH2, 3J 4.3 Hz), 2.85 t (2H, CH2, 3J 4.3 Hz), 3.71 m (2H, CH2), 4.30 m (4H, CH2), 6.99 d (2H, CHAr, 3J 8.4 Hz), 7.53 d (2H, CHAr, 3J 8.4 Hz), 7.94 s (С3HTrz), 8.19 s (С5HTrz). Mass spectrum(HRMS), m/z: 452.0321 [M + H]+ (calculated for C16H19BrN7S2+: 452.0327).

4-{4-[2-(1H-1,2,4-Triazol-1-yl)ethyl]piperazin-1-yl}-N-(4-methoxyphenyl)-5H-1,2,3-ditiazol-5-imine (3c). Yield 54% (eluent—dichloromethane–methanol, 4 : 1). 1Н NMR spectrum (CDCl3), δ, ppm: 2.64 t (4H, CH2, 3J 4.4 Hz), 2.87 t (2H, CH2, 3J 4.4 Hz), 3.73 m (7H, CH2, ОСН3), 6.96 d (2H, CHAr, 3J 8.8 Hz), 7.15 d (2H, CHAr, 3J 8.7 Hz), 7.94 s (С3HTrz), 8.21 s (С5HTrz). Mass spectrum (HRMS), m/z: 404.1322 [M + H]+ (calculated for C17H22N7OS2+: 404.1327).

4-{4-[2-(1H-Imidazol-1-yl)ethyl]piperazin-1-yl}-N-(4-chlorophenyl)-5H-1,2,3-ditiazol-5-imine (4a). Yield 30% (eluent—dichloromethane–methanol, 6 : 1). 1Н NMR spectrum (CDCl3), δ, ppm: 2.59 t (4H, CH2, 3J 4.3 Hz), 2.83 t (2H, CH2, 3J 4.3 Hz), 3.70 m (2H, CH2), 4.28 m (4H, 2CH2), 6.81 s (С4HIm), 7.02 br. s (3H, CHAr, С5HIm), 7.31 br. s (3H, CHAr, С2HIm). Mass spectrum (HRMS), m/z: 407.0874 [M + H]+ (calculated for C17H20ClN6S2+: 407.0879).