Thioamides are accessible and highly reactive compounds with both nucleophilic and electrophilic properties (the most significant reviews on the chemistry and application of thioamides, see [1–14]). This predetermines the variety of transformations of thioamides and their active use in complex formation reactions, in the synthesis of natural compounds and fine organic synthesis. One of the most intriguing and unpredictable reactions is the thioamides oxidation: depending on the conditions, the structure of the thioamide substrate, and the type of oxidant, the products can be the corresponding nitriles [15–17], disulfides [18, 19], carboxylic acids amides [4], benzothiasoles [ 20, 21], 1,2-dithiol derivatives [22, 23], 1,2,4-thiadiazoles [1–3], aminosulfines (thioamide-S-oxides) [24, 25], α-ketothioamides [3], and others (Scheme 1).

Scheme
scheme 1

1.

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Cyanothioacetamide 1 [6–9] and its reaction products with aldehydes, (E)-3-aryl-2-cyanoprop-2-entioamides (3-aryl-2-cyanothioacrylamides) 2 [26–29], are widely used in the synthesis of heterocyclic compounds [7–9]. Previously, it was shown that thioamides 2 can be oxidized with the hydrogen peroxide action to form S-oxides 3 [30], or or upon treatment with bromonitromethane [31], or using DMSO–HCl system [32] to form 1,2,4-thiadiazoles 4 (Scheme 2). It should be noted that 1,2,4-thiadiazoles are of considerable interest for pharmacology [33–35] due to their availability and biological activity profile, and also as starting compounds, for example, for the preparation of complex macrocyclic systems [36–38].

Scheme
scheme 2

2.

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Continuing research in the field of functional cyanothioacetamide derivatives with potential biological activity [39–43], we focused our attention on the possibility of obtaining new heterocyclic products through the oxidation of 2-cyanothioacrylamides 2 with sodium nitrite in an acidic medium. NaNO2 in the presence of acids can act as a nitrosating agent as well as mild oxidizing agent. Thus, unsubstituted cyanothioacetamide 1 reacts with NaNO2 in the presence of HCl to form α-isonitrosocyanothioacetamide 5 [44, 45] (Scheme 3). There are references that primary thioamides, when oxidized with nitrous acid, give nitriles, while secondary and tertiary thioamides are converted into the corresponding amides [4, 46]. However, according to [47, 48], primary aromatic thioamides are oxidized by HNO2 to 1,2,4-thiadiazoles 6. Alkyl nitrites react similarly: 1,2,4-thiadiazoles have been obtained from a wide range of primary thioamides in high yields [47, 49]. However, using other nitrosating agents and a number of N-substituted thioamides as an example, it was shown in [50, 51] that the nitrosation of thioamides can proceed in a more complex manner, with the formation of amides and bis(imidoyl)sulfides. At nitrosation conditions, 2,6-disubstituted thiobenzamides are predominantly converted into isothiocyanates or 1,2,4-thiadiazoles [52].

Scheme
scheme 3

3.

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It was found that the treatment of thioacrylamides 2a–2e with an aqueous solution of NaNO2 in hot acetic acid gave (2E,2′E)-2,2′-(1,2,4thiadiazol-3,5-diyl)bis[3-arylacrylonitriles] 4а–4e in 62–87% yield (Scheme 4).

Scheme
scheme 4

4.

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It should be noted that compounds 4a (Ar = 2-ClC6H4) and 4b (Ar = 4-ClC6H4) were previously obtained by oxidation of the corresponding thioamides 2 with bromonitromethane [31] and DMSO–HCl system [31, 32] in 21–65 yields (4a) and 58% (4b). At the oxidation with sodium nitrite in AcOH, the yields of thiadiazoles 4а and 4b were 87 and 62%, respectively. At the same time, thioacrylamides 2 containing an aromatic substituent with strong donor substituents [Ar = 4-HOC6H4, 4-MeOC6H4, 3,4-(MeO)2C6H3, 4-HO-3-MeOC6H3] could not be introduced into the reaction. In this case, resinification of the reaction mixture is observed, probably due to the occurrence of side reactions of oxidation and ring nitrosation. It should also be noted that at the oxidation with the action of BrCH2NO2 [31] or DMSO–HCl system [32], 2-cyanothioacrylamides 2 with donor substituents react smoothly to form the corresponding 1,2,4-thiadiazoles.

The probable reaction mechanism (Scheme 5) suggests nitrosation at the sulfur atom in accordance with the HSAB concept with the formation of A cations, which is consistent with the literature data [53, 54]. Cations A, by analogy with the available data [53–55], lose the NO molecule and undergo oxidative dimerization. The exact mechanism of this stage is unknown; however, according to the available data [54], a homolytic process with the formation of type C radicals and their dimerization with the formation of disulfides D is likely. Dimers D undergo intramolecular cyclization, presumably according to the previously described [56] scheme: through the possible formation of 1,2,4-dithiazole intermediates E and their recyclization to 1,2,4-thiadiazoles 4 through the formation of dithioperoxoimidate F and the elimination of hydrogen sulfide. The latter under reaction conditions undergo oxidation to elemental sulfur.

Scheme
scheme 5

5.

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We also made an attempt to oxidize the initial thioacrylamides 2 with the NaNO2–HCl system in ethanol. Due to the low solubility of thioacrylamides 2 in alcohol, the reaction is heterophasic and, according to TLC and NMR data, leads to a mixture of the expected 1,2,4-thiadiazole with the starting thioacrylamides 2. However, the addition of an excess of NaNO2 and hydrochloric acid leads to resinification of the reaction mixture. An attempt to carry out the synthesis in a one-pot variant, through the reaction of cyanothioacetamide 1 with aldehydes in EtOH in the presence of Et3N, followed by treatment with NaNO2–HCl without isolating the resulting thioacrylamide 2, also leads to resinification of the reaction mixture.

The structure of the obtained compounds was confirmed by spectral data and correlates with the results of earlier studies [31, 32]. Compounds 4 are finely crystalline powders, colored from pale yellow to orange, practically insoluble in EtOH, moderately soluble on heating in acetone, ethyl acetate, formic and acetic acids, and DMSO.

Taking into account the pharmacological activity of many 1,2,4-thiadiazole derivatives (see reviews [33–35]), it seemed appropriate to study the profile of possible biological action for the most soluble and, therefore, the most bioavailable compounds 4a, 4b, 4e by means of molecular docking. Possible protein targets for the obtained compounds were predicted using the new GalaxySagittarius protein ligand docking protocol [57] based on the GalaxyWeb web server [58, 59]. The 3D compounds structures were preliminarily optimized by means of molecular mechanics in the MM2 force field to optimize the geometry and minimize the energy. Docking using the GalaxySagittarius protocol was carried out in the Binding compatability prediction and Re-ranking using docking modes. Table S1 (see Supplementary materials) presents the results of docking for 1,2,4-thiadiazoles 4a, 4b, 4e for protein-ligand complexes with the lowest binding free energy ΔGbind and the best estimate of the target–ligand interaction. Predicted protein targets are identified by ID in the Protein Data Bank (PDB) and in the UniProt database. As you can see from Table S1, likely targets are non-receptor tyrosine-protein kinase TYK2 (PDB ID 5wal_A, UniProt ID P00519), ΔGbind = –19.5 to –21.1 kcal/mol, chaperone protein Hsp90 (PDB ID 5j20_A, UniProt ID P07900), ΔGbind = –20.5 to –23.9 kcal/mol, vascular endothelial growth factor receptor VEGF (PDB ID 3vo3_A, UniProt ID P35968), ΔGbind = –22.5 to –24.6 kcal/mol, eukaryotic translation initiation factor 4E (eIF4E, PDB ID 4tqb_A, UniProt ID P06730), ΔGbind = –19.6 to –21.8 kcal/mol, mitogen-activated protein kinase 14 (MAPK14, 3fly_A, UniProt ID Q16539), ΔGbind = –21.4 to –22.7 kcal/mol. Common targets for compounds 4a, 4b, 4e are cell proliferation regulators: ribosomal protein kinase S6 alpha-3 (RPS6KA3, PDB ID 4jg7_A, UniProt ID P51812), ΔGbind = –22.1 to –22.3 kcal/mol, platelet-derived growth factor A receptor (PDGFRα , PDB ID 5grn_A, UniProt ID P16234), ΔGbind = –23.6 to –24.3 kcal/mol, and mitogen-activated protein kinase 9 (MAPK9, PDB ID 3npc_A, UniProt ID P45984) ΔGbind = –23.5 to –25.3 kcal/mol. In general, for these compounds, screening in the direction of searching for antitumor drugs, as well as anti-inflammatory agents and regulators of antiviral immunity, is promising. Three-dimensional visualization of docking results (Figs. S1, S2, see Supplementary materials) was implemented using the UCSF Chimera software package [60, 61].

On the basis of the Federal Scientific Center for Biological Plant Protection (Krasnodar), we studied the antidote activity of the compounds against the herbicide 2,4-D (2,4-dichlorophenoxyacetic acid) on sunflower crops. It is known that 2,4-D has a rather high toxicity for sunflower: a dose of 15–18 g/ha according to the active substance leads to a 40–60% decrease in yield [62]. Herbicide antidotes are used to neutralize the negative effects of pesticides on crops. Antidotes do not affect the activity of herbicides against weeds and reduce the toxicity of the herbicide on the crop; they are harmless to the crop, or even additionally have a growth-promoting effect. The concept of herbicide antidotes was proposed by O. Hoffman in 1962 [63] and, despite the absence of a coherent theory of the mechanism of action, proved its effectiveness and economic significance (reviews on herbicide antidotes [64–66]). Under the laboratory experiment conditions, it was found that one of the compounds, 1,2,4-thiadiazole 4b, exhibits a pronounced antidote effect against 2,4-D on sunflower seedlings. Germinated seeds of sunflower cultivars Master were treated with 2,4-D herbicide (“herbicide” variant of the experiment), 2,4-D herbicide and then a potential antidote (“herbicide+antidote” variant), the control group of seeds was left without treatment. The antidote effect was determined by the increase in the length of the hypocotyl and root in the “herbicide+antidote” variant relative to the named values in the “herbicide” variant in percent. The results are summarized in Table 1. As can be seen, compound 4b reduced the negative effect of 2,4-D on the hypocotyls of sunflower seedlings by 24–42% and the roots of seedlings by 34–49%.

Table 1. Antidote activity of (2E,2′E)-2,2′-(1,2,4-thiadiazol-3,5-diyl)bis[3-(4-chlorophenyl)acrylonitrile] 4b against herbicide 2,4-D on sunflower seedling of cv. Master
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The evaluation of the antidote effect under the field experiment conditions was carried out on plots with an area of 2.8 m2 with five repetitions. The antidote effect was determined by the absolute value of the yield increase to the herbicidal standard and as a percentage by Eq. (1):

$${A_{\rm{x}}} = {{A - E} \over E} \times 100,$$
((1))

where Ax—the antidote effect, %; A—crop in the “herbicide+antidote” variant; E—crop in the variant standard (“herbicide”).

The results are presented in Table 2. In general, the using of thiadiazole 4b on sunflower plants as an antidote at a dose of 100 g/ha makes it possible to provide an antidote effect of 66%.

Table 2. Antidote activity of compound 4b against 2,4-D on sunflower cv. Master in the field experiment
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Thus, we succeeded in developing a new method for the preparation of (2Е,2′E)-2,2′-(1,2,4-thiadiazol-3,5-diyl)bis[3-arylacrylonitriles] based on the oxidation of 3-aryl-2-cyanothioacrylamides under the action of sodium nitrite in acetic acid. The reaction proceeds relatively smoothly only in the case of 3-aryl-2-cyanothioacrylamides having acceptor substituents in the aromatic ring. Therefore, despite the rather high yields of products, the new method still cannot be considered optimal for obtaining target thiadiazoles. Molecular docking in relation to a wide range of protein targets made it possible to identify the most priority areas for further screening. New 1,2,4-thiadiazoles are promising for the search for antitumor and anti-inflammatory agents. (2Е,2′Е)-2,2′-(1,2,4-Thiadiazol-3,5-diyl)bis[3-(4-chlorophenyl)acrylonitrile] exhibits a pronounced antidote effect against the herbicide 2,4-D in the conditions of laboratory experiment and small-plot experiment. In general, further search for optimal conditions for the oxidation of 3-aryl-2-cyanothioacrylamides seems to be appropriate in view of the pharmacological and agrochemical potential of the reaction products.

EXPERIMENTAL

IR spectra were obtained on a Bruker Vertex 70 spectrophotometer with an ATR attachment by the method of frustrated total internal reflection on a diamond crystal, error ± 4 cm–1. NMR spectra were recorded on Bruker Avance III HD 400MHz (400.17 and 100.63 MHz) and Agilent 400/MR (400 and 100 MHz, respectively) instruments in DMSO-d6 or CF3CO2D–CDCl3 (1 : 1) solutions. Residual solvent signals were used as a standard. Elemental analysis was carried out on a Carlo Erba EA 1106 instrument. The individuality of the obtained samples was controlled by TLC on Sorbfil-A plates (Imid, Krasnodar), eluent acetone–hexane (1 : 1), ethyl acetate–hexane (1 : 1), or acetone–chloroform (1 : 1), developer—iodine vapor, UV detector.

Cyanothioacetamide 1 [67] and 2-cyanothioacrylamides 2 [26–29] were obtained by known procedures.

Oxidation of 2-cyanothioacrylamides 2 with sodium nitrite in acetic acid (general procedure). A mixture of 1.5 mmol of the corresponding thioacrylamide 2a–f and 5 mL of acetic acid was heated with vigorous stirring to 100°C, then a solution of an excess (0.6 g, 9.0 mmol) of sodium nitrite in 3 mL of distilled water was added. Thioacrylamide 2 was dissolved, the reaction mass turned red and quickly turned cloudy due to the formation of colloidal sulfur. A precipitate of the product separated from the solution within 30 min [Attention! Release of nitric oxide(IV)!]. After cooling the mixture, the product was precipitated with 10 mL of distilled water, after which the precipitate was filtered off, washed with water and petroleum ether. The resulting product was recrystallized from a large volume of acetone, EtOAc or AcOH.

(2Е,2′Е)-2,2′-(1,2,4-Thiadiazol-3,5-diyl)bis[3-(2-chlorophenyl) acrylonitrile] (4а). Yield 87%, pale yellow fine crystalline powder. The spectra of the compound are identical to those described in [32].

(2Е,2′Е)-2,2′-(1,2,4-Thiadiazol-3,5-diyl)bis[3-(4-chlorophenyl) acrylonitrile] (4b). Yield 62%, pale yellow fine crystalline powder. IR spectrum, ν, cm–1: 2220 br. (C≡N). 1Н NMR spectrum (DMSO-d6), δ, ppm: 7.70 d (2H, Ar, 3JHH 8.6 Hz), 7.74 d (2H, Ar, 3JHH 8.6 Hz), 8.09–8.13 m (4Н, Ar, overlapping of two doublets), 8.63 s (1H, CH=), 8.65 s (1H, CH=). 1Н NMR spectrum (CF3CO2D–CDCl3), δ, ppm: 7.52 d (2H, Ar, 3JHH 8.6 Hz), 7.55 d (2H, Ar, 3JHH 8.6 Hz), 7.96 d (2H, Ar, 3JHH 8.6 Hz), 8.00 d (2H, Ar, 3JHH 8.6 Hz), 8.38 s (1H, CH=), 8.60 s (1H, CH=). 13С NMR spectrum (CF3CO2D–CDCl3), δC, ppm: 100.7 (CC≡N), 103.2 (CC≡N), 115.0 (C≡N), 115.1 (C≡N), 129.8 (СAr), 129.9 (2CHAr), 130.2 (2CHAr), 130.4 (СAr), 131.8 (2CHAr), 132.3 (2CHAr), 139.7 (СAr), 140.8 (СAr), 149.6 (CH=), 151.3 (CH=), 168.89 (C3thiadiazole), 184.0 (C5thiadiazole). Found, %: С 58.57; H 2.54; N 13.68. C20H10Cl2N4S. Calculated, %: C 58.69; H 2.46; N 13.69. M 409.29.

(2Е,2′Е)-2,2′-(1,2,4-Thiadiazol-3,5-diyl)bis[3-(2-nitrophenyl) acrylonitrile] (4c). Yield 70%, yellow orange powder. IR spectrum, ν, cm–1: 2222 br, 2233 br (C≡N), 1522 s, 1344 s (NO2). 1Н NMR spectrum (DMSO-d6), δ, ppm: 7.87–8.03 m (8Н, Ar), 8.33 s (1H, CH=), 8.35 s (1H, CH=). The 13С NMR spectrum could not be recorded due to insufficient solubility of the substance in DMSO-d6. Found, %: С 55.70; H 2.49; N 19.46. C20H10N6О4S. Calculated, %: C 55.81; H 2.34; N 19.53. M 430.40.

(2E,2′E)-2,2′-(1,2,4-Thiadiazol-3,5-diyl)bis[3-(4-hydroxy-3-methoxy-5-nitrophenyl) acrylonitrile] (4d). Yield 64%, orange powder. IR spectrum, ν, cm–1: 3192 br. s (O–H), 2229 w (C≡N), 1547 s, 1335 s (NO2). 1Н NMR spectrum (DMSO-d6), δ, ppm: 3.94 br. s (6Н, MeO), 7.58 br. s (2Н, Н2Ar), 8.09 br. s (2Н, Н6Ar), 9.83 s (1H, CH=), 9.84 s (1H, –CH=), 10.80 very br. s (2Н, OH, the integrated signal intensity is underestimated due to deuterium exchange). The 13С NMR spectrum could not be recorded due to insufficient solubility of the substance in DMSO-d6. Found, %: С 50.42; H 2.76; N 16.20. C22H14N6О8S. Calculated, %: C 50.58; H 2.70; N 16.09. M 522.45.

(2Е,2′Е)-2,2′-(1,2,4-Thiadiazol-3,5-diyl)bis[3-(4-bromophenyl) acrylonitrile] (4e). Yield 71%, yellow fine crystalline powder. IR spectrum, ν, cm–1: 2218 br (C≡N). 1Н NMR spectrum (CF3CO2D–CDCl3), δ, ppm: 7.68 d (2H, Ar, 3JHH 8.6 Hz), 7.71 d (2H, Ar, 3JHH 8.6 Hz), 7.88 d (2H, Ar, 3JHH 8.6 Hz), 7.91 d (2H, Ar, 3JHH 8.6 Hz), 8.35 s (1H, CH=), 8.56 s (1H, CH=). 13С NMR spectrum (CF3CO2D–CDCl3), δC, ppm: 100.9 (CC≡N), 103.5 (CC≡N), 115.1 (C≡N), 115.2 (C≡N), 128.1 (C–Br), 129.3 (C–Br), 130.2 (С1Ar), 130.8 (С1Ar), 131.8 (2CHAr), 132.2 (2CHAr), 132.9 (2CHAr), 133.2 (2CHAr), 149.4 (CH=), 151.0 (CH=), 168.8 (C3thiadiazole), 184.0 (C5thiadiazole). Found, %: С 48.17; H 2.13; N 11.25. C20H10Br2N4S. Calculated, %: C 48.22; H 2.02; N 11.25. M 498.19.

Evaluation of the antidote activity of compound 4b. Germinated seeds of sunflower cv. Master with an embryonic root length of 2–4 mm were placed for 1 h in a 2,4-D solution at a concentration of 10–3%, calculated for 40–60% inhibition of hypocotyl growth. After the herbicide treatment, the seedlings were washed with water and placed in a solution/thin suspension of compound 4b at concentrations of 10–2, 10–3, 10–4, and 10–5% (“herbicide+antidote” variant). After 1 hour, the seeds were washed with water and laid out on filter paper strips (size 10×75 cm), 20 pieces each, which were rolled up and placed in glasses with 50 mL of water. Further germination of seeds was carried out in a thermostat for 3 days at 28°C. The temperature of solutions and washing water is 28°C. Seeds of the “herbicide” variant (reference standard) were kept for 1 h in a 2,4-D solution at a concentration of 10–3% and then for 1 h in water. The seeds of the control variant were kept in water for 2 h. The experiment was repeated three times. In each repetition, 20 seeds were used. The antidote effect (%) was determined by the increase in the length of the hypocotyl and root in the “herbicide+antidote” variant relative to the length of the hypocotyl and root in the “herbicide” variant. Statistical processing of experimental data was carried out using Student's t-test at P 0.95.

The evaluation of the antidote activity of compound 4b under field experimental conditions was carried out in the experimental field of the Federal Scientific Center for Biological Plant Protection (Krasnodar). Sunflower plants of the cv. Master in the phase of 10–16 leaves were treated with an aqueous solution of 2,4-D at a dose of 18 g/ha. After 3 days, an antidote was applied to the site in the form of a thin aqueous suspension at a dose of 100 g/ha with a working fluid consumption rate of 300 L/ha. The experiment was carried out in the following variants: “control”—untreated plants; “herbicide” (reference)—plants treated only with 2,4-D; “herbicide+antidote”—plants treated with 2,4-D and then with an antidote. The experiments were carried out on plots with an area of 2.8 m2 with five repetitions. Sunflower harvesting was carried out at the time of full ripening of seeds. The antidote effect (%) was determined by the absolute value of the yield increase to the herbicidal standard according to the Eq. (1). The obtained data were statistically processed using Student’s t-test.