6-Amino-4-aryl-7-phenyl-3-(phenylimino)-4,7-dihydro-3H-[1,2]dithiolo[3,4-b]pyridine-5-carboxamides: Synthesis, Biological Activity, Quantum Chemical Studies and In Silico Docking Studies

New [1,2]dithiolo[3,4-b]pyridine-5-carboxamides were synthesized through the reaction of dithiomalondianilide (N,N′-diphenyldithiomalondiamide) with 3-aryl-2-cyanoacrylamides or via a three-component reaction involving aromatic aldehydes, cyanoacetamide and dithiomalondianilide in the presence of morpholine. The structure of 6-amino-4-(2,4-dichloro- phenyl)-7-phenyl-3-(phenylimino)-4,7-dihydro-3H-[1,2]dithiolo[3,4-b]pyridine-5-carboxamide was confirmed using X-ray crystallography. To understand the reaction mechanism in detail, density functional theory (DFT) calculations were performed with a Grimme B97-3c composite computational scheme. The results revealed that the rate-limiting step is a cyclization process leading to the closure of the 1,4-dihydropyridine ring, with an activation barrier of 28.8 kcal/mol. Some of the dithiolo[3,4-b]pyridines exhibited moderate herbicide safening effects against 2,4-D. Additionally, ADMET (Absorption, Distribution, Metabolism, Excretion, Toxicity) parameters were calculated and molecular docking studies were performed to identify potential protein targets.

The compounds 3 are colored in shades of yellow (from canary yellow to mustard yellow), sparingly soluble in boiling acetone or ethyl acetate, soluble in DMF or DMSO and insoluble in alcohols.

The Studies of the Reaction Mechanism
In recent papers [30][31][32], we have described the preparation of [1,2]dithiolo [3,4-b]pyridines via the reaction of dithiomalondianilide 1 with activated Michael substrates, such as substituted acrylonitriles 4,5 (Scheme 4), but no detailed study of the reaction mechanism has been carried out.
Here, we would like to consider a possible mechanism for the formation of the [1,2]dithiolo [3,4-b]pyridine ring system using the reaction of compound 1 with 2-cyanoacrylamides 2 as an example.It is obvious that an oxidizing agent is required for the successful formation of the 1,2-dithiol ring.Two reagents, either oxygen in the air or an unsaturated nitrile, can play the role of an oxidizing agent.In a number of studies concerning the synthesis of pyridine derivatives starting from unsaturated nitriles (e.g., arylmethylidene malononitriles 4), it was shown that the partially saturated pyridine intermediate easily reacts with unsaturated nitriles to yield α,β-saturated nitriles and their corresponding oxidation products, such as nicotinonitriles [64][65][66][67][68] (see Scheme S1, Electronic Supplementary Material File).The compounds 3 are colored in shades of yellow (from canary yellow to mustard yellow), sparingly soluble in boiling acetone or ethyl acetate, soluble in DMF or DMSO and insoluble in alcohols.

The Studies of the Reaction Mechanism
In recent papers [30][31][32], we have described the preparation of [1,2]dithiolo [3,4b]pyridines via the reaction of dithiomalondianilide 1 with activated Michael substrates, such as substituted acrylonitriles 4,5 (Scheme 4), but no detailed study of the reaction mechanism has been carried out.
Here, we would like to consider a possible mechanism for the formation of the [1,2]dithiolopyridine ring system using the reaction of compound 1 with 2-cyanoacryl amides 2 as an example.It is obvious that an oxidizing agent is required for the successful formation of the 1,2-dithiol ring.Two reagents, either oxygen in the air or an unsaturated nitrile, can play the role of an oxidizing agent.In a number of studies concerning the synthesis of pyridine derivatives starting from unsaturated nitriles (e.g., arylmethylidene malononitriles 4), it was shown that the partially saturated pyridine intermediate easily reacts with unsaturated nitriles to yield α,β-saturated nitriles and their corresponding oxidation products, such as nicotinonitriles [64][65][66][67][68] (see Scheme S1, Electronic Supplementary Material File).It was reported that when unsaturated nitriles were reacted in a two-fold excess, the yields of the target pyridines were increased sharply [67,68].Although no formation of disulfides was observed in the above reactions, activated nitriles can potentially play the role of oxidizing agents for 1,2-dithiol ring closure reactions.
As a model reaction, we studied the reaction of 2-cyano-4-(methoxyphenyl)acrylamide 2f with dithiomalondianilide 1 in the presence of morpholine in EtOH.Compound 2f was prepared beforehand via a Knoevenagel reaction of 4-methoxybenzaldehyde with cyanoacetamide in the presence of morpholine in EtOH at 50 °C.
To elucidate the nature of the oxidizing agent, the reaction between 1 and 2f was carried out using four different variants: (1) Under argon flow to prevent the effects of oxygen, at a ratio of 2f:1 = 2:1; (2) Under an air stream at a ratio 2f:1 = 1:1 (Table 1, entry  We found that in reaction (1), only trace amounts of product 3f was detected via TLC (thin layer chromatography) and NMR after 3 h.In experiments ( 2) and (3), we observed the formation of a precipitate of product 3f, though in reaction (3), TLC and HRMS showed the presence of considerable amounts of starting acrylamide 2f in the crude product.Finally, in experiment (4), no formation of 3f was detected, and the starting material remained unreacted.The yields of pure dithiolopyridine 3f in experiments ( 2) and (3) were 54% and 55%, respectively.Thus, no significant increase in the yields was observed when a twofold excess of acrylamide 2f was used.This fact indirectly negates the role of acrylamide 2f as an oxidant in this reaction.Overall, from the conducted experiments (1-4), we can conclude that a) oxygen in the air is the oxidizing agent in this reaction and that b) the formation of the pyridine ring precedes the oxidation that forms the 1,2-dithiol fragment (since the product of dithiomalondianilide 1 oxidation, 1,2-dithiol 6, did not react with acrylamide 2f under the reported conditions).
With these preliminary data in hand, we investigated the reaction mechanism in more detail.Quantum chemical study of the mechanism was performed using the ORCA It was reported that when unsaturated nitriles were reacted in a two-fold excess, the yields of the target pyridines were increased sharply [67,68].Although no formation of disulfides was observed in the above reactions, activated nitriles can potentially play the role of oxidizing agents for 1,2-dithiol ring closure reactions.
As a model reaction, we studied the reaction of 2-cyano-4-(methoxyphenyl)acrylamide 2f with dithiomalondianilide 1 in the presence of morpholine in EtOH.Compound 2f was prepared beforehand via a Knoevenagel reaction of 4-methoxybenzaldehyde with cyanoacetamide in the presence of morpholine in EtOH at 50 • C.
To elucidate the nature of the oxidizing agent, the reaction between 1 and 2f was carried out using four different variants: (1) Under argon flow to prevent the effects of oxygen, at a ratio of 2f:1 = 2:1; (2) Under an air stream at a ratio 2f:1 = 1:1 (Table 1 It was reported that when unsaturated nitriles were reacted in a two-fold excess, the yields of the target pyridines were increased sharply [67,68].Although no formation of disulfides was observed in the above reactions, activated nitriles can potentially play the role of oxidizing agents for 1,2-dithiol ring closure reactions. As a model reaction, we studied the reaction of 2-cyano-4-(methoxyphenyl)acrylamide 2f with dithiomalondianilide 1 in the presence of morpholine in EtOH.Compound 2f was prepared beforehand via a Knoevenagel reaction of 4-methoxybenzaldehyde with cyanoacetamide in the presence of morpholine in EtOH at 50 °C.
To elucidate the nature of the oxidizing agent, the reaction between 1 and 2f was carried out using four different variants: (1) Under argon flow to prevent the effects of oxygen, at a ratio of 2f:1 = 2:1; (2) Under an air stream at a ratio 2f:1 = 1:1 (Table 1 We found that in reaction (1), only trace amounts of product 3f was detected via TLC (thin layer chromatography) and NMR after 3 h.In experiments ( 2) and (3), we observed the formation of a precipitate of product 3f, though in reaction (3), TLC and HRMS showed the presence of considerable amounts of starting acrylamide 2f in the crude product.Finally, in experiment (4), no formation of 3f was detected, and the starting material remained unreacted.The yields of pure dithiolopyridine 3f in experiments ( 2) and (3) were 54% and 55%, respectively.Thus, no significant increase in the yields was observed when a twofold excess of acrylamide 2f was used.This fact indirectly negates the role of acrylamide 2f as an oxidant in this reaction.Overall, from the conducted experiments (1-4), we can conclude that a) oxygen in the air is the oxidizing agent in this reaction and that b) the formation of the pyridine ring precedes the oxidation that forms the 1,2-dithiol fragment (since the product of dithiomalondianilide 1 oxidation, 1,2-dithiol 6, did not react with acrylamide 2f under the reported conditions).
With these preliminary data in hand, we investigated the reaction mechanism in more detail.Quantum chemical study of the mechanism was performed using the ORCA We found that in reaction (1), only trace amounts of product 3f was detected via TLC (thin layer chromatography) and NMR after 3 h.In experiments ( 2) and (3), we observed the formation of a precipitate of product 3f, though in reaction (3), TLC and HRMS showed the presence of considerable amounts of starting acrylamide 2f in the crude product.Finally, in experiment (4), no formation of 3f was detected, and the starting material remained unreacted.The yields of pure dithiolopyridine 3f in experiments ( 2) and (3) were 54% and 55%, respectively.Thus, no significant increase in the yields was observed when a twofold excess of acrylamide 2f was used.This fact indirectly negates the role of acrylamide 2f as an oxidant in this reaction.Overall, from the conducted experiments (1-4), we can conclude that a) oxygen in the air is the oxidizing agent in this reaction and that b) the formation of the pyridine ring precedes the oxidation that forms the 1,2-dithiol fragment (since the product of dithiomalondianilide 1 oxidation, 1,2-dithiol 6, did not react with acrylamide 2f under the reported conditions).
With these preliminary data in hand, we investigated the reaction mechanism in more detail.Quantum chemical study of the mechanism was performed using the ORCA 5.0.4 software package [70,71].The search of transition states, determination of reaction trajectories, calculations of vibrational frequencies and Gibbs free energy were examined using density functional theory (DFT) with Grimme's B97-3c composite calculation scheme [72,73], which is based on the combination of the B97 GGA functional and the def2-mTZVP basis set with the D3BJ dispersion correction [74].Transition states were searched using relaxed scan and nudged elastic band (NEB) methods [75].The geometry of transition states was confirmed by the presence of an imaginary vibrational frequency corresponding to the reaction coordinate.All the calculations were performed with non-specific solvation using the CPCM (Conductor-like Polarizable Continuum Model) model (with ethanol as the solvent) [76].ChemCraft 1.8 software was used to visualize the molecular geometry and vibrational frequencies.According to the results of the quantum chemical studies, we proposed a mechanism for the formation of the [1,2]dithiolo [3,4-b]pyridine system (Scheme 6).
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 6 of 20 5.0.4 software package [70,71].The search of transition states, determination of reaction trajectories, calculations of vibrational frequencies and Gibbs free energy were examined using density functional theory (DFT) with Grimme's B97-3c composite calculation scheme [72,73], which is based on the combination of the B97 GGA functional and the def2-mTZVP basis set with the D3BJ dispersion correction [74].Transition states were searched using relaxed scan and nudged elastic band (NEB) methods [75].The geometry of transition states was confirmed by the presence of an imaginary vibrational frequency corresponding to the reaction coordinate.All the calculations were performed with non-specific solvation using the CPCM (Conductor-like Polarizable Continuum Model) model (with ethanol as the solvent) [76].ChemCraft 1.8 software was used to visualize the molecular geometry and vibrational frequencies.According to the results of the quantum chemical studies, we proposed a mechanism for the formation of the [1,2]dithiolo [3,4-b]pyridine system (Scheme 6).As we can see, the entire process of the formation of the bicyclic ring system can be divided into two main stages: heterocyclization to form dihydropyridine ring and oxidative cyclization to form the cyclic disulfide moiety.Since the reaction was carried out in the presence of excessive amounts of base (morpholine), we considered the model system of dithiomalondianilide 1 + the Michael acceptor (2-cyano-3-(4-methoxyphenyl)acrylamide 2f) + morpholine at the first stage.An oxygen molecule was added to the model system in the second stage.In order to determine the optimal geometry of the initial state, the most stable conformation of dithiomalondianilide 1 was determined.
The first step of the cascade reaction mechanism involves the deprotonation of the C27 atom (Scheme 6, Figure 2) of the dithiomalondianilide 1 molecule followed by the Michael addition of the resulting anion to the C39 atom of 2-cyano-3-(4-methoxyphenyl)-Scheme 6.The proposed mechanism of the formation of the [1,2]dithiolo [3,4-b]pyridine ring system.
As we can see, the entire process of the formation of the bicyclic ring system can be divided into two main stages: heterocyclization to form dihydropyridine ring and oxidative cyclization to form the cyclic disulfide moiety.Since the reaction was carried out in the presence of excessive amounts of base (morpholine), we considered the model system of dithiomalondianilide 1 + the Michael acceptor (2-cyano-3-(4-methoxyphenyl)acryl-amide 2f) + morpholine at the first stage.An oxygen molecule was added to the model system in the second stage.In order to determine the optimal geometry of the initial state, the most stable conformation of dithiomalondianilide 1 was determined.
The first step of the cascade reaction mechanism involves the deprotonation of the C27 atom (Scheme 6, Figure 2) of the dithiomalondianilide 1 molecule followed by the Michael addition of the resulting anion to the C39 atom of 2-cyano-3-(4-methoxyphenyl)acrylamide 2f.The molecular structures of the intermediates (I) and transition states (TS) are given in Figure 2.  Next, within the resulting Michael adduct I-2, an intramolecular proton transfer from the N35 atom to the N48 atom occurs, yielding an eight-membered cyclic transition state TS3.This process has the highest activation energy and, therefore, should be considered the rate-limiting step.After the protonation of the N48 atom, an intramolecular nucleophilic attack of the N35 atom on the C46 atom occurs to close a pyridine ring.Thus, the resulting anion I-5 then undergoes a secondary protonation of the N48 atom to form intermediate I-6.The energy profile of this process is shown in Figure 3, and the energy profile for oxidative cyclization to form the 1,2-dithiol fragment is given in Figure 4. Molecules not involved in a particular elementary act are not shown in the figure for ease of perception and better visualization.
Next, within the resulting Michael adduct I-2, an intramolecular proton transfer from the N35 atom to the N48 atom occurs, yielding an eight-membered cyclic transition state TS3.This process has the highest activation energy and, therefore, should be considered the rate-limiting step.After the protonation of the N48 atom, an intramolecular nucleophilic attack of the N35 atom on the C46 atom occurs to close a pyridine ring.Thus, the resulting anion I-5 then undergoes a secondary protonation of the N48 atom to form intermediate I-6..The energy profile of this process is shown in Figure 3, and the energy profile for oxidative cyclization to form the 1,2-dithiol fragment is given in Figure 4.The oxidation of intermediate I-5 (Scheme 6, Figure 3) involves an initial deprotonation of the C27 atom (intermediate I-6 is formed) followed by the addition of molecular oxygen to the S38 atom to form a peroxysulfenic acid (intermediate I-7) (Figure 5).After the deprotonation of the CSNHPh N20 nitrogen (intermediate I-8 is formed), an intra- Molecules not involved in a particular elementary act are not shown in the figure for ease of perception and better visualization.
Next, within the resulting Michael adduct I-2, an intramolecular proton transfer from the N35 atom to the N48 atom occurs, yielding an eight-membered cyclic transition state TS3.This process has the highest activation energy and, therefore, should be considered the rate-limiting step.After the protonation of the N48 atom, an intramolecular nucleophilic attack of the N35 atom on the C46 atom occurs to close a pyridine ring.Thus, the resulting anion I-5 then undergoes a secondary protonation of the N48 atom to form intermediate I-6..The energy profile of this process is shown in Figure 3, and the energy profile for oxidative cyclization to form the 1,2-dithiol fragment is given in Figure 4.The oxidation of intermediate I-5 (Scheme 6, Figure 3) involves an initial deprotonation of the C27 atom (intermediate I-6 is formed) followed by the addition of molecular oxygen to the S38 atom to form a peroxysulfenic acid (intermediate I-7) (Figure 5).After the deprotonation of the CSNHPh N20 nitrogen (intermediate I-8 is formed), an intra-  The oxidation of intermediate I-5 (Scheme 6, Figure 3) involves an initial deprotonation of the C27 atom (intermediate I-6 is formed) followed by the addition of molecular oxygen to the S38 atom to form a peroxysulfenic acid (intermediate I-7) (Figure 5).After the deprotonation of the CSNHPh N20 nitrogen (intermediate I-8 is formed), an intramolecular nucleophilic substitution at the S38 sulfur atom of the hydroperoxide anion occurs to form a disulfide bond in the final product 3f.In the process of nucleophilic substitution, the hydroperoxide anion is protonated, releasing hydrogen peroxide, which also acts as an oxidizing agent.Overall, during the first stage of dihydropyridine ring formation, the highest activation energy (24.8 kcal/mol) corresponds to the intramolecular cyclization that occurred at the cyano group.Apparently, this is the rate-limiting step for the entire process since the highest activation barrier at the second stage (the formation of the disulfide bond/1,2-dithiol ring closure) is somewhat lower (23.5 kcal/mol).
The analysis for compliance with Lipinski's "rule of five" is intended to predict drug -likeness and bioavailability for an oral therapeutic agent using its physicochemical properties.The following parameters were calculated: cLogP (logarithm of the distribution coefficient between n-octanol and water, log(c octanol /c water ), solubility (log S), topological polar surface area (TPSA), a number of toxicological characteristics, including the risks of side effects (mutagenic, oncogenic and reproductive effects) and a similarity parameter with known drugs (drug-likeness); a general assessment of the pharmacological potential of the compound (drug score) was also carried out.Since (R)-and (S)-stereoisomers of compound 3 revealed the same values in the in silico calculations, the results presented in Table S12 (Electronic Supplementary Material File) apply equally to both enantiomers.
As we can see from the Table S12, for all compounds except 3b and 3d, the cLogP values do not exceed 5.0, which indicates good absorption and permeability (which was expected) [77].However, most of the compounds 3 do not fit the Lipinski's rule of five in terms of molecular weight (MW ≥ 500 Da).Furthermore, these compounds 3 had poor calculated solubility (logS < −4) and did not pass the Veber filter (TPSA ≥ 140 Å 2 ) [78].The results of the ADMET calculations are given in Tables S13-S24 (Electronic Supplementary Material File).In general, good gastrointestinal absorption and blood-brain barrier permability were predicted for all the compounds 3a-f.However, the calculations revealed probable hepatotoxicity and mitochondrial toxicity.At the same time, low acute oral toxicity was predicted.The calculation derived from the GUSAR service assigns all compounds to categories 4 (LD 50 > 300-2000 mg/kg) or 5 (LD 50 > 2000-5000 mg/kg) according to OECD criteria [79].Overall, these compounds 3 do not meet certain oral availability criteria as lead molecules, e.g., poor solubility, some violations for Ghose filter (MW > 480, molar refractivity (MR) > 130), Veber filter (violation: TPSA > 140 Å 2 ), Egan filter (violation: TPSA > 131.6 Å 2 ), Muegge filter (violation: TPSA > 150 Å 2 ) and insaturation parameter (fraction of Csp 3 < 0.25).
For both the (R)-and (S)-enantiomers of each of the 3H- [1,2]dithiolo [3,4-b]pyridines 3a-f, the most likely protein targets were predicted using the GalaxyWEB server (https://galaxy.seoklab.org/index.html,accessed on 30 December 2023) [80,81].We used GalaxySagittarius-AF, the newest protein-ligand docking protocol that uses AlphaFold models for drug-like compounds.The 3D structures of (R)-and (S)-enantiomers of compounds 3a-f were preoptimized via molecular mechanics in the MM2 force field for geometry optimization and energy minimization.The molecular docking studies were performed in binding compatibility prediction mode using PDB + AlphaFold models and re-ranking using docking mode.Table S25 (Supplementary Materials File) shows the docking results for 25 target-ligand complexes for both isomers of 3a-f with the minimum binding free energy ∆G bind and the best protein-ligand interaction score.The predicted protein targets are marked using identifiers in the Protein Data Bank (PDB) and in the UniProt database.
As we can see from Table S25, for both the (R)-and (S)-enantiomers of 3H-[1,2]dithiolo pyridines 3a-f, a similar pool of protein targets is predicted, but the scoring function values are different.The most common targets for 3H- [1,2] geometry optimization and energy minimization.The molecular docking studies were performed in binding compatibility prediction mode using PDB + AlphaFold models and re-ranking using docking mode.Table S25 (Supplementary Materials File) shows the docking results for 25 target-ligand complexes for both isomers of 3a-f with the minimum binding free energy ΔGbind and the best protein-ligand interaction score.The predicted protein targets are marked using identifiers in the Protein Data Bank (PDB) and in the UniProt database.
2,4-D (2,4-dichlorophenoxyacetic acid) is an herbicide that displays a relatively low toxicity in humans and is actively used for the chemical weeding of cultivated plants [85].The chemical weeding of crops is an important element in the protection of agricultural crops from weeds because weeds reduce the yield of the most important crops by 15-25%.However, it should be noted that herbicides, as plant-killing agents, are toxic not only to weeds but also to the sunflower, and the widely used 2,4-dichlorophenoxyacetic acid is highly toxic to the latter.For example, a dose of 0.5-0.8kg/ha of the active ingredient 2,4-D is recommended for weed control in crops of resistant cereals, and for the sunflower, a dose as small as 15-18 g/ha of the active ingredient leads to a 40-60% reduction in the crop yield.

Materials and Methods
1 H and 13 C DEPTQ NMR spectra and 2D NMR experiments were recorded in solutions of DMSO-d6 on Bruker AVANCE-III HD (Bruker BioSpin AG, Fällanden, Switzerland) and Agilent 400/MR (Agilent Technologies, Santa Clara, CA, USA) instruments (at 400 MHz for 1 H and 101 MHz for 13 C nuclei).Residual solvent signals were used as internal standards in DMSO-d6 (2.49 ppm for 1 H and 39.50 ppm for 13 C nuclei).Single crystal X-ray diffraction analysis of compound 3d was performed on an automatic four-circle diffractometer Agilent Super Nova, Dual, Cu at zero, Atlas S2.High-resolution mass spectra (HRMS) were registered with a Bruker MaXis Impact (Bruker Daltonics, Bremen, Germany) spectrometer (electrospray ionization; HCO2Na-HCO2H was used for calibration).The samples were dissolved in MeCN under moderate heating (37-38 °C) and ultrasonication.See Electronic Supplementary Material file for NMR, FTIR and HRMS spectral charts and X-ray analysis data.
FT-IR spectra were measured on a Bruker Vertex 70 instrument (Bruker Optics GmbH & Co. KG, Ettlingen, Germany) equipped with an ATR sampling module.Elemental analyses were carried out using a Carlo Erba 1106 Elemental Analyzer (Carlo Erba Strumentazione, Cornaredo, Italy).Reaction progress and purity of isolated compounds were controlled via TLC on Sorbfil-A plates (Imid Ltd., Krasnodar, Russia), and the eluent was acetone:hexane 2:1 or ethyl acetate.Developed TLC plates were stained with UV light and iodine vapors.were prepared via slow evaporation of saturated solution in DMSO.The crystal was kept at 100.01 (10) K during data collection.Using Olex2 [91], the structure was solved with the olex2.solvestructure solution program using Charge Flipping and refined with the SHELXL [92] package using Gauss-Newton minimisation.
A full set of crystallographic data has been deposited in the Cambridge Crystallographic Data Center (CCDC 2310349).

Herbicide Safening Effect Studies
Germinated sunflower seeds (cv.Master) with 2-4 mm long embryo roots were placed in a solution of 2,4-D (10 -3 % by weight) for 1 h to achieve 40-60% inhibition of hypocotyls' growth.After treatment, the seedlings were washed with pure water and placed into a solution of the corresponding compounds 3b, 3d and 3f (concentrations 10 -2 , 10 -3 , 10 -4 or 10 -5 % by weight, "herbicide + antidote" experiments).After 1 h, the seedlings were washed with pure water and placed on paper stripes (10 × 75 cm, 20 seeds per stripe).The stripes were rolled and placed into beakers with water (50 cm 3 ).The reference group of seedlings ("herbicide" experiments) was kept in 2,4-D solution (10 -3 %) for 1 h and then in water for 1 h.The "control" seedlings were kept in water for 2 h.The temperature of all solutions was maintained at 28 • C. The seedlings were then thermostated for 3 days at 28 • C. Each experiment was performed in triplicate, and 20 seeds were used in each experiment.The results are given in Table 2.

Conclusions
In summary, we have synthesized new [1,2]dithiolo [3,4-b]pyridine-5-carboxamides using a base-promoted reaction of dithiomalondianilide with 3-aryl-2-cyanoacrylamides. The structure of the compounds was confirmed using spectral data and X-ray diffraction analysis.The mechanism of formation of the dithiolopyridine bicyclic ring system was examined in detail through DFT calculations with Grimme's B97-3c composite scheme based on the combination of the B97 GGA functional and the def2-mTZVP basis set with the D3BJ dispersion correction.It was shown that the rate-limiting step is the closure of the pyridine ring, whereas further oxidation and the closure of 1,2-dithiol ring with oxygen in the air results in a lower energy barrier.
The calculation of ADMET parameters for the new compounds was carried out.It was shown that dithiolopyridines 3 do not fully meet the criteria of oral bioavailability.The results of molecular docking studies and a search for possible protein targets for new [1,2]dithiolo [3,4-b]pyridine-5-carboxamides show that the compounds are of interest as promising antitumor agents or as regulators of blood coagulation factor VII. Finally, in contrast to close structural analogs, [1,2]dithiolo [3,4-b]pyridine-5-carboxamides 3 were found to exhibit only moderate herbicide safening effects against 2,4-D in the laboratory experiments with sunflower seedlings.

Figure 2 .
Figure 2. Molecular structures of intermediates I-1-I-5 and transition states TS1-TS5 (geometry and energy optimized at the B97-3c level).Molecules not involved in a particular elementary act are not shown in the figure for ease of perception and better visualization.

20 Figure 2 .
Figure 2. Molecular structures of intermediates I-1-I-5 and transition states TS1-TS5 (geometry and energy optimized at the B97-3c level).Molecules not involved in a particular elementary act are not shown in the figure for ease of perception and better visualization.

Figure 6 .
Figure 6.Position of compound (R)-3a in the site of coagulation factor VII (PDB ID 4yt6_H) via molecular docking.

Figure 6 .
Figure 6.Position of compound (R)-3a in the site of coagulation factor VII (PDB ID 4yt6_H) via molecular docking.

1 H
and13 C DEPTQ NMR spectra and 2D NMR experiments were recorded in solutions of DMSO-d6 on Bruker AVANCE-III HD (Bruker BioSpin AG, Fällanden, Switzerland) and Agilent 400/MR (Agilent Technologies, Santa Clara, CA, USA) instruments (at 400 MHz for 1 H and 101 MHz for 13 C nuclei).Residual solvent signals were used as internal standards in DMSO-d6 (2.49 ppm for 1 H and 39.50 ppm for 13 C nuclei).Single crystal X-ray diffraction analysis of compound 3d was performed on an automatic four-circle diffractometer Agilent Super Nova, Dual, Cu at zero, Atlas S2.High-resolution mass spectra (HRMS) were registered with a Bruker MaXis Impact (Bruker Daltonics, Bremen, Germany) spectrometer (electrospray ionization; HCO2Na-HCO2H was used for calibration).The samples were dissolved in MeCN under moderate heating (37-38 °C) and ultrasonication.See Electronic Supplementary Material file for NMR, FTIR and HRMS spectral charts and X-ray analysis data.FT-IR spectra were measured on a Bruker Vertex 70 instrument (Bruker Optics GmbH & Co. KG, Ettlingen, Germany) equipped with an ATR sampling module.Elemental analyses were carried out using a Carlo Erba 1106 Elemental Analyzer (Carlo Erba

.
The structure and yields of compounds 3.

Table 1 .
The structure and yields of compounds 3.

Table 1 .
The structure and yields of compounds 3.

Table 1 .
The structure and yields of compounds 3.

Table 1 .
The structure and yields of compounds 3.

Table 1 .
The structure and yields of compounds 3.

Table 1 .
The structure and yields of compounds 3.

Table 2 .
The antidote effects of the compounds 3b, 3d and 3f with respect to herbicide 2,4-D.

Table 2 .
The antidote effects of the compounds 3b, 3d and 3f with respect to herbicide 2,4-D.

Table 2 .
The antidote effects of the compounds 3b, 3d and 3f with respect to herbicide 2,4-D.