New 6′-Amino-5′-cyano-2-oxo-1,2-dihydro-1′H-spiro[indole-3,4′-pyridine]-3′-carboxamides: Synthesis, Reactions, Molecular Docking Studies and Biological Activity

The purpose of this work was to prepare new isatin- and monothiomalondiamide-based indole derivatives, as well as to study the properties of the new compounds. The four-component reaction of 5-R-isatins (R = H, CH3), malononitrile, monothiomalonamide (3-amino-3-thioxo- propanamide) and triethylamine in hot EtOH yields a mixture of isomeric triethylammonium 6′-amino-3′-(aminocarbonyl)-5′-cyano-2-oxo-1,2-dihydro-1′H- and 6′-amino-3′-(aminocarbonyl)- 5′-cyano-2-oxo-1,2-dihydro-3′H-spiro[indole-3,4′-pyridine]-2′-thiolates. The reactivity and structure of the products was studied. We found that oxidation of spiro[indole-3,4′-pyridine]-2′-thiolates with DMSO-HCl system produced only acidification products, diastereomeric 6′-amino-5′-cyano-5-methyl-2-oxo-2′-thioxo-1,2,2′,3′-tetrahydro-1′H-spiro-[indole-3,4′-pyridine]- 3′-carboxamides, instead of the expected isothiazolopyridines. The alkylation of the prepared spiro[indole-3,4′-pyridine]-2′-thiolates upon treatment with N-aryl α-chloroacetamides and α-bromoacetophenones proceeds in a regioselective way at the sulfur atom. In the case of α-bromoacetophenones, ring-chain tautomerism was observed for the S-alkylation products. According to NMR data, the compounds consist of a mixture of stereoisomers of 2′-amino-6′-[(2-aryl-2-oxoethyl)thio]-3′-cyano-2-oxo-1′H-spiro[indoline-3,4′-pyridine]-5′-carboxamides and 5′-amino-3′-aryl-6′-cyano-3′-hydroxy-2-oxo-2′,3′-dihydrospiro[indoline-3,7′-thiazolo[3,2-a]pyridine]-8′-carboxamides in various ratios. The structure of the synthesized compounds was confirmed by IR spectroscopy, HRMS, 1H and 13C DEPTQ NMR studies and the results of 2D NMR experiments (1H-13C HSQC, 1H-13C HMBC). Molecular docking studies were performed to investigate suitable binding modes of some new compounds with respect to the transcriptional regulator protein PqsR of Pseudomonas aeruginosa. The docking studies revealed that the compounds have affinity for the bacterial regulator protein PqsR of Pseudomonas aeruginosa with a binding energy in the range of −5.8 to −8.2 kcal/mol. In addition, one of the new compounds, 2′-amino-3′-cyano-5-methyl-2-oxo-6′-{[2-oxo-2-(p-tolylamino)ethyl]thio}-1′H-spiro-[indoline-3,4′-pyridine]-5′-carboxamide, showed in vitro moderate antibacterial effect against Pseudomonas aeruginosa and good antioxidant properties in a test with 1,1-diphenyl-2-picrylhydrazyl radical. Finally, three of the new compounds were recognized as moderately active herbicide safeners with respect to herbicide 2,4-D in the laboratory experiments on sunflower seedlings.


Introduction
Nicotinonitriles, nicotinamides and their partially saturated analogs represent a promising class of heterocyclic compounds with an interesting profile of biological activity (for reviews, see [1][2][3][4][5][6][7][8][9][10][11]). However, while nicotinonitriles have been fairly well studied, the related synthetic nicotinamides and 1,4-dihydronicotinamides have been less studied and require further investigation. One of the most accessible and efficient approaches to the synthesis of functionalized nicotinamides is based on the reaction of active methylene malonamides and malonthioamides with 1,3-C3 dielectrophiles [12][13][14][15][16][17][18][19][20][21][22]. Monothiomalonamide 1 (3-amino-3-thioxopropanamide) is known as a very convenient reagent for the preparation of substituted nicotinamides. It can be easily synthesized by passing H2S through a hot saturated solution of cyanoacetamide in pyridine in the presence of Et3N [23,24]. Substituted nicotinamides can be prepared by the reaction of monothiomalonamide 1 with malononitrile and aldehydes (or with arylmethylene malononitriles originated from Knoevenagel-type condensation) (Scheme 1). At the same time, it was noted that the structure of the reaction products strongly depends on the conditions. Thus, the paper [25] describes the preparation of 1,4-dihydropyridines 2 by reaction of monothiomalonamide 1 with aldehydes and malononitrile in the presence of Et3N in hot EtOH solution. However, other authors demonstrated the formation of nicotinamides 3 under similar reaction conditions [26]. As shown in paper [27], the reaction of thioamide 1 with benzylidene malononitrile may result in the formation of three different products, depending on the temperature, base and solvent used: the Michael adducts 4, 1,4-dihydropyridin-2-thiolates similar to compounds 2, or their 3,4-dihydro isomers 5 (Scheme 1). The formation of 3,4-dihydronicotinamides was also reported in other papers [13,21,28] Scheme 1. Possible pathways for reaction of monothiomalonamide 1 with malonononitrile and aldehydes.
Isatins have not previously been reacted with monothiomalonamide 1 and malononitrile. The expected products of spiro[indoline-3,4′-pyridine] structure are of interest as promising biologically active agents.
Isatins have not previously been reacted with monothiomalonamide 1 and malononitrile. The expected products of spiro[indoline-3,4 -pyridine] structure are of interest as promising biologically active agents.
This work presents our research results concerning the reaction of monothiomalonamide 1 with isatins and malononitrile under basic conditions, as well as reactivity features, molecular docking studies and biological activities of the prepared 2 -amino-3 -cyano-2oxospiro[indoline-3,4 -pyridine]-5 -carboxamides.
Overall, the NMR spectra of the compounds 13 and 14 show a complex picture. It is noteworthy that in some cases the signals of the minor 3 H-isomers 13b,14b exhibit signal doubling, apparently due to the splitting of the signals of two diastereomeric pairs (Scheme 4): Full signal assignment for 13a + 13b and 14a + 14b mixtures was based on an analysis of the IR and NMR spectroscopy data, including 13 C DEPTQ and 2D 1 H-13 C HSQC and 1 H-13 C HMBC experiments (Supplementary Data File). Thus, the 1 H NMR spectra of 13a + 13b and 14a + 14b revealed a double set of aromatic proton signals and two peaks of NH2 protons (for major 1′H isomer at δ 5.36-5.37 ppm and for minor 3′H isomer at δ 6.23-6.25 ppm), as well as a common signal for NH isatin protons (δ 9.44-9.55 ppm) and one peak of pyridine NH proton of the major 1′H isomer at δ 7.67-7.69 ppm.
Noteworthy is the fact that the signals of the CONH2 protons were observed as two peaks with a very significant difference in chemical shifts (∆δ ≥ 4 ppm). Thus, for the major 1′H isomer, CONH2 protons appeared at δ 5.75-5.77 ppm and δ 10.25-10.26 ppm, while the peaks of the minor 3′H isomer were observed at δ 6.32-6.37 ppm and δ 10.19-10.30 ppm. We assume that the S-C=C-C(O)NH2 fragment is planar because of conjugation. The observed difference in chemical shifts can be explained by the presence of an intramolecular hydrogen bond between one of the CONH2 protons and the negatively charged sulfur atom on the one hand, and the anisotropic shielding effect of the carbonyl group on the second proton on the other (Scheme 5).

Scheme 5.
The local shielding/deshielding effects on the CONH2 protons.
Due to the presence of a number of active functional groups, the resulting thiolates 13 and 14 seem to be attractive molecules for further transformations. Thus, in order to explore the reactivity of spiro[indole-3,4′-pyridine]-2′-thiolates, we attempted to oxidize thiolates 13 and 14 with dimethyl sulfoxide (DMSO)-HCl system. The oxidation of 2-mercaptonicotinamides and related compounds was reported as a general method for the synthesis of 3-oxo-2,3-dihydroisothiazolo [5,4-b]pyridines [24,[81][82][83]. Such compounds are direct structural analogues of the practically important thieno [2,3-b]pyridines [84]. However, isothiazolo [5,4-b]pyridines are much less studied, although some compounds showed anti-tuberculosis [85] effects, were reported as COX-1 cyclooxygenase inhibitors [86], analgesics [87] and strong inhibitors of histone acetyltransferases with potential anticancer effects [88]. Previously, we have reported that the DMSO-HCl sys- Noteworthy is the fact that the signals of the CONH 2 protons were observed as two peaks with a very significant difference in chemical shifts (∆δ ≥ 4 ppm). Thus, for the major 1 H isomer, CONH 2 protons appeared at δ 5.75-5.77 ppm and δ 10.25-10.26 ppm, while the peaks of the minor 3 H isomer were observed at δ 6.32-6.37 ppm and δ 10.19-10.30 ppm. We assume that the S-C=C-C(O)NH 2 fragment is planar because of conjugation. The observed difference in chemical shifts can be explained by the presence of an intramolecular hydrogen bond between one of the CONH 2 protons and the negatively charged sulfur atom on the one hand, and the anisotropic shielding effect of the carbonyl group on the second proton on the other (Scheme 5). Full signal assignment for 13a + 13b and 14a + 14b mixtures was based on an analysis of the IR and NMR spectroscopy data, including 13 C DEPTQ and 2D 1 H-13 C HSQC and 1 H-13 C HMBC experiments (Supplementary Data File). Thus, the 1 H NMR spectra of 13a + 13b and 14a + 14b revealed a double set of aromatic proton signals and two peaks of NH2 protons (for major 1′H isomer at δ 5.36-5.37 ppm and for minor 3′H isomer at δ 6.23-6.25 ppm), as well as a common signal for NH isatin protons (δ 9.44-9.55 ppm) and one peak of pyridine NH proton of the major 1′H isomer at δ 7.67-7.69 ppm.
Overall, the NMR spectra of the compounds 13 and 14 show a complex picture. It is noteworthy that in some cases the signals of the minor 3′H-isomers 13b,14b exhibit signal doubling, apparently due to the splitting of the signals of two diastereomeric pairs (Scheme 4): Noteworthy is the fact that the signals of the CONH2 protons were observed as two peaks with a very significant difference in chemical shifts (∆δ ≥ 4 ppm). Thus, for the major 1′H isomer, CONH2 protons appeared at δ 5.75-5.77 ppm and δ 10.25-10.26 ppm, while the peaks of the minor 3′H isomer were observed at δ 6.32-6.37 ppm and δ 10.19-10.30 ppm. We assume that the S-C=C-C(O)NH2 fragment is planar because of conjugation. The observed difference in chemical shifts can be explained by the presence of an intramolecular hydrogen bond between one of the CONH2 protons and the negatively charged sulfur atom on the one hand, and the anisotropic shielding effect of the carbonyl group on the second proton on the other (Scheme 5).

Scheme 5.
The local shielding/deshielding effects on the CONH2 protons.
Molecules 2023, 28, x FOR PEER REVIEW 5 of 20 tem can be successfully used as a mild oxidizing agent to convert 2-mercaptopyridin-3-carboxamides into isothiazolo [5,4-b]pyridines [72,89]. Nevertheless, upon treatment of thiolates 14a + 14b with excess HCl in DMSO solution, only corresponding 2-thioxopyridine 16 was isolated in 88% yield instead of the expected products 15. According to NMR spectroscopy data, compound 16 exists in the solution as a mixture of diastereomers in the ratio ~ 3:5 (Scheme 6).

Scheme 6.
Synthesis and stereochemical features of 2-thioxopyridine 16. An asterisk (*) marks the chiral centers in the molecules.
Alkylation of thiolates is a well-known approach to the diversity of heterocyclic thio ethers. Therefore, we decided to study the alkylation of thiolates 13 and 14 with some active α-halo carbonyls. It is well known that the alkylation of pyridine-2-thiolates and related compounds proceed in a regiospecific way at the sulfur atom. The S-alkylation products have been proven to be multifunctional reagents for fine organic synthesis and were recognized as biologically active compounds (for reviews, see [1,6,[9][10][11][90][91][92]).
As expected, upon treatment with N-aryl α-chloroacetamides and α-bromoacetophenones, thiolates 13 and 14 underwent regiospecific alkylation to give exclusively S-alkylation products in high yields. The structure of the products was confirmed by means of IR, 1 H NMR, 13 C NMR DEPTQ and HRMS spectroscopy. Analysis of spectral data of compounds 17 (Scheme 7) showed that the relevant products had the structure of 1′H-isomers only, while 3′H-isomers were not detected. This can be explained by the ease of prototropic migration 3′H→1′H with the formation of thermodynamically more stable products; a similar phenomenon was previously observed in some other S-alkylation reactions [13,28]. In actuality, there are two chiral centers in the molecule 16; therefore, the compound can exist as a mixture of two diastereomeric pairs-(3S,3 R)/(3R,3 S) and (3S,3 S)(3R,3 R) (Scheme 6). The H-3 proton signal of the minor isomers (δ 4.06 ppm) is noticeably shifted downfield with respect to the H-3 signal of the major isomers (δ 3.78 ppm). Hence, we believe that the H-3 proton of major isomer is affected by the anisotropic shielding effect of indoline C-2 carbonyl group, while the minor isomer H-3 proton is not. Therefore, the (3S,3 S)/(3R,3 R) configuration should be assigned to the major diastereomers, and the (3S,3 R)(3R,3 S) configuration to the minor diastereomeric pair.
Alkylation of thiolates is a well-known approach to the diversity of heterocyclic thio ethers. Therefore, we decided to study the alkylation of thiolates 13 and 14 with some active α-halo carbonyls. It is well known that the alkylation of pyridine-2-thiolates and related compounds proceed in a regiospecific way at the sulfur atom. The S-alkylation products have been proven to be multifunctional reagents for fine organic synthesis and were recognized as biologically active compounds (for reviews, see [1,6,[9][10][11][90][91][92]).
As expected, upon treatment with N-aryl α-chloroacetamides and α-bromoacetophenones, thiolates 13 and 14 underwent regiospecific alkylation to give exclusively S-alkylation products in high yields. The structure of the products was confirmed by means of IR, 1 H NMR, 13 C NMR DEPTQ and HRMS spectroscopy. Analysis of spectral data of compounds 17 (Scheme 7) showed that the relevant products had the structure of 1 H-isomers only, while 3 H-isomers were not detected. This can be explained by the ease of prototropic migration 3 H→1 H with the formation of thermodynamically more stable products; a similar phenomenon was previously observed in some other S-alkylation reactions [13,28]. It should also be noted that the spectra of carboxamides 17a,b revealed the expected patterns, whereas the spectral picture of the alkylation products with α-bromacetophenones was very complex and contained signals of at least three different compounds. This pattern can be explained by the ring tautomerism of compounds 17c,d in a solution. In addition, cyclic tautomers-thiazolo[3,2-a]pyridines 17c-cycl and 17d-cycl each have two stereogenic centers and are mixtures of two diastereomeric pairs (Scheme 8). It is noteworthy that similar ring-chain tautomerism was also previously observed in a number of related compounds [28,93].
The signals in the 1 H NMR spectra of ring and chain forms of 17c,d were assigned based on the integral intensities of SCH2 signals as well as on the presence of OH and pyridine NH proton signals. The major cyclic tautomers, thiazolo [  Nevertheless, the unambiguous assignment of signals in the NMR spectra of all tautomers and stereoisomers in the 17c,d system seems difficult. Additionally, the question of which tautomeric form of 17c,d is preferred in the solid state remains unclear; the presence of a strong absorption band at ν 1697-1701 cm −1 , which may be assigned to keto carbonyls, and the absence of band doubling indirectly indicate the preference of noncyclic forms. However, other authors [93] showed, using an X-ray method, that compounds It should also be noted that the spectra of carboxamides 17a,b revealed the expected patterns, whereas the spectral picture of the alkylation products with α-bromacetophenones was very complex and contained signals of at least three different compounds. This pattern can be explained by the ring tautomerism of compounds 17c,d in a solution. In addition, cyclic tautomers-thiazolo[3,2-a]pyridines 17c-cycl and 17d-cycl each have two stereogenic centers and are mixtures of two diastereomeric pairs (Scheme 8). It is noteworthy that similar ring-chain tautomerism was also previously observed in a number of related compounds [28,93].
The signals in the 1 H NMR spectra of ring and chain forms of 17c,d were assigned based on the integral intensities of SCH 2 signals as well as on the presence of OH and pyridine NH proton signals. The major cyclic tautomers, thiazolo [ It should also be noted that the spectra of carboxamides 17a,b revealed the expected patterns, whereas the spectral picture of the alkylation products with α-bromacetophenones was very complex and contained signals of at least three different compounds. This pattern can be explained by the ring tautomerism of compounds 17c,d in a solution. In addition, cyclic tautomers-thiazolo[3,2-a]pyridines 17c-cycl and 17d-cycl each have two stereogenic centers and are mixtures of two diastereomeric pairs (Scheme 8). It is noteworthy that similar ring-chain tautomerism was also previously observed in a number of related compounds [28,93].
The signals in the 1 H NMR spectra of ring and chain forms of 17c,d were assigned based on the integral intensities of SCH2 signals as well as on the presence of OH and pyridine NH proton signals. The major cyclic tautomers, thiazolo [  Nevertheless, the unambiguous assignment of signals in the NMR spectra of all tautomers and stereoisomers in the 17c,d system seems difficult. Additionally, the question of which tautomeric form of 17c,d is preferred in the solid state remains unclear; the presence of a strong absorption band at ν 1697-1701 cm −1 , which may be assigned to keto carbonyls, and the absence of band doubling indirectly indicate the preference of noncyclic forms. However, other authors [93] showed, using an X-ray method, that compounds Nevertheless, the unambiguous assignment of signals in the NMR spectra of all tautomers and stereoisomers in the 17c,d system seems difficult. Additionally, the question of which tautomeric form of 17c,d is preferred in the solid state remains unclear; the presence of a strong absorption band at ν 1697-1701 cm −1 , which may be assigned to keto carbonyls, and the absence of band doubling indirectly indicate the preference of noncyclic forms. However, other authors [93] showed, using an X-ray method, that compounds with such type of ring-chain tautomerism in the solid state exist as thiazolo [3,2-a]pyridines. In general, this question requires further exploration.

Docking Studies
First, we used the Osiris property explorer on-line tool [94] for in silico prediction of the drug likeness of the synthesized compounds. Salts 13 and 14, as well as tautomeric mixtures 17c,d, were excluded from further consideration. All the molecules 16,17a,b were found to be non-toxic in terms of mutagenicity, carcinogenicity, skin irritancy and reproductive effects.
Many indoles/isatins, nicotinonitriles and hybrid compounds combining fragments of the above systems are of interest as antimicrobial agents. Recently, indole-containing compounds have been reported to have antibacterial activity as well as activity as resistancemodifying agents [95][96][97][98]. It is noteworthy that infections caused by Pseudomonas aeruginosa pose a real problem, especially in critically ill and immunodeficient patients. The main cause of high mortality is the emergence of drug-resistant strains [99].
For this reason, we decided to investigate the antibacterial potential of the new compounds 16,17a,b against Pseudomonas aeruginosa. The LysR-type transcriptional regulator protein PqsR (PDB ID 4JVC) was chosen as a target. This bacterial transcriptional regulator protein is a key component of alkyl-quinolone-dependent quorum sensing in Pseudomonas aeruginosa and could be considered as a potential target for new antibacterial agents that attenuate infection by blocking virulence [100].
The energy-minimized 3D structures for each individual stereoisomer of ligand molecules 16,17a,b were docked using Autodock Vina against the bacterial regulator protein PqsR of Pseudomonas aeruginosa (PDB ID 4JVC) at standard precision mode (SP). The docking score of all the ligand molecules 16,17a,b were found to be in the range of −5.8 to −8.2 kcal/mol.
The docking results are presented in Table 1. As follows from Table 1, both the (R)and (S)-enantiomers of compound 17a had the minimum value of the scoring function and hence showed the best affinity to the protein target. The major interactions between the ligands with the target protein can be categorized as "hydrogen bonding, hydrophobic", which was crucial to stabilize the inhibitors inside the binding pocket of the receptor. The most important interactions are shown in Figures 1 and 2.

Docking Studies
First, we used the Osiris property explorer on-line tool [94] for in silico prediction of the drug likeness of the synthesized compounds. Salts 13 and 14, as well as tautomeric mixtures 17c,d, were excluded from further consideration. All the moleϲules 16,17a,b were found to be non-toxiϲ in terms of mutageniϲity, carcinogenicity, skin irritanϲy and reproduϲtive effects.
Many indoles/isatins, nicotinonitriles and hybrid compounds combining fragments of the above systems are of interest as antimicrobial agents. Recently, indole-containing compounds have been reported to have antibacterial activity as well as activity as resistance-modifying agents [95][96][97][98]. It is noteworthy that infections caused by Pseudomonas aeruginosa pose a real problem, especially in critically ill and immunodeficient patients. The main cause of high mortality is the emergence of drug-resistant strains [99].
For this reason, we decided to investigate the antibacterial potential of the new compounds 16,17a,b against Pseudomonas aeruginosa. The LysR-type transcriptional regulator protein PqsR (PDB ID 4JVC) was chosen as a target. This bacterial transcriptional regulator protein is a key component of alkyl-quinolone-dependent quorum sensing in Pseudomonas aeruginosa and could be considered as a potential target for new antibacterial agents that attenuate infection by blocking virulence [100].
The energy-minimized 3D structures for each individual stereoisomer of ligand molecules 16,17a,b were docked using Autodock Vina against the bacterial regulator protein PqsR of Pseudomonas aeruginosa (PDB ID 4JVC) at standard precision mode (SP). The docking score of all the ligand molecules 16,17a,b were found to be in the range of -5.8 to -8.2 kcal/mol.
The docking results are presented in Table 1. As follows from Table 1, both the (R)and (S)-enantiomers of compound 17a had the minimum value of the scoring function and hence showed the best affinity to the protein target. The major interaϲtions between the ligands with the target protein ϲan be ϲategorized as "hydrogen bonding, hydrophobiϲ", whiϲh was crucial to stabilize the inhibitors inside the binding poϲket of the reϲeptor. The most important interaϲtions are shown in Figures 1 and 2.

Docking Studies
First, we used the Osiris property explorer on-line tool [94] for in silico prediction of the drug likeness of the synthesized compounds. Salts 13 and 14, as well as tautomeric mixtures 17c,d, were excluded from further consideration. All the moleϲules 16,17a,b were found to be non-toxiϲ in terms of mutageniϲity, carcinogenicity, skin irritanϲy and reproduϲtive effects.
Many indoles/isatins, nicotinonitriles and hybrid compounds combining fragments of the above systems are of interest as antimicrobial agents. Recently, indole-containing compounds have been reported to have antibacterial activity as well as activity as resistance-modifying agents [95][96][97][98]. It is noteworthy that infections caused by Pseudomonas aeruginosa pose a real problem, especially in critically ill and immunodeficient patients. The main cause of high mortality is the emergence of drug-resistant strains [99].
For this reason, we decided to investigate the antibacterial potential of the new compounds 16,17a,b against Pseudomonas aeruginosa. The LysR-type transcriptional regulator protein PqsR (PDB ID 4JVC) was chosen as a target. This bacterial transcriptional regulator protein is a key component of alkyl-quinolone-dependent quorum sensing in Pseudomonas aeruginosa and could be considered as a potential target for new antibacterial agents that attenuate infection by blocking virulence [100].
The energy-minimized 3D structures for each individual stereoisomer of ligand molecules 16,17a,b were docked using Autodock Vina against the bacterial regulator protein PqsR of Pseudomonas aeruginosa (PDB ID 4JVC) at standard precision mode (SP). The docking score of all the ligand molecules 16,17a,b were found to be in the range of -5.8 to -8.2 kcal/mol.
The docking results are presented in Table 1. As follows from Table 1, both the (R)and (S)-enantiomers of compound 17a had the minimum value of the scoring function and hence showed the best affinity to the protein target. The major interaϲtions between the ligands with the target protein ϲan be ϲategorized as "hydrogen bonding, hydrophobiϲ", whiϲh was crucial to stabilize the inhibitors inside the binding poϲket of the reϲeptor. The most important interaϲtions are shown in Figures 1 and 2.

Docking Studies
First, we used the Osiris property explorer on-line tool [94] for in silico prediction of the drug likeness of the synthesized compounds. Salts 13 and 14, as well as tautomeric mixtures 17c,d, were excluded from further consideration. All the moleϲules 16,17a,b were found to be non-toxiϲ in terms of mutageniϲity, carcinogenicity, skin irritanϲy and reproduϲtive effects.
Many indoles/isatins, nicotinonitriles and hybrid compounds combining fragments of the above systems are of interest as antimicrobial agents. Recently, indole-containing compounds have been reported to have antibacterial activity as well as activity as resistance-modifying agents [95][96][97][98]. It is noteworthy that infections caused by Pseudomonas aeruginosa pose a real problem, especially in critically ill and immunodeficient patients. The main cause of high mortality is the emergence of drug-resistant strains [99].
For this reason, we decided to investigate the antibacterial potential of the new compounds 16,17a,b against Pseudomonas aeruginosa. The LysR-type transcriptional regulator protein PqsR (PDB ID 4JVC) was chosen as a target. This bacterial transcriptional regulator protein is a key component of alkyl-quinolone-dependent quorum sensing in Pseudomonas aeruginosa and could be considered as a potential target for new antibacterial agents that attenuate infection by blocking virulence [100].
The energy-minimized 3D structures for each individual stereoisomer of ligand molecules 16,17a,b were docked using Autodock Vina against the bacterial regulator protein PqsR of Pseudomonas aeruginosa (PDB ID 4JVC) at standard precision mode (SP). The docking score of all the ligand molecules 16,17a,b were found to be in the range of -5.8 to -8.2 kcal/mol.
The docking results are presented in Table 1. As follows from Table 1, both the (R)and (S)-enantiomers of compound 17a had the minimum value of the scoring function and hence showed the best affinity to the protein target. The major interaϲtions between the ligands with the target protein ϲan be ϲategorized as "hydrogen bonding, hydrophobiϲ", whiϲh was crucial to stabilize the inhibitors inside the binding poϲket of the reϲeptor. The most important interaϲtions are shown in Figures 1 and 2.

Antibacterial and Antioxidant Activity of Compound 17a
The leader compound 17a was also tested in vitro to determine its ability to inhibit pathogen growth by the agar well-diffusion method using Mueller-Hinton agar at 100 mg/mL concentration. Antibacterial activity was tested against two strains of Gramnegative bacteria: Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853. The reference drug was Ciprofloxacin. The results of in vitro studies showed that 17a efficiently suppresses Gram-negative bacteria P. aeruginosa ATCC 27853, but has no effect against Gram-positive bacteria. Compound 17a also demonstrated a moderate inhibitory effect against E. coli. Then, the minimum inhibitory concentration (MIC) value for 17a was determined based on the results of the examination according to the protocol described in [101]. Ciprofloxacin was used as the reference drug. We found that compound 17a showed moderate antibacterial activity with MIC 12.5 µg/mL (Ciprofloxacin-1 µg/mL).
The ability of 17a to scavenge DPPH radical (1,1-diphenyl-2-picrylhydrazyl) was examined by the method proposed by Blois [102]. Antioxidant activity was calculated in % of inhibition according to Equation (1): where I-inhibition effect, %; A control -absorption in control experiment; and A test -absorption in test with compound 17a.
The results are shown in Table 2. As follows from Table 2, compound 17a showed antioxidant effects comparable to those of ascorbic acid.

Agrochemical Studies
Some of the new compounds were tested as herbicide safeners with respect to 2,4-dichlorophenoxyacetic acid (2,4-D). 2,4-D is widely used and non-toxic for human herbicides [103]. However, 2,4-D is toxic for sunflowers. If the recommended dose for controlling weeds in resistant cereal crops is 0.5-0.8 kg/ha of 2,4-D by the active substance, the 15-18 g/ha dose for sunflower leads to a 40-60% decrease in yield.
One of the most effective approaches to increase plant resistance towards herbicides is the activation of metabolic processes affected by herbicide safeners (also called herbicide antidotes or detoxifiers) [104,105]. Herbicide safeners can be defined [106] as agrochemicals suitable for neutralization of phytotoxins in plants, thus protecting crop plants from herbicide injury. Safeners are harmless to crop plants (or even have a growth-stimulating effect), but do not affect the activity of herbicides against weeds.
It is known that some functionalized pyridines are efficient herbicide safeners and plant growth regulators [107][108][109][110][111] . Nicotinamides 16a and 17a,b were studied as 2,4-D antidotes with respect to sunflower seedlings using the reported procedure [107] (see the Materials and Methods). The antidote effect A was determined as a ratio of the hypocotyl (or root) length of sunflower seedlings in the "herbicide + antidote" experiments to the length in the reference group (where the seedlings were treated with 2,4-D only) (Equation (2)): where L exp is an organ length (mm) in the group of seedlings treated with 2,4-D and tested compound, and L ref is an organ length (mm) in the reference group of sunflower seedlings. We found that compounds 16a and 17a,b showed moderate 2,4-D antidote effect in the laboratory experiments (Table 3). As we can see, spiro[indole-3,4 -pyridines] 16a and 17a,b reduced the negative effect of 2,4-D on sunflower seedling hypocotyls by 16-33% and by 12-25% on sunflower seedling roots. where Lexp is an organ length (mm) in the group of seedlings treated with 2,4-D and tested compound, and Lref is an organ length (mm) in the reference group of sunflower seedlings. We found that compounds 16a and 17a,b showed moderate 2,4-D antidote effect in the laboratory experiments (Table 3). As we can see, spiro[indole-3,4′-pyridines] 16a and 17a,b reduced the negative effect of 2,4-D on sunflower seedling hypocotyls by 16-33% and by 12-25% on sunflower seedling roots.

Materials and Methods
1 H and 13 C DEPTQ NMR spectra and 2D NMR experiments were recorded in solutions of DMSO-d6 on a Bruker AVANCE-III HD instrument (at 400.40 or 100.61 MHz, respectively). 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. HRMS spectra were recorded using a Bruker MaXis Impact quadrupole time-of-flight mass spectrometer equipped with an electrospray ionization source in positive ion detection mode. The voltage at the ionization source was 3.5 kV, the drying gas flow rate was 8 L/min, the spray gas pressure was 2 bar, the temperature of the ionization source was 250 °C, the mass scanning range (m/z) was 50-1000 and the scanning speed was 3 Hz. The data were processed using Bruker Data Analysis 4.1 software. See Supplementary Materials File for NMR, FTIR and HRMS spectral charts.
FT-IR spectra were measured on a Bruker Vertex 70 instrument equipped with an ATR sampling module. Elemental analyses were carried out using a Carlo Erba 1106 Elemental Analyzer. Reaction progress and purity of isolated compounds were controlled by TLC on Sorbfil-A plates, eluent-acetone:hexane 1:1 or ethyl acetate:light petroleum 3:1, the spots were visualized in UV-light and iodine vapors. Monothiomalonamide 1 (3-amino-3-thioxopropanamide) was prepared from α-cyanoacetamide and hydrogen sulfide as described earlier [23,24]. Isatins, triethylamine, malononitrile and solvents were purchased from commercial vendors.
The reaction of isatins, malononitrile and monothiomalonamide. In a 50 mL beaker, 0.5 g (7.57 mmol) of malononitrile, 7.57 mmol of the corresponding isatin (1.11 g of isatin or 1.22 g of 5-methylisatin) and 20 mL of EtOH were added. Two drops of triethylamine were added to the suspension under heating (50 °C) and stirring. The reaction mixture became dark red due to the formation of the corresponding Knoevenagel con- where Lexp is an organ length (mm) in the group of seedlings treated with 2,4-D and tested compound, and Lref is an organ length (mm) in the reference group of sunflower seedlings. We found that compounds 16a and 17a,b showed moderate 2,4-D antidote effect in the laboratory experiments (Table 3). As we can see, spiro[indole-3,4′-pyridines] 16a and 17a,b reduced the negative effect of 2,4-D on sunflower seedling hypocotyls by 16-33% and by 12-25% on sunflower seedling roots.

Materials and Methods
1 H and 13 C DEPTQ NMR spectra and 2D NMR experiments were recorded in solutions of DMSO-d6 on a Bruker AVANCE-III HD instrument (at 400.40 or 100.61 MHz, respectively). 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. HRMS spectra were recorded using a Bruker MaXis Impact quadrupole time-of-flight mass spectrometer equipped with an electrospray ionization source in positive ion detection mode. The voltage at the ionization source was 3.5 kV, the drying gas flow rate was 8 L/min, the spray gas pressure was 2 bar, the temperature of the ionization source was 250 °C, the mass scanning range (m/z) was 50-1000 and the scanning speed was 3 Hz. The data were processed using Bruker Data Analysis 4.1 software. See Supplementary Materials File for NMR, FTIR and HRMS spectral charts.
FT-IR spectra were measured on a Bruker Vertex 70 instrument equipped with an ATR sampling module. Elemental analyses were carried out using a Carlo Erba 1106 Elemental Analyzer. Reaction progress and purity of isolated compounds were controlled by TLC on Sorbfil-A plates, eluent-acetone:hexane 1:1 or ethyl acetate:light petroleum 3:1, the spots were visualized in UV-light and iodine vapors. Monothiomalonamide 1 (3-amino-3-thioxopropanamide) was prepared from α-cyanoacetamide and hydrogen sulfide as described earlier [23,24]. Isatins, triethylamine, malononitrile and solvents were purchased from commercial vendors.
The reaction of isatins, malononitrile and monothiomalonamide. In a 50 mL beaker, 0.5 g (7.57 mmol) of malononitrile, 7.57 mmol of the corresponding isatin (1.11 g of isatin or 1.22 g of 5-methylisatin) and 20 mL of EtOH were added. Two drops of triethylamine were added to the suspension under heating (50 °C) and stirring. The reaction mixture became dark red due to the formation of the corresponding Knoevenagel con- where Lexp is an organ length (mm) in the group of seedlings treated with 2,4-D and tested compound, and Lref is an organ length (mm) in the reference group of sunflower seedlings. We found that compounds 16a and 17a,b showed moderate 2,4-D antidote effect in the laboratory experiments (Table 3). As we can see, spiro[indole-3,4′-pyridines] 16a and 17a,b reduced the negative effect of 2,4-D on sunflower seedling hypocotyls by 16-33% and by 12-25% on sunflower seedling roots.

Materials and Methods
1 H and 13 C DEPTQ NMR spectra and 2D NMR experiments were recorded in solutions of DMSO-d6 on a Bruker AVANCE-III HD instrument (at 400.40 or 100.61 MHz, respectively). 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. HRMS spectra were recorded using a Bruker MaXis Impact quadrupole time-of-flight mass spectrometer equipped with an electrospray ionization source in positive ion detection mode. The voltage at the ionization source was 3.5 kV, the drying gas flow rate was 8 L/min, the spray gas pressure was 2 bar, the temperature of the ionization source was 250 °C, the mass scanning range (m/z) was 50-1000 and the scanning speed was 3 Hz. The data were processed using Bruker Data Analysis 4.1 software. See Supplementary Materials File for NMR, FTIR and HRMS spectral charts.
FT-IR spectra were measured on a Bruker Vertex 70 instrument equipped with an ATR sampling module. Elemental analyses were carried out using a Carlo Erba 1106 Elemental Analyzer. Reaction progress and purity of isolated compounds were controlled by TLC on Sorbfil-A plates, eluent-acetone:hexane 1:1 or ethyl acetate:light petroleum 3:1, the spots were visualized in UV-light and iodine vapors. Monothiomalonamide 1 (3-amino-3-thioxopropanamide) was prepared from α-cyanoacetamide and hydrogen sulfide as described earlier [23,24]. Isatins, triethylamine, malononitrile and solvents were purchased from commercial vendors.
The reaction of isatins, malononitrile and monothiomalonamide. In a 50 mL beaker, 0.5 g (7.57 mmol) of malononitrile, 7.57 mmol of the corresponding isatin (1.11 g of isatin or 1.22 g of 5-methylisatin) and 20 mL of EtOH were added. Two drops of triethylamine were added to the suspension under heating (50 °C) and stirring. The reaction mixture became dark red due to the formation of the corresponding Knoevenagel con-

Materials and Methods
1 H and 13 C DEPTQ NMR spectra and 2D NMR experiments were recorded in solutions of DMSO-d 6 on a Bruker AVANCE-III HD instrument (at 400.40 or 100.61 MHz, respectively). Residual solvent signals were used as internal standards in DMSO-d 6 -2.49 ppm for 1 H, and 39.50 ppm for 13 C nuclei. HRMS spectra were recorded using a Bruker MaXis Impact quadrupole time-of-flight mass spectrometer equipped with an electrospray ionization source in positive ion detection mode. The voltage at the ionization source was 3.5 kV, the drying gas flow rate was 8 L/min, the spray gas pressure was 2 bar, the temperature of the ionization source was 250 • C, the mass scanning range (m/z) was 50-1000 and the scanning speed was 3 Hz. The data were processed using Bruker Data Analysis 4.1 software. See Supplementary Materials File for NMR, FTIR and HRMS spectral charts.
FT-IR spectra were measured on a Bruker Vertex 70 instrument equipped with an ATR sampling module. Elemental analyses were carried out using a Carlo Erba 1106 Elemental Analyzer. Reaction progress and purity of isolated compounds were controlled by TLC on Sorbfil-A plates, eluent-acetone:hexane 1:1 or ethyl acetate:light petroleum 3:1, the spots were visualized in UV-light and iodine vapors. Monothiomalonamide 1 (3-amino-3-thioxopropanamide) was prepared from α-cyanoacetamide and hydrogen sulfide as described earlier [23,24]. Isatins, triethylamine, malononitrile and solvents were purchased from commercial vendors.
The reaction of isatins, malononitrile and monothiomalonamide. In a 50 mL beaker, 0.5 g (7.57 mmol) of malononitrile, 7.57 mmol of the corresponding isatin (1.11 g of isatin or 1.22 g of 5-methylisatin) and 20 mL of EtOH were added. Two drops of triethylamine were added to the suspension under heating (50 • C) and stirring. The reaction mixture became dark red due to the formation of the corresponding Knoevenagel condensation product. Stirring at 50 • C was continued for another 0.5 h, after which 0.89 g (7.57 mmol) of monothiomalonamide 1 and 1.6 mL (11.35 mmol, 1.5 eq.) of triethylamine were added. The mixture was stirred under vigorous heating (50 • C) for 2 h, then cooled and kept in a freezer overnight. The precipitate was filtered off and washed with acetone until colorless washings were obtained and then dried at 60 • C.

Antibacterial Studies
A suspension of pathogens isolated from a swollen ear canal was prepared by transferring a loop full of isolated colony to 5 mL of normal saline in tubes. The turbidity of the bacterial growth in tubes was then compared with the turbidity of 0.5 McFarland suspension to give an approximate number of live cells equal to 0.5 × 10 8 CFU/mL. Then, 0.1 mL of the bacterial suspension was transferred and spread homogeneously over the Mueller-Hinton agar with a sterile swab and the plate was allowed to stand for 10 min at room temperature to dry. Using a sterile cork borer, a 6 mm diameter hole was made in each prepared plate. A solution of compound 17a at 100 mg/mL concentration was prepared by dissolving 17a in dimethyl sulfoxide (DMSO). Then, 100 µL of the solution was added using a micropipette in each hole under sterile conditions; DMSO was also added as control. The plate was kept for 24 h at 37 • C in an incubator. The diameter of the inhibitory zone around each hole was measured in millimeters.
The minimum inhibitory concentration (MIC) against P. aeruginosa ATCC 27,853 was determined using the classical broth dilution method [101] by preparing differently concentrated solutions of 17a using nutrient broth along with positive and negative controls for each pathogenic bacteria. The turbidity of P. aeruginosa suspension was brought to 0.5 Mc-Farland and 100 µL of bacterial suspension was added to the solutions of 17a. The positive control consisted of 1 mL of nutrient broth and 100 µL of bacterial suspension, and 1 mL of nutrient broth was used only as a negative control. All tubes were incubated at 37 • C for 24 h. After incubation, 100 µL of suspension was dropped onto sterile Mueller-Hinton agar and the plates were incubated at 37 • C for 24 h.

Antioxidant Activity of Compound 17a
The solutions of different concentrations (100,000, 10,000, 1000 and 100 µg/mL) were prepared from compound 17a. Methanolic solution of DPPH was prepared by dissolving 4 mg of DPPH in 100mL of MeOH. Then, 1 mL of DPPH methanolic solution was added to 1 mL of each solution of 17a. The reaction mixture was incubated for 30 min in the dark at room temperature. The absorption of the resulting solutions was recorded at 517 nm. The DPPH color changes from deep violet (free radical form) to pale yellow (1,1-diphenyl-2-picryl hydrazine, reduced form). Ascorbic acid was used as a positive control and methanolic solution was used as a negative control.

Herbicide-Safening Effect Studies
The 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 hypocotyl growth. After treatment, the seedlings were washed with pure water and placed into a solution of the corresponding compound 16a and 17a,b (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 strips (10 × 75 cm, 20 seeds per strip). The strips 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; 20 seeds were used in each experiment. The results are given in Table 3.