Synthesis and Anticancer Evaluation of Novel 7-Aza-Coumarine-3-Carboxamides

Herein, we report the design and synthesis of novel 7-aza-coumarine-3-carboxamides via scaffold-hopping strategy and evaluation of their in vitro anticancer activity. Additionally, the improved non-catalytic synthesis of 7-azacoumarin-3-carboxylic acid is reported, which features water as the reaction medium and provides a convenient alternative to the known methods. The anticancer activity of the most potent 7-aza-coumarine-3-carboxamides against the HuTu 80 cell line is equal to that of reference Doxorubicin, while the selectivity towards the normal cell line is 9–14 fold higher.


Introduction
Coumarins are widespread structural motifs in natural products and exhibit a broad range of biological activities [1][2][3]. Their anticancer properties [4][5][6] have attracted considerable interest and have been extensively studied by multiple research groups. One of the promising classes of coumarin derivatives is coumarin-3-carboxamides, which have been the object of numerous in-depth investigations [7][8][9][10][11]. A large series of coumarin-3-carboxamides have been obtained and tested against various types of cancer cell lines so far. Among them, promising candidates for further development have been reported (Scheme 1A) [11,12].
A powerful strategy for discovering novel biologically relevant compounds is scaffold hopping [13,14], a concept that was first introduced by Schneider and coauthors [15] in 1999. Scaffold-hopping methods imply the modification of the core structure of the molecule with known activity to give a novel chemotype while trying to conserve the biological properties of the parent compound. The simplest modification is replacing or swapping carbon and heteroatoms in a backbone ring (1 • hop, according to Sun and coworkers [14]), which in the case of coumarins, would derive their heterocyclic analogs. The incorporation of a nitrogen atom into aromatic moiety is of special interest. It may decrease compounds' susceptibility to oxidative metabolism [16], thus improving their bioactivity profile. Additionally, the ability of nitrogen atoms to form extra hydrogen bonds may also play a role in binding to the active site of a biological target. For example, the replacement of aromatic carbon atoms by nitrogen has been successfully employed for the Scheme 1. Examples of coumarin-3-carboxamides with antitumor activity and scaffold hopping to nitrogen analogs (A); the only known 7-aza-coumarin derivatives (B); synthesis of novel 7-aza-cou marin-3-carboxamides proposed in this work and the most potent compound (C).

Chemistry
We initiated our studies by searching the optimal conditions for the synthesis of a key intermediate, 7-aza-coumarin-3-carboxylic acid 3a, using easily available pyridoxal 1 or pyridoxal hydrochloride 1·HCl as a starting compound. As was mentioned above, th synthesis of compound 3a was achieved earlier via base-promoted Knoevenagel conden sation of pyridoxal with malononitrile and the subsequent acidic hydrolysis of intermedi ate nitrile [29]. We speculated that the usage of malonic acid esters instead of malononitril would allow us to obtain acid 3a directly, thus avoiding a hydrolysis step. Pleasingly simply keeping the water solution of pyridoxal or pyridoxal hydrochloride and Mel drum's acid at room temperature furnished the desired 7-aza-coumarin-3-carboxylic acid 3a in fairly high yield (Table 1, entry 1). The structure of acid 3a was confirmed by x-ray analysis of its potassium salt (see Supplementary Information, Table S1, Figure S7 for ad ditional details and x-ray data). Moreover, the phosphoryl derivative 3b could be obtained Scheme 1. Examples of coumarin-3-carboxamides with antitumor activity and scaffold hopping to nitrogen analogs (A); the only known 7-aza-coumarin derivatives (B); synthesis of novel 7-azacoumarin-3-carboxamides proposed in this work and the most potent compound (C).
Thus, we wanted to investigate if a similar replacement of aromatic carbon by nitrogen would allow us to enhance the anticancer properties of coumarin-3-carboxamides. Notably, azacoumarins represent an interesting yet underexplored family of compounds. Although multiple regioisomeric azacoumarins are possible, only a few of them have been obtained so far. The vast majority of available bioactivity data refers to 1-azacoumarins (2-quinolones) [20], which have been extensively studied due to their remarkable anticancer properties [21][22][23][24][25].
Taking all of the above into account, we first aimed at the synthesis of hitherto unknown 7-azacoumarin-3-carboxamides and the evaluation of their cytotoxicity towards cancer cell lines. The literature survey revealed that only a limited number of 7-azacoumarins are known until now (Scheme 1B). Lebeau and coworkers reported the 4-stage synthesis of methyl ester of 7-azacoumarin-3-carboxylic acid starting from 3,5-dichloropyridine in 25%

Chemistry
We initiated our studies by searching the optimal conditions for the synthesis of a key intermediate, 7-aza-coumarin-3-carboxylic acid 3a, using easily available pyridoxal 1 or pyridoxal hydrochloride 1·HCl as a starting compound. As was mentioned above, the synthesis of compound 3a was achieved earlier via base-promoted Knoevenagel condensation of pyridoxal with malononitrile and the subsequent acidic hydrolysis of intermediate nitrile [29]. We speculated that the usage of malonic acid esters instead of malononitrile would allow us to obtain acid 3a directly, thus avoiding a hydrolysis step. Pleasingly, simply keeping the water solution of pyridoxal or pyridoxal hydrochloride and Meldrum's acid at room temperature furnished the desired 7-aza-coumarin-3-carboxylic acid 3a in fairly high yield (Table 1, entry 1). The structure of acid 3a was confirmed by X-ray analysis of its potassium salt (see Supplementary Information, Table S1, Figure S7 for additional details and X-ray data). Moreover, the phosphoryl derivative 3b could be obtained from pyridoxal-5-phosphate using the same procedure (Table 1, entry 2). We tried to replace the Meldrum's acid with more accessible malonic acid, however, without success. Although both non-catalytic and catalytic variants were tested, the yield of compound 3a did not exceed 10% (Table 1, entry 3). We also tested dimedone in this reaction, which allowed us to obtain 7-azacoumarin 4 (Table 1, entry 4). Notably, the reaction of acetylacetone resulted in either 7-azacoumarin derivative 5 or fluoropyridine 6, depending on the reaction conditions (Table 1, entries 5,6). Table 1. Development of the synthesis of key 7-aza-coumarin-3-carboxylic acid 3a. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 4 of 19 allowed us to obtain 7-azacoumarin 4 (Table 1, entry 4). Notably, the reaction of acetylacetone resulted in either 7-azacoumarin derivative 5 or fluoropyridine 6, depending on the reaction conditions (Table 1, entries 5,6). The structures of compounds 4 and 5 were additionally confirmed by X-ray analysis (see Supplementary Information, Table S1, Figures S1-S3 for detailed X-ray data). Interestingly, compound 4 exhibited axial chirality similar to previously described 3-azaxanthene derivatives [33] and was isolated as 1:1 mixtures of diastereomers.
Having optimized the synthesis of 7-azacoumarin-3-carboxylic acid 3a, we screened the most optimal method of its conversion to the target amides. A number of amide bondforming reactions and coupling reagents have been developed so far [34][35][36], including the electrosynthetic [37] and photocatalytic [38] methods. However, the most straightforward and mature approach via transient acyl halide formation was proven to be the most effective in terms of reagents availability, reaction scope, and product yield in our case. Preliminary studies with compound 3a and aniline indicated that the phosphorus oxychloride was superior to other chlorinating reagents tested (thionyl halides and ox- allowed us to obtain 7-azacoumarin 4 (Table 1, entry 4). Notably, the reaction of acetylacetone resulted in either 7-azacoumarin derivative 5 or fluoropyridine 6, depending on the reaction conditions (Table 1, entries 5,6). The structures of compounds 4 and 5 were additionally confirmed by X-ray analysis (see Supplementary Information, Table S1, Figures S1-S3 for detailed X-ray data). Interestingly, compound 4 exhibited axial chirality similar to previously described 3-azaxanthene derivatives [33] and was isolated as 1:1 mixtures of diastereomers.
Having optimized the synthesis of 7-azacoumarin-3-carboxylic acid 3a, we screened the most optimal method of its conversion to the target amides. A number of amide bondforming reactions and coupling reagents have been developed so far [34][35][36], including the electrosynthetic [37] and photocatalytic [38] methods. However, the most straightforward and mature approach via transient acyl halide formation was proven to be the most effective in terms of reagents availability, reaction scope, and product yield in our case. Preliminary studies with compound 3a and aniline indicated that the phosphorus oxychloride was superior to other chlorinating reagents tested (thionyl halides and ox- allowed us to obtain 7-azacoumarin 4 ( Table 1, entry 4). Notably, the reaction of acetylacetone resulted in either 7-azacoumarin derivative 5 or fluoropyridine 6, depending on the reaction conditions (Table 1, entries 5,6). The structures of compounds 4 and 5 were additionally confirmed by X-ray analysis (see Supplementary Information, Table S1, Figures S1-S3 for detailed X-ray data). Interestingly, compound 4 exhibited axial chirality similar to previously described 3-azaxanthene derivatives [33] and was isolated as 1:1 mixtures of diastereomers.
Having optimized the synthesis of 7-azacoumarin-3-carboxylic acid 3a, we screened the most optimal method of its conversion to the target amides. A number of amide bondforming reactions and coupling reagents have been developed so far [34][35][36], including the electrosynthetic [37] and photocatalytic [38] methods. However, the most straightforward and mature approach via transient acyl halide formation was proven to be the most effective in terms of reagents availability, reaction scope, and product yield in our case. Preliminary studies with compound 3a and aniline indicated that the phosphorus oxychloride was superior to other chlorinating reagents tested (thionyl halides and ox- allowed us to obtain 7-azacoumarin 4 ( Table 1, entry 4). Notably, the reaction of acetylacetone resulted in either 7-azacoumarin derivative 5 or fluoropyridine 6, depending on the reaction conditions (Table 1, entries 5,6). The structures of compounds 4 and 5 were additionally confirmed by X-ray analysis (see Supplementary Information, Table S1, Figures S1-S3 for detailed X-ray data). Interestingly, compound 4 exhibited axial chirality similar to previously described 3-azaxanthene derivatives [33] and was isolated as 1:1 mixtures of diastereomers.
Having optimized the synthesis of 7-azacoumarin-3-carboxylic acid 3a, we screened the most optimal method of its conversion to the target amides. A number of amide bondforming reactions and coupling reagents have been developed so far [34][35][36], including the electrosynthetic [37] and photocatalytic [38] methods. However, the most straightforward and mature approach via transient acyl halide formation was proven to be the most effective in terms of reagents availability, reaction scope, and product yield in our case. Preliminary studies with compound 3a and aniline indicated that the phosphorus oxychloride was superior to other chlorinating reagents tested (thionyl halides and ox- <10 4 Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 4 of 19 allowed us to obtain 7-azacoumarin 4 ( Table 1, entry 4). Notably, the reaction of acetylacetone resulted in either 7-azacoumarin derivative 5 or fluoropyridine 6, depending on the reaction conditions (Table 1, entries 5,6). The structures of compounds 4 and 5 were additionally confirmed by X-ray analysis (see Supplementary Information, Table S1, Figures S1-S3 for detailed X-ray data). Interestingly, compound 4 exhibited axial chirality similar to previously described 3-azaxanthene derivatives [33] and was isolated as 1:1 mixtures of diastereomers.
Having optimized the synthesis of 7-azacoumarin-3-carboxylic acid 3a, we screened the most optimal method of its conversion to the target amides. A number of amide bondforming reactions and coupling reagents have been developed so far [34][35][36], including the electrosynthetic [37] and photocatalytic [38] methods. However, the most straightforward and mature approach via transient acyl halide formation was proven to be the most effective in terms of reagents availability, reaction scope, and product yield in our case. Preliminary studies with compound 3a and aniline indicated that the phosphorus oxychloride was superior to other chlorinating reagents tested (thionyl halides and ox- allowed us to obtain 7-azacoumarin 4 ( Table 1, entry 4). Notably, the reaction of acetylacetone resulted in either 7-azacoumarin derivative 5 or fluoropyridine 6, depending on the reaction conditions (Table 1, entries 5,6). The structures of compounds 4 and 5 were additionally confirmed by X-ray analysis (see Supplementary Information, Table S1, Figures S1-S3 for detailed X-ray data). Interestingly, compound 4 exhibited axial chirality similar to previously described 3-azaxanthene derivatives [33] and was isolated as 1:1 mixtures of diastereomers.
Having optimized the synthesis of 7-azacoumarin-3-carboxylic acid 3a, we screened the most optimal method of its conversion to the target amides. A number of amide bondforming reactions and coupling reagents have been developed so far [34][35][36], including the electrosynthetic [37] and photocatalytic [38] methods. However, the most straightforward and mature approach via transient acyl halide formation was proven to be the most effective in terms of reagents availability, reaction scope, and product yield in our case. Preliminary studies with compound 3a and aniline indicated that the phosphorus oxychloride was superior to other chlorinating reagents tested (thionyl halides and ox-89 5 lacetone resulted in either 7-azacoumarin derivative 5 or fluoropyridine 6, depending on the reaction conditions (Table 1, entries 5,6). The structures of compounds 4 and 5 were additionally confirmed by X-ray analysis (see Supplementary Information, Table S1, Figures S1-S3 for detailed X-ray data). Interestingly, compound 4 exhibited axial chirality similar to previously described 3-azaxanthene derivatives [33] and was isolated as 1:1 mixtures of diastereomers.
Having optimized the synthesis of 7-azacoumarin-3-carboxylic acid 3a, we screened the most optimal method of its conversion to the target amides. A number of amide bondforming reactions and coupling reagents have been developed so far [34][35][36], including the electrosynthetic [37] and photocatalytic [38] methods. However, the most straightforward and mature approach via transient acyl halide formation was proven to be the most effective in terms of reagents availability, reaction scope, and product yield in our case. Preliminary studies with compound 3a and aniline indicated that the phosphorus oxychloride was superior to other chlorinating reagents tested (thionyl halides and ox- allowed us to obtain 7-azacoumarin 4 (Table 1, entry 4). Notably, the reaction of acetylacetone resulted in either 7-azacoumarin derivative 5 or fluoropyridine 6, depending on the reaction conditions (Table 1, entries 5,6). The structures of compounds 4 and 5 were additionally confirmed by X-ray analysis (see Supplementary Information, Table S1, Figures S1-S3 for detailed X-ray data). Interestingly, compound 4 exhibited axial chirality similar to previously described 3-azaxanthene derivatives [33] and was isolated as 1:1 mixtures of diastereomers.
Having optimized the synthesis of 7-azacoumarin-3-carboxylic acid 3a, we screened the most optimal method of its conversion to the target amides. A number of amide bondforming reactions and coupling reagents have been developed so far [34][35][36], including the electrosynthetic [37] and photocatalytic [38] methods. However, the most straightforward and mature approach via transient acyl halide formation was proven to be the most effective in terms of reagents availability, reaction scope, and product yield in our case. Preliminary studies with compound 3a and aniline indicated that the phosphorus oxychloride was superior to other chlorinating reagents tested (thionyl halides and ox- lacetone resulted in either 7-azacoumarin derivative 5 or fluoropyridine 6, depending on the reaction conditions (Table 1, entries 5,6). The structures of compounds 4 and 5 were additionally confirmed by X-ray analysis (see Supplementary Information, Table S1, Figures S1-S3 for detailed X-ray data). Interestingly, compound 4 exhibited axial chirality similar to previously described 3-azaxanthene derivatives [33] and was isolated as 1:1 mixtures of diastereomers.
Having optimized the synthesis of 7-azacoumarin-3-carboxylic acid 3a, we screened the most optimal method of its conversion to the target amides. A number of amide bondforming reactions and coupling reagents have been developed so far [34][35][36], including the electrosynthetic [37] and photocatalytic [38] methods. However, the most straightforward and mature approach via transient acyl halide formation was proven to be the most effective in terms of reagents availability, reaction scope, and product yield in our case. Preliminary studies with compound 3a and aniline indicated that the phosphorus oxychloride was superior to other chlorinating reagents tested (thionyl halides and ox-82 a Isolated yield. The structures of compounds 4 and 5 were additionally confirmed by X-ray analysis (see Supplementary Information, Table S1, Figures S1-S3 for detailed X-ray data). Interestingly, compound 4 exhibited axial chirality similar to previously described 3-azaxanthene derivatives [33] and was isolated as 1:1 mixtures of diastereomers.
Having optimized the synthesis of 7-azacoumarin-3-carboxylic acid 3a, we screened the most optimal method of its conversion to the target amides. A number of amide bondforming reactions and coupling reagents have been developed so far [34][35][36], including the electrosynthetic [37] and photocatalytic [38] methods. However, the most straightforward and mature approach via transient acyl halide formation was proven to be the most effective in terms of reagents availability, reaction scope, and product yield in our case. Preliminary studies with compound 3a and aniline indicated that the phosphorus oxychloride was superior to other chlorinating reagents tested (thionyl halides and oxalylchloride). Importantly, this approach allowed the simultaneous replacement of the hydroxyl group by chlorine atom in compound 7a, which is useful if further modification via nucleophilic substitution is considered. Interestingly, the phosphoryl-substituted acid 3b could also be used instead of compound 3a, leading to the same amide 7a.
With these conditions in hand, we expanded the scope of the reaction using various aromatic and heteroaromatic amines (Scheme 2, see Supplementary Information, Figures S8-S55 for the NMR spectra of the obtained compounds). Both electron-donating and electronwithdrawing substituents in anilines were well tolerated, resulting in the compounds 7b,c in a 77-91% yield. More sterically demanding diphenylamine also provided compound 7f in high yield. Amino-and diaminopyridines furnished the desired amides 7d,e in good yields (66-79%). Notably, only one amino group was substituted in the case of diaminopyridines. Given the importance and versatility of alkyne-azide cycloaddition in current medicinal chemistry and drug design, we additionally tested a propargylamine in these conditions. Pleasingly, the triple bond remained intact under reaction conditions, and the corresponding amide 7g was isolated in a 45% yield. A series of amides 7h-l possessing aryl-and heteroarylsulfonylamide moieties were successfully obtained using 4-aminobenzenesulfonamides as starting compounds. To further expand the scope of the reaction, acid hydrazides were employed instead of amines under the same conditions. The reaction proceeded smoothly to give corresponding bis(hydrazides) 7m,n in 72-76% yield. Finally, phenylenediamines were used as reagents to give compounds 7o-q with two 7azacoumrin moieties in excellent yields (91-97%). Similarly, 3,3 -and 4,4 -sulfonyldianilines furnished bis(7-azacoumarins) 7r,s in a 67-78% yield. The structures of compounds 7a,f were additionally confirmed by X-ray analysis (see Supplementary Information, Table S1, Figures S4 and S5 for detailed X-ray data).
S8-S55 for the NMR spectra of the obtained compounds). Both electron-donating and elec tron-withdrawing substituents in anilines were well tolerated, resulting in the compound 7b,c in a 77-91% yield. More sterically demanding diphenylamine also provided com pound 7f in high yield. Amino-and diaminopyridines furnished the desired amides 7d,e in good yields (66-79%). Notably, only one amino group was substituted in the case o diaminopyridines. Given the importance and versatility of alkyne-azide cycloaddition in current medicinal chemistry and drug design, we additionally tested a propargylamine in these conditions. Pleasingly, the triple bond remained intact under reaction conditions and the corresponding amide 7g was isolated in a 45% yield. A series of amides 7h-l pos sessing aryl-and heteroarylsulfonylamide moieties were successfully obtained using 4 aminobenzenesulfonamides as starting compounds. To further expand the scope of the reaction, acid hydrazides were employed instead of amines under the same conditions The reaction proceeded smoothly to give corresponding bis(hydrazides) 7m,n in 72-76% yield. Finally, phenylenediamines were used as reagents to give compounds 7o-q with two 7-azacoumrin moieties in excellent yields (91-97%). Similarly, 3,3′-and 4,4′-sul fonyldianilines furnished bis(7-azacoumarins) 7r,s in a 67-78% yield. The structures o compounds 7a,f were additionally confirmed by X-ray analysis (see Supplementary Infor mation, Table S1, Figures S4,S5 for detailed X-ray data).

Scheme 2. Synthesis of amides 7 [a] [a] Isolated yield is given [b]
, and it was obtained from 3b via the same procedure.
The evaluation of the cytotoxicity of acids 3a,b was also of importance for comparison purposes. Additionally, we synthesized a series of ammonium salts 8a-f (Scheme 3, see Supplementary Information, Figures S56-S69 for the NMR spectra of the obtained com pounds). Not only would this allow us to evaluate the effect of counterion on the cytotox icity of acids 3, but also to estimate the importance of covalent amide bonds for anticance Scheme 2. Synthesis of amides 7 [a] [a] Isolated yield is given [b], and it was obtained from 3b via the same procedure.
The evaluation of the cytotoxicity of acids 3a,b was also of importance for comparison purposes. Additionally, we synthesized a series of ammonium salts 8a-f (Scheme 3, see Supplementary Information, Figures S56-S69 for the NMR spectra of the obtained compounds). Not only would this allow us to evaluate the effect of counterion on the cytotoxicity of acids 3, but also to estimate the importance of covalent amide bonds for anticancer activity. The structure of the salt 8b was additionally proved by X-ray analysis, which confirmed its ionic character (see Supplementary Information, Table S1, Figures S6 and S7 for detailed X-ray data).
activity. The structure of the salt 8b was additionally proved confirmed its ionic character (see Supplementary Information, detailed X-ray data).

Biological Studies
With a series of 7-azacoumarin-3-carboxamides 7 in hand, w against normal (Chang liver) and tumor (M-HeLa, HuTu 80) ce 1-100 µM ( Table 2). In general, the cytotoxicity of all the tested c higher towards the HuTu 80 cell line than M-Hela cells. The cy ids 3a,b and their ammonium salts 8b,e appeared to be close to bly lower than the cytotoxicity of reference compounds 5-fluo Carboxamides 7h-l and 7r,s possessing sulfonylamide moiety toxic compared to others. The sulfonylamide 7h and bis(7-azac 7o,r,s were the most cytotoxic towards the HuTu 80 cells. The pounds was comparable to that of Doxorubicin (IC50 = 2.9-5.5 µ compound 7s (IC50 = 13.8 µM). However, in sharp contrast to D city towards the normal cell line was much lower (selectivi liver)/IC50(HuTu 80) = 3.8-14 vs. SI = 1 for Doxorubicin). Notably the least selective (SI = 3.8), despite being one of the most active. exhibited much higher selectivity (SI = 9-14). Thus, one can sp mide moiety is crucial for both the activity and the selectivity of ing into account both the selectivity and cytotoxicity toward pounds 7h and 7r can be considered the most potent and prom

Biological Studies
With a series of 7-azacoumarin-3-carboxamides 7 in hand, we tested their cytotoxicity against normal (Chang liver) and tumor (M-HeLa, HuTu 80) cell lines at concentrations of 1-100 µM ( Table 2). In general, the cytotoxicity of all the tested compounds was somewhat higher towards the HuTu 80 cell line than M-Hela cells. The cytotoxicity of carboxylic acids 3a,b and their ammonium salts 8b,e appeared to be close to each other and considerably lower than the cytotoxicity of reference compounds 5-fluorouracil and Doxorubicin. Carboxamides 7h-l and 7r,s possessing sulfonylamide moiety appeared to be more cytotoxic compared to others. The sulfonylamide 7h and bis(7-azacoumarin-3-carboxamides) 7o,r,s were the most cytotoxic towards the HuTu 80 cells. The cytotoxicity of these compounds was comparable to that of Doxorubicin (IC 50 = 2.9-5.5 µM vs. 3.0 ± 0.2 µM), except compound 7s (IC 50 = 13.8 µM). However, in sharp contrast to Doxorubicin, their cytotoxicity towards the normal cell line was much lower (selectivity index, SI = IC 50 (Chang liver)/IC 50 (HuTu 80) = 3.8-14 vs. SI = 1 for Doxorubicin). Notably, bis(carboxamide) 7o was the least selective (SI = 3.8), despite being one of the most active. The sulfonylamides 7h,r,s exhibited much higher selectivity (SI = 9-14). Thus, one can speculate that the sulfonylamide moiety is crucial for both the activity and the selectivity of carboxamides 7h,r,s. Taking into account both the selectivity and cytotoxicity towards HuTu 80 cells, the compounds 7h and 7r can be considered the most potent and promising.
Induction of apoptosis is one of the most important mechanisms of anticancer activity. The apoptosis-inducing effect in HuTu 80 cells was studied by flow cytometry using sets of annexin V binding to the apoptosis marker phosphatidylserine. On the surface of the membranes of healthy cells, phosphatidylserine is contained in a minimal amount. Therefore, the interaction of annexin V with these cells is negligible. During apoptosis, phosphatidylserine molecules appear on the cell surface and can interact with the protein. This interaction leads to an increase in the fluorescence intensity of apoptotic cells, which is recorded by a flow cytometer. The apoptosis-inducing effect was evaluated using the example of the leader compound 7r at IC50/2 and IC50 concentrations on the HuTu 80 cell line (Figure 1). After 24-h incubation in the presence of 7r, apoptotic effects were registered in human duodenal adenocarcinoma cells, which were more pronounced at the early stage of apoptosis. Induction of apoptosis is one of the most important mechanisms of anticancer activity. The apoptosis-inducing effect in HuTu 80 cells was studied by flow cytometry using sets of annexin V binding to the apoptosis marker phosphatidylserine. On the surface of the membranes of healthy cells, phosphatidylserine is contained in a minimal amount. Therefore, the interaction of annexin V with these cells is negligible. During apoptosis, phosphatidylserine molecules appear on the cell surface and can interact with the protein. This interaction leads to an increase in the fluorescence intensity of apoptotic cells, which is recorded by a flow cytometer. The apoptosis-inducing effect was evaluated using the example of the leader compound 7r at IC50/2 and IC50 concentrations on the HuTu 80 cell line (Figure 1). After 24-h incubation in the presence of 7r, apoptotic effects were registered in human duodenal adenocarcinoma cells, which were more pronounced at the early stage of apoptosis.  Violation of the functions of the mitochondria of the cell is one of the most common signs of apoptosis inherent in eukaryotic organisms [39]. With mitochondrial dysfunction, pro-apoptotic factors are released into the cytoplasm-cytochrome c, AIF, Smac/DIABLO, endonuclease G, as well as proforms of caspases 2, 3, and 9-inducing the cascade [40]. The release of these protein factors can be associated both with the rupture of mitochondrial membranes and with the activation of specific channels in the outer mitochondrial membrane. This usually leads to a change in the mitochondrial membrane potential (∆Ψm) due to a change in the permeability of the inner mitochondrial membrane for H+ protons. Methods for studying the membrane potential of mitochondria using flow cytometry are based on the use of cationic lipophilic fluorescent dyes. The principle of operation of these dyes is determined by their ability to spontaneously penetrate through the cytoplasmic membranes of cells, as well as the outer and inner membranes of mitochondria and accumulate in areas with a high concentration of protons, that is, under the inner mitochondrial membrane. This effect is accompanied by a change in the intensity of cell fluorescence, which is recorded by cytofluorimetry. In this study, the fluorescent dye JC-10 from the Mitochondria Membrane Potential Kit was used to evaluate the change in ∆Ψm under the action of compound 7r at IC 50 /2 and IC 50 concentrations on the HuTu 80 cell line. JC-10 accumulates in the mitochondrial matrix and forms aggregates (J-aggregates) with red fluorescence in normal cells with a high mitochondrial membrane potential. Membrane mitochondrial potential is reduced in apoptotic cells. In this case, JC-10 begins to diffuse out of the mitochondria and turns into a monomeric form (J-monomer), and emits green fluorescence. A decrease in the mitochondrial membrane potential of HuTu 80 cells was observed after 24-h incubation with the leader compound 7r. The process of depolarization of the mitochondrial membrane increased with increasing compound concentrations up to IC 50 (Figure 2). The results obtained suggest that the mechanism of the cytotoxic action of 7r may be associated with the induction of apoptosis via the intrinsic mitochondrial pathway. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 8 of 19 Data are presented as mean ±SD of three independent experiments * Values indicate p < 0.05 compared to control.
Violation of the functions of the mitochondria of the cell is one of the most common signs of apoptosis inherent in eukaryotic organisms [39]. With mitochondrial dysfunction, pro-apoptotic factors are released into the cytoplasm-cytochrome c, AIF, Smac/DIABLO, endonuclease G, as well as proforms of caspases 2, 3, and 9-inducing the cascade [40]. The release of these protein factors can be associated both with the rupture of mitochondrial membranes and with the activation of specific channels in the outer mitochondrial membrane. This usually leads to a change in the mitochondrial membrane potential (ΔΨm) due to a change in the permeability of the inner mitochondrial membrane for H+ protons. Methods for studying the membrane potential of mitochondria using flow cytometry are based on the use of cationic lipophilic fluorescent dyes. The principle of operation of these dyes is determined by their ability to spontaneously penetrate through the cytoplasmic membranes of cells, as well as the outer and inner membranes of mitochondria and accumulate in areas with a high concentration of protons, that is, under the inner mitochondrial membrane. This effect is accompanied by a change in the intensity of cell fluorescence, which is recorded by cytofluorimetry. In this study, the fluorescent dye JC-10 from the Mitochondria Membrane Potential Kit was used to evaluate the change in ΔΨm under the action of compound 7r at IC50/2 and IC50 concentrations on the HuTu 80 cell line. JC-10 accumulates in the mitochondrial matrix and forms aggregates (J-aggregates) with red fluorescence in normal cells with a high mitochondrial membrane potential. Membrane mitochondrial potential is reduced in apoptotic cells. In this case, JC-10 begins to diffuse out of the mitochondria and turns into a monomeric form (J-monomer), and emits green fluorescence. A decrease in the mitochondrial membrane potential of HuTu 80 cells was observed after 24-h incubation with the leader compound 7r. The process of depolarization of the mitochondrial membrane increased with increasing compound concentrations up to IC50 (Figure 2). The results obtained suggest that the mechanism of the cytotoxic action of 7r may be associated with the induction of apoptosis via the intrinsic mitochondrial pathway.  Increased generation of reactive oxygen species (ROS) by the test compounds can also characterize the development of apoptosis along the mitochondrial pathway. Mitochondria are both a potential source and target of ROS. An increase in ROS production leads to the disruption of mitochondrial functions and, subsequently, to irreversible damage and cell death. In this regard, the effect of lead compound 7r at IC50/2 and IC50 concentrations on ROS production in HuTu 80 cells was investigated using a flow cytometry assay and the CellROX ® Deep Red flow cytometry kit. The data presented in Figure 3 show a significant increase in CellROX ® Deep Red fluorescence intensity when 7r is added at any concentration compared to the control (unstained cells). This indicates an increase in ROS production in the presence of the test compound. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 9 of 19 Increased generation of reactive oxygen species (ROS) by the test compounds can also characterize the development of apoptosis along the mitochondrial pathway. Mitochondria are both a potential source and target of ROS. An increase in ROS production leads to the disruption of mitochondrial functions and, subsequently, to irreversible damage and cell death. In this regard, the effect of lead compound 7r at IC50/2 and IC50 concentrations on ROS production in HuTu 80 cells was investigated using a flow cytometry assay and the CellROX ® Deep Red flow cytometry kit. The data presented in Figure 3 show a significant increase in CellROX ® Deep Red fluorescence intensity when 7r is added at any concentration compared to the control (unstained cells). This indicates an increase in ROS production in the presence of the test compound. Figure 3. Induction of ROS production by compound 7r at concentration IC50/2 (2.8 µM) and at concentration IC50 (5.5 µM). Data are presented as mean ± SD of three independent experiments * Values indicate p < 0.05 compared to control.

Chemistry
3.1.1. General Methods 1 H, 13 C, and 31 P NMR spectra were recorded on a Bruker Avance 400 (Bruker, Billerica, Massachusetts, USA) spectrometer (at the frequencies of 400.05, 100.61, and 161.94 MHz, respectively) and on a Bruker Avance 600 spectrometer (at the frequencies of 600.1, 150.9, and 242.0 MHz, respectively). Values of the chemical shifts for the 1 H and 13 C nuclei are reported relative to the residual signals of the solvent (DMSO-d6), and those for the 31 P nuclei are given relative to the used standard (H3PO4, dP = 0.00). IR spectra were recorded in KBr pellets on a Bruker 3/5 E2_76513 Tensor-27 spectrometer in the range of 400-3600 cm -1 . Mass spectra were recorded on an Ultraflex III TOF/TOF Bruker instrument (p-nitroaniline as the matrix) and an AmaZon X Bruker instrument. Elemental analysis was performed using a Carlo Erba EA 1108 (Carlo Erba, Cornaredo, Italy) instrument. Commercially available pyridoxal hydrochloride (abcr Gute Chemie, Karlsruhe, Germany), pyridoxal 5′-phosphate monohydrate (Sigma-Aldrich, St. Louis, MO, USA), Meldrum's acid (TCI, Tokyo, Japan), amines and diamines (Acros organics, Geel, Belgium) were used in the synthesis without additional purification.

Materials and Methods
3.1. Chemistry 3.1.1. General Methods 1 H, 13 C, and 31 P NMR spectra were recorded on a Bruker Avance 400 (Bruker, Billerica, MA, USA) spectrometer (at the frequencies of 400.05, 100.61, and 161.94 MHz, respectively) and on a Bruker Avance 600 spectrometer (at the frequencies of 600.1, 150.9, and 242.0 MHz, respectively). Values of the chemical shifts for the 1 H and 13 C nuclei are reported relative to the residual signals of the solvent (DMSO-d 6 ), and those for the 31 P nuclei are given relative to the used standard (H 3 PO 4 , dP = 0.00). IR spectra were recorded in KBr pellets on a Bruker 3/5 E2_76513 Tensor-27 spectrometer in the range of 400-3600 cm −1 . Mass spectra were recorded on an Ultraflex III TOF/TOF Bruker instrument (p-nitroaniline as the matrix) and an AmaZon X Bruker instrument. Elemental analysis was performed using a Carlo Erba EA 1108 (Carlo Erba, Cornaredo, Italy) instrument. Commercially available pyridoxal hydrochloride (abcr Gute Chemie, Karlsruhe, Germany), pyridoxal 5 -phosphate monohydrate (Sigma-Aldrich, St. Louis, MO, USA), Meldrum's acid (TCI, Tokyo, Japan), amines and diamines (Acros organics, Geel, Belgium) were used in the synthesis without additional purification.
The IC 50 values were calculated using the online calculator MLA-Quest Graph™ IC50 Calculator (AAT Bioquest, Inc., Pleasanton, CA, USA). Statistical analysis was performed using the Mann-Whitney test (p < 0.05). Tabular and graphical data are presented as mean ± SD of three independent experiments.

Conclusions
In conclusion, a series of novel, hitherto unknown 7-azacoumarin-3-carboxamides were obtained via a two-stage, two-step procedure. Additionally, the improved synthesis of 7-azacoumarin-3-carboxylic acid has been developed based on the reaction of pyridoxal with Meldrum's acid. The proposed method does not require any catalysts, proceeds in a water medium at room temperature, and provides a target compound in high yield. The applicability of this method to the synthesis of other 7-azacoumarin derivatives has also been demonstrated. The cytotoxicity of 7-azacoumarin-3-carboxamides toward cancer and normal cell lines has been evaluated. The most potent compounds exhibited activity towards the HuTu 80 cell line equal to that of Doxorubicin. In contrast to Doxorubicin, they are much more selective and less toxic to normal cell lines (selectivity index, SI = 9-14), thus representing a novel promising scaffold for the design of the anticancer drug. The cytotoxic effect of the 7r leader compound may be due to the induction of apoptosis through an intrinsic pathway associated with impaired mitochondrial function.

Data Availability Statement:
The data presented in this study are contained within the article or in Supplementary Materials or are available upon request from the corresponding author Almir Gazizov.