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Article

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

by
Alexey V. Trifonov
1,
Almir S. Gazizov
1,*,
Anipa S. Tapalova
2,
Lyudmila K. Kibardina
1,
Nurbol O. Appazov
2,
Alexandra D. Voloshina
1,
Anastasiia S. Sapunova
1,
Anna P. Luybina
1,
Gulmira M. Abyzbekova
2,
Alexey B. Dobrynin
1,
Igor A. Litvinov
1,
Akerke K. Tauekel
3,
Sholpan O. Yespenbetova
2,
Alexander R. Burilov
1 and
Michail A. Pudovik
1
1
Arbuzov Institute of Organic and Physical Chemistry, FRC Kazan Scientific Center, Russian Academy of Science, Arbuzova Str., 8, Kazan 420088, Russia
2
Korkyt Ata Kyzylorda University, Aiteke Bi Street, 29A, Kyzylorda 120014, Kazakhstan
3
Department of Oil, Chemistry and Nanotechnology, Kazan National Research Technological University, Karl Marx Str., 68, Kazan 420015, Russia
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2023, 24(12), 9927; https://doi.org/10.3390/ijms24129927
Submission received: 22 May 2023 / Revised: 4 June 2023 / Accepted: 5 June 2023 / Published: 9 June 2023
(This article belongs to the Section Molecular Biology)
Browse Figures
Versions Notes

Abstract

:
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.

1. 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 development of anticancer agents based on azaindole [17], imidazo[1,2-a]pyridinone [18], and pyrido[1,2-a]pyrimidin-4-one [19] scaffolds.
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% overall yield [26]. Casas and coworkers described the thallium(III) [27] and gold(I) [28] complexes of the 3-mercapto-7-azacoumarin derivative. However, the ligand was not isolated in pure form. Finally, Pizzo and Fringuelli developed the method of synthesis of 3-substituted 7-azacoumarins, including 7-azacoumarin-3-carboxylic acid, via one-pot two-stage Knoevenagel condensation of pyridoxal with CH-acids [29].
Based on these data, we proposed the synthetic route to desired compounds as depicted in Scheme 1C. As a result of our studies, herein we report a two-stage synthesis of diverse 7-azacoumarin-3-carboxamides and evaluation of their cytotoxicity towards tumor and normal cell lines. Taking into account that cervical cancer is one of the most common gynecologic cancers worldwide [30,31], the M-HeLa cell line was chosen for testing. Additionally, the cytotoxicity towards HuTu 80 cells was evaluated. Although duodenal adenocarcinoma is a rare type of tumor, its aggressive progression makes surgical resection the most used therapy [32]. Certainly, a lack of chemotherapeutic treatment for this type of cancer is a serious problem. Additionally, both M-HeLa and HuTu 80 cell lines are widely used for cytotoxicity evaluation, which facilitates the comparison of the obtained data with literature values. Doxorubicin and 5-fluorouracil were chosen as reference compounds. This choice was guided by following considerations: (a) both Doxorubicin and 5-fluorouracil are widely used for the treatment of various types of cancer, so a comparison of novel compounds with well-known marketed drugs can be made; (b) there are many published studies in which these compounds were employed as reference, thus a comparison with literature data becomes possible.
The cytotoxicity of the most potent compounds was comparable to that of “golden standard” Doxorubicin (IC50 2.9–5.5 µM vs. 3.0 ± 0.2 µM for Doxorubicin), while the toxicity towards normal cell line was much lower (selectivity index, SI = 10–14 vs. SI = 1 for Doxorubicin). Additionally, we report an improved synthesis of 7-azacoumarin-3-carboxylic acid, which features a simpler and “greener” procedure than known ones, and it does not require any catalysts and uses water as a reaction medium.

2. Results and Discussion

2.1. 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).
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 bond-forming 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 electron-withdrawing 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 7-azacoumrin 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).
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).

2.2. 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 (IC50 = 2.9–5.5 µM vs. 3.0 ± 0.2 µM), except compound 7s (IC50 = 13.8 µM). However, in sharp contrast to Doxorubicin, their cytotoxicity towards the normal cell line was much lower (selectivity index, SI = IC50(Chang liver)/IC50(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.
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.

3. Materials and Methods

3.1. Chemistry

3.1.1. General Methods

1H, 13C, and 31P 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 1H and 13C nuclei are reported relative to the residual signals of the solvent (DMSO-d6), and those for the 31P 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.

3.1.2. Procedures for the Synthesis of Compounds 3–6

5-(hydroxymethyl)-8-methyl-2-oxo-2H-pyrano[2,3-c]pyridin-7-ium-3-carboxylate 3ª. To a solution of pyridoxal 1 (0.82 g, 4.91 mmol) or pyridoxal hydrochloride 1·HCl (1 g, 4.91 mmol) in distilled water (10 mL), Meldrum’s acid (0.707 g, 4.91 mmol) was added. The reaction mixture was stirred at room temperature for 12 h. The precipitate was filtered off, washed consequently with dry ethanol (2 × 5 mL) and diethyl ether (10 mL), and dried in a vacuum to give compound 3a as a white solid. Yield: 0.79 g (69%); mp: 249–250 °C. IR (ν cm−1): 646, 733, 810, 908, 975, 1006, 1038, 1088, 1153, 1253, 1293, 1360, 1409, 1772, 3450. 1H NMR (DMSO-d6) δ ppm: 2.57 (s, 3H, CH3); 4.77 (s, 2H, CH2OH), 8.36 (s, 1H, CHarom) 8.73 (s, 1H, CH). 13C NMR (DMSO-d6) δ ppm: 18.98, 58.54, 121.54, 123.77, 132.25, 143.10, 143.43, 146.94, 148.13, 155.80, 164.18. MALDI TOF—MS m/z: 235.1 [M]+. Calculated for C11H9NO5, %: C, 56.18; H, 3.86; N, 5.96. Found, %: C,56.27; H, 3.75; N, 5.83.
8-methyl-2-oxo-5-((phosphonooxy)methyl)-2H-pyrano[2,3-c]pyridin-7-ium-3-carboxylate 3b. A suspension of pyridoxal-5′-phosphate monohydrate (0.54 g, 2.04 mmol) and Meldrum’s acid (0.29 g, 2.04 mmol) in distilled water (5 mL) was stirred for 4 h at room temperature. The formed precipitate was filtered off, washed with absolute ethanol (2 × 5 mL), then with diethyl ether (10 mL), and dried in a vacuum to give the compound 3b as a light yellow solid. Yield: 0.55 g (86%); mp: >300 °C. IR (ν cm−1): 726, 806, 823, 962, 1025, 1074, 1095, 1137, 1161, 1220, 1239, 1297, 1425, 1751, 1770, 2418, 3508, 3548. 1H NMR (DMSO-d6) δ ppm: 2.60 (s, 3H, CH3), 5.19 (d, 2H, J = 7.7 Hz, CH2O), 8.42 (s, 1H, CHarom), 8.65 (s, 1H, CH). 13C NMR (DMSO-d6) δ ppm: 19.06, 62.04, 121.67, 124.56, 127.56, 142.43, 144.12, 147.89, 148.27, 155.63, 164.07. 31P NMR (DMSO-d6) δ ppm: −1.88. MALDI TOF—MS m/z: 315.9 [M+H]+. Calculated for C11H10NO8P, %: C,41.92; H, 3.20; N, 4.44; P, 9.83. Found, %: C,41.57; H, 3.25; N, 4.53; P, 9.48.
5-(2-hydroxy-4,4-dimethyl-6-oxocyclohex-1-en-1-yl)-4-(hydroxymethyl)-1,8,8-trimethyl-5,7,8,9-tetrahydro-6H-chromeno[2,3-c]pyridin-6-one 4. A solution of pyridoxal (0.5 g, 2.99 mmol) and dimedone (0.84 g, 5.99 mol) was stirred at 60 °C for 4 h. The precipitate was filtered off. The filtrate was evaporated; the residue was refluxed in ethanol: diethyl ether mixture (1:4, 5 mL). The undissolved precipitate was filtered off. The precipitates were merged to give 1.1 g (89%) of target compound 4 as a light yellow solid; mp: 219–224 °C. IR (ν cm−1): 655, 918, 938, 974, 1005, 1043, 1064, 1130, 1150, 1171, 1199, 1220, 1234, 1267, 1368, 1407, 1604, 1643, 2956, 3100. 1H NMR (DMSO-d6) δ ppm: 0.87 (s, 6H, 2CH3), 0.93 (s, 3H, CH3), 1.06 (s, 3H, CH3), 2.03 (d, 1H, J = 16.0 Hz, CH2), 2.11 (br.s, 4H, 2CH2), 2.26 (d, 1H, J = 15.9 Hz, CH2), 2.38 (d, 1H, J = 15.2 Hz, CH2), 2.40 (s, 3H, CH3), 2.60 (d, 1H, J = 17.3 Hz, CH2), 4.23 (d, 1H, J = 14.5 Hz, CH2OH), 4.51 (d, 1H, J = 14.5 Hz, CH2OH), 5.00 (s, 1H, CH), 8.14 (s, 1H, CHarom). 13C NMR (DMSO-d6) δ ppm: 19.08, 23.45, 26.28, 28.11, 29.69, 31.84, 32.07, 41.06, 50.99, 58.23, 110.59, 129.58, 134.12, 142.40, 143.50, 145.63, 164.98, 196.01. MALDI TOF—MS m/z: 412.4 [M+H]+. Calculated for C24H29NO5, %: C, 70.05; H, 7.10; N, 3.40. Found, %: C, 70.17; H, 7.15; N, 3.43.
1-(2-hydroxy-5-(hydroxymethyl)-2,8-dimethyl-2H-pyrano[2,3-c]pyridin-3-yl)ethan-1-one, compound 5. To a suspension of pyridoxal (0.6 g, 3.59 mmol) in dry ethanol (4 mL), acetylacetone (0.5 g, 5 mmol) was added, followed by piperidine (0.006 g, 0.07 mmol) and acetic acid (0.004 g, 0.07 mmol). The reaction mixture was refluxed for 2 h, and then kept at room temperature for an additional 12 h. The precipitate of compound 5 was filtered off. The evaporation of the filtrate and recrystallization of the viscous residue from absolute ethanol (3 mL) provided an additional sample of compound 5. Light yellow solid. Yield: 0.7 g (79%); mp: 174 °C. IR (ν cm−1): 563, 606, 664, 747, 857, 885, 901, 923, 949, 967, 983, 1034, 1066, 1088, 1099, 1150, 1185, 1211, 1242, 1293, 1338, 1370, 1386, 1410, 1458, 1484, 1630, 1678, 2749, 2922, 3030, 3268. 1H NMR (DMSO-d6) δ ppm: 1.86 (s, 3H, CH3), 2.39 (s, 3H, CH3), 2.46 (s, 3H, CH3), 4.67 (dd, 2H, J = 5.6, 2.3 Hz, CH2OH), 5.35 (t, 1H, J = 5.5 Hz, CH2OH), 7.17 (s, 1H, OH), 7.72 (s, 1H, CHarom), 8.05 (c, 1H, CH). 13C NMR (DMSO-d6) δ ppm: 19.40, 27.91, 28.72, 59.17, 98.33, 123.77, 129.30, 131.92, 138.66, 140.93, 146.74, 147.32, 197.85. MALDI TOF—MS m/z: 250.2 [M+H]+. Calculated for C13H15NO4, %: C, 62.64; H, 6.07; N, 5.62. Found, %: C, 62.57; H, 6.15; N, 5.53.
1,3-bis(7-hydroxy-6-methyl-1,3-dihydrofuro[3,4-c]pyridin-1-yl)propan-2-one 6. To a solution of acetylacetone (0.19 g, 1.9 mmol) in a mixture of ethanol (30 mL) and 40% aqueous solution of KOH (10 mL) pyridoxal (0.64 g, 3.8 mmol) was added. The reaction mixture was stirred at room temperature for 24 h. Then, concentrated hydrochloric acid was added to the resulting solution (pH = 7), and the precipitated potassium chloride was filtered off. The filtrate was evaporated, and pure compound 6 was isolated by recrystallization from anhydrous ethanol. Light orange solid. Yield: 0.56 g (82%); mp: 157–160 °C. IR (ν cm−1): 516, 570, 652, 778, 840, 985, 1048, 1199, 1237, 1312, 1386, 1423, 1533, 1617, 1712, 2861, 3100, 3255, 3392. 1H NMR (DMSO-d6) δ ppm: 2.37 (s, 6H; 2CH3), 2.77 (dd, 2H, J = 16.4, 9.3 Hz, CH2), 3.06 (dd, 2H, J = 16.5, 2.5 Hz, CH2), 4.92 (d, 2H, J = 12.4 Hz, CH2O), 5.01 (dd, 2H, J = 12.4, 2.5 Hz, CH2O,), 5.68 (d, 2H, J = 9.2 Hz, CH), 7.89 (s, 2H, 2CHarom). 13C NMR (DMSO-d6) δ ppm: 19.89, 47.63, 70.92, 78.71, 133.22, 135.62, 136.36, 146.25, 146.62, 206.32. MALDI TOF—MS m/z: 357.2 [M+H]+. Calculated for C19H20N2O5, %: C 64.04; H 5.66; N 7.86. Found, %: C 63.97; H 5.63; N 4.91.

3.1.3. General Procedure for the Synthesis of Compounds 7

The mixture of the acid 3a (0.3 g, 1.28 mmol), the corresponding amine (2.56 mmol, 1.28 mmol in case of diamines), and 1.25 mL of phosphorus oxychloride was heated at 90 °C for 1 h. With heating, the precipitate gradually dissolved, and the reaction mixture acquired a dark purple color. Upon completion of the heating, the reaction mixture was cooled, and a small amount of ice was added. Then, with cooling and stirring, 10% aqueous ammonia solution was added dropwise until pH = 6–7. The formed precipitate was filtered off and washed consequently with hot water (2 × 5 mL), ethyl alcohol (2 × 5 mL), and diethyl ether (5 mL). If additional purification was required, the precipitate was refluxed in 10 mL of water and then in 10 mL of ethanol and filtered off. The resulting precipitate was dried in a vacuum to give title compounds 7.

3.1.4. Characterization Data for Compounds 7

5-(Chloromethyl)-8-methyl-2-oxo-N-phenyl-2aH-pyrano[2,3-c]pyridine-3-carboxamide 7a
Yellow solid. Yield: 90%; mp: 240 °C dec. IR (ν cm−1): 629, 703, 762, 791, 1054, 1214, 1255, 1285, 1412, 1444, 1491, 1550, 1595, 1616, 1666, 1723, 3242. 1H NMR (DMSO-d6) δ ppm: 2.66 (s, 3H, CH3), 5.24 (s, 2H CH2Cl), 7.17 (t, 1H, J = 7.4 Hz, Ph), 7.41 (t, 2H, J = 7.7 Hz, Ph), 7.73 (d, 2H, J = 8.0 Hz, Ph), 8.57 (s, 1H, CHarom), 8.87 (s, 1H, CH), 10.62 (s, 1H, NH). 13C NMR (DMSO-d6) δ ppm: 19.21, 40.19, 120.35, 122.08, 125.00, 126.77, 128.55, 129.53, 138.32, 140.87, 144.88, 147.69, 148.82, 158.76,160.01. MALDI TOF—MS m/z: 329.4 [M]+. Calculated for C17H13ClN2O3, %: C,62.11; H, 3.99; Cl, 10.78; N, 8.52. Found, %: C, 62.09; H, 4.01; Cl, 10.63; N, 8.87.
5-(Chloromethyl)-N-(2-methoxyphenyl)-8-methyl-2-oxo-2H-pyrano[2,3-c]pyridine-3-carboxamide 7b
Orange solid. Yield: 91%; mp: 214–5 °C. IR (ν cm−1): 717, 751, 788, 885, 972, 1022, 1114, 1145, 1229, 1256, 1284, 1305, 1389, 1435, 1467, 1486, 1541, 1598, 1622, 1670, 1732, 3233. 1H NMR (DMSO-d6) δ ppm: 2.66 (s, 3H, CH3), 3.93 (s, 3H, CH3), 5.26 (s, 2H, CH2Cl), 7.00 (ddd, 1H, J = 8.5, 5.9, 3.0 Hz, Ph), 7.07–7.15 (m, 2H, Ph), 8.42–8.46 (m, 1H, Ph), 8.59 (s, 1H, CHarom), 9.05 (s, 1H, CH), 11.14 (s, 1H, NH). 13C NMR (DMSO-d6) δ ppm: 18.74, 39.65, 56.59, 111.55, 120.03, 121.15, 122.69, 124.57, 125.27, 127.47, 128.95, 142.33, 143.92, 147,94, 148.60, 149.00, 158.49, 159.84. MALDI TOF—MS m/z: 359.1 [M]+. Calculated for C18H15ClN2O4, %: C, 60.26; H, 4.21; Cl, 9.88; N, 7.81. Found, %: C, 60.22; H, 4.15; Cl, 9.53; N, 7.99.
N-(2-Bromo-4-nitrophenyl)-5-(chloromethyl)-8-methyl-2-oxo-2H-pyrano[2,3-c]pyridine-3-carboxamide 7c
Light orange solid. Yield: 77%; mp: 240 °C dec. IR (ν cm−1): 690, 714, 742, 764, 793, 837, 891, 1057, 1118, 1159, 1215, 1268, 1313, 1341, 1395, 1413, 1514, 1547, 1584, 1613, 1677, 1727, 3167, 3391, 3486. 1H NMR (DMSO-d6) δ ppm: 2.66 (s, 3H, CH3), 5.29 (s, 2H, CH2Cl), 8.35 (dd, 1H, J = 9.2, 2.7 Hz, Ph), 8.57 (d, J = 2.6 Hz, Ph), 8.61 (s, 1H, CHarom), 8.76 (d, 1H, J = 9.2 Hz, Ph), 9.13 (s, 1H, CH), 11.47 (s, 1H, NH). 13C NMR (DMSO-d6) δ ppm:19.21, 40.19, 113.54, 113.94, 121.67, 122.02, 123.60, 124.83, 125.57, 128.62, 128.79, 129.34, 142.11, 143.88, 143.94, 145.01, 147.86, 149.14, 160.12, 160.15. Calculated for C17H11BrClN3O5, %: C, 45.11; H, 2.45; Br, 17.65; Cl, 7.83; N, 9.28. Found, %: C, 44.95; H, 2.63; Br, 17.38; Cl, 7.95; N, 9.13.
5-(Chloromethyl)-8-methyl-2-oxo-N-(pyridin-2-yl)-2H-pyrano[2,3-c]pyridine-3-carboxamide 7d
Black solid.Yield: 79%; mp: >300 °C. IR (ν cm−1): 700, 764, 793, 972, 990, 1055, 1148, 1220, 1265, 1293, 1305, 1378, 1415, 1435, 1534, 1575, 1591, 1671, 1723, 3282, 3435. 1H NMR (DMSO-d6) δ ppm: 2.66 (s, 3H, CH3), 5.25 (s, 2H, CH2Cl), 7.22 (dd, 1H, J = 7.2, 5.1 Hz, Py), 7.91 (t, 1H, J = 7.9 Hz, Py), 8.25 (d, 1H, J = 8.1 Hz, Py), 8.40 (d, 1H, J = 4.0 Hz, Py), 8.58 (s, 1H, CHarom), 9.00 (s, 1H, CH), 11.07 (s, 1H, NH). 13C NMR (DMSO-d6) δ ppm: 19.23, 40.19, 114.24, 121.08, 122.02, 128.61, 129.76, 132.08, 139.21, 144.89, 148.95, 151.17, 159.83. Calculated for C16H12ClN3O3, %: C, 58.28; H, 3.67; Cl, 10.75; N, 12.74. Found, %: C, 58.18; H, 3.81; Cl, 10.65; N, 13.01.
N-(2-Aminopyridin-3-yl)-5-(chloromethyl)-8-methyl-2-oxo-2H-pyrano[2,3-c]pyridine-3-carboxamide 7e
Dark brown solid. Yield: 66%; mp: >300 °C. IR (ν cm−1): 701, 762, 789, 889, 946, 1055, 1149, 1228, 1294, 1376, 1414, 1450, 1549, 1592, 1626,1666, 1733, 3233, 3350, 3424. 1H NMR (DMSO-d6) δ ppm: 2.66 (s, 3H, CH3), 5.25 (s, 2H, CH2Cl), 5.98 (br.s, 2H, NH2), 6.68 (dd, 1H, J = 7.7, 5.0 Hz, Py), 7.77 (dd, 1H, J = 7.6, 1.7 Hz, Py), 7.89 (dd, 1H, J = 5.0, 1.7 Hz, Py), 8.58 (s, 1H, CHarom), 8.91 (s, 1H, CH), 9.95 (s, 1H, NH). 13C NMR (DMSO-d6) δ ppm: 19.75, 41.12, 113.36, 118.78, 122.53, 126.62, 129.13, 133.52, 141.62, 145.43, 145.60, 148.22, 149.40, 154.50, 159.44, 161.07. MALDI TOF—MS m/z: 345.1 [M]+. Calculated for C16H13ClN4O3, %: C, 55.74; H, 3.80; Cl, 10.28; N, 16.25. Found, %: C, 55.89; H, 3.97; Cl, 10.43; N, 16.25.
5-(Chloromethyl)-3-(diphenylcarbamoyl)-8-methyl-2-oxo-2H-pyrano[2,3-c]pyridin-7-ium chloride 7f
Yellow solid. Yield: 78%; mp: 201–5 °C. IR (ν cm−1): 695, 766, 799, 1055, 1079, 1150, 1202, 1263, 1280, 1309, 1352, 1407, 1453, 1491, 1550, 1592, 1651, 1723, 3429. 1H NMR (DMSO-d6) δ ppm: 2.52 (s, 3H, CH3), 5.08 (s, 2H, CH2Cl), 7.23–7.41 (m, 10H, 2Ph), 8.46 (s, 1H, CHarom), 8.61 (s, 1H, CH). 13C NMR (DMSO-d6) δ ppm: 19.03, 40.06, 121.54, 127.01, 127.46, 128.26, 128.56, 129.07, 129.73, 130.00, 130.34, 138.41, 142.09, 144.79, 147.16, 148.65, 156.16, 163.65. MALDI TOF—MS m/z: 427.1[M-Cl+Na]+. Calculated for C23H18Cl2N2O3, %: C, 62.60; H, 4.11; Cl, 16.07; N, 6.35. Found, %: C, 62.42; H, 4.01; Cl, 15.93; N, 6.14.
5-(Chloromethyl)-8-methyl-2-oxo-N-(prop-2-yn-1-yl)-2H-pyrano[2,3-c]pyridine-3-carboxamide 7g
Brown solid. Yield: 45%; mp: 161 °C dec. IR (ν cm−1): 546, 562, 657, 699, 799, 957, 978, 1069, 1157, 1230, 1266, 1293, 1343, 1419, 1477, 1513, 1544, 1595, 1611, 1666, 1715, 2122, 2937, 3055, 3291, 3314. 1H NMR (DMSO-d6) δ ppm: 2.62 (s, 3H, CH3), 3.16 (t, 1H, J = 2.5 Hz, CH), 4.14 (dd, 2H, J = 5.7, 2.5 Hz, CH2), 5.21 (s, 2H, CH2Cl), 8.54 (s, 1H, CHarom), 8.86 (s, 1H, CH), 8.98 (t, 1H, J = 5.7 Hz, NH). 13C NMR (DMSO-d6) δ ppm: 19.76, 29.91, 41.13, 74.36, 81.50, 122.52, 125.23, 129.05, 142.46, 145.20, 148.37, 149.39, 159.35, 161.48. MALDI TOF—MS m/z: 291.1 [M]+. Calculated for C14H11ClN2O3, %: C, 57.84; H, 3.81; Cl, 12.19; N, 9.64. Found, %: C, 57.96; H, 4.10; Cl, 12.13; N, 9.58.
5-(Chloromethyl)-8-methyl-2-oxo-N-(4-sulfamoylphenyl)-2H-pyrano[2,3-c]pyridine-3-carboxamide 7h
Peach solid. Yield: 65%; mp: >300 °C. IR (ν cm−1): 541, 552, 597, 682, 706, 763, 792, 837, 889, 1046, 1098, 1163, 1188, 1217, 1253, 1316, 1340, 1407, 1438, 1547, 1593, 1616, 1675, 1728, 3068, 3109, 3242, 3281, 3318. 1H NMR (DMSO-d6) δ ppm: 2.65 (s, 3H, CH3), 5.23 (s, 2H CH2Cl), 7.32 (s, 2H, NH), 7.87 (q, 4H, J = 8.8 Hz, Ph), 8.57 (s, 1H, CHarom), 8.87 (s, 1H, CH), 10.87 (s, 1H, NH). 13C NMR (DMSO-d6) δ ppm: 19.75, 41.07, 120.69, 122.52, 127.23, 127.92, 129.12, 140.63, 141.66, 145.46, 148.24, 149.40, 159.06, 161.16. ESI—MS m/z: 408.1 [M]+.Calculated for C17H14ClN3O5S, %: C, 50.07; H, 3.46; Cl, 8.69; N, 10.30; S, 7.86. Found, %: C, 50.09; H, 3.54; Cl, 8.63; N, 10.47; S, 7.75.
N-(4-(N-Acetylsulfamoyl)phenyl)-5-(chloromethyl)-8-methyl-2-oxo-2H-pyrano[2,3-c]pyridine-3-carboxamide 7i
Coral solid. Yield: 72%; mp: >300 °C. IR (ν cm−1): 548, 576, 595, 623, 636, 685, 704, 762, 779, 791, 842, 862, 949, 996, 1055, 1091, 1159, 1187, 12,19, 1294, 1315, 1337, 1406, 1442, 1476, 1536, 1591, 1617, 1680, 1735, 3065, 3103, 3277. 1H NMR (DMSO-d6) δ ppm: 1.93 (s, 3H, CH3), 2.65 (s, 3H, CH3), 5.22 (s, 2H CH2Cl), 7.94 (s, 4H, Ph), 8.57 (s, 1H, CHarom), 8.86 (s, 1H, CH), 10.96 (s, 1H, NH), 12.03 (s, 1H, NH). 13C NMR (DMSO-d6) δ ppm: 19.73, 24.21, 41.05, 120.62, 122.49, 127.29, 129.14, 130.06, 135.32, 141.66, 143.28, 145.45, 148.24, 149.40, 158.91, 161.41, 169.73. ESI—MS m/z: 450.2 [M]+. Calculated for C19H16ClN3O6S, %: C, 50.73; H, 3.59; Cl, 7.88; N, 9.34; S, 7.13. Found, %: C, 50.69; H, 3.54; Cl, 7.73; N, 9.47; S, 7.25.
5-(Chloromethyl)-8-methyl-2-oxo-N-(4-(N-(pyrimidin-2-yl)sulfamoyl)phenyl)-2H-pyrano[2,3-c]pyridine-3-carboxamide 7j
Rosy brown solid. Yield: 73%; mp: >300 °C. IR (ν cm−1): 569, 581, 623, 633, 682, 705, 763, 794, 840, 939, 1014, 1055, 1090, 1158, 1186, 1217, 1256, 1293, 1317, 1344, 1407, 1435, 1461, 1540, 15910, 1620, 1678, 1725, 3042, 3116, 3292, 3437. 1H NMR (DMSO-d6) δ ppm: 2.64 (s, 3H, CH3), 5.21 (s, 2H CH2Cl), 7.05 (t, 1H, J = 4.9 Hz, CHPyr), 7.90 (d, 2H, J = 8.6 Hz, Ph), 8.02 (d, 2H, J = 8.6 Hz, Ph), 8.51 (d, 2H, J = 4.9 Hz, CHPyr), 8.56 (s, 1H, CHarom), 8.84 (s, 1H, CH), 10.91 (s, 1H, NH). 13C NMR (DMSO-d6) δ ppm: 19.74, 41.05, 116.79, 120.42, 122.47, 127.38, 129.10, 130.04, 136.48, 141.51, 142.67, 145.47, 148.22, 149.39, 157.90, 158.89, 159.34, 161.34. ESI—MS m/z: 486.2 [M]+. Calculated for C21H16ClN5O5S, %: C, 51.91; H, 3.32; Cl, 7.30; N, 14.41; S, 6.60. Found, %: C, 51.99; H, 3.44; Cl, 7.27; N, 14.50; S, 6.47.
5-(Chloromethyl)-8-methyl-N-(4-(N-(4-methylpyrimidin-2-yl)sulfamoyl)phenyl)-2-oxo-2H-pyrano[2,3-c]pyridine-3-carboxamide 7k
Rosy brown solid. Yield: 70%; mp: >300 °C. IR (ν cm−1): 546, 571, 582, 624, 681, 704, 762, 794, 841, 889, 964, 1054, 1092, 1158, 1186, 1217, 1257, 1318, 1345, 1405, 1439, 1498, 1543, 1591, 1679, 1727, 3064, 3287, 3393. 1H NMR (DMSO-d6) δ ppm: 2.33 (s, 3H, CH3), 2.64 (s, 3H, CH3), 5.21 (s, 2H CH2Cl), 6.90 (d, 1H, J = 5.2 Hz, CHPyr), 7.89 (d, 2H, J = 8.9 Hz, Ph), 8.02 (d, 2H, J = 8.8 Hz, Ph), 8.32 (d, 1H, J = 5.1 Hz, CHPyr), 8.56 (s, 1H, CHarom), 8.85 (s, 1H, CH), 10.90 (s, 1H, NH). 13C NMR (DMSO-d6) δ ppm: 19.73, 24.23, 41.06, 115.79, 120.48, 122.48, 127.33, 129.10, 130.24, 136.69, 141.54, 142.52, 145.46, 148.22, 149.39, 157.51, 158.93, 161.29. ESI—MS m/z: 500.2 [M]+. Calculated for C22H18ClN5O5S, %: C, 52.86; H, 3.62; Cl, 7.09; N, 14.01; S, 6.41. Found, %: C, 52.94; H, 3.51; Cl, 7.13; N, 14.11; S, 6.37.
5-(Chloromethyl)-N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)-8-methyl-2-oxo-2H-pyrano[2,3-c]pyridine-3-carboxamide 7l
Rosy brown solid. Yield: 66%; mp: >300 °C. IR (ν cm−1): 546, 562, 586, 624, 633, 680, 707, 763, 793, 840, 861, 974, 1015, 1057, 1083, 1159, 1185, 1217, 1256, 1315, 1350, 1384, 1404, 1416, 1498, 1543, 1594, 1678, 1729, 3066, 3110, 3247, 3286. 1H NMR (DMSO-d6) δ ppm: 2.26 (s, 6H, 2CH3), 2.64 (s, 3H, CH3), 5.21 (s, 2H CH2Cl), 6.75 (s, 1H, CHPyr), 7.88 (d, 2H, J = 8.7 Hz, Ph), 8.02 (d, 2H, J = 8.5 Hz, Ph), 8.56 (s, 1H, CHarom), 8.85 (s, 1H, CH), 10.89 (s, 1H, NH). 13C NMR (DMSO-d6) δ ppm: 19.73, 23.82, 41.06, 120.06, 122.49, 127.31, 129.10, 130.43, 141.53, 142.32, 145.45, 148.22, 149.39, 157.15, 158.95, 161.24, 167.53. ESI—MS m/z: 514.2 [M]+. Calculated for C23H20ClN5O5S, %: C, 53.75; H, 3.92; Cl, 6.90; N, 13.63; S, 6.24. Found, %: C, 53.84; H, 3.98; Cl, 7.01; N, 13.74; S, 6.30.
N′-Benzoyl-5-(chloromethyl)-8-methyl-2-oxo-2H-pyrano[2,3-c]pyridine-3-carbohydrazide 7m
Indianred solid. Yield: 75%; mp: 240 °C dec. IR (ν cm−1): 693, 715, 765, 929, 995, 1022, 1050, 1144, 1215, 1241, 1288, 1410, 1446, 1476, 1526, 1546, 1605, 1621, 1755, 3491. 1H NMR (DMSO-d6) δ ppm: 2.66 (s, 3H, CH3), 5.29 (s, 2H, CH2Cl), 7.68–7.71 (m, 3H, Ph), 8.14–8.17 (m, 2H, Ph), 8.58 (s, 1H, CHarom), 9.03 (s, 1H, CH). 13C NMR (DMSO-d6) δ ppm: 19.20, 40.22, 117.50, 121.68, 123.32, 127.41, 128.40, 130.06, 133.03, 140.12, 144.87, 147.81, 148.84, 154.83, 160.47, 165.25. MALDI TOF—MS m/z: 372.1[M]+. Calculated for C18H14ClN3O4, %: C, 58.15; H, 3.80; Cl, 9.54; N, 11.30. Found, %: C, 58.39; H, 3.71; Cl, 9.81; N, 10.99.
5-(Chloromethyl)-N′-isonicotinoyl-8-methyl-2-oxo-2H-pyrano[2,3-c]pyridine-3-carbohydrazide 7n
Peach solid. Yield: 72%; mp: >300 °C. IR (ν cm−1): 701, 754, 790, 846, 1061, 1192, 1220, 1252, 1296, 1315, 1414, 1480, 1507, 1552, 1617, 1638, 1653, 1722, 3233. 1H NMR (DMSO-d6) δ ppm: 2.65 (s, 3H, CH3), 5.25 (s, 2H, CH2Cl), 7.84 (dd, 2H, J = 4.4, 1.7 Hz, Ph), 8.57 (s, 1H, CHarom), 8.80 (dd, 2H, J = 4.4, 1.7 Hz, Ph), 8.86 (s, 1H, CH), 10.67 (s, 1H, NH), 11.27 (s, 1H, NH). 13C NMR (DMSO-d6) δ ppm: 19.25, 39.73, 121.83, 121.91, 124.59, 128.52, 139.71, 142.18, 144.76, 147.91, 149.00, 150.90, 158,24, 160.21, 163.96. MALDI TOF—MS m/z: 373.1[M]+. Calculated for C17H13ClN4O4, %: C, 54.78; H, 3.52; Cl, 9.51; N, 15.03. Found, %: C, 54.65; H, 3.64; Cl, 9.33; N, 14.79.
N,N′-(1,2-Phenylene)bis(5-(chloromethyl)-8-methyl-2-oxo-2H-pyrano[2,3-c]pyridine-3-carboxamide) 7o
Brown solid. Yield: 91%; mp: 239–241 °C. IR (ν cm−1): 704, 760, 792, 886, 957, 1015, 1054, 1083, 1146, 1220, 1266, 1292, 1412, 1456, 1479, 1518, 1547, 1600, 1676, 1722, 3233. 1H NMR (DMSO-d6) δ ppm: 2.64 (s, 6H, 2CH3), 5.25 (s, 4H, 2CH2Cl), 7.36 (dd, 2H, J = 6.1, 3.5 Hz, Ph), 7.89 (dd, 2H, J = 6.0, 3.5 Hz, Ph), 8.59 (s, 2H, 2CHarom), 9.02 (s, 2H, 2CH), 10.63 (s, 2H, 2NH). 13C NMR (DMSO-d6) δ ppm: 19.22, 40.13, 121.97, 125.03, 125.23, 126.84, 128.60, 130.98, 142.44, 144.88, 147.81, 148.99, 159.10, 160.23. MALDI TOF—MS m/z: 601.6 [M+Na-H]+. Calculated for C28H20Cl2N4O6, %: C, 58.05; H, 3.48; Cl, 12.24; N, 9.67. Found, %: C, 57.89; H, 3.59; Cl, 12.33; N, 9.82.
N,N′-(1,3-Phenylene)bis(5-(chloromethyl)-8-methyl-2-oxo-2H-pyrano[2,3-c]pyridine-3-carboxamide) 7p
Brown solid. Yield: 97%; mp: 274 °C dec. IR (ν cm−1): 689, 793, 879, 952, 1055, 1082,1144, 1184, 1229, 1293, 1413, 1491, 1548, 1608, 1671, 1722, 3307, 3504. 1H NMR (DMSO-d6) δ ppm: 2.66 (s, 6H, 2CH3), 5.23 (s, 4H, 2CH2Cl), 7.40–7.44 (m, 1H, Ph), 7.52 (d, 2H, J = 8.0 Hz, Ph), 8.22 (s, 1H, Ph), 8.57 (s, 2H, 2CHarom), 8.87 (s, 2H, 2CH), 10.70 (s, 2H, 2NH). 13C NMR (DMSO-d6) δ ppm: 19.24, 40.20, 111.77, 116.44, 122.10, 126.97, 128.58, 130.18, 138.93, 140.82, 144.93, 147.72, 148.86, 158.70, 160.25. MALDI TOF—MS m/z: 602.0[M+Na]+. Calculated for C28H22Cl2N4O6, %: C, 57.84; H, 3.81; Cl, 12.19; N, 9.64. Found, %: C, 58.09; H, 3.61; Cl, 12.04; N, 9.79.
N,N′-(1,4-Phenylene)bis(5-(chloromethyl)-8-methyl-2-oxo-2H-pyrano[2,3-c]pyridine-3-carboxamide) 7q
Brown solid. Yield: 95%; mp: >300 °C. IR (ν cm−1): 743, 792, 837, 951, 1055, 1083, 1150, 1217, 1259, 1292, 1314, 1411, 1516, 1561, 1616, 1721, 3294. 1H NMR (DMSO-d6) δ ppm: 2.66 (s, 6H, 2CH3), 5.24 (s, 4H, 2CH2Cl), 7.77 (s, 4H, Ph), 8.57 (s, 2H, 2CHarom), 8.87 (s, 2H, 2CH), 10.65 (s, 2H, 2NH). 13C NMR (DMSO-d6) δ ppm: 19.25, 40.21, 121.02, 122.12, 126.76, 128.58, 134.84, 140.88, 144.93. 147.72, 148.86, 158.79, 159.89. MALDI TOF—MS m/z: 602.0 [M+Na]+. Calculated for C28H20Cl2N4O6, %: C, 58.05; H, 3.48; Cl, 12.24; N, 9.67. Found, %: C, 58.12; H, 3.31; Cl, 12.13; N, 9.81.
N,N′-(Sulfonylbis(4,1-phenylene))bis(5-(chloromethyl)-8-methyl-2-oxo-2H-pyrano[2,3-c]pyridine-3-carboxamide) 7r
Apricot solid. Yield: 78%; mp: >300 °C. IR (ν cm−1): 548, 570, 595, 623, 668, 690, 718, 762, 794, 839, 887, 953, 1013, 1055, 1084, 1106, 1150, 1184, 1217, 1256, 130,5 1352, 1404, 1438, 1537, 1590, 1618, 1678, 1728, 3061, 3115, 3286. 1H NMR (DMSO-d6) δ ppm: 2.63 (s, 6H, 2CH3), 5.21 (s, 4H, 2CH2Cl), 7.97 (q, 8H, J = 8.7 Hz, 2Ph), 8.55 (s, 2H, 2CHarom), 8.85 (s, 2H, 2CH), 10.97 (s, 2H, 2NH). 13C NMR (DMSO-d6) δ ppm: 19.72, 41.04, 121.21, 122.44, 127.17, 129.11, 129.71, 137.36, 141.73, 143.27, 145.46, 148.22, 149.39, 158.89, 161.40. ESI—MS m/z: 718.2 [M-H]+. Calculated for C34H24ClN4O8S, %: C, 56.75; H, 3.36; Cl, 9.85; N, 7.79; S, 4.46. Found, %: C, 56.91; H, 3.34; Cl, 9.90; N, 7.81; S, 4.52.
N,N′-(Sulfonylbis(3,1-phenylene))bis(5-(chloromethyl)-8-methyl-2-oxo-2H-pyrano[2,3-c]pyridine-3-carboxamide) 7s
Rosewood solid. Yield: 67%; mp: >300 °C. IR (ν cm−1): 523, 563, 583, 613, 687, 716, 761, 794, 875, 894, 951, 970, 1015, 1054, 1084, 1101, 1152, 1217, 1304, 1413, 1479, 1546, 1592, 1616, 1673, 1723, 3063, 3240, 3285. 1H NMR (DMSO-d6) δ ppm: 2.64 (s, 6H, 2CH3), 5.21 (s, 4H, 2CH2Cl), 7.68 (t, 2H, J = 7.9 Hz, Ph), 7.75–7.77 (m, 2H, Ph), 7.90–7.93 (m, 2H, Ph), 8.49 (t, 2H, J = 2.0 Hz, Ph), 8.56 (s, 2H, 2CHarom), 8.87 (s, 2H, 2CH), 10.93 (s, 2H, 2NH). 13C NMR (DMSO-d6) δ ppm: 19.74, 41.03, 119.12, 122.46, 124.11, 125.84, 127.27, 129.12, 131.75, 139.98, 141.66, 142.59, 145.47, 148.22, 149.39, 158.75, 161.39. ESI—MS m/z: 741.2 [M-H+Na]+. Calculated for C34H24ClN4O8S, %: C, 56.75; H, 3.36; Cl, 9.85; N, 7.79; S, 4.46. Found, %: C, 56.87; H, 3.42; Cl, 9.98; N, 7.71; S, 4.32.

3.1.5. General Procedure for the Synthesis of Compounds 8

Method A. An aqueous solution of the acid 3a (0.3 g, 1.28 mmol) and the corresponding amine (1.28 mmol, 0.64 mmol in case of diamines) was stirred at room temperature for 2 h. The precipitate was filtered off and dried in a vacuum to give a title compound 8.
Method B. An aqueous solution of the acid 3a (0.3 g, 1.28 mmol) and the corresponding amine (1.28 mmol, 0.64 mmol in case of diamines) was stirred at room temperature for 2 h. The solvent was evaporated, and the residue was taken up in a ethanol:diethyl ether mixture (1:1, 4 mL). The precipitate was filtered off and dried in a vacuum to give a title compound 8.

3.1.6. Characterization Data for Compounds 8

Pyridin-2-aminium 5-(hydroxymethyl)-8-methyl-2-oxo-2H-pyrano[2,3-c]pyridine-3-carboxylate 8a
Method A. White solid. Yield: 96%; mp: 245 °C. IR (ν cm−1): 606, 623, 750, 773, 813, 843, 951, 984, 1017, 1072, 1148, 1208, 1256, 1370, 1413, 1491, 1603, 1637, 1666, 1746, 3089, 3325. 1H NMR (DMSO-d6) δ ppm: 2.56 (s, 3H, CH3), 4.76 (s, 2H, CH2OH), 6.53—6.61 (m, 2H, Py), 7.50 (t, 1H, J = 7.0 Hz, Py), 7.90 (d, 1H, J = 4.5 Hz, Py), 8.33 (s, 1H, CHarom), 8.58 (s, 1H, CH). 13C NMR (DMSO-d6) δ ppm: 18.98, 58.56, 109.94, 112.28, 121.87, 126.30, 132.04, 139.24, 141.23, 143.02, 144.93146.76, 147.94, 156.24, 158.94, 165.42. MALDI TOF—MS m/z: 235.4 [M-94 (C5H6N2—2-aminopyridine)]+. Calculated for C16H15N3O5, %: C, 58.36; H, 4.59; N, 12.76. Found, %: C, 58.15; H, 4.41; N, 12.69.
Cyclohexanaminium 5-(hydroxymethyl)-8-methyl-2-oxo-2H-pyrano[2,3-c]pyridine-3-carboxylate 8b
Method A. White solid. Yield: 95%; mp: 213 °C. IR (ν cm−1): 569, 677, 746, 929, 1037, 1066, 1087, 1173, 1264, 1387, 1413, 1562, 1628, 1708, 2931, 3410. 1H NMR (DMSO-d6) δ ppm: 1.08–1.13 (m, 1H, C6H11), 1.21–1.32 (m, 4H, C6H11), 1.57 (d, 1H, J = 13.0 Hz, C6H11), 1.71 (d, 2H, J = 12.2 Hz, C6H11), 1.92 (d, 2H, J = 11.2 Hz, C6H11), 2.54 (s, 3H, CH3), 2.94 (td, 1H, J = 10.5, 10.0, 5.4 Hz, C6H11), 4.70 (s, 2H, CH2OH), 8.07 (s, 1H, CHarom), 8.25 (s, 1H, CH). 13C NMR (DMSO-d6) δ ppm: 18.96, 24.31, 25.11, 30.90, 49.67, 58.60, 123.00, 131.43, 142.70, 146.14, 147.35, 157.57, 165.87. MALDI TOF—MS m/z: 235.1 [M-99 (C6H13N—cyclohexylamine)]+. Calculated for C17H22N2O5, %: C, 61.07; H, 6.63; N, 8.38. Found, %: C, 60.76; H, 6.75; N, 8.13.
Pyridine-2,3-diaminium 5-(hydroxymethyl)-8-methyl-2-oxo-2H-pyrano[2,3-c]pyridine-3-carboxylate 8c
Method A. Brown solid. Yield: 80%; mp: 184–5 °C. IR (ν cm−1): 645, 733, 772, 811, 844, 1014, 1038, 1086, 1151, 1254, 1290, 1375, 1410, 1577, 1681, 1750, 3191, 3321, 3449. 1H NMR (DMSO-d6) δ ppm: 2.56 (s, 6H, 2CH3), 4.76 (s, 4H, 2CH2OH), 6.65 (dd, 1H, J = 7.6, 5.9 Hz, Py), 6.99 (dd, 1H, J = 7.7, 1.4 Hz, Py), 7.26 (dd, 1H, J = 6.0, 1.4 Hz, Py), 8.32 (s, 2H, 2CHarom), 8.53 (s, 2H, 2CH). 13C NMR (DMSO-d6) δ ppm: 18.95, 58.53, 113.63, 119.63, 121.97, 124.39, 127.28, 131.92, 133.36, 140.31, 142.94, 146.65, 146.67, 147.82, 156.40, 165.92. MALDI TOF—MS m/z: 235.2 [M-109 (C5H7N3—2,3-diaminopyridine)]+. Calculated for C27H25N5O10, %: C, 55.96; H, 4.35; N, 12.08. Found, %: C, 55.31; H, 3.89; N, 12.63.
Pyridine-2,6-diaminium 5-(hydroxymethyl)-8-methyl-2-oxo-2H-pyrano[2,3-c]pyridine-3-carboxylate 8d
Method A. Palegreen solid. Yield: 95%; mp: 198 °C. IR (ν cm−1): 643, 733, 749, 777, 812, 908, 982, 1037, 1072, 1152, 1263, 1291, 1366, 1415, 1570, 1638, 1741, 3217, 3377, 3447. 1H NMR (DMSO-d6) δ ppm: 2.56 (s, 6H, 2CH3), 4.75 (s, 4H, 2CH2OH), 5.79 (d, 2H, J = 8.0 Hz, Py), 7.32 (t, 1H, J = 8.0 Hz, Py), 8.32 (s, 2H, 2CHarom), 8.52 (s, 2H, 2CH). 13C NMR (DMSO-d6) δ ppm: 18.99, 58.55, 95.47, 122.01, 127.34, 131.96, 140.25, 142.99, 143.08, 146.68, 147.85, 155.14, 156.45, 165.85. MALDI TOF—MS m/z: 236.0 [M-109 (C5H7N3—2,6-diaminopyridine)]+. Calculated for C27H25N5O10, %: C, 55.96; H, 4.35; N, 12.08. Found, %: C, 56.02; H, 3.94; N, 11.98.
Pyridin-2-aminium 8-methyl-2-oxo-5-((phosphonooxy)methyl)-2H-pyrano[2,3-c]pyridine-3-carboxylate 8e
Method B. White solid. Yield: 90%; mp: 203 °C. IR (ν cm−1): 495, 515, 562, 627, 732, 771, 804, 849, 963, 1052, 1161, 1243, 1299, 1442, 1488, 1636, 1668, 1751, 2745, 3089. 1H NMR (DMSO-d6) δ ppm: 2.56 (s, 3H, CH3), 5.09 (d, 2H, J = 7.5 Hz, CH2O), 6.67 (t, 1H, J = 6.5 Hz, Py), 6.76 (d, 1H, J = 8.7 Hz, Py), 7.69 (t, 1H, J = 7.8 Hz, Py), 7.82 (d, 1H, J = 6.0 Hz, Py), 8.36 (s, 1H, CHarom), 8.63 (s, 1H, CH). 13C NMR (DMSO-d6) δ ppm: 19.04, 61.50, 61.55, 112.15, 112.19, 121.93, 125.51, 128.35, 128.43, 139.8, 139.95, 141.68, 141.92, 141.96, 143.93, 147.70, 147.85, 155.87, 156.46, 156.49, 164.69. 31P NMR (DMSO-d6) δ ppm: -0.13. MALDI TOF—MS m/z: 315.3[M-94 (C5H6N2—2-aminopyridine)]+. Calculated for C16H16N3O8P, %: C, 46.95; H, 3.94; N, 10.27; P, 7.57. Found, %: C, 46.81; H, 4.16; N, 10.01; P, 7.51.
Pyridine-2,6-diaminium 8-methyl-2-oxo-5-((phosphonooxy)methyl)-2H-pyrano [2,3-c]pyridine-3-carboxylate 8f
Method B. White solid. Yield: 89%; mp: 215 °C. IR (ν cm−1): 477, 500, 565, 623, 726, 806, 953, 1029, 1073, 1096, 1162, 1219, 1297, 1415, 1549, 1652, 1771, 3089, 3348. 1H NMR (DMSO-d6) δ ppm: 2.57 (s, 6H, 2CH3), 5.11 (d, 4H, J = 7.7 Hz, 2CH2O), 5.82 (d, 2H, J = 8.2 Hz, Py), 7.42, (t, 1H, J = 8.2 Hz, Py), 8.38 (s, 2H, 2CHarom), 8.65 (s, 2H, 2CH). 13C NMR (DMSO-d6) δ ppm: 19.54, 62.14, 95.66, 122.36, 125.60, 128.76, 128.84, 142.55, 144.55, 145.41, 148.31, 148.40, 153.31, 156.33, 164.97. 31P NMR (DMSO-d6) δ ppm: 0.95. MALDI TOF—MS m/z: 236.0 [M-109 (C5H7N3—2,6-diaminopyridine)]+. Calculated for C27H27N5O16P2, %: C, 43.85; H, 3.86; N, 9.47; P, 8.38. Found, %: C, 43.30; H, 4.01; N, 10.20; P, 8.51.

3.2. Biology

Cells and Materials. For the experiments, we used cancer cell cultures M-HeLa clone 11 (epithelioid carcinoma of the cervix, subline HeLa., clone M-HeLa), HuTu 80, human duodenal adenocarcinoma from the collection of the Institute of Cytology, Russian Academy of Sciences (St. Petersburg, Russia); human liver cells (Chang liver) from the collection and the Research Institute of Virology of the Russian Academy of Medical Sciences (Moscow, Russia).
Cytotoxic Assay. The cytotoxic effect on cells was determined using the colorimetric method of cell proliferation—the MTT test [34]. The cells were cultured in a standard Eagle’s nutrient medium manufactured at the Chumakov Institute of Poliomyelitis and Virus Encephalitis (PanEco company, Moscow, Russian Federation) and supplemented with 10% fetal calf serum and 1% nonessential amino acids. Cells were seeded on a 96-well Nunc plate at a concentration of 5 × 103 cells per well in a volume of 100 μL of medium and cultured in a CO2 incubator at 37 °C until a monolayer was formed. Then the nutrient medium was removed, and 100 µL of solutions of the test drug in the given dilutions were added to the wells, which were prepared directly in the nutrient medium with the addition of 5% DMSO to improve solubility. After 24 h of incubation of the cells with the tested compounds, the nutrient medium was removed from the plates, and 100 µL of the nutrient medium without serum with MTT at a concentration of 0.5 mg/mL was added and incubated for 4 h at 37 °C. Formazan crystals were added at 100 µL of DMSO to each well. Optical density was recorded at 540 nm on an Invitrologic microplate reader (Russia). The experiments for all compounds were repeated three times.
The selectivity index was determined as a ratio of the half-maximal inhibitory concentration towards the normal cell line to the half-maximal inhibitory concentration towards the tumor cell line. E.g., for HuTu 80 cell line SI = IC50(Chang liver)/IC50(HuTu 80).
Flow Cytometry Assay
Cell Culture. HuTu 80 cells at 1 × 106 cells/well in a final volume of 2 mL were seeded into six-well plates. After 24 h of incubation, solutions of compound 7r at concentrations IC50/2 (2.8 µM) and IC50 (5.5 µM) were added to wells.
Cell Apoptosis Analysis. HuTu 80 cells were incubated with compound 7r at concentrations IC50/2 (2.8 µM) and IC50 (5.5 µM) for 24 h. The cells were harvested at 2000 rpm for 5 min and then washed twice with ice-cold PBS, followed by resuspension in binding buffer. Next, the samples were incubated with 5 μL of annexin V- Alexa Fluor 647 (Sigma-Aldrich, St. Louis, MO, USA) and 5 μL of propidium iodide (Sigma-Aldrich, St. Louis, MO, USA) for 15 min at room temperature in the dark. Finally, the cells were analyzed using flow cytometry (Guava easy Cyte, MERCK, Darmstadt, Germany) within 1 h. The experiments were repeated three times.
Mitochondrial Membrane Potential. HuTu 80 cells were incubated with compound 7r at concentrations IC50/2 (2.8 µM), and IC50 (5.5 µM) for 24 h Cells were harvested at 2000 rpm for 5 min and then washed twice with ice-cold PBS, followed by resuspension in JC-10 (10 µg/mL) and incubation at 37 °C for 10 min. After the cells were rinsed three times and suspended in PBS, the JC-10 (Sigma-Aldrich, St. Louis, MO, USA) fluorescence was observed using flow cytometry (Guava easy Cyte, MERCK).
Detection of Intracellular ROS
HuTu 80 cells were incubated with compound 7r at concentrations IC50/2 (2.8 µM) and IC50 (5.5 µM) for 24 h. ROS generation was investigated using a flow cytometry assay and CellROX® Deep Red flow cytometry kit. For this, M-HeLa cells were harvested at 2000 rpm for 5 min and then washed twice with ice-cold PBS, followed by resuspension in 0.1 mL of medium without FBS, to which we added 0.2 μL of CellROX® Deep Red (Thermo Fisher Scientific, Waltham, MA, USA) and incubated at 37 °C for 30 min After three times washing the cells and suspending them in PBS, the production of ROS in the cells was immediately monitored using flow cytometer Guava easy Cyte, MERCK).
Statistical Analysis
The IC50 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.

4. 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.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms24129927/s1.

Author Contributions

Synthesis—A.V.T., A.S.T., N.O.A., L.K.K., G.M.A., A.K.T. and S.O.Y.; writing—review and editing—A.R.B.; writing—original draft preparation, conceptualization—A.S.G.; biological studies—A.S.S. and A.P.L.; biological studies supervision—A.D.V.; X-ray studies—A.B.D. and I.A.L.; project administration, supervision—A.R.B. and M.A.P. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by the Russian Science Foundation, grant number 21-13-00022, https://rscf.ru/en/project/21-13-00022/ (accessed on 30 April 2023).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

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.

Acknowledgments

The authors are grateful to the Collective Spectral-Analytical Center of the Federal Research Center of the Kazan Scientific Center of RAS for technical support of the studies. X-ray studies were performed using the equipment of the Federal Research Center Kazan Scientific Center of RAS.

Conflicts of Interest

The authors declare no conflict of interest.

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Ijms 24 09927 sch001 550
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-coumarin-3-carboxamides proposed in this work and the most potent compound (C).
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-coumarin-3-carboxamides proposed in this work and the most potent compound (C).
Ijms 24 09927 sch001
Ijms 24 09927 sch002 550
Scheme 2. Synthesis of amides 7[a] [a] Isolated yield is given [b], and it was obtained from 3b via the same procedure.
Scheme 2. Synthesis of amides 7[a] [a] Isolated yield is given [b], and it was obtained from 3b via the same procedure.
Ijms 24 09927 sch002
Ijms 24 09927 sch003 550
Scheme 3. Synthesis of ammonium salts 8.
Scheme 3. Synthesis of ammonium salts 8.
Ijms 24 09927 sch003
Ijms 24 09927 g001 550
Figure 1. (A) Induction of apoptosis in HuTu 80 cells incubated with compound 7r at concentration IC50/2 (2.8 µM) and at concentration IC50 (5.5 µM) in HuTu 80 cells; L—living cells; D—dead cells; Ea.—early apoptotic cells; La.—late apoptotic cells; (B) Percentage of early apoptotic cells (cyan) and late apoptotic cells (magenta) in control HuTu 80 cells and HuTu 80 cells treated with compound 7r. Data are presented as mean ± SD of three independent experiments. * Values indicate p < 0.05 compared to control.
Figure 1. (A) Induction of apoptosis in HuTu 80 cells incubated with compound 7r at concentration IC50/2 (2.8 µM) and at concentration IC50 (5.5 µM) in HuTu 80 cells; L—living cells; D—dead cells; Ea.—early apoptotic cells; La.—late apoptotic cells; (B) Percentage of early apoptotic cells (cyan) and late apoptotic cells (magenta) in control HuTu 80 cells and HuTu 80 cells treated with compound 7r. Data are presented as mean ± SD of three independent experiments. * Values indicate p < 0.05 compared to control.
Ijms 24 09927 g001
Ijms 24 09927 g002 550
Figure 2. (A) Effects on the mitochondrial membrane potential by compound 7r at concentration IC50/2 (2.8 µM) and at concentration IC50 (5.5 µM); (B) percentage of J-aggregates (red) and J-monomers (green) in control HuTu 80 cells and HuTu 80 cells treated with compound 7r. Data are presented as mean ± SD of three independent experiments. * Values indicate p < 0.05 compared to control.
Figure 2. (A) Effects on the mitochondrial membrane potential by compound 7r at concentration IC50/2 (2.8 µM) and at concentration IC50 (5.5 µM); (B) percentage of J-aggregates (red) and J-monomers (green) in control HuTu 80 cells and HuTu 80 cells treated with compound 7r. Data are presented as mean ± SD of three independent experiments. * Values indicate p < 0.05 compared to control.
Ijms 24 09927 g002
Ijms 24 09927 g003 550
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.
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.
Ijms 24 09927 g003
Table
Table 1. Development of the synthesis of key 7-aza-coumarin-3-carboxylic acid 3a.
Table 1. Development of the synthesis of key 7-aza-coumarin-3-carboxylic acid 3a.
Ijms 24 09927 i001
Entry2ConditionsProductYield, % a
1Ijms 24 09927 i002H2O, rt, 12 hIjms 24 09927 i00369 (3a)
286 (3b)
3Ijms 24 09927 i004H2O, catalyst, rt, 12 h
catalyst: citric acid, piperidine		Ijms 24 09927 i005<10
4Ijms 24 09927 i006EtOH or H2O, 60 °C, 4 hIjms 24 09927 i00789
5Ijms 24 09927 i008EtOH, piperidine (2% mol), AcOH (2% mol), reflux, 2 hIjms 24 09927 i00979
6EtOH/H2O (3/1), KOH (40% mol), rt, 24 hIjms 24 09927 i01082
a Isolated yield.
Table
Table 2. Cytotoxic effects of 7-azacoumarins 7 on cancer and normal human cell lines a.
Table 2. Cytotoxic effects of 7-azacoumarins 7 on cancer and normal human cell lines a.
CompoundsIC50 (µM)
Cancer Cell LinesNormal Cell Lines
M-HeLaHuTu 80Chang Liver
3a75.5 ± 6.057.6 ± 4.658.0 ± 4.7
3b70.6 ± 5.760.5 ± 5.370.0 ± 5.6
7a>10059.4 ± 4.856.6 ± 4.5
7b84.4 ± 5.555.3 ± 3.946.0 ± 3.2
7c67.4 ± 4.643.0 ± 3.589.4 ± 6.3
7d84.0 ± 5.962.0 ± 4.388.0 ± 6.2
7e54.3 ± 3.672.0 ± 5.255.3 ± 3.7
7f32.0 ± 2.217.4 ± 1.348.4 ± 3.3
7g14.1 ± 1.211.2 ± 0.913.9 ± 0.3
7h14.8 ± 1.32.9 ± 0.2, SI = 10 b29.8 ± 2.0
7i77.8 ± 5.431.0 ± 1.9116.0 ± 8.1
7j73.4 ± 5.232.0 ± 2.173.0 ± 5.5
7k47.0 ± 3.118.1 ± 1.461.3 ± 4.2
7l62.4 ± 4.452.1 ± 3.4126.0 ± 8.8
7m61.1 ± 4.249.7 ± 3.562.4 ± 4.9
7n93.1 ± 6.661.2 ± 4.264.1 ± 4.8
7o22.5 ± 1.83.1 ± 0.2, SI = 3.8 b11.8 ± 0.9
7p63.0 ± 4.481.5 ± 5.737.0 ± 2.6
7q84.3 ± 5.995.4 ± 6.5>100
7r64.5 ± 4.55.5 ± 0.4, SI = 14 b78.0 ± 5.9
7s80.0 ± 5.713.8 ± 1.1, SI = 9 b125.0 ± 11
8b70.4 ± 5.778.2 ± 6.381.0 ± 6.5
8d>10088.4 ± 7.064.1 ± 5.1
8e99.5 ± 7.159.0 ± 4.178.4 ± 6.3
5-fluorouracil62.0 ± 4.765.2 ± 5.486.3 ± 6.5
Doxorubicin3.0 ± 0.23.0 ± 0.23.0 ± 0.1
a Three independent experiments were carried out; b selectivity index, SI = IC50(Chang liver)/IC50(HuTu 80).
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Trifonov, A.V.; Gazizov, A.S.; Tapalova, A.S.; Kibardina, L.K.; Appazov, N.O.; Voloshina, A.D.; Sapunova, A.S.; Luybina, A.P.; Abyzbekova, G.M.; Dobrynin, A.B.; et al. Synthesis and Anticancer Evaluation of Novel 7-Aza-Coumarine-3-Carboxamides. Int. J. Mol. Sci. 2023, 24, 9927. https://doi.org/10.3390/ijms24129927

AMA Style

Trifonov AV, Gazizov AS, Tapalova AS, Kibardina LK, Appazov NO, Voloshina AD, Sapunova AS, Luybina AP, Abyzbekova GM, Dobrynin AB, et al. Synthesis and Anticancer Evaluation of Novel 7-Aza-Coumarine-3-Carboxamides. International Journal of Molecular Sciences. 2023; 24(12):9927. https://doi.org/10.3390/ijms24129927

Chicago/Turabian Style

Trifonov, Alexey V., Almir S. Gazizov, Anipa S. Tapalova, Lyudmila K. Kibardina, Nurbol O. Appazov, Alexandra D. Voloshina, Anastasiia S. Sapunova, Anna P. Luybina, Gulmira M. Abyzbekova, Alexey B. Dobrynin, and et al. 2023. "Synthesis and Anticancer Evaluation of Novel 7-Aza-Coumarine-3-Carboxamides" International Journal of Molecular Sciences 24, no. 12: 9927. https://doi.org/10.3390/ijms24129927

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