The reactivity and structure of the products was studied. We found that oxidation of spiro-2′-thiolates with DMSO-HCl system produced only acidification products, diastereomeric 6′-amino-5′-cyano-5-methyl-2-oxo-2′-thioxo-1,2,2′,3′-tetrahydro-1′H-spiro-- 3′-carboxamides, instead of the expected isothiazolopyridines. The alkylation of the prepared spiro-2′-thiolates upon treatment with N-aryl α-chloroacetamides and α-bromoacetophenones proceeds in a regioselective way at the sulfur atom. In the case of α-bromoacetophenones, ring-chain tautomerism was observed for the S-alkylation products. According to NMR data, the compounds consist of a mixture of stereoisomers of 2′-amino-6′-[(2-aryl-2-oxoethyl)thio]-3′-cyano-2-oxo-1′H-spiro-5′-carboxamides and 5′-amino-3′-aryl-6′-cyano-3′-hydroxy-2-oxo-2′,3′-dihydrospiropyridine]-8′-carboxamides in various ratios. The structure of the synthesized compounds was confirmed by IR spectroscopy, HRMS, 1H and 13C DEPTQ NMR studies and the results of 2D NMR experiments . Molecular docking studies were performed to investigate suitable binding modes of some new compounds with respect to the transcriptional regulator protein PqsR of Pseudomonas aeruginosa. The docking studies revealed that the compounds have affinity for the bacterial regulator protein PqsR of Pseudomonas aeruginosa with a binding energy in the range of −5.8 to −8.2 kcal/mol. In addition, one of the new compounds, 2′-amino-3′-cyano-5-methyl-2-oxo-6′-{[2-oxo-2-(p-tolylamino)ethyl]thio}-1′H-spiro--5′-carboxamide, showed in vitro moderate antibacterial effect against Pseudomonas aeruginosa and good antioxidant properties in a test with 1,1-diphenyl-2-picrylhydrazyl radical. Finally, three of the new compounds were recognized as moderately active herbicide safeners with respect to herbicide 2,4-D in the laboratory experiments on sunflower seedlings.The purpose of this work was to prepare new isatin- and monothiomalondiamide-based indole derivatives, as well as to study the properties of the new compounds. The four-component reaction of 5-R-isatins (R = H, CH Thus, t13 and 14 show a complex picture. It is noteworthy that in some cases the signals of the minor 3′H-isomers 13b,14b exhibit signal doubling, apparently due to the splitting of the signals of two diastereomeric pairs . Thus, for the major 1′H isomer, CONH2 protons appeared at δ 5.75–5.77 ppm and δ 10.25–10.26 ppm, while the peaks of the minor 3′H isomer were observed at δ 6.32–6.37 ppm and δ 10.19–10.30 ppm. We assume that the S-C=C-C(O)NH2 fragment is planar because of conjugation. The observed difference in chemical shifts can be explained by the presence of an intramolecular hydrogen bond between one of the CONH2 protons and the negatively charged sulfur atom on the one hand, and the anisotropic shielding effect of the carbonyl group on the second proton on the other –HCl system. The oxidation of 2-mercaptonicotinamides and related compounds was reported as a general method for the synthesis of 3-oxo-2,3-dihydroisothiazolopyridines -2′-thiolate 13b.Triethylammonium 6′-amino-3′-(aminocarbonyl)-5′-cyano-2-oxo-1,2-dihydro-1′H-spiro--2′-thiolate  Off-white solid, yield was 2.74 g (88%). FTIR, νmax, cm−1: 3395, 3323, 2710 ; 2170 (C≡N); 1715, 1684 (C=O).1H NMR : the signals of major isomer 13a—1.10 , 2.90–2.95 , 5.37 , 5.77 NH2), 6.55 , 6.70–6.74 , 6.78 , 6.89–6.93 , 7.69 , 9.55 , 10.25 NH2); the observed signals of minor isomer 13b—1.10 , 2.90–2.95 , 3.80 , 6.25 , 6.34–6.37 NH2), 6.75–6.76 , 6.93–6.96 , 7.10–7.14 , 9.55 , 10.30 NH2). NH+ signal was not observed, probably due to the fast H–D exchange. According to the integral signal intensities in the 1H NMR spectrum, the ratio of 1′H-isomer 13a to 3′H-isomer 13b is about 7:1.13C DEPTQ NMR : the signals of major isomer 13a—9.3* (NCH2CH3), 45.7 (NCH2CH3), 52.2 (C spiro), 56.5 (C-5′), 100.4 (C-3′), 107.8* (CH-7 indole), 120.2* (CH-5 indole), 121.1 (C≡N), 122.3* (CH-4 indole), 125.7* (CH-6 indole), 141.0 (C-3a indole), 141.5 (C-7a indole), 150.3 (C-6′), 159.1 (C-2′), 170.0 (CONH2), 182.2 (C=O); the observed signals of minor isomer 13b—9.3* (NCH2CH3), 45.7 (NCH2CH3), 58.9* (CH-3′), 109.1* (CH-7 indole), 119.9 (C≡N), 121.8* (CH-5 indole), 124.2* (CH-4 indole), 128.1* (CH-6 indole), 135.3 (C-3a indole), 137.2 (C-7a indole), 151.8 (C-6′). *Negatively phased signals.20H27N6O2S [M + H]+: 415.1916, found 415.1912 (Δ 1.01 ppm).HRMS (ESI) m/z: calculated for C14a and isomeric 6′-amino-3′-(aminocarbonyl)-5′- cyano-5-methyl-2-oxo-1,2-dihydro-3′H-spiro-2′-thiolateTriethylammonium 6′-amino-3′-(aminocarbonyl)-5′-cyano-5-methyl-2-oxo-1,2-dihydro- 1′H-spiro-2′-thiolate  14b. Off-white solid, yield was 2.68 g (83%). FTIR, νmax, cm−1: 3406, 3377, 2711 ; 2172 (C≡N); 1676, 1666 (C=O).1H NMR : the signals of major isomer 14a—1.11 , 2.17 , 2.93–2.98 , 5.36 , 5.75 NH2), 6.44 , 6.61 , 6.72 , 7.67 , 9.44 , 10.26 NH2); the observed signals of minor isomer 14b—1.11 , 2.20 , 2.93–2.98 , 4.35 , 6.23 , 6.32–6.35 NH2), 6.64 , 6.76 , 6.93 , 9.44 , 10.19 NH2); NH+ signal was not observed, probably due to the fast H–D exchange. According to the integral signal intensities in the 1H NMR spectrum, the ratio of 1′H-isomer 14a to 3′H-isomer 14b is about 7:3.13C DEPTQ NMR : the signals of major isomer 14a—9.2* (NCH2CH3), 20.8* (ArCH3), 45.7 (NCH2CH3), 52.2 (C spiro), 56.7 (C-5′), 100.5 (C-3′), 107.6* (CH-7 indole), 121.2 (C≡N), 123.1* (CH-4 indole), 126.0* (CH-6 indole), 128.6 (C-5 indole), 139.0 (C-7a indole), 141.1 (C-3a indole), 150.3 (C-6′), 159.0 (C-2′), 170.0 (CONH2), 182.2 (C=O); the observed signals of minor isomer 14b—9.2* (NCH2CH3), 20.6* (ArCH3), 45.7 (NCH2CH3), 53.1* (CH-3′), 107.5 (C-5′), 108.9* (CH-7 indole), 120.0 (C≡N), 124.8* (CH-4 indole), 128.4* (CH-6 indole), 130.5 (C-5 indole), 135.4 (C-7a indole), 139.1 (C-3a indole), 151.7 (C-6′), 178.4 (C=O); *Negatively phased signals.m/z: calculated for C21H29N6O2S [M + H]+: 429.2073, found 429.2069 (Δ 0.86 ppm).HRMS (ESI) Synthesis of 6′-amino-5′-cyano-5-methyl-2-oxo-2′-thioxo-1,2,2′,3′-tetrahydro-1′H- spiro-3′-carboxamide 16. A 10 mL beaker was charged with 300 mg (0.7 mmol) of thiolate 14a + 14b and 2 mL of DMSO. To the solution formed, 0.2 mL of concentrated hydrochloric acid was added dropwise under vigorous stirring. Gas evolution (2S!Caution! Me) and formation of a precipitate were observed. The mixture was stirred for 10 min, diluted with EtOH up to a volume of 10 mL, filtered off, washed with EtOH and petroleum ether and dried at 60 °C. Compound 16 was obtained as a yellow finely crystalline powder, yield 201 mg (88%).max, cm−1: 3425, 3317, 3207 (N–H); 2193 (C≡N); 1705, 1653 (C=O).FTIR, ν1H NMR : the signals of major / diastereomers of 16—2.22 , 3.78 , 6.32 , 6.72 , 6.90–6.97 , 7.03 , 7.15 , 7.33 , 10.40 , 11.59 NH); the signals of minor  diastereomers of 16—2.23 , 4.06 , 6.35 , 6.62 , 6.90–6.97 , 7.27 , 10.06 , 11.55 NH).13C DEPTQ NMR : the signals of major / diastereomers of 16—20.92* (ArCH3), 48.8 (C spiro), 56.59 (C-5′), 59.0* (CH-3′), 109.3* (CH-7 indole), 118.9 (C≡N), 126.6* (CH-4 indole), 127.7 (C-3a indole), 129.4* (CH-6 indole), 130.3 (C-5 indole), 139.7 (C-7a indole), 152.1 (C-6′), 166.6 (CONH2), 178.5 (C=O), 198.2 (C=S); the signals of minor  diastereomers of 16—20.85* (ArCH3), 47.9 (C spiro), 56.64 (C-5′), 60.2* (CH-3′), 109.1* (CH-7 indole), 118.2 (C≡N), 124.1* (CH-4 indole), 129.1* (CH-6 indole), 129.6 (C-3a indole), 129.8 (C-5 indole), 140.5 (C-7a indole), 151.6 (C-6′), 166.9 (CONH2), 177.2 (C=O), 199.1 (C=S). *Negatively phased signals.m/z: calculated for C15H14N5O2S [M + H]+: 328.0868, found 328.0864 (Δ 1.22 ppm).HRMS (ESI) Preparation of compounds 17a–d. General procedure. A 25 mL beaker was charged with 300 mg of thiolates 13a + 13b (0.72 mmol) of 14a + 14b (0.70 mmol), DMF (5 mL) and water (3 mL). A hot (50 °C) solution of 0.75 mmol of the corresponding alkylating agent in 10 mL EtOH was added to the resulting solution of thiolates 13,14. A mixture was stirred for another 1 h at 40–50 °C until the reaction completion (TLC) and left for 24 h at room temperature. The precipitated solid was filtered off, washed with EtOH and dried at 60 °C.2′-Amino-3′-cyano-5-methyl-2-oxo-6′-{[2-oxo-2-(p-tolylamino)ethyl]thio}-1′H-spiro--5′-carboxamide 17a. Off-white solid, yield 92%.max, cm−1: 3476, 3406, 3358, 3298, 3175 (N–H); 2191 (C≡N); 1678, 1651 (C=O).FTIR, ν1H NMR : 2.15 , 2.26 , 3.86 , 5.70 , 6.54 , 6.75 , 6.87 , 7.00 , 7.14 , 7.45–7.49 , 8.74 , 9.90 , 10.25 NHAr).13C DEPTQ NMR : 20.3* (ArCH3), 20.8* (ArCH3), 37.3 (SCH2), 52.2 (C spiro), 56.4 (C-3′), 108.6* (CH-7 indole), 113.3 (C-5′), 119.7 (C≡N), 119.8* , 124.9* (CH-4 indole), 128.4* (CH-6 indole), 129.3* , 128.9 (C-Ar), 129.9 (C-Ar), 133.0 (C-Ar), 135.1 (C-Ar), 135.9 (C-Ar), 139.4 (C-2′), 151.9 (C-6′), 166.6 (CONH), 167.1 (CONH), 179.3 (C=O indoline); *Negatively phased signals.m/z: calculated for C24H23N6O3S [M + H]+: 475.1552, found 475.1551 (Δ 0.21 ppm).HRMS (ESI) 2′-Amino-3′-cyano-5-methyl-2-oxo-6′-{[2-oxo-2-(o-tolylamino)ethyl]thio}-1′H-spiro--5′-carboxamide 17b. Off-white solid, yield 82%. The compound is sparingly soluble in DMSO. FTIR, νmax, cm−1: 3437, 3358, 3302, 3283, 3236, 3165 (N–H); 2168 (C≡N); 1699, 1684 (C=O).1H NMR : 2.13 , 2.22 , 3.94 , 5.73 , 6.54 , 6.77 , 6.87 , 6.97 , 7.12–7.20 , 7.25 , 7.36 , 7.52 , 8.80 , 9.80 , 9.89 NHAr).13C DEPTQ NMR : 17.8* (ArCH3), 20.8* (ArCH3), 36.6 (SCH2), 49.9 (C spiro), 55.9 (C-3′), 108.7* (CH-7 indole), 112.2 (C-5′), 119.7 (C≡N), 124.8* (CH-4 indole), 125.6* (CH ArNH), 126.0* (CH ArNH), 126.1* (CH ArNH), 128.3* (CH-6 indole), 129.8 (C Ar), 129.9 (C Ar), 130.5* (CH ArNH), 133.4 (C-Ar), 135.1 (C-Ar), 135.9 (C-Ar), 138.3 (C-2′), 152.0 (C-6′), 166.6 (CONH), 167.5 (CONH), 179.3 (C=O indoline); *Negatively phased signals.24H23N6O3S [M + H]+: 475.1552, found 475.1551 (Δ 0.21 ppm).HRMS (ESI) m/z: calculated for C17c and 5′-amino-6′-cyano-3′-hydroxy-5-methyl-2-oxo-3′-phenyl- 2′,3′-dihydrospiropyridine]-8′-carboxamide2′-Amino-3′-cyano-5-methyl-2-oxo-6′-[(2-oxo-2-phenylethyl)thio]-1′H-spiro-5′-carboxamide  17c-cycl. Light brown solid, yield 93%. According to 1H NMR, the compound exists as a mixture of linear and cyclic tautomers with a predominance of the 17c-cycl form in a ratio ~3:7. The compound is sparingly soluble in DMSO. FTIR, νmax, cm−1: 3441, 3368, 3308, 3234, 3200, 3161 (N–H); 2178 (C≡N); 1697, 1680, 1649 (C=O).1H NMR : the signals of major cyclic tautomeric form 17c-cycl; signals of 17c-cycl are doubled due to existence of diastereomeric forms in 1:1 ratio—2.28 , 2.31 , 3.18–3.22 , 3.54–3.58 , 6.10 , 6.17 , 6.72–6.75 , 6.88–6.89 , 7.03–7.05 , 7.49–7.45 , 7.51–7.52 , 7.56–7.60 , 8.57 , 8.67 , 10.34 , 10.35 ; The observed signals of minor non-cyclic tautomer 17c—2.23 , 4.76 ), 5.68 , 6.56 , 6.99 , 7.25 , 7.69–7.73 Ph), 8.02 Ph), 8.53 , 9.91 .13C DEPTQ NMR  : 21.22* (CH3), 21.24* (CH3), 21.3* (CH3), 41.4 (SCH2), 42.46 (SCH2), 43.1 (SCH2), 56.4, 110.2*(CH-7 indole), 110.3* (CH-7 indole), 119.5 (CN), 124.0 (C Ar), 125.1* (CH Ar), 125.34* (CH Ar), 125.5* (CH Ar), 128.9* (CH Ar), 129.0* (CH Ar), 129.1* (CH Ar), 129.2* (CH Ar), 129.5* (CH Ar), 129.9* (CH Ar), 130.1 (C Ar), 130.3 (C Ar), 131.8 (C Ar), 131.9 (C Ar), 135.66 (C Ar), 135.68 (C Ar), 142.1 (C Ar), 151.8 (C-6′), 159.9 (C), 167.1 (CONH), 167.8 (CONH), 179.4 (C=O indoline), 179.6 (C=O indoline), 180.5 (C=O indoline), 185.6 (C(O)Ph); *Negatively phased signals.23H20N5O3S [M + H]+: 446.1289, found 446.1283 (Δ 1.35 ppm); calculated for C23H19N5NaO3S [M + Na]+: 468.1106, found 468.1101 (Δ 1.07 ppm).HRMS (ESI) m/z: calculated for C2′-Amino-6′-[(2-(4-bromophenyl)-2-oxoethyl)thio]-3′-cyano-2-oxo-1′H-spiro-5′-carboxamide 17d and 5′-amino-3′-(4-bromophenyl)-6′-cyano-3′-hydroxy-2-oxo- 2′,3′-dihydrospiropyridine]-8′-carboxamide 17d-cycl. Beige solid, yield 92%. According to 1H NMR, the compound exists as a mixture of linear and cyclic tautomers with a predominance of the 17d-cycl form in a ratio ~1:4. The compound is sparingly soluble in DMSO. FTIR, νmax, cm−1: 3450, 3337, 3312, 3202, 3163 (N–H); 2181 (C≡N); 1701, 1680 (C=O).1H NMR : the signals of major cyclic tautomeric form 17d-cycl; signals of 17d-cycl are doubled due to existence of diastereomeric forms in 1:1 ratio—3.19–3.26 , 3.55–3.58 , 6.08 , 6.14 , 6.81–6.83 , 6.99–7.28 , 7.44 Ar), 7.55 Ar), 7.63 Ar), 7.71 Ar), 8.69 , 8.79 , 10.38 , 10.41 ;17d—4.71 ), 5.70 , 6.67 , 6.88–6.92 , 7.80 Ar), 7.94 Ar), 8.53 , 10.02 .The observed signals of minor non-cyclic tautomer 13C DEPTQ NMR  : 41.8 (SCH2), 41.6 (SCH2), 42.5 (SCH2), 58.8, 61.6, 96.3, 100.0, 108.9* (CH-7 indole), 109.8* (CH-7 indole), 109.9* (CH-7 indole), 118.8 (CN), 118.9 (CN), 121.3* (CH Ar), 121.9 (C Ar), 122.0 (C Ar), 122.1 (C Ar), 122.3* (CH Ar), 122.5* (CH Ar), 124.3* (CH Ar), 124.4* (CH Ar), 127.0* (CH Ar), 127.3* (CH Ar), 128.1* (CH Ar), 128.9* (CH Ar), 129.5 (C Ar), 130.5* (CH Ar), 131.5* (CH Ar), 131.7* (CH Ar), 132.1* (CH Ar), 134.2 (C Ar), 134.5 (C Ar), 134.7 (C Ar), 134.8 (C Ar), 1401.0 (C Ar), 141.1 (C Ar), 141.6 (C Ar), 141.9 (C Ar), 146.6 (C Ar), 151.2 (C), 151.6 (C), 152.0 (C), 167.1 (CONH), 167.3 (CONH), 178.49 (C=O indoline), 179.1 (C=O indoline), 188.7 (C(O)Ar); *Negatively phased signals.22H17BrN5O3S [M + H]+: 510.0236, found 510.0235 (Δ 0.1 ppm); calculated for C22H15BrN5O2S [M + H–H2O]+: 492.0130, found 492.0130 (Δ 0 ppm).HRMS (ESI) m/z: calculated for C8 CFU/mL. Then, 0.1 mL of the bacterial suspension was transferred and spread homogeneously over the Mueller–Hinton agar with a sterile swab and the plate was allowed to stand for 10 min at room temperature to dry. Using a sterile cork borer, a 6 mm diameter hole was made in each prepared plate. A solution of compound 17a at 100 mg/mL concentration was prepared by dissolving 17a in dimethyl sulfoxide (DMSO). Then, 100 μL of the solution was added using a micropipette in each hole under sterile conditions; DMSO was also added as control. The plate was kept for 24 h at 37 °C in an incubator. The diameter of the inhibitory zone around each hole was measured in millimeters.A suspension of pathogens isolated from a swollen ear canal was prepared by transferring a loop full of isolated colony to 5 mL of normal saline in tubes. The turbidity of the bacterial growth in tubes was then compared with the turbidity of 0.5 McFarland suspension to give an approximate number of live cells equal to 0.5 × 10P. aeruginosa ATCC 27,853 was determined using the classical broth dilution method thio}- 1′H-spiro-5′-carboxamide 17a were recognized. The antibacterial effect of 17a was confirmed in experiments in vitro. Additionally, compound 17a revealed a pronounced antioxidant effect comparable with that for ascorbic acid in the experiments with 1,1-diphenyl-2-picrylhydrazyl radical. Finally, spiro 16a and 17a,b were recognized as moderately active 2,4-D herbicide safeners and reduced the negative effect of 2,4-D on sunflower seedling hypocotyls by 16–33% and by 12–25% on sunflower seedling roots.In summary, we studied for the first time the reaction between isatin, malononitrile and monothiomalonamide in the presence of triethylamine. It was found that the reaction proceeds with the formation of two isomeric 1′H and 3′H products—triethylammonium 6′-amino-3′-(aminocarbonyl)-5′-cyano-2-oxo-1,2-dihydro-1′H-spiro--2′-thiolate and 6′-amino-3′-(aminocarbonyl)-5′-cyano-2-oxo-1,2-dihydro-3′H-spiro--2′-thiolate. These results are in good agreement with the results of other works on the preparation of related nicotinamides, starting from monothiomalonamide. We failed to oxidize the above thiolates to isothiazolopyridines upon treatment with DMSO-HCl system. Instead, diastereomers of 6′-amino-5′-cyano-5-methyl-2-oxo-2′-thioxo-1,2,2′,3′-tetrahydro-1′H- spiro-3′-carboxamide were isolated. The alkylation of a mixture of the above 1′H/3′H thiolates proceeded in a regionspecific way on the sulfur atom and led to the formation of only 1′H derivatives in high yields. In the case of the reaction involving α-bromoketones, the isolated alkylation products were found to exist as a mixture of linear and chained  tautomers. The structure of the new compounds was thoroughly investigated using IR, HRMS and NMR spectroscopy, including DEPTQ and 2D


The structures of all undescribed components (1–8) were determined by analysis of IR, mass, NMR, and UV spectroscopic data. The absolute configuration of new compounds was determined by using circular dichroism and DP4+ calculations. The cytotoxic activities of all isolated marine natural products were evaluated. Compound 7 showed a significant cytotoxic effect against oral cancer cell line (Ca9-22) with an IC50 value of 7.26 ± 0.17 μg/mL.Five new eudensamane-type sesquiterpene lactones, clasamanes A–E ( In south–central Asia, people are susceptible to oral cancers because of the usage of areca nuts as chewing gum. Taiwan has one of the world’s highest incidences of oral cancer, which ranks fourth in the cause of cancer death among male Taiwanese . In TaiwClavularia have been found to have different kinds of MNPs, such as diterpenoids pentadeca-8,13-dien-13-yl)-propan-2-ol (19) pentadec-5-en-15-yl)-propan-2-ol (20) + ion at m/z 387.1414, suggesting the molecular formula of C19H24O7 with seven indices of hydrogen deficiency. The 1H and 13C data , 3.15 (m) (H2-1′); δC 59.1 (C-1′); δH 1.21 (t) (H3-2′); δC 15.1 (C-2′)) and acetoxy (δC 170.7 (C-1″); δH 2.17 (s) (H3-2″); δC 20.8 (C-2″)) groups. A detailed analysis of COSY and HMBC spectra /H-2 (δ 6.74)/H-3 (δ 6.39) and H-5 (δ 2.53)/H2-6 (δ 2.61 and δ 2.47) were found. These two proton spin systems and the HMBC correlations from H3-14 (δ 1.61) to C-1 (δ 79.5), C-5 (δ 42.5), C-9 (δ 43.8), and C-10 (δ 38.7), from H2-15 (δ 4.43 and δ 4.37) to C-3 (δ 129.2), C-4 (δ 79.8), and C-5, from H2-9 (δ 2.26 and δ 1.40) to C-8 (δ 105.3), from H2-6 to C-7 (δ 155.8), C-8, and C-11 (δ 124.2), and from H3-13 (δ 1.79) to C-7, C-11, and C-12 (δ 171.0) can be used to assemble the framework of eudensamane-type sesquiterpene lactone. The HMBC correlations from H2-1′ to C-8 and from H2-15 to C-1″ indicated the connection of ethoxy and acetoxy groups, respectively. The above findings accounted for six of the seven indices of hydrogen deficiency, which implied an additional ring should exist in 5. Considering the molecular formula, two oxygen atoms were not assigned yet, and the carbon chemical shift of C-1 and C-4 suggested that these two carbons are oxygen bearing. Thus, a peroxide bridge between C-1 and C-4 was allocated. This assignment was also confirmed by the down-field shifted signals of H-2 and H-3 +. The presence of one bromine atom was confirmed by the equal intensity between [M + Na]+ and [M + 2 + Na]+ in the mass spectrum. The presence of hydroxy functionality was confirmed by the IR absorption at 3432 cm−1. The 1H NMR data  (H3-20), δ 1.21 (s) (H3-19), and δ 0.86 (s) (H3-15)), an oxymethylene , δ 3.65  (H2-17)), and an exomethylene (δ 5.03 (s), δ 4.84 (s) (H2-16)). The twenty carbon signals of 6 could be clearly separated into one exocyclic C=C double bond (δ 113.8 (C-16) and δ 147.4 (C-4)), one oxymethylene (δ 67.0 (C-17)), three oxygen-bearing quaternary carbons (δ 76.2 (C-11), δ 75.4 (C-18), and δ 75.0 (C-8)), one quaternary carbon (δ 44.6 (C-1)), seven methylenes (δ 42.4 (C-2), δ 38.8 (C-14), δ 35.4 (C-5), δ 33.3 (C-9), δ 29.7 (C-6), δ 27.7 (C-13), and δ 25.1 (C-3)), three methines (δ 63.0 (C-7), δ 54.7 (C-10) and δ 50.2 (C-12)), and three methyls (δ 29.6 (C-19), δ 26.0 (C-20) and δ 24.1 (C-15)) by using 13C NMR /H2-3 (δ 2.11 and δ 1.63), H2-5 (δ 2.42 and δ 2.28)/H2-6 (δ 2.11 and δ 1.90)/H-7 (δ 4.04), and H2-9 (δ 2.25)/H-10 (δ 2.86) were observed from the COSY spectrum. Those findings and the HMBC correlation from H2-16 to C-3, C-4, and C-5; from H2-17 to C-7, C-8, and C-9; from H2-9 to C-8 and C-11; and from H3-15 to C-1, C-2, and C-11 could build the cycloundecane moiety (C-1 to C-11) of 6, and it could confirm the presence of exomethylene (H2-16) connecting at C-4, an oxymethylene attaching at C-8, and a methyl (H3-15) connecting at C-1. In addition, the COSY correlations of H-12 (δ 2.21)/H2-13 (δ 1.90 and δ 1.63)/H2-14 (δ 1.76) together with the HMBC correlations from H-12 to C-11 and from H3-15 to C-1, C-11, and C-14 could establish the cyclopentane ring of 6. The HMBC correlations from H-12 and the geminal methyls (H3-19 and H3-20) to C-3 constructed the isopropyl alcohol group. The planar structure of 6 was found to be similar to that of clavinflol B (13) except that the chlorine atom in the molecular formula (C20H33BrO4) of 13 was replaced by a bromine atom in 6. The halogen atom of 6 was allocated at C-7, the same as that of 13, due to the downfield shifted carbon chemical shift at this position (δ 66.2 in 13 and δ 63.0 in 6). The relative configuration of 6 was determined through the NOESY spectrum  compared with the 1R*,7R*,8S*,10S*,12R* isomer (0%), which confirmed the configuration of 6.The planar structure of elations . Three pspectrum . The bro7) was found to possess the molecular formula C20H33IO4 according to the sodium adduct ion at m/z 487.1318  in the HRESIMS. The UV, IR, and NMR data  revealed that the bromine atom at C-7 in 6 was replaced by an iodine atom in 7. Since the specific rotation, ECD, and NOESY data of 7 also resembled those of 6, and the stereochemistry of 7 was thus assigned identically.Clabellane B (NMR data  were qui8) has the molecular formula C20H32O4 (IHD = 5), as deduced from HRESIMS and NMR spectrometric data. The IR spectrum of 8 indicated the presence of hydroxy (3378 cm−1) and exomethylene (1643 cm−1) functionalities. The 1H and 13C NMR data of 8 were analogous to those of 6, suggesting it is also a dolabellane-type diterpenoid. Four proton sequences of H2-2 (δ 1.74 and δ 1.32)/H2-3 (δ 2.04 and δ 1.79), H2-5 (δ 2.46 and δ 2.27)/H2-6 (δ 1.96 and δ 1.49)/H-7 (δ 3.13), H2-9 (δ 2.58 and δ 1.96)/H-10 (δ 4.37), and H2-13 (δ 2.35 and δ 2.20)/H2-14 (δ 1.74 and δ 1.43) were observed by the cross-peaks in the COSY spectrum. Moreover, the above finding and the HMBC correlations from H2-16 (δ 4.71 and δ 4.67) to C-3 (δ 29.9), C-4 (δ 150.3), and C-5 (δ 28.7); from H2-17 (δ 3.98 and δ 3.32) to C-7 (δ 60.2), C-8 (δ 63.8), and C-9 (δ 37.8); from H-10 to C-1 (δ 53.1), C-11 (δ 141.2), and C-12 (δ 144.7); from H3-15 (δ 1.05) to C-1 (δ 53.1), C-2 (δ 35.7), C-11, and C-14 (δ 32.7); and from H3-19 (δ 1.35) to C-12, C-18 (δ 72.3), and C-20 (δ 29.7) could establish the planar structure of 8. Three hydroxy groups allocated at C-10 (δ 65.0), C-17 (δ 68.6), and C-18 were assured by virtue of their downfield shifted carbon chemical shifts. The aforementioned data accounted for four of the five indices of hydrogen deficiency, suggesting an additional ring remained in 8. An epoxy group was assigned to C-7 and C-8 by their downfield shifted carbon chemical shifts. Therefore, the planar structure was established. The relative configuration of 8 was determined by interpretation of NOESY data (3-15/H-2β (δ 1.74) indicated the β-orientation of these protons. The presence of NOESY cross-peaks between H-7 and H-2α (δ 1.32) indicated they are α-orientated. The configuration assignment was confirmed by the 100% possibility of DP4+ analysis. On the basis of the data described above, the configuration of 8 was established to be 1S*,7S*,8R*,10S*.Clabellane C  was seven times higher than that of 16 (IC50 = 166.7 μM), which implied the position of the C=C bond might change the bioactivity dramatically. For the halogenated dolabellanes 6, 7, and 13, the iodinated one showed the best cytotoxic activity and the chlorinated one was the weakest. Moreover, clasamane E (5) having a peroxide bridge showed a relatively good cytotoxic activity against the Ca9-22 cell among all isolated eudensamane-type sesquiterpene lactones.In our earlier investigation, we found that the methanol extract of er cells . Hence, 18, phenyl-hexyl, and biphenyl columns were used for high-performance liquid chromatography (HPLC). The Shimadzu  HPLC instrument consisted of an LC-40D solvent delivery module, DGU-405 degassing unit, CBM-40 system controller, CTO-40S column oven, SPD-M40 photo diode array detector, and FRC-10A fraction collector. A Jasco  V-650 spectrophotometer was used for measuring UV data. A Jasco FT/IR-4X spectrophotometer was chosen for measuring IR data. A Jasco J-815 CD spectrometer was used for recording the circular dichroism data. Specific optical rotation was measured by a Jasco P-2000 polarimeter. NMR spectra were obtained from Varian  Mercury Plus 400 MHz and VNMRS 600 MHz FT-NMR spectrometers. A Bruker  APEX II spectrometer was used for detecting HRSIMS.Merck KGaA  cellite 545 (0.02–0.1 mm) and silica gel 60 (0.015–0.040 mm) were used for dry sample and flash column chromatography, respectively. Phenomenex  CClavularia spp. by Dr. Yuan-Bin Cheng. A voucher specimen (code: CI2021) was given, and the specimens were deposited at the Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung, Taiwan. It is noted that the coral materials were previously identified as Clavularia inflata + .Clasamane A (2): colorless oil; max 2925, 1752, 1552, 1441, 1383, 1294, 1235, 1168, 1114, 1043 cm−1; ECD λmax(Δε) 248 (−0.60), 214 (−1.26) nm; 1H and 13C NMR data are presented in m/z 299.1616 [M + Na]+ .Clasamane B (3): colorless oil; max 2925, 2858, 1758, 1448, 1373, 1228, 1175, 1125, 1083, 1025 cm−1; ECD λmax(Δε) 274 (−4.58), 234 (+1.60), 209 (−1.59) nm; 1H and 13C NMR data are presented in m/z 341.1362 [M + Na]+ .Clasamane C (4): colorless oil; max 2975, 2928, 1763, 1443, 1378, 1320, 1233, 1175, 1088, 1026 cm−1; ECD λmax(Δε) 274 (−10.61), 234 (+3.04), 211 (−3.36) nm; 1H and 13C NMR data are presented in m/z 355.1518 [M + Na]+ .Clasamane D (5): colorless oil; max 2928, 1758, 1448, 1378, 1295, 1237, 1171, 1113, 1042 cm−1; ECD λmax(Δε) 290 (−0.08), 246 (+1.24), 207 (−1.81) nm; 1H and 13C NMR data are presented in m/z 387.1414 [M + Na]+ .Clasamane E (6): colorless oil; max 3432, 2966, 1725, 1646, 1447, 1383, 1252, 1169, 1131 cm−1; ECD λmax(Δε) 204 (+1.38) nm; 1H and 13C NMR data are presented in m/z 439.1455 [M + Na]+ .Clabellane A (7): colorless oil; max 3412, 2963, 2930, 1726, 1645, 1451, 1381, 1243, 1171, 1126, 1045 cm−1; ECD λmax(Δε) 208 (+1.68) nm; 1H and 13C NMR data are presented in m/z 487.1318 [M + Na]+ .Clabellane B (8): colorless oil; max 3412, 2963, 2930, 1726, 1645, 1451, 1381, 1243, 1171, 1126, 1045 cm−1; ECD λmax(Δε) 203 (−2.81) nm; 1H and 13C NMR data are presented in m/z 359.2193 [M + Na]+ .Clabellane C (50) at 72 h of oral cancer Ca9-22 cells  [The cell viability   was asse, Japan) ,20 and mClavularia spp. started last century [1–5 and 9–12) and dolabellane-type diterpenes (6–8 and 13–22). In terms of bioactivity, the dolabellane-type diterpenes demonstrate better cytotoxic activity than eudensamane-type sesquiterpene lactones. Our findings support our previous investigation and prove that the marine soft coral of the genus Clavularia is a rich source for the identification of cytotoxic compounds.Although the natural product investigation of octacoral  century , new mar century ,21,22. I


The catalytic activity of RL BF Fe-based MOF@CuO NC was explored in a facile and green methodology to prepare diverse N‑amino-2-pyridones by one-pot four component reactions comprising aromatic aldehyde, malononitrile, methyl cyanoacetate and hydrazine hydrate within mild and solvent-free conditions. This protocol enjoys features like providing the final products during low reaction times in excellent yields under solvent-free conditions. The use of easily available and inexpensive reactants for the synthesis of the catalyst, environmental compatibility, low catalyst loading, fast and clean work-up and reusability of catalyst for several cycles with consistent activity are counted as the outstanding features of this procedure.In this study, a new and efficient Rod-like bifunctional Fe-based MOF@CuO nanocomposites (RL BF Fe-based MOF@CuO NC) were synthesized as new and efficient heterogeneous catalyst through a simple method from easily available 1,3,5-benzenetricarbocylic acid linker, nitrate ferric as a source of iron and copper oxide (CuO) nanoparticles under microwave irradiation. The synthesized nanocatalysts were characterized with different techniques such as Brunauer–Emmett–Teller (BET), energy dispersive spectroscopy (EDS), field emission scanning electron microscopy (FE-SEM), mapping, transmission electron microscopy (TEM), X-ray diffraction (XRD), vibrating sample magnetometer (VSM), thermogravimetric analysis (TGA) and Fourier transform infrared spectroscopy (FT-IR). The RL BF Fe-based MOF@CuO NC had relatively high specific surface area (203 m The advantages of MCRs over conventional linear syntheses that cause MCRs being systems of extremely eco-friendly and ideal including, solvents, reagents, time and energy saving, quick and simple implementation, bond-forming efficiency, no production of by-products, minimize waste, lower costs, high degree of atom economy since most of the atoms of substrates will present in the target product, avoidance of complex purification procedure, high total yield and high selectivity10. The combination the merits of MCRs with the advantages of solvent-free conditions and easily recovered heterogeneous catalyst can meet the requirements of green chemistry4.Multicomponent reactions (MCRs) are becoming important tools for the efficient and rapid synthesis of new diverse complex products in chemistry, especially in pharmaceuticals and drug discovery with attractive biological properties11 with adjustable pore size12, high surface area12, thermal stability and good chemical13. They can be modified through several post-synthesis treatments or on-site methods to improve their performance14. They have gained widespread interest in various applications such as sensors15, storage or gas separation15, removal of pesticides16 and organic pollutants17, electronic and optical devices18, drug delivery15 and biomedical purposes19. MOFs and their nanocomposites have advanced catalytic features20.Metal–organic frameworks (MOFs) refer to porous hybrid materials22.The high surface-to-volume ratio of these structures results in promoted the activity of nanoparticles, shorter reaction time, enhanced selectivity, and convenient recovery from reaction mixtures using uncomplicated filtration techniques23. In addition, metal oxides have very active sites and high electronic conductivity but the low surface area restricts the direct use of metal oxides as catalyst. On the other side, MOFs usually have high surface area but poor electronic conductivity and low active site. Thus, the composite materials uniting the MOFs with metal oxides can provide the increased surface area as well as active metal sites. Thus, the synergetic action of both the MOF and metal oxides overcomes the shortcoming of individual frameworks and makes the bridge between these materials24.In turn, MOFs have catalytic features but the introduction of metal oxides enhances the catalytic efficiency of MOF to the next level12. Various morphologies of CuO nanostructures such as nanosheets, hollow-spherical, nano-needles, nanofibers, nanowires, nanofilaments have been utilized in multidimensional applications including catalysis, sensors, magnetic storage media, Li batteries, field emission, solar energy transformation, electronics and semiconductors26.Copper oxide (CuO) is an intermediate metal oxide with a narrow band gap (1.2–1.4 eV). It is a promising p-type material whose physicochemical properties strongly depend on its size and morphology28. Besides, the different numbers of active sites and crystal plane effects explained why the nanorods showed higher activity than the corresponding nanoparticles30.Recently, the synthesis of one-dimensional (1D) nanostructures has attracted considerable attention because 1D nanostructures have potential applications in wide-ranging sectors including catalysis, sensing, electronics and photoelectronics, with performances that are anticipated to be superior to those of their bulk counterpartsThe presence of versatile synthons of N-Amino-2-pyridone in complex biological scaffolds has resulted in a vast range of biological and pharmaceutical properties.β-lactam, quinolizidine, pyridine, piperidine, as well as indolizidine alkaloid31 with a wide spectrum of significant biological and pharmacological properties and physiological activities such as antibacterial32, antifungal33, antitumor35, cardiotonic36, psychotherapeutic37, and possible HIC-1 specific transcriptase inhibition38.Among a large diversity of heterocyclic compounds, the synthetic development of 2-pyridone derivatives has currently changed into an interesting challenge and an ongoing field of concern owing to the presence this valuable synthons in natural compound, which can lead to the synthesis of various nitrogen-containing heterocyclic structures, including 39, pyrazolotriazolopyridines40, pyridotriazines41, as well as pyridotriazepines39.Ful synthetic intermediates of 1,6-diamino-2-oxo-4-phenyl-1,2-dihydro pyridine-3,5-dicarbonitrile are an N‑amino-2-pyridone subset, representing a combination of valuable synthons of 2-oxo-3-cyanopyridines and 3,5-dicyanopyridines with divers and significant biodynamic, biological and pharmaceutical properties which led to this type of pyridine derivatives with high reactivity and vast medicinal utility with a wide spectrum of synthetic N-heterocycles, including 1,2,4-triazolopyridinesConsidering the significance of 2-pyridone skeleton, modern organic chemical sciences seek to develop effective, novel, and eco-friendly synthesis protocols for preparing 1, 6-diamino-2-oxo-4-phenyl-1,2-dihydropyridine-3,5-dicarbonitrile derivatives.42, piperidine43, magnesium oxide as an extremely powerful heterogeneous base catalytic agent42, polyaniline44, K2CO345 and poly(4-vinylpyridine)44. The above methods took the advantage of 2-cyanoacetohydrazide as the starting material to synthesize N-amino-2-pyridones. The current study thus sought to synthesize derivatives of 1,6-diamino-2-oxo- 4-phenyl-1,2-dihydropyridine-3,5-dicarbonitrile by the four-component one-pot coupling of aromatic aldehydes, malononitrile, methyl cyanoacetate and hydrazine hydrate using a solvent-free and reflux conditions in the presence of RL TF Fe-based MOF@CuO NC.A few catalysts were previously employed to synthesize N‑amino-2-pyridones such as bismuth (III) nitrate pentahydrate as an efficiently practical Lewis acid catalytic agent1H NMR) was obtained using a Bruker Avance 400 MHz NMR spectrometer in d6-DMSO at ambient temperature. The crystallinity, phase structure, and crystallite size of RL TF Fe-based MOF@CuO NC were determined by a PC-APD X-ray diffractometer and Kα radiation  and graphite mono-chromatic Cu radiation  . The X’Pert HighScore Plus software was used for data analysis. The XRD pattern was obtained in the range 2°–80° 2θ, with a step size of 0.016°. Then, SEM and EDS (KYKY & EM 3200) were applied to investigate RL TF Fe-based MOF@CuO NC. Thermal behavior analysis was carried out in N2 atmosphere in the temperature range of room temperature to 350 °C using a STA-1500 thermoanalyzer. A Lakeshore (model 7407) was employed to evaluate magnetization within magnetic fields under room temperature.High-purity chemical substances were bought from Sigma Aldrich and used with no additional purification. Physical constants of the products were compared with the authentic samples and FT-IR spectroscopy for threir characterization. Thin layer chromatography (TLC) on Silica G60 F254 (Merck) TLC plates was used to monitor the reaction progress and determine the substrate purity. An Electrotermal 9100 device in open capillary tubes was employed to measure melting points, the values of which were left uncorrected. FTIR spectra were recorded for KBr pellets of the samples using a JASCO FT-IR-4000 spectrophotometer. NMR spectroscopy (4·5H2O) was dissolved in deionized water. Then, saturated NaOH solution was added until the pH of the solution reached 8.0; where the resulting precipitates were collected. Raw materials were removed from the obtained product by three times washing with ethanol, followed by drying at 80 °C for 12 h. The resulting precipitates were calcinated under 400 °C within a 1-h period.First, 0.1 M copper (II) sulfate pentahydrate (CuSO4·5H2O) was dissolved in deionized water. Then, saturated NaOH solution was added until the pH of the solution reached 8.0; where the resulting precipitates were collected. Raw materials were removed from the obtained product by three times washing with ethanol, followed by drying at 80 °C for 12 h. The resulting precipitates were calcinated under 400 °C within a 1-h period.First, 0.1 M copper (II) sulfate pentahydrate , and bentonite  were dissolved in ion-free water (solution B). Then, solution B was added to A. The obtained mixture was heated to 80 °C for 2 h. The developed MOF sediments were collected after overnight refrigeration. Raw materials were removed from the obtained product by three times washing with boiling water. Finally, the MOF precipitates were calcined for one hour in an oven at 150 °C.0.092 mmol (0.026 g) of surfactant (sodium dodecyl sulfate) was dispersed in N-hexane (solution A). Fe-MOF , Fe52.Deionized water was used to disperse 3 mmol of dried TF Fe-based MOF at 80 °C in a beaker. Next, 1 mmol of CuO nanoparticles were added and the mixture was stirred for 10 min to achieve a homogeneous solution. The final powder mixture was delivered to a glassy vial, which was promptly transferred into the microwave oven (300 W) and microwaved for 60 min. Acetic acid was used to wash the obtained products, to remove the raw materials. The dried powder was finally calcined at 190 °C for 70 min1H NMR and 13C NMR spectra were considered to examine the structural characteristics of the products.A combination of hydrazine hydrate , methyl cyanoacetate , malononitrile , aromatic aldehydes (3 mmol), and RL TF Fe-based MOF@CuO NC (10 w%) was heated under solvent-free and reflux conditions and stirred for a determined period, as shown in Table 1,6-diamino-4-(4-methoxyphenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile (5a): m.p: 220–222 °C (lit.47 222–224 °C); Yield: 98%; 1H NMR : δ 3.62 , 3.85 , 5.51 , 6.66–7.24 , 8.85  ppm; 13C NMR : δ 55.1, 55.5, 66.1, 105.0, 118.9, 119.4, 130.7, 132.0, 159.3, 161.4, 162.2, 164.9, 174.6. ppm.1,6-diamino-4-(4-chlorophenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile (5b): m.p: 340–343 °C (lit.47 341–342 °C); Yield: 87%; 1H NMR  δ: 5.71 , 7.60 , 7.92 , 8.74  ppm; 13C NMR : δ = 68.7, 80.6, 115.2, 116.4, 128.3, 129.1, 130.0, 132.6, 136.0, 153.8, 158.2, 160.7 ppm.1,6-diamino-4-(4-nitrophenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile (5c): m.p: > 340 °C (lit.47 > 340 °C); Yield: 91%; 1H NMR : δ 5.79 , 7.63–8.44 , 8.75  ppm; 13C NMR : δ 53.6, 106.4, 114.9, 124.4, 131.7, 137.2, 149.2, 152.8, 153.3, 161.7 ppm.1,6-diamino-4-(2-fluorophenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile (5d): mp: 247–249 °C (lit.48 247–249 °C); Yield: 89%; 1H NMR : δ 5.62 , 7.34–7.67 , 8.63  ppm; 13C NMR : δ = 87.7, 116.2, 116.4, 122.2, 124.6, 130.1, 133.1, 133.2, 158.0, 160.5, 163.1, 164.1 ppm.1,6-diamino-4-(2-nitrophenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile (5e): m.p: 234–235 °C (lit.48 234–236); Yield: 90%; 1H NMR : δ 5.54 , 7.55–7.89 , 8.21  ppm; 13C NMR : δ 87.2, 98.0, 113.7, 114.3, 119.0, 125.0, 126.4, 129.4, 132.1, 134.3, 149.3, 155.3, 158.7 ppm.1,6-diamino-2-oxo-4-phenyl-1,2-dihydropyridine-3,5-dicarbonitrile (5f): m.p = 238–240 °C (lit.47 237–240); Yield: 87%; 1H NMR : δ 5.69 , 7.51–7.63 , 8.51  ppm; 13C NMR : δ 74.3, 86.4, 115.4, 116.3, 128.0, 128.6, 130.2, 134.6, 156.6, 159.2, 159.5.1,6-diamino-4-(4-(dimethylamino)phenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile (5g): m.p = 248–250 °C (lit.47 249–251); Yield: 99%; 1H NMR  δ: 3.07 , 5.67  ppm, 6.86 , 7.41 , 8.38  ppm; 13C NMR : δ = 73.6, 85.5, 111.1, 111.7, 116.2, 120.6, 128.9, 129.5, 151.5, 156.7, 159.5, 159.6 ppm.1,6-diamino-4-(4-hydroxyphenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile (5h): m.p: 324–326 °C (lit.47 325–327 °C); Yield: 99%,; 1H NMR  δ: 5.64 , 6.90 , 6.95 , 7.34 , 8.02 , 8.40 , 10.05  ppm; 13C NMR : δ 74.1, 115.2, 116.4, 122.5, 124.8, 129.9, 134.0, 154.8, 156.6, 159.3, 162.9 ppm.1,6-diamino-4-(3-nitrophenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile (5i): m.p: 250–251 °C (lit.49 249–251 °C); Yield 98%; 1H NMR  δ: 5.68 , 6.42 , 6.66 , 8.83  ppm. 13C NMR : δ 76.2, 91.4, 116.7, 125.8, 130.6, 134.4, 135.2, 148.1, 160.3, 160.5, 160.6 ppm.1,6-diamino-4--2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile (5j): m.p: 252–253 °C (lit.42 251–253); Yield: 99%, 1H NMR : δ 3.62 , 3.85 , 5.51 , 6.66–7.24 , 8.85  ppm; 13C NMR : δ 55.1, 55.5, 66.1, 105.0, 118.9, 119.4, 130.7, 132.0, 159.3, 161.4, 162.2, 164.9, 174.6 ppm.1,6-diamino-2-oxo-4-(p-tolyl)-1,2-dihydropyridine-3,5-dicarbonitrile (5k): m.p: 238–240 °C (lit.47 240–241 °C); Yield: 97%; 1H NMR : δ 5.66 , 7.35–7.40 , 8.45  ppm. 13C NMR : δ 20.9, 74.2, 86.3, 115.5, 116.4, 127.9, 129.1, 131.6, 140.0, 156.6, 159.3, 159.6 ppm.1,6-diamino-4-(furan-2-yl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile (5l): m.p: 323–326 °C (lit.47 325–327 °C); Yield: 98%; 1H NMR  δ: 5.79 , 7.49–7.62 , 8.17  ppm; 13C NMR : δ = 80.0, 114.4, 137.4, 139.2, 143.5, 146.5, 148.2, 150.2, 155.9, 164.1, 171.6 ppm.1',6'-diamino-2'-oxo-1',2'-dihydro--3',5'-dicarbonitrile (5m): m.p: 296–297 °C; Yield: 90%; 1H NMR : δ 5.79 , 7.64–7.88 , 8.81  ppm; 13C NMR : δ 85.2, 113.9, 121.8, 123.7, 129.6, 134.7, 140.9, 156.5, 161.6, 183.7, 187.7, 188.2 ppm.3)3∙9H2O as iron precursor and 1,3,5-benzenetricarboxylic acid as organic ligand CuO nanostructures were added to modify the surface of metal–organic nano-framework. The mentioned nanostructures were placed on the metal–organic nano-framework by co-precipitation method to achieve Rod-like trifunctional Fe-based MOF@CuO nanocomposites (RL TF Fe-based MOF@CuO NC) as nano-catalyst.After preparing metal–organic framework nanostructures (Fe-MOF) via co-precipitation method using Fe , Hydrazine hydrate (1 mmol), malononitrile (1 mmol) and 4-methoxybenzaldehyde (1 mmol) to achieve optimal reaction conditions. Investigations were primarily performed to determine the contribution of the solvent to the reactions through the model reaction. Hence, this step was carried out using different solvents, including water, ethanol, methanol, acetonitrile and In the next step, the effect of reaction temperature was tested considering room temperature, 50 and reflux conditions. Temperature decline below 50 ºC decremented the yield of the product and increased the reaction time. Moreover, increasing temperature from 50 °C to reflux conditions was improved the progress of the reaction , methyl cyanoacetate (3 mmol), hydrazine hydrate (3 mmol) and malononitrile (3 mmol), conducted as a model reaction in optimized reaction conditions. Noteworthy, the product yield was constant after recycling . It is possible to reuse RL BF Fe-based MOF@CuO NC until three runs with no significant decline in its catalytic function. In the recycling procedure, hot acetone addition aimed at diluting the model reaction mixture following the reaction completion and stirring the mixture over a 5-min period. The catalyst was insoluble in acetone and underwent several washings with acetone for its separation by filtration. The above steps were followed by drying at 40 °C within an 8-h period and reusing the catalyst with no considerable decline in its catalytic effects  formation was done from the Knoevenagel condensation aromatic aldehydes and malononitrile when RL BF Fe-based MOF@CuO NC was present. Meantime, methyl cyanoacetate reacts with hydrazine hydrate to form intermediate (B). Then, interaction of the enolizable cyanoacetic acid hydrazide (C) with arylidenemalononitrile (A) through Michael addition, followed by booting of the intermediate’s intramolecular cyclization by nano-catalyst result in the final product yields.The proposed mechanisms were used to prepare N-amino-2-pyridones in the presence of RL BF Fe-based MOF@CuO NC as shown in Fig. In summary, an effective technique was reported for the generation of useful RL TF Fe-based MOF@CuO NC nano-catalyst using 1,3,5-Benzenetricarboxylic acid linker via microwave irradiation. The catalytic activity of RL TF Fe-based MOF@CuO NC was explored in one-pot synthesis of N-amino-2-pyridones by facile, rapid and versatile multi-component reaction of malononitrile, diverse aromatic aldehydes, hydrazine hydrate and ethyl cyanoacetate under mild and solvent-free conditions. The procedure offers several advantages including environmentally friendly, excellent yields with short reaction times, simple work up procedure and the ability of recyclability catalyst for three times with no considerable functional loss.Supplementary Information.


N-acyl thiourea derivatives (1a–1o), incorporating thiazole or pyridine nucleus in the same molecule and showing antimicrobial potential previously predicted in silico. The compounds have been physiochemically characterized by their melting points, IR, NMR and MS spectra. Among the tested compounds, 1a, 1g, 1h, and 1o were the most active against planktonic Staphylococcus aureus and Pseudomonas aeruginosa, as revealed by the minimal inhibitory concentration values, while 1e exhibited the best anti-biofilm activity against Escherichia coli . The total antioxidant activity (TAC) assessed by the DPPH method, evidenced the highest values for the compound 1i, followed by 1a. A routine quality control method for the separation of highly related compounds bearing a chlorine atom on the molecular backbone  has been developed and validated by reversed-phase high-performance liquid chromatography (RP—HPLC), the results being satisfactory for all validation parameters recommended by the ICH guidelines  and recommending it for routine separation of these highly similar compounds.The present study aimed to synthesize, characterize, and validate a separation and quantification method of new  They have been reported to possess antimicrobial + C22H22N3O2S+ = 392.14272, found 392.14171  calcd for [M − H]+ C22H20N3O2S+ = 390.12707, found 390.12683 , calcd for C16H18NO2+ = 256.13321, found 256.13301 , calcd for C16H15O2+ = 239.10666, found 239.10655 , calcd for C8H8NO+ = 134.06004, found 134.06000 .Chemical Formula: CN-((pyridin-3-yl)carbamothioyl)benzamide (1c)2-((4-Ethylphenoxy)methyl)-1h-NMR : 12.32; 11.97; 8.63; 8.44; 8.01; 7.63; 7.60; 7.57; 7.47; 7.44; 7.09; 6.90; 5.28; 2.51; 1.12 (1h-NMR spectrum).13C-NMR : 180.23(C-16); 169.99(C-1); 156.27(C-9); 147.00(C-20); 146.14(C-21); 136.20(Cq); 135.84(Cq); 134.87(Cq); 133.25(Cq); 132.41(C-18); 131.02(C-5); 128.57; 128.45(C-4); 128.34(C-7); 127.71(C-6); 123.25(C-18); 114.55; 67.47(C-8); 27.23(C-15); 15.76(C-15′) (13C-NMR spectrum).−1): 3128w; 3032w; 2956m; 2912m; 2868m; 1673m; 1530vs; 1510vs; 1382m; 1284m; 1233s; 1199m; 1168s; 1120m; 1029m; 881w; 821m; 746s; 707m; 675m; 648w; 603w : m/z calcd for [M + H]+ C22H22N3O2S+ = 392.14272, found 392.14227 , calcd for [M − S − H]+ C22H20N3O2+ = 358.15500, found 358.15497 , calcd for C16H18NO2+ = 256.13321, found 256.13303 , calcd for C16H15O2+ = 239.10666, found 239.10638 , calcd for C6H5N2S+ = 137.01680, found 137.01651 , calcd for C8H8NO+ = 134.06004, found 134.05977 .Chemical Formula: CN-((3-methylpyridin-2-yl)carbamothioyl)benzamide (1d)2-((4-Ethylphenoxy)methyl)-1h-NMR : 12.11; 11.94; 8.31; 7.73; 7.63; 7.60; 7.57; 7.47; 7.30; 7.09; 6.89; 5.27; 3.31; 2.51; 1.14 (1h-NMR spectrum).13C-NMR : 180.02(C-16); 169.87(C-1); 156.21(C-9); 150.04(C-17); 146.15(C-C-21); 133.39(C-19); 136.18(Cq); 135.78(Cq); 133.34(Cq); 130.99(C-5); 130.53(Cq); 128.64(C-4); 128.59; 128.53(C-7); 127.74(C-6); 123.20(C-20); 114.66; 67.48(C-8); 27.24(C-15); 17.03(C-18′); 15.77(C-15′) (13C-NMR spectrum).−1): 3149m; 3062w; 2960m; 2929m; 2870m; 1672s; 1604w; 1581w; 1504vs; 1454vs; 1373m; 1326m; 1235s; 1161s; 1114m; 1019m; 954w; 882w; 816w; 734m; 667m; 582w : m/z calcd for [M + H]+ C23H24N3O2S+ = 406.15837, found 406.15779 , calcd for [M − H]+ C23H22N3O2S+ = 404.14272, found 404.14235 , calcd for C16H18NO2+ = 256.13321, found 256.13296 , calcd for C16H15O2+ = 239.10666, found 239.10636 , calcd for C7H7N2S+ = 151.03245, found 151.03220 , calcd for C8H8NO+ = 134.06004, found 134.05982 .Chemical Formula: CN-((4-methylpyridin-2-yl)carbamothioyl)benzamide (1e)2-((4-Ethylphenoxy)methyl)-1h-NMR : 13.01; 11.94; 8.60; 8.24; 7.63; 7.60; 7.57; 7.47; 7.10; 7.09; 6.89; 5.27; 2.49; 2.36; 1.09 (1h-NMR spectrum).17 bond and instead, the protons in the pyridine ring are on the time scale of the NMR experiment. By heating to 70 °C, the respective signals become sharper and present a hyperfine structure. The signals of carbon atoms in the pyridine ring appear broadened for the same reason.The formation of hydrogen bonds inhibits the free rotation around the SC–N–C13C-NMR : 177.52(C-16); 170.14; 156.16(C-9); 151.28(C-17); 148.93(C-19); 147.90(C-21); 136.11(Cq); 135.92(Cq); 133.26(Cq); 131.08(C-5); 128.51(C-7); 128.50; 128.39(C-4); 127.71(C-6); 122.18(C-20); 115.79(C-18); 114.54; 67.42(C-8); 27.18(C-15); 20.88(C-19′); 15.67(C-15′) (13C-NMR spectrum).−1): 3348w; 3162w; 3035w; 2968w; 2922w; 2872w; 1675m; 1606m; 1507vs; 1446m; 1378m; 1326s; 1289m; 1225s; 1151s; 1026m; 897w; 828m; 793w; 735m; 668m; 596w : m/z calcd for [M + H]+ C23H24N3O2S+ = 406.15837, found 406.15710 , calcd for [M − H]+ C23H22N3O2S+ = 404.14272, found 404.14241 , calcd for C16H18NO2+ = 256.13321, found 256.13269 , calcd for C16H15O2+ = 239.10666, found 239.10630 , calcd for C7H7N2S+ = 151.03245, found 151.03225 .Chemical Formula: CN-((5-methylpyridin-2-yl)carbamothioyl)benzamide (1f)2-((4-Ethylphenoxy)methyl)-1h-NMR : 12.86; 11.82; 8.54; 8.23; 7.77; 7.65; 7.60; 7.57; 7.47; 7.05; 6.88; 5.28; 2.49; 2.30; 1.11 (1h-NMR spectrum).At 30 °C, the spectrum is composed of broad lines, and we preferred the temperature of 60 °C for simulating the molecular movement.13C-NMR : 177.47(C-16); 169.86; 156.24(C-9); 149.05(Cq); 148.03(C-21); 138.15(C-19); 136.21(Cq); 135.94(Cq); 133.32(Cq); 131.03(C-5); 130.52(Cq); 128.46(C-7); 128.41(C-4); 128.38; 127.66(C-6); 115.09(C-18); 114.70; 67.56(C-8); 27.16(C-15); 17.27(C-20′); 15.52(C-15′) (13C-NMR spectrum).−1): 3237s; 3040m; 2963m; 2929m; 2869m; 1661m; 1605w; 1580m; 1526vs; 1471s; 1382m; 1344s; 1291m; 1248m; 1213s; 1149s; 1078w; 1047w; 1014m; 961w; 898w; 823w; 783m; 756m; 724m; 642w; 614w : m/z calcd for [M + H]+ C23H24N3O2S+ = 406.15837, found 406.15724 , calcd for C16H18NO2+ = 256.13321, found 256.13262 , calcd for C16H15O2+ = 239.10666, found 239.10710 , calcd for C7H7N2S+ = 151.03245, found 151.03261 .Chemical Formula: CN-((5-chloropyridin-2-yl)carbamothioyl)benzamide (1g)2-((4-Ethylphenoxy)methyl)-1h-NMR : 13.08; 12.13; 8.76; 8.45; 8.03; 7.65; 7.60; 7.57; 7.47; 7.03; 6.86; 5.27; 2.47; 1.08 (1h-NMR spectrum).13C-NMR : 177.83(C-16); 170.23; 156.15(C-9); 148.68(Cq); 146.75(C-21); 137.62(C-19); 136.11(Cq); 135.91(Cq); 133.09(Cq); 131.10(C-5); 130.52(Cq); 128.48(C-7); 128.47; 128.46(C-4); 128.35(C-6); 116.38(C-18); 114.49; 67.41(C-8); 27.17(C-15); 15.66(C-15′) (13C-NMR spectrum).−1): 3168m; 3027m; 2963m; 2926m; 2863m; 1675m; 1508vs; 1447s; 1379m; 1327s; 1293m; 1246s; 1219s; 1149s; 1106m; 1028m; 956m; 925w; 860m; 767m; 723m; 662m; 624w : m/z calcd for [M + H]+ C22H21ClN3O2S+ = 426.10375, found 426.10380 , calcd for C16H18NO2+ = 256.13321, found 256.13355 , calcd for C16H15O2+ = 239.10666, found 239.10708 , calcd for C8H8NO+ = 134.06004, found 134.06022 .Chemical Formula: CN-((2-chloropyridin-3-yl)carbamothioyl)benzamide (1h)2-((4-Ethylphenoxy)methyl)-1h-NMR : 12.52; 12.16; 8.40; 8.32; 7.65; 7.60; 7.57; 7.50; 7.47; 7.08; 6.90; 5.28; 2.51; 1.12 (1h-NMR spectrum).13C-NMR : 180.26(C-16); 170.28(C-1); 156.20(C-9); 147.05(C-19); 145.27(Cq); 136.29(C-21); 136.17(Cq); 135.81(Cq); 133.14(Cq); 132.57(Cq); 131.07(C-5); 128.51; 127.76(C-6); 122.93(C-20); 114.54; 67.52(C-8); 27.21(C-15); 15.75(C-15′) (13C-NMR spectrum).−1): 3098w; 3006m; 2962m; 2931m; 1672m; 1590m; 1516vs; 1451s; 1404m; 1380w; 1325m; 1302m; 1269m; 1245s; 1213m; 1157vs; 1119m; 1064m; 1010m; 951w; 870w; 823w; 755s; 730m; 662w; 640w : m/z calcd for [M + H]+ C22H21ClN3O2S+ = 426.10375, found 426.10289 , calcd for [M − Cl]+ C22H20N3O2S+ = 390.12707, found 390.12641 , calcd for C17H18NO2+ = 268.05391, found 268.05347 , calcd for C16H18NO2+ = 256.13321, found 256.13290 , calcd for C16H15O2+ = 239.10666, found 239.10637 .Chemical Formula: CN-((2-chloropyridin-4-yl)carbamothioyl)benzamide (1i)2-((4-Ethylphenoxy)methyl)-1h-NMR : 12.64; 12.11; 8.38; 8.08; 7.71; 7.62; 7.58; 7.57; 7.47; 7.05; 6.87; 5.27; 2.49; 1.10 (1h-NMR spectrum).13C-NMR : 179.20(C-16); 169.24(C-1); 156.22(C-9); 150.42(C-19); 150.17(C-20); 147.52(Cq); 136.16(Cq); 135.96(Cq); 132.99(Cq); 131.16(C-5); 128.52; 128.48(C-4); 128.33(C-7); 127.71(C-6); 116.48(C-21); 116.44(C-18); 114.48; 67.41(C-8); 27.19(C-15); 15.69(C-15′) (13C-NMR spectrum).−1): 3334w; 3112w; 2954m; 2926m; 2868w; 1682m; 1574s; 1506vs; 1447m; 1378m; 1320m; 1293m; 1224s; 1145s; 1074w; 1056w; 1022m; 982m; 875w; 860w; 831m; 779w; 730m; 676m : m/z calcd for [M + H]+ C22H21ClN3O2S+ = 426.10375, found 426.10331 , calcd for C16H18NO2+ = 256.13321, found 256.13294 , calcd for [C16H15O2]+ = 239.10666, found 239.10626 .Chemical Formula: CN-((6-chloropyridin-3-yl)carbamothioyl)benzamide (1j)2-((4-Ethylphenoxy)methyl)-1h-NMR : 12.30; 12.04; 8.48; 8.09; 7.55; 7.62; 7.58; 7.57; 7.47; 7.05; 6.87; 5.28; 2.51; 1.12 (1h-NMR spectrum).13C-NMR : 180.40(C-16); 169.89(C-1); 156.25(C-9); 146.82(C-20); 146.25(C-21); 136.21(C-18); 136.20(Cq); 135.88(C-q); 134.54(Cq); 133.15(Cq); 131.07(C-5); 128.56; 128.45(C-4); 128.34(C-7); 127.70(C-6); 123.74(C-19); 114.55; 67.44(C-8); 27.22(C-15); 15.76(C-15′) (13C-NMR spectrum).−1): 3170s; 3010m; 2958m; 2928m; 2886m; 1752w; 1691s; 1609w; 1574m; 1513vs; 1451s; 1393m; 1314m; 1275m; 1239s; 1166s; 1098m; 1024m; 824m; 735m; 690m; 652w; 608w : m/z calcd for [M + H]+ C22H21ClN3O2S+ = 426.10375, found 426.10364 , calcd for C16H18NO2+ = 256.13321, found 256.13373 , calcd for C16H15O2+ = 239.10666, found 239.10722 .Chemical Formula CN-((5-bromopyridin-2-yl)carbamothioyl)benzamide (1k)2-((4-Ethylphenoxy)methyl)-1h-NMR : 13.07; 12.14; 8.71; 8.53; 8.14; 7.64; 7.60; 7.57; 7.47; 7.03; 6.86; 5.27; 2.47; 1.09 (1h-NMR spectrum).13C-NMR : 177.81(C-16); 170.22(C-1); 156.15(C-9); 150.02(Cq); 148.94(C-21); 140.39(C-19); 136.12(Cq); 135.92(Cq); 133.09(Cq); 131.11(C-5); 128.49; 128.48(C-7); 128.36(C-4); 127.69(C-6); 116.82(C-18); 115.56(C-20); 114.49; 67.41(C-8); 27.18(C-15); 15.68(C-15′) (13C-NMR spectrum).−1): 3164m; 3026m; 2961m; 2926m; 2860w; 1674m; 1508vs; 1447s; 1376m; 1326m; 1290m; 1246s; 1220s; 1152s; 1090m; 1027m; 996m; 955w; 926w; 886w; 859w; 824m; 762m; 732s; 658m; 617w : m/z calcd for [M + H]+ C22H21BrN3O2S+ = 472.05119, found 472.05057 , calcd for C16H18NO2+ = 256.13321, found 256.13312 , calcd for C16H15O2+ = 239.10666, found 239.10666 , calcd for C5H6BrN2+ = 172.97089, found 172.97096 , calcd for C8H8NO+ = 134.06004, found 134.06001 , calcd for [Me2SO+H]+ C2H7SO+ = 79.02121, found 79.02091 .Chemical Formula: CN-((6-bromopyridin-2-yl)carbamothioyl)benzamide (1l)2-((4-Ethylphenoxy)methyl)-1h-NMR : 12.97; 12.16; 8.69; 7.85; 7.64; 7.60; 7.57; 7.51; 7.47; 7.03; 6.86; 5.28; 2.47; 1.09 (1h-NMR spectrum).13C-NMR : 177.98(C-16); 170.11(C-1); 156.16(C-9); 151.18(Cq); 141.03(C-19); 138.73(Cq); 136.13(Cq); 135.97(Cq); 133.02(Cq); 131.01(C-5); 128.53(C-7); 128.50; 128.30(C-4); 127.68(C-6); 125.07(C-20); 114.55; 114.38(C-18); 67.40(C-8); 27.19(C-15); 15.70(C-15′) (13C-NMR spectrum).−1): 3210m; 3172m; 3026m; 2971m; 29286m; 2872w; 1672m; 1556s; 1509vs; 1451m; 1421s; 1322s; 1255m; 1223s; 1170s; 1144m; 1120s; 1069w; 1026m; 980w; 820m; 762m; 725m; 660m : m/z calcd for [M + H]+ C22H21BrN3O2S+ = 472.05119, found 472.04990 , calcd for C16H18NO2+ = 256.13321, found 256.13287 , calcd for C16H15O2+ = 239.10666, found 239.10634 .Chemical Formula: CN-carbamothioyl)benzamide (1m)2-((4-Ethylphenoxy)methyl)-1h-NMR : 12.23; 12.09; 8.55; 8.35; 7.65; 7.60; 7.57; 7.47; 7.09; 6.90; 5.26; 2.52; 1.14 (1h-NMR spectrum).13C-NMR : 180.54(C-16); 169.84(C-1); 156.18(C-9); 147.43(Cq); 145.62(C-21); 137.97(C-19); 136.18(Cq); 135.84(Cq); 133.07(Cq); 131.11(C-5); 129.95(Cq); 129.00(Cq); 128.59(C-7); 128.56; 128.55(C-4); 127.74(C-6); 114.70; 67.45(C-8); 27.25(C-15); 15.79(C-15′) (13C-NMR spectrum).−1): 3129m; 3027m; 2962m; 2927m; 2868w; 1671m; 1560m; 1504vs; 1438m; 1231s; 1151s; 1114s; 1050m; 953w; 895w; 853w; 823m; 777m; 743w; 708w; 659w; 610w : m/z calcd for [M + H]+ = C22H20Cl2N3O2S+ 460.06478, found 460.06464 , calcd for C16H18NO2+ = 256.13321, found 256.13330 , calcd for C16H15O2+ = 239.10666, found 239.10677 , calcd for C8H8NO+ = 134.06004, found 134.06010 .Chemical Formula: CN-carbamothioyl)benzamide (1n)2-((4-Ethylphenoxy)methyl)-1h-NMR : 12.66; 12.19; 8.01; 7.65; 7.60; 7.57; 7.47; 7.05; 6.87; 5.26; 2.50; 1.10 (1h-NMR spectrum).13C-NMR : 179.38(C-16); 169.85(C-1); 156.21(C-9); 149.60(Cq); 149.23; 136.16(Cq); 135.99(Cq); 132.910(Cq); 131.22(C-5); 128.50(C-7); 128.49; 128.36(C-4); 127.74(C-6); 116.01; 114.45; 67.39(C-8); 27.18(C-15); 15.68(C-15′) (13C-NMR spectrum).−1): 3279m; 3116m; 2958w; 2927m; 2869m; 1681m; 1574s; 1503vs; 1372m; 1296s; 1232s; 1163s; 1123m; 1097m; 1043m; 1002w; 884w; 846w; 820m; 773w; 738w; 678m; 612w : m/z calcd for [M + H]+ C22H20Cl2N3O2S+ = 460.06478, found 460.06400 , calcd for [M − S + OH]+ C22H20Cl2N3O3+ = 444.08762, found 444.08749 , calcd for C16H18NO2+ = 256.13321, found 256.13300 , calcd for C16H15O2+ = 239.10666, found 239.10649 , calcd for C5H5Cl2N2+ = 162.98243, found 162.98230 , calcd for C8H8NO+ = 134.06004, found 134.05984 , calcd for [Me2SO+H]+ C2H7SO+ = 79.02121, found 79.02081 .Chemical Formula: CN-carbamothioyl)benzamide (1o)2-((4-Ethylphenoxy)methyl)-1h-NMR : 12.24; 12.09; 8.65; 8.54; 7.63; 7.61; 7.57; 7.48; 7.10; 6.91; 5.27; 2.53; 1.14 (1h-NMR spectrum).13C-NMR : 180.38(C-16); 169.81(C-1); 156.18(C-9); 148.98(C-17); 148.34(C-21); 143.47(C-19); 136.16(Cq); 135.85(Cq); 133.04(Cq); 131.13(C-5); 128.62(C-7); 128.57; 127.75(C-6); 119.54(C-20); 118.52(C-18); 114.70; 67.39(C-8); 27.25(C-15); 15.79(C-15′) (13C-NMR spectrum).−1): 3130m; 3027w; 2965w; 2844w; 1673m; 1546m; 1504vs; 1430m; 1360m; 1323w; 1236s; 1150vs; 1043m; 952w; 825w; 737m; 696w; 661w : m/z calcd for [M + H]+ C22H20Br2N3O2S+ = 549.96170, found 549.96069 , calcd for C14H9BrN3OS+ = 345.96442, found 345.96356 , calcd for C16H18NO2+ = 256.13321, found 256.13354 , calcd for C16H15O2+ = 239.10666, found 239.10707 , calcd for C8H8NO+ = 134.06004, found 134.06015 , calcd for [Me2SO + H]+ C2H7SO+ = 79.02121, found 79.02081 .Chemical Formula: CStaphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853. Fresh microbial cultures grown for 18–24 h on Plate Count Agar were used to prepare suspensions in sterile PBS . The 0.5 McFarland standard was used as a reference to adjust the turbidity of the microbial suspensions to 1.5 × 108 colony-forming units (CFU/mL). The compounds were solubilized in DMSO at a stock concentration of 10 mg/mL. Then, serial binary dilutions were prepared in liquid culture medium (Mueller–Hinton) in 96-well plates, starting from 5 mg/mL (the compound testing concentrations were between 5 and 0.009 mg/mL). The medium containing different concentrations of the compounds was further inoculated with the microbial suspensions at a final density of 106 CFU/mL. The inoculated 96 well-plates were incubated for 18–24 h at 37 °C. The positive control was represented by ciprofloxacin (5 µg/mL). Growth control was represented by the broth inoculated only with standard microbial suspension. The determinations were performed in triplicate to confirm the MIC (minimum inhibitory concentration) values. The MIC values were determined by visual inspection, as the lowest concentrations of tested compounds that inhibited the microbial growth in the liquid medium .The in vitro evaluation of the antimicrobial activity of the compounds was performed using the following standard microbial strains: The compounds’ antibiofilm activity was investigated using the crystal violet microtiter assay. After the determination of the MIC, the 96-well plates were emptied and washed three times with sterile saline to remove non-adherent microbial cells. Microbial cells adhering to the walls of the wells were fixed with 150 µL methanol for 5 min, then stained with 150 µL 1% violet crystal solution (prepared in distilled water) for 20 min. The dye was removed, and the plates were washed using sterile saline. The microbial biofilms formed on the inert substrate were suspended in 33% acetic acid, then the absorbance was read at a wavelength of 490 nm. The minimum inhibitory concentration of the total biofilm mass development (MBIC) was defined as the lowest concentration of compound that induced a decrease in the stained biofilm mass and, consequently, of the absorbance value, at levels similar to those of the sterility control.−4 M in acetone was prepared. Ascorbic acid was used as a reference. Stock solutions of ascorbic acid and compounds 1a–1o were prepared in acetone at a standard concentration of 2 mg/mL. For TAC measurements, 1 mL of stock solution of DPPH was added to 1 mL of stock solution of each compound, and the mixture was kept for 30 min in the dark, followed by the absorbance measuring at 517 nm. A UVD-3500 UV-Vis spectrophotometer was used for this purpose. The TAC percentage was calculated by Equation (1):i is the initial absorbance of the mixture, and the Abs30 min is the absorbance measured after 30 min + cation. In the mass spectra of compounds 1a, 1d, 1k, and 1m, the base peak is related to the [C16H15O2]+ fragment , whose structure is depicted on the scheme below .The results of the quantitative evaluation of the antimicrobial activity of the analyzed compounds are presented in Regarding the influence of the obtained compounds on bacterial adherence, the results showed a low antibiofilm activity, by comparison with the antibiotic control, with MBICs values of >5000–312 µg/mL (results attached in 1i (87%), followed by 1a (44%), while for the other compounds, TAC values were between 0 and 29%. The TAC value of every compound was calculated with regard to the antioxidant capacity of the reference .The total antioxidant capacity (TAC) was measured using the well-known DPPH method, as mentioned previously. Ascorbic acid was used as a reference, with a determined antioxidant capacity of 100%. The obtained results are shown in 1h, 1j, 1i, 1m, 1g, and 1n were injected in the chromatographic system. The retention time and UV spectra were extracted for peak identification  was injected, the chromatogram registered .A solution that contained 5 µg/mL of each of the related compounds  was in the range of 1.02–1.07, confirming the Gaussian symmetry of the peaks . The degThe interference study aims to demonstrate the lack of interference between the solvent used at solution preparation and the main peaks on the chromatogram. 1h, 1j, 1i, 1m, 1g, and 1n.The results sustain the hypothesis that the optimized analytical RP—HPLC method is well suited for the proper separation of the chemically related compounds The calibration curve was traced by preparing increasingly concentrated solutions for every related compound, in the range of 0.06 µg/mL–0.5 µg/mL.2) greater than 0.99, for each series of solutions  greater than 0.99  values of the analytes in the sample solutions and their determined concentrations. The accuracy parameter was performed for every compound, over three concentration levels, six replicates each . Consequently, the accuracy was expressed as the percentage recovery of the determined amount of each analyte in the sample solutions, along with the relative standard deviations and confidence interval , that incorporate both thiazole or pyridine nucleus in the same molecule. The present research is built on our previous in silico prediction and molecular docking studies, suggesting the antimicrobial potential of the designed compounds [1a–1o, through in silico computational approaches, to predict their drug-like, absorption, distribution, metabolism, elimination, and toxicity (ADMET). According to Lipinski’s rule, a promising chemical candidate with oral absorption and adequate permeability is characterized by a molecular mass not more than 500 Da, having maximum 10 H-bond acceptors, 5 H-bond donors and partition coefficient (log P) of 5 [1a, 1b, 1c, 1e, and 1f), 1o showed two violations , and the others showed one violation from the rule (log P > 5).The current research paper is a continuation of our previous efforts in this area , synchroompounds . First,  P) of 5 ,37,38. I1a–1o has been previously checked through molecular docking studies [S. aureus DNA gyrase B and E. coli DNA gyrase B), has been confirmed by the presence of H-bonds established with the amino acid scaffolds of the selected receptors, and satisfactory docking scores. Consequently, the results obtained after generating the in silico studies provided an impetus to synthesize and evaluate the in vitro biological activities of chemicals 1a–1o, on bacterial strains which are among the most threatening and challenging because of their resistance to all currently available antibiotics and high biofilm development ability. Among Gram-positive bacteria, S. aureus and E. faecalis are two of the most frequent Gram-positive opportunistic and nosocomial pathogens, exhibiting resistance to penicillin, methicillin, quinolones, aminoglycosides, macrolides, glycopeptides, and often harboring multi-drug resistance (MDR) phenotypes [E. coli) and non-fermentative (P. aeruginosa), are involved in endogenous and exogenous infections that are very difficult to treat due to their multiple intrinsic and acquired resistance sometimes to the majority (extended-drug resistance) or even all tested antibiotics (pan-drug resistance), including last resorts such as plasmid-mediated colistin resistance [The biological activity of the studied compounds  studies . The mosenotypes . The Grasistance ,41. The sistance ,43. In asistance .1a, 1g, 1h, and 1o had better activity in the series against S. aureus ATCC 25923, P. aeruginosa ATCC 27853 with moderate MIC values of 625 µg/mL.Among the tested compounds, 1a was designed with the thiazole ring on the backbone molecule. Structurally, thiazole has an electron-donating group (-S-) and an electron-accepting group (-C=N-). The thiazole nucleus demonstrated its activity against multi-drug resistant Gram-negative bacteria (P. aeruginosa) in recent studies [The compound  studies ,46.1g: 5-chloro; 1h: 2-chloro) and bromine  atoms on the pyridine ring distinguished in the series through higher activity. Thus, it may be inferred that the position of the halogen substituents improved the activity of the resulting compounds, in comparison with other designed isomers.The analogues substituted with halogens in the pyridine group showed similar antimicrobial activity against the tested bacterial strains. The derivatives designed with chlorine . The relationship among compounds with respect to the chemical structure and their molecular properties inspired us to evaluate their similar chromatographic characteristics via the HPLC technique. In such cases of related substances, the phenomenon of peak co-elution becomes challenging. As the peak overlapping represents an issue in chromatography, in the current study, an RP-HPLC method is proposed and optimized to simultaneously separate and quantify the chemically related compounds 1g, 1h, 1i, 1j, 1n, 1m.Remarkably, the compounds containing chlorine atoms in the molecule are designed as position isomers . Solvent A was represented by 6.8 g/L potassium dihydrogen phosphate R, while acetonitrile R, HPLC grade, represented solvent B. The method has also been validated and proved to be specific, precise, accurate, and linear in a particular range of concentrations, confirming that it is suited for the determination and quantification of 2-((4-methoxyphenoxy)methyl)-N-((6-methylpyridin-2-yl)carbamothioyl)benzamide.For the quantification of us study . The anaN-acyl-thiourea derivatives.Evaluating the data, both methods give similar results in terms of recovery (%), precision, and linearity. Despite these aspects, the newly developed and optimized method (as compared against the previously mentioned method) as well the increased simplicity of the experimental setup, i.e., the reduced number of steps for mobile phase preparation (water and acetonitrile R), the protection of the HPLC system by avoiding the use of phosphates for the mobile phase preparation and their precipitation during long analyses, the lower back pressure in the system, reduced consumption of reagents, and shorter turnaround time are reasons to support the use of the current optimized method for separation and quantification of the mentioned To demonstrate that the developed method fits its intended purpose, the validation parameters such as specificity, precision (intra-assay and intermediate precision), limits of detection (LOD) and quantification (LOQ), linearity, accuracy, and robustness have been completed, as per ICH guideline requirements . The valAs for the specificity parameter, no interfering peaks were observed from the diluent. Although the peaks are closely chemically related, the chromatographic analytical method ensured their separation properly, avoiding a potential co-elution. The reasonable separation was sustained by resolution values higher than 2 between two consecutive peaks, along with the purity determination by purity angles < purity threshold.The degree of repeatability was evaluated within the precision parameter. The results proved to be close to one another for six replicates of solutions (intra-assay precision), prepared on two different days (intermediate precision); specifically, the relative standard deviation values were less than 2, meeting the set acceptance criterion.1h, 1j, 1i, 1m, 1g, and 1n. Six regression lines corresponding to each analyte were traced for eight increasingly concentrated solutions. In every case, the analytes in the tested solutions proved to be linearly proportional to their concentration. The linearity was confirmed for every calibration plot, by evaluation of correlation coefficient, with satisfactory values (R2 ≥ 0.999).The lowest distinguished and determined concentration for every analyte in the sample was revealed by LOD—LOQ analysis, using dispersion characteristics of the regression line. For the six related compounds, the LOD limits were calculated in the range of 0.0184 µg/mL–0.0252 µg/mL, while LOQ ranged in an interval of 0.0614 µg/mL–0.0797 µg/mL. The linearity was demonstrated for each compound, covering the concentrations ranging from calculated LOQ to 6 µg/mL solutions of 1h, 1j, 1i, 1m, 1g, and 1n. The standard deviation, relative standard deviation, and confidence interval were calculated for six replicate samples. The determined concentrations were reported as percent recovery of the known added amount of compound, as per ICH Q2(R1) and the results were within 98–102%.The accuracy of the analytical procedure was determined over three concentration levels , namely 4 µg/mL, 5 µg/mL, and 6 µg/mL solutions of By altering the parameters that may affect the analytical procedure, namely mobile phase composition, flow rate, and column temperature, the method proved to be robust and reliable during normal use.1h, 1j, 1i, 1m, 1g, and 1n in a mixture.The analytical method was found satisfactory for validation parameters, following ICH guidelines requirements. Hence, it can be routinely used for the simultaneous separation and quantification of compounds N-acyl thiourea derivatives (1a–1o), incorporating both the thiazole or pyridine nucleus in the same molecule and showing a predicted antimicrobial potential previously studied by in silico approaches .The present study aimed to synthesize and characterize new After synthesis, the compounds have been physiochemically characterized by their melting points, IR, NMR, and MS spectra.Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa) in planktonic  and biofilm  growth state. Among the tested compounds, 1a, 1g, 1h and 1o displayed better activity against planktonic Staphylococcus aureus and Pseudomonas aeruginosa, as revealed by the MIC values determined by the broth microdilution assay, while 1e revealed the lowest antibiofilm activity against Escherichia coli.The in vitro evaluation of the antimicrobial activity has been performed against problematic opportunistic Gram-positive and Gram-negative bacterial strains , attached to the molecular backbone played a moderate role in inhibiting the microorganism’s activity. The unsatisfactory inhibition of the microbial strains may be attributed to the poor penetration of the bacterial membrane. N-acyl thiourea derivatives may be the focus of further investigation, in which case, we aim to evaluate the molecular properties that impact the mechanism of action of the compounds against microbial strains, by designing a more advanced number of molecules so that the corroboration of the data can reach a statistically relevant estimation.The thiazole ring or the pyridine nucleus substituted with halogens , assessed by the DPPH method, evidenced the highest values for the compound 1g, 1h, 1i, 1j, 1m, 1n). To demonstrate that the developed method fits its intended purpose, the validation parameters such as specificity (no interfering peaks were observed from the diluent), precision , limits of detection (LOD)  and quantification (LOQ) (ranging in 0.0614–0.0797 µg/mL) interval, linearity , accuracy , and robustness  have been assessed and found satisfactory, according to ICH guideline requirements. Hence, the analytical method can be routinely used for the simultaneous separation and quantification of compounds 1h, 1j, 1i, 1m, 1g, and 1n in a mixture.Also, another purpose of our research was to optimize and validate a separation and quantification method for highly related compounds bearing a chlorine atom on the molecular backbone (


Planar structures of the metabolites were elucidated by 1D and 2D NMR in addition to HRESIMS data. While ROESY data assigned relative configurations, absolute configurations were determined by the synthesis of MTPA esters of 1, 3, and 5. The [6.3.0] undecane core structure of compounds 3–5 is of the asteriscane-type, however, the scaffold of 1 and 2 with their bicyclo [5.3.0] decane core and germinal methyl substitution is, to our knowledge, unprecedented. Together with several new compounds that were previously isolated from solid cultures of this strain, Heimiomyces sp. showed an exceptionally high chemical diversity of its secondary metabolite profile.With heimionones A–E ( Armillaria, which are known for their large amount of structurally closely related sesquiterpenoids aryl esters  decane and [6.3.0] undecane core structures, respectively, that were isolated from submerged cultures of Heimiomyces sp. alongside the previously described heimiomycins A–B (11–12) +  indicating nine degrees of unsaturation. 1H NMR and HSQC data , 1.13 , 1.40 , 1.77 , 1.82 , and 2.31 , three oxymethines at δH 4.70 , 5.14 , and 6.12 , and five olefinic methines at δH 5.81 , 6.07 , 7.35 , 7.56 , and 7.69 . The 13C and HMBC NMR data ; ten sp2-hybridized carbons, comprising five nonprotonated carbons  and five methines ; one quaternary carbon ; three methines ; and six methyl carbons . The carbonyl and sp2-hybridized carbons accounted for seven degrees of unsaturation, suggesting two rings in the scaffold of heimionone A (1). By analyzing the 1H-1H COSY data, the first spin system was given due to correlations between H3-13, H-5, H-4, H-14, and H3-15. Correlations between H3-11/H3-12, H2-9, and H-1 led to the identification of the second spin system. HMBC correlations from H3-13 to C-4/C-5/C-6/C-7/C-8 and H-4 to C-2/C-3/C-5/C-6/C-7/C-8 revealed a cyclohepta-3,5,8-triene-7-one ring. The 14-hydroxyethyl moiety was deduced from the HMBC correlations of H3-15 to C-3/C-14. Further HMBC correlations from H3-11 and H3-12 to C-1/C-9/C-10, from H-9 to C-2/C-8/C-10/C-11, and from H-1 to C-2/C-8/C-9/C-10/C-11 led to the identification of a dimethylcyclopentane substructure that was fused to the cyclohepta-3,5,8-triene-7-one ring across C-2 and C-8. HMBC correlations from H3-6′ to C-3′/C-4′/C-5′/C-7′ and from H-3′ to C-1′/C-2′/C-4′/C-5′/C-7′ revealed the presence of a 4′-methylhexa-2′,4′-dienoic acid partial structure that was fused to C-1 according to an HMBC correlation from H-1 to C-1′. The E ∆2′,3′ configuration was assigned according to the large coupling constants (J = 15.7 Hz) between the olefinic methines H-2′ and H-3′, while the ∆4′,5′ configuration was assigned as E due to the ROESY correlation between H-3′ and H-5′. Furthermore, this structural elucidation was supported by the comparison of the 1H and 13C data to the ones of daldinin F  to its corresponding R- and S-MTPA esters at position C-9 and C-14 was done with S- and R-MTPA chloride. The pattern of ∆δSR, chemical shifts , which is closely related to 1, was obtained as a yellow oil from the supernatant extracts of liquid cultures. Its HRESIMS data indicated the same molecular formula (C22H28O5) as obtained for 1. 1H and 13C data showed high similarities to 1. They differ by the relocation of the hydroxy function from C-14 for 1 to C-6 (δC 78.7) for 2, whereas the oxymethine at C-14 was replaced by an olefinic methine . The cyclohepta-4,8-diene-7-one scaffold was confirmed by HMBC correlations of H3-13 to C-5/C-6/C-7 and H-4 to C-2/C-3/C-6. Relative configuration was assigned according to the ROE correlations. In particular, interactions between H-9β and H3-11 and between H-1α and H3-12 implied the same configuration as 1. Additionally, correlations among H-9α/H3-11/H3-13 defined the 6R configuration. The absolute configuration of 2 was confirmed by comparison to heimionone A (1) as 1S,9R,6R.Heimionone B (3) was isolated from the mycelial extracts of liquid cultures with a molecular formula of C22H30O4, implying eight degrees of unsaturation. Analysis of the 1H and 13C spectra showed similarities to those of 1 and 2. HMBC and COSY interactions confirmed the presence of the previously described dimethylcyclopentane substructure carrying a 4′-methylhexa-2′,4′-dienoc acid partial structure that was fused to C-1. A spin system was given based on 1H-1H COSY correlations among H3-14, H-6, H2-5, H2-4, H-3, and H3-15. Furthermore, HMBC correlations from H3-14 to C-6/C-7/C-8, H2-4 to C-3/C-5, and H3-15 to C-2/C-3/C-4 led to the identification of the 3,7-dimethylcycloocta-6,9-dien-8-one ring that was fused to the dimethylcyclopentane ring across C-2 and C-9 according to correlations from H-1/H-3/H3-15 to C-2 and from H-1/H-10/H3-12 to C-9. The relative configuration was deduced by an analysis of ROE data. Due to the key correlations among H-1α/H-4α/H-5α/H3-13/H3-15, these protons were arbitrarily assigned to the α face of the molecule. Correlations between H-3β/H-4β/H-5β and between H-10β/H3-12 indicated a β orientation of these protons. Finally, the absolute configuration was determined after heimionone C (3) was derivatized to its corresponding S- and R-MTPA esters at position C-10 in the course of Mosher’s method. The ∆δSR chemical shift pattern  was obtained as a yellow oil from the mycelial extracts of liquid cultures with a molecular formula of C22H30O3. The 1D and 2D NMR data of 4 suggested a high similarity to 3. They only differ in the absence of a hydroxy function, which was replaced by a methylene . This was confirmed by the HMBC correlations from H-1, H3-12, and H3-13 to C-10 (δC 47.8). The ROESY correlations among H-1α/H-4α/H-5α/H-10α/H3-13/H3-15 implied 4 has the same configuration as heimionone C (3).Heimionone D (5) was isolated as a yellow oil from the mycelial extracts of liquid cultures. Its NMR spectroscopic data were highly similar to the ones of heimionone D (4). They only differ in the absence of the 4′-methylhexa-2′,4′-dienoc acid moiety that was replaced by the hydroxy function at position C-1. Finally, the absolute configuration was determined as 1R,3SR after heimionone E (5) was derivatized to its corresponding S- and R-MTPA esters at position C-1 using Mosher’s method with both R- and S-MTPA chloride.Heimionone E   and 6) 25D +250 ; UV/vis  λmax(logε): 325 (3.99), 268 (4.48), 247 (4.48), 202 (3.82) nm; ECD  λ(Δε): 265 (+19.8), 243 (−18.0), 220 (−1.2), 202 (−7.6) nm, 1H and 13C NMR data (MeOH-d4), m/z 373.19 [M + H]+, 371.24 [M − H]−; HRESIMS m/z 373.2012 [M + H]+ ; tR = 9.2 min .Heimionone A (2): yellow oil; [α]25D +230 ; UV/vis  λmax(logε): 329 (3.70), 267 (4.23), 248 (4.22), 198 (4.73) nm; ECD  λ(Δε): 266 (+10.2), 241 (−7.7), 222 (+0.1) nm, 1H and 13C NMR data (MeOH-d4), m/z 373.21 [M + H]+, 371.04 [M − H]−; HRESIMS m/z 373.2011 [M + H]+ ; tR = 9.3 min .Heimionone B (3): yellow oil; [α]25D +156 ; UV/vis  λmax(logε): 376 (3.18), 269 (4.35), 198 (4.41) nm; ECD  λ(Δε): 270 (+8.9), 241 (−0.6), 219 (+0.2), 205 (−10.8) nm, 1H and 13C NMR data (acetonitrile-d3), m/z 341.25 [M − H2O + H]+, 375.28 [M – H + H2O]−; HRESIMS m/z 381.2034 [M + Na]+ ; tR = 11.9 min .Heimionone C (4): yellow oil; [α]25D +72 ; UV/vis  λmax(logε): 268 (3.68), 198 (4.50) nm; ECD  λ(Δε): 270 (+1.9), 247 (−0.7), 218 (+0.6), 200 (−3.7) nm, 1H and 13C NMR data (acetonitrile-d3) m/z 365.20 [M + Na]+; HRESIMS m/z 365.2086 [M + Na]+ ; tR = 14.1 min .Heimionone D (5): yellow oil; [α]25D +20 ; UV/vis  λmax(logε): 266 (3.83), 202 (3.82) nm; ECD  λ(Δε): 404 (+0.2), 361 (−1.0), 319 (+0.2), 255 (−2.0), 225 (+1.0), 204 (−6.8) nm, 1H and 13C NMR data ; tR = 8.1 min .Heimionone E (R)-(−)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride were added into the solution and left for 15 min at room temperature. The reaction was monitored by analytical HPLC/MS. Another 2 µL of R-MTPA were added if the compounds were not completely converted into the corresponding Mosher-ester. Immediately after Mosher esterification, the samples were transferred into 3.0 mm NMR tubes. This was followed by measurements of 1H NMR -(+)- α-methoxy- α-(trifluoromethyl)phenylacetyl chloride. Evaluation of the obtained ΔδSR values was conducted as described by Hoye et al.  decane and [6.3.0] undecane core structures, respectively, were isolated from submerged cultures of Heimiomyces sp. (MUCL 56078). In association with the previously described hispidin (6), hypholomin B (7), heimiocalamenes C–E (8–10), and heimiomycins A–C (11–13), as well as bis-heimiomycins A–D (14–17), heimiomycins D–E (18–19), and heimiocalamenes A–B (20–21), it becomes clear that Heimiomyces sp. is capable of producing a chemically very diverse spectrum of secondary metabolites. On top of that, a high number of as yet unidentified minor peaks were observed in the UV/Vis- and MS-spectra of the extracts obtained from Heimiomyces sp., therefore especially this strain should be considered for further analysis of its secondary metabolism, particularly for cultivation in more different media. Since all isolated compounds did not show outstanding activities in the antimicrobial and cytotoxicity assays, they can be considered for other assays with different targets. However, our findings once again show the importance of examining unexplored species from the tropics for their secondary metabolism during the search for novel natural products.Five new terpenoids, namely heimionones A-E (


The results obtained from in vitro experiments confirmed the ability of compound 9a to reduce the intracellular pyruvate level in A549 lung cancer cells with simultaneous impact on cancer cell viability and cell-cycle phase distribution. Moreover, compound 9a exhibited more cytotoxicity on cancer cells than normal cells, pointing to high selectivity in the mode of action. These findings indicate that the introduction of another quinolinyl fragment to the modulator molecule may have a significant impact on pyruvate levels in cancer cells and provides further directions for future research to find novel analogs suitable for clinical applications in cancer treatment.Cancer cells need to carefully regulate their metabolism to keep them growing and dividing under the influence of different nutrients and oxygen levels. Muscle isoform 2 of pyruvate kinase (PKM2) is a key glycolytic enzyme involved in the generation of ATP and is critical for cancer metabolism. PKM2 is expressed in many human tumors and is regulated by complex mechanisms that promote tumor growth and proliferation. Therefore, it is considered an attractive therapeutic target for modulating tumor metabolism. Various modulators regulate PKM2, shifting it between highly active and less active states. In the presented work, a series of 8-quinolinesulfonamide derivatives of PKM2 modulators were designed using molecular docking and molecular dynamics techniques. New compounds were synthesized using the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. Compound  Cinchona L.), such as quinine and its right-handed stereoisomer quinidine. Quinidine has anti-arrhythmic activity, while quinine exhibits antimalarial activity and antipyretic, anti-inflammatory, and analgesic properties. Reports in the contemporary literature also describe the antiproliferative activity of quinine in relation to breast cancer cells (MCF-7) carbonyl}phenyl)quinoline-8-sulfonamide 1. Therefore, we synthesized a small series 8-quinolinesulfonamido-1,2,3-triazoles and identified compound with confirmed anticancer activity. To our knowledge, this represents the first successful attempt to click 8-sulfamoylquinoline with 1,2,3-triazoles as potent PKM2 modulators.As examples, quinoline sulfonamide derivatives belonging to the activator group ,19 or naThe aim of the present study was to discover novel PKM2 modulators among novel 8-quinolinesulfonamide derivatives. From an in-house library of quinolinesulfoamoyl compounds, five derivatives were selected for synthesis  using GOThe single monomer of pyruvate kinase M2 is made of 531 amino acids and is composed of four domains: A, B, C, and one small N-terminal domain. The dimeric form is formed between the A domains of each monomer, whereas the tetrameric form exists due to dimerization along with the C subunit interfaces of each dimer. Activators bind to the active binding site of PKM2 (dimer), which is located between two A domains and is surrounded by key amino acid residues such as Phe26, Gly315, Tyr390, Leu394, and Leu353. On the other hand, the ATP binding site is a druggable pocket for PKM2 inhibitors. The amino acid residues corresponding to this region are Thr50, Ile51, Gly52, Pro53, His78, and Tyr83 .In our in silico research, we used the PKM2 complex with an activator (PDB ID: 4G1N). At the same time, the PKM2 complex with ATP (PDB ID: 4FXF) was used to determine the potential inhibitory activity.1–6 and derivatives of quinolinesulfonamide 9a–e ranked by GOLD are presented in Pyruvate kinase M2 modulators In this case, the more negative the ∆G value of the binding reaction, the higher the binding affinity of the ligand for its specific target protein.9a–e show similar affinity values as the reference ligand 1 (NZT), with derivative 9a having the lowest ∆G value . It should be noted that the remaining reference ligands 2–6 show a significantly lower affinity in relation to ligands 1 and 9a–e. In the case of the 4FXF complex, the compound 9a shows the lowest ∆G value and the highest binding affinity to target protein . The remaining derivatives 9b–e also show a significantly higher affinity compared to the reference shikonin (5).Comparing the docking score values obtained for 4G1N, it can be concluded that compounds 1 (NZT) sits on a flat and mainly apolar surface defined by residues Phe26 and Leu394 from chain A and Phe26, Leu27, and Met 30 from chain B. One of the two oxygen atoms of the sulfonamide group accepts a hydrogen bond from the oxygen of Tyr390. Adding a methyl group to the sulfonamide nitrogen causes a steric clash with the oxygen of Leu353, and lack activity of methyl analogue of compound 1 carbonyl}phenyl)quinoline-8-sulfonamide 1  for the 4G1N complex. Default values of all other parameters were used, and the complexes were submitted to 100 genetic algorithm runs using the GOLDScore fitness function. After calculations, only the ten poses that scored the highest were returned as a docking result for the ligand–cavity configuration. All obtained results were ranked according to their score value and presented in GOLD arbitrary units (a.u.). Molecular docking details were visualized using the BIOVIA Discovery Studio virtual environment +, Calcd. 247.0541; Found 247.0553; C12H10N2NaO2S [M + Na]+, Calcd. 269.0361; Found 269.0362.Yield: 91 %, m.p. 127–128 °C. 8b)8-N-methyl-N-(prop-2-ynyl)quinolinesulfonamide : 2.02 , 3.00 , 4.40 , 7.52 , 7.63 dd, J = 8.1 Hz, J = 8.1 Hz, 1H, H-6), 8.05 , 8.25 , 8.49 , 9.06 . 13C NMR : 34.6 (C-9), 40.5 (C-10), 72.6 (C-12), 78.3 (C-11), 122.0 (C-3), 125.5 (C-6), 128.9 (C-4a), 133.1 (C-7), 133.6 (C-7), 136.4 (C-4), 136.8 (C-8), 144.2 (C-8a), 151.2 (C-2). HRMS (ESI) m/z: C13H13N2O2S [M + H]+, Calcd. 261.0698; Found 261.0699.Yield: 95 %, m.p. 111–112 °C. Procedure A:8a or 8b) in 5 mL of DMF was added 0.75 mmol of the 4-azido-7-chloroquinoline or 3,28-di-O-acetyl-30-azidobetulin. Meanwhile, solutions were prepared: 0.02 g, 0.10 mmol of sodium ascorbate in 0.5 mL of H2O and 0.0125 g (0.05 mmol) of CuSO4 · 5H2O in 0.5 mL of H2O. The aqueous solutions were then mixed and added to the DMF solution of the starting materials. The mixture was stirred overnight at room temperature, and then the contents of the flask were poured into 50 mL of water and filtered. Products 9a or 9e were purified by column chromatography with SiO2 and ethyl acetate was used as mobile phase.To a solution of 0.5 mmol of the respective sulfonamide (9a)8-N-{methyl}quinolinesulfonamide : 4.58 , 7.68 , 7.90–8.02 , 8.38 , 8.44–8.51 , 8.63 , 9.23 , 9.27 . 13C NMR  d: 38.7 (C-10), 117.0 (C-3′), 120.4 (C-4a’), 122.9 (C-3), 125.8 (C-5′), 126.1 (C-6 and C-12), 128.6 (C-8′), 128.7 (C-4a), 129.3 (C-6′), 131.1 (C-7), 134.1 (C-5), 135.9 (C-7′), 137.1 (C-8), 137.4 (C-4), 140.4 (C-4′), 143.0 (C-8a), 144.4 (C-11), 149.8 (C-8a’), 151.7 (C-2), 152.8 (C-2′). HRMS (ESI) m/z: C21H16ClN6O2S [M + H]+, Calcd.451.0744; Found 451.0741.Yield: 90 %, m.p. 181–182 °C, t9e)8-N-methyl-N--en-30-yl]-1H-1,2,3-triazol-4-yl}methyl)-quinolinesulfonamide : 0.79–0.87 , 0.98 , 1.04 , 1.05–1.32 , 1.40–1.50 , 1.63–1.89 , 2.05 , 2.08 , 2.27-2.38 , 2.89 , 3.50 , 3.74 , 4.24-4.63 , 4.85-5.05 , 7.56 , 7.66 , 7.71 , 8.07 , 8.30 , 8.51 , 9.09 . 13C NMR  d: 14.7 (C-27′), 16.0 (C-24′), 16.2 (C-26′), 16.5 (C-25′), 18.1 (C-6′), 20.9 (C-11′), 21.1 (C-32′), 21.4 (C-34′) 23.7 (C-2′), 26.8 (C-12′), 27.0 (C-15′), 28.0 (C-23′), 29.8 (C-16′), 31.2 (C-21′), 34.1 (C-7′), 34.4 (C-22′), 35.0 (C-9), 37.1 (C-10′), 37.4 (C-13′), 37.8 (C-4′), 38.4 (C-1′), 40.9 (C-8′), 42.7 (C-14′), 43.9 (C-19′), 46.4 (C-17′), 46.7 (C-9), 49.9 (C-18′), 50.1 (C-9′), 54.6 (C-30′), 55.3 (C-5′), 62.4 (C-28′), 80.9 (C-3′), 122.2 (C-3), 123.5 (C-12), 125.6 (C-6), 129.0 (C-4a), 133.2 (C-7), 133.6 (C-5), 136.6 (C-4), 136.9 (C-8), 144.1 (C-8a), 145.5 (C-11), 148.8 (C-20′), 151.3 (C-2), 171.0 (C-33′), 171.5 (C-31′). HRMS (ESI) m/z: C47H66N5O6S [M + H]+, Calcd. 828.4734; Found 828.4732.Yield: 71 %, m.p. 136–137 °C, t2O and 0.0125 g (0.05 mmol) of CuSO4 · 5H2O in 0.5 mL of H2O. The aqueous solutions were then mixed and added to the DMF solution of the starting materials. The mixture was stirred overnight at room temperature, and then the contents of the flask were poured into 50 mL of water and filtered. Products 9b–d were purified by column chromatography with SiO2, and ethyl acetate was used as mobile phase.To a solution of 0.5 mmol of the respective bromide in 5 mL of DMF, 0.036 g (0.55 mmol) of the sodium azide was added. The mixture was stirred overnight at room temperature, and then respective sulfonamide (0.5 mmol) was added. Meanwhile, solutions were prepared: 0.02 g (0.10 mmol) of sodium ascorbate in 0.5 mL of H9b)Diethyl 2-{4-}ethylphosphonate : 1.20 , 2.20 , 3.96 , 4.17 , 4.25 , 7.62 , 7.68-7.76 , 8.25-8-29 , 8.52 , 9.05 . 13C NMR  d:16.6 , 26.4 , 39.0 (C-10), 44.0 (C-13), 61.8 , 122.9 (C-3), 123.6 (C-12), 126.1 (C-6), 128.8 (C-4a), 131.0 (C-7), 134.0 (C-5), 137.0 (C-8), 137.4 (C-4), 143.0 (C-8a), 143.8 (C-11), 151.7 (C-2). HRMS (ESI) m/z: C18H24N5NaO5PS [M + Na]+, Calcd. 476.1133; Found 476.1136.Yield: 85 %, oil, t9c)Ethyl 3-{[4-(8-sulfamoylquinolyl)methyl]-1H-1,2,3-triazol-1-yl}propanoate : 1.20 , 2.77 , 4.04 , 4.17 , 4.34 , 7.66-7.74 , 8.25-8.31 , 8.52 , 9.05 . 13C NMR  d:14.5 (C-17), 34.3 (C-13), 39.0 (C-10), 45.3 (C-14), 60.8 (C-16), 122.9 (C-3), 123.7 (C-12), 126.1 (C-6), 128.8 (C-4a), 131.0 (C-7), 134.0 (C-5), 137.0 (C-8), 137.4 (C-4), 143.0 (C-8a), 143.8 (C-11), 151.7 (C-2), 170.7 (C-15). HRMS (ESI) m/z: C17H20N5O4S [M + H]+, Calcd. 390.1236; Found 390.1239.Yield: 81 %, oil, t9d)8-N-{methyl}quinolinesulfonamide : 0.83 , 1.06 , 1.50 , 4.07 , 4.18 , 7.56 , 7.64-7.73 , 8.24-8.32 , 8.51 , 9.04 . 13C NMR  d:13.7 (C-16), 19.4 (C-15), 32.0 (C-14), 390 (C-10), 49.1 (C-13), 122.9 (C-3), 123.2 (C-12), 126.1 (C-6), 128.8 (C-4a), 130.9 (C-7), 134.0 (C-5), 137.0 (C-8), 137.4 (C-4), 143.0 (C-8a), 143.6 (C-11), 151.7 (C-2). HRMS (ESI) m/z: C16H20N5O2S [M + H]+, Calcd. 346.1338; Found 346.1336.Yield: 94 %, m.p. 80-81 °C, tDulbecco’s phosphate-buffered saline, Halt Protease Inhibitor Cocktail, Halt Phosphatase Inhibitor Single-Use Cocktail, Pierce BCA Protein Assay Kit, and Trypsin/EDTA solution were purchased from Thermo Fisher Scientific . Amphotericin B, penicillin G, and RIPA Buffer were purchased from Sigma-Aldrich Inc. . NC-Slides A8, Solution 3, and Via1-Cassettes were obtained from ChemoMetec . Growth media (DMEM and RPMI 1640) and fetal bovine serum were acquired from CytoGen . Neomycin sulfate was obtained from 1Amara . The remaining chemicals were purchased from POCH S.A. .2 incubator.All tested cell lines were purchased from ATCC (USA). C32 cells (human amelanotic melanoma), MDA-MB-321 cells (human breast carcinoma), U87-MG cells (human glioblastoma), and A549 cells (human lung carcinoma) were cultured in DMEM medium. COLO 829 cells (human melanotic melanoma) were cultured in RPMI 1640 medium. Both media were supplemented with 10% fetal bovine serum, penicillin (100 U/mL), neomycin (10 µg/mL), and amphotericin B (0.25 µg/mL). Cells were maintained at 37 °C in a 5% CO9a–e on C32, COLO 829, MDA-MB-321, U87-MG, and A549 cell viability was assessed using a WST-1 reagent  according to the manufacturer’s protocol. In brief, cells were seeded into 96-well microplates (2.5 × 103 cells/well) and grown for 24 h. Then, the cells were treated with the compounds in the concentration range from 0.1–200 µg/mL The impact of compounds 50 values were extrapolated from the obtained cell viability data using GraphPad Prism software.(0.444 mM) for 72 h. WST-1 reagent was added 2 h before the end of the incubation time. The absorbance was measured at 440 nm (with reference wavelength of 650 nm) using a microplate reader Infinite 200 Pro. GI9a on pyruvate kinase activity was determined using commercially available enzyme assay kit: Pyruvate Assay Kit , according to the manufacturer’s instruction. In brief, pyruvate concentration in cell lysates obtained from A549 cells treated with compound 9a  for 72 h was measured by a coupled enzyme assay, which resulted in a fluorometric (λex = 535/λem = 587 nm) product, proportional to the pyruvate present. The fluorescence of the samples was measured using a microplate reader Infinite 200 Pro. The results were calculated per mg of protein and expressed as percentage of the untreated control.The impact of After treatment of A549 cells with 9a in a concentration 200 μg/mL (0.444 mM) for 72 h, the cells were analyzed in terms of cell cycle progression using the fluorescence image cytometer NucleoCounter NC-3000 , according to the previously described method . In brie9a, in contrast to other studied derivatives, was characterized by an improved profile against the tumor-cell-specific M2 isoform of pyruvate kinase as a biological pathway target, and thus exerted ability to reduce lung cancer cell viability and apoptosis induction. Moreover, we demonstrated that the introduction of another quinolinyl fragment to the modulator molecule may have a significant impact on the greater activity of the 9a derivative compared to other compounds. In addition, it may comprise the basis and further directions for extensive research to find novel analogs suitable for clinical applications in cancer treatment.Herein, for the first time, we synthesized new quinoline-8-sulfonamide derivatives with the capacity to inhibit the tumor-cell-specific M2 isoform of pyruvate kinase. Basing on both in silico and in vitro studies, the compound