N-acyl-α-amino acids and N-acyl-α-amino ketones classes, and heterocycles from the large family of 1,3-oxazole-based compounds, were synthesized. The structures of the new compounds were established using elemental analysis and spectral  data, and their purity was checked by reversed-phase HPLC. The newly synthesized compounds were evaluated for their antimicrobial and antibiofilm activities, for toxicity on D. magna, and by in silico studies regarding their potential mechanism of action and toxicity. The 2-aza-3-isopropyl-1-[4-(phenylsulfonyl)phenyl]-1,4-butanedione 4b bearing a p-tolyl group in 4-position exhibited the best antibacterial activity against the planktonic growth of both Gram-positive and Gram-negative strains, while the N-acyl-α-amino acid 2 and 1,3-oxazol-5(4H)-one 3 inhibited the Enterococcus faecium biofilms. Despite not all newly synthesized compounds showing significant biological activity, the general scaffold allows several future optimizations for obtaining better novel antimicrobial agents by the introduction of various substituents on the phenyl moiety at position 5 of the 1,3-oxazole nucleus.In order to develop novel bioactive substances with potent activities, some new valine-derived compounds incorporating a 4-(phenylsulfonyl)phenyl fragment, namely, acyclic precursors from  Many heterocyclic compounds are very important in medicinal chemistry since they exhibit remarkable and various pharmacological activities, being present as active substances in the composition of numerous potent drugs.Among them, the synthetic heterocycles containing 1,3-oxazole nucleus have a wide range of biological activities, such as antimicrobial  , antican2 (from group A streptogramin antibiotics) phenyl}-1,3-benzoxazole-4-carboxylic acid A-33853) and of a number of its analogues, and discovered novel antileishmanial chemotypes . In addi53 and oftivities .N-acyl-α-amino acid, 1,3-oxazol-5(4H)-one, N-acyl-α-amino ketone, and 1,3-oxazole analogs derived from valine containing the 4-(phenylsulfonyl)phenyl substituent.The aim of the present work was to continue our previous research on 1,3-oxazole derivatives ,43,44,452–5 were prepared using the multi-step synthetic strategy presented in The new compounds 1 [N-acylation of L-valine -2-amino-3-methylbutanoic) to new 3-methyl-2-[4-(phenylsulfonyl)benzamido]butanoic acid 2. Intramolecular cyclodehydration of compound 2, using ethyl chloroformate in the presence of 4-methylmorpholine (in the molar ratio of 1:1:1), in anhydrous dichloromethane, at room temperature, led to 4-isopropyl-2-[4-(phenylsulfonyl)phenyl]-1,3-oxazol-5(4H)-one 3. Friedel–Crafts acylation, catalyzed by aluminum trichloride of aromatic hydrocarbons  with 2-aryl-4-isopropyl-1,3-oxazol-5(4H)-one 3 yielded the N-(1-aryl-3-methyl-1-oxobutan-2-yl)-4-(phenylsulfonyl)benzamides 4a,b. These intermediates underwent Robinson–Gabriel cyclization using phosphoryl trichloride at reflux with the preparation of 5-aryl-4-isopropyl-2-[4-(phenylsulfonyl)phenyl]-1,3-oxazoles 5a,b. Elemental and spectral  analyses were used to characterize the new compounds.Synthesis of the new compounds started from the known acyl chloride 1 ,46, whic2–5 showed the E band at λmax = 202.6 nm and the B band in the range of 241.0–252.0 nm. Furthermore, 2,5-diaryl-4-isopropyl-1,3-oxazoles 5a,b spectra presented a third absorption band at a longer wavelength: 331.3 (5a) or 336.6 (5b) nm, due to the appearance of the 1,3-oxazole chromophore, which determined the extension of the π electrons conjugation.The UV-Vis spectra of new compounds 2 and 4a,b, a characteristic absorption band due to N-H stretching vibration, ν(N-H), was registered in the range of 3302–3424 cm−1. In the IR spectrum of N-acyl-α-amino acid 2, the peak due to carbonyl valence vibration, ν(O=C-C), was recorded at 1720 cm−1, and amidic carbonyl absorption, ν(O=C-N), at 1674 cm−1. These two carbonyl absorption bands are overlapped in the case of N-acyl-α-amino ketones 4a,b, as suggested by the single very strong peak present in their spectra at 1657 (4b) or 1662 (4a) cm−1. Representative bands for the N-acyl-L-valine 2 associated by hydrogen bonds are also: a strong, broad absorption peak between 2500 and 3300 cm−1, and two medium broad bands at 2686 and 2609 cm−1 due to O-H stretching vibration, ν(O-H).For acyclic intermediates 3 and 5a,b differ significantly from the corresponding spectra of their open-chain precursors  and this demonstrates that intramolecular cyclocondensation reactions occurred. In the IR spectrum of saturated azlactone 3, the band due to carbonyl valence vibration, ν(C=O), is shifted at a higher wavenumber (1827 cm−1), compared with the corresponding absorption band in the compound 2 spectrum. The IR spectra of heterocycles 3 and 5a,b presented a peak due to C=N valence vibration, ν(C=N), at 1649 cm−1 (3), and 1597 (5b) or 1603 (5a) cm−1. Moreover, the band due to C-O-C symmetrical stretching vibration, νsym(C-O-C), was registered at 1037 cm−1 in the 1,3-oxazol-5(4H)-one 3 spectrum, and at 1095 (5b) or 1098 (5a) cm−1 in 1,3-oxazoles spectra. In the spectrum of 3, the peak due to C-O-C asymmetrical stretching vibration, νas(C-O-C), was observed at 1244 cm−1, while in compounds 5a,b spectra, it is overlapped with the asymmetric sulfonyl stretch, νas(SO2) from 1292 (5b) and 1294 cm−1 (5a), respectively.The IR spectra of heterocyclic compounds The NMR spectral data also proved the structures of the new compounds .2–5 is presented in The numbering of atoms used for assigning the NMR signals of the compounds 1H-NMR spectra of new compounds 2 and 4a,b, for the deshielded proton of NH group, respectively H-3, the signal was registered as a doublet at δH = 8.38 ppm (2) and 8.96 (4b) or 8.99 (4a) ppm, due to the vicinal coupling to H-4. In the 1H-NMR spectrum of the N-acyl-α-amino acid 2, a doublet of doublets signal at 4.26 ppm was assigned to the H-4 proton, coupled to the proton of the NH group and the H-18 methine proton. In the case of N-acyl-α-amino ketones 4a,b, the H-4 proton signal appeared as a triplet at 5.35 (4b) or 5.38 (4a) ppm, due to the coupling to H-3 and H-18. In the 1H-NMR spectra of acyclic intermediates 2 and 4a,b, for the isopropyl group, an octet or multiplet signal was recorded in the 2.12–2.26 ppm range due to the proton of the methine group (H-18) and two strongly shielded doublet signals at δH = 0.89  or 0.90 (4a) ppm and in the interval: 0.90–0.92 ppm, respectively, corresponding to the H-19 and H-20 nonequivalent protons from the two methyl groups.In 2 and 4a,b is represented by the absence in 1H-NMR spectra of heterocyclic compounds 3 and 5a,b of the signal assigned to the NH proton.A proof for the intramolecular cyclocondensations of precursors H)-one 3, the H-4 signal was recorded at 4.32 ppm as a doublet due to coupling to the adjacent methine proton and the signal of H-18 at 2.39 ppm as a heptet of doublets, this proton being coupled to H-19 and H-20 protons (with 3J = 6.9 Hz) and H-4 (with 3J = 4.7 Hz). In the 1H-NMR spectra of 1,3-oxazoles 5a,b, for the isopropyl group, a heptet signal appeared at δH = 3.26 (5b) or 3.28 (5a) ppm due to the H-18 proton , and a strongly shielded doublet at δH = 1.35 (5b) or 1.36 (5a) ppm, corresponding to protons of the two CH3 groups.In the case of 4-isopropyl-1,3-oxazol-5-one 3, the C-2 atom resonated at 160.44 ppm, being more shielded with 5.43 ppm than the corresponding carbon of intermediate 2, and the C-5 at 177.05 ppm, being shifted downfield with 4.24 ppm than the corresponding atom of 2.The C-4 signal was shifted downfield with 12.59 ppm after cyclodehydration of compound 13C-NMR spectra of 1,3-oxazoles 5a,b, the C-4 signal registered at δC = 143.56 (5b) or 144.15 (5a) ppm was more deshielded with ≈84.70 ppm by comparison of the signal of the corresponding carbon atom of 4a,b observed at δC = 59.09 (4b) or 59.22 (4a) ppm and this is an additional indication that the formation of the 1,3-oxazole ring took place. The signal attributed to C-2 of 1,3-oxazoles 5a,b was recorded at δC = 157.65 (5b) or 157.93 (5a) ppm, while the corresponding carbon signal of N-acyl-α-amino ketones 4a,b appeared at δC = 165.61 (4b) or 165.65 (4a) ppm. The C-5 atom of 1,3-oxazoles 5a,b resonated at δC = 145.41 (5a) or 145.60 (5b) ppm, whereas the corresponding carbon of 4a,b at δC = 198.66 (4b) or 199.22 (4a) ppm, revealing an upfield shift for this carbon atom, as a confirmation that the intramolecular cyclocondensation occurred.In the 2, 3, 4a, and 5a.The mass spectra recorded by the L-ESI-MS/MS technique had an additional contribution to the elucidation of the structures of compounds In this study was used the ability of ionization at atmospheric pressure of electrospray ionization (ESI) type for amino acid derivatives analysis. Further, a special advantage of the ESI source is that negative ions can also be analyzed.2 and 4a, dissolved in methanol/water with 0.1% ammonium carbonate , were ionized positively and negatively, respectively. For compounds 3 and 5a, methanol/water (containing 0.1% ammonium formate and 1% formic acid) 9:1 (v/v) was used as the solvent mixture. After evaporation of the solvents, the ions obtained were introduced into the mass spectrometer.In our experiments, compounds + for positive ionization or the deprotonated molecular ion [M−H]- for negative ionization. Non-covalent dipole–dipole interactions can be highlighted by using ESI so that in the compounds spectra appeared combinations like [2M + H]+ and [2M−H]−, respectively .ESI is considered a mild source of ionization. Consequently, the mass spectra of these compounds are very simple and consist mainly of the protonated molecular ion [M + H]2), carbon monoxide (for 3), or methane loss (for 5a), and in the negative ionization mode with carbon dioxide (for 2) or isopropyl radical loss (for 4a). Protonated and/or deprotonated molecular ions and the main fragments of these compounds are reported in the Materials and Methods section.Valuable structural information can be obtained from the fragmentation by the collision of pseudo-molecular ions with an inert gas (argon), as fragmentation in positive-ion mode can be different from fragmentation in negative-ion mode. In the positive ionization mode, the first fragmentation occurred with water (for H)-one 3 which was also achieved by GC-EI-MS analysis, the molecular ion, [M]+, being unstable, did not show a signal. 1,3-Oxazol-5(4H)-one 3 was first fragmented at the side chain from 4-position with the removal of a propene molecule and the formation of cation-radical with m/z = 301, which is the base peak (BP). Other main fragments of 3 are indicated in Materials and Methods.In the mass spectrum of 1,3-oxazol-5 +; 344 (14.9) [M+H-H2O]+; 316  [M+H-H2O-CO]+; 245 (15.4) [C6H5SO2C6H4CO]+.+ESI-MS/MS : 360  [M-H]-; 316 (73.1) [M-H-CO2]−; 288 (10.1) [M-H-CO2-C2H4]−; 245 (5.2) [C6H5SO2C6H4CO]−; 217 (10.3) [C6H5SO2C6H4]−.−ESI-MS/MS : purity = 99.99%; tR = 3.78 min.RP-HPLC : C, 59.82; H, 5.30; N, 3.88; S, 8.87. Found: C, 59.87; H, 5.28; N, 3.89; S, 8.85.Elemental analysis (%): Calculated for C2  was suspended at room temperature, under magnetic stirring in 50 mL anhydrous dichloromethane and an equimolar quantity of 4-methylmorpholine  was added. Ethyl chloroformate  was then added slowly to the formed solution. The reaction mixture was further stirred at room temperature for another 30 min and then poured over 100 mL of the ice–water mixture. The organic phase was separated and washed with 5% sodium hydrogen carbonate solution, then with water and dried over anhydrous magnesium sulfate. After concentration by distillation under reduced pressure, the solid product was purified by recrystallization from cyclohexane as white crystals; yield = 90% (3.24 g); m.p. = 115–117 °C.3-Methyl-2-[4-(phenylsulfonyl)benzamido]butanoic acid 3OH, λ nm) (lg ε): 202.6 (4.48); 247.6 (4.32).UV-Vis (CHν cm−1): 3093w; 3066m; 2964s; 2931m; 2875m; 1827vs; 1649vs; 1599w; 1584w; 1570m; 1492w; 1466m; 1447s; 1326vs; 1309vs; 1295vs; 1244s; 1159vs; 1037vs; 843s.FT-IR : 0.99 ; 1.14 ; 2.39 ; 4.32 ; 7.54 ; 7.61 ; 7.90 ; 8.07 ; 8.15 .13C-NMR : 17.65 (C-19); 18.89 (C-20); 31.40 (C-18); 71.02 (C-4); 127.99 ; 128.18 ; 128.87 ; 129.63 ; 130.32 (C-6); 133.82 (C-15); 140.85 (C-12); 145.38 (C-9); 160.44 (C-2); 177.05 (C-5).m/z, rel. abund. %): 344 [M+H]+; 316  [M+H-CO]+; 245 (19.0) [C6H5SO2C6H4CO]+.+ESI-MS/MS : 301  [M-C3H6]+; 245 (37.71) [C6H5SO2C6H4CHNH]+ or [C6H5SO2C6H4CO]+; 218 (17.37) [C6H5SO2C6H5]+; 152 (6.57); 132 (12.92); 125 (38.56) [C6H5SO]+; 104 (7.42) [C6H4CHNH]+; 97 (4.24) [C3H7CNCO]+; 77 (25.85) [C6H5]+; 76 (9.75) [C6H4]+; 51 (10.17) [C4H3]+; 44 (17.80) [C3H8]+ or [CO2]+; 43 (21.82) [C3H7]+; tR = 26.25 min.GC-EI-MS : purity = 91.85%; tR = 3.47 min.RP-HPLC : C, 62.96; H, 4.99; N, 4.08; S, 9.34. Found: C, 62.89; H, 4.95; N, 4.12; S, 9.28.Elemental analysis (%): Calculated for CH)-one 3  in 25 mL of anhydrous aromatic hydrocarbon (benzene or toluene). Stirring was continued for 20 h until hydrogen chloride emission ceased, then the reaction mixture was poured over 100 mL of ice water acidified with 5 mL of 37% hydrochloric acid. The precipitate was filtered off, washed with cold water, then with a cold mixture of ethanol–water . The aqueous phase was extracted with 2 × 15 mL dichloromethane. Combined organic layers were washed with water, dried over anhydrous sodium sulfate, and concentrated by distillation under reduced pressure, leaving the second fraction of the raw product. Purification by recrystallization from ethanol leads to compound 4 as colorless crystals.An excess amount of anhydrous aluminum trichloride  was added in portions, under stirring, at room temperature to crude 4-isopropyl-2-[4-(phenylsulfonyl)phenyl]-1,3-oxazol-5; m.p. = 162–164 °C.Compound 3OH, λ nm) (lg ε): 202.6 (4.49); 246.7 (4.19).UV-Vis (CHν cm−1): 3304m; 3088w; 3061m; 3039w; 2970m; 2935m; 2878m; 1662vs; 1595m; 1570m; 1528s; 1486m; 1468m; 1447m; 1324s; 1310m; 1300s; 1162s; 857m.FT-IR : 0.90 ; 0.92 ; 2.23 ; 5.38 ; 7.53 ; 7.63 ; 7.64 ; 7.71 ; 7.99 ; 8.02 ; 8.05 ; 8.07 ; 8.99 .13C-NMR : 18.37 (C-19); 19.74 (C-20); 29.51 (C-18); 59.22 (C-4); 127.47 ; 128.26 ; 128.83 ; 128.99 ; 129.85 ; 133.46 (C-24); 133.98 (C-15); 136.18 (C-21); 138.63 (C-6); 140.66 (C-12); 143.28 (C-9); 165.65 (C-2); 199.22 (C-5).m/z, rel. abund. %): 843 [2M+H]+; 422 [M+H]+; 245  [C6H5SO2C6H4CO]+; 161 (36.4) [C6H5COCHCH(CH3)2]+; 125 (65.8) [C6H5SO]+; 118 (15.7) [C6H5COCH]+; 105 (11.3) [C6H5CO]+.+ESI-MS/MS : 841 [2M-H]−; 420 [M-H]−; 377  [M-H-C3H7]−; 300 (22.3) [M-H-C3H7-C6H5]−; 217 (36.0) [C6H5SO2C6H4]−; 141 (57.5) [C6H5SO2]−.−ESI-MS/MS : purity = 94.95%; tR = 3.90 min.RP-HPLC : C, 68.39; H, 5.50; N, 3.32; S, 7.61. Found: C, 68.45; H, 5.48; N, 3.31; S, 7.64.Elemental analysis (%): Calculated for C4b was obtained by reaction of 3 with toluene; yield = 74% (1.61 g); m.p. = 155–156 °C.Compound 3OH, λ nm) (lg ε): 202.6 (4.49); 252.0 (4.21).UV-Vis (CHν cm−1): 3302s; 3091w; 3064w; 2965m; 2933w; 2875w; 1657vs; 1605s; 1570m; 1532s; 1484m; 1466m; 1447m; 1319s; 1308s; 1294s; 1158vs; 847m.FT-IR : 0.89 ; 0.91 ; 2.26 ; 2.36 ; 5.35 ; 7.33 ; 7.63 ; 7.71 ; 7.96 ; 7.98 ; 8.02 ; 8.07 ; 8.96 .13C-NMR : 18.40 (C-19); 19.77 (C-20); 21.20 (CH3); 29.62 (C-18); 59.09 (C-4); 127.50 ; 128.44 ; 129.01 ; 129.42 ; 129.89 ; 133.66 (C-21); 134.01 (C-15); 138.69 (C-6); 140.70 (C-12); 143.29 (C-24); 143.98 (C-9); 165.61 (C-2); 198.66 (C-5).v/v; 1 mL/min; 250 nm): purity = 92.70%; tR = 4.08 min.RP-HPLC : C, 68.94; H, 5.79; N, 3.22; S, 7.36. Found: C, 68.91; H, 5.73; N, 3.19; S, 7.33.Elemental analysis (%): Calculated for CN-(1-aryl-3-methyl-1-oxobutan-2-yl)-4-(phenylsulfonyl)benzamide 4 (10 mmol) in phosphoryl trichloride  was refluxed for 4 h. Excess of POCl3 was distilled under vacuum. The oily residue was slowly and carefully poured into a crushed ice–water mixture and extracted twice with dichloromethane (20 mL). Combined organic phases were washed with 5% sodium hydrogen carbonate solution, then with water and dried over anhydrous sodium sulfate. After removal of the solvent by distillation under reduced pressure, crude solid 5 was purified by recrystallization from ethanol, as colorless crystals.Raw 5a was obtained from 4.22 g of 4a; yield = 79% (3.19 g); m.p. = 168–169 °C.Compound 3OH, λ nm) (lg ε): 202.6 (4.48); 241.4 (4.06); 331.3 (4.23).UV-Vis (CHν cm−1): 3085w; 3055m; 2966m; 2929m; 2871m; 1603m; 1589m; 1547w; 1495m; 1483m; 1465m; 1447s; 1323s; 1312s; 1294s; 1160vs; 1098s; 842m.FT-IR : 1.36 ; 3.28 ; 7.37 ; 7.47 ; 7.54 ; 7.58 ; 7.64 ; 7.97 ; 8.02 ; 8.21 .13C-NMR : 22.07 ; 26.12 (C-18); 126.26 ; 127.01 ; 127.78 ; 128.28 ; 128.36 (C-24); 128.90 (C-21); 129.01 ; 129.49 ; 132.11 (C-6); 133.46 (C-15); 141.54 (C-9); 142.29 (C-12); 144.15 (C-4); 145.41 (C-5); 157.93 (C-2).m/z, rel. abund. %): 404 [M+H]+; 388 (13.1) [M+H-CH4]+; 262 (42.0) [C6H5SO2C6H4CONH3]+; 248 (30.6) [M+H-CH4-C6H5SO2+H]+; 245 (38.1) [C6H5SO2C6H4CO]+; 125  [C6H5SO]+.+ESI-MS/MS : purity = 99.99%; tR = 5.08 min.RP-HPLC : C, 71.44; H, 5.25; N, 3.47; S, 7.95. Found: C, 71.39; H, 5.23; N, 3.48; S, 7.93.Elemental analysis (%): Calculated for C5b was obtained from 4.36 g of 4b; yield = 76% (3.17 g); m.p. = 206–207 °C.Compound 3OH, λ nm) (lg ε): 202.6 (4.47); 243.2 (4.07); 336.6 (4.17).UV-Vis (CHν cm−1): 3055m; 3030m; 2970m; 2932m; 2872m; 1597s; 1547m; 1508s; 1481m; 1464m; 1450s; 1322vs; 1292s; 1157vs; 1095vs; 842m.FT-IR : 1.35 ; 2.40 ; 3.26 ; 7.27 ; 7.52 ; 7.53 ; 7.58 ; 7.96 ; 8.02 ; 8.20 .13C-NMR : 21.46 (CH3); 22.07 ; 26.08 (C-18); 126.20 ; 126.93 ; 127.76 ; 128.25 ; 129.48 ; 129.69 ; 132.17 (C-6); 133.45 (C-15); 138.43 (C-21); 141.54 (C-9); 142.11 (C-12); 143.56 ; 145.60 (C-5); 157.65 (C-2).v/v; 1 mL/min; 335 nm): purity = 99.99%; tR = 5.70 min.RP-HPLC : C, 71.92; H, 5.55; N, 3.35; S, 7.68. Found: C, 71.98; H, 5.53; N, 3.36; S, 7.68.Elemental analysis (%): Calculated for CThe antimicrobial activity of the compounds was investigated using the agar disc-diffusion method, broth microdilution, and microtiter plate assay.Bacillus subtilis 6683, Staphylococcus aureus ATCC 6538, Enterococcus faecium E5, two Gram-negative bacteria: Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 27853, and a fungal strain: Candida albicans 393.Microbial strains used in this study included three Gram-positive bacteria: The qualitative assessment of the antimicrobial activity of the tested compounds was performed using the agar disc-diffusion method. The Mueller Hinton (MH) agar plates are inoculated with a standardized microbial inoculum prepared in phosphate-buffered saline and adjusted to 0.5 McFarland scale. Then, five microliters of the solution of the tested compound (of 5000 µg/mL concentration in DMSO) were pipetted on the inoculated agar surface. After incubation of the plates for 24 h at 37 °C, the diameters of inhibition growth zones were measured.2 and 3 were achieved in sterile liquid culture medium; the obtained tubes were inoculated with 0.5 McFarland suspensions obtained from the 24 h microbial culture of E. faecium E5. A control of DMSO  and control of microbial growth  were also prepared. The inoculated tubes were incubated at 37 °C for 24 h. The microbial cultures obtained in the presence of subinhibitory concentrations of tested compounds 2 and 3, DMSO and control cultures, respectively, were used to determine the influence of the tested compounds on the antibiotic susceptibility spectrum of the studied strain. Thus, the liquid microbial cultures were sedimented by centrifugation at 10,000× g for 5 min, and the obtained cellular sediment was washed 3 times in sterile saline by centrifugation at 10,000× g for 5 min. The cell pellet was resuspended in sterile saline until a turbidity corresponding to the 0.5 McFarland standard was obtained and the classical Kirby–Bauer method was then performed to assess the susceptibility to the following antibiotics : ampicillin, penicillin, linezolid, vancomycin for the E. faecium E5 strain. The results were recorded after 24 h of incubation at 37 °C. The diameters of the areas of inhibition of bacterial growth were interpreted according to the recommendations of the current edition of the Clinical and Laboratory Standards Institute (CLSI).Subinhibitory concentrations (250 µg/mL) of tested compounds Quantitative assessment of the antimicrobial activity of the tested compounds was performed using broth microdilution, in 96-well microtiter plates. The tested concentrations of the solutions of the different compounds in DMSO achieved through double serial dilutions, in columns 1–10, were between 500–0.97 µg/mL. Then, the wells were inoculated with 10 µL microbial suspension prepared in the same medium after dilution (1:100) of standardized microbial inoculum adjusted to 0.5 McFarland scale. Column 11 contained 10 µL of standardized inoculum and 90 μL of Mueller Hinton Broth, and column 12 contained 100 µL of Mueller Hinton Broth (as a control to monitor sterility). Ciprofloxacin  and fluconazole (Sigma–Aldrich) served as positive controls. The microtiter plates were incubated without agitation for 24 h at 37 °C. In order to confirm the MIC value, the assays were performed in triplicate. The MIC was determined as the lowest concentration of tested compound that inhibited the growth of the microorganism as detected spectrophotometrically at 620 nm with an Apollo LB 911 ELISA Reader  .Evaluation of the antibiofilm activity of tested compounds was carried out using the microtiter biofilm inhibition assay. Briefly, in a 96-well polystyrene microtiter plate containing 90 μL of Mueller Hinton Broth, 90 μL of the tested compound solution was added in column 1. Serial two-fold dilutions were performed in columns 1–10. A volume of 10 μL microbial suspension , prepared from an overnight culture grown in TSB into MH broth, was added. Column 11 contained 10 µL of standardized inoculum and 90 μL of Mueller Hinton Broth, and column 12 contained 100 µL of Mueller Hinton Broth (as a control to monitor sterility). Ciprofloxacin (Sigma–Aldrich) and fluconazole (Sigma–Aldrich) served as positive controls. Microplates were incubated for 24 h at 37 °C under static conditions to allow for microbial growth and biofilm maturation. The wells of the microplate were emptied and washed twice with phosphate-buffered saline. The biofilms formed on the walls of wells of the microplate were fixed with methanol for 5 min and stained with 1% crystal violet solution for 15 min, then rinsed three times with distilled water to remove the unbound dye. The fixed dye was resuspended in 33% acetic acid and the A492 was recorded with an Apollo LB 911 ELISA Reader. The amount of biofilm inhibition was calculated relative to the amount of biofilm that was grown in the absence of the tested compound and the media sterility control. The minimal biofilm eradication concentration (MBEC) was determined to be the lowest concentration of the tested compounds at which the decrease in absorbance value, measured at 492 nm, was observed in comparison to the positive control. Results from at least three separate biological replicates were averaged .D. magna Straus was maintained parthenogenetically  at 25 °C with a photoperiod of 16 h/8 h light/dark cycle in a Sanyo MLR-351 H climatic chamber . With 24 h prior to the determination, young daphnids were selected according to their size and maintained for 24 h in an artificial medium. Ten daphnids/replicate were used, and the determination was performed in tissue culture plates with 12 wells (Greiner Bio-One) [L-Valine—control 1 (2.5–50 µg/mL) and 4-(phenylsulfonyl)benzoic acid—control 2 (1.1–22 µg/mL) were used as positive controls, and a 1% DMSO solution as a negative control. The concentration ranges were selected based on the solubility and a pre-screening assay. All determinations were performed in duplicate. The lethality was evaluated after 24 and 48 h of exposure. LC50 and 95% confidence intervals (95% CI) were calculated using the least square fit method. All calculations were performed using GraphPad Prism v 5.1 software . Freely available online GUSAR software  was used to predict the LC50 values for 48 h exposure of the new compounds [Bio-One) ,59. Eachompounds .A virtual screening was performed using the software PASS (Prediction of Activity Spectra for Substances), an application designed to evaluate the pharmacological potential of newly synthesized compounds. The structures were inputted in PASS as SMILES and the results were analyzed if the Pa values were above the corresponding Pi values.A similarity search was performed on the ChEMBL database for the newly synthesized compounds using a 50% threshold . The resN-acyl-α-amino acids, 1,3-oxazol-5(4H)-ones, N-acyl-α-amino ketones, and 1,3-oxazoles classes, that incorporate into the structure a 4-(phenylsulfonyl)phenyl fragment, were synthesized, and physicochemically characterized. N-Acyl-α-amino acid 2 was produced by Steiger acylation of L-valine with acyl chloride 1. Saturated 2-aryl-4-isopropyl-1,3-oxazol-5(4H)-one 3 was prepared from open-chain intermediate 2 by intramolecular cyclodehydration. N-Acyl-α-amino ketones 4a,b were synthesized by treatment of 2,4-disubstituted 1,3-oxazol-5(4H)-one 3 with arenes, in presence of aluminum trichloride. The 2,5-diaryl-4-isopropyl-1,3-oxazoles 5a,b were generated from acyclic precursors 4a,b under the action of phosphoryl trichloride. The structures of the newly synthesized compounds were confirmed through spectral and elemental analysis data. The antimicrobial activity evaluation demonstrated that compound 4b exhibited inhibitory effects against the planktonic growth of both Gram-positive and Gram-negative strains, while compounds 2 and 3 have inhibited the E. faecium biofilm development on the inert substrate. Despite the low antimicrobial effect of the newly synthesized compounds, the general scaffold allows several future optimizations for obtaining better novel antimicrobial agents.In this paper, new derivatives from H)-one Derivatives Containing a Diaryl sulfonyl Substituent in Position 2 with Antimicrobial Action, published in RO-BOPI, 8/2020 from 28 August 2020.Patent application a201900668: Theodora-Venera Apostol, Stefania-Felicia Barbuceanu, Laura-Ileana Socea, Ioana Saramet, Constantin Draghici, Valeria Radulescu, Mariana Carmen Chifiriuc, Luminita Gabriela Marutescu, Octavian Tudorel Olaru, George Mihai Nitulescu, 4-Isopropyl-1,3-oxazol-5(4


The designed, synthesized, and characterized compounds (11–20) were screened against monoacylglycerol lipase (MAGL) in order to find potential inhibitors. Compounds 19 (4-NO2 derivative) and 20 (4-SO2NH2 derivative), with an IC50 value of 8.4 and 7.6 nM, were found most active, respectively. Both of them showed micromolar potency  against a close analogue, fatty acid amide hydrolase (FAAH), therefore considered as selective inhibitors of MAGL. Molecular docking studies of compounds 19 and 20 revealed that carbonyl of 2-pyrrolidinone moiety sited at the oxyanion hole of catalytic site of the enzyme stabilized with three hydrogen bonds (~2 Å) with Ala51, Met123, and Ser122, the amino acid residues responsible for the catalytic function of the enzyme. Remarkably, the physiochemical and pharmacokinetic properties of compounds 19 and 20, computed by QikProp, were found to be in the qualifying range as per the proposed guideline for good orally bioactive CNS drugs. In formalin-induced nociception test, compound 20 reduced the pain response in acute and late stages in a dose-dependent manner. They significantly demonstrated the reduction in pain response, having better potency than the positive control gabapentin (GBP), at 30 mg/kg dose. Compounds 19 and 20 were submitted to NCI, USA, for anticancer activity screening. Compounds 19 (NSC: 778839) and 20 (NSC: 778842) were found to have good anticancer activity on SNB-75 cell line of CNS cancer, exhibiting 35.49 and 31.88% growth inhibition (% GI), respectively.Ten benzoxazole clubbed 2-pyrrolidinones ( N-arachidonoyl ethanolamine  functions as partial agonist on CB1 and CB2 receptors. It has low affinity for CB2 and moderate affinity for CB1. Endocannabinoids, 2-arachidonoylglycerol (2-AG) function as full agonist and have moderate affinity for both the receptors. Interestingly, 2-AG is the major endocannabinoid and is found to be approximately 170-fold higher in concentration than AEA, in the brain , 144 nM) , SPB01400 31 µM) , JZL184  1.1 nM) , KML29 ( 3.6 nM) , ML30 (I0.54 nM) , JJKK-04 363 pM) , ABX14310 14 nM) , R(3t) (50 3 nM) , a benzo0 80 nM) , and a b34.1 nM) . JJKK-0434.1 nM) ,35. In a34.1 nM) ,36,39,4034.1 nM) . Our gro 9.4 nM) ,43,44,4525 and compound R-3t), and the basic structural requirement for MAGL inhibitors was kept in mind to design novel pyrrolidin-2-one linked benzoxazole derivatives (11–20) is presented in The binding pattern of pyrrolidin-2-one derivatives , was successfully prepared by fusion of benzylamine and methylidenesuccinic acid in water, while compounds 2–10 were synthesized as reported by us in our previous publication oxazol-2-yl)-1-benzylpyrrolidin-2-one) exhibited specific IR bands at 1621 cm−1 (C=N of benzoxazole) and 1703 cm−1 (C=O of pyrrolidin-2-one). The two protons of CH2 of benzyl appeared at δ 3.95–4.01 as a multiplet. Two COCH2 protons of the 2-pyrrolidinone ring was assigned at δ 2.60–2.77 as a multiplet. The multiplets located at δ 3.13–3.18 and 3.81–3.86 were ascribed to one CH proton and two NCH2 protons of the 2-pyrrolidinone ring, correspondingly. Six aromatic protons were found resonating at δ 7.38–7.58 as a multiplet. The doublet resonating at δ 7.73–7.78 was assigned to the aromatic protons of benzoxazole. The other two protons of benzoxazole were found resonating at δ 7.94–7.96 as doublet. The M+ (molecular ion) peak of compound 11 was found at 292.14, validating its successful synthesis.The prototype intermediate compound 11–20) were screened for hMAGL inhibitory potential. The substituted phenyl derivatives (13–20) were established to reduce the MAGL activity at 100 μM concentration below 50%. Compound 19 (4-NO2 derivative) and compound 20 (4-SO2NH2 derivative) were the most potent, with an IC50 of 8.4 and 7.6 nM, correspondingly. The structure–function relationship of benzoxazole derivatives is displayed hMAGL inhibitory activity as follows: 4-SO2NH2 > 4-NO2 > 3-Cl,4-F > 4-OCH3 > 4-Cl > 4-OH > 4-CH3 > 2-CH3 > phenyl/benzyl. The IC50 of standard controls, selective MAGL inhibitors, CAY10499 (IC50 = 415 nM), and for JZL184 (IC50 = 10 nM) were comparable to the reported values +; Anal. calcd. For C12H13NO3: C, 65.74; H, 5.98; N, 6.39. Found: C, 65.80; H, 5.85; N, 6.50.2–10) were synthesized as per the procedure reported in our previous publication oxazol-2-yl)-1-benzylpyrrolidin-2-one (11), pale-yellow solid; yield: 57%; m.p. 160–162 °C; IR: 1370 (C-N), 1555 (C=C), 1621 (C=N), 1703 (C=O), 3077 (Ar C-H); 1H-NMR (DMSO-d6) δ (ppm): 2.60–2.77 , 3.13–3.18 , 3.81–3.86 , 3.95–4.01 , 7.38–7.58 , 7.73–7.78 , 7.94–7.96 ; ESI-MS (m/z): 292.14 [M]+, 293.14 [M + H]+; Anal. calcd. for C18H16N2O2: C, 73.95; H, 5.52; N, 9.58. Found: C, 74.16; H, 5.70; N, 9.82.4-(Benzo[d]oxazol-2-yl)-1-phenylpyrrolidin-2-one (12), pale-yellow solid; yield: 67%; m.p. 148–150 °C; IR: 1395 (C-N), 1552 (C=C), 1615 (C=N), 1698 (C=O), 3070 (Ar C-H); 1H-NMR (DMSO-d6) δ (ppm): 2.86–3.20 , 4.16–4.40 , 7.16–8.18 ; ESI-MS (m/z): 278.13 [M]+, 279.13 [M + H]+; Anal. calcd. for C17H14N2O2: C, 73.37; H, 5.07; N, 10.07. Found: C, 73.60; H, 5.18; N, 10.33.4-(Benzo[d]oxazol-2-yl)-1-(o-tolyl)pyrrolidin-2-one (13), pale-yellow solid; yield: 68%; m.p. 162–164 °C; IR: 1404 (C-N), 1568 (C=C), 1607 (C=N), 1691 (C=O), 3079 (Ar C-H); 1H-NMR (DMSO-d6) δ (ppm): 2.18 , 2.88–3.17 , 4.12–4.18 , 4.26–4.39 , 7.18–7.21 , 7.36–7.54 , 7.98–8.01 , 8.09–8.12 ; ESI-MS (m/z): 292.13 [M]+, 293.13 [M + H]+; Anal. calcd. for C18H16N2O2: C, 73.95; H, 5.52; N, 9.58. Found: C, 74.21; H, 5.82; N, 9.77.4-(Benzo[d]oxazol-2-yl)-1-(p-tolyl)pyrrolidin-2-one (14), pale-yellow solid; yield: 67%; m.p. 164–166 °C; IR: 1411 (C-N), 1562 (C=C), 1598 (C=N), 1697 (C=O), 3077 (Ar C-H); 1H-NMR (DMSO-d6) δ (ppm): 2.28 , 2.94–3.13 , 4.13–4.35 , 7.17–7.20 , 7.34–7.42 , 7.54–7.57 , 7.70–7.74 ; ESI-MS (m/z): 292.13 [M]+, 293.13 [M + H]+; Anal. calcd. for C18H16N2O2: C, 73.95; H, 5.52; N, 9.58. Found: C, 74.18; H, 5.78; N, 9.80.4-(Benzo[d]oxazol-2-yl)-1-(4-chlorophenyl)pyrrolidin-2-one (15), pale-yellow solid; yield: 70%; m.p. 174–176 °C; IR: 758 (C-Cl), 1396 (C-N), 1568 (C=C), 1612 (C=N), 1689 (C=O), 3086 (Ar C-H); 1H-NMR (DMSO-d6) δ (ppm): 3.01–3.09 , 4.20–4.33 , 7.35–7.43 , 7.68–7.72 ; ESI-MS (m/z): 312.11 [M]+, 314.10 [M + 2]+; Anal. calcd. for C17H13ClN2O2: C, 65.29; H, 4.19; N, 8.96. Found: C, 65.52; H, 4.35; N, 9.03.4-(Benzo[d]oxazol-2-yl)-1-(3-chloro-4-fluorophenyl)pyrrolidin-2-one (16), pale-yellow solid; yield: 68%; m.p. 187–189 °C; IR: 765 (C-Cl), 1388 (C-N), 1545 (C=C), 1613 (C=N), 1690 (C=O), 3098 (Ar C-H); 1H-NMR (DMSO-d6) δ (ppm): 2.92–3.19 , 4.13–4.21 , 4.25–4.41 , 7.41–7.60 , 8.05–8.08 , 8.11–8.14 ; ESI-MS (m/z): 330.10 [M]+, 332.09 [M + 2]+; Anal. calcd. for C17H12ClFN2O2: C, 61.73; H, 3.66; N, 8.47. Found: C, 62.07; H, 3.85; N, 8.61.4-(Benzo[d]oxazol-2-yl)-1-(4-hydroxyphenyl)pyrrolidin-2-one (17), pale-yellow solid; yield: 62%; m.p. 242–245 °C; IR: 1407 (C-N), 1535 (C=C), 1597 (C=N), 1698 (C=O), 3092 (Ar C-H), 3410 (O-H); 1H-NMR (DMSO-d6) δ (ppm): 2.91–3.20 , 4.12–4.23 , 4.25–4.39 , 5.64 , 7.41–7.60 , 7.72–7.77 , 7.96–7.99 , 8.08–8.10 ; ESI-MS (m/z): 294.12 [M]+, 295.12 [M + H]+; Anal. calcd. for C17H14N2O3: C, 69.38; H, 4.79; N, 9.52. Found: C, 69.66; H, 4.97; N, 9.78.4-(Benzo[d]oxazol-2-yl)-1-(4-methoxyphenyl)pyrrolidin-2-one (18), pale-yellow solid; yield: 66%; m.p. 177–179 °C; IR: 1412 (C-N), 1546 (C=C), 1600 (C=N), 1690 (C=O), 3081 (Ar C-H); 1H-NMR (DMSO-d6) δ (ppm): 2.88–3.18 , 3.67 , 4.08–4.16 , 4.22–4.39 , 7.16–7.19 , 7.42–7.58 , 7.95–7.98 , 8.09–8.12 ; ESI-MS (m/z): 308.13 [M]+, 309.13 [M + H]+; Anal. calcd. for C18H16N2O3: C, 70.12; H, 5.23; N, 9.09. Found: C, 70.35; H, 5.38; N, 9.18.4-(Benzo[d]oxazol-2-yl)-1-(4-nitrophenyl)pyrrolidin-2-one (19), yellow solid; yield: 62%; m.p. 276–278 °C; IR: 1405 (C-N), 1550 (C=C), 1595 (C=N), 1695 (C=O), 3089 (Ar C-H); 1H-NMR (DMSO-d6) δ (ppm): 2.89–3.15 , 4.11–4.16 , 4.24–4.37 , 6.79–6.82 , 7.43–7.48 , 8.01–8.18 ; ESI-MS (m/z): 323.11 [M]+, 324.11 [M + H]+; Anal. calcd. for C17H13N3O4: C, 63.16; H, 4.05; N, 13.00. Found: C, 63.35; H, 4.27; N, 13.23.4-(4-(Benzo[d]oxazol-2-yl)-2-oxopyrrolidin-1-yl)benzenesulfonamide (20), yellow solid; yield: 60%; m.p. 227–229 °C; IR: 1416 (C-N), 1564 (C=C), 1607 (C=N), 1704 (C=O), 3095 (Ar C-H); 1H-NMR (DMSO-d6) δ (ppm): 2.91–3.19 , 4.15–4.22 , 4.25–4.38 , 5.67 , 7.42–7.59 , 8.09–8.16 ; ESI-MS (m/z): 357.12 [M]+, 358.12 [M + H]+; Anal. calcd. for C17H15N3O4S: C, 57.13; H, 4.23; N, 11.76. Found: C, 57.36; H, 4.36; N, 11.95.11–20) for their capability to reduce hMAGL activity was performed according to the information leaflet provided with Cayman’s assay kit  by the reported method [The screening of the synthesized compounds  for their potential to inhibit hFAAH was performed according to the information leaflet provided with Cayman’s assay kit  by the reported method [The screening of the selected compounds . The .pdb file of hMAGL X-ray crystal structure was downloaded from protein data bank having ID 5ZUN  for molecular docking study [19 and 20) structures were prepared with the help of LigPrep 2.6 with Epik 2.4 at pH 7.0 ± 2.0. The methodology was validated by docking the cocrystallized ligand with Glide XP docking protocol [Glide executed on Maestro 9.4  was utilized for Glide XP docking of active compounds (ng study . The proprotocol .19 and 20), QikProp 3.6 module of Schrodinger was utilized. The generated data was then matched with the qualifying range as per the suggested guideline for good orally bioactive CNS drugs.Guidelines, concerning the validation and optimization of orally active CNS compounds, were developed by Ghose et. al. by analyzing 35 characteristic features of orally bioavailable 317 CNS and 626 non-CNS drugs . For com19 and 20) were evaluated for their absorption and toxicity profile by a bioinformatics tool admetSAR [19 and 20) were also evaluated.The selected compounds , were statistically compared with the control group.Formalin-induced analgesic test was executed by the procedure described by Coderre and Laughlin ,52 as di19 and 20 were supplied to National Cancer Institute , for in vitro sulforhodamine B (SRB) assay, anticancer screening on 60 cell lines of cancers of leukemia, melanoma, and tumors of the kidney, brain, breast, lung, colon, ovary, and prostate, as per their standard protocol [19 and 20 are provided in the Compounds protocol ,54. One The statistical study of the data was accomplished by GraphPad Prism . The dose response of the test compounds was compared with that of control, in formalin-induced analgesic test, by analysis of variance (ANOVA) followed by Dunnett’s test. Outcomes are communicated as mean ± SEM.11–20) as the inhibitors of MAGL were designed, synthesized, characterized, and assayed against MAGL and FAAH enzymes, in order to find potential small molecule selective MAGL inhibitor. Compounds 19 (4-NO2 derivative) and 20 (4-SO2NH2 derivative) were found most potent and selective, with an IC50 of 8.4 and 7.6 nM, respectively. The binding patterns of compounds 19 and 20 in the catalytic site of MAGL were found as expected and similar as those of the reported inhibitors.Ten benzoxazole clubbed 2-pyrrolidinone derivatives (19 and 20 were found to be almost in the qualifying range as per the proposed guideline for good orally bioactive CNS drugs. Compound 20 significantly demonstrated the reduction in pain response, having better potency than the positive control GBP, at the dose of 30 mg/kg. The present work concluded that compound 20 is the potential lead compounds that can be further studied and optimized at points 1 and 4 of the 2-pyrrolidinone moiety for the discovery and development of more selective and potent inhibitors of MAGL for neuropathic pain. The physiochemical and pharmacokinetic properties of compounds


Coccoloba cowellii Britton  is an endemic and critically endangered plant species that only grows in the municipality of Camagüey, a province of Cuba. A preliminary investigation of its total methanolic extract led to the discovery of promising antifungal activity. In this study, a bioassay-guided fractionation allowed the isolation of quercetin and four methoxyflavonoids: 3-O-methylquercetin, myricetin 3,3′,4′-trimethyl ether, 6-methoxymyricetin 3,4′-dimethyl ether, and 6-methoxymyricetin 3,3′,4′-trimethyl ether. The leaf extract, fractions, and compounds were tested against various fungi and showed strong in vitro antifungal activity against Cryptococcus neoformans and various Candida spp. with no cytotoxicity (CC50 > 64.0 µg/mL) on MRC-5 SV2 cells, determined by a resazurin assay. A Candida albicans SC5314 antibiofilm assay indicated that the antifungal activity of C. cowellii extracts and constituents is mainly targeted to planktonic cells. The total methanolic extract showed higher and broader activity compared with the fractions and mixture of compounds. Candida, Aspergillus, and Cryptococcus, representing over 90% of mycotic deaths − and m/z 179 . On the other hand, the peak 2 and 3 data implied the presence of a quercetin-O-pentoside following similar fragmentation behaviour to compound 1, but with the particularity that both homolytic and heterolytic loss of the sugar could be documented by the presence of ions at m/z 300 [Y0-H]−· and m/z 301 [Y0]−, respectively. It was impossible to differentiate between these possible isomers; therefore, they were labelled as quercetin-O-pentoside 1 and 2, respectively.MS spectral data from peaks 1–3 matched with those previously reported for the total extract . Peak 1 m/z 195  indicates that the 8-phenolic end (A− fragment) corresponds to a G unit  −, m/z 613 (B−), m/z 565 (B−-H2O-CH2O), m/z 417 (second B−) and m/z 195 (A−). These fragments  with yields of 2.6, 4.0, and 1.3 mg, respectively . The anat ratios a. The lot ratios  and the I) and an unidentified impurity (minor) . The NMR data  and 6-methoxymyricetin 3,4′-dimethyl ether  . The position of substituents was corroborated through 2JH-C and 3JH-C HMBC correlations  − (C15H9O7).Quercetin ): O-Methylquercetin ): 1H NMR : δ 3.79 ; 6.22 ; 6.41 ; 6.92 ; 7.54 ; 7.64 . 13C NMR : δ 60.5 (OCH3), 94.7 (C-8), 99.8 (C-6), 105.9 (C-10), 116.5 (C-2′), 116.5 (C-5′), 122.3 (C-6′), 123.0 (C-1′), 139.7 (C-3), 146.5 (C-3′), 150.2 (C-2), 150.0 (C-4′), 158.5 (C-9), 163.1 (C-5), 166.0 (C-7). ESI-MS (negative mode): m/z 315 [M-H]− (C16H11O7).3-1H NMR : δ 3.81; 3.89 ; 3.90 ; 6.52 ; 7.19 ; 7.19 . 13C NMR : δ 60.7 (OCH3), 60.8 (OCH3), 61.0 (OCH3), 95.0 (C-8), 106.4 (C-10), 109.2 (C-2′), 109.2 (C-6′), 127.0 (C-1′), 132.7 (C-6), 139.6 (C-4′), 140.1 (C-3), 152.0 (C-3′), 152.0 (C-5′), 153.9 (C-9), 157.5 (C-2), 158.9 (C-7), 180.5 (C-4). ESI-MS (negative mode): m/z 375 [M-H]− (C18H15O9).6-Methoxymyricetin 3,4′-dimethyl ether ): 1H NMR : δ 3.83 ; 3.89 ; 3.89 ; 3.93 ; 6.54 ; 7.30 ; 7.30 . 13C NMR : δ 55.2 (OCH3), 59.4 (OCH3), 59.6 (OCH3), 59.6 (OCH3), 93.7 (C-8), 103.9 (C-6′), 105.1 (C-10), 109.6 (C-2′), 125.5 (C-1′), 131.1 (C-6), 138.6 (C-3), 139.0 (C-4′), 150.5 (C-3′), 152.7 (C-9), 153.2 (C-5′), 155.8 (C-2), 157.9 (C-7), 179.0 (C-4). ESI-MS (negative mode): m/z 389 [M-H]− (C19H17O9).6-Methoxymyricetin 3,3′,4′-trimethyl ether ): 1H NMR : δ 3.83 ; 3.89 ; 3.93 ; 6.23 ; 6.43 ; 7.30 ; 7.30 . 13C NMR : δ 55.2 (OCH3), 59.4 (OCH3), 59.6 (OCH3), 93.5 (C-8), 98.6 (C-6), 103.9 (C-6′), 109.6 (C-2′), 125.8 (C-1′), 138.6 (C-3), 139.0 (C-4′), 150.5 (C-3′), 153.2 (C-5′), 155.8 (C-2). ESI-MS (negative mode): m/z 359 [M-H]− (C18H15O8).Myricetin 3,3′,4′-trimethyl ether ): GraphPad Prism V8 Software for Windows  was employed for all statistical analyses. The results were analysed and expressed as the means ± standard deviation (SD) of three different replicates.C. cowellii by means of a combined methodology of NMR and MS analysis. All five are reported here for the first time for both the plant and the genus. Another 16 compounds were tentatively characterized employing UHPLC-HRMS. C. cowellii extract was confirmed to have good antifungal activity against a second fungal/yeast panel, while fractions and mixtures of compounds obtained from the bioassay-guided fractionation showed acceptable activity specifically against C. glabrata and C. neoformans. These results highlight the possible use of this plant as a natural antifungal and contribute to a better understanding of the phytochemistry and biological activities of the genus Coccoloba. At the same time, they suggest the probable synergistic effect that the combination of different types of polyphenols may show in inhibiting fungal growth.In this study, five secondary metabolites were isolated and characterized from the MeOH90-F fraction of the total extract of


All the newly synthesized compounds were evaluated for their inhibitory activity towards PARP-1 and examined for their anti-proliferative activity against MCF-7 and HCT116 human cancer cell lines. The synthesized compounds showed promising activity where compounds S2 and S7 emerged as the most potent PARP-1 inhibitors with an IC50 value of 4.06 ± 0.18 and 3.61 ± 0.15 nM, respectively compared to that of Olaparib 5.77 nM and high cytotoxicity against MCF-7 with IC50 2.65 ± 0.05 and 1.28 ± 1.12 μM, respectively (Staurosporine 7.258 μM). Compound S8 remarkably showed the highest cell growth inhibition against MCF-7 and HCT116 with an IC50 value of 0.66 ± 0.05 and 2.76 ± 0.06 μM, respectively. Furthermore, molecular docking of the compounds into the PARP-1 active site was performed to explore the probable binding mode. Finally, most of the synthesized compounds were predicted to have good pharmacokinetics properties in a theoretical kinetic study.Poly(ADP-ribose) polymerases-1 (PARP-1) are involved in DNA repair damage and so PARP-1 inhibitors have been used as potentiators in combination with DNA damaging cytotoxic agents to compromise the cancer cell DNA repair mechanism, resulting in genomic dysfunction and cell death. In this study, we report the synthesis of a novel series of pyrano[2,3- PARP-1 are involved in DNA repair damage and so PARP-1 inhibitors have been used as potentiators in combination with DNA damaging cytotoxic agents to compromise the cancer cell DNA repair mechanism, resulting in genomic dysfunction and cell death. They regulate a number of cellular processes including surveillance of genome integrity, cellular differentiation, regulation of gene transcription, inflammation, mitosis, cell cycle progression, initiation of DNA damage response and apoptosis.2 PARP-1 is a known sensor of DNA damage as it is responsible for DNA base excision repair (BER) and DNA single-strand break (SSB) repair.3 Damaged DNA activates PARP-1 to cleave its substrate nicotinamide adenine dinucleotide (NAD+) and to catalyze the addition of ADP-ribose units to it and to nuclear target proteins to recruit BER components to facilitate DNA repair process and cell survival.4–7 Increased PARP-1 expression is sometimes observed in melanomas, breast cancer, lung cancer, and other neoplastic diseases.8PARP-1 has received great attention as a promising anti-cancer therapeutic target.9 So PARP-1 inhibitors have shown success when used as monotherapy for treating genetically DNA repair-defective cancers. PARP-1 inhibitors have not only been used in BRCA1/2 deficient cancers but also used in combination therapy with DNA-damaging therapeutics to improve their potencies by blocking DNA-repairing process.It has been disclosed that in BRCA1/2-mutant cancer cells, inhibition of PARP1 is synthetically lethal due to their dependence on PARP-1 activity for DNA (base excision) repair and subsequently survival.e.g., Olaparib I,10 Niraparib II,11 Rucaparib III12 and Talazoparib IV  and the other is the adenine-ribose binding site (AD site). Most of the reported PARP-1 inhibitors can bind the NI site through hydrogen bonds with residues Ser904 and Gly863, and additional pi–pi stacking interaction with Tyr907. Compared with the NI site, the AD site is large enough to accommodate diverse structure motifs.d] pyrimidine 2,4 dione analogues as a core structure to occupy the NI site and to interact with Ser904 and Gly863 through hydrogen bonds in an attempt to discover novel PARP-1 inhibitors.Herein, we employed pyranopyrimidine-6-carbonitrile (1) by treating cyclic compounds containing active methylene group with thiophen-2-carbaldehyde and malononitrile in solution of water : ethanol (1 : 1 ratio) as three component system for 2 h. The structure of the obtained products was confirmed using elemental analysis, spectroscopic data as well as chemical reaction -2,3,4,5-tetrahydro-1reaction .18–23H-pyranodipyrimidine-7,9dione (2). In compound 2 stretching vibration was found in IR analysis of the (NH2) and 2(NH) groups at ν 3420.87, 3281.06, 3256.68 and 3163.37 cm−1. This IR pattern appears to be consistent with the assigned structure.Compound (1) was fused with thiourea then dissolved in a solution of sodium hydroxide to produce the corresponding 4-amino-2-mercapto-10-(thiophen-2-yl)-6H-pyranopyrimidine-6-carbonitrile (1) with 2,4 dinitrophenyl hydrazine in 1,4-dioxane forming the corresponding new derivative 3-amino-1--9-(thiophen-2-yl)-7,9-dihydropyrazolopyrano pyrimidine-6,8-dione (3). The structure of compound (3) was characterized using spectroscopic and elemental analysis. IR spectroscopy of compound (3) showed the presence of two NH2 bands at 3420.17, 3365.15 cm−1, two NH bands at 3256.68 and 3163.37 cm−1 and two carbonyl bands at 1752.22 and 1694.82 cm−1 indicating the presence of unreacted cyano group. Furthermore, 1H-NMR spectra verified the structure by the appearance of three exchangeable signals at 9.12, 10.57 ppm for two NH and signals at 9.42–9.46 ppm for NH2.New pyrazolo derivatives were prepared by reacting 7-amino-2,4-dioxo-5-(thiophen-2-yl)-2,3,4,5-tetrahydro-1−1 assignable to NH and NH2 group at 3470.68, 3249.52 cm−1, are good evidence for the structure given to this compound. While IR spectrum of compound (5) revealed bands at 3470.68, 3249.52, 3214.28 and 3111.74 cm−1 characteristic for 4NH groups, respectively, while it's 1H-NMR spectrum showed an exchangeable singlet signal of the NH proton at δ 7.34, 11.236, 11.277 ppm. Moreover, refluxing compound 1 with acetic anhydride, afforded the 6-(methoxyamino)-2,4-dioxo-5-(thiophen-2-yl)-2,3,4,5-tetrahydro-1H-pyranopyrimidine-7-carbonitrile (6). IR spectroscopy of (6) showed the presence of three NH bands at 3468.7, 3254.05 and 3196.21 cm−1, two carbonyl bands at 1730.52, 1690.42 cm−1 and one CN band at 2074.78 cm−1, indicating open chain structure. Furthermore, 1H-NMR spectra verified the structure by the appearance of three exchangeable NH signals at 2.47, 11.23 and 11.27 ppm. Compound (1) was subjected to reaction with phenyl isothiocyanate in dry pyridine to furnish the corresponding 3-phenyl-10-(thiophen-2-yl)-2-thioxo-2,3-dihydro-1H-pyranodipyrimidine-4,7,9-trione (7). It has been observed in compound (7) the absence of the cyanide peak in their IR spectra and the appearance of the imino group of pyrimidine moiety which proves the formation of cyclic compound (The transformation of (1) into pyranodipyrimidine derivatives was achieved by its reaction with formamide and carbon disulfide respectively, under reflux. The structure of compound (4) was confirmed by their IR spectra through disappearance of CN with the appearance of two bands at 3214.28, 3111.74 cmcompound  -5-(thiophen-2-yl)-5,5a-dihydro-1H-pyridopyranopyrimidine-2,4-dione (8). The formation of compound (8) was confirmed by the appearance of a signal at 6.95, 7.72 , 7.98 , 8.18–8.36  in 1H-NMR spectra, respectively, also reaction of compound (1) with triethylorthoformate, yielded (E)-ethyl N--2,3,4,5-tetrahydro-1H-pyranopyrimidin-7-yl)formimidate (9). In the 1H-NMR spectra of (9) a singlet peak at 8.71, 11.23 ppm  were found. Furthermore, in the IR spectra, the bands at 2069.45 cm−1 (CN) and two bands at 3199.9, 3147.11 cm−1 (NH).Also cyclization of compound (1) with H-pyrano dipyrimidine-2,4-dione (10). 1H-NMR showed two exchangeable signals at 11.23, 11.27 ppm referred to NH and OH. IR spectroscopy provided two NH bands at 3125.16, 3040.10 cm−1 and one OH bands at 3450 cm−1. Moreover, two carbonyl bands appeared.New pyrimidine derivatives were prepared by treatment of compound (1) with a mixture of hydrochloric acid and acetic acid (1 : 3) giving 6-hydroxy-8-methyl-5-(thiophen-2-yl)-1a-dihydro-4aH-pyrimidopyranotriazine-6,8-dione (11). The structure was confirmed by infrared spectrum which revealed no absorption in the CN region; furthermore, it displayed absorption bands at 3199.19 and 3442.41 cm−1 as a broad band of NH, OH.Diazotization of compound (1) with sodium nitrite and conc. Hydrochloric acid in the presence of acetic acid led to the formation of 4-hydroxy-5-(thiophen-2-yl)-9,102.2.The biological evaluation was accomplished through testing both enzyme inhibition activity and anti-proliferative activity. The enzymatic activity of the synthesized compounds was assessed against PARP-1.2.2.150 values ranging from 3.61 nM to 114 nM compared to the reference drug Olaparib. Compounds S2 and S7 showed a higher potency than Olaparib. S4, S5, S8 and S10 showed a slightly less potency then Olaparib. Compounds S1, S6 and S9 showed the least potencies. The obtained result suggesting that the presence of a heterocycle fused with the pyranopyrimidine 2,4 dione enhances the potency.The synthesized compounds were evaluated for their PARP-1 inhibitory activity. As shown in 2.2.250 ranging from 0.66 ± 0.05 to 12.68 ± 0.12 μM. Also, the compounds showed good inhibition against HCT116 cells ranging from 2.76 ± 0.06 to 46.86 ± 1.82 μM  is shown in d]pyrimidine 2,4 dione derivatives, we obtained and analyzed the docked structures of the synthesized compounds within the catalytic site of PARP-1 and compared it with the that of Olaparib.The crystal structure of the ligand Olaparib complexed with PARP-1  are not expected upon administration of these compounds.In HIA plot, most of compounds fell inside the 99% ellipse, thus estimated to have good human intestinal absorption except for S3 and S8 which showed poor absorption. Aqueous solubility level of most of the compounds was found to be 3 or 2 which indicates low aqueous solubility. The hepatotoxicity level of all compounds was 1. Hence the compounds are predicted to possess hepatotoxicity. Further experimental studies are required to determine the hepatotoxic dose levels. Most of the compounds are predicted as noninhibitors of CYP2D6. Hence the side effects  using TMS as a reference standard and the coupling constants J values are given in Hz. The progress of the reactions was determined using TLC aluminum silica gel plates 60 F245. IR, the analysis  were done on the Main Chemical Warfare Laboratories, Chemical Warfare Department, Egypt.Melting points of all the new compounds are listed uncorrected and were measured by a Reichert Thermovar apparatus. Yields registered are of the new compounds. The IR spectra were determined using Perkin-Elmer spectrometer (KBr disc), model 1720 FTIR. 5.1.1−1 (νNH2), 3203.12, 3148.84 cm−1 (νNH), 2070.14 cm−1 (νCN), 1751.88, 1694.47 cm−1 (νOC). 1H-NMR (DMSO-d6): 7.32, 7.34 , 8.71  7.93, 11.27 . Anal. calculated for C12H8N4O3S (288.28): C, 50.00; H, 2.80; N, 19.43; S, 11.12. Found: C, 49.98; H, 2.69; N, 19.41; S, 11.06.18,19A mixture of thiophen-2-carbaldehyde , malononitrile  barbituric acid  and triethylamine (2–3 drops) in a solution of water: ethanol (1 : 1 ratio) 30 ml : 30 ml were heated under reflux for 2 h. The solid separated and collected after concentration and cooling of the solvent at room temperature, dried and crystallized from butanol to give 1 as yellow crystals; mp: 295–298 °C, yield 95%. IR (KBr) 3449.18 cm5.1.2ν/cm−1): 3420.87, 3281.06 cm−1 (νNH2); 3256.68, 3163.37 cm−1 (2νNH), 2687.10 cm−1 (νSH), 1752.22, 1694.82 cm−1 (2 νOC). 1H-NMR : δ 4.03 ,7.029 , 7.341–7.319 , 9.594 ppm , 10.005 ppm , 11.271 ppm . 13C-NMR  δ (ppm): 39.31 (CH), 112.02 (CC); 19.5; 112.02; 128.83 (2CH); 136.76; 142.57; 146.16 (2 C–O), 146.29 (C–N); 150.69 (C–NH2); 163.48, 163.97 (CO), 184.27 (C–SH), Anal. calculated for C13H9N5O3S2 (347.37): C, 44.95; H, 2.61; N, 20.16; S, 18.46, found: C, 44.85; H, 2.83; N, 19.44; S, 17.37.A mixture of compound (1)  with thiourea  was fused for 2 h, then dissolved in a solution of sodium hydroxide (10%) with stirring for another 2 h, then neutralized by HCl to obtain solid product which was filtered off, washed with water, dried and recrystallized from acetic acid to give 2. Yields 86%, mp 170–172 °C, yellow powder; IR   and 2,4 dinitrophenylhydrazine  in 1,4-dioxane (30 ml) was refluxed in water bathe for 3 h, cooled, filtered off, dried and recrystallized from methanol to give 3. Yields 86%, mp 225 °C, orange powder; IR : 3420.17, 3365.15 cm−1 (ν2NH), 3256.68, 3163.37 cm−1 (2νNH); 1752.22, 1694.82 cm−1 (νOC), 1656.12 cm−1 (νCC), 1589.47 cm−1 (νNC), 1468.39 cm−1 (νSC). 1H-NMR : δ 3.82 , 6.69–7.21 , 7.94 , 8.01  8.2 , 9.42, 9.46 ppm , 9.12, 10.57 ppm . 13C-NMR  δ (ppm): 31.72 (CH), 112.02 (C), 116.73, 123.50, 123.54 (CH), 128.71, 128.82, 130.27 (CH); 130.43 (C–O); 132.19 (CH); 136.77 (C–N); 130.30, 138.85 (CC), 142.57, 144.60 (C–NO2), 150.69 (C–NH), 159.71 (C–NH2), 163.96 (CO). Anal. calculated for C18H11N7O7S (469.39): C, 46.06; H, 2.36; N, 20.89; S, 6.83, found: C, 45.85; H, 2.43; N, 19.94; S, 7.02.A mixture of 7-amino-2,4-dioxo-5-(thiophen-2-yl)-2,3,4,5-tetrahydro-15.1.4H-pyranopyrimidine-6-carbonitrile (1)  and formamide (20 ml) for was refluxed for 2 h, then cooled, poured into crushed-ice, filtered off, dried and recrystallized from methanol to give 4. Yields 76%, mp over 300 °C, black powder; IR : 3470.68, 3249.52 cm−1 (νNH2); 3214.28, 3111.74 cm−1 (2νNH), 1751.15, 1689.78 cm−1 (νOC), 1557.04 cm−1 (νNC). 1H-NMR : δ 4.10 , 6.90–7.377 , 7.96 , 9.121, 9.426 , 6.626, 10.575 ppm . Anal. calculated for C13H9N5O3S (315.31): C, 49.52; H, 2.88; N, 22.21; S, 10.17, found: C, 49.85; H, 2.83; N, 21.94; S, 10.09.A solution of 7-amino-2,4-dioxo-5-(thiophen-2-yl)-2,3,4,5-tetrahydro-15.1.5H-pyranopyrimidine-6-carbonitrile (1)  and C2S (10 ml) in dry pyridine (25 ml) was heated under reflux in water bath for 6 h, cooled, poured into ice-water, acidified with glacial acetic acid then filtered, dried and recrystallized from ethanol to give 5. Yields 85%, mp over 300 °C, greenish yellow powder; IR : 3470.68, 3249.52, 3214.28, 3111.74 cm−1 (4νNH); 1751.16, 1689.78 cm−1 (2νOC), 1658.59 cm−1 (νCC). 1H-NMR : δ 3.99 , 7.30–7.32 , 7.34, 11.236, 11.277 ppm , 13C-NMR  δ (ppm): 39.33 (CH), 112.02 (C), 128.83, 142.57 (CH); 136.77, 146.16 (2CH), 146.28, 150.69 (C–NH), 163.48 (2CO), 163.96 (2CS). Anal. calculated for C13H8N4O3S3 (364.42): C, 42.85; H, 2.21; N, 15.37; S, 26.40, found: C, 42.65; H, 2.18; N, 15.44; S, 26.38.A mixture of 7-amino-2,4-dioxo-5-(thiophen-2-yl)-2,3,4,5-tetrahydro-15.1.6H-pyranopyrimidine-6-carbonitrile (1)  and acetic anhydride (20 ml) was refluxed for 4 h. The solid separated and collected after concentration and cooling of the solvent to room temperature, dried and crystallized from butanol to give 6 as yellow powder; mp: over 300 °C, yield 90%. IR (KBr) 3468.7, 3254.05 and 3196.21 cm−1 (3νNH), 2927.27 cm−1 (νCH3), 2074.78 cm−1 (νCN), 1730.52, 1690.42 cm−1 (2νOC). 1H-NMR : δ 3.38 , 3.99 , 2.47 , 7.32–8.54 , 11.23, 11.27 . Anal. calculated for C13H10N4O4S (318.31): C, 49.05; H, 3.17; N, 17.60; S, 10.07. Found: C, 49.55; H, 3.18; N, 17.52.A mixture of 7-amino-2,4-dioxo-5-(thiophen-2-yl)-2,3,4,5-tetrahydro-15.1.7H-pyranopyrimidine-6-carbonitrile (1)  and phenyl isothiocyanate  in dry pyridine (20 ml) was heated under reflux for 4 h. After cooling, the reaction mixture was diluted with ice cold water, acidified to litmus paper with glacial acetic acid, filtered and crystallized from butanol to give 7 as white powder; mp: 235–237 °C, yield 70%. IR (KBr): 3464.92, 3329.11, 3062.09 cm−1 (3νNH), 1717, 1694.58 cm−1 (2νOC). 1H-NMR : δ 3.99 , 2.066, 6.89 , 6.90–6.94 , 7.22–7.46 , 9.19 . Anal. calculated for C19H12N4O4S2 (424.45): C, 53.76; H, 2.85; N, 13.20; S, 15.11. Found: C, 53.55; H, 2.76; N, 12.82, S, 14.91.A mixture of 7-amino-2,4-dioxo-5-(thiophen-2-yl)-2,3,4,5-tetrahydro-15.1.8H-pyranopyrimidine-6-carbonitrile (1) , p-nitroacetophenone  and 2–3 drops of triethylamine in 1,4-dioxane (30 ml) was heated under reflux for 5 h then cooled and poured into ice-water, filtered off, dried and recrystallized from 1,4-dioxane to give 8 as orange crystals; mp: 130 °C, yield 82%. IR (KBr): 3201.92 cm−1 (ν2NH), 3147.46, 3108.03 cm−1 (2νNH), 1751.88, 1688.98 cm−1 (2νOC), 1552.69 cm−1 (ν2NO). 1H-NMR : δ 3.85 , 6.95 , 7.11–7.34 , 7.72 , 7.98 , 8.18–8.36 , 13C-NMR  δ (ppm): 39.33 (CH), 40.59 (C); 124.39, 125.13, 129.08, 129.09 (CH); 126.73, 129.75, 129.97, 130.22 (CH); 114.49, 129.08 (CC); 130.39 (CN); 136.08 (C–NO2), 141.60 (C–NH), 143.05 (CO), 146.08 (C–NH2). Anal. calculated for C20H15N5O5S (437.43): C, 54.92; H, 3.46; N, 16.01; S, 7.33. Found: C, 54.23; H, 3.04; N, 15.099; S, 6.82.A mixture of 7-amino-2,4-dioxo-5-(thiophen-2-yl)-2,3,4,5-tetrahydro-15.1.9H-pyrano pyrimidine-6-carbonitrile (1)  and  triethylorthoformate was refluxed for 7 h. The solid separated and collected after concentration and cooling of the solvent to room temperature was dried and crystallized from butanol to give 9 as yellow crystals; mp: 293–295 °C, yield 90%. IR (KBr): 3199.9, 3147.11 cm−1 (2νNH), 2069.45 cm−1 (νCN), 1751.42, 1665.32 cm−1 (νOC). 1H-NMR : δ 1.036 , 3.25 , 3.69 , 7.32–7.34 , 8.71 ,11.23 , 13C-NMR  δ (ppm): 39.33 (CH), 39.96, 40.59 (C); 112.03  scale" fill="currentColor" stroke="none"><path d="M80 600 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z M80 440 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z M80 280 l0 -40 600 0 600 0 0 40 0 40 -600 0 -600 0 0 -40z"/></g></svg>N); 128.82 (CH); 136.78 (CC); 142.56 (2C–NH), 146.15 (CN), 146.27 (2CH), 150.69, 163.48 (C–N), 163.96 (2CO). Anal. calculated for C15H12N4O4S (344.35): C, 52.32; H, 3.51; N, 16.27; S, 9.31. Found: C, 51.78; H, 3.23; N, 16.06; S, 9.11.A mixture of 7-amino-2,4-dioxo-5-(thiophen-2-yl)-2,3,4,5-tetrahydro-15.1.10H-pyrano pyrimidine-6-carbonitrile (1)  with a hydrochloric acid and acetic acid (1 : 3) (10 : 30 ml) was refluxed for 4 h. The solid separated, collected after concentration and cooling of the solvent to room temperature, dried and crystallized from butanol to give 10 as pale-yellow powder; mp: 298 °C; yield 90%. IR (KBr): 3450 cm−1 (νOH), 3125.16, 3040.10 cm−1 (2νNH), 1736.04, 1686.84 cm−1 (νOC). 1H-NMR : δ 2.48 , 3.91 , 7.32–7.34 , 11.23 , 11.27 , 13C-NMR  δ (ppm): 39.33, 40.58 (CH); 112.02, 128.82 (CH); 136.77 (2CC); 142.56 (C–CH3), 146.15 (2C–NH), 146.28 (C–O), 150.69, 163.48 (2CO), 163.96 (C–OH). Anal. calculated for C14H10N4O4S (330.32): C, 50.91; H, 3.05; N, 16.96; S, 9.71. Found: C, 50.57; H, 2.98; N, 16.34; S, 9.10.A mixture of 7-amino-2,4-dioxo-5-(thiophen-2-yl)-2,3,4,5-tetrahydro-15.1.11H-pyranopyrimidine-6-carbonitrile (1)  in 45 ml of a (1 : 2) mixture of HCl (15 ml) and acetic acid (30 ml) which was then stirred at room temperature for 2 h. The crude product obtained was recrystallized from acetic acid. Yields 72%, mp over 300 °C, yellow powder; IR : 3442.41 cm−1 (νOH), 3199.19, 3145.80 cm−1 (2νNH); 1750.04, 1693.81 cm−1 (2νOC), 1653.22 cm−1 (νNC). 1H-NMR : δ 2.066 ppm , 3.87 , 7.324–7.347 , 11.237, 11.176 , 13C-NMR  δ (ppm): 39.33 (CH); 112.03 (C), 128.83, 136.77 (CH); 142.57 (C), 146.16 (2C–O), 146.27 (C–NH), 163.48 (C–OH), 150.69, 163.96 (2CO). Anal. calculated for C12H9N5O4S (319.30): C, 45.14; H, 2.84; N, 21.93; S, 10.04, found: C, 45.04; H, 2.79; N, 20.99; S, 10.00.A solution of sodium nitrite  in 10 ml of water was added to a cold solution of 7-amino-2,4-dioxo-5-(thiophen-2-yl)-2,3,4,5-tetrahydro-15.2.5.2.12O (v/v), 20 μl of 1× PARP buffer was added to the wells designated as “blank”. The amount of PARP-1 required for the assay was then calculated. The reaction was initiated by adding 20 μl of diluted PARP1 enzyme to the wells designated “positive control” and “test inhibitor control”. The strip wells were incubated at room temperature for 1 hour. The strip wells were then washed three times with 200 μl PBST buffer. Then, 50 μl of 50 times diluted Streptavidin-HRP with blocking buffer was added to each well, and the strips were further incubated at room temperature for 30 min. After washing the wells three times with 200 μl PBST buffer, HRP colorimetric substrate was added to each well and the plate was incubated at the room temperature until a blue color is developed in the positive control well. Then reaction was quenched with 100 ml per well of 2 M sulfuric acid, and absorbance at 450 nm was determined. Carrier solvents were assayed as negative controls. All assays were performed in triplicate. To determine the IC50 value for each inhibitor, the average absorbance of each inhibitor concentration was plotted against the log of the concentration of each respective inhibitor and the IC50 value for each plot was obtained using computer-assisted non-linear regression analyses. Data presented are the results of at least two independent experiments done in triplicate. The results of these studies are presented as mean IC50 (nM).24PARP-1 enzyme inhibition activity was measured for using a colorimetric 96-well PARP-1 assay kit  (BPS Bioscience), according to the manufacturer's protocol. Briefly, the histone mixture was diluted 1 : 5 with 1× PBS, 50 μl of histone solution was added to each well and incubated at 4 °C overnight. The plate was washed three times using 200 μl PBST buffer (1× PBS containing 0.05% Tween-20) per well. Liquid was removed from the wells by tapping the strip wells on clean paper towels. To each well, 200 μl of blocking buffer was added, followed by 60–90 min incubation at room temperature. Then 25 μl of PARP master mixture (consisting of 2.5 μl 10× PARP buffer + 2.5 μl 10× PARP assay mixture + 5 μl activated DNA + 15 μl distilled water) was added to each well. Olaparib was used as a positive control. 5 μl of inhibitor solution of each well labeled as “test inhibitor” was added. For the “positive control” and “blank”, 5 μl of the same solution without inhibitor was added. 1× PARP buffer was prepared by adding 1 part of 10× PARP buffer to 9 parts H5.2.225 Only in live cells, mitochondrial enzymes can transform MTT into insoluble formazan. After incubated with serially diluted inhibitors for 96 h, cell cultures were incubated with MTT solution (5 mg ml−1) for 4 h at 37 °C. Then discard the medium and DMSO was added to solubilize the reaction product formazan by shaking for 10 min. Absorbance at 492 nm was measured with a microplate reader . Cell viability was expressed as an IC50 value.We used the MTT   assay to measure cell growth inhibition, which is based on the conversion of MTT to formazan crystals by mitochondrial dehydrogenases.5.3.5.3.1http://www.rscb.org/pdb (PDB ID: 5DS3) in PDB format. Computational docking is an automated computer-based algorithm designed to estimate two main terms.26 The first is to determine the suitable position and the orientation of certain test set molecule's pose inside the binding site in comparison to that of the X-ray crystallographic enzyme–substrate complex. The second term is the calculation of the estimated protein ligand interaction energy which is known as docking scoring.Molecular modeling simulation study was performed through docking of the target compounds in the binding site of PARP-1 enzyme using C-Docker protocol in Discovery Studio 4.0 Software. The X-ray crystal structure of Olaparib in complex with PARP-1 was downloaded from 5.3.2A log P98), 2D polar surface area (ADMET 2D PSA), absorption level (Absorb LEV), aqueous solubility level (AQ SOl LEV), Blood Brain Barrier value (BBB), Blood Brain Barrier Level (BBB LEV), Cytochrome P450 2D6 (CYP2D6), Cytochrome P450 2D6 Probability (CYP PROB), hepatotoxicity (HEPATOX), Hepatotoxicity Probability (HEPATOX PROB) and plasma protein binding logarithmic level (PPB LEV).27Computer aided ADMET study was performed by using the software: Accelrys Discovery studio 2.5. These studies are based on the chemical structure of the molecule and involves the calculation of certain parameters including; atom based log P98 (The authors declare no conflict of interest, financial or otherwise.RA-011-D0RA10321G-s001


Coreopsis lanceolata L. The dried flowers of C. lanceolata were extracted with 70% EtOH, and the obtained extract was divided into CH2Cl2, EtOAc, n-BuOH, and H2O fractions. The CH2Cl2 fraction was separated using silica gel and C-18 column chromatography to yield phenylheptatriyne (1), 2′-hydroxy-3,4,4′-trimethoxychalcone (2), and 4′,7-dimethoxyflavanone (3). Additionally, the EtOAc fraction was subjected to silica gel, C-18, and Sephadex LH-20 column chromatography to yield 8-methoxybutin (4) and leptosidin (5). All the compounds isolated from C. lanceolata inhibited the production of nitric oxide (NO) in LPS-induced BV2 and RAW264.7 cells. In addition, phenylheptatriyne and 4′,7-dimethoxyflavanone reduced the secretion of inflammatory cytokines, tumor necrosis factor alpha (TNF-α), and interleukin (IL)-6. Among them, phenylheptatriyne was significantly downregulated in the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2). Subsequently, phenylheptatriyne also effectively inhibited nuclear factor-kappa B (NF-κB) activation in LPS-stimulated BV2 and RAW264.7 cells. Based on these results, the anti-neuroinflammatory effect of phenylheptatriyne isolated from C. lanceolata was confirmed, which may exert a therapeutic effect in treatment of neuroinflammation-related diseases.Aging is associated with immune disregulation and oxidative stress which lead to inflammation and neurodegenerative diseases. We have tried to identify the anti-neuroinflammatory and anti-inflammatory components of  The 13C-NMR data showed the presence of 17 signals, including a carbonyl carbon (δ 190.9), three oxygenated aromatic carbons , an oxygenated methine carbon (δ 79.8), two methoxy carbons (δ 55.4 and 55.6), and a methylene carbon (δ 44.1). From the analysis of the 1H- and 13C-NMR data, the structure of this compound was conjectured to have a flavanone skeleton. Based on comparison with 1H- and 13C-NMR data in the literature, the structure of compound 3 was determined to be 7-methoxy-2-(4-methoxyphenyl)chromen-4-one  -6-hydroxy-7-methoxy-1-benzofuran-3-one (leptosidin) [Compound tosidin) .2) are secreted. Suppressing the secretion of active inflammatory substances can prevent cell damage. We, therefore, evaluated the neuroinflammation inhibitory effect of the compounds on BV2 microglia and RAW264.7 macrophage induced by LPS.Macrophages and microglia are activated in the body in the presence of harmful stimuli and play an important role in maintaining homeostasis in the body. However, when abnormally activated by external stimuli, pro-inflammatory mediators, ROS, and inflammatory cytokines are secreted . The sec4 and 5 in RAW264.7 macrophages  was used as a positive control. The results revealed that compound 1 upregulated the expression of HO-1 (Hemeoxygenase-1 (HO-1), a target of nuclear factor E2-related factor 2 (Nrf2), a powerful antioxidant signaling system in cells, is an enzyme that promotes free iron in carbon monoxide (CO), billiberdin, and heme . HO-1 re of HO-1 a,b.1 on nitrite production was dependent on HO-1. The investigation method used involved protoporphyrin IX (SnPP), a potent inhibitor of HO-1 activity, to determine whether HO-1 mediated the protective effect of compound 1 against the inflammatory response caused by LPS. The compound 1 treatment group showed a significant inhibitory effect on nitrite production in both LPS-induced BV2 microglia and RAW264.7, but the simultaneous treatment with SnPP showed no inhibitory effect on nitrite production  [Coreopsis lanceolata is a plant of the family Compositae. Many natural compounds, most of them flavonoids, have been reported from the Compositae family. C. lanceolata has been reported to contain many flavonoids [C. lanceolata, and in particular, the number of studies on the isolation of active compounds with anti-neuroinflammatory and anti-inflammatory effects are limited. In this study, we investigated the anti-neuroinflammatory and anti-inflammatory effects of five compounds isolated from the 70% EtOH extract of C. lanceolata flowers.Inflammation is a biological response to infection and damage in certain tissues in the body, the main targets of which are immune cells. Inflammation is classified as acute or chronic according to the size of the affected area and the damage to the tissues of the body. When macrophages are stimulated by lipopolysaccharides (LPS), known as endotoxins, an inflammatory response occurs, and inflammatory cytokines such as interleukin (IL)-6 and IL-1β are released. In addition, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression trigger the inflammatory responses by producing various inflammatory mediators such as nitric oxide (NO) and prostaglandin E2 (PGE2) ,35,36. Iavonoids ,15,16 anC. lanceolata were identified to be phenylheptatriyne (1), 2′-hydroxy-3,4,4′-trimethoxychalcone (2), 4′,7-dimethoxyflavanone (3), 8-methoxybutine (4), and leptosidin (5) (1) has previously been obtained from Bidens alba var. Other reports have confirmed the antifungal, phototoxin, and antiviral effects of phenylheptatriyne isolated from Bidens pilosa L. [C. lanceolata, and thus far, there have been no reports of its anti-inflammatory activity. 2′-Hydroxy-3,4,4′-trimethoxychalcone (2) is a compound with a chalcone skeleton, and it has been reported to have many active effects, such as anti-cancer and anti-inflammatory effects [3) is a compound with a flavanone skeleton, reported to have anticancer effects [4) and leptosidin (5) are known to have whitening and antioxidant effects and have been reported to be isolated from C. lanceolata [The compounds isolated from idin (5) . Phenylhilosa L. ,38,39. H effects ,41. 4′,7 effects , howevernceolata .2, like NO, is an important inflammatory mediator primarily involved in inducing pain and fever in damaged areas or tissues and is synthesized by COX-2. Excessive production of PGE2 causes excessive immune reactions, sclerosis, Parkinson’s disease, Alzheimer’s disease, and other inflammatory diseases [1) and 4′,7-dimethoxyflavanone (3) showed excellent inhibitory effects on NO production. Further investigation of the phenylheptatriyne (1) and 4′,7-dimethoxyflavanone (3) revealed similar effects on PGE2, TNF-α, and IL-6. However, since the yield of 4′,7-dimethoxyflavanone (3) was 0.8 mg, further studies could not be performed . Therefore, only phenylheptatriyne (1) was chosen for investigation of additional anti-inflammatory effects.NO is a free radical in the cardiovascular, nervous, and immune systems. It maintains intracellular homeostasis, transports neurotransmitters, and regulates anti-inflammatory activity and cytotoxicity . Howeverdiseases . Tumor ndiseases . In our 1) inhibits the expression of iNOS and COX-2, which produce NO and PGE2. As a result, phenylheptatriyne (1) inhibited the expression of the inflammatory proteins iNOS and COX-2  inhibits intranuclear translocation of IκB-α and p65 in LPS-stimulated BV2 and RAW264.7 cells. Phenylheptatriyne (1) inhibited the translocation of NF-κB in a concentration-dependent manner  expresses the antioxidant enzyme HO-1, and whether the expression of HO-1 was increased (1) was found to induce the expression of HO-1, and it was necessary to investigate whether the expression of HO-1 exhibits anti-inflammatory and anti-neuroinflammatory effects. Therefore, we investigated whether phenylheptatriyne (1) exhibited anti-inflammatory and anti-neuroinflammatory effects through HO-1. As a result, it was confirmed that only the phenylheptatriyne (1) treatment group inhibited nitrite compared to the phenylheptatriyne (1) treatment group treated with the HO-1 inhibitor SnPP in combination (1), isolated from C. lanceolata flowers, through HO-1 expression.HO-1 is known as a representative antioxidant enzyme which plays an important role in protecting cells from oxidative stress and is regulated by the transcriptional activity of nuclear factor erythroid 2-related factor 2 (Nrf2) ,54. Therncreased . Phenylhbination . This isC. lanceolata (CL19-001) were collected in August 2019 in Bulgap-myeon, Yeonggwang-si, Jeollanam-do. A voucher specimen (CL19-001) was deposited at the Natural Products Chemistry Laboratory, Chosun University .The flowers of 1H, 100 MHz for 13C) were obtained on a JNM ECP-400 spectrometer . Correlation spectroscopy (COSY), distortionless enhancement by polarization transfer (DEPT), heteronuclear single quantum correlation (HSQC), and heteronuclear multiple bond correlation (HMBC) were recorded using standard JEOL pulse sequences. 600 MHz NMR spectra were recorded at KBSI-Gwangju Center on a VNMRS 600 MHz NMR spectrometer  and chemical shifts are given in ppm (δ). The solvents used in the analysis, CDCl3 and DMSO-d6 were purchased from Sigma-Aldrich.Resins used for open column chromatography (C. C.) were silica gel 60 , LiChroprep RP-18 , Sephadex LH-20  and ODS-A  was used, and sand  was used to place the sample. Nuclear magnetic resonance (NMR) spectra  400 MHz (400 MHz for C. lanceolata (400 g) was extracted twice with 3 L of 70% ethanol at 80 °C to obtain 40 g of extract. The extract was sequentially fractionated using CH2Cl2, EtOAc, n-BuOH, and H2O to obtain 4.482 g of the CH2Cl2 fraction, 3.663 g of the EtOAc fraction, 8.051 g of the n-BuOH fraction, and 22.273 g of H2O fraction.Dried 2Cl2 fraction (4445 mg) was subjected to silica gel column chromatography (C.C.), and elution was performed using a hexane–acetone gradient system  to obtain seven subfractions (DM1-DM7). Fraction DM-1 was further chromatographed on silica gel C.C. by using a CHCl3-acetone isocratic system  to obtain four subfractions (DM11-DM14). Fraction DM11 was further chromatographed on silica gel C.C. by using a CHCl3-acetone isocratic system  to obtain compound 1 . Subsequently, the fraction DM-2 was further chromatographed on a silica gel C.C. by using a hexane-acetone gradient system  to obtain seven subfractions (DM21-DM27). Fraction DM24 was further chromatographed on a silica gel C.C. by using a hexane-acetone isocratic system  to obtain seven subfractions (DM241-DM247). Fraction DM241 was further chromatographed on a C-18 C.C. by using a MeOH-H2O gradient system  to obtain two subfractions (DM2411-DM2412). Fraction DM2411 was further chromatographed on a C-18 C.C. using a MeOH-H2O gradient system  to obtain seven subfractions (DM24111-DM24117), and compound 2  was obtained. Subsequently, fraction DM24116 was obtained by HPLC combined with a Kinetex® 5 µm EVO C18 100 Å LC column using a MeOH-H2O isocratic system  to obtain compound 3  (The CH 0.8 mg) .3-MeOH-H2O gradient system  to obtain two subfractions (EA1 and EA2). Fraction EA-1 was further chromatographed on ODS-A C.C. using a MeOH-H2O gradient system  to obtain six subfractions (EA11-EA16), including compound 4 . Subsequently, fraction EA14 was further chromatographed on ODS-A C.C. using a MeOH-H2O isocratic system  to yield nine subfractions (EA141-EA149). Fraction EA142 was further chromatographed on LH-20 C.C. using a CHCl3-MeOH isocratic system  to obtain four subfractions (EA1421-EA1424). Fraction EA1422 was further chromatographed at C-18 C.C. using a MeOH-H2O isocratic system  to obtain compound 5  (The EtOAc fraction (1500 mg) was subjected to silica gel C.C. by elution with a CHCl20.3 mg) .1 was obtained as a yellow gum. Based on the analysis of 1H- and 13C- NMR data, the molecular formula was determined to be C13H8. 1H-NMR : δ: 7.51-7.49 , 7.39-7.29 , 2.0 ; 13C-NMR : δ: 4.7 (C-1), 75.3 (C-2), 65.0 (C-3), 67.5 (C-4), 59.0 (C-5), 74.7 (C-6), 78.3 (C-7), 133.0 (C-2′), 128.5 (C-3′), 129.5 (C-4′), 128.5 (C-5′), 133.0 (C-6′).Compound 2 was obtained as a green gum. Based on the analysis of 1H- and 13C-NMR data, the molecular formula was determined to be C18H18O5. 1H-NMR : δ: 13.54 , 7.86 , 7.84 , 7.44  7.26 , 7.17 , 6.50 , 6.49 , 3.97 , 3.94 , 3.87 ; 13C-NMR : δ: 127.8 (C-1), 151.6 (C-4), 149.3 (C-5), 144.6 (C-7), 123.3 (C-8), 191.7 (C-9), 166.1 (C-1′), 168.6 (C-2′), 101.1 (C-3′), 107.6 (C-4′), 114.1 (C-5′), 110.1 (C-6′).Compound 3 was obtained as a yellow gum. Based on the analysis of 1H- and 13C- NMR data, the molecular formula was determined to be C17H16O4. 1H-NMR : δ: 7.87 , 7.41 , 6.96 , 6.62  6.48 , 5.42 , 3.84 , 3.83 , 3.06 , 2.80 ; 13C-NMR : δ: 79.8 (C-2), 44.1 (C-3), 190.9 (C-4), 128.7 (C-5), 110.2 (C-6), 166.1 (C-7), 100.8 (C-8), 163.6 (C-9), 114.7 (C-10), 130.6 (C-1′), 127.7 (C-2′ and C-6′), 114.0 (C-3′ and C-5′), 159.9 (C-4′), 55.6 (7′-OCH3), 55.4 (4′-OCH3).Compound 4 was obtained as a red gum. Based on the analysis of 1H- and 13C-NMR data, the molecular formula was determined to be C16H14O6. 1H-NMR : δ: 7.40 , 6.92 , 6.77 , 6.57  5.42 , 3.70 , 3.05 , 2.66 ; 13C-NMR : δ: 79.8 (C-2), 43.7 (C-3), 190.8 (C-4), 122.6 (C-5), 110.8 (C-6), 157.2 (C-7), 135.9 (C-8), 155.3 (C-9), 114.9 (C-10), 130.5 (C-1′), 118.3 (C-2′), 115.9 (C-3′), 145.7 (C-4′), 146.2 (C-5′), 114.8 (C-6′), 60.7 (OCH3).Compound 5 was obtained as a red gum. Based on the analysis of 1H- and 13C-NMR data, the molecular formula was determined to be C16H12O6. 1H-NMR : δ: 10.84 , 9.73 , 9.35 , 7.45 , 7.34 , 7.26 , 6.86 , 6.78 , 4.03 ; 13C-NMR : δ: 181.9 (C-3), 158.6 (C-8), 158.3 (C-6), 148.7 (C-2), 146.14 (C-3′), 146.06 (C-4′), 132.8 (C-7), 125.1 (C-6′), 123.8 (C-1′), 119.9 (C-2′), 118.4 (C-5′), 116.6 (C-4), 115.3 (C-9), 113.9 (C-10), 112.8 (C-5), 61.3 (7-OCH3).Compound 6 cells/dish) were seeded in 100 mm dishes in α-MEM (BV2) or RPMI-1640 (RAW264.7) containing streptomycin (100 μg/mL), 10% heat-inactivated FBS, and penicillin G (100 units/mL), and then incubated at 37 °C in a humidified atmosphere (5% CO2 and 95% air). All compounds were dissolved in dimethyl sulfoxide (DMSO) and used in each experiment. To determine cell viability, cells were maintained at a density of ~2 × 104 cells/well and then treated with compounds. After incubation for the indicated times, the cell culture medium was removed from each well and replaced with 200 μL of fresh medium in each well. The cells were incubated with 0.5 mg/mL of 3--2,5-diphenyltetrazolium bromide (MTT) for 1 h, and the formed formazan was dissolved in DMSO. The absorbance of the dissolved formazan was measured at a wavelength of 540 nm by using an ELISA microplate reader .Murine microglia BV2 cells and RAW264.7 cells were donated by Prof. Youn-Chul Kim, Wonkwang University . Cells , mouse microglia BV2 cells and mouse macrophage RAW 264.7 cells were treated with lipopolysaccharide (LPS) and the five compounds. After incubation for 18 h, the supernatant was mixed with the Griess Reagentand reacted. The measurements were then performed using an ELISA microplate reader at a wavelength of 570 nm (Molecular Devices).2 present in each sample was determined using a commercially available kit from R&D Systems . The assay was performed according to the manufacturer’s instructions.The culture medium was collected, and the level of PGEThe culture medium was collected, and the levels of IL-6 and TNF-α present in each sample were determined using a commercially available kit from BioLegend . The assay was performed according to the manufacturer’s instructions.Western blotting was performed as follows. After harvesting, the cells were pelleted by centrifugation for 3 min. Next, the cells were washed with PBS and lysed in 20 mM Tris-HCl buffer (pH 7.4) containing a protease inhibitor mixture . Protein concentration was determined using a Lowry protein assay kit (Sigma-Aldrich). Protein (30 mg) from each sample was resolved using 7.5% and 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then electrophoretically transferred onto a Hybond enhanced chemiluminescence (ECL) nitrocellulose membrane . The membrane was blocked with 5% skimmed milk and sequentially incubated with the primary antibody and a horseradish peroxidase-conjugated secondary antibody, followed by ECL detection . The following antibodies were used: IκBα, NF-κB p65, β-actin , HO-1 (Merck), iNOS , PCNA , and Anti-COX2/cyclooxygenase 2 . In addition, the cytosolic and nuclear fractions were extracted using the Cayman Nuclear Extraction Kit , and each fraction was lysed according to the manufacturer’s instructions.p < 0.05.All data were acquired from three independent experiments and expressed as mean ± SD. Statistical analysis was performed using the GraphPad Prism software version 3.03 . The mean difference was determined by one-way ANOVA, followed by Tukey’s multiple comparison test, and statistical significance was defined as C. lanceolata L. and investigated their anti-neuroinflammatory and anti-inflammatory effects on BV2 microglia and RAW264.7 macrophages. The isolated compounds were identified as phenylheptatriyne (1), 2′-hydroxy-3,4,4′-trimethoxychalcone (2), 4′, 7-dimethoxyflavanone (3), 8-methoxybutin (4), and leptosidin (5) through NMR analysis. Of these, phenylheptatriyne (1) was the most effective at inhibiting nitrite and PGE2 production in BV2 microglia and RAW264.7 macrophages. In addition, it was confirmed that phenylheptatriyne (1) has an inhibitory effect on the production of the inflammatory cytokines IL-6 and TNF-α. Phenylheptatriyne (1) also inhibited the expression of the pro-inflammatory proteins iNOS and COX-2 and prevented the translocation of p-IκBa and p65. It was also revealed that phenylheptatriyne (1) exhibits anti-inflammatory effects by inducing the expression of HO-1. Thus, phenylheptatriyne (1) has been proven to be beneficial as an anti-neuroinflammatory and anti-inflammatory agent.In this study, we isolated five compounds from the flowers of


Molecular structure reactivity and virtual screening were performed to check their biological efficacy as an anti-oxidant, anti-cancer and anti-diabetic agent. These studies were supported by in vitro analysis and QSAR studies. Results: After combined experimental and virtual screening 5c, 5g and 5e could serve as lead compounds, having low IC50 and high binding affinity. Objective: In this study, small molecules possessing tetrahydropyrimidine derivatives have been synthesized having halogenated benzyl derivatives and carboxylate linkage. As previously reported, FDA approved halogenated pyrimidine derivatives prompted us to synthesize novel compounds in order to evaluate their biological potential. Methodology: Eight pyrimidine derivatives have been synthesized from ethyl acetoacetate, secondary amine, aromatic benzaldehyde by adding catalytic amount of CuCl Heterocyclic compound chemistry has revolutionized the modern and versatile medicinal chemistry . These hd] pyrimidine structural framework.DHPMs become part of many medicinal and therapeutic agents which interact with DNA and RNA of the infected cells and terminate the cell division of infected cells by seizing DNA and RNA biosynthesis of these cells, as these DHPMs are structurally related to nucleotide bases of infected cells . PyrimidGenerally, in the past conventional methods for the synthesis of heterocyclic compounds were applied which was less efficient, time consuming and provided less yield . Followi2·2H2O as catalyst. Under these conditions, excellent yield and rapid products were obtained. Moreover, this synthetic method was advantageous over the conventional methods owing to its less solvent, economic, less time consuming and possessing single step properties [We have reported the synthesis of tetrahydrpyrimidine derivatives from the reaction mixture of urea/thiourea, substituting benzaldehydes and ethyl acetoacetate utilizing CuCloperties ,15.Targeted synthesized compounds were characterized to confirm their structures through the NMR spectral data  having fluoro substitution at para position with respect to pyrimidine moiety having oxo group showed best radical scavenging activity [5g with (IC50 = 6.539 µM) having meta substituted chloro benzyl derivative attached with pyrimidine ring with oxo linkage showed highest inhibitory potential for alpha amylase of all synthesized compounds which is attributed to its electron withdrawing effect [5e with meta substituted fluoro derivatives having thio linkage possessed the highest IC50 compared to control, as shown in According to limited structure activity relationship, all the activity . Compoung effect . Overall5a–h by docking with tyrosine kinase. Results obtained after docking analysis provide us with promising compound 5c, based on the best binding affinity value in comparison to standard , indicating a good binding affinity  are reactivity parameters. Electronegativity (X), electrophilicity index (ω), Dipole Moment (µ), Electronic energy (E), Ionization Potential (I), Chemical Potential (CP), Nucleophilicity Index (N) and electronic charges  or higher value of chemical potential (CP) refers to the electron delocalization. This means that a molecule can easily coordinate with a biological system by forming bonds. According to this criterion, increasing biological ranking should be as follows:5a has the highest nucleophilicity and lowest electrophilicity, it therefore showed seven hydrogen bonding interactions with highest MolDock compared to other compounds in case of protein ligand interaction with alpha amylase binding site.The next parameters are electrophilicity (ω) and nucleophilicity (N) indexes. An increase in biological activity is associated with an increasing value of nucleophilicity (N) index and decreasing value of electrophilicity (ω), therefore these molecules showed best binding affinities in case of alpha amylase protein interaction with these molecules for screening anti-diabetic activity virtually. Owing to this property, as The order of ranking should be:max is the charge of compounds. If ΔNmax increases then biological activities also increase. A high value of dipole moment of compound 5f is an indication of fine charge distribution and bond distance adjusted well. This explains that the molecule showing the process of oxidation reflects the best conductivity [ΔNuctivity ,42,43,445a–h is shown in Molecular electrostatic potential (MEP) helps us in assisting the inter and intra molecular interactions like hydrogen bonding, nucleophilic and electrophilic interactions with incoming molecules, and helps in recognition of biological interactions like molecular docking, drug protein interactions, etc. The MEP of the compounds 1H-NMR spectra were recorded on Bruker 300 MHz NMR spectrometer, and 13C-NMR on Bruker 25 and 175 MHz using DMSO-d6 as solvent and TMS as internal standard.The materials  used for synthesis were 95% pure of the brand Alfa Aesar Gemany and Daejung Korea, purchased from local vendor Musa ji Adam & Sons in Faisalabad, Pakistan. These were used as such, with no need for further purification. Aluminum pre-coated plates  were used for analytical thin layer chromatography with ethyl acetate/n-hexane (7:3) as eluent. The melting point of compounds was measured by Stuart melting point apparatus SP10. 2·H2O so that aromaticity could be achieved. Then the mixture obtained was left overnight for obtaining best results. After this the mixture was washed with cold water to remove excess copper salt, dried and re-crystallized with hot solvents to achieve pure products (5a–h). Progression of reaction was checked by TLC [For the synthesis of required compounds, ecofriendly, one-pot multi-component methodology was used with some modifications . In thisd by TLC ,47.2·2H2O by grinding for 7–0 min. After adding a few drops of HCl, they were again mixed for about 10 min. The mixture was then allowed to stayed overnight. Products obtained were purified by dissolving in methanol, by slightly heating the solution in a water bath. The solution was filtered off and allowed to re-crystallize. The fate of the performed reaction was determined with TLC.The appropriate amounts, i.e., 0.2 M (6.2 g) of fluoro-benzaldehyde, urea (3 g), thiourea (3.8 g) and ethyl acetoacetate (6.5 g), were mixed by adding a catalytic amount of CuCl5a): White shiny crystalline solid, soluble in DMSO, M.P = 230–233 °C; 1H-NMR : δ 9.26 , 7.70 , 7.30 , 7.26 , 7.11 , 7.17 , 5.44 , 3.94 , 2.26 , 1.05 ; 13C-NMR : δ 165.3 (C-1″), 100.3 (C-5), 60.2 (C-3″), 14.4 (C-4″), 17.6 (CH3), 146.1 (C-6), 54.07 (C-4), 152.0 (C-2), 143.9 (C-1′), 122.3 (C-2′), 161.3 (C-3′), 112.5 (C-4′), 130.2 (C-5′), 113.8 (C-6′); EIMS: m/z 278.12 (cacld. For C14H15FN2O3).4-(2-fluorophenyl)-6-methyl-2oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (5b): White shiny crystalline solid, soluble in DMSO, M.P = 210–213 °C; 1H-NMR: δ 9.27 , 7.81 , 7.40 , 7.36 , 7.28 , 6.98 , 5.17 , 4.03 , 2.50 , 1.10 ; 13C-NMR : δ 165.69 (C-1″), 152.41 (C-2), 149.19 (C-6), 144.21 (C-1′), 132.26 (C-3′), 129.35 (C-5′), 128.88 (C-4′), 128.66 (C-2′), 128.66 (C-6′), 99.29 (C-5), 59.77 (3″-CH2), 53.85 (C-4), 18.25 (CH3), 14.51 (4″-CH3); EIMS: m/z 278.12 (cacld. For C14H15FN2O3).ethyl 4-(3-fluorophenyl)-6-methyl-2oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (5c): White shiny crystalline solid, soluble in DMSO, M.P = 182–184 °C; 1H-NMR : δ 9.23 , 7.76 , 7.28 , 7.26 , 7.15 , 7.14 , 5.15 , 4.01 , 2.50 , 1.10 ; 13C-NMR : δ 165.70 (C-1″), 152.48 (C-2), 148.98 (C-6), 141.60 (C-1′), 128.66 (C-3′), 128.77 (C-5′), 115.70 (C-6′), 115.42 (C-2′), 160.17 (C-4′), 99.58 (C-5), 59.67 (3″-CH2), 53.81 (C-4), 18.23 (CH3), 14.49 (4″-CH3); EIMS: m/z 278.12 (cacld. For C14H15FN2O3).ethyl 4-(4-fluorophenyl)-6-methyl-2oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (5d): White shiny crystalline solid, soluble in DMSO, Decomposing temperature = 216–220 °C; 1H-NMR : δ 10.56 , 9.97 , 7.25 , 7.22 , 7.19 , 7.17 , 5.55 , 3.98 , 2.33 , 1.05 ; 13C-NMR : δ 165.02 (C-1″), 173.06 (C-2), 161.42 (C-6), 129.79 (C-1′), 115.24 (C-3′), 130.34 (C-4′), 125.24 (C-5′), 158.14 (C-2′), 130.52 (C-6′), 100.29 (C-5), 60.13 (C-4), 49.34 (C-3″); 17.56 (CH3), 14.24 (C-4″); EIMS: m/z 294.02 (cacld. For C14H15FN2O2S).ethyl 4-(2-fluorophenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (5e): White shiny crystalline solid, soluble in DMSO, M.P = 202–206 °C; 1H-NMR : δ 10.43 , 9.75 , 7.44 , 7.08 , 7.15 , 6.97 , 5.21 , 4.05 , 2.50 , 1.12 ; 13C-NMR : δ 165.43 (C-1″), 174.61 (C-2), 160.97 (C-6), 146.51 (C-1′), 164.21 (C-3′), 131.27 (C-5′), 113.73 (C-4′), 113.44 (C-2′), 122.87 (C-6′), 100.74 (C-5), 60.18 (3″-CH2), 53.99 (C-4), 17.67 (CH3), 14.46 (4″-CH3); EIMS: m/z 294.02 (cacld. For C14H15FN2O2S).ethyl 4-(3-fluorophenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (5f): White shiny crystalline solid, soluble in DMSO, M.P = 233–235 °C;1H-NMR : δ 10.43 , 9.77 , 7.27 , 7.24 , 7.18 , 7.15 , 5.20 , 4.04 , 2.30 , 1.11 ; 13C-NMR : δ 165.49 (C-1″), 174.57 (C-2), 163.60 (C-6), 140.23 (C-1′), 115.68 (C-3′), 115.68 (C-5′), 128.97 (C-6′), 128.86 (C-2′), 160.38 (C-4′), 101.95 (C-5), 60.09 (3″-CH2), 53.83 (C-4), 17.63 (CH3), 14.46 (4″-CH3); EIMS: m/z 294.02 (cacld. For C14H15FN2O2S).ethyl 4-(4-fluorophenyl)-6-methyl-2thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate  of chloro-benzaldehyde, urea (3 g), thiourea (3.8 g) and ethyl acetoacetate (6.5 g), were mixed by adding a catalytic amount of CuCl5g): White shiny crystalline solid, soluble in DMSO, M.P = 215–220 °C; 1H-NMR : δ 9.34 , 7.86 , 7.37 , 7.31 , 7.31 , 7.24 , 5.21 , 4.01 , 2.29 , 1.10 ; 13C-NMR : δ 165.6 (C-1″), 152.5 (C-2), 149.3 (C-6), 147.6 (C-1′), 133.4 (C-3′), 130.0 (C-5′), 127.6 (C-4′), 126.7 (C-2′), 125.3 (C-6′), 99.1 (C-5), 59.7 (3″-CH2), 54.1 (C-4), 18.2 (CH3), 14.4 (4″-CH3); EIMS: m/z 294.72 (cacld. For C14H15ClN2O3).ethyl 4-(3-chlorophenyl)-6-methyl-2oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (5h): White shiny crystalline solid, soluble in DMSO, M.P = 182–188 °C; 1H-NMR : δ 10.41 , 9.71 , 7.73 , 7.39 , 7.36 , 7.16 , 5.19 , 4.05 , 2.29 , 1.11 ; 13C-NMR : δ 174.5 (C-2), 165.3 (C-1″), 146.1 (C-6), 145.9 (C-1′), 133.5 (C-3′), 131.1 (C-5′), 128.1 (C-4′), 126.8 (C-2′), 125.5(C-6′), 100.6 (C-5), 60.2 (3″-CH2),54.0 (C-4), 17.6 (CH3), 14.4 (4″-CH3); EIMS: m/z 310.0 (cacld. For C14H15ClN2O2S).ethyl 4-(3-chlorophenyl)-6-methyl-2thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate  as free radical .5a–h having concentration 200 mM (0.022 g) were prepared. These stock solutions were then subjected to prepare their dilutions up to 250 µg, 200 µg, 150 µg, 100 µg, 50 µg and 25 µg respectively. DPPH solution of 4% was prepared by mixing 0.004 g of DPPH in 100 ml methanol . Ascorbic acid was used as control and its solution was prepared by mixing 0.0017612 g into 100 ml of water having 100 µg concentration.In order to perform anti-oxidant activity, stock solutions of compounds In test tubes 1 mL of sample from each diluted solution and 2 mL of DPPH solution was added. In the same way, 1 mL of the ascorbic acid was added along with 2 ml of DPPH as control and allowed to stand overnight. The next day, using Hitachi U-2900 spectrophotometer, readings at 517 nm were taken in triplicate and their inhibitory concentration calculated using formula .A=Acont5a–h were subjected to in vitro anti-diabetic activity using starch as substrate and alpha amylase as enzyme. Acarbose was used as standard drug [Synthesized compounds ard drug .In a test tube 0.5 mL of compound of each specific dilution and 0.5 mL of enzyme were added and kept in incubator at room temperature for 10 min, as done by our previous method . Reading2 in humidified atmosphere. Cells were treated with extracts/compound dissolved in DMSO with a final DMSO concentration of 0.05%. DMSO treated cells were used as control in all the experiments [The human HepG2 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fatal bovine serum (FBS), 100 unit’s/mL penicillin and 100 μg/mL streptomycin, and maintained at 37 °C with 5% COeriments .Cell viability was determined by MTT assay as described by us previously. Briefly, HepG2 cells were treated with different concentrations of compounds for 48 h. Following treatment, the MTT reagent was added (500 μg/mL) and cells were further incubated at 37 °C for 4 h. Subsequently, 150 μL DMSO was added to dissolve formazan crystals and absorbance was measured at 490 nm in a microplate reader . The percentage of cell viability was calculated .http://www.rcsb.org, accessed on 13–17 January 2021). PDB ID for anti-cancer activity is human serum albumin having ID (3B9L) with resolution of 2.60 Å. For antidiabetic activity, the crystal structure of human glucokinase having PDB ID (1V4S) with a resolution of 2.3 Å was used as target receptor site. In case of anti-oxidant activity, quercetin complex kinase having PDB ID (2HCK) with a resolution of 3.00 Å was used as target crystal structure. All these crystal structures were downloaded as PDB Format (gz) and then, after importing into MVD, their protein preparation was carried out before docking [All the synthesized compounds were drawn into 2D conformers by using ChemDraw Professional , then these structures were imported to chem3D Ultra . All the energy minimizations and other structural conformations, such as bond order, assigning missing bonds, explicit hydrogens and flexible torsional if absent in synthesized compounds, were optimized by using MM2 force field-Steepest Descent Algorithm in chem3D ,55. The  docking ,57,58,59Molecular docking studies based on synthesized compounds 5a-c was conducted by using Molegro Virtual Docker  and Discovery Studio Visualizer 2020. As studies revealed that MVD showed the best binding affinity in the form of Mol dock score as compared to other docking software like Surflex, Glide, Autodock Vina and FlexX ,60. PoseDensity functional language is a universal approach for defining the molecular structures of organic compounds and their activity relationships by making use of quantum-chemical descriptors and geometrical parameters . The mai5a–h was evaluated for their biological potential against diabetes, radical scavenge and cancer cell lines. After combinatorial in vitro and in silico analysis, compound 5c was pharmacologically active for anti-oxidant activity, 5e and 5c for cytotoxicity and 5g for combating diabetes. These potential leads had the best biological potential, and their biological activity can be enhanced by further futural derivatization, or by making their structural analogs. Hence, overall, the synthesized target molecules were proved to be potential therapeutic agents against different classes of inhibitor, thus opening new research interests regarding pyrimidine derivatives that may lead to evolution in medicinal systems and in the field of synthesis. The conclusion is that the synthetic pathway chosen here can be of primary interest for research communities and pharmacists, and the novel compounds synthesized here can be used in future as effective drugs due to their cell specificity, safety, inexpensiveness, sustainability, enhanced activity, eco-friendly nature and less time consuming and cost-effective synthetic method.Eight new compounds were synthesized in one-pot multi-component strategy by adopting solvent free methodology. All the compounds