Phaeosphaeria sp. LF5, an endophytic fungus with acetylcholinesterase (AChE) inhibitory activity isolated from Huperzia serrata, were investigated. Their structures and absolute configurations were elucidated by means of extensive spectroscopic data, including one- and two-dimensional nuclear magnetic resonance (NMR), high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) analyses, and calculations of electronic circular dichroism (ECD). A chemical study on the solid-cultured fungus LF5 resulted in 11 polyketide derivatives, which included three previously undescribed derivatives: aspilactonol I (4), 2-(1-hydroxyethyl)-6-methylisonicotinic acid (7), and 6,8-dihydroxy-3--isocoumarin (9), and two new natural-source-derived aspilactonols  . Moreover, the absolute configuration of de-O-methyldiaporthin (11) was identified for the first time. Compounds 4 and 11 exhibited inhibitory activity against AChE with half maximal inhibitory concentration (IC50) values of 6.26 and 21.18 µM, respectively. Aspilactonol I (4) is the first reported furanone AChE inhibitor (AChEI). The results indicated that Phaeosphaeria is a good source of polyketide derivatives. This study identified intriguing lead compounds for further research and development of new AChEIs.The secondary metabolites of  Natural products are important sources of active compounds and play important roles in modern drug research and development. Fungi are considered an important group of microorganisms in the production of antitumor, immunosuppressant, antibiotic, antifungal, antiparasitic, anti-inflammatory, enzyme-inhibiting, and other active secondary metabolites ,2. EndopHuperzia serrata is a member of the Lycophyllaceae family called shezucao in China +  in the HR-ESI-MS spectrum, indicating a molecular formula of C9H14O4, and two degrees of unsaturation. The 1H NMR data indicated two methyls at δH 1.20  and 1.39 ; one olefinic methane at δH 7.36 ; and three oxymethines at δH 3.58 , 3.60 , and 5.09 . The 13C-NMR spectra revealed one ester carbonyl (δC 176.5), one olefinic methine (δC 154.2), one nonprotonated sp2 carbon (δC 131.7), three oxygenated methines , one methylene (δC 29.6), and two methyls (δC 18.9 and 19.1).Compound δH 1.20)/C-8 (δC 74.8) and H-10 (δH 1.20)/C-9 (δC 71.6), as well as H-9 (δH 3.58)/C-7 (δC 29.6), H-9 (δH 3.58)/C-8, and H-9 (δH 3.58)/C-10. C-8 was correlated with C-3, C-7, and C-9 , C-9 was found to be connected to C-10, and C-8 was connected to C-9. Since C-8 and C-9 are methylene carbons and there is no nitrogen in the molecular formula, when combined with the chemical shift value, C-8 and C-9 were found to be connected to hydroxyl groups. H-7 is related to C-8, C-9, C-3, and C-4, and H-7 is methylene, but there were two groups of different H signals. Therefore, it can be inferred that one side of C-7 was connected to C-8. C-3 had the same characteristic signals as H-4, H-7, and H-8. The carbon shift signals of C-3 and C-4 were δC 131.7 and δC 154.2, respectively. HSQC indicated that C-4 was connected by protons, and it can be concluded that C-3 and C-4 were connected by a double bond and that C-4 was a quaternary carbon with two substitutions, one of which was connected with C-7. According to the HMBC cross-peak correlation of H-4/C-2 (δC 176.5), H-7/C-2, and C-3, we were able to infer that the side-chain fragment was attached to α,β-unsaturated-γ-lactone. HSQC indicated that C-2 was not connected to a proton. When combined with the molecular formula, C-4 was found to be a carbonyl group, H-4 was related to C-5, H-5 was related to C-6, and H-4 was related to HMBC. It was concluded that C-5 was connected to C-4, C-6 was connected to C-5, and the chemical shift value of C-5 was δC 79.8. When combined with the molecular formula, C-5 was also found to be connected to oxygen. Since the unsaturation degree of the compound was 2, the double bond between C-4 and C-3 occupied an unsaturation. An unsaturation remained, and there was no other double-bond carbon signal in the compound: hence, it is inferred that there was a cyclization system in which the chemical shift of C-2 was lower-field than that of the conventional carbonyl group. It was inferred that the other side of C-2 was connected to the oxygen and that C-5 and C-2 passed through the oxygen to form a lactone ring. The 1H  and 13C-NMR  data are listed in The HMBC spectrum showed correlations between H-10 /6-31G** level to obtain the accurate relative configuration of 4. In addition, the absolute configurations of 4  were established by comparing electronic circular dichroism (ECD) calculations at the PBE0/def2-TZVP level with the experimental one (R2) between experimental and calculated chemical shifts were shown, with 0.9985 for 4 -5--8, 9-dihydroxybutyl)-5-methylfuran-2(5H)-one, so compound 4 was named aspilactonol I, as shown in The ntal one . In addi85 for 4 , indicat7, a white powder soluble in MeOH, has the molecular formula of C9H11NO3 with four degrees of unsaturation from the protonated molecular ion at m/z 182.0810 [M + H]+ , as evidenced by HR-ESI-MS; the combination of 13C-NMR and 1H-NMR spectra showed that the compound contained six low-field carbon signals, including four substituted low-field carbons, two unsubstituted aromatic carbons, one submethyl carbon, and two methyl carbons, including one methyl carbon directly connected to the aromatic ring. The compound is an alkaloid, as verified by the bismuth potassium iodide reaction. It is inferred that the compound contained a nitrogen-containing heterocyclic ring. According to the HMBC signal (δH 7.67) was correlated with C-6, C-3, and C-9, and H-3 was strongly correlated with C-2, C-5, and C-9. Furthermore, the HMBC-related signal intensity was weakly correlated. Combined with hydrogen spectrum signal splitting, C-3 and C-5 were interpositionally substituted, and H-3 and H-5 were weakly correlated with the C-9 signal intensity. H-5 was correlated with C-6 and C-10, and H-10 was strongly correlated with C-6, indicating that C-10 was linked to C-6, and the chemical shift of C-6 was lower than that of conventional aromatic carbonization. It is inferred that C-6 was linked to heteroatom N, resulting in a low chemical shift field, and H-3 and C-2 (δC 166.9) were detected. The correlation signal of H-7 with C-3 and C-8 and the correlation hydrogen spectrum signal of H-8 split (d) indicated that C-2 was connected with a hydroxyethyl, and the abnormal chemical shift of C-2 indicates that it was connected to N. After assignment of the related signals, it was found that C-4 (δC 142.2) did not generate any related signals, and there were two oxygens in the molecular formula of the compound that had not been attributed. Thus, it was inferred that the compound contained carboxylic acid groups. Since the unsaturation degree of the compound was 4 and it was a nitrogen-containing alkaloid, the carboxylic acid groups should be connected with the pyridine ring  δH 7.89 , 7.67 , 4.89 , 2.59  and 1.46 ; 13C-NMR  δC 168.4 (C-9), 166.9 (C-1), 159.5 (C-6), 142.2 (C-4), 122.6 (C-5), 117.5 (C-3), 71.1 (C-3), 24.4 (C-8), and 23.7 (C-10).Compound C signal , H-5  and a positive Cotton effect at 286 nm (Δε +7.76), which displayed strong agreement with the calculated ECD curve of S-7 (7 was (7S). The structure of compound 7 was determined to be 2-(1-hydroxyethyl)-6-methylisonicotinic acid.To confirm the stereochemical assignments of e of S-7 . Thus, t9 was isolated as a light yellow powder, and the HR-ESI-MS data showed a molecule peak at m/z 253.0655 [M + H]+ , which indicated the molecular formula as C12H12O6 and 7 as the index of hydrogen deficiency; 1H-NMR  δH 1.12–1.14 , 3.77–3.85 , 3.97–4.00 , 3.75–3.77 , 5.64–5.55 , 6.34 , 6.44 , 6.61 , 10.85 , 11.00 ; and 13C-NMR  δC 165.5(C-1), 165.3 (C-6), 162.5 (C-8), 157.6 (C-3), 139.2 (C-4a), 104.4 (C-4), 102.9 (C-5), 101.5 (C-7), 97.8 (C-8a), 74.4 (C-1′), 67.4 (C-2′), and 19.7 (C-3′)  of the compound showed 12 carbon signals, including five oxygen-linked aromatic carbons, four oxygen-free aromatic carbons, two oxygen-linked methylene carbons, and one methyl carbon. The unsaturation of the compound was calculated as 6 according to the molecular formula of the compound, and it was inferred that the compound contained two amphioxic heterocyclic systems. H-7 (δ 6.33) was correlated with C-5 (δ 102.9), C-8a (δ 97.8), C-8 (δ 162.5), and C-6 (δ 165.3), and H-5 was correlated with C-4 (δ 104.4), C-6 (δ 165.3), and C-8a (δ 97.8). Among them, oxygen substitution existed in C-6 (δ 165.3), C-8 (δ 162.5), and C-1 (δ 165.54). According to a comparison of the hydrogen spectra of CD3OD, C-6 and C-8 are hydroxyl substitutions, and C-1 is the carbonyl group. The abnormal chemical shift of the C-8 hydroxyl group (δ 11.00) indicated that it was greatly shifted to the lower field under the influence of the neighboring carbonyl group. The low-field shift for C-3 (δ 157.6) and the HSQC signal suggested the existence of substitution. On the basis of a combination with the HMBC and chemical shift characteristics of C-3 and C-1, we inferred that the two carbons were connected by oxygen, resulting in a large chemical shift to the low field -isocoumarin (Compound 7 (C-3′) . The 1H-ow field . On the ulations . Finallycoumarin .1–3, 5–6, 8, and 10–11 by comparing their spectroscopic data to those reported in the literature. Details of NMR and MS data for compounds 1–11 were given in the In addition to the new compounds described above, eight known compounds obtained in this study were identified as compounds 1 was obtained as a colorless powder, and the HR-ESI-MS data showed a molecule peak at m/z 129.0552 [M + H]+ , which indicated a molecular formula of C6H8O3 with three degrees of unsaturation. In examining the proton nuclear magnetic resonance (1H-NMR) data, we found signals for methyl protons δH 1.41 , one methylene proton δH 4.28 , and two methines (two oxygenated sp3 and one sp2): δH 5.11–5.16  and δH 7.42–7.47 . The 13C-NMR spectra revealed six carbon signals: one ester carbonyl (δC 174.53), one olefinic methine (δC 152.93), one nonprotonated sp2 carbon (δC 134.75), two oxygenated methines (δC 80.13), one oxygenated methylene (δC 56.95), and methyls (δC 19.02). The structure of 1 was determined as 3-(hydroxymethyl)-5-methylfuran-2(5H)-one [Compound 2 was a white powder with the molecular formula (C7H8O4): HR-ESI-MS m/z 157.0493 [M + H]+ ; 1H-NMR  δH 1.33 , 5.05–5.09 , and 7.41–7.45 ; and 13C-NMR  δC 18.93 (C-CH3), 31.08 (C-2′), 80.00 (C-5), 127.99 (C-3), 155.38 (C-4), 173.10 (C-2), and 175.47 (C-1′). Thus, compound 2 was identified as [2--acetic acid] by comparing NMR reference data  by comparing NMR reference data + ; 1H-NMR  δH 2.11 , 2.30–2.34 , 3.67–3.70 , and 5.76 ; and 13C-NMR  δC 18.5, 44.6, 60.9, 120.4, 152.7, and 172.1. Compound 5 is E-Δ2-anhydromevalonic acid [Compound 6 yielded the following data: HR-ESI-MS m/z 113.0604 [M + H]+ ; 1H-NMR , δH 5.79 , 4.38 , 2.41 , and 2.02 ; and 13C-NMR  δC 22.6, 28.8, 65.6, 116.2, 158.0, and 164.4. Compound 6 is 4-methyl-5,6-dihydropyren-2-one +  and 13C-NMR data, corresponding to six degrees of unsaturation; 1H-NMR  δH 6. 21 , 6.20 , 4.63–4.67 , 2.92 , 2.82 , and 1.45 ; and 13C-NMR  δC 170.30 (C-1), 164.99 (C-8), 164.23 (C-6), 142.08 (C-4a), 106.53 (C-5), 100.82 (C-8a), 100.0 (C-7), 75.77 (C-3), 34.13 (C-4), and 19.44 (C-9). Compound 8 was identified as (R)-6-hydroxymellein + ; 1H-NMR  δH 6.39 , 6.29 , 6.24 , 3.78 , and 2.10 ; and 13C-NMR  δC 165.1 , 163.6 123 , 158.3 , 155.0 , 142.2 , 103.5 , 102.9 , 100.4 , 99.2 , 56.8 , and 19.4 . Compound 10 is 6-hydroxy-8-methoxy-3-methylisocoumarin + ; 1H-NMR , δH 6.40 , 6.37 , 4.69 , 2.59 , and 1.26 ; and 13C-NMR , δC 167.8 , 167.3 , 164.8 , 156.2 , 141.3 , 107.0 , 103.7 , 102.6 , 99.8 , 66.2 , 43.8 , and 23.3 . The absolute configuration of compound 11 was established to be 2′R by the ECD calculations  was first reported in 1988 [4 and isocoumarin compound 11 were found to have the potential to inhibit AChE. To the best of our knowledge, furanone compounds were reported here for the first time for their AChE inhibitory activity. Their mechanisms of action and structure–activity relationships in inhibiting AChE require further study by inhibition kinetics analysis and molecular docking methods.Structurally, almost all of the furanone compounds in this study contained an α,β-unsaturated carboxylic acid lactone moiety, which might be the key functional group for their biological activity. De- in 1988 ,35. It c in 1988 . AChEIs  in 1988 . Existin4), 2-(1-hydroxyethyl)-6-methylisonicotinic acid (7), and 6, 8-dihydroxy-3--isocoumarin (9), and two new natural-sources-derived aspilactonols   were isolated from an endophytic fungus Phaeosphaeria sp. LF5 of H. serrata. Their absolute configurations of three new compounds  and known compound 11 were determined by ECD calculations. Furanone compound 4 and isocoumarin compound 11 exhibited potent AChE inhibitory activities. This study indicates that Phaeosphaeria sp. LF5 from H. serrata may contain various AChEI compounds, which is a potential resource pool for bioprospecting and isolating AChEIs. Furthermore, this research also provided a material basis for the development of new and efficient AChEI drugs.To summarize, 11 polyketide derivatives, which included three new compounds, aspilactonol I (


H-indole derivatives (2–12) were designed by pairing the pyridine/pyrane/pyrimidine/pyrazole heterocycles with indole and thiophene to investigate their potential activities as  inhibitors. The purpose of these derivatives’ modification is to create high-efficiency antioxidants, especially against ABTS, as a result of the efficiency of this set of key heterocycles in the inhibition of ROS. Herein, 2D QSAR modeling was performed to recommend the most promising members for further in vitro investigations. Furthermore, the pharmacological assay for antioxidant activity evaluation of the yielded indole-based heterocycles was tested against ABTS ; by utilizing ascorbic acid as the standard. Candidate 10 showed higher antioxidant activity (IC50 = 28.23 μg/mL) than ascorbic acid itself which achieved (IC50 = 30.03 μg/mL). Moreover, molecular docking studies were performed for the newly designed and synthesized drug candidates to propose their mechanism of action as promising cytochrome c peroxidase inhibitors compared to ascorbic acid as a reference standard. Our findings could be promising in the medicinal chemistry scope for further optimization of the newly designed and synthesized compounds regarding the introduced structure-activity relationship study (SAR) in order to get a superior antioxidant lead compound in the near future.Novel candidates of 3-(4-(thiophen-2-yl)-pyridin/pyran/pyrimidin/pyrazol-2-yl)-1 Reactive oxygen species (ROS) are a normal product of cellular metabolism in a human cell. Some of these radicals are required for regular cell functions, including neurological signal transmission . OtherwiDuring normal physiological conditions, most species are formed on a small scale and are scavenged by intracellular antioxidant systems such as small molecules; vitamins C and E, and superoxide dismutase (SOD) . NeverthAscorbic acid (vitamin C) is present in many fruits and vegetables such as oranges, guava, lemons, berries, broccoli, mango, and peppers, which is crucial in the human diet. Szent-Györgyi first isolated ascorbic as an “acidic carbohydrate” from adrenal glands, lemons, cabbages, and oranges in 1928, and in 1933, Norman Haworth illustrated the chemical structure of vitamin C . In the Admittedly, heterocyclic moieties have vital biological activities, particularly that containing nitrogen, oxygen, and sulfur atoms ,11. BaseOne of the best-known computational tools applied in medicinal chemistry is the quantitative structure–activity relationship (QSAR). QSAR can predict the potential of various chemicals and their biological activities by statistical calculations. It gave impressive, truthful, and realistic results if the basic data were available for measurement on its basis in advance . AdditioFurthermore, we studied our compounds with molecular docking methodology, predicting the prevailing binding mode(s) of a ligand with a protein in the three-dimensional structure. Docking is an extremely useful strategy in optimizing the best candidates in the fastest and least expensive way by conducting virtual screening on large libraries of drugs, ranking the outcomes, and providing structural assumptions about how the ligands inhibit the target .Based on the aforementioned information, we designed and synthesized a new series of promising antioxidants by choosing different reducing moieties  which are corresponding to the reducing ascorbic acid ring and incorporating each in between two potential antioxidant moieties  using the molecular association approach in The molecular association was recommended to produce an additive or synergistic action. Moreover, it was approved to provide a broader spectrum of activity as well .1). The essential compound (1) that would be used in synthesizing the target indolyl derivatives (2–12); was conducted as shown in i) was prepared as reported previously through the Acylation of 1-(Pheny1sulfonyl)indole according to Friedel-Crafts method [H-indol-3-yl)-3-(thiophen-2-yl)prop-2-en-1-one (1) was developed by the condensation reaction of 3-acetyl indole (i) and thiophene-2-carbaldehyde (ii) in a basic medium (NaOH 40%). The chalcone structure 1 was elucidated with IR, 13C-NMR, 1H-NMR spectra, elemental analysis data, and mass spectroscopy.In our strategy to design and synthesize novel heterocyclic moieties as antioxidants, including indole nucleus, we synthesized a chalcone  at 7.17 and 7.6 ppm with coupling constant J = 8.0 and 7.5 Hz, respectively. 13C-NMR spectra showed carbonyl carbon at 183.6 ppm and α,β-unsaturated carbons at 128.9 and 135 ppm.The IR spectrum of 2, 3, and 4, respectively, as shown in 1H-NMR, 13C-NMR spectra and mass spectroscopy, and elemental analysis data. 4-(1H-indol-3-yl)-6-(thiophen-2-yl)pyrimidine-2-thiol (2) can be obtained by reacting thiourea with the start material under reflux for 13 h in the presence of sodium ethoxide as a base.In the present approach, we aimed to synthesize novel candidates over three preparing lines. Firstly, to discover a novel series of drugs containing a pyrimidine ring with indole moieties. We utilized highly functionalized reagents as thiourea, urea, and guanidine to give 2 illustrated a broad peak at 3350–3600 cm−1 due to 2NH groups stretching. The carbonyl of started chalcone disappeared, and the thione group that resulted from the formation of the pyrimidine ring via hetero-cyclization appeared at 860 and 825 cm−1. Moreover, the thiol group appeared at 2320 cm−1, which indicated that they are in tautomerism conformation. 1H-NMR of (2) showed a singlet peak for methine carbon (C11) at 8.65 ppm. While, NH, SH groups at 12.074 and 11.95 ppm, respectively. The structure of 4-(1H-indol-3-yl)-6-(thiophen-2-yl)pyrimidin-2-ol (3) was demonstrated by the IR spectrum. As OH and NH group depict stretching frequency in wide rang 3350–3560 cm−1 as a broadband. The presence of OH and NH groups was confirmed by 1H-NMR at values 10.76 and 12.08 ppm, respectively. Cyclization as pyrimidine ring illustrated the disappearance value of the ketonic-carbonyl group and the appearance of the carbinol (C20) at 162.4 ppm in 13C-NMR. 4-(1H-indol-3-yl)-6-(thiophen-2-yl)pyrimidin-2-ol (3) was afforded as yellow crystals by reacting the starting chalcone with urea using potassium hydroxide as a catalyst. On the other hand, 4-(1H-indol-3-yl)-6-(thiophen-2-yl)pyrimidin-2-amine (4) was synthesized by the reaction of the starting chalcone 1 with guanidine carbonate in dry ethanol and anhydrous sodium acetate. The IR spectrum estimated this compound’s structure by appearing as a broadband at 3400–3576 cm−1 due to NH and NH2 groups’ stretching frequency.The IR spectrum of α,β-ethylenic ketone 1 with hydrazine in the presence of glacial acetic acid under refluxing for 7 h yielded yellow crystals of 3-(5-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-3-yl)-1H-indole (5). Under the same condition, 3-(1-phenyl-5-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-3-yl)-1H-indole (6) was synthesized by refluxing phenylhydrazine with the start chalone 1. The chemical structures of compounds 5 and 6 were demonstrated with IR, 1H-NMR, 13C-NMR spectra, and mass spectroscopy, and elemental analysis data.The second line in our schematic strategy is to synthesize indolylpyrazoles, as shown in 5 was confirmed via IR spectrum through the stretching value of the carbonyl group and disappearance and methylene group (C11) appearance in 1H-NMR as a doublet peak at 3.48 ppm with a coupling constant J = 14.5 and 12.2 Hz. Additionally, the methine group (C12) appeared at 5.003 ppm with J = 7.0 Hz as a triplet peak. In the same strategy, the structure of 6 was elucidated. In 1H-NMR, the methylene carbon emerged at 3.32 ppm with J = 15.7 and 13.2 Hz as a doublet peak and methine carbon at 5.65 ppm and J = 6.5 Hz as a triblet peak.The structure of 7–10) was depicted in 7 and 10 were synthesized via cyclo-condensation of the chalcone 1 with different reagents in a sodium hydroxide (10%) basic medium as a catalyst. 6-(1-hydroxyethylidene)-3-(1H-indol-3-yl)-5-(thiophen-2-yl)cyclohex-2-en-1-one (7) was prepared by addition of acetylacetone to chalcone 1 in equilibrium quantities under refluxing for 5 h. Similarly, ethyl 4-(1H-indol-3-yl)-2-oxo-6-(thiophen-2-yl)cyclohex-3-ene-1-carboxylate (10) was synthesized under the same conditions but with using ethyl acetoacetate as a reagent. Whereas, the 2-pyranone ring 6-(1H-indol-3-yl)-2-oxo-4-(thiophen-2-yl)-2H-pyran-3-carbonitrile (8) was synthesized through the reaction of chalcone 1 with ethyl cyanoacetate in the presence of sodium ethoxide as a catalyst at room temperature. Treatment of compound 8 with hydrazine hydrate under refluxing for 6 h converted the pyranone ring to 2-pyridone to give 1-amino-6-(1H-indol-3-yl)-2-oxo-4-(thiophen-2-yl)-1,2-dihydropyridine-3-carbonitrile (9). The structures of compounds 7, 8, 9, and 10 were elucidated by IR, 1H-NMR, 13C-NMR spectra, and mass spectroscopy, also elemental analysis . The third synthetic approach to synthesize the functional compounds (H-indol-3-yl)-4-(thiophen-2-yl)-4H-pyran-3-carbonitrile (11) with a poly-functional pyrane ring, which can pave the way to synthesize a new series of novel compounds (12a–e) 11 with various aldehydes via different condensation reactions. Spectroscopic analysis  revealed that all derivatives formed via condensation of 11 with various aldehydes are open and not heterocyclized with the cyano group. Compound 11 was demonstrated by exhibiting the main functional groups in IR spectra at 2214 cm−1 (CN), and 3465–3584 cm−1 . Additionally, 1H-NMR spectra exhibited NH2 at δ 10.8 ppm, and (CH) methine groups at δ 4.51, 5.42 ppm. Furthermore, all carbons were observed in the 13C-NMR spectrum, as shown in the experimental part. In addition, all chemical structures of compounds 12a–e were illustrated in the IR spectrum by the observed imine group at the range of 1654 cm−1, and the cyano group still presented at 2212 cm−1. The experimental part observed further spectroscopic analysis .Finally, the reaction of malononitrile with the start chalcone yielded 2-amino-6- assay. The in vitro antioxidant assay (ABTS test) showed varied significant levels of free radical inhibition activity of indolyl derivatives H-indol-3-yl)-2-oxo-6-(thiophen-2-yl)cyclohex-3-ene-1-carboxylate 10 showed the highest antioxidant activity (IC50 = 28.23 μg/mL) which was found to be superior to the antioxidant activity of L-ascorbic acid (IC50 = 30.23 μg/mL), as a reference antioxidant compound. While 8, 3, 2, and 7 exhibited moderate antioxidant properties . Finally, the rest of the tested compounds displayed modest activities compared to L-ascorbic acid (IC50 = 30.03 μg/mL), as shown in The recorded results revealed that our candidates might be arranged into three levels according to their activities. Firstly, ethyl-4- as shown in On the other hand, compounds (1)10, 8, 3, 2, and 7) was found to be superior to its attachment to chalcone moieties .The introduction of simple six-membered rings in the linker region in between the indole and thiophene rings ((2)2), corresponding to compounds 3, 2, and 4, respectively.The introduction of hydrophilic functional groups in position 2 of the pyrimidine linker was observed to be in the following order (−OH > −SH > −NH(3)p-NO2 benzaldehyde derivative (12b) was better than the m-NO2 one (12d).The chalcone linker formed by (4)The presence of additional oxygen functional groups such as carbonyl or hydroxyl, even methoxy groups in the linker six-membered ring increases the antioxidant properties of these moieties.Regarding the structure-activity relationship study of the newly designed and synthesized candidates c peroxidase enzyme (PDB code: 2X08) [c peroxidase through the formation of four H-bonds with Gly41, His181, Arg184, and Val45 amino acids. Molecular docking results of the two most promising compounds (10 and 8) compared to the docked ascorbic acid as a reference standard were depicted in In our way to study the proposed mechanism of action for the newly designed and synthesized drug candidates as promising antioxidants compared to ascorbic acid as a reference standard, we performed the molecular docking study for the prepared database containing the previously mentioned new compounds besides the co-crystallized ascorbic acid extracted from cytochrome e: 2X08) . It was 10 and 8) got stabilized inside the binding pocket of cytochrome c peroxidase with very promising binding scores of −7.49 and −7.24 kcal/mol, respectively, compared to that of ascorbic acid . Moreover, ascorbic acid formed three H-bonds with Met172 and His175 at 3.21, 3.29, and 3.32 Å, respectively. On the other hand, compound (10) was stabilized inside the binding pocket of cytochrome c peroxidase by forming only one H-bond with Ser185 and one pi-pi interaction with His175 at 2.98 and 3.82, respectively, indicating a great binding affinity and an expecting intrinsic activity as well. However, compound (8) bound Arg184, Ser185, and His181 with three H-bonds at 2.89, 3.15, and 3.19 Å, respectively. Moreover, it formed a fourth H-pi interaction with His175 at 4.46 Å as represented in H-indol-3-yl) derivatives was developed [2 value of 0.09652. 50 are evaluated according to the QSAR model by the following equation:QSAR prediction is an in silico tool extensively, and it is applied to evaluate the structure-activity relationship of newly synthesized compounds. By using MOE software, a QSAR model of  can be calculated by the following equation:−1 on (Thermo-Fisher Scientific) FT-IR PLUS spectrometer (υ by cm−1) utilizing potassium bromide disks (KBr) at the microanalytical Laboratory, Faculty of Science, Cairo University, Egypt. C, H, and N analyses were carried out on a PerkinElmer CHN 2400. 1H and 13C-NMR spectra were carried out on a Bruker NMR spectrophotometer at 400 MHz in DMSO-6d using tetramethylsilane (TMS) as the internal reference standard, chemical shifts are expressed in δ which given in parts per million (ppm), and DMSO-6d was used as the solvent. All spectral analysis data are provided in the All chemicals included 3-Acetylindole were provided from Sigma-Aldrich , and all solvents included annular ethanol delivered from El-NASR Co., Egypt. All the reactions were carried out with readily available reagents that were utilized without additional purification as received. Digital Electrothermal IA 9100 Series apparatus Cole-Parmer, Beacon Road, Stone, Staffordshire, UK) used to measure the melting points that they are uncorrected. Mass spectra were recorded in the Thermo scientific GCMS model (ISQLT) via the direct probe controller inlet part to a single quadrupole mass analyzer utilizing Thermo X-Cali bur software, at Al-Azhar University, (RCMB), Naser City, Cairo. IR spectra were carried out in the range from 4000 to 400 cmH-indol-3-yl)-3-(thiophen-2-yl)prop-2-en-1-one (1)1-: 3400–3500 cm−1 , 3091 cm−1 (CH-Aryl), 2975 cm−1 (CH-Aliphatic), 1650 cm−1 (C=O). 1H-NMR : δ = 7.17 , 7.6 , 7.18–7.82 , 8.67 , and 12.1 . 13C-NMR : δ = 128.9 ppm (CO-CH=CH), 135 ppm (CO-CH=CH), 183.6 ppm (CO), 129, 131, 132, 140 ppm (thiophene), 112.66, 117.94, 122.22, 123.58, 123.68, 126.32, 137.33 ppm (indolyl carbons). MS (M/Z): M+• (253.20), Base-peak (57). Anal. Calcd. for C15H11NOS (253.32): C, 71.12; H, 4.38; N, 5.53; S, 12.66. Found: C, 71.31; H, 4.21; N, 5.65; S, 12.73.A few drops of 40% NaOH aqueous solution were added portion-wise to a mixture of 3-acetylindole  and thiophene-2-carbaldehyde  in 50 mL ethanol. The reaction mixture was stirred for 1h at room temperature. Filtration gave a pale-yellow precipitate. A 4% aqueous HCl was utilized to wash the precipitate and then were crystallized from ethanol to give chalcone H-indol-3-yl)-6-(thiophen-2-yl)pyrimidine-2-thiol (2)4- in 40 mL of absolute ethanol) was added to a mixture of Chalcone 1 (10 mmol) and thiourea (10 mmol). The reaction mixture was refluxed for 13 h. After cooling, the mixture was neutralized with diluted hydrochloric acid in crushed ice, filtered, washed with ethanol, and dried. Crystallization from ethanol afforded the pyrimidine-2-thiol 2 as yellow crystals. Yield 66%. m.p.: 250–255 °C. IR : 3350–3600 cm−1 , 3039 cm−1 (CH-Aryl), 2919 cm−1 (CH-Aliphatic), 2229 cm−1 (CN), 2320 cm−1 (thiol), 1588 cm−1 (C=C), 860, 825 cm−1 (thione group). 1H-NMR : δ = 8.65 , 7.46–7.51 , 7.58–7.81 , 12.074 , and 11.95 . 13C-NMR : δ = 181.4 ppm (C-SH), 127–142 ppm (thiophene), 111–136 ppm (indolyl carbons). MS (M/Z): M+• (309.00), Base-peak (253). Anal. Calcd. for C16H11N3S2 (309.41): C, 62.11; H, 3.58; N, 13.58; S, 20.72. Found: C, 62.26; H, 3.39; N, 13.46; S, 20.83.A freshly prepared EtOH-indol-3-yl)-6-(thiophen-2-yl)pyrimidin-2-ol (3)4-(11 (10 mmol) and urea (10 mmol). The reaction mixture was refluxed for 7 h. After cooling, the mixture was neutralized with diluted hydrochloric acid in crushed ice, filtered, washed with ethanol, and dried. Crystallization from ethanol afforded the pyrimidine-2-ol 3 as yellow crystals. Yield 82%. m.p.: 266–270°C. IR : 3350–3560 cm−1 , 3008 cm−1 (Aromatic-CH), 2975 cm−1 (CH-Aliphatic). 1H-NMR : δ = 7.18–7.51 , 7.58–8.30 , 10.76 , and 12.08 . 13C-NMR : δ = 162.4 ppm (C-OH), 130–141 ppm (thiophene), 115–132 ppm (indolyl carbons). MS (M/Z): M+• (294.15), Base-peak (253). Anal. Calcd. for C16H11N3OS (293.34): C, 65.51; H, 3.78; N, 14.32; S, 10.93. Found: C, 65.32; H, 3.91; N, 14.19; S, 11.09.A potassium hydroxide (10 mmol) was added to a mixture of Chalcone H-indol-3-yl)-6-(thiophen-2-yl)pyrimidin-2-amine (4)4-(11 with guanidine carbonate (10 mmol) in dry ethanol, then (10 mmol) of anhydrous sodium acetate was added. The reaction mixture was refluxed on a water bath for 4 h. After cooling, the mixture was neutralized with diluted hydrochloric acid in crushed ice, filtered, then washed with ethanol, and finally dried. After that, afforded precipitate was crystallized from ethanol to give the pyrimidine-2-amine 4 as pale-yellow crystals. Yield 75%. m.p.: 270–273 °C. IR : 3400–3576 cm−1 , 3148 cm−1 , 3039 cm−1 (CH-Aryl), 2918 cm−1 (CH-Aliphatic). 1H-NMR : δ = 8.64 , 7.14–7.81 , 7.48–8.57 , 12.04 , and 7.08 . 13C-NMR : δ = 111.2, 114.3, 123.5, 126.02, 128.2, 128.9, 132.4, 134.7, 136.5, 138.4, 140.1, 142.3, 160.4, and 163.5 ppm. MS (M/Z): M+• (292.56), Base-peak (253). Anal. Calcd. for C16H12N4S (292.36): C, 65.73; H, 4.14; N, 19.16; S, 10.97. Found: C, 65.57; H, 4.29; N, 19.31; S, 10.79. By mixing (1 mmol) of chalcone H-pyrazol-3-yl)-1H-indole (5)3-(5-(thiophen-2-yl)-4,5-dihydro-11 (10 mmol) in 30 mL absolute ethanol, hydrazine hydrate , and a few drops of glacial acetic acid were added, followed by refluxing the reaction mixture for 7 h. After cooling, the mixture was poured onto ice water. The crude product was filtered off and crystallized from ethanol to give pyrazolo indole 5 as yellow crystals. Yield 60%. m.p.: 210–215 °C. IR : 3465–3534 cm−1 , 3039 cm−1 (CH-Aryl), 2972 cm−1 (CH-Aliphatic). 1H-NMR : δ = 3.48 , 5.003 , 6.96–7.1 , 7.18–7.59 , 8.12 , and 11.39 . 13C-NMR : δ = 44.2, 53.7, 112.2, 116.20, 122.5, 125.46, 127.5, 128.9, 133.4, 134.8, 136.05, 138.4, 142.1, and 145.3 ppm. MS (M/Z): M+• (267.15), Base-peak (253). Anal. Calcd. for C15H13N3S (267.35): C, 67.39; H, 4.90; N, 15.72; S, 11.99. Found: C, 67.21; H, 5.01; N, 15.65; S, 12.08.To a solution of the started chalcone H-pyrazol-3-yl)-1H-indole (6)3-(1-phenyl-5-(thiophen-2-yl)-4,5-dihydro-11 (10 mmol) in 30 mL glacial acetic acid, phenylhydrazine (40 mmol) was added, then refluxing the reaction mixture for 7 h. After that, the reaction mixture was cooled then poured onto ice water. After filtration, the product was washed with water till neutralization, then recrystallized from ethanol to give pyrazolo indole 6 as yellow crystals. Yield 60%. m.p.: 210–215 °C. IR : 3403 cm−1 , 3038 cm−1 (CH-Aryl), 2973 cm−1 (CH-Aliphatic). 1H-NMR : δ = 3.32 , 5.65 , 6.68–7.47 , 7.51–8.34 , and 12.08 . 13C-NMR : δ = 42.2, 58.7, 112.4, 113.7, 116.7, 119.2, 121.4, 122.2, 124.46, 126.03, 127.5, 128.9, 133.4, 134.8, 136.05, 138.4, 141.1, and 152.3 ppm. MS (M/Z): M+• (343.75), Base-peak (253). Anal. Calcd. for C21H17N3S (343.45): C, 73.44; H, 4.99; N, 12.24; S, 9.33. Found: C, 73.31; H, 5.08; N, 12.34; S, 9.25.To a solution of the started chalcone H-indol-3-yl)-5-(thiophen-2-yl)cyclohex-2-en-1-one (7)6-(1-hydroxyethylidene)-3-(11 (8 mmol) and acetylacetone (8 mmol) in ethanol (20 mL) in the presence of 10% NaOH (1 mL) was refluxed for 5 h. After cooling, the product was filtered and recrystallized from ethanol to yield the yellow crystals of 7. Yield 80%. m.p.: 240–242 °C. IR : 3423 cm−1 , 3074 cm−1 (Aromatic-CH), 2974 cm−1 (CH-Aliphatic). 1H-NMR : δ = 2.4 3CH), 2.49;2.51 , 3.1 , 6.9–7.46 , 7.1–8.3 , and 10.7 . 13C-NMR : δ = 45.1, 47.6, 52.4, 57.6, 112.4, 113.7, 115.7, 120.2, 121.1, 122.4, 123.46, 125.03, 128.5, 129.9, 131.4, 135.8, 137.05, 138.4, 141.1, 158.4, and 161.3 ppm. MS (M/Z): M+• (336.10), Base-peak (253). Anal. Calcd. for C20H17NO2S (335.42): C, 71.62; H, 5.11; N, 4.18; S, 9.56. Found: C, 71.41; H, 5.38; N, 4.04; S, 9.71.A mixture of chalcone H-indol-3-yl)-2-oxo-4-(thiophen-2-yl)-2H-pyran-3-carbonitrile (8)6- and ethyl cyanoacetate  in 50 mL ethanol was stirred at room temperature. A freshly prepared EtO−Na+ solution  in 40 mL of absolute ethanol) was added to this mixture. The reaction mixture was refluxed for 7 h. After cooling, the formed solid was collected by filtration, then washed with water, dried, and finally recrystallized from ethanol to create compound 8 as greenish-yellow crystals. Yield: 70%. m.p.: 288–290 °C. IR : 3400–3500 cm−1 , 3039 cm−1 (CH-Aryl), 2920 cm−1 (CH-Aliphatic), 2283 cm−1 (CN), 1724 cm−1 (C=O for 2-pyrone cyclization), 1552 cm−1 (C=C). 1H-NMR : δ = 6.95 , 8.32–8.65 , 7.20–7.83 , and 12.03 . 13C-NMR : δ = 115.8 ppm (CN), 127–130 ppm (thiophene), 110–130 ppm (indolyl carbons). MS (M/Z): M+• (318.30), Base-peak (57). Anal. Calcd. for C18H10N2O2S (318.35): C, 67.91; H, 3.17; N, 8.80; S, 10.07. Found: C, 68.14; H, 2.99; N, 8.91; S, 9.95.A mixture of chalcone H-indol-3-yl)-2-oxo-4-(thiophen-2-yl)-1,2-dihydropyridine-3-carbonitrile (9)1-amino-6-(18 (2 mmol) in 30 mL of ethanol. The mixture was heated under reflux for 6 h. after cooling, the formed solid product was filtered off, dried, and then recrystallized from ethanol to give compounds 9 as a yellow powder. Yield: 75%. m.p.: 298–300 °C. IR : 3350–3600 cm−1 , 3039 cm−1 (CH-Aryl), 2975 cm−1 (CH-Aliphatic), 2229 cm−1 (CN), 1640 cm−1 (C=O for cyclic amide), 1588 cm−1 (C=C). 1H-NMR : δ = 6.54 , 8.32–9.6 , 7.13–7.91 , 11.54 , and 6.23 . 13C-NMR : δ = 116.4 ppm (CN), 164.21 (CO), 129–132 ppm (thiophene), 111–128 ppm (indolyl carbons). MS (M/Z): M+• (333.05), Base-peak (57). Anal. Calcd. for C18H12N4OS (332.38): C, 65.05; H, 3.64; N, 16.86; S, 9.65. Found: C, 64.91; H, 3.76; N, 16.95; S, 9.51.Hydrazine hydrate (2 mmol) was added to the pyranone H-indol-3-yl)-2-oxo-6-(thiophen-2-yl)cyclohex-3-ene-1-carboxylate (10)Ethyl 4-(11 (8 mmol), and ethylacetoacetate (8 mmol) in ethanol (20 mL) in the presence of 10% NaOH (1 mL) was refluxed for 5 h. After cooling, the product was filtered and recrystallized from ethanol to yield the yellow crystals of 10. Yield 85%. m.p.: 270–272 °C. IR : 3461 cm−1 , 3005 cm−1 (CH-Aryl), 2974 cm−1 (CH-Aliphatic), 1725 cm−1 (C=O on cyclohexanone), 1735 cm−1 (C=O in ester form). 1H-NMR : δ = 1.10 , 2.50 , 3.08 , 3.43 , 4.78 , 7.16–7.46 , 7.50–7.81 , and 10.72 . 13C-NMR : δ = 21.2, 36.4, 43.2, 58.7, 65.7, 67.4, 112.4, 113.7, 118.6, 119.2, 121.4, 122.2, 124.46, 126.03, 127.5, 128.9, 133.4, 134.8, 138.4, 173.1, and 184.3 ppm. MS (M/Z): M+• (364.95), Base-peak (57). Anal. Calcd. for C21H19NO3S (365.45): C, 69.02; H, 5.24; N, 3.83; S, 8.77. Found: C, 69.17; H, 5.09; N, 3.91; S, 8.59.A mixture of chalcone H-indol-3-yl)-4-(thiophen-2-yl)-4H-pyran-3-carbonitrile (11)2-amino-6-(11 (10 mmol) with malononitrile (10 mmol) and ammonium acetate (5 mmol) in ethanol 30 mL was refluxed for 6 h. The reaction mixture was cooled and poured into ice-cold acidified water. After neutralization, the crude precipitate was filtrated, then dried and recrystallized from ethanol to yield reddish-yellow crystals 11. Yield 85%. m.p.: 245–250 °C. IR : 3465–3584 cm−1 , 3003 cm−1 (CH-Aryl), 2971 cm−1 (CH-Aliphatic), 2214 cm−1 (CN). 1H-NMR : δ = 4.51 =C), 5.42 , 7.17–7.27 , 7.49–7.79 , 10.8 , and 12.10 . 13C-NMR : δ = 30.67, 112.6, 117.9 (CN), 122.25, 122.33, 123.7, 126.35, 128.9, 129.35, 131.66, 132.9, 135.0, 137.35, 140.69, 183.63 (O-C-NH2) ppm. MS (M/Z): M+• (319.55), Base-peak (57). Anal. Calcd. for C18H13N3OS (319.38): C, 67.69; H, 4.10; N, 13.16; S, 10.04. Found: C, 67.81; H, 3.99; N, 13.01; S, 10.12.A mixture of chalcone H-indol-3-yl)-4-(thiophen-2-yl)-4H-pyran-3-carbonitrile derivatives (12a–e)General synthesis of 6-(111 (10 mmol) with few drops of piperidine (0.5 mL) in ethanol (20 mL). The reaction mixture was heated under reflux for 4 h, cooled and poured onto cold water, and filtered. The obtained solid was recrystallized from dioxane to give 12a-e.An appropriate aromatic aldehyde (10 mmol) was added to compound H-indol-3-yl)-4-(thiophen-2-yl)-4H-pyran-3- carbonitrile (12a)2-((4-cyanobenzylidene)amino)-6-: 3392–3437 cm−1 , 3003 cm−1 (CH-Aryl), 2929 cm−1 (CH-Aliphatic), 2212 cm−1 (CN), 1654 cm−1 (C=N). 1H-NMR : δ = 2.5 =C), 3.35 , 7.2–8.16 , 11.95 . 13C-NMR : δ = 30.67, 112.6, 117.9, 118.2 (CN), 122.25, 122.33, 123.7, 126.35, 128.9, 129.35, 131.66, 132.9, 135.0, 137.35, 140.69, 183.63 (O-C-NH2) ppm. Anal. Calcd. for C26H16N4OS (432.50): C, 72.20; H, 3.73; N, 12.95; S, 7.41. Found: C, 72.39; H, 3.61; N, 13.01; S, 7.29.Yield 65%. m.p.: 285–290 °C. IR (ν/cmH-indol-3-yl)-2-((4-nitrobenzylidene)amino)-4-(thiophen-2-yl)-4H-pyran-3-carbonitrile (12b)6-: 3368–3586 cm−1 , 2920 cm−1 (CH-Aliphatic), 2201 cm−1 (CN), 1610 cm−1 (C=N). 1H-NMR : δ = 2.5 =C), 3.34 , 7.1–8.52 , 11.75 . 13C-NMR : δ = 42.51, 115.6 (CN), 121.25, 122.53, 125.7, 127.35, 128.56, 129.87, 130.66, 132.9, 134.0, 137.53, 150.69, 186.63 (O-C-NH2) ppm. MS (M/Z): M+• (451.55), Base-peak (311). Anal. Calcd. for C25H16N4O3S (452.49): C, 66.36; H, 3.56; N, 12.38; S, 7.09. Found: C, 66.19; H, 3.71; N, 12.22; S, 7.21.Yield 65%. m.p.: 285–290 °C. IR (ν/cmH-indol-3-yl)-4-(thiophen-2-yl)-4H-pyran-3-carbonitrile (12c)2-((4-chlorobenzylidene)amino)-6-: 3395–3646 cm−1 , 3050 cm−1 ( Aromatic-CH), 2921 cm−1 (CH-Aliphatic), 2201 cm−1 (CN), 1593 cm−1 (C=N). 1H-NMR : δ = 4.47 =C), 5.04 , 7.35–7.93 , 10.76 . 13C-NMR : δ = 30.67, 118.4 (CN), 120.25, 121.36, 123.7, 125.52, 127.9, 128.35, 131.53, 134.9, 135.25, 136.35, 146.69, 165.63 (O-C-NH2) ppm. Anal. Calcd. for C25H16ClN3OS (441.93): C, 67.95; H, 3.65; Cl, 8.02; N, 9.51; S, 7.25. Found: C, 67.82; H, 3.79; Cl, 8.21; N, 9.37; S, 7.19. Yield 65%. m.p.: 285–290 °C. IR (ν/cmH-indol-3-yl)-2-((3-nitrobenzylidene)amino)-4-(thiophen-2-yl)-4H-pyran-3-carbonitrile (12d)6-: 3390–3674 cm−1 , 3010 cm−1 (CH-Aryl), 2922 cm−1 (CH-Aliphatic), 2205 cm−1 (CN), 1609 cm−1 (C=N). 1H-NMR : δ = 2.9 =C), 3.7 , 7.15–8.52 , 10.93 . 13C-NMR : δ = 37.67, 116.6 (CN), 123.56, 123.88, 124.15, 124.36, 126.35, 129.10, 129.35, 131.66, 132.53, 136.0, 137.55, 145.69, 180.63 (O-C-NH2) ppm. MS (M/Z): M+• (452.15), Base-peak (311). Anal. Calcd. for C25H16N4O3S (452.49): C, 66.36; H, 3.56; N, 12.38; S, 7.09. Found: C, 66.41; H, 3.43; N, 12.19; S, 7.21.Yield 65%. m.p.: 285–290 °C. IR (ν/cmH-indol-3-yl)-2-((4-methoxybenzylidene)amino)-4-(thiophen-2-yl)-4H-pyran-3-carbonitrile (12e)6-: 3370–3693 cm−1 , 3102 cm−1 ( Aromatic-CH), 2924 cm−1 (CH-Aliphatic), 2206 cm−1 (CN), 1595 cm−1 (C=N).1H-NMR : δ = 3.6 =C), 3.88 , 6.99–8.68 , 11.87 . 13C-NMR : δ = 47.67, 116.5 (CN), 122.55, 122.78, 123.56, 126.44, 128.10, 129.10, 131.23, 132.41, 135.53, 137.45, 148.69, 167.63 (O-C-NH2) ppm. MS (M/Z): M+• (436.10), Base-peak (311). Anal. Calcd. for C26H19N3O2S (437.52): C, 71.38; H, 4.38; N, 9.60; S, 7.33. Found: C, 71.51; H, 4.21; N, 9.73; S, 7.18.Yield 65%. m.p.: 285–290 °C. IR (ν/cm·+). ABTS·+ can react with any compound that generates a hydrogen atom (H-Donor) or an electron such as phenols and thiols, where ABTS·+ reacts as H or an electron acceptor as shown in All chemicals, including L-ascorbic acid, were provided from Sigma-Aldrich , and all solvents, including annular ethanol, delivered from El-NASR Co., Egypt. This assay follows the methodology of Re et al. . ABTS me·+ solution from a dark green one to a colorless solution. Equal amounts of ABTS·+ and potassium persulphate K2S2O8  were added from their stock solutions to make the ABTS·+ standard solution. The yielded mixture was left to stand at room temperature in the darkness for 13–15 h overnight. The ABTS·+ stock solution is appropriate for use after the completion of the reaction. The evidence of finishing the reaction is the stability of the spectrophotometric absorbance of the ABTS·+ solution at a wavelength of 735 nm. ABTS·+ stock solution may be stored at room temperature in the darkness for about 2–3 days and will still be valid over this period to use. The present assay was prepared the ABTS·+ working solution from stock one by dilution in annular ethanol to get an absorbance Ablank of 0.7 ± 0.02 at a wavelength of 735 nm. In an incubator, the produced solution was controlled at a temperature of 30 °C and was equilibrated. In this assay, Ablank was adapted to be accurately 0.7 at time = 0, i.e., before determining the absorbance for all the targeted compounds. In order to measure the scavenging activities of the targeted compounds against the free radicals, 1.5 mL of dark blue ABTS·+ working solution was mixed with 10 µL of the compounds solutions . These steps are repeated using various concentrations of the tested compounds solutions over a range of 10–300 µM. These various concentrations are achieved by dilution using distilled water and annular ethanol or both, based on their solubility degree. After adding ABTS·+ solution with different tested candidates, the change in absorbance value was determined over various rang of time at 0, 0.5, 1, and 5 min in order to manage to achieve the steady-state value of absorbance. In our present assay, the steady-state value was achieved after 15 min. By generalization, Atest, absorbance value for each tested candidate, was reported after 15 min of addition ABTS·+ solution tested candidate solution. The mean of the values was recorded. Each concentration for each tested sample at a specific time was recorded separately for all compounds, then every three separate measurements were determined, and the mean was taken. The antioxidant activity of each compound against ABTS·+ was calculated from the percent reduction in absorbance values, according to the following equation:test or A15 = The absorbance value for each tested candidate after 15 min of addition ABTS·+ solution. Ablank or A0 = The absorbance value of ABTS·+ itself before adding the tested candidates.This transformation of hydrogen or electron converts ABTSblank was adjusted to be 0.70)i.e., Time = 0.  of each of the test compounds was determined and calculated by Unico spectrophotometer 1200 USA and compared to that of L-ascorbic acid as the reference and standard.1–12a–e) together with the co-crystallized ascorbic acid as a reference standard. A general docking process was performed using the site of the co-crystallized ascorbic acid inside cytochrome c peroxidase as the docking site. Furthermore, all the other docking parameters were adjusted as previously discussed in detail [c peroxidase enzyme. A valid performance was confirmed by obtaining a low RMSD value (˂1) [A molecular docking study of the sixteen newly designed and synthesized indolic compounds was performed using the MOE 2019.0102 program . The newn detail ,44. Morelue (˂1) ,46.A set of 10 derivatives of 1H-indol-3-yl compounds from antioxidant activities was assisted and examined by 2D-QSAR to evaluate the effect of its structure varieties. For further validation and experimental data you can refer to the H-indole derivatives were designed and synthesized as promising antioxidant candidates based on the introduction of equivalent reducing heterocyclic rings comparable to that of ascorbic acid. Applying a quantitative analysis of the structure-activity relationship (2D-QSAR) on candidates exhibited a various range of potentially promising antioxidant activities. Concerning ascorbic acid antioxidant activity, these synthesized compounds were categorized into three featured groups of antioxidants based on the results of their biological scavenging abilities against the evaluated radicals in vitro. Surprisingly, compound 10 was found to be more potent than ascorbic acid with IC50 = 28.23 μg/mL compared to that of ascorbic acid (IC50 = 30.03 μg/mL). It could be a promising lead compound, which via structural modification, led to the design and synthesis of novel powerful antioxidants. Furthermore, the mechanism of action for the new compounds was proposed as cytochrome c peroxidase inhibitors via molecular docking compared to ascorbic acid as a reference standard.A new series of 3-(4-(thiophen-2-yl)-pyridin/pyran/pyrimidin/pyrazol-2-yl)-1


Development of a high affinity 18F radiotracer would enable the study of Huntington’s disease pathology using a non-invasive imaging modality, positron emission tomography (PET) imaging. Herein, we report the first synthesis of fluorine-18 imaging agent, 6-ethoxy)pyridin-2-yl)methoxy)benzo[d]oxazol-2-yl)-2-methylpyridazin-3(2H)-one ([18F]1), a radioligand for HD and its preclinical evaluation in vitro (autoradiography of post-mortem HD brains) and in vivo (rodent and non-human primate brain PET). [18F]1 was synthesized in a 4.1% RCY (decay corrected) and in an average molar activity of 16.5 ± 12.5 GBq/μmol (445 ± 339 Ci/mmol). [18F]1 penetrated the blood-brain barrier of both rodents and primates, and specific saturable binding in post-mortem brain slices was observed that correlated to mHTT aggregates identified by immunohistochemistry.Mutations in the huntingtin gene (HTT) triggers aggregation of huntingtin protein ( PET imaging is a highly sensitive and noninvasive technique for quantifying biological targets within a living human and enabling their use as biomarkers of a disease. Thus a PET imaging agent for mHTT could be expected to have similar benefits to the amyloid, tau and α-synuclein PET imaging agents currently used for dementia imaging (mHTT. The only example to date for imaging mHTT is with the 11C-labeled agents [11C]CHDI-180R and [11C]CHDI-626 which exhibit high affinity (low nanomolar IC50) toward mHTT and have selectivity over other protein aggregates like amyloid and tau  to image mHTT in HD patients (Huntington’s disease (HD)  is a neu protein . The pat regions . Design  imaging , allowin and tau . To the patients .18F-labeled analog is highly desirable because of the longer half-life of fluorine-18 (109.8 min)   comparedyl group  due to pyl group , nor on yl group . So, we yl group  to limit3) group is a common motif in small molecule-based drug scaffolds , and in vivo (rodent and nonhuman primate (NHP) PET imaging).The trifluoromethyl , in DMSO-d6 or CDCl3 unless otherwise indicated, δ in ppm rel. to tetramethylsilane (δ = 0), J in Hz. Mass spectra were measured on an Agilent Q-TOF HPLC-MS.Unless otherwise stated all the chemicals were purchased from commercial suppliers and used without purification. Automated flash chromatography was performed with a Biotage Isolera Prime system. High-performance liquid chromatography (HPLC) was performed using a Shimadzu LC-2010A HT. 5-pyridin-2-yl)methanol (3): 6-(Hydroxymethyl)pyridin-3-ol  was dissolved in anhydrous DMF (5 mL) and K2CO3  was added to the solution. The reaction mixture was stirred at room temperature for 15 min. Trifluoroethyl triflate  was added into the reaction mixture, and the resulting mixture was heated at 120°C for 24 h. Upon completion of the reaction, the mixture was cooled to room temperature, filtered to remove solids and quenched with sat. NH4Cl (5 mL). The quenched filtrate was extracted with ethyl acetate (EA) (3 × 50 mL), washed with H2O and dried over anhydrous Na2SO4. The crude reaction mixture was purified with silica gel flash chromatography using a hexane:EA gradient mobile phase. Product collected in 41% yield as a brown oil. 1H NMR /δ (ppm): 8.25 , 7.27 , 4.69 , 4.38 .13C NMR /δ (ppm): 153.81, 152.91, 136.52, 126.30, 124.09, 123.34, 121.87, 121.42, 66.69, 66.40, 66.11, 65.83, 63.98.19F NMR /δ (ppm):–73.99 . HRMS: Calculated for [M+H]+ (M = C8H9F3NO2) = 208.0575, actual m/z = 208.0585.oxy)pyridin-2-yl)methanol (4): pyridin-2-yl)methanol  was added to a flame-dried round bottom flask. Anhydrous THF (5 mL) was added into the flask and the resulting solution was cooled to –78°C over 15 min in a dry ice acetone bath. n-Butyl lithium in hexane  was added slowly to the reaction mixture and was stirred at the same temperature for 45 min. The reaction was quenched with THF:water  and warmed to room temperature. It was extracted with ethyl acetate (3 x 50 mL) and the combined organic fractions were rinsed with brine and dried over anhydrous Na2SO4. The crude reaction mixture was purified with silica gel flash chromatography using an EA:Hexane mobile phase gradient. The purified product was collected in 19% yield as a brown oil. 1H NMR /δ (ppm): δ 8.30 , 7.33 , 7.30–7.25 , 6.10 , 4.71 . 13C NMR /δ (ppm): δ 159.13, 156.90, 156.81, 154.58, 154.14, 152.69, 136.68, 121.34, 104.61, 104.48, 104.16, 104.04. 19F NMR /δ (ppm): δ–95.39,–95.42,–95.52,–95.55,–113.85 ,–113.98 . HRMS: Calculated for [M+H]+ (M = C8H8F2NO2) = 188.0511, actual m/z = 188.0523.d]oxazole (5):2-Methyl-5-pyridin-2-yl)methoxy)benzo+ (M = C16H14F3N2O3) = 339.0951, actual m/z = 339.0957.d]oxazole (6):5-oxy)pyridin-2-yl)methoxy)-2-methylbenzo+ (M = C16H12F2N2O3) = 319.0894, actual m/z = 319.0882.2-Amino-4-pyridin-2-yl) methoxy)phenol (7): 2-Methyl-5- pyridin-2-yl)methoxy)benzo[d]oxazole 5  was dissolved in ethanol (10 mL) and in conc. HCl (2.0 mL). The reaction mixture was heated at 70 °C for 16 h. The solvent was evaporated, and crude reaction mixture was used directly for the next reaction. Product collected in 100% yield. 1H NMR /δ (ppm): 8.74 , 8.38–8.32 , 8.15 , 7.17 , 7.11 , 7.01 , 5.42 , 4.93 . 13C NMR /δ (ppm): 132.29, 130.26, 126.72, 116.53, 111.30, 66.25, 48.09, 47.92, 47.89, 47.75, 47.58, 47.41, 47.24, 47.07. 19F NMR /δ (ppm):–75.51 . HRMS: Calculated for [M+H]+ (M = C14H14F3N2O3) = 315.0957, actual m/z = 315.0944.2-Amino-4-oxy)pyridin-2-yl) methoxy)phenol (8): 5-oxy)pyridin-2-yl)methoxy)-2-methylbenzo[d]oxazole 6  was dissolved in ethanol (10 mL) and in conc. HCl (2.0 mL). The reaction mixture was heated at 70 °C for 16 h. The solvent was removed under reduced pressure, and the crude reaction mixture was used directly in the next reaction. Product was collected in 100% yield as a brown solid. HRMS: Calculated for [M+H]+ (M = C14H12F2N2O3) = 295.0894, actual m/z = 295.0881.N-pyridin-2-yl)methoxy)phenyl)-1-methyl-6-oxo-1,6-di hydropyridazine-3-carboxamide (9): 2-Amino-4- pyridin-2-yl)methoxy)phenol 7  and 1-methyl-6-oxo-1,6-dihydropyridazine-3-carboxylic acid  were dissolved in pyridine (3.0 mL). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride  was added to the reaction mixture and the reaction mixture was stirred at room temperature for 22 h. H2O (5 mL) was added and the precipitate was collected by filtration. The solid was thoroughly dried, redissolved in 7N ammonia in methanol (10 mL) and stirred for an hour. The solvent was removed to yield the title compound. Product was collected in 54% yield as an off-white solid. 1H NMR /δ (ppm): 10.01 , 9.56 , 8.40 , 7.97 , 7.94 , 7.57 , 7.51 , 7.10 , 6.84 , 6.65 , 5.04 , 4.89 , 3.76 . 13C NMR /δ (ppm): 161.58, 159.21, 154.24, 151.84, 150.90, 142.44, 137.98, 137.20, 130.79, 129.75, 125.55, 122.03, 117.27, 110.41, 108.51, 74.87, 65.45, 65.18, 64.49, 64.05, 40.91.19F NMR /δ (ppm):–72.59 . HRMS: Calculated for [M+H]+ (M = C16H14F3N2O3) = 451.1229, actual m/z = 451.1223.N-oxy)pyridin-2-yl)methoxy)- 2-hydroxyphenyl)-1-methyl-6-oxo-1,6-dihydropyridazine-3-carboxamide (10): 2-Amino-4- oxy)pyridin-2-yl)methoxy)phenol 8  and 1-methyl-6-oxo-1,6-dihydropyridazine-3-carboxylic acid  were dissolved in pyridine (2.0 mL). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride  was added into the reaction mixture and the reaction mixture was stirred at room temperature for 22 h. H2O (5 mL) was added and the precipitate was collected by filtration. The solid was thoroughly dried, redissolved in 7N ammonia in methanol (10 mL) and stirred for an hour. The solvent was removed to yield the title compound in 26% yield as an off-white solid. 1H NMR  /δ (ppm): 9.31 , 8.46–8.34 , 8.02 , 7.67 , 7.58 , 7.38 , 7.03 , 6.95 , 6.71 , 6.17 , 5.25 , 3.92 . 19F NMR /δ (ppm):–95.11,–95.14,–95.24,–95.27,–113.53. HRMS: Calculated for [M+H]+ (M = C20H16F2N4O5) = 431.1167, actual m/z = 431.1158.H)-one (1):2-Methyl-6-pyridin-2-yl) methoxy)benzo[d]oxazol-2-yl)pyridazine 3pyridin-2-yl)methoxy) phenyl)-1-methyl-6-oxo-1,6-dihydropyridazine-3-carboxamide 9  was dissolved in anhydrous toluene (9 mL) and pTsOH.H2O  was added. The reaction mixture was refluxed using a Dean Stark trap for 24 h. It was extracted with EA (3 x 50 mL), washed with H2O and dried over anhydrous Na2SO4. The crude reaction mixture was purified via silica gel flash chromatography using a DCM:MeOH mobile phase gradient. Product was collected in 80% yield as an off-white solid. 1H NMR  δ 8.65 , 8.47 , 7.95–7.78 , 7.63 , 7.46 , 7.41 , 5.52 , 4.84 , 4.24 . 13C NMR /δ (ppm): 160.62, 157.81, 155.78, 153.69, 149.65, 144.78, 141.88, 136.54, 134.00, 131.17, 129.27, 123.94, 123.25, 115.98, 111.45, 104.44, 66.01, 65.93, 65.74, 40.69, 29.47.19F NMR /δ (ppm): δ-70.77 . HRMS: Calculated for [M+H]+ (M = C20H16F3N4O4) = 433.1124, actual m/z = 433.1113.d]oxazol-2-yl)-2-methylpyridazin-3 (2H)-one (11):6-oxy)pyridin-2-yl)methoxy) benzo+ (M = C20H15F2N4O4) = 413.1061, actual m/z = 413.1055.Unless otherwise stated, reagents and solvents were commercially available and used without further purification: sodium chloride, 0.9% USP, and sterile water for injection, USP, were purchased from Hospira; ethanol was purchased from American Regent; HPLC grade acetonitrile was purchased from Fisher Scientific. Other synthesis components were obtained as follows: sterile filters were obtained from Millipore; sterile product vials were purchased from Hollister-Stier; C18 Sep-Paks were purchased from Waters Corporation. C18 Sep-Paks were flushed with 10 mL of ethanol followed by 10 mL of water prior to use. Radio-HPLC was performed using a Shimadzu LC-2010A HT system equipped with a Bioscan B-FC-1000 radiation detector.18F]Fluoride was prepared using an automated GE TRACERLab FXFN synthesis module. The TRACERLab was configured as shown in 4Cl); Vial 6: HPLC buffer ; Vial 7: 0.9% sodium chloride for injection, USP (4.18 mL); Vial 8: ethanol (0.66 mL) and Tween-80 (0.16 mL); and Vial 9: sterile water for injection, USP (10 mL); round bottom flask: water (50 mL); product vial: 0.9% sodium chloride for injection, USP (5.0 mL).[via the 18O 18F nuclear reaction using a GE PET Trace cyclotron equipped with a high yield fluorine-18 target at 55 μA to produce 74 GBq (2 Ci) of fluorine-18. The [18F]Fluoride was delivered from the cyclotron (in a 2.5 ml bolus of [18O]H2O) and trapped on a QMA-Light Sep-Pak, which had been preconditioned with sodium bicarbonate, to remove [18O]H2O. [18F]Fluoride was then eluted into the reaction vessel using aqueous potassium carbonate (3.5 mg in 0.5 mL of water). A solution of kryptofix-2.2.2 (15 mg in 1.0 mL of acetonitrile) was then added to the reaction vessel and the [18F]fluoride was dried by azeotropic evaporation of the water-acetonitrile mixture. Evaporation was achieved by heating the reaction vessel to 100°C. The reactor was then cooled to 90°C, and precursor was added with stirring for 3 min. Subsequently, the reaction mixture was cooled to 50°C, followed by the addition of HPLC buffer (3.0 mL). The reaction was loaded onto a semipreparative column . The product peak .18F]1111fluoride at 90°C for 3 min (Entry 1). When subjected to these conditions, we obtained the desired product 18F]1[ in only 0.01% radiochemical yield (RCY) and in 1:10 ratio of trifluoromethyl (18F]1[)/difluoro-alkene (12) (via an elimination-addition mechanism. Further 18F]N-methyl-lansoprazole (NML) for clinical use (n = 3) radiochemical yield, and a satisfactory ratio of [18F]trifluoromethyl 18F]1/[[18F]gem-difluoroalkene 12 (1:3).To accomplish the radiolabeling to generate valuated . Gem-difene (12) . While lene (12)  explaineical use . The optical use . Gratify18F]11 as the stationary phase worked well in both the semipreparative and analytical separations (see representative HPLC traces in the Supporting Information). Purification of 18F]1[ was achieved using semipreparative HPLC conditions (see 18F]1[ (tR = 82–86 min) and [18F]gem-difluoroalkene 12 (tR = 73–80 min). While retention times are somewhat long, and there is likely further scope for optimization, this method provided adequate separation of 18F]11111[ in non-human primates (NHPs). PET imaging studies were performed for 18F]1111[ did not display any specific binding or prolonged retention in the brain. The performance is comparable with literature data for [11C]CHDI-180R [e.g., SUV 2.7–3.0 in NHP imaging studies , establish the metabolism, validate a synthesis to provide tracer suitable for clinical use and further validate the specificity of the signal for mHTT aggregates.In summary, an automated radiosynthesis of an The original contributions presented in the study are included in the article/The animal study was reviewed and approved by the University of Michigan IACUC.PS designed the research. TK, AB, AL, TD, JS, JA, and WW performed the research. TK and AB contributed new reagents and analytical tools. TK, AB, AL, WW, TD, and PS analyzed the data. TK, AB, and PS wrote the manuscript. All authors reviewed the manuscript.The authors declare that the research was conduscted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Furthermore, the thiosemicarbazide derivative 3 was reacted with different bromoacetyl compounds 10–12 to afford the respective thiazole derivatives 13–15. Chemical composition of the novel derivatives was established on bases of their spectral data  and microanalytical data. The newly synthesized derivatives were screened for their in vitro anti-hepatic cancer potency using an MTT assay. Moreover, an in silico technique was used to assess the interaction modes of the compounds with the active site of Rho6 protein. The docking studies of the target Rho6 with the newly synthesized fourteen compounds showed good docking scores with acceptable binding interactions. The presented results revealed that the newly synthesized compounds exhibited promising inhibition activity against hepatic cancer cell lines (HepG2).Thiazoles are important scaffolds in organic chemistry. Biosynthesis of thiazoles is considered to be an excellent target for the design of novel classes of therapeutic agents. In this study, a new series of 2-ethylidenehydrazono-5-arylazothiazoles  In the scope of our program, we are aiming to synthesize biologically active compounds from available inexpensive starting materials ,14,15,16Cancer is a disease characterized by uncontrolled cell growth with the potential to invade other parts of the body. Hepatic cancer is the most common type of primary liver cancer, which causes death in people with cirrhosis. The Rho family of GTPases is a family of small signaling G proteins. They are important regulators of cell cycle progression, and are responsible for gene expression ,30,31. HH-1,2,3-triazol-4-yl)-1-phenyl-1H-pyrazole-4-carbaldehyde 1 was reacted with thiosemicarbazide 2 to give the corresponding ethylidene thiosemicarbazone 3. We launched our research on the reactions of ethylidenethiosemi carbazone 3 with the appropriate α-keto hydrazonoyl halides 4a–d in dioxane with catalytic amount of triethylamine (TEA)  and (C=N) groups, respectively. In 1H-NMR spectra, all the products showed characteristic singlet signals in the region δ 11.34–11.73 ppm (D2O exchangeable), referred to as the −NH protons. Based on the demonstrated results, the products isolated from the reactions of 3 with 4a–h can be assigned (3-(1-(4-bromophenyl)-5-methyl-1ne (TEA) . The strassigned .5a–d could be prepared via alternative synthetic pathway. Here, ethylidenethiosemi carbazone 3 was reacted with chloroacetone under thermal conditions to give thiazole derivative 6. Coupling of the latter product 6 with the appropriate arene diazonium chloride give the respective authentic samples 5a–d (Authentic samples of les 5a–d .3 was reacted with ethyl (N-arylhydrazono)chloroacetates 7a–d in dioxane in the presence of TEA, affording in each case a single isolable product 8a–d. The structures of 8a–d were elucidated based on spectral data and elemental analysis . For instance, the IR spectra of the products showed, in each case, one carbonyl band at 1695–1710 cm−1 and two NH bands in the regions 3334–3325 and 3259–3250 cm−1. Their mass spectra of the latter products revealed in each case, the molecular ion peaks at the expected m/z values and their elemental analysis data were consistent with the assigned structures. The thiazolidinone compound 9 was obtained by reaction of the thiosemicarbazone derivative 3 with ethyl chloroacetate in ethanol, in the presence of anhydrous sodium acetate. Coupling of the latter product 9 with arenediazonium chloride in ethanol give products identical in all aspects with the respective authentic samples 8a–d  exhibited H-bonding and arene-cation interactions with Gln158 and Arg108 at 1.97 and 4.02 Å, respectively. Introducing of electron withdrawing groups on phenyl ring causes lower activity than electron donating groups . After the complete addition of the diazonium salt, the reaction mixture was stirred for a further 30 min in an ice bath. The solid that separated was filtered off, washed with water and finally recrystallized from EtOH to give products proved to be identical in all respects  with compounds 8a–d, obtained from reaction of 3 with 7a–d.H-1,2,3-triazol-5-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)-4-phenylthiazole (13a)2-(2-((3-(1-(4-bromophenyl)-4-methyl-1v/cm−1 3429 (NH), 3025, 2922 (CH), 1600 (C=N); 1H-NMR (DMSO-d6): δ 2.49 , 7.26–8.48 , 8.67, 8.81, 8.94, 11.52 ; δ 9.6, 119.6, 123.5, 126.2, 128.4, 129.3, 131.6, 133.5, 139.7, 143.3, 143.6, 147.9, 165.3; MS m/z (%):581 . Anal. Calcd for C28H21BrN8S (581.50): C, 57.83; H, 3.64; N, 19.27 Found C, 57.92; H, 3.59; N, 19.22%.Brown solid; (68% yield); mp 210–112 °C (Acetic acid); IR (KBr): 1H-1,2,3-triazol-5-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)-4-(4-chlorophenyl)thiazole (13b)2-(2-((3-(1-(4-bromophenyl)-4-methyl-v/cm−1 3435 (NH), 3029, 2927 (CH), 1610 (C=N); 1H-NMR (DMSO-d6): δ 2.59, 7.23–8.49 , 8.76, 8.87, 8.98 , 11.71 ; δ 9.8, 120.0, 123.7, 126.2, 128.4, 129.3, 131.6, 133.5, 139.7, 143.3, 143.6, 147.9, 165.5; MS m/z (%): 615 . Anal. Calcd for C28H20BrClN8S (615.94): C, 54.60; H, 3.27; N, 18.19 Found C, 54.69; H, 3.25; N, 18.12%.Brown solid; (68% yield); mp 228–230 °C (Acetic acid); IR (KBr): 1H-1,2,3-triazol-4-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)-4-(pyridin-2-yl)thiazole (14)2-(2-((3-(1-(4-bromophenyl)-5-methyl-v/cm−1 3395 (NH), 3029, 2921 (CH), 1600 (C=N); 1H-NMR (DMSO-d6): δ 2.49 , 7.37–8.59 , 8.72 , 8.91, 9.01, 11.52 ; 13C-NMR (DMSO-d6): δ 9.6, 119.6, 123.5, 126.2, 128.4, 129.3, 131.6, 133.5, 135.9, 139.7, 143.3, 143.6, 147.9, 165.3; MS m/z (%): 582 . Anal. Calcd for C27H20BrN9S (582.48): C, 55.67; H, 3.46; N, 21.64 Found C, 55.74; H, 3.42; N, 21.61%.Yellow solid; (62% yield); mp 192–194 °C (Acetic acid); IR (KBr): H-1,2,3-triazol-4-yl)-1-phenyl-1H-pyrazol-4-yl)methylene)hydrazinyl)thiazol-4-yl)-3H-benzochromen-3-one (15)2-(2-(2-((3-(1-(4-bromophenyl)-5-methyl-1v/cm−1 3384 (NH), 3027, 2915 (CH), 1605 (C=N); 1H-NMR (DMSO-d6): δ 2.50 , 7.41–8.45 , 8.49 , 8.81 , 8.93 , 9.40 , 11.53 ; 13C-NMR (DMSO-d6): δ 9.6, 114.3, 115.6, 123.5, 126.2, 128.4, 129.3, 131.5, 133.5, 135.9, 139.7, 143.3, 143.6, 147.9, 151.2, 162.9, 172.2; MS m/z (%): 699 . Anal. Calcd for C35H23BrN8O2S (699.59): C, 60.09; H, 3.31; N, 16.02 Found C, 60.17; H, 3.27; N, 15.96%.Yellow solid; (69% yield); mp 122–224 °C (EtOH); IR (KBr): www.rcsb.org/pdb/) [n-octanol and water log P should be <5; the number of hydrogen bond acceptors should be nOH 2.0–20.0; the number of hydrogen bond donors nOHNH should be 0.0–6.0; and the number of rotatable bonds should be ≤10 [In this work, the binding of newly synthesized compounds to Rho6 was theoretically investigated using a computer-based docking approach. The X-ray crystal structure of the target Rho6 is retrieved from the RCSB Protein Data Bank web server (rg/pdb/) . The tworg/pdb/) . The docrg/pdb/) . In a dorg/pdb/) . Predictrg/pdb/) . The evarg/pdb/) ,40. Thesd be ≤10 . CompounThe cytotoxic evaluation of the synthesized compounds was carried out at the Regional Center for Mycology and Biotechnology at Al-Azhar University, Cairo, Egypt, according to the reported methods ,43.


Trypanosoma cruzi, and in Central America, it is considered one of the four most infectious diseases. This study aimed to screen the anti-trypanosomal activity of plant species from Salvadoran flora. Plants were selected through literature search for plants ethnobotanically used for antiparasitic and Chagas disease symptomatology, and reported in Museo de Historia Natural de El Salvador (MUHNES) database. T. cruzi was incubated for 72 h with 2 different concentrations of methanolic extracts of 38 species, among which four species, Piper jacquemontianum, Piper lacunosum, Trichilia havanensis, and Peperomia pseudopereskiifolia, showed the activity (≤ 52.0% viability) at 100 µg/mL. Separation of the methanolic extract of aerial parts from Piper jacquemontianum afforded a new flavanone (4) and four known compounds, 2,2-dimethyl-6-carboxymethoxychroman-4-one (1), 2,2-dimethyl-6-carboxychroman-4-one (2), cardamomin (3), and pinocembrin (5), among which cardamomin exhibited the highest anti-trypanosomal activity (IC50 = 66 µM). Detailed analyses of the spectral data revealed that the new compound 4, named as jaqueflavanone A, was a derivative of pinocembrin having a prenylated benzoate moiety at the 8-position of the A ring.Chagas disease is caused by the protozoan parasite The online version contains supplementary material available at 10.1007/s11418-021-01562-6. Trypanosoma cruzi has been identified as the causal agent of Chagas disease. This protozoan parasite is mainly transmitted through contact with the feces of hematophagous triatomine insects and infects a broad range of mammalian species including humans. In Latin America, Chagas disease continues to be an important public and social health problem, and the World Health Organization  with those of pinocembrin (5) except that only a singlet [6.07 ] was observed for ring A, indicating that compound 4 is a derivative of pinocembrin (5) having a substituent on the A ring. The spectrum also showed signals of a tri-substituted benzene ring , a methoxy group [3.85 ], and a prenyl group . These signals, together with the 13C NMR signals and the HMQC correlations ] of the prenyl group to C-7, C-8 and C-9 carbons of the ring A of pinocembrin moiety . As pinocembrin isolated from this extract was a racemic mixture, the stereochemistry of C-2 seems racemic. However, the stereochemistry of the other carbon could not be determined.The new compound um Table  showed s50 values of the isolated compounds against epimastigotes of T. cruzi were 1.28 mM (1), > 2 mM (2), 66 µM (3), 100 µM (4), and 714 µM (5). Cardamomin (3) has been reported to show trypanocidal activity against Trypanosoma brucei  − ; 1H NMR  δ: 12.2 , 8.01 , 7.81 , 7.44 , 7.39 , 6.83 , 6.07 , 5.98 , 5.45 , 5.39 , 3.85 , 3.09 , 2.84 , 1.79 , 1.68 . 13C NMR  δ: 196.2 (C-4), 167.5 (C-1’), 162.7 (C-7), 162.5 (C-5), 159.6 (C-9), 158.2 (C-5’), 138.0 (C-11), 136.1 (C-10’), 131.3 (C-3’), 129.7 (C-7’), 128.9 (C-13), 128.5 (C-2’), 126.1 (C-12), 126.0 (C-14), 122.9 (C-9’), 122.1 (C-4’), 116.0 (C-6’), 108.8 (C-8), 103.5 (C-10), 97.7 (C-6), 79.9 (C-2), 52.0 (C-13’), 43.4 (C-3), 33.6 (C-8’), 25.9 (C-12’), 18.2 (C-11’).Supplementary file1 (PDF 3565 kb)Below is the link to the electronic supplementary material.


The chloro, methoxy, cholroacetoxy, imidazolyl, azide, and arylamino derivatives were prepared to obtain the pyridine-−C2 functionalized derivatives. The derived pyrazolpyridine-N-glycosides were synthesized via heterocyclization of the C2-thioxopyridine derivative followed by glycosylation using glucose and galactose. The furopyridine derivative 14 and the tricyclic pyridofuropyrimidine 15 were prepared via heterocyclization of the ester derivative followed by a reaction with formamide. The newly synthesized compounds were evaluated for their ability to in vitro inhibit the CDK2 enzyme. In addition, the cytotoxicity of the compounds was tested against four different human cancer cell lines . The CDK2/cyclin A2 enzyme inhibitory results revealed that pyridone 1, 2-chloro-6--4-(thiophen-2-yl)nicotinonitrile (4), 6--4-(thiophen-2-yl)-1H-pyrazolopyridin-3-amine (8), S-(3-cyano-6-(naphthaen-2-yl)-4-(thiophen-2-yl)pyridin-2-yl) 2-chloroethanethioate (11), and ethyl 3-amino-6--4-(thiophen-2-yl)furopyridine-2-carboxylate (14) are among the most active inhibitors with IC50 values of 0.57, 0.24, 0.65, 0.50, and 0.93 µM, respectively, compared to roscovitine (IC50 0.394 μM). Most compounds showed significant inhibition on different human cancer cell lines  with IC50 ranges of 31.3–49.0, 19.3–55.5, 22.7–44.8, and 36.8–70.7 μM, respectively compared to doxorubicin . Furthermore, a molecular docking study suggests that most of the target compounds have a similar binding mode as a reference compound in the active site of the CDK2 enzyme. The structural requirements controlling the CDK2 inhibitory activity were determined through the generation of a statistically significant 2D-QSAR model.New pyridine, pyrazoloyridine, and furopyridine derivatives substituted with naphthyl and thienyl moieties were designed and synthesized starting from 6--2-oxo-4-(thiophen-2-yl)-1,2-dihydropyridine-3-carbonitrile ( The cyclin-dependent kinases (CDK) (a serine/threonine kinase family)  are respRoscovitine, which is a CDK2 inhibitor undergoing clinical trials to treat cancers pyridine/pyrido30,20:4,5]furopyrimidine with different substitutions has gained renewed interest as a template for drug discovery. Furopyridine derivatives also demonstrated in vitro activity for tubulin polymerization ,20:4,5fu and Akt 1) prompted us to prepare some of its derivatives starting from available and inexpensive compounds. The reaction of compound 1 with chloroacetyl choloride yielded 3-cyano-6--4-(thiophen-2-yl)pyridin-2-yl-2-chloroacetate (2) via substitution reaction in 84% yield. The structure of compound 2 was elucidated based on elemental and spectral analyses. The IR spectrum of 2 revealed strong absorption bands at ν 2216 and 1697 cm−1 for CN and CO groups, respectively. The 1H-NMR of the same compound showed signals at δ 4.40 ppm referring to CH2Cl in addition to the signals of the aromatic protons. The formation of imidazolthione 3 was obtained through the heterocyclization of compound 2 by reaction with thiourea in DMF. Its IR spectrum showed the disappearance of the absorption band at ν 1697 cm−1 of the carbonyl group and the appearance of the characteristic bands at ν 3424 and 1260 cm−1 due to NH and C=S groups, respectively. The 1H-NMR spectrum of compound 3 exhibited very distinct singlet signals resonating at δ 4.13 (imidazole-H) and δ 12.98 (exchangeable NH) confirmed the successful formation of 3. 13C-NMR spectrum of compound 3 showed signals at δ 49.6, 168.1, and 188.4 ppm for imidazoles-C and C=S groups, respectively beside the aromatic carbons at their specific regions -2-oxo-4-(thiophen-2-yl)-1,2-dihydropyridine-3-carbonitrile -4-(thiophen-2-yl)nicotinonitrile (5) in 75% yield. Its 1H-NMR revealed a signal at δ 4.16 ppm due to methoxy protons. In addition, compound 4 was reacted with aniline to give 6--2-(phenylamino)-4-(thiophen-2-yl)nicotinonitrile (6). IR spectrum of 6 revealed the presence of a strong absorption band at ν 3428 cm−1 due to the NH group, and also, it appeared as a strong signal at δ 9.80 ppm in the 1H-NMR spectrum (Compound 4  underwenspectrum .8) in a good yield. The IR spectrum of 8 revealed bands at ν 3375–3228 cm−1 referred to NH2 and NH groups. The 1H-NMR exhibited a singlet signal at δ 6.45 ppm referring to the exchangeable NH2 protons and also a singlet signal at δ 11.65 ppm corresponding to the exchangeable NH proton. 13C-NMR spectra agreed with such investigation and confirmed their structures, which showed that C-pyrazoles have been revealed at δ 90.2, 151.3, and 152.2 ppm.On the other hand, pyridine-2(1H) thione derivative 7  was reac8 was further reacted with the monosaccharides such as d-glucose or d-galactose as six-carbon sugars in ethanol, and a few drops of glacial acetic acid afforded compounds 9 and 10, respectively, which have been shown to incorporate sugar moieties in the cyclic form. Their IR spectra showed strong and broad absorption bands characterizing the polyhydroxy chain and NH groups in the range ν 3520–3390 cm−1. The 1H-NMR spectra of the same compounds showed signals for NH (D2O exchangeable) and OH groups at their specific regions -4-(thiophen-2-yl)furopyridine-2-carboxylate (14). This compound might be subjected to further structural modification. The formation of the furan ring with two functionalities (the amino and the carboethoxy groups) resulted, whose further modifications could provide the opportunity for new cyclization processes. The IR spectrum of 14 showed absorptions bands at ν 3437 cm−1 referring to the NH2 group and a strong absorption band at ν 1739 cm−1 referring to the ester carbonyl group. The 1H-NMR spectrum exhibited a singlet signal of the amino group at δ 5.61 ppm, in addition to triplet and quartet signals at δ 1.2 and 4.3 ppm for CH3 and CH2 groups. Compound 14 was condensed with electrophiles namely, formamide, to afford the pyridofuropyrimidine 15. The chemical structure of 15 was elucidated by its correct elemental analysis and spectral data. Its IR spectrum showed absorption bands at ν 3419 and 1662 cm−1 for the imino (NH) and the amidic carbonyl group (C=O). The 1H-NMR spectrum revealed singlet signals at δ 8.26 and 8.34 ppm for pyridine and pyrimidine protons, multiplet signals in the range of δ 7.24–8.11 ppm for aryl protons, and a single signal at δ 11.18 corresponding to the imine proton NH (D2O exchangeable) (The ester ngeable) .1–5, 7, 8, 11, 13, and 14 were carried out using Promega Kinase-Glo Plus luminescence kinase kit. It measures kinase activity by quantitating the amount of ATP remaining in solution following a kinase reaction. The luminescent signal from the assay is correlated with the amount of ATP present and is inversely correlated with the amount of kinase activity. The percent inhibition of the tested compounds against CDK2/cyclin A2 protein kinase was compared to reference kinase inhibitor roscovitine at a single concentration of 10 μM. The computer software Graphpad Prism was used for analyzing the luminescence data. The difference between luminescence intensities in the absence of CDK2/cyclin A2 protein kinase (Lut) and in the presence of kinase (Luc) was defined as 100% activity (Lut − Luc). Using luminescence signal (Lu) in the presence of the compound, the percentage of activity was calculated as: % activity = {(Lut − Lu)/(Lut − Luc)} × 100%, where Lu = the luminescence intensity in the presence of the compound. The percentage of inhibition was calculated as: % inhibition = 100 (%) − % activities pyridine (9).3-((β-D-glucopyranosylamino)-6--4-(thiophen-2-yl)-7,7a-dihydro-1 Brown powder; Yield: 86%; m.p. 285–286 °C; IR : 3520–3390 (OH), 3419 (NH), 1592 (C=N); 1H-NMR (DMSO-d6): δ (ppm) 3.00–3.04 , 3.59–3.62 , 3.73–3.80 , 4.45–4.47 , 4.73–4.78 , 5.85 , 6.80–7.80 , 7.77 , 10.31, 11.29 ; 13C-NMR: 61.6 (C-6`), 70.7 (C-4`), 75.2 (C-2`), 77.2 (C-3`), 81.5 (C-5`), 90.2 (pyridine-C3), 92.6 (C-1`), 121.7 (pyridine-C5), 127.5–141.9 (Ar-C), 144.9 (pyridine-C4), 151.1 (pyridine-C2), 154.0 (pyrazole-C), 155.4 (pyridine-C6). Anal. Calc. for C26H24N4O5S (504.56): C, 61.89; H, 4.79; N, 11.10. Found: C, 61.94; H, 4.85; N, 11.26.H-pyrazolopyridine (10).3--6--4-(thiophen-2-yl)-7,7a-dihydro-1 Yellow powder; Yield: 82%; m.p. 281–282 °C; IR : 3510–3385 (OH), 3475 (NH); 1H-NMR (DMSO-d6): δ (ppm) 3.12–3.13 , 3.40–3.42 , 3.85–3.89 , 4.39–4.41 , 4.58–4.88 , 6.17 , 7.11–8.00 , 7.85 , 10.45, 11.07 . Anal. Calc. for C26H24N4O5S (504.56): C, 61.89; H, 4.79; N, 11.10. Found: C, 61.60; H, 5.29; N, 11.0.7  and anhydrous potassium carbonate  in dry acetone (30 mL), chloroacetyl chloride  was added drop wise. The reaction mixture was stirred at room temperature for about 8 h. Then, the mixture was poured onto water and extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated under vacuum to give compound 11.To a solution of compound −1): 2921 , 2209 (CN), 1698 (C=O), 1590 (C=N); 1H-NMR (DMSO-d6): δ (ppm) 4.30 , 7.34–8.31 , 8.19 ; 13C-NMR: 47.0 (CH2-Cl), 98.3 (pyridine-C3), 112.5 (CN), 120.7 (pyridine-C5), 126.1–138.6 (Ar-C), 147.3 (pyridine-C4), 159.5 (pyridine-C6), 184.2 (pyridine-C2), 189.0 (C=O). Anal. Calc. for C22H13ClN2OS2 (420.93): C, 62.78; H, 3.11; N, 6.66; S, 15.23. Found: C, 62.65; H, 3.34; N, 6.50; S, 15.22.Black powder; Yield: 89%; m.p. 160–161 °C; IR  and sodium azide  in DMF (20 mL) was stirred at room temperature for 12 h. The reaction mixture was diluted with cold water (10 mL) and extracted with chloroform (3 × 10 mL). The combined organic layer was concentrated to afford compound 12.A mixture of compound −1): 2972 , 2366 (N3), 2206 (CN), 1676 (C=O); 1H-NMR (DMSO-d6): δ (ppm) 2.42 , 7.01–8.06 , 7.95 ; 13C-NMR: 65.1 (CN3), 101.9 (pyridine-C5), 110.4 (pyridine-C3), 116.0 (CN), 127.4–135.4 (Ar-C), 147,8 (pyridine-C4), 171.0 (pyridine-C6), 191.0 (C=O). Anal. Calc. for C22H13N5OS2 (427.50): C, 61.81; H, 3.07; N, 16.38; S, 7.74. Found: C, 61.51; H, 3.23; N, 16.40; S, 7.75.Black crystals; Yield: 75%; m.p. 152–153 °C; IR  was heated under reflux in sodium ethoxide solution (0.02 g atom of sodium in 25 mL of absolute ethanol) for 30 min. After cooling, the solid product was filtered and recrystallized from ethanol to afford compound 14.Compound −1): 3437 (NH2), 2921 , 1739 (C=O); 1H-NMR (DMSO-d6): δ (ppm) 1.19–1.23 , 4.35–4.38 , 5.61 , 6.96–8.52 , 8.06 ; 13C-NMR: 13.6 (CH3), 58.1 (CH2), 105.6 (furan-C3), 116.6 (pyridine-C3), 121.9 (pyridine-C5), 126.4–132.1 (Ar-C), 144.2 (furan-C2), 145.1 (pyridine-C4), 155.1 (pyridine-C6), 159.0 (pyridine-C2), 162.0 (C=O). Anal. Calc. for C24H18N2O3S (414.48): C, 69.55; H, 4.38; N, 6.76. Found: C, 69.60; H, 4.34; N, 6.90.Brown powder; Yield: 60%; m.p. 185–186 °C; IR  and formamide (20 mL) was heated under reflux for 5 h. The solid product that formed on cooling was collected and recrystallized from DMF to give compound 15.A mixture of compound −1): 3419 (NH), 1662 (C=O); 1H-NMR (DMSO-d6): δ (ppm) 7.24–8.11 , 8.26 , 8.34 , 11.18 ; 13C-NMR: 103.4 (furan-C3), 116.2 (pyridine-C3), 122.7 (pyridine-C5), 126.0–140.9 (Ar-C), 144,4 (pyridine-C4), 146.1 (pyrimidine-H), 147.8 (furan-C2), 157.0 (pyridine-C6), 158.5 (pyridine-C2), 160.8 (C=O). Anal. Calc. for C23H13N3O2S (395.44): C, 69.86; H, 3.31; N, 10.63. Found: C, 69.67; H, 3.51; N, 10.70.Black powder; Yield: 77%; m.p. 197–198 °C; IR  and fetal bovine serum (FBS) were purchased from Gibco, Paisley, UK. Dimethyl sulfoxide (DMSO) was of HPLC grade, and all other reagents and chemicals were of analytical reagent grade.2.Cell cultures of HCT-116 , MCF-7 (human breast adenocarcinoma), HepG2 (human hepatocellular carcinoma), and A549 (human lung carcinoma) cell lines were purchased from the American Type Culture Collection  and maintained in DMEM medium that was supplemented with 10% heat-inactivated FBS , 100 U/mL penicillin, and 100 U/mL streptomycin. The cells were grown at 37 °C in a humidified atmosphere of 5% CO4 cells/well) and incubated at 37 °C with a series of different concentrations, in DMSO, of each tested compound or doxorubicin (positive control) for 48 h in a serum-free medium prior to the MTT assay. After incubation, media were carefully removed, and 40 µL of MTT (2.5 mg/mL) were added to each well and then incubated for an additional 4 h. The purple formazan dye crystals were solubilized by the addition of 200 µL of DMSO. The absorbance was measured at 570 nm using a Spectra Max Paradigm Multi-Mode microplate reader. The relative cell viability was expressed as the mean percentage of viable cells compared to the untreated control cells. All experiments were conducted in triplicate and repeated on three different days. All the values were represented as mean ± SD. IC50s were determined by probit analysis by SPSS Incprobit analysis .The anticancer activity against HCT-116, HepG2, A549, and MCF-7 human cancer cell lines was estimated using the 3-[4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, which is based on the reduction of the tetrazolium salt by mitochondrial dehydrogenases in viable cells ,51,52. Chttp://www.rscb.org/pdb (accessed on 9 March 2021) (PDB code 2A4L) in PDB format. Computational docking is an automated computer-based algorithm designed to estimate two main terms [Molecular modeling simulation study was performed through docking of the target compounds in the binding site of CDK2 enzyme using C-Docker protocol in Discovery Studio 4.0 Software. The X-ray crystal structure of Roscovitine in complex with CDK2 was downloaded from in terms . The firN-glycosides  were designed and synthesized. All compounds exhibited potent CDK2/cyclin A2 inhibitory activity with IC50 values ranging 3.52–0.24 μM compared to roscovitine (IC50 0.394 ± 0.0 μM). Amongst them, compound 4 revealed the highest IC50 of 0.236 μM. Most of the new compounds exhibited potent antiproliferative activity against HCT-116, MCF-7, HepG2, and A549 compared to doxorubicin. The results revealed agreement between both the experimental and estimated data through docking. The most active compounds 1, 4, 8, 11, and 14 showed high docking scores and low binding energy values, and their binding mode was compatible with the reported binding mode of the reference compound.A new series of substituted pyridine derivatives and its