Staphylococcus aureus (MRSA) strains and one vancomycin-resistant Enterococcus (VRE) strain. Only natural compounds 7,15-isopimaradien-19-ol (1) and 19-acetoxy-7,15-isopimaradien-3β-ol (6) showed promising results. Isopimarane (1) was the most active, showing MIC values between 6.76 µM against S. aureus (ATCC 43866) and 216.62 µM against E. faecalis (FFHB 427483) and E. flavescens (ATCC 49996). Compound (6) showed moderated activity against all tested microorganisms . These compounds were found to be active against the methicillin-sensitive strains of S. aureus (CIP 106760 and FFHB 29593), showing MIC values of 13.55 (1) and 22.54 (6) µM. Both compounds were also active against vancomycin-resistant E. faecalis (ATCC 51299) . In addition, the cytotoxicity of nine compounds 7,15-isopimaradien-3β,19-diol (2); mixture: 15-isopimarene-8β-isobutyryloxy-19-ol and 15-isopimarene-8β-butyryloxy-19-ol (3); 3β-acetoxy-7,15-isopimaradiene-19-ol (5); 19-acetoxy-7,15-isopimaradiene-3β-ol (6); 3β,19-diacetoxy-7,15-isopimaradiene (8); 15-isopimarene-8β,19-diol (9); 19-O-β-d-glucopyranoside-7,15-isopimaradiene (10); lagascatriol-16-O-β-d-glucopyranoside (11) and lagascatriol-16-O-α-d-mannopyranoside (12) was evaluated in the human breast cancer cell line MDA-MB-231. Isopimarane (2) was the only compound showing some cytotoxicity. The IC50 value of compound (2) was 15 µM, suggesting a mild antiproliferative activity against these breast cancer cells.The antimicrobial evaluation of twelve natural and hemisynthetic isopimarane diterpenes are reported. The compounds were evaluated against a panel of Gram-positive bacteria, including two methicillin-resistant  Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) are a worldwide problem in clinical medicine, being the former the leading cause of mortality due to bacterial infections [Antibiotic resistance is nowadays an increasingly serious threat to global public health, leading to longer hospital stays, higher medical costs and increased mortality in all parts of the world . A growifections . Therefofections . An impofections ,5.Pimaranes are tricycle diterpenes with different stereochemistry features and are biosynthetically related to labdane terpenoids. They have been found in several plants, namely in Lamiaceae and Asteraceae families ,7. CurreAeollanthus rydingianus (Lamiaceae) as well as their antimicrobial activity against a set of Gram-positive and Gram-negative bacteria. Two compounds, namely 7,15-isopimaradien-19-ol  and 19-acetoxy-7,15-isopimaradien-3β-ol (6) showed promising antimicrobial activity against Staphylococcus aureus and Enterococcus hirae [In a previous study, we reported the isolation and structure characterization of isopimarane-type diterpenes from Aeollanthus rydingianus: 7,15-isopimaradien-19-ol (1), 7,15-isopimaradien-3β,19-diol (2), mixture of 3β-isobutyryloxy- and 3β-butyryloxy-8β-hydroxy-15-isopimarene (3), 3β-acetoxy-7,15-isopimaradiene (4), 3β-acetoxy-7,15-isopimaradien-19-ol (5), 19-acetoxy-7,15-isopimaradien-3β-ol (6) [1 and 6 were found to be very active against the methicillin-sensitive strain of S. aureus (ATCC 25923), showing MIC values of 3.90 and 15.6 µg mL−1, respectively. Both compounds were also active against E. hirae .In a previous study, a preliminary assay was conducted to assess the antimicrobial activity of a set of isopimarane diterpenes isolated from 1  and 2  previously isolated [7  and 8 , and the glucosyl derivative 10 . Derivatives 11 (lagascatriol-16-O-β-d-glucopyranoside) and 12 (lagascatriol-16-O-α-d-mannopyranoside) were also obtained.In this work, and in order to study the effect of substituents on the antimicrobial activity of isopimarane-type diterpenes, compounds 1H- and 13C-NMR spectra of compounds 7 and 8 revealed additional signals of acetyl ester residues . The presence of glucosyl and mannosyl residues of compounds 10–12 were also confirmed by NMR experiments, namely monodimensional 1H- and 13C-NMR spectra that evidenced the characteristic signals corresponding to the anomeric protons H-1′  and anomeric carbons C-1′ . All the assignments were in agreement with COSY, HSQC and HMBC spectra. The connection of the sugar residues to the C-16 position of lagascatriol was further evidenced by the HMBC spectrum, which showed correlations between the C-1′ carbon (δC 102.4) and both C-16 protons (at δH 3.92 and 3.32), and between the H-1′ proton (δH 4.77) and the C-16 carbon (at δH 69.8).The structure elucidation of these new compounds was essentially based on comparison of NMR spectra with those of the corresponding parent alcohol. In this way, the 1/7, 2/8), on the antimicrobial activity. The effect of a high hydrophilicity, achieved through the introduction of O-monoglycosyl groups , was also evaluated.Acetyl derivatives were synthesized aiming to clarify the role of the 3- and 19-oxygen atoms (hydroxyl versus acetoxyl groups) as either hydrogen donor or acceptor atom  and one vancomycin-resistant E. fecalis (ATCC 51299). A previous antimicrobial screening  of compounds 1 and 6, against Gram-negative bacteria, Mycobacterium smegmatis and Candida albicans indicated that they were inactive.All compounds were tested against an enlarged panel of Gram-positive bacteria  against S. aureus (ATCC 43866) and 216.62 µM (62.50 µg mL−1) against E. faecalis (FFHB 427483) and E. flavescens (ATCC 49996) , respectively. This isopimarane bears a C-19 hydroxyl group to which the activity was attributed in the related ent-isopimara-9(11),15-diene-19-ol [O-acetyl ester 7 and its 19-O-glucosyde 10 were inactive against all the bacteria tested, except for S. aureus ATCC 43,866 strain, which compound 7 had an MIC of 11.83 (3.91 µg mL−1) . Despite this, natural 19-acetyl-3-ol 6 that has a C-3 hydroxyl group and a C-19 acetoxyl substituent instead a C-19 hydroxyl group showed high MIC values (22.54 to 45.07 µM) against all the Gram-positive bacteria assayed and an effective antibacterial active against the methicillin-sensitive strains of S. aureus (MICs of 22.54 µM) and vancomycin-resistant E. faecalis (MIC 45.07 µM). No other combination of the polar hydroxyl and acetoxyl groups on C-3 and C-19 resulted in significative bioactivity. When the acetylation occurs only in the 3-O position, as in the analogous 5, or in both 3- and 19-hydroxyl groups, as in compound 8, the activity significantly drops . Both 8-hydroxyisopimaranes 3 and 9, and the glycosyl derivatives of the lagascatriol 11 and 12, did not reveal any activity in the tests carried out (Isopimaradienol C 49996) . In geneµg mL−1) . In the ried out .1 to 3, 5, 6, 8 to 12 was evaluated in MDA-MB-231 cells, a human cell line routinely used as a model for aggressive breast cancer. For compounds 4 and 7, only little amounts were available, precluding their inclusion in the cytotoxicity assays. The compounds were initially screened at a final concentration of 10 μM. The compounds 1, 3, 5, 6, 8–12 were not cytotoxic to MDA-MB-231 cells under the conditions tested. Such results show that these compounds are devoid of considerable anticancer activity against MDA-MB-231 cells. However, for a complete screening of their anticancer properties, cytotoxic effects should be studied in additional cell lines, representative of other cancer types.The cytotoxicity of the compounds 1 was the only compound showing some cytotoxicity at 10 μM, the concentration-response profile of this compound was evaluated (data not shown). The IC50 value of compound 1 was 15 µM, suggesting a mild antiproliferative activity against these breast cancer cells. Interestingly, this compound has shown an IC50 value of 56 μM in normal-like cells MRC-5 (data not shown), indicating some selectivity for cancer cells.As 7,15-isopimaradien-19-ol n = 7) with MIC values showing activity against S. aureus (ATCC 25923) limits the identification of relevant molecular descriptors. As can be seen (p = 0.049) with the Log(MIC) values. Since the low aqueous solubility appears to be responsible for the reduction of oral bioavailability of the different compounds, a molecular optimization in this respect should take this into consideration.Although the presence of an -OH group at the 3′ or 4′ position of the molecule seems to be essential for the existence of antimicrobial activity, the reduced number of compounds , followed by heating. For preparative TLC chromatography, 20 × 20 cm × 0.5 mm silica plates were used (Merck 1.05774). NMR spectra were recorded on a Brüker ARX-400 NMR spectrometer , or on a Varian System NMR spectrometer  using CDCl3 or CD3OD, as solvents. All tested compounds were purified to ≥95% purity as determined by HPLC  Merck column. The volume of injection of the samples was of 20 μL and a gradient composed of solution A (methanol), solution B (acetonitrile) and solution C (0.3% trichloroacetic acid in water), as follows, was used to carry out the analysis: 0 min, 15% A, 5% B and 80% C; 20 min, 70% A, 30% B and 0% C; 25 min, 70% A, 30% B and 0% C; and 28 min, 15% A, 5% B and 80% C. The flow rate was set at 1 mL min−1. The time of analysis was of 33 min, including the stabilization of the RP-18 column. The quantification for the all samples was performed in triplicate.All solvents were dried according to published methods and distilled prior to use. All the other reagents were obtained from commercial suppliers and were used without further purification. Flash column chromatography (CC) was performed on silica gel (Merck 9385). Merck silica gel 60 F254 plates were used in analytical TLC, with visualization under UV light (λ = 254 and 366 nm) and by spraying with H1–6 and 9 were isolated from Aeollanthus rydingianus aerial parts [10–12 were prepared as described by Uriel et al. [7, 8 and 10–12 were prepared as described below. All compounds were solubilized in DMSO and further tested for their biological assays.Twelve diterpenes with isopimarane skeleton, whose structures are presented in al parts . The benl et al. . Compoun1 (1 eq.) or 2 (2 eq.) in dry pyridine and acetic anhydride was stirred at room temperature until the reaction was complete. The reaction mixture was then diluted with CH2Cl2 and successively washed with water and NaOH solution (5%), and the organic layer dried over anhydrous Na2SO4. The residue was purified by preparative TLC  to afford compounds 7 and 8, respectively. After purification, the compounds were extracted from silica gel with CH2Cl2.A solution of parental alcohols 19-acetoxy-7,15-isopimaradiene (7), 1H-NMR : δ 5.78 , 5.35 , 4.94 , 4.88 , 4.36 , 3.98 , 2.05 , 1.99 , 1.94 , 1.91 , 1.77 , 1.67 , 1.55 , 1.46 , 1.44 , 1.33 , 1.30 , 1.02 , 1.00 , 0.96 , 0.86 , 0.86 . 13C-NMR  δ171,3 (19-OCOCH3), 150.2 (C-15), 135.5 (C-8), 121.2 (C-7), 109,1 (C-16), 66.8 (C-19), 51.9 (C-9), 51.2 (C-5), 45.9 (C-14), 39.5 (C-3), 36.7 (C-13), 36.7 (C-13), 36.2 (C-4), 36 (C-1), 35.9 (C-12), 35.2 (C-10), 27.2 (C-18), 22.9 (C-6), 21.3 (C-17), 20.9 (19-OCOCH3), 20.2 (C-11), 18.2 (C-2), 16.0 (C-20).3β,19-diacetoxy-7,15-isopimaradiene (8), 1H-NMR : δ 5.79 , 5.35 , 4.94 , 4.88 , 4.59 , 4.47 , 4.24 , 2.06 , 2.04 , 2.04 , 1.94 , 1.92 , 1.90 , 1.68 , 1.66 , 1.54 , 1.47 , 1.38 , 1.35 , 1.28 , 1.01 , 0.88 , 0.85 . 13C-NMR  δ 171,1 (3-OCOCH3), 170.8 (19-OCOCH3), 150.3 (C-15), 135.6 (C-8), 121.2 (C-7), 109.5 (C-16), 80.5 (C-3), 64.8 (C-19), 51.9 (C-9), 51.2 (C-5), 45.9 (C-14), 40,8 (C-4), 37.7 (C-1), 36.9 (C-13), 36.1 (C-12), 35.2 (C-10), 24.2 (C-2), 23.7 (C-6), 22.8 (C-18), 21.6 (3-OCOCH3), 21.4 (19-OCOCH3), 21.3 (C-17), 20.5 (C-11), 15.6 (C-20).−Na+ in MeOH was added until pH 8, the reaction was stirred at room temperature and, when TLC showed the disappearance of the starting material, resine H+ (IR-120) was added until pH = 7. The mixture was filtered and concentrated. The residue was purified by flash silica gel column chromatography.The benzoylated glycoside was dissolved in MeOH:THF (1:1) (20 mL/mmol), a 0.1 M solution of MeO19-O-β-d-glucopyranosyl-7,15-isopimaradiene (10), 1H-NMR : δ 5.80 , 5.36 , 4.92 , 4.85 , 4.21 , 3.88 , 3.73 , 3.68 , 3.34–3.25 , 3.17 , 2.09 , 1.99 , 1.97 ,1.88 , 1.87 , 1.68 , 1.57 , 1.55 , 1.47 , 1.42 , 1.38 , 1.37 , 1.26 , 1.07 , 1.00 , 0.92 , 0.89 , 0.86 . 13C-NMR : 150.1 (C-15), 136.6 (C-8), 122.9 (C-7), 109.7 (C-16), 105.4 (C-1′), 78.2, 77.8 , 75.3 (C-2′), 73.6 (C-19), 71.7 (C-5′), 62.8 (C-6′), 53.7 (C-9), 53.1 (C-5), 47.3 (C-14), 41.1 (C-1), 38.4 (C-4 or C-10), 37.9 (C-10 or C-4), 37.4 (C-13), 36.8 (C-3), 36.5 (C-12), 28.3 (C-18), 24.2 (C-6), 22.0 (C-17), 21.5 (C-11), 19.5 (C-2), 16.7 (C-20).Lagascatriol-16-O-β-d-glucopyranoside (11), 1H-NMR : δ 5.50 , 4.26 , 3.87 , 3.80 , 3.66  3.58 , 3.36 , 3.27 , 3.21 , 2.55 , 2.18 , 1.78 , 1.67 , 1.63 , 1.20 , 1.05 , 1.04 , 0.98 , 0.70 , 1.01, 1.53, 1.40, 0.99, 1.32, 1.53 and 1.42 . 13C-NMR : δ 148.1 (C-5), 116.9 (C-6), 104.3 (C-1′), 79.8 (C-15), 78.8 (C-11), 78.0 (C-3′), 77.9 (C-5′), 75.1 (C-2′), 71.7 (C-16), 71.6 (C-4′), 62.7 (C-6′), 48.2 (C-10), 42.3 (C-3), 41.5 (C-9), 39.9 (C-14), 38.8 (C-13), 37.4 (C-8), 37.3 (C-4), 37.1 (C-12), 32.1 (C-1), 30.6 (C-18 and C-7), 29.0 (C-19), 23.5 (C-2), 20.4 (C-17), 6.6 (C-20).Lagascatriol-16-O-α-d-mannopyranoside (12), 1H-NMR : δ 5.49 , 4.77 , 3.92 , 3.86 , 3.83 , 3.71 , 3.69 , 3.58 , 3.58 , 3.53 , 3.32 , 2.54 , 2.18 , 1.78 , 1.67 , 1.63 , 1.53 , 1.42 , 1.41 , 1.30 , 1.20 , 1.05 , 1.03 , 1.00 , 0.98 , 0.97 , 0.69 . 13C-NMR : δ 148.1 (C-5), 116.9 (C-6), 102.4 (C-1′), 80.4 (C-15), 78.8 (C-11), 74.8 (C-5′), 72.6 (C-3′), 72.0 (C-2′), 69.8 (C-16), 68.7 (C-4′), 63.0 (C-6′), 48.2 (C-10), 42.3 (C-3), 41.5 (C-9), 39.9 (C-14), 39.6 (C-13), 37.4 (C-8), 37.3 (C-4), 37.1 (C-12), 32.1 (C-1), 30.6 (C-18), 29.6 (C-7), 29.0 (C-19), 23.5 (C-2), 20.3 (C-17), 6.6 (C-20).Staphylococcus aureus ATCC 25923, S. aureus ATCC 43866, S. aureus ATCC 700699, S. aureus CIP 106,760 (MRSA—methicillin-resistant S. aureus), S. aureus FFHB 29,593 (MRSA), Enterococcus faecalis ATCC 51,299 (low level VRE—vancomycin-resistant Enterococcus), E. faecalis FFHB 427483, E. faecalis CIP 106996, E. faecium FFHB 435628, E. flavescens ATCC 49996, E. hirae CIP 5855. FFHB species were identified and deposited on the Microbiology Laboratory of the Faculty of Pharmacy, Lisbon University, from clinical isolates of Hospital do Barreiro.The MIC values of compounds, against the tested strains, were performed by means of the twofold serial broth microdilution assay . The com2.MDA-MB-231 cells were cultured in DMEM  supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin . The cul3 cells in 190 μL of culture medium per well, were plated in 96-well plates, and allowed to grow for 24 h. Cells were then exposed to the different compounds for 48 h. The CV assay was carried out according to a previously described protocol [v/v) was used as positive control. At least two independent experiments were performed, each comprising four replicate cultures.Cell viability was evaluated by the crystal violet (CV) staining assay. Approximately 6 × 10protocol . DMSO 5%Staphylococcus aureus (ATCC 25923). MIC values were considered with units of µM and a logarithmic transformation was performed. Only compounds with activities <125 µg mL−1 were considered and linear relationships between the molecular descriptors and the Log (MIC) were calculated. Only non-correlated molecular descriptors  were considered in the analysis.A relationship between some molecular descriptors, namely constitutional, topological, geometrical, WHIM, functional group counts and molecular properties descriptors, was evaluated based on the activity of the compounds against 1 and 6 showed promising antibacterial activity, despite the efforts to find more potent antimicrobial analogous. The antimicrobial activity against S. aureus and E. hirae was already known [1 showed the most potent antibacterial activity of the set of tested compounds. Isopimarane 6 presented high antimicrobial activity against the Gram-positive assayed. Among the acetylated and glicosyl derivates, only 19-O-acetyl ester 7 and 3,19-diol 2 showed some antibacterial activity. These results indicate that the additional substituents incorporated did not lead to an improved antimicrobial activity, probably because the acetyl group drastically changes the donor/acceptor abilities and glycosyl substituents diminished the lipophilic properties of these molecules. Further studies are being carried out to improve the bioactivity. Isopimarane 1 showed some cytotoxicity. The IC50 value of compound 1 was 15 µM, suggesting a mild antiproliferative activity against these breast cancer cells. The compounds 1, 3, 5, 6, 8–12 were not cytotoxic to MDA-MB-231 cells under the conditions tested; however, for a complete screening of their cytotoxic effects, they should be studied in additional cell lines, representative of other cancer types.Only the pimaranes dy known ; however


Their structures were elucidated using 1D and 2D NMR spectroscopy and MS/MS as 2-(1-methylethenyl)-5-hydroxy-6-carbomethoxy-2,3-dihydro-benzofuran, 4,9-dihydroxy-3-methoxy-2-dimethylallylpterocarpan, and 4,9-dihydroxy-3′,3′-dimethyl-2,3-pyranopterocarpan.Three new compounds, a dihydrobenzofuran (coumaran) derivative (compound  Calicotome villosa (Poir.) Link. (Fabaceae) is a shrub that can reach 2 m in high, with grey-tomentose stems and sharp terminations, villous pods, trifoliate and oval leaves, and yellow and grouped flowers during the spring season + 100); 256.0718 (36.3); 225.0522 (27.9); 62.9816 (26.8). Compound 2 m/z (%): 377.1347 ([M+Na]+ 9.4); 308.0651 (29); 293.0407 (24.4); 164.9189 (30.1); 149.0592 (35.1); 105.0333 (17.6); 90.9757 (39.8); 69.0684 (8.3); 62.9804 (100); 56.0480 (49.5). Compound 3: m/z (%): 361.1050 ([M + Na]+ 46.3); 313.2039 (10.8); 164.9197 (26.4); 104.9923 (6.2); 91.0541 (5.0); 62.9803 (100)  at m/z = 415.211. Products were dissolved in methanol for direct introduction. Compound C. villosa growing wild in Corsica (France) and their structures elucidated as 2-(1-methylethenyl)-5-hydroxy-6-carbomethoxy-2,3-dihydrobenzofuran (1), 4,9-dihydroxy-3-methoxy-2-dimethylallylpterocarpan (2), and 4,9-dihydroxy-3′,3′-dimethyl-2,3-pyranopterocarpan (3). Various pterocarpan derivatives were identified in root extracts from Fabaceae, some of which are known as bioactive compounds. These compounds hold great potential for therapeutic treatment. Lastly, to the best of our knowledge, dihydrobenzofuran derivatives have rarely been identified in Calicotome species extracts.Three new compounds were isolated from the roots of


N-acetylcysteine (NAC) as an antiapoptotic reference. Compounds 6a–c were the most active antiapoptotic hybrids with significant measurements for malondialdehyde (MDA) and total antioxidant capacity (TAC) and the apoptotic biomarkers  in comparison to the reference. A preliminary mechanistic study was performed to improve the antiapoptotic activity through caspase-3 inhibition. A compound assigned as 6-methoxy-4-oxy)methyl)-1H-1,2,3-triazol-1-yl)quinolin-2(1H)-one (6c) was selected as a representative of the most active hybrids in comparison to NAC. Assay of cytochrome C for 6c revealed an attenuation of cytochrome C level about 3.54 fold, comparable to NAC (4.13 fold). In caspases-3,8,9 assays, 6c was found to exhibit more potency and selectivity toward caspase-3 than other caspases. The testicular histopathological investigation was carried out on all targeted compounds 6a–g, indicating a significant improvement in the spermatogenesis process for compounds 6a–c if compared to the reference relative to the control. Finally, molecular docking studies were done at the caspase-3 active site to suggest possible binding modes. Hence, it could conceivably be hypothesized that compounds 6a–c could be considered good lead candidate compounds as antiapoptotic agents.A series of novel 1,2,3-triazoles hybridized with two quinolin-2-ones, was designed and synthesized through click reactions. The structures of the synthesized compounds were elucidated by NMR, IR, and mass spectra in addition to elemental analysis. The synthesized compounds were assessed for their antiapoptotic activity in testis, as testicular torsion is the main cause of male infertility. This effect was studied in light of decreasing tissue damage induced by I/R in the testis of rats using  Apoptosis is an essential programmed biological process in cells during animal development,  homeostaThe caspase family has been an intensely studied target in drug design for many years . To dateCurrently, one of the urological emergency conditions in male infertility is testicular torsion, which is common in new-born kids, children, and adolescent boys ,19. TestOn the other hand, the discovery of quinolone-based drugs has attracted wide attention due to their diverse spectrum of biological activities  as an anH-imidazol-2-yl)-2H-l,2,3-triazoIe acting as antagonists of GABA receptors ..I as an III to be used as an antioxidant and antiapoptotic agent to prevent ototoxicity caused by cisplatin-induced oxidative stress  cycloadditions of 4-azidoquinolin-2(1H)-ones 4a–d with 4-(prop-2-yn-1-yloxy)quinolin-2(1H)-ones 5a–c to give the corresponding 4--1H-1,2,3-triazol-4-yl)methoxy)quinolin-2(1H)-ones 6a–g -one (5a)  and and6a–g mpound 7 . Also, t13C-NMR spectrum of 6b, two signals appeared at δC 163.16 and 160.83, for two carbonyl groups assigned as C-2′ and C-2, respectively. Also, the signal at δC 61.81, which gives HSQC correlation with the proton at δH 5.48, was assigned as C-4a″ (methylene group). The signal at δC 20.52 ppm, which gives HSQC and HMBC correlation with the proton signal at δH 2.31, and H–H correlation with the proton at δH 7.27, was assigned as C-6a (methyl group). Other signals in the 13C-NMR of 6b appeared at δC 161.75, 143.38, 126.73, 123.81, 117.81 and 97.93, and were assigned to C-4′, C-4″, C-5″, C-5, C-3 and C-3′, respectively  and 8.98 ; correlations with the proton signals led to the assignment of the carbons at δC 142.39 (C-4″), 126.73 (C-5″), and 61.81 (C-4a″). 15N-NMR showed three types of nitrogen. One appears at δN 248.0, which indicates an sp2 nitrogen; it was assigned as N-1″ and gives HMBC correlation with a proton at δH 8.98 (H-5″) but gives no HSQC correlation. The other two nitrogen atoms resonated at δN 151.6 and 144.0; their chemical shifts are characteristic of amide-type nitrogen atoms, and they give HSQC correlation with the attached protons at δH 12.29 and 11.43; therefore, these nitrogen atoms were assigned as N-1 and N-1′, respectively.In the ectively . Also, t6a–g then supported by further histological and molecular docking studies. The antiapoptotic effect of 6a–g was evaluated through evaluation of decreasing tissue damage induced by I/R in the testis of rats in a dose equivalent to 30 mg N-acetylcysteine (NAC) as an antiapoptotic reference [6a–g were administrated intra-peritoneal (i.p.) an hour before ischemia, which lasted for (0.5 h) then reperfusion (1 h) followed by cutting the organ  along with a significant increase in testicular TAC  relative to NAC  when compared to I/R group. Compounds 6e and 6g were still showing significantly not improved testicular MDA and TAC  . Excessi and TAC A.6d and 6f showed a significant decrease in testicular testosterone with a significant increase in TNF-α when compared to sham control and NAC groups  as shown in . Cell-based caspase-3, and proapoptotic caspase-8 and 9 cleave the synthetic substrate to release free AFC, which can then be quantified by fluorometry. Compounds to be screened can directly be added to the reaction, and the level of inhibition of caspase-3 activity can be determined by comparison of the fluorescence intensity in samples with and without the testing inhibitors. Compound shown in . Moreove control . The datC was assayed. It was reported that cytochrome C concentration in the cell has a critical role in the inactivation of caspases and prohibiting the intrinsic apoptosis pathway [C while proapoptotic members function as activators of its release. When cytochrome C level is maintained in mitochondria, caspase-8 and 9 will not be activated leading to caspase-3 inactivation, which in turn ceased apoptosis and reserved the cell from the programmed death [For more proof of caspase-3 inhibition by our synthesized compounds, cell-based cytochrome  pathway . Antiapoed death .6c was evaluated for cytochrome C against the SR human cell line and the results are listed in 6c caused down-expression of cytochrome C level about 3.54 fold, comparable to NAC (4.13 fold), and lower than control. The displayed results could be a good guide for suggesting that antiapoptotic activity may be attributed to the attenuation of cytochrome C and hence inactivation of the intrinsic apoptotic pathway induced by the tested compounds.The 1,2,3-triazole/biquinolinone derivative Throughout the previous work, results were consistent that motivated us to carry out a histopathological investigation of the tested compounds in the testis.6a–c showed no significant difference if compared to the reference (NAC) rats. On the other hand, compounds 6e and 6g showed weak significant differences if compared to the reference group. Whereas compounds 6d and 6f showed a highly significant histopathological difference compared to the reference group .The testicular I/R significantly made more histopathological changes A of the 6a–c showed more or less normal seminiferous tubules with germinal epithelium and Leydig cells (6e and 6g showed dilated seminiferous tubules. Distorted Leydig cells with dilated blood vessels (BV) were frequently seen among the sections  version 2014.09 . The crystal structure of caspase-3 protein taken from the protein data bank (PDB: 3GJQ) was used as a good template for many validation sets of structure-based pharmacophore modeling of caspase-3 inhibitors.Aiming to investigate the docking fitness scores of the bioactive conformations of the synthesized compounds and their specificity for caspase-3, docking of compounds 6a–c by caspase-3 inhibition activity.Molecular docking results were found to agree with the findings of biochemical and histopathological studies, for the highly active derivatives such as As a first step, validation of the docking protocol settings was done through the re-docking of the extracted co-crystallized ligand Ac-IEPD-Cho from the 3D structure using the same protocol for the docked compounds (RMSD 0.38 Å). The used docking protocol closely reproduced the bound structure confirming the confidence in our docking study. The docked compounds were built using the builder tool in MOE then subjected to an energy minimization followed by a systematic conformational search using the default force field and settings, then the lowest energy conformer of each molecule was used for the docking experiment.The docking study will be valid proof of the previously displayed mechanistic antiapoptotic study. Docking will be based on some aspects (i) binding with a caspase-3 catalytic binding site (ASP135 and LYS137), where the more binding, the more caspase inhibitory activity and the more antiapoptotic activity  along wi6b showed low RMSD refined (root mean square deviation refinement) values near to the pose of the co-crystallized ligand, which ensures both the validity of the docking protocol and the good interactions of the ligands and the main residues at the active site domain.Recently, it is reported that an RMSD < 2.0 Å corresponds to good docking solutions. On the other hand, docking solutions with RMSD between 2.0 and 3.0 Å deviate from the position of the reference, but they keep the desired orientation. Finally, docking solutions with RMSD > 3.0 Å are completely wrong . This co6a–g showed better mode of interactions than the reference NAC. The 2D and 3D diagrams of the compounds showed crucial binding with LYS137 GLY125 and THR140 through quinoline NH and triazole C=N functionality. The next study, therefore, moves on to discuss the hypothesis that says linking with ASP135 with good binging energy and a low RMSD value give good caspase inhibition. Furthermore, stabilization of the reference NAC within the active site occurred through two strong hydrogen bond interactions with amino acid residues ASP135 and LYS137. Compound 6c /1,2,3-triazole derivatives pound 6c  exhibite6f  possessions in addition to the costs needed for developing a new drug. Hence, design and applied new drugs are considered to be complicated. Therefore, evaluating the pharmacokinetic properties of a new drug is a critical step in the process of drug development and can directly contribute to optimization efforts into recovered analogs . Current2 and 10 or fewer rotatable bonds.The higher oral bioavailability exhibited by compounds with tPSA of fewer than 140 Ahttps://preadmet.bmdrc.kr/) [https://www.molinspiration.com/) [https://www.molsoft.com/about.html) [http://www.swissadme.ch/) software [6a–g obey the Lipinski’s rule with Log P values from 2.32 to 2.73 (<5), an MW range from 385.38 to 415.40 (<500), HBD from 1 to 2 (≤5) and HBA from 5 to 6 (<10). They would theoretically show a strong oral absorption and this property cannot be attributed to variations in their bioactivity. Furthermore, the topological PSA values of the compounds ring are between 94.80 and 114.89 A2 (<140 A2) and the corresponding percentage oral absorption was between 72.55 and 69.36%, exhibit strong permeability, absorption and transport across a biological membrane. Furthermore, the drug-likeness model score and solubility for compounds was confirmed by Molsoft software . Herein, we used Pre-ADMET (drc.kr/) , Molinspon.com/) , Molsoftut.html) , and Swisoftware  for predsoftware . Aqueous6a–g showed high human intestinal absorption values of 95.58 to 100%, indicating very well-absorbed compounds especially for 6a, which exhibited 100% intestinal absorption. The examined compounds were found to be highly-bound to human plasma proteins from 89.41 to 97.71% except for 6g that was low bounded to plasma protein 84.71%. Additionally, the following pharmacokinetic parameters were experimented with in silico using Pre-ADMET software: blood–brain barrier partition coefficient (BBB), cytochrome inhibition of cytochrome P4502D6 (CYP2D6), Caco2, coefficient (human colon adenocarcinoma), MDCK (Madin–Darby canine kidney cells) permeability coefficient, human intestinal absorption (HIA) and human plasma–protein binding (PPB). The results of the ADME parameters are shown in 1 and R3 as in compounds 6a–c, the antiapoptotic activity revealed the highest potency. Meanwhile, upon substitution of R3 with an electron-donating group such as methyl (compound 6e) or -OCH3 group (compound 6g), the antiapoptotic activity was particularly diminished to become abolished upon substitution either at R1 only (compound 6d) or at R2 and R3 for compound 6f. These biochemical findings can be explained as a substitution at both two quinoline-2-one rings, which would hinder interaction with the target protein that may be attributed to the resulting steric hindrance. Because the target compounds 6a–g revealed interesting downregulation of caspase-3 in testis, this work has undergone further histopathological mechanistic investigations to prove exactly the explained hypothesis. The in silico study revealed that all the target compounds 6a–g obey the Lipinski’s rule in addition to the drug-likeness model score and solubility for compounds was confirmed by Molsoft software. The ADME predictions displayed moderate to good parameters for 6a–g. These results confirm the successful design of the target compounds.Based on the previous results, we can deduce that when the two quinoline moieties are unsubstituted at position Rd6 on a Bruker AV-400 spectrometer  versus internal tetramethylsilane (TMS) = 0 ppm for 1H and 13C, and external liquid ammonia = 0 ppm for 15N. Coupling constants are stated in Hz. Correlations were established using 1H-1H COSY, and 1H-13C and 1H-15N HSQC and HMBC experiments. All 15N signals were observed indirectly, via HSQC or HMBC experiments. Chemical shifts (δ) are reported in parts per million (ppm) relative to tetramethylsilane (TMS) as internal standard, and the coupling constants (J) are reported in Hertz (Hz). Splitting patterns are denoted as follows: singlet (s), doublet (d), multiplet (m), triplet (t), quartet (q), doublet of doublets (dd), doublet of triplets (dt), triplet of doublets (td), and doublet of quartet (dq). Melting points (mp) were determined with a Stuart melting point instrument in the Chemistry Department, Florida Institute of Technology, 150 W University Blvd, Melbourne, FL, USA, and are expressed in °C as well as Mass spectra were recorded on a Finnigan Fab 70 eV at Al-Azhar University, Egypt. Elemental analyses were carried out on a Perkin Elmer device at the Microanalytical Institute of Organic Chemistry, Karlsruhe Institute of Technology, Karlsruhe, Germany.All reagents were used as purchased from Merck . The progress of all reactions was monitored with thin-layer chromatography (TLC) on Merck alumina-backed TLC plates and visualized under UV light. Spectra were measured in DMSO-H)-ones 4a–d were prepared according to the literature as 4-azidoquinolin-2(1H)-one (4a) [H)-one (4b) [H)-one (4c) [H)-one (4d) [All 4-azidoquinolin-2(1one (4a) ,69, 4-azone (4b) , 4-azidoone (4c)  and 4-azH)-one (4 mmol) in DMF (30 mL) and K2CO3  was added propargyl bromide  dropwise at 30–40 °C with stirring for 5 h and the reaction mixture was mentioned by TLC. Then the reaction mixture was poured into 200 g ice to give a white precipitate, which was filtered off and recrystallized from ethanol to give products 5a–c.To a solution of 6-substituted 4-hydroxy-quinolin-2-(1H)-one (5a). White solid, yield: 112 mg, 40%; mp 210–212 °C; 1H-NMR (DMSO-d6) δH 1.44 , 7.76 , 7.53 , 7.30 , 7.18 , 5.97 , 5.01 , 3.75 . 1H-NMR (CDCl3) δH 11.95 , 7.95 , 7.55 , 7.41 , 7.24 , 6.17 , 4.88 , 2.64 . 13C-NMR (DMSO-d6): δC 162.91 (C-2), 160.91 (C-4), 138.64 (C-8a), 131.04 (C-7), 122.11 (C-5), 121.44 (C-6), 115.22 (C-8), 114.29 (C-4a), 98.13 (C-3), 79.32 (C-4e), 77.89 (C-4d), 56.34 (C-4c). 13C-NMR (CDCl3): δC 163.11 (C-4), 138.10 (C-8a), 131.54 (C-7), 122.94 (C-5), 122.64 (C-6), 116.11 (C-8), 115.47 (C-4a), 97.17 (C-3), 77.17 (C-4e), 76.45 (C-4d), 56.55 (C-4c). 15N-NMR (DMSO-d6): δN 144.1 (N-1). 15N-NMR: (CDCl3): δN 145.3 (N-1). Anal. Calcd. for C12H9NO2: C, 72.35; H, 4.55; N, 7.03. Found: C, 72.44; H, 4.39; N, 6.99.4-(Prop-2-yn-1-yloxy)quinolin-2(1H)-one (5b). White solid, yield: 176 mg, 60%; mp 214–216 °C; 1H-NMR (DMSO-d6): δH 11.37 , 7.55 , 7.35 , 7.20 , 5.94 , 4.99 , 3.75 , 2.35 . 13C-NMR (DMSO-d6): δC 162.79 (C-2), 160.77 (C-4), 136.65 (C-8a), 132.21 (C-7), 130.47 (C-6), 121.55 (C-5), 115.18 (C-8), 114.19 (C-4a), 98.09 (C-3), 79.26 (C-4d), 77.93 (C-4c), 56.27 (-O-CH2), 20.45 (CH3). 15N-NMR (DMSO-d6): δN 144.1 (NH). Anal. Calcd. for C13H11NO2: C, 73.23; H, 5.20; N, 6.57. Found: C, 73.33; H, 5.08; N, 6.68.6-Methyl-4-(prop-2-yn-1-yloxy)quinolin-2(1H)-one (5c). White solid, yield: 155 mg, 50%; mp 220–222 °C; 1H-NMR (DMSO-d6): δH 11.36 , 7.50 , 7.35 , 7.23 , 5.91 , 4.99 , 3.75 , 3.73 . 13C-NMR (DMSO-d6): δC 162.59 (C-2), 160.76 (C-4), 151.35 (C-6), 135.50 (C-8a), 132.21 (C-7), 122.05 (C-5), 115.48 (C-8), 114.13 (C-4a), 97.99 (C-3), 79.22 (C-4d), 77.93 (C-4c), 55.98 (-O-CH2), 55.13 (-OCH3). 15N-NMR (DMSO-d6): δN 144.1 (NH). Anal. Calcd for C13H11NO3: C, 68.11; H, 4.84; N, 6.11. Found: C, 68.29; H, 4.77; N, 5.98.6-Methoxy-4-(prop-2-yn-1-yloxy)quinolin-2(15a–c in 20 mL dimethylformamide (DMF), CuI (10 mol%) was stirred for 10 min at room temperature. Then, 4-azido compounds 4a–d (1.0 mmol) were added to the mixture. The reaction mixture was allowed to stir at 50–60 °C for 24 h. The reaction was monitored with TLC. After completion, the reaction mixture was poured on 200 g ice and extracted by filtration.In a round bottom flask, mixing (1 mmol) of terminal alkynes H-1,2,3-triazol-4-yl)methoxy)quinolin-2(1H)-one (6a). Yellowish green solid, yield: 328 mg, 85%; mp 300–302 °C; 1H-NMR (DMSO-d6): δH 12.29 , 11.43 , 9.00 , 7.84 , 7.67 , 7.52-7.48 , 7.29-7.27 , 7.16 , 6.91 , 6.21 , 5.47 . 13C-NMR (DMSO-d6): δC 163.12 (C-2′), 161.75 (C-4′), 160.96 (C-2), 143.59 (C-8a), 142.41 (C-4″), 139.45 (C-4), 138.67 (C-8a′), 131.89 (C-7), 131.02 (C-7′), 126.74 (C-5″), 124.02 (C-5), 122.60, 122.50 , 121.32 (C-6′), 117.82 (C-3), 115.93 (C-4a/4a′), 115.15 (C-8), 114.46 (C-4a′/4a), 114.41 (C-8′), 97.93 (C-3′), 61.81 (-O-CH2). EI-MS : 385 . Anal. Calcd for C21H15N5O3: C, 65.45; H, 3.92; N, 18.17. Found: C, 65.60; H, 3.88; N, 18.29.4--1H-1,2,3-triazol-1-yl)quinolin-2(1H)-one (6b). Yellowish green solid, yield: 360 mg, 90%; mp 298 °C; NMR: see m/z,%): 399 . Anal. Calcd for C22H17N5O3: C, 66.16; H, 4.29; N, 17.53. Found: C, 66.09; H, 4.33; N, 17.69.6-Methyl-4-oxy)methyl)-1H-1,2,3-triazol-1-yl)-quinolin-2(1H)-one (6c). White solid, yield: 270 mg, 65%; mp 295–297 °C; 1H-NMR (DMSO-d6): δH 12.21 , 11.44 , 9.04 , 7.84 , 7.53 , 7.45 , 7.36 , 7.30 , 7.16 , 6.96 , 6.91 , 6.21 , 5.49 , 3.71 . 13C-NMR (DMSO-d6): solubility was low. Carbon signals were observed indirectly, using HSQC or HMBC experiments. δC 162.1 (C-4′), 154.9 (C-6), 143.4 (C-4), 142.8 (C-4″), 134.9 (C-8a), 131.4 (C-7′), 127.1 (C-5″), 123.0 (C-5′), 121.7 (C-7), 121.5 (C-6′), 118.7 (C-3), 117.9 (C-8), 115.5 (C-8′), 106.2 (C-5), 62.3 (-O-CH2), 55.8 (OCH3). C-2, 2′, 3′, 4a, 4a′, and 8a′ were not observed. EI-MS : 415 . Anal. Calcd for C22H17N5O4: C, 63.61; H, 4.12; N, 16.86. Found: C, 63.72; H, 4.29; N, 16.99.6-Methoxy-4-oxy)methyl)-1H-1,2,3-triazol-1-yl)quinolin-2(1H)-one (6d). White solid, yield: 270 mg, 68%; mp 285–287 °C; 1H-NMR (DMSO-d6): δH 11.43 , 8.99 , 7.84 , 7.80 , 7.74 , 7.52 , 7.51 , 7.36 , 7.30 , 7.16 , 7.04 , 6.22 , 5.48 , 3.74 . 13C-NMR (DMSO-d6): δC 163.12 (C-2′), 161.75 (C-4′), 160.30 (C-2), 142.59 (C-4), 142.41 (C-4″), 140.15 (C-8a), 138.67 (C-8a′), 132.33 (C-7), 131.02 (C-7′), 126.96 (C-5″), 124.50 (C-5), 122.75 (C-6), 122.49 (C-5′), 121.32 (C-6′), 117.30 (C-3), 115.57, 115.54 , 115.15 (C-8′), 114.41 (C-4a′), 97.93 (C-3′), 61.79 (-O-CH2), 29.59 (CH3). 15N-NMR (DMSO-d6): δN 247.0 (N-1″), 147.2 (N-1), 144.1 (N-1′). EI-MS : 399 . Anal. Calcd for C22H17N5O3: C, 66.16; H, 4.29; N, 17.53. Found: C, 65.99; H, 4.36; N, 17.49.1-Methyl-4-oxy)methyl)-1H-1,2,3-triazol-4-yl)methoxy)quinolin-2(1H)-one (6e). Yellowish green, yield: 320 mg, 80%; mp 272–274 °C; 1H-NMR (DMSO-d6): δH 12.29 , 11.35 ; 8.99 , 7.67 , 7.61 , 7.51 , 7.49 , 7.35 , 7.27 , 7.21 , 6.91 , 6.19 , 5.46 , 2.33 . 13C-NMR (DMSO-d6): δC 163.01 (C-2′), 161.62 (C-4′), 160.97 (C-2), 143.61 (C-4), 142.36 (C-4″), 139.45 (C-8a), 136.68 (C-8a′), 132.21 (C-7′), 131.90 (C-7), 130.38 (C-6′), 126.81 (C-5″), 124.03 (C-5), 122.61 (C-6), 121.81 (C-5′), 117.84 (C-3), 115.94 (C-8), 115.13 (C-8′), 114.47 (C-4a), 114.29 (C-4a′), 97.90 (C-3′), 61.68 (-O-CH2), 20.45 (CH3). 15N-NMR (DMSO-d6) δN 247.8 (N-1″), 152.2 (N-1), 143.2 (N-1′). EI-MS : 399 . Anal. Calcd for C22H17N5O3: C, 66.16; H, 4.29; N, 17.53. Found: C, 66.09; H, 4.44; N, 17.38.6-Methyl-4--1H-1,2,3-triazol-4-yl)meth-oxy)quinolin-2(1H)-one (6f). White solid, yield: 363 mg, 88%; mp 270–272 °C; 1H-NMR (DMSO-d6): δH 12.22 , 11.36 , 8.98 , 7.61 , 7.49 , 7.39 , 7.34 , 7.26 , 7.20 , 6.87 , 6.19 , 5.46 , 2.32 , 2.31 . 13C-NMR (DMSO-d6): δC 163.04 (C-2′), 161.61 (C-4′), 160.83 (C-2), 143.41 (C-4), 142.36 (C-4″), 137.53 (C-8a), 136.69 (C-8a′), 133.19 (C-7), 132.19 (C-7′), 131.79 (C-6), 130.37 (C-6′), 126.81 (C-5″), 123.14 (C-5), 121.81 (C-5′), 117.87 (C-3), 115.92 (C-8), 115.14 (C-8′), 114.40, 114.31 , 97.91 (C-3′), 61.71 (-O-CH2), 20.52 (C-6a), 20.45 (C-6a′). 15N-NMR (DMSO-d6): δN 248.0 (N-1″), 151.5 (N-1), 143.2 (N-1′). EI-MS : 413 . Anal. Calcd for C23H19N5O3: C, 66.82; H, 4.63; N, 16.94. Found: C, 66.98; H, 4.77; N, 16.99.6-Methyl-4--1H-1,2,3-triazol-4-yl)methoxy)quinolin-2-(1H)-one (6g). Pale yellow, yield: 270 mg, 65%; mp 300–302 °C; 1H-NMR (DMSO-d6) δH 12.29 , 11.33 , 8.99 , 7.66 , 7.49 , 7.26 , 7.21 , 6.90 , 6.22 , 5.49 , 3.77 . 13C-NMR (DMSO-d6): δC 162.67 (C-2′), 161.26 (C-4′), 160.95 (C-2), 153.97 (C-6′), 143.60 (C-4), 142.41 (C-4″), 139.45 (C-8a), 133.18 (C-8a′), 131.89 (C-7), 126.76 (C-5″), 123.98 (C-5), 122.58 (C-6), 119.90 (C-7′), 117.86 (C-3), 116.62 (C-8′), 115.94 (C-8), 114.82, 114.48 , 104.33 (C-5′), 98.41 (C-3′), 61.79 (-O-CH2), 55.51 (-OCH3). 15N-NMR (DMSO-d6): δN 247.9 (N-1″), 142.7 (N-1). EI-MS : 415 . Anal. Calcd for C22H17N5O4: C, 63.61; H, 4.12; N, 16.86. Found: C, 63.77; H, 4.10; N, 17.00.6-Methoxy-4--1TAC was assessed by the colorimetric technique using commercial kits  See . TesticuTNF-α was measured by ELISA kit . Caspase-3 was measured by ELISA kit Cusabio, Houston, TX, USA See . TestostThe Caspase-3 Inhibitor Drug Screening Kit  provides an effective means for screening caspase inhibitors using fluorometric methods. The assay utilizes synthetic peptide substrate DEVD-AFC . Active caspase-3 cleaves the synthetic substrate to release free AFC, which can then be quantified by fluorometry. Compounds to be screened can directly be added to the reaction and the level of inhibition of caspase-3 activity can be determined by comparison of the fluorescence intensity in samples with and without the testing inhibitors. See .Cells were collected from American Type Culture Collection to be grown in SR containing 10% fetal bovine serum at 37 °C, stimulated with the compounds to be tested for cytochrome C using Cytochrome C Human ELISA Kit   See Sup.Multiple testicular specimens were stained by hematoxylin-eosin  using Cosentino’s score  for semiAn Olympus light microscopy and digital camera , were used in this study. Images were executed using Adobe Photoshop.; Wayne Rasb and National Institutes of Health).Semi-quantitative data were measured and counted per section in ten randomly selected non-overlapping fields using power × 400 magnifications of the sections from each rat . The resp values < 0.05 were considered significant.Data were evaluated as means (standard error of the mean). One-way analysis of variance (ANOV) with the use of Turkey’s post-test was carried out for the analysis of the results to detect the significant difference statistically. ®) version 2014.09, Chemical Computing Group Inc., Montreal, QC, Canada). The computational software operated under “Windows XP” installed on an Intel Pentium IV PC with a 1.6 GHz processor and 512 MB memory. The target compounds were constructed into a 3D model using the builder interface of the MOE program and docked into the active site of caspase-3 (PDB: 3GJQ). Checking their structures and the formal charges on atoms by 2D depiction was carried out and the energy, was minimized until an RMSD (root mean square deviations) gradient of 0.01 Kcal/mol and RMS (Root Mean Square) distance of 0.1 A with MMFF94X (Merck molecular force field 94x) force-field and the partial charges were automatically calculated (See The docking simulation study was carried out using Molecular Operating Environment (MOEated See .http://www.swissadme.ch/index.php) and Molinspiration software (https://preadmet.bmdrc.kr/). In addition, Lipinski drug-likeness of the target compounds were obtained by using Molsoft software (https://www.molsoft.com/) and Swiss ADME software. The percent of absorption was calculated through this equation:The physicochemical and lipophilicity of the target compounds were collected using Swiss ADME software (Minia University Faculty of Medicine, Research Ethics Committee “BFMREC” approved this research proposal regarding the source of the animals, health status, inclusion criteria, exclusion criteria, caging, comfort, and the detailed experimental design and procedures. It was conducted in agreement with the NIH Guide for Care and Use of Laboratory Animals. The approval number is: 251-2/2018.6a–g have been synthesized and identified with different spectroscopic techniques. The antiapoptotic activity of the target compounds in the testis was biochemically evaluated compared with the reference NAC. The most robust compounds were 6a–c, revealing promising antiapoptotic and antioxidant readings. Moreover, they exhibited a caspase-3 inhibition level of about 2.66, 2.94, and 2.39 ng/mL, respectively. Compound 6c can be evaluated as a hit for further screenings to acquire more active antiapoptotic agents due to its selectivity toward caspase-3 rather than caspase-8 and -9 as well as a higher cytochrome C potency with conc 0.28 ng/mL. Further histopathological study was performed in a specimen of testis treated with the tested compounds. Furthermore, the molecular docking study was performed on the caspase-3 active site revealing a good interaction with the enzyme. Finally, a computational prediction of physicochemical properties, pharmacokinetics, and drug-likeness profile in silico revealed that compound 6a–c should be taken into consideration as good antiapoptotic candidates for further study.A series of biquinoline-2-one/1,2,3-triazole hybrids


MTH1 is an enzyme that hydrolyzes 8-oxo-dGTP, which is an oxidatively damaged nucleobase, into 8-oxo-dGMP in nucleotide pools to prevent its mis-incorporation into genomic DNA. Selective and potent MTH1-binding molecules have potential as biological tools and drug candidates. We recently developed 8-halogenated 7-deaza-dGTP as an 8-oxo-dGTP mimic and found that it was not hydrolyzed, but inhibited enzyme activity. To further increase MTH1 binding, we herein designed and synthesized 7,8-dihalogenated 7-deaza-dG derivatives. We successfully synthesized multiple derivatives, including substituted nucleosides and nucleotides, using 7-deaza-dG as a starting material. Evaluations of the inhibition of MTH1 activity revealed the strong inhibitory effects on enzyme activity of the 7,8-dihalogenated 7-deaza-dG derivatives, particularly 7,8-dibromo 7-daza-dGTP. Based on the results obtained on kinetic parameters and from computational docking simulating studies, these nucleotide analogs interacted with the active site of MTH1 and competitively inhibited the substrate 8-oxodGTP. Therefore, novel properties of repair enzymes in cells may be elucidated using new compounds. HR-ESI-MS (m/z) calcd. for C11H12N4O4Br2Na [M + Na]+: 444.9118, 446.9097, 448.9077, Found: 444.9112, 446.9078, 448.9067.7,8-di-Bromo-7-deaza-2′-deoxy-guanosine (3): Yield was 18% for two steps. 1H-NMR  δ (ppm): 10.62 , 6.29 , 6.24 , 5.19 , 4.94–4.92 , 4.39–4.36 , 3.76–3.75 , 3.65–3.64 , 3.57–3.52 , 3.16–3.08 , 2.01–1.98 . HR-ESI-MS (m/z) calcd. for C11H13N4O4I2Na [M + Na]+: 540.8841, Found: 540.8844.7,8-di-Iodo-7-deaza-2′-deoxy-guanosine  (0.24 mmol) were dissolved in anhydrous pyridine (1.2 mL) under an argon atmosphere at room temperature, and tert-butyldimethylsilyl chloride (0.34 mmol), imidazole (0.48 mmol), and dimethylaminopyridine (DMAP) (0.05 mmol) were then added to the reaction mixture. The resulting mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure, and the residue was purified by flash silica-gel chromatography  to give a 5′-OTBS protected compound as a white powder. Acetic anhydride (0.95 mmol) was dropped into the solution of the above compound (0.24 mmol) in anhydrous pyridine (2.4 mL), and the reaction mixture was stirred overnight. The solvent was concentrated under reduced pressure, and the residue was purified by flash silica-gel chromatography  to give 3′-OAc and 5′-OTBS-protected compounds as a white powder. This compound (0.24 mmol) was dissolved in dry THF (2 mL). Triethylamine trihydrofluoride (0.33 mmol) and triethylamine (0.19 mmol) were then dropped into the reaction mixture at room temperature under an argon atmosphere. The reaction solution was stirred at room temperature for 5 h. After the removal of the solvent under reduced pressure, the residue was purified by flash silica-gel chromatography  to give 14, 15, and 16 as a white powder.The corresponding diol compounds : Yield was 40% for three steps. 1H-NMR  δ (ppm): 10.98 , 6.46 , 6.36–6.29 , 5.31–5.29 , 4.92 , 3.96–3.93 , 3.66–3.62 , 3.60–3.54 , 3.27–3.23 , 2.28–2.24 , 2.07 . HR-ESI-MS (m/z) calcd. for C13H14N4O5Cl2Na [M + Na]+: 399.0234, 401.0204, 403.0175, Found: 399.0252, 401.0225, 403.0199.3′-O-Acetyl-7,8-di-bromo-7-deaza-2′-deoxy-guanosine (15): Yield was 59% for three steps. 1H-NMR  δ (ppm): 10.75 , 6.40 , 6.30–6.27 , 5.33–5.31 , 4.94 , 3.97–3.96 , 3.68–3.66 , 3.61–3.57 , 3.38–3.36 , 2.27–2.23 , 2.07 . HR-ESI-MS (m/z) calcd. for C13H15N4O5Br2 [M + H]+: 464.9404, 466.9383, 468.9363, Found: 464.9437, 466.9420, 468.9398.3′-O-Acetyl-7,8-di-iodo-7-deaza-2′-deoxy-guanosine (16): Yield was 18% for three steps. 1H-NMR  δ (ppm): 10.67 , 6.29–6.27 , 5.35–5.32 , 5.02 , 3.98–3.96 , 3.72–3.67, 3.63–3.58 , 3.44–3.38 , 2.23–2.19 , 2.07 . HR-ESI-MS (m/z) calcd. for C13H14N4O5Cl2Na [M + Na]+: 582.8946, found: 582.8971.3′-14, 15, 16, and 17 −: 412.9826, 414.9797, 416.9767, found: 412.9826, 414.9805, 416.9781.7,8-di-Chloro-7-deaza-2′-deoxy-guanosine monophosphate (6): Yield was 27% for two steps. 1H-NMR  δ (ppm) 6.54–6.51 , 4.87–4.84 , 4.17–4.11 , 4.00–3.98 , 3.34–3.28 , 2.36–2.31 . 31P-NMR  δ (ppm) 1.68. HR-ESI-MS (m/z) calcd. for C11H12N4O7Br2P [M − H]−: 500.8864, 502.8795, 504.8775, found: 500.8864, 502.8831, 504.8819.7,8-di-Bromo-7-deaza-2′-deoxy-guanosine monophosphate (7): Yield was 9% for two steps. 1H-NMR  δ (ppm) 6.48 , 4.87–4.84 , 4.17–4.09 , 3.99–3.94 , 3.36–3.30 , 2.34–2.28 . 31P-NMR  δ (ppm) 3.98. HR-ESI-MS (m/z) calcd. for C11H12N4O7I2P [M − H]−: 596.8538, found: 596.8579.7,8-di-Iodo-7-deaza-2′-deoxy-guanosine monophosphate (8): Yield was 30% for two steps. 1H-NMR  δ (ppm) 7.05 , 6.28 , 4.88–4.85 , 4.22–4.16 , 4.15–4.13 , 4.01–3.97 , 3.41–3.35 , 2.38–2.33 . 31P-NMR  δ (ppm) 3.42. HR-ESI-MS (m/z) calcd. for C11H13N4O7IP [M − H]−: 470.9572, found: 470.9591.8-Iodo-7-deaza-2′-deoxy-guanosine monophosphate  in DMF (0.35 mL) and tributyl amine (0.5 mL) were added dropwise to the solution of tributyl ammonium pyrophosphate (0.26 mmol) in DMF (0.35 mL) and stirred for 30 min under an argon atmosphere at room temperature. This reaction mixture was added to the solution of the corresponding diol compounds  (0.13 mmol) in DMF (0.1 mL). After stirring for 2 h, the oxidation reagent  was added to the reaction mixture and stirred for 10 min, and then the reaction was quenched by 5.0% NaHSO3 solution (4 mL). After ethanol precipitation, the resulting residue was purified by reversed-phase HPLC to give corresponding monophosphate compounds as a white material after lyophilization. ; UV detector: 254 nm; 35 °C).The solution of 2-chloro-4-9): Yield was 30%. 1H-NMR  δ (ppm) 6.50 , 4.87–4.84 , 4.33–4.31 , 4.22–4.17 , 3.32–3.28 , 2.36–2.31 . 31P-NMR  δ (ppm) −10.9, −11.3, −23.3. HR-ESI-MS (m/z) calcd. for C11H14N4O13Cl2P3 [M − H]−: 572.9195, 574.9123, 576.9093, found: 572.9149, 574.9122, 576.9090.7,8-di-Chloro-7-deaza-2′-deoxy-guanosine triphosphate (10): Yield was 14%. 1H-NMR  δ (ppm) 6.52 , 4.92–4.90 , 4.39–4.37 , 4.23–4.17 , 3.38–3.31 , 2.35–2.30 . 31P-NMR  δ (ppm) −9.4, −11.1, −23.0. HR-ESI-MS (m/z) calcd. for C11H14N4O13Br2P3 [M − H]−: 660.8142, 662.8122, 664.8102, found: 660.8171, 662.8152, 664.8132.7,8-di-Bromo-7-deaza-2′-deoxy-guanosine triphosphate (11): Yield was 14%. 1H-NMR  δ (ppm) 6.48 , 4.94–4.92 , 4.40–4.36 , 4.24–4.15 , 3.38–3.30 , 2.31–2.26 . 31P-NMR  δ (ppm) −9.9, −11.1, −23.0 HR-ESI-MS (m/z) calcd. for C11H14N4O13I2P3 [M − H]−: 756.7865, found: 756.7902.7,8-di-Iodo-7-deaza-2′-deoxy-guanosine triphosphate , 8 mM dithiothreitol (DTT), 10% glycerol; 0.5 pmol MTH1; 50 μM 7,8-dihalogenated-7-deaza-dGTP , 8-CF3-7-deaza-dGTP (4), and 8-oxo-dGTP). The resulting solutions were incubated at 37 °C for various times and quenched with 100 μL of stop solution (5 mM EDTA). All samples were loaded on ice before the analysis with HPLC ; UV detector: 254 nm; column oven: 35 °C).The reaction mixture was pre-incubated at 37 °C for 10 min before the addition of MTH1 to a final volume of 100 L , 10 μL (500 μM) of 8-oxo-dGTP, 55 μL of MilliQ, and 10 μL of various concentrations of substituted 7-deaza-dG derivatives were pre-incubated at 37 °C for 10 min  was 0, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, and 25 μM; 7,8-dihalogenated-7-deaza-dGMP and –dGTP were 0, 0.0125, 0.025, 0.05, 0.1, 0.25, 0.5, 1, and 2.5 μM). Five microliters of MTH1 (0.1 pmol/μL in MTH1 buffer) was added, and the reaction mixture was incubated at 37 °C for 10 min. The resulting solutions were quenched with 100 μL of stop solution (5 mM EDTA). All samples were loaded on ice before the analysis with HPLC ; UV detector: 254 nm; 35 °C). The relative hydrolysis ratio values (ratio with inhibitor/ratio without inhibitor) were plotted at each inhibitor concentration. IC50 values were assessed using images.Twenty microliters of MTH1 buffer  was used to remove the water and oxo-dGTP molecules, add hydrogen atoms, merge the non-polar hydrogen atoms, check and repair missing atoms, and assign Gasteiger charges and the AD4 atom type for the 5FSI crystal structure. The area of the protein involved in the docking calculation was also set with a ADT of 60 × 60 × 60 Å3 grid size and the center x, y, and z coordinates were set at 14.1, 19.6, and 9.2, respectively, to cover the whole protein (blind docking). The MTH1 structure prepared was then saved in the .pdbqt format. The structure of 8-oxo-dGTP was extracted from the 5FSI crystal structure and used as the standard in the docking study. ADT was then used to prepare the docking input file of the 8-oxo-dGTP structure, which included the detection of torsion angles, setting the number of torsions to the maximum, the assignment of Gasteiger charges, and conversion into the .pdbqt format for flexible ligand docking using AutoDock Vina. The processed oxodGTP structure was redocked back into its crystal structure using AutoDock Vina with an optimized exhaustiveness at 400, employing 5 different seedings of docking approaches with an output of 20 final conformations for each docking . The root-mean-square deviation (RMSD) values between the resulting redocked conformations and the original conformation in the crystal structure of the ligand were then calculated using Discovery Studio 4.5 (DS) software. These redocked conformations with RMSD values less than 5 Å, when compared to the 8-oxo-dGTP moiety in the 5FSI crystal structure, were grouped and defined as “Percentage of 8-oxo-dGTP-like Binding”. A conformation with the lowest aforementioned RMSD value was selected as the best conformation for the binding interaction analysis through a 2D-interaction diagram generated by DS. The value of the lowest binding energy corresponding to the best conformation, which represents its binding affinity, was also extracted from the docking result. The best performing triphosphate derivatives in vitro, 8-dihalogenated-7-deaza-dGTPs , were then selected and prepared by modifying 8-oxo-dGTP using DS for subsequent molecular docking to predict their binding properties.The three-dimensional (3D) crystal structure of MTH1 complexed with 8-oxo-dGTP (PDB ID: 5FSI) was downloaded from the Protein Data Bank (PDB). AutoDockTools 1.5.6  were not hydrolyzed by MTH1. The triphosphate part was not hydrolyzed, which is consistent with our previous findings and is a very important property. 7,8-Dichloro-7-deaza-dG derivatives , 7,8-dibromo-7-deaza-dG derivatives , and 7,8-diiodo-7-deaza-dG derivatives  exerted markedly stronger inhibitory effects on the hydrolytic activity of MTH1 than previously reported corresponding 8-iodo-7-deaza-dG derivatives . The present results confirmed that the involvement of phosphate groups was also very important for binding to MHT1. This result was supported by the Ki value. Furthermore, the inhibition mode was competitive inhibition with the substrate, according to steady-state kinetic parameters. The binding mode was also predicted by the computational molecular docking simulation, and these 7,8-dihalogenated-7-deaza-dGTP derivatives were able to bind at an active site of MTH1 and showed similar binding shapes with the substrate, 8-oxo-dGTP. The present results are the first example of the development of novel nucleotide analogues for strong binding to nucleotide repair enzymes. In the future, these compounds will be modified with membrane-permeable molecules and developed into chemical tools for the inhibition of repair enzyme activity, selectivity, and elucidation of detailed enzyme functions or their distribution in cells.We herein designed and successfully synthesized various 7-substituted and 7,8-dihalogenated 7-deaza-dG derivatives. These derivatives were designed as molecules that bind more strongly to the 8-oxo-dGTP repair enzyme, MTH1, based on the findings of our previous studies. We found that 7,8-dihalogenated 7-deaza-dGTPs (


Amycolatopsis mediterranei S699 ΔrifT was constructed by deleting the rifT gene. From this strain, eleven 8-deoxy-rifamycin derivatives (1–11) and seven known analogues (12–18) were isolated. Their structures were elucidated by extensive analysis of 1D and 2D NMR spectroscopic data and high-resolution ESI mass spectra. Compound 1 is a novel amide N-glycoside of seco-rifamycin. Compounds 2 and 3 feature conserved 11,12-seco-rifamycin W skeleton. The diverse post-modifications in the polyketide chain led to the production of 4–11. Compounds 2, 3, 5, 6, 13 and 15 exhibited antibacterial activity against Staphylococcus aureus  values of 10, 20, 20, 20, 40 and 20 μg/mL, respectively). Compounds 14, 15, 16, 17 and 18 showed potent antiproliferative activity against KG1 cells with IC50  values of 14.91, 44.78, 2.16, 18.67 and 8.07 μM, respectively.Proansamycin X, a hypothetical earliest macrocyclic precursor in the biosynthesis of rifamycin, had never been isolated and identified. According to bioinformatics analysis, it was proposed that RifT (a putative NADH-dependent dehydrogenase) may be a candidate target responsible for the dehydrogenation of proansamycin X. In this study, the mutant strain  Ansamycins are a type of macrocyclic antibiotics formed by an aromatic moiety bridged at nonadjacent positions by an aliphatic chain ,2, and eAmycolatopsis mediterranei S699 20D = +9.8 ; UV (MeOH) λmax (logε) 216 (4.43), 262 (4.22), 315 (3.81) nm; IR (KBr) νmax 3347, 2969, 2932, 2256, 2128, 1657, 1454, 1232, 1025, 997, 825 cm−1; 1H NMR data, 13C NMR data, m/z 790.4012 [M + H]+ , and 812.3817 [M + Na]+ .Compound 2: yellow powder; [α]20D = +17.3 ; UV (MeOH) λmax (logε) 212 (4.39), 272 (4.32), 311 (4.13) nm; IR (KBr) νmax 3366, 2969, 2931, 1655, 1498, 1339, 1123, 1061, 976 cm−1; 1H NMR data, 13C NMR data, m/z 642.2920 [M + H]+ , and 664.2734 [M + Na]+ .Compound 3: yellow powder; [α]20D = +19.7 ; UV (MeOH) λmax (logε) 211 (4.37), 277 (4.33), 307 (4.15) nm; IR (KBr) νmax 3362, 2970, 2936, 2878, 2359, 1655, 1500, 1332, 1210, 1109, 979 cm−1; 1H NMR data, 13C NMR data, m/z 664.2736 [M + Na]+ .Compound 4: tawny powder; [α]20D = −268.5 ; UV (MeOH) λmax (logε) 211 (4.37), 275 (4.32), 310 (4.05) nm; IR (KBr) νmax 3362, 2968, 2931, 1657, 1499, 1335, 1201, 1110, 977 cm−1; 1H NMR data, 13C NMR data, m/z 654.2924 [M + H]+ , and 676.2737 [M + Na]+ .Compound 5: brown powder; [α]20D = +65.7 ; UV (MeOH) λmax (logε) 225 (4.29), 271 (4.16), 309 (3.95) nm; IR (KBr) νmax 3359, 2968, 2931, 1658, 1494, 1339, 1261, 1110, 975 cm−1; 1H NMR data, 13C NMR data, m/z 682.3227 [M + H]+ , and 704.3047 [M + Na]+ .Compound 6: yellow powder; [α]20D = +81.5 ; UV (MeOH) λmax (logε) 225 (4.36), 275 (4.26), 307 (3.99) nm; IR (KBr) νmax 3361, 2969, 2933, 1656, 1495, 1340, 1153, 1062, 977 cm−1; 1H NMR data, 13C NMR data, m/z 640.3118 [M + H]+ , and 662.2936 [M + Na]+ .Compound 7: brown powder; [α]20D = +124.7 ; UV (MeOH) λmax (logε) 210 (4.51), 263 (4.30), 310 (4.13) nm; IR (KBr) νmax 3355, 2966, 2936, 1655, 1494, 1339, 1201, 1052, 968 cm−1; 13C NMR data, 1H NMR data, m/z 656.3066 [M + H]+ , and 678.2884 [M + Na]+ .Compound 8: brown powder; [α]20D = +168.4 ; UV (MeOH) λmax (logε) 212 (4.40), 273 (4.31), 313 (4.11) nm; IR (KBr) νmax 3361, 2970, 2934, 1657, 1495, 1340, 1223, 1054, 975 cm−1; 13C NMR data, 1H NMR data, m/z 656.3076 [M + H]+ , and 678.2891 [M + Na]+ .Compound 9: yellow powder; [α]20D = +146.8 ; UV (MeOH) λmax (logε) 229 (4.38), 270 (4.26), 308 (4.08) nm; IR (KBr) νmax 3370, 2972, 2936, 1656, 1494, 1339, 1221, 1063, 974 cm−1; 13C NMR data, 1H NMR data, m/z 656.3067 [M + H]+ , and 678.2884 [M + Na]+ .Compound 10: brown powder; [α]20D = +161.1 ; UV (MeOH) λmax (logε) 226 (4.43), 310 (4.07) nm; IR (KBr) νmax 3359, 2969, 2933, 1656, 1492, 1339, 1219, 1051, 970 cm−1; 1H NMR data, 13C NMR data, m/z 676.2731 [M + Na]+ .Compound 11: red-brown powder; [α]20D = −3620.7 ; UV (MeOH) λmax (logε) 226 (4.39), 310 (4.06) nm; IR (KBr) νmax 3345, 2973, 2937, 1644, 1492, 1341, 1214, 1071, 971 cm−1; 1H NMR data, 13C NMR data, m/z 654.2925 [M + H]+ , and 676.2737 [M + Na]+ .Compound 1–18 were assayed for their antimicrobial activity with the paper disc diffusion assay against Staphylococcus aureus ATCC 25923, Mycobacterium smegmatis mc2 155, Pseudomonas aeruginosa PA01 and Proteusbacillus vulgaris CPCC 160013 purchased from the China Center of Industrial Culture Collection  20D = +7.3~8.0 of α-L-rhamnose. The ansa chain was suggested to occur as retro-Claisen cleavage between C-5 and C-11 in consideration of the remaining aromatic hydrogen H-5 (δH 7.34) and chemical shift of C-11 (δC 169.3)  . The 24- carbons . The pre carbons , orange,cts of 1 . The 1H rhamnose . The [α]C 169.3) . The ste2 was determined to be C34H43NO11 by the HRESIMS quasi molecular ion peaks at m/z 642.2876 [M + H]+ and 664.2736 [M + Na]+ (2 was different in the C-11/C-12 cleavage and decarboxylation of C-34a. The ether linkage between C-11 (δC nda) and C-12 (δC 107.5) and the formation of a five-membered ring  was confirmed by decarboxylation and hydroxylation of C-28 (δC 71.2) in 2, and supported by the HMBC correlations of H-13 (δH 1.44) to C-12 (δC 107.5) and C-29 (δC 46.6), H-27 (δH 3.98) to C-12 and C-28, the 1H-1H COSY of H-28 (δH 4.39) with H-29  and the degrees of desaturation  with C-12 (δC 211.0), from H-29  with C-12 and C-28 (δC 81.5) as well as the remaining degrees of unsaturation and the molecular formula , H-34a (δH 5.12) and HMBC correlation of H-34a and C-25 (δC 73.7)   (SupplemδC 73.7) . Thus, 45 was elucidated as C37H47NO11  with C-35 (δC 173.0) and H-36 (δH 2.03) with C-35  and up-field chemical shifts of C-34a  revealed the hydroxylation of C-31     , 8 to beδC 65.4)  and 9 toδC 76.9) .10 and 11 had the same molecular formula of C35H43NO11, as indicated by their HRESIMS data  and C-23 (δC 211.3) , 13C NMR of C-20 (δC 82.3) and C-23 (δC 106.8) (Compounds IMS data . CompounC 211.3)  (SupplemC 211.3) , and 11 C 106.8)  (SupplemC 106.8) .12–19 were determined to be proansamycin B (12) . C. Cansa prifT gene is involved in the biosynthesis of rifamycins, and the 8-deoxy-rifamycin proansamycin B could undergo post-PKS modifications similar to that of its 8-hydroxyl analogue 34a-deoxyrifamycin W. Accordingly, eleven new derivatives of 8-deoxy-rifamycin were isolated and characterized, including a novel amide N-glycoside of seco-rifamycin 1, 2 and 3, which featured the third ansa chain cleavage pattern of rifamycins [2, 3, 5, 6, 13 and 15 exhibited antibacterial activity against Staphylococcus aureus. Compounds 14, 15, 16, 17 and 18 showed potent antiproliferative activity against KG1 cells, respectively.In summary, the results of in vivo gene inactivation and complementation indicated that the famycins . Compoun


Bacillus cereus toxin cereulide presents an enormous safety hazard in the food industry, inducing emesis and nausea after the consumption of contaminated foods. Additional to cereulide itself, seven structurally related isoforms, namely the isocereulides A–G, have already been elucidated in their chemical structure and could further be identified in B. cereus contaminated food samples. The newly performed isolation of isocereulide A allowed, for the first time, 1D- and 2D-NMR spectroscopy of a biosynthetically produced isocereulide, revealing results that contradict previous assumptions of an l-O-Leu moiety within its chemical structure. By furthermore applying posthydrolytical dipeptide analysis, amino acid and α-hydroxy acid analysis by means of UPLC-ESI-TOF-MS, as well as MSn sequencing, the structure of previously reported isocereulide A could be corrected. Instead of the l-O-Leu as assumed to date, one l-O-Ile unit could be verified in the cyclic dodecadepsipeptide, revising the structure of isocereulide A to [2].The emetic  Bacillus cereus   . In carr-l-Val)]  and furt1; m/z 1170.7125, [M+NH4]+), isocereulide A  was isolated to allow structure elucidation via UPLC-ESI-TOF-MS and 1D- and 2D-NMR spectroscopy. Cereulide (1) and isocereulide A (2), isolated from cell material originating from a culture of the B. cereus strain F4810/72, were traced in the following semi-preparative HPLC-fractions: cereulide , isocereulide A   . 1 and 22) [m/z 1184.7281 for the [M+NH4]+ pseudomolecular ion was observed in UPLC-ESI+-TOF-MS measurements, varying +14 Da from the [M+NH4]+ adduct of cereulide (m/z 1170.7125)  , a mass 70.7125) , resultiα-hydroxy acid composition of cereulide (1) , one of the amino acids or α-hydroxy acids present in the structure of 2 was likely exchanged by a 14 Da heavier homologous unit, as indicated by the correspondingly detected m/z for isocereulide A . Reference –, with an empirical elemental composition of C11H20NO4 in a ratio of 3:2:1  and m/z 116.0707 , respectively  + pseudomolecular ions 1205.7 → 1006.6 → 821.4 in the MS, MS2, and MS3 scans for 2 indicating the elimination of l-O-Val-l-Val and d-O-Leu-d-Ala. The following MS4 scan then exhibited the key fragment (821.4 → 722.3/608.3), giving the location of the l-O-Ile-l-Val unit. After that, MS5 and MS6 exhibited the loss of d-O-Leu-d-Ala followed by l-O-Val-l-Val showing the fragments of 608.3 → 423.1 → 323.1, thus confirming the general sequence of the to date reported isocereulide A (2) + and 554.2615, [M−H]-), which was supplied as 2 ng/µL solved in MeCN/0.1% HCOOH . Scan time for lock mass was set to 0.3 s, an interval of 10 s, and 3 scans to average, with a mass window of ± 0.5 Da. Calibration of the Synapt G2-S was performed in the range from m/z 50 to 1200 with a solution of sodium formate (5 mmol/L) in 2-propanol/H2O . Source parameters were applied according to Reference : δ (ppm) 0.90 , 0.97 , 1.07–1.22 , 1.38–1.50 , 1.68 , 1.72-1.83 , 1.88–2.03 , 2.06–2.16 , 2.27–2.33 ], 2.38–2.50 , 2.50–2.61 , 4.69–4.75 , 4.75-4.82 , 5.43 , 5.48 , 5.62–5.67 , 8.76–8.80 , 9.15 . DEPT135, HSQC, HMBC : δ (ppm) 11.6 [C(13d)], 15.5 [C(13c)], 17.18 [C(5a)], 17.24 [C(13b)], 17.5 [C(8b)], 19.06/19.10/19.5 [C(8c/11b/11c)], 21.4 [C(2c)], 23.4 [C(2d)], 24.79 [C(2b)], 24.84 [C(13b)], 30.0 [C(11a)], 30.4 [C(11a)], 30.5 C[(11a)], 31.2 [C(8a)], 37.4 [C(13a)], 41.4 [C(2a)], 49.71 [C(5)], 49.74 [C(5)], 49.77 [C(5)], 59.18 [C(11)], 59.25 [C(11)], 59.28 [C(11)], 73.3 [C(2)], 78.5 [C(13)], 79.0 [C(8)], 170.4 [C(9)], 170.6 [C(9)], 170.9 [C(9)], 171.1 [C(3)], 171.26 [C(3)], 171.34 [C(3)].B. cereus toxin cereulide and its isocereulides comprise a complex, three-dimensional dodecadepsipeptide structure  beforehand .The emetic tructure ,6,7,8. Fforehand , differi


II(cym)Cl (cym = η6-p-cymene) complexes of pyridinecarbothioamides have shown potential for development as orally active anticancer metallodrugs, underlined by their high selectivity towards plectin as the molecular target. In order to investigate the impact of the metal center on the anticancer activity and their physicochemical properties, the Os(cym), Rh- and Ir(Cp*) (Cp* = pentamethylcyclopentadienyl) analogues of the most promising and orally active compound plecstatin 2 were prepared and characterized by spectroscopic techniques and X-ray diffraction analysis. Dissolution in aqueous medium results in quick ligand exchange reactions; however, over time no further changes in the 1H NMR spectra were observed. The Rh- and Ir(Cp*) complexes were investigated for their reactions with amino acids, and while they reacted with Cys, no reaction with His was observed. Studies on the in vitro anticancer activity identified the Ru derivatives as the most potent, independent of their halido leaving group, while the Rh derivative was more active than the Ir analogue. This demonstrates that the metal center has a significant impact on the anticancer activity of the compound class. Ru I1 ,35. N- calculations in methanol and found that the deprotonated form was about 30 kcal mol−1 energetically less favorable than the protonated complex, independent of the metal center.The molecular structures of complexes geometry  with PCAomplexes . Accordi7 and 8 was investigated in an analogous manner as reported earlier for the Ru and Os congeners  chloride 2 and [chlorido(η6-p-cymene)(N-(4-fluorophenyl)pyridine-2-carbothioamide)osmium(II)] chloride 5 were synthesized by adapting reported procedures [6-p-cymene)ruthenium(II)] [6-p-cymene)osmium(II)] [5-pentamethylcyclopentadienyl)rhodium(III)] [5-pentamethylcyclopentadienyl)iridium(III)] [ocedures . The dimium(II)] , bis[dicium(II)] , bis[dicum(III)]  and bis[um(III)]  were syn1H, 13C{1H} and 2D  NMR spectra were recorded in DMSO-d6, MeOH-d4 or CDCl3 on a Bruker Avance AVIII400 MHz NMR spectrometer  at 400.13 (1H) or 100.61 MHz (13C{1H}) and ambient temperature. Chemical shifts are reported versus SiMe4 and are reported by reference to the residual solvent peaks.3, 4 and 6 were performed on a Bruker SMART APEX2  diffractometer with a CCD area detector using graphite monochromated Mo-Kα radiation (λ = 0.71073 Å). The molecular structures were solved and refined with the SHELXL-2016 [The mass spectra were recorded on a Bruker micrOTOF-QII  mass spectrometer in positive electrospray ionization (ESI) mode. Elemental analyses were carried out on an Exeter Analytical Inc-CE-440 Elemental Analyser . X-ray diffraction measurements of single crystals of LXL-2016   and the respective dimeric precursors [M(π-bound ligand)X2]2 (0.5 eq.). The reaction mixture was stirred for 4 h at room temperature under nitrogen atmosphere. The solvent was concentrated in vacuo to ca. 5 mL and n-hexane was added to initiate precipitation in the fridge. The solvent was decanted and subsequent drying in vacuo yielded analytically pure solid product.6-p-cymene)(N-(4-fluorophenyl)pyridine-2-carbothioamide)ruthenium(II)] bromide[Bromido(η (3). Compound 3 was synthesized following the general procedure using N-(4-fluorophenyl)-2-pyridinecarbothioamide 1  and [Ru(cym)Br2]2 . Yield: 121 mg (60%), red solid. Elemental analysis found: C, 42.34; H, 3.88; N, 4.54, calculated for C22H23Br2FN2RuS: C, 42.12; H, 3.70; N, 4.47. 1H NMR : δ = 9.67 , 8.44 , 8.29 , 7.83 , 7.64–7.68 , 7.35 , 6.04 , 5.92 , 5.89 , 5.68 , 2.80 , 2.28 , 1.21 , 1.15  ppm. 13C{1H} NMR : δ = 194.1 (C-6), 165.0 (C-10), 162.5 (C-5), 160.8 (C-1), 154.7 (C-7), 141.0 (C-3), 130.7 (C-2), 128.9 (C-9), 125.8 (C-11), 125.0 (C-4), 117.9 (C-8), 117.7 (C-12), 108.2 (C-16), 105.0 (C-13), 89.3 (C-15), 89.2 (C-17), 86.7 (C-18), 85.8 (C-14), 32.6 (C-21), 22.9 (C-20), 21.9 (C-22), 19.3 (C-19) ppm. MS (ESI+): m/z 467.0531 [3 − 2Br − H]+ (mex = 467.0526).6-p-cymene)(N-(4-fluorophenyl)pyridine-2-carbothioamide)ruthenium(II)] iodide[Iodido(η (4). Compound 4 was synthesized following the general procedure using N-(4-fluorophenyl)pyridine-2-carbothioamide 1  and [Ru(cym)I2]2 . Yield: 179 mg (87%), red solid. Elemental analysis found: C, 36.87; H, 3.20; N, 3.66, calculated for C22H23FI2N2RuS: C, 36.63; H, 3.21; N, 3.88. 1H NMR : δ = 9.64 , 8.42 , 8.26 , 7.76 , 7.63–7.68 , 7.35 , 6.03 , 5.89 , 5.86 , 5.71 , 2.89 , 2.37 , 1.21 , 1.17  ppm. 13C{1H} NMR : δ = 164.9 (C-10), 162.4 (C-5), 154.9 (C-7), 140.7 (C-3), 130.2 (C-2), 128.9 (C-9), 125.8 (C-11), 125.1 (C-4), 109.3 (C-16), 104.7 (C-13), 89.5 (C-15), 89.1 (C-17), 86.9 (C-18), 86.8 (C-14), 33.0 (C-21), 23.0 (C-20), 22.1 (C-22), 20.1 (C-19) ppm. MS (ESI+): m/z 467.0531 [4 − 2I − H]+ (mex = 467.0538).6-p-cymene)(N-(4-fluorophenyl)pyridine-2-carbothioamide)osmium(II)] iodide[Iodido(η (6). DCM solutions of N-(4-fluorophenyl)pyridine-2-carbothioamide 1  and [Os(cym)I2]2  were combined and stirred for 4 h at room temperature under nitrogen atmosphere. After completion of the reaction, the solid product was filtered followed by washing with dichloromethane (2 × 10 mL) and tetrahydrofuran (10 mL) and drying in vacuum. Yield: 130 mg (66%), black solid. Elemental analysis found: C, 32.82, H, 2.86, N, 3.37, S, 3.96, calculated for C22H23FI2N2OsS: C, 32.60; H, 2.86; N, 3.46 S, 3.96. 1H NMR : δ = 9.60 , 8.47 , 8.22 , 7.71 , 7.62–7.67 , 7.35 , 6.18 , 6.06 , 6.03 , 5.87 , 2.78 , 2.43 , 1.20 , 1.13  ppm. 13C{1H} NMR : δ = 164.8 (C-10), 162.6 (C-5), 140.6 (C-3), 131.2 (C-2), 128.9 (C-9), 128.8 (C-11), 125.4 (C-4), 117.9 (C-8), 117.7 (C-12), 100.5 (C-16), 97.1 (C-13), 81.5 (C-15), 81.5 (C-17), 78.9 (C-18), 78.1 (C-14), 32.9 (C-21), 23.2 (C-20), 22.1 (C-22), 20.0 (C-19) ppm. MS (ESI+): m/z 557.1103 [6 − 2I − H]+ (mex = 557.1115).5-pentamethylcyclopentadienyl)(N-(4-fluorophenyl)pyridine-2-carbothioamide)rhodium(III)] chloride[Chlorido(η (7). Compound 7 was synthesized following the general procedure using N-(4-fluorophenyl)pyridine-2-carbothioamide 1  and [Rh(Cp*)Cl2]2 . Yield: 268 mg (88%), orange solid. Elemental analysis found: C, 50.80; H, 4.70; N, 5.35, calculated for C22H24Cl2FN2RhS·0.3C6H14 C, 50.40; H, 5.01; N,4.94. 1H NMR 400.13 MHz, CDCl3, 25 °C): δ = δ = 9.28 , 8.76 , 8.13 , 7.77 , 7.62 , 7.15 , 1.68  ppm. 13C{1H} NMR : δ = 162.8 (C-10), 160.4 (C-5),153.8 (C-1), 140.1 (C-3), 128.8 (C-9/C-11), 127.1 (C-2), 126.6 (C-4), 116.2 (C-8), 116.0 (C-12), 97.5 , 9.1 (Cp*-CH3) ppm. MS (ESI+): m/z 469.0621 [7 − 2Cl − H]+ (mex = 469.0614).5-pentamethylcyclopentadienyl)(N-(4-fluorophenyl)pyridine-2-carbothioamide)iridium(III)] chloride[Chlorido(η (8). Compound 8 was synthesized following the general procedure using N-(4-fluorophenyl)pyridine-2-carbothioamide 1  and [Ir(Cp*)Cl2]2 . Yield: 176 mg (81%), red solid. Elemental analysis found: C, 42.71; H, 3.80; N, 4.31, calculated for C22H24Cl2FIrN2S·0.1C4H8O·0.1C6H14: C, 42.73; H, 4.09; N, 4.33. 1H NMR : δ = 9.30 , 8.74 , 8.08 , 7.74 , 7.56 , 7.15 , 1.70  ppm. 13C{1H} NMR : δ = 154.5 (C-1), 139.8 (C-3), 129.2 (C-9/C-11), 126.3 (C-2), 116.2 (C-4), 115.9 (C-8/C-12), 90.1 (Cp*-C), 8.8 (Cp*-CH3) ppm. MS (ESI+): m/z 559.1195 [8 − 2Cl − H]+ (mex = 559.1200).The in vitro cytotoxicity of the compounds was investigated in HCT116, SW480, NCI-H460, and SiHa cells, as described elsewhere .7 and 8 (1–2 mg) were dissolved in D2O and 1H NMR spectra were recorded over a period of 48 h. After 48 h, equimolar amounts of AgNO3 were added and another 1H NMR spectrum was recorded.For the stability studies, 7 and 8 (1–2 mg) were incubated with His or Cys in D2O and 1H NMR spectra were recorded over a period of 48 h. After 48 h, equimolar amounts of the respective amino acids were added and another 1H NMR spectrum was recorded.To investigate the reactions with amino acids, GAUSSIAN 09W  was usedII, OsII, RhIII and IrIII complexes of N-4-fluorophenyl pyridine-2-carbothioamide 1 containing different halido leaving groups  and characterized them spectroscopically and in case of the Ru(cym)Br, Ru(cym)I and Os(cym)I derivatives by X-ray diffraction analysis. Interestingly, the Ru(cym)I crystallized in the deprotonated, neutral form while the other two complexes were found to crystallize as their complex cations, which appear to be prevalent when isolated synthetically and were shown by DFT calculations to be energetically preferred. In vitro anticancer activity studies revealed the highest potency for the Ru complexes, independent of their halido ligand, while all derivatives showed cytotoxic activity in the low μM range of around 10 μM. We associate the similar potency of the Ru complexes to their ligand exchange properties in an aqueous environment, which results in the rapid formation of the respective aqua complexes and suggests that covalent bond formation is essential for the mode of action of the complexes. This is supported by the slightly lower activity of the isostructural Os complexes, a trend also reflected for the Rh and Ir analogs.The coordination of PCA ligands to metal ions has resulted in compounds with promising in vitro and in vivo anticancer activity ,35,36. H


Karenia mikimotoi. The planar structure of the glycolipids was elucidated using mass spectroscopy (MS) and nuclear magnetic resonance (NMR) analyses and comparisons to the known glycolipid to confirm its structure. The MGDG was characterized as 3-O-β-D-galactopyranosyl-1-O-3,6,9,12,15-octadecapentaenoyl-2-O-tetradecanoylglycerol 1. The MGMG and PUFAME were characterized as (2S)-3-O-β-D-galactopyranosyl-1-O-3,6,9,12,15-octadecapentaenoylglycerol 2 and Methyl -octadeca-3,6,9,12,15-pentaenoate 3, respectively. The isolation of the PUFAME strongly supports the polyunsaturated fatty acid (PUFA) fragment of these glycolipids. The relative configuration of the sugar was deduced by comparisons of 3JHH values and proton chemical shifts with those of known glycolipids. All isolated compounds MGDG, MGMG and PUFAME 1-3 were evaluated for their antimicrobial and anti-inflammatory activity. All compounds modulated macrophage responses, with compound 3 exhibiting the greatest anti-inflammatory activity.A new monogalactosyldiacylglycerol (MGDG), a known monogalactosylmonoacylglycerol (MGMG) and a known polyunsaturated fatty acid methyl ester (PUFAME) were isolated from the marine dinoflagellate  Karenia mikimotoi (Miyake et Kominami ex Oda) Hansen et Moestrup  is a harmful dinoflagellate species with a worldwide distribution that forms massive blooms in coastal waters in the temperate regions of Europe +, 703.9 [M + H-H2O]+ , 72.5 (C-2‴), 75.0 (C-3‴), 70.3 (C-4‴), 76.9 (C-5‴) and 62.5 (C-6‴), and a glycerol moiety with carbon signals at δC 64.5 (C-1), 71.8 (C-2) and 68.8 (C-3). In its 1H NMR spectrum, signals were observed corresponding to two methyls at δH 0.90  and 0.98 , numerous oxymethylenes and oxymethines between δH 3.36 and 4.44, one oxymethine at δH 5.27 , methylenic hydrogens between δH 1.20 and 3.15 and olefinic hydrogens between δH 5.35 and 5.56. The 13C and 1H NMR (1 were similar to those of (2S)-3-O-β-D-galactopyranosyl-1-O-3,6,9,12,15-octadecapentaenoylglycerol  of the glycerol was correlated to the carbonyl C-1’ (δC 173.1) of the fatty acid side chain. The 2-O-tetradecanoyl moiety was attached at C-2 by the proton chemical shift of C-2, δH , which appears in the down field shift. The observation of the fragment ion peak at m/z 331 corresponded to the loss of tetradecanoic acid and the sugar moiety, which clearly supports the PUFAs description 3,6,9,12,15-octadecapentaenoic acid; this PUFA moiety was also support by observation of an ion peak at m/z 257. The tetradecanoic acid was further support by fragment ion peak at m/z 285, which corresponded to the loss of 3,6,9,12,15-octadecapentaenoic acid and a sugar moiety  + . Analysed 1H NMR  signals r moiety  and Suppr moiety . The oveshown in . The ano2 was obtained as an optically active amorphous white solid, . The infrared (IR) spectrum of 2 revealed absorptions indicative of hydroxyl (3366 cm−1), ester (1726 cm−1) and glycosidic moieties (1084 cm−1). The molecular formula of 2 was established as C27H42O9 on the basis of (HRESITOF) mass spectroscopy ) (2 to those of previously reported spectra showed that the planar structure of 2 was identified as the know MGMG (2S)-3-O-β-d-galactopyranosyl-1-O-3,6,9,12,15-octadecapentaenoylglycerol + ; 1 H NMR : δH 5.55 , 5.39 , 5.36 , 5.28 , 4.43 , 4.27 , 4.23 , 3.99 , 3.82 , 3.77 , 3.75 , 3.51 , 3.51 , 3.44 , 3.15 , 2.84 , 0.97 ; 13C NMR : δC 172.0 (C-1’), 132.9 (C-16’), 132.6 (C-4’), 129.7 (X3), 129.3, 128.3, 128.2 (X2) , 122.5 (C-3’), 105.3 (C-1‴), 76.9 (C-5‴), 74.9 (C-3‴), 72.1 (C-2‴), 70.4 (C-4‴), 70.4 (C-2), 67.0 (C-3), 62.6 (C-6‴), 62.6 (C-1), 32.3 (C-2’), 26.7, 26.6 (X2), 26.5 , 21.6 (C-17’), 14.7 (C-18’)  , in agre1H NMR : 5.50-5.59 ; 5.24-5.43 , 3.13 ; 3.67 ; 2.78-2.84 , 2.08 ; 0.97 ; 13C NMR : δC 172.0 (C-1), 132.8 (C-16), 132.5 (C-4), 129.6, 129.5, 129.3, 129.0, 128.9, 128.4 , 128.2 (C-15), 122.4 (C-3), 52.3 (COOCH3), 33.4 (C-2), 26.4, 26.5, 26.5, 26.6 , 21.4 (C-17), 14.6 (C-18) , LR-ESI-MS m/z: 288 [M]+, 259 [M-Et]+.White solid; 6 (C-18) , in agreStaphylococcus aureus, the gram-negative bacteria Escherichia coli, and the fungi Candida albicans . Bacteria were inoculated in nutrient broth for 24 h at 37 °C, and fungi were inoculated in Sabourand broth for 24 h at 27 °C.Compounds were tested for antimicrobial activity using a Minimum Inhibitory Concentration (MIC) assay with the gram-positive bacteria 4 CFUs per well. Plates were incubated for 24 h at the corresponding temperature . Plates were read for OD at 600 nm to assay for bacteriostatic or fungistatic activity. To assess for bactericidal or fungicidal activity, wells were assayed using an XTT Cell Proliferation Assay Kit , according to the manufacturer’s instructions.Test compounds and positive controls (10–50 µg/mL doxycycline for bacteria and 50 µg/mL amphotericin B for fungi) were dissolved in methanol, and 100 μL were added to duplicate wells of a 96-well assay plate. Compounds and controls were serially diluted 1:2 in broth. Vehicle control (methanol only) and negative control (no compound) was added to control wells. Following compound/control dilutions, 10 μL of bacteria or fungi were added to each well for a final concentration of 5 × 10RAW 264.7 macrophages were seeded in 12-well plates and incubated at 37 °C until cells adhered and grew to confluence before being treated with LPS (50 ng/mL). Cells were incubated for 48 h prior to treatment with vehicle control (EtOH) or one of the three compounds for another 24 h . Cells were then harvested, centrifuged, and media removed for ELISA experiments. Cells were blocked with anti-goat serum to block Fc receptors for 15 min on ice, then stained for 1 h with TLR4, CD80, CD206 and CD124 antibody solution. Unbound antibody was removed and cells resuspended in cold phosphate buffered saline prior to analysis on a BD FACS Celesta flow cytometer . Expression of Toll-like receptor 4 (TLR4 or CD284/MD-2) was measured using BV650-conjugated rat anti-mouse antibodies directed against CD284/MD-2 . Expression of mannose receptors (CD206) was measured using Alexa Fluor 488-conjugated rabbit anti-mouse antibodies directed against CD206 . Expression of CD80 receptors was measured using phycoerythrin (PE)/CF594-conjugated hamster anti-mouse antibodies directed against CD80 . Expression of IL4Rα/CD124 receptors was measured using Alexa Fluor 647-conjugated rat anti-mouse antibodies directed against CD124 .Cell media was assayed for TNFα or IL-10 expression using commercially available ELISA kits  according to the manufacturer’s directions.K. mikimotoi was investigated. K. mikimotoi is a promising candidate strain for sustainable production of high value co-products including long chain polyunsaturated fatty acids and omega-3 fatty acids. A new monogalactosyldiacylglycerol 1 was isolated and characterized. To the best of our knowledge this is the first time monogalactosylmonoacylglycerol 2 and a polyunsaturated fatty acid methyl ester 3 were isolated from K. mikimotoi. Compound 3 may be an artifact of the isolation procedure, and may be formed by methanolysis of biological ester. Because of a limited amount of compound 1, we were not able to perform an acid hydrolysis; fortunately, the isolation of compound 3 further supported the fatty acid side chain of compounds 1 and 2. The biological activities of 1, 2 and 3 were investigated on three human pathogenic microorganisms. None of the compounds exhibited the ability to kill or inhibit growth of bacterial species or fungal species, but they did exhibit the ability to modulate macrophage activation and functionality, indicating a potential anti-inflammatory role for this family, without the negative side effects) observed with steroids (Cushing’s syndrome and bone loss) and non-steroidal anti-inflammatory drugs  when used to treat inflammation. If the active PUFAME isolated from K. mikimotoi proves to be an interesting anti-inflammatory drug target, the yield of the PUFAME from the dinoflagellate could be increased through two methods. First, the growth conditions could be manipulated for higher lipid yields. Both Leblond et al. and Okuyama et al. have shown that cold adapted dinoflagellates have higher abundances of 18:5n-3 and 22:6n-3 PUFAMEs than do warm adapted dinoflagellates [K. mikimotoi produces an abundance of MGDGs and MGMGs with the fatty acid side chain of 18:5. The glyceroglycolipids could be isolated and then undergo methanolic hydrolysis to liberate the FA as the target PUFAME, which would also increase the yield.In our aim for isolation of a new natural product, the glyceroglycolipids composition of marine dinoflagellate gellates ,38. Seco


Neohelicosporium griseum, two new polyketides, neogrisphenol A (1) and neogrisphenol B (2), one new isochroman-1-one, (S)-6-hydroxy-7-methoxy-3,5-dimethylisochroman-1-one (3), and four known compounds (4–7) were isolated. Their structures were determined using 1D- and 2D-NMR, mass spectrometry, and chemical calculations. The C-3~C-2′ polymerization mode between the two α-naphthalenone derivative moieties is uncommon in compounds 1 and 2. Meanwhile, compounds 1–2 and 5 exhibited antibacterial activity against Bacillus subtilis, Clostridium perfringens, Staphylococcus aureus, and Staphylococcus aureus, with MIC values ranging between 16 and 31 µg/mL. In addition, compound 5 showed antifungal activity against Sclerotinia sclerotiorum and Phytophthora nicotianae var. nicotianae, with respective IC50 values of 88.14 ± 2.21 µg/mL and 52.36 ± 1.38 µg/mL. Compound 1 showed significant cytotoxicity against A2780, PC-3, and MBA-MD-231 cell lines with respective IC50 values of 3.20, 10.68, and 16.30 µM, and the cytotoxicity against A2780 cells was even higher than that of cisplatin (CDDP). With an IC50 value of 10.13 µM, compound 2 also exhibited cytotoxicity against A2780. The in vitro results showed that compound 1 inhibited A2780 cell proliferation, induced apoptosis, and arrested the cell cycle at the S-phase in a concentration-dependent manner.From the rice fermentation product of a new record fungus,  Secondary metabolites of fungi have enormous potential, particularly for drug discovery . More thHelicosporium, Helicoma, Helicomyces, and Tubeufia, are capable of producing a variety of bioactive compounds  and four known compounds from the EtOAc extracts of the rice fermentation product of a new record fungus, Neohelicosporium griseum. Compounds 1 and 2 have a rare C-3~C-2′ linkage between the cyclohexanone ring of one α-naphthalone derivative and the cyclohexanone ring of another α-naphthalone derivative, which has not been reported in natural or synthetic products. The antimicrobial and cytotoxic activities of compounds 1–7 were evaluated. Compound 1 exhibited significant cytotoxicity against the A2780 cell line with IC50 values of 3.20 µM, higher than that of cisplatin (CDDP). The results showed that this compound might serve as a potential inhibitor of ovarian cancer.In this study, bioassay-guided isolation led to the discovery of three new compounds 28 D +52 , indicating 13 degrees of unsaturation. The 1D NMR data , 6.35 ], as well as four exchangeable protons . The COSY correlations of H-3 with H-2a and H-4 along with the HMBC correlations of H-2a/C-1, C-3, C-4, H-2b/C-1, C-3, C-4, H-3/C-1, and H-4/C-2, C-3, C-4a, C-5, and C-8a indicated the presence of a substituted tetralone moiety (α-(2H)-naphthalenone moiety  indicated a Jaa coupling between Hax-2a and Hax-3, and a Jae coupling between Hax-3 and Heq-2b (J = 3.9 Hz). The very small coupling constant of Hax-3 with H-4 (J = 2.0 Hz) indicated a Jae coupling between them. Thus, H-3 and 4-OH were in the opposite orientations of the substituted tetralone moiety plane. The NOESY correlation of H-2a with 4-OH further confirmed this deduction. However, since the C-3~C-2′ bond can rotate, the relative configuration of the right part of compound 1 cannot be determined. Thus, we performed theoretical NMR chemical shift calculations for the two diastereomers 1a and 1b of 1 using the gauge-independent atomic orbital (GIAO) 13C NMR calculations  were meta-coupling on the right benzene ring. The HSQC correlations of H-5′/C-5′, H-7′/C-7′, along with the HMBC correlations of H-5′/C-4′, C-7′, C-8a’, H-7′/ C-5, C-6, C-8, 6′-OH/C-5′, C-7′, 8′-OH/ C-7′, C-8′, C-8a’, and H-4/C-2, C-3, C-4a, C-5, and C-8a, indicated that 6′-OH was connected to C-6′ and 8′-OH connected to C-8′, respectively. The position of the carbon-carbon double bond (C3′~4′) in the right cyclohexanone ring is also the same as in compound 1, based on similar HMBC correlations -2 and named neogrisphenol B.Compound 2  was obtaeft part . The 1H-elations . In addielations , similartermined . In the ed as 2b . The relculation , and theculation . The sig3 , indicating 6 degrees of unsaturation. The 1D-NMR data and HSQC correlation showed signals for an aromatic proton, an exchangeable proton, a methoxyl, two methyls, one methylene, and an oxygenated methine. These data were close to those of the isochroman-1-one skeleton −, 183.1 [M+H]+, 205.1 [M + Na]+ ; 1H NMR  δ 10.69 , 6.16 , 3.79 , 2.27 ; 13C NMR  δ 170.2, 161.4, 161.0, 140.7, 110.2, 107.6, 100.5, 51.8, 22.1. Compound 4 was identified as methyl orsellinate −, 459.1 [2M − H]− . Compound 5 was identified as diorcinol − , 229.1 [M + Na]+ , 435.0 [2M + Na]+ . Compound 6 was identified as 6-hydroxy-8-methoxy-3-methyl-isocoumarin [Compound 6 , C11H10Ocoumarin .7  δ 5.57 , 5.39 , 5.20 , 3.64 , 2.48 , 2.28 , 2.05 , 2.00–1.94 , 1.93–1.86 , 1.85 , 1.81–1.56 , 1.54–1.43 , 1.38 , 1.34–1.31 , 1.31–1.29 , 1.29–1.22 , 1.04 , 0.96–0.89 , 0.83 , 0.63 ; 13C NMR  δ 141.5, 139.9, 135.7, 132.1, 119.7, 116.4, 70.6, 55.9, 54.7, 46.4, 43.0, 43.0, 41.0, 40.6, 39.2, 38.5, 37.2, 33.2, 32.2, 28.4, 23.2, 21.3, 21.2, 20.1, 19.8, 17.8, 16.4, 12.2. Compound 7 was identified as ergosterin [Compound 7 , C28H44ONeohelicosporium griseum showed cytotoxicity against PC3 cell lines, with IC50 values of 0.4 mg/mL, as part of our ongoing research into the medical applications of helicosporous hyphomycetes. To investigate the bioactivities of compounds 1–7, their antimicrobial and cytotoxic properties were evaluated. Compounds 1–2 and 5 exhibited moderate antibacterial activity against Bacillus subtilis, Clostridium perfringens, Staphylococcus aureus, and Staphylococcus aureus with MIC values between 16 and 31 µg/mL  extracts from the rice fermentation product of 31 µg/mL . Only co38 µg/mL . Compoun16.30 µM . Compoun10.13 µM .The cytotoxicity of NeoA toward cancer cells was evaluated using the MTT technique. According to Flow cytometry analysis showed that the number of apoptotic cells increased with the increase in NeoA concentration E. As shoThe percentage of cells in the G0/G1 phase dramatically dropped as NeoA concentration increased, whereas the number of cells in the S-phase sharply increased F. The reα-naphthalenone dimer are primarily derived from microbial metabolites. Most of them were isolated from the fungal genus Cladosporium, named cladosporols [α-naphthalenone derivatives into a dimer. Typically, they are connected through the polymerization of the benzene ring of one α-naphthalone derivative with the benzene ring of another α-naphthalone derivative or the benzene ring of one α-naphthalone derivative with the cyclohexanone ring of another α-naphthalone derivative. The biological activities of α-naphthalenone dimer derivatives include anticancer, antimicrobial, and so on [α-naphthalenone dimer derivatives were isolated and identified during the study of secondary metabolites of Neohelicosporium griseum. They have a rare 3~2′ polymerization mode between the cyclohexanone ring of one α-naphthalone derivative and the cyclohexanone ring of another α-naphthalone derivative. This study demonstrated that helicosporous hyphomycetes are worthy of study, and future research may uncover additional active secondary metabolites with novel structures. Neogrisphenol A is anticipated to be a new anticancer drug with a bright future in both development and application.Derivatives of the osporols , and to osporols ,25, thrond so on ,30,31,321–2) and (R)-6-hydroxy-7-methoxy-3,5-dimethylisochroman-1-one (3) are three new polyketone derivatives isolated from the rice fermentation product of a new record fungus for China viz Neohelicosporium griseum, which is a new record for China. NOE and 13C-NMR calculations determined the relative configurations of compounds 1–2. The absolute configurations of compounds 1–2 were determined through ECD calculations. Neogrisphenol A showed extensive antibacterial activity against Bacillus subtilis, Clostridium perfringens, Staphylococcus aureus, MRSA-strain Staphylococcus aureus, and Ralstonia solanacearum. Meanwhile, neogrisphenol B exhibited antibacterial activity against the MRSA-strain Staphylococcus aureus. Diorcinol showed antifungal and antibacterial activity against Phytophthora nicotianae var. nicotianae and Sclerotinia sclerotiorum and weak antibacterial activity against Clostridium perfringens. Neogrisphenol A also showed potent inhibition on A2780, PC-3, and MBA-MD-231 cell lines, particularly A2780 cell lines, with more potency than the positive drug cis-platinum. Neogrisphenol B also exhibited cytotoxicity against A2780. Neogrisphenol A dramatically reduced the rate of proliferation of A2780 cells, induced apoptosis, and prevented the S-phase of the cell cycle in a concentration-dependent manner. It is anticipated that neogrisphenol A will be further developed and utilized as a novel anticancer chemical entity.Neogrisphenol A-B (