Chemical structures of the isolated compounds were identified by spectroscopic/spectrometric methods, including NMR and HRESIMS, and the absolute configuration of the new compounds (1 and 2) was performed by electronic circular dichroism (ECD) studies. Further, the anticancer activities of the isolated compounds (1–22) were evaluated using the epidermal growth factor (EGF)-induced as well as the 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced cell transformation assay. Among the 22 compounds, compounds 4, 9, 11, 13–15, 17, 18, and 22 significantly inhibited both EGF- and TPA-induced colony growth. In particular, askoseoside D , apigenin , apigenin-7-O-β-d-glucuronopyranoside , and 1- cyclopentane-2,3-diol  showed higher potent activities.In total, four new eudesmane-type sesquiterpene glycosides, askoseosides A–D ( Aster koraiensis Nakai (syn. Asteromoea koraiensis and Gymnaster koraiensis), belonging to the family Compositae (Asteraceae), is an endemic plant in the Republic of Korea distributed throughout the Korean peninsula. The plant presents beautiful pale purple flowers, which are used as tea and the young shoots are used as a food ingredient. Traditionally, extracts of the aerial parts and roots of A. koraiensis are used to treat pertussis, pneumonia, and chronic bronchitis ; two methylene groups, ; three oxygenated methine protons, ; two methine protons, [δH 1.04 (H-7) and 2.03 (H-11)]; and a β-glucopyranosyl unit containing an anomeric proton, . The 13C NMR and HSQC spectra displayed signals for 21 carbons, including a carbonyl group, [δC 200.8 (C-3)]; two unprotonated sp2 carbons, [δC 134.1 (C-4) and 159.0 (C-5)]; and a quaternary carbon, [δC 46.6 (C-10)] -absolute configuration of 1 was identified by the calculated and experimental ECD spectra  was established as -1,6,9-trihydroxy-eudesm-4-en-3-one-6-O-β-d-glucopyranoside.The molecular formula (C (C-10)] . The 1D ectively . In the spectrum . The cor spectra . The sug2, C35H46O14, was determined by the HRESIMS data. The 1H NMR data of compound 2 displayed the signals of five methyl groups, ; three methine protons, ; a trans-olefinic group, [δH 6.32 (H-8″) and 7.61 (H-7″)]; p-disubstituted aromatic protons, [δH 6.80  and 7.47 ]; and a β-glucopyranosyl unit. In the 13C NMR spectrum (δC 168.7 (C-9″) and 172.7 (C-7′)]; three unprotonated sp2 carbons, ; and a quaternary carbon, [δC 42.2 (C-10)], were observed. The 1D NMR spectra of compound 2 were similar to those of reported analogue, -9-O-(E-p-coumaroyl)-1,6,9-trihydroxy-eudesm-3-en-6-O-β-d-glucopyranoside  in it. The positions of the β-glucopyranosyl, p-coumaroyl, and acetyl groups were confirmed as C-9, C-6, and C-6′, respectively, by HMBC cross peaks of H-9 with C-9″, H-1′ with C-6, and H-6′ with C-7′  and 2.73 ] were confirmed in the 1H NMR and HSQC spectra. The HMBC cross peaks of H-1 with C-1′′′ and H-2′′′ with C-1′′′, as well as the HRESIMS fragment data, indicated that the malonyl moiety is linked with the C-1 position -absolute configuration of 2 was determined by means of ECD study (2 (askoseoside B) was established as -1-O-malonyl-9-O-(E-p-coumaroyl)-1,6,9-trihydroxy-eudesm-3-ene-6-O-(6’-O-acetyl)-β-d-glucopyranoside.The molecular formula of compound spectrum , signalsith C-7′ . No morehanol-d4 , but conexpected . Therefo solvent . Additioposition . The NOECD study . Thus, c21H38O8, of compound 3 was deduced from the HRESIMS. Signals for four methyl groups, ; four methylene groups, ; and a β-glucopyranosyl unit were observed in the 1H NMR spectrum. An oxygenated quaternary carbon signal [δC 73.5 (C-4)] was detected in the 13C NMR data  is more deshielded than 6S-alcohols (δC 100) in methanol-d4  present in it  was proposed as -1,4,6-trihydroxy-eudesmane-1-O-α-l-rhamnopyranoside-6-O-β-d-glucopyranoside -1,6,9-trihydroxy-eudesm-3-ene-6-O-β-d-glucopyranoside (5) + .Amorphous powder; [α]22D 22.7 ; UV (MeOH) λmax (log ε) 208 (3.84), 228 (3.75), 314 (3.99) nm; ECD  Δε +7.7 (220), –8.2 (250), +4.4 (302); see 1H , 1H , 13C , and 13C  NMR data; HRESIMS m/z 691.2969 [M + H]+ .Amorphous powder; [α]22D –11 ; UV (MeOH) λmax (log ε) 207 (3.73) nm; see 1H  and 13C  NMR data; HRESIMS m/z 441.2462 [M + Na]+ .Amorphous powder; [α]22D –9 ; UV (MeOH) λmax (log ε) 209 (3.98) nm; see 1H  and 13C  NMR data; HRESIMS m/z 587.3036 [M + Na]+ .Amorphous powder; [α]1−4 (each 0.5 mg) were confirmed according to the method of Tanaka et al. [l-glucose, d-glucose, l- rhamnose, and d-rhamnose; each 1.0 mg, Merck) and hydrolyzed compounds 1−4 were reacted and then analyzed using a UPLC system. A Waters CSH C18 analytical column with 30% acetonitrile isocratic elution  was carried out for analysis. Standard derivatives were recorded at tR 4.056 min (l-glucose derivative), tR 4.427 min (d-glucose derivative), tR 3.950 min (d-rhamnose derivative), and tR 7.307 min (l-rhamnose derivative).Sugar units of new compounds a et al. ,47. The Conformer distribution, optimization, and ECD analysis were performed, as described previously . All con50 μM) and added to the bottom agar layer. The plates were incubated (37 °C) in a 5% CO2 incubator for two weeks. Colonies were visualized by a microscope  and the numbers analyzed using an Image-Pro Plus software ver.6.1 .To evaluate the cellular anchorage-independent growth, the soft agar colony formation assay was conducted according to modified protocol . A mouse9, 14, 22, and 5-fluorouracil  for 48 h. The cytotoxicity of each compound was measured using Quanti-MAX WST-8 Cell Viability Assay Kit reagent . According to the instructions, the absorbance was measured at 450 nm using the Multiskan SkyHigh Spectrophotometer .To estimate cytotoxicity of compounds, normal human dermal fibroblast  cells (5000 cells/well) were seeded into each well of 96-well plates. After 24 h incubation, the cells were treated with various concentration of the representative compounds 1–4) were isolated from the flowers of A. koraiensis and their structures were identified through spectroscopic studies. The anticancer activity of a total of 22 compounds (1–22) isolated in this study was evaluated by cell transformation assay, and most of them showed significant anti-carcinogenic activity. In particular, four compounds, askoseoside D (4), apigenin (9), apigenin-7-O-β-d-glucuronopyranoside (14), and 1- cyclopentane-2,3-diol (22), including new compound (4), showed higher activity than other compounds. In addition, compounds 9, 14, and 22 did not exhibit any toxic effects in the cell viability assay for NHDF, which is a normal skin cell line. This study not only expands the chemical composition of A. koraiensis, but also provides new information on their physiological activities. In addition, this study suggests the potential value of this plant and its active compounds as natural anticancer agents.In the current study, four new compounds (


Ficus sagittifolia (Moraceae) led to the isolation of four flavonoids: (2R)-eriodictyol (1), 2′- hydroxygenistein (2), erycibenin A (3), and genistein (4); a dihydrobenzofuran: moracin P (5); a coumarin: peucedanol (6); and an apocarotenoid terpenoid: dihydrophaseic acid (7). These were identified via 1D and 2D nuclear magnetic resonance spectroscopy (NMR) and ultra-high-resolution liquid chromatography–quadrupole time-of-flight mass spectroscopy (UHPLC-QTOF MS). Moracin P (5) is being reported for the first time in the genus Ficus, while the others are known compounds (1–4 and 6–7) isolated previously from the genus but being reported for the first time from the species F. sagittifolia. Their antimicrobial activity against various pathogens  was tested. The mixture of genistein and moracin P (4+5) exhibited strong activity against K. pneumoniae (MIC < 0.0039 mg/mL), whereas dihydrophaseic acid (7) was the most active against P. aeruginosa and A. niger . These compounds might be considered potential antimicrobial agents with the potential to be starting points for the development of antimicrobial drugs.The phytochemical investigation of the ethylacetate fraction of an ethanolic extract obtained from the stem bark of  Despite the many existing antibiotics, infectious diseases are consistently ranked second among causes of death worldwide . AntimicMedicinal plants contain bioactive organic chemical compounds, which play a defensive role for the plants but may also be utilized in medicine, e.g., against major chronic diseases in host–metabolic and genetic dysfunctional disorders as well as infectious disease ,4. MedicFicus sagittifolia Warburg. ex Mildbraed & Burret is a plant from the Mulberry family (Moraceae). It is an epiphyte shrub/tree, often on oil palms about 30 feet high. It is native to Sub-Saharan West Africa +; LC-UV(MeOH): 264 nm and 284 nm; 1H-NMR  δ (ppm): 8.04 , 7.07 , 6.42 , 6.41 , 6.39 , 6.26 ; 13C-NMR  δ (ppm): 182.3 (C-4), 165.6 (C-7), 163.3 (C-5), 161.3 (C-4′), 159.8 (C-2′), 159.3 (C-2), 157.4 (C-8a), 156.3 (C-3′), 132.8 (C-6′), 122.2 (C-3), 110.4 (C-1′), 107.7 (C-5′), 105.8 (C-4a), 103.9 (C-3′), 99.8 (C-6), 94.4 (C-8). The NMR data are in agreement with the literature [terature .Erycibenin A (3) HR-ESI-MS m/z 373.1308 [M + H]+; LC-UV(MeOH): 212 nm and 264 nm; 1H-NMR  δ (ppm): 8.06 , 7.37 , 6.84 , 6.43 , 3.64 , 3.07 , 2.72 , 1.26 ; 13C-NMR  δ (ppm): 182.4 (C-4), 164.5 (C-7), 161.1 (C-5), 158.8 (C-8a), 157.9 (C-4′), 154.7 (C-2), 131.4 C-2′, 6′), 124.7 (C-1′), 123.5 (C-3), 116.3 , 111.5 (C-6), 106.2 (C-4a), 94.6 (C-8), 79.7 (C-2′′), 74.0 (C-3′′), 25.8 (C-1′′), 25.6 (C-4′′), 25.2 (C-5′′). The NMR data are in agreement with the literature [Genistein (4) HR-ESI-MS m/z 271.0618 [M + H]+; LC-UV(MeOH): 260 nm and 320 nm; 1H-NMR  δ (ppm): 8.07 , 7.38 , 6.85 , 6.23 , 6.35 ; 13C-NMR  δ (ppm): 182.5 (C-4), 163.6 (C-7), 161.2 (C-5), 159.7 (C-8a), 159.5 (C-4′), 152.3 (C-2), 131.1 (C-2′), 124.9 (C-1′), 124.5 (C-3), 116.0 (C-3′), 104.6 (C-4a), 99.4 (C-6), 94.5 (C-8). The NMR data are in agreement with the literature [Moracin P (5) HR-ESI-MS m/z 327.1248 [M + H]+; LC-UV(MeOH): 220 nm and 320 nm, 1H-NMR  δ (ppm): 7.24 , 6.89 , 6.86 , 6.76 , 6.25 , 3.80 , 3.13 , 2.84 , 1.37 , 1.29 ; 13C-NMR  δ (ppm): 159.7 , 156.3 (C-2), 152.3 (C-7a), 151.7 (C-6), 133.4 (C-1′), 121.5 (C-4), 117.4 (C-5), 124.5 (C-3a), 104.6 , 103.7 (C-4′), 103.4 (C-3), 99.4 (C-7), 78.0 (C-3′′), 70.3 (C-2′′), 32.2 (C-1′′), 25.7 (C-4′′), 20.8 (C-5′′). The NMR data are in agreement with the literature [Peucedanol (6) HR-ESI-MS m/z 265.1109 [M + H]+; LC-UV(MeOH): 204 nm and 332 nm; 1H-NMR  δ (ppm): 7.84 , 7.40 , 6.73 , 6.17 , 3.62 , 3.08 , 2.55 , 1.26 , 1.25 ; 13C-NMR  δ (ppm): 165.2 (C-2), 162.7 (C-7),157.0 (C-8a), 152.3 (C-3a), 145.8 (C-4), 131.2 (C-5), 127.0 (C-6), 114.4 (C-3), 111.8 (C-4a), 102.7 (C-8), 78.8 (C-2′), 73.4 (C-3′), 33.0 (C-1′), 25.2 (C-4′), 24.7 (C-5′). The NMR data are in agreement with the literature [Dihydrophaseic acid (7) HR-ESI-MS m/z 281.1395 [M−H]−; LC-UV(MeOH): 268 nm; CD  λ nm (∆ε) 266 (−3.8). The CD data are in full agreement with literature [1H-NMR  δ (ppm): 7.99 , 6.53 , 5.77 , 4.12 , J = 10 Hz, 7 Hz, H-3), 3.81 , 3.72 , 2.09 , 2.04 , 1.86 , 1.74 , 1.66 , 1.15 , 0.94 .13C-NMR  δ (ppm): 169.3 (C-11), 151.2 (C-9), 134.9 (C-7), 131.5 (C-8), 118.9 (C-10), 87.5 (C-5), 83.0 (C-6), 77.0 (C-12), 65.7 (C-3), 49.2 (C-1), 45.7 (C-4), 44.3 (C-2), 21.0 (C-15), 19.4 (C-14), 16.1 (C-13). The NMR data are in agreement with the literature [terature . 1H-NMR R)-(−)-eriodictyol (1) and dihydrophaseic acid (7) were generated with the Molecular Operations Environment  using the MMFF94x force field. A conformational search was conducted in each case using the systematic search method and the same force field. The lowest-energy conformers within an energy window of 3 kcal/mol above the global minimum were energy minimized with the semi-empirical method AM1. In case of 1, the two lowest-energy conformers were used for the spectra simulation. In case of dihydrophaseic acid 7, only one model for the lowest energy conformer was analyzed, as described.Molecular models of (21 and 7. In case of (S)-1, the simulated spectra of two conformers with very similar energy  were Boltzmann-averaged based on the computed total energy of each conformer in a ratio of 0.64:0.36. The simulated spectra of (2S)- and (2R)-eriodictyol shown in 1 (in the 30:70 mixture with CD-inactive 2) were scaled by factor 0.15. In case of 7, the spectrum was simulated only for one single lowest-energy conformer in the manner described, but no intensity scaling was necessary to obtain the plot shown along with the experimental CD curve in The structures were saved in mol2-format and imported into Gaussian , where they were fully energy minimized using density functional theory (DFT) with the B3LYP density functional and the 6-31G basis set. Time-dependent DFT (TDDFT) calculations using the same functional and basis set were then performed on the minimized structures. In all cases, the first  30 electronic transitions were computed. The output from Gaussian for electronic transition energies (E in eV) and rotator strengths  was extracted, and ECD spectra were simulated by applying a Gaussian shape function with a band width at 1/e height σ = 0.15 and 0.20 eV, respectively, for compounds 1–7 were determined using the broth micro-dilution method (96-well plates).Minimum inhibitory concentration (MIC) and minimum bactericidal/fungicidal concentration (MBC/MFC) of compounds Staphylococcus aureus (ATCC 6571), Escherichia coli (ATCC 700728), Pseudomonas aeruginosa (ATCC 27853), Salmonella typhi (ATCC 14028), and Klebsiella pneumoniae (ATCC 700303) and two fungi: Candida albicans  and Aspergillus niger ) were provided by the Pharmaceutical Microbiology Department, University of Ibadan, Ibadan, Nigeria. All the microbes were sub-cultured from the original culture and incubated overnight at 37 °C for 24 h and at 25 °C for 48 h in the case of bacteria and fungi, respectively. The standard drugs, gentamycin and ketoconazole, were used as the positive controls for the bacteria and fungi, respectively, while 1% DMSO was used as a negative control.Five bacteria  for bacteria and fungi, respectively. Both standards were also serially diluted to their respective concentrations and tested in the same manner as described above.After incubation, 10 µL of 0.2 mg/mL R)-eriodictyol, 2′-hydroxygenistein, erycibenin A, genistein, moracin P, peucedanol, and dihydrophaseic acid) were isolated from the stem bark of the title plant and are reported for the first time as constituents of F. sagittifolia. Moracin P, known from other Moraceae, is reported for the first time from the genus Ficus. These isolated compounds justified the taxonomic placement of F. sagittifolia in the genus Ficus and, therefore, can be used as a chemotaxonomic marker for F. sagittifolia. The isolated compounds showed moderate activity against the tested bacteria and fungi compared to the reference drugs (gentamycin and ketoconazole). The isolated mixture of genistein and moracin P displayed strong activity against K. pneumoniae, while dihydrophaseic acid was the most active against P. aeruginosa and A. niger.Seven known compounds ((2F. sagittifolia could, hence, be considered as a potential source of such compounds with interesting antimicrobial activity.These compounds might possess a potential as starting points for the development of antimicrobial drugs, and


Naphtho-triazoles and thienobenzo-triazoles have so far proven to be very potent inhibitors of the enzyme butyrylcholinesterase (BChE). Based on these results, in this work, new thienobenzo-thiazoles were designed and synthesized, and their potential inhibitory activity was tested and compared with their analogs, naphtho-oxazoles. The synthesis was carried out by photochemical cyclization of thieno-thiazolostilbenes obtained in the first reaction step. Several thienobenzo-thiazoles and naphtho-oxazoles have shown significant potential as BChE inhibitors, together with the phenolic thiazolostilbene being the most active of all tested compounds. These results are significant as BChE has been attracting growing attention due to its positive role in the treatment of Alzheimer’s disease. Computational examination based on the DFT approach enabled the characterization of the geometry and electronic structure of the studied molecules. Furthermore, the molecular docking study, accompanied by additional optimization of complexes ligand-active site, offered insight into the structure and stabilizing interactions in the complexes of studied molecules and BChE. The thiazole ring represents a very important motif with great potential in the field of medicine ,13,14,15p-methoxybenzyl- and hydroxybuthyl-naphthotriazoles with chloro or methoxy groups showed the best TNFα production inhibition. The latest study on the new functionalized group of thienobenzo/naphtho-triazoles +calcd = 207.0176, [M + H]+measured = 207.0180.HRMS (2-isopropyl-5-(2-(thiophen-2-yl)vinyl)thiazole (2a) (73%). Column chromatography on silica gel using PE/E (20%) afforded 111 mg of a mixture of cis- and trans-isomer. Repeated column chromatography on silica gel using PE/E (10%) afforded pure trans-isomer in the first fractions and cis-isomer in the last fractions.(Z)-2-isopropyl-5-(2-(thiophen-2-yl)vinyl)thiazole (cis-2a): 44 mg (isolated yield 40%), yellow oil, fR(PE/E = 10%) = 0.53; UV (ACN) λmax/nm (ε/dm3 mol−1 cm−1) 316 ; 1H NMR  δ/ppm: 7.29 , 7.24 , 7.13 , 6.96 , 6.71 , 6.42 , 3.41–3.34 , 1.46 , 1.45 ; 13C NMR  δ/ppm: 177.1, 151.5, 139.7, 130.1, 126.8, 126.5, 123.7, 120.6, 116.4, 33.4, and 23.2; and MS (ESI) m/z : 236 (100); (E)-2-isopropyl-5-(2-(thiophen-2-yl)vinyl)thiazole (trans-2a): 27 mg (isolated yield 25%), yellow oil, fR(PE/E = 10%) = 0.56; UV (ACN) λmax/nm (ε/dm3 mol−1 cm−1) 319 ; 1H NMR  δ/ppm: 7.49 , 7.11 , 7.01 , 6.93–6.91 , 6.80 , 3.34–3.20 , 1.36 , 1.34 ; 13C NMR  δ/ppm: 177.2, 152.1, 141.6, 126.6, 125.4, 123.4, 123.1, 120.2, 112.8, 32.5, and 22.2; and MS (ESI) m/z : 236 (100); m/z) for the mixture of configurational isomers C12H13NS2: [M + H]+calcd = 235.0489, [M + H]+measured = 235.0490.HRMS (2-chloro-5-(2-(thiophen-2-yl)vinyl)thiazole (3a) (76%). Column chromatography on silica gel using PE/E (5%) afforded 295 mg of a mixture of cis- and trans-isomer. Repeated column chromatography on silica gel using PE/E (1%) afforded pure trans-isomer in the first fractions.(E)-2-chloro-5-(2-(thiophen-2-yl)vinyl)thiazole (trans-3a): 20 mg (isolated yield 40%), yellow oil, fR(PE/E = 10%) = 0.59; UV (ACN) λmax/nm (ε/dm3 mol−1 cm−1) 342 ; 1H NMR  δ/ppm: 7.45 , 7.24 , 7.07 , 7.00 , 6.92 , 6.89  δ/ppm: 149.6, 141.2, 139.7, 139.1, 127.6, 127.3, 125.6, 125.3, and 117.0; and MS (ESI) m/z : 227/229 (100); HRMS (m/z) for C9H7ClNS2: [M + H]+calcd = 226.9630, [M + H]+measured = 226.9633.5-(2-(thiophen-2-yl)vinyl)-2-(trifluoromethyl)thiazole (4a) (46%). Column chromatography on silica gel using PE/E (20%) afforded 66 mg of a mixture of cis- and trans-isomer of 4a for the photochemical experiment. m/z : 261 (100); HRMS (m/z) for the mixture of configurational isomers C10H6F3NS2: [M + H]+calcd = 260.9894, [M + H]+measured = 260.9896.MS (ESI) N-(5-(2-(thiophen-2-yl)vinyl)thiazol-2-yl)acetamide (5a) (30%). The reaction mixtures of cis- and trans-isomer were filtered under reduced pressure. Thiazol-2-acetamide 5a was synthesized from thiazol-2-amine 5a′ in a round flask (0.025 L) using acetic anhydride (0.0015 L) at room temperature overnight. The water was added (0.00075 L), the reaction mixture was filtered under reduced pressure, and the beige solid was dried under pressure in an exicator for 2 h.m/z : 209 (100); HRMS (m/z) for the mixture of configurational isomers of 5a′, C9H8N2S2: [M + H]+calcd = 208.0129, and [M + H]+measured = 208.0133.MS (ESI) m/z : 251 (100), 209 (15), 121 (25); HRMS (m/z) for the mixture of configurational isomers of 5a, C11H10N2OS2: [M + H]+calcd = 250.0234, and [M + H]+measured = 250.0235.MS (ESI) N-(4-bromo-5-(2-(thiophen-2-yl)vinyl)thiazol-2-yl)pivalamide (7a) (10%). Column chromatography on silica gel using PE/E (30%) afforded 30 mg of a mixture of cis-, trans-isomers, and starting aldehyde. m/z : 408/410 (20), 386/388 (10), 330/332 (100), 121 (55); HRMS (m/z) for the mixture of configurational isomers of 7a, C14H15BrN2O2S2: [M + H]+calcd = 385.9758, and [M + H]+measured = 385.9751.MS (ESI) 4-chloro-2-(pyrrolidin-1-yl)-5-(2-(thiophen-2-yl)vinyl)thiazole (10a) (61%). Column chromatography on silica gel using PE/E (20%) afforded 61 mg of a cis- and trans-isomer mixture. Repeated column chromatography on silica gel using PE/E (10%) afforded pure trans-isomer in the first fractions.(E)-4-chloro-2-(pyrrolidin-1-yl)-5-(2-(thiophen-2-yl)vinyl)thiazole (trans-10a): 8 mg (isolated yield 32%), yellow oil, fR(PE/E = 20%) = 0.83; UV (ACN) λmax/nm (ε/dm3 mol−1 cm−1) 373 ; 1H NMR  δ/ppm: 7.15–7.14 , 6.96 , 6.94 , 6.57 , 3.48 , 2.07–2.05  δ/ppm: 163.0, 142.8, 135.0, 127.6, 125.0, 123.8, 119.5, 117.9, 116.5, 49.3, and 25.7; MS (ESI) m/z : 297/299 (100); HRMS (m/z) for C13H13ClN2S2: [M + H]+calcd = 296.0209, and [M + H]+measured = 296.0206.6a and 9a were synthesized using 3a , XPhos , Pd(OAc)2 , and Cs2CO3,  dissolved in 2 mL of dioxane, and benzyl-amines  were added. The reaction mixtures were purged with argon and heated to 120 °C in sealed tubes for 24 h. The reactions were always performed starting from pure isomers of 3a, and the solvent was evaporated under pressure. The target isomer was purified by column chromatography on silica gel using E/EtOAc (0–60%) as eluent afforded in the first fractions traces of 3a and in the last fractions mixture of isomers for 6a  and pure trans-isomer for 9a.Thiazol-2-amines N-(piperidin-4-ylmethyl)-5-(2-(thiophen-2-yl)vinyl)thiazol-2-amine (6a) (7%). Column chromatography on silica gel using E/EtOAc (60%) afforded 7 mg of a trans-isomer and XPhos ligand mixture. N-(sec-butyl)-5-(2-(thiophen-2-yl)vinyl)thiazol-2-amine (trans-9a) (10%). Column chromatography on silica gel using DCM/E (40%) afforded pure trans-isomer.(E)-N-(sec-butyl)-5-(2-(thiophen-2-yl)vinyl)thiazol-2-amine (trans-9a): 10 mg (isolated yield 10%), yellow oil, fR(DCM/E = 40%) = 0.24; UV (ACN) λmax/nm (ε/dm3 mol−1 cm−1) 362 ; 1H NMR  δ/ppm: 7.12 , 7.05 , 6.97–6.93 , 6.90 , 6.59 , 5.55 , 3.54–3.46 , 1.67–1.56 , 1.27 , 0.98 ; 13C NMR  δ/ppm: 168.3, 142.9, 139.0, 127.6, 125.4, 124.8, 123.5, 119.5, 119.2, 53.8, 29.7, 20.3, and 10.36; MS (ESI) m/z : 265 (100); HRMS (m/z) for C13H16N2S2: [M + H]+calcd = 264.0755, and [M + H]+measured = 264.0753.8a was synthesized by McMurry reaction. The reaction apparatus was under argon, Ar atmosphere while adding the reactants. The reaction is carried out in a three-necked flask (100 mL) with a magnetic stirrer. The zinc powder (0.00985 mol) and 30 mL of THF were added to the flask. The mixture was cooled to −5 °C, and titanium (IV) chloride, TiCl4 (0.0045 mol), was slowly added by a syringe with a temperature kept under 0 °C. The reaction mixture was warmed to room temperature, stirred for half an hour, then heated at reflux for 3 h. The mixture was cooled again to −5 °C, and the solution of two corresponding aldehydes  dissolved in THF (12 mL) was added dropwise. After aldehydes addition, the reaction mixture was heated at reflux for 2 h, then at room temperature overnight. The reaction was quenched with 10% aqueous sodium bicarbonate, NaHCO3 solution. The mixture was extracted with EtOAc (3 × 25 mL). The organic layers were dried under anhydrous MgSO4. Product 8a was isolated by repeated column chromatography on silica gel using PE/E as a solvent system.Thiazolo-stilbene 2-(2-(2-methylthiazol-5-yl)vinyl)phenol (8a) (10%). Column chromatography on silica gel using PE/E (90%) afforded 21 mg of a cis- and trans-isomer mixture. Repeated column chromatography on silica gel using PE/E (80%) afforded pure trans-isomer in the first fractions.(E)-2-(2-(2-methylthiazol-5-yl)vinyl)phenol (trans-8a): 13 mg (isolated yield from the mixture 65%), yellow oil, fR(PE/E = 80%) = 0.50; UV (ACN) λmax/nm (ε/dm3 mol−1 cm−1) 345 , 332 , 315 , 300 , 285 ; 1H NMR  δ/ppm: 7.69 , 7.47 , 7.16–7.09 , 7.01 , 6.90 , 6.79 , 6.25 , 2.76 ; 13C NMR  δ/ppm: 116.3, 154.2, 153.8, 128.8, 127.5, 126.0, 124.3, 122.5, 120.8, 116.1, 114.6, and 19.3; MS (ESI) m/z : 218 (20); 158 (100); HRMS (m/z) for C12H11NOS: [M + H]+calcd = 217.0561, and [M + H]+measured = 217.0557.1a–10a were dissolved in toluene p.a. (~2.5 × 10−3 M) and transferred to a quartz vessel (50 mL) with the addition of a catalytic amount of I2 and irradiated with 10 UV lamps at 313 nm in a photochemical reactor Rayonet for 1–5 h to achieve almost complete conversions. After removing the solvent by a rotary evaporator under reduced pressure, the photoproducts 1–5, 7, and 9 were purified by column chromatography from the remains of the starting substrates or unreacted aldehyde from the Wittig reaction. Pure photoproducts 1–5 were isolated and completely spectroscopically characterized by NMR and HRMS measurements. Compounds 7 and 9 were not isolated in a pure form, and further purification was impossible due to the insufficient amount of samples. Irradiation reaction did not produce photoproducts 6, 8, and 10. Mixtures of previously synthesized compounds 2-methylthienobenzothiazole (1): 16 mg (isolated yield 30%), yellow oil, fR(PE/E = 5%) = 0.35; UV (ACN) λmax/nm (ε/dm3 mol−1 cm−1) 249 ; 1H NMR  δ/ppm: 7.91 , 7.60 , 7.44 , 2.89 ; 13C NMR  δ/ppm: 164.9, 151.3, 136.5, 132.7, 129.8, 128.2, 122.3, 120.3, 118.9, and 20.0; MS (ESI) m/z : 206 (100); HRMS (m/z) for C10H7NS2: [M + H]+calcd = 205.0020, and [M + H]+measured = 205.0023.2-isopropylthienobenzothiazole (2): 14 mg (isolated yield 47%), yellow oil, fR(PE/E = 10%) = 0.40; UV (ACN) λmax/nm (ε/dm3 mol−1 cm−1) 251 ; 1H NMR  δ/ppm: 7.95 , 7.89 , 7.59 , 7.45 , 3.52–3.45 , 1.53 , 1.51 ; 13C NMR  δ/ppm: 176.7, 151.0, 136.4, 132.9, 128.9, 128.1, 122.3, 120.2, 119.2, 34.0, and 23.1; MS (ESI) m/z : 234 (100); HRMS (m/z) for C12H11NS2: [M + H]+calcd = 233.0333, and [M + H]+measured = 233.0335.2-chlorothienobenzothiazole (3): 5 mg (isolated yield 70%), yellow oil, fR(PE/E = 10%) = 0.45; UV (ACN) λmax/nm (ε/dm3 mol−1 cm−1) 214 ; 1H NMR  δ/ppm: 8.04 , 7.99 , 7.80 , 7.72 ; 13C NMR  δ/ppm: 152.7, 146.0, 138.9, 133.8, 132.1, 127.9, 121.7, 120.4, and 116.5; MS (ESI) m/z : 225/227 (75), 121 (100); HRMS (m/z) for C9H4ClNS2: [M + H]+calcd = 224.9474, and [M + H]+measured = 224.9475.2-(trifluoromethyl)thienobenzothiazole (4): 4 mg (isolated yield 20%), yellow oil, fR(PE/E = 10%) = 0.58; UV (ACN) λmax/nm (ε/dm3 mol−1 cm−1) 213 ; 1H NMR  δ/ppm: 8.08 , 8.04 , 7.89 , 7.69 ; 13C NMR  δ/ppm: 128.4, 122.3, 122.0, and 117.1 ; MS (ESI) m/z : 259 (100); HRMS (m/z) for C10H4F3NS2: [M + H]+calcd = 258.9737, and [M + H]+measured = 258.9740.N-acetamide (5): 2 mg (isolated yield 43%), yellow oil, fR(E/EtOAc = 60%) = 0.65; UV (ACN) λmax/nm (ε/dm3 mol−1 cm−1) 221 ; 1H NMR  δ/ppm: 9.36 , 7.82–7.81 , 7.76 , 7.57 , 2.37 ; 13C NMR  δ/ppm: 126.9, 121.2, 116.5, 117.4, and 29.6 ; MS (ESI) m/z : 249 (100), 207 (25), 121 (50); HRMS (m/z) for C11H8N2OS2: [M + H]+calcd = 248.0078, and [M + H]+measured = 248.0079.Electrophorus electricus, Type: V-S), BChE , and Trisma base were purchased from Sigma-Aldrich , while Ellman’s reagent 5,50-dithiobis-(2-nitrobenzoic acid) (DTNB) was purchased from Zwijndrecht . The reaction mixture contained 180 µL Tris-HCl buffer  or phosphate buffer, 10 µL of the enzyme , 10 µL of the tested solution, and 10 μL of DTNB . The reaction started with adding 10 μL of ATChI/BTChI . The developing yellow color was measured at 405 nm over 6 min at room temperature using a 96-well microplate reader . The experiment was run in triplicate. The percentage of enzyme inhibition was calculated from measured data according to the equation: Inhibition (%) = [(Ac − AT)/A.C.]∙100, where A.C. is the enzyme activity without the test sample, and AT is the enzyme activity with the test sample, calculated as mean values ± standard deviation. In the control measurement, the tested compound was replaced by a buffer solution. Non-enzymatic hydrolysis was measured as blank for each measurement. The IC50 value was calculated by a nonlinear fit of compound concentration (log) values vs. response.AChE and BChE inhibition were determined using a modified spectrophotometric Ellman’s method . Acetyltε = 4.Geometry optimizations and UV-Vis spectra are calculated using the Gaussian16 program package . The geo1a–10a were obtained as mixtures of cis- and trans-isomers, some by Wittig reaction, while others by continued Buchwald-Hartwig amination or by McMurry reaction. From thieno-thiazolostilbenes 1a–10a, corresponding thienobenzo-thiazoles 1–5, 7, and 9 were successfully obtained by photochemical cyclization. All isolated pure compounds were evaluated for cholinesterases’ inhibitory activity. Key determinants in a structure-activity relationship were the type of heterocyclic ring, the molecule’s geometry, and, where possible, the type and position of a substituent. Among stilbene derivatives, the best result is achieved by the derivative with a phenolic ring trans-8a and its thiophene analog trans-1a. The phenolic thiazolostilbene is the most active of all tested compounds. Three of the five tested photocyclization thiazole products achieved an IC50 value, compound 1 with methyl substituent, 3 with chlorine, and molecule 4 with trifluoromethyl. The achieved inhibitory activity is very promising, with IC50 values in the range of common reversible inhibitor huperzine A. Comparing electrocyclic thiazoles and oxazoles leads to the observation that these two rings are similar: one of the similarities being the inactivity towards the AChE enzyme. As the tested structures showed drastically better inhibition of the BchE than AchE, these results are very important as BchE has been attracting growing attention due to its positive role in Alzheimer’s disease. A computational study revealed that thieno-benzo-thiazoles and naphtho-oxazole that showed the best inhibitory activity toward BchE owe that to the rigidity of the main scaffold that can block the esteratic subsite of the active site, accompanied by π-π stacking between the ligand and the residues of the active site, that additionally stabilizes the formed complex. For more flexible thieno-thiazolostilbene trans-8, the main stabilizing interaction responsible for the inhibitory activity is its ability to form H-bonds with residues of the active site (due to the presence of the hydroxyl group at the phenyl ring), accompanied by some hydrophobic interactions.New thieno-thiazolostilbenes and thienobenzo-thiazoles were designed and synthesized, and their potential inhibitory activity toward cholinesterases was tested and compared with their analogs, naphtho-oxazoles. Thieno-thiazolostilbenes


The incorporation of p-carborane and further substitution of the p-position resulted in four carborane-based di-tert-butylphenol analogs that showed no or weak COX inhibition but high 5-LO inhibitory activities in vitro. Cell viability studies on five human cancer cell lines revealed that the p-carborane analogs R-830-Cb, S-2474-Cb, KME-4-Cb, and E-5110-Cb exhibited lower anticancer activity compared to the related di-tert-butylphenols. Interestingly, R-830-Cb did not affect the viability of primary cells and suppressed HCT116 cell proliferation more potently than its carbon-based R-830 counterpart. Considering all the advantages of boron cluster incorporation for enhancement of drug biostability, selectivity, and availability of drugs, R-830-Cb can be tested in further mechanistic and in vivo studies.Targeting inflammatory mediators and related signaling pathways may offer a rational strategy for the treatment of cancer. The incorporation of metabolically stable, sterically demanding, and hydrophobic carboranes in dual cycloxygenase-2 (COX-2)/5-lipoxygenase (5-LO) inhibitors that are key enzymes in the biosynthesis of eicosanoids is a promising approach. The di- At 0 °C, a tetra-n-butylammonium fluoride (TBAF) solution  was added dropwise. The reaction mixture was stirred for 20 min at 0 °C. The reaction was then stopped by adding 20 mL distilled water. After dilution with 50 mL EtOAc, the phases were separated. The aqueous phase was extracted three times with 25 mL EtOAc. The combined organic phases were washed with 25 mL brine and dried over magnesium sulfate. The volatiles were removed under reduced pressure and the crude product was purified by column chromatography . Colorless solid, yield 90% ; m.p.: 131–132 °C; TLC : Rf = 0.56. 1H-NMR : δ [ppm] = 1.56–3.43 , 3.56 , 7.08 , 7.66 , 7.83 ; 13C{1H}-NMR : δ [ppm] = 72.1 (6-Cq), 110.5 (7-Cq), 128.4 (2-Cq), 136.1 (1-Cq), 136.4 (3-Cq), 140.3 (4-Cq), 179.6 (5-Cq); 11B{1H}-NMR : δ [ppm] = −15.1 , −13.1 ; FTIR (ATR): ṽ [cm−1] = 3328 , 2615 , 2601 , 1646 , 1629 , 1510 , 1449 , 1403 , 1355 , 1345 , 1261 , 1215 (s),1192 , 1115 (s), 1063 (m), 1035 (w), 1010 (s), 935 (m), 865 (m), 741 (s), 722 , 691 (s), 665 (s), 573 (w), 556 (w), 447 (m); HR-ESI-MS : m/z [M−H]− calcd. for C7H13B10O2S: 271.1572, found: 271.1570; elemental analysis: calcd. (%) for C7H14B10O2S: C 31.19, H 5.22, found: C 31.04, H 5.21.TBDMS (tert-Butyl-dimethylsiloxy)-1,12-dicarba-closo-dodecaborane(12)  was dissolved in 100 mL dry diethyl ether. At 0 °C, nBuLi  was added dropwise. Afterwards, the solution was stirred at room temperature for two hours. At −78 °C, methyl formate  was added dropwise. The solution was first stirred for one hour at −78 °C, then warmed to room temperature and stirred for 24 h. After addition of 30 mL 1 M HCl, the phases were separated. The aqueous phase was extracted three times with 50 mL diethyl ether, and the combined organic phases were washed twice with 25 mL brine and then dried over magnesium sulfate. The solvent was removed under reduced pressure and the crude product was purified by column chromatography . Colorless solid, yield 86% ; m.p.: 40–41 °C; TLC (hexane): Rf = 0.57. 1H-NMR : δ [ppm] = 0.09 , 0.75 , 1.30–3.36 , 8.96 ; 13C{1H}-NMR : δ [ppm] = −4.20 (4-CH3), 17.7 (5-Cq), 25.1 (6-CH3), 70.7 (2-Cq), 113.1 (3-Cq), 187.2 (1-Cq); 11B{1H}-NMR : δ [ppm] = −16.9 , −12.5 ; FTIR (ATR): ṽ [cm−1] = 2955 , 2930 , 2859 , 2609 , 1970 (w), 1735 , 1471 , 1463 ,1390 , 1362 , 1252 , 1168 , 1031 , 1004 (w), 962 (m), 781 (s), 730 , 669 (m), 642 (m), 575 (w), 557 (m); HR-ESI-MS : m/z [M+Cl]−, calcd. for C9H26B10ClO2Si: 339.2326, found: 339.2302; elemental analysis: calcd. (%) for C9H26B10O2Si: C 35.74, H 8.66, found: C 35.70, H 8.67.1- was added dropwise. Afterwards, the solution was stirred at −78 °C for 30 min. At −78 °C, aldehyde 2  was added dropwise. The solution was first stirred for 1.5 h at −78 °C, then it was warmed to room temperature and stopped by adding 15 mL 1 M HCl. The phases were separated, and the aqueous phase was extracted three times with 75 mL diethyl ether. The combined organic phases were washed twice with 25 mL brine and then dried over magnesium sulfate. The solvent was removed under reduced pressure and the crude product was purified by column chromatography . Colorless solid, yield 53% ; m.p.: 149–150 °C; TLC : Rf = 0.55. 1H-NMR : δ [ppm] = 0.09 , 0.74 , 1.29–3.33 , 1.18 , 2.22 , 2.37 , 2.97 , 3.08 , 4.33 ; 13C{1H}-NMR : δ [ppm] = −4.3 (9-CH3), 13.2 (1-CH3), 17.7 (10-Cq), 18.1 (4-CH2), 39.4 (5-CH), 44.4 (2-CH2), 60.8 (3-CH2), 68.1 (6-C-OH), 77.3 (7-Cq), 111.6 (8-Cq); 11B{1H}-NMR : δ [ppm] = −16.0 , −12.8 ; FTIR (ATR): ṽ [cm−1] = 3447 , 2958 , 2929 , 2858 , 2598 , 1466 , 1409 , 1361 , 1329 , 1284 , 1266 (m), 1216 (s), 1183 , 1129 , 1109 , 1078 (w), 1032 (s), 1004 (w), 989 (m), 953 (w), 940 (m), 887 (w), 837 (w), 783 (s), 741 , 707 (w), 672 (m), 656 (w), 640 (w), 613 (w), 579 (m), 503 (w), 482 (w), 452 (w); HR-ESI-MS : m/z [M + H]+, calcd. for C14H38B10NO4SSi: 454.3215, found: 454.3234; elemental analysis: calcd. (%) for C14H37B10NO4SSi: C 37.23, H 8.26, N 3.10, found: C 37.90, H 8.25, N 3.89.2-Ethylisothiazolidine-1,1-dioxide  was dissolved in 15 mL dry THF. At −78 °C, tert-Butyl-dimethylsiloxy)-12--1,12-dicarba-closo-dodecaborane(12) (3)  was dissolved in 7.00 mL dry THF. At 0 °C, NaH  was added. The reaction mixture was stirred for two hours at room temperature. At 0 °C, p-toluenesulfonyl chloride  was added and the solution was stirred for 24 h at room temperature. The reaction was then stopped by adding 30 mL of a 1 M HCl. After dilution with 75 mL diethyl ether, the phases were separated. The aqueous phase was extracted three times with 25 mL diethyl ether. The combined organic phases were washed with 25 mL brine and dried over magnesium sulfate. The volatiles were removed under reduced pressure and the crude product was purified by column chromatography . Colorless solid, yield 87% ; m.p.: 181–182 °C; TLC : Rf = 0.54. 1H-NMR : δ [ppm] = 0.05 , 0.72 , 1.09–3.24 , 1.00 , 2.26 , 2.44 , 2.71 , 2.82 , 2.92 , 3.21 , 5.29 , 7.31 , 7.77 ; 13C{1H}-NMR : δ [ppm] = −4.3 (13-CH3), 12.7 (1-CH3), 17.7 (14-Cq), 20.5 (4-CH2), 21.8 (10-CH3), 25.1 (15-CH3), 38.9 (3-CH2), 43.5 (2-CH2), 60.6 (5-CH), 70.1 (11-Cq), 77.3 (6-CH), 112.6 (12-Cq), 128.5 (9-CH), 129.4 (8-CH), 134.2 (10-CH3), 145.0 (7-Cq); 11B{1H}-NMR : δ [ppm] = −15.8 , −12.6 ; FTIR (ATR): ṽ [cm−1] = 2956 , 2930 , 2857 , 2619 , 2592 , 1595 (w), 1463 , 1450 , 1372 , 1341 , 1293 , 1254 , 1189 ,1172 , 1132 (s), 1109 (s), 1095 (w), 1076 (w), 1034 (w), 1019 (w), 1005 (w), 972 (s), 894 (m), 870 (w), 838 (s), 810 (s), 783 (s), 751 , 720 (w), 670 (s), 639 (w), 631 (w), 577 (m), 559 (m), 544 (s), 498 (w), 455 (w), 442 (w), 435 (w), 429 (w); HR-ESI-MS : m/z [M + H]+, calcd. for C21H44B10NO6S2Si: 608.3304, found: 608.3350; elemental analysis: calcd. (%) for C21H43B10NO6S2Si: C 41.63, H 7.51, N 2.31 found: C 42.18, H 7.28, N 2.22.1-((tert-Butyl-dimethylsiloxy)-12--1,12-dicarba-closo-dodecaborane(12) (4)  was dissolved in 3.80 mL dry THF. At 0 °C, NaH  was added. The reaction mixture was stirred for two hours at room temperature. The reaction was then stopped by adding 25 mL distilled water. After dilution with 25 mL EtOAc, the phases were separated. The aqueous phase was extracted three times with 25 mL EtOAc. The combined organic phases were washed with 25 mL brine and dried over magnesium sulfate. The volatiles were removed under reduced pressure and the crude product was purified by column chromatography . Colorless solid, yield 64% ; m.p.: 147–148 °C; TLC : Rf = 0.50. 1H-NMR : δ [ppm] = 0.08 , 0.75 , 1.14–3.34 , 1.23 , 2.89 , 3.08 , 3.14 , 6.09 ; 13C{1H}-NMR : δ [ppm] = −4.4 (9-CH3), 12.7 (1-CH3), 17.6 (10-Cq), 21.9 (4-CH2), 24.9 (11-CH3), 39.1 (2-CH2), 42.8 (3-CH2), 63.9 (7-Cq), 112.6 (12-Cq), 111.8 (8-Cq), 126.9 (6-CH), 140.5 (5-Cq); 11B{1H}-NMR : δ [ppm] = −15.0 , −12.4 ; FTIR (ATR): ṽ [cm−1] = 2948 , 2930 , 2859 , 2619 , 1667 , 1471 , 1461 , 1422 , 1390 , 1361 , 1309 , 1297 , 1262 , 1250 , 1208 ,1189 , 1172 (m), 1156 (m), 1136 (w), 1102 (w), 1073 (m), 1037 (s), 1023 (m), 1005 (w), 972 (w), 939 (m), 837 (s), 781 (s), 750 (w), 729 , 698 (m), 670 (m), 643 (w), 596 (m), 572 (m), 561 (m), 533 (s), 509 (s), 466 (m), 448 (m), 441 (w), 435 (w); HR-ESI-MS : m/z [M + H]+, calcd. for C14H36B10NO3SSi: 436.3110, found: 436.3120; elemental analysis: calcd. (%) for C14H35B10NO3SSi: C 38.77, H 8.13, N 3.23 found: C 39.49, H 8.27, N 2.98.1-E)-1-(tert-Butyl-dimethylsiloxy)-12--1,12-dicarba-closo-dodecaborane(12) (5)  was dissolved in 0.80 mL dry THF. At 0 °C, a TBAF solution  was added dropwise. The reaction mixture was stirred for 20 min at 0 °C. The reaction was then stopped by adding 10 mL distilled water. After dilution with 25 mL EtOAc the phases were separated. The aqueous phase was extracted three times with 25 mL EtOAc. The combined organic phases were washed twice with 20 mL brine and dried over magnesium sulfate. The volatiles were removed under reduced pressure and the crude product was purified by column chromatography . Colorless solid, yield 96% ; m.p.: 220–222 °C; TLC : Rf = 0.46. 1H-NMR : δ [ppm] = 1.48–3.30 , 1.17 , 2.95 , 3.00 , 3.20 , 5.96 ; 13C{1H}-NMR : δ [ppm] = 13.2 (1-CH3), 22.9 (4-CH2), 40.0 (2-CH2), 43.8 (3-CH2), 65.0 (7-Cq), 111.8 (8-Cq), 126.0 (6-CH), 143.6 (5-Cq); 11B{1H}-NMR : δ [ppm] = −14.7 , −12.7 ; FTIR (ATR): ṽ [cm−1] = 3284 , 2987 , 2933 , 2880 , 2611 , 2595 , 1665 , 1466 , 1454 , 1423 , 1389 , 1352 , 1278 , 1235 , 1151 , 1132 , 1098 (s), 1023 (s), 959 (m), 935 (w), 897 (w), 832 (m), 795 (m), 728 , 700 (w), 602 (m), 570 (w), 546 (s), 507 (s), 487 (m), 457 (m); HR-ESI-MS : m/z [M + H]+, calcd. for C8H22B10NO2S: 322.2245, found: 322.2255; elemental analysis: calcd. (%) for C8H21B10NO2S: C 30.08, H 6.63, N 4.39, found: C 30.05, H 6.53, N 4.42. and 3-(triphenylphosphoranylidene)-γ-butyrolactone were dissolved in 11 mL dry THF and 1.40 mL dry -dimethylformamide (DMF). The reaction mixture was heated under reflux for 48 h. After cooling to room temperature, the solvent was removed under reduced pressure. The residue was then dissolved in 100 mL EtOAc and 75 mL distilled water. The phases were separated and the aqueous phase was extracted three times with 50 mL EtOAc. The combined organic phases were washed twice with 50 mL brine and then dried over magnesium sulfate. The solvent was removed under reduced pressure and the crude product was purified by column chromatography . Colorless solid, yield 84% ; m.p.: 149–151 °C; TLC : Rf = 0.62. 1H-NMR : δ [ppm] = 0.08 , 0.74 , 1.34–3.31 , 2.95 , 4.30 , 6.38 ; 13C{1H}-NMR : δ [ppm] = −4.2 (8-CH3), 17.7 (9-Cq), 25.1 (3-CH3), 64.7 (6-Cq), 65.5 (1-CH2), 111.8 (7-Cq), 128.6 (4-Cq), 134.0 (5-CH), 170.8 (2-Cq); 11B{1H}-NMR : δ [ppm] = −15.1 , −12.5 ; FTIR (ATR): ṽ [cm−1] = 2925 , 2885 , 2856 , 2608 , 1725 , 1676 , 1470 , 1428 , 1380 , 1360 , 1350 , 1261 , 1261 , 1250 , 1207 (s), 1072 (m), 1029 (s), 965 (m), 939 (w), 836 (s), 779 (s), 781 (s), 736 , 695 (m), 646 (m), 628 (w), 574 (m), 517 (m), 461 (m); HR-ESI-MS : m/z [M + NH4]+, calcd. for C13H34B10NO3Si: 390.3233, found: 390.3228; elemental analysis: calcd. (%) for C13H30B10O3Si: C 42.14, H 8.16, found: C 42.22, H 8.04.Aldehyde E)-1-(tert-Butyl-dimethylsiloxy)-12-(methylene-[dihydrofuran-2′(3H)-one])-1,12-dicarba-closo-dodecaborane(12) (6)  was dissolved in 2.50 mL dry THF. At 0 °C, a TBAF solution  was added dropwise. The reaction mixture was stirred for 25 min at 0 °C. The reaction was then stopped by adding 20 mL distilled water. After dilution with 25 mL EtOAc the phases were separated. The aqueous phase was extracted three times with 25 mL EtOAc. The combined organic phases were washed with 20 mL brine and dried over magnesium sulfate. The volatiles were removed under reduced pressure and the crude product was purified by column chromatography . Colorless solid, yield 99% ; m.p.: 210–212 °C; TLC : Rf = 0.62. 1H-NMR : δ [ppm] = 1.47–3.27 , 3.03 , 4.35 , 6.20 ; 13C{1H}-NMR : δ [ppm] = 26.0 (3-CH3), 66.0 (6-Cq), 66.3 (1-CH2), 111.5 (7-Cq), 131.5 (4-Cq), 132.1 (5-CH), 170.9 (2-Cq); 11B{1H}-NMR : δ [ppm] = −14.8 , −12.6 ; FTIR (ATR): ṽ [cm−1] = 3157 , 2977 , 2920 , 2603 , 1727 , 1672 , 1577 , 1480 , 1452 , 1427 , 1387 (s), 1358 ,1282 , 1209 , 1088 (m), 1032 (s), 976 (m), 866 (w), 776 (w), 754 (w), 732 (w), 715 (w), 693 (m), 634 (m), 588 (w), 572 (w), 524 (w), 483 (w); HR-ESI-MS : m/z [M + H]+, calcd. for C7H17B10O3: 257.2102, found: 257.2184; elemental analysis: calcd. (%) for C7H16B10O3: C 32.80, H 6.29, found: C 32.19, H 6.20. were dissolved in 7.00 mL dry THF. The solution was stirred for 45 min at room temperature. Afterwards, a solution of aldehyde 2  in 2.00 mL dry DMF was added dropwise. The reaction mixture was stirred for 24 h at 75 °C. After cooling to room temperature, the solvent was removed under reduced pressure. The residue was then dissolved in 50 mL EtOAc and 30 mL distilled water. The phases were separated, and the aqueous phase was extracted three times with 25 mL EtOAc. The combined organic phases were washed twice with 30 mL brine and then dried over magnesium sulfate. The solvent was removed under reduced pressure and the crude product was purified by column chromatography . Colorless solid, yield 50% ; m.p.: 168–170 °C; TLC : Rf = 0.60. 1H-NMR : δ [ppm] = 0.07 , 0.74 , 1.42–3.30 , 2.79 , 3.50 , 3,78 , 6.19 ; 13C{1H}-NMR : δ [ppm] = −4.2 (9-CH3), 17.7 (10-Cq), 21.1 (4-CH2), 25.1 (11-CH3), 42.8 (3-CH2), 62.2 (1-CH3), 65.2 (7-Cq), 111.3 (8-Cq), 128.5 (6-Cq), 131.3 (5-Cq), 163.7 (2-Cq); 11B{1H}-NMR : δ [ppm] = −15.0 , −12.5 ; FTIR (ATR): ṽ [cm−1] = 2953 , 2932 , 2896 , 2858 , 2603 , 1994 (w), 1708 , 1672 , 1472 , 1461 , 1428 , 1399 , 1362 , 1344 , 1249 , 1119 (w), 1038 (s), 1003 (w), 970 (m), 926 (m), 837 (s), 779 (s), 731 , 694 (m), 666 (w), 628 (w), 603 (m), 521 (w), 482 (m), 466 (m), 449 (m), 431 (m), 461 (m); HR-ESI-MS : m/z [M + H]+, calcd. for C14H34B10NO3Si: 402.3233, found: 402.3248; elemental analysis: calcd. (%) for C14H33B10NO3Si: C 42.08, H 8.32, N 3.51, found: C 42.39, H 8.07, N 3.53.(1-Methoxy-2-oxo-3-pyrrolidinyl)triphenyl-phosphonium bromide  and NEtE)-1-(tert-Butyl-dimethylsiloxy)-12-(methylene-[1′-methoxypyrrolidine-2′-one])-1,12-dicarba-closo-dodecaborane(12) (7)  was dissolved in 1.50 mL dry THF. At 0 °C, a TBAF solution  was added dropwise. The reaction mixture was stirred for 30 min at 0 °C. The reaction was then stopped by adding 20 mL distilled water. After dilution with 25 mL EtOAc the phases were separated. The aqueous phase was extracted three times with 25 mL EtOAc. The combined organic phases were washed twice with 20 mL brine and dried over magnesium sulfate. The volatiles were removed under reduced pressure and the crude product was purified by column chromatography . Colorless solid, yield 93% ; m.p.: 222–224 °C; TLC : Rf = 0.52. 1H-NMR : δ [ppm] = 1.48–3.28 , 2.84 , 3.58 , 3.73 , 5.99 ; 13C{1H}-NMR : δ [ppm] = 21.9 (4-CH2), 43.3 (3-CH2), 62.1 (1-CH3), 66.4 (7-Cq), 111.1 (8-Cq), 126.8 (6-Cq), 134.5 (5-Cq), 163.2 (2-Cq); 11B{1H}-NMR : δ [ppm] = −14.7 , −12.6 ; FTIR (ATR): ṽ [cm−1] = 3165 , 2941 , 2603 , 1686 , 1662 , 1487 , 1458 , 1439 , 1393 , 1355 , 1286 (m), 1215 (s), 1126 (m), 1036 (s), 1003 (m), 955 (m), 923 (w), 894 (w), 840 (w), 749 , 692 (s), 618 (s), 571 (w), 522 (w), 489 (w); HR-ESI-MS : m/z [M + H]+, calcd. for C8H20B10NO3: 288.2368, found: 288.2388; elemental analysis: calcd. (%) for C8H19B10NO3: C 33.67, H 6.71, N 4.91, found: C 33.22, H 6.68, N 4.80., UPLC : binary solvent manager UPB, sample manager UPA, column manager UPM, and diode array detector PDA, γ detector Gabi Star (Raytest), flow rate 0.4 mL/min, eluent: (A) 0.1% trifluoroacetic acid in H2O, (B) MeCN; gradient: t0 min 95/5—t0.3 min 95/5—t5.3 min 5/95—t6.5 min 5/95—t6.8 min 95/5—t10 min 95/5). All compounds were found to have a purity of >97% ; column ODP-50 4B (Shodex Asahipak 50 × 4.6 mm) with C18 pre-column; eluent: MeOH/phosphate buffer , gradient t0 min 30/70—t25 min 95/5—t27 min 95/5—t28 min 30/70—t40 min 30/70, flow rate = 0.6 mL/min. Oxycarboxin  and triphenylene  served as references. Toluene was used as control and logD7.4 was found to be 2.72 (literature logD7.4 2.72) [The loged by us  utilizined by us . The fol.4 2.72) .COX inhibition activity against ovine COX-1 and human COX-2 was determined using the fluorescence-based COX assay COX Fluorescent Inhibitor Screening Assay Kit  according to the manufacturer’s instructions as previously reported by us. All compounds were screened at a concentration of 100 µM in duplicate .6) were re-suspended in phosphate-buffered saline (PBS) (pH 7.4) containing 1 mg/mL glucose and 1 µM CaCl2. After preincubation with R-830, S-2474, KME-4, and E-5110 in DMSO for 15 min at 37 °C, 5-LO product formation was stimulated by addition of calcium ionophore A23187 (2.5 µM in MeOH) and exogenous arachidonic acid (20 µM in EtOH). After 10 min at 37 °C, the reaction was stopped by addition of ice-cold methanol (1 mL). HCl , prostaglandin B1 (200 ng) and PBS (500 μL) were added and the formed metabolites were extracted and analyzed by HPLC as described previously [6 cells, which includes leukotriene B4 (LTB4), its all-trans isomers and 5-hydroperoxyicosatetraenoic acid (5-H(P)ETE). Cysteinyl LTs C4, D4, and E4 as well as oxidation products of LTB4 were not detected. Data were normalized to vehicle control (DMSO) and IC50 values and 95% confidence intervals (CIs) of at least three independent measurements were calculated  . After incubation, the dye was discarded. In order to dissolve the formed formazan crystals DMSO was added, and the absorbance was measured at λmax = 540 nm, with the reference/background wavelength of 670 nm. All results are expressed as a percentage of the control value which was arbitrary set to 100%. After 72 h treatment, cells were washed with 200 µL of PBS and fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature (RT). Next, cells were stained with 1% CV solution. After 20 min, cells were washed in tap water and dried on air. Dye was dissolved in 33% acetic acid and the absorbance was measured at λmax = 540 nm, with the reference/background wavelength of 670 nm. Results were expressed as a percentage of the control value which was arbitrary set to 100%.All cell lines were seeded overnight and treated with t-test was used and two-sided p values of less than 0.05 were considered statistically significant.All experiments were repeated at least three independent times and data presented represents the means  ±  SD of replicates. To evaluate the significance between groups Student’s Further materials, methods, and procedures for physicochemical characterization and biological evaluation: see tert-butylphenyl moiety in the potent dual COX-2/5-LO inhibitors R-830, KME-4, E-5110, and S-2474 by p-carborane gave rise to the carborane-based analogs R-830-Cb, KME-4-Cb, E-5110-Cb, and S-2474-Cb. Replacing the bulky but also hydrophobic di-tert-butylphenyl moiety with the hydrophobic boron cluster resulted in a significant decrease in lipophilicity, suggesting that intermolecular interactions, such as hydrogen bonds or dihydrogen bonds, are contributing to the compounds’ lipophilicity [tert-butylphenols.The substitution of the bulky di-hilicity . The car50 values in the nanomolar range, comparable to the respective organic counterparts, indicating that the bioisosteric replacement of the di-tert-butylphenyl motif by p-carborane is well tolerated.However, in vitro studies on intact cells revealed that all carborane-based analogs are strong inhibitors of 5-LO product formation with ICp-carborane generally decreased the cytotoxic potential of dual inhibitors, except in the case of R-830. Interestingly, the p-carborane analog R-830-Cb was non-toxic to primary cells but effective against tested cell lines derived from various types of inflammation-related tumors. Its cytotoxic potential was mediated by potent inhibition of ROS, resulting in inhibited proliferation and caspase-dependent apoptosis.Furthermore, the introduction of R-830-Cb is a promising candidate for further assessment and detailed mechanistic as well as in vivo studies.Thus, carborane-based analog


Amomum kravanh is a traditional Chinese medicine used to relieve hangovers. However, whether its bioactive components improve alcohol metabolism is not clear. In this study, ten new  and thirty-five known (11–45) compounds were isolated from the fruits of Amomum kravanh by an activity-guided separation. Ten novel compounds were identified as four sesquiterpenoids (1–4), three monoterpene derivatives (5–7), two neolignans , and a novel norsesquiterpenoid (10) with a new C14 nor-bisabolane skeleton. Their structures were determined by the comprehensive analysis of high-resolution electrospray ionization mass spectrometry (HRESIMS), nuclear magnetic resonance (NMR), and electronic circular dichroism (ECD) calculation. The effects of all isolated compounds on the activity of alcohol dehydrogenase were evaluated in vitro, and it was found that eight compounds  exhibited significant activation effects on the alcohol dehydrogenase at 50 μM.Alcoholism is a worldwide health problem, and diseases caused by alcoholism are killing people every year.  In the 1H NMR spectrum, three methyl signals at δH 0.93 , 1.68 , and 1.72 , two oxygenated protons at δH 4.46  and 4.66 , a group of extracyclic double bond resonances at δH 4.92 and 4.78 , and an olefinic proton at δH 5.16  were observed , two methines (δC 43.6 and 127.4), two oxygenated methines (δC 69.8 and 73.9), an olefinic methylene (δC 103.2), four methylenes , two olefinic quaternary carbons (δC 134.6 and 151.8), and one oxygenated quaternary carbon (δC 84.1). These NMR data indicated that 1 is a sesquiterpenoid. Two double bonds occupied two degrees of unsaturation, and the remaining two indexes of hydrogen deficiency required a dicyclic skeleton for 1. The A-ring fragment in the structure  was deduced, which framed a specific 1-oxaspiro [4.5] decane ring system of 1. Thus, the plane structure of the compound was determined.Compound observed . Its 13CEPT data  showed 1tructure  was estaelations  of H-2/Hβ-orientation. The α-orientation of H-9/H-7 was determined by the NOE correlations of H-5/H-9/H-7. Finally, the calculated ECD spectra of -1 and -1 were compared to the experimental ECD of 1 and found that it was matched well with that of -1 . Therefore, the structure of 1 was established as shown and named amomumol A.Meanwhile, the NOE correlations between H-2/H-3a/H-14 indicated that they are on the same side and were assigned 7R,9R)-1 . Thus, t2 is a yellowish oil and has a molecular formula C15H22O2 deduced from the [M + H]+ ion at m/z 235.1701 . The NMR data of 2 (R)-11-hydroxybisabol-2,7(14)-dien-4-one, a bisabolane sesquiterpenoid isolated from the aerial parts of Mikania shushunensis  in 2 was observed. The double bond was assigned at C9-C10 based on the HMBC correlations from H-12/H-13 to C-10 and 1H-1H COSY correlations of H-8/H-9/H-10 (2 was comparable to that of (-)-(6R)-11-hydroxybisabol-2,7(14)-dien-4-one, which indicated that they possess the same absolute configuration of C-4, i.e., 4R , confirmed by the HMBC correlations from H-15 to C-1, C-2, and C-6 -ar-turmerone + . 1H and 13C-NMR data of 4 -2,6-dihydroxylhumla-9E,3(12),7(13),9-triene , two methyl singlets [δH 1.83  and 1.81 ], three oxygenated protons , two olefinic protons . The 13C-NMR, DEPT, and HSQC spectra revealed 20 carbon resonances, including six methyls, four olefinic carbons, three oxygenated carbons, two methylenes, four methines, and a carbonyl carbon (5 is a monoterpenoid dimer. The HMBC correlations (1H-1H COSY correlations (5. Two monoterpenes were connected by an oxygen bridge at C4–O–C1′ based on the HMBC correlation (J = 9.4 Hz) with H-5 and H-4′ (J = 9.1 Hz) with H-5′ indicated that H-4 and H-4′ were β-oriented and H-5 and H-5′ were α-oriented, which are the same as those of monoterpenes reported in the literature + , corresponding to 5 degrees of unsaturation. Comprehensive 1H and 13C NMR data analysis revealed that 6 is a similar derivative of 5. A 1,4-dihydroxy-menth-3(2)-ene moiety was first deduced by HMBC correlations (1H-1H COSY correlations (1H and 13C NMR data (1H-1H COSY correlations of H-2′/H-3′/H-4′/H-5′ and H-3′/H-8′/H-9′ (H-10′) established another monoterpene fragment, which could be derived from 1,4-dihydroxy-menth-3(2)-ene after undergoing dehydration and oxidation ring-opening at C1–C2. The relative configuration of 6 was determined as the same as 5 due to the similar NMR data and biogenetic origination. The absolute configuration of 6 was determined as  by ECD calculation (Amomumol F (elations  from H-1elations  of H-3/HNMR data  showed tculation .7 (amomumol G) had a molecular formula C18H24O4 corresponding to its HRESIMS ion at m/z 327.1568 [M + Na]+ . NMR data showed that 7 is also a monoterpene derivative. The monoterpene moiety was easily determined as 1,4-dihydroxy-menth-3(2)-ene inferred by HMBC correlations  and 8.0 . Combining with HMBC correlations between the methoxy group  and C-5′, and between H-3′,7′ and the carbonyl carbon (δC 166.6) determined that the remaining substructure is a p-methoxy benzoic acid. The HMBC correlation from H-4 to C-1′ revealed that 7 is 1,4-dihydroxy-menth-3(2)-ene-4-p-methoxybenzoate. Similar to compound 6, the absolute configuration of 7 was determined as  by comparable experimental and calculated ECD curves with a positive cotton effect at 225 nm and a negative cotton effect at 256 nm (Compound elations  from H-1elations  of H-3/Ht 256 nm .8) was obtained as an amorphous powder, and its molecular formula was established to be C22H28O7 by the HRESIMS [M + Na]+ ion at m/z 427.1735 . The NMR data  and H-7 /H-9 , between H-8′  and H-7′ /H-9′ , and other 2D NMR data  indicated the erythro configuration of H-7/H-8  +. The 1H and 13C NMR data  of 2. The 1H-1H COSY correlations of H-2/H-3/H-4/H-5/H-6 and HMBC correlations from H-3 (δH 7.03) and H-5  to C-1(δC 202.7) formed the ring structure of 10. HMBC correlations from H-12  to C-10 (δC 76.6) and C-13 (δC 17.6) and 1H-1H COSY correlations of H-14/H-7/H-8/H-9/H-10 established the 2-methyl-3-hydroxy-hept-1-ene side train . In the 13C NMR spectrum of 10a, the split carbons were still observed. Unfortunately, the separation of 10a was not successive. Although the configuration of 10 has not been determined, compound 10 is the first example of norsesquiterpenoid with a new C14 nor-bisabolane skeleton.Compound NMR data  of 10 wede train . As with11–45): 2-methyl-6-(4-methyl-3-cyclohexen-1-yl)-6-heptene-2,3-diol (11) dioxin]-4-one (41) 22.5D −20.5 ; UV (MeOH) λmax (log ε) 195 (0.85) nm; IR (KBr) νmax 2926, 1685, 1452, 1369, and 1095 cm−1; 1H NMR  and 13C NMR  data are given in m/z 237.1848 [M + H]+ .Amomumol A (2): yellow oil. [α]22.5D −32.2 (c = 0.5 MeOH); UV (MeOH) λmax (log ε) 195 (0.55) nm; IR (KBr) νmax 3435, 2929, 1708, 1450, 1375, 1242, 1051, and 898 cm−1; 1H NMR  and 13C NMR  data are given in m/z 235.1701 [M + H]+ .Amomumol B (3): yellow oil. [α]22.5D −61.5 ; UV (MeOH) λmax (log ε) 195 (2.01), 217 (0.90) nm; IR (KBr) νmax 2924, 1707, 1458, 1211, 1043 cm−1; 1H NMR  and 13C NMR  data are given in m/z 250.1810 [M + NH4]+ .Amomumol C (4): yellow oil. [α]22.5D −46.1 ; UV (MeOH) λmax (log ε) 195 (0.95), 225 (0.55) nm; IR (KBr) νmax 2926, 1732, 1714, 1666, 1454, 1043, 979 cm−1; 1H NMR  and 13C NMR  data are given in m/z 235.1694 [M + H]+ .Amomumol D (5): yellow oil. [α]22.5D −75.9 ; UV (MeOH) λmax (log ε) 195 (1.00), 228 (0.78) nm; IR (KBr) νmax 2956, 1676, 1369, 1265, 1051 cm−1; 1H NMR  and 13C NMR  data are given in m/z 321.2429 [M + H]+ .Amomumol E (6): yellow oil. [α]22.5D −34.4 ; UV (MeOH) λmax (log ε) 195 (1.00), 228 (0.78) nm; IR (KBr) νmax 2958, 1728, 1371, 1255, 1165, 983 cm−1; 1H NMR  and 13C NMR  data are given in m/z 359.2184 [M + Na]+ .Amomumol F (7): colorless oil. [α]22.5D −122.4 ; UV (MeOH) λmax (log ε) 197 (2.13), 256 (1.02) nm; IR (KBr) νmax 2978, 1718, 1458 cm−1; 1H NMR  and 13C NMR  data are given in m/z 327.1568 [M + Na]+ .Amomumol G (8): yellow powder. [α]22.5D −21.1 ; UV (MeOH) λmax (log ε) 205 (1.05), 220 (0.80), 270 (0.50) nm; IR (KBr) νmax 2958, 1581, 1500, 1458, 1419, 1328, 1219, 1122 cm−1; 1H NMR  and 13C NMR  data are given in m/z 427.1735 [M + Na]+ .Amomumol H (9): yellow powder. [α]22.5D −30.8 ; UV (MeOH) λmax (log ε) 205 (1.05), 220 (0.80), 270 (0.50) nm; IR (KBr) νmax 2958, 1724, 1375, 1240, 1028 cm−1; 1H NMR  and 13C NMR  data are given in m/z 389.1961 [M + H]+ .Amomumol I (J (10): yellow oil. [α]22.5D −25.1 ; UV (MeOH) λmax (log ε) 198 (0.95), 225 (0.55) nm; IR (KBr) νmax 2958, 1737, 1714, 1371, 1240, 1029 cm−1; 1H NMR  and 13C NMR  data are given in m/z 223.1697 [M + H]+ .Amomumol J derivative (10a): yellow oil. 1H NMR  and 13C NMR  data are given in m/z 265.1801 [M + H]+ .Amomumol 10. Compound 10 (4 mg) was dissolved in 150 μL of pyridine, and then 50 μL of acetic anhydride was added to react at room temperature for 3 h. The solvent was recovered to obtain compound 10a (4.5 mg).This acetylation reaction used the traditional pyridine-acetic anhydride method to acylate the acetyl group to the OH-10 of + solution, 10 μL of ADH (0.1 mg/mL) solution, and 10 μL of different concentrations of test compounds (metadoxine as a positive control) were added into 60 μL of 32 mM sodium pyrophosphate buffer (pH 8.8). The negative control group used 10 μL buffer instead of the sample solution. After incubating at room temperature for 30 min, 10 μL of 5% alcohol was added to the mixture for 5 min. The absorbances at 340 nm wavelength were then taken using a microplate reader.A modified traditional Valle–Hoch method was used to measure ADH activity . BrieflyA. kravanh, we used in vitro assay to evaluate the ADH-promoting activity of the PE, EtOAc, and n-BuOH fractions of the extract of the fruits of A. kravanh and found that the PE and EtOAc fractions showed ADH enhancing activity. The following phytochemical investigation resulted in the isolation of four new sesquiterpenoids (1–4), three new monoterpene derivatives (5–7), two new neolignans (8 and 9), a novel norsesquiterpenoid (10), and thirty-five known compounds. The bioassay results showed that compounds 11, 12, 15, 18, 26, and 36–38 significantly enhanced alcohol dehydrogenase activity in a dose-dependent manner. These results give a new insight into the chemical diversity and the potential usage in the hangover cure of A. kravanh.To identify the bioactive compounds from


Euphorbia neriifolia L. is distributed widely in India, Thailand, Southeastern China, and Taiwan and used as a carminative and expectorant to treat several inflammation-related diseases, such as gonorrhoea, asthma, and cancer. In the course of our search for potential anti-inflammatory agents from the titled plant, 11 triterpenes from the stem of E. neriifolia were isolated and reported in our previous endeavor. Given its rich abundance in triterpenoids, the ethanolic extract in this follow-up exploration has led to the isolation of additional eight triterpenes, including six new euphanes—neritriterpenols H and J–N (1 and 3–7)—one new tirucallane, neritriterpenol I (2), and a known compound, 11-oxo-kansenonol (8). Their chemical structures were elucidated on the basis of spectroscopic data, including 1D- and 2D NMR, and HRESIMS spectra. The absolute stereochemistry of neritriterpenols was determined by single-crystal X-ray diffraction analysis, ICD spectra, and DP4+ NMR data calculations. Compounds 1–8 were also evaluated for their anti-inflammatory activity by using lipopolysaccharide (LPS)-stimulated IL-6 and TNF-α on RAW 264.7 macrophage cells. Intriguingly, the euphane-type triterpenes (1 and 3–8) showed an inhibitory effect on LPS-induced IL-6 but not on TNF-α, while tirucallane-type triterpene 2 showed strong inhibition on both IL-6 and TNF-α.The Euphorbiaceae plant  Euphorbia neriifolia L., commonly known as a hedge or an ornamental horticultural cactus-like fleshy plant, belongs to the spurge family (Euphorbiaceae), one of the largest families of flowering plants  of E. neriifolia is poisonous and irritating to the skin and eyes , indicating that Euphorbia triterpenes have potential applications in immunomodulatory, anti-inflammatory, and analgesic treatments -eupha-8,23-diene-3β,25-diol-7-one, (+)-(24S)-eupha-8,25-diene-3β,24-diol-7-one, (24R)-eupha-8,25-diene-3β,24-diol-7-one, and sooneuphanone B  and a new tirucallane-type triterpene 2 (neritriterpenol I), as well as a known compound, 11-oxo-kansenonol (8) , induced circular dichroism (ICD), DP4+ NMR data calculation analysis, and single-crystal X-ray diffraction data, as well as a spectroscopic comparison with the reported literature data. In addition, we also evaluated each anti-inflammatory activity of these isolated compounds by measuring the effect of 1‒8 on the suppression of IL-6 and TNF-α in LPS-stimulated RAW 264.7 macrophages.Over the last decade, several  in 2013 . Most ofbclasses . Recentleatments . In our hanone B . To eluconol (8) . Compoun18 gel , and Sephadex LH-20 (GE) were used for open-column chromatography. Thin-layer chromatography (TLC) analyses were conducted on pre-coated silica gel plates , sprayed with anisaldehyde–sulfuric acid reagent, and then heated at 100°C. Preparative HPLC was performed using a Shimadzu LC-8A pump and an SPD-20A UV detector, equipped with a COSMOSIL 5C18 AR-II column . A Bruker D8 VENTURE single-crystal XRD diffractometer equipped with Mo–Kα radiation sources was used to record single-crystal X-ray diffraction.Optical rotations were recorded using a JASCO P-2000 polarimeter. Infrared (IR) spectra were obtained using a Thermo Scientific Nicolet iS5 FTIR Spectrometer. The circular dichroism (CD) spectra were measured using a JASCO J-715 spectropolarimeter. Nuclear magnetic resonance (NMR) spectra were recorded using a Varian Unity Inova 500-MHz instrument with a 5-mm SWPFG/TRPFG probe. High-resolution electrospray ionization mass spectrometry (HRESIMS) data were measured using a Thermo Scientific Q Exactive Focus Orbitrap LC-MS/MS instrument with an UltiMate 3000 UHPLC System. Silica gel 60 , CE. neriifolia L. were purchased from the traditional herbal market, Tianshun Ginseng Medicine and Herbal Shop, Taipei, Taiwan. The material was identified by one of the authors Dr. Chia-Ching Liaw, the curator of the Herbarium of National Institute Research of Chinese Medicine, Taiwan. A voucher specimen (EN-2018–10-001) was deposited at the Herbarium of NRICM, Taipei, Taiwan.The fleshy stems of E. neriifolia L. (45 kg) were air-dried and chopped. The dry material (12.0 kg) was extracted with 95% EtOH (80 L) at 50°С/24 h three times, and the extracts were combined and concentrated under reduced pressure to obtain the crude extract. The EtOH extract (ca.1.9 kg) suspended in H2O was sequentially partitioned with hexanes and CH2Cl2 to yield three partition layers. The CH2Cl2 layer  was chromatographed by flash column chromatography on silica gel and eluted with CH2Cl2/MeOH  to yield three fractions (ENDD1∼3). The fraction ENDD2  was fractionated using the HPLC system and eluted with MeOH in H2O (60–100%) to obtain four subfractions (ENDD2.1∼2.4). The subfraction ENDD2.2  was chromatographed using the Sephadex LH-20 column and eluted with 100% methanol to obtain seven parts (ENDD2.2.1∼7). ENDD2.2.4 was subjected to RP-HPLC using a C18 column detected at 210 nm, and then, the subfractions were obtained (ENDD.2.2.4.1∼6). Compounds 5  and 6  were purified from the subfraction ENDD2.2.4.2 by HPLC and eluted with 40% acetonitrile (flow rate: 10.0 mL/min). The subfraction ENDD2.2.4.3 was purified by HPLC and eluted with 45% acetonitrile to obtain compounds 1  and 2 . The subfraction ENDD2.2.4.4 was also purified by HPLC and eluted with 50% acetonitrile to obtain 8 . Subfractions ENDD2.2.4.5 and ENDD2.2.4.6 were further purified by HPLC and eluted with 50% acetonitrile to yield 3 , 4 , and 7 .The fleshy stems of α] +28.9 ; νmax (KBr) 3429, 2927, 1672, 1465, 1376, 1270, 1183, and 1037 cm−1; CD  216 (+10.67), 265 (−11.65), and 349 (+5.26) nm; 1H- (500 MHz) and 13C- (125 MHz) NMR spectroscopic data (CDCl3) in m/z 495.3451 [M + Na]+ .White amorphous powder; [α] −8.3 ; νmax (KBr) 3442, 2967, 2932, 2871, 1716, 1660, 1458, 1376, 1272, 1183, 1121, 1077, and 1040 cm−1; CD  216 (+11.40) and 249 (+3.62) nm; 1H- (500 MHz) and 13C- (125 MHz) NMR spectroscopic data (CDCl3) in m/z 493.3287 [M + Na]+ .White amorphous powder; [α] +22.7 ; νmax (KBr) 3417, 2967, 2925, 1640, 1462, 1378, 1292, 1240, 1173, and 1072 cm−1; CD  215 (+11.94), 261 (+4.06), and 313 (−0.55) nm; 1H- (500 MHz) and 13C- (125 MHz) NMR spectroscopic data (CDCl3) in m/z 497.3618 [M + Na]+ .Colorless crystal; [α] +12.9 ; νmax (KBr) 3419, 2970, 2871, 1643, 1596, 1460, 1381, 1267, 1173, 1075, and 1025 cm−1; CD  215 (+11.73), 257 (+8.26), and 313 (−2.17) nm; 1H- (500 MHz) and 13C- (125 MHz) NMR spectroscopic data (CDCl3) in m/z 473.3638 [M − H]− .White amorphous powder; [α] +5.8 ; νmax (KBr) 3432, 2923, 2854, 1672, 1467, 1378, 1270, 1235, 1183, and 1037 cm−1; CD  216 (+11.49) and 270 (+19.50) nm; 1H- (500 MHz) and 13C- (125 MHz) NMR spectroscopic data (CDCl3) in m/z 511.3397 [M + Na]+ .Colorless crystal;  +9.2 ; νmax (KBr) 3439, 2975, 2873, 1670, 1462, 1416, 1381, 1267, 1233, and 1183 cm−1; CD  215 (+12.08) and 273 (+10.23) nm; 1H- (500 MHz) and 13C- (125 MHz) NMR spectroscopic data (CDCl3) in m/z 511.3399 [M + Na]+ .White amorphous powder; [α] +10.5 ; νmax (KBr) 3454, 2975, 2933, 2871, 1670, 1645, 1383, 1267, 1233, and 1188 cm−1; CD  219 (+10.54) and 271 (+16.17) nm; 1H- (500 MHz) and 13C- (125 MHz) NMR spectroscopic data (CDCl3) in m/z 515.3734 [M − H]− .White amorphous powder; [α] +18.5 ; νmax (KBr) 3412, 2970, 2360, 2341, 1668, and 1034 cm−1; CD  215 (+11.96) and 276 (+21.4) nm; 1H- (500 MHz) and 13C- (125 MHz) NMR spectroscopic data (CDCl3) in m/z 469.3323 [M - H]- .White amorphous powder; , b = 7.0406 (2) Å [β = 94.7800 (10)°], and c = 17.1863 (7) Å [γ = 90°]; volume = 1,454.70 (9) Å3; Z = 2; density  = 1.125 Mg/m3; absorption coefficient = 0.074 mm-1; F (000) = 544 and T = 200 (2)K. A total of 27,930 reflections were collected, of which 5,130 independent reflections [Rint = 0.0678] with I > 2σ (I) were used for the analysis. The final indices were R1 = 0.0392 and wR2 = 0.0878 with goodness-of-fit = 1.059. The absolute structure parameter is 0.1 (10). All data are shown in Crystallographic data on neritriterpenol J (5): C30H50O6; crystal size: 0.60 × 0.08 × 0.03 mm3; wavelength (λ) = 0.71073 Å; crystal system: orthorhombic; space group P212121; unit cell dimensions, a = 6.9896 (3) Å [α = 90°], b = 12.3010 (6) Å [β = 90°], and c = 33.8198 (17) Å [γ = 90°]; volume = 2,907.8 (2) Å3; Z = 4; density  = 1.157 Mg/m3; absorption coefficient = 0.079 mm-1; F (000) = 1,112 and T = 200 (2)K. A total of 15,236 reflections were collected, of which 5,135 independent reflections [Rint = 0.0676] with I > 2σ (I) were used for the analysis. The final indices were R1 = 0.0513 and wR2 = 0.0963 with the goodness-of-fit = 1.054. The absolute structure parameter is −1.0 (14). All data are shown in Crystallographic data on neritriterpenol L 4]. The tested compounds  were directly dissolved in a solution of Mo2(OAc)4 complex in DMSO (5 mg/10 mL) in a molar ratio of Mo2(OAc)4/compound of about 1:0.5, and the mixture was subsequently measured for the induced CD spectra without the preparation and isolation of the complexes.The determination of the absolute configuration of cyclic and acyclic 3 using the gauge-independent atomic orbital (GIAO) method at the PCM/mPW1PW91/6–31G+//B3LYP/6–31G(d) level with the Boltzmann population refined in the solvation model based on density (SMD) for MeOH or CHCl3 at a new level //B3LYP/6–31G(d)) (The conformers discovered utilizing the GMMX package implemented in Gaussian 16  with the–31G(d)) . The Exc–31G(d)) .2 in a humidified incubator. Cells were plated at a density of 5 × 104 cells per well in 96-well plates 24 h before treatment of lipopolysaccharides  with a vehicle or indicated compounds with doses of 5, 10, or 20 μM. After 24 h of treatment, cell viability was evaluated by MTT assay.RAW 264.7 macrophages were purchased from American Type Culture Collection . Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) plus 1% penicillin and streptomycin at 37°C with 5% CO5 cells/well and pre-incubated for 24 h. Then, cells were treated with LPS (1 μg/mL) alone, LPS plus dexamethasone , or LPS plus various doses of indicated compounds  for 24 h. The conditioned medium was collected for the determination of TNF-α and IL-6 levels using ELISA kits , according to the manufacturer’s instructions.RAW 264.7 cells were seeded in 24-well plates at a density of 2 × 10p < 0.05, **p < 0.01, and ***p < 0.001).The data presented are representative of at least three independent experiments and expressed as the mean ± standard deviations (SD). All statistical analyses were performed using Prism 7.0  for a one-way ANOVA, followed by Tukey’s multiple range tests if ANOVA was significant  (The ethanolic extract of the ds (1–8) . All iso1 was obtained as white amorphous powders with [α] +28.9 , and its molecular formula C30H48O4 on par with seven degrees of unsaturation (DOU) was deduced from the HRESIMS pseudomolecular ion peak at m/z 495.3451 [M + Na]+ . The IR spectrum showed absorption bands, indicating the existence of hydroxyl (3429 cm-1), alkane (2927 cm-1), carbonyl (1672 cm-1), methyl (1465 and 1376 cm-1), and C‒O (1037 cm-1) functional groups. The 13C- and DEPT-NMR spectra of 1 separated the 30 carbons into seven methyl , eight methylene , one sp2 methylene (δC 111.0), three methine , three oxymethine , four aliphatic quaternary carbon , and three sp2 quaternary carbon  groups, and one carbonyl (δC 200.1) group, while the 1H-NMR spectrum demonstrated obviously seven methyl  and three oxymethylene  groups, and one olefinic methylene [δH 4.88 (brs) and 4.97 (brs)] group. The aforementioned data indicated that 1 possesses a symbolic tetracyclic-fused ring moiety containing seven methyl groups, three hydroxyl groups, a conjugated double-ketone system, and a terminal olefin. According to the C‒H single-bond correlations of 1 by the HSQC spectrum, the 1H- and 13C-NMR assignments of 1 were determined (1 was elucidated by 1H–1H COSY and HMBC spectra (1H–1H COSY correlations of H2-1 (δH 1.58 and 2.47)/H2-2 /oxymethine H-3 (δH 3.34), H-5 (δH 1.68)/H2-6 (δH 2.44 and 2.50), oxymethine H-11 (δH 4.72)/H2-12 (δH 1.83 and 2.43), and H2-15 (δH 1.46 and 2.12)/H2-16 (δH 1.35 and 1.96)/H-17 (δH 1.62), as well as the HMBC correlations from Me-28 (δH 0.94) and Me-29 (δH 1.02) to oxymethine C-3 (δC 78.3), C-4 (δC 39.1), and C-5 (δC 49.3); from Me-19 (δH 1.28) to C-1 (δC C 33.7), C-5, olefinic quaternary carbon C-9 (δC 161.2), and C-10 (δC 39.6); from Me-18 (δH 0.74) to C-12 (δC 42.8), C-13 (δC 46.2), C-14 (δC 48.0), and C-17 (δC 48.5); from Me-30 (δH 1.17) to olefinic quaternary carbon C-8 (δC 140.4), and C-13, C-14, and C-15 (δC 31.8); and from H-5 (δH 1.68) and H2-6 (δH 2.44 and 2.50) to carbonyl C-7 (δC 200.1). Then, a 24-diol-25-ene-contained C8-side chain attached at C-17 was connected by the 1H–1H COSY correlations of H-17 (δH 1.62)/H-20 (δH 1.51)/Me-21 (δH 0.90)/H2-22 (δH 1.24 and 1.60)/H2-23 (δH 1.49 and 1.61)/oxymethine H-24 (δH 4.06) and the HMBC correlations from olefinic methylene H2–26 (δH 4.88 and 4.97) and Me-27 (δH 1.79) to oxymethine C-24 (δC 76.2) and olefinic quaternary carbon C-25 (δC 147.8).Compound termined , 2. The  spectra : a 3,11-1 indicates that compound 1 belongs to the lanostane- or euphane-type triterpene, a pair of stereoisomers having two different conformations at C-13 and C-14 in a rigid Δ8,24–6/6/6/5-tetracyclic triterpene , and the chirality of these two chiral carbons cannot be determined solely by the NOE correlation between Me-18 and Me-30. Based on the regular conformations of lanostane and euphane triterpenoids, the anti-parallel relationship between H-5 (α-oriented) and Me-19 (β-oriented) was established, whereas the ß-configuration of H-17 was assigned unequivocally. In the NOESY spectrum of 1 , Me-19/Hax-2 (δH 1.78), Me-28/Hax-2, Heq-1/Hax-2, Me-30/Hax-12 (δH 2.43), Me-30/H-17, Heq-16 (δH 1.96)/Me-21, and H-17/Me-21. Meanwhile, the NOE correlations of Me-29/H-3, Me-29/H-5, Me-29/Heq-6 (δH 2.44), H-3/H-5, H-3/Hax-1 (δH 1.58), Hax-1/H-11, H-11/Heq-12 (δH 1.83), H-11/Me-18, Me-18/Hax-15 (δH 1.46), Me-18/Hax-16 (δH 1.35), Me-18/H-20, and Hax-15/Me-29 indicated their inverse α-orientations for Hax-1, H-3, H-5, Heq-6, H-11, Heq-12, Me-18, Hax-15, Hax-16, H-20, and Me-29. Of the complete configuration assignment, the ß-form of Me-30 and the α-form of Me-18 illustrated the 13S,14S-chirality of 1, and thus, compound 1 was recognized as a euphane-type triterpene. Likewise, the ß-orientations of OH-3 and OH-11 in 1 were decided by the downward orientation of both H-3 and H-11 on the rigid tetracyclic moiety. The spatial configuration of 24-OH at C-24 was resolved as an R-form configuration by comparison of chemical shifts with those of known compounds, namely, (24S/R)-eupha-8,25-dien-3β,24-diol-7-one  and 2b (24S). The DP4+ calculated results showed that the probability of C-24R is 100% (1 was determined as a structure of (24R)-3β,11β,24β-trihydroxy-eupha-8,25-dien-7-one, named neritriterpenol H.The planar framework of rum of 1 , the ß-ool-7-one  and 3β,1en-7-one  (Supplemen-7-one . Furtherulations  at the m is 100% . Taken t2) has a molecular formula C30H46O4 with eight DOU established from its HRESIMS ion peak at m/z 493.3287 [M + Na]+ , and the ring carbon signals in 13C-NMR data , carbonyl (1716 cm-1), and olefin (1660 cm-1) groups, and the 1H–1H COSY and HMBC correlations  and two methylene (δC 26.0 and 34.0) groups, and one aliphatic methine (δC 35.6), one sp2 methine (δC 154.9), one sp2 quaternary carbon (δC 139.3), and one aldehyde (δC 195.3) group. The combination of the COSY fragment of H-17/Me-21/H-20/H2-22/H2-23/H-24 and the HMBC correlations from Me-27 to olefinic methine C-24, olefinic quaternary carbon C-25, and aldehyde C-26  of Me-21, the negative optical rotation (−8.3°), and the NOESY correlations of 2  level on two possible poses, the isomers of C-20, 2a (20S) and 2b (20R), and the calculated results suggested that 2a is the favorite structure by its almost 100% probability distribution term -3β,11β-dihydroxytirucalla-8,24E-dien-7,26-dione.Neritriterpenol I  and 4 (neritriterpenol K) were concerned, these two compounds were considered to be a pair of stereoisomers because of their extreme resemblance of 1H- and 13C-NMR data (30H50O4 (six DOU) deduced from the HRESIMS ion peaks of 3 and 4 at m/z 497.3618 [M + Na]+  and 473.3638 [M − H]− , respectively. Analyzing the 13C- and DEPT-NMR spectra of 3 and 4, both of their 30 carbons were found to be assembled by eight methyl, nine methylene, three aliphatic methine, two oxymethine, four aliphatic quaternary carbon, one oxygenated quaternary carbon, and two olefinic quaternary carbon groups, as well as one carbonyl group, revealing that 3 and 4 are also triterpenoids. Then, the juxtaposition of the 1H- and 13C-NMR data on 3 and 1 revealed several common features in these two compounds, a hydroxyl group , a tetrasubstituted double bond (δC 161.5 and 139.6), a conjugated ketone (δC 199.1), and a multi-methyl group, suggesting that compound 3 has a similar unsaturated conjugated 6/6/6/5-fused ring moiety to 1. The 6/6/6/5-fused tetracyclic ring of 3 is estimated to be an eupha-8-en-3-ol-11-one moiety through the HMBC correlations (δH 0.93) to C-12 (δC 51.1), C-13 (δC 44.7), C-14 (δC 51.2), and C-17 (δC 50.3); from Me-19 (δH 1.22) to C-1 (δC 34.1), C-5 (δC 51.7), C-9 (δC 139.6), and C-10 (δC 37.1); from Me-28 (δH 0.85) and Me-29 (δH 1.06) to oxymethine C-3 (δC 78.9), C-4 (δC 39.0), and C-5; and from Me-30 (δH 1.04) to C-8 (δC 161.5), C-13, C-14, and C-15 (δC 30.0). Moreover, the 24,25-diol-containing side chain attached at C-17 was illustrated by the HMBC correlations from Me-26 (δH 1.18) and Me-27 (δH 1.24) to oxymethine C-24 (δC 79.3) and oxygenated quaternary carbon C-25 (δC 73.2). Similar 1H–1H COSY and HMBC correlations for 4  solution for the configuration determination by X-ray crystallographic analysis -eupha-8-en-3β,24β,25-triol-11-one, whereas compound 4 is thus a 24R-epimer having a 24β-hydroxyl group. The 24R/S configuration was further confirmed by the dimolybdenum tetraacetate [Mo2(OAc)4]-induced circular dichroism (CD) method for the rapid configuration diagnosis of the acyclic 24,25-sec,tert-diols (R/S-epimers 3 and 4 can be chelated by Mo2(OAc)4 in the aqueous DMSO solution to form complexes of Mo2(OAc)4 with 1,2-diol-containing molecules, and one complex has two envisaged ligation poses, the “equatorial” (major) and “axial” (minor) positions of bulk with side-chain residues in the diol−dimolybdenum adducts  in the HRESIMS spectra of 5 and 6 deduced the same molecular formula, C30H48O5 (7 DOU), indicating that compounds 5 and 6 are a couple of stereoisomers. Additionally, their 1H- and 13C-NMR data  is oxidized into a ketone group (δC 199.9/200.0) in 5 and 6. Furthermore, the HMBC corrections from H2-6  to ketone C-7 (δC 199.9/200.0) illustrated the planar eupha-8-en-3,24,25-triol-7,11-dione of 5 and 6. The same chirality of the rigid ring moiety of 5 and 6 was decided by their NOESY corrections, whereas the orientations of 24,25-sec,tert-diols in 5/6 were recognized by the X-ray crystallographic analysis of 5 4]-induced CD results of 5 and 6 to those of 3 and 4. Finally, the NMR comparison allocated compound 5 in 24S-chirality, while 6 in 24R-distribution. Compounds 5 and 6 were named neritriterpenols L and M, respectively.Similarly, the sodium adduct ion peaks at NMR data , 2 were , according to the HRESIMS pseudomolecular ion at m/z 515.3734 [M − H]− . This compound was presumed to be an alkylated derivative of 5 and 6, an ethanoxylated C-25 of 5 and 6, by the observation of the 1H–1H COSY correlations of H2-1' (δH 3.45)/Me-2' (δH 1.18) and the HMBC correlations from H2-1′ to the C-25 (δC 77.4)  that is close to those of 3  and 5 , revealing that 7 has the same stereochemistry as neritriterpenol L (5) and named neritriterpenol N.Compound C 77.4) . Moreove8, it was considered a known triterpenoid, 11-oxo-kansenonol, determined by the comparison of previously reported data including NMR, HRESIMS, UV, and optical rotation -stimulated macrophages. RAW 264.7 macrophages were treated with LPS and different concentrations of compounds 1–8  for 24 h, followed by the conditioned medium collection. Cytotoxicity analysis showed that the isolated compounds have little to no effect on cell viability with the doses of 5, 10, and 20 μM in macrophages. All isolated compounds (1–8) exhibited inhibitory activity against IL-6 secretion in a dose-dependent manner in LPS-stimulated macrophages , except compound 2, showed weak or no activity on the suppression of LPS-stimulated TNF-α secretion in RAW 264.7 macrophages (2 showed a remarkable inhibitory effect on TNF-α secretion in LPS-stimulated cells similar to the DEX group.The anti-inflammatory activity of the isolated triterpenes rophages . Among trophages . Only 201 and 3–8) from the titled plant that possess potent pharmacological activity specifically on the suppression of IL-6 but not TNF-α, an encouragement to develop into more effective IL-6-specific inhibitors. On the other hand, javamide-II identified from coffee also showed a similar inhibitory activity against IL-6 but not TNF-α and IL-1β in macrophage-like THP-1 cells  and a tirucallane-type triterpene (compound 2), as well as a known compound, 11-oxo-kansenonol (8). Based on the anti-inflammatory assay, euphanes 3–8 showed selective inhibition on IL-6 in a dose-dependent manner with little or no effect on TNF-α in contrast to tirucallane 2 that exhibited strong inhibition against both IL-6 and TNF-α. It has been well-documented that dysregulated IL-6 may lead to over-reacted immune responses and thus results in unfavorable consequences. Suppressing IL-6 apparently is a good strategy to cope with dysregulated IL-6-associated diseases such as rheumatoid arthritis. Given that TNF-α and IL-6 each has its own independent role, they, however, are often regulated together and function co-operatively in many aspects of immune responses. It would not be surprising that most IL-6 inhibitors reported up until now suppress IL-6 and TNF-α as a whole, as tirucallane 2 exemplifies here (E. neriifolia L., especially compounds 4 and 8, selectively inhibit the IL-6 dysregulated immune responses. In conclusion, our study contributes to the expanding knowledge of anti-inflammatory compounds derived from E. neriifolia L.In our continuous search for anti-inflammatory components from the ethanolic extract of ies here . Despite


N-acylation/aza Diels-Alder cycloaddition/decarboxylation/dehydration)/click strategy, both step-by-step to optimize all involved experimental stages, and in one pot manner to evaluate the scope and sustainability of this polyheterocyclic-focused synthetic strategy. In both ways, the yields were excellent, considering the high number of bonds formed with release of only one carbon dioxide and two molecules of water. The Ugi-Zhu reaction was carried out using the 4-formylbenzonitrile as orthogonal reagent, where the formyl group was first transformed into the pyrrolopyridin-5-one core, and then the remaining nitrile group was further converted into two different nitrogen-containing polyheterocycles, both via click-type cycloadditions. The first one used sodium azide to obtain the corresponding 5-substituted-1H-tetrazolyl-pyrrolopyridin-5-one, and the second one with dicyandiamide to synthesize the 2,4-diamino-1,3,5-triazine-pyrrolopyridin-5-one. Both synthesized compounds may be used for further in vitro and in silico studies because they contain more than two heterocyclic moieties of high interest in medicinal chemistry, as well as in optics due to their high π-conjugation.A diversity-oriented synthesis (DOS) of two new polyheterocyclic compounds was performed via an Ugi-Zhu/cascade ( N-heterocycles are the 5-substituted-1H-tetrazoles (5S-1H-Ts), due mainly to their ability to surrogate the carboxylic acids (RCOOH) into living systems since they have close values of pKa , similar topologies, and practically the same electrostatic potential and electron density . Ano. AnoN-heamine 2,  6], is , is N-hetriazine . This cola carte . Finally67778 3,  9] whic whicN-he-one 3′,  10]. Th. ThN-hetMCRs are privileged synthetic tools that involve sequential additions of at least three reagents into the same reactor (one pot). Given the nature of MCRs, they can be accessed quickly, in comparison to linear synthesis, to a series of highly functionalized and structurally complex products, leading to high atomic economy, eventually in good chemical yields and in short reaction times, needing only one work-up and in most cases just one purification process . MCRs , may or may not require activation by an acid, thus favoring the α-nucleophilic attack of the isocyanides 7, leading to formation of nitrilium cations 8. Then, the intermediate 8′ rapidly tautomerizes via a non-prototropic chain-ring process to the corresponding oxazoles 9  , where pazoles 9 .b]pyridin-5-ones either bound, fused or linked to various heterocyclic frameworks of interest in optics and medicinal chemistry. However, for the present work, the synthetic strategy behind a couple of new tris-heterocycles, from the diversity-oriented synthesis (DOS) approach, started with an Ugi-Zhu reaction performed stepwise to first obtain a Schiff base, and then, the corresponding 5-aminooxazole. This pivotal heterocycle then reacts with maleic anhydride via a cascade process (aza Diels–Alder cycloaddition/N-acylation/decarboxylation/dehydration) to assemble the pyrrolopyridin-5-one core pyridin-5-one 17. Different entries were carried out using toluene as solvent but varying temperature, finding that at 80 °C in 20 minutes the reaction was accomplished in 96% yield  cycloadditions  cycloaddition occurred due to a less steric hindrance, but it was not easy. Thus, a first attempt was carried out using the methodology described by Harasawa and co-workers in 2013  cycloaddition assisted by microwave radiation. Thus, the reaction was carried out using DMSO as solvent and KOH as catalyst, according to the methodology reported by Fang and co-workers  cycloaddition, leading to the formation of the six-membered polyheterocycle 22. This intermediate subsequently undergoes a base-assisted aromatization to get finally the 2,4-diamino-1,3,5-triazine-pyrrolopyridin-5-one 21, after recovering a H-atom from water. It is noteworthy that despite 20D and 17, in fact the [4+2] cycloadditions can occur stepwise, even by non-polar [Although there are some reports about the synthesis of triazines from nitriles and dicyandiamide via [4+2] cycloadditions, to our best knowledge, there is no proposals of mechanisms. Thus, in on-polar , or polaon-polar  mechanis12, 14, 17 and 21, and with the idea of evaluating the robustness of this methodology from the one pot chemistry approach, the synthesis of the final compound 21 was carried out via a domino process coupling in sequential manner the Ugi-Zhu reaction with the N-acylation, decarboxylation, dehydration, and the [4+2] cycloaddition, yielding 19%. Of course, this yield seems to be low, but it is not true at all considering the high number of formed bonds, the atom economy (89%), and that only a couple of water molecules and one carbon dioxide were released in the entire domino process  spectra were acquired on a Bruker AMX Advance III spectrometer . The solvents used for NMR experiments were deuterated chloroform (CDCl3) and deuterated dimethyl sulfoxide (DMSO-6d). Chemical shifts are reported in parts per million (d/ppm). Coupling constants are reported in Hertz (J/Hz). The internal reference for NMR spectra was tetramethyl silane (TMS) at 0.00 ppm. Multiplicities of the signals are reported using the standard abbreviations: singlet (s), doublet (d), triplet (t), quartet (q), and multiplet (m). NMR spectra were analyzed using the MestReNova software Ver. 12.0.0-20080 . The infrared (IR) spectrum was acquired on a Perkin-Elmer 2000 spectrometer  using the attenuated total reflectance (ATR) method. The maximum absorbance peaks are reported in reciprocal centimeters (νmax/cm−1), uncorrected. The IR spectrum was analyzed using the Origin software . The high-resolution mass spectroscopy (HRMS) spectrum was acquired by electrospray ionization (ESI) on a Micro-TOF II spectrometer Bruker Daltonics GmbH . The sample was injected directly (Apollo source) and analyzed by the time-of-flight method (TOF). The HRMS spectrum was analyzed using the Compass software . Microwave-assisted reactions were performed in closed-vessel mode on a CEM Discover SP MW-reactor . Reaction progress was monitored by thin-layer chromatography (TLC), and the spots were visualized under ultraviolet (UV) light (254 or 365 nm). Glass preparative plates (20 × 20 cm) coated with silica-gel 60 doped with UV indicator (F254) were used to purify the products. All starting reagents and solvents were used as received . Chemical structures were drawn using the ChemDraw software . The purity of all synthesized products (>96%) was assessed by NMR.f = 0.04 (EtOAc); 1H NMR : δ 8.37 , 7.86 , 7.69 , 6.84–6.76 , 5.94 , 4.76  ppm; 13C NMR : δ 159.6 (C-12), 147.8 (C-4), 146.8 (C-5), 139.9 (C-13), 132.4 , 132.3 (C-8), 128.6 , 121.4 (C-7), 118.4 (C-19), 114.0 (C-16), 108.6 (C-6), 108.3 (C-9), 101.0 (C-2), 64.8 (C-10) ppm; HRMS: (ESI+) m/z calcd. for [M − H]+ C16H13N2O2+ 265.0972, found 265.0968 (error = 1.3 ppm); FT-IR , 1,3-benzodioxole-5-methylamine  and sodium sulfate anhydrous  were placed in a sealed CEM Discover microware reaction tube (10 mL) and diluted in anhydrous toluene (1.5 mL). The mixture was stirred and heated using microwave irradiation  for 30 min. Subsequently, the reaction mixture was filtered, and the resulting solid was washed with 15 mL of dichloromethane and 15 mL of acetone; then, it was dried under high vacuum, obtaining  of a light yellow solid, R2CO3(aq.) (0.4 M) solution was added, and liquid–liquid extractions were carried out three times. The organic phases were collected and washed with brine three times (3 × 10 mL). The organic phase was dried with anhydrous MgSO4 and concentrated to dryness under vacuum. The resulting crude was purified by column chromatography using a mixture of hexanes: ethyl acetate 3:1 (v/v) as eluent to obtain a slightly yellowish oil , Rf = 0.36 (Hex-EtOAc = 3:2 v/v); 1H NMR : δ 7.62 , 7.54 , 7.29–7.25 , 7.23–7.17 , 6.80 , 6.71 , 6.67 , 5.91 , 4.88 , 3.81 , 3.70–3.67 , 3.65–3.61 , 2.92–2.89  ppm; 13C NMR : δ 157.5 (C-9), 152.3 (C-7), 147.7 (C-17), 146.7 (C-21), 144.6 (C-31), 139.2 (C-25), 132.9 (C-15), 132.3 , 128.3 , 126.1 (C-28), 124.7 (C-37), 121.4 (C-22), 188.5 (C-11), 111.7 (C-31), 108.7 (C-16), 108.0 (C-23), 100.8 (C-19), 66.6 , 59.4 (C-12), 51.2 (C-14), 50.8 , 31.7 (C-24) ppm; HRMS: (ESI+) m/z calcd. for [M − H]+ C30H29N4O4+ 509.2183, found 509.2188 (error = 1.0 ppm); IR , 1,3-benzodioxole-5-methylamine  and sodium sulfate anhydrous  were placed in a sealed CEM Discover microware reaction tube (10 mL) and diluted in anhydrous toluene (1.5 mL). The mixture was stirred and heated using microwave irradiation  for 30 min, and ytterbium (III) triflate (0.08 equiv.) was added. The mixture was stirred and heated using microwave irradiation  for 5 min, and then 2-isocyano-1-morpholino-3-phenylpropan-1-one  was added. The new mixture was stirred and again heated using microwave irradiation  for 25 min, time it took to complete the reaction. Then, the solvent was evaporated under reduced pressure and the reaction crude was diluted with ethyl acetate. A Na2CO3(aq.) (3 × 10.0 mL), and then washed with brine (3 × 10 mL). The organic layer was dried using anhydrous MgSO4, filtered, and concentrated to dryness under vacuum. The new crude was purified by silica-gel column chromatography using mixtures of hexanes (Hex) and ethyl acetate (EtOAc) in 4:1 to 3:2 (v/v) proportions as mobile phase to isolate the corresponding pyrrolopyridin-5-one-7-yl benzonitrile  as yellow oil, Rf = 0.30 (Hex-EtOAc = 3:2 v/v); 1H NMR : δ 7.90 , 7.67–7.64 , 7.28–7.25 , 7.18–7.10 , 6.70 , 6.67 , 6.57 , 5.92 , 5.91 , 5.33 , 5.29 , 4.26 , 4.15 , 3.81 , 3.73 , 2.87–2.80  ppm; 13C NMR : δ 167.0 (C-13), 162.3 (C-8), 159.2 (C-10), 148.2 (C-7), 148.1 (C-32), 147.3 (C-36), 141.1 (C-23), 139.0 (C-17), 132.7 , 130.0 (C-30), 128.7 , 128.6 , 128.2 , 126.2 (C-20), 123.9 (C-15), 123.5 (C-14), 121.8 (C-38), 118.3 (C-40), 112.5 (C-26), 108.7 (C-31), 108.3 (C-37), 101.2 (C-34), 67.0 , 63.7 (C-11), 53.0 , 44.0 (C-29), 39.9 (C-16) ppm; HRMS: (ESI+) m/z calcd. for [M − H]+ C33H29N4O4+ 545.2183, found 545.2192 (error = 1.6 ppm); IR : 4-formylbenzonitrile , 1,3-benzodioxole-5-methylamine  and sodium sulfate anhydrous  were placed in a sealed CEM Discover microwave reaction tube (10 mL) and diluted in anhydrous toluene (1.5 mL). The mixture was stirred and heated using microwave irradiation  for 30 min, and ytterbium (III) triflate (0.08 equiv.) was added. The mixture was stirred and heated using microwave irradiation  for 5 min, and then 2-isocyano-1-morpholino-3-phenylpropan-1-one  was added. The new mixture was stirred and again heated using microwave irradiation  for 25 min, and then maleic anhydride  was added. Finally, the reaction mixture was stirred and heated using microwave irradiation  for 20 min. Then, the solvent was removed to dryness under vacuum. The crude was extracted using ethyl acetate (3 × 10.0 mL) and Na17 was synthesized, and once the reaction was finished, the solvent was evaporated to dryness, and the crude was transferred to a Pyrex pressure tube (5 mL), sodium azide  and zinc bromide  were added. The mixture was dissolved in 1.5 mL of H2O and 1.5 mL of isopropyl alcohol, and then the mixture of reaction was heated in an oil bath at 120 °C for 40 hours. Subsequently, the reaction mixture was allowed to cool to room temperature, and a mixture 1:1 v/v of hexanes with ethyl acetate was added at 0 °C, stirred for 10 min, and then filtered and washed with hot water and hot ethyl acetate. The resulting solid was dried under high vacuum overnight, obtaining 264.44 mg of a light brown solid (45% yield); mp > 250 °C; 1H NMR : δ 7.96 , 7.92 , 7.16 , 7.14–7.05 , 6.79 , 6.72 , 6.62–6.60 , 5.95 , 5.93 , 5.44 , 5.00 , 4.28 , 4.06 , 3.76 , 3.72–3.66 , 2.89–2.76  ppm; 13C NMR : δ 166.5 (C-13), 161.7 (C-8), 160.2 (C-10), 148.1 (C-7), 147.9 (C-32), 146.9 (C-36), 139.7 (C-17), 136.5 (C-26), 131.1 (C-30), 130.1 (C-40), 129.0 , 128.7 , 128.5 , 127.5 , 126.3 (C-20), 124.0 (C-15), 123.8 (C-14), 121.7 (C-38), 108.7 (C-31), 108.6 (C-37), 101.3 (C-34), 66.8 , 64.1 (C-11), 52.8 , 43.7 (C-29), 40.4 (C-16) ppm; HRMS: (ESI+) m/z calcd. for [M − H]+ C33H30N7O4+ 588.2354, found 588.2348 (error = 1.0 ppm); IR , and potassium hydroxide  were added. The mixture was stirred and heated using microwave irradiation  for 90 min. Subsequently, the reaction mixture was allowed to cool to room temperature. Then, hot water was stirred for 10 min, and then filtered and washed with hot water and hot ethyl acetate. The resulting solid was dried under a high vacuum overnight, obtaining 408.65 mg of a yellow solid (65% yield); mp > 250 °C; 1H NMR : δ 8.28 , 7.92 , 7.20 , 7.15–7.09 , 6.70 , 6.69 , 6.59 , 5.92 , 5.91 , 5.47 , 5.33 , 5.30 , 4.80 , 4.12 , 3.81–3.77 , 3.66 , 2.86–2.77  ppm; 13C NMR : δ 171.8 (C-40), 167.6 , 167.0 (C-13), 162.2 (C-8), 160.2 (C-10), 148.0 (C-17), 147.9 (C-32), 147.2 (C-36), 139.2 (C-7), 138.9 (C-23), 137.0 (C-26), 130.4 (C-30), 129.0 , 128.7 , 128.2 , 128.0 , 126.1 (C-20), 124.0 (C-15), 123.8 (C-14), 121.9 (C-38), 108.9 (C-37), 108.3 (C-31), 101.1 (C-34), 67.1 , 64.1 (C-11), 53.0 , 43.7 (C-29), 40.0 (C-16) ppm; HRMS: (ESI+) m/z calcd. for [M − H]+ C35H33N8O4+ 629.2619, found 629.2647 (error = 4.4 ppm); IR , and the generation of different molecular skeletons  through post-functionalization processes. In line with the diversity-oriented synthesis (DOS) approach, both polyheterocyclic compounds correspond to a reagent-based approach because the new, structurally diverse polyheterocycles were synthesized from common scaffolds, first the 5-aminooxazole and then the pyrrolopyridin-5-one.A couple of new polyheterocycles were synthesized via synthetic strategies that involve combinations of MCRs with cascade processes and click reactions. Such target compounds were designed and synthesized using 4-formylbenzonitrile as a pivotal