Their structures were elucidated on the basis of spectroscopic analyses using 1D and 2D NMR techniques and HRFABMS. Compounds 1–8 were evaluated for their effects on the production of NO in LPS-activated mouse peritoneal macrophages. The NO inhibitory assay suggested that Compounds 3, 6, and 8 may be valuable as potential inhibitors of macrophage activation.Two new phragmalin-type limonoids, Carapanosins A and B ( Carapa guianensis AUBLET (Meliaceae) is a popular medicinal plant known as “Andiroba” in Brazil, and is in the same family as mahogany. Andiroba is a tall rainforest tree that grows up to 40 m in height. It is in the same family as mahogany and has been called Brazilian mahogany or bastard mahogany due to their similarities. The andiroba tree produces a brown, ligneous, quadrilateral nut that is approximately 3 to 4 in. in diameter and has the appearance of a chestnut. The nut from andiroba contains several oil-rich kernels and seeds that are composed of an ~60% pale yellow oil. The seed oil of andiroba was previously reported to exhibit highly efficient analgesic , two acetyl groups , a propanoyl , a methyl ester , two sp3 methylenes, six sp3 methines including five oxymethines , and seven sp3 quaternary carbons including four oxycarbons , and the last three displacements have already been quoted above for the orthoester. I suggest seven sp3 quaternary carbons including one with a hydroxyl attached, rather than an oxygen bridge [δC 78.4], an αβ-unsaturated δ-lactone , and a furan ring . In the 1H-1H COSY spectrum, cross peaks were observed between H-5–H-6, H2-11–H-12, H-22–H-23, and H2-2′′′′–H3-3′′′′, as shown in boldface in Carapanosin A ( spectra  exhibiteH 4.57 (s)]/C-2 [δC 78.4 (s)], C-4, C-5, C-1’ [δC 171.1 (s)]; H-6 ; H-12 [δH 4.99 (dd)]/C-11, C-13, C-14 [δC 162.7 (s)], C-17 [δC 78.7 (d)], C-18, C-1′′′ [δC 172.3 (s)]; H-15 [δH 6.06 (s)]/C-8 [δC 83.0 (s)], C-13, C-14, C-16 [δC 159.6 (s)]; H-17 [δH 5.29 (s)]/C-12 [δC 68.8 (d)], C-13, C-14, C-20 [δC 121.7 (s)], C-21 [δC 141.7 (d)], C-22 [δC 110.2 (d)]; Me-18 [δH 1.47 (s)]/C-12, C-13, C-14, C-17; Me-19 [δH 1.34 (s)]/C-1 [δC 84.5 (s)], C-5, C-9 [δC 85.2 (s)], C-10; Me-28 [δH 0.89 (s)]/C-3 [δC 83.8 (d)], C-4, C-5, C-29, H3-1′′ [δH 3.78 (s)]/C-7[δC 173.6 (s)]. The relative structure of 1 was determined on the basis of NOESY correlations ] and H-11β, H-12β and H-17β; therefore, C-6 was presumed to be in an R-configuration, which was consistent with Carapanolide N14.In the HMBC spectrum , cross pelations . Intense2), a colorless amorphous, had the molecular formula of C38H44O17  as determined by HRFABMS. The IR spectrum showed the presence of hydroxyl, ester groups, and an αβ-unsaturated δ-lactone at νmax 3566, 1734, and 1663 cm−1; and the UV spectrum indicated the presence of a furan ring and an αβ-unsaturated δ-lactone at λmax 213 nm (log ε 3.84) and 237.5 nm (log ε 3.62). The 1H- and 13C-NMR spectra ], three acetyl groups , a propanoyl group , a methyl ester , a methylene , five sp3 methines including four oxymethines , seven sp3 quaternary carbons including five oxycarbons , an α,β-unsaturated δ-lactone , and a furan ring . The 1H and 13C-NMR spectra , δC 71.2 (d)]. In the NOESY spectrum, significant NOEs were observed between H-6 and H-11α, and Me-19, so the configuration of H-6 was determined to have the same R as Compound 1 and Carapanolide N , a secondary acetoxy group , αβ-unsaturated six-membered ring ketone , an αβ-unsaturated δ-lactone , an acetal carbon [δC 104.0 (s)] , suggesting a gedunin-type limonoid. In the HMBC spectrum, the following correlations were observed: Me-18 [δH 1.16 (s)]/C-12, C-13, C-14 [δC 170.3 (s)], and C-17 [δC 104.0 (s)]; Me-19 [δH 1.25 (s)]/C-1 [δC 156.4 (d)], C-5, C-9, and C-10; Me-30 [δH 1.36 (s)]/C-7 [δC 73.2 (d)], C-8, C-9, and C-14 [δC 170.3 (s)]. The 1H-1H COSY spectrum  revealed the positions of substituents (3 shown in 9) (m.p. 270–274 °C)  from the fruits of Azadirachta indica A. Juss (Neem) , H-15, and Me-19; H-21/H-12α, H-12β, and Me-18; and Me-18/H-9α, H-12α, H-15, H-21, and H-23. Compound 3  has not yet been isolated.Carapanosin C  than L-NMMA (IC50 23.9 μM). On the other hand, Compounds 4 and 7 showed moderate activity on NO production .Macrophages may be a potential therapeutic target for inflammatory diseases . Activat1H- and 13C-NMR spectra were obtained on an Agilent vnmrs 600 spectrometer  with standard pulse sequences, operating at 600 and 150 MHz, respectively. CDCl3 was used as the solvent and TMS as the internal standard.Melting points were determined on a Yanagimoto micro-melting point apparatus and were uncorrected. Optical rotations were measured with a JASCO DIP-1000 digital polarimeter. IR spectra were recorded on a PerkineElmer 1720X FTIR spectrophotometer . UV spectra were measured on a HITACHI U-2000 spectrometer using EtOH as a solvent. 18-MS column  (25 cm × 20 mm i.d.)] and a UV detector (220 nm) with 70% MeOH (isocratic) at a flow rate 4.0 mL/min. Injector fitted with a 100 μL loop. Fractions obtained from column chromatography were monitored by TLC .FABMS were recorded on a JEOL JMS-7000 mass spectrometer . Column chromatography was performed over silica gel , while medium pressure liquid chromatography (MPLC) was conducted with silica gel . HPLC was carried out using an ODS column + (100), 671 (12), 95 (17); HR-FAB-MS m/z 731.2551 [M + H]+ .Carapanosin A (2): Colorless amorphous. c 0.05, EtOH); UV (EtOH) λmax (log ε): 237.5, 213 ; IR : 3566, 1734, 1663, 1039. FAB-MS m/z (rel.int.): 773 [M + H]+ (49), 715 (65), 699 (77), 43 (100); HR-FAB-MS m/z 773.2659 [M + H]+ .Carapanosin B (3): Colorless crystal; m.p. 236–239 °C; c 0.13, EtOH); UV (EtOH) λmax (log ε): 238.5 (log ε 3.74); IR : 3566 (OH), 1734, 1699, 1668, 1240, 1171; FAB-MS m/z (rel.int.): 505 [M + Na]+ (50), 483 [M + H]+ (77), 465 (23), 423 (52), 405 (14), 328 (25), 176 (37), 95 (100); HR-FAB-MS m/z: 483.2388 [M + H]+ .Carapanosin C (2 humidified incubator.RAW264.7 cells (mouse macrophages) ) were grown in DMEM. The medium was supplemented with 10% FBS and antibiotics (100 units/mL penicillin and 100 μg/mL streptomycin). The cells were incubated at 37 °C in a 5% CO4 cells in 100 μL) were seeded onto a 96-well microplate, and incubated for 24 h. DMEM containing test samples  dissolved in DMSO  was added. After treatment for 24 h, MTT solution was added. After a 3 h incubation, 20% sodium dodecyl sulfate in 0.1 M HCl was added to dissolve the formazan produced in the cells. The absorbance of each well was read at 570 nm using a microplate reader. The optical density of vehicle control cells was assumed to be 100%. Values are expressed as the mean ± standard error of the mean (S.E.M.). One-way ANOVA, followed by Dunnett’s test, was used for statistical analysis. Significant differences from the vehicle control (0 μM) group shown as *: p < 0.05 and **: p < 0.01.RAW264.7 cell proliferation was examined in accordance with a method reported previously . Briefly1) and B (2) as well as a gedunin-type limonoid, Carapanosin C (3) were isolated from the seeds of Carapa guianensis. Their structures were elucidated by spectroscopic analyses. In the NO inhibitory assay, Compounds 3, 6, and 8 exhibited similar NO inhibitory activities  to L-NMMA (IC50 23.9 μM) without cytotoxicity. These results suggest that Compounds 3, 6, and 8 have potential as anti-inflammatory disease agents.Two new phragmalin-type limonoids, Carapanosins A (


All structures were elucidated on the basis of spectroscopic analysis, including 1D- and 2D-NMR techniques and MS analysis. Selected isolated compounds were evaluated for in vitro cytotoxicity against four human cancer cell lines and antioxidant scavenging effects on DPPH. (−)--threo-carolignan X (3) exhibited cytotoxicity against Hep2, WiDr, Daoy, and MCF-7 cell lines with ED50 values of 3.60, 4.45, 6.07, and 13.7 μg/mL, respectively. Turformosin A (1), (−)--threo-carolignan X (3), methoxyhydroquinone-4-β-D-glucopyranoside (5), and methoxy-hydroquinone-1-β-D-glucopyranoside (6), exhibited similar anti-oxidative activity. Hep2 cells treated with 10 μg/mL of 3 showed elevation of sub-G1 population (from 20% at 8 h to 60% at 48 h), and activation of caspase-9/caspase-3/PARP cascade. Compound 3 induced intrinsic apoptotic pathway in Hep2 cells with dose and time dependence (10 μg/mL for 8 h).One new phenylpropanoid, turformosin A ( Turpinia formosana Nakai (Staphyleaceae) is an endemic plant which is widely found in the southern Taiwanese mountains. The roots have been used in Taiwanese traditional medicine to activate blood circulation, reduce swelling, and relieve pain and splenomegaly , suggested the presence of a (4-hydroxy-3-methoxyphenyl)-7,8,9-propantriol moiety in the molecule  and a propanol unit . Thus, the second aromatic unit was established as a (4-hydroxy-3-methoxyphenyl)-9'-propanol system , a methoxy group (δH 3.84), a pair of trans-vinyl groups [δH = 7.56  and 6.32 ], and an ester carbonyl carbon [δC 169.3 (C-9'')] [2B2 coupling pattern [δH 7.32  and 6.78 ] linked to a pair of trans-vinyl protons [δH 7.36  and 6.14 ], and an ester carbonyl carbon [δC 168.9 (C-9''')]. The fourth aromatic system was therefore identified as a 4'''-hydroxycinnamoyl unit. Compound rum of 1  showed fmolecule . A seconelations , this phl system . A third(C-9'')] . The rem13C-NMR --threo-carolignan X (3), except for the position of feruloyl and 4-hydroxycinnamoyl units and the stereochemistry of the 7,8,9-propantriol moiety -3'-methoxyphenoxy}-1-propanol. Due to the presence of two chiral carbons in the 7,8,9-trioxygenated propane moiety of 1, two possible diastereomers (erythro and threo) are possible for 1. Comparison of the chemical shift and coupling constant data of H-7 and H-8 with 3 and 1 indicated that 1 belongs to the erythro series -3'-methoxyphenoxy}-1-propanol and has been named as turformosin A.Compound l moiety . Inspecto series ,16. When2 was obtained as a colorless amorphous solid. A molecular ion peak at m/z 502.3290 [M]+ in the HR-ESI-MS spectrum suggested its molecular formula to be C30H46O6 . The 1H-NMR spectrum ], two hydroxymethylene groups at δH 3.48 and 3.33 , 3.19 , one olefinic proton [δH 5.24 ], and an oxygen-bearing methine at δH 3.81 . The 13C-NMR  and 144.4 (C-13), are indicative of an olen-12-en D25: +16.6 ; UV (MeOH) λmax (log ε) 229 (4.25), 289 (4.13), 317 (4.30) nm; IR (neat) vmax 3,398, 2,938, 2,839, 1,687, 1,631, 1,603, 1,508 cm−1; HR-ESI-MS m/z 723.2420 [M+Na]+ ; 1H- and 13C-NMR data, see Turformosinic acid (2): colorless amorphous solid; [α]D25: +122.3 ; IR (neat) vmax 3,384, 2,938, 2,868, 1,566, 1,550, 1,376, 1,048; HR-ESI-MS m/z 502.3290 [M]+ ; 1H- and 13C-NMR data, see 2 incubated at 37 °C. Test samples and the control drug standard were prepared at concentrations of 1, 10, 20, and 40 μg/mL. After seeding 2,880 cells/well in a 96-well microplate for 4 h, 20 μL of sample or standard agent was placed in each well and incubated at 37 °C for 3 days. Twenty μL of MTT were added, and incubation continued for 5 h. After removing the medium and adding DMSO (200 μL/well) into the microplate with shaking for 10 min, the formazan crystals (the product of MTT reacting with dehydrogenase existing in mitochondria) were re-dissolved, and their absorbance was measured on a model MR 7000 microtiter plate reader  at a wavelength of 550 nm. The ED50 was defined as the concentration of test sample resulting in 50% reduction of the absorbance found with the untreated cells.Cytotoxicity against Hep2 , WiDr (human colon adenocarcinoma), Daoy (human medulloblastoma), and MCF-7 (human breast adenocarcinoma) cells was measured using a 3--2,5-diphenyltetrazolium bromide (MTT) assay, based on reported methods . Briefly1–6, 9–16, and 18) (120 μL) were added to 30 μL of DPPH (0.75 mM). After 30 min at room temperature, the absorbance was recorded at 520 nm. The experiment was repeated three times. Radical-scavenging activity (%) was calculated by the following formula: {[Ab − (A − As)]/Ab} × 100, where Ab is the absorbance without sample, A is the absorbance with compound and DPPH, and As is the absorbance with compound only  and incubated for 10 min at 4 °C. Cells were homogenized with 15 strokes of a prechilled homogenizer, and the homogenates were sequentially centrifuged at 1,000 × g and 12,000 × g for 5 and 30 min, respectively, at 4 °C. The supernatant was collected as the cytosolic fraction. The pellet was re-suspended with ice-cold lysis buffer  and incubated for 20 min at 4 °C. The lysates were centrifuged at 15,000 × g for 5 min, and the supernatant was collected as the mitochondrial fraction.After being cultured overnight, cells were incubated with fresh medium containing compound Samples were separated by SDS-PAGE and transferred to PVDF sheets. Membranes were blocked with 5% non-fat milk dissolved in PBST buffer  for 1 h and incubated with the primary antibody at 4 °C overnight. Then, membranes were washed thoroughly with PBST buffer and incubated for 1 h with the secondary antibody at room temperature. Immunoreactive protein was visualized using Immobilon western chemiluminescent HRP substrate according to the manufacturer’s protocol . Antibodies against Bcl-2, Bcl-xL, and Bax were purchased from Santa Cruz Biotechnology . Antibodies against caspase-9 and PARP were obtained from BD Pharmingen . The anti-caspase-3 antibody was purchased from Millipore . Horse radish peroxidase (HRP)-conjugated anti-mouse IgG and HRP-conjugated anti-rabbit IgG were obtained from Jackson ImmunoResearch, Inc. .T. formosana Nakai was partitioned successively with EtOAc and n-BuOH. The EtOAc portion was fractionated by column chromatography on Sephadex LH-20 and silica gel, as well as separated by HPLC, to afford one new phenylpropanoid, turformosin A (1), one new triterpenoid, turformosinic acid (2), and two known compounds, (−)--threo-carolignan X (3) and 2α,3β,19β-23-tetrahydroxy-olean-12-en-28-oic acid (4). From the n-BuOH layer, fourteen known compounds, methoxyhydroquinone-4-β-D-glucopyranoside (5), methoxyhydroquinone-1-β-D-gluco-pyranoside (6), 3,5-dimethoxybenzyl alcohol 4-O-β-D-glucopyranoside (7), 4-(3-hydroxy-1-propenyl)-2,6-dimethoxyphenyl β-D-glucopyranoside (8), d-threo-guaiacylglycerol-O-β-D-glucopyranoside (9), -syringoylglycerol-7-O-β-D-glucopyranoside (10), 6-O-(p-hydroxybenzoyl)-D-glucose (11), d-glucose-6(4-hydroxy-3-methoxybenzoate) (12), dihydrophaseic acid 4′-O-β-D-glucopyranoside (13), 3′-O-methyl ellagic acid 4-O-β-D-xylopyranoside (14), 3-O-methylellagic acid 4-O-α-L-rhamnopyranoside (15), uridine (16), adenosine (17), and β-sitosteryl-3-O-β-D-glucopyranoside (18), were obtained. Their structures were determined based on extensive spectroscopic analyses, especially 2D NMR  as well as HRMS. On the antioxidation screening effects on DPPH for the isolated compounds, compounds 1, 3, 5, and 6 showed significant antioxidant effects. On the other hand, compound 3 exhibited most potent cytotoxicity against WiDr and Hep2 cancer cell lins with IC50 of 4.45 and 3.60 μg/mL, respectively. Compound 3 modulates intrinsic apoptotic pathway proteins in Hep2 cells, as shown by change expression of Bcl-2 family. In addition, compound 3 triggers caspase activation and also induces the release of cytochrome c and activation of caspase-9/caspase-3/PARP cascade. The EtOH extract of


The azo dyes prepared in this work may exist in three tautomeric forms. We found that the tautomerism is influenced mainly by the nature of substituent at the para position of the aniline coupling component. This tautomerisation was observed in the NMR spectra of the dyes. The dyes were characterized by IR, 1H-NMR, 13C-NMR and MS spectroscopic techniques.New 1,2,4-triazole colorants were obtained, in high yields, by coupling 3-ethylthio-5-cyanomethyl-4-phenyl-1,2,4-triazole ( Since the tautomeric ratio is important for the industrial application of azo dyes, determination of azo-hydrazone tautomerism (AHT) in the solid state and in solution is of interest both from a theoretical and practical aspects because the two tautomers have different technical properties and dyeing performance . Therefo1 with benzenediazonium chloride (2). Thus, coupling of diazonium salt 2 with compound 1 in aqueous ethanol in the presence of a buffered sodium acetate solution gave 5-ethylthio-N',4-diphenyl-4H-1,2,4-triazole-3-carbohydrazonoyl cyanide (3B), as the only isolable product, in excellent yield  and the (M+ + 1) peak were observed. In addition, the spectrum showed characteristic peaks at m/z values corresponding to C6H5NH (resulting from cleavage at the N-N bond), C8H5N3 and C10H10N3S ion fragments. The latter two fragments correspond to 4-phenyl-1,2,4-triazole and 3-ethylthio-4-phenyl-1,2,4-triazole residues, respectively. Moreover, the base peak that appeared at m/z 77 with relative intensity of 100% is due to cleavage of the phenylium cation (Ph+) from M+. Taken together the data is in good agreement with the proposed hydrazone structure. The 1H-NMR spectral data shows that two tautomeric forms 3B (hydrazone form) and 3C (azo-enamine form) are present in CDCl3 solution with relative intensities of 1:3  and the other downfield singlet at δ = 13.71 ppm [3C (75%). Tautomeric ratios were calculated from their 1H-NMR integrals by comparison of the NH signal of the hydrazone form 3B and NH signal of the azo-enamine form 3C. Therefore, 1H-NMR chemical shift data can readily be employed to study the tautomeric equilibria quantitatively. Also, the 13C-NMR spectrum of this product in CDCl3 displayed signals in agreement with the mixture of two tautomers, hydrazone form 3B and azo-enamine form 3C. The spectrum showed besides the signals due to aromatic, ethyl, cyano and triazole carbones, two characteristic signals at δ = 99.05 and 140.75 ppm attributable to the carbon atom at position 6 in both tautomeric forms 3C and 3B, respectively . Due to the novelty of this product, the 13C-NMR chemical shifts values were assigned for these carbon atoms by comparing the experimental data in the 13C-NMR spectrum of the product with the 13C-NMR chemical shifts of theoretical results for molecular modeling using ChemBio3D Ultra 12.0 [In the 8.83 ppm  is due t3.71 ppm  was assitra 12.0 .para-position of the diazonium salt benzene ring on the equilibrium between the three forms A–C , under similar reaction conditions as above, afforded 5-ethyl-thio-3-(1-(4-methylphenylazo)-4-phenyl-acetonitrile)-4H-1,2,4-triazole (5A), in 82% yield . Structure 5A seemed to be logical according to the IR spectrum (in KBr) which disclosed no amino group (NH) absorption band and the presence of intense cyano and azo (‑N=N-) [−1, respectively. The mass spectral data of azo dye 5A showed a molecular ion peak (M+) at m/z 362 (40%) which was in concordance with the molecular mass (362) of the product (C19H18N6S). In addition, cleavage at one of the C-N bonds in the azo tautomer 5A led to the appearance of the base peak at m/z 91 (CH3-C6H4) with relative intensity of 100%. Moreover, the spectrum showed characteristic peaks at m/z 119 (14%), 143 (6%), 156 (11%) and 243 (4%) corresponding to CH3-C6H4-N=N (resulting from cleavage at the CN bond), C8H5N3 , C8H5N3-CH , and C10H10N3S-CH-CN  residues, respectively. This would suggest that compound 5A exist almost exclusively in the azo form. Interestingly, the 1H-NMR spectral data shows that the hydrazone form 5B and azo-enamine tautomeric form 5C are present in CDCl3 solution with relative intensities of 1:4 . Also, the spectrum do not show any more signals around 4.5 ppm which is usually reported for the methine proton of azo form 5A of the annulated similar compounds [13C-NMR spectrum of this product in CDCl3 showed signals in accordance with the mixture of two tautomers, hydrazone form 5B and azo-enamine form 5C. The spectrum showed besides the signals due to aromatic, ethyl, methyl, cyano and triazole carbones, two characteristic signals at δ = 98.34 and 139.57 ppm attributable to carbon atom at position 6 in both tautomeric forms 5C and 5B, respectively .The structure of this azo dye was verified by elemental analyses and spectroscopic methods   bands ats of 1:4 , Table 1ompounds . In addipara position of benzene ring with compound 1. Surprisingly, when compound 1 was coupled with diazotized 4-chloroaniline (6), under the same reaction conditions as above, it afforded two tautomers, 7A (major product) and 8B (minor product)  , which r7A was supported by spectroscopic data. For example, its mass spectrum showed a molecular formula C18H15ClN6S (M+ 382) and peaks at 139  and 111  confirming its presumed structure . The IR spectrum showed no amino group (NH) absorption, but absorption bands for CN and -N=N- groups were observed at v = 2217 and 1547 cm−1, respectively. Interestingly, the 1H-NMR spectral data shows that the azo-enamine tautomeric form 7C and hydrazone form 7B are present in CDCl3 solution with relative intensities of 1:1.5 . The structure of the minor product 8B was fully confirmed with the help of analytical and spectroscopic data. Particularly, the IR spectrum showed an amino group (NH) absorption band. Moreover, its structure is supported by its mass spectrum which showed the molecular ion peak at m/z 382 (29%), which is consistent with its structural formula. Other prominent peaks that observed at m/z 126  and 111  confirming its presumed structure . However, the 1H-NMR spectrum revealed two singlet signals for the N-H proton of the hydrazone form 8B and the triazole N-H in the azo-enamine form 8C with relative intensities of 1:5 . Unfortunately, we did not succeed in growing the single crystal of compounds 3, 5, 7 and 8 suitable for X-ray crystallographic analysis.To the best of our knowledge, this is the first reported isolation of two isomers in solid state in such reactions. The identity of major product of 1:1.5 , Table 1d 8C cf. . Also, t−1. 1H and 13C-NMR spectra were recorded on a Bruker DPX (600 MHz for 1H-NMR and 150 MHz for 13C-NMR) spectrometer in CDCl3 using TMS as an internal standard; the chemical shifts are given in δ units (ppm). Mass spectra were performed on a Shimadzu GCMS-QP 1000 EX mass spectrometer at 70 eV. Analytical thin-layer chromatography (TLC) was performed on aluminum sheets precoated with silica gel . Visualization was accomplished by UV light. Microanalytical data were obtained from the Microanalytical Data Unit at Cairo University, Egypt.Melting points were measured on a Gallenkamp apparatus and are not corrected. IR spectra (KBr) were recorded with a Nicolet Magna 520FT IR spectrophotometer. Peaks are reported in cm2 and 4 in 2 mL of 6 M hydrochloric acid) with continuous stirring. The resulting solution of the aryl diazonium salt was then added carefully to a cold solution of 1,2,4-triazole derivative 1 (4 mmol) in ethanol (40 mL) containing sodium acetate (0.66 g in 2 mL H2O). The reaction mixture was stirred at room temperature for 24 h and the resulting solid product was collected by filtration, washed well with H2O and dried to afford compounds 3B and 5A, respectively, which were purified by preparative TLC using silica gel plates , followed by recrystallization from EtOH. In the case of the reaction of 1 with 6, the resulting solid product was chromatographed on a preparative TLC plate using 10:3 toluene/acetone as eluent to give two zones. Extraction with acetone followed by recrystallization from EtOH gave compounds 7A and 8B, respectively.A cold solution of aryldiazonium salt (4 mmol) was prepared by adding a sodium nitrite solution  to a pre-cooled solution of arylamine hydrochloride -2-(phenylhydrazono)acetonitrile (3B). Yellow crystals. Yield ; m.p.: 172–174 °C. IR (KBr): v = 3236 (NH), 2937 , 2213 (CN), 1594 (C=N), 1231 (N-N) cm−1; 1H-NMR: δ = 1.44 , 1.48 , 3.28–3.34 , 6.38 , 6.99 , 7.12–7.15 , 7.31–7.32 , 7.35–7.42 , 7.59–7.63 , 7.65–7.68 , 8.83 , 13.71 ; 13C-NMR: δ = 14.65 (CH3), 14.67 (CH3), 26.61 (CH2), 26.75 (CH2), 99.05 (=C-CN in azo-enamine form), 114.27 (CN), 114.30 (CN), 115.53 (3 Ar-C), 123.97 (1 Ar-C), 124.80 (2 Ar-C), 127.35 (1 Ar-C), 128.20 (3 Ar-C), 129.27 (1 Ar-C), 129.53 (3 Ar-C), 129.82 (1 Ar-C), 130.09 (3 Ar-C), 131.54 (2 Ar-C), 131.59 (1 Ar-C), 134.54 (1 Ar-C), 140.75 (CN-C=N-NH in hydrazone form), 141.81 (2 Ar-C), 148.47 (triazole C-3), 148.60 (triazole C-3), 154.14 (triazole C-5), 154.97 (triazole C-5); MS m/z (rel. int. %) 349 , 348 , 347 (22), 320 (9), 319 (7), 290 (4), 244 (3), 243 (4), 242 (6), 215 (9), 204 (2), 155 (6), 149 (6), 143 (3), 129 (6), 128 (5), 105 (19), 92 (6), 91 (8), 77 (100), 76 (67), 65 (20), 64 (14), 63 (7), 61 (6), 52 (5), 51 (26), 50 (14); Anal. Calcd. for C18H16N6S (348.42): C, 62.05; H, 4.63; N, 24.16; S, 9.20. Found: C, 61.91; H, 4.78; N, 24.35; S, 9.06.2--2-(4-methylphenyldiazenyl)acetonitrile (5A). Yellow crystals. Yield ; m.p.: 169–170 °C. IR (KBr): v = 2980, 2920 , 2217 (CN), 1594 (C=N), 1547 (-N=N-) cm−1; 1H-NMR: δ = 1.43 , 1.47 , 2.26 , 2.34 , 3.28-3.34 , 6.27 , 6.94 , 7.18 , 7.30-32 , 7.35–7.37 , 7.59–7.63 , 7.65–7.67 , 8.78 ; 13.70 ; 13C-NMR: δ = 14.66 (CH3), 14.68 (CH3), 20.70 (CH3), 20.92 (CH3), 26.60 (CH2), 26.76 (CH2), 98.34 (=C-CN in azo-enamine form), 114.21 (CN), 114.50 (CN), 115.48 (4 Ar-C), 127.37 (1 Ar-C), 128.22 (3 Ar-C), 129.78 (2 Ar-C), 130.04 (2 Ar-C), 130.07 (4 Ar-C), 131.50 (2 Ar-C), 131.64 (1 Ar-C), 133.75 (1 Ar-C), 134.58 (1 Ar-C), 134.64 (2 Ar-C), 138.48 (1 Ar-C), 139.57 (CN-C=N-NH in hydrazone form), 148.55 (triazole C-3), 148.73 (triazole C-3), 153.97 (triazole C-5), 154.75 (triazole C-5); MS m/z (rel. int. %) 363 , 362 , 361 (21), 348 (5), 347 (6), 346 (8), 345 (6), 335 (6), 334 (16), 333 (10), 306 (9), 305 (11), 274 (4), 273 (5), 257 (5), 243 (4), 242 (6), 231 (4), 230 (5), 215 (14), 188 (4), 157 (8), 156 (11), 149 (11), 148 (7), 144 (6), 143 (6), 128 (8), 119 (14), 118 (13), 117 (9), 106 (8), 105 (22), 104 (13), 103 (11), 97 (6), 92 (14), 91 (100), 90 (26), 77 (49), 76 (17), 66 (6), 65 (27), 64 (21), 63 (12), 61 (6), 60 (9), 59 (13), 56 (9), 51 (26); Anal. Calcd. for C19H18N6S (362.45): C, 62.96; H, 5.01; N, 23.19; S, 8.85. Found: C, 63.14; H, 4.87; N, 23.30; S, 9.01.2-(4-Chlorophenyldiazenyl)-2-acetonitrile (7A). Yellow crystals. Yield ; m.p.: 118–120 °C. IR (KBr): v = 2925 , 2217 (CN), 1597 (C=N), 1547 (-N=N-) cm−1; 1H-NMR: δ = 1.43 , 1.48 , 3.27–3.35 , 6.30 , 7.10 , 7.30–7.36 , 7.57–7.68 , 8.91 , 13.74 ; 13C-NMR: δ = 14.65 (2 CH3), 26.62 (CH2), 26.76 (CH2), 99.68 (=C-CN in azo-enamine form), 108.93 (CN), 114.02 (CN), 115.46 (2 Ar-C), 116.65 (2 Ar-C), 127.33 (3 Ar-C), 128.17 (2 Ar-C), 128.99 (1 Ar-C), 129.28 (4 Ar-C), 129.61 (1 Ar-C), 129.85 (1 Ar-C), 130.12 (4 Ar-C), 131.48 (1 Ar-C), 131.61 (1 Ar-C), 134.54 (1 Ar-C), 139.48 (1 Ar-C), 140.44 (CN-C=N-NH in hydrazone form), 148.30 (triazole C-3), 148.49 (triazole C-3), 154.37 (triazole C-5), 155.11 (triazole C-5); MS m/z (rel. int. %) 384 , 382 , 381 (20), 356 (4), 355 (7), 354 (13), 353 (15), 352 (5), 328 (7), 327 (10), 326 (13), 325 (17), 324 (7), 293 (7), 244 (4), 243 (9), 242 (15), 241 (7), 232 (3), 231 (3), 215 (15), 214 (10), 213 (7), 192 (3), 191 (8), 183 (4), 182 (6), 181 (7), 167 (5), 157 (10), 156 (25), 155 (13), 149 (12), 148 (5), 143 (3), 142 (6), 141 (9), 140 (4), 139 (17), 138 (11), 129 (13), 128 (12), 127 (10), 126 (11), 125 (9), 119 (5), 118 (17), 113 (32), 112 (17), 111 (100), 110 (78), 105 (23), 104 (15), 103 (12), 102 (13), 100 (6), 99 (16), 92 (8), 91 (30), 87 (3), 78 (11), 77 (73), 76 (31), 75 (37), 66 (4), 65 (23), 64 (17), 63 (24), 62 (9), 61 (14), 60 (10), 52 (9), 51 (42), 50 (26); Anal. Calcd. for C18H15ClN6S (382.87): C, 56.47; H, 3.95; Cl, 9.26; N, 21.95; S, 8.37. Found: C, 56.66; H, 3.83; Cl, 9.40; N, 22.04; S, 8.54.2-(4-Chlorophenylhydrazono)-2-acetonitrile (8B). Reddish crystals. Yield ; m.p.: 219–221 °C. IR (KBr): v = 3240 (NH), 2924 , 2220 (CN), 1595 (C=N), 1233 (N-N) cm−1; 1H-NMR: δ = 1.44 , 1.48 , 3.28–3.37 , 6.29 , 7.10 , 7.31–7.38 , 7.57–7.69 , 8.79 , 13.74 ; 13C-NMR: δ = 14.69 (2 CH3), 26.65 (CH2), 26.78 (CH2), 99.71 (=C-CN in azo-enamine form), 114.05 (CN), 115.45 (CN), 116.68 (3 Ar-C), 127.36 (1 Ar-C), 128.20 (4 Ar-C), 129.02 (1 Ar-C), 129.34 (1 Ar-C), 129.65 (4 Ar-C), 129.87 (1 Ar-C), 129.93 (1 Ar-C), 130.16 (4 Ar-C), 131.52 (1 Ar-C), 131.64 (2 Ar-C), 139.44 (1 Ar-C), 140.47 (CN-C=N-NH in hydrazone form), 148.30 (triazole C-3), 148.52 (triazole C-3), 154.40 (triazole C-5), 155.05 (triazole C-5); MS m/z (rel. int. %) 384 , 382 , 381 (11), 362 (3), 356 (3), 355 (3), 354 (5), 353 (4), 352 (2), 348 (3), 328 (3), 327 (4), 326 (4), 325 (7), 324 (2), 319 (6), 317 (8), 316 (5), 293 (3), 244 (2), 243 (3), 242 (7), 241 (3), 231 (2), 215 (7), 214 (4), 213 (2), 157 (4), 156 (9), 155 (6), 149 (4), 143 (2), 142 (4), 141 (9), 140 (4), 139 (29), 129 (5), 128 (8), 127 (5), 126 (12), 125 (7), 119 (2), 118 (6), 113 (31), 112 (20), 111 (100), 110 (27), 105 (8), 104 (4), 103 (4), 102 (5), 101 (8), 100 (3), 99 (17), 91 (13), 90 (12), 87 (3), 78 (4), 77 (31), 76 (17), 75 (36), 74 (13), 66 (2), 65 (9), 64 (12), 63 (17), 62 (6), 61 (7), 60 (4), 52 (5), 51 (23), 50 (17); Anal. Calcd. for C18H15ClN6S (382.87): C, 56.47; H, 3.95; Cl, 9.26; N, 21.95; S, 8.37. Found: C, 56.61; H, 4.04; Cl, 9.33; N, 21.87; S, 8.48.para-position of the diazonium salt benzene ring have some effect on the ratio of the resulting tautomers. 5-ethylthio-N',4-diphenyl-4H-1,2,4-triazole-3-carbohydrazonoyl cyanide (3B) (hydrazone form) was obtained by coupling 1 with benzenediazonium salt 2, while the azo dye 5A was obtained by coupling 1 with diazotized 4-methylaniline (4). Interestingly, coupling of 1 with diazotized 4-chloroaniline (6) afforded two isomeric products, 7A (azo form) and 8B (hydrazone form). To the best of our knowledge, this is the first reported isolation of two isomers in the solid state in such reactions. Analysis of the 1H-NMR data shows that the hydrazone and azo-enamine forms are the only two tautomers present in CDCl3 solution and the ratio of these tautomers depends on the electron-donating and electron-withdrawing properties of the substituent present at the para-position of the aryldiazonium salt.In conclusion, we have synthesized new azo dyes utilizing 3-ethylthio-5-cyanomethyl-4-phenyl-1,2,4-triazole as a coupling component. The experimental results show that the substituents at the


Here, we report the design, synthesis, pharmacological and in vitro absorption, distribution, metabolism, excretion, toxicity (ADMET) properties of a series of compounds related to 4. Starting from 4, a series of analogs were structurally modified and their ERRγ inverse agonist activity was measured. A key pharmacophore feature of this novel class of ligands is the introduction of a heterocyclic group for A-ring substitution in the core scaffold. Among the tested compounds, several of them are potent ERRγ inverse agonists as determined by binding and functional assays. The most promising compound, 15g, had excellent binding selectivity over related subtypes . Compound 15g also resulted in 95% transcriptional repression at a concentration of 10 μM, while still maintaining an acceptable in vitro ADMET profile. This novel class of ERRγ inverse agonists shows promise in the development of drugs targeting ERRγ-related diseases.GSK5182 ( Estrogen-related receptors (ERRs), comprising ERRα, ERRβ, and ERRγ, are constitutively active nuclear receptors closely related to the estrogen receptors (ER), ERα and ERβ, with high levels of sequence identity . They ar1, 4-OHT) [2, DES) [3), was reported as an ERRγ agonist with improved binding selectivity over the classical ER, yet expressed mixed ERRγ/ERRβ functional activities in cell-based assay systems [4) was developed as a 4-OHT (1) analog that showd better selectivity vs. the structurally related nuclear receptor ERα [Based on these findings, ERRγ has emerged as a promising target in the treatment of certain metabolic disorders and cancers. However, despite its biological versatility, only a handful of studies examining ERRγ ligands that modulate downstream signals have been performed in the past two decades. Consequently, to date, there are few synthetic compounds targeting ERRγ with interesting pharmacological activity . The fir, 4-OHT)  and diet[2, DES) , were id systems . This maptor ERα .4 alleviates diabetes through the inhibition of hepatic gluconeogenesis in a PGC-1α-dependent manner [4 has antimicrobial effects by reducing ERRγ-mediated hepcidin mRNA expression [4 enhances the responsiveness of radioiodine therapy by modulating sodium iodide symporter (NIS) function in ATC cells via the regulation of ERRγ and the MAP kinase signaling pathway [Several lines of evidence have revealed that the ERRγ inverse agonist t manner . Additiopression . More re pathway .In spite of these findings, the discovery of newly synthesized ERRγ ligands displaying better selectivity and potency is required not only to better understand the essential biological roles of ERRγ but also to eventually develop novel therapeutic agents. Therefore, the purpose of this study was to identify new ERRγ inverse agonists with increased potency and selectivity than those currently available.in vitro binding and functional characterization of novel ERRγ selective inverse agonists bearing comparable drug-like properties to those of 4 on the basis of in vitro ADMET considerations. Inspired by the early docking studies suggesting that the A-ring of 4 had structural binding flexibility in the ERRγ binding pocket, our initial strategy was focused on synthesizing A-ring analogs of 4. Thus, the lead optimization efforts were focused on modification of the A-ring phenyl group with various hydroxyaryl and heterocyclic substituents, in order to improve the in vitro ADMET profile good enough for subsequent in vivo assessment.In this article, we describe the discovery and 13a–c were synthesized according to the well-established methodology detailed in 8 was prepared in three steps by employing Wittig chemistry [8 and the corresponding boronic acids/esters was then performed to attach various aromatic groups to the A-ring (9a–c). Compounds 11a–c were derived by reduction of the methyl ester group with LiAlH4, which were subsequently treated with BBr3 to give compounds 12a–c. A Williamson ether synthesis was carried out with 2-chloro-N,N-dimethylethylamine to provide compounds 13a–c after further purification using preparative HPLC from a mixture of E/Z isomers and undesired di-alkylated product  among those was applied for the next reaction.Compounds  product . The brotography  and only15a–j were prepared using a cross-coupling reaction between 14 and the corresponding boronic acids/esters, along with reduction of the methyl ester group with LiAlH4 or DIBAL-H  led to a significant loss of affinity, while among the bicyclic system-adopted compounds 15g–j, an analog with the Me-indazole group (compound 15g) displayed decent binding affinity.While exploring the effects of R1 substitutions on binding affinity in the screening campaign of compounds 13b,c, and 17). The –OMe series showed no affinity at the maximum concentration of the assay (10 μM); the only notable difference occurred with compound 13a,which had a slightly improved affinity (IC50 = 0.99 μM at the ERRγ). Furthermore, by comparing compound 13a with 16, we determined that –CF3 substitution to the ortho position of the N,N-dimethylaminoethoxy group resulted in no affinity at the screening concentration.Incorporation of a methoxy group as a terminal –OH substitution resulted in significant loss of affinity in several heteroaryl-bearing compounds (50 > 10 μM) were found to be moderate  or to inactivate ; transcriptional repression of 13b,c, 15c, 17 at the screening concentration of 10 μM is around 50% and that of 15a,i, 16 is around 0%. However, compounds 15f,h deviated from the trend established by other compounds with poor activity in the binding assay, showing partial agonistic activity in the cellular system. Nonetheless, compounds 13a, 15e,g displayed comparable regression of the ERRγ transcriptional activity compared with the control ligand, 4.Along with the binding assay campaign, functional activity was determined in order to validate the utility of the binding affinity in a cellular context as described in the Experimental . The res15g to ERRγ. The docking study revealed that compound 15g may bind to the active site via hydrogen bonding with Asp 273, Glu 275, and Asn 346 of ERRγ, respectively. The docking study results suggest that compound 15g overlaps the active site of ERRγ in a similar manner as 4 . CYP inhibition was determined against five major subtypes, including 1A2, 2C9, 2C19, 2D6, and 3A4, which are primarily involved in drug metabolism. Compound 15g maximally inhibited the CYP 2C9 subtype by 17% at a concentration of 10 μM and showed nearly no inhibition against other subtypes. In comparison, compound 4 generally inhibits the CYP enzymes by 15%–27%  (Compound  >30 μM) .3 or d-MeOH and the spectra were acquired at 25 °C. In the 2D experiments, the 1H chemical shifts (δ) were referenced to the residual solvent peak: d-MeOH, δ = 3.31 (1H), δ = 49.2 (13C). 13C-heteronuclear single-quantum-correlated (HSQC) and 13C-heteronuclear multiple-bond-correlated (HMBC) spectra were recorded for the assignment of carbon and proton resonances. The 1H-1H 2D NOESY with mixing times of 300 ms, 600 ms were performed to obtain information on proton distance. Mass spectra were measured in positive electrospray ionization (ESI) mode on LCMS-2020 system . Column chromatography was performed using a CombiFlash® Rf system with RediSep® Rf . For the final compounds, further purification was performed by preparative HPLC on Kinetex® 5 μm Biphenyl 100 Å  with ACN/H2O as eluent. The purity of the target compounds was determined to be >95% by analytical HPLC using dual different wavelength UV detector. Starting materials were obtained from Aldrich , or Alfa Aesa . Solvents were obtained from Fisher Scientific  or Aldrich and were used without further purification unless noted otherwise.All NMR experiments were carried out using an Avance III 400 MHz NMR spectrometer equipped with a 5-mm broadband-observed probe head . The NMR spectrum optimization was conducted using the Bruker Topspin 3.1 software, and all parameters were set in it. The compounds were dissolved in CDCl2Cl2. The organic extracts were discarded and the aqueous layer was acidified with 12 N HCl and extracted with CH2Cl2. The organic layer was washed with brine (2 times) and dried over MgSO4. The crude compound was purified by column chromatography to give the desired product .In an inert atmosphere, NaH  was added in dry DMSO (50 mL). The mixture was stirred at 70 °C for 1 h. The mixture was cooled to 20 °C and 4-(bromotriphenylphosphoranyl)-butanoic acid  was added in several portions over 5 min. The red solution was stirred at 20 °C for 15 min, then a solution of bis(4-methoxyphenyl)methanone  in dry THF (33 mL) was added at such a rate. The mixture was stirred at rt for 19 h, then it was diluted with ice water and extracted with CH2  was added dropwise. The reaction mixture was heated to reflux for 2 h. The reaction mixture was removed and added water. The mixture was extracted with EtOAc and washed with aq. K2CO3, brine and dried over MgSO4. The solvent was removed under reduced pressure which was used for the next step without further purification .To a solution of 5,5-bis(4-methoxyphenyl)pent-4-enoic acid  in MeOH (60 mL) stirred at 0 °C, SOCl4 (45 mL) was added copper(II) bromide  at rt. The reaction mixture was heated to reflux at 83 °C for 12 h, cooled to room temperature and filtered off through a Celite pad. The solvent was removed and reaction mixture was dissolved in EtOAc. The mixture was washed with water, brine and dried over MgSO4. The crude compound was purified by column chromatography to give the brown oil product .To a solution methyl 5,5-bis(4-methoxyphenyl)pent-4-enoate  in CCl8 (1 eq) in DMF/H2O (50:1) was added corresponding boronic esters or boronic acid (1.2 eq), PdCl2(dppf)·CH2Cl2 (0.1 eq) and K3PO4 (3 eq) at rt. The reaction mixture was heated to 82 °C for 12 h. The reaction mixture was cooled to rt and was quenched by adding water. The mixture was extracted with EtOAc and washed with brine and dried over MgSO4, filtered, and concentrated. The residue was purified by column chromatography to give the desired product.To a solution Methyl 5,5-bis(4-methoxyphenyl)-4-(pyridin-3-yl)pent-4-enoate (9a). . 1H-NMR  δ 8.34 , 7.45 , 7.12 , 6.89 , 6.76 , 6.56 , 3.83 , 3.68 , 3.56 , 2.83 , 2.32 .Methyl 5,5-bis(4-methoxyphenyl)-4-(pyridin-4-yl)pent-4-enoate (9b). . 1H-NMR  δ 8.40 , 7.12 , 7.03 , 6.89 , 6.76 , 6.58 , 3.83 , 3.70 , 3.57 , 2.82 , 2.30 .Methyl 5,5-bis(4-methoxyphenyl)-4-(1-methyl-1H-pyrazol-4-yl)pent-4-enoate (9c). . 1H-NMR  δ 7.07 , 7.03 , 6.98 , 6.87 , 6.84 , 6.73 , 3.80 , 3.76 , 3.75 , 3.61 , 2.71 , 2.46 .4 solution in THF (1.5 eq) stirred at 0 °C, 9a–c (1 eq) in THF was added dropwise. The solution was stirred at 0 °C for 1 h and was quenched by adding H2O/5 N NaOH/H2O (1:1:3). The reaction mixture was filtered off through celite pad, washed with EtOAc and concentrated in vacuo. The crude compound was purified by column chromatography to give the desired product.To a solution of 1 M LiAlH5,5-bis(4-Methoxyphenyl)-4-(pyridin-3-yl)pent-4-en-1-ol (10a). . 1H-NMR  δ 8.35 , 8.33 , 7.43 , 7.16 , 7.10 , 6.89 , 6.77 , 6.56 , 3.83 , 3.68 , 3.54 , 2.56 , 1.60 .5,5-bis(4-Methoxyphenyl)-4-(pyridin-4-yl)pent-4-en-1-ol (10b). . 1H-NMR  δ 8.37 , 7.14 , 7.03 , 6.89 , 6.78 , 6.58 , 3.83 , 3.70 , 3.55 , 2.55 , 1.59 .5,5-bis(4-Methoxyphenyl)-4-(1-methyl-1H-pyrazol-4-yl)pent-4-en-1-ol (10c). . 1H-NMR  δ 7.11 , 7.07 , 6.99 , 6.85 , 6.83 , 6.74 , 3.80 , 3.76 , 3.74 , 3.58 , 2.46 , 1.75 .10a–c (1 eq) in CH2Cl2 stirred at 0 °C, 1 M BBr3 solution in CH2Cl2 (10 eq) was added dropwise. The solution was refluxed at 80 °C for 12 h and was quenched by adding H2O at 0 °C. The reaction mixture was diluted with EtOAc and washed with H2O and brine. The organic layer was dried MgSO4 and concentrated in vacuo. The residue was purified by column chromatography to give the desired product.To a solution of 4,4′-(5-Bromo-2-(pyridin-3-yl)pent-1-ene-1,1-diyl)diphenol (11a). . 1H-NMR  δ 9.53 , 9.35 , 8.59 , 8.51 , 8.24 , 7.81 , 7.02 , 6.77 , 6.66 , 6.49 , 3.44 , 2.64 , 1.82 .4,4′-(5-Bromo-2-(pyridin-4-yl)pent-1-ene-1,1-diyl)diphenol (11b). . 1H-NMR  δ 9.59 , 9.47 , 8.61 , 7.60 , 7.02 , 6.78 , 6.66 , 6.50 , 3.43 , 2.66 , 1.79 .4,4′-(5-Bromo-2-(1-methyl-1H-pyrazol-4-yl)pent-1-ene-1,1-diyl)diphenol (11c). . 1H-NMR  δ 7.27 , 6.92 , 6.81 , 6.71 , 6.69 , 6.62 , 3.68 , 3.44 , 2.41 , 1.91 .11a–c (1 eq) in MeOH stirred at 0 °C, 5 M NaOCH3 solution in MeOH (10 eq) was added dropwise. The solution was stirred at rt for 12 h and quenched by adding 6 N HCl at 0 °C. The reaction mixture was diluted with MeOH and filtered. The residue was purified by column chromatography to give the desired product.To a solution of 4,4′-(5-Methoxy-2-(pyridin-3-yl)pent-1-ene-1,1-diyl)diphenol (12a). . 1H-NMR  δ 9.53 , 9.43 , 8.60 , 8.51 , 8.23 , 7.81 , 7.02 , 6.78 , 6.65 , 6.49 , 3.21 , 3.17 , 2.55 , 1.50 .4,4′-(5-Methoxy-2-(pyridin-4-yl)pent-1-ene-1,1-diyl)diphenol (12b). . 1H-NMR  δ 9.60 , 9.48 , 8.62 , 7.63 , 7.01 , 6.78 , 6.65 , 6.51 , 3.21 , 3.13 , 2.58 , 1.47 .4,4′-(5-Methoxy-2-(1-methyl-1H-pyrazol-4-yl)pent-1-ene-1,1-diyl)diphenol (12c). . 1H-NMR  δ 7.24 , 6.92 , 6.79 , 6.69 , 6.67 , 6.61 , 3.68 , 3.21 , 3.14 , 2.30 , 1.59 .12a–c (1 eq) in acetone/H2O (10:1) was added 2-chloro-N,N-dimethylethylamine hydrochloride (2 eq) and K2CO3 (2 eq). The mixture was refluxed at 63 °C for 4 h and concentrated under reduced pressure. The crude E/Z mixture (1:1) was purified by preparative HPLC using eluent A (0.1% TFA H2O) and eluent B (0.1% TFA ACN) (A/B = 70/30) to give the desired Z isomer product.To a solution of (Z)-4-(1-(4-(2-(Dimethylamino)ethoxy)phenyl)-5-methoxy-2-(pyridin-3-yl)pent-1-en-1-yl)phenol (13a). . 1H-NMR  δ 8.23 , 8.18 , 7.68 , 7.28 , 7.16 , 6.96 , 6.68 , 6.46 , 4.14 , 3.28 , 3.21 , 2.81 , 2.57 , 2.39 , 1.58 . MS m/z (ESI) 433.2 (M + 1)+ .(Z)-4-(1-(4-(2-(Dimethylamino)ethoxy)phenyl)-5-methoxy-2-(pyridin-4-yl)pent-1-en-1-yl)phenol (13b). . 1H-NMR  δ 8.32 , 7.21 , 7.17 , 6.99 , 6.72 , 6.50 , 4.18 , 3.30 , 3.25 , 2.87 , 2.60 , 2.43 , 1.60 . MS m/z (ESI) 433.2 (M + 1)+ .(Z)-4-(1-(4-(2-(Dimethylamino)ethoxy)phenyl)-5-methoxy-2-(1-methyl-1H-pyrazol-4-yl)pent-1-en-1-yl)-phenol (13c). . 1H-NMR  δ 7.16 , 7.09 , 6.91 , 6.90 , 6.85 , 6.63 , 4.11 , 3.73 , 3.35 , 3.25 , 2.80 , 2.42 , 2.37 , 1.70 . MS m/z (ESI) 436.3 (M + 1)+ .(Z)-Methyl 4-bromo-5-(4-(2-(dimethylamino)ethoxy)phenyl)-5-(4-hydroxyphenyl)pent-4-enoate (14). To a solution of 8  in CH2Cl2 (80 mL) was added 1M BBr3 in CH2Cl2  was added dropwise at 0 °C. The mixture was stirred at rt for 3 h and quenched by adding sat. NaHCO3 solution at 0 °C. The mixture was extracted with CH2Cl2 and extracted with CH2Cl2. The combined organic layer was concentrated under reduced pressure and crude residue was purified by column chromatography. . To a solution of 2-(dimethylamino)ethanol  and triphenylphosphine  in CH2Cl2 (50 mL) stirred at 0 °C. DIAD  was added dropwise. After 10 min, a solution of methyl 4-bromo-5,5-bis(4-hydroxyphenyl)pent-4-enoate  in CH2Cl2 (50 mL) was added slowly to the reaction mixture at 0 °C. The reaction mixture was stirred for 12 h at rt and was removed under reduced pressure. The crude E/Z mixture (1:1) was purified by column chromatography. . 1H-NMR  δ 7.18 , 6.98 , 6.72 , 4.34 , 3.63 , 3.58 , 2.98 , 2.87 , 2.62 .(E)-Methyl 4-bromo-5-(4-(2-(dimethylamino)ethoxy)phenyl)-5-(4-hydroxyphenyl)pent-4-enoate. 1H-NMR  δ 7.13 , 7.01 , 6.70 , 4.36 , 3.63 , 3.59 , 2.98 , 2.82 , 2.62 .14 (1 eq) in DMF was added corresponding boronic esters or boronic acid (1.5 eq), Pd(dppf)Cl2·CH2Cl2 (0.1 eq) and 2 M Na2CO3 (3 eq). The mixture was refluxed at 85 °C for 4 h. The reaction mixture was cooled to rt and was quenched by adding water. The mixture was extracted with EtOAc and dried over MgSO4. The crude compound was purified by column chromatography. The obtained product was dissolved in THF and added 1 M LiAlH4 in THF (1.5 eq) dropwise at 0 °C. The solution was stirred at 0 °C for 1 h and was quenched by adding H2O/5 N NaOH/H2O (1:1:3). The reaction mixture was filtered off through a Celite pad, washed with EtOAc and concentrated in vacuo. The crude compound was purified by preparative HPLC using eluent A (0.1% TFA H2O) and eluent B (0.1% TFA ACN) (A/B = 70/30) to give the desired product.To a solution of (E)-4-(1-(4-(2-(Dimethylamino)ethoxy)phenyl)-5-hydroxypent-1-en-1-yl)phenol (15a). . 1H-NMR  δ 7.14 , 6.94 , 6.86 , 6.78 , 5.94 , 4.20 , 3.53 , 3.16 , 2.65 , 2.16 , 1.64 . MS m/z (ESI) 342.2 (M + 1)+ .(Z)-4,4′-(1-(4-(2-(Dimethylamino)ethoxy)phenyl)-5-hydroxypent-1-ene-1,2-diyl)diphenol (15b). . 1H-NMR  δ 7.15 , 6.99 , 6.92 , 6.82 , 6.75 , 6.66 , 6.58 , 6.43 , 4.32 , 4.18 , 3.47 , 3.40 , 2.89 , 2.84 , 2.46 , 1.55 . MS m/z (ESI) 434.2 (M + 1)+ .(Z)-4-(1-(4-(2-(Dimethylamino)ethoxy)phenyl)-5-hydroxy-2-(6-methoxypyridin-3-yl)pent-1-en-1-yl)phenol (15c). . 1H-NMR  δ 7.77 , 7.51 , 7.17 , 7.01 , 6.84 , 6.77 , 6.69 , 6.48 , 4.30 , 4.17 , 3.83 , 3.82 , 3.44 , 3.39 , 3.32 , 2.83 , 2.78 , 2.51 , 1.56 . MS m/z (ESI) 449.2 (M + 1)+ .(Z)-4-(1-(4-(2-(Dimethylamino)ethoxy)phenyl)-5-hydroxy-2-(pyridin-3-yl)pent-1-en-1-yl)phenol (15d). . 1H-NMR  δ 8.21 , 8.16 , 7.68 , 7.28 , 7.04 , 6.78 , 6.64 , 4.00 , 3.43 , 2.81 , 2.58 , 2.38 , 1.55 . MS m/z (ESI) 419.2 (M + 1)+ .(Z)-4-(1-(4-(2-(Dimethylamino)ethoxy)phenyl)-2-(2-fluoropyridin-4-yl)-5-hydroxypent-1-en-1-yl)phenol (15e). . 1H-NMR  δ 7.93 , 7.15 , 7.03 , 6.96 , 6.81 , 6.70 , 6.50 , 4.16 , 4.04 , 3.44 , 2.92 , 2.86 , 2.58 , 2.46 , 2.42 , 1.54 . MS m/z (ESI) 437.2 (M + 1)+ .(Z)-4-(1-(4-(2-(Dimethylamino)ethoxy)phenyl)-5-hydroxy-2-(6-morpholinopyridin-3-yl)pent-1-en-1-yl)phenol (15f). . 1H-NMR  δ 8.54 , 7.76 , 7.42 , 7.00 , 6.84 , 6.75 , 6.67 , 4.09 , 3.75 , 3.42 , 3.37 , 3.07 , 2.58 , 2.49 , 1.56 . MS m/z (ESI) 504.3 (M + 1)+ .(Z)-4-(1-(4-(2-(Dimethylamino)ethoxy)phenyl)-5-hydroxy-2-(1-methyl-1H-indazol-6-yl)pent-1-en-1-yl)phenol (15g). . 1H-NMR  δ 7.87 , 7.50 , 7.30 , 7.06 , 6.94 , 6.83 , 6.78 , 6.60 , 4.06 , 3.93 , 3.43 , 3.11 , 2.64–2.61 , 1.57 . 13C-NMR  δ 157.59, 156.08, 142.01, 140.09, 139.71, 139.01, 136.32, 134.57, 132.03, 131.62, 130.25, 123.53, 122.14, 119.89, 114.54, 113.02, 109.45, 64.11, 61.59, 57.32, 43.90, 33.92, 32.23, 31.75. MS m/z (ESI) 472.3 (M + 1)+ , (472.60), .(Z)-4-(1-(4-(2-(Dimethylamino)ethoxy)phenyl)-5-hydroxy-2-(1-methyl-1H-indol-6-yl)pent-1-en-1-yl)phenol (15h). . 1H-NMR  δ 7.33 , 7.19 , 7.14 , 7.05 , 7.01 , 6.91 , 6.65 , 6.34 , 6.28 , 4.36 , 3.7 , 3.58 , 3.38 , 2.97 , 2.51 , 1.55 . MS m/z (ESI) 471.3 (M + 1)+ .(Z)-4-(1-(4-(2-(Dimethylamino)ethoxy)phenyl)-5-hydroxy-2-pent-1-en-1-yl)phenol (15i). . 1H-NMR  δ 8.55 , 8.22 , 7.69 , 7.50 , 7.36 , 7.12 , 7.04 , 6.91 , 6.78 , 6.71 , 4.17 , 3.45 , 2.84 , 2.57 , 1.61 . MS m/z (ESI) 458.2 (M + 1)+ .(Z)-4-(1-(4-(2-(Dimethylamino)ethoxy)phenyl)-5-hydroxy-2-(isoquinolin-7-yl)pent-1-en-1-yl)phenol (15j). . 1H-NMR  δ 9.03 , 8.53 , 8.32 , 7.87 , 7.69 , 7.53 , 7.08 , 6.82 , 6.79 , 6.58 , 3.99 , 3.43 , 2.90 , 2.68 , 2.45 , 1.57 . MS m/z (ESI) 469.2 (M + 1)+ .(Z)-4-(1-(4-(2-(Dimethylamino)ethoxy)-3-(trifluoromethyl)phenyl)-5-hydroxy-2-(pyridin-3-yl)pent-1-en-1-yl)phenol (16). To a solution of 13a  and zinc trifluoromethanesulfinate  in CH2Cl2 (2.5 mL)/H2O (1.0 mL) was added TFA  and tert-butyl hydroperoxide (70% in H2O)  at 0 °C. The reaction mixture was stirred at rt for 12 h. Upon consumption of the starting material, the reaction was partitioned between CH2Cl2 (2 mL) and sat. NaHCO3 solution (2 mL). The organic layer was separated, and the aqueous layer was extracted with CH2Cl2 and dried over MgSO4. The crude product was purified by preparative HPLC using eluent A (0.1% TFA H2O) and eluent B (0.1% TFA ACN) (A/B = 70/30) to give the desired product. . 1H-NMR  δ 8.24 , 8.18 , 7.70 , 7.28 , 6.94 , 6.83 , 6.71 , 4.11 , 3.28 , 3.21 , 3.16 , 2.65 , 2.57 , 1.58 . MS m/z (ESI) 487.2 (M + 1)+ .(Z)-4-(1-(4-(2-(Dimethylamino)ethoxy)phenyl)-2-(6-isopropylpyridin-3-yl)-5-methoxypent-1-en-1-yl)phenol (17). To a solution of 13a  and zinc isopropylsulfinate  in DMSO (1.5 mL) was added TFA  and tert-butyl hydroperoxide (70% in H2O)  at 0 °C. The reaction mixture was stirred at rt for 12 h. Upon consumption of the starting material, the reaction was partitioned between EtOAc (5 mL) and sat. NaHCO3 solution (5 mL). The organic layer was separated, and the aqueous layer was extracted with EtOAc and dried over MgSO4. The crude product was purified by preparative HPLC using eluent A (0.1% TFA H2O) and eluent B (0.1% TFA ACN) (A/B = 70/30) to give the desired product. . 1H-NMR  δ 8.04 , 7.60 , 7.17 , 7.03 , 6.79 , 6.64 , 6.45 , 3.99 , 3.27 , 3.21 , 2.94 , 2.77 , 2.55 , 2.36 , 1.57 , 1.22 . MS m/z (ESI) 475.3 (M + 1)+ .50 value was calculated with the Prism 6 software [ERRγ binding assay was conducted with Lantascreen assay system , which was based on TR-FRET, followed by manufacturer’s instruction. Briefly, compounds were serially diluted with 2-fold staring from 10 μM and 10 μL of each diluent was added in 384 well plate. Then, GST-conjugated ERRγ LBD (ligand-binding domain) was added to be 5 nM. Next, the mixture of fluorescien-conjugated coactivator PGC1a  and Tb-a-GST antibody  was added. The reaction mixture was incubated at rt in dark state for 1 h. TR-FRET activity was measured at 340 nm excitation and 495 nm/520 nm dual emission using microplate reader  and ICsoftware ,16,17.ERRs and ERα binding assay used same assay system as ERRγ’s, Lanthascreen assay system. Therefore, procedure was same as ERRγ binding assay except tested enzymes . Used reference compounds were 4-OHT 1 for ERα and ERRβ binding assay, and XCT790 for ERRα binding assay.2 in a humidified incubator [AD293 cells were cultured in Dulbecco’ modified Eagle medium (DMEM) High glucose supplemented with 0.5% FBS . After 24 h, culture media was changed with DMEM High glucose containing 10% FBS. DNA constructs, which includes pCMX-Gal4-ERRγ, pFR-luciferase reporter plasmid, pCMV-β-gal were transiently transfected into AD293 cells using TransIT-LT1 transfection reagent  for 24 h. Then, transfected cells were treated with compounds for 24 h and harvested for detection of luciferase activity and β-gal activity. All cells were incubated at 37 °C under 5% COg for 5 min at 4 °C. Aliquots of the supernatant were injected onto an LC-MS/MS system. Incubations terminated prior to addition of NADPH regeneration system (time point 0 min) were used as standards, defined as 100%. Percent of the parent compound remaining was calculated by comparing peak areas [The metabolic stability assay was performed by incubation of human and selected animal liver microsomes , at 37 °C with a test compound at a final concentration of 1 μM, in the presence of 0.5 mg/mL microsomal protein and NADPH regeneration system, in a total volume of 100 μL of 100 mM phosphate buffer, pH 7.4. The incubation was started by the addition of NADPH regeneration system and terminated with adding 40 μL of ice-cold acetonitrile at 0 and 30 min. Precipitated proteins were removed by centrifugation at 10,000× ak areas .O-deethylase, tolbutamide 4-hydroxylase, S-mephenytoin 4-hydroxylase, dextromethorphan O-demethylase and midazolam 1′-hydroxylase activities were determined as probe activities for CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A, respectively, using cocktail incubation and tandem mass spectrometry. Briefly, incubation reaction was performed with 0.25 mg/mL human liver microsomes in a final incubation volume of 100 μL. The incubation medium contained 100 mM phosphate buffer (pH 7.4) with probe substrates. The incubation mixture containing various inhibitors (10 μM) was pre-incubated for 5 min. After pre-incubation, an NADPH regenerating system was added. After incubation at 37 °C for 15 min, the reaction was stopped by placing the incubation tubes on ice and adding 40 μL of ice-cold acetonitrile. The incubation mixtures were then centrifuged at 10,000× g for 5 min at 4 °C. Aliquots of the supernatant were injected onto an LC-MS/MS system. The CYP-mediated activities in the presence of inhibitors were expressed as percentages of the corresponding control values [All incubations were performed in duplicate, and the mean values were used for analysis. The assays of phenacetin 50, compounds were serially diluted  and then followed by reactions for 4 h at 25 °C in a reaction mixture containing hERG membrane, fluorescence tracer red dye and fluorescence polarization buffer. Fluorescence intensity  was measured using a multi-mode microplate reader Synergy Neo . E-4031 was used as the reference positive standard (IC50 = 10–90 nM) [hERG channel binding assay was performed using predictor hERG fluorescence polarization assay  according to the manufacturer’s instructions. Briefly, for measuring IC0–90 nM) .15g with ERRγ, we used the crystal structure of human ERRγ and its complex with 4 from Protein Data Bank (PDB code: 2EWP). The protein structure was minimized using the Protein Preparation Wizard by applying an OPLS force field. After complete preparation of the ligands and protein for docking, receptor-grid files were generated. Ligand docking into the active site of ERRγ was carried out using the Schrödinger docking program, Glide. The energy minimized compound 15g was docked into the prepared receptor grid. The best-docked pose was selected as the lowest Glide score. The molecular graphics for the agonist binding pocket and refined docking model for the selected compounds was generated using PyMol [For the prediction of docking model of compound ng PyMol .4 as ERRγ inverse agonists. The A-ring in the 4 scaffold was mainly modified to generate drug candidates that were then tested using in vitro binding and functional screening assays. Among the compounds tested, compound 15g represents a new structural class of promising 4 analogs that are fully subtype selective ERRγ inverse agonists. In addition, these compounds have acceptable ADMET profiles and show potential for the treatment of a variety of ERRγ-related disorders. Additional studies analyzing compound 15g in animal disease models are underway.In this report, we describe the discovery and structural exploration of new analogs of compound


Hypericum scabrum L. delivered eight pure phenolic compounds 1–8. The pure compounds were identified through physico-chemical, NMR  and mass spectrometric studies as: 3-8′′-bisapigenin (1), quercetin (2), quercetin-3-O-α-l-arabinofuranoside (3), quercetin-3-O-α-l-rhamnoside (4), quercetin-3-O-β-d-glucopyranoside (5), quercetin-3-O-β-d-galactopyranoside (6), (−)-epicatechin (7), (+)-catechin (8). Total polyphenolic compounds and total flavonoids contents were determined in the extract as 0.107 mg∙mg−1 and 0.023 mg∙mg−1 of the dried extract, respectively. Antioxidant activity using DPPH free radical scavenging assay delivered very strong activity for compounds 2 and 5, 6 and crude extract 10. Protein tyrosine phosphatase 1B (PTP-1B) inhibition experiment of isolated compounds and crude extracts resulted in significant inhibition activity for samples 2, 7a, 8a, 11 and 12 with IC50 values ranging from 1.57 to 2.91 µM. Antimicrobial activity of the pure compounds and extracts produced average results against Staphylococcus aureus, Escherichia coli and Candida albicans strains. From our literature survey, it appears that all pure compounds except 2 were isolated and reported for the first time in H. scabrum.Phytochemical investigations of ethyl acetate-soluble part of the aerial part of  At low field of the 1H-NMR spectra of 1 showed two broad singlet signals attributed to the six hydroxyl functionalities (δ 13.88 and 12.23 ppm). A pair of meta coupling proton signals observed at 6.75  and 6.81  for the A ring and another four doublets of symmetric proton signals at 8.01 , 7.13 , 7.94  and 7.16  showed the presence of an AA′BB′ coupling system, which indicated that 4′,4′′′-positions were substituted on the B and B′ rings, respectively. Two aromatic protons gave singlet signal at 6.85 ppm attributed to H-3′′ and H-6′′. The 13C-NMR data of 1 showed the presence of 30 carbon signals, which corresponded to 12 methines , 95.29 (C-8), 131.39 , 116.89 , 104.53 (C-3′′), 129.31 , 117.53 ), 18 quaternary carbons (δC 165.22 (C-2), 112.54 (C-3), 182.71 (C-4), 163.90 (C-5), 166.68 (C-7), 159.24 (C-9), 105.01 (C-10), 125.06 (C-1′), 162.20 (C-4′), 165.30 (C-2′′), 183.57 (C-4′′), 164.83 (C-5′′), 163.63 (C-7′′), 101.56 (C-8′′); 157.02 (C-9′′), 105.91 (C-10′′), 123.07 (C-1′′′), 163.23 (C-4′′′)). The linkage of two flavone units confirmed by analysis of the HMBC spectrum, which showed the cross-peak through four bond between H-6′′ (δH 6.85) and C-3 (δC 112.54). Furthermore, HMBC correlations were observed from H-2′, H-6′ to C-2, C-4′; H-6 to C-5, C-7, C-8, C-10; H-8 to C-8, C-7, C-9, C-10; H-3′′ to C-2′′, C-4′′, C-1′′′; H-6′′ to C-7′′, C-8′′, C-10′′. The above mentioned results revealed that the two flavone units were connected at C-3 and C-8′′ positions by C-C linkage in compound 1.The structures of compounds 1H-NMR spectrum of 2  showed three aromatic protons signals at 8.64 , 7.41  and 8.14  in the form of an ABC spin-system suggesting a flavonol with 3′,4′-disubstituted B-ring and showed a pair of meta coupling proton signals at 6.74  and 6.78  for the A ring. The 13C-NMR spectra supported this hypothesis and showed 15 signals including carbonyl signal at (δC 177.77 (C-4). It revealed chemical shifts of carbon nucleus at δC 138.40 (C-3), 162.92 (C-5), 166.01 (C-7), 147.59 (C-3′), 150.14 (C-4′) that suggested the 3,5,7,3′,4′-oxygenated flavone nucleus. ESI-MS of compound 2 in negative ionization mode presented a molecular ion [M − H]− at m/z value 301.0, suggesting the molecular formula C15H10O7 with the combination of 1H-NMR and 13C-NMR.The 1H-NMR  of compound 3, gave two meta-coupled doublets each with (J = 2.1), at 6.20 and 6.40, representing H-6 and H-8 of the A-ring. Three proton signals observed at 8.29 , 7.35  and 7.99 , correspond to the H-2′, H-5′ and H-6′ protons of B-ring, respectively. The anomeric proton signal appeared as broad singlet at 6.52 ppm assigned to α-arabinose moiety. Two double of doublets corresponded to methylene protons at 4.10 , 4.18 , respectively. The connection of sugar moiety to quercetin was confirmed by HMBC correlation between H-1′′ and C-3. The negative ESI-MS of compound 3 gave the quasi-molecular ion at m/z 433.4 [M − H]−. Thus, its molecular formula was deduced to be C20H18O11 and structure of compound 3 was determined to be quercetin-3-O-α-l-arabinofuranoside.The 1H-NMR  spectrum of compound 4, three broad singlet signals and two doublets were observed in aromatic region at 8.04, 6.72, 6.67, 7.73 and 7.32 which were assigned to H-2′, H-6, H-8, H-6′ and H-5′, respectively. In addition, 1H-NMR spectrum of compound 4 shows singlet signal at δ 6.30  for anomeric proton of α-rhamnose moiety. In the high field of spectrum appeared the methyl proton signal as doublet at 1.50 . The 13C-NMR  spectrum revealed four carbon signals in the region of 72 to 74 ppm and one methyl carbon signal at 18.92 ppm typical for rhamnose unit. On the basis of HMBC correlation from H-1′′ to C-3, we established the connection of sugar moiety to aglicon at position C-3 of compound 4. The structure was also confirmed by determination of ESI-MS (negative mode) giving [M − H]− ion at m/z 447.5. Therefore, its molecular formula was deduced to be C21H20O11 with the combination of 1H-NMR and 13C-NMR.In 1H-NMR spectrum of 5 exhibited signals from the H-6 and H-8 position of the flavone molecule at δ 6.20  and 6.41 , respectively. One broad singlet at δ 7.58 , two doublets (J = 8.6 Hz) at δ 6.85 and 7.59 ppm indicated the presence of H-2′, H-5′ and H-6′, respectively. The proton of hydroxyl group typical for 5-OH at δ 12.66  and seven broad singlet signals resonance at δ 10.88, 9.76, 9.25, 5.32, 5.10, 4.98, 4.30 ppm indicated the presence of hydroxyl groups in molecule. In addition, 1H-NMR signal at δ 5.48 was assigned to anomeric proton of sugar moiety. The sugar moiety was deduced to be attached at position C-3, on the basis of HMBC experiment cross-peak between H-1′′ (δ 5.48) and C-3 (δ 133.30). The anomeric configuration of glucose in 5 was concluded to be β-form from value of the coupling constant of H-1′′ . In 13C-NMR spectrum of 5, six carbon signals of glucose moiety were observed along with signals of quercetin. The molecular ion observed at m/z 463.5 in the ESI-MS negative mode. The above results support the structure of 5 as quercetin-3-O-β-d-glucopyranoside.The 6 was obtained as a yellow amorphous powder, m. p. 245–246 °C. Comparison of the 1H- and 13C-NMR data for 5 and 6 indicated a close relationship for the two structures, although 5, 6 were measured in DMSO-d6. The major difference between 5, 6 were the value of Rf (0.48 for 5 and 0.45 mm for 6), the proton multiplicity of sugar moiety and carbon chemical shift values of sugar moiety. Aglycon part of 1H-NMR spectrum of compound 6 displayed the typical signals for quercetin. The carbon signals were elucidated to be galactose unit. The position of the sugar moiety in quercetin was established from HMBC cross-peak between H-1′′ (δ 5.38) and C-3 (δ 133.45). Thus, the structure of compound 6 was determined to be quercetin-3-O-β-d-galactopyranoside.Compound 7, 8 were obtained as a pale red powder. It gave one spot on TLC chromatogram . The compounds 7, 8 on the basis 1H- and 13C-NMR were identified as a mixture of epicatechin (75%) and catechin (25%). The quantity of compounds 7 and 8 in sample were determined on the basis of integral intensities of the signals in 1H-NMR spectrum. Melting point of the mixture of standard epicatechin and catechin with the same ratio delivered the same result as of our purified compound.The compounds 1–6) and seven crude extracts  were determined. Pharmacological activities of the two pure compounds (7) and (8) could not be performed due to their low amounts which is not sufficient for these in vitro assays.Antidiabetic, antimicrobial and antioxidant activities of the six pure compounds (1–6) and the crude extract for their in vitro evaluation of percentage inhibition of the enzyme PTP-1B. Crude extracts 7a, 8a, 9a, and 10–13 induced a PTP-1B enzymatic inhibition in a concentration-dependent manner with IC50 values 2.03 µM, 2.16 µM, 4.91 µM, 2.91 µM, 2.10 µM, 1.57 µM and 14.29 µM, respectively. The individual flavonoids except quercetin  have not shown good effect for PTP-1B inhibition. IC50 values of the all the samples are presented in 12) showed highest antidiabetic activity (PTP-1B inhibition) with an IC50 1.57 µM among the crude extracts and even more than the PTP-1B inhibitor. This significant activity in the initial extract with low activity in the fractionated extracts proved the synergistic relationship among the compounds in the 70% ethanol extract, which weakened after the separation of the synergistically bound analytes.We have determined the effect of the pure isolated flavonoids , Escherichia coli ATCC 11229 (Gram negative bacteria) and Candida albicans ATCC 10231 (Fungi) strains. All the pure compounds except the 3 and 4 were active against SA delivering highest zones of inhibition 12 for compound 2. Zones of inhibition of compounds 1, 5 and 6 were 5.5, 11 and 11, respectively, against SA. Compounds 3, 4, and 6 showed activity against EC with inhibition zones as: 5.5, 8 and 6.5, respectively. Among the six pure compounds, only compounds 2 and 4 delivered mild results for the inhibition of CA with zones of inhibition as 6.5 and 5.5, respectively. 8a, 9a, 11–13 did not show activity against any microbial strain while the crude extract 7a showed activity with zones of inhibitions 7, 10 and 7 against SA, EC and CA, respectively. Sample 10 was only active against the EC and showed highest activity with zones of inhibitions as 12 among the crude extracts. The obtained results suggest that the tested compounds showed average and mild activity in comparison to the reference drugs Ampicillin and Amphotericin B.The pure compounds in vitro by 2,2-diphenyl-2-picrylhydrazyl (DPPH) assay. A lower IC50 value implied a better antioxidant activity of the tested samples. Quercetin (2) and quercetin glycoside (5) were the most active, with an IC50 value of 24.88 μM and 24.78 μM, respectively, which were even better than that of Vitamin C (IC50 30.32 µM). Other glycosides also showed significant antiradical activities. The crude extracts 10 showed potent antiradical activities with IC50 value of 1.19 μg∙mL−1 and 8a, 9a, 11–13 showed relatively average antiradical activities with IC50 values 13.86, 24.62, 6.18, 10.81 and 16.29, respectively. The results of the antioxidant activity determined by the DPPH assay procedure using the standard Vitamin C, in pure compounds and crude extract with IC50 values are presented in 10) delivered highest antioxidant activity with IC50 value of 1.19 μg∙mL−1 followed by n-butanol extract (sample 11), which is completely in agreement with the previous literature −; 1H-NMR : δ 6.75 , 6.81 , 8.01 , 7.13 , 6.85 , 7.94 , 7.16 , 13.88 , 12.23 ; 13C-NMR : δ 165.22 (C-2), 112.54 (C-3), 182.71 (C-4), 163.90 (C-5), 100.70 (C-6), 166.68 (C-7), 95.29 (C-8), 159.24 (C-9), 105.01 (C-10), 125.06 (C-1′), 131.39 , 116.89 , 162.20 (C-4′), 165.30 (C-2′′), 104.53 (C-3′′), 183.57 (C-4′′), 164.83 (C-5′′), 100.70 (C-6′′), 163.63 (C-7′′), 101.56 (C-8′′), 157.02 (C-9′′), 105.91 (C-10′′), 123.07 (C-1′′′), 129.31 , 117.53 , 163.23 (C-4′′′). The 1H-NMR and 13C-NMR spectral data of compound 1 were consistent with the reported literature −; 1H-NMR : δ 6.74 , 6.78 , 7.41 , 8.14 , 8.64 , 13.36 ; 13C-NMR : δ 148.21 (C-2), 138.40 (C-3), 177.77 (C-4), 162.92 (C-5), 99.70 (C-6), 166.01 (C-7), 94.78 (C-8), 157.94 (C-9), 104.94 (C-10), 124.33 (C-1′), 117.1 , 147.59 (C-3′), 150.14 (C-4′), 121.53 (C-6′). The 1H-NMR and 13C-NMR spectral data of compound 2 were consistent with the reported literature −; 1H-NMR : δ 6.72 , 6.67 , 7.35 , 7.99 , 8.29 , 13.31 , 6.52 , 5.19 , 4.90 , 4.82 , 4.10 , 4.18 ; 13C-NMR : δ 158.53 (C-2), 135.20 (C-3), 179.75 (C-4), 163.33(C-5), 100.24 (C-6), 166.41 (C-7), 95.08 (C-8), 158.17 (C-9), 105.72 (C-10), 122.90 (C-1′), 117.61 (C-2′), 147.61 (C-3′), 151.15 (C-4′), 117.14 (C-5′), 122.85 (C-6′), 110.29 (C-1′′), 83.94 (C-2′′), 79.44 (C-3′′), 89.13 (C-4′′), 62.96 (C-5′′) [identical with the literature] −; 1H-NMR : δ 6.72 , 6.67, 7.32 , 7.73 , 8.04 , 13.41 , 6.30 , 4.67 , 4.41 , 4.32 , 5.10 , 1.50 ; 13C-NMR : δ 158.69 (C-2), 136.53 (C-3), 179.59 (C-4), 163.47 (C-5), 100.25 (C-6), 166.44(C-7), 95.06 (C-8), 158.20 (C-9), 105.92 (C-10), 122.80 (C-1′), 117.58 (C-2′), 147.84 (C-3′), 151.09 (C-4′), 117.00 (C-5′), 122.68 (C-6′), 104.54 (C-1′′), 73.07 (C-2′′), 72.61 (C-3′′), 73.87 (C-4′′), 72.53 (C-5′′), 18.92 (C-6′′) [identical with the literature] −; 1H-NMR : δ 6.20 , 6.41, 6.85 , 7.59 , 7.58 , 12.66 , 10.88 , 9.76 , 9.25 , 5.32 , 5.10 , 4.98 , 4.30 , 5.48 , 3.23 , 3.08 , 3.32 , 3.58 ; 13C-NMR : δ 156.17 (C-2), 133.30 (C-3), 177.45 (C-4), 161.26 (C-5), 98.67 (C-6), 164.13(C-7), 93.51(C-8), 156.33 (C-9), 103.99 (C-10), 121.17 (C-1′), 116.20 (C-2′), 144.83 (C-3′), 148.47 (C-4′), 115.21 (C-5′), 121.62 (C-6′), 100.82 (C-1′′), 74.10 (C-2′′), 76.51 (C-3′′), 69.94 (C-4′′), 77.63 (C-5′′), 60.98 (C-6′′) [identical with the literature] −; 1H-NMR : δ 6.20 , 6.40, 6.81 , 7.67 , 7.52 , 12.64 , 10.89 , 9.76 , 9.18 , 5.15 , 4.88 , 4.45 , 5.38 , 3.57 , 3.37 , 3.65 , 3.33 , 3.29 , 3.45 ; 13C-NMR : δ 156.20 (C-2), 133.45 (C-3), 177.46 (C-4), 161.22 (C-5), 98.68 (C-6), 164.20 (C-7), 93.50 (C-8), 156.29 (C-9), 103.88 (C-10), 121.08 (C-1′), 115.90 (C-2′), 144.83 (C-3′), 148.46 (C-4′), 115.17 (C-5′), 122.01 (C-6′), 101.76 (C-1′′), 71.19 (C-2′′), 73.17 (C-3′′), 67.91 (C-4′′), 75.85 (C-5′′), 60.13 (C-6′′). The 1H-NMR and 13C-NMR spectral data of compound 6 were consistent with the reported literature −; 1H-NMR : δ 5.40 , 4.75 , 3.44 , 3.57 , 6.71 , 6.69, 7.30 , 7.37 , 7.95 , 11.26 , 11.47 ; 13C-NMR : δ 80.59 (C-2), 67.43 (C-3), 30.18 (C-4), 159.20 (C-5), 97.14 (C-6), 159.12 (C-7), 96.28 (C-8), 158.12 (C-9), 100.68 (C-10), 132.64 (C-1′), 116.85 (C-2′), 147.43 (C-3′), 147.33 (C-4′), 116.60 (C-5′), 119.89 (C-6′). The 1H-NMR and 13C-NMR spectral data of compound 7 were consistent with the reported literature −; 1H-NMR : δ 5.26 , 4.66 , 3.37 , 3.73 , 6.73 , 6.68, 7.26 , 7.68 , 11.56 ; 13C-NMR : δ 83.79 (C-2), 68.71 (C-3), 30.24 (C-4), 158.89 (C-5), 97.14 (C-6), 159.27 (C-7), 96.02 (C-8), 157.85 (C-9), 101.53 (C-10), 132.67 (C-1′), 116.85 (C-2′), 147.69 (C-3′), 147.67 (C-4′), 116.65 (C-5′), 120.22 (C-6′). The 1H-NMR and 13C-NMR spectral data of compound 8 were consistent with the reported literature  × 100. IC50 was calculated from the percentage inhibition values.The individual compounds 1 to 6 and crude extracts 7a, 8a, 9a; 10 to 13 were measured using the agar well diffusion method. Bacterial and fungal pathogens such as S. aureus (ATCC 6538), E. coli (ATCC 11229) and C. albicans (ATCC 10231) were used as indicator strains for this analysis. These microorganisms were aseptically inoculated into appropriate liquid media and incubated at 37 °C. After 16 h, the cells were centrifuged at 6000 rpm for 10 min and then suspended in sterile water. The different cells (1 mL) were added to appropriate agar media (100 mL) prior to plating, and the wells were made using an agar well borer. To these wells, different concentrations of compounds 1 to 6 and the extracts 7a, 8a, 9a; 10 to 13 were added and subsequently incubated at 37 °C for 24 h. Zones of inhibition were estimated by measuring the diameter of the microbial growth inhibition zones. Values were averaged from three independent experiments.Antimicrobial activity of compounds −1, respectively were made. Briefly DPPH solution (100 μL) and pure compound or crude extract in DMSO (100 μL) were added to each well of the micro plate and mixed. The mixture was shaken vigorously and left to stand at 37 °C for 30 min in dark. The absorbance of the solution was then measured at wavelength 515 nm using a micro plate spectrophotometer. Inhibition (%) of free radical (DPPH) was determined as [(Acontrol − Asample)/Acontrol] × 100, where: Acontrol is the absorbance of the control sample containing all reagents except the test sample and Asample is the absorbance of the test sample (pure compound or crude extract). Vitamin C was used as the positive control and tests were carried out in triplicate. The IC50 values were determined for all tested flavonoids, extract and control standard antioxidant. The IC50 value was defined as the concentration (in μM and μg∙mL−1) of pure compound and extracts that inhibits the formation of radicals by 50%.Radical scavenging assay was determined by a micro plate spectrophotometric method based on the reduction of DPPH according to the previous report . VariousH. scabrum in sufficient amounts to be isolated employing routinely applied chromatographic procedures. Seventy percent ethanol extract delivered best results for PTP-1B assay with IC50 1.57 µM among the crude extracts while quercetin showed significant inhibition activity (IC50 2.19 µM) for this enzyme. Quercetin (2) and quercetin glycoside (5) yielded strong DPPH radical scavenging activity among the isolated compounds. Our study suggests the use of aerial part of H. scabrum for antioxidant and antidiabetic formulations based on their proven activities. From our literature survey, it appears that all the pure compounds except 2 were isolated and reported for the first time in H. scabrum.The study confirmed the presence of bioactive compounds like quercetin glycosides, bisapigenin, catechin and epicatechin in the aerial part of


The optimal conditions of this reaction are proposed; they make it possible to prevent partial destruction of the original heterocyclic ester and thereby avoid formation of specific impurities of 7-hydroxy-2,3-dihydro-1H,5H-pyridoquinolin-5-one. To confirm the structure of all substances obtained, elemental analysis, nuclear magnetic resonance (NMR) spectroscopy, and mass spectrometry were used. Moreover, the peculiarities of their 1H and 13C-NMR spectra, as well as their mass spectrometric behavior under conditions of electron impact ionization, were discussed. The effect of pyridoquinoline-6-carboxanilides on the urinary function of the kidneys was studied in white rats of both genders by the standard method of oral administration at a dose of 10 mg/kg. Testing was conducted in comparison with hydrochlorothiazide, as well as with structurally close pyrrolo quinoline-5-carboxanilides studied earlier with the same substituents in the anilide fragments. It was found that addition of one methylene unit to the heterocycle partially hydrogenated and annelated with the quinolone core has a positive impact on biological properties—most of the substances studied exhibit a statistically significant diuretic effect exceeding the activity of not only hydrochlorothiazide, in some cases, but also the action of the structural analogs. The important structural and biological regularities, which are common with pyrroloquinolines and introduced by a chemical modification, were revealed. The importance of the presence in the structure of terminal amide fragments of tricyclic quinoline-3-carboxamides of a 4-methoxy-substituted aromatic ring was particularly marked. The expediency of further study of pyridoquinolines as promising diuretic agents has been shown.Continuing a targeted search for new leading structures with diuretic action among tricyclic derivatives of hydroxyquinolines, which are of interest as potential inhibitors of aldosterone synthase, the synthesis of a series of the corresponding pyrido[3,2,1- Currently, arterial hypertension is considered to be one of the most globally prevalent and important medical and social problems of mankind. The insidiousness of this disease lies not only in the fact that its manifestations significantly reduce the life quality of patients suffering from high blood pressure ; the numOver the last 10–15 years, the leading role in the drug therapy of hypertension, as well as in ischemic heart disease and chronic heart failure, has quite reasonably belonged to diuretics. These drugs are not directly antihypertensive agents. However, they eliminate large amounts of liquid from the body, and thus help to reduce blood pressure to the physiological norm ,3,4,5. TFinally, the third subgroup—inhibitors of aldosterone synthase—is unfortunately not yet represented by drugs officially approved for medical use. However, exactly this direction is considered to be the most promising in creating new agents for the treatment of hypertension, since it has a simple and, at the same time, very original idea: not to block the access of aldosterone to the corresponding receptors or, if necessary, even to stop its production at all . TheoretI -quinoline-6-carboxylate (1) was carried out by the method in the study quinoline-6-carboxamide (2a). Yield: 96%; m.p. 178–180 °C, 179 °C +, 227 (24), 93 (100). Analytically calculated  for C19H16N2O3: C, 71.24; H, 5.03; N, 8.74%. Found: C, 71.35; H, 4.94; N, 8.67%., 179 °C , 177–178, 179 °C ; 1H-NMR N-(2-Hydroxyphenyl)-7-hydroxy-5-oxo-2,3-dihydro-1H,5H-pyridoquinoline-6-carboxamide (2b). Yield: 88%; m.p. 281–283 °C; 1H-NMR : δ 16.84 , 12.59 , 9.73 , 8.24 , 7.97 , 7.42 , 7.16 , 6.92–6.07 , 6.76 , 4.16 , 3.00 , 2.12 . 13C-NMR : δ 170.9 (7-С-ОH), 168.7 (CONHAr), 160.8 (5-C=O), 153.0 (2′-C-OH), 147.3, 136.2, 130.9, 125.9, 124.4, 122.3, 121.6, 120.9, 119.0, 114.8, 114.2, 96.2 (6-C), 41.4 (3-CH2), 26.9 (1-CH2), 19.9 (2-CH2). EI-MS : 336 (81) [M]+, 227 (28), 109 (100). Anal. Calcd. for C19H16N2O4: C, 67.85; H, 4.79; N, 8.33%. Found: C, 67.92; H, 4.86; N, 8.27%.N-(3-Hydroxyphenyl)-7-hydroxy-5-oxo-2,3-dihydro-1H,5H-pyridoquinoline-6-carboxamide (2c). Yield: 93%; m.p. 192–194 °C; 1H-NMR : δ 16.57 , 12.58 , 9.25 , 7.96 , 7.50 , 7.23 , 7.19 , 7.10 , 6.96 , 6.54 , 4.14 , 3.01 , 2.10 . 13C-NMR : 170.8 (7-С-ОH), 168.6 (CONHAr), 161.4 (5-C=O), 157.9 (3′-C-OH), 143.2, 136.1, 132.7, 129.6, 125.9, 122.2, 121.4, 120.3, 117.0, 116.1, 112.4, 96.3 (6-C), 41.5 (3-CH2), 26.4 (1-CH2), 19.6 (2-CH2). EI-MS : 336 (87) [M]+, 227 (35), 109 (100). Anal. Calcd. for C19H16N2O4: C, 67.85; H, 4.79; N, 8.33%. Found: C, 67.93; H, 4.85; N, 8.37%.N-(4-Hydroxyphenyl)-7-hydroxy-5-oxo-2,3-dihydro-1H,5H-pyridoquinoline-6-carboxamide (2d). Yield: 97%; m.p. 208–210 °C, 208 °C +, 227 (39), 109 (100). Anal. Calcd. for C19H16N2O4: C, 67.85; H, 4.79; N, 8.33%. Found: C, 67.80; H, 4.82; N, 8.26%., 208 °C ; 1H-NMR N-(2-Methoxyphenyl)-7-hydroxy-5-oxo-2,3-dihydro-1H,5H-pyridoquinoline-6-carboxamide (2e). Yield: 84%; m.p. 223–225 °C; 1H-NMR : δ 16.69 , 12.73 , 8.37 , 7.98 , 7.43 , 7.16 , 7.05 , 6.96 , 6.92 , 4.17 , 3.98 , 3.01 , 2.14 . 13C-NMR : δ 170.3 (7-С-ОH), 164.6 (CONHAr), 161.5 (5-C=O), 152.2 (2′-C-OMe), 150.0, 145.1, 139.0, 136.5, 133.1, 127.2, 122.3, 121.1, 115.4, 114.0, 111.5, 95.8 (6-C), 56.1 (OCH3), 41.8 (3-CH2), 26.9 (1-CH2), 20.0 (2-CH2). EI-MS : 350 (73) [M]+, 227 (22), 123 (100). Anal. Calcd. for C20H18N2O4: C, 68.56; H, 5.18; N, 8.00%. Found: C, 68.45; H, 5.07; N, 7.92%.N-(3-Methoxyphenyl)-7-hydroxy-5-oxo-2,3-dihydro-1H,5H-pyridoquinoline-6-carboxamide (2f). Yield: 94%; m.p. 176–178 °C, 175 °C +, 227 (27), 123 (100). Anal. Calcd. for C20H18N2O4: C, 68.56; H, 5.18; N, 8.00%. Found: C, 68.47; H, 5.11; N, 8.09%. , 175 °C ; 1H-NMR N-(4-Methoxyphenyl)-7-hydroxy-5-oxo-2,3-dihydro-1H,5H-pyridoquinoline-6-carboxamide (2g). Yield: 97%; m.p. 180–182 °C; 1H-NMR : δ 16.65 , 12.48 , 7.97 , 7.58 , 7.44 , 7.17 , 6.86 , 4.14 , 3.79 , 3.00 , 2.13 . 13C-NMR : δ 172.6 (7-С-ОH), 165.4 (CONHAr), 159.1 (5-C=O), 156.4 (4′-C-OMe), 146.1, 134.0, 132.3, 126.4, 123.4, 122.5 , 120.0, 118.9, 114.6 , 96.2 (6-C), 57.8 (OCH3), 41.6 (3-CH2), 26.7 (1-CH2), 19.8 (2-CH2). EI-MS : 350 (91) [M]+, 227 (38), 123 (100). Anal. Calcd. for C20H18N2O4: C, 68.56; H, 5.18; N, 8.00%. Found: C, 68.61; H, 5.26; N, 7.94%. N-(4-Ethoxyphenyl)-7-hydroxy-5-oxo-2,3-dihydro-1H,5H-pyridoquinoline-6-carboxamide (2h). Yield: 92%; m.p. 189–191 °C; 1H-NMR : δ 16.70 , 12.47 , 7.97 , 7.56 , 7.43 , 7.18 , 6.83 , 4.14 , 4.02 , 3.01 , 2.13 , 1.41 . 13C-NMR : δ 173.2 (7-С-ОH), 169.4 (CONHAr), 162.2 (5-C=O), 155.1 (4′-C-OEt), 148.0, 139.7, 137.1, 126.9, 124.1, 121.4 , 120.5, 118.3, 116.8 , 95.2 (6-C), 63.7 (OCH2), 41.8 (3-CH2), 26.1 (1-CH2), 19.7 (2-CH2), 14.8 (CH3). EI-MS : 364 (79) [M]+, 227 (34), 137 (100); Anal. Calcd. for C21H20N2O4: C, 69.22; H, 5.53; N, 7.69%. Found: C, 69.15; H, 5.45; N, 7.78%.N-aryl-7-hydroxy-5-oxo-2,3-dihydro-1H,5H-pyrido-quinoline-6-carboxamides on the excretory function of the kidneys was studied in white outbred rats of both genders with weights of 180–200 g by the standard method quinoline-5-carboxanilides . The values of excretion were registered after 4 h and compared with the control, as well as a known diuretic, hydrochlorothiazide quinolin-5-one (3) as an impurity.As is known, the reactivity of the lower alkyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylates remains high, regardless of the structure of the benzene moiety of the molecule and the nature of the substituents at the nitrogen atom . Their t0–140 °С ,29. UnliN-aryl-7-hydroxy-5-oxo-2,3-dihydro-1H,5H-pyridoquinoline-6-carboxamides (2a–h) synthesized in such a way are colorless or white crystalline substances with a yellowish tint and a narrow interval of melting points . At room temperature, they are moderately soluble in DMF and dimethyl sulfoxide, sparingly soluble in ethanol, and practically insoluble in diethyl ether, hexane and water.All 1Н and 13С), and mass spectrometry were used. Interpretation of 1H NMR spectra of anilides 2a–h does not cause any difficulties. Even in the aromatic region of the spectrum, where there are simultaneously signals of 7–8 protons in the area of less than 1.4 ppm, coincidences of the resonance frequencies are not observed. The signals of aromatic protons of the pyridoquinoline core are typical for this chemical environment; they consist of two doublets (H-8 and H-10) and a triplet (H-9). In 1Н-NMR spectra, they remain in the same positions regardless of the substituents in the anilide fragment of the molecule  in DMSO-d6 at room temperature, it is unfortunately not always possible to register their 13С-NMR spectra with high quality. Nevertheless, this method allows us to unambiguously confirm the presence of the tetrahydropyridine cycle, alkoxy- and carbonyl groups, and other functional fragments in the compounds studied .Because of the relatively low solubility of 2a–h are very stable compounds and form high-intensity peaks of molecular ions that allow the determination of the molecular weight of the substances tested. The primary fragmentation of molecular ions is shown in the example of anilide 2a, it proceeds by the ketene type characteristic for 4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxamides and is accompanied by the breaking of the acyclic amide bond with formation of tricyclic ketene 4, which is common to all samples, with m/z 227 and the corresponding aniline 5  synthesized on the urinary function of the kidneys are presented in H-pyrroloquinoline-5-carboxylic (VI) and 6-hydroxy-2-methyl-4-oxo-2,4-dihydro-1H-pyrroloquinoline-5-carboxylic (VII) acids previously tested in similar conditions with the same substituents in the arylamide fragments. In the comparative analysis of these data, the attention was immediately drawn to the pronounced diuretic effect of the unsubstituted anilide 2а on the background of the moderately intense antidiuretic effect of its pyrroloquinoline analogs VI and VII. 3-Hydroxy- and 2-methoxy groups significantly changed the biological properties of the substances studied for the better—anilides 2c and 2е by their diuretic activity already exceeded not only the structural analogs VI and VII, respectively, but also hydrochlorothiazide, and in a much lower dose. The position of the methoxy group in the anilide moiety of the molecule interestingly affected the activity. If its gradual increase was previously observed while moving the specified substituent from the ortho- to the meta- and further to the para-position, then there was no direct dependence with transition to pyridoquinolines: the initially high diuretic effect of ortho-isomer 2e was immediately lost in meta-analog 2f, but then again, it recovered to the previous level in para-derivative 2g.The results of the experimental study of the effect of all VI and VII, N-(4-methoxyphenyl)-7-hydroxy-5-oxo-2,3-dihydro-1H,5H-pyridoquinoline-6-carboxamide (2g) appeared to be the most active of the whole group. In other words, another experimental confirmation of a positive effect on the diuretic properties of tricyclic quinolinecarboxamides was obtained. This was precisely due to the presence of the 4-methoxysubstituted aromatic ring in the structure of their terminal amide fragments. However, the 4-ethoxy group in all examples considered had a stable, but quite opposite effect. Its presence almost completely deprived the molecule of any significant effect on the urinary function of the kidneys, no matter which cycle was annelated with the hydroquinolone core on its edges i and j—tetrahydropyridine (anilide 2h), trihydropyrrole (VI) or 2-methyltrihydropyrrole (VII)  see . TherefoN-aryl-7-hydroxy-5-oxo-2,3-dihydro-1H,5H-pyrido-quinoline-6-carboxamides obtained within the framework of the integrated program on synthesis, studies of the reactivity, structure, chemical and biological properties of 4-hydroxyquinoline-2-one derivatives and structurally related heterocycles. Being close analogs of the pyrroloquinolone diuretics previously described, these compounds, as the objects of study, are of interest as a source of not only important regularities of the “structure-activity” relationship, but probably new leading structures. The chemical structure of all N-aryl-7-hydroxy-5-oxo-2,3-dihydro-1H,5H-pyridoquinoline-6-carboxamides has been reliably confirmed by elemental analysis, NMR (1Н and 13С) and mass spectra. According to the results of pharmacological tests, among the compounds synthesized, the samples that can actively enhance the urinary function of the kidneys have been identified. Based on these results, the transformation of the trihydropyrrole heterocycle annelated with the hydroxyquinolone core into the tetrahydropyridine heterocycle is recommended as one of the possible variants for optimization of quinoline diuretics.This report presents a series of new


O-[β-d-apiofuranosyl-(1''''→5''')-O-β-d-apiofuranosyl-(1'''→6'')]-β-d-glucopyranoside, namely sympracemoside (1), was isolated from the aerial parts of Symplocos racemosa along with 15 known flavonoids (2–16). Their structures were characterized by Q-TOF mass, optical rotation, UV, 1D and 2D-NMR spectroscopic data. Compounds 3, 9, 16 showed moderate inhibitory activities against NO production with IC50 value of 88.2, 42.1 and 74.3 μM, respectively.A novel isoflavone glycoside, peseudobatigenin 7- Symplocos racemosa Roxb. is a small evergreen tree with a broad crown and stems grows up to 6 m high -β-d-glucopyranoside, namely sympracemoside . 1 revealing [M+Na-Api]+ (m/z 599.1356) and [M+Na-2Api-Glc]+ (m/z 305.0477). The 1H-NMR spectrum of 1 showed a characteristic signal for the H-2 of isoflavone skeleton at δ 8.28 , two sets of 1,3,4-trisubstituted aromatic rings at δ 8.16 , 7.28 , 7.23 , 7.09 , 7.02 , and 6.89 , and the methylenedioxy group at δ 5.99 . The methylenedioxy group  showed HMBC cross peaks at δ 149.2 (C-3') and 149.3 (C-4'), demonstrating the presence of 3',4'-methylenedioxy moiety. From a comparison of the 1H and 13C- NMR data for 1 with literature values, 1 was determined to be a pseudobaptigenin  derivative -β-d-glucopyrano- side, namely sympracemoside.Main text paragraph Compound i]+ m/z 59.1356 aneriments B; H-1'''O-α-l-rhamnopyranoside (2) + ; 1H-NMR : δ 3.51–3.71 , 3.54 , 3.54 , 3.62 , 3.76 , 3.76 , 3.78 , 3.93 , 3.96 , 3.97 , 4.03 , 4.07 , 4.97 , 5.07 , 5.99 , 7.02 , 7.09 , 7.23 , 7.28 , 8.16 , 8.28 ; 13C-NMR : δ 65.5 (C-5''''), 69.1 (C-6''), 71.8 (C-5'''), 71.8 (C-4''), 74.9 (C-2''), 75.2 (C-4''''), 75.3 (C-4'''), 77.4 (C-5''), 78.0 (C-2''''), 78.1 (C-3''), 78.6 (C-2'''), 79.5 (C-3'''), 80.6 (C-3''''), 102.0 (C-1''), 102.7 (-O-C2H-O-), 105.2 (C-8), 109.4 (C-5'), 110.9 (C-2'), 111.0 (C-1'''), 111.0 (C-1''''), 117.4 (C-6), 120.4 (C-10), 123.9 (C-6'), 126.1 (C-3), 127.1 (C-1'), 128.5 (C-5), 149.2 (C-3'), 149.3 (C-4'), 155.7 (C-2), 159.4 (C-9), 163.7 (C-7), 178.0 (C-4).1–16 against NO production were evaluated using lipopolysaccharide-induced RAW 264.7 cells. Among the tested compounds, mearnsetin-3-O-α-l-rhamnopyranoside (3), kaempferol-3-O-β-d--gulucopyranoside (9) and 3'-O-methycatechin-7-O-β-d-glucopyranoside (16) showed weak inhibitory activities against NO production with IC50 value of 88.2, 42.1 and 74.3 μM, respectively. The positive control, i.e., dexamathasone, showed an IC50 value of 24.4 μM. The other compounds were found to be inactive against NO production in lipopolysaccharide-induced RAW 264.7 cells (The inhibitory effects of compounds .7 cells .1H-NMR and 13C-NMR spectra were recorded on a Bruker AscendTM 500 spectrometer. Mass spectra (Q-TOFMS) were obtained using an Agilent 6530 ESI-Q-TOF mass spectrometer. Optical rotations were recorded on a JASCO P-2000 polarimeter. UV spectra were measured using a Shimadzu UV-1800 spectrometer. The compounds were isolated using a Gilson preparative HPLC system  was applied to isolate compounds and equipped with binary pumps, an UV/VIS-155 detector, and an GX-271 liquid handler. The semi-preparative HPCCC instrument used in this study was a Spectrum  combined to an IOTA S 300 pump , a Foxy R2 fraction collector , and a CCA-1111 circulatory temperature regulator .Organic solvents for column chromatography were analytical grade and obtained from Daejung Chemical and Metals . Acetonitrile, methanol, and water for HPLC were purchased from Fisher Scientific Korea . Silica gel and reversed-phase silica gel were purchased from Merck (Germany), and Sephadex LH-20 was obtained from Pharmacia Co. (Sweden). HPLC was performed using an YMC-Pack ODS-A column .S. racemosa were collected at Popa Mountain National Park  in August 2011, and identified by Prof. Young Dong Kim . A voucher specimen (# MM-0097) was deposited at herbarium of National Institute of Biological Resources (NIBR) in Korea. Aerial parts of Symplocos racemosa (1012 g) were ground into fine powder, and extracted with methanol under ultrasoniation (3 × 3 h) to yield a methanol extract (52.7 g). The methanol extract was suspended in water and partitioned with n-hexane (7.0 g), ethyl acetate (12.3 g), and n-butanol (9.1 g). The ethyl-acetate fraction was subjected to silica-gel column chromatography (CC) with a step gradient elution of chloroform-methanol  to obtain four subfractions (fractions E1–E4). Fraction E3 (2.2 g) was chromatographed on Sephadex LH-20 CC with a methanol to yield four subfractions (Fractions E3-1–E3-4), and E3-1 was further subjected to reversed-phase (RP) HPLC using an acetonitrile-water mixture (30:70 v/v) to give quercetin-3-O-α-l-rhamnopyranoside (1.6 mg) and mearnsetin-3-O-α-l-rhamnopyranoside (8.0 mg). Kaempferol-3-O-β-d-glucopyranoside, (22 mg) and quercetin-3-O-β-d--glucopyranoside (5.5 mg) were isolated from E3-2 (226 mg) through Sephadex LH-20 CC with methanol followed by repetitive RP- HPLC using acetonitrile-water mixture (30:70 v/v). Fraction E3-3 (610 mg) were subjected to RP-HPLC  to give kaempferol-3-O-β-d-galactopyranoside (1.9 mg), quercetin-3-O-β-d-galactopyranoside (11.9 mg) and quercetin-3-O-β-d-glucopyranoside (16.4 mg). Kaempferol-3-O-β-d--glucopyranoside (2.9 mg) was separated from E4 via Sephadex LH-20 column CC using methanol as the mobile phase, followed by RP-HPLC .The aerial parts of n-butanol (9 g) soluble fraction was subjected to Diaion HP-20 CC to give two fractions [water fraction (BW) and methanol fraction (BM)]. BM (2.7 g) was chromatographed using high-performance countercurrent chromatography (HPCCC) with ethyl acetate–n-butanol–water system  to give fraction BM1–BM4. BM2 (208 mg) was separated by Sephadex LH-20 CC using methanol as an eluent followed by RP-HPLC to yield 3'-O-methylepicatechin-7-O-β-d-glucopyranoside (5.9 mg), 3'-O-methycatechin-7-O-β-d-glucopyranoside (1.6 mg), quercetin-3-O-rutinoside (14.4 mg) and quercetin-3-O-(2G-β-d-xylopyranosylrutinoside (20.2 mg). BM3 (123 mg) was subjected to RP-HPLC using a gradient elution of acetonitrile-water mixture (15%→30% acetonitrile) to yield (−)-epiafzelechin-7-O-β-d-glucopyranoside (2.4 mg), afzelechin-4'-O-β-d-glucopyranoside (1.3 mg), sympracemoside (2.3 mg).The 5 cells/mL in 96-well culture plates. After pre-incubation for 18 h, the cells were pretreated for 30 min with compounds and then stimulated with LPS (500 ng/mL) for 24 h. The supernatant was mixed with an equal volume of Griess reagent  and then incubated at room temperature for 5 min. The concentration of nitrite was determined by measuring the absorbance at 570 nm and comparing the values to a standard curve generated using sodium nitrite (NaNO2).NO production was assayed by measuring the amount of nitrite in the supernatants of cultured RAW 264.7 cells. Brieﬂy, the cells were seeded at a density of 5 × 10


We first reported the new application of a translate metal chelating ligand α-benzoin oxime for improving Cu-catalyzed C-N coupling reactions. The system could catalyse coupling reactions of (hetero)aryl halides with a wide of nucleophiles  in moderate to excellent yields. The protocol allows rapid access to the most common scaffolds found in FDA-approved pharmaceuticals. N-Arylated compounds are ubiquitous synthons in numerous natural products and functional molecules +, see ole (3a) : colorle1-Phenyl-1H-pyrrole (3b) +.ole (3b) : white s3-(1H-Pyrazol-1-yl)benzaldehyde (3c) +.yde (3c) : off whi2-(1H-Pyrazol-1-yl)benzoic acid (3d) +.cid (3d) : white s2-(1H-Pyrazol-1-yl)aniline (3e) +.ine (3e) : brown o1-(4-(1H-Pyrazol-1-yl)phenyl)ethan-1-one (3f) +.one (3f) : yellow Ethyl 3-(1H-pyrazol-1-yl)benzoate (3g) +.ate (3g) : yellow Methyl 4-(1H-pyrazol-1-yl)benzoate (3h) +.ate (3h) : white s2-(1H-Pyrazol-1-yl)benzonitrile (3i) +.ile (3i) : yellow 2-(1H-Pyrrol-1-yl)pyridine (3j) +.ine (3j) : colorle2-(1H-Imidazol-1-yl)pyridine (3k) +.ine (3k) : white s2-(2-Methyl-1H-imidazol-1-yl)pyridine (3l) +.ine (3l) : colorle2-(1H-Imidazol-1-yl)pyrimidine (3m) +.ine (3m) : white s2-(2-Methyl-1H-imidazol-1-yl)pyrimidine (3n) +.ine (3n) : white s2-(Piperidin-1-yl)pyridine (4a) +.ine (4a) : colorle2-(Pyrrolidin-1-yl)pyridine (4b) +.ine (4b) : colorle4-(Piperidin-1-yl)pyridine (4c) +.ine (4c) : colorle4-(Pyrrolidin-1-yl)pyridine (4d) +.ine (4d) : colorle2-(Pyrrolidin-1-yl)pyrimidine (4e) +.ine (4e) : colorle2-(Piperidin-1-yl)pyrimidine (4f) +.ine (4f) : colorle1-(2-Methoxyphenyl)pyrrolidine (4g) +.ine (4g) : colorle1-(2-Methoxyphenyl)piperidine (4h) +.ine (4h) : colorle4-(Pyrrolidin-1-yl)benzaldehyde (4i) +.yde (4i) : white s4-(Piperidin-1-yl)benzaldehyde (4j) +.yde (4j) : white sN,N-Diethylaniline (4k) +.ine (4k) : yellow 2-((4-Nitrophenyl)amino)ethan-1-ol (4l) +.-ol (4l) : yellow tert-Butyl phenyl-D-valinate (5a) +.ate (5a) : white s(4-Nitrophenyl)glycine (5b) +.ine (5b) : brown sPhenyl-D-phenylalanine (5c) +.ine (5c) : white sPhenyl-L-alanine (5d) +.ine (5d) : white sPyrimidin-2-yl-D-valine (5e): white solid , 113–115 °C. 1H-NMR  δ (ppm): 11.24 , 8.25 , 7.22 , 6.55 , 4.62–4.59 , 2.37–2.29 , 1.06 . MS (ESI) m/z: 196.16 [M + H]+, 218.11 [M + H]+. 13 C-NMR  δ (ppm): 176.0, 161.2, 110.4, 59.4, 31.0, 18.9, 18.2.Pyrimidin-2-ylmethionine (5f): white solid . 1H-NMR  δ (ppm): 13.32 , 8.29 , 7.92 , 6.62 , 4.88–4.84 , 2.70–2.64 , 2.37–2.22 , 2.11 . MS (ESI) m/z: 228.13 [M + H]+. 13 C-NMR  δ (ppm): 175.8, 160.3, 110.3, 53.7, 31.9, 30.0, 15.4.N-aryl compounds. This transformation occurs with good to excellent yields. A variety of substituted (hetero)aryl halides can be used as electrophiles, and azoles, piperidine, pyrrolidine, and amino acids, etc. function as nucleophiles. The key to this discovery was the identification of benzoin oxime ligand that can promote the (hetero)aryl halides to the corresponding N-arylation compounds. Efforts to apply our Cu-based system to other catalytic reactions and to expand the scope of the N-nucleophiles to other classes of nucleophiles are currently underway in our laboratory.In summary, a highly effective coupling reaction has been developed for the preparation of