Their structures were determined on the basis of 1D and 2D NMR spectroscopy and HRFABMS. Compounds 1–4 were evaluated for their effects on the production of NO in LPS-activated mouse peritoneal macrophages.A novel gedunin and two novel phragmalin-type limonoids, named carapanolides J–L (compounds  Carapa guianensis Aublet (Meliaceae), known locally as andiroba, is widely distributed in the Amazonas State of Brazil and its wood is extensively used as commercial timber . . Carapa 1) was determined as C26H30O7 ([M + H]+m/z 455.2075) based on HRFABMS. The IR and UV spectra showed bands assignable to a hydroxy group (υmax 3503 cm−1), a six-membered ring ketone (υmax 1727 cm−1), and an α,β-unsaturated six-membered ring ketone . The 1H and 13C NMR spectra , 4.47 (ddd), and 5.49 (s)]; five sp3 quaternary carbons, including an oxycarbon [δC 65.4 (s)]; an α,β-unsaturated six-membered ring ketone ; a saturated ketone [δC 207.7 (s)]; δ-lactone ; and furan ring . In the HMBC spectrum, long-range correlations were observed between Me-18 (δH 1.21) and C-12, C-13, C-14 [δC 65.4 (s)], and C-17 [δC 77.6 (d)]; between Me-19 (δH 1.56) and C-1 (δC 160.2), C-5, C-9, and C-10; between Me-28 (δH 1.17) and C-3 (δC 203.0), C-4, C-5, and C-29; between Me-29 (δH 1.16) and C-3, C-4, C-5, and C-28; between Me-30 (δH 1.28) and C-7 (δC 207.7), C-8, C-9, and C-14; between H-11 (δH 4.47) and C-8, C-9, C-10, C-12, and C-13; between H-15 (δH 3.88) and C-8, C-13, C-14 and C-16 (δC 166.4); and between H-17 (δH 5.49) and C-12, C-13, C-14, C-16, C-18, C-20 [δC 120.0 (s)], C-21 [δC 141.1 (d)], and C-22 [δC 109.7 (s)]  revealed the partial structure shown in 1H-1H COSY spectra revealed that 1 was a 11-hydroxy-7-deacetoxy-7-oxogedunin [H 4.47 (ddd)] was determined to have an α  orientation because significant NOEs were observed between H-11 and Me-19, and Me-30, while coupling constants were observed between H-11β and H-9α ; H-11β and H-12α ; H-11β and H-12β . Therefore, compound 1 was determined to be 11α-hydroxy-7-deacetoxy-7-oxogedunin, which has been thus isolated in Nature for the first time.The molecular formula of carapanolide J ( spectra  exhibite9.7 (s)] . An anal2), which was isolated as a colorless amorphous solid, had the molecular formula C39H50O13  as determined by HRFABMS. The IR spectrum showed the presence of a hydroxyl at υmax 3446 cm−1, and ester groups at υmax 1766, 1735, and 1698 cm−1. The 1H -and 13C-NMR spectra ], an acetyl group , 2-methylbutanoyl group , tigloyl group , methoxycarbonyl group , δ-lactone , two tertiary hydroxyl groups , a tetrasubstituted double bond , and furan ring . In the HMBC spectrum, cross-peaks were observed between Me-18 [δH 1.09 (s)] and C-12, C-13, C-14 [δC 135.4 (s)], and C-17 [δC 80.3 (d)]; between Me-19 [δH 1.14 (s)] and C-1 [δC 83.6 (s)], C-5, C-9, and C-10; between Me-28 [δH 0.83 (s)] and C-3 [δC 88.2 (d)], C-4, C-5, and C-29; between H-3 [δH 4.73 (s)] and C-1, C-2 [δC 77.0 (d)], C-4, C-5, C-28, C-29, C-30 [δC 69.7 (d)], and C-3' [δC 168.5 (s)]; between H-5 [δH 2.88 (dd)] and C-1, C-3, C-4, C-6, C-7 [δC 174.2 (s)], C-10, C-19, C-28, and C-29; between H-30 [δH 5.41 (s)] and C-1, C-2, C-3, C-8 [δC 134.9 (s)], C-9, and C-30' [δC 176.3 (s)]. The positions of the hydroxyl, 2-methylbutanoyl, methoxycarbonyl, and tigloyl groups were identified by detailed 1H-1H COSY and HMBC correlations (C 134.9 (s)]; between H-30, H-15, and C-14 [δC 135.4 (s)] revealed that compound 2 was a phragmalin-8(14)-ene derivative [H 4.73 (s)] and H-29 pro-S, H-30, and Me-28; between H-5 [δH 2.88 (dd)] and Me-28 and H-30; between Me-18 and H-11α and H-12α; between Me-19 and H-11α, between H-15 and H-17β, H-30, H-3', H-5', and H-2'''; therefore, the 2-methylbutanoyl group at C-30 and acetoxy group at C-15 were all α while the tigloyl group at C-3 had a β orientation. The configuration of the 2-methylbutanoyl group at C-30 was deduced to be R because the chemical shift value and NOESY correlation were very similar to that of carapanolide F , an acetyl , propanoyl , and orthoacetyl group , four methylenes, including an oxymethylene , a furan ring , seven sp3 quaternary carbons, including four oxycarbon , two ester carbonyls , and two lactone carbonyl . An analysis of the 1H-1H COSY spectrum of 3 revealed the partial structures shown in bold face in H 1.13 (s)] to C-12, C-13, C-14, and C-17 (δC 78.4); from Me-28 [δH 1.00 (s)] to C-3, C-4, C-5, and C-29; from H-30 [δH 5.71 (s)] to C-1 [δC 85.4 (s)], C-2 [δC 79.5 (s)], C-3 [δC 83.9 (d)], C-8 [δC 86.4 (s)], and C-9 [δC 86.3 (s)] from H-14 [δH 1.00 (s)] to C-8, C-9, C-12, C-13, C-15, and C-16 [δC 169.8 (s)]. Therefore, the planar structure of 3 was established as phragmalin-1,8,9-orthoacetate  and H-29 pro-S, H-30, and Me-28; between H-5β [δH 2.68 (dd)] and H-12β, Me-28, and H-30; between H-15β [δH 3.19 (dd)] and H-30; between H-17β [δH 5.35 (s)] and H-12β, H-15β, H-22, and H-30β, between Me-18 [δH 1.13 (s)] and H-11α, H-12α and Me-32. Therefore, the relative structure of 3 was established as shown in Carapanolide L  inhibitor, were evaluated for their inhibitory effects on NO production in LPS-stimulated RAW264.7 cells . In the inhibitory assay of NO production, compound 1 showed similar inhibitory activities  to the positive control, l-NMMA , with no cytotoxicities. Compound 4 exhibited superior inhibitory activities on NO production at non-toxic concentrations  to those of l-NMMA. These results suggested that compound 1 may be valuable as potential inhibitors of NO production.Physiological nitric oxide (NO) is involved in blood pressure regulation and blood flow distribution, whereas its overexpression may induce tissue injury, multiple organ dysfunction, and death, as well as systemic inflammatory responses in sepsis, such as hypotension, cardiodepression, and vascular hyporeactivity . In the .7 cells . To dete1H- 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. FABMS were recorded on a JEOL-7000 mass spectrometer. Column chromatography was carried out over silica gel  and MPLC was carried out with silica gel . HPLC was run on a JASCO PU-1586 instrument equipped with a differential refractometer (RI 1531). Fractions obtained from column chromatography were monitored by TLC . Melting points were determined on a Yanagimoto micro-melting point apparatus and were uncorrected. Optical rotations were measured using a JASCO DIP-1000 digital polarimeter. IR spectra were recorded using a Perkin-Elmer 1720X FTIR spectrophotometer. Carapa guianensis AUBLET (Meliaceae) was collected in the Amazon, Brazil, in March 2013. Kindly provided by Mr. Akira Yoshino (who is a representative of the NGO “Green Heart Love Amazon Project”). A voucher specimen (CGS-01-2) was deposited in the Herbarium of the Laboratory of Medicinal Chemistry, Osaka University of Pharmaceutical Sciences.The oil of (2.03 kg) Carapa guianensis AUBLET (Meliaceae) (2.03 kg) was dissolved in CHCl3 (1 L) and the CHCl3 solution was subjected to CC (silica gel 14 kg), to afford seven fractions: Fraction A  was eluted with n-hexane-CHCl3 = 1:1, B  was eluted with CHCl3, C  was eluted with CHCl3-EtOAc = 5:1, D  was eluted with CHCl3-EtOAc = 2:1, E  was eluted with CHCl3-EtOAc = 2:1, F  was eluted with EtOAc, G  was eluted with EtOAc:MeOH = 5:1, and H  was eluted with MeOH. Residue D was rechromatographed over a silica gel column (CC)  eluted with n-hexane- EtOAc (1:1) to give 13 fractions: D1 , D2 , D(3) , D(4) , D(5) , D(6) , D(7) , D(8) , D(9) , D(10) , D(11) , and D(12) . Fraction D(6) was subjected to CC  eluted with n-hexane–EtOAc (3:1) to give an amorphous solid (24.1 mg) that was separated by HPLC  to give compounds 2 (6.2 mg) and 3 (1.79 mg). Fraction D(8) was subjected to CC  eluted with n-hexane–EtOAc (3:1) to give an amorphous solid (34.0 mg) that was subjected to CC  eluted with n-hexane–EtOAc (3:1) to give an amorphous solid that was purified by HPLC  to give compounds 1 (7.5 mg) and 4 (3.8 mg). Fraction D(9) was subjected to CC  eluted with n-hexane–EtOAc (3:1) to give an amorphous solid (25.5 mg) that was separated by HPLC  to give compound 3 (6.2 mg).The seed oil of 1. Colorless crystals; mp 172–174 °C (from MeOH-CHCl3); c 0.1, CHCl3); HRFABMS m/z: 455.2075 [M+H]+ ; UV (EtOH) λmax nm (log ε): 230 (3.85), 237 (3.80), 248 (3.63); IR (KBr) υmax cm−1; 3503 (OH), 2926, 1727 (O-C=O), 1671 (C=C-C=O); 1H- and 13C-NMR, see  m/z (rel. int.): 477 , 455 , 83 (100).Compound 2. Colorless amorphous solids; c 0.1, CHCl3); HRFABMS m/z: 749.3152 [M+Na]+ ; UV λmax (EtOH) nm (log ε): 227 (4.19), 304 (3.98), 315 (4.00), 334 (3.72); IR (KBr) υmax cm−1: 3446 (OH), 2967, 1766 and 1735, 1698; 1H- and 13C-NMR, see  m/z (rel. int.): 749 (33) , 727 .Compound 3. Colorless amorphous solids; c 0.1, CHCl3); HRFABMS m/z: 643.2391 [M+H]+ ; UV λmax (EtOH) nm (log ε): 208 (1.26), IR (KBr) υmax cm−1: 3352 (OH), 1742 (O-C=O); 1H- and 13C-NMR, see  m/z (rel. int.): 665 (33) , 643 .Compound 4 cells in 100 μL) were seeded onto 96-well microplates, and incubated for 24 h. D-MEM (100 μL) containing test samples  dissolved in DMSO  was added. After the cells had been treated for 24 h, the MTT solution was added. After 3 h of incubation, 20% sodium dodecyl sulfate (SDS) in 0.1 M HCl was added to dissolve the formazan produced by 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%.RAW264.7 cell proliferation was examined according to a method reported previously  with som4 cells in 100 μL) were seeded onto 96-well microplates, and incubated for 24h. D-MEM (100 μL) containing test samples  dissolved in DMSO  and LPS  were added. After cells had been treated for 24 h, 50 μL of 0.1% N-(1-naphtyl)ethylenediamine in H2O and 50 μL of 1% sulfanylamide in 5% phosphoric acid were added. After being incubated for 30 min, 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%.An inhibitory assay of nitric oxide production was performed according to a method reported previously  with sli1–3), as well as a known gedunin-type limonoid 4 were isolated from the seeds of Carapa guianensis (andiroba). Their structures were determined by spectroscopic analyses. Compound 1 showed similar inhibitory activities  to positive control, l-NMMA , with no cytotoxicity. Known compound 4 exhibited superior inhibitory NO production activities at non-toxic concentrations  to those of l-NMMA. These results suggest that compound 1 may be a valuable potential inhibitor of NO production.A novel gedunin and two novel phragmalin-type limonoids, named carapanolides J–L (compounds


Sarcophyton sp. This approach facilitated the isolation of five new cembrane-type diterpenoids (1–5), along with two known analogs (6 and 7), as well as the identification of 19 further, known compounds. The chemical structures of the new compounds were elucidated while using comprehensive spectroscopic analyses, including one-dimensional (1D) and two-dimensional (2D) NMR and HRMS. All of the isolated cembranoids (1–7) showed moderate in vitro antiproliferative activity against a human breast cancer cell line (MCF-7), with IC50 ranging from 22.39–27.12 µg/mL. This class of compounds could thus serve as scaffold for the future design of anticancer leads.The combination of liquid chromatography coupled to high resolution mass spectrometry (LC-HRESMS)-based dereplication and antiproliferative activity-guided fractionation was applied on the Red Sea-derived soft coral  Sarcophyton  contains 46 species with typically mushroom- or toadstool-shaped appearance and it is considered one of the most abundant Red Sea soft corals , and five methylene groups . HMBC correlations between H2-14 and δC 161.4 (C-1), H-3 and δC 35.5 (C-5), H2-5 and δC 142.0 (C-4) and H2-6 and δC 35.5 (C-5) and H2-6 /H2-5 and δC 74.4 (C-7) established the carbon linkages from C-14 to C-7. Furthermore, correlations between H2-9 and δC 74.3 (C-8)/124.5 (C-10)/140.0 (C-11); H-10 and δC 74.3 (C-8)/74.4 (C-12); H2-13 and δC 74.4 (C-12)/140.0 (C-11)/23.0 (C-14); and finally, H2-14 and δC 161.4 (C-1)/79.2 (C-2) established the connectivity of the 14-membered ring. The positions of the methyl groups were recognized through HMBC correlations between δH 1.85  and C-3 and C-4; δH 1.20  and C-9; δH 1.36  and C-11 and C-12; and, δH 1.86  and C-1, C-15, and C-16. These NMR spectroscopic features were very similar to the reported spectroscopic data of the known compound sinumaximol G, which was previously isolated from the Soft Coral Sinularia maxima , along with the HMBC correlations of H3-2’/C-1’, H-7/C-1’, and H-7/C-19, suggested that the OH group in sinumaximol G at C-7 has been acetylated in compound 1. The relative configuration of 1 was determined on the basis of coupling constants and NOESY experiments (δH 5.48) and H-3 (δH 4.81), together with the 1H-1H NOESY correlations (3-18 (δH 1.85), and both H-2 (δH 5.48) and H-7 (δH 4.82), suggested a β-orientation of both H-2 and H-7. Moreover, the 1H-1H NOESY correlations between H-3 (δH 4.81) and H-20 (δH 1.36) suggested that H3-20 were in the α-orientation. Alterations in the 1H NMR chemical shift of H3-19 (δH 1.2), together with the 1H-1H NOESY correlations between H3-19 (δH 1.2) and H3-18 (δH 1.85), indicated changes in the orientation of the methyl group at C-8 to be in the β-orientation. Therefore, compound 1 was assigned as a new natural product and given the name 7-acetyl-8-epi- sinumaximol G (1).Compound IMS data  that shospectrum , togethespectrum , were in species ,18. 1H N 13C NMR , togethemoieties  H-5/C-4,eriments . The vicelations  between 6) (C20H30O5); m/z 351.2160 [M + H]+ ion  (1 and sinumaximol G (6) + ion at m/z 333.2035   + ion at m/z 333.2035   an α,β-unsaturated-γ-lactone functionality: δC 124.0 (C-15), δC 163.4 (C-1) δC 175.1 (C-16). (ii) olefinic carbons at δC 120.2/144.0 (C-3/C-4) and δC 123.9/135.1 (C-11/C-12), and (iii) a ketone functional group at δC 213.7 (C-7) and alcohol functionalities at δC 79.3 (C-2) and δC 78.2 (C-8). The COSY spectrum  and 4.95 ; δH 2.22 /2.47  and 2.76 /2.93 ; δH 1.98 , 2.20/2.44  and 4.70  and δH 1.81/1.95  and 2.21/2.44 . HMBC correlations (δC 163.4 (C-1), H-2 and δC 120.2 (C-3), H-2 and δC 144.0 (C-4), H-3 and δC 17.8 (C-18), H3-18 and δC 120.2 (C-3), H3-18 and δC 31.5 (C-5), H2-5 and δC 144.0 (C-4), H2-5 and δC 34.2 (C-6) and H2-6/H2-5 and δC 213.7 (C-7); these correlations confirmed the linkage from C-1 to C-7. Additional correlations between H3-19 and δC 213.7 (C-7), H2-9, and δC 78.2 (C-8), H2-10 and δC 35.8 (C-9), H-11 and δC 25.7 (C-10), H3-20 and δC 123.9 (C-11), H2-10 and δC 135.1 (C-12), H3-20 and δC 39.4 (C-13), H2-13 and δC 163.4 (C-1), and H2-14 and δC 163.4 (C-1) confirmed the 14-membered ring skeleton. 1H-1H NOESY correlations (3-18 (δH 1.89) and H-2 (δH 5.44), and between H3-19 (δH 1.26) and both H-3 (δH 4.95) and H-20 (δH 1.53) suggested a β-orientation of H-2 and a α-orientation of H-19. The chemical shifts of H-3 at δH 4.95 and H-11 at δH 4.70 are very characteristic for E configuration at the C-11/C-12 and C-3/C-4 double bonds  x100. The IC50 values (the concentrations of compound required to produce 50% inhibition of cell growth) were also calculated.The human breast cancer cell line (MCF-7) was obtained from the American Type Culture Collection . They were seeded in 96 well microtiter plates at a concentration of 1000–2000 cells/well, 100 µL/well. After 24 h, the cells were incubated for 72 h with the compounds to be tested. Dulbecco’s Modified Eagle Medium (DMEM) with 10% foetal calf serum, sodium pyruvate, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37 °C and 5% COSarcophyton. LC-HRESMS-based dereplication, in combination with biological activity-guided fractionation, allowed for the accelerated characterization of further new bioactive cembrane-type diterpenoids. All of the isolated compounds demonstrated moderate in vitro antiproliferative activity against breast cancer cell line MCF-7. This finding adds to our existing knowledge and understanding regarding the cytotoxic activity of cembranoid diterpenes, indicating that this class of compounds can be utilized as a potential scaffold for the future design of potent anticancer agents.The present study provides an additional chemical characterization of the Red Sea soft coral


Sorbus commixta Hedl. (Rosaceae) has been traditionally used in oriental countries for the treatment of asthma and other bronchial disorders. In this study, a chalcone glycoside was isolated from the ethyl acetate extract of the fruits of this plant. The compound was identified as neosakuranin based on the spectroscopic analysis and comparion with literature data. This is the first report of isolation of neosakuranin from Sorbus commixta. Sorbus commixta Hedl. (Rosaceae) has been used for the treatment of cough, asthma, and other bronchial disorders in East Asian countries, including Korea, China and Japan + ; 1H and 13C-NMR data for 1. 1H-NMR: δ 8.01 , 7.70 , 7.62 , 6.84 , 6.34 , 6.15 , 5.19 , 3.39 - 3.45 and 3.48-3.54 , 3.72 , 3.91 , 3.83 ; 13C-NMR: δ 124.45 (C-α), 143.31 (C-β), 193.43 (C=O), 127.11 (C-1), 130.58 , 159.88 (C-4), 115.60 , 107.00 (C-1’), 160.06 (C-2’), 93.72 (C-3’), 165.87 (C-4’), 95.23 (C-5’), 166.27 (C-6’), 100.63 (C-1"), 73.74 (C-2"), 77.17 (C-3"), 69.94 (C-4"), 77.26 (C-5"), 61.11 (C-6"), 54.85 (OMe).Sorbus commixta fruits led to the isolation of the known chalcone glycoside neosakuranin. This is the first report of the presence of this compound in this species. Phytochemical study of


Stevia rebaudiana and its diterpene glycosides are one of the main focuses of food companies interested in developing novel zero calorie sugar substitutes since the recognition of steviol glycosides as Generally Recognized as Safe (GRAS) by the United States Food and Drug Administration. Rebaudioside A, one of the major steviol glycosides of the leaves is more than 200 times sweeter than sucrose. However, its lingering aftertaste makes it less attractive as a table-top sweetener, despite its human health benefits. Herein, we report the purification of two novel tetra-glucopyranosyl diterpene glycosides 1 and 3 (rebaudioside A isomers) from a commercial Stevia rebaudiana leaf extract compounds, their saponification products compounds 2 and 4, together with three known compounds isolated in gram quantities. Compound 1 was determined to be 13-[(2-O-β-d-glucopyranosyl-6-O-β-d-glucopyranosyl-β-d-glucopyranosyl) oxy]ent-kaur-16-en-19-oic acid-β-d-glucopyranosy ester (rebaudioside Z), whereas compound 3 was found to be 13-[(2-O-β-d-glucopyranosyl-3-O-β-d-glucopyranosyl-β-d-glucopyranosyl) oxy]ent-hydroxyatis-16-en-19-oic acid -β-d-glucopyranosy ester. Two new tetracyclic derivatives with no sugar at position C-19 were prepared from rebaudiosides 1 and 3 under mild alkaline hydrolysis to afford compounds 2 13-[(2-O-β-d-glucopyranosyl-6-O-β-d-glucopyranosyl-β-d-glucopyranosyl) oxy]ent-kaur-16-en-19-oic acid (rebaudioside Z1) and 4 13-[(2-O-β-d-glucopyranosyl-3-O-β-d-glucopyranosyl-β-d-glucopyranosyl) oxy]ent-hydroxyatis-16-en-19-oic acid. Three known compounds were purified in gram quantities and identified as rebaudiosides A (5), H (6) and J (7). Chemical structures were unambiguously elucidated using different approaches, namely HRESIMS, HRESI-MS/MS, and 1D-and 2D-NMR spectroscopic data. Additionally, a high-quality crystal of iso-stevioside was grown in methanol and its structure confirmed by X-ray diffraction. Diabetes is a chronic disease that affected 422 million of people worldwide in 2014 and is increasing considerably every year. Diabetes is characterized by insulin deficit or insulin insensitivity and consequently produces high blood sugar levels which are associated with other disorders 25D −28.0 . HR-ESIMS and HR-ESIMS/MS data of compound 1 showed a molecular ion at m/z 965.4208 [M − H]− , suggesting a molecular formula C44H70O23. Both, the deprotonated molecular ion and an intense product ion m/z = 803.3703 Da resulted from the loss of one hexose at C-19 ([M − H] − H2O − 162 Da)− were observed at 10 eV. Further sequential loss of three hexoses ([M − H] − H2O – 3 × 162 Da)−m/z = 641.3185, m/z = 479.2635 and m/z = 317.2146 from the C-13 moiety was observed at 70 eV. Acid hydrolysis of 1 furnished a mixture of aglycones and only D-glucose which was identified by comparison of the HPLC retention times of thiocarbamoyl thiazolidine derivatives prepared from sugar standards as previously described ent-kaur-16-en-19-oic acid-β-D-glucopyranosy ester (rebaudioside Z) and assigned the structure shown in 1H- and 13C-NMR chemical shifts are shown in ent-kaurene core [The positions of attachment of the sugar moieties were established based on the 2D-NMR HMBC spectrum. 2′′′ δ 4. ppm and 2 was prepared through mild alkaline hydrolysis of 1 to afford a new rebaudioside B isomer with a Glcβ(1-6)[Glcβ(1-2)]Glcβ1- arrangement at C-13 with retention of 9.32 min in the RP-C18 HPLC method that did not match with any of the previously reported steviol-C13 oligosaccharides [2 showed a molecular ion at m/z 803.3721 [M − H]− , suggesting a molecular formula C38H60O18. A sequential loss of three glucoses units at C-13 portion were observed at 70 eV collision energy: m/z 641.3196 (−162 Da), m/z 479.2648 (−162 Da) and m/z 317.2115 Da. 1H-NMR showed three anomeric protons corresponding to the oligosaccharide portion linked at position C-13 . The free carboxylic acid at position C-19 was evidenced by the signal at 184.1 ppm in the 13C-NMR, less shielded than the observed for compound 1. Full structural assignment was performed using 1D- and 2D-NMR  experiments. Compound 2 was named as 13-[(2-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-β-D-glucopyranosyl) oxy]ent-kaur-16-en-19-oic acid (rebaudioside Z1). 1H and 13C chemical shifts are presented in Compound charides . HRESIMS3 was purified as an amorphous off-white solid with [α]25D −20.0 . HR-ESIMS/MS data of compound 3 showed a molecular ion at m/z 965.4297 [M − H]− , suggesting a molecular formula C44H70O23. Both, the deprotonated molecular ion and an intense product ion m/z = 803.3735 Da resultant from the loss of one hexose at C-19 [M – H − H2O − 162 Da]− were observed at 10 eV. Further sequential loss of three hexoses [M – H − H2O – 3 × 162 Da]−m/z = 641.3195, m/z = 479.27.0 and m/z = 317.2141 from C-13 moiety was observed at 70 eV. Compound 3 showed a HPLC retention time of 4.35 min that did not match with any diterpene glycosides in the RP-C18 method previously reported ent-hydroxyatis-16-en-19-oic acid-β-D-glucopyranosy ester and assigned structurally as shown in Four anomeric protons at δng times . Additiog times, . Compoun4 was prepared through alkaline hydrolysis of 3 to afford a rebaudioside B isomer with a with a Glcβ(1-3)[Glcβ(1-2)]Glcβ1- arrangement at C-13 with retention of 10.5 min that did not match with any of the previously reported diterpene glycosides with a free carboxylic acid previously reported in the RP-C18 HPLC method [4 showed a molecular ion at m/z 803.3682 [M − H]− , suggesting a molecular formula C38H60O18. A sequential loss of three glucoses units at C-13 portion at collision energy of 70 eV was observed: m/z 641.3162 (−162 Da), m/z 479.2703 (−162 Da) and m/z 317.2111 Da. 1H-NMR showed three anomeric protons corresponding to the oligosaccharide portion linked at position C-13 . The free carboxylic acid at position C-19 was evidenced by the signal at 184.4 ppm in the 13C-NMR, less shielded than that observed for compound 3. Full structural assignment was performed using 1D- and 2D-NMR experiments. Compound 4 was named as 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]ent-hydroxyatis-16-en-19-oic acid. 1H- and 13C-NMR chemical shifts are presented in − of compounds 1-5 including other related isomers were presented in Compound 5), H (6) and J (7). The purification of the degradation products formed under acidic condition from rebaudioside A and stevioside was recently reported -Glcβ1- were reported [1- at C-13 and 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl) oxy]ent-hydroxyatis-16-en-19-oic acid-β-D-glucopyranosy ester Glcβ(1-3)[Glcβ(1-2)]-Glcβ1- at C-13 with a different aglycone may serve as models to provide important findings to better understand the relationship between sugar arrangement and positions with sweet/bitter flavors. To date, several steviol glycoside isomers have been isolated from S. rebaudiana mainly with different sugar arrangements, position of the attachment to the aglycone and also with different aglycones as is the case of stevioside (with Glcβ(1-2)Glcβ1- at C-13)/12-α-[(2-O-β-D-glucopyranosyl-β-D-glucopyranosyl) oxy]ent-kaur-16-en-19-oic acid-β-D-glucopyranosyl ester (Glcβ(1-2)Glcβ1- at C-12) [1- at C-19) /rebaudioside Y (Glcβ(1-6)Glcβ1- at C-19) [1- at C-13)/rebaudioside R (Glcβ(1-2)[Glcβ(1-3)]-Xylβ1- at C-13)/rebaudioside F isomer (with Glcβ(1-2)[Xylβ(1-3)]-Glcβ1- at C-13) [1- at C-19)/rebaudioside I (Glcβ(1-3)Glcβ1- at C-19) [1- at C-13)/6-deoxyGlcβ(1-2)[Glcβ(1-3)]Glcβ1-/rebaudioside F  with additional 10 parts methanol and 10 parts water and 5 parts acetic acid). Column analysis . 1 × 1 L forerun and 48 × 120 mL fractions were collected and analyzed by HPLC. Column analysis allowed us to select five main pools. Chromatography 5, fraction 1.5 (67.1 mg); fraction 2.5 (135 mg); fraction 3.5 (226 mg); fraction 4.5 (179 mg) and fraction 5.5 (179 mg). Compound 1  was obtained from fraction 3.5.The supernatant second crop (7.02 g) was absorbed onto 70 g of Celite and subjected to a high efficiency normal-phase chromatography  with acetonitrile: Hanalysis . Chromatanalysis . Chromatanalysis  allowed 2O:AcOH (100:0.1 v/v/%) and ACN:H2O:AcOH (5:95:0.1 v/v/%) to afford seven main fractions. Chromatography 6, fraction 1.6 (175 mg); fraction 2.6 (120 mg); fraction 3.6 (108 mg); fraction 4.6 (169 mg); fraction 5.6 ; fraction 6.6 (156 mg) and fraction 7.6 (13 mg). Chromatography 7, fraction 1.6 (175 mg) was submitted to a RP-C18  chromatography using H2O: AcOH (100:0.1) and ACN: H2O: AcOH (10:90:0.1) to afford two main fractions., fraction 1.7  and fraction 2.7 (48.3 mg). Compound 3 was obtained from fraction 1.7. Additionally, several hundred grams from the initial crystallization and subsequent chromatographies of rebaudioside A after processing 1.5 kg of commercial extract. Rebaudiosides J (1 g) and H (1.8 g) were also isolated.Fractions rich in rebaudioside H were pooled (3.7 g) and chromatographed over a RP-C18 (7.5 i.d. ×50 cm) column with H1 (40 mg) and 3 (30 mg) were heated individually with NaOH (1 N) at 80 °C over 1 h. Each reaction mixture was cooled over 5 min and neutralized with two or three drops of acetic acid glacial 25D −28.0 . HR-ESIMS/MS m/z 965.4222 [M − H]− , m/z 803.3703 at 10 eV collision energy, loss of one hexose (−162 Da) from C-19 moiety, m/z 641.3185 (−162 Da), 479.2635 (−162 Da) and 317.2146 (−162 Da), loss of three hexoses from C-13 moiety at 70 eV collision energy. 1H- and 13C-NMR spectroscopic data are shown in 1Rebaudioside Z (2): Amorphous off-white solid; [α]25D −46.0 . HR-ESIMS/MS m/z 803.3721 [M − H]− , m/z 641.3196 (−162 Da), 479.2648 (−162 Da) and 317.2115 (−162 Da), loss of three hexoses from C-13 moiety at 70 eV collision energy. 1H- and 13C-NMR spectroscopic data are shown in d-glucopyranosy ester13-[(2-O-β-D-Glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl) oxy]ent-hydroxyatis-16-en-19-oic acid-β- (3): Amorphous off-white solid; [α]25D −22.0 , HR-ESIMS/MS m/z 965.4297 [M − H]− , m/z 803.3735 at 10 eV collision energy (−162 Da), loss of one hexoses from C-19 moiety, m/z 641.3195 (−162 Da), 479.2703 (−162 Da) and 317.2141 (−162 Da), loss of three hexoses from C-13 moiety at 70 eV collision energy. 1H- and 13C-NMR spectroscopic data are shown in 13-[(2-O-β-D-Glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl) oxy]ent-hydroxyatis-16-en-19-oic acid (4): Amorphous off-white solid; [α]25D −20.0 . HRESIMS/MS m/z 803.3682 [M − H]− , m/z 641.3162 (−162 Da), m/z 479.2703 (−162 Da) and m/z 317.2111 Da at 70 eV collision energy. 1H- and 13C-NMR spectroscopic data are shown in 1–4 were injected in the RP C-18 Luna (2), Phenomenex  column at 30 °C. The elution was performed using gradient of elution as follows: 0–5 min, 32% B; 5–13 min, 32–41% B; 13–16 min, 41–43% B; 16–17 min: 43–50% B; 17–23 min, 50% B. The chromatogram was recorded at 205 nm and the flow rate set at 1 mL/min ent-hydroxyatis-16-en-19-oic acid-β-D-glucopyranosy ester (3) were isolated from a partially processed commercial extract of S. rebaudiana and two new rebaudioside B isomers, rebaudiosides Z1 and 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl) oxy]ent-hydroxyatis-16-en-19-oic acid were prepared and purified for the first time, respectively. An additional three known compounds, rebaudiosides A, H and J, were purified in gram quantities. Scarce rebaudioside A isomers have been reported from Stevia rebaudiana differing in sugar arrangement and type of sugar at position C-13 (Glcβ(1-6)[Glcβ(1-3)]-Glcβ1-) and Glcβ(1-2)[Fruβ(1-3)]-Glcβ1-. However, herein we describe the occurrence of two rebaudioside A isomers with (Glcβ(1-6)[Glcβ(1-2)]-Glcβ1-) and (Glcβ(1-3)[Glcβ(1-2)]-Glcβ1-) at position C-13. However, compound 3 showed a different aglycone and was found to be a 13(S)-hydroxyatisenoic acid type. This finding may contribute to better understanding of the relationship between sweet/bitter taste of Stevia glycosides with their sugar arrangements. Several new compounds have been reported in recent years, however, a systematic study comparing organoleptic properties with structure of diterpene glycosides is still not available in literature.Two new rebaudioside A isomers, rebaudiosides Z (


Some of the obtained compounds exhibited at milimolar range moderate enzyme inhibitory properties against homoaconitase and/or homoisocitrate dehydrogenase of Candida albicans. The structural basis for homoisocitrate dehydrogenase inhibition was revealed by molecular modeling of the enzyme-inhibitor complex. On the other hand, the trimethyl ester forms of some of the novel compounds exhibited antifungal effects. The highest antifungal activity was found for trimethyl trans-homoaconitate, which inhibited growth of some human pathogenic yeasts with minimal inhibitory concentration (MIC) values of 16–32 μg/mL.Thirteen structural analogs of two initial intermediates of the  Microbial resistance to antibiotics and synthetic drugs is an emerging challenge for clinicians and the pharmaceutical industry. The multidrug-resistant fungi are the major cause of failure in chemotherapy of disseminated mycoses ,2. Thus,l-Lysine is an essential amino acid for humans, while bacteria, plants and fungi have developed pathways of lysine biosynthesis. There are two versions of these anabolic routes: a diaminopimelic acid pathway, which is characteristic for bacteria, plants and lower fungi and an α-aminoadipate pathway (AAP) for higher fungi, Ascomycetes and Basidomycetes, including most of the human pathogenic yeasts and filamentous fungi. Enzymes that catalyze the reactions of the above mentioned first pathway are considered promising molecular targets for antibacterial chemotherapy . Rf = 0.54 (solvent system A). 1H-NMR (CDCl3) δ [ppm]: 2.54 ; 3.09 ; 3.67 ; 3.78 ; 3.82 ; 6.82 ; 13C-NMR (CDCl3) δ[ppm]: 23.58; 33.28; 51.92; 52.14; 52.91; 127.94; 146.12; 166.01; 167.10; 173.08. Elemental anal. (%), calcd. for C10H14O6: C, 52.17; H, 6.09; found: C, 52.14; H, 5.93.Trimethyl cis-homoaconitate . Rf = 0.47 (solvent system A). 1H-NMR (CDCl3) δ [ppm]: 2.55 ; 2.69 ; 3.69 ; 3.73 ; 3.83 ; 5.90 ; 13C-NMR (CDCl3) δ [ppm]: 29.16; 31.49; 51.86; 51.91; 52.44; 120.86; 147.67; 165.21; 168.51; 172.08. Elemental anal. (%), calcd. for C10H14O6: C, 52.17; H, 6.09; found: C, 52.07; H, 5.97.Tripotassium trans-homoaconitate . Trimethyl trans-homoaconitate  was dissolved in anhydrous diethyl ether (20 mL). Potassium trimethylsilanolate  was added to the solution and the mixture was stirred for 24 h. The solvent was removed by evaporation under reduced pressure, the residue was washed with anhydrous diethyl ether and finally dried under reduced pressure. The product was obtained as a slightly brownish powder . 1H-NMR (D2O) δ [ppm]: 2.54 ; 2.74 ; 6.51 . 13C-NMR (D2O) δ [ppm]: 23.54; 33.32; 51.82; 52.18; 52.99; 127.90; 146.02. Elemental anal. (%), calcd. for C7H5O6K3: C, 27.63; H, 2.30; found: C, 27.69; H, 2.27.Trimethyl (1E)1,2-epoxy-butane-1,2,4-tricarboxylate (3). Tripotassium trans-homoaconitate  was dissolved in water (5 mL) and tungstic acid  and 30% H2O2 (300 μL) were added. The mixture was stirred for 2 h at 85 °C. Then 10 M HCl (0.35 mL) was added and the reaction mixture was subjected to continuous extraction with diethyl ether for several hours. The organic solvent was removed from the extract by evaporation, to give 172 mg of a slightly yellowish oil. The ethereal solution of diazomethane was then added dropwise at 0 °C until pH of the solution reached 7.0, then the mixture was left overnight at room temperature. After solvent evaporation, 216 mg of the crude oily product was obtained, that was further purified by column silica gel chromatography eluting with hexane/AcOEt (5:1), followed by AcOEt. Finally, after solvent evaporation, a slightly yellow oily product was obtained ; Rf = 0.57 (solvent system B). 1H-NMR (CDCl3) δ [ppm]: 2.39–2.33 ; 2.63–2.53 ; 2.75–2.67 ; 3.67 ; 3.69 ; 3.70 ; 3.74 ; 13C-NMR (CDCl3) δ [ppm]: 31.57; 45.64; 52.19; 52.92; 52.97; 53.21; 60.05; 168.16; 170.82; 173.23. Elemental anal. (%), calcd. for C10H14O7: C, 48.78; H, 5.69; found: C, 48.67; H, 5.63.Tripotassium (1E)1,2-epoxy-butane-1,2,4-tricarboxylate (4). Trimethyl (1E)1,2-epoxybutane-1,2,4-tricarboxylate  was dissolved in anhydrous diethyl ether (10 mL). Potassium trimethylsilanolate  was added to the solution and the mixture was stirred for 24 h. The solvent was removed by evaporation under reduced pressure, the residue was washed with anhydrous diethyl ether and finally dried under reduced pressure. The product was obtained as a yellow amorphous powder . 1H-NMR (D2O) δ [ppm]: 2.29–2.35 ; 2.57–262 ; 2.66–2.71 ; 3.78 ; 13C-NMR (CDCl3) δ [ppm]: 31.32; 45.68; 51.88; 53.15; 53.24; 53.48; 61.60. Elemental anal. (%), calcd. for C7H5O7K3: C, 27.27; H, 1.62; found: C, 27.27; H, 1.65.General procedure for the synthesis of compounds5a–c. n-BuLi  was added dropwise to a stirred solution of diisopropylamine (62.6 mmol) in THF (38 mL) at −78 °C under argon. After 1 h, a solution of dimethyl (S)-malate  in THF (6 mL) was added, while the temp. was kept at −78 °C. The mixture was then warmed to −25 °C and stirred for 1 h. After that time, the mixture was again chilled to −78 °C and propyl, butyl or allyl bromide (53 mmol) was added dropwise. The resulting solution was stirred for 2 h at −25 °C and then left overnight at 4 °C. The mixture was chilled to −78 °C, acetic acid (6.4 mL in 10 mL of Et2O) was added and the resulting mixture was combined with Et2O (250 mL) and water (30 mL). The organic layer was washed with the saturated solution of sodium carbonate and with saline. Aqueous layers were combined and extracted three times with Et2O. Combined ethereal layers were dried over anhydrous MgSO4 and the solvent was evaporated. The crude products were purified by flash silica-gel chromatography with hexane/AcOEt (5:1) as an eluent to afford the final products.Dimethyl -2-hydroxy-3-propylsuccinate (5a). Oil, final yield 58%. [α3). 1H-NMR (CDCl3) δ [ppm]: 0.95 , 1.41 , 1.66 , 1.83 , 3.69 ; 3.80 ; 4.29 ; 13C-NMR (CDCl3) δ [ppm]: 14.08; 20.79; 30.36; 48.54; 52.18; 52.93; 71.22; 173.74; 174.18. Elemental anal. (%), calcd. for C9H16O5: C, 52.94; H, 7.84; found: C, 52.87; H, 7.90.Dimethyl -2-hydroxy-3-butylsuccinate (5b). Oil, final yield 65%. [α3). 1H-NMR (CDCl3) δ [ppm]: 0.94 , 1.39 , 1.68 , 1.89 , 3.70 ; 3.77 ; 4.29 ; 13C-NMR (CDCl3) δ [ppm]: 12.01; 18.45; 21.01; 30.63; 48.91; 51.65; 52.98; 70.88; 173.11; 174.55. Elemental anal. (%), calcd. for C10H18O5: C, 55.05; H, 8.26; found: C, 54.98; H, 8.30.Dimethyl -2-hydroxy-3-allylsuccinate (5c). Oil, final yield 62%. [α3). 1H-NMR (CDCl3) δ [ppm]: 2.45 ; 2.63 ; 2.99 CHCO2CH3); 3.69 ; 3.81 ; 4.31 CHCO2CH3); 5.19–5.11 ; 5.82 ; 13C-NMR (CDCl3) δ [ppm]: 32.36; 48.38; 52.27; 52.99; 70.36; 118.30; 134.91; 172.78; 174.21. Elemental anal. (%), calcd. for C9H14O5: C, 53.47; H, 6.93; found: C, 53.37; H, 7.01.General procedure for the synthesis of compounds6a–c. DAST  was dissolved in anhydrous methylene chloride (30 mL) and the resulting solution was chilled to −78 °C under argon. An analogously chilled solution of one of the compounds 5a–c (4.4 mmol) in methylene chloride (10 mL) was added dropwise using a cannulated syringe and the resulting mixture was stirred for 1 h at −78°C and for 2 h at room temperature. Chloroform (40 mL) was added and the resulting solution was washed with 5% NaHCO3, water and saline. The solvent was evaporated and the crude products were purified by flash silica-gel chromatography with hexane/AcOEt (9:1) to afford the final products.Dimethyl -2-fluoro-3-propylsuccinate (6a). Oil, final yield 92%. [α3). 1H-NMR (CDCl3) δ [ppm]: 0.93 , 1.32 , 1.43 , 1.62 , 1.82 , 2.98 , 3.74 ; 3.82 ; 5.16 ; 13C-NMR (CDCl3) δ [ppm]: 14.07; 20.67; 29.30; 47.91 (2JFC = 23.8 Hz); 52.43; 52.85; 88.92 (JFC = 197.3 Hz); 169.04; 172.21. 19F-NMR (CDCl3) δ[ppm]: −197.51 . Elemental anal. (%), calcd. for C9H15O4F: C, 52.43; H, 7.28; found: C, 52.34; H, 7.35.Dimethyl -2-fluoro-3-butylsuccinate (6b). Oil, final yield 88%. [α3). 1H-NMR (CDCl3) δ [ppm]: 0.93 , 1.24 , 1.32 , 1.41 , 1.53 , 1.62 ; 1.82 ; 2.99 , 3.74 ; 3.82 ; 5.17 ; 13C-NMR (CDCl3) δ [ppm]: 12.01; 18.45; 21.01; 30.63; 48.91; 51.65; 52.98; 70.88; 173.11; 174.5514.07; 20.67; 29.30; 47.91 (2JFC = 23.8 Hz); 52.43; 52.85; 88.92 (JFC = 197.3 Hz); 169.04; 172.21. 19F-NMR (CDCl3) δ [ppm]: −197.46 . Elemental anal. (%), calcd. for C10H17O4F: C, 54.55; H, 7.73; found: C, 54.48; H, 7.73.Dimethyl -2-fluoro-3-allylsuccinate (6c). Oil, final yield 89%. [α3). 1H-NMR (CDCl3) δ [ppm]: 2.46 ; 2.60 ; 3.10 CHCO2CH3), 3.73 ; 3.83 ; 5.11 ; 5.18 ; 5.24 ; 5.76 ; 13C-NMR (CDCl3) δ [ppm]: 38.21; 47.74 (2JFC = 21.5 Hz); 52.13; 52.62; 88.20 (JFC = 190.2 Hz); 119.57; 133.08; 165.68; 169.06; 19F NMR (CDCl3) δ[ppm]: -198.16 . Elemental anal. (%), calcd. for C9H13O4F: C, 52.94; H, 6.37; found: C, 53.02; H, 6.40.Dipotassium -2-fluoro-3-allylsuccinate (7c). Dimethyl -2-fluoro-3-allylsuccinate  was dissolved in anhydrous diethyl ether (20 mL). Potassium trimethylsilanolate  was added to the solution and the mixture was stirred for 24 h. The solvent was removed by evaporation under reduced pressure, the residue was washed with anhydrous diethyl ether and finally dried under reduced pressure. The product was obtained as a slightly yellow amorphous powder . 1H-NMR (CDCl3) δ [ppm]: 2.44 ; 2.59 ; 3.14 CHCO2CH3), 5.11 ; 5.20 ; 5.25 ; 5.75 ; 13C-NMR (CDCl3) δ[ppm]: 38.44; 47.07 (2JFC = 23 Hz); 52.35; 53.62; 88.20 (JFC = 192 Hz); 118.22; 135.69; 19F-NMR (CDCl3) δ [ppm]: −197.55 . Elemental anal. (%), calcd. for C7H7O4FK2: C, 33.33; H, 2.78; found: C, 33.39; H, 2.79.Dimethyl -2-fluoro-2-deoxyhomoisocitrate (8). BH3 × SMe2  was added dropwise to a solution of dimethyl -2-fluoro-3-allyl succinate  in methylene dichloride (8 mL) at 0 °C. The reaction mixture was left at room temperature with stirring for 2 h. Then pyridinium dichromate  in anhydrous DMF (30 mL) was slowly added and stirring was continued for 48 h. After that time, the mixture was transferred to the flask containing water (300 mL) and the whole solution was stirred for another 16 h. The resulting mixture was extracted with diethyl ether (3 × 250 mL) and the aqueous layer was further extracted with ethyl acetate (2 × 170 mL). The organic layers were combined and dried over anhydrous MgSO4. The drying agent was removed and the organic solvents were evaporated under reduced pressure to give 1.2 g of the crude product that was further purified by flash chromatography on silica gel with stepwise elution with hexane/AcOEt (5:1), AcOEt and MeOH to give 0.87 g (73%) of the purified product. 1H-NMR (CDCl3) δ [ppm]: 2.02 ; 2.21 ; 2.48 ; 3.03 ; 3.76 ; 3.83 ; 5.32–5.22 ; 13C-NMR (CDCl3) δ [ppm]: 23.59; 31.80; 47.77; 51.98; 52.34; 53.09; 172.66; 173.52. 19F-NMR (CDCl3) δ [ppm]: −196.23 . Elemental anal. (%), calcd. for C9H13O6F: C, 45.76; H, 5.50; found: C, 45.71; H, 5.47.Trimethyl -2-fluoro-2-deoxyhomoisocitrate . Dimethyl -2-fluoro-2-deoxyhomoisocitrate  was treated with diazomethane solution in diethyl ether under argon. The mixture was left overnight at room temperature. The solvent was then removed by evaporation under reduced pressure. The residue was washed twice with diethyl ether. The crude oily product was purified by flash chromatography on silica gel with hexane/AcOEt (2:1) as an eluent. The final product was obtained as a colorless oil . [α3). 1H-NMR (CDCl3) δ [ppm]: 2.04 ; 2.21 ; 2.48 ; 3.03 ; 3.71 ; 3.72 ; 3.83 ; 4.63 ; 13C-NMR (CDCl3) δ [ppm]: 23.59; 31.80; 47.77; 51.98; 52.34; 53.09; 71.34; 172.66; 173.52; 173.84. 19F-NMR (CDCl3) δ [ppm]: −193.44 . Elemental anal. (%), calcd. for C10H15O6F: C, 50.00; H, 6.25; found: C, 50.07; H, 6.22.Tripotassium -2-fluoro-2-deoxyhomoisocitrate . Conversion of the trimethyl ester 9 into the respective tripotassium salt was achieved upon treatment with potassium trimethylsilanolate under condition described above for preparation of compound 4. The product was obtained as a yellowish amorphous powder . 1H-NMR (D2O) δ [ppm]: 2.02 ; 2.25 ; 2.52 ; 3.01 ; 4.68 ; 13C-NMR (CDCl3) δ [ppm]: 23.48; 31.85; 47.72; 51.95; 52.38; 53.00; 71.31. 19F-NMR (CDCl3) δ[ppm]: −192.58 . Elemental anal. (%), calcd. for C7H6O6FK3: C, 48.58; H, 4.86; found: C, 48.51; H, 4.92.in vitro activity was determined by the modified M27-A3 method specified by the CLSI [Candida albicans ATCC 10231, Candida glabrata DSM 11226, Candida tropicalis KKP 334 Candida krusei DSM 6128, Candida lusitaniae DSM 70102 and Saccharomyces cerevisiae ATCC 9763. Wells containing serially diluted examined compounds and compound-free controls were inoculated with 12 h cultures of tested strains to the final concentration of 104 colony-forming units (cfu)/mL. Plates were incubated for 24 h at 37 °C and growth was then quantified by measuring an optical density at 531 nm, using a microplate reader . The MIC was defined as the lowest drug concentration at which at least 80% decrease in turbidity, in comparison to the drug-free control, was observed.Antifungal the CLSI  in RPMI-C. albicans homoaconitase (HA) and homoisocitrate dehydrogenase (HIcDH) containing oligoHis N-terminal tags were constructed and purified to near homogeneity as described earlier [M = 1.5 mM for (R)-homocitrate and recombinant HIcDH showed KM = 74 μM for -homoisocitrate and KM = 1.1 mM for NAD+.Recombinant  earlier ,17,18. B2, 5 mM NAD+ and 0.1 mM -homoisocitrate in 50 mM Hepes-NaOH (pH 7.8) buffer. The reaction mixture with all required components except for the enzyme was preincubated for 3 min and the reaction was started by an addition of the recombinant His6-HIcDH (50 μg). The formation of NADH was measured for 30 s at 20 °C to determine initial reaction velocity. The initial rates were determined using a linear portion of the reaction progress curve.Reaction catalyzed by homoisocitrate dehydrogenase (HIcDH) was monitored by measuring the NADH absorption at 340 nm. The standard assay mixture contained 10 mM KCl, 5.0 mM MgCl+ to NADH at 340 nm [2, 20 mM dithiothreitol, 1 mM NAD+ and 30 μg/mL of His6-HIcDH in a total volume of 0.9 mL were degassed under argon for 10 min, after which 0.05 mL of His6-HAC solution was added to a final concentration of 10 μg/mL. The reaction was initiated with addition of 0.05 mL of 50 mM (R)-homocitrate. The formation of NADH was measured for 30 s at 20 °C to determine initial reaction velocity. The initial rates were determined using a linear portion of the reaction progress curve. Activity of homoaconitase (HA) was measured by a coupled reaction with HIcDH by monitoring the reduction of NADt 340 nm . Reactio50 values, i.e., concentrations causing 50% inhibition of enzyme activity in relation to control. All determinations were performed in triplicate. The effects of novel compounds on the HA and HIcDH activity was determined by measuring the enzyme activity in standard assay mixtures containing various concentration of an inhibitor (0–10 mM). Inhibitory potential was expressed as ICSchisosaccharomyces pombe HIcDH [Model of the receptor (CaHIcDH) was built using the template of be HIcDH , as descbe HIcDH . Dockingbe HIcDH . Initialcis-homoaconitate, namely the tripotassium salts of trans-homoaconitate and (1E)1,2-epoxybutane-1,2,4-tricarboxylate were found to inhibit activity of C. albicans HA, while tripotassium -2-fluoro-3-allylsuccinate and tripotassium -2-fluoro-2-deoxyhomo-isocitrate inhibited HIcDH from the same source. The trimethyl ester derivatives of these compounds inhibited growth of some human pathogenic yeast, including C. albicans. We have been thus able to demonstrate for the first time that inhibitors of particular enzymes catalyzing initial steps of fungi-specific AAP pathway may exhibit antifungal activity after conversion into more lipophilic, membrane-permeable forms. The compounds described in this work may serve as leads for further studies on the design of compounds targeting AAP as potential antifungals. In light of our results, it seems very likely that the ester forms of strong inhibitors of the enzymes of AAP may exhibit good antifungal potential, so that a further search for such compounds as potential antifungal pro-drugs is undoubtedly worth pursuing.Two analogs of The authors declare no conflict of interests.


An improved Helferich method is presented. It involves the glycosylation of 4-methyl-umbelliferone with glycosyl acetates in the presence of boron trifluoride etherate combined with triethylamine, pyridine, or 4-dimethylaminopyridine under mild conditions, followed by deprotection to give fluorogenic 4-methylumbelliferyl glycoside substrates. Due to the use of base, the glycosylation reaction proceeds more easily, is uncommonly α- or β-stereoselective, and affords the corresponding products in moderate to excellent yields (51%–94%) under appropriate conditions. Salmonella and enterohemorrhagic Escherichia coli, is essential for realizing and managing microbiological risks to ensure food safety, a worldwide public health concern + calcd for C23H24NaO12: 515.1160; found: 515.1164. 1H- and 13C-NMR, and HRMS spectrograms are seen in the ); {lit.  mp: 189–4-Methylumbelliferyl acetate (5) Yellowish white powder; mp: 148–150 °C; fR = 0.35 . 1H-NMR : δ = 7.54 , 7.04 , 7.01 , 6.20 , 2.36 , 2.27 . 13C-NMR : δ = 168.78 (OAc), 160.51 (C-2), 154.16 (C-7), 153.04, 151.95, 125.41, 118.12, 117.85, 114.52, 110.47 , 21.13 (C-Me), 18.74 (C-Me). HRMS (ESI): m/z [M + Na]+ calcd for C12H10NaO4: 241.0471; found: 241.0477. 1H- and 13C-NMR, and HRMS spectrograms are seen in the d-glucopyranoside4′-Methylumbelliferyl 2,3,4,6-tetra-O-acetyl-β- (13b) Compound 13b was prepared according to the general procedure for the glycosylation step using 4-MU , glycosyl acetate , dry ClCH2CH2Cl (3 mL), corresponding molar equivalents of pyridine and BF3·OEt2. The mixture was stirred for 5 h at 60 °C ; fR = 0.27 ; {lit. + calcd for C24H26NaO12: 529.1316; found: 529.1323. 1H- and 13C-NMR, and HRMS spectrograms are seen in the ); {lit.  mp: 144 3); lit.  mp: 144–d-glucopyranoside4′-Methylumbelliferyl 2,3,4,6-tetra-O-acetyl-α- (23b) White powder; mp: 132–133 °C; c = 0.30, CHCl3); fR = 0.31 ; {lit. + calcd for C24H26NaO12: 529.1316; found: 529.1321. 1H- and 13C-NMR, and HRMS spectrograms are seen in the ); {lit.  mp: 131–d-galactopyranoside4′-Methylumbelliferyl 2,3,4,6-tetra-O-acetyl-α- (3c) Compound 3c was prepared according to the general procedure for the glycosylation step using 4-MU , β-d-galactose pentaacetate , dry ClCH2CH2Cl (6 mL), TEA  and BF3·OEt2 . The mixture was stirred for 5 h at 60 °C ; fR = 0.32 ; {lit. + calcd for C24H26NaO12: 529.1316; found: 529.1323. 1H- and 13C-NMR, and HRMS spectrograms are seen in the ); {lit.  mp: 186.d-mannopyranoside4′-Methylumbelliferyl 2,3,4,6-tetra-O-acetyl-α- (3d) Compound 3d was prepared according to the general procedure for the glycosylation step using 4-MU , β-d-mannose pentaacetate , dry ClCH2CH2Cl (9 mL), pyridine  and BF3·OEt2 . The mixture was stirred for 5 h at 60 °C ; fR = 0.28 ; {lit. + calcd for C24H26NaO12: 529.1316; found: 529.1322. 1H- and 13C-NMR, and HRMS spectrograms are seen in the ); {lit.  mp 161–14′-Methylumbelliferyl 2,3,4-tri-O-acetyl-β-D-xylopyranoside (3e) Compound 3e was prepared according to the general procedure for the glycosylation step using 4-MU , β-d-xylose tetraacetate , dry CH2Cl2 (6 mL), TEA  and BF3·OEt2. The mixture was stirred for 5 h at 20–21 °C (as shown in 3). fR = 0.33 ; {lit. + calcd for C21H22NaO10: 457.1105; found: 457.1112. 1H- and 13C-NMR, and HRMS spectrograms are seen in the ); {lit.  mp: 158 d-ribofuranoside4′-Methylumbelliferyl 2,3,5-tri-O-acetyl-β- (3f) Compound 3f was prepared according to the general procedure for the glycosylation step using 4-MU , β-d-ribose tetraacetate , dry CH2Cl2 (6 mL), TEA  and BF3·OEt2 . The mixture was stirred for 3 h at 20–21 °C . Little purer colorless syrup obtained by HPLC separation was analyzed by NMR spectrometers and HRMS. 1H-NMR : δ = 7.45 , 6.92–6.85 , 6.10 , 5.63 , 5.47–5.42 , 4.38 , 4.31 , 3.98 , 2.33 , 2.10, 2.04, 1.87 . 13C-NMR : δ = 170.47, 169.75, 169.57 (3× OAc), 161.04 (C-2′), 158.71 (C-7′), 154.80, 152.36, 125.71, 114.96, 113.27, 112.84 , 103.95 (C-8′), 102.90 (C-1), 79.57, 74.78, 70.71, 63.22 , 20.64, 20.58, 20.51 (3× OAc), 18.67 (C-Me′). HRMS (ESI): m/z [M + Na]+ calcd for C21H22NaO10: 457.1105; found: 457.1111. 1H- and 13C-NMR, and HRMS spectrograms are seen in the d-glucopyranosiduronic acid4-Methylumbelliferyl β- (4a) To a stirred suspension of Ba(OH)2·H2O  in MeOH (6 mL) and distilled water (2.4 mL) in an ice-water bath under an argon atmosphere was added compound 3a . The mixture was stirred for 4 h in an ice-water bath, then glacial acetic acid was added carefully into to adjust the pH value to 7.5–8.0. The straw yellow solid precipitation was filtered off, washed with MeOH, then further purified by recrystallization using MeOH, and dried. The obtained barium salt product (105 mg) was added to fresh MeOH (5 mL) in an ice-water bath again, acidified with H2CO4·2H2O . After stirring for 0.5 h, the mixture was filtered. The filter residue was washed with 2–3 mL MeOH, and the merged filtrate was evaporated to a syrup under reduced pressure. The final product was crystallized from anhydrous ether, washed with a small account of cold acetone, and dried. White powder; yield: 42 mg (47%); mp: 140–144 °C; c = 0.20, H2O); {lit. + calcd for C16H16NaO9: 375.0687; found: 375.0692. 1H- and 13C-NMR, and HRMS spectrograms are seen in the ); {lit.  mp: 139–e); lit.  mp: 139–O); lit.  mp: 139–O); lit.  mp: 140–d-glucopyranoside4-Methylumbelliferyl β- (14b) Compound 13b  was O-deacetylated for 1 h at room temperature with KOH  in anhydrous MeOH (3 mL). Concentration of the solution gave a crystalline material that was filtered off, washed with ethanol until the washings were no longer fluorescent, and dried. White crystal; yield: 56 mg (83%); mp: 209–211 °C; c = 0.30, H2O); {lit. + calcd for C16H18NaO8: 361.0894; found: 361.0900. 1H- and 13C-NMR, and HRMS spectrograms are seen in the O); lit.  mp: 211–d-galactopyranoside4-Methylumbelliferyl α- (4c) Compound 3c  was O-deacetylated for 15 min at room temperature with KOH  in dry tetrahydrofuran (8 mL) and anhydrous MeOH (4 mL). The white solid precipitate was filtered off, washed with ethanol until the washings were no longer fluorescent, and dried. White powder; yield: 214 mg (80%); mp: 222–223 °C; c = 0.30, H2O); {lit. + calcd for C16H18NaO8: 361.0894; found: 361.0897. 1H- and 13C-NMR, and HRMS spectrograms are seen in the ); {lit.  mp: 212–d-mannopyranoside4-Methylumbelliferyl α- (4d) Compound 3d  was O-deacetylated for 45 min at room temperature with KOH  in anhydrous MeOH (2 mL). The beige solid precipitate was filtered off, washed with ethanol until the washings were no longer fluorescent, and dried. Beige powder; yield: 106 mg (80%); mp: 213–214 °C; c = 0.35, H2O); {lit. + calcd for C16H18NaO8: 361.0894; found: 361.0900. 1H- and 13C-NMR, and HRMS spectrograms are seen in the ); {lit.  mp: 220–O); lit.  mp: 222–-d-xylopyranoside4-Methylumbelliferyl β (4e) Compound 3e  was O-deacetylated for 25 min at room temperature with KOH  in anhydrous MeOH (5 mL). The white solid precipitate was filtered off, washed with ethanol until the washings were no longer fluorescent, and dried. White powder; yield: 112 mg (79%); mp: 217–218 °C; c = 0.10, H2O); {lit. + calcd for C15H16NaO7: 331.0788; found: 331.0793. 1H- and 13C-NMR, and HRMS spectrograms are seen in the ); {lit.  mp: 213–d-ribofuranoside4-Methylumbelliferyl β- (4f) Compound 3f  was O-deacetylated for 25 min at room temperature with KOH  in anhydrous MeOH (5 mL). The white solid precipitate was filtered off, washed with ethanol until the washings were no longer fluorescent, and dried. White powder; yield: 112 mg (79%); mp: 167–169 °C; c = 0.15, H2O). 1H-NMR : δ = 7.67 , 7.00–6.96 , 6.22 , 5.60 , 5.43 , 5.08 , 4.71 , 4.08–4.00 , 3.93 , 3.55 , 3.38–3.29 , 2.38 . 13C-NMR : δ = 160.02 (C-2′), 159.31 (C-7′), 154.26, 153.25, 126.41, 113.81, 113.32, 111.54 , 105.06 (C-8′), 103.26 (C-1), 84.82, 74.47, 70.41, 62.52 , 18.07 (C-Me′). HRMS (ESI): m/z [M + Na]+ calcd for C15H16NaO7: 331.0788; found: 331.0791. 1H- and 13C-NMR, and HRMS spectrograms are seen in the d-glucopyranosiduronic acid and 4-methylumbelliferyl β-d-glucopyranoside are widely used for detection of Escherichia coli and enterococci, respectively, and 4-methylumbelliferyl α-d-galactopyranoside can be used to help differentiate between streptococci and enterococci [We have developed a useful method for the synthesis of 4-methylumbelliferyl glycosides. Notable benefits, such as the easy availability of glycosyl acetates, α-/β-stereoselectivity, moderate to excellent yields (51%–94%), and easy operation, make this method a practical approach for obtaining 4-methylumbelliferyl glycosides. These compounds have been often applied to detect the activities of glycosidases from different sources, particularly microorganisms. For example, 4-methy-lumbelliferyl β-erococci . The sea


E)-2-(benzo[d]thiazol-2-yl)-3-heteroarylacrylonitriles are described as a new class of selective inhibitors of acetylcholinesterase (AChE). The most potent compound in the series exhibited good AChE inhibitory activity (IC50 = 64 µM). Compound 7f was found to be more selective than galanthamine in inhibiting AChE and it showed a moderate selectivity index. Kinetic studies on AChE indicated that a competitive type of inhibition pattern exist for these acrylonitrile derivates. Molecular docking models of the ligand-AChE complexes suggest that compound 7g is located on the periphery of the AChE active site.( These products were obtained in moderate yields and good purity after short reaction times. In general, in terms of biological activities they are versatile precursors that have been widely used in the synthesis of heterocyclic compounds and as intermediates for the development of new molecules with potential biological or pharmaceutical interest [d]thiazol-2-yl)acetonitrile moiety and evaluate them as AChE inhibitors.We have previously reported  the syntinterest ,8,9. Speinterest ,12,13,14interest , which minterest . In thisA characteristic important of the Knoevenagel adducts of that they are used as intermediates for the development of new molecules with potential biological or pharmaceutical interest, as it is the case of the AChE inhibitors .E)-2-(benzo[d]thiazole-2-yl)-3-heteroarylacrylonitriles 7a–k obtained by Knoevenagel condensation between equimolar amounts of 2-(benzo[d]thiazol-2-yl)acetonitrile (5) and different substituted hetarylaldehydes 6a–k  substituent. The presence of other substituents instead of furan-2-yl such as 3-isoxazolyl , phenyl , 1H-imidazol-2-yl , and pyridinyl  decreased the AChE inhibitory activity. Furthermore, when the 4-chlorophenyl group, at position 5 of furan-2-yl substituent in compound 7g, is removed (compound 7i) the activity also decreases (IC50 = 1982 µM). This suggests that chlorophenyl group in 7g is accommodated along the gorge of AChE. In vitro BuChE inhibition was also determined using the same method and all compounds that were tested showed poor inhibition against BuChE. The compound 7g was also the most active compound against BuChE (IC50 = 474 µM) with a moderate selectivity for AChE over BuChE (selectivity index = 7.40). Interestingly, the compound 7f (IC50 = 155 µM against AChE) has a better selectivity index (22.20) for AChE over BuChE. The selectivity toward AChE is interesting for these two compounds when comparing with the selectivity of galanthamine (selectivity index = 16.29).Compounds 7g). Compound 7g is a long chain molecule and we expected that its binding mode, within the AChE binding site, would share some common characteristics with the AChE–donepezil complex.With the aim to understand the ligand–protein interactions at atomic level detail, molecular docking studies were performed for the most potent AChE inhibitor reported in this work -2-(benzo[d]thiazol-2-yl)-3-heteroarylacrylonitrile derivatives form interactions in accordance with those previously described for donepezil and other AChE inhibitors thiazole scaffold in its binding to the peripheral site of AChE, via the π–π stacking interaction with Trp279 , doublet (d), triplet (t), quadruplet (q), multiplet (m). High-resolution mass spectrometry ESI-MS and ESI-MS/MS analyses were conducted in a high-resolution hybrid quadrupole (Q) and orthogonal time-of-flight (TOF) mass spectrometer  with a constant nebulizer temperature of 100 °C. The experiments were carried out in positive ion mode, and the cone and extractor potentials were set at 10 and 3.0 V, respectively, with a scan range of m/z 100–600. MS/MS experiments were carried out by mass selection of a specific ion in Q1, which was then submitted to collision-induced dissociation (CID) with helium in the collision chamber. The product ion MS analysis was accomplished with the high-resolution orthogonal TOF analyzer. The samples were directly infused into the ESI source, via a syringe pump, at flow rates of 5 µL min−1, via the instrument’s injection valve. The elemental analyses have been obtained using a LECO CHNS-900 and Thermo Finnigan FlashEA1112 CHNS-O (STIUJA) elemental analyzers. TLC was done on pre-coated silica gel 60 F254 plates (Merck).All solvents used were of analytical grade. Melting points were recorded on a Buchi apparatus and are uncorrected, IR spectra, KBr pellets, 500–4000 cmGeneral procedure for the preparation of the (E)-2-(benzo[d]thiazol-2-yl)-3-arylacrylonitriles7a–k. A mixture of 2-(benzo[d]thiazol-2-yl)acetonitrile  and commercial aromatic aldehydes 6a–k with electron donating/withdrawing groups (1 mmol) were reacted with stirring at room temperature for 3 to 10 min using ethanol (10 mL) as solvent and catalytic amounts of triethylamine (0.2 mL). The reactions were monitored by TLC, in order to verify the consumption of the precursors. The solid products were isolated by simple filtration and washed with a mixture of hexane/ethanol (7:3) to give the corresponding compounds.(E)-2-(Benzo[d]thiazol-2-yl)-3-(5-methylisoxazol-3-yl)acrylonitrile  (7a). White solid. Yield 84%. mp 209–201 °C. IR (cm−1): 3236, 3072, 2991, 2227 (CN), 1596, 1429, 1312, 893, 762. 1H-NMR (CDCl3): δ ppm 8.19 , 8.14 , 7.93 , 7.55 , 7.48 , 7.02 , 2.54 ; 13C-NMR (CDCl3): δ ppm 171.95 (C5isoxazole), 161.20 (C2′BT), 158.40 (C3isoxazole), 153.96 (C8′BT), 135.53 (C3), 134.84 (C9′BT), 127.63 (C5′BT), 127.20 (C6′BT), 124.69 (C4′BT), 122.16 (C1CN), 115.33 (C7′BT), 112.34 (C4isoxazole), 100.97 (C2), 12.83 (CH3); EI-MSMS (m/z): 267 , 252 (30), 226 (46), 199 (20). Anal. Calcd for C14H9N3OS: C, 62.91; H, 3.39; N, 15.72. Found: C, 62.72; H, 3.66; N, 15.83.(E)-2-(Benzo[d]thiazol-2-yl)-3-(5-phenylisoxazol-3-yl)acrylonitrile  (7b). Green solid. Yield 87%. mp 241–243 °C. IR (cm−1): 3066, 3060, 2912, 2220 (CN), 1570, 1450, 1310, 943, 756. 1H-NMR (CDCl3): δ ppm 8.39 , 8.26 , 8.17 , 7.99 , 7.66 , 7.60–7.58 , 7.56 ; 13C-NMR (CDCl3): δ ppm, 163.52 (C5isoxazole), 161.40 (C2′BT), 159.33 (C3isoxazole), 155.45 (C8′BT), 137.07 (C9′BT), 130.05 (Cp), 129.42 (Ci), 128.15 (Cm), 127.99 (C5′BT), 126.15 (Co), 126.09 (C6′BT), 122.25 (C3), 121.80 (C4′BT), 121.22 (C2), 120.95 (C7′BT), 119.32 (C1CN), 100.02 (C4isoxazole); EI-MS (m/z): 329 , 318 (50), 302 (20), 274 (100), 171 (8), 218 (10). Anal. Calcd for C19H11N3OS: C, 69.28; H, 3.37; N, 12.76. Found: C, 69.33; H, 3.27; N, 12.98.(E)-2-(Benzo[d]thiazol-2-yl)-3-[5-(4-fluorophenyl)isoxazol-3-yl]acrylonitrile (7c). White solid. Yield 85%. mp 228–230 °C. IR (cm−1) 3127, 3018, 2229 (CN), 1602, 1500, 1438, 1165, 948, 841. 1H-NMR (DMSO-6d): δ ppm 8.39 , 8.26 , 8.17 , 8.06 , 7.65–7.58 , 7.55 , 7.45 ; 13C-NMR (CDCl3): δ ppm 163.47 (C5isoxazole), 161.34 (C2′BT), 159.35 (C3isoxazole), 158.99 (Cp), 155.43 (C8′BT), 137.05 (C9′BT), 127.99 (C5′BT), 127.55 (Co), 126.09 (C6′BT), 125.19 (Ci), 122.25 (C3), 121.90 (C4′BT), 121.04 (C2), 120.90 (C7′BT), 119.25 (C1CN), 116.39 (Cm), 100.05 (C4isoxazole); EI-MS (m/z): 347 , 330 (8), 318 (18), 302 (10), 274 (28), 217 (7), 187 (12), 162 (25), 168 (100). Anal. Calcd for C19H10FN3OS: C, 65.70; H, 2.90; N, 12.10. Found: C, 65.92; H, 2.85; N, 12.50.(E)-2-(Benzo[d]thiazol-2-yl)-3-(4-morpholin-4-ylphenyl)acrylonitrile  (7d). Orange solid. Yield 40%. mp 244–246 °C. IR (cm−1): 3053, 2959, 2843, 2214 (CN), 1575, 1509, 1199, 1116. 1H-NMR (CDCl3): δ ppm 8.12 , 8.04 , 8.00 , 7.89 , 7.52 , 7.39 , 6.93 , 3.88 , 3.37 ; 13C-NMR (CDCl3): δ ppm 153.74 (C2′BT), 153.44 (C8′BT), 146.49 (C3), 134.73 (C9′BT), 133.13 (Cp), 132.77 (Co), 126.70 (C5′BT), 125.40 (C6′BT), 123.09 (C4′BT), 122.78 (Ci), 121.55 (C1CN), 117.66 (C7′BT), 113.86 (Cm), 100.07 (C2), 66.48 (C2morpholine and C6morpholine), 47.15 (C3morpholine and C5morpholine); EI-MS (m/z): 347 , 321 (13), 262 (7), 226 (50), 210 (100), 191 (12). Anal. Calcd for C20H17N3OS: C, 69.14; H, 4.93; N, 12.09. Found: C, 69.01; H, 4.94; N, 12.01.(E)-2-(Benzo[d]thiazol-2-yl)-3-(1H-imidazol-2-yl)acrylonitrile  (7e). White solid. Yield 80%. mp 190–192 °C. IR (cm−1): 2930, 2223 (CN), 1436, 1302, 1089, 942, 754; 1H-NMR (CDCl3): δ ppm 10.74 , 8.10 , 8.08 , 7.90 , 7.53 , 7.48–7.36 ; 13C-NMR (CDCl3): δ ppm 166.06 (C2′BT), 154.68 (C8′BT), 154.29 (C2imidazole), 135.22 (C9′BT), 133.66 (C3), 127.46 (C5′BT), 126.84 (C6′BT), 124.32 (C5imidazole), 122.71 (C1CN), 122.05 (C4′BT), 120.90 (C7′BT), 120.21 (C4imidazole), 117.41 (C2); EI-MS (m/z): 252 , 226 (18), 174 (8). Anal. Calcd for C13H8N4S: C, 61.89; H, 3.20; N, 22.21. Found: C, 61.70; H, 2.99; N, 22.10.(E)-2-(Benzo[d]thiazol-2-yl)-3-(pyridin-3-yl)acrylonitrile  (7f). Green solid. Yield 66%. mp 155–157 °C. IR (cm−1): 3055, 2989, 2215 (CN), 1551, 1465, 1437, 1313, 1189, 983; 1H-NMR (DMSO-6d): δ ppm 9.14 , 8.75 , 8.52 , 8.51 , 8.23 , 8.12 , 7.66 , 7.61 , 7.54 ; 13C-NMR (DMSO-d6): δ ppm 163.03 (C2′BT), 153.17 (C8′BT), 152.61 (C4pyridinyl), 151.81 (C6pyridinyl), 145.68 (C2pyridinyl), 136.36 (C9′BT), 134.80 (C3), 128.97 (Ci), 127.64 (C5′BT), 126,97 (C6′BT), 124.46 (C4′BT), 123,70 (C5pyridinyl), 122.98 (C1CN), 116.10 (C7′BT), 108.13 (C2); EI-MS (m/z): 263 , 226 (20), 210 (38). Anal. Calcd for C15H9N3S: C, 68.42; H, 3.45; N, 15.96. Found: C, 68.20; H, 3.70; N, 15.86.(E)-2-(Benzo[d]thiazol-2-yl)-3-[5-(4-chlorophenyl)furan-2-yl]acrylonitrile  (7g). Yellow solid. Yield 59%. mp 185–187 °C. IR (cm−1): 3122, 3054, 2916, 2211 (CN), 1588, 1463, 109. 1H-NMR (CDCl3): δ ppm 8.05 , 8.02 , 7.91 , 7.81 , 7.51 , 7.45 , 7.41 , 7.24 , 6.90 ; 13C-NMR (CDCl3): δ ppm 162.56 (C2′BT), 157.64 (C9′BT), 153.78 (C5furanyl), 148.63 (C2furanyl), 135.37 (C8′BT), 135.17 (Cp), 130.33 (Ci), 129.41 (Cm), 127.48 (C3), 126.92 (C5′BT), 126.35 (Co), 125.71 (C6′BT), 123.24 (C3furanyl), 122.72 (C4′BT), 121.68 (C1CN), 116.89 (C7′BT), 109.15 (C4furanyl), 100.54 (C2); EI-MS (m/z): 363 , 321 (13), 261 (8), 210 (100), 193 (42), 152 (27), 115 (20). Anal. Calcd for C20H11ClN2OS: C, 66.21; H, 3.06; N, 7.72. Found: C, 66.10; H, 3.12; N, 7.55.(E)-2-(Benzo[d]thiazol-2-yl)-3-(5-chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)acrylonitrile  (7h). Green solid. Yield 59%. mp 188–190 °C. IR (cm−1): 3053, 2917, 2213 (CN), 1589, 1510, 1409, 1309, 896; 1H-NMR (CDCl3): δ ppm 8.14 , 8.10 , 7.92 , 7.60 , 7.54 ; 7.60–7.42 , 2.56 ; 13C-NMR (CDCl3): δ ppm 162.20 (C2′BT), 153.63 (C9′BT), 149.88 (C5pyrazole), 137.43 (C3pyrazole), 136.81 (Ci), 135.07 (C8′BT), 129.32 (Cp), 129.23 (Cm), 128.97 (C3), 126.97 (C5′BT), 126.03 (C6′BT), 124.99 (Co), 123.63 (C4′BT), 121.69 (C1CN), 116.28 (C7′BT), 113.67 (C4pyrazole), 107.93 (C2), 15.05 (CH3); EI-MS (m/z): 377 , 341 (19), 340 (15), 274 (23), 246 (45), 205 (22), 168 (100), 135 (22), 102 (90). Anal. Calcd for C20H13ClN4S: C, 63.74; H, 3.48; N, 14.87. Found: C, 63.71; H, 3.51; N, 14.83.(E)-2-(Benzo[d]thiazol-2-yl)-3-(2-furyl)acrylonitrile  (7i). Yellow solid. Yield 51%. mp 138–140 °C. IR (cm−1): 3120, 3035, 2914, 2214 (CN), 1607, 1452, 1305, 882; 1H-NMR (CDCl3): δ ppm 8.07 , 8.05 , 7.90 , 7.73 , 7.52 , 7.41 , 7.32 , 6.65 ; 13C-NMR (CDCl3) δ ppm 162.28 (C2′BT), 153.69 (C9′BT), 149.27 (C2furanyl), 147.15 (C5furanyl), 135.08 (C8′BT), 131.69 (C3furanyl), 126.92 (C3), 125.82 (C5′BT), 123.38 (C6′BT), 121.67 (C4′BT), 119.66 (C1CN), 116.35 (C7′BT), 113.63 (C4furanyl), 101.44 (C2); EI-MS (m/z): 252 (23), 134 (8), 124 (70), 110 (52), 102 (100). Anal. Calcd for C14H8N2OS: C, 66.65; H, 3.20; N, 11.10. Found: C, 66.62; H, 3.10; N, 10.99.(E)-2-(Benzo[d]thiazol-2-yl)-3-acrylonitrile  (7j). Green solid. Yield 84%. mp 191–193 °C. IR (cm−1): 3057, 3011, 2216 (CN), 1583, 1480, 1302; 1H-NMR (CDCl3): δ ppm 8.28 , 8.12 , 8.08 , 7.92 , 7.76 , 7.66 , 7.54 , 7.50–7.39 ; 13C-NMR (CDCl3): δ ppm 162.86 (C2′BT), 153.63 (C9′BT), 146.33 (C3), 144.94 (C1CN), 139.56 (C′i), 134.98 (C8′BT), 131.26 (Ci), 130.98 (Co), 129.03 (C′o), 128.39 (C′p), 127.77 (C′m), 127.16 (Cm), 126.95 (C5′BT), 125.98 (C6′BT), 123.57 (C4′BT), 121.68 (C1CN), 116.66 (C7′BT), 105.08 (C2); EI-MS (m/z): 338 , 246 (20), 203 (7), 162 (6), 124 (100), 101 (41). Anal. Calcd for C22H14N2S: C, 78.08; H, 4.17; N, 8.28. Found: C, 78.05; H, 4.14; N, 8.27.(E)-2-(Benzo[d]thiazol-2-yl)-3-(pyridin-2-yl)acrylonitrile (7k). Green solid. Yield 83%. mp 158–160 °C. IR (cm−1): 3052, 2992, 2217(CN), 1557, 1469, 1433, 1313, 1171, 980; 1H-NMR (CDCl3): δ ppm 8.84 , 8.29 , 8.12 , 7.94 , 7.89 , 7.84 , 7.55 , 7.46 , 7.40 ; 13C-NMR (CDCl3): δ ppm 162.89 (C2′BT), 154.05 (C8′BT), 151.20 (C4pyridinyl), 150.81 (C6pyridinyl), 145.30 (C3pyridinyl), 137.38 (C9′BT), 135.75 (C3), 127.45 (Ci), 126.71 (C5′BT), 126.43 (C6′BT), 125.85 (C4′BT), 124.28 (C5pyridinyl), 122.18 (C1CN), 116.44 (C7′BT), 109.43 (C2); EI-MS (m/z): 263 , 262 (70), 200 (37), 180 (20), 152 (33), 185 (4). Anal. Calcd for C15H9N3S: C, 68.42; H, 3.45; N, 15.96. Found: C, 68.40; H, 3.41; N, 15.93.2HPO4, 2.3 mM NaH2PO4, 150 mM NaCl, and 0.05% Tween 20 at pH 7.6) and an AChE/BuChE solution  from Electroporus electricus and equine serum, respectively, in the same phosphate buffer, were added. The assay solutions, except substrate, were preincubated with the enzyme for 30 min at room temperature. After preincubation, the substrate was added. The substrate solution consists of Na2HPO4 (40 mM), acetylthiocholine/butyrylthiocholine (0.24 mM) and 5,5′-dithio-bis-(2-nitrobenzoic acid) . Absorbance of the yellow anion product, due to the spontaneous hydrolysis of substrate, was measured at 405 nm for 5 min on a Microtiter plate reader . The AChE/BuChE inhibition was determined for each compound. The enzyme activity was calculated as a percentage compared to a control using only the buffer and enzyme solution. The compounds were assayed in the dilution interval of 500 to 15 μg/mL, and the alkaloid galanthamine was used as the reference compound. Each assay was run in triplicate and each reaction was repeated at least three independent times. The IC50 values were calculated by means of regression analysis. The IC50 value of galanthamine was developed in three plates in a range 250 μg/mL to 0.122 μg/mL, in the first plate the value was 0.179 μg/mL (0.486 µM), in the second was 0.213 μg/mL (0.578 µM), and the third was 0.193 μg/mL (0.524 µM), which gives an average value of 0.54 μM.In 96-well plates, the sample (50 μL) was dissolved in phosphate buffer  plot was constructed using Sigma Plot 10.0 version software [Kinetic characterization of AChE was performed by means of Ellman’s method. The effect of inhibitor software .Docking was used to suggest the binding modes of some of the reported inhibitors. Docking was performed using Glide . The Gli7b and 7g were sketched with Maestro software. The extra-precision (XP) module of Glide was used. A grid box of 30 Å × 30 Å × 30 Å was first centered on the center of mass of the donepezil in PDB 1EVE. Default docking parameters were used [The protein coordinates were extracted from the X-ray crystal structure of the AChE–donepezil complex [accession code in the Protein Data Bank (PDB): 1EVE]. The structures of the compounds ere used . The docere used  force fiere used . The docE)-2-(benzo[d]thiazol-2-yl)-3-heteroarylacrylonitrile derivatives were designed, synthesized, and evaluated as AChE inhibitors. Structure–activity studies showed that AChE inhibitory activity of compounds was influenced by the substituent pattern at position 3 of acrylonitrile. The most potent compound (compound 7g) contains a 5-(4-chlorophenyl)furan-2-yl group at this position. The molecular modeling study of compound 7g indicated that it is accommodated inside the active-site gorge of AChE, adopting the same interactions that were described for the AChE–donepezil complex. The results suggest that rational modifications of the substituent in the scaffold (E)-2-(benzo[d]thiazol-2-yl)-3-(2-furyl)acrylonitrile would provide a rational basis for the development of novel AChE inhibitors. Further studies on this series of derivatives are in progress and will be reported in due course.In summary, a series of (


Treatment of 4 with ethyl acetoacetate, acetic anhydride, formic acid, urea and thiourea gave the corresponding pyrido  pyrimidine derivatives 7–10a,b. A new series of 6-substituted-4-methyl-2-phenyl-5-pyridine carbonitriles 11–13 has been synthesized via reaction of 4 with phenyl isothiocyanate, benzenesulphonyl chloride and acetic anhydride. Treatment of 4 with malononitrile gave 1,8-naphthyridine derivative 14. The reactivity of hydrazide 5 towards acetic acid, phenylisothiocyanate and methylacrylate to give pyrazolo--pyridine derivatives 15–17 was studied. Treatment of 5 with acetic anhydride, phthalic anhydride and carbon disulphide gave pyridine derivatives 18,19 and 1,2,4-triazolo--pyridine derivative 20.The reaction of 4-methyl-2-phenyl-1,2-dihydro-6-oxo-5-pyridine- carbonitrile ( A large number of heterocyclic compounds containing pyridine rings are associated with diverse pharmacological properties such as antimicrobial ,2, anticPrompted by recent literature observations, some new pyridine derivatives were synthesized, leading to interesting heterocyclic scaffolds that are particularly useful for the creation of diverse chemical libraries of drug-like molecules for biological screening.1 was prepared by condensing benzoylacetone with cyanoacetamide in the presence of sodium ethoxide -pyrimidine-4-one (7). The IR spectra of 7 contains bands at 3299, 1686 and 1660 cm−1 corresponding to (NH) group, and the amide and ketone carbonyl groups. The 1H-NMR spectrum exhibits a broadened signlet at δ 12.34 belonging to the (NH) group. The 13C-NMR spectra of carboxamide 6 and pyrido--pyrimidin-4-one 7 agree with their assigned structures.The target pyrido  pyrimidine-4-one derivatives were synthesized by the reactions depicted in 4 with acetic anhydride and formic acid afforded pyrido--pyrimidine-4-one derivatives 8 and 9. This reaction was carried out thermally in the absence of solvent. The structure of compounds 8 and 9 were inferred from their IR, 1H-NMR spectral data and elemental microanalysis. The spectral data of the products 8 and 9 revealed the disappearance of the NH2 group, indicating their involvement in the cyclization process. The characteristic IR stretching band at 1680 and 1688 cm–1 shows the occurrence of (C=O) groups due to the pyridopyrimidinone systems. Formation of the compounds 8 and 9 is thought to occur through the expected intermediates 7′ and 8′ which were not isolated -pyrido--pyrimidine-2-one (or thione) 10a,b. The IR spectra of 10a,b showed the disappearance of the C≡N group and showed absorptions bands at 3320, 3250, 3330, 3280 for NH2 groups. The 1H-NMR spectra exhibited broad signals at δ 8.91 and 10.68 ppm due to NH protons.Thermal cyclocondensation of 2-aminopyridine derivative isolated . The 1H-4, upon treatment with phenyl isothiocyanate afforded the corresponding thiourea derivative 11. The spectral data are in agreement with the assigned structure . The 1H-NMR spectrum of compound 11 showed two singlet signals at δ 11.08 and 13.26 ppm due to the protons at two (NH) groups, in addition to a MS peak at m/z = 344 . The possibility of formation of the product 11′ is excluded on the basis of the spectral data.Moreover, compound tructure . Thus th4 with benzenesulfonyl chloride led to the formation of 6-benzene sulfonylamino-4-methyl-2-phenyl-5-pyridinecarbonitile (12). IR spectrum of 12 displayed absorption bands at 3250, 2215, 1360 and 1150 cm–1 which are characteristic for NH, C≡N and SO2 asym, SO2 sym groups. The 1H-NMR spectrum of 12 showed a signal at δ 8.50 ppm for the proton of a (SO2NH) group. However, upon heating compound 4 in refluxing acetic anhydride for 15 min. it afforded 6-acetylamino-4-methyl-2-phenyl-5-pyridinecarbonitrile (13). IR spectrum of 13 showed absorption bands at 3280, 2220 and 1720 cm–1 which are characteristic of NH, C≡N and C=O groups. The 1H-NMR spectrum of 13 showed two singlet signals at δ 2.50 and 3.33 ppm corresponding to the protons of two methyl groups, and a singlet at δ 10.22 ppm for a (NH) group. Treatment of compound 4 with malonitrile in refluxing ethanol in the presence of triethylamine gave 2,4-diamino-3-cyano-5-methyl-7-phenyl-1,8-naphthyridine (14), whose structure is supported by elemental analysis, its IR spectrum, which showed bands at 3450, 3400, 3340, 3250 cm–1 (2NH2) and 2230 cm–1 (C≡N) and the 1H-NMR spectrum that showed two singlet at δ 4.42, 5.33 ppm for two amino groups.The reaction of compound Hydrazide derivative 5 proved to be versatile compounds by virtue of its vicinal cyano functions as confirmed by its reactivity in several cyclization reaction performed with the aim of obtaining new heterocycles with a conserved pyrazolo--pyridine motif .H-pyrazolo--pyridine (15), through intramolecular cyclization via addition of the NH2 functional group at the C≡N group. The proposed structure for 15 was supported by its independent synthesis from 5 by refluxing with DMF (melting point and mixed melting point was not depressed)  corresponding to its molecular ion.Thus, reaction of compound 5 with acetic acid afforded the corresponding 3-amino-4-methyl-6-phenyl-1pressed) . The IR 5 seemed to be a useful candidate for further chemical transformations. Thus, the reaction of 5 with phenyl isothiocyanate in absolute ethanol at refluxing temperature afforded the corresponding thiosemicarbazide derivatives 15′, which underwent a further an intramolecular cyclization to afford 3-amino-4-methyl-6-phenyl-1H-pyrazolo--pyridine-2-yl-phenyl thioamide (16), identified by the absence of the cyano group signal in its IR and the presence of a signal of the amino group at δ 4.80 ppm and the broad band of the (NHPh) proton at 11.80 ppm in its 1H-NMR spectrum, as well as the characteristic absorption band at 3261 cm–1 for NH in its IR spectrum. Its mass spectrum showed the molecular ion peak at m/z 359 (8.11) and the most intense peak at m/z 135, corresponding to [M-PhNCS]+.Interestingly, compound 5 with methyl methacrylate in acetic acid at reflux afforded 3-amino-4-methyl-6-phenyl-1H-pyrazolo--pyridine-2-yl-methyl propionate (17). The IR spectrum is characterized by the presence of strong NH2 group absorption bands at 3384 and 3280 cm–1 and a carbonyl group at 1710 cm–1. The 1H-NMR spectrum showed a singlet at 2.48 (CH3-pyridine), a singlet at 3.38 (OCH3), two triplet signals at δ 4.50, 4.80 (2CH2 groups). The mass spectrum exhibited the molecular ion peak at m/z 310  in addition to another peaks at 75 (100).Treatment of compound 5 in refluxing acetic anhydride was identified as the triacetyl derivative 18 . Its 1H-NMR spectrum exhibited signals at 2.50, 2.80, 3.10 for (3COCH3).However, the reaction product upon heating compound ative 18 . The strm/z 350  and a base peak at m/z 77 (100). 1-[3-Cyano-4-methyl-6-phenyl]-pyridine-2-yl-(2H)-phthalazine-3,8-dione (19) was obtained via reaction of compound 5 with phthalic anhydride . When 5 was allowed to react with CS2, 8-cyano-7-methyl-5-phenyl-2,3-dihydro-2-thioxo-1,2,4-triazolo--pyridine (20) was obtained in a good yield . The mass spectrum of 20 showed a molecular ion peak at m/z 266  with a base peak at m/z 75 (100).Theod yield . The str1H-NMR and 13C-NMR spectra were recorded on a Bruker 400 MHz instrument with DMSO-d6 as solvent and tetramethylsilane as an internal standard; chemical shifts are as δ units (ppm). The mass spectra were recorded on a MS-S988 instrument operating at 70 eV. Elemental analysis was determined using a Perkin-Elmer 240C microanalyser.All melting points were recorded on a Gallenkamp melting apparatus and are uncorrected. The IR spectra were recorded on a Pye-Unicam Sp-3-100 spectrophotometer using the KBr wafer technique. 8-Amino-7-cyano-1-oxo-6-aryl-3-phenyl-1,2-dihydroisoquinolines . A solution of compound 1 (10 mmol) in pyridine (30 mL) was treated with an arylidene malononitrile (10 mmol). The reaction mixture was refluxed for 6 h, left to cool to room temperature, poured into ice-cold water, and neutralized with HCl (10%). The solid product was filtered off and recrystallized from ethanol. 2a: Yield 60%; m.p. > 360 °C; IR cm–1: 3434, 3329 (NH2) 3169 (NH), 3050 (CH–Ar), 2221 (C≡N), 1672 (C=O); 1H-NMR: δ 4.22 , 6.80  7.22–7.72 , 9.80 ; 13C-NMR: δ 115.00 (C≡N), 188.00 (C=O), 111.00–142.90 (Ar–C); MS m/z (%): 327 . Anal. For: C20H13N3O2: Calcd. C, 73.39; H, 3.97; N, 12.84 Found: C, 73.40, H, 3.88; N, 13.00. 2b: Yield 62%; m.p. 280 °C; IR cm–1: 3430, 3333 (NH2) 3199 (NH), 3050 (CH–Ar), 2215(CN), 1666 (C=O); 1H-NMR: 4.82 , 6.64  7.88  9.12 , 7.64 , 9.30 ; 13C-NMR: δ 118.00 (C≡N), 188.00 (C=O), 101.00-139.63 (Ar-C); MS m/z (%): 382 , . Anal. For: C22H14N4O3: Calcd. C, 69.10; H, 3.66; N, 14.65; Found: C, 69.30, H, 3.80; N, 14.80.6-Chloro-4-methyl-2-phenyl-5-pyridinecarbonitrile (3). A mixture of 1 (116 mmol) and phosphorus oxychloride  in the presence of triethylamine (3 mL) was stirred at 105 °C for 3 h. The mixture was poured onto crushed ice (800 g) and neutralized with 10% sodium hydroxide. The extract was washed with water 5% aqueous sodium bicarbonate and again with water and recrystallized from ethanol. 3: Yield 72%, m.p. 180–182 °C, IR cm–1: 3033 (CH–Ar), 2999 , 2222 (C≡N), 1644 (C=N); 1H-NMR: δ 2.44 , 6.80 , 7.22–7.48 ; 13C-NMR: δ 116.22 (C≡N), 128.34–148.00 (Ar–C); MS m/z (%): 228.5 (3.28), 230.5 (1.11), 57 (100). Anal. For: C13H9ClN2: Calcd. C, 68.27, H, 3.93, N, 12.25, Cl, 15.53. Found. C, 68.30; H, 2.00; N, 12.50; Cl, 15.98.6-Amino-4-methyl-2-phenyl-5-pyridinecarbonitrile (4). Method A: A mixture of compound 1 (0.005 mL) and ammonium acetate (0.005 mol) in pyridine (30 mL) was refluxed for 8 h. The reaction mixture was cooled at room temperature, poured into ice water, and neutralized with dilute HCl. The isolated solid was filtered off and recrystallized from ethanol to afford 4: Yield 75%; m.p. 250–252 °C; IR cm–1: 3350, 3292 (NH2), 3100 (CH–Ar), 2900 , 2220 (C≡N), 1644 (C=N); 1H-NMR: δ 2.48 , 4.44 , 6.80 , 7.22–7.68 ; 13C-NMR: δ 115.72 (C≡N), 122.42–152.23 (Ar–C); MS m/z (%): 209 , 75 (100). Anal. For: C13H11N3: Calcd. C, 74.64; H, 5.26; N, 20.09; Found: C, 74.71, H, 5.22, N, 20.38. Method B: To a solution of compound 3 (9.58 mmL) in DMF (25 mL) was added NH3   and the resulting mixture was stirred at room temperature for 12 h. The mixture was extracted with ethylacetate (3 × 50 mL) and the combined organic layer was dried over Na2SO4, filtered and concentrated under vacuum and the resulting solid was recrystallized from ethanol to afford 4 in a low yield.6-Hydrazide-4-methyl-2-phenyl-5-pyridinecarbonitrile (5). A mixture of compound 3 (0.01 mmol) and hydrazine hydrate  in ethanol (20 mL) was heated under reflux for 6 h. The excess of solvent was removed under reduced pressure, and the resulting precipitate was filtered off, washed with ethanol and recrystallized from methanol to give 5, yield 62%; m.p. 100–102 °C; IR cm–1; 3428–3298 , 3046 (CH–Ar), 2921 , 2220 (C≡N), 1632 (C=N); 1H-NMR: δ 2.43 , 5.14 , 6.71 , 7.33–7.80 , 9.12 , 13C-NMR: δ 118.64 (C≡N), 126.88–155.43 (Ar–C); MS m/z (%): 244 , 75 (100). Anal. For: C13H12N4: Calcd. C, 69.64; H, 5.35; N, 25.00; Found: 69.70, H, 5.50; N, 24.80.2-(3-Cyano-4-methyl-6-phenyl-pyridine)-2,4-dioxo butanamide (6). A mixture of compound 4 (5.62 mmol) and (8.00 mL) of ethyl acetoacetate was heated for 6 h at 140–150 °C. Excess ethyl aceto- acetate was distilled off under reduced pressure, and the residue was purified by recrystallization from ethanol. 6: Yield 75%; m.p. 110–112 °C; IR cm–1: 3210 (NH), 3032 (CH–Ar), 2990–2828 , 2215 (C≡N) 1710 , 1680 ; 1H-NMR: δ 2.48 , 3.80 , 4.24 , 7.12–7.48 , 11.55 ; 13C-NMR: δ 14.11 (CH3), 64.19 (CH2), 115.00 (C≡N), 112.88–156.09 (Ar–C), 168.50 (CO–CH3), 182.41 (CO–NH); MS m/z (%): 293 (9.80), 75 (100). Anal. For: C17H15N3O2: C, 69.62; H, 5.11; N, 14.33; Found: C, 69.60, H, 5.22, N, 14.50.5-Methyl-7-phenyl-2-propanone-3H-pyrido  pyrimidine-4-one (7). Compound 6 (0.01 mol) was treated with concentrated hydrochloric acid (3 drops) in ethanol (30 mL) and refluxed for 6 h. The reaction mixture was left to cool, and then poured into ice-cold water. The precipitate was collected and recrystallized from ethanol. 7: Yield 55%; m.p. 222–224 °C, IR cm–1: 3299 (NH), 3112 (CH–Ar), 2989–2885 , 1686 (C=O) 1660 (C=O); 1H-NMR: δ 2.48 , 3.11 , 4.17 , 7.22–7.78 , 12.34 ; 13C–NMR: δ 14.36 (CH3), 16.50 (COCH3), 64.04 (CH2), 112.69–156.64 (Ar–C), 169.80 (C=O), 189.00 (C=O). MS m/z (%): 393 , . Anal. For: C17H15N3O2: Calcd. C, 69.62; H, 5.11; N, 14.33; Found: C, 69.50, H, 5.10, N, 14.30.2,5-Dimethyl-7-phenyl-3H-pyrido pyrimidine-4-one (8). Compound 4 (0.002 mol) in acetic anhydride (10 mL) was heated under reflux for 3 h. After cooling, the solvent was concentrated under reduced pressure, then the reaction mixture was poured into ice-water (40 mL) to give a solid precipitate which was filtered off and recrystallized from petroleum ether 60/80 to furnish 8, yield 55%; m.p. 300–302 °C; IR cm–1: 3300 (NH), 3033 (CH–Ar), 2928  1680 (C=O), 1644 (C=N); 1H-NMR: δ 2.50, 2.44 , 7.38–7.80 , 11.82 ; 13C-NMR: δ 13.02 (CH3), 14.22 (CH3), 112.70–148.12 (Ar–C), 170.80 (C=O); MS m/z (%): 251 (2.11), 77 (100). Anal. For: C15H13N3O: Calcd. C, 71.71; H, 5.17, N, 16.73; Found: C, 71.50, H, 5.20, N, 16.80.5-Methyl-7-phenyl-3H-pyrido  pyrimidin-4-one (9). Compound 4 (0.002 mol) in formic acid (10 mL) was heated under reflux for 6 h. After cooling the reaction mixture was poured into ice-water (40 mL) to give a solid precipitate which was filtered off and recrystallized from methanol to furnish 9, yield 45%; m.p. 180–182 °C; IR cm–1: 3330 (NH), 3100 (CH–Ar), 2900  1688 (C=O), 1640 (C=N); 1H-NMR: δ 2.55 , 6.80 , 7.28-7.84 , 8.28 . 13C-NMR: δ 14.62 (CH3), 110.18–155.80 (C–Ar), 188.01 (C=O); MS m/z (%): 237 (5.18) 75 (100). Anal. For: C14H11N3O: Calcd. C, 70.88; H, 4.64; N, 17.72. Found: C, 70.80; H, 4.70, N, 17.80.4-Amino-5-methyl-7-phenyl-1H-pyrido  pyrimidine-2-one(or thione) . A mixture of 4 (0.01 mol) and urea (or thiourea) (0.01 mol) was fused in an oil bath at 180 C, for 1h. After cooling and dilution with ethanol (30 mL) the solid product formed was filtered off and recrystallized from ethanol. 10a: Yield 70%; m.p. 320–322 °C; IR cm–1: 3483, 3320, 3250 (OH) , 3033 (CH–Ar), 2880 , 1683 (C=O), 1638 (C=N); 1H-NMR: δ 2.44 , 4.48 , 6.88–7.44 , 8.91 ; 13C-NMR: δ 11.12 (CH3), 202.32 (C=O), 112.43–148.91 (Ar–C); MS m/z (%): 252 (3.21) 75 (100). Anal. For: C14H12N4O: Calcd. C, 66.66; H, 4.76; N, 22.22; Found: C, 66.50; H, 4.80, N, 22.50. 10b: Yield 65%; 300–302 °C; IR cm–1: 3330, 3280, 3221 , 3052 (CH–Ar), 2988 , 1222 (C=S); 1H-NMR: δ 2.50 , 4.22  6.88–7.22 , 10.68 ; 13C-NMR: δ 13.43 (CH3), 212.98 (C=S), 112.65–144.34 (Ar–C); MS m/z (%): 268 (1.11), 77 (100). Anal. For: C14H12N4S: Calcd., C, 62.68; H, 4.47; N, 20.89, S, 11.94. Found: C, 62.80; H, 4.50, N, 2.80, S, 12.12.N-(Phenyl)-N′-[2(3-cyano-4-methylphenylpyridinyl] thiourea (11). A mixture of compound 4 (0.01 mol), finely divided sodium metal (0.01 mol) and phenyl isothiocyanate (0.01 mol) were refluxed for 6 h. in dry dioxane (50 mL). After cooling, the solvent was concentrated under pressure, then the reaction mixture was poured into ice-water (40 mL) to give a solid precipitate which was filtered off and recrystallized from petroleum ether 60/80 to furnish 11, yield 60%; m.p. 220–222 °C. IR cm–1: 3212 (NH), 2222 (CN); 1H-NMR: δ 2.55 , 11.08 , 13.26 , 7.58–7.69  13C-NMR: δ 115 (C≡N), 179.30 (C=S), 14.88 (CH3), 112.00–155.40 (C–Ar); MS m/z (%): 344 . Anal. For: C20H16N4S: Calcd. C, 69.76; H, 4.65; 16.27; S, 9.30; Found: C, 69.50; H, 4.70; N, 16.30, S, 9.50.6-Benzenesulfonylamino-4-methyl-2-phenyl-5-pyridinecarbonitrile (12). Compound 4 was taken in a mixture of pyridine (4 mL) and acetic anhydride (20 mL). To the solution formed, p-benzenesulphonyl chloride was added (0.01 mol) and the reaction mixture was reflux for 6 h., filtered and poured onto acidic crushed ice. The solid product 12 obtained was recrystallized from ethanol. 12: Yield 55%; m.p. 200–202 °C; IR cm–1: 3250 (NH), 3100 (CH–Ar), 2215 (C≡N), 2995 , 1632 (C=N), 1360 , 1150 ; 1H-NMR: δ 2.55 ; 6.88–7.67 , 8.50 ; 13C-NMR: δ 14.17 (CH3), 117 (C≡N), 110.00–155.50 (Ar–C). Anal. For: C19H15N3O2S: calcd. C, 65.32; H, 4.29; N, 12.03; S, 9.16; Found: C, 65.00, H, 4.80; N, 12.00, 55, 9.40.6-Acetyl amino-4-methyl-2-phenyl-5-pyridinecarbonitrile (13). A suspension of 4 (0.002 mol) in acetic anhydride (10 mL) was heated under reflux for 15 min. After cooling, the solvent was concentrated under reduced pressure, then the reaction mixture was poured into ice-water (40 mL) to give a solid precipitate which was filtered off and recrystallized from ethanol. 13: Yield 55%; m.p. 180–182 °C; IR cm–1: 4330 (OH), 3280 (NH), 3100 (CH–Ar), 2910 , 2220 (C≡N), 1720 (C=O); 1H-NMR: δ 2.50 , 3.33 , 6.82–7.72 , 10.22 ; 13C-NMR: δ 14.13 (CH3), 17.22 (CH3), 117 (C≡N), 122.00–156.11 (Ar–C), 168.33 (C=O). Anal. For: C15H13N3O. Calcd.: C, 71.71; H, 5.17; N, 16.73; Found: C, 71: 80; H, 5.00, N, 16.90.2,4-Diamino-3-cyano-5-methyl-7-phenyl-1,8-naphthyridine (14). A suspension of 4 (0.01 mol) in ethanol (20 mL) containing a catalytic amount of triethylamine (3 mL) was treated with malononitrile (0.01 mol). The reaction mixture was refluxed for 10 h. The separated solid was filtered off and recrystallized from ethanol. 14: Yield 72%; m.p. 300–302 °C; IR cm–1: 3450, 3400, 3340, 3250 (2NH2), 3100 (CH–Ar), 2990 , 2230 (C≡N); 1H-NMR: δ 2.48 , 4.42, 5.33  6.84–7.88 ; 13C-NMR: δ 13.42 (CH3), 118.95 (C≡N), 127.87–149.97 (Ar–C); MS: m/z (%): 275 (2.08), 77 (100). Anal. For: C16H13N5: C, 69.81; H, 4.72; N, 25.45; Found: C, 69.50; H, 4.80, N, 25.50.3-Amino-4-methyl-6-phenyl-1H-pyrazolo--pyridine (15). Method A: A mixture of compound 5 (0.001 mol) in ethanol and few drops of glacial acetic acid was refluxed for 8 h. The reaction mixture was poured into ice-cold water and the solid product was filtered off, washed with petroleum ether and recrystallized from ethanol. Yield 62%; Method B: Compound 5 (0.005 mol) in DMF 20 mL was refluxed for 12 h. and then allowed to cool. The solid product that precipitated on cooling was filtered off, dried and recrystallized from DMF. 15: Yield 50%; m.p 200–202 °C; IR cm–1: 3320, 3300, 3221 , 3090 (CH–Ar), 2980 , 1644 (C=N), 1H-NMR: δ 2.55  4.50 , 7.22–7.76 , 12.11 ; 13C-NMR: δ 11.18(CH3), 124.62–149.06 (Ar–C); MS m/z (%): 224 (6.88), 75 (100). Anal. For: C13H12N4. Calcd. C, 69.64; H, 5.35; N, 25.00; Found: C, 69.70, H, 5.50, N, 25.32.3-Amino-4-methyl-6-phenyl-1H-pyrazolo--pyridine-2-yl-phenylthioamide (16). A suspension of 5 (0.001 mol) and phenyl isothiocyanate (0.001 mol) in pyridine (10 mL) was heated under reflux for 6 h. After cooling the reaction mixture was poured into ice/water (30 mL) and neutralized with dilute 10% HCl to give a solid precipitate that was recrystallized from ethanol-DMF (3:1). 16: Yield 70%; m.p. > 360 °C; IR cm–1: 3330, 3280, 3261 , 3055 (CH–Ar), 2990 , 1333 (C=S); 1H-NMR: δ 2.50 , 4.80 , 6.88–7.76 , 11.80 ; 13C-NMR: δ 13.44 (CH3), 208.65 (C=S), 114.89–145.67 (Ar–C); MS m/z (%): 359 (8.11), 75 (100). Anal. For: C20H17N5S: Calcd. C, 66.85; H, 4.73; N, 19.49; S, 8.91. Found: C, 69.50; H, 5.00, N, 19.11.; S, 9.00.3-Amino-4-methyl-6-phenyl-1H-pyrazolo--pyridine-2-yl-methyl propionate (17). A mixture of compound 5 (0.01 mol) and methyl methacrylate (0.01 mol) in acetic acid (20 mL) containing 3 drops of concentrated hydrochloric acid was refluxed for 5 h. The reaction mixture was concentrated and allowed to cool and poured onto H2O (100 mL). The solid obtained was recrystallized from ethanol. 17: Yield 45%; m.p. 140–142 °C; IR cm–1: 3384, 3280 (NH2), 3099 (CH–Ar), 2888–2990 , 1710 (C=O); 1H-NMR: δ 2.48 , 3.38  4.50 , 4.80  5.80 , 6.82–7.78 ; 13C-NMR: δ 14.13 (CH3), 17.80 (OCH3) 40.81 (CH2), 60.38 (CH2), 112.8–153.11 (Ar–C); MS m/z (%): 310  75 (100). Anal. For: C17H18N4O2: Calcd. C, 65.80; H, 5.80; N, 18.06, Found: C, 65.38; H, 9.13, N, 18.00.4-Methyl-2-phenyl-6-triacetylhydrazide-5-pyridinecarbonitrile (18). Compound 5 (0.002 mol) in acetic anhydride (10 mL) was heated under reflux for 1h. After cooling, the solvent was concentrated under reduced pressure, then the reaction mixture was poured into ice-water (40 mL) to give a solid precipitate which was filtered off an recrystallized from methanol. 18: Yield 60%; m.p. 180–182 °C; IR cm–1: 3100 (CH–Ar), 2910 , 2215 (C≡N), 1681 (C=O); 1H-NMR: δ 2.48 , 2.50, 2.80, 3.10  7.52–7.78 , 13C-NMR: δ 13.22 (CH3) 17.11 (COCH3), 19.28 (COCH3), 21.00 (COCH3) 115 (C≡N), 186.00 (C=O), 180.11 (C=O), 110.66–153.18 (Ar–C); MS m/z (%): 350 , 75 (100). Anal. For: C19H18N4O3. Calcd. C, 65.14; H, 5.14; Nm 16.00; Found: C, 65.30; H, 5.25; N, 16.30.1-[3-Cyano-4-methyl-6-phenyl]-pyridine-2-yl-(2H)-phthalazine-3,8-dione (19). A mixture of compound 5 (0.01 mol) and phthalic anhydride (0.01 mol) in acetic acid (20 mL) was refluxed for 6 h. The reaction mixture was concentrated, allowed to cool and poured onto H2O (100 mL). The solid obtained was recrystallized from dioxane. 19: Yield 52%; m.p. 310–312 °C; IR cm–1: 3280 (NH), 3033 (CH–Ar), 2999 , 2222 (C≡N), 1739, 1714 (2C=O); 1H-NMR: δ 2.48 , 7.22–7.83 , 12.21 ; 13C-NMR: δ 11.82 (CH3), 186.98 (C=O), 202.65 (C=O), 118.76 (C≡N), 128.89–162.33 (Ar–C); MS m/z (%): 354 , 77 (100). Anal. For: C21H14N4O2: Calcd. C, 71.18; H, 3.95; Nm 15.81; Found: C, 71.30; H, 4.18, N, 15.50.8-Cyano-7-methyl-5-phenyl-2,3-dihydro-2-thioxo-1,2,4,triazolo--pyridine (20). To a stirred suspension of compound 5 (22 mmol) in ethanol (20 mL), ethanolic potassium hydroxide  and CS2 (2 mL) were added dropwise. The reaction mixture was then heated under reflux for 6 h. After cooling and evaporation of the solvent, the potassium salt obtained was dissolved in water and acidified with 2N aqueous HCl. The solid product formed was collected and recrystallized from ethanol. 20: Yield 72%; m.p. 160–162 °C. IR cm–1: 3220 (NH), 3055 (CH–Ar) 2880 , 2220 (C≡N), 1644 (C=N); 1H-NMR: δ 2.48 , 6.88–7.45  11.33 ; 13C-NMR: δ 14.58 (CH3), 118.67 (C≡N), 212.00 (C=S), 125.78–148.79 (Ar–C); MS m/z (%) 266  75 (100). Anal. For: C14H10N4S. Calcd. C, 63.15; H, 3.75; N, 21.05; S, 12.03; Found: C, 63.30; H, 4.00; N, 21.30; S, 12.11.4, 5, 11–13, 18 and 19 were synthesized starting from compounds 4 and 5. The present syntheses are one pot methods and afforded pyrido--pyrimidine and pyrazolo--pyridine derivatives in moderate to good yields. In addition, our study was extended to the synthesis of additional fused pyridine, such as isoquinolines 2a,b, 1,8-naphthyridine 14 and 1,2,4-trizolo--pyridine derivative 20. Spectral and analytical data of the newly synthesized compounds were all in good agreement with the proposed chemical structures.New pyridine derivatives


Their structures were established by spectroscopic analyses and comparisons with data from related compounds. A pilot pharmacological study showed that picrogentiosides A (1) and B (2) have an immunomodulatory effect in vitro.Three new secoiridoid glycosides, named picrogentiosides A ( Picrorhiza scrophulariiflora (Scrophulariaceae) grows in the high altitude regions  in the southeast of Tibet and the northwest of Yunnan in China. The roots of this plant, known to be rich in terpenoids, iridoid glycosides, phenolic glycosides and phenylethanoid glycosides 25D-153.7°  and its molecular formula was determined as C31H36O17 by HR-ESI-MS . UV  and IR  absorptions suggested the presence of hydroxyl, α, β-unsaturated ester and aromatic groups. Picrogentioside A , H-1], an oxygenated methylene proton , two olefinic protons , one methine proton , and one terminal vinyl group , due to one gentiopicroside moiety , and two H-atoms of an (trans)-configured C=C bond at δ(H) 6.18, 7.44 .The rum of 1  showed tδH 4.75  and δH 4.78 , which combined with the 13C-NMR signals at δc 101.6-60.7 -N-acetyl-α-methylbenzylamino]-1-deoxyalditol acetate derivative with the same derivative of the standard sugar 25D-144.3° . The HR-ESI-MS analysis established its molecular formula as C31H36O17. The UV, IR, 1H-NMR and the 13C-NMR spectral features of 2 were quite similar to those of 1 , revealing that the caffeoyl moiety should be linked to C-4' of the glucoside 1, as indicated by the HMBC crosspeak between this carbon (δC 165.9) and the anomeric proton (δH 4.61). Acid hydrolysis of 2 gave only d-glucose. Accordingly, the structure of 2 was elucidated to be as shown in Picrogentioside B  a yellow amorphous powder, [α]25D-208.3°. The molecular formula C32H42O18 was assigned for 3 on the basis of HR-ESIMS at m/z [M+Na]+ . The NMR spectroscopic data of 3 were similar to those of 1 , CH3O], an acetal proton , an oxygenated methylene proton , an olefinic proton , a methylene proton , two methine protons , and one terminal vinyl group . These signals indicated one secologanol moiety 25D-153.7° ; UV(MeOH) λmax nm (logε): 215 (3.20), 277 (3.11); IR (KBr) νmaxcm-1: 3522, 3437, 3297, 2899, 2257, 1712, 1645, 1606, 1510, 1403; for 1H- and 13C-NMR spectra, see m/z 703.1859 .Picrogentioside B (2): Yellow amorphous powder; [α]25D-144.3° ; UV (MeOH) λmax nm (logε): 208 (3.41), 271 (3.28); IR (KBr) νmaxcm-1: 3525, 3436, 3305, 2897, 2257, 1701, 1622, 1609, 1511,1408; for 1H- and 13C-NMR spectra, see m/z 703.1857 .Picrogentioside C (3): Yellow amorphous powder; [α]D-208.3°; UV (MeOH) λmax nm (logε): 209 (3.37), 287 (3.15); IR (KBr) νmaxcm-1: 3522, 3436, 3306, 2889, 2263, 1703, 1629, 1617, 1512, 1433, 1271, 1216, 1169, 966, 904, 835, 753, 711; for 1H- and 13C-NMR spectra, see m/z 737.2249 .Plantamajoside (4): White amorphous powder; 1H-NMR δ: 7.44 , 6.27 , 7.03 , 6.99 , 6.75 , 6.63 , 6.62 , 6.49 , 3.61 , 3.88 , 2.91 , 4.38 , 4.36 ; 13C-NMR δ: 129.1 (C-1), 116.3 (C-2), 144.9 (C-3), 143.5 (C-4), 115.4 (C-5), 121.1 (C-6), 35.0 (C-7), 70.1 (C-8), 125.6 (C-1′), 114.8 (C-2′), 145.5 (C-3′), 148.3 (C-4′), 115.7 (C-5′), 119.5 (C-6′), 114.2 (C-7′), 144.9 (C-8′), 165.7 (C-9′), 102.1 (C-1′′), 73.2 (C-2′′), 82.9 (C-3′′), 69.4 (C-4′′), 74.5 (C-5′′), 60.7 (C-6′′), 104.5 (C-1′′′), 74.4 (C-2′′′), 76.2 (C-3′′′), 69.7 (C-4′′′), 76.7 (C-5′′′), 60.7 (C-6′′′)Plantainoside D (5): White amorphous powder; 1H-NMR δ: 7.47 , 6.29 , 7.05 , 6.96 , 6.76 , 6.61 , 6.58 , 6.45 , 3.61 , 3.80 , 2.67 , 4.37 , 4.34 ; 13C NMR δ: 129.3 (C-1), 116.3 (C-2), 145.0 (C-3), 143.5 (C-4), 115.5 (C-5), 121.5 (C-6), 35.1 (C-7), 70.2 (C-8), 125.4 (C-1′), 114.9 (C-2′), 145.6 (C-3′), 148.6 (C-4′), 115.8 (C-5′), 119.5 (C-6′), 113.7 (C-7′), 145.4 (C-8′), 166.5 (C-9′), 102.2 (C-1′′), 73.2 (C-2′′), 87.6 (C-3′′), 68.6 (C-4′′), 73.9 (C-5′′), 63.3 (C-6′′), 104.1 (C-1′′′), 72.1 (C-2′′′), 76.1 (C-3′′′), 70.2 (C-4′′′), 76.9 (C-5′′′), 61.1 (C-6′′′)1, 2 and 3) (6 mg) in 2 N TFA (3 mL) was refluxed at 100°C for 3 h. The reaction mixture was extracted with EtOAc. The EtOAc extract of 1, 2 and 3 was proven to contain caffeic acid by direct TLC comparison with authentic samples. d-glucose was found as the only sugar present in the water part following the procedure of Oshima, Yamauchi and Kumanotani [2O layer was neutralized by passing through an ionexchange resin (Amberlite MB-3) column and concentrated under reduced pressure to dryness. The residue was dissolved in H2O (1 mL), to which L-(-)-α-methylbenzylamine (5 mg) and NaBH3CN (8 mg) in EtOH (1mL) were added. The mixture was stirred at 40 °C for 4 h, then acidified by addition of glacial HOAc (0.2 mL) and evaporated to dryness. The resulting solid was acetylated with Ac2O (0.3 mL) in pyridine (0.3 mL) for 24 h at room temperature. After evaporation, H2O (1 ml) was added to the residue. The solution was passed through a Sep-Pak C18 cartridge and washed with H2O and CH3CN. The CH3CN eluate was analyzed by HPLC under the following conditions: solvent, MeCN-H2O (2:3); flow rate, 0.8 mL/min; detection, UV 230 nm. The derivative of d-glucopyranose was detected with the tR of 17.3 min.A solution of each compound  were added to the culture of splenocytes of mice alone in control and combined with either Con A at a terminal concentration of 5 mg/L or LPS at 20 mg/L for 48 h. Cell proliferation was measured using the MTT assay [The compounds TT assay ,19.