The compounds were purified using several chromatographic methods. Structural elucidation of the compounds was performed using their 1H and 13C-NMR data, EI mass and UV spectral data. The compounds have been subjected to antimicrobial, antioxidant and cytotoxic activity. The diterpene showed higher cytotoxic activity than the flavones while the later compounds were better antioxidants compared with the isolated diterpene. Two flavones, ladanein and 6-hydroxy-5,7,4′-trimethoxyflavone and one labdane-type diterpene,  Salvia (common name: sage), the largest genus of the Lamiaceae family, includes about 900 species. The word Salvia was derived from the Greek word ‘‘Salvere: healer, curative” and refers to the numerous medicinal applications of the plants of this family. These plants grow in the temperate and warmer zones of the world. Fifty-eight species are found in Iran, among which 17 are endemic  free radical scavenging assays. Finally, the cytotoxic activity of isolated compounds has been tested against two human cancer cell lines: colon carcinoma (TH-29) and breast ductal carcinoma (T47D).nalgesic . To the Plant materialS. sharifii were collected during the full flowering stage in May 2011 from the mountain areas of the Geno protected area in 30 Km west north of Bandar Abbas  at an altitude of ca. 1800 m 27°29′ N and 55° 54′ E. It was identified by Dr. Mojtaba Asadollahi and a voucher specimen (NO. VC-19-8) was deposited at the Herbarium of Science and Research Branch, Islamic Azad University, Tehran, Iran. The aerial parts of the plant were air-dried at room temperature (25°C) in the shade for 5 days before the extraction.The aerial parts of the wild-growing Extraction and isolation2O (259 mL) and extracted successively with n-hexane (3 × 650) and CHCl3 (3 × 450 mL). The CHCl3 extract on evaporation at reduced pressure furnished a residue (10 g) which was then subjected to column chromatography on silica gel (200 g) using n-hexane with increasing amounts of EtOAc (0-100%) up to EtOAc : MeOH (9 : 1). Fifty-one fractions were collected which were monitored by silica gel-TLC. Fractions 10-12 exhibited two spots on TLC were combined and after repeated CC-chromatography purification, yielded the ent-13-epi-manoyloxide (169 mg). After being monitored by TLC, fractions 33-35 were combined and recrystallized from hexane-EtOAc to remove the pigments impurities. Recrystallization from n-heptane : EtOAc (3 : 1) afforded 54 mg of pure 6-hydroxy-5,7,4’-methoxy-flavone. Fraction 36 was subjected to CC on silica gel (230-400 mesh) using hexane : EtOAc (7 : 1) to yield 70 mg of ladanein.The separation process was carried out using several chromatographic methods . Ground Ent-13-epi-manoyloxide20H34O, White powder, m.p. 70-72ºC, 1H-NMR : 0.79 , 1.16 , 1.29 , 1.44  5.05, 5.23 , 5.94 ; 13C-NMR : C14 (145.8), C15 (111.2), C8 (76.7), C13 (77.0), C8 (76.7), C5 (61.5), C9 (56.0), C1 (44.95), C3 (44.4), C7 (41.9), C12 (39.6), C4 (33.3), C10(31.5), C16 (27.5), C18 (24.2), C19(22.6), C17 (21.4), C20 (20.5) C2 (18.4), C11 (18.3), C6 (15.3); MS (m/z) (rel.int.): 290 [M]+ (14), 275 [M-Me]+(7), 177 (88), 109 (68), 95 (84), 81 (80), 43 (100).C6-hydroxy-6,7,4′-trimethoxyflavone18H16O6, green crystals, m.p. 181-183ºC; 1H-NMR : 3.90 , 3.93 , 3.98 , 6.55 , 6.59 , 7.02 , 7.85 ; 13C-NMR : OMe-5 (55.54), OMe-7 (56.30), OMe-4 (60.86), C8(90.55), C3 (104.12), C10 (106.12), C5′ and C3′ (114.51), C1′ (123.53), C2′ and C6′ (127.99),C6 (132.81), C5 (153.03), C9 (153.22), C7 (158.71), C4′ (162.6), C2 (164.00), C4 (182.65); MS (m/z) (rel.int.): 328 [M]+ (85), 313 [M-Me]+(85), 285 [M-Me-CO]+(37),), 167 (52), 149 (100), 69(80), 57 (60), 43 (44).CLadanein17H14O6, White powder, m.p. 232-233ºC, 1H-NMR : 3.73 , 3.92 , 6.86 , 6.92 , 6.93 , 7.97 , 12.93 ; 13C-NMR : OMe-7(56.54), OMe-4 (60.04), C8(91.58), C3 (102.68), C10 (105.07), C5′ and C3′ (115.98), C1′ (121.01), C2′ and C6′ (128.58),C6 (131.85), C9 (152.09), C5 (152.63), C7 (158.62), C4′ (161.31), C2 (164.06), C4 (182.25); MS (m/z) (rel.int.): 313 [M]+ (29), 299 [M-Me]+(17), 279 (40), 214 (68), 167 (88), 149 (100), 57 (52), 43 (33).CAntimicrobial activityAntioxidant activityAntimicrobial tests were carried out by the disc diffusion method  and MIC The antioxidant activates of the isolated compounds were evaluated using 2, 2 diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assays. This test was determined using a published DPPH radical scavenging activity assay method  with minCytotoxic activityThe general method used for testing on antitumor properties of these compounds is the standard testing method that has been previously described in detail . IsolateThe isolated compounds were identified as ladanein, 6-hydroxy-5,7,4′ trimethoxyflavone, and ent-13-epi-manoyloxide . 1H- and 13C-NMR for the isolated flavones and diterpene have been published previously. Ladanein is a known methoxylated flavone that found abundantly in Salvia species. It has been isolated from S. hypoleuca, S. cyanescens, S. limbata and S. stenophylla  in terms of minimal inhibitory concentration (MIC) and zone of inhibition around the disc, while Escherichia coli with MIC of 32 μg/mL. This is particularly interesting from a medical point of view as this microbial agent is responsible for severe opportunistic infections. None of the isolated components showed significant activity against fungal microorganisms. We also screened the antioxidant activity of the isolated compounds from the aerial parts of S. sharifii by DPPH radical scavenging assay ( Both flavones were active against ng assay .50 = 5.3 ± 0.3 and 3.6 ± 0.1 mg/mL respectively) in comparison with the non-phenolic diterpene, ent-13-epi-manoyloxide (IC50 = 16.2 ± 0.8 mg/mL). In cytotoxic activity, the effects of these compounds on the proliferative response of the HT-29 and T47D cell lines have been analyzed by treating the cells with different concentrations of the compounds in DMSO and decrease in cell lines proliferation were observed. Ent-13-epi-manoyloxide exhibit high cytotoxic activity on T47D (IC50 = 80.89 ± 4.05 μg/mL) and HT-29 (IC50 = 31.30 ± 1.32 μg/mL) while this property in isolated flavones is less ( As expected for all phenolic compounds, ladanein and 6-hydroxy-5,7,4′-methoxy-flavone showed higher antioxidant activities (IC is less . These results showed that this medicinal plant has the required potential to be used in aromatherapy and pharmacy.


N--yl)-2-(2-oxo-2H-chromen-4-yloxy)acetamide 5 and N-(4-oxo-2-phenylthiazolidin-3-yl)-2-(2-oxo-2H-chromen-4-yloxy)acetamide 6 were studied with the DPPH, hydrogen peroxide and nitric oxide radical methods and compared with the known antioxidant ascorbic acid. Compounds 5 and 6 were synthesized in a good yield from the addition reaction of maleic anhydride or mercaptoacetic acid to compound 4, namely N′-benzylidene-2-(2-oxo-2H-chromen-4-yloxy)acetohydrazide. Compound 4 was synthesized by the condensation of compound 3, namely 2-(2-oxo-2H-chromen-4-yloxy) acetohydrazide, with benzaldehyde. Compound 3, however, was synthesized from the addition of hydrazine to compound 2, namely ethyl 2-(2-oxo-2H-chromen-4-yloxy)acetate, which was synthesized from the reaction of ethyl bromoacetate with 4-hydroxycoumarin 1. Structures for the synthesized coumarins 2–6 are proposed on the basis of spectroscopic evidence.The antioxidant activity of two synthesized coumarins namely,  Coumarin and its derivatives represent one of the most active classes of compound possessing a wide spectrum of biological activity –4. Many H-chromen-4-yloxy)acetate 2, which was used as a starting material for the synthesis of compounds 5 and 6, is presented in this study. The chemical structures of the synthesized compounds 2–6 were conducted and confirmed. The in vitro antioxidant activities were investigated for the synthesized compounds 5 and 6.The preparation of ethyl 2--yl)-2- (2-oxo-2H-chromen-4-yloxy)acetamide 5 and N-(4-oxo-2-phenylthiazolidin-3-yl)-2-(2-oxo-2Hchromen-4-yloxy)acetamide 6, is out lined in The reaction sequence for the synthesis of H-chromen-4-yloxy)acetate 2 was conducted via reflux of Ethyl bromoacetate with 4-hydroxycoumarin 1 in the presence of anhydrous potassium carbonate in acetone. Hydrazinolysis of compound 2 with hydrazine hydrate produced hydrazide 3 in good yield. The FT-IR spectrum of compound 3 showed absorption bands in the 3297.3, 3211 cm−1 (hydrazide NH-NH2), 1711 cm−1 (lactonic -C=O carbonyl stretching), and 1671.2 cm−1 (amide -C=O carbonyl stretching). The 1H-NMR spectrum exhibited a singlet due to the -CO-NH-NH2 proton at δ 8.89 ppm. Compound 3 was thencondensed with benzaldehyde to make compound 4 that can be cyclized with maleic anhydride (or mercaptoacetic acid)to yield compounds 5 (and 6). The IR spectrum of compound 4 showed characteristic bands at 3191.1 cm−1 (NH), 1734.3  cm−1, 1688 cm−1 . The 1H-NMR spectrum showed the absence of NH2 protons, and the presence of the N=CH proton at 8.23 ppm. Compound 5 shows pronounced peaks; a singlet due to the NH proton at 9.58 in addition to a singlet for 1H assigned to the (-C=C-H). The IR spectrum of compound 6 showed characteristic bands at 3198.7 cm−1, 29211731 cm−1, and 1689 and 1667 cm−1 (NH), ,  and  respectively. The 1H-NMR spectrum of compound 6 showed peaks at 5.87 ppm  =C-H and 3.91 .The synthesis of ethyl 2- −0.510] followed by the next charge value at [O(10) −0.509] and [O(15) (−0.437)]. These data show clearly that these three atoms are the most reactive toward the addition, substitution reactions and bonding with the metal. The determined bond angle and twist angle, stretch (1.4560), bend (4.3225), stretch-bend (−0.1213). The 3D geometrical structure indicates that this molecule is a non-planarmolecular and the stereochemistry is .An earlier study  has show5 and 6 were screened for in vitro antioxidant activity using  DPPH, Nitric oxide and hydrogen peroxide. They show good antioxidant activity against all methods  and used as supplied without further purifications. The FTIR spectra were recorded as KBr disc on FTIR 8300 Shimadzu Spectrophotometer. The UV-Visible spectra were measured using Shimadzu UV-VIS. 160A spectrophotometer. Proton NMR spectra were recorded on Bruker - DPX 300 MHz spectrometer with TMS as the internal standard. Elemental micro analysis was carried out using a CHN elemental analyzer model 5500-Carlo Erba instrument.All chemical1 (6.17 mmol) in acetone (30 mL) was refluxed with ethyl bromoacetate (9.15 mmol) and K2CO3  for 12 h. After cooling, the mixture was evaporated to dryness and the residue was partitioned between CHCl3 (50 mL) and water (50 mL). The organic phase was dried (Na2SO4), filtered and evaporated to dryness. The residue was recrystallized from acetone; yield 92%; m.p. 99.0 °C; 1H-NMR (CDCl3): δ 3.22 , δ 3.85 , δ 4.91  and δ 5.20, δ 5.250, δ 5.272  for CH2), δ 5.78  for -C=C-H), δ 7.291, δ 7.478, δ 7.80  for aromatic ring); 13C-NMR: 167.2; 165.1; 163.4, 155.9; 134.2; 121.8; 121.1; 119.0; 113.8; 100.9; 65.3; 54.7; 22.12; IR: 2987.3 cm−1 , 3089.5 cm−1 , 1759.3 cm−1 , 1717.6 cm−1 , 1629.2 cm−1 , 1577.6 cm−1 ; Theoretical Calculation for C13H12O5: C 62.90%, H 4.87%. Experimental: C 61.91% H 3.99%.A suspension of 4-hydroxycoumarins 2 (10 mmol) in ethanol 25 mL was refluxed with hydrazine hydrate (15 mmol) for 4 h. After concentrating the reaction mixture a solid mass separated out and recrystallized using ethanol, yield 51%, m.p. 228 °C; 1H-NMR (CDCl3): δ 4.48 , δ 4.65 , δ 8.89 , δ 4.92  and δ 5.210  for (O-CH2), δ 5.72  for (-C=C-H), δ 7.410, δ 7.521, δ 8.10  for aromatic ring; IR: 3297.3, 3211 cm−1 (N-H), 2906.0 cm−1 , 3072.7 cm−1 , 1711.5 cm−1 , 1671.2 cm−1 ; Theoretical Calculation for C11H10N2O4: C 56.41%, H 4.30%, N 11.96%. Experimental: C 57.13% H 4.01%, N 10.52%.A solution of compound 3 (0.2 mmol) in ethanol 25 mL was refluxed with benzaldehyde (0.2 mmol) for 20 h. After cooling to room temperature, a solid mass separated and recrystallized. Recrystallized from ethanol;yield 57%; m.p. 258 °C; 1H-NMR (CDCl3): δ 8.23, , δ 8.11 , δ 4.91  and δ 5.340  for O-CH2), δ 5.67  for (-C=C-H), δ 7.240, δ 7.312, δ 7.97  for aromatic ring; 13C-NMR (DMSO-d6): 170.0, 169.2, 164.1, 155.1,145.6, 136.9,129,8, 128, 125, 120.6, 119.9, 118.4,118.3, 117.9, 80.1, 65.2; IR: 3191.1cm−1 (N-H), 2901.0 cm−1 , 3058.2 cm−1 , 1743.3 cm−1 , 1688 cm−1 , 1613 cm−1 (C=N), 1627.7 cm−1 (C=C); Theoretical Calculation for C18H14N2O4: C 67.07%, H 4.38%, N 8.69%. Experimental: C 66.39% H 3.95%, N 7.97%.A solution of the compound 4 with 1 mmole of maleic anhydride in 50 mL of dry benzene was refluxed in water bath for 20 h. The solvent was removed and the precipitate was recrystallized from tetrahydrofuran; yield 44%; m.p. 201 °C; 1H-NMR (CDCl3): δ 3.93 and 4.22 , δ 9.58 , δ 4.81  and δ 5.350  for O-CH2), δ 6.11  for (-C=C-H), δ 7.320, δ 7.412, δ 7.79  for aromatic ring; 13C-NMR (DMSO-d6): 170.4, 167.2, 165.8, 160.6, 155.1, 155.4, 139.4, 136, 128.8, 125.8, 124.3, 120.6, 119.6, 117.1, 116.3 115.3, 113,100.6, 69.9, 55.4; IR: 3232.1cm−1 (N-H), 2979.5 cm−1 , 3097.3 cm−1 , 1765, 1741 cm−1 (C=O), 1671 cm−1 , 1617.8 cm−1 (C=N), 1632.6 cm−1 ; Theoretical Calculation for C22H16N2O7: C 62.86%, H 3.84%, N 6.66%. Experimental: C 61.99% H 3.72%, N 5.90%.Mixture of 1 mmole of compound 1H-NMR (CDCl3): δ 3.83 , δ 5.40 , δ 8.98 , δ 4.77  and δ 5.110  for O-CH2), δ 5.82  for (−C=C-H), δ 7.320, δ 7.441, δ 7.92  for aromatic ring; 13C-NMR (DMSO-d6): 171, 169.0, 167.8, 154.1, 159.8, 128.2, 127.8, 126.1, 124.3, 120.1, 117.1, 114.4, 112.9,110.1 101.4, 77.6, 65.4, 33.2; IR: 3198.7cm−1 (N-H), 2921 cm−1 , 3087.7 cm−1 , 1731 cm−1 , 1689, 1667 cm−1 , 1622 cm−1 (C=N), 1632.8 cm−1 ; Theoretical Calculation for C20H16N2O5S: C 60.60%, H 4.07%, N 7.07%. Experimental: C 60.41% H 3.91%, N 6.88%.Mixture of 0.01 mole of Schiff base with 0.01 of mercaptoacetic acid in 50 mL of dry benzene was refluxed in water bath for 20 hours., filtered off, washed with water, dried and recrystallized from dichloromethane; yield 70%; m.p. 261 °C; 5 and 6) were evaluated according to Soares, et al [The DPPH radical scavenging activities of the test samples compounds  or standard solution (0.5 mL) was incubated at 25 °C for 150 min. After the incubation, 0.5 mL of the reaction mixture containing nitrite was pipetted and mixed with 1 mL of sulphanilic acid reagent  and allowed to stand for 5 min for completing diazotization. Then, 1 mL of naphthylethylenediaminedihydrochloride (1%) was added, mixed and allowed to stand for 30 min. A pink-colored chromophore was formed in diffused light. The absorbance of these solutions was measured at 540 nm against the corresponding blank. Ascorbic acid was used as standard. Nitric oxide percentage scavenging activity was then calculated using Sodium nitroprusside in aqueous solution at physiological pH generates nitric oxide spontaneously; it interacts with oxygen to produce nitrite ions, which can be estimated by the use of GriessIllosvoy reaction . In the A solution of hydrogen peroxide 40 mM) was prepared in phosphate buffer (pH 7.4). Different concentrations ) of synthesized compounds (or ascorbic acid) were added to a hydrogen peroxide solution . Absorbance of hydrogen peroxide at 230 nm was determined after 10 min. against a blank solution containing phosphate buffer without hydrogen peroxide  mM was p. Hydroge3 model was employed to obtain quantum chemical parameters and optimization of the molecule geometry. Molecular atomic charges were calculated by Mulliken population analysis.The molecular sketch of the reference compound was plotted using Visualization Materials Studio 5.5 software. All quantum chemical calculations were performed using the Density Functional Theory (DFT) in the methodology. DMolN--yl)-2- (2-oxo-2H-chromen-4-yloxy)acetamide and N-(4-oxo-2-phenylthiazolidin-3-yl)-2-(2-oxo-2H-chromen-4-yloxy)acetamide, have been successively synthesized, and characterized by using various spectroscopic methods and elemental analysis techniques. Two postulated mechanisms have been proposed for the action of compound as antioxidant. The antioxidant activity of the compounds was initially tested and showed that the compounds have improved properties compared to ascorbic acid.In this study, the two coumarins, namely


Structures were established using IR, HRESIMS, 1D and 2D NMR, and single X-ray diffraction techniques. The isolated compounds were assessed for their cytotoxicity against a small panel of human cancer cell lines  with IC50 values in the range of 6–18 μM. In addition, compound 9 showed weak antifungal activity against Trichophyton rubrum and Microsporum gypseum with MIC values of 8 and 32 μg/mL, respectively, compound 10 displayed weak antifungal activity against fungi Candida albicans, Cryptococcus neoformans, T. rubrum, and M. gypseum with MIC values of 8, 8, 4, and 8 μg/mL, respectively.Seven new formamido-diterpenes, cavernenes A–D ( Acanthella have proven to be a rich source of new diterpenes and sesquiterpenes containing nitrogenous functional groups, including isonitrile (–NC), isothiocyanate (–NCS), isocyanate (–NCO), and formamide (–NHCHO) functionalities, which show various promising biological activities  and four olefinic proton signals resonated at δH 4.80 , 4.89 , 5.13 , and 5.24 . The 13C NMR and DEPT spectra of 1 displayed 21 carbon resonances for four methyls, seven methylenes (one olefinic), six methines (two olefinic and one formamido), and four quaternary carbons (three olefinic). The above moieties accounted for four of six degrees of unsaturation, indicating a bicyclic structure for 1. The COSY correlations of H-9/H-8/H-7/H-6/H-1/H-2/H-3/H-5/H-19, together with the HMBC correlations of H3-19/C-3, C-4, and C-5, H3-20/C-1, C-9, and C-10, H-5/C-1, C-3, C-6, and C-7, and CHO/C-10 indicated the presence of a decalin moiety (3-16 and H3-17) to C-14 and C-15, from terminal olefinic protons (H2-18) to C-7, C-11, and C-12, and from H-13 to C-11, C-12, C-14, and C-15 and COSY correlation between H-13 and H-14 revealed the presence of an isoprenoid unit homolog in 1 and connectivity of the two moieties between C-12 and C-7 through the quaternary carbon C-11.Cavernene A (1) was isolated as a colorless oil. A molecular formula of CNMR data , indicatn moiety . The HMB3-20 and H-6. In addition, the carbon resonances at δC 48.8 (C-1), 39.5 (C-6), 42.0 (C-9), and 19.0 (C-20) in 1 further confirmed the trans fusion of the decalin ring [1) can be envisaged as a decomposable intermediate product of isocyanobifloradiene epoxides in the plausible biogenetic pathway of kalihiprans [The relative configuration of 1 was established on the basis of NOESY data . The NOElin ring . Caverne2) was obtained as a colorless oil. Its molecular formula of C21H35ON was deduced from the HRESIMS (m/z 340.2617 [M + Na]+) combined with its NMR data . The relative configuration at C-11, however, could not be conclusively determined due to conformational flexibility between C-7 and C-11.Cavernene B , obtained as white needles, showed the same molecular formula of C21H35ON as 2 determined by pseudomolecular [M + Na]+ ion peak at m/z 340.2614 in HRESIMS. Its carbon skeleton was readily assignable as the same as 2 by HSQC, HMBC, and COSY spectra. In particular, the carbon resonances of the isoprenoid unit in 3 were almost superimposable on those of 2 , 38.4 (C-6), 41.8 (C-9), and 18.8 (C-20) in 2 were replaced by resonances at δC 45.6, 35.2, 33.0, and 27.2 in 3, respectively, indicating a cis fusion of the decalin ring [Cavernene C  displayed a HRESIMS [M + Na]+ peak at m/z 354.2407 corresponding to a molecular formula of C21H33O2N, implying six degrees of unsaturation. Many similarities of the 1H and 13C NMR data between 2 and 4 (δC 62.33 and 62.27) in 4 and the absence of two saturated carbons (δC 30.8 and 35.65) in 2. The C-11/C-12 position of the epoxy group was determined by a COSY correlation of H-12/H-13 and HMBC correlations of H3-18/C-7, C-11, and C-12 and H-13/C-11 and C-12. The relative configuration of the decalin ring of 4, found to agree with 2, was established by observation of NOESY correlations of NH/H-1, H-1/H-7, and H3-20/H-6. The relative configurations of C-7 and C-11 between the conjoined bicyclic ring systems in 4 were assigned as 7S* and 11 S*, respectively, based on NOESY correlations of H3-18/H-6, H3-18/H-8b, and H-8a/H-13, as shown in the Newman projection  was isolated as colorless needles (MeOH), and given a C21H34NO2Cl molecular formula with five degrees of unsaturation, based on the HRESIMS (m/z 368.2354 [M + H]+) and NMR spectra. The ESIMS of 5 showed a cluster of isotopic [M + H]+ ion peaks at m/z 368/370 in a ratio of ~3:1, indicating the presence of a chlorine atom in the molecule. The NMR spectra of 5 revealed the presence of five methyls , an olefinic methine , a chlorine-bearing methine [δH 3.93 /δC 64.2], and two oxygenated quaternary carbons (δC 74.2 and 76.9) for the major s-trans isomer  and Y (12) +) in combination with extensive NMR analysis. The NMR spectra of 6 , 1.20 , 1.31 , and 1.68 ], two trisubstituted double bonds , an oxymethine [δH 3.75 /δC 84.2], and an oxygenated quaternary carbons (δC 86.4) for the major s-trans isomer. The COSY correlations of H3-20/H-9/H-8/H-7/H-6/H-1/H-2/H-3 and H-6/H-5/H3-19, together with the HMBC correlations of H3-19/C-3, C-4, and C-5, H3-20/C-1, C-10, and C-9, H-5/C-1, C-3, and C-6, H-2 and H-3/C-4, and H-8/C-10 indicated the presence of a decalin moiety (δC 84.2 (C-14) and 86.4 (C-11), COSY correlations of H-12/H-13/H-14, HMBC correlations of H3-18/C-7, C-11, and C-12 and geminal methyls (H3-16 and H3-17)/C-14 and C-15, and NOESY correlation between H3-18 and H-14. The location of the formamide functionality was assigned to be at C-15 by the observation of doubled singlets for H3-16  and H3-17  and doubled triplets for H-14  +, indicating seven degrees of unsaturation. Its 1H and 13C NMR spectra , 1.62 , 1.78 ], two trisubstituted double bonds , a disubstituted double bond , an oxymethylene [δH 4.16 /δC 67.63], and an oxymethine δH [3.89 (br s)/δC 77.2] for s-cis isomer. Many similarities of the 1H and 13C NMR data between 7 and 8 suggested they were structural analogs  +25.0 ; UV (CH3CN) λmax (log ε) < 200 (2.45) nm; IR (KBr) νmax 3323, 3051, 2927, 2856, 1670, 1541, 1457, 1386 cm−1; 1H and 13C NMR data, see m/z 338.2461 [M + Na]+ .Cavernene A (2): colorless oil; [α] +46.2 ; UV (CH3CN) λmax (log ε) < 200 (2.79), 234  nm; IR (KBr) νmax 3295, 3053, 2960, 2926, 2869, 1681, 1537, 1453, 1382 cm−1; 1H and 13C NMR data, see m/z 340.2617 [M + Na]+ .Cavernene B (3): white needles (MeOH); m.p. 98.0–102.0 °C; [α] +20.0 ; UV (CH3CN) λmax (log ε) < 200 (2.95) nm; IR (KBr) νmax 3304, 3062, 2958, 2926, 2855, 1668, 1538, 1455, 1381 cm−1; 1H and 13C NMR data, see m/z 340.2614 [M + Na]+ .Cavernene C (4): colorless needles (MeOH); m.p. 112.0–115.0 °C; [α] +51.4 ; UV (CH3CN) λmax (log ε) < 200 (2.88) nm; IR (KBr) νmax 3296, 3055, 2928, 2857, 1681, 1538, 1453, 1383 cm−1; 1H and 13C NMR data, see m/z 354.2407 [M + Na]+ .Cavernene D (5): colorless needles (MeOH); m.p. 185.0–190.0 °C; [α] +25.0 ; UV (CH3CN) λmax (log ε) < 200 (2.94) nm; IR (KBr) νmax 3295, 3074, 2929, 2868, 1682, 1541, 1451, 1381 cm−1; 1H and 13C NMR data, see m/z 368.2354 [M + H]+ .Kalihinene E (6): colorless oil; [α] +2.5 ; UV (CH3CN) λmax (log ε) < 200 (2.63), 232  nm; IR (KBr) νmax 3216, 3057, 2966, 2929, 1686, 1451, 1379, 1311 cm−1; 1H and 13C NMR data, see m/z 354.2406 [M + Na]+ .Kalihinene F (7): colorless oil; [α] +24.6 ; UV (CH3CN) λmax (log ε) < 200 (2.87) nm; IR (KBr) νmax 3296, 3055, 2925, 2855, 1668, 1537, 1454, 1377 cm−1; 1H and 13C NMR data, see m/z 352.2255 [M + Na]+ .Kalihipyran C (5): Colorless needles, C21H34NO2Cl, MW = 367.94, monoclinic space group P21, a = 6.499 (2) Å, b = 7.952 (2) Å, c = 20.158 (4) Å, V = 1036.1 (4) Å3, Z = 2, Dcalcd = 1.179 g/cm3, and F (000) = 400. A single crystal of dimensions 0.05 × 0.13 × 0.18 mm was used for X-ray measurements and data collected on a Bruker SMART APEX-II CCD diffractometer using Cu Kα radiation and up to θ = 67.3 at 293 K. A total of 5789 reflections were collected, of which 3435 independent reflections were measured having an Rint of 0.0220, final R indices of I > 2σ (I), R1=0.0426, wR2 = 0.1163, R indices for all data R1 = 0.0445, and wR2 = 0.1184. The crystal structure solution was achieved using direct methods, as implemented with the SHELX-97 software program. The refinment method was full-matrix least-square on F2, goodness-of-fit on F2 was 1.042, and the largest difference peak and hole were 0.237 and −0.196 e. Å-3. The absolute structure was determined giving a Flack parameter of 0.07 (2). The X-ray diffraction material has been deposited in the Cambridge Crystallographic Data Center (CCCD No. 847695).X-ray Crystallographic Analysis Data of Kalihinene E  of these cell suspensions at a density of 5 × 10−4 cell mL−1 was plated in 96-well microtiter plates and incubated for 24 h under the above conditions. 2 μL of the test compound in DMSO at different concentrations was added to each well for 48 h, and then incubated with 1 mg/mL MTT for 4 h. The formazan dye product was measured by the absorbance at 570 nm on a microplate reader. IC50 values were calculated by Reed and Muench’s method.Cytotoxic activity was evaluated by a MTT method as described previously . Cells wCandida albicans, Candida parapsilosis, Candida glabrata, Cryptococcus neoformans, Trichophyton rubrum, Microsporum gypseum, and Aspergillus fumigates [1–12 prepared in RPMI 1640 were next inoculated with the microorganisms and incubated 24 h for C. albicans, C. parapsilosis, C. glabrata, and A. fumigatus, 72 h for C. neoformans, and 4–7 days for T. rubrum and M. gypseum. Broth tubes that appeared turbid were indicative of bacterial growth, while tubes that remained clear indicated no growth. The MIC, defined as the lowest concentration of inhibitory compound at which no growth was observed, was evaluated in triplicate for each compound (within the range 1.25–640 μg/mL). Cultures prepared under the same conditions but without compounds and cultures with the same proportions of DMSO (<1%) were used as controls. The growth of broth tubes without turbidity was further examined by counting the viable cells on the SDA plates.Antifungal activity was determined by the broth macrodilution method following the National Center for Clinical Laboratory Standards (NCCLS) recommendations against the following strains: umigates ,27. BrieAcanthella cavernosa led to the isolation of seven new fomamido-diterpenes, cavernenes A–D (1–4), kalihinenes E and F , and kalihipyran C (7), and five known compounds, kalihipyran A (8), 15-formamido-kalihinene (9), 10-formamido-kalihinene (10), and kalihinenes X (11) and Y (12). The absolute configuration of 5 was determined by single X-ray diffraction. The isolated compounds showed modest cytotoxicity against a small panel of human cancer cell lines, HCT-116, A549, HeLa, QGY-7701 and MDA-MB-231. Compounds 9 and 10 displayed antifungal activity against Trichophyton rubrum and Microsporum gypseum, Candida albicans, and Cryptococcus neoformans.A chemical investigation of the marine sponge of


Hyptis pectinata, popularly known in Brazil as “sambacaitá” or “canudinho,” is an aromatic shrub largely grown in the northeast of Brazil. The leaves and bark are used in an infusion for the treatment of throat and skin inflammations, bacterial infections, pain, and cancer. Analogues of rosmarinic acid and flavonoids were obtained from the leaves of Hyptis pectinata and consisted of two new compounds, sambacaitaric acid (1) and 3-O-methyl-sambacaitaric acid (2), and nine known compounds, rosmarinic acid (3), 3-O-methyl-rosmarinic acid (4), ethyl caffeate (5), nepetoidin A (6), nepetoidin B (7), cirsiliol (8), circimaritin (9), 7-O-methylluteolin (10), and genkwanin (11). The structures of these compounds were determined by spectroscopic methods. Compounds 1–5, and 7 were evaluated in vitro against the promastigote form of L. braziliensis, and the ethanol extract. The hexane, ethyl acetate, and methanol-water fractions were also evaluated. The EtOH extract, the hexane extract, EtOAc, MeOH:H2O fractions; and compounds 1, 2 and 4 exhibited antileishmanial activity, and compound 1 was as potent as pentamidine. In contrast, compounds 3, 5, and 7 did not present activity against the promastigote form of L. braziliensis below 100 µM. To our knowledge, compounds 1 and 2 are being described for the first time. Hyptis is one of the main genera of this subfamily and comprises 280 species. Of these species, 146 are endemic to Brazil + at m/z 359.0759 . The UV spectrum exhibited signals at 322, 296, and 239 nm, and the IR spectrum showed signals at 3435, 1628, 1524, and 1405 cm−1. The 1H and 13C NMR spectra of 1 were similar to those of rosmarinic acid. Upon extraction and fractionation, the leaves of  Figures . Compouned below . Compoun1H-NMR spectrum of 1, two sets of ABX proton signals at  and  and two olefinic proton signals at δ 7.34  and 6.18  were observed. In the aliphatic region, there were three proton signals at δ 4.85 (m), 3.01 (m), and 2.74 (m). The 13C-NMR spectrum showed 18 carbon signals. In the heteronuclear multiple bond correlation (HMBC) spectrum, the H-7 proton signal (δ 7.34) was long range coupled with aromatic carbons at δ 125.58 (C-1), 114.23 (C-2), and 119.4 (C-6), an olefinic carbon at δ 114.90 (C-8) and the carbonyl carbon at δ 166.22 (C-9)  was established to be isoferuloyl-4′-(3′-hydroxyphenyl)-(8′R)-lactic acid, and the compound was named sambacaitaric acid.  In the 22 (C-9) . The absc acid 3 , one wou1) was treated with Ac2O/pyridine to yield the peracetyl derivative (1a). The 1H and 13C NMR spectral data of 1a, obtained through the analysis of extensive 1D and 2D NMR experiments (1. Electrospray ionization mass spectroscopy (ESI-MS) of 1a showed the [M-H]+ at m/z 527, corresponding to the molecular formula C26H24O12. The sambacaitaric acid . Its molecular formula was deduced to be C19H18O8, which produced the [M-H]+ peak at m/z 373. The UV spectrum exhibited signal at 340 nm and the IR spectrum showed signals at 3420, 1680, and 1607 cm−1. The 1H and 13C NMR spectra were similar to those of 1 with the addition of a methoxyl group on the 3 position. Thus, the structure of this new compound was established as 3-O-methyl-sambacaitaric acid.  Compound 3), 3-O-methyl-rosmarinic acid (4) D = +30 , IR (KBr) νmax: 3435 (OH), 1658 (C=O), 1524, (C=C from aromatic rings). 1H NMR .Sambacaitaric acid (2), [α]D = +10 , 1H NMR : δ 7.35 ; 7.01 , 6.91 , 6.73 , 6.66 , 6.58 , 6.48 , 6.18 , 8.83 , 3.02 and 2.73 . 13C NMR : δ 172.6 (C-9′), 166.7 (C-9), 148.2 (C-4), 146.3 (C-3), 145.3 (C-3′), 144.5 (C-7), 143.89 (C-4′), 130.5 (C-1′), 126.1 (C-1), 121.3 (C-6), 120.1 (C-6′), 116.9 (C-2′), 116.3 (C-5), 115.8 (5′), 115.5 (C-2), 115.3 (C-8), 76.6 (C-8′), 56.9 (OCH3), 37.8 (C-7′). 3-O-methyl-sambacaitaric acid (3), [α]D = +10 , UV λmax 242, 324. IR (KBr) vmax: 3382 (OH), 1697 (C=O), 1606, 1522 (C=C from aromatic rings). LC-ESI-MS (negative mode) m/z 359 [M-H]+ (C18H16O8).Rosmarinic acid (O-methyl-rosmarinic acid (4), [α]D = +10 , UV λmax 253, 340. IR (KBr) vmax: 3394 (OH), 1692 (C=O), 1603, 1520 (C=C from aromatic rings). LC-ESI-MS (negative mode) m/z 373 [M-H]+ (C19H18O8).3-5) UV λmax 283, 337. IR (KBr) vmax: 3397 (OH), 1678 (C=O), 1605, 1520 (C=C from aromatic rings). LC-ESI-MS (negative mode) m/z 207 [M-H]+ (C11H12O4).Ethyl caffeate (6) UV λmax 249, 340. IR (KBr) vmax: 3418 (OH), 1680 (C=O), 1603, 1520 (C=C from aromatic rings). LC-ESI-MS (negative mode) m/z 313 [M-H]+ (C17H14O6).Nepetoidin A (7) UV λmax 251, 340. IR (KBr) vmax: 3382 (OH), 1701 (C=O), 1604, 1516 (C=C from aromatic rings). LC-ESI-MS (negative mode) m/z 313 [M-H]+ (C17H14O6).Nepetoidin B (8) UV λmax 272, 346. IR (KBr) vmax: 3419 (OH), 1650 (C=O), 1600, (C=C from aromatic rings). LC-ESI-MS (negative mode) m/z 329 [M-H]+ (C17H14O7).Cirsiliol (9) UV λmax 274, 336. IR (KBr) vmax: 3434 (OH), 1652 (C=O), 1599, (C=C from aromatic rings). LC-ESI-MS (negative mode) m/z 313 [M-H]+ (C17H14O6).Circimaritin (10) UV λmax 254, 349. IR (KBr) vmax: 3397 (OH), 1664 (C=O), 1601, 1507 (C=C from aromatic rings). LC-ESI-MS (negative mode) m/z 299 [M-H]+ (C16H12O6).7-O-methylluteolin (11) UV λmax 267, 338. IR (KBr) vmax: 3445 (OH), 1670(C=O), 1608, 1504 (C=C from aromatic rings). LC-ESI-MS (negative mode) m/z 283 [M-H]+ (C16H12O5).Genkwanin  and 3-O-methyl-sambacaitaric acid (2), and nine known compounds, rosmarinic acid (3), 3-O-methyl-rosmarinic acid (4), ethyl caffeate (5), nepetoidin A (6), nepetoidin B (7), cirsiliol (8), circimaritin (9), 7-O-methylluteolin (10), and genkwanin (11). The EtOH extract, the hexane, EtOAc, and MeOH:H2O fractions; and compounds 1, 2, and 4 exhibited antileishmanial activity; compound 1 was as potent as pentamidine. In contrast, compounds 3, 5, and 7 did not present activity against the promastigote form of L. braziliensis below 100 μM. The activity of the EtOAc fraction can be partially attributed to the isolated compounds 1, 2, and 4.The chemical study of leaves from


Objective(s): Nowadays, cancer is an important public health problem in all countries. Limitations of current chemotherapy for neoplastic diseases such as severe adverse reactions and tumor resistance to the chemotherapeutic drugs have been led to a temptation for focusing on the discovery and development of new compounds with potential anticancer activity.Materials and Methods: A new series of 1,3,4-thiadiazole-derived compounds (3a-3l) were synthesized. N--2-(4-methoxyphenyl) acetamide (2) was prepared through direct amidation of 4-methoxyphenylacetic acid (2) with 5-amino-1,3,4-thiadiazole-2-thiol using EDC (N-Ethyl-N-dimethylaminopropyl carbodiimide) and HOBt (Hydroxybenzotriazole). Then, various derivatives of benzyl chloride containing electron withdrawing and electron donating moieties were reacted with compound 2 to prepare compounds 3a-3l. In vitro cytotoxicity assessment using MTT method was applied and results are presented as IC50. Results: All the synthesized compounds were characterized by 1H-NMR and IR spectroscopy. Some of the synthesized compounds were also characterized using MS spectroscopy. Related melting points were also recorded. According to the obtained data from MTT assay, all compounds (3a-3l) demonstrated a higher cytotoxic activity against MDA-MB-231 breast cancer cell line in comparison with other cell lines. Conclusion: It is notable that four synthesized compounds 3h (IC50= 11 ± 0.18 µM), 3j (IC50= 10 ± 0.39 µM), 3k (IC50= 11 ± 0.77 µM) and 3l (IC50= 8 ± 0.69 µM) exhibited higher cytotoxic activity against MDA-MB-231 cell line compared to imatinib (IC50= 20 ± 0.69 µM) as the reference drug.  Cancer is a general name for a group of more than 100 diseases in which cells belonging to a part of the body, begin uncontrolled proliferation , 3. For in vivo stability of 1,3,4-thiadiazole nucleus are attributed to its aromaticity. 1,3,4-Thiadiazole has exhibited potential antiglaucoma, antiinflammatory, antitumor, antiulcer, antibacterial, antiviral, analgesic, antiepileptic, antifungal and radioprotective activities. Some marketed drugs like acetazolamide (diuretic), sulfaethidole , cefazolin , etc. have 1,3,4-thiadiazole ring .-17.10-17activity . Besidesive site , 14. In ChemistryAll chemicals including starter materials, reagents and solvents were bought from Merck and Sigma-Aldrich companies. Thin layer chromatography was done using TLC sheets from Merck Company. NMR spectra were obtained by Brucker 200  MHz and chemical shifts are expressed as δ (ppm) with tetramethylsilane (TMS) as internal standard. The IR spectra were obtained by Shimadzu 470 spectrophotometer  (potassium bromide disks). Melting points were determined using electrothermal melting point analyzer apparatus and are uncorrected. The mass spectra were run on a Finigan TSQ-70 spectrometer  at 70 eV. All cancerous cell lines were purchased from Pasteur Institute, Tehran, Iran.Synthesis of N--2-(4-methoxyphenyl) acetamide (2)1 ), EDC and HOBt were mixed and stirred in acetonitrile solvent for 30 min. Then, 5-amino-1,3,4-thiadiazole-2-thiol (2 ) was added to the mixture and stirring was continued for 24 hr. Acetonitrile was removed under reduced pressure, ethylacetate/water was added and aqueous layer was removed. Organic phase was washed three times by sodium bicarbonate, diluted sulfuric acid and brine. Drying was done by anhydrous sodium sulfate and filtration was applied for the removal of sodium sulfate. Organic layer was evaporated using rotary evaporator apparatus and a yellowish powder was obtained for compound 3. Diethyl ether was used for washing the obtained powder  δ: 3.35 , 3.38 , 3.79 , 7.76 , 8.02 , 13.1 . IR : 3421, 3147, 2924, 2854, 1697, 1570, 1512, 1303, 1249, 1064, 794. MS (m/z): M+: 281 (45), 238 (25), 148 (90), 121 (100), 91 (25), 78 (30).mp : 135-142General procedure of the synthesis of compounds3a-3l3a-3l derivatives were synthesized according to 3 and potassium hydroxide were mixed and heated for 5 min and then appropriate benzyl chloride derivative was added. Reflux condition was applied for 24 hr and thin layer chromatography (TLC) was used for determining the reaction end. The reaction medium was cooled by crushed ice and cool water. A white precipitate was obtained for all compounds 3a-3l.All intended N-(5-(2-Chlorobenzylthio)-1,3,4-thiadiazol-2-yl)-2-(4-methoxyphenyl) acetamide (3a)°C, Yield: 55%, 1H-NMR  δ: 3.38 , 3.37 , 4.58 , 6.93 , 7.27 , 7.37 , 7.48 , 12.89 . IR : 3440, 3155, 2921, 1690, 1567, 1509, 1321, 1247, 1134, 1031, 825, 760.mp: 190-192N-(5-(3-Chlorobenzylthio)-1,3,4-thiadiazol-2-yl)-2-(4-methoxyphenyl) acetamide (3b)°C, Yield: 56%, 1H-NMR  δ: 3.30 , 3.69 , 4.50 , 6.85 , 7.19 , 7.28 , 7.45 , 12.81 . IR : 3156, 2922, 1691, 1604, 1567, 1509, 1470, 1444, 1321, 1247, 1133, 1033, 826, 760.mp: 180-182N-(5-(4-Chlorobenzylthio)-1,3,4-thiadiazol-2-yl)-2-(4-methoxyphenyl) acetamide (3c)°C, Yield: 56%, 1H-NMR  δ: 3.38 , 3.76 , 4.50 , 6.92 , 7.26 , 7.39 , 12.86 . IR  ύ: 3375, 3125, 2924, 2850, 1693, 1512, 1458, 1296, 1253, 1159, 1051, 825. MS : M+: 405 (10), M++1: 406 (12), M++2: 407 (10), 148 (90), 121 (100), 91 (20), 83 (20), 69 (20), 57 (25). mp: 2102-(4-Methoxyphenyl)-N-(5-(2-nitrobenzylthio)-1,3,4-thiadiazol-2-yl) acetamide (3d)°C, Yield: 63%, 1H-NMR  δ: 3.38 , 3.76 , 4.78 , 6.92 , 7.26 , 7.69 , 8.09 , 12.88 . IR : 3430, 2923, 1685, 1610, 1558, 1523, 1341, 1306, 1036, 788.mp: 159-1622-(4-Methoxyphenyl)-N-(5-(3-nitrobenzylthio)-1,3,4-thiadiazol-2-yl) acetamide (3e)°C, Yield: 59%, 1H-NMR  δ: 3.35 , 3.65 , 4.63 , 6.88 , 7.22 , 7.79 , 7.86 , 8.14 , 8.30 , 12.84 . IR : 3443, 2901, 1694, 1553, 1519, 1439, 1351, 1247, 1170, 1089, 828, 715.mp: 2132-(4-Methoxyphenyl)-N-(5-(4-nitrobenzylthio)-1, 3, 4-thiadiazol-2-yl) acetamide (3f)°C, Yield: 72%, 1H-NMR  δ: 3.39 , 3.76 , 4.65, 6.92 , 7.26 , 7.70 , 8.22 , 12.87 . IR  ύ: 3260, 3150, 3050, 2926, 2854, 1689, 1568, 1519, 1348, 1301, 1257, 1150, 848. MS : M++1: 417 (10), M+: 416 (12), 148 (100), 121 (90).mp: 168-174N-(5-(3-Methoxybenzylthio)-1,3,4-thiadiazol-2-yl)-2-(4-methoxyphenyl) acetamide (3g)1H-NMR  δ: 3.39 , 3.76 , 4.48 , 6.88 , 6.95-7.01 , 7.26 , 12.86 . IR : 3432, 3155, 2904, 2839, 1703, 1612, 1562, 1514, 1435, 1301, 1249, 1170, 1087, 1047, 829.mp: 150-154°C, Yield: 59%, N-(5-(4-Methoxybenzylthio)-1,3,4-thiadiazol-2-yl)-2-(4-methoxyphenyl) acetamide (3h)1H-NMR  δ: 3.3 , 3.72 , 3.77 , 4.46 , 6.92 , 7.23-7.37 , 12.85 . MS (m/z): 401, 368, 281, 193, 148, 121, 78.mp: 165-170°C, Yield: 57%, N-(5-(2-Fluorobenzylthio)-1,3,4-thiadiazol-2-yl)-2-(4-methoxyphenyl) acetamide (3i)1H-NMR  δ: 3.38 , 3.76 , 4.51 , 6.92 , 7.30 , 7.28-7.52 , 12.88 . IR : 3430, 2922, 2853, 1692, 1564, 1510, 1248, 1136, 1031, 759. mp: 155-159°C, Yield: 54%, N-(5-(3-Fluorobenzylthio)-1,3,4-thiadiazol-2-yl)-2-(4-methoxyphenyl) acetamide (3j)1H-NMR  δ: 3.30 , 3.68 , 4.44 , 6.84 , 7.18 , 7.07-7.34 , 12.78 . IR : 3158, 2842, 1686, 1565, 1514, 1405, 1255, 1170, 827, 788.mp: 155-158°C, Yield: 56%, N-(5-(4-Fluorobenzylthio)-1,3,4-thiadiazol-2-yl)-2-(4-methoxyphenyl) acetamide (3k)1H-NMR  δ: 3.38 , 3.76 , 4.50 , 6.92 , 7.26 , 7.24 , 7.47 , 12.86 . IR  ύ: 3395, 3160, 2937, 2854, 1985, 1562, 1510, 1361, 1290, 1246, 1168, 1026, 817. MS : M+: 389 (30), 150 (40), 149 (100), 148 (90), 122 (80), 121 (90), 109 (40), 91 (20), 71 (60), 55 (30).mp: 184-186°C, Yield: 61%, N-(5-(Benzylthio)-1,3,4-thiadiazol-2-yl)-2-(4-methoxyphenyl) acetamide (3l)1H-NMR  δ: 3.29 , 3.68 , 4.42 , 6.84 , 7.26 , 7.16-7.36 . IR  ύ: 3350, 3150, 3030, 2945, 2887, 1716, 1666, 1556, 1396, 1325, 1244, 1165, 1124, 1080, 1022, 790, 696. MS : M++1: 372 (58), M+: 371 (60), 225 (20), 148 (100), 121 (90), 91 (40).mp: 177-180°C, Yield: 72%, MTT assay3a-3l) were assessed regarding their cytotoxic activity at 0.1-250 µg/ml in three human cancer cell lines; PC3 cell (prostate cancer), U87-C-531  and MDA-MB-231 (breast cancer). Cells from different cell lines were seeded in 96-well plates at the density of 10×104viable cells per well and incubated for 24 hr to allow cell attachment. Cells were then incubated for another 24 hr (depending on the cell cycle of each cell line) with various concentrations of compounds 3a-3l. Cells were then washed in PBS, and 20 μl of MTT -2,5-diphenyl tetrazolium bromide) solution (5 mg/ml) was added to each well. An additional 4 hr incubation at 37°C was done, and then the medium was discarded. Dimethyl sulfoxide (60 μl) was added to each well, and the solution was vigorously mixed to dissolve the purple tetrazolium crystals. The absorbance of each well was measured by ELISA plate reader  at the test wavelength of 550 nm against the standard reference solution at 690 nm. The amount of purple formazan production is proportional to the number of viable cells  as a yellowish powder and an average yield (54-72%) was obtained for final derivatives in the form of a creamy to white powder. Melting point of intermediate and final compounds was reported. A range of 154-213C was recorded for final compounds. 1H-NMR, IR and MS spectra for the intermediate and the final compounds were obtained. Compound 3a-3l afforded higher cytotoxic activity against MDA-MB-231, breast cancer cell line and lower cytotoxic activity against U87-C-531, glioblastoma cell line. Substitution of chlorine atom at position 3 (meta) of the phenyl ring resulted in a higher cytotoxic property than other positions of the phenyl ring. Compounds 3a-3c with chlorine moiety rendered better activity against MDA-MB-231 cell line in comparison with other cell lines. Replacement of chlorine moiety with nitro group decreased the cytotoxic effect of compounds 3d-3f against all three cell lines. Nitro group substituent can be more beneficial at positions 3 (meta) and 4 (para) of the phenyl ring compared to position 2 (ortho). Substitution of methoxy at position 4 in compound 3h (IC50= 11 ± 0.18 µM) led to a cytotoxic effect against MDA-MB-231 cell line compared to imatinib (IC50= 20 ± 0.82 µM). As with chlorine, a similar trend like was also observed for fluorine substituent. Position 3 (meta) of the phenyl ring was the best position for fluorine to render its cytotoxic activity in compounds 3i-3k. Compounds 3i-3k like other synthesized compounds in this series exhibited higher cytotoxic activity against MDA-MB-231 breast cancer cell line compared to cytotoxic effect against PC3 and U87-C-531 cell lines. Compound 3j (IC50= 10 ±0.39 µM) with ortho fluorine moiety and compound 3k (IC50= 11 ± 0.77 µM) with para fluorine moiety demonstrated higher cytotoxic activity against MDA-MB-231 cell line as compared with imatinib (IC50= 20 µM). Phenyl ring without any substituent (compound 3l) had the most beneficial cytotoxic effect (IC50= 8 ± 0.69 µM) in all synthesized compounds of this series. The cytotoxicity of all synthesized compounds against three cancerous cell lines was evaluated by MTT assay . Overallin vitro. Against PC3 cell line, Compound 3j with meta fluorine moiety showed the best cytotoxic effects. On the other hand, compound 3i with ortho fluorine substituent exerted the lowest anticancer activity in this series. This trend was also observed against U87-C-531 and MDA-MB-231 cell lines. U87-C-531 cell line was the most resistant cell line to the tested compounds in MTT assay and the recorded IC50 were not significant. A new series of 1,3,4-thiadiazole based compounds was synthesized and their anticancer property was assessed by MTT assay, ortho of the phenyl ring was not capable of enhancing the cytotoxic activity. Fluorine moiety as an electron withdrawing group demonstrated a high cytotoxic potency when substituted at positions meta and para. Interestingly, compound 3l without any electron withdrawing and donating moiety on the phenyl ring, rendered the highest anticancer potency toward MDA-MB-231, PC3 and U87-C-531 cell lines (IC50= 8 ± 0.69 µM). Methoxy moiety as applied in compound 3h as an electron donating group caused an increase in the cytotoxic potency against MDA-MB-231 cell line when it was located at para position of the phenyl ring. Overall, replacing electron withdrawing groups at position 3a-3l) were more toxic toward MDA-MB-231 breast cancer cell line in comparison with other cell lines. It is also notable that four synthesized compounds 3 hr (IC50= 11 ± 0.18 µM), 3j (IC50= 10 ± 0.39 µM), 3k (IC50= 11 ± 0.77 µM) and 3l (IC50= 8 ± 0.69 µM) exhibited higher cytotoxic activity against MDA-MB-231 cell line compared to imatinib (IC50= 20 ± 0.69 µM) as the reference drug.According to the obtained data from MTT assay, all compounds  cell line,


The structures and relative configurations of all the compounds were established by NMR spectroscopy and high-resolution mass spectrometry. The anticancer activity of sesquiterpenes 1, 3, 6–9, 11, 12, 14, and 16 was evaluated in vitro with the breast cancer cell lines HCC1937, JIMT-1, L56Br-C1, MCF-7, and SK-BR-3, and compared with the cytotoxicity in the non-cancerous breast epithelial cell line MCF-10A. All compounds were found to possess anticancer activity, and compound 1 was the most potent in all of the investigated cancer cell lines with IC50 values ranging between 2.0 and 6.2 μM. In order to demonstrate the importance of the α-methylene-γ-lactone/ester moiety present in all compounds for the effects on the cells, the methyl cysteine adduct 21 was prepared from 9 and found to be inactive or considerably less potent.Two new eudesmane derivatives ( Kaunia (Asteraceae: Eupatorieae) comprises only 14 species of which most grow in Bolivia, but that also are found in Argentina, Brazil, Peru, and Ecuador furan-6-yl acetate (+)-; IR vmax(cm−1) 3473, 2933, 2865, 2359, 2342, 1764, 1730, 1375, 1246, 1197, 1134, 1018, 966; 1H , and 13C  see + .Colourless gum; [α]S*,5aR*,6S*,9aS*,9bS*)-5a-Methyl-3,9-dimethylidene-2-oxododecahydro-naphthofuran-6-yl acetate ; IR vmax(cm−1) 2942, 1769, 1726, 1372, 1240, 1174, 1121, 1039, 1016, 969. 1H , and 13C  see m/z 291.1613 [M + H]+ .Colourless oil; [α]9 and 8.3 mg (0.048 mmol) methyl-L-cysteine ester hydrochloride were dissolved in methanol (1 mL) and the mixture was heated at 60°C and stirred. After 48 h, 1.6 mg (0.0093 mmol) methyl-L-cysteine ester was added and the reaction mixture was left for another 24 h. Then, the solvent was evaporated under reduce pressure and the dry residue dissolved in H2O (3 mL), and washed with CHCl3 (3 × 1.5 mL). The organic phase dried in vacuo yielded 15.5 mg of a yellow residue, which was purified by the Sephadex LH-20 (CHCl3:MeOH 1:1) to give 8.0 mg (0.021 mmol) of 21 as yellow oil. 1H NMR : δ 5.35 , 4.12 , 3.75 , 3.69 , 3.59 , 3.00 , 2.94 , 2.88 , 2.84 , 2.76 , 2.28 , 1.98 , 1.96 , 1.95 , 1.76 , 1.66 , 1.26 , 0.89 . 13C : δ 179.5 (C-12), 175.4 (C-3′), 134.7 (C-4), 122.7 (C-3), 82.8 (C-6), 75.9 (C-1), 55.0 (C-2′), 52.7 (C-4′), 52.2 (C-5), 51.8 (C-11), 47.0 (C-7), 41.9 (C-10), 38.6 (C-1′), 35.9 (C-9), 33.5 (C-2), 31.6 (C-13), 24.1 (C-8), 23.6 (C-15), 11.3 (C-14). HR-ESI-MS m/z 406.1703 [M + Na]+ .10.0 mg (0.04 mmol) of 2 in air. The MCF-10A cells have a population doubling time of 15 hours, the JIMT-1 cells 24 hours, and the other four cell lines around 35 hours.The L56Br-C1 cell line was established at the Department of Oncology, Clinical Sciences, Lund University, Sweden . The JIMThe L56Br-C1, MCF-7, SK-BR-3, and HCC1937 cells were cultured as described by Holst et al . JIMT-1 Stock solutions (10 or 100 μM) of the compounds were made in 100% DMSO. These were further diluted in PBS to obtain the correct concentrations used for the MTT assay. Appropriate DMSO controls were used. In general, the highest DMSO concentration was 0.1%, however, when treating with 100 μM and starting from a 10 μM stock in 100% DMSO, a final concentration of 1% DMSO was used as the control.2 incubator for 1 hour. The MTT-containing medium was removed. The blue formazan product formed by the reduction in live attached cells was dissolved by adding 100 μl of 100% DMSO per well after removal of the MTT-containing medium. The plates were swirled gently at room temperature for 10 minutes to dissolve the precipitate. Absorbance was monitored at 540 nm in a Labsystems iEMS Reader MF  using the DeltaSoft II v.4.14 software . Dose-response curves were drawn based on the % of the control in Excel. The IC50 was deduced from the curves.The MTT assay was performed as previously described . Briefly


This is a report of the complete genomic sequence of a rare rotavirus group A G3-P[9]-I2-R2-C2-M2-A3-N2-T1-E2-H3 strain designated RVA/Human-wt/USA/12US1134/2012/G3P[9]. Group A rotaviruses (RVAs) possess a segmented double-stranded RNA (dsRNA) genome composed of 11 segments encoding six structural proteins (VPs) and five or six nonstructural proteins (NSPs) . The majde novo assembly and subsequent mapping to a G3P[9] reference strain was used to obtain the full-length genome of strain 12US1134. The sizes of full-length segments 1 to 11 were 3,302, 2,687, 2,591, 2,359, 1,578, 1,356, 1,075, 1,059, 1,062, 751, and 667 bp, and the open reading frames (ORFs) for these segments were 3,267, 2,643, 2,508, 2,328, 1,476, 1,194, 942, 954, 981, 528, and 597 bp, respectively. The genotype assignment for each gene was accomplished using the RotaC online classification tool (http://rotac.regatools.be/) and BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi).Here, we report the full-genome sequence of rotavirus strain RVA/Human-wt/USA/12US1134/2012/G3P[9] (12US1134), detected in a stool sample collected through the National Rotavirus Strain Surveillance System  from a 6The full-genotype constellation for strain 12US1134 is G3-P9]-I2-R2-C2-M2-A3-N2-T1-E2-H3. This strain is nearly identical (98.9 to 99.8% nucleotide identity) to G3P[9] strain 0537, described previously (-I2-R2-C2Several full-genome sequences of G3P[9] RVA strains of feline or human origin have been reported , 10. G3PFull-genome studies help expand our knowledge of the genetic diversity and origin of uncommon rotavirus genotypes and highlight the need for continuous monitoring of RVA strains for timely recognition of novel or rare genotypes, including in postvaccine introduction settings.KF500514 to KF500524.The full-genomic sequence of the G3P[9] rotavirus strain 12US1134 has been deposited in GenBank under accession no.


The structures of cembranes 1–4 were elucidated by spectroscopic methods. The structure of 4, including its absolute stereochemistry, was further confirmed by single-crystal X-ray diffraction analysis. Cembrane 2 displayed a moderate inhibitory effect on the release of elastase by human neutrophils.Three new cembrane-type diterpenoids, flexibilins A–C ( Sinularia, have been demonstrated to be rich sources of bioactive natural products + at m/z 375.2144  identified by HRESIMS, as well as 13C NMR coupled with DEPT spectra , 142.7 (C-15) and 126.5 (CH2-17) and proton NMR signals appearing at δH 6.40  and 5.68  suggested the presence of a α-exomethylene-carboxylic acid moiety. The main carbon skeleton of 1 was elucidated by 1H–1H COSY and HMBC experiments . These data, together with the key HMBC correlations between protons and quaternary carbons, such as H2-2, H2-3, H2-6, H3-18/C-4; H2-6, H2-7, H2-10, H3-19/C-8; H2-10, H2-11, H-13, H2-14, H3-20/C-12; H2-2, H2-14, H2-17/C-15; and H2-17/C-16, permitted elucidation of the main carbon skeleton of 1. The tertiary methyls at C-4, C-8 and C-12 were confirmed by HMBC correlations between H3-18/C-3, -4, -5; H3-19/C-7, -8, -9; and H3-20/C-11, -12, -13. Thus, from the reported data, the skeleton of 1 was identified as a cembrane-type diterpenoid with two rings.Flexibilin A , had the molecular formula, C20H30O5, as deduced by HRESIMS  (6° of unsaturation). The IR spectrum of 2 revealed the presence of carboxylic acid (νmax 3700–2400 cm−1), hydroxy (νmax 3447 cm−1) and carbonyl (νmax 1713 cm−1) moieties. From the 13C NMR and DEPT spectra of 2 , a α-exomethylene-carboxylic acid , a keto carbonyl  and a trisubstituted epoxide  were observed. The coupling information in the 1H–1H COSY spectrum of 2 enabled identification of the H-13/H2-14/H-1/H2-2/H2-3, H2-6/H2-7, H-9/H2-10/H2-11 and H-9/H3-19  units. From these data, together with the results of an HMBC experiment for 2 , and there was a lack of correlation between H-9 and H3-19, which reflects the E geometry of the double bond between C-8/9. H3-20 showed correlations with H-9 and H-13, indicating that Me-20 is of a β-orientation at C-12. Furthermore, H-1 was found to be correlated with H2-3, and H3-18 exhibited a correlation with one of the C-3 methylene protons (δH 1.78), which indicated the β-orientation of Me-18 at C-4 by molecular modeling analysis. Based on the above information, the chiral carbons of cembrane 2 were assigned as 1R*, 4R*, 12R* and 13S*.The relative stereochemistry of periment . Due to 3) was obtained as a white powder. The molecular formula of 3 was established as C20H30O5 (6° of unsaturation) from a sodiated molecule at m/z 373 in the ESIMS spectrum and further supported by HRESIMS . The IR spectrum of 3 exhibited the presence of hydroxy (νmax 3431 cm−1) and carbonyl (νmax 1711 cm−1) groups. From the 13C NMR data of 3 (δC 168.0 (C-16), 143.3 (C-15), 126.0 (CH2-17), 90.0 (C-4), 37.4 (CH-1), 33.7 (CH2-3) and 29.7 (CH2-2) could be assigned to the α-exomethylene-ε-lactone moiety. Two additional unsaturated functionalities were indicated by 13C resonances at δC 209.3 (C-5), 134.7 (C-8) and 124.9 (CH-9), suggesting the presence of a keto carbonyl and a trisubstituted olefin. On the basis of the overall unsaturation data, 3 was concluded to be a diterpenoid molecule possessing two rings. The 1H NMR spectrum of 3 showed the presence of three methyl groups: two singlets at δH 1.27 and 1.49, representing the methyl groups on oxygenated quaternary carbons, respectively, and a vinyl methyl at δH 1.69. The 1H NMR coupling information in the 1H–1H COSY spectrum of 3 enabled identification of the H-13/H2-14/H-1/H2-2/H2-3, H2-6/H2-7, H-9/H2-10/H2-11 and H-9/H3-19  units, which were assembled with the assistance of an HMBC experiment  in the HMBC spectrum, which further supported the existence of the ε-lactone moiety in 3.Flexibilin C , but not with H3-19, which revealed the E geometry of the C-8/9 double bond. It was found that H-13 showed correlations with H-1 and H-9. From molecular modeling analysis, H-13 was found to be close to H-1 and H-9, when H-1 and H-13 were α-oriented. Correlations observed between H3-20/H-13 and H3-20/H2-10 reflected the β-orientation of Me-20. Furthermore, H3-18 exhibited correlations with C-3 methylene protons and the lack of correlation between H3-18 and H-1, indicating that Me-18 was positioned on the β-face in 3. Thus, the structure of 3 was established, and the chiral carbons of 3 were assigned as 1R*, 4R*, 12R* and 13S*.The relative stereochemistry of 4), was first isolated from a Chinese soft coral, Dendronephthya sp.  and 455 variable parameters. The absolute configuration was determined by Flack’s method, with the Flack’s parameter determined as 0.06(10) [Suitable colorless prisms of c. Elastase release experiments were performed using MeO-Suc-Ala-Ala-Pro-Valp-nitroanilide as the elastase substrate.Human neutrophils were obtained by means of dextran sedimentation and Ficoll centrifugation. Measurements of superoxide anion generation and elastase release were carried out according to previously described procedures ,31,32,33Sinularia flexibilis, collected from the waters of Taiwan, and compounds of this type have been shown to have the potential for use in medical applications. Our studies of S. flexibilis have led to the isolation of three new cembranes, flexibilins A–C (1–3), along with a known metabolite, (−)-sandensolide (4), and flexibilin B (2) displayed a moderate inhibitory effect on the release of elastase by human neutrophils. In addition to marine organisms, cembrane-type diterpenoids are also found in higher plants and bryophytes [1 and 2 are rarely found [S. flexibilis, has begun to be transplanted to culturing tanks with a flow-through seawater system located in the National Museum of Marine Biology and Aquarium, Taiwan, for exhibition and the extraction of additional natural products in order to establish a stable supply of bioactive material.Cembrane-type diterpenoids are the major component of the organic extract of the soft coral, yophytes ,41,42,43ly found ,44. The


H-[1]benzazepin-2-one-1-acetate under the influence of microwave irradiation are described. A comparative study of conventional and microwave assisted method is briefly discussed.Rapid and efficient methods for the synthesis of an important intermediate of benazepril ethyl 3-phthalimido-2,3,4,5-tetrahydro-1 H-[1]-benzazepin-2-one-1-acetate to be an important synthon which can be easily prepared from ethyl 3-phthalimido-2,3,4,5-benzazepin-2-one-1-acetate-benzazepin-2-one-1-acetate (4) from 3-bromo-2,3,4,5-tetrahydro-1H-H[1]-benzazepin-2-one (2). It was then treated with ethyl bromoacetate in presence of sodium t-butoxide in DMF to prepare (4). In route B, (1) was treated with ethyl bromoacetate in presence of sodium t-butoxide to prepare 3-bromo-1-ethoxycarbonylmethyl-2,3,4,5-tetrahydro-1H-1HNMR spectra).The present work involves study of the synthesis of ethyl 3-phthalimido-2,3,4,5-tetrahydro-1-1, 1774 cm-1, 1751 cm-1.1H NMR (CDCl3): δ 8.15 , 7.74-7.91 , 2.80 ), 2.38 ), 2.20-2.32 ).Conventional synthesis of compounds (2) and (4) were carried out as follows. To a suspension of 10.3 g (0.046 mol) of compound (1) in 25 ml of anhydrous DMF, 9.9 g (0.0527 mol) of potassium phthalimide in 15 ml of anhydrous DMF was added. The suspension was stirred for 29 h at 75-80°, cooled to room temperature and DMF was removed under vacuum. It was then extracted with 3×25 ml ethyl acetate. The combined ethyl acetate extract was washed with water and dried over anhydrous magnesium sulphate. Evaporation of solvent under vacuum yielded compound (2). Yield: 12 g (84%) Melting point: 225° FT-IR: 3065 cmt-butoxide in 5 ml of anhydrous DMF was added. To this, 1.82 g (0.011 mol) of ethyl bromoacetate was added and resulting suspension was stirred for 1 h followed by heating at 80-85° for 18 h. It was cooled to room temperature and poured into 25 ml of cold water. The suspension was filtered and the residue was washed with 35 ml DMF-water (4:1) and 10 ml cold water and dried to yield compound (4). Yield: 1.66 g (65 %) Melting point: 96° FT-IR: 1772 cm-1, 1743cm-1, 1718 cm-1. 1HNMR (CDCl3): δ 7.73-7.92 , 4.45 , 4.23 , 2.80 , 2.40 ), 2.20-2.34 ), 1.29.To a suspension of 2 g (0.0098 mol) of compound (2) in 25 ml of anhydrous DMF, a solution of 1.05 g (0.011 mol) of sodium 1HNMR spectral data was found similar to as obtained in conventional synthesisMicrowave assisted synthesis of compounds 2, 3 and 4 were carried out as follows. In a small pear shaped flask, a suspension of 100 mg (0.4 mmol) of compound (1) in 1 ml anhydrous DMF was treated with 95 mg (0.5 mmol) of potassium phthalimide and this reaction mixture was heated in a domestic microwave oven at 70W for 3 min. The compound (2) was obtained after similar workup as in conventional synthesis. Yield: 100 mg (77 %). IR and t-butoxide in 2 ml anhydrous DMF was added. To this, 28.75 mg (1.4 mmol) of ethyl bromoacetate was added and resulting suspension was irradiated in conventional microwave at 70W for 4 min. The compound (4) was obtained after similar workup as given in conventional synthesis. Yield: 40 mg (67%) IR and 1HNMR spectral data was found similar to as obtained in conventional synthesisTo 50 mg (1.3 mmol) of compound (2), a solution of 17.5 mg (1.4 mmol) of sodium -1, 1684 cm-1, 1705 cm-1, 555 cm-1(C-Br stretch).To 100 mg of compound (1), a solution of 60 mg (0.6 mmol) of sodium t-butoxide in 2 ml DMF was added. To this, 0.1 ml (0.6 mmol) of ethyl bromoacetate was added. The resulting suspension was irradiated in domestic microwave oven at 70W for 4 min. Usual workup gave compound (3). Yield: 120 mg (85 %) Melting point: 114° FT-IR: 1745 cm1HNMR spectral data was found similar to as obtained in conventional synthesis.To a suspension of 100 mg (0.32 mmol) of compound (3) in 1 ml anhydrous DMF, 70 mg of potassium phthalimide was added. The reaction mixture was irradiated in conventional microwave oven at 70W for 4 min. The compound (4) was obtained after usual workup. Yield: 80 mg (65 %). IR and Microwave assisted synthesis thus provided a rapid and efficient method for synthesis of an intermediate of benazepril. Rate acceleration was observed in microwave assisted methods of synthesis.


Dorema ammoniacum D. Don had shown acetylcholinesterase (AChE) inhibitory activity in a previous study. The aim of this study was the isolation and characterization of the active compounds from this resin. The extract was investigated by a respective colorimetric microplate assay and the active zones were identified via TLC bioautography and isolated using several chromatographic techniques. The structures of the active components were characterized by one- and two-dimensional 1H and 13C NMR spectroscopy and mass spectrometry as -2′-ethenyl-5′-(3-hy-droxy-6-methyl-4-oxohept-5-en-2-yl)-7-methoxy-2′-methyl-4H-spiro-2,4-dione (1), which is an analogue of doremone A and a new natural compound, and as -2′-ethenyl-5′-[-4-hydroxy-6-methyl-3-oxohept-5-en-2-yl]-7-methoxy-2′-methyl-4H-spiro-2,4-dione (2 = doremone A), -1--5,9,13-trimethyltetradeca-4,8,12-trien-1-one (3 = dshamirone), and 4,7-dihydroxy-3--2H-chromen-2-one (4 = am-moresinol). Dshamirone turned out to be the most active compound with an IC50 value for AChE inhibitory activity of 23.5 μM, whereas the other substances showed weak activity. The concentrations of the analytes in the resin were determined by HPLC as 3.1%, 4.6%, 1.9%, and 9.9%, respectively.The use of herbal medicinal preparations in dementia therapy has been studied based on experience from traditional medicine. A dichloromethane extract of gum ammoniacum, the gum-resin from  It is commonly accepted that a cholinergic deficit correlates with the severity of Alzheimer’s disease . Thus, rGalanthus nivalis L. (snowdrop) has been identified as a reversible, competitive AChE inhibitor and allosteric potentiating ligand of nicotinic acetylcholine re-ceptors and is today one therapeutic option in the treatment of Alzheimer’s disease + ion at m/z 427.2125 for compound 1, which is in agreement with the molecular formula C25H30O6 . In the positive ion mode ESI-MS3 spectra, major fragment ions were obtained at m/z 326.7 and 258.7 for 1 and 2, respectively.ESI-MS measurements yielded a molecular weight of 426.0 Da for both 1H and 13C NMR analyses of 2, together with numerous connectivities derived from 2D NMR spectra like COSY, TOCSY-NOESY, HSQC, as well as HMBC, resulted in the structure of a spiro-sesquiterpenoidic chromane-2,4-dione derivative, namely -2′-ethenyl-5′-[-4-hydroxy-6-methyl-3-oxohept-5-en-2-yl]-7-methoxy-2′-methyl-4H-spiro-2,4-dione which is known as doremone A -2′-ethenyl-5′-(3hydroxy-6-methyl-4-oxohept-5-en-2-yl)-7-methoxy-2′-methyl-4H-spiro-2,4-dione -1--5,9,13-trimethyltetradeca-4,8,12-trien-1-one (dshamirone) and 4 as 4,7-dihydroxy-3--2H-chromen-2-one (ammoresinol) (3 were in agreement with an earlier publication +; ESIMS2 (427.0 →) m/z 408.8 (100), 326.7 (14); ESIMS3 (427.0 → 408.8 →) m/z 326.7 (100), 298.7 (12), 284.7 (51), 204.6 (19), 150.7 (13); (−)ESIMS m/z 425.0 [M-H]−; ESIMS2 (425.0 →) m/z 380.8 (11), 362.8 (16), 204.6 (100); HR-ESI-MS m/z 427.2125 [M+H]+ ; CD (CHCl3) Δɛ253 +1.92, Δɛ275 −4.24, Δɛ297 +0.24, Δɛ320 −2.59; UV λmax (CHCl3) 240.1, 275.0, 308.2 (sh) nm; IR νmax 3184, 2359, 1626, 1550, 1296, 1037, 663 cm−1.Compound 2: (+)ESIMS m/z 427.0 [M+H]+; ESIMS2 (427.0 →) m/z 408.8 (100), 258.7 (11); ESIMS3 (427.0 → 408.8 →) m/z 390.8 (57), 352.7 (38), 334.7 (25), 326.7 (63), 310.6 (21), 298.7 (14), 284.7 (27), 270.6 (17), 258.6 (100), 240.6 (11), 230.7 (11), 218.6 (18), 216.7 (15), 204.6 (63), 192.7 (22), 174.8 (11), 150.7 (18); (−)ESIMS m/z 425.0 [M-H]−; ESIMS2 (425.0 →) m/z 380.8 (28), 362.8 (15), 256.7 (100), 166.6 (20), 150.7 (24).Compound 3: (+)ESIMS m/z 357.1 [M+H]+; ESIMS2 (357.1 →) m/z 339.1 (100), 229.1 (16), 203.1 (22), 165.0 (11), 149.0 (20), 147.1 (10), 137.0 (11), 123.1 (29); (−)ESIMS m/z 355.1 [M-H]−; ESIMS2 (355.1 →) m/z 217.9 (10), 149.9 (100), 135.9 (12), 122.0 (26); ESIMS3 (355.1 → 149.9 →) m/z 122.0 (100).Compound 4: (+)ESIMS m/z 383.1 [M+H]+; ESIMS2 (383.1 →) m/z 190.9 (100); ESIMS3 (383.1 → 190.9 →) m/z 162.9 (17), 123.0 (100); (−)ESIMS m/z 381.1 [M-H]−; ESIMS2 (381.1 →) m/z 254.9 (17), 243.9 (34), 242.9 (33), 228.9 (16), 202.9 (11), 189.9 (51), 188.9 (100), 175.9 (62).Compound 1H, 13C, 19F, 31P) with z axis gradients and automatic tuning and a matching accessory. The resonance frequency for 1H NMR was 600.13 MHz, for 13C NMR 150.92 MHz. All measurements were performed on a solution in CDCl3 at 298K. Standard 1D and gradient-enhanced (ge) 2D experiments, like double-quantum filtered (DQF) COSY, TOCSY, NOESY, HSQC, and HMBC, were used as supplied by the manufacturer. Chemical shifts are referenced internally to the residual, non-deuterated solvent signal for 1H (δ = 7.26 ppm) or to the carbon signal of the solvent for 13C (δ = 77.0 ppm). The 1H and 13C NMR data are summarized in All NMR spectra were recorded on a Bruker Avance DRX 600 NMR spectrometer using a 5 mm switchable quadruple probe .A quantitative colorimetric assay based on Ellman’s method was used to measure the AChE inhibitory activities of the substances , 21. BriThe DCM extract of gum ammoniacum and the isolated compounds were analyzed by HPLC using gradient elution on the Hypersil BDS-C18 column (250×4 mm id) at 35°C. The mobile phase consisted of acetonitrile (A) and 0.05% formic acid in water (B) at a flow rate of 0.75 mL/min. After an isocratic step at 50% A for 10 min, the concentration of solvent A increased up to 75% in 25 min. The mobile phase was kept at this concentration for 10 min, increased to 90% A in 10 min, and was kept again at 90% for a further 5 min. Elution was monitored at 260 nm.


The structures of these new compounds were determined through spectroscopic and MS analyses. Among the isolated compounds, decarine (4), (−)-syringaresinol (6), (+)-episesamin (8), glaberide I (9), (−)-dihydrocubebin (10), and xanthyletin (11) exhibited potent inhibition  of superoxide anion generation by human nutrophils in response to N-formyl-l-methionyl-l-leucyl-l-phenylalanine/cytochalasin B (fMLP/CB). Compounds 4, 8, and 11 also inhibited fMLP/CB-induced elastase release with IC50 values ≤ 5.48 μg/mL.A new benzo[ Eleven known compounds (4–14), have been isolated and identified from the twigs of Z. ailanthoides and their structures are depicted in In our studies of Formosan plants for 1 through 3, and the inhibitory activities of all isolates on superoxide generation and elastase release by neutrophils.This paper describes the structural elucidation of the compounds numbered 2.1) was isolated as a white amorphous powder. Its molecular formula, C20H15NO5, was determined on the basis of the quasi-molecular ion at m/z 372.0846  in the HR-ESI-MS spectrum (positive-ion mode) , 7.43 , 7.46 , 7.51 , 8.02 , 8.09 , two methoxy groups , a methylenedioxy group [δH 6.13 ], and an NH group [δH 9.14 ]. Comparison of the 1H- and 13C-NMR data (1 with those of oxychelerythrine [H 9.14) of 1 replaced the N-Me group [δH 3.89 ] of oxychelerythrine + at m/z 305  , 2.88 , 3.09 , 3.74 , 4.11 ; δC 14.1 (C-5′), 42.0 (C-3′), 52.0 (OMe-4′), 69.8 (C-1′), 71.8 (C-2′), 175.7 (C-4′)] at C-4 of 2 replaced the 4-hydroxy group [δH 6.58 ] of methyl 4-hydroxybenzoate +  in the HR-ESI mass spectrum -methyl 4-(4-(3-hydroxypropyl)phenoxy)-2-methylbut-2-enoate  at C-1 of 3 replaced 3-hydroxypropyl group of (E)-methyl 4-(4-(3-hydroxypropyl)phenoxy)-2-methylbut-2-enoate phenanthridines, decarine (4) phenanthridine derivative 4 {with 8-hydroxy group and double bond [N(5)=C(6)]} exhibited more effective inhibition than its analogues 4  and 5  against fMLP-induced O2•− generation and elastase release; (d) Glaberide I (9) (with a 6-oxo group) exhibited more effective inhibition than its analogue 6  Decarine (4) and (+)-episesamin (8) were the most effective among the isolated compounds, with IC50 values of 1.31 ± 0.18 and 1.42 ± 0.16 μg/mL, respectively, against fMLP-induced superoxide anion generation and elastase release.Human neutrophils are known to play an important roles in host defense against microorganisms and in pathogenesis of various diseases. In response to different stimuli, activated neutrophils secrete a series of cytotoxins, such as the superoxide anion radical  and phosphatidylinositol 3-kinase/Akt are the downstream signaling of fMLP in human neutrophils +. HR-ESI-MS: m/z = 372.0846 [M + Na]+ .White amorphous powder. UV (MeOH): λ3.3.2.max (log ɛ) = 254 (3.96) nm. IR (neat): υmax 3480 (OH), 1728 (C=O), 1714 (C=O) cm−1. 1H-NMR: see 13C-NMR: see m/z = 305 [M + Na]+. HR-ESI-MS: m/z = 305.1003 [M + Na]+ .Colorless oil. UV (MeOH): λ3.3.3.max (log ɛ) = 296 (4.18) nm. IR (KBr): υmax = 1715 (C=O) cm−1. 1H-NMR : δ = 1.93 , 3.73 , 3.76 , 4.69 , 5.83 , 6.86 , 6.89 , 6.93 , 7.69 . 13C-NMR : δ = 13.0 (C-5′), 51.3 (OMe-9), 51.5 (OMe-4′), 64.8 (C-1′), 114.2 (C-3), 114.2 (C-5), 116.7 (C-8), 127.3 (C-1), 129.6 (C-3′), 132.2 (C-2), 132.2 (C-6), 137.0 (C-2′), 143.6 (C-7), 159.2 (C-4), 166.9 (C-9), 167.8 (C-4′). ESI-MS: m/z = 313 [M + Na]+. HR-ESI-MS: m/z = 313.1055 [M + Na]+ .Amorphous powder. UV (MeOH): λ3.4.The effect of the isolated compounds on neutrophil pro-inflammatory response was evaluated by monitoring the inhibition of superoxide anion generation and elastase release in fMLP/CB-activated human neutrophils in a concentration-dependent manner. The purity of the tested compounds was >98% as identified by NMR and MS. LY294002  was used as a positive control.3.4.1.2+)-free HBSS buffer at pH 7.4 and were maintained at 4 °C prior to use.Human neutrophils from venous blood of healthy, adult volunteers (20–28 years old) were isolated using a standard method of dextran sedimentation prior to centrifugation in a Ficoll Hypaque gradient and hypotonic lysis of erythrocytes . Purifie3.4.2.c phenanthridine (1) and two new benzenoids (2 and 3), were isolated from the twigs of Z. ailanthoides. The structures of these compounds were established on the basis of spectroscopic data. Reactive oxygen species (ROS)  and granule proteases  produced by human neutrophils contribute to the pathogenesis of inflammatory diseases. The effects on neutrophil pro-inflammatory responses of isolates were evaluated by suppressing fMLP/CB-induced O2•− generation and elastase release by human neutrophils. The results of anti-inflammatory experiments indicate that compounds 4, 6, and 8–11 can significantly inhibit fMLP-induced O2•− generation and/or elastase release. Decarine (4) and (+)-episesamin (8) were the most effective among the isolated compounds, with IC50 values of 1.31 ± 0.18 and 1.42 ± 0.16 μg/mL, respectively, against fMLP-induced O2•− generation and elastase release. Compounds 4, 8, and 11 (10 μg/mL) caused a significant reduction of the phosphorylation of Akt and MAPks in fMLP-induced neutrophils. Thus, the anti-inflammatory effects of compounds 4, 8, and 11 are through the inhibition of activation of MAPKs and Akt in fMLP-activated neutrophils. Our study suggests Z. ailanthoides and its isolates  could be further developed as potential candidates for the treatment or prevention of various inflammatory diseases.Fourteen compounds, including a new benzo[


Clostridium difficile infections(CDI) epidemiology has changed:elevation in rate and severity of infection and increase in disease among outpatientsAim of study was to evaluate the epidemiology of CDI in North Tenerife Area.>3 co-morbidities,inflammatory boweldisease(IBD),solidorgan transplant(OST), immunocompromised states(IC),treatment previous,treatment of CDI,developed to Pseudo-membranous colitis(PMC),mortality due to CDI.The episodes were classificated as nosocomial, healthcare associated (HCA),community, indeterminate, and recurrence.This is an epidemiological study performed in the Hospital Universitario de Canarias a tertiary care institution during 2011-2012.Studied population was outpatients or inpatients attended in this hospital with CDI suspected.Diagnostic procedures were based in GDH and later Toxin A/B detection as CDI confirmation in stool samples by EIA.Medical charts of patient were reviewed to collect: demographic variables, underlying diseases (diabetes(D), renal disease(RD),liver disease(LD),respiratory disease(ReD),cardiopathy(C),neoplasia(N)),4 patient–day.The services distribution was:Internal Medicine 6/12(33/27%), Nephrology 6/4(33/9%), Hematology 2/5(11/11%).Episodes:Community 4/7(22/15%), Nosocomial 14/27(78/60%), HCA 0/6(0/13%), Indeterminate 0/1(0/2%) and recurrences 0/4 (0/9%).In Nosocomial the time average between admission and CDI diagnostic was 10,6+9,1/24+29 d.Underlying diseases:C 10/9(55/20%),RD 5/10,LD 2/4(11/9%), ReD 2/2(11/4%), N2/10(11/22%).>3 co-morbidities 3/3(17/7%). IBD 1/1(6/2%), OST 7/3,IC 10/20(55/44%),previous treatment:Omeprazol 6/10(33/22%), Ranitidine 3/1(17/2%),Aciclovir 1/2,Carbapenems 6/21(33/47%),Fluorquinolones 5/13(28/29%),Cephalosporins (3-4ª) 4/11 (22/24%),Vancomycin(VA) 3/6(17/13%), Amoxicillin-clavunate 2/7(11/16%).CDI Treatment:Metronidazole 18/39(100/87%), VA 3/9 (17/20%), developed PMC 2/5(11/11%), death for CDI 1/0.In 2011/2012 a total 18/45 episodes (17/41 patients) were diagnosticated. 50/62% were man and 7/22 (39/49%),<65 years.HCA and nosocomial CDI incidence were:0,7/1,7 case/10In our hospital there has been an increase in nosocomial CDI adquisition overtime and a high percentage in young patients.At 2012 OST patients declined and HCA episodes were increased thus we observed that CDI is not confined to hospitals.None declared


The compounds were tested for analgesic activity by acetic acid induced writhing method. Compounds IIh, IIe, IIk and IIl showed excellent to good analgesic activity. Other compounds showed moderate analgesic activity. The observed analgesic activity is mainly because of inhibition of the peripheral pain mechanism by the title compounds.A series of 2-methylthio-1,4-dihydropyrimidine derivatives (II) were synthesized in good yields by alkylation of 1,2,3,4-tetrahydropyrimidines (I) with methyl iodide in the presence of pyridine. Their structures were confirmed by elemental analysis, IR and  Multicomponent reactions (MCRs) are of increasing importance in organic and medicinal chemistry. Multicomponent condensations involve three or more compounds reacting in a single event to form a product, which contains the essential parts of all the starting materials. Among the wide range of heterocycles explored to develop pharmaceutically important molecules, pyrimidine has played an important role in medicinal chemistry. Dihydropyrimidines are being looked as an important class of molecules since many of them are clinical candidates. In recent years acid-catalyzed cyclocondensation of acetoacetate with aldehydes and (thio) ureas, known as the Biginelli reaction, has attracted significant attention -9. The rPain is defined as neuralgia, an unpleasant sensory experience associated with tissue damage, such as injury, inflammation or cancer, but severe pain can arise independently of any obvious predisposing cause, or persist long after the precipitating injury has healed. Acetic acid induced writhing model represents pain sensation by triggering localized inflammatory response. Such pain stimulus leads to the release of free arachidonic acid from tissue phospholipids . The abdPyrimidine and condensed pyrimidine derivatives possessing anti-inflammatory and analgesic activities are well documented in the literature . In the 1H NMR spectra. The IR spectra were recorded on a JASCO FT-IR 4100 spectrometer, using KBr discs. The 1H NMR spectra were obtained on a Varian-NMR-mercury300 spectrometer in DMSO-d6 as solvent and TMS as internal standard, chemical shifts are given in ppm.Melting points were determined in open capillaries and are uncorrected. All compounds were characterized by elemental analysis, IR and General procedure for the synthesis of compounds (IIa-IIl)A mixture of appropriate aldehyde (0.02 mole), acetoacetate (0.02 mole), thiourea (0.03 mole), catalyst aluminium chloride (0.01 mole) in methanol (10 mL) and concentrated hydrochoric acid (2 drops) was placed in round bottom flask. The mixture was stirred well and then refluxed. The completion of reaction was monitored by thin layer chromatography. After cooling, precipitate was formed which was filtered and washed with cold methanol (I).Compound I (0.01 mole), methyl iodide (0.011 mole) in methanol (20 mL) was placed in round bottom flask and refluxed for 2 h. Pyridine (0.037 mole) was then added and refluxed again for 10 min After cooling, the reaction mixture was poured onto crushed ice (200 g) and stirred for 5 min. Compound II obtained was filtered.Ethyl 6-methyl-2-(methylthio)-4-phenyl-1,4-dihydropyrimidine-5-carboxylate (IIa) -1: 3318.89 (NH), 2371.05 (S-CH3), 1659.45 (C=O), 1573.63 (C=N); 1H NMR : 14.18 , 7.75-7.20 , 5.988 , 3.98 , 2.35 , 2.193 , 1.11 .Yield: 82.10 %; m.p. 160-162ºC; IR-(KBr) cmMethyl 6-methyl-2-(methylthio)-4-phenyl-1,4-dihydropyrimidine-5-carboxylate (IIb) -1: 3316.96 (NH), 2362.37 (S-CH3), 1708.62 (C=O), 1646.91 (C=N); 1H NMR : 14.876 , 7.751-7.201 , 5.988 , 3.71 , 2.351 , 2.193 .yield: 67.51 %; m.p. 100-102ºC; IR-(KBr) cmEthyl 4-(4-methoxyphenyl)-6-methyl-2-(methylthio)-1,4-dihydropyrimidine-5-carboxylate (IIc) -1: 3318.89 (NH), 2345.98 (S-CH3), 1649.8 (C=0), 1150.33(C-O); 1H NMR : 14.876 , 7.68-7.069 , 5.988 , 3.98 , 3.605 , 2.351 , 2.193 , 1.11 .yield: 78.94 %; m.p. 116-117ºC; IR-(KBr) cmMethyl 4-(4-methoxyphenyl)-6-methyl-2-(methylthio)-1,4-dihydropyrimidine-5-carboxylate (IId)-1: 3318.89 (NH), 2364.3 (S-CH3), 1654.62 (C=O), 1249.65 (C-O); 1H NMR :14.87 , 7.68-7.069 , 5.988 , 3.708 , 3.605 , 2.351 , 2.193 .yield: 73.68 %; m.p. 146-147ºC; IR-(KBr) cmEthyl 4-(4-chlorophenyl)-6-methyl-2-(methylthio)-1,4-dihydropyrimidine-5-carboxylate (IIe)-1: 3341.07 (NH), 2358.52 (S-CH3), 1674.87 (C=O), 1574.59 (C=N), 745.35 (C-Cl); 1H NMR : 14.876 , 7.778-7.396 , 5.988 , 3.98 , 2.351 , 2.193 , 1.11 .yield: 92.98 %; m.p. 138-139ºC; IR-(KBr) cmMethyl 4-(4-chlorophenyl)-6-methyl-2-(methylthio)-1,4-dihydropyrimidine-5-carboxylate (IIf) -1: 3308.29 (NH), 2372.01 (S-CH3), 1659.45 (C=O), 1561.09 (C=N), 782.95 (C-Cl); 1H NMR :14.876 , 7.778-7.396 , 5.988 , 3.708 , 2.351 , 2.193 .yield: 57.89 %; m.p. 130-132ºC; IR-(KBr) cmEthyl 4-[4-(dimethylamino)phenyl]-6-methyl-2-(methylthio)-1,4-dihydropyrimidine-5-carboxylate (IIg)-1 3334.32 (NH), 2371.05 (S-CH3), 1654.62 (C=O), 1514.81 (C=N); 1H NMR :14.876 , 7.532-6.577 , 5.988 , 3.98 , 2.831 2), 2.351 , 2.193 , 1.11 ,.yield: 62.93 %; m.p.124-126ºC; IR-(KBr) cmMethyl 4-[4-(dimethylamino)phenyl]-6-methyl-2-(methylthio)-1,4-dihydropyrimidine-5-carboxylate (IIh)-1: 3327.57 (NH), 2358.52 (S-CH3), 1668.12 (C=O), 1555.31 (C=N); 1H NMR : 14.876 , 7.532-6.577 , 5.988 , 3.708, 2.831  2), 2.351 , 2.193 ,.yield: 75.75 %; m.p. 214-216ºC; IR-(KBr) cmEthyl 6-methyl-2-(methylthio)-4-(4-nitrophenyl)-1,4-dihydropyrimidine-5-carboxylate (IIi)-1: 3235.97 (NH), 2372.01 (S-CH3), 1693.19 (C=O), 1528.31 (Asy Ar-NO2), 1349 (Sym Ar-NO2), 855.27 (C-N); 1H NMR : 14.876 , 7.89-8.10 , 5.988 , 3.98 , 2.351 , 2.193 , 1.11 .yield: 83.68 %; m.p.180-182ºC; IR-(KBr) cmEthyl 6-methyl-2-(methylthio)-4--1,4-dihydropyrimidine-5-carboxylate (IIj) -1: 3288.04 (NH), 2345.02 (S-CH3), 1669.09 (C=O), 1573.63 (C=N),1184, 1123 (C-O); 1H NMR : 14.87 , 5.874 , 3.98 , 3.83 , 2.351 , 2.193 , 1.11 .yield: 75.14 %; m.p.206-208ºC; IR-(KBr)cmEthyl 6-methyl-2-(methylthio)-1,4-dihydropyrimidine-5-carboxylate (IIk)-1: 3209.93 (NH), 2352.73 (S-CH3), 1659.45 (C=O), 1501.31 (C=N); 1H NMR : 15.179 , 4.723 , 4.089 , 2.402 , 2.173 , 1.222 .yield: 65.47 %; m.p. 210-211ºC; IR-(KBr) cmMethyl 6-methyl-2-(methylthio)-1,4-dihydropyrimidine-5-carboxylate (IIl)-1): 3318.07 (NH), 2358.52 (S-CH3), 1668.12 (C=O), 1594.84 (C=N); 1H NMR : 15.17 , 4.723 , 3.705 , 2.402 , 2.173 .yield: 75.14 %; m.p. 198-200ºC; IR-KBr . 0.1 mL of a 0.6 % aqueous acetic acid solution was injected intraperitoneally (IP) as writhing inducing agent. In each group six mice were kept. Mice were kept individually in test cage, before acetic acid injection. Screening of analgesic activity was performed after oral administration of test compounds at a dose of 50 mg/kg. All compounds were dissolved in sterile water for injection (SWF). Diclofenac was used as reference drug. After 1 h of drug administration 0.10 mL of 0.6 % acetic acid solution was given to mice intraperitoneally. Stretching movements consisting of arching of the back, elongation of body and extension of hind limbs were counted for 10 min of acetic acid injection. The analgesic activity was expressed in terms of percentage inhibition. Percentage analgesic activity was calculated as follows:Analgesic activity (%inhibition) = (n-n’ / n) ×100Where, n = Mean number of writhes of control group.n’ = Mean number of writhes of test group.Statistical analysisValues are expressed as mean ± SEM and data was analyzed by ANOVA followed by Dunnet’s test. p< 0.01 was considered as significant.The 1,2,3,4-tetrahydropyrimidine-2-thione derivatives were prepared by Biginelli three-component reaction of appropriate aldehydes, acetoacetate and thiourea. Alkylation reaction was used for the formation of C-2 modified DHPM derivatives of type II. The target molecules, 2-methylthio-1,4-dihydropyrimidine derivatives were synthesized from the respective tetrahydropyrimidine-2-thiones by reaction with methyl iodide in the presence of pyridine , Table 11H NMR spectra. The amount of carbon, hydrogen and nitrogen found by elemental analysis is in good agreement with calculated. The purity of the compounds was checked by thin layer chromatography. Their structures were confirmed by elemental analysis, IR and The analgesic activity of the synthesized compound (IIa-IIl) was evaluated by acetic acid induced writhing test . The comet al.  and thus it appears that the test compounds inhibits predominantly the peripheral pain mechanism.


H-benzo[b]pyrazolo-1,6-naphthyridines. In the course of our search for new 1,6-naphthyridines derivatives with potential activity against HSV-1, we have synthesized and evaluated new 3H-benzo[b]pyrazolo-1,6-naphthyridines (1a-k) and 3H-pyridopyrazolo-1,6-naphthyridines (2a-c).Herpes simplex virus type-1 (HSV-1) is the primary cause of facial lesions  in humans. The widespread use of acyclovir and nucleoside analogues has led to emergence of HSV strains that are resistant to these drugs. Recently, non-nucleoside anti-HSV compounds have received considerable attention. 1,6-Naphthyridines are a class of heterocyclic compounds that exhibit a broad spectrum of biological activities such as inhibitor of HIV-1 integrase, HCMV, FGF receptor-1 tyrosine kinase, and the enzyme acetylcholinesterase. We previously reported the synthesis, SAR studies, and evaluation anti-HSV-1 activity of 3H-benzo[b]pyrazolo-1,6-naphthyridines (1a-k) and 3H-pyridopyrazolo-1,6-naphthyridines (2a-c), starting from ethyl 4-chloro-1-phenyl-1H-pyrazolopyridine-5-carboxylate (7). All compounds were identified by FTIR, 1H NMR, and mass spectrometry. The antiviral effect on HSV-1 virus replication was determined.A known synthetic approach was used for preparing new 31d, 1f, 1g, and 1h exhibited the highest anti-HSV-1 activity. In general, 3H-benzo[b]pyrazolo-1,6-naphthyridines were more effective inhibitors than their corresponding 3H-pyridopyrazolo-1,6-naphthyridines. The compound 1h reduced the virus yield in 91% at 50 μM and exhibited a low cytotoxicity (CC50 600 μM).The compounds  Herpes simplex virus type-1 (HSV-1) is a large enveloped virus containing double-stranded DNA genomes of approximately 152 kb in size. HSV-1 is the primary cause of facial lesions  in humans -5. Howev1,6-Naphthyridines are a class of heterocyclic compounds that exhibit a broad spectrum of biological activities such as inhibitor of HIV-1 integrase -15, HCMVH-benzo[b]pyrazolo-1,6-naphthyridines derivatives I  and 3H-pyridopyrazolo-1,6-naphthyridines (2a-c)  and 3H-pyridopyrazolo-1,6-naphthyridines (2a-c), starting from ethyl 4-chloro-1-phenyl-1H-pyrazolopyridine-5-carboxylate (7) (Scheme α-carboethoxy-β-(5-pyrazolylammonium)acrylate (8) was prepared by the condensation between 5-amino-1-phenyl-1H-pyrazole (9) and diethyl ethoxymethylenemalonate, in ethanol. The cyclization of the acrylate 8 was carried out by refluxing in phosphorus oxychloride to afford 4-chloro1-phenyl-1H-pyrazolopyridine-5-carboxylate (7) in 75% yield pyridine-5-carboxylates (5a-k) in yields 52-82% pyridine-5-carboxylates (6a-c) in yields 50-60%. These were achieved by heating at 140°C without solvents for 2-4 h an equimolar mixture of the appropriate aniline or aminopicoline and the compound 7. However, better results were obtained when these reactions were carried out in solvents such as DMF pyridine-5-carboxylic acids (3a-k) and 4-[(methylpyridin-2-yl)amino]-1-phenyl-1H-pyrazolopyridine-4-carboxylic acids (4a-c), in high yields, 86-93 and 80-93%, respectively [H-benzo[b]pyrazolo-1,6-naphthyridines (1a-k) and 3H-pyridopyrazolo-1,6-naphthyridines (2a-c), the respective carboxylic acids 3a-k and 4a-c were cyclized with phosphorus oxychloride at 110°C over a period of 3 h pyrazolo-1,6-naphthyridines (1a-k) and 3H-pyridopyrazolo-1,6-naphthyridines (2a-c) were evaluated for inhibition of HSV-1 replication in infected Vero cells. Results are shown in Table 1d, 1f, 1g, and 1h exhibited the highest anti-HSV-1 activity. Compound 1h reduced the virus yield in 91% at 50 μM. In general, 3H-benzo[b]pyrazolo-1,6-naphthyridines (1a-k) were more effective inhibitors than their corresponding 3H-pyridopyrazolo-1,6-naphthyridines (2a-c).The targets 350 and the selectivity index (SI) were determined in parallel. Several of the new compounds prevented the cytopathic effect of HSV-1 in Vero cells, at micromolar concentrations, and were minimally toxic to Vero cells resulting in a good SI. The MTT assay indicated that compound 1h exhibited a low cytotoxicity (CC50 600 μM). Trypan blue and MTT showed similar results (data not shown). ACV results have been included for comparison purposes  and 3H-pyridopyrazolo-1,6-naphthyridine (2a-c) were synthesized and some of them were potent anti-HSV-1 agents. The compounds 1d, 1f, 1g, and 1h exhibited the highest anti-HSV-1 activity, being the 3H-benzo[b]pyrazolo-1,6-naphthyridine derivatives, in general, more effective inhibitors than their corresponding 3H-pyridopyrazolo-1,6-naphthyridines. The compound 1h reduced the virus yield in 91% at 50 μM and exhibited a low cytotoxicity (CC50 600 μM). The mechanism of antiviral activity of these compounds is under investigation.In summary, a new series of 31H NMR spectra were recorded on a Varian Unity Plus spectrometer for 300 MHz, with tetramethylsilane as the internal standard. Chemical shifts (δ) are reported in parts per million (ppm) and the coupling constants (J) in Hertz (Hz). Fourier transform infrared absorption spectra were recorded in a Perkin-Elmer Spectrum One FTIR spectrophotometer. The solid samples were measured using potassium bromide (KBr) pellets. Thin-layer chromatography was performed on Uniplates (silica gel). All chemicals were reagent grade. High-resolution mass spectral analysis was recorded using a Finingan MAT 711A.Melting points were determined on a Fisatom 430D and are uncorrected. H-pyrazolopyridine-5-carboxylate (7) was prepared according to literature [7 (4 mmol) and anilines or aminopicolines in 10 mL DMF was heated under reflux for 2-4 h. The reaction mixture, after cooling, was poured into 50 mL of ice-water. The precipitated was filtered, dried, and recrystallized from a mixture of ethanol and water. The compounds obtained 5a-k and 6a-c were reacted with 10 mL NaOH (20%) and 10 mL of ethanol under reflux for 1-3 h. On cooling to room temperature, the mixture was acidified with diluted hydrochloric acid (1:3), and the precipitate was filtered and recrystallized from DMF and water. A mixture of the acids 3a-k and 4a-c (1 mmol), and phosphorus oxychloride (5 mL) was heated under reflux for 3 h. The reaction mixture was inverted over crushed ice. In some cases the excess of phosphorus oxychloride was removed under reduced pressure before inverting over crushed ice and neutralized. The new compounds 1a-k and 2a-c were isolated in yields 60-70%. The resulting precipitate was collected and purified by flash column chromatography . The structures of the compounds were elucidated by FTIR, 1H NMR, and mass spectrometry.The key intermediate ethyl 4-chloro-1-phenyl-1terature -28. An e-1) νmax C-H 3084, C = C 1596, C = N 1502; 1H NMR  δ 8.92 , 8.40 , 7.95 , 7.70 , 7.81 , 9.40 , 8.33 , 7.50 , 7.52 ; EI (70eV) m/z (%): M+ 330.00761 (100).Yield 70%; mp 259-260°C; IR  νmax C-H 3083, C = C 1595, C = N 1504; 1H NMR  δ 8.99 , 7.26 , 7.78-7.56 , 9.36 , 8.31 , 4.07 ; EI (70 eV) m/z (%): M+. 360.07037 (100).Yield 68%; mp > 300°C; IR  νmax C-H 3083, C = C 1595, C = N 1503; 1H NMR  δ 8.99 , 7.79-7.57 , 9.42 , 8.40 , 1.39 ; EI (70eV) m/z (%): M+. 344.81022 (100).Yield 65%; mp > 300°C; IR  νmax C-H 3082, C = C 1598, C = N 1503; 1H NMR  δ 9.08 , 7.99-7.53 , 9.38 , 8.41 ; EI (70 eV) m/z (%): M+. 364.01693 (100).Yield 60%; mp > 300°C; IR  νmax C-H 3084, C = C 1598, C = N 1503; 1H NMR  δ 8.97 , 7.87 , 7.89 , 8.22 , 9.31 , 8.27 , 7.46 , 7.65 ; EI (70 eV) m/z (%): M+. 364.01789 (100).Yield 68%; mp > 300°C; IR  νmax C-H 3084, C = C 1596, C = N 1503; 1H NMR  δ 8.92 , 8.84 , 8.05 , 8.02 , 9.40 , 8.31 , 7.52 , 7.71 ; EI (70 eV) m/z (%): M+. 375.03743 (100).Yield 60%; mp > 300°C; IR  νmax C-H 3100, C = C 1592, C = N 1500; 1H NMR  δ 8.90 , 8.12 , 8.80 , 8.89 , 9.42 , 8.30 , 7.51 , 7.68 ; EI (70 eV) m/z (%): M+. 375.70261 (100).Yield 60%; mp 280-281°C; IR  νmax C-H 3100, ν C = C 1600, ν C = N 1503; 1H NMR  δ 9.34 , 8.45 , 7.83-7.62 , 9.02 , 9.49 , 8.20 , 7.25 , 7.41 ; EI (70 eV) m/z (%): M+. 348.04188 (100).Yield 62%; mp 275-277°C; IR  νmax C-H 3051, C = C 1598, C = N 1503; 1H NMR  δ 9.06 , 8.03 , 8.05 , 8.02 , 9.37 , 8.32 , 7.51 , 7.69 ; EI (70 eV) m/z (%): M+. 348.09473 (100).Yield 65%; mp 278-279°C; IR  νmax C-H 3052, C = C 1593, C = N 1503; 1H NMR  δ 9.05 , 8.09 , 7.90-7.50 , 9.30 , 8.30 ; EI (70 eV) m/z (%): M+. 409.96178 (100).Yield 60%; mp > 300°C; IR  νmax C-H 3085, C = C 1596, C = N 1501; 1H NMR  δ 8.95 , 7.92-7.52 , 8.42 , 9.40 , 8.30 ; EI (70 eV) m/z (%): M+. 409,67991 (100).Yield 68%; mp > 300°C; IR  νmax C-H 3085, C = C 1596, C = N 1501; 1H NMR  δ 8.72 , 8.26 , 7.68 , 9.44 , 8.26 , 7.50 , 7.68 , 1.37 ; EI (70eV) m/z (%): M+. 345.74391 (100).Yield 60%; mp 230-232°C; IR  νmax C-H 3084, C = C 1596, C = N 1502; 1H NMR  δ 8.72 , 8.92 , 7.91 , 9.58 , 8.42 , 7.59 , 7.78 , 1.42 ; EI (70 eV) m/z (%): M+. 345.78660 (100).Yield 65%; mp 255-256°C; IR  νmax C-H 3084, C = C 1596, C = N 1502; 1H NMR  δ 8.83 , 7.67 , 7.93 , 9.41 , 8.39 , 7.56 , 7.75 , 1.39 ; EI (70 eV) m/z (%): M+. 345.76572 (100).Yield 62%; mp 247-249°C; IR . Cells grown in 96-well microtitre were inoculated with virus at input 1 PFU (plaque-forming unit)/cell for 2 h at 37°C. After virus adsorption, virus inoculum was replaced by a culture medium containing quinolone acyclonucleobases carboxylic acid and their correspondent esters at the concentration of 50 μM. Control cultures were incubated with media without compounds. After 3 days of incubation at 37°C in 5% CO2 atmosphere, the culture medium was harvested and the virus titre of each sample was determined in terms of 50% tissue culture dose (TCID 50/mL) by endpoint dilution.Compounds were tested as inhibitor of HSV replication in Vero  cells. They were grown in DMEM (Gibco Laboratories) supplemented with 2% heat-inactivated fetal bovine serum (purchased from Fazenda Pig), 8% calf serum (purchased from Centro Pan-Americano de Febre Aftosa), 2.25% sodium bicarbonate, 500 U/mL penicillin, 100 μg/mL streptomycin, and 2.5 μg/mL amphotericin B. HSV-1 (ACR-29 strain) was kindly provided by Marcia Wigg  and was routinely propagated in Vero cells. Virus stocks were stored at -70°C until use. ACV was purchased from Sigma (A 4669). It was dissolved in sterile deionized water and further diluted in culture medium. MTT was purchased from Sigma. Virus infectivity was measured by a dilution method using a 96-well microtitre plate and expressed as 50% tissue culture infections dose (TCID50) was calculated by linear regression analysis of the dose-response curves generated from these data. In the second method, monolayer of Vero cells in 96-multiwell plates were incubated with MTT (5 μg/mL) at 37°C for 4 h. After this period, SDS 10% and 0.01 N HCl were added to each well and incubated overnight. The plates were read using an automatic plate reader with a 540-nm test wavelength and a 690-nm reference wavelength. Plaque reduction assay was performed utilizing Vero cells at a density of 3 × 105 infected with various dilutions of the supernatant from a yield reduction assay for 1 h at 37°C and 5% CO2. After adsorption, the plates were washed and the medium was replaced with DMEM containing methylcellulose 1% and fetal bovine serum 5%. After incubation for 72 h, the monolayers were fixed with 1% formaldehyde in PBS, methylcellulose removed, and cell stained with a 0.1% solution of crystal violet in 70% methanol. The virus yield assay was performed as follows. Confluent Vero cells were washed with PBS and infected with HSV-1 at moi of 1 PFU/cell for 1 h at 37°C. The infected cells were washed with PBS and covered with a culture medium containing either no compounds or a different concentration of compounds. 20 h after adsorption, cells were lysed by freezing and thawing (three times), and the supernatant consisting of culture medium and lysed cells was obtained by centrifugation at 400g for 10 min at 4°C. Virus titre was determined by the plaque assay in Vero cells as described above. Data were statistically analyzed by Student's t-test for a significance level of p < 0.05.The cytotoxicity of the compounds was tested in Vero cells using two methods, namely, MTT and trypan blue dye exclusion assay. Monolayers of uninfected cells were incubated with culture medium containing different concentrations of compounds for 72 h at 37°C. The medium was then removed, the cells trypsinized and viable cells counted by trypan blue dye exclusion test. The 50% cytotoxic concentration -2,5-diphenyl tetrazolium bromide; PBS: phosphate buffered saline; PFU: plaque-forming unit; SDS: sodium dodecyl sulphate.DMEM: Dulbecco's modified Eagle's medium; DMF The authors declare that they have no competing interests.