Strobilanthes crispus of the Acanthaceae family exhibit potent anticancer properties in vitro and are non-toxic in vivo. S. crispus was also reported to be protective against chemical hepatocarcinogenesis. We previously showed that a bioactive fraction of S. crispus leaves also synergized with tamoxifen to cause apoptosis of human breast cancer cell lines without damaging non-malignant epithelial cells. The present study aimed to evaluate the antitumor effect of S. crispus dichloromethane fraction (F3) using N-methyl-N-Nitrosourea (NMU)-induced rat mammary tumor model. Tumor regression was observed in 75% of the rats following 8-week oral administration of F3 with no secondary tumour formation and no signs of anemia or infection. However, no improvement in the liver and renal function profiles was observed. Major constituents of F3 were identified as lutein, 131-hydroxy-132-oxo-pheophytin a, campesterol, stigmasterol, β-sitosterol, pheophytin a and 132-hydroxy-pheophytin a. These compounds however, may not significantly contribute to the antitumor effect of F3.Cancer patients seek alternative remedies such as traditional medicinal plants for safe and effective treatment and help overcome the side effects of conventional therapy. Current knowledge indicates that extracts of  An example of such traditional medicinal plants believed to have anticancer and general health-giving properties is Strobilanthes crispus (S. crispus) . The measures taken to minimize unnecessary pain and distress on the animals include utilization of small sample size, adequate housing and care, short term treatment, use of purely inbred strain to minimize variability of data, frequent monitoring of the animals with high chances of tumor ulceration and early humane end point for the morbid animals among others, all of which are in keeping with the principle of humane experimental technique . Total leukocyte count, differential leukocyte  counts and platelet (PLT) count were also measured.Biochemical parameters for liver and kidney function impairment were analyzed using automated chemistry analyzer  according to the manufacturer’s guidelines. The parameters measured were serum total protein (TP), alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea, creatinine, uric acid, calcium and electrolytes.3 (4 to 100%). A total of six sub fractions were obtained. From there, the third sub fraction was repeatedly chromatographed on silica gel preparative-TLC using the mobile phase that consisted of CHCl3-EtOAc  to afford compound 1 which was obtained as yellowish-orange crystals and compound 2 which was a greyish green powder. Chromatography of the second sub fraction of F3 on silica gel 60 using an isocratic mobile phase of hexane-CHCl3-EtOAc  yielded white solid that consisted of a mixture of compounds 3, 4 and 5.F3 was chromatographed on silica gel by flash column chromatography. Approximately 2 g of F3 was loaded onto the column. The mobile phase was made up of a gradient composition of EtOAc in CHCl6 and 7 present in F3 were also found to be present in higher abundance in the preceding fraction, F2. Hence, in order to get adequate yield of both compounds for further work, isolation of these two compounds was carried out from F2. The fraction (2 g) was subjected to flash column chromatography using a step gradient of hexane-EtOAc  to yield twenty sub fractions. The eleventh sub fraction was further purified by silica gel flash column chromatography using increasing amount of CHCl3 in hexane (0 to 100%) and subsequently developed twice on silica gel preparative-TLC using hexane-CHCl3-EtOAc  and hexane-CHCl3-EtOH , respectively, to afford the two pure compounds.Compounds 1, 2, 6 and 7 was done by nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). NMR was carried out on Avance 500 NMR spectrometer  operating at 25°C. The samples were dissolved in deuterated chloroform (CDCl3) and the chemical shifts were recorded with reference to that of tetramethylsilane δ . Mass spectrometry (MS) was carried out using AmaZon X mass spectrometer for nominal mass . Accurate mass analysis was acquired using MicroTOF-Q II 10269 system . Compound ionization was achieved by electrospray ionization (ESI) in the positive mode. Identification of compounds 3–5 was done through hyphenated gas chromatography mass spectrometry (GC-MS) . Separation was achieved on a HP-5MS column, 30 m × 0.25 μm, 0.25 m, with helium gas as the carrier at a flow rate of 0.5 ml/min. Mass acquisition was performed in the range of 40–550 a.m.u. using electron impact ionization at 70 eV. Compound identity was determined by matching the mass spectra of each compound with the MS data in the National Institute of Standards and Technology (NIST) database. Complete NMR spectra of all seven isolated compounds are provided in Structure elucidation of compounds Data were analyzed using SPSS Version 20  and results were expressed as median and interquartile range (IQR). Repeated measure analysis of variance (ANOVA) was used for comparison of average tumor number and tumor volume between F3-treated and untreated groups while significant differences in liver and kidney function parameters were computed using Mann-Whitney U-test. p < 0.05 was considered statistically significant.S. crispus to induce cell death was evaluated on MDA-MB-231 and MCF-7 breast cancer cells. At the test concentration of 100 μg/ml, DCM extract was significantly cytotoxic towards both MDA-MB-231 and MCF-7 cells with approximately 50% and 63% growth inhibition within 48 h, respectively : Yellowish-orange crystals. ESI-MS molecular ion peak at m/z 568.3 [M]+ corresponding to C40H56O2. 1H NMR : δ 6.65 , 6.36 , 6.27 , 6.14 , 5.54 , 5.43 , 4.25 , 4.00 , 2.40 , 2.39 , 2.05 , 1.97 *, 1.96 *, 1.91 , 1.84 , 1.77 , 1.74 , 1.62 , 1.48 , 1.36 , 1.07 , 1.00 , 0.85 . 13C NMR : 138.51 (C-8), 138.01 (C-6), 137.77 (C-5’), 137.74 (C-12), 137.58 (C-8’)a, 137.57 (C-12’)a, 136.51 (C-13)b, 136.43 (C-13’)b, 135.71 (C-9), 135.09 (C-9’), 132.6 , 131.32 (C-10)c, 130.82 (C-10’)c, 130.10 (C-15)d, 130.05 (C-15’)d, 128.75 (C-7’), 126.18 (C-5), 125.60 (C-7), 124.95 (C-11), 124.8 (C-11’), 124.49 (C-4’), 65.9 (C-3’), 65.1 (C-3), 54.97 (C-6’), 48.43 (C-2), 44.66 (C-2’), 42.56 (C-4), 37.14 (C-1), 34.05 (C-1’), 30.28 (C-17), 29.51 (C-17’), 28.74 (C-16), 24.28 (C-16’), 22.89 (C-18’), 21.64 (C-18), 13.13 (C-19’), 12.83 , 12.78 (C-19). a-dAssignments of resonances may be interchanged between those labelled with the same alphabet.Lutein . 1H NMR spectrum of the compound exhibited three downfield singlets at δ 9.76, 9.56 and 8.70 ppm corresponding to three aromatic methine protons of the porphyrin located at H-10, H-5 and H-20, respectively. Three sets of coupling doublet of doublets at δ 8.02, 6.34 and 6.17 ppm that had a correlation with the quarternary carbon at δ 136.55 ppm in HMBC were characteristic of the exocyclic ethylene group attached to C-3. The presence of four isolated methyl protons at H-121, H-134, H-21 and H-71 were indicated by four singlets at δ 3.90, 3.74, 3.44 and 3.28 ppm, respectively. Their positions were determined based on HMBC data. A pair of coupling signals made up of a quartet (δ 3.75 ppm) and a triplet (δ 1.72 ppm) that correlated to the carbon at δ 146.00 ppm in HMBC was typical of an ethyl group substituted on C-8. The presence of two coupling upfield methine proton multiplets at δ 5.14 (H-17) and δ 4.44 ppm (H-18) indicated that the structure assumed a chlorin moiety whereby one of the pyrrolic ring (ring D) is saturated at the δ position (positions 17 and 18). The doublet at δ 1.63 ppm which corresponded to the methyl protons H-181, four multiplets in the region of δ 2.4–2.8 ppm that belonged to two pairs of diastereotopic protons H-171a & b and H-172a & b which coupled to the carbonyl carbon at δ 173.32 ppm (C-173), together with a large number of overlapping proton signals in the region of δ 1.6–0.7 ppm belonging to the aliphatic methylene and methyl functions of phytyl further indicated that the structure was close to that of pheophytin a (6). However, the absence of H-132 signal and the significant downfield shift of C-132 signal to δ 161.04 ppm in compound 2 indicated that the 132 position in this compound consisted of a carbonyl group and a C = OCOOCH3 functionality must exist. Furthermore, to match the molecular formula, an additional OH group should exist in compound 2. This hydroxyl group was most probably attached to C-131 to form a COOH functionality so that the compound has a cleaved E ring structure as shown in 2 was identified as 131-hydroxy-132-oxo-pheophytin a (synonym: purpurin 7-monomethyl phytyl ester).The second compound was isolated as a greyish green solid. HR-ESI-MS analysis in the positive mode showed the molecular ion mass of m/z 903.5630 [M+H]3), stigmasterol (4) and β-sitosterol (5). The identity of these compounds was determined by GC-MS analysis and matching of the mass spectral data with that in the NIST database. The MS data of these compounds are given in 6) and 132-hydroxy-pheophytin a (7)Another three compounds isolated as a mixture from F3 were campesterol (6): Dark green powder. ESI-MS molecular ion peak at m/z 871.5 [M+H]+ corresponding to C55H75N4O5. 1H NMR : δ 9.49 , 9.35 , 8.55 , 7.97, 6.27 , 6.26 , 6.16 , 5.13 , 4.49 , 4.45, , 4.21 , 3.88 , 3.68 , 3.67 , 3.39 , 3.21 , 2.63 , 2.48 , 2.34 , 2.20 , 1.87 , 1.80 , 1.68 , 1.57 , 1.49 , 0.85 , 0.80 , 0.78 , 1.4–0.9 (H-P5-P14), -1.46 , -1.65 . 13C NMR : δ 189.66 (C-131), 173.02, (C-173), 172.23 (C-19), 169.62 (C-133), 161.24 (C-16), 155.60 (C-6), 150.97 (C-9), 149.65 (C-14), 145.21 (C-8), 142.86 (C-P3), 142.03 (C-1), 137.93 (C-11), 136.50 (C-3)a, 136.26 (C-4)a, 136.16 (C-7)a, 131.84 (C-2), 129.08 (C-31)b, 129.04 (C-12)b, 129.00 (C-13)b, 122.71 (C-32), 117.70 (C-P2), 105.21 (C-15), 104.42 (C-10), 97.52 (C-5), 93.12 (C-20), 64.70 (C-132), 61.47 (C-P1), 52.86 (C-134), 51.12 (C-17), 50.11 (C-18), 39.78–24.41 (C-P4-P14), 31.18 (C-172), 29.81 (C-171), 28.00 (C-P15), 23.07 (C-181), 22.71 (C-P16)c, 22.54 (C-P151)c, 19.71 (C-P111)d, 19.62 (C-P71)d,19.44 (C-81), 17.42 (C-82), 16.28 (C-P31), 12.12 (C-121), 12.10 (C-21), 11.23 (C-71). a-dAssignments of resonances may be interchanged between those labelled with the same alphabet.Pheophytin a (2-hydroxy-pheophytin a (7): Greenish dark brown powder. ESI-MS molecular ion peak at m/z: 909.4 [M+Na]+ corresponding to C55H74N4O6Na. 1H NMR : δ 9.61 , 9.47 , 8.63 , 8.02 , 6.32 , 6.21 , 5.51 , 5.21 , 4.56 , 4.41 , 4.15 , 3.74 , 3.71 , 3.61 , 3.42 , 3.25 , 2.94 , 2.55 , 2.28 , 1.91 , 1.71 , 1.61 , 1.60 , 1.50 , 1.37–0.9 (H-P4-P14), 0.85 , 0.80 *, 0.79 *, -1.83 . 13C NMR : δ 192.02 (C-131), 173.60 (C-173), 172.81 (C-133), 172.43 (C-19), 162.49 (C-16), 155.4 (C-6), 151.06 (C-9), 149.86 (C-14), 145.24 (C-8), 142.78 (C-P3), 142.04 (C-1), 137.83 (C-11), 136.54 (C-7), 136.30 (C-3)a, 136.23 (C-4)a, 131.77 (C-2), 129.43 (C-12), 129.10 (C-31), 126.98 (C-13), 122.89 (C-32), 117.89 (C-P2), 107.70 (C-15), 104.28 (C-10), 97.90 (C-5), 93.66 (C-20), 88.99 (C-132), 61.57 (C-P1), 53.42 (C-134), 51.85 (C-17), 50.34 (C-18), 39.84–24.44 (C-P4-P14), 31.60 (C-172), 31.16 (C-171), 29.97 (C-P15), 22.72 (C-P16)b, 22.66 (C-P151)b, 22.63 (C-181), 19.71 (C-P111)c, 19.62 (C-P71)c, 19.51 (C-81), 17.47 (C-82), 16.34 (C-P31), 12.31 (C-121), 12.13 (C-21), 11.30 (C-71). a-dAssignments of resonances may be interchanged between those labelled with the same alphabet.13The compounds isolated from F3 were evaluated for their cyotoxicity against MCF-7 cells. Among these compounds, lutein appeared to be most cytotoxic, exhibiting approximately 40% inhibition within 24 h of treatment . The resS. crispus extracts inhibited the growth of various human cancer cell lines [in vivo cancer therapeutic potential of this plant has never been reported. In the current study, the DCM extract of S. crispus was chromatographed into five subfractions. The third subfraction (F3) was highly cytotoxic to MCF-7 and MDA-MB-231 breast cancer cells with 80–100% cell death incurred following treatment at the concentration of 100 μg/ml F3. We then further determined the anticancer potential of F3 in NMU-induced breast cancer animal model and analysed the major constituents of this fraction.Previous studies showed that ll lines –32. A frll lines . HoweverS. crispus leaves, indicated that doses of up to 600 mg/kg [Acute oral toxicity studies using crude ethanol extract and juice of 00 mg/kg  and 4,9000 mg/kg  body wei00 mg/kg . Neverthet al., [The hematological profile of F3–treated animals showed no signs of anemia, infection or other adverse effects when compared to the normal rats, further supporting the non-toxic nature of F3. The ability of F3 to return the NMU-induced high WBC count towards normal value indicates its potential immunological role in protection against foreign body invasion. On the other hand, significantly low MCH and high platelet counts could also be due to the effect of host factors associated with NMU induction , 37. In et al., , we alsoS. crispus extracts. Suherman and colleagues [S. crispus water extract on rat liver or plasma microsomal γ-glutamyl transpeptidase (GGT) activities. More recently, a 14-day acute toxicity study using a 5 g/kg body weight daily dose of S. crispus ethanol extract showed no clinical signs of toxicity in the experimental animals [et al, [S. cripus extract is also reported to be protective against chemical hepatocarcinogenesis. Supplementation of the extract to Sprague-Dawley rats induced with diethylnitrosamine and 2-acetylaminofluorene showed reduced effect of hepatocarcinogenesis on liver GGT and ALP activities [Other studies also indicated non-toxicity of lleagues  reported animals . This wa [et al,  whereby tivities  as well tivities .Membrane-bound (ALP) or cytosolic (ALT & AST) enzymes of liver and kidney cells are used as indices of liver damage in both animals and humans . VariablUrea, creatinine and uric acid are metabolic by-products of the excess protein, creatine phosphate and purine in the liver and muscle, which are passed into the bloodstream and excreted by kidneys. Low serum concentration of these metabolic waste products could be multifactorial and usually indicates that the kidneys are hyperactive in removing excess fluids and wastes from the blood stream. A similar metabolic state is also obtainable in the presence of impaired liver function . F3 treain vitro and in vivo studies in the past had provided some supporting evidences concerning its use against cancer, phytochemical constituents which are responsible for the anticancer effect of S. crispus remained unclear. The presence of catechins and alkaloids contributes to the high antioxidant capacity of S. crispus [S. crispus leaves were found to be cytotoxic to human cancer cell lines [Although several  crispus  that mayll lines . In the Lutein was the predominant compound in F3 as it was isolated in high abundance compared to the other chemical constituents. This compound was also the most active constituent among the other compounds identified in this fraction. Lutein belongs to the xanthophyll family of carotenoids. It differs from other carotenoids in that it has two hydroxyl groups, one on each side of the molecule. These hydroxyl groups were found to play a significant role in the cytotoxic activity of lutein as di-acetylation of the hydroxyl groups resulted in a drop in activity against KB epithelial cancer cells . There i1-hydroxy-132-oxo-pheophytin a (synonym: purpurin-7-methyl phytyl ester), pheophytin a and 132-hydroxy-pheophytin a Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 Fig(PDF)Click here for additional data file.S7 Fig(PDF)Click here for additional data file.S8 Fig(PDF)Click here for additional data file.S9 Fig(PDF)Click here for additional data file.


Recent studies have indicated that 1, 4-dihydropyridine-3, 5-dicarboxamide derivatives show significant anti-tubercular activity. In this research, new derivatives of 1, 4-dihydropyridine were designed and synthesized using Hantzsch condensation in which dicyclohexyl and different dicyclohexylcarbamoyl were substituted at C-3 and C-5 positions of the DHP ring. In addition, 4 (5)-chloro-2-methyl-5 (4)-imidazolyl moiety was substituted at C-4 position of DHP. The structure of synthetized compounds were characterized by TLC, IR, elemental analysis and proton NMR. Based on the in vitro screening data, all of the designed and synthetized compounds (3a-3g) showed a good ability to inhibit the mycobacterium tuberculosis growth in terms of MIC. Aromatic carboxamide containing compounds were more potent than cyclohexyl derivative and the most potent compound was 3a (4-nitrophenyl derivative). The experimental data are in agreement with our computational predictions in terms of partial atomic charge of carbonyl moieties at the C-3 and C-5 positions of DHP ring and partition coefficient of the molecules. The feaBased on the MDRr -12 and aIn our previous studies we confirmed that in DHPs, 4 -chloro-2Chemistry1HNMR spectra were recorded on a Bruker FT-500 spectrometer and TMS was used as an internal standard. Infrared spectra were acquired on a Nicolet 550-FT spectrometer. Elemental analysis was carried out with a Perkin-Elmer model 240-C apparatus. The results of elemental analysis  were within ± 0.4% of the calculated amounts.Reagents and solvents were obtained from MERCK . All of the compounds were characterized by TLC, IR, elemental analysis and proton NMR. Melting points were determined using a Thomas- Hoover capillary apparatus and were uncorrected. Symmetrical dicarboxamides 3a-g  were synGeneral procedure for the prepartation of 3-oxo-N-aryl  butanamide (1a-g) A solution of an amine and dioxin in xylene was placed in an Erlenmeyer flask. The flask was immersed into an oil bath that had been preheated to 150°C, and the solution was vigorously stirred. The evolution of acetone became apparent within several minutes and heating was continued for around 4 h. The xylene was then removed, and the product was filtered and recrystallized from appropriate organic solvents.N-(4-nitrophenyl)-3-oxobutanamide (1a)−1 (CO). 1H-NMR (CDCl3): δ 9.70 , 8.20 , 7.80 , 3.65 , 2.30 ppm .Using the general procedure and 4-nitroaniline provided the title compound after 4 h of reflux: Yellow crystals, Yield 70%, mp 142-143°C. IR (KBr): ν 3325(NH), 1719, 1685 cmN-(4-fluorophenyl)-3-oxobutanamide (1b)−1 (CO). 1H-NMR (CDCl3): δ 9.40 , 7.56-7.78 , 7.15-7.25 , 3.70 , 2.29 ppm .Using the general procedure and 4-fluoroaniline provided the title compound after 3 h of reflux: White crystals, Yield 88%, mp 98-101°C. IR (KBr): ν 3315(NH), 1716, 1671 cmN-(4-chlorophenyl)-3-oxobutanamide (1c)−1 (CO). 1H-NMR (CDCl3): δ 9.37 , 7.50 , 7.23 , 3.58 , 2.30 ppm .Using the general procedure and 4-chloroaniline provided the title compound after 3 h of reflux: White crystals, Yield 79%, mp 127-129°C. IR (KBr): ν 3300 (NH), 1720, 1670 cmN--3-oxobutanamide (1d)−1 (CO). 1H-NMR (CDCl3): δ 9.45 , 8.05 , 7.30-7.47 , 3.65 , 2.39 ppm .Using the general procedure and 3, 4-dichloroaniline provided the title compound after 4 h of reflux: White crystals, Yield 65%, mp 79-81°C. IR (KBr): ν 3378 (NH), 1710, 1665 cmN-(4-bromophenyl)-3-oxobutanamide (1e)−1 (CO). 1H-NMR (CDCl3): δ 9.65 , 7.62 , 7.40 , 3.65 , 2.28 ppm .Using the general procedure and 4-bromoaniline provided the title compound after 3 h of reflux: Light yellow crystals, Yield 75%, mp 135-136°C. IR (KBr): ν 3260 (NH), 1716, 1680 cmN-(3-bromophenyl)-3-oxobutanamide (1f)−1 (CO). 1H-NMR (CDCl3): δ 9.25 , 7.78 , 7.18-7.45 , 3.54 , 2.29 ppm .Using the general procedure and 3-bromoaniline provided the title compound after 3 h of reflux: Light yellow crystals, Yield 80%, mp 98-101°C. IR (KBr): ν 3300 (NH), 1712, 1673 cmN-(cyclohexyl)-3-oxobutanamide (1g)−1 (CO). 1H-NMR (CDCl3): δ 9.40 , 3.65 , 2.15-2.40 , 1.30-1.75 ppm .Using the general procedure and cyclohexylamine provided the title compound after 3 h of reflux: Light yellow crystals, Yield 69%, mp 98-100°C. IR (KBr): ν 3310(NH), 1710, 1665 cmGeneral procedure for preparation of diaryl  4-(4 (5)-chloro-1H-imidazol-5 (4)-yl)-2, 6-dimethyl-1, 4-dihydropyridine-3, 5-dicarboxamide (3a-g)A solution of compound 2 , ammonium acetate , and compounds 1a-g (2.076 mmol) in methanol (3 mL) was refluxed. The solvent was removed under reduced pressure and the residue was crystallized from appropriate solvent to give the title compounds.4-(4 (5)-chloro-2-methyl-1H-imidazol-5 (4)-yl)-2, 6-dimethyl-N3, N5-bis (4-nitrophenyl)-1, 4-dihydropyridine-3, 5-dicarboxamide (3a)oC (ethyl acetate-methanol). IR (KBr): ν 3257 (NH), 1680, 1645 (CO), 1501, 1321 cm−1 (NO2). 1H-NMR (CDCl3 + DMSO- d6): δ 2.27 , 5.25 , 7.61 , 7.82 , 8.13 . 8.6 , 9.4 ppm . Molecular formula: C25H22ClN7O6; Calculated = C (54.40%) H (4.02%) N (17.76%); Found = C (54.49%) H (4.03%) N (17.79%).Compound 3a was provided from compound 1a using general procedure after 35 h reflux: Yellow crystals, yield 85%; mp 2504-(4 (5)-chloro-2-methyl-1H-imidazol-5 (4)-yl)-2, 6-dimethyl-N3, N5-bis (4-fluorophenyl)-1, 4-dihydropyridine-3, 5-dicarboxamide (3b)oC (methanol). IR (KBr): ν 3436, 3293, 3216 (NH), 1685, 1634 cm−1 (CO). 1H-NMR (CDCl3 + DMSO- d6): δ 2.21 , 2.30 , 5.19 , 6.94 , 7.49-7.7 , 8.10 , 9.4 ppm . Molecular formula: C25H22ClF2N5O2; Calculated = C (60.30%) H (4.45%) N (14.07%); Found = C (60.37%) H (4.46%) N (14.05%).Compound 3b was provided from compound 1b using general procedure after 38 h reflux: White crystals, yield 62%; mp 2664-(4(5)-chloro-2-methyl-1H-imidazol-5(4)-yl)-2, 6-dimethyl-N3, N5-bis (4-chlorophenyl)-1, 4-dihydropyridine-3, 5-dicarboxamide (3c)oC (methanol). IR (KBr): ν 3445, 3398 (NH), 1676, 1661 cm−1 (CO). 1H-NMR (DMSO- d6): δ 2.01 , 2.11 , 5.15 , 7.28 , 7.6 , 8.38 , 9.3 ppm . Molecular formula: C25H22Cl3N5O2; Calculated = C (56.57%) H (4.18%) N (13.19%); Found = C (56.62%) H (4.17%) N (13.22%).Compound 3c was provided from compound 1c using general procedure after 30 h reflux: White crystals, yield 80%; mp 2724-(4 (5)-chloro-2-methyl-1H-imidazol-5 (4)-yl)-2, 6-dimethyl-N3, N5-bis -1, 4-dihydropyridine-3, 5-dicarboxamide (3d)oC (methanol-ethyl acetate). IR (KBr): ν 3431, 3365 (NH), 1664 cm−1 (CO). 1H-NMR (CDCl3 + DMSO- d6): δ 2.13 , 5.19 , 7.32 , 7.68 , 8.42 , 9.44 ppm . Molecular formula: C25H20Cl5N5O2; Calculated = C (50.07%) H (3.36%) N (11.68%); Found = C (50.04%) H (3.37%) N (11.71%).Compound 3d was provided from compound 1d using general procedure after 40 h reflux: White crystals, yield 60%; mp 2444-(4 (5)-chloro-2-methyl-1H-imidazol-5 (4)-yl)-2, 6-dimethyl-N3, N5-bis (4-bromophenyl)-1, 4-dihydropyridine-3, 5-dicarboxamide (3e)oC (methanol-ethyl acetate). IR (KBr): ν 3452, 3367 (NH), 1655 cm−1 (CO). 1H-NMR (CDCl3 + DMSO- d6): δ 2.10 , 2.8 , 5.11 , 7.08-7.28 , 7.3-7.6 , 8.33 , 9.31 ppm . Molecular formula: C25H22 Br2ClN5O2; Calculated = C (48.45%) H (3.58%) N (11.30%); Found = C (48.51%) H (3.59%) N (11.27%).Compound 3e was provided from compound 1e using general procedure after 37 h reflux: White crystals, yield 64%; mp 2744-(4(5)-chloro-2-methyl-1H-imidazol-5(4)-yl)-2, 6-dimethyl-N3, N5-bis (3-bromophenyl)-1, 4-dihydropyridine-3, 5-dicarboxamide (3f)oC (methanol-ethyl acetate). IR (KBr): ν 3146(NH), 1649 cm−1 (CO). 1H-NMR (CDCl3 + DMSO- d6): δ 2.10 , 2.12 , 5.11 , 7.00-7.63 , 8.35 , 9.31 ppm . Molecular formula: C25H22 Br2ClN5O2; Calculated = C (48.45%) H (3.58%) N (11.30%); Found = C (48.41%) H (3.59%) N (11.33%).Compound 3f was provided from compound 1f using general procedure after 38 h reflux: Green crystals, yield 40%; mp 2834-(4(5)-chloro-2-methyl-1H-imidazol-5(4)-yl)-2, 6-dimethyl-N3, N5-bis (4-cyclohexyl)-1, 4-dihydropyridine-3, 5-dicarboxamide (3g)oC (ethyl acetate-methanol). IR (KBr): ν 3267 (NH), 2924, 2852 , 1664 cm−1 (CO). 1H-NMR (CDCl3+DMSO- d6): δ 0.62-1.93 , 1.97 , 2.24 , 5.95 , 8.34 , 9.26 ppm . Molecular formula: C25H36ClN5O2; Calculated = C (63.34%) H (7.65%) N (14.77%); Found = C (63.40%) H (7.66%) N (14.81%).Compound 3g was provided from compound 1g using general procedure after 39 h reflux: Light yellow crystals, yield 30%; mp 253Computational studies -1. The optimized conformer was transferred to Gaussian software to calculate HOMO, LUMO and partial atomic charge (Muliken) using RHF method and 3-21G basis set.The chemical structures of desired DHPs 3a-g were built and optimized using HyperChem software . Optimization of the compounds was performed through MM+ and PM3 methods and total energy gradient was calculated as a root mean square (RMS) value, until the RMS gradient was 0.01 kcal molIn-vitro evaluation of anti-mycobacterial activityThe test compounds 3a-g, were initially dissolved in DMSO to give a concentration of 1 or 2 mg/L. Except first column, all wells of micro plates were received 100 µL of freshly prepared Middle broke 7H9 medium . 200 µL of distilled water was added to the first column of 96 well plates to minimize evaporation of the medium in the test wells during incubation. Then 100 µL of test compounds with desired concentrations (1000 or 2000 µL) were added to the wells of the first row (each concentration was assayed in duplicate) and serial dilution was made from the first row to the last. Microbial suspension of BCG (1173P2) (100 µL), which had been prepared with standard concentration of 0.5 McFarland and diluted with 1:10 proportion by the distilled water, was added to all test wells. Plates were then sealed and incubated for 4 days at 37°C. After that, 12µl Tween 80 10% and 20µl Alamar blue 0.01%  were added to each test well. The results were assessed after 24 and 48 h. A blue color was interpreted as no bacterial growth, and color change to pink was scored as bacterial growth. Wells with a well-defined pink color were scored as positive for growth. The MIC (minimum inhibitory concentration) was defined as the lowest drug concentration, which prevented a color change from blue to pink. Ethambutol  and DMSO were used as positive control and negative controls respectively .ChemistrySeven new derivatives of dihydropyridine, compounds 3a-g , were syComputational studies Based on the subjects that mentioned in the introduction section, Partial atomic charge (PAC) of carbon atom of carbonyl moiety at the C-3 and C-5 position of dihydropyridine ring and the lipophilicity (log p) of DHPs 3a-3g was calculated using Gaussian and HyperChem software. To calculation the PAC, at first all of the compounds were optimized using HyperChem with molecular mechanics (MM+) and semi-empirical (PM3) methods. To finding the global minima, the best conformer from the previous stage was transferred to Gaussian and more optimization was performed using RHF method and 3-21G basis set. Confirmer with the global minima was used to calculation of partial atomic charges. Results of calculated PAC are presented in Anti-tubercular activity2, F, Cl and Br respectively) at the para position of phenyl ring, reveals that the compound 3a which contains more electronegative group is the more active than compounds 3c, 3e and 3b. Comparison of the MIC of compound 3e (4-Br) with 3f (3Br) and compound 3c (4-Cl) with 3d , indicate that existence of the substitution at the 3 position of phenyl ring results in to reduce the activity, probably in order to their hindrances effect. The ability of DHPs 3a-g to inhibition of mycobacterium tuberculosis growth was determined using in vitro assay. The results are summarized in the  Comparison of compounds 3a-f with compound 3g reveals existence of the electron donating group (cyclohexyl) at C-3 and C-5 of DHP ring result in to reduce the activity probably in order to low values of partial atomic charge of carbonyl moieties. Based on the PAC, it was expected that compound 3b be more active than 3c, but experimental data was not confirmed that, may be because of their low partition coefficient (log P) and consequently low penetration into the mycobacterium cells.1HNMR. The elemental analysis has confirmed the purity of products. The in vitro activities of all compounds against mycobacterium were investigated. Based on the in vitro screening data, all the designed and synthesized compounds (3a-3g) had good ability to inhibit mycobacterium tuberculosis growth in terms of MIC. The most potent compound was 3a, 4-nitrophenyl carboxamide derivative of DHPs, which was predicted in our computational studies based on PAC.Seven DHPs analogs were synthesized and characterized by TLC, FT-IR and The results so far indicated that the activity of these ligands against the mycobacterium can significantly be influenced by log p of molecules and partial atomic charge of carbon atom of carbonyl moiety at C-3 and C-5 position of DHPs ring. Currently, our research group is exploring this idea to design newer ligands with better anti-tubercular activity.


Polyporus ellisii led to the isolation of a novel compound 3β,9α,15α-trihydroxy--10(5→4)-abeo-ergosta-6,8(14),22-trien-5-one (1) with a new 5/7/6/5 ring system of ergosterol skeleton. In addition, five new steroids, 5β,6β-epoxy-3β,7α,9α-trihydroxy--ergosta-8(14),22-dien-15-one (2), 5β,6β-epoxy-3β,7α-dihydroxy--ergosta-8(14),22-dien-15-one (3), 5α,6α-epoxy-3β,9α,15α-trihydroxy--ergosta-8(14),22-dien-7-one (4), 15α-acetoxy--ergosta-4,6,8(14),22-tetraen-3-one (5), 15β-methoxy--ergosta-4,6,8(14),22-tetraen-3-one (6), along with four known ergosterols (7–10), were obtained. All structures were elucidated based on 1D and 2D NMR spectral data. New compounds were evaluated for cytotoxicity against five human cancer cell lines, only compound 4 was found to exhibit a favorable cytotoxicity profile toward all tested tumor cell lines.Investigation of the culture of basidiomycete Supplementary material is available for this article at 10.1007/s13659-012-0058-4 and is accessible for authorized users. Supplementary material, approximately 6.54 MB.


Pharmacokinetic studies on scutellarin showed that scutellarin (1) is readily converted into its metabolites in vivo. In this paper, a new and practical synthetic method for the synthesis of 6-O-methyl-scutellarein (3) (one metabolite of scutellarin in vivo) is reported. The benzyl bromide was firstly used to selectively replace the acetyl group at C-7 in 7, and was then used to protect the hydroxy groups at C-4' in 10, 6-O-methyl-scutellarein (3) is obtained in high yield through these methods.Scutellarin ( Pharmacological studies found that scutellarin (1) demonstrated antioxidant and anticoagulant activities to attenuate neuronal damage, thus had a wide range of benefits to brain injury caused by cerebral ischemia/reperfusion . Th. ThO-met2) would hamper the large-scale preparation of 6-O-methyl-scutellarein (3). After an extensive study, we have exploited a new and practical synthesis of 6-O-methyl-scutellarein (3) by first using acetic anhydride as the protecting group instead. Herein, we report this synthetic route in detail.However, the reaction condition using dichlorodiphenylmethane at 175 °C  to proteO-methyl-scutellarein (3) was synthesized from scutellarin (1) in seven steps. According to our previous method +.5,6,7-triacetoxy-2-(4-acetoxyphenyl)-4H-chromen-4-one (7) Pyridine (5 mL) and 4-dimethylaminopyridine (DMAP)  were added to a stirring solution of 2  in acetic anhydride (5 mL) at 0 °C, then the mixture was warmed to room temperature. After 4 h, the reaction mixture was poured into 100 mL water, and the solid obtained was filtered and purified by silica gel column chromatographic, using 25% ethyl acetate in petroleum ether, to afford compound 7  as a yellow solid, mp 251–252 °C. 1H NMR  δ 2.32 , 2.35 , 2.37 , 2.38 , 6.95 , 7.36 , 7.84 , 8.14 ; ESI-MS: m/z 455 [M + H]+.7-(benzyloxy)-5,6-diacetoxy-2-(4-acetoxyphenyl)-4H-chromen-4-one (8) K2CO3  and benzyl bromide  were added to a stirring solution of 7  in dry acetone (20 mL), and then the mixture was refluxed for 6 h and partitioned between 100 mL ethyl acetate and 100 mL water. The ethyl acetate layer was then washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated. The crude material was purified by column chromatography (25% ethyl acetate in petroleum ether) to yield 8  as a yellow solid, mp 190–191 °C. 1H NMR  δ 2.31 , 2.32 , 2.34 , 5.36 , 6.87 , 7.36–7.45 , 7.60 , 8.13 ; ESI-MS: m/z 503 [M + H]+.7-(benzyloxy)-5,6-dihydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-one (9) A solution of sodium hydroxide  was added to a stirring mixture of 8  in methanol (10 mL) at 0 °C under N2 atmosphere, after 1h, and the mixture was partitioned between 100 mL ethyl acetate and 100 mL water. The ethyl acetate layer was then washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated. The crude material was purified by column chromatography (25% ethyl acetate in petroleum ether) to yield 9  as a yellow solid, mp 198–200 °C. 1H NMR  δ 5.28 , 6.80 , 6.92 , 7.00 , 7.35–7.45 , 7.51–7.54 , 7.94 , 8.72 , 10.33 , 12.68 ; ESI-MS: m/z 377 [M + H]+.7-(benzyloxy)-2-(4-(benzyloxy)phenyl)-5,6-dihydroxy-4H-chromen-4-one (6) K2CO3  and benzyl bromide  were added to a stirring solution of 9  in dry DMF (20 mL) at 0 °C, and then the mixture was warmed to room temperature. After 12 h, the reaction mixture was partitioned between 100 mL ethyl acetate and 100 mL water. The ethyl acetate layer was then washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated. The crude material was purified by column chromatography (25% ethyl acetate in petroleum ether) to yield dibenzylether 6  as a yellow solid, mp 139–141 °C. 1H NMR  δ 5.03 , 5.23 , 6.62 , 6.87 , 7.18 , 7.31–7.53 , 8.04 , 10.82 , 13.11 ; ESI-MS: m/z 465 [M − H]−.7-(benzyloxy)-2-(4-(benzyloxy)phenyl)-5-hydroxy-6-methoxy-4H-chromen-4-one (10) K2CO3  and iodomethane  were added to a stirring solution of 6  in dry DMF (20 mL) at room temperature. After 12 h, the reaction mixture was then partitioned between 100 mL ethyl acetate and 100 mL water. The ethyl acetate layer was then washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated. The crude material was purified by column chromatography (25% ethyl acetate in petroleum ether) to yield 10  as a yellow solid, mp 196–198 °C. 1H NMR  δ 4.00 , 4.95 , 5.28 , 6.62 , 6.94 , 7.00 , 7.32–7.42 , 7.44–7.54 , 7.94 , 12.69 ; ESI-MS: m/z 481 [M + H]+.5,7-dihydroxy-2-(4-hydroxyphenyl)-6-methoxy-4H-chromen-4-one (3) Ten percent Pd/C (2 mg) was added to a solution of 10  dissolved in ethanol (10 mL) and THF (10 mL) with vigorous stirring. The reaction vessel was then evacuated and the atmosphere replaced with hydrogen. After 8 h, the reaction mixture was filtered and the filtrate was concentrated. The crude material was purified by column chromatography (50% ethyl acetate in petroleum ether) to yield 3  as a yellow solid, mp 172–174 °C. 1H NMR  δ 3.72 , 6.58 , 6.89 , 7.11 , 7.87 , 10.19 , 10.72 , 12.76 ; ESI-MS: m/z 299 [M − H]−.O-methyl-scutellarein (3) from scutellarin (1) in high yield. Firstly, the acetic anhydride was used to protect all the hydroxyl groups in scutellarein (2), and then the benzyl bromide was used to selectively substitute the acetic group at C-7 position in 7. Based on the their reactive activity of the other three hydroxyl groups were different and they were in the preferential order 4' > 6 > 5, benzyl group was used again to protect the C-4' hydroxyl group. This efficient and practical method could be applied to the selective synthesis of other O-methylflavonoid isomers, as well as the other flavonoid sulfate- and glucuronide-metabolites in vivo. In conclusion, we have efficiently and practically synthesized 6-


The structures of the synthesized compounds were assigned on the basis of elemental analysis, IR, 1H NMR, and mass spectral data. All the newly synthesized compounds were screened for anti-inflammatory and analgesic activities.A series of 3-[2′-(Substitutedbenzylideneamino)phenyl]-2-methyl-6-substituted quinazolin-4-ones ( H)-quinazolinone, 4-thiazolidinone, and azetidinones, we report here the synthesis of a new class of heterocyclic molecules in which all of these moieties are present and try to develop potential bioactive molecules. The structures of the compounds synthesized were assigned on the basis of elemental analysis, IR, 1H NMR, and Mass spectral data. These compounds were evaluated for their anti-inflammatory and analgesic activities.Quinazolinone derivatives represent one of the most active classes of compounds possessing a wide spectrum of biological activity. They are widely used in pharmaceuticals and agrochemicals. Several reports have been published on the biological activities of quinazolinone derivatives, including their anti-inflammatory –7, antim3,4). Compounds  when treated with different substituted aromatic aldehydes formed various substituted arylidene derivatives (5–10). These arylidene derivatives on treatment with chloroacetylchloride in presence of triethylamine yielded 3-[2′-(3′′-chloro-2′′-oxo-4′′-substituted phenylazetidin-1′′-yl)phenyl]-2-methyl-6-substitutedquinazolin-4-ones (11–16). On the other hand, these arylidene derivatives on reaction with thioglycolic acid and anhydrous ZnCl2 furnished 3-[2′-phenyl]-2-methyl-6-substitutedquinazolin-4-ones. (17–22). The structures of the compounds synthesized were assigned on the basis of elemental analysis, IR, 1H NMR, and Mass spectral data.The synthetic routes of compounds are outlined in The experiments were performed with albino rats of the Charles-foster stain of either sex, excluding pregnant females, of 70–95 d. weighting 100–150 g. Food  and water were given to the animals ad libitum. All the compounds were dissolved in propylene glycol. Phenylbutazone was used as reference drug.Anti-Inflammatory Activity against Carrageenan-Induced Rat's Paw OedemaVt and Vc are the mean increase in paw volume of rats of the treated and the control group, respectively. Results were statistically analyzed.This study was done with the procedure of Winter et al. . The ratAnalgesic ActivityThis activity was assessed following the method of Berkowitz et al. . This me−1. The 1H NMR spectra were recorded in CDCl3 on a Bruker Avance II 400 NMR spectrometer (400 MHz). The 13C NMR spectra were recorded in CDCl3 on a Bruker Avance II 400 NMR spectrometer operating at 100 MHz. The chemical shifts are reported in part per million (δ ppm) using trimethylsilane (TMS) as an internal standard. Elemental analysis  was performed on Perkin-Elmer 2400 analyzer, and values were either in ±0.4% of the calculated. Melting points were determined in one-end-open capillary tube on Thomas Hoover melting apparatus and are uncorrected. The purities of compounds were checked by thin layer chromatography (TLC) using silica gel-G coated plats. The spots were developed in an iodine chamber and visualized under ultraviolet (UV) lamp. Infrared (IR) absorption spectra were recorded on Perkin-Elmer RX-1 FTIR spectrometer using potassium bromide (KBr) pellet, and wave numbers were given in cm3-(2′-Aminophenyl)-2-Methyl-Quinazolin-4-one ( 3)3 (78%). m.p. 99°C. IR (KBr) νmax in cm−1: 3033 (CH aromatic), 2920 , 1690 (C=O), 1595 (C=N); 1HNMR (CDCl3) δ in ppm: 9.10 , 6.89–7.61 , 2.29 ; Anal. Calcd. for C15H13N3O: C, 71.71; H, 5.18; N, 16.73: Found: C, 71.85; H, 5.20; N, 16.68: MS [M]+ at m/z 251.A mixture of 2-methyl-4H-3,1-benzoxazin-4-one (1) (0.01 mole) with 2-aminophenylamine (0.01 mole) in acetic acid (40 mL) was refluxed for 7 hr. in the presence of sodium acetate. The solvent was removed under reduced pressure. The solid thus obtained was filtered off and recrystallized from toluene to give compound 3. The following compound was prepared using a similar procedure described for compound 3-(2′-Aminophenyl)-2-Methyl-6-Bromoquinazolin-4-one ( 4)νmax in cm−1: 3030 (CH aromatic), 2925 , 1695 (C=O), 1598 (C=N); 1H NMR (CDCl3) δ in ppm: 9.06 , 6.85–7.51 , 2.24 ; Anal. Calcd. for C15H12N3OBr: C, 54.55; H, 3.64; N, 12.73: Found: C, 54.38; H, 3.65; N, 12.76: MS [M]+ at m/z 330.(75%) (Benzene) m.p. 199°C, IR (KBr) 3-[2′-(Benzylideneamino)Phenyl]-2-Methyl-Quinazolin-4-one ( 5) 3 (0.01 moles) in ethanol (55 mL) was refluxed with benzaldehyde (0.01 moles) separately in the presence of glacial acetic acid (2.5 mL) for 11 hr. The reaction mixture was concentrated, cooled, and then poured into ice water. The separated solid was filtered and recrystallized from methanol to give compound 29 (63%) m.p. 118°C: IR (KBr) νmax in cm−1: 3028 (CH aromatic), 2930CH aliphatic), 1705 (C=O), 1610 (C=N); 1HNMR (CDCl3) δ in ppm: 6.79–7.63 , 6.35 , 2.21 ; Anal. Caled. for C22H17N3O: C, 77.88; H, 5.01; N, 12.39: Found: C, 77.96; H, 5.00; N, 12.41: MS [M]+ at m/z. 339.A solution of compound5.The following compounds were prepared using a similar procedure described for compound 3-[2′-(p-Chlorobenzylideneamino)Phenyl]-2-Methyl-Quinazolin-4-one ( 6)νmax in cm−1: 3028 (CH aromatic), 2929 , 1705 (C=O), 1598 (C=N) 788 (C–Cl); 1H NMR (CDCl3) δ in ppm: 6.82–7.65 , 6.38 , 2.24 ; Anal. Caled. for C22H16N3OCl: C,70.68; H, 4.28; N, 11.24: Found: C, 70.79; H, 4.27; N, 11.26: MS [M]+ at m/z. 373.5.(65%). (Benzene) m.p. 134°C: IR (KBr) 3-[2′-(p-Methoxybenzylideneamino)Phenyl]-2-Methyl-Quinazolin-4-one ( 7)νmax in cm−1: 3035(CH aromatic), 2932 , 1700 (C=O), 1600 (C=N); 1HNMR (CDCl3) δ in ppm: 6.80–7.61 , 6.40 , 3.48  2.26 ; Anal. Caled. for C23H19N3O2: C, 74.80; H, 5.15; N, 11.38: Found: C, 74.96; H, 5.17; N, 11.29: MS [M]+ at m/z. 369.(60%). (Methanol) m.p. 148°C: IR (KBr) 3-[2′-(Benzylideneamino)Phenyl]-2-Methyl-6-Bromoquinazolin-4-one ( 8)νmax in cm−1: 3033 (CH aromatic), 2926 , 1698 (C=O), 1606 (C=N); 1H NMR (CDCl3) δ in ppm: 6.83–7.60 , 6.43 , 2.22 ; Anal. Caled. for C22H16N3OBr: C,63.16; H, 3.83; N, 10.05: Found: C, 63.29; H, 3.82; N, 10.07: MS [M]+ at m/z. 418.(64%). (Ethanol) m.p. 215°C: IR (KBr) 3-[2′-(p-Chlorobenzylideneamino)Phenyl]-2-Methyl-6-Bromoquinazolin-4-one ( 9)νmax in cm−1: 3030 (CH aromatic), 2930 , 1695 (C=O), 1601 (C=N), 785 (C–Cl); 1HNMR (CDCl3) δ in ppm: 6.81–7.59 , 6.41 , 2.25 ; Anal. Caled. for C22H15N3OBrCl: C, 58.34; H, 3.31; N, 9.28: Found: C, 58.55; H, 3.32; N, 9.30: MS [M]+ at m/z. 452.5.(65%). (Ethanol) m.p. 234°C: IR (KBr) 3-[2′-(p-Methoxybenzylideneamino)Phenyl]-2-Methyl-6-Bromoquinazolin-4-one ( 10)νmax in cm−1: 3033 (CH aromatic), 2926 , 1698 (C=O), 1595 (C=N); 1HNMR (CDCl3) δ in ppm: 6.83–7.61 , 6.44 , 3.44  2.25 ; Anal. Caled. for C23H18N3O2Br: C, 61.61; H, 4.02; N, 9.38: Found: C, 61.52; H, 4.01; N, 9.36: MS [M]+ at m/z. 448(60%). (Acetone) m.p. 223°C: IR (KBr) 3-[2′-(3′′-Chloro- 2′′-Oxo- 4′′-Phenylazetidin- 1′′-Yl)-Phenyl]-2-Methyl-Quinazol-In-4-one ( 11)5 (0.01 mole) and triethylamine (0.5 mL) in ethanol (55 mL) was added in monochloroacetylchloride (0.014 mole) at 50°C. The reaction mixture was stirred for 45 min. at room temperature and refluxed for 8 hr. The reaction mixture was filtered to removed triethylamine hydrogen chloride and the resultant solution was poured onto crushed ice with constant stirring. The solid thus obtained was recrystallized from acetone to give desired compound 11 (60%) m.p. 153°C. IR (KBr): νmax in cm−1: IR (KBr) νmax in cm−1: 3030 (CH aromatic), 2925 , 1700 (C=O), 1595 (C=N); 1HNMR (CDCl3) δ in ppm: 6.81–7.98 , 6.51 , 4.60 , 2.21 ;. Anal. Calcd. for C24H18N3O2Cl: C, 69.31; H, 4.33; N, 10.11: Found: C, 69.47; H, 4.32; N, 10.14: MS [M]+at m/z 415.5.To a solution of compound 11. The following compounds were prepared using a similar procedure described for compound 3-[2′-(3′′-Chloro- 2′′-Oxo- 4′′-(p-Chlorophenyl)Azetidin- 1′′-yl)-Phenyl]-2-Methyl Quinazolin-4-one ( 12)νmax in cm−1: IR (KBr) νmax in cm−1: 3033 (CH aromatic), 2928 , 1705 (C=O), 1591 (C=N), 788 (C–Cl); 1HNMR (CDCl3) δ in ppm: 6.83–7.95 , 6.56 , 4.62 , 2.25 ;. Anal. Calcd. for C24H17N3O2Cl2: C, 64.00; H, 3.78; N, 9.33: Found: C, 64.15; H, 3.80; N, 9.30: MS [M]+at m/z 450.(58%) (Methanol) m.p. 198°C. IR (KBr): 3-[2′-(3′′-Chloro- 2′′-Oxo- 4′′-(p-Methoxyphenyl)Azetidin- 1′′-yl)-Phenyl]-2-Methyl Quinazolin-4-one ( 13)νmax in cm−1: IR (KBr) νmax in cm−1 : 3028 (CH aromatic), 2930 , 1700 (C=O), 1595 (C=N); 1HNMR (CDCl3) δ in ppm: 6.81–7.93 , 6.54 , 4.65 , 3.47  2.23 ;. Anal. Calcd. for C25H20N3O3Cl: C, 67.34; H, 4.49; N, 9.43: Found: C, 67.15; H, 4.50; N, 9.40: MS [M]+at m/z 445.5.(56%) (Ethanol)m.p. 212°C. IR (KBr): 3-[2′-(3′′-Chloro-2′′-Oxo-4′′-Phenylazetidin-1′′-yl)-Phenyl]-2-Methyl-6-Bromo-Quinazolin-4-one ( 14)νmax in cm−1: IR (KBr) νmax in cm−1 : 3030 (CH aromatic), 2925 , 1695 (C=O), 1595 (C=N); 1HNMR (CDCl3) δ in ppm: 6.80–7.92 , 6.54 , 4.64 , 2.22 ;. Anal. Calcd. for C24H17N3O2ClBr: C, 58.24; H, 3.44; N, 8.49: Found: C, 58.36; H, 3.43; N, 8.51: MS [M]+at m/z 494.5.(58%) (Methanol) m.p. 176°C. IR (KBr): 3-[2′-(3′′-Chloro-2′′-Oxo-4′′-(p-Chlorophenyl)Azetidin-1′′-yl)-Phenyl]-2-Methyl-6-Bromo Quinazolin-4-one ( 15)νmax in cm−1: IR (KBr) νmax in cm−1: 3027 (CH aromatic), 2930 , 1710 (C=O), 1598 (C=N), 785 (C–Cl); 1HNMR (CDCl3) δ in ppm: 6.79–7.90 , 6.55 , 4.61 , 2.20 ;. Anal. Calcd. for C24H16N3O2Cl2Br: C, 54.44; H, 3.02; N, 7.94: Found: C, 54.61; H, 3.01; N, 7.95: MS [M]+at m/z 529.(54%) (Ethanol) m.p. 204°C. IR (KBr): 3-[2′-(3′′-Chloro-2′′-Oxo-4′′-(P-Methoxyphenyl)Azetidin-1′′-yl)-Phenyl]-2-Methyl Quinazolin-4-one ( 16)νmax in cm−1: IR (KBr) νmax in cm−1: 3030 (CH aromatic), 2930 , 1700 (C=O), 1590 (C=N); 1HNMR (CDCl3) δ in ppm: 6.80–7.92 , 6.52 , 4.62 , 3.49  2.25 ;. Anal. Calcd. for C25H19N3O3ClBr: C, 57.20; H, 3.62; N, 8.01: Found: C, 57.31; H, 3.61; N, 8.06: MS [M]+at m/z 524.5.(60%) (Acetone) m.p. 246°C. IR (KBr): 3-[2′-Phenyl]-2-Methyl-6-Quinazolin-4-one ( 17)2 was refluxed for 18 hr. The solvent was removed under reduced pressure. The solid thus obtained was filtered and washed with water. The product finally obtained was dried and recrystallized from ethanol to yield compound 17 (55%) m.p. 164°C. IR (KBr): νmax in cm−1: IR (KBr) νmax in cm−1 : 3032 (CH aromatic), 2931 , 1705 (C=O), 1595 (C=N),685 (C–S–C); 1HNMR (CDCl3) δ in ppm: 6.80–7.97 , 4.55 , 4.25  2.25 ;. Anal. Calcd. for C24H19N3O2S: C, 69.73; H, 4.60; N, 10.17: Found: C, 69.82; H, 4.61; N, 10.22: MS [M]+at m/z 413.A mixture of compound 5(0.01 moles), thioglycolic acid (0.012 moles) in absolute dioxane (75 mL) and in the presence of anhydrous ZnCl3--2-Methyl-Quinazolin-4-one. ( 18)νmax in cm−1: IR (KBr) νmax in cm−1 : 3030 (CH aromatic), 2930 , 1700 (C=O), 1598 (C=N), 784 (C–Cl), 688 (C–S–C); 1HNMR (CDCl3) δ in ppm: 6.83–7.96 , 4.58 , 4.21  2.22 ;. Anal. Calcd. for C24H18N3O2SCl: C, 64.36; H, 4.02; N, 9.39: Found: C,64.41; H, 4.03; N, 9.35: MS [M]+at m/z 447.5.(49%) (Methanol) m.p. 192°C. IR (KBr): 3--2-Methyl Quinazolin-4-one ( 19)νmax in cm−1: IR (KBr) νmax in cm−1 : 3032 (CH aromatic), 2927 , 1700 (C=O), 1600 (C=N), 690 (C–S–C),; 1HNMR (CDCl3) δ in ppm: 6.81–7.95 , 4.52 , 4.23  3.51 , 2.20 ;. Anal. Calcd. for C25H21N3O3S: C, 67.72; H, 4.74; N, 9.48: Found: C,67.88; H, 4.73; N, 9.51: MS [M]+at m/z 443.(50%) (Acetone) m.p. 204°C. IR (KBr): 3-[2′-Phenyl]-2-Methyl-6-Bromo Quinazolin-4-one ( 20)νmax in cm−1: IR (KBr) νmax in cm−1: 3028 (CH aromatic), 2933 , 1705 (C=O), 1600 (C=N), 690 (C–S–C); 1H NMR (CDCl3) δ in ppm: 6.81–7.94 , 4.56 , 4.22  2.24 . Anal. Calcd. for C24H18N3O2SBr: C, 58.54; H, 3.66; N, 8.54: Found: C,58.66; H, 3.67; N, 8.56: MS [M]+at m/z 492.5.(55%) (Ethanol) m.p. 236°C. IR (KBr): 3--2-Methyl-6-Bromo Quinazolin-4-one ( 21)νmax in cm−1: IR (KBr) νmax in cm−1: 3033 (CH aromatic), 2925 , 1705 (C=O), 1595 (C=N), 784 (C–Cl), 685 (C–S–C); 1HNMR (CDCl3) δ in ppm: 6.80–7.93 , 4.52 , 4.24  2.26 ;. Anal. Calcd. for C24H17N3O2SClBr: C,54.70; H, 3.23; N, 7.98: Found: C, 54.86; H, 3.22; N, 7.97: MS [M]+at m/z 526.5.(48%) (Methanol) m.p. 267°C. IR (KBr): 3--2-Methyl-6-Bromo Quinazolin-4-one ( 22)νmax in cm−1: IR (KBr) νmax in cm−1: 3031 (CH aromatic), 2928 , 1710 (C=O), 1605 (C=N), 685 (C–S–C); 1HNMR (CDCl3) δ in ppm: 6.81–7.92 , 4.59 , 4.25 , 3.49 , 2.26 ;. Anal. Calcd. for C25H20N3O3SBr: C, 57.47; H, 3.83; N, 8.05: Found: C, 57.62; H, 3.82; N, 8.03: MS [M]+at m/z 522.(55%) (Ethanol) m.p. 254°C. IR (KBr): 5–22) were screened for anti-inflammatory activity at 50 mg/kg p.o. using phenylbutazone as reference drug. All the tested compounds exhibited 15.1 to 32.5% edema inhibition at the dose of 50 mg/kg p.o. When the compounds were substituted with p-chlorophenyl group showed better anti-inflammatory activity than phenyl group. Compounds (5–10) showed anti-inflammatory from 15.1 to 20.4%. Compound 9, which was substituted by 2′-(p-chlorobenzylideneamino)phenyl group at the IIIrd position of quinazolinone moiety showed better anti-inflammatory activity (20.4%) than compounds 5, 6, 7, 8, and 10. Cyclizations of these compounds into azetidinones and thiazolidinones have shown better anti-inflammatory activity than corresponding arylidene derivatives. Azetidinones (11–16) and thiazolidinones (17–22) have shown the anti-inflammatory activity from (24.6–27.3%) and (22.9–32.5%) respectively. In azetidinones, compound 15 showed better anti-inflammatory activity at the dose of 50 mg/kg p.o. than compounds 11, 12, 13, 14, and 16. Compound 11 exhibited lower percentage inhibition of oedema (24.6%) among azetidinones. Thiazolidinones (17–22) generally showed better anti-inflammatory then corresponding azetidinones (11–16). Among all the tested compounds, 3--2-methyl-6-bromo quinazolin-4-one (21) showed better anti-inflammatory activity (32.5%). The anti-inflammatory activity of newly synthesis compounds is showed in 5–10) have shown moderate analgesic activity (11.6–19.3%). The compound 9 showed good analgesic activity (19.3%) then compounds 5, 6, 7, 8, and 10. The formation of azetidinones (11–16) and thiazolidinones (17–22) from compounds (5–10) showed better analgesic activity than corresponding arylidene derivatives. Among these, compound 21 showed the better analgesic activity (29.6%) than other tested compounds. The results of the biological study are depicted in Compounds  and analgesics (29.6%) activities than other compounds of series.


Their structures were elucidated based on the spectroscopic data. The two new isolates were no inhibitory activity for the acetylcholinesterase (AChE).Carinatine A ( Lycopodium alkaloids, possessing diverse skeletons and interesting biological activities, have attracted great interest from biogenetic, synthetic and biological perspectives + , indicating 8° of unsaturation. Analysis of the 1H and 13C NMR spectra of 1 revealed 16 carbon signals due to four quaternary carbons, five tertiary carbons, six methylenes, and one methyl group (Table 2 quaternary carbons were attributable to two amide groups (δC 165.0 and 156.2). The 1H-1H COSY cross-peaks of 1 disclosed the presence of three structural fragments, a (C-1–C-3), b (C-9–C-11), and c (C-6–C-8–C-15–C-14 and C-15–C-16), as shown in Fig. δC 165.0) and from H-2 to C-4 (δC 141.8) indicated the presence of a pyridine ring which was formed by the connectivities of C-1 and C-5 through a nitrogen atom. While the connectivities between C-9 (δC 59.0) and C-13 (δC 156.2) through a nitrogen atom was revealed by the HMBC cross-peak from H-9 to C-13. An HMBCs from H-7 to C-5 indicated the connectivity of C-5 and C-6. Finally, the connectivity of C-13 and C-14 and the connectivities of C-4, C-7, C-11, and C-13 through C-12 were elucidated by HMBCs from H-11 and H-14 to C-13 and from H-3, H-6, H-8, H-10, and H-14 to C-12. Thus, the gross structure of 1 was established , a white amorphous powder, had a molecular formula C16H23NO3 as established by HREIMS at m/z 277.1667 [M]+ , suggesting 6° of unsaturation. IR absorptions implied the existence of hydroxy (3425 cm−1) group. Analysis of the 1D and 2D NMR spectra revealed the existence of 16 carbons due to two carbonyl carbons (δC 220.5 and 214.0), two sp3 quaternary carbons (δC 62.4 and 66.0), three sp3 methines , eight sp3 methylenes, and one methyl , b (C-9–C-11), and c , as shown in Fig. δC 214.0) and from H-8, H-10, and H-11 to C-12 (δC 66.0) indicated the connection of C-13 with C-14 (δC 49.0), and the connections of C-7 (δC 40.0), C-11 (δC 33.2) and C-13 through C-12. While the connectivities of C-1 (δC 60.2) and C-9 (δC 76.8) through a nitrogen atom was revealed by HMBC from H-9 to C-1. At last, the HMBCs from H-3 and H-7 to C-5 combining with the HMBCs of H-2, H-3, H-6, H-7, and H-10 to C-4 (δC 62.4) constructed the linkage of C-5 (δC 220.5) and C-6 (δC 47.0) and the connections of C-3, C-5, C-9, and C-12 through C-4. Therefore, the planar structure of 2 was established as a new C16N-type Lycopodium alkaloid with a 5/5/6/6 tetracyclic ring system, the first derivative of lycojaponicumins C D26.6−94.4 ; UV (MeOH) λmax (log ε): 192 (3.54), 245 (3.86) nm; IR (KBr) νmax 3424, 2925 and 1630 cm−1; 1H and 13C NMR see Table m/z 257 [M + H]+; HREIMS m/z 256.1579.Colorless oil; D24.6−56.9 ; UV (MeOH) λmax (log ε): 201 (3.10) nm; IR (KBr) νmax 3425, 2927 and 1630 cm−1; 1H and 13C NMR see Table m/z 278 [M + H]+; HREIMS m/z 277.1667.White powder; [6−56.9 c .5, MeOH;


Cephalantheropsis gracilis afforded five new compounds: cephalanthrin-A (1), cephalanthrin-B (2), cephathrene-A (3), cephathrene-B (4), methyl 2-(aminocarbonyl)phenylcarbamate (5), and 52 known compounds. The structures of the new compounds were determined by spectroscopic analysis. Among the compounds isolated, tryptanthrin (6), phaitanthrin A (7), cephalinone D (19), and flavanthrin (30) showed significant cytotoxicity against MCF-7, NCI-H460, and SF-268 cell lines. Cephalantheropsis  belongs to the Orchidaceae family and is comprised of eight species distributed in Southeast Asia. The plant, Cephalantheropsis gracilis (Lindl.) Shiu-Ying Hu var. gracilis, is an orchid native to Taiwan and grows in forests at altitudes of 500–1500 m throughout the island quinazoline-6,12-dione , except that the carbonyl C-6 is replaced by a saturated quaternary carbon (δ 75.9). NMR spectroscopic data also allowed us to determine the remaining two substituents on C-6 being an –OH group (δ 3.81) and a –CH2COOH group ; δC 43.6 (C-1') and 170.9 (C-2')). The methylene protons adjacent to a quaternary carbon (C-6) could be split due to either chirality or steric hindrance. HMBC correlations from H-1 to C-12, H-7 to C-6 and H-1' to C-5a, C-6 and C-6a, as well as the NOE correlations between H-1' and H-7, supported a structure of 6-hydroxy-6-(carboxymethyl)-tryptanthrin for cephalanthrin-A (1).Cephalanthrin-A , also isolated as an optically active yellow amorphous powder, was determined to have a molecular formula of C18H14N2O4. By comparison of the 1H and 13C NMR spectra of 2 (6) and cephalanthrin-A (1), an oxygenated quaternary carbon (δ 87.4) was shown to replace the carbonyl C-12 to form a 12,12-disubstituted tryptanthrin  and methoxycarbonyl  substituents were also identified, and their presence was confirmed by HMBC correlations from both H-1 and 12-OCH3 to C-12 and the NOE correlations between H-1, H-10 and 1'-OCH3, 12-OCH3. Hence, a structure of 12-methoxy-12-(methoxycarbonyl)-tryptanthrin was deduced for cephalanthrin-B (2). Although compound 2 has been synthesized by Cornforth et al. [Cephalanthrin-B (tra of 2  with thotanthrin . Methoxy1 ([α]D +8.0°) and 2 ([α]D +3.0°), we suspected they might be not optically pure compounds. The configuration of compounds 1 and 2 has not been determined due to the isolation of insufficient amounts of these materials. However, we adopted the similar structure of phaitanthrin A (7) as a model. First, our attempts to synthesize a pair of diastereomeric esters by acylating 7 with (+)-α-methoxy-α-trifluoromethylphenylacetyl chloride [(+)-MTPACl] [d- and l-camphorato]europium(III) [(R)- and (S)-Eu(tfc)3] [Due to the small specific rotations of compounds -MTPACl] , even winthrin A  as a mod3) was isolated as a white amorphous powder with a molecular formula of C17H18O5 as determined by the molecular ion peak at m/z 302.1153 in HREIMS data. UV absorptions at 267 and 304 nm indicated the presence of a benzene system, and the IR spectrum revealed an OH absorption band at 3404 cm−1. From the 1H NMR spectrum, two mutually-coupled aromatic protons at δ 6.86  and 7.93  and a proton at δ 6.63  suggested the presence of 1,2,3,4-tetrasubstituted and pentasubstituted benzene rings, respectively  was assigned the structure 2,7-dihydroxy-3,4,8-trimethoxy-9,10-dihydrophenanthrene.Cephathrene-A  was isolated as a white amorphous powder. The HREIMS showed a molecular ion consistent with the molecular formula C18H20O6. The spectral data showed a resemblance to compound 3 .Cephathrene-B , 7.49 , 7.83 , and 8.38  for an o-disubstituted benzene. The HMBC correlations from H-3 to an amide carbon (δ 172.0) and from OCH3 (δ 3.71) to a carbamate carbon (δ 154.6) and the NOE correlation between H-6 and an amine proton (δ 11.29) suggested the structure of methyl 2-(aminocarbonyl)phenylcarbamate for 5. The 1H and 13C NMR spectra of 5 and the acylation product of o-aminobenzamide with methyl chloroformate (ClCO2CH3) were identical and further confirmed the structure of 5 (Equation (1)).Compound spectrum  showed rC. grilis including 7 quinazolines, tryptanthrin (6) D +8.0° ; UV λmax (log ε) CH3OH 206 (4.0), 261 (3.3), 302 (3.0), 314 (3.1), 329 (3.0) nm; IR νmax (KBr) 3000 (br), 1652, 1464 cm−1; EIMS m/z (rel. int.) 308 , 250 (100), 219 (8), 119 (19); HREIMS m/z 308.0794 [M]+ .Cephalanthrin-B (2). Yellow amorphous powder, mp 215–217 °C; [α]D +3.0° ; UV λmax (log ε) CHCl3 259 (3.2), 276 (3.0), 316 (2.9), 442 (2.9) nm; IR νmax (KBr) 1754, 1722, 1643, 1607, 1592 cm−1; EIMS m/z (rel. int.) 322 , 291 (5), 263 (100); HREIMS m/z 322.0955 [M]+ .Cephathrene-A (3). White amorphous powder, mp 96–98 °C; UV λmax (log ε) CHCl3 267 (3.3), 304 (3.2) nm; IR νmax (KBr) 3404, 1582, 1483, 1467 cm−1; EIMS m/z (rel. int.) 302 , 255 (35), 184 (10); HREIMS m/z 302.1153 [M]+ .Cephathrene-B (4). White amorphous powder; UV λmax (log ε) CHCl3: 265 (3.2), 316 (3.0) nm; IR νmax (KBr) 3400, 1606, 1501, 1464 cm−1; EIMS m/z (rel. int.) 332 , 331 (100), 302 (24), 285 (17); HREIMS m/z 332.1262 [M]+ .Methyl 2-(Aminocarbonyl)phenylcarbamate (5). White amorphous powder, mp 200–202 °C; UV λmax (log ε) CH3OH 212 (2.9), 228 (3.0), 257 (2.9), 289 (2.7), 311 (2.7) nm; IR νmax (KBr) 3417, 3211, 1726, 1686, 1626, 1598, 1531 cm−1; 1H NMR (acetone-d6) δ 3.71 , 6.99 and 7.74 , 7.05 , 7.49 , 7.83 , 8.38 , 11.29 ; 13C NMR (acetone-d6) δ 52.2 (OCH3), 119.2 (C-2), 119.6 (C-6), 122.2 (C-4), 129.1 (C-3), 133.4 (C-5), 141.7 (C-1), 154.6 (NC=O), 172.0 (2-C=O); EIMS m/z (rel. int.) 194 , 162 (32), 146 (100), 118 (9); HREIMS m/z 194.0693 [M]+ .The cytotoxicity assay was carried out according to the procedure described in the literature .1), cephalanthrin-B (2), cephathrene-A (3), cephathrene-B (4), methyl 2-(aminocarbonyl)phenylcarbamate (5), and 52 known compounds were isolated from Cephalantheropsis gracilis. Cephalinone D (19) showed the strongest cytotoxicity against the tested tumor cell lines, with IC50 values ranging from 7.6 to 42.9 μM. The modifications using Cephalinone D as template are being studied in our laboratories, aiming to discover the derivatives with strong anticancer activity.Five new compounds, cephalanthrin-A (


Castanea mollissima Blume (Chinese chestnut), as a food product is known for its various nutrients and functional values to the human health. The present study was carried out to analyze the anti-diabetic complications and anti-cancer activities of the bioactive compounds present in C. mollissima.C. Blume were extracted with 90% alcohol. The water suspension of these dried alcohol extracts were extracted using EtOAc and n-BuOH successively. The n-BuOH fraction of CI (CI-B) was isolated by silica gel column, Sephadex LH 20 column and preparative HPLC. The isolated compounds were identified by 1H-NMR, 13C-NMR, HMBC, HMQC and ESI-Q-TOF MS, All the fractions and compounds isolated were evaluated on human recombinant aldose reductase (HR-AR) assay, advanced glycation end products (AGEs) formation assay and human COLO 320 DM colon cancer cells inhibitory assay.The kernels (CK), shells (CS) and involucres (CI) parts of O-[6''-O-(E)-p-coumaroyl]-β-D-glucopyranoside (2), kaempferol-3-O-[6''-O-(E)-p-coumaroyl]-β-D-galactopyranoside (3), kaempferol-3-O-[2''-O-(E)-p-coumaroyl]-β-D-glucopyranoside (4), kaempferol-3-O--β-D-glucopyranoside (5) and kaempferol-3-O--β-D-galactopyranoside (6), casuariin (7), casuarinin (8) and castalagin (9). Compounds 2–9 were found to show higher activity than quercetin (positive control) in the AR assay. Compounds 3–6, 8, and 9 showed stronger inhibitory effects than amino guanidine (positive control) on AGEs production. Compounds 4–6, 7, and 8 showed much higher cytotoxic activity than 5-fluorouracil (positive control) against the human COLO 320 DM colon cancer cells.CI-B was found to show a significant inhibitory effect in above biological screenings. Six flavonoids and three polyphenolic acids were obtained from CI-B. They were identified as kaempferol (1), kaempferol-3-Castanea mollissima Blume.Our results suggest that flavonoids and polyphenolic acids possesses anti-diabetes complications and anti-cancer properties, and they were presumed to be the bioactive components of The online version of this article (doi:10.1186/1472-6882-14-422) contains supplementary material, which is available to authorized users. Castanea mollissima Blume (family Fagaceae), with the Chinese name “Ban li” (chestnut) is widely distributed in Asian areas, such as China, Korea and Viet Nam. Chestnut fruits are highly regarded and widely consumed throughout Asia, Europe and America. Various commercial forms are available. Ban li is not only used as a food product for its various nutrients, but also used as a traditional Chinese medicine, including the flowers, leaves, and twigs to have been used to treat gastroenteritis, bronchitis and regurgitation for hundreds years -β-D-galactopyranoside (6) was obtained as a yellow powder with a negative optical rotation. Its structure identification was achieved by mass and NMR spectroscopy . 1H- and 13C NMR spectral data of 6  with C-3 (104.65). The attachments for two p-coumaroyl groups at C-2″ and C-6″ were based on correlations between δH 5.38  with δC 168.23 (C-9″′), and, δH 4.19, 4.36  with δC 168.21 (C-9""), respectively. The trans form configurations for their double bonds at C-7''', 8''' and C-7'''', 8'''' positions were confirmed by the coupling constant values 15.6 Hz for H-7''' and H-7'''' -β-D-galactopyranoside at finally.Kaempferol-3-n-BuOH and water part) of kernels , shells  and involucres  were evaluated on AR, AGEs and the growth inhibitory activity aginst human COLO 320 DM colon cancer cells. Among these fractions, the n-BuOH soluble fraction of involucres (CI-B) was found to be significantly more active than others , kaempferol-3-O-[6"-O-(E)-p-coumaroyl]-β-D-glucopyranoside (2), kaempferol-3-O-[6"-O-(E)-p-coumaroyl]-β-D-galatopyranoside (3), kaempferol-3-O-[2"-O-(E)-p-coumaroyl]-β-D-glucopyranoside (4), kaempferol-3-O--β-D-glucopyranoside (5), kaempferol-3-O--β-D-galactopyranoside (6), and three polyphenolic acids, casuariin (7), casuarinin (8), and castalagin (9) were obtained. Structures of compounds -p-coumaroyl]-β-D-glucopyranosidekaempferol-3-(2). Yellow powder; m.p.223-225°C; [α]D23 -39.0° . UV λmax (MeOH) nm (logϵ): 316 (2.25). FAB-MS m/z: 595 [M + H]+ and 593 [M-H]-. 1H-NMR and 13C-NMR spectra data see Table O-[6''-O-(E)-p-coumaroyl]-β-D-galatopyranosidekaempferol-3-3). Yellow powder (40.0 mg). m.p.245-247°C. [α]D22 -43.0° . UV λmax (MeOH) nm (logϵ): 318 (2.09). FAB-MS m/z: 595 [M + H]+ and 593 [M-H]-. 1H-NMR and 13C-NMR spectra data see Table . Yellow O-[2''-O-(E)-p-coumaroyl]-β-D-glucopyranosidekaempferol-3-4). Yellow powder. m.p.245-247°C. [α]D20 -74.1° . UV λmax (MeOH) nm (logϵ): 316 (2.74). FAB-MS m/z: 595 [M + H]+ and 593 [M-H]-. 1H-NMR and 13C-NMR spectra data see Table -p-coumaroyl]-β-D-glucopyranosidekaempferol-3-5). Yellow powder. m.p.196-199°C. [α]D22 -119.6° . UV λmax (MeOH) nm (logϵ): 330 (2.38). FAB-MS m/z: 741 [M + H]+. 1H-NMR and 13C-NMR spectra data see Table -p-coumaroyl]-β-D-galactopyranosidekaempferol-3-6). Yellow powder. m.p.200-203°C. [α]D22 -62.2° . UVλmax (MeOH) nm (logϵ): 323 (2.51); FAB-MS m/z: 741 [M + H]+, 739 [M-H]- and 763 [M + Na]+; ESI-Q-TOF MS m/z 741.1824 [M + H]+ . 1H-NMR, 13C-NMR and 2D-NMR spectra data see Table . Off-white amorphous powder. m.p.245-248°C. FAB-MS m/z: 783 [M-H]-. 1H-NMR ((CD3)2CO + D2O) δ: 6.81 , 6.62, 6.47 , 5.64 , 4.72 , 5.51 , 5.13 , 4.18 , 3.93 , 4.72 . 13C-NMR ((CD3)2CO + D2O) δ: 115.9 (s), 115.6 (s), 115.6 (s), 114.5 (s), 126.8 (s), 126.5 (s), 124.6 (s), 119.4 (s), 116.6 (d), 108.2 (d), 106.6 (d), 104.8 (d), 145.5 (s), 145.2 (s), 144.7 (s), 144.6 (s), 143.9 (s), 143.8 (s), 143.2 (s), 143.0 (s), 138.3 (s), 136.2 (s), 135.2 (s), 134.4 (s), 169.8 (s), 169.3 (s), 168.3 (s), 165.3 (s), 66.6 (d), 76.6 (d), 70.3 (d), 76.5 (d), 67.8 (d), 67.9 (q).(casuarinin8). Off-white amorphous powder. m.p.244-246°C. FAB-MS m/z: 935 [M-H]-. 1H-NMR ((CD3)2C O + D2O) δ: 6.91 , 6.64 , 6.57 , 7.16 , 5.70 , 4.73 , 5.47 , 5.52 , 5.40 , 4.20 , 4.89 . 13C-NMR ((CD3)2CO + D2O) δ: 115.6 (s), 115.4 (s), 115.3 (s), 114.3 (s), 126.3 (s), 125.8 (s), 123.8 (s), 119.1 (s), 117.0 (s), 108.2 (d), 106.7 (d), 105.0 (d), 145.2 (s), 145.1 (s), 144.6 (s), 144.6 (s), 143.9 (s), 143.2 (s), 142.7 (s), 138.3 (s), 136.4 (s), 135.3 (s), 134.3 (s), 169.2 (s), 169.1 (s), 168.4 (s), 165.6 (s), 119.6 (s), 109.8 (d), 139.0 (s), 166.0 (s), 66.1 (d), 76.5 (d), 69.2 (d), 73.6 (d), 70.5 (d), 64.3 (q).. Off-white amorphous powder. m.p.243-245°C. FAB-MS m/z: 935 [M + H]+ and 933 [M-H]-. 1H-NMR((CD3)2CO + D2O) δ: 6.66 , 6.65 , 6.52 , 5.57 , 4.97 , 4.92 , 5.04 , 5.44 , 3.94 , 4.80 . 13C-NMR ((CD3)2CO + D2O) δ: 121.3 (s), 124.4 (s), 124.4 (s), 125.8 (s), 126.9 (s), 107.2 (d), 107.9 (d), 108.4 (d), 115.7 (s), 115.5 (s), 146.1 (s), 144.9 (s), 144.7 (s), 144.6 (s), 144.2 (s), 144.0 (s), 143.9 (s), 143.8 (s), 143.6 (s), 143.1 (s), 137.5 (s), 136.2 (s), 136.0 (s), 135.4 (s), 134.5 (s), 112.4 (s), 113.9 (s), 113.9 (s), 114.5 (s), 115.4 (s), 168.9 (s), 166.9 (s), 166.2 (s), 165.1 (s), 164.7 (s), 66.0 (d), 73.8 (d), 66.6 (d), 68.9 (d), 70.9 (d), 65.2 (q). and LZ (Libin Zhan) are associate professor and professor at Dalian medical University, respectively, Academy of Integrative Medicine and the Second Affiliated Hospital. HG and LW are associate professor and professor at Shenyang pharmaceutical University, respectively, Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education. MB, YO and TO are associate professor, professor and professor at Meiji Pharmaceutical University, respectively, Department of Natural Medicine and Phytochemistry.The 1H-NMR spectrum of compound 6.(JPEG 41 KB)Additional file 1:The 13C-NMR spectrum of compound 6.(JPEG 47 KB)Additional file 2:The 13C-NMR DEPT spectrum of compound 6.(JPEG 1 MB)Additional file 3:The HMBC spectrum of compound 6.(JPEG 832 KB)Additional file 4:The HMQC spectrum of compound 6.(JPEG 778 KB)Additional file 5:The 1H–1H NOESY spectrum of compound 6.(JPEG 1 MB)Additional file 6:The Mass spectrum of compound 6.(JPEG 72 KB)Additional file 7:


Neosartorya pseudofischeri was isolated from Acanthaster planci from the South China Sea. In a preliminary bioactivity screening, the crude methanol extract of the fungal mycelia showed significant inhibitory activity against the Sf9 cell line from the fall armyworm Spodoptera frugiperda. Five novel compounds, including 5-olefin phenylpyropene A (1), 13-dehydroxylpyripyropene A (4), deacetylsesquiterpene (7), 5-formyl-6-hydroxy-8-isopropyl-2- naphthoic acid (9) and 6,8-dihydroxy-3--penta-1,3-dien-1-yl)isochroman-1-one (10), together with eleven known compounds, phenylpyropene A (2) and C (3), pyripyropene A (5), 7-deacetylpyripyropene A (6), -1,8a-dihydroxy-2-acetoxy-3,8-dimethyl-5- (prop-1-en-2-yl)-1,2,4a, 5,6,7,8,8a-octahydronaphthalene (8), isochaetominine C (11), trichodermamide A (12), indolyl-3-acetic acid methyl ester (13), 1-acetyl-β-carboline (14), 1,2,3,4-tetrahydro-6-hydroxyl-2-methyl-l,3,4-trioxopyrazino-indole (15) and fumiquinazoline F (16), were obtained. The structures of these compounds were determined mainly by MS and NMR data. The absolute configuration of 9 was assigned by the single-crystal X-ray diffraction studies. Compounds 1–11 and 15 showed significant cytotoxicity against the Sf9 cells from S. frugiperda.The marine fungus  Spodoptera frugiperda is a pest feeding on the gramineous plants occurring worldwide and causing serious damage to several economically-important crops, such as the maize, sorghum, sugarcane, as well as the cotton, cruciferous and Cucurbitaceae plants. Although other alternative methods have been investigated on the biological control, as well as the development of resistant transgenic plants, the control of S. frugiperda still relies mainly on the use of chemical pesticides, easily inducing resistance and causing severe damage to the environment and consumers. However, some natural products with higher selectivity and safety become an important potential source of new biopesticides for the control of S. frugiperda -indole (15) and fumiquinazoline F (16)  media . During e F (16) . Compoun1) was isolated as a yellowish oil. The molecular formula was established as C32H36O9 based on the analysis of 13C-NMR data and HR(+)ESIMS m/z 565.2448 [M + H]+ , implying fifteen degrees of unsaturation (−1) and a benzene ring . UV maxima at 232, 278 and 322 nm supported the long conjugated system containing a benzene ring. The 13C-NMR and DEPT spectra displayed six methyls, four methylenes, ten methines and twelve quaternary carbons. The diagnostic aryl protons at δH 7.81  and 7.44  revealed a monosubstituted benzene ring. Two alkenyl protons at δH 6.46 (s) and 6.36 (s) indicated there were two trisubstituted double bonds in the molecule. Three methyl group singlets at δH 2.16, 2.10 and 2.04 showed HMBC correlations with the carbonyl groups at δC 169.9, 171.1 and 170.4, respectively, confirming there are three acyloxy groups. The other three methyl group singlets at δH 1.57, 1.25 and 0.87 are connected with the quaternary carbons at δC 21.2, 24.2 and 13.3, respectively. In the 1H–1H COSY spectrum, the cross peaks of H-1/H-2, H-2/H-3, H-7/H-8, H-8/H-9, H-2′′(H-6′′)/H-3′′(H-5′′) and H-3′′(H-5′′)/H-4′′ revealed the partial structure of –CHCH2CH2–, –CHCH2CH– and –CHCHCHCHCH– in the molecule  was obtained as a white powder. It showed a molecular formula of C31H37NO9 determined by the 13C-NMR data and HR(+)ESIMS peak at m/z 568.2549 [M + H]+  (13C-NMR and DEPT spectra (5) , corresponding to one methine group  connected to one hydroxyl group in pyripyropene A  . The 13C spectra  displayectra (5)  17], a , a 4) waropene A . The relposition .6 was elucidated as 7-deacetyl pyripyropene A  in 80% methanol [Compound ropene A . 6 was omethanol . However7) was isolated as a yellowish oil. The molecular formula was determined as C15H24O3 from the 13C-NMR data and the HR(+)ESIMS peak at m/z 275.1603 [M + Na]+ , indicating four degrees of unsaturation  and the methine  formed a trisubstituted double bond. The quaternary carbon (δC 147.2) and the methylene  constructed a terminal double bond. Thus, Compound 7 must be bicyclic to account for the four double bond equivalents required by the molecular formula. The 1H–1H COSY cross peaks of H-1/H-2, H-4/H-4a, H-4a/H-5, H-5/H-6, H-6/H-7, H-7/H-8 and H-8/H-13 established two partial structures, –CHCH– and –CHCHCHCH2CH2CHCH3 (δC 74.6), C-2 (δC 74.2) and C-8a (δC 73.8). The NOESY correlations of H-1/H-12, H-7a/H-12, H-6e/H-12, H-2/H-4a, H-4a/H-5, H-4a/8a-OH and H-6a/H-13 revealing 1-OH, 8a-OH, H-5 and H-13 were α-oriented; however, 2-OH was β-oriented . The NMR spectroscopic data of Compound 8 were very similar to those of Compound 7, except that the C-2 hydroxyl group in 7 was replaced by an acetoxy group in 8  and O4-H...O3 (carbonyl) hydrogen bonds to form a zigzag chain running parallel to the b-axis. Neighboring chains of molecules are packed closely together with the hydrophobic methyl groups pointing towards each other. The absolute configuration of 8 was determined as 1S, 2R, 4aR, 5R, 8R, 8aR with the Flack parameter value −0.16(17) by single-crystal X-ray diffraction analysis -1,8a-dihydroxy-2-acetoxy- 3,8-dimethyl-5-(prop-1-en-2-yl)-1,2,4a,5,6,7,8,8a-octahydronaphthalene, was previously obtained as the degradation product of the natural product CJ-12662, which was obtained from the fermentation broth of Aspergillus fischeri var. thermomutatus ATCC 18,618 − , which required nine degrees of unsaturation (−1) and an aldehyde group (2812 cm−1). Two methyl groups at δH 1.45  showed 1H–1H COSY correlations with the methine group at δH 3.90  and formed an isopropyl fragment , 8.22  and 8.77  were located at the 1,3,4-position on a phenyl ring. HMBC correlations of H-1/C-2, H-1/C-8a, H-3/C-2, H-4/C-4a, H-4/C-5, H-7/C-5, H-7/C-6, H-11/C-7 and H-11/C-8 indicated the existence of a naphthyl ring, accounting for seven degrees of unsaturation. The remaining carboxyl group , the aldehyde , phenolic hydroxyl group ESIMS peak at m/z 245.0815 [M − H]+ , established the molecular formula as C14H14O4  and 6.18 , indicating the presence of a tetrasubstituted aromatic ring. Two phenolic hydroxyl groups at δH 11.06 (brs) were connected to C-6 (δC 163.4) and C-8 (δC 164.8). Additionally, two pairs of trans-oriented olefinic protons at δH 5.71 , 6.35 , 6.10 , 5.81 , one oxymethine at δH 5.13 , one methylene at δH 3.00 , 2.93  and one methyl group at δH 1.74  were connected in sequence and formed a pentadienyl group based on their 1H–1H COSY correlations. The remnant quaternary carbon (δC 169.1) connected with C-8a (δC 100.1) and the oxymethine and formed a lactone. The HMBC correlations of H-4/C-4a, H-4/C-8a, H-5/C-6, H-7/C-8 and H-7/C-8a revealed a 3,6,8-trisubstituted isocoumarin ring system -penta-1,3- dien-1-yl)isochroman-1-one.Compound C14H14O4 . The 13Cg system . The abs11)  and [14C] precursors. Pyripyropene A (5) is derived from three mevalonates, five acetates and one nicotinic acid. The pyridino-α-pyrone moiety is produced via condensation of a primer nicotinic acid with two acetates in a “head-to-tail” fashion; an all-trans farnesyl pyrophosphate is produced via the mevalonate pathway; the two parts are linked and cyclized to form the core skeleton, and then three acetyl residues from the acetates are introduced into the skeleton to yield 5 +. HR(+)ESIMS m/z 565.2448 [M + H]+ .5-Olefin phenylpyropene A (4): white powder; c = 0.1, CHCl3); white powder; UV (MeOH) λmax (ɛ) 216 , 246 , 278 (4038) nm; IR νmax 2960, 2921, 2851, 1713, 1464, 1377, 1260, 1035, 810, 721 cm−1. For 1H and 13C-NMR data, see m/z 568.2 [M + H]+. HR(+)ESIMS m/z 568.2549 [M + H]+ .13-Dehydroxylpyripyropene A (6): white powder; for 1H and 13C-NMR data, see 7-deacetylpyripyropene A (7): yellowish oil; c = 0.058, MeOH); UV (MeOH) λmax (ɛ) 212 (136) nm; IR νmax 3443, 2959, 2926, 2856 1721, 1675, 1609, 1455, 1376, 1260, 1095, 1029, 992, 887, 756 cm−1. For 1H and 13C-NMR data, see m/z 275.1603 [M + Na]+ .Deacetylsesquiterpene (8): white crystal. c = 0.079, MeOH); IR (MeOH) νmax 3677, 3436, 3317, 2928, 2862, 1731, 1642, 1444, 1405, 1369, 1238, 1030, 967, 945, 927, 885, 720, 609 cm−1. For 1H and 13C-NMR spectra, see m/z 294.1828 [M]+ . The crystal of 8 was obtained from MeOH solution. C17H26O4, M = 294.38, colorless block, Orthorhombic system, P 21 21 21 (19), a = 8.3364 (1), b = 9.3709 (2), c = 21.3150 (5) Å. V = 1665.12 (6) Å3, Z = 4. Dcalcd = 1.174 g/cm3, crystal size 0.41 mm × 0.45 mm × 0.43 mm, F(000) = 640, T = 173(2) K.-1,8a-dihydroxy-2-acetoxy-3,8-dimethyl-5-(prop-1-en-2-yl)-1,2,4a,5,6,7,8, 8a-octahydronaphthalene (9): white powder; IR νmax 3283, 3020, 2953, 2928, 2867, 2812, 2720, 1671, 1616, 1601, 1512, 1458, 1422, 1393, 1289, 1026, 1208, 1148, 1109, 1080, 1008, 985, 921, 796 cm−1; for 1H and 13C-NMR data, see m/z 257.0817 [M − H]− .5-formyl-6-hydroxy-8-isopropyl-2-naphthoic acid (10): white powder; c = 0.1, MeOH); UV (MeOH) λmax (ɛ) 232 (6312), 268 (3427), 302 (2060) nm; IR νmax 3528, 3400, 2953, 2926, 2855, 1656, 1622, 1460, 1374, 1247, 1110, 1003, 772 cm−1. For 1H and 13C-NMR data, see m/z 245.0815 [M − H]− .6,8-dihydroxy-3--penta-1,3-dien-1-yl)isochroman-1-one (S. frugiperda. Sf9 cells were maintained at 27 °C in TC-199-MK medium supplemented with 10% FCS (v/v), 1% L-glutamine 200 mmol/L and penicillin-streptomycin-neomycin solutions (v/v). The cell growth inhibition was measured by the MTT method. Cells were seeded in 96-well microtitration plates at the exponential growth phase. Different concentrations of compounds diluted with medium were added at their log-phase growth stage. Additionally, 0.2 μL of solvent (DMSO) only was added as the control (CK). The final concentration of solvent in the cultures assayed was 1%. Compounds were solubilized with DMSO at concentrations of 10 and 50 mg/L. The rotenone was used as the positive control. After 6 h, 12 h, 24 h and 48 h of treatment, 5 mg/mL MTT were dissolved in PBS, and 20 μL of this stock solution were added to the culture cells. After an additional 3 h of incubation, the medium was discarded, and the 96-well plates were dried in the air. Then, 100 μL of DMSO were added to dissolve the formazan crystals, and the absorbance was measured at 570 nm by a microplate reader .To evaluate the biological activities of these compounds, cytotoxic assays were carried out with insect cultured cell line Sf9 from The cytotoxic effect was expressed as the relative percentage of inhibition calculated as follows: cell growth inhibition rate (%) = [(A control − A treatment)/A control] × 100 .1, 4, 7, 9 and 10), were obtained from the mycelium of marine fungus N. pseudofischeri. Their structure types include phenylpyripyropenes, pyripyropenes, sesquiterpenoids, isocoumarin and alkaloids. Our findings provide further evidence that the marine fungus N. pseudofischeri has the tremendous potential of biosynthesis. The potent cytotoxicity of these compounds against the Sf9 cells revealed the prospect to develop biorational pesticides.In summary, sixteen compounds, including five new compounds (


The structures of all compounds were elucidated through spectroscopic analysis including 2D-NMR and HR-MS. The absolute configuration of these compounds was established by X-ray diffraction analysis, comparison of NOESY spectra and biogenetic means. The cytotoxities of compounds 2–8 were evaluated in vitro against HL-60, SMMC-7721, A549, MCF-7 and SW480 cell lines. Compounds 5–7 exhibited the highest activities with IC50 values of 11.8–30.1 μM in most cases. The SAR revealed that the introduction of hydroxyl group was able to significantly improve the activities of the compounds for most of the cell lines.Five new ( It is noteworthy that most of the natural dihydro-β-agarofurans originated mainly from plants of the Celastraceae family. Only a minority were found in other family plants such as Hippocrateaceae and Lamiaceae D21 +10.45° . The ions at m/z: 551 [M + H]+ in ESI-MS and 573.2107 [M + Na]+ in HR-ESI-MS of 3 afforded a molecular formula of C31H34O9. The IR showed the presence of hydroxyl (3493 cm−1), carbonyl  and aromatic groups .Compound 13C NMR and DEPT spectra of 3 indicated 27 carbon signals comprising three ester carbonyl carbons, one ketone carbonyl carbon, one quaternary sp3 carbon, three tertiary carbons including two ones linked to an oxygen atom, five secondary carbons including four ones linked to an oxygen atom, eight sp2 carbons including six ones linked to a hydrogen atom, one methylene carbons, and five methyl carbons , δC 20.0 (CH3CO2–), δC 169.5 (CH3CO2–); EI-MS m/z: 43 [Ac]+] and two benzoate esters .The  carbons . Its NMR1H and 13C NMR, DEPT and HSQC spectra showed the structural characteristics of a dihydro-β-agarofuran ketone skeleton consisting of four methyl carbons , one methylene carbon [δH 2.29  and 3.39 /δC 44.0], six methine carbons with four linked to an oxygen atom , three quaternary carbons with two linked to an oxygen atom  and one ketone carbonyl carbon (δC 204.6).Except for the signals of the ester groups, H 5.98 /δC 169.5 (CH3CO2–), between δH 6.27 /δC 165.5 (PhCO2–), and between δH 4.97 /δC 165.5 (PhCO2–) showed that the acetoxy group was at C-1 and two benzoyloxy groups were at C-6 and C-9, respectively. The correlation between δH 5.98  and δC 204.6 (C=O) as well as the lowfield chemical shift and singlet of H-1 showed that the 2 position was a ketone carbonyl group. The correlations between δH 4.97 , 2.72 /δC 74.5 (C-8) showed that the 8 site was a hydroxymethine group. The signal at δH 4.48 (s) was assigned to H-8 by the HSQC spectrum.In the HMBC spectrum , the corH 1.46  and/1.06 , 6.27 , 4.97  showed that 6-benzoyloxy group and 9-benzoyloxy group were equatorial and axial, respectively, and the correlations between δH 5.98 /3.39 , /8.03  showed that 1-acetoxy group was equatorial. The axial assignment of 8-OH was supported by the NOE effect between δH 4.48 / 1.50 . The above NOE cases were the same as that of 1, indicating that the space orientations of the substituents in 3 were identical to that of the substituents at the same site in 1. Thus, 3 was identified as -1-acetoxy-6,9-dibenzoyloxy-8-hydroxydihydro-β-agarofuran-2-one.In the NOESY spectrum , the cor4 was obtained as a white powder. Its molecular formula was established as C31H34O8 by HR-MS (m/z: 557.2144 [M + Na]+) and 13C NMR data, which had one less oxygen atom compared with that of 3. The IR spectrum showed the presence of carbonyl  and aromatic  functionalities. The UV spectrum exhibited an absorption maximum at 244, 274, 283 nm, suggesting the existence of aromatic moieties.Compound 13C NMR and DEPT spectra /δH 5.63 , between δC 165.2 (PhCO2–)/δH 5.08 ,/δH 8.05 , and between δC 169.5 (CH3CO2–)/δH 6.01 , /δH 1.73  suggested that the acetyl ester and the two benzoyl esters were positioned at C-1, C-6 and C-9, respectively.The carbonyl signals and regiosubstitution of three ester groups were assigned by HMBC . The corH 1.34, s)/H-9 , H-6 /H-14 , between Hax-3 /H-1 (δH 6.01), and between H-6 /H-7 , /Hax-8  showed that the acetyl group and the benzoate ester at the 6 position were equatorial, while Me-14, Me-15 and the benzoate ester at the 9 position were axial. These results were the same as that of 3. Taking into account biosynthetic relationship between 4 and 3, 4 was elucidated as -1-acetoxy-6,9-dibenzoyloxydihydro-β-agarofuran-2-one because the only difference of 4 from 3 is the absence of one hydroxyl group at the 8 position. The findings of 3 and 4 have a special significance; up to date, there have been few of examples of natural dihydro-β-agarofuran ketones.In the NOESY spectrum , the cor5 was isolated as a white solid, [α]D15 +43.34° . Its molecular formula was established as C31H36O8 by HR-MS (m/z: 559.2316 [M + Na]+) and 13C NMR data, which only has two additional hydrogen atoms than that of 4. The IR spectrum showed the presence of hydroxyl , carbonyl (1718 cm−1) and aromatic  functionalities. The 1H and 13C NMR spectra of 5 were similar to those of 4. Compared with 4, 5 only had one additional oxygenated methine group and one less ketone carbonyl group.Compound C 167.0 (PhCO2–)/δH 5.73 , 8.07 , between δC 166.8 (PhCO2–)/δH 5.02 , /8.03 , and between δC 172.2 (MeCO2–)/δH 5.44 , /1.69  suggested that one acetyl ester and two benzoyl esters were positioned at C-1, C-6 and C-9, respectively. In the 1H-1H COSY spectrum, the correlation between δH 4.42 /δH 5.44  revealed that the only hydroxyl group was at 2 position. The results above were further supported by the upfield chemical shifts of δH-1, δH-3, δH-4, δC-1, δC-3 and δC-4 in 5 relative to those in 4. Thus, 5 was identified as the derivative of ketone carbonyl reduction of 4.The regiosubstitution of three ester groups were determined by HMBC experiment . The corH 1.62, s)/H-14 , /H-6 , /H-9 , between H-1 /H-12 , /Hax-3  showed that the acetyl group at 1 position and the benzoate ester at 6 position were equatorial, while Me-14, Me-15 and the benzoate ester at 9 position were axial. NOE correlations between H-2 /H-1 , /Hax-3  showed that 2-OH was axial. As a result, compound 5 was elucidated as -1-acetoxy-6,9-dibenzoyloxy-2-hydroxydihydro-β-agarofuran. NOE correlations between H-15 , was assigned the molecular formula C33H38O10, as deduced from HR-MS data (m/z: 617.2377 [M + Na]+) and 13C NMR data. Its IR absorptions were indicative of the presence of hydroxyl (3511 cm−1), aromatic  and ester (1746 and 1722 cm−1) groups. The 1H and 13C NMR, EI-MS, IR and UV spectra displayed the presence of two acetate esters, two benzoate esters and one hydroxyl group. The 1H and 13C NMR spectra of 6 ], one additional acetate ester  and one less methylene.Compound tra of 6  were simH 5.42 , 1.49 /δC 171.7, between δH 5.49 , 1.92 /δC 172.0, between δH 6.30 , 7.97 /δC 167.0 (PhCO2–), between δH 4.93 , 7.91 /δC 166.8 (PhCO2–) revealed that two acetyl esters were located at C-1 and C-2, respectively, while two benzoyl esters were positioned at C-6 and C-9, respectively. The correlations between δH 4.93 /δC 75.2 (C-8) in the HMBC and the coupling between δH 2.46  and δH 4.20  in the 1H-1H COSY spectrum showed that the hydroxyl group was at 8 position.In the HMBC spectrum , the corH 5.52, d, J = 3.2 Hz), H-2 , H-6  and H-9 , the stereochemical assignments of C-1, C-2, C-4, C-6 and C-9 were in agreement with the stereochemistry observed at these positions in 5. NOE effect between H-8 /H-12 [δH 1.36 ] showed that the hydroxyl group at the 8 position was axial. Thus, 6 was elucidated as -1,2-diacetoxy-6,9-dibenzoyloxy-8-hydroxydihydro-β-agarofuran.Based on NOE effects  and the 7 was isolated as a white powder, [α]D15.2 +8.50° , possessed a molecular formula C31H36O9, as deduced from HR-MS data (m/z: 575.2269 [M + Na]+) and 13C NMR data. The IR spectrum showed the presence of hydroxyl (3503 cm−1), carbonyl (1721 cm−1) and aromatic  functionalities. The 1H NMR, 13C NMR, EIMS, IR and UV spectra displayed the presence of one acetate ester, two benzoate esters and two hydroxyl groups. The 1H and 13C NMR spectra , 1.97 /δC 172.6 (CH3CO2–), between δH 6.35 /δC 166.8 (PhCO2–), and between δH 4.98 /δC 166.7 (PhCO2–) showed that the acetyl ester and two benzoyl esters were located at C-2, C-6 and C-9, respectively. The splitting patterns and coupling constants between δH 4.45  and 5.25 , and between δH 4.31  and 2.50  in the 1H NMR spectrum showed that two hydroxyl groups were located at C-1 and C-8, respectively. The above results were further confirmed by HMBC spectrum -2-acetoxy-6,9-dibenzoyloxy-1,8-dihydroxydihydro-β-agarofuran.Compound  spectra  of 7, inspectrum . In the 8 was isolated as a white solid [α]D15 +11.80°  and had the molecular formula C31H36O9, as deduced from HR-ESI-MS data (m/z: 575.2271 [M + Na]+), which was identical to that of 7. The IR spectrum showed the presence of hydroxyl (3520 cm−1), aromatic  and carbonyl  groups. The 1H NMR  and 4.42  were assigned as H-1 and H-2, respectively. In the HMBC spectrum, the correlations between δH 5.37 (H-1), 1.69 /δC 172.2 (CH3CO2–) showed that the acetoxy group was located at one site.Compound e 1H NMR , 13C NMR-1-acetoxy-6,9-dibenzoyloxy-2,8-dihydroxydihydro-β-agarofuran.In the NOESY spectrum, the NOEs of ose of 7 . As the P. wightiana Wall, the compounds 2–8 were evaluated for in vitro cytotoxic properties against HL-60, SMMC-7721, A549, MCF-7 and SW480 cell lines by MTT assays. Because of poor solubility, compound 1 was not tested for cytotoxicity. The cytotoxicity data are shown in cis-diaminodichloroplatinum was used as a positive control.To explore the potential bioactivities of the isolates from 3, 5–8 exhibited moderate activities with IC50 values ranging from 11.8 to 30.6 μM, while 2 and 4 displayed poor activities with IC50 values >40 μM in most cases. It was worth noting that 4 showed higher activity against A-549 (IC50 = 17.4 μM). For each of the tested cell lines, 5, 6 or 7 were the most cytotoxic.All the test compounds showed the definite activities in varying degrees against all tested cell lines at the concentration of 40 μM. For most of the cell lines, compounds 3, 5–8) had greater cytotoxicity than the compounds without hydroxyl groups . The reduction of ketone carbonyl group in 4 to hydroxyl group such as 5 or the introduction of 8-OH into 4 such as 3 led to a significant improvement of activity, except against A-549 cells, showing that 2-OH and 8-OH were beneficial for the improvement of activity against most of the cell lines, and the presence of 2-carbonyl group was able to increase activity against A-549 cells. However, the 2,8-dihydroxy compound 8 did not demonstrate higher activity than 2-hydroxy compound 5 or 8-hydroxy compound 3. The higher activity of 7 than 8 showed that the combination of 1,8-dihydroxy groups led to higher activity than that of 2,8-dihydroxy groups. In addition, the 1,8-dihydroxy compound 7 demonstrated higher activity than the corresponding 1-acetoxy-8-hydroxy compound 6 against HL-60, MCF-7 and SW480 but lower activity against SMMC-7721 and A-549.Preliminary analysis of the structure–activity relationship revealed that the compounds with hydroxyl groups (−1) on a Bruker TENSOR 27 transform infrared spectrophotometer  with KBr disks. 1H NMR spectra at 400 or 500 MHz and 13C NMR spectra at 100 or 125 MHz were recorded with a Bruker AVANCE III spectrometer  with TMS as internal standard. Coupling constants were reported in hertz (Hz). Electrospray ionization (ESIMS) was measured on a 4000 QTRAP LC-MS instrument . MS spectra were obtained from a 4000 QTRAP LC-MS in MS mode. High-resolution mass spectra (HR-MS) were determined on a Thermo LTQ XL Orbitrap instrument . Analytic Preparative HPLC was carried out using a Shimadzu LC-20AD instrument  equipped with an Agilent ZORBAX Eclipse Plus C18 column  and SPD-20A UV/vis detector , and using methanol-water (70:30) as a mobile phase. Preparative HPLC was carried out using a Shimadzu LC-8A instrument  equipped with a SHIM-PACK PRC-ODS column  and SPD-M10Avp photodiode array detector and using methanol-water (80:20) as a mobile phase. Column chromatographic (CC) separations were performed using silica gel H60 (300–400 mesh) , and Sephadex LH-20  as packing materials.Melting points were determined on an XT-4 micro-melting point apparatus and were uncorrected. Optical rotations were measured with an Autopol V instrument . Infrared (IR) spectra were recorded in wave numbers  is deposited in botanic specimen center of Northwest A&F University, Yangling, China.The entire plants of Parnassia wightiana Wall and Wight and Arn were successively extracted with petroleum ether (bp 60–90 °C) at room temperature three times (3 × 72 h). After removal of solvent in vacuo, the black viscous residue (43.5 g) was treated with methanol to remove insoluble black oily deposits. The methanol-soluble fraction was subjected to silica gel column chromatography eluting with petroleum ether-ethyl acetate  and finally methanol (500 mL). Nine fractions (F1–F9) were obtained. Compound 1 was precipitated from F7 corresponding to the eluent of petroleum ether-ethyl acetate (1:1), and recrystallized in ethyl acetate to afford pure 1 (250 mg). The mother solution after removal of 1 was evaporated to dryness. The resulting residue (8.23 g) was chromatographed on a silica gel column eluting with petroleum ether-ethyl acetate  to afford six subfractions (F71–F76) by HPLC analysis. F73 (0.54 g) was separated by preparative HPLC using a gradient of MeOH-H2O  to yield 5 (76.8 mg), 6 (25.4 mg) and 3 (180.7 mg). The same steps as described for F73 were used to separate F74 (0.784 g), and yielded 8 (156.4 mg) and 7 (84.4 mg), and F6 (7.02 g) to afford 4 (14.0 mg), 2 (55.8 mg) and 3 (83.7 mg).Powdered and air-dried materials of 1: a colorless crystal; mp 236–238 °C; [α]D17 0.923° ; Its [α]Dt value was not provided in the literature + .Compound terature . UV  of 1 was grown by slow evaporation in AcOEt solution. Diffraction intensity data were acquired with a CCD area detector with graphite monochromated Cu Kα radiation (λ = 1.54184 Å). Crystal data for 1: C32H40O13, Mr = 632.64, trigonal, space group P31, Z = 3, a = 15.04493(18), b = 15.04493(18), c = 12.5329(2) Å, α = 90.00°, β = 90.00°, γ = 120.00°, V = 2456.76(6) Å3, T = 291(2) K, μ (Cu Kα) = 0.837 mm−1, 21,172 reflections measured, 6040 independent reflections (Rint = 0.0191), S = 1.029. The final R1 values were 0.0361 and Rw = 0.098 (I > 2σ (I)). The final R1 values were 0.0377 and Rw = 0.0997 . Flack parameter = −0.06(12). Crystallographic data for 1 have been deposited with the Cambridge Crystallographic Data Centre (deposition number CCDC 979627). Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB21EZ, UK.Crystallographic Data and X-ray Structure Analysis of 2: a white solid; [α]D21 +44.50 ; lit. [α]D14 +44.49°  [14a]; UV (CHCl3) λmax (log ε) 244 (3.68), 276 (3.12), 283 (3.04) nm; IR νmax (KBr) 3063, 2972, 2935, 1748, 1716, 1603, 1585, 1453, 1438, 1369, 1279, 1246, 1143, 1092, 1040, 1024, 876, 714 cm−1; 1H NMR  (ppm): δ 8.02 , 7.43–7.45 , 7.56 , 5.97 , 5.71 , 5.41 , 5.07 , 4.37 , 2.50–2.54 , 2.45–2.47 , 2.38–2.40 , 2.25 , 2.19 , 1.77–7.80 , 1.45 , 1.54, 2.08, 2.10, 2.25 , 1.43 , 1.18 ; 13C NMR  (ppm): δ 170.5, 170.0, 169.9, 169.3, (4 × Ac), 165.3 (C6H5CO), 133.5, 130.1, 129.1, 128.3, 89.2 (C-5), 82.7 (C-11), 78.1 (C-6), 71.5 (C-1), 69.51 (C-9), 69.45 (C-2), 65.5 (C-13), 53.3 (C-10), 48.8 (C-7), 34.9 (C-8), 33.1 (C-4), 30.8 (C-3), 30.3 (C-14), 25.9 (C-15), 21.35, 21.29, 21.2, 20.3, 17.8 (C-12); The NMR data were in agreement with the literature values [m/z: 574 [M]+, 559 [M − Me]+, 531[M − Ac]+, 105 [C6H5CO]+, 43 [Ac]+; (+)-ESI-MS m/z: 597 [M + Na]+, 539 [M + Na-Ac-Me]+.Compound 3: a white solid; mp 133–135 °C; [α]D21 +10.45° ; UV (MeOH) λmax (log ε) 202 (4.58), 232 (4.61), 274 (3.60), 280 (3.54) nm; IR νmax (KBr) 3493, 3063, 2970, 2930, 1752, 1724, 1601, 1452, 1386, 1370, 1271, 1231, 1107, 1070, 1025, 712 cm−1; 13C NMR and 1H NMR data, see m/z: 535 [M − Me]+, 105 [C6H5CO]+, 77 [C6H5]+, 43 [Ac]+; (+)-ESI-MS m/z: 551 [M + H]+, 573 [M + Na]+; (+)-HR-ESI-MS m/z: 573.2107 [M + Na]+ .Compound 4: a white powder; mp 120–122 °C; [α]D21 +13.23° ; UV (CHCl3) λmax (log ε) 244 (4.05), 275 (3.51), 283 (3.43) nm; IR νmax (KBr) 3062, 2966, 2930, 1753, 1720, 1601, 1451, 1387, 1314, 1273, 1229, 1105, 1070, 1025, 712 cm−1; 13C NMR and 1H NMR data, see m/z: 534 [M]+, 105 [C6H5CO]+, 77 [C6H5]+, 43 [Ac]+; (+)-HR-ESI-MS m/z: 557.2144 [M + Na]+ .Compound 5: a white solid; mp 110–112 °C; [α]D17 +43.34° ; UV (MeOH) λmax (log ε) 202 (4.75), 231 (4.87), 274 (3.69), 281 (3.60) nm; IR νmax (KBr) 3530 (br), 3063, 3016, 2970, 2928, 1718, 1602, 1585, 1480, 1452, 1387, 1368, 1276, 1176, 1149, 1107, 1069, 1025, 1014, 969, 712 cm−1; 13C NMR and 1H NMR data, see m/z: 536 [M]+, 105 [C6H5CO]+, 77 [C6H5]+, 43 [Ac]+; (+)-HR-ESI-MS m/z 559.2316 [M + Na]+ .Compound 6: a white powder; mp 129–131 °C; [α]D14.1 +11.63° ; UV (MeOH) λmax (log ε) 202 (4.68), 231 (4.68), 274 (3.61), 280 (3.54) nm; IR νmax (KBr) 3511 (br), 3062, 3032, 2972, 2930, 1746, 1722, 1602, 1585, 1452, 1387, 1367, 1273, 1178, 1107, 1069, 1024, 983, 892, 712 cm−1; 13C NMR and 1H NMR data, see m/z: 579 [M − Me]+, 105 [C6H5CO]+, 77 [C6H5]+, 43 [Ac]+; (+)-HR-ESI-MS m/z: 617.2377 [M + Na]+ .Compound 7: a white powder; mp 128–130 °C; [α]D15.2 +8.50° ; UV (MeOH) λmax (log ε) 202 (4.66), 231 (4.70), 273 (3.61), 280 (3.54) nm; IR νmax (KBr) 3503, 3062, 3034, 3010, 2970, 2930, 1721, 1602, 1584, 1452, 1386, 1367, 1316, 1274, 1231, 1177, 1144, 1109, 1069, 1027, 982, 712 cm−1; 13C NMR and 1H NMR data, see m/z: 537 [M − Me]+, 430 [M − C6H5CO2H]+, 105 [C6H5CO]+, 77 [C6H5]+, 43 [Ac]+; (+)-HR-MS m/z: 575.2269 [M + Na]+ .Compound 8: a white solid; mp 134–136 °C; [α]D15 +11.80° ; UV (MeOH) λmax (log ε) 202 (4.76), 231 (4.81), 274 (3.86), 313 (3.25) nm; IR νmax (KBr) 3520 (br), 3063, 2972, 2929, 1746, 1722, 1602, 1585, 1452, 1370, 1316, 1274, 1178, 1146, 1097, 1070, 1027, 1007, 982, 712 cm−1; 13C NMR and 1H NMR data, see m/z: 105[C6H5CO]+, 43[Ac]+; (+)-HR-ESI-MS m/z: 575.2271 [M + Na]+ .Compound 2 in RPMI 1640 medium supplemented with 10% fetal serum and dispersed in replicate 96-well plates. Eight concentrations of test compounds (dissolved in 0.5% DMSO) encompassing a 128-fold range were added to each cell line. After 48 h exposure to the compounds, cells’ viability was determined by the [3--5(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium] (MTS) cytotoxicity assay by measuring the absorbance at 490 nm with a microplate spectrophotometer. Each test was performed in triplicate. The IC50 was defined as the concentration of the test compound resulting in a 50% reduction of absorbance compared to untreated cells in the MTT assay. The anticancer agent DDP (cis-diamminedichloroplatinum) and 0.5% DMSO were used as the positive control and solvent control, respectively. The cytotoxicity assay was performed according to the modified MTT colorimetric method . HL-60 cParnassia wightiana and evaluated their cytotoxicities in vitro against five cancer cell lines. The structures of all the compounds were established through spectroscopic analysis, and their absolute configurations were established by X-ray diffraction analysis and biogenetic means. For most of tested cell lines, 3, 5, 6 and 7 exhibited moderate activity with IC50 values of 11.8–30.1 μM. In addition, the structure–activity relationship was discussed as well.In conclusion, we isolated five new and three known dihydro-β-agarofuran sesquiterpene polyesters from