Their structures were elucidated by 1D and 2D NMR spectroscopy, as well as HR-EI-MS data.Three new lanostanoid triterpenes—ganotropic acid ( Ganoderma, the major genus in the family Ganodermataceae, are widely used to cure various chronic diseases such as hypertension, diabetes, hepatitis and cancers +). The IR spectrum revealed the presence of hydroxyl (3743 and 3442 cm−1), carbonyl (1768 cm−1) and double bond (1652 cm−1) absorptions. The 1H-NMR spectrum , 1.01 , 1.02 , 1.14 , 1.24 , 1.26 , 1.32 ). The 13C-NMR and DEPT spectroscopic data (1 had the lanostane skeleton as ganoderic acid C2 +), as well as its 13C NMR and DEPT spectroscopic data ] with C-26 (δC 177.6) and C-22 (δC 46.7). Compound 2 had the same basic lanostane configuration as that of ganoderic acid C2. The β-orientations of 3-OH and 7-OH were determined by ROESY correlations of H-3 [δH 3.15 dd ] with H-5, and H-7 [δH 4.53 dd ] with Me-28. The α-orientation of 15-OH was assigned from ROESY correlation of H-15 [δH 4.77 dd ] with Me-18. Based on above evidence, compound 2 was elucidated as 3β,7β,15α,24-tetrahydroxy-11,23-dioxo-lanost-8-en-26-oic acid.Compound pic data  revealin3 was assigned the molecular formula C30H44O8 by analyses of its HREIMS (m/z 532.3030 [M]+) and 13C-NMR spectroscopic data revealing 30 carbon resonances. The 1H-NMR spectrum . Resemblance of its NMR spectroscopic data (C 66.2 (C-28) in compound 3 instead of a methyl in 3β,7β,15β-trihydroxy-11,23-dioxolanost-8,16-dien-26-oic acid, indicating that compound 3 was derived from the latter with an additional hydroxyl group attached to C-28. This was further confirmed by the HMBC correlation of H-29  with C-28. The stereo-configuration of compound 3 with a lanostane skeleton was confirmed by ROESY experiments. The β-orientations of 3-OH and 7-OH were determined by cross-peaks of H-3/H-5, H-7/H-5 and H-5/H-28 and the α-orientation of 15-OH was deduced by a H-15/H-18 cross-peak in the ROESY experiment. Accordingly, the structure of compound 3 was identified as 3β,7β,15α,28-tetrahydroxy-11,23-dioxo-lanost-8,16-dien-26-oic acid.Compound spectrum  of compopic data  with tho1–3 against AChE were tested using a spectrophotometric method + 539 (100); HREIMS .White power;c 0.2, MeOH); UV (MeOH) λmax (log ε) 253 (2.24), 201 (2.69); IR (KBr) vmax cm−1 3734, 2973, 1701, 1684, 1541, 1457, 1396, 1062, 992, 669; for 1H- and 13C-NMR spectroscopic data, see m/z [M+Na]+ 557 (100); HREIMS .Yellow oil;c 0.2, MeOH); UV (MeOH) λmax (log ε) 255 (2.67), 205 (3.35); IR (KBr) vmax cm−1 3730, 2926, 1734, 1652, 1541, 1457, 1396, 873, 669 ; for 1H and 13C NMR spectroscopic data, see m/z [M+Na]+ 555 (100); HREIMS .Yellow oil;2HPO4  and NaH2PO4  were mixed to prepare phosphate buffer . Compounds were dissolved in DMSO (2% in PB). The reaction mixture contained PB (110 μL), test compound solution  and acetyl cholinesterase solution , which were mixed and incubated for 20 min (30 °C). The reaction was initiated by the addition of DTNB  and acetylthiocholine iodide . The hydrolysis of acetylthiocholine was monitored at 405 nm every 30 s. Tacrine was used as positive control. All reactions were performed in triplicate. The percentage inhibition was calculated as follows: % age inhibition = (E − S)/E × 100 (E is activity of the enzyme without test compound and S is the activity of enzyme with test compound).AChE inhibitory activity of the three compounds was assayed by the spectrophotometric method developed by Ellman ,20. AcetG. tropicum led to the isolation of three new lanostanoid triterpenes: ganotropic acid (1), 3β,7β,15α,24-tetrahydroxy-11,23-dioxo-lanost-8-en-26-oic acid (2) and 3β,7β,15α,28-tetrahydroxy-11,23-dioxo-lanost-8,16-dien-26-oic acid (3). Ganotropic acid possessed two oxygenic five-membered rings system in the side chain of lanostane skeleton. The evaluation on activities of these new compounds against AChE showed no significant inhibitory activity.The present study of the mushroom


Juss. The structures of the new compounds 1–6 were confirmed by spectroscopic methods, including IR and HRESIMS, as well as 1D and 2D NMR, and by comparisons with the data of known analogues. All compounds were tested for α-glucosidase and acetylcholinesterase inhibitory activities. However, none of the compounds was active against α-glucosidase and acetylcholinesterase in vitro.Six new phragmalin limonoids, named moluccensin Z1 ( Chukrasia tabularis A. Juss (Meliaceae) are distributed over the tropical areas of Asia, and its root bark has been used as a traditional medicine for dispelling wind and heat from the body in the Hainan province of China for a long time  to obtain compound 1  and compound 2 , respectively. Fr.15-9-2-4 (400 mg) was chromatographed on a reversed-phase C18 column eluted with MeOH-H2O (from 5:6 to 7:3) to give six subfractions (Fr.15-9-2-4-1–Fr.15-9-2-4-6). Fr.15-9-2-4-6 (8 mg) was separated by preparative HPLC  to obtain compound 3 . Fr.15-9-2-3 (31 mg) was separated by preparative HPLC  to obtain compound 4 . Fr.15-9-2-8 (170 mg) was chromatographed on a reversed-phase C18 column eluted with MeOH-H2O (from 6:4 to 7:3) to give six subfractions (Fr.15-9-2-8-1–Fr.15-9-2-8-6). Fr.15-9-2-8-5 (16 mg) was further separated by preparative HPLC  to obtain compound 5 . Fr.15-9-2-11 (300 mg) was chromatographed on silica gel eluted with a petroleum ether–CHCl3–isopropanol system  to yield three subfractions (Fr.15-9-2-11-1–Fr.15-9-2-11-3). Fr.15-9-2-11-2 (78 mg) was further separated by preparative HPLC  to obtain compound 6 . Fr.15-9-2-11-3 (58 mg) was separated by preparative HPLC  to obtain compound 8 . Fr.15-9-2-5 (770 mg) was first subjected to a reversed-phase C18 column eluted with MeOH-H2O (from 5:5 to 7:3) to give eight subfractions (Fr.15-9-2-5-1–Fr.15-9-2-5-8). Fr.15-9-2-5-6 (88 mg) was separated by preparative HPLC  to obtain compound 7 .The dried stems of Moluccensin Z1 (1): White amorphous powder; m.p. 153–157 °C; c 0.08, MeOH); IR (KBr) νmax 3528, 2924, 1731, 1457, 1372, 1260, 1094, 801, 736 cm−1; 1H- and 13C-NMR data see m/z 711.2274 [M + Na]+ .Moluccensin Z2 (2): White amorphous powder; m.p. 155–157 °C; c 0.08, MeOH); IR (KBr) νmax 3545, 2925, 1734, 1458, 1371, 1260, 1027, 801, 737 cm−1; 1H- and 13C-NMR data see m/z 711.2260 [M + Na]+ .Carapanolide Y (3): White amorphous powder; m.p. 120–123 °C; c 0.08, MeOH); λmax (log ε) 306 (2.61) nm; IR (KBr) νmax 3557, 2924, 1736, 1467, 1372, 1260, 1026, 801 cm−1; 1H- and 13C-NMR data see m/z 797.3017 [M + Na]+ .Tabulalin N (4): White amorphous powder; m.p. 162–164 °C; c 0.20, MeOH); λmax (log ε) 264 (2.92) nm; IR (KBr) νmax 2923, 1750, 1372, 1217, 1026, 801 cm−1; 1H- and 13C-NMR data see m/z 825.2588 [M + Na]+ .Chukvelutilide A1(5): White amorphous powder; m.p. 139–142 °C; c 0.08, MeOH); λmax (log ε) 203 (3.86), 237 (3.32), 268 (3.57) nm; IR (KBr) νmax 3451, 2918, 1743, 1373, 1218, 1026, 801 cm−1; 1H- and 13C-NMR data see m/z 911.2932 [M + Na]+ .Velutinasin J (6): White amorphous powder; m.p. 169–171 °C; c 0.20, MeOH); λmax (log ε) 234 (3.43), 269 (3.80) nm; IR (KBr) νmax 3481, 2923, 1748, 1604, 1371, 1224, 1027, 800 cm−1; 1H- and 13C-NMR data see m/z 893.2822 [M + Na]+ .


Their structures were elucidated by means of various spectroscopic analyses. The cytotoxicities of 1 and 2 were tested by the MTT method, and both compounds showed no significant cytotoxic activities against the A549 and A2780 human cancer cell lines. This is the first time that aryltetralin-type lignans were isolated from the genus Dolomiaea.Two new aryltetralin-type lignans, dolomiaeasin A ( Dolomiaea souliei (Franch.) Shih belongs to the Dolomiaea genus in the family Compositae, and is mainly distributed in western Sichuan and eastern Tibet −4.0° . The HR-ESI-MS spectrum  indicated the molecular formula of 1 to be C20H24O8. The 1H and 13C-NMR (APT) data of 1 showed the presence of a 1,3,4-trisubstituted benzene moiety , a 1,2,4,5-tetrasubstituted benzene moiety , two methoxyl groups  and other aliphatic signals . The NMR signals were assigned with the aid of HSQC and HMBC spectra, and cross-peaks observed in the HMBC  indicated that 1 resembled the structure of (+)-cycloolivil  being very similar to that of (+)-cycloolivil 6-O-β-D-glucoside which has the same chiral centers −16.3° . The HR-ESI-MS spectrum  indicated the molecular formula of 2 to be C20H24O8. The NMR signals of 2 were assigned with the aid of HSQC and HMBC spectra and by comparison with the signals of 1. The spectroscopic data of 2 suggested that it was another aryltetralin-type lignin, exhibiting an identical skeleton of 1.Compound 2. A positive Cotton effect at 291 nm revealed that H-7' was β (R conﬁguration at C-7') : −4.0° ; UV λmax (log ε) nm (MeOH): 207 (4.46), 283 (3.60); CD nm (Δε) : 290 (−1.8), 271 (+0.5), 237 (+0.7); IR νmax cm−1 (KBr): 3392, 2928, 1647, 1516, 1445, 1383, 1261, 1126, 1100, 1033, 798, 762, 652, 601; 1H-NMR  δ: 6.86 , 3.76 , 6.76 , 6.78 , 4.38 , 3.49 , 3.59 , 6.16 , 3.82 , 6.66 , 2.56 , 3.34 , 3.85 ; 13C-NMR  δ: 134.0 (C-1'), 117.0 (C-2'), 148.7 (C-3'), 56.6 (3'-OCH3), 146.6 (C-4'), 115.5 (C-5'), 126.2 (C-6'), 48.6 (C-7'), 75.1 (C-8'), 65.0 (C-9'), 126.7 (C-1), 132.7 (C-2), 118.0 (C-3), 145.4 (C-4), 147.7 (C-5), 56.7 (5-OCH3), 113.2 (C-6), 37.0 (C-7), 76.2 (C-8), 68.3 (C-9).Dolomiaeasin B (2): HR-ESI-MS spectrum ; [α]: −16.3° ; UV λmax (log ε) nm (MeOH): 210 (4.8), 284 (3.99); CD nm (Δε) : 291 (+3.1), 273 (−1.2), 230 (+1.9); IR νmax cm−1 (KBr): 3419, 2954, 1652, 1520, 1456, 1373, 1260, 1127, 1097, 1033, 803, 773, 645, 597; 1H-NMR  δ: 6.78 , 3.78 , 6.72 , 6.63 , 4.06 , 3.55 , 3.96 , 6.30 , 3.83 , 6.67 , 3.02 , 3.34 , 3.91 ; 13C-NMR  δ: 133.3 (C-1'), 117.0 (C-2'), 148.4 (C-3'), 56.6 (3'-OCH3), 146.7 (C-4'), 115.5 (C-5'), 125.7 (C-6'), 56.3 (C-7'), 77.1 (C-8'), 67.0 (C-9'), 127.5 (C-1), 131.4 (C-2), 117.6 (C-3), 145.8 (C-4), 148.2 (C-5), 56.6 (5-OCH3), 112.9 (C-6), 39.2 (C-7), 77.6 (C-8), 67.7 (C-9).1 and 2 were assessed by the MTT method using the A549 and A2780 human cancer cell lines. Cells were seeded in 96-well plates and incubated at 37 °C, 5% CO2 for 24 h. Then 150 μL of five different concentrations  for each compound (dissolved in DMSO) were added to each well and incubated for another 24 h. After removing the supernatant, 150 μL of MTT (0.5 mg/mL) were added to each well and incubated for 4 h. Finally, the liquid in the wells was removed, DMSO (150 μL) was added, and the absorbance at 570 nm was recorded on a microplate reader .Compounds 1) and dolomiaeasin B (2), were isolated from the roots of Dolomiaea souliei. Both compounds showed no significant cytotoxicities against the A549 and A2780 human cancer cell lines. To the best of the authors’ knowledge, this is the first report of aryltetralin-type lignans from the genus Dolomiaea.Two new aryltetralin-type lignans, dolomiaeasin A (


The structures of 1 and 2 were elucidated on the basis of extensive spectroscopic analysis and comparisons with related known compounds. Antibacterial activities of two new compounds and four known compounds were tested.Two new lanostane triterpenoids, 20-hydroxyeuphorbol-7-one ( Polyathia in the world, of which only 17 species are distributed in China and Polyalthia obliqua is one of the seven Polyalthia species growing on Hainan Island, P.R. China. The genus is known for its folk medicine applications against a number of ailments such as stomach ache, dysmenorrhea and pharynx neurosis + indicative of the molecular formula of C31H50O3, indicating seven degrees of unsaturation. Its IR spectrum exhibited the presence of hydroxyl  and carbonyl groups .Compound 1H-NMR spectrum, six methyl singlet signals at δH 0.83 , 0.88 , 0.94 , 1.00 , 1.16  and 1.28 , two methyl doublet signals at δH 1.01  and 1.03 , one oxymethine signal at δH 3.26  and two olefinic proton signals at δH 4.67  and 4.73 , and a series of overlapped signals were observed. The 13C-NMR and DEPT spectra of 1 displayed 31 carbon signals which were assigned to eight methyls, ten methylenes, four methines, and nine quaternary carbons (including one carbonyl group at δC 199.2). The two olefinic carbon signals at δC 106.5 (CH2) and 156.3 (C) were characteristic of the exomethylene moiety. The other two olefinic carbon signals appearing at δC 138.5 (C) and 164.8 (C) corresponded to one double bond. The 13C-NMR chemical shifts at δC 75.3 (C) and 78.1 (CH) confirmed the presence of two oxygenated carbons. The aforementioned data indicated that 1 was most likely a lanostane triterpenoid with a C9 side-chain moiety at C-17 containing a CH2 substituent at C-24 , indicating a hydroxyl group was located at C-20. The location of the hydroxyl group at C-20 was further confirmed by the HMBC correlations from H-17, C-21, C-22 to C-20. The planar structure of 1 was further determined by the 2D NMR spectra including 1H-1H COSY and HMBC experiments  between H-3 and Hβ-2 clearly indicated that the 3-OH group was in equatorial β-position. The NOESY correlations , indicating eight degrees of unsaturation. Its IR spectrum showed an absorption at 3441 cm−1 for hydroxyl groups and 1669 cm−1 for carbonyl. The general features of its NMR spectroscopic data (β)-3-hydroxy-24-methylenelanost-8-ene-7,11-dione  in 2 instead of a methylene signal at δH 2.13–2.16  in (3β)-3-hydroxy-24-methylenelanost-8-ene-7,11-dione, and in the 13C-NMR spectrum a methine group at δC 72.3 (CH) for C-15 was observed in 2, instead of a methylene group at δC 32.2 for C-15 in (3β)-3-hydroxy-24-methylenelanost-8-ene-7,11-dione. These results suggested that there was a hydroxyl group at C-15. The location of the hydroxyl group at C-15 was confirmed by the HMBC correlation of H-15 to C-30. The gross structure of 2 was further confirmed by 1H-1H COSY and HMBC spectra  between H-3 and Hβ-2 clearly indicated that the 3-OH group was in equatorial β-position. The NOESY correlations  +; HRESIMS m/z [M+H]+: 471.3826 .15α-Hydroxyeuphorbol-7,11-dione (2): White amorphous powder (8.0 mg); IR (KBr): 3441, 1629, 1398, 1088, 475 cm−1; 1H-NMR and 13C-NMR: m/z: 507.6 [M+Na]+; HRESIMS m/z [M+H]+: 485.3627 .Staphylococcus aureus (ATCC 8032), Micrococcus luteus (ATCC 4698), Micrococcus tetragenus (ATCC 13623), Staphylococcus albus (ATCC 8032), Bacillus cereus (ACCC 11077) and Escherichia coli (ATCC 25922), were assayed using standard agar diffusion tests [Antimicrobial activity against six bacterial strains on tests . CiproflP. oblique afforded two new triterpenoids, 20-hydroxyeuphorbol-7-one (1); and 15-hydroxyeuphorbol-7,11-dione (2); together with four known triterpenoids, euphorbol-7-one (3); friedelin (4); stigmast-4-ene-6α-ol-3-one (5); stigmasta-4-en-3,6-dione (6). Compounds 4 and 5 showed antibacterial activities against E. coli and M. tetragenus with the same MIC values of 5 μg/mL.In conclusion, our phytochemical investigation of the branches and leaves of


Penicillium commune, which was isolated from the semi-mangrove plant Hibiscus tiliaceus, afforded one new compound 1-O--glycerol (1) along with thirteen known products, including 1-O-acetylglycerol (2), N-acetyltryptophan (3), 3-indolylacetic acid methyl ester (4), 1-ethanone (5), 2-acetic acid (6), -5-hydroxyhexan-4-olide (7), thymidine (8), uracil (9), thymine (10), ergosterol (11), β-sitosterol (12), β-daucosterol (13), and ergosta-7,22-dien-3β,5α,6β-triol (14). The structures of these compounds were established by detailed NMR spectroscopic analysis, as well as by comparison with literature data or with authentic samples.Cultivation of the endophytic fungus  Eurotium rubrum have led to the isolation of a variety of structurally unique metabolites with different biological activities , weO--glycerol (1), together with thirteen known products 2–14 of diverse molecular types from the solid cultures of the endophytic fungal strain P. commune that was isolated from the semi-mangrove plant Hibiscus tiliaceus. The fungal species P. commune are widely spread in nature, being isolated usually from foods and plant rhizospheres. The earlier studies showed that strains of P. commune mainly produce indole alkaloids such as cyclopiazonic acid 25D: + 15° ; IR (KBr) cm–1: 3,440, 3,274, 3,070, 2,939, 1,716, 1,623, 1,450, 1,381, 1,323, 1,261, 1,192, 1,115; UV λmax (MeOH) nm (log ε): 215 (4.63), 264 (4.40); 1H-NMR and 13C-NMR: see +, 507 [2M + Na]+; HR-ESI-MS: m/z 265.0689 [M + Na]+, calcd. for C11H14O6Na+, 265.0688.


The structures of the new saponins were elucidated as 26-O-β-d-glucopyranoside-3β,26-dihydroxy-(25R)-5α-furostan-22-methoxyl-6-one-3-O-α-l-arabinopyranosyl-(1→6)-β-d-glucopyranoside (1), 26-O-β-d-glucopyranoside-3β,26-dihydroxy-(25R)-5α-furostan-22-methoxyl-6-one (2), 26-O-β-d-glucopyranoside-3β,26-dihydroxy-(25R)-5α-furostan-20(22)-en-6-one (3), 26-O-β-d-glucopyranoside-3β,23,26-trihydroxy--5α-furostan-20(22)-en-6-one (4) on the basis of spectroscopic analysis. The isolated saponins were evaluated for cytotoxic activity against two human cancer cell lines including Hela  and SMMC-7221 (hepatocellular carcinoma). Compounds 1 and 7 demonstrated cytotoxicity against the tested cell lines.Four new furostanol saponins  Smilax (family Liliaceae) is mainly distributed in the tropical, subtropical and temperate areas of the world +  in the HR-ESI-MS spectrum and supported by the ESI-MS (m/z 941.4 [M+Na]+ and m/z 917.3 [M−H]−) spectrum. Its IR spectrum displayed strong absorption bands for hydroxyl groups at 3408 cm−1, for carbonyl group at 1707 cm−1 and absorption bands of alkyl groups at 2927 cm−1. The 1H- and 13C-NMR assignments of 1 were based on the DEPT and 2D-NMR  experiments and with the positive red colour reaction in Ehrlich’s test suggested 1 was a furostanol saponin. To be specific, The 1H-NMR spectrum of 1 showed four methyl proton signals including two tertiary methyl groups at δH0.86  and 0.80  and two secondary methyl groups at δH 1.04  and 0.98 , corresponding to C-atom signals at δC 15.5, 12.1, 14.7 and 15.9 in HSQC spectrum, typical steroid methyl signals. Besides, a signal for carbonyl at δC 212.0 was present in the 13C-NMR. A correlation signal between H-7 at δH 2.16 and C-6 at δC 212.0 was observed in the HMBC spectrum  was also observed in the 1H-NMR spectrum. In the HMBC spectrum, the correlations from OCH3 at δH 3.17 to C-22 at δC 112.6 to suggested the OCH3 was linked to the C-22 (H 2.40 and the H-9 proton at δH 1.40 was consistent with the 5α configuration. A NOESY cross-peak between H-5 (δH 2.40) and H-3 (δH 3.36) indicated that H-3 was α configuration. In addition, The NOE correlation between H-18  and H-20  suggested that the C-21 methyl group was α-configuration. Thus, the α-configurations of H-17 and the methoxy at C-22 position were unambiguously deduced based on the strong NOE correlations of H-21/H-17 and H-21/OCH3. The 14α configuration was further confirmed by the NOE correlations of H-14/H-16 and H-16/OCH3 + at m/z 647.3762, consistent with the molecular formula C34H56O10 , which was supported by the ESI-MS (m/z 647.4 [M+Na]+ and m/z 623.4 [M–H]−) spectrum. In the IR spectrum, the glycosidic nature of 2 was shown by strong hydroxyl groups absorptions at 3423 cm−1, a carbonyl group at 1713 cm−1 and the alkyl groups at 2927, 2872 and 2853 cm−1 was also displayed in the IR spectrum. Compound 2 was suggested to be a furostanol glycoside with one sugar moiety by Ehrlich’s test and the 1H- and 13C-NMR assignments of 2 based on the DEPT and 2D-NMR  experiments. The aglycone of 2 was deduced as the same as that of 1 by comparison the 1H- and 13C-NMR spectra of 2 with those of 1. This was further confirmed by comparison Rf values of the aglycones obtained by acid hydrolysis of 1 and 2 on TLC. The only one sugar moiety was assigned to C-26 by a glycosylation shift of C-26 at δC 74.6 and the HMBC correlation between H-1 of the glucopyranosyl at δH 4.26 and C-26 at δC 74.6  of the anomeric proton. The glucopyranose moiety was determined as d-glucose by acid hydrolysis of 2 and then trimethylsilylation and GC analysis on a chiral column in the same manner. The 25R configuration of 2 was deduced by the difference (∆ab = 0.35) of the chemical shifts of the H2-26 geminal protons. Thus, the structure of 2 was elucidated as 26-O-β-d-glucopyranoside-3β,26-dihydroxy-(25R)-5α-furostan-22α-methoxyl-6-one.Compound  δC 74.6 . The rel3 was obtained as a white amorphous powder and positive ion HR-ESI-MS provided an [M+Na]+ ion m/z 615.3510, corresponding to a molecular formula of C33H52O9 , which was supported by m/z 615.3 [M+H] + and m/z 591.3 [M–H]− in the ESI-MS spectrum. The IR spectrum showed the presence of hydroxyl groups at 3421 cm−1, a carbonyl group at 1697 cm−1 and alkyl groups at 2943 cm−1, 2915 cm−1and 2865 cm−1. It was apparent from the NMR spectroscopic data  of 3 that this compound differed from 2 only by the presence of the ∆20,22 double bond. The occurrence of a ∆20,22 double bond was confirmed from the HMBC spectrum which showed significant correlation peaks between the methyl proton signal of CH3-21 with the carbon resonances of C-20 (δC 103.6) and C-22 (δC 151.8)  of the anomeric proton. Its absolute configuration was also a d- as determined by acid hydrolysis of 3 and then trimethylsilylation and GC analysis on a chiral column. The α-configuration of H-17 was determined by the NOE correlations of H-14/H-17. The 25R configuration of 3 was assigned by the observed difference (∆ab = δa − δb = 0.32) of the 1H-NMR chemical shifts of the H2-26 geminal protons . Therefore, compound 3 was identified as 26-O-β-d-glucopyranoside-3β,26-dihydroxy-(25R)-5α-furostan-20(22)-en-6-one.Compound C 151.8) . The sug4, a white amorphous powder, was assigned as C33H52O10 on the basis of its HR-ESI-MS data [M+Na]+m/z 631.3450  and its ESI-MS data m/z 631.55 [M+Na]+, 609.34 [M+H]− and 1215.03 [2M−H]−. The IR spectrum showed absorptions for hydroxyl groups (3422 cm−1), carbonyl group (1708 cm−1) and alkyl groups (2934 cm−1 and 2870 cm−1). The 1D and 2D NMR spectroscopic data of 4 were in good agreement with those of 3 except for noticeable differences in the δH-23 and δC-23 of the aglycone. The sugar moiety also was determined as β-d-glucopyranose by the coupling constant  of the anomeric proton and GC analysis of hydrolysis followed trimethylsilylation of 4 in comparison with an authentic sample. The configurations of H-17 and the C-25 of 4 was assigned as the same as those of 3 by the NOE correlation of H-14/H-17 and the ∆ab = 0.24 of the two geminal protons of H2-26. In the 1H-NMR spectrum, the proton signal at δH 4.91 , corresponded C-atom signal at δC 63.6 in the HSQC spectrum showed the presence of the hydroxyl at the C-23. On the basis of this evidence, the structure of 4 was revised as 26-O-β-d-glucopyranoside-3β,23,26-trihydroxy-(25R)-5α-furostan-20(22)-en-6-one. Besides the four new furostanol saponins, three known steroid saponins were isolated from this plant. Their structures were identified by compared the NMR properties with the reported data.Compound 1–7 were tested against human cervical carcinoma cells (Hela) and human hepatocellular carcinoma cells (SMMC-7221) by the MTT method. The results revealed that compounds 2–6 were inactive (IC50 > 100 µM), while compounds 1 and 7 displayed cytotoxicity against Hela carcinoma cell lines with IC50 values of 18.79 ± 1.12 μM and 9.73 ± 1.64 μM, respectively and against SMMC-7221 cancer cell lines with IC50 values of 28.57 ± 1.57 μM and 21.54 ± 1.64 μM, respectively. Cisplatin used as the positive control had the IC50 values of 5.12 ± 0.26 μM (Hela) and 14.23 ± 0.44 μM (SMMC-7221). This investigation indicated that compound 7 which displays a spirostanol saponin structure showed stronger cytotoxic activity, whereas compound 1, with a furostanol saponin structure, displayed moderate cytotoxic activity. Thus, according to our results, the spirostanol saponin skeleton exhibits higher cytotoxic activity than furostanol saponins, which is consistent with the literature + and 917.3 [M−H]−; HR-ESI-MS: m/z 941.4727 [M+Na]+ .d-Glucopyranoside-3β,26-dihydroxy-(25R)-5α-furostan-22-methoxyl-6-one26-O-β- (2). White amorphous powder, mp > 290 °C;3OH); IR υmax (KBr): 3423, 2927, 2872, 2853, 1713 and 1042 cm−1; 1H-NMR and 13C-NMR data see m/z 647.4 [M+Na]+ and 623.4 [M−H]−; HR-ESI-MS: m/z 647.3762 [M+Na]+ .d-Glucopyranoside-3β,26-dihydroxy-(25R)-5α-furostan-20(22)-en-6-one26-O-β- (3). White amorphous powder, mp > 290 °C;3OH); IR υmax (KBr): 3421, 2943, 2915, 2865, 1697 and 1071 cm−1; 1H-NMR and 13C-NMR data see +, 591.3 [M−H]− and 1183.5 [2M−H]−; HR-ESI-MS: m/z 615.3510 [M+Na]+ .d-Glucopyranoside-3β,23,26-trihydroxy--5α-furostan-20(22)-en-6-one26-O-β- (4). White amorphous powder, mp > 290 °C; [α]D20.8 = −18.485° ; IR υmax (KBr) (cm−1): 3422, 2934, 2870, 1708, and 1077 cm−1; 1H-NMR and 13C-NMR data see m/z 631.55 [M+Na]+, 609.34 [M+H]+ and 1215.03 [2M−H]−; HR-ESI-MS: m/z 631.3450 [M+Na]+ .d-Glucopyranosyl-3β,22ξ,26-trihydroxy-(25R)-5α-furostan-6-one-3-O-α-l-arabinopyranosyl-(1→6)-β-d-glucopyranoside26-O-β- (5). A white amorphous powder, molecular formula was C44H72O19. ESI-MS m/z 903.3 [M−1]−; 1H-NMR  δ: 2.41 , 1.40 , 1.45 , 4.61 , 1.83 , 0.85 , 0.80 , 1.04 , 3.41 , 3.75 , 0.97 , 4.42 , 4.10 , 3.83 , 4.35 , 4.26 ; 13C-NMR  δ: 36.3 (C-1), 28.6 (C-2), 76.7 (C-3), 26.0 (C-4), 56.0 (C-5), 212.0 (C-6), 46.1 (C-7), 37.4 (C-8), 53.4 (C-9), 40.8 (C-10), 21.0 (C-11), 39.2 (C-12), 41.1 (C-13), 56.0 (C-14), 31.2 (C-15), 80.6 (C-16), 62.6 (C-17), 15.5 (C-18), 12.1 (C-19), 39.4 (C-20), 14.4 (C-21), 110.5 (C-22), 35.6 (C-23), 27.2 (C-24), 33.5 (C-25), 74.6 (C-26), 16.0 (C-27), 100.9 (glc-C-1'), 73.7 (glc-C-2'), 76.6 (glc-C-3'), 70.4 (glc-C-4'), 75.5 (glc-C-5'), 68.2 (glc-C-6'), 103.8 (ara-C-1''), 71.0 (ara-C-2''), 72.8 (ara-C-3''), 68.0 (ara-C-4''), 65.2 (ara-C-5''), 103.2 (glc-C-1'''), 73.8 (glc-C-2'''), 77.1 (glc-C-3'''), 70.3 (glc-C-4'''), 76.5 (glc-C-5'''), 61.4 (glc-C-6''').d-Glucopyranosyl-3β,26-dihydroxy-(25R)-5α-furostan-20(22)-en-6-one-3-O-α-l-arabinopyranosyl-(1→6)-β-d-glucopyranoside26-O-β- (6). A white amorphous powder, molecular formula was C44H70O18. ESI-MS m/z 885.3 [M−1]−; 1H-NMR  δ: 2.41 , 2.27 , 2.19 , 1.41 , 1.35 , 4.77 , 2.56 , 0.73 , 0.80 , 1.64 , 3.38 , 3.72 , 0.98 , 4.43 , 4.10 , 3.84 , 4.35 , 4.26 ; 13C-NMR  δ: 36.3 (C-1), 28.6 (C-2), 76.7 (C-3), 26.0 (C-4), 56.0 (C-5), 212.0 (C-6), 46.3 (C-7), 37.3 (C-8), 53.4 (C-9), 40.8 (C-10), 21.2 (C-11), 39.0 (C-12), 43.7 (C-13), 54.6 (C-14), 33.5 (C-15), 84.0 (C-16), 64.0 (C-17), 13.3 (C-18), 12.1 (C-19), 103.6 (C-20), 10.5 (C-21), 151.8 (C-22), 22.7 (C-23), 30.6 (C-24), 32.7 (C-25), 74.4 (C-26), 16.0 (C-27), 100.9 (glc-C-1'), 73.7 (glc-C-2'), 76.5 (glc-C-3'), 70.3 (glc-C-4'), 75.4 (glc-C-5'), 68.2 (glc-C-6'), 103.8 (ara-C-1''), 71.0 (ara-C-2''), 72.8 (ara-C-3''), 68.1 (ara-C-4''), 65.3 (ara-C-5''), 103.1 (glc-C-1'''), 73.7 (glc-C-2'''), 77.1 (glc-C-3'''), 70.1 (glc-C-4'''), 76.5 (glc-C-5'''), 61.4 (glc-C-6''').l-arabinopyranosyl-(1→6)-β-d-glucopyranosideSieboldogenin-3-O-α- (7). A white amorphous powder, molecular formula was C38H60O14. 1H-NMR  δ: 2.33 , 1.30 , 1.32 , 4.31 , 0.73 , 0.67 , 0.92 , 3.41 , 3.51 , 3.73 , 3.49 , 4.23 , 3.89 , 3.49 , 4.21 ; 13C-NMR  δ: 36.5 (C-1), 29.1 (C-2), 76.1 (C-3), 26.6 (C-4), 55.7 (C-5), 210.4 (C-6), 46.4 (C-7), 37.2 (C-8), 53.0 (C-9), 40.8 (C-10), 21.0 (C-11), 39.4 (C-12), 40.9 (C-13), 56.0 (C-14), 31.6 (C-15), 80.6 (C-16), 62.1 (C-17), 16.6 (C-18), 13.3 (C-19), 42.0 (C-20), 15.0 (C-21), 109.4 (C-22), 26.7 (C-23), 21.3 (C-24), 35.2 (C-25), 60.5 (C-26), 60.3 (C-27), 100.9 (glc-C-1'), 73.9 (glc-C-2'), 77.1 (glc-C-3'), 70.7 (glc-C-4'), 75.9 (glc-C-5'), 68.7 (glc-C-6'), 103.9 (ara-C-1''), 71.0 (ara-C-2''), 73.0 (ara-C-3''), 67.7 (ara-C-4''), 65.2 (ara-C-5'').1–4 (30 mg each) in 1 M HCl  were heated under reflux for 8 h. After removal of the solvent, the residue was partitioned between CHCl3 and H2O. The water layer was neutralized with 5% NaOH and desalted . The desalted segment was found to contain d-glucose and l-arabinose by TCL comparison with authentic sample (EtOAc–MeOH–H2O–AcOH = 6.5:2.0:1.5:1.5) and by measurement of their optical rotation  for compound 1, and was only found to contain d-glucose for compounds 2–4.Compounds 1–4 (each 4 mg) in 1 M HCl  were heated at 95 °C for 6 h, respectively. The reaction mixtures were evaporated in vacuo. The residues were dissolved in water and then extracted with CHCl3 for three times, respectively. After evaporating the aqueous phases to dryness in vacuo, the residues were dissolved in pyridine (5 mL) and 1-(trimethylsilyl)-imidazole (0.5 mL) at room temperature for 30 min. The reaction mixtures were dried with a stream of N2. The residues were partitioned between CHCl3 and H2O. The organic layers were subjected to GC analysis using an l-Chirasil-Val column (0.32 mm × 25 m)  assay  assay , with ci1–4 were isolated and identified from the rhizomes and roots of Smilax scobinicaulis. Besides, two known furostanol saponins 5–6 and one known spirostanol saponin 7 are described for the first time in this plant. Among the isolated saponins, compounds 1 and 7 displayed cytotoxicity against the Hela and SMMC-7221 cancer cell lines. The discovery of these new bioactive compounds further expands our knowledge of the structural diversity of the bioactive products produced by the plant Smilax scobinicaulis, and lays a chemical foundation for its pharmacological application.Four new furostanol saponins


R)-heteroarylpyrimidine analogs is described here, which involves introduction of a chiral group, formation and separation of diasteroisomers and final transformation of an amide to an ester. The absolute configuration of the enantiopure HAPs is confirmed by X-ray analysis of their intermediates.An efficient synthesis of enantiopure ( Z060228, another novel HAP derivative found by our laboratory, exhibits excellent activity against HBV replication at submicromolar concentration and is currently under preclinical study . How. HowR)-eC method . Herein,R)-HAPs involved resolution of the racemic-HAPs I via diastereomeric salts using camphorsulfonic acid as resolution reagent and direct enantioselective synthesis in the chiral environment of quinidine or quinine, but the results were not satisfactory. Then we attempted an indirect method with a chiral auxiliary group to synthesize (R)-HAPs as shown in Y was introduced in starting material and a couple of diasteroisomers II were formed by the Biginelli reaction. The enantiopure IIa and IIb could be separated by taking advantage of the differences in their physiochemical properties and then the enantiopure IIa could be transformed into (R)-HAPs after getting rid of the introduced chiral group Y. Apparently, the choice of chiral group Y is key for the whole strategy. According to their cost and practical properties, several chiral agents such as menthol, and mandelic acid were adopted. When menthol was used, the enantiopure IIa or IIb were not crystallized easily from common solvents. When mandelic acid was used, racemization was found to occur under basic conditions. By comparison, (R)-1-phenylethanamine was proved to be the suitable chiral agent, which was enantiomerically stable in the subsequent reaction steps and was removed conveniently after enantiopure intermediate IIa was separated.In general, the methods frequently used for the synthesis of enantiopure 1,4-dihydropyridines are the resolution of racemic dihydropyridines, separation via diastereomeric esters, enantioselective synthesis with chiral auxiliary groups, chemoenzymatic separation of dihydropyridines, and chromatographic separation of enantiomers ,12,13. TR)-Z060228( has been synthesized by the synthetic route shown in R)-1-phenylethanamine  with diketene (2) easily gave the intermediate 3 in 95% yield . Us. Us(R)-Z, CH3OH) .1H-NMR and 13C-NMR spectra were recorded at 400 MHz and 100 MHz on a JNM-ECA-400 instrument with tetramethylsilane as an internal standard in the DMSO. ESI-MS (high resolution) mass spectra were obtained by using a Waters Xevo G2 Qtof (ESI) mass spectrometer. Melting points were determined using a RY-1 apparatus and are uncorrected.R,R-N-(1-Phenylethyl)-4-(2-chloro-4-fluorophenyl)-2--6-methyl-1,4-dihydro-pyrimidine-5-carboxamide (4a) and R,S-N-(1-phenylethyl)-4-(2-chloro-4-fluorophenyl)-2--6-methyl-1,4-dihydropyrimidine-5-carboxamide (4b). (R)-(+)-N-(1-phenylethyl)-3-oxobutyramide  and 2-chloro-4-fluorobenzaldehyde  are dissolved in anhydrous alcohol (85 mL) containing acetic acid (1 mL) and piperidine (1 mL) as catalysts. After stirring at R.T. for 24 h, 2,4,6-trifluorobenzimidamide acetate  and sodium acetate  are added. After refluxing for 20 h, the reaction mixture is concentrated and the residue is dissolved in water (50 mL) and extracted with ethyl acetate (3 × 25 mL). The combined organic extracts are washed with brine (20 mL × 3) and dried over anhydrous sodium sulfate. After evaporation of the solvent in vacuo, a 1:1 mixture of diastereomeric amides 4a and 4b is obtained and subjected to recrystallization from ethyl acetate and pentane (50:50), 4a  is isolated as white particles . Rf = 0.34 (ethyl acetate-petroleum ether = 2:1). Mp = 208–210 °C. 1H-NMR (DMSO-6d) δ: 1.29–1.33 ; 1.97 ; 4.83–4.87 ; 5.97 ; 7.04–7.06 ; 7.12–7.24 ; 7.25–7.27 ; 8.08–8.10 ; 9.24 ; HRMS (ESI) m/z: Calcd for C26H21ClF4N3O, 502.1304 [M+H]+; Found: 502.1299.4b is crystallized  as white plates . Rf = 0.36 (ethyl acetate-petroleum ether = 2:1). Mp = 197–199 °C. 1H-NMR (DMSO-6d) δ: 1.17–1.25 ; 2.12 ; 4.85–4.88 ; 6.01 ; 7.15–7.20 ; 7.86–7.87 ; 7.94–7.95 ; 9.37 ; HRMS (ESI) m/z: Calcd for C26H20N3OF4Cl, 502.1304 [M+H]+; Found: 502.1299.The mother liquor was concentrated and redissolved in anhydrous alcohol, whereby R,R-N-(1-phenylethyl)-N-1-acetyl-6-(2-chloro-4-fluorophenyl)-2--4-methyl-1,6-dihydropyrimidine-5-carboxamide (5). A solution of diastereomeric amide 4a  and anhydrous triethylamine  in dry DMF (45 mL) is stirred at 0 °C, and then acetyl chloride  is added dropwise. After stirring for 6 h, the reaction mixture is quenched with water (60 mL), and extracted with ethyl acetate (3 × 60 mL). The combined organic phase is dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue is purified by column chromatography on silica gel (ethyl acetate-pentane = 1:3) to give 5  as a white powder. Rf = 0.4 (ethyl acetate-petroleum ether = 1:1). Mp: 183–184.2 °C. 1H-NMR (DMSO-6d) δ: 1.39–1.41 , 2.07 , 2.10 , 4.95–4.99 , 6.47 , 7.08–7.12 , 7.15–7.30 ; 7.45–7.51 , 8.70–8.72 . HRMS (ESI) m/z: Calcd for C28H23N3O2ClF4, 544.1409 [M+H]+; Found: 544.1398.Ethyl 4-(R)-(2-chloro-4-fluorophenyl)-2--6-methyl-1,4-dihydropyrimidine-5-carboxylate(R)-Z060228. To a solution of 5  in dichloromethane (50 mL) at 0 °C is added dinitrogen tetroxide . The solution is stirred under nitrogen at 0 °C for 20h and then poured over ice, and extracted with cold dichloromethane (2 × 200 mL), the organic part is concentrated in vacuum in an ice water bath to yield a yellow oil, which is dissolved in cold dry DMF , and sodium ethoxide  is added. The mixture is stirred for 15 min under nitrogen atmosphere and then quenched with water (200 mL) neutralized with 4 M HCl, and extracted with ethyl acetate (150 mL × 3). The combined extracts are dried over anhydrous sodium sulfate, and concentrated at reduced pressure. The residue is purified by column chromatography on silica gel (ethyl acetate-dichloromethane 4:100) to give R)-Z060228 as a white powder.  = −92.4 , 1H-NMR (DMSO-6d) δ: 1.02–1.05 ; 2.32 ; 3.92–3.96 ; 5.97 ; 7.21–7.45 ; 9.86 . 13C-NMR (DMSO-6d) δ: 14.4, 17.9, 56.0, 59.6, 96.2, 101.3, 101.5, 101.8, 115.2, 116.6, 116.8, 131.2, 131.2, 132.6, 139.8, 142.0, 147.6, 160.0, 162.4, 166.1. HRMS (ESI) m/z: Calcd for C20H15N2O2F4Cl, 427.0831 [M+H]+; Found: 427.0827.Ethyl 4-(R)-(2-chloro-4-fluorophenyl)-2-(thiazol-2-yl)-6-methyl-1,4-dihydropyrimidine-5-carboxylate(R)-Bay39-5493.  = −54.6 . 1H-NMR (DMSO-6d) δ: 1.01–1.04 ; 2.50 ; 3.90–3.94 ; 5.99 ; 7.36–7.43 ; 7.90–7.91 ; 7.97–7.98 ; 9.96 ; HRMS (ESI) m/z: Calcd for C17H15N3O2FSCl, 380.0630 [M+H]+; Found: 380.0628.In summary, a novel and efficient approach to the synthesis of enantiomerically pure HAPs is accomplished from inexpensive starting materials. Key feature of this synthesis include an introduction of another chiral group and the alcoholysis to yield the final product. The method described herein could be an attractive alternative for the synthesis of chiral HAPs.


Rauvolfia caffra Sond was determined by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Column chromatography and preparative thin layer chromatography were used to isolate three indole alkaloids  and one triterpenoid (lupeol). The antiplasmodial activity was determined using the parasite lactate dehydrogenase (pLDH) assay. The UPLC-MS profile of the crude extract reveals that the major constituents of R. caffra are raucaffricine (m/z 513.2) and spegatrine (m/z 352.2). Fraction 3 displayed the highest antiplasmodial activity with an IC50 of 6.533 μg/mL. However, raucaffricine, isolated from the active fraction did not display any activity. The study identifies the major constituents of R. caffra and also demonstrates that the major constituents do not contribute to the antiplasmodial activity of R. caffra.In this study, the chemical profile of a crude methanol extract of  In many countries worldwide medicinal plants remain the dominant form of medicine for the treatment and prevention of a wide range of diseases. Medicinal plants used as alternative drugs are indicative of the vital role that plants play in many developing countries, and are also sources of novel plant-derived constituents that could be leads for treatment of malaria and other diseases. Malaria is a major public health problem in the world, responsible for the death of millions, particularly in sub-Saharan Africa ,2. TodayTreatment, management, and control of malaria are still a challenge to the medical community and safer, cheaper and more accessible alternatives are needed to efficiently limit the problem of this ailment . FurtherRauvolfia caffra Sond is a plant species belonging to the family Apocynaceae + ion peak at m/z 513.2237 which matched the molecular formula C27H32N2O8. The IR showed characteristic absorption frequencies at 3312.90, 2942.44 and 2831.65 cm−1 typical of the O-H, C-H and C-H bond vibrations, respectively. This was supported by the appearance of 27 carbon signals in its 13C-NMR spectrum, including six quaternary carbons, fifteen methines, four methylenes and two methyl groups at δ 21.32  and 13.28 , respectively. The 1H-NMR spectrum showed an ortho-disubstituted aromatic ring with δH 7.57 to 7.2 ppm, which included two sets of doublets at δH 7.55 and 7.49 ppm corresponding to protons at positions 9 and 12, a doublet of doublets at δH 7.39 ppm corresponding to a proton at position 10 and another doublet of doublets at δH 7.23 ppm corresponding to a proton at position 11. The spectrum also showed the existence of an ethylidene group with a doublet at δH 1.67 ppm assigned to position 18 and a quartet at δH 5.65 ppm assigned to a proton at position 19. The 1H-NMR also revealed several other important features, including one methyl signal at δH 2.16 (s) corresponding to OCOCH3 and three methylene signals at δH 4.42 ppm, δH 1.87 and δH 1.77 ppm, and δH 2.68 and δH 1.45 ppm, corresponding to 6′-H, 14-H (α and β), and 6-H (β and α), respectively. Moreover, a glucopyranosyl moiety was indicated by the signal of the anomeric proton at δH 5.03  and in agreement with carbon signals at δC 99.32 , 77.48 , 74.26 , 70.58  and 61.56 . The complete assignment of the 1H-NMR and 13C-NMR chemical shifts was done using 2D-NMR  spectroscopic techniques. These results enabled us to confirm the 1H-NMR and 13C-NMR chemical attributions of raucaffricine as previously reported + ion peak at m/z 325.1912 in its HRMS spectrum, suggesting the molecular formula C20H25N2O2+. The 13C-NMR displayed 20 non-equivalent carbon signals, which was consistent with the HRMS result. The 1H-NMR showed an active hydroxyl proton at δ 1.97 ppm, attached to C-10 (δC 150.93). The 1H-NMR was further characterized by a doublet at δH 7.24 ppm , corresponding to a proton at position 12, another doublet at δH 6.90 ppm  corresponding to a proton at position 9 and a doublet of doublets at δH 6.78 ppm  corresponding to a proton at position 11. Furthermore, the 1H-NMR showed an ethylidene group at δH 1.74 ppm  associated with position 18 and a quartet at δH 5.68 ppm  assigned to a proton at position 19. A N+-Me group at δH 3.12 ppm (s) which is a strong electron withdrawing group let the 21-H protons appear at δH 4.45 ppm  and at δH 4.23 ppm , respectively. Another signal shifted downfield was found at δH 3.58 ppm  corresponding to two protons at C-17 (δC 62.40). This condition may be due to the presence of a hydroxyl group attached to C-17 (δC 62.40). Total assignment was done by a close examination of the 1D-NMR (1H-NMR and 13C-NMR), 2D-NMR , UPLC-MS and literature data.Spegatrine  with an IC50 value of 6.533 μg/mL, whereas the reference drug chloroquine showed an IC50 value of 0.032 µg/mL.As shown in All chemicals used were analytical grade purchased from Sigma-Aldrich . Silica gel (0.063–0.2 mm) or Sephadex LH20 was used as stationary phases and solvent mixtures described below were used as mobile phase in the chromatographic separations. Thin layer chromatography plates packed with silica gel , were used to locate major components of the fractions.A Waters Synapt G2 Quadrupole time-of-flight (QTOF) mass spectrometer (MS) connected to a Waters Acquity ultra-performance liquid chromatograph (UPLC)  was used for direct injection high resolution mass spectrometric analysis. 1 μL of sample was injected into a stream of 60% acetonitrile/40% 0.1% formic acid in water. This conveyed the sample directly to the QTOF mass spectrometer where data was acquired using both positive and negative electrospray ionisation. The following MS settings were used: cone voltage of 15 V, desolvation temperature of 275 °C, desolvation gas at 650 L/h, and the rest of the MS settings optimized for best resolution and sensitivity.ATR Infrared (IR) spectra were recorded on an Alpha FTIR spectrometer .1H- and 13C-nuclear magnetic resonance (NMR) spectra were recorded at 400 MHz and 100 MHz, respectively, with an Avance 400 spectrometer (Bruker) using residual undeuterated solvent as the internal standard.18, 2.1 × 100 mm, 1.7 μm column into the flow stream. This facilitated separation of compounds prior to data acquisition using a photodiode array detector (PDA), followed by the QTOF MS where data was acquired by scanning from m/z 150 to 1500 m/z in resolution mode as well as in MSE mode. In MSE mode two channels of MS data were acquired, one at a low collision energy (4 V) and the second using a collision energy ramp (40−100 V) to obtain fragmentation data as well. Leucine enkephalin was used as lock mass (reference mass) for accurate mass determination and the instrument was calibrated with sodium formate. An injection volume of 3 μL was used and the mobile phase consisted of 0.1% formic acid (solvent A) and acetonitrile containing 0.1% formic acid as solvent B.The instrumentation used for ultra-performance liquid chromatography MS (UPLC-MS) was identical to that described above, except for the inclusion of a Waters BEH CRauvolfia caffra Sond was collected at the University of Venda Campus  in January 2016. Botanical identification was provided by Prof. Tshisikhawe, a botanist in the Department of Botany at the University of Venda, and a voucher specimen (BD 001) was deposited. The plant samples were air-dried for two months and the dry samples were ground to fine powder using an industrial blender (mill).The stem bark of R. caffra were soaked with 2 L methanol for 48 h at room temperature. The methanol extract was filtered, and then concentrated using a rotary evaporator to obtain 49.2 g of dried extracts. The crude methanol extract (49.2 g) was subjected to column chromatography over silica gel.About 1.7 kg ground stems of A–FH. FA was obtained with hexane/ethyl acetate (70:30); FB, FC were obtained with hexane/ethyl acetate (30:70); FD was obtained with ethyl acetate (100%); FE, FF were obtained with ethyl acetate/methanol (70:30); and FG, FH were obtained with ethyl acetate/methanol (30:70). The collected fractions were monitored on TLC plates.The extract was eluted initially with hexane and the polarity was gradually increased with ethyl acetate and finally methanol, yielding 17 fractions. Fractions with similar TLC profile were combined and concentrated to dryness on a rotary evaporator giving a total of 8 fractions coded as FB, FE, FF and FG were further fractionated using column chromatography since they contained a large amount of material compared to fractions FA, FC, FD and FH.Fractions FB (1.42 g) was subjected to Sephadex LH-20 column chromatography; the column was eluted with CH2Cl2/MeOH (50:50) followed by an increasing gradient of CH2Cl2/MeOH (up to 10:90) to obtain F1 (1 g). Fraction FE (4 g) was subjected to silica gel column chromatography; the column was eluted using CH2Cl2/MeOH (50:50) followed by an increasing gradient of CH2Cl2/MeOH (up to 10:90) to obtain 2 subfractions, F2 (1.51 g) and F3 (1.55 g). Fraction FF (4 g) was also subjected to silica gel column chromatography and the column was eluted using n-C6H12/EtOAc (50:50) followed by an increasing gradient of n-C6H12/EtOAc (up to 30:70) to obtain 1 subfraction, F4 (3 g). Fraction FG (5.6 g) was subjected to Sephadex LH-20 column chromatography; the column was eluted using CH2Cl2/MeOH (50:50) followed by an increasing gradient of CH2Cl2/MeOH (up to 10:90) to obtain F5 (5 g). The collected fractions were monitored on TLC plates. The thin layer chromatograms were developed in a solvent system of ethyl acetate/methanol/water (EMW 81:11:8). A natural product staining solution  was used to visualize compounds on a TLC plate.Fraction F1 (0.5 g) was subjected to preparative TLC  to obtain compound 1 (0.230 g). Fraction F2 (0.5 g) was also subjected to preparative TLC (reversed phase) to obtain fraction F2a (0.2102 g). Fraction F3 (0.5 g) was further purified using semi-preparative HPLC to yield compound 2 (0.019 g). Fraction F4 (0.5 g) was also subjected to preparative TLC (reversed phase) to obtain compound 3 (0.1238 g). F5 (1 g) was further purified using semi-preparative HPLC to obtain compound 4 (0.086 g).FLupeol (1): IR: 3000 cm−1 (O-H). 1H-NMR : δH 4.7 , 4.6 , 3.18 , 2.38 , 1.91 , 1.70 , 1.60 , 1.39 , 1.30 , 1.04 , 1.0 , 0.97 , 0.86 , 0.77  ppm. 13C-NMR (CD3OD): δC 150.95 (C-20), 108.54 (C-29), 78.27 (C-3), 55.48 (C-5), 50.64 (C-9), 49.16 (C-18), 42.17 (C-14), 40.53 (C-8), 38.67 (C-4), 38.54 (C-1), 38.17 (C-13), 37.00 (C-10), 36.91 (C-16), 34.21 (C-7), 30.43 (C-21), 29.52 (C-23), 29.27 (C-2), 27.19 (C-15), 25.54 (C-12), 22.79 (C-11), 20.70 (C-30), 18.17 (C-6), 18.03 (C-28), 15.32 (C-26), 14.69 (C-24), 13.67 (C-27) ppm.Raucaffricine (2): IR: νO-H at 3421.5 cm−1 (O-H) and νC=O at 1662.0 cm−1 (C=O). 1H-NMR : δH 7.55 , 7.49 , 7.39 , 7.23 , 5.65 , 5.17 , 5.03 , 4.57 , 4.42 , 3.9–3.6 , 3.18 , 3.08 , 2.68 , 2.34 , 2.16 , 1.87 , 1.77 , 1.67 , 1.45  ppm. 13C-NMR (DMSO-d6): δC 184.42 (C-2), 170.13 (C-22), 156.86 (C-13), 137.94 (C-20), 137.09 (C-8), 127.06 (C-11), 125.81 (C-10), 124.29 (C-9), 122.76 (C-19), 120.85 (C-12), 99.32 (C-1′), 88.22 (C-21), 77.48 , 77.21 (C-17), 74.26 (C-2′), 70.58 (C-4′), 65.19 (C-7), 61.56 (C-6′), 55.24 (C-5), 50.45 (C-3), 48.49 (C-16), 37.32 (C-6), 27.47 (C-15), 24.67 (C-14), 21.32 (C-23), 13.28 (C-18) ppm. HRMS [M]+: m/z 513.2241; calcd. for C27H32N2O8: 513.2237.N-Methylsarpagine (3): IR: 3312.90 (O-H), 2942.44 (C-H) and 2831.65 cm−1 (C-H). 1H-NMR : δH 7.11 , 6.77 , 6.68 , 5.59 , 4.41 , 4.16 , 3.48 , 3.28 , 3.15 , 3.0 , 2.95 , 2.9 , 2.4 , 2.12–2.0 , 1.82 , 1.63  ppm. 13C-NMR (CD3OD): δC 150.91 (C-10), 132.10 (C-20), 131.68 (C-13), 127.67 (C-8), 126.83 (C-2), 120.72 (C-19), 112.34 (C-12), 111.75 (C-11), 102.14 (C-9), 99.77 (C-7), 65.45 (C-5), 64.39 (C-17), 62.40 (C-21), 61.07 (C-3), 43.63 (C-16), 32.01 (C-14), 26.01 (C-15), 23.87 (C-6), 11.57 (C-18) ppm.Spegatrine (4): IR: 3352.1 cm−1 (O-H) and 1638.8 cm−1 (N-H). 1H-NMR : δH 7.24 , 6.90 , 6.78 , 5.68 , 4.45 , 4.23 , 3.58 , 3.27 , 3.15 , 3.12 , 3.04 , 2.54 , 2.23–2.13 , 1.97 , 1.74  ppm. 13C-NMR (CD3OD): δC 150.93 (C-10), 132.10 (C-20), 131.66 (C-13), 127.68 (C-8), 126.83 (C-2), 120.71 (C-19), 112.35 (C-12), 111.73 (C-11), 102.13 (C-7), 99.77 (C-9), 65.46 (C-5), 64.38 (C-21), 62.40 (C-17), 61.08 (C-3), 46.67 (N+- CH3), 43.62 (C-16), 32.00 (C-14), 26.01 (C-15), 23.87 (C-6), 11.56 (C-18) ppm. HRMS [M]+: m/z 325.1912; calcd. for C20H25N2O2+: 325.1911.Plasmodium falciparum chloroquine-sensitive strain 3D7, obtained from the Malaria Research and Reference Reagent Resource Center, Manassas, VA, USA) were maintained in RPMI 1640 medium containing 2 mM l-glutamine and 25 mM Hepes . The medium was further supplemented with 5% Albumax II, 20 mM glucose, 0.65 mM hypoxanthine, 60 µg/mL gentamycin and 2–4% hematocrit human red blood cells. The parasites were cultured at 37 °C under an atmosphere of 5% CO2, 5% O2 and 90% N2 in sealed T75 culture flasks. For screening compounds against malaria parasites, 20 μM for pure compounds (2 and 4) or 25 μg/mL of the crude extract and fractions F1, F2, F3, F4 and F5 were added to parasite cultures  in 96-well plates and incubated for 48 h at 37 °C in a sealed chamber suffused with the above gas mixture. After 48 h the plates were removed from the incubator and parasite levels in the individual wells determined using the parasite lactate dehydrogenase (pLDH) assay [v/v] Triton X-100, pH 9.0) and 25 µL NBT/PES solution  in a fresh 96-well plate. After incubation for 30 min, colour development was measured as absorbance at 620 nm (Abs620) in a Spectramax M3 plate reader . After subtracting background readings obtained from control wells (uninfected red blood cells), Abs620 values were converted to % parasite viability relative to untreated control cultures. For dose-response analysis, the above procedure was repeated by incubating parasite cultures in 96-well plates with 3-fold serial dilutions of the crude extract, fractions  and compounds 2 and 4. Log (compound concentration) was plotted against % parasite viability and IC50 values calculated by non-linear regression using GraphPad Prism . Chloroquine  was used as a positive control drug standard for antimalarial activity.Malaria parasites (H) assay . 20 µL cR. caffra. Raucaffricine, a rare glycoalkaloid of the monoterpenoid indole class and spegatrine, an indole alkaloid isolated for the first time from Rauvolfia verticillata. This is the first report of the isolation of spegatrine from R. caffra. Lupeol, a pentacylic triterpenoid was previously isolated from various medicinal plants while N-methylsarpagine, an indole alkaloid, was first isolated from Rauvolfia vomitoria. This is the first report of the isolation of lupeol and N-methylsarpagine from the genus Rauvolfia and R. caffra species. The UPLC-MS chromatogram indicated that raucaffricine is a major alkaloid from R. caffra but did not contribute to the antiplasmodial activity of the fraction from which it was isolated.Four compounds were isolated from the stem bark methanol extract of


Their structures were elucidated by spectroscopic analyses, and they were further evaluated for their cytotoxic activities against human cancer cells . Compounds 1–4 showed differential cytotoxic effects on the target cancer cells with IC50 values in the range of 0.9–33.3 μM.Two new sesquiterpenoids, siegenolides A ( Siegesbeckia glabrescens Makino (Compositae) is an annual herb that has been used as a Chinese medicine to treat inflammatory disease, asthma, paralysis and allergic disorders -γ-lactone having an exocyclic double bond in lactone ring. We also found the HMBC correlations between oxy-methine H-4 and C-6, and between H-5 and C-3. In 1H-1H COSY spectrum, we found another AMX spin system from the correlations of H-14 (δ 3.94) with H-13 (δ 6.63) and H-15 (δ 9.43). From the coupling constant of H-15 (J = 2.0 Hz) and chemical shift of C-15 (δ 196.8) and the HMBC correlations between H-15 (δ 9.43) and C-14 (δ 79.4), we identified the presence of an aldehyde group that is linked to C-14. In HMBC spectrum, we also found correlation between a methoxyl protons (δ 3.10) and C-14 (δ 79.4), and correlation between H-13 (δ 6.63) and C-3 (δ 51.9). The long range allylic coupling was observed between H-13  and H-9. These results indicated that an oxy-carbon C-13 is linked to the eight-membered ring at C-10. From the coupling constant between H-3 and H-4  we postulated the configurations of H-3 and H-4 based on Karplus relationship and the reported values of several bicycle [6.3.0]-γ-lactone derivatives (1H-1H COSY correlation of an olefinic methine H-3' (δ 6.10) with methyl protons (δ 1.93) and the HMBC correlations of methyl protons (δ 1.88) with C-2' (δ 128.9) and C-1' (δ 168.4), and the correlations of methyl protons (δ 1.93) with C-2' (δ 128.9) and C-3' (δ 138.9). The HMBC correlation of an oxy-proton H-13 with carbonyl ester C-1' confirmed the esterification of 2-methylbut-2-enoyl group at C-13. We found NOESY correlations of H-13 (δ 6.63) with H-3 (δ 2.82) and H-11a (δ 6.15) that indicates the orientation of H-13 as β that is located close to H-3 and H-11a. NOESY correlations was also observed between H-9 (δ 6.97) and H-15 (δ 9.43) implying the orientation of methoxyl group as β. This orientation was confirmed by the coupling constant between H-13 and H-14 as 8.2 Hz. Thus, compound 1 was identified as 2-methylbut-2-enoic acid 1--2-methoxy-3-oxo-propyl ester. This structure is new and we named compound 1 as siegenolide A.Compound δ 141.5) . These divatives  8]. Fur. Fur1 wa2 was obtained as an amorphous solid. The HREIMS spectrum suggested a molecular formula as C20H24O7. The 13C-NMR, DEPT, and HSQC spectra showed similar signals as those of compound 1 except showing one terminal olefinic methylene signals of H2-3'  instead of the methyl protons (H3-4') of compound 1. The relative stereochemistry of compound 2 was determined by the analysis of NOESY spectra and coupling constants, which was same as compound 1. Thus, the structure of compound 2 was determined as 2-methyl-acrylic acid 1--2-methoxy-3-oxo-propyl ester, which was a new structure and named as siegenolide B.Compound 3 and 4 were identified as 2-methylbut-2-enoic acid,2,3,3a,4,5,8,9,10,11,11a-decahydro-6,10-bis(hydroxymethyl)-3-methylene-2-oxocyclodeca[b]furan-4-yl ester (3) and 2-methylacrylic acid, 2,3,3a,4,5,8,9,10,11,11a-decahydro-6,10-bis(hydroxymethyl)-3-methylene-2-oxocyclodeca[b]-furan-4-yl ester (4), respectively, by comparison with the reported spectral data  than that of compounds 1 and 2 . The four sesquiterpenoids ll lines . All of 2 showed relatively high cytotoxicity, and against MCF-7 cells all compounds showed moderate cytotoxicity. All of these compounds displayed weak cytotoxicity against HeLa cells, with IC50 values (12.3–33.3 μM) compared to that of cisplatin (0.9 μM). Cytotoxicity of sesquiterpenes with α, β-unsaturated lactone structure was well known. Compounds 1 and 2 are uncommon sesquiterpenoids with eight-membered ring and they also showed same type of cytotoxicity as reported + ; EIMS m/z 390 [M]+.Compound 2: amorphous solid; max (log ε) 253 (3.20), 245 (3.18) nm; IR  3500, 2931, 1766, 1722, 1686, 1157. 1H- and 13C-NMR data, see m/z 376.1518 [M]+ ; EIMS m/z 376 [M]+.Compound 1–4 against human breast cancer (MCF-7), pancreatic cancer (AsPC-1), colon cancers (SW480 and HCT 116), hepatoma (HepG2), and cervical carcinoma (HeLa) were assessed by the MTT method [1–4 for 3 days, and then treated with MTT (5 mg/mL) solution for 4 h and lysed with DMSO. Absorbance at 540 nm was measured by using a microplate reader . Cisplatin (purity > 98%)  was used as a positive control.The cytotoxicity of compounds


The cytotoxic effects of these compounds along with the lead compound curcumin (7) and their effect on the production of the reactive oxygen species nitric oxide and pro-inflammatory cytokines IL-1β, TNF-α and chemokine CXCL-8 were evaluated using human monocytic THP-1 and colon adenocarcinoma CACO-2 cell lines. All of the nitric oxide donating curcuminoids 9a–d and the thiophene curcuminoids 10a–c and 11 were non-cytotoxic to THP-1 cells over a concentration range of 10-100 μM and compared with curcumin compounds 10b and 10c, were more toxic. In CACO-2 cells, 10b and 11 appeared to be non-toxic at 10 to 50 μM, whereas 10a and 10c were non-cytotoxic at 10 μM only. These results clearly indicate that the introduction of a nitroxybutyl moiety to curcumin and replacement of phenyl rings with thiophene units reduces the cytotoxic effect of the parent curcumin, whereas a methyl substituted thiophene increases the cytotoxic effects. In THP-1 cells, drugs 10a and 11 significantly decreased IL-1-β production at their non-cytotoxic concentrations, whereas, they did not decrease TNF-α production in CACO-2 cells. Compound 11 showed a significant decrease in CXCL-8 production.In search of better NSAIDs four novel nitric oxide donating derivatives of curcumin (compounds  IR (ν) 3075 (aromatic C-H stretch), 2932 , 2759 , 1687 (conjugated >C=O stretch), 1598 and 1577 (aromatic C=C stretch), 1242 (asymmetric C-O-C stretch), 1042 (symmetric C-O-C stretch), 758 cm−1 (ortho di-substituted out of plane C-H stretch); 1H-NMR δ 1.89–2.17 , 3.50 , 4.11 , 6.95–7.05 , 7.53 , 7.82 , 10.5 ; EIMS m/z 256 [M 79Br]+ (12%), 258 [M 81Br]+. (12%), 227 [M−CHO]+. (3%), 135 [C4H879Br]+ (62%), 137 [C4H881Br]+ (59%), 121 [C7H5O2]+ (85%); Accurate mass found: m/z 256.0093 (Br 79), calculated for C11H13O279Br: 256.0099.All four compounds 3-(4-Bromobutoxy)benzaldehyde (6b). Yield: 79%, as a pale yellow oil, Rf 0.50 . IR (ν) 3067 (aromatic C-H stretch), 2945 , 2729 , 1696 (conjugated C=O stretch), 1596 and 1585 (aromatic C=C stretch), 1262 (asymmetric C-O-C stretch), 1043 cm−1 (symmetric C-O-C stretch); 1H-NMR δ 1.90–2.16 , 3.48 , 4.04 , 7.11–7.46 , 9.95 ; EIMS m/z: 256 [M 79Br]+. (7%), 258 [M 81Br]+. (7%), 135 [C4H879Br]+ (71%), 137 [C4H881Br]+ (69%), 121 [C7H5O2]+ (69%); Accurate mass found: m/z 256.0089, calculated for C11H13O279Br: 256.0099. 4-(4-Bromobutoxy)benzaldehyde (6c). Yield: 81%, as golden yellow oil, Rf 0.47 . IR (ν) 3074 (aromatic C-H stretch), 2945 , 2738 , 1690 (conjugated C=O stretch), 1600 and 1577 (aromatic C=C stretch), 1255 (asymmetric C-O-C stretch), 1040 (symmetric C-O-C stretch), 832 cm−1 (para disubstituted out of plane C-H stretch); 1H-NMR δ 1.85–2.13 , 3.49 , 4.08 , 6.98 , 7.82 , 9.87 ; EIMS m/z 256 [M 79Br]+ (6%), 258 [M 81Br]+. (6%), 135 [C4H879Br]+ (58%), 137 [C4H881Br]+ (56%); Accurate mass found: m/z 257.0176, calculated for C12H14O279Br: 257.0171.4-(4-Bromobutoxy)-3-methoxybenzaldehyde (6d). Yield: 50%, as a white solid, Rf 0.33 , m.p. 49.7 °C. IR (ν) 3081 (aromatic C-H stretch), 2872 and 2933 , 2756 and 2821 , 1679 (conjugated C=O stretch), 1596 and 1584 (aromatic C=C stretch), 1263 (asymmetric C-O-C stretch), 1046 cm−1 (symmetric C-O-C stretch); 1H-NMR δ 2.06–2.08 , 3.51 , 3.92 , 4.14 , 6.96 , 7.40–7.46 , 9.85 ; EIMS m/z 286 [M 79Br]+. (9%), 288 [M 81Br]+. (8%), 135 [C4H879Br]+ (54%), 151 [C4H881Br]+ (54%); Accurate mass found: m/z 286.0200 (79Br), calculated for C12H15O379Br: 286.0205.Method A: In this procedure the synthesis of curcuminoids was carried out according to Pabon  as eluent.The crude products -1,7-Bis(2-94-bromobutoxy)phenyl)-5-hydroxyhepta-1,4,6-trien-3-one (8a). Yield: 34% (method A), 21% (method B), a dark brown gum, Rf 0.44 . IR (KBr pellet) ν 3032 (aromatic C-H stretch), 2924 , 1620 (H-bonded >C=O stretch), 1595 and 1570 (aromatic C=C stretch), 1244 (asymmetric C-O-C stretch), 1046 (symmetric C-O-C stretch), 750 cm−1 (ortho disubstituted out of plane C-H stretch); 1H-NMR δ 1.98–2.20, , 3.55 , 4.10 , 5.87 , 6.75 , 6.91 , 6.98 , 7.33 , 7.57 , 7.97 ; 13C-NMR δ 28.1, 29.8, 33.7, 67.7, 101.9, 112.4, 121.2, 124.5, 125.2, 129.1, 131.5, 136.0, 157.9, 184.0; EIMS m/z 576 [M 79Br]+. (4%), 580 [M 81Br]+. (6%), 577 [M+H]+., (2%), 578, [M+2H]+., (7%), 135 [C4H879Br]+ (39%), 137 [C4H881Br]+ (37%); Accurate mass found: m/z 577.0583, calculated for C27H31O479Br2: 577.0589. -1,7-Bis(3-(4-bromobutoxy)phenyl)hepta-1,6-dien-3,5-dione (8b). Yield: 24% (method A) 21% (method B), as a dark brown solid, Rf 0.41 , m.p. 74.6–75.9 °C. IR (ν) 3426 (OH stretch), 3052 (aromatic C-H stretch), 2947 , 1625 (H-bonded >C=O stretch), 1596 and 1579 (aromatic C=C stretch), 1508 (enol), 1243 (asymmetric C-O-C stretch), 1044 (symmetric C-O-C stretch), 885, 791, 677 cm−1 (meta disubstituted out of plane C-H stretch); 1H-NMR δ 1.95–2.18 , 3.51 , 4.04 , 5.85 , 6.62 , 6.92 , 7.07 , 7.16 , 7.31 , 7.63 ; 13C-NMR δ 28.1, 29.8, 33.6, 67.2, 102.0, 113.9, 116.7, 121.2, 124.7, 130.2, 136.7, 140.8, 159.5, 183.5; ESMS m/z 576 [M 79Br]+., 577 [M 79Br+H]+., 578 [M 79Br+2H]+., 580 [M 81Br]+., 581 [M 81Br+H]+., 582, [M 81Br+2H]+..-1,7-Bis(4-(4-bromobutoxy)phenyl)hepta-1,6-dien-3,5-dione (8c). Yield: 66% (method A), 41% (method B), as a bright yellow solid, Rf 0.42 , m.p. 145–146.9 °C. IR (ν) 3433 (OH stretch), 3035 (aromatic C-H stretch), 2945 , 1628 (H-bonded >C=O stretch), 1600 (aromatic C=C stretch), 1510 (enol), 1420 (olefinic in plane bending vibration), 1257 (asymmetric C-O-C stretch), 1046 (symmetric C-O-C stretch), 837 cm−1 (para disubstituted out of plane C-H stretch); 1H NMR δ 1.92–2.14 , 3.50 , 4.04 , 5.78 , 6.50 , 6.90 , 7.50 , 7.62 ; 13C-NMR δ 29.7, 28.1, 67.3, 101.6, 115.1, 122.1, 128.3, 130.0, 140.3, 160.8, 183.6; EIMS m/z 576 [M 79Br]+ (3%), 578 [M 79Br+2H]+ (8%), 135, [C4H879Br]+(100%), 137 [C4H881Br]+ (98%); Accurate mass found: m/z 576.0535, calculated for C27H30O479Br2 : 576.0511). -1,7-Bis(4-(4-bromobutoxy)-3-methoxyphenyl)hepta-1,6-dien-3,5-dione (8d). Yield: 57% (method A), as a dark yellow solid between EtOAc and aqueous layers during work-up and no further purification was required; Rf 0.38 , m.p. 124.4–125.4 °C. IR (ν) 3548-3235 (OH enolic), 3003 (aromatic C-H stretch), 2955 and 2870 , 1620 (H-bonded >C=O), 1597 and 1581 (aromatic C=C stretch), 1508 (enol), 1422 cm−1 (olefinic in plane bending vibration); 1H-NMR δ 2.00–2.17 , 3.51 , 3.92 , 4.10 , 5.82 , 6.50 , 6.87 , 7.08 , 7.13 , 7.61 ; EIMS m/z 637 [M+H]+ (18%), 619 [M+H−H2O]+ (31%), 324 [C15H18O379Br+H]+ (31%), 326 [C15H18O381Br+H]+ (29%), 324 [C14H16O379Br]+ (20%), 313 [C14H16O381Br]+ (18%), 135 [C4H879Br]+ (100%), 137 [C4H881Br]+ (99%); Accurate mass found: m/z 636.0714 (Br79), calculated for C29H34O679Br2 : 636.0722.9a–d is illustrated for the formation of -1,7-bis(2-(4-butoxynitrato)hepta-1,6-diene-3,5-dione (9a). In a one-neck round bottom flask, a mixture of silver nitrate  in acetonitrile (3 mL) was stirred for 30 min. and then a solution of 8a (0.86 mmol) in acetonitrile (2 mL) was added. The reaction mixture was refluxed under stirring for 5 h at 80 °C. An aluminium sheet was wrapped round the flask to protect it from light and the mixture was allowed to stand overnight. Water (5 mL) was added and after filtration the mixture was extracted with EtOAc (2 × 30 mL). The organic layers were combined, dried over MgSO4, filtered under gravity and the solvent was removed on the rotary evaporator to yield pure 9a (90%) as a dark brown gum, Rf 0.43 , m.p. 136.0–136.8 °C. IR (KBr pellet) ν 3035 (aromatic C-H stretch), 2926 and 2878 , 1625 (conjugated >C=O stretch), 1596 and 1488 (aromatic C=C stretch), 1472 and 1280 , 1455 (CH2 bending absorption), 1244 (asymmetric C-O-C stretch), 1049 (symmetric C-O-C stretch), 753 cm−1 (ortho disubstituted out of plane C-H stretch); 1H-NMR δ 1.99–2.04 , 4.10 , 4.58 , 5.83 , 6.74 , 6.91 , 6.99 ; 13C-NMR δ 24.2, 25.9, 67.8, 73.1, 102.1, 112.4, 121.3, 124.6, 125.2, 129.0, 131.5, 135.8, 157.7, 183.9; ESMS m/z 543 [M+H]+., 332 [C17H18O6N]+; Accurate mass found: m/z 543.1964, calculated for C27H31N2O10: 543.1973. The general procedure for the synthesis of butoxy-nitrate curcuminoids -1,7-Bis(3-(4-butoxy-nitrate)hepta-1,6-diene-3,5-dione (9b). Yield: 58% as a dark brown solid, Rf 0.46 , m.p. 133.9–134.9 °C. IR (ν) 3414 (OH stretch), 3020 (aromatic C-H stretch), 2929 and 2875 , 1638 (conjugated >C=O stretch), 1596 and 1489 (aromatic C=C stretch), 1473 and 1279 , 1458 (CH2 bending absorption), 1247 (asymmetric C-O-C stretch), 1041 cm−1 (symmetric C-O-C stretch); 1H-NMR δ 1.95 , 4.04 , 4.56 , 5.85 , 6.61 , 6.91 , 7.06 , 7.16 , 7.31 , 7.62 ; 13C-NMR: δ 24.2, 25.9, 67.3, 73.1, 102.1, 113.9, 116.6, 121.3, 124.7, 130.3, 136.7, 140.7, 159.4, 183.5; ESMS m/z 543 [M+H]+., 565 [M+Na]+; Accurate mass found: m/z 543.1985, calculated for C27H31N2O10: 543.1973. -1,7-Bis(4-(4-Butoxy-nitrate)hepta-1,6-diene-3,5-dione (9c). Yield: 95%, golden yellow solid, Rf 0.35 , m.p. 112.6–113 °C. IR (ν) 3412 (OH stretch), 3040 (aromatic C-H stretch), 2936 , 1620 (conjugated >C=O stretch), 1603 and 1511 (aromatic C=C stretch), 1472 and 1287 , 1256 (asymmetric C-O-C stretch), 1056 (symmetric C-O-C stretch), 837 cm−1 (para disubstituted out of plane C-H stretch); 1H-NMR δ 1.95 , 4.06 , 4.56 , 5.79 , 6.51 , 6.90 , 7.51 , 7.63 ; 13C-NMR: δ 24.1, 25.8, 67.4, 73.1, 101.6, 115.1, 122.2, 128.3, 130.0, 140.3, 160.7, 183.6; EIMS m/z 543 [M+H]+, (42%). -1,7-Bis(4-(4-butoxy-nitrate)-3-methoxyphenyl)hepta-1,6-diene-3,5-dione (9d). Yield: 92% as an orange solid, Rf 0.43 , m.p. 101.6–102.6 °C. IR (ν) 3436 (OH stretch), 2954 (aromatic C-H stretch), 2931 and 2873 , 1621 (conjugated >C=O stretch), 1458 (CH2 bending absorption), 1512 (aromatic C=C stretch), 1257 (asymmetric C-O-C stretch), 1027 (symmetric C-O-C stretch). 1H-NMR δ 1.63 , 3.91 , 4.10 , 4.58 , 5.83 , 6.50 , 6.87 , 7.09 , 7.13 , 7.61 ; EIMS m/z 603 [M]+ (38%), 604 [M+H]+., (12%); Accurate mass found: m/z 603.2162, calculated for C29H35N2O10: 603.2184.1,7-Bis(thiophen-3-yl-)-1,6-heptadien-3,5,dione (11). Yield: 36% (method A) purified by column chromatography , 23% yield (method B), yellow solid, Rf 0.48 , m.p. 140.1–141.3 °C.; IR (ν) 3435 (OH stretch), 3092 (aromatic C-H stretch), 1624 (H-bonded >C=O), 1584 (conjugated C=C), 1507 (enol), 1412 cm−1 (olefinic in plane bending vibration); 1H-NMR δ 5.79 , 6.45 , 7.33–7.38 , 7.52 , 7.66 ; EIMS m/z 288 [M]+ (60%), 270 [M−H2O]+. (8%), 192 [C10H8O2S]+ (4%), 179 [C9H7O2S]+ (5%), 151 [C8H7OS]+ (21%), 137 [C7H5OS]+ (100%), 109 [C6H5S]+ (43%), 97 [C5H4S]+ (21%); Accurate mass found: m/z 288.0277, calculated for C15H12O2S2: 288.0279. i.e., thawing, passaging, plating as well as respective compound treatments applied to the cells and cytotoxicity using 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assays were performed under sterile conditions using a laminar flow cabinet. In these experiments the colorimetric assay CellTiter 96® AQueous one solution cell proliferation in vitro was used to examine the effects of the synthesized curcumin (7) and curcuminoids on cell viability [Escherichia coli O127:B8 (cell culture tested) and L-methionine sulfoximine (MS) were obtained from Sigma-Aldrich ). All incubations were carried out in a humidified incubator maintained at 37 °C with 5% (v/v) CO2 and 95% air. Experiments were performed in duplicate wells of the 96-well plates and were repeated at least four times unless otherwise stated in the figure legend. Human monocytic leukemia (THP-1) cell line and human caucasian colon adenocarcinoma (CACO-2) cell lines were obtained from the European Collection of Cell Cultures (ECACC) . Preparation of drug treatments and general procedures for cell culture were as previously described ,28,29,30iability . For exp9a–d these were complexed with hydroxypropyl-γ-cyclodextrin (HP-γ-CD) for improved solubilisation. A 100 mM stock solution was prepared using DMSO (100%). The drug/ HP-γ-CD complex (1:2 molar ratio) was diluted to 10, 50 and 100 µM (relative to the drug concentration) [For all MTS, nitric oxide and ELISAs, the stock solutions (33 mM) of all the compounds were prepared in DMSO (100%) followed by appropriate dilutions. For the determination of cytotoxic effects of the compounds tration) . The determination of nitrite in biological samples was measured by the spectrophotometric method using Griess reagent. NO levels are quantified by estimating its stable end product (nitrite anion) which is formed as a result of nitric oxide oxidation . Experim® 20 in PBS). After the last wash, the plate was dried by blotting against clean paper towel, blocked by adding 300 µL/well of reagent diluent (RD) (1%).Sandwich ELISAs are a widely used standard method for the quantitative detection of cytokines and other specific proteins in serum samples. Substrate was added which reacted with the enzyme producing a colour change, the intensity of which is directly proportional to the concentration of antigen in incubation mixture allowing its spectrophotometric detection and quantification. Standard proteins of known concentrations are incorporated into the assay to allow quantification of specific proteins to be determined ,33,34. T2O2) is added. TMB reacts with H2O2 in the presence of HRP enzyme to produce a water-soluble, blue coloured by-product the intensity of which is proportional to the amount of HRP activity, which in turn is related to the levels of target analyte in the experimental sample. Upon acidification with sulphuric-acid (stop solution) the colour changes from blue to yellow, enabling accurate measurement of the intensity at 450 nm using a plate-reader. The 96-well microtitre plate is coated with capture antibody (1st antibody) and the target protein or antigen is added. Detection antibody (2nd antibody) is added followed by the addition of enzyme, horseradish peroxidase (HRP)-linked antibody. Substrate solution containing colorimetric substrate, 3,3’,5,5’-tetramethylbenzidine (TMB) and hydrogen peroxide (H


The structures of the newly synthesized compounds were confirmed by IR, 1H-, 13C-NMR, mass spectral data and elemental analysis. The antibacterial and antifungal activities of all the synthesized compounds were evaluated. The results indicated that compounds 9, 12, and 19 were found to be more potent than the standard drug Amphotericin B against Aspergillus fumigates. Additionally, compound 12 exhibited higher activity than the standard drug Amphotericin B against Syncephalastrum racemosum.Several new pyrazole, pyridine, triazolopyrimidine, benzimidazo[1,2- The str1 with 2-aminobenzimidazole (5) under the same experimental conditions gave only one isolable product, as examined by TLC. The structure of the obtained products were established as ethyl 5--4-phenyl-2-(phenylamino)thiophene-3-carboxylate (7) (1 with the 4-chloroaniline (8) gave the corresponding thiophene derivative 9 . For example, its proton NMR spectrum revealed the absence of the protons of the dimethylamino group present in the spectrum of the enaminone 1.In a similar manner, the reaction of the enaminone late (7) . The sugvative 9 . The str1 with 2,4-pentanedione in glacial acetic acid in the presence of ammonium acetate gave the pyridine derivative 12  [1H-NMR and MS) data. The structure of the isolated cycloadduct was assigned as ethyl 5-(3-acetyl-1-phenyl-1H-pyrazole-4-carbonyl)-4-phenyl-2-(phenylamino)thiophene-3-carboxylate (16) based on its elemental analyses and spectroscopic data. The 1H-NMR spectrum of the formed cycloadduct showed a singlet signal at δ 7.46, which corresponds to H-5 of the pyrazole ring in the product. This assignment is based on the fact that in the pyrazole ring, H-4, is more shielded than H-5. Thus, the signal of H-5 usually appears at δ 8.66–8.69 [1 with hydrazonoyl chloride 14 is regiospecific.Treatment of the enaminone ide (14) ,24 by th.66–8.69 , whereas.66–8.69 ,27. The 16, we suggested that the reaction proceed via 1,3-dipolarcycloaddition of the imine 13, derived from hydrazonoyl chloride 14, to the activated double bond in enaminone 1 to give the respective nonisolable cycloadduct 15, which underwent aromatization via elimination of the dimethylamine molecule to give the final pyrazole derivative 16 -4-phenyl-2-(phenylamino)thiophene-3-carboxylate (19)  . The rea(19)  operating at 400 MHz or on a JEOL ECP 300 NMR spectrometer operating at 300 MHz in deuterated chloroform (CDCl3) or dimethyl sulphoxide (DMSO-d6). Chemical shifts are referred to in terms of ppm and J-coupling constants are given in Hz. Mass spectra were recorded on a Shimadzu GCMS-QP 1000 EX mass spectrometer  at 70 eV. Elemental analysis was carried out on a Perkin Elmer 2400 Elemental Analyzer; CHN mode. The biological evaluations of the products were carried out in the Medical Mycology Laboratory of the Regional Center for Mycology and Biotechnology of Al-Azhar University, Cairo, Egypt.All the chemicals were purchased from various suppliers, including Sigma-Aldrich , and were used without further purification, unless otherwise stated. Melting points were measured on a Gallenkamp melting point apparatus  in open glass capillaries and are uncorrected. Infrared spectra (IR) were recorded using the KBr disc technique using a Perkin Elmer FT-IR spectrophotometer 1000 . 1  and the appropriate amine derivatives 2 or 5 or 8 (1 mmol each), in absolute ethanol (20 mL) were heated under reflux for 6 h and then left to cool. The precipitated solid product was filtered off, dried and recrystallized from EtOH to afford the corresponding thiophene derivatives 4, 7 and 9, respectively.General procedure: A mixture of enaminone Ethyl 5--4-phenyl-2-(phenylamino)thiophene-3-carboxylate (4). Yield (90%), yellow powder, m.p. 230–232 °C; IR (KBr) v 3422 (NH), 1619 (C=O) cm−1; 1H-NMR  δ 0.71 , 3.90 , 6.5 , 7.02–8.01 , 10.62 ; MS m/z (%): 442 (2.7%), 441 , 440 (5.5%), 194 (99.9%). Anal Calcd. for C24H19N5O2S (441.50): C, 65.29; H, 4.34; N, 15.86. Found: C, 65.18; H, 4.22; N, 15.92%.Ethyl 5--4-phenyl-2-(phenylamino)thiophene-3-carboxylate (7). Yield (85%), yellow powder, m.p. 214–216 °C; IR (KBr) v 3428 (NH), 1632 (C=O) cm−1; 1H-NMR  δ 0.71 , 3.89 , 7.19–7.35 , 7.74 , 8.12 , 10.62 ; MS m/z (%): 490 , 368 (99.9%), 255 (19.9%), 105 (35.9%). Anal Calcd. for C29H22N4O2S (490.58): C, 71.00; H, 4.52; N, 11.42. Found: C, 71.12; H, 4.48; N, 11.35%.Ethyl 5-(3-(4-chlorophenylamino)acryloyl)-4-phenyl-2-(phenylamino)thiophene-3-carboxylate (9). Yield (92%), yellow powder, m.p. 222–225 °C; IR (KBr) v 3426 (NH), 1652, 1622 (2 C=O) cm−1; 1H-NMR  δ 0.73 , 3.90 , 4.63 , 6.82–7.44 , 10.68 , 11.70 ; MS m/z (%) 502 , 460 (46.5%), 353 (99.9%), 309 (15.5%). Anal Calcd. for C28H23ClN2O3S (503.01): C, 66.86; H, 4.61; N, 5.57. Found: C, 66.75; H, 4.48; N, 5.65%.Ethyl 5-(5-acetyl-6-methylpyridin-2-yl)-4-phenyl-2-(phenylamino)thiophene-3-carboxylate (12). Ammonium acetate  was added to a solution of enaminone 1  and 2,4-pentanedione  in glacial acetic acid (10 mL). The reaction mixture was heated under reflux for 6 h. After cooling, the reaction mixture was poured onto ice water mixture then the formed solid product was filtered and recrystallized from ethanol to afford compound 12 as deep yellow needles, in yield (97%), m.p. 197–200 °C; IR (KBr) v 3424 (NH), 1708, 1675 (2C=O) cm−1; 1H-NMR  δ 0.74 , 2.44 , 2.72 , 3.92 , 6.24 , 7.11–7.46 , 10.54 ; MS m/z (%) 456 , 259 (99.9%), 238 (27.9%), 109 (55.1%), 55 (66.2%). Anal Calcd. for C27H24N2O3S (456.56): C, 71.03; H, 5.30; N, 6.14. Found: C, 71.15; H, 5.21; N, 6.22%.Ethyl 5-(3-acetyl-1-phenyl-1H-pyrazole-4-carbonyl)-4-phenyl-2-(phenylamino)thiophene-3-carboxylate (16). Triethylamine (2 mmol) was added to a mixture of enaminone 1  and the hydrazonoyl chloride 14 in dry benzene (20 mL). The resulting reaction mixture was heated under reflux for 8 h then left to cool. The solvent was distilled under reduced pressure. The residue was triturated with methanol and the solid product was collected by filtration, washed with methanol and recrystallized from ethanol to afford the pyrazole derivative 16 as pale brown powder, in yield (89%), m.p. 205–206 °C; IR(KBr) v 3445 (NH), 1684, 1653, 1619 (3C=O) cm−1; 1H-NMR  δ 0.6 , 2.64 , 3.8 , 6.89–7.43 , 7.46 , 10.78 ; MS m/z (%) 535 , 456 (99.9%), 413 (68.4%), 217 (51.1%). Anal Calcd. for C31H25N3O4S (535.61): C, 69.52; H, 4.70; N, 7.85. Found: C, 69.38; H, 4.56; N, 7.86%.Ethyl 5--4-phenyl-2-(phenyl amino)thiophene-3-carboxylate (19). To a cold solution of the enaminone 1  in pyridine (10 mL) was added portion wise the diazonium salt of aminotriazole 17  in concentrated HNO3 (2 mL) with a solution of sodium nitrite  in H2O (2 mL). After complete addition of the diazonium salt the reaction mixture was stirred in an ice bath for 30 min then filtered off, dried, and recrystallized from EtOH to afford the title compound as deep yellow powder (92% yield), m.p. >300 °C; IR (KBr) v 3418 (NH), 1657, 1622 (2C=O) cm−1; 1H-NMR  δ 0.71 , 3.88 , 7.50–7.25 , 8.45 , 9.00 , 10.61 ; MS m/z (%) 470  for C24H18N6O3S, 455 (75.3%), 227 (35.4%), 155 (99.9%), 91 (91.1%), 69 (35.2%). Anal Calcd. for C24H18N6O3S (470.50): C, 61.27; H, 3.86; N, 17.86. Found: C, 61.38; H, 3.77; N, 17.93%.(E)-Ethyl 4-phenyl-2-(phenylamino)-5-thiophene-3-carboxylate (21). A mixture of enaminone derivative 1  and triethylorthoformate (2 mL) was heated under reflux for 2 h. After cooling, ethanol was added and the formed solid product was filtered off and recrystallized from ethanol to afford pale red needles (86% yield), m.p. 204–206 °C; IR (KBr) v 3462 (NH), 1658, 1619 (2C=O), 1554 (C=C) cm−1;  δ 0.66 , 1.24 , 3.3 , 3.85 , 4.31 , 5.11 , 7.2–7.4 , 10.28 ; 13C-NMR  179.9, 162.4, 157.8, 149.7, 147.3, 140.4, 135.9, 133.0, 128.4, 127.9, 127.8, 126.5, 126.1, 120.8, 118.4, 117.8, 92.6, 58.9, 53.4, 22.7, 12.2; MS m/z (%) 523 , 511 (16%), 270 (40%), 180 (29.8%), 91 (99.9%). Anal Calcd. for C29H33NO6S (523.64): C, 66.52; H, 6.35; N, 2.67. Found: C, 66.48; H, 6.42; N, 2.55%.The microorganism inoculums were uniformly spread using sterile cotton swabs on a sterile Petri dish malt extract agar (for fungi) and nutrient agar (for bacteria). One hundred microliters of each sample were added to each well . Then, the systems were incubated for 24–48 h at 37 °C (for bacteria) and at 28 °C (for fungi). After incubation, the microorganism’s growth was observed. Inhibition of the bacterial and fungal growth were measured in mm. Tests were performed in triplicate .9, 12, and 19 showed a higher activity towards Aspergillus fumigates than the standard drug Amphotericin B.In conclusion various thiophene derivatives were synthesized in efficient and easy protocol. Antimicrobial studies revealed that compounds


N-substituted homophthalimides. Bromination of the latter produces 4,4-dibromo-homophthalimide derivatives that can be used as precursors for spiro-derivatives. The dibromo derivatives react with different binucleophilic reagents to produce several spiro-isoquinoline derivatives. Reaction of the dibromo derivatives with malononitrile produces dicyanomethylene derivatives which react with different binucleophiles to produce new spiro-derivatives. Structures of the newly synthesized compounds are proved using spectroscopic methods such as IR, 1H-NMR and 13C-NMR. The newly synthesized compounds were tested for their antimicrobial and antioxidant activities, showing weak or no antimicrobial activity. On the other hand select compounds showed promising antioxidant activities.Homophthalic anhydride reacts with different aromatic amines to produce  Spiro-compounds form a group of generally less investigated compounds. However, recently growing efforts have been made to synthesize and characterize these compounds. Many spiro-compounds possess very promising biological activities as anticancer agents ,2, antibThe microwave technique has several advantages over traditional methods of synthesis. Reduced reaction times ,22,23,241) was reacted with aromatic amines, namely aniline and p-anisidine, to afford N-arylhomophthalimide derivatives 2a,b, respectively, which were used as precursors for preparing new spiro-isoquinolines dione derivatives 3a,b. The mass spectrum of compound 3a displayed the expected molecular ion peaks at m/z 394 (3.2%), 395 (6.7%), 396 (3.0%). Compound 3b gave molecular ion peaks at 424 (7.3%), 425 (15.1%), 426 (6.8%). Homophthalic anhydride -dione (4a) and 2'-phenyl-1'H,3H-spiro-1',3'(2'H)-dione (4b), respectively. Compounds 4a and 4b are new ring systems. Synthesis of compounds 4a,b was carried out under two different reaction conditions, namely by conventional heating and using microwave irradiation conditions. Thus, when the reaction was carried out in a refluxing ethanolic sodium ethoxide solution for 5 h under TLC monitoring, the product 4a was obtained in 42% yield, while 4b was obtained in 48% yield. However, when the same reaction was carried out by heating at 140 °C in a microwave oven for 15 min, the yields of 4a and 4b were 81% and 85%, respectively. It was then concluded that using microwave as a source of heat not only improves the reaction yield, but also significantly reduces the reaction time. The IR and 1H-NMR spectra of compounds 4a and 4b agreed with the proposed structures. Their mass spectra showed the molecular ion peaks at m/z 341 (12.3%) and 342 (8.6%), respectively.Compound 3a,b reacted with thiosemicarbazide under both conventional and microwave reaction conditions and produced 2-phenyl-5'-thioxo-1H-spirotriazolidine]-1,3(2H)-dione (5a) and 2-(4-methoxyphenyl)-5'-thioxo-1H-spiro--triazol-idine]-1,3(2H)-dione (5b), respectively. Compounds 5 also have a new ring system. Again, comparing the reaction times and overall yields for both traditional and microwave methods showed that traditional methods took 4 h, to give 55% and 48% yields of 5a and 5b, respectively, while the microwave method needed only 15 min to give 88% and 79% yields of 5a and 5b, respectively. The comparison again proves the advantages of using the microwave technique. Analytical and spectral data of 5a and 5b were in agreement with the proposed structures.Similarly, compounds 3a,b reacted with malononitrile, as an activated methylene compound, by refluxing under TLC monitoring in absolute ethanol containing a catalytic amount of piperidine for 3 h, to produce -ylidene)-malononitrile (6a) and -malononitrile (6b), respectively  and 6'-amino-1,3-dioxo-2-phenyl-2'-thioxo-2,2',3,5'-tetrahydro-1H,3'H-spiro--5'-carbonitrile (8), respectively. Compound 8 has a new ring system. Synthesis of compounds 7 and 8 was performed under TLC monitoring in refluxing ethanol solution containing catalytic amounts of piperidine for 4 h and the yields were 53% and 59%, respectively. Carrying out the same reaction under microwave assisted conditions reduced the reaction time to 15 min and increased the yields to 84% and 86%, respectively, for 7 and 8. Acceptable analytical data were obtained for the new compounds 7 and 8.Compound Escherichia coli, Pseudomonas putida, Bacillus subtilis, Streptococcus lactis, Aspergillus niger, Penicillium sp. and Candida albicans. The preliminary screening of the investigated compounds was performed using the filter paper disc-diffusion method. The most active compounds were 4a, 5a, 5b and 8, which were slightly inhibitory to the microorganisms. The rest of compounds showed no sensitivity at all to the tested organisms, and the results are summarized in The newly synthesized heterocyclic compounds listed in 7 and 8 manifested potent anti-oxidative activity in the lipid peroxidation assay and considerable inhibitory activity in the hemolysis assay. 7 and 8 showed interesting anti-oxidant activity. 4a and 4b have moderate anti-oxidant properties. All compounds have been tested on bleomycin-dependent DNA damage. The results indicate that some compounds, namely 4a, 4b, 7 and 8, may have some protective activity towards DNA from the damage induced by bleomycin (Table 4).The newly synthesized compounds were tested for anti-oxidant activity as reflected in the ability to inhibit lipid peroxidation in rat brain and kidney homogenates and rat erythrocyte hemolysis. The pro-oxidant activities of the aforementioned compounds were assessed by their effects on bleomycin-induced DNA damage. 1H-NMR and 13C-NMR spectra were recorded on a Varian Mercury 300 MHz, and a Varian Gemini 200 MHz. spectrometers using TMS as an internal standard and DMSO-d6 as solvent. Chemical shifts were expressed as δ (ppm) units. Mass spectra were recorded at 70 eV on a Shimadzu GCMS-QP1000EX using an inlet type injector. All reactions were followed by TLC . The Microanalytical Center of Cairo University performed the microanalyses. Microwave reactions were performed with a Millstone Organic Synthesis Unit  with a continuous focused microwave power delivery system in a pressure glass vessel (10 mL) sealed with a septum under magnetic stirring. The temperature of the reaction mixture was monitored using a calibrated infrared temperature control under the reaction vessel, and control of the pressure was performed with a pressure sensor connected to the septum of the vessel.Melting points were determined in open glass capillaries on a Gallenkamp melting point apparatus and are uncorrected. IR spectra (KBr discs) were recorded on a Shimadzu FTIR-8201PC spectrophotometer. 2a  or 2b  in glacial acetic acid (20 mL) was heated under reflux with bromine  for 2 h. After cooling, the reaction mixture was poured onto ice-water and the solid that precipitated was filtered off, dried and crystallized from the proper solvent.A solution of either of 4,4-Dibromo-2-phenylisoquinoline-1,3-dione (3a): crystallized from ethanol in 65% yield as white crystals, m.p. 236-237 °C; 1H-NMR: 7.10-8.30 ; 13C-NMR: 70.2 (sp3 C-4), 120.5, 123.2, 124.3, 126.1, 127.4, 130.0, 131.5, 134.6, 135.2, 136.4 (aromatic C), 158.4, 167.6 (2 CO); IRν: 3066 cm−1 (aromatic CH), 1645 , 1605, 1500 (aromatic C=C); MS: M+m/z 394 (3.2%), 395 (6.7%), 396 (3.0%); Anal. Calcd. for C15H9Br2NO2 (395.04): C (45.60%), H (2.30%), Br (40.45%), N (3.55%); Found: C (45.3%), H (2.4%), Br (45.5%), N (3.7%).4,4-Dibromo-2-(4-methoxyphenyl)isoquinoline-1,3-dione (3b): crystallized from dilute dioxane in 58% yield as white crystals, m.p. 255-256 °C; 1H-NMR: 3.60 , δ 6.80-7.90 ppm ; 13C-NMR: 57.1 (OCH3), 71.0 (sp3 C-4), 115.3, 118.2, 124.2, 126.0, 127.2, 130.3, 131.6, 134.4, 135.1, 152.2 (aromatic C), 155.4, 163.6 (2 CO); IRν: 3060 cm−1 (aromatic CH), 1640 , 1605, 1500 (aromatic C=C); MS: M+m/z 424 (7.3%), 425 (15.1%), 426 (6.8%); Anal. Calcd. for C16H11Br2NO3 (425.07): C (45.21%), H (2.61%), Br (37.60%), N (3.30%); Found: C (45.1%), H (2.5%), Br (45.0%), N (3.6%).Method A: Compound 3a  was heated under reflux with either of o-phenylenediamine  or o-aminophenol  in ethanolic sodium ethoxide solution [prepared by dissolving metallic sodium  in absolute ethanol (25 mL)] for 5 h, under TLC monitoring. The reaction mixture was then cooled, acidified with few drops of conc. hydrochloric acid and the solid that precipitated was filtered at the pump and crystallized from the appropriate solvent.Method B: The same reactants of method A were heated in microwave at 500 W and 140 °C for 15 min. The reaction mixture was treated in a similar manner to method A to obtain compounds 4a,b.2'-Phenyl-1,3-dihydro-1'H-spiro-1',3'(2'H)-dione (4a): Crystallized from dimethylformamide as grey crystals, m.p. 218-219 °C, in 42% yield (Method A) or 81% yield (Method B); 1H-NMR: 3.80 , 6.40-7.70 ; 13C-NMR: 83.4 (sp3 spiro C), 113.0, 115.9, 120.2, 122.9, 127.3, 127.8, 128.3, 128.9, 131.2, 133.7, 135.1, 135.9, 136.8 (aromatic C), 155.4, 163.6 (2 CO); IRν: 3180 cm−1 , 3065 (aromatic CH), 1645,1655 (2C=O), 1605, 1500 (aromatic C=C); MS: M+m/z 341 (12.3%); Anal. Calcd. for C21H15N3O2 (341.36): C (73.89%), H (4.43%), N (12.31%); Found: C (74.1%), H (4.4%), N (12.1%).2'-Phenyl-1'H,3H-spiro-1',3'(2'H)-dione (4b): Crystallized from dioxane as white crystals, m.p. 237-238 °C, in 48% yield (Method A) and 85% yield (Method B); 1H-NMR: 4.30 , 6.60-7.80 ; 13C-NMR: 83.4 (sp3 spiro C), 116.0, 117.9, 118.2, 121.2, 124.9, 125.8, 127.5, 128.1, 128.5, 129.9, 132.1, 134.5, 135.4, 136.3, 138.8, 144.9 (aromatic C), 160.0, 168.9 (2 CO); IRν: 3210 cm−1 (NH), 3065 (aromatic CH), 1655,1670 (2C=O), 1605, 1500 (aromatic C=C); MS: M+m/z 342 (8.6%); Anal. Calcd. for C21H14N2O3 (342.35): C (73.68%), H (4.12%), N (8.18%); Found: C (73.3%), H (4.2%), N (7.9%).Method A: Each of compounds 3a  and 3b  was heated under reflux with thiosemicarbazide  in ethanolic sodium ethoxide solution  for 4 h, under TLC monitoring. The reaction mixture was then cooled, acidified with few drops of conc. hydrochloric acid and the solid that precipitated was filtered at the pump and crystallized from the appropriate solvent.Method B: The same reactants of method A were heated in microwave at 500 W and 140 °C for 15 min. The reaction mixture was treated in a similar manner to method A to give compounds 5a,b.2-Phenyl-5'-thioxo-1H-spirotriazolidine]-1,3(2H)-dione (5a): Crystallized from ethanol as white crystals, m.p. 188-189 °C, in 55% yield (Method A) and 88% yield (Method B); 1H-NMR: 2.10 , 2.30 , 2.40 , 7.10-8.30 ; 13C-NMR: 77.7 (sp3 spiro C), 121.1, 123.4, 127.6, 128.4, 128.8, 129.8, 132.1, 134.0, 135.2, 136.8 (aromatic C), 154.9 (CS), 159.5, 166.9 (2 CO); IRν: 3220, 3185, 3150 cm−1 (NH), 3060 (aromatic CH), 1645,1670 (2C=O), 1600, 1490 (aromatic C=C); MS (70 eV): M+m/z 324 (11.3%); Anal. Calcd. for C16H12N4O2S (324.36): C (59.25%), H (3.73%), N (17.27%), S (9.89%); Found: C (59.5%), H (3.9%), N (16.9%), S (10.0%).2-(4-Methoxyphenyl)-5'-thioxo-1H-spiro-triazolidine]-1,3(2H)-dione (5b): Crystallized from dilute dioxane as white crystals, m.p. 202-203 °C, in 48% yield (Method A) and 79% yield (Method B); 1H-NMR: 2.10 , 2.30 , 2.40 , 3.70 , 7.10-8.30 ; IR ν: 3220, 3185, 3150 cm−1 (NH), 3060 (aromatic CH), 1640, 1660 (2C=O), 1600, 1490 (aromatic C=C); MS: M+m/z 354 (13.8%). Anal. Calcd. for C17H14N4O3S (354.38): C (57.62%), H (3.98%), N (15.81%), S (9.05%); Found: C (57.3%), H (3.9%), N (16.1%), S (9.3%).3a  and 3b  in absolute ethanol (30 mL) containing a catalytic amount of piperidine was added malononitrile . The reaction mixture was heated under reflux for 3 h, under TLC monitoring, then cooled and poured onto ice-cold water. The solid product that separated was filtered off, dried and crystallized from ethanol.To a solution of each of compounds -ylidene)-malononitrile (6a): Obtained in 60% yield as pale yellow crystals, m.p. 224-225 °C; 1H-NMR: 7.00-8.10 ; 13C-NMR: 83.1 (ethylenic C), 112.0 (CN), 122.4, 124.7, 127.1, 127.8, 128.2, 128.6, 129.9, 131.0, 133.1, 133.9 (aromatic C), 147.3 (C3), 158.1, 163.0 (2 CO); IRν: 3060 cm−1 (aromatic CH), 2185 (CN), 1710, 1660 (2C=O), 1600, 1490 (aromatic C=C); MS: M+m/z 299 (28.2%); Anal. Calcd. for C18H9N3O2 (299.28): C (72.24%), H (3.03%), N (14.04%); Found: C (72.0%), H (3.3%), N (14.3%).malononitrile (6b): Obtained as yellow crystals in 67% yield, m.p. 241-242 °C; 1H-NMR: 3.70 , 6.70-8.00 ; 13C-NMR: 57.3 (OCH3), 83.0 (ethylenic C), 113.1 (CN), 115.2, 124.1, 126.4, 127.2, 127.9, 128.3, 129.5, 131.0, 133.1, 144.2 (aromatic C), 146.9 (C3), 158.4, 163.2 , 2195 (CN), 1710, 1670 (2C=O), 1600, 1490 (aromatic C=C); MS: M+m/z 329 (24.8%); Anal. Calcd. for C19H11N3O3 (329.31): C (69.30%), H (3.37%), N (12.76%); Found: C (69.5%), H (3.3%), N (12.4%).Method A: A solution of 6a  in absolute ethanol (30 mL) containing a catalytic amount of piperidine was heated under reflux with hydrazine hydrate  for 4 h under TLC monitoring. The reaction mixture was then cooled, poured onto ice-cold water. The solid that separated was filtered off, dried and crystallized from dilute dimethylformamide.Method B: The same reactants of method A were heated in microwave at 500 W and 140 °C for 15 min. The reaction mixture was treated in a similar manner to method A to obtain compound 7.7 was obtained as white crystals, m.p. 254-255 °C, in 53% yield (Method A) and 84% yield (Method B); 1H-NMR: 2.10 , 3.50 , 6.10  6.90-8.10 ; 13C-NMR: 44.1 (pyrazole C4), 55.3 (sp3 spiro C), 116.1 (CN), 120.2, 123.1, 126.9, 128.2, 128.9, 129.1, 129.7, 132.0, 135.1, 144.4 (aromatic C), 150.9 (pyrazole C3), 155.3, 169.2 (2 CO); IR ν: 3340, 3315, 3260 cm−1 (NH2 and NH), 3080 (aromatic CH), 2180 (CN), 1670,1640 (2C=O), 1600, 1490 (aromatic C=C); MS: M+m/z 331 (7.8%); Anal. Calcd. for C18H13N5O2 (331.33): C (65.25%), H (3.95%), N (21.14%); Found: C (65.5%), H (4.1%), N (21.5%).Compound Method A: A solution of 6a  in absolute ethanol (30 mL) containing a catalytic amount of piperidine was heated under reflux with thiourea  for 4 h under TLC monitoring. The reaction mixture was then cooled, poured onto ice-cold water. The solid that separated was filtered off, dried and crystallized from dilute dimethylformamide. Method B: The same reactants of method A were heated in microwave at 500 W and 140 °C for 15 min. The reaction mixture was treated in a similar manner to method A to obtain compound 8.8 was obtained as white crystals, m.p. 243-244°C, in 59% yield (Method A) and 86% yield (Method B); 1H-NMR: 2.10 , 2.70 , 3.30 , 6.90-7.90 ; 13C-NMR: 39.3 (pyrimidine C5), 54.9 (sp3 spiro C), 118.4 (CN), 121.1, 124.6, 126.3, 128.8, 129.4, 129.8, 130.76, 132.7, 135.9, 143.6 (aromatic C), 150.9 (pyrimidine C4), 158.1 (CS), 161.3, 169.2 (2 CO); IR ν: 3300, 3285, 3180 cm−1 (NH2 and NH), 3070 (aromatic CH), 2150 (CN), 1670,1645 (2C=O), 1600, 1490 (aromatic C=C); MS: M+m/z 375 (5.8%); Anal. Calcd. for C19H13N5O2S (375.40): C (60.79%), H (3.49%), N (18.66%), S (8.54%); Found: C (61.0%), H (3.6%), N (18.5%), S (8.6%).Compound Escherichia coli and Pseudomonas putide; (b) Gram-positive: Bacillus subtilis and Streptococcus lactis; (c) Fungi: Aspergillus niger and Penicillium sp.; (d) Yeast: Candida albicans. Media: Three types of specific media were used in this study: The newly synthesized heterocyclic compounds were tested for their antimicrobial activity against the following microorganisms: (a) Gram-negative: Medium 1: For bacteria (Nutrient Medium), consisting of (g/L distilled water): peptone, 5 and meat extract, 3. pH was adjusted to 7.0. Medium 2: For fungi (Potato Dextrose Medium), consisting of (g/L distilled water): Infusion from potatoes, 4 and D(+)glucose, 20. pH was adjusted to 5.5. Medium 3: For yeast , consisting of (g/L distilled water): yeast extract, 3; malt extract, 3; peptone, 5 and glucose, 10. pH was adjusted to 5.5. For solid media, 2% agar was added. All media were sterilized at 121 °C for 20 min. E. coli, P. putida, B. subtilis, S. lactis, A. niger, Penicillium sp. and C. albicans.Proper concentrations of microbial suspensions were prepared from 1 (for bacteria) to 3 (for yeast and fungi)-day-old liquid stock cultures incubated on a rotary shaker (100 rpm). In the case of fungi, five sterile glass beads were added to each culture flask. The mycelia were then subdivided by mechanical stirring at speed No. 1 for 30 min. Turbidity of microorganisms was adjusted with a spectrophotometer at 350 nm to give an optical density of 1.0. Appropriate agar plates were aseptically surface inoculated uniformly by a standard volume (ca. 1 mL) of the microbial broth culture of the tested microorganism, namely i.e. the discs should be separated by a distance that is equal to or slightly greater than the sum of the diameters of inhibition produced by each disc alone. Each test compound was conducted in triplicate. Plates were kept in the refrigerator at 5 °C for 1 h to permit good diffusion before transferring them to an incubator at 37 °C for 24 h for bacteria and at 30 °C for 72 h for yeast and fungi.Whatman No. 3 filter paper discs of 10 mm diameter were sterilized by autoclaving for 15 min at 121 °C. Test compounds were dissolved in 80% ethyl alcohol to give final concentration of 5 μg/mL. The sterile discs were impregnated with the test compounds (5 μg/disc). After the impregnated discs have been air dried, they were placed on the agar surface previously seeded with the organism to be tested. Discs were gently pressed with forceps to insure thorough contact with the media. Three discs were arranged per dish, suitably spaced apart, Blood was obtained from rats by cardiac puncture and collected in heparinized tubes. Erythrocytes were separated from plasma and the buffy coat and washed three times with 10 volumes of 0.15 M NaCl. During the last washing, the erythrocytes were centrifuged at 2,500 rpm for 10 min to obtain a constantly packed cell preparation. Erythrocyte hemolysis was mediated by peroxyl radicals in this assay system . A 10% s2 suspension, all prepared in phosphate buffer . The mixture was shaken, centrifuged, filtered to remove excess MnO2, and the absorbance (Acontrol) of the resulting green-blue solution  was adjusted at ca. 0.5 at λ 734 nm. Then, 50 μL of (2 mM) solution of the test compound in spectroscopic grade MeOH/ phosphate buffer (1:1) was added. The absorbance (Atest) was measured and the reduction in color intensity was expressed as % inhibition. The % inhibition for each compound is calculated from the following equation  solution (60 mM) was added to MnOequation : % InhibAscorbic acid (vitamin C) was used as standard anti-oxidant (positive control). Blank sample was run without ABTS and using MeOH/phosphate buffer (1:1) instead of sample. Negative control sample was run with MeOH/phosphate buffer (1:1) instead of tested compound.et al. [2 (5 mM), FeCl3 (50 μM) and samples to be tested at different concentrations. L-Ascorbic acid was used as a positive control. The mixture was incubated at 37 °C for 1 h. The reaction was terminated by addition of 0.05 mL EDTA (0.1 M). The color was developed by adding 0.5 mL thiobarbituric acid (TBA)  and 0.5 mL HCl  followed by heating at 80 °C for 10 min. After centrifugation, the extent of DNA damage was measured by increase in absorbance at 532 nm.The assay was done according to Aeschlach et al.  with min7 and 8 showed promising antioxidant activity.New spiro-compounds have been synthesized using both traditional methods and microwave-assisted conditions. The latter methods proved much more efficient in reducing reaction times as well as increasing the overall yield of the reactions. The newly synthesized compounds were tested for their antimicrobial and antioxidant activities. Some compounds showed moderate or weak antimicrobial activity, whereas compounds