Careful analyses of the 13C spectra of (–)-gusanlung A and natural 8-oxyberberrubine also cast doubt on the correctness of the structures previously assigned to these two compounds. (±)-Gusanlung A and (±)-gusanlung D were inactive against Staphylococcus aureus ATCC25932, Escherichia coli ATCC10536 and Candida albicans ATCC90028.(±)-Gusanlung A, 8-oxyberberrubine and (±)-gusanlung D have been synthesized by radical cyclisation of the corresponding 2-aroyl-1-methylenetetra- hydroisoquinolines. The  Acangelisia gusanlung H. S. Lo (Menispermaceae), is the first natural 8-oxotetrahydroprotoberberine alkaloid with an unoxygenated ring D  and 8-oxyberberrubine [1], for which structures 2 and 3 were proposed based on spectral analysis, were isolated from Acangelisia gusanlung H. S. Lo. In view of the uncertainty regarding the correct structure of (–)-gusanlung D (1), it was therefore highly desirable to also confirm the correctness of the structures proposed for (–)-gusanlung A (2) and 8-oxyberberrubine (3) by total syntheses.Furthermore, two new related alkaloids: (–)-gusalung A  with sodium chlorite gave 2-benzyloxy-6-bromo-3-methoxybenzoic acid (4b), whose acid chloride (4c) was then reacted with 6,7-methylenedioxy-1-methyl-3,4-dihydroisoquinoline (5) [8] in the presence of triethylamine to give thee moderately stable compound 6a. Treatment of 6a with tributyltin hydride in the presence of a catalytic amount of 2,2'-azobis(isobutyronitrile) gave a 31.3% yield of a mixture of (±)-9-benzylgusanlung A (7a) and 9-benzyl-8-oxyberberrubine (8a) in a ratio of 78:22 according to 1H-NMR analysis. Catalytic hydrogenolysis of the mixture of 7a and 8a to remove the benzyl protecting group also resulted in the concurrent hydrogenation of the C-C double bond to give pure (±)-gusanlung A (2). On the other hand, oxidation of the mixture of 7a and 8a with iodine gave 9-benzyl-8-oxyberberrubine (8a), whose benzyl protecting group was removed by acid treatment to give 8-oxy-berberrubine (3).Thus, oxidation of 2-benzyloxy-6-bromo-3-methoxybenzaldehyde (1H-NMR data of synthetic (±)-gusanlung A (2) were in reasonably good agreement with those reported for natural (–)-gusanlung A (2). However, a number of carbons in the 13C-NMR spectrum of natural (–)-gusanlung A (2) were found to have quite different chemical shifts from the corresponding carbons in the spectrum of (±)-gusanlung A (2). We therefore carried out 1H-1H-COSY, HMQC and HMBC experiments to allow complete assignments of chemical shifts of (±)-gusanlung A (2). Details of the HMBC correlations are shown in 1H-NMR spectral data of natural 8-oxyberberrubine (3) were found to be in good agreement with those of synthetic 8-oxyberberrubine (3). However, from HMBC correlation experiment, it was possible to establish that the chemical shifts of H-1 and H-13 previously assigned should be interchanged. On the other hand, the 13C spectrum of natural 8-oxyberberrubine (3) had a number of features which were quite different from those of synthetic 8-oxyberberrubine (3). These differences were highlighted and the HMBC correlations were shown in 1H-NMR analysis lent good support to the structures proposed for (–)-gusanlung A (2) and 8-oxyberberrubine (3), in view of the discrepancies in a number of carbon chemical shifts in the 13C-NMR spectra of (-)-gusanlung A (2) versus those of (±)-gusanlung A (2) on the one hand, and natural 8-oxyberberrubine (3) versus synthetic 8-oxyberberrubine (3) on the other, no definite conclusions can be drawn at this time concerning the correctness of the structures previously assigned to (–)-gusanlung A (2) and 8-oxyberberrubine (3).The 1) was uneventful. Thus, 2-iodobenzoyl chloride (4d) was reacted with 5 [8] in the presence of triethylamine to give the highly unstable 2-(2'-iodobenzoyl)-1-methylene-6,7-methylenedioxy-1,2,3,4-tetrahydroisoquinoline (6b). Treatment of 6b with tributyltin hydride in presence of a catalytic amount of 2,2'-azobis(isobutyronitrile) gave a 39.0% yield of a mixture of 1 and 8b in a ratio of 87:23 from 1H-NMR analysis. Treatment of the mixture with hydrazine and palladium/charcoal gave (±)-gusanlung D (1), whose 1H- and 13C-NMR data were in good agreement with those of (±)-gusanlung D (1) and (–)-gusanlung D obtained from previous syntheses   and triethylamine (1.0 g) in dry benzene (20 mL), then the mixture was refluxed for 2 h. On cooling, the precipitated triethylamine hydrochloride was filtered off. The filtrate was evaporated under vacuum to give enamide 6a as a yellow oil  which was unstable and decomposed on standing. It was immediately used in the next step without further purification. 1H-NMR: δ 7.38-7.23 , 7.18, 6.89 , 6.75 , 6.41 , 5.90 , 5.14 , 5.00 , 4.81 , 4.13-4.02, 3.57-3.50 , 3.80 , 2.90-2.59 ; 13C-NMR: δ 165.0 (C), 152.1 (C), 147.8 (C), 146.5 (C), 145.3 (C), 141.4 (C), 137.4 (C), 134.3 (C), 129.0 (C), 128.4 (CH), 128.1 (CH), 127.9 (CH), 127.7 (CH), 125.1 (C), 113.4 (CH), 110.0 (C), 108.4 (CH), 104.4 (CH2), 103.8 (CH), 101.1 (CH2), 75.4 (CH2), 55.9 (OCH3), 41.6 (CH2), 28.8 (CH2).(±)-Gusanlung A (1) and9-benzyl-8-oxyberberrubine (8a). A solution of enamide 6a , tributyltin hydride  and 2,2'-azobis(isobutyronitrile)  in dry benzene (50 mL) was refluxed with stirring for 3 h., then the solvent was removed under vacuum. The residue was washed with hexane (4 × 15 mL) and dissolved in chloroform (30 mL). The chloroform layer was washed with brine, then dried over anhydrous sodium sulfate. The solvent was removed under vacuum to give a yellow solid which was recrystallized from ethanol to give a 31.3% yield of a mixture of (±)-9-benzylgusanlung A (7a) and 9-benzyl-8-oxyberberrubine (8a) in a ratio of 78:22 from 1H-NMR analysis.8a and 7a  in ethanol (50 mL) was hydrogenated over 10% Pd/C (30.4 mg) at atmospheric pressure for 48 h. The catalyst was fittered off and the solvent was removed under vacuum to give a crude yellow solid. Recrystallization of the crude solid from ethanol gave (±)-gusanlung A (2) as a pale yellow soild , m.p. 188-189 °C; UV (MeOH) λmax nm (log ε): 219 (4.54), 271sh (3.87), 281 (3.98), 308 (4.16), 319 (4.15); IR νmax (film): 3737, 3650, 3585, 2919, 2852, 1748, 1634, 1615, 1581, 1542, 1506, 1488, 1456, 1386, 1356, 1336, 1315, 1262, 1239, 1154, 1084, 1069, 1037, 1001, 933, 858, 804, 792, 728 cm-1; MS (EI) m/z (%): 339 , 176 (100). 1H-NMR see 13C-NMR and HMBC see A solution of the mixture of 7a and 8a  and sodium acetate (1.5 g) in dioxane (50 mL), then the mixture was refluxed for 6 h. On cooling, the sodium acetate was filtered off and the precipitate was washed with chloroform (100 mL). The chloroform layer was washed with 5% NaHSO3 (100 mL), dilute NH3 (30 mL), H2O (100 mL) then dried over anh. Na2SO4. Removal of the solvent under vacuum gave a red solid which was recrystallized with ethanol to give 9-benzyl-8-oxyberberrubine (8a) as red crystals , m.p. 190-192 °C. UV (MeOH) λmax nm (log ε): 206sh (4.62), 224 (6.31), 255sh (5.78), 312 (5.76), 342 (6.03), 369 (5.86), 387 (5.71); IR νmax (film): 2938, 2898, 2841, 1651, 1619, 1599, 1494, 1484, 1386, 1372, 1317, 1277, 1225, 1176, 1100, 1083, 939, 871, 834, 777, 734 cm-1; 1H-NMR: δ 7.73-7.68 ; 7.43-7.32 ; 7.32-7.28 ; 7.22 , 6.72 ; 6.70 ; 6.00 ; 5.16 ; 4.31 ; 3.88 ; 2.88 ; 13C-NMR: δ 160.2 (C), 151.7 (C), 148.4 (C), 148.2 (C), 147.3 (C), 138.1 (C), 135.6 (C), 132.4 (C), 130.1 (C), 128.7 (CH), 128.2 (CH), 127.7 (CH), 123.8 (C) , 122.5 (CH), 119.8 (C), 119.1 (CH), 107.9 (CH), 104.7 (CH), 101.4 (CH2), 101.3 (CH), 75.7(CH2), 56.9 (OCH3), 39.5 (CH2), 28.7 (CH2). A solution of iodine  in dioxane (100 mL) was added dropwise over 30 min. to a refluxing solution of the mixture of 8-Oxyberberrubine (3). A solution of 8a  in ethanol (30 mL) and conc. HCl (30 mL) was refluxed for 3 h. On cooling, the solution was extracted with chloroform (50 mL). The extract was washed with water (50 mL), then dried over anh. Na2SO4. Removal of the solvent under vacuum gave a yellow solid which was recrystallized with ethanol to give 8-oxyberberrubine (3) as pale yellow crystals , m.p. 238-239 °C (Lit.  m.p. 175-177 °C). UV (MeOH) λmax nm (log ε): 206 (6.27), 230 (5.78), 253sh (5.42), 290 (5.42), 335 (5.02), 365 (4.77); IR νmax (film): 2922, 1646, 1602, 1576, 1487, 1412, 1362, 1333, 1285, 1241, 1218, 1178, 1141, 1038, 936, 906, 853, 743, 636, 505 cm-1. 1H-NMR and 13C-NMR see A mixture of 2) and (±)-gusanlung D (1) were determined by NCCLS microbroth dilution methods [2) and (±)-gusanlung D (1) were weighed and dissolved in DMSO to make a solution of concentration 2.56 mg/mL. From this stock solution two-fold serial dilution has been carried out to give a series of solutions from 256 μg/mL to 0.50 μg/mL with culture medium in 96-well microplates . Three different microorganisms were selected viz.Staphytolcoccus aureus ATCC25932, Escherichia coli ATCC10536 and Candida albicans ATCC90028. They were subcultured on nutrient broth supplemented with 10% glucose (NBG) (for bacteria) or Sabouraud glucose broth (for yeast) and incubated at 37 °C for 24 h. A final concentration of 1 x 105 cfu/mL of test bacteria or yeast was added to each dilution. The plates were incubated at 37 °C for 48 h. MIC was defined as the lowest concentration of test agent that inhibited bacterial or yeast growth, as indicated by the absence of turbidity. Test agent-free broth containing 5% DMSO was incubated as growth control. MIC of (±)-gusanlung A ( methods . (±)-Gus


All active compounds were tested in a second panel of clinical isolates of C. albicans and non-albicans Candida spp., Cryptococcus neoformans and dermatophytes. The eugenol derivative 4-allyl-2-methoxy-5-nitrophenol (2) was the most active structure against all strains tested, and therefore it was submitted to targeted assays. These studies showed that the antifungal activity of 2 was not reversed in the presence of an osmotic support such as sorbitol, suggesting that it does not act by inhibiting the fungal cell wall synthesis or assembly. On the other hand, the Ergosterol Assay showed that 2 did not bind to the main sterol of the fungal membrane up to 250 µg mL−1. In contrast, a 22% of fungal membrane damage was observed at concentrations = 1 × MIC and 71% at 4× MIC, when 2 was tested in the Cellular Leakage assay. The comparison of log P and MICs for all compounds revealed that the antifungal activity of the eugenol analogues would not to be related to lipophilicity.Twenty one phenylpropanoids (including eugenol and safrole) and synthetic analogues, thirteen of them new compounds, were evaluated for antifungal properties, first with non-targeted assays against a panel of human opportunistic pathogenic fungi. Some structure-activity relationships could be observed, mainly related to the influence of an allyl substituent at C-4, an OH group at C-1 and an OCH Fungi have emerged over the past two decades as major causes of human infections, especially among immunocompromised hosts, having an enormous impact on morbidity and mortality ,2. A mat1)  and analogues, have led to contradictory results. Zemek et al.  exhibited a moderate inhibitory effect on the same fungi with MICs 100–250 µg mL−1 in broth dilution methods.Some studies on the antifungal activity of eugenol   possess moderate activity against S. cerevisiae, Candida utilis, Pityrosporum ovale, and Penicillum chrysogenum, with MICs between 100 to 800 µg mL−1 in broth dilution methods with shaking, being P. ovale the most sensitive fungus. In a second report, Kubo et al.  according to their 4-substituent  + 210.0688, found 210.0692. 1H-NMR: 7.64 ; 7.26 ; 5.96 ; 5.67 ; 5.10 ; 3.98 ; 3.68  13C-NMR: 150.3 (C-2); 143.9 (C-1); 141.9 (C-5); 135.6 (C-2′); 129.0 (C-4); 116.7 (C-3′); 112.5 (C-3); 111.6 (C-6); 56.3 (OCH3); 37.4 (C-1′).2-Methoxy-3-nitro-4-allylphenol (4). A solution containing 2-methoxy-3-nitro-4-allylphenyl acetate  in methanol (20 mL) and K2CO3  was stirred overnight at r.t. Then, 0.1 M HCl was added to the mixture (until pH 2) and the organic phase was extracted with CH2Cl2 (3 × 15 mL), washed with water, dried over Na2SO4, filtered and evaporated. Subsequently, the mixture was chromatographed by CC eluting with petroleum ether/EtOAc mixtures of increasing polarity to give a quantitative yield (0.17 g) of a yellow oil corresponding to the desired product 4. IR (cm−1): 3,448 (OH); 1,531 (N-O); 1,504 (C=C); 1,372 (N-O); 1,280 (C=O); 830 (C-H). HRMS (EI): m/z calcd. for C10H11NO4 [M+1]+ 210.0688, found 210.0690. 1H-NMR: 7.03 ; 6.94 ; 5.84 ; 5.65 ; 5.09 ; 3.90 ; 3.03  13C-NMR: 148.0 (C-2); 138.9 (C-1 and C-3); 134.8 (C-2′); 126.1 (C-5); 124.2 (C-4); 118.1 (C-6); 117.2 (C-3′); 62.7 (OCH3); 34.9 (C-1′).2-Methoxy-3,5-dinitro-4-allylphenol (5). Compound 14  dissolved in CH2Cl2 (15 mL) was added to a mixture containing KHSO4 , NaNO3  and wet silica 50% W/W (0.549 g). The mixture was stirred 3 days at r.t., then filtered, and the solid was washed with CH2Cl2 and the solvent was evaporated under vacuum to give a reddish oil. Pure compound 5  was as a red oil obtained by repeated CC eluted with petroleum ether/EtOAc mixtures of increasing polarity. IR (cm−1): 3,450 (OH); 1,540 (N-O); 1,500 (C=C); 1,365 (N-O); 1,299 (C=O); 810 (C-H). HRMS (EI): m/z calcd. for C10H11NO4 [M+1]+ 255.0539, found 255.05942. 1H-NMR: 9.64 ; 6.90 ; 5.85 ; 5.17 ; 4.00 ; 3.35  13C-NMR: 150.8 (C-2); 143.8 (C-1); 133.6 (C-5); 125.5 (C-2′); 118.6 (C-4); 115.7 (C-3′); 117.2 (C-3); 57.0 (OCH3); 35.3 (C-1′).4-Allyl-1,2-phenyldiacetate (9). To a cold suspension of anhydrous AlCl3  in anhydrous CH2Cl2 (10 mL), a cold solution of safrole  in anhydrous CH2Cl2 (10 mL) was added dropwise under a nitrogen atmosphere and the reaction was stirred for 2 h at −10 °C. Then, the ice bath was removed and cold water (80 mL) was added, maintaining the stirring 24 h. The reaction mixture was poured into a saturated NaHCO3 solution and extracted with EtOAc (3 × 100 mL). The organic layer was washed with water, then dried over anhydrous MgSO4, filtered, evaporated and re-dissolved in acetone (5 mL). Subsequently, it was adsorbed on a silica gel column and chromatographed eluting with mixtures of petroleum ether/EtOAc of increasing polarity (17.0:3.0→15.0:5.0) to give an oil (0.310 g), which corresponded to a mixture of compounds. This mixture was dissolved in anhydrous CH2Cl2 (50 mL) and DMAP (3.06 mg) and Ac2O  were added. The reaction mixture was stirred for 2 h at r.t., cooled to 0 °C and KHSO4  were added. Then, the mixture was extracted with EtOAc (3 × 50 mL) washed with water, dried over anhydrous MgSO4, filtered, evaporated and re-dissolved in CH2Cl2 (5 mL). Subsequently, it was adsorbed on silica gel and chromatographed eluting with petroleum ether/EtOAc mixtures of increasing polarity to give 9 as an oil ; IR (cm−1): 2,957 (=C-H); 1,768 (C=O); 1,636 (C=C); 1,371 (CH3); 1,232 (C-O); 905 (-CH=CH2). MS : [M]+ 234 (13.0); 192 (54.9); 175 (26.6); 152 (25.4); 150 (100); 133 (21.5); 131 (21.6); 123 (27.7); 116 (24.3); 104 (19.6); 91 (18.7); 77 (15.4). HRMS (EI): m/z calcd. for C10H11NO4 [M+1]+ 235.0892, found 235.0895. 1H-NMR: 7.08 ; 7.00 ; 5.93 ; 5.12 ; 5.09 ; 3.38 ; 2.28 .  13C-NMR: 168.4 (CH3CO); 168.3 (CH3CO); 141.8 (C-4); 140.2 (C-2); 138.9 (C-1); 136.4 (C-2′); 126.6 (C-5); 123.3 (C-3); 123.1 (C-6); 116.6 (C-3′); 39.4 (C-1′); 20.6 (2× CH3CO).4-Allyl-5-nitro-1,2-phenyldiacetate (10). A solution containing compound 7 , DMAP (3.75 mg) and of Ac2O  in anhydrous CH2Cl2 (50 mL) was stirred 2 h at r.t. Then, the mixture was cooled to 0 °C and a 10% KHSO4 solution (50 mL) was added. The organic phase was extracted with EtOAc (3 × 50 mL), washed with water, dried over anhydrous MgSO4, filtered, evaporated and re-dissolved in CH2Cl2 (5 mL). Subsequently, it was adsorbed on silica gel and chromatographed (CC) eluting with petroleum ether/EtOAc mixtures of increasing polarity to afford 10 as a brown solid ; m.p. 62.0–63.7 °C. IR (cm−1): 3,083 (=C-H); 2,938 (C-H); 1,779 (C=O); 1,639 (C=C); 1,527 (C=C); 1,370 (CH3); 1,272 (C-O-C). MS : [M]+ 279 (<1%); 237 (18.4); 220 (25.2); 195 (48.1); 179 (12.9); 178 (100); 165 (40.1); 164 (21.8); 161 (25.0); 149 (11.3); 147 (13.3). HRMS (EI): m/z calcd. for C10H11NO4 [M+1]+ 280.0743, found 280.0748. 1H-NMR: 7.87 ; 7.21 ; 5.92 ; 5.12 ; 3.67 ; 2.30 .  13C-NMR: 167.5 (CH3CO); 167.3 (CH3CO); 145.7 (C-5); 145.5 (C-2); 140.3 (C-1); 134.2 (C-2′); 134.0 (C-4); 126.2 (C-3); 120.6 (C-3′); 117.9 (C-6); 36.5 (C-1′); 20.4 (CH3CO); 20.3 (CH3CO).4-Allyl-5-nitrobenzene-1,2-diol (11). A solution of 13  in CH2Cl2 (7.0 mL) was slowly added to a cold suspension (0 °C) of AlCl3  in CH2Cl2 (5.0 mL) under nitrogen atmosphere. The resulting mixture was stirred 2 h at −10 °C and cold water (approx. 10 mL) was added to the mixture, which was then stirred for 18 h at r.t. under nitrogen and then poured into brine and extracted with EtOAc (3 × 100 mL). The organic layer was washed with brine and then dried over anhydrous MgSO4, filtered, evaporated and re-dissolved in acetone (5 mL). Then, it was adsorbed on silica gel and chromatographed (CC) eluting with mixtures of petroleum ether/EtOAc of increasing polarity (17.0:3.0→15.0:5.0) to give 11 as an oil . IR (cm−1): 3,311 (O-H); 2,907 (C-H); 1,598 (C=C); 1,526 (NO2); 1,495 (C=C); 1,429 (-CH2); 1,326 (N=O); 1,275 (C-O); 1,045 (-C-OH); 809 (-C-H). 1H-NMR: 8.99 ; 7.57 ; 6.84 ; 5.95 ; 5.03 ; 3.61 ; 13C-NMR: 151.3 (C-2); 144.4 (C-1); 141.4 (C-5); 137.1 (C-2′); 129.7 (C-4); 118.4 (C-3′); 116.3 (C-3); 113.0 (C-6); 37.6 (C-1′).4-Allyl-5-nitro-1,2-methylenedioxy benzene (13). To a cold (−5 °C) solution of safrole  in acetic acid (8 mL), a mixture of nitric and sulfuric acids  was slowly added dropwise at −5 °C. and then stirred 4 h at −10 °C. Water (10 mL) was added and the mixture was extracted with EtOAc (3 × 50 mL). The aqueous layer was discarded and the organic layer was neutralized with a saturated solution of NaHCO3. The organic layer was dried over anhydrous MgSO4, filtered, evaporated and re-dissolved in CH2Cl2 (5 mL). Subsequently, it was adsorbed on silica gel and chromatographed (CC) eluting with mixtures of petroleum ether/EtOAc of increasing polarity (19.8:0.2→17.8:2.2) to give 13  as a viscous oil. IR (cm−1): 3,081 (=C-H); 2,912 (C-H); 1,616 (C=C); 1,523 (-NO2); 1,480 (C=C); 1,421 (-CH2); 1,328 (N=O); 1,257 (C-O-C); 927 (-C-O-C-); 817 (-C-H). MS : [M+1]+ 208 (2.8); [M]+ 207 (23.0); 190 (100); 177 (21.0); 176 (17.5); 173 (50.2); 162 (16.9); 160 (23.0); 132 (29.7); 103 (24.9); 102 (51.3). HRMS (EI): m/z calcd. for C10H9NO4 [M+1]+ 208.0532, found 280.0535. 1H-NMR: 7.49 ; 6.76 ; 6.09 ; 5.95 ; 5.10 ; 3.65 ; 13C-NMR: 151.7 (C-2); 146.5 (C-5 and C-1); 135.2 (C-2′); 132.2 (C-4); 117.0 (C-3′); 110.4 (C-3); 105.7 (OCH2O); 102.7 (C-6); 37.6 (C-1′).4-(3-Hydroxypropyl)-5-nitrobenzene-1,2-diol (14). BH3·DMS in THF  was added dropwise under a nitrogen atmosphere at −10 °C to compound 11  with stirring. Then, the reaction was allowed to reach r.t. and it was maintained 1 h at this temperature. The resulting intermediate was oxidized with a solution of NaBO3·4H2O  in water (100 mL). The mixture was stirred 2 h at r.t., the organic phase was extracted with ether (4 × 50 mL), washed with water, dried over anhydrous MgSO4, filtered, evaporated and re-dissolved in acetone (5 mL). Subsequently, it was subjected to CC eluting with mixtures of petroleum ether/EtOAc to give a yellow solid which upon recrystallization from MeOH/ethyl ether, gave pure 14 ; m.p.: 98.9-99.8 °C. IR (cm−1): 3,458 (O-H); 2,921 (=C-H); 1,593 (C=C); 1,532 (NO2); 1,457 (CH2); 1,385 (CH3); 1,331 (N=O); 880 (-C-H). 1H-NMR: 9.24 ; 8.88 ; 7.53 ; 6.85 ; 5.89 ; 4.39 ; 2.88 , 1.79 . 13C-NMR: 151.3 (C-2); 144.2 (C-1); 141.5 (C-5); 132.2 (C-4); 118.5 (C-3); 112.9 (C-6); 61.8 (C-3′); 34.4 (C-2′); 30.1 (C-1′).4-[3-(Acetyloxy)propyl]-1,2-phenyl diacetate (15). To compound 9  BH3·DMS in THF  was added slowly under a nitrogen atmosphere, over a period of 15 min, making sure to keep the temperature at −10 °C. Then, the reaction was taken to r.t. and maintained 1h at this temperature. The resulting organoborane intermediate was oxidized with a solution containing NaBO3·4H2O  in water (100 mL). The mixture was stirred 2 h at r.t. The organic phase was extracted with ether (4 × 50 mL), dried over anhydrous Na2SO4, filtered, evaporated and re-dissolved in 5 mL of acetone. Subsequently, it was subjected to CC eluting with mixtures of petroleum ether/EtOAc yielding 147.4 mg of a solid which corresponded to a mixture of compounds. The mixture was dissolved in anhydrous CH2Cl2 (50 mL) and DMAP (1.02 mg) and Ac2O  were added. The reaction was stirred 2 h at r.t. and then cooled at 0 °C. KHSO4  was added, and the mixture was extracted with EtOAc (3 × 50 mL), washed with water, dried over MgSO4, filtered, evaporated and re-dissolved in CH2Cl2 (5 mL). Subsequently, it was adsorbed on silica gel and chromatographed by CC eluting with petroleum ether/EtOAc mixtures of increasing polarity to give a colorless oil which corresponded to the desired product 15 . IR (cm−1): 2,925 (=C-H); 1,731 (C=O); 1,561 (C=C); 1,429 (-CH2); 1,214 (C-O-Ar); 1,076 (AcO); 896 (C-H). MS : [M]+ 294 (25.7); 282 (18.4); 281 (64.2); 267 (11.5); 222 (18.7); 221 (80.6); 207 (33.6); 147 (78.6); 73 (100). HRMS (EI): m/z calcd. for C10H9NO4 [M+1]+ 295.1103, found 295.1106. 1H-NMR: 7.08 ; 7.01 ; 4.10 ; 2.68 ; 2.26 ; 2.05 ; 1.98 . 13C-NMR: 171.1 (CH3CO); 168.4 (CH3CO); 168.3 (CH3CO); 141.9 (C-2); 140.2 (C-1); 140.1 (C-4); 126.5 (C-5); 123.2 (C-3); 123.1 (C-6); 63.6 (C-3′); 31.6 (C-2′); 29.9 (C-1′); 20.9 (CH3); 20.6 (2× CH3).3-phenylpropanol (16). Compound 12 , was hydroborated with a 2.0 M solution of BH3·DMS/THF (0.67 mL) added dropwise during 15 min under a nitrogen atmosphere at −10 °C. Then, the mixture was stirred 1 h at r.t. The resulting organoborane was oxidized with sodium perborate  in water (100 mL). The mixture was stirred 2 h at r.t. Then, it was extracted with ethyl ether (4 × 50 mL) and the layers were separated. The organic layer was dried over anhydrous MgSO4, filtered, evaporated and re-dissolved in CH2Cl2 (5 mL). It was adsorbed on a silica gel column and chromatographed eluting with mixtures of petroleum ether/EtOAc of increasing polarity (18.8:1.2→17.6:2.4) to give 0.66 g (59.4%) of compound 16 as a viscous oil; IR (cm−1): 3,330 (O-H); 2,909 (C-H); 1,495 (C=C); 1,439 (-CH2); 1,245 (C-O-C); 1,039 (-C-OH); 932 (C-O-C); 811 (-C-H). MS : [M+1]+ 181 (6.2); [M]+ 180 (51.6); 136 (51.2); 135 (100); 119 (5.4); 106 (9.5); 105 (7.8); 104 (5.1); 91 (10.5. HRMS (EI): m/z calcd. for C10H12O3 [M+1]+ 181.0786, found 181.0790. 1H-NMR: 6.73 ; 6.69 ; 6.64 ; 5.91 ; 3.65 ; 2.62 ; 1.84 ; 1.56 ; 13C-NMR: 147.5 (C-2); 145.6 (C-1); 135.6 (C-4); 121.1 (C-5); 108.8 (C-6); 108.1 (C-3); 100.7 (OCH2O); 62.1 (C-3′); 34.4 (C-1′); 31.7 (C-2′).3-phenyl propanol (17). A 2.0 M solution of BH3·DMS/THF (0.27 mL) was added dropwise over 15 min at −10 °C to compound 13  under a nitrogen atmosphere, and the mixture was stirred 1 h at r.t. The resultant organoborane was oxidized with sodium perborate  in water (100 mL) and then the mixture was stirred 2 h at r.t. Then, it was extracted with ethyl ether (4 × 50 mL) and the layers were separated. The organic layer was dried over MgSO4, filtered, evaporated and re-dissolved in CH2Cl2 (5 mL). It was adsorbed on silica gel, and chromatographed eluting with mixtures of petroleum ether/EtOAc of increasing polarity (16.0:4.0→14.0:6.0) to give compound 17  as a yellow solid; m.p. (85.9–87.9 °C); IR (cm−1): 3,211 (O-H); 2,907 (C-H); 1,613 (C=C); 1,521 (NO2); 1,419 (-CH2); 1,337 (N=O); 1,260 (C-O-C); 1,045 (-C-OH); 922 (C-O-C); 825 (-C-H). HRMS (EI): m/z calcd. for C10H11NO5 [M+1]+ 226.0637, found 226.0639. 1H-NMR: 7.46 ; 6.76 ; 6.08 ; 3.71 ; 2.96 ; 1.90  1.50 ; 13C-NMR: 151.7 (C-2); 146.3 (C-1); 142.8 (C-5); 134.4 (C-4); 110.6 (C-3); 105.7 (C-6); 102.7 (OCH2O); 62.0 (C-3′); 33.4 (C-2′); 30.1 (C-1′).3-phenyl propyl acetate (18). To a solution of 17  in dry CH2Cl2 (30 mL), DMAP (0.98 mg) and Ac2O  were added and the mixture was stirred 2 h at r.t. A solution of 10% KHSO4 (approx. 50 mL) was then added to this mixture. The aqueous layer was discarded and the organic layer was taken to neutrality with a saturated solution of NaHCO3 and water. It was dried over MgSO4, filtered, evaporated and re-dissolved in CH2Cl2 (5 mL), then chromatographed (CC) eluting with petroleum ether/EtOAc mixtures of increasing polarity (19.8:0.2→19.0:1.0) to give 18 as an oil . IR (cm−1): 2,778 (C-H); 1,735 (C=O); 1,619 (C=C); 1,516 (NO2); 1,425 (-CH2); 1,379 (CH3); 1,330 (N=O); 1,260 (C-O-C); 1,255 (C-O-C); 928 (C-O-C); 817 (-C-H). MS : [M]+ 267 (<1%); 208 (16.0); 191 (13.6); 190 (100); 189 (14.5); 178 (23.1); 173 (9.2); 163 (19.9); 148 (13.7); 136 (13.3); 135 (13.1); 132 (15.7); 104 (9.9); 77 (12.2). HRMS (EI): m/z calcd. for C12H13NO6 [M+1]+ 268.0743, found 268.0747. 1H-NMR: 7.43 ; 6.69 ; 6.05 ; 4.07 ; 2.89 ; 2.03 ; 1.93 . 13C-NMR: 170.4 (CH3CO); 151.6 (C-2); 146.3 (C-1); 142.6 (C-5); 133.5 (C-4); 110.6 (C-3); 105.6 (OCH2O); 102.7 (C-6); 63.4 (C-3′); 30.5 (C-2′); 29.3 (C-1′); 20.8 (CH3CO).4-(3-Acetoxypropyl)-5-nitro-1,2-phenyl diacetate (19). DMAP (3.75 mg) and Ac2O  were added to a solution of 14  in dry CH2Cl2 (60 mL) and the mixture was stirred 2 h at r.t. A solution of 10% KHSO4 (approx. 50 mL) was then added to this mixture. The aqueous layer was discarded and the organic layer was washed to neutrality with a saturated solution of NaHCO3 and water. Then, it was dried over MgSO4, filtered, evaporated and re-dissolved in CH2Cl2 (5 mL). Subsequently, it was adsorbed on a silica gel column and chromatographed with petroleum ether/EtOAc mixtures of increasing polarity (19.8:0.2→16.4:3.6) to give 19,  as a yellow solid; m.p. (62.0–63.7 °C); IR (cm−1): 3,083 (=C-H); 2,938 (C-H); 1,779 (C=O); 1,639 (C=C); 1,527 (C=C); 1,370 (CH3); 1,272 (C-O-C). MS : [M]+ 337 (<1%) 237 (18.4); 220 (25.2); 195 (48.1); 179 (12.9); 178 (100); 165 (40.1); 164 (21.8); 161 (25.0); 149 (11.3); 147 (13.3). HRMS (EI): m/z calcd. for C16H19NO7 [M+1]+ 338.1162, found 338.1166. 1H-NMR: 7.87 ; 7.21 ; 5.92 ; 5.12 ; 3.67 ; 2.30 . 13C-NMR: 167.4 (2× CH3CO); 145.7 (C-5); 145.5 (C-2); 140.3 (C-1); 134.2 (C-2′); 134.0 (C-4); 126.2 (C-3); 120.6 (C-3′); 117.9 (C-6); 36.5 (C-1′); 20.4 (2× CH3CO).C. albicans ATCC 10231, S. cerevisiae ATCC 9763, C. neoformans ATCC 32264, Aspergillus flavus ATCC 9170, A. fumigatus ATTC 26934, A. niger ATCC 9029, Trichophyton rubrum C 113, T. mentagrophytes ATCC 9972, and M. gypseum C 115.For the antifungal evaluation, standardized strains from the American Type Culture Collection , and the Center of Reference in Mycology  were used in a first instance of screening: Candida spp. ; nine strains of C. neoformans; six strains of T. rubrum andsix of T. mentagrophytes. The voucher specimen numberx of each isolate are presented in 3 cells/spores with colony forming units (CFU) mL−1 [Then, active compounds were tested against clinical isolates from CEREMIC and the Malbrán Institute [M, Buenos Aires, Argentina). The isolates included 12 strains of FU) mL−1 ,28.Minimum Inhibitory Concentration (MIC) of each compound was determined by using broth microdilution techniques according to the guidelines of the CLSI for yeasts (M27-A3) and for filamentous fungi (M 38 A2) ,17. MIC −1  and a final DMSO concentration ≤1%. A volume of 100 µL of inoculum suspension was added to each well with the exception of the sterility control where sterile water was added to the well instead. Ketoconazole, terbinafine, amphotericin B and itraconazole were used as positive controls.For the assay, stock solutions of pure compounds were diluted two-fold with RPMI from 250–0.98 µg mL100) of visual growth compared to the growth in the control wells containing no antifungal. MIC80 and MIC50 were defined as the lowest concentration of a compound that showed 80% or 50% reduction of the growth control respectively (culture media with the microorganism but without the addition of any compound) and was determined spectrophotometrically with the aid of a VERSA Max microplate reader . Endpoints were defined as the lowest concentration of drug resulting in total inhibition  of each compound against each isolate was also determined as follows: after determining the MIC, an aliquot of sample (5 µL) was withdrawn from each clear well of the microtiter tray and plated onto a 150-mm RPMI-1640 agar plate buffered with MOPS . Inoculated plates were incubated at 30 °C, and MFCs were recorded after 48 h. The MFC was defined as the lowest concentration of each compound that resulted in total inhibition of visible growth.(a) Sorbitol protection assay: MIC values were determined using C. albicans ATCC 10231 and C. neoformans ATCC 32264, by the standard broth microdilution procedure described above. Duplicate plates were prepared: one of them containing two-fold dilutions of 2 from 250 to 0.98 µg mL−1 and the other one, containing 2 at the same concentrations plus 0.8 mol L−1 sorbitol, in each well, as osmotic support. MICs were read at 2 and 7 days [(b) Ergosterol Effect Assay: MIC of 2 against C. albicans (ATCC 10231) was determined following the guidelines of CLSI as explained above, in the absence and in the presence of different (50–250 µg mL−1) concentrations of ergosterol  added to the assay medium, in different lines of the same microplate [croplate . Amphote(c) Cellular Leakage Assay: Cells of C. albicans ATCC 10231 cultured by shaking at 30 °C to early stationary phase (18 h growth), were washed with MOPS and re-suspended in MOPS to prepare the inoculums [4 cells mL−1) and compound 2, at 1×, 4× MIC were left from 2 to 48 h. At 2, 4, 6, 24 and 48 h, eppendorfs were centrifuged  and the supernatants (200 µL) were drawn on the wells of a 96-wells-microplate and thoroughly mixed. The extractable 260 nm-absorbing materials were determined by duplicate in a Beckman Coulter DTX 880 Multimode Detector, considering 100% release the absorbance produced by cells treated with 1.2 mol L−1 HClO4 at 100 °C, 30 min [noculums ,33. Eppe, 30 min ,33. Resup < 0.05 was considered significant.Data were statistical analyzed by the Student’s test. A albicans and non-albicans Candida strains, Cryptococcus neoformans and dermatophytes. The eugenol derivative 4-allyl-2-OMe-5-NO2-phenol (2) possesses a high activity in these second panels, and therefore it was submitted to targeted assays to gain insight into its mode of action. Results showed that the antifungal activity of 2 was not reversed in the presence of an osmotic support such as sorbitol, suggesting that it does not act by inhibiting the fungal cell wall synthesis or assembly. On the other hand, 2 did not show to bind to ergosterol up to 250 µg mL−1 in the Ergosterol Effect Assay, while a 22% of fungal membrane damage at concentrations = 1× MIC and 71% at 4× MIC, were observed at 48 h in the Cellular Leakage Assay. A series of twenty-one phenylpropanoids including eugenol, safrole and synthetic analogues, were evaluated for antifungal properties in a first instance of screening with CLSI standardized non-targeted assays against a panel of human opportunistic pathogenic fungi. Based on MIC results, some structure-activity relationships could be established. All active compounds were tested in a second panel of clinical isolates of log P and MIC for each compound revealed that the antifungal activity of the eugenol analogues studied here, would not to be related to lipophilicity.Regarding the influence of compounds’ solubility on the antifungal behavior, the comparison of


Wedelia trilobata (L.) Hitchc, a notoriously invasive weed in South China, led to the isolation of twenty-six ent-kaurane diterpenoids, including seven new ones 1–7. Their structures and relative configuration were elucidated on the basis of extensive spectroscopic analysis, including 1D- and 2D-NMR experiments. The antimicrobial activities of all isolated diterpenoids were evaluated against a panel of bacteria and fungi.Continued interest in the metabolites of  Wedelia trilobata is a notoriously invasive weed in a wide range of tropical and subtropical areas + . The 1H- and 13C-NMR data of 6 ,16-dien-19-oic acid. As compared with compound 20, the obvious difference was due to the presence of one more cinnamoyloxy group at C-3 in 6. HMBC correlations from H-3, H-2′, and H-3′ to C-1′ further validated the conclusion above. The ROESY correlations of H-3 with H-5 and H3-18 suggested that the cinnamoyloxy was α-orientated. Consequently, the structure of 6 was determined as 3α-cinnamoyloxy-ent-kaura-9(11), 16-dien-19-oic acid.The molecular formula of compound ata of 6  showed m7 was isolated as a white powder, and its molecular formula was determined as C29H36O6 by HREIMS based on m/z 480.2508 [M]+ . The IR spectrum showed absorptions at 3441 (OH), 1701 (C = O), and 1639 and 1449 cm−1 (aromatic C = C). The presence of a cinnamoyloxy moiety was deduced by comparison with the NMR data ; δC 134.2 and 149.2), a carboxyl group (δC 176.0), an O-bearing methylene group  and 4.14 ; δC 60.9), and an O-bearing quaternary carbon (δC 75.6). Further analyses demonstrated that compound 7 showed a closely similar NMR pattern to that of 6, indicating that compound 7 was a structural analogue of ent-kaurane-19-oic acid. The double bond was located between C-15 and C-16 by the HMBC cross-peaks of H-15 with C-8, C-14, C-16 and C-17. Meanwhile, the O-bearing methylene group was only connected to C-17 by the HMBC correlations from H2-17 to C-14, C-15, and C-16. At last, the O-bearing quaternary carbon could be attributed to C-9 due to the HMBC correlations of H2-7, H2-11, and H3-20 to C-9. The relative configuration of 7 was shown to be identical with that of 6 by NMR analysis. Thus, compound 7 was determined as 3α-cinnamoyloxy-9β, 17-dihydroxy-ent-kaur-15-en-19-oic acid.Compound NMR data  of compoent-kaurane derivatives, namely 16α-hydroxy-ent-kauran-19-oic acid (8) +, 859 [2M + Na]+, HR-EIMS m/z 418.2722 M+ ; 1H-NMR  and 13C-NMR , see 3α-Angeloyloxy-16α,17-dihydroxy-ent-kauran-19-oic acid (2): white powder, c 0.09, MeOH); UV (MeOH) λmax (log ε) 216 (3.79) nm; IR (KBr) νmax 3432, 2931, 2869, 2853, 1707, 1641, 1454, 1356, 1236, 1021, 988 cm−1; ESIMS m/z 457 [M + Na]+; HREIMS m/z 434.2672 [M]+ ; 1H- and 13C-NMR data see 3α-Tigloyloxy-16α-hydroxy-ent-kauran-19-oic acid (3): white powder, c 0.1, MeOH); UV (MeOH) λmax (log ε) 210.2 (3.6) nm; IR (KBr) νmax 3442, 2927, 2854, 1705, 1624, 1446, 1383, 1122, 1080, 1007 cm−1; positive-ion ESI-MS m/z 441 [M + Na]+, 859 [2M + Na]+, HR-EIMS m/z 418.2723 M+ ; 1H-NMR  and 13C-NMR , see 3α-Tigloyloxy-16α,17-dihydroxy-ent-kauran-19-oic acid (4): white powder, c 0.10, MeOH); UV (MeOH) λmax (log ε) 216 (3.84) nm; IR (KBr) νmax 3431, 2929, 2868, 2851, 1706, 1649, 1464, 1384, 1270, 1151, 1126, 1083, 1053 cm−1; ESIMS m/z 457 [M + Na]+; HREIMS m/z 434.2677 [M]+ ; 1H- and 13C-NMR data see 3α-Diydrocinnamoyloxy-ent-kaur-16-en-19-oic acid (5): white powder, c 0.14, MeOH); UV (MeOH) λmax (log ε) 204 (3.96), 261 (2.57) nm; IR (KBr) νmax 3441, 2927, 2855, 1732, 1703, 1631, 1453, 1181 cm−1; ESIMS m/z 473 [M + Na]+; HREIMS m/z 450.2781 [M]+ ; 1H- and 13C-NMR data see 3α-Cinnamoyloxy-ent-kaura-9(11),16-dien-19-oic acid (6): white powder, c 0.14, MeOH); UV (MeOH) λmax (log ε) 203 (3.74), 277 (3.67) nm; IR (KBr) νmax 3450, 2931, 2866, 1636, 1387, 1173 cm−1; ESIMS m/z 469 [M + Na]+; HREIMS m/z 446.2463 [M]+ ; 1H- and 13C-NMR data see 3α-Cinnamoyloxy-9β,17-dihydroxy-ent-kaur-15-en-19-oic acid (7): white powder, c 0.11, MeOH); UV (MeOH) λmax (log ε) 205 (4.21), 215 (4.20), 277 (4.28) nm; IR (KBr) νmax 3441, 2925, 2863, 1701, 1639, 1449, 1311, 1281, 1204, 1185 cm−1; ES−–MS m/z 479 [M − H]−; HREIMS m/z 480.2508 [M]+ ; 1H- and 13C-NMR data see Pseudomonas aeruginosa (ATCC 27853), Staphyloccocus aureus (ATCC 25923) and Escherichia coli (ATCC 25922)  and the fungus Monilia albicans (ATCC Y0109) (grown on Sabauraud′s medium). For the agar plate punch assay [9 cfu/mL; cfu = colony forming unit). All active compounds with a diameter of inhibition greater than 10 mm were submitted to minimum inhibitory concentration testing. The MICs of compounds 2, 4, 7, 10, 12, and 13 against M. albicans, 1R , 2R, 3R, 4R, 5R, and 535R were determined using a 2-fold dilution method [4 cfu/mL of bacterial culture was added to each well (100 μL/well) to final concentrations in a range of 1.95−250 μg/mL. After incubating at 37 °C for 18 h, the lowest concentration without any colony growth was recorded as the MIC value. The resulting values were compared with the value for a positive control  under the same conditions.The strains used in antimicrobial tests were obtained from the Research Center of Natural Medicine, Clinical School of Kunming General Hospital of Chengdu Military Command. The test organisms in this bioassay were the bacteria  ATCC 259 and Esch ATCC 259 and Eschent-kaurane diterpenoids together with nineteen known ent-kaurane derivatives from Wedelia trilobata (L.) Hitchc, a notoriously invasive weed in South China. The structures of the new compounds were identified based on detailed spectroscopic analysis and comparison with the published data of analogues. All ent-kaurane derivatives isolated were tested against Pseudomonas aeruginosa, Staphyloccocusaureus, Monilia albicans and Escherichia coli, and compounds 2, 4, 7, 10, 12, and 13 showed weak activities against M. albicans. Taken together with data from prior research [W. trilobata can be drawn and they may be significant as chemical defenses allowing this notoriously invasive weed to adapt to varying surroundings rapidly and effectively.A systematic chemical search was performed and resulted in the separation of seven new


E)-3-hex-4-enoic acids (4) by kinetically controlled bromolactonization with N-bromosuccinimide (NBS). γ-Bromo-δ-lactones (5) were isolated as the major products. Absolute configurations of stereogenic centers of γ-bromo-δ-lactones (5) were assigned based on X-ray analysis; configurations of cis δ-bromo-γ-lactones (6) and trans δ-bromo-γ-lactones (7) were determined based on mechanism of bromolactonization. Synthesized compounds exhibited significant antiproliferative activity towards the four canine cancer cell lines  and one human cancer line (Jurkat). Classifying the compounds in terms of activity, the most active were enantiomers of trans δ-bromo-γ-lactones (7) followed by enantiomers of cis isomer (6) and enantiomeric γ-bromo-δ-lactones (5). Higher activity was observed for all stereoisomers with S configuration at C-4 in comparison with their enantiomers with 4R configuration. Synthesized compounds did not induce hemolysis of erythrocytes. The results of the interaction of bromolactones with red blood cell membranes suggest that these compounds incorporate into biological membranes, concentrating mainly in the hydrophilic part of the bilayer but have practically no influence on fluidity in the hydrophobic region. The differences in interactions with the membrane between particular enantiomers were observed only for γ-lactones: stronger interactions were found for enantiomer 4R,5R,6S of cis γ-lactone (6) and for enantiomer 4S,5R,6S of trans γ-lactone (7).Three novel enantiomeric pairs of bromolactones possesing a 2,5-dimethylphenyl substituent at the β-position of the lactone ring have been synthesized from corresponding enantiomeric ( Cytotoxic activity against different cancer cell lines is one of the most characteristic biological activities of lactones possessing an aromatic ring in their structures. Among this diverse group of compounds many reports concern antiproliferative activity of natural lactones i.a. coumarin , campthotrans-δ-iodo-γ-lactones possessing 2,5-dimethylphenyl ring were proved to induce caspase-dependent apoptosis through downregulation of the expression of antiapoptotic proteins Bcl-xL and Bcl-2  were more active than their antipodes derived from (S)-acid 4. This correlation is particularly observed for the activity of enantiomeric trans δ-bromo-γ-lactones 7 towards D17 cell line, in this case enantiomer 4S,5R,6S (entry 6) inhibited the proliferation whereas 4R,5S,6R was inactive (entry 5). For all tested enantiomeric pairs of bromolactones significant correlation between configurations of their stereogenic centers and cytotoxic effect was also found for GL-1 cell line; in this case the differences in activity between enantiomers were around 50%.All tested bromolactones (entries 1–6) exhibited noticeable concentration-dependent activity against examined hematopoietic cancer cell lines whereas only isomer cis (6) and trans δ-bromo-γ-lactones (7) with their iodo analogs 9,10 reported earlier  in 10% H2SO4.Analytical Thin Layer Chromatography (TLC) was carried out on aluminium plates covered with a silica gel (DC-Alufolien Kieselgel 60 F−1), 200–300 °C (30 °C min−1), final column temperature 300 °C (1 min). Total time of analysis 9.3 min.Gas chromatography (GC) was performed on Agilent Technologies 6890N instrument  equipped with autosampler and FID detector using hydrogen as a carrier gas. Progress of bromolactonization and the purity of isolated compounds was checked on DB-5HT column  under the following conditions, injector 200 °C, detector 280 °C, initial column temperature: 100 °C, 100–200 °C (20 °C min−1), final column temperature 200 °C (1 min). Total time of analysis 301 min.Chiral Gas Chromatography (CGC) was carried out on CP Chirasil-DEX CB column  with a temperature program as follows, injector 280 °C, detector 250 °C; initial column temperature 50 °C, 50–200 °C  were recorded on a Bruker Avance II 600 MHz spectrometer  for CDCl3 solutions, with signals of residual solvent  as references for chemical shifts. Infrared spectroscopy (IR) was conducted using Nicolet iS10 FTIR Spectrometer  equipped with monolithic diamond ATR crystal attachment. High-resolution mass spectra (HRMS) were recorded using electron spray ionization (ESI) technique on spectrometer Waters ESI-Q-TOF Premier XE . Nuclear magnetic resonance spectra -5 and (−)--5 reported in this paper, have been deposited with the Cambridge Crystallographic Data Centre (CCDC) as supplementary publication numbers 1874584 and 1874585, respectively. Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB12 1EZ, UK (fax +44-1223-336033 or e-mail: deposit@ccdc.cam.ac.uk).X-ray data were collected on a Xcalibur Sapphire2 diffractometer . X-ray data were collected at 100 K using an Oxford Cryosystem device. Data reduction and analysis were carried out with the CrysAlis ‘RED’ or CrysAlisPro program , 2001, 2003). Analytical numeric absorption correction using a multifaceted crystal model based on expressions derived by Clark and Reid  was appl program . Non-hyd4 (6.9 mmol), NBS (14 mmol) and a drop of acetic acid were dissolved in 70 mL of THF and the reaction mixture was stirred at room temperature. When the substrate reacted completely , the mixture was successively washed with saturated solutions of NaHCO3 and NaCl. Organic layer was separated and dried over anhydrous MgSO4. After evaporation of solvent in vacuo the products were separated by silica gel column chromatography using hexane followed by hexane/acetone, 20:1, as elution systems.Acid S,E)-3-hex-4-enoic acid (4)  afforded products with physical and spectral data given below:Bromolactonization of (+)-((+)--5-t-Bromo-4-r--6-c-methyltetrahydropyran-2-one (5): Yield 24% (0.486 g), colourless crystals, mp = 113–120 °C, Rf = 0.33 , ee > 99%; tR = 213.3 min, 2Cl2); IR : 1720 (s), 1506 (w), 1231 (s), 1037 (s), 972 (m), 820 (m), 632 (m); 1H NMR , δ 1.68 , 2.32, 2.33 , 2.63 , 2.97 , 3.74 , 4.05 , 4.68 , 6.92 , 7.01 , 7.08 ; 13C NMR  δ 19.2 (CH3-2′), 20.7 (C-7), 21.2 (CH3-5′), 37.7 (C-3), 41.8 (C-4), 54.2 (C-5), 80.1 (C-6), 125.6 (C-6′), 128.3 (C-4′), 130.7 (C-3′), 132.6 (C-2′), 136.5 (C-5′), 138.8 (C-1′), 169.2 (C-2); HRMS: calcd for C14H17BrO2 [M + Na]+: 319.0309, found 319.0307.R,5R,6S)-5: C14H17BrO2, M = 297.18, orthorhombic, P212121, a = 6.129(2), b = 10.839(3), c = 19.596(3) Å, V = 1301.8(6) Å3, Z = 4, Dc = 1.516 Mg·m−3, T = 100(2) K, R = 0.024, wR = 0.044 (3637 reflections with I > 2σ(I)) for 154 variables, Flack parameter: 0.000(4); CCDC 1874584.Crystal data for (+)-(4(−)-cis--5-(1-Bromoethyl)-4-dihydrofuran-2-one (6): Yield 7% (0.135 g); colourless solid; mp = 110–115 °C, Rf = 0.47 , ee > 99%, tR = 196.6 min; c 0.5, CH2Cl2); IR : 1774 (s), 1506 (w), 1185 (s), 1147 (s), 989 (m), 823 (m), 629 (m); 1H NMR  δ 1.80 , 2.29 , 2.37 , 2.61 , 3.09 , 3.80 , 4.11 , 4.75 , 6.86 , 7.00 , 7.06 ; 13C NMR  δ 19.9 (CH3-2′), 21.2 (CH3-5′), 23.4 (C-7), 38.4 (C-3), 38.5 (C-4), 45.3 (C-6), 86.8 (C-5), 126.4 (C-6′), 128.5 (C-4′), 130.9 (C-3′), 133.3 (C-2′), 136.1 (C-5′), 136.4 (C-1′), 176.4 (C-2); HRMS: calcd for C14H17BrO2 [M + Na]+: 319.0309, found 319.0305.(+)-trans--5-(1-Bromoethyl)-4-dihydrofuran-2-one (7): Yield 11% (0.233 g); dense, colourless liquid; Rf = 0.38 , ee = 99%, tR = 212.9 min; c 1.0, CH2Cl2); IR : 1779 (s), 1505 (w), 1151 (s), 1006 (s), 811 (m), 634 (m); 1H NMR  δ 1.69 , 2.34 , 2.38 , 2.54 , 3.14 , 3.95 , 4.33 , 4.67 , 7.01–7.03 , 7.09 ; 13C NMR  δ 19.4 (CH3-2′), 21.1 (CH3-5′), 21.5 (C-7), 37.7 (C-3), 38.9 (C-4), 50.1 (C-6), 88.6 (C-5), 126.3 (C-6′), 128.1 (C-4′), 130.8 (C-3′), 131.9 (C-2′), 136.8 (C-5′), 139.8 (C-1′), 175.2 (C-2); HRMS: calcd for C14H17BrO2 [M + Na]+: 319.0309, found 319.0311.−)--3-hex-4-enoic acid 4  afforded products with following data:Bromolactonization of (5):(−)--5-t-Bromo-4-r--6-c-methyltetrahydropyran-2-one ( Yield 16% (0.75 g); colourless crystals, mp 121−125 °C; ee > 99%; tR = 212.7 min, c 0.5, CH2Cl2); spectroscopic data identical to those reported herein for (+)--5.S,5S,6R)-5: C14H17BrO2, 297.18, orthorhombic, P212121, a = 6.142(2), b = 10.872(3), c = 19.627(3) Å, V = 1310.6(6) Å3, Z = 4, Dc = 1.506 Mg·m−3, T = 100(2) K, R = 0.046, wR = 0.111 (5312 reflections with I > 2σ(I)) for 154 variables, Flack parameter: – 0.007(8); CCDC 1874585.Crystal data for (−)-(4(+)-cis--5-(1-Bromoethyl)-4-dihydrofuran-2-one (6): Yield 10% (0.156 g); colourless solid, mp 113−119 °C; ee > 99%; tR = 194.8 min, c 0.5, CH2Cl2); spectroscopic data identical to those reported herein for (−)--6.(−)-trans--5-(1-Bromoethyl)-4-dihydrofuran-2-one (7): Yield 11% (0.174 g); dense, colourless liquid; ee = 99%; tR = 211.1 min, c 1.0, CH2Cl2); spectroscopic data identical to those reported herein for (+)--7.2 incubator in a humidified atmosphere at 37 °C using RPMI 1640 medium  supplemented with 2 mM l-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% fetal bovine serum (FBS)  for GL-1, D17 and Jurkat cells and 20% FBS for CLBL-1 and CLB70 cells. To keep at an optimal density (50–70% confluence) cells were cultured in 25 cm2 cell flasks  and subcultivated every other day. D17 cell line (canine osteosarcoma) was obtained from American Type Culture Collection . The B cell lymphoma cell line (CLBL-1) was obtained from Barbara C. Rütgen, Institute of Immunology, Department of Pathobiology of the University of Veterinary Medicine, Vienna, Austria . The canCytotoxicity of the tested bromolactones was determined using MTT assay. Initial solutions of these compounds at a concentration of 10 mg/mL in DMSO  were further diluted using the culture media as a solvent to prepare solutions within a concentration range of 0.05 to 50 μg/mL (DMSO concentration was less than 1% in each dilution). The details of the procedure were described in our previous work .In order to determine the cytotoxicity of tested compounds, a hemolytic activity test was performed using pig red blood cells (RBCs). The method applied was based on that described by Łuczyński et al. . The hemThe interaction of tested compounds with red blood cells membranes (RBCM) was investigated based on a fluorimetric method described earlier by Włoch et al. . RBCM weE)-3-hex-4-enoic acids (4) afforded three enantiomeric pairs of β--bromolactones: γ-bromo-δ-lactone (5), cis δ-bromo-γ-lactone (6), and trans δ-bromo-γ-lactone (7), with defined configurations of stereogenic centers. They showed significant cytotoxicity against canine cancer cell lines, the most potent were enantiomers of trans γ-lactone 7. In all cases higher activity was found for enantiomers obtained from (R)-acid 4. On the other hand, all tested bromolactones did not exhibit cytotoxic activity towards erythrocytes. The results of fluorescence spectroscopy suggest that these compounds concentrate mainly in the hydrophilic part of erythrocyte membrane but have practically no influence on fluidity in the hydrophobic region. The differences in interactions with the membrane between particular enantiomers were observed only for γ-lactones; stronger interactions were found for enantiomer 4R,5R,6S of cis γ-lactone 6 and for enantiomer 4S,5R,6S of trans γ-lactone 7Bromolactonization of enantiomeric (Preliminary studies presented in this work indicate that tested bromolactones are potentially good candidates as anticancer drugs and could be used in future in pharmacological preparations.


Nocardia ignorata, isolated from the terrestrial lichen Collema auriforme, was studied. The strain was cultivated in a BioFlo 115 bioreactor, and the culture medium was extracted using an XAD7HP resin. Five known diketopiperazines: cyclo (l-Pro-l-OMet) (1), cyclo (l-Pro-l-Tyr) (2), cyclo (d-Pro-l-Tyr) (3), cyclo  (4), cyclo (l-Pro-l-Leu) (5), and one auxin derivative: indole-carboxaldehyde (8) were isolated, along with two new brominated diketopiperazines: cyclo (d-Pro-l-Br-Tyr) (6) and cyclo (l-Pro-l-Br-Tyr) (7). Structure elucidation was performed using HRMS and 1D and 2D NMR analysis, and the synthesis of compounds 6 and 7 was carried out in order to confirm their structure.Actinobacteria are well known for their potential in biotechnology and their production of metabolites of interest. Lichens are a promising source of new bacterial strains, especially Actinobacteria, which afford a broad chemical diversity. In this context, the culture medium of the actinobacterium  Proteobacteria, Firmicutes, and Actinobacteria + .l-prolinateMethyl ((S)-3-(3-bromo-4-hydroxyphenyl)-2-((tert-butoxycarbonyl)amino)propanoyl)- (13b): prepared from 11  and 12b  in 50 mL of solvent ; white oil; c 0.77, CHCl3), 1H-NMR  mixture of rotamers δ 7.55 and 7.37 , 7.25 and 7.10 , 7.04 and 6.89 , 4.82 and 4.60 , 4.40 and 4.50 , 3.62 and 3.75 , 3.40, 3.21 and 3.67–3.55 , 3.35 and 3.02 , 3.17 and 2.82 , 2.18 and 2.05 , 1.96 and 1.81 , 2.02–1.88 and 1.74 , 1.39 3), 13C-NMR  δ 172.5 and 172.2 (C-15), 170.2 and 170.1 (C-9), 155.2 (Boc C = O), 148.5 and 148.2 (C-1), 134.7 and 133.1 (C-3), 129.7 (C-4), 130.3 and 130.1 (C-5), 123.3 and 116.4 (C-6), 109.8 and 109.7 (C-2), 80.1 (Boc C-(CH3)3), 59.2 and 53.1 (C-14), 54.6 and 52.8 (C-8), 52.5 and 52.4 (C-16), 47.2 (C-11), 38.3 and 37.3 (C-7), 30.7 and 29.2 (C-12), 28.4 (Boc C-(CH3)3), 25.0 and 22.4 (C-13), HRESIMS m/z 493.0941 [M + Na]+ .13a  and 13b ) were poured into a reactor with respectively 15 mL and 3 mL of water and heated to 130 °C during 4 h as described in literature for a one-pot deprotection and cyclization + .a CDCl3, 5 MHz as l-Pro-l-Tyr)Cyclo ( (2): 5.7 mg, white amorphous powder; r.t.: 20.7 min, c 1, MeOH), 1H-NMR  and 13C-NMR data  as described in the literature [m/z 261.1225 [M + H]+ .terature , HRESIMSd-Pro-l-Tyr)Cyclo ( (3): 8.5 mg, white amorphous powder; r.t.: 21.1 min, c 1, MeOH), 1H-NMR  and 13C-NMR data  as described in the literature [m/z 261.1225 [M + H]+ .l-Pro-l-Val)Cyclo ( (4): 1.1 mg, white amorphous powder; r.t.: 21.9 min, c 1, CHCl3), 1H-NMR  and 13C-NMR data  as described in the literature [m/z 197.1282 [M + H]+ .terature , HRESIMSl-Pro-l-Leu)Cyclo ( (5): 4.3 mg, white amorphous powder; r.t.: 24.1 min, 1H-NMR  and 13C-NMR data  as described in the literature [m/z 211.1438 [M + H]+ .terature ,15, HRESd-Pro-l-Br-Tyr)Cyclo ( (6): 1.4 mg, white amorphous powder; r.t.: 23.4 min, c 0.77, MeOH), 1H-NMR  and 13C-NMR data  as described in m/z 339.0329 [M + H]+ .l-Pro-l-Br-Tyr)Cyclo ( (7): 2.1 mg, white amorphous powder; r.t.: 23.9 min, c 0.77, MeOH), 1H-NMR  and 13C-NMR data  as described in m/z 339.0329 [M + H]+ .Indole-Carboxaldehyde (8): 5.0 mg, white amorphous powder; r.t.: 26.2 min, 1H-NMR  and 13C-NMR data  as described in the literature [m/z 146.0598 [M + H]+ .terature , HRESIMSNocardia ignorata. The synthesis of the new compounds was realized, and confirmed the structure elucidated by 2D NMR data and HRMS. None of the compounds isolated—except the auxin derivative—presented cytotoxic activity, but these compounds are well known for playing a role in bacterial communication. The role of diketopiperazines in quorum sensing is currently described. These compounds could thus interact with N-acylhomoserine lactone (AHL) biosensor and in this way they can modulate bacterial characters such as bioluminescence (via LuxR from Vibrio fischeri) [Serratia liquefaciens [In this study two new diketopiperazines as well as five known diketopiperazines and one known auxin derivative have been isolated from the culture medium of our strain ischeri) , swarminefaciens , pathogeefaciens ,24, and efaciens . These tefaciens . The two


XZ-01 of Camptotheca acuminate. Their structures were elucidated by spectroscopic analyses, including 1H- and 13C-NMR, 2D NMR  and HR-FT-MS. Cytotoxic activities of these compounds were evaluated. Some of them showed weak selective activities.Four new metabolites, including three new oblongolides named C1, P1, and X1 ( Camptotheca acuminate Decne (Cornaceae), numerous new compounds were obtained , quaternary carbon  and methylene  in 1 were replaced by oxymethylene , methine , oxymethine , and there was an acetyl group in 2. Key HMBC correlations from H-8 to C-8a, C-1′ and C-9a, from H-1′ to C-6, C-7 and C-8, from H-1″ to C-1, C-3a, C-9a and C-9b indicated the planar structure of 2. We determined the relative configuration of 2 by analysis of the NOESY spectrum. The NOE correlations between H-8 and H-1′, between H-8 and H-9a, between H-8 and H-9β, and between H-1′ and H-6β established the β-orientations of H-1′, H-8 and H-9a. The NOE correlations between H-3a and H-1″, between H-3α and H-3a and between H-1″ and H-5a indicated the α-orientations of H-1″, H-3a and H-5a. A comparison of the 1H- and 13C-NMR data of 2 with those of oblongolide P  was replaced by a quaternary carbon . Key HMBC correlations from H-15 to C-5, C-6 and C-7, from H-11 to C-1, C-2, C-9 and C-12, from H-16 to C-1, C-2 and C-3, from H-4 to C-2, C-5 and C-9 and from H-10 to C-3, C-6 and C-8 indicated the planar structure of 10. The relative configuration of 10 was deduced on the basis of NOESY spectroscopic data. The NOE correlations between H-10 and H-11, between H-2 and H-11, between H-13 and H-11, between H-15 and H-10 and between H-9 and H-16 indicated β-orientation of the hydroxyl group (6-OH) and the α-orientation of the side chain attached to C-1. Therefore, the structure of 10 was determined. We named it 6-hydroxyphomodiol ), indicating that XZ-01 belongs to this genus.The fungus (XZ-01) was isolated from current-year twigs  of XZ-01 was cultivated on potato dextrose agar at 28 °C. The agar blocks were chopped and transferred into Erlenmeyer flasks (10 × 3 L), each containing 1 L of potato dextrose broth (PDB), and then fermented at 28 °C on a rotary shaker (150 rpm) for 7d. The culture was filtered to separate broth and mycelia. The culture broth was extracted with EtOAc (6 × 10 L) for six times. The combined organic layer was concentrated under vacuum to afford 3.2 g of residue.2O . Fraction 3 (100 mg) was subjected to silica gel CC  to afford eleven fractions (3-1-3-11). Fractions 3-11 (4.9 mg) were further separated by silica gel CC  to yield 4 (2.3 mg). Fraction 5 (92.1 mg) was separated by Sephadex LH-20 (elution with 100% methanol) to give three subfractions (fraction 5-1–5-3). Fraction 5-2 (23.6 mg) was purified by silica gel CC  to produce fraction 5-2-1. Fraction 5-2-1 (3.7 mg) was separated by silica gel (eluted with 50% CHCl3 in petroleum ether) to afford 11 (2mg). Fraction 6 (225.8 mg) was fractionated by Sephadex LH-20 CC (elution with 100% MeOH) to provide nine fractions (6-1–6-9). Fraction 6-1 (28.8 mg) was further purified by silica gel CC  to furnish 6 (11.5 mg), 8 (2.6 mg) and 10 (6.4 mg). Fraction 7 (247.1 mg) was subjected to Sephadex LH-20 CC (elution with 100% MeOH) to give 5 fractions (7-1–7-5). Fraction 7-4 (36.1 mg) was purified by silica gel CC (elution with CHCl3) to yield 7 (3.1 mg). Fraction 10 (109 mg) was fractionated by Sephadex LH-20 CC (elution with 100% MeOH) to provide two fractions (10-1–10-2). Fraction 10-1 (72 mg) was further purified by silica gel CC  to give two subfractions (10-1-1 and 10-1-2). Fraction 10-1-2 (11.7 mg) was separated by silica gel CC (elution with 100% CHCl3) to yield 3 (3.8 mg). Fraction 11 (318.3 mg) was separated by Sephadex LH-20 (elution with 100% MeOH) to provide five fraction (11-1–11-5). Fraction 11-5 (23.9 mg) was further purified by silica gel CC (elution with 10% CHCl3 in petroleum ether) to afford 12 (22.8 mg). Fraction 11-3 (99 mg) was separated by silica gel CC  to give 5 (34 mg) and 9 (2.3 mg). Fraction 12 (117 mg) was fractionated by Sephadex LH-20 CC (elution with 100% MeOH) to provide three fractions (12-1–12-3). Fraction 12-1 (12.8 mg) was further separated by silica gel CC (elution with 33.3% CHCl3 in petroleum ether) to yield 2 (7.4 mg). Fraction 9 (232 mg) was separated by Sephadex LH-20 (elution with 100% MeOH) to give two fractions (9-1–9-2). Fraction 9-2 (38 mg) was purified by silica gel CC  to yield 1 (5.7 mg). The crude extract was separated into fifteen fractions (1-15) by column chromatography on RP-18 silica gel, eluted by methanol/HOblongolide C-1 (1): White needles; [α]D20: – 22.6 . IR (KBr) νmax 2919, 2359, 1219, 772, 668 cm–1. 1H- and 13C-NMR: see - .Oblongolide O-1 (2): White powder; [α]D20: – 72.2. IR (KBr) νmax 3344, 2922, 1588, 1383, 772 cm–1. 1H- and 13C-NMR: see + .Oblongolide X-1 (3): White oil; [α]D20: – 21.7. IR (KBr) νmax 3422, 1583, 773, 685 cm–1. 1H- and 13C-NMR: see - .6-Hydroxyphomodiol (10): Transparent oil; [α]D20: + 43.3. IR (KBr) νmax 2365, 1223, 771 cm–1. 1H- and 13C-NMR: see - .3/100 µL medium in 96-well microtiter plate and treated with the compounds at the concentration of 20 µg/mL. Viable cells were incubated with MTT (5 mg/mL) for 4 h and formazan precipitate was dissolved in 100 µL DMSO and the absorbance at 490 nm was measured by Multimode Detector DTX880 (Beckman Coulter).Cancer cell lines were derived from the cell bank of The Chinese Academy of Sciences. Cells were seeded at a density of 5 × 101), P1 (2), X1 (3), 6-hydroxyphomodiol (10), together with eight known compounds were isolated from the endophytic fungus Phomopsis sp. XZ-01. oblongolides C1 (1), P1 (2), X1 (3), and 6-hydroxyphomodiol (10) showed modest selective activities against HepG2 cancer cell lines. Oblongolide C (5) exhibited minor selective activity against A549. Four new compounds, oblongolides C1 (


Rennellia elliptica Korth. showed strong inhibition of Plasmodium falciparum growth in vitro with an IC50 value of 4.04 µg/mL. A phytochemical study of the dichloromethane root extract has led to the isolation and characterization of a new anthraquinone, 1,2-dimethoxy-6-methyl-9,10-anthraquinone (1), and ten known anthraquinones: 1-hydroxy-2-methoxy-6-methyl-9,10-anthraquinone (2), nordamnacanthal (3), 2-formyl-3-hydroxy-9,10-anthraquinone (4), damnacanthal (5), lucidin-ω-methyl ether (6), 3-hydroxy-2-methyl-9,10-anthraquinone (7), rubiadin (8), 3-hydroxy-2-methoxy-6-methyl-9,10-anthraquinone (9), rubiadin-1-methyl ether (10) and 3-hydroxy-2-hydroxymethyl-9,10-anthraquinone (11). Structural elucidation of all compounds was accomplished by modern spectroscopic methods, notably 1D and 2D NMR, IR, UV and HREIMS. The new anthraquinone 1, 2-formyl-3-hydroxy-9,10-anthraquinone (4) and 3-hydroxy-2-methyl-9,10-anthraquinone (7) possess strong antiplasmodial activity, with IC50 values of 1.10, 0.63 and 0.34 µM, respectively.Dichloromethane root extract of  Rennellia elliptica Korth. (Rubiaceae) is a tropical shrub locally known in Malaysia as Segemuk or Mengkudu Rimba. This plant is widely distributed, along riverbanks or lowland forest, in Peninsular Malaysia, Sumatra and Borneo + peak at 283.0968 [calc 283.3067] suggesting a molecular formula of C17H14O4. The absorption maxima in the UV spectrum were observed at 373, 341 and 257 nm, indicative of an anthraquinone moiety + ; For 1H-NMR (CDCl3) and 13C-NMR (CDCl3) data, see 1-Hydroxy-2-methoxy-6-methyl-9,10-anthraquinone (2). Red needles (CH2Cl2); m.p. 220-221 ºC. UV λmax (EtOH): 421, 278, 262 nm; IR νmax (KBr): 3,467, 1,653, 1,637 cm-1; HREIMS 269.0867 [M + H]+ ; For 1H-NMR (CDCl3) and 13C-NMR (CDCl3) data, see P. falciparum (3D7) clone was propagated in a 24-well culture plate in the presence of 10, 1, 0.1, 0.01 and 0.001 µg/mL range of concentrations of each compound. Chloroquine diphosphate was used as positive control. The growth of the parasite was monitored by making a blood smear fixed with MeOH and stained with Geimsa (Merck). The antiplasmodial activity of each compound was expressed as an IC50 value, defined as the concentration of the compound causing 50% inhibition of parasite growth relative to an untreated control. The antiplasmodial activity of dichloromethane extract and the isolated compounds were determined by methods as previously described . The samR. elliptica afforded a new anthraquinone, 1,2-dimethoxy-6-methyl-9,10-anthraquinone (1), together with ten known anthraquinones 2-11. Several anthraquinones strongly inhibited in vitro growth of a chloroquine sensitive strain of Plasmodium falciparum (3D7), with the strongest inhibition shown by compound 7. The antiplasmodial data suggested that the roots extract of R. elliptica is a potential source of new antiplasmodial agents.A phytochemical study on dried roots of


Dryopteris crassirhizoma’s antiplatelet activity. A water extract of D.crassirhizoma (WDC) was partitioned into dichloromethane (DCM), ethyl acetate, n-butyl alcohol, and water. Among these four fractions, the DCM fraction potently inhibited the collagen-stimulated platelet aggregation in a concentration-dependent manner. From this fraction, five different acylphloroglucinol compounds and one flavonoid were isolated by activity-guided column chromatography. They were identified by comparing their mass, 1H-, and 13C-NMR spectral data with those reported in the literature. Quantifying the six compounds in WDC and its DCM fraction by high-performance liquid chromatography (HPLC) revealed that butyryl-3-methylphloroglucinol (compound 4) was the most abundant in these samples. Additionally, butyryl-3-methylphloroglucinol showed the strongest inhibitory activity in the collagen- and arachidonic acid (AA)-induced platelet aggregation, with inhibition ratios of 92.36% and 89.51% in the collagen and AA-induced platelet aggregation, respectively, without cytotoxicity. On the active concentrations, butyryl-3-methylphloroglucinol significantly suppressed the convulxin-induced platelet activation. Regarding the structure–activity relationships for the five acylphloroglucinol compounds, our results demonstrated that the functional butanonyl, methoxy, and hydroxy groups in butyryl-3-methylphloroglucinol play important roles in antiplatelet activity. The findings indicate that acylphloroglucinols, including butyryl-3-methylphloroglucinol from D.crassirhizom, possess an antiplatelet activity, supporting the use of this species for antiplatelet remedies.Platelets are an important component of the initial response to vascular endothelial injury; however, platelet dysfunction induces the acute clinical symptoms of thrombotic disorders, which trigger severe cardiovascular diseases such as myocardial infarction, ischemia, and stroke. In this study, we investigated the  D. crassirhizoma, called “Gwanjung” in Korea (“Guan Zhong” in China and “Oshida” in Japan), has been extensively used to treat parasitic infestation, hemorrhage, epidemic flu, cold, and cancer +; 1H-NMR  δ: 5.98 , 3.79 , 3.09 , 1.89 , 1.13 ; 13C-NMR  δ: 208.3 (C=O), 164.9 (C-6), 163.6 (C-4), 161.9 (C-2), 104.6 (C-1), 90.9 (C-5), 55.8 (-OCH3), 38.2 (C-9), 19.1 (C-10), 7.2 (C-7).1--propan-1-one —Red powder; LC-ESI MS (positive mode) m/z: 225.1 [M + H]+; 1H-NMR  δ: 5.99 , 3.80 , 3.04 , 1.90 , 1.68 , 0.97 ; 13C-NMR  δ: 207.8 (C=O), 164.9 (C-6), 163.7 (C-4), 161.9 (C-2), 104.6 (C-1), 90.9 (C-5), 55.9 (-OCH3), 47.0 (C-9), 19.4 (C-10), 7.2 (C-7).1--butan-1-one —Red powder; LC-ESI MS (negative mode) m/z: 195.1 [M − H]−; 1H-NMR  δ: 5.88 , 3.07 , 1.90 , 1.13 ; 13C-NMR  δ: 208.0 (C=O), 164.9 (C-6), 163.7 (C-4), 161.4 (C-2), 103.6 (C-1), 94.8 (C-5), 38.1 (C-9), 9.4 (C-10), 7.4 (C-7).1--propan-1-one —Red powder; LC-ESI MS (positive mode) m/z: 209.2 [M − H]−; 1H-NMR  δ: 5.87 , 3.01 , 1.90 , 1.68 , 0.96 ; 13C-NMR  δ: 207.4 (C=O), 164.9 (C-6), 163.7 (C-4), 161.4 (C-2), 103.6 (C-1), 94.8 (C-5), 46.9 (C-9), 19.6 (C-10), 14.4 (C-11), 7.4 (C-7).1-butan-1-one —Red powder; LC-ESI MS (negative mode) m/z: 195.1 [M − H]−; 1H-NMR  δ: 5.79 , 5.79 , 3.01 , 1.68 , 0.96 ; 13C-NMR  δ: 207.4 (C=O), 166.0 (C-4), 165.9 (C-2), 165.9 (C-6), 105.5 (C-1), 95.8 (C-3), 95.8 (C-5), 46.9 (C-8), 19.6 (C-9), 14.5 (C-10).1-butan-1-one —Yellow powder; LC-ESI MS (negative mode) m/z: 287.1 [M − H]−; 1H-NMR δ: 1H-NMR  δ: 5.89 , 5.84 , 5.28 , 3.09 , 2.71 , 6.91 , 6.78 , 6.78 ; 13C-NMR  δ: 197.8, 168.4, 165.5, 164.9, 146.9, 146.5.3′,4′,5,7-Tetrahydroxyflavanone  using 0.1% TFA water (v/v); solvent A and acetonitrile; solvent B as the mobile phases at a flow rate of 1 mL/min. The HPLC elution condition was optimized as follows: 0–60 min, 10–70% B; 60–70 min, 100% B. The column oven and auto-sampler injection volume were wet to 40 °C and 10 μL, respectively. The detected wavelength was set at 280 nm and the total run time was 70 min.The analyses of the compounds isolated from the DCM fraction of WDC were carried out via an HPLC analysis using a Dionex UltiMate 3000 system equipped with a binary pump, auto-sampler, column oven and diode array UV/VIS detector . The chromatograms of the detector were registered using a Chromeleone software system . The chromatographic separation was achieved on an AcclaimY = Ax + B, where A is the slope of the calibration curve, B is the y-intercept of the calibration curve, x is the concentration of the marker components, and Y is the peak area. The correlation coefficients (R2) were calculated to assess the linearity. The limit of detection (LOD) and limit of quantification (LOQ) were determined at signal-to-noise (S/N) ratios of 3 and 10, respectively.The method validation for the selectivity, linearity, limit of detection (LOD), and limit of quantification (LOQ) in the present study was performed according to the Validation of Analytical Procedures: Text and Methodology Q2 (R1) Guideline of the International Conference on Harmonization (ICH). The standard calibration curve for the linearity assay was prepared with five different concentrations of diluted standard solutions and performed in triplicate. To demonstrate the linearity of the analytical method, calibration curves were generated by plotting the chromatographic peak area as a function of the analyte concentration. The linear regressions of these data were expressed as New Zealand white rabbits (2.5–3 kg) were purchased from Sam-Tako Animal Co.  and acclimated for 1 week at a temperature of 24 ± 1 °C and humidity of 55 ± 5%. The animals had free access to a commercial pellet diet obtained from Samyang Co.  and drinking water. The animal studies have been carried out in accordance with the Korea Institute of Oriental Medicine Care Committee Guidelines, and were approved by the Korea Institute of Oriental Medicine Care and Use Committee. The animals were cared for in accordance with the dictates of the National Animal Welfare Law of Korea (No.14651).v/v) of anticoagulant citrate dextrose (ACD) solution that contained 0.8% citric acid, 2.2% trisodium citrate, and 2% dextrose (w/v). Washed platelets were prepared as previously described [2O-soluble fractions, and isolated compounds from the DCM fraction, respectively. After 3 min of pre-incubation, the platelet aggregation was induced by the addition of collagen (5 µg/mL), arachidonic acid (100 µM) and thrombin (0.1 U/mL), respectively. Then extent of the platelet aggregation is expressed as a percentage of the control.Blood was withdrawn from the ear artery of the male New Zealand white rabbits and collected into 0.15 . A value of D. crassirhizoma, possess an antiplatelet activity, which could contribute to the use of D. crassirhizoma for antiplatelet remedies.In this study, we revealed that the DCM fraction from WDC contains acylphloroglucinol compounds, which enhance antiplatelet activities by inhibiting the platelet aggregation. We also found that butyryl-3-methylphloroglucinol from the DCM fraction strongly inhibited the collagen- and AA-induced platelet aggregation compared to other acylphloroglucinol compounds. Based on the SAR study, we interpreted that this is related to the antiplatelet efficacy. Therefore, we suggest that acylphloroglucinols, including butyryl-3-methylphloroglucinol from


Cissampelos capensis, commonly known by the Afrikaans name “dawidjies” or “dawidjieswortel”, is the most important and best known medicinal plant of the family Menispermaceae used by the Khoisan and other rural people in the western region of South Africa. The main alkaloids in the leaves, stems and rhizomes were isolated and identified. Several of the main compounds were previously found in species of the related genus Antizoma and this similarity indicates that the two genera are closely related if not congeneric. Bulbocapnine , dicentrine  and salutaridine  were the main alkaloids in the leaves, while bulbocapnine, cissacapine, cycleanine and insularine  are the major compounds in the stems. The rhizome contains mostly bisbenzyltetrahydroisoquinoline alkaloids, with 12-O-methylcurine, cissacapine and cycleanine as the main ones. Alkaloids appear to be quite variable within different plant parts and different provenances, as confirmed by the difference in alkaloid patterns between coastal and inland forms of Cissampelos capensis . The variety of alkaloids identified may contribute to the medicinal value of this species. The data on alkaloidal variation in the species has potential value and practical applications in chemotaxonomy, toxicology and pharmacognosy. Cissampelos capensis L.f. (Menispermaceae) are reported for the first time. Despite its importance in traditional medicine, almost nothing has yet been published on the alkaloids of this species. Accurate identification of the main alkaloids was considered to be an important first step to gain deeper insight into the value of the plant in traditional medicine. A further aim was to investigate possible geographical variation in the alkaloids found in different populations (provenances) as well as possible chemical differences between various plant parts, especially rhizomes – the part that is mainly used in traditional medicine – and leaves, which are reported to be toxic to cattle , glaziovine , lauroscholtzine  and pronuciferine . Leaves  collected from the coast yielded pure samples of bulbocapnine  and salutaridine . Glaziovine (2) was also isolated from Antizoma angustifolia [5) from A. miersiana [The organic extract was filtered through a glass column, packed with a coarse celite-577. Removal of the solvent under reduced pressure yielded the crude alkaloidal extracts. Alkaloids from the bulk rhizome and leaf extracts were isolated by column chromatography. Leaves  collected from the inland yielded pure samples of dicentrine (stifolia  and bulbiersiana .Dicentrine (1). 1H-NMR (CDCl3) δ: 7.65 , 6.76 , 6.49 , 6.05 , 5.91 , 3.90 , 3.89 , 3.09 , 2.62 , 2.53 . 13C-NMR (CDCl3) δ: 148.1 (C-9), 147.6 (C-10), 146.5 (C-2), 141.7 (C-1), 128.2 (C-7a), 126.5 (C-3a), 126.2 (C-1b), 123.4 (C-11a), 116.5 (C-1a), 111.2 (C-11), 110.4 (C-8), 106.7 (C-3), 100.6 (OCH2O), 62.4 (C-6a), 56.1 (OMe), 55.9 (OMe), 53.6 (C-5), 43.9 (N-Me), 34.2 (C-7), 29.2 (C-4). MS m/z: 39 , 178 (24), 97 (23), 85 (22), 83 (33), 81 (37), 71 (38), 70 (21), 69 (93), 67 (24), 60 (24), 59 (44), 57 (84), 56 (28), 55 (80), 45 (33), 44 (30), 43 (100), 41 (97).Lauroscholtzine (3). 1H-NMR (CDCl3) δ: 7.90 , 6.77 , 6.63 , 3.92 , 3.89 , 3.58 , 2.52 . MS m/z: 341 , 340 (25), 335 (20), 168 (82), 147 (44), 119 (22), 97 (30), 83 (22), 71 (25), 69 (100), 57 (50), 55 (40), 44 (53), 43 (55), 41 (44).Pronuciferine (4). 1H-NMR (CDCl3) δ: 7.02 , 6.86 , 6.61 , 6.38 , 6.27 , 3.78 , 3.57 , 2.34 . 13C-NMR (CDCl3) δ: 186.1 (C-10), 153.5 (C-12), 153.3 (C-1 or 2), 150.0 (C-8), 134.3 (C-3a), 132.7 (C-7c), 128.2 (C-9), 127.7 (C-7b), 127.4 (C-11), 111.7 (C-3), 65.7 (C-6a), 61.1 (OMe), 56.3 (OMe), 54.9 (C-5), 51.2 (C-7a), 47.5 (C-7), 43.6 (N-Me), 27.5 (C-4). MS m/z: 312 (22), 311 , 335 (20), 310 (20), 169 (77), 147 (41), 119 (23), 97 (26), 83 (24), 71 (24), 69 (100), 59 (28), 57 (40), 55 (37), 44 (51), 43 (53), 42 (23), 41 (34).Salutaridine (6). 1H-NMR (CDCl3) δ: 7.51 , 6.71 , 6.62 , 6.29 , 3.85 , 3.72 , 3.65 , 3.29 , 2.94 , 2.57 , 2.43 , 2.41 , 2.33 , 1.73 .13C-NMR (CDCl3) δ: 181.3 (C-7), 161.5 (C-14), 150.8 (C-6), 145.2 (C-3), 143.2 (C-4), 129.6 (C-11), 123.8 (C-12), 122.1 (C-8), 120.3 (C-5), 118.7 (C-1), 109.4 (C-2), 61.0 (C-9), 56.3 (OMe), 54.8 (OMe), 47.0 (C-16), 43.7 (C-13), 41.7 (NMe), 37.7 (C-15), 32.6 (C-10). MS m/z: 328 (22), 327 , 327 (83), 312 (44), 299 (73), 285 (25), 284 (100), 268 (27), 256 (23), 242 (30), 226 (23), 169 (61), 147 (28), 97 (24), 85 (32), 83 (59), 70 (21), 69 (64), 57 (30), 55 (33), 44 (59), 43 (37), 42 (54), 41 (26).7; 31 mg), which was also isolated from Antizoma miersiana [t and UV spectra (HPLC-DAD) with reference compounds. Cissacapine (8), crotsparine (9) and insularine (10) were isolated from Antizoma angustifolia [O-methylcurine (11) and reticuline (12) were isolated from Cissampelos hirta and insulanoline (13) from A. miersiana [The rhizome  sample collected from the coast yielded pure sample of cycleanine (iersiana . In the sularine  were isoylcurine  and reti6) in the leaves of plants from inland localities). Traditional healers are often not aware that the same species at different localities are chemically different and therefore perhaps also different in its biological activity and potential toxicity. The fact that some of the leaf alkaloids of C. capensis are known to be toxic to animals indicate that the alkaloidal data may be of practical value in forensic studies in cases of human and animal poisoning. Most of the medicinal uses of this species can probably be attributed to its rich diversity of alkaloids, especially the predominance of bisbenzyltetrahydroisoquinoline alkaloids in the rhizome . The three major alkaloids in the leaves are known to be biologically active. Bulbocapnine (5) has antimicrobial activity [1) has antibacterial and antifungal activity [6) has anti-inflammatory activity [7) could possibly explain the medicinal uses of the rhizome. This bisbenzyltetrahydroisoquinoline alkaloid is known for its analgesic, muscle relaxant and anti-inflammatory effects [The extreme variation in alkaloids should be taken into account if the plant is collected for medicinal purposes  and bulbocapnine (5) as main compounds in the leaves, stems and rhizomes; A. angustifolia has crotsparine (9) as major constituent in leaves, stems and rhizomes, while this compound has so far only been detected in the leaves of C. capensis and A. miersiana but not in their stems or rhizomes. With the exception of cycleaneonine  that the two genera are closely related and that Antizoma should perhaps be subsumed under Cissampelos. In order to use alkaloids for chemotaxonomic purposes it is important to take into account the plant part used, the different stages of development and genetic variation of plants, the time of the year and the geographical distribution. From a chemotaxonomic point of view, the qualitative and quantitative patterns of alkaloids in ma Miers ,11. Ciss


The compounds which possessed appropriate properties were additionally subjected to UVA irradiation at 365 nm. The ability of most of the compounds to photocleave DNA was high at 312 nm, whereas higher concentrations were required at 365 nm as a result of their lower UV absorption. The affinity of selected compounds to calf-thymus (CT) DNA was studied by UV spectroscopy, viscosity experiments and competitive studies with ethidium bromide (EB) revealing that all compounds interacted with CT DNA. The fluorescence emission spectra of the pre-treated EB-DNA exhibited a moderate to significant quenching in the presence of the compounds indicating the binding of the compounds to CT DNA via intercalation as concluded also by DNA-viscosity experiments. For the oxime esters the DNA photocleavage and affinity studies aimed to clarify the role of the oxime nature  and the role of the pyridine and p-nitrophenyl moieties both as oxime substituents and ester conjugates.Compared to standard treatments for various diseases, photochemotherapy and photo-dynamic therapy are less invasive approaches, in which DNA photocleavers represent promising tools for novel “on demand” chemotherapeutics. A series of  Photochemotherapy and photodynamic therapy are, compared to standard treatments for various types of cancer, less invasive approaches ,2,3,4,5.I, II and III, respectively) retain a complementary position in drug design and discovery since they are individually considered as pharmacophores. They, additionally, participate as parent compounds in multiple transformations leading to biologically interesting derivatives  (representatively shown in b constants of the compounds are relatively high with compound 21 bearing the highest Kb constant (=1.85(±0.18) × 106 M−1) among the studied compounds. The Kb constants of most of the compounds are higher than that of the classical intercalator EB (=1.23(±0.07) × 105 M−1) as it was previously calculated  = 20 µM) with CT DNA ([DNA] = 26 µM) for ~1 h. The fluorescence emission spectra  with the linear Stern-Volmer equation (Equation (4))  as indicompounds  are modeA system , the queompounds  are signechanism .260/A280) of 1.85–1.88, indicating that the DNA was sufficiently free of protein contamination + 316.0928; found 316.0925.(Z)-4-Nitro-N'-((4-nitrobenzoyl)oxy)benzimidamide (5). Solvent: THF; R.T.: 4 h; amine: Et3N; light yellow crystals, yield 79%, mp 243.8 °C (ethyl acetate); IR (KBr): 3490, 3360, 3111, 1725, 1627 cm−1; 1Η-NMR (CDCl3 + DMSO-d6) δ 8.45 , 8.29 , 8.27 , 8.04 , 7.25  ppm; 13C-NMR (CDCl3 + DMSO-d6) δ 160.2, 154.1, 148.4, 147.0, 136.0, 133.0, 129.3, 126.6, 121.6, 121.5 ppm; HRMS (ESI) Calc C14H11N4O6 [M + H]+ 331.0673; found 331.0675.(Z)-N'-(Isonicotinoyloxy)picolinimidamide (6). Solvent: CHCl3; R.T.: 5 h; amine: DIPEA; white crystals, yield 77%, mp 161.1 °C (ethyl acetate); IR (KBr): 3493, 3380, 3054, 1732, 1632 cm−1; 1Η-NMR (CDCl3 + DMSO-d6) δ 8.72 , 8.56 , 8.07 , 8.00 , 7.76 , 7.40 , 6.82  ppm; 13C-NMR (CDCl3 + DMSO-d6) δ 161.2, 153.5, 149.1, 147.2, 146.5, 135.6, 135.4, 124.4, 121.7, 119.9 ppm; HRMS (ESI) Calc C12H11N4O2 [M + H]+ 243.0877; found 243.0876.(Z)-N'-(Isonicotinoyloxy)nicotinimidamide (7). Solvent: CHCl3; R.T.: 3 h; amine: DIPEA; white crystals, yield 73%, mp 155.9 °C (ethyl acetate); IR (KBr): 3445, 3313, 3166, 1726, 1634 cm−1; 1Η-NMR  δ 8.93 , 8.73 , 8.63 , 8.08 , 8.02 , 7.37 , 7.00  ppm; 13C-NMR  δ 161.0, 154.5, 149.8, 148.9, 146.6, 135.2, 133.3, 126.2, 121.7, 121.5 ppm; HRMS (ESI) Calc C12H11N4O2 [M + H]+ 243.0877; found 243.0872.(Z)-N'-(Isonicotinoyloxy)isonicotinimidamide (8). Solvent: CHCl3; R.T.: 3 h; amine: Et3N; column chromatography (10% MeOH in ethyl acetate); Light yellow crystals, yield 70%, mp 155 °C (ethyl acetate); IR (KBr): 3459, 3366, 1731, 1627 cm−1; 1Η-NMR (DMSO-d6) δ 8.82 , 8.72 , 8.09 , 7.74 , 7.32  ppm; 13C-NMR (DMSO-d6) δ 162.4, 155.7, 150.6, 150.1, 139.0, 136.3, 122.9, 121.2 ppm; HRMS (ESI) Calc C12H11N4O2 [M + H]+ 243.0877; found 243.0872.(Z)-N'-(Isonicotinoyloxy)-4-methoxybenzimidamide (9). Solvent: THF; R.T.: 7 h; amine: Et3N; white crystals, yield 87%, mp 165.8 °C (ethyl acetate); IR (KBr): 3492, 3366, 1728, 1618 cm−1; 1Η-NMR (CDCl3 + DMSO-d6) δ 8.49 , 7.66 , 7.45 , 6.63 , 5.88 , 3.55  ppm; 13C-NMR (CDCl3 + DMSO-d6) δ 162.1, 161.0, 157.3, 149.8, 136.4, 127.9, 122.7, 122.1, 113.1, 54.7 ppm; HRMS (ESI) Calc C14H14N3O3 [M + H]+ 272.1030; found 272.1032.(Z)-N'-(Isonicotinoyloxy)-4-nitrobenzimidamide (10). Solvent: THF; R.T.: 7 h; amine: Et3N; yellow crystals, yield 85%, mp 176.2 °C (ethyl acetate); IR (KBr): 3536, 3420, 3120, 1730, 1649 cm−1; 1Η-NMR (CDCl3 + DMSO-d6) δ 8.72 , 8.19 , 8.00 , 7.99 , 6.90  ppm; 13C-NMR (CDCl3 + DMSO-d6) δ 161.4, 155.0, 149.2, 147.8, 136.6, 135.5, 127.3, 122.1, 121.8 ppm; HRMS (ESI) Calc C13H11N4O4 [M + H]+ 287.0775; found 287.0773.(E)-1-(Pyridin-4-yl)ethan-1-one O-(4-nitrobenzoyl) oxime (11). Prepared according to reference + 242.0924; found 242.0922.(E)-Picolinaldehyde O-4-nitrobenzoyl oxime (13). Prepared according to reference + 301.0819; found 301.0810.p 171 °C . Solvent(E)-Picolinaldehyde O-isonicotinoyl oxime (17). Solvent: THF; R.T.: 24 h; amine: DIPEA; beige crystals, yield 85%, mp 154 °C (hexanes/ethyl acetate); IR (KBr): 1754, 1612 cm−1; 1Η-NMR (CDCl3) δ 8.84 , 8.69 , 8.64 , 8.16 , 7.93 , 7.79 , 7.39  ppm; 13C-NMR (CDCl3) δ 162.3, 158.2, 150.8, 150.0, 149.4, 136.8, 135.6, 125.8, 122.8, 122.3 ppm; HRMS (ESI) Calc C12H10N3O2 [M + H]+ 228.0768; found 228.0765.(E)-Nicotinaldehyde O-isonicotinoyl oxime (18). Solvent: THF; R.T.: 24h; amine: DIPEA; off white crystals, yield 82%, mp 152 °C (ethyl acetate); IR (KBr): 1749, 1612 cm−1; 1Η-NMR (DMSO-6d) δ 9.05 , 8.95 , 8.88 , 8.75 , 8.24 , 7.95 , 7.58  ppm; 13C-NMR (DMSO-d6) δ 162.0, 156.7, 152.7, 151.0, 149.7, 135.3, 134.9, 126.0, 124.3, 122.6 ppm; HRMS (ESI) Calc C12H10N3O2 [M + H]+ 228.0768; found 228.0766.(E)-Isonicotinaldehyde O-isonicotinoyl oxime (19). Solvent: THF; R.T.: 24 h; amine: DIPEA; beige crystals, yield 77%, mp 138 °C (hexanes/ethyl acetate); IR (KBr): 1753, 1596 cm−1; 1Η-NMR (CDCl3) δ 8.85 , 8.76 , 8.55 , 7.92 , 7.67  ppm; 13C-NMR (CDCl3) δ 162.0, 155.6, 150.8, 150.8, 137.0, 135.4, 122.8, 121.9 ppm; HRMS (ESI) Calc C12H10N3O2 [M + H]+ 228.0768; found 228.0764.(E)-4-Methoxybenzaldehyde O-isonicotinoyl oxime (20). Solvent: CHCl3; R.T.: 4 h; amine: Et3N; column chromatography (hexanes/ethyl acetate); beige crystals, yield 83%, mp 141 °C (hexanes/ethyl acetate); IR (KBr): 1743, 1605 cm−1; 1Η-NMR (CDCl3) δ 8.81 , 8.48 , 7.90 , 7.73 , 6.95 , 3.85  ppm; 13C-NMR (CDCl3) δ 162.7, 162.6, 157.1, 150.7, 136.1, 130.4, 122.8, 122.0, 114.5, 55.4 ppm; HRMS (ESI) Calc C14H13N2O3 [M + H]+ 257.0921; found 257.0920.(E)-4-Nitrobenzaldehyde O-isonicotinoyl oxime (21). Solvent: CHCl3; R.T.: 4 h; amine: Et3N; yellow crystals, yield 79%, mp 164 °C (hexanes/ethyl acetate); IR (KBr): 1757, 1594 cm−1; 1Η-NMR (CDCl3) δ 8.86 , 8.67 , 8.33 , 8.01 , 7.94  ppm; 13C-NMR (CDCl3) δ 162.1, 155.2, 150.9, 149.8, 135.6, 135.4, 129.4, 124.2, 122.8 ppm; HRMS (ESI) Calc C13H10N3O4 [M + H]+ 272.0666; found 272.0663.The reaction mixtures (20 μL) containing supercoiled circular pBluescipt KS II DNA stock solution , compounds, and Tris buffer  in Pyrex vials were incubated for 30 min at 37 °C, centrifuged, and then irradiated with UV light  under aerobic conditions at room temperature for 15 min. For the case of pBR322 plasmid DNA, the above mentioned procedure has been followed. After centrifugion, the samples were irradiated with UVA light  under aerobic conditions at room temperature for 120 min. After addition of the gel-loading buffer (6X Orange DNA Loading Dye 10 mM Tris-HCl (pH 7.6), 0.15% orange G, 0.03% xylene cyanol FF, 60% glycerol, and 60 mM EDTA, by Fermentas), the reaction mixtures were loaded on a 1% agarose gel with EB staining. The electrophoresis tank was attached to a power supply at a constant current (65 V for 1 h). The gel was visualized by 312 nm UV transilluminator and photographed by an FB-PBC-34 camera vilber lourmat. Quantitation of DNA-cleaving activities was performed by integration of the optical density as a function of the band area using the program “Image J” version 1.50.I .ss% and ds% damages were calculated according to the following Equations (1) and (2):As Form II we consider Form II of each series minus Form II of the irradiated control DNA. As Form I we consider Form I of each series. The amount of supercoiled DNA was multiplied by factor of 1.43 to account for reduced ethidium bromide intercalation into supercoiled DNA.0) calculated according the general equation: D0 = E0 + E0 − E0(molecule), where E0 is the calculated electronic energy corrected with zero point vibrational energy (ZPE) /[DNA] mixing ratios (=r) as well as the UV spectra of the compounds (10–100 μM) in the presence of increasing amounts of CT DNA (r’ = 1/r = [DNA]/[compound] mixing ratios). Control experiments with DMSO were performed and no changes in the spectra of CT DNA were observed.The interaction of compounds b constants (in M−1) were obtained by monitoring the absorbance changes at the corresponding λmax with increasing concentrations of CT DNA and were calculated by the ratio of slope to the y intercept in plots [DNA]/(εA − εf) versus [DNA], according to the Wolfe-Shimer equation [A = Aobsd/[compound], εf = the extinction coefficient for the free compound and εb = the extinction coefficient for the compound in the fully bound form.The Kequation :(3) = 0.1 mM) in buffer solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0) was measured in the presence of increasing amounts of the compounds . All measurements were performed at room temperature. The obtained data are presented as  for 1 h. The possible intercalating effect of the compounds was monitored by recording the variation of fluorescence emission spectra (excitation wavelength at 540 nm) of the DNA-EB system upon stepwise addition of a solution of the compound.SV, in M−1) is used to evaluate the quenching efficiency for each compound according to the Stern-Volmer equation  is the concentration of the quencher, τo = the average lifetime of the emitting system without the quencher and kq = the quenching constant. KSV was obtained from the Stern–Volmer plots by the slope of the diagram Io/I versus [Q]. Taking τo = 23 ns as the fluorescence lifetime of the EB-DNA system [q, in M−1s−1) of the compounds was determined.The Stern-Volmer constant (Kequation :(4)IoI=A system , the quep-nitrophenyl moieties as both oxime substituents and/or ester conjugates were examined as DNA photocleavers. Both moieties were found to be important for the activity. Those compounds exhibited good to excellent binding to DNA and efficient DNA photocleavage, at 312 nm at a concentration of 100 μM . Some selected compounds were able to photocleave DNA at 365 nm . Their mode of action seems to follow the N-O bond homolytic cleavage with the generation of active p-nitrobenzoyl or p-pyridine carbonyloxyl radicals which attack DNA. Since however, DNA photo-cleavage is a rather complicated phenomenon, some observed differences could be attributed to other factors such as the presence of oxygen (which may produce reactive oxygen species), the mode of DNA affinity or the existence of specific structures facilitating H-abstraction. In general, for compounds with comparable structures, it seems that the lower the BDE the higher the % DNA photocleavage. Compounds 4, 9 and 16, as well as 6–8, although they possessed adequate to sufficient DNA binding activities, were found to lack DNA photocleavage activity, at the studied conditions. Nevertheless, their affinity to DNA may also lead to other cancer therapeutic approaches than photocleavage. Thus, the studied oxime esters emerge as lead compounds for the investigation of cancer therapeutics, in general, and in the field of photo-chemotherapy and photo-dynamic therapy, in particular.Oxime esters are easily accessible on a large scale and can be stored for prolonged periods of time. Aldoxime, ketoxime and amidoxime esters bearing the pyridine and