N-myristoyltransferase (NMT) from Trypanosoma brucei has been validated both chemically and biologically as a potential drug target for human African trypanosomiasis. We previously reported the development of some very potent compounds based around a pyrazole sulfonamide series, derived from a high-throughput screen. Herein we describe work around thiazolidinone and benzomorpholine scaffolds that were also identified in the screen. An X-ray crystal structure of the thiazolidinone hit in Leishmania major NMT showed the compound bound in the previously reported active site, utilising a novel binding mode. This provides potential for further optimisation. The benzomorpholinone was also found to bind in a similar region. Using an X-ray crystallography/structure-based design approach, the benzomorpholinone series was further optimised, increasing activity against T. brucei NMT by >1000-fold. A series of trypanocidal compounds were identified with suitable in vitro DMPK properties, including CNS exposure for further development. Further work is required to increase selectivity over the human NMT isoform and activity against T. brucei.The enzyme  Trypanosoma brucei gambiense and T. brucei rhodensiense, which are transmitted to the human host by the bite of an infected tsetse fly. If left untreated the disease is often fatal. There is a clinical need for more effective drug therapies, because current treatments are unsatisfactory due to toxicity, treatment failures, and inappropriate dosing regimens for a rural African setting.oxazin-3(4H)-one.H-benzo[b]oxazin-3(4H)-one (0.67 mmol) was taken into DMF (5 mL). Alkyl halide (0.74 mmol) and anhydrous K2CO3 (1.01 mmol) were added and stirred at 60 °C overnight. The reaction mixture was concentrated in vacuo, the residue partitioned between CH2Cl2 and saturated aqueous NaHCO3, the organics dried (MgSO4) and concentrated to dryness. Purification by column chromatography (0–5 % MeOH/CH2Cl2) gave the title compounds as solids.Appropriately substituted-23-Benzyl-2-(4-hydroxy-3-methoxyphenyl)thiazolidin-4-one (1): Prepared using benzylamine  and vanillin . Compound 1 was obtained as an off-white solid : 1H NMR : δ=9.23 , 7.33–7.24 , 7.11–7.07 , 6.81 , 6.77–6.70 , 5.48 , 4.76 , 3.93 , 3.78–3.67 ppm ; LC–MS: m/z=316 [M+H].3-(Furan-2-ylmethyl)-2-(4-hydroxy-3-methoxyphenyl)thiazolidin-4-one (2): Prepared with furfurylamine  and vanillin . Compound 2 was obtained as an off-white solid : 1H NMR : δ=9.23 , 7.58 , 6.85 , 6.75–6.71 , 6.38 , 6.15 , 5.51 , 4.74 , 3.93 , 3.77–3.67 ppm ; LC–MS: m/z=306 [M+H].3-Ethyl-2-(4-hydroxy-3-methoxyphenyl)thiazolidin-4-one (3): Prepared using 2 m ethylamine in THF  and vanillin . Compound 3 was obtained as an off-white solid : 1H NMR : δ=9.22 , 6.94 , 6.81 , 6.77 , 5.74 , 3.82 , 3.77 , 3.64 , 3.44 , 2.67 , 0.91 ppm ; LC–MS: m/z=254 [M+H].2-(4-Hydroxy-3-methoxyphenyl)-3-(pyridin-2-yl)thiazolidin-4-one (4): Prepared using 2-aminopyridine  and vanillin . Compound 4 was obtained as an off-white solid : 1H NMR : δ=9.04 , 8.31 , 7.91–7.86 , 7.85–7.81 , 7.18–7.13 , 6.92 , 6.75 , 6.70 , 6.62 , 4.08 , 3.87 , 3.71 ppm ; LC–MS: m/z=303 [M+H].3-(Furan-2-ylmethyl)-2-(3-methoxyphenyl)thiazolidin-4-one (5): Prepared using Furfurylamine  and 3-methoxybenzaldehyde . Compound 5 was obtained as a clear colourless oil : 1H NMR : δ=7.58 , 7.38 , 6.95–6.86 , 6.38 , 6.17 , 5.58 , 4.80 , 4.77 , 3.94 , 3.91 , 3.70–3.60 ppm ; LC–MS: m/z=290 [M+H].H-indazol-6-yl)thiazolidin-4-one (6)3-Benzyl-2- and indazole-5-carbaldehyde . Compound 6 was obtained as an off-white solid : 1H NMR : δ=13.15 , 8.07 , 7.68 , 7.35–7.25 , 7.10 , 7.33 , 5.68 , 4.84 , 4.01 , 3.87 , 3.71 ppm ; LC–MS: m/z=303 [M+H].3-(2-Chlorophenyl)-2-(pyridin-2-yl)thiazolidin-4-one (11): Prepared using 3-chloroaniline  and pyridine-2-carbaldehyde . Compound 11 was obtained as an off-white solid : 1H NMR : δ=8.58 , 7.78–7.82 , 7.53–7.59 , 7.31–7.38 , 7.25 , 7.05 , 6.14 , 4.09 , 3.82 ppm ; LC–MS: m/z=291 [M+H].3-(4-Chlorophenyl)-2-(pyridin-2-yl)thiazolidin-4-one (10): Prepared using 4-chloroaniline  and pyridine-2-carbaldehyde . Compound 10 was obtained as a beige solid : 1H NMR : δ=8.49 , 7.58 , 7.13–7.20 , 6.02 , 4.01 , 3.73 ppm ; LC–MS: m/z=291 [M+H].2-Phenyl-3-(pyridin-2-ylmethyl)thiazolidin-4-one (12): A solution of 2-pyridylmethylamine  and benzaldehyde  in EtOH (12 mL) was heated in a microwave at 100 °C for 10 min. Thioacetic acid (2 mmol) was added to the reaction, which was heated for a further 10 min at 100 °C. Zinc chloride (2 mmol) was then added and the reaction mixture was heated at high absorbance setting (in microwave) at 100 °C for 15 min, before being cooled and transferred into a separating funnel. EtOAc (30 mL) and 1 n sodium hydroxide (30 mL) was added to the separating funnel, which was then shaken. The organic layer was collected and washed with brine (2×30 mL), then dried with MgSO4. The solvent was removed in vacuo and the crude mixture was purified by column chromatography (0–6 % MeOH/CH2Cl2) to yield compound 12 as an off-white solid : 1H NMR : δ=8.5 , 7.76 , 7.32–7.40 , 7.29 , 7.20 , 5.79 , 4.87 , 3.97 , 3.76–3.82 ppm ; LC–MS: m/z=271 [M+H].2-(4-Hydroxy-3-methoxyphenyl)-3-(pyridin-2-ylmethyl)thiazolidin-4-one (13): Prepared as for 12, using 2-pyridylmethylamine  and 4-hydroxy-3-methoxybenzaldehyde  in EtOH (12 mL). Purification by column chromatography (0–6 % MeOH/CH2Cl2) gave compound 13 as an off-white solid : 1H NMR : δ=9.21 , 8.49 , 7.73–7.76 , 7.26–7.29 , 7.19 , 6.86 , 6.72 , 5.70 , 4.79 , 3.76–3.94 , 3.72 ppm ; LC–MS: m/z=317 [M+H].7, 8 and 9 were purchased from ChemDiv.Compounds H-benzo[b]oxazin-4(3H)-yl)acetate (14)Ethyl 2--one , ethyl bromoacetate  and anhydrous K2CO3  were taken up in DMF (5 mL) and stirred at 60 °C overnight. The reaction mixture was concentrated in vacuo, the residue partitioned with CH2Cl2 and saturated aqueous NaHCO3. The organic layer was dried (MgSO4) and concentrated to dryness. Purification by column chromatography (0–5 % MeOH/CH2Cl2) gave compound 14 as a white solid : 1H NMR : δ=7.03–7.09 , 4.73 , 4.71 , 4.16 , 1.21 ppm ; LC–MS: m/z=236 [M+H].N,NH-benzo[b]oxazin-4(3H)-yl)acetamide (19)-Dimethyl-2--one , dimethylcarbamic chloride  and anhydrous K2CO3  were treated as described for the synthesis of 14. After workup a solid was obtained, trituration with Et2O gave compound 19 as a white solid : 1H NMR : δ=6.99–7.02 , 6.76–7.79 , 4.70 , 4.69 , 3.15 , 3.02 ; LC–MS: m/z=235 [M+H].d]oxazol-2-ylmethyl)-2H-benzo[b]oxazin-3(4H)-one (22)4--one , 2-(chloromethyl)benzo[d]oxazole  and anhydrous K2CO3  were treated as described for the synthesis of 14 to give compound 22 as a white solid : 1H NMR : δ=7.70–7.74 , 7.50–7.54 , 7.33–7.37 , 7.10–7.13 , 7.02–7.04 , 6.98–7.02 , 5.42 , 4.78 ppm ; LC–MS: m/z=281 [M+H].H-benzo[b]oxazin-4(3H)-yl)propanoate (16)Ethyl 3--one , ethyl 3-bromopropanoate  and anhydrous K2CO3  were treated as described for the synthesis of 14 to give compound 16 as a white solid : 1H NMR : δ=7.04–7.06 , 7.01–7.03 , 4.61 , 4.23–4.27 , 4.15 , 2.68–2.72 , 1.26 ppm ; LC–MS: m/z=250 [M+H].H-benzo[b]oxazin-3(4H)-one (17)4-(3-Oxo-3-(pyrrolidin-1-yl)propyl)-2: Step 1: Ethyl 3--yl)propanoate (16)  was taken up in THF (100 mL), treated with 2 n aqueous KOH (100 mL), and the reaction was stirred at RT for 1 h. The reaction was then heated at 60 °C, and stirring continued for 2 h. The solvent was removed in vacuo and the aqueous phase was acidified to pH 2 using concentrated HCl. The aqueous phase was then extracted with EtOAc (2×50 mL), and the combined extracts were dried in vacuo to give key intermediate 3--yl)propanoic acid : 1H NMR : δ=12.4 , 7.25 , 7.07 , 7.02 , 4.62 , 4.14 , 2.54 ppm ; LC–MS: m/z=221 [M+H].H-benzo[b]oxazin-4(3H)-yl)propanoic acid  was taken into CH2Cl2 (5 mL), added carbonyldiimidazole . Stirred for 15 min then added 1.2 equiv amine . Stirred for 18 h at RT. Transferred reaction mixture to a separating funnel, added CH2Cl2 (30 mL) and washed the organic layer with 1 n HCl (20 mL), 2N NaHCO3 (20 mL) and then brine (30 mL). Dried organic layer with MgSO4, and removed the solvent in vacuo. Purification by column chromatography (0–6 % MeOH/CH2Cl2) yielded compound 17 as a white solid : 1H NMR : δ=7.24 , 7.06 , 7.01 , 4.63 , 4.11 , 3.34 , 3.28 , 2.57 , 1.84 , 1.75 ppm ; LC–MS: m/z=275 [M+H].Step 2: 3--one (21)4-(2-Oxo-2-(pyrrolidin-1-yl)ethyl)-2: The carboxylic acid intermediate was prepared from 14, as in step 1 for the synthesis of 17. The free acid was then taken into CH2Cl2 and reacted with carbonyldiimidazole and pyrrolidine as in step 2 for the synthesis of 17. Workup and purification as described for the preparation of 17 afforded compound 21 as a white solid (51 %): 1H NMR : δ=6.95–7.03 , 4.68 , 3.58 , 3.32 , 1.95 , 1.81 ppm ; LC–MS: m/z=261 [M+H].N,NH-benzo[b]oxazin-4(3H)-yl)propanamide (18)-Dimethyl-3--yl)propanoic acid  and dimethylamine (1.64 mmol), to give compound 18 as an off-white solid : 1H NMR : δ=7.22 , 7.06 , 7.02 , 4.63 , 4.09 , 2.92 , 2.82 , 2.63 ppm ; LC–MS: m/z=249 [M+H].b]oxazin-3(4H)-one (23)4-((5-Methylisoxazol-3-yl)methyl)-2H-benzo[: 2H-benzo[b]oxazin-3(4H)-one , 3-(chloromethyl)-5-methylisoxazole  and anhydrous K2CO3  were treated as described for the synthesis of 14. A solid was obtained by trituration with Et2O to give compound 23 as a white solid : 1H NMR : δ=7.21–7.26 , 6.98–7.03 , 5.98–5.99 , 5.14 , 4.68 , 2.38–2.39 ppm ; LC–MS: m/z=245 [M+H].H-benzo[b]oxazin-3(4H)-one (24)4-((2-Methylthiazol-4-yl)methyl)-2: 2H-benzo[b]oxazin-3(4H)-one , 4-(chloromethyl)-2-methylthiazole hydrochloride  and anhydrous K2CO3  were treated as described for the synthesis of 14 to give compound 24 as an off-white solid : 1H NMR : δ=7.18–7.24 , 6.99–7.03 , 6.95–6.97 , 5.23 , 4.69 , 2.74 ppm ; LC–MS: m/z=261 [M+H].NH-benzo[b]oxazin-4(3H)-yl)acetamide (20)-Methyl-2--one , ethyl bromoacetate  and anhydrous K2CO3  were taken up in DMF (30 mL) and stirred at 50 °C for 3 h. The reaction mixture was concentrated in vacuo, the residue partitioned with CH2Cl2 and saturated aqueous NaHCO3. The organic layer was collected, dried (MgSO4) and concentrated to dryness to give intermediate ethyl 2--yl)acetate as a clear gum : 1H NMR : δ=7.00–7.04 , 6.75–6.79 , 4.69 , 4.66 , 4.26 , 1.29 ppm .H-benzo[b]oxazin-4(3H)-yl)acetate  was taken up in THF (20 mL), KOH  in water (5 mL) was added and the reaction mixture stirred at 60 °C for 3 h. Solvent was removed in vacuo, the residue acidified to pH 3–4 by addition of 1 m HCl, then extracted into EtOAc. The organic layer was collected, then dried (MgSO4) and concentrated to dryness to give intermediate 2--yl)acetic acid as a white solid : 1H NMR : δ=13.11 , 7.02–7.08 , 4.71 , 4.65 ppm .Step 2: Ethyl 2--yl)acetic acid  was taken up in CH2Cl2 (10 mL), 2 m methylamine in THF  and carbonyldiimidazole  was added and the reaction mixture was stirred at RT for 18 h. The reaction mixture was then diluted with further CH2Cl2 (50 mL) and partitioned with saturated aqueous NaHCO3. The organic layer was collected, dried (MgSO4) and concentrated to dryness. The resulting solid was filtered off and triturated with Et2O to give compound 20 as an off-white solid : 1H NMR : δ=7.16–7.19 , 7.05–7.08 , 7.00–7.04  6.18 , 4.70  4.54 , 2.82 ppm ; LC–MS: m/z=221 [M+H].Step 3: 2--one (25)4-(Pyridin-2-ylmethyl)-2: 2H-benzo[b]oxazin-3(4H)-one , 2-(chloromethyl)pyridine hydrochloride  and anhydrous K2CO3  were treated as described for the synthesis of 14 and purified by column chromatography (EtOAc/Hexane) to give compound 25 as a white solid : 1H NMR : δ=8.58–8.60 , 7.64 , 7.23–7.26 , 7.19–7.22 , 7.03–7.06 , 6.97–7.02 , 6.91–6.95 , 5.28 , 4.75 ppm ; LC–MS: m/z=241 [M+H].H-benzo[b]oxazin-3(4H)-one (26)4-(Pyridin-4-ylmethyl)-2: 2H-benzo[b]oxazin-3(4H)-one , 4-(chloromethyl)pyridine hydrobromide  and anhydrous K2CO3  were treated as described for the synthesis of 14 and purified by column chromatography (0–20 % EtOAc/Hexane) to give compound 26 as a white solid : 1H NMR : δ=8.56–8.59 , 7.16–7.19 , 7.05 , 7.01 , 6.91–6.95 , 6.75 , 5.17 , 4.76 ppm ; LC–MS: m/z=241 [M+H].H-benzo[b]oxazin-3(4H)-one (27)4-methyl)-2: 2H-benzo[b]oxazin-3(4H)-one , 5-(chloromethyl)-3-methyl-1,2,4-oxadiazole  and anhydrous K2CO3  were treated as described for the synthesis of 14 to give compound 27 as an off-white solid (54 mg 33 %): 1H NMR : δ=6.99–7.08 , 6.86–6.89 , 4.74 , 2.40 ppm ; LC–MS: m/z=246 [M+H].H-benzo[b]oxazin-3(4H)-one (28)4-methyl)-2: 2H-benzo[b]oxazin-3(4H)-one , 3-(chloromethyl)-5-methyl-1,2,4-oxadiazole  and anhydrous K2CO3  were treated as described for the synthesis of 14 to give compound 28 as a white solid : 1H NMR : δ=6.99–7.04 , 5.24 , 4.72 , 2.58 ppm ; LC–MS: m/z=246.0 [M+H].H-benzo[b]oxazin-3(4H)-one (29)4-methyl)-2: 2H-benzo[b]oxazin-3(4H)-one , 3-(chloromethyl)-5-isobutyl-1,2,4-oxadiazole  and anhydrous K2CO3  were treated as described for the synthesis of 14 to give compound 29 as a white solid : 1H NMR : δ=6.98–7.03 , 5.25 , 4.72 , 2.75 , 2.19 , 0.98 ppm ; LC–MS: m/z=288 [M+H].H-benzo[b]oxazin-3(4H)-one (30)4-((5-Isobutylisoxazol-3-yl)methyl)-2: 2H-benzo[b]oxazin-3(4H)-one , 3-(chloromethyl)-5-isobutylisoxazole  and anhydrous K2CO3  were treated as described for the synthesis of 14 to give compound 30 as an off-white solid : 1H NMR : δ=7.21–7.25 , 6.98–7.03 , 5.97 , 5.15 , 4.68 , 2.58 , 2.01 , 0.93 ppm ; LC–MS: m/z=287 [M+H].b]oxazin-3(4H)-one (31)4-((6-Methylpyridin-2-yl)methyl)-2H-benzo[: 2H-benzo[b]oxazin-3(4H)-one , 2-(bromomethyl)-6-methylpyridine  and anhydrous K2CO3  were treated as described for the synthesis of 14 to give compound 31 as a white solid : 1H NMR : δ=7.50 , 7.04–7.06 , 6.95–7.02 , 6.91–6.95 , 5.24 , 4.74 , 2.58 ppm ; LC–MS: m/z=255 [M+H].H-pyrazol-3-yl)methyl)-2H-benzo[b]oxazin-3(4H)-one (32)4- was added to a solution of (1-methyl-1H-pyrazol-3-yl)methanol  in CHCl3 and heated at 60 °C for 2 h. The solvent was removed in vacuo and the resulting crude 3-(chloromethyl)-1-methyl-1H-pyrazole hydrochloride material used without purification in the next step: LC–MS: m/z=131.1 [M+H].H-benzo[b]oxazin-3(4H)-one , crude 3-(chloromethyl)-1-methyl-1H-pyrazole hydrochloride (assumed 0.89 mmol) and anhydrous K2CO3  were treated as described for the synthesis of 14 to give compound 32 as a gum : 1H NMR : δ=7.30–7.33 , 7.26–7.27 , 6.96–7.01 , 6.17 , 5.12 , 4.67 , 3.88 ppm ; LC–MS: m/z=244 [M+H].Step 2: 2H-benzo[b]oxazin-4(3H)-yl)propanoate (15)Ethyl 2--one , ethyl 2-bromopropanoate  and anhydrous K2CO3  were treated as described for the synthesis of 14 then purified by column chromatography (0–20 % EtOAc/hexane) to give compound 15 as an off-white solid : 1H NMR : δ=6.99–7.05 , 7.83–7.86 , 5.36 , 4.62 , 4.18–4.25 , 1.64 , 1.20 ppm ; LC–MS: m/z=250 [M+H].H-benzo[b]oxazin-3(4H)-one (38)8-Bromo-4-((5-methylisoxazol-3-yl)methyl)-2: Step 1: 2-chloroacetyl chloride  was added dropwise to a mixture of 2-amino-6-bromophenol  and K2CO3  in DMF (40 mL). The reaction mixture was heated at 80 °C for 3 h then the solvent removed in vacuo. The residue was partitioned between CH2Cl2 and water, the organic layer was collected, dried with MgSO4 and the solvent was then removed in vacuo. The resulting solid was filtered off and triturated with Et2O to give intermediate 8-bromo-2H-benzo[b]oxazin-3(4H)-one as an off-white solid : 1H NMR : δ=8.67 , 7.23  6.86 , 6.79 , 4.74 ppm ; LC–MS: m/z=226/228 [M+H].H-benzo[b]oxazin-3(4H)-one , 3-(chloromethyl)-5-methylisoxazole  and anhydrous K2CO3  were treated as described for the synthesis of 14 to give compound 38 as a pale-yellow solid : 1H NMR : δ=7.24 , 7.20 , 6.89 , 5.98 , 5.13 , 4.77 , 2.39 ppm ; LC–MS: m/z=322/324 [M+H].Step 2: 8-bromo-2H-benzo[b]oxazin-3(4H)-one (34)2-Methyl-4-((5-methylisoxazol-3-yl)methyl)-2: Step 1: 2-chloropropanoyl chloride  was added dropwise to a mixture of 2-aminophenol  and K2CO3  in DMF (50 mL) at 0 °C. The reaction mixture was heated at 60 °C for 2 h then the solvent removed in vacuo. The residue was partitioned between CH2Cl2 and water, the organic layer was collected, dried with MgSO4 and the solvent was then removed in vacuo. The resulting solid was filtered and triturated with Et2O to give intermediate 2-methyl-2H-benzo[b]oxazin-3(4H)-one as a colourless gum : 1H NMR : δ=8.69 , 6.95–7.02  6.82–6.86 , 4.68 , 1.60 ppm ; LC–MS: m/z=164 [M+H].H-benzo[b]oxazin-3(4H)-one , 3-(chloromethyl)-5-methylisoxazole  and anhydrous K2CO3  were treated as described for the synthesis of 14, then purified by column chromatography (0–20 % EtOAc/hexane) to give compound 34 as an off-white solid : 1H NMR : δ=7.18–7.21 , 6.98–7.02 , 5.95–5.96 , 5.18 , 5.08 , 4.69 , 2.38 , 1.60 ppm ; LC–MS: m/z=259 [M+H].Step 2: 2-methyl-2H-benzo[b]oxazin-3(4H)-one (37)7-Bromo-4-((5-methylisoxazol-3-yl)methyl)-2: Step 1: 2-chloroacetyl chloride , 2-amino-5-bromophenol  and K2CO3  were treated as described in step 1 for the synthesis of 38 to give intermediate 7-bromo-2H-benzo[b]oxazin-3(4H)-one as an off-white solid. The product was purified as described in step 1 of the synthesis of compound 38 : 1H NMR : δ=7.91 , 7.15–7.16  7.10 , 6.68 , 4.63 ppm ; LC–MS: m/z=226/228 [M+H].H-benzo[b]oxazin-3(4H)-one , 3-(chloromethyl)-5-methylisoxazole  and anhydrous K2CO3  were treated as described for the synthesis of 14 to give compound 37 as a white solid : 1H NMR : δ=7.14–7.15 , 7.11–7.14 , 7.08–7.11 , 5.97–5.98 , 5.11 , 4.67 , 2.39 ppm ; LC–MS: m/z=322/324 [M+H].Step 2: 7-bromo-2H-benzo[b]oxazin-3(4H)-one (35)6-Chloro-4-((5-methylisoxazol-3-yl)methyl)-2: Step 1: 2-amino-4-chlorophenol , 2-bromoacetylbromide  and K2CO3  were treated as described in step 1 for the synthesis of 38 to give intermediate 6-chloro-2H-benzo[b]oxazin-3(4H)-one : LC–MS: m/z=185 [M+H].H-benzo[b]oxazin-3(4H)-one , 3-(chloromethyl)-5-methylisoxazole  and anhydrous K2CO3  were treated as described for the synthesis of 14 to give compound 35 as an off-white solid : 1H NMR : δ=7.23 , 7.07 , 6.16 , 5.17 , 4.77 , 2.37 ppm ; LC–MS: m/z=280/281 [M+H].Step 2: 6-chloro-2H-benzo[b]oxazin-3(4H)-one (36)7-Chloro-4-((5-methylisoxazol-3-yl)methyl)-2: Step 1: 2-amino-5-chlorophenol , 2-bromoacetylbromide  and K2CO3  were treated as described in step 1 for the synthesis of 38 to give intermediate 7-chloro-2H-benzo[b]oxazin-3(4H)-one : LC–MS: m/z=185 [M+H].H-benzo[b]oxazin-3(4H)-one , 3-(chloromethyl)-5-methylisoxazole  and anhydrous K2CO3  were treated as described for the synthesis of 14 to give compound 36 as an off-white solid : 1H NMR : δ=7.16 , 7.12 , 6.16 , 5.15 , 4.80 , 2.36 ppm ; LC–MS: m/z=280/281 [M+H].Step 2: 7-chloro-2H-benzo[b]oxazin-3(4H)-one (39)4-((5-Methylisoxazol-3-yl)methyl)-8-(4-(piperidin-4-yl)butyl)-2: Step 1: tert-butyl 4-(but-3-en-1-yl)piperidine-1-carboxylate , 9-BBN  and 38  were treated as described in step 1 for the synthesis of 40. The Suzuki reaction was carried out at 90 °C for 1 h, then at 110 °C for 30 min, to give intermediate tert-butyl 4-(4-(4-((5-methylisoxazol-3-yl)methyl)-3-oxo-3,4-dihydro-2H-benzo[b]oxazin-8-yl)butyl)piperidine-1-carboxylate: LC–MS: m/z=484 [M+H].tert-butyl 4-(4-(4-((5-methylisoxazol-3-yl)methyl)-3-oxo-3,4-dihydro-2H-benzo[b]oxazin-8-yl)butyl)piperidine-1-carboxylate was taken up in CH2Cl2 (10 mL), trifluoroacetic acid  was added dropwise and the reaction mixture was stirred at RT for 3 h. The solvent was removed in vacuo, the residue was taken into CH2Cl2 (30 mL) then partitioned between CH2Cl2 and saturated aqueous NaHCO3. The organic layer was collected, dried (MgSO4) and concentrated in vacuo. The solid obtained was filtered off and triturated with Et2O to give compound 39 as a white solid : 1H NMR : δ=6.98 , 6.94 , 6.91 , 6.14 , 5.13 , 4.74 , 2.87–2.94 , 2.55–2.60 , 2.37 , 1.75–1.83 , 1.54–1.62 , 1.47–1.54 , 1.27–1.34 , 1.13–1.25 , 0.91–1.10 ppm ; LC–MS: m/z=384 [M+H].Step 2: H-benzo[b]oxazin-8-yl)butyl)homopiperazine-1-methyl (40)4-(4-(4-((5-Methylisoxazol-3-yl)methyl)-3-oxo-3,4-dihydro-2: Step 1: Following the methodology reported by Billotte,oxazin-8-yl)butyl)homopiperazine-1-carboxylate : LC–MS: m/z=499 [M+H].Billotte, a solutitert-butyl 4-(4-(4-((5-methylisoxazol-3-yl)methyl)-3-oxo-3,4-dihydro-2H-benzo[b]oxazin-8-yl)butyl)homopiperazine-1-carboxylate  was stirred overnight at RT with trifluoroacetic acid (2 mL) in CH2Cl2 (10 mL). The reaction mixture was then concentrated in vacuo, and taken up in MeOH (10 mL). The product was purified using an SCX column, eluting with 2 n NH3/MeOH, followed by silica chromatography with 0–10 % MeOH/(0.1 %) NH3/CH2Cl2 eluent to give intermediate 4-(4-(4-((5-methylisoxazol-3-yl)methyl)-3-oxo-3,4-dihydro-2H-benzo[b]oxazin-8-yl)butyl)homopiperazine : LC–MS: m/z=399 [M+H].Step 2: H-benzo[b]oxazin-8-yl)butyl)homopiperazine  was stirred in CHCl3 with paraformaldehyde (5 equiv) for 1 h at RT. Sodium cyanoborohydride (5 equiv) was then added and the reaction stirred overnight at RT. A further 2 equivalents of paraformaldehyde and sodium cyanoborohydride were added and the reaction stirred for a further 18 h: LC–MS showed product at m/z=413 [M+H]. The product was recovered by SCX purification, eluting with 2 n NH3/MeOH. The solvent was removed in vacuo to give the title compound (40) as an off-white solid : 1H NMR : δ=7.06 , 6.86–6.93 , 5.98 , 5.12 , 4.74 , 4.65 , 2.71 , 2.63 , 2.49 , 2.38 , 2.37 , 1.82 , 1.54 ppm ; LC–MS: m/z=413 [M+H].Step 3: 4-(4-(4-((5-methylisoxazol-3-yl)methyl)-3-oxo-3,4-dihydro-2H-benzo[b]oxazin-3(4H)-one (41)4-((5-Methylisoxazol-3-yl)methyl)-8-(4-(1-methylpiperidin-4-yl)butyl)-2: 4-(but-3-en-1-yl)-1-methylpiperidine , 9-BBN  and 38  were treated as described in step 1 for the synthesis of 40 to give compound 41 as an off-white solid : 1H NMR : δ=7.09 , 6.94 , 6.89 , 6.00 , 5.14 , 4.67 , 2.84 , 2.62 , 2.40 , 2.27 , 1.86–1.91 , 1.53–1.70 , 1.35 , 1.21–1.31 ppm ; LC–MS: m/z=398 [M+H].R)-4-((5-Methylisoxazol-3-yl)methyl)-7-(3-((1-methylpyrrolidin-3-yl)methoxy)propyl)-2H-benzo[b]oxazin-3(4H)-one (42)(: Step 1: (R)-tert-butyl 3-methyl)pyrrolidine-1-carboxylate , 9-BBN  and 37  were treated as described in step 1 for the synthesis of 40. The Suzuki reaction was carried out at 120 °C for 90 min, to give (R)-tert-butyl 3-((3-(4-((5-methylisoxazol-3-yl)methyl)-3-oxo-3,4-dihydro-2H-benzo[b]oxazin-7-yl)propoxy)methyl)pyrrolidine-1-carboxylate, which was used without further purification: LC–MS: m/z=486 [M+H].R)-tert-butyl 3-((3-(4-((5-methylisoxazol-3-yl)methyl)-3-oxo-3,4-dihydro-2H-benzo[b]oxazin-7-yl)propoxy)methyl)pyrrolidine-1-carboxylate (assumed 0.62 mmol) was deprotected as described for the synthesis of 39 to give (R)-4-((5-methylisoxazol-3-yl)methyl)-7-(3-(pyrrolidin-3-ylmethoxy)propyl)-2H-benzo[b]oxazin-3(4H)-one, which was used without further purification: LC–MS: m/z=386 [M+H].Step 2: (R)-4-((5-methylisoxazol-3-yl)methyl)-7-(3-(pyrrolidin-3-ylmethoxy)propyl)-2H-benzo[b]oxazin-3(4H)-one (assumed 0.62 mmol) was taken up in dry CHCl3 (5 mL), paraformaldehyde  and sodium triacetoxyborohydride  added and the reaction mixture stirred overnight at RT. Water (5 mL) was added, the layers separated and the organics concentrated to dryness and purified by column chromatography 0–10 % MeOH/(0.1 %) NH3/CH2Cl2 eluent to give compound 42 as a white solid : 1H NMR : δ=7.09–7.13 , 6.79–7–6.84 , 5.97–5.98 , 5.12 , 4.65 , 3.54–3.59 , 3.40–3.46 , 3.20–3.25 , 2.57–2.67 , 2.52 , 2.34–2.41 , 2.37–2.38 , 1.95–2.03 , 1.81–1.90 , 1.73–1.81 , 1.63–1.71 ppm ; LC–MS: m/z=400 [M+H].Step 3: -one (43)4-((5-Methylisoxazol-3-yl)methyl)-7-(3-(piperidin-4-yloxy)propyl)-2: Step 1: 4-hydroxy-N-Boc-piperdine  was taken into THF (150 mL) and cooled to 0 °C. NaH   was added slowly, and the reaction was warmed to RT (10 min) then cooled back to 0 °C. Allyl bromide  was added and the reaction was stirred for 18 h at RT. The reaction was diluted with EtOAc (80 mL) and transferred to separating funnel. The organic layer was washed with water (50 mL), brine (50 mL) then dried (MgSO4) and concentrated in vacuo. Purification by column chromatography (5–30 % EtOAc/pet ether) yielded intermediate tert-butyl 4-piperidine-1-carboxylate : 1H NMR : δ=5.95 , 5.31 , 5.20 , 4.04 , 3.80 , 3.52 , 3.10 , 1.85 , 1.54 , 1.48 ppm ; LC–MS: m/z=142 [M+H].tert-butyl 4-piperidine-1-carboxylate , 9-BBN  and 37  were treated as described in step 1 for the synthesis of 40. Purification by column chromatography (5–30 % EtOAc/pet ether) gave intermediate tert-butyl 4-(2-(4-((5-methylisoxazol-3-yl)methyl)-3-oxo-3,4-dihydro-2H-benzo[b]oxazin-7-yl)ethoxy)piperidine-1-carboxylate : LC–MS: m/z=472 [M+H].Step 2: tert-butyl 4-(2-(4-((5-methylisoxazol-3-yl)methyl)-3-oxo-3,4-dihydro-2H-benzo[b]oxazin-7-yl)ethoxy)piperidine-1-carboxylate  was taken into CH2Cl2 (6 mL), trifluoroacetic acid (1 mL) was added and the reaction was stirred at RT for 2 h. Solvent was removed in vacuo, and the residue was dissolved up in MeOH (10 mL) and purified on an SCX column, elution with 2 n NH3/MeOH. This yielded compound 43 as an off-white solid : 1H NMR : δ=6.79 , 6.63 , 5.89 , 5.20 , 4.88 , 4.48 , 3.60 , 3.04–3.16 , 2.65 , 2.30 , 2.15 , 2.12 , 1.50 , 0.98 ppm ; LC–MS: m/z=372 [M+H].Step 3: H-benzo[b]oxazin-3(4H)-one (44)4-((5-Methylisoxazol-3-yl)methyl)-7-(4-(1-methylpiperidin-4-yl)butyl)-2: 4-(but-3-en-1-yl)-1-methylpiperidine , 9-BBN  and 37  were treated as described in step 1 for the synthesis of compound 40 to give compound 44 as a white solid : 1H NMR : δ=7.01–7.04 , 6.86–6.87 , 6.83–6.86 , 6.13–6.14 , 5.12 , 4.72 , 2.68–2.73 , 2.48–2.51 , 2.37 , 2.12 , 1.74–1.80 , 1.54–1.60 , 1.24–1.31 , 1.17–1.23 , 1.04–1.14 ppm ; LC–MS: m/z=398 [M+H].LmNMT protein–ligand complexes were determined using methods described previously.) of each test compound was then calculated using Equation (V [mL (mg protein)−1] is the incubation volume per mg protein added, and microsomal protein yield is taken as 52.5 mg protein per gram of liver. Verapamil (0.5 μm) was used as a positive control to confirm acceptable assay performance.XLfit  was used to calculate the exponential decay and consequently the rate constant . Membranes (12–14 kDa cutoff) were conditioned in deionised water for 60 min, followed by conditioning in 80:20 deionised water/EtOH for 20 min, and then rinsed in isotonic buffer before use. Female CD1 mouse plasma was removed from the freezer and allowed to thaw on the day of experiment. Thawed plasma was then centrifuged , spiked with test compound (10 μg g−1), and 150 μL aliquots (n=6 replicate determinations) loaded into the 96-well equilibrium dialysis plate. Dialysis versus isotonic buffer (150 μL) was carried out for 5 h in a temperature-controlled incubator at ∼37 °C  using an orbital microplate shaker at 125 rpm (Barworld Scientific). At the end of the incubation period, aliquots of plasma or buffer were transferred to micronic tubes , and the composition in each tube was balanced with control fluid such that the volume of buffer to plasma was the same. Sample extraction was performed by the addition of 400 μL acetonitrile containing an appropriate internal standard. Samples were allowed to mix for 1 min and then centrifuged at 3000 rpm in 96-well blocks for 15 min . All samples were analysed by UPLC–MS/MS on a Quattro Premier XE Mass Spectrometer . The unbound fraction was determined as the ratio of the peak area in buffer to that in plasma.Mouse brain penetration studies: Compounds 41 and 44 were dosed as a bolus solution intravenously at 1 mg free base per kg  to female NMRI mice (n=6). At 5 and 30 min (compound 41 only) following intravenous bolus injection of test compound, mice (n=3 per time point) were placed under terminal anaesthesia with isofluorane. A blood sample was taken by cardiac puncture into two volumes of distilled water and the brain removed. After suitable sample preparation, the concentration of test compound in blood and brain was determined by UPLC–MS/MS using a Quattro Premier XE . For each mouse at each time point, the concentration in brain (ng g−1) was divided by the concentration in blood (ng mL−1) to give a brain/blood ratio. The mean value obtained was quoted.Ethics. All regulated procedures on living animals were carried out under the authority of a project license issued by the Home Office under the Animals (Scientific Procedures) Act 1986, as amended in 2012 (and in compliance with EU Directive EU/2010/63). License applications will have been approved by the University's Ethical Review Committee (ERC) before submission to the Home Office. The ERC has a general remit to develop and oversee policy on all aspects of the use of animals on University premises and is a subcommittee of the University Court, its highest governing body.Parasite and NMT assays: These were carried out as described previously.[eviously.–10


Arabidopsis thaliana is reported. Eight new DCCs were identified and characterized structurally. Strikingly, three of these DCCs carry stereospecifically added hydroxymethyl groups, and represent bilin-type linear tetrapyrroles with an unprecedented modification. Indeed, DCCs show a remarkable structural parallel, otherwise, to the bilins from heme breakdown.Colorless nonfluorescent chlorophyll (Chl) catabolites (NCCs) are formyloxobilin-type phyllobilins, which are considered the typical products of Chl breakdown in senescent leaves. However, in degreened leaves of some plants, dioxobilin-type Chl catabolites (DCCs) predominate, which lack the formyl group of the NCCs, and which arise from Chl catabolites by oxidative removal of the formyl group by a P450 enzyme. Here a structural investigation of the DCCs in the methylesterase16 mutant of  Two slightly less polar catabolites, At-mes16-9HM-DNCC-44  and At-mes16-7HM-iso-DNCC-46  were identified as isomers, as their mass spectra exhibited each an [M+H]+ ion at m/z 647.0, indicating a common molecular formula of C35H42N4O8. The molecular formula for the least polar nonfluorescent DCC, named At-mes16-DNCC-47 (6), was deduced as C34H40N4O7 from observation of the [M+H]+ ion at m/z 617.1. A fluorescent fraction at tR=48.2 min, and designated here as At-mes16-7HM-iso-DFCC (7), also exhibited a pseudo-molecular ion [M+H]+ at m/z 647.2 (corresponding to a molecular formula of C35H42N4O8 and identifying 7 as an isomer of 4 and of 5).The major nonfluorescent DCC fractions with retention times , (εrel). NMR: Bruker UltraShield 600 MHz Avance II+. ESI-MS:).At-mes16-9HM-DNCC-44 (4): tR=43.6 min; UV/Vis (c=3.8×10−5 m): λmax (εrel)=294 sh (0.12), 238 sh (1.00), 216 nm (1.50); ESI-MS: m/z (%): 685.3 ; 669.3 ; 649.1 (13), 648.1 (38), 647.0 ; 615.1 ; 524.0 .At-mes16iso-DNCC-46 (5)-7HM-: tR=45.7 min; UV/Vis (c=3.8×10−5 m): λmax (εrel)=284 sh (0.32), 238 sh (1.00), 216 nm (1.30); ESI-MS: m/z (%): 649.1 (13), 648.1 (41), 647.0 ; 617.0 ; 615.1 ; 585.2 ; 524.1 .At-mes16-DNCC-47 (6): tR=46.8 min; UV/Vis (c=3.2×10−5 m): λmax (εrel)=294 sh (0.23), 242 sh (1.00), 219 nm (1.36); ESI-MS: m/z (%): 619.1 (11), 618.1 (39), 617.2 ; 585.1 ; 494.1 +H]+).At-mes16iso-DFCC (7)–7HM-: tR=48.2 min; UV/Vis (c=1.8×10−5 m): λmax (εrel)=358 (1.00), 244 (1.28), 222 nm (1.34); ESI-MS: m/z : 685.1 ; 669.2 ; 649.1 (18), 648.2 (49), 647.2 ; 617.2 ; 615.0 ; 585.2 ; 494.1 ; 492.13 .


The structures of the new compounds were established using 1D and 2D NMR  spectroscopy, high resolution mass spectrometry, and chemical methods. The aglycones of 1–3 , 9 and 10 (the methoxyl substituent at C-23 position) were found, identified from natural products for the first time. Moreover, the anti-HIV activities of the isolated steroidal glycosides were assessed, and compounds 13, 14, 16 and 17 exhibited high active against HIV-1.Eleven new furostanol saponins, typaspidosides B-L ( Aspidistra typica Baill is a perennial herbaceous plant which belongs to the family Convallariaceae − .Typaspidoside B : White a2): White amorphous power; 1H-NMR and 13C-NMR data, see Tables m/z: 1277.5659 [M−H]− .Typaspidoside C : White a3): White amorphous power; 1H-NMR and 13C-NMR data, see Tables m/z: 1239.5238 [M−H]− .Typaspidoside D (4): White amorphous power; 1H-NMR and 13C-NMR data, see Tables m/z: 1225.5524 [M−H]− .Typaspidoside E (5): White amorphous power; 1H-NMR and 13C-NMR data, see Tables m/z: 1277.5681 [M−H]− .Typaspidoside F (6): White amorphous power; 1H-NMR and 13C-NMR data, see Tables m/z: 1225.5472 [M−H]− .Typaspidoside G (7): White amorphous power; 1H-NMR and 13C-NMR data, see Tables m/z: 1209.5521 [M−H]− .Typaspidoside H (8): White amorphous power; 1H-NMR and 13C-NMR data, see Tables m/z: 1223.5739 [M−H]− .Typaspidoside I (9): White amorphous power; 1H-NMR and 13C-NMR data, see Tables m/z: 1223.5660 [M−H]− .Typaspidoside J (10): White amorphous power; 1H-NMR and 13C-NMR data, see Tables m/z: 1223.5682 [M−H]− .Typaspidoside K (11): White amorphous power; 1H-NMR and 13C-NMR data, see Tables m/z: 1193.5667 [M−H]− .Typaspidoside L (12): White amorphous power; 1H-NMR and 13C-NMR data, see Tables m/z: 1209.5549 [M−H]− .Typaspidoside M (1–12 (2.0 mg each) were treated in 2 N CF3COOH-H2O (2 mL) at 95°C for 4 h. The reaction mixture was extracted with CH2Cl2 (2 ml) three times. The aqueous layer was repeatedly evaporated to dryness until neutral. Then, in the monosaccharide mixture, glucose and galactose were detected by TLC analysis on a cellulose plate using n-BuOH-EtOAc-C5H5N-H2O (6:1:5:4) as the developing solution and aniline-o-phthalic acid as the detection solution and were then compared with the control samples: xyloses (Rf 0.56), glucose (Rf 0.45) and galactose (Rf 0.48). The sugar residue in pyridine (1 mL) was added to L-cysteine methyl ester hydrochloride (3.0 mg), and the mixture was kept at 60°C for 1 h. Then, HMDS-TMCS (hexamethyldisilazane- trimethylchlorosilane) (0.6 mL) was added to the reaction mixture, which was then kept at 60°C for 0.5 h. The supernatant (1.0 mL) was analyzed by GC under the following conditions: Agilent Technologies 6890 gas chromatograph was the equipment, carrying a 5973 mass spectrograph detector, and a HP-5 capillary column (30 m × 0.25 mm × 0.25 μm) was used. The conditions were as follow: column temperature: 180°C/250°C; programmed increase, 15°C/min; carrier gas: N2 (1 mL/min); injection and detector temperature: 250°C; injection volume: 4.0 μL; and split ratio: 1/50. The absolute configurations of the monosaccharides were confirmed by comparison of the retention times of the monosaccharide derivatives with those of standard samples: D-glucose (17.95 min) xylose (12.97 min) and D-galactose (18.57 min).Compounds 5) were co-transfected with 0.4 μg of pHIT/G and 0.6 μg of pNL-Luc-E. After 48h, the VSV-G pseudotyped viral supernatant (HIV-1) was harvested by filtration through a 0.45 μm filter and the concentration of viral capsid protein was determined by p24 antigen capture ELISA (Biomerieux). SupT1 cells were exposed to VSV-G pseudotyped HIV-1 (MOI = 1) at 37.0°C for 48 h in the absence or presence of the test compounds (Efavirenz was used as positive control). Viral infectivity was determined by measurement of luciferase activity in the infected cell using a Luciferase Assay (Promega), which was used to calculate the inhibition rate [293T cells (2×10ion rate .1 was isolated as a white amorphous powder. The negative high resolution electronic spray ion mass spectrometry (HRESIMS) showed an [M–H]−ion peak at m/z 1227.5665, corresponding to the molecular formula C56H92O29. The negative ESIMS showed fragment ions at m/z 1095.5, 1065.5, 915.5, 753.4, 591.3 and 429.3, attributable to the sequential losses of one pentose and four hexose residues and one molecule of water. The 1H NMR spectrum of 1 showed three methyl singlet signals at δ 1.38 , 0.87  and 1.72 , one methyl doublet signal at δ 1.00 , two methine protons indicative of secondary alcohols at δ 3.86  and 4.83 , and one olefinic proton at δ 5.28 . The 13C NMR spectrum of 1 showed 56 carbons, including two olefinic carbon signals at δ 141.1 (C-5) and 121.6 (C-6) and one ketone group signal at δ 215.8 (C-22). The presence of one ketone group located at C-22 and one hydroxyl group located at C-20 were supported by the fact that the HMBC correlations between protons H2-23 (δ 3.13), H2-24 (δ 1.60 and 2.13) and H3-21 (δ 1.72) and the carbon C-22 (δ 215.8), between the proton H3-21 (δ 1.72) and carbons C-20 (δ 82.5) and C-17 (δ 59.9).The β orientation of the oxygen atom at C-3, C-16 and C-20 positions was confirmed by the NOE enhancements of H-3 (δ 3.86)/Ha-1 (δ 0.94)/Hb-4 (δ 2.63), H-16 (δ 3.86) /H-17 (δ 1.86)/H-21 (δ 1.72), and Hb-1 (δ 1.64)/Ha-4 (δ 2.39)/H-19 (δ 0.87), while H-17 did not possess a NOE correlation with H-18 (δ 1.38). NOE correlations were also observed between H3-19/H-11, H-8/H3-18 and H-9/H-14 suggesting the usual trans junction for the B/C and C/D rings (S) based on the difference of the chemical shifts of the geminal protons H2-26 (Δab = δ H26a –δ H 26b = 0.57 ppm) -(1→3)]-β-D-glucopyranosyl-(1→3)-β-D-galactopyranoside, named typaspidoside B.Compound /D rings . The C-2.83 o, H-, and onege sites . Thus, t2, a white amorphous powder, showed an [M–H]−ion peak at m/z 1227.5659 in the negative HRESIMS, corresponding to the molecular formula C56H92O29. The negative ESIMS showed fragment ions at m/z 1095.5, 1065.5, 933.5, 771.4, 657.3 and 459.3, attributable to the sequential losses of one pentose and three hexose residues and one molecule of C6H10O2. A detailed comparison of the NMR data of 2 with those of 1 revealed that they shared similar aglycone and sugar moieties, except at the E ring of aglycone. The fact that C-22 position of the aglycone of 2 is one carboxyl group instead of the ketone group was deduced from the chemical shift of its C15~C18 and C19~C24  (δ 1.43) with C-20 (δ 65.2) and C-17 (δ 62.4), and H2-24 (δ 1.58 and 1.99) with C-22 (δ 173.1) and C-27 (δ 16.9) revealed the ring crack was between C-20 and C-22 and the carboxyl group was substituted at C-22 of the aglycone. The NOE correlations between H-21 (δ 1.43) and H-17 (δ 1.58) and H-16 (δ 5.33) revealed the β orientation of the oxygen atom at C-16 and C-20. Thus, the aglycone moiety of 2 was deduced as -5-en-3β,20β,26-triol-20,22-secfurost-22-one, an unreported sapogenin. The sugar unit and its linkage site to the aglycone were identified as in 1. Thus, 2 was determined to be -26-O-β-D-glucopyranosyl-5-en-3β,20β,26-triol-20,22-secfurost-22-one-3-O-β-D-glucopyranosyl-(1→2)-[β-D-glucopyranosyl]-(1→3)]-β-D-glucopyranosyl-(1→3)-β-D-galactopyranoside, named typaspidoside C.Compound pound 1) . In the 3 was isolated as a white amorphous powder with a molecular formula of C56H88O30, as determined by HRESIMS (m/z: 1239.5238 [M−H]−). The negative ESIMS showed fragment ions at m/z 945.4, 813.3, 783.3, 651.3, 489.2 and 327.2. A detailed comparison of the NMR data of 3 with those of 2 revealed that they shared similar sugar moieties, but significant differences in the chemical shifts of B~E rings of the aglycone. The 1H NMR spectrum of 3 showed three methyl singlet signals at δ 1.52 , 0.90  and 2.26 , two methine protons at δ 3.27  and 5.62 . The 13C NMR spectrum of 3 showed two olefinic carbon signals at δ 141.0 (C-5) and 121.2 (C-6), one carboxyl group signal at δ 173.3 (C-22) and two ketone group signals at δ 211.9 (C-12) and 204.4 (C-20). The fact that two ketone groups were located at C-12 and C-20 were supported the long-range correlations of H2-11 (δ 2.57 and 2.23), H2-41 (δ 1.18), H-17 (δ 3.27) and H3-18 (δ 1.52) with C-12 (δ 211.9), and H-17 (δ 3.27) and H3-21 (δ 2.26) with C-20 (δ 204.4) in the HMBC spectrum. Thus, the aglycone moiety of 3 was deduced as (25S)-5-en-3β,26-diol-20,22-secfurost-12,20,22-trione -β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside, named typaspidoside D.Compound 2-trione ,17, an u4 displayed an [M−H]− ion peak at m/z 1225.5524 in the negative HRESIMS, indicating a molecular formula of C56H90O29. A detailed comparison of the MS and NMR data of 4 with those of 3 revealed that they shared similar structures but exhibited significant differences in the B ring of the aglycone. The lack of one ketone group at position C-12 in 4 was deduced from the chemical shift of its C-9~C-13 and C-17 -26-O-β-D-glucopyranosyl- 5-en-3β,26-diol-20,22-secfurost-20,22-dione-3-O-β-D-glucopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)]-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside, named typaspidoside E.Compound and C-17 . The sug5 was isolated as a white amorphous solid. Its molecular formula C56H92O29 was deduced from the pseudo-molecular ion peak at m/z 1227.5681 [M–H]−in the HRESIMS. The existence of four hexose units and one pentose unit was proved by the fragment ions at m/z 1095.5, 1065.5, 933.5, 771.4, 609.4 and 429.3 in the negative ESI-MS. The 1H NMR spectrum showed two singlet signals at δ 1.14  and 0.90 , two doublet signals at δ 1.59  and 1.01 , and three oxygenated methine protons at δ 3.84 , 3.57  and 5.02 . A detailed comparison of the NMR data of 5 with those of 4 revealed that they shared similar sugar moieties, except that the C~E rings of the aglycone lack three carbonyl groups. The long range correlations of C-12 (δ 79.1) with H2-11 (δ 1.67 and 1.85), H-14 (1.11), H-17 (2.30) and H-18 (1.14) in the HMBC spectrum and the cross-peak correlations between H-12 (δ 3.57) and H2-11 (δ 1.67 and 1.85) in the COSY spectrum indicated that the hydroxyl group was located at the C-12 of the aglycone. The β orientation of OH-12 was confirmed by the NOE correlations between H-12/H-9/H-14/H-11/H-17 in the ROESY spectrum -β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside, named typaspidoside F.Compound  δ 110.9 ,21. Ther6 showed an [M−H]− ion peak at m/z 1225.5472 in the negative HRESIMS, indicating a molecular formula of C56H90O29. In the 13C NMR spectrum, the carbon signals at δ 165.3 (C-5), 126.1 (C-6) and 201.0 (C-7) indicated the aglycone possess a conjugate ketone and double bond. In the HMBC spectrum, H2-4 (δ 2.44 and 2.73) and H-19 (0.95) showed long range correlations to C-5 (δ 165.3), and H-8 (δ 2.38) and H-14 (δ 1.48) showed long range correlations to C-7 (δ 201.0) indicated that the α,β-unsaturated carbonyl structure was located at the C-5~C-7 positions of the aglycone -β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside, named typaspidoside G.Compound 7 was isolated as a white amorphous powder. The negative HRESIMS showed an [M−H]− ion peak at m/z 1209.5521, corresponding to a molecular formula of C56H90O28. The 1H NMR spectrum of 7 showed three methyl singlet signals at δ 0.89 , 0.89  and 1.71 , one methyl doublet signal at δ 1.07 . Comparing the MS and NMR data with those of 5, compound 7 was determined to contain the similar sugar moieties, but significant differences in the chemical shifts of the aglycone. Through analysis of the 1D- and 2D-NMR data of 7 (7 could be deduced. This was confirmed by the HMBC correlations of H3-21 (δ 1.71) with C-17 (δ 67.9), C-20 (δ 76.7) with C-22 (δ 163.8), C-23 (δ 91.4) and H2-24 (δ 2.13 and 2.51) with H-25 (δ 2.09), and H-17 (δ 2.22) with C-12 (δ 39.3), C-13 (δ 40.4), C-20 (δ 76.7) and C-22 (δ 163.8). NOE correlations between H-21 and H-18 were indicative of α orientation of OH-20. The aglycone is same as the aglycone of compound 1 in the literature -β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside, named typaspidoside H.Compound ata of 7 , the lac8 showed an [M−H]− ion peak at m/z 1223.5739 in the negative HRESIMS, corresponding to a molecular formula of C57H92O28. A detailed comparison of the MS and NMR data of 8 with those of 7 revealed that they shared similar aglycone and sugar moieties, except at the E ring of the aglycone possess an additional methoxy at position C-20. This was confirmed by the HMBC correlation between H3-21 (δ 1.37) and C-17 (δ 66.7), C-20 (δ 82.4) and C-22 (δ 157.3), and OCH3-20 (δ 3.17) and C-20 (δ 82.4). NOE correlations of H-17/H-21/H-16 were indicative of β orientation of OCH3-20 -β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside, named typaspidoside I.Compound 9 and 10 showed the [M−H]− ion peaks at m/z 1223.5660 and 1223.5682 in the negative HRESIMS, indicating the same molecular formula of C57H92O28. A detailed comparison of the MS and NMR data of 9 and 10 with those of 8 revealed that they shared similar aglycone and sugar moieties, except at the E and F rings of the aglycone. In the HMBC spectrum, long rang correlations were observed between H3-21 (δ 1.73) and C-17 (δ 64.8), C-20 (δ 109.0) and C-22 (δ 15.7), and between H3-OCH3 (δ 3.35) and C-23 (δ 73.1) confirming the OCH3 at position C-23 and a double bond at position C-20(22) of the aglycone. Comparing the NMR data with those of dioscoresides C and E -β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside, named typaspidoside J.Compounds  C and E ,25, 9 wa10 was deduced from the chemical shift of its H-23 and H-24 . So, the structure of 10 was elucidated as 26-O-β-D-glucopyranosyl--furost-5,20(22)-dien-23-methoxy-3β,16β, 26-triol-3-O-β-D-glucopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)]-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside, named typaspidoside KThe different orientation of methoxy at C-23 in 11 displayed an [M−H]− ion peak at m/z 1193.5667 in the negative HRESIMS, indicating a molecular formula of C56H90O27. A detailed comparison of the NMR and MS data of 11 with those of 10 revealed that they shared similar aglycone and sugar moieties, except the lack of one methoxy at position C-23 of the aglycone in 11. So, the structure of 11 was defined as 26-O-β-D-glucopyranosyl-(25S)-furost-5,20(22)-dien-3β,16β,26-triol-3-O-β-D-glucopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→3)]-β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside, named typaspidoside L.Compound 12 was isolated as a white amorphous powder. The molecular formula was determined as C56H90O28 by the [M−H]− ion peak at m/z 1209.5549 in the negative HRESIMS. The 1H NMR spectrum of 12 showed four methyl signals at δ 0.74 , 0.86 , 1.09  and 1.31 , and three methine proton signals of CHOR at δ 3.86 , 4.43  and 4.80 . The hydroxyl group at the C-24 position was confirmed by the HMBC corrections of H3-27 (δ 1.31) with C-24 (δ 72.9), C-25 (δ 31.8) and C-26 (δ 64.2). A detailed comparison of the NMR and MS data of 12 with those of 10, terrestrinin I -β-D-glucopyranosyl-(1→4)-β-D-galactopyranoside, named typaspidoside M.Compound 1–17, typaspidoside A1 and timosaponin H1 [1~11, typaspidoside A1 and timosaponin H1 are furostanol saponins and 12~17 are spirostanol saponins. Compounds 13, 14 and 16 are the corresponding spirostanol type of 5, typaspidoside A1 and timosaponin H1, respectively. The proto-yamogenin may be the common evolutionary precursor for all compounds in this paper. Compounds 1, 2, 5 and 6 are the secondary oxidation level products of timosaponin H1, and 11 is a dehydroxylation product of timosaponin H1. Compound 7 was formed through dehydroxylation and hydroxylation from timosaponin H1. Compound 8 was a methylate of 7. Compounds 9 and 10 are hydroxylation and methylation products of 11. Compound 3 is the tertiary oxidation level product of timosaponin H1. Compounds 12, 13 and 14 are the secondary oxidation level products of 16, furthermore, 12 is a two chains saponin after glycosidation. The detail plausible biosynthetic relationship between the isolated compounds was shown in There are sixteen aglycones in compounds 1–17 were evaluated for anti-HIV activities using one-round infectivity assay and the results were shown in Table 5. All test compounds exhibited anti-HIV activities to different degrees, and 9 of them inhibited viral replication by more than 50% at a concentration of 30 uM. The spirostanol saponins showed the more strong activities than furostanol saponins. Especially, 4 compounds  showed potent activities against HIV-1with >95% inhibition of viral replication, which have the potential for anti-HIV drug development and are worth to exploit further.Compounds S1 File(DOC)Click here for additional data file.


KMM 4672. The structures of compounds 1–5 were determined based on spectroscopic methods. The absolute configuration of pretrichodermamide D (1) was established by a combination of modified Mosher′s method, NOESY data, and biogenetic considerations. N-Methylpretrichodermamide B (5) showed strong cytotoxicity against 22Rv1 human prostate cancer cells resistant to androgen receptor targeted therapies.Three new epidithiodiketopiperazines pretrichodermamides D–F ( N-methylation, C-4–C-5-epoxidation − and was in accordance with 13C NMR data. A thorough analysis of the 1H and 13C NMR data , 164.2 (C), 153.0 (C), 147.6 (C), 135.9 (C), and 116.3 (С), suggested the presence of two amide carbonyl carbons, three oxygenated, and one C-substituted sp2-carbons.The molecular formula of compound NMR data  of 1 wit1H and 13C NMR spectra of 1 (4) − and by 13C NMR analysis. The general features of the 1H and 13C NMR spectra  to C-6 (δC 129.7) and C-8 (δC 71.0), from H-9 (δH 3.83) to C-4 (δC 67.0) and C-8, and from 4-OH (δH 5.29) to C-3 (δC 39.0), C-5 (δC 131.8), and C-9 (δC 83.4) proved that the planar structure of 2 was identical with pretrichodermamide D (1). The vicinal coupling constant JH7-H8 (7.7 Hz) and JH8-H9 (10.7 Hz) according to Karplus equation  indicated the axial stereolocations of H-7, H-8 (δH 3.56), and H-9. These relative configurations were additionally proved by ROESY’s (H 4.64) and H-9. The absolute stereoconfigurations of 2 were assigned as for pretrichodermamide D (1) according to biogenetic considerations. Thus, compound 2 was determined as the C-7 epimer of pretrichodermamide D and named pretrichodermamide E.The molecular formula of compound  spectra  of 2 res ROESY’s  H-7 with3 was determined as C21H24N2O9S2 (the same as 1 and 2) on the basis of HRESIMS and 13C NMR spectra. The NMR data for this compound were very similar to those obtained for pretrichodermamide C (4) with the exception of proton and carbon signals at C-3, C-4, C-5, C-6, and C-9. The HMBC correlations from H-5 (δH 3.69) to C-3 (δC 35.6), C-4 (δC 67.4), C-6 (δC 126.9), and C-7 (δC 131.2), from H-8 (δH 4.16) to C-7 and C-9 (δC 83.5), and from H-9 (δH 3.97) to C-4 and C-8 (δC 64.6) established a planar structure of the cyclohexene ring with a double bond between C-6 and C-7. The mutual ROESY correlations (H 4.96), 5-OH (δH 5.19), and 8-OH (δH 5.15) showed α-orientation of 5-OH and detected 3 as the С-5-epimer of pretrichodermamide C (4). The relative configurations of the 1,2-oxazadecaline fragment were additionally suggested by a W-type coupling constant between H-9  and H-3β . The absolute configurations in compound 3 were proposed based on biogenetic relationships with compounds 1, 2, 4, and 5. Compound 3 was named pretrichodermamide F.The molecular formula of compound elations  from H-91–3), the known pretrichodermamide C (4) and N-methylpretrichodermamide B (5) were also isolated from this fungus. For the first time these compounds were found in Egyptian hyper saline lake fungus Penicillium sp. − .Pretrichodermamide D (2): white powder; c 0.4, MeOH); UV (MeOH) λmax (log ε) 205 (4.51) nm; ECD  λmax (Δε) 218 (−17.64), 258 (−6.04), 300 (+0.80) nm; IR (CHCl3) νmax 3514, 3000, 2842, 1694, 1617, 1509, 1466, 1346, 1274, 1097, 1056, 1029 cm−1; 1H and 13C NMR, see m/z 511.0869 [M − H]− .Pretrichodermamide E (3): white powder; c 0.4, MeOH); UV (MeOH) λmax (log ε) 206 (4.45) nm; ECD  λmax (Δε) 217 (−23.27), 260 (−6.64), 301 (+0.49) nm; IR (CHCl3) νmax 3515, 3001, 2842, 1692, 1617, 1509, 1466, 1347, 1277, 1098, 1054, 1036 cm−1; 1H and 13C NMR, see m/z 535.0815 [M + Na]+ .Pretrichodermamide F (4): white powder; c 0.09, MeOH)  + .Pretrichodermamide C ] ; UV (MeON-methylpretrichodermamide B (5): white powder; c 0.14, MeOH)  + ., MeOH)] ; UV (MeOR)-MTPA-Cl (5 μL) was added to a solution of the pretrichodermamide D (1.8 mg) in pyridine at room temperature and stirred for 4 h. After evaporation of the solvent, the residue was passed through a silica gel column (20% EtOAc-hexane) to afford the (S)-MTPA ester (1.0 mg). The (R)-MTPA ester (1.2 mg) was prepared in a similar manner using (S)-MTPA-Cl.4-dimethylaminopyridine  and (S)-MTPA ester of 1: 1H NMR  δ: 7.16 , 6.04 , 5.93 , 5.80 , 5.73 , 4.67 , 4.30 , 3.89 , 3.68 , 3.68 , 3.55 , 2.90 , 2.47 , 2.38 , 7.45–7.75 . The signals of H-2′ and H-3′ overlapped with solvent signals. HRESIMS m/z 979.1438 [M + Cl]– .(R)-MTPA ester of 1: 1H NMR  δ: 7.66 , 7.17 , 5.54 , 5.85 , 5.64 , 5.65 , 5.27 , 4.15 , 3.89 , 3.73 , 3.72 , 3.59 , 2.28 , 2.21 , 7.40–7.68 . The signals of H-2′, H-3′, and Me-10′ overlapped with solvent signals. HRESIMS m/z 979.1430 [M + Cl]– . with (for LNCaP) or without (for 22Rv1 and PC-3) 1 mM sodium pyruvate (Invitrogen). Cells were continuously kept in culture for a maximum of 3 months and were routinely inspected microscopically for stable phenotype and regularly checked for contamination with mycoplasma. Cell line authentication was performed by DSMZ  using highly polymorphic short tandem repeat loci .The in vitro cytotoxicity of individual substances was evaluated using the MTT -2,5-diphenyltetrazolium bromide) assay, which was performed as previously described . Cytotox5 cells/well in 2 mL/well). The medium was changed to fresh medium containing different concentrations of the substances. After 48 h of treatment, cells were harvested with a trypsin-EDTA solution, fixed with 70% EtOH, stained, and analyzed by FACS. The results were quantitatively analyzed using Cell Quest Pro software . Cells appeared at sub-G1 peak were assumed as apoptotic.The cell cycle distribution was analyzed by flow cytometry using PI staining as described before with slight modifications . In brieThe hemolytic activity was evaluated using CD-I mouse erythrocytes as previously described ,20.D–F (1–3) were isolated from the lipophilic extract of marine algae-derived fungus Penicillium sp. KMM 4672. Each new compound contains rare 1,2-oxazadecaline moieties [1 and 2 are the first isomers at oxazadecaline moiety among the related alkaloids. N-methylpretrichodermamide B (5), highly cytotoxic in 22Rv1 human prostate cancer cells, is resistant to androgen receptor-targeted therapies. At the same time, N-methylpretrichodermamide B was found to be cytotoxic for non-malignant cells (splenocytes and erythrocytes) only at high doses (ED50 62.1 and >100 µM). Therefore, this compound may be a promising candidate for the therapy of human drug-resistant prostate cancer.Three new epidithiodiketopiperazines, named pretrichodermamides D–F –3 were i


Aedes aegypti larvae, the vectorof dengue, hemorrhagic dengue, Zika and chikungunya fevers. Only the extract obtainedfrom seeds of Guarea kunthiana (Meliaceae) proved active (LC50 =169.93 μg/mL). A bioassay-guided investigation of this extract led to the isolationand identification of melianodiol, a protolimonoid, as the active constituent (LC50 =14.44 mg/mL). Meliantriol, which was also obtained from thebioactive fraction, was nevertheless devoid of any larval toxicity, even at thehighest concentration tested (LC50 > 100.0 mg/mL). These resultsindicate that the larvicidal activity of melianodiol stems from the presence of thecarbonyl moiety at C-3 in the 21,23-epoxy-21,24,25-trihydroxy-tirucall-7-ene-typeskeleton. The structures of both protolimonoids were established on the basis ofspectral methods (1H and 13C NMR and MS). This is the firstreport on the toxicity of melianodiol against Ae. aegypti larvae.Based on the results, melianodiol can be regarded as a potential candidate for use asan ecologically sound biocontrol agent for reducing the larval population of thisvector.A total of 36 ethanol extracts from different anatomical parts of 27 plant species(18 families), native to the Pantanal and Cerrado biomes in Midwest Brazil, wasassessed for their effect against  Aedes aegypti is the principal vector of arboviral diseases, such asdengue, hemorrhagic dengue, Zika, chikungunya, and yellow fevers. The incidence of denguefever has grown remarkably in recent decades worldwide  proved active, being therefore selected for a bioassay-guided fractionation.Herein, we report the isolation and structure elucidation of its bioactive constituent.Despite research advances from the pharmaceutical industry, developing dengue, Zika andchikungunya vaccines has proven a complex task and these vector-borne diseases remain aglobal public health challenge. Meanwhile, controlling the incidence of dengue, Zika andchikungunya fevers has largely depended on effectively fighting its main primary vector,nagement . Nonethenagement . In addinagement . As part aegypti , a total1H and 13C NMR spectra were obtained in CDCl3 on a Bruker DPX-300 spectrometer, operating at 300.13 MHz (1H)/75.47 MHz (13C).EIMS data were acquired at 70 eV on a Shimadzu  QP5000 DI-50 instrument.Pre-coated silica gel GF254 chromatoplates  were used foranalytical thin-layer chromatographic procedures. Silica gel 60 and silica gel 60 RP-18  were used for column chromatography.Plant material - The plants investigated  was extractedwith ethanol (4 x 2L) at room temperature. After concentration invacuo, the residue obtained from each extract was stored at -18ºC untilbiological screening was performed.Bioassay-guided fractionation of seeds of G. kunthiana - Fruits ofG. kunthiana were collected in August 2012 and their seeds were separated from the pulp and peel . The seeds were extracted with ethanol(4 x 4 L) for seven days and filtered. After concentration under reduced pressure, thebioactive ethanol extract was subsequently partitioned between MeOH-H2O (9:1)and hexane. Water was added to the hydromethanolic phase to yield a MeOH-H2O(1:1) mixture, which was partitioned with EtOAc. The hexane (9.0 g), EtOAc (14.0 g), andhydromethanolic (2.0 g) phases were tested against Ae. aegypti larvae,and the larvicidal activity was found to reside in the EtOAc solubles (LC50 =105.70 mg/mL). An aliquot of this bioactive phase (11.0 g) was then chromatographed onan RP-18 silica gel (350.0 g) column, using step gradient elution withMeOH-H2O , MeOH, and CHCl3 to give fivefractions (F1-F5) of 500 mL each. Testing for larvicidal activity showed fraction F3(2.0 g), containing two major components, to be bioactive (LC50 = 15.20mg/mL). An aliquot of this fraction (800.0 mg) was further subjected to columnchromatography on silica gel (30.0 g) using CHCl3 (750 mL),CHCl3-MeOH , CHCl3-MeOH ,CHCl3-MeOH , and CHCl3-MeOH ,yielding protolimonoids 1  and 2 , both of which were tested against Ae.aegypti larvae. Only compound 1 proved bioactive(LC50 = 14.44 mg/mL). Purity of protolimonoids 1 and2 was established on the basis of their 1H and 13CNMR spectra and by chromatographic techniques.Melianodiol (1) - colorless amorphous powder; EIMS (70 eV), m/z(rel. ab.): 488 , 470 (8) [M - H2O], 455 (25) [M -.CH3], 412 (17) [470 - CH3COCH3], 397(59) [455 - CH3COCH3/ 412 - .CH3], 379 (34)[ 397 - H2O], 365 (92) [397 - CH3OH], 297 (67) [379 -C5H6O], 59 (100) [ C3H7O+];1H NMR : δ 2.76 ; 5.25 ; 0.83 ; 1.01 ; 5.32; 4.38 ; 3.15 ; 1.26 ; 1.28 ; 1.03 ; 1.12 ; 1.03 ; 13C-NMR: δ 38.5 (C-1); 35.0 (C-2); 216.9 (C-3); 47.8 (C-4);52.3/52.4 (C-5); 24.3 (C-6); 118.0 (C-7); 145.6 (C-8); 48.3/48.8 (C-9); 35.1 (C-10);17.6 (C-11); 30.1/31.4 (C-12); 43.5/43.6 (C-13); 50.7 (C-14); 34.2 (C-15); 27.2 (C-16);45.3 (C-17); 21.5 (C-18); 12.7 (C-19); 45.2/46.3 (C-20); 96.7/102.2 (C-21); 30.2 (C-22);78.7 (C-23); 74.8/75.8 (C-24); 73.2 (C-25); 26.5 (C-26); 26.7 (C-27); 24.5 (C-28); 23.2(C-29); 27.4 (C-30). 1H and 13C NMR, and mass spectra ofmelianodiol are shown in Supplementary figures .Meliantriol(2) - colorless amorphous powder; EIMS (70 eV), m/z (rel. ab.): 490, 475 (19) [M - .CH3], 457 (40) [475 -H2O], 439 (61) [457 – H2O], 399 (39) [457 -CH3COCH3], 381 (40) [399 - H2O], 367 (100) [399 -CH3OH], 349 (20) [367- H2O], 281 (39) [ 381 -C5H8O2]; 1H NMR : δ 3.22 ; 5.21 ; 0.82; 0.72 ; 5.24 ; 4.34 ; 3.14 ; 1.26 ; 1.24 ; 0.84 ; 0.94 ; 0.96 ; 13C-NMR : δ 37.2 (C-1);27.5 (C-2); 79.1 (C-3); 39.0 (C-4); 50.7 (C-5); 24.0 (C-6); 118.2 (C-7); 145.4 (C-8);48.8 (C-9); 34.9 (C-10); 17.5 (C-11); 31.6 (C-12); 43.7 (C-13); 50.7 (C-14); 34.2(C-15); 27.3 (C-16); 50.7 (C-17); 23.2 (C-18); 13.1 (C-19); 45.3/46.5 (C-20); 97.1/102.3(C-21); 34.2 (C-22); 77.4/78.7 (C-23); 75.1 (C-24); 73.4 (C-25); 26.5 (C-26); 26.7(C-27); 27.5 (C-28); 14.7 (C-29); 27.3 (C-30). 1H and 13C NMR, andmass spectra of meliantriol are shown in Supplementaryfigures.Larvicidal activity assay against Ae. aegypti - The bioassays wereperformed according to World Health Organization protocols . For F3 (the bioactivefraction), serial diluting yielded test solutions of concentrations as low as 15 mg/mL.Compounds 1 and 2 were each initially tested at 100, 80, 60,40 and 20 mg/mL. Compound 1 (the bioactive protolimonoid) was subsequentlytested at concentrations as low as 15 mg/mL to allow calculating its LC50value. The purity of the isolated compounds tested was ≥ 95%. Ten third-instar larvaewere placed in a beaker containing 5 mL of test sample. For each concentration, 40larvae were exposed (four replicates of 10 larvae each). A set of controls was also run,using 0.5% DMSO in distilled water in four replicates. Each assay was repeated threetimes at different days and showed no significant variation. Larval mortality wasrecorded 24 h after treatment at 27 (± 2) ºC, during which no food was given to thelarvae. Larvae were considered dead when they did not respond to stimuli (gentle pokingwith a pipette). All the experiments were carried out at 27 (± 2) ºC and 70 (± 5) %relative humidity.rotocols  with slirotocols . Mosquit50 and LC90 values (µg/mL), slope and Chi-square value werecalculated using the Probit analysis on IBM SPSS Statistics 22.0. Comparison amongsamples in relation to the LC50 and LC90 values was performedusing the 95% confidence interval from LCs ratio  were obtained from a colony maintained at ourlaboratory , according to the Conselho Nacional de Controle de ExperimentaçãoAnimal - CONCEA/Ministério da Ciência, Tecnologia e Inovação-MCTI(http://www.mct.gov.br/index.php/content/view/310553.html). The authors declare noconflicts of interest.G. kunthianaseeds proved active against Ae. aegypti larvae (LC50 =169.93 μg/mL). No larval deaths occurred with any of the other extracts, not even thatprepared from G. kunthiana fruit peel and pulp, which hadLC50 values higher than 1,000 μg/mL  and meliantriol (2)  . Their s samples.Ae. aegypti larvae, only compound1 proved biologically active, with LC50 and LC90values of 14.44 and 17.54 mg/mL, respectively, as depicted in 2 had an LC50 value above 100mg/mL.After evaluation for toxicity against p values greater than 0.15 on chi-square test . All samples were also significantlydifferent with respect to the LC50 and LC90 values , and also presented decreasing LC values from crude extract to theisolated bioactive compound .All samples showed dose/response curve (slope) different from 0 degree (p < 0.001)and 1) was isolated as thelarvicidal constituent of the bioactive ethyl acetate phase obtained after partitioningof the ethanol extract of the seeds of G. kunthiana. Meliantriol(2), which was also obtained from the bioactive fraction, wasnevertheless devoid of any larval toxicity, even at the highest concentration tested.These results indicate that the larvicidal activity of 1 stems from thepresence of the carbonyl moiety at C-3 in the21,23-epoxy-21,24,25-trihydroxy-tirucall-7-ene-type skeleton, since its correspondingreduced derivative 2, which differs from 1 only for bearing ahydroxyl function at that same carbon, was found to be inactive.In the present study, the protolimonoid melianodiol  was obtained, despite the fact that limonoids are known for theirbiological, and particularly insecticidal, properties (Ae. aegypti larvae and pupae (Ae. aegypti larvae.Meliaceae, the family to which the genus imonoids . In a prAe. aegypti larvae in areaswhere dengue, Zika and chikungunya fevers are of concern. Also, melianodiol was isolatedfrom seeds, a feature that encourages the conservation and sustainable use of G.kunthiana as a source of this bioactive compound. The findings warrantinvestigating other Meliaceous species, as well as members of the Simaroubaceae andRutaceae, from which protolimonoids have also been obtained, for their potential towardsthe development of new biocontrol agents against Ae. aegyptilarvae.The foregoing results revealed melianodiol to be a potential candidate for thedevelopment of an ecologically sound alternative to environmentally hazardous syntheticinsecticides currently employed to control


S,Z)-4-methyl-2-(4-oxo-5-((5-substitutedphenylfuran-2-yl) methylene)-2-thioxothiazolidin-3-yl)pentanoic acids via a Knoevenagel condensation were synthesized and evaluated for their antibacterial activity in-vitro. The synthesized compounds were characterized by IR, 1H NMR and MS. The antibacterial test in-vitro showed that all of the synthesized compounds had good antibacterial activity against several Gram-positive bacteria  with minimum inhibitory concentration (MIC) values in the range of 2–4 µg/mL. Especially compounds 4c, 4d, 4e and 4f were the most potent, with MIC values of 2 µg/mL against four multidrug-resistant Gram-positive bacterial strains.The microbial resistance has become a global hazard with the irrational use of antibiotics. Infection of drug-resistant bacteria seriously threatens human health. Currently, there is an urgent need for the development of novel antimicrobial agents with new mechanisms and lower levels of toxicity. In this paper, a series of ( The microbial resistance, caused by the irrational use of antibiotics, has become a global hazard. Infection by drug-resistant bacteria seriously threatens human health. In the case of bacterial resistance growing, looking for antimicrobial agents with new mechanisms becomes an important issue to protect human life, and it has become the concern of the whole society. Therefore, the design and development of new antibacterial drugs have very important significance .Rhodanine-3-acetic acid and its derivatives bear broad biological activities as anti-epileptic, anti-bacterial, anti-viral and antidiabetic -8. For tIn the present work, we report the synthesis and antibacterial evaluation of compound II using compound I as the lead compound, which structure-based design mainly focused on the chalcone moiety, was replaced by its isostere 2-phenylfuran and simultaneously introduced by different substituents into the phenyl ring, expecting to get more optimized structures for binding to receptor easily, which consequently could result in their more potent activity. Moreover, the acetic acid group on the 3-position of the rhodanine was also substituted with an aliphatic side chain aiming to increase the bacterial cell wall penetration of these compounds and therefore potentially improve their anti-bacterial activities . Chemistry(S)-4-methyl-2-(4-oxo-2-thioxothiazolidin-3-yl) pentanoic acid (The target compounds (4a-4h) were synthesized according to the route depicted in 1H NMR, and high-resolution mass spectroscopy. Taking compound 4b as an example, the high-resolution mass spectrometry of 4b displayed a molecular ion signal at m/z 434.0284, which was corresponding to its theoretical value of 434.0293. In the 1H-NMR spectrum of compound 4b, in addition to the aromatic protons of benzene and furan ring protons (δ = 7.50–8.02 ppm), a sharp singlet due to vinylic hydrogen was observed at 7.81 ppm and a broad singlet due to methyne hydrogen linked to nitrogen-atoms was observed at 5.64 ppm. Meanwhile, it has been reported that the (Z)- or (E)-geometry was readily identified by 1H-NMR, as the vinylic proton is more deshielded in the (Z)-isomer than the (E)-isomer. In (Z)-form, vinylic proton appeared at 7.21 ppm due to the magnetic anisotropy effects of carbonyl group on the vinylic proton, while in (E)-form the resonance should be around 6.50 ppm -configuration as thermodynamically favored structures. The structures of the desired compounds were confirmed by IR, 6.50 ppm -20. In oThe physicochemical properties of the compounds synthesized are presented in the materials and methods section. Their antibacterial activities were all evaluated by a serial dilution method to obtain the Minimum Inhibitory Concentration (MIC) with different strains including multidrug-resistant clinical isolates.Materials and methods1H-NMR spectra were measured on a Bruker AV-300 spectrometer using tetramethylsilane as the internal standard. Mass spectra were measured on a micrOTOF-Q II mass spectrometer . Specific optical rotation was measured on a Digital automatic polariscope JASCO P-1020 . The major chemicals were purchased from Sigma-Aldrich  and Fluka Companies . Melting points were determined in open capillary tubes and are uncorrected. Reaction courses were monitored by TLC on silica gel-precoated F254 Merck plates. Developed plates were examined with Ultra Violet lamps (254 nm). IR spectra were recorded (in KBr) on a FTIR1730 . Preparation of compounds5-( Substituted-phenyl)furan-2-carbaldehydes (2a-2h)A mixture of aniline (30 mmol) and sodium nitrite (30.3 mmol) in hydrochloric acid (8 mL) and water (6 mL) was stirred for 1h at 0 °C. After filtering the reaction, acetone (30 mL), furfural (30 mmol) and cupric chloride (3 mmol) were slowly added to the filtrate, and the mixture was stirred for 12 h at 20 °C. The excess solvent was removed under reduced pressure and the residue was dissolved in ethyl acetate and extracted with water, dried over MgSO4, and the solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography (dichloromethane / methanol= 200:1) to obtain a brown solid. (S)-4-methyl-2-(4-oxo-2-thioxothiazolidin-3-yl) pentanoic acid (3)In a round-bottomed ﬂask equipped with a magnetic stirrer, L-leucine(30.3 mmol) was dissolved with sodium hydroxide (30.3 mmol) in water (25 mL). Then, carbon disulﬁde (30.3 mmol) was added to the reaction mixture, which was stirred vigorously overnight. An aqueous solution of sodium chloroacetate (30.3 mmol) was added and stirring was continued at 23 ?-2-(5-((5-(substituted-phenyl)furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-4-methylpentanoic acid 6-( phenyl)thiazolotriazoles (4a-4h)A mixture of compounds 2a-2h (2.2 mmol), compound 3 (2.0 mmol), 10 drops piperidine and 10 drops glacial acetic acid in ethanol (15 mL) was refluxed for 4 h. After cooling, the solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography (dichloromethane / methanol = 100:1) to afford a yellow solid. -2-furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-4-methylpentanoic acid (4a)α]     ：31.2 . IR (KBr) cm–1: 2963 (OH), 1715 (C=O). 1H NMR : δ 0.92 , 0.97 , 1.53 2), 2.05 , 2.25 , 5.62 , 7.49–7.99 , 7.74 , 13.41 . ESI-HRMS calcd for C20H16Cl2NO4S2- ([M-H]-): 467.9903; found: 467.9912.Yield 49%, m.p. 70-72 °C, Mol. weight: 468.99. [-2-(5-((5-(2-chlorophenyl)furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-4-methylpentanoic acid (4b)α]     ：35.4 . IR (KBr) cm–1: 2955 (OH), 1713 (C=O). 1H NMR : δ 0.91 , 0.96 , 1.52 2), 2.05 , 2.25 , 5.64 , 7.50–8.02 , 7.81 , 13.41 . ESI-HRMS calcd for C20H17ClNO4S2- ([M-H]-): 434.0293; found: 434.0284.Yield 38%, m.p. 71-73 °C, Mol. weight: 435.04. -): 434.0293; found: 434.0292.Yield 44%, m.p. 72-74 °C, Mol. weight: 435.04. IR (KBr) cm-2-(5-((5-(4-chlorophenyl)furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-4-methylpentanoic acid (4d)–1: 2955 (OH), 1713 (C=O). 1H NMR : δ 0.91 , 0.96 , 1.53 2), 2.05 , 2.23 , 5.64 , 7.47–7.95 , 7.77 , 13.39 . ESI-HRMS calcd for C20H18ClNNaO4S2+ ([M+Na]+): 458.0258; found: 458.0256.Yield 46%, m.p. 76-78 °C, Mol. weight: 435.04. IR (KBr) cm-2-(5-((5-(3-chloro-4-fluorophenyl)furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-4-methylpentanoic acid (4e)α]     ：36.9 . IR (KBr) cm–1: 2975 (OH), 1711 (C=O). 1H NMR : δ 0.91 , 0.96 , 1.52 2), 2.05 , 2.26 , 5.80 , 7.04–8.15 , 7.74 , 13.39 . ESI-HRMS calcd for C20H16ClFNO4S2- ([M-H]-): 452.0199; found: 452.0189.Yield 35%, m.p. 75-77 °C, Mol. weight: 453.03. [-2-(5-((5-(4-bromophenyl)furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-4-methylpentanoic acid(4f)α]     ：32.9 . IR (KBr) cm–1: 2964 (OH), 1711 (C=O). 1H NMR : δ 0.91 , 0.96 , 1.52 2), 2.05 , 2.24 , 5.63 , 7.47–7.85 , 7.77 , 13.38 . ESI-HRMS calcd for C20H17BrNO4S2- ([M-H]-): 477.9788; found: 477.9784.Yield 39%, m.p. 83-85 °C, Mol. weight: 478.99. [-2-(5-((5-(2-fluorophenyl)furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-4-methylpentanoic acid (4g)α]     ：46.1 . IR (KBr) cm–1: 2973 (OH), 1712 (C=O). 1H NMR : δ 0.91 , 0.96 , 1.55 2), 2.06 , 2.25 , 5.66 , 7.25–7.97 , 7.81 , 13.40 . ESI-HRMS calcd for C20H18FNNaO4S2+ ([M+Na]+): 442.0553; found: 442.0558.Yield 42%, m.p. 74-76 °C, Mol. weight: 419.07. [-4-methyl-2-(4-oxo-2-thioxo-5-((5-(4-(trifluoromethoxy)phenyl)furan-2-yl)methylene)thiazolidin-3-yl)pentanoic acid (4h)α]     ：30.7 . IR (KBr) cm–1: 2969 (OH), 1715 (C=O). 1H NMR : δ 0.91 , 0.97 , 1.53 2), 2.06 , 2.25 , 5.64 , 7.48–8.05 , 7.78 , 13.37 . ESI-HRMS calcd for C21H17F3NO5S2- ([M-H]-): 484.0506; found: 484.0510.Yield 48%, m.p. 79-81 °C, Mol. weight: 485.06. -). Cells at 80 to 90% confluence were split by trypsin , and the medium was changed at 24 h intervals. The cells were cultured at 37 °C in a 5% COEvaluation of anti-bacterial activity in-vitroS. aureus RN 4220, S. aureus KCTC 209 and S. aureus KCTC 503) and one Gram-negative strain (E. coli 1356) are shown in Staphylococcus aureus (MRSA CCARM 3167 and MRSA CCARM 3506) and quinolone-resistantStaphylococcus aureus (QRSA CCARM 3505 and QRSA CCARM 3519)) are shown in The results of compounds 4a-4h against three Gram-positive strains , but weaker than the positive control oxacillin (MIC = 1 µg/mL), gatifloxacin and moxifloxacin (MIC = 0.25 µg/mL). Compounds 4a-4h displayed good activity against S. aureus KCTC 503 with MIC values of 4 µg/mL. However, these compounds displayed moderate to weak potency against S. aureus KCTC 209 with MIC values ranging from 8-64 µg/mL. None of the compounds showed any inhibitory activity against the Gram-negative strain E. coli 1356 at 64 µg/mL. As shown in MRSA CCARM 3167 and 3506 strains. And they were 32-fold more potent than norfloxacin (MIC > 64 µg/mL) and 4 or 2-fold gatifloxacin and moxifloxacin (MIC = 8 or 4 µg/mL), but slightly weaker activity to oxacillin (MIC = 1 µg/mL) against the QRSA CCARM 3505 and 3519 strains. As shown in Evaluation of cytotoxicity in-vitro50 (12.41 µg/mL) of compound 4a is higher than its MIC values indicating that compound 4a is selectively toxic to bacteria. To see whether the antibacterial activity of compound 4a is selectively toxic to bacteria, its cytotoxicity was evaluated . As showS,Z)-2-(5-((5-(substituted-phenyl)furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)-4-methylpentanoic acid (4a-4h) were synthesized and evaluated for antibacterial activity. All of the compounds showed good antibacterial activities against multidrug-resistant strains of clinical isolates. Compounds 4c, 4d, 4e and 4f showed the most potent levels of activity (MIC = 2 µg/mL) against selected MRSA and QRSA strains. Cytotoxicity evaluation suggested that the promising antibacterial activity of these compounds is not attributed to their toxicity. While the mechanism of action of this compound remains unknown, efforts to establish the cause of their antibacterial activity are ongoing and will be reported in due course.A series of (


Current study on microbial transformation of dihydrotestosterone (DHT) (1) by using two fungal cultures resulted in potent butyrylcholinesterase (BChE) inhibitors. Biotransformation with Macrophomina phaseolina led to the formation of two known products, 5α-androstan-3β,17β-diol (2), and 5β-androstan-3α,17β-diol (3), while biotransformation with Gibberella fujikuroi yielded six known metabolites, 11α,17β-dihydroxyandrost-4-en-3-one (4), androst-1,4-dien-3,17-dione (5), 11α-hydroxyandrost-4-en-3,17-dione (6), 11α-hydroxyandrost-1,4-dien-3,17-dione (7), 12β-hydroxyandrost-1,4-dien-3,17-dione (8), and 16α-hydroxyandrost-1,4-dien-3,17-dione (9). Metabolites 2 and 3 were found to be inactive, while metabolite 4 only weakly inhibited the enzyme. Metabolites 5–7 were identified as significant inhibitors of BChE. Furthermore, predicted results from docking simulation studies were in complete agreement with experimental data. Theoretical results were found to be helpful in explaining the possible mode of action of these newly discovered potent BChE inhibitors. Compounds 8 and 9 were not evaluated for enzyme inhibition activity both in vitro and in silico, due to lack of sufficient quantities.Microbial transformation of DHT (1) with two fungal cultures produced eight known metabolites. Metabolites 5–7 effectively inhibited the BChE activity. Cholinesterase inhibition is among the key strategies in the management of Alzheimer’s disease (AD). The experimental findings were further validated by in silico inhibition studies and possible modes of action were deduced.Biotransformation of DHT ( Microbial transformation of steroids is being studied for decades but the need to develop new structural analogues remain strong due to multiple reasons, including quest for medicinally important novel steroids . In cont1) Figure DHT  Figure 1Butyrylcholine (BCh) is an acetylcholine-like compound Figure , hydrolyMacrophomina phaseolina and Gibberella fujikuroi and eight known metabolites 2–9 were obtained. Substrate 1 and its metabolites 2–7 were subjected to AChE and BChE inhibitory activity evaluation. All compounds were found to be inactive against AChE, while metabolite 5–7 have significantly inhibited the BChE. Compounds 8 and 9 were not subjected to the above mentioned activity due to lack of sufficient quantities.DHT was subjected to microbial transformation with two fungal cultures, 2–7 were also evaluated in silico to understand their mode of interaction with the BChE. Resolved crystal structure of BChE was used in molecular docking simulation studies. All biotransformed metabolites were docked within the binding pocket of the crystal structure of human BChE (PDB ID 1P0P: 2.30 Å), revealing structural features, responsible of observed enzyme inhibitory activities , was UV inactive, thus lacked α,β-unsaturated carbonyl system. The IR spectrum indicated the presence of -OH (3349 cm-1), but no ketonic absorption. This could be due to the reduction of the carbonyl group at C-3.The metabolite 1H-NMR spectral analysis indicated two hydroxyl-bearing methine-proton triplets at δ 3.94 , and 3.55 , with their respective carbons resonated at δ 67.2 and 82.6, respectively, in the 13C-NMR spectrum (HMQC). HMBC of H-5 (δ 1.48) with carbon at δ 67.2 indicated the reduction of C-3 carbonyl into an OH. The H-3 (δ 3.94) showed NOESY correlation with H-5 (δ 1.48). Interestingly H-5 did not show any NOESY cross peak with H-9 (δ 0.74), indicating that H-5 is not α-oriented. Comparison of the 1H- and 13C-NMR chemical shift data of C-3 and C–5 of metabolites 2 and 3 showed marked differences which may be due to differences in the stereochemistry at these positions . The presence of hydroxyl (3437 cm-1) and carbonyl (1667 cm-1) groups was inferred from the IR spectrum, while UV spectrum also indicated a conjugated ketone (λmax = 233 nm).The molecular formula C1H-NMR analysis of 4 displayed some new signals as compared to the starting material DHT (1). A downfield methine signal at δ 4.02  and its respective carbon at δ 68.9, indicated the hydroxylation of one of the methylene carbon atoms. A downfield olefinic proton singlet at δ 5.71 (s) and the corresponding carbon at δ 124.6 indicated the introduction of a C = C bond. The HMBC spectrum displayed long-range couplings of the hydroxyl-bearing methine proton (δ 4.02) with C-9 (δ 59.2), C-10 (δ 39.9), and C-13 (δ 43.7), which suggested the position of the -OH at C-11. The stereochemical assignments were based on NOESY interactions between H-11 (δ 4.02), H-8 (δ 1.56), Me-19 (δ 1.31) and Me-18 (δ 0.81). H-11 was thus deduced as β-oriented. The olefinic proton (δ 5.71) showed HMBC with C-10 (δ 39.9), and C-2 (δ 34.2). The enone system was thus deduced at C-3/C-4-C-5, with the olefinic proton at C-4  showed the presence of ketone (1730 cm-1) and a conjugated enone (1657 cm-1) in IR spectrum. UV spectrum showed a strong absorption for conjugated ketone (λmax = 243 nm).Metabolite 1H-NMR analysis of 5 showed three olefinic signals at δ 7.03 , 6.22  and 6.07 (s). Their corresponding carbons were resonated at δ 155.3, 127.7, and 124.2, respectively. The 13C-NMR spectrum was devoid of any OH-bearing carbon signal which suggested the oxidation of the hydroxyl at C-17 into a ketone carbonyl. The conjugated ketonic group, inferred from UV analysis, was placed at C-3. The 13C-NMR spectra  showed the UV absorption at 234 nm for a conjugated ketone. The IR spectrum of 6 showed absorptions at 1668 (C = C-C = O), 1730 (C = O), and 3451 cm-1 (OH).Metabolite W1/2 = 21.5 Hz) was observed in the 1H-NMR spectrum of 6, while its corresponding methine carbon resonated at δ 68.7 (C-11) in the 13C-NMR spectrum  was deduced from the HREI-MS of metabolite 7 . The UV analysis suggested a conjugated enone system (λmax 244 nm), while IR spectrum showed absorptions at 3386 (OH), 1731 (C = O) and 1658 cm-1 (C = C-C = O).Molecular formula C1H-NMR spectrum of metabolite 7 showed a downfield hydroxyl-bearing methine proton signal at δ 4.08, which was assigned to H/C-11. C-11 resonated at δ 67.7 in the 13C-NMR spectrum , 6.14  and 6.08 (s), with respective carbons resonated at δ 158.6, 125.1, and 124.8  showed UV absorption at 245 nm. The IR spectrum showed absorptions at 3389 (OH), 1730 (C = O) and 1658 cm-1 (C = C-C = O).Metabolite 7 (Table 8 (Table W1/2 = 13.0 Hz) was observed in the 1H-NMR of 8 (C at δ 86.0). The enone system in ring A of compound 8 showed the same HMBC interactions as in 7. The C-17 ketone carbon (δ 216.2) showed HMBC correlation with the downfield C-12 methine proton (δ 3.73). The OH-bearing methine C-12 (δ 86.0) showed HMBC correlations with H-9 (δ 1.26), H-14 (δ 1.48), and CH3-18 (δ 0.78). The H-12 (δ 3.73) showed NOESY cross peaks with H-14 (δ 1.48), and H-9 (δ 1.26), and thus supported an α-orientation of H-12 , showed UV of 245 nm, while the IR spectrum displayed the same pattern as 8 .Metabolite 1H-NMR spectra of 9 and 8 showed similarities, except for a methine signal at δ 4.36  in compound 9 which appeared at δ 3.73 in metabolite 8. The 13C-NMR spectrum , was correlated with C-13 (δ 38.7), C-14 (δ 47.3) and C-17 (δ 218.3) in HMBC spectrum. The methine C-15 (δ 71.1) showed HMBC correlations with H-14 (δ 1.50) and H2-15 . Based on the above observations, the hydroxyl-bearing methine carbon was assigned to C-16. The H-16 (δ 4.36) showed NOESY cross peaks with H-18 (δ 1.01), but no interaction with H-14 (δ 1.50). Therefore the C-16 proton was deduced to be β-oriented  from androst-1,4-dien-3,17-dione upon reaction with hypervalent iodine  (100), 277 (49), 259 (29), 248 (25), 233 (88), 215 (82), 201 (14), 175 (9), 165 (52), 121 (31), 107 (41), 55 (12).54–157°C ). [α]25D55, CHCl3). 1H-NMR5β-Androstan-3α,17β-diol (3). Colorless crystalline solid. M.p.: 229–231°C : δ 1.66, m, 1.82, dt  (H-1); 1.35, m; 1.69, m (H-2); 3.94, t (J = 2.4 Hz) (H-3); 1.45a, m; 1.59, m (H-4); 1.48a, m (H-5); 1.28a, m; 1.31, m (H-6); 1.27a, m; 1.39, m (H-7); 1.21, m (H-8); 0.74, m (H-9); 1.41, m; 1.47a, m (H-11); 0.99, m; 1.93, m (H-12); 0.89, m (H-14); 1.27a, m; 1.43, m (H-15); 1.46a, m; 1.56, m (H-16); 3.55, t  (H-17); 0.71, s (H-18); 0.82, s (H-19), (a = exchangeable assignments). 13C-NMR : See Table HREI-MSm/z : 292.2434 . EI-MSm/z : 292 [M+] (66), 277 (51), 259 (33), 241 (20), 217 (54), 215 (100), 201 (14), 173 (11), 165 (45), 147 (33), 107 (47), 93 (48), 55 (37).30–231°C ). [α]25D ethanol ). 1H-NMR1 (1.0 g) with G. fujikuroi. Fermentation medium was prepared as discussed earlier. Fermentation continued for 7 days. All the flasks were filtered, extracted with 12 litres of DCM and evaporated in vacuo. A brown extract (2.5 g) was obtained which was fractionated over a silica gel column with petroleum ether- acetone (P.E.- acetone) gradient solvent system. The fractions were compiled based on thin layer chromatography (TLC) profile to obtain five main fractions, which were again subjected to column chromatography (C.C.) (P.E.-acetone) and size exclusion high performance liquid chromatography (HPLC) by using a GS-320 column, and methanol as solvent system to afford pure metabolites 4–9.Two stage fermentation protocol (discussed above) was used for the fermentation of 13C-NMR chemical shifts of compounds 4–9 are presented in Table The 11α,17β-Dihydroxyandrost-4-en-3-one (4). Colorless crystalline compound. M.p.: 179–181°C : δ 2.62, dt , 1.98, td ; 2.41, m; 2.31, m (H-2); 5.71, s (H-4); 2.37, m; 2.27, m (H-6); 1.81, m; 1.02, m (H-7); 1.56a, m (H-8); 1.05a, m (H-9); 4.02, br. s (W1/2 = 22.0 Hz) (H-11); 1.17, m; 2.13, m (H-12); 1.07a, m (H-14); 1.58a, m; 1.27, m (H-15); 1.45, m; 2.08, m (H-16); 3.77, t  (H-17); 0.81, s (H-18); 1.31, s (H-19), (a = exchangeable assignments). 13C-NMR : See Table HREI-MSm/z : 304.2058 . EI-MSm/z : 304 [M+] (74), 286 (61), 253 (15), 180 (43), 163 (100), 147 (29), 137 (34), 124 (98), 109 (41), 91 (44), 79 (34), 55 (20).78–182°C ). [α]25D.1, CHCl3). 1H-NMRAndrost-1,4-dien-3,17-dione (5). Colorless crystals. M.p.: 140–142°C  : δ 7.03, d  (H-1); 6.22, d  (H-2); 6.07, s (H-4); 2.41a, m, 2.48, m (H-6); 1.12, m, 2.06, m (H-7); 1.80, m (H-8); 1.09, m (H-9); 1.67, m, 1.84, m (H-11); 1.27a, m, 1.87, m (H-12); 1.26a, m (H-14); 1.57, m, 1.95, m (H-15); 2.10, m, 2.43a, m (H-16); 0.92, s (H-18); 1.24, s (H-19), (a = exchangeable assignments). 13C-NMR : See Table HREI-MSm/z : 284.1726 . EI-MSm/z : 284 [M+] (45), 266 (8), 227 (6), 171 (8), 159 (66), 150 (26), 135 (31), 122 (100), 107 (36), 91 (46), 79 (19), 67 (10), 55 (10).41–142°C ). [α]25D17, CHCl3). 1H-NMR11α-Hydroxyandrost-4-en-3,17-dione (6). Colorless crystalline compound. M.p.: 238–239°C : δ 2.05, m; 2.65, dt  (H-1); 2.31a, m; 2.39, m (H-2); 5.72, s (H-4); 2.29a, m; 2.37, m (H-6); 1.12, m; 1.97a, m (H-7); 1.68, m (H-8); 1.15, m (H-9); 4.04, m (W1/2 = 21.5 Hz) (H-11); 1.30, m; 2.11a, m (H-12); 1.37, m (H-14); 1.53, m; 1.95a, m (H-15); 2.14a, m; 2.48, m (H-16); 0.92, s (H-18); 1.31, s (H-19), (a = exchangeable assignments). 13C-NMR : See Table HREI-MSm/z : 302.1852 . EI-MSm/z : 302 [M+] (24), 290 (35), 280 (21), 248 (51), 230 (95), 215 (21), 198 (19), 186 (28), 159 (31), 139 (100), 138 (88), 136 (52), 105 (29), 87 (65), 55 (18).40–241°C ). [α]25D11α-Hydroxyandrost-1,4-dien-3,17-dione (7). Colorless crystalline compound. M.p.: 208–210°C : δ 7.73, d  (H-1); 6.14, d  (H-2); 6.08, s (H-4); 2.41, m, 2.48, m (H-6); 1.15, m, 2.07, m (H-7); 1.82, ddd  (H-8); 1.12, m (H-9); 4.08, m (W1/2 = 19.6 Hz) (H-11); 1.25, m, 2.22, m (H-12); 1.35, m (H-14); 1.56, m, 1.95, m (H-15); 2.15, m, 2.50, m (H-16); 0.93, s (H-18); 1.31, s (H-19). 13C-NMR : See Table HREI-MSm/z : 300.1749 . EI-MSm/z : 300 [M+] (36), 282 (18), 231 (80), 161 (20), 124 (37), 109 (32), 84 (58), 55 (23).12–214°C ). [α]25D (+ 86.5 ). 1H-NMR12β-Hydroxyandrost-1,4-dien-3,17-dione (8). Colorless crystalline compound. M.p.: 163–165°C : δ 7.03, d  (H-1); 6.23, dd  (H-2); 6.03, br s (H-4); 2.39, m, 2.48, td  (H-6); 1.15, m, 1.92, m (H-7); 1.81, m (H-8); 1.26, m (H-9); 1.42, m, 2.03, m (H-11); 3.73, br s (W1/2 = 13.0 Hz) (H-12); 1.48, m (H-14); 1.75, m, 1.86, m (H-15); 1.90, m, 2.33, m (H-16); 0.78, s (H-18); 1.25, s (H-19). 13C-NMR : See Table HREI-MSm/z : 300.1737 . EI-MSm/z : 300 [M+] (66), 282 (8), 231 (100), 161 (20), 124 (37), 109 (32), 84 (58), 55 (43).64–166°C ). [α]25D.0, CHCl3). 1H-NMR16α-Hydroxyandrost-1,4-dien-3,17-dione (9). Colorless crystals. M.p.: 152–153°C. [α]25D : + 89.3 . 1H-NMR : δ 7.00, d  (H-1); 6.22, d  (H-2); 6.04, s (H-4); 2.35, m, 2.45, m (H-6); 1.32, m, 2.03, m (H-7); 1.77, m (H-8); 1.10, m (H-9); 1.67, m, 1.85, m (H-11); 1.37, m, 1.65, m (H-12); 1.50, m (H-14); 1.87, m, 1.98, m (H-15); 4.36, br s (W1/2 = 20.8 Hz) (H-16); 1.01, s (H-18); 1.24, s (H-19). 13C-NMR : See Table HREI-MSm/z : 300.1749 . EI-MSm/z : 300 [M+] (100), 272 (36), 246 (69), 239 (43), 216 (39), 200 (38), 190 (24), 165 (33), 147 (55), 108 (63), 92 (31), 81 (17).All experiments were performed in a 96-well microplate in triplicate on SpectraMax340 .bis (2-nitrobenzoic) acid were purchased from Sigma. Analytical grade buffers and other chemicals were used. The BChE inhibition was measured by a modified spectrophotometric method  (pH 8), 10 μL of test compound solution (0.2 mM), and 20 μL BChE solution were mixed together and incubated at 25°C for 15 minutes. The reaction was initiated by the addition of 10 μL DTNB and 10 μL of BCh. A yellow 5-thio-2-nitrobenzoate anion was formed by hydrolysis of the substrate as a result of the reaction of DTNB with thiocholine which was monitored spectrophotometrically at 412 nm. MeOH was used as a solvent to dissolve the test compounds and the controls.50 values were calculated by measuring the effect of varying concentrations of test compounds by using the EZ-Fit Enzyme Kinetic program. Galanthamine was used as a standard inhibitor of BChE.IC-1 Å-1 was reached. 3D structures of all metabolites 2–7 were drawn by molecule builder which is incorporated in MOE modeling package and the structures were subjected to MMFF94 for energy minimization. Subsequently for the evaluation of potential energy, partial charges were calculated by MMFF94 force field [Molecular modeling studies were conducted on a dual processor Intel® Xeon™ CPU 3.00 GHz LINUX work station running under SUSE 11.4. For the selection of best docking program with respect to the current biological system, GOLD , MOE 4242 and Suce field . Both prce field . A total1) for the first time by using suspension cultures of M. phaseolina and G. fujikuroi. Compounds 5–7 were found to be significant and specific inhibitors of the BChE, in comparison to standard drug, galanthamine. Current experimental studies revealed that the DHT derivatives actively participated in the inhibition of BChE. Additionally, experimental studies correlated well with theoretical studies thus indicating the behavior of different metabolites inside the binding pocket of BChE, and supported inhibition activity trend, followed by least active to highly active compounds. From the docking analysis, experimental inhibitory potency relationship of compounds 1–7 exhibited the fundamental role of α,β-unsaturated carbonyl moiety in ring “A”. In the bound state of metabolites with BChE, compounds 5–7 exhibited favorable hydrophilic, hydrophobic and hydrogen bond interactions with active site residues of the receptor protein. These compounds also participated in π-π interaction towards the TRP82.In summary, we report here the microbial transformation of dihydrotestosterone (DHT, The authors declare that they have no competing interests.in silico studies and wrote the simulation part of the manuscript. ZH supervised the computational studies. All authors read and approved the final manuscript.MIC envisioned the concept of the current study, supervised the practical work, helped in solving spectroscopic data and finalized the manuscript. SZ carried out the microbial transformation experiments, purified all the metabolites, interpreted the spectroscopic data and prepared the preliminary draft of the manuscript. KD conducted the biochemical assays for inhibition of BChE. UM conducted the 1H-, 13C- and 2D-NMR spectra of compound 2.Click here for file1H-, 13C- and 2D-NMR spectra of compound 3.Click here for file1H-, 13C- and 2D-NMR spectra of compound 4.Click here for file1H-, 13C- and 2D-NMR spectra of compound 5.Click here for file1H-, 13C- and 2D-NMR spectra of compound 6.Click here for file1H-, 13C- and 2D-NMR spectra of compound 7.Click here for file1H-, 13C- and 2D-NMR spectra of compound 8.Click here for file1H-, 13C- and 2D-NMR spectra of compound 9.Click here for file