This synthesis results in pure sildenafil with high yield. The structures of the compounds were determined with infrared (IR), 1H-NMR and 13C-NMR spectroscopic methods, and high resolution mass spectroscopy (HRMS) analysis.This paper describes the facile synthesis of sildenafil citrate ( Finally, poorly water soluble Sildenafil (4) is converted in to Sildenafil citrate (1)   by treatrate (1) .3 from compound 2 was critical as it highly influenced the yield and quality of the end product. The chlorination of compound 5 was a reversible reaction (2 into sulfonyl chloride (compound 3).In the above reported synthesis, preparation of compound General Procedures: All melting points were determined with the Polmon melting point apparatus. 1H-NMR and 13C-NMR spectra were recorded on a Bruker 300 & 500 spectrometer . Chemical shifts (δ) were reported in ppmdownfield with TMS as internal standard, multiplicities are described as s: singlet, d: doublet, t: triplet, dd: double doublet, m: multiplet, brs: broad singlet. The HRMS spectra were measured on the Perkin Elmer PE SCIEX-API 2000 mass spectrometer . An analytical HPLC  was run with the Symmetry C18, 210 × 4.6 mm column at 290 nm.n-propyl-1,6-dihydro-7H-pyrazolopyrimid-7-one  followed by thionyl chloride  were added to chlorosulfonic acid (50 mL) portion-wise at 0–10 °C. The reaction mass temperature was raised to 20–30 °C and stirred for 4 h to complete the reaction. The reaction mass was poured onto ice (~500 g) slowly and the product was extracted with dichloromethane (250 mL). The dichloromethane layer was separated and washed with 5% w/w aqueous sodium bicarbonate (100 mL). The dichloromethane layer containing compound 3 was taken to the next step as such.5-(2-ethoxyphenyl)-1-methyl-3-N-methylpiperazine  was added to the dichloromethane layer containing sulfonyl chloride compound 3 (obtained from example 1) and stirred for 1 h at 20–25 °C. Then the reaction mass was washed with 5% w/w aqueous sodium bicarbonate (100 mL) followed by demineralised (DM) water (100 mL). The dichloromethane layer was concentrated at <50 °C and methanol was added to the residue to crystallize the product. The product was filtered and dried at 55–60 °C under vacuum to obtain 34 g of pure sildenafil (90% yield).para-toluensulfonic acid  was added followed by hydrazine hydrate  over a period of 2 h at 10–15 °C. The reaction mass was stirred for 1 h and diluted with water (200 mL). The pH of the reaction mass was adjusted to 7.2 with 30% aqueous sodium hydroxide and the layers were separated. The bottom aqueous layer was extracted with ethyl acetate (100 mL) and mixed with the first ethyl acetate layer. The combined ethyl acetate layers were concentrated at 40–45 °C under reduced pressure to obtain the title compounds 7a–7c as an oily residue. To a mixture of diethyl oxalate  and appropriate ketone (685 mmol), powdered sodium methoxide  was added in portions at 20 °C. The reaction was heated to 50–55 °C and stirred for 1 h, the reaction was cooled to 20–25 °C, then diluted with ethyl acetate (200 mL) and DM water (200 mL). Further, the pH was adjusted to 1.8 with diluted hydrochloric acid and separated into two layers. The ethyl acetate layer was washed with 20% aqueous sodium chloride and dried over anhydrous sodium sulfate. The ethyl acetate layer was cooled to ~10 °C and 9a–9c as an oily mass. Five N aqueous sodium hydroxide  was added to the oily residue and the mixture was heated to 80 °C for 2 h. The reaction temperature was brought down to 25–30 °C and the pH was adjusted to ~1.0 with hydrochloric acid. The solid precipitate was collected by filtration, washed with water, and dried to obtain the carboxylic acid compounds 10a–10c. The residue was cooled to 20–25 °C and dimethyl sulphate  was added in 2 h at 20–25 °C. The temperature was raised to 55–60 °C and maintained for 2 h. The reaction mixture was cooled to 10–15 °C and poured into a mixture of dichloromethane (500 mL) and DM water (500 mL). The pH of the reaction was adjusted to 7.2 with 30% aqueous sodium hydroxide and the two layers were separated. The organic layer was concentrated at 40–45 °C to obtain compounds Preparation of 3-ethyl-1-methyl-1H-pyrazole-5-carboxylic acid (10a). Compound 10a was prepared from 2-butanone as a pale yellow solid. Yield 53%, 1H-NMR (DMSO-d6) δ: 1.131–1.182 , 2.510–2.549 , 3.759 , 6.91 . Mass: 155.217 (M+H)+.Preparation of 3-butyl-1-methyl-1H-pyrazole-5-carboxylic acid (10b). Compound 10b was prepared from 2-hexanone as a pale yellow solid. Yield 55%, 1H-NMR (DMSO-d6) δ: 0.86–0.91 , 1.27–1.34 , 1.50–1.57 , 2.49–2.54 , 3.99 , 6.60 , 13.18 . Mass: 183.22 (M + H)+.Preparation of 3-isopropyl-1-methyl-1H-pyrazole-5-carboxylic acid (10c). Compound 10c was prepared from 3-methyl-2-butanone as a pale yellow solid. Yield 56%, 1H-NMR (DMSO-d6) δ: 1.17–1.20 , 2.80–2.92 2), 4.0 , 6.63 . Mass: 169.27 (M + H)+.10a–10c, 380 mmol) was added portion–wise, keeping the temperature below 40 °C. After the addition, the reaction was heated at 60 °C and maintained for 18 h. The reaction was cooled to room temperature before it was poured onto ice, then filtration and drying gave 4-nitrocarboxylic acid (11a–11c) as a white solid.Sodium nitrate (380 mmol) was slowly added to sulfuric acid (192 mL) in about 30 min at 0–5 °C. The contents were heated to 25–30 °C and stirred for 1 h. Thereafter, carboxylic acid (Preparation of 3-ethyl-1-methyl-4-nitro-1H-pyrazole-5-carboxylic acid (11a). Compound 11a was prepared from 10a as a white solid. Yield 82%, 1H-NMR (DMSO-d6) δ: 1.17–1.26 , 2.807–2.857 , 3.894 . Mass: 200.27 (M + H)+.Preparation of 3-butyl-methyl-4-nitro-1H-pyrazole-5-carboxylic acid (11b). Compound 11b was prepared from 10b as a white solid. Yield 94%, 1H-NMR (DMSO-d6) δ: 0.874–0.966 , 1.328–1.397 , 1.546–1.646 , 2.766–2.817 , 3.912 . Mass: 228.22 (M + H)+.Preparation of 3-isopropyl-1-methyl-4-nitro-1H-pyrazole-5-carboxylic acid (11c). Compound 11c was prepared from 10c as a white solid. Yield 81%, 1H-NMR (DMSO-d6) δ: 1.23–1.29 , 3.32–3.41 2), 3.90 . Mass: 214.19 (M + H)+.11a–11c, 352 mmol) was added to thionyl chloride (200 mL) and the resulting mixture was heated under reflux for 3 h. Thereafter, excess thionyl chloride was removed by evaporation under vacuum. The oily residue was dissolved in acetone (750 mL) and the pH was adjusted to 10 with aqueous ammonia. Acetone was distilled off and the mass was diluted by adding DM water (750 mL). The resulting slurry was filtered and washed with DM water to provide 4-nitrocarboxamide (12a–12c) as a pale yellow solid.4-Nitrocarboxylic acid (Preparation of 3-ethyl-1-methyl-4-nitro-1-H-pyrazole-5-carboxamide (12a). Compound 12a was prepared from compound 11a as a pale yellow solid. Yield 92%, 1H-NMR (DMSO-d6) δ: 1.045–1.083 , 2.359–2.456 , 3.92 , 7.483–7.695 . Mass: 199.18 (M + H)+.Preparation of 3-butyl-1-methyl-4-nitro-1H-pyrazole-5-carboxamide (12b). Compound 12b was prepared from compound 11b as a pale yellow solid. Yield 90%, 1H-NMR (DMSO-d6) δ: 0.88–0.93 , 1.27–1.39 , 1.55–1.65 , 2.80–2.85 , 3.78 , 8.23, 8.42 . Mass: 227.3 (M + H)+.Preparation of 3-isopropyl-1-methyl-4-nitro-1H-pyrazoIe-5-carboxamide (12c). Compound 12c was prepared from compound 11c as a pale yellow solid. Yield 90%, 1H-NMR (DMSO-d6) δ: 1.23–1.26 , 3.34–3.51 2), 3.79 , 8.23, 8.43 . Mass: 213.21 (M + H)+.12a–12c, 94 mmol) in methanol (400 mL) and 5% Pd/C  was charged into a one-litre hydrogenator at 25–30 °C. The reaction mixture was hydrogenated with 4 kg/cm2 pressure at 25–30 °C for ~5 h. After completion of the reaction, the hydrogen pressure was released and carefully filtered to remove the catalyst under the nitrogen atmosphere. The filtrate was concentrated under reduced pressure at 40–45 °C and the product was crystallized by adding ethyl acetate (250 mL). The product was filtered and dried to obtain the 4-aminocarboxamide compound (13a–13c).A solution of 4-nitro carboxamide (Preparation of 3-ethyl-1-methyl-4-amino-1-H-pyrazole-5-carboxamide (13a). Compound 13a was prepared from compound 12a. Yield 84%, 1H-NMR (DMSO-d6) δ: 1.055–1.108 , 2.359–2.456 , 3.994 , 7.483–7.695 . Mass: 169.20 (M + H)+.Preparation of 3-butyl-1-methyl-4-amino-1-H-pyrazole-5-carboxamide (13b). Compound 13b was prepared from compound 12b. Yield 82%, 1H-NMR (DMSO-d6) δ: 0.86–0.93 , 1.27–1.35 , 1.48–1.53 , 2.42–2.50 , 3.85 , 4.09 , 7.48 . Mass: 197.25 (M + H)+.Preparation of 3-isopropyl-1-methyl-4-amino-1-H-pyrazole-5-carboxamide (13c). Compound 13c was prepared from compound 12c. Yield 81%, 1H-NMR (DMSO-d6) δ: 1.10–1.17 , 2.92–3.02 2), 3.94  4.08 , 7.50 . Mass: 183.1 (M + H)+.13a–13c, 50 mmol) and triethylamine  in dichloromethane (250 mL), 2-ethoxybenzoyl chloride  was added at 0–5 °C. The resulting mixture was allowed to warm to room temperature and it was stirred for a further 2 h. The reaction mixture was washed with DM water twice and the organic layer was concentrated under reduced pressure. Then n-hexane (250 mL) was added to the residue and stirred for 1 h to precipitate the product. The product was collected by filtration and dried to obtain pyrazole carboxamide, compounds (14a–14c).To a solution of 4-amino carboxamide (Preparation of 3-ethyl-4-(2-ethoxybenzamido)-1-methyl-1H-pyrazole-5-carboxamide (14a). Compound 14a was prepared from compound 13a. Yield 74%, 1H-NMR (DMSO-d6) δ: 1.11–1.16 , 1.37–1.41 , 2.45–2.51 , 3.92 , 4.17–4.23 , 7.03–7.08 , 7.16–7.19 , 7.47–7.53 , 7.64–7.67 , 7.37 & 7.76 , 9.49 . Mass: 317.35 (M + H)+.Preparation of 3-butyl-4-(2-ethoxybenzamido)-1-methyl-1H-pyrazole-5-carboxamide (14b). Compound 14b was prepared from compound 13b. Yield 83%, 1H-NMR (DMSO-d6) δ: 0.83–0.88 , 1.27–1.41 , 1.52–1.57 , 2.44–2.51 , 3.92 , 4.17–4.24 , 7.03–7.08 , 7.17–7.19 , 7.47–7.53 , 7.63 & 7.66 , 7.36 & 7.76 , 9.48 . Mass: 345.31 (M + H)+.Preparation of 3-isopropyl-4-(2-ethoxybenzamido)-1-methyl-1H-pyrazole-5-carboxamide (14c). Compound 14c was prepared from compound 13c. Yield 81%, 1H-NMR (DMSO-d6) δ: 1.14–1.22 , 1.37–1.41 , 2.88–2.97 2), 3.92 , 4.17–4.24 , 7.03–7.08 , 7.17–7.19 , 7.47–7.53 , 7.63–7.66 , 7.33 & 7.74 , 9.45 . Mass: 331.42 (M + H)+.14a–14c, 42.42 mmol) was added portion-wise to a solution of sodium hydroxide  and 30% hydrogen peroxide solution (10 mL) in DM water (200 mL). Ethanol (50 mL) was added and the resulting mixture was heated under reflux for 8 h. Thereafter, the reaction mixture was cooled and evaporated under vacuum. The resulting solid was treated with 2 N hydrochloric acid (100 mL) at 25–30 °C and the mixture was extracted with dichloromethane (2 × 200 mL). The combined organic extracts were washed successively with saturated aqueous sodium carbonate (100 mL) and saturated sodium chloride solution (100 mL). The dichloromethane layer was concentrated and n-hexane was added to the residue to crystallize the product. The product was collected by filtration and dried to obtain the cyclized compounds 2a–2c.Pyrazole carboxamide compound (Preparation of 3-ethyl-5-(2-ethoxyphenyl)-1-methyl-7H-pyrazolopyrimidin-7-one (2a). Compound 2a was prepared from compound 14a as a white solid. Yield 61%, 1H-NMR (DMSO-d6) δ: 1.26–1.35 , 2.78–2.86 , 4.09–4.14 , 4.15 , 7.04–7.09 , 7.14–7.16 , 7.46–7.51 , 7.65–7.68 , 11.97 . Mass: 299.1 (M + H)+.Preparation of 3-butyl-5-(2-ethoxyphenyl)-1-methyl-7H-pyrazolopyrimidin-7-one (2b). Compound 2b was prepared from compound 14b as a white solid. Yield 65%, 1H-NMR (DMSO-d6) δ: 0.88–0.93 , 1.31–1.39 , 1.68–1.73 , 2.77–2.82 , 4.09–4.12 , 4.15 , 7.04–7.09 , 7.14–7.16 , 7.45–7.51 , 7.64, 7.66 , 11.95, 9.48 . Mass: 327.2 (M + H)+.Preparation of 3-isopropyl-5-(2-ethoxyphenyl)-1-methyl-7H-pyrazolopyrimidin-7H-one (2c): Compound 2c was prepared from compound 14c as a white solid. Yield 71%, 1H-NMR (DMSO-d6) δ: 1.31–1.36 , 3.34–3.42 2), 4.09–4.15 , 4.16 , 7.04–7.09 , 7.14–7.16 , 7.45–7.50 , 7.65–7.68 , 11.95 . Mass: 313.1 (M + H)+.2a–2c, 80.13 mmol) followed by thionyl chloride  were added to chlorosulfonic acid (50 mL) portion-wise at 0–10 °C. The reaction temperature was raised to 20–30 °C and stirred for 4 h. The reaction mass was poured onto ice (500 g) and extracted with dichloromethane (250 mL). The dichloromethane layer was separated and washed with 5% w/w aqueous sodium bicarbonate (100 mL). The dichloromethane layer containing compound 3 was taken to next step as such.Cyclized compounds (Preparation of 5-(5-chlorosulfonyl-2-ethoxyphenyl)-1-methyl-3-ethyl-1,6-dihydro-7H-pyrazolopyrimidin-7-one (3a). Compound 3a was prepared from compound 2a. Dichloromethane solution containing compound 3a was used as such in the next step.Preparation of 5-(5-chlorosulfonyl-2-ethoxyphenyl)-1-methyl-3-butyl-1,6-dihydro-7H-pyrazolopyrimidin-7-one (3b). Compound 3b was prepared from compound 2b. Dichloromethane solution containing compound 3b was used as such in the next step.Preparation of 5-(5-chlorosulfonyl-2-ethoxyphenyl)-1-methyl-3-isopropyl-1,6-dihydro-7H-pyrazolopyrimidin-7-one (3c). Compound 3c was prepared from compound 2c. Dichloromethane solution containing compound 3c was used as such in the next step.N-methylpiperazine  was added to the dichloromethane layer, containing sulfonyl chloride  at 20–25 °C. The reaction was stirred for 4 h and then washed with 5% w/w aqueous sodium bicarbonate (100 mL) followed by DM water (100 mL). The dichloromethane layer was concentrated at <40 °C under reduced pressure to obtain a foamy material. Methanol was added to crystallize the product and it was dried to result in compounds 4a–4c.Preparation of 5-[2-ethoxy-5-(4-methylpiperazinylsulfonyl)phenyl]-1-methyl-3-ethyl-1,6-dihydro-7H-pyrazolopyrimidin-7-one . Sildenafil ethyl analogue (4a) was prepared from compound 3a. Yield: 91%, Melting point: 203–206 °C, HRMS for C21H28N6O4S (M + H)+ calc.: 460.1892, found: 460.1912, 1H-NMR (DMSO-d6) δ: 1.26–1.35 , 2.14 , 2.36 , 2.79–2.86 , 2.90 , 4.16 , 4.20–4.25 , 7.36–7.39 , 7.81–7.85 ,12.21 . 13C-NMR (DMSO-d6) δ:13.1, 14.2, 18.6, 37.8, 38.7, 39.2, 39.8, 40.3, 45.3, 45.7, 53.5, 64.8, 113.2, 123.7, 124.5, 126.2, 129.9, 131.4, 137.4, 146.1, 148.1, 153.7, 159.9. IR (KBr pellet): 3104, 2976, 2946, 2802, 1690, 1598, 1456, 1348, 1287, 1171 cm−1.Preparation of 5-[2-ethoxy-5-(4-methylpiperazinylsulfonyl)phenyl]-1-methyl-3-n-butyl-1,6-dihydro-7H-pyrazolopyrimidin-7-one . Sildenafil butyl analogue (4b) was prepared from compound 3b. Yield: 90%, Melting point: 189–192 °C, HRMS for C23H32N6O4S (M + H)+ calc.: 488.2205, found: 488.2310, 1H-NMR (DMSO-d6) δ: 0.88–0.93 , 1.31–1.39 , 1.70 , 2.14 , 2.36 , 2.50–2.80 , 2.90 , 4.16 , 4.20–4.23 , 7.36–7.39 , 7.81–7.85 ,12.19 .13C-NMR (DMSO-d6) δ:13.6, 14.2, 21.8, 24.7, 30.5, 37.8, 38.9, 39.2, 39.5, 39.8, 40.1, 40.3, 45.3, 45.7, 53.5, 64.8, 113.2, 123.7, 124.4, 126.2, 129.9, 131.5, 137.7, 145.1, 148.1, 153.7, 159.9. IR (KBr pellet): 3107, 2959, 2937, 2796, 1689, 1599, 1488, 1349, 1288, 1171 cm−1.Preparation of 5-[2-ethoxy-5-(4-methylpiperazinylsulfonyl)phenyl]-1-methyl-3-isopropyl-1,6-dihydro-7H-pyrazolopyrimidin-7-one . Sildenafil isopropyl analogue (4c) was prepared from compound 3c. Yield: 90%, Melting point: 212–215 °C, HRMS for C22H30N6O4S (M + H)+ calc.: 474.2049, found: 474.2105, 1H-NMR (DMSO-d6) δ: 1.31–1.36 , 2.14 , 2.36 , 2.91 , 4.16 , 4.18–4.23 , 7.36–7.39 , 7.82–7.85 , 12.20 . 13C-NMR (DMSO-d6) δ: 14.2, 21.8, 26.0, 37.8, 38.7, 38.9, 39.2, 39.8, 40.1, 40.3, 45.3, 45.7, 53.5, 64.8, 113.2, 123.7, 124.6, 126.3, 130.0, 131.4, 137.0, 147.8, 149.8, 149.9, 153.8, 159.9. IR (KBr pellet): 3147, 3078, 2980, 2937, 2797, 1689, 1602, 1457, 1349, 1289, 1168 cm−1.2 was converted into intermediate product sulfonic acid (5), then compound 5 was converted into sulfonyl chloride (compound 3) in the presence of excess of chlorosulfonic acid. We found that during the chlorosulfonation of compound 2 with chlorosulfonic acid to produce 5-(5-chlorosulfonyl-2-ethoxyphenyl)-1-methyl-3-n-propyl-1,6-dihydro-7H-pyrazolopyrimidin-7-one (3), about 10% of 5-[2-ethoxy-5-(sulfonyl)phenyl]-1-methyl-3-n-propyl-1,6-dihydro-7H-pyrazolopyrimidin-7-one (5) left as unreacted product, which was carried into sildenafil as an impurity. It was difficult to separate compound 5 from sildenafil and typically required repeated crystallizations to achieve the desired quality of sildenafil, which was not feasible for any generic pharmaceutical company in terms of cost and yield of the product. In view of this difficulty, we have designed the chlorosulfonation reaction in such a way that compound 2 is reacted with chlorosulfonic acid in the presence of thionyl chloride at room temperature and the reaction is monitored by HPLC until the sulfonic acid derivative, compound 5, is less than 2%. Addition of thionyl chloride as a chlorinating agent reduced the amount of unreacted sulfonic acid, compound 5, and provided consistent results. By this simple modification, the overall yield of this process increased to 90% from 70% pyrimidin-7-one ; (ii) 5-[2-ethoxy-5-(4-methylpiperazinylsulfonyl)phenyl]-1-methyl-3-n-butyl-1,6-dihydro-7H-pyrazolopyrimidin-7-one ; and (iii) 5-[2-ethoxy-5-(4-methylpiperazinylsulfonyl)phenyl]-1-methyl-3-isopropyl-1,6-dihydro-7H-pyrazolopyrimidin-7-one  using the improved chlorosulfonation reaction. All of these novel analogues were characterized by using IR, 1H-NMR, 13C-NMR, and elemental analysis.Several sildenafil analogues and related compounds were reported in the literature ,16,17,18


The structures of the new compounds were elucidated via extensive spectroscopic methods, including NMR and HRMS spectroscopic analyses.Six previously undescribed 5,6-The online version of this article (10.1007/s13659-018-0157-y) contains supplementary material, which is available to authorized users. Irpex lacteus has been used as a medicinal fungus for decades in China for the treatment of inflammation, bacterial/fungal infection, and urinary retention furan-2(3H)-one moiety which connected with part A through the double bond C-1–C-2 .The signals assigned for compound ) Tables . The 1H–1. The ROESY cross peaks of H-7/H-8β/H-15 led to the assignment of H-7 and 15-CH2OH as β orientation. The significant ROESY correlation between H-3 and H-12, H-10 and H-11 suggested that H-3 and H-12 were at the same side, and the C-1–C-2 double bond were Z configuration. Therefore, the structure of compound 1 was established as 11,12-epoxy-15-hydroxy-5,6-seco-tremula-1,6(13)-dien-5,12-olide . Careful analysis of the 2D NMR led to the conclusion that the only difference between 1 and 2 was the lactone group in 1 opened in 2 and the carbonyl group was methyl esterified, which was confirmed by the HMBC correlation from the methoxy (δH 3.65) to the carbonyl C-5 (δC 174.3) as well as the absence of HMBC correlation from H-12 to C-5. The relative configuration of 2 was identical with that of 1 by interpretation of the ROESY spectrum. The HRESIMS result of the mixture also gave a molecular formula of C16H24O5  which further reinforced the above assignment. Thus, compound 2 was deduced as methyl 12β,15-dihydroxy-5,6-seco-tremula-1,6(13)-dien-5-oate /H-8 , H-14/H-10 . As shown in Fig. 3 and 4 suggested that only when the 14-CH2OH adopted the equatorial direction, the above ROESY correlation could be seen. Therefore, compounds 3 and 4 were C-9 epimers of compounds 1 and 2, respectively. This conclusion was also evidenced by the large discrimination of 13C NMR chemical shifts of C-14 and C-15 between 1 and 3, 2 and 4. Thus, compounds 3 and 4 were identified as shown in Fig. seco-tremula-1,6(13)-dien-5,12-olide, and methyl 12β,14-dihydroxy-5,6-seco-tremula-1,6(13)-dien-5-oate.Compounds 5 was isolated as colourless oil. The HRESIMS result presented a molecular formula of C15H24O4 as indicated by the sodium adduct ion peak at m/z 291.1572 [M+Na]+ . The 1D NMR data of 5 (Tables 7) + ; 2: C16H24O5m/z 319.1529 [M+Na]+ .Colourless oil; mentclass2pt{minimc 0.10, MeOH); UV  λmax nm (Abs): 205 (0.4599), 238 (0.1222); IR (KBr) νmax 3425, 2927, 2860, 1728, 1631, 1032 cm−1; 1H NMR  data, see Table 13C NMR  data, see Table 3: C15H20O4m/z 287.1250 [M+Na]+ ; 4: C16H24O5m/z 319.1513 [M+Na]+ .Colourless oil; mentclass2pt{minim15H24O4; c 0.07, MeOH); UV (MeOH) λmax nm (log ε): 206 (4.13), 254 (2.97); IR (KBr) νmax 3422, 2952, 2930, 2868, 1750, 1632, 1383, 1193, 1022 cm−1; 1H NMR  data, see Table 13C NMR  data, see Table m/z 291.1572 [M +Na]+ .Colourless oil; C15H22O4; c 0.07, MeOH); UV (MeOH) λmax nm (log ε): 205 (3.46), 245 (2.18); IR (KBr) νmax 3432, 2927, 2859, 1631, 1385, 1031 cm−1; 1H NMR  data, see Table 13C NMR  data, see Table m/z 289.1416 [M+Na]+ .Colourless oil; CSupplementary material 1 (DOCX 7398 kb)Below is the link to the electronic supplementary material.


The N1-deprotonated anions of 7-azaindole or its derivatives (naza) are coordinated to the metal centre through the N1 atom of their pyrrole ring, as proved by a single crystal X-ray analysis of the complexes [Au(3I5Braza)(PPh3)] (7) and [Au(2Me4Claza)(PPh3)]·½H2O (8′). The in vitro cytotoxicity of the complexes 1–8 was studied against both the cisplatin-sensitive and -resistant variants of the A2780 human ovarian carcinoma cell line, as well as against the MRC-5 human normal fibroblast cell line. The complexes 4, 5, and 8, containing deprotonated 3-iodo-7-azaindole, 5-bromo-7-azaindole, and 2-methyl-4-chloro-7-azaindole (2Me4Claza), respectively, showed significantly higher potency (IC50 = 2.8–3.5 µM) than cisplatin (IC50 = 20.3 µM) against the A2780 cells and markedly lower effect towards the MRC-5 non-cancerous cells (IC50 = 26.0–29.2 µM), as compared with the mentioned A2780 cancer cells. The results of the flow cytometric studies of the A2780 cell cycle perturbations revealed a G2-cell cycle phase arrest of the cells treated by the representative complexes 1 and 5, which is indicative of a different mechanism of action from cisplatin (induced S-cell cycle phase arrest). The stability of the representative complex 8 in the water-containing solution as well as its ability to interact with the reduced glutathione, cysteine and bovine serum albumin was also studied using 1H and 31P-NMR spectroscopy (studied in the 50% DMF-7d/50% D2O mixture) and ESI+ mass spectrometry (studied in the 50% DMF/50% H2O mixture); DMF = dimethylformamide. The obtained results are indicative for the release of the N-donor azaindole-based ligand in the presence of the used biomolecules.A series of gold(I) complexes of the general composition [Au( O-acetyl-β-d-thiopyranosato)gold(I) complex (PEt3)]), are clinically used for the treatment of rheumatoid arthritis , that these agents release their S-donor ligands (auranofin [Au(SAtg)(PEt3)]) or chlorido ([AuCl(PPh3)]) ligand under the physiological conditions to form [Au(PR3)]+ species which react easily with various biomolecules, such as albumin (AlbH) or glutathione (GSH) [3)] with the mixture of GSH and 5,5′-dithiobis-2-nitrobenzoic acid (dtnb) provided the [Au(PPh3)(SG)] adduct , to form species, such as [Au(SG)2]− or [Au(Alb)2]−. On the other hand, several highly potent anticancer gold(I) complexes, e.g., [AuCl(NHC)] or [Au(NHC)(PPh3)]I were found to be inert to GSH  adduct . Interest to GSH ,17,18, iN6-benzyladenine-, hypoxanthine- and 9-deazahypoxanthine-derived N-donor ligands (HL), [Au(L)(PPh3)], which exhibited high cytotoxicity, ability to overcome acquired resistance to cisplatin, selectivity towards cancer cells over the normal ones and/or high anti-inflammatory activity [naza)(PPh3)] (1–8) containing the N1-deprotonated, C2-, C3-, C4-, and/or C5-substituted 7-azaindole-based ligands (Haza)2], cis-[Pt(3ClHaza)2Cl2] or [(6η-p-cym)Ru(Haza)Cl2]; ox = oxalate(2–), p-cym = p-cymene) [N1-monodentate- (PPh3)]; 1 in this work), or N1,N7-bidentate-coordinated 4(dmf)2]) N1-deprotonated 7-azaindolate anions  (1–8), which were synthesized, thoroughly characterized in the solid state as well as in solution, and their in vitro cytotoxicity against both cancer and normal  cell lines was studied, together with basic mechanistic aspects of their cytotoxic action. The complexes 1–8 were prepared in acetone from [AuCl(PPh3)] using a general procedure, as previously described in the literature for similar triphenylphosphanegold(I) complexes containing heterocyclic N-donor ligands different from 7-azaindole (naza) were deprotonated in situ by an equimolar amount of NaOH. The products were isolated in good yields (≈70%) and acceptable purity (>95%), as proved by 1H-NMR and 31P-NMR spectroscopy; NMR = nuclear magnetic resonance. The studied substances are well-soluble in acetone, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), chloroform, methanol, and ethanol, but the solubility in water was found to be very low. Complexes were stable in DMF-7d used for the NMR studies, because no changes were observed in the 1H-NMR and 31P-NMR spectra of 1–8 even after one week of standing at ambient temperature.In this work, we report on a series of eight triphenylphosphanegold(I) complexes of the general formula  complexes, as a consequence of deprotonation of these organic molecules by 1 M NaOH  atom, resulting in the negative coordination shift values . Regarding the 13C-NMR coordination shifts, high values (∆δ = 6.7–11.7 ppm), calculated for the C2 and C7a carbons adjacent to the N1 nitrogen atom (N1-coordination mode of the used naza ligands. The N1-coordination mode was further proved by higher 15N-NMR coordination shift of N1 (48.0 ppm) than N7 (−0.8 ppm), as observed for the representative complex 5. The 31P-NMR spectra of the studied complexes contained only one signal at ca. 33 ppm, assignable to the P-coordinated PPh3 molecule.The 1 M NaOH . The arogen atom , are con1–8 contained, except for the most intensive peaks of the [Au(PPh3)2]+ species (100% relative intensity) at ca. 721.2 m/z, the characteristic molecular peaks of {[Au(naza)(PPh3)] + H}+ (2%–10% relative intensity) and the peaks of [Au(PPh3)]+ and free {(Hnaza) + H}+ (see naza)2]− and (naza)− species were detected in the ESI− mass spectra of 1–8.The positive mode electrospray ionization (ESI+) mass spectra of the complexes  H}+ see . The pea1–8 contained, similarly to the spectra of uncoordinated Hnaza, the intensive peaks at 1574–1590 cm−1 and 1463–1481 cm−1, assignable to the ν(C–N)ring, and ν(C–C)ring stretching vibrations, respectively  (7) and [Au(2Me4Claza)(PPh3)]·½H2O (8′), were characterized by single crystal X-ray analysis. Crystal data and structure refinement parameters are given in 7 and 8′ are depicted in Two representative complexes, [Au] (1 in this work)   Å)  (2.2321(12) Å) do not differ markedly.Both the complexes lues see  fall wits, e.g., ,22,28, ds, e.g., ). For thse of 8′  and   dissolved in the same mixture of solvents  coordinated to the Au(I) atom through its sulphur atom, were detected immediately in the mixture of 8 and GSH (1 mol equiv), showing the formation of the [Au(PPh3)(SG)] adduct (SG)] adduct (2Me4Claza) was found at 6.35 ppm in the 1H-NMR spectra of the mixture of 8 and GSH (3)(SG)] + H}+ adduct (overlapped with {[Au(PPh3)(SG)] + 2H}+ in a 1.75:1.00 ratio) and the {[Au(PPh3)(SG)] + Na}+ adduct (overlapped with {[Au(PPh3)(SG)] + H + Na}+ in a 1.25:1.00 ratio) were found at 765.9 m/z  and 789.1 m/z , respectively, with the appropriate isotopic distribution pattern.The new ] adduct . The pos] adduct . The cha and GSH . Neither and GSH . The pea1H-NMR signals of β CH2 of the coordinated deprotonated cysteine (Cys−) are clearly detectable at 3.44 and 3.63 ppm. The 31P-NMR signal was detected at 40.6 ppm for the [Au(Cys−)(PPh3)] adduct, that is at negligibly higher field than for the [Au(PPh3)(SG)] adduct (see above). The C3–H 1H-NMR signal of the released H2Me4Claza was found at 6.36 ppm (8.Regarding the interaction studies with cysteine (Cys), the 6.36 ppm . Any oth8 with bovine serum albumin (BSA) resulted immediately in a white turbidity, which was subsequently removed by centrifugation . The 1H-NMR spectrum recorded on the obtained supernatant contained the signals of the released N-donor ligand, with the signal positions  being consistent with those detected for free H2Me4Claza in the same mixture of solvents  and one human non-cancerous cell line (MRC-5) (The complexes  (MRC-5) .1, 4, 5, and 8 were significantly more effective (p < 0.005) against the A2780 cells (IC50 = 2.8–3.8 μM) than conventional cisplatin (IC50 = 20.3 μM); IC50 stands for the half maximal inhibitory concentration. Contrary, the complexes 2, 3, 6, and 7 showed only comparable in vitro anti-tumor activity with cisplatin /IC50(A2780), equalled 1.2–4.6. The complexes 2, 3, 6, and 7 showed comparable or even higher potency against the A2780R cells as compared with A2780 ones, resulting in the RF values of 0.6–1.1 /IC50(A2780)) were calculated as being equal to ca. 1.2–9.6 for 1–8.The complexes isplatin . The in tors RF; , defined 0.6–1.1 . All the-5 cells . The val50 concentrations of the representative complexes 1 and 5 (and cisplatin for comparative purposes). The obtained results are depicted in The cell cycle modification was studied by flow cytometry (propidium iodide (PI) staining), using the A2780 cells treated by the IC1 and 5 caused a G2 cell cycle phase arrest (populations of 44.0% ± 2.3% for 1 and 30.4% ± 2.9% for 5), because the % of the populations were higher than in the case of the non-treated A2780 cells  (2-arrest was connected with a decrease of the G0/G1 cell cycle phase population of the treated A2780 cells (46.0% ± 2.8% for 1 and 58.1% ± 5.8% for 5), as compared with control (66.3% ± 3.8%). On the other hand, the S cell cycle phase populations of the cells treated by 1 (7.2% ± 0.9%) and 5 (9.1% ± 2.1%) were comparable with the control cells (9.0% ± 1.9%), but considerably different from cisplatin (44.8% ± 2.9%).Treatment of the A2780 cells by the representative complexes  ± 2.6%) . The G2-1 was previously reported by Chan et al. [1–8. The reported protocol [3)] after the addition of Haza deprotonated in situ by triethylamine. In contrast to this protocol, NaOH was used in this work to deprotonate the 7-azaindoles, allowing the isolation of NaCl, which formed as a side product of the performed reactions, as previously reported in the literature (PPh3)] (7) and [Au(2Me4Claza)(PPh3)]·½H2O (8′)  were consistent with the mentioned 7 and 8. On the other hand, the observation of the negative ∆δ values detected for all the aromatic C–H hydrogens of the naza ligands points out a different coordination mode from previously-reported platinum(II) or ruthenium(II) complexes containing the electroneutral 7-azaindole-based N-donor ligands coordinated through the N7 nitrogen atom  precursor was prevented by the low stability of [AuCl(PPh3)] in DMF-7d . With respect to this fact, the 31P-NMR spectra were recorded also for the CDCl3 solutions of the representative complex 8 and [AuCl(PPh3)], both showing one PPh3 signal at 33.6 ppm and 33.8 ppm, respectively, thus indicating a slight upfield shift as a consequence of the replacement of the chlorido ligand by 2Me4Claza anion.The H2O (8′) . Moreovegen atom ,26,27. Tauranofin, the mechanism of their action is connected with the ligand displacement − gold-glutathione adduct [3)], whose interaction with GSH provided the [Au(PPh3)(SG)] adduct, subsequently releasing its PPh3 ligand in the presence of GSH or 5,5′-dithiobis- ) or possible product of the decomposition of 8 (O=PPh3) dissolved in the same mixture of solvents ] + O + 2H}+ species detected in the ESI+ mass spectra at 477.0 m/z  resulted from the hydrolysis of 8, but its formation is most likely connected with the coordination of water molecule to the [Au(PPh3)]+ species under the electrospray ionization conditions.As it is known for biologically-active substituted-phosphanegold(I) complexes, such as lacement ,36,37,38d; dtnb) . For thesolvents . With re8 with GSH  indicated an immediate covalent bonding between both the compounds, probably connected with the formation of the [Au(PPh3)(SG)] adduct (SG)] adduct. The signal positions of the released N-donor ligand correlated well with those of free H2Me4Claza dissolved in the same mixture of solvents, as depicted in 3 ligand and formation of a [Au(SG)2]− adduct, because a) any new 1H-NMR signals were not detected for glutathione (SG)] adduct, even after 48 h of standing at ambient temperature. The results of ESI+ mass spectrometry are consistent with those of 1H and 31P-NMR spectroscopy and proved the covalent interaction of the complex 8 with GSH. Similar results were also obtained when the representative complex 8 interacted with another naturally occurring biomolecule, cysteine [2Me4Claza ligand by Cys (PPh3)] complexes containing different N-donor ligand  interacting with N-acetyl-l-cysteine (1 mol equiv) in CD3CN showed similar downfield shift from 30 to ca. 38 ppm [N-donor ligand 2Me4Claza in the structure of the representative complex 8 (7d/50% D2O).The results of the interaction experiment of ] adduct . This is signals ; the Cystathione , and b) cysteine . This ind by Cys , as prov. 38 ppm . The resomplex 8 , resulti1, 4, 5, and 8 (significantly more potent than cisplatin), and 2, 3, 6, and 7 (comparable potency with cisplatin) against the A2780 cells , the potency of the previously reported substituted-phosphanegold(I) complexes is usually comparable or only slightly higher than cisplatin  complexes , structurally similar to the herein studied complexes 1–8, at the HCT116 human colon carcinoma cells, as well as gold(I) complex of the bis-chelated gold(I)-diphosphane type at the A549 cancer cells, induced the similar increase of the G0/G1 cell cycle population ∙3H2O, triphenylphosphane (PPh3), 7-azaindole (Haza), 3-chloro-7-azaindole (H3Claza), 3-bromo-7-azaindole (H3Braza), 3-iodo-7-azaindole (H3Iaza), 5-bromo-7-azaindole (H5Braza), 3-chloro-5-bromo-7-azaindole (H3Cl5Braza), 3-iodo-5-bromo-7-azaindole (H3I5Braza), 2-methyl-4-chloro-7-azaindole (H2Me4Claza), NaOH, cisplatin, reduced glutathione (GSH), cysteine, BSA, Roswell Park Memorial Institute medium, fetal calf serum, glutamine, penicillin and streptomycin, and solvents  and solvents for NMR experiments  were supplied by Sigma-Aldrich Co.  and Fisher-Scientific Co. .H[AuCl3)], was prepared as described previously  (0.5 mmol) and the appropriate 7-azaindole-based ligand (0.6 mmol) in acetone (10 mL). After 48 h of stirring at 50 °C, the obtained mixtures, containing the white solid, were filtered. The volume of the clear colourless filtrates was reduced until the off-white precipitate formed . In the case of the complexes 5 and 7, the brown gel was obtained when the solvent was removed, which crystalized to the off-white precipitate under diethyl ether. The products (One mol of NaOH (0.6 mL) was poured into the mixture of [AuCl(PPhproducts  were col3)] (1): Anal. Calcd. for C25H20N2PAu: C, 51.7; H, 3.6; N, 4.8; Found: C, 51.5; H, 3.6; N, 4.4%. 1H-NMR : 8.14 , 7.90 , 7.73 , 7.52 , 6.89 , 6.38  ppm. 13C-NMR : 160.1 (C7a), 141.6 (C6), 137.7 (C2), 135.2, 135.1, 130.7, 130.6, 129.7 (PPh3), 127.7 (C4), 122.2 (C3a), 114.8 (C5), 99.8 (C3) ppm. 31P-NMR : 32.7 (P1) ppm.[Au(aza)(PPh3Claza)(PPh3)] (2): Anal. Calcd. for C25H19N2ClPAu: C, 49.2; H, 3.1; N, 4.6; Found: C, 48.9; H, 3.1; N, 4.5%. 1H-NMR : 8.24 , 7.89 , 7.75 , 7.63 , 7.03  ppm. 13C-NMR : 155.6 (C7a), 143.0 (C6), 135.4, 135.2 (PPh3), 133.3 (C2), 130.8, 130.6, 130.3, 129.7 (PPh3), 125.4 (C4), 119.5 (C3a), 115.6 (C5), 101.2 (C3) ppm. 31P-NMR : 32.9 (P1) ppm.[Au(3Braza)(PPh3)] (3): Anal. Calcd. for C25H19N2BrPAu: C, 45.5; H, 3.0; N, 4.2; Found: C, 45.0; H, 2.9; N, 4.0%. 1H-NMR : 8.23 , 7.83 , 7.74 , 7.65 , 7.04  ppm. 13C-NMR : 156.2 (C7a), 143.2 (C6), 136.9 (C7), 135.4, 135.3 (PPh3), 133.4 (C2), 130.8, 130.7, 130.4, 129.9 (PPh3), 126.2 (C4), 121.1 (C3a), 115.7 (C5), 86.3 (C3) ppm. 31P-NMR : 32.8 (P1) ppm.[Au(3Iaza)(PPh3)] (4): Anal. Calcd. for C25H19N2IPAu: C, 42.8; H, 2.7; N, 4.0; Found: C, 42.7; H, 2.4; N, 4.1%. 1H-NMR : 8.20 , 7.72 , 7.03  ppm. 13C-NMR : 156.8 (C7a), 143.0 (C6), 142.0 (C2), 135.1, 130.7, 130.6, 130.1, 129.7 (PPh3), 127.7 (C4), 124.2 (C3a), 116.0 (C5), 54.6 (C3) ppm. 31P-NMR : 32.8 (P1) ppm.[Au(5Braza)(PPh3)] (5): Anal. Calcd. for C25H19N2BrPAu: C, 45.8; H, 2.9; N, 4.3; Found: C, 45.6; H, 2.8; N, 4.0%. 1H-NMR : 8.10 , 8.10 , 7.71 , 7.70 , 6.65  ppm. 13C-NMR : 156.0 (C7a), 141.6 (C6), 139.9 (C2), 135.4, 135.2 (PPh3), 133.7 (C4), 130.8, 130.7, 129.7 (PPh3), 124.3 (C3a), 110.1 (C5), 99.9 (C3) ppm. 15N-NMR : 276.7 (N7), 188.9 (N1) ppm. 31P-NMR : 33.2 (P1) ppm.[Au(3Cl5Braza)(PPh3)] (6): Anal. Calcd. for C25H18N2BrClPAu: C, 43.5; H, 2.6; N, 4.1; Found: C, 43.2; H, 2.6; N, 3.9%. 1H-NMR : 8.26 , 8.02 , 7.72 , 7.72  ppm. 13C-NMR : 154.0 (C7a), 143.2 (C6), 135.4, 135.3 (PPh3), 133.4 (C2), 130.8, 130.6, 130.2, 129.8 (PPh3), 127.4 (C4), 121.1 (C3a), 110.8 (C5), 101.0 (C3) ppm. 31P-NMR : 33.1 (P1) ppm.[Au(3I5Braza)(PPh3)] (7): Anal. Calcd. for C25H18N2BrIPAu: C, 38.3; H, 2.4; N, 3.6; Found: C, 38.1; H, 2.1; N, 3.8%. 1H-NMR : 8.23 , 7.73  ppm. 13C-NMR : 152.2 (C7a), 144.2 (C6), 143.10 (C2), 135.3, 135.1 (PPh3), 132.8 (C4), 130.8, 130.7, 130.2, 129.8 (PPh3), 126.0 (C3a), 111.1 (C5), 55.6 (C3) ppm. 31P-NMR : 32.8 (P1) ppm.[Au(2Me4Claza)(PPh3)] (8): Anal. Calcd. for C25H19N2BrPAu: C, 50.0; H, 3.4; N, 4.5; Found: C, 49.7; H, 3.3; N, 4.4%. 1H-NMR : 7.97 , 7.74 , 6.93 , 6.19 , 2.59  ppm. 13C-NMR : 158.9 (C7a), 148.0 (C2), 141.0 (C6), 135.4, 135.3 (PPh3), 133.4 (C4), 130.8, 130.7, 130.4, 130.0 (PPh3), 122.3 (C3a), 114.3 (C5), 96.7 (C3), 18.1 (CH3) ppm. 15N-NMR : 269.2 (N7), 203.3 (N1) ppm. 31P-NMR : 33.1 (P1) ppm.) and calibrated against 85% H3PO4 used as an internal reference standard. Electrospray ionization mass spectrometry (ESI-MS) was performed by LCQ Fleet ion trap spectrometer  in both the positive (ESI+) and negative (ESI−) ionization modes on the methanol solutions. FTIR spectra were recorded by a Nexus 670 FT-IR spectrometer  at 150–600 cm−1 (far-IR) and 400–4000 cm−1 (mid-IR), using the ATR technique. Raman spectroscopy was performed on a NXR FT-Raman Module  between 250 and 3700 cm−1.Elemental analysis was carried out using a Flash 2000 CHNS Elemental Analyzer . 3I5Braza)(PPh3)] (7) and [Au(2Me4Claza)(PPh3)]·½H2O (8′) suitable for a single crystal X-ray analysis were prepared by slow evaporation of the solutions of the named complexes in the mixture of dichloromethane and n-hexane . X-ray data of both crystals were collected on a Bruker  D8 QUEST diffractometer equipped with a PHOTON 100 CMOS detector (Bruker) using Mo–Kα radiation. The APEX3 software package was used for data collection and reduction [The crystals of the complexes [Au(eduction . The streduction . The graeduction  and Merceduction  software8 and [AuCl(PPh3)] (for comparative purposes) were dissolved in 600 µL of the 50% DMF-7d/50% D2O mixture to give the solutions of ca. 1 mM concentration; a presence of DMF ensured the solubility of the complex 8, with respect to its low solubility in water. Similar solutions were prepared with 1 or 5 mol equivs of GSH, Cys, or BSA. 1H and 31P-NMR spectra were recorded at different time points over 48 h at 300 K. The obtained 1H-NMR spectra were calibrated against the residual signal of water found at 4.75 ppm, while the 31P-NMR spectra were referenced as described above.Complex Similar experiments were carried out also in non-deuterated solvents and evaluated by ESI+ mass spectrometry.cisplatin-resistant ovarian carcinoma and MRC-5 normal fibroblast cell lines were supplied by the European Collection of Cell Cultures (ECACC). The cell lines were cultured, according to the supplier´s instructions, in RPMI-1640 medium supplemented with 10% of fetal calf serum, 1% of 2 mM glutamine, and 1% penicillin/streptomycin. All cell lines were grown as adherent monolayers at 37 °C and 5% CO2 in a humidified atmosphere.Human A2780 ovarian carcinoma, A2780R 1–8 (or cisplatin involved in the study as standard) was dissolved in DMF to give the 50 mM stock solutions. The cells were seeded to 96-well culture plates, pre-incubated in drug-free medium at 37 °C for 24 h and treated with the 0.01–10.0 μM solutions (for the complex 1 being less soluble in the used medium) or 0.01–50.0 μM solutions (for sufficiently soluble complexes 2–8 and cisplatin) prepared from the stock solutions by dilution with medium, for 24 h at 37 °C. In parallel, the cells were also treated with vehicle  and Triton X-100  to assess the minimal and maximal cell damage, respectively. The MTT assay was used to determine the cell viability. A concentration of the formed dye was evaluated spectrophotometrically at 540 nm . The data were expressed as the percentage of viability, where 100% and 0% represent the treatments with negative and positive controls, respectively. The data were acquired from three independent experiments (conducted in triplicate) using cells from different passages. The resulting IC50 values (µM) were calculated from viability curves and the results are presented as the arithmetic mean ± SD.An appropriate amount of the complexes 6 per well) were pre-incubated in a six-well plate for 24 h, as described above. The selected complexes 1 and 5 were added at the concentrations equal to their IC50 (cisplatin was involved in the study for comparative purposes). After 24 h, floating cells were collected and attached cells were harvested using trypsin/EDTA in PBS. Total cells were washed twice with PBS and fixed in 70% ethanol. Cells were resuspended in PBS and DNA staining was achieved by a solution of propidium iodide (PI) supplemented with RNase A . After that, the cells were washed (PBS), resuspended (PBS), and DNA content was measured using flow cytometry  detecting emission of DNA-bound PI (maximum at 617 nm) after excitation at 535 nm. The data were analysed using CytExpertTM software (Beckman Coulter).A2780 cells (1.0 × 10p < 0.05 (*), 0.01 (**) and 0.005 (***) considered to be statistically significant. QC Expert 3.2 Statistical software  was used to perform the analysis.An analysis of variance (ANOVA) test was used for statistical analysis with the values of naza) were investigated. Complexes [Au(3I5Braza)(PPh3)] (7) and [Au(2Me4Claza)(PPh3)]·½H2O (8′) were crystallographically characterized, showing the used 7-azaindole derivatives as coordinated through their N1 atoms. The complexes 4, 5, and 8 exhibited low-micromolar in vitro anticancer potency against the A2780 cells , which is significantly higher than that of cisplatin. These complexes were markedly less effective (IC50 = 26.0–29.2 µM) against the MRC-5 cells, which is suggestive for promising selectivity towards cancer cells over the normal ones. The treating of the A2780 cells by the representative complexes 1 and 5 (IC50 concentrations were used) led to different cell cycle modification (G2-arrest) as compared with the cells treated by the conventional platinum-based anticancer drug cisplatin. The representative complex 8 was hydrolytically stable in the water-containing solution (50% DMF-7d/50% D2O mixture), while it released its N-donor ligand when mixed with relevant naturally occurring biomolecules, i.e., reduced glutathione, cysteine, or bovine serum albumin, as proved by the detailed 1H and 31P-NMR, and ESI+ MS studies.In the present work, eight triphenylphosphanegold(I) complexes containing the deprotonated 7-azaindole or its variously substituted derivatives (


Identification of intermediate metabolites is facilitated by their unusual MSn fragmentation patterns. Semiquantitative measurements are possible, but absolute values await the synthesis of isotope-labeled standards.7-Oxocholesterol (7-OC), 5,6-epoxycholesterol , and its hydrolysis product cholestane-3β,5α,6β-triol  are normally minor oxysterols in human samples; however, in disease, their levels may be greatly elevated. This is the case in plasma from patients suffering from some lysosomal storage disorders, e.g., Niemann-Pick disease type C, or the inborn errors of sterol metabolism, e.g., Smith-Lemli-Opitz syndrome and cerebrotendinous xanthomatosis. A complication in the analysis of 7-OC and 5,6-EC is that they can also be formed ex vivo from cholesterol during sample handling in air, causing confusion with molecules formed in vivo. When formed endogenously, 7-OC, 5,6-EC, and 3β,5α,6β-triol can be converted to bile acids. Here, we describe methodology based on chemical derivatization and LC/MS with multistage fragmentation (MS For apatients , 18. Theg., SLOS –21, strog., SLOS , 23, whig., SLOS , represeg., SLOS . Pulfer g., SLOS .5-BA and 5,6-EC and 3β,5α,6β-triol into 3β,5α,6β-triHBA, we have optimized a charge-tagging methodology to specifically identify 7-oxo-containing sterols and sterols with a 3β,5α,6β-triol function using chemical derivatization and LC/MS with multistage fragmentation (MSn). The resultant method is described below.To investigate how 7-OC is metabolized in vivo into 3βH,7O-ΔPlasma was from patients diagnosed with lysosomal storage disorders, their siblings, or parents. All participants or their parents provided written informed consent in accordance with the Declaration of Helsinki, and the study was conducted with institutional review board approval (REC08/H1010/63). National Institute of Standards and Technology (NIST) standard reference material , a pooled plasma sample representative of the US population , was use27 bile acids were from Avanti Polar Lipids Inc. ; C24 bile acids were a kind gift from Professor Jan Sjövall ; 3β,5α,6β-triHBA was a kind gift from Professor Douglas F. Covey (Washington University School of Medicine). See supplemental Table S1 for a list of oxysterols and bile acid with their common and systematic names, abbreviations, LipidMaps ID, and suppliers. Cholesterol oxidase (ChOx) enzyme from Streptomyces sp. was from Sigma-Aldrich Ltd. , [2H0]Girard P ([2H0]GP) reagent was from TCI Europe , and [2H5]GP was synthesized as described in ref. 18 (200 mg) and Oasis HLB (60 mg) solid phase extraction (SPE) columns were from Waters Ltd .Oxysterols and C2H7]7-OC ([2H7]7-OC), 5α,6-EC , 3β,5α,6β-triol , 7α-hydroxycholesterol ([2H7]7α-HC), 24R/S-hydroxycholesterol ([2H7]24R/S-HC), 7α,25-dihydroxycholesterol , 22R-hydroxycholest-4-en-3-one ([2H7]22R-HCO), 25-hydroxyvitamin D3, and cholesterol ([2H7]C). The solution was diluted to 70% ethanol with 0.35 ml of water, sonicated, and centrifuged to remove precipitated matter. To separate bile acids and oxysterols from cholesterol and similarly hydrophobic sterols, the sample solution was applied to a 200 mg Sep-Pack C18 column; cholesterol was absorbed while oxysterols and bile acids eluted in the flow-through and column wash . After a further column wash , cholesterol and sterols of similar hydrophobicity were then eluted in a separate fraction with absolute ethanol . The oxysterol/bile acid (SPE1-Fr1) and cholesterol-rich (SPE1-Fr3) fractions were then each divided into two equal aliquots (A) and (B) and lyophilized. After reconstitution in propan-2-ol (100 µl), ChOx (0.26 units) in 50 mM phosphate buffer (1 ml), pH 7, was added to subfractions (A). After 1 h at 37°C, the reaction was quenched with methanol (2 ml). Subfractions (B) were treated in an identical manner but in the absence of ChOx. Glacial acetic acid (150 µl) was added to each subfraction, followed by [2H5]GP as the bromide salt (190 mg) to subfractions (A) and [2H0]GP as the chloride salt (150 mg) to subfractions (B). The derivatization reactions were left to proceed overnight in the dark at room temperature. Excess derivatization reagent was removed by SPE using a recycling method. Each subfraction  was applied to a 60 mg Oasis HLB column and washed with 70% methanol (1 ml) and 35% methanol (1 ml). The combined effluent was diluted to 35% methanol and recycled through the column. This was repeated with dilution to 17.5% methanol and a further recycling, and the column was finally washed with 10% methanol (6 ml). At this point, all oxysterols/bile acids, or more hydrophobic sterols, were absorbed on the column while unreacted GP reagent elute to waste. Oxysterols/bile acids were then eluted with 100% methanol , while more hydrophobic sterols with 3 ml of 100% methanol (SPE2-Fr1+2+3). Just prior to LC/MS(MSn) analysis, equal volumes of subfractions (A) and (B) were combined and diluted to 60% methanol ready for injection. This allowed the simultaneous analysis of subfractions A and B.The sample preparation protocol is described in detail in ref. Analysis was performed on an Orbitrap Elite  exploiting electrospray ionization. Chromatographic separation was achieved with a reversed-phase Hypersil Gold column  using an Ultimate 3000 LC system (now Thermo Fisher Scientific) with the mobile phase and gradient described in refs. m/z 400) in the Orbitrap analyzer in parallel to four MS3 scan events in the linear ion-trap. Quantification was by isotope dilution or by using isotope-labeled structural analogs.For each injection, five scan events were performed: one high-resolution scan  fragmentation patterns for the 7-oxo derivatives are unlike those from compounds with GP derivatization at position C-3 . The suggested structures of these ions are shown in 2H7]7-OC , and unlike oxysterols/bile acids without an oxo group, will react with GP reagent in the absence of ChOx; hence, this compound and its metabolites possessing a 7-oxo group will be found in the (B) subfraction derivatized with the 7-OC  in the 17 min chromatographic gradient and is readily quantified by isotope dilution against [2H7]7-OC , but 26-2H7]7-OC , 29, is 2H7]7-OC . In the 3 fragment ion at m/z 426.3 ([M-Py-59]+) that is not present in the MS3 spectrum of coeluting 7αH,3O-CA  is present in the MS3 spectrum of 7αH,3O-CA  , is not αH,3O-CA . Thus, brevealed . TherefoαH,3O-CA , but notαH,3O-CA ; hence, 27 acid, dehydrogenation to a 24-carbonyl group, and then β-oxidation to a C24 acid , and 3βH,7O-Δ5-BA. Although authentic standards are not available for these metabolites, by analogy to the MS3 spectra of 7-OC, 26H,7-OC, and 3βH,7O-CA where standards are available, prominent patterns of distinguishing fragment ions corresponding to the neutral losses [M-Py-43]+, [M-Py-59]+, [M-Py-90]+, and [M-Py-98]+ are predicted to be present in the MS3 spectra of 3β,24-diH,7O-CA, 3βH,7,24-diO-CA, and 3βH,7O-Δ5-BA. Similar to the analysis of 3βH,7O-CA, a MS3 RIC for [M-Py-59]+  should reveal 3β,24-diH,7O-CA 26-oic  and 7α,25-dihydroxy-3-oxocholest-4-en-26-oic  acids, two commercially available isomers of 3β,24-diH,7O-CA . While for most plasma samples we have analyzed in recent times, the MS3 RIC channel for m/z 442.3 ± 0.3 is empty  reveals 3βH,7O-Δ5-BA in plasma samples from patients with elevated 7-OC  in the 17 min chromatographic gradient , 177.1 (*b2), 179.1 (*b3-28), and 231.1, (*c2+2-18) (m/z 157.1 which probably consists of the unsaturated diazacyclohexanone ring and remnants of the B-ring (While GP-derivatized sterols with a 7-hydroxy-3-oxo-4-ene structure give a characteristic pattern of ring fragment ions at c2+2-18) , 4E, 5C e B-ring , 5D. Thi2H7]7-OC, we can only make approximate or semiquantitative measurements. However, as all the 7-oxo compounds, except 3βH,7O-CA, are resolved from their 3-oxo isomers in either the 17 or 37 min chromatographic gradients, quantification is possible using the isotope-labeled internal standard [2H7]22R-HCO. In the absence of authentic standards, 3β,24-diH,7O-CA and 3βH,7O-Δ5-BA were quantified assuming the same response factors as for their structural analogs 7α,24-diH,3O-CA and 7αH,3O-Δ4-BA. As 3βH,7O-CA was not chromatographically resolved from 7αH,3O-CA, the MRM [M+]→[M-Py]+→[M-Py-59]+ was used for quantification of the former isomer.Using the methodology described, other than for 7-OC, where an isotope-labeled standard is available, i.e., [+ and [M-H2O]+ ions  spectrum of the 3β,5α,6β-triol is identical to the MS3 ([M]+→[M-Py]+→) spectrum of cholest-4-ene-3β,6β-diol (6β-HC), confirming dehydration through loss of the 5α-hydroxy group. An unusually prominent fragment-ion observed in the MS3 ([M-H2O]+→[M-H2O-Py]+→) spectrum of 3β,5α,6β-triol is at m/z 383.3, corresponding to [M-H2O-Py-72]+ . The availability of [2H7]3β,5α,6β-triol allows quantification by isotope dilution utilizing RICs for [M-H2O]+ ions. Similar to 3β,5α,6β-triol, 3β,5α,6β-triHBA, the end product of 3β,5α,6β-triol metabolism  spectrum of 3β,5α,6β-triHBA shows a prominent [M-H2O-Py-72]+ fragment-ion at m/z 371.3, [M-H2O-Py-90]+ at m/z 353.2, and the unusual neutral-loss [M-H2O-Py-100]+ at m/z 343.3 . In the absence of an isotope-labeled standard, approximate quantification of 3β,5α,6β-triHBA is made against the internal standard [2H7]24R/S-HC, taking into account relative response factors.Sterols with a 3β-hydroxy group and a planar A/B ring system are substrates for ChOx; these include cholest-5-en-3β-ols and 5α-cholestan-3β-ols . 3β,5α,6O]+ ions  33), wi, wi+ andtabolism , 16, giv2O]+ ion of cholestanetetrols  which has an identical mass. However, chromatographic resolution is sufficiently good to generate an MS3 (m/z 555.4→471.4→) spectrum from the apex of the new peak, which is entirely compatible with that expected for the [M-H2O]+ ion of cholestane-3β,5α,6β,26-tetrol , showing a prominent fragment-ion at m/z 399.3, i.e., [M-H2O-Py-72]+; a distinct ion at m/z 381.3, i.e., [M-H2O-Py-90]+; and a minor fragment at m/z 371.3, i.e., [M-H2O-Py-100]+ (3 (m/z 555.4→471.4→) spectrum of closely eluting 7α,25-diHC does not give a fragment ion at m/z 399.3 or 371.3 , so by generating a RIC for the fragment ion m/z 399.3 from the MS3 (m/z 555.4→471.4→) chromatogram, 3β,5α,6β,26-tetrol ([M-H2O]+) is resolved from 7α,25-diHC , i.e., the dihydroxycholesterols (diHC) 7α,12α-diHC, 7α,24S-diHC, 7α,26-diHC, 7β,25-diHC, and 7β,26-diHC, are all chromatographically resolved from 3β,5α,6β,26-tetrol ([M-H2O]+). It is only possible to make semiquantitative measurement of 3β,5α,6β,26-tetrol in the absence of an authentic standard and its incomplete chromatographic resolution from 7α,25-diHC. Semiquantification is made based on the [M-H2O]+ of 3β,5α,6β,26-tetrol against the internal standard [2H6]7α,25-diHC.In plasma samples high in 3β,5α,6β-triol , a new peak is evident in the RIC for the [M-HPy-100]+ . The MS327 acids proceeds through C-24 hydroxylation, C-24 dehydrogenation, and β-oxidation to generate 3β,5α,6β-triHBA as the ultimate product. The relevant pathway intermediates would be 3β,5α,6β,24-tetrahydroxycholestan-26-oic acid  and 3β,5α,6β-trihydroxy-24-oxocholestan-26-oic acid . By analogy to 3β,5α,6β-triol, 3β,5α,6β,26-tetrol, and 3β,5α,6β-triHBA, 3β,5α,6β-triHCa, 3β,5α,6β,24-tetraHCa, and 3β,5α,6β-triH,24O-Ca should give [M]+ and [M-H2O]+ products upon ChOx treatment and GP derivatization, and the MS3 spectra of the [M-H2O]+ ions ([M-H2O]+→[M-H2O-Py]+→]) are predicted to show neutral-loss fragment-ions [M-H2O-Py-72]+, [M-H2O-Py-90]+, and [M-H2O-Py-100]+.CYP27A1 is the enzyme likely to introduce the (25R)26-hydroxy group to the sterol side chain. This enzyme could then oxidize the primary alcohol to a carboxylic acid to give 3β,5α,6β-trihydroxycholestan-(25R)26-oic acid . As discussed above, peroxisomal side-chain shortening of C2O-Py-72]+ ion has an m/z of 413.3 . A RIC for the MRM transition [M-H2O]+→[M-Py-H2O]+→[M-H2O-Py-72]+ (m/z 569.4→485.3→413.3) reveals a new chromatographic peak in samples where the concentration of the 3β,5α,6β-triol is high , which is at, or below, the detection limit in normal plasma samples (3 ([M-H2O]+→[M-H2O-Py]+→) spectrum underlying the new chromatographic peak in plasma samples rich in 3β,5α,6β-triol is compatible with that predicted for 3β,5α,6β-triHCa, showing the predicted neutral-loss fragment ions [M-H2O-Py-72]+, [M-H2O-Py-90]+, and [M-H2O-Py-100]+ and is thus assigned to this acid 26-oic acid  and a second oxysterol with a probable 3β,22,25-trihydroxycholest-5-en-24-one structure, but neither compound gives a fragment ion at m/z 413.3 in their MS3 ([M]+→[M-Py]+→) spectra , unlike well-resolved 3β,7α-dihydroxycholest-5-en-(25S)26-oic and 3β,7α-dihydroxycholest-5-en-(25R)26-oic acids, where this ion is more abundant . In fact, the 25R and 25S epimers give identical MS3 spectra, but are chromatographically resolved. In the absence of an authentic standard of 3β,5α,6β-triHCa, only semiquantification is possible. This can be made using the RIC for the [M-H2O]+ ion in samples where coeluting compounds 3β,7β-diHCA(25S) and 3β,22,25-trihydroxycholest-5-en-24-one are minor , then determining an appropriate response factor for the MRM chromatogram 569.4→485.3→413.3 and using this for quantification of other samples.For 3β,5α,6β-triHCa, the [M-H2O-Py-72]+ fragment ion has an m/z of 429.3 . In the RIC for the MRM transition [M-H2O]+→[M-Py-H2O]+→[M-H2O-Py-72]+ (m/z 585.4→501.3→429.3) from samples where 3β,5α,6β-triol is abundant, new peaks appears in both the 17 min . Both fragment ions are essentially absent from MS3 spectra of the closely eluting peak  spectra is characteristic and valuable for metabolite identification via appropriate MRM ([M]+→[M-Py]+→[M-Py-59]+) RICs  leads to a characteristic neutral-loss fragment ion [M-H2O-Py-72]+  spectra, which are less structurally characteristic, making identification of intermediate metabolites difficult .Considering metabolites with a 7-oxo-5-ene structure, the fragment ion resulting from an [M-Py-59]ral loss  and appe]+) RICs , 5B. Theen atoms . With re-Py-72]+ . Again, abolites , 8B, E. e A-ring . BesidesAlthough the primary aim of this work was identification of 7-oxo-5-ene- and 3β,5α,6β-triol-containing metabolites, approximate or semiquantitative measurements can also be made. Accurate quantification, however, awaits further synthesis of authentic standards and their isotope-labeled analogs.


The three title compounds are centrosymmetric. Their packing features chains linked by either pairwise C—H⋯O or C—H⋯π interactions. E,2′E)-3,3′-bis[1-(2-methoxyphenyl)prop-2-en-1-one], C26H22O4 (I), -3,3′-bis[1-(3-methoxyphenyl)prop-2-en-1-one], C26H22O4 (II) and -3,3′-bis[1-prop-2-en-1-one], C28H26O6 (III), the asymmetric unit consists of a half-molecule, completed by crystallographic inversion symmetry. The dihedral angles between the central and terminal benzene rings are 56.98 (8), 7.74 (7) and 7.73 (7)° for (I), (II) and (III), respectively. In the crystal of (I), molecules are linked by pairs of C—H⋯π interactions into chains running parallel to [101]. The packing for (II) and (III), features inversion dimers linked by pairs of C—H⋯O hydrogen bonds, forming R22(16) and R22(14) ring motifs, respectively, as parts of [201] and [101] chains, respectively.In the title compounds, (2 The α,β-unsaturated enone C=C—C(=O)—C moiety in the chalcone structure plays an important role in the biological activities of these species -3,3′-bis(1-(2-methoxyphenyl)prop-2-en-1-one), C26H22O4 (I)E,2′E)-3,3′-bis(1-(3-methoxyphenyl)prop-2-en-1-one), C26H22O4 (II)E,2′E)-3,3′-bisprop-2-en-1-one), C28H26O6 (III)Chalcones and their derivatives are natural or synthetic 1,3-diaryl-2-propenones that may exist in The molecular structures of (I)–(III) are shown in Figs. 1ortho-O2 atom. Hence, the dihedral angle between the 2-methoxyphenyl and benzene rings in (I)Each compound is constructed from two aromatic rings , which are linked by a C=C—C(=O)—C enone bridge. Despite having an extra methoxy substituent, the conformation of compounds (II)A–C12A). This C—H⋯π interaction connects the molecules of (I)The packing of (I)ns Fig. 5 in (II),ns Fig. 5.et al., 2016E,2′E)-3,3′-bis(1-phenylprop-2-en-1-one) as the main skeleton, revealed the presence of four structures containing the bischalcone moiety with different substituents, similar to the title compounds in this study. These include 3,3′-bis[1-(X)prop-2-en-1-one], where X = 2-hydroxyphenyl  and terephthaldialdehyde (0.01 mol) was dissolved in methanol (20 ml). A catalytic amount of NaOH was added to the solution dropwise with vigorous stirring. The reaction mixtures were stirred for about 5–6 h at room temperature. The resultant crude products were filtered, washed successively with distilled water and recrystallized from ethanol to obtain the title compounds. Yellow blocks [(I) and (III)] and yellow needles (II))-3,3′-bis(1-(2-methoxyphenyl)prop-2-en-1-one), C−1]: 3010 , 2945 , 2842 , 1658 , 1598, 1417, 1245, 1055 . 1H NMR : δ ppm 7.677–7.632 , 7.536–7.504 , 7.496–7.437 , 7.095–7.065 , 7.048–7.031 , 3.944 . 13C NMR : δ ppm 192.61 (C7), 158.23 (C1), 141.48 (C9), 136.95 (C10), 133.09 (C3), 130.47 (C5), 129.17 (C6), 128.84 , 127.91 (C4), 120.84 (C8), 111.69 (C2), 55.80 (C13).Solvent for growing crystals: acetone; yield 85%, m.p. 429–431 K. FT–IR : 3074 , 2952 , 2839 , 1658 , 1582, 1414 , 1259, 1022 . 1H NMR : δ ppm 7.855–7.823 , 7.722  , 7.650–7.635 , 7.606–7.574 , 7.473–441 , 7.189–7.172 , 3.924 . 13C NMR : δ ppm 189.99 (C7), 159.99 (C2), 143.57 (C9), 139.46 (C10), 136.92 (C6), 129.65 (C5), 128.98 , 123.14 (C8), 121.08 (C3), 119.45 (C4), 112.96 (C1), 55.53 (C13)Solvent for growing crystals: chloroform and methanol; yield 85%, m.p. 444–446 K. FT–IR : 3018 , 2962 , 2836 , 1651 , 1592, 1418 , 1240, 1017 . 1H NMR : δ ppm 7.857–7.826 , 7.740–7.720 , 7.666 , 7.651–7.619 , 6.984–6.967 , 4.012–4.009 . 13C NMR : δ ppm 188.34 (C7), 153.47 (C3), 149.37 (C2), 142.80 (C9), 136.94 (C10), 131.22 (C6), 128.89 , 123.11 (C5), 122.62 (C8), 110.82 (C4), 110.01 (C1), 56.15 (C14), 66.10 (C13)Solvent for growing crystals: acetone; yield 85%, m.p. 479–481 K. FT–IR [ATR (solid) cmUiso(H) = 1.5Ueq(C-methyl) and 1.2Ueq(C) for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989017007460/hb7678sup1.cifCrystal structure: contains datablock(s) global, I, II, III. DOI: 10.1107/S2056989017007460/hb7678Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989017007460/hb7678IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989017007460/hb7678IIIsup4.hklStructure factors: contains datablock(s) III. DOI: Click here for additional data file.10.1107/S2056989017007460/hb7678Isup5.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989017007460/hb7678IIsup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989017007460/hb7678IIIsup7.cmlSupporting information file. DOI: 1449626, 1449625, 1449624CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Hyrtios digitatus has provided a perspective on the connection between the chemistry and biology of the puupehenones, a unique and unusual class of merosesquiterpenes. In this study, a new tetracyclic merosesquiterpene, 19-methoxy-9,15-ene-puupehenol (1) was isolated from the marine sponge Hyrtios digitatus along with the known 20-methoxy-9,15-ene-puupehenol (2). Their structures were elucidated on the basis of spectroscopic data (1H and 13C NMR) in combination with experimental electronic circular dichroism (ECD) data. Compounds 1 and 2 are active at 1.78 μM and 3.05 μM, respectively, on Scavenger Receptor-Class B Type 1 HepG2 (SR-B1 HepG2) stable cell lines, targeting atherosclerosis disease.A study of the chemical constituents from the Australian Sponge  From the 1H and 13C NMR spectra, 30 proton and 22 carbon resonances were observed. The 1H NMR spectrum of 1 (see H 3.68 ppm), four characteristic singlet methyl signals , two aromatic protons (δH 6.22 and 6.67 ppm), five methylenes , one olefinic proton (δH 6.11 ppm) and one hydroxyl proton (δH 8.98 ppm) (The freeze-dried nol (2),  24]. 19. 19Hyrtiof 1 see  displaye.98 ppm) .13C NMR and heteronuclear single quantum for proton-carbon correlation (gHSQC) spectra , two aromatic carbons (δC 103.9 and 110.9 ppm), one olefinic (δC 114.1 ppm), four methyl carbons , one methine (δC 32.6 ppm), one methoxy (δC 56.7 ppm), and five methylenes . Analysis of correlation spectroscopy (gCOSY) and gHSQC data led to the assignment of the ABCD ring system in compound 1. A gCOSY spectrum displayed correlations from methylene proton H-6 (δH 1.64 ppm) to H-7 (δH 1.88 ppm), indicating the connectivity of C-6 (δC 16.7 ppm) and C-7 (δC 30.6 ppm).Further analysis of the  spectra  indicateH 0.85 ppm) had gHMBC correlations with C-3 (δC 16.7 ppm), C-5 (δC 43.4 ppm), and C-12 (δC 16.7 ppm). gHMBC signals from proton H-12 (δH 0.92 ppm) to C-3, C-4 (δC 32.6 ppm), C-5, and C-11 (δC 33.4 ppm) suggested that H-11 and H-12 constituted a gem-dimethyl, connected at C-4 (δC 32.6 ppm). gHMBC correlations from H-2 to C-1 (δC 38.7 ppm), C-3 (δC 41.7 ppm), and C-4 (δC 32.6 ppm) and from H-14 (δH 1.14 ppm) to C-5 (δC 43.4 ppm) and C-10 (δC 38.1 ppm) completed the establishment of ring A. gHMBC analysis also indicated H-14 had correlations with C-9 (δC 148.7 ppm) and C-13 (δC 24.9 ppm), supporting the connection of rings A and B. A singlet methyl, H-13 (δH 1.25 ppm), displayed gHMBC correlations with C-7 (δC 30.6 ppm), C-8 (δC 75.9 ppm), and C-9 (δC 148.7 ppm). Two methylene signals H-6 and H-7 showed gHMBC correlations to carbons C-5, C-7, C-8, and C-6, C-14, respectively, and an olefinic proton signal H-15 (δH 6.11 ppm) had gHMBC correlations with C-8 (δC 75.9 ppm) and C-10 (δC 38.1 ppm), allowing for the connection of ring B and ring C.Detailed heteronuclear multiple bond correlation (gHMBC) analysis showed that H-11 (δC 115.0 ppm), C-17 (δC 145.9 ppm), and C-21 (δC 110.9 ppm). The methoxy proton signal H-22 (δH 3.68 ppm) was correlated with C-19 (δC 141.9 ppm), while a hydroxyl proton signal (δH 8.98 ppm) was correlated with C-18, C-19, and C-20. gHMBC correlations also observed from an aromatic proton signal, H-18 (δH 6.22 ppm) to C-17 (δC 145.9 ppm) and C-16 (δC 115.0 ppm), supported the C/D ring system. The relative stereochemistry of 1 was deduced on the basis of nuclear overhauser effect spectroscopy (NOESY) interactions (H 0.92 ppm) and H-14 (δH 1.14 ppm), which revealed their co-facial relationship and assignment as β-oriented. It indicated a trans fusion of rings A/B. Meanwhile, the NOESY correlation between a methine proton signal, H-5 (δH 1.37 ppm) and a methyl proton, H-13 (δH 1.25 ppm) implied a cis fusion of rings B/C. Taken together, the relative configuration of 1 was assigned as 5S*,8S*,10S* as shown in The establishment of ring C to ring D was confirmed by gHMBC correlations between the olefinic proton H-15 to C-16 (δractions . NOESY a2) was confirmed as C22H30O3 by accurate mass measurement and displayed a protonated molecular ion peak [M + H]+ at m/z 343.4532 in the (+)-HRESIMS spectrum c 0.2, MeOH), CD (MeOH) λmax (log ε) 238 (+3.98), 278 (+3.75), and 320 (+3.81) nm, UV (MeOH) λmax (log ε) 235 (+3.66), 279 (+3.75), and 325 (+3.81) nm, 1H  and 13C  NMR data are summarized in + at m/z 343.3418.-20-Methoxy-9,15-ene-puupehenol (2): pale yellow oil; [α]c 0.2, MeOH), CD (MeOH) λmax (log ε) 242 (+3.68), 283 (+3.75), and 325 (+3.81) nm, UV (MeOH) λmax (log ε) 230 (+3.65), 277 (+3.74), and 322 (+3.81) nm, 1H  and 13C  NMR data are summarized in + at m/z 343.4532.1) and 20-Methoxy-9,15-ene-puupehenol (2) were optimized using the OPLS-2005 force field method applying a 21 kJ/mol energy window [All molecular mechanics analyses and calculations were determined using Macromodel interfaced to the Maestro program ,34. The y window . The opty window . ECD spe2 in a humidified environment at 37 °C. Thirty microliters (30 μL) of media containing 10,000 cells were added to a 384 well microtitre white clear bottom plate  containing 0.3 μL of a compound. Final compound concentration range tested was 100 μM to 0.1 μM . Each concentration in the media was tested in triplicate and in two independent experiments. Cells and compounds were then incubated for 48 h at 37 °C, 5% CO2 and 80% humidity. Cell proliferation was measured with the addition of 30 μL of a Luciferase reagent  solution to each well of the microtitre plate. The plates were incubated at room temperature for 10 min. The luminescence of each well was read on the VICTOR X Multilabel Plate Readers (Perkin Elmer). Nine-point concentration-response curves were then analyzed using non-linear regression and EC50 values were determined by using GraphPad Prism 5 . Trichostatin-A (TSA) was used as a positive control compound.Hepatocellular carcinoma cells (HepG2) stably transfected with SR-B1 were kindly provided by the Malaysian Institute of Pharmaceuticals and Nutraceuticals (IPharm), Penang, Malaysia. SR-B1 HepG2 stable cell lines were grown in MEM media  supplemented with 10% fetal bovine serum (FBS). Cells were grown under 5% COHyrtios digitatus, a new -19-methoxy-9,15-ene-puupehenol (1) and the known -20-methoxy-9,15-ene-puupehenol (2) belonging to the merosesquiterpene structure class were identified. Structures of these compounds were determined by NMR spectroscopy and confirmed by ECD analysis. These two compounds satisfy properties for drug-like molecules with one violation (c logP) for oral bioavailability. This is the first time that the merosesquiterpene structure class has been reported to increase the activity of SR-B1 in a HepG2 cell line, indicating the potential role of these compounds in therapeutic intervention against atherosclerosis.Contributing to the chemical study on


Our results used to design new derivatives of Oxim with better efficacy than 2-PAM and obidoxime. Syntheses of some selected bis-pyridiniumoximes based on the nitrones are underway.Acetylcholinesterase has important role in synaptic cleft. It breaks down the acetylcholineat cholinergic synapsesand terminates the cholinergic effects. Some chemical agents like organophosphorus compounds (OPCs) including nerve agents and pesticides react with acetylcholinesteraseirreversibly. They inhibit normal biological enzyme action and result in accumulation of acetylcholineand show toxic effects andcholinergic symptoms. The process of Acetylcholinesterase (AChE) inhibition can be reversed by a nucleophilic agent to dephosphorylate and reactivate the enzyme. In this study, design and docking studies of 15 novel nitrone based onoximes as reactivators were performed by using AutoDock program. Then, more effective reactivatorsoximes in terms of binding energy and orientation within the active site were synthesized and evaluated  Acetylcholinesterase EC 3.1.1.7;AChE), one of the most important enzyme for human and other species, terminates cholinergic neurotransmission by splitting the neurotransmitter acetylcholine to choline and acetic acid .1.7;AChE. The cate.g., lacrimation, miosis, salivation) and nicotinic receptors , finally leading to death caused by respiratory failure (It is well known that organophosphorus compounds (OPCs) including nerve agents and pesticides react irreversibly with acetylcholinesterase by forming a covalent binding to the serine hydroxyl group within the active site , 8. Afte failure .e.g., diazepam), a para sympatholytic agent  and an oxime reactivator .-13.e.g.,in-vitro evaluation and the results were compared to those of obidoxime and 2-PAM.Due to their high nucleophilicity and secondary interactions of their cationic parts with the peripheral site of the enzyme, oximes can displace the phosphyl group from the catalytic serine and thus, restore the enzymeʹs vital function . DespiteMolecular modelingCrystallographic coordinates of mAChE phosphorylated by tabun were taken from the Brookhaven Protein Data Bank (PDB code: 2Jf0) . All hetChemistry1H and 13C NMR spectra were recorded on Bruker 250 and 62.90 MHz instrument respectively. NMR spectra were obtained in DMSO-d6 using TMS as internal standard. Four different selected nitron based oximes were synthesized on the basis of preliminary computational predictions which are presented in  and were used without further purification. The solvents were purchased from Merck (Germany) and dried and distilled before use. Diaminoglyoxim (DAG) was prepared according to the literature procedure . Obidoxiented in .In-vitro reactivation assayThe reactivation ability was measured at 412 nm by using UV spectrophotometer (PERKIN-ELMER Lambda 5 and CECIL 8000) with a modified Ellman protocol. Recombinant hAChE (Sigma-Aldrich) was used throughout experiments. Pesticide paraoxon, diethyl-O-(4-nitrophenyl) phosphate, was purchased from (Sigma–Aldrich). Phosphate buffer , acetylthiocholine (1 mM) as substrate and DTNB (1 mM) as chromogen were used throughout all experiments. Paraoxon was used as organophosphorus inhibitor and the AChE reactivating capability of novel oximes, 2-PAM and obidoxime were examined against paraoxon-inhibited AChE. The enzyme was inhibited via the addition of excess of a paraoxon solution. To remove excess inhibitor, the reaction mixture was partitioned with six folded volume of hexane -42, and r - Ei/E0 – Ei) × 100%Reactivation = -2-(1-pyridin-3-yl)-2, 5-dihydro-1H-imidazole 3-oxide (3)4-amino-5-(hydroxyimino)-2-(1-pyridin-3-yl)-2, 5-dihydro-1H-imidazole 3-oxide (3) was prepared by the reaction of DAG , pyridine-3-carboxaldehyde  and p-toluene sulphonic acid (p-TSA)  in ethanol. The reaction mixture was stirred at ambient temperature for 90 min after that white precipitates appeared in the reaction mixture. It was filtered and dried.-1): 3327-3242 (NH2), 3159 (OH), 2851 (CH-Imidazole), 1715-1693 (C=N), 1605-1480 (Py-ring), 1330 (N=O), 1229 (C-N), 935-924 (N-O); 1HNMR ; 5.63, 5.88 , 5.47, 5.55 ,7.42-8.71 , 10.06, 10.21  ppm. 13C NMR : 91.84, 124.24, 134.937, 135.59, 141.87, 148.64, 149.66, 150.93, 152.15 ppm.White powder; m.p 209-211 °C; yield 96%; IR  and corresponding alkyl or benzyl halides (7 mmol) in dried acetone. Reaction mixture was heated at 60 °C for 6-8 h while vigorously stirred. After cooling, the separated precipitates were collected, washed with dry ether (20 mL) and dried. The powder 3-formyl-1-methylpyridinium iodide, dried in vacuo over phosphorus pentoxide. 3-formyl-1-butylpyridinium bromide product was not obtained under various conditions examined.Compounds B3-Formyl-1-methylpyridinium Iodide (B1)1H NMR and 13C NMR spectra are consistent with the literature data 1H NMR : 1.55-1.61 , 4.70-4.79 , 8.33-8.38 , 8.98, 9.01 , 9.31, 9.33 , 9.56 , 10.17 ; 13C NMR : 16.61, 57.35, 128.98, 134.85, 145.06, 146.60, 148.46, 189.28.Yellow powder, m.p 132–135 °C, yield 76%, 3-Formyl-1-benzylpyridinium Bromide (B5)1H NMR : 6.00, 7.44-7.63 , 8.35-8.38 , 9.01, 9.04 , 9.43, 9.46 , 9.80 , 10.16 ; 13C NMR : 63.72, 129.30-129.93 (6C), 134.42, 135.24, 145.52, 146.76, 148.54, 189.10.White powder, m.p 140–142 °C, 92%, General procedure for the synthesis of oximes 1B-2B and 4B-5B First strategy3 , alkyl or benzyl halides (1.2 mmol) in DMF (3 mL) was stirred at room temperature for appropriate time. The progress of the reaction was monitored by TLC (ethyl acetate). Then, the mixture of the reaction was placed in a vacuum oven at 40 °C for 55 h to remove DMF. The precipitate was washed with dry acetonitrile and dry ethanol and then dried under vacuum.A solution of Second strategy1-B2 and B5 compounds (1.2 mmol) in dry ethanol under nitrogen atmosphere, which is sealed and heated at 40 °C with stirring for 40 h. The precipitates were collected, washed with dry ethanol and dried.Target oximes 1B-2B and 4B-5B were synthesized by the reaction between DAG  and corresponding BPrepared oximes by the first strategy4-amino-2-(1-methylpyridinium-3-yl)-5-(hydroxyimino)-2,5-dihydro-1H-imidazole 3-oxide Iodide (1B)-1): 3307, 3240 (NH2), 3153 (OH), 2856 (CH), 1715, 1683 (C=N), 1642, 1474 (Py-ring), 1352, 1293 (N=O), 1221 (C-N), 958, 911 (N-O); 1H NMR : 4.40 , 5.63 , 6.72 ,7.90-9.10 , 10.31  ppm; 13C NMR : 48.04, 89.04, 127.59, 139.59, 141.67, 142.56, 143.44, 145.86, 151.31 ppm.Yellow powder; m.p 158-160 °C; yield 64%; IR -5-(hydroxyimino)-2,5-dihydro-1H-imidazole 3-oxide Iodide (2B)-1): 3412, 3254 (NH2), 3159 (OH), 2980 (CH), 1714, 1693 (C=N), 1652, 1480 (Py-ring), 1357 (N=O), 1230 (C-N), 954, 924 (N-O); 1H NMR : 1.51-1.56 , 4.63-4.71 , 5.72, 5.90 , 6.70, 6.84 ,7.70-9.21 , 10.14, 10.27  ppm; 13C NMR : 16.42, 56.74, 89.45, 128.43, 140.57, 141.14, 142.96, 143.28, 145.24, 151.63 ppm.Yellowish powder; m.p 145-146 °C; yield 73%; IR -5-(hydroxyimino)-2,5-dihydro-1H-imidazole 3-oxide Bromide (4B)-1): 3335, 3241 (NH2), 3161 (OH), 2975 (CH), 1714, 1693 (C=N), 1606, 1434 (Py-ring), 1309 (N=O), 1230 (C-N), 935, 925 (N-O); 1H NMR : 0.83-0.96 , 1.23-1.39 , 1.93-1.98 White powder; m.p 169-171 °C; yield 52%; IR  4.77-4.80  6.15 , 7.76 , 8.32-9.97 , 10.54,  ppm; 13C NMR : 13.79, 19.18, 33.23, 79.40, 124.52, 129.25, 142.01, 144.01, 145.07, 148.19, 164.85, 171.20 ppm.-5-(hydroxyimino)-2,5-dihydro-1H-imidazole 3-oxide Bromide (5B)-1): 3392, 3290 (NH2), 3155 (OH), 3001 (CH), 1644 (C=N), 1578, 1473 (Py-ring), 1341 (N=O), 1255 (C-N), 951, 936 (N-O); 1H NMR : 6.03, 6.15 , 7.44-7.62 , 8.37-8.42 , 9.20, 9.23 , 9.41, 9.43 , 10.11 , 10.54  ppm; 13C NMR : 63.86, 124.54, 129.18-130.10 (6C), 133.93, 141.67, 144.23, 144.66, 147.74, 164.41, 170.77 ppm.White Powder; m.p 206-207 °C; yield 87%; IR , 124.54 (spiro carbon), 129.18-130.10 , 133.93-147.74  along with 164.41 and 170.77 that are assigned to the two alkene carbons at imidazole ring. The 1H and 13C NMR spectra of 1B-2B and 4B were more or less similar to those of 5B except for the alkyl substituents, which exhibited characteristic signals with appropriate chemical shifts. Also, it should be pointed out that compound 3 and butyl bromide reacted more smoothly than others, which need longer reaction time and gave lower yield of product.Afterward, the N-alkylation reactions of compound 3 with alkyl or benzyl halides were carried out under the mild condition, at room temperature, in DMF solvent for 2–12 h . The str1-B2 and B5 were synthesized by the N-alkylation reactions between pyridine-3-carboxaldehyde and corresponding alkyl or benzyl halides in dried acetone , 129.30-129.93 , 134.42-148.54 , 189.10 ppm (CHO) in agreement with the proposed structure. The 1H and 13C NMR spectra of B2 were similar to those of B5 except for the ethyl substituent, which exhibited characteristic signals with appropriate chemical shifts. Also, 1H NMR and 13C NMR results of B1 are consistent with literature data , 4B (1.4%), 5B (2.1%) and 1B, 2B (0%) were all inefficient reactivators at 10 µM. However, obidoxime was found to be potent paraoxon-reactivator (23.2%). While, all of our novel reactivators showed some reactivation ability at a concentration of 100 µM, their ability 1B (3.2%), 2B (3.4%), 4B (4.1%) and 5B (7.8%) were obviously less than obidoxime (60.3%) and 2-PAM (11.2%). Moreover, among the novel synthesized oximes, compound 5B was found to be the best but did not reach the reactivator ability of 2-PAM oxime.Finally, it might be of interest to note that although none of these novel oximes were able to surpass the reactivation efficacy of obidoxime and 2-PAM for the reactivation of hAChE inhibited by paraoxon at concentrations of 10 µM and 100 µM, bis-pyridinium derivatives of these newly introduced oximes might be promising. The preliminary docking studies on such bispyridinium species indicates that it is logical to practically evaluate this proposal.In summary, docking studies on some novel nitrone oximes and two current standard reactivatores, obidoxime and 2-PAM, were performed. The screening results showed that all of the new oximes possess better binding energies than 2-PAM and were comparable with obidoxime. Moreover, the binding energies of oximes 1-5 B were better than those of corresponding 1-5 A and 1-5 C in terms of binding energy and orientation within the active site.Superior oximes, then, were synthesized and evaluated for their reactivation efficacy against paraoxon-inhibited AChE. None of the tested oximes have shown better reactivation efficiencies than those of the standard oximes. It is suggested that bis-pyridinium derivatives of these oximes family may have higher potency than the reported cases here. The synthesis and evaluation of bis-pyridiniumoximes based on the nitrones are underway.