N′-substituted derivatives of 5-amino-N,3-dimethyl-1,2-oxazole-4-carbohydrazide (MM1–MM10) was synthesized in reaction of 5-amino-N,3-dimethyl-1,2-oxazole-4-carbohydrazide with relevant carbonyl compounds. The isoxazole derivatives were tested in several in vitro models using human cells. The compounds inhibited phytohemagglutinin A (PHA)-induced proliferation of peripheral blood mononuclear cells (PBMCs) to various degrees. The toxicity of the compounds with regard to a reference A549 cell line was also differential. 5-amino-N′-methylidene-N,3-dimethyl-1,2-oxazole-4-carbohydrazide (MM3) compound was selected for further investigation because of its lack of toxicity and because it had the strongest antiproliferative activity. The compound was shown to inhibit lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF α) production in human whole blood cell cultures. In the model of Jurkat cells, MM3 elicited strong increases in the expression of caspases, Fas, and NF-κB1, indicating that a proapoptotic action may account for its immunosuppressive action in the studied models.This work describes the synthesis of a new series of isoxazole derivatives, their immunosuppressive properties, and the mechanism of action of a representative compound. A new series of  Isoxazoles are an important class of heterocyclic compound, displaying a broad spectrum of biological activities. Modification in their structures has offered a high degree of diversity that has proved useful for the development of new therapeutic agents with improved potency and lower toxicity. Isoxazole derivatives display a wide array of pharmacological activities that have been successfully screened for anticancer , anti-inN-(4-methylbenzyl)-4-isoxazolecarboxamide, selected as the most interesting compound, exhibited strong anti-proliferative and anti-inflammatory properties. On the other hand, this compound, had the ability to enhance manifestation of the cellular immune response -3-methylisoxazol-5-yl}acetimidate) +m/z 197.1054, calc. m/z 197.1033, [M + Na]+m/z 219.0876, calc. m/z 219.0852.5-Amino-N′-(4-hydroxyphenyl)methylidene-N,3-dimethyl-1,2-oxazole-4-carbohydrazide (MM2). Anal. C13H14N4O3 (m.w. 274.275 g/mol); m.p. 149–151 °C; yield 71%; FTIR  vmax/cm−1: 1615 (C=O), 3446 (NH2). 1H NMR (DMSO-d6) δ (ppm): 2.09 , 3.33 , 6.79–6.82 , 7.07 , 7.50–7.53 , 7.88 , 9.807 . MS (ESI) m/z [M + H]+ 275.1157, calc. m/z 275.1139, m/z [M + Na]+ 297.0976, calc. m/z 297.0958.5-amino-N′-methylidene-N,3-dimethyl-1,2-oxazole-4-carbohydrazide (MM3). Anal. C13H14N4O4 (m.w. 290.275 g/mol); m.p. 211–212 °C; yield 73%; FTIR  vmax/cm−1: 1620 (C=O), 3442 (NH2). 1H NMR (DMSO-d6) δ (ppm): 2.08 , 3.33 , 6.28–6.32 , 7.11 , 7.36–7.39 , 8.05 , 9.78 , 10.25 . MS (ESI) m/z [M + H]+ 291.1114, calc. m/z 291.1087, m/z [M + Na]+ 313.0930, calc. m/z 313.0907.5-Amino-N,3-dimethyl-N′-(5-nitrothiophen-2-yl)methylidene-1,2-oxazole-4-carbohydrazide (MM4). Anal. C11H11N5O4S (m.w. 309.301 g/mol); m.p. 230–231 °C; yield 81%; FTIR  vmax/cm−1: 1628 (C=O), 3445 (NH2). 1H NMR (DMSO-d6) δ (ppm): 2.11 , 3.34 , 7.28 , 7.44–7.46 , 8.10–8.11 , 8.17 . MS (ESI) m/z [M + H]+ 310.0630, calc. m/z 310.0604, m/z [M + Na]+ 332.0449, calc. m/z 332.0424.5-Amino-N,3-dimethyl-N′-phenylmethylidene-1,2-oxazole-4-carbohydrazide (MM5). Anal. C13H14N4O2 (m.w. 258.276 g/mol); m.p. 134–135 °C; yield 75%; FTIR  vmax/cm−1: 1635 (C=O), 3443 (NH2). 1H NMR (DMSO-d6) δ (ppm): 2.09 , 3.37 , 7.13 , 7.40–7.43 , 7.67–7.70 , 7.97 . MS (ESI) m/z [M + H]+ 259.1215, calc. m/z 259.1189, m/z [M + Na]+ 281.1035, calc. m/z 281.1009.5-Amino-N′-(4-chlorophenyl)methylidene-N,3-dimethyl-1,2-oxazole-4-carbohydrazide (MM6). Anal. C13H13N4O2Cl (m.w. 292.721 g/mol); m.p. 210–211 °C; yield 69%; FTIR  vmax/cm−1: 1628 (C=O), 3447 (NH2). 1H NMR (DMSO-d6) δ (ppm): 2.08 , 3.32 , 7.13 , 7.48–7.51 , 7.68–7.71 , 7.97 . MS (ESI) m/z [M + H]+ 293.0811, calc. m/z 293.0800, m/z [M + Na]+ 315.0631, calc. m/z 315.0613.5-Amino-N′-(2-chlorophenyl)methylidene-N,3-dimethyl-1,2-oxazole-4-carbohydrazide (MM7). Anal. C13H13N4O2Cl (m.w. 292.721 g/mol); m.p. 208–209 °C; yield 65%; FTIR  vmax/cm−1: 1630 (C=O), 3438 (NH2). 1H NMR (DMSO-d6) δ (ppm): 2.09 , 3.39 , 7.19 , 7.367–7.41 , 7.51–7.52 , 7.85–7.88 , 8.06 . MS (ESI) m/z [M + H]+ 293.0811, calc. m/z 293.0800, m/z [M + Na]+ 315.0631, calc. m/z 315.0619.5-Amino-N′-(2-methoxyphenyl)methylidene-N,3-dimethyl-1,2-oxazole-4-carbohydrazide (MM8). Anal. C14H16N4O3 (m.w. 288.302 g/mol); m.p. 224–225 °C; yield 63%; FTIR  vmax/cm−1: 1625 (C=O), 3445 (NH2). 1H NMR (DMSO-d6) δ (ppm): 2.08 , 3.35 , 3.87 , 6.95–7.09 , 7.12 , 7.13 , 7.35–7.40 , 8.08 . MS (ESI) m/z [M + H]+ 289.1313, calc. m/z 289.1295, m/z [M + Na]+ 311.1132, calc. m/z 311.1115.5-Amino-N,3-dimethyl-N′-(4-nitrophenyl)methylidene-1,2-oxazole-4-carbohydrazide (MM9). Anal. C13H13N5O4 (m.w. 303.273 g/mol); m.p. 214–216 °C; yield 73%; FTIR  vmax/cm−1: 1635 (C=O), 3446 (NH2). 1H NMR (DMSO-d6) δ (ppm): 2.09 , 3.39 , 7.20 , 7.89–7.92 , 8.08 , 8.27–8.31 . MS (ESI) m/z [M + H]+ 304,1055, calc. m/z 304.1040, m/z [M + Na]+ 326.0875, calc. m/z 326.0860.5-Amino-N,3-dimethyl-N′-(3-nitrophenyl)methylidene-1,2-oxazole-4-carbohydrazide (MM10). Anal. C13H13N5O4 (m.w. 303.273 g/mol); m.p. 209–210 °C; yield 77%; FTIR  vmax/cm−1: 1638 (C=O), 3438 (NH2). 1H NMR (DMSO-d6) δ (ppm): 2.10 , 3.34 , 7.19 , 7.69–7.75 , 8.08–8.12 , 8.19–8.23 , 8.53 . MS (ESI) m/z [M + H]+ 304.1059, calc. m/z 304.1040, m/z [M + Na]+ 326.0876, calc. m/z 326.0860.Additional spectroscopic data is available in the Escherichia coli 0111:B4 (LPS), phytohemagglutinin (PHA), dimethyl sulfoxide (DMSO), and MTT  and all other reagents were from Sigma-Aldrich . Fetal calf serum (FCS), RPMI-1640, and Hanks’ medium were purchased from Biowest . Lipopolysaccharide from The compounds were dissolved in DMSO to a concentration of 10 mM and kept at 4 °C. Then, the compound solutions were incubated at 37 °C for 10 min with vigorous shaking and subsequently diluted in the culture medium to the respective concentrations used in the cell cultures.6 mL in a culture medium, referred to below as “the culture medium”, consisting of RPMI-1640 with the addition of 10% fetal calf serum (FCS), 100 U/mL of penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine. The compounds were tested at a concentration range of 100–12.5 µM and the MM3 compound at 250–1.9 µM. The test was performed in 96-flat bottom plates containing 2 × 105 cells incubated initially for 24 h in a cell culture incubator. Then, the supernatants were removed and to the cells, appropriate dilutions of the compounds (0.2 mL) were added in a cell culture medium containing 2% FCS. The cultures were incubated for 72 h followed by the determination of cell viability by a colorimetric MTT method. In parallel, control cultures containing appropriate dilutions of the solvent (DMSO) were also incubated. The results were presented as mean optical density (OD) values from four wells ± standard deviation.The cytotoxicity of the compounds was determined by measuring growth of human lung epithelial A549 cells (ATCC CCL 185). The cells were suspended in a density of 2 × 10g for 20 min at 4 °C. The interphase cells were then washed three times with Hanks’ medium and re-suspended in the culture medium at a density of 2 × 106 cells/mL.Venous blood from a single donor was withdrawn into heparinized syringes and diluted twice with phosphate buffered saline (PBS). PBMCs were isolated by centrifugation on a Ficoll-Uropoline gradient (density 1.077 g/mL) at 800× 5 cells/well). PHA was added at a concentration of 5 µg/mL. The compounds were tested at the following doses: 100, 50, and 25 uM. DMSO at appropriate dilutions served as the control. After three days of incubation in a cell culture incubator, the proliferative response of the cells was determined by colorimetric MTT [PBMC were distributed into 96-well flat-bottom plates in 100 µL aliquots , and the studied compound was added at concentrations of 50, 25, 12.5, and 6.15 µM. The control cultures contained DMSO in appropriate concentrations. After an overnight incubation, the supernatants were harvested and frozen at −80 °C until cytokine determination. TNF α concentrations were determined in the supernatants by using an ELISA kit from eBioscience in a presence of TNF α standard and originally expressed in pg/mL. The assay was performed according to . Briefly5/mL) in the culture medium were cultured overnight with the MM3 compound (50 μM) Total RNA isolation was carried with TRIzol Reagent (Ambion) according to the manufacturer’s recommendations. The cell pellet (2 × 106 cells) was suspended in 1 mL of TRIzol reagent, shaken, incubated for 10 min at room temperature (RT), supplemented with 0.2 mL of chloroform, shaken vigorously for 15 s, incubated for 3 min at RT, and centrifuged at 12,000× g for 15 min at 4 °C. The water phase was collected, transferred to a new tube, supplemented with 0.5 mL of isopropanol, incubated at RT for 10 min, and centrifuged at 12,000× g for 10 min at 4 °C. The RNA pellet was washed with 1 mL of 75% ethanol, dried in air, and dissolved in 20–30 μL of sterile diethylpyrocarbonate-treated Mili-Q water. RNA samples were stored at −20 °C. Jurkat cells (10Single stranded complementary DNA (cDNA) was synthesized with oligo (dT) 12–18 primers from 5 μg of total RNA using Novazym VerteKit, according to the manufacturer’s instruction.The expression of the genes  were measured using an APA SYBR FAST qPCR Kit. The sequences of primers are enclosed in the p < 0.05. Statistical analysis was performed using STATISTICA 6.1 for Windows.The results are presented as mean values ± standard deviation (SD). The Brown–Forsythe test was used to determine the homogeneity of the variance between the groups. When the variance was homogenous, analysis of the variance (one-way ANOVA) was applied, followed by post hoc comparisons with the Tukey test to estimate the significance of the difference between the groups. Nonparametric data were evaluated with the Kruskal–Wallis analysis of variance. The significance was determined at


Optical resolution of 2-methyl-2-nitrobut-3-en-1-ol has been accomplished using a “low-temperature lipase-catalyzed transesterification” carried out at −40 °C. The mixture was extracted with ethyl acetate and the combined organic layer was washed with brine (3 times) and dried over NaSO4; evaporation and silica gel flash column chromatography (hexane: ethyl acetate = 10:1 to 5:1) then afforded (±)-1  in 76% yield. 1H-NMR : 6.17–6.11 , 5.49 , 5.43 , 4.12–4.09 , 3.82–3.80 , 2.54  1.73 ; 13C-NMR  134.2, 119.0, 92.0, 67.5, 19.5; IR  3415, 3098, 2997, 2881, 2946, 1732, 1545, 1461, 1419, 1380, 1349, 1059, 944.Nitroethane  was reacted with acetaldehyde  in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)  at 0 °C and the mixture was stirred for 2 h at rt. The reaction was quenched by addition of 10 mL of 1 M HCl aqueous solution and 100 mL of diethyl ether, then the organic layer was washed with 1 M HCl (3 times) and brine (3 times) and dried over MgSO1 Dl3), lit  −14.0 (C(R)-benzyl (2-methyl-2-nitrobut-3-en-1-yl) carbonate ((R)-6): DMAP  and iPr2NEt  were added to a solution of (R)-1  in CH2Cl2 (30 mL). A solution of cbz-Cl  in CH2Cl2 (10 mL) was added to the solution dropwise. The mixture was stirred at room temperature for 48 h, then poured into 1 M HCl (20 mL). The organic layer was separated and the water layer was extracted with CH2Cl2 (3 × 30 mL). The organic phases were combined and dried over Na2SO4. After filtration, the filtrate was concentrated and the residue was purified by flash chromatography (hexane-EtOAc 3:1) to give (R)-6 in 95% yield (5.05 g) Oil. The enantiomeric excess of (R)-6 was determined by HPLC analysis, tR 23.9 min ((R)-6), tR 27.0 min ((S)-6)  as 96% ee: [α]D +24.8 ; 1H-NMR  7.39–7.34 , 6.11 , 5.46 , 5.46 , 5.16 , 4.69 , 4.42 , 1.75 ; 13C-NMR  154.43, 134.79, 133.54, 128.87, 128.77, 128.55, 120.00, 89.18, 70.52, 70.35, 19.61; Anal. Calcd. for C13H15NO5: C, 58.86; H, 5.70; N, 5.28. Found: C, 58.75; H, 5.77; N, 5.26.-benzyl (4-(4-(heptyloxy)phenyl)-2-methyl-2-nitrobut-3-en-1-yl) carbonate ((R)-8): A solution of (R)-6  in dry CH3CN (4 mL) was purged by N2 and NaOAc , Pd2dba3  and p-C7H15OC6H4N2BF4 (7) [R)-8 in 63% yield (216.0 mg). [α]D +80.2 . 1H-NMR  7.41–7.35 , 7.32 , 6.86 , 6.70 , 6.24 , 5.17 , 4.80 , 4.48 , 3.96 , 1.85 , 1.82–1.75 , 1.51–1.25 , 0.90 ; 13C-NMR  160.10, 154.54, 134.27, 128.86, 128.78, 128.56, 128.48, 121.48, 114.83, 89.30, 70.90, 70.30, 68.16, 31.80, 29.22, 29.08, 26.00, 22.63, 19.89, 14.12; HRMS (ESI M+H) m/z 456.2404. Calcd for C26H34NO5 456.2386.2BF4 (7)  (464.0 mPreparation of (R)-2-amino-2-methyl-4-(4-(heptyloxy)phenyl)butan-1-ol ((R)-5): (R)-8  was dissolved in MeOH (3 mL) and Pd-C  was added. The mixture was placed autoclave and stirred at room temperature under hydrogen atomosphere at 5 MPa for 50 h. After filtration, the filtrate was concentrated to give (R)-5 (130 mg) in 97% yield. Absolute configuration of (R)-5 was confirmed by comparison with the sign of specific rotation value of that reported: , lit 26D −16.5 , >99% ee.To a mixture of (S)-1  and vinyl acetate  in i-Pr2O (120 mL) of was added lipase QLM powder (1.70 g) and the mixture was stirred at −40 °C. After being stirred for 24 h, the reaction mixture was filtered through a glass sintered filter with a Celite pad to remove the enzyme. The filtrate was evaporated and chromatographed on a silica gel flash column (hexane: ethyl acetate = 10:1 to 5:1) to give (S)-2  and (S)-1 : (S)-1: [α]26D +76.8 , 94% ee. (S)-2 was converted to (S)-1 (18% ee) by acid hydrolysis and the resulting (S)-1 (2.34 g) was combined with (R)-1 (1.05 g), which was obtained by the upper route in ee substrate alcohol 1 ), then used for the next cycle of the lipase-catalyzed reaction.To a mixture of (1) with over 94% ee using lipase-catalyzed transesterification under low temperature reaction conditions. It was possible to apply the reaction protocol to the multi gram scale preparation and we succeeded in preparing the desired compounds easily. Synthetic application of a medicinal compound using optically active 1 is now ongoing in our laboratory.In summary, we established a convenient protocol to prepare both enantiomers of 2-methyl-2-nitrobut-3-en-1-ol (


S,12S)-4-methyl-11,12-epoxy-2-hydroxy-3-oxoursa-1,4-dine-28-oic acid γ-lactone (1), named ulmoidol A, together with ten known compounds: ulmoidol (2), corosolic acid (3), 2α,3α-dihydroxy-24-nor-4(23),12-oleanadien-28-oic acid (4), oleanolic acid (5), ursolic acid (6), cycloart-3β, 25-diol (7), foliasalacioside B1 (8), -9-hydroxy-4,7-megastigmadien-3-one-9-O-α-L-arabinopyranosyl-(1→6)-β-D-glucopyranoside (9), -9-hydroxy-4,7-megastigma-dien-3-one-9-O-β-D-xylopyranosyl-(1→6)-β-D-glucopyranoside (10), and quercetin 3-O-sambubioside (11) were isolated from the leaves of Eucommia ulmoides Oliv. The structure of compound 1 was determined by extensive spectroscopic analysis, and its absolute configuration was determined by CD experiments and a computational method. Compounds 3, 4, 7–10 were isolated from this plant for the first time. Compounds 3 and 4 showed inhibition to PTPIB activities, with IC50 values of 0.69 and 3.98 μM, respectively.A new ursane-type nortriterpenoid, (11 Eucommia ulmoides Oliv. mainly grows along the Yangtze River and in southern China. Its bark has traditionally been applied in China as an antihypertensive, diuretic, sedative, tonic and nourishing agent +, 467 [M+H]+; HRESIMS m/z: 467.2804 [M+H]+ . For 1H and 13C-NMR spectroscopic data, see E. coli and purified by GST affinity chromatography. The reagent pNPP was used as substrate for the measurement of PTP1B activity. Compounds were pre-incubated with the enzyme at room temperature for 5 min. Assay was performed in final volume of 100 μL in the active system containing 50 Mm HEPES, 5 mM DTT, 150 mM NaCl, 2 mM EDTA, and 2 mM pNPP (pH 7.0), incubated at 30 °C for 10 min, stopped by addition of 50 μl 3 M NaOH. Then, the absorbance was determined at 405 nm wavelength. The similar system without GST-PTP1B protein was used as blank. The effects of compounds 1–6, 8–10 on PTP1B activity were measured, and the IC50 value was calculated by nonlinear regression.Recombinant human GST-PTP1B protein was overexpressed by hGST-PTP1B-BL21 1), together with ten known compounds were isolated from the leaves of Eucommia ulmoides Oliv. The structure of compound 1 was determined by extensive spectroscopic analysis, and the absolute configuration was determined by CD experiments and computational methods. Compounds 3, 4, 7–10 were isolated from this plant for the first time. Compounds 1–6, and 8–10 were tested for inhibition of PTP1B activities, and compounds 3 and 4 showed inhibit activities with IC50 values of 0.69 and 3.98 μM, respectively.A new ursane-type nortriterpenoid, ulmoidol A (


Structures of 1–3 were determined by analysis of the spectroscopic data as well as chemical transformations. As a result, the structure of anthenoside A has been revised and the structures of 1 and 2 were established. Glycosides 1–3 contain a 2-acetamido-2-deoxy-4-O-methyl-β-d-glucopyranosyl residue, found in the starfish steroidal glycosides for the first time. All the isolated compounds slightly inhibited cell viability of human cancer T-47D cells and did not show cytotoxic effects against RPMI-7951 cells. Glycoside 1 slightly inhibited colony formation of human cancer RPMI-7951 cells by 16% while compound 2 decreased the number of colonies of T-47D cells by 40%.Two new polyhydroxysteroidal glycosides, anthenosides A A series of steroidal glycosides containing β-d-galactofuranosyl, 6-O-methyl-β-d-galactofuranosyl, 3-O-methyl-β-d-galactofuranosyl, 3-O-methyl-β-d-glucopyranosyl and 4-O-methyl-β-d-glucopyranosyl residues was isolated from the starfishes of the genus Anthenea + sodium adduct ion peak at m/z 686.4238 in the (+)HRESIMS , a 8(14) double bond , three oxygenated methines HC-3 , HC-6 , HC-7 , and one oxygenated HC-16 , the latter carbon being linked to a monosaccharide residue through an ether function (1 was determined as Δ8(14)-3α,6β,7β,16α-tetrahydroxysteroidal moiety glycosylated at C-16 position which is characteristic of starfish steroidal glycosides of the genus Anthenea , CH3-26 , CH3-27  and a 24(28) double bond − at m/z 662 contained the fragment ion peaks at m/z 258 [С9H17NO6 + Na]+ and 234 [С9H16NO6]−, respectively, which together with the NMR spectroscopic data confirmed the presence of a 2-acetamido-2-deoxy-O-methyl-hexose unit in 1. The chemical shifts and coupling constants of H-1′ − H-6′ of this monosaccharide unit were determined by irradiation of anomeric proton in a 1D TOCSY experiment. The 1H-1H COSY and HSQC correlations attributable to monosaccharide unit revealed the corresponding sequences of protons at C-1′ − C-6′ -16-O-(2-acetamido-2-deoxy-4-O-methyl-β-d-glucopyranosyl)-24-methyl-5α-cholesta-8(14),24(28)-diene-3α,6β,7β,16α-tetraol (1).The  δC 60.9 . The (+)′ − C-6′ . The key residue . The couobserved . Based o2 was determined to be C37H61NO9, the same as in the glycoside 1, from the [M + Na]+ sodium adduct ion peak at m/z 686.423941 in the (+)HRESIMS ; δC 24.4], CH3-26 , CH3-27 , CH3-28 , and a 22(23) double bond  -double bond followed from a J22,23 of 15.4 Hz -16-O-(2-acetamido-2-deoxy-4-O-methyl-β-d-glucopyranosyl)-24-methyl-5α-cholesta-8(14),22(23)-diene-3α,6β,7β,16α-tetraol (2).The molecular formula of compound )HRESIMS . The thoNMR data  of compoe chains . The NMR, 134.9] . The proeriments . The HMB 15.4 Hz . Thus, t1H- and 13C-NMR, and MS spectral data of steroidal glycoside 3 coincided with those reported earlier for anthenoside A  is on deposit at the marine specimen collection of the G.B. Elyakov Pacific Institute of Bioorganic Chemistry of the FEB RAS, Vladivostok, Russia.Specimens of A. aspera  were chopped into small pieces and extracted thrice with EtOH. The H2O/EtOH layer was evaporated, and the residue was dissolved in H2O (1.0 L). The H2O-soluble material was passed through a Polychrom-1 column (7 × 26.5 cm), eluted with distilled H2O (4.0 L) until a negative chloride ion reaction was obtained, and then eluted with EtOH (3.5 L). The combined EtOH eluate was evaporated to give a reddish residue (7.0 g). This material was chromatographed over a Si gel column (6 × 18.5 cm) using CHCl3/EtOH  to yield eight fractions, 1–8, which were then analyzed by TLC in the eluent system BuOH/EtOH/H2O . Fractions 1–6 mainly contained the polyhydroxysteroids and related glycosides and admixtures of pigments and concomitant lipids. HPLC separation of fraction 5 (212 mg) on a Diasorb-130-C16T column (2.5 mL/min) with EtOH/H2O  as an eluent system yielded pure 3  and sub-fractions 5.6 (4.0 mg) and 5.7 (13.0 mg), which were additionally submitted to a purification on Discovery C18 analytical column (1.0 mL/min) with EtOH/H2O  as an eluent system to give pure 1  and sub-fraction 5.72 (8.5 mg). HPLC separation of sub-fraction 5.72 on a Diasorb-130 Si gel analytical column (1.0 mL/min) with EtOAc/EtOH  as an eluent system yielded pure 2 .The fresh animals of 1Anthenoside A  (1): Amorphous powder; [α]c 0.1, MeOH); IR (KBr) νmax 3550, 3478, 3417, 2962, 2924, 2852, 1737, 1637, 1618, 1467, 1262, 1098, 1032, 801 cm−1; HRESIMS m/z 686.4238 [M + Na]+ ; ESIMS/MS of the ion at m/z 686: m/z 258 [С9H17NO6 + Na]+; ESIMS/MS of the ion at m/z 662: m/z 234 [С9H16NO6]−; 1H- and 13C-NMR data, see 2Anthenoside A  (2): Amorphous powder; [α]c 0.15, MeOH); IR (KBr) νmax 3550, 3478, 3417, 2962, 2924, 2858, 1735, 1637, 1618, 1467, 1262, 1098, 1033, 801 cm−1; HRESIMS m/z 686.4239 [M + Na]+ ; ESIMS/MS of the ion at m/z 686: m/z 258 [С9H17NO6 + Na]+; ESIMS/MS of the ion at m/z 662: m/z 234 [С9H16NO6]−; 1H- and 13C-NMR data, see Anthenoside A  (3): Amorphous powder; [α]D: –48.3 . 1H-NMR : δH 4.55 , 4.51 , 4.26 , 4.08 , 3.83 , 3.67 , 3.62 , 3.55 , 3.53 , 3.52 , 3.24 , 3.05 , 2.90 , 2.34 , 2.24 , 2.17 , 1.99 , 1.96 , 1.82 , 1.76 , 1.67 , 1.65 , 1.62 , 1.61 , 1.59 , 1.53 , 1.45 , 1.37 , 1.36 , 1.35 , 1.32 , 1.31 , 1.27 , 1.06 , 1.04 , 1.00 , 0.92 , 0.89 , 0.88 , 0.85 , 0.83 ; 13C-NMR : δC 173.8 (CH3-C=O), 147.4 (C-14), 128.0 (C-8), 100.9 (C-1′), 82.0 (C-4′), 79.7 (C-16), 77.3 (C-6 and C-5′), 76.2 (C-3′), 72.2 (C-7), 67.4 (C-3), 63.0 (C-6′), 62.7 (C-17), 60.9 (4′-OCH3), 58.4 (C-2′), 47.5 (C-24), 45.6 (C-9), 45.0 (C-13), 38.6 (C-10), 37.7 (C-5), 37.2 (C-12), 34.5 (C-1), 34.3 (C-15), 33.8 (C-20), 33.4 (C-4), 33.3 (C-22), 30.2 (C-25), 29.5 (C-2 and C-23), 24.1 (C-28), 23.2 (CH3-CO), 21.2 (C-21), 20.2 (C-18), 20.0 (C-26), 19.5 (C-11 and C-27), 15.4 (C-19), 12.7 (C-29); (+)-HRESIMS: m/z 702.4549 [M + Na]+ ; ESIMS/MS of the ion at m/z 702: m/z 258 [С9H17NO6 + Na]+.3 (1.5 mg) was carried out in a solution of 2 M trifluoroacetic acid  (0.75 mL) in a sealed vial on a H2O bath at 100 °C for 2 h. The H2O layer was washed with CHCl3 (3 × 1 mL) and concentrated in vacuo. One drop of concentrated TFA and 0.5 mL of (R)-(–)-2-octanol  were added to the sugar mixture, and the sealed vial was heated in a glycerol bath at 130 °C for 6 h. The solution was evaporated in vacuo and treated with a mixture of pyridine-acetic anhydride  for 24 h at room temperature. The acetylated 2-octylglycosides were analyzed by GC using the corresponding authentic samples prepared by the same procedure. The following peaks for monosaccharides were detected in the hydrolyzate of 3: 2-acetamido-2-deoxy-4-O-methyl-d-glucopyranose (Rt = 16.21 and 20.08 min). The retention times of the authentic samples were as follows: 2-acetamido-2-deoxy-4-O-methyl-d-glucopyranose (Rt = 16.20 and 20.09 min), and 2-acetamido-2-deoxy-4-O-methyl-β-l-glucopyranose (Rt = 16.07 and 20.00 min).Acid hydrolysis of O-methyl-α-d-glucopyranose was synthesized as described previously [The sample of 2-acetamido-2-deoxy-4-eviously .MTS -5-(3-carboxymethoxyphenyl)-2H-tetrazolium) assay kit was purchased from “Promega” .l-glutamine, gentamicin solution, trypsin, fetal bovine serum (FBS), sodium hydrocarbonate (NaHCO3), and agar were purchased from “Sigma” and “Gibco” . All other common chemicals, solvents and reagents were of the highest grade available from various commercial sources.The Basal Medium Eagle (BME), Minimum Essential medium (MEM), Roswell Park Memorial Institute medium (RPMI 1640), phosphate buffered saline (PBS), TM), and human breast cancer cells T-47D (ATCC # HTB-133TM) were obtained from the American Type Culture Collection .Human melanoma RPMI-7951 cells  were seeded in 96-well plates for 24 h at 37 °C in a 5% CO2 incubator. The attached cells were fed with fresh medium containing various concentrations of compounds 1–3 from (0–150 µM) for an additional 24 h. After, the cytotoxicity of 1–3 was measured using an MTS assay kit according to the manufacturer’s instructions. All the experiments were performed in triplicate, and the mean absorbance values were calculated. IC50 is the concentration of compounds that caused a 50% reduction of cell viability of human cancer cells after 48 h of treatment.To determine cytotoxicity, RPMI-7951 and T-47D cells (1 × 103 cells/mL) were treated with/without cisplatin (1 μM) and compounds 1–3 (50 µM) in 1 mL of 0.3% Basal Medium Eagle (BME) agar containing 10% FBS, 2 mM l-glutamine, and 25 µg/mL gentamicin. The cultures were maintained at 37 °C in a 5% CO2 incubator for 14 days, and the cell’s colonies were scored using a microscope «Motic AE 20» (China) and the Motic Image Plus computer program.RPMI-7951 and T-47D cells . A Student’s 1 (1) and A2 (2), and one previously known steroidal glycoside anthenoside A (3) were isolated from the alcoholic extract of the tropical starfish Anthenea aspera. Chemical structures of 1 and 2 were elucidated by extensive NMR and ESIMS techniques as well as by chemical transformations. The structure of the known glycoside 3 was revised by NMR spectroscopy and GC analysis of acetylated 2-octylglycosides. Glycosides 1–3 contain a new monosaccharide residue, 2-acetamido-2-deoxy-4-O-methyl-β-d-glucopyranose, never found before in the starfish polar steroidal compounds. It is worth noting that amino sugars are atypical monosaccharide residues in starfish steroidal glycosides but often found in the steroidal glycosides of sponges. Steroidal glycosides 1–3 slightly inhibited cell viability of breast cancer T-47D cells but they did not have cytotoxic effects against human melanoma RPMI-7951 cells. It was shown that only compound 1 slightly inhibited colony formation of RPMI-7951 cancer cells by 16%. On the other hand, the new glycoside 2 decreased the number of colonies of T-47D cells by 40%.Two new polyhydroxysteroidal glycosides, anthenosides A


O-α-d-manopyranosyl-(1 → 3)-α-l-rhamnopyranoside (compound 1) and 4-O-α-l-rhamnopyran-osyl-(1 → 6)-α-d-manopyranosyl-(1 → 3)-α-l-rhamnopyranoside (compound 2), and seven known compounds, compound 3, 6, 7 (acid components), compound 8, 9 (flavonoids), compound 4 (a coumarin) and compound 5 , were isolated from the 70% ethanol aqueous extract of the aerial parts of Melilotus officinalis (Linn.) Pall. The structures of all compounds were elucidated by use of extensive spectroscopic methods Infrared Spectroscopy (IR), High resolution electrospray ionization mass spectrometry (HR-ESI-MS), and 1H and 13C-NMR). Sugar residues obtained after acid hydrolysis were identified by high-performance liquid chromatography (HPLC). The antioxidant activity of all the compounds was evaluated by 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+) and 1,1-diphenyl-2-picrylhydrazyl (DPPH). The anti-inflammatory effects of the compounds were also evaluated in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. All compounds were shown to inhibit LPS-induced nitric oxide (NO) and prostaglandin E 2 (PGE 2) production by suppressing the expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2), respectively, in LPS-stimulated RAW 264.7 cells. The inhibitory effect of all the compounds on MCF-7 cells was determined by Cell Counting Kit-8 (CCK-8) method. The results showed that compounds 1, 2, 7, 8, 9 exhibited better antioxidant activity compared to the other compounds. compounds 1–9 had different inhibitory effects on the release of NO, TNF-α and IL-6 in LPS-stimulated RAW264.7 cells by LPS, of which compound 7 was the most effective against inflammatory factors. compounds 1 and 2 have better antitumor activity compared to other compounds. Further research to elucidate the chemical composition and pharmacological effects of Melilotus officinalis (Linn.) Pall is of major importance towards the development and foundation of clinical application of the species.Two new p-hydroxybenzoic acid glycosides, namely p-hydroxybenzoic acid-4- Melilotus officinalis (Linn.) Pall belongs to the genus Melilotus of Fabaceae family, and is an annual herb. It is also known as yellow sweet clover. It was first published in the “European Pharmacopoeia” eighth edition + at m/z 615.1807. In the NMR spectra  and 6.69 , and seven carbon signals at δC 121.1, 130.69, 130.7, 116.0, 116.0, 161.2, 175.8 showed the existence of the same aglycone (p-hydroxybenzoic acid) in the structure of compound 2 as in 13C-NMR spectrum of compound 2 showed three six-carbon units. Acid hydrolysis of compound 2 gave rhamnose and mannose which identified by TLC comparing with authentic samples. In comparison with the NMR data of compound 1, two of three six-carbon units were coincident with the sugars moiety of compound 1, except for carbon signal at δC 65.6 showing a significant downfield shift (Δδ = 5.9) than C-6 signal of d-mannosyl group of compound 1, which point to the existence of p-hydroxybenzoic acid-4-O-α-d-mannopyranosyl-(1 → 3)-α-l-rhamnopyranosyl moiety in the structure of compound 2, the 13C-NMR data  of the remainder six-carbon unit was coincident with these of methyl-O-α-l-rhamnoside reported + .White amorphous powder (Methanol); m.p. 214.0–216.5 °C; IR (KBr) νmax (cm−1): 3363, 1677, 1605, 1588, 1481, 1423, 1308, 1282, 1153, 1133, 1030, 854; 1H  and 13C-NMR  spectral data, see m/z 615.1807 [M + Na]+ .White amorphous powder(Methanol); m.p. 217.2–219.0 °C; IR (KBr) 7H6O3, m.p. 252~254 °C. 1H-NMRδ: 7.83 , 7.41 , 6.89 , 6.82 .White solid powder. The molecular formula was C9H6O2, m.p. 68~70 °C. EI-MS m/z: 146 [M+], 118 [M+-CO], 90; 1H-NMR δ: 6.43 , 7.48 , 7.52 , 7.71 .Colorless column crystal. The molecular formula was C5 included: White crystals, the formula was C5H11NO2, m.p. 301~305 °C. IRνKBrmax (cm−1): 3023, 2985, 1621, 1492, 1471, 1422, 1395, 1339, 1238, 1120, 982, 930, 870, 720, 603; MS m/z: 117 [M]+. 1H-NMRδ: 3.28 , 3.80 ; 13C-NMRδ: 97.0 (-N-CH3), 108.2 (-N-CH2), 210.2 (C=O).Characterizations of compound 4H4O4, m.p.: 296.8~299.2 °C. 1H-NMRδ: 13.10 , 6.64 ; 13C-NMRδ: 166.4 (C-1), 134.4 (C-2), 134.4 (C-3), 166.4 (C-4).Yellow block crystals, the formula was C1H-NMRδ: 7.51 , 7.03 , 6.92 , 6.72 , 6.22 . 13C-NMRδ: 171.6 (C-9), 149.4 (C-7), 148.6 (C-3), 145.4 (C-4), 128.3 (C-1), 126.5 (C-6), 115.1 , 113.7 (C-8).Light yellow powder, m.p. 199.1~201.4 °C. m/z: 286 [M+], 153, 134. 1H-NMRδ: 6.23 , 6.51 , 6.56 , 6.97 , 7.43 , 7.46 . Yellow needle crystal, m.p. 235~238 °C. EI-MS m/z: 301 [M−H]−; 1H-NMRδ:δ6.17 , 6.37 , 6.87 , 7.62 , 7.73 .ESIMS (-ve) 1 or 2) (5 mg) in a mixture of 1:2 (v/v) 1M H2SO4–MeOH (20 mL), was heated under reflux for 3 h in a water bath at 80 °C. The reaction mixture was evaporated to dryness in vacuo, dissolved in H2O (5 mL), and neutralized with NaOH. Then, the resulting samples were analyzed using high-performance liquid chromatography (HPLC) coupled with an ELSD detector according to the method of Yang [v/v) mixtures of water and acetonitrile (ACN) was used.A solution of each Compound  were added to 2 mL ATBS+ solution, respectively. The mixture, protected from light, was reacted for 30 min. The decrease of absorbance was monitored at 734 nm. The control was 0.2 mL of distilled water and 2 mL of ATBS+ solution. The same method was used in Vitamin C (Vc). The methods for determining DPPH· free radical scavenging activity was as follows. About 0.2 mL tested compounds at various concentrations  were added to 2 mL DPPH· solution (200 µM ethanol solution), respectively. The mixture, protected from light, was reacted for 30 min. The decrease of absorbance was monitored at 517 nm. The control was the DPPH· solution. The same method was used in Vc. The half maximal inhibitory concentration (IC50) was used to evaluated the ATBS+ free radical scavenging activity and the DPPH· free radical scavenging activity.The methods for determining ATBSl-glutamine (2 mM). The cells were grown in a humidified atmosphere containing 5% CO2 at 37 °C. RAW264.7 cells were seeded in 96-well plates at a density of 8 × 104 cells/well for 24 h. The cells were randomly divided into control group, LPS (1 μg/mL) group, LPS (1 μg/mL) + compound 1–9 (50 μg/mL) group. After adding the corresponding drug, the supernatant was used to detect NO, TNF-α and IL-6 after culturing for 24 h at 5% CO2 and 37 °C under saturated humidity.The rat macrophage RAW264.7 cell line was maintained in dulbecco’s modified eagle medium (DMEM) supplemented with 10% heat inactivated fatal bovine serun (FBS), penicillin G (100 units/mL), streptomycin (100 mg/mL) and 2, respectively. The cells of logarithmic growth phase were seeded into 96-well plates with density of 1 × 104 cells/well in 100 μL medium, respectively. The cells were treated with the tested compounds at various concentrations  and 5-FU as positive control, each of two parallel holes are located, then incubated for 72 h. Subsequently, remove the 96 well plate, add 10 μL of CCK-8; meanwhile, two separate holes for the blank control, only added to each well with 10 μL CCK-8 in DMEM 0.1 mL. Then incubate under the same conditions for 4 h. The optical density (OD) was measured at 490 nm using a Bio-red 550 . Reference wavelength was 620 nm. The experiment was repeated 3 times. Calculation of the impact of drugs on cell growth inhibition rate and IC50 values is performed with the following equation:0 is the OD value of the control wells, and D1 is the OD value of the samples wells.The cytotoxicity assay was carried out using CCK-8 method. MCF-7 and PC-3M cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 and DMEM at 37 °C in 5% CO


Maytenus robusta (Celastraceae) were subjected to phytochemical investigation mainly directed at the isolation of pentacyclic triterpenes. The compounds friedelin (1), β-friedelinol (2), 3-oxo-21β-H-hop-22(29)-ene (7), 3,4-seco-friedelan-3,11β-olide (8), 3β-hydroxy-21β-H-hop-22(29)-ene (9), 3,4-seco-21β-H-hop-22(29)-en-3-oic acid (10), 3,4-seco-friedelan-3-oic acid (11), and sitosterol were identified in the hexane extract of M. robusta leaves. Compounds 8 and 9 are described herein for the first time. The structure and stereochemistry of both compounds were experimentally established by IR, HRLC-MS, and 1D  and 2D  NMR data and supported by correlations with carbon chemical shifts calculated using the DFT method (BLYP/6-31G* level). Compounds 7 and 10 are also described for the first time, and their chemical structures were established by comparison with NMR data of similar structures described in the literature and correlations with BLYP/6-31G* calculated carbon chemical shifts. Compound 9, a mixture of 11 and sitosterol, and 3β,11β-dihydroxyfriedelane (4) were evaluated by the Ellman’s method and all these compounds showed acethylcholinesterase inhibitory properties.Leaves of  Secondary metabolites are isolated from plants and animals, and many of them have been used as sources of derivatives with a large spectrum of biological activities , includiMaytenus (Celastraceae) +, calc. 443.3922).3β-Hydroxy-21β-H-hop-22(29)-ene (9): white solid (103.0 mg); m.p. 217–221 °C; [α]2°D= +46 ; IR  ν 3488, 2931, 2870, 1640, 1445, 1372, 896, and 886; 1H-NMR  δH 4.79 , 3.23 , 2.67 , 1.97 (H-20β), 1.84 (H-20α), 1.75 (H-30), 1.74 (H-16α), 1.70 (H-1β), 1.65 (H-16β), 1.63 (H-2α and H-2β), 1.62 (H-7β), 1.60 (H-19α), 1.53 (H-6β), 1.51 (H-11α), 1.49 (H-12β), 1.47 (H-7α), 1.43 (H-12α), 1.42 (H-15α), 1.40 (H-6α), 1.39 (H-17β), 1.37 (H-13β), 1.32 (H-11β), 1.24 (H-9α and H-15β), 1.04 (H-19β), 1.02 (H-23), 0.97 (H-27), 0.94 (H-1α and H-26), 0.83 (H-25), 0.81 (H-24), 0.73 (H-28), and 0.69 (H-5α); the 13C-NMR data of 9 are shown in m/z 409.3855 [M+H−18]+, calc. 409.3834). 3,4-seco-21β-H-Hop-22(29)-en-3-oic acid (10): white solid obtained as a mixture with 11 (59.5 mg); 1H-NMR  δH 4.78 , 2.67 , 2.38 , 1.75–0.73 . The 13C-NMR data of 10 are shown in 3,4-seco-Friedelan-3-oic acid (11) [10 (59.5 mg); 1H-NMR  δH 2.38  and 1.75–0.73 . The 13C-NMR data of 11 are shown in cid (11) : white sE) of the optimized geometries was given in atomic unit (Hartree). This theoretical methodology has been efficiently employed in the study of different organic compounds, including terpenes [Theoretical studies were carried out using the Gaussian 03 software package . The geoterpenes ,36,37,38σC) were determined in relation to the corresponding calculated value for tetramethylsilane (σC 187.97). Correlations between σC values and experimental carbon chemical shifts (δC) were obtained using software package Origin™ Standard 7.5. The σC and δC values were plotted on the x and y axes, respectively. The σC/δC correlation curves were given as linear fits with correlation coefficients (R2) and slope of the R2 curve (α) furnished by the program. The BLYP/6-31G* calculations usually give satisfactory results of carbon chemical shifts, as have been obtained in previous works [The optimized geometries were used to calculate carbon chemical shifts at the same levels of theory. Values of calculated carbon chemical shift , B , and C  were prepared to study the in vitro AChE inhibitory activity. This activity was measured using a 96-well microplate reader based on an adapted Ellman’s method [The buffers A , DTNB , buffer B (50 μL), and sample  were added into each well of a 96-well microplate. Instead of adding the sample solution, a volume of 25 μL of buffer C was employed to prepare the blank sample. The positive control was prepared under the same conditions, using physostigmine (eserine) as standard. Tests were carried out in quintuplicate. The absorbance was measured at 405 nm every 60 s by eight times using a Elisa Thermoplate microplate reader. After addition of 25 μL of acetylcholinesterase solution (0.226 U/mL in buffer B), the absorbance was again read every 60 s for ten times. The increase in absorbance relative to substrate spontaneous hydrolysis was corrected by reaction rate variation before and after addition of the enzyme. The inhibition percentage was calculated by comparing the rates of the sample with the blank.M. robusta provided seven triterpenes. The triterpenes 1, 2, and 11 were also isolated in a previous phytochemical investigation. The triterpenes 8 and 9 are described for the first time in the literature. The triterpenes 7 and 10 are also new compounds, but both compounds were obtained as a mixture. Hopane and seco-hopane triterpenoids are not usual in species of the family Celastraceae. The combination of experimental NMR analyses with carbon chemical shift calculations was a useful procedure for the structural determination of these hopane and friedelane triterpenes. Compounds 4, 9, and the mixture of 11 and sitosterol showed acetylcholinesterase inhibitory properties. These compounds present hopane- and friedelane-type skeletons, suggesting biological potential of their derivatives for Alzheimer’s desease.The hexane extract of the leaves of


O-tosylation protocol is observed. The acceleration of the transformation by the piperazine unit is demonstrated. It is found that the reaction goes via the corresponding O-tosylate, which converts spontaneously into chloride with different rate depending on the substrate structure. In the case of pirlindole derivative, partially aromatized chloride formation was observed upon prolongation and/or increased excess of tosyl chloride.A direct conversion of piperazinyl ethanols into chlorides via a classical  The reaction mixture was poured into water. The products were partitioned between water and ethyl acetate. The organic phase was dried over MgSO4 and evaporated to dryness. Flash chromatography purification on silica gel by using a mobile phase with a gradient of polarity from DCM to 2% acetone in DCM gave:4c: colourless solid; recrystallization from heptane: m.p. 57.6–58.2°C (lit. [Rf 0.26 (1% acetone/DCM), Rf 0.58 (5% acetone/DCM); 1H NMR 2.653 , 2.766 , 3.188 , 3.600 , 6.847 , 6.908 , 7.248 ; 13C NMR 40.89 (CH2-Cl), 49.07 (CH2-3 + CH2-5 piperazine), 53.16 (CH2-2 + CH2-6 piperazine), 59.80 (CH2-N), 116.10 (o-Ph), 119.76 (p-Ph), 129.08 (m-Ph), 151.23 (i-Ph).°C (lit.  colourle4d: colourless oil (lit. 40–41°C [Rf 0.24 (5% acetone/DCM); 1H NMR 2.49 , 2.54 , 2.722 , 3.569 ; 13C NMR 40.83 (CH2-Cl), 52.72 (2 × CH2 piperazine), 52.89 (2 × CH2 piperazine), 59.70 (CH2-N), 62.85 (CH2-Ph), 127.23 (p-Ph), 128.26 (o-Ph or m-Ph), 129.33 (o-Ph or m-Ph), 137.38 (i-Ph). 40–41°C ); Rf 0.2Version 3: A solution of 2f (10 mmol) and TosCl (11 mmol) in pyridine (10 ml) was kept at rt. The reaction mixture was poured into water. The products were partitioned between water and ethyl acetate. The organic phase was washed with 5% aq. HCl, and then with brine, was dried over MgSO4, and evaporated to dryness. Flash chromatography purification on silica gel by using a mobile phase with a gradient of polarity from DCM to 2% acetone in DCM gave:4f: colourless oil (lit. m.p. 37.5–39.5°C [Rf 0.45 (DCM); 1H NMR 2.996 , 3.679 , 3.848 , 3.866 , 6.733 , 6.754 , 6.810 ; 13C NMR 38.78 (CH2-2′), 45.21 (CH2-3′), 55.81 (OCH3), 55.85 (OCH3), 111.19 (CH-5), 111.98 (CH-2), 120.80 (CH-6), 130.62 (Cq-1), 147.89 (Cq-4), 148.87 (Cq-3).5–39.5°C , colourl5–39.5°C ); Rf 0.43f: colourless solid; recrystallization from hexane: m.p. 46.1–46.7°C (lit. 48–50°C [Rf 0.16 (DCM); 1H NMR 2.419 , 2.887 , 3.797 (OCH3-3), 3.845 (OCH3-4), 4.189 , 6.591 , 6.654 , 6.745 , 7.265 , 7.660 ; 13C NMR 21.61 (CH3 Tos), 34.91 (CH2-2′), 55.73 (OCH3), 55.88 (OCH3), 70.89 (CH2-3′), 111.16 (CH-5), 111.90 (CH-2), 121.00 (CH-6), 127.79 (CH-2 + CH-6 Tos), 128.74 (Cq-1), 129.74 (CH-3 + CH-5 Tos), 132.85 (Cq-1 Tos), 144.71 (Cq-4 Tos), 147.91 (Cq-4), 148.84 (Cq-3). 48–50°C , 49–50°C 48–50°C ); Rf 0.13.2.2a, 4a, 4b and 5 were mounted on a glass capillary and all geometric and intensity data were taken from these crystals. Diffraction data were taken on an Agilent SuperNova Dual diffractometer equipped with an Atlas CCD detector using micro-focus Mo Kα radiation (λ = 0.71073 Å) at room temperature. The determination of the unit cell parameters, data collection and reduction were performed with Crysalispro software [F2 with ShelxS and ShelxL 2016/6 programs [Uiso(H) = 1.2Ueq(C). The most important crystallographic and refinement indicators are listed on The crystals of software . The strprograms . All non4.O-tosylation protocol is observed. It is found that:—2-Substituted ethanols can be easily converted into the corresponding ethyl chlorides via a simple cheap protocol.—O-tosylate formation.The reaction goes via initial —O-tosylate into chloride is very fast in piperazinyl ethanols and slower in aromatic ethanols.The presence of piperazine fragment at the end of ethanol unit speeds up the conversion. The transformation of —The protocol has practical value only when the product possesses limited solubility in water-pyridine system.—The prolongation of the reaction and/or increased excess of tosyl chloride lead to partial aromatization of pirlindole chloride. Tosyl chloride catalyses the transformation.—The particular conditions are not applicable to piperazinyl propanols.This study aims to warn the synthetic community about the eventual problems if trying to tosylate 2-hydroxyethyl derivatives, especially piperazinyl ethanols, and to inform on the possibility to convert directly ethanols into ethyl chlorides.A direct conversion of ethanols into chlorides via a classical


P. lanosocoeruleum KCH 3012 metabolizes androstanes to the corresponding lactones with high yield. In order to explore more thoroughly the factors determining steroid metabolism by this organism, the current study was initiated to delineate the specificity of this fungus with respect to the cleavage of steroid side chain of progesterone and pregnenolone The effect of substituents at C-16 in 16-dehydropregnenolone, 16α,17α-epoxy-pregnenolone and 16α-methoxy-pregnenolone on the pattern of metabolic processing of these steroids was also investigated.Metabolic activities of microorganisms to modify the chemical structures of organic compounds became an effective tool for the production of high-valued steroidal drugs or their precursors. Currently research efforts in production of steroids of pharmaceutical interest are focused on either optimization of existing processes or identification of novel potentially useful bioconversions. Previous studies demonstrated that d-lactones. The activity of 3β-HSD was a factor affecting the composition of the product mixtures. 16α,17α-epoxy-pregnenolone underwent a rare epoxide opening with retention stereochemistry to give four 16α-hydroxy-lactones. Apart from oxidative transformations, a reductive pathway was revealed with the unique hydrogenation of 5-ene double bond leading to the formation of 3β,16α-dihydroxy-17a-oxa-d-homo-5α-androstan-17-one. 16α-Methoxy-pregnenolone was transformed to the 20(R)-alcohol with no further conversion.All of the analogues tested (except the last of the listed) in multi-step transformations underwent the Baeyer–Villiger oxidation to their δ-P. lanosocoeruleum KCH 3012 has great multi-functional catalytic properties towards the pregnane-type steroids. Studies have highlighted that a slight modification of the d-ring of substrates may control metabolic fate either into the lactonization or reductive and oxidative pathways. Possibility of epoxide opening by enzymes from this microorganism affords a unique opportunity for generation of novel bioactive steroids.This work clearly demonstrated that The online version of this article (10.1186/s12934-018-0948-1) contains supplementary material, which is available to authorized users. Steroids are an important class of natural compounds, ubiquitous in nature and playing crucial functions for metabolism. Their physiological activity depends on the structure, primarily location and stereochemistry of the functional groups attached to the steroid nucleus. A variety of steroid compounds are widely used as anti-inflammatory, anti-allergic, immunosuppressive, anabolic, diuretic, progestational and contraceptive agents. They have also been used for the prevention of coronary heart disease, for the treatment of adrenal insufficiencies and some types of cancers . StructuBVO is an oxidation of ketones, in which cleavage of one of the C–CO–C bonds occurs with simultaneous insertion of an oxygen atom. The products of oxidation of acyclic ketones are esters, while cyclic ketones yield lactones. Oxidative factors employed for this reaction are most often hazardous peroxyacids or hydrogen peroxide. Toxicity and instability of these oxidizers, their explosive tendencies , and lack of selectivity, make chemical synthesis employing BVO seriously limited. In case the substrate is a compound containing other functions vulnerable to oxidation, an important issue is formation of byproducts . The enzymes performing the BVO belong to the group of so-called Baeyer–Villiger monooxygenases (BVMOs). It is a class of oxidoreductases using atmospheric oxygen as a “green and free oxidant”. Steroidal BVMOs catalyze mainly oxidation of ketones bound to ring D, i.e. at C-17 and/or C-20 carbonyl groups. Although research on BVO of steroids has long history , the subPenicillium lanosocoeruleum ATCC 48919, closely related to the one used in the current study, has been sequenced in the framework of the US Department of Energy Joint Genome Institute studies. Numerous enzymes possibly involved in steroid metabolism in this strain include, among others, 214 putative monooxygenases , 27 putative steroid reductases (including three 3-ketosteroid reductases). The function of these proteins was however assigned only by sequence similarity to fungal enzymes of other species. We are not aware of any report linking the sequence information with enzymatic activity of P. lanosocoeruleum towards steroids. In light of the large number of possibly involved enzymes that cannot be readily isolated, we have rather adopted the strategy of considering the fungal cell as a micro-bioreactor, and we concentrated on the ability of a given strain to carry out successful biotransformation.Although steroidal BVMO activity was found in many fungi, only two enzymes from bacteria and lower eukaryotes have been characterized until now , 9 and wAspergillus D20 − 32.1 (c = 1.8 EtOH) (ref.: [α]D20 − 33.0 (c = 2.0 EtOH) [t = 3.62 min.; 1H NMR  δH: 1.16 , 2.70–2.79 ; 3.16–3.25 , 3.65 , 3.86 , 4.87–4.97 , 6.22 , 6.25 ; 13C NMR : 20.4 (C-17), 23.8 (C-4), 24.9 (C-6), 27.7 (C-5), 33.0 (C-7), 40.4 (C-11), 44.6 (C-3), 73.8 (C-8), 102.7 (C-15), 112.2 (C-13), 120.9 (C-1), 137.2 (C-12), 159.4 (C-14), 161.1 (C-16), 172.8 (C-10), 209.7 (C-2). The spectroscopic data corresponded to those described in literature [S)-curvularin [After 96 h of fermentation, the following compounds were isolated (% mol): 79 mg 25%) of unreacted pregnenolone (.0 EtOH) ); Rt = 35% of unrrvularin .Fig. 2X-4) and 106 mg (73%) of 3β-hydroxy-17a-oxa-d-homo-androst-5-en-17-one (7): 1H NMR  δH: 0.96 , 1.30 , 3.53 , 5.33 , 13C NMR : 19.3 (C-19), 19.9 (C-15), 20.1 (C-18), 21.9 (C-11), 28.8 (C-16), 31.1 (C-8), 31.5 (C-7), 34.4 (C-2), 36.6 (C-10), 36.9 (C-1), 38.9 (C-12), 41.9 (C-4), 46.7 (C-14), 49.0 (C-9), 71.5 (C-3), 83.2 (C-13), 120.6 (C-6), 140.6 (C-5), 171.5 (C-17). The spectroscopic data are in agreement with those reported in literature [After 48 h of fermentation, the following compounds were isolated (% mol): 22 mg (16%) of testololactone  , 1H NMR  δH: 0.97 , 1.36 , 3.53 , 4.46 , 5.35 , 13C NMR : 19.2 (C-18), 19.3 (C-19), 21.9 (C-11), 30.6 (C-15), 30.8 (C-7), 31.4 (C-2), 35.2 (C-8), 36.5 (C-10), 36.8 (C-1), 39.0 (C-12), 41.8 (C-4), 46.5 (C-14), 48.6 (C-9), 64.6 (C-16), 71.4 (C-3), 85.1 (C-13), 120.5 (C-6), 140.5 (C-5), 175.2 (C-17); 16α-hydroxy-17a-oxa-d-homo-androst-4-en-3,17-dione (11) , 1H NMR  δH: 1.16 , 1.39 , 4.46 , 5.75 , 13C NMR : 17.3 (C-19), 19.2 (C-18), 21.8 (C-11), 30.2 (C-15), 30.6 (C-7), 32.2 (C-6), 33.7 (C-2), 35.4 (C-1), 38.3 (C-10), 38.6 (C-8), 39.0 (C-12), 45.7 (C-14), 52.1 (C-9), 64.4 (C-16), 84.5 (C-13), 124.2 (C-4), 168.9 (C-5), 174.9 (C-17), 199.0 (C-3); 3β,16α-dihydroxy-17a-oxa-d-homo-5α-androstan-17-one (12) , 1H NMR  δH: 0.77 , 1.34 , 3.58–3.63 , 4.46 , 13C NMR : 12.1 (C-19), 19.3 (C-18), 22.0 (C-11), 28.2 (C-6), 30.4 (C-7), 30.7 (C-15), 31.2 (C-2), 35.4 (C-10), 36.7 (C-1), 37.7 (C-4), 38.6 (C-8), 39.4 (C-12), 44.1 (C-5), 46.4 (C-14), 52.7 (C-9), 64.6 (C-16), 71.0 (C-3), 85.4 (C-13), 175.3 (C-17), and 16α-hydroxy-17a-oxa-d-homo-5α-androstan-3,17-dione (13) , 1H NMR  δH: 0.98 , 1.36 , 4.46 , 13C NMR : 11.3 (C-19), 19.3 (C-18), 22.1 (C-11), 28.4 (C-6), 30.1 (C-7), 30.7 (C-15), 35.5 (C-10), 37.9 (C-2), 38.1 (C-1), 38.5 (C-8), 39.2 (C-12), 44.2 (C-4), 45.8 (C-14), 46.2 (C-5), 52.2 (C-9), 64.5 (C-16), 85.1 (C-13), 175.1 (C-17), 211.1 (C-3).After 72 h of fermentation, the following compounds were isolated (% mol): 8 mg (5%) of unreacted substrate 17)  1H NMR  δH: 0.66 , 1.17 , 2.18 , 2.54 , 3.20 , 4.32–4.37 , 5.74 . 13C NMR : 14.6 (C-18), 17.3 (C-19), 20.7 (C-11), 31.7 (C-21), 31.9 (C-7), 32.6 (C-6), 33.9 (C-15), 33.9 (C-2), 35.1 (C-8), 35.6 (C-1), 38.5 (C-10), 38.6 (C-12), 44.3 (C-13), 53.4 (C-9), 53.6 (C-14), 57.2 (C-22); 71.4 (C-17), 81.3 (C-16), 124.0 (C-4), 170.5 (C-5), 199.3 (C-3), 207.9 (C-20), and (20R)-20-hydroxy-16α-methoxy-pregn-4-en-3-one (18) , 1H NMR  δH: 0.72 , 1.18 , 1.21 , 3.32 , 3.86–3.91 , 5.74 . 13C NMR : 13.7 (C-18), 17.3 (C-19), 20.3 (C-11), 23.1 (C-21), 30.6 (C-15), 31.8 (C-7), 32.7 (C-6), 33.9 (C-2), 34.9 (C-8), 35.6 (C-1), 38.4 (C-12), 38.5 (C-10), 42.0 (C-13), 53.1 (C-14), 53.6 (C-9), 57.0 (C-22), 64.6 (C-17), 69.6 (C-20), 86.6 (C-16), 124.0 (C-4), 170.8 (C-5), 199.4 (C-3).After 72 h of fermentation, the following compounds were isolated (% mol): 16α-methoxy-progesterone (1), pregnenolone (5) and 16-dehydro-pregnenolone (8) yielded known products: testololactone (4) and 3β-hydroxy-17a-oxa-d-homo-androst-5-en-17-one (7) which were identified by comparison of their spectroscopic data with the literature values [t from GC and Rf from TLC with standards available in our laboratory. The determination of the structure of the other metabolites was based primarily on their NMR data  were devoid of the side-chain methyl-ketone resonance signals in 1H NMR spectrum at δH 2.02 ppm (21-CH3) and in 13C NMR spectrum at δC 26.0 ppm (C-21) and δC 205.0 ppm (C-20). The absence of these signals fully supported the assumption that removal of the side-chain had occurred. A significant downfield shift in comparison with the substrate was observed for the 18-methyl resonance signals in the 1H NMR spectra of these products . It was consistent with an oxygen atom insertion into the ring-D. This was supported by the 13C NMR spectra in which C-13 resonance signal had undergone a downfield shift of ca. 43 ppm . The lactonization in the ring-D of these metabolites, via Baeyer–Villiger oxidation, was confirmed by the appearance of the signal at δC ca. 175 ppm (C-17). The epoxide 16β-proton (δH 3.67 ppm) was absent from the spectra of metabolites 10–13 indicating epoxide opening. This was supported by a new signal present in the 1H NMR spectra of these metabolites at δH ca.4.46 ppm and in the 13C NMR spectra at δC ca.64.6 ppm which demonstrated the presence of a hydroxyl group. The multiplicity of this signal in 1H NMR spectra suggested that it belongs to a proton coupled with only two protons which is consistent with a presence of 16-hydroxyl group. The stereochemistry of this hydroxyl group was further supported by NOESY spectra which showed correlation between H-16β signal and C-18 methyl group and H-15β signals . This data led to identification of 10 as 3β,16α-dihydroxy-17a-oxa-d-homo-androst-5-en-17-one. The absence of the 3α-H resonance (tt) from the 1H NMR spectrum of the metabolite 11 coupled with an increase in the C-19 methyl resonance signal (Δ 0.12 ppm relative to the substrate 9) and a shift of the signal of the olefinic proton from δH 5.32 ppm to δH 5.75 ppm indicated double bond migration into ring A (between C-4 and C-5) and oxidation of the C-3 alcohol to a ketone. Oxidation of the 3β-OH group was fully supported by the loss of a methine signal at δC 74.6 ppm in the starting material 13C NMR spectrum, being replaced by a new non-protonated signal in the spectrum of the product at δC 199.0 ppm. Thus, the structure of metabolite 11 was deduced to be 16α-hydroxy-17a-oxa-d-homo-androst-4-en-3,17-dione. The NMR spectra of product 12 revealed that there were two hydroxyl groups (multiplet at δH 3.58–3.63 ppm and doublet of doublets at 4.46 ppm) and no carbon–carbon double bond. In comparison to the 13C NMR spectrum of 10, the disappearance of resonance signal at δC 120.5 ppm and δC 140.5 ppm confirmed hydrogenation of this double bond. This was further supported by a new presence of methine C-5 and methylene C-6 signals at δC 44.1 ppm and δC 28.2 ppm, respectively. Hydrogenation resulting in 5α-stereochemistry at A/B rings was determined by chemical shift of C-19 methyl group signal, which was resonating at similar field (δC 12.1 ppm and δH 0.77 ppm) when compared with known 3β-hydroxy-17a-oxa-d-homo-5α-androstan-17-one [12 was proposed to be 3β,16α-dihydroxy-17a-oxa-d-homo-5α-androstan-17-one. The 13C NMR spectrum of 13 was similar to that of 12 with the exception of signals of the A-ring carbons. The absence of the C-3 methine signal at δC 71.0 ppm and the appearance an additional quaternary carbon signal at δC 211.1 ppm indicated the oxidation of the C-3 hydroxyl to a carbonyl group. Also, the signal of 3α-proton disappeared in the 1H NMR spectrum and the chemical shift of C-19 methyl group signal was resonating at similar field (0.98 ppm) when compared with known 17a-oxa-d-homo-5α-androstan-3,17-dione [13 as 16α-hydroxy-17a-d-homo-5α-androstan-3,17-dione.Transformation of progesterone -20-hydroxy-16α-methoxy-pregn-4-en-3-one (18) was provided by the 13C NMR spectrum with loss of the resonance signal at δC 207.9 ppm for the C-20 ketone and its replacement with a HCOH signal at δC 69.6 ppm. Also, the C-21-methyl signal of 18 underwent an upfield shift (Δ 8.6 ppm) accordingly to the presence of the less electronegative alcohol, and the multiplet visible at δH 3.89 ppm showed correlation with the carbon C-20 in HSQC spectrum. Additional confirmation of C-20 reduction was the upfield shift (Δ 6.8 ppm) of resonance for C-17. This was coupled with the significant upfield shift (Δ 0.46 ppm) of the 16β-H resonance signal in 1H NMR spectrum, and NOESY spectrum showed correlation of 16α-OCH3 signal with the proton signal of C-20.The resonance signals in both 19 was determined by 2D NMR experiments and supported by the single crystal X-ray analysis -curvularin published previously [The structure of the non-steroidal compound eviously , 32.P. lanosocoeruleum KCH 3012 are involved in the degradation of the C-17β-acetyl side chain of progesterone (1), pregnenolone (5), 16-dehydro- (8) and 16α,17α-epoxy-pregnenolone (9) via oxygenative esterification of these 20-ketosteroids and hydrolyze esters into alcohols and acetic acid. The subsequently formed 17-keto steroids could be then oxygenated by the lactonizing enzyme to their respective lactones  the multistep transformation led to afford testololactone (4) as a single product isolated with 82% yield. The same metabolite  was obtained from pregnenolone (5) and, as a minor metabolite, from 16-dehydro-pregnenolone (8). The main product of the conversion of 8 was lactone with conserved ring-A of the substrate—3β-hydroxy-17a-oxa-d-homo-androst-5-en-17-one (7).The results herein presented clearly show that enzymes of 5 and 8), we studied composition of mixtures sampled after various transformation periods (Table 5) was the oxidative conversion its 3β-hydroxy-5-ene group to 3-oxo-4-ene system which resulted in progesterone (1), the product of its 17β-acetyl chain cleavage—testosterone (2), and its subsequent oxidation—androstenedione (3). Since the moment of identification this 3-oxo-4-ene C19 metabolites, the content of testololactone (4)—product of BV oxidation of C-17 ketone significantly increased reaching a maximum at 72 h of the process. Only small amount of DHEA (6) and 3β-hydroxy-lactone (7) were identified in the reaction mixtures even if the substrate 5 was present throughout the studied period of transformation.In order to investigate metabolic pathways of these two structurally related compounds (ds Table . Their a8). Comparison of the course of transformation of both C21-20-ketones 5 and 8 in time indicated that 16-dehydro-analog of pregnenolone underwent conversion noticeably faster. Approximately 90% of the incubated 8 was affected by the enzymatic action of the fungus during first 24 h of transformation  were both lactones—3β-hydroxy-5-ene as well as 3-oxo-4-ene lactone  , whereas after transformation completion the amount of 4 was only twofold lower. Because at the same time the substrate 8 was not identified in the extracts, lactone 4 could be formed only via the conversion of hydroxylactone 7 (3β-HSD activity occurred following d-lactonization).The products of scission of 17β-acetyl group—DHEA and androstenedione were not identified during the transformation of 16-dehydropregnenolone (9) was incubated with P. lanosocoeruleum we obtained mixture of four hydroxylactones. The substrate underwent a rare epoxide opening [alpha stereochemistry to give 16α-hydroxy metabolites 10–13 . Other metabolites of 9 were mostly products of further transformations of this 16α-hydroxylactone. The observed reactions included the oxidation of hydroxyl group at C-3 to ketone and the subsequent isomerization of double bond C=C from C-5 to C-4 and hydrogenation of C5–C6 double bond. The time experiments indicated that the relative content of 10 was decreasing and 16α-hydroxy-17a-oxa-d-homo-androst-4-en-3,17-dione (11) began to dominate in the mixture of metabolites after 72 h of transformation (Table 14) nor 16α-hydroxyandrostenedione (15) were identified in any of the reaction mixtures, which suggests that 3β-HSD activity occurred here following ring d-lactonization. This is in contrast to previous studies in which 3β-HSD was active in the presence of a C-17 ketone as in DHEA [5). It is also interesting to note that the minor metabolites of 9 were 5α-saturated 16α-hydroxylactones (12 and 13). Although microbial hydrogenation of 4-en-3-keto steroids to 5-dihydrosteroids using Penicillium species has been described [When 16α,17α-epoxy-pregnenolone  resulted in a significantly different pattern of metabolism in comparison to other tested C21-20-ketones, notably being devoid of side chain degradation . The mixture after 12 h of reaction contained 66% of 17 and it was the highest content of 3-oxo-4-ene metabolite of tested 3β-hydroxy-5-ene steroids. Some amount of this derivative in the next hours was reduced to (20R)-20-hydroxy-16α-methoxy-pregn-4-en-3-one (18), whose content at the end of the transformation reached only 19%. The stereochemical reduction at C-20 of pregnanes is common to a wide range of fungi [R)-20-hydroxy-16α-methoxy-pregn-4-en-3-one (18) did not undergo further transformation, it can be assumed that in the presence of 16α-methoxy substituent the 20-hydroxy group could not be re-oxidized to the corresponding C-20 ketone and finally BVMO could not be activated. In this case it was probable that intramolecular hydrogen bonds may be inhibiting substrate binding to the oxidase. However, a physical ball-and-stick model demonstrated possibility of only a weak hydrogen interaction between the C-20 hydroxyl group and 16α-methoxy group (more than 3.2 Å), and this reason should be discarded. It is worth noting that we were unable to isolate the corresponding 20-alcohols from other tested steroids. This may indicate that progesterone (1) and pregnenolones 5, 8 and 9 do not activate reductase or that their molecular structure is precluding binding to this enzyme. On the other hand, the mentioned substrates were metabolized at a faster rate than 16 by side-chain degrading enzymes  we isolated in large yield a 12-membered macrolactone—(S)-curvularin (19). Curvularin and its structural relatives are produced by a number of phytopatogenic fungi from such genera as Curvularia, Alternaria and Penicillium [P. lanosocoeruleum. It is apparent that further investigation is required and we hope to provide detailed information of this finding in future reports.During the experiments carried out to elucidate the pattern of metabolic processing of steroidal compounds by icillium , but thiP. lanosocoeruleum KCH 3012 clearly demonstrated that this microorganism has potent multi-functional catalytic properties towards androstane- and pregnane-type steroids. It contains  an endonsformed  or with H moiety . The tes1), pregnenolone (5), 16α,17α-epoxy-pregnenolone (9), 16α,17α-epoxy-progesterone (14), and 16-dehydropregnenolone acetate (20) were purchased from Sigma-Aldrich Chemical Co. Testosterone (2), androstenedione (3), DHEA (dehydroepiandrosterone) (6) were purchased from Steraloids Inc. 16α-Hydroxyandrostenedione (15) was obtained in our previous work by transformation of testosterone using Aspergillus niger KCH910 [14 were used as analytical standards for the time course experiments. 16-Dehydro-pregnenolone (8) and 16α-methoxy-pregnenolone (16) (in a ratio of 3:7) were prepared from the 16-dehydropregnenolone acetate (20) by its saponification with potassium hydroxide in methanol [Progesterone  δH: 0.90 , 1.03 , 2.25 , 3.52 , 5.35 , 6.70 . 13C NMR : 15.7 (C-18), 19.3 (C-19), 20.7 (C-11), 27.1 (C-21), 30.2 (C-8), 31.5 (C-7), 31.6 (C-2), 32.2 (C-15), 34.6 (C-12), 36.7 (C-10), 37.1 (C-1), 42.2 (C-4); 46.1 (C-13); 50.4 (C-9), 56.4 (C-14), 71.7 (C-3), 121.1 (C-6), 141.3 (C-5), 144.5 (C-16), 155.6 (C-17), 196.9 (C-20). NMR data was found to be in good agreement with that described by Szendi [y Szendi .1H NMR  δH: 0.62 , 0.99 , 2.17 , 2.53 , 3.20 , 3.50–3.54 , 4.33–4.35 , 5.34 . 13C NMR : 14.4 (C-18), 19.3 (C-19), 20.7 (C-11), 31.4 (C-8), 31.5 , 31.7 (C-21), 31.9 (C-7), 36.5 (C-10), 37.1 (C-1), 38.8 (C-12), 42.2 (C-4), 44.4 (C-13), 49.8 (C-9), 54.4 (C-14), 57.1 (C-22), 71.6 , 81.4 (C-16), 121.2 (C-6), 140.7 (C-5), 208.1 (C-20). NMR data was found to be identical with that given by Wölfling [Wölfling .Penicillium lanosocoeruleum KCH 3012 used in this study was taken from the collection of the Department of Chemistry, Wrocław University of Environmental and Life Sciences . The fungus was maintained on Sabouraud 4% dextrose-agar slopes at 4 °C and freshly subcultured before use in the transformation experiments.The fungal strain General experimental and fermentation details were described previously . Each suin vacuo and analyzed by TLC and GC. Transformation products were separated by column chromatography on silica gel with ethyl acetate:methylene chloride:acetone (3:1:1 v:v:v) for progesterone (1) and pregnonolone (5), hexane:ethyl acetate:isopropyl alcohol (2:0.5:0.5 v:v:v) for 16-dehydro-pregnenolone (8), ethyl acetate:diethyl ether (2:1 v:v) for 16α,17α-epoxypregnenolone (9), or hexane:ethyl acetate:isopropyl alcohol (1:0.15:0.1 v:v:v) for 16α-methoxy-pregnenolone (16) as eluents. TLC was carried out with Kieselgel 60 F254 plates  using the same eluents. In order to develop the image, the plates were sprayed with solution of methanol in concentrated sulfuric acid (1:1) and heated to 120 °C for 3 min. GC analysis was performed using Hewlett Packard 5890A Series II GC instrument  with DB-5MS column cross-linked phenyl-methylsiloxane, 30 m × 0.32 mm × 0.25 μm film thickness. The following program was used in the GC analysis: 220 °C/1 min, gradient 4 °C/min to 280° and 30 °C/min to 300°/2 min  or 230 °C/1 min, gradient 4 °C/min to 280° and 30 °C/min to 300°/2 min (for 9 and 16); injector and detector temperatures were 300 °C. The NMR spectra were measured in CDCl3 or CD3OD and recorded on a DRX 300 MHz Bruker Avance spectrometer. Characteristic 1H- and 13C-NMR shift values in comparison to the starting compounds were used to determine structures of metabolites, in combination with DEPT analysis to identify the nature of the carbon atoms. Optical rotation measurements were carried out on Autopol IV automatic polarimeter (Rudolph).The products of biotransformation were extracted three times with ethyl acetate. The organic extracts were dried over anhydrous magnesium sulfate, concentrated For studying the time-dependent progress of the bioconversion and to determine the metabolic pathways of substrates, 5-mL samples of the broth were taken out at regular intervals from the reaction flask, extracted with ethyl acetate and analyzed by comparison of the GC and TLC data with those of authentic samples. Conditions of the reaction were identical to those in the main biotransformation experiments.Additional file 1.1H, 13C, DEPT and NOESY NMR spectra of the biotransformation products.


All the substrates were converted in high yield (70%–99%) into d ring δ-lactones. The oxidation of 1 produced 3α-hydroxy-17a-oxa-d-homo-5α-androstan-17-one (5). The oxidation of 2 led to 3β-hydroxy-17a-oxa-d-homo-5α-androstan-17-one (6). The biotransformation of 3 resulted in the formation of 3α-hydroxy-17a-oxa-d-homo-5α-androstan-17-one (5) and 17a-oxa-d-homo-5α-androstan-3,17-dione (7). An analysis of the transformation progress of the studied substrates as a function of time indicates that the Baeyer-Villiger monooxygenase of this fungus does not accept the 3β-hydroxy-5-ene functionality of steroids. In this microorganism steroidal 3β-hydroxy-dehydrogenase (3β-HSD) was active, and as a result DHEA (4) was transformed exclusively to testololactone (8). Apart from the observed oxidative transformations, a reductive pathway was revealed with the C-3 ketone being reduced to a C-3α-alcohol. It is demonstrated for the first time that the reduction of the 3-keto group of the steroid nucleus can occur in the presence of a ring-D lactone functionality.The biotransformation of androsterone ( Steroids represent an important class of natural products with varying pharmacological properties. Minor modifications in their structure may affect their bioactivity, consequently, research on the preparation of potentially useful steroid analogues is being continued for development of processes exploitable by the pharmaceutical industry. Biotransformations are powerful tools for generation of novel steroidal drugs, as well as for efficient production of steroidal key intermediates. In the majority of cases, these modifications have been achieved by the use of whole-cells microorganisms. The use of enzymes ensures the high regio- and stereoselectivity of the reaction to be performed, and biotransformations are more environmentally friendly in comparison with chemical syntheses. Microbial transformations of various steroids have been reviewed recently ,2.Pseudomonas sp., involved in the catabolism of cyclopentadecanone in Nature, was also found to accept steroids as substrates [Rhodococcus rhodochrous) in the biotransformation of progesterone afforded the final lactone (testololactone) with a moderate (19%) conversion [Penicillium [Aspergillus [Fusarium genera [21 as well as C19 steroids. Enzymes performing oxidation belong to the family of NADPH-dependent steroid Baeyer-Villiger monooxygenases (BVMOs) containing FAD as a cofactor. The catalytic activity of this class of enzymes is manifested in the transformation of C-20 ketones into the corresponding acetates, which then hydrolyze to the related C-17 alcohols, followed by oxidation to ketones, and finally ring-D Baeyer-Villiger oxidation gives lactones. Currently research efforts in production of steroids of pharmaceutical interest are focused on either optimization of existing processes or identification of novel potentially useful bioconversions. It is not surprising that several microorganisms have been reported to exhibit Baeyer-Villiger oxidation properties. Only in a few cases the isolated enzymes have been used ,3,4. Recicillium ,7,8,9,10ergillus ,18,19,20m genera ,22,23 arSteroidal lactones are important compounds due to their biological activity such as anticancer and antiandrogenic action. It was found that they could inhibit the 5α-reductase, and as a result of this, block the conversion of testosterone to 5α-dihydrotestosterone  and therPenicillium citreo-viride oxidized androstenedione, progesterone and DHEA (dehydroepiandrosterone) to testololactone, while pregnenolone was not transformed; conversion of DHEA to testololactone occurred through the 3β-hydroxysteroid dehydrogenase/5-ene-4-ene isomerase pathway resulting in the generation of a 4-en-3-oxo system in the steroid ring-A [P. griseopurpureum and P. glabrum [P. citreo-viride, BVMO(s) from P. lilacinum accepted 3β-hydroxy-5-ene substrates [d-homo-androst-5-en-17-one (a d-lactone with conserved ring-A moiety), whereas pregnenolone was also transformed to testololactone (a d-lactone with a 3-oxo-4-ene system). P. camemberti was able to carry out the oxidation of pregnenolone and DHEA to testololactone, which could be formed via two routes: through 4-en-3-ketone or 3β-hydroxylactone [Microbial steroid BVMOs are steroid-induced enzymes, which utilize atmospheric oxygen as oxidant. Their catalytic properties exhibit differences , even wid ring-A . This sa glabrum . In contbstrates . The enzylactone . In mostylactone ,19,23,29Penicillium lanosocoeruleum KCH 3012. To the best of the author’s knowledge, this is the first report of the use of P. lanosocoeruleum in biotransformations.In the light of our interest ,9,29 in P. lanosocoeruleum KCH 3012 strain produces a Baeyer-Villiger monooxygenase which is able to carry out the regioselective ring-D lactonization of 5α-dihydro-steroids (1–3) as well as unsaturated steroid 4  as the sole products from epimeric 3-hydroxy substrates—androsterone (1) and epiandrosterone (2). Two lactones: 3α-hydroxy-17a-oxa-d-homo-5α-androstan-17-one (5) and 17a-oxa-d-homo-5α-androstan-3,17-dione (7) were isolated after transformation of a 3,17-diketo compound—androstanedione (3). DHEA (4) was converted to testololactone (8).The teroid 4 , Table 11H-NMR and 13C-NMR). The assumed structures were confirmed by comparison of the characteristic shift values of selected, diagnostic signals of products and starting compounds. Resonance signals in both 1H-NMR and 13C-NMR spectra of all metabolites 5–8 suggested the formation of the ring-D lactones. The absence of the carbonyl group signals at δC ca. 220 ppm, the presence of new signals at δC ca. 171 ppm and downfield shifts of the C-13 resonance signals with respect to the substrates, confirmed insertion of an oxygen atom into the ring-D. The oxygen insertion into ring-D of all metabolites was supported by significant downfield shifts for both the 18-methyl protons’ signals in 1H-NMR and the C-18 signals in 13C-NMR. The bands at 1714–1720 cm−1 in the IR spectra of the products confirmed the δ-lactone structures. The absence of the 3α-H resonance (m) from the 1H-NMR spectrum of the metabolite 8 coupled with an increase in the C-19 methyl resonance signal (∆ 0.14 ppm relative to substrate 4) and a shift of the signal of olefinic proton from δH 5.36 ppm to δH 5.75 ppm indicated isomerization of the double bond with its formation between C-4 and C-5 and oxidation of the C-3 alcohol to a ketone. Double bond migration into ring-A was fully supported in the 13C-NMR spectrum with downfield shifts observed for C-4 (∆ 81.8 ppm) and C-5 (∆ 28.1 ppm). Signal C-5 was visible at δc 169.1 ppm and its position was consistent with the position of a β-carbon conjugated with the carbonyl group. Oxidation of the 3β-OH group was fully supported by the loss of a methine signal at δc 71.5 ppm in the starting material 13C-NMR spectrum, being replaced by a new non-protonated resonance signal in the product spectrum at δc 199.4 ppm. All these observations for metabolites 5–8 were in agreement with those reported in the literature [The structures of the obtained products were established by using spectroscopic techniques  was previously obtained in 53% yield by transformation of androsterone using Aspergillus tamarii KITA, but it was one of the six metabolites produced from this substrate. During that transformation, substrate as well as 3α-hydroxy-17a-oxa-d-homo-5α-androstan-17-one also underwent hydroxylation, giving a mixture of 1β- and 11β-hydroxy products [d-homo-5α-androstan-17-one (6) with 10% yield by Aspergillus terreus [d-homo-5α-androstan-3,17-dione (7) was previously obtained with 29% yield as one of the three metabolites by transformation of 5α-pregna-3,20-dione using Aspergillus tamarii KITA [8) was the only product of the transformation of C19 or C21 3-oxo-4-ene steroids or DHEA and androstenediol by P. citreo-viride [A. terreus [3α-Hydroxy-17a-oxa-products . Epiandr terreus . 17a-Oxa terreus . This la3 and 4 (3) contained, apart from the substrate, 3% of androsterone (1) and 2% of 3-keto-d-lactone 7. That result indicated that substrate 3 underwent transformation via two routes: through stereoselective ketone reduction at C-3 and Baeyer-Villiger oxidation in ring-D. The rapid increase in the contents of d-lactones 5 and 7 between 8 and 12 h of the incubation indicates that compound 1 as well as compound 3 are the inducers of the BVMO responsible for the lactonization of C-17-ketones. In the product mixture obtained after 12 h and 24 h of transformation of androstanedione (3) the 3-oxo-lactone 7 prevailed, but its content decreased with prolonged reaction time, and consequently after 48 h of incubation, the main metabolite of 3 was 3α-hydroxy-17a-oxa-d-homo-5α-androstan-17-one (5). Because the amount of accumulating androsterone (1) did not exceed 10%, it can be assumed that lactone 5 was formed mainly from lactone 7  indicated that the first stage of the process was the oxidative conversion of 3β-hydroxy-5-ene to 3-oxo-4-ene functionality (9) was the only metabolite identified after 4 h incubation of 4; the mixtures after 8 h and 12 h incubation contained a significant amount of 9. After 12 h of reaction, the content of androstenedione in the reaction mixtures started to decrease. Simultaneously, the content of testololactone (8) increased. The production of 8 reached a maximum at 24 h of the experiment. 3β-Hydroxy-17a-oxa-d-homo-androst-5-en-17-one was not identified in any of the reaction mixtures, which suggests that the BVMO from P. lanosocoeruleum does not accept 3β-hydroxy-5-ene steroids as substrates.The analysis of composition of mixtures after transformation of DHEA , epiandrosterone  and dehydroepiandrosterone  were purchased from Sigma-Aldrich . Androstanedione  was obtained by oxidation of androsterone (1) with Jones’ reagent according to the known procedure [19H28O2 calcd. C, 79.12; H 9.78; found. C, 79.36; H. 9.84%. 1H-NMR: 0.89 , 1.03 . 13C NMR: 11.4 (C-19), 13.8 (C-18), 20.7 (C-11), 21.7 (C-15), 28.6 (C-6), 30.5 (C-7), 31.4 (C-12), 34.9 (C-8), 35.8 (C-16), 35.8 (C-10), 38.0 (C-2), 38.4 (C-1), 44.5 (C-4), 46.6 (C-5), 47.7 (C-13), 51.2 (C-14), 53.8 (C-9), 211.6 (C-3), 220.9 (C-17). 1H- and 13C-NMR data were found to be identical to that of androstanedione reported in the literature [9) was purchased from Steraloids Inc. . 3β-Hydroxy-17a-oxa-d-homo-androst-5-en-17-one was obtained previously in our laboratory by transformation of DHEA by Penicillium lilacinum AM111 [The substrates androsterone . The fungus was maintained on Sabouraud 4% dextrose-agar slopes at 4 °C and freshly subcultured before use in the transformation experiments.The fungal strain Time course experiments were conducted in order to determine the metabolic pathways. Conditions were identical to those in the main biotransformation experiments. One flask was harvested after 4, 8, 12 h, and every consecutive flask—after another 24 h incubation of substrate. Reaction mixtures were extracted and analyzed by GC and TLC as described above.in vacuo and analyzed by TLC and GC. Transformation products were separated by column chromatography on silica gel with hexane/acetone (3:1 v:v) for androsterone (1), hexane/acetone/2-propanol (1:1:0.15 v:v:v) for epiandrosterone (2), hexane/acetone/ethyl acetate (1:0.6:0.3 v:v:v) for androstanedione (3) and hexane/acetone (1:1 v:v) for DHEA (4) and as eluents. TLC was carried out with Kieselgel 60 F254 plates  using the same eluents. In order to develop the image, the plates were sprayed with solution of methanol in concentrated sulfuric acid (1:1) and heated to 120 °C for 3 min. GC analysis was performed using Hewlett Packard 5890A Series II GC instrument  with a HP-1 column cross-linked methyl siloxane, 30 m × 0.53 mm × 1.5 μm film thickness. The following program was used in the GC analysis: 220 °C/1 min, gradient 4 °C/min to 260° and 30 °C/min to 300°/2 min; injector and detector temperatures were 300 °C. Elemental analysis was performed on vario EL III analyzer. IR spectra were recorded in KBr disc on a Mattson IR 300 Spectrometer. The NMR spectra were measured in CDCl3 and recorded on a DRX 300 MHz Bruker Avance spectrometer with TMS as internal standard. Characteristic 1H- and 13C-NMR shift values in comparison to the starting compounds were used to determine structures of metabolites, in combination with DEPT analysis to identify the nature of the carbon atoms.The products of biotransformation were extracted three times with ethyl acetate. The organic extracts were dried over anhydrous magnesium sulfate, concentrated d-homo-5α-androstan-17-one3α-Hydroxy-17a-oxa- (5). C19H30O3, Found: C, 74.51; H, 9.86. requires C, 74.47; H, 9.87; IR νmax(cm−1): 3462, 1718. 1H-NMR δ (ppm): 0.73 , 1.28 , 4.05 , 13C-NMR δ (ppm): 172.0 (C-17), 83.4 (C-13), 66.2 (C-3), 53.0 (C-9), 46.3 (C-14), 39.3 (C-12), 38.4 (C-5), 37.9 (C-8), 36.0 (C-10), 35.5 (C-4), 31.9 (C-1), 30.5 (C-7), 28.9 (C-2), 28.6 (C-16), 28.1 (C-6), 21.5 (C-11), 20.1 (C-18), 19.6 (C-15), 11.0 (C-19). The spectroscopic data corresponded to those described in literature [d-homo-5α-androstan-17-one3β-Hydroxy-17a-oxa- (6). C19H30O3, Found: C, 74.52; H, 9.89. requires C, 74.47; H, 9.87; IR νmax(cm−1): 3443, 1720. 1H-NMR δ (ppm): 0.76 , 1.29 , 3.56 , 13C-NMR δ (ppm): 171.6 (C-17), 83.4 (C-13), 71.2 (C-3), 52.9 (C-9), 46.1 (C-14), 44.3 (C-5), 39.2 (C-12), 37.7 (C-8), 37.6 (C-4), 36.5 (C-1), 35.4 (C-10), 31.2 (C-2), 30.6 (C-7), 28.7 (C-16), 28.3 (C-6), 22.0 (C-11), 20.1 (C-18), 19.6 (C-15), 12.1 (C-19). The spectroscopic data corresponded to those described in literature [9 3H, s, -CH3, 3.5d-homo-5α-androstan-3,17-dione17a-Oxa- (7). C19H28O3, Found: C, 74.88; H, 9.29. requires C, 74.96; H, 9.27; IR νmax(cm−1): 1721, 1714. 1H-NMR δ (ppm): 0.97 , 1.31 ; 13C-NMR δ (ppm): 211.7 (C-3), 171.3 (C-17), 83.3 (C-13), 52.2 (C-9), 46.0 (C-5), 45.9 (C-14), 44.0 (C-4), 39.0 (C-12), 38.0 (C-1), 37.9 (C-2), 37.6 (C-8), 35.4 (C-10), 30.2 (C-7), 28.6 (C-16), 28.5 (C-6), 22.2 (C-11), 20.1 (C-18), 19.5 (C-15), 11.2 (C-19). The spectroscopic data corresponded to those described in literature [d-homo-androst-4-en-3,17-dione)Testololactone (17a-oxa- (8). C19H26O3, Found: C, 75.48; H, 8.68. requires C, 75.46; H, 8.67; IR νmax(cm−1): 1717, 1664. 1H-NMR δ (ppm): 1.17 , 1.35 , 5.75 ; 13C-NMR δ (ppm): 199.4(C-3), 171.2 (C-17), 169.1 (C-5), 124.0 (C-4); 82.7 (C-13), 52.4 (C-9), 45.9 (C-14), 39.2 (C-12), 38.4 (C-10), 38.0 (C-8), 35.3 (C-1), 33.9 (C-2), 32.4 (C-6), 30.4 (C-7), 28.7 (C-16), 30.0 (C-11), 20.0 (C-18), 19.9 (C-15), 17.45 (C-19). The spectroscopic data were in agreement with those of authentic sample isolated by us in previous studies [P. lanosocoeruleum KCH 3012 strain is a promising fungus that may be used in commercial processes, and which offers a new route to potentially active steroidal lactones. Among all the microorganisms that have been examined so far, this strain demonstrates the highest activity of Baeyer-Villiger monooxygenase against 5α-saturated steroids of the androstane series. Taking into account the catalytic abilities of this strain explored so far and its high 3β-HSD activity, one should keep in mind that it can potentially modulate the estrogen/androgen balance in Nature.The results obtained in this study indicate that the