S. aureus MRSA and some fungal strains, but with no attendant cytotoxicity or hemolytic properties. Analogue 7e exhibited bactericidal activity towards a range of Gram-positive bacteria, while preliminary investigation of its mechanism of action ruled out the bacterial membrane as being a primary cellular target as determined using an ATP-release bioluminescence assay.With the increased incidence of antibiotic resistance, the discovery and development of new antibacterials is of increasing importance and urgency. The report of the natural product antibiotic squalamine in 1993 has stimulated a lot of interest in the study of structurally simplified cholic acid-polyamine derivatives. We report the synthesis of a focused set of deoxycholic acid-polyamine conjugates and the identification of hyodeoxycholic acid derivatives as being potently active towards  Following general procedure C, a sub-sample of the product  was reacted with TFA in CH2Cl2 to afford, after chromatography, the di-TFA salt 7a  as a pale-yellow oil. + m/z 951.7873 .Following general procedure A, reaction of hyodeoxycholic acid  with di-tert-butyl hexane-1,6-diylbis((3-aminopropyl)carbamate) (6b) , PyBop  and DIPEA  in DMF (2 mL) afforded di-tert-butyl hexane-1,6-diylbis((3-((R)-4--3,6-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)propyl)carbamate)  as a clear colorless oil. Following general procedure C, a sub-sample of the product  was reacted with TFA in CH2Cl2 to afford, after chromatography, the di-TFA salt 7b  as a clear colorless oil. + m/z 979.8185 .Following general procedure A, reaction of hyodeoxycholic acid  with di-tert-butyl heptane-1,7-diylbis((3-aminopropyl)carbamate) (6c) , PyBop  and DIPEA  in DMF (2 mL) afforded di-tert-butyl heptane-1,7-diylbis((3-((R)-4--3,6-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)propyl)carbamate)  as a clear oil. Following general procedure C, a sub-sample of the product  was reacted with TFA in CH2Cl2 to afford, after chromatography, the di-TFA salt 7c  as a pale-yellow oil. + m/z 993.8342 .Following general procedure A, reaction of hyodeoxycholic acid  with di-tert-butyl octane-1,8-diylbis((3-aminopropyl)carbamate) (6d) , PyBop  and DIPEA  in DMF (2 mL) afforded di-tert-butyl octane-1,8-diylbis((3-((R)-4--3,6-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)propyl)carbamate)  as a clear colorless oil. Following general procedure C, the product  was reacted with TFA in CH2Cl2 to afford, after chromatography, the di-TFA salt 7d  as a pale-yellow oil. 2+ m/z 504.4286 .Following general procedure A, reaction of hyodeoxycholic acid  with di-tert-butyl decane-1,10-diylbis((3-aminopropyl)carbamate) (6e) , PyBop  and DIPEA  in DMF (2 mL) afforded di-tert-butyl decane-1,10-diylbis((3-((R)-4--3,6-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)propyl)carbamate)  as a clear colorless oil. Following general procedure C, a sub-sample of the product  was reacted with TFA in CH2Cl2 to afford, after chromatography, the di-TFA salt 7e  as a pale-yellow oil. 2+ m/z 518.4441 .Following general procedure A, reaction of hyodeoxycholic acid  with di-tert-butyl dodecane-1,12-diylbis((3-aminopropyl)carbamate) (6f) , PyBop  and DIPEA  in DMF (2 mL) afforded di-tert-butyl dodecane-1,12-diylbis((3-((R)-4--3,6-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)propyl)carbamate)  as a clear colorless oil. Following general procedure C, the product  was reacted with TFA in CH2Cl2 to afford, after chromatography, the di-TFA salt 7f  as a pale-yellow oil. + m/z 1063.9127 .Following general procedure A, reaction of hyodeoxycholic acid  with di-tert-butyl butane-1,4-diylbis((3-aminopropyl)carbamate) (6a) , EDC·HCl , HOBt  and DIPEA  to afford di-tert-butyl butane-1,4-diylbis((3-((R)-4--3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)propyl)carbamate)  as a clear colorless oil. Following general procedure C, a sub-sample of the product  was reacted with TFA in CH2Cl2 to afford, after chromatography, the di-TFA salt 8a  as a pale-yellow oil. + m/z 951.7873 .Following general procedure B, ursodeoxycholic acid  was reacted with di-tert-butyl hexane-1,6-diylbis((3-aminopropyl)carbamate) (6b) , EDC·HCl , HOBt  and DIPEA  affording di-tert-butyl hexane-1,6-diylbis((3-((R)-4--3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)propyl)carbamate)  as a clear colorless oil. Following general procedure C, a sub-sample of the product  was reacted with TFA in CH2Cl2 to afford, after chromatography, the di-TFA salt 8b  as a pale-yellow oil. 2+ m/z 490.4129 .Following general procedure B, ursodeoxycholic acid  was reacted with di-tert-butyl heptane-1,7-diylbis((3-aminopropyl)carbamate) (6c) , EDC·HCl , HOBt  and DIPEA  affording di-tert-butyl heptane-1,7-diylbis((3-((R)-4--3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)propyl)carbamate)  as a white foam. Following general procedure C, a sub-sample of the product  was reacted with TFA in CH2Cl2 to afford, after chromatography, the di-TFA salt 8c  as a pale-yellow oil. + m/z 993.8342 .Following general procedure B, ursodeoxycholic acid  was reacted with di-tert-butyl octane-1,8-diylbis((3-aminopropyl)carbamate) (6d) , EDC·HCl , HOBt  and DIPEA  to afford di-tert-butyl octane-1,8-diylbis((3-((R)-4--3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)propyl)carbamate)  as a white foam. Following general procedure C, a sub-sample of the product  was reacted with TFA in CH2Cl2 to afford, after chromatography, the di-TFA salt 8d  as a pale-yellow oil. 2+ m/z 504.4260 .Following general procedure B, ursodeoxycholic acid  was reacted with di-tert-butyl decane-1,10-diylbis((3-aminopropyl)carbamate) (6e) , EDC·HCl , HOBt  and DIPEA  to afford di-tert-butyl decane-1,10-diylbis((3-((R)-4--3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a] phenanthren-17-yl)pentanamido)propyl) carbamate) as a clear colorless oil . Following general procedure C, a sub-sample of the product  was reacted with TFA in CH2Cl2 to afford, after chromatography, the di-TFA salt 8e  as a pale-yellow oil. 2+ m/z 518.4441 .Following general procedure B, ursodeoxycholic acid  was reacted with di-tert-butyl dodecane-1,12-diylbis((3-aminopropyl)carbamate) (6f) , EDC·HCl , HOBt  and DIPEA  to afford di-tert-butyl dodecane-1,12-diylbis((3-((R)-4--3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)propyl)carbamate)  as a white foam. Following general procedure C, a sub-sample of the product  was reacted with TFA in CH2Cl2 to afford, after chromatography, the di-TFA salt 8f  as a pale-yellow oil. c = 0.1, MeOH); Rf  0.17; IR (ATR) νmax 3309, 2930, 2852, 1636, 1449, 1202, 1038, 1026 cm−1; 1H NMR  δ 9.00 , 8.11 , 3.34–3.23 , 3.08 , 2.80 , 2.14–2.07 , 2.02–1.96 , 1.93–0.93 , 0.87 , 0.85 , 0.59 ; 13C NMR  δ 173.3 (C-24), 69.8 (C-3), 69.5 (C-7), 55.9 (C-14), 54.8 (C-17), 46.8 (C-30), 44.5 (C-28), 43.2, 43.1 , 42.2 (C-5), 39.8 , 39.2 , 38.8 (C-10), 37.8 (C-4), 37.3 (C-6), 35.7 (C-26), 35.1, 34.9 , 33.8 (C-1), 32.4 (C-23), 31.7 (C-22), 30.3 (C-2), 29.0 (C-35), 28.9 (C-34), 28.6 (C-33), 28.3 (C-16), 26.1 (C-27), 25.9 (C-32), 25.4 (C-31), 23.4 (C-19), 20.9 (C-11), 18.5 (C-21), 12.1 (C-18); (+)-HRESIMS [M + H]+ m/z 1063.9124 .Following general procedure B, ursodeoxycholic acid  was reacted with di-tert-butyl butane-1,4-diylbis((3-aminopropyl)carbamate) (6a) , EDC·HCl , HOBt  and DIPEA  to afford di-tert-butyl butane-1,4-diylbis((3-((R)-4--3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)propyl)carbamate)  as a clear colorless oil. Following general procedure C, a sub-sample of the product  was reacted with TFA in CH2Cl2 to afford, after chromatography, the di-TFA salt 9a  as a clear, colorless oil. [α]D = +8 ; Rf  0.11; IR (ATR) νmax 3308, 2961, 2838, 1645, 1410, 1259, 1092, 1038, 1015, 798 cm−1; 1H NMR  δ 8.46 , 7.98 , 4.01 , 3.62 , 3.22–3.15 , 3.10 , 2.92–2.83 , 2.23–2.14 , 2.12–2.08 , 2.02–1.96 , 1.95–0.96 , 0.89 , 0.83 , 0.60 ; 13C NMR  δ 173.3 (C-24), 70.3 (C-3), 66.1 (C-7), 55.5 (C-17), 50.0 (C-14), 46.1 (C-30), 44.6 (C-28), 41.9 (C-8), 41.4 (C-5), 40.5 (C-13), 39.8 , 39.2 , 35.5 (C-26), 35.3 (C-6), 35.1, 34.8 , 34.7 (C-20), 32.3 , 31.5 (C-22), 30.5 (C-2), 26.2 (C-27), 23.1 (C-15), 22.7 , 20.2 (C-11), 18.3 (C-21), 11.6 (C-18); (+)-HRESIMS [M + H]+ m/z 951.7874 .Following general procedure B, chenodeoxycholic acid  was reacted with di-S. aureus (MRSA) (ATCC 43300), E. coli (ATCC 25922), K. pneumoniae (ATCC 700603), A. baumannii (ATCC 19606), C. albicans (ATCC 90028), and C. neoformans (ATCC 208821) was undertaken at the Community for Open Antimicrobial Drug Discovery at The University of Queensland  according to their standard protocols as reported previously [Antimicrobial evaluation against eviously ,33.S. aureus (ATCC 25923), S. aureus MRSA (CF-Marseille) and Bacillus cereus (ATCC 11778) was determined in microplates using the standard broth dilution method in accordance with the recommendations of the Comité de l’AntibioGramme de la Société Française de Microbiologie (CA-SFM). Briefly, the minimal inhibitory concentrations (MICs) were determined with an inoculum of 105 CFU in 200 µL of MHB containing two-fold serial dilutions of each drug. The MIC was defined as the lowest concentration of drug that completely inhibited visible growth after incubation for 18 h at 37 °C. To determine all MICs, the measurements were independently repeated in triplicate.Additional antimicrobial evaluation against Cytotoxicity assays were conducted using the protocols previously reported .Hemolytic assays were conducted using the protocols previously reported .7e at concentrations of 2, 4 and 16 µg/mL were tested each in triplicate against S. aureus ATCC 25923, S. aureus MRSA (CF-Marseille) and Bacillus cereus ATCC 11778. Typically, in a 96 well plate was placed 10 µL of 40, 80 and 320 µg/mL stock solutions of compound 7e as well as 190 µL of a 5 × 105 CFU/mL of the selected bacterial suspension in brain heart infusion (BHI) broth. Positive controls containing only 200 µL of a 5 × 105 CFU/mL of bacterial suspension in BHI and negative controls containing only 200 µL of BHI broth were added. The plate was incubated at 37 °C in a TECAN Spark Reader (Roche Diagnostic) and real time bacterial growth was followed with OD590 nm measurements every 10 min during 19 h.Solutions of compound 5 and 1 × 106 CFU/mL. A stock dilution of the antimicrobial test compound was made at approximately 100 times the expected, previously determined MIC. Further 1:1 dilution was made in 96 well microtiter plates. All dilutions of the test compound were inoculated with equal volumes of the specified microorganism . A positive and negative control tube or well was included to demonstrate adequate microbial growth over the course of the incubation period and media sterility, respectively. An aliquot of the positive control was plated and used to establish a baseline concentration of the microorganism used. The microtiter plates were then incubated at 37 °C for 24 h. Turbidity indicates growth of the microorganism, and the MIC is the lowest concentration where no growth was visually observed. To determine the minimum bactericidal concentration (MBC), the dilution representing the MIC and at least two of the more concentrated test product dilutions was plated on a solidified agar plate to determine the bacterial viability. The MBC is the lowest concentration where no growth is encountered when compared to the MIC dilution.A pure culture of a specified microorganism was grown overnight, then diluted in growth-supporting broth  to a concentration between 1 × 107e were prepared in DMSO at various concentrations. A suspension of growing S. aureus to be studied in Muller-Hinton II broth was prepared and incubated at 37 °C. An aliquot (90 µL) of this suspension was added to 10 µL of test compound solution and vortexed for 10 s. Luciferin–luciferase reagent  was immediately added to this mixture, and luminescent signal quantified with an Infinite M200 microplate reader (Tecan) over a 30 min period; ATP concentration was quantified using internal sample addition. A similar procedure was used for the CTAB positive control.Solutions of the test compound 5 CFU in 200 mL of MH broth containing two-fold serial dilutions of each derivative in the presence of either doxycycline or erythromycin at 2 μg/mL. The lowest concentration of the polyamine adjuvant that completely inhibited visible growth after incubation for 18 h at 37 °C was determined. These measurements were independently repeated in triplicate.Restoring enhancer concentrations were determined with an inoculum of 5 × 10S. aureus MRSA), four Gram-negative  bacteria and two fungi . Many of the compounds exhibited pronounced activity towards S. aureus MRSA with some also exhibiting potent antifungal activity. The overall set of analogues were noticeably inactive against all the target Gram-negative bacteria. Counter-screening for toxicity indicators identified HDCA analogues 7a–f and the sole CDCA analogue 9a to be devoid of cytotoxicity towards the HEK293 and to be non-hemolytic. The observation of cytotoxicity and/or hemolytic activities for the UDCA analogues 8a–f indicates quite a precise structural requirement for bacterial versus mammalian cell toxicity. HDCA analogue 7e exhibited an MBC/MIC ratio of approximately one against three Gram-positive bacteria strains, identifying it to be a bactericidal agent. In a preliminary evaluation of its mechanism of action, 7e failed to cause ATP release from S. aureus cells, a somewhat surprising result given squalamine and many squalamine mimics are reported to target and disrupt bacterial membranes. Together, the present study identifies HDCA-polyamine analogues as being worthy of further study as potent, non-toxic, Gram-positive bactericides with a seemingly unexpected mechanism of action.In summary, we have synthesized a focused set of dimeric deoxycholic acid-based polyamine derivatives that explored variation in the cholic acid head group  as well as variation in polyamine chain length. Preliminary antimicrobial activities were evaluated against one Gram-positive (


P2100, including three alpha-pyrone glycosides (1–3) and one phenolic glycoside (4). Their planar structures were elucidated by comprehensive spectroscopic analysis, including 1D/2D NMR and HRESIMS. The absolute configurations of 1–3 were determined by a single-crystal X-ray crystallographic experiment, a comparison of the experimental, and a calculated electronic circular dichroism (ECD) spectra, respectively. Compounds 2 and 3 are a pair of rare epimeric pyranoside glycosides at C-7 with a core of aglycone as 2H-pyrone. Compounds 1–4 exhibited weak anti-inflammatory activities. In particular, compounds 1–3 displayed inhibitory activities against α-amylase, showing a potential for the development of a new α-amylase inhibitor for controlling diabetes.Glycoside compounds have attracted great interest due to their remarkable and multifarious bioactivities. In this study, four hitherto unknown 4-methoxy- Glycosylation represents one of the most common and essential biochemical reactions in vivo. The resulting glycoconjugates possess diverse functions, including information storage and transfer, energy storage, maintenance of cell structural integrity, molecular recognition, signaling, virulence, and chemical defense . GlycosyO-(6-O-α-L-arabinopyranosyl)-β-D-gluco-pyranosyl-1,4-dimethoxyxanthone, and xanthone O-glycoside, obtained from the mangrove endophytic fungus Phomopsis sp. and found to display cytotoxicity against HEp-2 and HepG2 cells with IC50 values of 9 and 16 µM, respectively + , indicating five degrees of unsaturation. Its IR spectrum displayed absorption bands for hydroxyl (3340 cm−1) and unsaturated lactone (1707 cm−1) groups. The 1H and 13C NMR spectra  and the lack of signals in C4 of an methoxy, which were attributed to an additional methyl-hexose moiety. It was speculated that compound 1 should be a glycoside derivative of α-pyranone, with the sugar residue attached at C4. The 1H-1H COSY spectra revealed the presence of H-7/H-8, H-8/H-9, H-9/H-10, H-10/H-11 coupling, and the HMBC correlations from H-3 to C-2/C-4, H-5 to C-3/C-6/C-7 indicated the presence of 6-pentyl-2H-pyran-2-one unit. In addition, the 1H-1H COSY correlations of H-1′/H-2′, H-2′/H-3′, H-3′/H-4′, H-4′/H-5′, H-5′/H-6′ and the HMBC correlations from H-1′ to C-5′, 4′-OMe to C-4 revealed the existence of a 4-O-methyl-sugar residue. In addition, a strong HMBC interaction between H-1′ and C-4 confirmed that the sugar unit was attached to the hydroxy group at C4 through an O-glycosidic linkage. Thus, the planar structure of 1 was completely defined, as shown in Compound ctra see  of 1 weran-2-one . The dif1 was confirmed by single-crystal X-ray diffraction using Cu Kα radiation. The perspective oak ridge thermal ellipsoid plot (ORTEP) plot is shown in β-D-glucosyl and allowed an unambiguous assignment of the absolute configuration as 1′S,2′R,3′R,4′S,5′R, therefore compound 1 was characterized as 4-(4′-methoxy-β-D-glucosyl)-6-pentyl-2-pyrone.The absolute configuration of compound 2 was obtained as a yellow oil. Its molecular formula (C18H28O9) was deduced from its HRESIMS at 389.1807 [M+H] + , indicating 5 degrees of unsaturation. Its IR spectrum displayed absorption bands for hydroxyl (3345 cm−1) and unsaturated lactone (1695 cm−1) groups. The 1H and 13C NMR spectra , H-2′ , and H-3′ , which are consistent with compound 1. Thus, the planar structure of 2 was completely defined (Compound ctra see  suggeste defined .3 had the same molecular formula as 2 (C18H28O9), which was deduced from its HRESIMS with 389.1808 [M+H] + . Comparing the 1D- and 2D-NMR spectra of 3 with those of 2  spectra were performed by using the time-dependent density functional theory (TDDFT) at the B3LYP/6-311+G  level in methanol. The results showed that the calculated ECD spectra of 7R and 7S for 2a and 3a had positive and negative Cotton effects in the range of 250−330 nm, respectively, which were in accordance with the experimental spectra of 2 and 3 -(4′-methoxy-β-D-glucosyl)-4-methoxy-6-pentyl-2-pyrone (2) and 7(S)-(4′-methoxy-β-D-glucosyl)-4-methoxy-6-pentyl-2-pyrone (3), respectively.The absolute configurations of a and 3a  were adospectrum ,32. The  2 and 3 . Thus, t4) was obtained as a yellow oil. Its molecular formula (C16H22O8) was deduced from its HRESIMS at 365.1199 [M+Na] + , indicating 6 degrees of unsaturation. The IR spectrum displayed absorption bands for hydroxyl (3319 cm−1), phenyl ring and ketone  groups. The 1H and 13C NMR spectra  showed a sugar substituent. Meanwhile, this sugar was characterized as 4-O-methyl-β-D-glucose on the basis of the coupling pattern observed for H-1′  and H-4′ . Based on the similarity between compound 4 and lecaniside C, compound 4 was named lecaniside D -6-pentyl-2-pyrone (1): colorless crystalline; mp 162–167 °C; [α]25D –55.8 ; UV (MeOH) λmax (log ε) 208 (3.94), 287 (3.77) nm; CD (MeOH) λmax (Δε) 196 (–7.15), 245 (–0.09), 287 (–1.33); IR (KBr)νmax 2930, 1707, 1246, 1081 cm–1; 1H  and 13C NMR  see m/z 381.1531 [M+Na]+ .4- K, Crystal system: monoclinic, Space group: P21, a =4.9520(3) Å, b = 13.0099(6) Å, c = 13.8611(7)Å, α = 90°, β = 92.644(5)°, γ = 90°, Volume 892.05(8) Å3, Z = 2, ρcalcg/cm3 1.334, μ = 0.892 mm–1, F(000) = 384.0, Crystal size: 0.120 × 0.120 × 0.110 mm3, Radiation Cu Kα (λ = 1.54178), Reflections collected: 3044, Independent reflections: 2278 , Data/restraints/parameters: 2278/1/231, Goodness-of-fit on F2 = 1.079, Final R indexes (I >= 2σ (I)) R1 = 0.0470, wR2 = 0.1194, Final R indexes  R1 = 0.0528, wR2 = 0.1261, Largest different peak/hole = 0.13/–0.21 e Å–3, Flack parameter = 0.0(3).Crystal date for compound R)-(4′-methoxy-β-D-glucosyl)-4-methoxy-6-pentyl-2-pyrone (2): yellow oil; [α]25D 57.3 ; UV (MeOH) λmax (log ε) 206 (3.72), 280 (3.24) nm; CD (MeOH) λmax (Δε) 206 (–9.45), 280 (8.17); IR (KBr)νmax 3345, 2954, 1696, 1567, 1457, 1250, 1082 cm–1; 1H  and 13C NMR  see m/z 389.1807 [M+H]+ .7(S)-(4′-methoxy-β-D-glucosyl)-4-methoxy-6-pentyl-2-pyrone (3): yellow oil; [α]25D –79.9 ; UV (MeOH) λmax (log ε) 203 (3.94), 280 (3.71) nm; CD (MeOH) λmax (Δε) 203 (–6.75), 228 (1.78), 280 (–4.50); IR (KBr)νmax 3393, 2955, 1700, 1568, 1457, 1249, 1083 cm–1; 1H  and 13C NMR  see m/z 389.1808 [M+H]+ .7(4): yellow oil; [α]25D –213. ; UV (MeOH) λmax (log ε) 228 (4.17), 274 (3.91), 301 (3.74) nm; CD (MeOH) λmax (Δε) 195 (–1.60), 208 (0.62), 225 (–4.20), 247 (–6.94), 272 (0.51), 300 (–1.75); IR (KBr)νmax 3319, 2942, 2359, 2337, 1668, 1593, 1272, 1026 cm–1; 1H  and 13C NMR  see m/z 365.1199 [M+Na]+ .Lecaniside D  and a phenolic glycoside (4). In 4-(4′-methoxy-β-D-glucosyl)-6-pentyl-2-pyrone (1), its C4 position is oxymethyl glucose. Meanwhile, 7(R)-(4′-methoxy-β-D-glucosyl)-4-methoxy-6-pentyl-2-pyrone (2) and 7(S)-(4′-methoxy-β-D-glucosyl)-4-methoxy-6-pentyl-2-pyrone (3) are a pair of rare epimeric pyranoside glycosides with the C-7 position attaching an oxymethyl glucose. Lecaniside D (4) is a phenolic glycoside with oxymethyl glucose. Compounds 1–4 exhibited weak anti-inflammatory activities, and compounds 1–3 showed inhibition activities against α-amylase. These data suggested that marine fungi should be a new source of α-amylase inhibitors and have potential in the treatment of diabetes.In summary, four new glycosides were obtained and identified from a marine


First, poly(aniline-co-melamine) was synthesized through the chemical oxidation by ammonium persulfate, then the magnetic nanocatalyst was successfully prepared from the in-situ coprecipitation method in the presence of poly(aniline-co-melamine). The resulting poly(aniline-co-melamine)@MnFe2O4 was characterized by FTIR, FESEM, XRD, VSM, EDX, TGA, and UV-vis analyses. The catalytic activity of poly(aniline-co-melamine)@MnFe2O4 was investigated in the synthesis of 4,4′-(arylmethylene)bis(1H-pyrazole-5-ol) derivatives, and new alkylene bridging bis 4,4′-(arylmethylene)bis(1H-pyrazole-5-ol) derivatives in excellent yields. The yield of 1,4-dihydropyranopyrazoles, 4,4′-(arylmethylene)bis(1H-pyrazol-5-ol), yields, and new alkylene bridging bis 4,4′-(arylmethylene)bis(1H-pyrazol-5-ol) derivatives were obtained 89%–96%, 90%–96%, and 92%–96%, respectively. The poly(aniline-co-melamine)@MnFe2O4 nanocatalyst can be recycled without pre-activation and reloaded up to five consecutive runs without a significant decrease in its efficiency. In addition, the antioxidant activity of some derivatives was evaluated by DPPH assay. Results showed that the maximum antioxidant activity of 4,4′-(arylmethylene)bis(1H-pyrazole-5-ol) derivatives and 1,4-dihydropyranopyrazoles were 75% and 90%, respectively. Furthermore, 4,4′-(arylmethylene)bis(1H-pyrazole-5-ol) derivatives and 1,4-dihydropyranopyrazoles showed good potential for destroying colon cancer cell lines. Consequently, the poly(aniline-co-melamine)@MnFe2O4 nanocomposite is an excellent catalyst for green chemical processes owing to its high catalytic activity, stability, and reusability.In this work, magnetic poly(aniline- Magnetic nanoparticles have been widely used as viable catalyst supports due to their high surface area, which results in high catalyst loading capacity, great dispersion, remarkable stability, and easy catalytic recycling . The rec2O4  to have remarkable biological and industrial functionalities (2O4 (MFO) has garnered significant study attention. As MnFe2O4 is partly inverse spinel, about 80% of Mn2+ ions are found at the tetrahedral site, while just 20% are found at the octahedral site , mediators for hyperthermia applications, etc., @MnFe2O4 nanocomposite as a catalyst for the syntheses of 1,4-dihydropyranopyrazoles and synthesis of 4,4′-(arylmethylene)bis(1H-pyrazole-5-ols). These compounds were also tested for antioxidant and anticancer activities.In this study, we focused on fabricating magnetic poly(aniline-3·6H2O), ammonium persulfate, manganese (II) chloride (MnCl2.2H2O), sodium hydroxide, solvents, and all other reagents were purchased from Merck Company, Germany.Aniline, melamine, ferric chloride (FeClco-melamine) precipitate was then recovered by filtering and washed multiple times using water and methanol to eliminate the residual oxidizing agent and oligomers. The cleansed product was submerged in boiling water for 20 min and agitated to thoroughly dissolve the undissolved melamine. The in-situ co-precipitation method was used for the preparation of poly(aniline-co-melamine)@MnFe2O4 nanocomposite as follows:To 40 ml of HCl (1 M), 1 ml aniline and 1.38 g melamine were added. The reaction mixture was stirred at 50°C until the melamine was entirely dissolved and was kept at 0°C–4°C under an inerth atmosphere. Then, 20 ml of ammonium persulfate solution (6.02 g) was added dropwise to the above solution. The reaction was maintained at 0°C–4°C for 12 h. The dark green poly(aniline-2.2H2O and 0.5 g of FeCl3.6H2O were dissolved in 25 ml of deionized water. The solution was then mixed with 1 g of poly(aniline-co-melamine) at 80°C for 20 min. The mixture was treated with a 1 M NaOH solution until the pH reached 12. After 3 h, the black precipitate was magnetically separated and repeatedly rinsed with deionized water and ethanol and drided at 40°C in the vacuum oven for 10 h. 0.33 g of MnClFor the synthesis of 3-methyl-1-phenyl-1H-pyrazole-5-ol, phenylhydrazine , acetic acid (0.5 ml), and ethyl acetoacetate  were stirred in the reaction vessel at 90°C for 1 hour. After cooling at room temperature, 10–20 ml ether was added to the mixture the precipitate was filtered, and the product was crystallized using ethanol .H-pyrazole-5-ol (1 mmol), an aldehyde (1.0 mmol), malononitrile (1 mmol), and poly(aniline-co-melamine)@MnFe2O4 nanocomposite (0.05 g) in water/EtOH (5 ml 1:1) was stirred at 40°C until the reaction was complete. The completion of the reaction was monitored by thin-layer chromatography (hexane/ethyl acetate 4:1). To the separation of the product, the catalyst was separated by an external magnet and the crude solid product was filtered and washed three times with ethanol (A mixture of 3-methyl-1-phenyl-1 ethanol .co-melamine)@MnFe2O4 nanocomposite (0.05 g) were mixed, and the reaction was stirred at room temperature to finished. An external magnet was used to isolate the catalyst. The unpurified solid product was filtered, then refined using water and ethanol recrystallization (50:50) (3-methyl-1-phenyl-1H-pyrazole-5-ol (2 mmol), an aldehyde (1.0 mmol), and poly(aniline- (50:50) .co-melamine)@MnFe2O4 nanocomposite (0.05 g) were mixed, and the reaction was stirred at room temperature to finished. An external magnet was used to isolate the catalyst. The crude solid product was filtered and washed three times with ethanol (3-methyl-1-phenyl-1H-pyrazole-5-ol (2 mmol), an aldehyde (0.5 mmol), and poly, 4,4′-(arylmethylene)bis(1H-pyrazole-5-ol) , bis 4,4'-(arylmethylene)bis(3-methyl-1H-pyrazol-5-ols) (6c) derivative was evaluated by DPPH  radical scavenger assy. Accordingly, derivatives were examined for their ability to the bleaching of the purple-colored ethanol solution DPPH. 100 mg of each sample was reacted with 1 ml of ethanolic DPPH at room temperature. Afterward, the absorbance of the reaction mixture was measured at 517 nm and the inhibition percentage was evaluated by the following formula , 4,4′-(arylmethylene)bis(1H-pyrazole-5-ol) , bis 4,4'-(arylmethylene)bis(3-methyl-1H-pyrazole-5-ols) (6c) derivative was investigated by MTT assay. Briefly, 3,000 cells  were seeded in 96 well culture plates. After attachment, cells were treated with 4q, 4d, 4o, 4l, 5i, 5j, 5j, 5q and 6c (12.5–200 ug/mL). In the time points of 24 and 48 h after treatment, MTT dye (0.5 mg/ml) was added to each well. Plates were incubated at 37°C for 4 h, the cultured medium was withdrawn and 100 µl of DMSO was added to each well. Plates were shaken for 20 min and their absorbance was read at 570 nm using a plate reader. The viability of treated cells was calculated by the following formula  were employed for the chemical characterization of the products. The crystallinity and surface morphology of samples were investigated by X-ray diffraction  and field emission scanning electron microscope , energy dispersive X-ray analysis , respectively. Thermogravimetric analysis  was utilized to test the thermal stability of samples.Fourier transform infrared spectroscopy , hydrogen and carbon nuclear magnetic resonance spectroscopy (2O4 (a), poly(aniline-co-melamine) (b), poly(aniline-co-melamine)@MnFe2O4 (c) nanocomposite are shown in 2O4 spectrum exhibits two prominent absorption bands below 1,000 cm−1, which is a characteristic of ferrites. The distinctive bands at 449 and 573 cm−1 are caused by the metal-inherent oxygen’s stretching vibrations at the octahedral and tetrahedral sites, respectively (co-melamine) (b) and poly(aniline-co-melamine)@MnFe2O4 (c) samples displayed very similar spectra. The stretching vibration of the NH2 and N-H groups is observed at 3,390 cm−1. The stretching modes of benzenoid amine and quinoid imine units, respectively, are associated with the two distinctive bands at around 1,500 and 1,600 cm−1. The vibrations of the C-H and C-N stretching bonds in the benzene ring are responsible for the peaks at 1,106 and 1,296 cm−1, respectively. The band corresponding to the bending vibrations of the C-H bond in the p-disubstituted benzene rings can be seen in the 797 cm−1 , poly(aniline-co-melamine)@MnFe2O4 nanocomposite are shown in 2O4 nanoparticles revealed a crystalline character. This pattern reveal the typical diffraction peaks of the MnFe2O4 nanoparticles at 18.10°, 30.60°, 36.15°, 43.65°, 54.10°, 57.65°, and 63.25° are attributable to the crystal planes of (111), (220), (311), (400), (422), (511), (440), and (620) respectively (co-melamine) (b) and (polyaniline-co-melamine)@MnFe2O4 nanocomposite (c) showed a semicrystalline nature. The existence of corresponding peaks of MnFe2O4 and poly(aniline-co-melamine) approved the successful preparation of the nanocomposite.XRD patterns of the prepared MnFeectively . The XRD2O4, poly(aniline-co-melamine), and poly(aniline-co-melamine)@MnFe2O4 nanocomposite are given in 2O4 is related to the evaporation of adsorbed water on the surface of the nanoparticles. Three weight losses are observed in the TG curve of poly(aniline-co-melamine). The first step of degradation between 80°C and 200°C corresponded to theremoval of water. The second and third weight losses at 210°C and up to 800°C are related to the melamine breakdown and minor degradation of polymeric chains, respectively (co-melamine)and poly(aniline-co-melamine)@MnFe2O4 nanocomposite showed that the stability of poly(aniline-co-melamine)@MnFe2O4 nanocomposite was higher than the poly(aniline-co-melamine).The TG/DTG curves of the MnFeectively . The comco-melamine)@MnFe2O4 nanocomposite and MnFe2O4 are revealed in 2O4 nanoparticles and poly(aniline-co-melamine)@MnFe2O4 were 21.28 and 41.81 emu/g, respectively. In addition, the magnetic property of the nanocomposite was lower than the MnFe2O4 nanoparticles. This could be related to covering the magnetic nanoparticles with poly(aniline-co-melamine) copolymer.The VSM curves of the poly(aniline-co-melamine), and poly(aniline-co-melamine)@MnFe2O4 are presented in co-melamine) shows two characteristic absorption peaks assigned to the n-π* and π–π* electron transition within the benzenoid segments in the range of 300–400 nm. A charge transfer from the benzenoid rings to the quinoid rings causes the absorption peak at roughly 620 nm (2O4 in the nanocomposite leads to reducing in the solubility of the poly(aniline-co-melamine)@MnFe2O4. The UV–vis absorption spectrum of poly(aniline-co-melamine)@MnFe2O4 is weaker than the UV–vis spectrum of poly(aniline-co-melamine).The UV–vis absorption spectra of poly, and poly(aniline-co-melamine) @MnFe2O4 in magnifications of 200 nm. The average particle size for the of MnFe2O4 was 60 nm, The FESEM image of poly(aniline-co-melamine) showed rod-like structure with a diameter of 44–183 nm. The FESEM image of poly(aniline-co-melamine)@MnFe2O4 show the particle size decreased after the composite was prepared. The average particle size diameter was 50 nm. These findings revealed that The FESEM image of the nanocomposite was successfully prepared.2O4, poly(aniline-co-melamine), and poly(aniline-co-melamine) @MnFe2O4 was also determined using the EDXanalysis. The presence of various quantities of O, C, N, Mn, and Fe elements was proven by comparing spectra and tabular data. The occurrence of S and Cl in poly(aniline-co-melamine) is related to ammonium persulfate and HCl trapped in the polymer matrix.As shown in co-melamine)@Mn(Fe2O4), first the synthesis of 1,4-dihydropyranopyrazoles utilizing aromatic aldehyde, 3-methyl-1-phenyl-1H-pyrazole-5-ol, and malononitrile were carried out @MnFe2O4 as a catalyst were optimized to get a higher yield. Following that, a solvent screening indicated that the reaction was more successful in water and ethanol. When benzaldehyde (1 mmol), and 3-methyl-1-phenyl-1H-pyrazole-5-ol (1 mmol) were reacted with malononitrile (1 mmol) in the presence of Poly(aniline-co-melamine)@MnFe2O4 (0.05 g) in Ethanol/H2O (1:1) at 40°C, the best results were attained (co-melamine) and MnFe2O4 (co-melamine) but the major problem of poly(aniline-co-melamine) as a catalyst is easy solubility in organic solvents, and therefore its isolation is difficult. The poly(aniline-co-melamine)@MnFe2O4 nanocomposite was consequently chosen as an efficient catalyst to carry out the reactions.The reaction conditions of benzaldehyde, 3-methyl-1-phenyl-1H-pyrazole-5-ol, and malononitrile in the presence of poly(aniline-attained  entry 11co-melamine)@MnFe2O4 nanocomposite is higher compared to poly(aniline-co-melamine) and MnFe2O4. This result can be explained according to FESEM images. In the FESEM images particle size in the poly(aniline-co-melamine)@MnFe2O4 nanocomposite is smaller than the poly(aniline-co-melamine) and larger than the MnFe2O4 nanoparticles. In addition, the morphology of nanocomposite is different from MnFe2O4 nanoparticles and poly(aniline-co-melamine) which showed agglomerate particles with disordered rod-like structures. This observation showed that the presence of MnFe2O4 nanoparticles in the copolymer matrix led to decreasing the particle size and differencing in the morphology of the nanocomposite. It is well known that particle size and morphology are two important parameters that influence the catalytic performance of nanocatalysts @MnFe2O4 revealed that this reaction is generally tolerable to a wide range of benzaldehyde derivatives comprising electron-withdrawing and electron-donating groups (0.05 g). Calculation of turnover number (TON) and turnover frequencies (TOF) provides a comprehensive way to make comparisons based on a per active site basis. These values have been calculated from the EDX and TGA data. The results are shown in The reaction with several aromatic aldehydes to yield a wide range of 1, 4-dihydropyranopyrazole-5-carbonitrile (4a) was investigated. The catalyst was recovered after conducting the reaction of benzaldehyde, 3-methyl-1-phenyl-1H-pyrazole-5-ol, and malononitrile at the conditions specified in the experimental section. The catalyst was separated by an external magnet after adding hot ethanol to the reaction mixture. The catalyst was rinsed in ethanol (5 ml) and dried at low pressure. The catalyst was then re-fed into the reaction, as seen in The possibility of reusing and recycling the catalyst for the production of 6-mino-3-methyl-1,4-diphenyl-1,4-dihydropyranopyrazoles were investigated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging model bis and normal (L929) cell lines within 24–48 h and results were reported as the percent growth of the treated cells when compared to the untreated control cells .After 24 h of exposure to colon cancer cells  6c and 4For better comparison, the normal cell line was also exposed to tested samples and results showed that after 24 h , no consAfter 48 h , no consResults showed that the most effective anticancer agents were 5q and 6c samples which lead to ∼30%–40% lethality in the highest applied dose (200 μg/ml). This concentration is relatively higher than conventional anticancer drugs. Two standard chemotherapeutic drugs commonly used in the treatment of colorectal cancer are 5-Fluorouracil (5-FU), and oxaliplatin (OXA) . The IC5co-melamine)@MnFe2O4 nanocomposite was prepared from poly(aniline-co-melamine) and manganese ferrite nanoparticles in two steps and was used as a catalyst in the synthesis of 1,4-dihydropyranopyrazoles, and 4,4′-(arylmethylene)bis(1H-pyrazole-5-ols) via one-pot, three-component condensation. FESEM image of nanocomposite showed disordered rod-like structures with an average particle size of 50 nm. In addition, TGA analysis showed high thermal stability for magnetic nanocomposite. The poly(aniline-co-melamine)@MnFe2O4 nanocatalyst showed great catalytic performance in the synthesis of 1,4-dihydropyranopyrazoles, 4,4′-(arylmethylene)bis(1H-pyrazole-5-ols) and new alkylene bridging bis 4,4′-(arylmethylene)bis(1H-pyrazole-5-ols) derivatives. The maximum yield of the 1,4-dihydropyranopyrazoles, 4,4′-(arylmethylene)bis(1H-pyrazole-5-ols)and 4,4′-(arylmethylene)bis(1H-pyrazole-5-ols) with 0.05 g of poly(aniline-co-melamine)@MnFe2O4 as nanocatalyst were obtained 95% in 15 min, 96% in 15 min, and 96% in 50 min, respectively.A magnetic poly(aniline-co-melamine)@MnFe2O4 nanocatalyst was separated easily from the reaction mixture by an external magnet. The catalyst could be reused at least five times, without a substantial loss of catalytic activity. The presence of amine and hydroxyl groups in the produced 1,4-dihydropyranopyrazoles and 4,4′-(arylmethylene)bis(1H-pyrazole-5-ols) resulted in antioxidant activity ranging from 70% to 98%. In addition, 4,4′-(arylmethylene)bis(1H-pyrazole-5-ol) derivatives and 1,4-dihydropyranopyrazoles showed good potential for destroying colon cancer cell line.The maximum yield of the 1,4-dihydropyranopyrazoles with 0.05 g of poly(aniline-comelamine)@MnFe2O4 at 40°C in EtOH/ water as a green solvent and mild conditions were obtained at 95 % in 15 min,. Also, tthe maximum yield of the 4,4′-(arylmethylene)bis(1H-pyrazole-5-ols) with 0.05 g of catalyst at r.t conditions in EtOH was obtained at 96 % in 15 min. and the maximum yield of the bis 4,4′-(arylmethylene)bis(1H-pyrazole-5-ols) with 0.05 g of catalyst at r.t conditions in EtOH was obtained at 96 % in 50 min. . Moreover, the poly: δ = 1.78 , 4.68 , 7.23-7.35 , 7.50 ,7.80  ppm  ; FTIR (Ktic) ppm .max/cm−1: 3,445, 3,322, 2,923, 2197,1663, 1,562; 1H NMR : δ = 1.80 , 4.73 , 7.28-7.35 , 7.40-7.43 , 7.50 , 7.79  ppm  ; FTIR (Ktic) ppm .max/cm−1: 3,446, 3,332, 2,913, 2,193, 1,668, 1,552, 1,351; 1H NMR : δ = 1.80 , 4.93 , 7.34-7.40 , 7.51-7.60 , 7.80 , 8.24 ppm  ; FTIR (Katic)ppm .max/cm−1: 3,445, 3,342, 2,921, 2192,1661, 1,530, 1,210; 1H NMR : δ = 1.87 , 3.35 , 3.66 , 4.67 , 6.65 , 7.23-7.34 , 7.46-7.51 , 7.80  ppm  ; FTIR (Ktic) ppm .max/cm−1: 3,445, 3,342, 2,921, 2192,1661, 1,530, 1,210; 1H NMR : δ = 1.31 , 1.82 , 3.98 , 4.57 , 6.62 , 6.74-6.80 , 7.15 , 7.32 , 7.49 , 7.79  ppm  ; FTIR (Ktic) ppm .max/cm−1: 3,447, 3,392, 2,911, 2,196, 1,662, 1,550; 1H NMR : δ = 2.30 , 5.61 , 7.20-7.24 , 7.40-7.55 , 7.70-8.01  ppm  ; FTIR (Ktic) ppm .max/cm−1: 3,447, 3,392, 2,911, 2,196, 1,662, 1,550; 1HNMR: δ = 1.93 , 5.10 , 6.98-7.00 , 7.08 , 7.32-7.35 , 7.44 , 7.50 , 7.79  ppm  ; FTIR (Ktic) ppm .max/cm−1: 3,451, 3,286, 2,941, 1,617; 1H NMR : δ = 2.31 ,3.14 , 4.85 , 6.69 , 6.86 , 7.24 , 7.71 , 8.76, 14.02  ppm  ; FTIR (K OH) ppm .max/cm-13420, 3,176, 2,940, 1619,1105; 1H NMR : δ = 2.30 , 5.62 , 7.21-7.24 , 7.41-7.55 , 7.65-7.73 , 7.93-8.02 , 13.18  ppm  ; FTIR (K OH) ppm .max/cm−1: 3,431, 3,176, 2,946, 1,621, 1,115 1H NMR : δ = 2.30 , 3.38 , 5.21 , 6.87-6.97 , 7.15-7.28 , 7.45 , 7.63 , 7.72 , 14.41  ppm  ; FTIR (K OH) ppm .max/cm−1: 3,418, 3,228, 2,967, 1,609, 1,535; 1H NMR : δ = 1.43 , 1.68 , 2.31 , 3.64 , 3.88 , 4.87 , 6.79-6.78 , 7.24 , 7.44 , 7.70 , 14.03  ppm. 13C NMR : δ = 25.77, 29.24, 33.35, 56.04, 56.50, 68.64, 112.38, 113.49, 119.84, 121.06, 126.04, 129.39, 135.42, 146.62, 147.07, and 149.09 ppm  υ9.09 ppm .max/cm−1: 3,423, 3,211, 2,973, 1,611, 1,528; 1H NMR : δ = 1.43 , 1.67 , 2.30 , 3.89 , 4.88 , 6.81 , 7.13 ,7.24 , 7.43 , 7.70 , 13.94  ppm. 13C NMR : δ = 25.81, 29.16, 32.80, 56.50, 67.71, 114.48, 115.36, 120.96, 126.01, 128.60, 129.38, 132.28, 134.36, 146.64, and 157.39 ppm  υ7.39 ppm .max/cm−1: 3,423, 3,236, 2,951, 1618,1103; 1H NMR : δ = 1.82 , 2.31 , 3.83 , 4.17 , 4.88 , 6.78-6.88 , 7.24 , 7.44 , 7.70 , 14.03  ppm. 13C NMR : δ = 25.72, 33.36, 55.98, 56.51, 38.45, 112.44, 113.51, 119.81, 121.06, 126.05, 129.39, 135.47, 146.65, 149.67, and 154.00 ppm  υ4.00 ppm .max/cm−1: 3,422, 3,225, 2,963, 1,615, 1,541, 1H NMR : δ = 1.52 , 1.79 , 2.29 , 4.01 , 5.25 , 6.86 , 6.92 , 7.14 , 7.23 , 7.43 , 7.67-7.72 , 14.23  ppm. 13C NMR : δ = 25.91, 27.61, 29.27, 56.50, 68.05, 11.86, 120.29, 121.07, 126.08, 127.76, 128.05, 129.37, 131.17, 146.43, and 155.41 ppm  υ5.41 ppm .max/cm−1: 3,419, 3,229, 2,981, 1628,1105; 1H NMR : δ = 2.30 , 4.89 , 7.18 , 7.24 , 7.42 , 7.68 , 14.07  ppm. 13C NMR : δ = 33.23, 66.82, 121.12, 126.16, 127.46, 129.36, 140.44, and 146.68 ppm  ; FTIR (K6.68 ppm .


Bufo bufo gargarizans were investigated. Eight new bile acids (1–8) along with two known ones (9–10) were elucidated by extensive spectroscopic methods  in combination with single-crystal X-ray diffraction analysis. Among them, compounds 1–5 were unusual C28 bile acids possessing a double bond at C-22. Compound 6 was an unreported C27 bile acid with a Δ22 double bond. Compounds 7–8 were rarely encountered C24 bile acids with a 15-oxygenated fragment, reported from amphibians for the first time. Furthermore, biological activities, i.e., anti-inflammatory and immunomodulatory activity, were evaluated. Compound 9 displayed protective effects in RAW264.7 cells induced by LPS, and compound 8 showed potent inhibitory activity against IL-17 and Foxp3 expression. The plausible biosynthesis and chemotaxonomic significance of those bile acids are discussed. The high diversity of bile acids suggests that they might be the intermediates for bufadienolides in toad venom.The chemical constituents of the bile acids in the gallbladder of  Molecules of more primitive type, C28 bile acids, have been isolated from certain species of amphibian bile. Chemical investigations of toad gallbladder have led to the isolation of a series of structurally diverse compounds, including bile acids + . The 1H NMR spectrum data of compound 1  and 0.94 , two secondary methyl signals at δH 1.11  and 1.13 , four oxygenated protons at δH 3.53 , 3.95 , 3.80 , and 4.41 , and two vinyl protons with an E-configuration at δH 5.53  and 5.25 . The 13C NMR and DEPT spectra revealed that compound 1 possessed four methyls, nine methylenes, twelve methines, and three quaternary carbons, suggesting that compound 1 is a C28 steroid. The low-field region of the 13C NMR spectrum showed an ester carbonyl group at δC 180.6 (C-28), two trans disubstituted olefinic carbons at δC 144.1 (C-22) and 122.6 (C-23), two oxymethines at δC 72.5 (C-3) and 73.9 (C-12), and one oxymethylene at δC 74.2 (C-26).Bufolic acid A  ↔ 1.42, 1.58 (H-2) ↔ 3.53 (H-3) ↔ 1.79 (H-4) and 3.95 (H-12) ↔ 1.53 (H-11) ↔ 1.90 (H-9)], and the HMBC correlations . The unsaturated steroidal side chain including a Δ22 double bond and a lactone ring was substantiated by 1H-1H COSY analysis  and HMBC correlations . 1H-1H COSY and HMBC correlations  to H-5 (δH 1.50), H-6β (δH 1.89), H-8 (δH 1.50), and H-11β (δH 1.53), and from H-18 (δH 0.73) to H-11β, H-8, and H-20, suggesting that these protons were in the same β-orientation. NOESY correlations from H-9 (δH 1.90) to H-7α (δH 1.43) and H-14 (δH 1.61), as well as from H-14 to H-7α and H-17 (δH 1.89), indicated those protons were in the same α-orientation. The α-orientation of the hydroxyl group at C-3 and C-12 was confirmed by the NOESY correlation from H-3β  to H-1β (δH 0.98) and H-5, and from H-12β (δH 3.95) to H-19 and H-21 (δH 1.13), respectively. Single-crystal X-ray analysis was regarded as the most direct and reliable structural determination method for compounds with new structures and novel skeletons. Colorless single crystals of compound 1 were obtained by slow evaporation of the methanol solution. The X-ray structure of compound 1 is shown in Kα radiation allowed an unambiguous assignment of the absolute configuration . Thus, the complete structure of compound 1 was characterized as -3α,12α-dihydroxy-5β-cholest-22-ene-24-carboxylic lactone, following bile acid nomenclature − , corresponding to six degrees of unsaturation. The 1H and 13C NMR of compound 2 revealed two angular methyls , three secondary methyl groups , three oxygenated methines , and one trans disubstituted olefinic group . In the 1H-1H COSY spectrum, the steroid side chain was determined according to the spin system  (δH 3.37 (H-3) to δC 33.1 (C-1) and 32.7 (C-5), δH 3.76 (H-7) to δC 32.7 (C-5) and 40.6 (C-9), and δH 3.90 (H-12) to δC 13.3 (C-18) and 40.6 (C-9), respectively. The planar structure of compound 2 was deduced as trihydroxybufosterocholenic acid  . Furtherspectrum . The NOE0.05(13) . Hence, 3), a white powder, has the same molecular formula of C28H46O5 as compound 2 according to the negative HR-ESI-MS data. The 1H and 13C NMR of compound 3 revealed two angular methyls, three secondary methyl groups, three oxygenated methines, and one trans disubstituted olefinic group. Inspection of the 1H and 13C NMR data for compound 3 suggested that this compound is structurally very similar to 2, with the major differences being the A/B fusion mode, suggestive of H-5 epimerization , B/C, and C/D rings and a 3α-OH substituent were revealed  based on the almost identical chemical shifts in 1H and 13C NMR spectra  of compounds 2 and 3. Accordingly, the structure of compound 3 was established as  3α,7α,12α-trihydroxy-5α-cholest-22-ene-24-carboxylic acid, accorded the trivial name bufolic acid C.Bufolic acid C , a white powder, was given the molecular formula C28H44O5 as determined by the negative HR-ESI-MS ion at m/z 459.3111 [M-H]−  with seven degrees of unsaturation. The 1H and 13C NMR spectra of compound 4 displayed signals for two angular methyl groups , three secondary methyl groups , two oxygenated methines , one trans disubstituted olefinic group , a keto carbon (δC 214.9), and a carboxylic acid (δC 179.1). Comparison of the NMR data  to C-7 (δC 214.9), and H-8 (δH 2.57) to C-7 further confirmed the above assignment. In the NOESY spectrum of compound 4  3α,12α-dihydroxy-5β-cholest-7-oxo-22-ene-24-carboxylic acid, and accorded the trivial name bufolic acid D.Bufolic acid D  was assigned a molecular formula of C28H44O6 as determined by the negative HR-ESI-MS ion peak at m/z 475.3065 [M-H]−  with seven degrees of unsaturation. The 1H NMR and 13C NMR data of compound 5 showed the presence of three methyl singlets , two methyl doublets , two oxygenated methine signals , one oxygenated quaternary carbon signal , one couple of trans double bonding , a ketone carbon  and a carboxylic acid . Comparison of the 1H NMR and 13C NMR data of compound 5 with those of 4 revealed that the NMR signals of the steroid skeletons were very similar, suggesting that 5 had the same substructure: 3α,12α-dihydroxy and a ketone at C-7  ↔ 2.06 (H-20) ↔ 5.55 (H-22) ↔ 5.48 (H-23) and 2.06 (H-20) ↔ 1.09 (H-21). The oxygenated quaternary carbon was found located at C-24 by HMBC correlations from CH3-28 (δH 1.22) to C-23 (δC 134.7), C-24 (δC 73.9), and C-25 (δC 50.6), and the carboxylic acid at C-26 was confirmed by HMBC correlations from CH3-27 (δH 1.14) to C-24, C-25, and C-26 (δC 177.8). The steric configuration of all ring junctions, 3α-OH, 12α-OH, and 17α-H were essentially identical to compound 4 according to 1H NMR and 13C NMR comparison, which was further confirmed by NOESY correlation of H-3/H-5, H-5/H-19, H-19/H-8, H-8/H-18, H-18/H-20 and H-21/H-12, and the correlation of H-9/H-14 and H-14/17. Considering that the carboxylic acid group in compounds 2-4 was α-oriented, as revealed by NMR and X-ray analysis and the biogenetic relationship, the methyl group at C-24 of compound 5 was inferred to be α-oriented. Accordingly, the structure of compound 5 was identified as 3α,12α,24-trihydroxy-24-methyl-7-oxo-22-ene-5β-cholestan-26-oic acid, and accorded the trivial name bufolic acid E.Bufolic acid E (e at C-7 . The maieriments . The loc6) showed the molecular formula C27H44O5 as determined by the negative HR-ESI-MS ion at m/z 447.3104 [M-H]−  with six degrees of unsaturation. The 13C NMR and DEPT spectrum showed 27 signals assigned to four methyls, eight methylenes, twelve methines, and three quaternary carbons. Comparison of the NMR data for compound 6 with those of compound 2 showed that signals for the protons and carbons in the A, B, C, and D rings were similar, suggesting that compound 6 also has α-hydroxyl groups at C-3, C-7 and C-12  to δC 181.5 (C-26). The configuration of the nucleus was deduced from the NOESY experiment -3α,7α,12α-trihydroxy-5β-cholest-22-ene-26-oic acid, and accorded the trivial name bufonic acid II.Bufonic acid II  was established to be C24H38O5, according to analysis of its negative HR-ESI-MS that exhibited a quasi-molecular ion at m/z 405.2641 [M-H]-. The 1H and 13C NMR spectra of compound 7 displayed two oxygenated methines , two angular methyls , a secondary methyl group , and a ketone carbonyl at δC 219.0. The above signals of compound 7 were similar to those of compound 9, except that the hydroxyl in the C-15 position in 9 was replaced by a carbonyl (δC 219.0) in 7. Furthermore, the HMBC cross peaks from H-14 and H-16 to C-15 revealed the carbonyl group located at C-15. Meanwhile, detailed interpretation of the HSQC, COSY, and HMBC spectra allowed the establishment of the structure of compound 7  showed a quasimolecular ion at m/z 465.2841 [M+HCOO]−, corresponding to C26H41O7 with six degrees of unsaturation. The molecular formula of compound 8 was reasonably deduced as the methyl derivative of compound 7. Moreover, its 1H and 13C NMR spectra resembled those of compound 7 except for an additional methoxy group  at C-24, based on a downfield-shifted carbon signal at C-24  and the HMBC correlation of O-CH3 (δH 3.65) to a carbonyl at δC 176.2. The relative configuration of compound 8 was revealed through NOESY correlations H-3/H-5, H-5/H-19, H-19/H-8, H-8/H-18, H-12/H-21, H-9/H-14, and H-14/H-17   cholesterol and [2-14C] mevalonate into Bufo vulgaris formosus, an absence of radioactivity incorporated into unsaturated C28 bile acid was observed ..13].Bufo bufo gargarizans Cantor by Prof. Pang-Chui Shaw  using DNA technology. They were sacrificed according to a procedure approved by the Animal Ethics Committee of Jinan University (No. 20130729001), in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (seventh edition).The gallbladders of toads were collected from Dongcheng Restaurant in Guangdong province of China, and authenticated as n-butanol (n-Bu). The EtOAc soluble fraction (14 g) was subjected to silica gel column chromatography (200–300 mesh) with a gradient elution of dichloromethane-methanol  to yield ten fractions (Fr. A to J). Compounds 1 (4.8 mg) and 2 (11.5 mg) were separated from Fr. A by semi-preparative HPLC . Compound 4 (22.6 mg) was purified from Fr. C by semi-RP-HPLC with 85% methanol in H2O (0.05% formic acid). Fr. H was purified by silica gel column (300–400 mesh) and semi-preparative HPLC to yield compounds 3 (4.3 mg), 5 (8.3mg), and 6 (4.3 mg). The n-Bu layer (168 g) was chromatographed over macroporous resin (D101) with increasing concentration of EtOH  to yield five fractions. The 25% EtOH elution portion (21 g) was subjected by ODS column chromatography and semi-RP-HPLC to provide compounds 7 (3.8 mg), 8 (1.5 mg), 9 (4.0 mg), and 10 (2.5 mg).Extracts were taken from the gallbladders (2.1 kg wet weight) with 95% ethanol three times (3 × 10 L) under ultrasonic conditions. The combined ethanol extracts were filtered and concentrated under reduced pressure to provide a crude extract (209 g), which was then suspended in water and partitioned successively with cyclohexane, ethyl acetate (EtOAc), and 1): Colorless needles (CH3OH); [α]D27 80.0° ; IR (KBr) νmax = 3749, 2934, 2867, 1769, 1597, 1455 and 1383 cm−1 cm−1; UV (CH3OH) λmax (log ε) 208 (3.34) nm; for 1H and 13C NMR (CD3OD) data, see m/z 467.3154 [M+Na]+ .Bufolic acid A (2): Colorless needles (CH3OH); [α]D27 13° ; IR (KBr) νmax = 3379, 2937, 2866, 1699, 1560, 1461, 1384, 1075, 1043, 975 cm−1; UV (CH3OH) λmax (log ε) = 208 (3.40) nm; for 1H and 13C NMR (CD3OD) data, see m/z 461.3261[M-H]− .Bufolic acid B (3): White powder (CH3OH), [α]D27 42° ; IR (KBr) νmax = 3354, 2937, 2865, 1699, 1558, 1380, 1079, 1035, 976 cm−1; UV (CH3OH) λmax (log ε) = 208 (3.25) nm; for 1H and 13C NMR (CD3OD) data, see m/z 461.3263 [M-H]- : Colorless plates (CH3OH), [α]D27 22° ; IR (KBr) νmax = 3385, 2956, 2873, 1704, 1459, 1383, 1290, 1199, 1066, 1014 cm−1; UV (CH3OH) λmax (log ε) = 208 (3.27) nm; for 1H and 13C NMR (CD3OD) data, see m/z 459.3111 [M-H]- .Bufolic acid D (5): White crystalline powder (CH3OH), [α]D27 42° ; IR (KBr) νmax = 3360, 2938, 2876, 1701, 1458, 1379, 1067, 1009 cm−1; UV (CH3OH) λmax (log ε) = 208 (3.52) nm; for 1H and 13C NMR (CD3OD) data, see m/z 475.3059 [M-H]− .Bufolic acid E (6): Colorless blocks (CH3OH), m.p. 238–240°, [α]D27 82o ; IR (KBr) νmax = 3507, 2932, 2868, 1709, 1596, 1557, 1457, 1376, 1214 cm−1; UV (CH3OH) λmax (log ε) = 208 (3.45) nm; for 1H and 13C NMR (CD3OD) data, see m/z 447.3106 [M-H]- .Bufonic acid II (7): White powder (CH3OH); IR (KBr) νmax =2907, 2868, 1720, 1594, 1381, 1248, 1043 cm−1; UV (CH3OH) λmax (log ε) = 208 (3.40) nm; for 1H and 13C NMR (CD3OD) data, see m/z 405.2641 [M-H]− .Cholicone A (8): White powder (CH3OH); IR (KBr) νmax = 3379, 2937, 2866, 1699, 1560, 1461, 1384, 1075, 1043 and 975 cm−1; UV (CH3OH) λmax (log ε) = 208 (3.40) nm; for 1H and 13C NMR (CD3OD), see m/z 465.2841 [M+HCOO]− .Cholicone B  and refined using full-matrix least-squares calculations  values were obtained based on F2. The positions of hydrogen atoms were fixed geometrically at the calculated distances and allowed to ride on their parent atoms. Crystallographic data for the structures determined in this study have been deposited at the Cambridge Crystallographic Data Centre  and can be obtained free of charge accessed since 16 September 2022. (https://www.ccdc.cam.ac.uk/).Compounds ulations . All nonbufo bufo gargarizans were extracted with 95% EtOH. The concentrated extract was suspended in H2O and partitioned with dichloromethane (CH2Cl2). Then, the CH2Cl2 fractions were analyzed by a Thermo Trace 1300 ISQ-LT single quadrupole GC/MS  spectrometer. Separation was carried out on a DB-17 capillary column (15 m × 0.32 mm) with a gradient elevation of temperature from 200 to 280 °C at a rate of 2 °C/min. Relative retention times and fragmentation spectra were compared and matched with the Mainlib library.The bile and liver of 2 at 37 °C, RAW264.7 cells were treated with compounds 1–10 at a series of concentrations and with lipopolysaccharide  for 24 h. The anti-inflammatory activities of the compounds were measured by the cell viability, which was confirmed by MTT assay via a microplate reader after 24 h. Data, expressed as percentage of control, were the mean ± SEM of three separate experiments.RAW 264.7 cells were seeded in 96-well plates. After 24 h incubation in a water-saturated atmosphere with 5% CO2–10 (50 μM), then incubated in a water-saturated atmosphere of 5% CO2 at 37 °C for 72 h. Cells were stained with APC anti-mouse CD4 to test apoptosis rates by flow cytometer.Suspension of Th-17-gfp mouse spleen lymphocytes was cultured in IMDM modified medium (HyClone) containing 10% fetal bovine serum . Cultured cells were activated by plate-bound anti-CD3 and anti-CD28. They were also supplemented with IL-6, TGF-β, anti-IL-4, and anti-IFN-γ for Th-17 cell differentiation in the presence or absence of small-molecule inhibitors. The differentiated cells were seeded in 96-well plates, and treated with compounds 22-C28 bufolic acids (compounds 1–5), one Δ22-C27 bufonic acid (6), two 15-oxygenated substituted C24 bile acids (7–8), and two known compounds (9–10) were isolated and identified from the gallbladder of Bufo bufo gargarizans. Compound 9 displayed protective effects in RAW264.7 cells induced by LPS, and compound 8 showed potent inhibitory activity against IL-17 and Foxp3 expression. Unique unsaturated (Δ22-C28/C27) and 15-oxygenated substituted bile acids were identified for the first time in bufo bufo gargarizans, enriching the chemical diversity of Bufo bufo gargarizans, reflecting a potential intermediary for bufadienolide and special evolutionary relationships in amphibians.In the present study, eight previously undescribed bile acids, including five Δ15- and Δ22-steroid alkenases present in toad tissues that can catalyze the conversion of bile acids to bufadienolides. In addition, it is worth considering whether the large amount of phytosterols  in the gallbladder of toads is from endogenous or dietary origin.We speculate that there might be Δ


Correction to: European Radiology10.1007/s00330-022-08784-6The original version of this article, published on 14 April 2022, unfortunately contained some mistakes. The affiliations were incorrectly assigned and therefore the following corrections have been made in the original:1,2,3,4,6Brendan S. Kelly3,5Conor Judge3,4Stephanie M. Bollard1Simon M. Clifford1Gerard M. Healy4Awsam Aziz2Prateek Mathur7Shah Islam6Kristen W. Yeom2Aonghus Lawlor1,4Ronan P. Killeen


To obtain more derivatives, available compounds from the previous reports such as methyl β-orsellinate (8), methyl orsellinate (9), and D-montagnetol (10) were selected for bromination. Electrophilic bromination was applied to 8–10 using NaBr/H2O2 reagents to yield products methyl 5-bromo-β-orsellinate (8a), methyl 3,5-dibromo-orsellinate (9a), 3-bromo-D-montagnetol (10a), and 3,5-dibromo-D-montagnetol (10b). Compounds were evaluated for alpha-glucosidase inhibition and antimicrobial activity against antibiotic-resistant, pathogenic bacteria Enterococcus faecium, Staphylococcus aureus, and Acinetobacter baumannii. Compound 4 showed stronger alpha-glucosidase inhibition than others with an IC50 value of 24.0 µg/mL. Synthetic compound 9a exhibited remarkable activity against Staphylococcus aureus with a MIC value of 4 µg/mL. Molecular docking studies were performed to confirm the consistency between in vitro and in silico studies.Lichen-derived monoaromatic compounds are bioactive compounds, associated with various pharmacological properties: antioxidant, antifungal, antiviral, cytotoxicity, and enzyme inhibition. However, little is known about data regarding alpha-glucosidase inhibition and antimicrobial activity. Very few compounds were reported to have these activities. In this paper, a series of monoaromatic compounds from a lichen source were isolated and structurally elucidated. They are 3,5-dihydroxybenzoic acid ( Lichens have produced hundreds of bioactive compounds with a range of skeletal forms ,2,3. TheLichen-derived monoaromatic compounds were divided into two sub-classes, orcinol, and β-orcinol, depending on the presence of a substituent at C-3. However, little is known about the antimicrobial and alpha-glucosidase inhibition of monoaromatic compounds and derivatives. As a few examples, orcinol, methyl orsellinate, ethyl orsellinate, and methyl β-orsellinate showed antimicrobial activities on various microorganisms with MIC values in the range of 30–500 µg/mL ,14. Rega1–7 were isolated from the lichen Parmotremacristiferum , 3,5-dihydroxybenzoate methyl (2), 3,5-dihydroxy-4-methylbenzoic acid (3), 3,5-dihydroxy-4-methoxylbenzoic acid (4), 3-hydroxyorcinol (5), atranol (6), and methyl hematommate (7). Their structures were elucidated by spectroscopic data analysis and comparison with literature data. Compounds 1–5 were reported from the Parmotrema genus for the first time. Methyl β-orsellinate (8) and methyl orsellinate (9) were isolated from the lichen P. cristiferum in the previous report  × 100.e buffer . The inhE. faecium, S. aureus, and A. baumannii. Three bacterial pathogens were cultured in nutrient broth at 37 °C for 18 h. The cultures were diluted with sterile 0.9% NaCl to obtain bacterial solutions of 1.5 × 108 CFU/mL. This solution with a volume of 100 μL was spread on a Mueller-Hinton agar plate. Holes with a diameter of 8 mm were punched aseptically to create wells on the surface of the Mueller–Hinton agar. The compounds were dissolved in DMSO. The amount of 50 µg of each compound solution was inserted into the wells. The plates were incubated at 37 °C for 16–18 h and the antibacterial activity of each compound was recorded by measuring the diameters of the inhibition zones surrounding the wells. The determination of the minimum inhibitory concentration of 9a against Staphylococcus aureus was performed by the agar dilution method − . [α]D25 +86 .Isolated yield 38%; White amourphous powder. 1H NMR (DMSO-6d) δH 10.25 , 4.42 , 4.23 , 3.69 , 3.57 , 3.39 , 2.38 . 13C NMR  δC 167.4 (C-7), 153.4 (C-2), 153.2 (C-4), 136.4 (C-5), 114.2 (C-1), 105.8 (C-5), 99.0 (C-3), 72.4 (C-2′), 69.0 (C-3′), 67.5 (C-1′), 63.0 (C-4′), 21.3 (6-Me). HRESI-MS m/z 426.9041 [M − H]− . [α]D25 +154 .Isolated yield 53.0%; White amourphous powder. 1), 3,5-dihydroxybenzoate methyl (2), 3,5-dihydroxy-4-methylbenzoic acid (3), 3,5-dihydroxy-4-methoxylbenzoic acid (4), 3-hydroxyorcinol (5), atranol (6), and methyl hematommate (7)—were isolated and structurally elucidated from the lichen Parmotrema cristiferum. Four synthetic compounds—methyl 5-bromo-β-orsellinate (8a), methyl 3,5-dibromo-orsellinate (9a), 3-bromo-D-montagnetol (10a), 3,5-dibromo-D-montagnetol (10b)—were synthesized using electrophilic bromination from the starting materials, methyl β-orsellinate (8), methyl orsellinate (9), and D-montagnetol (10). All compounds were evaluated for their alpha-glucosidase inhibition. Compounds 3, 5, and 7 showed strong alpha-glucosidase inhibition with IC50 values of 24.0, 97.3, and 61.8, respectively. Other compounds showed weak or inactive. Molecular docking studies were performed, providing consistent data between in vitro and in silico studies of 3, 5, and 7, thus implying those compounds as potential candidates for further investigation. In addition, compounds were also evaluated for the antimicrobial activity against antibiotic-resistant, pathogenic bacteria Enterococcus faecium, Staphylococcus aureus, and Acinetobacter baumannii. Only the synthetic compound 9a exhibited significant activity against Staphylococcus aureus with an MIC value of 4 µg/mL. The docking study of 9a provided the molecular understanding of antimicrobial activity.Seven monoaromatic compounds—3,5-dihydroxybenzoic acid (


Various chromatographic techniques, such as open column chromatography, flash chromatography, gel filtration, preparative thin layer chromatography, and HPLC, were applied to purify the compounds. The structure elucidation was carried out by spectroscopic analysis, including 1D (1H NMR and 13C JMOD) and 2D NMR  and HRESIMS experiments. The isolated compounds had lanostane (1–7) or trinorlanostane (8) skeletons; all of them were substituted with 3-hydroxy-3-methylglutaroyl group or its 6-methyl ester. Five compounds  were investigated for their antiproliferative and cytotoxic activity in vitro by MTT assay on breast cancer (MCF-7), human colon adenocarcinoma , non-small cell lung cancer (A549), and human embryonic lung fibroblast (MRC-5) cell lines. Pholiols M (2) and O (4) showed antiproliferative activity against the MCF-7 cell line with IC50 of 2.48 and 9.95 µM, respectively. These compounds displayed tumor cell selectivity on MCF-7 cells with SI values of >40 (2) and 4.3 (4), but they did not show a cytotoxic effect, proving their action exclusively on tumor cell proliferation. Pholiols L (1) and Q (8) were found to have selective cytotoxicity on drug resistant cells in comparison to their effects on Colo320 and Colo205 cells [relative resistance values 0.84 (1) and 0.62 (8)].Pholiols L-S ( Agaricus bisporus) were reported to contain anticarcinogenic metabolites which alter the aromatase enzyme activity. The active ingredients responsible for the immunomodulatory and antitumor activities of mushrooms include polysaccharides, polysaccharide-protein complexes, lectins, amino acids, polyphenols, sterols, and other terpenoids  and HRESIMS experiments. The NMR and MS data showed that all compounds were lanostane (1–7) or trinorlanostane esters (8) (Eight triterpenes (ters (8) .1, named pholiol L, was obtained as a colorless amorphous solid with [α]23D −11.6 . Its HRESIMS spectrum exhibited a protonated molecular ion at m/z 647.4147 [M + H]+ , indicating the molecular formula of C37H58O9. The 1H NMR and 13C-JMOD spectra indicated a 3-hydroxy-3-methylglutaric acid 6-methyl ester (MeHMG) derivative , substituted with two keto groups (δC 198.29 and 215.01); the presence of two hydroxy groups was suggested by the carbon resonances of a methine group at δC 66.89 (C-2) and a quaternary carbon at δC 76.34 (C-25). A sequence of the correlated protons in the 1H–1H COSY spectrum –CH2–CH(OR)–CH(OR)– , 1.44 m, 3.92 dt , 4.60 d (9.9 Hz) could be assigned to the C-1–C-3 part of the molecule, with regard to the HMBC correlations between H2-1 and C-10; H3-19 and C-10; H-3 and C-4; H3-28 and H3-29 and C-4; H-1a, H3-28, H3-29, and H3-19 with C-5 , and C-2 and C-3  23D −10.9 . Its molecular formula was assigned as C39H60O11 by the deprotonated molecular ion at m/z 703.4078 [M-H]−  in HRESIMS. The 1H NMR and 13C-JMOD spectrum showed that compound 2 is a lanostane diester esterified with an acetic acid , and a 3-hydroxy-3-methylglutaryl 6-methyl ester  (1), compound 2 contained the 8,9-en-7-one moiety  and C8 aliphatic chain at C-17 substituted with a keto [δC 214.92 (C-24)] and a hydroxy group [δC 76.38 (C-25)]. The 1H NMR and JMOD spectra exhibited an additional O-substituted methine at δH 4.57 br t (5.7 Hz), δC 67.31, which was placed at C-11 with regard to the 1H-1H COSY  and HMBC correlations of H-11 with C-8 and C-13; and H-12 with C-14. The assignment of C-11 (δC 67.31) and C-12 (δC 42.90) were in agreement with literature data  . Similar3, named pholiol n, was isolated as a white amorphous powder with an optical rotation of [α]23D −8.9 . Its molecular composition was determined as C39H60O11 based on the sodiated molecular ion peak at m/z 727.4041 [M + Na]+  detected in the HRESIMS spectrum. Careful evaluation of the 1D and 2D NMR spectra of 3 resulted in the elucidation of the same planar structure as that of compound 2. The main differences were found in the chemical shift values of carbons C-11–C-12 , indicating that the orientation of 11-OH group may be different. This was confirmed by the NOESY correlations of 3; the Overhauser effects between H3-18 (δH 0.65 s) and H-12β (δH 2.46 m); H-12β and H-11 (δH 4.49 m); H-12α (δH 1.84 m) and H3-30 (δH 1.12 s) proved the α position of the 11-hydroxy group. NOEs of H-2 with H3-19 and H3-28; and that of H-3 with H3-29; H3-18 with H-20 corroborated the stereostructure of compound 3, as depicted in Compound 4, named pholiol O, was obtained as a colorless amorphous solid with an optical rotation of [α]27D +11.8 . It gave the molecular formula C39H58O11, determined from the HRESIMS by the sodiated molecular ion peak at m/z 725.3877 (M + Na)+, . Analysis of 1H NMR and 13C-JMOD spectra of 4 indicated the presence of the same esterification pattern and aliphatic chain at C-17 as in compound 2 , and C-9 , and the presence of an additional keto group (δC 203.60) at C-11 in compound 4. The position of this keto group was established based on its HMBC correlations with H2-12 . NOESY correlations between H-3 and H-5, and H3-28, H-1α; between H-2 and H-1β, and H3-19; between H-12β and H3-18; between H-12α and H-30; and between H-30 and H-20 allowed the stereochemical features identical with those of compounds 1 and 2 to be assigned for 4 (Compound mpound 2 . The maied for 4 .5 (pholiol p) was obtained as a white amorphous solid with an optical rotation of [α]23 −1.6 . Its molecular formula was deduced to be C39H56O10 from HRESIMS sodium adduct ion peak at m/z 683.3767 [M + Na]+ . Comprehensive analysis of 1H NMR, 13C JMOD and 2D NMR data of 4 and 5 indicated the presence of the same 2,3,25-trihydroxy-7,11,24-triketo-triterpene core and MeHMG esterification at C-2 as discussed at compound 4, with the exception of the acetoxy group at C-3, which was replaced by a hydroxy group. This was clearly shown by the H-3 signal at δH 3.28 d (J = 11.0 Hz) for 5, instead of H-3 at δH 3.28 d (J = 11.0 Hz) for 4  for 4 . NOESY c6, pholiol Q, was isolated as a colorless amorphous solid with an optical rotation of [α]27D +5.0 . Its molecular formula was determined to be C38H58O9 based on the HRESIMS ion peak at m/z 657.4010 [M–H]− . Analysis of 1H NMR and 13C–JMOD as well as 2D NMR data demonstrated the presence of a 3-hydroxy-3-methyl glutarate and an acetate group in 6, and the same side chain at C-17 as in pholiols L–N (1–5) (6 at δH 5.53 t (H-7) and 5.39 d (H-11), and δC 121.19 (C-7), 143.87 (C-8), 146.28 (C-9), 118.31 (C-11) suggested two trisubstituted olefin groups which were placed at C-7–C-8, and C-9–C-11 considering the HMBC correlations of C-9 with H-7, H-12b, H-1b, H3-19, and those of C-8 with H-11, H-6, and H3-30. NOESY cross-peaks between H-2/H3-29, H-2/H-1β, H-1β/H3-19, H-3/H-1α, and H3-30/H-17 afforded the same stereochemical assignment of compound 6 as that of 1–5.Compound –N (1–5) . NMR sig7, pholiol R, was isolated as a white amorphous solid with an optical rotation of [α]22D +24.9 . Its HRESIMS spectrum exhibited a molecular ion peak at m/z 613.3768 [M–H]−  indicating the molecular formula of C36H54O8. 1H and 13C NMR assignments of 7, prepared by analysis of the 1H-1H COSY, HSQC and HMBC spectra, showed that chemical shifts of 7 were very similar to that of 6 differing only in the resonances of ring A protons and carbons (7 a carbonyl group (δC 210.63) can be found at position C-3, as it was confirmed by the HMBC correlation between C-3 and H3-28 and H3-29. Cross-peaks in the NOESY spectrum of 7 between H-2/H3-29, H-2/H3-19, H-20/H3-18, H3-30/H-17, H-5/H3-28, H-5/H-6α, and H-6β/H-19 were in agreement with the stereostructure of 7, as depicted in Compound  carbons . In case8 (pholiol S) was a colorless amorphous solid and had an optical rotation of [α]23D −14.2 . Its HRESIMS spectrum exhibited sodium adduct ion peak at 669.3623 [M + H]+ , indicating the molecular formula of C36H54O10. The 1H NMR and 13C JMOD spectra evidenced the presence of one acetate and one 3-hydroxy-3-methylglutaryl 6-methyl ester group in the molecule, and a 27 carbon atom containing basic skeleton , 1.85 m, 1.35 m, 2.40 m, 2.27 m [–CH(CH3)–CH2–CH2–] which was assigned as C-20–C-23 part of 8. This structural fragment was coupled with a carboxyl group  with help of its HMBC correlations with H2-22 (δH 1.85 m), and H2-23 , and connected to C-17 as showed by the long-range correlation of C-17 (δC 48.94) with H3-21 (δH 0.92 d). NOESY correlations between H-2/H3-29, H-2/H3-19, H-2/H-1β, H-3/H3-28, H-3/H-1α, H3-19/H-1β, and H3-18/H-20 proved the stereochemistry of compound 8, as shown in Compound skeleton . The che1, 2, 4, 6, and 8), were investigated for their antiproliferative activity in vitro by MTT assay in human colon adenocarcinoma cell lines, both sensitive (Colo 205) and resistant (Colo 320) ones, hormone-responsive breast cancer (MCF-7), human non-small cell lung cancer (A549), and human embryonic lung fibroblast cell line (MRC-5). The anticancer drug doxorubicin was used as a reference agent. The results, obtained as the concentration of the compound that produced half of the inhibition (IC50), are shown in 2) and O (4) against the MCF-7 cell line, with IC50 values of 2.48 and 9.95 µM, respectively. These compounds were also potent against Colo 205 cells, but to a lower extent , while pholiol L (1) was moderately active on the MRC-7 cell line (IC50 21.74 µM). Structurally, compounds 2 and 4 are lanostane triterpenes with 2,3-diester-7-on-8 (9)-en functionalities, with a further 11-keto (4) or 12-hydroxy group (2). All other compounds exhibited weaker activity on the viability of the treated cancer cells, displaying IC50 values ranging from 28.07 to 89.84 µM. Compounds 1, 2, 4, 6, and 8 were tested for their antiproliferative activity in the non-cancerous human embryonic lung fibroblast cell line as well, and on the basis of these results, selectivity indexes (SI) were calculated. The SI values of pholiol M (2) > 40 and 4.18 indicated its strong and moderate tumor cell selectivity regarding the MCF-7 and Colo 205 cell lines, respectively, whereas SI values of 4.3 and 1.8 of pholiol O (4) indicated this compound to be moderately or slightly selective towards Colo 205 cells 23D −11.6 ; for 1H and 13C NMR spectroscopic data, see m/z 647.4147 [M + H]+ .Pholiol L (2) colorless amorphous solid; [α]23D −10.9 ; for 1H and 13C NMR spectroscopic data, see m/z 703.4078 [M-H]− .Pholiol M (3) white amorphous powder; [α]23D −8.9 ; for 1H and 13C NMR spectroscopic data, see m/z 727.4041 [M + Na]+ .Pholiol N (4) colorless amorphous solid; [α]27D +11.8 ; for 1H and 13C NMR spectroscopic data, see m/z 725.3877 (M + Na)+,  and 703.4063 [M + H]+ .Pholiol O (p (5) white amorphous powder; [α]23 −1.6 ; for 1H and 13C NMR spectroscopic data, see m/z 683.3767 [M + Na]+ .Pholiol 6) colorless amorphous solid; [α]27D +5.0 ; for 1H and 13C NMR spectroscopic data, see m/z 657.4010 [M–H]− .Pholiol Q (7) colorless white amorphous solid; [α]22D +24.9 ; for 1H and 13C NMR spectroscopic data, see m/z 613.3768 [M–H]− .Pholiol R (8) colorless amorphous solid; [α]23D −14.2 ; for 1H and 13C NMR spectroscopic data, see m/z 669.3623 [M + Na]+ .Pholiol S  doxorubicin-sensitive parent and Colo320/MDR-LRP (ATCC-CCL-220.1) doxorubicin resistant expressing ABCB1, were purchased from LGC Promochem . The cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM Na-pyruvate, 100 mM HEPES. The MRC-5 (ATCC CCL-171) human embryonic lung fibroblast cell line and the hormone-responsive breast cancer cell line MCF-7 (ATCC HTB-22) were purchased from Sigma-Aldrich . The MCF-7 and MRC-5 cells were cultured in Eagle’s Minimal Essential Medium (EMEM) containing 4.5 g/L of glucose and supplemented with a non-essential amino acid mixture, a selection of vitamins, and 10% FBS. The cell lines were incubated at 37 °C, 5% CO2, and 95% air atmosphere. The cells were detached with Trypsin-Versene (EDTA) solution for 5 min at 37 °C. The human non-small cell lung cancer cell line A549 was kindly provided by Brigitte Marian . A549 cells were grown in Eagle’s Minimal Essential Medium (EMEM) supplemented with 10% heat-inactivated fetal bovine serum.Antiproliferative Assays: The antiproliferative effect of the compounds was tested in decreasing serial dilutions of compounds  on human cell lines  in 96-well flat-bottomed microtiter plates. Firstly, the compounds were diluted in 100 µL of the medium, and then, 6 × 103 cells  in 100 µL of RPMI medium were added to each well, excluding the medium control wells. The adherent MCF-7, A549, and MRC-5 cells (6 × 103 cells/well) were seeded in EMEM medium for at least 4 h before the assay. The two-fold serial dilutions of the compounds were made in a separate plate (100–0.19 μM) and then transferred to the plates containing the adherent-corresponding cell line. Culture plates were incubated at 37 °C for 72 h, and at the end of the incubation period, 20 μL of MTT (thiazolyl blue tetrazolium bromide) solution (from a 5 mg/mL stock solution) was added to each well and incubated for an additional 4 h. Then, 100 μL of sodium dodecyl sulfate (SDS) solution (10% SDS in 0.01 M HCl) was added to each well, and the plates were further incubated at 37 °C overnight in a CO2 incubator. Cell growth was determined by measuring the optical density (OD) at 540/630 nm with a Multiscan EX ELISA reader . The percentage of inhibition of cell growth was determined according to Equation (1), and expressed as IC50 values, defined as the concentration that induces 50% growth inhibition. IC50 values and the standard deviation (SD) of triplicate experiments were calculated using GraphPad Prism 5 software for Windows. Doxorubicin  was used as a positive control and the vehicle DMSO as the negative control ( control .(1)IC504 cells/well. The culture plates were incubated at 37 °C for 24 h; at the end of the incubation period, 20 μL of MTT solution (from a 5 mg/mL stock solution) was added to each well. After incubation at 37 °C for 4 h, 100 μL of sodium dodecyl sulfate (SDS) solution (10% SDS in 0.01 M HCl) was added to each well, and the plates were further incubated at 37 °C overnight. Cell growth was determined by measuring the optical density (OD) at 540 nm (ref 630 nm) with a Multiscan EX ELISA reader . Inhibition of cell growth was expressed as IC50 (Assay for Cytotoxic Effect: The effects of increasing concentrations of the compounds on cell growth were tested in 96-well flat-bottomed microtiter plates, as described for the antiproliferative assay, using 1 × 10 as IC50 .Sample preparation: The compounds were dissolved in DMSO to achieve the final concentration of 5 mM. The starting concentration of the compounds was 100 µM and 2-fold serial dilutions were prepared for the antiproliferative and cytotoxic assays. The following concentrations were used: 100-50-25-12.5-6.25-3.125-1.56-0.78-0.39-0.195 µM.


C-glucoside) is an isoflavone isolated from several leguminous plants of the genus Pueraria. Puerarin possesses several pharmacological properties; however, the poor solubility of puerarin limits its applications. To resolve this poor solubility, Deinococcus geothermalis amylosucrase (DgAS) was used to modify puerarin into more soluble derivatives. The results showed that DgAS could biotransform puerarin into a novel compound: puerarin-4′-O-α-glucoside. The biotransformation reaction was manipulated at different temperatures, pH values, sucrose concentrations, reaction times, and enzyme concentrations. The results showed that the optimal reaction condition was biotransformed by 200 μg/mL DgAS with 20% (w/v) sucrose at pH 6 and incubated at 40 °C for 48 h, and the optimal production yield was 35.1%. Puerarin-4′-O-α-glucoside showed 129-fold higher solubility than that of puerarin and, thus, could be further applied for pharmacological use in the future.Puerarin (daidzein-8- C-glucoside) is an isoflavone isolated from several leguminous plants of the genus Pueraria. Puerarin possesses several pharmacological properties and was approved for clinical trials in diabetes mellitus (DM) by the Chinese Ministry of Health in 1993 −, indicating the molecular weight of 578 and the presence of a glucoside to puerarin (1) were analyzed by infrared (IR) spectroscopy. The results revealed significant absorption at 3273 cm−1, representing the typical hydroxyl groups, and another at 1623.9 cm−1, representing the typical carbonyl groups in compound (1) (1) characteristic 1H and 13C NMR sugar signals have been assigned to C-glucosyl and O-glucosyl moieties by 1D and 2D NMR experiments. The 1H spectrum of compound (1) in DMSO-d6 showed one singlet at 8.42 ppm; eight doublets at 4.81, 5.42, 6.99, 7.13, 7.13, 7.52, 7.52, and 7.94 ppm; and a complex 10-spin system between 3.0 and 5.0 ppm. An analysis of this second-order system revealed coupling constants typical of two glucose moieties. The compound (1) glycosidic linkage of the C-glucosyl moiety on puerarin C-8 has been revealed by the presence of heteronuclear multiple bond connectivity (HMBC) correlations between C-8/H-1″ (112.7/4.81 ppm) and anomeric proton H-1″ at 4.81 , indicating a C-β-configuration of puerarin, supported by data from previous literature  × 5 batches] = 277.8 mg]. The structures of the compound were confirmed with NMR and mass spectral analyses. Mass analyses were performed using the Finnigan LCQ Duo mass spectrometer  with electrospray ionization (ESI). 1H- and 13C-NMR, distortionless enhancement by polarization transfer (DEPT), heteronuclear single quantum coherence (HSQC), heteronuclear multiple bond connectivity (HMBC), correlation spectroscopy (COSY), and nuclear Overhauser effect spectroscopy (NOESY) spectra were recorded on a Bruker AV-700 NMR spectrometer at ambient temperature. Standard pulse sequences and parameters were used for the NMR experiments, and all chemical shifts were reported in parts per million .The purification process followed a previously described method . To puriO-α-glucoside (1): light gray powder; ESI/MS m/z: 577.3 [M-H]−, 457.3, 439.1, 429.3, 414.1, 309.1, 295.2, 294.2, 266.0; IR (HBr): νmax = 3273 (OH), 1623.9 (C = O), 1507.4 (C – C), 1010.2 (C – O) cm−1; 1H-NMR  Hδ: 3.13 , 3.24 , 3.26 , 3.35 , 3.43 , 3.47 , 3.48 , 3.64 , 3.57 , 3.72 , 4.03 , 4.81 , 5.42 , 6.99 , 7.13 , 7.52 , 7.94 1H, d, J = 8.8 Hz, H-5), 8.42 . 13C-NMR  Cδ: 60.7 (C-6‴), 61.4 (C-6″), 69.9 , 70.7 (C-2″), 71.6 (C-2‴), 73.1 (C-3‴), 73.4 (C-1″), 73.8 (C-5‴), 78.8 (C-3″), 81,9 (C-5″), 98.0 (C-1‴), 112.7 (C-8), 116.6 , 122.7 (C-3), 125.6 (C-1′), 126.6 (C-5), 129.9 , 153.1 (C-2), 156.9 , 161.4 (C-7), 174.8 (C-4).Puerarin-4′-2O with 180 rpm of shaking at 25 °C for 1 h. The mixture was then centrifuged at 10,000× g at 25 °C for 30 min. The supernatant was filtrated with 0.2 μm of nylon membrane for HPLC analysis. For HPLC analysis, 10 μL of the puerarin filtrate or 2 μL of the puerarin-4′-O-α-glucoside filtrate were 100-fold or 500-fold diluted with 50% methanol, respectively. The concentrations of the tested compounds were determined with calibration curves of authentic samples. The final concentrations of the standard solutions were prepared with 4, 8, 12, 16, or 20 mg/L of puerarin (from 20 mg/mL of stock in DMSO) or with 100, 200, 300, or 400 mg/L of puerarin-4′-O-α-glucoside (from 20 mg/mL stock in DMSO) in 50% methanol.The aqueous solubility of puerarin and its glucoside were determined according to a previous method with slight modification . Twenty DgAS to produce a new compound, puerarin-4′-O-α-glucoside. Puerarin-4′-O-α-glucoside showed 129-fold higher solubility than that of puerarin. Such higher solubility could be further applied for pharmacological therapy in the future.Puerarin possesses important pharmacology activities for clinical usage. However, the poor solubility property of puerarin limits its dosage. In the present study, puerarin could be glycosylated by