The structures of the above compounds were established by a combination of spectroscopic methods, including HR-ESI-MS, 1H-NMR, 13C-NMR, HMQC and HMBC spectra. All compounds were isolated from and identified in this species for the first time and compounds 1-6 are new for the genus Croton.A new flavone, named crotoncaudatin ( Croton belongs to the family of Euphorbiaceae, with more than 700 species in the World, widely distributed throughout tropical and subtropical regions. There are 21 species in China, mainly growing in the southern provinces  to yield a new compound, named crotoncaudatin (1), along with nine known ones 2-10. The known flavonoids were readily identified as 3,5,6,7,8,3′,4′-heptamethoxyflavone (2) + ion peak at m/z 453.1127 and a [M+H]+ ion peak at m/z 431.1308, corresponding to the molecular formula C22H22O9 , possessing twelve degrees of unsaturation, which was further confirmed by the 1H- and 13C-NMR data , 3.88, 3.91 , 3.94 , 4.08 , and 4.08 , with the corresponding δC 61.9, 61.6, 56.2, 56.2, 61.6, and 61.6, respectively.], one oxygenated -CH2 group [δH 5.14  with the correlated δC 68.0 (C-5)], two aromatic protons , and thirteen quaternary carbons  including a carbonyl carbon at δC 169.4 (C-7). The above mentioned data suggested compound 1 to be a hexamethoxylated flavone. In the HMBC spectrum, correlations were observed between δH 3.85 and δC 149.0 (C-8), δH 3.88 and δC 144.5 (C-9), δH 3.91 and δC 152.8 (C-3), δH 3.94 and δC 150.4 (C-2), δH 4.08 and δC 151.6(C-10), δH 4.08 and δC 138.7 (C-11), confirming the locations of the methoxyl groups. The long range correlations between δH 5.14 (H-5) and δC 105.2 (C-1), 118.1 (C-12b), 126.2 (C-4a), and 136.8 (C-6a) suggested a cyclization between the ring-B and ring-C through a –OCH2 group. Other key HMBC correlations are shown in 1 was identified conclusively as 2,3,8,9,10,11-hexamethoxy-7-oxo-[2]benzopyrano + and 431.1308 [M+H]+, . 1H- and 13C-NMR spectral data see Crotoncaudatin  were obtained, consistent with some reports that polymethoxylated flavones were also presented in C. schiedeanus [C. ciliatoglanduliferus [C. brasiliensis [C. cajucara [Croton, e.g. nearly none of the American Croton species has been reported to produce diterpenoids except C. draco [Previous reports have shown that the species of the genus iedeanus , C. ciliiliensis , and C. cajucara . MoreoveC. draco . Thus, t


The structures of the new compounds were elucidated by detailed spectroscopic analyses, including 1D and 2D NMR techniques and chemical methods. The absolute configurations of the new compounds 5 and 6 were determined by comparing their optical rotation values with the hydrolysis products of the known compounds 3 and 4, respectively, derived from the same plant. On the basis of an analysis of their physical and chemical properties we show that the alkaline hydrolysis of 3 and 4 afforded the new compounds 5 and 6, respectively.Two new polyynes, named oplopantriol A ( Oplopanax, belonging to the family Araliaceae, comprise three species which are Oplopanax japonicus (Nakai) Nakai. uniquely found in Japan, Oplopanax elatus Nakai, only distributed in northeast China, and Oplopanax horridus (Smith) Miq. exclusively originated and grown in North America . Al. AlOplopO. horridus afforded two new polyynes 5, 6, along with four known compounds 1-4. The identification of 5 and 6 were made by spectroscopic data. The absolute configurations of the new compounds 5 and 6 were determined by comparing their optical rotation values with the hydrolysis products of the known compounds 3 and 4, respectively, derived from the same plant. By successive column chromatography (CC) on silica gel and octadecyl silica gel (ODS gel) and prep-HPLC, an 85% ethanol extract of air-dried root bark of 5 was obtained as a yellowish oil. The molecular formula of 5 was determined to be C18H26O3 on the basis of the HR-electrospray ionization (ESI)-MS spectrum . The UV  and IR absorptions (2250 and 1675 cm–1) indicated the presence of two C≡C bonds ¯ peak at 291.1966 , in the HR-ESI-MS spectrum. The UV  and IR absorptions (2232 and 1656 cm–1) suggested that compound 6 had the same structural skeleton as compound 5. The 1H and 13C NMR  and 1.74 ; δC 9.3 and 30.6] and the disappearance of the signals for the terminal double bond , suggesting that 6 was a dihydro derivative of 5. In the HMBC spectrum ] and C-17 (δC 30.6), C-18 (δC 9.3), C-15 (δC 80.9) and C-14 (δC 68.8), as well as between H-11 [δH 5.19 ] and C-10 (δC 134.1), C-9 (δC 128.0), C-12 (δC 79.1) and C-13 (δC 68.8) confirmed the structure as shown in 5 as cis and Z. According to the above results, the planar structure of 6 was elucidated to be 9-octadecaen-12,14-diyne-1,11,16-triol.Compound  13C NMR  spectra spectrum , the cor3 and 4 afforded their deacetyl derivatives 3a and 4a, respectively, which had the same retention times as 5 and 6 by Ultra Performance Liquid Chromatography (UPLC) analysis. Furthermore, the optical rotation values of 3a { + 189.3° } and 4a { + 230.6° } were identical with those of the new polyynes 5 { +194.4° }and 6{ + 233.0° }, respectively. The above evidence indicated that 5 and 6 should have the same absolute configurations with the known compounds 3 and4. Thus, the complete structures of the new polyynes, oplopantriol A (5) and oplopantriol B (6), were elucidated to be -9,17-octadecadien-12,14-diyne-1,11,16-triol and -9-octadecaen-12,14- diyne-1,11,16-triol, which were named as oplopantriol A (5) and oplopantriol B (6), respectively.Alkaline hydrolysis of Peucedanum oreoselinum was assigned as  by Lemmich in 1981 on the basis of chemical correlation studies + (100); Negative mode HR-ESI-MS m/z: 289.1867 [M-H]¯, Calcd for C18H25O3: 289.1804).NMR data ; PositivOplopantriolB (6), -9-octadecaen-12,14-diyne-1,11,16-triol yellowish oil;  + 233.0° ; UV(CHCl3) λmax (log ξ): 207 (1.07), 226 (1.19), 232 (1.17), 253 (4.02), 263 (3.98), 265 (3.95), 272 (4.01) and 288 (3.84) nm; IR (KBr) νmax : 3355, 3021, 2930, 2856, 2232, 2143, 1656, 1463, 1305, 1095, 1017, 970 and 866 cm-1; 1H and 13C NMR data (m/z: 315 [M+Na]+ (100); Negative mode HR-ESI-MS m/z: 291.1966 [M-H]¯, Calcd for C18H27O3: 291.1960).NMR data ; PositivO. horridus led to the isolation of two new polyynes -9,17-Octadecadien-12,14-diyne-1,11,16-triol and -9-octadecaen-12,14- diyne-1,11,16-triol named oplopantriol A (5) and oplopantriol B (6), along with four known polyynes -falcarindiol (1), oplopandiol (2), -9,17-octadecadiene-12,14-diyne-1,11,16-triol, 1-acetate (3) and oplopandiol acetate (4). A detailed phytochemical investigation on


N4-aminocytosine, 4-thiouracil as well as 2-pyridone, 4-chloro- and 4-bromo-2-pyridone as a nucleobase were accepted by TdT, thus allowing enzymatic synthesis of 3’-terminally modified ONs. The successful UV-induced cross-linking of 4-thiouracil-containing ONs to TdT was carried out. Enzymatic post-synthetic 3’-modification of ONs with various photo- and chemically-reactive groups opens novel possibilities for future applications, especially in analysis of the mechanisms of polymerases and the development of photo-labels, sensors, and self-assembling structures.The synthesis of novel modified nucleotides and their incorporation into DNA sequences opens many possibilities to change the chemical properties of oligonucleotides (ONs), and, therefore, broaden the field of practical applications of modified DNA. The chemical synthesis of nucleotide derivatives, including ones bearing thio-, hydrazino-, cyano- and carboxy groups as well as 2-pyridone nucleobase-containing nucleotides was carried out. The prepared compounds were tested as substrates of terminal deoxynucleotidyl transferase (TdT). The nucleotides containing  UV (CH3OH) λmax (log ε) 262 (9.60) nm. 1H-NMR (DMSO-6d): δ = 2.06 , 2.07 , 2.28–2.34 , 2.43–2.46 , 4.16–4.19 , 4.21–4.24 , 5.16–5.20 , 5.71 , 6.16 , 7.66 , 11.40 . 13C-NMR (DMSO-6d): δ = 21.05, 21.23, 36.18, 64.10, 74.38, 81.65, 84.91, 102.72, 140.83, 150.83, 163.44, 170.47, 170.61.(i) 3’,5’-Bis-O-acetyl-4-thio-2’-deoxyuridine (1ii). To a solution of 3’,5’-bis-O-acetyl-2’-deoxyuridine  in toluene (43 mL ) Lawesson’s reagent  was added. The reaction mixture was stirred for 2 h at 90 °C. After the reaction was completed (TLC), the mixture was quenched with sodium bicarbonate and extracted with chloroform. The organic phase was dried (Na2SO4) and evaporated under reduced pressure. The residue was purified by column chromatography . Yield 850 mg (98%), yellowish oil, Rf = 0.56 (CHCl3/MeOH 9:1). MS (ESI+): m/z 329.00 [M + H]+, 326.95 [M-H]−. UV (CH3OH) λmax (log ε) 330 (16.50) nm. 1H-NMR (DMSO-6d): δ = 2.05 , 2.06 , 2.36–2.39 , 2.45–2.48 , 4.20–4.27 , 5.17–5.22 , 6.07–6.11 , 6.37 , 7.57 , 12.78 . 13C-NMR (DMSO-6d): δ = 21.04, 21.22, 36.59, 64.02, 74.30, 82.10, 85.91, 113.39, 136.16, 148.08, 170.47, 170.60, 190.75.(ii) 3’,5’-Bis-2). A mixture of 3’,5’-bis-O-acetyl- 4-thio-2’-deoxyuridine  and 1M sodium methylate (5.1 mL) was stirred for 30 min at room temperature. The reaction was monitored with TLC. The crude reaction mixture was purified by reverse phase column chromatography . The solvents were removed under reduced pressure to afford yellowish solid reaction product, mp 147–150 °C. Yield 438 mg (70%), Rf = 0.19 (CHCl3/MeOH 9:1). MS (ESI+): m/z 242.95 [M − H]−. UV (H2O) λmax (log ε) 332 (16.50) nm. 1H-NMR (DMSO-6d): δ = 2.03–2.06 , 2.11–2.15 , 3.56–3.61 , 3.79–3.84 , 4.20–4.24 , 4.93 , 5.25 , 6.09 , 6.25 , 7.64 , 11.79 .13C-NMR (DMSO-6d): δ = 40.46, 49.05, 61.64, 70.70, 85.39, 88.00, 113.25, 135.39, 192.53. The NMR data were consistent with those reported previously +, 482.90 [M-H]−. UV (H2O) λmax (log ε) 332 (16.50) nm. 1H-NMR (D2O): δ = 2.18–2.26 , 2.21–2.25 , 4.12–4.15 , 4.50–4.54 , 6.19 , 6.57 , 7.75 . 31P-NMR (D2O): δ = –20.39 ; −10.85 ; –5.19 .To a suspension of synthesized 4-thio-2’-deoxyuridine (3) in water (2 mL) hydrazine hydrate  was added and the reaction mixture was stirred at room temperature for 18 h. After the reaction was completed (TLC), the reaction mixture was purified by ion exchange chromatography on DEAE-Sephadex A25 column (20 mL) with a linear gradient (0.05–0.4 M) of LiCl as the mobile phase. The product was eluted with 0.25–0.3 M LiCl, the solution was concentrated under reduced pressure to several mililiters and desalted using reverse phase column C-18. The synthesis and purification afforded 0.044 mmol (66% yield) of N4-amino-dCTP. Compound was quantified by the extinction coefficient of 13.700 M−1 × cm−1 at 274 nm. Rf = 0.12 (dioxane/iPrOH/H2O/NH4OH 4:2:5:1). MS (ESI+): m/z 483.00 [M+H]+, 480.95 [M − H]−. UV (H2O) λmax (log ε) 274 (13.70) nm. 1H-NMR (D2O-): δ = 2.60–2.68 , 2.73–2.81 , 4.31–4.39 , 4.43–4.41 , 6.38 , 6.63 , 8.09 . 31P-NMR (D2O-): δ = −19.09 ; −10.05 ; −5.86 . The NMR spectra were consistent with the data reported in −. UV (CH3OH) λmax (log ε) 277 (11.70) nm. 1H-NMR (DMSO-6d): δ = 2.09–2.13 , 3.54–3.58 , 3.60–3.64 , 3.86–3.90 , 4.23–4.27 , 6.09 , 8.64 . 13C- NMR (DMSO-6d): δ = 40.87, 61.97, 70.81, 88.19, 88.44, 90.73, 115.19, 146.71, 149.76, 160.51. The NMR spectra were in agreement with the data published in +, 271.00 [M − H]−. UV (H2O) λmax (log ε) 275 (9.70) nm. 1H-NMR (DMSO-6d): δ = 2.28–2.35 , 3.78–3.82 , 4.19–4.23 , 4.55–4.59 , 6.29 , 8.84 . 13C-NMR (DMSO-6d): δ = 39.17, 61.92, 70.81, 86.34, 87.73, 105.17, 148.71, 158.76, 162.53, 167.14. The 1H-NMR spectrum was consistent with the previously reported one −. UV (H2O) λmax (log ε) 277 (11.70) nm. 1H-NMR (D2O): δ = 2.30–2.35 , 2.65–2.72 , 4.01–4.04 , 4.16–4.20 , 4.52–4.56 , 6.12 , 8.52 . 31P-NMR (D2O): δ = −20.69 , –10.95 , −5.33 .-Carboxy-2’-deoxyuridine-5’-triphosphate (8). The synthesis and purification afforded 0.041 mmol (20.5% yield) of 5-carboxy-2’-dUTP. Compound was quantified by the extinction coefficient of 9.700 M−1 × cm−1 at 275 nm. Rf = 0.10 (dioxane/iPrOH/H2O/NH4OH 4:2:5:1). MS (ESI+): m/z 510.85 [M − H]−. UV (H2O) λmax (log ε) 275 (9.70) nm. 1H-NMR : δ = 2.31–2.37 , 2.65–2.72 , 4.01–4.04 , 4.10–4.14 , 4.49–4.53 , 6.16 , 8.43 . 31P-NMR : δ = −20.58 , −10.91 , −5.28 . The NMR spectra were consistent with those reported previously [5eviously .4OHTP), (5-(4-chloro-2-oxo-1-pyridyl)-3-hydroxytetrahydrofuran-2-yl)methyl triphosphate (dPyr4ClTP), (5-(4-bromo-2-oxo-1-pyridyl)-3-hydroxytetrahydrofuran-2-yl)methyl triphosphate (dPyr4BrTP), and (5-(5-carboxy-2-oxo-1-pyridyl)-3-hydroxytetrahydrofuran-2-yl)methyl triphosphate (dPyr5COOHTP) were prepared as described previously [(3-Hydroxy-5-(2-oxo-1-pyridyl)tetrahydrofuran-2-yl)methyl triphosphate (dPyrTP), (3-hydroxy-5-(4-hydroxy-2-oxo-1-pyridyl)tetrahydrofuran-2-yl)methyl triphosphate  using T4 polynucleotide kinase (Thermo Fisher Scientific) according to manufacturer’s recommendations. The 5’-labelled primer was desalted using ZebaTM Spin desalting columns . The reaction mixtures were prepared in a total volume of 10 µL by adding 5’-33P-labelled primer , 2 µL 5 × TdT reaction buffer  or 5 × glutamate buffer , modified dNTP , TdT  and distilled water. The reaction mixtures were incubated at 37 °C for 5 min. The reactions were quenched with 20 µL of loading solution . Reaction products were analyzed by denaturing PAGE  in TBE  buffer  and visualized using a FLA-5100 imaging system .The DNA primer (5’-TAATACGACTCACTATAGGGAGA-3’) was purchased from Metabion , TdT was purchased from Thermo Fisher Scientific . The primer sequence was 5’-2) 5 and 15 mm away from the surface of the light source, which provided dose of UV irradiation ~17.2 J/cm2 and ~4.6 J/cm2, respectively. A sheet of parafilm was placed over the top of 96-well plate, and taped to the plate on all four sides. Each well was pressed to create a shallow groove. The plate was kept on ice before and during irradiation.The cross-linking apparatus was constructed with slight modifications as described previously . It cons33P-labelled) to TdT was carried out after PEX. PEX were performed in Co2+-containing buffer as described above. Immediately after incubation, the reaction mixtures were chilled on ice and transferred as 10 µL drops to the wells on the parafilm tape. The ice container was placed underneath a 365 nm UV, so that the samples were 5 and 15 mm from the surface of the light source. Following incubation on ice the samples were irradiated at 365 nm for 5 min. Then the samples were transferred from parafilm wells to microtubes and quenched with 20 µL of loading solution. To verify the products of elongation reactions using 4-thio-dUTP and dUTP by TdT samples were examined on 15% polyacrylamide gel containing 8 M urea. Alternatively, to examine potential TdT-ON cross-links generated by irradiation, the samples were supplemented with SDS loading dye, heated for 5 min at 95 °C, and analyzed by electrophoresis on a 14% w/v SDS-PAGE gel. Proteins were stained with Coomasie Briliant Blue staining solution . TdT and radiolabelled-ONs cross-links were visualized using an FLA-5100 imaging system (FUJIFILM).UV cross-linking of 4-thio-dU-elongated DNA primer (5’-N4-amino-2’-deoxycytidine and 2-pyridone base-bearing nucleotides utilizing template-independent TdT, which produced the artificial DNA fragments tailed with several to tens nucleotides at the 3′-end. For example, 4-thio-dU-label has potential in studying specific proteins which interact exclusively with the DNA 3’-end, hydrazine-bearing ONs could be successfully applied in a conjugation to improve in vivo delivery of therapeutic ONs or immobilization, whereas halogenated-ONs seem to be irreplaceable radiosensitizing agents. Enzymatic post-synthetic modification of ONs with various photo- and chemically-reactive groups is in progress towards further use for the modification of DNA or RNA. Thus, this study would open many possibilities for future applications, especially in the development of photo-labels, sensors, and self-assembling structures.We have synthesized novel modified nucleotides that can be incorporated into DNA sequences by terminal deoxynucleotidyl transferase in the primer-extension reaction. We have succeeded in the enzymatic elongation by 4-thio-2’-deoxyuridine,


II-5 was identified as the most potent 5-HT (IC50 = 620 nM) and NE (IC50 = 10 nM) dual reuptake inhibitor. Compound II-5 exhibited potent antidepressant activity in the rat tail suspension test and showed an acceptable safety profile in a preliminary acute toxicity test in mice. Our results show that these arylamidine derivatives exhibit potent 5-HT/NE dual reuptake inhibition and should be explored further as antidepressant drug candidates.To improve the in vivo antidepressant activity of previously reported serotonin (5-HT) and norepinephrine (NE) dual reuptake inhibitors, three series of arylamidine derivatives were designed and synthesized. The in vitro 5-HT and NE reuptake inhibitory activities of these compounds were evaluated, and compound  Depression is a common mental illness that can be severe, chronic, and sometimes life-threatening ,2. AccorThe biogenic amine transmitters, serotonin (5-HT), norepinephrine (NE), and dopamine (DA), are closely related to symptoms of depression . First-gIn our previous study, we used compounds with 5-HT or NE reuptake inhibition activities to build a pharmacophore model of the characteristics of 5-HT and NE inhibitors  11]. A . A 11]. However, an in vivo study of antidepressant activity in rats showed that arylamidine derivatives were less potent than the commercial SNRI antidepressants venlafaxine and duloxetine. We suspected that the high rigidity of the planar structure in arylamidine derivatives, compared with the structures of other SNRI inhibitors, such as venlafaxine and duloxetine, was a main cause of the poor antidepressant activity in rats. To circumvent these problems with arylamidine derivatives, in this work, we designed and synthesized 32 compounds in three series (I–III) of optimized arylamidine derivatives. The pharmacological activity of the 32 compounds was evaluated in vitro and selected compounds were evaluated in vivo.2-) was inserted between the aromatic ring and nitrogen atom to form the structure of series I. Then, based on series I, another two series of derivatives were designed. In series II, the benzyl ring was replaced with a naphthalene ring and in series III, the amidine ring was replaced with a naphthalene ring.To increase the structural flexibility of the optimized compounds, we designed three series of compounds . A methyI-4 and II-4 are shown as examples of mapping on the pharmacophore model in I-4 was docked in the 5-HT model, and the two aromatic rings matched either the RA or HY features well. The nitrogen atoms of the piperazine ring were recognized as an HA feature, and the amidine imino nitrogen atom was recognized as a PI feature. Compound I-4 was also mapped on the NE model and the two aromatic rings matched the HY feature, the nitrogen atoms of the piperazine ring were recognized as the PI feature, and amidine imino nitrogen atom was recognized as the HA feature. We observed similar mapping for compound II-4 in both models. The compounds were docked into the 5-HT and NE pharmacophore models, and all three types of molecules matched the model well. Compounds E) configuration, would have to be retained. Only small changes to the structure of arylamidine derivatives are tolerated. Otherwise, the new compounds would lose their inhibitory activities against both 5-HT and NE reuptake. Thus, in the design, the key change was the addition of a methylene group, which broke the original planar configuration and increased the flexibility of the molecules. This design strategy balanced the pharmacological activity and the desired structural flexibility. Additionally, based on the pharmacophore models, the space for the aromatic ring in the arylamidine derivatives can tolerate a bigger aromatic fragment, such as an aphthalene ring. This might further favor the binding of the new compounds to either 5-HT or NE.Our experience suggested that for the design of a new series of compounds , the majI-4 and II-4 in both models. The docking results indicated that the designed compounds were likely to be good dual reuptake inhibitors of 5-HT and NE. In summary, the docking study showed that all of the designed compounds matched the 5-HT and NE pharmacophore models well. The fragments in the compounds matched the corresponding binding domains in both models. Similar binding modes were observed for the mapping of compounds 1) was used as the starting reagent to form an acyl chloride (2) with yields of 75–95%. The intermediate (2) was reacted with various aromatic methylamines to form a substituted amide (3). The most important step in the scheme was the synthesis of compound 4. All the final compounds had an (E) configuration, which was initially formed in the production of acyl chloride 4. (E)-acyl chloride 4 was obtained by the reaction between amide 3 and PCl5. During the synthesis of compound 5, piperazine could react with two molecules of compound 4 to produce disubstituted by-products. The amount of disubstituted by-products was reduced by increasing the amount of piperazine in this reaction. Subsequently, all target compounds were prepared as hydrochloride salts E)-I-II-III.1D-NOE NMR was used to confirm the configuration of the structure. The 1D-NOE spectrum of compound I-11  showed w1H-NMR spectra of several compounds, such as I-15, the Ar-CH2-N fragment exhibited a singlet, where as in the spectra of other compounds, such as I-14, the same fragment produced a double doublet. The only structural difference between I-14 and I-15 was the ortho- or para-chloro substituent on the amidine ring , and there would be no steric hindrance, the two Ar-CH2-N protons would not be magnetically inequivalent, and the double doublet would not appear. Consequently, the steric hindrance indirectly indicated that the absolute configuration of the final compound was (E).In the ine ring . The dif3H-5-HT and 3H-NE [3H]-Noradrenaline and 5-hydroxy-[3H]-tryptamine trifluoroacetate were obtained from Amersham Corporation. Duloxetine was used as the reference compound. All compounds were tested at a concentration of 10 μM, and potent compounds (inhibition >80%) were assayed to obtain IC50 values , and was comparable to the reference compound, duloxetine . In series II, compounds II-4  and II-5  were the most potent compounds. Most compounds in this series had improved dual reuptake inhibition activity. In series III, compound III-1 also showed good potency for 5-HT and NE transporters.In series I, most compounds exhibited moderate dual reuptake inhibition activity. Compound The series I compounds maintained the dual 5-HT and NE inhibitory activity after the insertion of a methylene into the arylamidine derivative structure, although the general inhibitory activity was moderate. Various substituents on the benzene ring did not dramatically improve the binding of these compounds with the targets. In series II, a naphthalene ring replaced the benzyl ring. Interestingly, some compounds in this series exhibited dramatically improved dual inhibitory activities. Thus, the addition of a big hydrophobic fragment, that is, the naphthalene ring, improved binding to the5-HT and NE transporters. I-19, II-4, and II-5 were selected for the tail suspension test (TST) in rats. The TST has been used widely as a preclinical model for screening antidepressant activity, and it is sensitive to commercially available antidepressant drugs. In the assay, rats are suspended by tail and the time that the animal is immobile is recorded. The compounds were administered to male rats at a dose of 30 mg/kg (PO). Duloxetine was used as the positive control . The compounds all reduced the immobility times of rats compared with the negative control vehicle group, indicating that all compounds exerted an antidepressant effect  (n = 6). Compound II-5 did not result in the death of mice at doses of 200 and 400 mg/kg after 20 h, and thus the LD50 was >400 mg/kg (PO), indicating that the toxicity was low and that the safety profile was acceptable  was smaller than that of leading antidepressant duloxetine , although they had similar in vitro activities. The less potent in vivo antidepressant effect of compound II-5 may arise from its in vivo metabolism, which needs to be examined and optimized. In summary, compound 2). 1H-NMR spectra were obtained on an NMR spectrometer . Melting points were measured with a melting point apparatus  and are not corrected. Electro spray ionization (ESI) mass spectra and high-resolution mass spectroscopy (HRMS) were performed with a liquid chromatograph/mass selective detector time-of-flight mass spectrometer . The yields are of the purified product and are not optimized. Silica gel column chromatography was performed with silica gel 60G . The procedure for preparing series I–III was the same as for compound I-1. Purity was determined using HPLC, LC/MS and NMR spectroscopy. All of the synthesized compounds have purity over 95%.Most reagents and solvents were obtained from commercial suppliers and used as received. Some reactions were performed under an inert atmosphere  were added dropwise to a solution of 4-methoxybenzoic acid  in dry CH2Cl2 (20 mL). The reaction mixture was stirred at room temperature for 2 h, and then distilled in vacuo to give the crude acyl chloride, which was dissolved in dry CH2Cl2 (10 mL). Dry triethylamine  was added to a solution of benzylamine  in dry CH2Cl2 (20 mL). The reaction mixture was cooled to 0 °C, and the acyl chloride solution was added dropwise. After stirring at 20–25 °C for 4 h, the reaction mixture was distilled in vacuo, and washed with a 10% solution of NaOH and water, and dried. Silica gel column chromatography (hexane/ethyl acetate) afforded I-1 as a white powder .One drop of dry N-Benzyl-4-methoxybenzamide  was dissolved in dry dichloroethane (10 mL), and the solution was heated to 50 °C for 30 min. PCl5  was added in one portion and the reaction was stirred at 90 °C for 2 h. After most of the solvent was removed in vacuo, the residue was dissolved in dry CH2Cl2 (10 mL) and was added dropwise to a solution of piperazine  in dry CH2Cl2 (10 mL) at 0 °C. The reaction mixture was stirred at room temperature for 2 h, and then distilled in vacuo. Et2O (50 mL) and saturated aqueous Na2CO3 (30 mL) were added to the mixture. After the aqueous extract was separated, the organic extract was washed twice with saturated aqueous Na2CO3 and 10% aqueous NaOH, and then extracted with 1 mol/L HCl. The aqueous extract was washed twice with CH2Cl2 and Et2O, and was treated with 10% aqueous NaOH to increase the pH to 10. The aqueous extract was re-extracted with Et2O (50 mL), and the Et2O extract was dried over Na2SO4. After Et2O was removed under vacuum, the residue was dissolved in dry ethanol (5 mL), and an ethanolic solution of HCl was added dropwise to adjust the pH to 2 to afford I-1 as a white powder . Mp: 195–197 °C. 1H-NMR : δ 3.37 , 3.61 , 3.66 , 3.89 , 4.13 , 4.44 , 7.10–7.16 , 7.35–7.41 . HRMS calcd for C19H23N3O (M + H)+, 310.1914; found, 310.1912.(E)-N-((4-Nitrophenyl)(piperazin-1-yl)methylene)-1-phenylmethanaminehydrochloride (I-2): Yield 40%, Mp: 230–231 °C. 1H-NMR : δ 3.37 , 3.61 , 3.66 , 4.13 , 4.44 , 7.10–7.16 , 7.35–7.41 . HRMS calcd for C18H20N4O2 (M + H)+, 325.1659; found, 325.1654.(E)-N-((2-Chlorophenyl)(piperazin-1-yl)methylene)-1-phenylmethanaminehydrochloride (I-3): Yield 18%, Mp: 142–144 °C.1H-NMR : δ 3.40 , 3.67 , 4.24 , 4.38 , 4.46 , 7.11–7.12 , 7.37–7.38 , 7.48 , 7.55–7.58 , 7.70–7.73 . HRMS calcd for C18H20ClN3 (M + H)+, 314.1419; found, 314.1421. (E)-N-((3-Chlorophenyl)(piperazin-1-yl)methylene)-1-phenylmethanaminehydrochloride (I-4): Yield 66%, Mp: 235–237 °C. 1H-NMR : δ 3.34 , 3.58–3.66 , 4.14 , 4.39 , 4.45 , 7.07–7.11 , 7.35–7.38 , 7.45 , 7.56–7.61 , 7.73 . HRMS calcd for C18H20ClN3 (M + H)+, 314.1419; found, 314.1411.(E)-N-((4-Chlorophenyl)(piperazin-1-yl)methylene)-1-phenylmethanaminehydrochloride (I-5): Yield 65%, Mp: 182–184 °C. 1H- NMR : δ 3.35 , 3.64 , 4.14 , 4.42 , 7.11 , 7.35–7.42 , 7.63 . HRMS calcd for C18H20ClN3 (M + H)+, 314.1419; found, 314.1414.(E)-N-((4-Fluorophenyl)(piperazin-1-yl)methylene)-1-phenylmethanaminehydrochloride (I-6): Yield 33%, Mp: 220–222 °C. 1H-NMR : δ 3.34 , 3.58 , 3.64 , 4.12 , 4.43 , 7.11 , 7.32–7.37 , 7.46–7.50 . HRMS calcd for C18H20FN3 (M + H)+, 298.1714; found, 298.1710.(E)-N-((2-Fluorophenyl)(piperazin-1-yl)methylene)-1-phenylmethanaminehydrochloride (I-7): Yield 22%, Mp: 182–184 °C. 1H-NMR : δ 3.34 , 3.59 , 3.71 , 4.17 , 4.42 , 4.49 , 7.08–7.10 , 7.34–7.39 , 7.43–7.45 , 7.72–7.80 . HRMS calcd for C18H20FN3 (M + H)+, 298.1714; found, 298.1706.(E)-4-((Benzylimino)(piperazin-1-yl)methyl)benzonitrilehydrochloride (I-8): Yield 38%, Mp: 205–207 °C. 1H-NMR : δ 3.36 , 3.61 , 4.17 , 4.40 , 7.04–7.08 , 7.34–7.36 , 7.60 , 7.98 . HRMS calcd for C19H20N4 (M + H)+, 305.1761; found, 305.1760.(E)-3-((Benzylimino)(piperazin-1-yl)methyl)benzonitrilehydrochloride (I-9): Yield 60%, Mp: 190–192 °C. 1H-NMR : δ 3.35 , 3.61 , 4.18 , 4.36 , 4.44 , 7.04–7.06 , 7.31–7.41 , 7.72–7.81 , 8.07 . HRMS calcd for C19H20N4 (M + H)+, 305.1761; found, 305.1758.(E)-1-Phenyl-N-(piperazin-1-yl(p-tolyl)methylene)methanaminehydrochloride (I-10): Yield 50%, Mp: 172–174 °C. 1H-NMR : δ 2.42 , 3.34 , 3.59 , 3.64 , 4.12 , 4.42 , 7.09–7.12 , 7.30–7.37 , 7.45 . HRMS calcd for C19H23N3 (M + H)+, 294.1965; found, 294.1962.(E)-N-((2-Chloro-4-nitrophenyl)(piperazin-1-yl)methylene)-1-phenylmethanaminehydrochloride (I-11): Yield 73%, Mp: 195–197 °C. 1H-NMR : δ 3.39 , 3.66 , 4.13 , 4.35 , 4.46 , 7.05 , 7.31–7.38 , 7.71 , 8.35 , 8.57 . HRMS calcd for C18H19ClN4O2 (M + H)+, 359.1269; found, 359.1277.(E)-1-(4-Chlorophenyl)-N-((4-nitrophenyl)(piperazin-1-yl)methylene)methanamine(hydrochloride I-12): Yield 22%, Mp: 172–174 °C. 1H-NMR : δ 3.35 , 3.65 , 4.18 , 4.39 , 7.01 , 7.34 , 7.67 , 8.42 . HRMS calcd for C18H19ClN4O2 (M + H)+, 359.1269; found, 359.1271.(E)-1-(4-Chlorophenyl)-N-((2-chlorophenyl)(piperazin-1-yl)methylene)methanaminehydrochloride (I-13): Yield 56%, Mp: 181–183 °C. 1H-NMR : δ 3.36 , 3.61 , 4.14 , 4.34 , 4.42 , 7.01 , 7.33 , 7.42 , 7.55 , 7.64–7.72 . HRMS calcd for C18H19Cl2N3 (M + H)+, 348.1029; found, 348.1032.(E)-1-(4-Chlorophenyl)-N-((3-chlorophenyl)(piperazin-1-yl)methylene)methanaminehydrochloride (I-14): Yield 53%, Mp: 218–220 °C. 1H-NMR : δ 3.34 , 3.63 , 4.14 , 4.35 , 4.35 , 7.03 , 7.33–7.37 , 7.40 , 7.55–7.61 , 7.72 . HRMS calcd for C18H19Cl2N3 (M + H)+, 348.1029; found, 348.1027.(E)-1-(4-Chlorophenyl)-N-((4-chlorophenyl)(piperazin-1-yl)methylene)methanaminehydrochloride (I-15): Yield 36%, Mp: 157–159 °C. 1H-NMR : δ 3.33 , 3.62 , 4.13 , 4.40 , 7.03 , 7.35 , 7.37 , 7.63 . HRMS calcd for C18H19Cl2N3 (M + H)+, 348.1029; found, 348.1027.(E)-1-(4-Chlorophenyl)-N-((4-fluorophenyl)(piperazin-1-yl)methylene)methanaminehydrochloride (I-16): Yield 20%, Mp: 165–167 °C. 1H-NMR : δ 3.34 , 3.62 , 4.13 , 4.41 , 7.05 , 7.31–7.37 , 7.42–7.47 . HRMS calcd for C18H19ClFN3 (M + H)+, 332.1324; found, 332.1323.(E)-1-(4-Chlorophenyl)-N-((2-fluorophenyl)(piperazin-1-yl)methylene)methanaminehydrochloride (I-17): Yield 21%, Mp: 215–217 °C. 1H-NMR : δ 3.26 , 3.52 , 3.70 , 4.18 , 4.41 , 4.47 , 7.02 , 7.32–7.38 , 7.41–7.43 , 7.72–7.79 . HRMS calcd for C18H19ClFN3 (M + H)+, 332.1324; found, 332.1321.(E)-1-(4-Chlorophenyl)-N-((4-cyanophenyl)(piperazin-1-yl)methylene)methanaminehydrochloride (I-18): Yield 39%, Mp: 180–182 °C. 1H-NMR : δ 3.35 , 3.62 , 4.16 , 4.37 , 7.01 , 7.34 , 7.58 , 7.97 . HRMS calcd for C19H19ClN4 (M + H)+, 339.1371; found, 339.1368.(E)-1-(4-Chlorophenyl)-N-((3-cyanophenyl)(piperazin-1-yl)methylene)methanaminehydrochloride (I-19): Yield 26%, Mp: 156–158 °C. 1H-NMR : δ 3.21 , 3.49 , 4.03 , 4.21 , 4.29 , 6.85 , 7.21 , 7.56–7.67 , 7.93 . HRMS calcd for C19H19ClN4 (M + H)+, 339.1371; found, 339.1366.(E)-1-(4-Chlorophenyl)-N-(piperazin-1-yl(p-tolyl)methylene)methanaminehydrochloride (I-20): Yield 57%, Mp: 183–185 °C. 1H-NMR : δ 2.41 , 3.32 , 3.58 , 3.63 , 4.11 , 4.40 , 7.04 , 7.27 , 7.35 , 7.43 . HRMS calcd for C19H22ClN3 (M + H)+, 328.1575; found, 328.1574.(E)-1-(4-Methoxyphenyl)-N-((4-nitrophenyl)(piperazin-1-yl)methylene)methanaminehydrochloride (I-21): Yield 15%, Mp: 210–212 °C. 1H-NMR : δ 3.33 , 3.59 , 3.81 , 4.15 , 4.35 , 6.90 , 6.97 , 7.66 , 8.42 . HRMS calcd for C19H22N4O3 (M + H)+, 355.1765; found, 355.1766.(E)-1--N-(phenyl(piperazin-1-yl)methylene)methanaminehydrochloride (II-1): Yield 34%, Mp: 187–189 °C. 1H-NMR : δ 3.33 , 3.54–3.68 , 4.12 , 4.82 , 7.22 , 7.40–7.44 , 7.51–7.57 , 7.60–7.68 , 7.87 , 7.87 . HRMS calcd for C22H23N3 (M + H)+, 330.1965; found, 330.1962.(E)-1--N-((4-nitrophenyl)(piperazin-1-yl)methylene)methanaminehydrochloride (II-2): Yield 11%, Mp: 202–204 °C. 1H-NMR : δ 3.34 , 3.60 , 4.21 , 4.84 , 7.20 , 7.38–7.43 , 7.46–7.53 , 7.56–7.61 , 7.84–7.89 , 8.17 . HRMS calcd for C22H22N4O2 (M + H)+, 375.1816; found, 375.1814.(E)-N-((4-Fluorophenyl)(piperazin-1-yl)methylene)-1-methanaminehydrochloride (II-3): Yield 24%, Mp: 193–195 °C. 1H-NMR : δ 3.36 , 3.58 , 3.65 , 4.12 , 4.87 , 7.22–7.28 , 7.41–7.48 , 7.52–7.60 , 7.65–7.68 , 7.91 , 7.95–7.98 . HRMS calcd for C22H22FN3 (M + H)+, 348.1871; found, 348.1873.(E)-N-((2-Fluorophenyl)(piperazin-1-yl)methylene)-1-methanaminehydrochloride (II-4): Yield 41%, Mp: 148–150 °C. 1H-NMR : δ 3.33 , 3.56 , 3.67 , 4.12–4.21 , 4.92 , 7.12 , 7.23–7.29 , 7.33–7.46 , 7.56 , 7.64–7.72 , 7.88–8.05 . HRMS calcd for C22H22FN3 (M + H)+, 348.1871; found, 348.1868.(E)-N-((2-Chlorophenyl)(piperazin-1-yl)methylene)-1-methanaminehydrochloride (II-5): Yield 58%, Mp: 165–167 °C. 1H-NMR : δ 3.34 , 3.63 , 4.15 , 4.89 , 7.07 , 7.37–7.49 , 7.55–7.65 , 7.78–7.81 , 7.91 , 7.98–8.04 . HRMS calcd for C22H22ClN3 (M + H)+, 364.1575; found, 364.1574.(E)-N-((3-Chlorophenyl)(piperazin-1-yl)methylene)-1-methanaminehydrochloride (II-6): Yield 34%, Mp: 198–200 °C. 1H-NMR : δ 3.31 , 3.65 , 4.12 , 4.78 ,7.16 , 7.29–7.32 , 7.39–7.62 , 7.87 , 7.93 . HRMS calcd for C22H22ClN3 (M + H)+, 364.1575; found, 364.1576.(E)-N-((4-Chlorophenyl)(piperazin-1-yl)methylene)-1-methanaminehydrochloride (II-7): Yield 41%, Mp: 210–212 °C. 1H-NMR : δ 3.19 , 3.43 , 3.50 , 4.12 , 4.75 , 7.09 , 7.20 , 7.30–7.35 , 7.38 , 7.41–7.54 , 7.76–7.85 . HRMS calcd for C22H22ClN3 (M + H)+, 364.1575; found, 364.1577.(E)-4-imino)(piperazin-1-yl)methyl)benzonitrilehydrochloride (II-8): Yield 30%, Mp: 158–160 °C. 1H-NMR : δ 3.34 , 3.63 , 4.18 , 4.84 , 7.19 , 7.40–7.63 , 7.77 , 7.88 , 7.94 . HRMS calcd for C23H22N4 (M + H)+, 355.1917; found, 355.1921.(E)-3-imino)(piperazin-1-yl)methyl)benzonitrilehydrochloride (II-9): Yield 11%, Mp: 168–170 °C. 1H-NMR : δ 3.16 , 3.45 , 4.04 , 4.72 , 4.78 , 6.85 , 7.01–7.05 , 7.24–7.29 , 7.35–7.52 , 7.73–7.90 . HRMS calcd for C23H22N4 (M + H)+, 355.1917; found, 355.1911.(E)-1--N-(piperazin-1-yl(p-tolyl)methylene)methanaminehydrochloride (II-10): Yield 49%, Mp: 178–180 °C. 1H-NMR : δ 2.38 , 3.19 , 3.42 , 3.51 , 3.96 , 4.74 , 7.10–7.15 , 7.21 , 7.30–7.35 , 7.37–7.44 , 7.46–7.52 , 7.77 , 7.83 . HRMS calcd for C23H25N3 (M + H)+, 344.2121; found, 344.2108.(E)-N-(piperazin-1-yl)methylene)-1-phenylmethanaminehydrochloride(III-1): Yield 14%, Mp: 173–175 °C. 1H-NMR : δ 3.14–3.26 , 3.51–-3.57 , 3.71 , 4.34 , 4.23 , 4.36 , 6.73 , 7.06–7.16 , 7.59 , 7.80–7.86 , 7.94 , 7.29 , 8.74 . HRMS calcd for C22H22N4O2 (M + H)+, 375.1816; found, 375.1815.g and 4 °C for 10 min. The supernatant was decanted and used for the uptake experiments. In the assay, the tissue suspension (200 µL) was incubated with 50 nmol/L 3H-NE or 3H-5-hydroxytryptamine (800 µL) in Krebs-Henseleit bicarbonate buffer and a solution of the drug at the appropriate concentration  or the vehicle (20 µL) at 37 °C for 10 min. For each assay, three tubes were incubated with the vehicle (20 µL) at 0 °C in an ice bath. After incubation, the tubes were immediately filtered with a cell harvester. The fiberglass filter membrane was washed three times with ice-cold saline, placed in a scintillation vial, and counted in liquid scintillation cocktail (4 mL). Active uptake was the difference between the counts per minute at 37 and 0 °C. The percent inhibition at each drug concentration was the mean of three measurements [All animal experiments were conducted in compliance with the Care and Use of Laboratory Animals, with the approval of Peking Union Medical College and Chinese Academy of Medical Sciences’ Animal Studies Committee . Adult male Wistar rats were used. Animals were killed by decapitation and the whole brain, with the exception of the brainstem and cerebellum, was quickly removed. The procerebrum region was prepared, weighed, and homogenized in 10 volumes of ice-cold 0.32 mol/L sucrose solution using a glass homogenizer. The homogenate was centrifuged at 1000× urements .I-19, II-4, and II-5 (30 mg/kg). For two days before the formal experiment, the rats in each group were given doses orally once a day in the morning and in the afternoon. The rats were given doses orally once in each group in the formal experiment. After 60 min of administration, the rats were suspended head-down more than 50 cm from the ground with a 12-cm-long piece of insulating tape at a distance of 1 cm from the tail tip. The distance between adjacent rats was 15 cm. The immobility time of the rats was recorded. The rate of change in the immobility time of the rats in the test drug group and the positive control group was calculated and compared with the negative control group. Statistical analysis was performed using Prism 7.0 . The data of the immobility time of vehicle, duloxetine, I-19, II-4, and II-5 were analyzed by the One-way ANOVA.Rats were administered the vehicle as a negative control, duloxetine (30 mg/kg) as a positive control, or test compounds II-5 with a single dose 200 and 400 mg/kg or vehicle control, respectively. The mouse death was monitored for 20 h after treatment. Male ICR mice (20–25 g) were used from Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing, China. Mice were randomly divided into two groups with six mice each. Mice were orally given II-5 was the most potent dual inhibitor . Compounds I-19, II-4, and II-5 were selected for TST profiling in rats to test the in vivo antidepressant effect. These three compounds reduced the immobility time in the TST, indicating in vivo antidepressant activity. Compound II-5 showed the most potent in vivo antidepressant activity and had an acceptable safety profile. These arylamidine derivatives are interesting compounds to explore further as potential antidepressant drug candidates.In this study, we designed and synthesized three series of arylamidine derivatives to discover potent reuptake inhibitors of 5-HT and NE transporters. Our results showed that series II compounds with a large aromatic ring exhibited improved in vitro 5-HT and NE inhibitory activity, and compound


Hedyotis diffusa Willd. has been using to treat malignant tumors, and many studies also showed that the extracts from Hedyotis diffusa Willd. possessed evident antitumor activities. Therefore, we carried out chemical study on Hedyotis diffusa Willd. and investigated the cytotoxicity of the obtained compounds on a panel of eight tumor cell lines. As a result, four new compounds were isolated from Hedyotis diffusa Willd., including three iridoid glycosides of Shecaoiridoidside A–C (1–3) and a cerebroside of shecaocerenoside A (4). Also, six known iridoid compounds (5–10) were also obtained. The cytotoxicity of all compounds against human tumor cell lines of HL-60, HeLa, HCT15, A459, HepG2, PC-3, CNE-2, and BCG-823 were also evaluated in vitro. New compound 3 exhibited evident cytotoxicity to all tumor cell lines except the Hela, and the IC50 values are from 9.6 µM to 62.2 µM, while new compound 4 showed moderate cytotoxicity to all the cell lines, and the IC50 values are from 33.6 µM to 89.3 µM. By contrast, new compound 1 and known compound 9 showed moderate cytotoxicity to HCT15, A459, and HepG2 selectively. Known compound 7 also exhibited moderate cytotoxicity to HCT15 and A459 selectively. As a TCM,  Hedyotis has been widely distributed in subtropical area of the world pyran-1-O-4-methylsenecioyloxyl-11-hydroxymethyl-3-en 11-O-β-d-galactopyranoside and named as Shecaoiridoidside A  determined the molecular formula of 2 as C21H32O13. Hydrolysis experiment of 2 liberated d-glucose and d-apiose which determined by GC-MS analysis. In the 1H-NMR spectrum of 2 , 3.87 , 3.08 , three methylene groups at δ 5.10 , 4.44 , 2.34 , 2.19 , 5.08 , 5.011 , and a methyl group at δ 1.59  could be observed. The β-configuration of glucopyranosyl moiety was confirmed by the coupling constant of H-1′ (J = 7.8 Hz). The 13C-NMR and DEPT spectra of 2 −C(5)−C(6)−C(7) −C(7) . The cycd at C-7 .2 was determined by NOESY spectrum. The NOE correlations -8-hydroxy-8-methyl-4-methylenehexahydrocyclo-penta[c]pyran-1(3H)-one 7-O-(6-O-β-d-apiofuranosyl)-β-d-glucopyranoside and named Shecaoiridoidside B  established the molecular formula of 3 as C23H28O10. Hydrolysis experiment of 3 liberated d-glucose which determined by GC-MS analysis. In the 1H-NMR of 3 , 3.76, 4.37  and 4.18 , a nonoxygenated methylene at δ 2.74  and 2.26 , a nonoxygenatedmethine at δ 3.25 , and three olefinic protons at δ 5.78 , 4.91  and 4.92 , and a p-substituted benzene protons at 7.88  and 6.81  were observed. The β-configuration of glucopyranosyl moiety was confirmed by coupling constant of H-1′ (J = 7.8 Hz). The 13C-NMR and DEPT spectra of 3  in patriridoside G was substituted by the signal at δC 59.3 (CH2-10) in 3, which indicated that CH3-10 of patriridoside G was substituted by a hydroxyl group.Compound NMR of 3 , signalstra of 3  showed 2doside G . The mai1H−1H COSY spectra of 3 showed the coupling sequences of C-3/C-4/C-11, C-11/C-4/C-5/C-6/C-7/C-8/C-10, C-2”/C-3”, and C-5”/C-6” -6-O-(6-O-4-hydroxybenzoyl-β-d-glucopyranosyl)-8-hydroxymethyl-4-methylene-4,5,6,9-tetrahydro-3H-cyclopenta[b]furan-9-yl-methanol and named Shecaoiridoidside C  determined the molecular formula of 4 as C48H93NO9. Methanolysis experiment of 4 liberated d-glucose which determined by GC-MS analysis. In 1H- and 13C-NMR spectra of 4, signals of anomeric proton δH  and δC  indicated the presence of a β-d-glucopyranosyl moiety. The characteristics of a cerebroside with a 2-hydroxy fatty acid fraction in 4 could be confirmed by analyzing its 1H- and 13C-NMR data  of the FAM -21-methyl-4-tetracosene-1,3-diol which was named as shecaocerenoside A  +; HRESIMS [M + Na]+m/z 511.1788 calc. 511.1791 for C22H32O12Na; 1H- and 13C-NMR data, see Shecaoiridoidside B (2). white amorphous powder, c 0.10, MeOH); IR (KBr) νmax 3462, 3430, 2974, 2858, 1712, 1648, 1428, 1373, 1235, 1104 cm−1; ESIMS m/z 515 (100) [M + Na]+; HRESIMS [M + Na]+m/z 515.1737, calcd. 515.1741 for C21H32O13Na; 1H- and 13C-NMR data, see Shecaoiridoidside C (3). white amorphous powder, c 0.12, MeOH); IR (KBr) νmax 3518, 3421, 2875, 1674, 1447, 1384, 1325, 1169, 1080, 891, 595 cm−1; ESIMS m/z 487 (100) [M + Na]+; HRESIMS [M + Na]+m/z 487.1576, calcd. 487.1580 for C23H28O10Na; 1H- and 13C-NMR data, see Shecaocerenoside A (4). white amorphous powder; c = 0.15, C5H5N); IR (KBr) νmax 3411, 2941, 2838, 1635, 1532, 1455, 1162, 724 cm−1; ESIMS m/z 828 (100) [M + H]+; HRESIMS [M + H]+m/z 828.6924 calc. 828.6929 for C48H93NO9H; 1H- and 13C-NMR data, see 1–3 (2.0 mg) with 2 mol/L H2SO4 (2.0 mL), and then treated with trimethylchlorosilane, respectively. The sugar derivatives were further analyzed by GC-MS. As a result, the sugar derivatives from compounds 1 and 3 were determined to be d-galactose (tR = 19.46 min) and d-glucose (tR = 11.33 min), respectively. The sugar derivatives from compound 2 was determined to be d-glucose (tR = 11.33 min) and d-apiose (tR = 14.53 min).Acid hydrolysis experiment was carried out as the method in reference . Briefly4 was carried out according to the previous study + and 366 [M − H2O + H]+ from ESIMS analysis led the LCB of 4 was identified as 2-aminotetracos-7-ene-1,3-diol -2,5-diphenyltetrazolium bromide (MTT) method in vitro. The assay protocol was conducted by previous published paper ,39. The H. diffusa Willd. based on its clinical application of treating malignant tumors and 10 compounds were obtained, including three new iridoid glycosides and a new cerebroside. The structures of new compounds were identified as -8-hydroxy-8-hydroxymethyl-6,7-epoxylcyclopenta[c]pyran-1-O-4-methylsenecioyl-oxyl-11-hydroxymethyl-3-en 11-O-β-d-glucopyranoside (1), -8-hydroxy-8-methyl-4-methylenehexahydrocyclopenta[c]pyran-1(3H)-one 7-O-(6-O-β-d-apiofuranosyl)-β-d-glucopyrano-side (2), -6-O-(6-O-4-hydroxybenzoyl-β-d-glucopyranosyl)-8-hydroxymethyl-4-methylene-4,5,6,9-tetrahydro-3H-cyclopenta[b]furan-9-yl-methanol (3), and 1-O-β-d-glucopyranosyl- -2-[(2′R)-2-hydroxyloctadecanamideamino]-21-methyl-4-tetracosene-1,3-diol (4), respectively. Antitumor assays in vitro discovered cytotoxic compounds 1, 3, 4, 7, and 9, especially found that new compound 3 exhibited evident cytotoxicity to all tumor cell lines except the Hela.We investigated the chemical constituents of


E-β-trans-5,8,11-trihydroxybergamot-9-ene (1) and β-trans-2β,5,15-trihydroxybergamot-10-ene (2), were isolated from the marine-derived fungus Aspergillusfumigatus YK-7, along with three known terpenoids 3–5. Their structures were determined by spectroscopic methods . Antiproliferative effects on human leukemic monocyte lymphoma U937 and human prostate cancer PC-3 cell lines were measured in vitro. Compound 4 exhibited potent activity against the U937 cell line with an IC50 value of 4.2 μM.Two new β-bergamotane sesquiterpenoids,  Aspergillusfumigatus has been found to generate many structurally and biologically diversified metabolites heptane skeleton. Additionally, the COSY correlations from H-8 to H-10, and the HMBC correlations of the olefinic proton H-9 (δH 5.66) with the oxygenated carbon C-11 (δC 70.9), and of the olefinic proton H-10 (δH 5.88) with C-12 (δC 29.8) and C-13 (δC 30.1) suggested the presence of a 1,4-dihydroxy-4-methylpent-2-enyl side chain in 1. The linkage of the two moieties was secured by the HMBC correlations of H-8 (δH 4.85) and H-9 (δH 5.66) with C-6 (δC 52.5). Therefore, compound 1 was established as a β-5,8,11-trihydroxybergamot-9-ene + . The IR, NMR, and HR-MS spectra of compound 1 can be found at -trans-2β,5,15-trihydroxybergamot-10-ene (2): colorless needles (MeOH); mp 115–116 °C;c 0.10, MeOH); IR(KBr) νmax 3405, 2921, 2852, 1642, 1452, 1383, 1150, 1052, 954 cm−1; 1H- and 13C-NMR data, see m/z 277.1737 [M + Na]+ . The IR, NMR, and HR-MS spectra of compound 2 can be found at β50, was calculated. Doxorubicin hydrochloride  was used as a positive control, and 0.1% DMSO was used as a negative control.The growth inhibitory assay was performed as described previously ,18. Huma


Five key intermediate nucleosides, having 6-methoxy, 7-iodo, and 2-bromo groups, were further derivatized to 23 final 8-aza-7-deazapurine nucleoside derivatives. The structures of N9- and N8-glycosylated products were assigned based on UV and NMR spectra. HMBC analysis of 2D NMR spectra and X-ray crystallographic studies of the representative compounds unambiguously verified the connection of ribose ring to N9- or N8-position of the purine ring. The anticancer activity of these new compounds was evaluated.Glycosylation of 6-amino-4-methoxy-1H-pyrazolo[3,4- Leishmania and trypanosome species [5 are powerful purine nucleoside antagonists [N8-2′-deoxy-adenine analogues show ambiguous base pairing when incorporated into oligonucleotide duplexes opposite to the four canonical DNA-constituents [5 (pK = 5.8) and 11 (pK = 6.4)   are poss1–3 and 5–13  [d]pyrimidine (purine base B) [d]pyrimidin-4(5H)-one (purine base C) . Su. SuA witectively . The 6-m8 showed the best inhibitory activity against A549 with an IC50 value of 7.68 μM. Compounds 14 and 16 showed slightly better activity than other modified derivatives, which did not show detectable activity against the tested tumor cell lines. By comparing the activity of tested analogs with different substituents, it suggested that electron-donating group at 6-position of the base may improve the activity. Further biological evaluation of these modified nucleosides is in progress and will be reported in due course.Newly synthesized compounds were tested for inhibitory activity against human lung carcinoma cell line A549 and the human breast cancer cell line MDA-MB-231 . We foun1H NMR spectra were obtained with a Bruker Avance 400 spectrometer using DMSO-d6 or CDCl3 (ppm) downfield with respect to an internal δ as solvents. Chemical shifts are reported as standard of tetramethylsilane (TMS). Product purity was tested by an Agilent 1260 analytical HPLC system. LC-MS spectra were measured on an Agilent 6120 LC–MS spectrometer. High-resolution mass spectra were obtained on a Micro-Q-TOF mass spectrometer. Single-crystal structure was determined by a Xcalibur, Eos, Gemini X-ray single crystal diffractometer. TLC was performed on silica gel GF254. Flash chromatography was performed on silica gel 200–300 mesh (Yantai Silica Gel Co. LTD). 6-Amino-4-methoxy-1H-pyrazolo[3–d]pyrimidine (purine base A), 6-amino-3-iodo-4-methoxy-1H-pyrazolopyrimidine (purine base B) and 3,6-dibromo-1H-pyrazolopyrimidin-4(5H)-one (purine base C) were prepared following literature procedures pyrimidineSynthesis of 6-amino-4-methoxy-1-: A suspension of 6-amino-4-methoxy-1H-pyrazolo[3.4-d]pyrimidine  and a catalytic amount of ammonium sulfate in hexamethyldisilazane  was refluxed for 6 h. The excess hexamethyldisilazane was removed by evaporation under reduced pressure, and the residue was dissolved in 1,2-dichloroethane (200 mL). l-O-Acetyl-2,3,5-tri-O-benzoyl-d-ribofuranose (ribose I)  was added at room temperature. The reaction mixture was cooled to 0 °C, and trimethylsilyl trifluoromethanesulfonate  was added dropwise for 30 min with stirring. The reaction mixture was stirred at room temperature overnight. Upon completion of the reaction as monitored by TLC, the mixture was diluted with dichloromethane (200 mL) and washed with saturated sodium bicarbonate solution. The aqueous layer was extracted with dichloromethane. The combined organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford 10.0 g main product N9 β-isomer 24 as a white solid in 30.3% yield with an HPLC purity of 96%; fR = 0.3 (petroleum ether–ethyl acetate = 1:1).d-ribofuranosyl)-2H-pyrazolopyrimidineSynthesis of 6-amino-4-methoxy-2-: 6-Amino-4-methoxy-1H-pyrazolopyrimidine  was suspended in 200 mL dry acetonitrile and 1-O-acetyl-2,3,5-tri-O-benzoyl-d-ribofuranose (ribose I)  was added. The reaction mixture was heated to reflux at 95 °C, and the freshly distilled BF3·OEt2  was then added with stirring. The reaction mixture became clear immediately and then slowly became dark. The mixture was stirred at this temperature for 15 min. Upon the completion of the reaction as monitored by TLC, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford 25.0 g N8-product 25 as a white solid in 83.3% yield with an HPLC purity of 96.8%. fR = 0.6 (dichloromethane–methanol = 30:1). UV–vis (MeOH) λmax: 225 nm; ESI-MS m/z: 610.6 [M + H]+.d-ribofuranosyl)-1H-pyrazolo-pyrimidineSynthesis of 6-amino-3-iodo-4-methoxy-1- d-ribofuranosyl)-1H-pyrazolopyrimidineand 6-amino-3-iodo-4-methoxy-1-(β- (3): 6-Amino-3-iodo-4-methoxy-1H-pyrazolopyrimidine  and 1-O-acetyl-2,3,5-tri-O-benzoyl-d-ribofuranose (ribose I)  were suspended in 200 mL of anhydrous acetonitrile. The freshly distilled BF3·OEt2 solution  was added in 30 min with stirring at room temperature, and the reaction mixture was stirred at the same temperature for 5 h. Upon the completion of the reaction as monitored by TLC, the reaction mixture was poured into 500 mL of saturated sodium bicarbonate solution and extracted with ethyl acetate. The organic phase was separated, and the aqueous phase was extracted with ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate. The drying agent was filtered off, and the filtrate was concentrated under reduced pressure to afford crude product 26, which was used directly without further purification. Rf = 0.2 (dichloromethane–methanol = 30:1). The crude compound 26 was dissolved in 100 mL of MeOH and 10 mL of THF solution. Then sodium methoxide  was added, and the mixture was stirred at room temperature for 5 h. Upon completion of the reaction as monitored by TLC, the mixture was neutralized with 2 N HCl solution and evaporated under reduced pressure. The resulting residue was purified by column chromatography to afford 1.8 g desired product 3 as a white solid in 25% overall yield with an HPLC purity of 98.5%. Rf = 0.2 (dichloromethane–methanol = 10:1). UV–vis (MeOH) λmax: 232, 278 nm; 1H NMR  δ 7.02 , 5.90 , 5.33 , 5.08 , 4.74 , 4.45–4.55 , 4.08–4.15 , 3.09 , 3.80–3.87 , 3.38–3.58 ; ESI-MS m/z: 424.3 [M + H]+, 446.2 [M + Na]+.Synthesis ofd-ribofuranosyl)-1H-pyrazolopyrimidine3,6-dibromo-4-hydro-1-: 3,6-Dibromo-1H-pyrazolopyrimidin-4(5H)-one  was suspended in 300 mL dry acetonitrile and 1-O-acetyl-2,3,5-tri-O-benzoyl-d-ribofuranose (ribose I)  was added. The reaction mixture was heated to reflux at 95 °C, and the freshly distilled BF3·OEt2  was then added with stirring. The reaction mixture became clear immediately and then slowly became dark. The mixture was kept stirred at this temperature for 20 min. Upon the completion of the reaction as monitored by TLC, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford 33 g product 27 as a white solid in 92% yield with an HPLC purity of 98%. fR = 0.5 (dichloromethane–methanol = 20:1). UV–vis (MeOH) λmax: 231, 275 nm; 1H NMR  δ 8.06–8.16 , 7.91–8.01 , 7.51–7.61 , 7.34–7.46 , 6.62 , 6.27–6.36 , 6.17–6.26 , 4.72–4.89 , 4.57–4.70 ; ESI-MS m/z: 761.0 [M + Na]+.d-ribofuranosyl)-4H-pyrazolopyrimidin-4-oneSynthesis of 6-amino-3-bromo-1,5-dihydro-1-(β- (4): Compound 27  was dissolved in NH3/MeOH  in high pressure reactor, and the reaction mixture was heated at 120 °C overnight. Upon completion of the reaction as monitored by TLC, the reaction solvent was evaporated under reduced pressure. The resulting precipitate was filtered off and recrystallized from water to afford 2.5 g compound 4 as a white solid in 56% yield with an HPLC purity of 98%. Rf = 0.3 (dichloromethane–methanol = 1:1). UV–vis (MeOH) λmax: 223, 255 nm; ESI-MS m/z: 363 [M + H]+.d-ribofuranosyl)-1H-pyrazolopyrimidineSynthesis of 6-amino-4-methoxy-1-(β- (1): Compound 24  was dissolved in a mixture of methanol (200 mL) and THF (50 mL). Sodium methoxide  was then added, and the mixture was stirred at room temperature. Upon completion of the reaction as monitored by TLC, the mixture was neutralized with 2 N HCl solution. Then the reaction mixture was evaporated under reduced pressure, and the resulting residue was purified by column chromatography to afford 6.2 g desired product 1 as a white solid in 87% yield with an HPLC purity of 98.2%. Rf = 0.2 (dichloromethane–methanol = 10:1). UV–vis (MeOH) λmax: 222, 252, 276 nm; 1H NMR  δ 7.90 , 6.86 , 5.97 , 5.32 , 5.05 , 4.74 , 4.46–4.56 , 4.13–4.21 , 3.97 , 3.82–3.89 , 3.49–3.60 , 3.36–3.47 ; ESI-MS m/z: 298.1 [M + H]+, 320.1 [M + Na]+.d-ribofuranosyl)-2H-pyrazolopyrimidineSynthesis of 6-amino-4-methoxy-2-(β- (2): As described for 1, 6-amino-4-methoxy-2--2H-pyrazolopyrimidine 25  was converted to 6.2 g of compound 2 as a white solid in 85% yield with an HPLC purity of 97.2%. Rf = 0.15 (dichloromethane–methanol = 10:1). UV–vis (MeOH) λmax: 222, 266, 296 nm; 1H NMR  δ 8.53 , 6.46 , 5.75 , 4.28–4.34 , 4.15 , 3.92–4.01 , 3.66–3.72 , 3.51–3.57 ; ESI-MS m/z: 297.9 [M + H]+. HRMS calcd for C11H16N5O5 298.1151 [M + H]+, found 298.1153.d-ribofuranosyl)-1H-pyrazolopyrimidineSynthesis of 4,6-diamino-1-(β- (5): Compound 1  was dissolved in 25% ammonium hydroxide (40 mL) in high pressure reactor, and the reaction mixture was heated at 80 °C overnight. Upon completion of the reaction as monitored by TLC, the reaction solvent was evaporated under reduced pressure. The resulting precipitate was filtered off and recrystallized from water to afford 900 mg of compound 5. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by column chromatography to afford 520 mg more desired product 5 as a white solid. Totally, 1.42 g desired product was obtained in 75% yield with an HPLC purity of 96.8%. Rf = 0.2 (dichloromethane–methanol = 4:1). UV–vis (MeOH) λmax: 223, 254, 275 nm; 1H NMR  δ 7.97 , 7.38 , 6.34 , 5.91 , 5.21–5.38 , 5.02–5.20 , 4.45–4.55 , 4.08–4.20 , 3.80–3.97 , 3.38–3.58 ; ESI-MS m/z: 283.1 [M + H]+.d-ribofuranosyl)-1H-pyrazolo-pyrimidineSynthesis of 6-amino-4-hydrazino-1-(β- (6): Compound 1  was dissolved in 80% hydrazine hydrate solution (30 mL), and the reaction mixture was heated at 70 °C for 3 h. Upon completion of the reaction as monitored by TLC, the reaction solvent was evaporated under reduced pressure. The resulting precipitate was filtered off and recrystallized from water to afford 850 mg compound 6. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by column chromatography to afford 550 mg desired product 6 as a white solid. Totally, 1.4 g desired product was obtained in 72% yield with an HPLC purity of 96%. Rf = 0.1 (dichloromethane–methanol = 3:1). UV–vis (MeOH) λmax: 225, 275 nm; 1H NMR  δ 8.98, 8.50 , 8.10, 7.86 , 6.23 , 6.01 , 5.93 , 5.25 , 5.01 , 4.78–4.96, 4.55–4.74 , 4.40–4.54 , 4.07–4.24 , 3.76–3.92 , 3.36–3.58 ; ESI-MS m/z: 298.1 [M + H]+. HRMS calcd for C10H16N7O4 298.1264 [M + H]+, found 298.1265.d-ribofuranosyl)-4H-pyrazolopyrimidin-4-oneSynthesis of 6-amino-1,5-dihydro-1-(β- (7): Compound 1  was dissolved in 2N NaOH solution (50 mL). The reaction mixture was stirred at room temperature overnight. Upon completion of the reaction as monitored by TLC, the reaction mixture was neutralized with 6 N HCl under cooling condition and then concentrated under reduced pressure. The resulting precipitate was filtered off and recrystallized from water to yielding 1.2 g of compound 7 as a white solid in 62% yield with an HPLC purity of 96.3%. Rf = 0.2 (dichloromethane–methanol = 4:1). UV–vis (MeOH) λmax: 219, 254 nm; 1H NMR  δ 10.61 , 7.85 , 6.70 , 5.88 , 5.31 , 5.05 , 4.73 , 4.42–4.52 , 4.10–4.20 , 3.80–3.90 , 3.38–3.62 ; 13C NMR  δ: 157.7 (C(4)), 155.9 (C(6)), 154.9 (C(2)), 135.4 (C(7)), 99.6 (C(5)), 87.5 (C(1′)), 84.8 (C(4′)), 73.0 (C(2′)), 70.8 (C(3′)), 62.4 (C(5′)); ESI-MS m/z: 284.1 [M + H]+.d-ribofuranosyl)-1H-pyrazolopyrimidineSynthesis of 4,6-diamino-3-iodo-1-(β- (8): As described for 5, 6-amino-3-iodo-4-methoxy-1-(β-d-ribofuranosyl)-1H-pyrazolopyrimidine 3  was converted to 860 mg of compound 8 as a white solid in 69% yield with an HPLC purity of 95.3%. Rf = 0.3 (dichloromethane–methanol = 6:1). UV–vis (MeOH) λmax: 229, 263, 278 nm; 1H NMR  δ 6.37 , 5.85 , 5.29 , 5.04 , 4.78 , 4.43–4.54 , 4.06–4.16 , 3.80–3.86 , 3.37–3.60 ; ESI-MS m/z: 409.0 [M + H]+, 431.0 [M + Na]+.d-ribofuranosyl)-4H-pyrazolopyrimidin-4-oneSynthesis of 6-amino-3-iodo-1,5-dihydro-1-(β- (9): As described for 7, 6-amino-3-iodo-4-methoxy-1-(β-d-ribofuranosyl)-1H-pyrazolopyrimidine 3  was converted to 1.0 g compound 9 as a white solid in 65% yield with an HPLC purity of 97%. Rf = 0.2 (dichloromethane–methanol = 6:1). UV–vis (MeOH) λmax: 220, 232, 256 nm; ESI-MS m/z: 410.1 [M + H]+.d-ribofuranosyl)-4H-pyrazolopyrimidin-4-oneSynthesis of 6-amino-2,5-dihydro-2-(β- (10): As described for 7, 6-amino-4-methoxy-2-(β-d-ribofuranosyl)-2H-pyrazolopyrimidine 2  was converted to 650 mg of compound 10 as a white solid in 67.5% yield with an HPLC purity of 98%. Rf = 0.15 (dichloromethane–methanol = 1:1). UV–vis (MeOH) λmax: 225, 280 nm; 1H NMR  δ 10.45 , 8.52 , 6.28 , 5.66 , 5.49 , 5.11 , 5.01 , 4.25–4.35 , 4.08–4.18 , 3.89–3.96 , 3.61–3.64 , 3.46–3.57 ; 13C NMR  δ: 160.8 (C(4)), 159.3 (C(6)), 153.7 (C(2)), 128.2 (C(7)), 102.2 (C(5)), 94.0 (C(1′)), 85.1 (C(4′)), 74.7 (C(2′)), 70.0 (C(3′)), 61.3 (C(5′)); ESI-MS m/z: 284.0 [M + H]+.d-ribofuranosyl)-2H-pyrazolopyrimidineSynthesis of 4,6-diamino-2-(β- (11): As described for 5, 6-amino-4-methoxy-2-(β-d-ribofuranosyl)-2H-pyrazolopyrimidine 2  was converted to 670 mg of compound 11 as a white solid in 70% yield with an HPLC purity of 96%. Rf = 0.15 (dichloromethane–methanol = 10:1). UV–vis (MeOH) λmax: 225, 262, 302 nm; 1H NMR  δ 8.42 , 7.79 , 6.58 , 5.73 , 4.90–5.54 , 4.34 , 4.11 , 3.97 , 3.45–3.74 ; ESI-MS m/z: 283.2 [M + H]+.d-ribofuranosyl)-2H-pyrazolopyrimidineSynthesis of 6-amino-4-hydrozino-2-(β- (12): As described for 6, 6-amino-4-methoxy-2-(β-d-ribofuranosyl)-2H-pyrazolopyrimidine 2  was converted to 695 mg of compound 12 as a white solid in 69% yield with an HPLC purity of 96%. Rf = 0.1 (dichloromethane–methanol = 1:2). UV–vis (MeOH) λmax: 223, 264, 298 nm; 1H NMR  δ 8.80, 8.35 , 6.89 , 6.64 , 5.68–5.84 , 5.38–5.60 , 4.92–5.24 , 4.26–4.43 , 4.04–4.25 , 3.86–4.02 , 3.16–3.70 , 3.46–3.57 ;ESI-MS m/z: 298.8 [M + H]+. HRMS calcd for C10H16N7O4 298.1264 [M + H]+, found 298.1263.d-ribofuranosyl)-2H-pyrazolopyrimidineSynthesis of 6-amino-4-hydroxyamino-2-(β- (13): Compound 2  was dissolved in 50% hydroxylamine solution , and the reaction mixture was heated at 60 °C. Upon completion of the reaction as monitored by TLC, the reaction solvent was evaporated under reduced pressure. The resulting precipitate was filtered off and recrystallized from water to yielding 506 mg of compound 13 as a white solid in 50% yield with an HPLC purity of 96%. Rf = 0.2 (dichloromethane–methanol = 1:1). UV–vis (MeOH) λmax: 223, 285 nm; 1H NMR  δ 9.97 , 9.86 , 8.16 , 6.81 , 5.59 , 5.46 , 5.14 , 4.94–5.08 , 4.26–4.33 , 4.07–4.16 , 3.87–3.94 , 3.56–3.64 , 3.45–3.53 ; ESI-MS m/z: 298.8 [M + H]+, 320.7 [M + Na]+. HRMS calcd for C10H14N6O5Na 321.0923 [M + Na]+, found 321.0920.d-ribofuranosyl)-1H-pyrazolopyrimidineSynthesis of 6-amino-3-(furan-2-yl)-4-methoxy-1-(β- (14): To a stirred mixture of 6-amino-3-iodo-4-methoxy-1-(β-d-ribofuranosyl)-1H- pyrazolopyrimidine 3  and bis(triphenylphosphine)-palladium (II) chloride  in 30 mL of anhydrous DMF was added 2-(tributylstannyl)furan  under an nitrogen atmosphere. The reaction mixture was stirred at 90 °C for 18 h and the concentrated to dryness under reduced pressure. The resulting residue was purified by column chromatography to afford 450 mg desired product 14 as a pale-yellow solid in 90% yield with an HPLC purity of 96%. Rf = 0.5 (dichloromethane–methanol = 10:1). 1H NMR  δ 7.77–7.84 , 7.21 , 6.96 , 6.61–6.69 , 6.02 , 5.34 , 5.09 , 4.76 , 4.54–4.61 , 4.17–4.26 , 4.05 , 3.84–3.91 , 3.41–3.64 ; ESI-MS m/z: 364.2 [M + H]+. HRMS calcd for C15H18N5O6 364.1257 [M + H]+, found 364.1258.d-ribofuranosyl)-1H-pyrazolopyrimidineSynthesis of 6-amino-4-methoxy-3-phenyl-1-(β- (15): To a stirred mixture of 6-amino-3-iodo-4-methoxy-1-(β-d-ribofuranosyl)-1H-pyrazolo pyrimidine 3 , phenylboronic acid  and potassium carbonate  in 60 mL of DME–water (2:1) was added Pd(PPh3)4 . The reaction mixture was stirred at 90 °C for 4 h. Upon completion of the reaction as monitored by TLC, the mixture was concentrated under reduced pressure. The residue was purified by flash chromatography on a silica gel column to provided 570 mg of product 15 as a white solid in 80% yield with an HPLC purity of 96%. Rf = 0.6 (dichloromethane–methanol = 10:1). 1H NMR  δ 7.90–8.00 , 7.39-7.54 , 6.92 , 6.07 , 5.36 , 5.08 , 4.76 , 4.54–4.62 , 4.21–4.31 , 3.99 , 3.85–3.92 , 3.43–3.65 ; ESI-MS m/z: 374.1 [M + H]+. HRMS calcd for C17H20N5O5 374.1464 [M + H]+, found 374.1461.d-ribofuranosyl)-1H-pyrazolopyrimidineSynthesis of 6-amino-3-ethynyl-4-methoxy-1-(β- (16): To a stirred mixture of compound 3 , Pd (PPh3)4 , CuI  and triethylamine  in DMF (20 mL) was added trimethylsilylacetylene . The reaction mixture was stirred at room temperature overnight. Upon completion of the reaction as monitored by TLC, the mixture was concentrated under reduced pressure. The resulting residue was directly deprotected by treatment with K2CO3  in MeOH (30 mL) at room temperature for 2 h. Upon completion of the reaction as monitored by TLC, the mixture was concentrated under reduced pressure. The residue was purified by flash chromatography on a silica gel column to provided 300 mg of product 16 as a white solid in 65% overall yield with an HPLC purity of 96%. Rf = 0.45 (dichloromethane–methanol = 10:1). 1H NMR  δ 7.03 , 5.95 , 5.37 , 5.10 , 4.75 , 4..45–4.55 , 4.10–4.17 , 3.98 , 3.81–3.90 , 3.37–3.58 . HRMS calcd for C13H16N5O5 322.1151 [M + H]+, found 322.1150.d-ribofuranosyl)-1H- pyrazolopyrimidineSynthesis of 6-amino-4-methoxy-3-[N-(trifluoroacetyl)aminopropyn-1-yl]-1-(β- (17): The mixture of compound 3 , Pd (PPh3)4 , CuI , N-(2-propynyl)-2,2,2-trifluoroacetamide  and triethylamine  in DMF (20 mL) was stirred at room temperature overnight. Upon completion of the reaction as monitored by TLC, the mixture was concentrated under reduced pressure. The residue was purified by flash chromatography on a silica gel column to provided 310 mg of product 17 as a white solid in 60% yield with an HPLC purity of 95%. Rf = 0.40 (dichloromethane–methanol = 10:1). 1H NMR  δ 10.16 , 7.03 , 5.95 , 5.36 , 5.10 , 4.74 , 4.44–4.55 , 4.07–4.17 , 3.96 , 3.76–3.88 , 3.37–3.56 . ESI-MS m/z: 447.1 [M + H]+.d-ribofuranosyl)-1H-pyrazolo pyrimidineSynthesis of 6-amino-4-methoxy-1--α- (28) d-ribofuranosyl)-1H- pyrazolopyrimidineand 6-amino-4-methoxy-1--β- (29): To a stirred suspension of tris[2-(2-methoxyethoxy)ethyl]amine (TDA-1)  and finely grounded KOH  in anhydrous acetonitrile (200 mL) was added 6-amino-4-methoxy-1H-pyrazolo-[3.4-d]pyrimidine . The reaction mixture was stirred for another 15 min. 2-Deoxy-3,5-di-O-(p-toluoyl)-α-D-erythro-pentfuranosyl chloride (ribose II)  was added, and the reaction mixture was stirred at room temperature for an additional 30 min. Upon completion of the reaction as monitored by TLC, the reaction mixture was diluted with ethyl acetate (100 mL) and washed with saturated ammonium chloride solution. The aqueous layer was extracted with ethyl acetate. The combined organic layer was washed with saturated brines solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by column chromatography to afford two products. The compound eluting first was β-isomer 29  as a white solid with an HPLC purity of 97.1%. Rf = 0.4 (petroleum ether–ethyl acetate = 3:1). The compound eluting last was α-isomer 28  as a white foam with an HPLC purity of 96%. Rf = 0.35 (petroleum ether–ethyl acetate = 3:1).d-ribofuranosyl)-1H-pyrazolopyrimidineSynthesis of 6-amino-4-methoxy-1-(2-deoxy-α- (18): As described for 1, 6-amino-4-methoxy-1--α-d-ribofuranosyl) -1H-pyrazolopyrimidine 28  was converted to 1.2 g of compound 18 as a white solid in 89% yield with an HPLC purity of 95%. Rf = 0.35 (dichloromethane–methanol = 10:1). 1H NMR  δ 7.94 , 6.88 , 6.27–6.45 , 5.60 , 4.72 , 4.10–4.20 , 3.98 , 3.87–3.95 , 3.35–3.56  2.51–2.73 ;d-ribofuranosyl)-1H-pyrazolopyrimidineSynthesis of 6-amino-4-methoxy-1-(2-deoxy-β- (19): As described for 1, 6-amino-4-methoxy-1--β-d-ribofuranosyl)- 1H-pyrazolo pyrimidine 29  was converted to 2.0 g of compound 19 as a white solid in 91% yield with an HPLC purity of 97%. Rf = 0.4 (dichloromethane–methanol = 10:1). UV–vis (MeOH) λmax: 224, 251, 285 nm; 1H NMR  δ 7.88 , 6.84 , 6.41 , 5.23 , 4.73 , 4.39 , 3.97 , 3.78 , 3.41–3.56  2.62–3.18 , 2.12–2.23 ; ESI-MS m/z: 282.8 [M + H]+.d-ribofuranosyl)-4H-pyrazolopyrimidin-4-oneSynthesis of 6-amino-1,5-dihydro-1-(2-deoxy-β- (20): As described for 7, 6-amino-4-methoxy-1-(2-deoxy-β-d-ribofuranosyl)-1H-pyrazolo pyrimidine 19  was converted to 990 mg of compound 20 as a white solid in 88% yield with an HPLC purity of 95%. Rf = 0.15 (dichloromethane–methanol = 5:1). UV–vis (MeOH) λmax: 257 nm; ESI-MS m/z: 267.9 [M + H]+, 289.9 [M + Na]+.d-ribofuranosyl)-1H-pyrazolopyrimidineSynthesis of 4,6-diamino-1-(2-deoxy-β- (21): As described for 5, 6-amino-4-methoxy-1-(2-deoxy-β-d-ribofuranosyl)-1H-pyrazolopyrimidine 19  was converted to 620 mg of compound 21 as a white solid in 65% yield with an HPLC purity of 96%. Rf = 0.2 (dichloromethane–methanol = 5:1). UV–vis (MeOH) λmax: 225, 257, 278 nm; ESI-MS m/z: 267.7 [M + H]+.d-ribofuranosyl)-1H-pyrazolopyrimidineSynthesis of 6-amino-4-hydrazino-1-(2-deoxy-β- (22): As described for 6, 6-amino-4-methoxy-1-(2-deoxy-β-d-ribofuranosyl)-1H-pyrazolopyrimidine 19  was converted to 690 mg of compound 22 as a white solid in 70% yield with an HPLC purity of 96%. Rf = 0.15 (dichloromethane–methanol = 5:1). UV–vis (MeOH) λmax: 226, 276 nm; 1H NMR  δ 9.04, 8.49 , 8.09, 7.80 , 6.36 , 6.05, 6.15 , 5.17 , 4.83 , 4.45–4.76 , 4.33–4.42 , 3.72–3.81 , 3.45–3.55 , 3.35–3.41 , 2.67–2.76 , 2.07–2.17 ; ESI-MS m/z: 282.8 [M + H]+.d-ribofuranosyl)-1H-pyrazolopyrimidineSynthesis of 6-amino-4-hydroxyamino-1-(2-deoxy-β- (23): As described for 13, 6-amino-4-methoxy-1-(2-deoxy-β-d-ribofuranosyl)-1H-pyrazolopyrimidine 19  was converted to 560 mg of compound 23 as a white solid in 56% yield with an HPLC purity of 95%. Rf = 0.2 (dichloromethane–methanol = 4:1). UV–vis (MeOH) λmax: 223, 276 nm; 1H NMR  δ 9.75, 9.53 , 7.50 , 6.68 , 6.25 , 5.18 , 4.78 , 4.32–4.45 , 3.72–3.85 , 3.35–3.53 , 2.61–2.75 , 2.07–2.17 ; ESI-MS m/z: 283.8 [M + H]+.3 cells per well) in the medium supplemented with 10% fetal bovine serum (FBS) and Dulbecco’s modified Eagle medium (DMEM) at 37 °C with a humidified 5% CO2 atmosphere. After 24 h, different concentrations of synthesized compounds  were added and the cells were exposed to compounds for 72 h. Cell proliferation was evaluated by MTT assay according to the manufacturer’s instructions and incubated for 4 h. The optical density was determined at 570 nm by Varioskan Flash Multimode Reader . The IC50 value, which refers to the concentration (μM) of the compound to cause 50% cell death with respect to the control, was calculated according to the inhibition ratios. The experiments were performed in triplicates to obtain the mean cell viability.Cells were plated in a 96-well plate  and 2-deoxy-3,5-di-O-(p-toluoyl)-α-D-erythro-pentafuranosyl chloride (ribose II), and further deprotection provided the corresponding key intermediates 1, 2, 3, and 19. These intermediates were further substituted with NH2, OH, NHNH2 and NHOH to furnish final products 5–13 and 20–23. The iodinated intermediate 3 was cross-coupled with aromatic and alkynyl reagents to provide final products 14–17. The newly synthesized compounds were all well characterized. The structures of N9- and N8-glycosylated products were assigned. HMBC analysis of 2D NMR spectra further verified the N9- and N8-products. X-ray crystallographic studies unambiguously confirmed the conclusion. Antitumor inhibitory test resulted in some good hits to guide further studies. The protocols discovered herein can be utilized to expand further research in related fields.The 8-aza-7-deaza-purine bases


E-7′R,8′-dihydroxy-4′,8′-dimethyl-3′-nonenyl)-2,5-dihydro-1,1-dioxo-thiophen 7′-O-β-d-glucopyranosyl-(1→4)-O-β-d-glucopyranosyl-(1→4)-O-β-d-glucopyranoside (1), 3--2,5-dihydro-1,1-dioxo-thiophen 7′-O-β-d-glucopyranosyl-(1→4)-O-β-d-glucopyranoside (2), and 3--2,5-dihydro-1,1-dioxo-thiophen 7′-O-β-d-glucopyranosyl-6′-O-acetyl-(1→4)-O-β-d-glucopyranosyl-(1→4)-O-β-d-glucopyranoside (3), respectively, were isolated from the rhizomes of Trillium tschonoskii. Their structures were established on the basis of spectroscopic data, including HR-ESI-MS, IR, 1D and 2D NMR. The cytotoxic properties of the three compounds were investigated using human hepatic L02 cells.Three new sesquiterpene glycosides, possessing a rare aglycone with a sulfonyl between C-1 and C-15 positions, named 3-(3′ Trillium tschonoskii Maxim is perennial herb, mainly distributed in Hubei, Shanxi and Anhui provinces of China at an altitude of 1600–3200 m − . The 1H-NMR (600 MHz) spectrum of 1 , two olefinic protons ), as well as signals for three anomeric protons at , 5.12 , and 5.15 ). The 13C combined with HSQC NMR spectra of 1 indicated a structure with a total of 33 C-atom signals. Fifteen of them were attributed to the aglycone carbons including four olefinic carbons , one oxygenated methine carbon (δC 90.0), one oxygenated quaternary carbon (δC 71.9), two sulfonated methylene carbons , four sp3 methylene carbons , and three tertiary methyl carbons , while the remaining carbon signals were characteristic to three glucosyl moieties. By comparing the NMR data of 1 with 3--2,5-dihydro-1,1-dioxo-thiophen − . Comparing the NMR and MS data of 2 with 1, it was determined that 2 had the same aglycone as 1. The 13C-NMR resonances of the sugar unit were identified by HSQC and further confirmed by HMBC experiments. The sugar moieties of 2 were the same as those of 7,11-dimethyl-3-methylene-1,6-dodecadien-10,11-diol 10-O-β–d-glucopyranosyl-(1→4)-O-β-d-glucopyranoside − . The NMR data of 3 were very similar to 1, except for the presence of the CH3CO group. Furthermore, the CH3CO group located at the OH group of C-6 position in the inner Glc′, which turned into ester, was supported by the HMBC correlations between H-6-Glc′  and C-CH3CO (δ 170.8). The sugar moieties of 3 were further assigned by the HSQC, HMBC, and 1H−1H COSY experiments. Therefore, 3 was defined as 3--2,5-dihydro-1,1-dioxo-thiophen 7′-O-β-d-glucopyranosyl-6′-O-acetyl-(1→4)-O-β-d-glucopyranosyl-(1→4)-O-β-d-glucopyranoside.Compound 1–3 were evaluated for cytotoxicity against L02 cells. All three compounds showed no cytotoxic activity at 100 μM.Compound 1H-NMR and 150 MHz for 13C-NMR, PaloAlto, CA, USA) in pyridine-d5 , respectively. HPLC analyses were performed on Agilent 1100 system  equipped with a Silgreen C18 column  and an Alltech 2000 evaporative light scattering detector . Preparative HPLC was performed on an NP7000 module  equipped with a Shodex RID 102 detector , and a Silgreen C18 column . Semi-preparative HPLC was performed on a Waters 515 pump  equipped with a Shodex RID 101 detector , using a Silgreen C18 column . TLC was performed on silica gel GF254 plates . Macroporous resin SP825 , silica gel H , and MCI gel  were applied to the performance of column chromatography.IR spectra, HR-ESIMS and NMR were recorded on a VERTEX 70 FT Infrared Spectrometer, Synapt MS  and Varian UNITYINOVA 600 spectrometer . A voucher specimen (No. 151010) was deposited in the author′s laboratory in the Beijing Institute of Radiation Medicine.The rhizomes of T. tschonoskii (5 kg) were crushed and extracted with 50% aq. EtOH at reflux three times . The filtered solution was concentrated in vacuo. The supernatants were applied to a macroporous resin SP825 column, eluted with EtOH/H2O  to yield five factions (Fr. A−Fr. E). Fr. C (120 g) was subjected to silica-gel CC with a gradient mixture of CHCl3:MeOH:H2O  as the eluent, and five subfractions were obtained (Fr. C-1−Fr. C-5). Fr. C-3 (35 g) was further subjected to MCI gel CC with a gradient mixture of acetone/H2O  as the eluent. As a result, a total of 30 fractions were collected (Fr. C-3-1−Fr. C-3-30). Fr. C-3-6 was purified by preparative HPLC with ACN/H2O  to obtain seven fractions (Fr. C-3-6-1−Fr. C-3-6-7). Fr. C-3-6-4 underwent semi-preparative HPLC with acetone/H2O  to yield 1 (55 mg) and 2 (12 mg). Fr. C-3-12 was purified by preparative HPLC with ACN/H2O  to yield five fractions (Fr. C-3-12-1~Fr. C-3-12-5). Fr. C-1-12-5 was purified by semi-preparative HPLC with ACN/H2O  to yield 3 (15 mg).The rhizomes of d-glucopyranosyl-(1→4)-O-β-d-glucopyranosyl--2,5-dihydro-1,1-dioxo-thiophen 7′-O-β-d-glucopyranoside→4)-O-β- (1): White amorphous power; c 0.085, MeOH); IR (KBr) νmax 3408, 2970, 2927, 1642, 1382, 1306, 1236, 1159, 1072, 1026; 1H-NMR and 13C-NMR spectroscopic data, see m/z 789.3241 [M + H]+ , 811.3036 [M + Na]+, 627.2696 [M + H − Glc]+, 465.2148 [M + H − Glc − Glc]+, 303.1644 [M + H − Glc − Glc − Glc]+.d-glucopyranosyl-(1→4)-O-β-d-glucopyranoside3--2,5-dihydro-1,1-dioxo-thiophen 7′-O-β- (2): White amorphous power; c 0.082, MeOH); IR (KBr) νmax 3433, 2928, 1642, 1383, 1306, 1237, 1074, 1041; 1H-NMR and 13C-NMR spectroscopic data, see m/z 627.2679 [M + H]+ , 649.2488 [M + Na]+, 465.2147 [M + H − Glc]+, 303.1637 [M + H − Glc − Glc]+.d-glucopyranosyl-6′-O-acetyl--2,5-dihydro-1,1-dioxo-thiophen 7′-O-β-d-glucopyranosyl-(1→4)-O-β-d-glucopyranoside→4)-O-β- (3): White amorphous power; c 0.088, MeOH); IR (KBr) νmax 3428, 2970, 2926, 1736, 1644, 1383, 1307, 1239, 1161, 1072, 1029; 1H-NMR and 13C-NMR spectroscopic data, see m/z 831.3329 [M + H]+ , 853.3165 [M + Na]+, 669.2820 [M + H − Glc]+, 507.2261 [M + H − Glc − Glc]+, 303.1612 [M + H − Glc − Glc − (Ac-Glc)]+.1 (1.4 mg), 2 (1.6 mg), and 3 (1 mg) were hydrolyzed with 2 N aq. CF3COOH (5 mL) for 5 h at 95 °C, respectively. After extraction with CH2Cl2 (3 × 5 mL), the aq. layer was repeatedly evaporated to dryness with EtOH until neutral, and then analyzed by TLC over silica gel  by comparison with authentic samples. Furthermore, the residue of sugars was dissolved in anhydrous pyridine (2 mL), and l-cysteine methyl ester hydrochloride (3 mg) was added. The mixture was stirred at 60 °C for 1 h, then 3 mL of HMDS-TMCS  was added, and the mixture was stirred at 60 °C for 30 min. The precipitate was centrifuged off, and the supernatant was analyzed by GC-MS (Agilent Technologies 5977A MSD). The absolute configurations were determined by comparing the retention times with derivatives prepared in a similar way from standard d-glucose (Sigma-Aldrich). Identification of d-glucose was carried out for compounds 1–3, giving two peaks at 3.75 min and 4.15 min which were two silylated derivatives , IR, and HR-ESI-MS. The aglycone of the compounds found in this study was a rare aglycone which contains a sulfonyl between C-1 and C-15 positions. Compounds 1–3 were investigated for their cytotoxic activity against L02 cells, and no obvious cytotoxic activity was found.In conclusion, three new sesquiterpene glycosides were isolated from the rhizomes of


Dihydroxyoxocholestenoic acids are intermediates in bile acid biosynthesis. Here, using liquid chromatography – mass spectrometry, we confirm the identification of 7α,24-dihydroxy-3-oxocholest-4-en-26-oic and 7α,25-dihydroxy-3-oxocholest-4-en-26-oic acids in cerebrospinal fluid (CSF) based on comparisons to authentic standards and of 7α,12α-dihydroxy-3-oxocholest-4-en-26-oic and 7α,x-dihydroxy-3-oxocholest-4-en-26-oic (where hydroxylation is likely on C-22 or C-23) based on exact mass measurement and multistage fragmentation. Surprisingly, patients suffering from the inborn error of metabolism cerebrotendinous xanthomatosis, where the enzyme CYP27A1, which normally introduces the (25 R)26-carboxylic acid group to the sterol side-chain, is defective still synthesise 7α,24-dihydroxy-3-oxocholest-4-en-26-oic acid and also both 25 R- and 25 S-epimers of 7α,12α-dihydroxy-3-oxocholest-4-en-26-oic acid. We speculate that the enzymes CYP46A1 and CYP3A4 may have C-26 carboxylase activity to generate these acids. In patients suffering from hereditary spastic paraplegia type 5 the CSF concentrations of the 7α,24- and 7α,25-dihydroxy acids are reduced, suggesting an involvement of CYP7B1 in their biosynthesis in brain.  •Dihydroxy-3-oxocholest-5-en-26-oic are found in human CSF and plasma.•Hydroxy groups may be at 7α,24, 7α,25, or 7α,12α.•Another acid is hydroxylated at 7α and in the side-chain probably at C-22 or C-23.•In patients with CTX acids with 25 R or 25 S stereochemistry are found.•In patients with SPG5 the concentrations of acids in CSF are reduced. Once 7α-hydroxylated, the acid is a substrate for the enzyme hydroxysteroid dehydrogenase (HSD) 3B7, which is a 3β-hydroxysteroid dehydrogenase Δ5,Δ4 isomerase, giving the product 7α-hydroxy-3-oxocholest-4-en-(25 R)26-oic acid (CA4-7α-ol-3-one) 24(R/S)-hydroxycholesterol, 2 ng [2H7]22 R-hydroxycholest-4-en-3-one, 2 ng [2H7]7α-hydroxycholesterol, 1.38 ng [2H6]7α,25-dihydroxycholesterol, 4 ng [2H6]25-hydroxyvitamin D3 and 800 ng [2H7]cholesterol. Each sample was sonicated for 5 min, after which 0.65 mL of water was added dropwise while continuing to sonicate. Sonication was continued for a further 5 min. The solution was centrifugation at 2400 g, 4 °C, for 30 min. The supernatant, now in 3 mL of 70% ethanol, was loaded onto a 200 mg Certified Sep-Pak C18 column . The 3 mL eluate was combined with a column wash of 4 mL of 70% ethanol to give SPE1-Fr1 (7 mL) in which oxysterols including cholestenoic acids elute.Two hundred and fifty μL of CSF was added dropwise to 2.1 mL of ethanol containing 2 ng of [Streptomyces sp  was added to sub-fraction A and incubated for 1 h at 37 °C. The reaction was quenched with 2 mL of methanol. Sub-fraction B was treated in an identical fashion but in the absence of cholesterol oxidase. To each sub-fraction 150 μL of glacial acetic acid was added followed by 190 mg of [2H5]Girard P (GP) reagent as the bromide salt to sub-fraction A and 150 mg of [2H0]GP as the chloride salt to sub-fraction B. The reaction proceeded at room temperature overnight in the dark. Excess derivatisation reagent was then removed by re-cycling solid phase extraction (SPE) on a 60 mg Oasis HLB column . Each eluate was diluted to half its organic content until 17.5% organic was achieved at which point derivatised oxysterols including cholestenoic acids were bound to the column and unreacted GP reagent was in the flow-through. After a wash with 10% methanol, derivatised oxysterols were eluted in 2 mL of methanol. LC-HRMS(MS)n was performed as described in Abdel-Khalik et al. [18 Hypersil Gold column (Thermo Fisher Scientific) and Orbitrap Elite mass spectrometer (Thermo Fisher Scientific). Quantification was by the isotope dilution method exploiting LC-HRMS data with [2H7]24(R/S)-hydroxycholesterol and [2H7]22 R-hydroxycholest-4-en-3-one as internal standards for sub-fractions A (with cholesterol oxidase) and B (without cholesterol oxidase), respectively. Plasma samples were prepared and analysed as described in Abdel-Khalik et al. [The oxysterol fraction was then derivatised and analysed as described in Abdel-Khalik et al. . In brie33.13.1.12H5]GP-derivatised (25 R)CA4-7α,24 S-diol-3-one gave rise to two peaks in the LC-HRMS reconstructed ion chromatogram (RIC) for its [M]+ ion at m/z 585.4059, corresponding to the syn and anti conformers of the derivative  ring  spectra acquired at 2.01 min from the CSF sample -like chromatogram 585.4→501.3→427.3 the syn and anti conformers of CA4-7α,24-diol-3-one are resolved from the chemical background of the CSF sample, the conformers are evident as peak at 1.99 and 2.79 min in m/z 427.3, probably results from loss of CO2 + H2 (46 Da) from the [M-Py-CO]+ ion at m/z 473.3 via a classical charge-remote fragmentation mechanism [When fragmented GP-derivatised sterols give intense [M-Py]ring see  11]. Th. Th+ ionF sample A and 2.0ol-3-one B were esechanism   spectra acquired at 3.47 min for CSF  a combination of dehydration (−18 Da) and decarbonylation (−28 Da) with loss of CO from the GP reagent and loss of the labile 25-hydroxy or 7α-hydroxy group, and/or (ii) loss of the C26 acid group as H2O plus CO with the second hydrogen abstracted from the C-25-hydroxy group. This reaction could proceed via a 6-electron classical charge-remote fragmentation mechanism + (m/z 483.3), [M-Py-28]+ (m/z 473.3), [M-Py-46]+ (m/z 455.3) are common to GP-derivatised acids (m/z 419.3 ([M-Py-82]+), 440.3 (M-Py-61) and 447.3 (+ ion, respectively. A further fragment-ion was observed at 465.3 [M-Py-36]+ indicating the presence of at least two labile hydroxy groups, supporting the suggestion of a dihydroxy-3-oxocholest-4-en-26-oic acid structure. While one of the hydroxy groups is at C-7, most likely of 7α-stereochemistry, the location of the second hydroxy group is more difficult to assign possibly at C-22, C-23 or less likely at C-27  spectrum acquired at 4.16 min  and 179.1 (*b3-28) is characteristic of 7-hydroxy-3-oxo-4-ene sterols and is observed in each spectrum, however, fragment ions at m/z 209.1 (*b3+2) and 279.2 (*c3+2), observed in the spectrum of the unknown compound shown in m/z range of this spectrum, a distinctive fragment-ion of m/z 422.3 is evident. This is analogous to an abundant ion at m/z 392.3 characteristic of 7α,12α-dihydroxycholest-4-en-3-one (2O (−79 Da) from the [M-Py]+ ion . By generating a MRM chromatogram 585.4→501.3→422.3, CA4-7α,12α-diol-3-one is highlighted eluting at 4.16 and 4.64 min  deficiency  ..16]. Def3.2.1m/z 585.4059, corresponding to the [M]+ ion of dihydroxy-3-oxocholest-4-en-26-oic acids, recorded from CSF of CTX patients (n = 6) gave rise to a different profile of peaks to that observed normally in CSF  and of CA4-7α,12α-diol-3-one (3.74 and 4.28 min) were detected. Note, the chromatograms depicted in 4-7α,24-diol-3-one  than in control CSF , where both epimers are expected, substantiating the current identification.The MS4.28 min F give siol-3-one C–F. Normtructure C. The fo4-7α,12α-diol-3-one, corresponding to the earlier eluting components in the twin pairs of peaks . The two epimers are interconvertible via the enzyme alpha-methylacyl-CoA racemase (AMACR) . Itol-3-one B. The sein brain . Howeverin brain . C5-3β,24-7α,x-diol-3-one, but the MS3 fragmentation pattern suggests that the second hydroxy group is located on C-22 or C-23. CYP3A4 has 23 R-hydroxylase activity [In the absence of additional authentic standards we cannot be sure of the structure of CAactivity , and is activity .4-7α,24-diol-3-one and CA4-7α,12α-diol-3-one were detected, the latter acid as both 25 S and 25 R-epimers. Shown in 4-7α,12α-diol-3-one in the absence of CYP27A1. CYP3A4 has been shown to have (25 S)26-hydroxylase activity [5-3β,7α-diol (unpublished data). Mast et al. noted that recombinant CYP46A1 could further hydroxylate C5-3β,24 S-diol to 24 S,26-dihydroxycholesterol  [4-7α,24 S-diol-3-one CA4-7α,12α-diol-3-one and CA4-7α,x-diol-3-one are formed even in the absence of CYP7B1 activity, with 7α-hydroxylation presumably being catalysed by CYP39A1 or CYP7A1 [4-7α,12α-diol-3-one, indicating that CYP7B1 has a role in their biosynthesis. In contrast to control samples where the concentrations of CA4-7α,24-diol-3-one, CA4-7α,25-diol-3-one and CA4-7α,x-diol-3-one are higher in CSF than plasma, the concentration of CA4-7α,24-diol-3-one is higher in SPG5 plasma than CSF. This suggests that CYP7B1 is important for the biosynthesis of CA4-7α,24-diol-3-one in brain.Analysis of CSF  and plasr CYP7A1 ,29. In CThe biological relevance of the dihydroxy-3-oxocholest-4-en-26-oic acids identified in this work is unknown. While cholestenoic acids with a 3β-hydroxy-5-ene structure are ligands to LXRs, sterols with a 3-oxo-4-ene group do not appear to activate these receptors.Although we have only limited clinical data for the CTX and SPG5 patients studied in this work the levels of the dihydroxy-3-oxocholest-4-en-26-oic acids determined in CSF do provide confirmatory diagnostic biomarkers of the two diseases. Whether or not the levels of these acids provide an indication of the severity of disease requires further study.


Magnolia flos with antiplasmodial, Ca2+-antagonistic, platelet activating factor-antagonistic, and chemopreventive activities. We investigated its inhibitory effects on the activities of eight major human cytochrome P450 (CYP) and uridine 5′-diphospho-glucuronosyltransferase (UGT) enzymes of human liver microsomes to determine if mechanistic aschantin–enzyme interactions were evident. Aschantin potently inhibited CYP2C8-mediated amodiaquine N-de-ethylation, CYP2C9-mediated diclofenac 4′-hydroxylation, CYP2C19-mediated [S]-mephenytoin 4′-hydroxylation, and CYP3A4-mediated midazolam 1′-hydroxylation, with Ki values of 10.2, 3.7, 5.8, and 12.6 µM, respectively. Aschantin at 100 µM negligibly inhibited CYP1A2-mediated phenacetin O-de-ethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-mediated bupropion hydroxylation, and CYP2D6-mediated bufuralol 1′-hydroxylation. At 200 µM, it weakly inhibited UGT1A1-catalyzed SN-38 glucuronidation, UGT1A6-catalyzed N-acetylserotonin glucuronidation, and UGT1A9-catalyzed mycophenolic acid glucuronidation, with IC50 values of 131.7, 144.1, and 71.0 µM, respectively, but did not show inhibition against UGT1A3, UGT1A4, or UGT2B7 up to 200 µM. These in vitro results indicate that aschantin should be examined in terms of potential interactions with pharmacokinetic drugs in vivo. It exhibited potent mechanism-based inhibition of CYP2C8, CYP2C9, CYP2C19, and CYP3A4.Aschantin is a bioactive neolignan found in  Magnoliaflos and Hernandia nymphaeifolia -mephenytoin 4′-hydroxylation, and CYP3A4-mediated midazolam 1′-hydroxylation, with IC50 values of 27.8, 40.5, 22.7, and 57.5 µM, respectively , indicating that it should be used carefully by patients taking CYP2C9 substrates such as celecoxib, diclofenac, glyburide, losartan, tolbutamide, torasemide, and S-warfarin, to avoid drug interactions -mephenytoin  were obtained from Corning Life Sciences . SN-38 was the product of Santa Cruz Biotechnology . 13C2,15N-acetaminophen , chenodeoxycholic acid-24-acyl-β-glucuronide , mycophenolic acid-β-d-glucuronide , and SN-38 glucuronide  were obtained from Toronto Research Chemicals . Acetonitrile and methanol (HPLC grade) were obtained from Burdick & Jackson Inc. ; all other chemicals were of the highest quality available.Acetaminophen , amodiaquine hydrochloride , coumarin , diclofenac sodium , 7-hydroxycoumarin , midazolam , 50 values) of aschantin toward CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 were evaluated using pooled human liver microsomes employing a cocktail of CYP substrates and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The incubation mixtures were prepared in total volumes of 100 µL, as follows: pooled human liver microsomes (0.2 mg/mL), 1.0 mM NADPH, 10 mM MgCl2, 50 mM potassium phosphate buffer (pH 7.4), various concentrations of aschantin in DMSO , and a cocktail of seven CYP probe substrates as defined in our previous report -mephenytoin, 5 µM bufuralol, and 2.5 µM midazolam. After 3 min pre-incubation at 37 °C, the reactions were initiated by addition of NADPH and incubation proceeded for 15 min at 37 °C in a shaking water bath. The reaction was stopped by placing the tubes on ice and adding 100 µL amounts of ice-cold methanol containing internal standards . Then the incubation mixtures were centrifuged at 13,000× g for 4 min at 4 °C. All assays were performed in triplicate and the average values were used in calculations. To measure the mechanism-based inhibition of CYP activities, various concentrations of aschantin (0.1–100 µM) were pre-incubated for 30 min with human liver microsomes in the presence of NADPH. Each reaction was initiated by adding the seven-CYP probe substrate cocktail. The degrees of inhibition . The metabolites formed from the seven substrates were simultaneously quantified using our previously described LC-MS/MS method . To this2, 50 mM potassium phosphate buffer (pH 7.4), various concentrations of aschantin (0.1–100 µM), pooled human liver microsomes (0.2 mg/mL), and a CYP2B6-selective substrate (50 µM bupropion), as reported previously -mephenytoin 4′-hydroxylation, and CYP3A4-mediated midazolam 1′-hydroxylation, with Ki values of 10.2, 3.7, 5.8, and 12.6 µM, respectively. It weakly inhibited UGT1A1-catalyzed SN-38 glucuronidation, UGT1A6-catalyzed N-acetylserotonin glucuronidation, and UGT1A9-catalyzed mycophenolic acid glucuronidation, with IC50 values of 131.7, 144.1, and 71.0 µM, respectively. The compound negligibly inhibited CYP1A2, CYP2A6, CYP2B6, CYP2D6, UGT1A3, UGT1A4, and UGT2B7. Aschantin should be examined in terms of potential in vivo pharmacokinetic drug interactions attributable to inhibition of CYP2C8, CYP2C9, CYP2C19, and CYP3A4.Aschantin exhibited potent mechanism-based inhibition of CYP2C8-mediated amodiaquine