Excoecaria formosana showed glycine N-methyltransferase (GNMT)-promoter-enhancing and nuclear factor erythroid 2-related factor 2 (NRF2)-suppressing activities. Further, the investigation of the whole plant of E. formosana led to the isolation of a new steroid, 7α-hydroperoxysitosterol-3-O-β-d-glucopyranoside (1); two new coumarinolignans, excoecoumarin A (2) and excoecoumarin B (3); a new diterpene, excoeterpenol A (4); and 40 known compounds (5–44). Among them, Compounds 38 and 40–44 at a 100 μM concentration showed a 2.97 ± 0.27-, 3.17 ± 1.03-, 2.73 ± 0.23-, 2.63 ± 0.14-, 6.57 ± 0.13-, and 2.62 ± 0.05-fold increase in GNMT promoter activity, respectively. In addition, Compounds 40 and 43 could reduce NRF2 activity, a transcription factor associated with drug resistance, in Huh7 cells with relative activity of 33.1 ± 0.2% and 45.2 ± 2.5%. These results provided the basis for the utilization of Taiwan agarwood for the development of anti-HCC agents.Hepatocellular carcinoma (HCC) is considered to be a silent killer, and was the fourth leading global cause of cancer deaths in 2018. For now, sorafenib is the only approved drug for advanced HCC treatment. The introduction of additional chemopreventive agents and/or adjuvant therapies may be helpful for the treatment of HCC. After screening 3000 methanolic extracts from the Formosan plant extract bank,  N-methyltransferase (GNMT) has been shown to decrease DNA adduct formation and cytotoxicity induced by carcinogens  of Compound 1 replaced the methylene group of -sitosterol-3-O-β-d-glucopyranoside. It was also supported by HMBC correlations of 1 from H-7 (δH 4.16) to C-5 (δC 148.2), C-6 (δC 120.4) and C-9 (δC 43.5). The relative configuration of OOH–C(7) was α–oriented, confirmed by the chemical shift of C-6 (δC 120.4) + peak at m/z  . Maximal UV absorptions at 2: 225 and 330 nm, and 3: 230 and 330 nm, and IR absorption at 2: 1702 cm−1, and 3: 1699 cm−1 suggested the existence of coumarin residue. Taking the 1H NMR data of Compound 2 (δH 6.26) and H-4 (δH 7.79) with a coupling constant of 9.6 Hz. The chemical shift of H-4, being smaller than δH 7.8, suggested that there was no substituent at C-5 but a proton appearing at δH 6.62  . The 1H NMR spectrum further showed two aromatic methoxyl group at  along with two propanoid moiety attached to aromatic ring and dioxane at  for H-7′, H-8′, and H-9, respectively. The presence of a 1,4-denzodioxane residue in these compounds was further supported by their 13C NMR spectra, showing typical signals at , respectively. In addition, the two oxymethines (H-7′/H-8′ and H-7′’/H-8″) were involved in ether linkages of benzene moiety, as determined by their chemical shifts, and were further confirmed with the HMBC spectrum /2-methoxy-3,4-dihydroxyphenyl-C3 (II) residue was similar to that of signals of the literature compound, (E)-5-(3-(hydroxymethyl)-7-(3-hydroxyprop-1-enyl)-5-methoxy-2,3-dihydrobenzo[b]dioxin2-yl)-3-methoxybenzene-1,2-diol de B (5) , seven b (5) [6) , methyl  [6) [7) , 4-metho [7) [8) , 3-hydro [8) [9) , 3-hydro[9) [10) , 2,3-dih10) [11) , and (2Ster (12) , a mixtu [13–16) , three c16) [17) , fraxeti17) [18) , and 6-h18) [19) , six cou [20–23) ,48,49, m23) [24) ,33, and 24) [25) ,33, threide 26) , excoeca26) [27) , and aga27) [28) , two fla28) [29)  and kaemide (30) , six steate (31) ,  , a mixturol (36) , and eigide (37) , 4-hydroide (38) , 3-methoide (39) , 1,2,3,4ose (40) , corilag40) [41) , 1,4,6-tose (42) , 1,3,6-tose (43) , and galose (44) . The phy1‒7, 9, 10, 12, 17‒20, 22, 24‒29, and 31‒44) were evaluated for their GNMT-promoter-enhancing activity  and ellagitannin (Compound 41) showed GNMT-promoter-enhancing activity in 100 μM concentrations, with a 2.97 ± 0.27-, 3.17 ± 1.03-, 2.73 ± 0.23-, 2.63 ± 0.14-, 6.57 ± 0.13-, and 2.62 ± 0.05-fold increase, respectively. Furthermore, only Compounds 40 and 43 exhibited NRF2-inhibitory effects in the Huh7 cells with a related activity of 33.1 ± 0.2% and 45.2 ± 2.5%.Thirty-five isolates  is a glycine N-methyltransferase (GNMT) inducer. They showed that PGG could not only induce apoptosis in Huh7 cells, but also sensitize Huh7 cells to sorafenib treatment [O-β-glucopyranoside (37) and 3-methoxy-4-hydroxyphenyl 1-O-β-d-glucopyranoside (39) only had one galloyl group connected with the glucose core, they did not show GNMT-promoter-enhancing activity. Gallic acid 4-O-β-d--glucose (44) is an unusual galloyl glucose compound with one ester linkage group and one ether linkage galloyl group connected with the glucose core. Surprisingly, Compound 44 also depicted GNMT-promoter-enhancing activity. Further evidence is required to support whether the numbers of the ether linkage galloyl group or the substituted position of the galloyl group on the glucose core affect GNMT-promoter-enhancing activity. Compounds with three galloyl groups connected with the glucose core, including gallotannins  and ellagitannin (Compound 41) showed GNMT-promoter-enhancing activity. Among them, 1,3,6-tri-O-linkage (43) was the most active compound, two-fold higher in induction activity than 1,4,6-tri-O-linkage (42) and positive control PGG. This finding suggested that the substituted position of galloyl groups is crucial for GNMT-promoter-enhancing activity. Gallotannin 43 showed better GNMT-promoter-enhancing activity than ellagitannin 41, suggesting different types of galloyl glucose change their GNMT-promoter-enhancing activity. Previous biological studies of tannin analogs were found to have antiviral, antibacterial, antioxidant, and antitumor activity [Previously, Kant and his colleagues reported that 1,2,3,4,6-penta-reatment . Since Pactivity ,66. Surp40 and 43, the two most potent GNMT inducers, also suppressed NRF2 activity in Huh7 cells. These results indicated that the sensitization ability of PGG could be attributed to its inhibitory effect on NRF2 activity. However, further investigations are needed to delineate the underlying mechanisms.Transcriptional factor NRF2, a master regulator for antioxidative and detoxification responses, is implicated in chemoresistance in several cancers . As ment2 and 3), with an unprecedented carbon skeleton, together with two new compounds, were obtained from the whole plant of E. formosana, and their structures were established by spectroscopic analysis. Coumarinolignans have many interesting biological activities, and their cytotoxic activity and hepatoprotective potential are globally known. Although the coumarinolignans in this study did not show biological effects on GNMT-promoter and NRF2 activity, this indicated that the coumarinolignans might exert its activity via mechanisms not involving these two proteins. To our knowledge, this is the first report on the GNMT-promoter-enhancing and NRF2 suppressing activities of this plant in Huh 7 cells. Further evidence is needed to support whether E. formosana is helpful to HCC patients.Two new coumarinolignans, excoecoumarins A and B  and 2D  NMR spectra on a Varian Germini-2000 spectrometer  operated at 200 (1H) and 50 MHz (13C), Varian Unityplus-400 spectrometer  operated at 400 (1H) and 100 MHz (13C), Varian Mercuryplus-400 spectrometer  operated at 400 (1H) and 100 MHz (13C), and Varian VNMRS-600 spectrometer  operated at 600 (1H) and 150 MHz (13C). Low-resolution mass spectra were obtained with POLARIS Q Thermo Finnigan , Water ZQ 4000 , and VG Quattro GC/MS/MS/DS  mass spectrometers. HRESIMS were recorded on a Bruker APEX II mass spectrometer . Silica gel  was used for column chromatography (CC), and silica gel 60 F254  and RP-18 F254S  were used for TLC and preparative TLC, respectively, visualized with a Ce2(SO4)3 aqueous solution. Further purification was performed by medium-performance liquid chromatography .All melting points were determined on a Yanaco micromelting apparatus  and were uncorrected. Optical rotations were measured on a Jasco P-2000 polarimeter , UV spectra were obtained with a Jasco-V-530 UV/vis spectrophotometer , and IR spectra (ATR) were acquired with a Jasco FT/IR-4600 spectrometer. We recorded 1D  was deposited with the herbarium of the College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan.The whole plant of E. formosana was extracted at room temperature with MeOH (30 L) three times to yield an MeOH extract (465 g) that was partitioned between EtOAc and H2O (1:1) to provide an EtOAc-soluble (110 g) and an H2O-soluble fraction. The H2O-soluble fraction was then partitioned between n-butanol and H2O (1:1) to obtain an n-butanol-soluble (100 g) and H2O-soluble layer (255 g). The EtOAc-soluble fraction (100 g) was subjected to column chromatography  to yield 12 subfractions (Fr. 1–12). Fr. 5 (18 g) was recrystallized from MeOH to give Compounds 33 and 34 . Fr. 9 (2.7 g) was subjected to MPLC  to give 12 fractions (Fr. 9-1–9-12). Fr. 9-5 was subjected to MPLC  to afford ten fractions (Fr. 9-5-1–9-5-10). Fr. 9-5-7 was further separated with prep. TLC (CH2Cl2/EtOAc = 40:1) to obtain Compound 26 . Fr. 9-11 was subjected to MPLC  to give ten fractions (Fr. 9-11-1–9-11-10). Fr. 9-11-8 was subjected to MPLC  to generate eight fractions (Fr. 9-11-8-1–9-11-8-8). Fr. 9-11-8-2 was subjected to MPLC  to furnish Compound 31 . Fr. 9-11-8-3 was subjected to MPLC  to yield four fractions (Fr. 9-11-8-3-1–9-11-8-3-4). Fr. 9-11-8-3-2 was subjected to MPLC  to provide eight fractions (Fr. 9-11-8-3-2-1–9-11-8-3-2-8). Fr. 9-11-8-3-2-3 was subjected to MPLC  to afford Compounds 1  and 32 . Fr. 9-11-3 was subjected to MPLC  to give nine fractions (Fr.9-11-3-1–9-11-3-9). Fr. 9-11-3-7 was subjected to MPLC  to obtain Compound 7 . Fr. 10 was subjected to subjected to silica gel column chromatography (column size: 5.5 cm × 70 cm) and eluted with CH2Cl2, EtOAc, and methanol, to which EtOAc and MeOH were gradually added to increase the polarity resulting in ten fractions (Fr. 10-1–10-10). Fr. 10-7 was subjected to MPLC  to give 11 fractions (Fr. 10-7-1–10-7-11). Fr.10-7-1 was separated with Sephadex LH-20 (column size: 1.5 cm × 45 cm) and eluted with MeOH to furnish Compounds 10  and 29 . Fr. 10-7-10 was subjected to MPLC  to yield 26 fractions (Fr. 10-7-10-1–10-7-10-26). Fr.10-7-10-5 was subjected to MPLC  to gain Compound 28 . Fr. 10-8 was subjected to MPLC  to gain 10 fractions (Fr. 10-8-1–10-8-10). Fr. 10-8-4 was subjected to MPLC  to obtain Compound 11 . Fr. 10-8-4-9 was subjected to MPLC  to obtain Compound 4 . Fr.10-4 was subjected to MPLC  to give ten fractions (Fr. 10-4-1–10-4-10). Fr.10-4-10 was subjected to MPLC  and separated with prep. TLC (CH2Cl2/acetone 30:1) to obtain Compound 19 . Separation of fraction 11 on a silica gel column  furnished 22 subfractions (Fr. 11-1–11-22). Fr. 11-12 was recrystallized from MeOH to give a mixture of Compounds 13–16 . Fr. 11-8 was subjected to MPLC  to generate 11 fractions (Fr. 11-8-1–11-8-11). Fr. 11-8-1 was separated with prep. TLC (CH2Cl2/acetone 8:1) to gain Compound 9 . Fr. 11-8-2 was subjected to MPLC  to afford three fractions (Fr. 11-8-2-1–11-8-2-3). Fr. 11-8-2-1 was separated with prep. TLC (CH2Cl2/acetone 8:1) to obtain Compound 17 . Fr. 11-8-3 was subjected to MPLC  to produce Compound 8 . Fr. 11-8-2-2 was recrystallized and washed by acetone, and the washing liquor (45.2 mg) was subjected to MPLC  to generate nine fractions (Fr. 11-8-2-2-1–11-8-2-2-9). Fr. 11-8-2-2-M-5 was separated with prep. TLC (CH2Cl2/acetone 8/1) to obtain Compound 23 . Fr. 11-8-2-2-C was separated with prep. TLC (CH2Cl2/acetone 8:1) to gain Compound 22 . Fr. 11-8-2-2-C-A was subjected to MPLC  to give Compounds 20  and 21 . Fr. 11-15 was subjected to MPLC  to generate 16 fractions (Fr. 11-15-1–11-15-16). Fr. 11-15-8 was subjected to MPLC  to give nine fractions (Fr. 11-15-8-1–11-15-8-9). Fr. 11-15-8-3 was subjected to MPLC  to obtain Compound 25 . Fr. 11-15-8-3-2 was purified by MPLC  to furnish Compound 24 . Fr. 11-15-10 was subjected to MPLC  to give six fractions (Fr. 11-15-10-1–11-15-10-6). Fr. 11-15-10-3 was subjected to MPLC  to afford 5 fractions. Fr.11-15-10-3-4 was subjected to HPLC  to obtain Compounds 2  and 3 . Fr. 11-15-5 was subjected to MPLC  to give eight fractions (Fr. 11-15-5-1–11-15-5-8). Fr. 11-15-5-3 was subjected to MPLC  to gain Compound 27 . Fr. 11-15-16 was recrystallized from CH2Cl2-MeOH to obtain Compounds 35 and 36 . Fr. 11-13 was subjected to MPLC  to afford five fractions (Fr. 11-13-1–11-13-5). Fr. 11-13-1 was subjected to Sephadex LH-20 (column size: 2 cm × 45 cm) and eluted with MeOH to give 11 fractions (Fr. 11-13-1-1– 11-13-1-11). Fr. 11-13-1-2 was separated by MPLC  to obtain Compound 5 . Fr. 11-13-1-4 was separated with prep. TLC  to furnish four fractions (Fr. 11-13-1-4-1–11-13-1-4-4). Fr. 11-13-1-4-A was subjected to HPLC  to give Compound 12 . Fr. 11-13-1-6 was subjected to MPLC  to produce two fractions (Fr. 11-13-1-6-1–11-13-1-6-2). Fr. 11-13-1-6-2 was separated with prep. TLC  to obtain Compound 18 . Fr. 11-20 was separated with Sephadex LH-20 (column size: 3 cm × 45 cm) and eluted with MeOH to provide 16 fractions. Fr.11-20-7 was subjected to MPLC  to afford Compound 37 . Fr. 11-20-13 was subjected to MPLC  to obtain Compound 38 . Fr. 11-20-13-13 was subjected to MPLC  to give 2 fractions (Fr. 11-20-13-13-1–11-20-13-13-2). Fr. 11-20-13-13-2 was separated with HPLC  to furnish Compound 30 . Fr. 11-20-10 was subjected to MPLC  to generate Compound 39 . Fr. 11-21 and 22 were separated with Sephadex LH-20 (column size: 3 cm × 45 cm) and eluted with MeOH to afford 11 fractions (Fr. 11-21-1–11-21-11). Fr. 11-21-11 was subjected to MPLC  to gain Compound 40 . Fr. 11-21-8 was subjected to MPLC  to generate Compound 42 . Fr. 11-21-8-5 was subjected to Sephadex LH-20 (column size: 2 cm × 45 cm) and eluted with H2O/EtOH 1:1 (column size: 2 cm × 45 cm) to give Compound 43 . Fr. 11-21-3 was subjected to Sephadex LH-20 (column size: 2 cm × 45 cm) and eluted with H2O /EtOH 1:1 (column size: 2 cm × 45 cm) to afford 7 fractions (Fr. 11-21-3-1–11-21-3-7). Fr. 11-21-3-6 was subjected to MPLC  to obtain Compound 6 . Fr. 11-21-4 was subjected to MPLC  to produce Compound 44 . Fr. 12 was subjected to a Diaion HP-20 (column size: 2 cm × 45 cm) and eluted with MeOH-H2O , , and methanol 400 mL to furnish three fractions (Fr. 12-1–12-3). Fr. 12-1 was subjected to Sephadex LH-20 (column size: 2 cm × 45 cm) and eluted with MeOH to generate Compound 41 .The dried whole plant  of 23D + 36 , IR υmax (ATR) 3387 (OH), 1730 (C=O) cm−1; 1H NMR and 13C NMR ; UV (MeOH) λmax (log ε) 220 (4.68), 320 (3.96) nm; UV (MeOH + KOH) λmax (log ε) 225 (4.68), 330 (3.91) nm; IR υmax (ATR) cm−1: 3404 (OH), 1702 (C=O), 1606, 1575, 1514 (aromatic ring); 1H NMR and 13C NMR ; UV (MeOH) λmax (log ε) 220 (4.76), 320 (4.07) nm; UV (MeOH + KOH) λmax (log ε) 230 (4.64), 330 (4.02) nm; IR υmax (ATR) cm−1: 3464 (OH), 1699 (C=O), 1608, 1575, 1514 (aromatic ring); 1H NMR and 13C NMR ; IR υmax (ATR): 3383 (OH), 1710 (C=O), 1578 (C=C) cm−1; 1H NMR and 13C NMR  in 96-well plate, then treated with indicated concentrations of compounds for 18 h. At the assay time point, resazurin  was added to a final concentration of 0.1 mg/mL and further incubated for 4 h at 37 °C. Fluorescence of the reduced resazurin (ex/em: 530/590 nm) was measured from the culture supernatant by using a Synergy HT Multi-Mode Reader  to determine cell viability. The cells were then harvested for luciferase activity measurements according to the manufacturer’s protocol . Relative luciferase activity was calculated by normalizing luciferase activity to cell viability. For GNMT promoter activity assay treatment, data were presented as the fold to DMSO solvent control; for NRF2-activity assay, DMSO solvent control was used as 100% activity.


The compounds were tested in vitro against a set of two amitochondriate protozoa: Giardia duodenalis and Trichomonas vaginalis. Compounds 1–10 showed strong antiprotozoal activities, with potency values in the low micromolar-to-nanomolar range, being more active than their parent drugs. Metronidazole carbamate (1) was the most active of the series, with nanomolar activities against G. duodenalis (IC50 = 460 nM) and T. vaginalis (IC50 = 60 nM). The potency of compound 1 was 10 times greater than that of metronidazole against both parasites. None of compounds showed in vitro cytotoxicity against VERO cells tested at 100 µM. Molecular dynamics of compounds 1–10, secnidazole, and metronidazole onto the ligand binding site of pyruvate–ferredoxin oxidoreductase of T. vaginalis and the modeled β-tubulin of G. duodenalis revealed putative molecular interactions with key residues in the binding site of both proteins implicated in the mode of action of the parent drugs.We prepared a series of 10 carbamates derivatives based on two common antiprotozoal drugs: metronidazole ( Giardia duodenalis is an intestinal parasite that infects several mammalian hosts including humans, and it is considered a leading cause of waterborne diarrheal disease and malabsorption syndrome [Trichomonas vaginalis is the etiologic agent of the most common non-viral sexually transmitted disease in humans [syndrome . Trichomn humans . The chen humans , such asG. duodenalis, urogenital tract protozoa T. vaginalis, the cytotoxicity over mammalian VERO cells, and the molecular docking and dynamics prediction of this mode of binding over PFOR and β-tubulin are reported in this work.Metronidazole and secnidazole are prodrugs that are reductively activated (the nitro group is reduced) under low oxygen tension to produce free radical intermediates that form adducts with numerous biomolecules such as nucleic acids, proteins, and membrane lipids . However1–10 were synthesized starting from metronidazole (11) and secnidazole (12), which were reacted with appropriated isocyanates 13–17  and compounds 2 and 10 , were the most potent of the series (IC50 ranging from 0.46 to 0.96 µM) against G. duodenalis. They were almost 5–10 times more potent than metronidazole and secnidazole, which were equipotent among them. All carbamates were much more lipophilic than metronidazole and secnidazole, with a calculated MlogP around 1.68–3.87. This physicochemical property is important for high permeability of compounds across the protozoal membrane [T. vaginalis, compounds 1 and 2 also exhibited nanomolar trichomonicidal effects . They were 10- to 15-fold more potent than metronidazole, which is the drug of choice for trichomoniasis. Secnidazole was the least potent compound against T. vaginalis, but all its carbamate derivatives 6–10 were more potent than this parent drug. This result is remarkable since if Trichomonas is more resistant to secnidazole treatment, its carbamate derivatives could be a possible therapeutic option for this protozoosis. In summary, the preliminary structure–activity relationship (SAR) analysis revealed that cyclic unsubstituted metronidazole carbamates 1 and 2 are the most potent compounds against both parasites. Of note, the SAR derived from the results in The biological activity of carbamates nidazole , which amembrane . In our 1–10, metronidazole, and secnidazole were evaluated for their intrinsic toxicity against mammalian VERO cell lines (50 > 100 µM). The selectivity index (SI) is the ratio of cytotoxicity to biological activities. If SI is greater than 10, it is typically an indicator that the underlying antiprotozoal activity is not due to the intrinsic cytotoxicity of a given compound [1–10 showed nanomolar to micromolar giardicidal activities and no observable cytotoxic effects at a 100 µM concentration, showing selectivity indexes prominently higher than 20. This implies that carbamates 1–10 are more selectively toxic against G. duodenalis than against mammalian cells. The same parasiticidal discrimination was observed between T. vaginalis and VERO cells.Compounds ll lines , showingcompound . Compoun1 and 2) were selected for further computational studies to explore their putative mechanism of action at the molecular level. Molecular modeling studies were performed with two antiprotozoal relevant molecular targets: PFOR and β-tubulin. These two targets were chosen based on the known relationship with the parent compounds. A preliminary molecular docking simulation was performed to assess the putative binding mode of compounds 1 and 2 with the proposed targets. Then, molecular dynamics simulations were conducted to determine the relative stability of in silico binding modes. We emphasize that the molecular modeling studies reported in this work are intended to hypothesize the ligand–target interactions with two putative molecular targets. However, we did not intend to provide a detailed explanation for experimental SAR based on the predicted binding models. This is because it is not feasible to establish a reliable correlation between the measured antiprotozoal activity in vitro using the entire parasite, with the binding models derived with isolated 3D coordinates of the putative and isolated molecular targets. In vitro experiments have a significantly larger number of variables that cannot be addressed with in silico studies.Based on the in vitro antiparasitic assays, the most active compounds . 1H NMR spectra were determined on Varian 600 MHz AR Premium Compact  and 13C NMR (150 MHz) instrument . Chemical shifts are reported in ppm in DMSO-d6 and CDCl3 as deuterated solvents. Mass spectrometry was obtained from a JEOL JMS-700 spectrometer by electronic impact .Solvents and reagents were acquired from Sigma-Aldrich . Melting points were obtained using a capillary apparatus from Stanford Research Systems . All reactions were monitored by thin layer chromatography (TLC) on 0.2 mm precoated silica gel 60 F13–17 , 10% triethylamine, and NH4Cl as catalysts at 25 °C. The mixture was stirred at reflux (110 °C) under a nitrogen atmosphere for 7–33 h. The solvent was removed using a high vacuum system, and the residue was suspended in cold water. The solids were recovered by filtration, dried in the hood and recrystallized from suitable solvent.To a solution of metronidazole or secnidazole (0.0023 mol) in toluene (5 mL), we dropwise added the suitable isocyanate H-imidazol-1-yl)ethyl cyclohexylcarbamate2-(2-methyl-5-nitro-1(1): 21 h, yield 67%, recrystallized from ethanol-water, white crystals, Mp 103.7 °C (dec.). 1H NMR  δ: 1.10–1.31 , 1.57–1.68 , 1.68–1.87 , 2.45 , 3.39–3.41 , 4.35 , 4.54 , 7.93 , 9.32  ppm. 13C NMR  δ: 14.19 (CH3), 24.7 , 25.4 (C-4´), 33.2 , 45.6 (N–CH2), 50.04 (C-1´), 62.4 (O–CH2), 133.1 (C-4), 138.5 (C-5), 150.8 (C-2), 154.6 (C=O) ppm. MS/EI: m/z (% int. rel). 296.32 , 225.20 .H-imidazol-1-yl)ethyl phenylcarbamate2-(2-methyl-5-nitro-1(2): 15 h, yield 86%, recrystallized from ethanol-water, white powder Mp 197.7 °C (dec.). 1H NMR  δ: 2.48 , 4.47 , 4.63 , 7.01 , 7.27 , 7.40 , 8.06 , 9.66  ppm. 13C NMR  δ: 19.1 (CH3), 50.6 (N–CH2), 67.4 (O–CH2), 123.7 (C-4´), 127.8 , 133.9 , 138.3 (C-4), 143.7 (C-5), 143.9 (C-1´), 156.8 (C-2), 158.12 (C=O) ppm. MS/EI: m/z (% int. rel). 290.27 , 170.05 .H-imidazol-1-yl)ethyl (4-chlorophenyl)carbamate2-(2-methyl-5-nitro-1(3): 7 h, yield 65%, recrystallized from ethanol, yellow crystals, Mp 175.0–177.3 °C; 1H NMR  δ: 2.45 , 4.45 , 4.58 , 7.29 , 7.39 , 8.02 , 9.77  ppm. 13C NMR  δ: 14.4 (CH3), 45.7 (N–CH2), 62.82 (O–CH2), 120.4 , 129.1 , 133.5 (C-1´), 138.2 (C-4), 138.9 (C-5), 152.1 (C-2), 153.3 (C=O) ppm. MS/EI: m/z (% int. rel). 324.71 , 325.16 .H-imidazol-1-yl)ethyl (4-fluorophenyl)carbamate2-(2-methyl-5-nitro-1 (4): 9 h, yield 77%, recrystallized from ethanol-water, yellow crystals, Mp 168.2–170.6 °C; 1H NMR  δ: 3.34 , 4.46 , 4.60 , 7.13 , 7.10 , 8.02 , 9.63  ppm. 13C NMR  δ: 14.4 (CH3), 45.8 (N–CH2), 62.7 (O–CH2), 115.7 , 120.6 (C-1), 133.5 , 135.4 (C-5), 138.9 (C-4), 152.07 (C-2), 153.5 (C=O), 158.2 . MS/EI: m/z (% int. rel). 308.26 , 137.13 .H-imidazol-1-yl)ethyl (4-nitrophenyl)carbamate2-(2-methyl-5-nitro-1(5): 10 h, yield 87%, recrystallized from ethanol, yellow crystals, Mp 253.9–256.0 °C. 1H NMR  δ: 2.48 , 4.50 , 4.85 , 7.62 , 8.14 , 8.28 , 9.69  ppm. 13C NMR  δ: 14.2 (CH3), 44.5 (N–CH2), 61.9 (O–CH2), 117.6 , 124.6 , 129.4 (C-4), 138.5 (C-5), 139.4 (C-1´), 141.2 (C-4´), 150.1 (C-2), 151.1 (C=O) ppm. MS/EI: m/z (% int. rel). 335.27 , 125.96 .H-imidazol-1-yl)ethyl cyclohexylcarbamate1-methyl-2-(2-methyl-5-nitro-1 (6): 21 h, yield 51%, recrystallized from ethanol-water, white crystals, Mp 219.2–222.7 °C. 1H NMR  δ: 1.10–1.26 , 1.22 , 1.46–1.49 , 1.57–1.71 , 2.42 , 4.18-4.22 , 4.48 , 5.04–5.07 , 7.93 , 9.32  ppm. 13C NMR  δ: 14.4 (CH3), 17.9 (CH3), 25.4 (C-4´), 25.8 , 33.1 , 48.6 (N-CH2), 50.1 (C-1´), 64.7 (O-CH), 128.8 (C-4), 134.6 (C-5), 153.4 (C-2), 155.7 (C=O) ppm. MS/EI: m/z (% int. rel). 310.34 , 264.13 , 139.11 .H-imidazol-1-yl)ethyl phenylcarbamate (7):1-methyl-2- δ: 1.31 , 2.47 , 4.57 , 5.18-5.20 , 6.92-695 , 7.23–7.26 , 7.42 , 7.99 , 9.52  ppm. 13C NMR  δ: 14.5 (CH3), 17.9 (CH3), 46.9 (N–CH2), 59.8 (O–CH), 118.3 , 122.3 (C-4´), 129.2 (C-3´-C-5´), 133.5 (C-4), 138.6 (C-1´), 140.1 (C-5), 152.9 (C-2), 161.1 (C=O) ppm. MS/EI: m/z (% int. rel). 304.30 , 258.08 , 139.11 .H-imidazol-1-yl)ethyl (4-chlorophenyl)carbamate (8):1-methyl-2-. 1H NMR  δ: 1.03 , 2.05 , 4.33 , 5.85-5.87 , 7.29, 7.45 , 8.80 , 9.52  ppm. 13C NMR  δ: 14.5 (CH3), 17.9 (CH3), 50.4 (N–CH2), 69.4 (O–CH), 120.5 , 126.8 (C-4´), 129.1 (C-3´-C-5´), 133.5 (C-4), 138.2 (C-1´), 139.1 (C-5), 152.1 (C-2), 152.9 (C=O) ppm. MS/EI: m/z (% int. rel). 338.74 , 152.01 .H-imidazol-1-yl)ethyl (4-fluorophenyl)carbamate (9):1-methyl-2- δ: 1.31 , 2.06 , 4.56 , 5.16–5.21 , 7.04–7.10 , 7.42 , 7.98 , 9.56  ppm. 13C NMR  δ: 14.5 (CH3), 17.9 (CH3), 50.4 (N–CH2), 69.2 (O–CH), 115.7 , 120.5 , 133.6 (C-4), 136.4 , 139.0 (C-5), 152.1 (C-2), 153.1 (C=O), 157.8  ppm. MS/EI: m/z (% int. rel). 322.29 , 276.07 .H-imidazol-1-yl)ethyl (4-nitrophenyl)carbamate (10):1-methyl-2-. 1H NMR  δ: 1.34 , 2.05 , 4.58 , 5.23–5.26 , 7.53 , 7.98 , 8.18 , 9.62  ppm. 13C NMR  δ: 14.5 (CH3), 17.8 (CH3), 50.22 (N–CH2), 7.01 (O–CH), 118.3 , 125.6 (C-3´–C-5´), 133.6 (C-4), 139.1 (C-1´), 142.3 (C-4´), 146.1 (C-5), 152.1 (C-2), 152.7 (C=O) ppm. MS/EI: m/z (% int. rel). 349.29 , 137.99 .G. intestinalis strain IMSS:0696:1 and T. vaginalis strain GT3 were cultured in TYI-S-33 medium, complemented with 10% calf serum and bovine bile [4 trophozoites of G. intestinalis or T. vaginalis, which were incubated at 37 °C for 48 h with cumulative concentrations of carbamates 1–10, metronidazole, and secnidazole, and also incubated alone in culture medium, with DMSO used as the solvent (0.05%). Subsequently, trophozoites were washed and subcultured for another 48 h in fresh medium without any drugs. Once this time was reached, trophozoites were counted and the median inhibitory concentration (IC50) was calculated. All the experiments were completed in triplicate.ine bile . In vitr4 VERO cells in a 96-well plate and incubated in DMEM media complemented with 10% fetal bovine serum, 100 µg/mL streptomycin, and 100 UI/mL penicillin. The culture was incubated at 37 °C in a 5% CO2 atmosphere with 95% humidity for 48 h. When cells reached >80% confluence, the culture media were replaced and the VERO cells were treated with carbamates 1–10, metronidazole, and secnidazole at 1–100 µM dissolved in DMSO at a maximum concentration of 0.05%. After 48 h of incubation, viability of the cells was estimated using the sulforhodamine B method [50) was calculated by nonlinear fit (GraphPad Prism 4 software). All concentrations were evaluated in triplicate.We cultivated 1.5 × 10B method . The conBos taurus, Sus scrufa, and Ovis aries as proper templates for the G. duodenallis tubulin model. The model was further refined with a short molecular dynamics simulation (500 ps) with the YASARA2 forcefield, which includes knowledge-based potentials. The simulation was conducted at 298 K and 1 atm (NPT ensemble), using an 8 Å cutoff for Van der Waals interactions. Coulombic interactions were computed using the particle mesh Ewald (PME) method [Z-score, QMEAN [https://servicesn.mbi.ucla.edu/SAVES/), which includes PROCHECK [The modeling was completed with YASARA v. 19.12.14) , followi.12.14 , ) method . The inte, QMEAN , and witPROCHECK , WHATCHEPROCHECK , ERRAT [PROCHECK , VERIFY PROCHECK , and PROPROCHECK  metrics http://www.rcsb.org/pdb) [http://www.chemcomp.com) version 2019.01 [The crystal structure of the PFOR (PDB-ID: 1L5P) at 2.2 Å resolution was obtained from the Protein Data Bank (org/pdb) . All doc 2019.01 . All wat 2019.01 . As a va 2019.01  and cons 2019.01 . After tNon-equilibrium Langevin dynamics were used to determine the putative stability of binding modes obtained from docking with Desmond .Using the top poses of the most active compounds as starting point, protein–ligand complexes were prepared in Maestro (19-2), with the System Builder utility. The complexes were buffered in a truncated octahedron box with a 14.0 Å solvent shell around the protein, the system was neutralized, and NaCl was added to obtain a 0.15 M concentration. The system was parameterized with the OPLS 2005 force field. Each system was minimized in three steps, using Brownian dynamics under NVT ensemble at 10 K with a small time-step to avoid numerical errors , first wGiardia duodenalis and Trichomonas vaginalis, with no observable cytotoxic effects in mammalian VERO cells. The giardicidal effect of carbamates 1–10 was improved compared to the two first-line commercial drugs: metronidazole and secnidazole. All compounds showed trichomonicidal effects greater than secnidazole. The antiprotozoal effect could be related to the higher lipophilicity of the 10 compounds, because they could penetrate the protozoal membrane more effectively. The most active compounds were 1 and 2, which are metronidazole cyclohexylcarbamate and phenylcarbamate, respectively. The plausible modes of action of compounds 1 and 2 involve inhibition of PFOR and β-tubulin as suggested by docking and molecular dynamics.We reported the one-step preparation of 10 carbamate derivatives of metronidazole and secnidazole in modest yields, which showed strong nanomolar and micromolar antiprotozoal activity against two amitochondriate parasites,


Allium tuberosum was performed, leading to the isolation of 27 steroidal glycosides (SGs 1–27). The structures of SGs were identified mainly by nuclear magnetic resonance and mass spectrometries as well as the necessary chemical evidence. In the SGs, 1–10 and 22–26 are new steroidal saponin analogues. An in vitro bioassay indicates that 1, 2, 7, 8, 10, 13–15, 20, 23, and 26 display promotional roles in testosterone production of rat Leydig cells with the EC50 values of 1.0 to 4.5 μM, respectively.A systematic phytochemical study on the components in the seeds of  Dioscorea zingiberensis are widely used for preventing cardiovascular diseases -β-d-glucopyranoside (17) [18) [20) [21) [27) [19 has been already registered in CAS with the numbers of 1493828-40-2 and also reported as 3-O-α-l-rhamnopyranosyl(1→4)-[β-d-glucopyranosyl(1→2)]-β-d-glucopyranosyl-26-O-β-d-glucopyranosyl-(25R)-5β-furostane-3β,22α,26-triol in the literature −  together with its 13C NMR data , 1.17 , 1.33 , 1.05 , and two anomeric proton signals at δ 4.83  and 5.11  are observed. Its 13C NMR spectrum exhibits total 39 carbon resonances including 27 ones due to the aglycone part and twelve ones attributed to the two hexoses. Comparison of NMR data suggests that 1 has the identical planar structure of aglycone skeleton with 26-O-β-d-glucopyranosyl-(25S)-3β,5β,6α,22,26-pentahydroxyl-5β-furostane 3-O-α-l-rhamnopyranosyl-(1→4)-β-d-glucopyranoside -β-D-glucopyranoside, named allituberoside B.Compound 3 displays a molecular formula of C50H84O23 as confirmed by HR-ESI-MS. Its NMR data suggests that 3 has the identical structure with 2 except for the terminal sugar unit at C-4′. The neutral missing fragment of 136 Da presented on the mass spectrum of 3 suggests that 3 has a xylose moiety, and detailed analyses of 1H-1H COSY, HSQC, and HMBC spectra finally identified the structure of the C-3 sugar chain of 3 to be 3-O-β-d-xylopyranosyl-(1→4)-[α-l-rhamnopyranosyl-(1→2)]-β-d-glucopyranoside. The chemical shifts of H-26a (δ 4.11) and H-26b (δ 3.49) (Δab > 0.57) further confirm the C-25S configuration of 3. Therefore, the structure of 3 is elucidated as 26-O-β-d-glucopyranosyl-(25S)-5α-furost-2α,3β,22α,26-tetrol 3-O-β-d-xylopyranosyl-(1→4)-[α-l-rhamnopyranosyl-(1→2)]-β-d-glucopyranoside, named allituberoside C.Compound 4 has a molecular formula of C50H82O22 as determined by HR-ESI-MS. Its NMR data suggests that the structure of 4 is closely similar to that of 3 except for the different substructure surrounding C-20 and C-22 positions. In its 13C NMR spectrum, the characteristic carbon signals of δ 103.6 and 152.4 which are the same as those of 15–17 indicate the existence of the Δ20(22)-ene substructure in 4, which is supported by the HMBC correlations of δ 1.62 (H-21)/103.6 (C-20) and 152.4 (C-22). By detailed analysis of 1H-1H COSY, HSQC, and HMBC spectra, the structure of 4 is further confirmed, and its C-25S configuration is deduced according to the chemical shifts of H-26a (δ 4.10) and H-26b (δ 3.49) (Δab > 0.57). Subsequently, the structure of 4 is elucidated as 26-O-β-d-glucopyranosyl-(25S)-5α-furost-Δ20(22)-ene-2α,3β,26-triol 3-O-β-d-xylopyranosyl-(1→4)-[α-l-rhamnopyranosyl-(1→2)]-β-d-glucopyranoside, named allituberoside D.Compound 5 has a molecular formula of C45H74O18 as established by HR-ESI-MS. Its NMR data suggests that except for the C-3 sugar chain, 5 and 4 have the same structure. Analyses of the 1H-1H COSY spectrum give the structures of the glucose and rhamnose comprising the C-3 sugar chain, and the HMBC correlations between δ 5.92 (H-1′′ of Rha) and 78.3 (C-4′ of 3-O-Glc) confirm their connectivity. By detailed analyses of 1H-1H COSY, HSQC, and HMBC spectra, the structure of 5 is further confirmed. The C-25S configuration is deduced according to the chemical shifts of H-26a (δ 4.10) and H-26b (δ 3.49) (Δab > 0.57). Thus, the structure of 5 is elucidated as 26-O-β-d-glucopyranosyl-(25S)-5α-furost-Δ20(22)-ene-2α,3β,26-triol 3-O-α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranoside, named allituberoside E.Compound 6 has a molecular formula of C51H84O22 as identified by HR-ESI-MS. Its NMR data suggest that 6 has the identical aglycone structure with 5 and has the same C-3 sugar chain as 2. According to its 1H-1H COSY, HSQC and HMBC spectra, the structure of 6 is confirmed. The chemical shifts of H-26a (δ 3.96) and H-26b (δ 3.63) (Δab < 0.48) further deduce its C-25R configuration -β-D-glucopyranoside, named allituberoside F.Compound 7 with a molecular formula of C45H76O19 confirmed by HR-ESI-MS has the same sugar moieties as 5 by detailed comparison of the NMR data. Further comparison of the NMR data of 7 and 18 deduces that they share the same aglycone skeleton. According to its 1H-1H COSY, HSQC, and HMBC spectra, the structure of 7 is confirmed. The chemical shifts of H-26a (δ 4.10) and H-26b (δ 3.49) (Δab > 0.57) further confirm the C-25S configuration of 7. Finally, the structure of 7 is elucidated as 26-O-β-d-glucopyranosyl-(25S)-5β-furost-2β,3β,26-triol 3-O-α-l-rhamnopyranosyl-(1→4)-β-d-glucopyranoside, named allituberoside J.Compound 8 has the same molecular formula of C45H76O19 as 7 determined by HR-ESI-MS together with its 13C NMR data. The same 13C NMR data suggests that 8 and 7 are C-25R/S isomers of each other. The chemical shifts of H-26a (δ 3.95) and H-26b (δ 3.63) (Δab < 0.48) exhibit that the C-25 configuration is R. Consequently, the structure of 8 is elucidated as 26-O-β-d-glucopyranosyl-(25R)-5β-furost-2β,3β,26-triol 3-O-α-l-rhamnopyranosyl-(1→4)-β-d-glucopyranoside, named allituberoside H.Compound 9 has a molecular formula of C45H76O20 as confirmed by HR-ESI-MS along with its 13C NMR data  and 83.1 (C-2′) confirm their connectivity. By detailed analyses of 1H-1H COSY, HSQC, and HMBC spectra, the structure of 9 is further confirmed. The chemical shifts of H-26a (δ 4.10) and H-26b (δ 3.50) (Δab > 0.57) exhibit that the C-25S configuration. Consequently, the structure of 9 is elucidated as 26-O-β-d-glucopyranosyl-(25R)-5β-furost-2β,3β,26-triol 3-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside, named allituberoside I.Compound NMR data . The NMR10 has a molecular formula of C45H74O18 as assigned by HR-ESI-MS. Its NMR data suggest that it shares the identical structure with 7 except for the differences surrounding the C-20 and C-22 positions. The characteristic carbon signals of δ 103.6 and 152.4 indicate the existence of the Δ20(22)-ene substructures in the molecule. Detailed analysis of 1H-1H COSY, HSQC, and HMBC spectra allows the structure of 10 to be further confirmed. The chemical shifts of H-26a (δ 4.09) and H-26b (δ 3.49) (Δab > 0.57) exhibit that the C-25S configuration of 10. Thus, the structure of 10 is elucidated as 26-O-β-d-glucopyranosyl-(25S)-5β-furost-Δ20(22)-ene-2α, 3β, 26-diol 3-O-α-l-rhamnopyranosyl-(1→4)-β-d-glucopyranoside, named allituberoside J.Compound 22 is isolated in the form of white powders with a molecular formula of C45H74O18 based on HR-ESI-MS and 13C NMR data. In its 1H NMR spectrum, the anomeric proton signals at δ 4.87 , 5.93 , and 5.47  suggest that 22 has three sugar units. Comparing the NMR data of 22 and 18 reveals that they share the same C-3 sugar chain together with the substructure of A-E rings. Only three typical methyl proton signals at δ 0.85 , 0.97 , 1.18  are observed in the 1H NMR spectrum of 22, and its 13C NMR spectrum shows that the chemical shift of C-27 (δ 64.1) shifted to a lower field, suggesting the linkage of hydroxyl group to the C-27. By further comparing the NMR data, it is deduced that 22 had the same F-ring structure as (25S)-27-hydroxypenogenin-3-O-α-l-rhamnopyranosyl-(1→2)-O-β-d-glucopyranoside -β-d-glucopyranoside, named allituberoside K.Compound ranoside . Thus, t23 has a molecular formula of C51H84O24 as measured by HR-ESI-MS and 13C NMR data. In the 1H NMR spectrum, the anomeric proton signals at δ 4.94 , 5.45 , and 5.90  suggest that it has four sugar units. According to its NMR data, 23 has the same C-3 sugar chain as 22, and shares the identical A-E rings substructure with 7–9. The 1H NMR spectrum of 23 shows only three typical methyl proton signals at δ 0.78 , 0.98 , 1.09 , suggesting that its C-27 is substituted by an additional glucose unit, and that is also supported by the HMBC correlations of δ 4.37 (H-27a), 3.94 (H-27b)/21.4 (C-24), 33.5 (C-25), 61.0 (C-26), and of δ 4.94 (H-1′′′′)/69.5 (C-27). While the different NMR data of F-ring (C-22~C-27) between 23 and 27 suggest that they might have different C-25 configurations. The key carbon signals of δ 27.1 (C-23), 21.4 (C-24), and 61.0 (C-26) which are in accordance with those of trikamsteroside A -β-d-glucopyranoside, named allituberoside L.Compound 24 with a molecular formula of C45H74O19 as deduced by HR-ESI-MS and 13C NMR data has the identical sugar chain at C-3 position and A-E rings substructure with 11–13 and shares the same F-ring including the 27-O-β-d-glucopyranose with 23 by comparing their NMR data. A detailed analysis of 1H-1H COSY, HSQC, and HMBC spectra allows the structure of 24 to be further confirmed as 27-O-β-d-glucopyranoside-(25R)-5α-spirost-2α,26,27-triol 3-O-α-l-rhamnopyranosyl-(1→4)-[β-d-glucopyranosyl-(1→2)]-β-d-glucopyranoside, named allituberoside M. Compound 25 has a molecular formula of C51H84O23 as determined by HR-ESI-MS and 13C NMR data. The NMR data suggests that, except for the different sugar chain at C-3 position, it has the identical structure with 24. Comparison of NMR data further reveals that it has the same C-3 sugar chain as 27. Detailed analysis of 1H-1H COSY, HSQC, and HMBC spectra finally confirms the structure of 25 to be 27-O-β-d-glucopyranoside-(25R)-5α-spirost-2α,26,27-triol 3-O-α-l-rhamnopyranosyl-(1→4)-[α-l-rhamnopyranosyl-(1→2)]-β-d-glucopyranoside, named allituberoside N. Compound 26 has a molecular formula of C50H82O23 as revealed according to HR-ESI-MS. Its NMR data suggests that 26 shares the identical structure with 24 except for the difference in structure of sugar chain at C-3. Further comparison of the NMR data reveals that the C-3 sugar chain of 26 is same as that of 3. According to its 1H-1H COSY, HSQC, and HMBC spectra data, the whole structure of 26 is finally confirmed. Therefore, the structure of 26 is confirmed to be 27-O-β-d-glucopyranoside-(25R)-5α-spirost-2α,26,27-triol 3-O-β-d-xylopyranosyl-(1→4)-[β-d-glucopyranosyl-(1→2)]-β-d-glucopyranoside, named allituberoside O.Compound 1–27 for evaluating their promotional roles in testosterone production of rat Leydig cells. The results of 3β-HSD staining show that Leydig cells are successfully isolated from testes   .® IV polarimeter. HR-ESI-MS was recorded on a Synapt MS . The NMR experiments were performed on Varian UNITY INOVA 600 spectrometer (600 MHz for 1H NMR and 150 MHz for 13C NMR). The optical rotations were measured with a JASCO J-810 polarimeter. HPLC analysis is performed on an Agilent 1100 system equipped with an Alltech 2000 evaporative light scattering detector. Semi-preparative HPLC is performed on an NP7000 module  equipped with a Shodex RID 102 detector . Silgreen HPLC C18 columns  were used for HPLC and Semi-preparative HPLC. Silica gel H , AB-8 macroporous adsorption resin , SP825 macroporous adsorption resin , MCI gel , and ODS silica-gel  were applied for column chromatography. Optical rotations were recorded on a Rudolph AutopolThe dried ATSs were purchased from Shoguang City of Shandong Province in Oct 2018, and identified by Professor Baolin Guo . A voucher specimen was deposited in the authors’ lab.Allium tuberosum seeds (50 kg) were crushed and extracted with 70% aq. CH3CH2OH (400 L) at reflux three times (1 h for each time). The filtered solution was concentrated in vacuo to get the supernatants and sediments. The supernatants were subjected to an AB-8 macroporous adsorption resin column eluted with CH3CH2OH-H2O  to yield five fractions (Fr.A~Fr.D). Fr.B were further subjected to a SP825 macroporous adsorption resin column eluted with CH3CH2OH-H2O  to yield three fractions (Fr.B1~Fr.B3). Fr.B3 was suspended with water and partitioned with ethyl acetate and butanol in turn to yield Fr.B3-A and Fr.B3-B. Fr.B3-B was subjected to silica-gel column chromatography eluted with a gradient mixture of CHCl3-CH3OH  to yield 66 subfractions (Fr.B3-B-S1~Fr.B3-B-S66). Fr.B3-B-S36–40 was subjected to ODS column chromatography eluted with CH3OH--H2O  to afford 35 subfractions (Fr.B3-B-S36–40-O1~Fr.B3-B-S36–40-O35). Among them, Fr.B3-B-S36–40-O16–17 was separated by semi-preparative HPLC with CH3CN-H2O  to afford 1 (12.5 mg); Fr.B3-B-S36–40-O20–23 was separated by semi-preparative HPLC with CH3CN-H2O  to afford 7 (225.7 mg), 8 (13.5 mg), and 11 (125.6 mg); Fr.B3-B-S36–40-O30–34 was separated by semi-preparative HPLC with CH3CN-H2O  to afford 10 (78.5 mg); Fr.B3-B-S36–40-O35–36 was separated by semi-preparative HPLC with CH3CN-H2O  to afford 5 (18.7 mg) and 22 (4.5 mg). Fr.B3-B-S48–54 was subjected to ODS column chromatography eluted with methanol-H2O  to afford 30 subfractions (Fr.B3-B-S48–54-O1~Fr.B3-B-S48–54-O30). Among them, Fr.B3-B-S48–54-O6–8 was separated by semi-preparative HPLC with CH3CN-H2O  to afford 2 (18.3 mg), 12 (122.6 mg), 13 (20.7 mg) and 24 (20.8 mg); Fr.B3-B-S48–54-O18–20 was separated by semi-preparative HPLC with CH3CN-H2O  to afford 14 (87.8 mg) and 20 (18.4 mg); Fr.B3-B-S48–54-O26–29 was separated by semi-preparative HPLC with CH3CN-H2O  to afford 15 (48.5 mg); Fr.B3-B-S48–54-O34–38 was separated by semi-preparative HPLC with CH3CN-H2O  to afford 17 (84.2 mg) and 21 (13.0 mg). Fr.B3-B-S55–65 was subjected to ODS column chromatography eluted with CH3OH-H2O  to afford 39 subfractions (Fr.B3-B-S55–65-O1~Fr.B3-B-S55–65-O39). Among them, Fr.B3-B-S55–65-O7-9 was separated by semi-preparative HPLC with CH3CN-H2O  to afford 9 (45.8 mg) and 18 (19.2 mg); Fr.B3-B-S55–65-O14–15 was separated by semi-preparative HPLC with CH3CN-H2O  to afford 2 (53.3 mg), 3 (18.1 mg), 25 (25.6 mg), and 26 (49.6 mg); Fr.B3-B-S55–65-O22–25 was separated by semi-preparative HPLC with CH3CN-H2O  to afford 18 (134.5 mg), 19 (16.5 mg), and 27 (18.3 mg); Fr.B3-B-S55–65-O33 was separated by semi-preparative HPLC with CH3CN -H2O  to afford 14 (25.6 mg); Fr.B3-B-S55–65-O34–37 was separated by semi-preparative HPLC with CH3OH-H2O  to afford 4 (9.4 mg), 6 (7.2 mg), and 16 (36.5 mg). Fr.B3-B-S66 was subjected to ODS column chromatography eluted with CH3OH-H2O  to afford 7 subfractions (Fr.B3-B-S66-O1~Fr.B3-B-S66-O7). Among them, Fr.B3-B-S66-O4 was separated by semi-preparative HPLC with CH3CN-H2O  to afford 23 (14.0 mg).1): C39H66O16; white amorphous powder; [α]c 0.036, CH3OH); 1H NMR  δ 4.71 , 0.88 , 1.17 , 1.33 , 4.09 , 3.50 , 1.05 , 5.11 , 4.83 ; 13C NMR  spectroscopic data see m/z 789.4272 [M − H]− .Allituberoside A (2): C51H86O23; white amorphous powder; [α]c 0.028, CH3OH); 1H NMR : δ 3.89 , 0.87 , 0.90 , 1.31 , 4.10 , 3.49 , 1.03 , 5.03 , 6.40 , 5.86 , 4.82 ; 13C NMR  spectroscopic data see m/z 1065.5466 [M − H]− .Allituberoside B (3): C50H84O23; white amorphous powder; [α]c 0.037, CH3OH); 1H NMR  δ 3.88 , 0.87 , 0.91 , 1.31 , 4.11 , 3.49 , 1.03 , 5.03 , 6.26 , 5.04 , 4.82 ; 13C NMR  spectroscopic data see m/z 1051.5316 [M − H]− .Allituberoside C (4): C50H82O22; white amorphous powder; [α]c 0.028, CH3OH); 1H NMR  δ 3.88 , 0.68 , 0.92 , 1.62 , 4.10 , 3.49 , 1.04 , 5.04 , 6.27 , 5.05 , 4.85 ; 13C NMR  spectroscopic data see m/z 1033.5208 [M − H]− .Allituberoside D (5): C45H74O18; white amorphous powder; [α]c 0.024, CH3OH); 1H NMR  δ 3.86 , 0.69 , 0.75 , 1.62 , 4.10 , 3.49 , 1.04 , 5.04 , 5.92 , 4.85 ; 13C NMR  spectroscopic data see m/z 901.4807 [M − H]− .Allituberoside E (6): C51H84O22; white amorphous powder; [α]c 0.030, CH3OH); 1H NMR  δ 3.89 , 0.70 , 0.91 , 1.63 , 3.96 , 3.63 , 1.03 , 5.03 , 6.41 , 5.87 , 4.86 ; 13C NMR  spectroscopic data see m/z 1047.5400 [M − H]− .Allituberoside F (7): C45H76O19; white amorphous powder; [α]c 0.029, CH3OH); 1H NMR : δ 3.90 , 0.87 , 0.85 , 1.33 , 4.10 , 3.49 , 1.04 , 4.94 , 5.92 , 4.83 ; 13C NMR  spectroscopic data see m/z 919.4893 [M − H]− .Allituberoside G (8): C45H76O19; white amorphous powder; [α]c 0.025, CH3OH); 1H NMR  δ 3.90 , 0.87 , 0.85 , 1.34 , 3.95 , 3.63 , 1.04 , 4.94 , 5.92 , 4.84 ; 13C NMR  spectroscopic data see m/z 919.4892 [M − H]− .Allituberoside H (9): C45H76O20; white amorphous powder; [α]c 0.038, CH3OH); 1H NMR  δ 4.26 , 0.87 , 1.00 , 1.32 , 4.10 , 3.50 , 1.04 , 5.04 , 5.39 , 4.83 ; 13C NMR  spectroscopic data see m/z 935.4837 [M − H]− .Allituberoside I (10): C45H74O18; white amorphous powder; [α]c 0.031, CH3OH); 1H NMR  δ 4.47 , 0.68 , 0.87 , 1.62 , 4.09 , 3.49 , 1.04 , 4.96 , 5.93 , 4.86 ; 13C NMR  spectroscopic data see m/z 901.4787 [M − H]− .Allituberoside J (22): C45H74O18; white amorphous powder; [α]c 0.025, CH3OH); 1H NMR  δ 4.26 , 0.85 , 0.97 , 1.18 , 4.17 , 3.93 , 4.87 , 5.47 , 5.93 ; 13C NMR  spectroscopic data see m/z 901.4800 [M − H]− .Allituberoside K (23): C51H84O24; white amorphous powder; [α]c = 0.024, CH3OH); 1H NMR  δ 4.27 , 0.78 , 0.98 , 1.09 , 4.37 , 3.94 , 4.94 , 5.45 , 5.90 , 4.94 ; 13C NMR  spectroscopic data see m/z 1079.5267 [M − H]− .Allituberoside L (24): C45H74O19; white amorphous powder; [α]c 0.039, CH3OH); 1H NMR  δ 3.93 , 0.77 , 0.89 , 1.08 , 4.36 , 3.92 , 5.10 , 6.39 , 4.93 ; 13C NMR  spectroscopic data see m/z 917.4756 [M − H]− .Allituberoside M (25): C51H84O23; white amorphous powder; [α]c 0.032, CH3OH); 1H NMR  δ 3.88 , 0.77 , 0.89 , 1.07 , 4.37 , 3.92 , 5.03 , 6.40 , 5.86 , 4.93 ; 13C NMR  spectroscopic data see m/z 1063.5320 [M − H]− .Allituberoside N (26): C50H82O23; white amorphous powder; [α]c 0.026, CH3OH); 1H NMR  δ 3.88 , 0.77 , 0.89 , 1.07 , 4.36 , 3.93 , 5.03 , 6.26 , 5.04 , 4.93 ; 13C NMR  spectroscopic data see m/z 1049.5180 [M − H]− .Allituberoside O (1–10 and 20–26 (each 1.0 mg) were individually hydrolyzed by heating in 1 mL of 6 M TFA at 90 °C for 2 h. After cooling, the reaction mixture was extracted with CHCl3. Then, each aqueous layer was evaporated to dryness, and the residue was dissolved in 1 mL of pyridine containing 1 mg of L-cysteine methyl ester hydrochloride and further heated at 60 °C for 1 h. Following, o-tolyl isothiocyanate (5 μL) was added to each mixture, and heated at 60 °C for 1 h. Standard sugars (each 5 mg) and L-cysteine methyl ester hydrochloride (5 mg) was dissolved in pyridine (5 mL) and heated to 60 °C for 1 h. Then o-tolyl isothiocyanate (10 μL) was added to the mixture and refluxed for 1 h. The reaction mixture was analyzed by HPLC. As a result, the D-configurations for glucoses and xylose, L-configrations for rhamnoses in the corresponding compounds were identified by comparing the retention time with the standards. Compounds g at 4 °C, and cells localized between Percoll gradient 70 and 58% were isolated (the second layer). After the repeating wash steps of the medium, the Leydig cells were incubated in the DMEM-F12 culture medium.Leydig cells were isolated from 50–70-day-old Sprague Dawley rats followed the procedure described in the literature with some modifications . In a nuβ-hydroxysteroid dehydrogenase (3β-HSD) histochemical staining [2. The staining solution contained PBS supplemented with 0.1 mg/mL nitro-blue tetrazolium (Biosharp), 1.0 mg/mL nicotinamide adenine dinucleotide (Sigma-Aldrich), 0.1 mg/mL dehydroepiandrosterone , and 0.1 mg/mL niacinamide for 90 min. The positive cells were stained a dark blue.The purity of Leydig cells were determined by 3staining . Leydig Animal experiments were approved by the Institutional Animal Care and Use Committee and the local experimental Ethics Committee . Male Sprague-Dawley rats were purchased from the Hubei Provincial Center for Disease Control and Prevention .3/mL) were cultured in 96-well plates at 37 °C with 5% CO2 for 48 h. The cells were afterward cultured in serum-free medium containing different doses of compounds, forskolin, HCG (1 IU/mL) for 24 h. Cellular viability was evaluated using the MTT proliferation assay. The MTT (Sigma-Aldrich) solution was maintained for 4 h, then 100 μL DMSO was added. Finally, the absorbance was measured at 570 nm by a microplate reader (Synergy HT). Testosterone secreted into the culture medium was measured using ELISA kits according to the manufacturer’s instructions .Purified Leydig cells (5 × 10This phytochemical work presented a study on ATSs leading to the isolation of 27 steroidal saponins, which facilitates understanding the structural composition of steroidal saponins as the main constituents in ATSs. The subsequent activity assay shows that nearly half of the isolated steroidal glycosides can remarkably promote the testosterone production of rat Leydig cells, proving that the steroidal saponin could be considered as the basis of active material of this traditional herb medicine for playing a role in treating both impotence and nocturnal emissions. The result of this work reveals the active substance basis of ASTs to some extent. Meanwhile, this work clarifies the structure of steroidal saponin in ASTs, establishing a foundation for the quality control research of this traditional medicine.


Mycobacterium tuberculosis (TB). To identify new antimicrobial agents with reduced propensity for the development of resistance, a series of dual-acting nitroimidazole-oxazolidinone conjugates were designed, synthesized and evaluated for their antimicrobial activity. Compounds in this conjugate series have shown synergistic activity against a panel of anaerobic bacteria, including those responsible for serious bacterial infections.The introductions of the bicyclic 4-nitroimidazole and the oxazolidinone classes of antimicrobial agents represented the most significant advancements in the infectious disease area during the past two decades. Pretomanid, a bicyclic 4-nitroimidazole, and linezolid, an oxazolidinone, are also part of a combination regimen approved recently by the US Food and Drug Administration for the treatment of pulmonary, extensively drug resistant (XDR), treatment-intolerant or nonresponsive multidrug-resistant (MDR)  Recently, a three-drug combination containing both pretomanid and linezolid was approved by the US Food and Drug Administration for the treatment of pulmonary XDR, treatment-intolerant or nonresponsive MDR-TB oxazin-6-ol core (6) was prepared according to literature procedures oxazin-7-yl)methanol (17a) and methanol (17b) were prepared according to a procedure described in the literature oxazin-6-yl)oxy)pyridin-3-yl)phenyl)-5-(hydroxymethyl)oxazolidin-2-one (8a) oxazine  and (R)-3-phenyl)-5-(hydroxymethyl)-oxazolidin-2-onein DMF (30 mL) was added a solution of K2CO3  in water (1 mL) and the mixture was purged with nitrogen. Pd(PPh3)4  was then added and the mixture was warmed with stirring under nitrogen at 80 °C for 3 h. The solvents were completely removed under reduced pressure and the residue was partitioned between EtOAc and water. The extract was worked up and chromatographed on silica. Elution with EtOAc gave four fractions, then elution with (1:20) MeOH/DCM gave 8a as a yellow powder . This general procedure of Suzuki coupling reaction was also applied for the synthesis of 8b, 11b, 14a–d, and 19a–c. 1H-NMR  δ 8.38 , 8.06, 7.94–7.91 , 7.63 , 7.57 , 7.46–7.43 , 6.97 , 5.76 , 5.24 , 4.74–4.67 , 4.48–4.38 , 4.13–4.09 , 3.86 , 3.70–3.64 , 3.56–3.52 , 3.14 . LC-MS (ESI): m/z = 472 (M + H)+.one (8a) . To a so(R)-3-(3-fluoro-4-(2-(((S)-2-nitro-6,7-dihydro-5H-imidazooxazin-6-yl)oxy)pyrimidin-5-yl)phenyl)-5-(hydroxymethyl)oxazolidin-2-one (8b) oxazine was used instead of (S)-6-((5-bromopyridin-2-yl)oxy)-2-nitro-6,7-dihydro-5H-imidazooxazine. . 1H-NMR  δ 8.86 , 8.06 , 7.68 , 7.66 , 7.47 , 5.74 , 5.23 , 4.77–4.70 , 4.47 , 4.11 , 3.86 , 3.71–3.65 , 3.57–3.53 , 3.38–3.35 . LC-MS (ESI): m/z = 473 (M + H)+.one (8b) . The titN-(((S)-3-(2-fluoro-4′-((((S)-2-nitro-6,7-dihydro-5H-imidazooxazin-6-yl)oxy)methyl)--4-yl)-2-oxooxazolidin-5-yl)methyl)acetamide (11a) oxazine  and (S)-N-phenyl)-2-oxooxazolidin-5-yl) methyl)acetamide  in DMF (8 mL) was added a solution of Na2CO3  in water (1 mL) and the mixture was purged with nitrogen. Pd(PPh3)4  was then added and the mixture was warmed with stirring under nitrogen at 85 °C for 90 min. The solvents were completely removed under reduced pressure and the residue was partitioned between EtOAc and water. The extract was worked up and chromatographed on silica. Elution with EtOAc gave four fractions, then elution with (1:9) MeOH/EtOAc gave 11a as an off-white powder. . 1H-NMR  δ 8.22 , 8.07 , 7.60–7.50 , 7.39 , 7.15 , 5.29 , 4.79–4.70 , 4.70–4.64 , 4.42 , 4.35 , 4.16 , 40.03 , 3.78 , 3.43 , 1.84 . LC-MS (ESI): m/z = 526 (M + H)+.cetamide a [11]. T(R)-3-(3-fluoro-4-(6-((((S)-2-nitro-6,7-dihydro-5H-imidazooxazin-6-yl)oxy)methyl)pyridin-3-yl)phenyl)-5-(hydroxymethyl)oxazolidin-2-one (11b) oxazine was used instead of (S)-6-((5-bromopyridin-2-yl)oxy)-2-nitro-6,7-dihydro-5H-imidazooxazine. . 1H-NMR  δ 8.68 , 8.04 , 7.96 , 7.65 , 7.60 , 7.46 , 5.74 , 5.24 , 4.77–4.69 , 4.48 , 4.35–4.22 , 4.12 , 3.86 , 3.70–3.65 , 3.58–3.52 , 3.16–3.12 . LC-MS (ESI): m/z = 486 (M + H)+.in-2-one b [11]. T(5R)-3-methoxy)--4-yl)-5-(hydroxymethyl)oxazolidin-2-one (14a). The title compound was prepared by following the same procedure as that described for the preparation of 8a, except (S)-6-((5-bromopyridin-2-yl)methoxy)-2-nitro-6,7-dihydro-5H-imidazooxazine was used instead of (S)-6-((5-bromopyridin-2-yl)oxy)-2-nitro-6,7-dihydro-5H-imidazooxazine . 1H-NMR  δ 8.14 , 7.67–7.26 , 6.96 , 5.20 , 4.69 , 4.39–4.28 , 4.17 , 4.07 , 3.82 , 3.65 , 3.58–3.48 , 1.66 . LC-MS (ESI): m/z = 485 (M + H)+.(5R)-3-methoxy)--4-yl)-5-(hydroxymethyl)oxazolidin-2-one (14b). The title compound was prepared by following the same procedure as that described for the preparation of 8a, except 2-((4-bromo-3-chlorophenoxy)methyl)-2-methyl-6-nitro-2,3-dihydroimidazooxazole was used instead of (S)-6-((5-bromopyridin-2-yl)oxy)-2-nitro-6,7-dihydro-5H-imidazooxazine . 1H-NMR  δ 8.13 , 7.55 , 7.37 , 7.28 , 7.13 , 6.93 , 5.20 , 4.69 , 4.41–4.29 , 4.16 , 4.08 , 3.82 , 3.69–3.60 , 3.58–3.49 , 1.65 . LC-MS (ESI): m/z = 567 (M + H)+.(5R)-3-methoxy)pyridin-3-yl)phenyl)-5-(hydroxymethyl)oxazolidin-2-one (14c). The title compound was prepared by following the same procedure as that described for the preparation of 8a, except 2-(((5-bromopyridin-2-yl)oxy)methyl)-2-methyl-6-nitro-2,3-dihydroimidazooxazole was used instead of (S)-6-((5-bromopyridin-2-yl)oxy)-2-nitro-6,7-dihydro-5H-imidazooxazine . 1H-NMR  δ 8.34 , 8.15 , 7.94–7.87 , 7.68–7.55 , 7.45 , 6.86 , 5.22 , 4.74 , 4.63 , 4.41 , 4.25–4.09 , 3.88 , 3.70 , 3.58 , 1.71 . LC-MS (ESI): m/z = 486 (M + H)+.(5R)-3-methoxy)pyridin-2-yl)phenyl)-5-(hydroxymethyl)oxazolidin-2-one (14d). The title compound was prepared by following the same procedure as that described for the preparation of 8a, except 2-(((6-bromopyridin-3-yl)oxy)methyl)-2-methyl-6-nitro-2,3-dihydroimidazooxazole was used instead of (S)-6-((5-bromopyridin-2-yl)oxy)-2-nitro-6,7-dihydro-5H-imidazooxazine . 1H-NMR  δ 8.32 , 8.16 , 7.90 , 7.69 , 7.58 , 7.48–7.38 , 5.21 , 4.70 , 4.40 , 4.17 , 4.08 , 3.83 , 3.65 , 3.59–3.49 , 1.67 . LC-MS (ESI): m/z = 486 (M + H)+.(5R)-3-methoxy)pyridin-2-yl)oxy)phenyl)-5-(hydroxymethyl)oxazolidin-2-one (16).2-(((5-bromopyridin-2-yl)oxy)methyl)-2-methyl-6-nitro-2,3-dihydroimidazooxazole , (R)-3-(3-fluoro-4-hydroxyphenyl)-5-(hydroxymethyl)oxazolidin-2-one , Potassium carbonate , Cuprous iodide  and N,N,N′,N′-Tetramethylethylenediamine (TMEDA)  were added in N,N-dimethylformamide (5 mL). The mixture was stirred at 90 °C for 12 h. The mixture was cooled to room temperature, then poured into water (50 mL), the solvent was filtered and the filtered solid was pump dried. The crude product was further purified by silica chromatography column (DCM:MeOH = 100:2) to give (5R)-3-methoxy)pyridin-2-yl)oxy)phenyl)-5-(hydroxymethyl)oxazolidin-2-one. 1H NMR  δ 8.29 , 8.05 , 7.88 , 7.64–7.50 , 7.41 , 6.92 , 4.70 , 4.48 , 4.23–4.01 , 3.83 , 3.65 , 3.53 , 2.40–2.29 , 2.22–2.09 , 1.46 . LC-MS (ESI): m/z = 502 (M + H)+.(5R)-3-methoxy)pyridin-3-yl)phenyl)-5-(hydroxymethyl)oxazolidin-2-one (19a). The title compound was prepared by following the same procedure as that described for the preparation of 8a, except 7-(((5-bromopyridin-2-yl)oxy)methyl)-2-nitro-6,7-dihydro-5H-imidazooxazine was used instead of (S)-6-((5-bromopyridin-2-yl)oxy)-2-nitro-6,7-dihydro-5H-imidazooxazine . 1H-NMR  δ 8.32 , 8.06 , 7.91 , 7.58 , 7.42 , 6.97 , 5.21 , 4.91 , 4.71 , 4.57 , 4.21–4.01 , 3.89–3.80 , 3.66 , 3.60–3.49 , 2.29 , 2.18 . LC-MS (ESI): m/z = 486 (M + H)+.(5R)-3-methoxy)pyrimidin-5-yl)phenyl)-5-(hydroxymethyl)oxazolidin-2-one (19b). The title compound was prepared by following the same procedure as that described for the preparation of 8a, except 7-(((5-bromopyrimidin-2-yl)oxy)methyl)-2-nitro-6,7-dihydro-5H-imidazooxazine was used instead of (S)-6-((5-bromopyridin-2-yl)oxy)-2-nitro-6,7-dihydro-5H-imidazooxazine . 1H-NMR  δ 8.81 , 8.07 , 7.70–7.62 , 7.48–7.41 , 5.22 , 5.01–4.90 , 4.76–4.61 , 4.20–4.06 , 3.85 , 3.66 , 3.54 , 2.30 , 2.24–2.10 . LC-MS (ESI): m/z = 487 (M + H)+.(5R)-3-methoxy)pyridin-3-yl)phenyl)-5-(hydroxymethyl)oxazolidin-2-one (19c). The title compound was prepared by following the same procedure as that described for the preparation of 8a except 7-(((5-bromopyridin-2-yl)oxy)methyl)-7-methyl-2-nitro-6,7-dihydro-5H-imidazooxazine was used instead of (S)-6-((5-bromopyridin-2-yl)oxy)-2-nitro-6,7-dihydro-5H-imidazooxazine . 1H-NMR  δ 8.29 , 8.05 , 7.88 , 7.64–7.50 , 7.41 , 6.92 , 4.70 , 4.48 , 4.11 , 3.83 , 3.65 , 3.53 , 2.40–2.29 , 2.22–2.09 , 1.46 . LC-MS (ESI): m/z = 500 (M + H)+.Mycobacterium tuberculosis isogenic resistant mutant panel used for the mechanism of action study were generated and tested at Beijing Tuberculosis and Thoracic Tumor Research Institute . Strains of the spectrum panel were all ATCC stains, and testing was conducted at HD Biosciences . Strains of the anaerobic bacterial panel were ATCC strains, and the test was conducted at Micomyx Inc. .The bacterial strains used in the current study are listed in 4–105 CFU/mL of M. tuberculosis H37Rv (ATCC27294) in 7H9 liquid culture medium at logarithmic growth phase were evenly spread on a 7H11 solid medium containing 1× MIC (0.07 µg/mL) of pretomanid and blank control medium. After sealing, the plates were incubated at 37 °C under 5% CO2 for 3 to 4 weeks. Single colonies with good growth were collected from the drug-containing solid medium, milled and diluted to a concentration of 104–105 CFU/mL, and then inoculated onto 7H11 solid medium with pretomanid at 2× MIC (0.15 μg/mL) and blank control medium. The plates were then incubated under the same conditions for another 3 to 4 weeks to observe the growth of single colonies. A single colony with robust growth was collected from the drug-containing medium and further subcultured on the drug-containing solid medium with a 2-fold increase of concentration of pretomanid until the selection of a single colonies that grew well on solid medium with pretomanid concentration of 16× MIC. The phenotypic confirmation of the pretomanid resistant mutant strains was performed by the Alamar Blue double dilution method [ddn gene. A similar sequential drug selection method was applied to obtain linezolid resistant mutant trains. The genotypic change of the linezolid resistant mutant strains was found to be the point mutation of the rplC (T460C).The methods to induce and characterize the Mtb isogenic mutant strains resistant to pretomanid or linezolid have been described previously ,13. Brien method . The genv/v) laked horse blood , Lot No. 385663). Brucella broth (BD) supplemented with 10% fetal bovine serum  was used for testing of H. pylori. Cation-adjusted Mueller Hinton Broth (CAMHB) media was used for the spectrum determination against the spectrum panel. Difco Middlebrook  7H9 Broth  supplemented with 0.2% (v/v) glycerol, 0.05% Tween 80, and 10% (v/v) albumin-dextrosecatalase  ADC Enrichment, Catalog No. 212352) (7H9-ADC-T) was used for the MIC assay against the M. tuberculosis isogenic resistant mutant panel.Media were prepared according to guidelines from CLSI. The test medium used for the anaerobic bacterial was supplemented Brucella broth  containing 5 μg/mL hemin , Lot No. SLBC4685V), 1 mg/mL Vitamin K1 , and 5%  plates and tested with drug in CAMHB. Anaerobic bacterial 7 CFU/mL and diluted 1:20. Twofold dilutions of testing compounds and comparators were prepared in 7H9-ADC-TG in volumes of 100 µL in 96-well, black, clear-bottom microplates . Bacterial cells (100 µL containing 2 × 105 CFU) was added, yielding a final testing volume of 200 µL. The plates were incubated at 37 °C, and on day 7, 12.5 µL of 20% Tween 80 and 20 µL of Alamar blue were added to all wells. After incubation at 37 °C for 16 to 24 h, the fluorescence was read at an excitation of 530 nm and an emission of 590 nm. The MIC was defined as the lowest concentration effecting a reduction in fluorescence of ≥90% relative to the mean of replicate bacterium-only controls.The MIC assay against the Mtb isogenic resistant mutant strains was performed at BTTTRI by the microplate Alamar blue assay . Pretoma5 CFU/mL (5 × 104 CFU/mL for Clostridium spp.). For the H. pylori strain, colonies were picked from the primary plate and a suspension was prepared to equal a 2.0 McFarland turbidity standard. Suspensions were diluted 1:15 in Brucella broth with 10% FBS, and then transferred to compartments of sterile reservoirs divided by length (Beckman Coulter). The Biomek 2000  was used to inoculate the plates. Daughter plates were placed on the Biomek 2000 work surface in reverse orientation so that plates were inoculated from low to high drug concentration. The Biomek 2000 delivered 10 μL of standardized inoculum into each well of the appropriate daughter plate for an additional 1:20 dilution. Anaerobe plates were placed in an anaerobic box with GasPak sachets (BD), and were incubated anaerobically for 46–48 h at 35–37 °C. H. pylori was incubated for 72 h in a microaerophilic atmosphere using boxes with GasPak EZ Campy sachets (BD) prior to reading. After incubation, plates were viewed from the bottom using a plate viewer. An uninoculated solubility control plate was observed for evidence of drug precipitation. MIC values were read and recorded as the lowest concentration of drug that inhibited the visible growth of the organism.The MIC assay against anaerobic bacterial strains was performed at Micromyx by following the procedure described by CLSI ,16,17. A2 at 37 °C to reach confluent, and then diluted to 4 × 105 cells/mL. Threefold serial dilutions of the stock solutions resulted in final concentrations of 64 to 0.26 µg/mL in a final volume of 100 µL. Cytotoxicity testing was performed in a transparent 96-well microplate. After incubation at 37 °C for 48 h, the medium was removed, and the monolayers were washed twice with 100 µL of warm Hanks balanced salt solution (HBSS). Warm medium and freshly made methyl-thiazolyldiphenyl-tetrazolium bromide (MTT) were added to each well, and then the plates were incubated for 4 h, after which the absorbance was determined at 492 nm.The cytotoxicity assay followed the previously published protocol . BrieflyMycobacterium tuberculosis by utilizing an isogenic mutant panel resistant to either pretomanid or linezolid. Compounds in the series are highly active against a panel of clinically important anaerobic bacteria. A strong synergistic effect was observed compared to the combination of linezolid and pretomanid. It can be concluded that the nitroimidazole-oxazolidinone conjugate molecules hold potential for the treatment of anaerobic bacterial infections.A series of 4-nitroimidazole and oxazolidione conjugate molecules was designed, synthesized and evaluated based on previous structure-activity relationship information. The dual mechanism of action of this series was demonstrated against


Acari, Hydrachnidia), collected in the Anzihe and Qingliangfeng national nature reserves, R. P. China, are identified. Three species are described as new to science: Torrenticolapseudosiamis Gu & Guo, sp. nov., T.anziensis Gu & Guo, sp. nov., and Monatractidessichuanensis Gu & Guo, sp. nov. The other two species, M.macrocorpis Gu & Guo, 2019, M.xiaoxiensis Gu & Guo, 2019, are newly reported from Zhejiang Province. Descriptions and illustrations of these species are included.Five species of torrenticolid mites ( Torrenticolidae is one of the largest groups of water mites. Until now, the total number of torrenticolid mites is about 600 worldwide, but only 38 species are known from China, including the three new species added in this paper .All measurements of palp and legs are of the dorsal margin, given in micrometers (μm) and following Torrenticolidae Piersig, 1902Family Torrenticolinae Piersig, 1902Subfamily Torrenticola Piersig, 1896Genus Taxon classificationAnimaliaTrombidiformesTorrenticolidaeGu & Guosp. nov.E9AF6221-6BC9-539B-B90F-4B7297DF7346http://zoobank.org/C8F4222B-E74D-4A0E-BE01-80751961123FHolotype: ♀ (ZJ-TO-20180701), Qingliangfeng National Nature Reserve, Lin’an, Zhejiang Province, P. R. China , collected by Xinyao Gu, 31-VII-2018. Paratype: 2 ♀♀ , same data as holotype.4E at the same level as the 6th pair of acetabula; gnathosoma with a short rostrum, the lateral view of gnathosoma regular triangle-like.Idiosoma elliptical; dorsal plate 4+1; infracapitular bay U-shaped; n = 3)Female , W 586 (586–648), L/W ratio 1.4 (1.3–1.4). Dorsal plate 4+1 , Cx-II+III mL 33 (19–33); Gf pentagonal, L 220 (180–220), W 168 (168–192), L/W ratio 1.3 (1.1–1.3), distance between Gf and Ap 220 (156–220); 4E at the same level as the 6th pair of acetabula; Ap away from the line of primary sclerotization, on the same level of 1V and anterior to 2V , dL 151 (128–159), chelicera bs L 214 (202–251), claw L 41 (41–43). P-1 with one dorsal seta; P-2 with one dorsal and one ventral setae at the base of the ventral extension; P-3 with three dorsal setae and one long seta at the base of the ventral extension; P-4 with one dorsal seta in proximal half, two mediodistal setae and two ventral setae at the slight ventral extension ; P-2, 68 (65–68); P-3, 41 (37–48); P-4, 62 (59–72); P-5, 20 (19–25). Legs , 86 (86–87), 84 (82–92), 109 (105–123), 124 (116–124), 125 (115–125); II-L-1–6: 51 (44–51), 92 (89–116), 78 (76–86), 110 (110–132), 129 (129–149), 132 (127–142); III-L-1–6: 73 (54–73), 80 (80–108), 82 (72–86), 123 (105–123), 139 (107–139), 146 (121–146); IV-L-1–6: 126 (115–134), 119 (111–123), 118 (112–133), 169 (157–185), 169 (165–192), 167 (148–174).4+1 Fig. , dorsal  V2 Fig. . Gnathos V2 Fig. : the lation Fig. . dL of pMale. Unknown.Streamlet.Torrenticolasiamis Pešić & Smit, 2009  4E at the same level as the 6th pair of acetabula in the new species, but the 4th pair in T.siamis; (3) 2D on the same level with muscle scars in the new species, but 2D anterior to muscle scars in T.siamis.Due to the characteristic shape of gnathosoma and dorsal shield , this new species is similar to it, 2009 . Howeverpseudo-”, which means fake or simulated; this new species is named after its similar species, T.siamis.The specific name is from Latin affix: “China (Zhejiang).Taxon classificationAnimaliaTrombidiformesTorrenticolidaeGu & Guosp. nov.A677171E-416A-59C3-AA12-7F56487AF754http://zoobank.org/2BB83E40-0117-4BD6-8205-AF3A754EBD6BHolotype: ♀ (SC-TO-20160701), Anzihe, Chongzhou, Sichuan Province, P. R. China , collected by Zhuhui Ding, 29-VII-2016. Paratype: 1 ♀ (SC-TO-20160702), same data as holotype.4E at the same level as the 3rd pair of acetabula; 1V fused with the line of primary sclerotization; gnathosoma: rostrum long, dorsal apodemes short and sharp, ventral apodemes slender and sharp, claw short.Dorsal plate 4+1; infracapitular bay U-shaped and wide; genital flaps with six pairs of setae at the margins; n = 2)Female , W 583 (576), L/W ratio 1.4 (1.5). Dorsal plate 4+1 , Cx-II+III mL 99 (89); Gf L 200 (208), W 140 (144), L/W ratio 1.4 (1.4), genital flaps with six pairs of setae at the margins; distance between Gf and Ap 123 (134); 4E at the same level as the 3rd pair of acetabula; Ap on the same line with 2V, 1V fused with the line of primary sclerotization, and 1V anterior to 2V , dL 256 (242); chelicera bs L 378 (369), claw L 63 (58). P-1 with one long dorsal seta; P-2 with three dorsal setae and one ventral seta at the base of the ventral extension; P-3 with two dorsal setae and one long seta at the base of the ventral extension; P-4 with one mediodistal seta and two ventral setae at two ventral extensions ; P-2, 113 (109); P-3, 67 (66); P-4, 93 (95); P-5, 18 (16). Legs , 95 (102), 88 (85), 102 (108), 110 (116), 96 (101); II-L-1–6: 43 (45), 98 (102), 93 (94), 121 (118), 136 (129), 149 (139); III-L-1–6: 43 (45), 98 (102), 81 (85), 101 (108), 117 (115), 114 (121); IV-L-1–6: 107 (102), 118 (115), 125 (119), 161 (158), 179 (183), 166 (171).4+1 Fig. , dorsal  V2 Fig. . Gnathos V2 Fig. : rostrumons Fig. . dL of pMale. Unknown.Streamlet.Torrenticolahaliki Pešić & Smit, 2010  P-4 with one long and three short setae at the base of ventral extensions and one thick mediodistal seta in T.haliki, but only with two ventral setae and one fine mediodistal seta in this new species; (3) ratio Cx-I mL/Cx-II+III mL 1.2 , 1.7–2.3  in T.haliki, but 0.8  in this new species.Due to the characteristic shape of gnathosoma and dorsal shield , this new species is similar to it, 2010 . HoweverThis new species is named after Anzi (Anzi River), where the new species was collected.China (Sichuan).Taxon classificationAnimaliaTrombidiformesTorrenticolidaeGu & Guosp. nov.225CB29B-8954-5683-B54A-E43DE50DD47Ahttp://zoobank.org/8EFE370B-7D4D-46B4-9D87-2608AE618101Holotype: ♀ (SC-TO-20160703), Anzihe National Nature Reserve, Chongzhou, Sichuan Province, P. R. China , collected by Zhuhui Ding, 29-VII-2016. Paratype: 1 ♀ (SC-TO-20160704), same data as holotype.4E at the same level as the posterior margins of Gf; 1V fused with the line of primary sclerotization; 2D on the same level with muscle scars; dorsal and ventral apodemes of gnathosoma slender and sharp; dorsal seta on P-1 long.Infracapitular bay U-shaped and wide; the tip of Cx-I with a papillary cuticular extension, n = 2)Female , W 497 (469), L/W ratio 1.5 (1.5). Dorsal plate 4+1 , Cx-II+III mL 46 (31); Gf pentagonal, L 156 (159), W 122 (109), L/W ratio 1.3 (1.5), genital flaps with six pairs of setae at the margins; distance between Gf and Ap 222 (181); 4E at the same level as the posterior margins of Gf; 1V fused with the line of primary sclerotization, Ap at the same line with 2V , dL 177 (116); dorsal and ventral apodemes slender and sharp; claw short, chelicera bs L 159 (162), claw L 27 (22). P-1 with one long dorsal seta; P-2 with three dorsal and one ventral setae; P-3 with two long dorsal setae and one short ventral seta; P-4 with one short dorsal seta and two mediodistal setae ; P-2, 52 (48); P-3, 34 (32); P-4, 34 (39); P-5, 17 (14). Legs , 70 (71), 59 (62), 67 (80), 58 (72), 79 (83); II-L-1–6: 55 (61), 92 (85), 56 (60), 86 (75), 84 (92), 108 (96); III-L-1–6: 63 (51), 99 (92), 71 (69), 94 (95), 118 (112), 120 (115); IV-L-1–6: 113 (115), 97 (87), 115 (111), 138 (140), 151 (126), 144 (142).4+1 Fig. , dorsal  V2 Fig. . Gnathos V2 Fig.  vL 182  the margins of Cx-II and Cx-III sharp in M.harveyi, but blunt and flat in the new species; (3) 2D anterior to muscle scars in M.harveyi, but 2D on the same level with muscle scars in the new species.This new species is similar to it, 2012  in the gThis new species is named after Sichuan Province, where it was collected.China (Sichuan).Taxon classificationAnimaliaTrombidiformesTorrenticolidaeGu & Guo, 201982581258-93B6-5DBD-AA47-83BC97F5EDC630°6'44"N, 118°53'36"E, 940 m a.s.l.), collected by Xinyao Gu, 31-VII-2018, 1 ♀(ZJ-TO-20180704), 2 ♂♂ .Qingliangfeng National Nature Reserve, Lin’an, Zhejiang Province, P. R. China (n = 1)Male .Female , W 945 (949), L/W ratio 1.2 (1.3). Dorsal plate , Cx-II+III mL 68 (38); Gf L 256 (267), W 253 (236), L/W ratio 1.0 (1.1); distance between Gf and Ap 292 (248). Gnathosoma , dL 246 (262), claw short L 32 (29), chelicera bs L 246 (262). dL of palp segments: P-1, 32 (34); P-2, 90 (91); P-3, 51 (58); P-4, 77 (83); P-5, 21 (32). Legs: dL of leg segments: I-L-1–6: 100 (77), 114 (137), 121 (120), 153 (157), 147 (149), 127 (123); II-L-1–6: 90 (78), 134 (148), 99 (98), 154 (175), 195 (203), 168 (176); III-L-1–6: 101 (-), 161 (170), 119 (120), 177 (193), 214 (228), 217 (203); IV-L-1–6: 144 (159), 181 (160), 188 (192), 235 (243), 245 (261), 230 (220).ate Fig. , dorsal oma Fig.  vL 257 .Taxon classificationAnimaliaTrombidiformesTorrenticolidaeGu & Guo, 201957566E15-04FD-502A-B9BF-847E1350D36E30°6'12"N, 118°55'12"E, 440 m a.s.l.), female, collected by Xinyao Gu, 31-VII-2018, 3 ♀♀ (ZJ-TO-20180707 – ZJ-TO-20180709).Qingliangfeng National Nature Reserve, Lin’an, Zhejiang Province, P. R. China (n = 3)Female , W 473 (472–491), L/W ratio 1.6 (1.5–1.6). Dorsal plate 4+1 , Cx-II+III mL 37 (37–42); Gf L 132 (132–151), W 116 (115–118), L/W ratio 1.1 (1.1–1.3), distance between Gf and Ap 197 (197–217); Ap away from the line of primary sclerotization, on the same line with 1V and anterior to 2V , dL 117 (113–121); claw short L 23 (19–23), chelicera bs L 159 (159–172). dL of palp segments: P-1, 23 (20–23); P-2, 52 (43–52); P-3, 34 (34–38); P-4, 44 (43–44); P-5, 15 (15–19). dL of leg segments: I-L-1–6: 66 (60–66), 71 (71–81), 76 (67–76), 97 (90–98), 97 (94–97), 92 (88–94); II-L-1–6: 63 (58–63), 80 (72–85), 68 (60–76), 93 (88–93), 107 (96–107), 109 (107–115); III-L-1–6: 54 (54–58), 93 (87–93), 71 (71–74), 99 (97–109), 127 (114–127), 117 (112–127); IV-L-1–6: 102 (99–114), 94 (88–108), 115 (109–118), 145 (133–154), 154 (151–156), 147 (135–149).4+1 Fig. , dorsal  V2 Fig. . Gnathos V2 Fig.  vL 152 .