•A series of peptidomimetic aldehydes were discovered as EV71 3Cpro inhibitors.•Many inhibitors exhibited potent 3Cpro inhibitory and anti-EV71 activity in vitro.•Docking studies predicted the binding mode of some inhibitors bound to EV71 3Cpro.•5x (IC50 = 0.10 ± 0.02 μM, EC50 = 0.11 ± 0.07 μM) exhibited the best efficacy.A series of peptidomimetic aldehydes were designed, synthesized, and evaluated for their biochemical activity against 3C protease (3Cpro) and anti-enterovirus 71 (EV71) activity in vitro. Molecular docking revealed that 5s (IC50 = 0.22 ± 0.07 μM, EC50 = 0.18 ± 0.05 μM) could bind well to the active site of EV71 3Cpro, which was consistent with the biological data compared to reference 5a (IC50 = 0.54 ± 0.02 μM, EC50 = 0.26 ± 0.07 μM). Structure and relationship study led to the discovery of aldehyde 5x (IC50 = 0.10 ± 0.02 μM, EC50 = 0.11 ± 0.07 μM), which exhibited the most potent 3Cpro inhibitory and antiviral activity.

•Indole derivatives were discovered as HCV inhibitors.•SAR studies resulted in the most potent and the least cytotoxic compound (R) -10m.•R-enantiomer showed better anti-HCV activity compared with S-enantiomer.•NINS derivatives interfere with the viral entry step.In this study, a library of in-house small molecule was screened using a HCV cell-based assay and a compound (1) containing an N-protected indole scaffold (NINS) was identified as a novel anti-HCV inhibitor. Through structure activity relationship (SAR) study, it was observed that the racemic inhibitor (10m) displayed good anti-HCV activity (EC50 = 1.02 ± 0.10 μM) with the excellent selectivity index (SI = 45.56). Interestingly, R-enantiomer ((R)-10m) showed better anti-HCV activity and lower cytotoxicity than S-enantiomer ((S)-10m). (R)-10m gave the best anti-HCV potency (EC50 = 0.72 ± 0.09 μM) with the highest selectivity index (SI > 69.44). In addition, the mechanism of action study of NINS derivatives demonstrated that NINS derivatives interfere with the early step (viral entry) of the HCV life cycle.

α-Keto amide derivatives as enterovirus 71 (EV71) 3C protease (3Cpro) inhibitors have been synthesized and assayed for their biochemical and antiviral activities. structure–activity relationship (SAR) study indicated that small moieties were primarily tolerated at P1′ and the introduction of para-fluoro benzyl at P2 notably improved the potency of inhibitor. Inhibitors 8v, 8w and 8x exhibited satisfactory activity (IC50 = 1.32 ± 0.26 μM, 1.88 ± 0.35 μM and 1.52 ± 0.31 μM, respectively) and favorable CC50 values (CC50 > 100 μM). α-Keto amide may represent a good choice as a warhead for EV71 3Cpro inhibitor.

2′-α-C-Methyl-2′-β-C-fluorouridine and its phosphoramidate prodrugs were synthesized and evaluated for their inhibitory activity against HCV. The structure–activity relationship analysis of the phosphoramidate moiety found that 17m, 17q, and 17r exhibit potent activities against HCV, with EC50 values of 1.82 ± 0.19, 0.88 ± 0.12, and 2.24 ± 0.22 μM, respectively. The docking study revealed that the recognition of the 2′-β-F by Arg158, 3′-OH by N291, and the Watson–Crick pairing with the template allowed 23 to form the in-line conformation necessary for its incorporation into the viral RNA chain.Keywords: docking; hepatitis C virus; Hint 1; inhibitor; phosphoramidate prodrugs;

•Online SPE LC–MS/MS methods are developed for highly polar nucleoside drugs in plasma.•Methods are sensitive, accurate, reliable and high-throughput.•MCX cartridge is used for lamivudine, didanosine, entrictabine and zidovudine.•PBA cartridge packed by our lab for ribavirin and taribavirin.•Methods may provide a practical solution for clinical research.The bioanalysis and especially the sample preparation of nucleoside drugs in complex media, such as human plasma, has been challenging due to the high polarity and high solubility of these drugs in water. Online solid phase extraction (SPE) offers significant advantages, such as automation and timesaving. Thus, several types of SPE columns have been developed for compounds with different polarities. In this study, SPE was applied to overcome the issue of sample pretreatment of nucleoside drugs in human plasma, with the final aim of establishing a robust analytical platform for drugs with similar structures. A simple, easy-to-use, and efficient method is described for the simultaneous determination of lamivudine, zidovudine, didanosine and emtricitabine in human plasma via online SPE and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Following a simple centrifugation step, a 10 μL plasma sample was injected directly onto the HPLC system. The Oasis MCX cartridge was washed, and the analytes were removed by back-flushing directly onto the analytical column. The analytes were quantified using a triple-quadrupole tandem mass spectrometer in multiple-reaction monitoring mode. Similarly, with the development and application of a Bond Elut phenylboronic acid (PBA) SPE cartridge, a fully automated online SPE-HPLC-MS/MS method was established for the simultaneous determination of ribavirin and taribavirin in human plasma. Linear calibration curves were obtained over the range of 0.5–2000 ng mL−1, and the limit of quantification ranged from 0.5 ng mL−1 to 10 ng mL−1, which is sensitive enough for clinical drug monitoring. The intra- and inter-day precisions were in the range of 0.2–8.9%, and the trueness ranged between 88.9% and 113.1%. Excellent recoveries from plasma were achieved with a range between 86.7% and 105.1%. This procedure is easier to perform and requires less sample handling compared to methods previously described in the literature. This high-throughput method involving the direct injection of plasma samples may provide a practical solution for the analysis of multiple nucleoside drugs in clinical research. The method was tested in plasma samples from some patients and showed good performance.

A direct aldol reaction employing 2,4-thiazolidinediones as nucleophilic donors was performed to modify peptides and protein under mild conditions. Various functional groups could be readily introduced into protein without conformation change.

Cyanohydrin derivatives as enterovirus 71 (EV71) 3C protease (3Cpro) inhibitors have been synthesized and assayed for their biochemical and antiviral activities. Compared with the reported inhibitors, cyanohydrins (1S,2S,2′S,5S)-16 and (1R,2S,2′S,5S)-16 exhibited significantly improved activity and attractive selectivity profiles against other proteases, which were a result of the specific interactions between the cyanohydrin moiety and the catalytic site of 3Cpro. Cyanohydrin as an anchoring group with high selectivity and excellent inhibitory activity represents a useful choice for cysteine protease inhibitors.

The olanzapine–fluoxetine augmentation strategy has been proved efficacious for treatment-resistant depression, psychotic depression and bipolar depression. To achieve efficient therapeutic drug monitoring (TDM), we develop an automated online SPE-LC-MS/MS method for the simultaneous quantification of olanzapine, fluoxetine and norfluoxetine in human plasma. After adding an internal standard of diphenhydramine and centrifugation, a 10 μL plasma sample was directly injected into the SPE cartridge. While the analytes were retained on the SPE cartridge, the endogenous materials were washed out by the loading solvent. Following the valve switching, the analytes were eluted from the SPE cartridge to the analytical column by gradient elution. The analytes were quantified using a triple-quadrupole tandem mass spectrometer. Calibration curves were linear over the concentration range of 0.25–50.00 ng mL−1 for olanzapine, and 0.50–100.00 ng mL−1 for fluoxetine and norfluoxetine. The intra- and inter-day precisions were within 1.17% and 4.63%. And the accuracies were between 95.60% and 101.48%. The mean matrix effect was in the range of 90.35% to 99.89% and mean recovery was in the range of 90.35% to 96.99%. This method has been successfully applied to two Chinese schizophrenia patients. The online SPE-LC-MS/MS method allows sensitive and robust quantification of olanzapine, fluoxetine and norfluoxetine for routine TDM in clinic.

A simple, easy to use and efficient method was described for simultaneous determination of ten cardiovascular drugs with a broad range of physicochemical properties in rat plasma via online solid phase extraction (online SPE) and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Following a simple centrifugation step, a 10 μL aliquot of the plasma sample was injected directly onto the HPLC system. The LiChrospher® RP-18 ADS (25 mm × 4 mm, 25 μm, Merck) cartridge was washed with 10 mM ammonium acetate buffer (pH 9.5) for 1 min, after which time the analytes were removed by back-flushing directly onto the analytical column (Acclaim 120 C18 column, 150 mm × 4.6 mm, 5 μm) with gradient elution using acetonitrile–10 mM ammonium acetate buffer (pH 3.5) as mobile phase. The flow rate through both columns was 1 mL min−1, and the analytes were quantified using a triple-quadrupole tandem mass spectrometer in multiple-reaction monitoring mode. Linear calibration curves were obtained over the range of 0.2–100 ng mL−1, and the method limits ranged from 0.2 ng mL−1 to 1 ng mL−1 which is sensitive enough for clinical drug monitoring. The intra- and inter-day precisions were in the range of 0.20–2.32%, and the accuracies were between 93.33% and 114.60%. Excellent recoveries from plasma were achieved with a range from 83.52% to 107.38%. The procedure was easier to execute and required less sample handling than methods previously described in the literature. This easy to use and high-throughput method with direct injection of plasma samples for the analysis of multiple cardiovascular drugs may provide a practical solution for tailoring drug dosage in a rational manner to rapidly achieve optimal efficacy and safety of medication.

Captopril is a New Delhi metallo-β-lactamase-1 (NDM-1) inhibitor with an IC50 value of 7.9 μM. It is composed of two units: a 3-mercapto-2-methylpropanoyl fragment and a proline residue. In this study, we synthesized simple amide derivatives of 3-mercapto-2-methylpropanoic acid, and then tested them as NDM-1 inhibitors in order to identify the pharmacophore for NDM-1 inhibition. We found that the lead compound 22 had an IC50 value of 1.0 μM. Further structure simplification provided compounds 31 and 32, which had IC50 values of 15 and 10 μM, respectively. As compound 32 is a clinically used antidote for metal poisoning, it has great potential to be repurposed to treat bacterial infections.Captopril is a New Delhi metallo-β-lactamase-1 (NDM-1) inhibitor with an IC50 value of 7.9 μM. It is composed of two units: a 3-mercapto-2-methylpropanoyl fragment and a proline residue. In this study, we synthesized simple amide derivatives of 3-mercapto-2-methylpropanoic acid, and then tested them as NDM-1 inhibitors in order to identify the pharmacophore for NDM-1 inhibition. We found that the lead compound 22 had an IC50 value of 1.0 μM. Further structure simplification provided compounds 31 and 32, which had IC50 values of 15 and 10 μM, respectively. As compound 32 is a clinically used antidote for metal poisoning, it has great potential to be repurposed to treat bacterial infections.

Response surface methodology (RSM) was utilized for rapid and systematic optimization of on-line solid-phase extraction (SPE) parameters to maximize the response and separation of WM-5. The optimization was performed with Box–Behnken designs. Four major parameters were investigated for their contributions to the response and separation of WM-5, with a total of 29 experiments being performed for each instrument, respectively. Quantitative determination of WM-5 in mouse plasma was performed to evaluate the statistical significance of the parameters on chromatographic response. A fully automated on-line SPE and high-performance liquid chromatography (HPLC) with diode array detection (DAD) method was developed for the determination of WM-5 in mouse plasma. Calibration curve with good linearity (r = 0.9989) was obtained in the range of 20–4000 ng/mL in mouse plasma. The limit of detection (LOD) and lower limit of quantification (LLOQ) of the assay were 6 ng/mL and 20 ng/mL, respectively. The overall intra-day and the inter-day variations were less than 1.90%. The recovery of the method was in the range of 93.74–96.33% with RSD less than 3.06%. The optimized method demonstrated good performance in terms of specificity, LLOQ, linearity, recovery, precision and accuracy, and was successfully applied to quantify WM-5 in mouse plasma to support the pharmacokinetic study.Highlights► Fully automated on-line SPE–HPLC utilized for PK evaluation of WM-5. ► Response surface methodology used for optimization of on-line SPE parameters. ► On-line SPE removed the time-consuming, tedious and costly manual process. ► The method is simple, fast, specific and sensitive.

A fully automated on-line solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) with diode array detection (DAD) method was developed for determination of bavachinin in mouse plasma. Analytical process was performed on two reversed-phase columns (SPE cartridge and analytical column) connected via a Valco 6-port switching valve. Plasma samples (10 μL) were injected directly onto a C18 SPE cartridge (MF Ph-1 C18, 10 mm × 4 mm, 5 μm) and the biological matrix was washed out for 2 min with the loading solvent (5 mM NaH2PO4 buffer, pH 3.5) at a flow rate of 1 mL/min. By rotation of the switching valve, bavachinin was eluted from the SPE cartridge in the back-flush mode and transferred to the analytical column (Venusil MP C18, 4.6 mm × 150 mm, 5 μm) by the chromatographic mobile phase consisted of acetonitrile-5 mM NaH2PO4 buffer 65/35 (v/v, pH 3.5) at a flow rate of 1 mL/min. The complete cycle of the on-line SPE purification and chromatographic separation of the analyte was 13 min with UV detection performed at 236 nm. Calibration curve with good linearity (r = 0.9997) was obtained in the range of 20–4000 ng/mL in mouse plasma. The intra-day and inter-day precisions (RSD) of bavachinin were in the range of 0.20–2.32% and the accuracies were between 98.47% and 102.95%. The lower limit of quantification (LLOQ) of the assay was 20 ng/mL. In conclusion, the established automated on-line SPE-HPLC-DAD method demonstrated good performance in terms of linearity, specificity, detection and quantification limits, precision and accuracy, and was successfully utilized to quantify bavachinin in mouse plasma to support the pharmacokinetic (PK) studies. The PK properties of bavachinin were characterized as rapid oral absorption, high clearance, and poor absolute bioavailability.Highlights► Fully automated on-line SPE-HPLC utilized for PK evaluation of bavachinin. ► On-line SPE removed the time-consuming, tedious and costly manual process. ► The method is simple, fast, specific and sensitive.