Nicotinic acetylcholine receptor antagonists alter the function and expression of serine racemase in PC-12 and 1321N1 cells

Western blot analysis demonstrated that PC-12 cells express monomeric and dimeric forms of serine racemase (m-SR, d-SR) and that 1321N1 cells express m-SR. Quantitative RT-PCR and functional studies demonstrated that PC-12 cells express homomeric and heteromeric forms of nicotinic acetylcholine receptors (nAChR) while 1321N1 cells primarily express the α₇-nAChR subtype. The effect of nAChR agonists and antagonists on SR activity and expression was examined by following concentration-dependent changes in intracellular d-Ser levels and SR protein expression. Incubation with (S)-nicotine increased d-Ser levels, which were attenuated by the α₇-nAChR antagonist methyllycaconitine (MLA). Treatment of PC-12 cells with mecamylamine (MEC) produced a bimodal reduction of d-Ser reflecting MEC inhibition of homomeric and heteromeric nAChRs, while a unimodal curve was observed with 1321N1 cells, reflecting predominant expression of α₇-nAChR. The nAChR subtype selectivity was probed using α₇-nAChR selective inhibitors MLA and (R,S)-dehydronorketamine and α₃β₄-nAChR specific inhibitor AT-1001. The compounds reduced d-Ser in PC-12 cells, but only MLA and (R,S)-dehydronorketamine were effective in 1321N1 cells. Incubation of PC-12 and 1321N1 cells with (S)-nicotine, MEC and AT-1001 did not affect m-SR or d-SR expression, while MLA and (R,S)-dehydronorketamine increased m-SR expression but not SR mRNA levels. Treatment with cycloheximide indicated that increased m-SR was due to de novo protein synthesis associated with phospho-active forms of ERK1/2, MARCKS, Akt and rapamycin-sensitive mTOR. This effect was attenuated by treatment with the pharmacological inhibitors U0126, LY294002 and rapamycin, which selectively block the activation of ERK1/2, Akt and mTOR, respectively, and siRNAs directed against ERK1/2, Akt and mTOR. We propose that nAChR-associated changes in Ca^2 + flux affect SR activity, but not expression, and that MLA and (R,S)-dehydronorketamine bind to allosteric sites on the α₇-nAChR and promote multiple signaling cascades that converge at mTOR to increase m-SR levels.     

Patent Reports

We synthesized a hydroquinone glucoside (HG) as a potential skin-whitening agent using Leuconostoc mesenteroides (B-1299CB BF563) dextransucrase with hydroquinone (HQ) as an acceptor and sucrose as a donor. The product was purified using butanol partitioning and silica gel column chromatography. The structure of the purified HG was determined by nuclear magnetic resonance and the ionic product was observed at m/z 295 (C12, H16, O7 Na)⁺. HG was identified as 4-hydroxyphenyl-α-d-glucopyranoside. The optimum condition of HG synthesis, determined using a response surface methodology, was 450 mM HQ, 215 mM sucrose, and 0.55 U/mL dextransucrase; the final HG produced was 544 mg/L. The IC₅₀ of diphenylpicryl-hydrazyl scavenging activity was 3.85 mM indicating a higher antioxidant activity compared to β-arbutin (IC₅₀ = 6.04 mM). HG-mediated inhibition of lipid peroxidation was 3.51% that of HQ (100%) and much higher than that of β-arbutin (0.81% of HQ). In addition the IC₅₀ value of nitrite-scavenging activity was 14.76 mM showing a superior scavenging activity to that of β-arbutin (IC₅₀ = 27.09 mM).     

Study on alanine aminotransferase kinetics by microchip electrophoresis

Alanine aminotransferase (ALT), which catalyzes the reversible conversion between l-glutamic acid (l-Glu) and l-alanine (l-Ala), is one of the most active aminotransferases in the clinical diagnosis of liver diseases. This work displays a microanalytical method for evaluating ALT enzyme kinetics using a microchip electrophoresis laser-induced fluorescence system. Four groups of amino acid (AA) mixtures, including the substrates of ALT (l-Glu and l-Ala), were effectively separated. Under the optimized conditions, the quantitative analysis of l-Glu and l-Ala was conducted and limits of detection (signal/noise = 3) for l-Glu and l-Ala were 4.0 × 10^?7 and 2.0 × 10^?7 M, respectively. In the reaction catalyzed by ALT, enzyme kinetic constants were determined for both the forward and reverse reactions by monitoring the concentration decrease of substrate AAs (l-Ala and l-Glu), and the K_m and V_max values were 10.12 mM and 0.48 mM/min for forward reaction and 3.22 mM and 0.22 mM/min for reverse reaction, respectively. Furthermore, the applicability of this assay was assessed by analysis of real serum samples. The results demonstrated that the proposed method could be used for kinetic study of ALT and shows great potential in the real application.     

The kinetics and regulation of phosphofructokinase from Teladorsagia circumcincta

Phosphofructokinase (PFK-1) activity was examined in L₃ and adult Teladorsagia circumcincta, both of which exhibit oxygen consumption. Although activities were higher in the adult stage, the kinetic properties of the enzyme were similar in both life cycle stages. T. circumcincta PFK-1 was subject to allosteric inhibition by high ATP concentration, which increased both the Hill coefficient (from 1.4 ± 0.2 to 1.7 ± 0.2 in L₃s and 2.0 ± 0.3 to 2.4 ± 0.4 in adults) and the K_½ for fructose 6 phosphate (from 0.35 ± 0.02 to 0.75 ± 0.05 mM in L₃s and 0.40 ± 0.03 to 0.65 ± 0.05 mM in adults). The inhibitory effects of high ATP concentration could be reversed by fructose 2,6 bisphosphate and AMP, but glucose 1,6 bisphosphate had no effect on activity. Similarly, phosphoenolpyruvate had no effect on activity, while citrate, isocitrate and malate exerted mild inhibitory effects, but only at concentrations exceeding 2 mM. The observed kinetic properties for T. circumcincta PFK-1 were very similar to those reported for purified Ascaris suum PFK-1, though slight differences in sensitivity to ATP concentration suggests there may be subtle variations at the active site. These results are consistent with the conservation of properties of PFK-1 amongst nematode species, despite between species variation in the ability to utilise oxygen.     

Analytical measurement of discrete hydrogen sulfide pools in biological specimens

Hydrogen sulfide (H₂S) is a ubiquitous gaseous signaling molecule that plays a vital role in numerous cellular functions and has become the focus of many research endeavors, including pharmacotherapeutic manipulation. Among the challenges facing the field is the accurate measurement of biologically active H₂S. We have recently reported that the typically used methylene blue method and its associated results are invalid and do not measure bona fide H₂S. The complexity of analytical H₂S measurement reflects the fact that hydrogen sulfide is a volatile gas and exists in the body in various forms, including a free form, an acid-labile pool, and bound as sulfane sulfur. Here we describe a new protocol to discretely measure specific H₂S pools using the monobromobimane method coupled with RP-HPLC. This new protocol involves selective liberation, trapping, and derivatization of H₂S. Acid-labile H₂S is released by incubating the sample in an acidic solution (pH 2.6) of 100 mM phosphate buffer with 0.1 mM diethylenetriaminepentaacetic acid (DTPA), in an enclosed system to contain volatilized H₂S. Volatilized H₂S is then trapped in 100 mM Tris–HCl (pH 9.5, 0.1 mM DTPA) and then reacted with excess monobromobimane. In a separate aliquot, the contribution of the bound sulfane sulfur pool was measured by incubating the sample with 1 mM TCEP (tris(2-carboxyethyl)phosphine hydrochloride), a reducing agent, to reduce disulfide bonds, in 100 mM phosphate buffer (pH 2.6, 0.1 mM DTPA), and H₂S measurement was performed in a manner analogous to the one described above. The acid-labile pool was determined by subtracting the free hydrogen sulfide value from the value obtained by the acid-liberation protocol. The bound sulfane sulfur pool was determined by subtracting the H₂S measurement from the acid-liberation protocol alone compared to that of TCEP plus acidic conditions. In summary, our new method allows very sensitive and accurate measurement of the three primary biological pools of H₂S, including free, acid-labile, and bound sulfane sulfur, in various biological specimens.     

Synthesis,and docking studies of phenylpyrimidine-carboxamide derivatives bearing 1H-pyrrolo[2,3-b]pyridine moiety as c-Met inhibitors

Four series of phenylpyrimidine-carboxamide derivatives bearing 1H-pyrrolo[2,3-b]pyridine moiety (14a–e, 15a–g, 16a–e and 17a–g) were designed, synthesized and evaluated for the IC₅₀ values against three cancer cell lines (A549, PC-3 and MCF-7). Four selected compounds (15e, 16a–b and 17a) were further evaluated for the activity against c-Met kinase, HepG2 and Hela cell lines. Most of the compounds showed excellent cytotoxicity activity and selectivity with the IC₅₀ valuables in single-digit μM to nanomole range. Eleven of them are equal to more active than positive control Foretinib against one or more cell lines. The most promising compound 15e showed superior activity to Foretinib against A549, PC-3 and MCF-7 cell lines, with the IC₅₀ values of 0.14 ± 0.08 μM, 0.24 ± 0.07 μM and 0.02 ± 0.01 μM, which were 4.6, 1.6 and 473.5 times more active than Foretinib (0.64 ± 0.26 μM, 0.39 ± 0.11 μM, 9.47 ± 0.22 μM), respectively. Structure–activity relationships (SARs) and docking studies indicated that the replacement of phenylpicolinamide scaffold with phenylpyrimidine fragment of the target compounds was benefit for the activity. What’s more, the introduction of fluoro atom to the aminophenoxy part played no significant impact on the activity and any substituent group on aryl group is unfavourable for the activity.     

Design,synthesis, and docking studies of phenylpicolinamide derivatives bearing 1H-pyrrolo[2,3-b]pyridine moiety as c-Met inhibitors

Four series of phenylpicolinamide derivatives bearing 1H-pyrrolo[2,3-b]pyridine moiety (12a–e, 13a–f, 14a–f and 15a–i) were designed, synthesized and evaluated for the IC₅₀ values against three cancer cell lines (A549, PC-3 and MCF-7) and c-Met kinase. Five selected compounds (13b, 15b, 15d, 15e and 15f) were further evaluated for the activity against HepG2 and Hela cell lines. Eighteen of the compounds showed excellent cytotoxicity activity and selectivity with the IC₅₀ valuables in single-digit μM to nanomole range. Seven of them are equal to more active than positive control Foretinib against one or more cell lines. The most promising compound 15f showed superior activity to Foretinib, with the IC₅₀ values of 1.04 ± 0.11 μM, 0.02 ± 0.01 μM and 9.11 ± 0.55 μM against A549, PC-3 and MCF-7 cell lines, which were 0.62 to 19.5 times more active than Foretinib (IC₅₀ values: 0.64 ± 0.26 μM, 0.39 ± 0.11 μM, 9.47 ± 0.22 μM), respectively. Structure–activity relationships (SARs) and docking studies indicated that replacement of quinoline nucleus of the previous active compounds with 1H-pyrrolo[2,3-b]pyridine moiety maintained even improved the potent cytotoxic activity. The results suggested that the introduction of fluoro atoms to the aminophenoxy part of target compounds or the phenyl group of pyrimidine substituted on C-4 position was benefit for the activity.     

Online immobilized enzyme microreactor for the glucose oxidase enzymolysis and enzyme inhibition assay

An online immobilized glucose oxidase (GOx) capillary microreactor was developed based on an enzymatic redox reaction with 1,4-benzoquinone as an acceptor of electrons, replacing the molecular oxygen typically used in a GOx reaction to achieve direct ultraviolet detection without derivation. A high efficiency of enzymolysis was obtained at 1 mg ml^?1 1,4-benzoquinone for 5 min of incubation at 25 °C, and baseline separation of the substrate and product could be achieved with a resolution of 3.85 by employing 20 mM phosphate buffer (pH 8.0) containing 40 mg ml^?1 sulfated β-cyclodextrin as an additive, a constant voltage of 15 kV, and a detection wavelength of 220 nm. In addition, an online enzyme inhibition study was performed on the immobilized GOx microreactor with metal ions Ag⁺ and Cu²⁺ used as model inhibitors. The results indicate that Ag⁺ (IC₅₀ = 69.16 μM) has a markedly higher inhibitory effect than Cu²⁺ (IC₅₀ = 1.33 mM). The protocol described can be applied in high-throughput screening of enzyme reactions and inhibitors.     

Design, synthesis and inhibitory effect of pentapeptidyl chloromethyl ketones on proteinase K

The synthesis and proteolytic inhibitor function of new modified pentapeptide MeOSuc-AAAPF-CH₂Cl 6 is described. The efficacy of 6 in inhibiting the proteolytic activity of proteinase K at a concentration of 0.10 mM allows a signal to be obtained for an exogenous target (‘Xeno RNA’) at 29 PCR cycles (i.e., Ct = 29), whereas the control MeOSuc-AAAPV-CH₂Cl 1 requires a 7.5-fold higher concentration (0.75 mM) to produce the same Ct.     

Dual role of imidazole as activator/inhibitor of sweet almond (Prunus dulcis) β-glucosidase

The activity of Prunus dulcis (sweet almond) β-glucosidase at the expense of p-nitrophenyl-β-d-glucopyranoside at pH 6 was determined, both under steady-state and pre-steady-state conditions. Using crude enzyme preparations, competitive inhibition by 1–5 mM imidazole was observed under both kinetic conditions tested. However, when imidazole was added to reaction mixtures at 0.125–0.250 mM, we detected a significant enzyme activation. To further inspect this effect exerted by imidazole, β-glucosidase was purified to homogeneity. Two enzyme isoforms were isolated, i.e. a full-length monomer, and a dimer containing a full-length and a truncated subunit. Dimeric β-glucosidase was found to perform much better than the monomeric enzyme, independently of the kinetic conditions used to assay enzyme activity. In addition, the sensitivity towards imidazole was found to differ between the two isoforms. While monomeric enzyme was indeed found to be relatively insensitive to imidazole, dimeric β-glucosidase was observed to be significantly activated by 0.125–0.250 mM imidazole under pre-steady-state conditions. Further, steady-state assays revealed that the addition of 0.125 mM imidazole to reaction mixtures increases the K_m of dimeric enzyme from 2.3 to 6.7 mM. The activation of β-glucosidase dimer by imidazole is proposed to be exerted via a conformational transition poising the enzyme towards proficient catalysis.     

Measurement of plasma hydrogen sulfide in vivo and in vitro

The gasotransmitter hydrogen sulfide is known to regulate multiple cellular functions during normal and pathophysiological states. However, a paucity of concise information exists regarding quantitative amounts of hydrogen sulfide involved in physiological and pathological responses. This is primarily due to disagreement among various methods employed to measure free hydrogen sulfide. In this article, we describe a very sensitive method of measuring the presence of H₂S in plasma down to nanomolar levels, using monobromobimane (MBB). The current standard assay using methylene blue provides erroneous results that do not actually measure H₂S. The method presented herein involves derivatization of sulfide with excess MBB in 100 mM Tris–HCl buffer (pH 9.5, 0.1 mM DTPA) for 30 min in 1% oxygen at room temperature. The fluorescent product sulfide-dibimane (SDB) is analyzed by RP-HPLC using an eclipse XDB-C18 (4.6 × 250 mm) column with gradient elution by 0.1% (v/v) trifluoroacetic acid in acetonitrile. The limit of detection for sulfide-dibimane is 2 nM and the SDB product is very stable over time, allowing batch storage and analysis. In summary, our MBB method is suitable for sensitive quantitative measurement of free hydrogen sulfide in multiple biological samples such as plasma, tissue and cell culture lysates, or media.     

Synthesis and biological evaluation of novel C6-amino substituted 4-azasteroidal purine nucleoside analogues

Novel C6-amino substituted purine nucleoside analogues (2–12) bearing a modified pyranose-like D ring of the 4-azasteroid moiety were efficiently synthesized through nucleophilic substitution at C6 position of the steroidal nucleoside precursors (1a, b) with versatile amines. All the synthesized new compounds were evaluated for their anticancer activity in vitro against Hela, PC-3 and MCF-7 cell lines. Among them, compounds 4b, 7b and 9b exhibited significant cytotoxicity with the IC₅₀ values of 2.99 μM (PC-3), 2.84 μM, (PC-3) and 2.69 μM (Hela), respectively.     

A novel disintegrin protein from Naja naja venom induces cytotoxicity and apoptosis in human cancer cell lines in vitro

Animal venoms and toxins are potential bioresources that have been known to mankind as a therapeutic tool for more than a century through folk and traditional medicine. The purified “disintegrin protein” (64 kDa) from the venom of the Indian cobra snake (Naja naja) exhibited cytotoxic effects of various types of human cancer cell lines such as breast cancer (MCF-7), lung cancer (A549) and liver cancer (HepG2). In vitro cytotoxicity, DNA fragmentation, an apoptotic assay and a cell cycle analysis were performed to evaluate the anticancer activity of disintegrin against the above cell lines. The IC₅₀ value of disintegrin was determined to be 2.5 ± 0.5 μg/mL, 3.5 ± 0.5 μg/mL, and 3 ± 0.5 μg/mL for the MCF-7, A549 and HepG2 cell lines respectively. Moreover, the increased distribution of G0/G1 and S phase led to decreased populations of cells in the G2/M phase of MCF-7, HepG2 and A549 cells.     

Capillary electrophoresis separation and matrix-assisted laser desorption/ionization mass spectrometry characterization of bovine serum albumin–fluorescein isothiocyanate conjugates

A protocol using enzymatic digestion, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and capillary electrophoresis with laser induced fluorescence detection (CE-LIF) for the investigation of the binding of the fluorescent contact allergen fluorescein isothiocyanate (FITC) to the 66 kDa large protein bovine serum albumin (BSA), as a model system for protein–hapten binding in the skin, is presented. Mass spectra of BSA–FITC digestions, using trypsin and chymotrypsin, respectively, provided sequence coverage of 97%. To investigate the number of FITC-bound peptides using CE-LIF separation, three different buffer salts at four different pH levels were evaluated. The use of 20 mM sodium citrate pH 6.5 as well as 20 mM sodium phosphate pH 6.5 or pH 7.5 as background electrolyte revealed high numbers of peptides with at least one bound FITC. The effect of the electrolyte counter ion on MALDI-MS was investigated and was found to have effect on the MALDI spectra signal-to-noise (S/N) at 50 mM but not at 10 mM. Of the 60 theoretical FITC-binding sites in BSA this MALDI-MS protocol presents 30 defined, 28 possible and 2 non-binding sites for FITC.     

The effect of cysteine and glutathione on sperm and oxidative stress parameters of post-thawed bull semen

The aim of this study was to determine the effects of antioxidants such as reduced glutathione (GSH) and cysteine in Laiciphose® extender on semen parameters, fertilizing ability, lipid peroxidation (LPO) level and glutathione peroxidise (GPx) activity of post-thawed bull semen. Totally 54 ejaculates of three bulls were used in the study. Five groups, namely; GSH (0.5 and 2 mM), cysteine (5 and 10 mM) and control group, were conducted to test the antioxidants in Laiciphose®. Insemination doses were processed that each 0.25-mL straw contained 15 × 10⁶ sperm. The addition of antioxidants did not present any significant effect on the percentages of post-thaw sperm morphology (acrosome and total abnormalities), subjective, CASA and progressive motilities, as well as sperm motility characteristics (VAP, VSL, VCL, LIN and ALH), compared to the control groups (P > 0.05). GSH 0.5 mM (55.5 ± 7.38%) and cysteine 10 mM (48 ± 5.65%) led to lower rates of DNA damage, compared to control (P < 0.05). As regards to MDA level, cysteine at 10 mM dose gave the highest level (4.99 ± 0.44 nmol/L) (P < 0.001). GPx activity was demonstrated to be higher level upon the addition of 5 mM cysteine when compared to the other groups (P < 0.05). With respect to fertility results based on 60-day non-returns, the supplementation of antioxidants did not present significant differences (P > 0.05). The results of this study may provide an useful information for the future studies in this area. So, further studies could be suggested to achieve better information in terms of the DNA damage and fertilizing capacity of bull sperm frozen with effective antioxidants.     

The increase in rat ventricular automaticity induced by salbutamol is mediated through β(1)- but not β(2)-adrenoceptors: role of phosphodiesterases

AimsWhile β₂-adrenoceptor (AR) agonists are useful bronchodilators, they also produce cardiac arrhythmias. These agents are not fully selective and also activate β₁-AR, but the involvement of β₁-AR and β₂-AR in the observed pro-arrhythmic effect has not been established. We studied the effect of β₁-AR and β₂-AR activation on ventricular automaticity and the role of phosphodiesterases (PDE) in regulating this effect.Main methodsExperiments were performed in the spontaneously beating isolated right ventricle of the rat heart. We also measured cAMP production in this tissue.Key findingsThe β₂-AR agonist salbutamol (1-100 μM) produced a concentration-dependent increase in ventricular automaticity that was not affected by 50 nM of the β₂-AR antagonist ICI 118551. This effect was enhanced by the non-selective PDE inhibitor theophylline (100 μM) and by the selective PDE4 inhibitors rolipram (1 μM) and Ro 201724 (2 μM), but not modified by the selective PDE3 inhibitors cilostamide (0.3 μM) or milrinone (0.2 μM). The effects of salbutamol alone and in the presence of either theophylline or rolipram were virtually abolished by 0.1 μM β₁-AR antagonist CGP20712A. Salbutamol (10 μM) increased the cAMP concentration, and this effect was abolished by CGP 20712A (0.1 μM) but enhanced by theophylline (100 μM) or rolipram (1 μM). Cilostamide (0.3 μM) failed to modify the effect of salbutamol on cAMP concentration.SignificanceThese results indicate that the increase of ventricular automaticity elicited by salbutamol was exclusively mediated through β₁-AR and enhanced by non-selective PDE inhibition with theophylline or selective PDE4 inhibition. However, PDE3 did not appear to regulate this effect.     

μ1- and 5-HT1-dependent mechanisms in the anterior pretectal nucleus mediate the antinociceptive effects of retrosplenial cortex stimulation in rats

AimThis study examines if injection of cobalt chloride (CoCl₂) or antagonists of muscarinic cholinergic (atropine), μ₁-opioid (naloxonazine) or 5-HT₁ serotonergic (methiothepin) receptors into the dorsal or ventral portions of the anterior pretectal nucleus (APtN) alters the antinociceptive effects of stimulating the retrosplenial cortex (RSC) in rats.Main methodChanges in the nociceptive threshold were evaluated using the tail flick or incision pain tests in rats that were electrically stimulated at the RSC after the injection of saline, CoCl₂ (1 mM, 0.10 μL) or antagonists into the dorsal or ventral APtN.Key findingsThe injection of CoCl₂, naloxonazine (5 μg/0.10 μL) or methiothepin (3 μg/0.10 μL) into the dorsal APtN reduced the stimulation-produced antinociception from the RSC in the rat tail flick test. Reduction of incision pain was observed following stimulation of the RSC after the injection of the same substances into the ventral APtN. The injection of atropine (10 ng/0.10 μL) or ketanserine (5 μg/0.10 μL) into the dorsal or ventral APtN was ineffective against the antinociception resulting from RSC stimulation.Significanceμ₁-opioid- and 5-HT₁-expressing neurons and cell processes in dorsal and ventral APtN are both implicated in the mediation of stimulation-produced antinociception from the RSC in the rat tail flick and incision pain tests, respectively.     

Bisphenol A inhibits compound action potentials in the frog sciatic nerve in a manner independent of estrogen receptors

Although the endocrine disruptor bisphenol A (BPA) is reported to inhibit nerve conduction, the underlying mechanisms are unclear. Therefore, in the present study, we examined the effect of BPA on compound action potentials (CAPs) recorded from the frog sciatic nerve using the air-gap method. Treatment of the sciatic nerve with BPA (0.5 mM) for 20 min reduced the peak amplitude of the CAP by approximately 60% in a partially reversible manner. The reduction in the CAP peak amplitude was concentration-dependent, with a half-maximal inhibitory concentration (IC₅₀) value of 0.31 mM. This effect of BPA was unaffected by an estrogen-receptor antagonist, 4-hydroxytamoxifen, which by itself reduced CAP peak amplitude, with an IC₅₀ value of 0.26 mM (comparable to that of BPA). The natural estrogen 17β-estradiol, at the highest dissolvable concentration (0.05 mM), had an effect similar to that of BPA. The IC₅₀ value of BPA was comparable to those of some local anesthetics in inhibiting frog CAPs. Our findings suggest that BPA inhibits nerve conduction in a manner independent of estrogen receptors. This action of BPA may underlie, at least in part, the neurotoxicity of the compound.     

Anti-HBV agents. Part 2: Synthesis and in vitro anti-hepatitis B virus activities of alisol A derivatives

Chemical modifications were performed on hydroxyl groups at C-11,23,24,25 positions and C-13(17) double bond of alisol A for structure–activity relationship study. Forty-one derivatives of alisol A were synthesized and assayed for their in vitro anti-hepatitis B virus (HBV) activities and cytotoxicities. Of them, 14 compounds were active against HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) secretion in HepG 2.2.15 cells, and the most promising compound 25 exhibited high activities against secretion of HBsAg (IC₅₀ = 0.028 mM), HBeAg (IC₅₀ = 0.027 mM) and remarkable selective indices (SI_HBsAg >90, SI_HBeAg >93).