Synthesis and properties of 3′-azido-3′-deoxythymidine derivatives of glycerolipids

A promising approach to enhancing the bioavailability of hydrophilic therapeutic agents including anti-HIV nucleosides is designing their pseudotriglyceride derivatives that may mimic natural lipids and take advantage of their metabolic pathways resulting in improved delivery to target cells. The synthesis of a series of new 1,3-diglycerides and AZT conjugates employing various linkage types between the pharmacophore residue and the spacer part of the hydrophobic moiety (ester or phosphodiester) is described. The hydrolytic properties of the synthesized liponucleosides (in buffer solutions and under the action of pancreatic lipase) have been studied. Liposomes based on these AZT-containing prodrugs have been obtained.     

Exploration of 3-Aminoazetidines as Triple Reuptake Inhibitors by Bioisosteric Modification of 3-α-Oxyazetidine

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Cross immunoreactivity of monoclonal antibody 3F3 to Torpedo acetylcholinesterase

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Binding studies and quantitative structure-activity relationship of 3-amino-1H-indazoles as inhibitors of GSK3β

Docking of 3-amino-1H-indazoles complexed with glycogen synthase kinase 3 beta (GSK3β) was performed to gain insight into the structural requirements and preferred conformations of these inhibitors. The study was conducted on a selected set of 57 compounds with variation in structure and activity. We found that the most active compounds established three hydrogen bonds with the residues of the hinge region of GSK3β, but some of the less active compounds have other binding modes. In addition, models able to predict GSK3β inhibitory activities (IC(50) ) of the studied compounds were obtained by 3D-QSAR methods CoMFA and CoMSIA. Ligand-based and receptor-guided alignment methods were utilized. Adequate R(2) and Q(2) values were obtained by each method, although some striking differences existed between the obtained contour maps. Each of the predictive models exhibited a similar ability to predict the activity of a test set. The application of docking and quantitative structure-activity relationship together allowed conclusions to be drawn for the choice of suitable GSK3β inhibitors.     

Synthesis and pharmacology of 3-hydroxy-Δ-isoxazoline-cyclopentane analogues of glutamic acid

The synthesis and pharmacology of two potential glutamic acid receptor ligands are described. Preparation of the bicyclic 3-hydroxy-Δ²-isoxazoline-cyclopentane derivatives (±)-7 and (±)-8 was accomplished via 1,3-dipolar cycloaddition of bromonitrile oxide to suitably protected 1-amino-cyclopent-3-enecarboxylic acids. Their structure was established using a combination of ¹H NMR spectroscopy and molecular mechanics calculations carried out on the intermediate cycloadducts (±)-11 and (±)-12. Amino acid derivatives (±)-7 and (±)-8 were assayed at ionotropic and metabotropic glutamic acid receptor subtypes and their activity compared with that of trans-ACPD and cis-ACPD. The results show that the replacement of the ω-carboxylic group of the model compounds with the 3-hydroxy-Δ²-isoxazoline moiety abolishes or reduces drastically the activity at the metabotropic glutamate receptors. Conversely, on passing from cis-ACPD to derivative (±)-8, the agonist activity at NMDA receptors is almost unaffected.     

Docking-based 3D-QSAR modeling of the inhibitors of IMP metallo-β-lactamase

IMP metallo-β-lactamases (MBLs) confer broad-spectrum resistance to β-lactam antibiotics such as imipenem and escape the action of almost all clinically used β-lactamase inhibitors. We conducted three-dimensional quantitative structure–activity relationships (3D-QSAR) for a series of IMP-1 MBL inhibitors with the aid of docking-based alignment. While the 3D-QSAR models were created based on a training set of 41 compounds, their external predictivity was validated using a test set of eight compounds. The study has resulted in two types of satisfactory 3D-QSAR models for predicting the biological activity of new compounds: CoMFA model (r ² = 0.989; $ r_{\text{pre}}^{ 2} = 0. 8 4 3 $ ) and CoMSIA model (r ² = 0.968; $ r_{\text{pre}}^{ 2} = 0. 9 5 7 $ ). Our work will facilitate the design and optimization of new potential inhibitors.     

Binding of 3H-clonidine to an α-adrenoceptor in membranes of guinea-pig ileum

Summary The binding of ³H-clonidine to membrane particles from guinea-pig ileum was investigated. The specific binding, i.e. the binding that could be inhibited by high concentrations of unlabeled clonidine or noradrenaline, was of high affinity, K _D3 nM. The number of sites was approximately 25 fmol/mg protein. Rate constants of association and dissociation were 5.3×10⁷ M^–1 min^–1 and 0.18 min^–1, respectively. Affinites of various drugs to the binding site were determined by measuring their effect on the binding of ³H-clonidine. The affinity of adrenergic agonists decreased in the order clonidine = tramazoline > (–)-erythro--methylnoradrenaline > (–)-noradrenaline (–)-phenylephrine. (–)-Noradrenaline had about 20 times more affinity than the (+)-isomer. The affinity of -adrenoceptor antagonists decreased in the order phentolamine > rauwolscine = yohimbine > WB 4101 > pseudoyohimbine > prazosin = corynanthine. Yohimbine and rauwolscine had about 100 times more affinity than their stereoisomer corynanthine. Serotonin 10 M and metiamide 10 M did not affect the binding, and propranolol inhibited it only at high concentrations. — The results indicate that ³H-clonidine labels an ₂-adrenoceptor in guinea-pig ileum. The orders of affinity of -adrenoceptor agonists and antagonists agree well with their orders of potency in functional tests, namely as modulators of cholinergic transmission in the guinea-pig ileum and as modulators of noradrenaline release in the rabbit pulmonary artery. An -adrenoceptor should be classified as ₂ when the affinities of clonidine, tramazoline and -methylnoradrenaline greatly exceed the affinity of phenylephrine, and when the affinities of rauwolscine and yohimbine exceed those of prazosin and corynanthine.     

Dexamethasone inhibits the formation of multinucleated osteoclastsvia down-regulation of β3 integrin expression

Although glucocorticoids are known to affect osteoclast differentiation and function, there have been conflicting reports about the effect of glucocorticoids on osteoclast formation, leading to the assumption that microenvironment and cell type influence their action. We explored the effect of the synthetic glucocorticoid analog dexamethasone on the formation of osteoclasts. Dexamethasone inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts without affecting the formation of TRAP-positive mononuclear cells in a coculture of mouse osteoblasts and bone marrow cells. Dexamethasone did not inhibit mRNA expression levels of the receptor activator of nuclear factor-kappaB ligand and osteoprotegerin, the essential regulators of osteoclastogenesis. Dexamethasone down-regulated the expression of beta3 integrin mRNA and protein but did not alter expression of other osteoclast differentiation marker genes. Both dexamethasone and echistatin, a beta3 integrin function blocker, inhibited TRAP-positive multinucleated osteoclast formation but not TRAP-positive mononuclear cell formation. These results suggest that dexamethasone inhibits the formation of multinucleated osteoclasts, at least in part, through the down-regulation of beta3 integrin, which plays an important role in the formation of multinucleated osteoclasts.     

Investigation of Cardiovascular Effects of Tetrahydro-β-carboline sstr3 antagonists

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A synergetic role of 1,25-dihydroxyvitamin D(3) in 17β-estradial induced-proliferation and differentiation of osteoblastic MC3T3-E1 cells

Although the effect of 17β-estradial, a polyphenolic phytoestrogen, on bone cell function has been studied in numerous cell models, the synergetic role of 1, 25-dihydroxyvitamin D(3) on 17β-estradial induced-proliferation and differentiation of osteoblastic cells, and the underlying mechanism are obscure. Here, we investigated the in vitro effect of 17β-estradial on cell proliferation and osteoblastic maturation in MC3T3-E1 cells. 17β-estradial could promote the proliferation and viability of MC3T3-E1 cells, associated with upregulation of cyclin E and proliferation cell nuclear antigen (PCNA) mRNA expression, and downregulation of cyclin-dependent kinase inhibitor 2b (Cdkn2b) mRNA expression. Moreover, 17β-estradial also could stimulate osteoblastic differentiation and bone formation as assessed by alkaline phosphatase (ALP) and Alizarin Red S staining, through induction of the expression of osteoblastic markers, including ALP, osteopontin and type I collagen in MC3T3-E1 cells. However, 1,25-dihydroxyvitamin D(3) treatment alone showed no effect on proliferation and differentiation of MC3T3-E1 cells, but could coordinately augment effects of 17β-estradial on MC3T3-E1 cells. The mechanism conducted demonstrated that 17β-estradial activated ERK1/2 but not JNK and p38, and U0126, an ERK1/2 pathway inhibitor, significantly downregulated vitamin D receptor expression induced by 17β-estradial in MC3T3-E1 cells. Thus, our data demonstrated a synergistical role of 1,25-dihydroxyvitamin D(3) and 17β-estradial in proliferation and differentiation of osteoblasts, and this coordinated regulation might depend on the upregulation of vitamin D receptor in osteoblasts by 17β-estradial. Moreover, during the process of vitamin D receptor upregulation by 17β-estradial, ERK1/2 signaling is involved.     

Comparison of 3 risk factor–based screening tools for the identification of prediabetes

ObjectiveTo compare risk factor–based screening tools for identifying prediabetes.MethodsParticipants in an employer-based wellness program were tested for glycosylated hemoglobin (A1C) at a regularly scheduled appointment, and prediabetes risk factor information was collected. The likelihood of having prediabetes and the need for laboratory testing were determined based on 3 risk factor–based screening tools: the Prediabetes Screening Test (PST), Prediabetes Risk Test (PRT), and 2016 American Diabetes Association guidelines (ADA2016). The results from the screening tools were compared with those of the A1C test. The predictive ability of the PST, PRT, and ADA2016 were compared using logistic regression. Results were validated with data from a secondary population.ResultsOf the 3 risk factor–based tools examined, the PRT demonstrated the best combination of sensitivity and specificity for identifying prediabetes. From July 2016 to March 2017, 740 beneficiaries of an employer-sponsored wellness program had their A1C tested and provided risk factor information. The population prevalence of prediabetes was 9.3%. Analysis of a second independent population with a prediabetes prevalence of more than 50% of confirmed PRT’s superiority despite differences in the calculated sensitivity and specificity for each population.ConclusionBecause PRT predicts prediabetes better than PST or ADA2016, it should be used preferentially.     

A convenient semisynthesis of myricetin-3-O-β-d-glucuronide

Myricetin-3-O-β-d-glucuronide, a bioactive flavonol glycoside, was synthesized effectively starting from myricetrin in a total yield of 49.2%. The structures of all synthetic compounds were confirmed by ¹H, ¹³C NMR, and HR-MS techniques.     

Pharmacokinetics of 2′,3′-dideoxyadenosine in dogs

The pharmacokinetics of 2,3-dideoxyadenosine (ddAdo) and 2-3-dideoxyinosine (ddIno) were determined after intravenous bolus administration and long-term intravenous infusion of ddAdo in dogs. ddAdo was rapidly deaminated to ddIno and ddAdo plasma concentrations were only a fraction of ddIno concentrations. The total body clearance of ddAdo exceeded the literature value for the cardiac output of the dog, indicating an extremely rapid metabolism, and the existence of extrahepatic metabolism. Urinary excretion of unchanged ddAdo was a minor route of elimination ( 1%). The pharmacokinetics of ddIno was determined assuming complete conversions of ddAdo to ddIno. ddIno elimination was dose-dependent with total body clearance ranging from 4 to 55 ml/min/kg in individual animals. The plasma half-life was approximately 30 min after most routes of administration, but increased to approximately 60 min in two animals receiving a large intravenous dose of 500 mg/kg. ddIno penetrated into the cerebrospinal fluid to a limited extent, reaching concentrations of 3–11% of those in plasma. Urinary excretion of unchanged ddIno accounted for approximately 20% of the administered dose of ddAdo, while uric acid and hypoxanthine were minor urinary metabolites.Concentrations exceeding the in vitro minimal viral inhibitory concentration (2.4 g/mL) could be safely maintained in plasma for a 10-day period. Infusions which gave cerebrospinal fluid concentrations of 12 to 17 Lg/mL resulted in dose limiting myelosuppression and intestinal toxicity, after less than 10 days of infusion. Orally administered ddAdo was absorbed as ddIno, with bioavailabilities ranging from 28 to 93% in experiments where no emesis occurred. These studies indicate the rapid in vivo conversion of ddAdo to ddIno, and support the selection of ddIno over ddAdo for further drug development.     

Synthesis of 2′,3′-dideoxyisoguanosine from guanosine

2,3'-Dideoxyisoguanosine was synthesized from guanosine via intermediate 6-[(4-methylphenyl)thio]-2-oxo-9-(2',3',5'-tri-O-acetyl-beta-D-ribofuran osyl)-2,3-dihydropurine (4). The 2-oxo, 6-amino and 5'-hydroxy triprotected isoguanosine derivative was utilized to reduce high polarity and promote poor solubility of intermediates. The protecting groups for oxo and 6-amino were easily removed in reduction of olefin in ribose without additional reaction steps. 2',3'-Vicinal diol in ribose sugar moiety was transformed to olefin with Bu3SnH by radical reaction via bisxanthate. Removing 5'-O-TBDMS protecting group gave final product, 2',3'-dideoxyisoguanosine (12) in a 10% overall yield.     

Energy-dependent extrusion of cyclic 3′,5′-adenosine-monophosphate

Summary In reticulocyte-rich suspensions of red blood cells from rats extrusion of cAMP as a regulatory mechanism of intracellular cAMP was investigated.In response to isoprenaline and/or the phosphodiesterase inhibitors Ro 20-1724 and rolipram extrusion of cAMP increases dependent on the concentration of the drugs and time of exposure. However, these drugs exert their effects on the extrusion of cAMP only indirectly, i.e. via increased intracellular levels of cAMP, since the respective EC₅₀-values of the drugs for intracellular accumulation and extrusion of cAMP are identical (isoprenaline: 50 nM; rolipram: 1 M; Ro 20-1724: 15 M).The dependence of the rate of extrusion on intracellular levels of cAMP is characterized by a typical concentration-effect relationship from which a maximal capacity of cAMP extrusion of 3–6 nmol/10 min/10⁹ cells and a half maximal effective intracellular cAMP concentration of 40–50 nmol/10⁹ cells can be derived. This relationship has been inferred from either kinetic or steady-state approaches. At rapidly changing intracellular levels of cAMP an apparent time lag of extracellular cAMP accumulation is obligatorily conditioned by this relationship. Vasodilating drugs which lower the ATP content of the cells as well as the uncoupler of oxidative phosphorylation, FCCP, inhibit the extrusive process (papaverine > FCCP > dipyridamole > dilazep hexobendine carbocromene) leading to a 3–5-fold increase of the intrato extracellular concentration gradient of cAMP.It is concluded that extrusion of cAMP is a saturable and energy-dependent process which regulates the intracellular cAMP concentration independent of the activities of adenylate cyclase and phosphodiesterase.This work was supported by a grant from the Deutsche Forschungsgemeinschaft     

Specificity evaluation of antibodies against human β3-adrenoceptors

β(3)-Adrenoceptors are a promising drug target for the treatment of urinary bladder dysfunction, but knowledge about their expression at the protein level and their functional role is limited, partly due to a lack of well validated tools. As many antibodies against G-protein-coupled receptors, including those against β(3)- and other β-adrenoceptor subtypes, lack selectivity for their target, we have evaluated the specificity of five antibodies raised against the full-length protein of the human β(3)-adrenoceptor (H155-B01), its N-terminus (LSA4198 and TA303277) and its C-terminus (AB5122, Sc1472) in immunoblotting and immunocytochemistry. Our primary test system were Chinese hamster ovary cells stably transfected to express each of the three human β-adrenoceptor subtypes at near physiological levels (100-200?fmol/mg protein). None of the five antibodies exhibited convincing target specificity in immunoblotting with Sc1472 apparently being least unsuitable. In immunocytochemistry, LSA4198 and Sc1472 appeared most promising, exhibiting at least some degree of specificity. As these two antibodies have been raised against different epitopes (N- and C-terminus of the receptor, respectively), we propose that concordant staining by both antibodies provides the most convincing evidence for β(3)-adrenoceptor labelling in cyto- or histochemistry studies.