Stereoselective synthesis of Psi[CH(2)O] pseudodipeptides and conformational analysis of a PhePsi[CH(2)O]Ala containing analogue of the drug desmopressin

A method for synthesis of XaaPsi[CH(2)O]Ala/Gly pseudodipeptides in good yields and excellent diastereoselectivity from azido alcohols and (R)-2-chloropropionic acid or tert-butyl bromoacetate has been developed. Insertion of one of the pseudodipeptide building blocks in the peptide drug desmopressin revealed that methylene ether isosteres may have only a minor influence on the secondary structure of peptides.     

Synthesis of a [Glu5, Ala12, Ala18, Ala21]sheep insulin-A-chain by fragment condensation on a polymer support (author's transl)]

The synthesis of a [Glu5, Ala12, Ala18,-Ala21]sheep insulin-A-chain by condensation of 5 fragments on a polymer support is described. The 5 fragments Boc-Gly-Ile-Val-Glu(gammaOBut)-Glu(gammaOBut) (V), Boc-Cys(SiPr) (IV), Boc--Cys(SiPr)-Ala-Gly-Val (III), Boc-Cys(SiPr)-Ala-Leu-Tyr-Gln-Leu (II) and Boc-Glu(gammaOBut)-Ala--Tyr(Bzl)-Cys(SiPr)-N2H3 (I) were synthesized by conventional methods and coupled with dicyclohexylcarbodiimide/1-hydroxybenzotriazol (II, III, IV, V) and by the azide method (I) with coupling yields of 60-98% on an Ala-polymer. The failure sequence peptides were separated by ion exchange chromatography on DEAE-Sephadex and by chromatography on Biogel P4. The A-chain was obtained in 9% yield, which, after combinations with natural B-chain, gave insulin activities comparable to that obtained with natural A-chain. These results demonstrate that fragment condensation by the solid-phase method together with simple techniques for purification can be used for the synthesis of longer peptides.     

Conformational change of a synthetic amyloid analogue des[Ala21,3O]A42 upon binding to octyl glucoside micelles

The secondary structure of a synthetic amyloid fragment des [Ala^21,30]A42 was studied by circular dichroism and Fourier transformed infrared spectroscopy. Measurements were performed in trifluoroethanol/water and octyl -d-glucopyranoside solutions. The spectra of the peptide in trifluoroethanol indicate a high percentage of a-helical structure. However, in octyl glucoside, at and above the critical micelle concentration, the peptide adopts a -sheet conformation. Secondary structure analysis yields a predominant (> 70 %) -sheet content. Our data suggest that the peptide backbone or polar side groups of des[Ala^21,30]A42 interact with the sugar-coated surface of micelles, which promotes an a to conformational transition.     

Synthesis and anti-HIV activity of [d4U]-[trovirdine analogue] and [d4T]-[trovirdine analogue] heterodimers as inhibitors of HIV-1 reverse transcriptase

A series of eleven heterodimers containing both a nucleoside analogue (d4U, d4T) and a non-nucleoside type inhibitor (Trovirdine analogue) were synthesized and evaluated for their ability to inhibit HIV replication. Unfortunately, the (N-3)d4U-Trovirdine conjugates (9a-e) and (N-3)d4T-Trovirdine conjugates (10a-f) were found to be inactive suggesting that the two individual inhibitor compounds do not bind simultaneously in their respective sites.     

Stereoselective synthesis of a novel 2-aza-7-oxabicyclo[3.3.0]octane as neurokinin-1 receptor antagonist

A novel neurokinin-1 receptor antagonist, (±)-(1R^*,3S^*,4S^*,5S^*)-4-[(N-(2-methoxy-5-trifluoromethoxybenzyl)amino]-3-phenyl-2-aza-7-oxabicyclo[3.3.0]octane (1), was synthesized stereoselectively using Padwa’s intramolecular 1,3-dipolar cycloaddition methodology as the key step. Compound (±)-1 showed high affinity for the NK-1 receptors in human IM-9 cells with an IC₅₀ value of 0.22 nM. This new structural scaffold demonstrated significant in vivo antagonistic activity in the guinea pig ureter capsaicin-induced plasma extravasation model with an ED₅₀ value of 1–10 mg/kg, po.     

Identification of a pepducin acting as S1P3 receptor antagonist

Sphingosine‐1‐phosphate (S1P) is a bioactive lipid with key functions in the immune, inflammatory, and cardiovascular systems. S1P exerts its action through the interaction with a family of five known G protein‐coupled receptors, named S1P_1–5. Among them, S1P₃ has been implicated in the pathological processes of a number of diseases, including sepsis and cancer. KRX‐725 (compound 1) is a pepducin that mimics the effects of S1P by triggering specifically S1P₃. Here, aiming to identify novel S1P₃ antagonists, we carried out an alanine scanning analysis to address the contribution of the side chains of each amino acid residue to the peptide function. Then, deleted peptides from both the C‐ and N‐terminus were prepared in order to determine the minimal sequence for activity and to identify the structural requirements for agonistic and, possibly, antagonistic behaviors. The pharmacological results of the Ala‐scan derived compounds (2–10) suggested a high tolerance of the pepducin 1 to amino acid substitutions. Importantly, the deleted peptide 16 has the ability to inhibit, in a dose‐dependent manner, both pepducin 1‐induced vasorelaxation and fibroblast proliferation. Finally, a computational analysis was performed on the prepared compounds, showing that the supposed antagonists 16 and 17 appeared to be aligned with each other but not with the others. These results suggested a correlation between specific conformations and activities. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Biological activities of des-His[Glu]glucagon amide, a glucagon antagonist

Hyperglycemia in diabetes mellitus is generally associated with elevated levels of glucagon in the blood. A glucagon analog, des-His¹[Glu⁹] glucagon amide, has been designed and synthesized and found to be an antagonist of glucagon in several systems. It has been a useful tool for investigating the mechanisms of glucagon action and for providing evidence that glucagon is a contributing factor in the pathogenesis of diabetes. The in vitro and in vivo activities of the antagonist are reported here. The analog bound 40% as well as glucagon to liver membranes, but did not stimulate the release of cyclic AMP even at 10⁶ higher concentration. However, it did activate a second pathway, with the release of inositol phosphates. In addition, the analog enhanced the glucose-stimulated release of insulin from pancreatic islet cells. Of particular importance were the findings that the antagonist also showed only very low activity (<0.2%) in the in vivo glycogenolysis assay, and that at a ratio of 100:1 the analog almost completely blocked the hyperglycemic effects of added glucagon in normal rabbits. In addition, it reduced the hyperglycemia produced by endogenous glucagon in streptozotocin diabetic rats. Thus, we have an analog that possesses properties that are necessary for a glucagon antagonist to be potentially useful in the study and treatment of diabetes.     

Solution NMR evidence for a cis Tyr-Ala peptide group in the structure of [Pro93Ala] bovine pancreatic ribonuclease A

Proline peptide group isomerization can result in kinetic barriers in protein folding. In particular, the cis proline peptide conformation at Tyr92-Pro93 of bovine pancreatic ribonuclease A (RNase A) has been proposed to be crucial for chain folding initiation. Mutation of this proline-93 to alanine results in an RNase A molecule, P93A, that exhibits unfolding/refolding kinetics consistent with a cis Tyr92-Ala93 peptide group conformation in the folded structure (Dodge RW, Scheraga HA, 1996, Biochemistry 35:1548-1559). Here, we describe the analysis of backbone proton resonance assignments for P93A together with nuclear Overhauser effect data that provide spectroscopic evidence for a type VI beta-bend conformation with a cis Tyr92-Ala93 peptide group in the folded structure. This is in contrast to the reported X-ray crystal structure of [Pro93Gly]-RNase A (Schultz LW, Hargraves SR, Klink TA, Raines RT, 1998, Protein Sci 7:1620-1625), in which Tyr92-Gly93 forms a type-II beta-bend with a trans peptide group conformation. While a glycine residue at position 93 accommodates a type-II bend (with a positive value of phi93), RNase A molecules with either proline or alanine residues at this position appear to require a cis peptide group with a type-VI beta-bend for proper folding. These results support the view that a cis Pro93 conformation is crucial for proper folding of wild-type RNase A.     

Synthesis and binding characteristics of [3H] H-Tyr-Ticpsi[CH2-NH] Cha-Phe-OH, a highly specific and stable delta-opioid antagonist.

Substitution of the Phe3 aromatic ring in H-Tyr-Ticpsi[CH2-NH]Phe-Phe-OH with cyclohexylalanine (Cha) has been reported to result in a compound, H-Tyr-Ticpsi[CH2-NH]Cha-Phe-OH (TICP[psi]), showing substantially increased delta-opioid antagonist potency and high delta selectivity. TICP[psi] was radiolabeled by catalytic tritiation of its precursor Tyr(3',5'-I2)1TICP[psi]. Binding characteristics of the new tritiated pseudopeptide were determined using the radioligand binding assay in rat brain membranes. On the basis of the results of saturation binding studies performed at 25 degrees C, an equilibrium dissociation constant (Kd) of 0.35 nM and a receptor density (Bmax) of 112 fmol/mg protein were calculated. This new tritiated ligand exhibits high affinity for delta-opioid receptors, whereas its binding to mu and kappa receptors is weak. A study of [H3]TICP[psi] binding displacement by various receptor-selective opioids showed the following rank order of potency: delta > kappa = mu. These receptor binding characteristics of the ligand, together with its high specific radioactivity (41.3 Ci/mmol) and stability, makes it a useful tool for labeling delta-opioid receptors, both in vitro and in vivo.     

Selection of a 2-azabicyclo[2.2.2]octane-based alpha4beta1 integrin antagonist as an inhaled anti-asthmatic agent

The alpha4beta1 integrin, expressed on eosinophils and neutrophils, induces inflammation in the lung by facilitating cellular infiltration and activation. From a number of potent alpha4beta1 antagonists that we evaluated for safety and efficacy, 1 was selected as a lead candidate for anti-asthma therapy by the inhalation route. We devised an optimized stereoselective synthesis to facilitate the preparation of a sufficiently large quantity of 1 for assessment in vivo. Administration of 1 to allergen-sensitive sheep by inhalation blocked the late-phase response of asthma and abolished airway hyper-responsiveness at 24h following the antigen challenge. Additionally, the recruitment of inflammatory cells into the lungs was inhibited. Administration of 1 to ovalbumin-sensitized guinea pigs intraperitoneally blocked airway resistance and inhibited the recruitment of inflammatory cells.     

Synthesis and biological activity of analogs of dynorphin-A(1-13) substituted in positions 2 and 4: design of [Ala2,Trp4]-Dyn-A(1-13) as a putative selective opioid antagonist

Mono- and di-substituted analogs of dynorphin-A(1-13) (Dyn-A(1-13)) were synthesized by the solid-phase procedure. The products were purified and analyzed for their ability to inhibit the electrically evoked contractions of the guinea pig ileum (GPI) and mouse vas deferens (MVD) and to compete with the binding of [3H]etorphine ([3H]ET) and [3H]ethylketocyclazocine ([3H]EKC) to homogenates of rat brain (mu-, delta-, kappa 2-receptors) and guinea pig cerebellum (kappa-receptor), respectively. Introduction of Ala in position 2 caused a drastic decrease in the activity of the peptide on the smooth muscle preparations (IC50 of 104 and 2.250 nM in the GPI and the MVD as compared with 0.7 and 21 nM for the parent peptide, respectively). Conversely, this analog retained much of the opioid binding activity of Dyn-A(1-13) (relative binding potencies of 15 and 72% for the displacement of [3H]ET and [3H]EKC, respectively). The replacement of Phe4 by Trp also caused drastic decreases in the activity of the peptide in the smooth muscle preparations (relative potencies of 0.8 and 8.8% on the GPI and MVD) while much of the binding potency to the opioid receptors was retained (31 and 67% for the displacement of [3H]ET and [3H]EKC, respectively). [Ala2,Trp4]-Dyn-A(1-13) was the least potent peptide tested in the smooth muscle assays (relative potencies: 0.1 and 0.6%). However, this latter analog still retained some opioid binding activity in the displacement of [3H]ET to rat brain homogenates (3%).(ABSTRACT TRUNCATED AT 250 WORDS)     

The use of a prostacyclin analogue, [3H]iloprost,for studying prostacyclin-binding sites on human platelets and neuronal hybrid cells

The stable prostacyclin analogue [³H]iloprost has been used for labelling prostacyclin-binding sites on human platelets and NCB-20 neuronal hybrid cells. The ligand-binding properties of the sites have been determined and correlate well with stimulation of cAMP synthesis in NCB-20 cells and inhibition of aggregation in human platelets.     

Identification of ghrelin receptor blocker, D-[Lys3] GHRP-6 as a CXCR4 receptor antagonist

[D-Lys3]-Growth Hormone Releasing Peptide-6 (DLS) is widely utilized in vivo and in vitro as a selective ghrelin receptor (GHS-R) antagonist. Unexpectedly, we identified that DLS also has the ability to block CXCL12 binding and activity through CXCR4 on T cells and peripheral blood mononuclear cells (PBMCs). Moreover, as CXCR4 has been shown to act as a major co-receptor for HIV-1 entry into CD4 positive host cells, we have also found that DLS partially blocks CXCR4-mediated HIV-1 entry and propagation in activated human PBMCs. These data demonstrate that DLS is not the specific and selective antagonist as thought for GHS-R1a and appears to have additional effects on the CXCR4 chemokine receptor. Our findings also suggest that structural analogues that mimic DLS binding properties may also have properties of blocking HIV infectivity, CXCR4 dependent cancer cell migration and attenuating chemokine-mediated immune cell trafficking in inflammatory disorders.     

Synthesis and biodistribution of [11C]R107474, a new radiolabeled alpha2-adrenoceptor antagonist

R107474, 2-methyl-3-[2-(1,2,3,4-tetrahydrobenzo[4,5]furo[3,2-c]pyridin-2-yl)ethyl]-4H-pyrido[1,2-a]pyrimidin-4-one, was investigated using in vitro and in vivo receptor assays and proved to be a potent and relatively selective alpha(2)-adrenoceptor antagonist. Performed assays in vitro were inhibition of binding to a large number of neurotransmitter receptor sites, drug receptor binding sites, ion channel binding sites, peptide receptor binding sites, and the monoamine transporters in membrane preparations of brain tissue or of cells expressing the cloned human receptors. The compound has subnanomolar affinity for halpha(2A)- and halpha(2C)-adrenoceptors (K(i) = 0.13 and 0.15 nM, respectively) and showed nanomolar affinity for the halpha(2B)-adrenoceptors and 5-hydroxytryptamine(7) (h5-HT(7)) receptors (K(i) = 1 and 5 nM, respectively). R107474 interacted weakly (K(i) values ranging between 81 and 920 nM) with dopamine-hD(2L), -hD(3) and -hD(4), h5-HT(1D)-, h5-HT(1F)-, h5-HT(2A)-, h5-HT(2C)-, and h5-HT(5A) receptors. The compound, tested up to 10 microM, interacted only at micromolar concentrations or not at all with any of the other receptor or transporter binding sites tested in this study. In vivo alpha(2A)- and alpha(2C)-adrenoceptor occupancy was measured by ex vivo autoradiography 1h after subcutaneous (sc) administration of R107474. It was found that R107474 occupies the alpha(2A)- and alpha(2C)-adrenoceptors with an ED(50) (95% confidence limits) of 0.014 mg/kg sc (0.009-0.019) and 0.026 mg/kg sc (0.022-0.030), respectively. Radiolabeled 2-methyl-3-[2-([1-(11)C]-1,2,3,4-tetrahydrobenzo[4,5]furo[3,2-c]pyridin-2-yl)ethyl]-4H-pyrido[1,2-a]pyrimidin-4-one ([(11)C]R107474) was prepared and evaluated as a potential positron emission tomography (PET) ligand for studying central alpha(2)-adrenoceptors. [(11)C]R107474 was obtained via a Pictet-Spengler reaction with [(11)C]formaldehyde in 33 +/- 4% overall decay-corrected radiochemical yield. The total synthesis time was 55 min and the specific activity was 24-28 GBq/micromol. The biodistribution of [(11)C]R107474 in rats revealed that the uptake of [(11)C]R107474 after in vivo intravenous administration is very rapid; in most tissues (including the brain) it reaches maximum concentration at 5 min after tracer injection. In agreement with the known distribution of alpha(2)-adrenoceptors in the brain, highest uptake of radioactivity was observed in septum (3.54 +/- 0.52 ID/g, 5 min pi) and entorhinal cortex (1.57 +/- 0.10 ID/g, 5 min pi). Tissue/cerebellum concentration ratios for septum (5.38 +/- 0.45, 30 min pi) and entorhinal cortex (3.43+/-0.24, 30 min pi) increased with time due to rapid uptake followed by a slow washout. In vivo blocking experiments using the non-selective alpha(2)-adrenoceptor antagonist mirtazapine demonstrated specific inhibition of [(11)C]R107474 binding in selective brain areas. The receptor binding profile of mirtazapine is reported and the selectivity of inhibition of binding is discussed. These results suggest that [(11)C]R107474 deserves further investigation as a potential radioligand for studying alpha(2)-adrenoceptors using PET.     

An orally active corticotropin releasing factor 1 receptor antagonist from 8-aryl-1,3a,7,8-tetraaza-cyclopenta[a]indenes

8-Aryl-1,3a,7,8-tetraaza-cyclopenta[a]indenes represent a novel series of high-affinity corticotropin-releasing factor-1 receptor (CRF1R) antagonists. Herein we report the synthesis and SAR around the tricyclic core and the anxiolytic activity of an orally dosed exemplary compound 9d (K(i)=8.0 nM) in a mouse canopy model.     

Synthesis of [2H8]estradiol, [2H7]estrone, [2H6]2-hydroxyestrone and [2H6]4-hydroxyestrone as internal standards for selected ion monitoring

The synthesis of [²H₈]estradiol, [²H₇]estrone, [²H₆]2-hydroxyestrone and [²H₆]4-hydroxyestrone from estrone (as a source) is described. The high isotopical purity renders the labelled compounds as suitable carriers and internal standards for quantitative gas chromatography — mass spectrometry. The content of protonium-form (i.e. natural) estrogens in the labelled derivatives ranged from 0.12 % to 2.58 %. The perfcrmance of these compounds in quantitative assays using selected ion monitoring has been established; and this allows the determination of estrogens from biological material in the lower picogram range.     

2-Amino-[1,2,4]triazolo[1,5-a]pyridines as JAK2 inhibitors

The advancement of a series of ligand efficient 2-amino-[1,2,4]triazolo[1,5-a]pyridines, initially identified from high-throughput screening, to a JAK2 inhibitor with pharmacodynamic activity in a mouse xenograft model is disclosed.

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Synthesis and binding characteristics of a novel enkephalin analogue, [3H]Tyr-D-Ala-Gly-Phe-D-Nle-Arg-Phe

The endogenous opioid heptapeptide (Tyr-Gly-Gly-Phe-Met-Arg-Phe; MERF) has been shown to interact with multiple opioid as well as non-opioid sites in mammalian brain membranes. To increase the stability and bioavailability of MERF, new synthetic derivatives with D-amino acid substitutions were prepared and studied. One of the new compounds in this series, Tyr-D-Ala-Gly-Phe-D-Nle-Arg-Phe (DADN), had only moderate affinity in competing with [3H]MERF, whereas it displayed the highest potency in producing antinociception following intrathecal administration. DADN was radiolabeled with 41Ci/mmol specific activity. Specific binding of [3H]DADN was saturable, stereoselective and of high affinity. Chemical stability, increased micro-receptor selectivity, and hydrophobicity of the peptide all contribute to the effectiveness observed in biochemical and pharmacological studies.