Terrence R. Burke

A general method for the preparation of benzylic α,α-difluorophosphonic acids; non-hydrolyzable mimemtics of phosphotyrosine

Abstract Methodology is presented for the preparation of heretofore unreported benzylic α,α-difluorophosphonic acids. Oxidation of benzylic α-hydroxyphosphonates using MnO 2 , provided a new route for the synthesis of benzylic α-oxophosphonates, which were then fluorinated using DAST. Hydrolysis of ester protecting groups yielded α,α-difluorophosphonic acids. These represent non-hydrolyzable mimetics of arylphosphates which could be of value in the study of cellular phosphotyrosine-utilizing signal transduction pathways.

Morphine tolerance increases μ-noncompetative δ binding sites☆

Abstract In light of more recent knowledge concerning endogenous opioid peptides and their multiple opiate receptors, we reevaluated the effects of morphine tolerance on opiate receptor binding parameters. Rats were implanted with morphine or placebo pellets, and [ 3 H][D-Ala 2 ,D-Leu 5 ]enkephalin ([ 3 H]DADL) was used to label brain membranes. Utilizing the technique of binding surface analysis, we observed a selective 47% up-regulation of lower affinity [ 3 H]DADL binding sites (μ-noncompetitive δ binding sites) in morphine pelleted rats. To corroborate these results, we treated brain membranes with the site directed alkylating agent FIT (N-phenyl-N-[1-(2-p-isothiocyanato)phenyl-ethyl)-4-piperidinyl]propanamide), which results in membranes highly enriched with the lower affinity [ 3 H]DADL binding site. Scatchard plots of [ 3 H]DADL binding to FIT-treated membranes also revealed that chronic morphine treatment produced a 60–65% up-regulation of the μ-noncompetitive δ binding site. These data indicate that chronic morphine alters a selective subpopulation of opiate receptors that may play a role in the mechanisms of opiate tolerance and physical dependence.

Synthesis of 4-phosphono(difluoromethyl)-D,L-phenylalanine and N-boc and N-Fmoc derivatives suitably protected for solid-phase synthesis of nonhydrolyzable phosphotyrosyl peptide analogues

Abstract Synthesis of 4-phosphono(difluoromethyl)-D,L-phenylalanine as well as its diethyl phosphonate analogues bearing either Boc or Fmoc-amino protection are reported. The latter two derivatives were utilized for the solid-phase synthesis of SH2-related peptides containing nonhydrolyzable phosphotyrosyl mimetics.

Enantioselective synthesis of N-boc and N-fmoc protected diethyl 4-phosphono(difluoromethyl)-L-phenylalanine; agents suitable for the solid-phase synthesis of peptides containing nonhydrolyzable analogues of O-phosphotyrosine

Abstract Enantioselective convergent syntheses of N-Boc and N-Fmoc protected diethyl 4-phosphono(difluoromethyl)-L-phenylalanine are reported.

Synthesis of a difluorophosphonomethyl-containing phosphatase inhibitor designed from the X-ray structure of a PTP1B-bound ligand

Abstract Protein-tyrosine phosphatase (PTP) inhibitors are potentially valuable pharmacological tools for studying cellular signal transduction and for therapeutic intervention. Small peptides containing the non-hydrolyzable phosphotyrosyl mimetic difluorophosphonomethyl phenylalanine (F2Pmp) have been shown to be extremely potent PTP inhibitors, with the fluorines increasing inhibitory potency 1000-fold relative to the unfluorinated species. The high PTP affinity of the phosphonodifluoromethyl pharmacophore has allowed the preparation of small molecule inhibitors containing this moiety, which lack any peptide component. The X-ray structure of one such inhibitor. 2-difluoromethylnaphthylphosphonic acid (6) complexed to the catalytic site of PTP1B has recently been solved. Computer assisted molecular modelling of this complex indicates that enhanced binding interactions may result by introduction of hydroxyl functionality onto the naphthalene ring system. Herein is reported the synthesis of one such inhibitor 1,1-difluoro-1-[2-(4-hydroxynaphthalenyl)]methyl¦phosphonic acid (7), which is prepared in 12 steps from commercially available 1,3-dihydroxynaphthalene. The synthetic approach relies on selective hydroxyl protection and Pd-catalyzed carbonylation to introduce functionality which is subsequently elaborated into the difluorophosphonate structure. The techniques reported herein may be applicable to the preparation of other PTP inhibitors.

Solid-phase synthesis of viscosin, a cyclic depsipeptide with antibacterial and antiviral properties

Viscosin, a cyclic depsipeptide (containing a peptide lactone) has been prepared by solid-phase chemistry using an Fmoc-protocol starting with an acid sensitive resin. Cyclization with the activating agent BOP-CI gave a product which was indistinguishable from natural viscosin thereby supporting the proposed structure.

Preparation of rat brain membranes highly enriched with opiate kappa binding sites using site-directed acylating agents: Optimization of assay conditions

The goal of this study was to determine optimal conditions with which to measure opiate kappa binding sites in rat brain. Membranes were pretreated with mu-selective (BIT) and delta-selective (FIT) site-directed acylating agents (Rice et al., Science 220, 314-316), and the binding of [3H]bremazocine to the residual binding sites was defined as the kappa binding site. The binding of [3H]bremazocine to BIT/FIT-treated membranes was greatly increased by conducting the assay at 0 degrees C in the presence of 0.4 M NaCl. Using this 0 degrees C/NaCl assay condition, the binding of [3H]bremazocine was best described by a one-site binding model with a KD of 0.45 nM and a Bmax of 378 fmol/mg protein. Autoradiographic studies demonstrated that, using this assay condition, [3H]bremazocine densely labeled the deep layers of guinea pig cortex, an area known to be enriched with kappa binding sites. These and additional data suggest that the binding of [3H]bremazocine to the kappa binding site of rat brain is optimally assayed at 0 degrees C in the presence of 0.4 M NaCl using BIT/FIT-treated membranes and that rat brain is endowed with a high level of kappa binding sites.

Solid-phase synthesis of nonhydrolyzable phosphotyrosyl peptide analogues with Nα-Fmoc-(O,O-di-t-butyl)phosphono-p-methylphenylalanine

Abstract A new, protected derivative of phosphonomethylphenylalanine is used to synthesize nonhydrolyzable analogs of phosphotyrosyl peptides for use as inhibitors and affinity ligands of proteins that recognize phosphotyrosyl sequences.

Pentafluorophenyl ester activation for the preparation of N,N′-diaroylhydrazines

Abstract Procedures are reported for the preparation of N,N′-diaroylhydrazines using pentafluorophenyl (Pfp) ester activation of aryl carboxylic acids. Mild conditions which avoid intermediate protection of ring substituents, allows the synthesis of both symmetrical and unsymmetrical diaroylhydrazines in high yields. The recent discovery of potent HIV-1 integrase inhibition by N,N′-bis-salicylhydrazine (1) highlights the potential importance of this class of compounds. The stability of pre-activated Pfp ester intermediates and the facility with which N,N′-diaroylhydrazines can be synthesized using this procedure (stirring at room temperature in DMF) may make the method particularly attractive for synthesis of hydrazide libraries.

QSAR of conformationally flexible molecules: Comparative Molecular Field Analysis of protein-tyrosine kinase inhibitors

SummaryComparative Molecular Field Analysis (CoMFA) has been applied to a study of quantitative structureactivity relationships (QSAR) of conformationally flexible molecules. The relationship between three-dimensional structure and activity of 20 styrene derivatives which inhibit protein-tyrosine kinase was determined. A technique was developed that allows accurate prediction of the inhibitory activity of these molecules and identification in each case of the active conformation. The problem of multiple energetically acceptable conformations was approached in an iterative procedure. Use was made of the varying degrees of symmetry among the molecules. First, CoMFA QSAR models were developed using only those compounds that possess a symmetrical substituent pattern on the phenyl ring. These CoMFA models were then used to select the active conformers of the less symmetrical compounds in the set. Allowing multiple conformers for each compound in the dataset yielded higher crossvalidated r2 values and better predictivity of the QSAR models. Different probe atoms (C+, O−, neutral C) were explored, the O− probe atom exhibiting the highest selectivity in the conformer selection process.