Stereocontrolled synthesis of cis-dibenzoquinolizine chlorofumarates: curare-like agents of ultrashort duration.

The quaternizations of dibenzoquinolizines 9 and 14 with 3-halo-1-propanols are highly cis-selective (94-100% cis), results consistent with the N-methylation of O-methylcapaurine (7b), but in contrast to the proposed trans-stereochemistry of dibenzo[a,h]quinolizine methiodide 10 and the analogous quaternizations of 1-benzyl- and 1-phenylisoquinoline congeners 5b and 5c. In this report, we describe stereoselective preparation of the unique cis-dibenzoquinolizinium propanols 15 and 16and their transformation into bis- and mixed-onium chlorofumarates 19, 20ab, and 26. Dibenzo[a,g]quinolizinium propanol 15 was prepared enantioselectively in three steps from dihydroisoquinoline 11. Asymmetric transfer hydrogenation of 11 in the presence of triethylamine/formic acid and Noyori's chiral ruthenium catalyst 12 produced R-(-)-5',8-dimethoxynorlaudanosine (13) in 98% yield and 87% ee. Pictet-Spengler cyclization of 13 in formalin/formic acid afforded the dibenzo[a,g]quinolizine 14 in 65% yield. Quaternization of 14 with 3-chloro-1-propanol under Finkelstein conditions generated cis-dibenzoquinolizinium propanol 15 in 85% yield with >94% cis-selectivity. The cis-dibenzo[a,h]quinolizinium propanol 16 was obtained as a single stereoisomer by reaction of the known tetramethoxyquinolizine 9 with neat 3-iodo-1-propanol. Bis-onium chlorofumarates 18 and 19 and the mixed-onium derivative 20ab were prepared by a pool synthesis procedure from (1R)-trans-6a, 16, and chlorofumaryl chloride (17). Mixed-onium alpha-chlorofumarate 26 was synthesized from (1S)-trans-6d, 15 and (+/-)-trans-2,3-dichlorosuccinic anhydride (22), employing a recently disclosed chlorofumarate mixed-diester synthesis. The title compounds (19, 20ab, and 26) displayed curare-like effects of ultrashort duration in rhesus monkeys.

Using one- and two-dimensional NMR techniques to characterize reaction products bound to Chiron SynPhase crowns.

Solid-phase MAS techniques have proved to be very useful in characterizing compounds bound to resin; however, little has been reported on using NMR to characterize compounds attached to Chiron SynPhase crowns. We have used proton, carbon, and COSY spectra obtained with a Varian Nano.nmr probe to characterize products from a published reaction sequence attached to MD (methacrylic acid/dimethylacrylamide copolymer) crowns. We have also performed solvent surveys to determine the best solvents for acquiring spectra of materials bound to both MD and PS (polystyrene) crowns.

A convenient method for determining cyclic peptide conformation from 1D 1H-NMR information.

A rapid and convenient method for determining the backbone conformation of cyclic peptides results from the combination of 1D 1H NMR information and molecular modeling. phi Angle torsional constraints calculated from 3JHN.H alpha coupling constants are used to determine the position of multiple-welled potential energy penalty functions that are imposed on the force field used in the structure refinement (Amber* with GB/SA solvation model). Monte Carlo searches and minimizations lead to a collection of structures that are clustered by backbone similarity and then filtered according to hydrogen-bonding constraints determined by the chemical shift temperature dependencies of the amide protons. This approach was applied to five cyclic peptides whose structures had been determined previously using more extensive 2D NMR techniques, and the importance of the torsional, H-bonding, and solvation restraints were assessed. For the four peptides that adopt a predominant conformation, this method reproduced the reported structures closely; lack of convergence for the fifth structure reflected the multiple backbone conformations that this macrocycle adopts.

Functional activity of new C-terminal cyclic-neurotensin fragment analogs.

Neurotensin (NT, pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu) is a tridecapeptide that displays a wide spectrum of biological actions. Cyclic derivatives of a hexapeptide NT [(8-13)] (N alpha MeArg-Lys-Pro-Trp-Tle-Leu, Tle = tert-leucine) were designed and prepared by a combination of solution and solid-phase peptide synthetic methodologies. As reported previously, several analogs possessed nanomolar binding affinities for NT receptors in newborn (10-day-old) mouse brain membrane preparations. In this study, we determined the functional ability of these analogs to mobilize intracellular free calcium, [Ca2+]i, in HT-29 cells (human colonic adenocarcinoma). Of greatest interest were the cyclic compounds 2, 6 and 9 that had Ki values of 0.19, 3.50 and 4.18 microM for [3H]NT labeled receptors in the HT-29 cell membrane assay, respectively. In the functional assay, compounds 2 and 6 mobilized [Ca2+] with EC50 values of 0.13 and 20 microM, respectively. In comparison, Compound 9 blocked the NT-induced mobilization of [Ca2+]i, with an IC50 of 1.70 microM. The present findings indicate that small molecule cyclic analogs, that possess functional activity, can be designed and may have therapeutic utility in the treatment of schizophrenia and possibly other neurological disorders.

Structure-activity relationships of the potent combined endothelin-A/endothelin-B receptor antagonist Ac-DDip16-Leu-Asp-Ile-Ile-Trp21: development of endothelin-B receptor selective antagonists.

The endothelins (ETs) are a family of bicyclic 21-amino acid-containing peptides that are highly potent and prolonged vasoconstrictors. The discovery of potent ET antagonists will facilitate the understanding of the physiological and/or pathophysiological role of ET. Structure-activity studies have revealed the importance of the C-terminal hexapeptide (residues 16-21) of ET (His16-Leu17-Asp18-Ile19-Ile20-Trp21) to the development of potent antagonists at both receptor subtypes (ETA and ETB). In particular, it has been shown that Ac-DDip16-Leu-Asp-Ile-Ile-Trp21 (Dip = 3,3-diphenylalanine) has low nanomolar affinity for the two endothelin receptor subtypes and is a functional antagonist of ET activity, both in vitro and in vivo at both receptors. Herein, we will describe the structure-activity relationships of Ac-DDip16-Leu-Asp-Ile-Ile-Trp21 (PD 142893) with a particular emphasis on modifications that lead to enhanced receptor affinity and/or individual receptor subtype selectivity. In particular, we will demonstrate how we utilized PD 142893 to develop ETB receptor selective ligands and the pharmacological differences that exist between species ETB receptors with respect to their affinity for C-terminal hexapeptide antagonists.

Design and structure-activity relationships of C-terminal cyclic neurotensin fragment analogues.

Neurotensin (NT) is a linear tridecapeptide with a broad range of central and peripheral pharmacological effects. The C-terminal hexapeptide of NT (NT8-13) has been shown to possess similar properties to NT itself, and in fact, an analogue of NT8-13 (N alpha MeArg8-Lys-Pro-Trp-Tle-Leu13, Tle = tert-leucine) has been reported to possess central activity after peripheral administration. Cyclic derivatives of this hexapeptide were synthesized by a combination of solution and solid-phase peptide synthetic methodologies, and several analogues had low nanomolar binding affinity for the NT receptor. In particular, cyclo[Arg-Lys-Pro-Trp-Glu]-Leu (cyclized between the alpha amine of Arg and the gamma carboxylate of Glu) possessed 16 nM NT receptor affinity and was determined to be an agonist in vitro. 1H-NMR and 13C-edited 1H-NMR spectroscopy were performed on this and related cyclic analogues to help identify structural properties which may be important for receptor recognition. These cyclic peptides represent novel molecular probes to further investigate NT receptor pharmacology, as well as to advance our understanding of the structure-conformation relationships of NT and to help establish a working basis for additional pharmacophore mapping studies.

Differentiation of peptide molecular recognition by phospholipase C gamma-1 Src homology-2 domain and a mutant Tyr phosphatase PTP1bC215S.

Activated epidermal growth factor receptor (EGFR) undergoes autophosphorylation on several cytoplasmic tyrosine residues, which may then associate with the src homology-2 (SH2) domains of effector proteins such as phospholipase C gamma-1 (PLC gamma-1). Specific phosphotyrosine (pTyr)-modified EGFR fragment peptides can inhibit this intermolecular binding between activated EGFR and a tandem amino- and carboxy-terminal (N/C) SH2 protein construct derived from PLC gamma-1. In this study, we further explored the molecular recognition of phosphorylated EGFR988-998 (Asp-Ala-Asp-Glu-pTyr-Leu-Ile-Pro-Gln-Gln-Gly, I) by PLC gamma-1 N/C SH2 in terms of singular Ala substitutions for amino acid residues N- and C-terminal to the pTyr (P site) of phosphopeptide I. Comparison of the extent to which these phosphopeptides inhibited binding of PLC gamma-1 N/C SH2 to activated EGFR showed the critical importance of amino acid side chains at positions P+2 (Ile994), P+3 (Pro995), and P+4 (Gln996). Relative to phosphopeptide I, multiple Ala substitution throughout the N-terminal sequence, N-terminal sequence, N-terminal truncation, or dephosphorylation of pTyr each resulted in significantly decreased binding to PLC gamma-1 N/C SH2. These structure-activity results were analyzed by molecular modeling studies of the predicted binding of phosphopeptide I to each the N- and C-terminal SH2 domains of PLC gamma-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure-activity and conformational studies of a series of modified C-terminal hexapeptide neurotensin analogues.

Neurotensin (NT), is a linear tetradecapeptide (pGlu1-Leu2-Tyr3-Glu4-Asn5- Lys6-Pro7-Arg8-Arg9-Pro10-Tyr11-Ile12-Leu13) that has been found in the central nervous system and peripheral tissues and appears to have a variety of physiological properties. A C-terminal hexapeptide analogue [N alpha Me-Arg-Lys-Pro-Trp-Tle-Leu, (1) Tle = tert-leucine] has recently been reported to have high affinity for the NT receptor and appears to possess central activity after systemic administration. In an effort to probe the structure-activity and conformational properties of the dipeptide, Pro-Trp for binding and functional activity, these residues have been substituted with several natural and unnatural amino acids. Some of these analogues have binding affinities similar to compound 1, while in other cases, such as D-amino acid substitutions, the peptides had negligible binding affinity. In general, the Pro10 position seems more tolerant of substitution by amino acids that favor a reverse turn, rather than those that favor an extended conformation. The Trp11 position accepted extra steric bulk more readily than conformational constraints.

A continuous spectrophotometric and fluorimetric assay for protein tyrosine phosphatase using phosphotyrosine-containing peptides.

Two continuous assays for protein tyrosine phosphatases (PTPases) have been developed using phosphotyrosine containing peptide substrates. These assays are based on the marked differences in the spectra of the peptide before and after the removal of the phosphate group. The increase in the absorbance at 282 nm or the fluorescence at 305 nm of the peptide upon the action of PTPase can be followed continuously and the resulting progress curve (time course) can be analyzed directly using the integrated form of the Michaelis-Menten equation. The procedure is convenient and efficient, since both kcat and Km values can be obtained in a single run. The difference absorption coefficient (delta epsilon) at 282 nm is relatively insensitive to the pH of the reaction media. These techniques were applied to two homogeneous recombinant PTPases employing six phosphotyrosine-containing peptides. Km and kcat values obtained from the progress curve analysis were similar to those determined by the traditional initial rate inorganic phosphate assay. The peptides corresponding to autophosphorylation sites in Neu, p56lck, and p60src proteins show distinct behavior with the Yersinia PTPase, Yop51*, and the mammalian PTPase (PTP1U323). In both cases, the kcat values were relatively constant for all the peptides tested whereas the Km values were very sensitive to the amino acid sequence surrounding the tyrosine residue, especially in the case of Yop51*. Thus, both Yop51* and PTP1U323 show differential recognition of the phosphotyrosyl residues in the context of distinct primary structure of peptide substrates.

Substrate specificity of the protein tyrosine phosphatases.

The substrate specificity of a recombinant protein tyrosine phosphatase (PTPase) was probed using synthetic phosphotyrosine-containing peptides corresponding to several of the autophosphorylation sites in epidermal growth factor receptor (EGFR). The peptide corresponding to the autophosphorylation site, EGFR988-998, was chosen for further study due to its favorable kinetic constants. The contribution of individual amino acid side chains to the binding and catalysis was ascertained utilizing a strategy in which each amino acid within the undecapeptide EGFR988-998 (DADEpYLIPQQG) was sequentially substituted by an Ala residue (Ala-scan). The resulting effects due to singular Ala substitution were assessed by kinetic analysis with two widely divergent homogeneous PTPases. A "consensus sequence" for PTPase recognition may be suggested from the Ala-scan data as DADEpYAAPA, and the presence of acidic residues proximate to the NH2-terminal side of phosphorylation is critical for high-affinity binding and catalysis. The Km value for EGFR988-998 decreased as the pH increased, suggesting that phosphate dianion is favored for substrate binding. The results demonstrate that chemical features in the primary structure surrounding the dephosphorylation site contribute to PTPase substrate specificity.