Hydrophobic peptides that exhibit significant beta-turn structure in solution are more lipophilic as measured by log Po/w and more readily permeate Caco-2 cell monolayers via the transcellular route than hydrophobic peptides that lack this type of solution structure. The ability of these peptides to permeate Caco-2 cell monolayers via the transcellular route also exposed them to metabolism, presumably by cytosolic endopeptidase. Similar secondary structural features in hydrophilic peptides do not appear to sufficiently alter the physicochemical properties of the peptides so as to alter their paracellular flux through unperturbed Caco-2 cell monolayers.
The coumarinic acid-based cyclic DADLE (H-Tyr-D-Ala-Gly-Phe-D-Leu-OH) prodrug 1a exhibited more favorable physicochemical properties than did DADLE for permeation across the intestinal mucosa. However, prodrug 1a, whose bioconversion to DADLE was slow, was subject to extensive biliary clearance when administered to rats in vivo. To increase the rate of esterase-catalyzed bioconversion of prodrug 1a, thus decreasing its biliary clearance, the oxymethyl-modified prodrug 1, in which an aldehyde equivalent is inserted between the phenolic group of the promoiety and the carboxylic acid group of the peptide, was synthesized from benzofuran-2-carboxylic acid 16 via a nine-step procedure. Briefly, phenacyl-protected 3-(2-hydroxyphenyl)-propynoic acid 17 was coupled with Boc-d-Leu-OCH(2)I 5 to give the intermediate 18, which was further elaborated and conjugated with tetrapeptide 4 to give linear precursor 2. Precursor 2 was then deprotected and cyclized to obtain compound 1 using a high dilution technique. In an attempt to investigate the effect of the physicochemical properties and the conformation of prodrug 1 on its permeation characteristics, we calculated its physicochemical parameters and determined its solution conformation using spectroscopic techniques (CD and NMR) and molecular dynamics simulations. Prodrug 1 showed a cLogP value and a molecular size similar to that of prodrug 1a. The deconvoluted CD spectra indicated that prodrug 1 has more random component (71%) than prodrug 1a (42%). 2D-NMR studies of prodrug 1 showed no signals for amide-amide hydrogen interactions and few ROE cross-peaks in ROESY spectra. Using distance restraints constructed from ROESY spectra, molecular dynamics simulations of prodrug 1 generated five conformation families. One family satisfied most of the distance restraints and all of the dihedral angles measured by NMR coupling constants. In summary, prodrug 1 showed favorable physicochemical properties for permeation of the intestinal mucosa. Prodrug 1 adopted a more random conformation in solution than prodrug 1a. These differences in solution conformation could affect the permeation of the prodrugs across the intestinal mucosa by passive diffusion and/or their ability to interact with efflux transporter(s) that would limit their transcellular permeation.
Chemical, conformational and structure-activity studies of taxol and related taxanes are detailed. Semisynthetic methodology for the preparation of taxol and related analogues with modified C-l3 phenylisoserine side chains was developed and analogues, modified at the C-3' phenyl group and the N-benzoyl group, were prepared. 3'-Cyclohexyl and 2-cyclohexylcarbonyl taxol analogues and C-13 side chain homologated derivatives were synthesized. Methods for the selective hydrolysis of all ester groups in baccatin ΙΠ and the conversion of 4-deacetylbaccatin III to 4-deacetyltaxol are reported. Reduction of taxanes with samarium diiodide provided 10-deacetyl derivatives as well as 9-dihydrotaxanes. Conformational analysis of taxol and other bioactive derivatives demonstrated the formation of hydrophobically clustered conformations in aqueous solvents.The discovery by the Potier group that 10-deacetylbaccatin III (4) can be isolated in significant quantities from a regenerable source, the needles of the European yew tree Taxus baccata L., was the most significant finding in the attempt to secure the long term supply of the anticancer agent taxol (1) through semisynthesis (Fig. 1) (7). Extraction of the fresh needles yields 4 in amounts of up to lg/kg, which is about ten times the amount of taxol isolated from the bark (0. lg/kg). It is of importance to note that the needles are a fully regenerable source and that their harvest does not threaten the survival of the yew species. The availability of 4 also facilitated semisynthetic studies directed at the elucidation of the taxol pharmacophore (2,3). Synthesis and Biological Evaluation of C-13 Chain Modified Taxol AnaloguesSince the C-13 Af-benzoyl-3-phenylisoserine side chain of taxol is of crucial importance for taxol's cytotoxicity (4), efficient methodology for the asymmetric synthesis of the C-13 side chain 2 and its attachment to baccatin III required development (5). NOTE: Paclitaxel is the generic name for Taxol, which is now a registered trademark. 0097-6156/95/0583-0217$08.00/0
Cyclization of the linear hexapeptides increased their lipophilicities. The increased permeation characteristics of the cyclic hexapeptides as compared to their linear analogs appears to be due to an increase in their flux via the transcellular route because of these increased lipophilicities. Structural analyses of the cyclic Asp-containing hexapeptide suggest that its well-defined solution structure and, specifically the existence of two beta-turns, explain its greater lipophilicity.
In an earlier study using Caco-2 cells, an in vitro cell culture model of the intestinal mucosa, we have shown that the coumarinic-based (3 and 4) and the phenylpropionic acid-based (5 and 6) cyclic prodrugs were more able to permeate the cell monolayers than were the corresponding opioid peptides, [Leu5]-enkephalin (1, H-Tyr-Gly-Gly-Phe-Leu-OH) and DADLE (2, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH). In an attempt to explain the increased permeation of the cyclic prodrugs, we have determined the possible conformations of these cyclic prodrugs in solution, using spectroscopic techniques (2D-NMR, CD) and molecular dynamics simulations. Spectroscopic as well as molecular dynamic studies indicate that cyclic prodrug 4 exhibits two major conformers (A and B) in solution. Conformer A exhibited a type I beta-turn at Tyr1-D-Ala2-Gly3-Phe4. The presence of a turn was supported by ROE cross-peaks between the NH of D-Ala2 and the NH of Gly3 and between the NH of Gly3 and the NH of Phe4. Conformer B of cyclic prodrug 4 consisted of type II beta-turns at the same positions. The type II turn was stabilized by hydrogen bonding, thus forming a more compact structure, whereas the type I turn did not exhibit similar intramolecular hydrogen bonding. Spectroscopic data for compounds 3, 5 and 6 are consistent with the conclusion that these cyclic prodrugs have solution structures similar to those observed with cyclic prodrug 4. The increased lipophilicity and well-defined secondary structures in cyclic prodrugs 3-6, but not in the linear peptides 1 and 2, could both contribute to the enhanced ability of these prodrugs to permeate membranes.
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