Synthesis and conformational analysis of a coumarinic acid‐based cyclic prodrug of an opioid peptide with modified sensitivity to esterase‐catalyzed bioconversion

Abstract

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.

Cyclization-activated Prodrugs

“…Another approach, quite similar to the "trimethyl lock" concept, for the design of two-step prodrugs has been based on coumarinic acid derivatives, as these equally undergo facile lactonization [83]. In this case, the "conformation lock" that favors lactonization is due to the cis double bond of the coumarinic acid aliphatic chain (Scheme 15B), and enzyme-sensitive two-step prodrugs can be conceived in a virtually superimposable way to that depicted above in Scheme 13 for "trimethyl lock"-based pro-prodrugs [6,[83][84][85][86][87][88]. This approach yielded model two-step prodrugs of amines that were released upon incubation in the presence of porcine liver esterase, with t 1/2 between 1.7 and 35 min [6,84].…”

“…Scheme 15. Cyclic two-step prodrugs of peptides based on: (A) the "trimethyl lock" [80,81] and, (B) coumarinic acid derivatives [85][86][87][88][89][90][91]. Another approach, quite similar to the "trimethyl lock" concept, for the design of two-step prodrugs has been based on coumarinic acid derivatives, as these equally undergo facile lactonization [83].…”

“…This approach yielded model two-step prodrugs of amines that were released upon incubation in the presence of porcine liver esterase, with t 1/2 between 1.7 and 35 min [6,84]. The most relevant example of application of such coumarinic acid derivatives concerns cyclic proprodrugs of the opioid peptide DADLE (22), depicted in Scheme 15B [85,86]. This approach was also employed to oxymethyl-modified cyclic pro-prodrugs of DADLE (23, Scheme 15B), designed to promote better intrinsic cell permeation while preserving the desired sensitivity to esterases [87][88][89][90][91], and to RGD peptidomimetics [92].…”

Evaluation of the Permeation Characteristics of a Model Opioid Peptide, H-Tyr-d-Ala-Gly-Phe-d-Leu-OH (DADLE), and Its Cyclic Prodrugs across the Blood-Brain Barrier Using an In Situ Perfused Rat Brain Model

“…1). Unlike DADLE, which is hydrophilic and charged, AOA-DADLE (Bak et al, 1999b), CA-DADLE (Wang et al, 1999), and OMCA-DADLE (Ouyang et al, 2002a) are lipophilic and uncharged. The physicochemical properties of these cyclic prodrugs of DADLE are indicative of solutes that have good cell membrane permeation characteristics (Pauletti et al, 1997).…”

“…In contrast, GF-120918 had no effect on the P app value of DADLE. In addition, the observed bioconversions of the prodrugs to DADLE in the rat brains after 240-s perfusion were very low (5.1% from AOA-DADLE, 0.6% from CA-DADLE, and 0.2% from OMCA-DADLE), which was consistent with the in vitro bioconversion rates determined previously in rat brain homogenates.In an attempt to improve the blood-brain barrier (BBB) permeation of H-Tyr-D-Ala-Gly-Phe-D-Leu-OH (DADLE), an opioid peptide (Hill and Pepper, 1978;Iyengar et al, 1987;Prokai-Tatrai et al, 1996), our laboratory has synthesized cyclic prodrugs of this peptide using an acyloxyalkoxy (AOA) linker (Bak et al, 1999b), a coumarinic acid (CA) linker (Wang et al, 1999), and an oxymethyl-modified coumarinic acid (OMCA) linker (Ouyang et al, 2002a) (Fig. 1).…”