Lipid II: total synthesis of the bacterial cell wall precursor and utilization as a substrate for glycosyltransfer and transpeptidation by penicillin binding protein (PBP) 1b of Escherichia coli.

An essential feature in the life cycle of both gram positive and gram negative bacteria is the production of new cell wall. Also known as murein, the cell wall is a two-dimensional polymer, consisting of a linear, repeating N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) motif, cross-linked via peptides appended to MurNAc. The final steps in the maturation of murein are catalyzed by a single, bifunctional enzyme, known as a high MW, class A penicillin binding protein (PBP). PBPs catalyze polymerization of the sugar units (glycosyltransfer), as well as peptide cross-linking (transpeptidation) utilizing Lipid II as substrate. Detailed enzymology on this enzyme has been limited, due to difficulties in obtaining sufficient amounts of Lipid II, as well as the availability of a convenient and informative assay. We report the total chemical synthesis of Lipid II, as well as the development of an appropriate assay system and the observation of both catalytic transformations.

Identification of selective inhibitors of cyclin dependent kinase 4.

A new structural type of kinase inhibitor, containing a benzocarbazole nucleus, has been identified. Members of the series are selective for inhibition of the cyclin dependent kinase family of enzymes. Although the cdks are highly homologous, representatives of the series showed intra-cdk selectivities, especially for cdk4. SAR studies elucidated the important features of the molecules for inhibition.

Synthesis and biological activities of potential metabolites of the non-nucleoside reverse transcriptase inhibitor efavirenz.

Studies on the biotransformation of the clinically important non-nucleoside reverse transcriptase inhibitor efavirenz have shown that oxidation and secondary conjugation are important components of the processing of this molecule in vivo. We have synthesized metabolites of efavirenz to confirm their structure and to evaluate their activity as antivirals.

3,3a-Dihydropyrano[4,3,2-de]quinazolin-2(1H)-ones are potent non-nucleoside reverse transcriptase inhibitors.

A series of unique 3,3a-dihydropyrano[4,3,2-de]quinazolin-2(1H)-ones and a 2a,5-dihydro-2H-thieno[4,3,2-de]quinazo-line-4(3H)-thione were found to be HIV-1 non-nucleoside reverse transcriptase inhibitors. One of these compounds, as the racemate, possessed an IC90 = 4.6 nM against wild-type virus in a whole cell antiviral assay and had an IC90 = 76 and 897 nM against the clinically significant K103N and K103N/L100I mutant viruses, respectively.

The species-dependent metabolism of efavirenz produces a nephrotoxic glutathione conjugate in rats.

Efavirenz, a potent nonnucleoside reverse transcriptase inhibitor widely prescribed for the treatment of HIV infection, produces renal tubular epithelial cell necrosis in rats but not in cynomolgus monkeys or humans. This species selectivity in nephrotoxicity could result from differences in the production or processing of reactive metabolites, or both. A detailed comparison of the metabolites produced by rats, monkeys, and humans revealed that rats produce a unique glutathione adduct. The mechanism of formation and role of this glutathione adduct in the renal toxicity were investigated using both chemical and biochemical probes. Efavirenz was labeled at the methine position on the cyclopropyl ring with the stable isotope deuterium, effectively reducing the formation of the cyclopropanol metabolite, an obligate precursor to the glutathione adduct. This substitution markedly reduced both the incidence and severity of nephrotoxicity as measured histologically. Further processing of this glutathione adduct was also important in producing the lesion and was demonstrated by inhibiting gamma-glutamyltranspeptidase with acivicin pretreatment (10 mg/kg, IV) prior to dosing with efavirenz. Again, both the incidence and severity of the nephrotoxicity were reduced, such that four of nine rats given acivicin were without detectable lesions. These studies provide compelling evidence that a species-specific formation of glutathione conjugate(s) from efavirenz is involved in producing nephrotoxicity in rats. Mechanisms are proposed for the formation of reactive metabolites that could be responsible for the renal toxicity observed in rats.

Synthesis and evaluation of analogs of Efavirenz (SUSTIVA) as HIV-1 reverse transcriptase inhibitors.

Efavirenz (SUSTIVA) is a potent non-nucleoside reverse transcriptase inhibitor. Due to the observation of breakthrough mutations of the reverse transcriptase enzyme during Efavirenz therapy, we sought to develop an optimized second generation series. To that end, SAR of the substituents on the aromatic ring was undertaken and the results are summarized here. The 5,6-difluoro (4f) and the 6-methoxy (4m) substituted benzoxazinones were determined to be equipotent, and as a result such substitution patterns will be incorporated in second generation scaffolds.

Identification and characterization of efavirenz metabolites by liquid chromatography/mass spectrometry and high field NMR: species differences in the metabolism of efavirenz.

Efavirenz (Sustiva, Fig. 1) is a potent and specific inhibitor of HIV-1 reverse transcriptase approved for the treatment of HIV infection. To examine the potential differences in the metabolism among species, liquid chromatography/mass spectrometry profiles of efavirenz metabolites in urine of rats, guinea pigs, hamsters, cynomolgus monkeys, and humans were obtained and compared. The metabolites of efavirenz were isolated, and structures were determined unequivocally by mass spectral and NMR analyses. Efavirenz was metabolized extensively by all the species as evidenced by the excretion of none or trace quantities of parent compound in urine. Significant species differences in the metabolism of efavirenz were observed. The major metabolite excreted in the urine of all species was the O-glucuronide conjugate (M1) of the 8-hydroxylated metabolite. Efavirenz was also metabolized by direct conjugation with glucuronic acid, forming the N-glucuronide (M2) in all five species. The sulfate conjugate of 8-OH efavirenz (M3) was found in the urine of rats and cynomolgus monkeys but not in humans. In addition to the aromatic ring-hydroxylated products, metabolites with a hydroxylated cyclopropane ring (at C14) were also isolated. GSH-related products of efavirenz were identified in rats and guinea pigs. The cysteinylglycine adduct (M10), formed from the GSH adduct (M9), was found in significant quantities in only rat and guinea pig urine and was not detected in other species. In vitro metabolism studies were conducted to show that the GSH adduct was produced from the cyclopropanol intermediate (M11) in the presence of only rat liver and kidney subcellular fractions and was not formed by similar preparations from humans or cynomolgus monkeys. These studies indicated the existence of a specific glutathione-S-transferase in rats capable of metabolizing the cyclopropanol metabolite (M11) to the GSH adduct, M9. The biotransformation pathways of efavirenz in different species were proposed based on some of the in vitro results.

Cyclic HIV protease inhibitors: synthesis, conformational analysis, P2/P2' structure-activity relationship, and molecular recognition of cyclic ureas.

High-resolution X-ray structures of the complexes of HIV-1 protease (HIV-1PR) with peptidomimetic inhibitors reveal the presence of a structural water molecule which is hydrogen bonded to both the mobile flaps of the enzyme and the two carbonyls flanking the transition-state mimic of the inhibitors. Using the structure-activity relationships of C2-symmetric diol inhibitors, computed-aided drug design tools, and first principles, we designed and synthesized a novel class of cyclic ureas that incorporates this structural water and preorganizes the side chain residues into optimum binding conformations. Conformational analysis suggested a preference for pseudodiaxial benzylic and pseudodiequatorial hydroxyl substituents and an enantiomeric preference for the RSSR stereochemistry. The X-ray and solution NMR structure of the complex of HIV-1PR and one such cyclic urea, DMP323, confirmed the displacement of the structural water. Additionally, the bound and "unbound" (small-molecule X-ray) ligands have similar conformations. The high degree of preorganization, the complementarity, and the entropic gain of water displacement are proposed to explain the high affinity of these small molecules for the enzyme. The small size probably contributes to the observed good oral bioavailability in animals. Extensive structure-based optimization of the side chains that fill the S2 and S2' pockets of the enzyme resulted in DMP323, which was studied in phase I clinical trials but found to suffer from variable pharmacokinetics in man. This report details the synthesis, conformational analysis, structure-activity relationships, and molecular recognition of this series of C2-symmetry HIV-1PR inhibitors. An initial series of cyclic ureas containing nonsymmetric P2/P2' is also discussed.

Synthesis and NMR conformational analysis of a beta-turn mimic incorporated into gramicidin S. A general approach to evaluate beta-turn peptidomimetics.

The solution structure of a gramicidin S (GS) analog containing a beta-turn mimic [BTD4-5, Lys2.2']GS has been compared to that of native GS. The linear [BTD4-5, Lys2.2']GS was synthesized by solid phase methodology and the cyclized peptide was analyzed by NMR. In the peptide portion of [BTD4-5, Lys2.2']GS, the intramolecular hydrogen bonding pattern, inter-residue NOEs, including a transannular H alpha-H alpha NOE, and JN alpha coupling constants all describe a solution structure which is equivalent to that of native GS. These data confirm that the BTD group is a competent Type II' beta-turn mimic since it does not disrupt the native conformation of GS. It also supports the use of GS as a conformational model in which to test beta-turn mimics.