WRIGHT, GEORGE E
Scope of overall project and anticipated contribution of supplement.
The overall project is to advance a hybrid antibiotic "AU-FQ" compound consisting of a Gram+ DNA polymerase III inhibitor and a fluoroquinolone, to IND status with the FDA. Experiments include selection of a lead compound and its formulation, studies of efficacy, toxicity and PK in the rat, and the GLP toxicology and pharmacology and GMP production necessary to complete FDA-mandated preclinical studies. The compound family is targeted to infections with antibiotic-resistant Gram+ bacteria, such as MRSA and VRE.
During the PK studies of these compounds in mice and rats, we identified a major metabolite that appears to be a glucuronide of the parent drug. The science educator and student were to devote their summer to confirm the structure of this metabolite derived from our preclinical candidate "259C" by chemical synthesis of this compound, independent determination of its PK properties, and determination if the metabolite has antibacterial activity. Understanding the behavior of this metabolite in vivo will add to our understanding of the dynamics and utility of the parent compound, increase the quality of the IND, and add to the investment/licensing package that we will prepare for financing clinical trials.
A direct approach to the identification of the metabolite is the chemical synthesis of the acyl glucuronide.
The allyl ester of glucuronic acid was first prepared. A suspension of 9.25 mmole (1 eq) of glucuronic acid 20 ml dry DMF was treated with 1.23 eq of the base DBU. After 15 minutes stirring 1.37 eq of allyl bromide was added, and the reaction mixture was stirred overnight. The product was purified by silica gel chromatography and mixed-bed ion chromatography, providing 0.77 g (36% yield) of the product.
The coupling of this ester to 259C was attempted using the Mitsunobu method (triphenylphosphine, DIAD). Chromatography of reaction mixtures gave fractions that were (LC/MS) shown to be the starting material, 259C.
An experiment was designed to determine the identity of the 259C glucuronide. The drug was treated with human liver microsomes under conditions that afford the maximum amount of glucuronide metabolite. After working up the sample, a 2 M solution of TMS-diazomethane in ether (which furnishes diazomethane on hydrolysis) was added. The sample was analyzed by LC/MS. The expectation was that an acyl glucuronide would take up one CH2 and show an increase in mass of 14 because a single carboxyl group is available, but a N-glucuronide would take up two CH2’s to add 28 mu because 2 carboxyl groups would be available.
LC/MS analysis of the samples showed a new peak at 7.25 minutes, with m/z 851.4 (- mode) and 853.4 (+ mode). This is consistent with the addition of just one CH2 to the 259C glucuronide (exact mass of which is 838.3). A search through the chromatogram for a peak with the mass of a di-methylated peak (ie, 865) yielded no results. Although the rat liver microsomal metabolism sample showed 259C glucuronide (m/z 838.4), no significant peaks with a mass corresponding to the addition of CH2 (or two CH2’s) were seen after addition of TMS-diazomethane. A small peak was seen corresponding to the glucuronide.
Given literature precedent, the carboxylic acid is the most likely spot for methylation with TMS-diazomethane.
The identification of the glucuronide of 259C was attempted by the chemical synthesis of the acyl glucuronide. Although seemingly straightforward, purification of the product proved difficult, and must be addressed in future. An indirect method suggested that the metabolite is indeed the acyl glucuronide.