Stability Study of Phoslactomycin B and Analysis of Degradation Products

STABILITY STUDY OF PHOSLACTOMYCIN B AND ANALYSIS OF DEGRADATION PRODUCTS. By Suparna Das Choudhuri, M.S. A thesis submitted in the partial fulfillment of the requirements for the degree of Masters of Science from Virginia Commonwealth University. Virginia Commonwealth University, 2005 Chairperson of the Graduate CommitteeDr. Kevin A. Reynolds, PhD Department of Medicinal Chemistry. Phoslactomycin B (PLM-B), a potent and selective inhibitor of serine threonine phosphatase is of interest for its antitumor, antifungal and antiviral activity. The objective of this study was to evaluate the stability of phoslactomycin B at various pH and temperature conditions. Phoslactomycin B was produced from the mutant strain NP1 of Streptomyces sp.HK-803 and was purified by semi-preparative HPLC . A study of PLM-B degradation was carried out in the pH range of 2-10 at 30oC and 50°C using HPLC. The PLM-B decomposition was observed to exhibit a U-shaped pH profile and demonstrated both acid and base-catalyzed decomposition. The decomposition could be described by the equation kOBS = kH x 10 + xv kOH x 10 pH14 (kH = 45±7 M h; kOH = 448± 73 M h). PLM-B was found to be most stable at pH 6.63. The degradation products in both acidic and basic pH have been collected and analyzed. Under basic condition, mass spectroscopic analysis revealed addition of water and NMR was consistent with the major products being formed due to lactone ring opening and/or a Micheal type addition reaction. Under acidic condition MS revealed loss of water for all three compounds. NMR analysis of one product (product 8) was consistant with C9C11 phosphorinane derivative of PLM-B. The remaining compounds were shown to be mixture of various dehydration products. The degradation products despite containing small structural changes to PLM-B lost all detectable levels of antifungal activity. INTRODUCTION Streptomyces produce a series of structurally novel antifungal and antitumor agents named phoslactomycins (PLMs) [1], phospholine, phosphazomycin A, leustroducsins and fostriecin. These agents are structurally related and contain an unusual phosphate ester, an α,βunsaturated δ-lactone and either a conjugated diene or triene chain. PLMs (Figure 1) were isolated from several Streptomyces strains including Streptomyces. sp HK 803 [2]. The principal structural differences distinguishing PLMs from fostriecin [3] are replacement of the C-8 methyl substituent by ethylamine and the terminal allylic triene alcohol by a cyclohexane diene. All the above compounds including fostriecin (2) are produced in multiple forms (see Figure 1). For phoslactomycins, PLM-B [1] contains a fully reduced cyclohexyl ring, [4] whereas other PLMs and leustroducsins contain a hydroxyl substituent at (C-18) that is esterified with a wide array of carboxylic acids of length 4 to 9 carbons. Antifungal activity of PLMs The PLMs and phosphazomycins were isolated based on their antifungal activity. All the analogs were found to exhibit comparable antifungal activity [5]. PLM-E was shown to be active against various fungi, particularly phytopathogenic fungi (Botrytis cinerea, Alternaria kikuchiana, Alternaria mali, Mycosphaerella melonis, Chaetomium globosum, Verticillium albo-atrum and Pseudcercisporella herpotrichiodes). PLMs A, B, C, D, E, and F on comparison were also found to have almost the same antifungal activity [6]. The difference in the chemical structures between each compound is the substituent bound to a cyclohexane ring. This substituent would appear not to be important for antifungal activity.