SB-253514 and Analogues. Novel Inhibitors of Lipoprotein Associated Phospholipase A2 Produced by Pseudomonas fluorescens DSM 11579. III. Biotransformation Using Naringinase.

The Journal of Antibiotics
2000.0

Abstract

Lipoprotein associated phospholipase A2 (LpPLA2) converts phosphatidylcholine to lysophosphatidylcholine and oxidised free fatty acids, which are potent chemoattractants for circulating monocytes and contribute to macrophage proliferation and endothelial dysfunction, making LpPLA2 inhibition an attractive strategy for treating atherosclerosis. Two isomeric series of LpPLA2 inhibitors were isolated from culture broths of Pseudomonas fluorescens DSM11579, with the most active compounds based on a 5:5 bicyclic carbamate function and rhamnose-substituted β-hydroxy fatty acid derived side chains. The most abundant inhibitor, SB-253514 (1), was a highly selective competitive inhibitor of LpPLA2, active when administered intravenously to Watanabe heritable hyperlipidaemic (WHHL) rabbits but inactive when dosed orally. Its stereochemistry was undetermined as crystals for X-ray crystallography could not be obtained. Deglycosylation of 1, SB-253517 (2), and SB-253518 (3) was attempted to improve bioavailability and obtain crystalline products. Acid and base catalysed hydrolysis failed due to carbamate sensitivity, so enzymatic methods were explored. Among six enzymes screened, only naringinase (a crude preparation from Penicillium decumbens with α-rhamnosidase activity) showed desired activity, with 15% conversion initially. Optimisation via a designed array experiment identified a system with 1 mg/ml substrate in 10% methanolic buffer (pH 4.8, 48°C) and 3U enzyme per mg substrate, yielding 63% conversion. Adding bisulphite (Na2S2O5) to complex liberated rhamnose increased conversion to >98%. Larger scale reactions produced the deglycosylated compound SB-311009 (4). Deglycosylation increased potency of saturated side-chain species (1, 3) but reduced that of unsaturated 2. SB-311009 exhibited improved oral bioavailability in WHHL rabbits with significant and prolonged LpPLA2 inhibition, and crystallised successfully from methanol at 7°C, allowing stereochemistry determination via X-ray crystallography.

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