Enhanced production of Lovastatin by filamentous fungi through solid state fermentation.

Lovastatin is a natural competitive inhibitor of 3-hydroxy-3-methyl glutaryl coenzyme-A (HMG-CoA) reductase and inhibits specifically rate limiting step in cholesterol biosynthesis. Further, lovastatin in comparison with synthetic drugs has no well-reported side effects. Four pure isolated filamentous fungal strains including Aspergillus niger IBL, Aspergillus terreus FFCBP-1053, Aspergillus flavus PML and Aspergillus nidulans FFCBP-014 have been cultured by solid state fermentation (SSF) using rice straw as substrate for the synthesis of lovastatin. After selecting Aspergillus terreus FFCBP-1053 as the best producer of lovastatin, various selected physical parameters including pH, temperature, inoculums size and moisture content were optimized through response surface methodology (RSM) under center composite design (CCD) for lovastatin hyper production. Maximum lovastatin production of 2070±91.5 was predicted by the quadratic model in the medium having moisture content 70% and pH 4.5 at 35°C which was verified experimentally to be 2140±93.25µg/g DW of FM (microgram/gram dry weight of fermentation medium), significantly (P<0.05) high as compared to un-optimized conditions while it was noted that lovastatin production is independent on inoculum size (P>0.05) measured by spectrophotometer at 245 nm against standard. It was determined that optimized conditions for the hyper-production of lovastatin from fungal sources have a significant effect.

Chemoenzymatic synthesis of statine side chain building blocks and application in the total synthesis of the cholesterol-lowering compound solistatin.

The synthesis and enzymatic reduction of several 6-substituted dioxohexanoates are presented. Two-step syntheses of tert-butyl 6-bromo-3,5-dioxohexanoate and the corresponding 6-hydroxy compound have been achieved in 89% and 59% yield, respectively. Regio- and enantioselective reduction of these diketones and of the 6-chloro derivative with alcohol dehydrogenase from Lactobacillus brevis (LBADH) gave the (5S)-5-hydroxy-3-oxo products with enantiomeric excesses of 91%, 98.4%, and >99.5%, respectively. Chain elongation of the reduction products by one carbon via cyanide addition, and by more than one carbon by Julia-Kocienski olefination, gave access to well-established statine side-chain building blocks. Application in the synthesis of the cholesterol-lowering natural compound solistatin is given.

Application of a 3,3-diphenylpentane skeleton as a multi-template for creation of HMG-CoA reductase inhibitors.

Based on our hypothesis that the 3,3-diphenylpentane (DPP) skeleton is useful as a multi-template for creation of various biologically active compounds and acts as a steroid skeleton substitute, we designed and synthesized novel HMG-CoA reductase inhibitors with a DPP skeleton. Among them, sodium (E,3R,5S)-7-(2-(4-fluorophenyl)-4-(3-phenylpentan-3-yl)phenyl)-3,5-dihydroxy-hept-6-enoate showed potent HMG-CoA reductase-inhibitory activity comparable with that of clinically useful mevastatin.

Formal enantioselective synthesis of (+)-compactin.

[reaction: see text] The challenging structural features and important biological activity of (+)-compactin (1) explain the substantial synthetic interest that it has generated. We report a novel enantioselective approach to the advanced intermediate 2a, which constitutes a formal synthesis of (+)-1. The sequence utilizes MacMillan's organocatalytic Mukaiyama-Michael reaction, which stereoselectively adds the silyloxyfuran 6 to alpha,beta-unsaturated aldehyde 7. The chirality generated in this reaction guides the formation of the other three consecutive stereocenters found in 2a.

Remote asymmetric induction in an intramolecular ionic Diels-Alder reaction: application to the total synthesis of (+)-dihydrocompactin.

The total synthesis of (+)-dihydrocompactin via an intramolecular ionic Diels-Alder reaction that proceeds with remote stereocontrol is described. This reaction proceeds by an intermediate vinyl-oxocarbenium ion (6), the conformational constraints of which lead to the observed asymmetric induction. The sense of asymmetric induction appears contrasteric and is explained by the proposed reactive conformation shown in Figure 1.

Natural insights for chemical biologists.

Chemical biologists studying natural-product pathways encoded in genomes have unearthed new chemistry and insights into the evolution of biologically active metabolites.

Chelation-controlled reduction: stereoselective formation of syn-1,3-diols and synthesis of compactin and mevinolin lactone.

Chelation-controlled reduction of chiral beta-alkoxy ketones containing a competing beta'-oxygen functionality has been investigated. Various syn-1,3-diols were prepared conveniently by reduction of beta-alkoxy ketones with LiI/LiAlH(4) (syn:anti selectivity up to >99:1). The corresponding beta-alkoxy ketones were derived from nitro-aldol reactions of chiral alkoxy aldehydes with a series of nitro compounds. This methodology is utilized in a short and efficient synthesis of the delta-lactone moiety of the HMG-CoA reductase inhibitors compactin and mevinolin.

Aza-analogue of mevinolin: synthesis of substituted octahydroisoquinoline, the bottom half of aza-analogue of mevinolin.

trans-Ethyl 1,2,3,4a,5,6,8a-octahydro-2-benzyl-4-hydroxy-6-methyl-5- isoquinoline-carboxylate was prepared by intramolecular Diels-Alder reaction as a precursor of newly designed aza-analogue of mevinolin.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors. 9. The synthesis and biological evaluation of novel simvastatin analogs.

Substitution of hydroxy and hydroxyalkyl functionality at C-7 of the hexahydronaphthalene nucleus of simvastatin has provided novel analogs. The synthetic strategy employed epoxidation or Lewis acid-catalyzed aldol reaction of the 8-keto silyl enol ether as a key reactive intermediate. These analogs were evaluated as potential hypocholesterolemic agents via initial determination of their ability to inhibit HMG-CoA reductase in vitro. Oral activity of these compounds was determined in an acute rat model and a three-week study in cholestyramine-primed dogs. Compounds were identified that possessed in vitro and in vivo activity comparable to that of simvastatin.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors. 8. Side chain ether analogues of lovastatin.

A general route for preparing side chain ether analogues of lovastatin is presented. These analogues proved to be weaker inhibitors of HMG-CoA reductase than the corresponding side chain ester analogues. Interestingly, inhibitory potency was enhanced markedly when the 4-fluoro group was incorporated in the aromatic moiety of the side chain benzyl group of 2d.

3-Hydroxy-3-methylglutaryl-coenzyme a reductase inhibitors. 7. Modification of the hexahydronaphthalene moiety of simvastatin: 5-oxygenated and 5-oxa derivatives.

Modification of the hexahydronaphthalene ring 5-position in simvastatin 2a via oxygenation and oxa replacement afforded two series of derivatives which were evaluated in vitro for inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and acutely in vivo for oral effectiveness as inhibitors of cholesterogenesis in the rat. Of the compounds selected for further biological evaluation, the 6 beta-methyl-5-oxa 10 and 5 alpha-hydroxy 16 derivatives of 3,4,4a,5-tetrahydro 2a, as well as, the 6 beta-epimer 14 of 16 proved orally active as hypocholesterolemic agents in cholestyramine-primed dogs. Subsequent acute oral metabolism studies in dogs demonstrated that compounds 14 and 16 evoke lower peak plasma drug activity and area-under-the-curve values than does compound 10 and led to the selection of 14 and 16 for toxicological evaluation.

Biochemical aspect of HMG CoA reductase inhibitors.

Subsequent to the discovery of compactin (ML-236B) as a specific inhibitor of HMG CoA reductase, a series of compactin analogs have been either isolated or synthesized. Several of these compounds, which include compactin, mevinolin (monacolin K) and CS-514, have been extensively studied. The inhibition of HMG CoA reductase by these compounds is reversible and competitive (Ki = approximately 1 nM). The 3', 5'-dihydroxypentanoic acid portion of the acid form of compactin analogs, which resembles the HMG portion of HMG CoA, plays a crucial role in inhibitory activity. These inhibitors block sterol synthesis both in cultured mammalian cells and in animals. Strong inhibition of sterol synthesis results in a marked increase in HMG CoA reductase activity both in vitro and in vivo. These compounds strongly lower plasma LDL-cholesterol levels in animals and humans. The lowering of LDL-cholesterol levels occurs by an inhibition of LDL synthesis and/or by an elevation of the receptor-mediated LDL catabolism in the liver.

Synthesis of arylnaphthalenyl derivatives: inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme a reductase.

Trans-tetrahydro-4-hydroxy-6-[1-aryl-7-naphthalenyl]-2H-pyran-2-ones of general structure 4 were prepared and tested for inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase in vitro. In contrast to previously described biphenyl lactones (2) containing an ethenyl linkage, linkage of the pyranone ring to the aryl moiety in these compounds is via a rigid aromatic ring system, which still allows free rotation of the aryl rings. The imposed conformational constraint is compatible with activity, since members of the series had activity in range 1-20 microM (IC50 values).

Total synthesis and biological evaluation of structural analogues of compactin and dihydromevinolin.

The full experimental details for the total synthesis of (+)-compactin and 19 structural analogues are reported. We have evaluated three classes of analogues as inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase: (1) functional and stereoisomeric analogues that possess the full carbon skeleton of compactin or dihydromevinolin, (2) functional analogues in which one carbon of the skeleton has been replaced by oxygen, and (3) analogues in which all of the 3,5-dihydroxyvaleric acid moiety has been omitted. Our most potent inhibitors belong to the first class of analogues. Compounds 42 (5-ketocompactin) and 69 (5-ketodihydromevinolin) are as active as the natural products compactin and dihydromevinolin, respectively (I50 = 1-20 nM). The corresponding enones 37 and 68 are less active, having I50 values 20-30 times larger. Inverting the stereochemistry at C-3 or C-5 or about the hexahydronaphthalene ring of compactin results in the elevation of the I50 to values in the micromolar range, comparable to the KM of the natural substrate 3-hydroxy-3-methylglutaryl coenzyme A. Class 2 analogues are active in this concentration range also. The synthetic sequence developed for compactin and its analogues includes a new method that permits the selective preparation of either the R or the S epimer at C-3 of the 3,5-dihydroxyvaleric acid moiety. This entails the reaction of anhydride 9 with either (R)- or (S)-1-phenylethanol in the presence of 4-(N,N-dimethylamino)pyridine and triethylamine. The prochiral recognition is surprisingly high; under optimum conditions, the reaction of 9 with (R)-1-phenylethanol leads to a 15:1 ratio of diesters 17 and 18.