Synthesis and Metabolism of Drugs by Means of Enzyme-Catalysed Reactions
Total Page：16
File Type：pdf, Size：1020Kb
ADVANCED BIOTECHNOLOGY 536 CHI MIA 1999, 53, No. 11 Chimia 53 (1999) 536-540 © Neue Schweizerische Chemische Gesellschaft ISSN 0009-4293 Synthesis and Metabolism of Drugs by Means of Enzyme-Catalysed Reactions Hans Georg W. Leuenberger*), Peter K. Matzinger, and Beat Wirz Abstract. The usefulness of enzyme catalysed-reactions is exemplified by recent results from research at Roche. Sequences of enzyme reactions, well organised in metabolic pathways of selected microorganisms, lead to secondary metabolites with innovative chemical structures. An example is the pancreas-lipase inhibitor lipstatin produced by Streptomyces toxytricini. Hydrogenation of lipstatin yields tetrahydrolipstatin, the active substance of the anti-obesity drug Xenical™. The biosynthetic pathway has been elucidated and an improved fermentation process for the production of lipstatin has been developed. Intermediates of the primary metabolism can be valuable building blocks for the chemical synthesis of drugs. Examples are quinic acid and shikimic acid, which are both suitable starting materials for the synthesis of the neuraminidase inhibitor GS 4104. Metabolic engineering of Escherichia coli with the goal to overproduce these two substances is briefly described Microorganisms or enzymes derived thereof are used in drug synthesis to catalyse single, highly specific reaction steps (biotransformations). Three examples yielding chiral precursors of a protein-kinase inhibitor, a collagenase inhibitor, and an antifungal compound are discussed Recombinant Escherichia coli strains expressing human drug-metabolising enzymes are suited to mimic drug metabolism and to produce intermediates of human drug metabolism. The desired hydroxylated drug derivatives could be obtained after incubation of drug substances with strains coexpressing one specific human cytochrome P450 isozyme together with human reductase. 1. Introduction catalyse single, highly specific reac- the anti-obesity drugXenical™. Tetrahy- tion steps (biotransformations) diffi- drolipstatin can be obtained by chemical Microorganisms with their great variety cult to perform by chemical methods. synthesis [3] as well as by fermentative of highly specific enzymes have proven to - Recombinant microorganisms express- production of lips tat in followed by hydro- be useful catalysts in medicinal chemis- ing human drug-metabolising enzymes genation. However, original fermentation try: are used to mimic drug metabolism yields of lipstatin amounted only to a few - Sequences of enzyme reactions, well and to produce intermediates of human milligrams per litre. organised in metabolic pathways, lead liver drug metabolism. The biosynthesis of lipstatin was in- to a variety of secondary metabolites Recent applications from the pharmaceu- vestigated in Streptomyces toxytricini by with innovative chemical structures. tical research at Hoffmann-La Roche will trace studies with crude (U_13C)-lipid mix- Many such secondary metabolites ex- be discussed. tures obtained from algal biomass cul- hibit pharmaceutical activities, e.g. as tured with 13C02. The data showed that antibiotics or enzyme inhibitors. the carbon backbone oflipstatin is built up - Intermediates of the primary metabo- 2. Lipstatin, the Precursor of by Claisen condensation of a C14- and an lism can be valuable building blocks XenicafTM Cg moiety before the L-leucine residue is for the chemical synthesis of drugs. added [4]. Their overproduction in suitable mi- Lipstatin is a potent, irreversible inhib- The fermentation process for lipstatin croorganisms can be initiated by ge- itor of pancreas lipase, the key enzyme in could be significantly improved by estab- netic engineering. dietary fat absorption. It has been detected lishing a fed-batch process with feeding of - Microorganisms or enzymes derived in a screening for lipase inhibition in broths linoleic acid (which is readily degraded by thereof are used in drug synthesis to from soil microorganisms. Lipstatin is pro- twofold {J-oxidation into the required Cl4 duced by Streptomyces toxytricini [1][2]. unit), caprylic acid (C8 precursor) and L- The molecule consists of a hydrocarbon leucine [5]. Feeding of these precursors to 'Correspondence: Dr. H.G.W. Leuenberger backbone bearing two double bonds, a {3- a typical run with 81 of starting volume in F. Hoffmann-La Roche Ltd. Department PRPB lactone structure essential for the biologi- a 141 reactor was initiated after a growth p.e.Box cal activity and a hydroxy group substitut- phase of 47 h. In order to avoid inhibitory CH-4070 Basel ed with N-formyl-L-leucine (Fig.). effects, fatty acids were added in a way Tel.: into +41 61 6884561 Fax: int +41 61 6881673 Hydrogenation of lipstatin yields tet- that the concentration of each remained E-Mail: [email protected] rahydrolipstatin, the active substance of below 70 mg/I. Lipstatin concentration in ADVANCED BIOTECHNOLOGY 537 CHI MIA 1999, 53. No. 11 such a fed-batch experiment was 150 mg/ Iafter an incubation period of 138 hours [5]. Further strain improvement and proc- ess optimisation is in progress. ••.. NHCHO 3. Quinic Acid and Shikimic Acid as Starting Materials for the Synthesis of the Neuraminidase Inhibitor GS 4104 Neuraminidase is an enzyme essential for the propagation of the influenza virus. The sialic-acid analogue substance GS 4104 (Ro 64-0796; Tamiflu™) is a potent inhibitor of neuraminidase and therefore a ••--NHCHO promising drug substance for the preven- tion and treatment of influenza [6]. It is o under advanced development jointly by 9 Hoffmann-La Roche Ltd. and Gilead Sci- ences Inc. .J~ Starting materials for the synthesis of GS 4104 [6-8] are either quinic acid (QA) Figure. Structures of the pancreas-lipase inhibitors lipstatin (a fermentation product from Strep- or shikimic acid (SA) (Scheme 1) which tomyces toxytricim) and tetrahydrolipstatin (XenicaI™) are both available by extraction from plant sources. Scheme 1. (-)-Quinic Acid (QA) or (-)-Shikimic Acid (SA) as Precursors of the Neuraminidase QA and SA are both intermediates in Inhibitor GS-4104 (for details, see [6-8]) the common biosynthetic pathway lead- ing to aromatic amino acids (Scheme 2). Therefore, it was obvious to consider mi- ,')(,ooH crobial fermentation as a production meth- QA od. H(t'~OH Recombinant Escherichia coli strains • which overproduce QA or SA have been 5H eOOEI engineered by streamlining carbon flow into the respective metabolic pathway, by repeated cloning of the genes encoding for -- ~","H2H3P04 rate-limiting key enzymes as well as by eOOH knocking out the genes encoding the two ) ~HAC isozymes of shikimate kinase [9]. The latter step prevents further processing of GS-41 04-02 the metabolic intermediate SA (or its pre- SA Ro 64-0796/002 cursor QA) to shikimk acid-3-phosphate H~~ and further towards aromatic amino acids OH (Scheme 2). The overproduced intermedi- ates, QA or SA, are excreted and the strains become auxotrophic for aromatic of a precursor of protein-kinase inhibitor brevis, yielding the isolated product in amino acids. Resulting strains produced in Ro 43-2759. The synthetic pathway (de- 82% from regioisomerically pure substrate a fed-batch process 60 gIl ofQA within 60 signed by Ulrich Zutter, Roche, Basel la. h (side product: 3-dehydro-QA) or 27.2 gI [10]) is based on asymmetric microbial For economic reasons, the attempt was I of SA in 42 h (side products: QA and 3- reduction of the fl-keto ester la to the made to use directly the unpurified keto dehydro-SA) [9]. Further strain and proc- respective cis-(3R,4S)-configured hydroxy ester substrate containing la and about ess development is in progress. ester 2 (Scheme 3). The theoretical yield is 25% of the unwanted regioisomer lb. If 100% because of in situ racemisation of the microorganism is able to reduce selec- substrate la. tively the desired regioisomer la, the re- 4. Biotransformations A microbial screening with various maining regioisomer lb can easily be re- yeasts and fungi was carried out. Most of moved by decarboxylation under alkaline 4.1. Microbial Hydrogenation of the strains tested afforded preferentially conditions and subsequent extraction of Racemic p-Keto Ester 1a in the the cis-configured product. The best five the reaction product. Kloeckera brevis Synthesis of the Protein-Kinase strains generated both centres of asymme- conveniently did exhibit this additional Inhibitor Ro 43-2759 try with excellent specificity. The enanti- regioselectivity: only the desired isomer A microbial hydrogenation with a use- omeric excess as well as the cis/trans- la was reduced and afforded the cis-hy- ful combination of regio- and enantiose- purity was 97% or better. The best result droxy ester in excellent steric purity. In a lectivity was employed for the synthesis was obtained with the yeast Kloeckera typical fermentation run, 29 g of 1 (3.5 g/ ADVANCED BIOTECHNOLOGY 538 CHIMIA 1999, 53, No. 11 Scheme 2. Metabolic Pathway Towards Aromatic Amino Acids. DAHP: 3-Deoxy-o-arabino- I) were fed to a pre-grown stirred culture of heptulosonic acid-7-phosphate; DHQ: 3-Dehydro-quinic acid; DHS: 3-Dehydro-shikimic acid; Kloeckera brevis supplemented with 5% S3P: Shikimic acid-3-phosphate glucose. After] 2 h of incubation at 27 ec, the culture broth was extracted with ethyl OOH0,\ XOOH H acetate. The yield of isolated product 2 /HXO was 80% (98% ee and 99% cis-purity). Thus, Kloeckera brevis exhibited excel- . H ~H HO·l.AH lent regio- and stereoselectivity in this H203P;+6H 6H 6H hydrogenation reaction and afforded the DAHP DHO OA desired product 2 in high chemical yield t and steric purity. t 4.2. Enzymatic Resolution of t Racemic Ester 3 Yielding a Chiral glucose Precursor of the Collagenase XH Inhibitor Ro 32-3555 Collagenase inhibitor Ro 32-3555 had to be prepared in larger amounts as a ~H candidate for clinical trials. Among sever- 6H al synthetic approaches, the most advanced DHS at that time proceeded via the benzyI- protected (R)-cyclopentyllactate (R)-3. En- zymatic kinetic resolution of racemic es- ]' ter 3 with preferential hydrolysis of the (S)-ester was investigated as an approach to produce (R)-3 (Scheme 4).