Strategic use of preparative chiral chromatography for the synthesis of a preclinical pharmaceutical candidate

The modern use of preparative chromatography in pharmaceutical development is illustrated by the case of a recent preclinical candidate from these laboratories. The synthesis of the candidate employed a coupling of two enantiopure intermediates, each of which could be resolved using preparative chiral chromatography. SFC screening was employed to identify the enantioselective stationary phases, and semipreparative SFC methods derived from this screening were used to produce gram amounts of enantiopure intermediate for initial studies. However, initial larger scale resolution required the translation of the SFC methods to HPLC conditions. Preparative chiral HPLC on a 30-cm i.d. column was then used to produce enantiopure intermediates which were coupled to give 170 g of the preclinical candidate. Subsequent preparation of the candidate at larger scale for later-stage clinical evaluation employed an improved synthesis in which one component was constructed by asymmetric synthesis. Resolution of the other component, now a more advanced intermediate, was carried out using newly obtained large-scale SFC equipment. Some discussion is presented on the varying strategies whereby preparative chiral chromatography can be used to support either short-term or long-term synthetic goals in preclinical pharmaceutical development.     

Biocatalysts for the pharmaceutical industry created by structure-guided directed evolution of stereoselective enzymes

Enzymes have been used for a long time as catalysts in the asymmetric synthesis of chiral intermediates needed in the production of therapeutic drugs. However, this alternative to man-made catalysts has suffered traditionally from distinct limitations, namely the often observed wrong or insufficient enantio- and/or regioselectivity, low activity, narrow substrate range, and insufficient thermostability. With the advent of directed evolution, these problems can be generally solved. The challenge is to develop and apply the most efficient mutagenesis methods which lead to highest-quality mutant libraries requiring minimal screening. Structure-guided saturation mutagenesis and its iterative form have emerged as the method of choice for evolving stereo- and regioselective mutant enzymes needed in the asymmetric synthesis of chiral intermediates. The number of (industrial) applications in the preparation of chiral pharmaceuticals is rapidly increasing. This review features and analyzes typical case studies.     

酶法制备羟甲基戊二酰CoA还原酶抑制剂手性中间体的研究进展

羟甲基戊二酰CoA(HMG-CoA)还原酶抑制剂是降血脂药物。综述了近年来国内外酶法制备HMG-CoA还原酶抑制剂手性中间体的研究进展,从不对称合成和外消旋体拆分2个方面介绍了酶法制备HMG-CoA还原酶抑制剂手性中间体的工艺路线和研究水平,对其工业化前景进行了展望。     

Development of a cyclosporin A derivative with excellent anti-hepatitis C virus potency

Synthetic modification of cyclosporin A at P3-P4 positions led to the discovery of NIM258, a next generation cyclophilin inhibitor with excellent anti-hepatitis C virus potency, with decreased transporter inhibition, and pharmacokinetics suitable for coadministration with other drugs. Herein is disclosed the evolution of the synthetic strategy to from the original medicinal chemistry route, designed for late diversification, to a convergent and robust development synthesis. The chiral centers in the P4 fragment were constructed by an asymmetric chelated Claisen rearrangement in the presence of quinidine as the chiral ligand. Identification of advanced crystalline intermediates enabled practical supply of key intermediates. Finally, macrocyclization was carried out at 10% weight concentration by a general and unconventional “slow release” concept.     

Synthesis of 7-thiaarachidonic acid as a mechanistic probe of prostaglandin H synthase-2

The mechanism by which prostaglandin synthase converts arachidonic acid to prostaglandin G(2), creating five new chiral centers in the process, is still incompletely understood. The first radical intermediate has been characterized by EPR spectroscopy but subsequent proposed intermediates have not succumbed to detection. We report the synthesis of 7-thiaarachidonic acid designed to stabilize one of the proposed radical intermediates, which may allow its detection.     

Microbial conversion of indene to indandiol: a key intermediate in the synthesis of CRIXIVAN

Indene is oxidized to mixtures of cis- and trans-indandiols and related metabolites by Pseudomonas putida and Rhodococcus sp. isolates. Indene metabolism is consistent with monooxygenase and dioxygenase activity. P. putida resolves enantiomeric mixtures of cis-1,2-indandiol by further selective oxidation of the 1R, 2S-enantiomer yielding high enantiomeric purity of cis-(1S, 2R)-indandiol, a potential intermediate in the synthesis of indinavir sulfate (CRIXIVAN), a protease inhibitor used in the treatment of AIDS. Molecular cloning of P. putida toluene dioxygenase in Escherichia coli confirmed the requirement for the dihydrodiol dehydrogenase in resolving racemic mixtures of cis-indandiol. Rhodococcus sp. isolates convert indene to cis-(1S, 2R)-indandiol at high initial enantiomeric excess and one isolate also produces trans-(1R, 2R)-indandiol, suggesting the presence of monooxygenase activity. Scale up and optimization of the bioconversions to these key synthons for chiral synthesis of potential intermediates for commercial manufacture of indinavir sulfate are described.     

基于噁唑烷酮的不对称催化合成构建多官能化手性氨基酸类化合物

多官能化手性氨基酸及其衍生物是一类重要的手性药物以及合成手性药的关键中间体,如现在大量用于临床的左甲状腺素、赖诺普利、阿莫西林、缬沙坦、头孢氨苄以及青霉素等。进行多官能化手性氨基酸类化合物的不对称催化合成,可为新型化学药的设计与发现开辟新的视野。噁唑烷酮(Azlactone)被证明是合成四取代氨基酸衍生物的优秀底物。可通过不对称催化手段向其中引入需要的基团,再经多取代的噁唑烷酮直接开环得到一系列的目标化合物。本文主要综述了近年来基于恶唑烷酮的不对称催化反应构建四取代氨基酸类化合物的研究。     

Synthesis and structure-activity relationships of 1-arylmethyl-3-(1-methyl-2-amino)ethyl-5-aryl-6-methyluracils as antagonists of the human GnRH Receptor

A new class of small molecule GnRH antagonists, the 1-arylmethyl-3-(1-methyl-2-amino)ethyl-5-aryl-6-methyluracils, was designed and a novel stereoselective synthesis for these compounds was developed. The stereochemical integrities of key intermediates (S)-6 and (R)-6 were confirmed by a combination of X-ray crystallography and chiral HPLC determinations. SAR studies were performed, which allowed the identification of derivatives (R)-9f, (R)-9h and (R)-12 as potent hGnRH antagonists (K(i)=20 nM).     

微生物酶转化合成手性药物的研究进展

通过微生物酶催化不对称合成反应或拆分外消旋体合成医药手性中间体具有独特的优势。结合作者自身近年来在该技术领域的实践对相关课题作了介绍,总结了微生物酶催化不对称反应和拆分反应得到手性药物的研究进展。     

Continuous production of erythrulose using transketolase in a membrane reactor

Transketolase can be used for synthesis of chiral intermediates and carbohydrates. However the enzyme is strongly deactivated by the educts. This deactivation depends on the reactor employed. An enzyme membrane reactor allows the continuous production of L-erythrulose with high conversion and stable operational points. A productivity (space-time yield) of 45g L d was reached.     

Biocatalytic synthesis of atorvastatin intermediates

Atorvastatin is a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, and this drug leads to decreased levels of low density lipoprotein (LDL) cholesterol. Lower LDL cholesterol has direct relationship in reducing mortality from coronary heart diseases. Lipitor® (atorvastatin calcium) was the first drug to reach the annual sales of 10 billion dollars in USA and currently is the top selling pharmaceutical product globally. Atorvastatin has a side chain containing two chiral centers as its pharmacophore and it can be synthesized either from chiral pool precursors, by using metal catalysts; or more preferably by the application of free or immobilized enzymes and whole cell biocatalysts for carrying out either asymmetric synthesis or racemic resolution. Biocatalytic synthesis methods for chiral atorvastatin intermediates employ a wide variety of biocatalysts such as alcohol dehydrogenase, 2-deoxy-d-ribose 5-phosphate aldolase, nitrilase, lipase, etc. and each of these biocatalytic processes is discussed in detail in this paper.     

Highly Enantioselective Production of Chiral Secondary Alcohols Using Lactobacillus paracasei BD101 as a New Whole Cell Biocatalyst and Evaluation of Their Antimicrobial Effects

Chiral secondary alcohols are valuable intermediates for many important enantiopure pharmaceuticals and biologically active molecules. In this work, we studied asymmetric reduction of aromatic ketones to produce the corresponding chiral secondary alcohols using lactic acid bacteria (LAB) as new biocatalysts. Seven LAB strains were screened for their ability to reduce acetophenones to their corresponding alcohols. Among these strains, Lactobacillus paracasei BD101 was found to be the most successful at reducing the ketones to the corresponding alcohols. The reaction conditions were further systematically optimized for this strain and high enantioselectivity (99%) and very good yields were obtained. These secondary alcohols were further tested for their antimicrobial activities against important pathogens and significant levels of antimicrobial activities were observed although these activities were altered depending on the secondary alcohols as well as their enantiomeric properties. The current methodology demonstrates a promising and alternative green approach for the synthesis of chiral secondary alcohols of biological importance in a cheap, mild, and environmentally useful process.     

Functional characterization of salt‐tolerant microbial esterase WDEst17 and its use in the generation of optically pure ethyl (R)‐3‐hydroxybutyrate

The two enantiomers of ethyl 3‐hydroxybutyrate are important intermediates for the synthesis of a great variety of valuable chiral drugs. The preparation of chiral drug intermediates through kinetic resolution reactions catalyzed by esterases/lipases has been demonstrated to be an efficient and environmentally friendly method. We previously functionally characterized microbial esterase PHE21 and used PHE21 as a biocatalyst to generate optically pure ethyl (S)‐3‐hydroxybutyrate. Herein, we also functionally characterized one novel salt‐tolerant microbial esterase WDEst17 from the genome of Dactylosporangium aurantiacum subsp. Hamdenensis NRRL 18085. Esterase WDEst17 was further developed as an efficient biocatalyst to generate (R)‐3‐hydroxybutyrate, an important chiral drug intermediate, with the enantiomeric excess being 99% and the conversion rate being 65.05%, respectively, after process optimization. Notably, the enantio‐selectivity of esterase WDEst17 was opposite than that of esterase PHE21. The identification of esterases WDEst17 and PHE21 through genome mining of microorganisms provides useful biocatalysts for the preparation of valuable chiral drug intermediates.     

Enzymatic synthesis of chiral intermediates for pharmaceuticals

There has been an increasing awareness of the enormous potential of microorganisms and enzymes for the transformation of synthetic chemicals with high chemo-, regio- and enatioselective manner. Chiral intermediates are in high demand by pharmaceutical industries for the preparation bulk drug substances. In this review article, microbial/enzymatic processes for the synthesis of chiral intermediates for antihypertensive drugs, melatonin receptor agonists, and beta3-receptor receptor agonists are described.     

Determination of enantiomeric excess of ethyl 3,5-dihydroxy-6-benzyloxy hexanoate by chiral reverse phase high performance liquid chromatography

A simple and reliable chiral HPLC method was developed for the determination of enantiomeric excess of a chiral dihydroxy intermediate for the chemoenzymatic synthesis of side chain of statin drugs. After evaluating different columns and conditions, the four stereoisomers of ethyl 3,5-dihydroxy-6-benzyloxy hexanoate were well resolved by a simple gradient elution on OD-RH column, and the enantiomeric excess of the desired 3R,5S-enantiomer was accurately measured. This study provides a simple, rapid, accurate, and reliable method to assess the enantiomeric quality of such important intermediates.     

Biocatalysis for pharmaceuticals--status and prospects for a key technology

In reviewing how biocatalysis can be applied to improve chiral synthesis for pharmaceuticals it becomes clear that there will be many opportunities using a simple enzyme system but that many of the more useful applications will require the whole cell because of the requirement for cofactors. An assessment is made of the opportunities to apply metabolic engineering to construct de novo metabolic pathways for the biosynthesis of useful advanced intermediates and a conceptual example is provided for the biosynthesis of cis-aminoindanol. We predict that in the future novel pathways will be assembled for a one-step biosynthesis of many semisynthetic natural products.     

Biocatalytic synthesis of C3 chiral building blocks by chloroperoxidase‐catalyzed enantioselective halo‐hydroxylation and epoxidation in the presence of ionic liquids

The optically active C3 synthetic blocks are remarkably versatile intermediates for the synthesis of numerous pharmaceuticals and agrochemicals. This work provides a simple and efficient enzymatic synthetic route for the environment‐friendly synthesis of C3 chiral building blocks. Chloroperoxidase (CPO)‐catalyzed enantioselective halo‐hydroxylation and epoxidation of chloropropene and allyl alcohol was employed to prepare C3 chiral building blocks in this work, including (R)‐2,3‐dichloro‐1‐propanol (DCP*), (R)‐2,3‐epoxy‐1‐propanol (GLD*), and (R)‐3‐chloro‐1‐2‐propanediol (CPD*). The ee values of the formed C3 chiral building blocks DCP*, CPD*, and glycidol were 98.1, 97.5, and 96.7%, respectively. Moreover, the use of small amount of imidazolium ionic liquid enhanced the yield efficiently due to the increase of solubility of hydrophobic organic substrates in aqueous reaction media, as well as the improvement of affinity and selectivity of CPO to substrate. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:724–729, 2015     

Stereoselective synthesis of cyclometalated iridium(III) complexes: Characterization and photophysical properties

The stereoselective synthesis of a highly luminescent neutral Ir(III) complex comprising two bidentate chiral, cyclometalating phenylpyridine derivatives, and one acetylacetonate as ligands is described. The final complex and some intermediates were characterized by X-ray structural analysis, NMR-, CD-, and CPL-spectroscopy.     

Biocatalysis for synthesis of pharmaceuticals

Chirality is a key factor in the safety and efficacy of many drug products and thus the production of single enantiomers of drug intermediates and drugs has become important and state of the art in the pharmaceutical industry. There has been an increasing awareness of the enormous potential of microorganisms and enzymes (biocatalysts) for the transformation of synthetic chemicals with high chemo-, regio- and enatioselectivities providing products in high yields and purity. In this article, biocatalytic processes are described for the synthesis of key chiral intermediates for development pharmaceuticals.     

Biocatalytic synthesis of intermediates for the synthesis of chiral drug substances

There has been an increasing awareness of the enormous potential of microorganisms and enzymes for the transformation of synthetic chemicals with high chemo-, regio- and enantioselectivity. Chiral intermediates and fine chemicals are in high demand, both from the pharmaceutical and agrochemical industries, for the preparation of bulk drug substances and agricultural products. Biocatalytic processes have been described for the synthesis of chiral intermediates for beta3- and beta2-receptor agonists, antihypertensive drugs, antiviral agents, melatonin receptor agonists, anticholesterol and anticancer drugs, and drugs to treat Alzheimer's disease.