Chapter 15
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Ramesh N. Patel and RonaldL.Hanson Department of Enzyme Technology, Process Research, Bristol-Myers Squibb Pharmaceutical Research Institute,P.O.Box 191, New Brunswiick,NJ08903 Chiral intermediates were prepared by enzymatic process using oxidoreductases for the chemical synthesis of pharmaceutical drug candidates. These includes: (1) the microbial reduction of 1-(4-fluorophenyl)-4-[4-(5-fluoro-2-pyrimidinyl)-1piperazinyl]-1-butanone 1 to R-(+)-1-(4-fluoro-phenyl)-4-[4-(5-fluoro-2-pyrimidinyl)1-piperazinyl]-1-butanol [R-(+)-BMY 14802], a antipsychotic agent; (2) the reduction of N-(4-(1-oxo-2-chloroacetyl ethyl) phenyl methane sulfonamide 3 to corresponding chiral alcohol 4, an intermediate for D-(+)-N-[4-[1-Hydroxy-2-[(methylethyl)amino]ethyl]phenyl] methanesulfonamide [D-(+) sotalol], a β-blocker with class III antiarrhythmic properties; (3) biotransformation of Νε-carbobenzoxy (CBZ)-L-lysine 7 to CBZ-L-oxylysine 5 an intermediate needed for synthesis of (S)-1[6-amino-2-[[hydroxy(4-phenylbutyl) phosphinyl]oxy] 1-oxohexyl]-L-proline [ceronapril], a new angiotensin converting enzyme [ACE] inhibitor 6 (4) enzymatic synthesis L-β-hydroxyvaline 9 from α-keto-β-hydroxy isovalerate 16. L-β-Hydroxy valine 9 is a key chiral intermediate needed for the synthesis of [S-(Z)]-[[[1-(2-Amino4-thiazolyl)-2- [[2,2-dimethyl-4-oxo-1-(sulfooxy)-3-azetidinyl] amino]-2oxoethylidene] amino]oxy]acetic acid [tigemonam] 10, a orally active monobactam, (5) enzymatic synthesis of L-6-hydroxynorleucine 17, and (6) enzymatic synthesis of (S)-2-amino-5-(1,3-dioxolan-2-yl)-pentanoic acid (allysine ethylene acetal, 21), one of three building blocks used for an alternative synthesis of omapatrilat, a vasopeptidase inhibitor.
Introduction Recently much attention has been focused on the interaction of small molecules with biological macromolecules. The search for selective enzyme inhibitors and receptor agonists or antagonists is one of the keys for target-oriented research in the pharmaceutical industry. Increasing understanding of the mechanism of drug interaction on a molecular level has led to the wide awareness of the importance of chiraiity as the key to the efficacy of many drug products. It is now known that in many cases only one stereoisomer of a drug substance is required for efficacy and the other stereoisomer is either inactive or exhibits considerably reduced acitivity.
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© 2001 American Chemical Society
In Applied Biocatalysis in Specialty Chemicals and Pharmaceuticals; Saha, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2001.
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217 Pharmaceutical companies are aware that, where appropriate, new drugs for the clinic should be homochiral to avoid the possibility of unnecessary side effects due to an undesirable stereoisomer. In many cases where the switch from racemate drug substance to enantiomerically pure compound is feasible, there is the opportunity to extend the use of an industrial process. The physical characteristic of enantiomers versus racemates may confer processing or formulation advantages. Chiral drug intermediates can be prepared by different routes. One is to obtain them from naturally occuring chiral synthons mainly produced by fermentation processes. The chiral pool primarily refers to inexpensive, readily available, optically active natural products. Second is to carry out the resolution of racemic compounds. This can be achieved by preferential crystallization of stereoisomers or diastereoisomers and by kinetic resolution of racemic compounds by chemical or biocatalytic methods. Finally, chiral synthons can also be prepared by asymmetric synthesis by either chemical or biocatalytic processes using microbial cells or enzymes derived therefrom. The advantages of microbial or enzyme catalyzed reactions over chemical reactions are that they are stereoselective, can be carried out at ambient temperature and atmospheric pressure. This minimizes problems of isomerization, racemization, epimerization and rearrangement that generally occur during chemical processes. Biocatalytic processes are generally carried out in aqueous solution. This will avoid the use of environmentally harmful chemicals used in the chemical processes and solvent waste disposal. Furthermore, microbial cells or enzymes derived therefrom can be immobilized and reused many cycles. Recently, a number of review articles (1-9) have been published on the use of enzymes in organic synthesis. This report provides some specific examples of the use of oxidoreductases in stereoselective catalysis and preparation of chiral drug intermediates required for our antipsychotic, antiarrhythmic (β-Blocker), antihypertensive and antibacterial agents (10-13).
Antipsychotic Drug(BMY-14802) Stereoselective Microbial Reduction of l-(4-Fluorophenyl)-4-[4-(5-Fluoro-2Pyrimidinyl)-l-Piperazinyl]-l-Butanone 1. During the past few years, much research effort has been directed towards the understanding of the Sigma receptor system in brain and endocrine tissue. This effort has been motivated by the hope that the Sigma site may be a target for a new class of antipsychotic drugs (14-17). The characterization of the Sigma system helped to clarify the biochemical properties of the distinct haloperidol-sensitive Sigma binding site, the pharmacological effects of Sigma drugs in several assay systems, and the transmitter properties of a putative endogenous ligand for the Sigma site (18-21). R (+) compound 2 ( B M Y 14802) is a Sigma ligand and has a high affinity for Sigma binding sites and can selectively inhibit conditioned avoidance and apomorphine-induced stereotype in rats predictive of antipsychotic efficacy (22,23). The dopamine cell firing in the Substantia Nigra caused by the putative Sigma receptors was inhibited by (+)-3H-3-(3-hydroxyphenyl)-N-(l-propyl) piperidine (17, 18). In this section, we are describing the stereoselective microbial reduction of l-(4fluorophenyl)-4-[4-(5-fluoro-2-pyrimidinyl)-l-piperazinyl]-l-butanone 1 to yield R (+) B M Y 14802 2 [Scheme 1] In Applied Biocatalysis in Specialty Chemicals and Pharmaceuticals; Saha, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2001.
In Applied Biocatalysis in Specialty Chemicals and Pharmaceuticals; Saha, B., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2001.
SCHEME 1
2 ANTIPSYCHOTIC DRUG
R-(+)-BMY 14802
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219 Among microorganisms evaluated for the reduction of compound 1 to 2, M. ramanniana A T C C 38191 predominately reduced compound 1 to R (+) compound 2 and Pullularia pullulans A T C C 16623 reduced compound 1 to S (-) compound 2. The enantiomeric excess (ee) of > 98% was obtained in each reaction (Table 1). Further research was conducted using M ramanniana A T C C 38191 to convert compound 1 to R (+) compound 2. Cells of M. ramanniana A T C C 38191 were grown in a 280-L fermentor containing 250-L of medium. During growth, cells were harvested periodically, and used to conduct the reduction of compound 1 by cell suspensions of microorganisms to determine optimum time to harvest cultures. As shown in the Table 2, cells harvested after 31 hours growth and used in the bioreduction of compound 1 gave higher reaction yield (89%) and the ee (> 98%) of R (+) compound 2. Cells harvested from a 380-L fermentor were evaluated for the reduction of compound 1 in a 5-L and 15-L fermentors using 3-L and 10-L cell-suspensions (20% w/v, wet cells), respectively. Compound 1 was supplied at 2 g/L concentration. Glucose was supplemented at 70 g/L concentration. After a 24-hour reaction period, about 99% yield (99 % ee) of R (+) compound 2 was obtained from both batches. The kinetics of transformation of compound 1 are as shown in Table 3. Isolation of R (+) compound 2 from the 10-L fermentation broth gave 14.2 gram of product in overall 70 M % yield and 99% H P L C area % purity. Isolated R (+)-compound 2 gave a melting point of 115°C, specific rotation
