Enzymatic reduction method for the preparation of compounds useful for preparing taxanes

An enzymatic reduction method, particularly a stereoselective enzymatic reduction method, for the preparation of compounds useful as intermediates in the preparation of taxanes.

FIELD OF THE INVENTION 
The present invention relates to an enzymatic reduction method for the 
preparation of compounds useful as intermediates in the preparation of 
taxanes, and particularly to the stereoselective preparation of such 
compounds. 
BACKGROUND OF THE INVENTION 
Taxanes are diterpene compounds which find utility in the pharmaceutical 
field. For example, taxol, a taxane having the structure: 
##STR1## 
where Ph is phenyl, Ac is acetyl and Bz is benzoyl, has been found to be 
an effective anticancer agent. 
Naturally occurring taxanes such as taxol may be found in plant materials, 
and have been isolated therefrom. Such taxanes may, however, be present in 
plant materials in relatively small amounts so that, in the case of taxol, 
for example, large numbers of the slow-growing yew trees forming a source 
for the compound may be required. The art has thus continued to search for 
synthetic, including semi-synthetic routes for the preparation of taxanes, 
such as taxol and analogs thereof, as well as routes for the preparation 
of intermediates used in the preparation of these compounds. Methods 
allowing efficient preparation of chiral intermediates, providing final 
taxane products having a desired stereoconfiguration, are particularly 
sought. 
SUMMARY OF THE INVENTION 
The present invention provides a method for the enzymatic reduction, 
preferably, the stereoselective enzymatic reduction, of keto 
group-containing compounds to form hydroxyl group-containing stereoisomers 
useful as intermediates in the preparation of taxanes such as taxol. 
Specifically, the present invention provides a method for the enzymatic 
reduction of a compound of the formula I or a salt thereof: 
##STR2## 
to form a compound of the formula II or a salt thereof: 
##STR3## 
where 
W is 
(a) --NHR.sup.3 ; or 
(b) --N.sub.3 ; 
R.sup.1 is 
(a) aryl; 
(b) alkyl; 
(c) alkenyl; or 
(d) alkynyl; 
R.sup.2 is 
(a) hydrogen; or 
(b) R.sup.4 ; 
R.sup.3 is 
(a) hydrogen; 
(b) R.sup.4; 
(c) --C(O)--OR.sup.4 ; or 
(d) --C(O)--R.sup.4 ; and 
R.sup.4 is 
(a) alkyl; 
(b) aryl; 
(c) cycloalkyl; 
(d) alkenyl; 
(e) alkynyl; 
(f) cycloalkenyl; or 
(g) heterocyclo; 
where, with respect to the chiral center marked with an asterisk, said 
compound of the formula I may be present as a single isomer or as a 
mixture of both R and S isomers (for example, as a racemate), 
comprising the step of contacting said compound of the formula I or salt 
thereof with an enzyme or microorganism capable of catalyzing said 
reduction, and effecting said reduction. 
In a preferred embodiment of the present invention, the compound of the 
formula I or salt thereof is reduced to preferentially form the following 
compounds IIa and/or IIb or salts thereof: 
##STR4## 
A particularly preferred embodiment of the present invention provides a 
method for the stereoselective enzymatic reduction of a compound of the 
formula I or salt thereof to form a compound of the formula IIa or IIb or 
a salt thereof, comprising the step of contacting said compound of the 
formula I or salt thereof with an enzyme or microorganism capable of 
catalyzing said stereoselective reduction, and effecting said reduction.

DETAILED DESCRIPTION OF THE INVENTION 
The methods of the present invention are described further as follows. 
In the compounds of formula II, the group W and the hydroxyl group are 
bonded to asymmetric carbon atoms. Thus, the following four stereoisomers 
may be formed as the compound of the formula II: 
##STR5## 
As used herein, "preferential" formation of the compounds of the formulae 
IIa and/or IIb denotes the formation of one or both of these compounds 
preferentially relative to formation of the compounds of the formulae IIc 
and/or IId. 
The terms "stereoselective enzymatic reduction" and "stereoselective 
reduction", as used herein, refer to the preferential formation of a 
single enantiomer of the compound of the formula II (that is, IIa, IIb, 
IIc or IId) relative to other stereoisomers thereof. Thus, for example, 
stereoselective reduction of the compound of the formula I to form a 
compound of the formula IIa denotes prefential formation of the compound 
of the formula IIa relative to the formation of compounds of the formulae 
IIb, IIc and IId. Stereoselective reduction of the compound of the formula 
I to form a compound of the formula IIb denotes preferential formation of 
the compound of the formula IIb relative to compounds of the formulae IIa, 
IIc and IId. 
Compounds of the formula IIa have the same absolute stereoconfiguration, at 
the carbon atom bearing the group W and the carbon atom bearing the 
hydroxyl group formed by the reduction process, as the compound 
(2R,3S)-(-)-N-benzoyl-3-phenylisoserine ethyl ester. Compounds of the 
formula IIb have the same absolute stereoconfiguration at the 
corresponding carbon atoms as the compound 
(2S,3S)-(-)-N-benzoyl-3-phenylisoserine ethyl ester. 
With respect to the chiral center marked with an asterisk, the starting 
compound of formula I may be present as a single isomer having the R or S 
configuration, or as a mixture of the R and S isomers, for example, as a 
racemate. 
The term "mixture", as said term is used herein in relation to 
stereoisomeric, such as enantiomeric compounds, includes mixtures having 
equal (i.e. racemic for an enantiomeric mixture) or non-equal amounts of 
stereoisomers. 
The terms "enzymatic process" or "enzymatic method", as used herein, denote 
a process or method of the present invention employing an enzyme or 
microorganism. 
The terms "alkyl" or "alk", as used herein alone or as part of another 
group, denote optionally substituted, straight and branched chain 
saturated hydrocarbon groups, preferably having 1 to 10 carbons in the 
normal chain. Exemplary unsubstituted such groups include methyl, ethyl, 
propyl, isopropyl, n-butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, 
heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, 
undecyl, dodecyl and the like. Exemplary substituents may include one or 
more of the following groups: halo, alkoxy, alkylthio, alkenyl, alkynyl, 
aryl, cycloalkyl, cycloalkenyl, hydroxy or protected hydroxy, carboxyl 
(--COOH), alkyloxycarbonyl, alkylcarbonyloxy, carbamoyl (NH.sub.2 --CO--), 
amino (--NH.sub.2), mono-or dialkylamino, or thiol (--SH). 
The terms "lower alk" or "lower alkyl" as used herein, denote such 
optionally substituted groups as described above for alkyl having 1 to 4 
carbon atoms in the normal chain. 
The terms "alkoxy" or "alkylthio" denote an alkyl group as described above 
bonded through an oxygen linkage (--O--) or a sulfur linkage (--S--), 
respectively. The term "alkyloxycarbonyl", as used herein, denotes an 
alkoxy group bonded through a carbonyl group. The term "alkylcarbonyloxy", 
as used herein, denotes an alkyl group bonded through a carbonyl group 
which is, in turn, bonded through an oxygen linkage. The terms 
"monoalkylamino" or "dialkylamino" denote an amino group substituted by 
one or two alkyl groups as described above, respectively. 
The term "alkenyl", as used herein alone or as part of another group, 
denotes such optionally substituted groups as described above for alkyl, 
further containing at least one carbon to carbon double bond. 
The term "alkynyl", as used herein alone or as part of another group, 
denotes such optionally substituted groups as described above for alkyl, 
further containing at least one carbon to carbon triple bond. 
The term "cycloalkyl", as used herein alone or as part of another group, 
denotes optionally substituted, saturated cyclic hydrocarbon ring systems, 
preferably containing 1 to 3 rings and 3 to 7 carbons per ring. Exemplary 
unsubstituted such groups include cyclopropyl, cyclobutyl, cyclopentyl, 
cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, cyclododecyl, and 
adamantyl. Exemplary substituents include one or more alkyl groups as 
described above, or one or more groups described above as alkyl 
substituents. 
The term "cycloalkenyl", as used herein alone or as part of another group, 
denotes such optionally substituted groups as described above for 
cycloalkyl, further containing at least one carbon to carbon double bond 
forming a partially unsaturated ring. 
The terms "ar" or "aryl", as used herein alone or as part of another group, 
denote optionally substituted, homocyclic aromatic groups, preferably 
containing 1 or 2 rings and 6 to 12 ring carbons. Exemplary unsubstituted 
such groups include phenyl, biphenyl, and naphthyl. Exemplary substituents 
include one or more, preferably three or fewer, nitro groups, alkyl groups 
as described above or groups described above as alkyl substituents. 
The terms "heterocyclo" or "heterocyclic", as used herein alone or as part 
of another group, denote optionally substituted fully saturated or 
unsaturated, aromatic or non-aromatic cyclic groups having at least one 
heteroatom in at least one ring, preferably monocyclic or bicyclic groups 
having 5 or 6 atoms in each ring. The heterocyclo group may, for example, 
have 1 or 2 oxygen atoms, 1 or 2 sulfur atoms, and/or 1 to 4 nitrogen 
atoms in the ring. Each heterocyclo group may be bonded through any carbon 
or heteroatom of the ring system. Exemplary heterocyclo groups include the 
following: thienyl, furyl, pyrrolyl, pyridyl, imidazolyl, pyrrolidinyl, 
piperidinyl, azepinyl, indolyl, isoindolyl, quinolinyl, isoquinolinyl, 
benzothiazolyl, benzoxazolyl, benzimidazolyl, benzoxadiazolyl, and 
benzofurazanyl. Exemplary substituents include one or more alkyl groups as 
described above or one or more groups described above as alkyl 
substituents. 
The terms "halogen" or "halo", as used herein alone or as part of another 
group, denote chlorine, bromine, fluorine, and iodine. 
The term "taxane moiety", as used herein, denotes moieties containing the 
core structure: 
##STR6## 
which core structure may be substituted and which may contain ethylenic 
unsaturation in the ring system thereof. 
The term "taxane", as used herein, denotes compounds containing a taxane 
moiety as described above. 
The term "hydroxy protecting group", as used herein, denotes any group 
capable of protecting a free hydroxyl group which, subsequent to the 
reaction for which it is employed, may be removed without disturbing the 
remainder of the molecule. Such groups, and the synthesis thereof, may be 
found in "Protective Groups in Organic Synthesis" by T. W. Greene, John 
Wiley and Sons, 1981, or Fieser & Fieser. Exemplary hydroxyl protecting 
groups include methoxymethyl, 1-ethoxyethyl, 1-methoxy-1-methylethyl, 
benzyloxymethyl, (.beta.-trimethylsilylethoxy)methyl, tetrahydropyranyl, 
2,2,2-trichloroethoxycarbonyl, t-butyl(diphenyl)silyl, trialkylsilyl, 
trichloromethoxycarbonyl, and 2,2,2-trichloroethoxymethyl. 
The term "salt" includes acidic and/or basic salts formed with inorganic 
and/or organic acids and bases. 
Starting Materials 
The starting materials employed in the present reduction method may be 
obtained according to the following Reaction Scheme, and as described by 
Charles et al., J. C. S. Perkin I, 1139 (1980). 
##STR7## 
According to the above Reaction Scheme, compounds of the formula I may be 
prepared by reacting a compound (i) with an oxalyl chloride ester of the 
formula Cl--C(O)--C(O)--OR.sup.2, where R.sup.2 is preferably 
unsubstituted lower alkyl such as ethyl or methyl, for example, in 
anhydrous tetrahydrofuran (THF) in the presence of 4-dimethylaminopyridine 
(DMAP) and pyridine, to form a compound (ii). Compounds of the formula (i) 
and esters of the formula Cl--C(O)--C(O)--OR.sup.2 are commercially 
available or may readily be prepared by one of ordinary skill in the art. 
In the compound (i), W is preferably an amide group --NH--C(O)--R.sup.4, 
such as benzoylamino, or a urethane group --NH--C(O)--OR.sup.4, such as 
where R.sup.4 is unsubstituted alkyl (for example, the urethane group 
t-butyloxycarbonylamino (BOC)), which may be prepared by reacting the 
corresponding compound (i) where W is amino (--NH.sub.2) with the reagent 
R.sup.4 --C(O)--Cl or [R.sup.4 --C(O)].sub.2 O. 
A racemate of a compound of the formula I may then be prepared from the 
compound (ii), for example, by heating the compound (ii) in ethanol in the 
presence of anhydrous NaHCO.sub.3 or other mild bases. Starting materials 
which are other than racemic may be obtained, for example, by separation 
of the isomers of the racemate prepared above, or by addition of one or 
both of the enantiomers of the compound of formula I in unequal portions 
to a racemic mixture thereof. 
The present invention provides novel compounds of the formula (ii), where 
R.sup.1, R.sup.2 and W are as defined above, except that, when W is 
--NH--C(O)--R.sup.4 and R.sup.2 is ethyl, (1) R.sup.4 is not isobutyl, 
n-propyl, cyclopentyl or phenyl when R.sup.1 is methyl, and (2) R.sup.4 is 
not n-propyl when R.sup.1 is phenyl. Preferably, in the compounds of the 
formula (ii), R.sup.1 is aryl such as phenyl, W is arylcarbonylamino such 
as benzoylamino or alkyloxycarbonylamino such as t-butyloxycarbonylamino 
(BOC), and R.sup.2 is alkyl such as unsubstituted lower alkyl (e.g. methyl 
or ethyl). All stereoisomers, such as cis- and trans-isomers, of the novel 
compounds of the formula (ii), alone or in admixture, are contemplated. 
The present invention also provides novel compounds of the formula I, where 
R.sup.1, R.sup.2 and W are as defined above, except that, when W is 
--NH--C(O)--R.sup.4 and R.sup.2 is ethyl, (1) R.sup.4 is not isobutyl, 
n-propyl, cyclopentyl or phenyl when R.sup.1 is methyl, and (2) R.sup.4 is 
not n-propyl when R.sup.1 is phenyl. Preferably, in the compounds of the 
formula I, R.sup.1 is aryl such as phenyl, W is arylcarbonylamino such as 
benzoylamino or alkyloxycarbonylamino such as t-butyloxycarbonylamino, and 
R.sup.2 is alkyl such as unsubstituted lower alkyl (e.g. methyl or ethyl). 
All stereoisomers of the novel compounds of the formula I, alone or in 
admixture (e.g. racemates), are contemplated. 
Preferred Compounds 
It is preferred to prepare, according to the present invention, compounds 
of the formula II in which: W is --NHR.sup.3, R.sup.1 is aryl, especially 
phenyl, R.sup.2 is alkyl, especially unsubstituted lower alkyl such as 
ethyl or methyl, and R.sup.3 is arylcarbonyl, especially benzoyl, or 
alkyloxycarbonyl, especially t-butyloxycarbonyl. It is further preferred 
to stereoselectively prepare compounds of the formula IIa or IIb. 
Enzymes and Microrganisms 
The enzyme or microorganism employed in the present invention may be any 
enzyme or microorganism capable of catalyzing the enzymatic reduction, 
preferably the stereoselective enzymatic reduction, described herein. The 
enyzmatic or microbial materials may be employed in the free state or 
immobilized on a support such as by physical adsorption or entrapment. 
Suitable enzymes, regardless of origin or purity, include those enzymes 
referred to as oxido-reductases or dehydrogenases. The enzyme employed 
may, for example, be an enzyme isolated from a microorganism such as by 
homogenizing cell suspensions, followed by disintegration, centrifugation, 
DEAE-cellulose chromatography, ammonium sulfate fractionation, 
chromatography using gel filtration media such as Sephacryl (cross-linked 
co-polymer of allyl dextran and N,N'-methylene bisacrylamide) 
chromatography, and ion exchange chromatography such as Mono-Q (anion 
exchanger which binds negatively charged biomolecules through quaternary 
amine groups) chromatography. Exemplary such enzymes include 
L-2-hydroxyisocaproate dehydrogenase, lactic acid dehydrogenase, yeast 
enzyme concentrate (may be obtained from Sigma), .beta.-hydroxybutyrate 
dehydrogenase, glucose dehydrogenase, alcohol dehydrogenase, glycerol 
dehydrogenase, formate dehydrogenase, pyruvate dehydrogenase, hydroxy 
steroid dehydrogenase, and those enzymes derived from the microorganisms 
described following. 
With respect to the use of microorganisms, the methods of the present 
invention may be carried out using any suitable microbial materials 
capable of catalyzing the enzymatic reduction, preferably the 
stereoselective enzymatic reduction, described herein. For example, the 
cells may be used in the form of intact wet cells or dried cells such as 
lyophilized, spray-dried or heat-dried cells, or in the form of treated 
cell material such as ruptured cells or cell extracts. Suitable 
microorganisms include genera from bacteria, yeasts and fungi such as 
Achromobacter, Acinetobacter, Actinomyces, Alkaligenes, Arthrobacter, 
Azocobacter, Bacillus, Brevibacterium, Corynebacterium, Flavobacterium, 
Methylomonas, Mycobacterium, Nocardia, Pseudomonas, Rhodococcus, 
Streptomyces, Xanthomonas, Aspergillus, Candida, Fusarium, Geotrichum, 
Hansenula, Kloeckera, Penicillium, Pichia, Rhizopus, Rhodotorula, 
Saccharomyces, Trichoderma, Mortierella, Cunninghamella, Torulopsis, Mucor 
and Rhodopseudomonas. 
The use of genetically engineered organisms is also contemplated. The host 
cell may be any cell, e.g. Escherichia coli, modified to contain a gene or 
genes for expressing one or more enzymes capable of catalysis as described 
herein. 
Preferred microorganisms include Artrobacter simplex, Nocardia restricta, 
Rhodococcus fascians, Mycobacterium vacca, Nocardia meditteranei, Nocardia 
autotrophica, Rhodococcus equi, Candida albicans, Pichia pastoris, Pichia 
methanolica, Torulopsis polysporium, Torulopsis glabrata, and 
Acinetobacter calcoaceticus, and especially Mortierella alpina (e.g. ATCC 
32221), Nocardia globerula (e.g. ATCC 21505), Cunninghamella chinulata 
(e.g. ATCC 26269), Nocardia salmonicolor (e.g. ATCC 19149), Geotrichum 
candidum (e.g. ATCC 34674), Candida guilliermondii (e.g. ATCC 20318 and 
ATCC 9058), Aspergillus versicolor (e.g. ATCC 26268), Penicillium thomii 
(e.g. ATCC 14974), Rhodococcus erythropolis (e.g. ATCC 4277), Rhodococcus 
rhodochorus (e.g. ATCC 19150 and ATCC 14342), Saccharomyces cerevisiae 
(e.g. ATCC 24702), Pseudomonas putida (e. g. ATCC 11172), Mortierella 
rammanianna (e.g. ATCC 38191), Mucor hiemalis (e.g. ATCC 6977B), Pichia 
pinus (e.g. ATCC 28780), Hansenula anomala (e.g. ATCC 8170), Hansenula 
fabianii (e.g. ATCC 58045) and Hansenula polymorpha, (e.g. ATCC 26012) . 
Particularly preferred organisms for the preparation of compounds of the 
formula IIa are microorganisms of the species Hansenula polymorpha, 
especially the strain Hansenula polymorpha ATCC 26012, and the species 
Hansenula fabianii, especially the strain Hansenula fabianii ATCC 58045. 
It is also particularly preferred to employ cell extracts or isolated 
enzymes from these organisms. 
The term "ATCC" as used herein refers to the accession number of the 
American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 
20852, the depository for the organism referred to. 
The enzymatic reduction method of the present invention may be carried out 
subsequent to the fermentation of the microorganism employed (two-stage 
fermentation and reduction), or concurrently therewith, that is, in the 
latter case, by in situ fermentation and reduction (single-stage 
fermentation and reduction). In the single-stage process, the 
microorganisms may be grown in an appropriate medium until sufficient 
growth of the microorganisms is attained. A compound of the formula I may 
then be added to the microbial cultures and the enzymatic reduction 
continued with the fermentation, preferably until complete conversion is 
obtained. 
In the two-stage process, the microorganisms may, in the first stage, be 
grown in an appropriate medium for fermentation until exhibiting the 
desired enzymatic (e.g. oxido-reductase) activity. Subsequently, the cells 
may be harvested by centrifugation and microbial cell suspensions prepared 
by suspending harvested cells in an appropriate buffered solution. Buffers 
such as tris-HCl, phosphates, sodium acetate and the like may be used. 
Water may also be used to prepare suspensions of microbial cells. In the 
second stage, the compound I may be mixed with the microbial cell 
suspensions, and the enzymatic reduction of compound I catalyzed by the 
microbial cell suspensions. The reduction is preferably conducted until 
all or nearly all of the compound I is reduced. 
Growth of the microorganisms may be achieved by one of ordinary skill in 
the art by the use of an appropriate medium. Appropriate media for growing 
microorganisms include those which provide nutrients necessary for the 
growth of the microbial cells. A typical medium for growth includes 
necessary carbon sources, nitrogen sources, and trace elements. Inducers 
may also be added. The term "inducer", as used herein, includes any 
compound enhancing formation of the desired enzymatic (e.g. 
oxido-reductase) activity within the microbial cell, such as those 
compounds containing keto groups. Formula I compounds may be added as 
inducers during growth of the microorganisms. 
Carbon sources may include sugars such as maltose, lactose, glucose, 
fructose, glycerol, sorbitol, sucrose, starch, mannitol, propylene glycol, 
and the like; organic acids such as sodium acetate, sodium citrate, and 
the like; amino acids such as sodium glutamate and the like; and alcohols 
such as ethanol, propanol and the like. 
Nitrogen sources may include N-Z amine A, corn steep liquor, soy bean meal, 
beef extracts, yeast extracts, molasses, baker's yeast, tryprone, 
nutrisoy, peptone, yeastamin, sodium nitrate, ammonium sulfate and the 
like. 
Trace elements may include phosphates and magnesium, manganese, calcium, 
cobalt, nickel, iron, sodium and potassium salts. 
The medium employed may include more than one carbon or nitrogen source or 
other nutrient. 
Preferred media include aqueous media containing the following (in weight 
%): 
______________________________________ 
Medium 1 
Malt Extract 1% 
Yeast Extract 1% 
Peptone 1% 
Glucose 2% 
pH 7.0 
Medium 2 
Peptone 0.3% 
Glycerel 4% 
Malt Extract 1% 
Yeast Extract 1% 
pH 7.0 
Medium 3 
Peptone 0.3% 
Fructose 2% 
Malt Extract 1% 
Yeast Extract 1% 
pH 7.0 
Medium 4 
Sodium Succinate 2% 
Malt Extract 1% 
Yeast Extract 1% 
Peptone 0.3% 
pH 7.0 
______________________________________ 
The pH of the medium is preferably adjusted to about 6 to 8, most 
preferably to 6.5, sterilized, e.g. at a temperature of 121.degree. C. for 
30 minutes, and then adjusted to a pH of about 6.5 to 7.5, preferably 7.0, 
after sterilization. The pH of the medium is preferably maintained between 
4.0 and 9.0, most preferably between 6.0 and 8.0, during the growth of 
microorganisms and during the reduction process. 
Temperature is a measure of the heat energy available for the reduction 
process, and should be maintained to ensure that there is sufficient 
energy available for this process. A suitable temperature range is from 
about 15.degree. C. to about 60.degree. C. A preferred temperature range 
is from about 25.degree. C. to about 40.degree. C. 
The agitation and aeration of the reaction mixture affects the amount of 
oxygen available during the reduction process, which may be conducted, for 
example, in shake-flask cultures or fermentor tanks during growth of 
microorganisms in a single-stage or two-stage process. The agitation range 
from 50 to 1000 RPM is preferable, with 50 to 500 RPM being most 
preferred. Aeration of about 0.1 to 10 volumes of air per volume of media 
per minute (i.e., 0.1 to 10 v/vt) is preferred, with aeration of about 5 
volumes of air per volume of media per minute (i.e., 5 v/vt) being most 
preferred. 
Complete conversion of the compound I may take, for example, from about 12 
to 48 hours, such as 4 to 24 hours, measured from the time of initially 
treating the compound I with a microorganism or enzyme as described 
herein. 
The enzymatic reduction method of the present invention may be carried out 
using a co factor such as nicotinamide adenine dinucleotide (NADH), 
especially when an isolated enzyme is employed. NADH, for example, may 
thereafter be regenerated and reused. A further enzyme that regenerates 
the NADH in situ may be employed such as formate dehydrogenase. Suitable 
hydrogen donors include molecular hydrogen, a formate (e.g. an alkali 
metal or ammonium formate), a hypophosphite or an electrochemical 
reduction in the presence of a viologen, for example methyl viologen. It 
is also possible to regenerate NADH without further enzymes using, for 
example, ethanol or formate. 
It is preferred to employ an aqueous liquid as the reaction medium, 
although an organic liquid, or a miscible or immiscible (biphasic) 
organic/aqueous liquid mixture may also be employed. 
It is preferred to employ 0.1 to 25 weight % of the compound I starting 
material based on the combined weight of compound I and reaction medium. 
The amount of enzyme or microorganism employed relative to the starting 
material is selected to allow catalysis of the enzymatic reduction of the 
present invention. 
It is preferred to employ parameters, such as enzymes and microorganisms, 
which provide a stereoselective reduction. A stereoselective reduction is 
advantageous in that an efficient conversion of substrate may be achieved, 
and in that the procedures which may be employed in the subsequent 
separation of the desired enantiomer of the formula II from the remaining 
components of the reaction medium may be minimized. It is particularly 
preferred to employ parameters which provide a reaction yield greater than 
about 80%, most preferably greater than about 90%, and an optical purity 
greater than about 90%, most preferably greater than about 99%, of a 
desired enantiomer of the formula II. To obtain stereoselective reduction 
of the substrate compound I, it is desirable to employ the enzymes and 
microorganisms indicated above as preferred. 
Separation 
The products of the reduction process of the present invention may be 
isolated and purified, for example, by methods such as extraction, 
distillation, crystallization, and column chromatography. 
For example, a preferred method for separating the compound IIa from the 
remaining components of the reaction medium is by extraction. An exemplary 
extraction technique, such as where 
(2R,3S)-(-)-N-benzoyl-3-phenylisoserine ethyl ester is prepared by whole 
cell suspensions, is that where the reaction medium, containing the 
aforementioned suspensions, is extracted with ethyl acetate, the organic 
layer is washed with brine, and the solvent is then removed under reduced 
pressure to generate an oily liquid which is chromatographed on silica to 
produce the desired product compound IIa. 
Taxanes are diterpene compounds containing the taxane moiety: 
##STR8## 
described above. Of particular interest are taxanes containing a taxane 
moiety in which the 11,12-positions are bonded through an ethylenic 
linkage, and in which the 13-position contains a sidechain, which taxanes 
are exemplified by taxol. Pharmacologically active taxanes such as taxol 
may be used as antitumor agents to treat patients suffering from cancers 
such as breast, ovarian, colon or lung cancers, melanoma and leukemia. 
The compounds of the formula II obtained by the reduction method of the 
present invention are particularly useful as intermediates in forming the 
aforementioned sidechain on the taxane moiety. The addition of such a 
sidechain, in and of itself, may impart an increased or more desirable 
pharmacological activity to the taxane product, or may form a taxane 
product which is more readily converted to a taxane having an increased or 
more desirable pharmacological activity than the starting compound. 
The compounds of the formula II prepared according to the reduction method 
of the present invention may optionally be modified prior to use in 
sidechain formation. For example, compounds containing an azide group 
(N.sub.3) as the group W may be treated with a reducing agent to form an 
amine group, the latter which may be substituted to form the group 
--NHR.sup.3. 
The compounds of the formula II obtained by the method of the present 
invention may, for example, be used in the preparation of 
sidechain-bearing taxanes such as those described in European Patent 
Publication No. 400,971, U.S. Pat. No. 4,876,399, U.S. Pat. No. 4,857,653, 
U.S. Pat. No. 4,814,470, U.S. Pat. No. 4,924,012, and U.S. Pat. No. 
4,924,011, all incorporated herein by reference. 
For example, taxanes bearing a hydroxyl group at C-13, such as those 
described in the aforementioned European Patent Publication No. 400,971, 
may be coupled with an optionally modified compound of the formula II in 
the presence of a condensing agent, for example, a carbodiimide such as 
dicyclohexylcarbodiimide or a reactive carbonate such as di-2-pyridyl 
carbonate, as well as a tertiary amine activating agent, for example, a 
dialkylaminopyridine such as 4-dimethylaminopyridine. An inert solvent 
such as benzene, toluene, a xylene, ethylbenzene, isopropylbenzene or 
chlorobenzene, and a temperature of from about 60.degree. C. to about 
90.degree. C., may be employed. 
Coupling may be conducted as described by Ojima et al., J. Ora. Chem., 56, 
1681 (1991), incorporated herein by reference. See also Denis et al., J. 
Am. Chem. Soc., 110, 5917 (1988), also incorporated herein by reference. 
Taxol is preferably ultimately prepared as the sidechain-bearing taxane. 
Salts or solyates such as hydrates of reactants or products may be employed 
or prepared as appropriate in any of the methods of the present invention. 
The present invention is further described by the following examples which 
are illustrative only, and are in no way intended to limit the scope of 
the instant claims. 
EXAMPLE 1 
Preparation of Starting Material and Enzymatic Reduction 
Preparation of 2-Keto-3-(N-benzoylamino)-3-phenyl propionic acid ethyl 
ester: Racemic starting material 
(a) Benzoyl phenylalycine 
##STR9## 
To (DL)-phenylglycine (9 g, 60 mmole) in aqueous NaOH (1N, 180 ml) at 
0.degree. C. was added dropwise neat benzoyl chloride (PhCOCl) (7.73 ml, 
66 mmole) over a period of 5 minutes. The resulting solution was stirred 
for an additional 1 hour. The reaction solution was washed with ethyl 
acetate (EtOAc) (20 ml.times.2), then neutralized by 6N HCl and extracted 
with EtOAc (60 ml.times.2). The combined EtOAc layer was washed with brine 
(30 ml.times.2), dried over MgSO.sub.4, filtered and concentrated to give 
a residue. The residue was crystallized from EtOAc/hexane to give 10.65 g 
of benzoyl phenylglycine as a white solid (70% yield, first crops). (The 
title product is also commercially available.) 
(b) 3-Benzoylamino-3-phenyl-(ethyl, 2-oxalyl) propenoic acid, ethyl ester 
##STR10## 
To a stirred solution of benzoyl phenylglycine prepared in step (a) (6.12 
g, 24 mmole), 4-dimethylaminopyridine (100 mg, 0.82 mmole), and pyridine 
(5.86 ml, 72 mmole) in anhydrous tetrahydrofuran (THF) (24 ml) was added 
ethyl oxalyl chloride (5.35 ml, 48 mmole) at a rate to initiate gentle 
refluxing. (Refluxing at this point was not critical when sufficient 
refluxing (.about.3.5 h) as followed was employed). The mixture was then 
heated to maintain a gentle reflux for 3.5 hours. The reaction was 
monitored by thin layer chromatography (TLC) using 30% EtOAc in hexane as 
eluent (R.sub.f for the starting material was on the base line and R.sub.f 
for the products were 0.50 and 0.63 (E and Z isomers)). After cooling, the 
room temperature mixture was treated with water (48 ml) and stirred 
vigorously at room temperature for 1/2 hour. The resulting organic layer 
was separated and the aqueous layer was extracted with EtOAc (36 
ml.times.2). The combined organic layer was washed with brine (30 
ml.times.1), dried over Na.sub.2 SO.sub.4, filtered, concentrated, and 
crystallized from EtOAc/hexane to obtain 4.68 g of the enol ester title 
product (.about.63% yield, first crop--no attempt was made to get a second 
crop.) 
(c) Racemic 2-Keto-3-(N-benzoylamino)-3-phenylpropionic acid ethyl ester 
##STR11## 
To a suspension of the enol ester title product prepared in step (b) above 
(6.0 g, 14.6 mmole) in 20 ml ethanol (EtOH) was added anhydrous 
NaHCO.sub.3 (0.8 g, 9.49 mmole). The reaction mixture was refluxed for 1/2 
hour. The reaction was monitored by TLC using 2% acetone in CH.sub.2 
Cl.sub.2 as eluent (R.sub.f for the starting materials were 0.50 and 0.75 
(E & Z isomers) and R.sub.f for the product was 0.41). NaHCO.sub.3 was 
filtered (if any) and the filtrate was concentrated to an oil. It was 
purified by column chromatography ((CO2Et)2 was removed by column 
chromatography) (2% acetone/CH.sub.2 Cl.sub.2) to give 5.6 g of the title 
product (.about.100% yield). (Crystallization was used for purification in 
subsequent preparation of the title product.) When the compound was stored 
in the freezer, it solidified. 
m.p.: 80.degree.-83.degree. C. TLC: R.sub.f =0.43; Silica gel; 2% Acetone 
in CH2C12; UV and PMA Visualization. 
Enzymatic Reduction 
Use of Various Strains of Whole Cells 
The substrate for the following enzymatic reduction process was racemic 
2-keto-3-(N-benzoylamino)-3-phenylpropionic acid ethyl ester ("Compound 
A") having the structure set forth above. Of particular interest in this 
example was preparation of the compound having the following structure: 
##STR12## 
and the name (2R,3S)-(-)-N-benzoyl-3-phenylisoserine ethyl ester 
("Compound B"). The microorganisms which were employed in the reduction 
process are listed in Table 1 following. 
The microorganisms employed were maintained in a vial in liquid nitrogen. 
For routine development of inoculum, one vial was inoculated into 100 ml 
of Medium 1 (see above for the composition thereof) in a 500 ml flask and 
incubated at 28.degree. C. and 280 RPM on a shaker for 48 hours. After 
growth of the microorganism, 10 ml of culture was inoculated into a 500 ml 
flask containing 100 ml of Medium 1 and incubated at 28.degree. C. and 250 
RPM on a shaker. 
Cells were harvested and suspended in 100 mM potassium phosphate buffer pH 
6.0. 10 ml of 20% w/v cell-suspensions were prepared. Cell-suspensions 
were supplemented with 25 mg of substrate (Compound A) and 750 mg of 
glucose and the reductions ("biotransformations") were conducted at 
25.degree. C., 150 RPM for 72 hours. One volume of sample was taken and 
extracted with two volumes of ethyl acetate and the separated organic 
phase was filtered through a 0.2 .mu.m LID/x filter and collected. 
Samples were analyzed for substrate and product concentration by a Hewlett 
Packard 1070 HPLC System. A Phenomenex Cyanopropyl Column (150 .times.4.6 
mm, 5.mu.) was used. The mobile phase consisted of 5% isopropanol in 
hexane. The flow rate was 0.5 ml/min at ambient temperature. The detection 
wavelength was 230 nm. The retention times for substrate, syn diastereomer 
(both enantiomers) of product and anti diastereomer (both enantiomers) of 
product were 26.8 min., 20.4 min., and 22.2 min, respectively. 
The separation of the two enantiomers of the syn and anti diastereomers was 
achieved by HPLC using dual columns connected in a series. The first 
column was a Pirkler column (DNBPG, dinitrophenylglycine) (250.times.4.6 
mm, 5.mu.) and the second column was Chiralcel OB (250.times.4.6 mm, 
5.mu.) (both columns purchased from J. T. Baker, Inc., Phillipsburg, 
N.Y.). The mobile phase consisted of 25:2.5:2.5:70 of 
isopropanol:n-butanol:methanol: hexane. The flow rate was 0.5 ml/min. and 
the detector wavelength was 230 nm. The retention times for the two 
enantiomers of syn were 20.1 min. and 23.3 min., respectively. The 
retention times for the two enantiomers of anti were 22 min. and 27.8 
min., respectively. 
The results obtained by using various microorganisms grown on Medium 1 and 
following the above procedure are shown in Table 1. 
Batches were also further purified, subsequent to extraction, as 
exemplified by the following procedure: 
The reduction product subsequent to extraction isolation (0.822 g) was 
dissolved in hot acetonitrile (16.5 ml) and the solution was filtered hot 
through a "D" sintered glass funnel. The filtrate was allowed to stand at 
room temperature (crystals formed quickly) for 45 minutes, and was then 
allowed to stand at 4.degree. C. It was filtered and washed with cold 
acetonitrile. The crystals were air dried and weighed. .sup.1 H NMR in 
dimethylsulfoxide demonstrated that the crystals were essentially the syn 
material. (Crystal weight: 0.38 g; [.alpha.].sup.D.sub.20 (Cl, 
CHCl.sub.3)=-21.7; [.alpha.].sup.D.sub.20 (Cl, CH.sub.3 OH)=-36.5). The 
mother liquor residue (0.414 g) was dissolved in 7 ml of hot acetonitrile, 
filtered hot through a "D" sintered glass funnel and allowed to stand at 
room temperature for 2 hours. It was then placed in a cold room (4.degree. 
C.) and left overnight. The crystals were then filtered and washed with 
cold acetonitrile (3.times.0.5 ml) and air dried giving 0.072 g as a 
second crop. .sup.1 H NMR was consistent with &gt;98% syn. 
Analysis indicated that the crystals obtained were approximately 100% 
optically pure Compound B. 
TABLE 1 
______________________________________ 
Reaction Optical 
Yield Purity 
(syn com- (Compound 
Microorganism pounds) (%).sup.1 
B) (%).sup.2 
______________________________________ 
Candida guilliermondii ATCC 20318 
31 95 
Rhodococcus erythropolis ATCC 4277 
39 96 
Saccharomyces cerevisiae ATCC 24702 
35 94 
Hansenula polymorpha ATCC 26012 
98 99.5 
Pseudomonas putida ATCC 11172 
32 94 
Nocardia globerula ATCC 21505 
36 92 
Mortierella rammanianna ATCC 
35 97 
38191 
Hansenula fabianii ATCC 58045 
90 96 
______________________________________ 
.sup.1 Reaction yield calculated as: 
##STR13## 
.sup.2 Optical purity calculated as: 
##STR14## 
.sup.3 Compound C had the following structure: 
##STR15## 
EXAMPLE 2 
Use of Whole Cells: Variation in Reaction Time The substrate for this 
process was Compound A. Of particular interest in this example was 
preparation of Compound B. Both Compounds A and B are described in Example 
1. 
Cells of Hansenula polymorpha ATCC 26012 were grown in 100 ml of Medium 1 
combined in 500 ml flasks. Growth was carried out at 25.degree. C. for 48 
hours at 280 rpm. 100 ml of cultures were inoculated into 15 L of Medium 2 
(see above for the composition thereof) combined in a fermentor. Growth in 
the fermentor was carried out at 25.degree. C., 15 liters per minutes 
(LPM) aeration and 500 RPM agitation for 60 hours. Cells were harvested 
from the fermentor and used for the reduction ("biotransformation") of 
Compound A to Compound B. 
Cells (200 grams) were suspended in 1 liter of 100 mM potassium phosphate 
buffer, pH 6.0 and homogenous cell suspensions were prepared. 2.5 grams of 
Compound A and 35 grams of glucose were added to the cell suspensions and 
the biotransformation of Compound A to Compound B was carried out at 
22.degree. C., 160 RPM for 24 hours. After 24 hours, an additional 35 
grams of glucose were added and the biotransformation was continued for 72 
hours at 22.degree. C., 160 RPM. Samples were prepared and product yield 
and optical purity were determined as described in Example 1. The results 
obtained are summarized in Table 2 following. 
TABLE 2 
______________________________________ 
Reaction Time 
Yield of Optical Purity 
(Hours) syn compounds (%) 
of Compound B (%) 
______________________________________ 
24 32 -- 
48 65 -- 
72 90 99.5 
______________________________________ 
EXAMPLE 3 
Use of Cell Extracts and Co-factor 
The substrate for this process was Compound A as described above. Of 
particular interest in this example was preparation of Compound B also 
described above. 
Cells of Hansenula polymorpha ATCC 26012 were grown on Medium 1 and Medium 
2 as described in Example 2. 
Cells (150 grams) were suspended in 1.5 L of 0.2M potassium phosphate 
buffer, pH 6.0. The homogenized cell suspensions were disintegrated at 
4.degree. C. by a Microfluidizer at 13,000 psi pressure. The disintegrated 
cell suspension was centrifuged at 12,000 RPM for 30 minutes. The clear 
supernatant ("cell extract") was used for the biotransformation of 
Compound A to Compound B. 
One liter of cell extract was supplemented with 2.5 grams of substrate 
(Compound A), formate dehydrogenase (500 units), 0.7 mM NAD.sup.+ 
(nicotinamide adenine dinucleotide), and 25 grams of sodium formate. The 
reaction was carried out in a pH star at pH 6.0, 150 RPM agitation, and 
22.degree. C. Periodically, samples were taken and analyzed for the 
reaction yield and optical purity of Compound B as described in Example 1. 
The results obtained are those shown in Table 3 following. 
TABLE 3 
______________________________________ 
Reaction Time 
Compound B Yield Optical Purity 
(Hours) g/L (%) (%) 
______________________________________ 
48 2.2 88 &gt;99% 
______________________________________ 
In the above procedure, the NADH cofactor used for the biotransformation of 
Compound A to Compound B was, concurrent with the biotransformation, 
formed and regenerated using formate dehydrogenase, NAD.sup.+, and formate 
as shown below. 
##STR16## 
After complete conversion of Compound A to Compound B, the reaction mixture 
was adjusted to pH 7.0 and extracted three times with equal volumes of 
ethyl acetate. The organic phase was separated and washed twice with 0.7M 
sodium bicarbonate. The separated organic layer was dried over anhydrous 
sodium sulfate and ethyl acetate was removed under reduced pressure. The 
resulting oily residue was dried under vacuum at room temperature to 
recover a pale white solid in 85% yield (isolated) and 99% optical purity. 
EXAMPLE 4 
Use of Purified Oxido-Reductase 
The substrate for this process (Compound A) was described in Example 1. Of 
particular interest in this example was the preparation of Compound B also 
described in Example 1. 
Growth of Hansenula polymorpha ATCC 26012 was carried out on Medium 1 as 
described in Example 1. Cell extracts of Hansenula polymorpha ATCC 26012 
were prepared as described in Example 3. 
Cell extracts (700 ml) were loaded onto a DEAE-cellulose (DE-52) column and 
eluted with buffer containing sodium chloride in a linear gradient from 0 
to 0.5M . Fractions containing oxido-reductase activity were pooled and 
concentrated by ammonium sulfate precipitation (70% saturation). 
Precipitated material was collected by centrifugation, dissolved in 
buffer, and loaded onto a Sephacryl S-200 column. Fractions containing 
reductase activity were pooled after chromatography and loaded onto a 
Mono-Q column. Proteins bound on the Mono-Q column were eluted with a 
buffer containing sodium chloride in a linear gradient from 0 to 0.5M . 
Fractions having oxido-reductase activity were pooled and analyzed by 
sodium dodecyl sulfate (SDS) gel electrophoresis. The purified enzyme was 
homogeneous. Overall, 250 fold purification was achieved. 
The transformation of Compound A to Compound B was carried out by the 
purified enzyme (Mono-Q fraction). The reaction mixture in 20 ml of 0.1M 
potassium phosphate buffer (pH 6.0) contained 20 units of purified 
oxido-reductase enzyme, 200 mg of substrate (Compound A), 100 units of 
formate dehydrogenase, 1 gram of formate, and 50 mg of NAD.sup.+. The 
reaction was carried out in a pH star at pH 6.0, 100 RPM agitation and 
22.degree. C. for 48 hours. Product (Compound B) and substrate (Compound 
A) concentrations were determined by the procedures described in Example 
1. After 48 hours of reaction time, an 89% reaction yield and greater than 
99% optical purity of Compound B was obtained. 
EXAMPLE 5 
Enzymatic Reduction 
The substrate for this process (Compound A) is described in Example 1. Of 
particular interest in this example was the preparation of Compound B, 
also described in Example 1. 
Commmercially available oxido-reductases (5-20 units) were suspended in 10 
ml of 50 mM potassium phosphate buffer at pH 7.0. To the suspension NADH 
or NADPH (1.5 mg/ml) was added. The reaction was started by addition of 2 
mg/ml of Compound A. The reaction was carried out at 25.degree. C. at 150 
RPM agitation for 48 hours. After 48 hours, samples were taken and 
analyzed for the reaction yield of compound B as described in Example 1. 
The enzymes which produced Compound B and the reaction yields obtained are 
listed in the following Table 4. 
TABLE 4 
______________________________________ 
Reaction Yield 
(syn compounds) 
Enzyme % 
______________________________________ 
L-2-hydroxyisocaproate dehydro- 
20 
genase 
Lactic acid dehydrogenase 
10 
Yeast enzyme concentrate 
42 
.beta.-hydroxybutyrate dehydrogenase 
5 
______________________________________ 
EXAMPLE 6 
The substrate (Compound A) and method of bioreduction employed for this 
example were the same as those of Example 1. The organisms used and the 
results obtained are listed in the following Table 5. 
TABLE 5 
______________________________________ 
Organism Syn %.sup.1 
Anti %.sup.2 
______________________________________ 
Candida guilliermondii ATCC 9058 
18 82 
Candida guilliermondi ATCC 20318 
30.8 69.2 
Penicillium thomii ATCC 14974 
10 90 
Rhodococcus rhodochorus ATCC 19150 
14.4 85.6 
Rhodococcus rhodochorus ATCC 14342 
5.8 94.2 
Mortierella alpina ATCC 32221 
11 89 
Hansenula anomala ATCC 8170 
18.9 81.1 
Cunninghamella chinulata ATCC 26269 
5.6 94 
Pseudomonas putida ATCC 11172 
30. 1 69.9 
Nocardia salmonicolor ATCC 19149 
11 89 
Geotrichum candidum ATCC 34674 
12.1 87.9 
Nocardia globerula ATCC 21505 
35.5 64.5 
Pichia pinus ATCC 28780 
31 72 
Aspergillus versicolor ATCC 26268 
3.2 96 
Mucor hiemalis ATCC 6977B 
8 92 
______________________________________ 
##STR17## 
that is, [%(B + C)]- 
##STR18## 
that is, [%(D + E)]- 
.sup.3 Compound D had the structure: 
##STR19## 
.sup.4 Compound E had the structure: 
##STR20## 
EXAMPLE 7 
The substrate (Compound A) and method of enzymatic reduction employed for 
this example were those of Example 5. The commercially available enzymes 
used for this example, and the results obtained, are listed in the 
following Table 6. 
TABLE 6 
______________________________________ 
Enzyme Anti %.sup.1/ 
______________________________________ 
Glucose dehydrogenase 88 
Alcohol dehydrogenase 81 
Glycerol dehydrogenase 
72 
Formate dehydrogenase 78 
.beta.-hydroxybutyrate dehydrogenase 
90 
Pyruvate dehydrogenase 
92 
Hydroxy steroid dehydrogenase 
87 
______________________________________ 
.sup.1/ As defined above in Example 6.