1
0
However, due to a limited solubility of the acetate,
adequate stirring was necessary to obtain consistent results
and good yield. On the basis of the reaction rate and the
desired enantiomeric selectivity, we narrowed down the
choice of enzymes for gram-scale evaluation to cholesterol
esterases from either pseudomonas fluorescens or porcine
pancreas and lipase from porcine pancreas. All these enzymes
hydrolyzed preferentially the 1R,2S enantiomer; therefore,
CP-336156 can be isolated immediately from these enzy-
matic resolutions without further deacylation. In the large-
scale enzymatic resolution, cholesterol esterase from porcine
pancreas yielded encouraging and consistent results (Scheme
336156 was obtained in 96% ee. On the other hand, the
acylated enantiomer of CP-336156 was obtained in 98% ee10
and 76% theoretical yield when the reaction was stopped at
62% conversion. The excellent results are interesting because
the site of enzyme-catalyzed hydrolysis is remote from the
chiral centers; nevertheless, good yield and excellent ee
values still can be achieved. Also of note was that the lipase
from Mucor miehei preferred an enantiomer with 1S,2R
10,11
configuration as the hydrolyzing substrate
when butyrate
was used for the enzymatic resolution (Scheme 1); this
observation was contrary to the majority of enzymes that
we tested. The optical purity of CP-336156 obtained from
the enzymatic resolution can be further improved to greater
than 99% ee by recrystallization from 95:5 mixture of
absolute ethanol/water solution. The absolute configuration
of CP-336156 was determined through an X-ray diffraction
study of the corresponding hydrochloride salt and was
found to correspond to an estradiol-like absolute configu-
ration at C-1.
1). In this large scale esterase-catalyzed kinetic resolution,
a good E value (the ratio of the specificity constants of the
9
two enantiomers), 60, was obtained. When the reaction was
stopped at 35% conversion (70% theoretical yield), CP-
1
1
(
7) For reviews in this area, see: (a) Wong, C. H.; Whitesides, G. M.
Enzymes in Synthetic Organic Chemistry; Pergamon: New York, 1994.
b) Sugai, T. Curr. Org. Chem. 1999, 3, 373-406. (c) Santaniello, E.;
Ferraboschi, P.; Grisenti, P.; Manzocchi, A. Chem ReV. 1992, 92, 1071-
140. (d) Mori, K. Synlett 1995, 1097-1109. (e) Drauz, K.; Waldmann,
(
7
Investigations into various reaction parameters, such as
1
solvent and temperature effects, and into lipase-catalyzed
irreversible acylation of the parent phenol (in order to
improve the reaction yield and selectivity) were futile.
H. Enzyme Catalysis in Organic Synthesis; VCH: Weinheim, 1995. (f)
Carnell, A. J. Annual reports on the Progress of Chemistry: Organic
Chemistry 1998, 94, 39-49. (g) Faber, K. Biotransformations in Organic
Chemistry, 3rd ed.; Springer: Berlin, 1997. (h) Robert, S. M. J. Chem.
Soc., Perkin Trans. 1 1998, 157-169; 1999, 1-12.
In summary, even though the chiral centers are remote
from the center of enzymatic reaction, Lasofoxifene was
still efficiently prepared in good yield and excellent ee.
(8) The representative experimental procedure for enzymatic resolu-
1
2
tion: substrate (1 mg) and a enzyme (1 mg) were added to 1 mL of pH 7
phosphate buffer and stirred under room temperature. The reaction was
monitored on a chiral HPLC column for reaction conversion and the ee of
product and substrate10 without isolation. The enzyme was added accordingly
if the reaction was slow. Typically, the enzymatic reactions were stopped
OL006652+
9
at 30-70% conversions; then enzymes that gave good E values were picked
up for further evaluation. For large-scale reaction, the same process was
adopted except reactions were done in 0.1 M concentration and ethyl acetate
was added to extract compounds from aqueous solution. Product was
separated from substrate with silica gel column chromatography (3% MeOH,
(10) For ee determination: Chiralcel OD column; mobile phase ethanol:
hexane ) 95:5; flow rate 1 mL/min; wavelength 215 nm; rt; retention times
for CP-336156 and its enantiomer are 23 and 31 min; retention times of
the methyl acetates are 10 and 14 min.
(11) For determining the absolute configuration of CP-336156, see ref
6.
(12) For another example of enzymatic resolution in which the site of
enzymatic hydrolysis is remote from the chiral center, see: Horiuchi, S.;
Takikawa, H.; Mori, K. Bioorg. Med. Chem. 1999, 7, 723-726.
0
.5% NH4OH in CH3Cl). Subsequently, the ee was determined on the
isolated product and starting material again.
9) E is the ration of specificity constants of two enantiomers, see: Chen,
C. S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J. J. Am. Chem. Soc. 1982, 104,
294.
(
7
Org. Lett., Vol. 2, No. 25, 2000
4027
Yang, Xiaojing
