Note: Descriptions are shown in the official language in which they were submitted.
W0 95/32947 21913 6 3 r ~ 228
CHIRAL C:u~l~uu~vS AND THEIR RESOLUTION
Field of the Invention
This invention relates to chiral compounds that are
useful as intermediates in the synthesis of
5 pharmaceutically-active glutarimides, and to their
resolution .
Ba.,}.u~u--d of Invention
Racemates of 3,3-disubstituted glutarimides such as 3-
ethyl-3-(4-aminophenyl)piperidine-2,6-dione
10 (aminoglutethimide) and 3-ethyl-3- (4-pyridyl) piperidine-
2,6-dione (rogletimide) have been shown to be effective for
the treatment of h~L ,~ tlep~ Pnt breast cancer; see Smith
et al, Lancet ii:646 (1978), and Foster et al, J.Med. Chem.
28:200 (1985). The mode of action of these compounds is
15 considered to be inhibition of the enzyme aromatase that
catalyses the formation of e~LL~y~ s from androgens; thus
the ~ o~..ds inhibit tumours whose growth is promoted by
e"~, .,y~:l,5 .
McCague et al, J.Med.Chem. 35:3699-3704 (1992),
disclose that derivatives of rogletimide, including 5-21kyl
derivatives, may have i uved aromatase inhibition
activity. Aromatase inhibition by the enantiomers of
aminoglutethimide, rogletimide and also
cyclohexylaminoglutethimide, in vitro, is reported by
Ogbunude et al, chirality 6:623-626 (1994).
Graves et al, Endocrinology 105:52 (1979), disclose
that the (2)-enantiomers of these compounds are much more
potent as inhibitors of aromatase than the (s)-enantiomers.
Therefore, it is likely that the (R)-enantiomers are
essentially the active ~ --nts in the racemates, and so
a process for their preparation is desirable.
The separate enantiomers of aminoglute~h ~ P and
rogletimide have been prepared respectively by repeated
recrystallisation of tartrate salts, and by using camphor-
derived chiral All~i l; Aries; see Finch et al, Experientia
31:1002 (1975), and McCague et al, J.Chem.Soc.Perkin Trans.
1: 196-8 (1989) - Separation has also been accomplished by
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
W0 95/32947 21 9 ~ 3 b 3 r~ L~
chromatography on chiral stationary phases based on
tartramides or triacylcelluloses. However, these methods
are not ---hlP to economic large-scale working
appropriate for the manufacture of the bulk single-
enantiomer drug substance.
WO-A-9304058 discloses a process for the manufacture
of such glutarimide c - 'c, by way of biocatalytic
resolution of glutarate diesters. Only the less hindered
ester function is hydrolysed by an appropriate biocatalyst,
with a degree of enantiospecif icity showing that the
biocatalyst can dist;nqll;ch aryl, ethyl and carboxylic
ester substituents borne on a quarternary carbon atom.
While only moderate specificity was observed in the case of
precursors of rogletimide, the biotransf ormation products
were easily converted into rogletimide and means to then
increase the enantiomeric excess was found.
S -ry of the Invention
Whereas in WO-A-9304058 the substrate for
biotransformation is a diester, it has now been found that
a ~oLl-6~t~n~l;nq ester-nitrile (see formula II in claim 1,
but R i8 not H) i8 a satisfactory substrate. The desired
enantiomer can be separated from the unwanted Pn~ntit ~r,
and readily cyclised, e.g. using acid, to form the same
product (I) as in WO-A-9304058.
According to one aspect of the present invention,
effective biocatalytic resolution, using available
esterases, is pocc~hlp using ester-nitriles of formula II.
Good enantiospecificities have been obtained for
resolution by way of biocatalytic hydrolysis of the ester
function. Thus the appropriate enzyme is able to
distinguish between substituents, e. g . aryl, ethyl and
nitrile, borne on a quarternary carbon atom. More
particularly, racemic formula II compound may be contacted
with an enantiospecific esterase that enriches the mixture
in terms of one enantiomer, by reacting with the other
enantiomer to form the corresponding acid (II: R s H) which
may be separated; partial enrichment may be Pnh~nt~pd by
21 q 1 363 =
WO 95132947 P~ ~ /~1,; '.
further resolution with a tartaric acid or conventional
camphor-derived chiral auxiliary.
Descrimtion of the InYention
As a substrate for biotransformation, in formula II,
R is an esterifying group, suitably an alkyl residue
containing up to 10 carbon atoms, e. g . straight-chain
alkyl, branched alkyl, arylalkyl and aryl optionally
substituted with, for example, halogen. For the purpose of
the invention, the simplest alkyl group (R = methyl or
ethyl) is adequate, and in terms of simplifying the
chemical processing, is preferred. For cyclisation, after
biotransformation, R may be H; alternatively, depQn~inq on
the enantiomer that is desired, R may be l~nrh~nqQ~.
X and Z are each H or an organic group. X may be, for
example, C1 10 alkyl such as ethyl. Z is preferably H or a
Cl lO alkyl group, e.g. to give a s-alkyl product. The
' of formula I may be any aromatase inhibitor such
as aminoglutP~him~ X = ethyl, Y = 4-aminophenyl, Z -
H) or any analogue, e.g. the specific ~~ described
above, or isopropylglutethimide. The .u,,d of formula
I may also be an intermediate for hypotensive agents such
as verapamil. Y is thus defined; in general, Y (or Ar in
the Chart) is a cyclic group, either an aryl, carbocyclic
or heterocyclic radical, e.g. of up to 12 C atoms,
including any substituents. Y is preferably
dimethoxyphenyl, 4-pyridyl, 4-Am;nnphQnyl (optionally N-
protected), isopropyl-phenyl or cyclohexylphenyl.
F~:PQC~11Y as a precursor to Y=Am;nnrhQnyl, e.g. by
catalytic hydrogenation, Y may also be nitrophenyl; (R)-3-
3 o ethy 1- 3 - ( 4 -nitropheny 1 ) p iperi d ine- 3, 6 -dione is a novel
ul.d. c '- of formula II in which Y is
nitrophenyl give especially good biotransformation yields.
For the purpose of illustration only, the process
involved in the invention may be described with reference
to the production of, say, enantiomeric aminoglutethimide,
as outlined in the Chart. Compounds of formula II
(specifically formula 1) may be prepared by methods known
_ _ _ _ _ _ _ .
-
2~9~3~,3
Wo 95/32947 1 .~ '01~28
to those skilled in the art, and exemplified below. One
such method inYolves ISichael addition to an acrylate ester
(6ee Example 4).
The f irst step shown in the Chart is a characteristic
5 of the invention. It is based on the use of biocatalysts
that preferentially hydrolyse one enantiomer of a racemic
nitrile (1) to give optically-enriched residual ester (2)
and the acid (3). There are biocatalysts that produce the
R-enantiomer of the ester (i.e. biocatalysts A in the
10 Chart) and those that produce the S-enantiomer
(biocatalysts B).
Suitable esterase activities may be available from
acylase I (AspergiLlus), esterase 30,000, Rhizopus
Japonicus lipase, F3 lipase, A2 lipase (porcine pancreas),
15 F6 lipase (from Candida), pig liver esterase, CE lipase and
AY lipase. Cholesterol esterase is an alternative.
Examples of biocatalysts A are Candida cylindr2cae lipase
and enzyme activities of the genera in Examples 8 to 10.
Another example of a biocatalyst suitable for the
20 biotransfor~ation is the microbial strain P3U1, NCIMB
40517, which can produce ~-ester acid of greater than 60%
ee. Another suitable biocatalyst (of type B) is
Trichosporon ENZA I-3, IMI 348917, whose characteristics,
including its enantiospecificity for the conversion of
25 aralkanoic acid esters into the acid, e.g. (S)-ketoprofen,
are described in WO-A-9304189. ~-CIIy ~,y~sin is another
suitable biocatalyst of category B.
A further biocatalyst is obtainable from any fungus of
the type described in WO-A-9420634 for the enantiospecific
30 hydrolysis of arylpropionic acid esters. A specific fungus
of this type is Ophiostoma novo-ulmi, IMI 356050.
In specific examples of the biotransfor~ation, the
phenylqlutaronitrile ester (Ar = Ph, R = Me) with Candida
antarctica lipase gave hydrolysis of ~he ester function to
35 the acid with an enantiospecificity (E) = 12. The
nitrophenyl ~ _ ' (Ar = 4-nitrophenyl, R = Me) with ~-
~I.y LLy~sin gave a transformation with E = 39. The same
W095132947 2 l 91363 r~
substrate with esterase deriYed from the given fungus
Ophiostoma novo-ulmi also gave transformation with the
opposite specif icity.
Conversion of the biotransformation products, which
5 are readily separated by solvent extraction at neutral pH,
into enantiomerically-enriched glutarimide is by
conventional chemical techniques.
Conversion of such nitrile esters to the glutarimides
was accomplished easily under such conditions as heating
10 with acid, e.g. a mixture of acetic acid and sulphuric
aeid, to provide the optically-active glutarimide
c~ __ . These conditions are known in the eonversion of
racemic nitrile-esters into the racemic glutarimides,
aminoglutethimide and rogletimide.
The following Examples 1 and 4 illustrate the
preparation of nitrile-esters (II) that are substrates for
biotransformation, and Example 6 illustrates a relevant
reduction. Examples 2, 5 and 8 to lO illustrate
biotransformations, and Examples 3 and 7 illustrate
cyclisation reactions, in accordance with the invention.
Examples 1 to 3, and Examples 4 to 7, provLde different
routes to the same product.
r le 1 Methyl 4-cyano-4-(4-Amino~hPnyl)hexanoate
A 3 -necked round-bottomed f lask was charged with
methyl 4-cyano-4-(4-nitrophenyl)hexanoate (20.0 g), 90%
ethanol (lOOO ml) and PtOz (l. O g) . The vessel was then
evacuated and charged with nitrogen. ~he mixture was
stirred vigorously and subjected to H2 at ai _~^ric
~es,,uL~ supplied via a balloon. The catalyst was removed
by filtration through celite and the solvent removed under
reduced ~ s~ule to give methyl 4-cyano-4-(4-Am;no~hPnyl)-
hexanoate (18 g, 1009c) as a viscous, brown oil.
Exam~le 2
A 500 ml jacketed biotransformation vessel was charged
with O.lM KH2Po4, pH 7.0 (250 ml) and methyl 4-(4-
aminophenyl)4-cyAnoh~PyAn~ate (5.0 g, 20.3 mmol). Candida
eylindracea lipase (CCL; 5 . O g) was introdueed and the
2191363
Wo 95/32947 PCr/GB9~/01228
mixture was agitated using an overhead stirrer.
Temperature was maintained at 30C with the aid of a
thermocirculator and the pH controlled by a probe linked to
an autotitrator. The biotransformation was allowed to
proceed until 10 ml of lM NaOH had been addQd (e~uivalent
to 50% conversion). This took about 3 hours. At this
point, the biotransf ormation was quenched by the addition
of NaCl (25 g) and the resulting mixture was extracted with
diethyl ether (250 ml x 4). The pH of the aqueous solution
was then adjusted to 3 using conc. HCl and the mixture
extracted with ethyl acetate (400 ml x 3). The eYtracts
were pooled, dried and c~ ted under reduced pLes:,uLe,
yielding 1.8 g (38%) of 4-(4 - Aminnrhpnyl)-4 - cy~nnhpy~nnic
acid, enriched in the (R)-enantiomer, in the form o~ a
brown oil. Without further treatment, a sample of this
material was reacted as described in Example 3.
nle 3 (R)-Aminoglu~pthimid~
4-(4-Aminophenyl)-4-cy~nnhPY~noicacid (Example2; 1.8
g, 7.7 mmol), enriched in the (R)-enantiomer, was
dissolved in glacial acetic acid (6.0 ml) contained in a 25
ml round-bottomed flask. The resulting mixture was heated
to 60C with the aid of an oil bath followed by dropwisQ
addition of conc. HzSO,, (3 . O ml) . The solution was then
heated to 100C and maintained there for 30 minutes before
pouring onto ice (100 g). The pH is adjusted to 6 using 5M
NaOH followed by extraction with dichloromethane (3 x 200
ml). The extracts were pooled, dried (over MgSO4) and
concentrated under reduced pressure, giving (R)-amino-
glutethimide (1.75 g, 97%) as a brown oil. Chiral HPLC
analysis (Chiralcel OJ column; mobile phase 1:1 n-heptane-
isopropanol) indicated an ee of 78%.
Example 4~ 2-(4-Ni-Lu~hel~yl)butyronitrile
A 3-necked round-bottomed flask was charged with conc.
HNO3 (240 ml) and cooled to 10C with the aid of an
3S ice/acetone bath. Conc. H2SO4 (240 ml) was then added
slowly so as to maintain the temperature below 30C.
2-Phenylbutyronitrile (Aldrich, 110 ml) was introduced
Wo gs/32947 2 1 9 1 3 6 3 PCT/GB9S/01228
.
dropwise to the stirred solution over a period of 1 hour,
maintaining the temperature below 20C. The icelacetone
bath was then removed and the mixture stirred for a further
3 0 minutes at ambient temperature bef ore pouring it onto
5 crushed ice (100 g). The resulting mixture was extracted
with ethyl acetate (1500 ml x 2) and the extracts pooled,
washed with saturated bicarb. (1000 ml) and water (500 ~1) .
After drying over MgSO, the ethyl acetate was evaporated
under reduced pL~s,,uL~ to give crude 2-(4-nitrophenyl)-
butyronitrile as a yellow oil, crude yield 138 g, 98%.
Analysis by GC.MS indicated a para:meta ratio of 3.5:1.
EY~ le 4B Methyl 4-cyano-4- (4-nitrophenyl) hexanoate
A mixture of 2-(4-nitrophenyl)butyronitrile (10.0 g),
butanol (10 ml) and methyl acrylate (5.2 ml) was cooled to
10C in a 100 ml 3-necked round-bottomed flask, equipped
with a magnetic follower. A solution of potassium tert-
butoxide (0.6 g) in tert-butanol (10 ml) was added
dropwise, maintaining the temperature at approximately 10C
(solution turns purple). After addition was complete, the
mixture was allowed to reach ambient temperature and then
stirred for a further 2 hours. The reaction was worked-up
by partitioning between diethyl ether (400 ml) and 1 N
KH2PO4 (400 ml). The ether layer was washed with water (50
ml), dried (MgSO4) and concentrated under reduced pressure
to yieldmethyl 4-cyano-4-(4-nitrophenyl)hexanoate (14.1 g,
99%) as an orange oil.
Exam~le 5
A 1 1 jacketed biotransformation vessel was charged
with 0.05 N ~ zP04~ pH 7.5 (500 ml) and methyl 4-cyano-4-(4-
nitrophenyl)hexanoate (20 g, 72 mmol). ~-CI-y Lyl,sin (ex.
Aldrich; 4 g) was introduced and the mixture was agitated
using an overhead stirrer. Temperature was maintained at
37C with the aid of a thermocirculator and the pH
controlled by a probe linked to an autotitrator. The
biotransformation was allowed to proceed until 18 ml of lM
NaOH had been added (equivalent to 50% conversion). This
took about 68 hours, with addition of more ~ cl-y LLY~Sin
WO 95/32947 2 1 9 1 3 ~ 3 PCT/GBgS/01228
(1 g portions) after 24 hours and 50 hours. At this point,
the biotransformation was quenched by the addition of NaCl
(50 g) and the resulting mixture was extracted with diethyl
ether (500 ml x 3). The extracts were pooled, dried and
5 concentrated under reduced pressure, yielding 10 g (50%) of
(R)-methyl 4-cyano-4-(4-nitrophenyl)hexanoate, enriched in
the (R)-enantiomer, 70% ee by chiral HPLC analysis.
ExamPle 6 Nltroglutethimide
(R) -methyl 4-cyano-4- ~4-nitrophenyl) hexanoate (Example
5; 10 g, 36 mmol), enriched in the (R)-enantiomer to
approximately 70S ee, was dissolved in glacial acetic acid
(30.0 ml) contained in a 25 ml round-bottomed flask. The
resulting mixture was hQated to 60C with the aid of an oil
bath followed by dropwise addition of conc. H2SO4 (15.0 ml) .
The solution was then heated at 100C ~or 30 minutes before
pouring onto ice (100 g). The p~ was ad~usted to 6 using
5M NaOH and the mixture was then extracted with
dichloromethane (3 x 200 ml). The extracts were pooled,
dried (over MgSO4) and concentrated under reduced pL~::S~SU~,
giving (R) -nitroglutethimide of approximately 70% ee (8 . 3
g, 88%) as a brown oil.
e 7 (R)-Aminoglutethimide
A 3-necked round-bottomed flask was charged with (R)-
nitroglutethimide (ca. 70~ ee, 8.3 g, 32 mmol), 90% ethanol
(250 ml) and PtO2 (0.35 g). The vessel was then evacuated
and charged with nitrogen. The mixture was stirred
vigorously and subjected to Hz at atmospheric pL~:,uLc:
supplied via a balloon. The catalyst was removed by
f iltration through celite and the solvent removed under
reduced pressure to give (R)-aminoglutethimide (ca. 70% ee,
7.1 g, 96%) as a pale brown solid.
ExamPle 8
A loopful of Candida nlgosa, ATCC 10571, was used to
inoculate 50 ml of sterile pH 6 . 0 aqueous medium
[containing (g/1) yeast extract (5), (NH4)2SO4 (1), ~H2PO4
(5), MgSO4.7H2O (0.2) and glucose (10) ] in 500 ml Erlenmeyer
flasks shaken at 250 rpm with a one inch (25 mm) throw for
W0 95/32947 2 1 9 1 3 6 3 r~ /01228
.
24 hours at 25C. The cells were then harvested by
centrifugation at 1200 g for 10 minutes. The cells were
rPcllcpPnAP~l to one fifth of their original harvest volume
in 50 mM potassium phosphate pH 6 . 0. A 50 mg/ml emulsion of
5 ethyl 4-cyano-4-(4-nitrophenyl)hexanoate in 50 mM potassium
phosphate + 0.1% Tween 80 was prepared by sonication for 10
minutes (cycles of 10 seconds on, 3 seconds off) at an
amplitude of 18 ~m in a Soniprep 150. 400 ~1 of this
substrate emulsion was added to 1. 6 ml of the rPcllcppn~lpcl
10 cells in a 20 ml glass vial. The biotransformation
reaction mixture was then incubated with shaking at 25C,
250 rpm for 69 hours. After this time the reaction was
stopped by the addition of 2 ml ethyl acetate. The sample
was then analysed f or enantiomeric excess by chiral HPLC
(Chiralpak AD column; mobile phase 98.3% heptane-1.7%
isopropyl alcohol; flow rate was 2 ml/min). The quenched
reaction mixture was shaken vigorously and allowed to
separate and the ethyl acetate layer pipetted off.
Anhydrous magnesium sulphate was added. The dried ethyl
acetate was transferred to a fresh vial and 25 ~1
trimethylsilyl diazomethane was added. The sample was
mixed and left to stand for an hour at ambient temperature
prior to HPLC analysis, which indicated >99% ee (R)-4-
cyano-4- (4-nitrophenyl) hexanoic acid was produced in the
biotransformation, with residual substrate ee of 24S,
conversion 19%.
ExamP 1 e 9
Fusarium oxysporum IMI 329662 was cultured on 25 ml of
sterile pE 6.0 aqueous medium [containing (g/l) yeast
extract (20), (NH4)2SO4 (4), KH2PO4 (S), MgSO4.7H2O (0.3)
Na2HPO4.2H2O (5), CaCl2.2H2O (0.2) and glucose (40) ], in 250
ml point-baffled Erlenmeyer flasks shaken at 250 rpm with
a one inch (25 mm) throw for 72 hours at 25C. Cells were
harvested, res~cpPn~Pd to original volume in 50 mM
potassium phosphate and used in biotransformation as in
Example 8. The biotransformation was stopped after 24 hours
and chiral HPLC analysis was carried out as described in
Wo 95/32947 2 1 9 1 3 6 3 pcrlGB9~lolz28
Example 8. This indicated 95.9% ee (R)-4-cyano-4-(4-
nitrophenyl) hexanoic acid was produced in the
biotransf ormation .
T~mnle 10
5 Penicillium pinophilum I~I 114933 was cultured as
described in Example 9 but with the inclusion of 10 g/l
tributyrin in the medium. Cells were harvested,
rPcllcrpn~lpfl to original volume in 50 mM potassium phosphate
and used in biotransformation as in Example 8. The
10 biotransformation was stopped after 24 hours and chiral
HPLC analysis was carried out as described in Example 8.
This indicated 89% ee (R)-4-cyano-4-(4-nitrophenyl)-
hexanoic acid was produced in the biotransformation.
WO 95132947 2 1 9 1 3 6 3 PCTIGB9510122~
t
I~f Ar
COzR CN
racel-late (I)
biocalalys~ bioc~alyst
Et ~t , ~:~ Et
~r~ " Ar ~ ", Ar ~Ar
CO~R CN CO2H CN CO2R CN CO~H CN
(2a) (3b) (~b) (32)
(S)-enantiomer (R)-en2ntiomcr (R)-enantiomcr (S)-en~ntioln~r
t
~".. Ar
H
\ (R)-en~ntiomer (~2)/
~Ar
0~0
H
(S)-enantiomer (4b)
