CA2191363A1 - Chiral compounds and their resolution - Google Patents

Abstract

Enantiomeric glutarimides such as aminoglutethimide and rogletimide are prepared by cyclisation of a corresponding ester-nitrile which is a good substrate for biotransformation with an enantiospecific esterase.

Description

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 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)

Claims (18)

12

1. A method for preparing, in the form of at least predominantly one enantiomer thereof, a chiral compound of formula I
I
wherein X and Z are each H or an organic group and Y is a cyclic group, which comprises the steps of:
contacting a chiral ester of formula II
II

wherein R is an esterifying radical, in racemic form, with an enantiospecific esterase;
separating the ester of formula II from the corresponding acid (R = H) formed by the esterase; and cyclising this optically-enriched acid or ester.

2. A method according to claim 1, wherein X is C1-10 alkyl and R is H or C1-10 alkyl.

3. A method according to claim 2, wherein X is ethyl.

4. A method according to any preceding claim, wherein R
is methyl or ethyl.

5. A method according to any preceding claim, wherein Z
is H.

6. A method according to any preceding claim, wherein Y
is 4-pyridyl, phenyl, 4-nitrophenyl or optionally N-protected 4-aminophenyl.

7. A method according to claim 1, wherein Y is 4-nitrophenyl, and which comprises the additional step of 12a reduction, to give the corresponding compound wherein Y is 4-aminophenyl.

8. A method according to any preceding claim, wherein the compound of formula I is (R)-4-pyridoglutethimide or (R)-aminoglutethimide.

9. A method according to any preceding claim, wherein the compound of formula I is an aromatase inhibitor.

10. A method according to any preceding claim, wherein cyclisation comprises heating in an acidic medium.

11. A method according to any preceding claim, which comprises the prior steps of contacting racemic formula II
compound, wherein R is not H, with an enantiospecific esterase; further resolution (if desired); and separating the compound of formula II from the acid formed by the esterase reaction.

12. A method according to claim 11, wherein the esterase has the characteristics of a protease such as .alpha.-chymotrypsin or that obtainable in Ophiostoma novo-ulmi, IMI 356050.

13. A method according to claim 11 or claim 12, wherein further resolution is conducted, using a camphorsulphonate salt or other resolution agent.

14. A compound of formula II as defined in any of claims 1 to 9 in the form of one enantiomer substantially free of the other enantiomer.

15. A compound according to claim 14, wherein the one enantiomer is the (R)-enantiomer.

16. A compound according to claim 14 or claim 15, wherein X is ethyl and Y is as defined in claim 6.

7. (R)-3-ethyl-3-(4-nitrophenyl)piperidine-2,6-dione (nitroglutethimide).

18. A compound according to any of claims 14 to 17, in an enantiomeric excess of at least 50%.