Method for the Production of a Compound, Comprising a Free Hydroxyl Group and a Hydroxyl Group Which is Protected by an Ester Function by Enzymatic Reaction

Abstract

The invention relates to a method for the production of a compound comprising a free hydroxyl group and a hydroxyl group which is protected by an ester function by enzymatic reaction, using a lipase EC 3.1.1.3. The invention also relates to the use of the resultant compound as an intermediate for the production of medicaments and pharmaceutical products.

Description

EXAMPLE 1

Materials Used:

Lipase from Alcaligenes sp.: GLG, QLC or QL, sold by Meito-Sangyo, or Chirazyme L10™, sold by Roche (hereinafter referred to as L10);

Pancreatin lipase: porcine pancreatin, sold by Sigma;

Diol substrate: cis-4-cyclopentene-1,3-diol, sold by Fluka (compound of formula (I)).

4.21 g (0.042 mol) of 1,3-dihydroxycyclopent-2-ene are introduced with stirring into 30 ml of acetone (23.7 g) at ambient temperature (24-25° C.) in a 100 ml reactor.

After dissolution of the diol in the acetone, 18.08 g of vinyl acetate (0.21 mol; 5 molar equivalents with respect to the diol) are added, followed by 420 μl (10% by weight with respect to the diol) of demineralized water. The temperature of the medium is then set at 5° C.

100% by weight of pancreatin enzyme with respect to the diol or a predetermined percentage by weight of lipase enzyme from Alcaligenes sp. with respect to the diol are added. After 6.5 hours, 0.5 ml of reaction medium is withdrawn and centrifuged. 200 μl are withdrawn and diluted in 800 μl of acetone before injection in chiral gas chromatography.

The remainder of the reaction medium is filtered, so as to separate the lipase; the cake is washed with approximately 3 g of acetone. The filtrate, to which approximately 8 g of TMBE (tert-butyl methyl ether) have been added, is subsequently distilled under vacuum, so as to remove the acetone and the vinyl acetate. At the end of this first devolatilization, approximately 15 g of TMBE and active charcoal are added. The reaction medium is stirred and filtered through Clarsel. The filtrate is devolatilized. Heptane is subsequently added to crystallize the desired product and the temperature is reduced from 28 to 8° C. Crystallization is observed to begin at approximately 16° C. The desired compound, which exists in the form of white crystals, is subsequently filtered off. The crystals, comprising the R monoacetate (compound III) and the S monoacetate (compound II), are dried at ambient temperature under 50 mbar for 18 hours.

Gas chromatography (GC) is carried out using a Cyclodex B column composed of permethylated β-cyclodextrin deposited in a silicone oil composed of 86% of dimethylsiloxane units and of 14% of methyl(cyano-propyl)siloxane units. The column has a length of 30 m, an internal diameter of 250 μm and a thickness of silicone oil film of 0.25 μm. The diol (compound (I)) is eluted with a relative retention time of 1.00, the R monoacetate (compound (III)) with a relative retention time of 1.10, the S monoacetate (compound (II)) with a relative retention time of 1.13 and the diacetate (compound (IV)) with a relative retention time of 1.38.

The area percentage of the peaks extracted from the chromatograph is subsequently measured for the compounds (I), (II), (III) and (IV). The results appear in table I:

	TABLE I
	Pan-
	creatin	QLG	QLG	QLG	QLG	QLC	QL	L10
Degree of	20	78.9	88.6	93.5	98.5	98.7	99	99
conversion
of the
compound
(I) (%)
Enantiomeric	67	80	74	87	94	94	97	97
excess of
compound
(II) (%)
Selectivity	10.3	7.1	5.6	4.8	3.9	3.9	3.3	3.5
Yield of	16.5	61.9	69.1	72.5	75.8	76.4	75.5	76.4
compound
(II) (%)
Reaction	6.5	6.5	6.5	6.5	6.5	6.5	6.5	6.5
time
(hours)
Amount	100	3.6	5.3	7.0	10.0	10.0	5.0	5.0
of enzyme
(as % by
weight
with respect
to the diol)

The degree of conversion of the compound (I) (%) is calculated in the following way: (area percentage of the compound (I) at time 0 (beginning of the reaction)

the area percentage of the compound (I) at the end of the reaction)/area percentage of the compound (I) at time 0.

The enantiomeric excess of compound (II) (%) is calculated in the following way: (absolute value of the (area percentage of the compound (II)−area percentage of the compound (III)))/(area percentage of the compound (II)+area percentage of the compound (III)).

The selectivity is calculated in the following way: it corresponds to the (area percentage of the compounds (II)+(III))/(area percentage of the compound (IV)).

The yield of compound (II) (%) is calculated in the following way: (area percentage of the compound (II))/(area percentage of the compound (I) at time 0).

It is thus observed that the use of the lipase makes it possible to obtain a transesterification reaction of the diol to produce a compound (II) with a better performance than that obtained with an enzyme of animal origin, with markedly lower amounts.

As a reaction scheme of the type:

occurs, the low amount of the compound (IV) obtained at the end of the reaction using the enzyme pancreatin leads to a high selectivity measurement.

Claims

1. A process for the manufacture of a compound of formula (II):

2. The process as claimed in claim 1, wherein the lipase of the class EC 3.1.1.3 exhibits the following characteristics: an enantiomeric excess of compound (II) of greater than or equal to 50%;a selectivity for compounds (II) and (III) of greater than or equal to 2, wherein compound (III) has the formula:

3. The process as claimed in claim 1, wherein the lipase is selected from the group consisting of the QL lipase from Alcaligenes sp. PL-266, registered under the number FERM-P No. 3187, and the PL lipase from Alcaligenes sp. PL-679, registered under the number FERM-P No. 3783.

4. The process as claimed in claim 1, wherein the lipase is immobilized on an appropriate solid support.

5. The process as claimed in claim 4, the solid support is selected from the group consisting of DEAE cellulose, DEAE sepharose, diatomaceous earth, silica, alumina, and polypropylene and their mixtures.

6. The process as claimed in claim 1, wherein the lipase is selected from the group consisting of the QL, QLC, QLG, PL, PLC and PLG lipases.

7. The process as claimed in claim 1, wherein R is a hydrocarbon chain having at least one unsaturation.

8. The process as claimed in claim 1, wherein the compound of formula (I) is selected from the group consisting of the compounds of formula (V), (VI) and (VII):

9. The process as claimed in claim 1, wherein the proportion of lipase is between 0.1 and 30% by weight with respect to the weight of the compound of formula (I).

10. The process as claimed in claim 1, wherein the organic solvent is selected from the group consisting of: ketones, ethers, nitriles, and aromatic compounds.

11. The process as claimed in claim 1, wherein the reaction medium of stage a) comprises water.

12. The process as claimed in claim 1, wherein the acylating agent is a compound of formula (VIII): R1-COO-R3  (VIII)

13. The process as claimed in claim 1, wherein the acylating agent is selected from the group consisting of acetates, benzoates and isobutyrates.

14. The process as claimed in claim 1, wherein the acylating agent is selected from the group consisting of vinyl acetate, ethyl acetate, isopropyl acetate, 2,2,2-trifluoroethyl acetate and isopropenyl acetate.

15. The process as claimed in claim 1, wherein the reaction of stage a) is carried out at a temperature of between −5 and 40° C.

16. The process as claimed in claim 1, wherein the duration of the enzymatic reaction of stage a) is between 1 and 24 hours.

17. (canceled)

18. The process as claimed in claim 1, wherein the lipase is not immobilized on an appropriate solid support.

19. The process as claimed in claim 1, wherein the organic solvent is selected from the group consisting of acetone, methyl ethyl ketone, cyclohexanone, cyclopentanone, methyl isobutyl ketone (MIBK), methyl tert-butyl ether (MTBE), tetrahydrofuran (THF), acetonitrile and toluene.