GB2241953A

GB2241953A - Stereoselective hydrolysis

Info

Publication number: GB2241953A
Application number: GB9102131A
Authority: GB
Inventors: Michael John Nicholds; David Herbert George Crout; Ian Harvey
Original assignee: Imperial Chemical Industries Ltd
Current assignee: Imperial Chemical Industries Ltd
Priority date: 1990-03-12
Filing date: 1991-01-31
Publication date: 1991-09-18
Also published as: GB9102131D0; GB9005467D0

Abstract

An optically active alcohol of formula (II): <IMAGE> is prepared by stereoselective hydrolysis of a racemic ester derived from (II) and acids of formula RCO2H, where R is C1-20 alkyl group, in the presence of a lipase.

Description

PROCESS FOR THE PREPARATION OF OPTICALLY ACTIVE INTERMEDIATES FOR INSECTICIDAL COMPOUNDS This invention relates to a process for the preparation of optically active intermediates for insecticidal compounds.

European Patent Application No. EP-A-211S61 discloses insecticidal activity for compounds of formula (I):

wherein Y represents a substituted aryl group where each substituent is selected from halo, alkyl, aryl, aralkyl, aryloxy and arylamino. These compounds exhibit asymmetric substitution at the carbon atom marked * in formula (I).

In common with many biologically active molecules exhibiting asymmetric substitution, one enantiomeric form of these compounds exhibits higher levels of activity than the other.

The resolution of compounds into their individual enantiomers is described in EP-A-211561 by use of high pressure liquid chromatography on a chiral support.

Although such chromatographic methods are effective on a small scale, they are unsuitable for operation on a large scale owing to high expense and the requirement for large volumes of eluent.

The compounds of formula (I) may be prepared by processes described in EP-A-211561 from the alcohol intermediate 2-(4-ethoxyphenyl)-3,3,3-trifluoropropanol of formula (II):

The compound of formula (II) shares the same asymmetric centre as the insecticidal products derived from it.

The present Applicants have sought to overcome the requirement for chromatographic resolution of compounds of formula (I) by developing a process for the preparation of compounds of formula (II) in optically active form, that is to say, having a high excess of one enantiomer.

Optically active alcohols may be prepared by stereoselective hydrolysis of esters derived therefrom by treatment with enzymic esterases. Many such esterases are known in the literature, but their suitability for specific substrates cannot be readily predicted because of the highly specific nature of the steric and electronic interaction between the active site of the esterase and its substrates. By the use of a screening process in which both the hydrolysis of esters derived from the compounds of formula (II) in the presence of a wide range of esterases, and the enantiomeric excess of the resultant alcohol of formula (II) may be monitored, the present Applicants have identified that stereoselective hydrolysis may be achieved by the use of lipases of various origins.

Accordingly, the invention provides a process for the preparation of an optically active alcohol of formula (11):

by stereoselective hydrolysis of a racemic ester of formula (III):

wherein R represents a C1 20 straight or branched chain alkyl group, in the presence of a lipase.

Lipases from a variety of origins may be used to effect the stereoselective hydrolysis. In particular there may be mentioned lipases from the following sources: porcine pancreas, wheat germ, Aspergillus niger, Candida cylindracea, Candida lypolytica, Geotrichum candidum, lipozyme, Mucor javanicus, Penicillium cyclopium, Penicillium roqueforti, Pseudomonas florescens, Rhizopus arrhizus, Rhizopus delemar, Rhizopus javanicus and Rhizopus niveus.

Preferred lipases according to the invention are those derived from porcine pancreas, Mucor javanicus, Penicillium rogueforti, Rhizopus arrhizus and Rhizopus delemar. Porcine pancreatic lipase has been found to be a particularly effective stereoselective hydrolytic agent for the esters of formula (III).

The process according to the invention may be performed under any of the conditions well-known to be suitable for enzyme-catalysed reactions. Thus, for example, the ester of formula (zit) may be mixed with the lipase in a buffered medium at a temperature within the range 20 OC - 500C , and preferably within the range 300C - 400C . The preferred pH value of the buffered medium will depend on the optimum value for the chosen lipase, and will be readily determinable by the skilled operator.

Typically, the prefered value will be approximately pH 7.

In a preferred embodiment of the invention, the ester and lipase are mixed in a two-phase system comprising an aqueous, buffered phase and a substantially water-immiscible organic phase. Suitable organic phases which may be employed are C6 10 liquid hydrocarbons such as iso-octane, hexane or cyclohexane; ethers such as diethyl ether or diisopropyl ether; and halogenated hydrocarbons such as carbon tetrachloride or chloroform.

Iso-octane is particularly suitable for use as a co-solvent in such a two-phase system.

The rate of hydrolysis and enantiomeric excess of the hydrolysis product have been found to vary with the concentration of substrate in the reaction mixture, particularly in two-phase systems. Thus, stereoselective hydrolysis of esters of formula (II) may be optimal over a concentration range of 0.01 to 5 moles of ester per litre of medium, and preferably within the range of 0.025 to 0.5 moles per litre. Progress of the reaction may be monitored by any of the standard chromatographic techniques capable of distinguishing the esters of formula (II) and the alcohol hydrolysis product, for example, gas liquid chromatography (GLC) and high pressure liquid chromatography (HPLC). The rate of hydrolysis is also dependent on the choice of lipase, substrate and reaction medium. The degree of stereoselectivity of the hydrolysis reaction is also dependent on the factors mentioned above.

Optimum results have been found by the use of porcine pancreatic lipase in a two-phase reaction medium comprising 30-80% of iso-octane and 70-20% of an aqueous phosphate buffer maintained at pH7 during the hydrolysis procedure, using an ester substrate of formula (11) at 0.025-0.1 mole/litre. Hydrolysis rates of 1-4% per hour are achieved in such a medium.

The degree of stereoselectivity of the hydrolysis reaction may be measured by the excess of one enantiomeric form of the hydrolysis product present in the reaction mixture. The enantiomeric excess, (ee) expressed as a percentage figure, may be determined by HPLC using a chiral column packing.

The overall effectiveness of the stereoselective hydrolysis reaction may be measured in terms of the Specificity Ratio (E) may be calculated as shown below: E - Ln (1 - c (lee? Ln (1 - c (l+ee) where : c represents the total amount of ester hydrolysed, expressed as a fraction, and ee represents the enantiomeric excess of the alcohol hydrolysis product, expressed as a fraction.

Specificity Ratios in excess of 10.0 represent an effective combination of hydrolysis and stereoselectivity and are of particular importance as the basis of hydrolysis reactions suitable for scaling up alcohol enantiomer production to a technical or semi-technical scale.

Separation of the reaction product and unhydrolysed ester may be achieved by any suitable standard procedure known in the art. Suitable examples include separation by distillation following extraction of the ester and alcohol mixture from the reaction medium. Alternatively, the extracted mixtures may be separated by preparative scale high pressure liquid chromatography. The separation process leads to an alcohol hydrolysis product which is rich in one enantiomeric form, and an ester which is rich in the other enantiomeric form. Hydrolysis of the ester, for example by reaction with an alkali metal hydroxide in an aqueous alkanol mixture, has been found to proceed with retention of the enantiomeric excess, thus allowing the preparation of the alcohol of formula (II) having a high excess of either enantiomeric form.The desired enantiomeric form may then be converted to the insecticidal compounds of formula (I) by processes described in EP-A-211561 with retention of the enantiomeric excess.

The lipases used in the process of the invention may be employed in their crude form or may be purified by fractionation. Increased Specificity Ratios may be obtained using purified enzymic extracts.

The racemic esters of formula (III) used as substrates for the stereoselective hydrolysis reaction may be prepared by reaction of (RS)-2-(4-ethoxyphenyl)-3,3,3-trifluoropropan-l-ol with an acid halide of formula (IV):

where R is a C1-20 straight or branched chain alkyl group and Hal is a halogen atom, for example chlorine or bromine. The reaction may be carried out in the presence of a base at temperatures in the range -150C to 40 CC. The reaction may be suitably performed in an inert organic solvent, or may be carried out in a liquid tertiary amine acting as both base and solvent, for example pyridine.

Reaction rates may be increased by heating to the reflux temperature of the solvent.

The following examples illustrate various aspects of the invention. Porcine pancreatic lipase used in Examples 2-8 was obtained from the Sigma Chemical Company Ltd, Poole, Dorset, UK.

EXAMPLE 1 Preparation of the racemic esters of formula (III):

General Method (RS)-2-(4-ethoxyphenyl)-3,3,3,3-trifluoropropan-l-ol (1 molar equivalent) was dissolved in pyridine and the solution cooled to OOC by external cooling. The appropriate acid chloride of formula R-COC1 (1.5 molar equivalents) was added to the stirred solution, and the progress of the reaction monitored by chromatography.

When all of the alcohol had been consumed, the reaction mixture was distilled under reduced pressure to remove volatile materials and the crude product partitioned between water and dichloromethane. The organic layer was separated and washed with dilute hydrochloric acid solution to remove traces of pyridine, then dried over anhydrous magnesium sulphate. Evaporation of the solvent under reduced pressure gave the crude ester which was purified by chromatography. The following esters were prepared and characterised by 1R and 19F Nuclear magnetic resonance spectroscopy:

R CH3 CH2CH3 CH2CH2CH3 CH(CH3)2 -(CH2 )17CH3 EXAMPLE 2 Screening assay for stereoselective hydrolysis by esterases.

The following procedure forms the basis of the screening assay used to investigate the suitability of esterases for stereoselective hydrolysis.

The enzyme was added to an aqueous phosphate buffer solution (5cm3, 0.067moles, pH7.0) at 370C, and the mixture stirred vigorously.

(RS)-2-(4-ethoxyphenyl)-3,3,3-trifluoropropyl acetate (0.25 mmoles) was added to the stirred mixture and the temperature maintained at 370C for the duration of the reaction. As the hydrolysis proceeded, the pH of the mixture fell, but was maintained at pH 7.0 by the automated addition of sodium hydroxide solution. The reaction was terminated after the consumption of 0.4-0.6 molar equivalents of sodium hydroxide, or at 24 hours, whichever was earlier. The substrate and hydrolysis product were extracted into hexane (5cm3) and analysed by chiral high pressure liquid chromatography using a D-PGI (Pirkle type IA) column.

The extent of the reaction (measured as a percentage of the racemic ester hydrolysed) and the stereospecificity (measured as enantiomeric excess of the alcohol hydrolysis product) are recorded in Table I.

TABLE I

Enzyne Source Supplier Hydrolysis Enantiomeric Enantiomer4 (%) Excess(%) Lipase Porcine Pancreas S 27 21 2 " " " S 685 435 2 " Wheatgerm S 7 8 2 " Aspergillus niger A 89 20 1 " " " B 34 9 1 " Candida cylandracea A 58 3 2 " " " S 25 4 2 " " " S 525 35 2 " Candida lypolytica B 37 2 1 " Geotrichum candidum B 64 15 1 " lipozyme N 13.5 12 1 " " N 54 10 1 " Mucor javanicus A 60 42 2 " " " B 59 26 2 TABLE I (contd)

Enzyme Source Supplier Hydrolysis Enantiomeric Enantiomer4 (%) Excess(%) Lipase Penicillium cyclopuim B 56 21 2 " Penicillium roqueforti B 71 26 1 " Pseudomonas florescens A 33 6 2 " " " B 37 3 2 " Rhizopus arrhizus B 24 21 2 " Rhizopus delmar B 18 31 2 " " " B 135 235 2 " " javanicus A 37 6 1 " " " B 12 7 2 " " niveus A 17 8 2 " " " B 12 17 2 Footnotes S = Sigma Chemical Company; A = Amano; B = Biocatalysts; N = Novo 2 Extent of Hydrolysis as determined by NaOH uptake 3 Of alcohol, determinated by chiral HPLC 4 Refers to order of elution from HPLC column 5 10% (volume/volume) iso-octane added to reaction mixture EXAMPLE 3 Use of a two-phase system for stereoselective hydrolysis.

A solution of (RS)-2-(4-ethoxyphenyl)-3,3,3trifluoropropyl acetate (0.25mmoles) in iso-octane (5cm3) was added to a solution of the crude lapase in an aqueous phosphate buffer (5cm3, 1M, 7.0). The reaction vessel was stoppered to prevent evaporation and the two-phase mixture stirred at 370C. Samples of the organic phase were withdrawn periodically and the progress of the stereoselective hydrolysis monitored by chiral HPLC, the extent of the reaction and the enantiomeric excess (ee) of the hydrolysis product being determined.The overall effectivness of the stereoselective hydrolysis was calculated in terms of the Specificity Ratio (E) according to the formula: E - ln (1 - c (l+ee)) ln (1 - c (1+eye)) where c represents the proportion of ester hydrolysed (expressed as a fraction) and ee represents the enantiomeric excess of the alcohol (expressed as a fraction). Results are shown in Table II.

TABLE II

Lipase TIME (Days) 1 2 5 7 porcine hydrolisis(%) 2 3 6 8 pancreatic ee(%) 66 69.4 7 74 lipase E 4.95 5.66 7.01 7.13 Mucor Hydrolysis(%) 1 6 18 25 javanicus ee(%) 27 30 28 28 lipase E 1.74 1.89 1.89 1.94 TABLE II (contd)

Lipase TIME (Days) 1 2 5 7 Penicillium Hydrolysis(%) 1 2 3 3 roqueforti ee(%) 42 42 36 36 E 2.46 2.48 2.15 2.15 Penicillium Hydrolysis(%) 2 3 4 8 arrhizus ee(%) 28 28 15 22 E 1.28 1.79 1.36 1.59 Penicillium Hydrolysis(%) 3 6 41 48 delemar ee(%) 19 19 9.5 11.8 E 1.48 1.47 1.29 1.40 EXAMPLE 4 Effectiveness of cosolvents in two-phase systems for stereoselective hydrolysis using porcine pancreatic lipase.

Following the general method of Example 3, the effect of variation of co-solvent and proportion of organic to non-organic phase was studied for (RS)-2-(4-ethoxyphenyl)-3,3,3-trifluoropropyl acetate and (RS)-2-(4-ethoxyphenyl)-3,3,3-trifluoropropyl valerate.

The buffer medium employed was an aqueous phosphate buffer (5cm3, 0.067M, pH 7.0). The vessels were not stoppered in this example. The rate of hydrolysis was determined by reference to the take up of sodium hydroxide solution required to maintain the pH at 7.0, and was controlled and monitored by autotitration. In each case, a lag time was observed before hydrolysis began. The observed Specificity Ratio (E - calculated as in Example 3), hydrolysis rate and lag time are recorded in Tables lIlA and IIIB.

TABLE III Two-phase hydrolysis of acetate ester by porcine pancreatic lipase.

Solvent Proportion of E Hydrolysis lag solvent to aqueous rate time phase (% volume) (% per hour) minutes None - 1.6 - iso-octane 33 8.5 4 45 " 50 7.6 2.3 90 " 75 10.0 1.3 60 hexane 50 8.3 4.0 45 cyclohexane 50 6.2 1.9 30 diethyl ether 50 10.6 3.6 15 diisopropylether 50 8.1 2.7 30 CCl4 50 10.0 2.9 30 CHCl3 50 12.7 3.4 20 TABLE IIIB Two-phase hydrolysis of valerate ester by porcine pancreatic lipase.

Solvent Proportion of E Hydrolysis lag solvent to aqueous rate time phase (% volume) (% per hour) minutes CHCl3 50 3.2 4.8 60 iso-octane 50 3.8 2.7 30 iso-octane 50 5.8 - 60 Footnote : Reaction temperature 20 C.

EXAMPLE 5 Effect of substrate concentration.

A two phase mixture of porcine pancreate lipase (0.lug) in an aqueous phosphate buffer solution (5cm3, 0.067N, pH 7.0) and (RSJ-2-(4-ethoxyphenyl)-3,3,3- trifluoropropyl acetate (0.25mmol) in iso-octane (5cm3) was stirred at 370C, and the hydrolysis rate measured by autotitrat on as described in Example 4. The experiment was repeated using 0.1 mmol of the acetate ester in the same volumes of aqueous and non-aqueous phases. Results are shown in Table IV, and illustrate that the Specificity Ratio increases with increasing concentration of the substrate.

TABLE IV

Amount Concentration E Hydrolysis substrate scaling rate (mmol) factor (mmol/hour) 0.25 x1 7.6 1 1.0 x4 10.6 2.3 Footnote : corresponds to 17% hydrolysis and 80% ee EXAMPLE 6 Variation of the ester group.

Stereoselective hydrolysis of a number of esters of (RS)-2-(4-ethoxyphenyl)-3,3,3-trifluoropropanol was carried out by reaction in a two-phase system comprising iso-octane (5cm3) and an aqueous phosphate buffer solution 3 (5cm , 0.067M, pH 7.0). In each case, 0.25mmol of ester was hydrolysed in the presence of 0.lug of porcine pancreatic lipase. The hydrolysis rate was monitored by auto-titration as illustrated in Example 4. Results are shown in Table V.

TABLE V

Ester Carbon chain E Hydrolysis lagtime length rate (%/hour) (minutes) acetate 2 7.6 2.3 90 propionate 3 13.0 2.6 0 butyrate 4 12.4 6.5 20 iso-butyrate 4 (branched) 3.1 6.5 300 valerate 5 3.8 2.7 30 stearate 18 2.8 0.65 120 EXAMPLE 7 Effect of pH on stereoselective hydrolysis.

Hydrolysis of (RS)-2-(4-ethoxyphenyl)-3,3,3trifluoropropyl acetate by porcine pancreatic lipase was carried out under the conditions described in Example 6, with the exception that the pH of the buffer medium was maintained at alternative values. Results are shown in Table VI.

TABLE VI

pH E Hydrolysis ee (%) Extent of rate hydrolysis (%) (%/hour) 6.0 6.00 2.8 55 51 7.0 7.60 2.3 - 8.0 5.23 1.0 63 22 EXAMPLE 8 Analysis of enantiomeric excess of residual esters.

Following stereoselective hydrolysis of (RS)-2-(4-ethcxyphenyl) 3,3,3-trifluoropropyl acetate by porcine pancreatic lipase under the conditions described in Example 6, the organic phase was separated and the alcohol hydrolysis product and residual ester were isolated by flash column chromatography. Analysis of the reaction products by chiral HPLC showed 58% hydrolysis, and enantiomeric exesses for the alcohol of 61% and for the residual ester of 85%. The calculated specificity ratio was 10.59.

Optical rotations for the separated materials were determined as follows: residual acetate: [ ]24 = + 14.320 D hydrolysis product (alcohol) [ ]24 =-10.1 D The residual acetate was hydrolysed in a potassium hydroxide aqueous ethanol mixture, and shown to give an alcohol with a retained enantiomeric excess of > 70%.

In a repeat of this procedure, the following values were obtained: enantiomeric excess of alcohol: 65% extent of hydrolysis: 57% enantiomeric excess of residual ester: 86% E: 12.48 [a] 24 residual acetate: + 15.250 D [ ] 24 alcohol: - 21.30 D enantiomeric excess of hydrolysed residual acetate: > 70%

Claims

CLAIMS 1. Process for the preparation of an optically active alcohol of formula (II):

by stereoselective hydrolysis of a racemic ester of formula (III):

wherein R represents a C1 20 straight or branched chain alkyl group, in the presence of a lipase.
2. Process according to claim 1 wherein the lipase is derived from a source selected from porcine pancreas, wheat germ, Aspergillus niger, Candida cylindracea, Candida lypolytica, Geotrichum candidum, lipozyme, Mucor javanicus, Penicillium cyclopium, Penicillium roqueforti, Pseudomonas florescens, Rhizopus arrhizus, Rhizopus delemar, Rhizopus javanicus and Rhizopus niveus.
3. Process according to claim 1 wherein the lipase is derived from a source selected from porcine pancreas, Mucor javanicus, Penicillium roqueforti, Rhizopus arrhizus and Rhizopus delemar.
4. Process according to claim 1 wherein the lipase is porcine pancreatic lipase.
5. Process according to any preceding claim performed in a buffered medium at a temperature within the range 200C-500C.