MXPA00002250A - Improvements in or relating to the preparation of lactam compounds - Google Patents

Abstract

Lactams, for example 6-aminopenicillanic acid (6-APA), may be prepared by enzymatic conversion of a first compound, for example penicillin-G, in a solvent mixture comprising water and a non-aqueous organic solvent, especially 1,1,1,2-tetrafluoroethane. The 6-APA can be caused to precipitate, isolated by filtration and optionally derivatised to produce a desired compound. A by-product of the enzymatic conversion, phenylacetic acid, can be isolated by solvent extraction, suitably using a solvent which also comprises 1,1,1,2-tetrafluoroethane.

Description

IMPROVED PROCEDURE FOR THE PREPARATION OF LACTAMA COMPOUNDS X.ESCJB.IPTION OF THE INVENTION This invention relates to the preparation of a compound, especially an active pharmaceutical compound. Particularly, although not exclusively, the invention relates to the preparation of lactams, for example penicillins and cephalosporins and / or derivatives thereof. 6-aminopenicillanic acid (6-APA) _ and 7-aminodesacetoxycephalosporanic acid (7-ADCA) are intermediates used for the manufacture of most of the semisynthetic ß-lactam antibiotics. The commercially preferred method for the manufacture of 6-APA is by means of biochemical deacylation of benzyl-penicillin, commonly known as Pen-G or the equivalent enzymatic deacylation of phenoxymethyl-penicillin, commonly known as Pen-V. This is accomplished by using an enzyme such as penicillin acylase, which has been immobilized on an insoluble matrix such as polystyrene or polyacrylate polymers or copolymers. REF .: 32825 Various processes that use this technique are illustrated in the scientific literature. In such processes, penicillin G is isolated from a fermentation liquor as a solid intermediate using known means, and is then dissolved in water in a relatively dilute solution (for example 5% w / v). The enzyme reaction (shown in reaction scheme 1 below where R represents a potassium or sodium ion) is carried out at an elevated temperature (for example 35 to 40 ° C). The phenylacetic acid (PAA) produced is neutralized by the continuous addition of dilute aqueous sodium hydroxide (for example to 5% w / v) to maintain a pH of around 8.0. The isolation of 6-APA is usually carried out by the concentration of the liquors of the enzyme reaction, for example up to 15% with respect to 6-APA, in order to maximize the yield, followed by the precipitation with an acid diluted inorganic, such as 5% nitric acid or sulfuric acid. The PAA is removed by extraction in a non-miscible organic solvent, such as methyl isobutyl ketone or butyl acetate. The 6-APA is finally removed by filtration, washed with acetone then vacuum dried. A standard conversion performance acceptable for the deacylation process described is 94 to 96% - and is 82 to 86% for the precipitation and isolation stage.

REACTION SCHEME 1 C s COONa PENICILLIN G 6 -APA PAA The immobilized enzymes used in the above-described process may be sensitive to the inhibition of the product. Hence, the reaction is carried out normally in relatively dilute solutions. A drawback of this is that the solubility of 6-APA in water is about 2%, which means that a concentration step is necessary in order to minimize the product losses in the mother liquors. An expensive concentration step is therefore often applied to increase the 6-APA content to 12-16%.

PAA is one of the ingredients normally used in the fermentation of penicillin G. Therefore, it is commercially advantageous to recover PAA from the reaction effluent so that it can be recycled. Conventional methods used commercially for the recovery of PAA employ a multi-step process involving a combination of two or more of the following techniques: vacuum distillation, purification (for example carbon treatment), extraction in an aqueous phase, chromatography , precipitation, filtration and drying. These techniques are relatively expensive and environmentally problematic. An objective of this invention is to face the problems described above. According to a first aspect of the invention, there is provided a method for preparing a second compound by the catalytic conversion of a first compound, the method comprising contacting the first compound and a catalyst in a solvent mixture comprising water and a first non-aqueous solvent. Unless stated otherwise, or the context requires otherwise, a reference any compound herein includes a reference to a salt of the compound. Said catalyst is preferably an enzyme. The enzyme can be sensitive to the second compound, for example in the sense that relatively high concentrations of the second compound can reduce the ability of the enzyme to effect the conversion - an effect known as "product inhibition." Said enzyme is preferably capable of catalyzing a deacylation reaction This can suitably comprise an acylase which can be produced from any penicillin-acylase producing microorganism such as Escherichia, especially Esch eri chi a co li, Ps eudomonas, Streptomyces, Pro t eus and My crococus. Said enzyme is preferably immobilized, suitably by physical absorption or binding to an insoluble, solid matrix. The first compound is preferably of the general formula R1NHQ wherein R1 represents an optionally substituted alkylcarbonyl group and Q represents an optionally substituted cyclic group, especially an optionally substituted lactam, for example a β-lactam group.

Unless stated otherwise, an optionally substituted alkyl group may have up to 12, preferably up to 8, more preferably up to 6, especially up to 4, carbon atoms. Optional substituents of an alkyl group include the aryl, alkenyl, alkynyl, acyl, nitro, cyano, alkoxy, hydroxyl, amino, alkylamino, sulfinyl, alkylsulfinyl, sulfonyl, alkylsulfonyl, sulfonate, amido, alkylamido, alkoxycarbonyl, halocarbonyl and haloalkyl groups optionally substituted, and halogen, especially fluorine, chlorine or bromine. Preferred optional substituents of the alkylcarbonyl group include optionally substituted, especially unsubstituted phenyl, carboxyl and amino groups. In the preferred embodiments, the group R1 may be an optionally substituted benzylcarbonyl group or a group C (COOH) (NH2) HCH2CH2CH2C (0) - or a salt thereof. Said group Q may comprise a lactam fused to another optionally substituted cyclic portion which may be a 5 or 6 membered ring.

The first compound can be a natural product, especially a product of a fermentation reaction or a derivative thereof. The first compound is preferably an antibiotic. The method of the first aspect can include the step of preparing the first compound by a biochemical process, especially a fermentation. The ratio of the weight of the first solvent to that of the water in the solvent mixture can be at least 2, preferably at least 5, more preferably at least 7, especially at least 10. In some cases, this can be at least 15 or more. 20. Said proportion may be less than 100, preferably less than 50, more preferably less than 30, especially 20 or less. The proportion referred to is suitably present at any time during the conversion reaction, but preferably refers to the ratio at the start of the reaction. The ratio, as% w / v, of the first compound to the water present in said mixture may be at least 10, suitably at least 20, preferably at least 30, more preferably at least 40, especially 50 or more. The proportion can be less than 100, adequately less than 90, preferably 80 or less. The aforementioned ratio is suitably present at any time during the conversion reaction, but preferably it refers to the ratio at the start of the reaction. Said first non-aqueous solvent may have a boiling point, at atmospheric pressure, less than 80 ° C, suitably less than 60 ° C, preferably less than 40 ° C, more preferably less than 20 ° C. Especially preferred solvents have a boiling point lower than 0 ° C, preferably lower than -10 ° C. The boiling point can be higher than -90 ° C, preferably higher than -70 ° C, more preferably greater than -50 ° C. The first solvent is preferably organic. This may be aromatic, but is preferably aliphatic. This may include less than 10, suitably less than 8, preferably less than 6, more preferably less than 4, especially 2 or fewer carbon atoms. This may be an optionally substituted alkane, alkene or alkyne. Alkanes of 1 to 4 optionally substituted carbon atoms are preferred. It is preferably halogenated. This can include less than 10, suitably less than 8, preferably less than 6, more preferably less than 5, especially 4 or less halogen atoms. Preferred halogen atoms include fluorine, chlorine and bromine, with the fluorine and chlorine atom being preferred and the fluorine atoms being especially preferred. Preferably, the first solvent is non-chlorinated. It preferably comprises one or more carbon atoms, fluorine and hydrogen, only. Preferably, the first solvent is tetrafluoroethane, with 1,1,1,2-tetrafluoroethane which is especially preferred. The solvent mixture may include other solvents. The second compound, particularly when it is in the form of a free acid (for example when it is not a salt), can be at least 0.1%, suitably at least 0.5%, preferably at least 1.0%, more preferably at least 1.5%, especially approximately 2% soluble in water. The above-mentioned solubilities are preferably measured at 5 ° C. The second compound in the salt form, for example as an alkali metal salt, may have a solubility in water of at least 5% w / v, preferably at least 10% w / v, more preferably at least 15% w / v. The method preferably includes the step of adjusting the pH of the reaction mixture during the conversion reaction, suitably to maintain it within a physiologically acceptable pH range. The pH is preferably in the range of 7 to 9, more preferably in the range of 7.8 to 8.2. In a first embodiment, the method can include the isolation of the second compound from the other compounds / solvents. Isolation may include causing precipitation of the second compound and subsequent isolation of the precipitate by filtration. Precipitation can be caused by adjusting the pH at or near the PKa value of the second compound. The suitable pH may be less than 5, preferably less than 4. The suitable pH may be above 2.5, preferably above 3.5. Suitably, a substantially insoluble form of the second compound is produced. The pH can be conveniently adjusted by the addition of an aqueous solution of an acid such as 1 M or 2 M nitric acid or preferably 1 M or 2 M sulfuric acid. second filtrate can be washed with a second solvent. The traces of the second solvent can be substantially or completely removed from the filtrate by evaporation. In a second embodiment of the invention, the method can include adjusting the pH in the presence of a second solvent to a value at which the second compound is substantially soluble in the aqueous phase of the solvent mixture, while the by-products of the reaction They are substantially soluble in the second non-miscible solvent phase of the solvent mixture. The suitable pH may be less than 2.5, more suitably less than 2. The pH may suitably be 1 or higher. The solution of the second solvent can be separated from the aqueous phase containing the second compound, by sedimentation and physical separation, whereby an aqueous reaction liquor containing the second compound is produced. The reaction liquor can be washed with an amount of the second solvent, in order to remove contaminating traces from the reaction by-products. The second compound can be precipitated from the reaction liquor by adjusting the pH with a suitable base to produce a form substantially insoluble of the second compound. A suitable base may be an aqueous solution of sodium hydroxide, potassium hydroxide or ammonia. The pH can be adjusted appropriately above 2.5, more adequately above 3.5. The pH can suitably be less than 5, preferably less than 4. The yield of the second compound in the first or second embodiments can be maximized by stirring at reduced temperature, preferably between 2 and 10 ° C. The second compound can be isolated by filtration, washing and drying . The second solvent used in the first and second embodiments described preferably includes the first non-aqueous solvent, preferably in combination with a co-solvent. The co-solvent may include another first solvent of a type described herein. Preferably, however, this is of a different type. Said co-solvent is selected to affect the boiling point and / or the dissolution properties of the solvent for the first material. The boiling point of said co-solvent may be lower than 60 ° C, preferably lower than 30 ° C, more preferably lower than 15 ° C, especially lower than ° C. The boiling point of the co-solvent may be greater than -90 ° C, preferably greater than -70 ° C, more preferably greater than -50 ° C. Preferably, the second solvent includes a larger portion of the first solvent in combination with a minor portion of the co-solvent. Preferably, at least 90% by weight, more preferably at least 93% by weight, especially at least 97% by weight of the second solvent is comprised by the first, non-aqueous solvent, especially by a hydrofluorocarbon solvent. The remainder is preferably constituted of one or more co-solvents, as described. The co-solvent can be selected from hydrocarbons and ethers. Preferred hydrocarbons have up to six carbon atoms. These can be alicyclic or, preferably, aliphatic. These are preferably alkanes with methane, ethane, propane, and butane which are preferred. Preferred ethers are dialkyl ethers, for example dialkyl ethers of 1 to 4 carbon atoms, with dimethyl ether which is especially preferred. In the purification of the second compound using the second solvent, the second compound may be precipitated, suitably by adjusting the pH as described above. In the method of the first aspect, the catalytic conversion can result in the preparation of the second compound and a third compound. The enzyme may be sensitive to the third compound, for example in the sense that relatively high concentrations of the third compound may reduce the ability of the enzyme to effect the conversion. Where the first compound is of the general formula R1NHQ./ said third compound may represent a compound of the formula R1COOH or a salt thereof. In this case, the second compound may represent a compound of the formula H2NQ or a salt thereof. The method of the first aspect may include the step of separating the second and third compounds from each other. This may involve the provision of a solvent (which may be the first or second solvents described) in which the second and third compounds (or salts thereof) have different solubilities and / or have different partition coefficients and use this property to effect the separation. The second and third compounds can be separated using a mixture comprising the solvent and water. The third compound, especially a free acid thereof, is preferably substantially soluble in the solvent used for the separation. After isolation of the second compound it can be derivatized, suitably to produce an antibiotic. The first compound can be a natreral penicillin, or a biosynthetic penicillin prepared by the addition of a precursor to penicillin fermentation broths, or a cephalosporin. Preferably, the first compound is selected from Penicillin G, Penicillin X (p-hydroxyphenylpenicillin), Penicillin V (phenoxymethylpenicillin) and cephalosporin G. The second compound is preferably 6-aminopenicillanic acid or 7-aminodesacetoxycephalosporanic acid; and the third compound can be optionally substituted, especially unsubstituted, phenylacetic acid. In one embodiment of the invention, the penicillin acylase enzyme which has been immobilized on an insoluble matrix is charged to a lined reaction vessel, followed by the required volume of water and penicillin G is added while stirring. The reaction vessel is then sealed and applied under vacuum to reach the pressure of 10 mbars or less. The first solvent is charged to the reaction mixture. An aqueous solution of sodium hydroxide (range of 2.5 to 20% w / v or more) is introduced into the reaction to maintain a pH range of 7.0 to 9.0 especially of about pH 8.0. The reaction temperature is maintained at an approximately constant level throughout. A preferred temperature range is 20 to 50 ° C, more preferably from 30 to 40 ° C. At the end of the reaction, which is indicated by a constant pH reading without the need for the subsequent addition of aqueous NaOH solution, the reaction liquors are charged from the bottom of the reaction vessel into a second lined vessel (the evaporation vessel) via a line filter. The immobilized enzyme is thus recovered, washed with water and stored for later use. The clear filtered solution is cooled by the flow of a refrigerant through the liner. The preferred temperature is from 0 to 20 ° C, more preferably from 2 to 10 ° C. An aqueous solution of an inorganic acid, such as 1-4 M nitric acid or 1-4 M sulfuric acid, is added slowly while stirring at a pH in the range of 3.5 to 4.0, with a pH of 3.8 which is ideal. During this operation, the phenylacetic acid (PAA) present as the sodium salt is converted to the free acid form which is soluble in the first solvent. While the PAA is extracted into the first solvent, the 6-APA (in the free acid form) is precipitated and is now present as a suspension. The reaction mixture can be stirred for an additional 30 minutes up to one hour, while keeping the pH and temperature constant. 6-APA can be convenient and simply isolated by charging the reaction mixture from the bottom of the evaporation vessel back into the reaction vessel via an in-line filter. Optionally, a filter element can be adjusted to the outlet of the bottom of the container, such as a wire mesh filter or a sintered glass. According to a second aspect of the invention, there is provided a method of removing a third compound (preferably an acid or acid salt, especially PAA), as described in present, from a mass of material containing the compound, the method comprises: a) contacting the mass of the material with a solvent (for example the first solvent or especially the second solvent described herein) for charging the solvent with the third compound; and b) the separation of the charged solvent from the rest of the mass of the material. The third compound contacted in the method is preferably a free acid. In the method, the third compound can be isolated by allowing the solvent to evaporate. Any feature of any aspect of any invention or embodiment described therein may be combined with any feature of any aspect of any other invention or embodiment described herein. The specific embodiments of the invention will now be described by way of example. The following terms are used hereinafter: Pen-G: refers to penicillin G as shown in Reaction Scheme 1. 6-PAA: refers to 6-aminopenicillanic acid.

PAA: refers to phenylacetic acid. MIBK: refers to methylbutyl ketone .. Phytosol: refers to 1,1,1,2-tetrafluoroethane. Fitosol D .: refers to a mixture comprising dimethyl ether (at 10% by weight) and 1,1,1,2-tetrafluoroethane (90% by weight) .. All the analyzes referred to herein were carried out using high performance liquid chromatography (HPLC) as follows: Mobile phase: 25 mM ammonium acetate in 1: 1 methanol / water solution + acetic acid at pH 6.0. Column: column of 3.9 x 300 mm, packing of Ease Reverse C18 of LQ mieras. Detection: 230 nm Injection: 10 μl The numbers of the examples prefixed with the letter * C "are comparative examples.

Example Cl - Standard (known) method for the preparation of 6-APA i). Enzymatic Disability of Pen-G 480 ml of water and 30 g of penicillin G were charged into a container in a water bath set at 37 ° C. The mixture was stirred gently until the temperature was stabilized at 37 ° C. 83 g of enzyme resin comprising penicillin-acylase was added on a polymer resin matrix, followed by 5% sodium hydroxide at pH 8.0. Agitation was continued while maintaining the pH of 8.0 and 37 ° C until a resting state was reached. This took approximately 2 hours. The total volume of 5% sodium hydroxide used was usually about 65 ml. The resin with enzyme was filtered through a sintered funnel and the resin was washed with water and stored in a refrigerator for later use. The solution containing 6-APA and PAA was processed as described in (ii) below. ii) Isolation of 6-APA The enzyme liquor containing 6-APA and PAA was concentrated to one quarter, then cooled to 5 ° C and an equal volume of MIBK was added. 2 M nitric acid was added dropwise until pH 3.8. The pH was maintained at 3.8 for 1 hour, during which time the free acid of 6-APA was precipitated. Then, the precipitate was filtered, washed with MIBK, then with acetone and dried.

Example 1 - General method according to the embodiment of the present invention The methods of (iii) and (iv) described hereinafter are alternative, which can be used for the isolation of 6-APA. i) Apparatus An apparatus for carrying out the method comprises a reaction vessel and an evaporation vessel, which are lined to provide a means for controlling the temperature. In addition, both containers are equipped with burettes for the addition of reagents, and a means for stirring, measuring temperature and pH. Both vessels communicate with each other and with a vacuum pump and a gas compressor, so that the reaction streams can be transferred from one vessel to another and a volatile solvent used (as described herein) can be transferred to and from both vessels and to a suitable solvent storage tank. In-line filters, one-way valves and pressure relief valves can be adjusted to allow convenient and safe operation of the device. ii) Enzymatic Disability of Pen-G 100 ml of water and 40 g of Pen-G were charged to the reaction vessel, followed by 50.6 g of enzyme resin. The container was sealed and evacuated to a pressure below 10 mbar and stirring was started. 1 to 2 kg of Fitosol A were loaded and the temperature stabilized at 37 ° C by flowing hot water through the liner. A pH of 8 was maintained by the addition of 5% sodium hydroxide via the reagent burette until a state of rest was reached. The total volume of the NaOH solution required was usually around 90 ml. iii) Isolation of 6-APA The enzyme liquor containing 6-APA and PAA was reloaded into the reaction vessel. 1 to 2 kg of Fitosol D was loaded and the stirring started. Cooling was applied by flowing cold water through the liner. 2 M nitric acid was slowly added via the reagent burette at pH 3.8 and stirring was continued for 1 hour while maintaining the pH of 3.8. During this time, the free acid of 6-APA was precipitated, while the PAA remained in the solution in the Fitosol D. The reaction mixture was charged to the evaporation vessel via an in-line filter which retained the precipitate 6-APA . The Fitosol D was then recirculated through the reaction vessel (to wash the 6-APA product) and the filter in line for 30 minutes after which the flow of Fitosol D was diverted to the storage cylinder. The precipitate of 6-APA was then isolated from the filter in line. When all the Fitosol D evaporated, the Remaining aqueous solution contained PAA co or an oily suspension. iv). Isolation of 6-APA (alternative method) The enzyme liquor containing 6-APA and PAA was stirred with either Fitosol A or Fitosol D or with an alternative solvent mixture comprising 1,1,1,2-tetrafluoroethane and a co-solvent, for example an aliphatic alcohol, ketone or ether. An inorganic acid, such as 1-4M nitric or sulfuric acid, was slowly added until a pH of less than 2.5 was reached. The pH is suitably adjusted to between 1.5 and 2.0. The temperature during the acid addition was reduced to within the range of 2 to 20 ° c. The two immiscible layers were separated by allowing the mixture to settle and the lower layer comprising the PAA solution in solvent, to be run off. The upper layer containing 6-APA was treated with an aqueous solution of a base such as sodium hydroxide or potassium hydroxide, while stirring at a reduced temperature until a pH of 3.8 was reached, at which pH the 6-APA free acid. The precipitated 6-APA was isolated by filtration. v) PAA insulation The aqueous solution comprising the oily suspension of PAA produced in step (iii) was extracted into the Fitosol solvent and the immiscible Fitosol layer containing PAA in solution was separated from the aqueous layer. The isolation of PAA was achieved by causing the Fitosol to evaporate. Alternatively, the PAA can be isolated after step (iv) by evaporating the solvent from the isolated PAA solution.

Example C2 - Preparation of 6-APA The method of Example Cl was carried out to produce a baseline for comparison with other examples. In the method 30 g of Pen-G were reacted with the immobilized enzyme and the final product (6-APA) was dried, weighed and evaluated using HPLC. The results were as follows: Weight of the Pen-G used = 30.0 g Weight of the immobilized enzyme = 38.0 g Volume of water used = 480 ml Volume of 5% NaOH used = 65 ml Volume of the enzyme enzyme produced = 550 ml Enzyme enzyme assay = 31,100 μg / ml as 6-APA Conversion Step Performance = 96% Weight of 6-APA produced = 13.23 g Full Performance from Pen-G = 74% Example 2 A suspension was prepared in the reaction vessel containing 40 g of Pen-G, 50 ml of water and 2 kg of Fitosol. 5% sodium hydroxide was added in 2 hours, during which time the pH was maintained at 8.0. The total volume of the sodium hydroxide solution used was 85 ml, indicating complete conversion. At the end of the reaction, the enzyme resin was filtered and the Fitosol evaporated back to its storage tank. Enzyme liquor volume = 160 ml Enzyme liquor test = 142,300 μg / ml Weight of 6-APA produced = 22.76 g Conversion yield = 98.2% 80 ml of the enzyme liquor was precipitated and the 6-APA was isolated following the method described in Example 1 (iii). Weight of 6-APA produced = 9.6 g Complete yield from Pen-G = 84.4% The product obtained was dried and it was not necessary to dry it in additional vacuum.

Example C3 To provide a direct comparison with the 6-APA isolation step of Example 2, a second 80 ml portion of the enzyme liquor was precipitated using MIBK, washed with acetone and dried in vacuo for 20 hours. Weight of 6-APA produced = 9.45 g Insulation Step yield = 83.0% Example 3 - Effect of pH on extraction of PAA This example was carried out to evaluate the efficiency and selectivity of Fitosol D in the extraction of PAA from the enzymatic liquor at different pH's.

The enzyme liquor was prepared as described in Example 1, using the enzyme resin of Example C2. Samples of 20 ml of liquor were extracted with 40 ml of Fitosol D, at various pH's, using the handheld device as follows: The apparatus consists of a 100 ml graduated glass tube, equipped with a filter assembly and a clamping ring which in turn is equipped with a needle valve. The material or solution to be extracted is loaded into the tube. The filter is then assembled with the aid of a sealing O-ring, followed by the clamping ring that ensures a firm fit. The Fitosol liguid-gas is introduced to the glass tube from an aerosol can via a needle valve. The contents of the tube are mixed by vigorous stirring, after which the tube is inverted and allowed to stand until the two layers separate. The Fitosol is then released via the needle valve in an evaporator bottle, taking care not to allow any of the aqueous layer to co-discharge. The results are given in the following table.

Example 4 - Effect of the presence of Fitosol A on the enzyme performance The experiment was carried out to evaluate the effect of the presence of Fitosol A on the performance of the enzyme - that is (6-APA produced in the enzyme) - (theoretical 6-APA). In the experiment, 30 g of Pen-G was dissolved in 300 ml of water and was deacylated as described in Example 1.

Volume of NaOH at 2.5% used = 134 ml Volume of enzyme liquor produced = 450 ml Enzyme liquor assay = 38,120 μg / ml as 6 -APA Enzyme yield 98.2% of the theoretical It will be noted that the yield is higher in the presence of Fitosol A (compare Example 4 and Example C2). - Example Effect of using a more concentrated Pen-G solution This experiment involved the conversion of a more concentrated solution of Pen-G, with a view to avoiding the need for additional concentration before the step of precipitation and isolation of 6-APA of Example 1 (iii). In the experiment, 30 g of Pen-G was dissolved in 150 ml of water and 2 kg of Fitosol A were used and the method was carried out as described above.

Final volume of the enzyme liquor = 350 ml Enzyme liquor assay 47,800 μg / ml as 6-APA Conversion yield = 96% 6-APA was isolated as described in Example 1 (iii) by the addition of the enzyme liquor to Fitosol D (2 kg) and acidification to pH 3.8 using 1 M nitric acid.

Product weight 6-APA isolated = 5.6 g It will be appreciated that the concentration of the enzyme liquor used is preferably as high as possible to minimize the losses of the desired 6-APA in the mother liquor.

Example 6 - Isolation of PAA A previously obtained aqueous effluent solution (300 ml) containing about 14 g of PAA as an oily suspension was charged to the reaction vessel. The Fitosol D was charged to the vessel and the mixture was stirred for 30 minutes. The two layers were allowed to settle by resting for 15 minutes, then separated with the aid of a sintered glass fitted to the lower outlet of the container. The Fitosol D was then evaporated and returned to the storage cylinder. The PAA was collected from the vessel as a blanquecino crystalline solid.

PAA weight recovered = 14.2 g Although it is difficult to accurately determine the PAA content of the initial solution, yields close to the theoretical appear to be possible.

Example 7 - Use of sulfuric acid for the adjustment of the Pl The enzyme reaction was carried out as described in general in Example 1 (ii) using 40 g of Pen-G, 61 g of enzyme resin, 50 ml of water, 5% NaOH to maintain pH 8.0 and at a temperature of 37 ° C. The results were as follows: Volume of enzyme liquor = 145 ml Concentration = 13.2% (by HPLC) Yield of enzyme passage = 83% Precipitation and isolation were carried out as described in general in Example 1 (iii), using a mixture of solvents comprising 2 kg of Fitosol and 30 ml of isopropanol. It caused the 6-APA to precipitate by the addition of 1 M sulfuric acid until pH 3.8, at 4 ° C. The 6-APA was isolated as a white solid. Yield by weight = 14.8 g HPLC analysis showed PAA as a very sticky trace. Preferred embodiments of the present invention may have the following advantages: - the imaging reaction may be carried out in a solution with a higher concentration of Pen-G than hitherto, thereby eliminating or reducing the need for a potentially expensive concentration step. A more efficient enzymatic reaction is achieved which involves a faster reaction and / or improved performance. Conversion yields of 98% or greater have been demonstrated. The activity of the enzyme is not damaged by the solvent system. The need for large quantities of organic solvents is eliminated or reduced, thereby eliminating the problems associated with such solvents such as storage, recovery and treatment of effluents before disposal.

- Fitosol shows good efficiency in the withdrawal of PAA while the solubility of 6-APA in the solvent is negligible. The crystalline forms can be manipulated by the use of a co-solvent during the precipitation of 6-APA, which can be advantageous in the downstream processing. Dry 6-APA is produced directly, without the need for subsequent drying of the product. - In general, the process is faster and cheaper than established industrial processes. Advantageously, the present invention in its broadest terms is not restricted to the dividing enzyme of penicillin and cephalosporin, but is relevant to other related enzymes, including acylases, amidases, proteases and esterases. The solvents described herein can be used to substantially improve reaction rates and isolation of the product in stream line. The reader's attention is directed to all documents that are presented concurrently with or prior to this specification, in relation to this application and which are open to public inspection with this specification, and The contents of all such documents are incorporated by reference herein. All the features described in this specification (including any claims, summaries and appended drawings), and / or all steps of any method or process described thus, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. Each characteristic described in this specification (including any claims, summary and attached drawings) may be replaced by alternative features that serve the same, equivalent or similar purpose, unless otherwise expressly stated. Thus, unless otherwise expressly stated, each characteristic described is an example only of a generic series of eguivalent or similar characteristics. The invention is not restricted to the details of the previous modality (s). The invention extends to any novel or novel combination of the features described in this specification (including any claims, summary and appended drawings), or any new or novel combination of the steps of any such method or process.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.

Claims (17)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A method for preparing a second compound by catalytic conversion of a first compound, the method is characterized in that it comprises contacting the first compound and a catalyst in a mixture of solvents comprising water and a first non-aqueous solvent.

2. A method according to claim 1, characterized in that the catalyst is an enzyme.

3. A method according to claim 2, characterized in that the enzyme is sensitive to the second compound, since relatively high concentrations of the second compound reduce the ability of the enzyme to effect the conversion.

4. A method according to claims 2 6 3, characterized by the enzyme is able to catalyze a deacylation reaction.

5. A method according to any of the preceding claims, characterized in that the first compound is of the general formula R1NHQ wherein R1 represents an optionally substituted alkylcarbonyl group and Q represents an optionally substituted cyclic group.

6. A method according to any one of the preceding claims, characterized in that the ratio of the weight of the first solvent to that of the water in the solvent mixture is at least 2.

7. A method according to any one of the preceding claims, characterized in that the ratio, as% p / v, of the first compound to the water present in the mixture, is at least 10.

8. A method according to any of the preceding claims, characterized in that the first non-aqueous solvent has a point of boiling, at atmospheric pressure, less than 80 ° C and greater than -90 ° C.

9. A method according to any of the preceding claims, characterized in that the first solvent is organic.

10. A method according to any one of the preceding claims, characterized in that the first solvent comprises an alkane of 1 to 4 carbon atoms optionally substituted.

11. A method according to any one of the preceding claims, characterized in that the first solvent is halogenated.

12. A method according to any one of the preceding claims, characterized in that the first solvent is non-chlorinated.

13. A method according to any of the preceding claims, characterized in that the first solvent is tetrafluoroethane.

14. A method according to any of the preceding claims, characterized in that it includes the step of isolating the second compound from the other compounds / solvents by causing the precipitation of the second compound and the subsequent isolation of the precipitate.

15. A method according to any one of claims 1 to 13, characterized in that it includes adjusting the pH in the presence of a second solvent to a value at which the second compound is substantially soluble in the aqueous phase of the solvent mixture. , while the by-products of the reaction are substantially soluble in the second non-miscible solvent phase of the solvent mixture.

16. A method according to any of the preceding claims, characterized in that the first compound is a natural penicillin or a biosynthetic penicillin prepared by the addition of a precursor to a fermentation broth of penicillin, or a cephalosporin, and the second compound is the acid 6-aminopenicillanic acid or 7-aminodesacetoxycephalosporanic acid.

17. A method for removing a third compound, for example an acid or acid salt, especially f.-acetylacetic acid, from a mass of material containing the compound, the method is characterized by comprising: a) contacting the mass of material with a solvent to charge the solvent with the third compound; and b) the separation of the charged solvent from the rest of the mass of the material. IMPROVED PROCEDURE FOR THE PREPARATION OF LACTAMA COMPOUNDS SUMMARY OF THE INVENTION Lactams, for example 6-aminopenicillanic acid (6-APA), can be prepared by the enzymatic conversion of a first compound, for example penicillin G, into a mixture of solvents comprising water and a non-aqueous organic solvent, especially 1 , 1, 1, 2- tetrafluoroethane. The 6-APA can be caused to precipitate, be isolated by filtration and optionally derivatized to produce a desired compound. A by-product of the enzymatic conversion, phenylacetic acid, can be isolated by solvent extraction, suitably using a solvent also comprising 1,1,1,2-tetrafluoroethane.