NO158541B - PROCEDURE FOR PREPARING A PENICILLIN. - Google Patents

Description

The present invention relates to a special method for the production of antibacterial agents of the class usually called semi-synthetic penicillins, and preferably of the subclass characterized by an oc-amino group on the acyl side in the 6-position, such as in ampicillin and amoxicillin.

The first commercial penicillin with an α-amino group on the 6-acylamidoside chain was ampicillin, which is 6-(D-α-amino-α-phenylacetamido)penicillanic acid (see US Patent 2,985,648).

Amoxicillin is an antibacterial agent that is used in human therapy and is marketed as a trihydrate of the free acid (ie the zwitterion). The connection is e.g. described in British Patent No. 978,178, in "J. Chem. Soc." (London), pp. 1920-1922 (1971) and in "Antimicrobial Agents and Chemotherapy", 1970, pp. 407-430 (1971). The chemical name is 6-[D-α-amino-α-(p-hydroxyphenyl)acetamido]penicillanic acid.

The use of amino acid chloride, hydrochlorides for the production of such penicillins is discussed in the patent literature, e.g. in GB-PS 938 321 and 959 853, under anhydrous conditions, (the latter applied protection during the acylation of the carboxy group of 6-aminopenicillanic acid with a silyl group, as also discussed in GB-PS 1 008 468 and US-PS 3 249 622) as well as in GB-PS 962 719, in cold aqueous acetone. These penicillins are amphoteric amino acids and therefore certain aliphatic unsymmetrical branched secondary amines (often called liquid amine resins), which had previously been used for the isolation of 6-aminopenicillanic acid which is also an amphoteric amino acid (see US-PS 3,008,956). Improved methods for isolating and purifying such penicillins are discussed, e.g. in US-PS 3,180,862 via p-naphthalenesulfonates and in TJS-PS 3,198,804 via intermediate isolation and subsequent light hydrolysis of hetacillin.

The use of a silyl group to protect the carboxy group of a natural penicillin during chemical cleavage to 6-aminopenicillanic acid is disclosed in US-PS 3,499,909. The use of silylated 6-aminopenicillanic acid during anhydrous acylation with amino acid chloride, hydrochloride is disclosed in numerous patents, e.g. US-PS 3,479,018, 3,595,855, 3,654,266, 3,479,338 and 3,487,073. Some of these patents also deal with the use of liquid amine resins, see also US-PS 3,912,719, 3,980,637 and 4 128,547.

GB-PS 1 339 605 contains various specific and detailed examples of the preparation of amoxicillin by reacting a silylated derivative of 6-aminopenicillanic acid with a reactive derivative (including chloride, hydrochloride) of D-(-)-α-amino-p-hydroxyphenylacetic acid , where the amino group is protected, then removal of the silyl group or groups by hydrolysis or alcoholysis and then, if possible, recovery of amoxicillin, usually as the crystalline trihydrate. Thus, crystalline amoxicillin was obtained in Ex. 1 by isoelectric precipitation from an aqueous solution, e.g. at a pH value of 4.7. Purification is probably achieved in this example by dissolving the crude product (before isoelectric precipitation) in water at an acidic pH value such as 1.0 (e.g. in aqueous hydrochloric acid) in the presence of a water-immiscible organic solvent, such as methyl isobutyl ketone ( 4-methylpentan-2-one). Essentially the same method was used in US-PS 3,674,776.

From "Synthesis", May 1970, pp. 259-260, a method for producing N-silyloxycarbonylamino acid derivatives from N-silylated amino acid derivatives by adding carbon dioxide is known. However, the article does not contain anything that could lead to the thought of applying such a reaction to a penicillin-1 compound to obtain a carbamate intermediate that withstands acylation during the formation of the desired penicillins, as it is known that the p<->lactam ring in penicillin is very sensitive.

The present invention aims to improve the known technique and thus relates to a method for producing a penicillin with the formula

where R-CO- is α-aminophenylacetyl where the phenyl ring may optionally be substituted with a hydroxy group in the para position, or phenyloxyacetyl, and this method is characterized by:

a) an N-silylated compound of the formula:

where B is an easily cleavable ester protecting group in the form asv trimethylsilyl, is converted before acylation into a compound with the formula:

where B has the above meaning, by adding dry carbon dioxide to a solution of the silylated compound in an anhydrous organic solvent at a temperature within the range 0 to 100°C until the reaction is complete,

b) after which the compound obtained is reacted with an acid chloride of the formula RCOC1, where R has the above

importance at a temperature above -10°C and preferably within the interval 0 to 20°C, after which the group B is converted to hydrogen.

One of the surprising features of the hitherto unknown method is the stability of the anhydrous acylation solution. This can be kept for long periods of time, even at room temperature, without noticeable decomposition of the penicillin molecule. This is in contrast to the behavior of acylation solutions in previously described methods. This stability advantage makes it possible to carry out the acylation reaction at much higher temperatures (room temperature was used here) than is usually used in the manufacture of ampicillin, which is normally below 0°C and characteristically approx. -10°C.

The surprising and unexpected features of the method according to the invention can be summarized as follows: a) The acylation can be carried out at a much higher temperature than usual without loss of yield; b) The acylation mixture is very stable and is kept for 24 hours at 5°C without any loss of yield. This is in stark contrast to other anhydrous acylation processes such as shows extensive degradation during this time at this temperature; c) Very high dividends of $85 or more are always achieved; d) The method can be used for the production of amoxicillin of very high quality and which does not contain toxic bases such as N,N-dimethylaniline or N-methylmorpholine. e) The carboxylated mixture containing the new intermediate has a practically infinite stability.

No loss of yield can be noted after storage at room temperature for several weeks;

f) No exothermic reaction occurs during the acylation, which would require cooling in the same way as with

other acylation processes.

The connection with the formula

are referred to here by various tivial names such as bis-silylated carbamate of 6-APA, SCA, 6-trimethylsilyloxycarbonylpenicillanic acid TMS-ester and TMSO2C, APA, TMS.

The very existence of this compound is surprising in view of the well-known fact that reaction of 6-APA with carbon dioxide destroys 6-APA and yields 8-hydroxypenicillanic acid as disclosed in US-PS 3,225,033.

One means of obtaining quantitative yields of 6-trimethylsilyloxycarbonylaminopenicillanic acid trimethylsilyl ester (TMS02C, APA, TMS) lies in completely preparing the 6-APA bis-TMS precursor in the first instance. This was achieved by reacting 6-APA with hexamethyldisilazane (HMDS) as in the following reaction core:

The completion of the bis-trimethylsilylation reaction can be easily followed using NMR. The 3-trimethylsilyloxycarbonyl group shows a methylsilyl singlet at 0.31 ppm (tetramethylsilane = 0), while the 6-trimethylsilylamino group shows a methylsilyl singlet at 0.09 ppm.

The 6-APA trimethylsilylation reaction has so far only been carried out in methylene chloride. Other solvents can, however, be used such as e.g. acetonitrile, dimethylformamide or even HMDS.

Conversion of the trimethylsilylamino group to the trimethylsilyloxycarbonylamino group is readily accomplished by bubbling dry CO 2 through the reaction solution. The transformation is easily followed by NMR because the trimethylsilylamino singlet at 0.09 ppm declines as a new singlet appears for the trimethylsilyloxycarbonylamino group at 0.27 ppm.

When the method according to the present invention is used for the production of ampicillin, ampicillin anhydrate, ampicillin trihydrate, amoxicillin and amoxicillin trihydrate, the end products are isolated and purified according to conventional methods which are well known in the art, as stated in US-PS 3 912 719, 3 980 637 and 4 128 547, as well as other patents and publications indicated therein.

Acid chlorides are normally prepared under vigorous conditions such as by treating the acid under reflux with thionyl chloride, but they can, when sensitive groups are present, including sensitive blocking groups, be prepared under practically neutral conditions by reacting a salt of the acid with oxalyl chloride.

The method according to the invention will be explained in more detail in the following examples.

Example 1

To a mixture of 6-aminopenicillanic acid (6-APA) and 10 ml of CH2CI2 and 1.13 ml of trimethylchlorosilane at a temperature of 25-27"C, 1.23 ml of triethylamine was added dropwise over the course of 30 minutes. Stirring was continued for another 2 hours. Dry carbon dioxide gas was then bubbled through the mixture for 2 hours. Dry carbon dioxide gas was then bubbled through the mixture for about 3 hours. At the end of this time, NMR spectra showed the presence of 60$ silylated carboxy-6-APA (SCA) with the structure:

The mixture was kept in the refrigerator overnight. The next morning, 0.77 ml of N,N-dimethylaniline was added and the mixture was cooled to -8°C. Then 1.2 g of D-(-)-p-hydroxy-2-phenylglycyl chloride, hydrochloride with a purity of 79% was added in portions as follows:

At the end of the 310 minute reaction period, thin layer chromatography (TLC) performed on a sample of the reaction mixture using a solvent system of 60% ethyl acetate, 20% acetic acid and 20% water shows the presence of an amoxicillin.

To a cold sample of 2 ml of the final reaction mixture was added 1.0 ml of D2O. After separation by centrifugation, the aqueous phase was shown by NMR to contain 78% amoxicillin and approx. 20$ 6-APA. The presence of amoxicillin was also confirmed by TLC.

Example 2

A mixture of 5.4 g or 0.025 mol of 6-aminopenicillanic acid and 6.2 ml of 93% hexamethyldisilazane (HMDS; 0.0275 mol), as well as 0.07 g or approx. 0.001 mol of imidazole in 40 ml of CH 2 Cl 2 was refluxed under a nitrogen purge for approx. 17.5 hours. At the end of this period, 0.13 ml or approx. 0.001 mol trimethylchlorosilane (TMCS). The resolution became unclear. The reflux continued for another 7 hours and deposits of NH 4 Cl were observed in the condenser. At this point, NMR spectra showed approx. 100% silylation of both the amino and carboxyl groups in 6-APA. 0.2 ml or 0.00125 mol (approx. 5 mol %) of HMDS and 0.06 ml or approx. 0.0005 mol TMCS, and the reflux under nitrogen purging was continued for another 17 hours. At this point, the NMR spectrum was the same as before, but with the addition of small amounts of HMDS and TMCS. Dry carbon dioxide was then bubbled through the reaction mixture at room temperature for 175 minutes, after which NMR spectra showed no HMDS and more than 92% silylated carboxy-6-APA (SCA). 4.45 ml or 0.035 mol of N,N-dimethylaniline (DMA) was then added and the mixture was cooled to -3°C. 5.65 g or 0.026 mol of D-(-)-2-phenylglycyl chloride with a purity of 95% was then added in portions as follows:

The reaction was followed by NMR spectra which showed very little change approx. 5 hours after the start of the reaction. The temperature was then 3°C. The reaction mixture was kept packed in ice for the next 16 hours. It was then removed from refrigeration and stirred for 3.5 hours at room temperature (about 20-24°C). A large amount of solids was still present. The reaction mixture was then stirred at room temperature, 22 - 24°C for approx. 63 hours. At the end of this period there was only a slight blur. Upon D20 extraction of a sample, NMR spectra showed ampicillin and 6-APA.

The reaction mixture was cooled to approx. 0°C and stirred for 5 minutes in the cold after adding 35 ml of ice water. After filtration, the mixture was washed with cold water and CH2Cl2. The aqueous phase showed, after separation by TLC, a large zone which was slower than ampicillin and 6-APA, and which represented a new intermediate X.

The aqueous phase was adjusted to a pH value of 3.0 with NH 4 OH and was inoculated with ampicillin. Methyl isobutyl ketone (MIBK) was added in an amount of 35 ml and the mixture was stirred, adjusted to a pH of 5.2 with more NH 4 OH, stirred at 20°C for 1 hour, stirred in an ice bath for another 1 hour and cooled overnight. The precipitate of ampicillin was collected by filtration, first washed with 25 ml of cold water and then with 40 ml of MIBK and finally with 40 ml of a mixture of 8 parts of isopropyl alcohol and 15 parts of water, dried at 50°C and found to weigh 4 .5 g as the identity as ampicillin was confirmed by TLC.

Example 3

A mixture of 5.4 g 6-APA, 6.2 ml 93% HMDS and 0.06 g imidazole in 50 ml CH 2 Cl 2 was refluxed and purged with nitrogen for 18 hours. Then 0.1 ml of TMCS was added which caused cloudiness. Refluxing for a further 2 hours gave a clear solution of NH4CI in the condenser. An additional 0.1 ml of TMCS was then added which left only a very slight cloudiness. The reflux was continued under a nitrogen purge for the next 65 hours. The mixture was then cooled to approx. 22°C and the addition of dry carbon dioxide was started. After 75 minutes, NMR spectra showed formation of more than 90% bis-silylated carbamate (SCA). 4.45 ml of DMA were then added and then 5.6 g of D-(-)-2-phenylglycyl chloride, hydrochloride with 97% purity in proportions as follows:

After this mixture was stirred for a further 17 hours, TLC was performed on samples of the reaction mixture and on a diluted reaction mixture (1 ml of the mixture diluted with 2 ml of CH2Cl2) and found in each a small zone of ampicillin and a large zone of a new intermediate X.

The reaction mixture was then cooled to 0°C, 40 ml of ice water was added and the mixture was stirred for 5 minutes, filtered and washed with water and with CH2C12- The aqueous phase was separated, 10% removed for testing and the remainder adjusted to a pH- value 3.0 with NH4OH, inoculated with ampicillin and stirred. After addition of another 40 ml of MIBK, the mixture was stirred and the pH was adjusted to 5.2 with NH 4 OH and stirred at room temperature for 1 hour and then in an ice bath for another 1 hour. Crystals were precipitated. After cooling overnight, the crystalline product was collected by filtration, washed successively with MIBK, water and MIBK and then with 40 ml of isopropanol-water in a ratio of 85:15 and dried at 45°C to obtain 6.25 g of ampicillin (6 .8 g corrected for sampling, corresponding to a yield of 68%).

Example 4

To a mixture of 1.0 g 6-APA and 1.13 ml TMCS in 10 ml CD 2 Cl 2 1.23 ml TEA was added dropwise over 30 minutes, and the mixture was stirred for a further 2 hours. Dry carbon dioxide was bubbled through for 4 hours. At this point, NMR showed approx. 55 - 60% carboxylylation. The mixture was kept in the refrigerator overnight. In the morning, 0.77 ml of DMA was added, the mixture was stirred, cooled to -8°C and 1.2 g of D_(- )-p-hydroxy-2-phenylglycyl chloride, hydrochloride was added in portions as follows:

At the end of the 310-minute period, NMR showed approx. 78% amoxicillin and approx. 20% 6-APA.

Example 5

10.0 g or 46.24 mmol corresponding to 1.0 equivalent of dry 6-aminopenicillanic acid was suspended in 175 ml of anhydrous methylene chloride with stirring at 25°C. 10.76 g or 106.23 mmol corresponding to 2.30 equivalents of triethylamine was added at 25°C followed by the addition of 11.70 g or 107.75 mmol corresponding to 2.33 equivalents of trimethylchlorosilane over 10

- 15 minutes, as the temperature was kept below approx. 30°C using the rate of addition of trimethylchlorosilane. After stirring for 20-30 minutes, the mixture containing precipitated triethylamine hydrochloride was analyzed for complete silylation by 80 MHz NMR. The mixture was then treated with carbon dioxide at 20°C for 2 hours and analyzed for complete carboxylation by 20 MHz NMR. Additional gas addition was sometimes necessary. The volume of the carboxylation mixture was, if necessary, readjusted to approx. 175 ml of dry methylene chloride. After complete carboxylation, the slurry was treated with 2.95 g or 3.56 ml or 50.87 mmol corresponding to 1.1 equivalent of propylene oxide and cooled to 0-5°C. D-(-)-2-(p-hydroxyphenyl)-glycyl chloride hydrochloride hemidioxane solvate was added in five portions to 2.71 g at ca. 2°C (total 13.54 g or 50.87 mmol corresponding to 1.1 equivalent). Each portion

acid chloride was allowed to dissolve before the next portion was added. (Stirring was stopped and the mixture examined for any solids at the bottom of the flask. The slurry must not be heated above 5°C for this test, as the results may be erroneous). This required approx. 20 minutes per portion. This portionwise addition was very important. The final acylation mixture was examined for any undissolved acid chloride hydrochloride. The mixture was kept at 0-5°C for 30 minutes and treated with cold (0-5'C) deionized water (DI) in an amount of 100 ml under very rapid stirring for 10 minutes. The mixture was allowed to separate and the lower methylene chloride phase was removed. The aqueous phase was filtered (very little solids) through a thin ("Dicalite") layer of diatomaceous earth and the cake was washed with cold (0-5°C) DI water in an amount of 15 ml. Any lower phase of organic layer was removed before crystallization. The clear, pale yellow, aqueous solution with a pH value of 2 - 2.5 was adjusted to pH 3.5 at 0 - 5°C and, if necessary, inoculated. The slurry was kept at 0-5°C for 40 minutes and the pH was adjusted to 4.8-5.0 with 6N ammonium hydroxide and crystallized for 2 hours. The slurry was filtered and the solid amoxicillin thus collected was washed with a cold (0-5°C) mixture of 1:1 isopropanol:water and the cake was washed with methylene chloride in an amount of 30 ml, giving approx. 13.5 or around 70% snow white amoxicillin trihydrate.

Example 6

108 gels of 0.5 mol 6-aminopenicillanic acid, 1.0 g or 0.017 ml of imidazole, 800 ml of dry methylene chloride and 120 ml or 0.56 mol of 98% HMDS were stirred and heated to reflux for 3.3 hours. The reaction mixture was flushed with dry nitrogen gas during reflux to remove the NH formed during the reaction. Then 2.0 ml or 0.016 mol of trimethylchlorosilane (TMSC) was added. Refluxing was continued with ^ flushing for a further 19 hours, and then in the condenser sublimed NH 4 Cl was removed and 2.6 ml

or 0.0206 mol of TMCS added to the reaction. Refluxing with ^ flushing was continued for a further 34 hours. The volume of the reaction mixture was brought to 1000 ml with dry methylene chloride. NMR then showed 100% silylation of the amino and carboxyl group on the 6-aminopenicillanic acid. The solution was covered with N2~ gas and kept like this for 9 days. NMR spectra confirmed the above and the stability. The solution was stirred and CO2 bubbled through for approx. 90 minutes. The temperature was 20 - 22"C. NMR showed 100% conversion of bis-trimethylsilyl-6-aminopenicillanic acid to bis-trimethylsilylcarboxy-6-aminopenicillanic acid (SCA).

This base mixture was used for the acylation experiments described below. The chemical compound in this solution had the formula:

NMR showed that bis-trimethylsilylcarboxy-6-aminopenicillanic acid was still stable after 9 days.

100 ml of the base mixture (SCA equivalent to 10.8 g of 6-aminopenicillanic acid, 0.05 mol) was stirred at 22°C and 8.0 g or 0.058 mol of TEA, HC1 and 4.2 m of 1 or 0, was added. 06 moles of propylene oxide (see US-PS 3,741,959). Some TEA, HC1 was precipitated. The mixture was stirred and cooled to +3°C. Then 15.5 g, or 0.55 mol of D-(-)-p-hydroxyphenyl-glycyl chloride-hydrochloride-hemidioxane solvate with a purity of 75% was added in portions as follows:

After a further 70 minutes, approx. 50 ml of dry methylene chloride to the reaction mixture to reduce the viscosity.

After another 160 minutes, a 2 ml sample was removed and added to 1.0 ml D2O. After centrifugation, NMR analysis of the aqueous phase showed approx. 6% non-acylated 6-aminopenicillanic acid. 10 minutes later, the reaction mixture was transferred to a 600 ml beaker and the transfer was terminated by washing with 50 ml of methylene chloride. While stirring in an ice bath, 60 ml of cold deionized ice water was added to produce a solution of two phases without solids content and with a pH value of 1.0.

15.0 ml of "LA-1" type liquid anion exchange resin was added to the two-phase system under stirring and inoculation at a pH value of 2.0. A crystallization began. An additional 10.0 ml of "LA-1" was slowly added over 5 minutes. Then 5.0 ml of "LA-1" was added and the pH value was 4.5. Stirring was continued and 1.0 g of NaHSO 3 , sodium bisulphite, 14.0 ml of water were added dropwise. Then 10.0 ml of "LA-1" was added and the pH continued to rise. A total of 40 ml of "LA-1" was added and the final pH value was 5.6. Then 5 ml of acetone was added. At this point, 1.5 g of NaHSO dissolved in 6.0 ml of water was added over 30 minutes. Stirring in an ice bath was continued.

The precipitated product was collected by filtration and the cake was washed successively with 50 ml of methylene chloride, 40 ml of water, 100 ml of isopropyl alcohol-water in a ratio of 80:20 and 100 ml of methylene chloride. The cake was then dried at atmospheric pressure and 45°C to obtain 18.2 g of amoxicillin trihydrate, which represented a yield of 87% calculated for 6-aminopenicillanic acid, corrected for 1% for testing the overall yield was approx. 88%.

"LA-1" liquid anion exchange resin is a mixture of secondary amines, each secondary amine having the formula

in which each R<*>, R<2> and R<3> is an aliphatic hydrocarbon group and where R<1>, R<2> and R<3> in the aggregate contain from 11 - 14 carbon atoms, this particular mixture of secondary amines sometimes referred to as "Liquid Amine Mixture No. I", is a clear amber liquid with the following physical properties: viscosity at 2'C equal to 70 eps; specific gravity at 20' C equal to 0.845; refractive index at 45°C equal to 1.467; distillation interval at 10 mm: up to 160°C - 4%, 160 - 210°C - 5%, 210-220°C - 75%, above 220°C - 17%.

Example 7

A methylene chloride solution (5.0 ml) of 0.54 g or 2.497 mmol of trimethylsilyl-6-trimethylsilyloxycarbonylaminopenicillinate was treated with 0.20 g or 1.45 mmol of triethylamine hydrochloride followed by 0.162 g or 2.75 mmol of propylene oxide at 25°C. The mixture was stirred at 25°C for 20 minutes to facilitate dissolution of most of the triethylamine hydrochloride. 0, 45 g or 2.75 mmol of phenoxyacetyl chloride was added dropwise at 25°C and the mixture was stirred at 25°C for 30 minutes. A sample was removed and analyzed by CMR analysis at 20.0 MHz. CMR (carbon-13 nuclear magnetic resonance) showed complete disappearance of the phenoxyacetyl chloride as well as the APA carbamate and the presence of penicillin V trimethylsilyl ester. The presence of penicillin V trimethylsilyl ester was demonstrated by spectral comparison with an identical sample prepared by silylation of the free penicillin V acid with triethylamine and trimethylchlorosilane. Based on the CMR spectrum, the yield was estimated to be 85 - 90%.

Claims (1)

Procedure for the preparation of a penicillin with the formula: where R-CO- is α-aminophenylacetyl where the phenyl ring may optionally be substituted with a hydroxy group in the para position or phenyloxyacetyl, characterized in that: a) an N-silylated compound with the formula: where B is an easily cleavable ester protecting group in the form of trimethylsilyl, is converted before acylation into a compound with the formula: where B has the above meaning, by adding dry carbon dioxide to a solution of the silylated compound in an anhydrous organic solvent at a temperature within the range 0 to 100°C until the reaction is complete, b) after which the obtained compound is reacted with an acid chloride of the formula RC0C1, where R has the above meaning, at a temperature above -10°C and preferably within the interval 0 to 20°C, after which group B is converted to hydrogen.