SE440506B - PROCEDURE FOR PREPARING ALFA-L-ASPARTYL-L-PHENYLALANINE METHYL ESTER - Google Patents

Description

7f714709-8 alanine. The amino protecting group may be any such group known to those skilled in the art, for example formyl, acetyl, benzoyl, substituted and unsubstituted carbobenzoxy, t-butoxycarbonyl and hydrohalide salts. Particularly preferred is N-formyl-L-aspartic anhydride.

The N-protected-oL-L-aspartyl-L-phenylalanine can be separated from the N-protected-β-L-aspartyl-L-phenylalanine and treated to remove the protecting group to give oz-L-aspartyl-L-phenylalanine as in equation 2). The previous process concerned the isolation of α-L-aspartyl-L-phenylalanine, which was then esterified with methanol, as in Equation 3), to form oL-APM.

As described in U.S. Pat. No. 3,933,781, the esterification reaction was preferably carried out "with as little water as possible present". Such an esterification reaction is described for illustrative purposes as carried out in methanol in the presence of hydrogen chloride. At this time, we considered that the presence of any significant amount of water during the esterification would tend to reduce the desired esterification by causing undesired deesterification reactions.

A preferred method of recovering oL-APM, prepared by the process of U.S. Pat. No. 3,933,781, was to convert it to the 1-ICl salt, which was recovered as a solid and converted to oL-APM.

Such a solid I-1Cl salt of oz-APM is also described in U.S. Pat. No. 3,798,207, which utilized it in a purification procedure to give oL-APM by separation from B-APM and other undesirable by-products. In both of these prior art methods, the 1CI salt was formed as a means of recovering α-APM after its preparation.

The main object of the invention is to provide an improved process for producing oL-APM. Other objects and advantages and aspects of the invention will become apparent from the following description. The distinctive features of the invention appear from the following claims.

The invention thus offers an improved process for the preparation of α-APM, in which oz-L-aspartyl-L-phenylalanine is allowed to come into contact with a reaction medium containing water, methanol and hydrogen chloride or hydrogen bromide, to form a solid hydrochloride. or hydrobromide salt of oL-APM, separates the solid hydrohalide salt and converts the separated salt to. a-APM. The oL-L-aspartyl-L-phenylalanine can be obtained by treating the N-protected-o-L-aspartyl-L-phenylalanine to remove the protecting group (Equation 2). Any method suitable for removing protecting groups from the amines is suitable. Examples of such methods are catalytic hydrogenation and treatment with mineral acids or bases. It is preferred to remove the protecting group, especially the formyl group, by acid hydrolysis. This hydrolysis can be performed in e.g. a dilute aqueous hydrochloric acid solution. The conversion to oL-L-aspartyl-L-phenylalanine is usually very high, i.e. of the order of 95% or more, based on the N-protected-oL-L-aspartyl-L-phenylalanine thus treated. Another medium for such treatment is an aqueous solution of acetic acid-hydrochloric acid, and L-aspartyl-L-phenylalanine can then be recovered by precipitation and liquid / solid separation. Such precipitation can e.g. achieved by pH adjustment when the protecting group has been removed in an acidic solution.

The main undesirable by-product remaining in the sodium hydroxide solution is β-L-aspartyl-L-phenylalanine, if its precursor is carried out from previous steps. It can be treated, e.g. by hydrolysis, for the recovery of L-aspartic acid and L-phenylalanine for recovery to previous steps. However, any β-L-aspartyl-L-phenylalanine can be carried into the process of the invention without elongation, since the hydrohalide salt formed of α-APM will allow adequate separation from this undesired isomer or its esters.

It is also possible with the process of the invention to use the N-protected-uL-aspartyl-L-phenylalanine to form the αL-aspartyl-L-phenylalanine in situ in the reaction medium or to form the oL-L-aspartyl-L-phenylalanine in a reaction medium without the need for isolation. An especially preferred N-protected-α-L-aspartyl-L-phenylalanine useful in this latter manner is N-formyl-α-L-aspartyl-L-phenylalanine.

The amount of hydrogen halide useful in the reaction medium is from 0.1 to about 0.80 moles per 10 g of reaction medium. A particularly useful amount of hydrogen halide is from about 0.3 moles to about 0.7 moles per 100 g of reaction medium. The amount of methanol useful in the reaction medium is from about 0.1 to about 1.1 moles per 100 g of reaction medium. A particularly useful amount of methanol is from about 0.4 to about 0.8 moles per 100 g of reaction medium. .

The remaining part of the reaction medium is water. Those skilled in the art will appreciate that other materials may be included, but that the above remarks describe the useful concentrations of the three component reaction medium.

The hydrogen halide must be present in the reaction medium in an amount of from at least 1.0 to about 20 moles of hydrogen halide per mole of oL-L-aspartyl-L-phenylalanine. An especially preferred amount is from about 1.15 to about 10.0 moles per mole of oL-L-aspartyl-L-phenylalanine. Hydrogen chloride is the preferred hydrogen halide.

Those skilled in the art will appreciate that the reaction medium must also contain at least 1.0 mole of methanol per mole of α-L-aspartyl-L-tenylalanine, and higher levels may also be used.

It is also understood that the concentrations and amounts of materials used in the reaction medium and of ol-L-aspartyl-L-phenylalanine cannot be practically carried out if undesired excessive mixing problems arise.

When the α-L-aspartyl-L-phenylalanine is to be formed in situ, it has been found advantageous to add less hydrogen halide, followed by heating to about 65 ° C and cooling. This causes hydrolysis of the N-protected-oz-L-aspartyl-L-phenylalanine to oL-L-aspartyl-L-phenylalanine. After such heating, additional aqueous hydrogen halide can be added to the reaction mass to form a reaction medium as described above, leading to the formation of the solid hydrogen halide salt of oL-APM.

The temperatures used should be up to about the boiling point of the reaction mass. From about 50 to about 50 ° C is preferred, especially from about 200 to about GOOC. Although the most preferred temperature is close to ambient temperature, it should be noted that higher temperatures tend to increase the rate of formation of oL-APM but have the disadvantages of causing decomposition reactions. and increase the solubility of the hydrogen halide salts of α-APM. On the other hand, lower temperatures tend to reduce the rate of formation of α-APM, inhibit decomposition reactions and give higher levels of solid hydrohalide salts of α-APM. Those skilled in the art will recognize the need to balance these considerations in order to achieve the most economical temperature for the concentrations in question.

In the reaction which takes place in the process according to the invention, the formation of the following undesired by-products is, so to speak, built-in. n CO-CH CH - I 2 39 NHZ-CH-C-NH-CH-CO-CHS nz 0 CHZ (hereinafter referred to as "the diester") and 7714709-8 O Il CH -CO-CHS 2 o N fl z-CH -C-NH-çß-E-OH O Clrlz (hereinafter referred to as "the aspartyl ester").

In addition to these two undesirable by-products, the reaction mass may also contain unesterified α-L-aspartyl-L-phenylalanine and small amounts of the β-form analogs, if this isomer is passed on in the synthesis. The reactions that lead to the desired product and to the by-products are all equilibrium reactions. In the process of our U.S. Pat. No. 3,933,781, the yields of the α-APM obtained were generally about 25 to 3096 based on α-L-aspartyl-L-phenylalanine.

It has now been found that the process according to the invention gives a highly isolated yield of oL-APM. For example, at room temperature, as much as about 55 to 60% yield of α-APM can be obtained, based on oi-L-aspartyl-L-phenylalanine.

This is particularly surprising in light of the yields that can be achieved with our previous methods.

The solid hydrohalide salt of oL-APM can be recovered by solid phase liquid separation. Substantially all of the other compounds remain in the mother liquor and can be hydrolyzed, recycled and / or fed back to the preceding reactions. The separated salt can then be converted to substantially pure α-APM, e.g. as shown in U.S. Pat. Nos. 3,798,207 and 3,933,781.

The following examples illustrate the present invention in detail. The specific details of the examples should not be construed as limiting the scope of the invention.

Materials and methods for the TLC analyzes in the examples are as follows: A. Plate Silica Gel F on. glass plate from Brinkman Instrument inc., Westbury, N.Y., ll 590, USA. 7714709-8 6 B. Solvent system 1. chloroorm 64% by volume. methanol 30 "acetic acid 2" dest. water 4 "2. n-propanol 70" dest. water 10 "methanol 10" formic acid 10 "C. Detection spray solutions 1. 0.3 g of ninhydrin dissolved in a mixture of 100 ml of n-butanol and 3 ml of glacial acetic acid. 2. 1 g of potassium iodide and 1 g of soluble starch dissolved in 100 ml of dist. water.

D. Methods After sampling and development in a suitable solvent system, the plate was air dried for 30 minutes.

Ninhydrin spray - The plate was sprayed and kept in a 100 ° C oven for 15 minutes.

Starch iodide spray - The plate was placed in a chamber saturated with t-butyl hypochlorite vapor for 15 minutes, air dried for 30 minutes. and then sprayed with freshly prepared starch iodide solution.

Example 1 140 ml of methanol and 420 ml of 9N hydrochloric acid were charged to a suitable vessel and cooled on an ice bath. The resulting solution was added with 113.8 g (0.4 mol) of α-L-aspartyl-L-phenylalanine (98.596 purity). Precipitation began shortly thereafter. The resulting mass was removed from the ice bath and stirred for 30 minutes, causing the temperature to rise to ZOOC. The resulting mass was cooled again with an ice bath, stirred for 1.5 hours, leading to significant precipitation, and then placed in a refrigerator overnight.

The next morning, the reaction mass was stirred for 1 hour in an ice bath, and the precipitate (130.5 g wet cake) was separated by filtration. The resulting cake was dissolved in 750 ml of deionized water at 40 ° C, and the pH was adjusted to 4.2 over a 1.5 hour period with 36.7 g of 50% aqueous sodium hydroxide. The resulting mass was cooled to about 5 ° C and kept at this temperature for 4 hours. The resulting precipitate was separated by filtration and washed with x 30 ml of deionized water at 5 ° C and dried. The resulting product was 51.8 g of oL-APM, a yield of 4496, based on the α-L-aspartyl-L-phenylalanine. 10 77147094 TLC and sodium chloride analysis confirmed that the purity of the oL-APM product was greater than 95%. Example 2 To a stirred solution of 34.2 ml (0.41 mol) of 3796 hydrochloric acid, 60 ml of water, and 40 ml of methanol was added 110 g (0.357 mol) of N-formyl-o-L-aspartyl-L-phenylalanine under a 20 minute period, with a temperature increase from 400 to 580C. The resulting mass was stirred at 58-60 ° C for 3 hours to allow elimination of the formyl group by hydrolysis.

The reaction mass was cooled to 25 ° C, 0 ° C 65.3 mL (0.79 mL) 3796 Hydrochloric acid was added over 10 minutes. A precipitate began to form shortly thereafter. The resulting mass was kept under stirring for 45 hours at ambient temperature and for 1.5 hours at 5 ° C, which caused further precipitation to form. The solid precipitate was separated by centrifugation, and the cake was washed with 100 ml of deionized water of 5 ° C. The wet cake (110.2 g) was dissolved in 410 ml of deionized water at 45 ° C. The pH was adjusted for 10 minutes to 2.5 with 80.1 g of 4.896 aqueous sodium hydroxide, and stirred for 1 hour at 40 ° C. While maintaining the temperature at 40-42 ° C, 151.9 g of 4.896 aqueous sodium hydroxide was added over 3 hours to raise the pH to 4.2. The mixture was stirred for 1 hour at 0.5 ° C, and the resulting feather-like crystals were separated by centrifugation. The cake was washed with 200 ml of deionized water of SOC and dried overnight in a vacuum oven at 55-60 ° C. The yield of α-APM was 58.33 65.596 based on N-formyl-α-L-aspartyl-L-phenylalanine [α] D + 16.2 ° (c = 4.15 N formic acid); TLC analysis - more than 9896 pure u-APM.

Example 3 Repeating essentially the same procedure as in Example 2, except that 32.5 ml of 37% hydrochloric acid and 33.3 ml of water were added to the reaction mass after elimination of the formyl group by hydrolysis and cooling to 25 ° C, gave 33.096 yields of α-APN ., based on N-formyl-cc-L-aspartyl-L-phenylalanine. - (o: lšo + 15.30 (C = 4, 15 N formic acid).

Example 4 Repeating essentially the same procedure as in Example 2, except that the time for elimination of the formyl group by hydrolysis was limited to 1 hour, gave 46,296 yields of ot-APM, based on N-formyl-oz-L-aspartyl-L-phenylalanine. [also + 1.55 ° (car, 15 N formic acid), Example 5 Repeat essentially the same procedure as in Example 2, except that 38.7 ml of 37% hydrochloric acid was used in the initial solution instead of 34.2 ml of 37% hydrochloric acid and that the acid added after the hydrolysis was reduced to 61.3 ml of 37% hydrochloric acid, gave 53,296 yields of oL-APM, based on N-formyl-oL-L-aspartyl-L-phenylalanine. (ajg) + 15.40 (c = 1 +, 15 N formic acid).

Example 6 Repeating essentially the same procedure as in Example 2, except that the time to cause a solid precipitate was increased to 4 days, gave 59,296 yields of o-APM, based on N-formyl-oz-L-aspartyl-L-phenylalanine. [oz 1%) + 15.2 (c = 1 +, 15 N formic acid).

Example 7 Repeating essentially the same procedure as in Example 2, except that the time to cause a solid precipitate was reduced to 1 day, gave 36,396 yields of α-APM, based on N-formyl-α-L-aspartyl-L-phenylalanine. (odâo + 15.5 (C = 11.5 N formic acid).

Examples 1 to 7 are summarized in Table 1, with information on the previously described parameters. Table 1 Example Methanol HCl (moles Painter per mole No. (moles per per 100 g oL-L-aspartyl-L-100greak reaction phenalanine ____, _ _____, g __ ”_ tion medium) medium) methanol 'in fi g' 1 0.75 0.65 9.45 10.95 2 0.148 0.56 3.28 2.8 3 0.49 0.45 2.58 2.8 4 0.08 0.56 3.28 2.8 0.48 0.56 3.28 2.8 6 0.48 0.56 3.28 2.8 7 0.148 0.56 3.28 2.8 The invention is of course not limited to the embodiments specifically shown above, but many variations and modifications are possible within the scope of the appended claims.

Claims (1)

A process for the preparation of αL-aspartyl-L-phenylalanine methyl ester, characterized in that aL-aspartyl-L-phenylalanine is contacted with a reaction medium which, per 100 g contains from 0.1 to 0.8 moles of hydrogen chloride or hydrogen bromide, and from 0.1 to 1,1 moles of methanol, the residue being water, at a temperature up to the boiling point of the reaction mass, the reaction medium containing at least 1.0 to 20.0 moles of hydrogen chloride or hydrogen bromide and at least 1.0 moles of methanol per mole of oL-L-aspartyl-L-phenylalanine, to form a solid hydrochloride or bromide of oz-L-aspartyl-L-phenylalanine methyl ester, separating it solid hydrochloride or hydrobromide, and converting the separated salt to oz-L-aspartyl-L-phenylalanine methyl ester. Characterized in that the αL-aspartyl-L-phenylalanine is formed in situ from The process according to claim 1, N-protected-oL-L-aspartyl-L-phenylalanine by carrying N-protected-αL-aspartyl-L- phenylalanine in contact with the reaction medium. 3. A characterizing device according to claim 2, The N-protected-o-L-aspartyl-L-phenylalanine is N-formyl-α-L-aspartyl-L-phenylalanine. ll. Process according to Claim 1, 2 or 3, characterized in that the reaction medium contains 0.3 to 0.7 moles of hydrogen chloride or bromide and 0.4 to 0.8 moles of methanol per 100 grams of reaction medium. 5. A process according to claim 1, characterized in that the oL-L-aspartyl-L-phenylalanine is prepared and contacted with the reaction medium in uninsulated form. 6. characterized in that the temperature does not exceed 50 ° C at the contact process according to any one of claims 1-5, between u-L-aspartyl-L-phenylalanine and the reaction medium, and is preferably from about about 50 ° C. 7. characterized in that the temperature during the elimination of the formyl group Process according to claim 3, does not exceed 65 ° C. s. 'k n n e t e c k n a t that the reaction medium contains hydrogen chloride. A method according to any one of the preceding claims,