DK149432B - PROCEDURE FOR PREPARING ALFA-L-ASPARTYL-L-PHENYLALANINE METHYL ESTER - Google Patents

Description

in 149432

The invention relates to a process of the kind set forth in the preamble of claim 1 for the preparation of α-L-aspartyl-L-phenylalanine methyl ester (α-ΑΡΜ, II), which is known for its utility as a sweetener.

Such a method is known from U.S. Patent No. 3,933,781, and can be illustrated by the following general reaction sequence:

CEUCOOH

0 I

ii O

1) CH -C I 11 I 2 ^ 0 + NHo-CH-COOH-? CH-C-NH-CH-COOH and / p-form7 PH-C · ^ ** * * * ./ II CH0 X-NH CHp x-nh o Γ2 / a-form7

CH5COOH CH2C00H

i 0 I o 2) CH-C-NH-CH-COOH a ^ block: Lng) CH-C-NH-CH-COOH (I) 1 i * * X-NH CH «NH? CH «6 6 CH2C00H gh2cooh I s -I? 3) CH-C-NH-CH-COOH + CH 2 OH- * CH-C-NH-CH-COCH-z

I I J T I D

NHp CHp NH «CH2 ό ό II, ./ά-ΑΡΜ7 i · In the above formulas, X represents an amino protecting group.

As shown in Scheme 1), the starting reactants 2 149432 are an N-protected L-aspartic anhydride and L-phenylalanine which react to form N-protected α-L-aspartyl-L-phenylalanine. The amino protecting group may be any of the commonly used, e.g. formyl, acetyl, benzoyl, substituted and unsubstituted carbobenzoxy, t-butoxycarbonyl and the hydrohalide salt. Particularly preferred is N-formyl-L-aspartic anhydride.

The N-protected α-aspartyl-L-phenylalanine can be separated from the N-protected β-L-aspartyl-L-phenylalanine 10 and treated to remove the protecting group to give α-aspartyl-L-phenylalanine (I) as indicated in Reaction Scheme 2). The prior process comprising the isolation of α-L-aspartyl-L-phenylalanine (I), which was then esterified with methanol, as indicated in Reaction Scheme 5, to form II or α-APM.

As described in U.S. Patent No. 3,933,781, the esterification reaction I + CH₂OH-- II was preferably carried out in the presence of as little water as possible. Thus, the esterification reaction was preferably carried out in methanol in the presence of hydrogen chloride, believing that the presence of any significant amount of water during the esterification would tend to reduce the desired esterification by inducing unwanted deesterification reactions.

A preferred method of recovering α-APM, prepared by the method of U.S. Pat.

3,933,781, was to convert it to the HCl salt which was recovered as a solid which was then converted to α-APM.

Such an HCl salt of α-APM is also disclosed in United States Patent No. 3,798,207, in which it was used in a purification method to prepare α-APM in separation from β-ΑΡΜ and other unwanted by-products. In both of the previous methods, the HCl salt was formed as a means of recovering α-APM after it was prepared.

It is an object of the invention to provide a process for the preparation of α-APM which is improved in the sense that it does not, as is known in the process of US Patent No. 3,933,781, employ the use of an organic solvent, but is carried out. in aqueous medium, so that all the disadvantages of the organic solvent, including its regeneration and the fire and explosion risk thereof, are totally eliminated, while still achieving almost as good or even better yields of the desired final product II.

The method according to the invention, which is of the kind set forth in the preamble of claim 1, is characterized by the method of claim 1.

Surprisingly, it has been found that this method 20 fulfills the object of the invention. The moment of surprise appears very clearly, on the basis of U.S. Patent No. 3,933,781, according to which the amount of water in the reaction mixture should be as small as possible, the bulk being organic solvent.

According to the invention, there is thus provided an improved process for the preparation of α-APM which comprises contacting aL-aspartyl-L-phenylalanine or an N-protected derivative thereof with a reaction medium comprising water, methanol and hydrogen halide which is hydro -30 gene chloride and / or hydrogen bromide to form a solid hydrogen halide salt of α-APM, to separate the solid hydrogen halide salt and to convert the separated salt of α-APM to α-APM.

A particularly preferred embodiment of the method according to the invention is characterized by the characterizing part of claim 2. This results in a particularly high yield.

A particularly preferred embodiment of the method according to the invention is characterized by the characterizing part of claim 3. This provides a particularly simple process, which can normally be carried out without heat supply, and which also produces a fully satisfactory yield.

The α-L-aspartyl-L-phenylalanine (I) can be prepared by treating N-protected α-L-aspartyl-L-phenylalanine 15 to remove the N-protecting group (Scheme 2). Any suitable method for removing protecting groups from amines can be used. Examples of such methods are catalytic hydrogenation and treatment with mineral acids or bases. It is preferred to remove the protecting group, which is especially the formyl group, by acid hydrolysis. This hydrolysis can e.g. is carried out in a dilute aqueous hydrochloric acid solution. The conversion to α-L-aspartyl-L-phenylalanine (I) is usually very high, i.e. of the order of 95% or more, relative to the N-protected α-L-aspartyl-L-phenylalanine. Another suitable medium for this treatment is an aqueous solution of acetic acid-hydrochloric acid.

α-L-aspartyl-L-phenylalanine (I) can then be recovered by precipitation and liquid-solid separation. Such a precipitate may e.g. is induced by pH adjustment when the protecting group has been removed in an acidic solution.

It is also possible in the process of the invention to use N-protected α-L-aspartyl-L-phenylalanine to form the α-L-aspartyl-L-phenylalanine (I) in situ 5 in the reaction medium or to form aL-aspartyl-L -phenyl-alanine (I) in a reaction medium without the need for isolation. A particularly preferred N-protected O 1 -L-aspartyl-L-phenylalanine useful in this last manner is, as stated, N-formyl-α-L-aspartyl-10 L-phenylalanine.

The amount of hydrogen halide useful in the reaction medium ranges from approx. 0.1 mole to approx. 0.8 moles per 100 grams of reaction medium. A particularly useful amount of hydrogen halide is between about 0.3 mol and approx. 0.7 moles per 100 grams of reaction medium. The amount of methanol useful in the reaction medium ranges from approx.

0.1 to approx. 1.1 moles per 100 grams of reaction medium. A particularly useful amount of methanol ranges from approx. 0.4 to approx. 0.8 moles per 100 grams of reaction medium.

The hydrogen halide is present in the reaction medium in an amount of from 1.0 to ca. 20.0 moles of hydrogen halide per mole of α-L-aspartyl-L-phenylalanine (I) contacted.

A particularly preferred amount ranges from approx. 1.15 to) approx. 10.0 moles per mole of α-L-aspartyl-L-phenylalanine (I).

Hydrogen chloride is the preferred hydrogen halide.

The reaction medium also contains at least 1.0 mole of methanol per mole of α-L-aspartyl-L-phenylalanine.

The reaction temperatures used are from ca. 0 ° C to approx. 6 ° C, especially from approx. 20 ° C to approx. 40 ° C. Although the 25 most preferred temperature is in the vicinity of ambient temperature, it should be noted that higher temperatures tend to increase the rate of formation of α-APM, but have the disadvantages of inducing decomposition reactions and increasing the solubility of hydrogen halide. the salt salts of α-APM. On the other hand, lower temperatures tend to reduce the rate of formation of α-APM, inhibit decomposition reactions and produce higher amounts of solid hydrogen halide 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 involved.

The reaction which occurs in the process of the invention leads to the formation of the following undesired by-products:

II

CHO C-O-CH

S

NHo-CH-C-NH-CH-C-O-CH, O CE0 6 (hereinafter referred to as "the diester") and 0

II

CH2-C-O-CH3 o

II

NH ^ CH-C-NH-CH-C-OH

^ II I

0 CE0 6 15 (herein called the "aspartyl ester").

7 149432

In addition to these two undesirable by-products, the reaction mixture may also contain unesterified α-L-aspartyl-L-phenylalanine (I) and optionally small amounts of the β-form analogue resulting from the preparation of the starting compound. The reactions leading to the desired product and the by-products are all equilibrium reactions.

It has been found that the process of the invention provides a large isolated yield of α-APM.

For example, at about room temperature, 10 can be obtained as much as approx. 55 to 60% yield of α-APM relative to α-L-aspartyl-L-phenylalanine (I).

The α-APM solid hydrogen halide salt can be recovered by liquid / solid type separation methods. Essentially all the other compounds remain in the mother liquor. The separated salt can then be converted to substantially pure α-APM, for example, as shown in United States Patent Nos. 3,798,207 and 3,933,781.

The following examples are given to illustrate the present invention in detail.

The materials and methods used in the thin layer chromatography (TLC) assays of the Examples are as follows: A. Plate

Silica gel F on glass plate supplied by Brinkman 25 Instrument In., Westbury, N.Y. 11590 8 149432 B. Solvent Systems 1. Chloroform 64? Ό (by volume) methanol 30% "acetic acid 2%" 5 distilled water 4% "2. n-propanol 70%" distilled water 10% "methanol 10%" formic acid 10% "10 C. Syringe Solutions for Detection 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 potassium iodide and 1 g soluble starch dissolved in 100 ml distilled water.

D. Methods

After staining and developing in the appropriate solvent system, the plate was air dried for 30 min.

Nine Hydrine Injection - The plate was sprayed and kept in a 100 ° C hot oven for 15 min.

20 Starch Iodide Spraying - The plate was introduced into 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 25 Into a suitable vessel was introduced 140 ml of methanol and 420 ml of 9 N hydrochloric acid which was cooled with an ice bath.

To the resulting solution was added 113.8 U943-29 g (0.4 mol) of α-L-aspartyl-L-phenylalanine (I) (98.5% purity). The settlement began shortly after. The resulting mass was removed from the ice bath and kept under stirring for 30 minutes causing the temperature to rise to 20 ° C. The resulting mass was again cooled with an ice bath, kept under stirring for 1.5 hours, resulting in substantial precipitation, and then placed in a refrigerator overnight.

The next morning, the reaction mass was kept under stirring 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 ca. 5 ° C. and kept at this temperature for 4 hours. The precipitate which formed was separated by filtration and washed with five 30 ml portions of 5 ° C deionized water and dried. The resulting product was 51.8 g of α-APM, corresponding to a 44% yield 20 relative to the α-L-aspartyl-L-phenylalanine (I). TLC and sodium chloride analysis confirmed that the purity of the α-APM product was greater than 95%.

EXAMPLE 2

To a stirred solution of 34.2 ml (0.41 mol) of 37% hydrochloric acid, 60 ml of water and 40 ml of methanol was added 110 g (0.357 mol) of N-formyl-αL-aspartyl-L-phenylalanine over a 20 ml solution. minute period with a temperature rise from 40 ° C to 58 ° C. The resulting mass was kept under stirring at 58 - 60 ° C for 3 hours to effect the removal of the formyl group by hydrolysis.

The reaction mass was cooled to 25 ° C and 65.8 ml (0.79 mol) of 37% hydrochloric acid was added over 10 minutes.

149432 ίο

Shortly thereafter, a precipitate begins to form.

With stirring, the resulting mass was maintained for 45 hours at ambient temperature and 1.5 hours at 5 ° C, causing another 5 precipitates to form. The solid precipitate was separated by centrifugation and the cake was washed with 100 ml of 5 ° C hot deionized water. The wet cake (110.2 g) was dissolved in 410 ml of 45 ° C hot deionized water. The pH was adjusted over a 10 minute period to 2.5 with 80.1 g of 4.8% aqueous sodium hydroxide and kept under stirring for 1 hour at 40 ° C. Keeping the temperature at 40 - 42 ° C, 151.9 g of 4.8% aqueous sodium hydroxide was added over 3 hours to raise the pH to 4.2. The mixture was kept under stirring for 1 hour at 0-5 ° C and the resulting feathery crystals were separated by centrifugation. The cake was washed with 200 ml of 5 ° C hot, deionized water and dried overnight in a vacuum oven at 55 - 60 ° C. The yield of α-APM was 58.3 g (55.5% relative to N-formyl-α-L-aspartyl-L-phenylalanine); [A] p: (c = 4, 15 N formic acid); TLC analysis - over 98% pure a-APM.

EXAMPLE 3

Following essentially the same method as in Example 2, except that 32.5 25 ml of 37% hydrochloric acid and 33.3 ml of water were added to the reaction mass

after removal of the formyl group by hydrolysis and cooling to 25 ° C, a 33.0% yield of α-APM was obtained

relative to N-formyl-α-L-aspartyl-L-phenylalanine. on [α] ρ: + 15.3 ° (c = 4, 15 N formic acid.

EXAMPLE 4

Following essentially the same method as in Example 2, except that the time allowed to remove the formyl group by hydrolysis was limited to 1 hour, a 46.2% yield of α was obtained. -ΑΡΜ relative to N-formyl-αL-aspartyl-L-phenylalanine. [α] p: + 15.5 ° (c = 4, 15 N formic acid).

EXAMPLE 5

Following essentially the same method as in Example 2, except that 38.7 ml of 37 SS hydrochloric acid is used in the starting solution instead of 34.2 ml of 37% hydrochloric acid and reducing the amount of acid 10 After the hydrolysis, to 61.3 ml of 37% hydrochloric acid, a 53.2% yield of α-APM relative to N-formyl-αL-aspartyl-L-phenylalanine was obtained. [α] D: + 15.4 ° (c = 4, 15 N formic acid).

EXAMPLE 6 Following essentially the same method as in Example 2, except that the residence time to form a solid precipitate is increased to 4 days, a 59.2% yield of α-APM relative to N is obtained. -formyl-αL-aspartyl-L-phenylalanine. [α] 25 S + 15.2 ° (c = 4, 15 N formic acid).

EXAMPLE 7

By essentially following the same method as in Example 2, except that the holding period to cause a solid precipitate to form is reduced to 1 day, a 36.3% yield of α-APM is obtained relative to N-formyl-αL-aspartyl-L-phenylalanine. [α] p: + 15.5 ° (c = 4, 15 N formic acid).

Examples 1 to 7 are summarized in Table 1 using the previously described parameters.

12 TABLE 1

Methanol HCl (mol mol per mol α-L- (mol per 100 g aspartyl-L-phe-

Example 100 g of Reaction Reaction Nylalanine (I) No. reaction medium) Methanol HCl 1 0.75 0.65 9.45 10.95 2 0.48 0.56 3.28 2.8 30 49 0.45 2.58 2.8 4 0.48 0.56 3.28 2.8 10 5 0.48 0.56 3.28 2.8 6 0.48 0.56 3.28 2.8 7 0.48 0.56 3.28 2.8