CH616649A5 - Process for the preparation of N-acylamino acid polyethylene glycol esters - Google Patents

Description

616 649

Patent claim Process for the preparation of N-acylamino acid poly-ethylene glycol esters, characterized in that polyethylene glycol in solvents suitable for peptide chemistry with N-acylamino acids, dicyclohexylcarbodiimide and a substance of the formula (I)

(I)

implemented in which Xn represents hydrogen, halogen, trifluoromethyl, nitro, sulfonic acid amide, sulfonic acid methylamide, sulfonic acid diethylamide, carboxamide, cyano, lower alkyl or alkoxy groups and n represents the numbers 1 to 4, with the polyethylene glycol before or after the dicyclohexylcar-bodiimide addition to the reaction mixture.

Polyethylene glycol (PEG) is used in peptide chemistry as an ester component in the synthesis of peptides [Nature 273, pages 512 to 513, (1972) 1-

Amino acids and peptides can be used as amino acid and peptide esters of PEG by both ultrafiltration and crystallization [Angew. Chemie, 86, page 101 (1974)] are separated from low molecular weight substances.

So far, the ester bond between polymer and acylamino acid has mainly been produced using dicyclohexylcarbodiimide (DCCI). Either the symmetrical anhydrides of N-acylamino acids were prepared using DCCI and allowed to react with the polyethylene glycol, or the N-acylamino acids were reacted directly with the polyethylene glycol and DCCI.

In this method of esterifying polyethylene glycol,

However, urethane acylation and, as a result, dipeptide formation was observed, which reached 30% in extreme cases.

The invention relates to a process for the preparation of N-acylamino acid polyethylene glycol esters, which is characterized in that polyethylene glycol in solvents suitable for peptide chemistry with N-acylamino acids, dicyclohexylcarbodiimide and a substance of the formula (I)

implemented in which Xn represents hydrogen, halogen, trifluoromethyl, nitro, sulfonic acid amide, sulfonic acid methylamide, sulfonic acid diethylamide, carboxamide, cyano, lower alkyl or alkoxy groups and n represents the numbers 1 to 4, with the polyethylene glycol before or after the dicyclohexylcar-bodiimide addition is added to the reaction mixture.

The intermediate N-acylamino acid esters with the various N-hydroxy compounds of formula (I) can optionally be isolated.

The process according to the invention is advantageously carried out as follows:

N-acylamino acids, an optionally nucleus-substituted 1-hydroxybenzotriazole and dicyclohexylcarbodiimide are reacted in suitable solvents. Polyethylene glycol is added to the reaction mixture either at the beginning of the reaction before the addition of dicyclohexylcarbodiimide or after a pre-activation phase, after which the sparingly soluble dicyclohexylurea has been filtered off. The individual components are used in molar ratios or in excess, based on polyethylene glycol (see Tables 1 and 2).

As nucleus-substituted 1-hydroxy-benzotriazole compounds of formula (I) come, for. B. l-hydroxy-6-benzotriazole-sulfonic acid diethylamide l-hydroxy-6-benzotriazole-sulfonic acid methylamide l-hydroxy-6-benzotriazole-sulfonic acid amide l-hydroxy-6-trifluoromethyl-benzotriazoI l-hydroxy-6-methoxy-benzotriazole 1 -hydroxy -5-methoxy-benzotriazole l-hydroxy-4-methyl-benzotriazole l-hydroxy-5-methyl-benzotriazole l-hydroxy-5,6-dimethyl-benzotriazole 1 -hydroxy-6-nitro-benzotriazole 6-chloro-1 - hydroxy-benzotriazole

5-chloro-l-hydroxy-benzotriazole 5,6-dichloro-l-hydroxy-benzotriazole

6-bromo-1-hydroxy-benzotriazole

4-chloro-1-hydroxy-7-methyl-6-nitro-benzotriazole 6-chloro-1-hydroxy-5-methyl-benzotriazole l-hydroxy-6-methyl-5-benzotriazole-carbonitrile l-hydroxy-4-methyl -6-nitro-benzotriazole 6-chloro-l-hydroxy-5-isopropylbenzotriazole and 4,5,6,7-tetrachloro-1-hydroxy-benzotri azole in question.

As a solvent, polar solvents, such as. As dimethylformamide and tetramethylurea, use, methylene chloride and tetrahydrofuran can also be used. A large excess of acylating agents is not necessary, rather a 100% excess is generally sufficient to achieve at least 80% loading. If necessary, the reagents can be added again after a single precipitation and the reaction repeated.

The esterification of polyethylene glycol with N-acylamino acids and DCCI or their symmetrical anhydrides, which has been carried out to date, in many cases either proceeds with unsatisfactory yields or one is forced to use long reaction times or large excesses of expensive reagents. When using symmetrical anhydrides, the previous method is particularly unfavorable in that only 50% of the reagent can be used. For example, about 80% of Boc-Met-Oh can only be esterified with PEG (molecular weight 15,000) if a 10-fold excess and a 7-day reaction time are used [Chem. Ber. 107, pages 1344 to 1352 (1974)].

However, the addition of 1-hydroxybenzotriazole or nucleus-substituted 1-hydroxybenzotriazoles to the DCCI method significantly accelerates the esterification reaction. Under the influence of DCCI, the N-acylamino acids react with optionally substituted 1-hydroxybenzotriazoles in a very rapid reaction to the corresponding active esters, which then form the product with the polymeric alcohol (PEG) with alcoholysis. That actually this N-acylamino acid-l-hydroxybenzotriazole active ester as

2nd

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

3rd

616 649

Intermediates occur, is evidenced by the data in Table 1. In these experiments, the two reaction steps described were separated. In a pre-activation phase, the active ester is formed from the reagents, dicyclohexylurea can be filtered off, and in the second step, the clear solution is combined with the polyethylene glycol, the alcoholysis now taking place.

The active esters formed of the acylamino acids and the N-hydroxy compounds of the formula (I) make it possible to carry out the process according to the invention at higher temperatures, since they do not give rise to any thermal rearrangement products, which is the case when reacting with DCCI alone [J. Amer. Chem. Soc. 80, 6204 (1958)].

Up to now it was only known of esters of 1-hydroxybenztriazole that with their help the peptide bonds were formed more specifically and with fewer side reactions.

A higher specificity of the reaction means that aminolysis is preferred over hydrolysis or alcoholysis. However, it was not to be expected that in the absence of amines the alcoholysis would be accelerated and improved so much that the esters mentioned are even superior to activated derivatives such as symmetrical anhydrides.

The sterically strongly hindered acylamino acids Z-IIe-OH, Z-Pro-OH and Boc-Cys (Bzl) -OH can also be converted into the poly-ethylene glycol ester under somewhat more severe conditions (70 ° C), which after the previous uncatalyzed Procedure was not easily possible.

Another advantage of the method according to the invention is the suppression of side reactions. So z. B. from J. Org. Chem. 26 (1961), page 3256 known, caused by dicyclohexylcarbodiimide nitrile formation from Boc-Gln-OH and Boc-Asn-OH under the conditions according to the invention.

The activation of N-acylamino acids via their active esters with 1-hydroxybenzotriazole also rules out any noteworthy racemization, which is a further advantage of the process according to the invention.

Finally, the urethane acylation already mentioned as a side reaction is also suppressed, which is often observed when strongly activated alkyl or aralkyloxycarbonylamino acids have to be used in excess as acylating reagents [compare J. Org. Chem. 39, pages 660 to 668 (1974)] . The method according to the invention also has a particularly favorable effect here. With simultaneous yield

25th

30th

35

40

45

The urethane acylation remains below 1%. Even with repeated acylation, the by-product formation does not exceed 2%.

In addition to this great advantage, the esterification according to the invention proceeds more quickly and does not require large excesses of N-acylamino acids.

The acylamino acid esters produced according to the invention are intermediate products in the synthesis of therapeutically valuable peptides.

The following abbreviations are used:

PEG = polyethylene glycol -OPEG (4), (6), (10), (15) and (20)

= Polyethylene glycol ester with a molecular weight of 4000, 6000, 10,000, 15,000 and 20,000 DMF = dimethylformamide DMSO = dimethyl sulfoxide DMA = dimethylacetamide TMH = tetramethylurea THF = tetrahydrofuran HOBt = 1-hydroxybenzotriazole

The amino acids and their derivatives are designated according to JUPACJUB rules J. Biol. Chem. 247, pages 977 to 983 (1972).

Example 1 (see Table 1)

Esterification of polyethylene glycol with N-amino acid 1-hydroxybenzotriazole esters.

Acylamino acid, l-hydroxybenzotriazole (HOBt) and dicyclohexylcarbodiimide (DCCI) are dissolved in DMF in the stated proportions and stirred for 2 hours at room temperature (preactivation). The resulting dicyclohexylurea is then filtered off, the appropriate amount of polyethylene glycol is added to the solution of the 1-hydroxybenzotriazole ester and the reaction mixture is heated to 45 to 60 ° C. for 16 to 20 hours. After cooling, any dicyclohexylurea which has still precipitated is removed, the solvent is evaporated off and extracted from a methylene chloride or. Ethanol solution of the residue precipitated the product by adding ether. The precipitation process is repeated a number of times until thin-layer chromatography (butanol / glacial acetic acid / water 3: 1: 1) can no longer detect any low-molecular excesses. The degree of esterification is determined by amino acid analysis of a defined sample of the substance after acid hydrolysis.

Table 1

Esterification of polyethylene glycol with N-acylamino acid l-hydroxybenzotriazole esters

(see example 1)

conditions

product

Acyl - ** aminos.

HOBt * ®

DCCI **

solvent

Time / temp.

comment

Esterification

[Equiv.]

[Equiv.]

[Equiv.]

[g * / ml]

Degree

Boc-Gly-OPEG (6)

5

5

5.35

DMF / 0.2

16 h / 55 ° C

2 h / pre-act. 25 ° C

95.1%

Boc-Gly-OPEG (6)

3rd

3rd

3.3

DMF / 0.3

20 h / 45 ° C

2 h / pre-act. 25 ° C

86.4%

Boc-Gly-OPEG (6)

3rd

3rd

3.2

DMF / 0.3

16 h / 60 ° C

2 h / pre-act. 25 ° C

92.0%

Boc-Pro-OPEG (10)

5

5

5.3

DMF / 0.4

20 h / 55 ° C

2 h / pre-act. 25 ° C

78.5%

Z-Pro-OPEG (6)

5

5

5.3

DMF / 0.3

16 h / 60 ° C

2 h / pre-act. 25 ° C

85.6%

Z-Ser- (But) -OPEG (6)

5

5

5.3

DMF / 0.3

16 h / 60 ° C

2 h / pre-act. 25 ° C

87.4%

based on -OH groups of PEG's 9 based on PEG

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4th

Example 2 (see Table 2)

Esterification of polyethylene glycol with N-acylamino acids using dicyclohexylcarbodiimide with and without the addition of 1-hydroxybenzotriazole. s

PEG (of specified molecular weight), acylamino acid,

HOBt and DCCI are dissolved in the appropriate proportions in DMF, TMH, THF, CH2C12 or mixtures of these solvents and the solution is stirred at temperatures from 25 to 70 ° C for 24 hours. After cooling, the precipitated dicyclohexylurea is filtered off, the solvent is evaporated off, the residue is taken up in a little methylene chloride or ethanol and the product is precipitated with ether. In some cases, the product is redissolved in the appropriate solvent, new reagents are added, and the reaction is repeated under the same conditions. Otherwise, the products are freed from low-molecular substances by ether precipitation as in Example 1.

The comparative experiments were carried out in an analogous manner, but without HOBt.

Table 2

Esterification of polyethylene glycol with N-acylamino acids using dicyclohexylcarbodiimide with and without the addition of 1-hydroxybenzotriazole

conditions

Product Acyl- HOBt DCCI solution time / temp. Remark ver

aminos. medium esterification

[Equiv. | * [Equiv.] * [Equiv.] * [G / ml] * grad

Z-Gly-OPEG (6)

20th

20th

21.4

THF / 0.15

24 h / 55 ° C

74%

Z-Gly-OPEG (6)

20th

20th

21.4

DMF / 0.15

24 h / 55 ° C

78%

Z-Gly-OPEG (6)

20th

20th

21.4

TMH / 0.15

24 h / 55 ° C

90%

Z-Gly-OPEG (6)

20th

-

21.4

TMH / 0.15

24 h / 55 ° C

47%

Boc-Gly-OPEG (10)

2x2

2x2

2x2.1

CH2C12 /

2 x 24 h /

***

91%

0.125

25 ° C

for ether

precipitation will

repeated

Boc-Gly-OPEG (10)

3rd

-

3.1

CH2C12 /

24 h / 45 ° C

40%

0.16

Z-Ala-OPEG (6)

2X2

2x2

2x2.1

DMF / 0.16

2 x 24 h /

87%

50 ° C

for ether

precipitation will

repeated

Z-Phe-OPEG (6)

5

5

5.3

DMF / 0.16

24 h / 50 ° C

84.5%

Z-Phe-OPEG (4)

2.5

-

2.6

CH2C12 / n o

24 h / 45 ° C

27%

Z-Leu-OPEG (6)

5

5

5.3

u, e.g.

DMF / 0.16

24 h / 50 ° C

76%

Z-Pro-OPEG (6)

2x5

2x5

2x5.3

DMF / THF

2x24 h /

90%

(1; 1) / 0.15

o o

O

for ether

precipitation will

repeated

Z-Pro-OPEG (6)

5

-

5.3

CH2C12 /

7d / 25 ° C

16%

0.16

Z-Ile-OPEG (6)

2x5

2x5

2 x 5.3

DMF / 0.15

2 x 24 h /

75%

70 ° C

for ether

precipitation will

repeated

Z-Ile-OPEG (6)

5

-

5

CH2C12 /

7d / 25 ° C

6.59c

0.16

Z-Val-OPEG (6)

2x5

2x5

2x5.3

DMF / 0.15

2x24 h /

88%

70 ° C

Z-ASN-OPEG (6)

2nd

2nd

2.1

TMH / 0.16

24 h / 50 ° C

97%

Z-ASN-OPEG (6)

2nd

-

2.1

DMF / 0.15

24 h / 50 ° C

29%

Boc-Cys (Bzl) -

OPEG (6)

2nd

2nd

2.1

DMF / 0.15

24 h / 55 ° C

46%

Boc-Cys (Bzl) -

OPEG (6)

2nd

-

2.1

DMF / 0.15

24 h / 55 ° C

1.7

*** DCH is filtered beforehand; ** based on PEG; * based on PEG OH groups