DE2038121A1 - Acyl derivs of insulin - with changed solubility properties used as anti-diabetes agents - Google Patents

Abstract

Cpds. are of formula (I): (where A is 1-4C alkanoyl, phenyl-alkaoyl (1-3C alkanoyl), benzoyl, alkyl-oxycarbonyl or aralkyloxycarbonyl (1-4C alkyl), amino-acyl derived from natural alpha- or beta-amino acids or their D-enantiomers). They are prepd. by reacting insulin in a mixture of N,N-dialkylcarboxylic acid amide (total 3-6C) and aqs. buffer at pH 8-9 with excess of tert. butyloxycarbonyl azide and then with an activated ester of a carboxylic acid A - OH. The reaction product is reacted with a strong acid e.g. trifluoroacetic acid. (I) have full insulin activity but with changed physical properties such as change in iso-electric point.

Description

Acyl derivatives of insulin and process for their preparation are the subject matter of the invention are insulin derivatives of the general formula 1 H-GZy-Ile -., .. ,, .. (A chain) APhe-Val-.00 ... 00 (B-chain) ... Insulin (i) in the A is an alkanoyl radical with 1 to k carbon atoms, a phenylalkanoyl radical with 1 to 3 carbon atoms in the alkanoyl part, benzoyl or an alkyl-oxycarbonyl or aralkyloxycarbonyl radical, the alkyl part being 1 contains up to 4 carbon atoms, or an amino-acyl radical, which differs from the naturally occurring oC- or ß-amino acids or, if appropriate, their D-enantiomers, means.

The invention also relates to a method for production of these compounds, which is characterized by the fact that insulin is used in a mixture, from an N.N-dialkylcarboxamide with a total of 3 to 6 carbon atoms and one aqueous buffer solution with a pH of 8.0 to 9.0, with an excess on tert-butyloxycarbonyl azide at room temperature or slightly elevated temperature reacted, then with 2 to 4 equivalents of an activated ester of a carboxylic acid A-OH, where the acyl radical A has the above meaning, converts and the reaction product treated with a strong acid such as trifluoroacetic acid.

Various species of insulin can be used as the starting product, bovine or porcine insulin or a mixture of these two is preferably used Insulins in any mixing ratio.

The alkanoyl radical is preferably acetyl, propionyl or butyryl radical in question; as the aralkanoyl radical, for example the phenylacetyl radical; as aralkyloxycarbonyl radical the benzyloxycarbonyl radical is preferably used.

Comes as compounds to form the active esters of the present invention Hydroxy or imido compounds with a pK value between 4.0 and 8.0 are possible. In particular, the compounds customary in peptide chemistry, such as 4-nitrophenol, 2. 4-dinitrophenol, 2.4. 5-trichlorophenol, pentachlorophenol, pentafluorophenol, hydroxysuccinimide, Hydroxyphthalimide, saccharine, 1-hydroxybenzotriazole or 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine used.

At the first stage of the reaction sequence, the conversion of insulin with tert-butyloxycarbonyl azide, surprisingly arises among those mentioned Conditions predominate over the Nα (1), N # (B29) -di-tert-butyloxyearbonyl-131 insulin, whose still free amino group on the phenylalanine is now selectively linked to one of the can be implemented carboxylic acid active ester according to the invention. It is still there Surprisingly, despite the excess of active carboxylic acid ester used, none other functional groups that are abundant in the molecule are attacked will.

If the carboxylic acid on which the active ester is based is an amino acid, so the amino group of the amino acid must be protected during this reaction step will. The tert-butyloxycarbonyl radical or is used as a protective group other Protective groups that are split off under the same conditions as the tert-butyloxycarbonyl radical, e.g. the tert-amyloxycarbonyl, the adamantyloxycarbonyl or the isobornyloxycarbonyl radical.

The method according to the invention is carried out, for example, that 1 mmol of insulin is 30 to 70 times the amount by weight of an N.N-dialkylcarboxamide, J. which can contain up to 25% water, suspends or dissolves. As a dialkylcarboxamide are used, for example: dimethylformamide, dimethylacetamide, diethylformamide or N-methyl-pyrrolidone.

Then 20 to 75 mmol of tert-butyloxycarbonylazid and a buffer solution in 2.5 to 4 times the volume, based on the weight of the used insulin, added and then 3 to 6 hours at about 350 to 40 C stirred. As a buffer solution, e.g.

used in alkali bicarbonate or alkali phosphate buffers from pH 8.0 to 9.0 will. Of course, the buffer solution must not contain any components such as ammonia, contain primary or secondary amines that react with tert-butyloxycarbonyl azide would. The reaction temperature can also be increased to about 50 ° C., preferably however, are 350 to 110 C. The reaction already takes place at room temperature, however 4 to 6 times the time is then required.

The di-tert-butyloxycarbonyl-insulin formed is advantageously isolated. For this one either falls directly with ether or brings the solution to at most 500 C bath temperature in vacuo to dryness, if necessary after previous neutralization with acetic acid. Then with Ether rubbed with 5 to 10 times Amount by weight of 1 to 2 percent acetic acid and water digested and in vacuo dried over P205.

To react with the active barboxylic acid esters according to the invention dissolves the product is advantageous in about 20 to 50 times the amount about 60 to 95 percent Pyridines. Instead of pyridine, one of the above-mentioned N.N-dialkylcarboxamides can also be used be used. The reaction with the carboxylic acid active ester is expediently carried out for 3 to 20 hours, preferably 3 to 6 hours, at room temperature. at Any turbidity that may occur (indication of the beginning of denaturation) is ended the implementation. One can also shorten or lengthen the Reaction time at about 400 C or at 0 ° to 100 C work.

After the reaction has ended, the reaction product is precipitated with ether, Stored for 1 to 2 hours in about 10 times the amount by weight of trifluoroacetic acid, again precipitated with ether and in a known manner by adjusting to the isoelectric Point brought to crystallization.

The reaction products are chromatographic after crystallization once or twice and electrophoretically pure or

correspond in their purity to the insulin used.

The new insulin derivatives have full insulin activity, differentiate However, it differs from insulin due to changed physical properties, such as shift of the isoelectric point. This causes, for example, MonoN «(B1) acetyl-insulin pH 6.5 is still easily soluble. N and asparagyl insulin behave in the same way (B1) glutamyl.

On the other hand, N «(1) -lysyl- and arginyl-insulin are at pH 6 to 6.5 in the range of their lowest solubility.

The special one is based on these changed solubility properties Use of the compounds according to the invention as antidiabetic medicaments. With the help of the new compounds, preparations can be produced, with contain a more easily and a more difficultly soluble component in a preparation is, whereby a desired time-effect ratio can be set. These Preparations are also physically stable, making them therapeutic Purposes to be used instead of insulin itself or in combination with insulin Ri can.

EXAMPLE The following examples are intended to illustrate the invention explain in more detail. Paper electrophoresis was used to characterize the acylated insulins (6 hours 200 volts in acetic acid / formic acid pH 2. staining with bromophenol blue), paper chromatography and amino acid analysis.

Example 1 a) 1 g (0.167 mmol) of insulin in 70 ccm of 80 percent Dimethylacetamide added. 1.45 g (10 mmol) of tert-butylpxycarbonyl azide are added and 3.3 com (3.3 mmol) 1N sodium bicarbonate * and stir for 5 hours at 350 C.

Then the solution is in vacuo at a maximum of 50 ° C bath temperature constricted. The residue is triturated with ether and 1.275 g of salt is obtained Product which after digestion with 20 ccm 2 percent acetic acid 945 mg N (A1), N -Di-tert-butyloxycarbonyl-insulin gives.

b) This compound is dissolved in 6 cc of 95 percent pyridine. Man releases 0.6 g of N-hydroxysuccinimide acetate (prepared in a known manner Acetic acid and N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide) and stir for 6 hours at room temperature.

Then in a vacuum at a maximum of 500 C bath temperature to a small Valumen narrowed and the connection with ether precipitated.

Yield: 910 mg of N (A1), N, N (1), N 6 (B29) -Di-tert-butyloxycarbonyl-N (131) acetyl insulin.

* -solution c) The connection established according to 1 b) is kept in 8 cc trifluoroacetic acid for 1 hour. On the addition of 100 cc of ether 855 mg of Nα (B1) -acetyl-insulin precipitate in a known manner in water dissolved and crystallized at about pH 5 Example 2 The according to Example 1 a) Di-tert-butyloxycarbonyl-insulin produced is in 6 cc of 90 percent dimethylformamide Working up and splitting off overnight with 0.8 g of 4-nitrophenyl propionate the protective groups analogous to Example 1 b) and 1 yield: 870 mg Nα (B1) -propionyl-insulin, Example 3 The di-tert-butyloxycarbonyl-insulin prepared according to Example 1 a) is in 8 ccm of dimethylacetamide with 1.3 g of n-butyric acid pentachlorophenyl ester for 6 hours stirred at 250 to 300 C. Working up and splitting off the protective groups analogously Example 1 b) and 1 c).

Yield: 875 mg of N α (B1) -butyryl-insulin, Example 4 The after Example 1 a) Di-tert-butyloxycarbonyl-insulin produced is dissolved in 8 cc of n-methyllyl-pyrrolidone with 1.0 g of 4-nitrophenyl benzoate overnight at room temperature touched. Working up and splitting off of the protective groups analogously to Example 1 b) and 1- c).

Yield: 890 mg of Nα (B1) -benzoyl-insulin.

Example 5 The di-tert-butyloxycarbonyl-insulin prepared according to Example 1 a) is in 8 cc of 95 percent pyridine with 1.1 g of 4-nitrophenyl phenylacetate stirred overnight at room temperature. Working up and splitting off of the protective groups analogous to “Example 1 b) -and 1sc-).

Yield: 885 mg of N (1) -phenylacetyl-insulin.

Example 6 The di-tert-butyloxycarbonyl-insulin prepared according to Example 1 a) is in 6 ccm of dimethylformamide with 0.8 g of carbonic acid ethyl 4-nitrophenyl ester Stirred overnight at room temperature. Working up and splitting off of the protective groups analogous to example 1 b) and 1 c).

Yield: 875 mg of N (B1) -thyloxyearbonyl-insulin.

Example 7 The di-tert-butyloxycarbonyl-insulin prepared according to Example 1 a) is in 6 ccm of dimethylformamide with 1.2 g of carbonic acid-benzyl-4-nitrophenyl ester 20 Stirred for hours at room temperature. Working up and splitting off of the protective groups analogous to example 1 b) and 1 c).

Yield: 900 mg of N «(B1) -Benzyloxycarbonyl-insulin.

Example 8 The di-tert-butyloxycarbonyl-insulin prepared according to Example 1 a) is in 6 ccm of dimethylformamide with 1.2 g of tert-butyloxyearbonyl-ß-alanine hydroxysuccinimide ester Stirred for 12 hours at room temperature. Working up and splitting off of the protective groups analogous to example 1 b) and 1 c).

Yield: 890 mg of N «() -β-alanyl-insulin.

Example The preparation is analogous to the preparation of benzyloxycarbonyl chloride the oily isobornyloxycarbonyl chloride is produced from isoborneol and phosgene. In terms of sales this connection with glycine in a known way according to Schotten-Baumann isobornyloxyearbonyl-glycine, Melting point 1820-1830 C.

It is produced therefrom in a likewise known manner by reacting with N-hydroxysuccinimide in the presence of dicyclohexylcarbodiimide the isobornyloxycarbonyl-glycine-hydroxysuccinimide ester, Melting point 830-840 ° C.

1 g di-tert. -Butyloxycarbonyl-insuiin, prepared according to the example 1 a), is dissolved in 10 ccm of dimethylformamide with 1.75 g of isobornyl-vxyearbonyl-glycine-hydroxysuccinimide ester Stirred at 300 ° C. for 5 hours. Working up and splitting off the protective groups analogously Example 1 b) and 1 c), yield: 940 mg N (B1) -glycylinsulin example 10 1 g of di-tert-butyloxycarbonyl insulin, prepared according to Example 1 a), is in 12 cc of 95 percent pyridine with 2.4 g of N-tert-butyloxycarbonyl-L-glutamic acid oC-2. 4.5-trichlorophenyl ester- t-tert-butyl ester for 20 hours at room temperature touched. Working up and splitting off of the protective groups analogously to Example 1 b) and 1 c).

Yield: 1.00 g of N (B1 (-L-glutamyl-insulin.

Example 11 Analogous to Chem. Ber. 103 (1970), page 788, is issued 1.42 g of N-tert-butyloxycarbonyl-aspartic acid-t-tert-butyl ester, 675 mg of 1-hydroxy-benzotriazole and 1.05 g of dicyclohexyl carbodiimide in 10 cc of dimethylacetamide the corresponding Hydroxybenzotriazol ester and sets this after filtering off the precipitated Dicyclohexylurea without isolation with 1 g of di-tert-butyloxycarbonyl-insulin, which are added to the solution of the active ester for 3 hours at room temperature around.

Working up and splitting off of the protective groups analogously to Example 1 b) and 1 c).

Yield: 995 mg of N i (B1) -L-asparagyl-insulin, Example 12 1 g of di-tert-butyloxycarbonyl-insulin is in 10 com 95 percent pyridine / dimethylformamide (1: 1) for 8 hours at room temperature with 2.2 g of Nα, N 6-di-tert-butyloxycarbonyl-L-lysine hydroxysuccinimide ester touched. Work-up and splitting off of the protective groups analogously Example 1 b-) and 1 c). Purification by dissolving in dilute acetic acid and precipitation at pH 5.8 to 6.0.

Yield: 1.03 g of Nα (B1) -L-lysyl-insulin.

Example 13 1 g of di-tert-butyloxycarbonyl-insulin is dissolved in 15 cc of dimethylacetamide with 4.0 g of tri-adamantyloxycarbonyl-L-arginine-4-nitropheny, 1ester (manufactured according to Chem. Ber. 103 (1970), page 1727) for 20 hours at room temperature.

Working up and splitting off of the protective groups analogously to Example 1 b) and 1 c). Purification by dissolving in dilute acetic acid and precipitation at pH 6.0 up to 6.2.

Yield: 1.00 g of Nα (B1) -L-arginyl insulin.

Example 111 1 g of di-tert-butyloxycarbonyl-insulin is dissolved in 10 ccm Dimethylacetamide with 1.4 g of N-tert-butyloxycarbonyl-D-alanine hydroxysuccinimide ester Stirred for 5 hours at room temperature. Working up and splitting off of the protective groups analogous to example 1 b) and 1 c).

Yield: 980 mg of Nα (B1) -D-alanyl-insulin.

Claims (6)

P a t e n t a n s p r ü c h e 1. nsulin derivatives of the general formula I 0 II-Gly-Ile- ..... (A chain) ... Insulin (I) A-Phe-Val -....... (B chain) in which A is an alkanoyl radical with 1 to 4 carbon atoms, a phenylalkanoyl radical with 1 to 3 carbon atoms in the alkanoyl part, benzoyl or an alkyl-oxycarbonyl or aralkyloxycarbonyl radical, the alkyl part being 1 contains up to 4 carbon atoms, or an amino-acyl radical, which differs from the naturally occurring α- or ß-amino acids or, if appropriate, their D-enantiomers, means. 2. Insulin derivatives of the general formula I, in which A denotes the acetyl, Propionyl, butyryl, benzoyl, phenylacetyl, methyloxycarbonyl, ethyloxycarbonyl or benzyloxycarbonyl radical. 3. Insulin derivatives of the general formula 1, in which A is the glycyl, L-alanyl, D-alanyl, β-alanyl, L-glutamyl, L-asparagyl, L-lysyl or arginyl radical means. 4. Process for the preparation of insulin derivatives of the general Formula I H-Gly-Ile -... .. to. . (A-chain) .... insulin (I) A-Phe-Val -........ (B-chain) in the A an Alkanoylre.st with 1 to 4 carbon atoms, a phenylalkanoylrest with 1 to 3 carbon atoms in the alkanoyl part, benzoyl or an alkyl-oxyearbonyl or aralkyloxycarbonyl radical, whereby the alkyl part contains 1 to 4 carbon atoms, or an amino-acyl radical, which is different from the naturally occurring oC- or ß-amino acids or optionally their D-enant: iomers derives, means, characterized in that one insulin in a mixture that from a N.N-Dialkylcarbonsäurea'nid with a total of 3 to 6 carbon atoms and one aqueous buffer solution with a pII value of 8.0 to 9.0, with an excess an ter-butyloxycarbonyl azide at room temperature or slightly elevated temperature reacted, then with 2 to 4 equivalents of an activated ester of a carboxylic acid A-OH, where the acyl radical A has the above meaning, converts and the reaction product treated with a strong acid such as trifluoroacetic acid. 5. Process for the preparation of pharmaceutical preparations with antidiabetic Effect, characterized in that compounds of the general formula I, in which A has the above meaning, optionally in combination with insulin or its derivatives in a form suitable for therapeutic purposes. 6. Pharmaceutical preparations m antidiabetic effect, marked by a content of a compound of the general formula I in which A the above Has meaning.