DE2042299A1 - Acyl derivs of insulin - with modified solubility - Google Patents

Abstract

The insulin derivs. are of formula H-Gly-lleu.......(chain A) insulin X-Phe-Val.......(chain B) (in which X s 1-18C alkanoyl (opt. substd. by COOH, OH, lower alkoxy, phenoxy, benzyloxy, SH, lower alkylthio, phenylthio or benzylthio), 4-9C cycloalkanoyl, CF3CO, benzoyl phenyl-(1-3C)-alkanoyl (in which the phenyl grp. may be substd. by one or more halogen, CH3, C2H5, C6H5, NO2, OH, lower alkylthio, phenylthio, benzylthio, CN, COOH, 1-4C alkoxy-carbonyl, NH2 acylamino or sulphonyl grps) the radical of a pentagonal or hexagonal heteroaromatic carboxylic acid in which the heteroatoms are O,N or S and N, or alkoxycarbonyl, benzyloxycarbonyl or the radical of an aminocarboxylic acid derived from a natural alpha-amino acid or its D-enantiomers or 1-6 omega -amino-carboxylic acid (in which the amino grp. may be substd. by acryl 1-4C alkyl, phenyl or benzyl the OH or SH gps. are substd. by lower alkylphenyl or benzyl and other NH2 gps. are blocked by acyl) or X may be peptide radical. contg. up to 6 natural alpha-aminoacids found in peptides and proteins, with the exception of cysteine.

Description

Acyl derivatives of insulin and process for their production The invention relates to insulin derivatives of the general formula I H-Gly-Ile -.......... (A-Eette) ... (I)? A-Phe-Val-0 ..... 0 ... (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 alkyloxycarbonyl or aralkyloxycarbonyl radical, the alkyl part containing 1 to 4 carbon atoms, or an amino-acyl radical which is derived from the naturally occurring OC- or ß-amino acids or optionally their D-enantiomers.

The invention also relates to a method for production of these compounds, which is characterized in 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, beef or pig 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 as a compound for formation the active esters of the invention come with a hydroxy or imido compound pK value between 4.0 and 8.0 is possible. In particular, those in peptide chemistry common compounds such as 4-nitrophenol, 2.4-dinitrophenol, 2.4.5-trichlorophenol, Pentachlorophenol, pentafluorophenol, hydroxysuccinimide, hydroxyphthalimide, saccharin, 1-hydroxybenzotriazole or 3-hydroxy-4-oxo-3.4-dihydro-1 .2.3-benzotriazine is used.

At the first stage of the reaction sequence, the conversion of insulin with tet-butyloxycarbonyl azide, surprisingly arises among those mentioned Conditions predominantly the N α (A1), N # (B29) -di-tert-butyloxycarbonyl B1 insulin, whose still free amino group on the phenylalanine is now selectively linked with a the active carboxylic acid ester according to the invention can be reacted. It is there Furthermore, surprisingly, despite the excess of carboxylic acid active ester used no other functional groups, which 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 protective group used is the tert-butyloxycarbonyl radical or andore Protective groups that are split off under the same conditions as dex 'tert-butyloxycarbonyl radical, e.g.

the tert. -Miiyloxycarbonyl, the adamantyloxycarbonyl or the isobornyloxycarbonyl radical.

The method according to the invention is carried out, for example, that 1 mPlol of insulin is 30 to 70 times the amount by weight of an N.N-dialkylcarboxamide, 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 75mMol tert-Butyloxycarbonylazid and one Buffer solution in 2.5 to 4 times the volume, based on the weight of the one used Insulins, added and then stirred at about 350 to 40 ° C for 3 to 6 hours. 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 The reaction temperature can also be increased to about 500 ° C., preferred are, however, 35 to 400 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 in with the 5- to 10-fac1ien Amount by weight of 1 to 2 percent acetic acid and water digested and in vacuo dried over P205, for reaction - with the active carboxylic acid esters according to the invention dissolve the product advantageously in about 20 to 50 times the amount about 60 to 95 percent pyridine. Instead of pyridine, one of the above-mentioned can also be used N.N-dialkylcarboxamides can be used. The implementation with the carbolic acid active ester is expediently carried out for 3 to 20 hours, preferably 3 to 6 hours, at room temperature the end. If cloudiness occurs (indication of beginning denaturation) finished one the implementation. You can also shorten or lengthen it accordingly 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 after 1 to 2 times Crystallization chromatographically 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, Mono-N CC (B1) acetyl-insulin is still easily soluble at pH 6.5. Nα (B1) -glutamyl- or asparagyl insulin.

On the other hand, Nα (B1) -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 »hese Preparations are also physically stable, making them therapeutic Purposes instead of insulin itself or in combination with insulin can.

EXAMPLES The following examples are intended to illustrate the invention closer he clarify. 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. R means the speed of migration in paper electrophoresis, based on insulin = 1.0 Example 1 a) 1 g (0.167 mmol) of insulin are taken up in 70 cc of 80 percent dimethylacetamide. Man gives 1.45 g (10 mmol) of tert-butyloxycarbonyl azide and 3.3 cc (3.3 mmol) of 1N sodium bicarbonate solution and stirred for 5 hours at 35 ° C. Then the solution is in vacuo at a maximum of 50 ° C restricted bath temperature. The residue is rubbed in with ether and obtained 1.275 g of salty product, which after digestion with 20 ccm of 2 percent acetic acid 9145 mg Nα (A1) N, N insulin yields. RI 0.88; Amino acid analysis: = insulin.

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 Volume narrowed and the compound precipitated with ether.

Yield: 910 mg of Nα (A1), N carbonyl-Nα (B10-acetyl-insulin. RI: 0.82; Amino acid analysis: = insulin.

c) The compound prepared according to 1 b) is in 8 ccm of trifluoroacetic acid for 1 hour kept. With the addition of 100 ccm of ether, 855 mg of Nα (B1) -Acctyl-insulin fall which are dissolved in water in a known manner and crystallized at about pH 5. RI: 0.94; Amino acid analysis: = insulin.

Example 2 The di-tert-butyloxycarbonyl-insulin prepared according to Example 1 a) is in 6 ccm of 90 percent dimethylformamide with 0.8 g of 4-nitrophenyl propionate stirred overnight. Working up and splitting off of the protective groups analogously to the example 1 b) and 1 c).

Yield: 870 mg RI: - 0.94; Amino acid analysis: = 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 Nα (B1) -butyryl-insulin.

RI: 0.94; Amino acid analysis: = insulin.

Example 4 The di-tert-butyloxycarbonyl-insulin prepared according to Example 1 a) is in 8 ccm of n-methyl-pyrrolidone with 1.0 g of 4-nitrophenyl benzoate Night at room temperature touched. Processing and splitting off the Protective groups analogous to Example 1 b) and 1 c).

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

RI: 0.93; Amino acid analysis: = 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 1 c).

Yield: 885 mg of Nα (B1) -phenylacetyl-insulin.

RI: 0.93; Amino acid analysis: = 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 α (B1) -ethyloxycarbonyl-insulin.

R1: 0.914; Amino acid analysis: = 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 α (B1) -benzyloxycarbonyl-insulin.

RI: ° X93i amino acid analysis: = insulin Example 8 That according to the example 1 a) produced di-tert-butyloxycarbonyl-insulin is dissolved in 6 ccm of dimethylformamide with 1.2 g of tertO-butyloxyearbonyl-ß-alanine-hydroxysuccinimide ester for 12 hours Stirred at room temperature, working up and splitting off of the protective groups analogously to the example 1 b) and 1 c).

Yield: 890 mg RI: 1.00; Amino acid analysis: ß-Ala Ber. 1.00 Found 1.04 Example 9 The production of benzyloxycarbonyl chloride is exhibited Isoborneol and phosgene produce the oily isobornyloxyearbonyl chloride. When it comes to sales of this Combination with glycine in the known manner according to Schotten-Baumann isobornyloxycarbonyl-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 of di-tert-butyloxycarbonyl insulin, prepared according to Example 1 a), is in 10 ccm of dimethylformamide with 1.75 g of isobornyl-oxycarbonyl-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 of N NO (1) glycyl insulin.

RI: 1.00; Amino acid analysis: Gly Ber. to, 5.00 found. 4.98 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-ter.-butyloxycarbonyl-L-glutamic acid-α-2.4.5-trichlorophenyl ester- r, -tert. -Butyl ester stirred at room temperature for 20 hours3 work-up and cleavage the protective groups analogous to Example 1 b) and 1 c).

Yield: 1.00 g of N α (B1) -L-giutamyl-insulin.

RI: 0.99; Amino acid analysis: Glu Ber. 8.00 Gef, 8, o6 Example 11 Analog Chem. Ber. 103 (1970), page 788, is made from 1.42 g of N-tert-butyloxycarbonyl-aspartic acid f, -tert. butyl ester, 675 mg of 1-hydroxy-benzotriazole and 1.05 g of dicyclohexyl-carbodiimide in 10 cc of dimethylacetamide and sets the corresponding hydroxybenzotriazol ester this after filtering off the precipitated dicyclohexylurea without isolation with 1 g of di-tert-butyloxycarbonyl-insulin, which is added to the solution of the active ester be around for 3 hours at room temperature. Processing and splitting off the Protective groups analogous to Example 1 b) and 1 c).

Yield: 995 mg of Nα (B1) -L-asparagyl-insulin.

RI: 0.99; Amino acid analysis: Asp Ber. 4.00 Found 4.05 Example 12 1 g of di-tet.-butyloxycarbonyl-insulin is dissolved in 10 cc of 95 percent pyridine / dimethylformamide (1: 1) 8 hours at room temperature with 2.2 g of N α, N # -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 N () -L-Lysyl-insulill.

RI: 1.05; Amino acid analysis: Lys Ber. 2.00 Found 2.03 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-nitrophenyl ester (prepared according to Chem. Ber. 103 (1970), page 1727) for 20 hours at room temperature touched.

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 RI: 1.06; Amino acid analysis: Arg Ber. 2.00 found 2.01 Example 14 1 g of di-tert-butyloxycarbonyl-insulin is dissolved in 10 cc of dimethylacetamide with 1.4 g of N-tert. -Butyloxycarbonyl-D-alanine hydroxysuccinimide ester for 5 hours Room temperature stirred. Working up and splitting off of the protective groups analogously to the example 1 b) and 1 c).

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

RI: 1.00; Amino acid analysis: Ala Ber. 4.00 Found 4.02

Claims (6)

P atent claims 1. Insulin derivatives of the general formula I H-Gly-Ile -........ (A chain) A-Phe-Val -........ (B-chain) ... # insulin (I) 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 alkyloxycarbonyl or aralkyloxycarbonyl radical, the alkyl part containing 1 to 4 carbon atoms, or an amino-acyl radical which is derived from the naturally occurring t- or ß-amino acids or, if appropriate, their D-enantipmeres. 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 I, in which A is the glycyl, L-alanyl, D-alanyl, ß-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 -........ (A-chain) .... # insulin (I) A-Phe-Val -........ (B chain) in which A is an alkanoyl radical with 1 to 4 carbon atoms, a phenylalkanoyl rust with 1 to 3 carbon atoms in the alkanoyl part, benzoyl or an alkyloxycarbonyl or aralkyloxycarbonyl radical, the alkyl part containing 1 to 4 carbon atoms, or an amino-acyl radical which is derived from the naturally occurring Cc- or ß-amino acids or optionally their D-enantiomers, characterized in that insulin is used in a mixture consisting of an NN-dialkylcarboxamide with a total of 3 to 6 C- Atoms and an aqueous buffer solution with a pH of 8.0 to 9.0, reacted with an excess of tert-butyloxycarbonyl azide at room temperature or slightly elevated temperature, then with 2 to 4 equivalents of an activated ester of a carboxylic acid A. -OlI, where the acyl radical A has the above meaning, is reacted and the reaction product is treated with a strong acid, such as, for example, 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 with an antidiabetic effect, marked by a content of a compound of the general formula 1 in which A has the above Has meaning.