NO843799L - INSULIN DERIVATIVES MODIFIED IN POSITION B30, PROCEDURE FOR THEIR PREPARATION AND THEIR USE, AND PHARMACEUTICAL MEDICINE TO TREAT DIABETE MELLITUS - Google Patents

Abstract

1. Claims for the Contracting States : BE CH DE FR GB IT LI LU NL SE A medicament having a delayed action for the treatment of diabetes mellitus, comprising at least one insulin derivative of the formula I see diagramm : EP0137361,P16,F1 in which R**1 denotes H or H-Phe R**30 represents the radical of a neutral, genetically codable L-aminoacid whose OH group, where present, can be free or protected by a physiologically acceptable group, and R**31 denotes a physiologically acceptable neutral group blocking the carboxyl group, with the exception of des-B30 human insulin Leu**B30 -NH2 and des-B30 human insulin Gly**B30 -NH2 and des-B30 human insulin Phe**B30 -NH2 and a pharmaceutically acceptable carrier. 1. Claims for the Contracting State : AT A process for the preparation of a medicament having a delayed action for the treatment of diabetes mellitus, which comprises bringing into a suitable dosage form at least one insulin derivative of the formula I see diagramm : EP0137361,P17,F1 in which R**1 denotes H or H-Phe R**30 represents the radical of a neutral, genetically codable L-aminoacid whose OH group, where present, can be free or protected by a physiologically acceptable group, and R**31 denotes a physiologically acceptable neutral group blocking the carboxyl group, with the exception of des-B30 human insulin Leu**B30 -NH2 and des-B30 human insulin Gly**B30 -NH, and des-B30 human insulin Phe**B30 -NH2 Phe**B30 -NH2 and a pharmaceutically acceptable carrier.

Description

In the treatment of diabetes mellitus, preparations of the glucose-lowering hormone insulin are usually administered parenterally today. The special nature of insulin and its metabolism means that the duration of action of a simple solution is only very short, so for sustained blood sugar control in diabetics, either a permanent infusion must be applied with dosing devices several times a day infusions or delayed effective insulin preparation. As delay principles, such states of insulin which are poorly soluble at the injection site (e.g. crystalline or amorphous) are therefore of particular importance. Examples of this include zinc-insulin crystals or protamine-insulin crystals which, during their slow re-dissolution, release insulin over a certain period of time.

Now, in therapy, it has proven to be very helpful to have different insulin preparations available which, in their action characteristics, come as close as possible to the individual patient's needs. In the context of non-optimal settings, immediate effects such as hyper-

or hypoglycaemia, especially the diabetic late complications which include retinopathy, neuropathy, nephropathy, micro-

and macroangiopathy.

The lack of insulin in diabetics means that the body can no longer achieve its natural and hormonal equilibrium.

The task of the invention is to provide an insulin derivative or a corresponding pharmaceutical agent with which one can better approach the natural hormonal balance in a diabetic state and by which this can be better maintained than by administering insulin in the hitherto usual forms.

This task is solved according to the invention by means of insulin derivatives, whose C-terminal amino acid in position B30 is esterified respectively with a pharmaceutical agent which is characterized by containing this insulin derivative which

active substance.

Some esterified human insulins in position B30 and methods for their preparation have already been discussed as intermediates in the semisynthesis of human insulin. thus, human insulin ThrB^^>-OBut is known, for example, from US-PS 4,320,196 and 4,320,197. In GB-A 2,069,502, human insulin Thr B3 0 -OMe, human insulin Thr B3 0 -OEt, human insulin Thr<B30->O-(2,4,6-trimethylbenzyl) and human insulin ThrB (But)-OBu<t> were mentioned. . Human insulin (B30)amide is known from EP-A 45,187.

The invention relates to the insulin derivative of formula I

in which

a) R1 means H,

R means the residue of a neutral, genetically codeable L-amino acid, if any OH group present can be free or protected with a physiologically tolerable group and

R means a physiologically tolerable carboxy group blocker

N

neutral group S,

b) R 1 means H-Phe,

bl)R~^ means the remainder of a neutral, genetically codeable L-amino acid with the exception of Thr, as any OH group present may be free or protected with a physiologically tolerable group and

31

R has the meaning specified under a),

b2) R<30>means Thr,

B3 0

b2.1) as the OH group of Thr is unprotected

and J.R31 means a physiologically tolerable neutral group -NRaR

or -0R°, in which

R and R are the same or different and mean (C1-C6)-alkyl, (C3-C6)-cycloalkyl, (C6-C1Q)-aryl, (C7-C11)-aralkyl, (C3-C8)-heteroaryl or - (CH2-CH2-0-)mR with m = 1 to approx. 120 and R = (C^-C^)-alkyl, since Ra can also be hydrogen and since these groups in the aryl part can also be protected with one or more identical or different substituents from the series halogen, nitro, (C^-C^ )-alkoxy, methylenedioxy and (C^-C^ )-alkyl or Ra and R together mean -(CH2)n- with n = 4-6, in which a methylene group can also be replaced by 0 or NH and

R<c>means propyl, isopropyl, butyl, isobutyl, sec-butyl, (C5-C8)-alkyl, (C3-C8)-cycloalkyl, (C6-C10)-aryl, (Cy-C8)-aralkyl, (C3 -C 8 )-heteroaryl or (CH 2 -CH 2 -O-)mR with m = 1 to approx. 120 and R = (C^-C^)-alkyl, as these groups in the aryl part can also be substituted with one or two identical or different substituents from the range of halogen, nitro, (C^-C^)-alkoxy, methylenedioxy and ( (C 1 -C 4 )-alkyl,

B3 0

b2.2) as Thr is present as tert-butyl ether and R<3>means a physiologically tolerable neutral group -NRaR

c ci b

or -OR , in which R and R have the meaning specified under section b2.1) and R can also mean hydrogen and R<c>means (C-^-C^ )-alkyl, butyl, isobutyl, sec-butyl, (C5-C6)-alkyl, (C3-C8)-cycloalkyl, (C8-C10)-aryl, (C7-C11)-aralkyl, (C3-C8)-heteroaryl or

(CH2-CH2-0-)mR, in which m and R have the above-mentioned meaning, as these groups in the aryl part can be substituted with one or more substituents from the series halogen, nitro, (C-L-C4 )-Alkoxy, methylenedioxy and (C^ -C 1 )-alkyl,

b2.3) as the OH group of Thr B3 0 can be substituted with a physiologically tolerable group from the series (C^-C^)-alkyl, butyl, isobutyl, sec-butyl, (C5~Cg)-alkyl, (C3 -C 8 )-cycloalkyl, (C 8 -C 10 )-aryl, (C 8 -C 8 )-aralkyl, (C 8 -C 8 )-heteroaryl, formyl, (C 8 -C 8 )-alkanoyl and (C 7 -C 11 )- aroyl, which in the aryl part can be substituted as indicated under section b2.1) for Ra and R<b>or another common physiologically tolerable residue in peptide chemistry is protected and

R<3>^ has the meaning specified under point a) or

B3 0

b2.4) as the OH group of Thr is protected by means of acetyl or benzyl and

R 31 means a physiologically tolerable, neutral group -NR ci Rb or -0R<C>, in which Ra and R<b>, except for R<b>= (C?-C^g)-aryl, it has under point b2.1) given meaning and in which R<c>has the meaning given under point b2.2), as well as their physiologically tolerable salts.

Genetically codeable are the following L-amino acids:

Gly, Ala, Ser, Thr, Val, Leu, Ile, Asp, Asn, Glu, Gin, Cys, Met, Arg, Lys, His, Tyr, Phe, Trp, Pro (neutral amino acids underlined).

The neutral groups S N, which block the free carboxy function at the C-terminal end of the B chain in the compound according to the invention, are understood as physiologically tolerable uncharged groups, above all ester and amide groups or other COOH protecting groups, as for example described in Bodanszky et al., "Peptide Synthesis", 2nd Edition (1976)

John Wiley & Sons, preferably groups of the formula -NRaR or -OR, in which R and R are the same or different and mean hydrogen, (C-^-Cg )-alkyl, (C^-Cg)-cycloalkyl, (Cg-C1Q )-aryl, (C -C 11 )-aralkyl, (C 1 -C 8 )-heteroaryl or

-(CH2-CH2-0)mR with m = 1 to approx. 120 and R = (C^-C^)-alkyl, as these groups in the alkyl part can also be substituted with one or more identical or different substituents from the series halogen, nitro, (C-^-C^ )-alkyloxy, methylenedioxy

and (C1~C4)-alkyl or R å and R b together mean -(CH2)n- with n = 4-6, in which a methylene group can also be replaced by 0 or NH and R<C>means (C^-Cg )-alkyl, (C3-Cg)-cycloalkyl, (Cg-C10)-aryl, (C- j-cn )-aralkyl, (C3~Cg )-hetero-

aryl or the above-mentioned residue (CH2CH2-0-)mR, the aryl residue can be substituted as in Ra and R°.

As OH-protecting group for Ser, Thr resp. Tyr, we are talking about physiologically tolerable groups from the series (C-^-Cg)-alkyl, (C3-Cg)-cycloalkyl, (Cg-C10)-aryl, (Cg-C^ )-aralkyl, (C3-C9 ) -heteroaryl, (C-^-Cg )-alkanoyl and (C^-C^)-aroyl or is protected by another physiologically tolerable residue common in peptide chemistry (cf. e.g. Bodanszky et al., cited above).

In the above connection and also in the following, (C-^-Cg)-alkyl means, for example, methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, amyl or hexyl,

with (C3~Cg)-cycloalkyl, for example cyclopropyl, cyclo-butyl, cyclohexyl, etc.,

with (C, -C, n)-aryl, for example phenyl or naphthyl, preferably 11)

show phenyl,

with (C^-C-^)-aralkyl, for example benzyl, phenethyl, etc.,

with (C3~Cg)-heteroaryl, for example pyridyl, pyrrolidyl, pyrimidinyl, morpholinyl, pyrazinyl, imidazolyl, indolyl or quinolinyl,

with (C-j^-Cg)-alkanoyl, for example formyl, acetyl, propionyl, butyryl, etc. and

with (C-y-C^)-aroyl, for example benzoyl, naphthoyl or toluyl.

Insulin derivatives which have phenylalanine in position B1 are particularly preferred. Furthermore, those that have Ala, Thr or Ser in position B30 are preferred.

The A-chain and the chain (B2-30) of the compound according to the invention conveniently have the sequence of bovine or porcine insulin, especially, however, human insulin.

Also preferred are insulin derivatives in which R<31> means alkoxy or aralkyl resp. -O(CH2CH2-O)mR.

Preferred OH-protecting groups for Ser, Thr or Tyr are (C 1 -C 8 )-alkyl, (C 1 -C 8 )-alkanoyl or (C 1 -C 8 )-aralkyl.

In the range of insulin derivatives according to the invention, the following compounds should be mentioned, for example, without the invention being limited to these: Bl t

Des-Phe -porcine insulin-(B30)-OBu

Bl t

Des-Phe -human insulin-(B30)-OBu

Bl

Des-Phe -porcine insulin-(B30)-OCH.,

Bl

Des-Phe -human insulin-(B30)-OBZL

Porcine insulin-(B3 0)-O-cyclohexyl

Human insulin-(B30)-

Bovine insulin-(B3 0)~0CH3

Pig insulin<->(B3 0)-0-(CH2-CH2-0-)2u<C>2<H>5 Human insulin- (B30 ) -NH-C-H,-

B30 B30

Des-Thr -human insulin-Val -O-C-H-B30 B30

Des-Thr -human insulin-Val -NH_

Human insulin

Human insulin-(B3 0)-NH-(CH2-CH2-0)3Q-CH3 Human insulin-(B3 0)-N(CH3)

Human insulin-(B30)-OBZL

Human insulin

Human insulin-Thr (Et)OEt

Human insulin-Thr<B3>°(CH3)0CH3

Human insulin-(B30)-O-(CH2)5CH3

Human insulin-Thr(Ac)O-Et

Human insulin-Thr(Bz)0Bu<t>

Human insulin-(B3 0)■

Human insulin-(B3 0)-

B3 0

Human insulin-Thr (FOR)-OPr

Human insulin-(B3 0)-NH2

The invention also relates to a method for producing insulin derivatives with formula I, the method being characterized by

a) a Des-octapeptide (B23-30) insulin of formula II,

in which R"'" means Phe or a bond and S means a proton-solvolytic or B-elimination cleavable amino protecting group such as tert-butyloxycarbonyl-(Boe), tert-amyloxycarbonyl-(Aoc) or methylsulfonylethyloxycarbonyl-(Msc) -rest,

is condensed with a peptide of formula III

in which R31"1 or R<3>^ has the above meaning, means hydrogen, Bzl or Bu~ and S3 means a urethane protecting group such as Boe, Moe, Fmoc or Z and the protecting groups present are removed in a manner known per se or b) a Des-B30 insulin of formula I, wherein R means H or H-Phe and the C-terminus R^-R3"*" together means OH, is reacted in the presence of trypsin or a trypsin-like endopeptidase with a compound of formula IV

wherein R3^ and R<3>"'" have the above meaning and

then any protective group present is cleaved off in a manner known per se and

the compounds obtained according to point a) or b) are optionally transferred to their physiologically tolerable salts.

In method variant a) one converts, for example, N<0>^,

ctBl

N -Bis-Boc derivative of a Des-octapeptide (B23-30)-insulin analogous to the method described in US-PS 4,029,642 directly with an equivalent of a compound of formula III, dicyclohexylcarbodiimide serving as condensation agent in small excess in the presence of 1-hydroxybenzotriazole.

Since in this process variant no protection of the carboxyl group is usually necessary, the damage to the insulin derivative is normally eliminated both during the esterification and also during the alkaline saponification. Due to the different molecular size and charge number, unreacted Des-octapeptide and a peptide resulting from the condensation of IV with Asp -OH are easily separable by partition chromatography on "Sephadex"-LH20 or by gel chromatography on "Sephadex"-G75 or G50 superfine.

To remove the tert-butyl protecting group, the reaction product only has to be treated with trifluoroacetic acid for 30

60 minutes at room temperature. This reaction does not damage the insulin derivative. If the methylsulfonylethyloxycarbonyl residue is selected as the N-protecting group, an alkali treatment is necessary for cleavage by 6-elimination. The reaction conditions are thus (e.g. 0.IN NaOH, 0°C, 5 sec.), that the insulin derivative is not damaged. The starting product NaAl, NaB1-Bis-Boc-Des-B23_30-octapeptide insulin from pigs is prepared, for example, in the following way: Pig insulin is reacted in a mixture of dimethylformamide, dimethylsulfoxide and water in the presence of N-ethylmorpholine with excess tert-butyloxycarbonyl N-hydroxysuccinimide ester. Thereby the expected NaA1, NaBl, N<eB29->Tris-Boc insulin occurs.

To the dissolution of this compound in dimethylformamide

and tris buffer (pH 7.5), trypsin is added in small portions until the starting product is no longer visible in electrophoresis. N01^,N^^-Bis-Boc-Des-B^^Q-octapeptide-insulin is purified by partition chromatography on "Sephadex" LH20.

This compound is now brought into reaction with one mole of the peptide of formula III, which is prepared in a manner known per se according to the methods from peptide chemistry, 1-2 moles of 1-hydroxybenzotriazole and approx. 0.9 mol of dicyclohexylcarbodiimide in dimethylformamide at approx. pH 7-8 (cf. Chem. Ber. 103 (1970), page 788).

The crude product is purified by partition chromatography and freed from the protecting groups by treatment with trifluoroacetic acid/anisole at room temperature. After precipitation with ether, isoelectric precipitation from water and chromatography on "Sephadex" G75 or G50 superfine, the compound is electrophoretically pure and can be brought to crystallization in a known manner. The insulin derivative produced in this way is biologically fully active.

The des-Phe B1 insulin as starting compounds for the method according to the invention is known, for example, from DE-PS 20 05 658 or from EP-A 46,979.

The Des-B30 insulins used as starting compounds in process variant b) are known, for example, from EP-A 46,979 or Hoppe-Seyler's Z. Physiol. Chem. 359 (1978) 799. The starting material of formula IV used in variant b) is prepared in a manner known per se according to methods from peptide chemistry.

The des-B30 insulin and the compound of formula IV are condensed with each other analogously to the method described in US-PS 4,320,196 in the presence of trypsin or a trypsin-like endopeptidase in an organic-aqueous solvent system at pH 5-9 and a temperature from 20- 40°C. The insulin derivative formed can be isolated according to the usual methods from peptide chemistry.

Furthermore, the compounds according to the invention can be synthesized analogously to the semi-synthetic methods for producing the known insulin described, for example, in BG-A 2,069,502, EP-A 56,951, WO 82/4069, DE-A 31.29.404 or WO 83/1074 -(B30)-ester.

31

Ammo acid esters of formula IV, in which R means an alkyl-polyoxyethyleneoxy chain, are prepared analogously to the method described in EP-A 27,161. Other compounds of formula IV are known or can be prepared analogously to the known methods.

All the aforementioned insulin derivatives with formula I have one thing in common

that by blocking the (B30) carboxy group, the molecule effectively gains an additional positive charge, which gives it an isoelectric point shifted in the direction of the neutral point.

Depending on the derivative, isoelectric points are measured between 5.8

and 7.3 in the isoelectric focusing. Thus, the derivatives are less soluble in the neutral range than native insulin or proinsulin, which have their isoelectric point and thus the area of maximum insolubility at pH = 5.4, while in the neutral range they are normally dissolved.

The solubility properties of insulin and proinsulin can be affected by the addition of zinc ions in the area above the isoelectric point, i.e. in the therapeutically particularly interesting neutral area. Zinc thereby acts as a depot principle so that it stabilizes the hexameric state of the insulin as well as its crystallization tendency.

In subcutaneous tissue, these aggregates dissolve again.

A further common depot principle is crystallization of insulin or proinsulin as a complex with a basic protein, e.g. globin or protamine.

When using proinsulins in solution or in connection with one of the described depot principles, a further proteolytic breakdown is required to release native, fully active insulin. Intact proinsulin has only approx. 1/8 of the biological effect of the insulin, because thus the representation, part of the biologically active surface region, the receptor-binding region is masked with the C-peptide present in the proinsulin. As proinsulin, it is true that for the diabetes therapy only homologues are mentioned, that is

only those with human sequence (cf. e.g. DE-A1

32 32 036). Heterologous proinsulin has a significant immunogenicity. In this connection, it is noteworthy that human proinsulin can also have variations in the C-peptide part.

Surprisingly, it was now found that the insulin derivatives of formula I whose B-chain is blocked C-terminally in contrast to proinsulin in equimolar amounts have approximately the same high biological activity as native insulin.

The invention now further relates to the use of the insulin derivative of formula I, wherein

R31"* means the residue of a neutral, genetically codeable L-amino acid, if any OH group present can be free or protected with a physiologically tolerable

group and

R3"*" means a physiologically tolerable carboxy group blocker

neutral group SN,

as medicine.

The invention also relates to drugs for the treatment of Diabetes mellitus of a pharmaceutically acceptable carrier and an insulin derivative with formula I as active substance.

These drugs are completely new delay principles that can be brought into effect without depot aids such as zinc or protamine sulfate. The depot effect goes back to an inherent, protein-chemically conditioned, physical principle, namely the insulin derivative's heavy solubility at its isoelectric point. Its re-dissolution under physiological conditions is possibly achieved by cleavage of the additional group(s), e.g. by means of enzymes with esterase activity. The group(s) each time split off are either pure physiological metabolites or also easily metabolizable, physiologically tolerable substances.

The insulin derivatives as active substances of these new drugs also differ from the intermediates mentioned in the literature, which also contain parts of the heterologous C-peptide, usually no more immunogenic than the corresponding insulin itself.

The agent according to the invention contains as active substance one or more of the new insulin derivatives with formula I.

They preferably have a pH value between 2.5 and 8.5, contain a suitable isotonic agent, a suitable preservative and optionally a suitable buffer for a pH range between 5.0

and 8.5.

A typical form of application for the mentioned derivatives are preparations which exist below the isoelectric point as solutions in a physiologically harmless carrier. Thereby, the pH of the solution can typically amount to pH 4.5, which is clearly higher than that of acidic old insulin (typical pH value = 3.0). A more neutral injection solution meanwhile offers clear advantages with regard to their tolerability.

Another typical form of application is suspensions of amorphous

and crystalline precipitates of the mentioned derivatives in a physiologically harmless carrier of approx. neutral pH.

However, it is also possible to enhance the inherent heavy solubility in the derivatives in the physiological pH range by additional depot principles such as e.g. by adding zinc or protamine sulphate. The added amount of zinc can thereby amount to up to 100 |ig Zn 2 +/100 insulin units, typically approx.

50 |ig Zn 2 4-/100 insulin units. The amount of protamine can be between 0.28 mg and 0.6 mg per 100 units (referred to protamine sulphate). In this way, particularly long-acting preparations can be prepared, for which in the future a wider application is given than in the past, since precisely a basal amount of insulin appeared to be therapeutically advantageous. This is already a realization from the therapy with insulin dosing devices.

A sterile aqueous solution, which is made isotonic to blood in the usual way, is suitable as a carrier medium that is physiologically harmless and compatible with the insulin derivative, e.g. in the case of glycerol, table salt, glucose, and also contains a common preservative e.g. phenol, m-cresol or p-hydroxybenzoic acid ester. The carrier medium can also contain a buffer substance, e.g. sodium acetate, sodium citrate, sodium phosphate. Diluted acids (typically HC1) or lye (typically NaOH).

In the agents according to the invention, the insulin derivatives can also be used as alkali or ammonium salts. A desirable amount of one or more insulin derivatives of formula I

or an insulin derivative of formula I can be present in a mixture of further of these insulin derivatives independently of each other, resp. in dissolved, amorphous and/or crystalline form.

It is often advantageous to add a suitable amount of a suitable stabilizer to the preparations which prevents the precipitation of protein during thermal-mechanical stress in contact with different materials. Such stabilizers are known, for example, from EP-A 18,609, DE-A 32 40 177 or from WO-83/00288.

With the agent according to the invention which can also contain one of the known delay principles such as e.g. protamine sulfate, globin or zinc in suitable amounts, such a delay principle can be used in combination with the overall active substance part or parts thereof or one or more insulin derivatives with formula I in a mixture. An agent may contain different insulin derivatives of formula I in combination with several different delay-acting excipients.

The drugs can next to the insulin derivatives with formula

In also contain native insulin and/or proinsulin and/or des-Phe-insulin independently of each other or in dissolved, amorphous and/or crystalline form.

With the therapeutic agents, it is thus clearly achievable diverse and very fine-tunable action characteristics, thus, according to the remarks at the beginning, progress should be connected, especially with regard to late diabetic complications.

The invention will be explained in more detail with the help of some examples.

Manufacturing example 1

Human insulin-(B30)-0-CH2-CH2~CH3

5 g of porcine insulin is dissolved in 45 ml of dimethylformamide, 25 ml of dimethyl sulphoxide, 0.5 ml of N-ethylmorpholine and 2.5 ml of water. While stirring, 1.5 g of tert-butyloxycarbonyl-N-hydroxysuccinimide is added at room temperature and allowed to react for 6 hours. It is then stopped by adding a drop of glacial acetic acid, the product is combined with ether and filtered off. The residue is dissolved in 360 ml of dimethylformamide and diluted

with 320 ml of Tris buffer (0.05M, 0.01M of CaCl2, pH 7.5).

At 36°C, portions of 20 mg of trypsin are added at intervals of 1 hour.

After a total of 12 additions, the pH is adjusted to 4.5 with acetic acid and the solution is evaporated. The subsequent purification of the material on a "Sephadex" LH20 column (8 x 200 cm) by means of partition chromatography in the system n-butanol-glacial acetic acid-water (2:1:10) yields 3.25 g of N<aA1>, N <aB1->Bis-Boc-Des-B23_3Q-octapeptide-insulin (porcine) which the acid and basic electrophoresis no longer shows the starting material. Amino acid analysis of the substance is correct. After a tentative removal of the Boc groups, there is no longer any insulin activity to be found. This material (3.25 g) is dissolved in 30 ml of dimethylformamide together with 100 mg of 1-hydroxybenzotriazole, 750 mg of HC1•Gly-Phe-Phe-Tyr(Bu<fc>)-Thr-Pro-Lys(Boe)-Thr( Bu<fc>)-OCH 2 CH 2 -CH 3 and 0.5 ml of N-ethylmorpholine. 120 mg of dicyclohexylcarbodiimide are then added at room temperature and the reaction is stirred for 24 hours. The separated dicyclohexylurea is filtered off, the product is combined with the addition of ether.

The precipitate is filtered off, washed with ether and dried. The substance is purified by partition chromatography on "Sephadex" LH20 in the above-mentioned system. 2.6 g of material from the main peak is isolated by precipitation with acetone/ether. The dried, still unprotected derivative is allowed to react with a mixture of 5 ml of trifluoroacetic acid and 1 ml of anisole for 60 minutes at room temperature. The raw material is then precipitated from the ice-cooled solution by adding ether. The dried precipitate is dissolved in water, precipitated with aqueous ammonia and centrifuged. The product's purification takes place in 10% acetic acid over "Sephadex" G50 superfine or G75. Human insulin-(B30)-OCH2CH2CH3 can be isolated from the fractions of the desired peak by freeze-drying (yield after crystallization 1.2 g). The insulin derivative produced in this way shows a human insulin-equivalent effect in a biological test.

The production of octapeptides with formula III takes place according to the following condensation core according to usual peptide condensation methods:

Synthesis core for the octapeptide of formula III

Amino acid and elemental analysis corresponding to the theoretical.

Drug

Example 1

Porcine insulin-(B30)-OCH^ (semi-synthetic from Des-B30 porcine insulin) in weakly acidic, dissolved formulation with 40 I.U. per

ml and its depot effect:

Dissolve in a total volume of 10 ml of water:

The pH value is set by adding IN HC1 or 1 N NaOH to pH = 4.5.

Such a solution shows, at a dosage of 0.4 I.E./kg in rabbits, a distinct depot effect. The area under the blood sugar curve is the same as for a standard preparation with 40 IU/ml.

Example 2

Human insulin-ThrB3^( Bu~)OBUt (Ph=6.8) prepared by semi-synthesis from porcine insulin with a neutral formulation of 40 I.U./ml and its depot action:

Dissolve in a total volume of 10 ml of water:

The pH value is set by adding IN HC1 or 1 N NaOH to pH = 7.3.

Such a suspension shows, at a dosage of 0.4 I.E./kg in rabbits, a distinct depot effect.

Example 3

Human insulin-(B30)-NH2 prepared from porcine insulin by semi-synthesis in the form of a crystalline NPH preparation of 40 I.U./ml and its greatly delayed action:

Dissolve in a total volume of 10 ml with water:

The pH value is set by adding IN HC1 or 1 N NaOH to pH = 7.3.

Such a crystal suspension shows, at a dosage of 0.4 I.E./kg in rabbits, a strongly delayed effect.

Example 4

B31 B30 t Mixture of human insulin-Arg- -OH and human insulin-Thr Bu -

(OBu<t>) both produced by semi-synthesis from porcine insulin in the form of a zinc-containing suspension with 40 I.U./ml and the greatly delayed effect:

Dissolve in a total volume of 10 ml with water:

The pH value is set by adding lN HC1 or 1N NaOH at pH = 7.0.

Such a suspension shows a strong delayed effect at a dosage of 0.4 I.E./kg in rabbits.

Example 5

Human insulin- (B30 ) -O- ("CH-CH--0-) „„C.H, , prepared by semi-2 2 20 2 o

synthesis of porcine insulin in the form of a neutral preparation with 100 I.U./ml and their delayed action:

Dissolve in a total volume of 10 ml with water:

When adding IN HC1 resp. IN HaOH set pH = 7.0..

Such a suspension shows a delayed effect on rabbits.

Claims (20)

1. Insulin derivative of formula I in which a) R1 means H, R 3<^ means the residue of a neutral, genetically codeable L-amino acid, if any OH group present can be free or protected with a physiologically tolerable group and R 31 means a physiologically tolerable carboxy group blocker N neutral group S, b) R<1> means H-Phe, bl)R<31> " <1> means the residue of a neutral, genetically codeable L-amino acid with the exception of Thr, since any OH group present can be free or protected with a physiologically tolerable group and R<3> ^ has the meaning specified under point a), b2) R <30> means Thr, B 3 0 b2.1) as the OH group of Thr is unprotected and R<31> means a physiologically tolerable neutral group -NR <a> R <b> or -OR c, wherein R ci and R b are the same or different and mean (C1 -C6 )-alkyl, (C3-C8 )-cycloalkyl, (C8-C10 )-aryl, (C7 -C11 )-aralkyl, (C3-C9 )-heteroaryl or -(CH2 -CH2 -0-)mR with m = 1 to approx. 120 and R = (C^-C4 )-alkyl, since Ra can also be hydrogen and since these groups in the aryl part can also be substituted with one or more identical or different substituents from the series halogen, nitro, (C-^-C^ )-alkoxy, methylenedioxy and (C^ -C^)-alkyl or Ra and R <b> together mean -(CH2) - with n = 4-6, in which a methylene group can also be replaced by = or NH and R <c > means propyl, isopropyl, butyl, isobutyl, sec-butyl, (C5 -C8)-alkyl, (C3 -C8)-cycloalkyl, (C8-C8)-aryl, (C?-C11)-aralkyl, (C3 -C9 )-heteroaryl or (CH2 -CH2 -O-)mR with m = 1 to approx. 120 and R = (C^-C^)-alkyl, as these groups in the aryl part can also be substituted with one or two identical or different substituents from the range of halogen, nitro, (C^-C4 )-alkoxy, methylenedioxy and (C ^ -C 4 )-alkyl, B 3 0 b2.2) as Thr is present as tert-butyl ether and R3"*" means a physiologically tolerable neutral group -NR <a> R <b> or -0R <C> , in which Ra and R <b> have the meaning specified under section b2.1) and R<b> can also mean hydrogen and R <c> means (C^-C^ )-alkyl, butyl , isobutyl, sec-butyl, (C5 -Cg)-alkyl, (C3 -Cg)-cycloalkyl, (Cg-C10 )-aryl, (C7~ C11 )-aralkyl, (C3~ Cg)-heteroaryl or (CH2- CH2-0-) R, in which m and R have the above-mentioned meaning, as these groups in the aryl part can be substituted with one or more ssubstituents from the series halogen, nitro, (C1-C4 )-alkoxy, methylenedioxy and (C-^ -C ^)-alkyl, B3 0 b2.3) as the OH group of Thr can be protected with a physiologically tolerable group from the series (C^ -C3 )-alkyl, butyl, isobutyl, sec-butyl, (C5 -Cg)-alkyl, (C3 -Cg)-cycloalkyl, (Cg-C10 )-aryl, (Cg-C ^ )-aralkyl, (C3 -Cg)-heteroaryl, formyl, (C3 -Cg)-alkanoyl and ^ c- j~ cn^~ aroyl, which in the aryl part may be substituted as stated under b2.1) for Ra and R <b> or another common physiologically tolerable residue in peptide chemistry and R3"*" has the meaning specified under point a) or B3 0 b2.4) as the OH group of Thr is protected with acetyl or benzyl and R3"*" means a physiologically tolerable, neutral group -NRaR or -0R <C> , in which Ra and R <b> , apart from Rb = (C7~ C^Q )-aryl, has the meaning given under point b2.1) and in which R is has the meaning under point b2 .2) stated meaning, as well as their physiologically tolerable salts. 2. Insulin derivative according to claim 1, characterized in that R3"*" means alkyloxy or aralkyloxy, which is stated in claim 1. 3. Insulin derivative of formula I, wherein R<1> means H-Phe, R<3> ^ means the residue of a neutral, genetically codeable L-amino acid, if any OH group present can be free or protected with a physiologically tolerable group and R3"*" means a physiologically tolerable, carboxy group blocker neutral group S and their physiologically tolerable salts for use as medicine. 4. Insulin derivative according to one of claims 1 to 2, characterized in that R means H. 5 . Insulin derivative according to one of claims 1 to 4, characterized in that R 3 ^ means Ala, Thr or Ser. 6. Insulin derivative according to one of claims 1 to 5, characterized in that the A-chain and the chain (B2-29) have the sequence of human insulin. 7 . Insulin derivative according to one of claims 1 or 3 to 6, characterized in that R<31> means -0-(CH2 -CH2 -0-)mR, where m and R have the meaning stated in claim 1. 8. Insulin derivative according to one of the claims 3 to 6, characterized in that R 31 means (C 1 -C 8 )-alkoxy or (C 1 -C 3 )-araloxy. 9 . Insulin derivative according to one of claims 1 to 8, characterized in that an optionally present OH group in R3 ^ )-aralkyl. 10 . Method for producing an insulin derivative of formula I according to claim 1 or 3, characterized by ata) a Des-octapeptide (B23-30) insulin of formula II in which R"*" means Phe or a bond and S means a proton-solvolytic or by 6-elimination cleavable amino protecting group such as tert-butyloxycarbonyl-(Boe), tert-amyloxycarbonyl-(Aoc) or methylsulfonylethyloxycarbonyl-(M sc ) residue is condensed with a peptide of formula III 2 2 3 30 31 H-Gly-Phe-Phe-Tyr(S )-Thr(S )-Pro-Lys(S )-R -R (III) in which R^® or R3 has the meaning specified in claims 1 and 3, 2 t 3 S means hydrogen, Bzl or Bu and S means a urethane protecting group, such as Boe, Moe, Fmoc or Z and protecting groups present are removed in a manner known per se orb) a Des-B30 insulin of formula I, in which R"* " means H or H-Phe or the C-terminus R3^-R together means OH, is reacted in the presence of trypsin or a trypsin-like endopeptidase with a compound of formula IV in which R <3> ^ and R3"*" have the meaning stated in claim 1 or 3 and then any protective group present is cleaved off in a manner known per se, and the compounds formed according to point a) or b) are optionally transferred in their physiologically tolerable salts. 11. Medicine consisting of a pharmaceutical harmless carrier and an active substance according to one of claims 1 to 9. 12 . Agent according to claim 11, characterized in that it has a pH value between 2.5 and 8.5, contains a suitable isotonic agent and a suitable preservative and that the insulin derivative of formula I is present dissolved and/or in suspension. 13 . Agent according to one of claims 11 or 12, characterized in that it contains a suitable buffer and the pH value is between 5.0 and 8.5. 14 . Agent according to one of claims 11 to 13, characterized in that it contains between 0 and 100 µg zinc/100 I.U. 15. Agent according to one of claims 11 to 14, characterized in that the active substance of formula I is in the form of an alkali salt or ammonium salt. 16. Agent according to one of claims 11 to 15, characterized in that the desired amount of one or more insulin derivatives of formula I or an insulin derivative of formula I is present in a mixture of further of these insulin derivatives independently of each other or in dissolved, amorphous and/or crystalline form. 17. Agent according to one of claims 11 to 16, characterized in that it contains a suitable amount of an aid with delaying effect. 18. Agent according to claim 17, characterized in that this delay principle is used in combination with the overall active substance part or with parts thereof or with several insulin derivatives of formula I in a mixture. 19. Agent according to one of claims 11 to 18, characterized in that it contains different insulin derivatives of formula I in combination with several different delaying auxiliary substances. 20 . Method for producing an agent according to et of claims 11 to 19, characterized in that the active substances are brought in a suitable administration form.