CZ171793A3 - Novel analogs of carboxythermal sequence of human insulin b-chain (b23-b30) - Google Patents

Abstract

Analogues of the carboxyterminal sequence of human insulin with an alternation of amino acids in positions B<24> and B<25> of the chain and methylated nitrogen of the peptide bond created by amino acids in the abovementioned positions of the general formula IGly - X<1> - X<2> Y23 24 25 26 - 30 (I)where X<1> is L-Phe or D-Phe,X<2> is L or D-(Me)PheHN orL or D-(Me)Phe, where (Me)Phe is N-methylphenylalanine,Y is H or L-Tyr-L-Thr-L-Pro-L-N(Pac)Lys-L-ThrOH where Pac is the phenylacetyl group.

Description

Variations in position B ²⁴ -B ^{23 of} the carboxyterminal sequence of insulin are shown to be among the most important as regards the size and nature of the biological response. Our previous results (PV 604-92 and PV 701-92) yielded new analogs of sequence B ²³ -B ^{y <J} with increased lipophilicity in the region B ~ ⁴ -B ~ * ^: ', increased stability in this region (stereoschange in B ²⁴ and ^B23 ), as well as the new amino-protecting group of the lysine at position ^B2SI .

The presented results show at. the possibility of eliminating the terminal pentapeptide of the B-chain of insulin provided the carboxyl of the phenylalanine in B ²³ is converted to the amide form. (WH, Fischer et al., Biol. Chem., Hoppe Seyler 366, 521, 1985; Casaretto et al., Biol. Chem. Hoppe Seyler 368, 709, 1987). ^{S * * * * * * * * * * * *}

With this in mind, we have prepared a series of tri- and octapeptides as precursors for enzyme semisyntheses in which amino acids at position B ³⁴ and B ^{3S are} exchanged for their stereoisomers and still substituted by hydrogen at the nitrogen of the peptide bond between amino acids B ²⁴ and B ³⁵ methyl. This substitution should significantly contribute to the metabolic stability in the region.

Object of the invention

1. The subject invention are analogs carboxyterminal sequence of human insulin with the amino acid variation at positions B ²⁴ and B ²³ chain and the methylated nitrogen of the peptide bond formed by the amino acids at the positions listed.

* A ¹ 2 t

Gly-X-X-Y;

24 25 25 - 30 where.

Substance X ¹ X ² '  Y AND L-Phe L- (Me) Phe NH * II L-Phe D - (Me) PheN: H H III D-Phe D- (Me) Phe NH H IV D-Phe L- (Me) Phe NH H in L-Phe L- (Me) Phe NH L-Tyr-L-Thr -N (Pac) -Lys-L-ThrOH VI D-Phe L- (Me) Phe as in VII L-Phe D- (Me) Phe as in VIII D-Phe D- (Me) Phe as in

2. The present invention relates to the above tripeptides which are prepared by condensing L- or D- (Me) PheQMe with Boc-Gly-L-Phe or Boc-Gly-D-Phe. Boc-tripeptides-QMe were converted to amides by dissolution in methanolic ammonia. After standing at room temperature for 48 hours, methanol was evaporated and the residue crystallized from ether-petroleum ether and the peptide was further purified by HPLC using a water-methanol gradient. The Boc-group was removed from the tripeptidamides. with trifluoroacetic acid.

3. The present invention provides synthetic octapeptide prepared by solid phase synthesis on esterified Merrifield resin (15fe DVB), based on a carrier treatment protocol: 3 x dimethylformamide x trifluoroacetic acid and condensation of the following amino acid in the form of a 1-hydroxybenzotriazole ester in presence of a base, 20% diisopropylethylamine solution.

4. SUMMARY OF THE INVENTION Synthetic octapeptides in which the amino acid at position B ^2S is condensed to the amino group of the peptide chain using 2- (1-H-benzotriazol-1-yl) -1,1,3,3-tetramethylammonium tetrafluoroborate in the presence of diisopropylamine .

The present invention provides synthetic octapeptides wherein the amino acid at position B ² * is condensed with the amino group of the peptide chain using Br-tris-pyrrolidinophosphonium hexafluorophosphate in dimethylformamide in a 20% solution of diisopropylethylamine in dimethylformamide.

t iř

6. The present invention relates to octapeptides which have been prepared by solid-phase synthesis using amino acids whose side functional groups have been protected as follows; hydroxyl tyrosine by O-Br-benzyloxycarboxylic acid, hydroxyl threonine by benzyl group and epsilon amine lysine by renyiacetyl group.

7. The present invention is concerned with the construction by the hydrogen fluoride synthesis method. presence of cleavage of protecting groups from

8. The subject of the invention are human insulin B-chains, high-performance chromatography, said octapeptides which are released in the solid phase by liquid anisole, which also leads to hydroxyl tyrosine and threonine.

analogues of the C-terminal sequence which is ultimately purified and using a methanol gradient.

Example 1.

Preparation of Boc-N-methyl-D-phenylalanine methyl ester

To a mixture of 50.4 ml of tetrahydrofuran and 5.6 ml of dimethylformamide was added 5.3 g of Boc-D-phenylalanine. 2.88 g of an oil suspension of sodium hydride were added with stirring and after 10 minutes 10 ml of methyl iodide. The entire reaction mixture was heated at 80 ° C for 24 hours. Hydrolysis was then carried out and the product was shaken out to a pH of 50. diethyl ether. Etheric anhydrous magnesium sulfate and ether were distilled off in vacuo. To mL of dioxane and 20 mL of 2M NaOH. The reaction solution was filtered off the residue, dried over dessicant, added to the room, then shaken until dried over anhydrous dessic. The mixture was stirred at acid temperature for 2 hours with citric acid and ethyl acetate product. The ethyl acetate product was magnesium sulfate and, after filtration, the solvent was distilled off. The residual oil was dissolved in ether and the product precipitated as a salt with dicyclohexylamine.

Boc-N-methyl-D-phenylalanine was further treated with diazomethane in ether. The reaction was stopped with the appearance of a yellow color. Excess diazomethane was removed by addition of acetic acid. After evaporation of the ether, the Boc-N-methyl-D-phenylalanine methyl ester was further purified by HPLC.

Ct

Preparation of Boc-N-methyl-L-phenylalanine-methyl stearate 8.0 g of Boc-D-phenylalanine were added with a mixture of 75.6 ml of tetrahydrofuran and 8.4 ml of dimethylformamide. While stirring, 4.32 g of an oil suspension of sodium hydride was added and after 10 minutes T5 ml of methyl iodide was added. The entire reaction mixture was heated to hydrolysis the solution was filtered off the residue and the mixture was at a

80 ° C for the product of dried desiccant added 30 stirred for 2 hours. The mixture was shaken into diethyl ether, anhydrous magnesium sulfate, and the ether was distilled off in vacuo. To mL of dioxane and 30 mL of 2M NaOH. The reaction hours at room temperature were shaken to dry with anhydrous desiccants, acidified with citric acid and ethyl acetate. The ethyl acetate product was magnesium sulfate and the solvent was distilled off after filtration. The residual oil was dissolved in ether and the product precipitated as a salt with dicyclohexylamine.

Boc-N-methyl-L-phenylalanine was further treated with diazomethane in ether, the reaction was stopped when a yellow color appeared. Excess diazomethane was removed by addition of acetic acid. After evaporation of the ether, the product Boc-N-methyl-D-phenylalanine methyl ester was further purified by HPLC.

Example 3

Preparation of Gly-L-Phe-L- (Me) PheNH 2

The peptide was prepared by condensing L- (Me) PheOMe with Boc-Gly-L-Phe. Condensation was carried out in methylene chloride using N, N - dicyclohexylcarbodiimide as a condensing agent. After Ν, Ν'-dicyclohexylurea was filtered off, the filtrate was shaken with a 20% citric acid solution followed by

After drying the methylene chloride solution in vacuo ether-petroleum ether. was dissolved in methanol at room temperature for 48 hours.

sodium bicarbonate solution with anhydrous magnesium sulfate was evaporated and the residue recrystallized from Boc-Gly-L-Phe-L- (Me) PheOMe ammonia and left at

The methanol was then evaporated and the residue crystallized from ether-petroleum ether. The purity of the protected tripeptidamide was checked by elemental analysis and HPLC. The Boc-group was removed from the tripeptidamides by treatment with trifluoroacetic acid for 30 minutes. The product in the form of the trifluoroacetate salt was precipitated with ether after evaporation of the free trifluoroacetic acid and isolated.

Example 4

Preparation of Gly-L-Phe-D- (Me) PheNH 2

The peptide was prepared by condensing D- (Me) PheOMe with Boc-Gly-L-Phe. The procedure was as in case 3.

Example 5

Preparation of Gly-D-Phe-D- (Me) PheNH2 peptide was prepared by condensation of D- (Me) PheOMe with

Boc-Gly-D-Phe. Proceed as in case 3.

Example 6

Preparation of Gly-D-Phe-L-fMejPheNHa

The peptide was prepared by condensing L- (Me) PheOMe with Boc-Gly-D-Phe. Proceed as in case 3.

Example 7

Preparation

Gly-L-Fhe-L-N (Me) -Phe-L-Tyr-L-Thr-L-Pro-L-N (Fac) -Lys-L-Thr

The peptide was synthesized on a semiautomatic synthesizer of constitutional production. The alpha amino group of the amino acids used was protected with a tert-butyloxycarbonyl group, the side chains were protected as follows: O-Br-benzyloxycarbonyl group for Tyr, benzyl group for Thr, and phenylacetyl group for Lys. The esterified Merrifield resin (1% DVB.) With a substitution of 0.77 mmol / g was used for the synthesis.

The synthetic process consisted of resin handling:

x washing with dimethylformamide with 2 x trifluoroacetic acid condensation with another amino acid.

Diisopropylethylamide in DMF was added in situ to the activated amino acid in an amount of preparation of 1 mmol of 1-hydroxybenzotriazole esters peptides. amino acids in position 2a 3. attached with 0.81 g hexafluorophosphate (PyBroP) in the following step requirement used prepared in dichloromethane) 2.15 ml of 20¾ solution Except for condensation In position 2 the amino acid bromo-tris-pyrrolidino-phosphonium DMF and 3, 0 ml of a 20¾ solution of diisopropylethylamine in DMF. Condensation at position 3 was performed using 1.12 with 2- ((-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium borate (TBTU) and 1.5 mL base. Condensation was controlled by Kaiser test. Upon completion of the synthesis, the peptide was released from the resin by liquid HF in a special Teflon vessel using anisole as a free radical scavenger. The crude peptides were purified on a preparative column packed with RP HPLC Separon SGX C-18 in a methanol / water gradient with the addition of 0.1¾ TFA in both phases.

satisfying »(were in

The peptide yield was 1.32% of theory.

Example 8

Preparation _ η _ ό κ o _ τ -> tfm £> \ _ τ "m · ,. w. _ r «_ t _ π - - _ ^ τ _- w./ji ev. v r r <<υ Γ υ υ υ ur ur ur ii ii \ \ \ \ \ \; 14 / a

The corresponding amino acids and procedure of Example 7 were used to prepare the peptide.

The yield of the peptide was 2.33% of theory.

Example 9

Preparation

Gly-L-Phe-D-N-Me-Phe-L-Tyr-L-Thr-L-Pro-L-N-Pac-Lys-L-Thr

The corresponding amino acids and procedure of Example 7 were used to prepare the peptide.

The peptide yield was 3.29% of theory.

Example —10— .... Preparation

Gly-D-Phe-D-N (Me) -Phe-L-Tyr-L-Thr-L-Pro-L-N (Pac) -Lys-L-Thr

The corresponding amino acids and procedure of Example 7 were used to prepare the peptide.

The peptide yield was 4.3% of theory.

Claims (3)

Patent Claims. ^R £ 1. Analogs carboxyterminal sequence of human insulin variation | amino acids in position B ²⁴ and B ^{25 of the} chain and methylated nitrogen (peptide bonds consisting of amino acids in the above positions of formula I Gly - X ¹ - X ² - Y (I) wherein X ¹ is L-Phe or D-Phe X ² is L or D- (Me) PheNH or L or D- (Me) Phe Y is H or L-Tyr-L-Thr-L-Pro-L-N (Pac) -Lys-L-ThrOH A process for the preparation of compounds according to claim 1, wherein Y is H, characterized in that L- or D- (Me) PheOMe is condensed with Boc-Gly-L-Phe or Boc-Gly-D-Phe formed by Boc- The tripeptides-OMe are converted to amides, whereupon the Boc group is removed from the tripeptidamides, for example by treatment with trifluoroacetic acid. A process for the preparation of compounds according to claim 1, wherein Y is L-Tyr-L-Thr-L-Pro-LN (Pac) Lys-L-ThrOphi, characterized in that the individual amino acids of YB ²⁶ to B ^{30 are} condensed in in the form of a 1-hydroxybenzotriazole ester in the presence of a base on an esterified Merrifield resin, to the N-amino group of the prepared peptide is condensed with L or D- (Me) Phe using 2- (1-H-benzotriazol-1-yl) -1,1 3,3-tetramethylammonium tetrafluoroborate in the presence of diisopropylamine, further coupled by L or D-Phe condensation with the methylated amino group of the peptide chain using Br-tris-pyrrolidinophosphonium hexafluorophosphate in dimethylformamide, then terminating the chain formation with N-protected glycine; releasing the resulting octapeptide from the resin with liquid hydrogen fluoride in the presence of anisole.