Protected derivative of octreotide

 

(57) Abstract:

Described derivative of octreotide General formula (1), where (D)represents A residue of D-(Nindole-formyl)tryptophan; R1is a hydrogen atom or a tert-butoxycarbonyl group or a group-CO-OH1where X1can take values: 2-alkylsulfonates, 2-phenylsulfonyl, 2-(4-substituted aryl)sulfonylated; R2= -CO-OX2where X2can take values: 2-alkylsulfonates, 2-phenylsulfonyl, 2-(4-substituted aryl)sulfonylated; R3and R4at the same time take the values-CH2-NH-CO-Y, where Y is methyl, alkyl WITH1-C5, phenyl, substituted phenyl, or together form a disulfide bond; R5represents a hydrogen atom or a group-CO-OH3where X3can take values: 2-alkylsulfonates, 2-phenylsulfonyl, 2-(4-substituted aryl)sulfonylated. New protected derivative of octreotide are the parent compounds for a more effective and simple receiving octreotide with improved output. table 2.

The invention relates to new original compounds used for receiving octreotide, namely to secure the peptides of General FD hydrogen or tert-butoxycarbonyl group, or a group-CO-OH1where X1can take values: 2-alkylsulfonates, 2-phenylsulfonyl, 2-(4-substituted aryl)sulfonylated;

R2=-CO-OX2where X2can take values: 2-alkylsulfonates, 2-phenylsulfonyl, 2-(4-substituted aryl)sulfonylated;

R3and R4at the same time take the values-CH2-NH-CO-Y, where Y is methyl, alkyl (C1-C5, phenyl, substituted phenyl, or together form a disulfide bond;

R5represents a hydrogen atom or a group-CO-OH3where X3can take values: 2-alkylsulfonates, 2-phenylsulfonyl, 2-(4-substituted aryl)sulfonylated.

Octreotide I is a synthetic analogue of somatostatin, which has a similar profile of pharmacological activity, but far exceeds the natural peptide in strength and duration of action. Like it inhibits somatostatin secretion of peptide hormones gastropancreatic endocrine system (insulin, glucagon, gastrin and other), and growth hormone.

Octreotide is used as a drug for the treatment of acromegaly, tumors gastropancreatic endocrine system, and the cataloguing of octreotide, radiolabelled, for example, indium, rhenium and technetium, are used for the localization of tumors by computer scintigraphy.

Octreotide is a cyclic oktapeptid the following structure:

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The peculiarities of its structure are:

- the presence of two D-amino acids;

- the presence of the disulfide loop;

- restored the C-terminal residue of threonine (threonine);

- high content of hydrophobic aromatic amino acids.

Significant from the point of view of chemical synthesis is the presence of unstable to the action of oxidizing agents and strong acids tryptophan residue. Synthesis of octreotide may be implemented as tverdofaznym method and classical methods of peptide synthesis in solution.

U.S. patent 4395403 describes a method of synthesis of octreotide in the solution. By this method from the protected dipeptide segments synthesized protected octreotide, with the remaining tert-butoxycarbonyl-lysine and cysteine residues blocked p-methoxybenzylidene groups, which is then subjected to a full release by the action of triptoreline boron and thioanisole in triperoxonane acid and oxidation oxygen is in organic solvents (>1 l/g) to allocate the released peptide; in addition, there is a risk of partial degradation of the tryptophan residue under the action of strong acids on the stage of the final release.

The main problems of the synthesis of octreotide solid-phase method associated with the presence of a molecule in its C-terminal residue threonine. Threonine't carboxyl group, which makes it impossible to use traditional methods of joining the first (C-terminal) amino acids to the polymer matrix. In the work of W. B. Edwards, et al. (J. Med. Chem. 1994, 37, 3749) the synthesis was carried out starting with the penultimate residue Cys(Acm) attached to the polymer ester bond. After Assembly of the peptide was oxidized to the disulfide on the polymer, then received protected [D-Trp(Boc)4, Lys(Boc)5, hr(VIt)6]-octreotide by aminolysis peptidyl-polymer excess threonine. Aminals was very slow, and the overall yield of the protected peptide was 14%.

More effectivnes was way Y. Arano, et al. (Bioconjugate Chem. 1997, 8, 442), which was synthesized octreotide, starting with residue Fmoc-Thr(But)-ol, attached to 2-chloro-triticina the polymer. However, the synthesis necessary for this purpose protected derivative threonine represents a different challenge.

In paragraph who eat the formation of a cyclic acetal with polymer-bound terephthalic aldehyde. Such cyclic acetal provides simultaneous protection of both hydroxyl groups threonine and easily cleaved by acid reagents in terms of the removal of the protective groups of the tert-Putilkovo type.

It should be noted that the known solid-phase methods of obtaining of octreotide developed mainly for the subsequent synthesis of conjugates with chelates, Biotin and other markername molecules, implemented at the microscale (0.1 to 0.25 mmol) and practically not suitable for the synthesis of octreotide in gram and more quantities, as they imply the use of inaccessible and expensive raw materials - protected amino acids, specialty polymers and condensing reagents for solid-phase synthesis.

Thus, there is a need for efficient and scalable ways of receiving octreotide.

The aim of the invention is to create new protected peptides that could be used as starting compounds for efficient and scalable synthesis of octreotide.

This goal is achieved by the fact that these are new compounds, namely, protected peptides of the General formula:

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where (D)A is carbonilla group, or a group-CO-OH1where X1can take values: 2-alkylsulfonates, 2-phenylsulfonyl, 2-(4-substituted aryl)sulfonylated;

R2=-CO-OH2where X2can take values: 2-alkylsulfonates, 2-phenylsulfonyl, 2-(4-substituted aryl)sulfonylated;

R3and R4at the same time take the values-CH2-NH-CO-Y, where Y is methyl, alkyl (C1-C5, phenyl, substituted phenyl, or together form a disulfide bond;

R5represents a hydrogen atom or a group-CO-OH3where X3can take values: 2-alkylsulfonates, 2-phenylsulfonyl, 2-(4-substituted aryl)sulfonylated.

The subject of the present invention are thus derivative of octreotide II and III, containing the Nindole-formirovanii the residue is D-tryptophan, protected-amino group in the lysine residue, S-secured (II) or oxidized to the cyclic disulfide (III) residues of cysteine

H-D-Phe-Cys(R3)-Phe-D-Trp(For)-Lys(R2)-Thr-Cys(R4)-Thr-ol (II)

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where R2, R3, R4take the above values.

The subject invention are also derived IV-IX, which may contain DOPOLNITEL groups C-terminal residue threonine:

R1-D-Phe-Cys(R3)-Phe-D-Trp(For)-Lys(R2)-Thr-Cys(R4)-Thr-ol; (IV)

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H-D-Phe-Cys(R3)-Phe-D-Trp(For)-Lys(R2)-Thr-Cys(R4)-Thr-ol(R5)2; (VI)

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R1-D-Phe-Cys(R3)-Phe-D-Trp(For)-Lys(R2)-Thr-Cys(R4)-Thr-ol(R5)2; (VIII)

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where R1N, and the group R2-R5take the above values.

Protective group, R1(for the case when R1=-CO-OX1), R2, R5are removed by reason group 2-alkyl - or 2-arylsulfonamides type: urethane (urethane) for N-terminal-amino group and residue Lys (R1= -CO-OX1, R2=-CO-OX2) and carbonate to hydroxyl groups of the residue threonine (R5= -CO-OH3). It is obvious that 2-alkyl(aryl)sulfonylation deputies1-X3can be used in various combinations, can be the same or different, but in practice it is preferable to use a uniform protective group (X1= X2= X3), for example, as described in the literature (table 1).

Protective group, R3and R4for cysteine residues are selected from among acylaminoacyl (S,N-acetaline) groups capable of dissociation under the action of okiki the receiving disulfide. Such protective groups, for example acetamidomethyl (AFM), benzamidomethyl (Bzm), tert-butylacetamide (Tacm), as well as methods for their introduction and removal are described in the literature. Group R3and R4may be different, but it is preferable to use the same protective group (R3=R4).

Protected peptides II-IX are new, not previously described substances.

For the synthesis of peptides II-IX can be used techniques and methods peptide synthesis described in the literature.

Peptide IV can be synthesized, for example, by the method of stepwise extension of the peptide chain from the C-end to N-with N-protected amino acids; start-end balance in this case can be threonines with unprotected hydroxyine groups. As a temporary N-protection you can apply, remove soft acidolysis, for example tert-butoxycarbonyl, tert-aryloxyalkyl, 4-methoxybenzylideneamino or other known protective group. To activate the carboxyl groups of amino acid residues introduced into the peptide chain, you can use a variety of methods described in the literature, for example, a method activated inalsa group R1N. Alternative amino acid sequence of oktapeptid can be divided into segments of varying lengths, each of which is synthesized separately, and then these segments going full peptide chain. The release and oxidation of cysteine residues in the peptide IV, for example, by the action of iodine or other reagents mentioned above, leads to the peptide V.

If the peptides IV and V R1represents a urethane group, a removable acidolysis, for example tert-butoxycarbonyl, its removal by the action of acid gives the peptides II and III, respectively. Alternative peptide III can be obtained by the release and oxidation of cysteine residues in peptide II as described above.

When using as the starting C-terminal residue threonine containing hydroxyl groups blocked carbonate protective groups R5using methods similar to those described above for peptides II-V, can be derived peptides VI, VII, VIII, IX.

Protected peptides III, V, VII, IX (R1=-CO-OH1can be used directly to obtain octreotide. For this purpose in these peptides need to remove existing protective group, R1oC, the mixture is neutralized, for example, by adding an excess of acetic acid, and then separated from the resulting solution octreotide by known methods, for example, ion exchange or/and obremenitve chromatography.

The essence of the invention is illustrated by examples. In the description of examples, the following abbreviations and symbols:

DMF - dimethylformamide

DCGC - dicyclohexylcarbodiimide

MBT - 1-hydroxybenzotriazole

TFU - triperoxonane acid

HPLC - high performance liquid chromatography

AST - atsetamidometil

Bzm - benzamidomethyl

Vos - tert-butoxycarbonyl

Thr-ol - residue L-threonine [(2S,3R)-1,3-dihydroxy-2-aminobutane]

Abbreviations of amino acids and the protective groups used in accordance with the recommendations of the Commission on biochemical nomenclature at IUPAC-IUB, published in Eur. J. Biochem., 1984, v. 138, No. 1, pp. 9-37. Abbreviations 2-alkyl(aryl)sulfonylating protective groups are shown in table 1. Opti is the situation.

The values of the chromatographic mobility of Rfrefer to plates for thin-layer chromatography Aluflien Kieselgel 60 F254(Merck, Germany) in the system chloroform-methanol-acetic acid, 95:5:3 (A) or 90:10:3 (B); ethyl acetate-pyridine-acetic acid-water, 60:5:15:10 (). Detection of the spots on the plates was performed in UV-light and ninhydrin reagent after warming up. The masses of the molecular ions (M+H)+measured by time-of-flight mass spectrometer MSBH-1 (NGOs "Electron", Ukraine) or mass spectrometer MALDI-TOF VISION 2000 (Thermo Bioanalysis, England). The analysis of amino acids was performed on the analyzer Biotronik LC5001 after acid hydrolysis of the samples of peptide material in sealed ampoules (3 M methansulfonate, 1% phenol, 24-hour reception, 110oC).

Example 1. Boc-D-Phe-Cys(Bzm)-Phe-D-Trp(For)-Lys(Psc)-Thr-Cys(Bzm)-Thr-ol (peptide IVa).

a. Boc-Cys(Bzm)-Thr-ol. To a solution of 4.4 g of triptoreline threonine and 10.5 g of pentafluorophenyl ester of Boc-Cys(Bzm) in 100 ml DMF added to 3.75 ml of N, N-diisopropylethylamine and stirred mixture of 4 h at room temperature. The mixture is evaporated in vacuo to an oil, the residue is dissolved in 200 ml ethyl acetate, washed with 280 ml Polynesians aqueous NaCl solution and evaporated to dryness. The residue is treated with ether and obtain 7.2 g of the target is between 50 ml and cooled in an ice bath, TFUK, after 20 min at 0oWith evaporated to oil, perevarivat with 250 ml of toluene. The resulting triptorelin H-Cys(Bzm)-Thr-ol are dissolved in 100 ml of DMF, add 4 ml of N,N-diisopropylethylamine, then under stirring 6.6 g of pentafluorophenyl ester of BOC-threonine. The mixture is stirred for 2 h at room temperature and evaporated in vacuo to an oil. The residue is dissolved in 200 ml ethyl acetate, washed with 280 ml Polynesians aqueous NaCl solution and evaporated to dryness. After treatment with ether to obtain 7.9 g of the target compound; Rf0,35 (B); m/z=542,6, M+N+(calculated 543,7).

C. Boc-Lys(Psc)-Thr-Cys(Bzm)-Thr-ol. With 7.5 g of Boc-Thr-Cys(Bzm)-Thr-ol remove the BOC-protection, as described in example 1B, the received triptorelin dissolved in 100 ml of DMF, add 4 ml of N,N-diisopropylethylamine and 0.9 g of MBT, then under stirring 9.6 g of 2,4,5-trichlorphenol ester Boc-Lys(Psc). The mixture is stirred for 5 h at room temperature and evaporated in vacuo to an oil. The residue is treated with ether, the precipitate filtered off, washed with ether and obtain 10.6 g of the target compound; Rf0,45 (B); m/z=883,6, M+N+(calculated 884,1).

, Boc-D-Trp(For)-Lys(Psc)-Thr-Cys(Bzm)-Thr-ol. With 10.5 g of Boc-Lys(Psc)-Thr-Cys(Bzm)-Thr-ol remove the BOC-protection, as described in example 1B, the received triptorelin dissolved in 100 ml of DMF, add 4 is peremeshivayte 10 h at room temperature and evaporated in vacuo to an oil. To the residue is added ethyl acetate, precipitated precipitate is filtered off, washed with ether and obtain 11.9 g of the target Pentapeptide; Rf0,45-50 (B); m/z=1082,6, M+H+(calculated 1083,3).

D. Boc-D-Phe-Cys(Bzm)-Phe-OH. To a solution of 3.3 g of phenylalanine and 3.5 ml of triethylamine in 20 ml of water and 50 ml of DMF with vigorous stirring, in portions a solution of 7.9 g of pentafluorophenyl ester of Boc-Cys(Bzm) in 30 ml DMF and stirred mixture of 4 h at room temperature. The mixture is evaporated in vacuo to an oil, the residue is added 200 ml of ethyl acetate and 100 ml of 1 M aqueous KHSO4. The organic layer was washed with 280 ml of saturated aqueous NaCl solution and evaporated to dryness. The residue is treated with petroleum ether and receive Boc-Cys(Bzm)-Phe-OH, Rf0,35 (A). The resulting dipeptide was dissolved in 50 ml TFUK, after 10 minutes the solution is evaporated in vacuum and the residue is treated with ether. The precipitate was separated, dissolved in 30 ml of water and 50 ml of DMF, added with stirring, 2.5 ml of triethylamine and 4.9 g of p-nitrophenylthio ester Boc-D-Phe. The mixture is stirred 18 h at room temperature, then evaporated in vacuo to an oil, the residue is added 200 ml of ethyl acetate and 100 ml of 1 M aqueous KHSO4. The organic layer is washed with water, saturated aqueous NaCl and evaporated to dryness. The remainder of obrabatyvajushhee 649,8).

that is, Boc-D-Phe-Cys(Bzm)-Phe-D-Trp(For)-Lys(Psc)-Thr-Cys(Bzm)-Thr-ol (peptide IIA). 2.17 g of Pentapeptide Boc-D-Trp(For)-Lys(Psc)-Thr-Cys(Bzm)-Thr-ol (example 1G) was dissolved in 15 ml of cold TFUK, after 20 minutes the solution is evaporated in vacuum and the residue is treated with ether. The precipitate was separated, dissolved in 20 ml of DMF, add 0.4 ml of N,N-diisopropylethylamine, 0.4 g of MBT, 1.47 g of Boc-D-Phe-Cys(Bzm)-Phe-OH (example 1D), followed by cooling and stirring 0.51 g DCGK. The mixture is stirred for 3 h at 0oC and 18 h at room temperature, filtered, the filtrate evaporated in vacuo to an oil. The residue is treated with 100 ml of ethyl acetate, precipitated precipitate is washed with ethyl acetate, ether and obtain 3.0 g of the peptide IVa; Rf0,25-30 (B), 0.65 (); m/z=1615,1, M+N+(calculated 1614,0).

Example 2. Boc-D-Phe-Cys(Acm)-Phe-D-Trp(For)-Lys(Nsc)-Thr-Cys(Acm)-Thr-ol (peptide IV).

Peptide IV get similar to that described in Example 1; Rf0,15-20 (B) Of 0.55 (In); m/z=1535,4, M+N+(calculated 1534,9).

Example 3. Nsc-D-Phe-Cys(Bzm)-Phe-D-Trp(For)-Lys(Msc)-Thr-Cys(Bzm)-Thr-ol (peptide IVC).

Peptide IVC get similar to that described in Example 1; Rf0,10-20 (B) Of 0.60 (C); m/z=1561,4, M+N+(calculated 1561,8).

Example 4.

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To a solution of 1.62 g of peptide IVa (Example 1) in 700 ml of acetic acid and 200 ml of water with stirring, poured a solution of 1.5 g of iodine in 80 ml of the UKS and stirred until the discoloration. Zinc sludge is filtered off, the filtrate is evaporated under reduced pressure to a volume of 15-20 ml, to the residue was added 100 ml of water. The precipitation was separated, washed with water and dried in air. The output of the peptide Va 1.20 g; Rf0,40 (C); m/z= 1345,4, M+H+(calculated 1345,7).

Example 5. H-D-Phe-Cys(Bzm)-Phe-D-Trp(For)-Lys(Psc)-Thr-Cys(Bzm)-Thr-ol (peptide IIA).

0,81 g peptide IVa (Example 1) dissolved in 10 ml chilled to 0oWith TFUK, after 20 minutes the solution is evaporated and the residue is treated with ether. Get 0,80 g trifenatate peptide IIA. Rf0,35 (C); m/z=1513,4, M+H+(calculated 1513,9).

Example 6.

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Triptorelin peptide IIIa is obtained from Va peptide similar to that described in Example 5; Rf0,25 (C); m/z=1245,6, M+N+(calculated 1245,3).

Example 7.

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To a solution of 1.54 g of peptide IV (Example 2) in 600 ml of acetic acid and 300 ml of water with stirring, poured a solution of 1.5 g of iodine in 80 ml of acetic acid and 20 ml of water and leave the mixture for 10 h at room temperature. To the mixture add a solution of 2.5 g of ascorbic acid in 50 ml of water and stirred until the discoloration, then evaporated under reduced pressure to a volume of 25-30 ml, to the residue, add 150 ml of water. The precipitation was separated, washed with water and dried in the BR>
Triptorelin IIIB peptide derived from the peptide V similar to that described in Example 5; Rf0,20 (C); m/z=1290,3, M+N+(calculated 1290,6).

Example 9.

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Peptide VC is obtained from peptide IVC as described in example 7.f0,30 (C); m/z=1292,9, M+N+(calculated 1293,5).

Example 10. Boc-D-Phe-Cys(Bzm)-Phe-D-Trp(For)-Lys(Psc)-Thr-Cys(Bzm)-Thr-ol(Psc)2(peptide VIIIa).

a. Boc-Thr-ol(Psc)2. To a solution of 2.1 g of the BOC-threonine in 20 ml of dichloromethane and 3 ml of pyridine while cooling in an ice bath and stirring, 5.9 g of 2-vinylsulfonylacetamido ether harpalinae acid (Psc-Cl), the mixture is stirred 2 h at 0oC and 15 hours at room temperature. To the mixture add 120 ml of ethyl acetate and 100 ml of 2 M KHSO4, the organic layer is separated, washed with 2100 ml of 2 M KHSO4, 50 ml saturated aqueous NaCl solution and evaporated to dryness. The remainder chromatographic on a column of Kieselgel 60 (Merck, Germany), using as eluent ethyl acetate and obtain 4.4 g of the target dicarbonate in the form of oil; Rf0,70 (A).

B. Boc-Cys(Bzm)-Thr-ol(Psc)2. With 4.4 g of Boc-Thr-ol(Psc)2remove the BOC-protection, as described in example 1B, and the resulting triptorelin dissolved in 20 ml of DMF. To the solution was added 2.65 g of Boc-Cys(Bzm)-OH, of 1.75 ml of N,N-diisopropylethylamine, 0.95 g of MBT,the temperature value, then filtered and the filtrate evaporated in vacuo to an oil. The residue is dissolved in 100 ml ethyl acetate, washed with water, saturated aqueous Panso3a 2 m solution of KHSO4, saturated aqueous NaCl and evaporated to dryness. The residue is treated with petroleum ether and obtain 5.6 g of target compound; Rf0,65 (A); m/z=865,7, M+N+(calculated 865,0).

C. Boc-Thr-Cys(Bzm)-Thr-ol(Psc)2. With 5.6 g of Boc-Cys(Bzm)-Thr-ol(Psc)2remove the BOC-protection, as described in example 1B, and the resulting triptorelin dissolved in 25 ml of DMF. To the solution was added 1.55 g of Boc-Thr-OH, 1.5 ml of N, N-diisopropylethylamine, 0,80 g MBT, followed by cooling to 0oWith, 1.55 g DCGK. The mixture is stirred 1 h at 0oC and 5 h at room temperature, then filtered and the filtrate evaporated in vacuo to an oil. The residue is dissolved in 100 ml ethyl acetate, washed with water, saturated aqueous NaHCO3a 2 m solution of KHSO4, saturated aqueous NaCl and evaporated to dryness. The residue is treated with ether and receive 5.3g target Tripeptide; Rf0,45 (A); after removal of the BOC-group m/z=866,4, M+N+(calculated 865,9).

, Boc-Lys(Psc)-Thr-Cys(Bzm)-Thr-ol(Psc)2. With 5.3 g of Boc-Thr-Cys(Bzm)-Thr-ol(Psc)2remove the BOC-protection, as described in example 1B, treat the ether is then under stirring of 3.9 g of 2,4,5-trichlorphenol ester Boc-Lys(Psc).The mixture is stirred for 5 h at room temperature and evaporated in vacuo to an oil. The residue is treated with ether, the precipitate filtered off, washed with ether and receive 6,53 g tetrapeptide; Rf0,35-0,40 (A) 0,60 (B); after removal of the BOC-group m/z=1206,6, M+H+(calculated 1206,4).

D. Boc-D-Trp(For)-Lys(Psc)-Thr-Cys(Bzm)-Thr-ol(Psc)2. With 6.50 g of Boc-Lys(Psc)-Thr-Cys(Bzm)-Thr-ol(Psc)2remove the BOC-protection, as described in example 1B, treated with ether, the precipitate of triptoreline dissolved in 30 ml of DMF, add 1.3 ml of N, N-diisopropylethylamine and 0.70 g of MBT, then with stirring, 2.7 g of 2,4,5-trichlorphenol ester Boc-D-Trp(For). The mixture was stirred for 12 h at room temperature and evaporated in vacuo to an oil. To the residue is added ether, precipitated precipitate is filtered off, washed with ether and obtain 7.6 g of the target Pentapeptide; Rf0,45-55 (B); after removal of the BOC-group m/z=1407,6, M+N+(calculated 1407,8).

that is, Boc-D-Phe-Cys(Bzm)-Phe-D-Trp(For)-Lys(Psc)-Thr-Cys(Bzm)-Thr-ol(Psc)2(peptide VIIIa). 3.1 g of Pentapeptide Boc-D-Trp(For)-Lys(Psc)-Thr-Cys(Bzm)-Thr-ol(Psc)2dissolved in 15 ml of cold TFUK, after 20 minutes the solution is evaporated in vacuum and the residue is treated with ether. The precipitate was separated, dissolved in 20 ml of DMF, add 0.4 ml of N,N-diisopropylethylamine, 0.4 g of MBT, 1.47 g of Boc-D-Phe-Cys(Bzm)-Phe-OH (example 1D), followed by cooling and stirring 0.51 g DCGK. The mixture is stirred for 3 h at 0of0,35-45 (B) Of 0.75 (In); after removal of the BOC-group m/z=1940,0, M+H+(calculated 1938,5).

Example 11. The Psc-D-Phe-Cys(Bzm)-Phe-D-Trp(For)-Lys(Psc)-Thr-Cys(Bzm)-Thr-ol(Psc)2(peptide VIII).

Peptide VIII get similar to that described in Example 10; Rf0,35-40 (B) Of 0.75 (In); m/z=2167,9, M+H+(calculated 2168,7).

Example 12.

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Peptide IXa obtained from peptide VIIIa as described in example 4. Rf0,55 (C); m/z =1768,6, M+H+(calculated 1768,1).

Example 13.

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Peptide HB obtained from peptide VIII as described in example 4. Rf0,55 (C); m/z=1900,0, M+N+(calculated 1898,2).

Example 14. H-D-Phe-Cys(Bzm)-Phe-D-Trp(For)-Lys(Psc)-Thr-Cys(Bzm)-Thr-ol(Psc)2(peptide VIa).

Triptorelin peptide VIa derived from peptide VIIIa similar to that described in Example 5; Rf0,35 (C); m/z=1940,3, M+N+(calculated 1938,5).

Example 15

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Triptorelin VIIa peptide derived from the peptide Ha similar to that described in Example 5; Rf0,25 (C); m/z=1668,3, M+H+(calculated 1668,0).

Example 16. Receiving octreotide (I).

145 mg (100 μmol) of triptoreline IIIB peptide dissolved in 10 ml of a mixture of DMF-water (1: 2). To the solution at strong paramasivam add 0.5 ml of acetic acid. The mixture is diluted with water to 50 ml and applied to a column 25100 mm cellulose CM-52 (Whatman, England), balanced 0.03 M ammonium acetate (pH of 5.9). Spend the elution gradient from 0.05 to 0.5 M ammonium acetate (pH 5,9), the fractions containing the desired product are pooled and lyophilized. Obtain 59 mg of octreotide acetate; chromatographic purity by HPLC 94%, the mass content of peptide material (octreotide diacetate) 82% (exit 42 µmol). Mass spectrum: (M+N)+1019,8 (calculated: 1020,31); amino acid composition: Thr 0,94 (1); Phe 2,04 (2); Trp 0,92 (1); Lys 1,00 (1); Cys is not defined.

Similarly receive octreotide releasing other above protected peptides (table 2).

The peptides of General formula

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where (D)represents A residue of D-(Nindole-formyl)tryptophan;

R1is a hydrogen atom or a tert-butoxycarbonyl group or a group-CO-OH1where X1can take values: 2-alkylsulfonates, 2-phenylsulfonyl, 2-(4-substituted aryl)sulfonylated;

R2= -CO-OH2where X2can take values: 2-alkylsulfonates, 2-phenylsulfonyl, 2-(4-substituted aryl)sulfonylated;

R3and R4at the same time take the values-CH2-NH-CO-Y, GD is SUP>5
represents a hydrogen atom or a group-CO-OH3where X3can take values: 2-alkylsulfonates, 2-phenylsulfonyl, 2-(4-substituted aryl)sulfonylated.

 

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The invention relates to medicine, in particular to the creation of analogues of hormonal drugs protein nature

The invention relates to therapeutic peptides

The invention relates to compounds of the formula X1-Leu-His-Lys(R1)-Leu-Gln-Thr-Tyr(R2)-Pro-Y, where X1- H-, A1O-CO-, H-Lys(R3)-Leu-Ser-Gln-Glu(B4)-, A1O-CO-Lys(R3)-Leu-Ser-Gln-Glu(B4)-; Y is-OH, -Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2;1there is tert-butyl; R1- protective group of the formula IN the1O-CO - Epsilon-amino group of the Lys residue, R2- protective group of the formula IN the2About-WITH - hydroxyl group of residue Taut, R3- protective group of the formula IN the3O-CO - Epsilon-amino group of the Lys residue IN4- protective group for the gamma-carboxyl group of Glu residue, and1IN2IN3and4may be the same or different and are selected from the range: 2-alkylsulfonates, 2-phenylsulfonyl, 2-(substituted aryl)sulfonylated

The invention relates to an improved process for the preparation of collagen from solubilizing and purified, may pasensyahan extract non-sterile native or telopeptide collagen, including: i) the stage of mixing and shearing of the extract in mixer with dual lateral incisors with the gradual increase of the initial rate of mixing at 500-1000 rpm without exceeding the speed of 10000 rpm and the gradual increase of temperature for 2-10oWith, preferably 3-5oWith, thus, to increase the initial ambient temperature of the extract to the maximum controlled temperature, component not exceeding 50oWith, and then (ii) the stage of sterilization in the liquid environment of the extract with obtaining sterile collagen in native or telopeptides, native or telopeptide collagen type I, obtained with the above method, with the following characteristics or properties: ratio2(I)1/1(I)2from 0.48 to 0.52; sterility in accordance with the standard of the European Pharmacopoeia; total nitrogen from 17,0 to 18.7%; hydrox

The invention relates to the field of biotechnology and biochemistry, and can be used in medicine

The invention relates to a method for cyclosporine And high purity by purification of the crude product containing cyclosporiasis complex by multi-step chromatography on silica gel at high load columns from 10 to 52%, using as eluent a mixture of toluene with acetone in an amount of from 10 to 30 vol.% or toluene with ethyl acetate in an amount of from 10 to 35 vol.%, cyclosporine And high purity with content cyclosporine L, U and D less than 0.05% and the content of cyclosporine and < 0,02% vol., industrial method of purification of cyclosporin a from a crude product containing complex cyclosporiasis

The invention relates to method modification, facilitating access to the treated (poly)peptides and to a method of cleaning using this method modifications
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