Rgd-containing peptides

 

(57) Abstract:

The invention relates to a group of new protected linear peptides containing the amino acid sequence, which can be used as starting compounds to obtain RGD-containing cyclopeptides, the General formula

R3-Arg-Gly-Asp(OR1)-OR2,

where R1is benzyl or tert-butyl; R2not equal to R1and is selected from the group of tert-butyl; benzyl; 4-methoxybenzyl; 4-nitrobenzyl; diphenylmethyl; 2,2,2-trichloroethyl; 2,2,2-trichloro-1,1-dimethylethyl; allyl; 9-fluorenylmethyl; carboxamidine; substituted 2-sulfonylated type AND-SO2-CH2-CH2- where a is substituted or unsubstituted phenyl or benzyl; R3is a hydrogen atom or a urethane protective group of the B1O-CO-, where1not equal to R1and can take values tert-butyl, benzyl, 4-methoxybenzyl, 9-fluorenylmethyl, 2-(4-nitrophenyloctyl)ethyl; or is a peptidyl containing from one to three amino acid residues; and the peptides, where R3- peptidyl structure E-Z-Y-X-, in which E is a hydrogen atom or a urethane protective group IN2O-CO-, where2not equal to R1and can take values tert-, la, Val, Ile, Pro, Lys(G) or Orn(G), where G is a urethane protective group IN3O-CO-, in which3= R1attached to the omega-amino group; Z can take values Phe or D-Phe. 1 C.p. f-crystals.

The invention relates to the field of peptide chemistry, namely:

1. The peptides of General formula

R3-Arg-Gly-Asp(OR1)-OR2,

where R1there is benzyl or tert-butyl;

R2not equal to R1and is selected from the group of tert-butyl; benzyl; 4-methoxybenzyl; 4-nitrobenzyl; diphenylmethyl; 2,2,2-trichloroethyl; 2,2,2-trichloro-1,1-dimethylethyl; allyl; 9-fluorenylmethyl; carboxamidine; substituted 2-sulfonylated type A-SO2-CH2-CH2- where a represents a substituted or unsubstituted phenyl or benzyl; R3represents a hydrogen atom or a urethane protective group IN1O-CO-, where1not equal to R1and can take the values: tert-butyl, benzyl, 4-methoxybenzyl, 9-fluorenylmethyl, 2-(4-nitrophenyloctyl)ethyl; or is a peptidyl containing from one to three amino acid residues.

2. The peptides mentioned formula, where R3is a peptidyl structure E-Z-Y-X-, where

E is a hydrogen atom or urethane protection of the sludge; 4-methoxybenzyl; 9-fluorenylmethyl; 2-(4-nitrophenyloctyl)ethyl; X is absent or equal to Gly;

Y is Gly, Ala, Val, Ile, Pro, Lys(G) or Orn(G), where G is a urethane protective group IN3O-CO-, in which3=R1attached to the omega-amino group;

Z can take the values: Phe or D-Phe.

Amino acid sequence Arg-Gly-Asp or RGD in single-letter encoding is universal structural motive of the majority of intercellular matrix proteins, mediating the adhesion of different cell types in vivo. Found that RGD-motif is the main structural element of matrix proteins directly involved in binding to cellular receptors. Various RGD-binding cellular receptors have a significant structural similarity and belong to the superfamily of integrins and membrane proteins with subunit structure . Integrins, in turn, are divided into several groups (families) depending on the type included in their composition-subunit. Each integrin can bind one ligand (e.g., fibronectin receptor) or more different proteins, ligands (e.g., glycoprotein gpIIb/IIIa platelet), and the specificity of binding of ligands to the keep the same binding site RGD. This, apparently, is due to the high sensitivity of the receptors to the conformation of the binding site in the protein-ligand (E. Ruoslahti, Ann. Rev. Biochem., 1988, 57, 375; R. Hynes, Cell, 1992, 69, ll).

Synthetic peptides containing the RGD-motif, open the possibility of selective modulation of cell-matrix and cell-cell interactions and therefore are currently considered as a promising new class of drugs for research, diagnosis, treatment and prevention of diseases associated with such interactions, in particular cardiovascular diseases, thrombosis, inflammation and autoimmune disorders, certain types of tumors.

Directional changes in receptor specificity of RGD-containing peptides can be achieved by changing the preferred conformations or restrict the conformational mobility of the RGD site in the design of the peptide. An effective way to do this is the cyclization of the peptide, which may be implemented by various methods. Known methods of cyclization RGD-peptides through the formation of disulfide bonds between specially introduced in the peptide residues of cysteine or its analogues (for example, European patent EP 0275748; U.S. patent 5643872; patent what xerophagy the side chains of amino acids (for example, U.S. patent 521210). A number of five - and six-membered RGD-containing cyclopeptides obtained by cyclization of the main peptide chain "head to tail" - intramolecular condensation of an amino group with a carboxyl group of the linear precursor (U.S. patent 5849692, 5866540, 6127335), some of these peptides showed high selectivity towards certain types integranova receptors. In particular, the peptide cyclo(Arg-Gly-Asp-D-Phe-Val) (R. Haubner, et al. , J. Am. Chem. Soc., 1996, 118, 7461; U.S. patent 5866540) was active and highly selective antagonist integranova receptors V3/V5playing an important role in angiogenesis induced by tumors and is considered as a promising anticancer drug.

Obviously, there is an urgent need for efficient and versatile methods of chemical synthesis of RGD-containing cyclopeptides to make different peptides of the specified type for laboratory research and clinical trials.

In U.S. patent 5849692 described the synthesis of five - and six-membered rings of cyclopeptides by cyclization in solution of linear predecessors in the form of H-Arg(Mtr)-Gly-Asp-Aaa1-Aaa2-(Aaa3)-OH (AAA1, AAA21-Aaa2-(Aaa3)-OH, obtained obviously by synthesis in solution. The feasibility of these methods is questionable, because the original peptides, in addition to end-carboxyl groups, also contain free carboxyl group of the Asp residue, and attempted cyclization under the action of condensing agents must inevitably lead to a complex mixture of products.

In the mentioned work (R. Haubner, et al., J. Am. Chem. Soc. 1996, 118, 7461) five-membered cyclopeptide synthesized by cyclization in solution of the linear peptides of the form of H-Asp(OtBu)-Aaa1-Aaa2-Arg(Mtr)-Gly-OH, which was obtained by solid-phase synthesis of 2-chlorotitanium the polymer. In the future, this approach has been improved mainly due to the replacement of Mtr-Arg protection on more kislotolabilen group PMC or Pbf and optimization of reagents and conditions cyclization (X. Dai, et al., Tetrahedron Lett., 41, 6295; D. Boturyn, P. Dumy, 2001, Tetrahedron Lett., 42, 2787). This method is suitable for scale, but this scale involves the use of expensive raw materials - protected amino acids, specialty polymers and condensing reagents for solid-phase synthesis.

The aim of the invention is to create new linear peptides that could be used as starting compounds for the synthesis of RGD-containing cyclopeptides.

In accordance with the above purpose, the subject of the invention are new tripeptides General formula

R3-Arg-Gly-Asp(OR1)-OR2, (Ia,b)

which have C-terminal residue of aspartic acid in the form of diapir. The amino group of these peptides can be free (Ib; R3=N) or blocked urethane protective group IN1O-CO- (Ia; R3=B1O-CO-). Deputy R1represents a protective group for carboxyl of the Asp residue of the number usually used for this purpose in peptide synthesis - benzyl or tert-boutelou. The protective group R2for-carboxyla Asp different from R1and is selected so that it can be selectively removed without affecting the group R1.

For R1=benzyl, R2chosen from among the groups removed by acidolysis under mild conditions (tert-butyl; 4-methoxybenzyl; diphenylmethyl), chemical recovery (2,2,2-trichlorethyl; 2,2,2-trichloro-1,1-dimethylethyl; 4-nitrobenzyl) catalyzed base-elimination (9-funny or unsubstituted phenyl or benzyl), either under the action of complexes of Pd(0) (allyl).

If R1= tert-butyl, R2can be selected from among the groups that are removed by catalytic hydrogenolysis (benzyl, 4-methoxybenzyl; diphenylmethyl, 4-nitrobenzyl), chemical recovery (2,2,2-trichlorethyl; 2,2,2-trichloro-1,1-dimethylethyl; 4-nitrobenzyl) catalyzed base-elimination (9-fluorenylmethyl; substituted 2-sulfonylated type A-SO2-CH2-CH2- where a represents a substituted or unsubstituted phenyl or benzyl), alkaline hydrolysis under mild conditions (carboxamidates), or under the action of complexes of Pd(0) (allyl).

The group R3in peptides Ia is chosen in such a way that it can be removed or selectively, without affecting the groups R1and R2or simultaneously with the group R2. Depending on the combination of the groups R1and R2the protective group R3you can choose from a number of well-known urethane protective groups such as tert-butoxycarbonyl, benzyloxycarbonyl, 4-methoxybenzylideneamino, 9-fluorenylmethoxycarbonyl, 2-(4-nitrophenyloctyl)ethoxycarbonyl.

The tripeptides of formula Ia,b are universal source connections to receive the ohms all invariant amino acid sequence RGD; secondly, they can be built from N-Terminus by the addition of amino acid residues or peptide segments to the linear precursor of the desired structure; thirdly-amino and-carboxyl groups of the synthesized linear precursors can be selectively released for subsequent directed cyclization.

Another object of the present invention are linear peptides that can be obtained by increasing the above tripeptides of formula Ia, b with the N-terminal 1-3 amino acid residue and next used for obtaining physiologically active cyclic RGD-peptides by cyclization and subsequent release. This increase may be effected using methods and techniques known in the chemistry of peptides. The subject of the invention, in particular, are peptides of General formula

E-Z-Y-X-Arg-Gly-Asp(OR1)-OR2(II)

in which E is a hydrogen atom or a urethane protective group of the B2O-CO-, where2not equal to R1and can take the values: tert-butyl; benzyl; 4-methoxybenzyl; 9-fluorenylmethyl; 2-(4-nitrophenyloctyl)ethyl;

X is absent or equal to Gly;

Y is Gly, Ala, Val, Ile, Pro, Lys(G) or Orn(G), where G is ureterovaginal values: Phe or D-Phe.

The synthesis of the tripeptides of formula Ia can be realized, since, diapir aspartic acid of the formula H-Asp(OR1)-OR2(III) where R1and R2take the above values. Many diesters III are known compounds described in the literature, some diesters are new substances, but they can be obtained using known methods and techniques. In particular, the diesters III can be obtained by esterification-carboxyl group is benzyl or t-butyl ether N-protected aspartic acid, followed by removal of the N-protection. The etherification can be made by condensing the ether N-protected aspartic acid with the appropriate alcohol under the action of a condensing reagent, or by alkylation of salt-ester of N-protected aspartic acid corresponding alkylhalogenide or other alkylating derivative. Methods, reagents and conditions for carrying out these reactions are described in the literature (e.g., P. Kocienski, Protecting Groups, Corrected Edition, Thieme, Stuttgart-N.-Y., 2000). N-Protective group for the source-ester of aspartic acid is chosen in such a way that it can be removed selectively without affecting groups1and R2

The tripeptides Ia then synthesized by sequential attachment of the amino group received diapir III residues Gly and Arg in the form of N-protected derivatives. N-Protective group for this purpose are chosen as described above to obtain diesters III. Alternative tripeptides Ia was prepared by condensation of diesters III dipeptides of the formula R3-Arg-Gly-OH (IV) wherein R3can take the values shown above for tripeptides Ia. A dipeptide IV, in turn, can be obtained by condensation of N-protected derivatives of arginine R3-Arg-OH with esters of glycine, followed by cleavage of the ester group in the resulting ester of the dipeptide.

The tripeptides Ib with a free-amino group receive selective removal of N-protective group from compounds Ia.

Peptides II can be synthesized by sequential attachment of the amino group of Tripeptide Ib desired amino acid residues in the form of N-protected derivatives. N-Protective group for this purpose p is insatia tripeptides Ib pre-synthesized peptide segments of the form E-Z-Y-X-OH. Group E is selected so that it can be removed or selectively, without affecting the groups R1and R2or simultaneously with the group R2. Depending on the combination of the groups R1and R2the protective group can be selected from among known urethane protective groups such as tert-butoxycarbonyl, benzyloxycarbonyl, 4-methoxybenzylideneamino, 9-fluorenylmethoxycarbonyl, 2-(4-nitrophenyloctyl)ethoxycarbonyl.

If the lateral radicals attached amino acid residues or peptide segments contain functional groups that need blocking, such as amino group, carboxyl group, tirinya group, the protective group for their block is chosen so that these protective groups were stable in terms of removal of Nprotective groups in sequential (stepwise) the extension of the peptide chain, as well as in terms of the removal of groups E and R2. In practice, it is advisable that these side protective group can be removed after cyclization of the linear peptide simultaneously with the group R1. Side protection group, select from among known protective groups used in the synthesis of peptide is, amoxiline in the form of benzyl ester, tirinya - 4-methoxybenzylidene, diphenyl-methyl, or trailmeme protections; when R1=tert-butyl can be used, respectively, N-tert-butoxycarbonyl, tert-butylene and S-triteleia protective group. An adequate choice of the group R1and other side protective groups for specific combinations of groups E and R2or, on the contrary, the necessary combinations of groups E and R2for a specific value of the group R1and type of side protection can be carried out by a specialist in the field of peptide chemistry without further invention, based on the rules set forth in the present description, and the information that is known about the stability of the protective groups and methods for their removal.

By selective removal of groups E and R2of peptides II get a linear peptide with a free amino and a-carboxyl groups suitable for subsequent cyclization with the aim of obtaining cyclic RGD-peptides. The cyclization should be carried out in dilute solution at the initial concentration of the linear peptide from 0.1 to 10 mm, preferably from 0.4 to 2 mm. As a solvent used aprotic solvents capable of dissolving the original reagents and prochlorper and other solvents. The cyclization is carried out under the action of known condensing reagents such as dicyclohexylcarbodiimide, diphenylphosphinite, hexaflurophosphate benzotriazolyl-1-oxy-Tris(dimethylamino)phosphonium (THIEF-reagent), hexaphosphate or tetrafluoroborate benzotriazolyl-1-hydroxy-tetramethylurea, or other reagents. A variety of methods, conditions, and reagents for carrying out the cyclization of the peptides described in the literature.

Upon completion of the cyclization reaction of the reaction mixture to remove the solvent, for example, by distillation under reduced pressure, allocate protected cyclopeptide and expose it to release, then purify the target cyclic RGD-peptide by known methods, such as adsorption, reverse-phase or ion-exchange chromatography, or a combination of chromatographic methods. Alternative secure cyclopeptide can be purified chromatographically to stage release. Reagents and conditions for release are chosen depending on the nature of the group R1and other protective groups. In particular, if R1=benzyl, the release can be accomplished by catalytic hydrogenolysis or by the action of strong celtificate acid; solutions of hydrogen bromide in acetic or triperoxonane acid. If R1=tert-butyl, is used to release acidosis, for example, with trifluoroacetic acid or its solution in methylene chloride, solutions of hydrogen chloride in acetic acid, dioxane, tetrahydrofuran or ethyl acetate. The release can also be applied to other known reagents designed to remove the protective groups are benzyl or tert-Putilkovo type.

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

DIEA - N,N-diisopropylethylamine

DMF - dimethylformamide

DCGC - dicyclohexylcarbodiimide

MBT - 1-hydroxybenzotriazole

THF - tetrahydrofuran

TFU - triperoxonane acid

The tea - triethylamine

HPLC - high performance liquid chromatography

4-DMAP - 4-dimethylaminopyridine

The all-allyl

Vos - tert-butoxycarbonyl

Bzl is benzyl

Cam carboxamidates

Cbz - benzyloxycarbonyl

Cpse - 2-(4-chlorophenylsulfonyl)ethyl

Dpm - diphenylmethyl

Fm - 9-fluorenylmethyl

Fmoc - 9-fluorenylmethoxycarbonyl

Mbzl-l)ethyl

Nsc - 2-(4-nitrophenyloctyl)etoxycarbonyl

Pfp - pentafluorophenyl

Phse - 2-phenylsulfonyl

Pse - 2-(4-feniletinilpireny)ethyl

tBu is tert-butyl

Tce - 2,2,2-trichlorethyl

Tctbu - 2,2,2-trichloro-tert-butyl

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, 138, 1, pp. 9-37. Optically active amino acids and their derivatives, are given in the descriptions of the examples, by default, have the L-configuration.

The values of the chromatographic mobility of Rfrefer to plates for thin-layer chromatography Alufolien 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 on the 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 HR, 110oC).

oC and stirring, add 2.4 g DCGK. Continue stirring 3 h at cooling and a further 2 hours at room temperature. The precipitation is filtered off, washed with ethyl acetate, the filtrate is extracted sequentially with 5% aqueous solution Panso3, 0.5 m solution of KHSO4and saturated NaCl solution, dried over anhydrous sodium sulfate and evaporated under reduced pressure. The residue is dissolved in 30 ml of 2 n solution model HC1 in dioxane, incubated for 40 min at room temperature, then evaporated to dryness. The residue is treated with ether and obtain 3.65 g of the target diapir III-1 in the form of hydrochloride. Rf0.25 (B); m/z=391.8, M+N+(calculated 392.5).

Similarly the received hydrochloride diesters:

H-Asp(OBzl)-OPse, III-2; Rf0.30 (B); m/z=510.2, M+H+(calculated 510.6).

H-Asp(OBzl)-ONse, III-3; Rf0.15 (B); m/z=436.9 M+H+(calculated 437.5).

H-Asp(OBzl)-OTce, III-4; Rf0.25 (B); m/z=355.3 M+H+(calculated 355.6).

H-Asp(OBzl)-OTctbu, III-5; Rf0.28 (B); m/z= 383.5 M+H+(calculated 383.7).

H-Asp(OBzl)-OFm, III-6; Rf0.35 (B); m/z=401.8, M+H+(calculated 402.5).

B. -4-Nitrobenzyloxy A add 2.0 ml DIEA. The mixture was stirred for 12 h at room temperature, then add 100 ml of water and 80 ml of ethyl acetate. The organic phase is separated and extracted successively with water, 5% aqueous solution Panso3, 0.5 m solution of KHSO4and saturated NaCl solution, dried over anhydrous sodium sulfate and evaporated under reduced pressure. The residue is dissolved in 30 ml of 2 n Hcl solution in dioxane, incubated for 40 min at room temperature, then evaporated to dryness. The residue is treated with ether and obtain 3.32 g of the target diapir III-7 in the form of hydrochloride. Rf0.20 (B); m/z=359.1, M+N+(calculated 359.4).

century-4-Methoxybenzyloxy ether-benzenepropanoic acid (H-Asp(OBzl)-OMbzl, III-8). To a solution of 4.81 g Nsc-Asp(OBzl)-OH, 1.5 g of 4-methoxybenzylamine alcohol and 0.12 g of 4-DMAP in 30 ml of THF while cooling to 0oC and stirring, add 2.4 g DCGK. Continue stirring 3 h at cooling and another 4 h at room temperature. The precipitation is filtered off, washed with THF, the filtrate is evaporated. The residue is dissolved in 100 ml of ethyl acetate, extracted sequentially with 5% aqueous solution of NaHCO3, 0.5 m solution of KHSO4and saturated NaCl solution, dried over anhydrous sodium sulfate and evaporated under reduced pressure. The rest of rest the Ute dry. The remainder perevarivat with toluene and receive in the form of oil 5.1 g equimolecular mixture target diapir III-8 and N, N-diethyl-N-2-(4-nitrophenyloctyl)ethylamine, which is used without further separation. Rf0.30 (B); m/z=344.2 M+H+(calculated 344.4).

Similarly, the resulting diesters:

H-Asp(OBzl)-ODpm, III-9; Rf0.35 (B); m/z=390.2, M+N+(calculated 390.5).

H-Asp(OtBu)-OTce, III-10; Rf0.25 (B); m/z= 321.0 M+H+(calculated 321.6).

H-Asp(OtBu)-ODpm, III-11; Rf0.25 (B); m/z= 356.6, M+N+(calculated 356.5).

H-Asp(OtBu)-OMbzl, III-12; Rf0.25 (B); m/z= 310.1, M+H+(calculated 310.4).

g-4-Nitrobenzyloxy ether-tert-butyl-aspartic acid (H-Asp(OtBu)-ONb, III-13). To a solution of 4.46 g Nsc-Asp(OtBu)-OH and 2.20 g of 4-nitrobenzylamine in 30 ml of DMF, add 2.0 ml DIEA. The mixture was stirred for 12 h at room temperature, then add 100 ml of water and 80 ml of ethyl acetate. The organic phase is separated and extracted successively with water, 5% aqueous solution of NaHCO3, 0.5 m solution of KHSO4and saturated NaCl solution, dried over anhydrous sodium sulfate and evaporated under reduced pressure. The residue is dissolved in 30 ml of a mixture of THF and diethylamine (1:1 by volume) and incubated for 60 min at room temperature, then evaporated DOS is 3 and N,N-diethyl-N-2-(4-nitrophenyloctyl)ethylamine, which is used without further separation. Rf0.20 (B); m/z=325.2, M+N+(calculated 325.4).

Similarly, the resulting diesters:

H-Asp(OBzl)-OAll, III-14; Rf0.25 (B); m/z= 263.0 M+H+(calculated 263.3).

H-Asp(OtBu)-OAll, III-15; Rf0.25 (B); m/z= 230.1 M+H+(calculated 230.3).

H-Asp(OtBu)-OCam, III-16; Rf0.15 (B); m/z= 247.6, M+N+(calculated 247.3).

D. -(2-Phenylsulfonyl)ethyl ether tert-butylacetophenone acid (H-Asp(OtBu)-OPhse. III-17). To a solution of 3.24 g of Cbz-Asp(OtBu)-OH, 2.2 g of 2-(phenylsulfonyl)ethanol and 0.12 g of 4-DMAP in 30 ml of THF while cooling to 0oC and stirring, add 2.4 g DCGK. Continue stirring 3 h at cooling and another 4 h at room temperature. The precipitation is filtered off, washed with THF, the filtrate is evaporated. The residue is dissolved in 100 ml of ethyl acetate, extracted sequentially with 5% aqueous solution Panso3, 0.5 m solution of KHSO4and saturated NaCl solution, dried over anhydrous sodium sulfate and evaporated under reduced pressure. The residue is dissolved in 100 ml of ethanol and hydronaut hydrogen at atmospheric pressure over 0.3 g 10% Pd/C for 2 hours, the Catalyst was separated by filtration, the filtrate is evaporated to dryness and obtain 3.1 g of the target diapir III-17 in the form of oil. Rf0.2 f 0.20 (B); m/z= 392.7, M+N+(calculated 393.0).

Example 2. Getting dipeptides R3Arg-Gly-OH (IV)

a. Boc-Arg-Gly-OH, (IV-1). To a solution of 2.9 g of Boc-Arg-OH and 3.37 g of H-Gly-OBzl x TsOH in 30 ml DMF added 2.7 g of MBT, then, under stirring and cooling to 0oWith 2.4 g DCGK. The mixture is stirred for 18 h at 0oWith deposited precipitate is filtered off and the filtrate evaporated in vacuum. The residue is dissolved in 100 ml of chloroform, the solution is extracted with water (4 x 50 ml), dried over anhydrous sodium sulfate and evaporated under reduced pressure. The residue is dissolved in 100 ml of ethanol and hydronaut hydrogen at atmospheric pressure over 0.3 g 10% Pd/C for 3 hours, the Catalyst was separated by filtration, the filtrate is evaporated to dryness and treat the residue with ether. Obtain 4.2 g of the target dipeptide IV-1 p-toluensulfonate. Rf0.35 (In); m/z=331.8, M+N+(calculated 332.4).

B. Nsc-Arg-Gly-OH, (IV-2). To a solution of 4.32 g Nsc-Arg-OH and 1.9 g of H-Gly-OtBu model HC1 x 30 ml DMF added 2.7 g of MBT, then, under stirring and cooling to 0oWith 2.4 g DCGK. The mixture is stirred for 18 h at 0oWith deposited precipitate is filtered off and the filtrate evaporated in vacuum. The residue is treated with ethyl acetate, the precipitate is separated, washed with ether, dried and dissolved in 30 ml of 2 n Hcl solution in Axum and obtain 4.65 g of the target dipeptide IV-2 in the form of hydrochloride. Rf0.25 (C); m/z=489.1, M+H+(calculated 489.5).

Similarly the received hydrochloride of dipeptides:

Cbz-Arg-Gly-OH, IV-3; Rf0.35 (In); m/z=365.8 M+N+(calculated 366.4).

Fmoc-Arg-Gly-OH, IV-4; Rf0.40 (C); m/z=454.1, M+N+(calculated 454.5).

Example 3. Getting tripeptides R3-Arg-Gly-Asp(OR1)-OR2(Ia)

a. Boc-Arg-Gly-Asp(OBzl)-OPhse. (Ia-1). To a solution of 5.04 g of Boc-Arg-Gly-OH x TsOH (IV-1, example 2a) and 3.60 g of H-Asp(OBzl)-OPhse x HCl (III-l, example la) in 50 ml of DMF under stirring and cooling to 0oTo add 1.6 ml of tea, 1.35 g of MBT and 2.4 g DCGK. The mixture is stirred for 18 h at 0oWith deposited precipitate is filtered off and the filtrate evaporated in vacuum. The residue is dissolved in 150 ml of chloroform, the solution is extracted with water (4 x 50 ml), dried over anhydrous sodium sulfate and evaporated under reduced pressure. The residue is treated with ether and receive 7.42 g of the target Tripeptide Ia-1 p-toluensulfonate. Rf0.55-0.60 (C); m/z=705.3, M+N+(calculated 705.8); amino acid composition: Asp 0.87, Gly 1.00, Arg 1.04.

Similarly, the obtained p-toluensulfonate tripeptides:

Boc-Arg-Gly-Asp(OBzl)-OPse, Ia-2; Rf0.65 (C); m/z=824.1, M+H+(calculated 824.0).

Boc-Arg-Gly-Asp(OBzl)-ONb, Ia-3; Rf0.55 (In); m/z=672.3, M+N+(calculated 672.8).

Boc-Arg-Gl, +H+(calculated 750.8).

Boc-Arg-Gly-Asp(OBzl)-OTctbu, Ia-6; Rf0.60 (C); m/z= 697.7, M+H+(calculated 697.0).

Boc-Arg-Gly-Asp(OBzl)-OTce, Ia-7; Rf0.55 (In); m/z=669.4, M+H+(calculated 669.0).

B. Nsc-Arg-Gly-Asp(OBzl)-OMbzl, (Ia-8). To a solution of 5.0 g of a mixture of diapir III-8 and N,N-diethyl-N-2-(4-nitrophenyloctyl)ethylamine (example 1B), 4.15 g Nsc-Arg-Gly-OH x HCl (IV-2, example 2B) and 2.7 g of MBT in 50 ml of DMF under stirring and cooling to 0oWith add 2.0 g DCGK. The mixture is stirred for 18 h at 0oWith deposited precipitate is filtered off and the filtrate evaporated in vacuum. The residue is treated with ethyl acetate, the precipitate is separated, washed with ethyl acetate and ether. After the resultant deposition rates from ethanol-ether to obtain 5.4 g of Tripeptide Ia-8 in the form of hydrochloride. Rf0.50-0.55 (In); m/z=815.3, M+N+(calculated 814.9); amino acid composition: Asp 0.92, Gly 1.00, Arg 0.93.

Similarly the received hydrochloride tripeptides:

Nsc-Arg-Gly-Asp(OBzl)-OtBu, Ia-9; Rf0.55 (In); m/z=751.3, M+N+(calculated 750.8).

Nsc-Arg-Gly-Asp(OBzl)-ODpm, Ia-10; Rf0.60 (C); m/z= 861.3, M+H+(calculated 860.9).

Nsc-Arg-Gly-Asp(OtBu)-OBzl, Ia-11; Rf0.55 (B); m/z= 750.8, M+H+(calculated 750.8).

Nsc-Arg-Gly-Asp(OtBu)-OAll, Ia-12; Rf0.45 (C); m/z= 702.0, M+H+(calculated 700.8).

Nsc-Arg-Gly-Asp(OtBu)-OCam, Ia-13; R 792.1).

Fmoc-Arg-Gly-Asp(OtBu)-OMbzl, Ia-15; Rf0.65 (B); m/z=745.3, M+H+(calculated 745.9).

C. Cbz-Arg-Gly-Asp(OtBu)-OPhse, (Ia-16). To a solution of 3.0 g of diapir III-17 (example 1D), 3.22 g of Cbz-Arg-Gly-OH x HCl (IV-3, example 2B) and 1.2 g of MBT in 30 ml of DMF under stirring and cooling to 0oWith add 2.0 g DCGK. The mixture is stirred for 18 h at 0oWith deposited precipitate is filtered off and the filtrate evaporated in vacuum. The residue is treated with ether, the precipitate is separated and washed with ether. After the resultant deposition rates of acetic acid ether obtain 5.1 g of Tripeptide Ia-16 in the form of hydrochloride. Rf0.50-0.55 (In); m/z= 706.6, M+N+(calculated 705.8); amino acid composition: Asp 1.02, Gly 1.00, Arg 0.93.

Similarly obtained hydrochloride Tripeptide

Cbz-Arg-Gly-Asp(OtBu)-OCpse, Ia-17; Rf0.55 (In); m/z= 740.0. M+N+(calculated 740.3).

Example 4. Getting tripeptides R3-Arg-Gly-Asp(OR1)-OR2(R3=H, Ib)

a. Removal of the BOC-protected peptides Ia-1 - Ia-7 (R3=Vos). p-Toluensulfonate Tripeptide dissolved in 2 N. a solution of HCl in acetic acid (3 ml/mmol) and incubated for 40 min at room temperature. To the solution was added 1 EQ. monohydrate p-toluoyltartaric and evaporated in vacuo, the residue is treated with ether. Get peptides 1b-1 - 1b-7 in the form of di-p-tolua is ptid dissolved in a mixture of DMF-piperidine (2:1 by volume, 3 ml/mmol) and incubated for 30 min at room temperature. The solution is evaporated in vacuum, the residue is treated with ether. Get peptides 1b-8 - Ib-15 as hydrochloride.

C. Removal of Cbz-protected peptides Ia-16 Ia - 17 (R3=Cbz). Hydrochloride Tripeptide dissolved in ethanol (10 ml/mmol) and hydronaut hydrogen at atmospheric pressure over 10% Pd/C (30-60 mg/mmol) for 2-4 hours, the Catalyst was separated by filtration, the filtrate is evaporated to dryness, the residue is treated with ether. Get peptides 1b-16 1b-17 in the form of hydrochloride.

Example 5. Obtaining peptides E-Z-Y-X-Arg-Gly-Asp(OR1)-OR2(II)

a. Boc-D-Phe-Val-Gly-Arg-Gly-Asp(OBzl)-OPhse, (II-1). To a solution of 0.95 g of H-Arg-Gly-Asp(OBzl)-OPhse x 2 TsOH (Ib-1) and 0.5 g of Boc-D-Phe-Val-Gly-OH in 10 ml of DMF under cooling and stirring, add 0.15 g of MBT, 0.22 ml DIEA and 0.25 g DCGK. The mixture is stirred for 2 hours while cooling and 5 h at room temperature, the precipitate is filtered off. The filtrate is evaporated in vacuo, the residue is treated with a mixture of ethyl acetate and ether, the precipitate washed with ether. Obtain 1.05 g of the Hexapeptide II-1 p-toluensulfonate; m/z=1009.8, M+H+(calculated 1009.2); amino acid composition: Asp 0.90, Gly 1.87, Phe 1.06, Val 1.00, Arg 1.07.

Similarly obtained:

Boc-Phe-Val-Gly-Arg-Gly-Asp(OBzl)-OtBu (II-2), hydrochloride, 1b-9, and Boc-Phe-Val-Gly-OH; m/z=91.5, M+H+(calculated 1290.6).

Cbz-D-Phe-Pro-Arg-Gly-Asp(OtBu)-OBzl (II-4), hydrochloride, Ib-11 and Boc-D-Phe-Pro-OH; m/z=871.6, M+H+(calculated 872.1).

Boc-D-Phe-Orn(Cbz)-Gly-Arg-Gly-Asp(OBzl)-OMbzl (II-5), hydrochloride, 1b-8, and Boc-D-Phe-Orn(Cbz)-Gly-OH; m/z=1010.0, M+H+(calculated 1010.3).

Boc-Phe-Ala-Arg-Gly-Asp(OBzl)-ODpm (II-6), the hydrochloride of the Ib-10 and Boc-Phe-Ala-OH; m/z=923.0, M+H+(calculated 922.1).

Boc-D-Phe-Ile-Gly-Arg-Gly-Asp(OBzl)-OTce(II-7), p-toluensulfonate, 1b-7, and Boc-D-Phe-Ile-Gly-OH; m/z=987.1, M+H+(calculated 986.4).

Boc-Phe-Gly-Arg-Gly-Asp(OBzl)-ONse (II-8), p-toluensulfonate, Ib-5, and Boc-Phe-Gly-OH; m/z=954.0 M+H+(calculated 955.1).

B. Nsc-D-Phe-Val-Arg-Gly-Asp(OBzl)-OPhse, (II-9). To a solution of 0.95 g of H-Arg-Gly-Asp(OBzl)-OPhse x 2 TsOH (Ib-1) 0.22 ml DIEA in 5 ml of DMF added 0.45 g of Boc-Val-OPfp. The mixture is stirred for 2 h and evaporated in vacuo, the residue is treated with ethyl acetate and ether and receive Boc-Val-Arg-Gly-Asp(OBzl)-OPhse x TsOH. It removes the BOC-protection, as described in example 4A, and condense in a similar way with 0.75 g Nsc-D-Phe-OPfp. Obtain 1.11 g of p-toluensulfonate of Pentapeptide II-9; m/z=1108.6, M+N+(calculated 1109.3); amino acid composition: Asp 0.90, Gly 0.89, Phe 1.03, Val 1.00, Arg 1.10.

Similarly obtained:

Fmoc-Phe-Val-Arg-Gly-Asp(OBzl)-OFm (II-10), p-toluensulfonate, Ib-9; m/z=1085.1, M+N+(calculated 1084.3).

Mcbz-D-Phe-Lys(Boc)-Arg-Gly-Asp(OtBu)-OBzl (II-11), p-toluensulfonate, Ib-11;9.5, M+H+(calculated 1089.3).

Nsc-D-Phe-Ile-Arg-Gly-Asp(OtBu)-OCam (II-13), hydrochloride, Ib-13, Nsc-Ile-OPfp and Nsc-Phe-OPfp; m/z=979.1, M+N+(calculated 978.1).

Example 6. Deleting groups R2and E peptides E-Z-Y-X-Arg-Gly-Asp(OR1)-OR2(II)

Group R2and E are removed simultaneously or sequentially; the sequential removal is preferable to delete the group R2before the removal or Acidolysis in the conditions of Example 4A are removed following groups: R2=tBu, Mbzl, Dpm; F=Vos, Mcbz. The elimination of bases in the conditions of Example 4B removed the following groups: R2=Fm, Nse, Phse, Cpse, Pse (for the last three groups, the reaction time 1.5 h); E=Fmoc, Nsc. Catalytic hydrogenolysis in the conditions of Example 4B removed the following groups: R2=Bzl, Mbzl, Dpm, Nb; E=Cbz, Mcbz.

Deleting groups Tce and Tctbu reductive elimination. 1 mmol of the peptide with R2=TSE or Tctbu dissolved in a mixture of 5 ml of DMF, 10 ml THF, 3 ml of water and 1 ml of acetic acid and with vigorous stirring, add 1 g of activated zinc powder. Stirring is continued for 30 minutes, then zinc sludge is filtered off, the filtrate is diluted with 40 ml of water and applied on the column with 30 ml of Lichroprep RP18 (Merck KG, Darmstadt, Germany), equilibrated with water containing 2% acetic acid. Colo To the eluate was added 1 mmol monohydrate p-toluenesulfonic acid and evaporated to dryness. The residue is treated with ether, and get the target peptide with R2=N.

Deleting a group Nb reductive elimination (U.S. patent 5817758). 1 mmol of the peptide with R2=Nb dissolved in 10 ml of DMF, was added 1 ml of acetic acid and 1 g of SnCl2and stirred for 5 hours the Mixture is diluted with 40 ml of water and was isolated by chromatography of the target peptide with R2=N, as described above.

Delete group Cam. 1 mmol of the peptide with R2=Cam dissolved in 10 ml of DMF and added dropwise with vigorous stirring, 10 ml of 10% aqueous piperidine. After 10 min the mixture was evaporated in vacuum to dryness, treated the residue with ether, and get the target peptide with R2= H (these conditions result in the simultaneous removal of Nsc-group).

Example 7. Receiving RGD-containing cyclopeptides (R1=Bzl) [for example, cyclo(Arg-Gly-Asp-D-Phe-Val)].

With 0.64 g of p-toluensulfonate peptide II-9 (Example 5B) remove the Nsc group (E) and Phse (R2as described in Example 6 (Example 4B). The resulting peptide (0.41 g) was dissolved in 300 ml of DMF and added dropwise with stirring, 0.4 ml DIEA, 0.135 g of MBT and 0.45 g of hexaflurophosphate benzotriazolyl-1-oxy-Tris(dimethylamino)phosphonium. The mixture is stirred for 24 h at room temperature, then add 3 ml of water and evaporated to dryness in HAC.5 g of sodium acetate and chromatographic in column (2 x 15 cm with Lichroprep RP18 (25-40 μm) in a gradient from 0 to 50% acetonitrile with 2% acetic acid. The fractions containing pure by HPLC peptide cyclo[Arg-Gly-Asp(OBzl)-D-Phe-Val] , evaporated to a minimum volume, dissolved in 20 ml of a mixture of ethanol-water-acetic acid (70: 20:10) and hydronaut as described in example 4B. The resulting solution was evaporated to dryness, dissolve the residue in 10 ml of water and lyophilized. Receive 240 mg acetate, cyclo(Arg-Gly-Asp-D-Phe-Val) (67% II-9); purity by HPLC 96%; m/z=576.1, M+N+(calculated 575.6); amino acid composition: Asp 0.93, Gly 1.91, Phe 1.03, Val 1.00, Arg 1.07 (weight content of peptide material 80%).

Example 8. Receiving RGD-containing cyclopeptides (R1=tBu) [for example, cyclo(Arg-Gly-Asp-D-Phe-Lys)].

With 0.52 g of the hydrochloride of peptide II-11 (Example 5B) delete group Mcbz (E) and (Bzl (R2as described in Example 6b (Example 4B). The resulting peptide (0.40 g) was dissolved in 300 ml of DMF and added dropwise with stirring, 0.4 ml DIEA, 0.135 g of MBT and 0.45 g of hexaflurophosphate benzotriazolyl-1-oxy-Tris(dimethylamino)phosphonium. The mixture is stirred for 24 h at room temperature, then add 3 ml of water and evaporated to dryness in a vacuum. The residue is treated with ether, the precipitate is dissolved in 10 ml of 20% aqueous acetic acid, 0.5 g of sodium acetate and chromatographic in column (2 x 15 cm with Lichroprep RP18 (25-40 μm) in a gradient from 0 to 50% acetonitrile dissolved in 10 ml of 2 N. HCl in acetic acid. After 30 minutes the solution is evaporated to dryness, dissolve the residue in 10 ml of water and lyophilized. Obtain 228 mg of cyclo(Arg-Gly-Asp-D-Phe-Lys) x 2hcl (62% II-11); purity by HPLC 98%; m/z=605.0 M+H+(calculated 604.7); amino acid composition: Asp 0.96, Gly 1.90, Phe 1.00, Lys 1.03, Arg 1.07 (weight content of peptide material 82%).

1. The peptides of General formula

R3-Arg-Gly-Asp (OR1)-OR2,

where R1is benzyl or tert-butyl;

R2not equal to R1and is selected from the group of tert-butyl; benzyl; 4-methoxybenzyl; 4-nitrobenzyl; diphenylmethyl; 2,2,2-trichloroethyl; 2,2,2-trichloro-1,1-dimethylethyl; allyl; 9-fluorenylmethyl; carboxamidine; substituted 2-sulfonylated type AND-SO2-CH2-CH2- where a is substituted or unsubstituted phenyl or benzyl;

R3is a hydrogen atom or a urethane protective group IN1O-CO-, where1not equal to R1and can take values tert-butyl, benzyl, 4-methoxybenzyl, 9-fluorenylmethyl, 2-(4-nitrophenyl-sulfonyl)ethyl, or peptidyl containing from one to three amino acid residues.

2. Peptides under item 1, where R3- peptidyl structure

E-Z-Y-X-,

in which E is a hydrogen atom or a urethane protective group IN2-O-CO-, where
X is absent or equal to Gly;

Y is Gly, Ala, Val, Ile, Pro, Lys(G) or Orn(G), where G is a urethane protective group IN3O-CO-, in which3=R1attached to the omega-amino group;

Z values can be Phe or D-Phe.

 

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