TW201730203A - Method for preparation of RADA-16 - Google Patents

Abstract

The invention discloses a method for the preparation of RADA-16, which is a 16-mers peptide with the sequence Ac-(Arg-Ala-Asp-Ala)4-NH2,by liquid phase peptide synthesis.

Description

Method for preparing RADA-16

The present invention discloses a method for preparing RADA-16 by liquid phase peptide synthesis, which is a 16 peptide having the sequence Ac-(Arg-Ala-Asp-Ala) ₄ -NH ₂ .

In the following, the following abbreviations and definitions are used unless otherwise indicated.

RADA-16 is a synthetic amphiphilic peptide designed to self-assemble into fibers and a highly ordered structure in a controlled manner according to pH, such as a hydrogel having an extremely high water content. This biocompatible material has been found to have potential applications in medical, biotechnology, nanotechnology and biology. Examples include three-dimensional cell culture, tissue repair and tissue engineering, the cosmetic industry, drug release and drug delivery, biosurface engineering, separation of substrates, and membrane protein stabilization. Moreover, self-complementing peptides represent a useful model system for investigating the in vitro formation of amyloid fibrils involved in some neurodegenerative diseases.

A survey based on its performance is disclosed by Paolo Arosio et al. in Biophysical Journal 2012, 102, 1617-1626.

SPPS is known to date for RADA-16. RADA-16 shows a low yield in SPPS. Paradis-Bas, M., et al., Eur. J. Org. Chem., 2013, 26, 5871-5878, discloses that SPPS results in a rather poor final purity of the crude product and that the formed impurities are not easily removed. SPPS has the disadvantage that a larger volume is costly. In SPPS Fmoc chemistry, Arg is usually protected with Pbf. Pbf generally results in cleavage of, for example, derived from an incomplete cleavage and by-products of competing reactions SO ₃ derivative. Moreover, it has been observed that the coupling is completed in SPPS for a considerable period of time, 3-4 hours. In particular, the coupling of Fmoc-Arg (Pbf) at position 1 is very difficult and takes a very long time.

The use of NO ₂ protection of Arg in SPPS is in principle possible, but not in combination with Bzl protection of Asp in SPPS Fmoc chemistry, as it leads to the formation of high levels of aspartimide. And related impurities. Therefore, when combined with NO ₂ on Arg, Asp will be protected using tBu. However, if Arg is protected with NO ₂ , two deprotection steps are required to catalytically cleave NO ₂ and acid cleave tBu.

Further, in SPPS, the tetramer e.g. Fmoc-RADA-OH tetramer conjugated to another when the NH Arg ₂ -RADA- resin, will produce a relatively high content of Ala racemization.

Paradis-Bas, M., et al., Eur. J. Org. Chem., 2013, 26, 5871-5878, discloses a method for preparing RADA-16 in Scheme 1, in which the fragment F1 Fmoc-Arg (Pbf) is obtained three times. -Ala-Asp(tBu)-Ala-OH is coupled to the resin-binding fragment H-Arg(Pbf)-Ala-Asp(tBu)-Ala-NH-Rink Chemmatrix (CM) resin, which is called " Solid phase fragment condensation". In this method, the fragment F1 is prepared by SPPS, requiring a 3-fold molar excess of amino acids 2 to 4 (Asp(tBu), Ala and Arg(Pbf)).

For the preparation of peptide resin II ([RADA] _{2 -} CM resin), which is the first of the three on the coupling of the resin fragments, 3 equivalents of fragment F1 are required.

For the preparation of peptide resin III ([RADA] _{3 -} CM resin), which is the second of the three on the coupling of the resin fragments, it is required to couple the fragment F1 three times, 1.5 equivalents of fragment F1 each time, That is, a total of 4.5 equivalents of fragment F1 was obtained to obtain a negative for the ninhydrin test.

Preparation of Ac- (RADA) ₄ -NH ₂ with respect to which is the third of the three coupling segments in the resin, the coupling of the two fragments required F1, 1.5 equivalents of each fragment F1, i.e., a total of 3.0 Equivalent fragment F1 was obtained to be negative for the ninhydrin test.

In Scheme 2, fragment condensation of two octamer fragments F2 and F3 in solution is disclosed, and these fragments F2 and F3 are prepared by SPPS. Furthermore, a three-fold molar excess of each Fmoc-amino acid 2 to 8 is used, which is subsequently coupled to the ever-expanding peptide chain after the first amino acid Ala.

It has been reported that the crude product purity is 58% after the global deprotection in the fragment condensation method.

Wang Jun, “Synthesis of short peptide RADA for molecular self-assembly”, China Excellent Master's Thesis Full-text Database (Engineering Science and Technology Series I), Issue 02, February 15, 2007 (15.02.2007) in the abstract and The tetramer Boc-Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-Obzl is disclosed on page 18, line 11 to page 25, line 4. Due to the Bzl protecting group of the COOH of the C-terminal Ala, the tetramer is not suitable as a building block for RADA-16.

There is a need for a process for the preparation of RADA-16 in high yields which has less impurities or by-products, can be adapted for bulk production, and does not require a relatively large excess of substrate or building block relative to each other.

It was unexpectedly found that a pure LPPS-based preparation method satisfies these needs: the tetrapeptide of the sequence RADA prepared by LPPS is coupled to the octapeptide of the sequence (RADA) 2, which are coupled to provide the desired RADA-16. This LPPS method allows the fragment strategy to produce a lower racemization level of Ala than the resin-on-resin coupling disclosed by Paradis-Bas.

Similarly, some racemization was still observed in LPPS: When the protected form of Arg, Arg(NO ₂ ), was used, two isomers were observed during the coupling reaction of the two octapeptides and were overall deprotected. Two isomers were also observed afterwards. LC-MS (liquid chromatography-mass spectrometry) spectra showed that the two pairs of peaks have similar molecular weights. Such impurities generally represent isomers derived from racemization. After the overall deprotection, two corresponding isomers are still present in the LC-MS spectrum. Even if separation is entirely possible, the isomers derived from racemization are difficult to separate.

When the alternative form of protection to unprotected Arg (NO ₂₎ used in the form Arg, Ala in the method RADA-16 in the final level of racemization of 1% or less accidentally.

Furthermore, the LPPS strategy of the present invention uses an allyl group for the protection of the C-terminal COOH residue of the primary tetrameric fragment such that the primary tetrameric fragment (which is prepared to have the allyl protection) Base) can be used for both transformations, ie for the conversion of the C-terminal COOH residue to the guanamine residue, and for the unprotected C-terminal COOH (which is the fragment Conversion of the required). This represents a rational strategy for preparing only this one major tetrameric fragment, which can then be used for derivatization as a tetrameric fragment substrate for construction of the octamer fragment as outlined herein. .

The present invention provides a novel preparation method based on pure LPPS. This method is necessary for production on a ton scale, while in the known SPPS strategy, lower conversions and the need for more substrates, materials and intermediates are observed. In the coupling reaction in LPPS, a stoichiometric or near stoichiometric amount of building blocks can be used.

When Arg was used in its protected form Arg(NO ₂ ), the crude purity after total deprotection was 58%, which was higher than the 41% purity reported by Paradis-Bas for the fragment coupling strategy on the resin, respectively. The purity was 46% and was similar to the purity of the condensation of the fragments of the two octamer fragments F2 and F3 in solution reported by Paradis-Bas. When Arg is used in its unprotected form, even 46% purity is obtained.

The subject of the invention is a process for the preparation of said peptide (RADA) ₄ ; wherein an octapeptide having the sequence (RADA) ₂ is coupled in the liquid phase to provide said peptide (RADA) ₄ ; by having a sequence RADA The tetrapeptide is coupled in a liquid phase to prepare the octapeptide; the tetrapeptide having the sequence RADA is prepared in a liquid phase.

The octapeptide is the fragment FRAG2.

FRAG2 and (RADA) ₄ were prepared in the liquid phase, ie by LPPS.

The tetrapeptide having the sequence RADA is the fragment FRAG1.

FRAG1 is prepared in the liquid phase, ie by LPPS.

Preferably, any coupling is carried out in the liquid phase, i.e. by LPPS. This means that solid phase coupling is not used in any coupling, ie no SPPS is used.

Preferably, FRAG1 is prepared by stepwise coupling of a single amino acid; more preferably, said stepwise coupling is initiated by the coupling of D with said C-terminal A to provide DA as a starting, then a second A and The DA is coupled to provide ADA, and finally the N-terminal R is coupled to the ADA to provide RADA.

Within the scope of the invention and the definition of the present specification, any amino acid or amino acid residue in the peptide is represented by its one-letter code, unless otherwise specified, any amino acid or amino acid residue in the peptide. any N- any α-amino acid residues of any amino acid residue COOH, NH ₂ terminus of the peptide and peptide residue COOH any C- terminal residues may be protected or unprotected.

Within the definition of the invention and the present specification, the term fragment refers to a peptide derived from a fragment of (RADA) ₄ .

Preferably, any Asp is used in its side chain protected form Asp (OBzl).

Preferably, any Arg is used only in the form of an unprotected side chain or any Arg is only used in its side chain protected form Arg(NO ₂ ); therefore, more preferably, provided by the method of the invention (Arg (NO ₂ )-Ala-Asp(OBzl)-Ala) ₄ , or (Arg-Ala-Asp(OBzl)-Ala) ₄ is provided by the method of the present invention.

Even more preferably, any Arg is used only in the form of unprotected side chains, thus providing (Arg-Ala-Asp(OBzl)-Ala) ₄ by the method of the invention.

Preferably, the coupling of the tetrapeptide is carried out at a temperature below 5 ° C (preferably below 3 ° C) to provide (RADA) ₂ .

Preferably, where Arg is used only in the form of unprotected side chains, the coupling of the octapeptide is carried out at a temperature below 5 ° C (preferably below 3 ° C) to provide (RADA) ₄ .

More preferably, the coupling of the octapeptide is carried out at a temperature below 5 ° C (preferably below 3 ° C) to provide (RADA) ₄ .

Preferably, any protection of the N-terminal alpha amino residue during any coupling is carried out by using Boc or Ac.

Preferably, in any arbitrary C- terminal COOH is protected during the coupling residue is All or allyl amine to acyl C (O) NH ₂ in the form of.

Preferably, the peptide (RADA) ₄ is provided in the form of the peptide PEP-AC-NH2; PEP-AC-NH2 is Ac-(RADA) ₄ -NH ₂ ; that is, its N-terminal alpha amino group Ac residues are protected, and its C- terminus of Amides COOH C (O) NH ₂ form.

Preferably, the method comprises the step STEPB; in step STEPB, the PEP-AC-NH2 is prepared by coupling the fragment FRAG2-AC-OH with the fragment FRAG2-H-NH2; the FRAG2-AC-OH is Ac- (RADA) ₂ -OH; FRAG2-H-NH2 is H-(RADA) ₂ -NH ₂ .

FRAG2-AC-OH and FRAG2-H-NH2 are embodiments of FRAG2.

Preferably, the method comprises the step STEPA1; in STEPA1, the FRAG2-H-NH2 is prepared; STEPA1 comprises a fragment FRAGA1-Boc-OH coupled with the fragment FRAGA1-H-NH2, followed by cleavage of the N-terminal Boc residue FRAGA1-Boc-OH is Boc-RADA-OH and FRAGA1-H-NH2 is H-RADA-NH ₂ .

Preferably, the method comprises the step STEPA2; in STEPA2, the FRAG2-AC-OH is prepared; STEPA2 comprises a fragment FRAGA2-Ac-OH coupled to the fragment FRAGA2-H-OAll, and comprising subsequently cleaving the C- End All residue; FRAGA2-Ac-OH is Ac-RADA-OH; FRAGA2-H-OAll is H-RADA-OAll.

FRAGA1-Boc-OH, FRAGA1-H-NH2, FRAGA2-Ac-OH, and FRAGA2-H-OAll are embodiments of FRAG1.

Preferably, for the preparation of FRAG1, ie for the preparation of said tetrapeptide having the sequence RADA, said three amino acids R, A and D are used as Boc-Ala-OH, Boc-Asp(OBzl)-OH and Boc-Arg(NO ₂ )-OH; or used as Boc-Ala-OH, Boc-Asp(OBzl)-OH and Boc-Arg-OH.

Preferably, Boc-Arg-OH is used in the form of Boc-Arg-OH.HCl.H ₂ O.

FRAG1 is used as an intermediate for the process of the invention, and another preferred embodiment thereof is Boc-RADA-OAll; especially in the form of Boc-Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-OAll or Boc -Arg-Ala-Asp(OBzl)-Ala-OAll form.

Preferably, cleavage of any of the side chain protecting groups is carried out after coupling of the octapeptide, i.e., after STEPB.

Preferably, any of the NO ₂ protecting groups of Arg and the Bzl protecting group of Asp are cleaved by catalytic hydrogenation. Catalytic hydrogenation can be achieved by elemental hydrogen, or a suitable hydrogen donor, such as formic acid, ammonium formate, 1,3-cyclohexadiene, 1,4-cyclohexadiene or a borane adduct (eg borane) Tert-butylamine (tert-BuNH ₂ .BH ₃ )). Suitable hydrogenation catalysts are, for example, noble metal-based hydrogenation catalysts, especially metals known as platinum group metals, namely ruthenium, rhodium, palladium, osmium, iridium and platinum. Conveniently, the hydrogenation catalyst is on a support such as carbon.

In particular, the cleavage of NO ₂ and Bzl is carried out simultaneously.

In particular, the cleavage of NO ₂ and Bzl was carried out using the catalyst Pd/C.

In particular, the cleavage of NO ₂ and Bzl is carried out in TFA, formic acid or a mixture thereof as a solvent, more particularly in TFA.

Preferably, the cleavage of NO ₂ and Bzl is carried out at atmospheric pressure or above atmospheric pressure. The pressure is usually from 1 to 100 bar, preferably from 1 to 70 bar, more preferably from 1 to 10 bar.

Preferably, the reaction temperature for the cleavage of NO ₂ and Bzl is -20 to 70 ° C; more preferably 0 to 60 ° C, even more preferably 10 to 40 ° C.

Preferably, the reaction time for the cleavage of NO ₂ and Bzl is from 1 to 6 hours, more preferably from 1 to 5 hours.

Preferably, All is used as a C-terminal protecting group for any Ala that is required to protect its COOH residue, and as a C-terminal protecting group for any C-terminal Ala of any FRAG1 or FRAG2.

Preferably, All is attached to the COOH residue to be protected, the linkage being achieved by a reaction REACALL between the COOH residue and the allyl donor, preferably the allylic donor 3-bromopropene.

Preferably, the molar amount of the allyl donor is from 1 to 2 times the molar amount of the COOH residue.

Preferably, REACALL is carried out in the presence of a base, preferably in the presence of K ₂ CO ₃ .

Preferably, the molar amount of the base is from 1 to 2 times the molar amount of the COOH residue.

Preferably, REACALL is carried out in a solvent, preferably in DMF.

Preferably, the REACALL is carried out under atmospheric pressure.

Preferably, the reaction temperature of the REACALL is -20 to 70 ° C, more preferably -15 to 60 ° C.

Preferably, the reaction time of the REACALL is from 1 to 48 hours.

Preferably, any cleavage of All is carried out using the reaction CLEAVALL using the catalyst Pd(PPh ₃ ) ₄ .

Preferably, the molar amount of Pd(PPh ₃ ) ₄ is 0.01 to 0.05 times the molar amount of the All residue.

CLEAVALL can be carried out in the presence of the additive ADDALL selected from the group consisting of morpholine, 1,3-dimethylbarbituric acid, pyrrolidine, triphenylphosphine, dimethanone, and mixtures thereof .

Preferably, the molar amount of ADDALL is from 2 to 7 times the molar amount of the All residue.

Preferably, CLEAVALL can be carried out in any inert solvent that will dissolve the reactants. Preferred solvents for CLEAVALL are dimethyl hydrazine (DMSO), dioxane, tetrahydrofuran (e.g., tetrahydrofuran (THF) or methyltetrahydrofuran (methyl-THF)), 1-methyl-2-pyrrolidone ( NMP), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), dichloromethane (DCM), ethyl acetate, acetonitrile or any mixture thereof.

More preferably, CLEAVALL is carried out in DMF, acetonitrile or dioxane.

Preferably, CLEAVALL is performed at atmospheric pressure.

Preferably, the reaction temperature of CLEAVALL is from -20 to 70 ° C; more preferably from -15 to 60 ° C.

Preferably, the reaction time of CLEAVALL is from 1 to 48 hours.

Preferably, the process of REACALL and CLEAVALL is monitored by HPLC to determine the necessary reaction time.

Preferably, Boc is used as an N-terminal protecting group.

Any cleavage of the N-terminal Boc residue was carried out using the reaction CLEAVBOC. CLEAVBOC can be carried out using reaction conditions known in the art of peptide synthesis. Preferably, the cleavage is effected by an acid, preferably trifluoroacetic acid or HCl, which may be used alone in the case of TFA, or as a mixture with an inert solvent, or as a gas in the case of HCl. The inert solvent is, for example, toluene or dioxane or a mixture thereof.

Typically, CLEAVBOC is carried out in a solvent, which may be any solvent that does not interfere with the reactants, such as a chlorinated hydrocarbon such as dichloromethane or dichloroethane; an aromatic hydrocarbon such as toluene; an ether such as THF or Dioxane; or any mixture thereof.

Preferably, CLEAVBOC is carried out in toluene or dioxane.

Preferably, CLEAVBOC is carried out at atmospheric pressure.

Preferably, the reaction temperature of CLEAVBOC is -20 to 70 ° C; more preferably -15 to 60 ° C.

Preferably, the reaction time of CLEAVBOC is from 0.5 to 12 hours.

Preferably, the process of CLEAVBOC is monitored by HPLC to determine the necessary reaction time.

Preferably, any coupling can be carried out using reaction conditions known in the art of peptide synthesis, including the stepwise coupling of a single amino acid providing FRAG1 or the coupling of the tetrapeptide of the octapeptide. Or the coupling of the octapeptide.

Preferably, the in situ coupling reagent for the coupling is, for example, a hydrazine coupling reagent or a urea hydrazine coupling reagent or a carbodiimide coupling reagent, and the hydrazine coupling reagent or the urea hydrazine coupling reagent is For example, benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol-1-yloxy-tris(pyrrolidinyl)phosphonium hexafluorophosphate ( PyBOP), O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), O-(6-chlorobenzotriazole- 1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), O-(6-chlorobenzotriazol-1-yl)-1,1,3,3 -tetramethyluronium tetrafluoroborate (TCTU), O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate ( HATU), O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TATU), O-(benzotriazole) -1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU), O-[cyano(ethoxycarbonyl)methyleneamino]-1,1,3, 3-tetramethyluronium tetrafluoroborate (TOTU) and (1-cyano-2-ethoxy-2-oxoethylaminooxy)dimethylamino-morpholine-carbon hexafluoro Phosphate (COMU); the carbonized secondary The coupling reagent is, for example, diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimide (DCC) and water-soluble carbodiimide (WSCDI), such as 1-ethyl-3-(3- Dimethylaminopropyl)carbodiimide (EDC), optionally in the form of a salt such as the hydrochloride salt.

Other coupling techniques use pre-prepared active esters (eg, N-hydroxysuccinimide (HOSu) and p-nitrophenol (HONp) esters), pre-prepared symmetric anhydrides, asymmetric anhydrides (eg, N-carboxy anhydrides) NCAs)) and hydrazine halides (such as decyl fluoride or hydrazine chloride).

Preferred coupling reagents are carbodiimide coupling reagents and hydrazine coupling reagents. The preferred coupling reagents are selected from the group consisting of PyBOP, DCC, DIC and EDC; more preferably, the coupling reagent is PyBOP, DCC or EDC.

Preferably, EDC is used as a salt, and more preferably, as EDC.HCl.

Preferably, the molar amount of the coupling reagent is from 1 to 10 times, more preferably from 1 to 5 times the molar amount of the substrate (for example, AA or a fragment to be coupled, respectively).

Any coupling can be carried out in the presence of hydrazine, preferably a tertiary amine base which deprotonates the COOH residue of the carboxylic acid moiety in the coupling and neutralizes the coupling Any counterion of the NH ₂ residue of the amino moiety in the middle, thus promoting the coupling reaction.

Suitable bases are, for example, trialkylamines, for example N,N-diisopropylethylamine (DIPEA) or triethylamine (TEA); N,N-dialkylanilines, for example N,N-diethylaniline 2,4,6-trialkylpyridine, such as 2,4,6-trimethylpyridine; and N-alkylmorpholine, such as N-methylmorpholine.

Preferably, the coupling is carried out in the presence of DIPEA as a base.

Preferably, the molar amount of base is from 1 to 3 times the molar amount of the substrate (for example, AA or a fragment to be coupled, respectively).

Since the auxiliary nucleophile as an additive has a positive effect of suppressing undesirable side reactions, any coupling can be carried out in the presence of an auxiliary nucleophile as an additive. Any known auxiliary nucleophile can be used.

An example of a suitable auxiliary nucleophile is 1-hydroxybenzotriazole (HOBt), N-hydroxysuccinimide (HOSu), N-hydroxy-3,4-dihydro-4-oxo-1,2 , 3-benzotriazine (HOOBt) and 1-hydroxy-7-azabenzotriazole (HOAt).

Preferably, HOBt or HOAt is used as an auxiliary nucleophile.

Preferably, the molar amount of the auxiliary nucleophile is 0.5 to 3 times the molar amount of the AA or fragment to be coupled, respectively.

In particular, any coupling is carried out using DCC/HOBt, EDC/HOBt, PyBOP/HOBt or EDC/HOAt.

Preferably, any coupling can be carried out in any inert solvent capable of dissolving the reactants. Preferred solvents for the coupling are water-miscible solvents, and water-immiscible solvents and any suitable mixture of water-miscible solvents and water-immiscible solvents, including mixtures with water. The water-miscible solvent is, for example, dimethyl hydrazine (DMSO), dioxane, tetrahydrofuran (for example, tetrahydrofuran (THF) or methyltetrahydrofuran (methyl-THF)), 1-methyl-2-pyrrolidone (NMP), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), or any mixture thereof; the water-immiscible solvent is, for example, dichloro Methane (DCM), ethyl acetate or any mixture thereof.

More preferably, the coupling is carried out in a solvent selected from the group consisting of dichloromethane, methyl-THF, DMF, NMP, and mixtures thereof.

Preferably, the coupling is carried out at atmospheric pressure.

Preferably, the reaction temperature of the coupling is -20 to 70 ° C, more preferably -15 to 40 ° C, even more preferably -15 to 30 ° C.

Preferably, the coupling has a reaction time of from 1 to 48 hours.

Preferably, the coupling process is monitored by HPLC to determine the necessary reaction time.

In the sense of the present invention, any amide amination of the C-terminal COOH residue or the COOH residue of an amino acid is also a coupling, which is an even bond between the C-terminal COOH residue and the ammonium donor. Union. Thus, all embodiments described for coupling are also suitable for the amide amination.

Preferably, the ammonium donor is NH ₃ or a salt thereof, such as a hydrochloride, acetate or trifluoroacetate salt thereof.

Preferably, the ammonium donor is NH ₄ Cl or NH ₄ OAc.

Preferably, the molar amount of ammonium donor is from 3 to 7 times the molar amount of the COOH residue to be amidated.

Preferably, the guanylation is carried out using EDC/HOBt or EDC/HOAt.

Preferably, Ac on the N-terminal NH ₂ residue or on the alpha amino residue of the amino acid is introduced by reaction ACETYLA, wherein the N-terminal NH _{2 is} reacted with an acetamyl donor.

The ethyl thiol donor can be acetic anhydride or acetic acid.

Preferably, the molar amount of the ethyl hydrazide donor is from 1 to 2 times the molar amount of the amino residue to be ethoxylated.

In the sense of the present invention, in the case of acetic acid, ACETYLA actually coupling, which is coupled between the N- terminal residue COOH NH ₂ or an amino acid residue of the α-amino acid residues. Therefore, all embodiments described for coupling are also applicable to ACETYLA.

Preferably, ACETYLA is performed using EDC/HOBt or EDC/HOAt, more preferably EDC/HOBt.

Preferably, ACETYLA is carried out in the presence of a base, preferably in the presence of diisopropylethylamine.

Preferably, ACETYLA is carried out in a solvent, preferably in DMF or CAN.

Preferably, ACETYLA is carried out under atmospheric pressure.

Preferably, the reaction temperature of ACETYLA is from -20 to 70 ° C, more preferably from -15 to 60 ° C, even more preferably from -10 to 40 ° C.

Preferably, the reaction time of ACETYLA is from 1 to 48 hours, more preferably from 1 to 12 hours.

Preferably, the process of ACETYLA is monitored by HPLC to determine the necessary reaction time.

RADA-16, when it is obtained in crude form after cleavage of any of the side chain protecting groups, can be purified by conventional methods known to those skilled in the art, for example by HPLC.

Preferably, RADA-16 obtained after the cleavage of any of the side chain protecting groups is a TFA salt. If a hydrochloride or a different salt is desired, conventional salt exchange methods known to those skilled in the art can be employed.

Preferably, any purification or salt exchange is carried out using MeOH or EtOH as one of the mobile phases.

In the general schemes 1, 2, 3 and 4, it is preferred to prepare Ac-[Arg-Ala-Asp-Ala] ₄ -NH ₂ .TFA using Arg in the side chain protected form of Arg (NO ₂ ). route.

General scheme 1 .

General scheme 2 .

General scheme 3 .

General scheme 4 .

In Formula Scheme A, B, C and D illustrate the preparation of side chain unprotected Arg use of Ac- [Arg-Ala-Asp- Ala] 4 -NH 2 .TFA preferred route.

General scheme A .

General scheme B .

General formula C .

General scheme D . Example

material

Boc-Ala-OH, Boc-Asp(OBzl)-OH and Boc-Arg(NO ₂ )-OH and Boc-Arg-OH.HCl.H ₂ O were purchased from GL Biochem.

Pd(PPh ₃ ) _{4 was} purchased from Alfa Aesar.

Pd/C was purchased from Johnson Matthey; product model number A109047-5.

method

The purity was determined by HPLC.

HPLC conditions for program control (IPC) and intermediates: Waters 2695 or cooperative VWD HPLC column: Nucleosil 100-5 C18, 250 mm x 4.6 mm, 5 μm mobile phase: A = 0.1% aqueous solution of TFA B = 0.1% TFA acetonitrile solution Wavelength: 215nm Flow rate: 1.0mL/Min Temperature: 15-25°C Detector: Waters 2996 Gradient: From 5% B to 85% B in 20 minutes, then held for 5 minutes.

HPLC conditions for the final crude peptide instrument: Waters 2695 or cooperative VWD HPLC column: Nucleosil 100-5 C18, 250 mm x 4.6 mm, 5 μm mobile phase: A = 0.1% aqueous solution of TFA B = 0.1% TFA acetonitrile solution wavelength : 215nm Flow rate: 1.0mL/Min Temperature: 15-25°C Detector: Waters 2996 Gradient: From 5% B to 40% B in 25 minutes, then hold for 5 minutes.

Examples 1 to 4 show the synthesis of Ac-[Arg-Ala-Asp-Ala] ₄ -NH ₂ .TFA using NO ₂ -protected Arg as a starting material.

Examples 5 and 6 show the synthesis of Ac-[Arg-Ala-Asp-Ala] ₄ -NH ₂ .TFA using unprotected Arg as a starting material.

Example 7 shows the purification and exchange of TFA to HCl. Example 1

The fragment Boc-Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-OAll was synthesized according to the general formula 1.

Synthesis of 1(1) Boc-Ala-OAll Boc-Ala-OH+ Br-CH ₂ -CH=CH ₂ -> Boc-Ala-OAll

K ₂ CO ₃ (0.134 kg, 1.50 equivalents) was added to a mixture of DMF (0.61 kg) and Boc-Ala-OH (1.0 eq., 0.122 kg) at 25 °C. After stirring at 25 ° C for 0.5 hour, 3-bromopropene (0.122 kg, 1.56 equivalent) was added dropwise while maintaining the temperature at 25 ± 5 °C. After about 3 hours, the reaction was complete as indicated by HPLC monitoring. The reaction mixture was then filtered. The wet cake was washed twice with ethyl acetate (0.73 kg each, for a total of 1.46 kg). The filtrate and washing solution were combined and mixed with deionized water (1.46 kg), and the resulting mixture was extracted three times with ethyl acetate (0.61 kg each, a total of 1.83 kg). All organic phases were combined and washed twice with saturated brine (0.14 kg each, for a total of 0.28 kg). The organic phase was dried over anhydrous MgSO ₄ (0.061 kg) was dried for 1 hour and dried organic phase was then filtered and the wet cake was washed twice with ethyl acetate (each 0.61 kg, a total of 1.22 kg). The filtrate and the washing solution were combined, concentrated under vacuum at 40 ± 5 ° C, and then azeotropically distilled twice with dioxane (0.122 kg each time, a total of 0.244 kg) to obtain a concentrated oily product Boc-Ala-OAll. , which is used in the next step. HPLC purity: 94.3%.

Boc-Ala-OAll is a compound of formula (1). .

1(2) Synthesis of HCl.H-Ala-OAll Boc-Ala-OAll + HCl Gas -> HCl.H-Ala-OAll

The concentrated oil containing Boc-Ala-OAll was diluted with dioxane (620 ml). To the mixture was added HCl gas (186 g) by bubbling while maintaining the temperature at 20 ± 5 °C. After 1 hour, the reaction mixture was evaporated in vacuo and then dried then evaporated. The isolated HCl.H-Ala-OAll was taken up in the oil and used directly in the next coupling step with Boc-Asp(OBzl)-OH.

1(3) Synthesis of Boc-Asp(OBzl)-Ala-OAll Boc-Asp(OBzl)-OH + HCl.H-Ala-OAll + DCC/HOBt -> Boc-Asp(OBzl)-Ala-Oall

An oil containing HCl.H-Ala-OAll (200 g, 1.00 eq.) was used in diisopropylethylamine (91.7 g) in dichloromethane (1000 ml) at 0 °C compared to Boc -Ala-OH, a mixture of 1.10 equivalents) and 13% by weight of brine (600 ml). The organic phase was dried over anhydrous MgSO ₄ (100 g) and dried. After 1 hour, the mixture was filtered and the wet cake was washed twice with dichloromethane (70 ml each time, a total of 140 ml). The filtrate and washing solution were combined. Boc-Asp(OBzl)-OH (175 g, 0.84 equivalents) and HOBt.H ₂ O (83 g, 1.00 equivalents) were continuously added. A solution of DCC (111 g, 1.00 equiv) in dichloromethane (400 ml) was then added while maintaining the temperature at -6 °C ± 5 °C. The mixture was stirred at -6 °C ± 5 °C for 1 hour and then warmed to 20 °C. After stirring at 20 ° C for about 1 hour, the reaction was completed as indicated by HPLC monitoring. The reaction mixture was then filtered. The filtrate was then washed twice with 5 wt% NaHCO ₃ aqueous solution (600 ml each time, 1200 ml total), washed twice with 10 wt% KHSO ₄ aqueous solution (600 ml each time, total 1200 ml), and washed twice with 20 wt% saline. (600 ml each time, a total of 1200 ml). The organic phase was then concentrated in vacuo at a jacket temperature of 40 ° C ± 5 ° C and dried by azeotropic distillation twice with dioxane (400 ml each time, a total of 800 ml) to give an oil. In the next step.

HPLC purity: 92.28%.

1(4) Synthesis of HCl.H-Asp(OBzl)-Ala-OAll Boc-Asp(OBzl)-Ala-OAll + HCl Gas -> HCl.H-Asp(OBzl)-Ala-OAll

The oil containing Boc-Asp(OBzl)-Ala-OAll was diluted with dioxane (500 ml). To the mixture was added HCl gas (225 g) by bubbling while maintaining the temperature at 20 ± 5 °C. After 1 hour, the reaction mixture was evaporated in vacuo, then the residue was dried by azeotrope twice with dioxane (150 ml each time, a total of 300 ml). The oily residue was used directly in the next coupling step.

1(5) Synthesis of Boc-Ala-Asp(OBzl)-Ala-OAll DCC/HOBt Boc-Ala + HCl.H-Asp(OBzl)-Ala-OAll -> Boc-Ala-Asp(OBzl)-Ala- OAll

The oily residue containing HCl.H-Asp(OBzl)-Ala-OAll (148 g, 1.0 eq.) was used in diisopropylethylamine in dichloromethane (750 ml). g, 1.10 equivalents) and 13% by weight of brine (450 ml) were neutralized. The organic phase was dried over anhydrous MgSO ₄ (75 g) and dried. After 1 hour, the solid was removed by filtration, and the wet cake was washed twice with dichloromethane (55 ml each time, a total of 110 ml). The filtrate and washing solution were combined. Boc-Ala-OH (78 g, 1 eq.) and HOBt.H ₂ O (63 g, 1.00 eq.) were continuously added to the H-Asp(OBzl)-Ala-OAll dichloromethane solution. A solution of DCC (102 g, 1.20 equivalents) in dichloromethane (320 ml) was then slowly added while maintaining the temperature at -6 °C ± 5 °C. The mixture was stirred at -6 °C ± 5 °C for 1 hour and then warmed to 20 °C. After about 8 hours, the coupling was completed as indicated by HPLC monitoring. The salt and the resulting DCU are then removed by filtration. The filtrate was washed twice with 5 wt% NaHCO ₃ (450 ml each time, 900 ml total), washed twice with 10 wt% KHSO ₄ (450 ml each time, 900 ml total), and washed twice with 20 wt% saline (each time) 450 ml for a total of 900 ml). The organic phase is then concentrated under vacuum at a jacket temperature of 40 ° C ± 5 ° C and dried by azeotropic distillation twice with dioxane (300 ml each time, a total of 600 ml) to obtain an oil directly. Used for the next deprotection step.

HPLC purity: 93.3%.

1(6) Synthesis of HCl.H-Ala-Asp(OBzl)-Ala-OAll HCl gas Boc-Ala-Asp(OBzl)-Ala-OAll -> HCl.H-Ala-Asp(OBzl)-Ala-OAll

An oil containing Boc-Ala-Asp(OBzl)-Ala-OAll (14 g, 1.00 equivalent) was dissolved in dioxane (100 ml). To the mixture was added HCl gas (21.0 g) by bubbling while maintaining the temperature at 20 ± 5 °C. After 1 hour, the reaction mixture was evaporated in vacuo and then the residue was dried with <RTI ID=0.0>> The oily product was used directly in the next coupling reaction with Boc-Arg(NO ₂ )-OH.

1(7) Synthesis of Boc-Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-OAll Boc-Arg(NO ₂ )-OH + HCl.H-Ala-Asp(OBzl)-Ala-OAll DCC / HOBt ->Boc-Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-OAll

The oily product containing HCl.H-Ala-Asp(OBzl)-Ala-OAll (116 g, 1.00 eq.) was used in diisopropylethylamine in methyl-THF (1080 ml). 39.7 ml, 1.10 equivalents) and 13 wt% saline (300 ml) were neutralized. The organic phase was dried over anhydrous MgSO ₄ (58 g) and dried. After 1 hour, the solid was removed by filtration, and the wet cake was washed twice with methyl-THF (70 ml each time, a total of 140 ml). The filtrate and washing solution were combined. To the H-Ala-Asp(OBzl)-Ala-OAll in a methyl-THF solution, Boc-Arg(NO ₂ )-OH (86 g, 1 equivalent) and HOBt.H ₂ O (41.4 g, 1.00) were continuously added. equivalent). A solution of DCC (61 g, 1.10 eq.) in DMF (120 ml) and methyl-THF (270 ml) was then slowly added while maintaining the temperature at -6 °C ± 5 °C. The mixture was stirred at -6 °C ± 5 °C for 1 hour and then warmed to 20 °C. After about 2 hours at 20 ° C, the coupling was completed as indicated by HPLC monitoring. The salt and the resulting DCU were removed by filtration. The filtrate was washed twice with 5 wt% NaHCO ₃ (220 ml each time, 440 ml total), washed twice with 10 wt% KHSO ₄ aqueous solution (220 ml each time, a total of 440 ml), and washed twice with 20 wt% saline (per 220 ml, a total of 440 ml). The organic phase was then concentrated in vacuo at a jacket temperature of 40 ° C ± 5 ° C until an oily residue was obtained. The oily residue was then heated to 50 ± 5 ° C, diisopropyl ether (200 ml) was added dropwise over 1 hour, followed by the addition of diisopropyl ether (200 ml) and methyl group over 1 hour. a mixture of -THF (330 ml). After the addition of the mixed solvent, the suspension was cooled to 35 ± 5 ° C in 1 hour. The suspension was then stirred for 1 hour and filtered. The wet cake was washed three times with diisopropyl ether (480 ml each time, a total of 1440 ml). The wet cake was dried under vacuum at 35 ± 5 °C.

Purity: 92.2%.

Global yield: 28%. Example 2

According to the general scheme 2, the fragment Boc-Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-OAll was used to synthesize TFA.H-[Arg(NO ₂ )-Ala-Asp(OBzl)-Ala] ₂ - NH ₂ , which is a protected C-terminal dimer of the target product.

2(1) Synthesis of Boc-Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-OH Pd(PPh ₃ ) ₄ Boc-Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-OAll -> Boc-Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-OH

Boc-Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-OAll (40 g, 1.00 equivalent) prepared according to Example 1 (7) was dissolved in DMF (160 ml) at 20 ± 5 °C. In acetonitrile (100 ml). Then morpholine (24.7 g, 5.00 equivalents) was added all at once. N ₂ was added to the mixture by bubbling to drive off residual oxygen in the mixture. After 0.5 hours, Pd(PPh ₃ ) ₄ (1.3, 0.02 equivalent) was added. The mixture was stirred under a N ₂ atmosphere. After about 1 hour, the reaction was complete as indicated by HPLC monitoring. The organic mixture was washed three times with a 10% aqueous KHSO ₄ solution (80 ml each time, 240 ml total) and washed twice with 20% brine (80 ml each time, a total of 160 ml). The organic phase was then concentrated in vacuo at a jacket temperature of 40 ± 5 ° C and dried by azeotropic distillation with acetonitrile (100 ml) to give an oil.

The oil is used in step (2) and step (2a), respectively.

2(2) Synthesis of Boc-Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-NH ₂ Boc-Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-OH -> Boc-Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-NH ₂

The oil containing Boc-Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-OH (21.5 g, 1.00 equivalent) prepared according to Example 2 (1) was dissolved in DMF at 20 ± 5 °C. (80 ml) and acetonitrile (50 ml). Ammonium acetate (12.4 g, 5.00 equivalents), HOBt.H ₂ O (4.9 g, 1.00 equivalents) and EDC.HCl (8 g, 1.3 equivalents) were continuously added. After stirring at 20 ° C for about 24 hours, the hydrazide reaction was completed as indicated by HPLC monitoring. The reaction mixture was concentrated in vacuo <RTI ID=0.0></RTI> to <RTIgt; The solution was washed twice with 5% NaHCO ₃ (45 ml each time, 90 ml total), washed twice with 10% aqueous KHSO ₄ (45 ml each time, 90 ml total) and washed twice with 20% brine ( 45 ml each time, a total of 90 ml). The organic phase was then concentrated in vacuo at a jacket temperature of 40 ° C ± 5 ° C to give an oily residue. The residue was dissolved in DMF (45 ml) and the mixture was heated to 50 ± 5 °C. A mixture of diisopropyl ether (120 ml) and ethyl acetate (270 ml) was added while maintaining the temperature at 50 ± 5 °C. The resulting suspension was cooled to 10 ± 5 ° C over 2 hours and stirred for 1 hour and filtered. The wet cake was washed three times with diisopropyl ether (60 ml once and 180 ml twice). The wet cake was dried under vacuum at 35 ± 5 °C.

Purity: 94.7%.

Yield: 38%.

2(3) Synthesis of HCl.H-Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-NH ₂ Boc-Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-NH ₂ -> HCl. H-Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-NH ₂

Boc-Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-NH ₂ (14.3 g, 1 equivalent) prepared according to Example 2 (2) was mixed with dioxane (150 ml). To the mixture was added HCl gas (21.5 g) by bubbling while maintaining the temperature at 20 ± 5 °C. After 1 hour, the supernatant was discarded and the remaining oily residue was evaporated and dried by azeotropic distillation twice with dioxane (15 ml each time, 30 ml total).

2(4) Boc-[Arg(NO ₂ )-Ala-Asp(OBzl)-Ala] Synthesis of ₂ -NH ₂ Boc-Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-OH + HCl.H -Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-NH ₂ EDC/HOBt ->Boc-[Arg(NO ₂ )-Ala-Asp(OBzl)-Ala] ₂ -NH ₂

HCl.H-Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-NH ₂ (2.3 g, 1.00 equivalent) prepared according to Example 2 (3) was dissolved in DMF at 20 ± 5 °C (15) In ml). Diisopropylethylamine (0.9 ml, 1.4 equivalents), HOBt.H ₂ O (0.9 g, 0.9 eq.) and Boc-Arg(NO ₂ )-Ala-Asp prepared according to Example 1 (1a) were continuously added. (OBzl)-Ala-OH (2.3 g, 0.9 eq.). The mixture was then cooled to 0 ° C and EDC.HCl (1.1 g, 1.5 eq.) was then charged. The reaction mixture was allowed to reach room temperature (20 ° C) and stirred for about 20 hours to achieve complete coupling. The reaction mixture was concentrated in vacuo at 55 ± 5 °C until 7 ml of distillate was collected. Then at 20 ± 5 ° C The residue was poured into 2wt% NaHCO ₃ solution (35 ml). The resulting suspension was then filtered and the wet cake was washed three times with deionized water (5 ml each time, a total of 15 ml). The wet cake was dried under vacuum at 35 ± 5 °C.

Purity: 83%.

Yield: 71%.

2(5) TFA.H-[Arg(NO ₂ )-Ala-Asp(OBzl)-Ala] Synthesis of ₂ -NH ₂ TFA/Toluene Boc-[Arg(NO ₂ )-Ala-Asp(OBzl)-Ala ] ₂ -NH ₂ -> TFA.H-[Arg(NO ₂ )-Ala-Asp(OBzl)-Ala] ₂ -NH ₂

Boc-[Arg(NO ₂ )-Ala-Asp(OBzl)-Ala] ₂ -NH ₂ (2 g, 1.00 equivalent) prepared according to Example 2 (4) and toluene (12) at 20 ± 5 °C Ml) mixed. To the mixture was added dropwise TFA (8 ml) while maintaining the temperature at 20 ± 5 °C. After 1 hour, diisopropyl ether (40 ml) was added over 1 hour, then the resulting suspension was filtered and the wet cake was washed three times with diisopropyl ether (5 ml each, 15 ml total). The wet cake was dried under vacuum at 35 ± 5 °C.

Purity: 85.7%.

Yield: 100%. Example 3

According to the general scheme 3, the fragment Boc-Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-OAll is used to synthesize Ac-(RADA) ₂ -OH, which is the protected N-terminal dimerization of the target product. body.

3(1) Synthesis of HCl.H-Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-OAll Boc-Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-OAll -> HCl.H- Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-OAll

Boc-Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-OAll (10 g, 1 equivalent) prepared according to Example 1 (7) was mixed with dioxane (100 ml). To the mixture was added HCl gas (15 g) by bubbling while maintaining the temperature at 20 ± 5 °C. After 1 hour, the reaction was completed. The supernatant was discarded and the remaining oily residue was evaporated and dried by azeotropic distillation twice (10 ml each time, 20 ml total) with dioxane under vacuum. The oily residue was used directly.

Synthesis of 3(2) Ac-Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-OAllAc ₂ O HCl.H-Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-OAll -> Ac -Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-OAll

The oily residue (11 g, 1.00 equivalent) containing HCl.H-Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-OAll was dissolved in DMF (55 ml) at 20 ± 5 °C. The solution was cooled to 0 ± 5 °C. Diisopropylethylamine (3.2 g, 1.40 equivalents) and acetic anhydride (4.1 g, 1.40 equivalents) were added continuously while maintaining the temperature at 0 ± 5 °C. After the addition of acetic anhydride, the reaction mixture was warmed to 20 ± 5 °C. As indicated by HPLC monitoring, the reaction is usually completed in 3 hours at 20 °C. The reaction mixture was used directly in the next step.

Synthesis of 3(3) Ac-Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-OH Pd(PPh ₃ ) ₄ Ac-Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-OAll -> Ac-Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-OH

To the reaction mixture containing Ac-Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-OAll (10 g, 1 eq.) was added morpholine (6.7 g, 5.00 eq.) at 20 ± 5 °C. During 0.5 hour, N ₂ was bubbled through the solution to remove traces of oxygen. Pd(PPh ₃ ) ₄ (0.35 gg, 0.02 eq.) was added. The reaction mixture was stirred under a N ₂ atmosphere for about 2 hours. The reaction mixture was concentrated in vacuo at 55 ± 5 ° C until about 25 mL of residue remained. Methyl-THF (120 ml) was added at 20 ± 5 ° C to dissolve the residue, and then the solution was washed with 10 wt% aqueous KHSO ₄ (30 ml). The aqueous phase was extracted twice with methyl-THF (25 ml each time, a total of 50 ml). All organic phases were combined and concentrated in vacuo at 40 ± 5 °C until about 80 ml of residue was obtained. Ethyl acetate (60 ml) was added and the suspension was cooled to 10 ± 5 °C over 1 hour. The suspension was filtered. The filter cake was washed twice with ethyl acetate (40 ml each time, a total of 80 ml) and washed with diisopropyl ether (40 ml). The wet cake was dried under vacuum at 35 ± 5 °C.

Purity: 96.1%.

The overall yield of the three steps 3(1), 3(2) and 3(3) was 71%.

3(4) Synthesis of Ac-[Arg(NO ₂ )-Ala-Asp(OBzl)-Ala] ₂ -OAll Ac-Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-OH + HCl.H- Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-OAll EDC/HOBt ->Ac-[Arg(NO ₂ )-Ala-Asp(OBzl)-Ala] ₂ -OAll

An oily residue comprising HCl.H-Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-OAll (6.3 g, 1.00 equivalent) prepared according to Example 3(1) at 20 ± 5 °C Dissolved in a mixture of 0.4 M LiCl in DMF (42 ml). Diisopropylethylamine (1.5 g, 1.20 equivalents), HOBt.H ₂ O (1.6 g, 1.00 equivalents) and Ac-Arg(NO ₂ )-Ala-Asp prepared according to Example 3 (3) were continuously added. (OBzl)-Ala-OH (6 g, 1.00 equivalent). The mixture was then cooled to 0 ° C and EDC.HCl (2.3 g, 1.2 eq.) was added. The reaction mixture was warmed to 20 ° C and stirred for about 4 hours until the reaction indicated by HPLC monitoring was completed. The reaction mixture was concentrated in vacuo at 55 ± 5 ° C until 18 ml of distillate was collected. The residue was then poured into a 2% aqueous NaHCO3 solution (90 ml) at 20 ± 5 °C. The resulting suspension was then filtered. The filter cake was washed twice with water (12 ml each time, 24 ml total) and washed twice with ethyl acetate (15 ml each time, 30 ml total). The wet cake was dried under vacuum at 35 ± 5 °C.

Purity: 82.1%.

Yield: 83%.

3(5) Synthesis of Ac-[Arg(NO ₂ )-Ala-Asp(OBzl)-Ala] ₂ -OH Ac-[Arg(NO ₂ )-Ala-Asp(OBzl)-Ala] ₂ -OAll -> Ac-[Arg(NO ₂ )-Ala-Asp(OBzl)-Ala] ₂ -OH

Ac-[Arg(NO ₂ )-Ala-Asp(OBzl)-Ala] ₂ -OAll (6.3 g, 1.00 equivalent) prepared according to Example 3 (4) was dissolved in DMF at 80 ± 5 °C. In ml), then 1,3-dimethylbarbituric acid (2.5 g, 3.00 equivalents) was added. N _{2 was} bubbled through the solution during 0.5 hours to remove oxygen. Pd(PPh ₃ ) ₄ (0.1 g, 0.02 eq.) was added. The reaction mixture was stirred under N ₂ atmosphere over a period of about 2 hours until the reaction as indicated by HPLC monitoring was completed. The reaction mixture was concentrated in vacuo at 55 ± 5 ° C until the residual volume was about 25 ml. Deionized water (100 ml) was added to promote precipitation. The resulting suspension was filtered. The wet cake was washed twice with deionized water (20 ml each time, 40 ml total) and washed three times with ethyl acetate (25 ml each time, a total of 75 ml). The wet cake was dried under vacuum at 35 ± 5 °C.

Purity: 85.2%.

Yield: 72%. Example 4

4 The protected N- and C- terminal dimer connection scheme according to formula and deprotected by catalytic hydrogenation to produce Ac- [Arg-Ala-Asp- Ala] 4 -NH 2 .TFA.

4(1) Synthesis of Ac-[Arg(NO ₂ )-Ala-Asp(OBzl)-Ala] ₄ -NH ₂ Ac-[Arg(NO ₂ )-Ala-Asp(OBzl)-Ala] ₂ -OH + TFA.H-[Arg(NO ₂ )-Ala-Asp(OBzl)-Ala] ₂ -NH ₂ ->Ac-[Arg(NO ₂ )-Ala-Asp(OBzl)-Ala] ₄ -NH ₂

The TFA.H-[Arg(NO ₂ )-Ala-Asp(OBzl)-Ala] ₂ -NH ₂ (0.2 g, 1.00 equivalent) prepared according to Example 2 (5) was dissolved at 20 ± 5 °C. In NMP (3 ml). Ac-[Arg(NO ₂ )-Ala- prepared according to Example 3 (5) was continuously added with diisopropylethylamine (0.065 ml, 2.30 equivalent), HOBt.H ₂ O (0.026 g, 1.00 equivalent). Asp(OBzl)-Ala] ₂ -OH (0.188 g, 1.00 equivalent) and PyBOP (0.128 g, 1.50 equivalents). After stirring at 20 ° C for about 40 hours, the reaction was completed as indicated by HPLC monitoring. The reaction mixture was concentrated under vacuum at 55 ± 5 °C until 0.6 ml of distillate was collected. Then at 20 ± 5 ° C The residue was poured into 2wt% NaHCO ₃ aqueous solution (3 ml). The resulting suspension was filtered, and the filter cake was washed twice with deionized water (0.4 ml each time, a total of 0.8 ml) and twice with ethyl acetate (0.4 ml each time, a total of 0.8 ml). The wet cake was dried under vacuum at 35 ± 5 °C.

Purity: 68%.

Yield: 78%.

Synthesis of 4(2) Ac-[Arg-Ala-Asp-Ala] ₄ -NH ₂ as a crude TFA salt Ac-[Arg(NO ₂ )-Ala-Asp(OBzl)-Ala] ₄ -NH ₂ -> Ac -[Arg-Ala-Asp-Ala] ₄ -NH ₂ .TFA

The Ac-[Arg(NO ₂ )-Ala-Asp(OBzl)-Ala] ₄ -NH ₂ (0.5 g, 1.00 equivalent) prepared according to Example 4 (2) was dissolved in a flask at 20 ± 5 °C. In TFA (5 ml), Pd/C (load: 5 wt%, water content: 53 wt%) (0.71 g) was then added. At 20 ± 5 ° C, the flask was evacuated and refilled three times with H ₂ and then pressurized with 1.1 atmospheres of H ₂ . After stirring for about 2 hours, the reaction was completed as indicated by HPLC monitoring. The reaction mixture was then filtered through diatomaceous earth to remove Pd/C particles. TFA (3 ml) was added to wash the wet diatomaceous earth filter cake. The TFA filtrates were combined and concentrated in vacuo at 45 ± 5 °C until the residual volume was about 2 ml. The residue was poured into diisopropyl ether (7 ml) at 20 ± 5 °C. The resulting suspension was filtered. The filter cake was washed three times with diisopropyl ether (1.5 ml each time, a total of 4.5 ml) and dried under vacuum at 35 ± 5 °C.

Purity: 58%.

Yield: 62%. Example 5

The synthetic fragment Boc-Arg-Ala-Asp(OBzl)-Ala-OAll (1) was assembled by stepwise assembly according to the general scheme A.

5(1) Synthesis of Boc-Arg-Ala-Asp(OBzl)-Ala-OAll EDC / HOBt Boc-ArgOH + HCl.Ala-Asp(OBzl)-Ala-OAll -> Boc-Arg-Ala-Asp(OBzl )-Ala-OAll

The residue containing HCl.H-Ala-Asp(OBzl)-Ala-OAll (79.5 g, 1.0 eq.) prepared according to Example 1 (6) was dissolved in DCM (400 ml). The solution was washed with an aqueous solution of 5wt% NaHCO _3. The pH of the aqueous phase was adjusted to 7.5-7.8. The organic phase was then separated and concentrated in vacuo at 35 °C. Add DMF (290 ml). The resulting solution was cooled to 0 °C. HOBt.H ₂ O (19.9 g, 1.0 eq.) and Boc-Arg-OH.HCl.H ₂ O (50.9 g, 1.1 eq.) were then continuously added. EDC.HCl (49.5 g, 1.3 eq.) was slowly added while maintaining the internal temperature below 3 °C. The reaction mixture was then stirred at 3 ° C for 2 hours. The reaction mixture was then warmed to 20 ° C over 1 hour. After stirring at 20 ° C for about 2 hours, the reaction was completed as confirmed by HPLC monitoring. The mixture was then concentrated to an oily residue under vacuum at 45 ± 5 °C. DCM (360 ml) was added to dilute the oily residue. The solution was washed three times with 20 wt% saline (870 ml each time, a total of 2610 ml). The organic phase was concentrated in vacuo at 35 °C. Then add DIPE (1850 ml). The mixture was stirred at room temperature for at least 2 hours until the mixture became a homogeneous suspension. The mixture was filtered. The filter cake was washed with DIPE (220 ml). The filter cake was dried overnight at 40 °C.

Crude yield: 92.8%.

Purity: 97.7%. Example 6: Synthesis of Ac-(RADA) ₄ -NH ₂

The fragment Ac-(Arg-Ala-Asp(OBzl)-Ala) _2- OH was prepared according to the general scheme B.

The fragment TFA.H-(Arg-Ala-Asp(OBzl)-Ala) ₂ -NH _{2 was} prepared according to the general scheme C.

The fragment Ac-(Arg-Ala-Asp-Ala) ₄ -NH ₂ .TFA was prepared according to the general scheme D.

6(1) Synthesis of TFA.H-Arg-Ala-Asp(OBzl)-Ala-OAll Boc-Arg-Ala-Asp(OBzl)-Ala-OAll -> TFA.H-Arg-Ala-Asp(OBzl) -Ala-OAll

TFA (150 ml) was cooled to 10 °C. Then, Boc-Arg-Ala-Asp(OBzl)-Ala-OAll (25 g) prepared according to Example 5 (1) was slowly added while maintaining the temperature below 20 °C. The resulting mixture was then stirred at 20 ± 5 ° C for 1 hour. Then DCM (50 ml) was added to quench the reaction. The mixture was then concentrated in vacuo at 30 ± 5 °C. The residue was azeotroped twice with ACN (380 ml) to remove most of the residual TFA. DIPE (240 ml) was added to the residue. The mixture was stirred at room temperature for 30 minutes. Then the upper liquid phase is poured out. DIPE (340 ml) was added to the residue. The mixture was stirred at room temperature for at least 2 hours until the mixture became a homogeneous suspension. The mixture is then filtered. The filter cake was washed with DIPE (68 ml). The filter cake was dried overnight at 40 °C.

Crude yield: 98.5%.

Purity: 96.5%.

6(2) Synthesis of Ac-Arg-Ala-Asp(OBzl)-Ala-OAll TFA.H-Arg-Ala-Asp(OBzl)-Ala-OAll -> Ac-Arg-Ala-Asp(OBzl)-Ala -OAll

TFA.H-Arg-Ala-Asp(OBzl)-Ala-OAll (23.3 g, 1.0 eq.) prepared according to Example 6 (1) was dissolved in ACN (280 ml). The solution was cooled to 0 °C. Then DIPEA (7.4 ml, 1.2 eq.) was added while maintaining the temperature below 10 °C. Acetic acid (2.53 g, 1.2 equiv) was added HOBt.H ₂ O (4.7 g, 1.0 equiv). EDC.HCl (7.9 g, 1.3 eq.) was then added while maintaining the temperature below 3 °C. The reaction mixture was then stirred at 3 ° C for 2 hours. The reaction mixture was then warmed to 20 °C over 1 hour. After stirring for about 1 hour, the reaction was completed as indicated by HPLC monitoring. The mixture was then concentrated to an oil under vacuum at 35 ± 5 °C. DCM (230 ml) and DIPE (230 ml) were added to the residue. The resulting mixture was stirred at room temperature for 30 minutes. Pour out the upper liquid phase. DCM (230 ml) was added to dilute the residue. The mixture was stirred at room temperature for about 2 hours until the mixture became a homogeneous suspension. The mixture was filtered. The filter cake was washed with DCM (35 ml). The filter cake was dried overnight at 40 °C.

Crude yield: 109.7%.

Purity: 98.24%.

Synthesis of 6(3) Ac-Arg-Ala-Asp(OBzl)-Ala-OH Ac-Arg-Ala-Asp(OBzl)-Ala-OAll -> Ac-Arg-Ala-Asp(OBzl)-Ala-OH

Ac-Arg-Asp(OBzl)-Ala-OAll (25 g, 1.0 equivalent) prepared according to Example 6 (2) was dissolved in DMF (260 ml). 1,3-Dimethyl-barbituric acid (19.5 g, 3.0 eq.) was added followed by Pd(PPh ₃ ) ₄ (1.4 g, 0.04 eq.). The resulting mixture was degassed and backfilled in a nitrogen atmosphere three times. After stirring at 20 ° C for about 3 hours, the reaction was completed as indicated by HPLC monitoring. The mixture was then concentrated to an oil under vacuum at 45 ± 5 °C. To the residue were added DCM (250 ml) and DIPE (250 ml). The resulting mixture was stirred at room temperature for 30 minutes. Pour out the upper liquid phase. Add DCM (150 ml). The mixture was stirred at room temperature for at least 2 hours until the mixture became a homogeneous suspension. Filter the solution. The filter cake was washed with DCM (40 ml). The filter cake was dried overnight at 40 °C.

Crude yield: 86.1%.

Purity: 96.77%.

6(4) Synthesis of Ac-(Arg-Ala-Asp(OBzl)-Ala) ₂ -OAll Ac-Arg-Ala-Asp(OBzl)-Ala-OH + TFA.H-Arg-Ala-Asp(OBzl) -Ala-OAll -> Ac-(Arg-Ala-Asp(OBzl)-Ala) ₂ -OAll

TFA.H-Arg-Ala-Asp(OBzl)-Ala-OAll (25 g, 1.0 eq.) prepared according to Example 6 (1) was dissolved in DMF (100 ml). The solution was cooled to 0 °C. Then add DIPEA (8.1 ml) while keeping the temperature below 10 °C. A solution of Ac-Arg-Ala-Asp(OBzl)-Ala-OH (23 g, 1.0 eq.) prepared in Example 6 (3) in DMF (120 mL). HOBt.H ₂ O (4.6 g, 1.0 eq.) was then added followed by EDC.HCl (8.3 g, 2 eq.) while maintaining the temperature below 3 °C. The mixture was stirred at 0 ° C for 2 hours. The mixture was then warmed to 20 °C over 1 hour. After stirring for an additional 2 hours at 20 ° C, the reaction was completed as indicated by HPLC monitoring. The mixture was then concentrated under vacuum at 45 ± 5 °C. DCM (240 ml) and DIPE (270 ml) were added to the residue. The mixture was stirred at room temperature for 30 minutes. The upper clear liquid phase is then poured out. Additional DCM (360 ml) was added to the residue. The mixture was stirred at room temperature for about 2 hours until the mixture became a homogeneous suspension. The suspension was filtered. The filter cake was washed with DCM (30 mL) and dried overnight at 40 °C.

Crude yield: 77.0%.

Purity: 90.03%.

6(5) Synthesis of Ac-(Arg-Ala-Asp(OBzl)-Ala) ₂ -OH Pd(PPh ₃ ) ₄ Ac-(Arg-Ala-Asp(OBzl)-Ala) ₂ -OAll -> Ac- (Arg-Ala-Asp(OBzl)-Ala) ₂ -OH

Ac-(Arg-Ala-Asp(OBzl)-Ala) ₂ -OAll (28 g, 1 equivalent) prepared according to Example 6 (4) was dissolved in DMF (750 ml). 1,3-Dimethyl-barbituric acid (11.8 g, 3 equivalents) and Pd(PPh ₃ ) ₄ (0.9 g, 0.03 equivalents) were continuously added. Nitrogen gas was bubbled through the solution to remove traces of oxygen. The reaction mixture was then stirred at room temperature for 3 hours and the completion of the reaction was confirmed by HPLC. The mixture was concentrated under vacuum at 40 ± 5 °C. DIPE (500 ml) was added to the residue. The mixture was stirred at room temperature for 30 minutes. The mixture was filtered. The filter cake was washed with DIPE (80 ml) and suspended in DCM (400 ml). The mixture is then filtered. The filter cake was washed with DCM (40 ml) and evaporated in ACN (360 ml). The mixture is then filtered. The filter cake was washed with ACN (50 ml), filtered and dried overnight at 40 °C.

Crude yield: 84.4%.

Purity: 91.91%.

6(6) Synthesis of Boc-Arg-Ala-Asp(OBzl)-Ala-OH Pd(PPh ₃ ) ₄ Boc-Arg-Ala-Asp(OBzl)-Ala-OAll -> Boc-Arg-Ala-Asp( OBzl)-Ala-OH

Add 1 to a solution of Boc-Arg-Asp(OBzl)-Ala-OAll (50 g, 1.0 eq.) prepared in accordance with Example 5 (1) in dioxane (780 ml) and DMF (210 ml). 3-Dimethyl-barbituric acid (35.5 g, 3.0 eq.). Then Pd(PPh ₃ ) ₄ (3.5 g, 0.04 equivalent) was added. The resulting mixture was degassed and backfilled in a nitrogen atmosphere three times. The mixture was then stirred at room temperature for 3 hours and the completion of the reaction was confirmed by HPLC. The mixture was then concentrated to an oil under vacuum at 45 ± 5 °C. To the residue were added DCM (500 ml) and DIPE (500 ml). The resulting mixture was stirred at room temperature for 30 minutes. Pour out the upper liquid phase. Add ACN (500 ml). The mixture was stirred at room temperature for 30 minutes. Pour out the liquid phase. Then add ACN (250 ml). The mixture was stirred at room temperature for 30 minutes. Pour out the liquid phase. Add MeOH (650 ml). The mixture was stirred until dissolved. Filter the solution. The filtrate was collected and concentrated under vacuum at 35 ± 5 °C. The solid was collected and dried overnight at 40 °C.

Crude yield: 86.1%.

Purity: 97.4%.

6(7) Synthesis of Boc-Arg-Ala-Asp(OBzl)-Ala-NH ₂ Boc-Arg-Ala-Asp(OBzl)-Ala-OH -> Boc-Arg-Ala-Asp(OBzl)-Ala- NH ₂

Boc-Arg-Ala-Asp(OBzl)-Ala-OH (20 g, 1 equivalent) prepared according to Example 6 (6) was dissolved in a mixture of dioxane (250 ml) and acetonitrile (100 ml). . Solution in water (30 ml) was then added NH ₄ Cl (8.6 g, 5 eq). The resulting mixture was cooled to 5 °C. HOAt (4.4 g, 1 eq.) was added followed by EDC.HCl (37 g, 5 eq.). During the addition, the temperature was kept below 10 °C. The mixture was then stirred at 5 ° C for 2 hours. The mixture was then warmed to 20 ° C over 1 hour. After stirring at 20 ° C for an additional hour, HPLC monitoring showed the reaction was complete. The mixture is then filtered. The filter cake was washed with MeOH (150 mL). The filtrate was collected and concentrated under vacuum at 35 ± 5 °C. Then 20 wt% saline (240 ml) was added to the residue. The mixture was extracted three times with 10 wt% MeOH in DCM (980 ml each time, a total of 2940 ml). The organic phase was dried over MgSO ₄ (120 g) 3 hours. The mixture is then filtered. The filtrate was collected and concentrated under vacuum at 35 ± 5 °C. The resulting solid was dried overnight at 40 °C.

Crude yield: 121.6%.

Purity: 90.18%.

6(8) TFA.H-Arg-Ala-Asp(OBzl)-Ala-NH ₂ Synthesis Boc-Arg-Ala-Asp(OBzl)-Ala-NH ₂ -> TFA.H-Arg-Ala-Asp(OBzl )-Ala-NH ₂

The TFA (240 ml) was cooled to 10 °C in an ice bath. Boc-Arg-Ala-Asp(OBzl)-Ala-NH ₂ (31.5 g) prepared according to Example 6 (7) was then added while maintaining the temperature below 20 °C. The resulting mixture was then stirred at 20 ± 5 ° C for 1 hour. Then add DCM (60 ml). The mixture was then concentrated under vacuum at 35 ± 5 °C. The residue was dissolved in acetonitrile (450 mL) and evaporated to dryness to remove most of the residual TFA. The removal operation of the residual TFA is performed once more. DIPE (300 ml) was added to the residue. The mixture was stirred at room temperature for 30 minutes. Then the upper liquid phase is poured out. DCM (225 ml) was added to the residue. The mixture was stirred at room temperature until the mixture was a homogeneous suspension. The mixture was then filtered. The filter cake was washed with DCM (45 ml) and dried overnight at 40 °C.

Crude yield: 85.5%.

Purity: 89.15%.

6(9) Boc-[Arg-Ala-Asp(OBzl)-Ala] Synthesis of ₂ -NH ₂ Boc-Arg-Ala-Asp(OBzl)-Ala-OH + TFA.H-Arg-Ala-Asp(OBzl )-Ala-NH ₂ EDC/HOBt -> Boc-[Arg-Ala-Asp(OBzl)-Ala] ₂ -NH ₂

TFA.H-Arg-Ala-Asp(OBzl)-Ala-NH ₂ (17.9 g, 1.0 eq.) prepared according to Example 6 (8) was dissolved in DMF (160 ml) and cooled to 0. Then DIPEA (6.2 g, 1.5 eq.) was added while maintaining the temperature below 10 °C. A solution of Boc-Arg-Ala-Asp(OBzl)-Ala-OH (20.4 g, 1.0 eq.) prepared in Example 6 (6) in DMF (160 ml) was added. HOBt.H ₂ O (4.3 g, 1.0 eq.) was then added followed by EDC.HCl (12.3 g, 2 eq.) while maintaining the temperature below 3 °C. The mixture was then stirred at 0 ° C for 2 hours. The mixture was then warmed to 20 ° C over 1 hour. After stirring at 20 ° C for about 1 hour, HPLC monitoring showed the reaction was completed. The mixture was then concentrated under vacuum at 45 ± 5 °C. DCM (170 ml) and DIPE (190 ml) were added to the residue. The mixture was stirred at room temperature for 30 minutes. Then the upper liquid phase is poured out. DCM (250 ml) was added to the solid. The mixture was stirred at room temperature for 30 minutes. The solid was then suspended in ACN (170 ml) and stirred at room temperature until the mixture became a homogeneous suspension. The mixture was filtered. The filter cake was washed with ACN (40 mL), filtered and dried overnight at 40 °C.

Crude yield: 71.84%.

Purity: 90%.

6(10) TFA.H-[Arg-Ala-Asp(OBzl)-Ala] Synthesis of ₂ -NH ₂ TFA Boc-(Arg-Ala-Asp(OBzl)-Ala) ₂ -NH ₂ -> TFA.H -(Arg-Ala-Asp(OBzl)-Ala) ₂ -NH ₂

Cool TFA (100 ml) to 10 °C. Boc-(Arg-Ala-Asp(OBzl)-Ala) ₂ -NH ₂ (26 g) prepared according to Example 6 (9) was added while maintaining the temperature below 20 °C. The resulting mixture was stirred at 20 ± 5 ° C for 1 hour. Then add DCM (45 ml). The mixture was then concentrated under vacuum at 3 ± 5 °C. The residue was dissolved in ACN (350 mL) and evaporated to dryness to remove residual TFA. The removal operation of the residual TFA is performed once more. ACN (270 ml) was added to the residue, and the mixture was stirred at room temperature until a homogeneous suspension was obtained. After filtration, the filter cake was suspended in ACN (60 mL) filtered and dried overnight at 40 °C.

Crude yield: 100%.

Purity: 90.17%.

6(11) Ac-(Arg-Ala-Asp(OBzl)-Ala) Synthesis of _4- NH ₂ Ac-(Arg-Ala-Asp(OBzl)-Ala) ₂ -OH + TFA.H-(Arg-Ala -Asp(OBzl)-Ala) ₂ -NH ₂ ->Ac-(Arg-Ala-Asp(OBzl)-Ala) ₄ -NH ₂

Ac-(Arg-Ala-Asp(OBzl)-Ala) ₂ -OH (20.5 g, 1 equivalent) prepared according to Example 6 (5) was dissolved in DMF (1700 ml). TFA.H-(Arg-Ala-Asp(OBzl)-Ala)2-NH2 (24.1 g, 1 equivalent) prepared according to Example 6 (10) was dissolved in DMA (500 ml). The two solutions were combined and cooled to 0 °C. Then DIPEA (2.7 g, 1.05 equivalents) and HOAt (2.2 g, 1 equivalent) were added. EDC.HCl (6.1 g, 2 equivalents) was then added while maintaining the temperature below 3 °C. The mixture was then stirred at 0 ° C for 2 hours. The mixture was then warmed to 20 ° C over 1 hour. After stirring at 20 ° C for about 40 hours, HPLC monitoring showed the reaction was complete. The mixture was then concentrated under vacuum at 55 ± 5 °C. ACN (480 ml) and DIPE (530 ml) were added to the residue. The mixture was stirred at room temperature for 30 minutes. Then the upper liquid phase is poured out. ACN (500 ml) was added to the residue. The mixture was stirred at room temperature until the mixture became a homogeneous suspension. The mixture was then filtered and the filter cake was washed with ACN (50 mL). The filter cake was dried overnight at 40 °C.

Crude yield: 103.8%.

Purity: 81.35%.

6(12) Ac-(Arg-Ala-Asp-Ala) ₄ -NH ₂ . Synthesis of TFA Ac-(Arg-Ala-Asp(OBzl)-Ala) ₄ -NH ₂ -> Ac-(Arg-Ala- Asp-Ala) ₄ -NH ₂ .TFA

Ac-[Arg-Ala-Asp(OBzl)-Ala] ₄ -NH ₂ (33.7 g, 1.00 equivalent) prepared according to Example 6 (11) was dissolved in TFA (900 ml) at 20 ± 5 °C. Then, Pd/C (2.5 g, loading: 5% Pd, water content 53%) was added. The resulting mixture was degassed and backfilled with a hydrogen atmosphere three times. The reaction mixture was then stirred at 20 ± 5 ° C for 4 hours under 1.1 atmospheres of H ₂ . The mixture was then filtered through diatomaceous earth to remove Pd/C particles. The diatomaceous earth filter cake was washed with TFA (20 ml). The TFA filtrates were combined and concentrated in vacuo <RTI ID=0.0> The residual TFA was removed by azeotropic distillation with DCM (280 ml) and the procedure was carried out again. DIPE (300 ml) was then added to the residue. The mixture was stirred at room temperature for 30 minutes. Pour out the liquid phase. DCM (480 ml) was added to the solid. The mixture was stirred at room temperature until the mixture was a homogeneous suspension. The mixture is then filtered. The filter cake was washed with DCM (50 mL) filtered and dried overnight at 40 &lt;0&gt;C.

Crude yield: 104.3%.

Purity: 46.52%. Example 7

7(1) Preliminary purification Each Ac-(Arg-Ala-Asp-Ala) ₄ -NH ₂ .TFA prepared according to Example 4 (2) and according to Example 6 (12) was purified.

7(2) Salt exchange Salt exchange from TFA salt to HCl salt was carried out by chromatography under the following conditions:

An Amberchrom® HP10 column or any conventional hydrophobic stationary phase column such as a C8 to C18 column can also be used as a fix for the purification or salt exchange.

no

no

Claims (19)

A method of preparing a peptide (RADA) ₄ , wherein an octapeptide having a sequence (RADA) ₂ is coupled in a liquid phase to provide the peptide (RADA) ₄ ; the octapeptide is obtained by having a sequence in a liquid phase The tetrapeptide coupling of RADA is prepared; the tetrapeptide having the sequence RADA is prepared in a liquid phase. The method according to claim 1, wherein the tetrapeptide having the sequence RADA is prepared by stepwise coupling of a single amino acid. The method according to claim 1 or 2, wherein Asp is used in its side chain protection form Asp (OBzl). The method according to one or more of claims 1 to 3, wherein Arg is used only in the form of an unprotected side chain or Arg is used only in its side chain protected form Arg(NO ₂ ). The method of claim 4, wherein Arg is used only in the form of unprotected side chains. The method of one or more of claims 1 to 5, wherein the coupling of the tetrapeptide is carried out at a temperature below 5 °C. The method of one or more of claims 1 to 6, wherein the coupling of the octapeptide is carried out at a temperature below 5 °C. The method of one or more of claims 1 to 7, wherein any protection of the N-terminal alpha amino residue during any coupling is carried out by using Boc or Ac. The method of one or more of claims 1 to 8, wherein any protection of the C-terminal COOH residue during any coupling is carried out by using All or in the form of indoleamine C(O)NH ₂ . The method according to claim 9, wherein any cleavage of All is carried out by using the reaction CLEAVALL of the catalyst Pd(PPh ₃ ) ₄ . The method of claim 10, wherein the CLEAVALL is carried out in the presence of the additive ADDALL selected from the group consisting of morpholine, 1,3-dimethylbarbituric acid, and mixtures thereof. The method of one or more of claims 1 to 11, wherein the peptide (RADA) ₄ is provided in the form of the peptide PEP-AC-NH2; the PEP-AC-NH2 is Ac-(RADA) ₄ -NH ₂ . The method of claim 12, wherein the method comprises the step STEPB; in STEPB, the PEP-AC-NH2 is prepared by coupling the fragment FRAG2-AC-OH with the fragment FRAG2-H-NH2; FRAG2- AC-OH is Ac-(RADA) ₂ -OH; FRAG2-H-NH2 is H-(RADA) ₂ -NH ₂ . The method of claim 13, wherein the method comprises the step STEPA1; preparing the FRAG2-H-NH2 in STEPA1; STEPA1 comprises coupling the fragment FRAGA1-Boc-OH to the fragment FRAGA1-H-NH2, and subsequently The N-terminal Boc residue is cleaved; FRAGA1-Boc-OH is Boc-RADA-OH; FRAGA1-H-NH2 is H-RADA-NH ₂ . The method of claim 13 or 14, wherein the method comprises the step STEPA2; preparing FRAG2-AC-OH in STEPA2; STEPA2 comprises coupling the fragment FRAGA2-Ac-OH to the fragment FRAGA2-H-OAll, and including The C-terminal All residue is subsequently cleaved; FRAGA2-Ac-OH is Ac-RADA-OH; FRAGA2-H-OAll is H-RADA-OAll. The method of one or more of claims 1 to 15, wherein for the preparation of the tetrapeptide having the sequence RADA, the three amino acids R, A and D are as Boc-Ala-OH, Boc-Asp (OBzl -OH and Boc-Arg(NO ₂ )-OH are used; or as Boc-Ala-OH, Boc-Asp(OBzl)-OH and Boc-Arg-OH. The method of one or more of claims 3 to 16, wherein the cleavage of any of the side chain protecting groups is carried out after the coupling of the octapeptide. The method of one or more of claims 4 to 17, wherein the NO ₂ protecting group of Arg and the Bzl protecting group of Asp are cleaved by catalytic hydrogenation. The method according to one or more of claims 1 to 18, wherein Boc-Arg(NO ₂ )-Ala-Asp(OBzl)-Ala-OAll or Boc-Arg-Ala-Asp(OBzl)-Ala-OAll As an intermediate.