KR20090091863A - Peptide derivatives and method for preparing thereof - Google Patents

Abstract

A peptide derivative of the chemical formula (I) and its manufacturing method are provided to induce two carbon chains at the N-terminal using AKD (alkyl ketene dimer) and produce the novel peptide derivative. A peptide derivative of the chemical formula (I) is produced by reacting AKD (alkyl ketene dimer) and peptide on the organic solvent phase. In the chemical formula (I), R1 and R2 is C1-C20 saturated or unsaturated alkyl group of linear or branched type, X is O or N, R is H or alkyl group of C1-C10. The peptide derivative of the chemical formula (I) is excellent in permeability on cell or skin.

Description

Peptide Derivatives and Methods for Preparing Thereof

The present invention relates to peptide derivatives of the general formula (I) More specifically, the present invention relates to a peptide derivative of formula (I) and a method of preparing a peptide derivative of formula (I) comprising the step of reacting a peptide with an alkyl ketene dimer (AKD).

Formula (I)

Wherein R 1 and R 2 are alkyl groups having straight or branched C ₁ to C ₂₀ saturated or unsaturated bonds; X is O or N; R is H or a C ₁ to C ₁₀ alkyl group; PEPTIDE is a peptide in which one or more amino acids are bound.

Chemical synthesis of peptides can be largely divided into solution phase synthesis and solid phase synthesis. Solution-phase synthesis is a classical chemical synthesis method in which all reactants are dissolved in a solvent. The reaction rate is high, but the separation and purification are difficult.

Solid phase synthesis has been developed since R. B. Merrifield's thesis on solid phase peptide synthesis, and has the advantage of easy separation and automation.

Since Merrifield's research, support resins for numerous peptide synthesis have been developed and many peptides have been synthesized using them. The chloromethyl polystyrene resin developed by Merrifield, and Wang resin having a structure of 4-alkoxybenzyl alcohol, which is developed to compensate for the disadvantages of this resin, are widely used.

Since then, support resins have been continuously developed to compensate for their shortcomings. Among them, 2-chlorotrityl resin having a trityl structure and linkamide resin which can obtain the carboxyl terminal of the peptide in amide form are representative peptide synthesis. It is support resin for reaction. Using these support resins, simple peptides were initially synthesized, but gradually complex peptides having physiological activity were synthesized.

It is a primary object of the present invention to provide peptide derivatives of formula (I) having high cell and skin permeability.

Formula (I)

Wherein R 1 and R 2 are alkyl groups having straight or branched C ₁ to C ₂₀ saturated or unsaturated bonds; X is O or N; R is H or a C ₁ to C ₁₀ alkyl group; PEPTIDE is a peptide in which one or more amino acids are bound.

Still another object of the present invention is to provide a method for preparing a peptide derivative of formula (I), which comprises reacting a peptide with an alkyl ketene dimer (AKD).

The basic object of the present invention described above can be achieved by providing a peptide derivative of formula (I) having high cell and skin permeability.

Formula (I)

Wherein R 1 and R 2 are alkyl groups having straight or branched C ₁ to C ₂₀ saturated or unsaturated bonds; X is O or N; R is H or a C ₁ to C ₁₀ alkyl group; PEPTIDE is a peptide in which one or more amino acids are bound.

Another object of the present invention described above can be achieved by providing a method for preparing a peptide derivative of formula (I), comprising the step of reacting a peptide with an alkyl ketene dimer (AKD).

The peptide may be prepared on a polymer support or in solution according to a known synthesis method as in Scheme 1 below.

Scheme 1

In Scheme 1, Y represents an amine protecting group, Z represents a side chain protecting group of an amino acid, and X represents oxygen or nitrogen. W represents a reactive functional group of the polymer support, and halogen, hydroxy, amine, thiol, and the like may be used.

R in solution phase synthesis represents an alkyl group, indicating that the C-terminus of the amino acid is an ester.

Such peptide synthesis reactions can generally employ known peptide synthesis methods (Synthetic Peptides: A User's Guide, G.R.Grant, ed., Freeman & Co., 1992, pp.77-183).

Coupling of amino acids is carried out using active esters of each amino acid or by using dicyclohexyl carbodiimide (DCC), diisopropyl carbodiimide (DIC), benzotriazole-1-yl-oxy-tris- (dimethylamino) phosphonium hexafluorophosphate (BOP), and benzotriazole-1- (PyBOP). yl-oxytripyrrolidinophosphonium hexafluorophosphate), HBTU (o-benzotriazole-N, N, N ', N'-tetramethyluronium hexafluorophosphate), TBTU (o- (benzotriazole-1-yl) -N, N, N', N'-tetramethyluronium tetrafluoroborate ), HATU (2- (1H-7-azabenzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate), TATU (2- (1H-7-azabenzotriazol-1-yl) -1,1, 3,3-tetramethyluronium tetrafluoroborate (CDI), carbonyl diimidazole (CDI) and the like can be optionally used in coupling reagents, the amount is used 1 to 10 equivalents, preferably 1.5 to 3 equivalents. The amount of the amino acid derivative is used in the range of 1 to 10 equivalents, preferably 1.5 to 3 equivalents, based on the substitution rate of the resin.

The amino acid active ester is preferably used 1 to 10 equivalents of a substance that activates the C-terminus of the amino acid, such as symmetric anhydride, mixed anhydride, pentafluoro phenyl ester of the amino acid derivative.

The desired peptide can be obtained by repeating the coupling step and the amine protecting group removal step.

The polymeric support resin is selected from polystyrene, polyamide, glass and silica.

The reaction is any one selected from the group consisting of methanol, ethanol, dichloromethane, chloroform, dichloroethane, dimethylformamide, dimethylacetamide, N-methylpyrrolidinone, tetrahydrofuran or dioxane. Is used as a reaction solvent.

The reaction temperature of the reaction is not limited, but is preferably in the range of 0 ° C to 150 ° C, more preferably 20 ° C to 70 ° C.

In addition, the reaction time of the reaction is in the range of 10 minutes to 48 hours, preferably in the range of 0.5 hours to 6 hours, considering the reactivity of each reactant and the productivity of the product after the reaction. However, when the reaction does not proceed as desired, the same reaction may be performed two to five times more to increase the reaction yield.

The peptide prepared in Scheme 1 was reacted with an alkyl ketene dimer (AKD) prepared in Scheme 2 in an organic solvent (Scheme 3) to prepare a novel peptide derivative.

Scheme 2

Scheme 3

In Scheme 2, AKD may be a single AKD or mixed AKD, and an alkyl group may be a saturated carbon chain or an unsaturated bond. The single AKD is AKD synthesized from a single acyl chloride (ie, R 1 = R 2), and the mixed AKD is AKD synthesized from two kinds of acyl chlorides (ie, R 1? R 2).

In Scheme 3, AA represents an amino acid, Y represents a side chain protecting group of an amino acid, X represents oxygen or nitrogen, and R represents hydrogen or an alkyl group. That is, the C-terminus of the peptide is carboxylic acid or amide.

As the organic solvent used in Scheme 3, tertiary amines such as TEA (triethylamine), TMA (trimethylamine), DIEA (diisopropylethylamine), NMP (N-methylmorpholine) or pyridine can be used.

The present invention relates to a method for introducing a double carbon chain at the N-terminus of a peptide synthesized in a liquid phase and a solid phase, and to a peptide derivative synthesized by the method. A peptide is prepared by a generally known method, and an alkyl ketene dimer is prepared. ) Will introduce two carbon chains at the N-terminus. Using this method, various types of carbon chains can be introduced effectively, enabling the production of novel peptide derivatives in an effective and novel way that has not been attempted in the prior art.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following description of the embodiments is only intended to specifically describe the specific embodiments of the present invention, it is not intended to limit or limit the scope of the present invention to the contents described therein.

Example 1 Preparation of Single AKD

12.6 g of acyl chloride (C ₁₈ ) was dissolved in 150 mL of tetrahydrofuran, and 12 mL of pyridine was added thereto, followed by reaction for 12 hours with stirring. After removing the solvent by rotary evaporation, the white solid obtained was dissolved in reflux in 250 mL of ethanol, and cooled to give 4.4 g (yield 70%) of white AKD (C ₁₈ ).

Example 2. Preparation of Peptides (AKD-VGVAPG-OH)

Fmoc was removed by treating 1 g of 2-chlorotrityl resin having a substitution rate of 0.4 mmol / g with fluorenylmethoxycarbonyl-glycine bound to 20% piperidine / DMF solution. The resin was washed with DMF and methanol and then swollen in 10 mL of DMF. Thereafter, 387 mg (1.3 mmol) of Fmoc-Pro-OH, 201 μL (1.3 mmol) of DIC, and 176 mg (1.3 mmol) of HOBt were added and reacted for 1.5 hours.

The resulting resin was treated with 20% piperidine to remove the Fmoc group, followed by 405 mg (1.3 mmol) of Fmoc-Ala-OH, 201 μL (1.3 mmol), and 176 mg (1.3 mmol) of HOBt. It was reacted using. The obtained resin was treated with 20% piperidine to remove the Fmoc group. In the same manner, Fmoc-Val-OH 441 mg (1.3 mmol), DIC 201 μL (1.3 mmol) and HOBt 176 mg (1.3 mmol) were reacted and the resin was washed again and treated with 20% piperidine to give Fmoc The group was removed.

In the same manner, the resin was washed with 387 mg (1.3 mmol) of Fmoc-Gly-OH, 201 μL (1.3 mmol) of DIC, and 176 mg (1.3 mmol) of HOBt, followed by 20% piperidine treatment with Fmoc. The group was removed. In the same manner, 859 mg (1.3 mmol) of Fmoc-Val-OH, 201 μL (1.3 mmol) of DIC, and 176 mg (1.3 mmol) of HOBt were reacted and the resin was washed again. Treatment with 20% piperidine removes the Fmoc group.

The resin was washed with DMF and methanol and then swollen in 10 mL of DMF. Thereafter, 693 mg (1.3 mmol) of AKD prepared in Example 1 and 244 μL (1.3 mmol) of DIEA were dissolved in tetrahydrofuran and reacted for 3 hours, followed by washing.

     For 70 mg of peptide resin, 2 mL of 1% TFA / DCM was added to the resin and eluted to release the peptide from the resin and receive the filtrate. The process was repeated five times. The resin was washed with DCM and methanol, collected together with the filtrate, the combined solution was concentrated by evaporation and ether was added to obtain peptide precipitate. The obtained peptide was separated using a reversed phase column to give a yield of 85%.

Example  3. Of the peptide (AKD-KTTKS-OH)  Produce

1 g of 2-chloro trityl chloride resin having a substitution rate of 0.6 mmol / g combined with Fmoc-Ser (tBu) was washed with DMF and methanol, and then treated with 20% piperidine to remove Fmoc. In the same manner as in Example 2, reaction was performed using 843 mg (1.8 mmol) of Fmoc-Lys (Boc) -OH, 279 μL (1.8 mmol) of DIC, and 243 mg (1.8 mmol) of HOBt.

The obtained resin was treated with 20% piperidine to remove the Fmoc group. In the same manner, Fmoc-Thr (tBu) -OH was reacted with 715 mg (1.8 mmol), DIC 279 μL (1.8 mmol), HOBt 243 mg (1.8 mmol), and the resin was washed again, followed by 20% piperidine. Treatment removes the Fmoc group.

In the same manner, the reaction was carried out using 715 mg (1.8 mmol) of Fmoc-Thr (tBu) -OH, 279 μL (1.8 mmol) of DIC, and 243 mg (1.8 mmol) of HOBt, and the resin was washed again and treated with 20% piperidine. Fmoc group was removed.

In the same manner, 843 mg (1.8 mmol) of Fmoc-Lys (Boc) -OH, 279 μL (1.8 mmol) of DIC, and 243 mg (1.8 mmol) of HOBt were reacted and the resin was washed again. Treatment with 20% piperidine removes the Fmoc group.

After washing the resin with DMF and methanol and swelling in 10 mL of DMF, 959 mg (1.8 mmol) of AKD prepared in Example 1 and 338 μL (1.8 mmol) of DIEA were dissolved in tetrahydrofuran and reacted for 3 hours. Washed.

For 70 mg of peptide resin, 2 mL of 1% TFA / DCM was added to the resin and eluted to release the peptide from the resin and receive the filtrate. This process was repeated five times. The resin was washed with DCM and methanol, the filtrates were collected together, the combined solution was concentrated by evaporation, and ether was added to obtain peptide precipitates. The obtained peptide was separated using a reversed phase column to give a yield of 82%.

Example  4. Of the peptide (AKD-GPQGPQ-OH)  Produce

1 g of 2-chloro trityl resin having a substitution rate of 0.6 mmol / g combined with Fmoc-Gln (Trt) was washed with DMF and methanol, and then treated with 20% piperidine to remove Fmoc. In the same manner as in Example 2, 607 mg (1.8 mmol) of Fmoc-Pro-OH, 279 μL (1.8 mmol) of DIC, and 243 mg (1.8 mmol) of HOBt were used.

The obtained resin was treated with 20% piperidine to remove the Fmoc group. In the same manner, Fmoc-Gly-OH 535 mg (1.8 mmol), DIC 279 μL (1.8 mmol) and HOBt 243 mg (1.8 mmol) were reacted and the resin was washed again and treated with 20% piperidine to give Fmoc The group was removed.

In the same manner, the reaction was performed using 1.1 g (1.8 mmol) of Fmoc-Gln (Trt) -OH, 279 μL (1.8 mmol) of DIC, and 243 mg (1.8 mmol) of HOBt, and the resin was washed again and treated with 20% piperidine. Fmoc group was removed.

In the same manner, 607 mg (1.8 mmol) of Fmoc-Pro-OH, 279 μL (1.8 mmol) of DIC, and 243 mg (1.8 mmol) of HOBt were reacted and the resin was washed again.

In the same manner, Fmoc-Gly-OH 535 mg (1.8 mmol), DIC 279 μL (1.8 mmol) and HOBt 243 mg (1.8 mmol) were reacted and the resin was washed again and treated with 20% piperidine to give Fmoc The group was removed.

After washing the resin with DMF and methanol and swelling in 10 mL of DMF, 959 mg (1.8 mmol) of AKD prepared in Example 1 and 338 μL (1.8 mmol) of DIEA were dissolved in tetrahydrofuran and reacted for 3 hours. Washed.

     For 70 mg of peptide resin, 2 mL of 1% TFA / DCM was added to the resin and eluted to release the peptide from the resin and receive the filtrate. This process was repeated five times. The resin was washed with DCM and methanol, collected together with the filtrate, the combined solution was concentrated by evaporation and ether was added to obtain peptide precipitate. The obtained peptide was separated using a reversed phase column to give a yield of 85%.

Example  5. Of the peptide (AKD-EEMQRR-OH)  Produce

Fmoc was removed by washing 1 g of 2-chloro trityl resin having a substitution rate of 0.6 mmol / g with Fmoc-Arg (Pbf), using DMF and methanol, followed by 20% piperidine treatment. The reaction was carried out in the same manner as in Example 2, using 1.2 g (1.8 mmol) of Fmoc-Arg (Pbf) -OH, 279 μL (1.8 mmol) of DIC, and 243 mg (1.8 mmol) of HOBt. The obtained resin was treated with 20% piperidine to remove the Fmoc group.

In the same manner, the reaction was performed using 1.1 g (1.8 mmol) of Fmoc-Gln (Trt) -OH, 279 μL (1.8 mmol) of DIC, and 243 mg (1.8 mmol) of HOBt, and the resin was washed again, followed by 20% piperidine. Treatment removes the Fmoc group.

In the same manner, 669 mg (1.8 mmol) of Fmoc-Met-OH, 279 μL (1.8 mmol) of DIC, and 243 mg (1.8 mmol) of HOBt were reacted and the resin was washed again and treated with 20% piperidine. Removed.

In the same manner, 766 mg (1.8 mmol) of Fmoc-Glu (otBu) -OH, 279 μL (1.8 mmol) of DIC, and 243 mg (1.8 mmol) of HOBt were reacted and the resin was washed again. Treatment with 20% piperidine removes the Fmoc group.

In the same manner, 766 mg (1.8 mmol) of Fmoc-Glu (otBu) -OH, 279 μL (1.8 mmol) of DIC, and 243 mg (1.8 mmol) of HOBt were reacted and the resin was washed again. Treatment with 20% piperidine removes the Fmoc group.

After washing the resin with DMF and methanol and swelling in 10 mL of DMF, 959 mg (1.8 mmol) of AKD prepared in Example 1 and 338 μL (1.8 mmol) of DIEA were dissolved in tetrahydrofuran and reacted for 3 hours. Washed.

For 70 mg of peptide resin, 2 mL of 1% TFA / DCM was added to the resin and eluted to release the peptide from the resin and receive the filtrate. This process was repeated five times. The resin was washed with DCM and methanol, collected together with the filtrate, the combined solution was concentrated by evaporation and ether was added to obtain peptide precipitate. The obtained peptide was separated using a reversed phase column to obtain a yield of 80%.

Claims (6)

Peptide derivatives of formula (I) Formula (I) Wherein R 1 and R 2 are alkyl groups having straight or branched C ₁ to C ₂₀ saturated or unsaturated bonds; X is O or N; R is H or a C ₁ to C ₁₀ alkyl group; PEPTIDE is a peptide in which one or more amino acids are bound. A method for preparing a peptide derivative of formula (I) comprising reacting a peptide with an alkyl ketene dimer (AKD) in an organic solvent: Formula (I) Wherein R 1 and R 2 are alkyl groups having straight or branched C ₁ to C ₂₀ saturated or unsaturated bonds; X is O or N; R is H or a C ₁ to C ₁₀ alkyl group; PEPTIDE is a peptide in which one or more amino acids are bound. The method of claim 2, wherein the AKD is a single AKD. The method of claim 2, wherein the AKD is a mixed AKD. The method of claim 2, wherein the organic solvent is a tertiary amine. The method of claim 5, wherein the tertiary amine is characterized in that any one selected from the group consisting of TEA (triethylamine), TMA (trimethylamine), DIEA (diisopropylethylamine), NMP (N-methylmorpholine) and pyridine Process for the preparation of peptide derivatives of formula (I).