JPS5830299B2 - Tripeptides and their derivatives - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to novel tripeptides and derivatives thereof.

The tripeptide of the present invention is a novel peptide that can suppress intracellular synthesis of collagen, thereby regulating and limiting the formation of scar tissue due to wounds.

These peptides can therefore be used to treat diseases including fibrosis, such as atherosclerosis, liver cirrhosis, keloids, scleroderma, rheumatoid arthritis, osteoarthritis, pulmonary fibrosis, and organs with excessive collagen accumulation, such as ichthyoderma. It is useful as a medicine or veterinary medicine that can be used for.

To explain the present invention in detail, the tripeptide which is the compound of the present invention is represented by the following general formula (I).

A-Pro-B-C-D (I)(
In the above formula, A is a hydrogen atom, a benzoyl group or a dansyl group, Pro is an N-terminal amino acid residue L-7' loline residue, B is L-leucine, L-phenylalanine,
L-methionine, L-cysteine, L-glutamic acid or L-tyrosine residue, C is glycine residue, D is pyrrolidine residue).

That is, the tripeptide and its derivatives according to the present invention have L-proline residue, C-
These are tripeptides having a glycine residue as the terminal amino acid residue, and derivatives in which the hydrogen atom of the terminal amino group and/or the hydroxyl group of the terminal carboxy group of the tripeptide are substituted.

Next, the method for producing the tripeptide and its derivatives, which are the compounds of the present invention, will be explained by giving one example.

That is, a partial peptide constituting a part of the amino acid sequence of general formula (I) or an activated terminal group and/or a protected terminal group (amino group and/or a protected terminal group constituting a part of the amino acid sequence) A partial peptide or amino acid constituting the remainder of the amino acid sequence of the above tripeptide, or an activated terminal group and/or a protected terminal constituting the remainder of the amino acid sequence. groups (amino groups and/or
or a carboxy group), the above tripeptide or its derivative can be obtained.

In general, in peptide synthesis, many methods have been established to form the desired amino acid sequence by protecting amino groups or carboxy groups that do not participate in the condensation reaction in the raw material amino acids or peptides, and then carrying out the condensation reaction. However, any of these methods may be used to synthesize the compound of the present invention.

Advantageous examples include the following conventional methods.

(1) Reaction of free amino group and free carboxyl group An amino acid ester or peptide ester having a free amino group is reacted with another amino acid or peptide having a protected amino group and a free carboxyl group in the presence of a condensing agent. Make it react.

As the condensing agent, N,N'-dicyclohexylcarbodiimide, N,N'-carbonyldiimidazole, tetraethylpyrophosphite, etc. are used.

(2) Reaction of free amine group and activated carboxy group (in the above formula, R1 represents a substituent for activating the carboxy group).

) Reacting an amino acid or peptide with a free amino group and a protected or free carboxy group with another amino acid or peptide with an activated carboxy group and a protected amino group.

Examples of the activated carboxy group include activated esters such as cyanomethyl ester, P-nitrophenyl ester, P-nitrothiophenol ester, and thiophenol ester, acid azides, acid chlorides, mixed acid anhydrides, and the like.

(3) Reaction of activated amine group and free carboxy group (in the above formula, R2 represents a substituent for activating the amino group).

) Reacting an amino acid or peptide with an activated amino group and a protected carboxy group with another amino acid or peptide with a free carboxy group and a protected amine group.

Examples of activated amino groups include phenylthiocarbonyl derivatives, phenoxycarbonyl derivatives, P-nitrophenoxycarbonyl derivatives, diphenylaminocarbonyl derivatives, N-carboxylic acid anhydrides, and the like.

As mentioned above, the carboxy or amino groups of amino acids and peptides that do not participate in the reaction are generally protected.

Specifically, the carboxy group is a lower alkyl ester (for example, t-butyl ester), a lower aralkyl ester (
For example, it is protected by converting it into a benzyl ester) or an amide.

These protecting groups are generally removed by alkaline hydrolysis, but the protecting groups are easily removed from t-butyl esters by acid hydrolysis and from benzyl esters by hydrogenolysis.

On the other hand, amino groups are protected by acetylation, fosmylation, phthaloylation, trifluoroacetylation, P-methoxybenzyloxycarbonylation, benzoylation, benzyloxycarbonylation, t-butyloxycarbonylation, or tritylation.

These protective groups can be removed by hydrolysis for acetyl and benzoyl groups, hydrogenolysis on a palladium catalyst for benzyloxycarbonyl groups or ring removal using sodium in liquid ammonia, and catalytic hydrogenation for trityl groups. The t-butyloxycarbonyl group is easily removed with cold trifluoroacetic acid.

The synthetic reaction of peptides, amino acids, or their derivatives is usually carried out at -15 to 60°C, preferably 0 to 10°C, and usually at 1 to 10°C.
It is carried out for 24 hours, preferably 3 to 6 hours.

By appropriately selecting the terminal group protection method, the protecting group removal method, and the condensation conditions described above, novel tri- and tetrapeptides and derivatives thereof, which are the objects of the present invention, can be obtained.

Next, a preferred embodiment of the method for producing the compound of the present invention will be described.
Taking o-Glu-Gly-Pyrr as an example, it is shown in the diagram below.

In addition, in the above and following explanations, Bz is a benzoyl group, DNS is a dansyl group, BOC is a t-butyloxycarbonyl group, Bzl is a benzyl group, Pyrr is a 1-pyrrolidinyl group, Z is a benzyloxycarbonyl group, DC
C is dicyclohexylcarbodiimide, Pro is L-proline residue, GIy is glycine residue, Leu is L-leucine residue, Phe is L-phenylalanine residue, Met
is L-methionine residue, Gys is L-cystine residue,
Glu represents an L-glutamic acid residue, and Try represents an L-tyrosine residue.

In addition, it is of course possible to use other methods explained in detail above in place of the terminal group protection method, protective group removal method, condensation method, etc. shown in the above diagram, and it is also possible to use the other methods described in detail above. You can also change its position.

The tripeptide which is the compound of the present invention is protocollagen proline hydroxylase (hereinafter abbreviated as PPH).

) in a cell-free system, and has the effect of inhibiting collagen synthesis.

It is thought that collagen production progresses through the following stages.

(See The New England Journal of Medicine, Vol. 286, p. 242 (1972), Ibid., Vol. 286, p. 291 (1972)) ■ Protocollagen is synthesized on intracellular ribosomes.

■ Proline and lysine at specific sites in the synthesized protocollagen are hydroxylated to become hydroxyproline and hydroxylysine.

■ Sugars such as galactose and glucose bind to hydroxylysine.

■ Sugar-bound protocollagen is secreted extracellularly as soluble collagen.

■ Outside the cells, soluble collagen takes on a network structure and transforms into insoluble collagen.

Therefore, if it is possible to inhibit PPH, which is one of the enzymes that control the steps shown in The synthesis of protocollagen is suppressed, and as a result, the synthesis of collagen is suppressed.

Several synthetic peptides that inhibit PPH are already known, but most of them can also serve as synthetic substrates for PPH, and their inhibitory function is thought to be antagonism with protocollagen. Most of these synthetic peptides are polymeric substances, and hexapeptides have been shown to have the least PPH inhibitory activity, although it is weak.

(Archives of Biochemistry and Biophysics, Vol. 125, p. 779 (1968)) However, the tripeptide and its derivatives, which are the compounds of the present invention, have much stronger PP than these known peptides.
It was confirmed through the following experiment that it exhibits H inhibitory activity.

That is, the PPH inhibitory activity of various peptides, which are compounds of the present invention, in a cell-free system was measured by the following method.

Test method ■ Preparation of substrate 3H-protocollagen was prepared according to the method of Prock, op. (Archives of Biochemistry and Biophysics, Vol. 118, p. 611 (1968)).

That is, 100 tibiae of chicken egg embryos were precultured in Krebs' solution for 90 minutes, and α,α'-dipyridyl was added to incubate F.
Except for e+1, the uniform label 3H-proline is 2
Add 50 microcuries and incubate for 180 minutes.

Homogenize the tibia and ultracentrifuge (100,000, 9x60 minutes)
The supernatant was collected and subjected to running water dialysis for 48 hours at 100°C.
The mixture was heat-treated for 5 minutes and ultracentrifuged again (100,000 g x 40 minutes), and the supernatant was used as H-protocollagen.

(2) Preparation of zymogen Add 0.9% NaCl to 1 g of hamster liver, homogenize, centrifuge at high speed (50,000 g x 30 minutes), and use the supernatant as zymogen.

■Measurement method Uden f r i end method (Analytical Biochemistry Vol. 16, p. 384 (1966)
).

That is, α-ketoglutaric acid 0.5 mmol, FeS
The zymogen was added to 40.05 mmol of O, 2.0 mmol of vitamin C, 50 mmol of Tris buffer (pH 7.8), and 3H-protocollagen (100,000 cpm) to give 0.05 mmol of vitamin C.
6 ml, put this in a round bottom 798 stool scoop, and start the reaction at 37°C.

The reaction was stopped by quenching with liquid nitrogen, and HTO (t
-IJ thiumated water) from the round-bottomed neck flask to the other tube, and the HTO is collected and counted in a liquid scintillation counter.

Inhibitors were added to the reaction system at concentrations of 0.02 and 0.35 mmol, respectively, and the inhibition rate was determined.

Note that water was used as a control.

The results are shown in Table-1.

The manufacturing method of the compounds of the present invention will be specifically explained in Examples below, but the compounds included in the present invention are not limited to the compounds synthesized in the Examples below unless it goes beyond the gist of the present invention. do not have.

In order to synthesize tripeptides and derivatives thereof, which are compounds of the present invention, partial peptides shown in the following reference examples were synthesized.

Reference Example I Synthesis of Z-Gly-Pyrr 22.7 g of Z-e+y-OH and 11 g of N-hydroxysuccinimide are dissolved in 1001 rLl of dichloromethane, and 23 g of DCC is added at 0 to 5°C and shaken for 2 hours.

After further stirring at room temperature for 2 hours, 7.5 g of pyrrolidine was gradually added at 0 to 5°C and the mixture was shaken for 2 hours.

- After standing overnight, dicyclohexylurea is filtered off.

The solvent was distilled off under reduced pressure, and the oily residue was dissolved in ethyl acetate and washed with 10% hydrochloric acid, a saturated aqueous solution of hydrogen carbonate, and then water.

After drying over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure to obtain an oily residue.

When about 10 ml of water is added to this, it crystallizes.

This was collected by filtration and recrystallized from ethyl acetate to give Z - G l
Obtain y −P y r r.

Yield: 25g (90%) Elemental analysis: H as C14H16N202/H2O
N Calculated value (fund) 61.00 7.02 10.13 Experimental value C%) 59.98 7.19 9.99 Reference example 2 Z-Gly-Pyrr-H20 obtained by Synthesis of Gly Pyrr Reference Example 1
7 g is dissolved in 100 TLl of methanol, a catalytic amount of palladium black is added and hydrogen gas is passed through for about 5 hours.

When the catalyst and solvent are distilled off, oily e+yPyrr is obtained.

b-p・130'C/8關Hg Yield 3g Elemental analysis value: HN as C6H1□N20 Calculated value (%) 56.22 9.68 21.52 Experimental value (fund), 56.22 9.44 21. 86 Reference Example 3 Synthetic amino acid derivative BOC-B-OH of BOC-B-Gly-Pyrr (B in the formula corresponds to B in Table 2 below).

) o, oi mol and Gly-Pyrr obtained in Reference Example 2
Dissolve 0.01 mol in 50 ml of dichloromethane, cool with water to 0-5°C, add 2.3 g of DCC, and stir for 1 hour.

- Dicyclohexylurea precipitated after standing overnight is filtered off, and the solvent -> rice is distilled off under reduced pressure.

The residue was dissolved in 50 rI'Ll of ethyl acetate, and the ethyl acetate layer was washed with 3% hydrochloric acid, a saturated aqueous sodium bicarbonate solution, and then water.

After drying with sodium sulfate, the solvent was distilled off under reduced pressure to obtain the partial peptide derivative BOC-B-Gly-P shown in Table 2 below.
yrr is obtained as crystals.

This is purified by recrystallization from ethyl acetate.

Example I Synthesis of A-Pro-B-Gly-Pyrr Partial peptide BOC-B having the amino acid residue shown by B in Table 3
-Gly-Pyrr O. 1 mol is suspended in 50 ml of ethyl acetate containing 10% hydrogen chloride and shaken for 2 hours.

When the solvent was distilled off under reduced pressure, the oily substance B-Gly-Py
rr-HCIJ is obtained.

This and the proline derivative A-Pro-OH (A is B
z or DNS.

)0.01 mol was condensed in the presence of DCC according to the method of Reference Example 3, A Pro B shown in Table 3 was obtained.
Gly-Pyrr is obtained.

Example 2 Synthesis of Bz-Pro-Glu-Gly-Pyrr Bz-Pro-Glu (OB
z I )-Gly-Pyrr O, 01 mol to 50
ml of methanol, add a catalytic amount of palladium black, and pass hydrogen gas through thin layer chromatography until no spots of raw material remain.

The crystals or oily residue obtained by distilling off the catalyst and solvent are dissolved in 30 ml of a saturated aqueous sodium bicarbonate solution, the insoluble materials are filtered, and the aqueous layer is washed with 50 mA of ethyl acetate.

The aqueous layer is brought to pH 2 with 3% hydrochloric acid.

The aqueous layer is then extracted three times with 50 ml of dichloromethane.

After drying with sodium sulfate and removing the solvent under reduced pressure, B
z-Pro-Glu-Gly-Pyrr is obtained in quantitative yield.

Melting point 112-115℃ Elemental analysis value: C23H2ON4
HN as o6 Calculated value (%) 60.25 6.60 12.22 Experimental value (%;) 60,01 6.86 12.03 Example 3 Synthesis Example 2 of Bz-Pro-Tyr-Gly-Pyrr In the method shown, B z-P obtained in Example 1 using methanol containing 1N hydrogen chloride as the solvent.
Bz-Pr by performing catalytic hydrogenation of ro-Tyr(Bzl)-Gly-Pyrr.

Tyr-Gay-P yrr is obtained in quantitative yield.

Melting point: 102-105°C Elemental analysis value: CHN calculated value as C2□H32N405 (%;) 65.83 6.55], 1. ．．
38 Experimental Values (Capital) 65.1.3 6.43 11.
51 Example 4 Synthesis of Bz-Pro-Cys-Gay-Pyrr Bz-Pro-Cys (Bzl
) Gly-Pyrr l & is stirred with 5 TL of anhydrous hydrogen fluoride in the presence of 0.5 parts of anisole for 2 hours at 10° C. or lower.

After distilling off the volatile portion under reduced pressure, ether is added to the oily residue to form a powder.

This is dissolved in a small amount of methanol and reprecipitated by adding ether.

Yield 0.3g Melting point 85-90'C Elemental analysis value: C2
1H28N4045-HN as H2O

Claims (1)

[Claims] 1 General formula % Formula % () (In the above formula, A is a hydrogen atom, a benzoyl group or a dansyl group, Pro is an L-proline residue which is an N-terminal amino acid residue, and B is an L- Leucine, L-phenylalanine, L
- methionine, L-cysteine, L-glutamic acid or L-tyrosine residue, C represents a glycine residue, and D represents a pyrrolidine residue. ) and its derivatives.