JPH02115198A - Animal collagenase-inhibiting agent - Google Patents

Abstract

NEW MATERIAL:Benzylocxycarbonyl-L-Glu-L-Leu-L-Ala-Gly and a salt thereof. USE:An animal collagenase-inhibiting agent. Useful for treating and preventing rheumatic arthritis, dental periostitis, the infiltration of tumor cells, etc. PREPARATION:For example, glycine ethyl ester hydrochloride is condensed with Boc-Ala-OH (Boc is tertiary butyloxycarbonyl) in the presence of activators, N,N'-dicyclohexylcarbodiimide and 1-hydroxybenzotriazole, in a solvent such as DMF and the reaction product is treated with trifluoroacetic acid to give H-Ala-Gly-OEt trifluoroacetic acid salt. The obtained salt is condensed with Boc-Leu-H2O in the same method as the above-mentioned method and further with benzyloxycarbonyl-Glu(OBZl)(r-benzylester) to provide the subject inhibitor.

Description

Detailed Description of the Invention [Industrial Field of Application] The present invention provides benzyloxycarbonyl-L-Glu-L-
Leu-L-Ala-Gly and its salts with bases, and animal collagenase inhibitors containing them as active ingredients, for the treatment of collagenase-related diseases such as rheumatoid arthritis, periodontitis, tumor cell infiltration, etc. It is expected to be useful for prevention.

[Conventional technology]

Collagen, which accounts for 30% of the protein in higher animals, forms a right-handed triple helical structure consisting of three α chains.

The α chain is a polypeptide chain consisting of approximately 1000 amino acid residues, and its triple helical structure is stable under physiological conditions and shows resistance to common proteases. It is only degraded by bacterial collagenase, which recognizes and cleaves the y sequence, or animal collagenase, which cleaves collagen molecules 3:1.

Animal collagenase was first discovered in 1962 in the tissue culture fluid of tadpole tail fins, and it was later revealed that it is distributed not only in amphibians but also in various tissues including the skin and bones of mammals.

In addition, during developmental differentiation and recombination of tissues and organs during growth,
High collagenase activity has been observed in the synovial fluid of patients with tumors and rheumatism, and this enzyme has come to be considered to be closely related to connective tissue metabolism under physiological conditions and in pathological conditions.

Therefore, in recent years, collagenase inhibitors have been synthesized for the purpose of treating rheumatoid arthritis, periodontal ligament disease, tumor cell infiltration, etc. (Japanese Unexamined Patent Publication No. 57-212157, Biochemistry, 1962 (1987)).

In addition, a polypeptide derived from soybean protein has been discovered as a collagenase inhibitor derived from natural products (Japanese Unexamined Patent Application Publication No. 57-1999).
-7490).

However, the reality is that the collagenase inhibitors known to date do not have very high inhibitory activity.

[Problem to be solved by the invention]

Under the above circumstances, collagenase inhibitors for the treatment and prevention of rheumatoid arthritis and the like are currently in wide demand. Therefore, an object of the present invention is to provide a peptide having an excellent collagenase inhibitory effect and a salt thereof with a base, and a collagenase inhibitor containing such a peptide or a salt thereof with a base as an active ingredient.

[Means to solve the problem]

Animal collagenase is a metalloprotease with zinc in its active center, and has some similarities to thermolysin, a metalloprotease derived from microorganisms. The present inventors focused on this point and first sought a new thermolysin inhibitor in natural products, and then attempted to synthesize a new collagenase inhibitor based on the thermolysin inhibitor. In addition, unless otherwise specified, the following amino acids are assumed to be in the L form. That is,
Peptide Gln-Thr- having thermolysin inhibitory activity in chymotrypsin hydrolyzate of non-0 albumin
Ala-Ala-Asp-Gln-Ala-Arg-G
discovered lu-Leu and deduced that the vicinity of its carboxyl terminus is important for its activity. So this Glu-L
As a result of trying to bond various amino acids to the eu moiety, the hensyloxycarbonyl-Glu-Le of the present invention was obtained.
It has been discovered that u-Ala-Gly inhibits animal collagenase. That is, the present invention provides benzyloxycarbonyl-Glu- which is a novel collagenase inhibitory peptide.
Leu-Alacty and its salt with a base, and benzyloxycarbonyl-Glu-Leu-Ala-Gly
The present invention relates to an animal collagenase inhibitor containing a salt thereof as an active ingredient.

Here, salts with bases include pharmaceutically acceptable salts with bases (inorganic bases and organic bases), such as sodium salts, potassium salts, ammonium salts, calcium salts, magnesium salts,
Includes salts with inorganic bases such as aluminum salts, and salts with organic amines such as basic amino acids (eg arginine, lysine).

Benzyloxycarbonyl-Glu-Leu- of the present invention
Ala-Gly is mainly produced by a method of stepwise introduction of amino acids using an organic chemical synthesis method.

It can also be produced by a peptide synthesis method that utilizes the reverse reaction of a hydrolase. That is, in organic chemical synthesis methods, individual amino acids are usually condensed sequentially, and this condensation usually involves combining an amino acid with a protected α-amino group and an active terminal carboxyl group with a free α-amino group and a protected terminal carboxyl group. The reaction is carried out by reacting the amino acid with the amino acid in an appropriate solvent. Although the synthesis may be performed in any order, it is preferable to connect the amino acids sequentially from the C-terminal side. The protecting group for the α-amine group includes various amino protecting groups used in peptide synthesis, such as tertiary-butyloxycarbonyl (BOC). The protecting group for the terminal carboxyl group includes various carboxyl protecting groups used in peptide synthesis, and examples include a methyl ester group and an ethyl ester group. In glutamic acid, the γ-carboxyl group is also usually protected, and a benzyloxycarbonyl group or the like is used as the protecting group. Activation of the terminal carboxyl group may be performed by a method commonly used in peptide synthesis, for example, using an activating agent such as N,N'-dicyclohexylcarbodiimide (DCC) or 1-hydroxybenzotriazole (HOBt). be able to. It is preferable to use DCC and 11OBt in combination. Dimethylformamide (DM
F) etc. are used. The condensation reaction is usually carried out at 0°C to room temperature.
Perform for ~30 hours. Removal of the protecting group and purification of the product may be carried out by a conventional method for peptide synthesis, for example, by the method shown in the Examples.

A salt of the present peptide with a base can be produced by a conventional method.

This peptide has an inhibitory effect on animal collagenase, and is effective in treating rheumatoid arthritis, periodontal ligament disease, and other diseases in mammals including humans.
It is expected to be useful for treating or preventing invasion of l1fi tumor cells.

The peptides and their salts with bases are used as such or in pharmaceutical compositions, usually mixed with at least one pharmaceutical auxiliary. The present peptide and its salt with a base can be administered parenterally (ie, intravenous injection, direct application) or orally, and can be formulated into a form suitable for each administration method.

- The formulation as an injection usually contains a sterile aqueous solution. The above-mentioned formulations also contain buffering agents/pH adjusting agents (sodium hydrogen phosphate, citric acid, etc.), tonicity agents (sodium chloride, glucose, etc.), preservatives (methyl p-hydroxybenzoate, p-hydroxybenzoic acid, etc.), and preservatives (methyl p-hydroxybenzoate, p-hydroxybenzoic acid, Other pharmaceutical adjuvants other than water may be included, such as propyl (propyl, etc.). The formulation can be sterilized by filtration through a bacteria-retaining filter, by incorporating a disinfectant into the composition, by irradiating or heating the composition. The preparation can also be prepared as a seedling-killing solid composition and dissolved in sterilized water or the like before use.

Orally administered preparations are formulated in a form suitable for absorption by the gastrointestinal tract. Tablets, capsules, granules, granules and powders are prepared with the usual pharmaceutical auxiliaries, such as binders (syrup, gum arabic,
gelatin, sorbitol, tragacanth, ponyvinyl pyrrolidone, hydroxypropyl cellulose, etc.), excipients (
Lactose, sucrose, corn starch, potato starch, sorbitol, crystalline cellulose, etc.), lubricants (magnesium stearate, talc, polyethylene glycol, silica, etc.), disintegrants (potato starch, carboxymethylcellulose, etc.), wetting agents (sodium lauryl sulfate) etc.) can be included.

Tablets can be coated by conventional methods.

Oral liquid preparations can be made into aqueous solutions or dry products. Such oral solutions may contain conventional additives such as preservatives (methyl or propyl p-hydroxybenzoate, sorbic acid, etc.).

The amount of the present peptide or its salt with the base in the present collagenase inhibitor can be varied, but is usually 5 to 100%.
χ(w/v4), especially 10 to 60χ(weak/v4) is suitable. The appropriate dosage of this collagenase inhibitor is 10 to 200 mg/kg/day as the active ingredient.

〔Example〕

Next, the present invention will be explained by examples.

Example Benzyloxycarbonyl-Glu-Leu
-Synthesis of Ala-Gly and collagenase inhibitory activity A) Synthesis of benzyloxycarbonyl- 1) II-Synthesis of Ala-Gly-OEt It-G
ly-OEt-Hci (glycine ethyl ester hydrochloride) 1.4 g (10 mmol), Boc-Ala-O
1.9 g (10 mmol) of H and 1.35 g (10 mmol) of 1-hydroxybenzotriazole (HOBt) were dissolved in 10 ml of dimethylbormamide (DMF). 9? Ice-cold ditriethylamine 1 d and dicyclohexylcarbodiimide (DC
C) 2.06g was added and then stirred overnight while maintaining at 5'C.

The generated dicyclohexylurea was filtered off, the filtrate was concentrated to dryness, and the residue was dissolved in ethyl acetate. This solution was thoroughly washed with a 10% aqueous citric acid solution, water, a 4% aqueous sodium bicarbonate solution, and then water, and dried under reduced pressure. This substance was dissolved in a mixture of trifluoroacetic acid 25 and anisole (1 ml) and left at room temperature for 20 minutes. Next, the reaction mixture was concentrated under reduced pressure, and the residue was washed three times with ether, and then the ether was distilled off to obtain H-^1a-Gly-OEt as a trifluoroacetate salt (yield: 3 mmol).

2) Synthesis of H-Leu-Ala-Gly-OEt
-8la-Gly-OEt trifluoroacetate (3
mmol), Boc-Leu-Hzo 0.75g (3
mmol) and HOBt (0.41 g (3 mmol)) were dissolved in DMF5d, and this solution was neutralized with O''C water-cooled ditriethylamine, then 0.62 g of DCC was added, and the mixture was stirred overnight while being maintained at 5°C. The generated dicyclohexylurea was filtered off, the filtrate was concentrated to dryness, and the residue was dissolved in ethyl acetate.This solution was diluted with 10%
Citric acid aqueous solution, water, 4% sodium bicarbonate aqueous solution,
Then, it was thoroughly washed with water and dried under reduced pressure. This material was dissolved in a mixed solution of 25 d of trifluoroacetic acid and 0.5 d of anisole and left at room temperature for 20 minutes. Next, the reaction mixture was concentrated under reduced pressure, and the residue was washed twice with ether, and then the ether was distilled off to obtain H-Leu-Ala-Gly-OEt as a trifluoroacetate (yield: 0.5 mmol).

3) Synthesis of benzyloxycarbonyl-Gly H-Leu-Ala-Gly-OEt-)lifluoroacetate (0.5 mmol), benzyloxycarbonyl Glu(OBzl) (T benzyl ester) 0.1
9 g (0.5 mmol) and lIOBt O. 068
D
0.10 g of CC was added, and the mixture was stirred overnight while being maintained at 5°C.

The generated dicyclohexylurea was filtered off, the filtrate was concentrated to dryness, and the residue was dissolved in ethyl acetate. This solution was thoroughly washed with a 10% aqueous citric acid solution, water, a 4% aqueous sodium bicarbonate solution, and then water, and dried under reduced pressure. This substance was mixed with 48 d of methanol, 24 d of dioxane, and 24 d of IN Na.
After dissolving in a mixed solution of OH4- and leaving it at room temperature for 5 hours, water was added thereto and washed with ether, and then Na'' ions were adsorbed using a cation exchange resin (AG50W-X8).
By making the solution acidic, the target befutide and benzyloxycarbonyl were recovered as a single peptide by further performing gel filtration with Sephadex L11-20.

This peptide was analyzed using HPLC (high performance liquid chromatography)
A single peak was observed at an elution time of 2.9 minutes. I
The elution conditions for IPLc are shown below.

Column: Waters Radial Pack Cartridge C18 Eluent: Phosphate buffer (10mM KI1.PO., 50
mMNa. S.O. .

pH2.5) Niacetonitrile-4=6 flow rate: 1 relative/
min detection: ultraviolet absorption at 210 nm Next, various analytical values of this peptide are shown.

Amino acid analysis: Glu (0.98), Gly (0.
97), Ala (1.00) Leu (0.97) () The molar ratio analysis using 8 la as 1 was carried out after hydrolysis with 6N hydrochloric acid at 110"C for 24 hours.

Specific light intensity: [α) = -60° (C = 0.1, Hz
O) Mass spectrometry: m/z 523 (M+H)”
B) Collagenase inhibitory activity Measured using JEOL HX 110, FAB-MS B) Collagenase inhibitory activity The collagenase inhibitory activity of the present peptide obtained as described above was determined by R. D. The method of Gray et al. (Biochen+.

Biophys. Res. Commun. 101
1251 (1981)).

That is, 0.05M Tris-HCl buffer (pH 7.0
.

0.5M substrate peptide (DNP-Pro-
Gln-Gly- 11e-Ala-Gly-Gin-
D-Arg. (purchased from Peptide Institute, Inc.) and use this as a substrate solution.

Then, Tadpol collagenase (2+ng/rn1) purchased from New England Nuclear was added.
) was diluted to 1710 with the above buffer solution and used as an enzyme solution.

First 10μ! The peptide of the present invention and 45 μl of the substrate solution were placed in a small test tube and kept at 37°C. Then 45μ2
The enzyme solution was added and the mixture was kept at 37°C. 5,
At 15 and 25 minutes, 10 μ2 of the reaction solution was collected and immediately injected into an HPLC apparatus, and DNP-Pro-Gin-Gly produced by decomposition of the substrate was quantified.

The HPLC conditions are shown below.

Column: Waters Radial Back Cartridge C8 Eluent: Phosphate buffer (10mM KIlzPO4,50
(mM NazSO4 + pH 2.5) Niacetonitrile = 7:3 Flow rate: 2/11 in Detection: Absorption at 375 nm A plurality of such experiments were conducted, and the inhibition rate was calculated from the following formula.

A: DNP-Pro-Gin- without inhibitor
Amount of Gly B: DNP-Pro-Gln-Gl when inhibitor is added
The amount of y and the concentration of this peptide when the inhibition rate is 50% I
C. The value was found to be 450 μm.

〔Effect of the invention〕

The present peptide and its salt with a base have animal collagenase inhibitory activity and are more useful than known inhibitors because they can be produced more easily.

Claims (1)

[Claims] 1. Benzyloxycarbonyl-L-Glu-L-Le
u-L-Ala-Gly and its salt with a base. 2. Benzyloxycarbonyl-L-Glu-L-Le
An animal collagenase inhibitor containing uL-Ala-Gly or a salt thereof with a base as an active ingredient.