JPH0832722B2 - Peptide and anti-dementia drug containing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a peptide having a nootropic effect and therefore useful as a medicine, particularly as an anti-dementia agent.

[PRIOR ART] Vasopressin has been known for a long time as having a cognitive effect, but a peptide which can be considered as a fragment of vasopressin recently, for example, It has been reported that the peptide represented by [6] has a nootropic effect similar to vasopressin [Science 2
21 , 1310-1312 (1983)].

Also, It has also been reported that the peptide represented by the formula also has a nootropic effect (Japanese Patent Laid-Open No. 59-93036).

[Problems to be Solved by the Invention] An object of the present invention is to provide a novel peptide having a better cognitive activity than such vasopressin and vasopressin fragment peptide.

[Means for Solving the Problem] The present invention has the general formula (I): A-Ser-Pip-Arg (I) (wherein A represents Pro-Asn-, Asn- or Pro-). It relates to the represented peptide or a derivative in the functional group thereof, or a pharmaceutically acceptable salt thereof.

Furthermore, the present invention provides an effective amount of a peptide represented by the above general formula (I) or a derivative in a functional group thereof, or a pharmacologically acceptable salt thereof, and a pharmacologically acceptable carrier or diluent. The present invention relates to an anti-dementia drug contained.

The peptide represented by the above general formula (I) in the present invention is a compound having an amino acid sequence which is completely different from that of the above-mentioned known peptide.

The derivative in the functional group of the peptide represented by the above general formula (I) means the following.

a) N-acyl derivatives derived from aliphatic carboxylic acids having 1 to 6 carbon atoms, b) amides or mono-alkyl or di-alkyl substituted amides having an alkyl group of 1 to 6 carbon atoms, And c) an ester derived from an alcohol having 1 to 18 carbon atoms, preferably an aliphatic alcohol having 1 to 6 carbon atoms.

Examples of the pharmacologically acceptable salt of the peptide or a derivative of the functional group thereof include an acid addition salt and a basic salt. Such acid addition salts include inorganic acids (eg, hydrochloric acid, sulfuric acid, phosphoric acid) or organic acids (eg,
Salts such as acetic acid, propionic acid, citric acid, tartaric acid, malic acid, oxalic acid, and methanesulfonic acid) can be mentioned. Examples of the basic salt include a sodium salt, a potassium salt, a triethylamine salt and the like.

In the present specification, amino acids, peptides, protecting groups, solvents and the like are abbreviations commonly used in the art, or IU
The abbreviations adopted by the PAC-IUB naming committee are used. For example, the following abbreviations are used. Amino acids mean L-form.

Arg: Arginine Asn: Asparagine Pip: Pipecolic acid Pro: Proline Ser: Serine Boc: t-Butoxycarbonyl Z: Benzyloxycarbonyl NO ₂ : Nitro Bzl: Benzyl OBzl: Benzyl ester OSu: N-Hydroxysuccinimide ester DCC: N , N'-dicyclohexylcarbodiimide DC Urea: N, N'-dicyclohexylurea HOBt: 1-hydroxybenzotriazole Et ₃ N: triethylamine NMM: N-methylmorpholine TFA: trifluoroacetic acid AcOEt: ethyl acetate DMF: N, N-dimethylformamide MeOH: Methanol The compound of the present invention is a method commonly used in peptide chemistry, for example, Schrder and Lbke, "The Peptides," Volume 1, Academic Press, New Y.
ork, USA (1965), Nobuo Izumiya et al., “Basic and Experimental Peptide Synthesis,” Maruzen Co., Ltd. (1985). It can be manufactured by any of them.

As a condensation method for forming a peptide bond, an azide method, an acid chloride method, an acid anhydride method, a mixed acid anhydride method,
Carbodiimide method, carbodiimide-additive method,
The active ester method, the carbonyldiimidazole method, the redox method, the method using Woodward reagent K and the like can be mentioned.

Before performing the condensation reaction, by a means known per se,
A carboxyl group or amino group that does not participate in the reaction may be protected, or a carboxyl group or amino group that participates in the reaction may be activated.

Examples of carboxyl protecting groups include, for example, methyl,
Ethyl, benzyl, p-nitrobenzyl, t-butyl,
Esters such as cyclohexyl may be mentioned.

Examples of the amino group-protecting group include a benzyloxycarbonyl group, a t-butoxycarbonyl group, an isobornyloxycarbonyl group and a 9-fluorenylmethoxycarbonyl group.

Examples of the guanidino group-protecting group include a nitro group and
Examples thereof include a benzyloxycarbonyl group, a tosyl group, a p-methoxybenzenesulfonyl group and a 4-methoxy-2,3,6-trimethylbenzenesulfonyl group.

Examples of the hydroxyl-protecting group include a t-butyl group, a benzyl group, a tetrahydropyranyl group, and an acetyl group.

Examples of activated carboxyl groups include corresponding acid anhydrides, azides, active esters [alcohols (eg, pentachlorophenol, 2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, N- Hydroxy-5-norbornene-2,3-dicarboximide, N-hydroxysuccinimide, N-
Hydroxyphthalimide, ester with 1-hydroxybenzotriazole)] and the like. Activated amino groups include, for example, the corresponding phosphoric amides.

The reaction is usually performed in a solvent, for example, a solvent such as chloroform, dichloromethane, ethyl acetate, N, N-dimethylformamide, dimethyl sulfoxide, pyridine, dioxane, tetrahydrofuran, water, methanol, or a mixture thereof. be able to.

The reaction can be carried out at a temperature generally ranging from about -30 ° C to about 50 ° C.

The protective group elimination reaction of the peptide of the present invention depends on the type of the protective group used, but it does not affect the peptide bond and it is necessary to remove the protective group.

Examples of the method for removing the protecting group include, for example, acid treatment with hydrogen chloride, hydrogen bromide, hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid, or a mixture thereof, Other examples include reduction with sodium or palladium carbon in liquid ammonia. In the above deprotection reaction by acid treatment, it is effective to add a cation scavenger such as anisole, phenol or thioanisole.

The peptide of the present invention thus produced is, after the reaction is completed, a peptide separation means known per se, for example,
It can be obtained by extraction, partitioning, reprecipitation, recrystallization, column chromatography and the like.

Further, the peptide of the present invention can be obtained by a method known per se, as described above, a derivative of the functional group, or
These can be pharmacologically acceptable salts.

The peptides of the invention show a strong nootropic action in the passive avoidance test in rats.

Examples of useful target disease names of the peptide derivative of the present invention include senile dementia (Alzheimer type dementia), cerebrovascular dementia, and Alzheimer's disease, Pick's disease, Huntington's chorea, Creutzfeldt-Jakob's disease, Parkinson's disease, Examples thereof include dementia based on cerebellar spinal degeneration and the like, and can be used for prevention or treatment of these diseases.

The toxicity of the peptide derivative of the present invention is extremely low, and there is no death even at a dose far exceeding the pharmaceutically effective dose.

The peptide derivative of the present invention can be administered as a free form or as a derivative at a functional group thereof. Regarding the dose, whether it is the free form or a salt thereof, an appropriate amount of the free form is generally in the range of 0.1 ng to 1 mg / day.
Particularly, for parenteral administration and nasal administration, 0.1 ng to 100 μg / day is preferable, and for oral administration and rectal administration, parenteral administration is 10 to 10 μg / day.
It is preferable to administer 100 times. The peptide derivative of the present invention is mainly administered parenterally (eg, intravenous or subcutaneous injection, intracerebroventricular or intrathecal administration, nasal administration, rectal administration). Good.

Dosage forms include, for example, injections, suppositories, powders, nasal drops, pills, tablets and the like. Although the peptide derivative of the present invention can be stored as a solution of physiological saline,
A lyophilized ampule can be prepared by adding mannitol and sorbitol, and can be dissolved at the time of use.

 Examples will be shown below.

In each example, the developing solvent for thin layer chromatography was as follows, and TLC plate silica gel 60F ₂₅₄ manufactured by Merck was used.

R _f ¹ : Chloroform-methanol-acetic acid-water (80: 20: 2.
5: 5) Lower layer R _f ² : Chloroform-methanol-water (70: 30: 5) R _f ³ : n-butanol-acetic acid-water (2: 1: 1) Purification by high performance liquid chromatography : ΜBondapak C ₁₈ 1.9 × 15 cm Mobile phase: A) 0.05% TFA, B) Acetonitrile was used.

[Example 1] H-Asn-Ser- Pip-Arg-OH · acetate (1) Boc-Pip-Arg (NO 2) -OBzl H-Arg (NO 2) -OBz10.6g ice cooling DMF100ml solution of Boc-Pip-OH 7.1g, HOBt 7.1g and DCC6.7g were added under below, and after stirring at room temperature for 18 hours, DCUrea was separated by filtration and DMF was distilled off.

The residue was dissolved in AcOEt, washed successively with saturated aqueous sodium hydrogen carbonate solution, diluted hydrochloric acid solution and water, and dried over anhydrous sodium sulfate.

AcOEt was distilled off, and the residue was purified by silica gel column using CHCl ₃ -acetone to obtain the title compound as an oil.

Yield: 5.5 g R _f ¹ : 0.68, R _f ² : 0.80 [α] _D : −24.0 ° (c = 1.0, DMF) (2) Boc-Ser (Bzl) -Pip-Arg (NO ₂ ) -OBzl Boc the _{-Pip-Arg (NO 2) -OBzl5.2g} , 4NHCl-AcOEt25
After standing in ml for 30 minutes at room temperature, the solvent was distilled off.

The residue was dried under reduced pressure, dissolved in 50 ml of DMF, and then E under ice cooling.
t ₃ N1.4ml, and Boc-Ser and (Bzl) -OSu3.9g added and stirred for 18 hours at room temperature.

DMF was distilled off, the residue was dissolved in AcOEt, washed successively with saturated aqueous sodium hydrogen carbonate solution, diluted hydrochloric acid solution and water, and then dried over anhydrous sodium sulfate.

AcOEt was distilled off, and the residue was purified by silica gel column using CHCl ₃ -acetone to obtain the title compound as an oil.

Yield: 2.8 g R _f ¹ : 0.74, R _f ² : 0.86 [α] _D : -42.6 ° (c = 1.0, DMF) (2) Boc-Asn-Ser (Bzl) -Pip-Arg (NO ₂ )- OBzl Boc-Ser (Bzl) -Pip -Arg (NO 2) 4NH the -OBz1.6g
After standing in 6 ml of Cl-AcOEt for 30 minutes at room temperature, the solvent was distilled off.

2-Butanol-CH ₂ Cl ₂ (5: 1 v / v) and saturated aqueous sodium hydrogen carbonate were added to the residue, the organic layer was separated, washed with saturated brine and dried over anhydrous sodium sulfate.

The solvent was distilled off and the residue was dissolved in 20 ml of DMF.
-Asn-OH 0.53g, HOBt 0.53g and DCC 0.52g were added.

After stirring at room temperature for 18 hours, DCUrea was filtered off and DMF was distilled off.

The residue was dissolved in AcOEt, washed successively with saturated aqueous sodium hydrogen carbonate solution, diluted hydrochloric acid solution and saturated brine, and dried over anhydrous sodium sulfate.

The solvent was evaporated, and the residue was purified by silica gel column with CHCl ₃ -MeOH to give the title compound as an oil.

Yield: 1.0 g R _f ¹ : 0.57, R _f ² : 0.74 [α] _D : -37.8 ° (c = 1.0, DMF) (4) H-Asn-Ser-Pip-Arg-OH-acetate Boc-Asn -Ser (Bzl) -Pip-Arg ( NO 2) -OBz150m
After leaving g in 0.5 ml of 4N HCl-AcOEt at room temperature for 30 minutes, the solvent was distilled off.

The residue was stirred in 20 ml of 80% acetic acid in the presence of 10% palladium on carbon for 18 hours in a stream of hydrogen.

After the palladium carbon was filtered off, the solvent was distilled off, the residue was dissolved in water and freeze-dried.

Furthermore, after high-performance liquid chromatography purification with (A) (mobile phase) with a linear gradient of 0 to 10% (B) for 20 minutes at 12 ml / min (flow rate), Dowex 1 × 2 (acetate type) treatment and freezing Dry to give the title compound.

^{_{Yield: 63mg R f 3: 0.19 [}} α] D: -46.2 ° (c = 0.5, water) FAB mass spectrum (M + 1): 487 [Example 2] H-Pro-Ser- Pip-Arg-OH · acetate (1) Z-Pro-Ser (Bzl) -Pip-Arg (NO 2) -OBzl Boc-Ser (Bzl) -Pip-Arg (NO 2) -OBz1.0g, 4NH
Cl-AcOEt5ml, from Et ₃ N0.2ml and Z-Pro-OSu0.49g,
The title compound was obtained as an oil in the same manner as in Example 1- (2).

Yield: 0.9 g R _f ¹ : 0.72, R _f ² : 0.82 [α] _D : -51.2 ° (c = 1.0, DMF) (2) H-Pro-Ser-Pip-Arg-OH-acetate Z-Pro -Ser (Bzl) -Pip-Arg ( NO 2) -OBz150mg
After palladium carbon reduction in the same manner as in Example 1- (4), high performance liquid chromatography was performed using 12 ml / min (flow rate), 2 to 12% (B) 20 minute linear gradient (A) (mobile phase). The product was purified by chromatography, treated with Dowex 1 × 2 (acetate type) and then lyophilized to obtain the title compound.

Yield: 31 mg R _f ³ : 0.18 [α] _D : -76.5 ° (c = 0.5, water) FAB mass spectrum (M + 1): 470 [Example 3] H-Pro-Asn-Ser-Pip-Arg-OH acetate (1) Z-Pro-Asn -Ser (Bzl) -Pip-Arg (NO 2) -O
Bzl Boc-Asn-Ser (Bzl ) -Pip-Arg (NO 2) -OBzl0.68
g, 4NHCl-AcOEt3ml, NMM0.14ml and Z-Pro-OSu0.32g
From the above, the title compound was obtained in the same manner as in Example 1- (2).

Yield: 0.7 g Melting point: 95-97 ° C. R _f ¹ : 0.57, R _f ² : 0.74 [α] _D -48.2 ° (c = 1.0, DMF) (2) H-Pro-Asn-Ser-Pip-Arg -OH · acetates Z-Pro-Asn-Ser ( Bzl) -Pip-Arg (NO 2) -OBz
150 mg after palladium on carbon reduction as in Example 1- (4), 12 ml / min (flow rate), 0 to 20% (B) 20.
The product was purified by high performance liquid chromatography using (A) (mobile phase) with a linear gradient, treated with Dowex 1 × 2 (acetate type) and then lyophilized to obtain the title compound.

Yield: 60 mg R _f ³ : 0.14 [α] _D : -76.4 ° (c = 0.5, water) FAB mass spectrum (M + 1): 584 Next, pharmacological test examples showing the efficacy of the peptide of the present invention will be shown. .

[Pharmacological test example] The effect on memory fixation was examined by using male Wistar rats, [Burbach] et al. [Science (Scienc
e), 221 , 1310-1312 (1983)]. The experimental apparatus consists of a light room and a dark room, and the floor is made of a stainless steel grid. Rats placed in the lightroom are free to move to the darkroom. Using this device, rats undergo a single electrical shock when they enter the darkroom. The retention of the passive avoidance behavior to the electric shock was determined by the time (reaction latency) until a rat placed again in the light room after a certain period of time entered the dark room.

Examination of the effect of cycloheximide on the improvement of experimental retrograde amnesia After the subcutaneous administration of the peptide or physiological saline of the present invention, an electric shock (0.5 mA) was experienced 1 hour later, and immediately after that, cycloheximide 2.7 to 3.0 mg / kg or physiological saline was subcutaneously administered, and a memory retention test was performed 48 hours later. Rats to which only saline was administered generally exhibited a response latency of about 300 seconds, and rats of the control group to which cycloheximide alone was administered exhibited a response latency of about 50 seconds, and developed retrograde amnesia.

The average value of the reaction latency of the peptide-administered group of the present invention was compared with that of the control group. The number of rats used in the tests in each group is 6-8. The maximum measurement time was 600 seconds.

For the peptide obtained in Example 1, the dose was 1n
The effect at g / kg (the ratio of the response latency of the peptide-administered group to the response latency of the control group is shown in%) was 235%.

From the above test results, the peptide of the present invention showed an excellent improving effect on retrograde amnesia.

Next, formulation examples of the drug containing the peptide of the present invention will be shown.

[Formulation Example 1] (Injection) In 100 ml of distilled water for injection, 0.1 mg of the peptide derivative obtained in Example 1 and 0.9 g of sodium chloride were added, and p
An aqueous solution in which H was adjusted to 6.0 to 8.0 with sodium hydroxide was prepared. This was recently filtered, then filled in a 1 ml ampoule, sealed and heat sterilized to prepare an injection.

[Formulation Example 2] (Freeze-dried preparation) 5 ml of the peptide derivative obtained in Example 1 and 5 g of D-mannite were added to 100 ml of distilled water for injection, and the pH was adjusted to 6.0 to 8.0 with a phosphate buffer. A conditioned aqueous solution was prepared.
This was subjected to bacterial filtration, dispensed in a vial (1 ml), and lyophilized to produce a lyophilized injection.

[Formulation Example 3] (Nasal drops) 10 mg of the peptide derivative obtained in Example 1 was added to 100 ml of physiological saline, and the pH was adjusted to 3.0 to 6.0 with a citrate buffer.
To 50 μg in a single dose of 0.5 ml to prepare a nasal drop.

[Formulation Example 4] (suppository) Hard fat (triglyceride of saturated fatty acid) 9
After adding 0.5 g of egg yolk lecithin to 8.5 g and melting at 40 to 45 ° C., 5 mg of the peptide derivative obtained in Example 1 was added to PEG400.
After adding 1g of the liquid dissolved in this and stirring and dispersing,
1 g thereof was poured into a suppository mold, and after solidification, it was separated from the mold to produce a suppository.

EFFECT OF THE INVENTION The peptide of the present invention is a novel compound, has an excellent nootropic action, and is useful as a medicine.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Reference number within the agency FI Technical indication location C07K 7/16 (72) Inventor Yoshikazu Isowa 4-6-7 Shimoochiai, Shinjuku-ku, Tokyo Fujirebio Incorporated (72) Inventor Yoshiaki Sato 4-6-7 Shimoochiai, Shinjuku-ku, Tokyo Fuji Rebio Inc. (72) Inventor Yoshiharu Nakajima 4-6-7 Shimoochiai, Shinjuku-ku, Tokyo Fuji Rebio Co., Ltd.

Claims (2)

[Claims] 1. A peptide represented by the general formula (I): A-Ser-Pip-Arg (I) (wherein A represents Pro-Asn-, Asn- or Pro-) or a functional group thereof. Derivatives, or pharmacologically acceptable salts thereof. 2. A peptide represented by the general formula (I): A-Ser-Pip-Arg (I) (wherein A represents Pro-Asn-, Asn- or Pro-) or a functional group thereof. An effective amount of a derivative or a pharmacologically acceptable salt thereof,
And a pharmacologically acceptable carrier or diluent.