JPH02273697A - Peptide and antidemential agent containing the same - Google Patents

Abstract

NEW MATERIAL:The peptide of formula A-Ser-Pip-Arg (A is Pro-Asn, Asn or Pro; Pip is pipecolic acid), its derivative or their salt. EXAMPLE:The compound of formula I. USE:An antidemential agent useful for the prevention and treatment of Alzheimers disease, etc. It exhibits strong nootropic action and has extremely low toxicity. PREPARATION:The exemplified compound can be produced e.g. by reacting a compound of formula II (Boc is t-butoxycarbonyl; Bzl is benzyl; OBzl is benzyl ester) with a compound of formula Z-Pro-OSn (Z is benzyloxycarbonyl; OSu is N-hydroxysuccinimide ester) in a mixture of hydrochloric acid and ethyl acetate in the presence of triethylamine and demasking the resultant compound of formula III by reducing with Pd-carbon, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to peptides that have nootropic activity and are therefore useful as medicines, particularly as anti-dementia agents.

[Prior Art J It has been known for a long time that vapressin has a nootropic effect, but recently peptides that can be considered to be fragments of vapressin, such as the peptide expressed by H-Cy s -011, have also been shown to have the same therapeutic effect as vapressin. It has been reported that there is a cognitive effect [Science
ce) 221.1110-1:+12 (1983)
].

Also, (pGlu-^5n-Cys-Pro-8rg-Gly
-Nl+) It has also been reported that the peptide represented by 2 has nootropic effects (Japanese Unexamined Patent Publication No. 59-93036).

[Problems to be Solved by the Invention] An object of the present invention is to provide a novel peptide having even better nootropic activity than the pap pressin and vasobrecin fragment peptides.

[Means for Solving the Problems] The present invention provides general formula (1): % formula % () (wherein A is Pro-^5n-1Asn- or Pro
The present invention relates to a peptide represented by - or a functional group derivative thereof, or a pharmacologically acceptable salt thereof.

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

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

The functional group derivative of the peptide represented by the above general formula (1) means the following.

a) N-acyl derivatives derived from aliphatic carboxylic acids with 1 to 6 carbon atoms; b) amides or 1,000-alkyl- or di-alkyl-substituted amides with alkyl groups of 1 to 6 carbon atoms; and c) esters derived from alcohols having 1 to 18 carbon atoms, preferably aliphatic alcohols having 1 to 6 carbon atoms.

The pharmacologically acceptable salts of the peptides or functional group derivatives thereof include acid addition salts and basic salts. Such acid addition salts include:
Examples include salts of inorganic acids (eg, hydrochloric acid, sulfuric acid, phosphoric acid) or organic acids (eg, acetic acid, propionic acid, citric acid, tartaric acid, malic acid, oxalic acid, methanesulfonic acid). Further, examples of the basic salt include sodium salt, potassium salt, triethylamine salt, and the like.

In this specification, amino acids, peptides, protecting groups, solvents, etc. are referred to by abbreviations commonly used in the art, or IU
Abbreviations adopted by the PAC-IUB nomenclature committee are used. For example, the following abbreviations are used: In addition, amino acids shall mean L-type amino acids.

＾rg: Arginine Asn: Asparagine Pip: Pipecolic acid Pro Nibroline Ser: Serine Bac: t-Butoxycarbonyl 2: Benzyloxycarbonyl N02: Nitro Bzl: Benzyl 0 Bzl: Benzyl ester O20: N-hydroxysuccinimide ester DCC
: N,N'-dicyclohexylcarbodiimide D (:
Urea: N,N'-dicyclohexylurea 11
0BL: 1-hydroxybenzotriazole Et3N:
) ethylamine NMM: N-methylmorpholine.

TFA: trifluoroacetic acid 8cOEt: ethyl acetate DMF: N,N-dimethylformamide Me011
:methanol The compounds of the present invention can be prepared using methods commonly used in peptide chemistry, such as 5chr5der and Ljbk.
“The Peptides” Volume 1 by e, Acade-mic Press, New Yo
It can be produced by the methods described in ``Basics and Experiments of Peptide Synthesis'' by Nobuo Izumiya et al. (1985), etc., and liquid phase method and solid phase method. It can be manufactured by any of the phase methods.

Condensation methods for forming peptide bonds include azide method, acid chloride method, acid anhydride method, mixed acid anhydride method, carbodiimide method, carbodiimide-additive method, active ester method, carbonyldiimidazole method, redox method, Examples include a method using Woodward reagent.

Before carrying out the condensation reaction, carboxyl groups, amino groups, etc. that do not participate in the reaction may be protected, or carboxyl groups and amino groups that participate in the reaction may be activated by means known per se.

Examples of the carboxyl protecting group include esters such as methyl, ethyl, benzyl, p-nitrobenzyl, t-butyl, and cyclohexyl.

Examples of protecting groups for amino groups include benzyloxycarbonyl group, t-butoxycarbonyl group, isobornyloxycarbonyl group, and 9-fluorenylmethoxycarbonyl group.

Examples of the protecting group for the guanidino group include a nitro group, 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 t-butyl group, benzyl group, tetrahydropyranyl group, and acetyl group.

Examples of activated carboxyl groups include:
Corresponding acid anhydrides, azides, active esters [alcohols (e.g., pentachlorophenol, 2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, N-hydroxy-5-norbornene-2,3-dicarboximide) , N-hydroxysuccinimide, N-
esters with hydroxyphthalimide, 1-hydroxybenzotriazole), and the like. Examples of activated amino groups include the corresponding phosphoric acid amides.

The reaction is usually carried out in 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 temperature can be within the commonly used range of about -30°C to about 50°C.

The protective group removal reaction for the peptide of the present invention varies depending on the type of protective group used, but it does not affect the peptide bond and requires that the protective group be removed.

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

After the reaction, the peptide of the present invention produced in this manner can be obtained by peptide separation methods known per se, such as extraction, partitioning, reprecipitation, recrystallization, column chromatography, and the like.

Furthermore, the peptide of the present invention can be made into a derivative at its functional group or a pharmacologically acceptable salt thereof, as described above, by a method known per se.

The peptides of the present invention exhibit strong nootropic effects in passive avoidance tests in rats.

Useful target diseases for the peptide derivatives of the present invention include:
For example, senile dementia (Alzheimer's type dementia), cerebrovascular dementia, and dementia based on Alzheimer's disease, Pick's disease, Huntington's chorea, Kreuffelt-Jakob disease, Parkinson's disease, myelinopathy of the cerebellum, etc. can be used for the prevention or treatment of these diseases.

The toxicity of the peptide derivative of the present invention is extremely low, and there have been no cases of death even at doses far exceeding the therapeutically effective dose.

The peptide derivatives of the invention can be administered as free forms or as derivatives at their functional groups.

The appropriate dosage for the administration is generally in the range of 0.1 ng to 1 mg/day, regardless of whether it is the free form or its salt. In particular, parenteral and nasal administration
It is preferably 0.1 ng to 100 μg for 7 days, and when administered orally or rectally, it is preferably administered 10 to 100 times as much as parenterally.

The peptide derivative of the present invention is mainly administered parenterally (e.g., intravenous or subcutaneous injection, intraventricular or intrathecal administration, nasal administration, rectal administration), but in some cases, it may be administered orally. Good too.

Examples of dosage forms include injections, flat tablets, powders, nasal sprays,
Examples include pills and tablets. The peptide derivative of the present invention can be stored as a solution in physiological saline, but it can also be prepared into a lyophilized ampoule by adding manlnitol and sorbitol, and dissolved at the time of use.

Examples are shown below.

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

Ry': Chloroform-methanol-acetic acid-water (80:
20:2.5:5) Lower layer R, 2, chloroform-methanol-water (70:30:
5) R,3: n-butanol-acetic acid-water (2:l:l) Also, special preparation by high performance liquid chromatography is as follows: Column: μBondapak Cla 1.9
x15cm mobile phase: A) 0.05! I, TFA, B)
This was done using acetonitrile.

[Example 1] H-Asn-5er-Pip-Arg-011-acetate (1) Boc-Pip-Arg (No2)-0Bzl
II-Arg (No2)-0Bzl Io, 6g DM
Add Boc-Pip-OH7 to F100ml solution under water cooling.
,1g, 1lOBt 7.1g and DCC6,7g
was added and stirred at room temperature for 18 hours, then DC:Ure
a was filtered off, and DMF was distilled off.

The residue was dissolved in Ac0EL, washed successively with saturated aqueous sodium bicarbonate, diluted hydrochloric acid, and water, and then dried over anhydrous sodium sulfate.

Ac0EL was distilled off, and the residue was purified on a silica gel column using Cl1C13-acetone to obtain the title compound as an oil.

Yield Jl: 5.5g R,': 0.68. Ry”: o, 8° [α
]. : -24,0' (cd, o, DMF) (2)
Boc-5er(Bzl)-Pip-Arg(NO,)
-0BzlBoc-Pip-Arg(No2)-0Bz
5.2 g of 4 N HCl-Ac0Et 25
After standing in mIt for 30 minutes at room temperature, the solvent was distilled off.

After drying the residue under reduced pressure, it was dissolved in DMF50mj2 and mixed with Et, N1.4m It, and Boc-5er under water cooling.
(Bzl) -0Su 3.9g was added, and 18
Stir for hours.

DMF was distilled off, the residue was dissolved in AcOEt, and washed sequentially with saturated sodium bicarbonate water, dilute hydrochloric acid water, and water.
It was dried with anhydrous sodium sulfate.

Ac0Et was distilled off, and the residue was purified on a silica gel column using CHCl3-acetone to obtain the title compound as an oil.

Yield 11: 2.8g R,l: 0.74. R,2: 0.86 [α]. Knee 42.6° (c-1,0,DMF) (3) Boc-
Asn-5er(Bzl)-Pip-Arg(NO,)
-0BzlBoc-5er(Bzl)-Pip-Arg
(N(h)-0Bzl 1.6g to 4N[1-AcOE
After standing for 30 minutes in t6ml at room temperature, the solvent was distilled off.

2-butanol-(:H2(:12 (5:
1 v/v) and saturated sodium bicarbonate water were added, and the organic layer was separated, washed with saturated brine, and dried over anhydrous sodium sulfate.

The solvent was distilled off, the residue was dissolved in 20 mL of DMF, and under ice cooling Boa-Asn-OHO, 53 g, HOBt O,
53g and DC (: 0.52g) were added.

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

The residue was dissolved in Ac0Et, washed successively with saturated aqueous sodium bicarbonate, diluted hydrochloric acid, and saturated brine, and then dried over anhydrous sodium sulfate.

The solvent was distilled off, and the residue was purified on a silica gel column using CHCl3-Meoll to obtain the title compound as an oil.

Yield: 1. Og R,': 0.57. R,2: 0.74 [α]. Knee 37.8° (c-1,0,DMF) (4)l+-^
sn-5er-Pjp-Arg-OH-acetate Boc-
Asn-5er (Bzl)-Pip-Arg(NO□
)-0Bzl 150mg to 4N HCl-Ac0
After standing for 30 min at room temperature in EtO, 5 m It,
The solvent was distilled off.

The residue was stirred in 20 m2 of 80% acetic acid in the presence of 10% palladium on carbon for 18 hours in a stream of hydrogen.

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

Furthermore, 12 ml 1 minute (flow rate), 0 to 10% (B) 2
Dowexlx2 (
acetate form) and lyophilization to give the title compound.

Yield: 63 mg R,': 0.19 [α]I, knee 46.2° (c-0,5, water) FAB mass spectrum (M+1): 487 [Example 2] tl-Pro-5er-Pip- Arg-OH/acetate (1) Z-Pro-5er (Bzl)-Pip-Ar
g(NO2)-0BzlBoc-5er(Bzl)-P
ro-Arg(NO2)-0Bzl 1. Og, 4NH
CI-AcOEt 5 m IL, EtzN 0.2
From 49 g of m 11 and Z-Pro-O5uO, the title compound was obtained as an oil in the same manner as in Example 1-(2).

Yield: 0.9g Ry': o, 72. Re2: 0.82 [a]
o Knee 51.2° (c-1,0, DMF) (2) l
t-Pro-5er-Pip-^rg-OH-acetate Z
-Pro-5er(Bzl)-Pip-Arg(NOx
)-0Bzl 150 mg was reduced with palladium on carbon as in Example 1-(4), then 12 ml 1 min (flow rate) of 2 to 12% (B) 20 min linear gradient (
A) (mobile phase) was used for high-performance liquid chromatography, treated with DowexlX2 (acetate type), and then lyophilized to obtain the title compound.

Yield: 31 mg R, 3:0.18 [α]. Knee 76.5° (cJ, 5.water) FAB mass spectrum (M+1): 470 [Example 3] H-Pro-Asn-5er-Pip-Arg-OH-
Acetate (1) Z-Pro-Asn-5er (Bzl)-
Pip-Arg(NO2)-0BzlBoc-Asn-
5er(Bzl)-Pip-Arg(NO2)-0Bz
lO, 68g, 4NH(:1-AcOEt3
m It, NMM O, 14 m j2 and Z-Pro
-O5u From 32 g of O, the title compound was obtained in the same manner as in Example 1-(2).

Yield: 0.7g Melting point: 95-97°C R, 12 0. 57. R, 2:0.
74[αko. Knee 48.2° (cd, 0.D
MF) (2) H-Pro-Asn-5er-Pip-
Arg-OH-acetate Z-Pro-Asn-5er (B
zl)-Pip-Arg(NOx)-0Bzl 150
After palladium carbon reduction in the same manner as in Example 1-(4), 12 ml 1 min (flow rate) of 50 or 620% (
B) In (A) (mobile phase) of a 20 minute linear gradient,
Purified by high performance liquid chromatography and purified with Dowexlx2
(acetate type) treatment followed by lyophilization to obtain the title compound.

Yield: 60 mg R,': 0.14 [α]o knee 76.4° (c-0,5, water) FAB mass spectrum (M+1): 584 Next, pharmacology showing the effectiveness of the peptide of the present invention A practical test example is shown below.

[Pharmacological test example] The effect on memory consolidation was determined using Wistar male rats by Burbach et al.
Science), 221.1310-1312 (1
This study was conducted using a trial passive avoidance experiment based on the method of [983]. The experimental setup consisted of a light room and a dark room, and the floor was made of stainless steel grids. Rats placed in the light room can freely move to the dark room. Using this device, rats are given a single electric shock when they enter a dark room.

Retention of passive avoidance behavior in response to electric shock was determined by the time it took for the rat, placed in the light room again after a certain period of time, to enter the dark room (response latency).

Examination of the effect of cycloheximide on improving experimental retrograde amnesia One hour after the peptide or physiological saline of the present invention was subcutaneously administered, an electric shock (0.5 mA) was administered, and immediately thereafter, cycloheximide 2 was administered subcutaneously. .7 to 3.0 mg/kg or physiological saline was administered subcutaneously, and a memory retention test was conducted 48 hours later. Rats given only saline generally
The rats in the control group to which only cycloheximide was administered showed a response latency of around 50 seconds and developed retrograde amnesia.

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

Regarding the peptide obtained in Example 1, its administration ffi
The effect at lng/kg (expressed as a percentage of the response latency of the peptide-administered group relative to the response latency of the control group) was 235%.
Met.

The above test results showed that the peptide of the present invention had an excellent improvement effect on retrograde amnesia.

Next, examples of formulations of drugs containing the peptide of the present invention will be shown.

[Formulation Example 1] (Injection) 0.1 mg of the peptide derivative obtained in Example 1 and 0.9 g of sodium chloride were contained in 100 mL of distilled water for injection, and the pH was adjusted to 6.0-6.0 with sodium hydroxide. An aqueous solution adjusted to 8.0 was prepared. After bacterial filtration, this
The mixture was filled into a JZ ampoule, sealed and sterilized by heating to produce an injection.

[Formulation Example 2] (Freeze-dried preparation) Distilled water for injection 100mj! An aqueous solution was prepared in which 5 mg of the peptide derivative obtained in Example 1 and 5 g of D-mannitol were contained, and the pH was adjusted to 6.0 to 8.0 with a phosphate buffer. This is filtered for bacteria and placed in a vial.
After dispensing 1 ml, freeze-drying was performed to produce a freeze-dried injection.

[Formulation Example 3] (Nasal Drop) 10 mg of the peptide derivative obtained in Example 1 was contained in 100+nJ2 of physiological saline, and the pH was adjusted to 3.0 to 6.0 with a citric acid buffer. Dose 0.5ml1
A nasal spray containing 50 μg was produced.

[Formulation example 4] (Planning) Hard fat (triglyceride of saturated fatty acids) 98
．． After adding 5 g of egg yolk lecithin O to 5 g and melting at 40 to 45°C, the peptide derivative 5 obtained in Example 1 was added.
A solution obtained by dissolving 1 mg of PEG400 in 1 g of PEG400 was added thereto, stirred and dispersed, and then 1 g of the solution was poured into a planing mold, and after solidification, it was separated from the mold to produce a planing.

[Effects of the Invention] The peptide of the present invention is a novel compound, has excellent nootropic activity, and is useful as a medicine.

Patent applicant: Nippon Chemifa Co., Ltd. Patent applicant: Fujirebio Co., Ltd. Representative Patent Attorney Yasuo Yanagawa

Claims (1)

[Claims] 1. General formula (I): A-Ser-Pip-Arg (I) (wherein A is Pro-Asn-, Asn- or Pro
- represents a peptide represented by the following formula or a derivative in its functional group, or a pharmacologically acceptable salt thereof. 2. General formula (I): A-Ser-Pip-Arg (I) (wherein A is Pro-Asn-, Asn- or Pro
an effective amount of a peptide represented by - or a functional group derivative thereof, or a pharmacologically acceptable salt thereof;
and an anti-dementia agent comprising a pharmacologically acceptable carrier or diluent.