JPH02273699A - Peptide and antidemential agent containing the same - Google Patents

Abstract

NEW MATERIAL:The peptide (derivative or salt) of formula I ((m) and (n) are 1 or 0). USE:An antidemential agent. PREPARATION:The objective peptide of formula I can be produced e.g. by adding and reacting a compound of formula Boc-Pro-OSu (OSu is N- hydroxysuccinimide ester; Boc is t-butoxycarbonyl) to a THF solution of a compound of formula H-Arg(NO2)-OBzl (Bzl is benzyl) under ice cooling, remov ing alpha amino-protecting group, reacting with Boc-Ser(Bz1)-OH in DMF to elimi nate alpha -aminoprotecting group. reacting with Boc-Asn-OH to obtain a protected- peptide of formula II, reacting the resultant peptide with Z-Pro-OSu (Z is benzyloxycarbonyl). deprotecting in hydrogen gas stream in the presence of 10% Pd-carbon and purifying the isolated peptide by high performance liquid chromatography.

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] It has been known for a long time that vanpressin has a nootropic effect, but recently peptides that can be considered as fragments of vanpressin, such as pGIu-Asn-Cys-Pro-Arg-Gly-
It has been reported that the peptide represented by NH2O-Cys-OH also has a nootropic effect similar to vanpressin [Science
ce) 221.1310-1312 (I983)
].

Also, (pGIu-Asn-C7g-Pro-Arg-GI7
It has also been reported that the peptide represented by -Mn2)2 has nootropic effects (Japanese Patent Application 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 even better nootropic activity than such vanpressin and vasobrecin fragment peptides.

[Means for Solving the Problems] The present invention provides general formula (I): Pro-(Asn)*-5er-L-(D-)Pro
-Arg-(Gly) n (I) (in the formula, 1m and n are each independently 1 or 0) 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 (I) means the following.

a) N-acyl derivatives derived from aliphatic carboxylic acids having 1 to 6 carbon atoms; b) amides or mono- or di-alkyl substituted amides having alkyl groups of 1 to 6 carbon atoms; ,as well as.

c) Esters derived from alcohols having 1-18 carbon atoms, preferably aliphatic alcohols having 1-6 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
The abbreviations adopted by the PAC'-IUB nomenclature committee are used. For example, the following abbreviations are used. Furthermore, if the optical configuration is not indicated, the amino acid shall mean the L-type.

Arg: Arginine Asn: Asparagine G! ! =Glycine P'0 Nibroline Ser: Serine Hoe: t-Butoxycarbonyl Z: Penzyloxycarbonyl N02: Nitro Bzl: Benzyl 0 Bzl: Benzyl ester O9u: N-hydroxysuccinimide ester DC:
C: N,N'-dicyclohexylcarbodiimide DC
Urea: N,N'-dicyclohexylurea HO
Bt: 1-hydroxybenzotriazole EtzN:
) ethylamine NNM: N-methylmorpholine TFA: trifluoroacetic acid HF: tetrahydrofuran Ac0Et: ethyl acetate DMF: N,N-dimethylformamide NeOH: methanol The compounds of the present invention can be prepared by methods commonly used in peptide chemistry, e.g. and Li1
“The Peptides” by bke
J Volume-, Acade-mic Press, New
York, U. S. A. (1965), Nobuo Izumiya et al., "Fundamentals and Experiments of Peptide Synthesis" Marukubi (1985), etc.
It can be produced by both liquid phase method and solid phase method.

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, A method using Woodward reagent K, etc. can be mentioned.

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, inbornyloxycarbonyl group, and 9-fluorenylmethoxycarbonyl group.

Examples of protecting groups for guanidino groups include nitro group, benzyloxycarbonyl group, tosyl group, p-methoxybenzenesulfonyl group, and 4-methoxy-2,3,8-)limethylbenzenesulfonyl 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-21,3-dicarboximide) , N-hydroxysuccinimide, N
-hydroxyphthalimide, l-hydroxybenzotriazole)] and the like. Examples of activated amine 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 protecting group removal reaction for the peptide of the present invention varies depending on the type of protecting group used, but it is necessary that the protecting group be removed without affecting the peptide bond.

Examples of methods for removing the protective group include acid treatment with hydrogen salt, hydrogen bromide, anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid, or a mixture thereof. In addition to this, reduction with sodium in liquid ammonia, palladium on carbon, etc. may also be mentioned. 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.

The names of diseases for which the peptide derivatives of the present invention are useful are 1.
Examples include senile dementia (Alzheimer's dementia), cerebrovascular dementia, and dementias caused by Alzheimer's disease, Pick's disease, Huntington's chorea, Kroinfeldt-Jakob disease, Parkinson's disease, myelinopathy of the cerebellum, etc. and 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 medicinally 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 ug/day, and in oral administration or rectal administration, it is preferable to administer 10 to 100 times as much as in parenteral administration.

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

Dosage forms include, for example, injections, tablets, powders, nasal drops,
The peptide derivative of the present invention, including pills, tablets, etc., can be stored as a solution in physiological saline, but it can also be prepared into a freeze-dried ampoule by adding mannitol or 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.

R (I: chloroform-methanol-acetic acid-water (80:
20:2.5:5) Lower layer Rf2: Chloroform-methanol-water (70:30:
5) Rt3: n-butanol-acetic acid-water (2:1:1) Also, for purification by high performance liquid chromatography, Column: μBondapak CIB 1.9
X15cm mobile phase: A) 0.05$ TFA, B) 7
C)-to! This was done using J.

[Example 1] H-Pro-Agn-Set-Pro-Arg-OH・
Acetate (I) Boc-Pro-Arg(NOz)-0
BzlH-Arg(NO2)-0Bzl 15 g T
Add Boc-Pro-O3 to 250 mJL of HF solution under water cooling.
15 g of the mixture was added, and the mixture was stirred at room temperature for 18 hours.

T) IF was distilled off, and the residue was dissolved in AcOEt, washed successively with diluted hydrochloric acid, saturated sodium bicarbonate, and water, and then dried over anhydrous sodium sulfate.

Ac0Et was distilled off, and the residue was purified on a silica gel column using GHGI3-MeOH to obtain the title compound as an oil.

Yield: 22g Rr': 0.61. R(2:0.77[(XI o
: -37, 1' (c=1.0. DMF) (2)
Hoe-Ser(Bzl)-Pro-Arg(NOz)
-0BzlHoe-Pro-Arg(NOz)-0Bz
l 22 g, 4NHCI-AcOEt 110 m
After standing for 30 minutes at room temperature in 100 ml of water, the solvent was distilled off.

After drying the residue under reduced pressure, it was dissolved in DIIF150+nJ1 and diluted with [3% 9 rrl fl , Bo
c-Ser(Bzl)-0H12,8g, HOBt
Add 9.4 g of lOg and IICC and stir at room temperature for 18
Stir for hours.

It(IJrea) was filtered off, DMF was distilled off, and the residue was dissolved in Ac0Et, washed successively with saturated aqueous sodium bicarbonate, diluted hydrochloric acid, and water, and then dried over anhydrous sodium sulfate.

Ac0Et was distilled off, and the residue was crystallized from Ac0Et-ether and collected by filtration to obtain the title compound.

Yield: 21g Melting point: 80-82°C Rr': 0.67, Rr2: 0.83 [α] o
Knee 30.8° (c-1,0, DNF) (3) Bac-
Asn-Ser(Bzl)-Pro-Arg(NO2)
-0BzlBoc-Ser(Bzl)-Pro-Arg
(NO2)-0Bzl 4. Og4NHCI-AcO
After standing in 20 m fl of Et at room temperature 't' for 30 minutes, the solvent was distilled off.

Add 2-butanol (J2CI2 (5: 1 m/m) to the residue.
M) 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, and the residue was dissolved in 60 m of DMF, and cooled with water.
．． 34 g and ncc l-32 g were added.

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

The residue was diluted with 2-butanol-GH2CI2 (5:1
After washing successively with saturated sodium bicarbonate water, salt-saturated dilute hydrochloric acid water, and saturated salt water, it was dried over anhydrous sodium sulfate.

The solvent was distilled off, and Ac0Et was added to the residue to crystallize it, and the title compound was collected by filtration.

Yield: 4.3g Melting point: 186-187°C Rrl: 0.55. Rr2: 0.73[α]o knee 34.6° (c=1.0, DMF) (4) Z-Pr
o-AsIII-5sr(Bzl)-Pro-Arg(
NOz)-0BzlHoe-Asn-Ser(Bzl)
-Pro-Arg(NO2)-0Bzl 3.5g to 4
NHCl-Ac0Et in 15 ml at room temperature.
After standing for a minute, the solvent was distilled off.

After drying the residue under reduced pressure, it was dissolved in DMF, and then dissolved in NM under water cooling.
M 0.8 ml and Z-Pro-O9u 1.52 g
added.

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

The residue was dissolved in 2-butanol-GH2Gh (5:1 m/
After washing successively with saturated sodium bicarbonate water, salt-saturated dilute hydrochloric acid water, and saturated salt water, it was dried over anhydrous sodium sulfate.

The solvent was distilled off, and the residue was purified with a silica gel column using CHCh-methanol, and then ether was added to crystallize, and the title compound was collected by filtration.

Yield: 3.2g Melting point: 94-96°C Rrl: 0.55. Rr2: 0.75 [α] o
Knee 45.5° (c-1,0, D) fF) (5) H-
Pro-Asn-Set-Pro-Arg-OH*Acetate Z-Pro-Asn-Set (Bzl)-Pro-A
150 mg of rg(NO2)-0Bzl was dissolved in 20 ml of 80% acetic acid in the presence of 10% palladium on carbon.
The mixture was stirred in a hydrogen stream for 8 hours.

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 mJL/min (flow rate), 0 to 10% (B)2
DowexlX2 (
Acetate ff1) treated.

Lyophilization gave the title compound.

Yield: 96 mg Rr3: 0.10 [αlo knee 91.0° (c=0.5, water) FAB mass spectrum (M+1): 570 [Example 2] H-Pro-5et-Pro-Arg-OHm acetate (
I) Z-Pro-'Jer(Bzl)-Pro-Ar
g(NO2)-0BzlBoc-Ser(Bzl)-P
ro-Arg(NO2)-0Bzl 4. Og, 4NH
2.2 g of CI-AcOEt, NNNlmJL, and 2.2 g of Z-Pro-O9u were treated in the same manner as in Example 1-(4), and then crystallized by adding ether to obtain the title compound.

Yield: 4.9g Melting point = 72-74°C Rr': 0.66, Rr2: 0.82 [α] D
Knee 45.8° (c=1.o, DMF) (2) H
-Pro-9et-Pro-Arg-OH11 acetate Z
-Pro-9et(Bzl)-Pro-Arg(NO2
) -0 Bzl 150 mg was reduced to palladium on carbon as in Example 1-(5), then 12 m/min (flow rate), 0 to 10% (B) of a 20 min linear gradient (
The product was purified by high performance liquid chromatography using A) (mobile phase), treated with DowexlX2 (acetate type), and then lyophilized to obtain the title compound.

Yield near 5 mg 11r3:0.13 [(XI D knee 97-4° (csO, 5, water) FA
B mass spectrum (M+1): 456 [Example 3] H-Pro-3et-Pro-Arg-Gly-Nl2
m acetate (I) Boc-Arg(NO2)-Gly
-MH2Boc-Arg(NOz)-0H10g c7
) NMN 3.5 in a DMF 80 ml solution under water cooling.
ml and 3.1 ml of ethyl chlorocarbonate were added and stirred for 15 minutes.

Then 3-5 g of H-Gly-MHz hydrochloride, NMM
A mixture of 3.5 ml (I)DNF and 20 ml was added thereto, and the mixture was stirred for 3 hours under water cooling, and then [lNF was distilled off.

Convert the residue into 2-B)) /-RueC)12c12 (5:
1 v/v) and saturated sodium bicarbonate water 1
After sequentially washing with salt-saturated dilute hydrochloric acid and saturated brine, it was dried over anhydrous sodium sulfate.

The solvent was distilled off, and Ac0Et was added to the residue to crystallize it, and the title compound was collected in pieces.

Yield near, 4g Melting point = 160-162°C Rr': 0.21. Rr2:0.42[α]D:+
3.2° (c=11.0, DNF) (2) Bac
-Pro-Arg(NO2)-G! y-NH2Boc-
Arg(NO2)-Gly-MHz G, Og,
4 NHCI-AcOEt40mJ1. EhN 3.4
mJl and 5.1 g of Boc-Pro-OSu,
The title compound was obtained in the same manner as in Example 1-(4).

Yield: 6.5g Melting point = 109-111"C Rr': 0.23. Rr2: 0.45 [α] D
Knee 30, 5° (c=1.0, DMF) (3)
Boc-Ser (Bzl)-Pro-Todoroki rg (NOz
)-Gly-MH2Boc-Pro-Arg(NOz)
-Gly-MHz 6. Og, 4NMCI-
AcOEt 35ml, EhN 1.8ml
and Boc-5er (Bzl)-OSu 5. t g
The title compound was obtained in the same manner as in Example 1-(4).

Yield: 6.7g Melting point = 109-113°C Rfl: 0.32. Rr2: 0.56 [αlo knee 29.2° (c-1,0, DNF) (4) Z-Pro
-Set(Bzl)-Pro-Arg(NOz)-CI
F-MH2Boc-Ser(Bzl)-Pro-Arg
(N(h)-Gly-MHz1.Og, 4N IC
l-Ac0Et 10 ml, INN O, 34
ml and 0.54 g of Z-Pro-OSu, the title compound was obtained in the same manner as in Example 1-(4).

Yield: 0.7g Melting point: 108-11"0 Ryl: 0.34. Rr2: 0.56 [Q] D
Knee 56.0° (c=1.0, DMF) (5) H
-Pro-5et-Pro-Arg-Gly-MHz
m acetate Z-Pro-Ser(Bzl)-Pro-Ar
After 150 mg of g(NO2)-Gly-MHz was reduced with palladium on carbon in the same manner as in Example 1-(5),
Purified by high performance liquid chromatography using (A) (mobile phase) with a linear gradient of 12 mJl/min (flow rate) from 0 to 10% (B) in 20 minutes, treated with DowexlX2 (acetate type), and lyophilized. The title compound was obtained.

Yield: 105mg Rr3: 0.10 [Q] o Knee 96.7° (cmo, 5, Thu) FA
B mass spectrum (M+1): 512 Next, pharmacological test examples showing the effectiveness of the peptide of the present invention will be shown.

[Pharmacological test example] The effect on memory consolidation was determined using Wistar male rats by Burbach et al.
Science), 221.1310-1312 (I
This study was conducted using a trial passive avoidance experiment based on the method used in 1983. 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 was administered subcutaneously. 2.7-3.01 g/kg or physiological saline was administered subcutaneously, and a memory retention test was conducted 96 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 2, the dosage was 0.
The effect at 1 mg/kg (expressed as a percentage of the response latency of the peptide-administered group relative to the response latency of the control group) was 213%.
Met.

From the above test results, the peptide of the present invention showed 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 11 (Injection) In 100 mJl of distilled water for injection, 0.1 mg of the peptide derivative obtained in Example 1 and 0.9 g of sodium chloride.
and adjust the pH to 6.0 to 8.0 with sodium hydroxide.
An aqueous solution was prepared. This was filtered for 1 m after bacterial filtration.
The mixture was filled into fL ampoules, sealed and sterilized by heat to produce an injection.

[Formulation Example 2] (Freeze-dried preparation) 5 mg of the peptide derivative obtained in Example 1 and 5 g of D-mannitol were contained in 100 ml of distilled water for injection, and the pH was adjusted to 6.0 with a phosphate buffer. An aqueous solution adjusted to 0 to 8.0 was prepared. This was subjected to bacterial filtration, dispensed in 1 mJL into vials, and then freeze-dried 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 mJL of physiological saline, and the pH was adjusted to 3.0 to 6.0 with a citric acid buffer; afI5t ,, single dose 0
．． A nasal spray containing 50 gg in 5 mJl was produced.

[Formulation example 4] (Planning) Hard fat (triglyceride of saturated fatty acids) 98
．． After adding 0.5 g of egg yolk lecithin to 5 g and melting at 40 to 45°C, the peptide derivative 5 obtained in Example 1 was added.
m g t-P EG 400 (F) 1 g was added thereto, stirred and dispersed, and then the I
g was injected into a layer mold, and after solidification, it was separated from the mold to produce a flat plate.

[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): Pro-(Asn)_m-Ser-L-(D-)Pro
-Arg-(Gly)_n(I) (wherein m and n are each independently 1 or 0) or a functional group derivative thereof, or a pharmacologically acceptable salt thereof . 2. General formula (I): Pro-(Asn)_m-Ser-L-(D-)Pro
-Arg-(Gly)_n(I) (wherein m and n are each independently 1 or 0) or a functional group derivative thereof, or a pharmacologically acceptable salt thereof an effective amount of
and an anti-dementia agent comprising a pharmacologically acceptable carrier or diluent.