JPH02273698A - Peptide and antidemential agent containing the same - Google Patents

Abstract

NEW MATERIAL:The peptide (derivative or salt) of formula (A is azetidine-2- carboxylic acid, D- or L-Pro, sarcosine or pipecolic acid; B is D- or L-Arg, citrulline, homoarginine. Lys or Orn: (n) is 1 or 0). USE:An antidemential agent. PREPARATION:An amino acid having protected alpha-amino group is condensed from the C terminal side according to the amino acid sequence of the objective peptide by solid-phase technique using a 2.4-dimethoxybenzhydrylamine resin as a carrier and the alpha-amino-protecting group is eliminated. The above procedures are repeated to obtain a peptide resin. The resin is dried and treated with trifluoroacetic acid to remove the whole protecting groups and separate the peptide from the resin. The separated peptide is purified by high performance liquid chromatography to obtain the objective peptide of formula.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] 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 nootropic effects, but recently peptides that can be regarded as fragments of vanpressin, such as peptides, have been developed.

pGlu-As! I-Cys-Pro-Arg-Gly
It has been reported that the peptide represented by -NH4I H-Gys-OH also has a nootropic effect similar to pap pressin [Science
ce) 221.1310-1312 (1983)
].

Also, (pGlu-1sn-Cys-Pro-Arg-Gly
It has also been reported that the peptide represented by -NO3)2 also has a nootropic effect (Japanese Patent Laid-Open No. 59-93036).

[Problem to be Solved by the Invention 1] The object of the present invention is to provide a novel peptide having an even better nootropic effect than such vanpressin and pappressin fragment peptides.

[Means for solving the problem] The present invention provides general formula (I): pGlu-Asn-Ssr-A-B-(Gly)
n (I) (wherein A represents Aze, D- or L-Pro, Pip or Sar, B
is D- or L-Arg, Git, Har,
Lyt or Orn, n is 1 or 0) or its functional group derivatives, or pharmacologically acceptable salts 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 heptanoalkyl or di-alkyl substituted amides having alkyl groups of 1 to 6 carbon atoms; and 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
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 Aze: Azetitan-2-carboxylic acid Cit Niditrulline Glj Nigericine Har: Homoarginine Lys: Lysine Orn: Ornithine pGlu: Pyroglutamic acid Pip: Pipecolic acid Pro Nibroline Sar: Sarcosine Sir: Serine Boc : t-butoxycarbonyl Z: penzyloxycarbonyl Fmac: 9-fluorenylmethoxycarbonyl BuL
: t-Butyl Ntr: 4-methoxy-2,3,6-trimethylbenzenesulfonyl No2 dinitro Bzl: Benzyl 0 Bzl: Benzyl ester OSu: N-hydroxysuccinimide ester DCC
: N,N'-dicyclohexylcarbodiimide DCU
rea: N,N'-dicyclohexylurea DIG
: N,N'-diisopropylcarbodiimide HOB
t:1-Hydroxybenzotriazole EtzN: Triethylamine DIIF: N,N-dimethylformamide TFA:
Trifluoroacetic acid THF: Tetrahydrofuran Ac0Et: Ethyl acetate MeOH: Methanol The compounds of the present invention can be prepared by methods commonly used in peptide chemistry, such as 5 childr and Lube.
“The Peptides” J by Ke.
Volume-, Acade-mic Press, New Y
ork, υ, S, A, (1965), "Fundamentals and Experiments of Peptide Synthesis" by Nobuo Izumiya et al., 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 = 7 date method, active ester method, carbonyldiimidazole method, and redox method. , a method using Woodward reagent K, and the like.

Before carrying out the condensation reaction, carboxyl groups, amino groups, etc. that do not participate in the reaction may be protected, or carboxyl groups, amine 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, E-butyl, and cyclohexyl.

Examples of protecting groups for amino groups include benzyloxycarbonyl group, t-butoxycarbonyl group, inbornyloxycarbonyl 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-2,3,6-1-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-2,3-dicarboximide) , N-hydroxysuccinimide, N-
esters with hydroxyphthalimide, l-hydroxybenzotriazole), and the like. Examples of activated amide 7 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, etc., or a mixture thereof. be able to.

The reaction temperature is generally used from about -30"C to about 50"C.
This can be done within the range of

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 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 a peptide separation method known per se, such as extraction and distribution, reprecipitation, recrystallization, column chromatography, etc.

Further, the peptide of the present invention can be prepared by a method known per se, as described above, or a derivative of the functional IL group.
They can be made into pharmaceutically acceptable salts.

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, Kroinfeldt-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 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 gg/day, 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.

Dosage forms include, for example, injections, halves, powders, nasal sprays,
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 "1: Chloroform-methanol-acetic acid-water (80:
20:2.5:5) Lower layer 1 ((2: Chloroform-methanol-water (70:30)
:5) Rr3: n-butanol-acetic acid-water (2:1:l) Also, purification by high performance liquid chromatography.

Column: JLBondapak Cps 1.9
X15cm mobile phase: A) 0.05X TFA, B)
7-4 = Performed using tonitrile.

[Example 1] pGlu-Asn-9et-Pro-Arg-Gly-
MHz Acetate 2,4-dimethoxybenzhydrylamine resin (0,24 mmol MH2/g) in The Journal of Organic Chemistry (J, Org,
Cbe layer).

52.1197 (1987), and the peptide chain was extended by repeating the condensation step and N''-deprotection step shown below.

Condensation process reagent/solvent Time (minutes) x number of times 1, DMF
1X32, F set OC-amino acid + HOBt+ OH: 120 (3 equivalents each) - DMF 3, DMF 1X34, Impropatool 1X35, CH2C exchange 2 1 x 3N-Deprotection process reagent/solvent time (minutes) x number of times 6, DMF
lX37.20% piperidine-DMF 18.20% piperidine-DM
F209,DMF
lX310, CH2C cross 2 l
×3 The condensation reaction was confirmed by Kaiser test, and the above steps 1 to 5 were repeated as necessary. First, 1 g of 2,4-dimethoxybenzhydrylamine resin was added with Fmac-
F-oc-GI2-resin was prepared by condensation steps 1 to 5 using 214 mg of GI7-OH, 110 mg of HOBt, and 12 ml of DIGO.

Next, the protecting groups were removed in NQ'-deprotection steps 6 to 10 to obtain an nc+7-resin.

By repeating the condensation and Na-deprotection steps in the same manner, H-A
sn-3er(Bu')-Pro-Arg(Mtr)
-Gly- resin, condensation steps 1 to 5 using pGlu-OH are performed to form pGlu-Asn-3er (Bu
[)-Pro-Arg(Mtr)-Gly-resin was prepared. After drying the resin, TFA-anisole (
The mixture was stirred for 4 hours at room temperature in a 10-Im vacuum chamber, and the resin was filtered off and washed with TFA.

After the TFA solution was further left at room temperature for 2 hours, TFA was distilled off, ether-water was added to the residue, and the aqueous layer was separated.
It was treated with DowexlX2 (acetate type) and freeze-dried.

Furthermore, 12 mJ1/min (flow rate), 0 to 10% (B)2
After high performance liquid chromatography purification using (A) (mobile phase) of a 0 minute linear gradient, DowexlX2
acetate form) and lyophilization to give the title compound.

Yield +69mg Rr3: 0.14 [αlo knee 91.8° (c-0,5, water) FAB mass spectrum (M+1): 640 [Example 2] pGlu-Asn-Ser-D-Pro-Arg-G
ly-NH2 acetate 2,4-dimethoxybenzhydrylamine resin Ig in the same manner as in Example 1.
pGlu-Asn-Ser(Bu-11-Pro-A
After preparing rg(Ntr)-Gly-resin and treating it with TFA, it was similarly purified by high-performance liquid chromatography and ion-exchanged, followed by freeze-drying to obtain the title compound.

Yield near 4 mg Rr3: 0-15 [αJρ knee 13.9° (c=0.5, water) FAB
Mass spectrum (M+1): 640 [Example 3] pG l u-Asn-5e r-Pro-D-A r
pGlu-Asn-Ser (Buly-Pro
-D-Arg(Mtr)-G17- resin was prepared and T
After the FA treatment, high performance liquid chromatography purification and ion exchange treatment were performed in the same manner, followed by lyophilization to obtain the title compound.

Yield: 49 mg Rr: 0.15 [a] knee 67.4° (c-0,5, Thursday) FAB mass spectrum (M+1): 640 [Example 4] pGlu-ASn-Ser-D-Pro-D -Arg-
From 1 g of Gly-MH211 acetate 2.4-dimethoxybenzhydrylamine resin, pGlu-Asn-Ser(Bu L)-〇-Pr was prepared in the same manner as in Example 1.
An o-D-Arg(Mtr)-Gly-resin was prepared, treated with FA, followed by high performance liquid chromatography purification, ion exchange treatment, and lyophilization to obtain the title compound.

Yield: 62 mg Rr3: 0.16 [alo: +15.3° (c-0,5, water) FAB mass spectrum (M+1): 640 [Example 5] pGlu-Asn-Ser-Pro-Arg-OH (1
) Hoe-Pro-Arg(NO2)-0BzlH-
Arg(N(h)-0Bzl l 5 g(1)TH
Add Boa-Pro-O to 250 ml of F solution under water cooling.
3 ul 5 g was added and stirred at room temperature for 18 hours.

↑) 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 CHG13-MeOH to obtain the title compound as an oil.

Yield: 22g Rrl: 0.61. Rr2:0.77[Q]D
Knee 37.1° (c=1.0, DMF) (2) B
oc-Ser(Bzl)-Pro-Arg(NO2)-
0BzlBoc-Pro-Arg(NO2)-0Bzl
22 g of 4NH (IAcOEt 110 ml
After being left in the solution for 30 minutes at room temperature, the solvent was distilled off.

After drying the residue under reduced pressure, it was dissolved in 150 ml of DMF and washed with 9 ml of Et3N under water cooling in an AC-3ER (Bz
l)-0H 12.8g, HOBtlOg and [1009.4g were added and stirred at room temperature for 18 hours.

The CUrea was filtered, [1MF was distilled off, and the residue was
The solution was dissolved in c0Et, 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 280-82°C Rrl: 0.67, Rf2: 0.83 [α] o nee 3o, a° (c-1,0, DNF) Z-pGlu-Ag
n-8er(Bzl)-Pro-Arg(NO2)-0
BzlBoc-Set(Bzl)-Pro-Arg(N
oz)-〇Hz12.3g to 4NH(I-AcOEt
After standing for 30 minutes at room temperature in 10 ml, the solvent was distilled off.

2-Butanol-GIhl (I2 (5:1 m/m)) and saturated sodium bicarbonate water were added to the residue, and the organic layer was separated, washed with saturated brine, and then dried over anhydrous sodium sulfate.

The solvent was distilled off, the residue was dissolved in 20 mM DNF, and 1-4 g of Z-pGiu-Asn-OH, HOBt was added under ice cooling.
0.7 g and DCCO, 8 g were added.

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

The residue was dissolved in 2-butanol-GH2C12 (5:1 maha), washed successively with saturated aqueous sodium bicarbonate, 1 sodium chloride, saturated dilute 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-HeOH, and then ether was added to crystallize, and the title compound was collected by filtration.

Yield: 1.9g Melting point = 122-125°C Rrl: 0.46. R(2:0.64 [Q] o
: -38,6' (c=1.0.DMF) (4)
pGlu-Asn-Ser-Pro-Arg-OH2-
pGlu-Asn-Ser(Bzl)-Pro-Arg
(NOz)-0Bzl 100mg in 80% acetic acid 20m
The mixture was stirred in a hydrogen stream for 18 hours in the presence of 10% palladium on carbon in Jl.

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

Furthermore, 12mM/min (flow rate), 0 to 10% (B)20
After purification by high performance liquid chromatography using fractional linear gradient (A) (mobile phase), the product was treated with DowexlX2 (acetate type) and lyophilized to obtain the title compound.

Yield: 55 mg Rr3: 0.18 [α]o knee 85.5° (c-0,5, water) FAB mass spectrum (M+1) +584 Next, pharmacological test examples showing the effectiveness of the peptide of the present invention shows.

[Pharmacological test example] The effect on memory consolidation was determined using 11 star 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 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 cycloheiimide on improving experimental retrograde amnesia The peptide of the present invention or physiological saline was administered subcutaneously, and 1 hour later, an electric shock (0.5 mA) was experienced, and immediately thereafter, cycloheiimide was administered subcutaneously. 2.7-3, 0 mg/kg
Alternatively, physiological saline was administered subcutaneously, and a memory retention test was conducted 48 hours later. Rats administered only with physiological saline generally exhibited a response latency of around 300 seconds, while rats in the control group administered only with cycloheximide exhibited 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, the dosage was 0.
The effect at 1 ng/kg (expressed as a percentage of the response latency of the peptide-administered group relative to the response latency of the control group) was 353%.
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 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.
Fill into JL ampoule.

The mixture was melt-sealed and heat sterilized to produce an injection.

[Formulation Example 2] (Lyophilized preparation) 5 mg of the peptide derivative obtained in Example 1 and 5 g of D-mannitol were added to 100 m 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 into 1 ml vials, and then lyophilized to produce a lyophilized injection.

[Formulation Example 3] (Nasal Drop) 100m of physiological saline! 10 mg of the peptide derivative obtained in Example 1 was added to L, and the pH was adjusted to 3.0 to 6.0 with a citric acid buffer. +,, single dose 0.
A nasal spray containing 50 g in 5 mJZ was prepared.

[Formulation example 4] (Half tone) 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.
A solution obtained by dissolving PEG400 in Ig was added thereto, stirred and dispersed, and then the Ig was injected into a planing plate, and after solidification, it was separated from the mold to produce a half-tone.

[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): pGlu-Asn-Ser-A-B-(Gly)_n(
I) (wherein A is Aze, D- or L-Pro
, Pip or Sar, and B is D- or L-
represents Arg, Cit, Har, Lys or Orn,
(n is 1 or 0) or a functional group derivative thereof, or a pharmacologically acceptable salt thereof. 2. General formula (I): pGlu-Asn-Ser-A-B-(Gly)_n(
I) (wherein A is Aze, D- or L-Pro
, Pip or Sar, and B is D- or L-
represents Arg, Cit, Har, Lys or Orn,
an effective amount of a peptide represented by (n is 1 or 0) or a derivative in its functional group, or a pharmacologically acceptable salt thereof;
and an anti-dementia agent comprising a pharmacologically acceptable carrier or diluent.