JPH02273696A - Peptide and antidemential agent containing the same - Google Patents

Abstract

NEW MATERIAL:The peptide of formula I [when A is D- or L-PrO, B is homoarginine or citrulline, or when B is D- or L-Arg, A is pipecolic acid, Arg, etc.), its derivative or their salt. EXAMPLE:The compound of formula II. USE:An antidemential agent. It has strong nootropic action and extremely low toxicity. PREPARATION:The exemplified compound can be produced e.g. by successively condensing amino acids to 2,4-dimethoxybenzhydrylamine resin to form pGlu- Asn-Cys(Trt)-Pro-Cit-Gly-resin (Trt is trityl), treating the product with trifluoroacetic acid, etc., to separate the resin and reacting the obtained peptide with cystine monoxide.

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 vapressin has a nootropic effect, but recently it has been discovered that peptides that can be considered fragments of vapressin, such as the peptide represented by , also have a nootropic effect like vapressin. Reported [Science
ce) 221.1310-1312 (I983)
].

Also, (pGlu-Asn-Cys-Pro-Arg-Gly
It has also been reported that the peptide represented by -Ntf□)2 also has a nootropic effect (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 that has a more superior nootropic effect than such pap pressin and pap pressin fragment peptides.

[Means for Solving the Problems] The present invention is based on the general formula (I): % formula % () (wherein A and B are Har or Cit when A is D- or L-Pro) Yes, A is D-Pr
o, then B is D-Arg, or when B is G or L-Arg, A is Sar, Pip,
The present invention relates to a peptide represented by the combination of Aze or Arg, a derivative thereof in a functional group, 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 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 1,000-alkyl- or di-alkyl-substituted amides having 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 technical field, or by I
The abbreviations adopted by the UPAC-I UB naming committee are used. For example, the following abbreviations are used: Furthermore, amino acids that do not show an optical configuration are assumed to be L-type.

Arg: arginine Asn: asparagine Aze: azethian-2-carboxylic acid Cit niditrullin Gys nigistein cty nigericin 11ar: homoarginine pGlu: pyroglutamic acid Pip: pipecolic acid Pro nibroline Sar: sarcosin DIG: N,N'-diisopropylcarbodi imide) 10Bt:1 -Hydroxybensitriazonyl Mtr
: 4-Methoxy-2,3,6-)lyntylbenzenesulfonyl Trt : 'p-lytyl Fmoc: 9-fluorenylmethoxycarbonyl DMF
: N,N-dimethylformamide TH^ Nitrifluoroacetic acid Compounds of the present invention can be prepared by methods commonly used in peptide chemistry, e.g. 5chr5der and Li1b
“The Peptides” by ke
Volume-, Acade-mic Press, New Y
ork, Ll, S, ^, (I965), "Basics and Experiments of Peptide Synthesis" by Nobuo Izumiya et al. (I985)
It can be manufactured by the method described in et al., and can be manufactured by both a liquid phase method and a 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.

Among the amino acids constituting the peptides of the present invention in the condensation reaction, for the cystine site, cystine derivatives are used, or
Alternatively, it is also possible to form a peptide chain as a cysteine residue and then convert it to cystine by a known method.

Before carrying out the condensation reaction, carboxyl groups, amino groups, etc. that do not participate in the reaction may be protected, or carboxyl groups, 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-fluorenylmethodicarbonyl 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 protecting groups for mercapto groups include trityl group,
Examples include acetamidomethyl group, benzyl group, p-methoxybenzyl group, 3-nitro-2- and lysine sulfenyl 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-
and droxyphthalimide, 1-hydroxybenzotriazole). 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, etc.
Alternatively, it can be carried out in a mixture of these.

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 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 disease groups for the peptide derivatives of the present invention include:
Examples include senile dementia (Alzheimer's dementia), cerebrovascular dementia, and dementias caused by Alzheimer's disease, Vick's disease, Huntington's disease, Creutzfeldt-Jakob disease, Parkinson's disease, myelin degeneration 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 in terms of the amount of the free form, whether it is the free form or its salt. In particular, parenteral and nasal administration
It is preferably 0.1 ng to 100 μg/day, and in oral administration or rectal administration, it is preferable to administer 10 to 100 times as much as parenteral administration.

The peptide derivative of the present invention is mainly administered parenterally (e.g., 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,
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 mannitol or sorbitol, and then dissolved at the time of use.

Examples are shown below.

In each example, the developing solvent for thin layer chromatography was n-butanol-acetic acid-water (2:1:1),
TLC plate silica gel 60F254 manufactured by Merck was used.

In addition, for loading by high performance liquid chromatography, column: μBondapak C, 81, 9X15
cm mobile phase: A) 0.05! k TFA, B) Performed using acetonitrile.

The solid-phase production of peptides was performed using 2,4-dimethoxyhenzhydrylamine resin [The Journal of Organic Chemistry (J, Org.

Chem, ), 52.1197 (I987)] was used as Ig (0.24 mmol NH2/g), and the peptide chain was sequentially extended by the operations shown below.

Condensation step; he-3 ne 1222)-da 1
, Day
X times 1, DMF
lX32, Fmoc-amino acids + lOBt+DI to 120 (3 equivalents each) -
DMF 3, DMF
1x34, isoproper tool
1×35, CH2Cl12
1x3N (X-deprotection step left 1) 1 ■, day 'X times 6, DMF

1x37.20% piperidine=DMF 18
．． 20% Piperidine-DMF 209, DMF
lX310, CH2Cl
□ The 1×3 condensation reaction was confirmed by Kaiser test, and the above steps 1 to 5 were repeated as necessary.

[Example 1] H-Cys-OH pGlu-Asn-Cys-5ar-^rg-Gly-
N)12 ・Fmoc-Gly-011214m to 1 g of acetate 2.4-dimethoxyhenzhydrylamine resin
g,ll0BL! 10mg and DIG O,12
m 11 and Fmoc-Gl by condensation steps 1 to 5.
A yl-resin was prepared.

Next, the protecting groups were removed by N''-deprotection steps 6 to 10, resulting in u-G+y=resin.

Similarly, the condensation and N''-deprotection steps were repeated to obtain 1 to 85
n-Cys (Trt)-5ar-Arg (Mtr)
-G Iy = After making resin, condensation steps 1 to 5 using pGlu-Off are performed, and pGlu-Asn-Cys
(Trt)-5ar-Arg (Mtr)-Gly
- Prepared the resin. After drying the resin, it was stirred in TFA-anisole-thiophenol (I0-1-1mu) for 4 hours, and the resin was filtered off and washed with TFA.

After leaving the TF 8-warm solution at room temperature for 2 hours, TFA was distilled off, ether-water was added to the residue, and the aqueous layer was separated.
Lyophilized.

The obtained lyophilized peptide was added to 0.05% T'F810m
40mg of cystine monooxide dissolved in water and cooled with water.
was added and further stirred for 30 minutes.

Then, 12mff1/min (flow rate), 0 to 10% (B
) After high performance liquid chromatography purification with (A) (mobile phase) of a 20 minute linear gradient, DowexlX2
(acetate form) and freeze-drying to give the title compound.

Yield: 98mg R,:O,t.

[a]D: -137,8° (c-0,5, water) FA
B mass spectrum (M+1) near 49 [Example 2] tl -Cys -OH pGlu-Asn-Gys-Pro-Har-Gly-
From 1 g of NH□ acetate 2,4-dimethoxybenzhydrylamine resin, pGI was prepared in the same manner as in Example 1.
u-Asn-Cys (Trt) -Pro-Har
(Mtr)-Gly-resin was prepared, treated with TFA, and then reacted with cystine monoxide.

Further, high performance liquid chromatography (Special) and ion exchange treatment were performed in the same manner, followed by lyophilization to obtain the title compound.

Yield: 108 mg R, +0.10 [α]. Knee 161.6° (c-0,5, water) FAB
Mass spectrum (M+1) Near 89 [Example 3] From 1 g of 2.4-dimethoxybenzhydrylamine resin, pGIu-Asn-C was prepared in the same manner as in Example 1.
ys(Trt)-Pro-Cit-Gly-resin was prepared, treated with TFA, and then reacted with cystine monoxide.

Furthermore, high performance liquid chromatography, ion exchange treatment, and lyophilization were performed to obtain the title compound.

Yield: 92 mg Rf: 0.12 [α]. : -165,6" (c=0.5, water)
FAB mass spectrum (M+1) near 76 [Example 4] H-Gys-OH pGIu-Asn-Cys-Arg-Δrg-Gly-
From 1 g of NH2-acetate 2,4-dimethoxybenzhydrylamine resin, pGI was prepared in the same manner as in Example 1.
u-Asn-Gys (Trt)-Arg (Mtr)
-8rg(Mtr)-Gly-resin was prepared and TFA
After treatment, it was reacted with cystine monoxide.

Furthermore, high performance liquid chromatography purification and ion exchange treatment were performed in the same manner, followed by lyophilization to obtain the title compound.

Yield: 99 mg Rf: 0.06 [α]. Knee 108.8° (c-0,5, water) FAB
Mass spectrum (M+1): 834 [Example 5] It -Cys -OH pGIu-^sn-Cys-Pip-Arg-Gly-
From 1 g of Nl2 acetate 2,4-dimethoxybenzhydrylamine resin, pGI was prepared in the same manner as in Example 1.
u-Asn-Gys (Trt) -P ip-Arg
(Mtr)-Gly-resin was prepared, treated with TFA, and then reacted with cystine monoxide.

Furthermore, high-performance liquid chromatography loading and ion exchange treatment were carried out in the same manner, followed by freeze-drying to obtain the title compound.

Yield: 55mg R,: 0.09 [α], Knee 134.4° (C・0.5.Water) FA
B mass spectrum (M+1) near 89 [Example 6] HC:ys-OH pGIu-Asn-Cys-Aze-Arg-Gly-
From 1 g of Nl2-acetate 2,4-dimethoxybenzhydrylamine resin, pGI
u-Asn-(:ys (Try)-＾ze-Arg
(I4tr)-Gly-resin was prepared, treated with TFA, and then reacted with cystine monoxide.

Furthermore, high-performance liquid chromatography loading and ion exchange treatment were carried out in the same manner, followed by freeze-drying to obtain the title compound.

Acquisition f1: 98 m g R,: 0. os [α]. Knee 159.0° (c=0.5, water) FA
B Mass Spectrum (M+1) Near 61 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 Purbach et al. [Science, February 2, 1310-1312]
This study was conducted using a trial passive avoidance experiment based on the method of [I983]. 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,
When a rat enters a dark room, it experiences a single electric shock. 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 improving experimental retrograde amnesia by cycloheximide (cyclohexistone 1de) 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 after that, cycloheximide was administered subcutaneously. Cymid 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 the test figure in each group is 6-8. The maximum measurement time was 600 seconds.

For the peptides obtained in each example, the dosage and effects (the ratio of each individual response latency to the response latency of the control group is shown in %) are shown in Table 1.

Table 1 The above test results show that the peptide of the present invention has 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) 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 jL ampoules, sealed and heat sterilized to produce an injection.

[Formulation Example 2] (Freeze-dried preparation) Distilled water for injection 100ml! 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 mIL was dispensed, freeze-drying was performed to produce a freeze-dried injection.

[Formulation Example 3] (Nasal Drop) Physiological saline 100mj! A nasal spray containing 10 mg of the peptide derivative obtained in Example 1, the pH adjusted to 3.0 to 6.0 with a citrate buffer, and 50 μg per 0.5 ml dose. was manufactured.

[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.
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.

[Effect of the invention] The peptide of the present invention is It is a new compound, Excellent It has a nootropic effect, Useful as a medicine It is.

Claims (1)

[Claims] 1. General formula (I): ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) (In the formula, A and B are Har when A is D- or L-Pro. or Cit and A is D-Pr
o, then B is D-Arg, or when B is D- or L-Arg, A is Sar, Pip, Az
(representing a combination of e or Arg) or a derivative in its functional group, or a pharmacologically acceptable salt thereof. 2. General formula (I): ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) (In the formula, A and B are Har or Cit when A is D- or L-Pro, is D-Pr
o, then B is D-Arg, or when B is D- or L-Arg, A is Sar, Pip, Az
an effective amount of a peptide represented by (representing a combination of e or Arg) 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.