JPS5978151A - Novel peptide and antischizophrenic agent containing the same - Google Patents

Abstract

NEW MATERIAL:A peptide of formula I [R is HOOC(CH2)3CO- or pGlu], and a salt thereof. USE:An antischizophrenic agent having improved central nervous system depressing action and further more powerful antidopamine action than the compound of formula II. PROCESS:A peptide of formula III is preferably sulfated with a complex of pyridine with sulfuric acid anhydride, and the resultant sulfuric acid ester is then desalted and purified to afford the aimed compound of formula I . The sulfating reaction in the compound of formula I is carried out by directly sulfating the peptide of formula III, and purifying the resultant sulfuric acid ester rapidly after the reaction to give the compound of formula I . The above-mentioned method is advantageous due to the following facts; Minimized decomposition of the sulfuric acid ester bonds and little by-products.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel peptide and an antischizophrenic drug containing the same.

In this specification, when amino acids, peptides, protective groups, active groups, etc. are expressed by abbreviations, IUPACXIU
The regulations in Section B or the symbols commonly used in the field shall be followed, examples of which are listed below. However, Lsp... Aspartic acid Tyr... Tyrosine Met... Methionine Gly... Glycine Trp... Tryptophan Phe... Phenylalanine pGlu... Pyroglutamic acid The peptide of the present invention is novel and has the following: General formula % Formula % [In the formula, R is HOOC(CH2) a CO- or pGl
Indicates u. ] The peptide of the present invention has an excellent central nervous system depressing effect. That is, the peptide of the present invention exhibits an inhibitory effect on the locomotor hyperactivity in mice induced by administration of thyrotropin-releasing hormone and methamphetamine, significantly prolongs the anesthesia time of mice induced by administration of bendoparbital and halothane, This drug inhibits apomorphine-induced hounding behavior and methamphetamine-induced lightning syndrome, and increases dopamine metabolism in certain brain regions of rats. Furthermore, in a test for the excitatory response caused by the administration of pargyline and L-dopa, which are monoamine oxidase inhibitors, in a so-called L-dopa enhancement test, the peptide of the present invention suppresses the dopa-enhanced response in mice. Therefore, it is clear that the peptide of the present invention has an antidopaminergic effect. As a peptide similar to the peptide of the present invention, for example, a peptide represented by the following formula (2) (hereinafter referred to as "CCK-8") is known.

Asp-Tyr(SO3H)-Met-Gly-Tr
p -Met-Asp-Phe-NH2f2) However, (a) the peptide of the present invention suppresses apomorphine-induced hounding behavior in mice, but CCK-8 does not suppress it, and (b) CCK-8 does not suppress this behavior. Unlike the peptide of the invention, it does not increase dopamine metabolism in the rat brain (c)
In experiments on yawning behavior, which is thought to be caused by a decrease in dopamine release, CCK-8 did not induce yawning behavior at all, whereas the peptide of the present invention induced yawning behavior. The peptide of the present invention suppresses the yawning behavior caused by administration of apomorphine, which is a receptor stimulant, but does not suppress CCK-8.The peptide of the present invention has a stronger anti-dopamine effect than CCK-8. It is. The peptide of the present invention can be used to treat various symptoms of chronic patients, especially autistic symptoms, for which conventional anti-schizophrenic drugs are not effective.
It is expected to be more effective than K-8. In addition, in terms of side effects, CCK-8 exhibits a catalepsy-inducing effect in animal experiments, whereas the peptide of the present invention does not exhibit an 8-catalepsy-inducing effect. It is thought that extrapyramidal side effects, which have become a problem, will not occur. Therefore, the peptide of the present invention is extremely effective as a therapeutic agent for schizophrenia.

[The peptide (
The Peptides) Volume 1 (1966) [5chr2ider and Luhke, A
Academic Press.

N6w York, U, S, A, ] or “peptide synthesis”, Azumaya et al., Maruzen Co., Ltd. (1975
For example, the azide method, chloride method, acid anhydride method, mixed acid anhydride method, DCC method, active ester Ffi (P-nitrophenyl ester method, N-hydroxysuccinimide ester method) , cyanomethyl ester method, etc.), method using Woodward reagent K, carbodiimidazole method, redox method, DDC/additive (HONB, HOBtXHO5u')4-Usually, the peptide of the general formula +1) can be It is produced according to the peptide synthesis method, for example, by a so-called stepwise method in which amino acids are sequentially condensed one by one to the terminal amino acid, or by a method in which several fragments are coupled together. More specifically, the above-mentioned peptide consists of a raw material having a reactive carboxyl group corresponding to one of two types of fragments that are divided into two at an arbitrary position of the bond, and a raw material having a reactive amino group corresponding to the other fragment. is condensed using conventional methods of peptide synthesis,
When the resulting condensate has a protecting group, it can be produced by removing the protecting group by conventional means. In addition, general formula +
When using aspartic acid in the reaction step to produce peptide 11, it is often desirable to protect it, and in the final step, it is usually removed from the protected peptide in which at least one of the constituent amino acid residues of the peptide is protected. Remove all protecting groups.

In addition, in the synthesis reaction step of the peptide of the above general formula f1),
Functional groups that should not participate in the reaction are protected by conventional protecting groups, and the protecting groups are removed after the reaction is completed. Furthermore, the functional groups involved in the reaction are usually activated. These reaction methods are known, and the reagents used therein can be appropriately selected from known methods.

Examples of protecting groups for amino groups include carbobenzoxy,
tert-butyloxycarbonyl (hereinafter abbreviated as "Bocj"), tert-amyloxycarbonyl, inbornyloxycarbonyl, p-methoxybenzyloxycarbonyl, 2-chloro-benzyloxycarbonyl, adamantyloxycarbonyl, trifluoroacetyl,
Examples include phthalyl, formyl, 0-nitrophenylsulfenyl, diphenylphosphinothioyl, and the like. Examples of protective groups for carboxyl groups include alkyl esters (e.g. methyl, ethyl, propyl, butyl, tert-
ester groups such as butyl), benzyl ester, p-nitrobenzyl ester, p-methoxybenzyl ester, p-chlorobenzyl ester, benzhydryl ester, carbobenzoxy hydrazide, tert-butyloxycarbonyl hydrazide, trityl hydrazide, etc. .

Examples of activated carboxyl groups include the corresponding acid chlorides, acid anhydrides or mixed acid anhydrides, azides, active esters (methyl alcohol, ethyl alcohol, benzyl alcohol, pentachlorophenol, p
- ditrophenol, N-hydroxysuccinimide,
N-hydroxybenztriazole, N-hydroxy-
Examples include esters with 5-norbornene-2,3-dicarboximide, etc. In some cases, the peptide bond forming reaction can be carried out in the presence of a condensing agent such as a carbodiimide reagent such as dicyclohexylcarbodiimide or carbodiimidazole, or tetraethylpyrophosphite.

A preferred production example of the peptide of the above general formula ft) is as follows.

The compound of the general formula f1+ is the general formula (3) R-Asp -Tyr -Met-Gly -Trp
-Met-Asp -Phe -NFT2 f31 [
In the formula, R is the same as above.] It is produced by reacting the peptide represented by the following with a pyridine-sulfuric anhydride complex, converting it into a sulfuric acid ester, and further desalting and purifying it.

As shown in the general formula (1), as in the present invention, an acyl group (HOOC(CH2') acO-') or pGl
For compounds in which u is located at the N-terminus and the α-amino group is masked, the sulfuric acid esterification reaction is preferably carried out in the final step. That is, a method in which the peptide of general formula (3) is directly subjected to a sulfuric acid esterification reaction and then quickly purified after the reaction to obtain the peptide of the general formula fl) is a method in which the peptide of general formula (3) is subjected to a sulfuric acid esterification reaction, and an intermediate substance with a protecting group is subjected to a sulfuric acid esterification reaction. , then deprotected,
Furthermore, compared to known methods of acylation or peptide chain elongation, this method has the advantage of minimizing decomposition of sulfate ester bonds and producing fewer by-products, resulting in products with higher yields and better purity.

The peptide of general formula (3) can be produced according to known peptide synthesis methods. For example, the protected hexapeptide BoC-
Met-Gly-Trp-Met-Asp-P
he-NH2 (M, A.

0ndetti et, al, Journ
a/ of the American C
hanica/5ocity 9-2.195 (
1970)) with an acid such as trifluoroacetic acid, and R
Asp'hr -NHNH2[R is produced by condensing the same 1 as described above by the azide method. Also, octapeptide H-Asp-Tyr-Met-Gly-T
rp-Met-Asp-Phe-MH2-(M,A
, 0ndettI et, at.

Journa/ of the American C
hemica/ 5ociety 92. 195 (1
970)) with an active ester of glutaric anhydride or pyroglutamic acid.

The reaction of sulfuric acid esterification is already known. For example, the peptide of general formula (3) is dissolved in an inert solvent such as dimethylformamide or pyridine, and about 10 times the amount of pyridine sulfuric anhydride complex is added thereto, and the reaction is carried out. This is done by the method of The reaction was initially carried out at low temperature and then at room temperature for 15-20
It is preferable to do it for an hour.

As a method for purifying this sulfate-esterified peptide, DEA
This can be easily carried out by ion exchange chromatography such as E-cellulose, partition chromatography such as Sephadex LH-20 or Sephadex G-25, high performance liquid chromatography, or the like.

Also, the method shown in Patent Application Publication No. 56-4953,
That is, the sulfuric acid esterification reaction solution is concentrated, and a solvent such as methanol, butanol, ethanol, dimethylformamide, or water and an aqueous solution of a water-soluble salt of a divalent metal such as calcium or zinc are added to the residue to form a peptide amide sulfate ester. Also suitable is a method in which the sulfuric acid produced from the unreacted pyridine/sulfuric anhydride complex is precipitated and removed as an insoluble divalent metal sulfate salt, and then purified.

The compound of general formula +11 thus obtained may be optionally treated with pharmaceutically acceptable alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium, and amine salts such as triethylamine and ammonium. can do.

The anti-schizophrenic drug of the present invention containing the peptide represented by the above general formula (1) as an active ingredient is usually in the form of a pharmaceutical composition together with a known pharmaceutical carrier. As carriers, fillers commonly used to prepare drugs according to the usage form,
Examples include diluents or excipients such as fillers, binders, wetting agents, disintegrants, surfactants, and lubricants.

As the dosage unit form of the above-mentioned anti-schizophrenic drug, various forms can be selected depending on the therapeutic purpose, and representative examples thereof include tablets, emulsions, powders, liquids, and suspensions. For example, lactose as a carrier,
White sugar, sodium chloride, glucose solution, urea, starch,
Binding of excipients such as calcium carbonate, kaolin, crystalline cellulose, silicic acid, water, ethanol, propatool, simple syrup, glucose, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, etc. agent, dried starch, sodium alginate, agar powder, laminaria powder, sodium bicarbonate, calcium carbonate, twin,
Disintegrants such as sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose, disintegration inhibitors such as sucrose, stearin, cocoa butter, hydrogenated oil, etc., absorption enhancers such as quaternary ammonium bases, sodium lauryl sulfate, glycerin, starch Moisturizers such as starch, lactose, kaolin, bentonite, colloidal silicic acid adsorbents,
Lubricants such as purified talc, stearate, boric acid powder, macrogol, 12-solid polyethylene glycol, etc. can be used. When forming into an emulsion, carriers such as excipients such as glucose, lactose, starch, cacao butter, hydrogenated vegetable oil, kaolin, and talc, binders such as gum arabic powder, tragacanth powder, gelatin, and ethanol, laminaria,
A disintegrant such as agar can be used. Furthermore, the tablets can be made into conventionally coated tablets, such as sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, film-coated tablets, double tablets, or multilayer tablets, if necessary. When forming into a suppository, for example, polyethylene glycol, cacao butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc. can be used as carriers.

When prepared as injections, solutions, emulsions and suspensions are preferably sterilized and isotonic with blood, and when formed into the form of solutions, emulsions and suspensions,
Diluents commonly used in this field include water, ethyl alcohol, propylene glycol, ethoxylated instearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol,
Sorbitan ester etc. can be used. In this case, the drug may contain a sufficient amount of salt, glucose, or glycerin to prepare an isotonic solution, and may also contain conventional solubilizing agents, buffers, soothing agents, preservatives, etc. Coloring agents, preservatives, fragrances, flavors, sweeteners, etc. and other pharmaceuticals may be included in the drug of the present invention, if necessary.

General formula to be contained in the antischizophrenic drug of the present invention +
The amount of the peptide 1) is not particularly limited and can be appropriately selected within a wide range, but it is usually 1 to 70% by weight based on the total composition.

Furthermore, there are no particular restrictions on the use of the above-mentioned anti-schizophrenic drugs, and they can be administered in a manner appropriate for various forms. For example, in the case of tablets, emulsions, solutions, suspensions, emulsions, granules, and capsules, they are administered orally; in the case of injections, they are administered alone or intravenously in a mixture with normal replacement fluids such as glucose and amino acids. If necessary, it can be administered alone intramuscularly, subcutaneously, subcutaneously, or intraperitoneally, and in the case of suppositories, it can be administered rectally. Moreover, in the case of a nasal administration agent, it is administered into the nasal cavity.

The dosage of the anti-schizophrenic drug of the present invention is determined by the intended use,
A pharmaceutical composition containing approximately 40 ft9 to 2 tzLy/kf/day of the peptide of the general formula fil, which is selected as appropriate depending on the symptoms and the like, may be administered in 3 to 4 divided doses.

Production Example I HOOC(CHg) aα)-Asl) TVr (
SOaH)-Met-Gly-Trp-Met-A
Method for producing sp-Pbe-NI2.

1) H-Asp-Tyr-Met-Gly
-Trp -Met -Asp -('he-N)I2
16.5y was dissolved in dimethyl 16-formamide 20077LA' containing 4.4 ml of triethylamine, and 8.55y of glutaric anhydride was added under water cooling. After stirring at 4°C for 17 hours, the solvent was distilled off under reduced pressure, assimilated with IN citric acid, and washed with water.

Recrystallized from methanol room, Glt-A
sp -T'lr -Met -Gly -Trp -
Met-Asp-Phe-NI217.26y
get. Yield 94.6% ■) 1.18y of the acylated peptide obtained in 1) above was dissolved in 20ml of dimethylformamide and 2ml of pyridine, 1.59y of pyridine sulfuric anhydride complex was added under water cooling, and the mixture was heated at 0°C. Stir for 30 minutes and then for 20 hours at room temperature.

The reaction solution was concentrated under reduced pressure, 0.05M ammonium carbonate aqueous solution (50mJ') was added to the residual solution, and the pH was evaporated with aqueous ammonia.
Adjust H to 8.5. The solution is purified by DEAE-cellulose column (4X12) chromatography. Adsorption and washing with 0.05M ammonium carbonate-ammonium bicarbonate buffer (pH 8.5) 1.51, and 0.05M ammonium carbonate-ammonium bicarbonate buffer (pH 8.5).
2M same buffer (p)H8,5)2 I! Elutes with HOOC(CH2)8Co-As was obtained by collecting active fractions, concentrating the solvent, and lyophilizing l6-
p-Thr(SOBH)-Met-Gly-Trp
801 my of -Met-Asp-Phe-NI (2 (hereinafter referred to as "peptide A") (yield 66.4%) is obtained.

Elemental analysis value (cs4 H68NIOolo sa -
[As 18・4H20] Calculated value: C, 48, 17
%iH, 5,91%iN, 11.44% Measured value: C
, 47,97%; H, 5,68% iN, 11.73% Amino acid analysis value by acid degradation Asp 1.98 (2), Gly 1.05 fl)
, Met 2.08 (2) Tyr O, 98fl)
, Phe O, 94 fl) Infrared absorption spectrum shows a strong peak specific to sulfate ester at 1050 ctrx'.

Production example 2 pGlu-Asp-Tyr (SOgH)-Met
-Gly-Trp -Met -Asp -Phe-
Nt(2 manufacturing method.

1) Boc-Met-Gly-Trp-M
et -Asp-Phe-NI210.27y in the presence of 0.2 ml of ethanedithiol in 30 ml of trifluoroacetic acid.
and process for 30 minutes at room temperature. Anhydrous ether 20
Add 0mj' to solidify, filter and dry.

Dimethylformamide containing 1.63 ml of triethylamine of this hexapeptide (pG
lu -Asp-4yr -NHNH2 (mp 213
-215° Two-element analysis cta H28N507 H2O
Calculated values: C, 49,20%; H, 5,73%
; N, 15.94% Measured value: C, 49.08%;
H, 5,51%iN, 15.94%)7.32y is condensed with isoamyl nitrite by the so-called azide method in which it reacts as an azide. The solvent was distilled off under reduced pressure, and the remaining liquid was
Add citric acid to solidify and wash with water. Recrystallized from dimethylformamide-methanol to give pGlu-Asp-
Tyr-Met-Gly-Trp-Met-
9.66y of Asp-Phe-Nl2 is obtained. Yield 70
．． 9% II) 4.70y of nonapeptide obtained in 1) above was mixed with 100yrtl of dimethylformamide and 10yrtl of pyridine.
Pyridine sulfuric anhydride complex 6.3 dissolved in 1nl under water cooling
Add 7y and stir at 0°C for 30 minutes and then at room temperature for 17 hours. The reaction solution was concentrated under reduced pressure, and 200ml of 0.05M ammonium carbonate was added to the residue! and adjust the pH to 8.5 by adding aqueous ammonia. The solution was added to a DEAE-cellulose column (5C77ZX25) in the same manner as in Production Example 1.
cm) Purify by ion exchange chromatography. By collecting the active fractions, concentrating the solvent, and freeze-drying, pGlu-Asp-'I7r(SOaH)-
Met-Gly-Trp-Met-Asp-Ph
e-N) 12 (hereinafter referred to as "peptide B") 8.30
9y (yield 66.0%) was obtained.

Elemental analysis value C54) Ta'r! 'Jo ots
Calculated value as a・NHB・4H20: C, 48, 28
%;H, 5,85%iN, 12.51%Measured value:
C, 48, 23% iH, 5, 45% i N, 12.3
Amino acid analysis value Asp 1-97 +21. Glu O, 98 (1
) + Gly 1.04 fl) Met 2.15 (
21, Tyr 0.97flL Phe O, 94f
l) Infrared absorption spectrum: 1050 cm-” Pharmacological test +11 Thyroid-stimulating hormone-releasing hormone (TRH)
ddy male mice (18-24.5y) are used.

19- Place the mouse in a clear acrylic cage (26X37X11 cm) placed on top of the Animex meter. Experiments are conducted with the locomotor activity of the mice sufficiently reduced by allowing the mice to acclimate for 2 hours. After acclimatization, peptide A1, peptide B or solvent (physiological saline containing 1 m?/ml of sodium bicarbonate) was subcutaneously administered to the mouse, and after 30 minutes, TRH5 m? A
f/ is administered intraperitoneally. The amount of exercise is measured for 60 minutes after TRH administration.

Furthermore, Student's t test is used for statistical processing. The results are shown in Table 1 below.

1st dose Momentum test compound (-y/ky) Number of times per 60 minutes 0, 1 936 ± 115 Peptide A O, 3613 ± 1141
148± 34 0.03 1009±165 Peptide B 0.1 601± 78
0, 3 626±143 −2 〇− Table 1 shows that peptide A and peptide B suppress the original locomotor activity caused by TRH.

(2) Effect of apomorphine on climbing behavior Using ddy male mice (20-30y), a group of 10
Conduct experiments as animals. 10trLt of rod with diameter 2mm
Mice are placed in individual cylindrical cages with a diameter of 12 ctrL and a height of 14 cm that are vertically spaced at n intervals and left for 2 hours. Peptide A1 peptide BXCCK-8 and the above vehicle were administered subcutaneously, and 30 minutes later, apomorphine 0.7
Administer 5 my/ky subcutaneously. After administration of apomorphine 1
0 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes and 4
Observe for 1 minute each from minute 0, and add up these seven scores. All experiments were conducted in a soundproof room, and scores were assigned as follows.

0: All four limbs are on the floor 1: Forelimbs are on the bar 2: Three or more limbs are on the bar For statistical analysis, Student's t test is used. The results are shown in Table 2 below.

Table 2 From Table 2, Peptide A and Peptide B are from Token (3)
Effect on amine metabolism in the brain Male Wistar rats (180-220y) are used without fasting. Peptide A
and Peptide B were subcutaneously administered at a dose of 1 m y/ky, and 1 hour later, the head was irradiated with microwaves to extract amine metabolites in the brain. First, the tissue was homogenized with 1M formic acid, and after centrifugation at 20.000Xp for 20 minutes,
1fnl of supernatant was shaken with 5ml of ethyl acetate to remove 3,4-dihydroxyphenylacetic acid (DO) in the ethyl acetate layer.
PAC), homovanillic acid (HVA) and 5
-Hydroxyindoleacetic acid (5-HIAA) by HPL
Analyze with C.

The analysis conditions are as follows. That is, Ch
emcosorb, 5-0DS, 20 cm x 4
．． Using 6 mm l, D, mobile phase (0,1 M
KH2PO4 (pH 5,0)-5mMEDTA-2Na
-5% acetonitrile) was flowed at a flow rate of 1 ml/11 min. The oxidation potential of the electrochemical detector was set at 10.8V.

28- Neither Peptide A nor Peptide B affected the content of these metabolites in the frontal cortex, limbic forebrain and stratum, but reduced DOPAC content by approximately 60% in the hypothalamus. significantly increased.

(4) Effect on yawning behavior Wistar male rats (200~aooy) were born with Lh
Serve. One group consists of 10 animals. The rat is placed in a plastic cage (17 x 24 x 12 cm) lined with bedding and allowed to acclimate for 30 minutes. Peptide A1, peptide B, CCK-8, and the above solvent are administered subcutaneously, and 30 minutes later, apomorphine 0.03 my/k f' is administered subcutaneously. The number of yawns produced by apomorphine in 30 minutes after administration is measured.

All tests were conducted in a soundproof room, and the Wilcoxon rank-sum test (Wilcox
on rank-sum test).

The results are shown in Table 3 below.

24-Table 3 From Table 3 above, it can be seen that peptide A and peptide B suppress yawning behavior caused by administration of apomorphine.

(that's all)

Claims (1)

[Claims] ■ General formula % formula % [In the formula, R is HOOC(CH2)a co- or pGlu
shows. ] and its salt. ■ General formula % Formula % [In the formula, R is HOOC(CH2)8 Co- or pGlu
shows. ] An antischizophrenic drug characterized by containing a peptide represented by the following or a salt thereof as an active ingredient.