JPH0564160B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel peptide represented by the following general formula () or a salt thereof. [In the formula, R ¹ represents hydrogen or a lower alkyl group, and R ² represents a lower alkyl group. ] Each of the amino acid residues constituting the above compound () may be in any of L form, D form, or racemic form. The object of the present invention is to provide a novel peptide () or a salt thereof useful for the treatment of various diseases belonging to the psychiatric and neurological fields, such as anesthetic addiction, schizophrenia, depression, ataxia caused by spinocerebellar degeneration, etc. Our goal is to provide the following. In this specification, for example, amino acids, peptides, residues of compounds, protective groups, solvents, etc. may be indicated by the following abbreviations. His: Histidine MeTac: 5(R)-methylthiazolidine-4
(R)-carboxylic acid Czc: Oxazolin-2-one-4(S) carboxylic acid MeOzc: 5(R)-methylthiazolidine-2-
One-4(S)carboxylic acid Boc: t-butoxycarbonyl Tos: Tosyl Z: Benzyloxycarbonyl DMF: N,N-dimethylformamide THF: Tetrahydrofuran HOBt: N-hydroxy-1,2,3-benzotriazole DCC: Dicyclohexylcarbodiimide HONB: N-hydroxy-5 -norbornene-2,3-dicarboximide VSCi: 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide The above abbreviations may also indicate residues that can form a peptide bond in the corresponding compound. be. In the above general formula (), examples of the lower alkyl group represented by R ¹ and R ² include linear or branched lower alkyl groups having 1 to 4 carbon atoms. Examples include methyl, ethyl, n-propyl, isopropyl, and butyl groups that are linear or branched at arbitrary positions, with methyl groups being particularly preferred. The object of the present invention () can be produced by known peptide synthesis methods. The introduction of a protecting group, the means for forming a peptide bond, the means for removing a protecting group, etc. are all known per se, and the product can be produced by either a liquid phase method or a solid phase method. Peptide synthesis means itself that can be applied to the production of the target product () include, for example, The
Peptides, Vol. 1 (1966), Schruder and
Lubke, Academic Press, New York, US
A.; Amino acids, Peptides and Proteins,
Vol.1−5, edited by GTYoung, The
Chemical Society, London; Nobuo Izumiya et al.'s book "Peptide Synthesis"(Maruzen); Fujino et al.'s US Patent No.
3870694, etc., such as the chloride method, acid anhydride method, mixed acid anhydride method, DCC method [including water-soluble carbodiimide (e.g. WSCi) method],
Active ester method, DCC/HONB method, DCC/
HOBt method, azide method, method using Woodward reagent K, carbodiimidazole method, redox method,
Examples include the EEDQ (1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline) method. The object of the present invention () can be produced by reacting raw materials corresponding to the parts divided by either of the two dotted lines shown below using the above-mentioned known means. Next, a specific example of the method for producing () is shown using a reaction formula. (1) (2) (3) [In the formula, P represents a protective group, and (P) represents that a protective group is not necessarily required. indicates a solid-phase method resin, and condensation includes not only condensation using a dehydrating condensation agent such as DCC, but also condensation via intermediates such as azide, chloride, and active ester. ] The amino acids that make up each part of the compound () are:
They can be synthesized by their own known methods or methods analogous thereto. For example, 5-alkylthiazolidine-4-carboxylic acids are listed in Bull.Chem.Soc.
It can be obtained by the method described in Tetra Hedron Vol. 28, 4503 (1972) from threo-3-methyl-cysteine synthesized by the method described in Jpn, 56, 1559-1560 (1983). Prior to the peptide bond-forming reaction for producing compound (), functional groups such as amino groups, carboxyl groups, and imino groups that do not participate in the reaction may be protected by known means and protective groups. The protecting group for the α-amino group that does not participate in the reaction of the raw material (for example, the α-amino group of the histidine moiety) may be a known protecting group, such as Z, BOC group, t-amyloxycarbonyl, isobornyloxycarbonyl, Examples include phthaloyl, trifluoroacetyl, and formyl groups. As the protecting group for the imidazole nucleus of the histidine moiety, any known one may be used.
Examples include tosyl, benzyl, 2,4-dinitrophenyl groups, etc., but protection thereof is not always necessary. Carboxyl groups that do not participate in the reaction of the raw materials may be protected with known protecting groups, such as esters (e.g. esters such as methyl, ethyl, benzyl, p-nitrobenzyl, t-butyl, t-amyl, etc.) or metal It may be protected in the form of a salt (eg, salts of sodium, potassium, etc.). The carboxyl groups involved in the reaction of the raw materials may be activated in a known manner, such as activated esters (e.g., pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, cyanomethyl alcohol, p -Nitrophenol, N-hydroxy-5-norbornene-
2,3-dicarboximide, N-hydroxysuccinimide, N-hydroxyphthalimide, ester with N-hydroxy-1,2,3-benzotriazole) or carboxylic acid anhydride, azide, etc. corresponding to the carboxylic acid as the raw material. It may be activated in the form of Among the above active esters, for example, N-hydroxy-5-norbornene-2,3-dicarboximide, N-hydroxy-1,2,3-benzotriazole, N-
When hydroxysuccinimide or the like condenses the histidine moiety, it may be advantageous because racemization is less likely. This condensation reaction is carried out in a non-reactive solvent (e.g. DMF, chloroform, dioxane, THF, etc.) for approximately 30 to 60 hours.
It can be carried out for about 2 to 24 hours at <0>C. When the peptide produced by this condensation reaction has a protecting group, the protecting group can be removed by known means. Protecting group removal methods include, for example, catalytic reduction using catalysts (e.g., palladium black, palladium on carbon, platinum), hydrolysis with acids (e.g., hydrogen fluoride, hydrogen bromide, hydrogen chloride, trifluoroacetic acid), liquid Examples include reduction by sodium in ammonia. Compound (2) can be separated and collected from the reaction product in its free form or in its acid salt form by conventional methods such as dissolution, extraction, chromatography, crystallization, and reprecipitation. Compound () is a pharmacologically acceptable inorganic acid (e.g., hydrochloric acid) or organic acid (e.g., acetic acid, tartaric acid,
Can form salts with citric acid). The compound () of the present invention or a salt thereof can be used in animals (e.g., mice, rats, cats, dogs, monkeys).
When administered to patients, it antagonizes pentobarbital anesthesia, so it can be used clinically to treat poisoning caused by an overdose of anesthetics and consciousness disorders caused by various brain disorders, as well as sleeping pill addiction, hyperactive children, and schizophrenia. It can also be used to treat depression, Parkinson's disease, and epilepsy. In addition, the compound () or a salt thereof has the effect of significantly reducing the number of falls per unit time in genetically ataxic mice, and is also useful for treating ataxia caused by spinocerebellar degeneration and the like. From the viewpoint of pharmacological action, the compound () or a salt thereof is most preferably in the L form, and then preferably in the racemic form. The administration can be, for example, by injection (eg, intravenously, intramuscularly, subcutaneously), orally, rectally, nasally, and the like. The dose of the compound () or its salt required to exhibit the above action varies depending on the type of the compound () or its salt, the type and health condition of the animal to which it is administered, the administration route, etc., but for example, in the case of injection, approximately
It can be appropriately selected from the range of 0.01 mg/Kg to 1 mg/Kg (one dose), and about 0.1 mg/Kg to 10 mg/Kg (one dose) for oral administration. Compound () or a salt thereof may be administered as is, or may be administered in the same dosage form as known TRH (eg, injection, powder, tablet). In all of the following examples and experimental examples of pharmacological effects, when optical isomers may exist for amino acids, peptides, and other compounds, the L-isomer is shown unless otherwise specified. Example 1 5-(R)-methyloxazolidin-2-one-
4-(S)carbonyl-L-histidyl-5(R)
-Methylthiazolidine-4(R)-carboxamide citrate MeOzc−His−MeTac・NH ₂・Citrate (a) Synthesis of Boc−MeTac・NH ₂ Boc−MeTac 200 mg (0.807 mmol), NH ₄
Cl52mg (1.2 equivalents), HOBt130mg (1.2 equivalents)
It was dissolved in 3 ml of THF/DMF (2/1 volume ratio), and 178 µ of WSCi (1.2 equivalents) was added while stirring at about -10°C, and the mixture was further stirred overnight. The solvent was distilled off, the residue was dissolved in 10 ml of ethyl acetate, and water, 5% sodium bicarbonate solution,
It was washed successively with saturated brine and dried over magnesium sulfate. Ethyl acetate was distilled off, the residue was dissolved in a small amount of ether, and then n-hexane was added to collect the precipitated solid. Yield 130mg. The Rf value of TLC with silica gel plate is 0.66 (solvent:
CHCl ₃ /MeOH/pyridine = 95/5/3 volume ratio)
It was hot. (b) Synthesis of Boc-His(Tos)-MeTac・NH ₂ Add 2 ml of trifluoroacetic acid to 125 mg (0.50 mmol) of BOC-MeTaC・NH ₂ obtained in (a) at −10°C for 10 min.
The mixture was added in minutes and allowed to react for an additional 50 minutes at room temperature. Add to this 6.9N HCl/dioxane 0.082ml
(1.2 equivalents) was added, and excess trifluoroacetic acid was distilled off. A small amount of ether/n-hexane (1:1 volume ratio) was added to the residue and the precipitated solid was washed by decanting and dried over sodium hydroxide in a desiccator. The entire amount of the powder thus obtained and 245 mg (1.2 equivalents) of Boc-His (Tos) were dissolved in 1 ml of DMF, and while cooling to -10°C, 0.110 ml of WSCi was added, and the mixture was left overnight. The next day, about 10 ml of ethyl acetate was poured into the reaction mixture, and the mixture was washed with water, 5% aqueous sodium bicarbonate, water, N-HCl, and water in this order, and dried by adding magnesium sulfate powder. Magnesium sulfate was removed by filtration, ethyl acetate was distilled off, and n-hexane was added to the oily residue to precipitate a solid. Yield 220mg. Rf value of TLC with silica gel plate (solvent:
CHCl ₃ /MeOH/CH ₃ COOH=85/10/5 volume ratio) was 0.60. (c) Synthesis of MeOzc−His−MeTac・NH ₂・Citrate Boc−His(Tos)−MeTac・NH ₂ 0.215 mg (0.40
1 ml of trifluoroacetic acid was added to the solution (mmol) over 10 minutes while cooling to -10°C, and further incubated at room temperature.
The reaction was allowed to proceed for 50 minutes. To this was added 0.12 ml (2 equivalents) of 6.9N HCl/dioxane, and excess acid was distilled off. 50 ml of ether was added, and the precipitated solid was collected and dried over sodium hydroxide in a desiccator. Obtained His(Tos)・MeTac・_NH2・HCl
95 mg (0.20 mmol) of powder, 44 mg (1.5 equivalents) of MeOzc, and 41 mg (1.5 equivalents) of HOBt were mixed in DMF/THF (1/
1 volume ratio), and 0.041 ml (1.1 equivalent) of WSCi was added under stirring while cooling to -10°C. Triethylamine was added to this to adjust the pH to 5, and the mixture was allowed to react overnight. The next day, the reaction solution tested negative for fluorescamine. DMF was distilled off from the reaction solution, the oily residue was dissolved in acetic acid, and 50 mg (1.2 equivalents) of powdered citric acid was added and dissolved. Acetic acid was distilled off, and acetonitrile was added to the residue to collect the precipitated solid. Yield approx.
100mg. Sephadex LH-
20 (φ1.8×40 cmH ₂ O), and the fraction containing the target product was lyophilized to obtain 17 mg of lyophilized powder. This is HPLC (high performance liquid chromatography)
When applied to Nucleozil 5C ₁₈ (φ4 x 150 mm, 3% acetonitrile/0.1% trifluoroacetic acid, 1 ml/min), elution was observed at a retention time of 7.8 minutes. The physical properties of this substance were as shown in Table 1.

[Table] Example 2 Oxazolidin-2-one-4(S)carbonyl-L-histidyl-5-(R)-methylthiazolidine-4(R)-carboxamide citrate Ozc-His-MeTac・NH ₂・Citrate His(Tos)-MeTac obtained in (c) of Example 1
NH ₂ HCl 90 mg (0.19 mmol), Ozc 38 mg (1.5
equivalent), HOBt40mg (1.5 equivalent) in DMF/THF
0.039 ml (1.1 equivalent) of WSCi was dissolved in 2 ml (1/1 volume ratio) and cooled to -10° C. with stirring, the pH was adjusted to 4 to 5 with triethylamine, and the mixture was left overnight. The next day, the fluorescamine test of the reaction solution was negative. DMF was distilled off from the reaction solution, the oily residue was dissolved in acetic acid, and then 50 mg (1.2 equivalents) of powdered citric acid was added. Acetic acid was distilled off, and acetonitrile was added to the residue to collect the precipitated solid. Yield approximately 100mg,
Sephadex LH20 (φ1.8×40
cm, H ₂ O), and 14 mg of lyophilized product was obtained from the fraction containing the target product. The physical properties of the obtained target product were measured in the same manner as in Example 1 and were as shown in Table 2.

[Table] Pharmacological effect experiment Compounds used in this experiment Compound 1 5(R)-methyloxazolidin-2-one-4
(S)-Carbonyl-L-Histidyl-5(R)-Methylthiazolidine-4(R)-Carboxamide Citrate Compound 2 Oxazolin-2-one-4(S)-Carbonyl-L-Histidyl-5(R)-Carboxamide Citrate Compound 2 )-Methylthiazolidine-4(R)-carboxamide citrate TRH・T Pyroglutamyl-histidyl-prolinamide tartrate 1 Anesthetic antagonism (pentobarbital sleep shortening effect) Male JCL/ICR mice (4 Each group used 8 or 16 mice. Pentobarbital sodium 55 mg/Kg was administered intraperitoneally to each mouse, and 10 minutes later, when the righting reflex disappeared, various doses of compounds 1, 2 and TRH-T were administered via the tail vein, and the control group received physiological saline. Water was administered, and the time until the righting reflex appeared again was measured, and this was determined as the sleeping time from the time the righting reflex disappeared. The results were expressed as a percentage reduction in the average sleep time of the control group. The concentration of the test drug was adjusted so that the administration volume was 0.2 ml/10 g body weight. The estimation process is
Student's t-test was conducted.

【table】

[Table] 2. Spontaneous locomotor activity enhancement In the experiments, 5 or 12 male JCL/ICR mice (4 weeks old) were used per group. Locomotor activity was measured using a grid-floor measuring device, and the amount of locomotor activity in the drug-administered group and the physiological saline-administered group was measured simultaneously. After acclimatization for 1 hour before drug administration, compound 1, compound 2 and TRH
-Various doses of T were administered intraperitoneally, immediately after administration.
The total amount of exercise for 120 minutes was determined and compared with a control group administered with physiological saline. The administration volume was 0.2 ml/10 g body weight, and the estimation process was based on Student's t-test. The results are shown in Table 4.

【table】

[Table] 3. Improving effect of Rolling Mouse Nagoya on ataxia Rolling Mouse Nagoya (Rolling Mouse Nagoya)
Nagoya, hereinafter abbreviated as RMN. ) is a genetically ataxic mouse that exhibits strong ataxia in its hind limbs from 10 to 14 days after birth, sways its body when walking, and frequently falls. This mouse is used as a pathological model animal for human spinocerebellar degeneration. In this experiment, 8
~12 week old male RMNs were used. Animal height/width 70cm
After each mouse was placed in an open field divided into seven equal parts and allowed to acclimate to the environment, the drug or physiological saline was injected intraperitoneally as a control. Immediately after administration, the amount of movement (1
The amount of movement [1] was defined as moving from one compartment to another) and the number of falls were measured over 20 minutes. A fall index (number of falls/amount of movement) was calculated as an index of ataxia. The results are shown in Table 5.

【table】

[Table] Intraperitoneal administration of Compounds 1 and 2 at 25 mg/Kg reduced the number of falls, lowered the fall index, and showed a marked ataxia-improving effect. The effects of these two compounds were stronger and somewhat longer lasting than those of conventionally known TRH derivatives.

Claims (1)

[Claims] 1. Peptide of the following formula [In the formula, R ¹ represents hydrogen or a lower alkyl group, and R ² represents a lower alkyl group. ] or its salt.