JP3354173B2 - Polypeptides and uses thereof - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel polypeptide having c-AMP producing activity or an amide, ester or salt thereof, and use thereof.

[0002]

2. Description of the Related Art As a new physiologically active peptide derived from the hypothalamus, testis and the like of the brain, a polypeptide consisting of 38 amino acids, PACAP38-, has been discovered in humans, sheep, rats and the like. The amino acid sequence of human, sheep and rat-derived PACAP38 is identical, and His-Ser-
Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Ly
s-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-
It is represented by Gly-Lys-Arg-Tyr-Lys-Gln-Arg-Val-Lys-Asn-Lys (SEQ ID NO: 1). PACAP38 has an activity of increasing intracellular c-AMP production of pituitary cells and an effect of increasing c-AMP production of astroglial cells to prolong the survival of nerve cells. In addition, a polypeptide consisting of 27 amino acids on the N-terminal side of PACAP38-PACA
P27 has also been found to have PACAP38 activity [[i) Biochemical and Biophysical Research Communications (Biochem. Biophys.
mmun.), 164, 567-574 (1989); (ii) European Patent Publication No. 404,652].
Polypeptide consisting of 26 to 23 amino acids on the terminal side
The same effect as PACAP27 has been confirmed for PACAP26, PACAP25, PACAP24 and PACAP23 [Japanese Patent Application No. 2-87959].

[0003]

The above-mentioned PACAP 38
Methionine, which is the amino acid at position 17 of -23, is susceptible to oxidation in and out of the body,
It is an object of the present invention to provide a polypeptide having an action equal to or greater than ACAP27.

[0004]

Means for Solving the Problems The present inventors have created a novel polypeptide which is less susceptible to oxidation and which has a c-AMP production activity equal to or higher than that of PACAP27. The present invention has been completed. That is, the present invention relates to the His-Ser-Asp-Gly-Ile-Phe-Thr-A
sp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-NH-CHX-CO-Ala-
Val-Lys-Lys-Tyr-Y [I] [wherein, X is substituted with a hydrogen atom, or a hydroxyl group, an optionally substituted amino group, a carboxyl group, a carbamoyl group or an optionally substituted aromatic ring group. A lower alkyl group which may be
Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-Tyr-Lys-Gln-Arg-Va
1 to 16 amino acids or peptides counted from the N-terminal side of 1-Lys-Asn-Lys], or an amide, ester or salt thereof, and
(2) a pharmaceutical composition containing the polypeptide represented by the formula [I] or an amide, ester or pharmacologically acceptable salt thereof. In the peptide in the present specification, the left end is the N-terminus (amino terminus) and the right end is the C-terminus (carboxyl terminus) according to the convention of peptide labeling.

In the formula [I], X is a hydrogen atom or a lower alkyl group which may be substituted with a hydroxyl group, an optionally substituted amino group, a carboxyl group, a carbamoyl group or an optionally substituted aromatic ring group. Is shown.
Here, the lower alkyl group may be a straight-chain or branched C
Preferably ₁ _ ₆ alkyl group, e.g., methyl, ethyl, n- propyl, isopropyl, n- butyl, isobutyl, sec- butyl, tert-butyl, n- pentyl, isoamyl, tertiary amyl, n- hexyl, isohexyl, etc. Is raised. These lower alkyl groups include a hydroxyl group, an optionally substituted amino group,
It may be substituted with one to three substituents selected from the group consisting of a carboxyl group, a carbamoyl group and an optionally substituted aromatic ring group. The lower alkyl substituted with a hydroxyl group the C ₁ _ ₆ alkyl group by hydroxyl (-
OH) is preferable, and examples thereof include hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 1-hydroxypropyl and the like. Substituted examples of the lower alkyl group substituted with an amino group optionally above C ₁ _ ₆ alkyl group is an amino group (-NH ₂₎ or a substituted amino group (e.g., mono C ₁ such as methylamino, ethylamino _ ₃ alkylamino group, di-C ₁ _ ₃ alkylamino group such as dimethylamino, diethylamino,
Guanidino group) are preferable, and examples thereof include 2-aminoethyl, 2- (methylamino) ethyl, 3-aminopropyl, 4-aminobutyl, and 3-guanidinopropyl. Are preferable as the substituted lower alkyl which C ₁ _ ₆ alkyl group described above is substituted with a carboxyl group (-COOH) at the carboxyl group, for example, carboxymethyl, 1-carboxyethyl, 2-carboxyethyl and the like are mentioned Can be As the lower alkyl group substituted by a carbamoyl group,
Is preferably _{one 1} _ ₆ alkyl group substituted with a carbamoyl group (-CONH _2), for example, carbamoylmethyl, 1
-Carbamoylethyl, 2-carbamoylethyl and the like. C ₁ _ ₆ alkyl group described above aromatic Hajime Tamaki as the lower alkyl group substituted with also an aromatic ring group optionally substituted [for example, C ₆ _ _{1 2,} such as phenyl, naphthyl
Aryl group, aromatic heterocyclic group, especially imidazolyl / pyrazolyl indolyl / dihydroindolyl, quinolyl /
Tetrahydroquinolyl / isoquinolyl / tetrahydro-
N, O and S such as isoquinolyl, benzazepinyl, tetrahydrobenzazepinyl, pyrrolyl, piperidyl, piperazinyl, morpholinyl, furanyl and thiophenyl
1-3 heteroatoms and an aromatic heterocyclic group of 5-9 membered containing carbon atoms] or a substituted aromatic Hajime Tamaki [e.g., C ₁ _ ₆ alkyl group selected from (eg, methyl, ethyl), halogen atom (e.g., chlorine, bromine), a hydroxyl group, a carboxyl group, an amino group, a mono- or dihydro C ₁ _ ₆ alkylamino (e.g., methylamino, ethylamino, dimethylamino, diethylamino), C ₁ _ ₆ alkoxycarbonyl group ( For example, formyl, methoxycarbonyl), C ₁ _
₆ alkoxy group (e.g., methoxy, ethoxy), 1-3 is substituted with a substituent the above C ₆ _ ₁₂ aryl group selected from the group consisting of a cyano group or a 5-9-membered aromatic heterocyclic ring, And the like, for example, benzyl, 1-phenylethyl, 2-phenylethyl, 1-naphthylmethyl, 2-naphthylmethyl, 4-hydroxyphenylmethyl, 1H-imidazol-4-ylmethyl, 1H -Indol-3-ylmethyl, N-methylindol-3-ylmethyl, 5-fluoro-1H
-Indol-3-ylmethyl and the like. X in the formula [I] is in the range described above. In other words, the group —NH—CHX—CO— is a glycine residue or α
-Indicates a residue of an amino acid. Such α-amino acids are preferably natural amino acids including glycine, for example, alanine, valine, leucine, isoleucine, serine, threonine, lysine, arginine, aspartic acid, asparagine, glutamic acid, glutamine, phenylalanine, tyrosine, histidine , Tryptophan and the like. Among them, serine and arginine are particularly preferred. These α-amino acids are L-form, D-form,
Although any of the DL-forms may be used, the L-form is particularly preferable.

In the formula [I], Y is Leu-Ala-Ala-Val-Leu
1 to 16 amino acids or peptides counted from the N-terminal side of -Gly-Lys-Arg-Tyr-Lys-Gln-Arg-Val-Lys-Asn-Lys are shown, in other words, Y is Leu, Leu- Ala, Leu-
Ala-Ala, Leu-Ala-Ala-Val, Leu-Ala-Ala-Val-Leu, Leu
-Ala-Ala-Val-Leu-Gly, Leu-Ala-Ala-Val-Leu-Gly-Ly
s, Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg, Leu-Ala-Ala-Va
l-Leu-Gly-Lys-Arg-Tyr, Leu-Ala-Ala-Val-Leu-Gly-Lys-
Arg-Tyr-Lys, Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-Tyr-L
ys-Gln, Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-Tyr-Lys-Gl
n-Arg, Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-Tyr-Lys-Gln
-Arg-Val, Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-Tyr-Lys-
Gln-Arg-Val-Lys, Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-T
yr-Lys-Gln-Arg-Val-Lys-Asn and Leu-Ala-Ala-Val-Le
u-Gly-Lys-Arg-Tyr-Lys-Gln-Arg-Val-Lys-Asn-Lys.

In the formula [I], the C-terminus is usually a carboxyl group (-
COOH) or carboxylate (-COO-), but C
The terminal may be an amide (-CONH2) or an ester (-COOR). Here, R of the ester group is methyl, ethyl, n-propyl, isopropyl or n-
C ₁ _ ₆ alkyl group, C ₃ _ ₈ cycloalkyl or butyl (e.g., cyclopentyl, cyclohexyl) cycloalkyl groups such as, C ₆ _ ₁₂ aryl group (e.g. phenyl, alpha
- naphthyl) aryl group such as, C ₇ _ ₁₄ aralkyl group (e.g. benzyl, phenyl -C ₁ _ such as phenethyl
₂ alkyl, or α- Nafuchirumeru α- naphthyl -C ₁ _ ₂ alkyl) other aralkyl groups such as such, pivaloyloxymethyl esters are frequently used as oral esters. When the polypeptide represented by the formula [I] has a carboxyl group or a carboxylate other than at the C-terminus, those in which these groups are amidated or esterified are also included in the polypeptide of the present invention. As the ester at this time, for example, the above-mentioned C-terminal ester and the like are used. In the present invention, those having an amide at the C-terminus are more preferable. The pharmacologically acceptable salts of the polypeptide [I] of the present invention include metal salts such as sodium salt, potassium salt, calcium salt and magnesium salt, and inorganic acid addition salts such as hydrochloride, sulfate and phosphate. , Acetate, propionate, citrate, tartrate, malate, oxalate and other organic acid salts.

In the present specification, when amino acids and peptides are indicated by abbreviations, IUPAC-IUB Com
It is based on the abbreviation by mission on Biochemical Nomenclature or the abbreviation commonly used in the relevant field, examples of which are described below. Gly: glycine Ala: alanine Val: valine Ile: isoleucine Leu: leucine Pro: proline Arg: arginine Lys: lysine His: histidine Asp: aspartic acid Asn: asparagine Glu: glutamic acid Gln: glutamine Ser: serine Thr: threonine Phe : Tyrosine Met: Methionine Met (O): Methionine-S-oxide Further, protecting groups and reagents commonly used in the present specification are represented by the following abbreviations. Z: benzyloxycarbonyl Boc: tert-butoxycarbonyl Bzl: benzyl Trt: trityl Bum: tert-butoxymethyl Br-Z: 2-bromobenzyloxycarbonyl Cl-Z: 2-chlorobenzyloxycarbonyl Cl2-Bzl: 2,6 -Dichlorobenzyl Tos: paratoluenesulfonyl Mts: 2,4,6-trimethylbenzenesulfonyl Bom: benzyloxymethyl Fmoc: 9-fluorenylmethyloxycarbonyl NO2: nitro DNP: dinitrophenyl PAM: phenylacetamidomethyl DEAE: diethylaminoethyl OBzl: benzyl ester OcHex: cyclohexyl ester DCC: N, N'-dicyclohexylcarbodiimide HOBt: 1-hydroxybenzotriazole HOOBt: 3-hydroxy-4-oxo-3,4-dihydrobenzotriazine HONB: N-hydroxy-5-nor Runen 2,3-dicarboximide DMF: N, N-dimethylformamide BHA: benzhydrylamine.

The polypeptide [I] of the present invention can be produced by a conventional means of peptide synthesis. The means for such peptide synthesis may be in accordance with any known method, for example,
M. Bodansky and MA Ondetti, Peptide Synthesis, Interscience, New York, 1966; FM Finn and K. Hofmann
Written by The Proteins, Volume 2, H. Nenr
ath, RL Hill editor, Academic Press Inc.,
New York, 1976; Nobuo Izumiya et al., "Basics and Experiments in Peptide Synthesis" Maruzen Co., Ltd. 1985; Haruaki Yajima, Shunpei Sakakibara et al., Biochemistry Experiment Course 1, Ed.
1977; Shun Kimura et al., Seminar on Seismic Chemistry 2, Tokyo Biochemical Society, 1987; JM Stewart and
JD Young, Solid Phase Peptide Synthesis (Solid Phase Peptide Synthesis), Pierce Chemical Company, Illinois, 1984 and other methods described, such as azide method, chloride method, acid anhydride method, mixed acid anhydride method, DCC method, Active ester method, method using Woodward reagent K, carbonylimidazole method, redox method, DCC / HONB method, method using BOP reagent and the like. The polypeptide [I] of the present invention is synthesized by a liquid phase synthesis method,
Although any of the solid phase synthesis methods may be used, the solid phase synthesis method is more preferred in the present invention. In the case of the solid phase synthesis method, an insoluble resin known in the art is used. Examples of such insoluble resins include chloromethyl resin,
Hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylphenylacetamidomethyl resin, polyacrylamide resin, 4- (2 '4,4'-Dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4-
(2 ′, 4′-dimethoxyphenyl-Fmoc · aminoethyl) phenoxy resin. Using these resins, the protected amino acids are sequentially condensed according to the conventional method in the order of the amino acid sequence from the C-terminal side of the polypeptide [I], and then subjected to a protecting group removal treatment described later to remove all protecting groups, thereby achieving the object. [I] can be synthesized. The polypeptide of the present invention can also be synthesized by amidating or esterifying the carboxyl group or carboxylate of the obtained polypeptide as necessary. Furthermore, the polypeptide of the present invention can also be synthesized by synthesizing one or more peptide fragments of the polypeptide of the present invention using the above method, and then condensing them.

For the condensation of protected amino acids, various activating reagents that can be used for peptide synthesis can be used, and carbodiimides are particularly preferable. Examples of carbodiimides include DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminopropyl) carbodiimide, and the like. For these activations, the protected amino acid is directly added to the resin together with a racemization inhibitor additive (eg, HOBt, HOOBt), or the protected amino acid is preliminarily activated as a symmetric anhydride or HOBt ester or HOOBt ester. Later it can be added to the resin. The solvent used for the activation of the protected amino acid and the condensation with the resin can be appropriately selected from solvents known to be usable for the peptide condensation reaction. For example, DMF, dimethyl sulfoxide,
Pyridine, chloroform, dioxane, methylene chloride,
Tetrahydrofuran, acetonitrile, ethyl acetate, N
-Methylpyrrolidone or an appropriate mixture thereof. The reaction temperature is appropriately selected from a range known to be usable for the peptide bond formation reaction, and is usually appropriately selected from a range of about -20 ° C to 30 ° C.
The activated amino acid derivative is usually used in a 1.5-4 fold excess. As a result of the test using the ninhydrin reaction, when the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When sufficient condensation cannot be obtained even by repeating the reaction, unreacted amino acids can be acetylated using acetic anhydride or acetylimidazole. As the protecting group for the amino group of the starting material, for example, Z, Boc,
Tertiary amyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, trifluoroacetyl, phthalyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinochi oil,
Fmoc and the like. Examples of the carboxyl-protecting group include alkyl esters (e.g., methyl,
Ester groups such as ethyl, propyl, butyl, tertiary butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl), benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, Benzhydryl ester, phenacyl ester, benzyloxycarbonyl hydrazide, tert-butoxycarbonyl hydrazide, trityl hydrazide and the like can be mentioned. The hydroxyl group of serine can be protected, for example, by esterification or etherification. Examples of groups suitable for the esterification include lower alkanoyl groups such as an acetyl group, aroyl groups such as a benzoyl group, and groups derived from carbonic acid such as a benzyloxycarbonyl group and an ethoxycarbonyl group. Examples of groups suitable for etherification include a benzyl group, a tetrahydropyranyl group, and a tertiary butyl group. Examples of the protecting group for the phenolic hydroxyl group of tyrosine include Bzl, Cl ₂ -Bzl, 2-nitrobenzyl, BrZ, tertiary butyl and the like. Examples of the imidazole protecting group for histidine include Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum, Boc, Trt, and Fmoc. Examples of the activated carboxyl group of the raw material include the corresponding acid anhydride, azide, active ester [alcohol
(For example, esters with pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, HOBt) ]. The activated amino group of the raw material includes, for example, a corresponding phosphoric amide.

As a method for removing (eliminating) the protecting group, for example, catalytic reduction in a stream of hydrogen in the presence of a catalyst such as Pd black or Pd-carbon, hydrogen fluoride anhydride, methanesulfonic acid, Acid treatment with trifluoromethanesulfonic acid, trifluoroacetic acid or a mixture thereof, and reduction with sodium in liquid ammonia can also be mentioned. The elimination reaction by the acid treatment is generally-
The treatment is performed at a temperature of 20 ° C to 40 ° C. In the acid treatment, anisole, phenol, thioanisole, metacresol, paracresol, dimethylsulfide, 1,4-
It is effective to add a cation scavenger such as butanedithiol or 1,2-ethanedithiol. In addition, the 2,4-dinitrophenyl group used as the imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan is 1,2-ethanedithiol, 1,4-butane described above. In addition to deprotection by acid treatment in the presence of dithiol or the like, it is also removed by alkali treatment with dilute sodium hydroxide, dilute ammonia or the like. Protection and protection of functional groups that should not be involved in the reaction of the raw materials, elimination of the protected groups, activation of the functional groups involved in the reaction, and the like can also be appropriately selected from known groups or known means. After completion of the reaction, the polypeptide [I] produced in this manner is collected by usual means for separating and purifying peptides, for example, extraction, distribution, reprecipitation, recrystallization, column chromatography, high performance liquid chromatography, and the like. . Since the polypeptide [I] of the present invention does not contain Met in the molecule, it is stable against oxidation at the stage of its synthesis as well as the product itself.

The polypeptide [I] of the present invention can be prepared by a method known per se such as a metal salt (eg, sodium salt, potassium salt, calcium salt, magnesium salt, etc.), a salt with a base or a basic compound (eg, ammonium salt, arginine salt). ), Especially as pharmacologically acceptable acid addition salts, eg, inorganic acids
(Eg, hydrochloric acid, sulfuric acid, phosphoric acid) or salts of organic acids (eg, acetic acid, propionic acid, citric acid, tartaric acid, malic acid, oxalic acid, methanesulfonic acid).
The amidation or esterification of the carboxyl group or carboxylate of the polypeptide represented by the formula [I]
It can be carried out according to a method known per se or a method analogous thereto.

The c-AMP production activity was measured using a subcultured cell derived from rat adrenal medulla PC12h, and the amount of c-AMP secreted extracellularly was measured using a c-AMP measurement kit. As a result, as shown in FIG. 1, the polypeptide [I] of the present invention exhibited activity equivalent to that of PACAP27.
Further, in order to prove that the polypeptide of the present invention is less susceptible to oxidation, the Ar obtained in Example 1 described below was used.
The oxidant content (%) was measured when g ¹⁷ -PACAP27 was freeze-dried and stored in an aqueous acetic acid solution. The oxidant content (%) was measured using high performance liquid chromatography (HPLC). Table 1 shows the results. HPLC conditions are as follows. Column: YMC ODS AM-301 (4.6 × 10
0mm) Eluent: solution A (0.1% -trifluoroacetic acid aqueous solution) Using solution B (acetonitrile containing 0.1% -trifluoroacetic acid), linear concentration gradient elution from solution A to solution B (50 minutes) Flow rate: 1.0 ml / Min Arg ^17- PACAP27 elution time: 19.9 minutes Table 1 At the time of synthesis of sample oxidant content (%) After lyophilization storage After storage in acetic acid aqueous solution From Table 1 of Arg ^17- PACAP20000 of Example 1, the poly (polyethylene glycol) of the present invention was obtained. It can be seen that the peptide is less susceptible to oxidation.

[0014]

[Action and Effect] The novel polypeptide [I] (including its amide, ester or salt) of the present invention enhances c-AMP production. Therefore, the novel polypeptide [I] of the present invention can be used, for example, as a nerve activator capable of extending the survival time of nerve cells.
Specifically, it can be used as a therapeutic agent for various neuropathies or a repair agent for damaged nerves in mammals (eg, sheep, rats, humans, etc.). Furthermore, the novel polypeptide [I] of the present invention also has an effect of suppressing gastric secretion. When the polypeptide [I] of the present invention is used as a therapeutic agent for neuropathy or a repair agent for injured nerve as described above, it may be used as it is or mixed with a pharmacologically acceptable carrier, excipient, or diluent to obtain a powder, granule or tablet , Capsules, injections, suppositories, ointments, sustained-release preparations and the like can be safely administered orally or parenterally. The polypeptide [I] of the present invention is mainly administered parenterally (for example, intravenous or subcutaneous injection, intracerebroventricular or intraspinal injection, nasal administration, rectal administration). There is also. Polypeptide [I] of the present invention
Is stable as a substance and can be stored as a physiological saline solution. Alternatively, mannitol and sorbitol can be added to make a lyophilized ampule, which can be dissolved at the time of use.
The polypeptide [I] of the present invention can be administered as a free form or as a base salt or an acid addition salt thereof. The dosage of the free form, the base salt and the acid addition salt of the peptide is generally in the range of 0.1 nanomol to 1 micromol per 1 kg of body weight as the free form. More specifically, the dose varies depending on the target disease, symptom, administration subject, administration method, and the like. For example, when administered to an adult patient by injection, usually the active ingredient (peptide [I]) 1
0.1 nanomol / kg to 1 micromol / kg body weight as a dose, more preferably 1 nanomol / kg to 0.1 micromol / k
It is convenient to administer about g body weight once to three times a day. Infusion is also effective, and the total dose in the case of infusion is the same as in the case of injection. When using this polypeptide as a therapeutic agent, care must be taken to ensure careful purification and the elimination of bacteria and pyrogens. No toxicity is found in the purified polypeptide of the present invention.

[0015]

The present invention will be described more specifically with reference to examples, reference examples and test examples. The amino acids in Examples and Reference Examples are all L-forms.

[0016]

Example 1 Arg ¹⁷ -PACAP27-NH ₂ (H-His-Ser-Asp-Gly
-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-A
rg-Ala-Val-Lys- Lys-Tyr-Leu-Ala-Ala-Val-Leu-NH 2)
Synthesis of [Ia] (SEQ ID NO: 2) Using a commercially available paramethyl BHA resin (Applied Biosystems, Inc.) 0.60 g (0.5 mmole), using a peptide synthesizer (Applied Biosystems, Inc. model 430A) did. HOBt / DCC for the first amino acid, Boc-Leu
After condensing to the resin, the Boc group on the resin is treated with 50% trifluoroacetic acid / methylene chloride to release the amino group, and the amino group is replaced with Boc-Val, Boc-Ala, Boc-Leu ,
Boc-Tyr (Br-Z), Boc-Lys (Cl-Z), Boc-Arg (Tos), Boc-Gl
n, Boc-Ser (Bzl), Boc-Gly, Boc-Asp (OcHex), Boc-Thr
(Bzl), Boc-Phe, Boc-Ile and Boc-His (Bom) were activated and condensed with HOBt / DCC in the order of the indicated amino acid sequence. After condensing again with the same amino acid derivative activated with DCC or HOBt / DCC, unreacted amino groups were acetylated with acetic anhydride to obtain 1.34 g of a protected peptide resin. 0.89 g of this protected peptide resin was treated with 10 ml of anhydrous hydrogen fluoride at 0 ° C. for 60 minutes in the presence of 1.52 g of paracresol, and then the hydrogen fluoride was distilled off under reduced pressure, and the residue was washed twice with 5 ml of ethyl ether. Thereafter, the residue was extracted with 5 ml of 50% aqueous acetic acid. The insoluble material was removed by filtration and washed with 5 ml of 50% aqueous acetic acid. The filtrate and washing solution were combined, concentrated under reduced pressure to 2-3 ml, and Sephadex G-25 (2x75
cm) and eluted with 50% acetic acid. After collecting and evaporating the main fraction under reduced pressure, the residue was dissolved in 100 ml of 0.1% aqueous trifluoroacetic acid, and the mixture was dissolved in a LiChroprep RP-18 resin column (2.6
x9.2 cm) and eluted with a linear gradient between 0.1% aqueous trifluoroacetic acid and 50% acetonitrile (containing 0.1% trifluoroacetic acid). Combine the main fractions and again LiChropr
The mixture was applied to an ep RP-18 column (2.6 x 9.2 cm), and a 20 to 40% aqueous solution of acetonitrile (containing 0.1% trifluoroacetic acid) was subjected to linear gradient elution. The main fraction was collected and freeze-dried. This was dissolved in 20 ml of 0.05 M aqueous ammonia acetate, and C
Attached to M-Cellulofine resin column (2.5 × 10 cm)
Elution was performed with a linear concentration gradient of 0.05 M to 0.5 M aqueous ammonia acetate. The main fractions were combined and lyophilized to give a white powder 9.
6 mg were obtained. Amino acid analysis value Asp 1.98 (2), Thr 0.90 (1), Ser 2.26 (3), Glu 1.10
(1), Gly 1.10 (1), Ala 2.95 (3), Val 1.93 (2), Ile 0.9
7 (1), Leu 2.00 (2), Tyr 2.87 (3), Phe 0.98 (1), Lys 2.
87 (3), His 1.02 (1), Arg 2.89 (3) Mass spectrometry (M + H) + 3171.8710 HPLC elution time 20.3 min Column conditions Column: Wakosil 5C18 (4.6 × 100 cm) Eluent: Solution A (0.1% -triol) (Aqueous fluoroacetic acid) Linear concentration gradient elution from solution A to solution B using solution B (acetonitrile containing 0.1% -trifluoroacetic acid) (50 minutes) Flow rate: 1.0 ml / min.

[0017]

Example 2 Ser ¹⁷ -PACAP27-NH ₂ (H-His-Ser-Asp-Gly
-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-S
er-Ala-Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-NH ₂ )
Synthesis of [Ib] (SEQ ID NO: 3) Using a commercially available paramethyl BHA resin (manufactured by Applied Biosystems) 0.74 g (0.5 mm mole), and using a peptide synthesizer (Applied Biosystems model 430A) did. HOBt / DCC for the first amino acid, Boc-Leu
After condensing to the resin, the Boc group on the resin is treated with 50% trifluoroacetic acid / methylene chloride to release the amino group, and the amino group is replaced with Boc-Val, Boc-Ala, Boc-Leu ,
Boc-Tyr (Br-Z), Boc-Lys (Cl-Z), Boc-Ser (Bzl), Boc-G
ln, Boc-Arg (Tos), Boc-Gly, Boc-Asp (OcHex), Boc-T
hr (Bzl), Boc-Phe, Boc-Ile and Boc-His (Bom) were activated and condensed with HOBt / DCC in the order of the indicated amino acid sequence. After condensing again with the same amino acid derivative activated with DCC or HOBt / DCC, unreacted amino groups were acetylated with acetic anhydride to obtain 2.82 g of a protected peptide resin. This protected peptide resin (1.01 g) was treated with anhydrous hydrogen fluoride (10 ml) at 0 ° C. for 60 minutes in the presence of paracresol (1.62 g), hydrogen fluoride was distilled off under reduced pressure, and the residue was washed twice with ethyl ether (5 ml). Thereafter, the residue was extracted with 5 ml of 50% aqueous acetic acid. The insoluble material was removed by filtration and washed with 5 ml of 50% aqueous acetic acid. The filtrate and washing solution were combined, concentrated under reduced pressure to 2-3 ml, and Sephadex G-25 (2x7
5 cm) and eluted with 50% acetic acid. After collecting the main fractions and distilling them under reduced pressure, the residue is dissolved in 100 ml of 0.1% aqueous trifluoroacetic acid, and the residue is dissolved in a LiChroprep RP-18 resin column.
(2.6 x 9.2 cm) and eluted with a linear gradient between 0.1% aqueous trifluoroacetic acid and 50% acetonitrile (containing 0.1% trifluoroacetic acid). Combine the main fractions and again LiCh
Attached to roprep RP-18 column (2.6x9.2 cm), aqueous solution of acetonitrile up to 20-40% (containing 0.1% trifluoroacetic acid)
, And the main fractions were collected and freeze-dried. This was dissolved in 20 ml of 0.05 M aqueous ammonia acetate, applied to a CM-Cellulofine resin column (2.5 × 10 cm), and eluted with a linear concentration gradient of 0.05 M to 0.5 M aqueous ammonia acetate. The main fractions were combined and lyophilized to give 17.7 mg of a white powder. Amino acid analysis value Asp 2.03 (2), Thr 0.98 (1), Ser 3.53 (4), Glu 1.13
(1), Gly 1.03 (1), Ala 3.13 (3), Val 1.93 (2), Ile 0.9
0 (1), Leu 2.00 (2), Tyr 3.01 (3), Phe 0.92 (1), Lys 2.
96 (3), His 1.10 (1), Arg 2.01 (2) Mass spectrometry (M + H) + 3102.7930 HPLC elution time 21.0 min Column conditions Column: Wakosil 5C18 (4.6 × 100 cm) Eluent: A solution (0.1% -triol) (Aqueous fluoroacetic acid) Linear concentration gradient elution from solution A to solution B using solution B (acetonitrile containing 0.1% -trifluoroacetic acid) (50 minutes) Flow rate: 1.0 ml / min.

[0018]

Example 3 Phe ¹⁷ -PACAP27-NH ₂ (H-His-Ser-Asp-Gly
-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-P
he-Ala-Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-NH ₂ )
Synthesis of [Ic] (SEQ ID NO: 4) Using a commercially available paramethyl BHA resin (manufactured by Applied Biosystems, Inc.) 0.72 g (0.5 mmole), using a peptide synthesizer (Applied Biosystems, model 430A) did. HOBt / DCC for the first amino acid, Boc-Leu
After condensing to the resin, the Boc group on the resin is treated with 50% trifluoroacetic acid / methylene chloride to release the amino group, and the amino group is replaced with Boc-Val, Boc-Ala, Boc-Leu ,
Boc-Tyr (Br-Z), Boc-Lys (Cl-Z), Boc-Phe, Boc-Gln, Bo
c-Arg (Tos), Boc-Ser (Bzl), Boc-Gly, Boc-Asp (OcHe
x), Boc-Thr (Bzl), Boc-Ile, and Boc-His (Bom) were activated and condensed with HOBt / DCC in the order of the indicated amino acid sequence. After condensing again with the same amino acid derivative activated with DCC or HOBt / DCC, unreacted amino groups were acetylated with acetic anhydride to obtain 2.96 g of a protected peptide resin. 0.98 g of this protected peptide resin was treated with 10 ml of anhydrous hydrogen fluoride at 0 ° C. for 60 minutes in the presence of 1.62 g of paracresol, hydrogen fluoride was distilled off under reduced pressure, and the residue was washed twice with 5 ml of ethyl ether. Thereafter, the residue was extracted with 5 ml of 50% aqueous acetic acid. The insoluble material was removed by filtration and washed with 5 ml of 50% aqueous acetic acid. The filtrate and washing solution were combined, concentrated under reduced pressure to 2-3 ml, and Sephadex G-25 (2x7
5 cm) and eluted with 50% acetic acid. After collecting the main fractions and distilling them under reduced pressure, the residue is dissolved in 100 ml of 0.1% aqueous trifluoroacetic acid, and the residue is dissolved in a LiChroprep RP-18 resin column
(2.6 x 9.2 cm) and eluted with a linear gradient between 0.1% aqueous trifluoroacetic acid and 50% acetonitrile (containing 0.1% trifluoroacetic acid). The main fractions were combined and lyophilized. Dissolve this in 20 ml of 0.05 M aqueous ammonia acetate,
The mixture was applied to a resin column (2.5 × 10 cm) of CM-Cellulofine, and eluted with a linear concentration gradient of 0.05 M to 0.5 M aqueous ammonium acetate. The main fractions were combined and lyophilized to give 24 mg of a white powder. Amino acid analysis value Asp 1.98 (2), Thr 0.90 (1), Ser 2.26 (3), Glu 1.10
(1), Gly 1.02 (1), Ala 3.10 (3), Val 1.88 (2), Ile 0.8
8 (1), Leu 2.00 (2), Tyr 3.09 (3), Phe 1.90 (2), Lys 2.
90 (3), His 1.00 (1), Arg 1.99 (2) Mass spectrometry (M + H) + 3162.6340 HPLC elution time 21.5 minutes Column conditions Column: Wakosil 5C18 (4.6 x 100 cm) Eluent: Solution A (0.1% -triol) (Aqueous fluoroacetic acid) Linear concentration gradient elution from solution A to solution B using solution B (acetonitrile containing 0.1% -trifluoroacetic acid) (50 minutes) Flow rate: 1.0 ml / min.

[0019]

Example 4 Leu ¹⁷ -PACAP27-NH ₂ (H-His-Ser-Asp-Gly
-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-L
eu-Ala-Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-NH ₂ )
Synthesis of [Id] (SEQ ID NO: 5) Using 0.60 g (0.5 mmole) of a commercially available paramethyl BHA resin (manufactured by Applied Biosystems), a peptide synthesizer (Model 430A manufactured by Applied Biosystems) was used. . HOBt / DCC for the first amino acid, Boc-Leu
After condensing to the resin, the Boc group on the resin is treated with 50% trifluoroacetic acid / methylene chloride to release the amino group, and the amino group is replaced with Boc-Val, Boc-Ala, Boc-Leu ,
Boc-Tyr (Br-Z), Boc-Lys (Cl-Z), Boc-Gln, Boc-Arg (To
s), Boc-Ser (Bzl), Boc-Gly, Boc-Asp (OcHex), Boc-T
hr (Bzl), Boc-Phe, Boc-Ile and Boc-His (Bom) were activated and condensed with HOBt / DCC in the order of the indicated amino acid sequence. After condensing again with the same amino acid derivative activated with DCC or HOBt / DCC, unreacted amino groups were acetylated with acetic anhydride to obtain 1.34 g of a protected peptide resin. 1.00 g of this protected peptide resin was treated with 10 ml of anhydrous hydrogen fluoride at 0 ° C. for 60 minutes in the presence of 1.61 g of paracresol, hydrogen fluoride was distilled off under reduced pressure, and the residue was washed twice with 5 ml of ethyl ether. Thereafter, the residue was extracted with 5 ml of 50% aqueous acetic acid. The insoluble material was removed by filtration and washed with 5 ml of 50% aqueous acetic acid. The filtrate and washing solution were combined, concentrated under reduced pressure to 2-3 ml, and Sephadex G-25 (2x75
cm) and eluted with 50% acetic acid. After collecting and evaporating the main fraction under reduced pressure, the residue was dissolved in 100 ml of 0.1% aqueous trifluoroacetic acid, and the mixture was dissolved in a LiChroprep RP-18 resin column (2.6
x9.2 cm) and eluted with a linear gradient between 0.1% aqueous trifluoroacetic acid and 50% acetonitrile (containing 0.1% trifluoroacetic acid). The main section was collected and lyophilized, dissolved in 20 ml of 0.05 M aqueous ammonia acetate, applied to a CM-Cellulofine resin column (2.5 x 10 cm), and the linear concentration of 0.05 M to 0.5 M aqueous ammonia acetate was added. Eluted with a gradient. The main fractions were combined and lyophilized to give 35.4 mg of a white powder. Amino acid analysis value Asp 1.97 (2), Thr 0.94 (1), Ser 2.46 (3), Glu 1.08
(1), Gly 1.02 (1), Ala 3.08 (3), Val 1.93 (2), Ile 0.9
4 (1), Leu 3.00 (3), Tyr 2.51 (3), Phe 0.94 (1), Lys 2.
91 (3), His 1.09 (1), Arg 1.99 (2) Mass spectrometry (M + H) + 3128.5970 HPLC elution time 21.5 minutes Column conditions Column: Wakosil 5C18 (4.6 × 100 cm) Eluent: Solution A (0.1% -triol) (Aqueous fluoroacetic acid) Linear concentration gradient elution from solution A to solution B using solution B (acetonitrile containing 0.1% -trifluoroacetic acid) (50 minutes) Flow rate: 1.0 ml / min.

[0020]

Example 5 Glu ¹⁷ -PACAP27-NH ₂ (H-His-Ser-Asp-Gly
-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-G
lu-Ala-Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-NH ₂ )
Synthesis of [Ie] (SEQ ID NO: 6) Using a commercially available paramethyl BHA resin (manufactured by Applied Biosystems) 0.74 g (0.5 mmole), using a peptide synthesizer (Applied Biosystems model 430A) did. HOBt / DCC for the first amino acid, Boc-Leu
After condensing to the resin, the Boc group on the resin is treated with 50% trifluoroacetic acid / methylene chloride to release the amino group, and the amino group is replaced with Boc-Val, Boc-Ala, Boc-Leu ,
Boc-Tyr (Br-Z), Boc-Lys (Cl-Z), Boc-Arg (Tos), Boc-Gl
n, Boc-Ser (Bzl), Boc-Gly, Boc-Asp (OcHex), Boc-Th
r (Bzl), Boc-Phe, Boc-Ile and Boc-His (Bom) were activated and condensed with HOBt / DCC in the order of the amino acid sequence shown in the table. After condensing again with the same amino acid derivative activated with DCC or HOBt / DCC, unreacted amino groups were acetylated with acetic anhydride to obtain 1.94 g of a protected peptide resin. 0.93 g of this protected peptide resin was treated with 10 ml of anhydrous hydrogen fluoride at 0 ° C. for 60 minutes in the presence of 1.52 g of paracresol, then hydrogen fluoride was distilled off under reduced pressure, and the residue was washed twice with 5 ml of ethyl ether. Thereafter, the residue was extracted with 5 ml of 50% aqueous acetic acid. The insoluble material was removed by filtration and washed with 5 ml of 50% aqueous acetic acid. Combine the filtrate and washings,
Concentrate the solution to 2-3 ml under reduced pressure and use Sephadex G-25 (2x75 cm)
And eluted with 50% acetic acid. After collecting and evaporating the main fraction under reduced pressure, the residue was washed with 0.1% aqueous trifluoroacetic acid 1
Dissolve in 100 ml, and add a LiChroprep RP-18 resin column (2.6 x 9.2
cm) and eluted with a linear concentration gradient between 0.1% aqueous trifluoroacetic acid and 50% acetonitrile (containing 0.1% trifluoroacetic acid). Combine the main fractions and reconstitute LiChroprep RP-
The column was attached to an 18 column (2.6 x 9.2 cm), and a linear concentration gradient elution of an aqueous solution of acetonitrile (containing 0.1% trifluoroacetic acid) up to 20 to 40% was performed. This was dissolved in 20 ml of 0.05 M aqueous ammonia acetate, and applied to a CM-Cellulofine resin column (2.5 × 10 cm).
Was eluted with a linear concentration gradient of 0.5 M aqueous ammonia acetate. The main fractions were combined and lyophilized to give a white powder 17.5 mg
I got Amino acid analysis value Asp 1.98 (2), Thr 0.94 (1), Ser 2.47 (3), Glu 2.27
(2), Gly 1.10 (1), Ala 3.28 (3), Val 1.92 (2), Ile 0.9
3 (1), Leu 2.00 (2), Tyr 2.93 (3), Phe 0.94 (1), Lys 2.
80 (3), His 0.98 (1), Arg 1.93 (2) Mass spectrometry (M + H) + 3144.6870 HPLC elution time 21.2 minutes Column conditions Column: Wakosil 5C18 (4.6 × 100 cm) Eluent: Solution A (0.1% -triol) (Aqueous fluoroacetic acid) Linear concentration gradient elution from solution A to solution B using solution B (acetonitrile containing 0.1% -trifluoroacetic acid) (50 minutes) Flow rate: 1.0 ml / min.

[0021]

Example 6 Gly ¹⁷ -PACAP27-NH ₂ (H-His-Ser-Asp-Gly
-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-G
ly-Ala-Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-NH ₂ )
Synthesis of [If] (SEQ ID NO: 7) Using a commercially available paramethyl BHA resin (manufactured by Applied Biosystems) 0.64 g (0.5 mmole) and a peptide synthesizer (Applied Biosystems model 430A) did. HOBt / DCC for the first amino acid, Boc-Leu
After condensing to the resin, the Boc group on the resin is treated with 50% trifluoroacetic acid / methylene chloride to release the amino group, and the amino group is replaced with Boc-Val, Boc-Ala, Boc-Leu ,
Boc-Tyr (Br-Z), Boc-Lys (Cl-Z), Boc-Gly, Boc-Gln, Bo
c-Arg (Tos), Boc-Ser (Bzl), Boc-Asp (OcHex), Boc-Thr
(Bzl), Boc-Phe, Boc-Ile and Boc-His (Bom) were activated and condensed with HOBt / DCC in the order of the indicated amino acid sequence. further
After condensing again with the same amino acid derivative activated with DCC or HOBt / DCC, unreacted amino groups were acetylated with acetic anhydride to obtain 2.75 g of a protected peptide resin. This protected peptide resin (1.01 g) was treated with anhydrous hydrogen fluoride (10 ml) at 0 ° C. for 60 minutes in the presence of paracresol (1.63 g), hydrogen fluoride was distilled off under reduced pressure, and the residue was washed twice with ethyl ether (5 ml). Thereafter, the residue was extracted with 5 ml of 50% aqueous acetic acid. The insoluble material was removed by filtration and washed with 5 ml of 50% aqueous acetic acid. Combine the filtrate and washings,
Concentrate the solution to 2-3 ml under reduced pressure and use Sephadex G-25 (2x75 cm).
And eluted with 50% acetic acid. The main fraction was collected and distilled under reduced pressure.
9.2 cm) and eluted with a linear gradient between 0.1% aqueous trifluoroacetic acid and 50% acetonitrile (containing 0.1% trifluoroacetic acid). Combine the main fractions and again LiChropr
The mixture was applied to an ep RP-18 column (2.6 x 9.2 cm), and a 20 to 40% aqueous solution of acetonitrile (containing 0.1% trifluoroacetic acid) was subjected to linear gradient elution. The main fraction was collected and freeze-dried. Dissolve this in 20 ml of 0.05 M aqueous ammonia acetate,
The mixture was applied to a CM-Cellulofine resin column (2.5 × 10 cm) and eluted with a linear concentration gradient of 0.05 M to 0.5 M aqueous ammonia acetate. The main fractions were combined and lyophilized to give 20.5 mg of a white powder. Amino acid analysis value Asp 1.97 (2), Thr 0.93 (1), Ser 2.44 (3), Glu 1.07
(1), Gly 1.97 (2), Ala 3.10 (3), Val 1.93 (2), Ile 0.9
4 (1), Leu 2.00 (2), Tyr 3.02 (3), Phe 0.96 (1), Lys 2.
92 (3), His 1.03 (1), Arg 1.96 (2) Mass spectrometry (M + H) + 3072.6120 HPLC elution time 20.7 min Column conditions Column: Wakosil 5C18 (4.6 × 100 cm) Eluent: Solution A (0.1% -triol) (Aqueous fluoroacetic acid) Linear concentration gradient elution from solution A to solution B using solution B (acetonitrile containing 0.1% -trifluoroacetic acid) (50 minutes) Flow rate: 1.0 ml / min.

[0022]

Example 7 Arg ¹⁷ -PACAP26-NH ₂ (H-His-Ser-Asp-Gly
-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-A
rg-Ala-Val-Lys- Lys-Tyr-Leu-Ala-Ala-Val-NH 2) [Ih]
Synthesis of (SEQ ID NO: 8) Using a paramethyl BHA resin and a peptide synthesizer,
The title polypeptide [Ih] is synthesized according to the method of Example 1. Boc-Val is used as the first amino acid instead of Boc-Leu.

[0023]

Example 8 Arg ¹⁷ -PACAP23-NH ₂ (H-His-Ser-Asp-Gly
-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-A
rg-Ala-Val-Lys- Lys-Tyr-Leu-NH 2) [Ii] ( SEQ ID NO:
9) Synthesis using a paramethyl BHA resin and a peptide synthesizer,
The title polypeptide [Ii] is synthesized according to the method of Example 1.

[0024]

Example 9 Arg ¹⁷ -PACAP38-NH ₂ (H-His-Ser-Asp-Gly
-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-A
rg-Ala-Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys
-Arg-Tyr-Lys-Gln-Arg-Val-Lys-Asn-Lys-NH ₂ ) [Ij]
Synthesis of (SEQ ID NO: 10) Using paramethyl BHA resin and a peptide synthesizer,
According to Example 1, the title polypeptide [I
j]. After the first amino acid, Boc-Lys (Cl-Z), is activated with an activating reagent and condensed on the resin, the Boc group on the resin is removed to release the amino group, and the amino group is Boc-As
n, Boc-Lys (Cl-Z), Boc-Val, Boc-Arg (Tos), Boc-Gln,
Boc-Tyr (Br-Z), Boc-Gly, Boc-Leu, Boc-Ala, Boc-Ser
(Bzl), Boc-Asp (OcHex), Boc-Thr (Bzl), Boc-Phe, Boc-
The protected peptide resin is obtained by condensing Ile and Boc-His (Bom) in the order of the indicated amino acid sequence while activating with an activating reagent. After treating the protected peptide resin with hydrogen fluoride, the residue is extracted with a solvent. The extract is applied to a resin column, and the operation of separation and purification is repeated. The main fraction is collected and lyophilized to obtain the desired product [Ij].

[0025]

Example 10 Ser ¹⁷ -PACAP38-NH ₂ (H-His-Ser-Asp-G
ly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln
-Ser-Ala-Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-L
ys-Arg-Tyr-Lys- Gln-Arg-Val-Lys-Asn-Lys-NH 2) [Ik]
Synthesis of (SEQ ID NO: 11) Using a commercially available paramethyl BHA resin and a peptide synthesizer, according to the method of Example 9, the title polypeptide [I
k].

[0026]

Example 11 Arg ¹⁷ -PACAP27-OH (H-His-Ser-Asp-Gl
y-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-
Arg-Ala-Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-OH)
Synthesis of [Il] (SEQ ID NO: 12) Using a commercially available Boc-Leu-OCH2-PAM resin, the Boc group on the resin was
After treatment with 0% trifluoroacetic acid / methylene chloride, the above-mentioned polypeptide [Il] is synthesized according to the method of Example 1 in the indicated amino acid sequence.

[0027]

[Reference example] Met (O) ¹⁷ -PACAP27-NH ₂ (H-His-Ser-Asp-Gl
y-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-
Met (O) -Ala-Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-N
H ₂₎ Synthesis PACAP27-NH2 and (10.15mg) was dissolved in 1N acetic acid (1 ml), and after the 30% aqueous hydrogen peroxide (0.1 ml) was added and stirred for 10 minutes under ice-cooling, 20
% Ascorbic acid aqueous solution (2.3 ml) was added. This was applied to a column (2 × 85 cm) of Sephadex G-25 packed with 1N acetic acid and developed with the same solvent. Fractions of 102-156 ml were collected and lyophilized to give a white powder (9.25 mg). Amino acid analysis value Asp 2.00 (2), Thr 0.93 (1), Ser 2.46 (3), Glu 1.09
(1), Gly 1.01 (1), Ala 3.11 (3), Val 1.91 (2), Ile 1.0
6 (1), Leu 1.99 (2), Tyr 2.36 (3), Phe 1.03 (1), Lys 2.
90 (3), His 0.83 (1), Arg 1.96 (2) Mass spectrometry (M + H) + 3163.6 HPLC elution time 20.4 min Column conditions Column: YMC ODS AM301 (4.6 × 100 cm) Eluent: A solution (0.1% -Aqueous trifluoroacetic acid) Linear concentration gradient elution from solution A to solution B using solution B (acetonitrile containing 0.1% -trifluoroacetic acid) (50 minutes) Flow rate: 1.0 ml / min.

[0028]

[Test Example] PC12h cell line derived from the adrenal medulla was cultured at 37 ° C in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, and 5x per well in a collagen-treated 48-well plate.
104 cells were seeded and cultured for 7 to 10 days. afterwards,
The medium is replaced with 500 μl of Hank's balanced salt solution at 37 ° C. for 30 minutes, and a test substance (Example compound [Ia-f], PACAP38-NH ₂ or PACAP27-NH ₂ ) dissolved in the same solution is added thereto. The cells were cultured at 37 ° C for 2 hours. Next, c-AM in this culture solution
The amount of P was measured using an Amersham c-AMP measurement kit. The result is as shown in FIG.

[0029]

[0030]

[Sequence List] SEQ ID NO: 1 Sequence length: 38 Sequence type: amino acid Topology: Linear Sequence type: Peptide Sequence His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr- Ser-Arg-Tyr-Arg-Lys- 5 10 15 Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg- 20 25 30 Tyr-Lys- Gln-Arg-Val-Lys-Asn-Lys 35.

SEQ ID NO: 2 Sequence length: 27 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser -Arg-Tyr-Arg-Lys-5 10 15 Gln-Arg-Ala-Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu 2025.

SEQ ID NO: 3 Sequence length: 27 Sequence type: amino acid Topology: linear Sequence type: peptide sequence His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser -Arg-Tyr-Arg-Lys-5 10 15 Gln-Ser-Ala-Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu 2025.

SEQ ID NO: 4 Sequence length: 27 Sequence type: amino acid Topology: linear Sequence type: peptide Sequence His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser -Arg-Tyr-Arg-Lys-5 10 15 Gln-Phe-Ala-Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu 2025.

SEQ ID NO: 5 Sequence length: 27 Sequence type: amino acid Topology: linear Sequence type: peptide sequence His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser -Arg-Tyr-Arg-Lys-5 10 15 Gln-Leu-Ala-Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu 2025.

SEQ ID NO: 6 Sequence length: 27 Sequence type: amino acid Topology: linear Sequence type: peptide Sequence His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser -Arg-Tyr-Arg-Lys-5 10 15 Gln-Glu-Ala-Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu 2025.

SEQ ID NO: 7 Sequence length: 27 Sequence type: amino acid Topology: linear Sequence type: peptide Sequence His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser -Arg-Tyr-Arg-Lys-5 10 15 Gln-Gly-Ala-Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu 2025.

SEQ ID NO: 8 Sequence length: 26 Sequence type: amino acid Topology: linear Sequence type: peptide sequence His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser -Arg-Tyr-Arg-Lys-5 10 15 Gln-Arg-Ala-Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val 20 25.

SEQ ID NO: 9 Sequence length: 23 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser -Arg-Tyr-Arg-Lys-5 10 15 Gln-Arg-Ala-Val-Lys-Lys-Tyr-Leu 20.

SEQ ID NO: 10 Sequence length: 38 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser -Arg-Tyr-Arg-Lys- 5 10 15 Gln-Arg-Ala-Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg- 20 25 30 Tyr-Lys-Gln -Arg-Val-Lys-Asn-Lys 35.

SEQ ID NO: 11 Sequence length: 38 Sequence type: amino acid Topology: linear Sequence type: peptide Sequence His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser -Arg-Tyr-Arg-Lys- 5 10 15 Gln-Ser-Ala-Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg- 20 25 30 Tyr-Lys-Gln -Arg-Val-Lys-Asn-Lys 35.

SEQ ID NO: 12 Sequence length: 27 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser -Arg-Tyr-Arg-Lys-5 10 15 Gln-Arg-Ala-Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu 2025.

[Brief description of the drawings]

FIG. 1 is a graph showing a comparison of the c-AMP production activities of compound [Ia-f], PACAP27-NH ₂ and PACAP38-NH ₂ of the present invention.

────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-3-22987 (JP, A) JP-A-3-123798 (JP, A) JP-A-62-246595 (JP, A) US Pat. , A) (58) Fields surveyed (Int. Cl. ⁷ , DB name) CA (STN) REGISTRY (STN) PubMed

Claims (8)

(57) [Claims] 1. The formula His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Ty
r-Arg-Lys-Gln-NH-CHX-CO-Ala-Val-Lys-Lys-Tyr-Y [where NH-CHX-CO is Glu, Gly, Ser or Arg residue
A group, Y is Leu-Ala-Ala-Val- Leu-Gly-Lys-Arg-Tyr-
1 counting from the N-terminal side of Lys-Gln-Arg-Val-Lys-Asn-Lys
Or 16 amino acids or peptides], or an amide, ester or salt thereof. 2. The polypeptide according to claim 1, wherein NH-CHX-CO is a Ser or Arg residue, or an amide, ester or salt thereof. 3. The polypeptide according to claim 1, wherein NH-CHX-CO is a Glu residue, or an amide, ester or salt thereof. 4. Y is Leu, Leu-Ala, Leu-Ala-Ala, Leu-Ala
The polypeptide according to claim 1, which is -Ala-Val or Leu-Ala-Ala-Val-Leu, or an amide, ester or salt thereof. (5) H-His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-
Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Arg-Ala-Val-Lys-Lys-Ty
polypeptide or its salt according to claim 1, wherein represented by r-Leu-Ala-Ala- Val-Leu-NH 2. (6) H-His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-
Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Ser-Ala-Val-Lys-Lys-Ty
polypeptide or its salt according to claim 1, wherein represented by r-Leu-Ala-Ala- Val-Leu-NH 2. 7. A pharmaceutical composition for a nerve activator, comprising the polypeptide according to claim 1 or an amide, ester or pharmacologically acceptable salt thereof. 8. A pharmaceutical composition for treating a neurological disorder or repairing an injured nerve, comprising the polypeptide according to claim 1 or an amide, ester or pharmacologically acceptable salt thereof.