JPH07116223B2 - Novel tripeptide and its use - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a nerve cell containing a novel tripeptide considered to be effective for preventing or treating nerve cell damage in senile dementia and the like as an active ingredient. It relates to a protrusion extender.

[Conventional technology]

The number of senile dementia patients requiring nursing is increasing with the increase of the aged population, and this countermeasure is greatly highlighted as a social problem, and the development of a preventive / therapeutic drug therefor is desired.

Senile dementia is an acquired intellectual disorder that occurs after adulthood and is considered to be caused by a brain organic disorder, an organ disorder, or a physical disorder caused by stress.

About 90% of dementia due to cerebral organic disorder considered to be irreversible disease is due to stroke (based on multiple cerebral infarction), Alzheimer-type dementia (based on degenerative degenerative disease of nerve cells in the brain) It is said to be occupied by these) and their mixed types, and the development of therapeutic agents for them is being attempted.

For the treatment of dementia due to organic disorders of the brain, repair of the damaged site,
It is considered necessary to recover the functional deterioration of the tissue around the damaged site, and in particular, it is considered necessary to develop a drug that promptly repairs the damaged site. Further, it is considered that the repair needs to be performed not only on the nervous system but also on the vascular system, glial system and the like which compose the brain. Therefore, it is considered that a drug that promotes cell proliferation is required for vascular cells and glial cells, and that a drug that enhances regeneration and plasticity of nerve cells and enhances colonization of transplanted nerve cells is necessary. Has been. It is considered necessary to develop a drug that recovers the functional decline of cells of the nervous system, vascular system, and glial system even in the case of peripheral functional decline.

In the treatment of senile dementia, in the future, a combination of drugs having the above-mentioned effects of promoting the repair of various damaged sites and recovering the functional deterioration of the surrounding tissues will be used, and the development of the therapy will progress. It is thought to do. And
To date, in the case of the nervous system, as a natural drug that is expected to prevent degenerative loss of nerve cells and quickly repair the damaged site, nerve growth factor (NGF) consisting of 118 amino acid residues is used. ) (The molecular weight is 13,000 for the monomer and 26,500 for the dimer.) But,
The NGF currently obtained is made of mouse, and even if it is effective as a drug, it has a problem in terms of production and antigenicity for use in human treatment.

Therefore, PC12 cells (AT
CC CRL 1721), a synthetic peptide CH ₃ CONH L CONH L CONH N CHO (where L is a leucine to a carboxyl group and It represents a leucine residue with one amino group removed. N represents a norleucine residue obtained by removing one carboxyl group and one amino group from norleucine. The number of protrusions from a nerve cell is usually two or more in NGF, whereas this tripeptide forms one or two protrusions and the length at that time is NG.
It will be longer than in the case of F. This tripeptide is considered to be less problematic in terms of antigenicity than mouse NGF. ] Have been found.

(Yumiko Saito and Seiichi Kawashima: Neuroscience
Letters, 89 , 102-107 (1988)].

[Problems to be solved by the invention]

An object of the present invention is to provide a neurite outgrowth drug containing, as an active ingredient, a novel tripeptide which is considered to be effective in preventing or treating nerve cell damage in senile dementia and the like.

[Means for solving problems]

The present inventor has conducted extensive studies to solve the above-mentioned problems, and as a result, has previously found a synthetic peptide CH ₃ CONH L CONH L C.
ONH N CHO [where L represents a leucine residue obtained by removing one carboxyl group and one amino group from leucine. N represents a norleucine residue obtained by removing one carboxyl group and one amino group from norleucine. ]
By substituting the acetyl group (CH ₃ CO) at the N-terminus of the benzyl group with a benzyloxycarbonyl group (C ₆ H ₅ CH ₂ OCO, hereinafter abbreviated as Z), the elongation action of neurites is remarkably promoted. Further, by substituting the C-terminal amino acid of the tripeptide having the benzyloxycarbonyl group with another amino acid residue represented by X with one carboxyl group and one amino group removed, the effect is further enhanced. This has led to the completion of the present invention.

That is, the present invention has the formula: in _{C 6 H 5 CH 2 OCONH L} CONH L CONH X CHO [wherein, L represents a leucine residue, respectively one removed carboxyl and amino groups leucine; X is
Norleucine, leucine, isoleucine, norvaline,
It represents an amino acid residue obtained by removing one carboxyl group and one amino group from an amino acid selected from the group consisting of valine and phenylalanine. ] It is related with the novel tripeptide shown by these.

Furthermore, the present invention relates to a neurite outgrowth drug containing the novel tripeptide as an active ingredient.

In a preferred aspect of the present invention, X in the novel tripeptide is norleucine (hereinafter abbreviated as Nle), leucine (hereinafter abbreviated as Leu), isoleucine (hereinafter abbreviated as Ile), A carboxyl group and an amino group are respectively selected from amino acids selected from the group consisting of norvaline (hereinafter abbreviated as Nva), valine (hereinafter abbreviated as Val), and phenylalanine (hereinafter abbreviated as Phe). One of the removed amino acid residues (hereinafter, Nle-derived amino acid residue is N , Leu-derived amino acid residue is L , and Ile-derived amino acid residue is
The amino acid residues derived from I and Nva are abbreviated to W , the amino acid residues derived from Val are abbreviated to V , and the amino acid residues derived from Phe are abbreviated to P. ).

Hereinafter, the present invention will be described in detail.

The novel tripeptide having neurite outgrowth action of the present invention includes the following formula: ZNH L CONH L CONH X CHO [wherein X is norleucine, leucine, isoleucine, norvaline, valine, and phenylalanine. It represents an amino acid residue obtained by removing one carboxyl group and one amino group from the selected amino acid. ] A tripeptide represented by, for example, ZNH L CONH L CONH N CHO ZNH L CONH L CONH L CHO ZNH L CONH L CONH I CHO ZNH L CONH L CONH W CHO ZNH L CONH L CONH V CHO or ZNH L CONH L CONH P CHO etc. can be mentioned.

Synthesis of the novel tripeptide of the present invention, ZNH L CONH and H ₂
N L COOMe and DCC method [JC Sheehan, GH Hess; J. Am. Chem.
Soc., 77 , 1067 (1955)] to give a protected dipeptide ester (ZNH L CONH L COOMe), which is then hydrazided to obtain the protected dipeptide hydrazide (ZNH L CONH L COMe).
ONHNH ₂ ) and the amino acid semicarbazone (H ₂ N X CHNNHCON
H ₂ ) is condensed by the azide method and ZNH L CONH L CONH X CHNNHCON
H ₂ ) can be obtained and the C-terminal can be converted to an aldehyde. Then, the novel tripeptide obtained by such synthesis can be confirmed by reverse phase high performance liquid chromatography, mass spectrum, 2,4-dinitrophenylhydrazine test, and the like.

The activity of the novel tripeptide of the present invention showing the neurite outgrowth effect can be measured using PC12 cells (ATCC CRL 1721) that respond to NGF (nerve growth factor), and the activity has been found conventionally. Used synthetic tripeptide CH ₃ CONH L CONH L CONH N CHO (Yumiko Saito and Seiichi Kawashima: Neuroscience
Letters, 89 , 102-107 (1988)].

The neurite outgrowth drug containing the novel tripeptide of the present invention as an active ingredient may contain the novel tripeptide of the present invention as a single component as a substance having physiological activity, or may have other physiological properties. It may contain an active substance.

〔Example〕

Hereinafter, the present invention will be specifically described with reference to examples. It should be noted that these examples are for illustrating the present invention and do not limit the scope of the present invention.

Example 1 [Preparation of Protected Amino Acid] Synthesis of ZNH L COOMe Leu (26.20 g, 0.20 mol) was dissolved in 2M aqueous sodium hydroxide solution (100 ml, 0.20 mol), and carbobenzoxy chloride (34.00 g was stirred with ice cooling. , 0.20 mol) and 4M aqueous sodium hydroxide solution (50 ml, 0.20 mol) were added simultaneously over 20 minutes. Then, the mixture was stirred at room temperature for 3 hours, the aqueous layer was washed with ether, and the pH was adjusted to 2-3 with 6M hydrochloric acid under ice cooling. After extraction with ethyl acetate (100 ml x 3), 5% citric acid solution (100
ml × 3), water (100 ml × 3), and dried over anhydrous sodium sulfate overnight. After the desiccant was filtered off, it was concentrated under reduced pressure,
Drying in a dessicator gave 41.88 g (94% yield) of ZNH L COOH.

Using ZNH L COOH obtained in this way,
ZNH L COOMe was synthesized.

First, methanol (100 ml) was cooled to −10 ° C., thionyl chloride (21.8 ml, 0.30 mol) was gradually added, and the mixture was stirred for 10 minutes. Then add ZNH L COOH (26.50g, 0.10mol),
The mixture was stirred at room temperature for 10 hours. After concentration under reduced pressure, the residue was dissolved in ether and washed with water. It was dried over anhydrous sodium sulfate overnight, the desiccant was filtered off, concentrated under reduced pressure, and dried in a dicator to give 25.39 g of ZNH L COOMe (yield: 91%).
%) I was able to get it.

Synthesis of ZNH N COOMe In the above synthesis of ZNH N COOH, NLe was used instead of Leu.
(3.93 g, 30 mmol), 2M aqueous sodium hydroxide solution (15 ml, 30 mmol), carbobenzoxy chloride (5.10
g, 30mmol), 4M aqueous sodium hydroxide solution (7.5ml, 30
As a result of synthesizing ZNH N COOH at 6.48 g (yield: 86
%) Dry sample could be obtained.

Using ZNH N COOH obtained in this way,
ZNH N COOMe was synthesized.

First, methanol (50 ml) was cooled to −10 ° C., thionyl chloride (4.36 ml, 60 mmol) was gradually added, and the mixture was stirred for 10 minutes. Then, ZNH N COOH (5.30 g, 20 mmol) was added, and the mixture was stirred at room temperature for 10 hours. After concentration under reduced pressure, the residue was dissolved in ether and washed with water. 5.19 g (93% yield) of ZNH N COOMe by drying overnight with anhydrous sodium sulfate, filtering off the desiccant, concentrating under reduced pressure, and drying in a desiccator.
I was able to get it.

Synthesis of ZNH I COOMe In the above synthesis of ZNH L COOH, ILe instead of Leu
(13.10g, 0.10mol), 2M sodium hydroxide aqueous solution (50ml, 0.10mol), carbobenzoxy chloride (17.
00g, 0.10mol), 4M aqueous sodium hydroxide solution (25ml,
As a result of synthesizing ZNH I COOH with 0.10 mol, 25.97 g (yield 9
It was possible to obtain a dry standard of 8%).

Using ZNH I COOH obtained in this way,
ZNH I COOMe was synthesized.

First, methanol (100 ml) was cooled to −10 ° C., thionyl chloride (10.9 ml, 0.15 mol) was gradually added, and the mixture was stirred for 10 minutes. Then, ZNH I COOH (13.25 g, 50 mmol) was added, and the mixture was stirred at room temperature for 10 hours. After concentration under reduced pressure, the residue was dissolved in ether and washed with water. After drying over anhydrous sodium sulfate overnight, the desiccant was filtered off, the filtrate was concentrated under reduced pressure and dried in a desiccator to obtain 10.60 g of ZNH I COOMe (yield: 76%).
%) I was able to get it.

Synthesis of ZNH V COOMe In the above synthesis of ZNH I COOH, Val instead of Ile was used.
As a result of synthesizing ZNH V COOH using (11.70 g, 0.10 mol), 21.08 g (yield 84%) of a dried sample could be obtained. Using the thus obtained ZNH V COOH, ZNH V COOMe was synthesized as follows.

First, in the synthesis of the above ZNH I COOMe, ZNH V COOH ( 12.55g, 50mmol) in place of ZNH I COOH by using, 6.63 g of ZNH V COOMe could (yield 50%) as possible.

Synthesis of ZNH W COOMe In the above synthesis of ZNH N COOH, Nva was used instead of Nle.
As a result of synthesizing ZNH W COOH using (3.51 g, 30 mmol),
6.70 g (yield 89%) of dry standard could be obtained.
Using ZNH W COOH obtained in this way,
ZNH W COOMe was synthesized.

First, in the synthesis of said ZNH N COOMe, ZNH W COOH ( 5.02g, 20mmol) in place of ZNH N COOH by using, 5.19 g of ZNH W COOMe could (yield 98%) as possible.

Synthesis of ZNH P COOMe In the above synthesis of ZNH I COOH, Phe instead of Ile
As a result of synthesizing ZNH P COOH using (16.50 g, 0.10 mol), 22.58 g (yield: 76%) of a dry sample could be obtained. Using the thus obtained ZNH P COOH, ZNH P COOMe was synthesized as follows.

First, in the synthesis of the above ZNH I COOMe, ZNH P COOH ( 14.95g, 50mmol) in place of ZNH I COOH by using, 13.15 g of ZNH P COOMe could (yield 84%) as possible.

Synthesis of H ₂ N L COOMe (hydrochloride) Methanol (200 ml) was cooled to −10 ° C., thionyl chloride (30 ml, 0.41 mol) was gradually added, and the mixture was stirred for 10 minutes. Then, Leu (26.20 g, 0.20 mol) was added, and the mixture was stirred at room temperature for 2 days. After concentration under reduced pressure, ether was added to the residue for crystallization to give 35.96 g of H ₂ N L COOMe (hydrochloride).
(Yield 99%) could be obtained.

Example 2 [Synthesis of protected dipeptide hydrazide] ZNH L COOH (7.95 g, 30 mmol) was dissolved in THF (tetrahydrofuran) (60 ml), dicyclohexylcarbodiimide (6.80 g, 33 mmol) was added with stirring under ice-cooling, and further 30 Stir for minutes.

Then, a THF solution (50 ml) of H ₂ N L COOMe (hydrochloride) (5.99 g, 33 mmol) and triethylamine (3.33 g, 33 mmol) was added, and the mixture was stirred for 1 hour under ice cooling. Then, at room temperature, 7
Stir for hours. After standing overnight, dicyclohexylurea was filtered, the filtrate was concentrated under reduced pressure, and the residue was ethyl acetate (300 ml).
Melted into This solution was added with 5% citric acid (100 ml x 3),
The extract was washed with saturated saline (100 ml × 3), 4% sodium hydrogen carbonate solution (100 ml × 3) and saturated saline (100 ml × 3) in this order, and dried over anhydrous sodium sulfate overnight. After the desiccant was filtered off, the mixture was concentrated under reduced pressure and crystallized using ether and petroleum ether to obtain 8.70 g (yield 74%) of ZNH L CONH L COOMe.

The protected peptide thus obtained (7.84 g, 2
(0 mmol) was dissolved in methanol (70 ml), hydrazine monohydrate (10.00 g, 0.2 mol) was added, and the mixture was stirred at room temperature for 1 day. Then, this solution is concentrated, water is added to crystallize,
7.76 g (yield 99%) of ZNH L CONH L CONHNH ₂ could be obtained.

Example 3 [Preparation of Z-α-aminoaldehyde semicarbazone and Z
Removal of group] Hereinafter, scaffold aldehyde semicarbazone (CHNNHCONH ₂ )
Is abbreviated.

Preparation of H ₂ N L -sc (TosOH salt) ZNH L COOMe (6.98 g, 25 mmol) was dissolved in anhydrous toluene (50 ml), and 1.0 M diisobutylaluminum hydride / hexane solution (50 ml, at −50 ° C. under a nitrogen atmosphere). 50 mmol) was added over 1 hour. After stirring at -50 ℃ for 1 hour, 2M
Hydrochloric acid (125 ml, 0.25 mol) was added carefully. The temperature was gradually raised to room temperature, the aqueous layer was extracted with ethyl acetate (100 ml × 3), combined with the organic layer, and washed with water (100 ml × 3).
After drying over anhydrous sodium sulfate overnight, the desiccant was filtered off, the temperature of the water bath was set to 40 ° C or lower, and the mixture was concentrated under reduced pressure. Dissolve the residue in 70% ethanol (100 ml) and add semicarbazide hydrochloride (3.12 g, 28 mmol) and sodium acetate (2.30 g, 28 mmo) at 80 ° C.
l) was added and stirred for 10 minutes. The mixture was allowed to cool to room temperature, the temperature of the water bath was set to 40 ° C or lower, and concentrated under reduced pressure. The residue was dissolved in ethyl acetate (300 ml), washed with water (100 ml × 3), anhydrous sodium sulfate was added, and the mixture was dried overnight. After the desiccant was filtered off, the temperature of the water bath was adjusted to 40 ° C. or lower and the mixture was concentrated under reduced pressure and decanted with petroleum ether. In addition, set the temperature of the water bath to 40
Concentrated under reduced pressure below ℃, dried in a desiccator,
7.19 g (yield 94%) of ZNH L -sc could be obtained.

Using this, H ₂ N L -sc (TosOH salt) was prepared as follows.
Was manufactured.

Palladium was activated by adding 10% palladium activated carbon (0.5 g) to methanol (50 ml), substituting with nitrogen and hydrogen, and stirred for 2 hours. After nitrogen substitution, ZNH L -sc (2.45g, 8m
A methanol solution (50 ml) of (mol) was added, the mixture was replaced with nitrogen and replaced with hydrogen, and the mixture was stirred for 5 hours. After purging with nitrogen, the solvent was removed by filtration, and p-toluenesulfonic acid monohydrate (3.04 g, 1
The 6 mmol) was added, and concentrated under reduced pressure, and crystallized from anhydrous ether, methanol - by recrystallization from ethanol, H ₂ N L -sc (TosOH salt) and 1.28 g (yield can 66%) to obtain It was

In H ₂ N L -sc method for manufacturing the H ₂ N L -sc (TosOH salt) of (TosOH salt), ZNH L
ZNH N COOMe (5.02 g, 18 mmol) was used instead of COOMe,
By using semicarbazide hydrochloride (2.23 g, 20 mmol) and sodium acetate (1.64 g, 20 mmol), 5.34 g (yield 97%) of ZNH N -sc could be obtained.
Using this, in the method for producing ZNH L -sc (TosOH salt), 2.59 g of H ₂ N N -sc (TosOH salt) was obtained by using ZNH N -sc instead of ZNH L -sc. The rate was 94%).

Production of H ₂ N I -sc (TosOH salt) In the production method of H ₂ N L -sc (TosOH salt), ZNH L
ZNH I COOMe (6.98 g, 25 mmol) was used instead of COOMe,
By using (3.12 g, 28 mmol) as the semicarbazide hydrochloride and (2.30 g, 28 mmol) as the sodium acetate, 3.21 g (yield 42%) of ZNH I -sc could be obtained.
Using this, in the above-mentioned method for producing H ₂ N L -sc (TosOH salt), by using ZNH I -sc instead of ZNH L -sc, H ₂ N I -sc (TosOH salt) was 1.21. g (44% yield)
I was able to get it.

In the method of manufacturing the H ₂ N L -sc (TosOH salt) of H ₂ N V -sc (TosOH salt), ZNH L
ZNH V COOMe (6.63 g, 25 mmol) was used instead of COOMe,
3.72 g (yield 51%) of ZNH V -sc could be obtained by using (3.12 g, 28 mmol) as the semicarbazide hydrochloride and (2.30 g, 28 mmol) as the sodium acetate.
Using this, in the method for producing H ₂ N L -sc (TosOH salt), 1.35 g of H ₂ N V -sc (TosOH salt) was obtained by using ZNH V -sc instead of ZNH L -sc. (Yield 51%) could be obtained.

Production of H ₂ N W -sc (TosOH salt) In the production method of H ₂ N L -sc (TosOH salt), ZNH L
ZNH W COOMe (4.77 g, 18 mmol) was used instead of COOMe,
By using semicarbazide hydrochloride (2.23 g, 20 mmol) and sodium acetate (1.64 g, 20 mmol), 4.94 g (yield 94%) of ZNH W -sc could be obtained.
Using this, in the method for producing H ₂ N L -sc (TosOH salt), by using ZNH W -sc instead of ZNH L -sc, 1.90 g of H ₂ N W -sc (TosOH salt) was obtained. (Yield 72%) could be obtained.

Production of H ₂ N P -sc (TosOH salt) In the method for producing H ₂ N L -sc (TosOH salt), ZNH L
ZNH P COOMe (5.33 g, 17 mmol) was used instead of COOMe,
By using semicarbazide hydrochloride (2.23 g, 20 mmol) and sodium acetate (1.64 g, 20 mmol), 5.17 g (yield 89%) of ZNH P -sc could be obtained.
Using this, in the manufacturing method of the H ₂ N L -sc (TosOH salt), by using a ZNH P -sc instead of ZNH L -sc, ZNH P (TosOH salt) and 1.65 g (yield 73%).

Example 4 [Synthesis of tripeptide aldehyde] Production of ZNH L CONH L CONH L CHO The protected dipeptide ZNH L CONH L CONHNH ₂ (0.78 g, 2
mmol) was dissolved in DMF (dimethylformamide) (10 ml), and after cooling to -50 ° C, 3.5M hydrogen chloride / dioxane (1.
71 ml, 6 mmol) and isoamyl nitrite (0.35 g, 3 mmol) were added.

The temperature was raised to −20 ° C. and stirred for 1 hour. When the hydrazine test became negative, the temperature was lowered to −60 ° C., neutralized with triethylamine (0.61 g, 6 mmol), and H ₂ N L -sc (Tos
OH salt) (0.69g, 2mmol) and triethylamine (0.20g, 2
mmol) in DMF (10 ml) was added, and the mixture was stirred at 0 ° C. for 2 days. The reaction solution was filtered, concentrated under reduced pressure, crystallized by adding water, and further recrystallized with methanol-ether to give 0.76 g of ZNH L CONH L CONH L -sc which is a semicarbazone of tripeptide aldehyde. (Yield 71
%) I was able to get it.

ZNH L CONH L CONH L -sc (0.53g, 1m
mol) was dissolved in methanol (20 ml), 37% formalin (2.5 ml) and 4M hydrochloric acid (1.25 ml, 5 mmol) were added, and the mixture was stirred at room temperature for 3 hours. After that, the temperature of the water bath was set to 40 ° C or lower and the mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate (200 ml),
It was washed with 00 ml × 3). Anhydrous sodium sulfate was added and the mixture was dried overnight, then the desiccant was filtered off, the temperature of the water bath was set to 40 ° C. or lower and the mixture was concentrated under reduced pressure, and the residue was dried in a desiccator. This residue was dissolved in 50% acetonitrile (0.1% trifluoroacetic acid) and the preparative ODS column (CAPCELL PAK C ₁₈ , 2) was used.
ZNH L CONH L CONH L CHO separated and purified by reverse phase high performance liquid chromatography using 0.0φ x 250 mm, Shiseido)
76 mg (yield 16%) of which the physicochemical properties are: -Molecular weight: [FBS-MS m / z: 476 (M + H) ⁺ ] -Molecular formula: (M ⁺ , C ₂₆ H ₄₁ O ₅ N ₃₎ · mp: 85-90 ° C. · Optical _{rotation: [α] D = -52.0 °} (c 0.1, MeOH) · Rf: 0.73 (CHCl 3: MeOH = 5: 1) · 2,4- dinitrophenylhydrazine The test was positive.

Production of ZNH L CONH L CONH N CHO In the production method of ZNH L CONH L CONH L CHO, H ₂ N L -sc
Use H ₂ N N -sc (TosOH salt) instead of (TosOH salt) to produce Z
It was possible to obtain 0.69 g (yield: 65%) of NH L CONH L CONH N -sc.

Furthermore, by using ZNH L CONH L CONH N -sc thus obtained, instead of ZNH L CONH L CONH L -sc
64 mg (14% yield) of NH L CONH L CONH N CHO can be obtained. Its physicochemical properties are: -Molecular weight: [FBS-MS m / z: 476 (M + H) ⁺ ] -Molecular formula: (M ⁺ , C ₂₆ H ₄₁ O ₅ N ₃ ) ・ Melting point: 68 to 73 ° C. ・ Optical rotation: [α] _D = −21.6 ° ( c 0.1, MeOH) ・ Rf: 0.68 (CHCl ₃ : MeOH = 5: 1) ・The 2,4-dinitrophenylhydrazine test was positive.

Production of ZNH L CONH L CONH I CHO In the production method of ZNH L CONH L CONH L CHO, H ₂ N L -sc
Use H ₂ N I -sc (TosOH salt) instead of (TosOH salt) to produce Z
NH L CONH L CONH I and 0.93g could (yield 87%) as possible.

Furthermore, instead of ZNH L CONH L CONH L -sc, by using ZNH L CONH L CONH I -sc thus obtained, Z
101 mg (yield 21%) of NH L CONH L CONH I CHO can be obtained. Its physicochemical properties are: -Molecular weight: [FBS-MS m / z: 476 (M + H) ⁺ ] -Molecular formula: (M ⁺ , C ₂₆ H ₄₁ O ₅ N ₃ ) ・ Melting point: 145-150 ° C. ・ Optical rotation: [α] _D = -58.2 ° ( c 0.1, MeOH) ・ Rf: 0.60 (CHCl ₃ : MeOH = 5: 1) ・The 2,4-dinitrophenylhydrazine test was positive.

Production of ZNH L CONH L CONH V CHO In the production method of ZNH L CONH L CONH L CHO described above, H ₂ N L -sc
Z with H ₂ N V -sc (TosOH salt) instead of (TosOH salt)
It was possible to obtain 1.00 g (yield 97%) of NH L CONH L CONH V -sc.

Furthermore, by using ZNH L CONH L CONH V -sc thus obtained, instead of ZNH L CONH L CONH L -sc
122 mg (36% yield) of NH L CONH L CONH V CHO can be obtained, and its physicochemical properties are: -Molecular weight: [FBS-MS m / z: 462 (M + H) ⁺ ] -Molecular formula: (M ⁺ , C ₂₅ H ₃₉ O ₅ N ₃ ) ・ Melting point: 147 to 153 ℃ ・ Optical rotation: [α] _D = -52.3 ° ( c 0.2, MeOH) ・ Rf: 0.61 (CHCl ₃ : MeOH = 5: 1) ・The 2,4-dinitrophenylhydrazine test was positive.

Production of ZNH L CONH L CONH W CHO In the production method of ZNH L CONH L CONH L CHO, H ₂ N L -sc
Use H ₂ N W -sc (TosOH salt) instead of (TosOH salt) to produce Z
It was possible to obtain 0.93 g (yield 90%) of NH L CONH L CONH W -sc.

Furthermore, by using ZNH L CONH L CONH W -sc thus obtained, instead of ZNH L CONH L CONH L -sc
70 mg (15% yield) of NH L CONH L CONH W CHO can be obtained, and its physicochemical properties are: -Molecular weight: [FBS-MS m / z: 462 (M + H) ⁺ ] -Molecular formula: (M ⁺ , C ₂₅ H ₃₉ O ₅ N ₃ ) -Melting point: 78 to 86 ° C-Optical rotation: [α] _D = -52.3 ° ( c 0.05, MeOH) -Rf: 0.63 (CHCl ₃ : MeOH = 5: 1)- The 2,4-dinitrophenylhydrazine test was positive.

Manufacture of ZNH L CONH L CONH P CHO In the above manufacturing method of ZNH L CONH L CONH L CHO, H ₂ N L -sc
Use H ₂ N P -sc (TosOH salt) instead of (TosOH salt) to produce Z
It was possible to obtain 0.17 g (yield 15%) of NH L CONH L CONH P -sc.

Further, by using ZNH L CONH L CONH P -sc thus obtained, instead of ZNH L CONH L CONH L -sc
55 mg (yield 54%) of NH L CONH L CONH P CHO can be obtained. Its physicochemical properties are: -Molecular weight: [FBS-MS m / z: 510 (M + H) ⁺ ] -Molecular formula: (M ⁺ , C ₂₉ H ₃₉ O ₅ N ₃ ) ・ Melting point: 61 to 67 ° C. ・ Optical rotation: [α] _D = -63.1 ° ( c 0.2, MeOH) ・ Rf: 0.73 (CHCl ₃ : MeOH = 5: 1) ・The 2,4-dinitrophenylhydrazine test was positive.

Example 5 [Neurite outgrowth effect of PC12 cells by tripeptide aldehyde] Dulbecco's modified Eagl containing 5% horse serum and 5% newborn calf serum obtained by heat-treating PC12 cells (ATCC CRL 1721).
The cells were grown in e's medium under the conditions of 90% oxygen / 10% carbon gas at 37 ° C.

Put these cells on a plastic plate coated with calf skin collagen so that 1 to 2 × 10 ⁴ cells / cm ²
Serum-free synthetic medium (N2 medium: transferrin (100 μg / m
l), progesterone (5 μg / ml), putrescine (5 μg)
/ ml), insulin (5 μg / ml), selenium (52 μg / m)
Ham's F12 / DMEM medium containing l), 37 ° C, 95%
The cells were cultured under oxygen / 5% carbon dioxide gas conditions for 2 hours.

When the cells adhered to the collagen matrix after 2 hours, the various tripeptides synthesized in Example 4 (for control, CH
₃ CONH L CONH L CONH N CHO) containing N2 medium (concentrations of various tripeptide aldehydes are 0, 0.005, 0.01,
0, 02, 0.03, 0.04, 0.05, 0.08, 0.10, 0.15, 0.20,
0.25, 0.30, 0.35, 0.40, 0.50, 0.75, 1.00, 1.30, 1.
It was set to 60 or 1.90 μM. ), And cultured under the same conditions for 48 hours. Then, after washing with physiological saline, fixing with 2% glutaraldehyde, and staining with crystal violet, randomly selected 400-800 cells that have at least one neurite longer than the cell diameter Were measured by a phase contrast microscope.

The neurite outgrowth effect of various tripeptides on PC12 cells was determined by determining the optimum concentration (μM) of various tripeptide aldehydes when the ratio of this number of cells to the total number of measured cells reached a maximum value (about 50%). investigated. The results are shown in Table 1.

Table 1 [Effects of the Invention] The novel tripeptide of the present invention is useful as a neurite outgrowth drug.

Claims (2)

[Claims] 1. A following formula: C ₆ H in _{5 CH 2 OCONH L CONH L CONH} X CHO [wherein, L represents a leucine residue, respectively one removed carboxyl and amino groups leucine; X is
Norleucine, leucine, isoleucine, norvaline,
It represents an amino acid residue obtained by removing one carboxyl group and one amino group from an amino acid selected from the group consisting of valine and phenylalanine. ] The novel tripeptide which is. 2. A neurite outgrowth drug comprising the novel tripeptide of claim 1 as an active ingredient.