JPH0940577A - Medicinal composition containing tripeptide or dipeptide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a medicinal composition, containing a tripeptide having a specific amino acid sequence or its salt as an active ingredient and extremely rarely causing dangerousness of developing adverse effects due to its carcinostatic effects and ability to hydrolyze into amino acids which are metabolized in living bodies. SOLUTION: This medicinal composition contains a tripeptide of the formula, Pro-A-B (A is phenylalanine, lysine, etc.; B is proline or tryptophan) or its salt as an active ingredient. The objective composition is preferably administered in a daily dose of 0.01-10g for an adult in one or several divided portions. Furthermore, the tripeptide is usually prepared by mutually condensing protected amino acids using a condensing agent such as dicyclohexylcrbodiimide in a solid or a liquid phase method according to the amino acid sequence. Protecting groups of the resultant tripeptide are removed by a trifluoromethanesulfonic acid method, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a pharmaceutical composition containing a tripeptide or dipeptide useful as an anticancer agent.

[0002]

2. Description of the Related Art Studies on inhibitors of thrombin activity are known for utilizing tripeptides and their derivatives as pharmaceuticals (Journal Medical Chemistry: vol.37, p.2122, 1994, same volume: vol. .36, p.300,19
93). In addition, research and development of relatively short peptides and derivatives thereof as pharmaceuticals include, for example, Japanese Patent Publication No. 4-502154, Japanese Patent Publication No. 4-502306, Japanese Patent Publication No. 4-502308, and Japanese Patent Publication No. See, for example, the 502309 publication. In these studies,
The amino acid sequence of the peptide contains a prolyl group, and the length of the sequence is 5 or more amino acids. These peptides are TN derived from tumor necrosis factor (TNF)
It can also be said to be an F-improved peptide. Thus, there have been some studies using short peptides as medicines. It is considered that short peptides are easily taken up into cells, and since they are decomposed into innocuous amino acids in the living body, it is considered that there is almost no side effect to administration to the living body. Therefore, these short peptides are considered to be promising agents in the future as therapeutic agents for other diseases including cancer. However, nothing has been known so far regarding the use of short peptides as anticancer agents.

[0003]

While investigating the growth mechanism of cancer cells, the present inventors came to discover that it is the protein-protein interaction that determines the important metabolic pathway. It was Generally, it is considered that the complex structure of the protein itself is involved in the protein-protein binding, but from the results of detailed studies of the binding domain between the protease and its inhibitor, the protein binding was confirmed. It was found that a small peptide chain of a small part of a large molecular weight protein is involved in the. From the above, when a peptide corresponding to the binding domain of a protein involved in the growth of cancer cells is allowed to coexist in the cell, the binding between the protein and other proteins can be blocked, and as a result, the function of the protein Therefore, such a short peptide was considered to be useful as a carcinostatic agent. An object of the present invention is to provide a short peptide having an anti-cancer effect, and more specifically, the object of the present invention is to provide a pharmaceutical composition containing a tripeptide, a dipeptide or a derivative thereof having a specific amino acid sequence. Another object of the present invention is to provide a pharmaceutical composition containing a physiologically acceptable salt of a tripeptide, a dipeptide or a derivative thereof having a specific amino acid sequence.

[0004]

The present inventors have proposed various oncogene products such as K-Sam, Yes, Ret, Kit, Fms, ErbB, Met, Ro as amino acid sequences of promising tripeptides.
s, Sea, Trk, Src, Fgr, Fyn, Lyn, Lck, Hck, Abl, Ar
We searched for consensus sequences of oncogene products that have a sequence called g, etc., or sark homology (SH) domain, and predicted sequences involved in protein binding through studies of tripeptides corresponding to binding domains. As a result, they have found that tripeptides having a certain amino acid sequence have the effect of inhibiting the growth of cancer cells, and have completed the present invention based on this finding.

That is, the present invention is Pro-AB (where A = Phe, Ly
s, Asn, Tyr, Thr; B = Pro, Trp. ), Or a pharmaceutical composition containing a physiologically recognized salt thereof as an active ingredient, a tripeptide represented by Thioproline-Thr-Trp (hereinafter, thioPro or P ′),
Or a pharmaceutical composition containing a physiologically recognized salt thereof as an active ingredient, a dipeptide represented by Glu-Arg, or a pharmaceutical composition containing a physiologically recognized salt thereof as an active ingredient, CD- Pro (however C = Tyr, Glu, Pro; D = A
sn), Ser, Arg, Tyr), or a physiologically acceptable salt thereof, as an active ingredient.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION Examples of the amino acid sequence of the tripeptide according to the present invention include Pro-Phe-Pro, Pro-Lys-Pro,
Pro-Tyr-Pro, Tyr-Ser-Pro, Glu-Arg-Pro, Pro-Tyr-Trp
And so on. The amino acid sequence of the dipeptide of the present invention is, for example, Glu-Arg. In the present invention, the N-terminal amino acid acylated product of the above tripeptide and dipeptide, the C-terminal amino acid amidated product, and both ends thereof modified as described above are also used. N- of tripeptide and dipeptide of the present invention
The acylated derivative is a formyl group, an acetyl group, an arylcarbonyl group, an aromatic carbonyl group derivative,
Examples of the C-amidated derivative include an amino group, an alkylamino group and an aromatic amino group derivative. Physiologically recognized salts of the tripeptides, dipeptides and derivatives thereof of the present invention include hydrochloric acid, citric acid, phosphoric acid, tartaric acid, lactic acid, acetic acid, formic acid, fumaric acid, maleic acid, succinic acid and the like.

The synthesis of tripeptides, dipeptides and their derivatives according to the present invention can be carried out by either solid phase method or liquid phase method as described in, for example, 2nd Biochemistry Laboratory, edited by The Biochemical Society of Japan. It can also be easily synthesized by the method. Purification of the tripeptide, dipeptide and derivatives thereof according to the present invention can be carried out by a usual method such as silica gel column chromatography, ion exchange column chromatography and the like. The eluate is not limited, and a common organic solvent such as water, methanol or chloroform can be used. To describe the synthesis of the tripeptide, dipeptide and derivatives thereof according to the present invention in more detail, first, a commercially available amino acid having a carboxyl group protected with a benzyl group (Bzl group) and 1-ethyl-3- (3-dimethylaminopropyl) are used. ) -Carbodiimide hydrochloride (WSCD HCl) or dicyclohexylcarbodiimide (DCCD) is used for condensation to obtain the dipeptide. The tripeptide of the present invention, as a solvent used in the condensation reaction of the dipeptide, N, N-dimethylformamide (DMF), dimethylsulfoxide (DMSO), acetonitrile, 1,4-dioxane, tetrahydrofuran (THF) and mixtures thereof. It is suitable. The tripeptide and dipeptide protecting groups can be removed by the trifluoromethanesulfonic acid method (TMSFA method), the hydrogen catalytic reduction method using palladium / carbon as a catalyst, or liquefied hydrogen fluoride (HF). As the solvent used in the hydrogen catalytic reduction method, there is no problem because it is a commonly used solvent. Methanol (hereinafter sometimes abbreviated as MeOH), acetic acid or a mixture thereof is suitable. To check the reaction progress and purity in each synthesis step, thin layer chromatography (TL
The general method of C) can be used. The developing solvent is chloroform-methanol system, n-butanol-acetic acid-
A pyridine-water (4: 1: 1: 2) system can be used, and the spots can be confirmed by the hydrogen bromide (HBr) -ninhydrin method.

The pharmaceutical composition of the present invention is not particularly limited except that it contains the tripeptides, dipeptides and derivatives thereof, or their physiologically recognized salts obtained as described above as active ingredients, Additives that are commonly used in formulating compounds may be included. The pharmaceutical composition of the present invention can be used as a cancer drug having excellent activity. The applicable cancer types are leukemia, osteosarcoma, breast cancer, ovarian cancer, gastric cancer, colon cancer, lung cancer and the like. The method of administration of the pharmaceutical composition of the present invention naturally varies depending on the symptoms of the subject to be administered, but 0.01 to 10 g per day for an adult is administered once or in several divided doses. The side effect of the pharmaceutical composition of the present invention does not cause any problem when it is used normally.

The determination of the carcinostatic effect is carried out by adding the tripeptide of the present invention and its derivative or a physiologically acceptable salt thereof to the culture medium of a cultured cell line which has been transformed into cancer by various oncogenes, and culturing under ordinary conditions. By culturing, changes in cell morphology, growth rate, survival rate, etc. can be observed. For example, it is convenient to use the 3- [4,5-dimethylthiazol-2-yl] -2,5 diphenyltetrazolium bromine salt method (MTT method) for measuring the effect of suppressing the growth rate of cancer cells. Examples of cancer cell lines include v-Ha-ras, Ki-ras, v-src, c-erbB-2, v-mos,
NIH3T3 fibroblasts and 3Y1 transformed by oncogenes such as SV40
An example is a cultured cell line such as rat fibroblasts. The present invention will be specifically described below with reference to examples.
The present invention is not limited to this.

[0010]

【Example】

Example 1 [Synthesis of Pro-Phe-Pro (PFP)] Boc-Phe 2.7 g (10 mmol), ProOBzl HCl 2.4 g (10 mmol), 1-hydroxybenzotriazole (HOBt) 1.5
g (11 mmol) was dissolved in 30 ml of DMF, and DMF was stirred under ice cooling.
1.7 g (11 mmol) of WSCD HCl suspended in 10 ml was added dropwise. 1.8 ml of triethylamine (TEA) was added, and after confirming that the pH was 7 to 8 with a universal pH test paper, the mixture was stirred overnight at room temperature. The deposited precipitate was removed by filtration, the filtrate was concentrated under reduced pressure, ethyl acetate (EtOAc) (300 ml) was added, and 4% sodium hydrogen carbonate (NaHCO ₃ ) in 10% sodium chloride (NaCl) aqueous solution, 10% NaCl aqueous solution, 0.4M. The citric acid was washed with a 10% NaCl aqueous solution and a 10% NaCl aqueous solution in this order. Anhydrous sodium sulfate (Na ₂ SO ₄ ) was added to the EtOAc layer for dehydration, the solvent was evaporated under reduced pressure, and the residue was dried under reduced pressure in a desiccator. 4N hydrochloric acid / dioxane 4
0 ml and thioanisole 4 ml were added, and the mixture was reacted under ice cooling for 2 hours. After this was concentrated under reduced pressure, 50 ml of dioxane was added,
Repeat the operation of concentrating twice.
The operation of adding (Et ₂ O) and concentrating was repeated twice. Z-Pro 1.5 g (6.2 mmol), HOB
0.84 g (6.8 mmol) of t was added and dissolved in 30 ml of DMF.
Under ice-cooling stirring, 1.2 g (6.8 mmol) of WSCD HCl suspended in 10 ml of DMF was added dropwise. After adjusting the pH to 7 to 8 with 1.6 ml of TEA, the mixture was reacted at room temperature for 1.5 hours. After reacting at 4 ° C. overnight, the deposited precipitate was removed by filtration, and the filtrate was concentrated under reduced pressure. Add 300 ml of EtOAc, add 4% NaHCO ₃ in 10% NaCl,
10% NaCl aqueous solution, 0.4 M citric acid 10% NaCl aqueous solution, 10% NaC
l Washed in order of aqueous solution. Anhydrous Na ₂ SO ₄ was added to the EtOAc layer for dehydration, the solvent was evaporated under reduced pressure, and the residue was dried under reduced pressure in a desiccator. Dissolve the dried product in a small amount of methanol (MeOH), and use Sephadex LH-20 column (internal diameter 3 cm x length 55 cm).
Separated and purified in. It was eluted with the same solvent and fractionated for 5 minutes (about 8 ml) per fraction. (The Kieselgel 60F ₂₅₄ developing solvent, using a system of chloroform -MeOH.) TLC for each fraction after checking in, and concentrated to dryness under reduced pressure The fractions containing the desired product. This is MeOH 20 ml, acetic acid (AcOH) 20
It was dissolved in a mixed solution of ml and stirred under a nitrogen stream. Stop stirring, 10% palladium / carbon (Pd / C) (51.60% water content)
After adding 2.0 g and passing a hydrogen stream, the reaction was started by vigorously stirring at room temperature and atmospheric pressure. When the absorption of hydrogen was completed, the stirring was stopped, the hydrogen gas was replaced with nitrogen gas, and the catalyst was filtered off. The filtrate was concentrated under reduced pressure, water was added, and the filtrate was concentrated again under reduced pressure. The concentrate was separated and purified on a Sephadex G-10 column (inner diameter 2.6 cm x length 60 cm). It was eluted with water and fractionated for 5 minutes (about 10 ml) per fraction. The purity of each fraction was measured by TLC (developing solvent: n-butanol: acetic acid: pyridine: water = 4: 1: 1: 2 (BAPW
System) was used. Was confirmed (Rf value 0.39), and fractions giving a single spot of the target substance were collected and dried under reduced pressure. The dried solid was dissolved in 15 ml of water and freeze-dried to obtain 1.0 g of a colorless amorphous target product. The total yield so far was 28%. Elemental analysis C ₁₉ H ₂₅ N ₃ O ₄ MW.359.43 Calculated value C 63.
49, H 7.01, N 11.69. Analytical value C 59.41, H 7.42, N 10.
66.

Example 2 [Synthesis of N-acetylated Pro-Phe-Pro (Ac-PFP)] 1.2 g (3.3 mmol) of Pro-Phe-Pro obtained in Example 1
Is dissolved in 10 ml of water, and N-acetylsuccinimide is added to this.
(N-ASI) (1.5 g, 10 mmol) was added, and the pH was adjusted to about 7 with a 1N aqueous potassium hydroxide solution, and the mixture was stirred at room temperature overnight. After concentrating the reaction solution, Sephadex G-10 column (inner diameter 2.6 cm ×
It was separated and purified with a length of 60 cm). Each fraction is confirmed by TLC, and when concentrated to dryness, it gives a single spot by TLC (developing solvent BAPW system Rf = 0.56).
1.2 g (yield 90%) was obtained. Elemental analysis C ₂₁ H ₂₇ N ₃ O ₅ M
W.401.46 Calculated C 62.83, H 6.78, N 10.47. Analytical value C
59.69, H 6.50, N 10.97.

Example 3 [C-amidated form of Pro-Phe-Pro]
Synthesis of (PFP NH ₂ )] Boc-Phe 2.3 g (8.8 mmol), Pro NH ₂ 1.0 g (8.8 mmol), HOBt 1.33 g (9.8 mmol) were dissolved in DMF 30 ml, and DMF 10 ml was stirred under ice cooling. Suspended WSCD HCl (MW115.
24 36.46) 1.7 g (8.8 mmol) was added dropwise. TEA 1.7ml
In addition, after confirming that the pH was 7 to 8 using a universal pH test paper, the reaction was carried out at 4 ° C. overnight. After the reaction, the same treatment as in Example 1 was carried out to obtain Boc-Phe-Pro NH ₂ as a colorless oil. 4N hydrochloric acid / dioxane 16ml, thioanisole 1.5ml
Was added, and the mixture was stirred under ice cooling for 1 hour. The same treatment as in Example 1 was performed, and the obtained residue was added with Z-Pro 2.0 g (7.9 mmol) and HOBt.
1.1 g (8.8 mmol) was added and dissolved in 20 ml DMF. Under ice-cooling stirring, 1.7 g (8.7 mmol) of WSCD HCl suspended in 10 ml of DMF was added dropwise. After adjusting the pH to 7-8 with 1.5 ml TEA,
The reaction was carried out at 0 ° C overnight. After the reaction, the same treatment as in Example 1 was performed to obtain an oily product of Z-Pro-Phe-Pro NH ₂ . Purification was carried out on a Sephadex LH-20 column in the same manner as in Example 1 to obtain an almost single spot by TLC, followed by deprotection reaction by a hydrogen catalytic reduction method, followed by purification on a Sephadex G-10 column to obtain E
Crystallized from t ₂ O, 0.2g colorless crystals (BAPW Rf = 0.48)
I got The total yield up to this point was 39%. Elemental analysis
C ₁₉ H ₂₆ N ₄ O ₃ MW.358.44 Calculated value C 60.37, H 7.31, N 15.
63. Analytical value C 60.48, H 7.38, N 14.55.

Example 4 [Synthesis of N-acetylated Pro-Phe-Pro, C-amidated product (Ac-PFP NH ₂ )] 1.0 g (2.8 mmol) of PFP NH ₂ obtained in Example 3 was used. 6 ml of water was added and dissolved, and by reacting and purifying in the same manner as in Example 2, 0.9 g of the target product (yield 80%) was obtained. This gave a single spot by TLC (developing solvent BAPW system Rf = 0.62). Elemental analysis C ₂₁ H ₂₈ N ₄ O ₄ MW.400.25 Calculated value C 62.98, H 7.05, N
13.99. Found C 58.91, H 6.68, N 13.01.

Example 5 [Synthesis of Pro-Lys-Pro (PKP)] 3.5 g (7.1 mmol) of Boc-Lys (Cl-Z) t-Bu NH ₂ in 400 ml.
Dissolved in EtOAc, 0.4 M citric acid in 10% NaCl, 10% N
It was washed in order of aCl aqueous solution. Anhydrous Na ₂ SO ₄ was added to the EtOAc layer for dehydration, and then the solvent was distilled off under reduced pressure to obtain amorphous Boc-L.
ys (Cl-Z) was obtained. ProOBzl HCl 1.7 g (7.1 mmol), HOBt 1.0 g (7.7 mmol) and WSCD HCl 1.5 g (7.8 mmol).
Mmol) and reacted and purified in the same manner as in Example 1 to obtain Boc-
Lys (Cl-Z) -ProOBzl was obtained. The obtained dried product was used in Example 1.
The Boc group was removed by acid treatment in the same manner as described above, and ZP was added to the resulting residue.
ro 1.7g (6.8 mmol), HOBt 1.0g (7.5 mmol),
WSCD HCl (1.5 g, 7.8 mmol) was added and the reaction and purification were carried out in the same manner as in Example 1 to obtain an oily Z-Pro-Lyz (Cl-Bzl) -ProOBzl. In addition, this is Sephadex LH-20 column (inner diameter 2.6 cm
X 60 cm) and purified by TLC to give a fraction that gives a nearly single spot and dried in vacuo. It was crystallized by adding Et ₂ O dissolved in a small amount of EtOAc and cooling. The crystals were collected by filtration and dried in a vacuum desiccator under reduced pressure to give colorless crystals (2.68 g). This crystal was mixed with AcOH 36ml, MeO
After dissolving in 24 ml of H 2, all protecting groups were removed by the hydrogen catalytic reduction method as in Example 1. Separation and purification were performed using a Sephadex G-10 column (inner diameter 2.6 cm x length 60 cm). Each fraction was confirmed by TLC (developing solvent BAPW system Rf = 0.009), the target fractions were collected, concentrated, and further lyophilized to obtain 1.4 g of a colorless amorphous target product. The total yield up to this point was 63%. Elemental analysis C ₁₆ H ₂₈ N ₄ O ₄ MW.340.42 Calculated C 56.45, H 8.29, N 16.34. Analytical value C 54.06, H
8.59, N15.27.

Example 6 [N-acetylation of Pro-Lys-Pro
Synthesis of Body (Ac-PKP)] Amorphous substance of Pro-Lys-Pro obtained in Example 5 1.
Dissolve 2 g (3.8 mmol) in 10 ml of water and adjust the solution to pH 7.
After that, 1.6 g (11.3 mmol) of N-ASI was added, and the mixture was stirred at room temperature overnight.
Stirred. After the reaction mixture was concentrated to dryness, the residue was
Holm (CHCl _Three) Dissolved in CHCl_Three: MeOH = 10: 1 solution
Made silica gel C-200 column (inner diameter 2.2 cm x length 33 cm)
After layering on CHCl_Three: MeOH = 10: 1 in 300 ml of solution, and MeOH
Elute with only 10 ml and fractionate into approximately 10 ml fractions.
Check the traction with TLC and give the spot of the target.
Eluate fractions 102 to 115 are concentrated and concentrated to dryness.
Was. After dissolving this in a small amount of water and lyophilizing it, TLC
Colorless to give a single spot (developing solvent BAPW system Rf = 0.40)
Amorphous substance 1.25 g (76%) was obtained. Nuclear magnetic
The position of the signal was satisfied by the sound spectrum (NMR). 1H NM
R (DMSO-d₆, δ) 1.82 (3H, s) 2.15 (3H, s).

Example 7 [Pro-Lys-Pro NH ₂ (PKP NH ₂ )
Synthesis of Boc-Lys (Z) 3.5 g (9.2 mmol), Pro NH ₂ 1.1 g (9.2
Mmol), HOBt 1.34 g (9.9 mmol), WSCD HCl 1.
95 g (10 mmol) was reacted and purified in the same manner as in Example 1 to obtain the desired product Boc-Lys (Z) -Pro NH ₂ as a colorless oil. Z-Pro 1. was added to the residue obtained by treating in the same manner as in Example 1.
5g (6.1 mmol), HOBt 0.85g (6.3 mmol), WSCD
HCl (1.3 g, 6.8 mmol) was added, and the reaction was carried out in the same manner as in Example 1 to obtain Z-Pro-Lys-Pro NH ₂ as an oily substance, which was treated in the same manner as in Example 1 with Sephadex LH. -20
Purified by column to give an almost single spot by TLC. After deprotection by hydrogen catalytic reduction method using 10% Pd / C, it was dissolved in a small amount of water, purified by Sephadex G-10 column and lyophilized to give a single spot (developing solvent BAPW system Rf = 0.17) was obtained. The total yield up to this point was 35%. C ₁₆ H ₂₉ N ₅ O
₃ MW 339.44 Calculated C 56.62, H 8.61, N 20.63. Found C 51.56, H 8.83, N 18.63.

Example 8 [Synthesis of Pro-Tyr-Pro (PYP)] Boc-Tyr (Bzl) 2.0 g (5.3 mmol), ProOBzl HCl 1.3 g
(5.38 mmol), HOBt 0.81 g (6.0 mmol), WSCD
1.14 g (5.9 mmol) of HCl was reacted in the same manner as in Example 1 to obtain the desired product Boc-Tyr (Bzl) -ProOBzl. The dried product was treated with acid in the same manner as in Example 1 to remove the Boc group, and the resulting residue was Z-Pro 1.3 g (5.1 mmol) and HOBt 0.76 g (5.
6 mmol) and 1.1 g (5.6 mmol) of WSCD HCl were added dropwise and reacted in the same manner as in Example 1, then Sephadex L
Separation and purification was carried out using an H-20 column (inner diameter 2.6 cm x length 60 cm). After concentration under reduced pressure, this was dissolved in a small amount of EtOAc, added with Et ₂ O, and cooled to crystallize. The crystals were collected by filtration and dried under reduced pressure to give 2.68 g. The crystals were deprotected by the hydrogen catalytic reduction method and then purified on a Sephadex G-10 column. After concentration, the residue was dissolved in a small amount of MeOH, Et ₂ O was added to crystallize it, and after cooling, the crystal was collected by filtration and dried to obtain 1.16 g. The total yield up to this point was 55%. This gave a single spot by TLC (developing solvent BAPW system Rf = 0.32). Elemental analysis C ₁₉ H ₂₅ N ₃ O ₅ MW375.43 Calculated value C 60.7
9, H 6.71, N 11.19. Analytical value C57.54, H 6.94, N 10.1
9.

Example 9 [N-acetylation of Pro-Tyr-Pro
Synthesis of Form (Ac-PYP)] 1 g (2.6 mmol) of Pro-Tyr-Pro obtained in Example 8 had N
-ASI 1.1 g (8 mmol) was added and the reaction was carried out as in Example 2.
And extract the reaction solution with 20 ml of chloroform 5 times.
Repeated. Chloroform layer with Na₂SO_FourAfter dehydration at reduced pressure
did. Dissolve this in a small amount of water, lyophilize and use TLC.
Colorless that gives a single spot (developing solvent BAPW system Rf = 0.51)
0.68 g (yield 61%) of an amorphous substance was obtained. Elemental analysis
C_{twenty one}H ₂₇N_ThreeO₆ MW417.46 Calculated value C 60.42, H 6.52, N 10.0
7. Analytical value C 54.52, H 6.58, N 11.04.

Example 10 [Pro-Tyr-Pro NH ₂ (PYP N
Synthesis of H ₂ ) Boc-Tyr (Bzl) 3.25 g (8.8 mmol), Pro NH ₂ 1.0 g
(8.8 mmol), HOBt 1.2 g (8.9 mmol) to WSCD HCl
1.7 g (9.0 mmol) was added and the reaction and purification were carried out in the same manner as in Example 1 to obtain Boc-Tyr (Bzl) -Pro NH ₂ as a colorless oil. The dried product thus obtained was treated with acid in the same manner as in Example 1 to produce Boc.
The group was removed, the solvent was concentrated under reduced pressure and Et ₂ O was added to precipitate crystals. The crystals were collected by filtration and dried under reduced pressure in a vacuum desiccator. Z-Pro 2.0 g (7.9 mmol) and HOBt 1.2 g (8.9 mmol) were added to the dried crystals, and WSCD HCl 1.7 was added.
g (9.0 mmol) was added dropwise and the reaction and purification were carried out in the same manner as in Example 1 to obtain an oily substance. Dissolve this in a small amount of methanol and use Sephadex LH-20 column (inner diameter 2.6 cm x length 60 c
m) and eluted with the same solvent. T the desired fraction
It was searched for by LC, collected into one, and dried under reduced pressure to give 4.9 g of the amorphous target product. After deprotection by hydrogen catalytic reduction method, it was purified by Sephadex G-10 column and freeze-dried to give a single spot by TLC (developing solvent BAPW system Rf = 0.42).
2.9 g was obtained. The total yield up to this point was 87%. Elemental analysis C ₁₉ H ₂₆ N ₄ O ₄ MW 377.44 Calculated value C 60.95, H 7.60, N
14.96. Analytical values C 56.07, H 7.19, N 13.84.

Example 11 [Synthesis of N-acetylated Pro-Tyr-Pro, C-amidated product (Ac-PYP NH ₂ )] To 1.0 g (2.6 mmol) of PYP NH ₂ obtained in Example 10 N
-ASI 1.0 g (7 mmol) was added, reacted and purified in the same manner as in Example 2, and almost single spot by TLC (developing solvent BAPW).
The fractions giving the system Rf = 0.61) were collected, concentrated and freeze-dried to obtain 1.05 g of the colorless amorphous target product. The yield up to this point was 96%. Elemental analysis C ₂₁ H ₂₈ N ₄
O ₅ MW 416.48 Calculated C 60.56, H 6.78, N 13.45. Analytical value C 55.11, H 6.01, N 13.09

Example 12 Synthesis of Tyr-Ser-Pro (YSP) Boc-Ser (Bzl) 2.0 g (6.8 mmol), ProOBzl HCl 1.6 g
(6.8 mmol), HOBt 1.0 g (7.5 mmol) DMF 20 m
W, suspended in 10 ml of DMF while stirring under ice-cooling.
Boc-Ser (Bzl) -ProOBzl was obtained by adding 1.4 g (7.3 mmol) of SCD HCl and reacting in the same manner as in Example 1. As in the method described in Example 1, the removal of Bo by acid treatment was performed.
c-group reaction was performed, and 2.6 g (6.3%) of Z-Tyr (Bzl) was added to the resulting residue.
, HOBt 0.85g (6.3 mmol), and added WSCD
HCl (1.3 g, 6.8 mmol) was added dropwise, and the reaction was carried out in the same manner as in Example 1. The obtained residue was dissolved in a small amount of MeOH and purified by a Sephadex LH-20 column. The target fraction was searched for by TLC, collected and dried under reduced pressure. This is EtOAc 50
After dissolving in ml, 250 ml of Et ₂ O was added and left in the refrigerator to crystallize. The crystals were collected by filtration and dried under reduced pressure (2.
40g). This crystal was subjected to a deprotection reaction by a hydrogen catalytic reduction method. Purified by Sephadex G-10 column and lyophilized, single spot by TLC (developing solvent BAPW system Rf = 0.
As a result, 1.70 g of a colorless amorphous target product which gives 30) was obtained. The total yield up to this point was 68%. Elemental analysis C
₁₇ H ₂₃ N ₃ O ₆ MW 365.38 Calculated C 56.03, H 6.63, N 15.3
7. Found C 55.91, H 6.81, N 13.82.

Example 13 [Synthesis of N-acetylated form of Tyr-Ser-Pro (Ac-YSP)] 1.0 g of YSP (2.7 mmol of YSP) obtained in Example 12 was replaced with N.
-1.1 g (8 mmol) of ASI was added and reacted at room temperature for 2 hours
(pH about 8). After concentrating the reaction solution, it was purified with Sephadex G-10 column and single spot was analyzed by TLC (developing solvent BAPW system R
0.6 g of the target product giving f = 0.44) was obtained. The total yield so far was 54%. Elemental analysis C ₁₉ H ₂₅ N ₃ O ₇ MW 407.
41 Calculated C 56.01, H 6.18, N 10.31. Analytical value C 54.9
5, H 5.37, N 10.11.

Example 14 [Tyr-Ser-Pro NH₂(YSP N
H₂)) Boc-Ser (Bzl) 2.6 g (8.8 mmol), Pro NH₂ 1.0g
(8.8 mmol), HOBt1.30 g (9.7 mmol) WSCD H
Cl 2.0 g (10 mmol) was added and the reaction was carried out as in Example 1.
Let's Boc-Ser (Bzl) -Pro NH₂Was obtained as an oil. this
The oil was treated with acid and dissolved in the same manner as in Example 1.
After concentrating the medium, Et₂Add O and settle by decantation
A precipitate was obtained. This was dried under reduced pressure, and Z-Tyr (Bzl) 3.2 g (7.9
Limol), 1.2 g (8.7 mmol) of HOBt, and suspend in DMF.
1.7 g (8.8 mmol) of cloudy WSCD HCl was added dropwise. solvent
Was concentrated, dissolved in a small amount of EtOAc, and then Et.₂Add O and cool
It crystallized. The crystals were collected by filtration and dried under reduced pressure,
3.25 g was obtained. Deprotection of this crystal by hydrogen catalytic reduction method
React, purify on Sephadex G-10 column and purify by TLC
Gives almost a single spot (developing solvent BAPW system Rf = 0.44)
The target product fraction is collected, concentrated, and freeze-dried.
Then, 1.7 g of a colorless amorphous substance was obtained. So far
The total yield of was 53%. Elemental analysis C₁₇H_{twenty four}N_FourO _Five MW 36
4.39 Calculated C 56.03, H 6.63, N 15.37. Analytical value C 5
3.91, H 6.71, N 13.82.

Example 15 [Synthesis of N-acetylated Tyr-Ser-Pro, C-amidated product (Ac-YSP NH ₂ )] 0.8 g (2.2 mmol) of YSP NH ₂ obtained in Example 14
1.0 g (7 mmol) of N-ASI was added to and treated and reacted in the same manner as in Example 2 to obtain a dried product. However, since it was estimated that the condensing agent-derived substance was mixed in this, it was dissolved in a small amount of water, and extraction was repeated 5 times with 10 ml of EtOAc, and the aqueous layer was freeze-dried.
%)was gotten. This gave a single spot by TLC (developing solvent BAPW system Rf = 0.56).

Example 16 [Synthesis of Glu-Arg-Pro (ERP)] 3.7 g (10 mmol) of Boc-Arg (NO ₂ ) 1 / 2AcOEt 1 / 4H ₂ O, P
2.1 g (11.0 mmol) of WSCD HCl was added to 2.4 g (10 mmol) of roOBzl HCl and 1.5 g (11 mmol) of HOBt, and the reaction was carried out in the same manner as in Example 1 to treat Boc-Arg (NO ₂ ) -ProOBzl. Obtained as an oil. The obtained oily substance was treated with an acid as in Example 1. 1.7 g (5.0 mmol) of Z-Glu (Bzl), 0.75 g (5.7 mmol) of HOBt, and 1.1 g of WSCD HCl were added to the obtained oil.
(5.5 mmol) was added dropwise and the reaction was carried out in the same manner as in Example 1. The obtained residue was dissolved in a small amount of EtOAc, Et ₂ O was added, and the mixture was crystallized when cooled. The crystals were collected by filtration and dried under reduced pressure to give 2.3 g of crystals. This crystal is subjected to a reaction to remove all protectors by a hydrogen catalytic reduction method, and the obtained residue is purified by a Sephadex G-10 column to give a single spot by TLC (developing solvent BAPW system Rf = 0.007). Fractions are collected, concentrated, freeze-dried, and colorless amorphous target product
Obtained 1.3 g. The total yield up to this point was 32%. Elemental analysis C ₁₆ H ₂₈ N ₆ O ₆ MW 400.44 Calculated value C 47.99, H 7.05,
N 20.99. Analytical values C 45.84, H 7.55, N 19.52.

Example 17 [Synthesis of N-acetylated Glu-Arg-Pro (Ac-ERP)] 1.1 g (2.7 mmol) of ERP obtained in Example 16 was replaced with N-
1.1 g (7.1 mmol) of ASI was added, and the mixture was stirred overnight at room temperature. After the reaction solution was concentrated, it was purified by a Sephadex G-10 column, and each fraction was collected, concentrated and freeze-dried to obtain 0.80 g of the colorless amorphous target product. The total yield up to this point was 67%. This gave a single spot by TLC (developing solvent BAPW system Rf = 0.16). Elemental analysis C ₁₈
H ₃₀ N ₆ O ₇ MW442.47 Calculated C48.86, H6.83, N18.99. Analytical values C45.89, H7.03, N18.07.

Example 18 [Synthesis of C-amidated product (ERP NH ₂ ) of Glu-Arg-Pro] Boc-Arg (NO ₂ ) 1 / 2AcOEt 1 / 4H ₂ O 3.7 g (10.0 mmol), ProOBzl HCl 2.4 g (10 mmol), HOBt 1.49 g
2.1 g (11 mmol) of WSCD HCl was added to (11 mmol), the reaction was carried out in the same manner as in Example 1, and chloroform was used instead of EtOAc. To the residue obtained by concentrating the chloroform layer under reduced pressure, EtOAc was added, resulting in white turbidity. Et ₂ O was further added and the mixture was allowed to stand overnight at 4 ° C. to obtain a gummy precipitate, which was decanted and dried under reduced pressure. The obtained dried product was treated with an acid in the same manner as in Example 1 to remove the Boc group, and a precipitate formed during the reaction was obtained by decantation and dried under reduced pressure. Z-Glu (Bzl) 1.7 g (5.0 mmol), HOBt 0.75 g (5.5
(1.0 mmol) and 1.1 g (5.7 mmol) of WSCD HCl were added dropwise, the reaction was carried out in the same manner as in Example 1, and after treatment, the product was purified by a column to obtain a colorless oil. This oily substance was subjected to a hydrogen catalytic reduction method to remove all protecting groups, and then Sephadex
After purification by G-10 column, a fraction of almost a single spot (developing solvent BAPW system Rf = 0.21) was collected by TLC to obtain a crystalline residue. To this was added cold MeOH to loosen the crystals and collect by filtration to obtain 0.2 g. The total yield up to this point was 6%. Elemental analysis C ₁₆ H ₂₉ N ₇ O ₅ MW399.45 Calculated value C 48.11, H
7.32, N 24.55. Analytical values C 46.61, H6.92, N22.34.

Example 19 [Synthesis of Pro-Thr-Trp (PTW)] Boc-ThrOSu 3.7 g (12 mmol), TrpOBzl HCl 3.8 g (1
TEA was added to a DMF solution of
After confirming that it was H7-8, the reaction was carried out overnight. Example 1
Post-treatment was carried out in the same manner as above to obtain Boc-Thr-TrpOBzl as an oil. Subsequently, acid treatment was carried out in the same manner as in Example 1 to remove the Boc group, and 2.8 g (11 mmol) of Z-Pro was added to the resulting residue,
DCCD 2.5g (12mmol) in DMF 10ml solution was added dropwise at 4 ℃.
And reacted overnight at. After filtering off the precipitate, the filtrate was concentrated under reduced pressure and treated in the same manner as in Example 1 to give 4.21 g of an amorphous substance.
was gotten. The protective group was removed from 2 g of this by hydrogen catalytic reduction method, the resulting residue was dissolved in a small amount of water and purified by Sephadex G-10 column, and almost a single spot by TLC (developing solvent BAPW system Rf = 0.33) was collected and dried under reduced pressure. A small amount of MeOH was added to the residue for crystallization, and after cooling, the crystals were collected by filtration and dried to obtain 1.40 g. The total yield up to this point was 30%. Elemental analysis C ₂₀ H ₂₅ N ₄ O ₅ MW
402.22 Calculated C 59.69, H 6.51, N 13.91. Analytical value C 5
5.90, H 6.41, N 12.75.

Example 20 [Pro-Thr-Trp NH ₂ (PTW N
Synthesis of H ₂ )] 6.0 g (19 mmol) of Boc-ThrOSu was dissolved in 50 ml of DMF.
rp NH ₂ hydrochloride 4.6 g (19 mmol) and HOBt 2.9 g (21 mmol) were added as powders and dissolved with stirring. Under ice cooling
After adding TEA and confirming that the pH is 7-8, WSCD HCl 4.1
A solution of g (21 mmol) in DMF (10 ml) was added, and the mixture was reacted at 4 ° C. overnight. The precipitate was filtered off, the filtrate was concentrated under reduced pressure and then EtOAc 500
ml was added and treated in the same manner as in Example 1 to obtain 8.3 g (87%) of the desired product Boc-Thr-Trp NH ₂ as a colorless oil. This was reacted and treated in the same manner as in Example 1 to give an oily substance.
7.2 g (16 mmol, 98%) were obtained. Next to this Z-Pro
4.1 g (16 mmol), HOBt 2.5 g (18 mmol), WSCD H
3.5 g (18 mmol) of Cl was added and reacted and treated in the same manner as in Example 1 to obtain 7.3 g (70%) of Z-Pro-Thr-Trp NH ₂ as an amorphous substance. 2.5g of this amorphous material
After reacting and treating in the same manner as in Example 1 by adding 1 g of 10% Pd / C,
The deprotection reaction was performed. The obtained product was dissolved in a small amount of water, purified on a Sephadex G-10 column, and freeze-dried to obtain 1.0 g of the target product consisting of a single spot (developing solvent BAPW system Rf = 0.47). Elemental analysis C ₂₀ H ₂₇ N ₅ O ₄ MW.401.
33 Calculated C 59.80, H 6.78, N 17.46. Analytical value C 57.6
3, H 6.94, N 16.27.

Example 21 [Pro-Thr-Trp NH₂N-asset
Chilled product (Ac-PTW NH₂Synthesis of)] PTW NH obtained in Example 20₂ To 0.95 g (2.4 mmol)
N-ASI 0.9g (6mmol) was added and reacted overnight at room temperature.
After that, it was concentrated to dryness under reduced pressure. In a little chloroform
Dissolve and mix with chloroform: methanol = 30: 1
Silica gel C-200 (manufactured by Wako Pure Chemical Industries) column (inner diameter
2.6 cm x 45 cm in length), chloroform: methanol
= 30: 1 mixed solution 400 ml, 10: 1 mixed solution 300 ml, methano
Eluate only 500 ml and each fraction (about 10 ml)
After checking with TLC, collect fractions 72 to 75
Was. After concentration to dryness, the residue is dissolved in a small amount of water and freeze-dried.
And 0.8 g of a colorless amorphous substance was obtained. Up to here
The total yield was 75%. This is a single spot on TLC
(Developing solvent BAPW system Rf = 0.64). Elemental analysis C _{twenty two}H
₂₉N_FiveO_Three MW443.50 Calculated C 59.58, H 6.59, N 15.79.
Analytical values C 56.98, H 6.54, N 15.34.3 g were obtained.

Example 22 [thioPro-Thr-Trp (P'T
Synthesis of W)] Boc-ThrOSu 3.7 g (12 mmol), TrpOBzl HCl 3.8 g (1
TEA was added to a DMF solution of
After confirming that it was H7-8, it was reacted overnight. Post-treatment was carried out in the same manner as in Example 1 to obtain Boc-Thr-TrpOBzl as an oil. Subsequently, the Boc group was removed by acid treatment in the same manner as in Example 1, and 2.8 g (12 mmol) of Boc-thioPro was added to the obtained residue to give 1.784 g of HOBt (13.2 mmol) and 2.53 g of WSCD HCl.
A solution of (13.2 mmol) in DMF (10 ml) was added dropwise, and the mixture was reacted overnight at 4 ° C. After filtering off the precipitate, the filtrate was concentrated under reduced pressure and treated in the same manner as in Example 1 to obtain 6.3 g of an amorphous substance. Boc-thioPro-Thr-TrpO obtained by coupling
Cleavage of the protecting groups with HF from Bzl 6.3 g was performed as follows. 6.3 g of Boc-thioPro-Thr-TrpOBzl was divided into 10 equal parts, and each was cut with HF. That is, Boc-thioPro-Thr-TrpOBz
l was put in a reaction vessel, 11 ml of thioanisole was added, and the mixture was left at room temperature for 1 hour. While cooling the reaction vessel in a dry ice-acetone bath, 100 ml of HF was introduced, and the reaction was carried out at 0 ° C. with stirring for 1 hour. HF was removed under reduced pressure with a water stream aspirator, and further dried with a vacuum pump. The obtained residue was dissolved in 100 ml of 10% acetic acid aqueous solution, and 2 ml was added with 50 ml of diethyl ether.
It was washed twice to remove the remaining thioanisole. The acetic acid aqueous solution fraction was neutralized and concentrated to dryness, 3.2 g of which was dissolved in a small amount of a chloroform-methanol mixed solution (5: 1) and purified by silica gel column chromatography to obtain the desired product. Yield 0.6g. TLC (BAPW system Rf = 0.62) gave a single spot. Elemental analysis C ₁₉ H ₂₅ N ₄ O ₅ S MW420.53
Calculated C 54.22, H 5.99, N 13.35. Analytical value C 55.90, H
6.41, N 12.75.

Example 23 [D-Pro-Thr-Trp NH ₂ (D-
Synthesis of PTW NH ₂ : Pro is D)] Boc-ThrOSu 3.7g (12 mmol), TrpOBzl HCl 3.8g (1
TEA was added to a DMF solution of
After confirming that it was H7-8, it was reacted overnight. Post-treatment was carried out in the same manner as in Example 1 to obtain Boc-Thr-TrpOBzl as an oil. Subsequently, acid treatment was carried out in the same manner as in Example 1 to remove the Boc group, 2.8 g (11 mmol) of ZD-Pro was added to the resulting residue, and 2.5 g (12 mmol) of DCCD in 10 ml of DMF was added dropwise. The reaction was carried out at 4 ° C overnight. After filtering off the precipitate, the filtrate was concentrated under reduced pressure and treated in the same manner as in Example 1 to give an amorphous substance.
4.21 g was obtained. 2 g of this was removed by the catalytic reduction method to remove the protecting group, the resulting residue was dissolved in a small amount of water, purified by Sephadex G-10 column, and the fractions giving almost a single spot by TLC were collected and dried under reduced pressure. Solidified A small amount of MeOH was added to the residue for crystallization, and after cooling, the crystals were collected by filtration and dried to obtain 1.40 g (total yield 30%) (BAPW system Rf = 0.3.
3). Elemental analysis C ₂₀ H ₂₅ N ₄ O ₅ MW 402.22 Calculated C 59.69,
H 6.51, N 13.91. Analytical value C55.90, H 6.41, N 12.75.

Example 24 [Glu-D-Arg (EDR: Arg
Is a D-form) Z-Glu (Bzl) OH 3.4 g (10 mmol), D-Arg (NO ₂ ) OBzl To
s 4.82 g (10 mmol), HOBt 1.5 g (11 mmol) in DM
Dissolved in 100 ml of F, under ice cooling, WSCD HCl 2.1 g (11 mmol)
Was added, the reaction was carried out in the same manner as in Example 1, post-treatment was carried out,
5.3 g (8 mmol) of an oily product of Z-Glu (Bzl) -D-Arg (NO ₂ ) OBzl was obtained. This oily substance is subjected to a deprotection reaction by a catalytic reduction method, purified on a Sephadex G-10 column, and a fraction of a target substance giving a nearly single spot by TLC is collected.
It concentrated and freeze-dried. As a colorless amorphous substance
1.82 g (6 mmol) was obtained (TLC: BAPW system Rf = 0.5
8). Elemental analysis C ₁₁ H ₂₁ N ₅ O ₅ , MW 303.33 Calculated value C 43.5
2, H 6.92, N 23.14. Analytical value C 42.98, H 6.88, N 2
2.76

Test Example 1 [Focus formation inhibition test] NIH3T3 cells transformed with the protooncogene erbB-2 were placed in an 8-well Labtech chamber (slide glass size), and 1
Samples were added to 7 wells, leaving the wells as controls, cultured in a 5% carbon dioxide incubator at 37 ° C, and observed every 24 hours for 5 days with a light microscope to observe the cell growth state (focus number) and photographed each time. (Sample final concentration 2
50 μg / ml) A part of the result is shown in Table 1 (Table 1).

[0035]

[Table 1]

Test Example 2 [MTT assay method] NI transformed with DME medium containing 10% bovine serum with various oncogenes
Suspend H3T3 cells at 5 × 10 ⁴ cells / ml and
l was dispensed into each well of a 96-well flat bottom plate. 3 of these
Only the same amount of culture solution was added to the well to serve as a negative control. After culturing this plate at 37 ° C. in a 5% carbon dioxide gas incubator overnight, water or acetone at a concentration of 880 μg / ml, for example, acetone, dimethylformamide, dimethyl sulfoxide,
10 μl of a solution of the tripeptide of the present invention and its derivative prepared in an organic solvent such as alcohol was added to each well (final concentration 80 μg / ml). After culturing this in a 5% carbon dioxide incubator at 37 ° C for 2 hours, a 5 mg / ml concentration of MTT or PBS (-) solution
10 μl was added to all wells and cultured at 37 ° C. in a 5% carbon dioxide gas incubator for 2 hours. Each plate was centrifuged at 3000 rpm for 15 minutes using a cooling low speed centrifuge equipped with a plate holder. The plate was taken out from the centrifuge, the supernatant of each well was removed by suction, 150 μl of DMSO was added, the mixture was stirred with a mixer, and then the absorbance at 540 nm was measured with an immunoreader (colorimetric determination). A part of the result is shown in Table 2 (Table 2). F25, PH1, S59, erbB-2, vs in Table 2
rc, v-mos, SV-40 are cH-ras protooncogene, vHr, respectively
NIH3T3 cells cancerous with as protooncogene, KH-ras protooncogene, erbB-2 protooncogene, v-src protooncogene, v-mos protooncogene, SV-40 protooncogene ..

[0037]

[Table 2]

[0038]

INDUSTRIAL APPLICABILITY The present invention provides a completely novel pharmaceutical composition containing a tripeptide. The active ingredient of the pharmaceutical composition of the present invention is a peptide, which is decomposed into amino acids and metabolized in vivo. Therefore, the pharmaceutical composition of the present invention has an extremely low risk of causing side effects when administered to a living body, and is effective as a drug.

Claims (7)

[Claims] 1. Pro-AB (where A = Phe, Lys, Asn, Tyr, Th
r; B = Pro, Trp is shown. ) A pharmaceutical composition containing the tripeptide represented by the formula (4) or a physiologically recognized salt thereof as an active ingredient. 2. CD-Pro (where C = Tyr, Glu, Pro; D = Asn, S
er, Arg, Tyr), or a physiologically recognized salt thereof, as an active ingredient. 3. A pharmaceutical composition containing a tripeptide represented by Thioproline-Thr-Trp or a physiologically recognized salt thereof as an active ingredient. 4. A pharmaceutical composition containing a dipeptide represented by Glu-Arg or a physiologically acceptable salt thereof as an active ingredient. 5. The tripeptide according to any one of claims 1 to 4, which is an acylated derivative at the N-terminus or a physiologically acceptable salt thereof.
The pharmaceutical composition according to item. 6. The derivative according to claim 1, which is a derivative in which the C-terminal of the tripeptide is amidated, or a physiologically acceptable salt thereof.
The pharmaceutical composition according to item. 7. The pharmaceutical composition according to claim 1, which is an anticancer agent.