JPH0321039B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel bioactive peptide that has the activity of inhibiting alkaline phosphatase (derived from bovine liver) and is useful as an anticancer agent, and also relates to a method for producing the same and an anticancer agent containing the same as an active ingredient. Specifically, the present invention relates to the general formula (wherein R represents hydrogen or a lower alkyl group, particularly a methyl group), pharmaceutically acceptable salts and hydrates thereof (hereinafter referred to as
The present invention also relates to compounds of the present invention (sometimes simply referred to as compounds of the present invention), methods for producing them, and anticancer agents containing them. The present inventors have succeeded in newly synthesizing the compound of formula (1), and have demonstrated that the compound of the present invention has physiological activity that strongly inhibits alkaline phosphatase present in bovine liver, and anticancer activity. I found out. Alkaline phosphatase exists mainly in cell membranes, and is also found in the endoplasmic reticulum, lysosomes, mitochondrial outer membrane, Golgi apparatus, nuclear membrane, etc. The original physiological significance of alkaline phosphatase has not yet been established. However, the fact that many of these enzymes are membrane-bound and their localization suggests that they play some role in membrane functions (e.g., synthesis, decomposition, transport, etc.) (Niichi Komoda, Yoshikatsu Sakata: Protein , Nucleic Acids, Enzymes 24 , 131-144 (1979)). It has been suggested that alkaline phosphatase inhibitors may affect cellular functions including immunity after binding to enzymes or binding proteins present in cell membranes. The anti-alkaline phosphatase activity of the compound of the present invention is measured by the following method. Namely, paranitrophenyl phosphate disodium salt (manufactured by Daiichi Chemical Co., Ltd.)
This is a colorimetric method for quantifying paranitrophenol, which is hydrolyzed and liberated using alkaline phosphatase (Sigma, USA) obtained from bovine liver as a substrate. Dissolve paranitrophenyl phosphate disodium salt in water to make a 0.1M solution. Add 0.3M 2-amino-2- to 0.04ml of this substrate solution.
Methyl-1,3-propanediol buffer (PH
9.0) Add 0.32 ml, 0.02 ml of 0.2M magnesium chloride, and 0.1 ml of distilled water or an aqueous solution containing the specimen, and warm at 37°C for 3 minutes, then prepare a solution of 30 μg of alkaline phosphatase dissolved in 1 ml of distilled water. Add 0.02ml, react at 37℃ for 20 minutes, add 1.5ml of 0.15N caustic soda to stop the reaction, and rotate at 3000 revolutions per minute.
The absorbance (a) at 420 nm of the supernatant obtained by centrifugation for 5 minutes was measured. At the same time, the absorbance (b) of a control containing only distilled water, which did not contain the above specimen, was measured, and the inhibition rate was calculated as (ba)/b×100. In this method, the compound of the present invention, isoleucyl-isoleucyl-phosphoserine, is obtained.
Methyl ester is 5.6×10 ^-6 M, isoleucyl-
Isoleucyl-phosphoserine is 6.3×10 ^-6 M
showed a 50% inhibitory concentration (IC ₅₀ ). Currently, phorhenisin, levamisole, and the like are known as inhibitors of alkaline phosphatase, and all of these compounds have immunostimulatory effects. Although the compound of the present invention has no cytotoxicity, it exhibits anticancer activity in animal experiments, and it also has alkaline phosphatase inhibitory activity, suggesting that the immune system is involved in its anticancer activity, and it may be useful as a pharmaceutical agent. It is a compound useful as an anticancer agent in the field of cancer. The present invention further provides the following general formula (In the formula, R ¹ is a protected or unprotected amino group, R ² is a protected or unprotected carboxyl group) is reacted with phosphoric acid or a reactive derivative thereof, and then general formula for (In the formula, R ¹ and R ² have the same meanings as above,
R ³ represents hydrogen or a protecting group for the phosphoric acid group), and further, when the produced phosphoric ester of formula (3) contains a protecting group, from the ester of formula (3) The following general formula characterized by removing the protecting group (In the formula, R represents hydrogen or a lower alkyl group.)
It also relates to a method for producing a peptide represented by The method for producing the compound of the present invention will be explained below. The N-protected tripeptide in the case where the serine side chain of the starting compound of formula (2) is unprotected can be easily produced from, for example, N-protected isoleucyl isoleucine and carboxyl group-protected serine by a normal peptide synthesis method. can. In this case, the preferred α-amino protecting groups used are alkoxycarbonyl groups such as t-butoxycarbonyl (Boc) groups and aralkyloxycarbonyl groups such as benzyloxycarbonyl (Z) groups, but commonly used amino protecting groups can be used. can be used; for example, a divalent phthaloyl group can also be used. All known carboxyl protecting groups can be used to protect the carboxyl group of the C-terminal serine residue of starting compound (2), but it is usually protected as an ester. Preferred carboxyl protecting groups include alkyl groups such as methyl, ethyl, t-butyl, and aralkyl groups such as benzyl. Phosphoric acid can also be used to introduce a phosphoric acid group into the starting compound (2), but in terms of reactivity, ease of purification and deprotection, reactions that act equally with phosphoric acid such as phosphoryl fluoride are recommended. Derivatives can be used.
Preferably, the formation of the phosphate ester (3) is carried out by means of their protective forms, for example dibenzylphosphoryl chloride. However, all known methods for producing phosphoric acid esters can be used. The product with a phosphate group introduced has the following formula: (In the formula, R ¹ is an optionally protected amino group, R ²
is a carboxyl group that may be protected, and R ³ is a protecting group for a phosphoric acid group, such as a benzyl group). Deprotection from this protected peptide (3) can be easily carried out by combining hydrolysis with trifluoroacetic acid, catalytic reduction with hydrogen at normal pressure, alkaline saponification, etc. as necessary depending on the type of protecting group used. be able to. The compound of the present invention thus obtained can be easily isolated and purified by conventional means commonly used to separate peptides, preferably column chromatography using Dowex 50. The compound of the present invention of general formula (1) is an amphoteric substance that can be named isoleucyl-isoleucyl-phosphoserine when R is hydrogen, so it can easily form a salt with a pharmaceutically acceptable acid or base. , such salts and hydrates thereof, as well as esters are included in the compounds of the present invention. Examples of suitable salts include salts with pharmaceutically non-toxic inorganic cations such as sodium, potassium, magnesium, calcium, etc. or salts with organic amines such as guanidine, or mineral acids such as hydrochloric acid, sulfuric acid, trifluoroacetic acid, etc. There are acid addition salts with organic acids. Next, the antitumor effects of the compounds of the present invention will be described. IMC carcinoma cells 2×10 ^6
24 hours after transplantation, once a day for 10 days, a total of 10
Isoleucyl-isoleucyl-phosphoserine dissolved in sterile physiological saline was administered intraperitoneally. Tumor nodules were excised on the 21st day after transplantation, and the growth inhibition rate (IR%) was calculated using the following formula. (1-T/C) x 100 = IR (%) T: Administration group average tumor weight C: Control group average tumor weight The results are shown in Table 1.

[Table] As is clear from Table 1, this substance is 0.1mg/
Growth inhibition rate is observed at doses of 0.1 to 1 mg/Kg or more, and a dose of 0.1 to 1 mg/Kg is particularly optimal. When the compound of the present invention is used as an anticancer agent, it can be formulated by mixing with a pharmaceutically acceptable liquid or solid carrier by conventional pharmaceutical techniques. Therefore, according to the third invention, general formula (I)
An anticancer agent containing a physiologically active peptide or a salt or hydrate thereof as an active ingredient is provided. In addition, to examine the acute toxicity of the compounds of the present invention, isoleucyl-isoleucyl-phosphoserine was administered intravenously, intraperitoneally, or orally to the following animals, namely ddB mice (male, weighing 20 to 25 g). The LD ₅₀ value was measured. The results are shown in the table below.

[Table] The anticancer agent containing the compound (1) of the present invention as a main ingredient may contain any of its pharmacologically acceptable salts, hydrates of the compound, or hydrate salts. good. The dosage form may be oral, injection, or rectal suppository. When preparing an injection, a PH adjuster, buffer, stabilizer, excipient, etc. may be added to the above-mentioned main drug, and further A lyophilized injection can be made by freeze-drying using the method, and by adding PH regulators, buffers, stabilizers, isotonic agents, local narcotics, etc. to the main drug, it can be administered subcutaneously or intramuscularly using a conventional method. It can also be made into an intravenous injection. When preparing oral solid preparations, add excipients to the main drug, and if necessary, binders, disintegrants, lubricants, colorants, flavoring agents, and flavoring agents.
Tablets, coated tablets, granules, powders, capsules, etc. can be made by conventional methods. When preparing oral liquid preparations, syrups and dry syrups can be prepared by adding flavoring agents, buffering agents, stabilizers, flavoring agents, etc. to the main drug by conventional methods. When preparing a rectal suppository preparation, an excipient and, if necessary, a surfactant are added to the main drug, and then a suppository can be prepared by a conventional method. The dosage of the compound of the present invention varies depending on the symptoms, but
Normally, the single dose for adults is 0.02mg to 200mg of the compound represented by general formula (1), once a day.
It is advisable to administer the drug twice a day or more than once a day depending on the symptoms. Production examples of the compounds of the present invention are listed below as examples, but the present invention is not limited thereto. In addition,
When using abbreviations for amino acids, IUPAC,
Follow IUB regulations or symbols commonly used in the field. Example 1 Production of isoleucyl-isoleucyl-phosphoserine methyl ester 4.28 g of Nt-butoxycarbonyl isoleucyl-isoleucine (Boc-Ile-Ile-OH) was dissolved in 100 ml of acetonitrile to obtain 1.94 g of serine methyl ester hydrochloride. g was added. Add 1.72 ml of triethylamine while stirring under ice-cooling, and then add 1.72 ml of triethylamine.
-2.50 g of hydroxybenzotriazole and N,
3.04 g of N'-dicyclohexylcarbodiimide were added in this order. After stirring for 1 hour under ice-cooling and overnight at room temperature and drying under reduced pressure, it was purified by silica gel column chromatography (developing solvent: chloroform:methanol = 20:1) to obtain N-t-butoxycarbonyl isoleucyl-iso. 3.0 g of leucylserine methyl ester (Boc-Ile-Ile-Ser-OMe) was obtained. 3.34 g of dibenzyl phosphite was dissolved in 15 ml of dry benzene, and while stirring at room temperature, 1.6 g of N-chlorosuccinimide was added while being careful not to allow the temperature to rise above 35°C. After further stirring at room temperature for 1 hour, the precipitate was filtered out using a glass filter.
The resulting filtrate containing dibenzylphosphoryl chloride was concentrated under reduced pressure, and then added to a 7 ml pyridine solution of 2.7 g of Boc-Ile-Ile-Ser-OMe prepared as above with stirring under ice cooling. did. After stirring for 16 hours under ice cooling, the reaction solution was poured into 100 ml of ice water, extracted twice with 200 ml of ethyl acetate, washed with water, dried the ethyl acetate layer with Glauber's salt, dried under reduced pressure, and subjected to silica gel column chromatography. (Developing solvent:
It was purified with chloroform:methanol=40:1) to give N-t-butoxycarbonyl-isoleucyl-isoleucyl-dibenzylphosphoserine.
2.26g of methyl ester was obtained. N-t-butoxycarbonyl-isoleucyl-isoleucyl-dibenzylphosphoserine methyl ester 500mg
was dissolved in 3.0 ml of trifluoroacetic acid under ice-cooling.
After reacting for 30 minutes (removal of Boc group), it was dried under reduced pressure.
It was dried under reduced pressure in a desiccator. The obtained residue was dissolved in 15 ml of ethanol, 1 ml of water was added, 200 mg of 10% palladium-carbon was added,
Hydrogen was introduced for 3 hours at normal pressure for reduction (removal of benzyl group). Thereafter, the reaction solution was filtered to obtain 382 mg of viscous solid. After dissolving this residue in 400 ml of water and filtering off insoluble materials, it was added to Dowex 50 (H ⁺ ).
It was adsorbed onto a column, washed with water, and then eluted with 0.2M ammonia. Fractions positive for the Lydon-Smith reaction were collected and concentrated to dryness under reduced pressure to obtain 192 mg of isoleucyl-isoleucyl-phosphoserine methyl ester. This substance against alkaline phosphatase
The IC ₅₀ value was 5.6×10 ⁻⁶ M. Example 2 Production of isoleucyl-isoleucyl-phosphoserine 192 mg of isoleucyl-isoleucyl-phosphoserine methyl ester was dissolved in 10 ml of 1N NaOH and left at room temperature for 1 hour to perform hydrolysis. Dissolve the reaction solution in 300ml of water and add Dowex 50 (H ⁺ ).
It was adsorbed on and eluted with water. Fractions positive for the Lydon-Smith reaction were collected and concentrated to dryness under reduced pressure.
isoleucyl-isoleucyl-phosphoserine
142.5 mg was obtained. Elemental analysis value (%) Measured value: C, 43.64 H, 7.49 N,
10.12 Calculated value (as C ₁₅ H ₃₀ N ₃ O ₈ P): C, 43.75 H,
7.29 N, 10.21 Effect of this substance on alkaline phosphatase
The IC ₅₀ value was 6.3×10 ⁻⁶ M.

Claims (1)

[Claims] First-order general formula (In the formula, R represents hydrogen or a lower alkyl group.)
Physiologically active peptides represented by the following, pharmaceutically acceptable salts thereof, and hydrates thereof. 2nd order general formula (wherein R ¹ represents a protected or unprotected amino group and R ² represents a protected or unprotected carboxyl group) is reacted with phosphoric acid or a reactive derivative thereof, and then general formula for (In the formula, R ¹ and R ² have the same meanings as above,
R ₃ represents hydrogen or a protecting group for the phosphoric acid group), and further, when the produced phosphoric ester of formula (3) contains a protecting group, The following general formula is characterized by removing the protecting group from (In the formula, R represents hydrogen or a lower alkyl group)
A method for producing a peptide represented by 3rd order general formula (In the formula, R represents hydrogen or a lower alkyl group)
An anticancer agent characterized by containing a peptide represented by the following formula or a salt or hydrate thereof as an active ingredient.