JPH0249314B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to novel water-soluble porphyrin derivatives. Generally, porphyrins (protoporphyrin,
When hematoporphyrin, uroporphyrin, etc.) are intravenously injected into tumor-bearing animals, cancer cells take up about twice as much of this substance as normal cells, and the cancerous tissue
When irradiated with 405nm ultraviolet light, 630 and 690nm
Because it emits red fluorescent light, it is used to detect and judge cancerous tissue. In addition, porphyrins have a stronger affinity for cancer cells than normal cells, and by irradiating cancer cells that have incorporated porphyrins with 630 nm light,
Cancer cells die completely after 3 days. Therefore, porphyrins are useful substances for eradicating cancer cells. However, such porphyrins are generally solid and fat-soluble substances, and when administered to animals orally or parenterally, their rate of movement to any part of the animal is slower than that of water-soluble substances, and their uptake is slow. The amount released is also at a low level. To increase the transfer rate, porphyrins are solubilized with organic solvents such as ethanol and dimethyl sulfoxide, and CMC,
Dispersion has been done using emulsifiers such as sucrose esters and monoglycerides, but special techniques are required for solubilization, emulsification, and dispersion. Therefore, the present inventors conducted extensive research aimed at making the porphyrin derivative itself water-soluble, and as a result, they succeeded in obtaining a new water-soluble porphyrin derivative. That is, the water-soluble porphyrin derivative of the present invention has a structure represented by the following formula (). (In the formula, R ₁ is H ₂ or

[Formula] R ₂ is −C ₂ H ₅ or

[Formula] represents, and R ₁ is

When [formula], R ₂ is

[Formula]. EN represents an ethylenediamine residue (-NH- _CH2 - _CH2 - _NH2 ). ) Specific examples of the water-soluble porphyrin derivative represented by the above general formula () include the following compounds. (1) Methylpyropheophorbide ethylenediamine hydrochloride (hereinafter abbreviated as MPPEH): (2) Methylpheophorbide ethylenediamine hydrochloride (hereinafter abbreviated as MPEH): (3) Methyl mezopyropheophorbide ethylenediamine hydrochloride (hereinafter abbreviated as MMPPEH): The water-soluble porphyrin derivative of the present invention described above can be produced by the method shown below. First, a chlorophyll derivative represented by the following general formula () is used as a starting material. (In the formula, R ₃ is H ₂ or −COOCH ₃ , R ₄ is −CH
= _CH2 or _-C2H5 , and when _R3 is _{-COOCH3 , R4} is -CH= _CH2 . ) Specific examples of the chlorophyll derivative represented by the above general formula () include the following compounds. (i) Methylpyropheophorbide: (ii) Methylpheophorbide: (iii) Methyl mezopyropheophorbide: Among the above, methylpheophorbide is a substance that can be easily and inexpensively obtained by dissociating Mg from chlorophyll and replacing the phytyl ester at the 7-position carbon with a methyl ester. Methods for producing methylpheophorbide include acid-catalyzed transesterification using a solvent containing a highly concentrated mixture of methanol and a mineral acid such as hydrochloric acid or sulfuric acid, or acid-catalyzed transesterification using an enzyme such as chlorophyllase. Although a method such as transesterification in water-containing methanol can be adopted as the present invention, it cannot be produced industrially and efficiently by a method already filed for a patent by the same applicant as the present invention (Japanese Patent Laid-Open No. 147286/1986). Can be done. According to this first-to-file method,
By transesterifying phytol bound as a propionyl ester in the molecules of chlorophyll a or b, pheophytin a or b, or their derivatives with methanol using a porous strongly acidic ion exchange resin as a catalyst. , methylpheophorbide is produced. Methylpyropheophorbide can be produced by decarburizing the methylpyrofluoride obtained in this way, and methylpyrofluoride can be produced by hydrogenating the methylpyrofluoride. Bide can be produced. All of these chlorophyll derivatives are solid and fat-soluble substances. According to the method of the present invention, a chlorophyll derivative represented by the above general formula () is reacted with ethylenediamine under anhydrous conditions. None of the reaction products obtained at this time are water-soluble as they are;
It becomes water-soluble only when it is made acidic by adding hydrochloric acid to form a hydrochloride salt. In this case, even if an inorganic acid such as sulfuric acid or phosphoric acid or an organic acid such as oxalic acid, succinic acid or tartaric acid is added to make the solution acidic, a stable water-soluble compound cannot be obtained. By using methylpyropheophorbide as a starting material in the process of the invention
MPPEH by using methylpheophorbide and MMPPEM by using methyl mezopyropheophorbide.
are obtained as the water-soluble porphyrin derivatives of the present invention. Table 1 shows the results of examining the solubility of the thus obtained water-soluble porphyrin derivatives in various solvents.

【table】

[Table] The novel water-soluble porphyrin derivative according to the present invention is a compound useful for detecting cancer tissue and eradicating cancer cells by ultraviolet irradiation, similar to the general porphyrins mentioned above, Since it is water-soluble, whereas it is fat-soluble, it is easy to use and has a good rate of movement within the body. Further, by utilizing the reactivity of ethylenediamine hydrochloride introduced into the porphyrin derivative of the present invention, it can be expected to be used as an intermediate for obtaining various useful compounds. Furthermore, as a result of experiments, it has been found that the water-soluble porphyrin derivative of the present invention has an antibacterial effect that inhibits the growth of certain microorganisms. The antibacterial test method is shown below. The composition of the microbial culture medium is as follows: Meat extract 1% Peptone 1% NaCl 0.5% Agar 1.5% PH 7.0 The above medium was pre-coated on a shear dish, and test tube cultures of various microorganisms were spread radially from the center of the shear dish. It was applied to. The water-soluble porphyrin derivatives of the present invention, MPPEH and MPEH, were prepared in a 0.1M aqueous solution, and MMPPEH was added in a 0.01M aqueous solution.
A solution containing 0.5% Tween 80 was used as the test solution, and a paper disk (9φ) impregnated with this test solution was placed in the center of the shear plate, and heated at 30°C.
Microorganisms were cultured for 24 hours under 60W light bulb irradiation, and the ability of the disc to inhibit microbial growth was investigated. The results are shown in the table below.

【table】

[Table] As can be seen from the table, the porphyrin derivative of the present invention does not exhibit antibacterial properties against yeast and mold.
Shows antibacterial properties selectively against bacteria. Furthermore, among bacteria, there is a difference in antibacterial properties between Gram-negative and Gram-positive bacteria, with stronger antibacterial properties against Gram-positive bacteria, and MMPPEH in particular does not show antibacterial properties against Gram-negative bacteria. , which has the specificity of acting only on Gram-positive bacteria. The present invention will be specifically described below with reference to Examples, but the present invention is not limited to these Examples unless the gist of the invention is exceeded. Example 1 A jacketed glass column was filled with 50 ml of a porous strongly acidic ion exchange resin "Revacht SP120" (trade name, manufactured by Bayer AG), and the solvent was replaced with acetone. A solution of 5 g of chlorophyll paste obtained from spirulina in acetone was passed through this as a raw material. After sufficient adsorption and washing, the solvent is replaced with methanol and washing is carried out again until the eluate becomes colorless. Next, heat the resin bath by running warm water at 60°C into the jacket and leave it as is for several hours. After the reaction is completed, the resin bath is cooled to room temperature and washed with 200 ml of methanol. Here, 3% MgCl ₂ -methanol solution was used as the elution solvent SV=
1 to 2, 300 ml of liquid was passed through and methylpheophorbide was recovered. Add 150ml of water to the obtained eluate,
Methylpheophorbide and inorganic substances are separated by liquid-liquid separation using 200 ml of ether, and the ether phase is washed several times with 50 ml of water and then evaporated to dryness. Next, methylpheophorbide with a purity of 98.5% was obtained by crystallization in an organic solvent and purification by column treatment. The thus obtained methylpheophorbide 1
Add 50g of ethylenediamine to g and add it in a nitrogen stream.
Heat to reflux at 120°C and react for 3 hours. Next, ethylenediamine was distilled off under reduced pressure, and 3%
Add hydrochloric acid water to adjust the pH to 5.0 and solubilize to obtain a solution of water-soluble MPEH. This aqueous MPEH solution is passed through a column filled with 200 ml of porous resin (HP20 manufactured by Mitsubishi Kasei Corporation), unreacted hydrochloric acid and ethylenediamine are removed by washing with water, and MPEH is adsorbed onto the column. MPEH is eluted when either methanol or acetone is used as an eluent to pass through the column. When the eluent in the eluate is removed by vacuum distillation, about 1 g of MPEH is obtained. The elemental analysis values are as follows. Theoretical value Actual value C 57.80% 56.95% H 6.74 6.79 N 16.86 16.73 Cl 12.82 13.15 O 5.78 6.29 Moreover, the ultraviolet-visible absorption spectrum is as shown in FIGS. 1 and 2. Example 2 50 g of ethylenediamine was added to methylpyropheophorbide obtained by decarboxylating 1 g of methylpyropheophorbide (prepared and purified in the same manner as in Example 1) in pyridine, and the mixture was heated to 120 g in a nitrogen stream. The mixture was heated to reflux at ℃ and allowed to react for 3 hours. Thereafter, in the same manner as in Example 1, ethylenediamine was distilled off, hydrochloride was generated by adding hydrochloric acid, and purification was performed using a porous resin column to obtain about 1 g of MPPEH. The elemental analysis values are as follows. Theoretical value Actual value C 62.71% 60.34% H 6.92 7.10 N 15.82 15.51 Cl 10.03 12.50 O 4.52 4.51 Moreover, the ultraviolet-visible absorption spectrum is as shown in FIG. Example 3 1 g of methylpheophorbide (prepared and purified in the same manner as in Example 2) was dissolved in Pd-
50 g of ethylenediamine is added to methyl mezopyropheophorbide obtained by hydrogenation in the presence of catalyst C, and the mixture is heated to reflux at 120° C. in a nitrogen stream and reacted for 3 hours. Thereafter, in the same manner as in the example, ethylenediamine was distilled off, hydrochloride was generated by adding hydrochloric acid, and purified using a porous resin column, and approximately 1 g
Obtained MMPPEH. The elemental analysis values are as follows. Theoretical value Actual value C 68.46% 67.24% H 6.85 6.98 N 13.69 13.85 Cl 5.79 6.19 O 5.21 5.67 Moreover, the ultraviolet-visible absorption spectrum is as shown in FIG.

[Brief explanation of drawings]

FIG. 1 shows the ultraviolet-visible absorption spectrum of MPEH in an aqueous solvent, and FIG. 2 shows the ultraviolet-visible absorption spectrum of MPEH in an alcoholic solvent. Figure 3 shows the ultraviolet-visible absorption spectrum of MPPEH in ethanol solvent. Figure 4 is
The ultraviolet-visible absorption spectrum of MMPPEH in ethanol solvent is shown.

Claims (1)

[Claims] 1. General formula (In the formula, R ₁ represents H ₂ or [formula] R ₂ represents -C ₂ H ₅ or [formula], and when R _{1 is [formula], R 2 is} [formula]. EN is an ethylenediamine residue A water-soluble porphyrin derivative represented by (-NH-CH ₂ -CH ₂ -NH ₂ ). 2 General formula () is the following formula The compound according to claim 1, which is methylpyropheophorbide ethylenediamine hydrochloride represented by: 3 General formula () is the following formula The compound according to claim 1, which is methylpheophorbide ethylenediamine hydrochloride represented by: 4 General formula () is the following formula The compound according to claim 1, which is methyl mezopyropheophorbide ethylenediamine hydrochloride represented by: 5 General formula (In the formula, R ₃ is H ₂ or −COOCH ₃ , R ₄ is −CH
= _CH2 or _-C2H5 , and when _R3 is _{-COOCH3 , R4} is -CH= _CH2 . ) is reacted with ethylenediamine and then with hydrochloric acid to form an adduct. (In the formula, R ₁ represents H ₂ or [formula] R ₂ represents -C ₂ H ₅ or [formula], and when R _{1 is [formula], R 2 is} [formula]. EN is an ethylenediamine residue (-NH- _CH2 - _CH2 - _NH2 ).) A method for producing a water-soluble porphyrin derivative represented by: 6 The general formula () is the following formula It is methylpyropheophorbide represented by, and the general formula () is the following formula: The manufacturing method according to claim 5, which is methylpyropheophorbide ethylenediamine hydrochloride represented by: 7 General formula () is the following formula It is methylpheophorbide represented by
The general formula () is the following formula The manufacturing method according to claim 5, which is methylpheophorbide ethylenediamine hydrochloride represented by: 8 General formula () is the following formula It is methyl mezopyropheophorbide represented by, and the general formula () is the following formula: The manufacturing method according to claim 5, which is methyl mezopyropheophorbide ethylenediamine hydrochloride represented by.