JPS63290881A - Pheophorbide derivative - Google Patents

Abstract

NEW MATERIAL:A pheophorbide derivative expressed by formula III [R1 is formula IV (n is 2-6); R2 is CH3, CHO or CH2OH; R3 is H or CH3] or salt thereof. EXAMPLE:2-(1-Methoxyethyl)perpurine. USE:A medicine useful as diagnosis and remedy for cancer having specific affinity to tumor tissue and photosensitizing action. PREPARATION:A pheophorbide A expressed by formula I is reacted with an acetic solution of hydrogen bromide in a solvent and the resultant product is successively reacted with a compound expressed by the formula HOR1 to afford a compound expressed by formula II, which is then subjected to air oxidation with an alcoholic alkali such as KOH-n-PrOH in a solvent, neutralized with an acid and then subjected to esterification with e.g. diazomethane-ether solution to give the compound expressed by formula III (R2 is CH3; each R3 is CH3). The compound expressed by III is hydrolyzed with an acid or alkali to provide the compound expressed by formula I (R2 is CH3; each R3 is H).

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to novel pheophorbite derivatives used in the diagnosis and treatment of cancer.

(Prior art) The so-called photodynamic diagnostic treatment method, in which a photosensitizer that has an affinity for cancer cells is intravenously administered and accumulated in the cancer cells, and then irradiated with laser light to diagnose and treat cancer tissue, is It is highly effective in the diagnosis and treatment of cancer (T, D
eighty ed “PorphyrinLoc”
Alization and Treatment o
f Tumors” pp75-78 (1984)].

The photosensitizers used in this process include porphyrins such as hematoporphyrin (hereinafter abbreviated as CHp), and Hp is treated with a decomposing acid in acidic vinegar, followed by alkaline hydrolysis and neutralization. A hematoporphyrin derivative (hereinafter abbreviated as (HpD)) produced through the above process is used.

(Problem to be solved by the invention) However, it has been difficult to obtain pure Hp in the past.
he porph-yrins” vol.1.1 pl)
297-298 (1978)), and HpD produced using it is known to be a mixture of several types of porphyrins, and a few putative structures have been proposed for its essential active substance. It's just that. It is also known that large amounts are taken up not only by cancer tissues but also by normal tissues, especially the liver.

These problems are major obstacles to clinical use.

(Means for Solving the Problem) The present inventors searched for a highly safe photosensitizer that has a specific affinity for tumor cells, and found a pheophorbite derivative derived from plant chlorophyll, which is a porphyrin-related compound. The present invention was completed based on the discovery that the compound exhibits high affinity for cancer tissues and photosensitivity.

The present invention is based on the general formula [wherein R1 is (CH20H20) nCHg (n represents an integer of 2 to 6), R2 is OH3, OHO or C
H2oH1R8 represents H or OH8] A pheophorbite derivative or a salt thereof.

An example of the synthetic route for the pheophorbite derivative of the present invention is shown below.

o2cn8 O2H After treating pheophorbite-a represented by formula (1) with an acetic acid solution of hydrogen bromide in the presence of a solvent, it is treated with the general formula HORt (in the following formulas, each symbol is the same as above). A compound represented by general formula (2) is obtained by reacting the represented compound. Examples of the solvent used in this reaction include methylene chloride, chloroform (not containing ethanol), and ethylene dichloride.

The hydrogen bromide concentration of the hydrogen bromide acetic acid solution is not particularly limited, but it is usually preferably 30%, and the reaction with HORI is 0 to 1%.
It can be completed in 5 minutes to 5 hours at 50℃, but it is usually completed at around 80℃.
A reaction time of about hours is preferred.

A compound represented by general formula (2) is air oxidized with an alcoholic alkali in a solvent, neutralized with an acid, and then esterified with an esterifying agent to obtain a compound represented by general formula (3). Examples of the solvent used in this reaction include ether and tetrahydrofuran.

Examples of the alcohol include methanol, ethanol, and n-propanol, with n-propanol being preferred, and examples of the alkali include sodium hydroxide, potassium hydroxide, and the like, with potassium hydroxide being preferred.

As the esterifying agent, a diazomethane-ether solution, a trifluoro-borane ether complex-methanol, or sulfuric acid-methanol is used, but a diazomethane-ether solution is particularly preferred, and the reaction is completed in several minutes at room temperature.

A compound represented by general formula (4) is obtained by subjecting the compound represented by general formula (3) to acid or alkali hydrolysis by a conventional method. Acids usually used for hydrolysis include mineral acids such as hydrochloric acid and sulfuric acid, or acetic acid,
Organic acids such as p-toluenesulfonic acid are used, and the alkalis include sodium hydroxide, potassium hydroxide,
Ammonia and the like are used, but potassium hydroxide is usually preferred.

This reaction is completed in 5 minutes to 48 hours at 0 to 150°C.
For example, in a reaction using potassium hydroxide in methanol, 8
Complete in 2 hours at 0-85°C.

Post-reaction treatment and purification of these new pheophorbite derivatives are carried out by conventional methods, such as extraction, recrystallization, column chromatography, etc.

The pheophorbite derivatives of the present invention can be used in free or salt form. The derivative is an acid, e.g.
It forms salts with hydrochloric acid, and those with a carboxyl group also form salts with bases, such as sodium and potassium.

The compound of the present invention has the following features in cancer diagnosis and treatment.

That is, when the compound of the present invention is administered to a tumor-bearing animal,
It specifically accumulates in cancer tissues, and when irradiated with light, it produces a fluorescence spectrum characteristic of the compound. Therefore, by measuring its fluorescence spectrum, the location and size of cancer tissue can be determined.

On the other hand, when irradiated with laser light of an appropriate wavelength, the present compound can selectively cause necrosis of only cancer cells since it generates singlet oxygen which has a cell-killing effect.

Various methods can be used to administer the compounds of the present invention, including intravenous, subcutaneous, intraperitoneal, oral and rectal administration.

The dosage is i -850m, p per 1K11 body weight.
However, it is not limited to this range depending on the treatment content.

(Examples) Hereinafter, the present invention will be explained in detail with reference to Examples, but the present invention is not limited thereto.

Example 1 Synthesis of 8-(1-methoxyethyl)-purpurin-7-drimethyl ester (R1= (OH20H20)nOH8 in general formula (1)
Compound where n=0R2=CHB, R5=C1Ha) Example 1-A Methylpheophorbite-a200mji was added to anhydrous methylene chloride aomt! Add 5 ml of 80% hydrogen bromide and acetic acid solution, and react for 6 hours with stirring at room temperature. The solvent was distilled off under reduced pressure, 10 ml of methanol was added to the residue, and the mixture was refluxed for 6 hours. Diluted with 100ml of methylene chloride, 100ml of water! After washing twice with water, dry with anhydrous sodium sulfate. The residue after evaporation of the solvent was subjected to alumina chromatography using methylene chloride as the eluent (activity I to ■).

140 m, 9
8-(1-methoxyethyl)methylpheophorbite-a is obtained.

405.508.7,585,604,661 Infrared absorption spectrum JL/ (cm', KBr) 8400.29
50,2980,2860°1740.1710,16
20 Nuclear magnetic resonance spectrum (δppm, CD01g) 9.6
2,9.88,8.51,6.18 (s, each
11′f), 5.80(m, IE[), 8.76(q.

2H), 8.89 (s, 8H), 8.60 (s.

8H), 8.56 (S, 6H), 8.41 (S.

8H), 8.28 (s, 8H), 2.87 (m.

4H), 2.18 (d, 8H), 1.80 (d.

8H), 1.65(t, 8H), -1,70(br
.

S, 2H) Example 1-B 265 mIi of 8-(1-methoxyethyl)methylpheophorbite-a obtained in Example 1-A was dissolved in a small amount of pre-warmed pyridine, and 125 m1 of ether was dissolved. Dilute with While introducing air, add 7 ml of potassium hydroxide 2I! Add a solution dissolved in normal propatool. After introducing air for 30 minutes, the reaction solution was
Extract twice with water from Step 6. Discard the ether layer and replace the aqueous layer with 10ml of water! 2 ml of concentrated sulfuric acid diluted with! After acidifying with water, extract with methylene chloride. The extract is immediately treated with an excess of an ethereal solution of diazomethane (prepared from 1,F nitrodimethyurea and 7 ml of 20% (w/v) potassium hydroxide solution). After standing at room temperature for 10 minutes, the solvent was distilled off, and the residue was purified by alumina column chromatography (activity 1 ml, Mark 50.!7) to give 1
40 m, 9+ 3-(1-methoxyethyl)-purpurin-7-drimethyl ester is obtained.

1m Electronic spectrum (λ, ClIC31B) ax 408.505.8.540.4.604.

Infrared absorption spectrum (am, [, Br) 8500.
29.60.2940.2880.

1780.1600 Nuclear magnetic resonance spectrum (δppm, CD0Is)9
．． 50 (S, 2E[), 8.88 (S, IH).

5.58 (m, IH), 4.70 (m, LH).

4.80 (m, IH), 4.18 (s, 8H).

8.81 (8, 8H), 8.61 (S, 8H).

8.50 (S, 6I (), 8.81 (S, 8H).

8.15 (S, 8H), 2.10 (m, 4H).

2.09 (d, 3H), 1.80 (d, 8T ().

1.68 (t, 8H), -0,1(br,s
, 2H) Example 2 60 rl of 3-(1-methoxyethyl)purpurin-7-drimethyl ester obtained in Example 1-B was dissolved in 1% KOH-
Dissolve in 20ml of MeOH, add 1 drop of distilled water, and heat under reflux for 2 hours. After cooling, the solvent was distilled off and the residue was evaporated to 2 Q m
lf: Soluble in water. 0.1ml of acetic acid! was added, the precipitated crystals were collected, washed with water, and dried to obtain 2-(1
-methoxyethyl) purpurin was obtained.

1m Electronic spectrum (λ, PBS) aX 896.4.500.546.608 (sh).

Infrared absorption spectrum (cm, KBr) 8820.2
960.2925.2860.

1780 (sh), 1710.1600 Example 3 Synthesis of 8-(1-(2-methoxyethyloxy)ethyl)-purpurin-7-drimethyl ester (R1=(CH2CH20)no, R3 in general formula (1)
Compound where n=lR2=CHB, R3=OHs) Example 8-A After treating 200 m, p methylpheophorbite-a with a hydrobromide acetic acid solution in methylene chloride in the same manner as in Example 1-A, Dissolve this in 20ml of methylene chloride! , 1ml
! Add ethylene glycol monomethyl ether of 8
Heat and stir at 0°C for 6 hours.

After cooling, methylene chloride 100 mI! diluted with distilled water 10
0m1! Wash 6 times with After drying over anhydrous sodium sulfate, the solvent was distilled off and the residue was subjected to alumina column chromatography using methylene chloride as an eluent (activity 1 to I[, 10
110m, 9's 8-(1
-(2-methoxyetheroxy)ethyl)-pheophorbite-a was obtained.

nm.

Electronic spectrum (λ, CHClg) aX 405.504.585.5.608.5.

Infrared absorption spectrum (cm-', KBr) 8400
．． 2960.2980.2870.

1740. 1690. 1620 Nuclear magnetic resonance spectrum (δppm, CDOIg)9
-67 m 9-42 * 8.47 * 6-22
(s, eachIH), 5.98 (m, IH), 4
．． 40~4.10 (m, 4H), 8.90~
8.50 (m.

2H), 4.17(8,8I(), 8.64(8°8,
H), 8.62(s, 81F(), 8.54(s.

8H), 8.85 (S, 8H), 8.28 (8.

LH), 2.80 (m, 4J'I), 2.13 (d.

8H), 1.87 (d, 8H), 1.67 (t.

8H), -1,80(br,s,2I() Example 8-B 8-(1-(2-methoxyethyloxy)ethyl)-pheophorbite-a t4om obtained in Example 2-A
Dissolve y in a small amount of pre-warmed pyridine and add 60 ml of ether! Dilute with While introducing air, a solution of potassium hydroxide II in 8.5 liters of normal propatool is added. After introducing air for 80 minutes, 50ml of reaction solution was added! Extract twice with water. Discard the ether layer and add 5 mI of water to the aqueous layer! Add 1 m/ml of concentrated sulfuric acid diluted with water to make it acidic, and then extract with methylene chloride. The extract is immediately treated with excess diazomethane in ether and left at room temperature for 10 minutes. The residue after evaporation of the solvent was collected by alumina column photography (activity I to ■).

Mark, 25Ii) with 40m# 8-(1-(
2-Methoxyethyloxy)ethyl)purpurin-7-
Obtain dolimethyl ester.

8m Electronic spectrum (λ, 0HC13) ax 408.504.6.589.8.604.

Infrared absorption spectrum (Cm-1, KBr) 8400.
2960, 2920, 2860°1789. 1700
．． 1 620 Nuclear magnetic resonance spectrum (δppm, CD01a)9.
70, 9.2B, 8.50, 6.20 (s,
eachIH), 6.01 (m, IH),
4.88-8.98 (m, 4H), 8.80 (Q, 2H
).

4.15 (8, 8H), 8.79 (S, 8H).

8.60 (S, 8H), 8.52 (S, 8H).

8.50 (S, 8) I), 8.88 (S, 8H).

8.14 (S, 8H), 2.1'8 (m, 4FI).

2.08 (d, 8T (), 1.79 (d, 8H).

1.67 (t, 3H), -0,2(br, s,
2H) Example 4 8-(1-(2-methoxyethyloxy)ethyl)-7-barbrin trimethyl ester 6 obtained in Example 2-B
0m, 9 in the same manner as in Example 2, 8 of 23rQ
-(1-(2-methoxyethyloxy)ethyl)-7-
Got Purpurin.

8m Electronic spectrum (λ, PBS) ax 896.6,499.8,545,604° Infrared absorption spectrum (cm', xnr) 8870.2960.29
20.2870.

1720. 1610 Reference example 3 week old BALB/C! IX 10-1 m-8A cells derived from mouse renal fibrosarcoma were transferred to the back of the mouse, and two to three weeks later, the pheophorbite derivative obtained in the present invention was transferred to the back of the mouse at a concentration of 20 m, p per mouse body weight IKII. PB8
The solution was administered intravenously in the tail vein. After 24 hours, each organ and cancer cells were removed, and an excimer dye laser endoscope diagnostic device (Katsuo Otozawa et al., Journal of the Laser Medical Society, Vol. 5, pp. 68-68 (1)
984)) at a wavelength of 405 nm, 600-8
The fluorescence spectrum caused by the pheophorbite derivative at 00 nm was measured.

The results are shown in Table 1 as the ratio of the fluorescence intensity at the normal site to the fluorescence intensity at the tumor site and the fluorescence intensity at the tumor site.

The compound obtained in Example 2 is referred to as (1).

EIp is hematoporphyrin% HpD is the Por described above.
phyrin Localization and T
reatment of Tumors, 75-78
This is a porphyrin derivative obtained according to the description of P., (1984).

Table 1 (Effects of the Invention) The compounds according to the present invention are useful compounds as diagnostic and therapeutic agents for cancer, having specific affinity for tumor tissues and photosensitizing action.

Claims (1)

[Claims] 1) General formula▲There are mathematical formulas, chemical formulas, tables, etc.▼ [In the formula, R_1 is (CH_2CH_2O)_nCH_3
(n indicates an integer from 2 to 6), R_2 is CH_3, CH
O or CH_2OH, R_3 represents H or CH_3] or a salt thereof.