JPH01221382A - Novel chlorin derivative and diagnosticum for tumor - Google Patents

Abstract

NEW MATERIAL:A compound expressed by formula I (X represents H or alkali metal such as Na and K). USE:A diagnosticum for tumor. PREPARATION:A product (chlorin-e6) from hydrolysis of a chlorophyll a expressed by formula II under alkaline conditions is dissolved into a 10-20 times amounts of acetic acid, to which a chloride, sulfate or acetylacetonate of V is added, and the resultant solution is refluxed at 130-140 deg.C for 1-3hr, whereby a metal ion is introduced into chlorin-e6 and the reaction liquid is changed from dark brown to dark green. After the reaction is completed, the solvent is distilled off under reduced pressure and the residue is extracted with a 0.1% NaOH/methanol solution to extract a metal chelate compound of chlorin- e6 and separate the unreacted metal salt. The extract is distilled under reduced pressure to afford a green - dark green compound expressed by formula I.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a novel lolin derivative and a tumor 1g1 diagnostic agent containing the same as an active ingredient.

[Conventional technology]

It is generally known that porphyrin compounds have an affinity for tumor tissues. Utilizing this property, a diagnostic method has been put into practical use in which porphyrin compounds, especially hematoporphyrin derivatives, are administered to a patient, and after a certain period of time, the fluorescent light produced by irradiation with laser light is detected to confirm the site of the tumor. I am "being".

However, ItjI occurs deep where it is difficult for the irradiation light to reach.
t Jj& was difficult to detect.

[Problem to be solved by the invention]

The present invention has 1lff! of the prior art! The purpose of the present invention is to synthesize a new lolin derivative to improve its shortcomings as a tumor diagnostic agent, and to provide this as a tumor diagnostic agent.

[Means to solve the problem]

The present inventors have already found that the vO chelate compound of pheophorbite-a represented by formula (11) accumulates in the IIIJI tissue, and that the 41yo chelate compound has the effect that the 41yo chelate compound emits γ
We have discovered that it is possible to detect tumors that occur deep within the body by using T-ray cameras and positron cameras, and we are currently applying for a patent [Patent Application No. 198247/1983]
However, the compound represented by formula (n) is insoluble in water, and when using the compound as an injection, emulsification techniques such as liposome formation are required.

While continuing this research, the present inventors found the following formula (
A vO chelate compound of an alkali hydrolysis product of chlorophyll-a (chlorin-ei), represented by ■ Coo\ [in the formula, represents ■ or an alkali metal such as Na or K] solves the above problem. This discovery led to the completion of the present invention.

That is, the present invention provides a novel chlorin derivative represented by the formula [I], [wherein X represents H or an alkali metal such as Na or K], and a tumor containing the compound represented by the formula [I] as an active ingredient. It is a diagnostic agent.

The drug according to the present invention is also useful as a tumor therapeutic agent for inhibiting tumor growth and killing tumors by administering the drug to accumulate it in tumors, and by the radiation action of β1 rays and γ rays emitted. .

A general method for producing the compound of formula (I) will be described below.

Dissolve the compound of formula (I[l) in 10 to 20 times the amount of acetic acid, add an appropriate amount of V chloride, sulfate, or acetylacetonate, and heat this solution at 130 to 140°C for 1 to 3 hours. When refluxed, metal ions are converted to chlorin-e.

After the reaction, the color of the reaction solution changed from dark brown to dark green, the solvent was distilled off under reduced pressure and 0.1% Na011 was added to the residue.
The chlorin-eb gold chelate compound is extracted using a methanol solution and separated from unreacted metal salts.

When this is distilled under reduced pressure, a green to dark green compound of formula (1) is obtained.

The compound of formula (1) is soluble in water, methanol, and acetic acid, but is insoluble in acetone, benzene, chloroform, and the like.

Moreover, the acetic acid solution of the compound of formula (1) has a visible part of 640 to 6
'50, and has an absorption maximum at 405 to 415 nm, indicating that it retains a chlorin ring, which is the basic skeleton of chlorophyll derivatives.

Next, the tumor diagnostic effect using the compound of the present invention will be explained.

[Experimental Example 1] Mouse breast cancer (FM3A) cells were subcutaneously implanted into the back of 8×10 cells/individual, and solid tumors were generated by raising them for about 2 hours, and the cells were treated with vO-pheophorbite-a and vO
-Chlorin-e& was intravenously injected into each animal through the tail vein at a dose of 5 μ/kg body weight.

After administration, the cells were cultured at 2 hours, 16 hours, 24 hours, and 48 hours, and the uptake into each organ was measured.

The measurement method is as follows.

vO-Chlorin-e& is prepared by preparing organ tissues in 10 times the amount of acetone: methanol: 0.9N perchloric acid (45: 10:
Homogenize in a 10V/V) solution and obtain a supernatant by centrifugation. After drying this supernatant, it was extracted with methanol and used as a sample for high performance liquid chromatography (hereinafter abbreviated as +1PLC). Also, vO-pheophorbite-a
Homogenize the organ tissue in 10 times the volume of 85% acetone and centrifuge to obtain the supernatant. Add half the amount of ether and a small amount of water to this supernatant, shake it, and add vO to the ether.
-Pheophorbite-a was extracted, the ether was dried, and then dissolved in methanol to prepare a sample for II PLC.

These samples were measured using the IIPLc analysis method, and from the absolute calibration curve of each standard, each 'W? The amount of uptake into organ A tissue was calculated.

The results are shown in Tables 1 and 2.

(Margins below this page) Table 1: Organ side distribution of vO-pheophorbite-a
iver 21.49±1.17 11.24±1.
48 5.68±0.44 4.40±0.46Kid
ney 10.72±0.43 7.51±0.54
4.11±0.28 1.65±0.13Splee
n 7.39±1.35 5.13±0.49 3.
95±0.11 1.34±0.08Muscle
1.21±0.26 0.92±0.07 0.84±
0.09 0.60±0.08ff12 Table Organ side distribution of vO-chlorin-e Liver 7.70±0
．． 37 3.04±0.16 1.14±0.01 0
．． 65±0.02 Kidney 8.26±0.29
2.14±0.20 1.32±0.23 N,
DSpleen 6.55±0.57 1.80±0
．． 11 1.44±0.19 0.88±0. ■OMu
scle 2.60±0.20 0.45±0.07
0.1B±0.02N, DPecas 10
．． 92±0.19 11.25±0.16 5.84±
0.44 3.18±0.15vO-Pheophorbite-a is highly taken up in the liver, kidneys, and lungs, but decreases significantly over time. Uptake into tumors is initially small but increases over time. , and reached its highest level 24 hours later. That is, vO-pheophorbite-
Regarding the in vivo kinetics of a, uptake peaked after 2 hours in normal tissues and was highest in the liver. While excretion from normal tissues was very smooth, it tended to accumulate gradually in tumors.

Uptake and excretion into normal tissues is extremely rapid for vO-chlorin-e6. It can be seen that although it gradually decreases over time in tumor tissue, the persistence is higher than in normal tissue.

By the way, the degree of persistence in the tumor tissue after 24 hours is vO-
In pheophorbite a, 2.13 times that of the liver;
2.95 times that of kidney, 3.07 times that of lung, 14.4 times that of muscle.
Compared to 2 times, vO-chlorin-eb has 4 times more in liver.
．． It was 69 times larger, 4.05 times larger than a kidney, 3.72 times larger than a lung, and 29.72 times larger than a muscle. This is radionuclide 4@
In the detection of 1lffl 7B tissue from outside the body by positron tomography using a vo chelate compound, vO-chlorin-○ has been shown to be able to visualize tumors with better contrast than vO-phe1-phorbite-a.

As shown in the above experimental examples, the compound represented by formula [I], Jlvo-chlorin-e6, can detect tumor sites in any part of the body due to its tumor tissue affinity and radioactivity. .

Note that nuclides other than 41y include Zn, Cu, Ni, and C.
o, Fe.

Mn, Ag, In, l1g, TI, Sn,
Pt, Rh, Ir, Cd+ Si, Ge+ p
b, Ga, Cr.

Mo, Zr, old, Eu, Pr, Yb, YtTh, Ta,
W, Re, Os, Nb, Sb, old.

Chlorine, a radioactive isotope of elements such as Re, Ru, Ti, etc.
e.

JIIV, although complexes also satisfy the objects of the invention.

4SH is the most effective.

In particular, S S Co, S 2 p, which are frequently used in nuclear medicine.
e, 54 p.e.

44Sc, "Mn1" Cr+ "Cu, a to 6ff
, 6nGa1 ”Get+11■,, 9(I+/,
ZI03i, 2 (IIH etc. are valid, 111
1n, 90Y, ! ++1Bi is promising as a therapeutic agent.

Next, the acute toxicity of the compound represented by 2 [I] was tested on rats, and the results were shown as LD. (mg/kg body weight) as shown in Table 3.

Table 3 Animal Administration Compound t, aS. (mg/k
g body weight) Raft ♂ II Intracavitary administration VO-Chlorin-e, 270m5Next, the compound represented by formula (I) is mixed with a suitable solvent, adjuvant,
It can be made into an injection using a filler, a carrier, etc. according to conventional methods for manufacturing pharmaceuticals. This stuff includes veins, muscles,
Can be administered subcutaneously, subcutaneously, or intraperitoneally.

The effective dose of the compound represented by formula (I) is appropriately selected depending on the intended use, but is usually 10 to 10 mg per dose.
A range of about 8 to 500 μg/m is considered appropriate.

〔Example〕

Next, examples of the present invention will be shown. Naturally, the following examples are merely preferred embodiments of the present invention, and the present invention is not limited in any way by these examples.

Example 1 Vanadyl acetylacetonate ((CIhCOCHCO
CIIz) xvO] 3■ is dissolved in 1.0-acetic acid, and 5■ of chlorin-eb-Na is added thereto. The mixed solution is heated and stirred at 130 to 140"C for 1 hour. After the reaction, acetic acid is distilled off under reduced pressure, and the residue is extracted using 10 III l of a 0.1% Na011 methanol solution. After distilling the extract under reduced pressure, The residue was crystallized from hot ethanol, (1)
3.7 μm of dark green crystals were obtained. The yield was 74%.

The physical and chemical properties of this substance are as follows.

・Visible spectrum (in acetic acid solvent): As shown in Figure 1 ・Thin layer chromatogram: As shown in Figure 2. When analyzed by silica gel thin layer chromatography using aqueous ammonia (2:1:1 (V/V)), as shown in FIG.
A spot was given near Rf 0.4.

Furthermore, as shown in Figure 1, the UV absorption spectrum of the acetic acid solution of the generated vO-chlorin-e & (500-60
0 nm) have disappeared, and the spectrum has shifted to the shorter wavelength side. Furthermore, it does not emit fluorescence even when irradiated with an ultraviolet lamp. The above facts indicate that vanadyl acetylacetonate reacts with chlorin-eh to form a vanadyl chlorin-eh complex, and the structure of vanadyl chlorin-e is composed of oxyvanadine and chlorin. It is recognized that the formula (1) is as follows.

Example 2 5 mg of vO-chlorin-e6Q was dissolved in 5 rnl of a phosphate buffer solution at pH 7.2 and filtered through a Membrane filter with a pore size of 0.45 μI to prepare an injection solution.

〔Effect of the invention〕

According to the present invention, vO-chlorin-eh, like vO-pheoforoid-a, can be used to treat tumors that develop deep within the body using γ-ray cameras and positron cameras when administered at low concentrations. It is a more useful tumor diagnostic agent in that it can detect the site, visualize tumors with better contrast than vO-pheoforoid-a, and since it is water-soluble itself, it can be made into an injection without emulsification.

[Brief explanation of the drawing]

Figure 1 shows the visible spectra of the cyO-chlorin-e6 obtained in Examples and chlorin-e6 represented by the formula (I[I), and Figure 2 shows the visible spectra of the same compound and chlorin-e6 represented by the formula (Ill). This is a thin layer chromatogram of .

Claims (1)

[Claims] 1. A novel chlorin derivative represented by formula [I]. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [I] [In the formula, X represents H or an alkali metal such as Na, K, etc.] 2. A tumor diagnostic agent containing a compound represented by the formula [I] as an active ingredient. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [I] [In the formula, X represents H or an alkali metal such as Na, K, etc.]