JP2638042B2 - Novel chlorin derivatives and tumor diagnostics - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a novel chlorin derivative and a tumor diagnostic agent containing the same as an active ingredient.

[Conventional technology]

In general, porphyrin compounds are known to have an affinity for tumor tissues. Utilizing this property, a diagnostic method has been put to practical use in which a porphyrin-based compound, particularly a hematoporphyrin derivative, is administered to a patient, and after a certain period of time, fluorescence generated by irradiation with laser light or the like is detected, thereby confirming the site of tumor occurrence. ing.

However, it has been difficult to detect deeply developed tumors that are difficult to reach with irradiation light.

[Problems to be solved by the invention]

An object of the present invention is to synthesize a novel chlorin derivative in order to improve the disadvantages of the prior art as a tumor diagnostic agent, and to provide this as a tumor diagnostic agent.

[Means for solving the problem]

The present inventors have already accumulated a VO chelate compound of pheophorbide-a represented by the formula (II) in tumor tissue, In addition, ⁴⁸ VO chelate compound has been applied for a patent, and it has been discovered that the γ-rays emitted from the VO chelate compound can be used to detect tumors that have developed deep inside the body using a γ-ray camera or a positron camera. 61-198247]. However, the compound represented by the formula (II) is insoluble in water,
When the compound is used as an injection, an emulsification technique such as liposome formation is required.

The present inventors further continued this research, and the following formula [III] [Wherein X represents H or an alkali metal such as Na, K] The VO chelate compound of an alkali hydrolyzate of chlorophyll-a (chlorin-e ₆ ) represented by The present invention has been completed.

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 diagnostic agent comprising a compound represented by the formula [I] as an active ingredient.

The drug according to the present invention is also useful as a tumor therapeutic drug for inhibiting the growth and killing of tumors by administering the drug, accumulating the drug in the tumor, and irradiating β ⁺ rays and γ rays emitted from the drug. It is.

Hereinafter, a general method for producing the compound of the formula [I] will be described.

A compound of the formula (III) is dissolved in 10 to 20 times the amount of acetic acid, an appropriate amount of chloride, sulfate or acetylacetonate of V is added thereto, and the solution is heated and refluxed at 130 to 140 ° C. for 1 to 3 hours. , metal ions are introduced into the chlorin -e _6, the reaction solution changed from dark brown to dark green. After the reaction, the solvent is distilled off under reduced pressure, and the chlorin-e ₆ metal chelate compound is extracted from the residue using a 0.1% NaOH methanol solution, and separated from unreacted metal salts.

When this is distilled under reduced pressure, a green or dark green place of the formula [I]
Is obtained.

The compound of the formula [I] is soluble in methanol and acetic acid, but insoluble in acetone, benzene, chloroform and the like.

An acetic acid solution of the compound of the formula [I] has a visible part of 640 to 65.
It has an absorption maximum at 0 and 405 to 415 nm, and indicates that the chlorophyll derivative retains a chlorin ring which is a substrate skeleton.

Next, the results of tumor diagnosis using the compound of the present invention will be described.

[Experimental Example 1] A mouse in which mouse breast cancer (FM3A) cells were subcutaneously transplanted to the back at 8 × 10 ⁶ cells / individually was bred for about 2 weeks to form a solid tumor, VO-pheophorbide-a, and VO-chlorin. the -e ⁶ from the tail vein, was injected intravenously each 5 mg / kg body weight.

Sacrifice 2 hours, 16 hours, 24 hours, 48 hours after administration,
The uptake into each organ was measured.

 The measuring method is as follows.

VO- chlorine -e ₆ is, organ tissues 10 times the amount of acetone:
Homogenize in a methanol: 0.9N perchloric acid (45:10:10 V / V) solution and obtain a supernatant by centrifugation. After the supernatant was dried, it was extracted with methanol and used as a sample for high performance liquid chromatography (hereinafter abbreviated as HPLC). In addition, VO-pheophorbide-a is obtained by homogenizing an organ tissue in a 10-fold amount of 85% acetone and centrifuging to obtain a supernatant. Add half amount of ether and a small amount of water to the supernatant, shake,
VO-Pheophorbide-a was extracted into ether, and the ether was evaporated to dryness and then dissolved in methanol to prepare a sample for HPLC.

These samples were measured by the HPLC analysis method, and the uptake amount into each organ tissue was calculated from the absolute calibration curve of each sample.

 The results are shown in Tables 1 and 2.

The biodistribution of VO-pheophorbide-a is highly taken up by the liver, kidney and spleen, but decreases significantly with time. Tumor uptake is low at first but rises over time, peaking after 24 hours. In other words, in vivo kinetics of VO-pheophorbide-a showed that the uptake reached a peak after 2 hours in normal tissues and was highest in the liver. Excretion from normal tissues was very smooth, whereas tumors tended to accumulate gradually.

In VO- chlorine -e _6, significantly faster uptake and excretion to normal tissue. It can be seen that although the tumor tissue also gradually decreases over time, the residual property is higher than that of the normal tissue.

By the way, the residual degree in the tumor tissue after 24 hours was 2.13 times that of the liver, that of the kidney in VO-pheophorbide-a.
Compared to 2.59 times, 3.07 times of spleen and 14.42 times of muscle, V
4.69 times the liver in O- chlorine -e _6, 4.05 times the kidney, 3.72 times that of the spleen, was as large as 29.72 times the muscle. This is because the detection of tumor tissue from outside the body by positron tomography using a radionuclide ⁴⁸ VO chelate compound shows that VO-
Chlorin -e ₆ shows can be good contrast draw further tumor from VO- pheophorbide carbide -a.

As shown in the above embodiments, compounds of Formula (I), ⁴⁸ VO- chlorin -e _6, by their tumor tissue affinity and radioactivity can be detected in tumor occurrence site in any part of the body It is.

As nuclides other than ⁴⁸ V, Zn, Cu, Ni, Co, Fe, Mn, Ag,
In, Hg, Tl, Sn, Pt, Rh, Ir, Cd, Si, Ge, Pb, Ga, Cr, Mo, Zr, Hf, E
u, Pr, Yb, Y, Th, Ta, W, Re, Os, Nb, Sb, Bi, Re, Ru, the desired chlorin -e ₆ complexes of radioactive isotopes of elements such as Ti likewise present invention Satisfied, but ⁴⁸ V, ⁴⁵ Ti is most effective.

In particular, ⁵⁵ Co, ⁵² Fe, ⁵⁴ Fe, ⁴⁴ Sc, ⁵² M frequently used in nuclear medicine
n, ⁵¹ Cr, ⁶⁴ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ⁷¹ Ge, ¹¹¹ In, ⁹⁰ Y, ²¹⁰ Bi, ²⁰¹ Ti and the like are effective, and ¹¹¹ In, ⁹⁰ Y, ²¹⁰ Bi is promising as a therapeutic agent.

Next, the results of an acute toxicity test of the compound represented by the formula [I] in rats are shown in Table 3 as LD ₅₀ (mg / kg body weight).

Next, the compound represented by the formula [I] can be made into an injection using a suitable solvent, an auxiliary agent, a bulking agent, a carrier and the like used in the preparation according to a conventional method of preparation of the preparation. It can be administered intravenously, intramuscularly, intradermally, subcutaneously or intraperitoneally.

The effective dose of the compound represented by the formula [I] is appropriately selected depending on the purpose of use.
A range of about 590 μg / kg is considered appropriate.

〔Example〕

Next, examples of the present invention will be described. Naturally, the following merely illustrates preferred embodiments of the present invention, and the present invention is not limited by these embodiments.

Example 1 Vanadyl acetylacetonate [(CH ₃ COCHCOCH ₃ ) ₂ V
O) was dissolved in 1.0 ml of acetic acid, and chlorin-e ₆ -Na was added thereto.
5 mg is added. This mixture is heated and stirred at 130 to 140 ° C. for 1 hour. After the reaction, acetic acid was distilled off under reduced pressure.
Extract with 10 ml of aOH methanol solution. After vacuum distillation of the extract, the residue is crystallized from hot ethanol,
3.7 mg of dark green crystals of [I] were obtained. The yield was 74%.

 Its physicochemical properties are as follows.

・ Visible spectrum (in acetic acid solvent): As shown in Fig. 1 ・ Thin layer chromatogram : As Figure 2 i.e., the VO- chlorine -e ₆ produced by this embodiment eluent (n- butanol: ethanol: 28% ammonia water = 2: 1: 1 (V / V)) silica gel thin layer When analyzed by chromatography, the product gave a spot near Rf 0.4, whereas chlorin-e ₆ near Rf 0.12, as shown in FIG.

The UV absorption spectrum of the acetate solution of the resulting VO- chlorin -e ₆ absorption characteristic Soret band porphyrin was observed at 410nm as shown in FIG. 1, Q band observed in chlorine -e ₆ free (500 to 600 nm), the absorption has 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-e ₆ to form a vanadyl chlorin-e ₆ complex, and the structure of vanadyl chlorin-e ₆ is based on chlorinated oxyvanazine. Is coordinated as in Formula [I].

Example 2 VO- chlorin -e ₆ 0.5 mg, phosphate buffer pH 7.2 5 ml
And filtered through a membrane filter having a hole diameter of 0.45 μm to obtain an injection solution.

〔The invention's effect〕

According to the present invention, VO-chlorin-e ₆ is capable of producing γ-
X-ray camera, a tumor generated deep inside the body using a positron camera can be detected, but the tumor can be drawn with better contrast than VO-pheophorid-a,
Since it is water-soluble in itself, it is a more useful tumor diagnostic agent in that it can be used as an injection without emulsification.

[Brief description of the drawings]

Figure 1 is a visible spectrum of chlorin -e ₆ represented by obtained VO- chlorin -e ₆ and equation in Example (III), Figure 2 is chlorine -e represented by the compounds and the formula [III]
_{6 is} a thin layer chromatogram of FIG.

Claims (2)

(57) [Claims] 1. A novel chlorin derivative represented by the formula [I]. [Wherein X represents H or an alkali metal such as Na, K] 2. A diagnostic agent for a tumor comprising a compound represented by the formula [I] as an active ingredient. [Wherein X represents H or an alkali metal such as Na, K]