JPH0615545B2 - Metal pheophobide derivatives and metal porphyrin derivatives - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application The present invention relates to a novel cancer-directing preparation containing, as an active ingredient, a metal complex of pheophobide and a porphyrin derivative for the treatment, diagnosis, marker of cancer and missile therapy. .

(B) Conventional technology The present inventors have long been engaged in the research of chlorophyll-related compounds, and as a method for obtaining pheophobide, the cost of pheophobide, which is excellent in economic efficiency, is obtained by using a photosynthetic organism having a cheap and stable source material. The separation method was established (Japanese Patent Publication No. 58-69884).

The present inventors found for the first time that the plant-derived pheophobide derivative has the property of selectively binding to cancer tissue and reacting when exposed to light (Japanese Patent Application No. 58-198934,
59-June 22), and blood-derived porphyrin derivatives have conventionally been known to have these properties. However, it was not considered that the metal-coordinated pheophobide and porphyrin derivatives had the cancer concentration and the absence of the reaction to light.

The present inventors have previously applied for a pheophobide compound, that is, a forbin derivative derived from plant chlorophyll (Japanese Patent Application No. 58-198934 59-6-22, a prior application substance) and a porphyrin compound, that is, derived from animal hemoglobin. Porphyrin derivatives are known to have binding to cancer tissues and photodynamic properties, and are being put to practical use for cancer treatment.

Examples of analogs of metal pheophobide derivatives and metalloporphyrin derivatives (hereinafter referred to as “substances of the present application”) include animal-derived hemin (porphin Fe complex), plant-derived chlorophyll (phorbin Mg complex), Cu and Fe chlorophyllin (phorbin Cu and Fe complex), CO-protoporphyrin (Cu complex of porphine), Hg-porphyrin (Hg complex of porphine) [Tasaki et al., Cancer Chenmothe
rapy Reports, 13, 41 (1961)] etc. Moreover, in recent years, Fukuda et al.
7-31688), and Umino et al., A method for producing a hematoporphyrin Si derivative (Japanese Patent Publication No. 58-201793).

However, hemin is a blood component and chlorophyll is a plant component Cu.
And Fe chlorophyllin are colorants and deodorants of food additives, and their cancer affinity is not recognized. C
O-protoporphyrin has been approved as a carcinostatic drug until recently, but the Pharmaceutical Affairs Council (August 10, 1982).
The item was rejected in Japan. The Hg-porphyrin derivative also has an action as an anticancer agent, but the toxicity of Hg is unsuitable as a drug. Regarding Ge-protoporphyrin, it is described that it has an antitumor effect as well as an effect of improving liver dysfunction, but its data has not been published, and there is also a word on the point of selective accumulation in cancer cells. Not not. The hematoporphyrin Si derivative is an organometallic compound in which the Si metal is not coordinated to the nitrogen atom of the porphyrin skeleton, which is our objective, and the Si metal is directly bonded to the side chain of the skeleton, and therefore has phototoxicity. .

(C) Problems to be Solved by the Invention Pheophobide and porphyrin compounds are useful substances having all the selective accumulation in cancer cells, the reactivity to light, and the cancer-destroying property. On the other hand, the action, on the other hand, acts as phototoxicity, increasing the difficulty in actual treatment. That is, cancer patients have to live in the dark for 48 to 72 hours after administration of these drugs, which is extremely distressing. Further, depending on the patient, even after 1 to 2 weeks after the administration, phototoxicity may occur due to the high cancer accumulation of these compounds. Therefore, these compounds could not be used as diagnostic agents.

The present invention aims to eliminate the phototoxicity of these compounds and obtain new cancer therapeutic agents and diagnostic agents.

(D) Means for Solving the Problems Therefore, the inventors of the present invention can open up applications as new cancer therapeutic agents and diagnostic agents if these pheophobide and porphyrin derivatives are used as metal complexes and phototoxicity is eliminated. As a result of extensive studies, the metal complex of Mg, Si, Ni, Zn, Ga, Ge, In, Pt, etc. of the previous substance and the porphyrin derivative showed remarkable accumulation and external energy in cancer cells. It has strong pharmacological effects such as strong reactivity with and destruction, while the phototoxicity of pheophobide and porphyrin derivatives does not exist in this metal complex compound.
It was found to be extremely excellent.

Next, a method for producing the compound of the present invention will be described.

The formula (1), which belongs to the metal pheophobide derivative, is the method for producing the substance of the present invention.
Treated with ₄ , GaCl ₃ , GeCl ₄ , InCl ₃ or K ₂ PtCl ₆ , Si-9
-Desoxo-9-hydroxyphenophobide (hereinafter referred to as I), Ga-2-desethenyl-2- (1-
Acetyloxyethyl) -Pyropheophobide (hereinafter
II), Ga-ethylene glycol di-10b-pheophorbate (hereinafter III), Ge-2-desethenyl-2- (1-hydroxyoxy) -9-desoxo-9-hydroxy-pheophorbide (hereinafter IV)
, In-monoacetylethylene glycol, mono-10b-pheophorbate (hereinafter referred to as V), and Pt-pheophobide (hereinafter referred to as VI).

In the practice of the present invention, with respect to metal complexes such as Si, Ga and Ge, the substances of the prior application are dissolved in pyridine, then each metal salt is added and reacted under heating and stirring to obtain I, II, III and IV, respectively. For metal complexes of In, Pt, etc., the substances of the previous application are dissolved in acetic acid, and then each metal salt is added with heating and stirring to cause reaction V and VI. The end points of these reactions can be easily determined by thin layer chromatography or UV handcorp.

Further, in the general formula (1), the substance of the present application belonging to the metalloporphyrin derivative is obtained by subjecting the side chain of the porphyrin skeleton to hydrobromide treatment or the hydroxide obtained after the treatment in the same manner as in the case of obtaining the prior application substance. Acetylate and prepare hydroxide or acetylated porphyrin. Then,
The obtained compounds were added to Mg (ClO ₄ ) ₂ , GaCl ₃ , GeCl ₄ , Ni (O
Ac) ₂ , Zn (OAc) ₂ , InCl ₃ or Eu (AA) _{3 and} treated with Mg-di-2.4- (1-ethanealcoholoxyethyl) -deuteroporphyrin (hereinafter referred to as VII), Ga- The -2.4-
(1-ethane alcohol oxyethyl) -deuteroporphyrin (hereinafter referred to as VIII), Ge-di-2.4- (1-
Ethane acetate oxyethyl) -deuteroporphyrin (hereinafter referred to as IX), Ni-hematoporphyrin (hereinafter referred to as X), Zn-di-2.4- (1-ethane alcoholoxyethyl) -deuteroporphyrin (hereinafter referred to as XI), In -Hematoporphyrin (hereinafter referred to as XII), Eu
-Di2.4- (1-ethane alcohol oxyethyl) -deuteroporphyrin (hereinafter referred to as XIII) is produced respectively.

In the practice of the present invention, for metal complexes such as Mg, Ga and Ge, each porphyrin derivative is dissolved in pyridine, and then each metal salt is added and reacted under heating and stirring to obtain VII to IX, respectively. For metal complexes of Ni, Zn, In, etc., each porphyrin derivative is dissolved in acetic acid, and each metal salt is added with stirring under heating to react to obtain X to XII. For metal complexes such as Eu, each porphyrin derivative is dissolved in trichlorobenzene, then Eu (AA) ₃ is added and reacted with heating and stirring to obtain XIII.

As another method of producing the substance of the present application, a metal complex is formed at the stage of starting material methylpheophorbide or dimethylprotoporphyrin, and then the side chain of each skeleton is hydroxylated, acetylated, dimerized or derivatized. It is also possible to obtain desirable results. It is not limited to this combination as long as it is a hydroxylation reaction, a hydrolysis reaction, an esterification reaction, and a metal complexation reaction.

It is also preferable to add an antioxidant, a solvent and the like as appropriate and heat, but this is not an essential condition.

Examples of starting materials include plant-derived representatives such as pheophobide, and animal-derived representatives such as protoporphyrin, but the starting materials are not limited to these as long as they are obtained from photosynthetic organisms and blood components.

The data of the ultraviolet absorption spectrum of the substance of the present invention are shown in the first column of Table 1.

Table 1 of Table 1 shows the maximum absorption (λmax) nm of each substance.

(E) Action Next, the pharmacological action of the substance of the present application will be explained. The substance of the present invention selectively accumulates in cancer tissues, is slowly excreted from cancer cells, does not react even when exposed to light, and reacts with excitation by external energy other than light, that is, microwaves, electromagnetic waves, etc. Singlet oxygen, which has a strong action, is generated, and the action of this singlet oxygen destroys cancer cells. It is not excreted from normal organs and cells and does not damage normal cells.

Most of the pheophobide and porphyrin derivatives have a strong effect on light, but by using both of these derivatives as a metal complex, the advantageous property of selective accumulation in cancer cells remains, and there is a strong resistance to light. The action can be erased. Therefore, metal complex derivatives are effective as diagnostic agents, tumor markers, and missile therapy because they do not exhibit phototoxicity.

The substance of the present application is a useful substance having all of these properties (cancer affinity, non-phototoxicity, and cancer destructiveness to external energy).

(F) Example Hereinafter, the pharmacological effect and production method of the substance of the present invention will be described with reference to examples.

Example 1 Laser Irradiation in Excised Organs (Excited Fluorescent Pestol) A 14 to 21-day-old golden hamster (5 animals per group) transplanted with nitrosamine-carcining pancreatic cancer cells was diluted with physiological saline (1 ml). Analyte Ga-monoacetylethylene glycol mono-10b-Phoophorbate sodium salt, Mg-di-2.4- (1-ethanealcoholoxyethyl) -deuteroporphyrin (VII) disodium salt, Ge-di-2.4 -(1-Ethaneacetate oxyethyl) -deuteroporphyrin (IX) disodium salt and In-hematoporphyrin (XII) disodium salt were separately administered by 5 mg intravenous injection (1 ml), then cancer cells and other , The organs obtained by removing the N ₂ −
A pulsed laser (N ₂ , 337 nm, ₂ ns 400 to 1000 nm) was irradiated, the excitation fluorescence spectrum was measured, and wavelengths of 600 to 900 nm were examined with the NADH peak wavelength of 470 nm as a reference.

FIG. 1 shows that each drug significantly accumulated in cancer cells after 24 hours and did not remain in other cells.

In addition, the same test was conducted for other drugs.
The results shown in Table 2 were obtained. Table 2 of Table 1 shows the measured fluorescence spectra of each organ extracted after 24 hours.
600-90 with NADH peak wavelength of 70 nm as reference 1
The value which calculated the peak wavelength in 0 nm is shown.

As is clear from the above results, these metalloporphyrin-related compounds have a remarkable selective affinity for cancer cells.

Example 2. 1 g of 9-desoxo-9-hydroxy-pheophobide was dissolved in 90 ml of pyridine and then 0.5 g of SiCl ₄
(Dissolved in 10 ml of pyridine) is added, and the mixture is heated to 165 ° C. in a sealed tube and reacted for 2 hours. The end point of the reaction was determined by thin layer chromatography and an ultraviolet hand scope. After the reaction, add 100 ml of the reaction solution and water and extract with CHCl ₃ . C
The HCl ₃ layer is washed with water, dried and concentrated to obtain red Si-9-desoxo-9-hydroxy-phophophobide (I) (yield 0.7 g). This yield is 60%.

The obtained I is dissolved in methanol, and a 5% Na ₂ CO ₃ aqueous solution is added for neutralization to obtain a sodium salt of I. This yield is 98%.

Example 3. 2-Desethenyl-2- (1-acetyloxyethyl)
Dissolve 1 g of pyropheophorbide in 90 ml of pyridine, then add 0.5 g of GaCl ₃ (dissolved in 10 ml of pyridine), heat to 160 ° C. in a sealed tube, and react for 1 hour. Thereafter, the same procedure as in Example 2 is carried out to obtain a blue-green Ga-2-desethenyl-2- (1-acetyloxyethyl) -pyropheophobide (II) (yield 1 g). This yield is 82%
Is.

Example 4. 1 g of ethylene glycol di-10b-pheophorbate
Was dissolved in 90 ml of pyridine, 0.5 g of GaCl ₃ (dissolved in 10 ml of pyridine) was added, and the mixture was heated to 160 ° C. in a sealed tube and reacted for 1 hour. Thereafter, the same operation as in Example 3
Ga-ethylene glycol-di-10b-pheophorbate (III) is obtained (yield 0.9 g), the yield is 73%.

Example 5. 2-Desethenyl-2- (1-hydroxyethyl)
Dissolve 1 g of -9-desoxo-9-hydroxy-pheophobide in 90 ml of pyridine, then 0.5 g of GeCl ₄
(Dissolved in 10 ml of pyridine) is added, and the mixture is heated in a sealed tube at 180 ° C. and reacted for 2 hours. Thereafter, the same operation as in Example 3 is carried out to give red Ge-2-desethenyl-2- (1-hydroxyoxyethyl) -9-desoxy-9-hydroxy-.
Pheophobide (IV) is obtained (yield 0.8 g), the yield is 66%.

Example 6. 1 g of monoacetyl ethylene glycol mono-10b-pheophorbate was dissolved in 90 ml of acetic acid and then InCl
₃ 0.5 g (dissolved in 10 ml of acetic acid) and 0.5 g of NaCl are added, and the mixture is heated to reflux and allowed to react for 1 hour. The end point of the reaction can be determined by thin layer chromatography and a UV hand scope. After the reaction, add 100 ml of water to the reaction solution and extract with CH ₂ Cl ₂ .

The CH ₂ Cl ₂ layer was washed with water, dried and concentrated to give a green In-monoacetylethylene glycol mono-10b-phenophorbate.
(V) is obtained (yield 1 g). This yield is 80%.

Example 7. 1 g Pheophobide is dissolved in 95 ml acetic acid and then K ₂
Add 0.5 g of PtCl ₆ (dissolved in 5 ml of water) and add 1 g of sodium acetate and heat to reflux to react for 2 hours. Thereafter, the same procedure as in Example 6 is carried out to obtain pt-pheophobide (VI) (yield 1.3
g). This yield is 85%.

Example 8 di-2,4 (1 ethane alcohol oxyethyl) - the Deuteroxyl porphyrin 1g dissolved in pyridine 100 ml, followed by Mg (ClO ₄₎ was added and heated under reflux ₂ 0.5 g, allowed to react for 1 hour. Thereafter, the same operations as in Example 3 were carried out to obtain light pink Mg.
-Di-2.4- (1-ethane alcohol oxyethyl) deuteroporphyrin (VII) is obtained (yield 1 g).

This yield is 92%.

Example 9. 1 g of di-2.4- (1-ethanealcoholoxyethyl) -deuteroporphyrin was dissolved in 90 ml of pyridine,
Next, 0.5 g of GaCl ₃ (dissolved in 10 ml of pyridine) is added, and the mixture is heated to 160 ° C. in a sealed tube and reacted for 1 hour. Thereafter, the same operation as in Example 3 was carried out to obtain crimson Ga-2.4- (1-ethane alcohol oxyethyl) -deuteroporphyrin (V
III) is obtained (yield 1.1 g). This yield is 90%.

Example 10. 1 g of di-2.4- (1-ethaneacetate oxyethyl) -deuteroporphyrin was dissolved in 90 ml of pyridine,
Next, 0.5 g of GaCl ₄ (dissolved in 10 ml of pyridine) is added, and the mixture is heated to 180 ° C. in a sealed tube and reacted for 2 hours. Thereafter, the same procedure as in Example 3 was carried out to obtain light pink Ge-di-2.4- (1-ethaneacetateoxyethyl) -deuteroporphyrin (I
X) is obtained (yield 0.8 g). This yield is 67%.

Example 11. 1 g of hematoporphyrin was dissolved in 90 ml of acetic acid and then
Ni (OAc) ₂ 0.5g (dissolved in 10ml acetic acid), sodium acetate
Add 0.5 g and heat to reflux to react for 1 hour. Thereafter, the same operation as in Example 3 was performed to obtain crimson Ni-hematoporphyrin.
(X) is obtained (yield 1.1 g). This yield is 85%.

Example 12. di-2,4 (1 ethane alcohol oxyethyl) - CHCl ₃ 90m to Deuteroxyl porphyrin di Mel ester 1g
Dissolve in 1 l, then add 0.5 g of Zn (OAc) ₂ (dissolve in 10 ml of methanol) and heat to reflux to react for 1 hour. Thereafter, the same procedure as in Example 3 is carried out to obtain a Zn complex. The obtained complex was hydrolyzed with alkali by a conventional method and Zn di-2.4- (1-
Ethane alcohol oxyethyl) -deuteroporphyrin (XI) is obtained (yield 0.9 g). This yield is 73%.

Example 13. 1 g of hematoporphyrin was dissolved in 90 ml of acetic acid and then
Add 0.5 g of InCl ₃ (dissolved in 10 ml of acetic acid) and sodium acetate and heat to reflux to react for 1 hour. Thereafter, the same procedure as in Example 6 is carried out to obtain In-hematoporphyrin (XII) (yield
0.7 g). This yield is 51%.

Example 14. 1 g of 2.4- (1-ethanealcoholoxyethyl) -deuteroporphyrin dimethyl ester is dissolved in 100 ml of trichlorobenzene, 0.5 g of En (AA) ₃ is added and the mixture is heated under reflux and reacted for 2 hours. The obtained reaction liquid was subjected to silicic acid column chromatography. First, trichlorobenzene was removed with n-hexane, and then the desired product was eluted with ethyl acetate and then concentrated. The obtained Eu complex was hydrolyzed with alkali by a conventional method to give a red Eu-2.4-di- (1-ethanealcoholoxyethyl) -deuteroporphyrin.
(VIII) is obtained (yield 1 g). This yield is 65%.

(G) Effect of the Invention The compound of the present invention has pharmacological effects such as accumulation in cancer cells, reactivity to external energy, and destruction of cancer cells, and has no reactivity to light. This compound is extremely useful as a cancer therapeutic agent and cancer diagnostic agent, which is different from the conventional pheophobide and porphyrin derivatives.

The present invention is a metal complex of a porphyrin derivative in a broad sense,
Both porphyrins (broadly defined) of plant-derived chlorophyll (forvin derivative) and animal-derived hemoglobin (porphine derivative) having a cheap and stable source can be used as a raw material.

The substance of the present application is a metalloporphyrin (phorpine and porphine) related compound and has characteristic physiological activities such as cancer affinity, non-phototoxicity, and action against external energy. Therefore, it is used as a carrier for cancer diagnostic agents, tumors, markers and other anticancer agents. It is a very useful substance that is either used as a drug for missile therapy as it is or has almost unlimited uses as a raw material for producing them, and that it can be mass-produced by a simple and short-time operation, and thus is highly economical.

[Brief description of drawings]

Fig. 1 shows the excitation fluorescence spectra of each organ 24 hours after intravenous injection of each drug. Fig. 1 shows the sodium salt of Ga-monoacetylethylene glycol mono-10b-pheophorbate, Fig. 2. Is Mg-di-2.4- (1-ethane alcohol oxyethyl) -deuteroporphyrin (VI
I) the disodium salt, Fig. 3 shows the disodium salt of Ge-di-2.4- (1-ethaneacetateoxyethyl) -deuteroporphyrin (IX), and Fig. 4 shows the disodium salt of In-hematoporphyrin (XII). It is a chart regarding cancer cell affinity when each drug of sodium salt is used. 1. Saline (control) 2. Tumor 3. Liver 4. Kidney 5. Serum

Continuation of the front page (72) Inventor Natsuki Samejima 2-3-3 Midorigaoka, Asahikawa City, Hokkaido A-42 (72) Inventor Wakai Inohara 7-9, 3-9 Gomoncho, Fukuyama City, Hiroshima Prefecture (72) Inventor Hiroyuki Takada 2098, Satomi, Satosho-cho, Asaguchi-gun, Okayama (56) References JP-A-57-185220 (JP, A) JP-A-57-31688 (JP, A) JP-A-57-11985 (JP, A) ) JP-A-51-125757 (JP, A) JP-B-47-19919 (JP, B1)

Claims (1)

[Claims] 1. A general formula [Wherein, R ₁ and R ₆ are CH = CH ₂ , CH ₂ CH ₃ , CH (OH) CH ₃ , CH (OCH ₂ CH ₂ OH) CH ₃ or CH (OCOCH ₃ ) CH ₃ C, respectively.
H _3, R ₂ is CH _3, CHO or _{CH 2 OH, R 3 = O} , OHOCOCH 3 or H, _{R 4} is _{H, COOH, COOCH 3, COOCH} 2 CH 2 OH, COOCH 2 CH 2 OCOCH 3 or COOCH ₂ CH ₂ OCOA [A is a residue obtained by removing R ₄ from the compound of the formula (1)], R ₅ is COOH or COONa, provided that when R ₄ is H, R ₁ is CH (OCH ₂ CH ₂ OH) CH. ₃ , when R ₄ is COOCH ₃ , a compound having at least one oxygen atom in the formula of R ₁ is used, and when R ₄ is COOH, these two of the two in the formulas of R ₁ and R ₆ are In the formula, those having at least two oxygen atoms are represented. Further, neither R ₃ nor R ₄ is H. M is Si, Ga, Ge, In or Pt, X is a halogen atom, OH, pyridine or OCOCH ₃ , the dotted line bound to the γ position represents no bond or a single bond, and the dotted line between the 7th and 8th positions is single Indicates the presence of a bond or double bond. ] The metal pheophobide and metal porphyrin derivative shown by these. However, metal complexes of protoporphyrin and hematoporphyrin are excluded.