WO1995006473A2

WO1995006473A2 - Medical use of polysulfonic acid compound

Info

Publication number: WO1995006473A2
Application number: PCT/JP1994/001410
Authority: WO
Inventors: Hisao Ekimoto; Eri Iizuka; Tohru Tabei; Mineaki Okada; Masako Aono
Original assignee: Nippon Kayaku Kabushiki Kaisha
Priority date: 1993-09-01
Filing date: 1994-08-25
Publication date: 1995-03-09
Also published as: AU7508594A; WO1995006473A3

Abstract

Polysulfonic acid compounds shown by formula (1) or (2) or pharmaceutically acceptable salts thereof exhibit an in vivo antitumor effect on rat prostatic cancer and an antiangiogenic activity by the rat aorta ring assay and the rabbit corneal assay. The compounds are thus expected to be useful as antitumor and angiogenic inhibitors. In formula (1) and (2) M, X, X1, X2, Y1, Y2 and Z represent a specific group, respectively.

Description

DESCRIPTION MEDICAL USE OF POLYSULFONIC ACID COMPOUND

TECHNICAL FIELD

The present invention relates to medical use of a polysulfonic acid compound. The polysulfonic acid compound of the present invention possesses an antitumor and antiangiogenesis activity. The compound is thus expected as a drug for the treatment of malignant tumors, or as a drug for the prevention and treatment of diseases caused by abnormal growth of blood vessels, e.g., rheumatoid arthritis, diabetic retinopathy, retinopathy of prematurity, senile degeneration of macula lutea and excessive cicatrization during wound curing.

BACKGROUND ART

' Indometacin, medroxiprogesterone, cortison in combination with heparin, cartilage extract and the like are currently known to have an inhibitory action of angiogenesis. Applicability of FUMAGIRIN derivatives and sulfated polysaccharides to carcinostatic agents having an antiangiogenetic activity has been studied, _See Drug Design and Discovery, 8., 3-35, 1992.

However, none of these compounds having an antiangiogenetic activity has been provided so far actually for clinical use as drugs. It has thus been expected to develop a new drug having an antitumor activity and an angiogenesis inhibitory activity and having a minimized toxicity for practical use.

On the other hand, polysulfonic acid compounds which can be employed as azo dye compounds are described in Japanese Patent Application KOKAI (Laid-Open) Nos. 50-103527 and 60-86169. However, pharmacological activities of these polysulfonic acid compounds are totally unknown to date. Accordingly, an object of the present invention is to provide use of the polysulfonic acid compound as a medicament, particularly as a medicament for antitumor or inhibition of angiogenesis. Disclosure of the Invention A first aspect of the present invention relates to a polysulfonic acid compound for use as a medicament, said polysulfonic acid compound being selected from the group consisting of: a compound of formula (1)

wherein X represents 0 or COO and M represents a transition metal, and a pharmaceutically acceptable salt thereof ; and, a compound of formula (2)

wherein each of X_: and X₂ independently represents a substituted or unsubstituted phenyl or naphthyl group, each of Y_α and Y₂ independently represents a halogen atom, an amino group or a mono- or di-substituted amino group, and Z represents HNC₂H₄NH or a substituted or unsubstituted aromatic diamine residue, and a pharmaceutically acceptable salt thereof. A second aspect of the present invention relates to a pharmaceutical composition, which comprises the polysulfonic acid compound of formula (1) or (2) or the pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable carrier.

A third aspect of the present invention relates to use of the polysulfonic acid compound of formula (1) or (2) or the pharmaceutically acceptable salt thereof in the manufacture of a medicament for antitumor or inhibition of angiogenesis.

A fourth aspect of the present invention relates to a method for antitumor or inhibition of angiogenesis, which comprises administering to a human body the polysulfonic acid compound of formula (1) or (2) or the pharmaceutically acceptable salt thereof in an effective amount. A fifth aspect of the present invention relates to a method for preparing a pharmaceutical composition, which comprises a step of mixing the polysulfonic acid compound of formula (1) or (2) or the pharmaceutically acceptable salt with a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF DRAWINGS

Fig. 1 shows an antitumor effect of tetrasodium salt of 4,4 '-bis(8-amino-l-hydroxy-3, 6- disulfo-2-naphthylazo)-3,3'-dimethoxybiphenyl cuprate (ii) on nude mice bearing rat prostatic cancer (R3327-

G).

Fig. 2 shows an antitumor effect of hexasodium salt of 4,4 '-bis{4-morpholino-6- (7-phenylazo-8-hydroxy-

3, 6-disulfo-l-naphthyl)amino-1,3, 5-triazine-2- ylamino}stilbene-2,2'-disulfonate on nude mice bearing rat prostatic cancer (R3327-G) .

Fig. 3 shows an antitumor effect of hexasodium salt of 4,4 '-bis[4-chloro-6-{7- (2-sulfophenylazo) -8- hydroxy-3, 6-disulfo-l-naphthylJamino-l,3,5-triazine-2- yl]-phenylenediamine on nude mice bearing rat prostatic cancer (R3327-G) . Fig. 4 shows an antitumor effect of Suramin for control on nude mice bearing rat prostatic cancer (R3327-G) .

BEST MODE FOR CARRYING OUT THE INVENTION Although malignant tumors treated or prevented by the medicament of the present invention are not limited, the preferable tumors are solid cancer such as a lung cancer, a cancer of digestive system such as stomach cancer, cancer of liver and cancer of pancrease, and prostatic carcinoma.

The most preferable medicament^' of the present invention is a medicament for treatment or prevention of prostatic carcinoma.

As the transition metal shown by M in the formula (1) above, typical examples are Cu, Zn, Ni and Fe. Cu is generally preferable.

In the formula (2) above, representative examples of the phenyl or naphthyl group shown by X. and X₂ which may be substituted include unsubstituted phenyl, unsubstituted naphthyl and a substituted phenyl or substituted naphthyl group which is substituted with the following substituent(s) .

Representative examples of the substituent are a sulfonic acid group, a halogen atom such as chlorine, an alkyl group having 1 to 3 carbon atoms, an alkoxy group having 1 to 3 carbon atoms, carboxyl, hydroxy, β- hydroxyethylsulfonyl and β-sulfatoethylsulfonyl. Among them, a sulfonic acid group is preferred. The number of the substituent is not particularly limited but 1 to 3 substituents are generally preferred.

In the groups shown by Y, and Y₂ in the formula (2), examples of the halogen atom are chlorine, bromine and fluorine; examples of the mono- or di-substituted amino group are a morpholino group, an anilino group and a naphthylamino group, wherein the alkyl, phenyl or naphthyl group on the amino group may further substituted with a carboxyl group, a sulfonic acid group, a lower alkyl group, a lower alkoxy group, a hydroxy group, a halogen atom, a β-hydroxyethylsulfonyl group or a β-sulfatoethylsulfonyl group. Among others, a halogen atom and a morpholino group are preferred as Y₁ and Y₂. Where a lower alkyl group or a lower alkoxy group is referred to in the specification, the alkyl moiety means to have 1 to 5 carbon atoms, preferably 1 to 3 carbon atoms.

The aromatic diamine residue shown by Z in the formula (2) which may optionally be substituted with a substituent is a residue obtained by removing one hydrogen atom from each of the two amino groups in the aromatic diamine. These diamine residues preferably contain 1 or 2 benzene rings. Where the diamine residue has two benzene rings, these benzene rings may be bound to each other directly or via a saturated or unsaturated carbon chain having preferably 1 to 4 carbon atoms. Examples of such diamine residue include residues of diaminobenzene, diaminobiphenyl, diaminostilbene and diaminobiphenylamine.

The benzene ring(s) of these groups may be substituted; examples of the substituent are a sulfonic acid group, a carboxyl group, a lower alkyl group and a lower alkoxy group. Among them, preferred are residues of diaminobenzene or disulfonic acid-substituted diaminostilbene or sulfonate residues thereof. In the present invention, the polysulfonic acid compounds of the formula (1) wherein X is 0 and M is a copper atom as the transition metal, or pharmaceutically acceptable salts thereof are preferable. In the formula (2), preferred are the polysulfonic acid compounds wherein each of X. and X₂ independently represents an unsubstituted phenyl group or a phenyl group substituted with a sulfonic acid group, which may be the same or different; each of Y. and Y₂ independently represents a halogen atom or a morpholino group and Z represents a phenylenediamine residue or a diaminostilbene residue, which may have a sulfonic acid residue as a substituent on the benzene nucleus, and pharmaceutically acceptable salts thereof. Preferred examples of the compounds shown by formula (1) and salts thereof include 4,4 '-bis(8-amino- l-hydroxy-3, 6-disulfo-2-naphthylazo)-3,3'- dimethoxybiphenyl copper complex tetrasodium salt (hereinafter referred to as Compound No. 1) .

Examples of the compounds shown by the formula (2) include hexasodium salt of 4,4 '-bis{4-morpholino-6- (7-phenylazo-8-hydroxy-3, 6-disulfo-1-naphthyl)amino- 1,3,5-triazine-2-ylamino}stilbene-2,2 '-disulfonate (hereinafter referred to as Compound No. 2) and hexasodium salt of 4,4 '-bis[4-chloro-6-{7- (2- sulfophenylazo)-8-hydroxy-3, 6-disulfo-l-naphthyl}amino- 1,3,5-triazine-2-yl]-phenylenediamine (hereinaf er referred to as Compound No. 3) .

Compound No. 1 may be synthesized by the method described in Japanese Patent Application KOKAI No. 50-103527, Example 1.

Compound No. 3 may be synthesized by the method described in Japanese Patent Application KOKAI No. 60-86169, Example 1.

Where the compounds of formula (1) or formula (2) (hereinafter collectively referred to as the compound of the present invention) are employed as anticancer agents or angiogenesis inhibitors, the compound of the present invention is administered in the form of injection, oral agents or suppositories, alone or as admixtures with excipient or carriers. As the excipient or carriers, pharmaceutically acceptable ones are chosen; the kind and composition are determined depending upon route or method of administration.

As a liquid carrier, there may be used water, an alcohol, animal and vegetable oil such as soybean oil, peanut oil, sesame oil, mineral oil, etc., or synthetic oil. As a solid carrier, there may be used sugars such as maltose or sucrose, an amino acid, a hydroxypropyl cellulose derivative, and an organic acid 5 salt such as magnesium stearate.

In the case of injection, the compound of the present invention may be freeze-dried, generally together with physiological saline, various buffer solutions, sugars such as glucose, inositol, mannitol,

10 mannose, maltose and sucrose, or excipient like amino acids such as phenylalanine. Upon use, the thus freeze- dried preparation is dissolved in an appropriate solvent for injection, e.g., sterile water, physiological saline, glucose solution, electrolyte solution, infusion

15. for intravenous administration such as amino acid infusion, etc., and the resulting solution is then injected.

The amount of the compound of the present invention contained in the preparation varies depending 0 upon the form of preparation; in general, the compound of the present invention may be contained generally in the range of 0.01 to 100 wt%, preferably 0.1 to 50 wt%. In the case of, e.g., injection, the compound may be contained, as the effective component, generally in the

25 range of 0.1 to 30 wt%, preferably 1 to 10 wt%. In the case of oral administration, the compound of the present invention is applied in the form of a tablet, a capsule, powders, granules, liquid or dry syrup, together with the solid carrier or liquid carrier described above. Such a capsule, tablet, granule and powder contain the effective component generally in the range of 5 to 100 wt%, preferably 25 to 98 wt%, respectively.

Dose may be determined depending on the age, body weight and condition of a patient or the purpose of treatment but is generally in the range of 50 to 500 mg/ body. The compounds of the present invention are prepared generally by the following methods.

The compounds of the present invention are known by Japanese Patent Application KOKAI Nos. 50- 103527, 52-78926 and 60-86169, or may be prepared by a modification of the methods described therein.

That is, the compound of formula (1) is obtained by converting dianisidine or benzidinedi- carboxylic acid into the corresponding tetrazo compound in a conventional manner, coupling the tetrazo compound with a compound shown by (1A) :

in the form of free acid under weakly acidic or alkaline conditions, and then treating the coupling compound with a metal-yielding agent to convert into a metal complex. If desired, the formation of the metal complex may be accelerated by adding a complex forming accelerator. The thus obtained compound of formula (1) is present in the form of a free acid or an alkali metal salt, alkylamine salt, alkanolamine salt or alkaline earth metal salt.

Specific examples of the compounds shown by formula (1A) include l-amino-8-naphthol-3, 6-disulfonic acid, l-amino-8-naphthol-4, 6-disulfonic acid, 1-amino-8- naphthol-2,4-disulfonic acid and 2-amino-8-naphthol-3, 6- disulfonic acid. Representative examples of the metal- yielding agent are copper (II) sulfate, copper (II) acetate, zinc (II) chloride, zinc (II) acetate, zinc (II) sulfate, nickel (II) chloride, iron (II) sulfate and iron (II) nitrate. Examples of the complex forming accelerator are tartaric acid, citric acid, salicylic acid or salts thereof, ammonia, monoethanolamine, N- methylethanolamine, diethanolamine and urotropin. The compounds of the formula (2) may be prepared, e.g., by the following methods A to D. Synthesis Method A

(1) Synthesis of compounds of formula (2) wherein Y. and Y represent a halogen atom One mole of a cyanuric halide is condensed with 1 mole each of a compound shown by formula (2A) :

wherein X_: has the same significance as defined hereinabove, and a compound of formula (2B) :

wherein X₂ has the same significance as defined hereinabove, respectively, under acidic to alkaline conditions, preferably pH of 2 to 9, at a temperature of 0 to 20'C in a conventional manner. The condensation products are condensed with 1 mole of a compound shown by HZH, wherein Z has the same significance as defined above, under acidic to alkaline conditions, preferably pH of 2 to 9, at a temperature of 20 to 70'C in a conventional manner to give a compound shown by formula (2C) :

wherein Q represents a halogen atom, and X,, X₂ and Z have the same significance as defined above.

To obtain the compound of formula (2C) , 2 moles of the cyanuric halide may be condensed with 1 mole of HZH, wherein Z has the same significance as defined above, followed by condensation of 1 mole of the compound of formula (2A) and 1 mole of the compound of formula (2B) .

(2) Synthesis of compounds of formula (2) wherein

Y- and Y represent a group other than a halogen atom

One mole of the compound of formula (2C) in Synthesis Method A (1) above is condensed with 1 mole each of Y_nH (wherein Y_u has the same significance as for Y_1# except for a halogen atom for Y_χ ) and Y₂₂H

(wherein Y₂₂ has the same significance as for Y , except for a halogen atom for Y₂) under acidic to alkaline conditions, preferably pH of 4 to 10, at a temperature of 70 to 100^*C in a conventional manner to give the compound of formula (2), wherein Y_χ and Y₂ represent a group other than a halogen atom. Synthesis Method B

(1) Synthesis of compounds of formula (2) wherein Y. and Y represent a halogen atom One mole of a cyanuric halide is condensed with 1 mole each of a compound shown by formula (2D) :

and a compound of formula (2E)

respectively, under acidic to alkaline conditions, preferably pH of 0.5 to 7, at a temperature of 0 to 20 ' C in a conventional manner. The condensation products are then subjected to coupling with 1 mole each of diazonium compounds, which are obtained by diazotization of the amines shown by X,NH₂ and X₂NH₂ wherein X, and X₂ have the same significance as defined above with nitrous acids (HN0₂) in a conventional manner, under slightly acidic to alkaline conditions, preferably pH of 7 to 9, at a temperature of 0 to 40^*C, preferably 10 to 20 'C, respectively, in a conventional manner. The thus obtained coupling compounds, each of which is used in an amount of 1 mole, are condensed with 1 mole of a compound shown by HZH, wherein Z has the same significance as defined above, under acidic to alkaline conditions, preferably pH of 2 to 9 and a temperature of 20 to 70'C, to give a compound shown by formula (2C) . (2) Synthesis of compounds of formula ( 2 ) wherein Y_η and Y represent a group other than a halogen atom

The thus obtained compound of formula (2C) is treated in the same way as the method (2) in Synthesis Method A as described above to give the captioned compound.

Synthesis Method C

(1) Synthesis of compounds of formula (2) wherein Y, and Y represent a halogen atom One mole of a cyanuric halide is condensed with 1 mole each of a compound shown by formula (2D) and a compound shown by formula (2E) in a conventional manner. The condensation products are condensed with 1 mole of a compound shown by HZH, wherein Z has the same significance as defined above, in a conventional manner to give a compound shown by formula (2F) :

wherein Q and Z have the same significance as defined above. To obtain the compound of formula (2F) , 2 moles of the cyanuric halide may be condensed with 1 mole of HZH, followed by condensation of 1 mole each of the compound shown by formula (2D) and the compound shown by formula (2E) . Then, the condensation product is subjected to coupling with 1 mole each of diazonium compounds, which are obtained by diazotization of the amines X..NH₂ and X₂NH₂ wherein X_χ and X₂ have the same significance as defined above with nitrous acids (HN0₂) in a conventional manner, under slightly acidic to alkaline conditions, preferably pH of 7 to 9, at a temperature of 0 to 40"C, preferably 10 to 20'C, in a conventional manner. Thus, the compound shown by formula (2C) is obtained.

(2) Synthesis of compounds of formula (2) wherein Yi and Y represent a group other than a halogen atom

The thus obtained compound of formula (2C) is treated in the same way as the method (2) in Synthesis Method A as described above to give the captioned compound. Synthesis Method D

(1) Synthesis of compounds of formula (2) wherein Y. and Y represent other than a halogen atom One mole of the compound of formula (2F) in Synthesis Method C above is condensed with 1 mole each of Y H and Y₂₂H under acidic to alkaline conditions, preferably pH of 4 to 10, at a temperature of 70 to 100^*C in a conventional manner to give the compound of formula (2G) :

wherein each of Y,,, Y₂₂ and Z have the same significance as defined above, respectively. To obtain the compound of formula (2G) , condensation between a cyanuric halide and the compounds of formula (2D) and formula (2E) and condensation of HZH, Y_χ,H and Y₂₂H may be made in an arbitrary order.

Then, the condensation product is subjected to coupling with 1 mole each of diazonium compounds, which are obtained by diazotization of the amines X,NH₂ and X₂NH₂ wherein X. and X₂ have the same significance as defined above with nitrous acids (HN0₂) in a conventional manner, under slightly acidic to alkaline conditions, preferably pH of 7 to 9, at a temperature of 0 to 40'C, preferably 10 to 20'C, in a conventional manner, to give the compound shown by formula (2C) .

The thus obtained compound of formula (2) is present in the form of a free acid, or in the form of an alkali metal salt, an alkylamine salt, an alkanolamine salt or an alkaline earth metal salt. As the cyanuric halide described above, there are, for example, cyanuric chloride and cyanuric fluoride.

As HZH described above, examples of the aromatic diamine which may be substituted are a diaminobenzene, diaminobiphenyl, diaminostilbene and diaminobiphenyla ine which may be substituted with a sulfonic acid group, carboxyl, methyl or ethoxy. Specific examples of such compounds are shown below.

.9 -

The unsubstituted or, mono- or di-substituted amine shown by Y_χH or Y₂H includes the following compounds.

(1) Examples of the mono- or di-substituted amine are a mono- or di-lower alkylamine preferably having 1 to 3 carbon atoms. The these alkylamine may be substituted with carboxyl a sulfonic acid , sulfato or hydroxy on the lower alkyl moiety thereof. Specific examples are given below.

CH 3, C ., 2H =_ C , 3,H,7 H H NH₂C₂H₄OH

NH₂, NH₂, NH₂, N(CH₃)₂, N(C₂H₅)_2ι

NH(C,H.OH),, NH,C,H.,OH, NH₂CH₂COOH,

NH₂CH₂CH₂S0₃H, NH₂CH₂CH₂OS0₃H (2) As the aniline or naphthylamine, there are, for example, unsubstituted aniline and naphthylamine, a substituted aniline and naphthylamine substituted on the benzene or naphthalene nucleus with carboxyl, a sulfonic acid, an alkyl group having 1 to 3 carbon atoms, an alkoxy group having 1 to 3 carbon atoms, hydroxy, chlorine, β-hydroxyethylsulfonyl or β- sulfatoethylsulfonyl. Specific examples are given below.

^QT -- κ- o ^*Λ -O "

Representative examples of the compounds represented by formulae (2A) and (2B) include 7- (2- sulfophenylazo) -8-hydroxy-3, 6-disulfo-l-naphthylamine and 7-phenylazo-8-hydroxy-3, 6-disulfo-l-naphthylamine. A representative example of the compound shown by formula (2C) includes 4, 4 '-bis [4-chloro-6-{7- (2- sulfophenylazo) -8-hydroxy-3, 6-disulfo-1-naphthyl}amino- 1,3, 5-triazine-2-ylamino]stilbene-2,2 ' -disulfonic acid.

A representative example of the compound shown by formula (2F) includes 4,4 ' -bis{4-chloro-6- (8-hydroxy- 3, 6-disulfo-1-naphthyl)amino-1,3, 5-triazine-2-ylamino}- stilbene-2,2 '-disulfonic acid.

A representative example of the compound shown by formula (2G) includes 4,4 '-bis[4-morpholino-6-{8- hydroxy-3, 6-disulfo-1-naphthyl)amino-1,3,5-triazine-2- yl}-phenylenediamine.

Representative examples of the compounds represented by formulae (2D) and (2E) include l-amino-8- naphthol-3, 6-disulfonic acid, l-amino-8-naphthol-4, 6- disulfonic acid, l-amino-8-naphthol-2,4-diεulfonic acid and 2-amino-8-naphthol-3, 6-disulfonic acid.

As the compounds represented by X,NH₂ and X₂NH₂, in addition to aniline or naphthylamine, there are, for example, a aniline and naphthylamine substituted on the benzene or naphthalene nucleus with a sulfonic acid, chlorine, an alkyl group having 1 to 3 carbon atoms, an alkoxy group having 1 to 3 carbon atoms, carboxyl, hydroxy, β-hydroxyethylsulfonyl or β-sulfatoethyl- sulfonyl. Specific examples are given below.

23

Among these compounds, preferred are aniline and 2-aminobenzenesulfonic acid.

Pharmacological activities of the compounds of the present invention are described below.

Samples

Compound Nos. 1 to 3 shown in Table 1 were employed for Test Examples 1 to 3 described below. For control, Suramin containing a sulfonic acid group was used. Suramin has an inhibitory activity on cell proliferation by growth facturs'and has been used for clinical trials on prostatic cancer in the United States.

Test Example 1

Antitumor effect of the compounds on rat prostatic cancer in nude mice Method Male BA B/c-nu/nu mice of 11 weeks age were subcutaneously transplanted with rat prostatic cancer R3327-G into the right flank. Seven days after transplanation, each compound dissolved in distilled water was mtraperitoneally administered consecutively once a day. A dose of each compound and schedule are shown below.

No. 1 : 9 mg/kg, once a day, consecutive 7 days No. 2 : 60 mg/kg, once a day, consecutive 10 days No. 3 : 60 mg/kg, once a day, consecutive 15 days Suramin for control :

60 mg/kg, once a day, consecutive 7 days

A long (L) and short (W) axis of the tumor was measured with passage of time and a tumor volume was calculated by the formula x W²/2. Based on a rate of each compound group to the control group in tumor volume, the antitumor effect was examined.

The maximum growth inhibition rate is determined as follows.

A growth inhibition rate at each measurement was determined by the following formula. During the obεervation period for 27 days, the numerical value showing the maximum growth inhibition rate was made the maximum growth inhibition rate.

Growth inhibition rate (%) = (1 - rate of the tumor volume in the compound group/rate of the tumor volume in the control group) x 100

The results are shown in Figs. 1 to 4. As shown in Figs. 1 to 4, Compound Nos. 1, 2 and 3 inhibited growth of rat prostatic cancer R3327-G. The maximum growth inhibition rate of each compound was:

No. 1 : 32.7%

No. 2 : 29.7%

No. 3 : 43.9%

Suramin : 35.8%

Test Example 2

Antiangiogenetic activity of the compounds (Rat Aorta

Ring Assay) Method

The aorta was obtained from the thoracic cavity of rats to prepare aortic rings each having a thickness of 0.5 mm. After the ring was preincubated in a 96-well plate laying fibrin gel for 24 hours, each compound was added in final concentrations of 10 and 100 μg/ml. Five days after the addition of the compound, the level of angiogenesis was observed and compared with the intact group. The level of angiogenesis was determined based on the following criteria.

- : 0 Vascular outgrowth from the aortic ring is not observed at all.

± : 1 Vascular short strands emerge from the aortic ring at several points.

+ : 2 Microvessels having a sufficient length are observed in such a density that each microvessel is clearly distinguishable.

++ : 3 Microvessels having a sufficient length are observed in such a high density that the microvessels are not distinguishable from one another.

The inhibition rate was determined by the following equation.

Inhibition rate (%) =

(1 - level of angiogenesis in the compound group/level of angiogenesis in the control group) x 100

The results are shown in Table 2. Table 2

Inhibitory activity of the compounds against angiogenesis with rat aortic ring

Concentra¬ Level of Inhibition tion (μg/ml) Angiogenesis Rate (%)

Control Group - 3.0 0.0

Compound 100 2.2 26.7 No. 1 10 2.7 10.0

Compound 100 2.3 23.3 No. 2 10 2.2 26.7

Compound 100 0.8 73.3 No. 3 10 1.8 40.0

Suramin 100 2.7 10.0 10 2.3 23.3

As demonstrated in Table 2, Compound Nos. 1 and 2 and Suramin showed only a weak angiogenesis inhibitory action at the doses used. However, Compound No. 3 showed an angiogenesis inhibitory activity dose- dependently.

Test Example 3

Antiangiogenic activity of the compounds: rabbit corneal assay by M.A. Gimbrone et al. (Journal National Cancer Institute, 52, 413, 1974) Method

A superficial incision, about 2 mm long, was made with a mess in the corneal dome to one side of its center to prepare a pocket in the cornea. A slow release pellet containing 100 μg of Ξhrlich's cancer extract (TAF pellet) previously prepared by the method of R. Langer et al. A slow release pellet containing 200 μg of each compound was implanted into the pocket in the cornea. Furthermore, the TAF pellet was implanted in contact with the sample pellet. Four days after the transplantation, the level of angiogenesis was observed. The length (L) of newly born microvessels is evaluated in 6 levels.

0 : Neovascularization is not observed at all.

1 : Vascular outgrowth is appreciable with difficulty only through a loupe (like buds of microvessels) 2 : Neovascularization is observed with the naked eye but microvessels do not reach the sample pellet. 3 : Microvessels reach the sample pellet but do not reach the TAF pellet. 4 _: Microvessels reach the TAF pellet.

5 : Microvessels outgrow over the TAF pellet and are extended far away.

The inhibition rate was determined by the following equation. Inhibition rate (%) =

(1 - length of angiogenesis in the compound group/length of angiogenesis in the control group) x 100

The results are shown in Table 3.

Table 3. Antiangiogenic active of the compounds against pellets containing Ehrlich's cancer extract implanted into the rabbit cornea

Inhibition

L* Rate (%)

Control Group 3.0 0.0

Compound No. 1 3.0 0.0

Compound No. 2 3.0 0.0

Compound No. 3 0.0 100.0

Suramin 2.0 33.3

Length of newly born microvessel from the limbus

As shown in Table 3, Compound Nos. 1, 2 and Suramin barely exerted the antiangiogenetic activity but Compound No. 3 markedly inhibited angiogenesis by the Ehrlich's cancer extract. Toxici ty

General toxicity in the mice treated with the compounds When 15 mg/kg of Compound No. 1 and 100 mg/kg each of Compound Nos. 2 and 3 and Suramin were mtraperitoneally administered to nude mice once a day for consecutive 7 days, death was noted in all of the administered groups. In these dead mice, hemorrhage in the abdominal cavity was noted in the Suramin group but no such condition was noted in the groups administered with Compound Nos. 1, 2 and 3.

Industrial Applicability

From the these results, the compounds of the present invention exhibit the antitumor effect and the antiangiogenetic activity and are thus expected to be useful as carcinostatic agents and angiogenetic inhibitors.

CLAIMS 1. A polysulfonic acid compound for use as a medicament, said polysulfonic acid compound being selected from the group consisting of: a compound of formula (1)

wherein X represents 0 or COO and M represents a transition metal, and a pharmaceutically acceptable salt thereof; and, a compound of formula (2)

wherein each of X_: and X₂ independently represents a substituted or unsubstituted phenyl or naphthyl group, each of Y and Y₂ independently represents a halogen atom, an amino group or a mono- or di-substituted amino group, and Z represents HNC₂H₄NH or a substituted or unsubstituted aromatic diamine residue, and a pharmaceutically acceptable salt thereof.

Claims

2. A polysulfonic acid compound according to claim 1, wherein said polysulfonic acid compound is a compound of formula (1), wherein X represents O and M represents copper atom, or a pharmaceutically acceptable salt thereof.

3. A polysulfonic acid compound according to claim 1, wherein said polysulfonic acid compound is a compound of formula (2), wherein each of X, and X₂ independently represents an unsubstituted phenyl group or a phenyl group substituted with a sulfonic acid group; each of Y_χ and Y₂ independently represents a halogen -atom or a morpholino group; and Z represents a phenylenediamine or diaminostilbene residue, each of which may be substituted at the phenyl ring with a sulfonic acid group; and a pharmaceutically acceptable salt thereof.

4. A polysulfonic acid compound according to claim 1, wherein said polysulfonic acid compound is a copper complex of 4,4 '-bis(8-amino-l-hydroxy-3, 6- disulfo-2-naphthylazo)-3, 3 ' -dimethoxybiphenyl or a salt thereof.

5. A polysulfonic acid compound according to claim 1, wherein said polysulfonic acid compound is 4,4'-bis [4-morpholino-6- (7-phenylazo-8-hydroxy-3, 6- disulfo-1-naphthyl)amino-1,3, 5-triazine-2-ylamino] stilbene-2,2 '-disulfonic acid or a salt thereof.

6. A polysulfonic acid compound according to claim 1, wherein said polysulfonic acid compound is 4,4'-bis[4-chloro-6- (7- (2-sulfophenylazo)-8-hydroxy-3, 6- disulfo-l-naphthyl)amino-l, 3,5-triazine-2-yl] - phenylenediamine or a salt thereof.

7. A polysulfonic acid compound according to any one of claims 1 to 6, wherein said medicament is for antitumor or inhibition of angiogenesis.

8. A polysulfonic acid compound according to claim 7, wherein said medicament is for the treatment of a disease caused by abnormal growth of blood vessel. 9. A polysulfonic acid compound according to claim 7, wherein said medicament is for the treatment of a solid cancer.

10. A pharmaceutical composition, which comprises a polysulfonic acid compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6 as an active ingredient, and a pharmaceutically acceptable carrier.

11. A pharmaceutical composition according to claim 10, wherein said composition is for antitumor or inhibition of angiogenesis.

12. A pharmaceutical composition according to claim 11, wherein said composition is for the treatment of a disease caused by abnormal growth of blood vessel.

13. A pharmaceutical composition according to claim 11, wherein said composition is for the treatment of a solid cancer.

14. Use of a polysulfonic acid compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6 in the manufacture of a medicament for antitumor or inhibition of angiogenesis.

15. Use according to claim 14, wherein said medicament is for the treatment of a disease cause by abnormal growth of blood vessel.

16. Use according to claim 14, wherein said medicament is for the treatment of a solid cancer.

17. A method for antitumor or inhibition of angiogenesis, which comprises administering to a human body a polysulfonic acid compound or a pharmaceutically acceptable salt thereof according to any of claims 1 to 7 in an effective amount.

18. A method according to claim 17, wherein said method is for the treatment of a disease caused by abnormal growth of blood vessel.

19. A method according to claim 17, wherein said method is for the treatment of a solid cancer.

20. A method for preparing a pharmaceutical composition, which comprises a step of mixing a polysulfonic acid compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, together with a pharmaceutically acceptable carrier.

21. A method according to claim 20, wherein said composition is for antitumor or inhibition of angiogenesis.

22. A method according to claim 20, wherein said composition is for the treatment of a disease caused by abnormal growth of blood vessel.

23. A method according to claim 20, wherein said composition is for the treatment of a solid cancer.