WO1993016064A1

WO1993016064A1 - Coumarin derivatives and their analogues inhibiting cell proliferation and tumour growth, pharmaceutical compositions containing them and process for preparing same

Info

Publication number: WO1993016064A1
Application number: PCT/HU1993/000010
Authority: WO
Inventors: György Kéri; Tamás BAJOR; László O^'RFI; Endre Tihanyi; Ágnes BALOGH; Gyöngyi Bökönyi; Anikó HORVÁTH; Miklós IDEI
Original assignee: BIOSIGNAL, Kutató-Fejleszto^' Kft.
Priority date: 1992-02-13
Filing date: 1993-02-12
Publication date: 1993-08-19
Also published as: AU3573893A; HUT63843A; HU9200438D0

Abstract

The invention relates to novel compounds of general formula (I), wherein R1 stands for -CN, -COOH, -CSOH, -COSH, -CONH2, -CSNH2, -NHCHO, -COOCH3, -CSOCH3, -COSCH3, -COOCH2CH3, -CSOCH2CH3, -COSCH2CH3, -NHCOCH3, -NHCSCH3, -CS-NH-CONH2, -CO-NH-CONH2, -CO-NH-CSNH2, -CS-NH-CSNH2, -C(NH2)=C(CN)2, -CH(CN)2, -CH(CN)-COOH, -CH(CN)-CONH2, -CH(CN)-CSNH2, -CH(CN)CSNH-C1-4 alkyl, -NO2 or -NO group; X means =O, =S or =NH group; Y means -O-, -S- or -NH- group; R2 and R4, independently from each other, represent hydrogen atom, -OH, -SH, -NH2, -CF3, C1-4 alkoxy or C1-4 alkylthio group; R3 stands for -OH, -SH, -NH2, -CF3, -NO, -O-SO2-V0, -O-CO-V0, -O-CS-V0, -S-CO-V0, -NH-CO-V0 or -CH=CH-V0 group, wherein V0 stands for 3,4,5-tri-V1-phenyl group; and the V1 substituents, independently from each other, mean hydrogen or halogen atom or -OH, -SH, -NH¿2?, -CF3, C1-4 alkoxy, C1-4 alkylthio or C1-4 alkylamino group; or V?1¿ bound in the 3- or 4-position may be -COOH, -CSOH, -COSH, -CONH¿2?, C1-4 dialkylamino, pyridyl, -CH2-2-pyridyl, -O-CH2-O- or -N(CH2-V?2)-CH¿2-V3 group, wherein V?2 and V3¿, independently from each other, stand for -OH, -SH, -NH¿2?, -CF3, C1-4 alkyl, C1-4 alkoxy or C1-4 alkylthio group; or V?2 and V3¿ together stand for -NH-CH¿2?-NH- or -N(CH3)-CH2-N(CH3)- group; R5 represents hydrogen, C1-4 alkyl, C1-4 alkoxy group or halogen; and the double bond belonging to the skeletal atom substituted by R1 can also be saturated when R3 is a substituent containing the -3,4,5-tri-V?1¿-phenyl moiety, as well as physiologically acceptable salts and complexes of these compounds. The invention furthermore relates to pharmaceutical compositions containing the above compounds as well as processes for the preparation of the above compounds and compositions. The compounds of formula (I) are prepared by condensing a phenol or benzaldehyde derivative with a bifunctional carboxylic acid derivative. The compounds of formula (I) show a tumour-inhibititing action: they inhibit the activity of tyrosine kinase enzymes playing a role in the signal transfer mechanism, but do not practically influence the activity of insulin-receptor tyrosine kinase.

Description

COUMARIN DERIVATIVES AND THEIR ANALOGUES INHIBITING CELL PROLIFERATION AND TUMOUR GROWTH, PHARMACEUTICAL COMPOSITIONS CONTAINING THEM AND PROCESS FOR PREPARING SAME

This invention relates to novel compounds of the general formula (I),

wherein

R₁ stands for -CN, -COOH, -CSOH, -COSH, -CONH₂,

-CSNH₂, -NHCHO, -COOCH₃, -CSOCH₃, -COSCH₃,

-COOCH₂CH₃, -CSOCH₂CH₃, -COSCH₂CH₃, -NHCOCH₃, -NHCSCH₃, -CS-NH-CONH₂, -CO-NH-CONH₂,

-CO-NH-CSNH₂, -CS-NH-CSNH₂, -C (NH₂)=C(CN)₂, -CH(CN)₂, -CH(CN)-COOH, -CH(CN)-CONH₂, -CH(CN)-CSNH₂, -CH(CN)CSNH-C_1-4 alkyl, -NO₂ or -NO group;

X means =O, =S or =NH group;

Y means -O-, -S- or -NH- group;

R₂ and R₄, independently from each other, represent hydrogen atom, -OH, -SH, -NH₂, -CF₃ , C_1-4 alkoxy or C_1-4 alkylthio group;

R₃ stands for -OH, -SH, -NH₂, -CF₃ , -NO, -O-SO₂-V⁰,

-O-CO-V⁰, -O-CS-V⁰, -S-CO-V⁰, -NH-CO-V⁰ or

-CH=CH-V⁰ group, wherein

V⁰ stands for 3,4,5-tri-V¹-phenyl group; and the

V¹ substituents, independently from each other. mean hydrogen or halogen atom or -OH, -SH, -NH₂, -CF₃, C_1-4 alkoxy, C_1-4 alkylthio or C_1-4 alkylamino group; or

V¹ bound in the 3- or 4-position may be -COOH, -CSOH, -COSH, -CONH₂, C_1-4 dialkylamino, pyridyl, -CH₂-2-pyridyl, -O-CH₂-O- or

-N(CH₂-V²)-CH₂-V³ group, wherein

V² and V³, independently from each other,

stand for -OH, -SH, -NH₂, -CF₃, C_1-4 alkyl, C_1-4 alkoxy or C_1-4 alkylthio group; or

V² and V³ together stand for -NH-CH₂-NH- or -N(CH₃)-CH₂-N(CH₃)- group;

R₅ represents hydrogen, C_1-4 alkyl, C_1-4 alkoxy

group or halogen; and

the double bond belonging to the skeletal atom

substituted by R₁ can also be saturated when R₃ is a substituent containing the -3,4,5-tri-V¹-phenyl moiety, as well as physiologically acceptable salts and complexes thereof and pharmaceutical compositions containing these compounds.

According to an other aspect of the invention there is provided a process for the preparation of the new compounds of general formula (I).

It is known that the intercellular communication plays a decisive role in the maintenance of differentiated functions of specialized cells in the organism. A defective function of this communication system may lead to various disorders, inter alia to tumour development in the organism. Today, the process of carcinogenesis can unanimously be comprehended as a result of the defective functioning of intercellular communication and intracellular signal transmission mechanism, which occurs as a consequence of false messages sent e.g. by hormones, growth factors and oncogenes or disturbation of the signal transfer mechanisms. In normal cases the regulation of cell division and differentiation is accomplished by a number of independent mechanisms and the simultaneous existence of several damaging factors is needed to stop this regulating process or to develop a tumorous transformation. Based on investigations recently carried out it can unambiguously be proven that the development of a tumour is a multistep process being the consequence of a cooperative interaction between activated oncogenes and inactivated antioncogenes. The processes of cell division and differentiation as well as the intricate functioning of specialized cells reveal a high degree of coordinated regulation depending on both the inner genetic program of the cell and the intercellular communication of the cells.

In the course of tumour formation the systems regulating the propagation and differentiation, which are connected in a complicated communication system under normal conditions, are disconnected and the proliferation becomes stimulated, this process leading to an unhindered cell division, i.e. tumorous transformation. A tumour begins to develop when viable cell variants are formed among these transformed tumour cells, which commence to multiply in the "hostile" microenvironment.

In comparison to normal cells, these cell variants have to possess specific growth and other selective properties for the survival in the competitive environment and for further cell division. In the middle of a constant interaction and control of microenvironmental factors, specific signal transfer mechanisms are activated in the surviving tumour cells, which are capable of stopping the feedback mechanisms and inhibitory effects. Recently it has been unambiguously shown that the signal transfer mechanisms regulating cell division are started by specific tyrosine kinases sub sequently activating the serine kinases (e.g. protein kinase C) and other routes of signal transmission. Based on the unambiguous correlation between the activity of tyrosine kinase and the extent of cell division, the preparation of active agents being capable of inhibiting the tyrosine-kinase activity of tumourous cells presents a new perspective in the tumour-biologic and -therapeutic researches.

During the past decades the research of drugs having antitumourous activity was concentrated to develop active agents destroying the tumourous cells. During this period a number of useful drugs were developed mostly by the systematic variation of the chemical structure of available active agents and testing of new compounds.

Since the proliferation of tumourous cells is generally an active division, the most active compounds proved to be compounds which were cytotoxic to cells under division; however, such substances exert many severe adverse effects chiefly because of inhibiting the division of healthy cells, too.

In the last two or three years several native active agents have been discovered and their analogues have also been worked out, but these active agents are useful only for the specific diminution of the activity of EGF-RTK tyrosine kinase (see the published Japanese patent applications Nos. 62/39523, 62/42923 and 62/42925 as well as the European patent specification No. 322,783).

The therapeutical utilization of coumarin derivatives of plant origin goes back to thousands of years. The preparation of coumarin derivatives was studied from the beginning of this century. The best known coumarin drugs are anticoagulants such as dicoumarol, ethyl bis-coumacetate, acenocoumarol, warfarin and phenprocoumon, the common structural characteristics of which are an aromatic substituent bound through a methylene bridge in 3-position and a hydroxy group in 4-position [A. Kleeman and J. Egel: Sostanze Farmaceutiche, OEMF-Milano (1988)]. Bucoumol containing a methyl group in 5-position and an amino-hydroxy-propyloxy substituent, characteristic of beta-blocking compounds, is a beta-adrenergic blocking agent (U.S. patent specification No. 3,663,570). Folescutol, chemically 6,7-dihydroxy-4-morpholinomethylcoumarin, shows a capillary-protecting action (French patent specification No. M 2035). Hymechromon, chemically 7-hydroxy-4-methylcoumarin, exerts a bile-secretion-promoting effect (French patent specification No. M 1430). Thioclomarol, chemically 4-hydroxy-3-[5-chloro-alpha- (p-chloro-beta-hydroxyphenethyl)-2-thienyl]-coumarin, is an antithrombotic agent [Drugs of Today 14, 383 (1978)].

The invention is aimed at developing novel antitumour compounds inhibiting the activity of tyrosine kinase enzymes playing a role in the signal transfer mechanism without any practical influence on the function of the tyrosine kinase enzyme of the insulin receptor.

The invention is based on the recognition that, due to their molecular structure and electron-arrangement properties, the members of the compound class of general formula (I), being new coumarin derivatives and their analogues, are capable of being non-covalently bound with the desired specifity in a suitable position to substructures recognizing the tyrosine side chain of the protein substrate at catalytic sites of the tyrosine kinase enzymes. Thus, they satisfy the principles discussed above and are useful to inhibit the proliferation of mammalian cells and the tumor growth.

According to the invention, the novel coumarin derivatives and their analogues of general formula (I) are prepared by a) condensing a benzaldehyde derivative of general formula (IV)

wherein Q means -OH, -SH or -NH₂ group and R₂, R₃, R₄ as well as R₅ are as defined for the formula (I), with a carboxylic acid derivative of general formula (V),

L1 — CH₂— R 1 (V) wherein L₁ stands for a -COOEt, -CSOEt or -CN group and R₁ is as defined for formula (I), in the presence of an acid catalyst; or

b) condensing a benzaldehyde derivative of general formula (IV), wherein Q, R₂, R₃, R₄ and R₅ are as defined above, with a carboxylic acid derivative of the general formula (V), wherein L₁ and R₁ are as defined above, in two steps, first in the presence of an alkaline catalyst and then in the presence of an acidic catalyst; or

c) condensing a benzaldehyde derivative of general

formula (IV), wherein Q, R₂, R₃, R₄ and R₅ are as defined above, with a carboxylic acid derivative of the general formula (V), wherein L₁ and R₁ are as defined above, in two steps, first in the presence of an alkaline or acid catalyst and then by a thermal reaction; or condensing a compound of general formula (II),

wherein Q means -OH, -SH or -NH₂ group, and R₂, R₃, R₄ as well as R₅ are as defined above, with a carboxylic acid derivative of the general formula (III),

wherein R stands for -COOC₂H₅ or -CSOC₂H₅ group, in the presence of an acid catalyst,

to obtain compounds of the general formula (la),

representing a preferred group of compounds of general formula (I), wherein R means -COOC₂H₅ or -CSOC₂H₅ group, and R₂, R₃, R₄, R₅, X and Y are as defined above; or

e) condensing a compound of the general formula (II), wherein Q, R₂, R₃, R₄ and R₅ are as defined above, with a carboxylic acid derivative of general formula (III), wherein R is as defined above, in two steps, first in the presence of an alkaline catalyst and then in the presence of an acidic catalyst,

to obtain compounds of the general formula (la) representing a preferred group of compounds of general formula (I), wherein R, R₂, R₃, R₄, R₅, X and Y are as defined above; or

f) condensing a compound of the general formula (II), wherein Q, R₂, R₃, R₄ and R₅ are as defined above, with a carboxylic acid derivative of general formula (III), wherein R is as defined above, in two steps, first in the presence of an alkaline or acidic catalyst and then by a thermal reaction, to obtain compounds of the general formula (la) representing a preferred group of compounds of general formula (I), wherein R, R₂, R₃, R₄, R₅, X and Y are as defined above.

In the above process a) the condensation is carried out at a temperature between 100 °C and 200 °C, optionally under a protective gas depending on the substituents. Depending on the starting substances, water or a polar solvent, preferably ethanol, is used as solvent. Phosphoric acid, hydrochloric acid or in some cases sulfuric acid may preferably be used as acid catalysts in an amount of 0.01 to 10.0 molar equivalent(s).

In the above process b) the condensation is carried out at a temperature between room temperature and 200 °C, optionally under a protective gas depending on the substituents. Alkali metal or alkali earth metal hydroxides taken in an amount of 0.001 to 1.0 molar equivalent are suitable alkaline catalysts. Preferred solvents are water or a polar solvent, suitably ethanol, depending on the starting substances. After neutralizing the reaction mixture, the ring is closed in a further condensation reaction in the presence of an acid catalyst. This condensation is accomplished between 100°C and 200 °C, optionally under a protective gas depending on the substituents. Suitable solvents depending on the starting substances are water or a polar solvent, preferably ethanol. Phosphoric acid, hydrochloric acid and in some cases sulfuric acid are preferably used as acid catalysts in an amount of 0.01 to 10.0 molar equivalent(s).

In the above process c) the condensation is carried out between room temperature and 100 °C, optionally under a protective gas depending on the substituents. Alkali metal or alkali earth metal hydroxides taken in an amount of 0.001 to 1.0 molar equivalent are suitable alkaline catalysts whereas 0.001 to 1.0 molar equivalent of phosphoric acid or hydrochloric acid or in some cases sulfuric acid may suitably be used as acid catalysts. Depending on the starting substances, water or a polar solvent, preferably ethanol, is used as solvent. After neutralizing the reaction mixture, the intermediate is taken up in an organic solvent being inert to the reaction conditions, then the ring is closed by thermal condensation between 100 °C and 200 °C, optionally under a protective gas depending on the substituents. Suitable organic solvents are alkanes, aromatic hydrocarbons containing no unsaturation in their side chain or some polar organic solvents such as chloroform or dimethylsulfoxide.

In the above process d) the condensation is realized at a temperature between 100 °C and 200 °C, optionally under a protective gas depending on the sub stituents. Depending on the starting substances, water or a polar solvent, suitably ethanol, may be used as solvent. Phosphoric acid or hydrochloric acid or in some cases sulfuric acid taken in an amount of 0.01 to 10.0 molar equivalent(s) may preferably be used as acid catalysts.

In the above process e) the condensation is carried out between room temperature and 200 °C, optionally under a protective gas depending on the substituents. Alkali metal or alkali earth metal hydroxides in an amount of 0.001 to 1.0 molar equivalent may be used as preferable alkaline catalysts. Depending on the starting substances water or a polar solvent, suitably ethanol, is used as solvent. After neutralizing the reaction mixture the ring is closed in a second condensation reaction in the presence of an acid catalysts. This condensation is accomplished at a temperature between 100 °C and 200 °C, optimally under a protective gas depending on the substituents. Depending on the starting substances, water or a polar solvent, preferably ethanol, is used as solvent. As acid catalyst 0.01 to 10.0 molar equivalent(s) of phosphoric acid or hydrochloric acid or in some cases sulfuric acid is (are) used.

In the above process f) the condensation is carried out between room temperature and 100 °C, optionally under a protective gas depending on the substituents. Alkali metal or alkali earth metal hydroxides taken in an amount of 0.001 to 1.0 molar equivalent are preferable alkaline catalysts whereas 0.001 to 1.0 molar equivalent of phosphoric acid or hydrochloric acid or in some cases sulfuric acid may suitably be used as acid catalyst. Depending on the starting substances water or a polar solvent, preferably ethanol, is used as solvent. After neutralizing the reaction mixture and then taking up the intermediate in an organic solvent being inert to the condensation conditions, the ring is closed by thermal condensation at a temperature between 100 °C and 200 °C, optionally under a protective gas depending on the substituents. Alkanes, aromatic hydrocarbons containing no unsaturation in their side chain or some polar organic solvents such as chloroform or dimethyl sulfoxide may be used as organic solvents.

After carrying out the condensation, the substituent R of the product of general formula (la) may be subjected to further transformations by using methods known from the literature to obtain other derivatives of the compounds of general formula (I). Thus, by subjecting to acidic hydrolysis a compound of the general formula (la) prepared as described above, wherein R means

-COOC₂H₅ or -CSOC₂H₅ group, a compound of the general formula (I) is obtained, wherein R₁ stands for -COOH or -CSOH group; and/or by saturating with gaseous ammonia an alcoholic solution of a carboxylic acid derivative thus prepared a compound of the general formula (I) is obtained, wherein R₁ means -CONH₂ or -CSNH₂ group; and/or by reacting a carboxylic acid amide derivative thus prepared e.g. with phosphorus oxychloride, a compound of the general formula (I) can be prepared, wherein R₁ stands for -CN group.

Compounds, wherein at least one of the R₂ and R₄ substituents is different from hydrogen, are preferred.

Compounds, wherein R₂ and R₃ are hydroxy groups and R₃ means an -O-SO₂-V⁰ type substituent, where V⁰ is as defined above, are very preferable.

The compounds of general formulae (II), (III), (IV) and (V) were prepared by using methods described in the literature cited hereinafter or analogues thereto. Compounds of the general formula (II): Beilsteins Handbuch der Organischen Chemie 6, 833, 1071, 1154; 6 (4.

Erganzungswerk) 722, 5633; 10 (2. Erganzungswerk), 333; 13, 826; 13 (4. Erganzungswerk) 1425. Compounds of the general formula (III): ibidem 3, 469. Compounds of the general formula (IV): ibidem 8, 388; 8 (1. Erganzungswerk), 684. Compounds of the general formula (V): ibidem 2, 232, 573, 589; 2 (4. Erganzungswerk), 1907; 3, 66; 4, 354; as well as Angew. Chem., Int. Ed. Engl. 16, 339.

The invention furthermore relates to pharmaceutical compositions inhibiting tumour growth and cell proliferation; these compositions contain the compounds of general formula (I) and/or their physiologically acceptable salts and/or complexes as active agents together with carriers and additives commonly used in the pharmaceutical industry. The invention also relates to the preparation of these compositions.

In the above pharmaceutical compositions either the compounds of general formula (I) themselves or their physiologically acceptable salts or complexes may be used.

The pharmaceutical compositions may contain any solvent being suitable for therapeutical use (such as water or aqueous solutions containing ethanol and/or a polyalcohol, e.g. polyethylene glycol and/or glycerol or the like); salts (e.g. sodium chloride for adjusting the physiological osmotic pressure or e.g. iron, cobalt, zinc or copper chloride and the like for supplementing trace elements); fillers and carriers (such as lactose, potato starch, talc, magnesium carbonate, calcium carbonate, waxes, vegetable oils, polyalcohols and the like); additives promoting dissolution (such as polar organic solvents, usually ethanol, polyalcohols, most frequently polyethylene glycol or glycerol and/or complex-forming agents, e.g. cyclodextrins, crown ethers, native proteins, saponins and the like in the case of water); tablet-disintegrating agents (inert artificial or native polymers strongly swelling in water, e.g. carboxymethyl cellulose); the usual complex-forming agents in composition with sustained release (such as water-insoluble or slightly soluble cyclodextrin derivatives, artificial and native polymers, crown ethers and the like); pH-adjusting substances such as mineral or organic buffers; savouring agents (such as cyclodextrins and/or crown ethers) and flavouring substances (such as beet sugar, fructose, dextrose, saccharin, invert sugar and the like); antioxidants (e.g. vitamin C); as well as substances promoting the development of effect of the compounds of general formula (I).

The compounds of general formula (I) can be used as active ingredients for the preparation of orally administered compositions such as tablets, pills,

dragees, hard or soft gel capsules, microcapsules, solutions, emulsions or suspensions; parenterally administered compositions such as injections, rapid or slow infusions; rectally administered compositions such as suppositories; as well as cremes or gels. It is also possible to formulate the pharmaceutical compositions developed for the above uses by incorporating to liposomes or microcapsules.

The compounds of general formula (I) are suitable to be used in the form of aerosol compositions aimed at resorption through the skin or lungs.

For the preparation of tablets, dragees or hard gel capsules e.g. lactose, maize, wheat or potato starch, talc, magnesium carbonate, magnesium stearate, calcium carbonate, stearic acid and its salts and the like can be used as carriers. Vegetable oils, fats, waxes or polyalcohols of suitable density are useful carriers for preparing soft gel capsules. Water, polyalcohols such as polyethylene glycol or glycerol, beet sugar, dextrose and the like may be used as carriers for the preparation of solutions and syrups. Parenterally administered composi tions may contain water, alcohol, polyalcohols or vegetable oils as carriers. Suppositories may contain e.g. oils, waxes, fats or polyalcohols of suitable density as carriers.

The compounds of general formula (I) or pharmaceutical compositions containing them, respectively, are useful also together with other artificial or native active agents for therapeutical use.

In the therapeutical compositions the compounds of general formula (I) may be used in a daily dose of 0.01 to 5000 mg/kg, preferably 0.1 to 250 mg/kg, but the dose is dependent on the sort of the tumour, degree of malignancy, state and age of the patient and the like. METHODS AND RESULTS OF BIOLOGICAL INVESTIGATIONS

Measurement of inhibition of cell division, I

The incorporation of [³H]-thymidine into tumour cells of various origin and measurement of the cell count were carried out by using the method of Kéri et al.

[Tumor Biology 9, 315-322 (1988)]. The biological activity of the analogues was expressed as te percentage of inhibition of the incorporation of labelled thymidine or cell count elevation measured on untreated cells used as control. HT-29 and SW-620 cell lines were employed in these experiments.

The measurement results on the effect of the compounds of Examples 1 to 3 on the division of HT-29 cell line are summarized in Figure 1. The measurements were carried out as mentioned above, except that it was also examined whether the results were influenced by adding to the cell culture various amounts of fetal calf serum (FCS) stimulating cell division. The degree of cell division was determined after 48-hour incubation. Each of the three active compounds was used in 100 μM

concentration. The measurement results on the effect of the compounds of Examples 1 and 2 on the division of SW-620 cell line are summarized in Figure 2. The measurements were carried out with various concentrations of the active compounds. The cell cultures were incubated for 24 hours in all experiments. The culture media contained also 10 % of FCS.

Measurement of tyrosine kinase activity

The acitivity of tyrosine kinase was also

determined by using the method of Kéri et al. [Tumor Biology 9, 315-322 (1988)]. The biological activity of analogues was also characterized on the basis of their effect inhibiting the incorporation of ³²P isotope in relation to the incorporation determined in untreated cells used as control.

The measurement results on the effect of the compounds of Examples 1 to 3 on the tyrosine kinase activity of cells of SW-620 cell line are summarized in Figure 3. The measurements were carried out as described above. The active compounds were used in various concentrations.

In vivo determination of the effect on tumour metastasis and growth in a spleen-liver metastasis model

The antimetastatic activity of compounds was investigated on Lewis lung tumour (LLT) cells in muscle-lung and spleen-liver metastasis models as well as on their immunoresistant cell variant (LLT-HH) of increased metastatizing ability. These experiments were accomplished on inbred C5781 mice of both sexes. LLT was transplanted into the muscle in the muscle-lung metastasis model whereas a suspension of the tumour cells was injected to the spleen in the spleen-liver metastasis model. The compounds to be tested were injected intra-peritoneally (ip.), intravenously (iv.), orally (po.) or subcutaneously (sc.) in doses of 0.1 to 10 mg/kg daily 1 to 3 times on the Sth to 13th days. The therapeutical effect was determined on the basis of the number of metastases in such a way that a sample was taken on the 10th and 14th days in the spleen-liver model, and on the 17th and 18th days in the muscle-lung model, then the macroscopic metastases were counted in the liver or lungs, respectively, under a stereomicroscope. The activity of the compounds was expressed as percentage of the number of metastases determined in the control animals.

The measurement results obtained with the compounds of Examples 1 to 3 in the LLT-HH spleen-liver metastasis model are summarized in Figure 4. The results were based on the data obtained after a 12-day treatment. The substances were used in various concentrations.

The measurement results obtained with the compound of Example 2 on the LLT-HH spleen-liver metastasis model are summarized in Figure 5. The results were based on the data obtained after a 10-day treatment. The substance was used in concentrations according to the measurement illustrated in Figure 4.

The measurement results obtained with the compounds of Examples 1 and 3 in the LLT-HH spleen-liver metastasis model are summarized in Figure 6. The results were based on data obtained after a 14-day treatment. The substances were used in various concentrations according to the measurement illustrated in Figure 4.

In vivo determination of tumour-growth inhibiting effect

Own outbred, inbred and hybrid mice, respectively, were used for establishing tumour models developed by transplanting tumours of animal origin. The tumours and mouse strains used will be described in detail hereinafter together with the individual measurement results. The effect of various treatments on the tumour growth was evaluated on the basis of alteration of the life span in relation to that of the tumorous control group or by measuring the tumour size in cases of the so-called solid tumours. The volume of the tumour was determined on the basis of the following correlation:

V = (Pi/6).L.D²

wherein

V means the tumour volume

Pi is Ludolf's number,

L is the longest diameter of the tumour and

D² is the square of the shortest diameter being perpendicular to L.

The experimental results showing the effect of compound of Example 1 on S-180 sarcoma tumour growth are summarized in Figure 7. (S-180 sarcoma was obtained from Chester Beatty Cancer Res. Inst., London). The tumour was transplanted by sc. tumour fragments. Outbred Swiss mice were used as host animals. Each experimental group consisted of 10 animals with a body weight of 21 to 23 g. The results were calculated from the data measured on the 10th, 12th and 14th days. The compound of Example 1 was administered in a daily dose of 5 or 10 mg/kg, respectively. The treatments were carried out 3 times, repeated every 2 days. The treatments were started two days after the tumour transplantation.

The experimental results on the effect of compound of Example 1 on B-16 melanoma tumour growth are summarized in Figure 8. (B-16 melanoma tumour was obtained from Karolinska Inst. Dep. of Tumor Biology, Stockholm.) The transplantations were carried out by using cell suspensions in sc. route. BDF₁ hybrid mice were used as host animals. Each experimental group consisted of 10 animals with a body weight of 21 to 23 g. The results were calculated from the data measured on the 10th, 12th and 14th day, resp. The compound of Example 1 was administered in a daily dose of 5 or 10 mg/kg, respectively. The treatments were carried out 3 times, repeated every second day. The treatments were started 2 days after tumour transplantation.

The experimental results showing the effect of the compound of Example 2 on the P-388 sc. leukemia tumour growth are summarized in Figure 9. (The P-388 lymphoid leukemia tumour was obtained from Arthur Little and Co., Cambridge MA, USA.) The transplantation was carried out in sc. route by using 1×10⁷ cells. BDF₁ hybrid mice were used as host animals. Each experimental group consisted of 10 animals with a body weight of 21 to 23 g. The results were calculated from the data measured on the 11th, 14th and 16th day, resp. The compound of Example 2 was administered in a daily dose of 0.1, 1.0 or 10.0 mg/kg, respectively. The treatments were carried out 3 times, repeated every second day. The treatments were started 2 days after tumour transplantation.

The results showing the effect of compound of Example 2 on the B-16 melanoma tumour growth are summarized in Figure 10. (B-16 melanoma tumour was obtained from Karolinska Inst. Dep. of Tumor Biology, Stockholm.) The transplantation was carried out by using a cell suspension in sc. route. BDF₁ hybrid mice were used as host animals. Each experimental group consisted of 10 animals with a body weight of 21 to 23 g. The results were calculated from the data measured on the 11th, 14th and 16th day, resp. The compound of Example 2 was administered in a daily dose of 0.1, 1.0 and 10.0 mg/kg, respectively. The treatments were carried out 3 times, repeated every second day. The treatments were started 2 days after tumour transplantation.

Measurement of inhibition of cell division, II

ZR-75.1 human breast cancer cell line and its form transformed by the human EGF receptor cDNA, i.e. the ZRHERc cell line (in the cells of which functional human EGF receptor tyrosine kinase is expressed), were used for these examinations. During these measurements the cancerous cells were incubated in serum or serum-free culture medium after adding a suitable amount of EGF for stimulating cell growth. The measurement results were obtained by determining the cell counts of the cultures usually on the 3rd, 6th and 10th days.

The measurement results showing the effect of compounds of Example 1 and 2 on the growth of ZR-75.1 cells are summarized in Figure 11. During this experiment the ZR-75.1 cells were incubated in serum. The concentration of compounds of both Examples 1 and 2 was 100 μM in the serum.

The experimental results showing the effect of compounds of Examples 1 and 2 on the growth of ZRHERc cells are summarized in Figure 12. During this examination the ZRHERc cells were incubated in serum. The concentration of compounds of both Examples 1 and 2 was 100 μM in the serum.

The results showing the effect of compound of

Example 1 on the cell growth are summarized in Figure 13. ZRHERc cell line was used for this investigation. The cells were incubated in a serum-fre nutritive solution together with EGF added in 5 nanogram (ng)/ml concentration in the presence of various concentrations of the active agents (10, 50 and 100 μM, respectively, of the compound of Example 1).

The results showing the effect of compound of Example 2 on the cell growth are summarized in Figure 14. ZRHERc cell line was used for this examination. The cells were incubated in a serum-free nutritive solution together with EGF added in a concentration of 5 ng/ml in the presence of various concentrations of the active agents (10, 50 and 100 μM, respectively, of the compound of Example 2). Measurement of the inhibition of cell transformation under the effect of H-ras oncogene

This study was carried out on NIH3T3 cells accoding to a method known from the literature [Seymour J. Garte et al.: Inhibition of H-ras Oncogene Transformation of NIH3T3 Cells by Protease Inhibitors; Cancer Research 47, 3159-3162 (1987)].

The measurement results showing the inhibitory effect of compounds of Examples 1 and 2 on the

transformation by H-ras oncogene of NIH3T3 cells are summarized in Figure 15. The compounds of Examples 1 and 2 were studied in 10 and 100 μM concentrations, resp.

Based on the experimental results it can be stated that the compounds according to the invention strongly inhibit the growth of cancerous cells.

According to Figure 1 the cell count of HT-29 cell line does not exceed the starting cell count even on the 2nd day in spite of increasing amounts of serum stimulating the cell growth.

It can be seen in Figure 2 that, depending on the concentration of the active agent, the ratio of number of living cells of serum-containing untreated cultures of the SW-620 cell line in relation to that of the treated culture significantly decreased after one day.

It is clear from Figure 11 that, with progressing time, the growth of ZR-75.1 cells in the pure serum falls far behind the growth of untreated cells.

It is obvious from Figure 12 that the growth of

ZRHERc cells cultivated in pure serum also lags behind the growth of untreated cells on effect of the treatment.

The experimental results prove that the compounds according to the invention inhibit the tyrosine kinase activity of cancerous cells.

It can be seen in Figure 3 that the total tyrosine kinase activity of SW-620 cancerous cells was diminished on increasing the concentration of the administered active agent.

Figures 13 and 14 illustrate how the growth of ZRHERc cells cultivated in serum-free nutritive solution with stimulation by EGF falls behind the growth of untreated cells with progressive time, as a consequence of increasing the amount of active agent, a process due to the inhibition of the EGF receptor tyrosine kinase enzyme.

The experimental results also prove that the number of metastases formed in the cancerous process is lowered by the compounds according to the invention.

It can be seen in Figures 4, 5 and 6 that, in the LLT-HH spleen-liver metastasis model, the number of metastases developed is significantly reduced by the active agents used in increasing doses in comparison to the number of metastases formed in the untreated group.

In addition, it is proven by the experimental results that the tumour growth is diminished by the compounds according to the invention.

It is evident from Figures 7, 8, 9 and 10 that, by increasing the doses of the active compound and progress of the treatment period, the growth of tumorous foci introduced to the experimental animals falls more and more behind the growth of tumours introduced to the untreated animals.

Based on the experimental results it can be stated that the development of several cancerous processes is inhibited by the compounds according to the invention.

It can be seen from Figure 15 that the malignisa- tion of NIH3T3 cells under the effect of the H-ras oncogene introduced is inhibited by the compounds of Examples 1 and 2.

The main advantages of the invention are as

follows: a) The novel active compounds inhibiting the specific signal transfer of tyrosine analogue tyrosine kinase do not inhibit the cell division in the traditional sense and therefore they do not considerably interfere with the division of normal cells. Thus, they are not encumbered by adverse side effects accompanied with severe complications being unavoidable concomitants of the classical chemotherapeutics.

b) The novel active compounds according to the invention do not inhibit the kinase activity from the side of the ATP binding site and therefore, they do not show the strong cytotoxicity which is characteristic of ATP analogues.

c) The novel active compounds according to the invention possess a broader spectrum of activity in relation to the known signal transfer-inhibiting active agents (which are EGF-RTK specific) while maintaining the favourable property that they do not interfere with the function of the insulin receptor tyrosine kinase.

The invention is illustrated in detail by the following non limiting Examples.

Example 1

Preparation of 3-cyano-7-hydroxycoumarin [compound of general formula (I), wherein R ₁ = -CN, X = O=, Y = -O-, R₂ = -H, R₃ = -OH, R₄ and R₅ = -H]

After dissolving 3.81 g of 2,4-dihydroxybenzaldehyde and 3.18 ml of ethyl cyanoacetate in 30 ml of ethanol and adding 0.2 ml of piperidine, the reaction mixture is stirred at room temperature for 5 hours. The crystalline precipitate is filtered and dried in air. After taking up the intermediate obtained in a 3-fold amount of dimethyl sulfoxide (DMSO), the reaction mixture is stirred on an oil bath at a temperature of 170-180 °C for 20 minutes. After cooling, the crystals are filtered and washed with alcohol to obtain 2.82 g of crude product. After recrystallization from alcohol while clearing with activated carbon, the aimed final product is obtained in a yield of 1.19 g, m.p.: 270-272 °C.

Example 2

Preparation of 7-hydroxycoumarin-3-thiocarboxylic acid amide [compound of general formula (I), wherein R₁ = -CSNH₂, X = O=, Y = -O-, R₂ = -H, R₃ = -OH, R₄ and R₅ = -H]

After reacting 4.5 g of 2,4-dihydroxybenzaldehyde, 3.28 g of alpha-cyanothioacetamide and 0.2 ml of triethyl amine in ethanol while stirring at room temperature for 5 hours, the crystalline precipitate is filtered, washed with ethanol and dried. The intermediate obtained is treated with a 40-fold amount of 5 % hydrocloric acid at 100 °C. After cooling down, the crystals are filtered, washed with water and again dried. In this way 4.81 g of crude product are obtained to give 3.51 g of the aimed final product after recrystallization from dimethylformamide (DMF), m.p.: 256-258 °C.

Example 3

Preparation of 3-acetylamino-7-hydroxycoumarin

[compound of general formula (I), wherein R₁ = = -NHCOCH₃, X = O=, Y = -O-, R₂ = -H, R₃ = -OH, R₄ and R₅ = -H]

A mixture containing 6.91 g of 2,4-dihydroxybenzaldehyde, 5.86 g of acetylglycne, 4.19 g of anhydrous sodium acetate and 15 ml of acetic anhydride is reacted at a temperature of 100 °C for 90 minutes. The precipitate is filtered, washed with ether and dried. The intermediate obtained is suspended in 60 ml of ethanol and, after adding 1.5 ml of hydrazine hydrate, the mixture is stirred at 25 °C for one hour. After filtering, the crystalline precipitate is washed with alcohol and water to give 2.29 g of crude product. After recrystallization from dimethylformamide, 1.69 g of the aimed final product are obtained, m.p.: 296-297 °C.

Example 4

Preparation of 3-ethoxycarbonyl-6,7,8-trihydroxycoumarin [compound of general formula (I), wherein R₁ = -COOC₂H₅, X = O=, Y = -O-, R₂, R₃ and R₄ =

-OH, R₅ = -H]

To a solution of 1.42 g of 1,2,3,4-tetrahydroxybenzene in 20 ml of abs. ethanol, 2.16 g of diethyl ethoxymethylene malonate are portionwise added in the presence of a catalytic amount of sodium ethoxide under vigorous stirring and cooling by ice. The reaction mixture is then left to stand at -4 °C for 2 days, thereafter it is neutralized with acetic acid and evaporated. The intermediate obtained is vigorously stirred with 85 % phosphoric acid at 100 °C for one hour, then poured onto 500 g of crushed ice. The product is extracted into ethyl acetate and then recrystallized from ethyl acetate to give 0.212 g of the aimed product, m.p.: 155-157 °C.

Example 5

Preparation of 3-carboxy-6,7,8-trihydroxycoumarin

[compound of general formula (I) wherein R₁ =

-COOH, X = O=, Y = -O-, R₂, R₃ and R₄ = -OH,

R₅ = -H]

A solution containing 0.266 g of 3-ethoxycarbonyl-6,7,8-trihydroxycoumarin (prepared according to Example 4) in 5 ml of 5% sodium hydroxide solution is stirred at room temperature until dissolution. The reaction mixture is stirred for additional two hours at room temperature and then acidified to pH 2 by 5 % hydrochloric acid.

After extracting the product twice with 5 ml of ethyl acetate each and evaporating, the product obtained is recrystallized from ethanol to give the aimed compound in a yield of 0.204 g, m.p.: 212-214 °C (with decomposition). Example 6

Preparation of 6,7,8-trihydroxycoumarin-3-carboxylic acid amide [compound of general formula (I), wherein R₁ = -CONH₂, X = O=, Y = -O-, R₂, R₃ and R₄ = -OH, R₅ = -H]

0.264 g of 3-ethoxycarbonyl-6,7,8-trihydroxycoumarin (prepared according to Example 4) is dissolved in 5 ml of ethanol at room temperature, then the solution is saturated with gaseous ammonia. The crystalline precipitate is filtered and recrystallized from ethanol to give 0.209 g of the aimed product, m.p.: 205-207 °C.

Example 7

Preparation of 3-cyano-6,7,8-trihydroxycoumarin

[compound of general formula (I), wherein R₁ = -CN, X = O=, Y = -O-, R₂, R₃ and R₄ are -OH, R₅ = -H]

After vigorously stirring a mixture of 0.236 g of 6,7,8-trihydroxycoumarin-3-carboxylic acid amide (prepared according to Example 6) and 2 ml of phosphorus oxychloride in a water bath of 60 °C for 2 hours, the reaction mixture is cooled to room temperature and poured into 20 g of crushed ice. The crystalline precipitate is filtered and recrystallized from benzene to obtain 0.151 g of the aimed product, m.p.: 158-160 °C.

By suitably changing the starting substances, the compounds of Nos. 8 to 41 listed in Table 1 hereinafter were prepared. For identification of the compounds (also including the aimed final products of Examples 1 to 7), the retention times of high-pressure liquid chromatography (HPLC) are indicated.

The characteristics of the HPLC systems used are as follows:

HPLC - A: 50 % H₂O, 50 % CH₃OH, isocratic, Nucleosil C10

C₁₈, 300x4.6 ID, 1.5 (ml/min), t_o = 120 (s); - B: 22 % H₂O, 78 % CH₃OH, isocratic, Nucleosil C10 C₁₈, 250×4 ID + 30×4 ID guard, 1 (ml/min). t_o = 150 (s).

Table 1

Example Substituents HPLC retention time No. (other than H) System t (min)

1 X:O=, Y:- O-, R₁:-CN, R₃:-OH A 5.85 2 X:O=, Y:- O-, R₁:-CSNH₂, R₃:-OH A 3.47 3 X:O=, Y:- O-, R₁:-NHCOCH₃, R₃:-OH A 4.97 4 X:O=, Y:- O-, R₁:-COOEt, R₂,R₃,R₄:-OH A 11.73 5 X:θ=, Y:- O-, R₁:-COOH, R₂,R₃,R₄:-OH A 6.47 6 X:O=, Y:- O-, R₁:-CONH₂, R₂,R₃,R₄:-OH A 3.10 7 X:θ=, Y:- O-, R₁:-CN, R₂,R₃,R₄:-OH A 7.91 8 X:NH=, Y:-O-, R₁:-CN, R₂,R₃: -OH A 0.23 9 X:O=, Y:-S-, R₁:-CN, R₃:-SH A 25.22

B 9.26

10 X:S=, Y:- O-, R₁:-CN, R₃ : -OH A 19.21 11 X:O=, Y:-NH-, R₁:-COOEt,

R₂:-OH, R₃:-NH₂, R₅:-F A 2.07

12 X:HN=, Y:-S-, R₁:C-N

R₂:-OCH₃, R₃:CF₃, R₄:-OCH₃ A 16.01

13 X:O=, Y:- O-, R₁:-NHCSCH₃, R₃:-OH A 2.71 14 X:O=, Y:-O-, R₁:-NHCHO, R₃ : -OH A 0.31 15 X:O=, Y:- O-, R₁:-CSNH-CONH₂

R₃:-OCH₃, R₅:-CH₃ A 0.22

16 X:O=, Y:- O-, R₁:-C(NH₂)=C(CN)₂,

R₃:-OCH₃, R₅:-Br A 0.23

17 X:O=, Y:- O-, R₁:-CH(CN)₂,

R₂, R₄:-CF₃, R₃:-OH A 10.24

18 X:O=, Y:- O-, R₁:-CH(CN)-CSNH₂ A 19.78

R₂, R₃, R₅:-OEt B 10.21

19 XrO=, Y:- O-, R₁:-NO A 28.30

R₂, R₄:-SEt, R₃:-OH B 11.56

20 X:O=, Y:- O-, R₁:-NO₂, R₃:-CF₃, R₄ : -NO A 6.85

21 X:O=, Y:- O-, R₁.-CN, (3,4-dihydro derivative)

R₃:-OSO₂-p-CF₃-phenyl A 17.63 Table 1 ( contd . )

Example Substituents HPLC retention time No . (other than H) System t (min)

22 X:O= , Y -O-, R₁:-CN, (3,4-dihydro derivative)

R₃:- OSO₂ (3-OH-4-COOH-phenyl) A 8.85

23 X:O= -O-, R₁:-CN

R₃:- OSO₂-(3,5-di-SCH₃-4-CSOH-phenyl) A 6.70

24 X:O= , Y: O- R₁: -CN A 29.31

R₃:- OSO₂ (3, 5-di-Br-4-OCH₃-phenyl) B 12.10

25 X:O= O- R₂:-CN

R₃:- OSO₂ p-NH₂-pheny1 A 1.25

26 X:O= , Y:- O-,

R₃:- OSO₂ p-NHEt-phenyl A 10.21

27 X:O= , Y:- O-, R₁:-CN, (3,4-dihydro derivative)

R₃ : - OSO₂ [3,4-(-O-CH₂-O-)phenyl] A 13.20

28 X:O=, Y:-O-, R₁:-CN, (3,4-dihydro derivative)

R₃:-OSO₂-p-(2-pyridyl)-phenyl A 11.45

29 X:O=, Y.-O-, R₁:-CN

R₃ : - OSO₂-[3-(o-CH₂-pyridyl)-4-benzamide] A 6.08

30 X:O=, Y L-O-, -CN, (3,4-dihydro derivative) A 19.24

R₃: -oso₂ -p-N(CH₂-CF₃)₂-phenyl B 9.61

31 X:O=- , Y: -O- -CN

R₃:-OSO₂-p-N(CH₂-NH₂)₂-phenyl A 0.31 32 X:O=, Y:-O-, R₁: -CN, (3,4-dihydro derivative)

R₃: -OSP₂ p-(HS-CH₂-N-CH₂-OH)-phenyl A 0.31

33 X:O=, Y :-O-, R_1:-CN, (3,4-dihydro derivative)

^R3: -OSO₂-p-(C₅H₁₂N₃)-Pheny; A 0.22

34 X:O=, Y :-O-, -CN, (3,4-dihydro derivative)

R₃: -O-CO-p-N(CH₂-OH)₂-pheny; A 1.66

35 X:O=, Y: -O-, -CN, (3,4-dihydro derivative)

R₃:-O-CS-p-N(CH₂-CH₃)₂-phenyl B 11.45

36 X:O=, Y -O-, -CN, (3,4-dihydro derivative)

R₃: S-CO-p-(C₅H₁₂N₃)-phenyl A 4.13 Table 1 (contd.)

Example Substituents HPLC retention time No. (other than H) System t(min)

37 X:O=, Y:-O-, R₁:-CN,

R₃:-NH-CO-p-(C₃H₆N₃) -pheny 1 A 0.37

38 X:O=, Y:-O-, R₁:-CN, (3,4-dihydro derivative) A 20.80

R₃:-HC=CH-3,4,5-tri-OH-phenyl B 10.35

39 X:O=, Y:-O-, R₁:-CN, (3,4-dihydro derivative) B 15.05

R₃:-OSO₂-3-(4-N(CH₂-S-C₂H₅)₂-phenyl]-4-F-phenyl

40 X:O=, Y:-O-, R₁:-CSNH₂, (3,4-dihydro derivative) A 18.71

R₃:-OSO₂-p-N(CH₂-CH₃)₂-phenyl B 9.43

41 X:O=, Y:-O-, R₁:-NH-CHO, (3,4-dihydro derivative) A 5.34

R₃:-OSO₂-p-N(CH₂-O-CH₃)₂-phenyl B 4.31

Claims

1. Compounds of the general formula (I)

wherein

R₁ Stands for -CN, -COOH, -CSOH, -COSH, -CONH₂,

-CSNH₂, -NHCHO, -COOCH₃, -CSOCH₃, -COSCH₃,

-CO-NH-CSNH₂, -CS-NH-CSNH₂, -C(NH₂)=C(CN)₂, -CH(CN)₂, -CH(CN)-COOH, -CH(CN)-CONH₂, -CH(CN)-CSNH₂, -CH(CN)CSNH-C_1-4 alkyl, -NO₂ or -NO group;

X means =O, =S or =NH group;

Y means -O-, -S- or -NH- group;

R₂ and R₄, independently from each other, represent hydrogen atom, -OH, -SH, -NH₂, -CF₃, C_1-4 alkoxy or C_1-4 alkylthio group;

R₃ Stands for -OH, -SH, -NH₂, -CF₃, -NO, -O-SO₂-V⁰,

-O-CO-V⁰, -O-CS-V⁰, -S-CO-V⁰, -NH-CO-V⁰ or

-CH=CH-V⁰ group, wherein

V⁰ stands for 3,4,5-tri-V¹-phenyl group; and the

V¹ substituents, independently from each other, mean hydrogen or halogen atom or -OH, -SH, -NH₂, -CF₃, C_1-4 alkoxy, C_1-4 alkylthio or C_1-4 alkylamino group; or

-N(CH₂-V²)-CH₂-V³ group, wherein

V² and V³, independently from each other,

stand for -OH, -SH, -NH₂ , -CF₃ , C_1-4 alkyl, C_1-4 alkoxy or C_1-4 alkylthio group; or V² and V³ together stand for -NH-CH₂-NH- or -N(CH₃)-CH₂-N(CH₃)- group;

R₅ represents hydrogen, C_{1- 4} alkyl, C_1-4 alkoxy

group or halogen; and

the double bond belonging to the skeletal atom

substituted by R₁ can also be saturated when R₃ is a substituent containing the -3,4,5-tri-V¹-phenyl moiety, as well as physiologically acceptable salts and complexes of these compounds.

2. A compound as claimed in claim 1, selected from the group consisting of 3-cyano-7-hydroxycoumarin, 7-hydroxycoumarin-3-thiocarboxylic acid amide, 3-acetylamino-7-hydroxycoumarin, 3-ethoxycarbonyl-6,7,8-trihydroxycoumarin, 3-carboxy-6,7,8-trihydroxycoumarin, 6,7,8-trihydroxycoumarin-3-carboxylic acid amide, 3-cyano-6,7,8-trihydroxycoumarin, 3-cyano-3,4-dihydro-7-hydroxy-coumarin-(3-hydroxy-4-carboxyphenyl-sulphonic acid) ester and 3-cyano-3,4-dihydroxy-7-hydroxy-coumarin-(3,4-methylendioxybenzosulphonic acid) ester and their salts and complexes.

3. A pharmaceutical composition, which c o m p r i s e s as active ingredient a novel compound of general formula (I), wherein R₁, R₂, R₃, R₄, R₅, X and Y are as defined in claim 1, or a physiologically acceptable salt and/or complex thereof in admixture with carriers and/or additives commonly used in the pharmaceutical industry.

4. A process for the preparation of novel compounds of the general formula (I)

wherein

R₁ Stands for -CN, -COOH, -CSOH, -COSH, -CONH₂,

-CSNH₂, -NHCHO, -COOCH₃, -CSOCH₃ , -COSCH₃,

-CO-NH-CSNH₂, -CS-NH-CSNH₂, -C(NH₂)=C(CN)₂, -CH(CN)₂, -CH(CN)-COOH, -CH(CN) -CONH₂, -CH(CN)-CSNH₂, -CH(CN)CSNH-C₁_₄alkyl, -NO₂ or -NO group;

X means =O, =S or =NH group;

Y means -O-, -S- or -NH- group;

R₂ and R₄, independently from each other, represent hydrogen atom, -OH, -SH, -NH₂, -CF₃, C₁_₄ alkoxy or C_1-4 alkylthio group;

R₃ stands for -OH, -SH, -NH₂, -CF₃, -NO, -O-SO₂-V⁰,

-O-CO-V⁰, -O-CS-V⁰, -S-CO-V⁰, -NH-CO-V⁰ or

-CH=CH-V⁰ group, wherein

V⁰ stands for 3,4,5-tri-V¹-phenyl group; and the

-N(CH₂-V²)-CH₂-V³ group, wherein V² and V³, independently from each other, stand for -OH, -SH, -NH₂, -CF₃, C_1-4 alkyl,

C_1-4 alkoxy or C_1-4 alkylthio group; or

V² and V³ together stand for -NH-CH₂-NH- or

-N(CH₃)-CH₂-N(CH₃)- group;

R₅ represents hydrogen, C_1-4 alkyl, C_1-4 alkoxy

group or halogen; and

the double bond belonging to the skeletal atom

substituted by R₁ can also be saturated when R₃ is a substituent containing the -3,4,5-tri-V¹-phenyl moiety, as well as physiologically acceptable salts and complexes of these compounds, which c o m p r i s e s a) condensing a benzaldehyde derivative of general formula (IV)

wherein Q means -OH, -SH or -NH₂ group, and R₂, R₃, R₄ as well as R₅ are as defined above, with a carboxylic acid derivative of general formula (V),

L1 — CH₂— R 1 (V) wherein L₁ stands for a -COOEt, -CSOEt or -CN group and R₁ is as defined above, in the presence of an acid catalyst; or

b) condensing a benzaldehyde derivative of general formula (IV), wherein Q, R₂, R₃ , R₄ and R₅ are as defined above, with a carboxylic acid derivative of the general formula (V), wherein L₁ and R₁ are as defined above, in two steps, first in the presence of an alkaline catalyst and then in the presence of an acidic catalyst; or

c) condensing a benzaldehyde derivative of general formula (IV), wherein Q, R₂, R₃, R₄ and R₅ are as defined above, with a carboxylic acid derivative of the general formula (V), wherein L₁ and R₁ are as defined above, in two steps, first in the presence of an alkaline or acid catalyst and then by a thermal reaction; or

d) condensing a compound of general formula (II),

to obtain compounds of the general formula (la),

5. A process as claimed in claim 4, which c o m p r i s e s carrying out the condensation reaction at a temperature between 20 °C and 200 °C under an inert gas atmosphere.

6. A process as claimed in claim 4 or 5, which c o mp r i s e s using phosphoric acid, hydrochloric acid, sulfuric acid and/or alkali metal hydroxides or alkali earth metal hydroxides as catalysts.

7. A process as claimed in claims 4 to 6, which c o mp r i s e s using water and/or an inert polar organic solvent.

8. A process as claimed in claim 7, which c o mp r i s e s using ethanol, dimethylsulfoxide or chloroform as polar organic solvent; or aliphatic or cyclic alkanes or aromatic hydrocarbons bearing saturated side chain as apolar solvents.

9. A process for the preparation of a pharmaceutical composition inhibiting tumour growth and cell proliferation, which c o m p r i s e s mixing as active ingredient a novel compound of formula (I), wherein R₁, R₂, R₃, R₄, R₅, X and Y are as defined in claim 1, or a physiologically acceptable salt or complex thereof, prepared by using any of the processes a) to f) claimed in claim 4, with carriers and/or additives commonly used in the pharmaceutical industry and transforming them to a pharmaceutical composition.

10. Method for treating mammals (including man) suffering from a tumorous disease, which c o m p r i s e s administering a therapeutically effective amount of a compound of general formula (I), wherein R₁, R₂, R₃, R₄, R₅, X and Y are as defined in claim 1, or a physiologically acceptable salt or complex thereof to a subject in need of such treatment.