Isoflavone derivatives, salts thereof, and oncostatic and immunosuppressive agents.

( Technical Field )

0001This invention relates to new isoflavone derivatives and salts thereof having cancerocidal and immunosuppressive actions.

( Background of the Invention )

0002We formerly found a cancerocidal substance in the product of Pseudomonas <img file="EP0245518A1_D0001.tif" />YO-0170 strain ( FERII-P8170 ) during a study in search of cancerocidal substances produced by microorganisms, identified it as genistein ( chemical name: 5,7,4'-trihydroxyisoflavone,) ― a compound reported in J.A.C.S ( on page 3447, 1951 ) and filed patent applications on the manufacturing process of this compound and anticancer agents containing the same ( Japanese Patent Publication No.88235 and No.89770, 1985 ).

0003The above paper reports that genistein has weak estro- genic action, but nothing is disclosed about cancerocidal or immunosuppressive action of this compound.

( Disclosure of the Invention)

0004A primary object of this invention is to provide useful compounds having cancerocidal and immunosuppressive actions. A further object of this invention is to provide useful compounds having inhibitory actions against tyrosine-specific protein kinase activity and against the growth of tumor cells, as well as imunosuppressive action. Another object of this invention is to provide drugs of low toxicity having cancerocidal and immunosuppressive actions.

0005In search for substances showing cancerocidal and immunosuppressive actions,' we synthesized various compounds having isoflavone skeletons and found, after screening tests, that the new isoflavone derivatives represented by the general formula (I) and salts thereof exhibit inhibitory action against tyrosine-specific protein kinase activity and immunosupressive action and hence are useful as cancerocidal and immunosuppressive agents. This invention was accomplished based on these findings.

0006Thus this invention relates to new isoflavone derivatives represented by the general formula (I) and salts thereof and to new cancerocidal and immunosuppressive agents • containing the same; <chemistry id="chem0001" num="0001"><img file="EP0245518A1_D0002.tif" /></chemistry> wherein R¹ is a radical <chemistry id="chem0002" num="0002"><img file="EP0245518A1_D0003.tif" /></chemistry>a radical -CONHR⁵ , a radical -A-S-R⁶ , or a radical -COOR⁷; A denotes a lower alkylene group; Rand R⁴ are independently a hydrogen atom, a lower alkyl , a cycloalkyl or a sulfur- and nitrogen-containing 5- or 6-membered heterocyclic group which groups may optionally be substituted by.one or two hydroxyl groups, or R³and R⁴ may be taken together to form, in conjunction with the adjacent nitrogen, a pyrrolidine, piperidine or morpholine ring; R⁵ stands for hydrogen atom or a lower alkyl which may be substituted by one or two hydroxyl groups; R⁶ represents a lower alkyl or a Gulfur-and nitrogen containing 5- or ,6-membered hetero- cyclic radical which may optionally be substituted by one or two hydroxyl, carboxyl, or lower alkoxycarbonyl groups; R⁷ is a lower alkyl which may be substitued by one or two hydroxyl or lower alkoxy groups ( a lower alkyl having a carbon number of 1 or 3 to 6, or a lower alkyl substituted by one or two hydroxyl or lower alkoxy groups, when R² is hydroxyl or a lower alkoxy group ); and R² is by- droryl, a lower alkoxy or an acyloxy group.

0007Unless otherwise specified, the term "lower" herein means a linear or branched chain of 1 to 6 carbon atoms, Hence, "lowor alkyl radicals" represent linear or branched alkyl groups of. 1 to 6 carbon atoms, illustrative examples including methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert- pentyl, 1-methylbutyl, 2-methylbutyl, 1,2-dimethylpropyl; 1-ethylpropyl, hexyl, isohexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 1-ethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl-2-methylpropyl and 1-ethyl-1-methylpropyl.

0008As examples of "lower alkoxy radicals" there may be mentioned linear and branched alkoxy groups of 1 to 6 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, isopentyloxy, hexyloxy and isohexyloxy groups.

0009Illustrative examples of "lower alkylene radicals" in- clude methylene, ethylene, methylethylene (^-Me-CH-), trimethylene, 1-propylene, 2-propylene, tetramethylene, 1-methyltrimethylene, 2-methyltrimethylene, 3-methyltrimethylene, 1-ethylethylene, 2-ethylethylepe, pentamethylene, 1-methyltetramethylene, 2-methyltetramethylene, 3-methyltetramethylene, 4-methyltetramethylene and hexamethylene .

0010"Lower alkyls substituted by one or two hydroxyl groups" means those lower alkyls as defined above in which any one or two of the hydrogen atoms are substituted by hydroxyl groups ( mono- or di-hydroxy lower alkyls ). Illustrative examples of "monohydroxy lower alkyls" include hydroxymethyl, 1-hydroryethyl, 2-hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl, 3-hydroxypropyl, 2-hydroxy-1-methylethyl, 1-hydroxybutyl, 2-hydroxybutyl, 3-hydroxybutyl, 4-hydroxybutyl, 3-hydroxy-2-methylpropyl, 3-hydroxy-1-methylpropyl, 2-hydroxy-1,1-dimethylethyl, 1-hydroxypentyl, 2-hydroxypentyl, 3-hydroxypentyl, 4-hydroxypentyl, 5-hydroxypentyl, 4-hydroxy-3-methylbutyl, 6-hydroxyhexyl and 5-hydroxy-4-methylpentyl. Preferred examples of "dihydroxy lower alkyls" include 1,2-dihydroxyethyl, 3,4-dihydroxybutyl, 1 -hydroxymethyl-3-hydroxypropyl, 2,2-bis(hydroxymethyl)ethyl, 4,5-dihydroxypentyl and 5,6-dihydroxyhexyl.

0011"Lower alkyls substituted by one or two lower alkoxy groups" means those lower alkyls as defined above in which any one or two hydrogen atoms are substituted by lower alkoxy groups ( mono- or di-lower-alkoxy lower alkyls ). Typical examples in which the lower alkoxy is methoxy and the lower alkyl is ethyl include 2-methoxyethyl and 1,2-dimethoxyethyl.

0012"Lower alkyls substituted by carboxyl or a lower alkoxycarbonyl group" means those lower alkyls in which any hydrogen atom is substituted by carboxyl or a lower alkoxycarbonyl group ( carboxy or lower-alkoxy-carbonyl lower alkyls ). Preferred examples of the lower alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl, pentyloxycarbonyl and hexyloxycarbonyl. Thus typical exampls of carboxy or lower-alkoxy-carbonyl lower alkyls in which the lower alkoxy is methoxy and the lower alkyl is ethyl include carboxyethyl and methoxycarbonylethyl.

0013Prefered examples of "cycloalkyls" include 5- and 6- membered cyclic alkyls, such as cyclohexyl and cyclopentyl.

0014"Sulfur- and nitrogen-containing, 5- or 6-membered heterocyclic radicals" means 5- or 6-membered heterocyclic radicals containing sulfur and nitrogen as hetero atoms, in which the hetero ring may be unsaturated, partially saturated or completely saturated. Particularly preferable examples include thiazolyl, isothiazolyl, thiazolinyl, isothiazol- inyl, thiazolidinyl, isothiazolidinyl, 2H-1,2-thiazinyl, 2H--1,3-thiazinyl, 4H-1,4-thiazinyl, dihydro-2H-1,2-thiazinyl, dihydro-2H-1,3-thiazinyl, dihydro-4H-1,4-thiazinyl, per- hydro-2H-1,2-thiazinyl, perhydro-2H-1,3-thiazinyl and per- hydro-1,4-thiazinyl radicals.,

0015As preferred "acyloxy radicals" there may be mentioned lower alkanoyloxy radicals, oxaloxy radical and lower alkoxyoxalyloxy radicals. Typical examples of lower alkanoyloxy radicals include acetyl, propionyl, butylyl, isobutylyl, valeryl, isovaleryl, pivaloyl and hexanoyl radicals. The lower alkoxyoxalyloxy radicals are oxalyloxy radical ( -O-COCO- ) to which a lower alkoxy group is attached. Illustrative examples include methoxalyloxy, ethoxalyloxy, propoxalyloxy, isopropoxalyloxy, butoxalyloxy, pentyloxalyloxy and hexyloxalyloxy radicals.

0016Some of the compounds represented by the general formula (I) exist in optical, tautomeric and other types of isomeric forms. This invention includes all of these isomers and combinations thereof.

0017Some of the compounds of this invention are capable of forming salts, and this invention also includes all of these salts. Typical examples include inorganic salts, such as sodium, potassium and ammonium salts; salts with organic bases, such as ethylamine, propylamine, diethylamine, triethylamine, morpholine, piperidine, N-ethylpiperidine, diethanolamine and cyclohexylamine; salts with basic amino acids, such as lysine and ornitine; salts with mineral acids, such as hydrochloric, sulfuric, phosphoric and hydrobromic acids; salts with organic acids, such as acetic, oxalic, citric, maleic, malic, fumaric, tartaric and methanesulfonic acids; and salts with acidic amino acids, such as glutamic and aspartic acids.

0018The compounds of this invention can be synthesized by various methods. Typical synthetic methods are shown below. <chemistry id="chem0003" num="0003"><img file="EP0245518A1_D0004.tif" /></chemistry><chemistry id="chem0004" num="0004"><img file="EP0245518A1_D0005.tif" /></chemistry><chemistry id="chem0005" num="0005"><img file="EP0245518A1_D0006.tif" /></chemistry><chemistry id="chem0006" num="0006"><img file="EP0245518A1_D0007.tif" /></chemistry><chemistry id="chem0007" num="0007"><img file="EP0245518A1_D0008.tif" /></chemistry>( wherein A, R² R³ R⁴ R⁵, R⁵, and R⁷ are as defined above; X¹ denotes a halogen atom or an organic sulfonic acid residue; M¹ stands for hydrogen atom or an alkali metal atom; X² represents a halogen atom; X³ expresses a halogen atom or a radical represented by formula R⁸-CO-O-; R⁸ is a lower-alkoxycarbonyl, a lower-alkylthio-lower-alkyl, .a cycloalkylthio-lower-alkyl or a sulfur- and nitrogen-containing 5- or 6-membered heterocyclic-thio-lower alkyl which may be substitued by one or two lower alkoxy groups; -and- _R²¹ is a lower alkoxy or an acyloxy group. )

0019Halogen atoms include iodine, bromine and chlorine; alkali metals include sodium and potassium; and organic sulfonic acid residues include alkanesulfonic acid residues ( e.g., methanesulfonic acid and ethanesulfonic acid residues), and aromatic sulfonic acid residues ( e.g., p-toluenesulfonic acid and benzenesulfonic acid residues ).

0020The synthetic methods 1 to 5 mentioned above are described below in more detail.

( Synthetic method 1 )

0021The substituted or unsubstituted amino-lower-alkyl isoflavone derivatives represented by the general formula (Ia) can be prepared by reaction of amine (III) with halogeno-lower-alkyl isoflavone derivative or sulfonyloxy-lower-alkyl isoflavone derivative (II).

0022The reaction is carried out preferably in an inert organic solvent, such as benzene, toluene, xylene, dimethylformamide, dichloromethane or chloroform, although it proceeds in the absence of solvent when a halogeno-lower-alkyl isoflavone derivative (II) is used. The amine (III) is preferably used in an amount not smaller than that of compound (II) on a molar basis. In some cases, addition of organic base ( e.g., pyridine, picoline, N,N-dimethylaniline, N-methylmorpholine or trimethylamine ) or inorganic base ( e.g., potassium carbonate, sodium carbonate or sodium bicarbonate ) is effective in ensuring smooth reaction.

0023The reaction is generally conducted at room temperature, but may also be carried out at elevated temperatures or under reflux.

0024The compcund (III) may be used for reaction with its amino group blocked with a protective group to avoid side reactions, followed by removal of the protective group after reaction. Examples of the protective group include N bromosuccinimide, N-bromophthalimide, toluenesulfonyl, acetyl, phenacylsulfonyl, trifluoromethanesulfonyl and bis- benzensulfonyl groups. These protective groups can be easily removed after reaction by conventional hydrolysis.

0025when a sulfonyloxy-lower-alkyl isoflavone derivative (II) is used as starting material, the reaction is preferably carried out at room temperature or under cooling in an inert solvent, such as diethyl ether, tetrahydrofuran, benzene, toluene, xylene, methanol or ethanol, using compound (III) in an amount not smaller than that of compound (II) on a molar basis.

( Synthetic method 2 )

0026The carboxamide compounds represented by the general formula (Ib) can be prepared by reaction of ammonia or amine (V) with carboxylic acid(IV), or salt or reactive derivative thereof.

0027Examples of reactive derivatives of compound (IV) include acid halides ( e.g., acid chloride and acid bromide ), acid azides, active esters with N-hydroxybenzotriazole and N-hydroxysuccinimide, symmetric. acid anhydrides, mixed anhydrides with alkyl carbonate, and mixed anhydrides with p-toluenesulfonic acid.

0028When compound (I_V) is used in the form of free carboxylic acid, the reaction is preferably carried out in the presence of a condensation reagent, such as dicyclohexyl carbodiimide or 1,1'-carbonyl-diimidazole.

0029When carboxylic acid compound (IV) is used in the form of a reactive derivative, the reaction is preferably carried out in an inert solvent, such as pyridine, tetrahydrofuran, dioxane, diethyl ether, benzene, toluene, xylene, dichloromethane, dichloroethane, chloroform, dimethylformamide, ethyl acetate or acetonitrile.

0030With some of the reactive derivatives, addition of a base, such as triethylamine, pyridine, picoline, lutidine, N,N-dimethylaniline, potassium carbonate or caustic soda, is effective in ensuring smooth reaction. Of these, pyridine serves also as reaction medium.

0031There is no specific limitation upon the reaction temperature, which should be properly selected depending on the type of reactive derivative used.,

0032When an ester ( methyl or ethyl ester) is used as starting material, intended amides ( primary or secondary ) can be obtained by ammonolysis, which is accelerated by the addition of water or glycol. Liquid ammonia may be employed for this purpose.

0033The reaction is carried out in the absence of solvent or in an organic solvent, such as methanol, ethanol, benzene, xylene, chloroform, dimethylsulfoxide or tetrahydrofuran.

0034Ammonium chloride; alkali metal alcoholate ( e.g., sodium methoxide ), sodium amide or butyl lithium may be added as catalyst. The reaction can thus be carried out at room temperature.

( Synthetic method 3 )

0035The substituted thio-lower-alkyl-isoflavone derivatives represented by the general formula (Ic) can be prepared by reaction of halogeno-lower-alkyl-isoflavone derivative (VI) with thiol or alkali metal salt thereof ( VII).

0036The reaction is carried out in water, organic solvent or a mixture thereof. Suitable organic solvents include dimethylformamide, dimethylsulfoxide, methanol, ethanol, propanol, acetone, methyl ethyl ketone, tetrahydro- furan, chloroform and dioxane.

0037When a free thiol is used as compound (VII), the reaction is conducted in the presence of a base, such as potassium carbonate, Triton B, caustic potash, caustic soda or sodium hydride.

0038There is no specific limitation upon the reaction temperature, but the reaction is generally carried out at room temperature or at elevated temperatures.

( Synthetic method 4 )

0039The compounds represented by the general formula (Id) can be prepared by esterification or ester exchange reaction between carboxylic acid of formula (IV),or salt, ester or reactive derivative thereof with. substituted or unsubstituted alkanol of formula (VIII) or reactive derivative thereof.

0040Suitable reactive derivatives of carboxylic acid (IV) include acid halides and acid anhydrides; and halides and sulfonates ( e.g., toluenesulfonates and methanesulfonctes) are commonly used as reactive derivatives of alkanol ( VIII).

0041When a substituted or unsubstituted alkanol (VIII) is used as alcohol component, it is allowed to react with free carboxylic acid (IV) in the presence of acid catalyst e.g., hydrochloric, sulfuric, trifluoroacetic, p-toluenesulfonic or benzenesulfonic acid in an organic solvent ( e.g., chloroform, carbon tetrachloride, dichloromethane, dichloroethane, benzene, toluene or xylene ), or is allowed to react with reactive derivative of carboxylic acid (IV) ( e.g., . acid halide or acid anhydride ) in the presence of base ( e.g., trimethylamine, triethylamine, dimethylaniline, pyridine, sodium carbonate or caustic potash ) in an organic solvent ( e.g., diethyl ether, tetrahydrofuran, ethyl acetate or acetonitrile ).

0042When a substituted or unsubstituted lower alkyl halide or sulfonate of alkanol (VIII) is used as alcohol component, it is allowed to react with free carboxylic acid (IV) at room temperature or at elevated temperatures in the presence of base ( e.g., caustic soda, caustic potash, potassium carbonate, sodium hydride or- triethylamine ) in an organic solvent ( e.g., methanol, ethanol, acetone, dimethylformamide, ethyl acetate, benzene, toluene or xylene ), or is allowed to react with a salt of carboxylic acid (IV) ( e.g., sodium or potassium salt) in an organic solvent mentioned above under the same temperature conditions as above.

0043Ester exchange reaction, on the other hand, may be effected by heating ester ( methyl or ethyl ester ) of carboxylic acid (IV) and unsaturated alkanol (VIII) in the presence of acid or base as catalyst. Suitable catalysts include mineral acids (hydrochloric and sulfurie acids ), p-toluenesulfonic acid and alkali metal alcoholates. ( Synthetic method 5 )

0044Isoflavone derivatives of this invention may be prepared by cyclization reactions. Of various types of cyclization, reaction of 2,4,6-trihydroxy-α-phenylacetophenone derivative (IX) with compound of formula (X) ( acid halide or acid anhydride ) to form isoflavone derivative of formula (Ie) is the most preferred because the substituent at position 2 can be simultaneously introduced.

0045The reaction is carried out in pyridine at room temperature or under cooling using an acyl halide (X), or using an acid anhydride (X). When R² is hydroxyl, R^2' in the reaction product will be an acyloxy radical represented by R⁸COO-.

0046The compounds of this invention thus prepared can be converted to corresponding salts by usual methods.

0047The isoflavorie derivatives and salts thereof thus formed can be isolated and purified by commonly used techniques, such as concentration, distillation, crystallization, various types of chromatography and recrystallization.

0048Compounds of this invention and salts thereof show inhibitory action against tyrosine-specific protein kinase activity and against the growth of tumor cells, and are hence useful as cancerocidal agents. What is important is that these compounds are of low toxicity, with no serious side effects as observed in conventional cancerocidal agents. Compounds of this invention also have immunosuppressive activity and are useful as immunosuppresive agents.

0049Described below are the cancerocidal and immunosuppressive actions of compounds represented by formula (I).

(1) Cancerocidal action

(a) Inhibitory action against tyrosine-specific protein kinase activity

0050The inhibitory action against tyrosine-specific protein kinase activity of compounds of this invention was measured according to the method described below using tyrosine-specific protein kinase derived from Rous sarcoma virus ( src gene pp60^src).

Method of measurement

0051Measurement of tyrosine-specific protein kinase activity ( src gene pp60^src derived from Rous sarcoma virus ) (refer to M.S. Colet, R.L. Ericson: Proceeding of the National Academy of Sciences of USA, 75, 2021-2024, 1978 )

00523Y1 cells ( fibroblasts derived from fetal rat kidney) transformed by Rous sarcoma virus ( RSV ) were cultured, the cultured cells were collected and washed, RIPA buffer solution [ containing 0.5% NP40, 0.1% sodium deoxycholate, 50mM Tris-HCl ( pII 7.2 ), 1 mM phenylmethylsulfonyl fluoride (PMSF) and 0.15H NaCl ]was added to the collected cells, the mixture was allowed to stand at 0°C for 30 minutes to effect solubilization, and the resulting mixture was centrifuged ( 100,000 x g, 20 minutes ). To the supernatant thus collected, was added antiserum obtained from a rabbit cancered by inoculation of RSV, the mixture was incubated at 0°C for 30 to 60 minutes to cause pp60^src to react with antibody, and the immune complex was isolated by mixing with protein A/Sepharose-4B ( Pharmacia ) and washed with RIPA buffer. The total complex thus obtained ( PP60^src/anti- body/protein A/Sepharose-4B) was treated in a solution containing 20mM Pipes/NaOH ( pH 7.2 ), 5mM MgCl₂, 1 mM DTT and 10,µM [γ=³²P]ATP ( 2 mCi/mMol ) at 30°C for five minutes to effect protein kinase reaction, which was terminated by adding terminating solution containing SDS followed by boiling for three minutes. The reaction mixture was subjected to electrophoresis on 8% SDS/polyacrylamide gel and then to autoradiography, and the radioactivity of cut-out pp60^src band was measured on a liquid scintillation counter, thus quantifying the protein kinase reaction and'calculating IC₅₀.

(b) Inhibitory action against the growth of tumor cells

0053The inhibitory action of compounds of this inven- tio_n against the growth of tumor cells was examined using cells of rats transformed by Rous sarcoma virus ( RSV-3Y1 cells).

Test method

0054RSV-3Y1 cells were cultured in Dulbecco's MEM ( Nippon Suisan) containing 10% fetal bovine serum ( Gibco ) and a compound of this invention at different concentrations ( ₁ µg/ml, 3 µg/ml, 10µg/ml, 30 µq/ml. and 100 µp/ml ), and the number of live cells in each dish after 1, 2, 3 and 4 days was measured using trypan blue to determine IC_50'

(2) Immunosuppressive action

Method of measurement

0055A 5-week-old, male ddy mouse was immunized by abdominal injection with 4 :: 10⁸ ovine erythrocytes ( Mihon Dio- material Center ), and a suspension of a compound of this invention (I) in 0.5% methylcellulose ( Shin-Etsu Chemical ) solution once a day over a period of four days ( two cays before immunization and two days after immunization). The spleen was taken out five days after immunization, and the number of antibody-forming cells contained in it was measured as follows according to the method of Cunningham [Cunningham A.J.: Nature, 207 , 1106 ( 1965 )]. The splenetic cells were suspended in Hagle's MEH ( Nissui Pharmaceutical ), ovine erythrocytes and guinea-pig complement were added to this suspension, the resulting mixture was put in a Cunningham chamber (with both ends sealed with paraffin ) and incubated at 37°C for 90 minutes, and the number of hemolytic plaques ( a ) ( the number of antibody-forming cells ) was counted.

0056Similarly, the number of hemolytic plaques ( b ) was measured with a mouse not injected with the compound of this invention ( control ). The antibody-formation inhibition rate was calculated according to the following equation:<maths id="math0001" num=""><img file="EP0245518A1_D0009.tif" /></maths>

0057Compounds of this invention exhibit inhibitory actions against the activity of tyrosine-specific protein kinase derived from oncogene and against the growth of tumor cells, and also show favorable immunosuppresive action.

0058Since the tyrosine-specific protein kinase is considered to participate in the growth of cancerous cells, the inhibitory action against the activity of this protein kinase exhibited by compounds of this invention supports their cancerocidal action.

0059Hence, compounds of this invention are useful as drugs for the treatment of human and animal cancer, . for the cure of diseases caused by cancer metastasis and for the prevention of their recurrence. These compounds are also useful as drugs for the treatment of autoimmune diseases; such as chronic articular rheumatism, systemic lupus erythematosus, chronic hepatitis and osteoporosis, and for the prevention of their recurrence.

0060The daily dosage of a compound of this invention (I) or a salt thereof is generally 200 to 1000 mg ( as active ingredient ) for adults, which should be subdivided into 1 to 4 doses. The optimum dose should be determined within the range mentioned above depending on the condition, age and other factors of the patient.

0061The compounds of this invention may be applied either alone or in combination with other types of chemotherapeutics or with immunotherapeutic agents. As examples of the other types of chemotherapeutics, there may be mentioned cyclophosphamide, vinblastine, vincristine, adriamycin, 6-mercaptopurine, 5-fluorburacil, mitomycin C, bleomycin, aclacinomycin, neocarzinostatin, cytosine arabinoside, actinomycin, and nitrosourea-related drugs. Examples of the immunotherapeutic agents to be used in combination include, among others, clistin, BCG, picibani1, lentinan, interferon and interleukin. A suitable amount of these agents to be used in combination is about 0.1 to 1000% based on the weight of the compound of this invention.

0062Drugs containing a compound of this invention or a salt thereof as an active ingredient may be administered orally ( in the form of tablets, capsules or solutions ) or parenterally ( in the form of rectal suppositories, injections or pellets ). These preparations can be made by usual methods using common carriers and excipents. For tablets, for example, water, glucose, maltose, gum arabic, gelatin, mannitol, starch paste, magnesium trisilicate, talc, cone starch,colloidal silica, potato starch and urea are used as carriers and excipients. Solutions include aqueous or oily suspensions, syrup and elixirs, which can be prepared by commonly used techniques. Suppositories are for rectal administration, which are prepared by using, as base, polyethylene glycol, lanolin, cacao butter, witeb Sol® (Dinamit Nobel) and others.

0063Shown below is an example of capsule formulation. <tables id="tabl0001" num="0001"><img file="EP0245518A1_D0010.tif" /></tables>

0064To a mixture of the active ingredient, maltose and crystalline cellulose, was added an aqueous solution of hydroxypropylcellulose, the mixture was kneaded and sujected to granulation, the granules thus obtained were dried and mixed with starch and magnesium stearate, and the resulting mixture was filled into No.1 gelatin capsules. ( Preferred Embodiments of the Invention)

0065The following Examples will further illustrate the invention.

0066Of the starting materials used for the synthesis of compounds of this invention, those represented by the general formula (II) are novel compounds, and their manufacturing methods are described in Reference Examples.

0067Ac, NBS, ag, Et and DMF used in these Examples and Reference Examples mean acetyl group, M-bromosuccinimide, aqueous, ethyl group and dimethylformamide, respectively.

Reference Example 1

0068<chemistry id="chem0008" num="0008"><img file="EP0245518A1_D0011.tif" /></chemistry>

0069A solution of 5,7-diacetoxy-4'-methoxy-2-methyliso- flavone ( 2 g ), benzoyl peroxide ( 0.1 g ) and N-bromosuccinimide ( 1.4 g ) in 120 ml carbon tetrachloride was heated under reflux for 20 hours, the solvent was distilled off under reduced pressure, and the solid residue was treated with water. The crude product collected by filtration was recrystallizcd from methanol, affording 2.7 g of 5,7-diacetoxy-4'-methoxy-2-bromomethylisolfavone. <ul id="ul0001" list-style="none"><li>Melting point: 181-183°C</li><li>Elemental analysis ( C₂₁H₁₇O₇Br) :<img file="EP0245518A1_D0012.tif" /></li><li>NHR spectrum. ( CDCl₃ ) : <ul id="ul0002" list-style="none"><li>δ 2.32 ( 6H, s )</li><li>3.80 ( 3H, s )</li><li>4.16 ( 2H, s )</li></ul></li></ul>

Reference example 2

0070<chemistry id="chem0009" num="0009"><img file="EP0245518A1_D0013.tif" /></chemistry>A solution of 4',5,7-triacetoxy-2-methylisoflavone ( 5 g) benzoyl peroxide ( 0.3 g ) and N-bromosuccinimide ( 3.9 g) in 300 ml carbon tetrachloride was heated under reflux for 12 hours, the solvent was distilled off under reduced pressure, water was added to the solid residue, the mixture was stirred well, and the crude product collected by fii- tration was recrystallized from a methanol/chloroform mixed solvent, affording 4.3 g of 4',5,7-triacetoxy-2-bromometh- ylisoflavone. <ul id="ul0003" list-style="none"><li>Melting point: 214-216°C</li><li>Elemental analysis ( C₂₂H₁₇O₈Br ):<img file="EP0245518A1_D0014.tif" /></li><li>NHR spectrum ( CDCl₃): <ul id="ul0004" list-style="none"><li>δ 2.34 ( 3H, s ), 4.18 ( 2H, s )</li><li>2.36 ( 3H, s ), 6.85 ( 1H, d )</li><li>2.38 ( 3H, s ), 7.30 ( 1H, d )</li></ul></li></ul>

Reference Example 3

0071<chemistry id="chem0010" num="0010"><img file="EP0245518A1_D0015.tif" /></chemistry>A mixture of 4',5,7-triacetoxy-2-bronomethylisoflavone ( 1 g ) and 48% hydrobromic acid ( 7 ml ) was heated at 100°C for one hour, the reaction mixture was cooled with stirring, and the solid which separated out was collected by filtration and washed with water, giving 0.7 g of 4',5,7-trihydroxy-2-bromomethylisoflavone. <ul id="ul0005" list-style="none"><li>Melting point: 213-21 5°C Elemental analysis ( C₁₆H₁₁O₅Dr):<img file="EP0245518A1_D0016.tif" /></li><li>NMR spectrum ( DMSO-d₆ ): <ul id="ul0006" list-style="none"><li>δ 4.34 ( 2H, s ), 6.33 ( 1H, d), 7.13 ( 2H, d )</li><li>6.20 ( 1H, d ), 6.83 ( 2H, d)</li></ul></li></ul>

Example 1

0072<chemistry id="chem0011" num="0011"><img file="EP0245518A1_D0017.tif" /></chemistry>

0073To a solution of 2, 4, 6-trihydroxy-α-p-hydroxyphenyl- acetophenone ( 4.7 g ) in 47 ml dry pyridine, was added dropwise 10 ml of ethyloxalyl chloride with stirring under ice cooling so that the temperature will not exceed 5°C, and stirring was further continued at 4°C for 12 hours. The reaction mixture was poured into ice water, the resulting mixture was extracted with chloroform, and the extract was washed with dilute hydrochloric acid and then water, dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography ( eluent: chloroform ), and the solid crude product thus obtained was recrystallized from ethanol, affording 3.0 g of ethyl 4'-ethoxalyloxy-5,7-di- hydroxy-2-isoflavonecarboxylate.

0074Melting point: 193-194°C

0075Elemental analysis ( C₂₂H₁₈O₁₀ ): <tables id="tabl0002" num="0002"><img file="EP0245518A1_D0018.tif" /></tables>

0076NMR spectrum ( DMSO-d₆ ): <ul id="ul0007" list-style="none"><li>δ 0.92 (3H, t ), 6.28 (1H, d )</li><li>1.14 ( 3H, t ), 6.45 ( 1H, d )</li><li>4.10 ( 2H, q ), . 7.32 ( 4H, s )</li><li>4.36 ( 2H, q )</li></ul>

Example 2 ,

0077<chemistry id="chem0012" num="0012"><img file="EP0245518A1_D0019.tif" /></chemistry>To a solution of 5,7-diacetoxy-4'-methoxy-2-bromometh- ylisoflavone ( 500 mg ) in 5 ml chloroform, was added 240 mg of 50% aqueous solution of dimethylamine at room temperature with stirring. After one hour, the solid which separated out was filtered off, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography ( eluent: chloroform ). To the liquid thus obtained was added ehanolic solution of hydrogen chloride, the solvent was ditilled off, and the solid which separated out was recrystallized from ethanol, giving 150 mg of 2-(dimethylaminomethyl)-5,7-dihydroxy-4'-methoxyiso- flavone hydrochloride.

0078Melting point: 262-266°C

0079Elemental analysis (C₁₉H₂₀NO₅Cl·0.4H₂O ): <tables id="tabl0003" num="0003"><img file="EP0245518A1_D0020.tif" /></tables>

0080NMR spectrum ( DHSO-d₆ ): <ul id="ul0008" list-style="none"><li>δ 2.72 ( 6H, s ) , 6.26 ( 1H, d ), 11.2 ( 1H, s )</li><li>3.80 ( 3H, s ), 6.61 ( 1H, d.), 12.6 ( 1H, s )</li><li>4.26 ( 2H, s ), 7.01 ( 2H, d ),</li><li>7.25 ( 2H, d ),</li></ul>

Examples 3 and 4

0081The compounds enumerated below were prepared in the same way as in Example 2. <tables id="tabl0004" num="0004"><img file="EP0245518A1_D0021.tif" /></tables>

Example 5

0082<chemistry id="chem0013" num="0013"><img file="EP0245518A1_D0022.tif" /></chemistry>4',5,7-Trihydroxy-2-bromomethylisoflavone ( 160 mg ) was dissolved in 2 ml N,N-dimethylformamide, 100 mg piperidine was added to the solution, and the mixture was stirred at room temperature for three hours. The solvent was distilled off under reduced pressure, the residue was purified by silca gel column chromatography ( chloroform: methanol = 96:4 ), and the solid thus obtained was recrystallized from ethanol, giving 150 mg of 4',5,7-trihydroxy-2-piperidinomethylisoflavone.

0083Melting point: 265-267°C

0084Elemental analysis ( C₂₁H₂₁NO₅ ): <tables id="tabl0005" num="0005"><img file="EP0245518A1_D0023.tif" /></tables>

0085NMR spectrum ( DMSO-d₆ ) <ul id="ul0009" list-style="none"><li>δ 1.36 ( 6H, m ), 6.36 ( 1H, d )</li><li>2.28 ( 4H, m ) , 6.77 ( 2H, d )</li><li>3.32 ( 2H, s ), 7.05 ( 2H, d )</li><li>6.18 ( 1H, d ), 12.88 ( 1H, s )</li></ul>

Example 6

0086The following compound was prepared in much the name manner as in Example 5, except that 2-aminothiazoline was used in place of piperidine. <tables id="tabl0006" num="0006"><img file="EP0245518A1_D0024.tif" /></tables>

Example 7

0087<chemistry id="chem0014" num="0014"><img file="EP0245518A1_D0025.tif" /></chemistry>

0088A mixture of ethyl 4',5,7-trihydroxy-2-isoflavonecar- boxylate ( 120 mg ) and 28% ammonia ( 2 ml ) was stirred at room temperature for three hours, water was distilled off under reduced pressure, and the solid residue was recrystallized from methanol, affording 65 mg of 4',5,7-trihy- droxy-2-isoflavonecarboxamide.

0089Melting point: >300°C

0090Elemental analysis ( C₁₆H₁₁NO_6.) : <tables id="tabl0007" num="0007"><img file="EP0245518A1_D0026.tif" /></tables>

0091NMR spectrum ( DMSO-d₆ ) : <ul id="ul0010" list-style="none"><li>δ 6.25 ( 1H, d ), 6.45 ( 1H, d ), 6.77 ( 2H, d ),</li><li>7.13 ( 2H, d ), 9.52 ( 1H, s ), 11.0 ( 1H, s ),</li><li>12.7 ( 1H, s )</li></ul>

Example 3

0092<chemistry id="chem0015" num="0015"><img file="EP0245518A1_D0027.tif" /></chemistry>A mixture of ethyl 4',5,7-trihydroxy-2-isoflavonecar- boxylate ( 100 mg ) and 30% methanolic solution of methylamine ( 5 ml ) was stirred at room temperature for 30 minutes, the solvent was distilled off under reduced pressure, and the solid residue was recrystallized from methanol, affording 50 mg of 4',5,7-trihydroxy-N-methyl-2-isoflavocecarboxamide.

0093Melting point: 193-195°C Elemental analysis ( C₁₇H₃NO₆ ) : <tables id="tabl0008" num="0008"><img file="EP0245518A1_D0028.tif" /></tables>

0094NMR spectrum ( DMSO-d₆ ): <ul id="ul0011" list-style="none"><li>δ 2. 32 ( 3H, s ) , 5.90 ( 1H, d ), 6.04 ( 1H, d ),</li><li>6.73 ( 2H, d ), 7.05 ( 2H, d )</li></ul>

Example 9

0095<chemistry id="chem0016" num="0016"><img file="EP0245518A1_D0029.tif" /></chemistry>

0096Ethanolamine ( 100 mg ) was added to a solution of 4',5,7-trihydroxyisoflavonecarboxylate ( 100 mg ) in 2 ml methanol, and the mixture was stirred at room temperature for 12 hours. The solvent was distilled off under reduced presure, the residue was purified by silica gel column chromatography ( chloroform : methanol = 95:5 ), and the solid thus obtained was recrystallized from a methanol/diethyl ether mixed solvent, giving 40 mg of 4',5,7-trihydroxy-N-(2-hydroxyethyl)-2-isoflavonecarboxamide.

0097Melting point: 259-262°C Elemental analysis ( C₁₈H₁₅NO₇ ) : <tables id="tabl0009" num="0009"><img file="EP0245518A1_D0030.tif" /></tables>

0098NMR spectrum ( DMSO-d₆ ): <ul id="ul0012" list-style="none"><li>δ 2.3-3.8 ( 4H, m ), 6.24 ( 1H, d ), 6.75 ( 2H, d ),</li><li>7.72 ( 1H, m ), 4.68 ( 1H, m ) , 6.44 ( 1H, d ),</li><li>7.13 ( 2H, d ), 12.7 ( 1H, s )</li></ul>

Example 10

0099<chemistry id="chem0017" num="0017"><img file="EP0245518A1_D0031.tif" /></chemistry>4',5,7-Trihydroxy-2-bromomethylisoflavone ( 40 mg ) was dissolved in 1 ml N,N-dimethylformamide, 15% aqueous solution of sodium methylmercaptide ( 90 mg ) was added to the solution, and the mixture was stirred at room temperature for 10 hours. The solvents were distilled off under reduced pressure, the residue was purified by silica gel column chromatography ( eluent: chloroform ), and the solid thus obtained was recrystallized from ethanol, affording 34 mg of 4',5,7-trihydroxy-2-(methylthiomethyl)isoflavone.

0100Melting point: 705-207°C

0101Elemental analysis ( C₁₇H₁₄O₅S): <tables id="tabl0010" num="0010"><img file="EP0245518A1_D0032.tif" /></tables>

0102NMR spectrum ( DMSO-d₆ ): <ul id="ul0013" list-style="none"><li>δ 2.04 ( 3H, s ), 6.20 ( 1H, d ), 5.81 ( 2H, d ),</li><li>12.83 ( 1H, s ), 3.52 ( 2H, s ), 6.34 ( 1H, d ),</li><li>7.05 ( 2H, d )</li></ul>

Example 11

0103<chemistry id="chem0018" num="0018"><img file="EP0245518A1_D0033.tif" /></chemistry>2-Mercaptothiazoline ( 35 mg ) was added to a mixture of 4',5,7-trihydroxy-2-bromomethylisoflavone ( 50 mg ), anhydrous potassium carbonate ( 21 mg ) and N,N-dimethylformamide ( 1 ml ), and the resulting mixture was stirred at room temperature for 10 hours. The solvent was distilled off under reduced presure, and the residue was purified by silica gel column chromatography ( eluent: chloroform ), giving 32 mg of 4',5,7-trihydroxy-2-[2-thiasolin-2-ylthio)-methyl]isoflavone as amorphous powder.

0104Elemental analysis ( C₁₉H₁₅NO₅S₂ ) : <tables id="tabl0011" num="0011"><img file="EP0245518A1_D0034.tif" /></tables>

0105NMR spectrum ( DMSO-d₆ ) : <ul id="ul0014" list-style="none"><li>δ 3.44 ( 2H, t ), 4.06 ( 2H, t ), 4.26 ( 2H, s ) ,</li><li>6.20 ( 1H, d ), 6.32 ( 1H, d ), 6.79 ( 2H, d ),</li><li>7.07 ( 2H, d ), 10.9 ( 1H, s ), 12.7 ( 1H, s )</li></ul>

Examples 12 and 13

0106The following compounds were prepared in much the same manner as in Example 8. <tables id="tabl0012" num="0012"><img file="EP0245518A1_D0035.tif" /></tables><tables id="tabl0013" num="0013"><img file="EP0245518A1_D0036.tif" /></tables>

Example 14

0107<chemistry id="chem0019" num="0019"><img file="EP0245518A1_D0037.tif" /></chemistry>β-Hercaptopropionic acid ( 60 mg ) was added to a mixture of 4',5,7-trihydroxy-2-bromomethylisoflavone ( 100 mg), anhydrous potassium carbonate ( 80 mg) and M,N-dimethylformamide ( 2 ml ), and the resulting mixture was stirred at room temperature for 12 hours. The solvent was distilled off under reduced pressure, the residue was dissolved in water, and the solution was acidified by adding 1N-HCl with stirring. The solid which separated out was collected by filtration, washed with water and recrystallized from ethanol, giving 90 mg of 3-[(4',5,7-trihy- droxy-2-isoflavonyl)methyl]thio]propionic acid.

0108Melting point: 206-208°C

0109Elemental analysis ( C₁₉H₁₆O₇S ): <tables id="tabl0014" num="0014"><img file="EP0245518A1_D0038.tif" /></tables>

0110NMR spectrum ( DMSO-d₆): <ul id="ul0015" list-style="none"><li>δ 2.36 ( 2H, t), 6.20 ( 1H, d), 7.10 ( 2H, d)</li><li>2.72 ( 2H, t). 6.36 ( 1H, d), .</li><li>3.60 ( 2H, s ), 6.81 ( 2H, d),</li></ul>

Example 15

0111<chemistry id="chem0020" num="0020"><img file="EP0245518A1_D0039.tif" /></chemistry>Ethyl 4',5,7-trihydroxy-2-isoflavonecarboxylate ( 100 mg ) was added to 10 ml of methanol saturated with HCl gas, and the mixture was heated at 70°C with stirring for five hours. The solvent was distilled off under reduced pressure, and the solid residue was recrystallized from methanol, giving 55 mg of 4',5,7-trihydroxy-2-isoflavone- carboxylate.

0112Melting point: 258-260°C

0113Elemental analysis ( C₁₇H₁₂O₇): <tables id="tabl0015" num="0015"><img file="EP0245518A1_D0040.tif" /></tables>

0114NMR spectrum₃( CDCl₃ + DMSO-d₆ ): <ul id="ul0016" list-style="none"><li>δ 3.70 ( 311, s), 6.37 ( 2H, d ), 10.33 ( 1H, s ),</li><li>6.32 ( 1H, d ), 7.09 ( 2H, d ), 12.48 ( 1H, s ),</li><li>6.42 ( 1H, d ), 9.04 ( 1H, s )</li></ul>

Example 16

0115<chemistry id="chem0021" num="0021"><img file="EP0245518A1_D0041.tif" /></chemistry>Ethyl 4',5,7-trihydroxy-2-isoflavonecarboxylate ( 100 mg ) was added to 10 ml of isopropanol saturated with HCl gas, and the mixture was heated at 70°C with stirring for five hours.. The solvent was distilled off under reduced pressure, the residue was purified by silica gel column chromatography ( eluent: chloroform ), and the solid thus obtained was recrystallized from a chlorofor/methanol mixed solvent, giving 60 mg of isopropyl 4',5,7-trihydroxy-2-isoflavonecarboxylate.

0116Melting point: 226-28°C

0117Elemental analysis ( C₁₉H₁₆O₇): <tables id="tabl0016" num="0016"><img file="EP0245518A1_D0042.tif" /></tables>

0118NMR spectrum ( DMSO-d₆): <ul id="ul0017" list-style="none"><li>δ 1.00 ( 6H, d ), 6.26 ( 1H, d), 6.70 ( 2H, d ),</li><li>4.90 ( 1H, m), 6.42 ( 1H, d ), 7.07 ( 2H, d)</li></ul>

Example 17

0119The following compound was prepared in much the same manner as in Example 16, except that HCl-saturated methoxyethanol was used in place of HCl-saturated isopropanol. <tables id="tabl0017" num="0017"><img file="EP0245518A1_D0043.tif" /></tables>

Example 18

0120The inhibitory actions of the compounds of Examples 1 to 17 against tyrosine-specific protein kinase activity and against the growth of RSV-3Y1 cells were measured according to the methods described earlier. The result is summarized in Table 1. <tables id="tabl0018" num="0018"><img file="EP0245518A1_D0044.tif" /></tables>

Example 19

0121The immunosuppressive action of the compounds of Examples 1, 12 and 13 was measured according to the method described earlier. The result is summarized in Table 2. <tables id="tabl0019" num="0019"><img file="EP0245518A1_D0045.tif" /></tables>

( Possible Industrial Use )

0122As may be apparent from the foregoing, the isoflavone derivatives and their salts of this invention are useful as drugs for the treatment of human and animal cancer, for the cure of diseases caused by cancer metastasis, and for the prevention their recurrence, and are also useful as drugs for the treatment of human immune diseases, such as chronic articular rheumatism, systemic lupus erythematosus, chronic hepatitis and osteoporosis, and for the prevention of their recurrence.