CH683593A5 - Pharmaceutical preparation for inhibiting undesirable cell growth - contains new and known 2,4-di:hydroxy-2H-1,4-benzoxazin-3(4H)-ones derivs., esp. useful for treating cancer - Google Patents

Abstract

Pharmaceutical preparation for inhibiting undesirable cell growth in vertebrates comprises a 2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one deriv. of formula (I), or a salt of (I), as active agent. R1 = H, 1-6C alkyl (opt. substd. by 1-3 halo, OH, 1-6C alkoxy, phenoxy, opt. esterified carboxy, or phenyl), or phenyl (opt. substd. by 1-3 1-6C alkyl, OH, alkoxy, acyloxy, halo or opt. esterified carboxy); R2 = H, COR1, a glycosidically linked sugar, 1-6C alkyl (opt. substd. by 1-3 halo, OH, 1-6C alkoxy, phenoxy, phenyl or opt. esterified carboxy), or phenyl (opt. substd. by 1-3, 1-6C alkyl, OH, alkoxy, acyloxy, halo or opt. esterified carboxy); R3 = H or COR1; n = 0-4; each R4 = 1-6C alkyl (opt. substd. by OH, alkoxy, phenyl or phenoxy), OR11, OCOR11; and R11 = R1, opt. esterified carboxy, or a glycosidically linked sugar; or two neighbouring R4 gps. form a gp. OCH2O or OCH2CH2O. Also new are cpds. (I), provided that when n is 0 or 1, R4 is 1-6C alkyl, 1-6C alkoxy, halo or opt. substd. phenyl, R3 is H and R2 is H, 1-6C alkyl or a glycosidically linked sugar, then R1 is not H. The most preferred cpds. of formula (I) are of formula (IA) where Q = H, or a sugar gp. (esp. a glucose or mannose residue). USE/ADVANTAGE - The preparations are esp. useful in treatment of cancers. Admin. is esp. oral. Dosage is, e.g. 0.2-20 (esp. 0.4-0.6) mg/kg/day. (I) are of low toxicity to healthy cells.

Description

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description

Undesirable cell proliferation is common in vertebrates, both cold-blooded vertebrates such as fish, amphibians and reptiles, and especially warm-blooded vertebrates such as birds and mammals. Unwanted high cell proliferation can be found especially in different types of cancer, such as the formation of cancer tumors. However, the undesirably high cell proliferation can also be benignly increased cell proliferation, such as is found, for example, in the case of increased growth of different tissues, for example an undesired swelling of the glands due to increased cell proliferation. A typical example of a benign, excessive cell proliferation is benign prostatic hyperplasia.

Although a large number of active substances for the treatment of malignant and benign, undesirably high cell proliferation are known, there is still a need for new pharmaceutical preparations which bring about a selective inhibition of the undesirably high cell proliferation, in particular the proliferation of cancer cells, and on the other hand one have low toxicity, or do not impair the growth of healthy, non-degenerate cells. Furthermore, there is a great need for corresponding pharmaceutical preparations which are particularly well suited for inhibiting special, undesirably high cell proliferation, for example for the treatment of specific tumors.

DESCRIPTION OF THE PRIOR ART

Various active substances with antitumor activity are known which are derived from derivatives of hydroxyurea or hydroxamic acid. In these known compounds the hydroxamic acid grouping is of the formula

O H II I -C-N-OH

bound to an organic radical, and accordingly they have the following basic structure A

OH -JL

Il I A

R-C-N-OH

on in which

R is an NH2 group (in this case the compound A is hydroxyurea) or the radical R denotes an aliphatic, an araliphatic, an aromatic or a heterocyclic radical. In this connection, for example, refer to the publication by Howard L. Elford and Bart Van't Riet in Pharmac. Ther., Volume 29, pages 239 to 254, 1985, in which a summary of various substances with antitumor activity is given, which correspond to the formula A given above.

Of the compounds with antitumor activity of the formula A, mainly those in which the radical R is a benzene nucleus which has hydroxyl groups, methoxy groups, nitro groups and amino groups as substituents, and as a specific example of such a compound, have been used for combating cancer Formula A is the compound called Didox (3,4-dihydroxybenzohydroxamic acid), which has the following formula

2nd

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CH 683 593 A5

0 NHOH DIDOX

having. This latter compound also has good iron chelating power and is also a radical scavenger.

In the already known antitumor agents of structure A above, however, a hydrogen atom is bonded to the nitrogen atom of the hydroxamic acid structure of the formula given above. The corresponding nitrogen atom is therefore not substituted with any organic radical.

In particular, no such hydroxamic acid derivatives have hitherto been used as active ingredients in pharmaceutical preparations in which a radical of the hydroxamic acid which is organically substituted on the nitrogen atom forms part of a corresponding heterocyclic structure, in which a divalent grouping of the structure in a heterocycle

* C = 0 I

, N

OH

is present.

However, several compounds in which the rest of the hydroxamic acid or a derivative thereof is a divalent structure which forms part of a heterocyclic skeleton have already been described in the literature. In this connection, reference is made in particular to US Pat. No. 3,862,180, in which derivatives of 2,4-dihydroxy-2H-1,4-benzoxazin-3 (4H) -ones are described, which have the following formula B

B

correspond.

In these known derivatives of 2,4-dihydroxy-2H-1,4-benzoxazin-3 (4H) -one, R denotes hydrogen, halogen, lower alkoxy, lower alkyl or aryl,

R2 is hydrogen, a lower alkyl radical or the residue of a protected glucoside and R3 means hydrogen, alkyl or benzyl, where, when R3 represents a hydrogen atom, the corresponding compounds can also be present in the form of the salts with metals of the transition elements of the periodic table.

The compounds of formula B described above in this US patent have an insecticidal activity.

Compounds corresponding to formula B given above have previously been isolated from plants, in particular from grasses and cereals, such as maize, rice, wheat and rye. In the products isolated from these plants, R2 and R3 represent a hydrogen atom, and R represents a hydrogen atom or a methoxy group. The corresponding compound with R in the meaning of a hydrogen atom is 2,4-dihydroxy-1,4-benzoxazin-3 ( 4H) -one, which is also abbreviated as DIBOA in the literature, and the compound of formula B in which R is methoxy is 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3 ( 4H) -on, which is also referred to in the literature as DIMBOA3

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is shortened. The glucosides corresponding to these two compounds mentioned were also isolated from the grasses and cereals, in which R2 is a giucosidically bonded glucose residue, R3 is hydrogen and R is hydrogen or a methoxy group. Furthermore, the benzoxazolinone of the following formula, which is believed to be a degradation product of DIBOA, was also found in the corresponding plants.

Of the large number of publications in which the products DIBOA and DIMBOA isolated from plants are described, reference is made, for example, to the following:

Virtanen et al., O. Suomen. Kemistilehti B, 32, 252 (1959);

Walroos et al., Acta. Chem. Scand., 13, 1906 (1959); and

Virtanen, Angew. Chem. International Edit., 1, 299 (1962).

US Pat. No. 3,862,180 also describes a process for the preparation of the compounds of structure B given above, in which the heterocyclic ring is formed, in which a corresponding benzene derivative, in which one is ortho-graded to the hydroxy-substituted amino group Methoxy group is bound, which has both a Niederaikoxysubstituenten and a carboxylic acid ester substituent, the heterocyclic ring takes place by formation of the internal acid amide between the carboxylic acid ester group bound to the methoxy substituents and the hydrogen atom of the hydroxy-substituted NH group.

A process leading to better yields and easier to carry out for the preparation of the special compound of formula B with the designation DIBOA is further described in the GDR patent 264 694. This GDR patent also emphasizes that, owing to its antibacterial, fungicidal and insecticidal activity, this compound is of particular value in agriculture, in particular in order to be able to use it to breed cultivars which are resistant to harmful influences. Furthermore, it is also known that DIBOA influences the growth of plants, both of dicotyledonous plants, such as cress, and of monocotyledonous plants, such as certain types of grass. Surprisingly, the growth of cress roots and seedlings is stimulated at very low concentrations (0.09 millimolar) of DIBOA, but is inhibited at higher concentrations (see the publication by Barnes, JP; Putnam, AR; Bur-ke, BA and Aasen, AJ in Phytochemistry, 26 (5), 1385-90 [see also CA107 (13): 112630q]).

Thus, although it has long been known that some of the compounds of the formula B given above, which are described in the literature, have a bactericidal and fungicidal activity in addition to their insecticidal activity, the corresponding active compounds, and in particular the one in this connection, have nevertheless been best investigated Natural substance DIBOA has so far been used exclusively for the treatment of plants, but not as an active ingredient in corresponding pharmaceutical preparations for the treatment of animals and humans.

It has now surprisingly been found that 2,4-dihydroxy-2H-1,4-benzoxazin-3 (4H) -one, namely both known compounds corresponding to formula B above and new derivatives, have the property of inhibiting an undesired cell proliferation of living cells of vertebrates, both a corresponding malignant cell proliferation and undesirably high benign cell proliferation.

DESCRIPTION OF THE INVENTION

Accordingly, the present invention relates to a pharmaceutical preparation for inhibiting undesired cell multiplication in vertebrates, which is characterized in that it contains at least one 2,4-dihydroxy-2H-1,4-benzoxyazin-3 (4H) -one or a as active ingredient Derivative thereof of formula I.

NH

/

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CH 683 593 A5

O -RJ

^ 0 -R2

R

contains, in this formula I

R1 is hydrogen, an unsubstituted alkyl radical with 1-6 carbon atoms, a substituted alkyl radical with 1-6 carbon atoms in the alkyl part which carries 1-3 substituents which are independently selected from the group consisting of halogen atoms, hydroxyl groups and alkoxy groups each having 1 -6 carbon atoms, phenoxy groups, carboxylic acid groups, esterified carboxylic acid groups and phenyl radicals, is an unsubstituted phenyl radical or a substituted phenyl radical which has 1-3 substituents which are selected from the group consisting of the substituents alkyl having 1-6 carbon atoms, hydroxy, Alkoxy, acyloxy, carboxylic acid groups and carboxylic acid ester groups and halogen atoms,

R2 is hydrogen, an acyl radical of the formula

11 1 -C-R,

in which R1 has the meaning given above, a glycosidically bound sugar radical, an unsubstituted alkyl radical with 1-6 carbon atoms, a substituted alkyl radical with 1-6 carbon atoms in the alkyl part, which carries 1 to 3 substituents which are selected independently of one another from the group which comprise halogen atoms, hydroxyl groups, alkoxy groups each having 1-6 carbon atoms, phenoxy groups, carboxylic acid groups, esterified carboxylic acid groups and phenyl radicals, an unsubstituted phenyl radical or a substituted phenyl radical which has 1 to 3 substituents which are selected independently of one another from the group, which comprises alkyl groups with 1-6 carbon atoms, hydroxyl groups, alkoxy groups, acyloxy groups, carboxylic acid groups, carboxylic ester groups and halogen atoms,

R3 represents a hydrogen atom or an acyl group of the formula

O

in which R1 has the meaning given above,

n is an integer in the range 0-4, and the substituents

R4 independently of one another for alkyl radicals with 1 to 6 carbon atoms, substituted alkyl radicals with 1-6 carbon atoms in the alkyl part which carry hydroxyl, alkoxy, phenoxy or phenyl radicals as substituents, free or etherified hydroxyl groups of the formula

-0-R1

or acylated hydroxy groups of the formula

O

H 1 -O-C-R

stand, in these groupings R1 has the meaning given above or free or esterified carboxyl groups, halogen atoms, unsubstituted phenyl radicals or substituted phenyl radicals which have 1-3 substituents which are selected from the group consisting of the substituents alkyl having 1-6 carbon atoms , Hydroxyl, alkoxy, acyloxy, carboxylic acid groups and carboxylic ester groups and halogen atoms, or the radical R4 denotes a glycosidically bonded sugar radical or 2 radicals R4 bonded to adjacent carbon atoms of the benzene nucleus together form a group -O-CH2-O- or -O-CH2- CH2-O, or that the pharmaceutical preparation contains a pharmaceutically acceptable salt of a compound of formula I.

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The active compounds of the formula I contained in the pharmaceutical preparations according to the invention include both new chemical compounds and those which have already been described in the literature, such as, for example, the compounds of the formula B given above which are described in US Pat. No. 3,862,180 .

If the pharmaceutical preparations according to the invention contain those active ingredients of the formula I which have already been described in the literature, these compounds of the formula I have hitherto only been used in agents for treating plants, for example as insecticides, herbicides or fungicides, but not as the active ingredient of any pharmaceutical preparations that can be used to treat undesirable conditions in vertebrates.

The pharmaceutical preparations according to the invention therefore also relate to a first medical indication of these active substances when active compounds of the formula I are used as the active substance.

The pharmaceutical preparations according to the invention are suitable for controlling unwanted cell proliferation in cold-blooded vertebrates, such as fish, amphibians and reptiles, but they are preferably used for inhibiting undesired cell proliferation in warm-blooded vertebrates, namely birds and mammals, including humans.

The pharmaceutical preparations according to the invention can advantageously be used for the treatment of cancer in order to inhibit the undesired multiplication of the malignant tumor cells, but not the multiplication of the healthy cells.

However, the pharmaceutical preparations according to the invention are also suitable for the treatment of benign, undesirably high cell proliferation, for example for the treatment of an undesirable enlargement of glandular tissue, for example for the treatment of benign prostatic hyperplasia.

The pharmaceutical preparations according to the invention preferably contain as active ingredient those compounds of the formula I which are unsubstituted in the benzene nucleus, in which n is 0, or which have 1 or 2 substituents R4 in the benzene nucleus, in which n is 1 or 2.

Of the compounds of the formula I substituted in the benzene nucleus, those are particularly preferred in which R4 is an unsubstituted alkyl radical having 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, and particularly preferably 1 or 2 carbon atoms. If the corresponding alkyl radical carries substituents, then hydroxyl groups, alkoxy groups and phenyl radicals are preferred, in which case the substituted alkyl radical preferably also has 1 or 2 carbon atoms in the alkyl radical, and accordingly there are examples of substituted and unsubstituted alkyl radicals which occur as substituents R4 in the benzene nucleus can, methyl radicals, ethyl radicals, isopropyl radicals, 2-hydroxyethyl radicals, methoxymethyl radicals, methoxyethyl radicals, and benzyl radicals.

Further preferred meanings for substituents R4 which can occur in the phenyl radical are free or etherified or acylated hydroxyl groups, ie substituents which have the following formulas

-0-R1,

respectively

»1 -O-C-R

have, or the residues R4 can also be glycosidically bound sugar residues, ie residues of the formula

-O-Glu,

wherein Glu represents a sugar residue bound via a glycoside bond, in particular a Glu-cos residue or a mannose residue. Examples of radicals R4 in the meaning of etherified hydroxyl groups of the formula given above are corresponding alkoxy groups with 1-6 carbon atoms, in particular those with 1-4 carbon atoms, preference being given to ethoxy groups and in particular methoxy groups, and also aralkoxy groups, in particular those with 1 or 2 carbon atoms in the alkyl part, particularly preferably the benzyloxy group. Furthermore, the etherified hydroxyl groups can also be phenoxy groups, the phenyl nucleus preferably being unsubstituted or carrying 1 or 2 substituents as explained in connection with the definition of the radical R1, in particular alkyl or alkoxy groups having 1-3 carbon atoms.

If the radicals R4 have the meaning of acylated hydroxyl groups of the formula given, corresponding acyl radicals which are derived from lower aliphatic carboxylic acids, such as formic acid, acetic acid and propionic acid, and acyl radicals which are derived from aromatic acids, such as benzoic acid or salicylic acid, are special prefers.

However, the R4 radicals can also mean free or esterified carboxyl groups. In the case of the esterified carboxyl groups, corresponding alkyl esters, in particular those with 1-6, are preferred

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1-3 carbon atoms, in the alkyl part, and furthermore esterified carboxyl groups, in which the alcohol part is derived from a phenyl-substituted alkyl radical, in particular benzyl, preferably, and furthermore those esterified carboxyl groups in which the ester-forming alcohol is a phenol or a substituted phenol.

If the substituents R4 have the meaning of halogen atoms, then these are preferably fluorine, chlorine or bromine atoms, chlorine substituents being particularly preferred. As already mentioned, the substituents R4 can also have the meaning of unsubstituted or substituted phenyl radicals, but preferably only one substituent R4 in the meaning of an unsubstituted or substituted phenyl radical is bonded to the benzene nucleus.

The substituents R4 can be bound to any position, that is to say positions 5, 6, 7 or 8 of the benzene nucleus, but for steric reasons the substituents are preferably bound to position 6 or 7. Also in the event that two R4 groups together form a methylenedioxy group of the formula

-O-CH2-O-

represent, this grouping is preferably bound to positions 6 and 7 of the benzene nucleus. Particularly preferably, radicals R4 in the meaning of lower alkyl groups or lower alkoxy groups are bonded to position 6, and in particular to position 7 of the benzene nucleus, such as, for example, an alkoxy radical bonded to the 7-position with 1-3 carbon atoms, in particular a corresponding one Methoxy residue.

As can be seen from the structural formula I, in the active compounds of the formula I which are contained in the pharmaceutical preparations according to the invention, a hydroxyl group which is optionally esterified, that is to say, must be bound to the nitrogen atom of the heterocyclic radical, that is to the 4 position Acyloxy group of the formula

O

»1 -O-OR

is present, in which acyloxy radical R1 has the meaning given above.

If in the compounds of the formula I R3 is an acyl radical, then this R1 preferably means an alkyl group with 1-6 carbon atoms, in particular an alkyl group with 1-3 carbon atoms, or a phenyl-substituted alkyl radical, in particular a benzyl radical, or else an optionally substituted phenyl radical . Accordingly, these acyl residues are preferably derived from formic acid, acetic acid or propionic acid or from phenylacetic acid, benzoic acid or a correspondingly substituted benzoic acid, for example salicylic acid.

In the active compounds of the formula I, a hydroxyl group or an etherified or esterified hydroxyl group must be bound to the carbon atom at position 2 of the benzene nucleus, the corresponding etherified hydroxyl groups also including those in which a sugar radical is bound to the corresponding oxygen atom via a glycoside bond, in which means the radical R2 has the meaning of glu, the corresponding sugar residue preferably being derived from glucose or mannose.

If an etherified hydroxy group is bonded to the carbon atom in the 2-position in the active compounds of the formula I, the corresponding radical R2 is preferably an unsubstituted or substituted alkyl radical having 1-6 carbon atoms, in particular one having 1-4 carbon atoms, and especially preferably a substituted or unsubstituted methyl, ethyl or propyl radical. Of the corresponding substituted alkyl radicals, preference is again given to those in which the substituents are alkoxy groups, phenoxy groups or phenyl radicals, such as, for example, a R2 radical meaning a methoxymethyl radical, methoxyethyl radical, phenoxymethyl radical or a benzyl radical.

If an acylated hydroxy group is bonded to the carbon atom 2 in the active compounds of the formula I, i.e. the radical R2 has the meaning of an acyl radical of the formula, preferred meanings for this acyl radical are again those which are derived from lower aliphatic or araliphatic or aromatic carboxylic acids, for example formic acid, acetic acid, propionic acid, phenylacetic acid, benzoic acid or Salicylic acid.

Accordingly, in particularly preferred active compounds of the formula I, R2 is a hydrogen atom, a lower alkyl radical, a lower aralkyl radical, a glycosidically bound sugar radical or an acyl group of a lower aliphatic carboxylic acid or at most an aromatic carboxylic acid.

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The second radical which is bonded to the carbon atom in the 2-position of the active compounds of the formula I, namely the radical R1, is preferably a hydrogen atom, an unsubstituted alkyl radical having 1-6 carbon atoms, in particular 1-4 carbon atoms, and particularly preferably 1 or 2 carbon atoms, or a correspondingly substituted lower alkyl radical, alkoxy groups, phenoxy groups and phenyl radicals being preferred among the substituents. Furthermore, the R1 radical can also be an unsubstituted or substituted phenyl radical in which the substituents are as defined above. Accordingly, particularly preferred meanings of the radical R1 in the active compounds of the formula I of the pharmaceutical preparations according to the invention are the radicals hydrogen, alkyl having 1-3 carbon atoms, in particular 1 or 2 carbon atoms, and particularly preferably methyl, methoxymethyl, methoxyethyl, phenoxymethyl, phenaikyl, in particular benzyl and possibly also a phenyl radical.

In preferred active compounds of the formula I which are used in the pharmaceutical preparations according to the invention, the substituents mentioned accordingly have the following meaning: n is 0 or 1,

R4 represents alkyl or alkoxy each having 1-3 carbon atoms, benzyloxy, phenoxy or hydroxy, R3 is hydrogen or a lower aliphatic acyl radical, the group of the formula

—O — R2

stands for a group of the formula

-0-R1

or

-O-C-R

or

-O-glu,

wherein in these groups R1 preferably denotes hydrogen, alkyl with 1-3 carbon atoms, phenylalkyl or phenyl and glu stands for a glycosidically bound sugar residue, in particular mannose or glucose.

As specific examples of compounds of the formula I which show the desired inhibition of undesired cell proliferation in vertebrates, there are corresponding compounds of the formula I in which the radicals R1, R2 and R3 are hydrogen and n is 0, that is to say 2,4-dihydroxy -1,4-benzox-azin-3 (4H) -one, which is also abbreviated as DIBOA, and furthermore analogue compounds in which n is 1 and R4 has the meanings given above, and in particular an alkyl substituent or alkoxy substituent having 1-6 carbon atoms, such as a corresponding methoxy substituent.

In the event that the substituent R4 is a methoxy substituent in the 7-position of the structure, the corresponding compound of the formula I is 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3 (4H) -one , which was also isolated from grass and grain a long time ago and is also abbreviated in the literature as DIMBOA.

Different compounds of formula I and pharmaceutically acceptable salts thereof, such as, for example, alkali metal salts of corresponding compounds, which have a carboxyl group in their structure, were tested for their inhibition of undesired cell growth.

The corresponding in vitro tests were carried out using cell lines derived from various human cancers, namely human cancer cells from bladder cancer, testicular cancer, bone cancer, liver cancer, larynx cancer and prostate cancer, both the cell line DU-145, which is derived from androgen-insensitive tissue of the prostate cancer, as well as the Zeil line with the name LNCaP, which is derived from the androgen-sensitive tissue of the prostate cancer. The remaining cell lines tested were RT112, TERRA, MG63, CHANG and HEP.

The cell line derived from human prostate carcinoma called DU-145 is a cell line from androgen-independent adenocarcinoma, which was originally isolated from metastases in the brain. In this context, it is due to the publication of K.R. Stone et al. in the int. J.Cancer 21, 274-281 (1978), in which the isolation of this human prostate carcinoma cell line with the designation DU-145 is described in more detail.

The inhibition of cell proliferation was tested by the method described by F.K. Habib, M. Rose, A.C. Buck, L. Ebeiing and A. Lewenstein on testing the cancer-inhibiting effect

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of an aqueous pollen extract was developed for the growth of these prostate cells, and that in British J. Urol. 66, pages 393-397, (1990).

The in vitro tests performed on the other cell lines mentioned were carried out under analogous conditions as described in the above publication by F.K. Habib et al. described.

With all the compounds and cell lines tested, it was found that in these in vitro tests at concentrations of only 0.1 to 1 ng of the compound of the formula I per ml of culture medium, no significant inhibition of the cell proliferation of these cancer cells could be found. However, as soon as the concentration of the active compound of the formula I was increased to 2-100 ng per ml, there was a strong inhibition, until finally a complete inhibition of the cell growth, in all the compounds and all the tumor cell lines tested.

With the help of the cell lines of the cancer cells mentioned, for example with DU-145, the inhibition of the undesired cell proliferation was also tested on the basis of the uptake of tritium-labeled thymidine, that is to say 3H-thymidine, by the cancer cells in question. The implementation of this in vitro test is described in more detail below, and the corresponding cultures were grown in the presence of an active ingredient of the formula I, or the cultures for comparison purposes, in the absence of such an active ingredient for three to nine days. In this test, it was found that in all of the active compounds of formula I tested and in all cell lines tested, there was a clear inhibition of thymidine uptake at concentrations of the active compound from 2 ng per ml to 100 ng per ml of the cell culture. In the DU-145 cells, the thymidine uptake at a concentration of the active ingredient of 10 ng per ml, depending on the active ingredient tested, had decreased to 10% to 60% of the increase, compared to the cultures without addition of active ingredient (100%).

In vivo testing of the inhibition of cell growth in benign prostatic hyperplasia in humans was carried out using the nude mouse model according to the method described by B. Wagner, U. Otto, H. Becker, S. Schröder and H. Kosterhalfen in the book « Benign Prostatopathies », ed. W. Vahlensieck and G. Rutishauser, G. Thieme Verlag, Stuttgart, pp. 129-133 (1992). In this procedure, male, three-month-old MNRI nu / nu mice have their testicles removed, and the day after, human tissue is implanted subcutaneously on both sides of the chest. The growth of the transplanted human tissue was stimulated hormonally with dihydrotestosterone and estradiol, the size of the tumors was tested in the test group with administration of the active compounds of the formula I and in the comparison group without administration of these active compounds. The active compounds of the formula I were administered orally to the animals in the test group. The corresponding active compounds of the formula I were administered to the animals in an amount of 0.2 mg-20 mg, e.g. 0.4-0.6 mg per kg body weight administered per day. Oral treatment was given one week after the transplant and continued until the sixth week after the transplant. A comparison of the hormone-stimulated group without administration with the hormone-stimulated group with the administration of the active substances of the formula I showed an increase in the transplanted tumors to more than twice the value without administration of the active substances, while the transplanted tumors to a volume below the volume before the treatment in the group with hormone stimulation plus administration of the active substances. In some cases, the tumor size had decreased to less than half the value before treatment, after four weeks or after six weeks of treatment.

As has already been mentioned, the active compounds of the formula I used in the agents according to the invention include those which have already been described in the literature and corresponding new chemical compounds. Another object of the present invention are accordingly new derivatives of 2,4-dihydroxy-2H-1,4-benzoxazin-3 (4H) -ones, which are characterized in that they have the following formula la la v

0 î

have, in this formula la

Ri 'hydrogen, an unsubstituted alkyl radical with 1-6 carbon atoms, a substituted alkyl radical with 1-6 carbon atoms in the alkyl part, which carries 1-3 substituents which are independently selected from the group consisting of halogen atoms, hydroxyl groups, alkoxy groups with each 1-6 carbon atoms, phenoxy groups, carboxylic acid groups, esterified carboxylic acid groups

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and phenyl radicals, is an unsubstituted phenyl radical, or a substituted phenyl radical which has 1-3 substituents selected from the group consisting of alkyl having 1-6 carbon atoms, hydroxy, alkoxy, acyloxy, carboxylic acid groups and carboxylic acid ester groups and halogen atoms ,

R2 'is hydrogen, an acyl radical of the formula in which R1' has the meaning given above, a glycosidically bound sugar radical, an unsubstituted alkyl radical with 1-6 carbon atoms, a substituted alkyl radical with 1-6 carbon atoms in the alkyl part, which has 1 to 3 substituents as substituents which are independently selected from the group comprising halogen atoms, hydroxyl groups, alkoxy groups each having 1-6 carbon atoms, phenoxy groups, carboxylic acid groups, esterified carboxylic acid groups and phenyl radicals, an unsubstituted phenyl radical or a substituted phenyl radical which has 1 to 3 substituents, which are independently selected from the group comprising alkyl groups with 1-6 carbon atoms, hydroxyl groups, alkoxy groups, acyioxy groups, carboxylic acid groups, carboxylic ester groups and halogen atoms,

R3 'represents a hydrogen atom or an acyl group of the formula in which R1' has the meaning given above,

n 'represents an integer in the range 0-4, and the substituents

R4 'independently of one another for alkyl radicals with 1 to 6 carbon atoms, substituted alkyl radicals with 1-6 carbon atoms in the alkyl part which carry hydroxyl groups, alkoxy groups, phenoxy groups or phenyl radicals as substituents, free or etherified hydroxyl groups of the formula

-0-R1 '

or acylated hydroxyl groups of the formula in which R1 'has the meaning given above or free or esterified carboxyl groups, halogen atoms, unsubstituted phenyl radicals or substituted phenyl radicals which have 1-3 substituents which are selected from the group consisting of the substituents Alkyl having 1-6 carbon atoms, hydroxy, alkoxy, acyloxy, carboxylic acid groups and carboxylic ester groups and halogen atoms, or the radical R4 'means a glycosidically bonded sugar radical or 2 radicals R4' bonded to adjacent carbon atoms of the benzene nucleus together form a group —O — GH2 —O— or —O — CH2 — CH2 — O—,

provided that when n 'is 0 or 1 and also R4' is an alkyl radical with 1-6 carbon atoms, an alkoxy radical with 1-6 carbon atoms, a halogen atom or an unsubstituted or substituted phenyl radical, and also R3 ' Means hydrogen atom and furthermore R2 'represents hydrogen, alkyl having 1-6 carbon atoms or a glycosidically bound sugar radical, the radical R1' must have a different meaning than that of a hydrogen atom, and salts of the compounds of the formula la.

The new compounds of the formula Ia can be prepared by processes which are already known for the preparation of such heterocyclic compounds which have both a nitrogen atom and an oxygen atom in their heterocyclic ring part. For example, the preparation can be carried out by the process described in US Pat. No. 3,862,180 for the already known compounds of structure B given above. However, the preparation is preferably carried out by the process described for the connection with the abbreviation DIBOA in the GDR patent 264 694. According to the manufacturing process described there, first of all by cyclization of a nitro compound of the formula II

-C-R

>

O

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, f ^ V-N02

II

R

î

a corresponding heterocyclic compound of formula III

O -H

III

li manufactured. This heterocyclic compound is then brominated by reaction with bromine to give the corresponding 2-bromine compound and then the bromine atom in the 2-position is replaced by a hydroxyl group by hydrolysis with moist silver carbonate, the corresponding 2,4-dihydroxy compound of the following formula being obtained. The compounds thus obtained can be used as active ingredients of the formula I in the pharmaceutical preparations according to the invention. The compounds obtained in this way also represent new derivatives of the formula Ia according to the invention if either the radical R1 'has a different meaning than that of a hydrogen atom and R4' and n 'has any of the meanings according to Claim 8, or if they contain R1 'is a hydrogen atom and n' either represents an integer in the range from 2-4, or n 'is 1 and, moreover, R4' has a different meaning than that of an alkyl radical having 1-6 carbon atoms, an alkoxy radical having 1-6 carbon atoms, a halogen atom or an unsubstituted or substituted phenyl radical.

The compounds of the formulas obtained in this way can be converted by further reactions into corresponding new compounds of the formula Ia in which R2 'has a different meaning than that of a hydrogen atom and is, for example, an acyl radical or in which R3' is an acyl group in connection with Formula la has structure given above. These further reactions for introducing the corresponding substituents can be carried out according to known working methods, for example by acylation.

The invention will now be explained in more detail with reference to the examples which now follow, which serve only to illustrate special types of embodiment of the pharmaceutical compositions according to the invention and are in no way to be regarded as restrictions.

example 1

Determination of the uptake of 3H-thvmidine by living cancer cells

In this example, human prostate carcinoma cells of the cell line DU-145 were examined. Analogous investigations were also carried out on six further Zeil lines, namely the Zeil lines RT112, Terra, MG63, Chang, Hep and LNCaP, which are also derived from human cancerous tumors, as will be explained in more detail above.

O - H

11

5

10th

15

20th

25th

30th

35

40

45

50

55

CH 683 593 A5

The cells were in the subconfluent stage and rinsed with Dulbecco "A" phosphate buffered saline and once with 0.25% trypsin and 0.2% EDTA. The cells were incubated at 37 ° C. for five minutes and then resuspended in a serum-free medium, which is abbreviated as “SFM”. The cells were counted in three parallel determinations using dishes and a hemocytometer chamber. 1.5 x 103 cells to 1.5 x 104 cells were plated overnight in SFM to which 0.5% fetal calf serum (available from Gibco) had been added to aid plating. The plating was carried out on culture plates with 96 wells (cell cult). The plating medium was then replaced by SFM (if the comparative tests were carried out) or by SFM, which also contained the active ingredient of the formula I to be tested of the pharmaceutical preparations according to the invention. The cultivation in the SFM, or in the SFM plus compound of the formula I, was carried out for 5-6 days. After this growth period, the tritiated thymidine was added, using methyl 3H-thymidine (37 kBq per well, specific activity 75 GBq mmol-1; Amersham International) in the culture medium RPMI 1640 from Gibco, and this treatment was carried out for at least four hours. The medium in each well was aspirated and resuspended before adding 10% ice cold trichloroacetic acid. The cells were then collected on filter mats two hours later using a device called Skatron Combi Cell Harvester. The cells were collected by washing each well three times with water and at the end of this collection of the cells, the filter mats were dried at 60 ° C. for 30 minutes. Each disc thus obtained on the filter mat (filter paper) contained the precipitable material and the determination of the tritium content was carried out by counting in a scintillation fluid. Overall, the cultivation was carried out in the presence of the compound of the formula i which inhibits cell proliferation, or in the absence of the same in the comparative experiments, for 3-9 days.

Example 2

The 2,4-dihydroxy-2H-1,4-benzoxazin-3 (4H) -one (ie DIBOA) was prepared by the process described in the GDR patent 264,694.

Prostate cancer cells of the cell line DU-145 were tested according to the method described in Example 1, 1500 cells being contained in each well of the test plate.

In the series for comparison purposes, the medium contained no DIBOA. In the test series, the medium contained 2 ng / ml, or 10 ng / ml, or 100 ng / ml of the synthetically produced compound DIBOA. The inclusion of tritiated thymidines in the group for comparison purposes was set at 100% on each of days 1-5 of incubation.

In the test group with a concentration of 1 / µg DIBOA per ml, the thymidine uptake on test days 1 and 2 was 80% of the value of the comparison group, then rose to 92% by day 4 and then decreased again to 87% on day 5 .

In the test group in which the concentration of DIBOA was 10 ng / ml, the thymidine uptake on test day 1 decreased to 48% compared to the group for comparison purposes, then increased slightly on test day 2 and finally reached on test days 3, 4 and 5 values in the range of 35-40% of the group for comparison purposes.

In the test group with a concentration of 100 ng / ml DIBOA, the thymidine intake had already decreased to 8% of the value of the comparison group on day 1, and decreased to 2% of the comparison group after a slight increase on day 2, finally on test day 5 .

In the active compounds of the formula I, the carbon atom in position 2 of the heterocyclic ring is an optically active carbon atom. With the synthetically produced DIBOA there is of course a racemate in which the two enantiomers are present in a ratio of 1: 1.

Tests similar to the synthetically produced DIBOA were carried out with DIBOA isolated from rye. It was found that the corresponding natural product was about twice as effective as the synthetically manufactured product. It can be assumed that the enantiomer ratio in the natural product is not 1: 1, but that there is a higher concentration at one of the two entantiomers, and that the natural product only consists of one of the two antipodes.

Corresponding attempts to separate the antipodes of the synthetically produced DIBOA are currently being carried out in order to be able to determine which of the two antipodes has the activity or the higher activity to inhibit the undesired cell proliferation.

Example 3

New derivatives of DIBOA synthetically prepared according to Example 2 were synthesized by subjecting the DIBOA to acetylation with acetic anhydride. This gave corresponding acetylation products of the formula I in which the radical R2 or radical R3 or both radicals R2 and R3, acetyl groups of the formula

12th

CH 683 593 A5

-CO-CH3.

In the test procedure described in Example 2, the acetylation products had a somewhat higher inhibitory effect than the synthetically produced DIBOA.

Example 4

Orientative preliminary tests indicate that the synthetically produced compound 10 DIBOA and derivatives thereof additionally have an antiviral activity.

Claims (7)

Claims 1. Pharmaceutical preparation for inhibiting undesired cell proliferation in vertebrates, characterized in that it has at least one 2,4-dihydroxy-2H-1,4-benzoxazin-3 (4H) -one or a derivative thereof of the formula I as active ingredient 20th 25th 30th 55 60 65 ^^ (3 ^ 0 -R2 R1 contains, in this formula I R1 is hydrogen, an unsubstituted alkyl radical with 1-6 carbon atoms, a substituted alkyl radical with 1-6 carbon atoms in the alkyl part which carries 1-3 substituents which are independently selected from the group consisting of halogen atoms, hydroxyl groups and alkoxy groups with 35 each 1-6 carbon atoms, phenoxy groups, carboxylic acid groups, esterified carboxylic acid groups and phenyl radicals, is an unsubstituted phenyl radical or a substituted phenyl radical which has 1-3 substituents selected from the group consisting of the substituents alkyl having 1-6 carbon atoms, hydroxy , Alkoxy, acyloxy, carboxylic acid groups and carboxylic ester groups and halogen atoms, 40 R2 hydrogen, an acyl radical of the formula 0 II! -C-R, 45 in which R1 has the meaning given above, a glycosidically bound sugar radical, an unsubstituted alkyl radical with 1-6 carbon atoms, a substituted alkyl radical with 1-6 carbon atoms in the alkyl part, which carries 1 to 3 substituents which are selected independently of one another from the group which comprises halogen atoms, hydroxyl groups, alkoxy groups each having 1-50 6 carbon atoms, phenoxy groups, carboxylic acid groups, esterified carboxylic acid groups and phenyl radicals, an unsubstituted phenyl radical or a substituted phenyl radical which has 1 to 3 substituents which are selected independently of one another from the group which comprises alkyl groups with 1-6 carbon atoms, hydroxyl groups, alkoxy groups, acyloxy groups, carboxylic acid groups, carboxylic ester groups and halogen atoms, R3 represents a hydrogen atom or an acyl group of the formula O II 1 -C-R in which R1 has the meaning given above, n is an integer in the range 0-4, and the substituents R4 independently of one another for alkyl radicals with 1 to 6 carbon atoms, substituted alkyl radicals with 1-6 carbon atoms in the alkyl part which carry hydroxyl, alkoxy, phenoxy or phenyl radicals as substituents, free or etherified hydroxyl groups of the formula 13 5 10th 15 20th 25th 30th 35 40 45 50 55 60 65 CH 683 593 A5 -0-R1 or acylated hydroxy groups of the formula O "1 -O-C-R stand, in these groupings R1 has the meaning given above or free or esterified carboxyl groups, halogen atoms, unsubstituted phenyl radicals or substituted phenyl radicals which have 1-3 substituents selected from the group which are substituted by alkyl having 1-6 carbon atoms , Hydroxy, alkoxy, acyloxy, carboxylic acid groups and carboxylic ester groups and halogen atoms, or the radical R4 means a glycosidically bonded sugar radical or 2 radicals R4 bonded to adjacent carbon atoms of the benzene nucleus together form a group -O-CH2-O- or -O-CH2-CH2-O-, or contain pharmaceutically acceptable salts of the compounds of formula I. 2. Pharmaceutical preparation according to claim 1, characterized in that it contains as active ingredient a compound of formula I in which n is 0 or 1 and R4 is alkoxy with 1-3 carbon atoms, preferably methoxy in position 7 or a pharmaceutically acceptable salt of the compound of formula I. 3. Pharmaceutical preparation according to claim 1 or 2, characterized in that it contains as active ingredient a compound of formula I in which R3 is hydrogen, or a pharmaceutically acceptable salt of this compound of formula I. 4. Pharmaceutical preparation according to one of claims 1 to 3, characterized in that it contains as active ingredient a compound of formula I in which R2 represents hydrogen or a glycosidically bound sugar residue or contains a pharmaceutically acceptable salt of this compound of formula I. 5. Pharmaceutical preparation according to one of claims 1 to 3, characterized in that it contains as active ingredient a compound of formula I in which R1 is hydrogen or an unsubstituted alkyl radical having 1 to 3 carbon atoms or a pharmaceutically acceptable salt of this compound of formula I. . 6. Pharmaceutical preparation according to one of claims 1 to 5, characterized in that it is the active ingredient of the formula I, the compound of the following structure OH or a glycoside of this compound represented by the following formula OH 1 contains wherein in the glycoside the symbol glu represents a sugar residue bonded via a glycoside bond, in particular a glucose or mannose residue. 7. Derivatives of 2,4-dihydroxy-2H-1,4-benzoxazin-3 (4H) -ones, characterized in that they have the following formula la 14 5 10th 15 20th 25th 30th 35 40 45 50 55 60 65 CH 683 593 A5 la have, in this formula la R'- hydrogen, an unsubstituted alkyl radical with 1-6 carbon atoms, a substituted alkyl radical with 1-6 carbon atoms in the alkyl part which carries 1-3 substituents which are independently selected from the group consisting of halogen atoms, hydroxyl groups, alkoxy groups each comprises 1-6 carbon atoms, phenoxy groups, carboxylic acid groups, esterified carboxylic acid groups and phenyl radicals, is an unsubstituted phenyl radical, or a substituted phenyl radical which has 1-3 substituents which are selected from the group consisting of the substituents alkyl having 1-6 carbon atoms, Hydroxy, alkoxy, acyloxy, carboxylic acid groups and carboxylic acid ester groups and halogen atoms, R2 'is hydrogen, an acyl radical of the formula î -C-R, in which R1 'has the meaning given above, a glycosidically bonded sugar radical, an unsubstituted alkyl radical with 1-6 carbon atoms, a substituted alkyl radical with 1-6 carbon atoms in the alkyl part, which carries 1 to 3 substituents independently of one another from the group are selected which comprises halogen atoms, hydroxyl groups, alkoxy groups each having 1-6 carbon atoms, phenoxy groups, carboxylic acid groups, esterified carboxylic acid groups and phenyl radicals, an unsubstituted phenyl radical or a substituted phenyl radical which has 1 to 3 substituents which are selected independently of one another from the group which comprises alkyl groups with 1-6 carbon atoms, hydroxyl groups, alkoxy groups, acyloxy groups, carboxylic acid groups, carboxylic ester groups and halogen atoms, R3 'represents a hydrogen atom or an acyl group of the formula in which R1' has the meaning given above, n 'represents an integer in the range 0-4, and the substituents R4 'independently of one another for alkyl radicals with 1 to 6 carbon atoms, substituted alkyl radicals with 1-6 carbon atoms in the alkyl part which carry hydroxyl groups, alkoxy groups, phenoxy groups or phenyl radicals as substituents, free or etherified hydroxyl groups of the formula -O-R1 ' or acylated hydroxy groups of the formula O l! 1 -O-C-R stand, in these groups R1 'have the meaning given above or free or esterified carboxyl groups, halogen atoms, unsubstituted phenyl radicals or substituted phenyl radicals which have 1-3 substituents which are selected from the group consisting of the substituents alkyl with 1-6 Carbon atoms, hydroxy, alkoxy, acyloxy, carboxylic acid groups and carboxylic acid groups and halogen atoms, or the radical R4 'means a glycosidically bound sugar radical or 2 radicals R4' bound to adjacent carbon atoms of the benzene nucleus together form a group -O-CH2-O- or -O -CH2-CH2-O-, provided that when n 'is 0 or 1 and also R4' is an alkyl radical with 1-6 15 5 10th 15 20th 25th 30th 35 40 45 50 55 60 65 CH 683 593 A5 Is carbon atoms, an alkoxy radical having 1-6 carbon atoms, a halogen atom or an unsubstituted or substituted phenyl radical, and R3 'is a hydrogen atom and R2' is hydrogen, alkyl having 1-6 carbon atoms or a glycosidically bound sugar radical, the radical R1 'Must have a different meaning than that of a hydrogen atom, and salts of the compounds of formula la. 16