WO1992000740A2

WO1992000740A2 - Bis-dioxopiperazines and their use as protection agents

Info

Publication number: WO1992000740A2
Application number: PCT/GB1991/001156
Authority: WO
Inventors: Andrew Malcolm Creighton
Original assignee: National Research Development Corporation
Priority date: 1990-07-13
Filing date: 1991-07-12
Publication date: 1992-01-23
Also published as: WO1992000740A3; GB9015435D0; GB9115156D0; GB2245832A

Abstract

Compounds of formula (II), wherein n is 0, 1 or 2, R1, R2, R3 and R4 are each separately selected from hydrogen, unsubstituted acyclic aliphatic hydrocarbon groups having a maximum of six carbon atoms and C1-6 alkyl groups substituted by a hydroxy group or by a C1-6 alkoxy group, or one of R1 and R2 and one of R3 and R4 is hydrogen and the others together form a trimethylene, tetramethylene or pentamethylene bridging group, R5 is hydrogen or an unsubstituted acyclic aliphatic hydrocarbon group having a maximum of six carbon atoms, and R6 is an acyclic aliphatic hydrocarbon group having a maximum of six carbon atoms or a group CH2R7 in which R7 is a C1-5 alkyl group substituted by a hydroxy group or by a C1-6 alkoxy group, or a salt thereof formed with a physiologically acceptable inorganic or organic acid, are of use in therapy, particularly as cardioprotective agents.

Description

BIS-DIOXOPIPERAZINES AND THEIR USE AS PROTECTION AGENTS

This invention relates to pharmaceutical compounds and to compositions containing them, being primarily concerned with substances of use as cardioprotective agents and in certain other protective roles.

Certain bis-dioxopiperazines of formula (I) are cytotoxic and have been used in the treatment of cancer. Thus UK patent 1,234,935 describes the compounds of formula (I) having R = CH₃ and

R' = R" = H (as the dl, d and 1 isomers); R = R' = R" = H;

R = R' = CH₃ and R" = H (as the meso isomer); and R + R' = -CH₂CH₂-and R" = H. Of these the first named compound has proved to be of most value although a further compound of formula (I) having

R = R' = H and has also been used in treating cancer.

Studies have been reported by various authors on the chelating properties of these bis-dioxopiperazines and the use in treatment of lead poisoning has been proposed by Wittig and Hultsch,

Int. Arch. Occup. Environ. Health, 1981, 48, 89, for the compound of formula (I) having R = R' = H and R" = CH₃ and by May et al, Agents and Actions, 1984, 15, 448 and Willes and Williams,

Plzen. Lek. Sborn., 1985, 42, 113, for the compound having

R = R" = H and R' = C₂H₅ in the dl form.

German Offenlegungschrift 2,511,891 and 2,511,892 describe the use of certain bis-dioxopiperazines of formula (I) for the treatment and prophylaxis of thrombosis and embolism. These compounds (I) include, inter alia, those in which R and R' are each separately selected from hydrogen and C_1-6 alkyl or together are linked so as to form a cycloalkyl residue and R" is C_1-6 alkyl, with the sum of the carbon atoms in R, R' and R" being greater than two. = In Research Communications in Chemical Pathology and

Pharmacology, 1985, 48, 39, Herman et al report tests on the protective effect against acute daunorubicin toxicity of a range of bis-dioxopiperazines of formula (I). They conclude that although the compound blmolane (R = R' = H and R" - -CH₂-N 0) and the compound having R = CH₃ and R' = R" = H (as the dl, d or 1 isomer) give protection against the lethal effects of daunorubicin, the remainder of the compounds tested (R = R' = R" = H; R = R^l = H and R" = CH₃; R = R" = CH₃ and R' = H (1); R = R' = CH₃ and R" = H (meso): R = C₂H₅ and R' = R" = H (d1); R = CH₃, R' = C₂H₅ and R" = H (dl-ervthro): and R + R' = -CH₂-CH₂- and R" = H; as well as the compound in which -CHR-CHR'- is replaced by -(CH₂ )₃-and the ring opened bis-diacid diamide compound having R = CH₃ and R' = H) all showed either no protective activity or only minimal protective activity.

It is the case that all of the bis-dioxopiperazines identified in this paper as exhibiting a useful level of cardioprotection against daunorubicin toxicity are cytotoxic. We have now found that, despite the Indications to the contrary in the paper, various bis-dioxopiperazines which are substantially non-cytotoxic are of interest as cardioprotective agents, as well as in other roles outside the area of lead poisoning. These non-cytotoxic

bis-dioxopiperazines are of interest for providing protection against the cardlotoxic effects of various anthracycHne drugs but particularly doxorublcin (adriamydn). In this context it is relevant that, in addition to the comments in the Herman et al

Research Communications in Chemical Pathology and Pharmacology paper, it is indicated by Herman et al in Advances in Pharmacology and Chemotherapy, 1982, 19, 249 that even the cardioprotective compound ICRF 159, which is the d1 isomer of the compound of formula (I) having R = CH₃ and R = R¹ = H, is consistently more effective in reducing high dose daunorubicin toxicity than

doxorubicin toxicity. Accordingly the present I nvention compri ses the use of a compound of formula (II) :

wherein n is 0, 1 or 2, R₁ , R₂, R₃ and R₄ are each separately selected from hydrogen, unsubstituted acyclic aliphatic hydrocarbon groups having a maximum of six carbon atoms and C_1-6 alkyl groups substituted by a hydroxy group or by a C_1-6 alkoxy group, or one of R₁ and R₂ and one of R₃ and R₄ is hydrogen and the others together form a trimethylene, tetramethylene or pentamethylene bridging group, R₅ is hydrogen or an unsubstituted acyclic aliphatic hydrocarbon group having a maximum of six carbon atoms, and R₆ is an acyclic aliphatic hydrocarbon group having a maximum of six carbon atoms or a group CH₂R₇ in which R₇ is a C_1-5 alkyl group substituted by a hydroxy group or by a C_1-6 alkoxy group, or a salt thereof formed with a physiologically acceptable inorganic or organic acid, for the manufacture of a medicament for use as a cardioprotective agent.

In the compounds (II) of the present invention the central grouping in the molecule has the form:

Although n may be 2 or more especially 1, each of R₁ to R₄ then conveniently being hydrogen, 1t 1s preferred that n is 0, in which latter case the grouping will be of the form:

As indicated, R₁, R₂, R₃ and R₄ may be hydrogen, an unsubstituted acyclic C_1-6 aliphatic hydrocarbon group or a C_1-6 alkyl group substituted by a hydroxy group or by a C_1-6 alkoxy group. The term acyclic aliphatic hydrocarbon group is used herein to include both branched and especially straight chain groups. The group may be unsaturated or especially saturated, conveniently containing one double or triple bond in the former case. Thus, in particular, unsubstituted groups may be alkenyl, alkynyl and particularly alkyl groups, which may be straight or branched chain. The aliphatic hydrocarbon groups conveniently contain a maximum of four or especially three carbon atoms, preferred groups therefore being C₁-C₄ or C₁-C₃ alkyl groups and C₂-C₄ or C₂-C₃ alkenyl and alkynyl groups.

As regards the substituted C_1-6 alkyl groups, these may be branched or especially straight chain alkyl groups substituted, particularly terminally, by a hydroxy group or particularly an alkoxy group. Conveniently the alkyl groups are of 1 to 3 or 1 to 4 carbon atoms, substituted ethyl and particularly substituted methyl groups being of most interest. Preferred alkoxy group substituents similarly contain 1 to 3 or 1 to 4 carbon atoms with ethoxy and particularly methoxy groups being of most Interest. Conveniently the total number of carbon atoms in such an alkoxyalkyl group is from 2 to 6, particularly 2 to 4 and especially 2 or 3. Examples of specific substituted groups R₁, R₂, R₃ and R₄ are the groups hydroxymethyl, 2-hydroxyethyl and methoxymethyl. Preferably no more than two of R₁ to R₄, for example R₁ and R₃ or R₂ and R4, and especially only one is a substituted alkyl group and when this Is the case the others of R₁ to R₄ are conveniently hydrogen.

It is generally preferred, however, that where they do not constitute trimethylene, tetramethylene or pentamethylene bridging groups the groups R₁, R₂, R₃ and R4 are selected from hydrogen and unsubstituted acyclic aliphatic hydrocarbon groups, especially from the group consisting of hydrogen, methyl, ethyl, n-propyl,

isopropyl, allyl and propargyl. Preferably R₁ is hydrogen and conveniently either R₂ is also hydrogen but R₃ and R₄ are not or, more usually, R₃ is also hydrogen whilst R₂ and R₄ are either hydrogen or not, for example conveniently being selected from the whole group specified above. Also of some particular interest, however, are compounds 1n which one of R₁ and R₂ and one of R₃ and R₄ is hydrogen and the others together form a bridging group, especially a tetramethylene group. Examples of compounds of particular Interest are those in which n is 0, R₁ and R₂ are each hydrogen and R₃ and R₄ are each methyl, or more particularly n is 0, R₁ and R₃ are each hydrogen and (a) R₂ is hydrogen and R4 is hydrogen, methyl, ethyl, n-propyl, Isopropyl, allyl or propargyl, (b) R₂ is methyl or ethyl and R₄ is methyl, ethyl, n-propyl, isopropyl, allyl or propargyl, or (c) R₂ and R₄ together form a tetramethylene bridging group.

Specific combinations of particular Interest are n « 0,

R₁ = R₂ = H, R₃ = R₄ = CH₃; n = 0, R₁ = R₂ = R₃ = H, R₄ = CH₂OH or CH₂OCH₃; n = 0, R₁ = R₃ = H, R₂ = R₄ = CH₃; n = 0, R₁ = R3 = H, n = 0, R₁ = R₂ = R₃ = H, R₄ = CH₃ or C₂H₅; particularly n = 0, R₁ = R₂ = R₃ = R₄ = H; and especially R₂ + R₄ = CH₂CH₂CH₂CH₂.

As regards the groups R₅ one preference is for these to be hydrogen but where this is not the case the groups may conveniently be a C_1-4 aliphatic hydrocarbon group, particularly a C_1-4 or C_1-3 alkyl group or, to a lesser extent, a C_2-4 or C_2-3 alkenyl or alkynyl group, an ethyl or especially a methyl group being of most Interest and constituting another preference. When R₅ is other than hydrogen each of R₁, R₂, R₃ and R₄ is preferably hydrogen or alternatively R₁, R₂ and R₃ are each hydrogen and R₄ is ethyl or especially methyl.

As regards the group Rg this is preferably selected from unsubstituted aliphatic hydrocarbon groups, the detailed

preferences among such groups being as described hereinbefore for the groups R₁ to R₄. Thus groups R₆ which are alkyl groups, particularly C_1-4 or C_1-3 alkyl groups, for example ethyl or particularly methyl are preferred, and where R_g is unsaturated It preferably has the form CH₂R₈ where R₈ is a C_2-5 alkenyl or alkynyl group, preferably a C₂ or C₃ group, for example CH₂R₈ being allyl or propargyl. As regards the groups R₆ of the form CH₂R₇, these may be branched or especially straight chain alkyl groups substituted, particularly terminally, by a hydroxy group or particularly by an alkoxy group. Conveniently the groups R7 are substituted alkyl groups of 1 to 2 or 1 to 3 carbon atoms, substituted ethyl and artlcularly substituted methyl groups being of most Interest.

Preferred alkoxy group substltuents similarly contain 1 to 3 or 1 to 4 carbon atoms with ethoxy and particularly methoxy groups being of most Interest. Conveniently the total number of carbon atoms in such an alkoxyalkyl group CH₂R₇ is from 3 to 6, especially 3 or 4.

Compounds of particular Interest are those in which Rg is selected from 2-hydroxyethyl, 2-methoxyethyl, particularly ethyl, n-propyl, isopropyl, and especially methyl, for example together with the combinations of R₁ to R₄ Indicated as preferred.

Specific preferred compounds according to the present invention are those having the specific combinations of n, R₁, R₂, R₃ and R₄ as Indicated previously, together with R₅ being ethyl, particularly methyl or especially hydrogen and with R₆ being ethyl or especially methyl. Among these compounds may particularly be mentioned those in which n = 0, R₁ = R₃ = H, R₂ = R₄ = CH₃, R₅ = H, R₆ = CH₂CH₂OH, particularly C₂H₅ or especially CH₃; n = 0, R₁ = R₂ = R₃ - R₅ = H, R₄ = CH₃ or especially C₂H₅, R₆ = CH₂CH₂OH, particularly C₂H₅ or especially CH₃; n = 0, R₁ = R₂ = R₃ = R₄ = H, R₅ = CH₃, C₂H₅ or especially H, and R₆ - CH₂CH₂OH, particularly C₂H₅ or

especially CH₃; and particularly n = 0, R₁ = R₃ = R₅ = H,

R₂ + R₄ = CH₂CH₂CH₂CH₂; R₆ = C₂H₅ or especially CH₃ or CH₂CH₂OH. Enhanced water solubility will result from the presence of hydroxyalkyl groups in the molecule, for example in compounds having R₆ = CH₂CH₂OH and also in compounds having R₄ = CH₂OH, for example in the latter instance with n = 0, R₁ = R₂ = R₃ = H, R₅ = H, CH₃ or C₂H₅ and R₆ = CH₃ or C₂H₅. The compounds may exist in various stereochemlcal forms, each of which is included by _the present invention. Thus, when neither of the groupings -CR₁R₂- and CR₃R₄- is -CH₂- the possibility of geometrical isomerlsm exists (the compounds being in the dl or threo configuration or alternatively in the meso or ervthro

configuration when n = 0). Moreover, compounds in which two of R₁ to R₄ provide a bridging group may be in the cis or trans form. It will also be appreciated that when the bridging grouping

-(CH₂)_n-CR₁R₂-CR₃R₄- does not have a centre of symmetry, the compounds can exist in enant1omorphic d and l forms. In addition, when R₅ is not hydrogen this will lead to the presence of two identically substituted asymmetric carbon atoms in the molecule with the possible stereochemlcal variations this produces. The invention includes the use of the various different isomers of the compounds. In some cases the optically active d- and l-1somers may have the advantage of significantly higher water solubility than the corresponding racemate and it may also be the case that the biological activity of the compound will differ as between the isomers. The invention does therefore extend to the use of such compounds not only as the dl-racemate but also in a form in which the amount of the compound in either the d or l configuration is greater than that in the 1 or d configuration, respectively

(Including amounts in that configuration present in the dl racemate). In particular the compound may be essentially in the form of the d or l isomer, for example being substantially free from (I.e. containing no more than 20% and conveniently no more than 10% of) the dl and l or dl and d isomers. However, where the advantage lies in enhanced solubility of the optically active Isomers compared with the racemate, rather than enhanced biological activity for one isomer, any enhancement of the proportion of one isomer should have some effect.

Of the specific compounds previously indicated as being particularly preferred (as the free compound or a salt),

l,3-bis(4-methyl-3,5-dioxopiperazin-l-yl)propane and

1,2-bis(4-methyl-3,5-dioxopiperazin-l-yl)ethane do not exist in optically active forms but 1,2-bis(4-methyl-3,5-dioxopiperazinl-yl)propane and 1,2-bis(4-methyl-3,5-dioxopiperazin-l-yl)butane and 1,2-bis(4-methyl-3,5-dioxopiperazin-l-yl)cyclohexane can do so. Compounds In which R₅ is methyl, for example the analogues of the four compounds just mentioned, can also exist in optically active forms.

It will be appreciated that certain of the compounds of formula (II) as defined hereinbefore are novel and that the present Invention extends to such compounds per se.

The compounds of formula (II) may conveniently be prepared by reaction of the corresponding tetra-acetic add of formula (III) with an N-subst1tuted formamide in which the nitrogen atom is substituted by a group R₆, this reaction being carrried out in an excess of the substituted formamide as the solvent where convenient and at an elevated temperature, preferably under nitrogen.

The tetra-acetic adds of formula (III) may conveniently be obtained from the corresponding dicarboxy acid of formula (IV).

Examples of such procedures and/or of intermediates of use in such procedures are to be found in UK patents 966,802, 978,724, 1,234,935 and 1,374,979, and in German Offenlegungschrift 2,511,891.

Moreover, Intermediates of formula (III) in which R₅ is other than hydrogen are described in UK patent 723,316. In general terms, a process for the preparation of a compound of formula (II) as defined hereinbefore and salts thereof formed with a physiologically acceptable inorganic or organic add comprises reacting a tetra-acetic add of formula (III)

1n which n has the same value as in the compound of formula (II) and R₁ to R₅ represent groups as in the compound of formula (II);

(i) as the tetra-acetic acid or as its bis anhydride by heating with an add amide of the general formula RCONH₆ in which R represents hydrogen, an aliphatic residue, for example an alkyl group, having one to four carbon atoms, or an amino group NH₂; or (ii) as the tetra-acetic add or as its bis anhydride, tetra hallde, for example the add chloride or bromide, or tetra ester, for example one formed with a lower aliphatic alcohol (particularly C_1-4 or a phenol, with an amlne of the general formula

H₂N-R₆

1n which R₆ represents a group as 1n the compound of formula (II); and, where appropriate, separating the required compound (II) from other Isomers thereof and/or converting the compound (II) to a physiologically acceptable addition salt thereof.

The preferred procedure is (i), particularly as described hereinbefore.

To obtain compounds (II) of the desired stereochemistry it is most convenient to use an intermediate compound having the equivalent stereochemistry, particularly as regards the preparation of geometrical Isomers, for example compounds in the dl or threo form as opposed to the meso or erythro form, but conveniently also as regards the preparation of optically active compounds. Thus the tetra-acetic add which is used conveniently has the same stereochemistry as is desired in the final product. When a d or l isomer is required rather than the dl racemate, however, an alternative to the preferred utilisation of a d or l intermediate compound is to effect a resolution of the compound (II), for example using an appropriate optically active acid to form a mixture of salts of the d and l forms of the compound (II) which are then separated.

Only certain of the compounds (II) have previously been

Indicated in the literature as being of use In therapy.

Accordingly the present invention further comprises a compound of formula (II) where this is not the case, for use in therapy.

The present invention also includes pharmaceutical compositions comprising as an active component such compounds of formula (II) where this is not the case, together with a physiologically acceptable diluent or carrier. In general, compositions containing the compounds of formula (II) which are of particular interest are those in which the diluent or carrier excludes any liquid which is not sterile and pyrogen free.

As Indicated, the compounds (II) may be formulated as salts formed with physiologically acceptable inorganic or organic adds and, when so formulated, it is preferred to use methane sulphonic acid, isethionic acid, tartaric add or another solubillsing acid.

The compounds of formula (II) may be formulated singly, or as a mixture of two or more compounds, for use as pharmaceuticals by a variety of methods. For instance, they may be applied as aqueous, oily (e.g. as a suspension in isopropyl myristate), or in some cases emulsified compositions for parenteral administration and therefore preferably sterile and pyrogen-free. Some of these compounds have rather low solubility In aqueous media and are therefore usually administered in the form of aqueous suspensions containing suitable surface active agents. It will be appreciated that the dosage levels used may vary over quite a wide range especially since certain of the compounds (II) are more active than others. However, without commitment to a rigid definition of dosages it may be stated that a daily dosage of active constituent (estimated as the free base), divided if necessary, of from 10 mg to 3 g is proposed for parenteral mammalian use. This dosage may conveniently be applied as a solution in 500-1000 ml of liquid for intravenous injection by slow Infusion, or as a solution or suspension in about 10 ml of liquid by the intramuscular route, or in small volumes subcutaneously. (Parenteral, particularly intravenous, administration is the route preferred for use in conjunction with the anthracycline drugs so that Injectable compositions are of especial Interest.) More particularly, with many compounds (II) the dally dose for a 70 kg human, administered parenterally, will often be in the range from about 100 mg to about 500 mg but with the more active compounds it may be less than this (the dose being varied pro rata for humans of a different weight or other mammals). When used in conjunction with an anthracycline drug, where a single administration of the drug and the compound (II) Is common, however, higher doses than this may often be employed, for example between about 500 mg and about 3 g, with doses of more than this being considered where appropriate in terms of the ratios of compound (II):anthracycline drug as discussed hereinafter.

Where appropriate, the substances may also be compounded for oral administration in dosages which may be similar but may often be somewhat higher, for example in a range from 100 mg to 1 g or even as high as 3 g for the daily dose for a 70 kg human for many compounds (II) but possibly somewhat less than this for the more active compounds. Such oral formulations may particularly take the form of tablets compounded in the presence of conventional solid carrier materials such as starch, lactose, dextrin and magnesium stearate, or of capsules or cachets. Suppositories, pessaries, aerosol and other formulations may also be employed. The compounds may be formulated in unit dosage form, i.e. in discrete portions each containing a unit dose, or a multiple or sub-multiple of a unit dose of the active Ingredient.

The compounds (II) as defined hereinbefore are primarily of value as cardioprotective agents and it should be noted that their potential in such a use extends not only to use in conjunction with drugs having a cardlotoxic side effect, these often being cytotoxlc agents such as the anthracycline drugs, which are of particular value in treating breast cancer, but also extends to pathological conditions where the heart is at risk. The term "anthracycline drug" is used herein to include not only natural and semi-synthetic anthracyclines such as eplrubldn, idarubicin, daunorubicin and especially doxorubidn (which names are used herein to include salts of these compounds), but also synthetic anthracyclines such as mitoxantrone. Indeed, the compounds (II) are of value in providing cardloprotectlon against the cardiotoxic side effect of various compounds containing a moiety

the toxic effect of such compounds being believed to derive from their chelating ability.

The compounds (II) also find a secondary use in protection against other toxic effects arising from natural diseases or induction by drugs, for example by various agents which are either toxic as such or when present in the body in excess, such agents including paracetamol (p-hydroxyacetanH1de).

The compounds (II) find most application in the treatment of humans although they can find veterinary use in certain other mammals such as dogs, rabbits, cattle, and horses.

When used as a cardioprotective agent in the context of a pathological condition where the heart is at risk as a result of that condition the compounds (II) are administered for a period dictated by the existence of this condition. When used in a cardioprotective role 1n conjunction with a drug having a

cardiotoxic side effect, the period of administration will be related to that of the use of the drug which will usually be administered at normal dosage rates and by the usual regimen, often parenterally. The compounds (II) may conveniently be administered before, together with or less often after the drug, the choice depending to some extent on the particular drug In question. In the first and third usages both the compound (II) and the drug will each be formulated separately, usually in a conventional manner, for example both being formulated as described above, although the two compositions may be packaged together for ease of sequential administration to the patient. A suitable time lapse between administration of the compound (II) and the drug in either order is quite short, being no more than about 1 to 4 hours, for

example 2 hours, and particularly being about 1 hour or somewhat less, depending on the drug In question.

When the compound (II) is administered together with the drug, the two may be formulated separately but it may be preferred to include the compound (II) and the drug in the same composition. Such a pharmaceutical composition may again conveniently take one of the forms described above for compositions containing only the compound (II) and may, if desired, contain more than one

compound (II) and/or more than one drug. The present invention thus includes (a) a pharmaceutical composition which comprises a compound of formula (II), as defined hereinbefore, and a drug having a cardiotoxic or other toxic side effect, for example an anthracycline drug, together with a physiologically acceptable diluent or carrier, and also (b) a kit comprising in association a compound of formula (II), as defined hereinbefore, and a drug having cardiotoxic or other toxic side effect.

As indicated, the compounds (II) are of particular interest for use with doxorubidn and the present invention therefore

particularly includes a pharmaceutical composition comprising a compound of formula (II) as defined hereinbefore and doxorubidn, together with a physiologically acceptable diluent or carrier.

In instances where a series of doses of the drug is administered it may not be necessary for each administration of the drug to be made concomltantly with, or at the Interval given above after or before the administration of the compound (II). It may be possible to administer the compound (II) alone or together with the drug, followed by one or more repeated spaced doses of the drug alone or, more often, in view of the more rapid metabollsation of the compound (II), to administer the drug alone or together with the compound (II), followed by one or more repeated spaced doses of the compound (II) alone. If the treatment with the drug is continued over an extended period repeat doses of the compound (II) are also likely to be required and one possible regimen would involve the administration of the drug and compound (II) together on certain occasions followed by the compound (II) alone on others.

As regards the relative amounts of the compound (II) and a drug to be used, this will depend on both the particular compound (II), the drug used and the regimen of use, a good Indication being provided, however, by the dosages indicated hereinbefore for the compounds (II) and the conventional doses used for the drug.

However, some additional comments may be made concerning the proportions of compound or compounds (II) to anthracycline drug which are used either singly or together in a pharmaceutical composition containing both a compound or compounds (II) and an anthracycline drug. Thus, by way of guidance it may be stated that a dose ratio of between 5:1 to 20:1 or even 25:1 w/w of compound or compounds (II) to drug, especially about 10:1 w/w, is often suitable. By way of further guidance, it may be mentioned that a normal single dosage of doxorubidn is in the range of about 0.75 to 2 mg/kg, i.e. about 50 to 150 mg for a 70 kg human being, but that the use of the compounds (II) is Intended to enable some increase in the dosage, for example to 4 or 5 mg/kg, if desired, in order to enhance the anti-cancer effect of the doxorubidn whilst its cardiotoxic side effects are controlled by the presence of the compound (II).

The exact dosage of an anthracycline drug such as doxorubidn which is used will depend on whether It is given with other anti-tumour agents. Thus anthracycline drugs are often given together with one or more of other such agents, for example fluorouradl and cyclophosphamide and where desired, a

pharmaceutical composition containing a compound or compounds (II) and an anthracycline drug can contain other such anti-tumour, agents. Moreover, it may be advantageous to administer a calcium supplement together with the compounds (II), this usually being administered separately. When used as a protective agent against the toxic effect of paracetamol, the compounds (II) may be used protectively before occurrence of the toxidty or following occurrence of the

toxicity. It may even be possible to formulate the compound (II) with paracetamol in order automatically to counter the effect of an overdose thereof. Broadly similar dosage levels may be used to those described hereinbefore although where the toxic effect is acute, as for example is usually the case following an overdose of paracetamol, higher dosages over a shorter period may be indicated.

Other forms of protection include the use of the compounds (II) in conjunction with any condition which is either "naturally occurring" or drug induced where free radical damage occurs (this may also be involved in some of the conditions described

hereinbefore such as a anthracycline drug-Induced damage), for example in reducing the dlabetogenlc effect of drugs such as alloxan which generate free hydroxyl radicals. The compounds (II) may once again be used in a broadly similar manner as when employed in cardloprotectlon, Including formulation together with the drug, and the dosage levels used.

The present Invention thus includes a method of providing protection against a toxic effect on the body, particularly a cardiotoxic effect, which comprises administering to a patient in need thereof a therapeutically effective amount of a compound (II) as defined hereinbefore.

The present invention is illustrated by the following Examples.

EXAMPLES

Example 1 : Preparation of 1,2-bis(4-methyl-3.5-dioxopiperazin-l-yl)ethane

1,2-Diaminoethane tetra-acetic add (20 g) and

N-methylformamide (50 ml) are heated together in an atmosphere of nitrogen at 160-170°C for 8 hours. The reaction mixture is cooled, filtered and the collected solid is washed with cold isopropanol and then petroleum ether (b.p. 60-80°C) to give

1,2-bis(4-methyl-3,5-dioxopiperazin-l-yl)ethane (16.5 g, 85%), m.p. 180-182°C, v_max (nujol) 1675, -720, 3350 cm^-1. Example 2 : Preparation of 1,2-bis(4-methyl-3.5-dioxopiperazln-1-yl)propane

(+)-, (-)-(R) and (+)-(S)-1,2-Diaminopropane tetra-acetic acid are each reacted with N-methylformamide and the reaction mixture is worked up In a similar manner to that described in Example 1. The resultant products are respectively (±)-1,2-bis(4-methyl-3,5-dioxoplperazin-1-yl)-propane obtained in 27% yield, m.p. 156-158°C from aqueous ethanol, (-)-(R)-1,2-bis(4-methyl-3,5-dioxopiperazin-1-yl)- propane obtained in 81% yield, m.p. 132-133°C from methanol, and (+)-(S)-1,2-bis(4-methyl-3,5-dioxopiperazin-1-yl)-propane obtained in 48% yield, m.p. 132-133°C from methanol, [α]^D ₂₆ = -9.4 in dimethylformamide. The solubilities of the two isomers in water isomers in water at 25°C are about 10 mg/ml for the (±)isomer and about 40 mg/ml for the (-)-(R) and (+)-(S) isomers. Example 3 : Preparation of meso-2,3-bis(4-mgthy1-3,5-dioxopiperazin-1-yl)butane

meso-2,3-Diaminobutane tetra-acetic acid is reacted with

N-methyl formamide and the reaction mixture is worked up in a similar manner to that described in Example 1 to give

meso-2,3-bis(4-methyl-3,5-dioxopiperazin-1-yl)-butane in 55% yield, m.p. 262-265°C from N-methylformamide.

Example 4 : Preparation of d1-trans-1,2-bis(4-methyl-3.5-dioxopiperazin-l-y1)cyclohexane

dl-trans-1,2-Diaminocyclohexane tetra-acetic acid (20 g)

(prepared essentially as described in Example 14 of U.S.

Patent 3,196,153) is heated with freshly distilled methylformamide (100 ml) under nitrogen at 150-160°C for 6 hours. The reaction mixture is then evaporated under reduced pressure to remove the excess methylformamide and the residue is taken up in hot methanol (50 ml). The solid obtained on cooling is recrystallised from ethanol to give d1-trans-1,2-bis(4-methyl-3,5-dioxopiperazin-1-yl)-cyclohexane (6.2 g, 33%), m.p. 145-147°C. Example 5 : Formulation of compounds

(A) Tablets of the following composition are prepared: mg/tablet Compound of Example 1 (micronlsed) 250 'AvlceT (mlcrocrystal line cellulose)* 38 polyvinylpyrrolidone 3 alglnic add 6 magnesium stearate 3

The compound of Example 1 is mixed with 'Avlcel 'and

polyvinylpyrrolidone is added, dissolved in sufficient industrial methylated spirits (74° OP) to produce a mass suitable for granulating. The mass Is granulated through a 20 mesh sieve and the resultant granules are dried at a temperature not

exceeding 50°C. The dried granules are passed through a 20 mesh sieve and the alglnic acid and magnesium stearate are then added and mixed with the granules. The product is compressed into tablets each weighing 300 mg on 3/8 inch flat bevelled edge divided punches.

(B) Tablets of the following composition are prepared: mg/tablet Compound of Example 1 (micronlsed) 250

'Avlcel' (mlcrocrystal line cellulose) 134 polyvinylpyrrolidone 4 alglnic add 8 magnesium stearate 4

The tablets are prepared by essentially the same procedure as described in (A) and are compressed at a tablet weight of 400 mg on 7/16 Inch flat bevelled edge punches.

* 'Avlcel' is a Registered Trade Mark or Service Mark. (C) Tablets of the following composition are prepared: mg/tablet Compound of Example 1 (micronlsed) 250 lactose (300 mesh) 19 maize starch 15 gelatine 10 magnesium stearate 6

The tablets are prepared by mixing the compound of Example 1 with lactose and half the total quantity of maize starch required, and adding to the mass a 5% solution of gelatine in water. The product is granulated through a 16 mesh sieve, and the resultant granules are dried to constant weight at a temperature not exceeding 50°C. The dried granules are passed through a 20 mesh sieve and mixed with magnesium stearate and the remainder of the maize starch. The product is compressed at a 300 mg tablet weight on 3/8 inch flat bevelled edge divided punches.

Similar procedures may be followed with the compounds of Examples 2, 3 and 4.

Example 6 : Cardioprotective effect of trans-1.2-bis(4-methyl-3.5-dioxopiperazin-l-yl)cyclohexane against the toxicity of doxorubidn The experiments were conducted as follows.

Animals

Mature male Sprague Dawley rats, 13-14 weeks old, weighing 400-500 gm were used. The animals were caged in groups of three and were kept in a constant environment. They were fed 41-B cubed diet and water M libitum. The animal house was maintained in an alternating 12-hour wake-sleep cycle.

Drug administration

The cardioprotective activity of tranns-1,2-bis(4-methyl-3,5-dioxopiperazin-l-yl)cyclohexane (hereinafter referred to as the compound) was studied in the rats. The compound was administrred as a saline solution. The solution was prepared shortly before use and was administered at a constant injection volume of 1 ml per 100 gm body weight. Doxorubidn (Farmitalia, Milan, Italy) was dissolved in sterile water (2 mg/ml) before injection.

A group of 8 animals received a combined treatment of the compound and doxorubidn. The compound was administered to animals as a single intraperitoneal (i.p.) injection into the lower right quadrant of the abdomen to avoid damge to vital organs. An hour after the administration of the compound, the animals received a single intravenous (i.v.) injection of doxorubidn (4 mg/kg), via the femoral vein. A group of 10 animals received an i.p. injection of saline (1 ml/100 gm) followed by an i.v. injection of saline (0.2 ml/100 gm) one hour later. These rats acted as control animals. Nine animals received an i.p. injection of saline one hour before an i.v. administration of doxorubidn (4 mg/kg). To minimise the effects of circadian timing, doxorubidn administration was carried out between 13.30 h and 15.30 h. During intravenous drug administration and for subsequent cardiac output measurements, animals were anaesthetised with choral hydrate (300 mg/kg).

Assessment of toxidty

The protective activity of the compound against doxorublcininduced cardlotoxidty 1n the rats was assessed at 8 weeks by measuring the cardiac output in animals receiving the treatment, using an external counting technique wherein a radioactive tracer, technetium (^99mTcO₄) was injected as a bolus into the femoral vein of an anaesthetised animal. The activity time curve over the heart was recorded at 0.1 second Intervals for 40 seconds, using an Nal detector connected to a multi-channel analyzer (ND-62, Nuclear Data). ECG and heart rate were determined concomitantly with the cardiac output measurement, using a human ECG monitor (Hewlett Packard 7830A) coupled to a scope memory (model VK-12-2, Seltek Instrument Ltd.) and a chart recorder.

Gross post-mortem examinations were carried out on the animal that died during the study as well as those killed at the end of experiment (8 weeks).

In this study statistical differences between group means were analysed using Student's t-test. Results

The results are summarised in the Table.

All animals showed a transient reduction 1n body weight in the first three weeks aftrer treatment. Animals receiving doxorubidn and pretreated with saline or the compound all showed a >10% reduction in body weight. Control animals, i.e. those receiving saline (i.p.) and saline (i.v.), showed a 3% reduction in body weight over this period; significantly less than animals receiving saline and doxorubidn (p<0.001).

Measurement in control animals at 8 weeks showed a mean cardiac output value of 222.4 ± 5.8 ml/min/kg and a mean heart rate of 441 ± 7 beats per minute. All control animals survived the course of study of 8 weeks. Animals receiving saline and doxorubidn showed a 60% reduction in cardiac output and a 35% reduction in heart rate. In this group of animals, one animal died just before 8 weeks. Information concerning this animal was loss due to severe post-mortem changes.

(1) CHF = total incidence of congestive heart failure over the

course of study of 8 weeks. Symptoms of congestive heart failure are defined by the appearance of general subcutaneous oedema, ascites and/or pleural effusion.

(2) Animal died just before 8 weeks. Information on this animal lost due to severe post-mortem changes.

Claims

1. The use of a compound of formula (II) :

wherein n 1s 0, 1 or 2, R₁, R₂, R₃ and R₄ are each separately selected from hydrogen, unsubstituted acyclic aliphatic hydrocarbon groups having a maximum of six carbon atoms and C_1-6 alkyl groups substituted by a hydroxy group or by a C_1-6 alkoxy group, or one of R₁ and R₂ and one of R₃ and R₄ is hydrogen and the others together form a trimethylene, tetramethylene or pentamethylene bridging group, R₅ is hydrogen or an unsubstituted acyclic aliphatic hydrocarbon group having a maximum of six carbon atoms, and R₆ is an acyclic aliphatic hydrocarbon group having a maximum of six carbon atoms or a group CH₂R₇ in which R₇ is a C_1-5 alkyl group substituted by a hydroxy group or by a C_1-6 alkoxy group, or a salt thereof formed with a physiologically acceptable inorganic or organic add, for the manufacture of a medicament for use as a protective agent in (a) cardloprotectlon, (b) protection against the toxic effects of paracetamol or (c) protection against damage caused by free radicals.

2. The use of a compound of formula (II): 6

wherein n is 0, 1 or 2, R₁, R₂, R₃ and R₄ are each separately selected from hydrogen, unsubstituted acyclic aliphatic hydrocarbon groups having a maximum of six carbon atoms and C_1-6 alkyl groups substituted by a hydroxy group or by a C_1-6 alkoxy group, or one of R₁ and R₂ and one of R₃ and R₄ is hydrogen and the others together form a trimethylene, tetramethylene or pentamethylene bridging group, R₅ is hydrogen or an unsubstituted acyclic aliphatic hydrocarbon group having a maximum of six carbon atoms, and R₆ is an acyclic aliphatic hydrocarbon group having a maximum of six carbon atoms or a group CH₂R₇ in which R₇ is a C_1-5 alkyl group substituted by a hydroxy group or by a C_1-6 alkoxy group, or a salt thereof formed with a physiologically acceptable inorganic or organic add, for the manufacture of a medicament for use as a cardioprotective agent.

3. The use according to Claim 1 or 2, in which R₁, R₂, R₃ and R4 are each separately selected from hydrogen and C_1-4 alkyl,

C_2-4 alkenyl and C_2-4 alkynyl groups or one of R₁ and R₂ and one of R₃ and R₄ is hydrogen and the others are a trimethylene,

tetramethylene or pentamethylene bridging group.

4. The use according to Claim 3, in which R₁, R₂, R₃ and R₄ are each separately selected from hydrogen, methyl, ethyl, n-propyl, isopropyl, allyl and propargyl or one of R₁ and R₂ and one of R₃ and R₄ is hydrogen and the others are tetramethylene.

5. The use according to any of Claims 1 to 4, in which n is 0.

6. The use according to Claim 1 or 2, 1n which n 1s 0, R₁ and R₂ are each hydrogen and R₃ and R₄ are each methyl, or n is O, R₁ and R₃ are each hydrogen and (a) R₂ is hydrogen and R₄ is hydrogen, methyl, ethyl, n-propyl, isopropyl, allyl or propargyl or (b) R₂ is methyl or ethyl and R₄ is methyl, ethyl, n-propyl, isopropyl, allyl or propargyl.

7. The use according to Claim 1 or 2, in which n = 0, R₁ = R₂ = H, R₃ = R₄ = CH₃; n = O, R₁ = R₂ = R₃ = H, R₄ = CH₂OH or CH₂OCH₃; n = O, R₁ = R₃ = H, R₂ + R₄ = CH₂CH₂CH₂CH₂;

n = O, R₁ = R₂ = R₃ = H, R₄ = CH₃ or C₂H₅; or

n = O, R₁ = R₂ = R₃ = R₄ = H.

8. The use according to any of Claims 1 to 7, in which R₅ is hydrogen.

9. The use according to any of Claims 1 to 7, in which R₅ is methyl or ethyl.

10. The use according to any of Claims 1 to 9, in which R₆ is a C_1-4 alkyl group, a group CH₂R₈ wherein R₈ is a C₂ or C₃ alkenyl or alkynyl group, or a group CH₂R₇ wherein R₇ is a C₁ or C₂ alkyl group substituted by a hydroxy, methoxy or ethoxy group.

11. The use according to Claim 10, 1n which Rg 1s methyl, ethyl, n-propyl, isopropyl, 2-hydroxyethyl or 2-methoxyethyl.

12. The use according to Claim 1, in which n = 0, R₁ - R₃ = H, R₂ = R₄ = CH₃, R₅ = H, R₆ = CH₃, C₂H₅ or CH₂CH₂OH;

n = 0, R₁ = R₃ = H, R₂ + R₄ = CH₂CH₂CH₂CH₂, R₅ = H, R₆ = CH₃, C₂H₅ or CH₂CH₂OH; n = 0, R₁ = R₂ = R₃ = R₅ = H, R₄ = CH₃ or C₂H₅, Rg = CH₃, C₂H₅ or CH₂CH₂OH; or n = 0, R₁ = R₂ = R₃ = R₄ = H, R₅ = H, R₆ = CH₃, C₂H₅ or CH₂CH₂OH.

13. The use according to Claim 1 or 2, 1n which the compound (II) is 1,2-bis(4-methyl-3,5-dioxopiperazin-1-yl)ethane,

1,2-bis(4-methyl-3,5-dioxopiperazin-1-yl)propane, or

1,2-bis(4-methyl-3,5-dioxopiperazin-1-yl)butane.

14. The use according to Claim 1 or 2, 1n which the compound (II) Is 1,2-bis(4-methyl-3,5-dioxopiperazin-1-yl)cyclohexane.

15. The use according to any of Claims 1 to 14, in which the compound is used in conjunction with an anthracycline drug.

16. The use according to Claim 15, in which the anthracycline drug 1s doxorubidn.

17. A pharmaceutical composition comprising a compound of

formula (II):

wherein n is 0, 1 or 2, R₁, R₂, R₃ and R₄ are each separately selected from hydrogen, unsubstituted acyclic aliphatic hydrocarbon groups having a maximum of six carbon atoms and C_1-6 alkyl groups substituted by a hydroxy group or by a C_1-6 alkoxy group, or one of R₁ and R₂ and one of R₃ and R₄ is hydrogen and the others together form a trimethylene, tetramethylene or pentamethylene bridging group, R₅ is hydrogen or an unsubstituted acyclic aliphatic hydrocarbon group having a maximum of six carbon atoms, and R₆ is an acyclic aliphatic hydrocarbon group having a maximum of six carbon atoms or a group CH₂R₇ in which R₇ is a C_1-5 alkyl group substituted by a hydroxy group or by a C_1-6 alkoxy group, or a salt thereof formed with a physiologically acceptable Inorganic or organic add, and an anthracycline drug, together with a

physiologically acceptable diluent or carrier.

18. A pharmaceutical composition according to Claim 17, in which the anthracycline drug is doxorubidn.

19. A pharmaceutical composition according to Claim 17 or 18 in a form suitable for parenteral administration.

20. The use of a compound of formula (II):

wherein n is 0, 1 or 2, R₁, R₂, R₃ and R₄ are each separately selected from hydrogen, unsubstituted acyclic aliphatic hydrocarbon groups having a maximum of six carbon atoms and C_1-6 alkyl groups substituted by a hydroxy group or by a C_1-6 alkoxy group, or one of R₁ and R₂ and one of R₃ and R₄ is hydrogen and the others together form a trimethylene, tetramethylene or pentamethylene bridging group, R₅ is hydrogen or an unsubstituted acyclic aliphatic hydrocarbon group having a maximum of six carbon atoms, and R₆ is an acyclic aliphatic hydrocarbon group having a maximum of six carbon atoms or a group CH₂R₇ in which R₇ is a C_1-5 alkyl group substituted by a hydroxy group or by a C_1-6 alkoxy group, or a salt thereof formed with a physiologically acceptable inorganic or organic add, for the manufacture of a medicament for providing protection against the toxic effects of paracetamol.

21. The use of a compound of formula (II):

wherein n is 0, 1 or 2, R₁, R₂, R₃ and R₄ are each separately selected from hydrogen, unsubstituted acyclic aliphatic hydrocarbon groups having a maximum of six carbon atoms and C_1-6 alkyl groups substituted by a hydroxy group or by a C_1-6 alkoxy group, or one of R₁ and R₂ and one of R₃ and R₄ Is hydrogen and the others together form a trimethylene, tetramethylene or pentamethylene bridging group, R₅ is hydrogen or an unsubstituted acyclic aliphatic hydrocarbon group having a maximum of six carbon atoms, and R₆ is an acyclic aliphatic hydrocarbon group having a maximum of six carbon atoms or a group CH₂R₇ in which R₇ is a C_1-5 alkyl group substituted by a hydroxy group or by a C_1-6 alkoxy group, or a salt thereof formed with a physiologically acceptable inorganic or organic add, for the manufacture of a medicament for providing protection against damage caused by free radicals.

22. The use according to Claim 20 or 21, 1n which the compound of formula (II) is as defined 1n any of Claims 3 to 13.

23. A method for the treatment of a patient in need of

cardloprotectlon or in need of protection against the toxic effects of paracetamol or against damage caused by free radicals which comprises administering to said patient a therapeutically effective amount of a compound of formula (II) as defined 1n any of Claims 2 to 13.