WO1992000951A1 - Pharmaceutical compositions - Google Patents

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 are a trimethylene, tetramethylene or pentamethylene bridging group, R5 is an acyclic aliphatic hydrocarbon group having a maximum of six carbon atoms or a group CH2R7 in which R is a C1-5 alkyl group substituted by a hydroxy group or by a C1-6 alkoxy group, and R6 is a group such that under physiological conditions R6OH undergoes elimination with the formation of a 3,5-dioxopiperazinyl ring N-substituted by a group R5, and salts thereof formed with a physiologically acceptable inorganic or organic acid, are of use in therapy, particularly as cardioprotective agents.

Description

PHARMACEUTICAL COMPOSITIONS

This invention relates to pharmaceutical compounds and to compositions containing them, being primarily concerned with substances of use as cardioprotectlve 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 l isomers); R = R' = R" = H;

R = R' = CH₃ and R" = H (as the mesois omer); 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 m 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 the

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 Hi lies and Williams,

Plzen. Lek. Sborn., 1985, 49, 113, for the compound having R = H and R' = C₂H₅ in the dl form. In Research Communications in Chemical Pathology and

Pharmacology, 1985, M, 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 bimolane (R = R' = H and ) and the

compound having R = CH₃ and R' = R" = H (as the 41, ύ or 1 isomer⁾ give protection against the lethal effects of daunorubicin, the remainder of the compounds tested (R = R' = R" = H; R = R' = H and

R" = CH₃; R = R" = CH₃ and R' = H (l); R = R' = CH₃ and

R" = H (meso): R = C₂H₅ and R' = R" = H ( dl) ; 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-diacld diamlde 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 theindications to the contrary in the paper, certain pro-drugs of various bis-dioxopiperazines which are substantially non-cytotoxic are ofinterest as cardioprotective agents, as well as in other roles. These non-cytotox1c bis-dioxopiperazine pro-drugs are ofinterest for providing protection against the cardiotoxic effects of various anthracycline drugs but particularly doxorubicln (adriamydn). In this context 11 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, 12, 249 that even the cardioprotective compound ICRF 159, which is the dl isomer of the compound of formula (I) having R = CH₃ and R = R' = H, is consistently more effective in reducing high dose daunorubicin toxicity than doxorublcin toxicity. Japanese Patent Appl i cation Number 47109752 di scloses a novel method for the manufacture of add amides , one example of whi ch i s the acid amide NN^,-di-(butoxycarbonylmethyl )-NN'-di-(butylamino- carbonylmethy)-1 ,2-di ami noethane of formula

However, no indication is given of the properties of these acid amides and in particular no suggestion is made that they may possess any physiological activity.

Accordingly the presentinvention comprises 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 R^ and R₂ and one of R₃ and R₄ is hydrogen and the others together are a trimethylene, tetramethylene or pentamethylene bridging group, R₅ 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, and R₆ is a group such that under physiological

conditions R₆OH undergoes elimination with the formation of a

3,5-d1oxopiperazinyl ring N-substituted by a group R₅, and salts thereof formed with a physiologically acceptable inorganic or organic acid, for use in therapy.

These compounds are novel per se with the exception of a compound of formula (II) wherein n = 0, R₁ = R₂ = R₃ = R₄ = H and R₅ = R₆ = a C₄ alkyl group.

The pro-drug compounds (II) which are the subject of the present invention do not undergo simple hydrolysis of the two similar ester groups in vivo which would result in a non-cyclic diamide diadcid compound of formula (IV) as described hereinafter but instead eliminate two R₆OH molecules with the formation of a compound of formula (III) containing two N-subst1tuted dioxopiperazlne rings

In contrast with bimolane and the compound of formula (I) having R = CH₃ and R' = R" = H, the compounds (III) produced in vivo from the pro-drug compounds (II) of the present invention have the particular advantage of being substantially non-cytotoxic. The compounds (II) are thus distinguished from the related pro-drugs which are the. subject of UK Patent GB 2173195 and which are converted in vivo to cytotoxic compounds having a formula (I) as given hereinbefore with R" being hydrogen and R and R' being as defined in that patent.

in the pro-drugs (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, it is 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 or an unsubsti tuted 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 toinclude both branched and especially straight chain groups. The aliphatic hydrocarbon 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 or particularly alkyl groups (the terms alkyl, alkenyl and alkynyl are used throughout this specification toinclude both straight and branched groups). 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 R₁ to R₄, these may be branched or especially straight chain alkyl groups

substituted, particularly terminally, by a hydroxy group or particularly an alkoxy group. Conveniently the 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. It is generally preferred, however, that where they do not constitute trimethylene, tetramethylene or particularly

pentamethylene bridging groups R₁ , R₂ R₃ and R₄ are selected from hydrogen and unsubsti tuted acyclic aliphatic hydrocarbon groups, especially from the group consisting of hydrogen, methyl, ethyl, n-propyl, isopropyl, allyl and propargyl. Preferably R₁ i s hydrogen and conveni ently ei ther R₂ i s 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 in 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, ally! or propargyl, or (c) R₂ and R₄ together form a tetramethylene bridging group, especially preferred combinations being n = 0, R₁ = R₂ = H, R₃ = R₄ = CH₃; n = 0, R₁ = R₂ = R₁ = H , R₄ = CH₂OH or CH₂OCH₃; particularly n = 0, R₁ = R₂ = R₃ = R₄ = H; n = 0, R₁ = R₃ = H, R₂ = R₄ = CH₃; n = 0, R₁ = R₂ = R₃ = H,

R₄ = CH₃ or C₂H₅; and especial ly n = 0, R₁ = R₃ = H,

R₂ + R₄ = CH₂CH₂CH₂CH₂.

As regards the group R₅ 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 R₅ which are alkyl groups, particularly C₁_₄ or C₁_₃ alkyl groups, for example ethyl or particularly methyl are preferred, and where R₅ is unsaturated it preferably has the form CH₂R₈ where R₈ is a C₂_₅ alkenyl or alkynyl group, preferably a C₂ or C₃ group, for example CH₂R₈ being allyl or propargyl. As regards the groups R₅ 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 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 CH₂R₇ is from 3 to 6,

especially 3 or 4.

Compounds of particular interest are those in which R5 is selected from 2-hydroxyethyl, 2-methoxyethyl, particularly ethyl, n-propyl, isopropyl, and especially methyl, for example together with the combinations of R₁ to R₄in dicated as preferred.

Asin dicated previously, the pro-drugs (II) are converted in vivo to the compounds (III). By modification of the nature of the ester groups CO.OR₆ it is possible to alter the water

solubility, lipophilicity and rate of conversion to the active molecules (III), the methyl, lower hydroxyalkyl and lower

alkoxyalkyl esters, for example, having a much greater water solubility than the cyclic diimides which they generate. Thus, the diimide (III) in which n = 0, R₁ = R₂ = R₃ = R₄ = H and R₅ = CH₃ has a very low water solubility at 20°C and pH 7.2 and is therefore difficult to use parenterally, whereas the corresponding

compounds (II) in which R₆ = CH₃ or CH₂CH₂OH have a much higher water solubility and can readily be used for parenteral

administration. Similarly, increased lipophilicity may be obtained in these pro-drugs by theintroduction of, for example, hydrocarbon groupings such asisobutyl or benzyl and this is reflected by relatively high Rf values in a 50% v/v chloroform:ethanol/SiO₂ thin layer chromatography system. An increased lipophilicity favours better absorption and changes in tissue distribution. Further, depending upon whether R₆ is an electron-withdrawing group, for example benzyl, propargyl or ethoxycarbonylmethyl, or an electron-repelling group, for example isobutyl, hydroxyethyl or methoxyethyl, the rate of conversion of the compound (II) to the corresponding compound (III) can be speeded up or slowed down.

Thus, by providing some control over the water solubility, lipophilicity and rate of generation of the active species, the pro-drug compounds of the presentinvention provide the ability to modify the tissue distribution and pharmacodynamics of the

dioxoplperazine protective drugs with consequential therapeutic benefit to the patient.

The group R₆ is eliminated under physiological conditions and, although it is possible that enzymic catalysis may be involved in some cases, the elimination will usually occur spontaneously and may be tested for in vitro by incubation of the compound (II) under physiological conditions of pH and temperature (i.e. pH 7.2, 37°C), for example as described in Example 11(B) of UK Patent GB 2173195. Whilst a very wide range of unsubstituted and substituted aliphatic hydrocarbon groups may conveniently be used as the group R₈, it has been found that certain groups R₆ such as t-butyl groups normally undergo S_N1 reactions which do not involve cyclisation so that no significant amount of the desired compound (III) is produced. For this reason, the unsubstituted and substituted aliphatic hydrocarbon groups R₆ of use in the present invention preferably contain a bonding carbon atom, i.e. that atom linked to the group

, which carries at least one hydrogen atom. Subject to this

preference, R₆ may conveniently be selected from aliphatic

hydrocarbon groups having a maximum of ten carbon atoms which may be either unsubstituted or substituted. Preferences as to the branching, degree of unsaturation and size of these aliphatic hydrocarbon groups are broadiy as discussed hereinbefore in relation to R₁, R₂, R₃ and R₄. As regards substitution, for example in a substituted alkenyl, alkynyl or particularly alkyl group, this may conveniently involve as substituents one or more groups selected from halogeno (for example fluoro, chloro or bromo), hydroxy, alkoxycarbonyl, benzyloxycarbonyl, cyano, amino (and mono- and di-alkylamino) groups, alkoxy, carboxy and oxo groups. The alkoxy and alkyl groups present as the whole or a part of a substituent in a substituted aliphatic hydrocarbon group are conveniently of one to ten, especially one to four carbon atoms. In general, substituted alkyl groups, especially the alkoxyalkyl groups which may conveniently contain a maximum of ten carbon atoms in total, are of particularinterest. Examples of groups

containing an oxo substituent are acetonyl and phenacyl, such groups providing ketonic esters, particularly β-keto esters. Also of some particular interest as substituted aliphatic hydrocarbon groups are aralkyl, aralkenyl and aralkynyl groups in which the aromatic part of the group may optionally be substituted by one or more, particularly one or two, substituents selected from halogeno, lower alkyl, lower alkoxy, amino (and mono- and di-alkylami no) and nitro groups or by one methyl enedioxy group, and the aliphatic hydrocarbon part of the group (again conveniently having a maximum of ten carbon atoms) may optionally be substituted by one

substituent selected from alkoxycarbonyl and cyano. The term 'lower' is used herein to denote a group of 1 to 4 carbon atoms. As regards the aromatic groups, the preferred form of group is an unsubstituted or substituted aromatic hydrocarbon group,

particularly a naphthyl or especially a phenyl group, although aromatic heterocyclic groups are also of interest, for example pyridyl groups such as pyrid-2-yl, pyrid-3-yl and pyrid-4-yl.

Among the various aliphatic hydrocarbon groups R₆, both unsubstituted and substituted, it is preferred for reasons of ease of synthesis and stability of the compound (II) prior to

administration that the bonding carbon atom of the group, as defined hereinbefore, is not linked to any atom which is not hydrogen or carbon and, moreover, conveniently also is not

unsaturated, i.e. is not linked to any adjacent atom by a double or particularly a triple bond. Moreover, while the groups, for example alkyl groups, may be substituted by one or more

substituents, groups containing one or two substituents are preferred, and conveniently only one substituent in most cases although with some substituent groups, such as alkoxycarbonyl groups, the presence of two substituents may be of value. Those comments also apply to the case of aliphatic hydrocarbon groups substituted by an aryl group which most usually contain two or particularly one aryl group, although groups such as the

diphenylmethyl group may be of interest in resisting the esterase activity which occurs in some animal species as discussed

hereinafter.

Although substituted aliphatic hydrocarbon groups R₆, for example substituted alkyl groups, are of more interest than is the case with R₁, R₂, R₃ and R₄, unsubstituted groups, particularly alkyl, alkenyl and alkynyl groups, are also of particular interest.

Specific examples of preferred groups R₆ are as follows (the terms ethyl, propyl and butyl used without qualification in the names of the substituted groups as usual indicate a substituted n-alkyl group (and similarly for alkoxy groups presentin a substituent) but, except where indicated, without any restriction upon the position of the substituent in the carbon chain of that alkyl group although, as mentioned hereinbefore, substitution upon the bonding carbon atom is generally of lesser interest,

substitution upon the terminal carbon of the chain usually being of most interest; in the cases where R₆ is a substituted benzyl group, substitution at the α-position is specifically indicated and where this is not done the substituent is located on the ring): methyl, ethyl, n-propyl, n-butyl, isobutyl, allyl, propargyl, benzyl, α-methyl benzyl , α-ethoxycarbonyl benzyl, nitrobenzyl, aminobenzyl, mono and dichlorobenzyl, chloro-3,4-methylenedioxy-benzyl, mono- and di-methoxybenzyl, mono- and di-methyl benzyl, cinnamyl, methoxyethyl, ethoxyethyl, propoxyethyl, butoxyethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, ethoxycarbony1methyl, ethoxycarbonylethyl, ethoxycarbonylpropyl, carboxymethyl,

carboxyethyl, carboxypropyl, benzyloxycarbonylmethyl,

benzyloxycarbonylethyl, benzyloxycarbonylpropyl,

t-butoxycarbonylmethyl, t-butoxycarbonylethyl,

t-butoxycarbonylpropyl, di-(ethoxycarbonyl)methyl,

cyanomethyl, acetonyl, phenacyl and 3-dimethylaminopropyl. Particularly preferred compounds according to the presentinvention are those having the specific combinations of n, R₁, R₂, R₃ and R₄ asin dicated previously, for example n = 0 or 1,

R₁ = R₂ = R₃ = R₄. H; n = 0, R₁ = R₂ = R₃ = H, R₄ = H or CH₃; n = 0, R₁ = R₃ = H, R₂ = R₄ = CH₃; particularly n = 0,

R₁ = R_{2 m} R₃. H, R₄ = C₂H₅; and especially n = 0, R₁ = R₃ = H, R₂ + R₄ = CH₂CH₂CH₂CH2; and in which R₅ is one of the six specific groups R₅ mentioned as of particular interest, for example

2-hydroxyethyl, particularly ethyl or especially methyl, whilst R₆ is selected from methyl, ethyl, isobutyl, allyl, propargyl, benzyl, α-methyl benzyl, α-ethoxycarbonylbenzyl, o-nitrobenzyl, amlnobenzyl, 2,6-dichlorobenzyl, methoxyethyl, ethoxyethyl, propoxyethyl, butoxyethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl,

ethoxycarbonylmethyl, di-(ethoxycarbonyl)methyl,

benzyloxycarbonylmethyl, ethoxycarbonylpropyl, carboxymethyl and acetonyl (as regards the substituted ethyl and propyl groups particularly those which are terminally substituted). Enhanced water solubility will result from the presence of hydroxyalkyl groups in the molecule, for example in compounds having

R₅ = CH₂CH₂OH and also in compounds having R₄ = CH₂OH, for exampl e with n = 0, R₁ = R₂ = R₃ = H, R₅ = CH₃ or C₂H₅ and R₆ as just defined.

The compounds may exist in various stereochemical 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 isomerism exists (meso/dl or erythro/threo when n = 0), for example where n = 0 and R₂ = R₃ = H and R₁ = R₄ = CH₃ or where n = 0 and R₂ = R₃ = H, R₁ = CH₃ and R₂ = C₂H₅, respectively, and when two of R₁ to R₄ provide a bridging group the compound may be in the cis or trans form. Moreover, when the bridging grouping -(CH₂)_n-CR₁R₂-CR₃R₄- does not have a centre of symmetry, the compounds can exist in enantiomorphic d and 1 forms. The invention includes the use of the various different isomers of the compounds. In some cases the optically active d- and 1-isomers 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 1 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 isomer, for example being substantially free from (i.e. containing no more than 20% and conveniently no more than 10% of) the dl and 1 or dl and d isomers. However, where the advantage lie's 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.

In a paper relating to chemical synthesis and containing no reference to any possible pharmaceutical use, Houghton and Williams (Journal of the Chemical Society, Perkin Transactions I, 1982, 2693), refer to the preparation of a compound of formula (II) having a structure in which n = 0, R₁ = R₂ = R₃ = R₄ = H, and R₅ = R₆ = CH₃, the copper chelate of the bis-cycl1c 1mide

(III, R₁ = R₂ = R₃ = R₄ = H and R₅ = CH₃) being reacted with an excess of methanol and the copper subsequently removed from the product with hydrogen sulphide.

The method described by Houghton and Williams may be applied to other bis-cyclic imides (III) in which R₅ is other than hydrogen but is most successful with methanol, being less successful with ethanol and becoming progressively more difficult with higher alcohols. For the preparation of compounds corresponding to the compounds (III but with R₅ = H we have found that it is advantageous to replace the cupric chloride used by Houghton and Williams to make the initial chelate by a cupric salt of a sulphonic add such as methane sulphonic or i sethioni c acid. These give more soluble intermediates which lead to more efficient reactions and, after treatment with hydrogen sulphide, to the direct isolation of more water soluble, pharmaceutically acceptable salt forms of the described products. It may sometimes also be advantageous to add about two equivalents of free sulphonic add, for example methane sulphonic acid, to the initial reaction mixture. Reasonable yields of diesters from higher alcohols such as 2-butoxyethanol may also be obtained by using an ester exchange reaction between the dimethyl ester, activated in the form of the copper chelate and an excess of the appropriate alcohol. Treatment with hydrogen sulphide may again be used to liberate the desired product. The use of other forms of activated ester for this purpose may also be employed.

A more generally useful method for the preparation of the compounds of the present invention where, unlike the case with the Houghton and Williams compound, R₅ is not hydrogen and which is also particularly appropriate where the alcohol is not a liquid, or is uneconomic to use in excess or is pH-labile, is neutral esterificatlon using caesium salts and the appropriate halide, which is more reactive than the corresponding alcohol as described for simple N-acyl amino adds by Wang et al, (Journal of Organic Chemistry, 1977, 42, 1286). In this procedure the appropriate bis-diacid diamlde (IV) (prepared as described by Huang et al,

Agents and Actions, 1982, 12, 536) is carefully neutralised with caesium bicarbonate (or caesium carbonate) and a solution, or more usually a suspension, of the dried salt in a neutral aprotic solvent such as dimethylformamide, is treated with a reactive halide such as benzyl bromide. The reaction is usually complete within a few hours at from 50 to 100°C. Alternative solvents Include hexamethylene phosphoramlde, dimethyl sulphoxide and

N-methylpyrrol!done and the caesium salts can generally be replaced by rubidium salts and, in favourable cases where the halide is particularly reactive such as with the benzyl halides, by salts of other metals including sodium or potassium as well as by salts of tertiary amines such as tr1ethylamine or 4-dimethylaminopyridine. Yet another procedure involves the use of an acetal of dimethylformamide of formula (R₆O)₂CH.N(CH₃)₂ which is reacted with the appropriate diacid diamide (IV), conveniently by heating the two reactants in a suitable mutual solvent, an excess of the acetal generally being employed. Reaction at 50 to 100°C is usually appropriate, refluxing benzene being suitable as the reaction medium in many cases. This reaction is particularly adapted to the preparation of compounds in which R₆ is an unsubsti tuted aliphatic hydrocarbon group, for example ethyl, methyl, isopropyl, n-propyl, n-butyl etc.

It will be appreciated that the present invention includes a process for the preparation 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 up to 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 are a trimethylene, tetramethylene or pentamethylene bridging group, R₅ is an acyclic aliphatic hydrocarbon group having a maximum of up to six carbon atoms or a group CH₂R₇ in which R is a C_{1 -5} alkyl group substituted by a hydroxy group or by a C_{1 -6} alkoxy group, and R₆ is a group such that under physiological conditions R₆OH undergoes elimination with the formation of a 3,5-dioxo- piperazi nyl ring N-substituted by a group R₅, but excluding specifically a compound of formula (II) wherein n = 0, R₁ = R₂ = R₃ = R₄ = H, when _R5 = R₆ = a C₄ alkyl group, and salts thereof formed with a physiologically acceptable inorganic or organic acid, which comprises reacting a compound of formula (IV):

in which n, R₁ , R₂, R₃, R₄ and R₅ are as defined for the compound of formula (II), or a related compound in which the carboxy groups are in derivative form, including that form in which the carboxy groups are derivatlsed by the amide groups to form 3,5-dloxo- piperazinyl rings N-subst1tuted by a group R₅ with an alcohol R₆OH in which R₆ is as defined for the compound of formula (II), or a derivative thereof, where appropriate using a compound of formula (IV) or a related compound as a salt formed with a physiologically acceptable inorganic or organic add or reacting the compound (II) from the reaction with the alcohol R₆OH or derivative thereof with such an add to form a salt.

As discussed hereinbefore, the alcohol R₆OH is preferably a primary or secondary one, I.e. the carbon atom joined to the hydroxy group carries one or two hydrogen atoms, in order to produce a compound (II)in which the bonding carbon atom of the group R₆ carries at least one hydrogen atom, which would not be the case with a tertiary alcohol. To obtain compounds (II) of the desired stereochemistry it is most convenient to use a compound (IV) or a related compound having the equivalent stereochemistry. When a d or 1 isomer is required rather than the dl isomer, however, an alternative to the uti l i sation of a d or 1 compound (IV), which is preferred, 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 1 forms of the compound (II) which are then separated.

The bis-diacid diamides (IV) may conveniently be prepared by the reaction of the corresponding bis-anhydride with a secondary amine R₅NH₂ to effect the reaction:

for each ring system.

It will be appreciated, however, that the methods described above are not the only ones which may be used for the preparation of the compounds of the present invention and of intermediates therefor, and that various alternative procedures including obvious chemical equivalents of those described herein may be used as will be apparent to those skilled in the art.

The present invention also includes pharmaceutical compositions comprising as an active component a compound of formula (II) as defined hereinbefore, in cluding compounds in which n = 0,

R₁ = R₂ = R₃ = R₄= H and R₅ = R₆ = a C₄ alkyl group, together with a physiologically acceptable diluent or carrier. As indicated, the compounds may be formulated as salts formed with physiologically acceptableinorganic or organic adds and, when so formulated, it is preferred to use methane sulphonic add,isethionic acid, tartaric acid or another solubilising add.

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 myri state), 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 (III) are more active than others and as the rate of formation of these compounds will depend upon the particular nature of the group R₆ in the

pro-drug (II) which is being used. Moreover, the amount of active compound (III) produced by a given weight of a pro-drug (II) will depend upon the nature of the groups R₆ therein and the following discussion is therefore particularly directed to the use of methyl esters. If the group R₆ is sufficiently large toin crease the molecular weight of the compound (II) significantly beyond that of the corresponding methyl ester then a corresponding increased dosage may well be appropriate. Without commitment to a ri gi d definition of dosages it may be stated that a dally dosage of active constituent (estimated as the free base), divided 1f 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 slowinfusion, 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 100 mg to 500 mg but with the more active compounds 1t 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.

It will be appreciated that certain formulations of the compounds (II) will tend to cyclise to the compounds (III) on storage if made up in advance. For this reason, although the compounds may conveniently be formulated in advance of their use as a solid composition it will usually be appropriate to prepare certain forms of liquid composition, particularly those containing an aqueous diluent, just prior to their use. Providing such steps are taken to avoid premature cycllsation before administration, however, it will be appreciated from the foregoing discussion that the compounds used may have a very wide range of half lives in vivo.

The pro-drug compounds (II) of the presentinvention are primarily of value as cardioprotectlve agents and it should be noted that their potential in such a use extends not only to use in conjunction with drugs having a cardiotoxic 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 toin clude not only natural and semi-synthetic anthracyclines such as epirubicin, idarubicin, daunorubicin and especially doxorubi ci n (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 cardioprotection 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 orinduction 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-hydroxyacetanilide) and various metals such as iron, zinc, cadmium, nickel and lead. In many of these cases, particularly when the toxic agent is a metal, the chelating ability of the compounds (III) produced in vivo by the pro-drugs (II) is often an important factor in achieving the protective effect.

The compounds (II) find most application in the treatment of humans and although they can find veterinary use in certain other mammals such as dogs, rabbits, cattle, and horses, their activity is not expressed in rodents such as rats and mice owing to an esterase activity existing in the plasma thereof which prevents cyclisation of the compounds (II) to the compounds (III).

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 in 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 conveni ently 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 thusin cludes (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 compri si ng 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 doxorubi ci n and the presentin vention therefore

particularly includes a pharmaceutical composition comprising a compound of formula (II) as defined hereinbefore, for example one in which n = 0, R₁ = R₂ = R₃ = R₄ = H and R₅ = CH₃ and doxorubicin, 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 concomitantly with, or at the i nterval 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 metabolisation 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) and 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 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 doxorubicin 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 doxorubicin whilst its cardiotoxic side effects are controlled by presence of the

compound (II).

The exact dosage of an anthracycline drug such as doxorubicin 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 fluorouracil 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 wih the compounds (II), this usually being administered separately. When used as a protective agent against the toxic effect of a metal, or an excess thereof, or against the toxic effect of paracetamol, the compounds (II) may be used protectively before occurrence of the toxicity or following occurrence of the

toxldty. 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 differences may arise as to whether the toxic effect is acute, as for example is usually the case following an overdose of paracetamol, or chronic, as will often be the case with conditions such as iron overload; higher dosages over a shorter period being indicated in the former type of case as compared with the latter.

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 bein volved in some of the conditions described

hereinbefore such as a anthracycline drug-induced damage), for example in reducing the diabetogenic 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 cardioprotection, including formulation together with the drug, and the dosage levels used.

The presentin vention thus includes a method of providing protection against a toxic effect on the body, particularly a cardiotoxic effect, which comprises admi ni steri ng to a patient in need thereof a therapeutically effective amount of a compound (II) as defined hereinbefore. Furthermore the invention includes the use of a compound (II) in the manufacture of a medicament for use in providing protection against a toxic effect on the body.

The present invention is illustrated by the following

Examples. Further exemplification of preparative procedures is provided by the Examples of UK Patent No. GB 2173195 which, although directed to the preparation of cytotoxic compounds, do involve reactions of a similar chemical nature. EXAMPLES

Example 1 : Preparation of NN'-Dimethoxycarbonylmethyl- NN'-di-(methvlaminocarbonylmethyl)-1.2-diaminoethane

A mixture of 1 ,2-bis-(4-methyl-3,5-dioxop1perazin-1-yl)-ethane (0.1 moles), anhydrous copper (II) chloride (0.2 moles) and dry methanol (600 ml) was stirred and heated under reflux for 24 hours. The mixture was then filtered and the solvent was removed from the filtrate under reduced pressure. The residue was dissolved in water (ca 200 ml), and the solution was saturated with hydrogen sulphide and then filtered. Evaporation of the solution under reduced pressure gave a solid hydrochloride which was treated with an excess of ammonia in chloroform. Concentration of the solution and cooling gave a residue which was recrystal l i sed from chloroform to provide 6% yi eld of NN'-dlmethoxycarbonylmethyl-NN'-di-dnethyl- aminocarbonyl-methyl)-1,2-diaminoethane having m.p. 125-126°C.

Example 2 : Formulation of compounds

(A) Tablets of the following composition are prepared: mg/tftblet

Compound of Example 1 (micronised) 250

'Avicel' (mlcrocrystal line cellulose)* 38 polyvlnylpyrrol idone 3

alginic add 6

magnesium stearate 3

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

polyvlnylpyrrolidone 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 alginic acid and magnesium stearate are then added

Avicel' is a Registered Trade Mark or Service Mark. and mixed with the granules. The product is compressed into tablets each weighing 300 mg on 3/8inch flat bevelled edge divided punches.

(B) Tablets of the following composition are prepared: mα/tablet

Compound of Example 1 (micronised) 250

'Avicel' (microcrystalline cellulose) 134 polyvinylpyrrolidone 4 alginic acid 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.

(C) Tablets of the following composition are prepared: mα/tablet

Compound of Example 1 (micronised) 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.

Claims

1. 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 are a trimethylene, tetramethylene or pentamethylene bridging group, R5 is an acyclic aliphatic hydrocarbon group having a maximum of six carbon atoms or a group CH2R₇ in which R is a C_1-5 alkyl group substituted by a hydroxy group or by a C_1-6 alkoxy group, and R₆ is a group such that under physiological conditions R₆OH undergoes elimination with the formation of a 3,5-dioxopiperazinyl ring N-substituted by a group R₅, but excluding specifically a compound of formula (II) wherein n = 0 and R₁ = R₂ = R₃ = R₄ = H when R₅ = R₆ = a C₄ alkyl group, and salts thereof formed with a physiologically acceptable inorganic or organic acid.

2. A compound according to Claim 1, 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.

3. A compound according to Claim 2, 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.

4. A compound according to any of Claims 1 to 3, in which n is 0.

5. A compound according to Claim 1, in which n is 0, R₁ and R₂ are each hydrogen and R₃ and R₄ are each methyl, or 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 or (b) R₂ is methyl or ethyl and R₄ is methyl, ethyl, n-propyl, isopropyl, allyl or propargyl.

6. A compound according to Claim 1, in which 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 = CH₂CH₂CH₂CH₂; n = 0,

R₁ = R₂ = R₃ = H, R₄ = CH₃ or C₂H₅; or n = 0,

R₁ = R₂ = R₃ = R₄ - H.

7. A compound according to any of Claims 1 to 6, in which R5 is a C_1-6 alkyl group, a group CH₂R₈ wherein R₈ is a C₂ or C₃ alkenyl or alkynyl group, or a group CH₂R₇ wherein R₇ is a C_1-C₂ alkyl group substituted by a hydroxy, methoxy or ethoxy group.

8. A compound according to Claim 7, in which R₅ is methyl, ethyl, n-propyl, isopropyl, 2-hydroxyethyl or 2-methoxyethyl.

9. A compound according to Claim 1, in which n = 0, R₁ = R₃ = H, R₂ = R₄ = CH₃, R₅ = CH₃, C₂H₅ or CH₂OH; n = 0, R₁ = R₃ = H,

R₂ + R₄ = CH₂CH₂CH₂CH₂, R₅ = CH3, C₂H₅ or CH₂OH; R₁ = R₂ = R₃ = H, n = 0, R₄ = CH₃ or C₂H₅, R₅ = CH₃, C₂H₅ or CH₂0H; or n = 0,

R₁ = R₂ = R_{3 β} R₄ = H, R₅ = CH₃, C₂H₅ or CH₂OH.

10. A compoupd according to Claim 1, in which n = 0,

R₁ = R₃ = H, R₂ + R₄ = CH₂CH₂CH₂CH₂, R₅ = CH₃,

R₁ = R₂ = R₃ = R. = H, R₅ = CH₃; n = 0, R₁ = R₂ = R3 = H,

R₄. R₅ = CH₃; or n = 0, R₁ = R₂ = R₃ = H, R₄ = C₂H₅, R₅ = CH₃.

11. A compound according to any of Claims 1 to 10, in which R₆ is an unsubstituted aliphatic hydrocarbon group having a maximum of ten carbon atoms, a substituted aliphatic hydrocarbon group which is (a) an aliphatic hydrocarbon group having a maximum of ten carbon atoms which is substituted by one or more groups selected from halogeno, hydroxy, alkoxy, carbonyl , benzyloxycarbonyl, cyano, amino, mono- and di-alkylamino, alkoxy, carboxy and oxo groups, or (b) an aralkyl, aralkenyl or aralkynyl group in which the aromatic part of the group may optionally be substituted by one or more substituents selected from halogeno, lower alkyl, lower alkoxy, amino, mono- and di-alkylamino, and nitro groups or by one methylenedioxy group, and the aliphatic hydrocarbon part of the group, which has a maximum of ten carbon atoms, may optionally be substituted by one substituent selected from alkoxycarbonyl and cyano.

12. A compound according to Claim 11,in which R₆ is a C_1-4 alkyl or C_2-4 alkenyl or alkynyl group, or a C_1-4 alkyl group substituted either as described under (a) or as described under (b) with the aromatic part of the group being a phenyl group or a substituted phenyl group.

13. A compound according to Claim 12, in which R₆ is methyl, ethyl, n-propyl, n-butyl,isobutyl, allyl, propargyl, benzyl, α-methylbenzyl, α-ethoxycarbonylbenzyl, nltrobenzyl, amlnobenzyl, mono- and di-chlorobenzyl, chloro-3,4-methylenedioxybenzyl, monoand di-methoxybenzyl, mono- and di-methylbenzyl, dnnamyl, methoxyethyl, ethoxyethyl, propoxyethyl, butoxyethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, ethoxycarbonylmethyl,

ethoxycarbonylethyl, ethoxycarbonylpropyl, carboxymethyl,

carboxyethyl, carboxypropyl, benzyloxycarbonylmethyl,

benzyloxycarbonylethyl, benzyloxycarbonylpropyl,

t-butoxycarbonylmethyl, t-butoxycarbonylethyl,

t-butoxycarbonylpropyl, di-(ethoxycarbonyl)methy1, cyanomethyl, acetonyl, phenacyl or 3-dimethylami nopropyl.

14. A compound according to Claim 1, in which n = 0,

R₁ = R₃ = H, R₂ + R₄ = CH₂CH₂CH₂CH₂, R₅ = CH₃, C₂H₅ or CH₂OH;

R₁ = R₂ = R₃ = H, R₄ = H, CH₃ or C₂H₅, R₅ = CH₃, C₂H₅ or CH₂OH; or n = 0, R₁ = R₃ = H, R₂ = R₄ = CH₃, R₅ = CH₃, C₂H₅ or CH₂OH;

and R₆ is selected from methyl, ethyl,isobutyl, allyl, propargyl, benzyl, α-methyl benzyl, ethoxycarbonyl benzyl, o-nitrobenzyl, amlnobenzyl, 2,6-dichlorobenzyl, methoxyethyl, ethoxyethyl, propoxyethyl, butoxyethyl, hydroxyethyl, hydroxypropyl ,

hydroxybutyl, ethoxycarbonylmethyl, di-(ethoxycarbonyl)methyl, benzyloxycarbonylmethyl, ethoxycarbonyl propyl, carboxymethyl and acetonyl .

15. A process for the preparation of a compound of formula (II) as defined in Claim 1 and salts thereof formed with a physiologically acceptable inorganic or organic add which comprises reacting a compound of formula (IV)

in which n, R₁ , R₂, R₃, R₄ and R₅ are as defined for the compound of formula (II) or a related compound in which the carboxy groups are in derivative form, including that form in which the carboxy groups are derivatised by the amide groups to form 3,5-dioxo-piperazinyl rings N-substituted by a group R₅, with an alcohol R₆OH or a derivative thereof in which R₆ is as defined for the compound of formula (II), where appropriate using a compound of formula (IV) or a related compound as a salt formed with a physiologically acceptable inorganic or organic acid or reacting the compound (II) from the reaction with the alcohol R₆OH or a derivative thereof with such an acid to form a salt.

16. A process according to Claim 15, in which the compound of formula (IV) is reacted with an alcohol derivative which is a dimethylformamide acetal of formul a (R₆O)₂CH.N(CH₃)₂.

17. A process according to Claim 15, in which the compound of formula (IV) or related compound is reacted in the form of a metal complex or a metal salt of the compound, the metal subsequently being removed.

18. A process according to Claim 15, in which the caesium or rubidium salt of the compound of formula (IV) is reacted with a halide R₆X, X being a halogeno group.

19. A process accordi ng to Claim 15, i n whi ch a copper compl ex of a related compound wherei n the carboxy groups are deri vati sed by the amide groups to form N-substi tuted 3,5-dioxopi perazi nyl rings i s reacted wi th an alcohol R₆OH.

20. A process according to Claim 15, in which a copper complex of a related compound wherein the carboxy groups are in the form of a methyl ester giving a compound of formula (II) 1n which R₆ i s methyl i s reacted wi th an alcohol R₆OH wherein R₆ i s other than methyl .

21 . A pharmaceutical composition compri sing 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 are a trimethylene, tetramethylene or pentamethylene bridging group, R₅ is an acyclic aliphatic hydrocarbon group having a maximum of six carbon atoms or a group CH₂R₇ in which R is a C_1-5 alkyl group substituted by a hydroxy group or by a C_{1 -6} alkoxy group, and R₆ is a group such that under physiological conditions R₆OH undergoes elimination with the formation of a 3,5-dioxopiperazinyl ring N-substituted by a group R₅, and salts thereof formed with a physiologically acceptableinorganic or organic acid, together with a physiologically acceptable diluent or carrier.

22. A pharmaceutical composition according to Claim 21, comprising a compound of formula (II) 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.

23. A pharmaceutical composi tion according to Claim 22, comprising a compound of formula (II) 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.

24. A pharmaceutical composition according to any of Claims 21 to 23, comprising a compound of formula (II) in which n is 0.

25. A pharmaceutical composition according to Claim 21, comprising a compound in which n is 0, R₁ and R₂ are each hydrogen and R₃ and

R₄ are each methyl, or 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 or (b) R₂ is methyl or ethyl and R₄ is methyl, ethyl, n-propyl, isopropyl, allyl or propargyl.

26. A pharmaceutical composition according to Claim 21, comprising a compound of formula (II) in which 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 = CH₂CH₂CH₂CH₂ ; n = 0,

R₁ = R₂ = R₃ = H, R₄ = CH₃ or C₂H₅; or n = 0,

R₁ = R₂ = R₃ = R₄ = H.

27. A pharmaceutical composition according to any of Claims 21 to 26, compri si ng a compound of formul a (II) i n whi ch R₅ i s 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-C₂ alkyl group substituted by a hydroxy, methoxy or ethoxy group.

28. A pharmaceutical composition according to Claim 27, comprising a compound of formula (II) in which R₅ is methyl, ethyl, n-propyl, isopropyl, 2-hydroxyethyl or 2-methoxyethyl.

29. A pharmaceutical composition according to Claim 21, comprising a compound of formula (II) in which n = 0, R₁ = R₃ = H,

R₂ = R₄ = CH₃, R₅ = CH₃, C₂H₅ or CH₂OH; n = 0, R₁ = R₃ = H,

R₂ + R₄ = CH₂CH₂CH₂CH₂, R₅ = CH₃, C₂H₅ or CH₂OH; R₁ = R₂ = R₃ = H, n = 0, R₄ = CH₃ or C₂H₅, R₅ = CH₃, C₂H₅ or CH₂OH; or n = 0,

R₁ = R₂ = R₃ = R₄ = H, R₅ = CH₃, C₂H₅ or CH₂OH.

30. A pharmaceutical composition accordi ng to Claim 21, comprising a compound of formula (II) in which n = 0, R₁ = R₃ = H,

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

31. A pharmaceutical composition according to any of Claims 21 to 30, comprising a compound of formula (II) in which R₆ is an unsubstituted aliphatic hydrocarbon group having a maximum of ten carbon atoms, a substituted aliphatic hydrocarbon group which is (a) an aliphatic hydrocarbon group having a maximum of ten carbon atoms which is substituted by one or more groups selected from halogeno, hydroxy, alkoxy, carbonyl, benzyloxycarbonyl, cyano, amino, mono- and di-alkylamino, alkoxy, carboxy and oxo groups, or (b) an aralkyl, aralkenyl or aralkynyl group in which the aromatic part of the group may optionally be substituted by one or more substituents selected from halogeno, lower alkyl, lower alkoxy, amino, mono- and di-alkyl amino, and nitro groups or by one

methylenedioxy group, and the al i phati c hydrocarbon part of the group, which has a maximum of ten carbon atoms, may optionally be substituted by one substituent selected from alkoxycarbonyl and cyano.

32. A pharmaceutical composition according to Claim 31, comprising a compound of formula (II) in which R₆ is a C_1-4 alkyl or C_2-4 alkenyl or alkynyl group, or a C_1-4 alkyl group substituted either as described under (a) or as described under (b) with the aromatic part of the group being a phenyl group or a substituted phenyl group.

33. A pharmaceutical composition according to Claim 32, comprising a compound of formula (II) in which R₆ is methyl, ethyl, n-propyl, n-butyl, isobutyl, allyl, propargyl, benzyl, α-methylbenzyl, α-ethoxycarbonylbenzyl, nitrobenzyl, amlnobenzyl, mono- and di-chlorobenzyl, chloro-3,4-methylenedioxybenzyl, mono- and di-methoxybenzyl, mono- and di-methylbenzyl, cinnamyl,

methoxyethyl, ethoxyethyl, propoxyethyl, butoxyethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, ethoxycarbonylmethyl,

ethoxycarbonylethyl, ethoxycarbonylpropyl, carboxymethyl,

carboxyethyl, carboxypropyl, benzyloxycarbonylmethyl,

benzyloxycarbonylethyl, benzyloxycarbonylpropyl,

t-butoxycarbonylmethyl, t-butoxycarbonylethyl,

t-butoxycarbonylpropyl, di-(ethoxycarbonyl)methyl, cyanomethyl, acetonyl, phenacyl or 3-dimethylaminopropyl.

34. A pharmaceutical composition according to Claim 21, comprising a compound of formula (II) in which n = 0, R₁ = R₃ = H,

R₂ + R₄ = CH₂CH₂CH₂CH₂, R₅ = CH₃, C₂H₅ or CH₂OH; R₁ = R₂ = R₃ = H, R₄ = H, CH₃ or C₂H₅, R₅ = CH₃, C₂H₅ or CH₂OH; or n = 0,

R₁ = R₃ = H, R₂ = R₄ = CH₃, R₅ = CH₃, C₂H₅ or CH₂OH;

and R₆ is selected from methyl, ethyl, isobutyl, allyl, propargyl, benzyl, α-methylbenzyl, ethoxycarbonylbenzyl, o-nitrobenzyl, amlnobenzyl, 2,6-dichlorobenzyl, methoxyethyl, ethoxyethyl, propoxyethyl, butoxyethyl, hydroxyethyl, hydroxypropyl,

hydroxybutyl, ethoxycarbonylmethyl, di-(ethoxycarbonyl)methyl, benzyloxycarbonylmethyl, ethoxycarbonylpropyl, carboxymethyl and acetonyl.

35. A pharmaceutical composition according to any of Claims 21 to 34 which further comprises an anthracycline drug.

36. A pharmaceutical composition according to Claim 35, in which the anthracycline drug is doxorubicin.

37. A compound of formula (II) as defined in any of Claims 21 to 34 for use In therapy.

38. The use of a compound of formula (II) as defined in any of Claims 21 to 34 for use in the manufacture of a medicament for use as a cardioprotective agent.

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

cardioprotection 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 therapeuti cal ly effective amount of a compound of formula (II) as defined in any of Claims 21 to 34.