WO1993019037A1

WO1993019037A1 - Compounds

Info

Publication number: WO1993019037A1
Application number: PCT/GB1993/000546
Authority: WO
Inventors: Jeffrey Cummings; David John Mincher
Original assignee: Imperial Cancer Research Technology Limited
Priority date: 1992-03-18
Filing date: 1993-03-17
Publication date: 1993-09-30
Also published as: GB9205859D0; JPH07507996A; AU3759793A; EP0631570A1

Abstract

A compound having structural formula (I) where Y and Y1 are independently hydrogen or hydroxyl, B and B1 are independently oxo or hydrogen, R5 is hydrogen or hydroxyl and X is the residue of an amino acid, joined to the ring shown via the nitrogen atom of the amino acid adjacent the acid group thereof, or a derivative of a said amino acid. The compounds are useful as anticancer drugs and dyes.

Description

COMPOUNDS

The present invention relates to compounds which are based on an anthraquinone nucleus.

The inhibition of DNA topoisomerases, particularly topoisomerase II (topo II) is now considered to be an important component in the mechanism of action of a large number of the most clinically active anticancer drugs presently available including doxorubicin, mitoxantrone, VP16, eamptolhecin. topotecan. M-AM SA , VM26 and the ellipiticines. These drugs are believed to inhibit topo II by stabilising a protein/drug/nucleic acid ternary complex termed the cleavable complex.

However, whilst targeting topoisomerases, these drugs also exhibit a number of other mechanisms of action, such as generation of free radicals and formation of DNA covalent adducts which contribute to their overall toxicity and poor therapeutic index. Additionally, the failure of these agents to produce long term cures in the major malignancies is probably exacerbated by the presence of de novo resistance and the development of acquired drug resistance.

US-A-4 894 451 describes asymmetrically substituted anthracene- 1 ,4-dione compounds of Formula (A):

where B is a lower dialkyl amino group, n is 3-5 and R is hydrogen, alkanoyl or alkylsulphonyl. These compounds were proposed for use against tumours. The present invention seeks to provide monosubstitυted compounds. At least where the left hand ring shown in Formula (A) is unsubstituted, this has not previously been possible.

It is an object of this invention to provide improved clinically active drugs. It is a further object of the invention to provide coloured compounds useful as dyestuffs.

In one aspect the invention provides a compound having the structural formula (I):

where Y and Y¹ are independently hydrogen or hydroxyl. B and B¹ are independently oxo or hydrogen, R⁵ is hydrogen or hydroxyl and X is the residue of an α amino acid or a derivative of an a amino acid, joined to the ring shown via the nitrogen atom of the amino acid adjacent the acid group thereof.

Clearly, when B or B¹ are oxo, the single line to the ring represents a double bond. By "α amino acid" , we mean any compound having a group where R¹ is the residual group of an amino acid,

for example hydrogen, straight or branched C_1-6 alkyl (such as methyl, isopropyl, 2-methylpropyl or 1-methylpropyl), hydroxyalkyl (such as -CH₂OH or 1-hydroxyethyl), aralkyl (such as benzyl or 4-hydroxy-benzyl), thiolalkyl (such as -CH₂SH), alkylthioalkyl (such as -CH₂CH₂SCH₃), acyl (such as -CH₂COOH or -CH₂CH₂COOH). amidalkyl (such as -CH₂CO.NH₂ or -CH₂CH₂CO.NH,) or linear or cyclic, aromatic or non aromatic, nitrogen-containing heterocyclic groups such as the groups forming part of tryptophan, lysine. arginine or histidine; and R² is a group—C(=O)R³ wherein R³ is —OH, or any —O— linked or —N— linked radical, for example —O— alkyl, -O- alkylaminoalkyl, -O-alkoxyalkyI or—NH—NHR⁴ wherein R⁴ is straight or branched alkyl, optionally substituted by -CN or -OH, an amide group (such as -CONH₂) or a hydrazine group (such as -(CH₂)₂NH(CH₂)₂OH). Examples of alkylaminoalkyl groups include CH₃(CH₃)NCH₂CH₂- and

CH₃(CH₃)NCH₂CH₂NHCH₂CH₂-.

By "alkyl", we include branched or straight chain alkyl of up to 20 carbon atoms, preferably 1-10 carbon atoms, more preferably 1 -6 or 1 -4 carbon atoms. We include all of the 20 α-amino acids commonly found in naturally-occurring proteins and their D-isomers; less common naturally-occurring α-amino acids found in proteins, such as 4-hydroxyproline, 5-hydroxylysine, desmosine,∈-N-methyllysine, 3-methylhistidine and isodesmosine and their D-isomers: naturally-occurring amino acids not found in proteins, such as β-alanine, γ-aminobutyric acid, homocysteine, homoserine, citrulline, ornithine. canavaninc. djenkolic acid and β-cyanoalanine and their D-isomers; and di-, tri-. tetra-. penta-, oligo- or polypeptides based on these or other amino acids (providing that the amino acid joined to the anthracenyl ring is an a amino acid) which peptides may optionally include non-amino acid residues or side elements such as sugar residues. Preferably, there is only a single amino acid group.

Thus, R¹ may be: hydrogen; straight or branched chain C_1-4 alkyl (for example methyl, isopropyl, isobutyl or sec-butyl); aryl-C_1-4-alkyl (for example benzyl, β-indolylmethyl, 4-hydroxybenzyl or 4-imidazolylmethyl); C_1-4-alkylthio-C_1-4-alkyl (for example methylthioethyl); hydroxy-C_1-4-alkyl (for example hydroxymethyl or 1-hydroxyethyl); mercaptomethyl (for example -CH₂SH); C_1-4 amide (for example -CH₂C(O)NH₂ or -CH₂CH₂C(O)NH₂); C_1-4 alkyl carboxylate (for example -CH₂C(O)OH or -CH₂CH₂C(O)OH); C_1-6 alkylamine

(for example (CH₂)₄NH₂); and imino(C_1-6)alkyl-amine (for example - (CH₂)₃NHC( =NH)NH₂).

By "derivatives" of the amino acids, we include salts (acid or base addition), esters, amides, hydrazides and hydroxamic acids and other derivatives which do not diminish to an unacceptable extent the fundamental topoisomerase- inhibiting or colouring properties of the compounds.

Salts which may be conveniently used in therapy include physiologically acceptable base salts, for example, derived from an appropriate base, such as an alkali metal (eg sodium), alkaline earth metal (eg magnesium) salts, ammonium and -NX₄ ⁺ (wherein X is C_1-4 alkyl) salts. Physiologically acceptable acid salts include hydrochloride, sulphate, mesylate, besylate, phosphate and glutamate.

Salts according to the invention may be prepared in conventional manner, for example by reaction of the parent compound with an appropriate base to form the corresponding base salt, or with an appropriate acid to form the corresponding acid salt.

It has been found that the compounds of the invention may be prepared as substantially pure optical isomers.

Preferably, X is

wherein R₁ = -CH₂C₆H₄OH, -CH₃, or -CH₂OH and R₂ = -COOC₂H₅, -COOCH₃ or -CONHNH₂, or a said derivative thereof.

Preferably, only one of Y and Y¹ (preferably Y¹) is hydrogen. The compounds where both Y and Y¹ are hydrogen are believed to be novel as a class. At least some of the compounds where at least one of Y and Y¹ is -OH are novel and it is believed that, as a class, they have not previously been proposed for use against tumours. Preferably, B = B¹ = oxo.

Preferred compounds are those in which R¹ = -CH₂OH and R² = -CONHNH₂ and it is further preferred, when R¹ and R² are -CH₂OH and -CONHNH₂ respectively, that B = B¹ = oxo and either Y = Y ¹ = H or Y = -OH and Y¹ = H.

In a further aspect the invention provides a pharmaceutical preparation comprising a pharmaceutically acceptable carrier and a compound of the above structure. Any suitable pharmaceutically acceptable carrier can be used. The preparation should be suitable for administration in the chosen manner. In particular, it should be sterile and, if intended for injection, non-pyrogenic.

The aforementioned compounds of the invention or a formulation thereof maybe administered by any conventional method including enteral (for example oral and rectal) or parenteral (for example delivery into the nose or lung or injection into the veins, arteries, brain, spine, bladder, peritoneum, muscles or subcutaneous region. The compounds may be injected directly into the tumour. The treatment may consist of a single dose or a plurality of doses over a period of time. The dosage will be determined by the physician but may be between 0.01 mg and 1.0 g/kg/day, for example between 0.1 and 500 mg/kg/day. At least some compounds of the invention have a particularly low toxicity to normal mammalian cells and could be given in quite high doses, for example 50-300 mg/kg. (Compare the doxorubicin maximum dose of 5 mg/kg in rodents and 1-2 mg/kg in man.)

Whilst it is possible for a compound of the invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers. The carrier(s) must be "acceptable" in the sense of being compatible with the compound of the invention and not deleterious to the recipients thereof. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy . Such methods include the step of bringing into association the active ingredient (compound of the invention) with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Formulations in accordance with the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (eg povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (eg sodium starch glycollate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide desired release profile. Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient: and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of an active ingredient.

It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.

At least some of the compounds are useful as anticancer, antiviral and/or antiparasitic drugs and at least some of the anticancer compounds can be used against most malignancies. Particular tumours suitable for treatment in accordance with the invention include leukaemias, and cancers of the uterine cervix, head, neck, brain gliomas, breast, colon, lung, prostate, skin, mouth, nose, oesophagus, stomach, liver, pancreas and metastatic forms of any of these.

Particular viral infections suitable for treatment in accordance with the invention include those caused by the viruses herpes simplex virus I (HSV I); herpes simplex virus II (HSV II); varicella-zoster virus/Ellen (VZV Ellen); bovine papilloma virus (BPV); and human immunodeficiency virus (HIV).

Particular protozoal infections suitable for treatment in accordance with the invention include trichomoniasis; malaria (especially that caused by Plasmodium falciparum); trypanosomiasis (caused by Trypanosoma brucei and T. cruzi); and leishmaniasis.

At least some of the compounds are useful as antibacterial agents.

At least some of the compounds are useful as dyestuffs and may be combined with a carrier, diluent or mordant for dyeing purposes, for example for dyeing cotton, nylon and paper.

In a still further aspect, the invention provides a method of preparing a compound of the above structure comprising the steps of (a) oxidising the product obtainable by reacting leucoquinizarin (or a 5'-OH and/or 8'-OH derivative thereof) and an amino acid or derivative thereof having an -NH₂ group alpha to a carboxyl group to join the amino acid or derivative thereof to the leucoquinizarin ring at one of the carbonyl groups thereof; (b) converting one compound of Formula (I) to a second compound of Formula (I); or (c) when R⁵ = -OH, B² = B² = oxo and Y = Y¹ = H, by deprotection of a compound of Formula (IA), namely a compound corresponding to the final product of Formula (I) but in which R⁵ is a protected -OH group, for example alkoxy, such as methoxy.

Leucoquinizarin is the compound of Formula (II) below in which Y = Y² = H and D = D¹ = -OH. It may be reacted with the amino acid in an inert solvent such as dimethylformamide (DMF) at 80-100°C with a K₂CO₃ catalyst.

The reaction of the leucoquinizarin and the amino acid is typically carried out at -10°C to.95°C. Step (a) is carried out with any suitable oxidising agent, preferably oxygen, hydrogen peroxide or chloranil, in any miscible organic solvent/water system, for example ethanol/water, acetonitrile/water or dimethylsulphoxide/water or DMF/water at -10°C to 95°C, preferably 0-40°C. The addition of a small amount of alkali (for example NaOH) speeds up the oxidation.

Step (b), for example, consists of converting an ester to an amide, hydrazide, acid, salt or hydroxamic acid adjacent the carbonyl group α to the nitrogen atom joined to the anthraquinone ring, or converting any of those said possibilities to another of the said possibilities or to the ester. Such conversion may, for example, be carried out by reaction of the ester with ammonia (NH₃, sealed tube, 100°C) to produce the corresponding amide; by reaction with hydroxylamine (⁺NH₃OHCl-/NaOMe, MeOH, r.t., 1 week) to give the hydroxamic acid; and by reaction with hydrazine hydrate (in excess, MeOH, reflux, 2-3 h) or other asymmetric monosubstituted hydrazine (NH₂NHR⁴) to furnish the hydrazides. It is preferable to prepare these classes of compounds of the invention from the ester rather than to use a hydrazide, hydroxamic acid or amide derivative of an amino acid as the starting material.

Compounds of the invention in which B and/or B¹ are hydrogen are best prepared from (I) where B = B¹ = O by a 2 stage process. In stage 1, partial reduction is carried out using a modification of a standard literature procedure for reduction to a mixture of anthrones (L. Liebermann & A. Gimbel Ber. 1887, 20, 1854) with tin and HCl in glacial acetic acid to give a mixture of anthrones in which only one of B and B¹ is oxo.

In stage 2 (ref L. Cagliotti (1972) Organic Syntheses 52, 122), the C = O group in the 2 compounds of the aforementioned mixture is converted into a mixture of tosylhydrazones by reaction with tosylhydrazine (NH₂NH-tosyl) to give the corresponding mixture, which can be prepared in situ and not isolated.

Reaction of the desired tosylhydrazones with a reducing agent such as sodium cyanoborohydride (Na(CN)BH₃) in DMF at 50-100°C then affords the desired structure (I; B = B¹ = hydrogen), which is a 9, 10-dihydro-anthracene. The product of the reaction of the leucoquinizarin or derivative thereof and the amino acid or derivative thereof may be isolated by acidifying the reaction mixture and adding excess water followed by chloroform extraction. Evaporation of the chloroform extract affords the crude leuco form, which in some cases can be crystallised from toluene or benzene. These intermediates form a further aspect of the invention. They may be oxidised to yield the final compound of the invention. These intermediates are believed to have the structure of Formula (III):

wherein Y, Y¹, R⁵ and X are as defined in relation to Formula (I) and E and E¹ are independently hydrogen or hydroxyl. The compound is fully aromatic.

The compounds of Formula (III) where E = E¹ = OH can be isolated as above or they can be deliberately synthesised by a reduction reaction upon (I), for example by using sodium dithionite in aqueous DMF and NaOH or NaHCO₃ at 0°C-100°C, or alternatively by using zinc dust in glacial acetic acid-ethanol at room temperature (reflux). These are standard procedures for reducing anthraquinones (I) to 9.10-dihydroxyanthracenes (III): the dithionite reaction is disclosed in E. Grandmougin Ber (1906) 39, 3563 and the zinc reaction in H.M. Crawford et al (1952) J. Amer. Chem. Soc. 74, 4087. Where in (III) E = E¹ = H, these compounds could never be isolated from a reaction mixture described in the general procedure - but could be produced only by a reduction of (I)→ (III) as above. Methods exist for such a conversion for anthracene from anthraquinone, for example using a NH₃/zinc reduction: J.T. Traxler (1977) "Synthesis of Anthracenes from Anthraquinones" Synthetic Communications 161-166.

Step (c) may be carried out using any one of a variety of Lewis acids, for example boron trichloride (or tribromide or trifluoride) in chloroform or dichloromethane at -78°C (dry ice) for 30-60 minutes.

The protected compound (IA) can be made by a variation of the conversion of compound II→ compound IV in US Patent No 4 894 451 to Krapcho et al (incorporated herein by reference), namely by using an amino acid derivative (having a free primary amine group) as described above in place of the primary amine (III) in the said patent. The Roman numbers in the preceding sentence refer to the formulae of the said patent and not to the formulae of this specification. To avoid confusion they will hereinafter be referred to as K-II etc (K for Krapcho). The compound of K-II may be any 1,4-dialkoxy-anthracene-9,10-dione or similar 1,4-substituted compound and may be made. by the method disclosed in Krapcho et al (1984) J. Org. Chem. 49, 5254. The reaction of K-II with the amino acid to give K-IV may be achieved photochemically in, for example, a methylene chloride solution, using a 2-5 molar excess of the amino acid derivative. The solution is irradiated, the solvent removed and the product purified by column chromatography on silica gel and recrystallised from a suitable solvent. The K-IV compound (ie the compound of our formula IA) may then be converted into a compound of Formula (I) as above.

The invention will now be described in detail with reference to the accompanying drawings in which :-

Figure 1 shows the in vitro chemosensitivity results for the compound of

Example 10 (24 hour incubation);

Figure 2 shows the in vitro chemosensitivity results for the compound of Example 9 (24 hour incubation);

Figure 3 shows the results of cross-resistance tests with the compound of

Example 10 (24 hour incubation);

Figure 4 shows the results of the topoisomerase II decatenation assay;

Figure 5 shows the results of the topoisomerase II relaxation assay; and Figure 6 shows the results of the topoisomerase I relaxation assay.

General Synthetic Chemistry

The N-anthracenyl amino acid derivatives of the present invention may be made by reacting an alpha-amino acid with [2H,3H]-9,10-dihydroxyanthracene-1 ,4- dione or [2H]-9, 10-dihydroxyanthracene-1-one (Formula II below) and then oxidising the intermediate product as shown below.

wherein D and D¹ are independently hydrogen or hydroxyl and Y and Y¹ are as defined above.

The K₂CO₃ is used to liberate the free amine NH₂CHR¹R² from its hydrochloride salt. It may therefore be replaced by a variety of other bases including Na₂CO₃, pyridine, triethylamine or diisapropylamine etc (but not alkali hydroxides or any other base likely to cause hydrolysis of the ester function in R² of some of the amines).

The DMF may be replaced by DMA (dimethylacetamide), THF (tetrahydrofuran), dioxan, HMPA (hydroxymethylphosphoramide) or any other aprotic polar solvent. Alcoholic solvents can also be used but the scale of reaction is limited by the poor solubility of leucoquinizarin (II, D = OH, D¹ = OH, Y = H, Y¹ = H) in these solvents.

The product obtained has been found to be mono substituted, uncontaminated by bis-substitution products and obtained in high yield. These highly crystalline and substantially optically pure N-anthracenyl amino acid derivatives can be characterised by a range of analytical and spectroscopic methods.

General Procedure Leucoquinizarin is obtainable commercially, for example from Yorkshire Chemicals, Leeds, UK, or it may be made by reducing quinizarin (available from Aldrich), for example with sodium dithionite in DMF and NaHCO₃.

A solution of [2H,3H]-2,3-dihydro-9, 10-dihydroxyanthracene-1 ,4-dione (leucoquinizarin), 2.4 g (0.01 rnol) (obtainable from Yorkshire Chemicals plc, Leeds. UK) in dimethylformamide (40 cm³) was heated at 95 °C with a mixture of the appropriate a-amino acid derivative NH₂CH(R¹)R² (0.01 rnol ) and anhydrous potassium carbonate (0.05 mol) for 1 h under argon. The reaction mixture was cooled to 25°C, ethanol (20 cm³) and water (2 cm³) were added and the solution was aerated for 5h.

The solution was diluted with water (70 cm³) and extracted several times with chloroform. The extracts were combined, dried (MgSO₄), filtered and evaporated to a purple solid. A solution of the foregoing solid in the minimum amount of toluene-ethyl acetate (9: 1 to 4: 1) was applied to a silica gel column (5 × 25 cm) and eluted by the same solvent mixture. Chloroform:methanol (9: 1) or chloroform:ethyl acetate (4: 1) can be used instead. The fractions containing the product were combined, filtered and evaporated to a solid residue which gave an analytically pure sample upon recrystallisation from the appropriate solvent. For esters, ethanol is the preferred solvent. Example 1: (S)-N- (4'-hydroxy-9' , 10'-dihydro-9',10'-dioxo-1 '-anthryl)-L-alanine methyl ester

The general method given above was followed, using L-alanine methyl ester (obtainable from Aldrich Chemical Co, Poole, Dorset, UK) as the α-amino acid derivative starting material.

Yield 58 %; m.p. 170°C.e.i. mass spectrum m/z = 325 (M⁺), 266 (100%) M, 325. ¹H nmr spectrum (CDCl₃; 270 MHz) δ: 1.7 (3H, d, CHCH₃); 3.8 (3H, s, OCH₃); 4.4 (1H, m, CH); 7.05 (1H, d. H-2'): 7.27 (1H, d. H-3'): AA'.BB'-Signal [δ_A 7.7 - 7.9 (H-6,H-7): δ_B 8.2 - 8.4 (H - 5',H-8')]; 10.5 (1H, d, NH); 13.6 (1H, s, 4'-OH)

Anal. Calcd. for C₁₈H₁₅NO₅ C: 66.46: H: 4.62; N: 4.31 %

Found: C: 66.45, H: 4.35, N: 4.25% Example 2: (S)-N-(4'-hydroxy-9' ,10'-dihydro-9 '10'-dioxo-1'-anthryl)-L-serine ethyl ester The same general method was followed, using L-serine ethyl ester (obtainable from Aldrich) as the starting material.

Yield 63 % m.p. 189°C (crystals from ethanol) d.c.i. mass spectrum m/z = 356, (M⁺ + 1)

1H nmr spectrum (CDCl₃; 270MHz)

δ: 1.3 (3H, t, CH₃); 2.65 (1H, br s, CH₂OH, exch. D₂O); 4.15 (2H, d, CH₂OH); 4.3 (2H, q, CH₂CH₃); 4.5 (1 H, m, CH); 7.13 (1H, d, H-2'); 7.21 (1H, d, H-3'); AA'BB'-Signal - [δ_A, 7.72 - 7.82 (2H, m. H-6',H-7'); δ_B, 8.28 - 8.34 (2H, m, H-5', H-8')]; 10.75 (1H, d, NH, exch. D₂O): 13.52 (1H, s, 4'- OH, exch. D₂O). Anal. Calcd for C₁₉H₁₇O₆N C: 64.23; H: 4.79, N: 3.94% . Found: C: 64.25; H: 4.65; N: 3.85 % .

Example 3:

For preparation of the sodium salt series, the required amine is NH₂CH(R¹)R² where R¹ = ONa. The amine was obtained by the addition of 1 equivalent of sodium hydroxide in water to the α-amino acid and used directly. Example 4:

Acid compounds were prepared by neutralisation of the sodium salts prepared as in Example 3 followed by recrystallisation from aqueous ethanol. Example 5: N-(4'-hydroxy-9' ,10'-dihydro-9' ,10'-dioxo-1 '-anthryl)-glycylglycine

This was prepared by the direct coupling method, in other words a dipeptide derivative was used as the starting material.

The requisite amine was used as the sodium salt (prepared by addition of one equivalent of sodium hydroxide to the dipeptide glycylglycine, (gly-gly), NH₂CH₂CONHCH₂COOH), giving the required NH₂CH₂CONHCH₂COONa. After aeration, the crude product was isolated as a precipitate upon pouring the reaction mixture into 2M hydrochloric acid

Yield, 47% , m.p. 205°C

1H nmr spectrum (d₆-DMSO, 270 MHz)

δ: 3.6 (2H, m, CH₂COOH); 4.15 (2H, d, NH-CH₂), 7.28 (2H. m. H-2', H- 3'); 7.8 (2H, m, H-6', H-7;); 8.23 (2H, m. H-5', H-8'; 10.4,(1H, m, Ph-NH); 11.4 (1H, m, unresolved, CO-NH); 13.0 (2H, m, unresolved. COOH and 4'-OH, each exch. D₂O).

Anal. Calcd. C₁₈H₁₄O₆N₂, C: 61.02, H: 3.95, N: 7.91 % ; Found C: 61.15. H: 3.90, N: 7.85%

Example 6: (S)-N-(4',8'-dihydroxy-9^,,10'-dihydro-9' 10^,-dioxo-1 '-anthryl)-L-alanine This was an example of an alternative route to molecules with remote functionality, eg an 8'-OH group (ie Y is -OH).

1 ,4,5-trihydroxyanthracene-9, 10-dione (4.0 g) was part dissolved in a mixture of ethanoic acid (50 cm³) and dimethylformamide (150 cm³). Zinc powder was added and the mixture was stirred at 40°C for 1.5h, under nitrogen. The mixture was filtered and the filtrate was evaporated to dryness. Small portions of added ethanol were repeatedly evaporated from the residue to afford the leuco form of the starting material. The foregoing leuco form was dissolved in dimethylacetamide (50 cm³) and a solution of L-alanine ( 1.2 g) in dimethylacetamide containing sodium hydroxide (0.47 g) was then added. The resulting mixture was heated at 95°C for 3 h, under nitrogen. A steady stream of oxygen was bubbled through the solution at room temperature for 4 h after which the solution was poured, with stirring, into hydrochloric acid (2M; 300 cm³) at 0°C. The resultant purple precipitate was collected by filtration and air-dried. The crude product was dissolved in the minimum amount of chloroform and applied to a silica-gel column, eluting firstly with chloroform and then chloroform-methanol (9: 1). The fractions containing the product were combined, filtered, dried (MgSO₄) and evaporated to dryness. The residue was recrystallised from propan-2-ol to afford the title compound as purple platelets. Yield 37% . m.p. 238°C ¹H nmr spectrum (d₆ - DMSO; 270 MHz)

δ: 1.43 (3H, d, CH₃); 4.21 (1H, m, CH)

7.01 (1H, m, H-7'); 7.1-7.4 (2H, m, H-2' , H-5'); 7.53-7.90 (2H, m, H-3', H- 6'); 10.62 (1H, m, NH); 13.61 , 13.97, 14.43 (3H, 4'-OH, 8'-OH, COOH; each exch. D₂O).

e.i. mass spectrum, 314 (M⁺), 299 (M-CH₃)

i.r. spectrum V_max (KBr)cm^-1 : 3400 (OH)

2920 (N-H), 1590 (quinone)

Anal. C₁₆H₁₂O₆N, Found: C : 61 .1 ; H: 3.75; N: 4.45 %

Example 7: (S)-N-(4'-hydroxy-9',10'-dihydro-9' , 10'-dioxo- 1 '-anthryl)-L-tyrosine ethyl ester

This was made from L-tyrosine ethyl ester as in Example 1 above.

Example 8: (S)-N-(4'-hydroxy-9' ,10'-dihydro-9', 10'-dioxo-1'-anthryl)-L- alanine hydrazide

This was made from L-alanine hydrazide as in Example 1 above.

Example 9: (S)-N-(4'-hydroxy-9', 10'-dihydro-9' , 10'-dioxo-1 '-anthryl)-L- tyrosine hydrazide

This was made from L-tyrosine hydrazide as in Example 1 above. (S)-N-(4'- hydroxy-9' , 10'-dihydro-9' , 10'-dioxo- 1 '-anthryl)-L-tyrosine ethyl ester (0.19 g; 4.46 × 10^-4 mol) was dissolved in methanol (20 cm³). Hydrazine hydrate (0.045 g; 9 × 10^-4 mol) was added and the solution was heated under reflux for 3 h. The solution was evaporated to dryness and the residue was recrystallised from absolute alcohol to afford an analytically pure sample of the title compound in 87% yield, m.p. 284°C.

i.r. spectrum ʋ_max (Nujol) cm^-1 3320, 3200 (NHNH₂) 1640 (CO) Anal. Cald. C: 66.35; H: 4.57, N: 10.10% for C₂₃H₁₉O₅N₃; Found C: 66.35: H: 4.35.. N: 10.05% . M₁ 416.

Example 10: (S)-N-(4'-hydroxy-9', 10'-dihydro-9', 10'-dioxo-1 '-anthryl)-L-serine hydrazide This was made from L-serine hydrazine as in Example 1 above.

Example 11; (S)-N-(9' , 10'-dihydro-9', 10'-dioxo- 1'-anthryl)-L-serine ethyl ester (Procedure A) This is an example of a compound in which, in Formula (I), Y = Y¹ = R⁵ = H.

1-Chloroanthracene-9, 10-dione (0.01 mol) was dissolved in diemthylsulphoxide (50 cm³) and heated to 95°C. Anhydrous potassium carbonate (0.05 mol) was added followed by L-serine ethyl ester hydrochloride (0.03 mol) and heating was continued for 1 h under nitrogen. The crude product was precipitated from the reaction mixture upon addition of water, recovered by filtration and airdried. The residue was dissolved in toluene-ethyl acetate (9: 1), applied to a silica-gel column and eluted by the same solvent mixture. Fractions containing the product were combined, filtered and evaporated to yield a red residue which gave the title compound as analytically pure crystals from ethanol m.p. 158 °C: Yield 24% . Example 12: (S)-N-(9', 10'-dihydro-9' , 10'-dioxo-1'-anthryl)-L-serine ethyl ester (Procedure B)

1-Hydroxyanthraquinone (5 g) was dissolved in 2% aqueous sodium hydroxide solution (500 cm³) at 80°C under nitrogen. Sodium dithionite (8 g) was added and heating was continued for a further 45 min. Addition of excess 5 M hydrochloric acid gave a precipitate of crude leuco-1-hydroxyanthraquinone.

The precipitate was filtered, washed with water and dried in vacuo. The foregoing leuco derivative was dissolved in dimethylformamide (50 cm³) and heated at 95°C with L-serine ethyl ester hydrochloride (3.8 g) and anhydrous potassium carbonate (5 g) for 1 h under nitrogen. The reaction mixture was then aerated for 4 h at 25°C and diluted with water (500 cm³) . Chloroform extraction gave the title compound in crude form after evaporation. At this point the work-up procedure was as in Example 1 1 , m.p. 158°C; Yield 54% . Anal. cald. C: 67.26; H: 5.01 ; N: 4.13 % . Found: C: 67.35; H: 4.95; N:

3.95 % . The d.c.i. Mass Spectrum had m/z = 340 (M⁺ + 1), 40%); 310

(70%); 224 (100% , 210 (60%).

Example 13: (S)-N-(4'-hydroxy-9' , 10'-dihydro-9' , 10'-dioxo-1'-anthryl)-L- phenylalanine methyl ester.

The compound was made by the method of Example 1 , using L-phenylalanine methyl ester (Aldrich) as the starting material. e.i. mass spectrum m/z = 401 (M⁺); 310 (100%) M-CH₂Ph; 250 (90%), M- (CH₂Ph, and COOCH₃). ¹H nmr spectrum (200 MHz) in CDCl₃, h: 3.28 (2H, m, CH₂); 3.75 (3H, s, CH₃); 4.6 (1H, q, CH); 6.98 (1H, d, H-2'); 7.18 (1H, d, H-3'); 7.3 (5H, s, Ph); AA'BB' Signal [δ_A 7.68-7.84 (H-6 ', H-7'); δ_B 8,27- 8.40 (H-5' , H-8')]; 10.6 (1 H, d, NH, exch. D₂O); 13.55 (1H, s, OH exch. D₂O). m.p. 204°C. Example 14: (S)-N-(4',8'-dihydroxy-9' , 10'-dihydro-9' ,10'-dioxo- 1'-anthryl)-L-serine ethyl ester

The compound was prepared as in Example 6 using L-serine ethyl ester and is structure (I) where: Y = OH; Y¹ = H; R⁵ = OH; B = B¹ = oxo and X =

-NHCH-R² where R¹ = CH₂OH and R² = COOEt

R¹

d.c.i. mass spectrum had m/z = 372, (M⁺ + 1). ¹H nmr spectrum (CDCl₃; 270 MHz) δ: 1.29 (3H, t, CH₃); 2.5 (1H, s. CH₂OH, exch. D₂O); 4.10 (2H, d. CH₂OH): 4.25 (2H, q, CH₂CH₃); 4.55 ( 1H, m, CH); 7.05 (1H, m, H-7'): 10.5 (1H, m, NH); 7.12-7.4 (2H, m. H-2', H-5'); 14.3 (2H, 4'-OH, 8'-OH, each exch. D₂O). mp. 220°C.

Example 15: (S)-N-(4',8'-dihydroxy-9', 10'-dihydro-9', 10'-dioxo-1 '-anthryl)- L-serine hydrazide Prepared from the ethyl ester Example 14 according to the method described in Example 9.

Example 15 is structure (I), where Y = OH; Y¹ = H; R⁵ = OH; B = B¹ = oxo and X = -NHCH-R² where R¹ = CH₂OH and R² = CONHNH₂.

R¹

m.p. 276°C. i.r. spectrum ʋ_max (Nujol) cm^-1 3320, 3195 (NHNH₂), 1642 (CO). Anal. Cald. C: 57.14; H: 4.76, N: 11.76 % for C₁₇H₁₅O₆N₃; Found C: 57.15, H: 4.55, N: 11.65%, M. 357. d.c.i. mass spectrum m/z = 358 (M⁺ + 1). Example 16: (S)-N-(4'-hydroxy-9', 10'-dihydro-9', 10'-dioxo-1 '-anthryl)-L-valine methyl ester

Prepared from L-valine methyl ester according to Example 1 . ¹H nmr spectrum (CDCL₃; 270 MHz), δ: 1 .05 (3H, d. CH₃): 1.15 (3H, d, CH₃); 3.7 (OCH₃); 4.05 (1H, m, CH); 7.1 (2H, unresolved, H-2', H-3'); 7.5-7.8 (2H, m, H-6', H-7'); 8.1-8.3 (2H, m, H-5' , H-8'); 10.5 (1 H, d, NH): 13.45 (1H, s, OH). i.r. spectrum had ʋ_max (KBr), cm^-1 1745 (COOMe), 1620 (quinone). e.i.. mass spectrum had m/z = 353 (M ⁺). Biological Examples

The following experiments demonstrate the actions of seven compounds of the invention. In vitro Chemosensitivity

Seven anthracenyl-peptides were screened for in vitro activity against a panel of 4 Chinese hamster ovary (CHO) cell lines and 2 human ovary (A2780) cell lines whose characteristics and special features are summarised in Table 1. An MTT assay was employed with a drug exposure time of 24 hr. In preliminary experiments a linear or near linear relationship between absorbance and cell number was established for all 6 cell lines.

Topoisomerase Assays

Drug inhibition assays were performed on purified human placental topo I and II obtained from Topogen (Ohio. USA) and Hela cell topo II purified in house; For all assays drugs at a concentration of 1 μg/ml-50 μg/ml (approx 3 μM-150 μM) were incubated with enzyme and DNA substrate for 30-45 min at 37°C in a pH 7.5 buffer system. The reaction was stopped by adding loading buffer and samples were then run on a 1 % agarose gel, stained with ethidium bromide and photographed. For decatenation assays, DNA extracted from the kinetoplast of C. fasiculata (kDNA) was the substrate; for relaxation assays supercoiled plasmid RGY was the substrate. Appropriate positive control known inhibitors were run: doxorubicin for topo II decatenation. VM 26 for topo II relaxation and camptothecin for topo I relaxation. Results

In vitro Chemosensitivity

IC₅₀ and IC₉₀ values for the newly synthesised anthracenyl-peptides against all six cell lines are shown in Table 2. In general, the compounds had values in the μM range making them 10-100 fold less potent than doxorubicin. The compound of Example 10 was significantly more effective in ADR-1 (the line hypersensitive to topo II inhibitors, see Figure 1) whereas the other compounds with activity were not (see Figure 2). On the whole, the compounds were non- cross resistant in the MDR models (see Figure 3) and this may be related to their high lipophilicity.

The compound of Example 15 was significantly more effective in the ADR-r cell-line (the resistant line due to decreased topo II) which has elevated topo I activity.

Topoisomerase Assays

Inhibition data for seven compounds of the invention are summarised in Table 3 and illustrated in Figures 4. 5 and 6 for topo II decatenation, topo II relaxation and topo I relaxation respectively. The compound of Example 10 inhibited topo II decatenation significantly (see Figure 5) with an approximate IC₅₀ concentration of 5 μg/ml (15.2 μM) which is very similar to its IC₅₀ in the cell lines and is also similar to the IC₅₀ value for inhibition of decatenation by doxorubicin (approx 4 μM). The compound of Ex. 15 was a particularly good inhibitor of topo II (IC₅₀ = 4 μM), and topo I, and proved to be most active with an IC₅₀ for cell kill against A2.780 in the low micromolar range (see Table 2).

It was found that one compound (Ex. 10) proved to be a good inhibitor of purified human topo II and produced a pattern of activity against a panel of cell lines consistent with a mechanism of action involving topo II. Additionally, it proved to be either completely non-cross resistant or considerably less crossresistant in models of MDR. One compound (Ex. 15) proved 10 be a particularly good inhibitor of purified human topo II and topo I and was significantly more effective against a cell line with increased topo I activity.

One compound (Ex. 9) was a poor inhibitor of both topo I and II but displayed in vitro activity against the cell lines.

The following examples illustrate pharmaceutical formulations according to the invention in which the active ingredient is a compound of any of the above structures.

Example A: Tablet

Active ingredient 100 mg

Lactose 200 mg

Starch 50 mg

Polyvinylpyrrolidone 5 mg

Magnesium stearate 4 mg

359 mg

Tablets are prepared from the foregoing ingredients by wet granulation followed by compression. Example B: Ophthalmic Solution

Active ingredient 0.5 g

Sodium chloride, analytical grade 0.9 g

Thiomersal 0.001 g

Purified water to 100 ml

pH adjusted to 7.5

Example C: Tablet Formulations

The following formulations A and B are prepared by wet granulation of the ingredients with a solution of povidone, followed by addition of magnesium stearate and compression. Formulation A

mg/tablet mg/tablet

(a) Active ingredient 250 250

(b) Lactose B.P. 210 26

(c) Povidone B.P. 15 9

(d) Sodium Starch Glycollate 20 12

(e) Magnesium Stearate 5 3

500 300

Formulation B

mg/tablet mg/tablet

(a) Active ingredient 250 250

(b) Lactose 150 -

(c) Avicel PH 101* 60 26

(d) Povidone B.P. 15 9

(e) Sodium Starch Glycollate 20 12 (f) Magnesium Stearate 5 3

500 300

Formulation C

mg/tablet

Active ingredient 100

Lactose 200

Starch 50

Povidone 5

Magnesium stearate 4

359

The following formulations, D and E, are prepared by direct compression of the admixed ingredients. The lactose used in formulation E is of the direction compression type.

Formulation D

mg/capsule

Active Ingredient 250

Pregelatinised Starch NF15 150

400

Formulation E

mg/capsule

Active Ingredient 250

Lactose 150

Avicel ^x 100

500 Formulation F (Controlled Release Formulation)

The formulation is prepared by wet granulation of the ingredients (below) with a solution of povidone followed by the addition of magnesium stearate and compression.

mg/tablet

(a) Active Ingredient 500

(b) Hydroxypropylmethylcellulose 1 12

(Methocel K4M Premium)^x

(c) Lactose B. P. 53

(d) Povidone B.P.C. 28

(e) Magnesium Stearate 7

700 Drug release takes place over a period of about 6-8. hours and was complete after 12 hours.

Example D: Capsule Formulations Formulation A

A capsule formulation is prepared by admixing the ingredients of Formulation D in Example C above and filling into a two-part hard gelatin capsule. Formulation B {infra) is prepared in a similar manner.

Formulation B

mg/capsule

(a) Active ingredient 250

(b) Lactose B.P. 143

(c) Sodium Starch Glycollate 25 (d) Magnesium Stearate 2

420

Formulation C

mg/capsule

(a) Active ingredient 250

(b) Macrogol 4000 BP 350

600 Capsules are prepared by melting the Macrogol 4000 BP. dispersing the active ingredient in the melt and filling the melt into a two-part hard gelatin capsule.

Formulation D

mg/capsule

Active ingredient 250

Lecithin 100

Arachis Oil 100

450 Capsules are prepared by dispersing the active ingredient in the lecithin and arachis oil and filling the dispersion into soft, elastic gelatin capsules.

Formulation E (Controlled Release Capsule) The following controlled release capsule formulation is prepared by extruding ingredients a, b, and c using an extruder, followed by spheronisation of the extrudate and drying. The dried pellets are then coated with release-controlling membrane (d) and filled into a two-piece, hard gelatin capsule. mg/capsule

(a) Active ingredient 250

(b) Microcrystalline Cellulose 125

(c) Lactose BP 125

(d) Ethyl Cellulose 13

513

Example E: Injectable Formulation Active ingredient 0.200 g

Sterile, pyrogen free phosphate buffer (pH7.0) to 10 ml

The active ingredient is dissolved in most of the phosphate buffer (35-40°C), then made up to volume and filtered through a sterile micropore filter into a sterile 10 ml amber glass vial (type 1 ) and sealed with sterile closures and overseals.

Example F: Intramuscular injection Active ingredient 0.20 g

Benzyl Alcohol 0.10 g

Glucofurol 75^x 1 .45 g

Water for Injection q.s. to 3.00 ml The active ingredient is dissolved in the glycofurol. The benzyl alcohol is then added and dissolved, and water added to 3 ml. The mixture is then filtered through a sterile micropore filter and sealed in sterile 3 ml glass vials (type 1). Example G: Syrup Suspension

Active ingredient 0.2500 g

Sorbitol Solution 1.5000 g

Glycerol 2.0000 g

Dispersible Cellulose 0.0750 g

Sodium Benzoate 0.0050 g

Flavour, Peach 17.42.3169 0.0125 ml

Purified Water q.s. to 5.0000 ml

The sodium benzoate is dissolved in a portion of the purified water and the sorbitol solution added. The active ingredient is added and dispersed. In the glycerol is dispersed the thickener (dispersible cellulose). The two dispersions are mixed and made up to the required volume with the purified water. Further thickening is achieved as required by extra shearing of the suspension.

Example H: Suppository

mg/suppository

Active ingredient (63 μm)* 250

Hard Fat, BP (Witepsol H15 - Dynamit Novel) 1770

2020

The active ingredient is used as a powder wherein at least 90% of the particles are of 63 μm diameter or less.

One fifth of the Witepsol H15 is melted in a steam-jacketed pan at 45°C maximum. The active ingredient is sifted through a 200 μm sieve and added to the molten base with mixing, using a silverson fitted with a cutting head, until a smooth dispersion is achieved. Maintaining the mixture at 45°C. the remaining Witepsol H15 is added to the suspension and stirred to ensure a homogenous mix. The entire suspension is passed through a 250 μm stainless steel screen and, with continuous stirring, is allowed to cool to 40°C. At a temperature of 38°C to 40°C 2.02 g of the mixture is filled into suitable plastic moulds. The suppositories are allowed to cool to room temperature.

Example I: Pessaries

mg/pessary

Active ingredient 250

Anhydrate Dextrose 380

Potato Starch 363

Magnesium Stearate 7

1000

The above ingredients are mixed directly and pessaries prepared by direct compression of the resulting mixture.

Example J: Dyestuff

1.0 g of the compound of Example 1 is dissolved in 100 ml an aqueous solution of ethanol (50% v/v) and applied to a cotton web. Legend to Figure 4: (inhibition of topoisomerase II mediated decatenation by newly synthesised anthracenyl peptides); Lane 1 kDNA standard; Lanes 2 and 12 Topo II+ kDNA; Lanes 6-8, 1-50μg/ml Example 10; Lanes 9-11, 1-50μg/ml Example 9. Legend to Figure 5: (inhibition of topoisomerase II mediated relaxation by newly synthesised anthracenyl peptides); Lane 1 supercoiled, Lane 2 Topo II + supercoiled; Lanes 4-5 VM 26; Lanes 6-8, 1-50μg/ml Example 10; Lanes 9-11, 1-50 μg/ml Example 9. Legend to Figure 6: (inhibition of Topoisomerase I mediated relaxation by newly synthesised anthracenyl peptides); Lane 1 supercoiled, Lane 2 Topo I + supercoiled; Lanes 4+5 Camptothecin; Lanes 6-8, 1-50μg/ml Example 10; Lanes 9-11, 1-50μg/ ml Example 9.

Claims

A compound having the structural formula (I):

where Y and Y¹ are independently hydrogen or hydroxyl, B and B¹ are independently oxo or hydrogen, R⁵ is hydrogen or hydroxyl and X is the residue of an amino acid, joined to the ring shown via the nitrogen atom of the amino acid adjacent the acid group thereof, or a derivative of a said amino acid.

2. A compound according to Claim 1 wherein X is a group:

-N(R¹)H-C(H) -R²

wherein R₁ = CH₂C₆H₄OH, -CH₃, or -CH₂OH and R₂ = -COOC₂H₅, - COOCH₃ or -CONHNH₂, or a derivative thereof.

3. A compound according to Claim 2 wherein R₁ = CH₂OH and R₂ = CONHNH₂, or a derivative thereof.

4. A compound according to any one of the preceding claims wherein B

= B¹ = oxo.

5. A compound according to any one of the preceding claims wherein Y = Y¹ = H.

6. A pharmaceutical preparation comprising a pharmaceutically acceptable carrier and a compound according to any one of Claims 1 to 5.

7. A compound according to any one of Claims 1 to 5 for use in medicine.

8. The use of a compound according to any one of Claims 1 to 5 in the manufacture of a medicament for treating cancer in humans or mammals.

9. A dyestuff composition comprising a compound according to any one of Claims 1 to 5 and a dyestuff composition diluent, carrier or mordant.

10. A process for preparing a compound according to any one of Claims 1 to 5 comprising the steps of:

(a) oxidising the product obtainable by reacting leucoquinizarin or a 5'- OH, 8'-OH or 5' ,8'-(-OH)₂ derivative thereof and an amino acid or derivative thereof having an -NH₃ group alpha to a carboxyl group to join the amino acid or said derivative thereof to the leucoquinizarin ring at one of the carbonyl groups thereof; or (b) converting one compound of Formula (I) to a second compound of

Formula (1).

11. A compound of formula (III):

wherein Y, Y¹, R⁵ and X are as defined in Claim 1 and E and E¹ are independently hydrogen or hydroxyl.