Anthracene derivatives for use as anticancer agents

A compound having the structural formula, ##STR1## where R.sup.1 and R.sup.2 are independently hydrogen or hydroxyl. R.sup.2 and R.sup.3 are independently oxo or hydrogen, one of R.sup.5 and R.sup.6 is A--B and the other is hydrogen, hydroxyl, or a group A, wherein each A is a spacer group providing --NH-- or --CO-- in the bond with B (if present); at least one A group does not provide the residue of an .alpha.-amino acid adjacent the anthraquinone nucleus and the A of any A--B moiety is joined to the anthraquinone nucleus via an --NH-- bond, and each B is a peptide group or a physiologically acceptable derivative thereof. The compounds are useful as antitumor compounds.

This application is a 371 of PCT/GB94/02128 filed Sep. 30, 1994. 
The present invention relates to compounds which are based on an 
anthraquinone nucleus, for use in medicine or as dyes. 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, camptothecin, 
topotecan, M-AMSA, VM26 and the ellipiticines. These drugs are believed to 
inhibit topo II by stabilising a protein/drug/nucleic acid ternary complex 
termed the clearable 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. 
U.S. Pat. No. 4,894,451 describes asymmetrically substituted 
anthracene-1,4-dione compounds of Formula (A): 
##STR2## 
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 co-pending WO 93/19037 describes compounds having the structural 
formula: 
##STR3## 
where Y and Y.sup.1 are independently hydrogen or hydroxyl, B and B.sup.1 
are independently oxo or hydrogen, R.sup.5 is hydrogen or hydroxyl and X 
is the residue of an .alpha. amino acid or a derivative of an .alpha. 
amino acid, joined to the ring shown via the nitrogen atom of the amino 
acid adjacent the acid group thereof. These compounds provide clinically 
active drugs and coloured compounds useful as drugs. 
EP-A-0 295 316 discloses symmetrically-substituted compounds for use in 
anti-tumour therapy, namely 1,4-bis(aminoalkyl- and 
hydroxyaminoalkyl)-amino!-5,8 -dihydroxyanthraquinones. 
A peptidic DNA-binding motif, the tetrapeptide SPKK (Ser-Pro-Lys-Lys) has 
been proposed (Suzuki (1989) EMBO J. 8, 797). SPKK dimers and hexamers 
were shown to compete with the AT-specific DNA-binding dye Hoechst 33258; 
nevertheless, they present a lower degree of specificity than Hoechst 
(Churchill & Suzuki (1989) EMBO J. 8, 4189; Suzuki (1989) EMBO J. 8, 797). 
Statistical studies suggested that SPKK forms a .beta.-turn stabilized by 
an additional hydrogen bond between the Ser side chain OH group and the 
main chain NH group of the third residue Lys. A model was devised for the 
S.sub.2 peptide, SPKKSPKK, in which the amides of Ser (the only amides to 
be free in the .beta.-turn/Asx-turn mixed conformation) were relatively 
well overlapped onto the amides of netropsin known to form three centered 
hydrogen bonds with DNA. Thus, a succession of SPKK motifs may bind to 
AT-rich sequences in the minor groove by adopting a crescent shape similar 
to that of netropsin and also by using the same specific hydrogen bonds. 
Bailly et al (1992) Anti-Cancer Drug Design 7, 83-100 describe 
anilinoacridine derivatives containing the nucleic acid-binding unit SPKK. 
The peptide is joined to the acridine heterocyclic ring system at the 
position opposite the N heteroatom in the middle ring. 
A series of papers from Morier-Teissier et al (1989) Anti-Cancer Drug 
Design 4, 37-52; ibid. (1990), 5, 291-305; (1993) J Med Chem 36, 
2084-2090! disclosed various copper-chelating asymmetric 
peptide-anthraquinone compounds using a Gly-His-Lys moiety, or sometimes 
just the initial Gly, attached directly to the 4-position of the 
anthraquinone ring, with the 1-position being substituted by a hydroxyl 
group. 
JP,A,82, 141456 describes 
N-4-(9,10-dihydro-9,10-dioxo-1-anthracenyl)amino!carbonyl!benzoyl!-D,L-a 
lanine as a dyestuff. 
Morier-Teissier et al (1993) J. Med. Chem. 36, 2084-2090 describes the 
synthesis of anthraquinone bisubstituted by the copper chelating peptide 
Gly-Gly-His. 
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): 
##STR4## 
where R.sup.1 and R.sup.2 are independently hydrogen or hydroxyl, R.sup.3 
and R.sup.4 are independently oxo, hydroxyl or hydrogen, 
one of R.sup.5 and R.sup.6 is A--B and the other is hydrogen, hydroxyl, or 
a group A, wherein, the or each A is independently a spacer group 
providing --NH-- or --CO-- in the bond with B (if present), at least one A 
group does not provide the residue of an .alpha.-amino acid adjacent the 
anthraquinone nucleus and the A of any A--B moiety is joined to the 
anthraquinone nucleus via an --NH-- bond, and 
B is a peptide group; 
or a physiologically acceptable derivative thereof. 
Clearly, when R.sup.3 or R.sup.4 are oxo, the single line to the ring 
represents a double bond. 
By a "spacer group" A we mean a bifunctional group providing either 
--NH.sub.2 or --COOH at its extremities, but having an --NH-- link to the 
ring. 
It is preferred if a given spacer group is internally symmetrical as this 
avoids mixtures of products. 
It is further preferred if the spacer groups are derived from 
.alpha.,.omega.-diamines. A .alpha.,.omega.-dicarboxylic acid can be used 
to extend the diamine group. 
The .alpha.,.omega.-diamine may be an .alpha.,.omega.-diamino alkane. 
.alpha.,.omega.-Diamino alkanes include, for example NH.sub.2 
(CH.sub.2).sub.n NH.sub.2 wherein n is 1 to 12, polyamines, for example 
diethylenetriamine NH.sub.2 CH.sub.2 CH.sub.2 NHCH.sub.2 CH.sub.2 NH.sub.2 
and chains including other heteroatoms for example NH.sub.2 CH.sub.2 
CH.sub.2 OCH.sub.2 CH.sub.2 NH.sub.2 or NH.sub.2 CH.sub.2 CH.sub.2 
SCH.sub.2 CH.sub.2 NH.sub.2. 
Examples of suitable .alpha.,.omega.-diamines are shown in the table. 
TABLE I 
______________________________________ 
.alpha.,.omega.-Diamine 
______________________________________ 
H.sub.2 N(CH.sub.2).sub.n NH.sub.2,n = 2,3,4 
H.sub.2 N(CH.sub.2).sub.n NH.sub.2,n = 7,8,9 
H.sub.2 N(CH.sub.2).sub.n NH.sub.2,n = 5,6 
H.sub.2 N(CH.sub.2).sub.2 SS(CH.sub.2).sub.2 NH.sub.2 (cystamine) 
H.sub.2 N(CH.sub.2).sub.2 NH(CH.sub.2).sub.2 NH.sub.2 
H.sub.2 N(CH.sub.2).sub.2 O(CH.sub.2).sub.2 NH.sub.2 
H.sub.2 N(CH.sub.2).sub.3 NH(CH.sub.2).sub.3 NH.sub.2 
H.sub.2 N(CH.sub.2).sub.2 NHCOCONH(CH.sub.2).sub.2 NH.sub.2 
H.sub.2 N(CH.sub.2).sub.2 NH(CH.sub.2).sub.2 NH(CH.sub.2).sub.2 NH.sub.2 
(TET) 
H.sub.2 N(CH.sub.2).sub.3 N(CH.sub.2 CH.sub.2).sub.2 N(CH.sub.2).sub.3 
NH.sub.2 
______________________________________ 
1,3-diaminopropane and 1,6-diaminohexane are preferred. 
Other suitable diamines include those with alkene double bonds or saturated 
ring systems or substituted (para, ortho or meta) aryl groups. These 
provide conformationally restrained structures. For example cis and trans 
versions of NH.sub.2 --(CH.sub.2).sub.n CH.dbd.CH--(CH.sub.2).sub.m 
NH.sub.2 and NH.sub.2 (CH.sub.2).sub.n C.sub.6 H.sub.4 (CH.sub.2).sub.m 
NH.sub.2 (para, ortho or meta) and 
##STR5## 
Conveniently m is 1-10 and n is 1-10. It is preferred if m=n. 
.alpha.,.omega.-Dicarboxylic acids wherein the --NH.sub.2 functional group 
of the aforementioned .alpha.,.omega.-diamines are replaced by --COOH are 
also useful in the present invention to extend the spacer group adjacent 
the ring. Suitable .alpha.,.omega.-dicarboxylic acids include succinic 
acid, malonic acid, fumaric acid, maleic acid and the like. The peptide is 
joined to such an extender group via a nitrogen, instead of an acid group, 
of an amino acid. 
The peptide group B may be a single amino acid residue or an oligopeptide 
or polypeptide of up to 100 amino acid residues, preferably no more than 
50, more preferably no more than 10 and especially 1, 2 or 3. The peptide 
group may contain spacer groups between the amino acids thereof. If 
present, such spacer groups are preferably selected from the same 
possibilities as group A and may alternate with the amino acid residues. 
The amino acids in the peptide group are preferably .alpha.-amino acids. 
By ".alpha.amino acid", we mean any compound having a group 
##STR6## 
where R.sup.7 is the residual group of an amino acid, for example 
hydrogen, straight or branched C.sub.16 alkyl (such as methyl, isopropyl, 
2-methylpropyl or 1-methylpropyl), hydroxyalkyl (such as --CH.sub.2 OH or 
1-hydroxyethyl), aralkyl (such as benzyl or 4-hydroxy-benzyl), thiolalkyl 
(such as --CH.sub.2 SH), alkylthioalkyl (such as --CH.sub.2 CH.sub.2 
SCH.sub.3), acyl (such as --CH.sub.2 COOH or --CH.sub.2 CH.sub.2 COOH), 
amidalkyl (such as --CH.sub.2 CO.NH.sub.2 or --CH.sub.2 CH.sub.2 
CO.NH.sub.2) or linear or cyclic, aromatic or non aromatic, 
nitrogen-containing heterocyclic groups such as the groups forming pan of 
tryptophan, lysine, arginine or histidine. 
We include all of the 20 .alpha.-amino acids commonly found in 
naturally-occurring proteins and their D-isomers; less common 
naturally-occurring .alpha.-amino acids found in proteins, such as 
4-hydroxyproline, 5-hydroxylysine, desmosine, .epsilon.-N-methyllysine, 
3-methylhistidine and isodesmosine and their D-isomers; 
naturally-occurring amino acids not found in proteins, such as 
.beta.-alanine, .gamma.-aminobutyric acid, homocysteine, homoserine, 
citrulline, ornithine, canavanine, djenkolic acid and .beta.-cyanoalanine 
and their D-isomers; and di-, tri-, tetra-, penta-, oligo- or polypeptides 
based on these or other amino acids which peptides may optionally include 
non-amino acid residues or side elements such as sugar residues. For 
example, aspartic acid and glutamic acid can be incorporated containing 
lower alkyl, benzyl, or 4-nitrobenzyl esters as part of the side chain 
carboxyl group, or lysine and ornithine can contain carbobenzyloxy, 
tertiary-butyloxy, fluorenylmethoxycarbonyl protecting groups on the 
side-chain amino functionality, or arginine can be incorporated containing 
carbobenzyloxycarbonyl, tertiary-butyloxycarbonyl or nitro protection of 
the guanidinium functionality, or cysteine can be incorporated with 
tertiary-butyl or acetyl groups on the side-chain sulfhydryl group. 
Thus, R.sup.7 may be: hydrogen; straight or branched chain C.sub.1-4 alkyl 
(for example methyl, isopropyl, isobutyl or sec-butyl); aryl-C.sub.1-4 
-alkyl (for example benzyl, .beta.-indolylmethyl, 4-hydroxybenzyl or 
4-imidazolylmethyl); C.sub.1-4 -alkylthio-C.sub.1-4 -alkyl (for example 
methylthioethyl); hydroxy-C.sub.1-4 -alkyl (for example hydroxymethyl or 
1-hydroxyethyl); mercaptomethyl (for example --CH.sub.2 SH); C.sub.1-4 
amide (for example --CH.sub.2 C(O)NH.sub.2 or --CH.sub.2 CH.sub.2 
C(O)NH.sub.2); C.sub.1-4 alkyl carboxylate (for example --CH.sub.2 C(O)OH 
or --CH.sub.2 CH.sub.2 C(O)OH); C.sub.1-6 alkylamine (for example 
(CH.sub.2).sub.4 NH.sub.2); and imino(C.sub.1-6)alkyl-amine (for example 
--(CH.sub.2).sub.3 NHC(.dbd.NH)NH.sub.2). 
The di-, tri-, tetra-, penta-, oligo and polypeptides may be of any 
suitable amino acid sequence. 
It is preferred if the peptide has the sequence (Ser-Pro-Lys-Lys)n wherein 
n is 1 to 10. It is further preferred if n is 1 to 6 and still further 
preferred if n=1 or 2. 
Useful intermediates in the synthesis of Ser-Pro-Lys-Lys peptides include 
N.alpha.-Z-N.epsilon.-BOC-L-lysyl-N.epsilon.-BOC-L-lysine methyl ester, 
N.epsilon.-BOC-L-lysyl-N.epsilon.-BOC-L-lysine methyl ester, 
N.alpha.-Z-(O-t-butyl)-1-seryl-1-proline methyl ester, 
Na-Z-(O-t-butyl)-L-seryl-L-prolyl-N.epsilon.-BOC-L-lysyl-N.epsilon.-BOC-L- 
lysine methyl ester, 
N.alpha.-Z-(O-t-butyl)-L-seryl-L-prolyl-N.epsilon.-BOC-L-lysyl-N.epsilon.- 
BOC-L-lysine, L-Seryl-L-prolyl-L-lysyl-L-lysine hydrobromide, 
N.alpha.-BOC-(O-t-butyl)-L-seryl-L-proline methyl ester, 
N.alpha.-BOC-(O-t-butyl)-L-seryl-L-proline, 
N.alpha.-BOC-(O-t-butyl)-L-seryl-L-prolyl-N.epsilon.-BOC-L-lysyl-N.epsilon 
.-BOC-L-lysine methyl ester, 
N.alpha.-BOC-(O-t-butyl)-L-seryl-L-prolyl-N.epsilon.-BOC-L-lysyl-N.epsilon 
.-BOC-L-lysine(O-t-butyl)-L-seryl-L-prolyl-N.epsilon.-BOC-L-lysyl-N.epsilon 
.-BOC-L-lysine methyl ester, 
N.alpha.-BOC-(O-t-butyl)-L-seryl-L-prolyl-.epsilon.-BOC-L-lysyl-.epsilon. 
-BOC-L-lysyl-.epsilon.-BOC-L-lysine!.sub.2 methyl ester, 
N.alpha.-BOC-(O-t-butyl)-L-seryl-L-prolyl-.epsilon.-BOC-L-lysyl-.epsilon. 
-BOC-L-lysine!.sub.2, (L-seryl-L-prolyl-L-lysyl-L-lysine).sub.2, 
hydrochloride. The syntheses of these compounds are described in detail in 
Bailly et al (1992) Anti-Cancer Drug Design (1992) 7, 83-100 incorporated 
herein by reference. 
More generally, peptides may be synthesised by the Fmoc-polyamide mode of 
solid-phase peptide synthesis as disclosed by Lu et al (1981) J. Org. 
Chem. 46, 3433 and references therein. Temporary N-amino group protection 
is afforded by the 9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive 
cleavage of this highly base-labile protecting group is effected using 20% 
piperidine in N,N-dimethylformamide. Side-chain functionalities may be 
protected as their butyl ethers (in the case of serine threonine and 
tyrosine), butyl esters (in the case of glutamic acid and aspartic acid), 
butyloxycarbonyl derivative (in the case of lysine and histidine), trityl 
derivative (in the case of cysteine) and 
4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case of 
arginine). Where glutamine or asparagine are C-terminal residues, use is 
made of the 4,4'-dimethoxybenzhydryl group for protection of the side 
chain amido functionalities. The solid-phase support is based on a 
polydimethylacrylamide polymer constituted from the three monomers 
dimethylacrylamide (backbone-monomer), bisacryloylethylene diamine (cross 
linker) and acryloylsarcosine methyl ester (functionalising agent). The 
peptide-to-resin cleavable linked agent used is the acid-labile 
4-hydroxymethyl-phenoxyacetic acid derivative. All amino acid derivatives 
are added as their preformed symmetrical anhydride derivatives with the 
exception of asparagine and glutamine, which are added using a reversed 
N,N-dicyclohexyl-carbodiimide/1-hydroxybenzotriazole mediated coupling 
procedure. All coupling and deprotection reactions are monitored using 
ninhydrin, trinitrobenzene sulphonic acid or isotin test procedures. Upon 
completion of synthesis, peptides are cleaved from the resin support with 
concomitant removal of side-chain protecting groups by treatment with 95% 
trifluoroacetic acid containing a 50% scavenger mix. Scavengers commonly 
used are ethanedithiol, phenol, anisole and water, the exact choice 
depending on the constituent amino acids of the peptide being synthesised. 
Trifluoroacetic acid is removed by evaporation in vacuo, with subsequent 
trituration with diethyl ether affording the crude peptide. Any scavengers 
present are removed by a simple extraction procedure which on 
lyophilisation of the aqueous phase affords the crude peptide free of 
scavengers. Reagents for peptide synthesis are generally available from 
Calbiochem-Novabiochem (UK) Ltd, Nottingham NG7 2QJ, UK. Purification may 
be effected by any one, or a combination of, techniques such as size 
exclusion chromatography, ion-exchange chromatography and (principally) 
reverse-phase high performance liquid chromatography. Analysis of peptides 
may be carried out using thin layer chromatography, reverse-phase high 
performance liquid chromatography, amino-acid analysis after acid 
hydrolysis and by fast atom bombardment (FAB) mass spectrometric analysis. 
It will be appreciated by those skilled in the art that the peptide 
derivatives of the invention may be synthesised by chain extension on the 
spacer group or the peptide may be coupled to the spacer group once it has 
been synthesised. 
Conveniently the anthroquinone nucleus is attached to a support and the 
spacer and amino acid added by solid phase synthesis. 
By "derivatives" of the compounds of the invention, we include salts (acid 
or base addition), esters, amides, hydrazides and hydroxamic acids of the 
peptide group and other derivatives which do not diminish to an 
unacceptable extent the fundamental anti-turnout 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 NX.sub.4 .sup.+ (wherein X is C.sub.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. 
Especially preferred derivatives include those in which functional groups 
on the peptide group (which may be side groups or the terminal group) are 
capped and the terminal --NH-- or --CO-- on a spacer group which does not 
carry a peptide group is capped. 
Suitable chemical groups to cap --NH-- include H, --COCH.sub.3, 
tertiary-butoxycarbonyl, benzyloxycarbonyl and other groups known in the 
art. 
Suitable chemical groups to cap --CO-- include --OH or any --O-linked or 
--N-linked radical, for example --O-alkyl, --O-benzyl, 
--O-alkylaminoalkyl, --O-alkoxyalkyl or --NH--NHR.sup.4 wherein R.sup.4 is 
straight or branched alkyl, optionally substituted by --CN or --OH, an 
amide group (such as --CONH.sub.2) and other groups known in the art. 
Examples of alkylaminoalkyl groups include CH.sub.3 (CH.sub.3)NCH.sub.2 
CH.sub.2 --, --(CH.sub.2).sub.2 NH(CH.sub.2).sub.2 OH and CH.sub.3 
(CH.sub.3)NCH.sub.2 CH.sub.2 NHCH.sub.2 CH.sub.2 --. 
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. 
A useful discussion of alternative protective groups for amino acids (all 
types) and the scope of coupling reagents and deprotection reactions is to 
be found on pages 153-184 of a section called "chemical synthesis of 
peptides" in chapter 3 "Amino acids and Peptides" by R. S. Davidson and J. 
B. Hobbs in: "Natural Products, their Chemistry and Biological 
Significance", Authors: J. Mann, R. S. Davidson, J. R. Hobbs, D. V. 
Banthorpe & J. B. Harborne, publ. Longman Scientific and Technical (1994), 
incorporated herein by reference. 
It has been found that the compounds of the invention may be prepared as 
substantially pure optical isomers. 
Preferably B is a residue of alanine, phenylalanine, glycine, proline, 
valine, leucine, methionine, tyrosine or glycyl-glycine. The L-isomer is 
preferred in each case, although D-Phe is also preferred. 
Preferably the capping entity is simply a hydrogen atom, an O-benzyl group 
or a tert-butoxy-carbonyl group. 
Preferably R.sup.3 .dbd.R.sup.4 =oxo. 
Preferably R.sup.1 .dbd.R.sup.2 =hydrogen. 
Preferably A is 1,3-bisiminopropano, 1,4-bisiminobutano or 
1,6-bisiminohexano. 
It is further preferred that if B is a residue of Ala or Phe then A is 
1,3-bisiminopropano or 1,6-bisiminohexano, the cap is hydrogen, R.sup.3 
.dbd.R.sup.4 =oxo, and R.sup.1 .dbd.R.sup.2 =hydrogen. 
Preferred sub-formulae of the invention are: 
##STR7## 
where n is 1 to 6, preferably 3, 4 or 6; "peptide" is a peptide of 1 to 5 
amino acid residues, preferably only one or two; and "cap" one or more of 
is hydrogen, N-tBOC and O-benzyl; and 
##STR8## 
where x and y are independently 1 to 6, preferably 1, 2 or 3, and 
preferably x=y; and "peptide" and "cap" are as defined above in relation 
to formula (V). 
A further aspect of the invention provides a process for preparing a 
compound of the invention comprising: a) reacting a compound of Formula 
(IV) (IV) 
##STR9## 
where Q is a reactive group such as --Cl or --Br, R.sup.3 .dbd.R.sup.4 
=oxo and R.sup.1 and R.sup.2 are as defined above, with a compound A--B, 
wherein B is as defined above and A is a .alpha.,.omega.-diaminoalkane 
having a free --NH.sub.2 group for coupling to the ring in compound (IV); 
(b) reacting a compound of Formula (IV) 
##STR10## 
where R.sup.3 .dbd.R.sup.4 .dbd.Q=--OH and R.sup.1 .dbd.R.sup.2 .dbd.--H, 
with a compound A--B, wherein B is as defined above and A is an 
.alpha.,.omega.-diaminoalkane having a free-NH.sub.2 group for coupling to 
the ring in compound (IV); (c) reacting a compound of Formula (IV) 
##STR11## 
wherein Q is a group A as defined above and R.sup.1 to R.sup.6 are as 
defined above, with a compound B where B is a peptide as defined above, 
having an activated acid group on the .alpha.-amino acid which is to be 
coupled to spacer group A; or (d) conversion of one compound of Formula 
(I) to another compound of Formula (I). Referring to reaction (a) above, 
the compound of Formula (IV) is commercially available. The reaction 
generally proceeds in an aprotic solvent (eg DMSO or DMF). The amine 
(which can be used in excess) can form the solvent as well as being a 
reagent. The compound A--B used in reactions (a) and (b) may be made by 
reacting the .alpha.,.omega.-diaminoalkane spacer compound A with an 
activated acid derivative of peptide B, for example a pentafluorophenolate 
ester thereof, to form a complex primary amine. Reaction (b) generally 
proceeds in an inert atmosphere with excess primary amine above 50.degree. 
C. for 1-2 hours, followed by cooling and aerial oxidation. 
Similarly, in reaction (c) an activated acid derivative of the peptide B is 
reacted with the anthraquinone-spacer (mono- or disubstituted) compound. 
Compound (IV) and the activated, protected amino acid are reacted at 1:1 
molar ratio in an inert solvent such as ethyl acetate, dichloromethane, 
chloroform or DMF, usually at -10.0.degree. C. to room temperature (eg 
0.degree. C.) in an inert atmosphere. If A terminates in --COOH, it is 
activated (eg with pentafluorophenol), and coupled (eg with DCC, 
dicyclohexylcarbodiimide) to the N of an amino acid with a protected C 
terminus. 
One compound of the invention can be converted to another by, for example, 
oxidising --H at R.sup.1 and/or R.sub.2 to --OH; oxidising --H at R.sup.3 
and/or R.sup.4 to --OH; oxidising --OH at R.sup.3 and/or R.sup.4 to oxo, 
for example in an aerial oxidation or using chloranil; or reducing oxo at 
R.sup.3 and/or R.sup.4 to --OH (for example with sodium dithionite or 
zinc/acetic acid) or onward to --H. The sodium dithionite reaction is 
described in Marschalk et al (1936) Bull. Soc. Chim. Fr. 3, 1545, and the 
Zn/CH.sub.3 COOH reaction in Morris, G. A. et at (1986) Tetrahedron 42, 
3303. Another conversion of one compound of the invention to another 
involves extending the B group by removing any cap which is present and 
adding one or more amino acid residues. 
Compounds of structure (II): 
##STR12## 
are readily available. R.sup.8 is conveniently X.sup.1 --NH.sub.2 wherein 
X.sup.1 is --CH.sub.2 --X.sup.2 --CH.sub.2 --and X.sup.2 is a divalent 
radical. 
It is preferred if X.sup.1 is --(CH.sub.2).sub.n --NH.sub.2 wherein n is 1 
to 20, preferably 1 to 10, more preferably 1 to 5. 
Compound (II) is readily synthesized from 1,4-dichloroanthroquinone and an 
appropriate diamine of structure NH.sub.2 --X.sup.1 --NH.sub.2. 
Alternatively, compound (II) can be made using compound (III): 
##STR13## 
and aerially oxidizing within the presence of R.sup.8 NH.sub.2. 
Suitably R.sup.9 .dbd.R.sup.10 .dbd.OH or H. When R.sup.9 .dbd.R.sup.10 
.dbd.H then compound (II) is leucoquinizarin and when R.sup.9 =R.sup.10 
=OH then compound (II) is dihydroxyquinizarin. 
Compounds of structure (II) can be readily synthesised from leucoquinizarin 
or dihydroxyquinizarin using the methods described in C. W. Greenhalgh and 
N. Hughes (1968) J. Chem. Soc. (C), 1284; K. C. Murdock et al (1979) J. 
Med. Chem. 2, 1024; and L. P. G. Wakelin et al (1987) J. Med. Chem. 30, 
855, all incorporated herein by reference. 
In a general scheme, the anthraquinone, di-substituted with spacer groups 
A, is dissolved in a solvent such as dimethylformamide containing an 
organic base such as triethylamine or diisopropylethylamine, or 
alternatively, in dry tetrahydrofuran in the presence of trimethylsilyl 
chloride and triethylamine. The solution is chilled, and to this is added 
the first amino acid, activated through its carboxyl group as its 
corresponding hydroxysuccinimide ester, or as the isobutyryl 
chloroformate, or as a mixed or symmetrical anhydride, or as any one of a 
number of carboxyl activating functionalities known to those skilled in 
the art of peptide synthesis. For the asymmetric compounds of the present 
invention, only one equivalent of the amino acid is added. 
The .alpha.-amino group of the activated acid must be protected at this 
point by a group such as tertiary-butyloxycarbonyl, benzyloxycarbonyl, 
fluorenylmethoxycarbonyl, and the like, to avoid interference during 
condensation with the anthraquinones of this invention. Similarly, those 
amino acids which contain functionality in their side-chains in general 
also need to have the functionality protected, and are selected as 
described previously. The protecting groups used on the side chain can be 
the same or different than those used to protect the .alpha.-amino 
radical. 
The activated, protected amino acid is dissolved in the same solvent used 
to dissolve the anthraquinone, and addition is done dropwise with 
stirring. The reaction is stirred at 0.degree. to 40.degree. C., 
preferably at room temperature, for about 24 hours, then filtered and the 
desired amide is isolated either by precipitation with a solvent of low 
polarity, or by evaporation. The protecting group on the .alpha.-amine is 
then removed such that elongation of the peptide chain can be achieved if 
desired. For example, the tertiary-butyloxycarbonyl group can be removed 
by dissolving the compound in anisole, cooling the solution in an ice bath 
and adding trifluoroacetic acid. The solution is stirred briefly in the 
cold, then warmed to room temperature for 1-24 hours. The desired 
deprotected product is isolated by diluting the reaction with a solvent in 
which the product is not soluble, for example diethyl ether, and the 
precipitate collected by filtration. Alternatively, the 
tertiary-butyloxycarbonyl group can be removed by dissolving the compound 
in a mixture of acetic acid and anisole, then hydrogen chloride gas is 
bubbled into the solution for a few minutes. After standing at room 
temperature for 1-24 hours, the absence of the compound is determined by a 
technique such as thin layer chromatography or analytical high pressure 
liquid chromatography, then the deprotected product is isolated by 
precipitation as described above. A benzyloxycarbonyl group can be removed 
by dissolving in a solvent such as acetic acid, cooling (but not freezing) 
the solution, and bubbling in gaseous hydrobromic acid. After the reaction 
has remained at room temperature for 1-24 hours and absence of starting 
material has been determined, the deprotected product is isolated by 
precipitation. Alternatively, the benzyloxycarbonyl group can be removed 
by hydrogenation of the protected compound in the presence of a noble 
metal catalyst such as palladium on carbon, but in this case re-oxidation 
of the reduced anthraquinone ring is generally necessary. A 
fluorenylmethoxycarbonyl group can be removed by dissolving the protected 
compound in a polar solvent such as dimethylformamide and adding a 
secondary amine such as dimethylamine. The deprotected product is again 
isolated either by precipitation or by evaporation of the reaction 
solution. 
If desired, the next amino acid fragment is then added by repeating the 
sequence of reacting the deprotected compound with an .alpha.-amino and 
side-chain protected, carboxy group activated amino acid derivative, 
isolating the intermediate and removing the .alpha.-amino protecting group 
as described above. The process is repeated by judicious manipulation of 
the above conditions, or by applying conditions familiar to those skilled 
in the art of peptide synthesis until the entire desired peptide sequence 
has been assembled. 
Alternatively, the entire peptide sequence can be assembled prior to 
formation of the amide bond between the peptide carboxy terminus and the 
anthraquinone nucleus. This can be accomplished by applying the solution 
techniques described above or assembling the peptide chain using any one 
of the techniques which have been developed as modifications of the 
Merrifield solid phase peptide synthesis procedure. 
Removal of side-chain protecting groups, where desirable, and where these 
groups are the tertiary-butyloxycarbonyl, benzyloxycarbonyl or 
fluorenylmethoxycarbonyl radicals is accomplished as described above. In 
addition, the tertiary-butyloxy group required for protection of aspartic 
and glutamic acids is removed under the acid hydrolysis conditions 
described for cleavage of the tertiary-butyloxycarboxy protecting group. 
Most other groups are removed by slight modifications of the 
above-described procedures. 
The starting materials where the spacer is hydroxyethyl-diaminoalkyl are 
described in U.S. Pat. No. 4,197,249. 
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 may 
be 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 sub-cutaneous 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. In terms of dose per square meter of body surface, 
the compound can be administered at 1.0 mg to 1.5 g per m.sup.2 per day, 
for example 3.0-200.0 mg/m.sup.2 /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. A 
unit dosage form may comprise 2.0 mg to 2.0 g, for example 5.0 mg to 300.0 
mg of active ingredient. 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 carder, diluent or mordant for dyeing purposes, for example for 
dyeing cotton, nylon and paper.