WO2006097472A2

WO2006097472A2 - Novel inhibitors of glutathione-s-transferase

Info

Publication number: WO2006097472A2
Application number: PCT/EP2006/060707
Authority: WO
Inventors: Katja Becker-Brandenburg; Herbert Zimmermann; Karin Fritz-Wolf
Original assignee: Universität Giessen; MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
Priority date: 2005-03-14
Filing date: 2006-03-14
Publication date: 2006-09-21
Also published as: WO2006097472A3; EP1865942A2

Abstract

The invention relates to novel methods of preventing, treating, or ameliorating medical conditions, including cancer, drug resistance, and parasite infections such as malaria, by administering compounds that are capable of inhibiting glutathione-S-transferase, as well as to the use of said compounds for preparing pharmaceutical compositions for preventing, treating, or ameliorating said medical conditions. Furthermore, the invention relates to novel GST inhibitor compounds and pharmaceutical compositions comprising said GST inhibitors, optionally comprising further compounds known to be effective in treating said medical conditions.

Description

Novel Inhibitors of Glutathiones-Transferase

Field of the Invention

The present invention relates to compounds that are capable of inhibiting glutathione-S-transferase (GST). These GST inhibitor compounds are thus useful for the prevention, treatment and amelioration of a variety of medical conditions including cancer, multi-drug resistance and parasitic diseases such as malaria.

Background of the Invention

Glutathione-S-transferases .

Glutathione-S-transferases ("GSTs") form a large enzyme family ubiquitously expressed in animals and plants. GSTs are involved in the cellular defense against a broad spectrum of cytotoxic agents (Gate and Tew, 2001). Up until today, over

400 different GST sequences have been identified. Based on their genetic characteristics and substrate specificity, the various GSTs can be classified in at least four different classes α, μ, π, and θ (Manncrvik et al., Biochem. J. 282:305, 1992), wherein each allelic variant encoded at the same gene locus is distinguished by a letter. In humans, five α-class genes

(GSTA1, GSTA2, GSTA3, GSTA4 and GSTω ); five μ-class genes (GSTM1, GSTM2,

GSTM3, GSTM4 and GSTM5); fourπ-class genes (GSTP1*A, GSTP1*B, GSTP1*C and

GSTP 1*D); and two θ-class genes (GSTTl and GSTT2) are currently known.

The various GST isozymes occur as dimeric proteins formed by the intra-elass binary combinations of GST monomers. The monomeric proteins in a dimeric GST isozyme assume a fixed geometry with respect to one another (Fritz-Wolf et al., 2003).

The GSTs are an extended family of Phase II xenobiotic or "drug" metabolizing enzymes that catalyze the conjugation of glutathione ("GSH") to a wide variety of exogenous electrophilic compounds including anticancer drugs, pesticides and genotoxic molecules. The first step in this process is the conversion of the GSH thiol group to a thiolatc which then attacks an electrophilic site of the substrate, leading to the formation of a glutathione-substrate conjugate. GSH conjugates of such compounds are generally more hydrophilic and less toxic and may therefore be excreted more easily. The GSTs contain two functional regions that participate in the conjugation to the electrophilic substrate. These include the N-teiminal, hydtophilic domain that binds GSH (the "G-site"), and the C-terminal hydrophobic domain that binds the substrate (the "H-site") (cf. Dirr et al., Eur. J. Biochem. 220:645, 1994 and Armstrong, Chem. Res. Toxicol. 10:2, 1997). An additional function attributed to GSTs relates to the protection of cells against autocatalytic lipid peroxidation brought on by oxidative stress, This glutathione peroxidase activity, with the GSTAI isozyme providing the greatest specific activity (Hurst et al., Biochem. J. 332:97, 1998) plays a critical role in signaling and apoptosis triggered by lipid peroxidation products such as 4-hydroxynonenal (sec Yang, et al., Acta Biochimica Polonica 50(2):319, 2003; Yang, et al., J. Biol Chem. 276(22); 19220, 2001; and Yang, ct al., Invest. Ophthalmol. Vis. Sci. 43:434, 2002). GSTs therefore have a variety of beneficial physiologic functions that range from detoxification to regulation of cell signaling pathways, GST also binds to olbcr cellular proteins and is capable of regulating their biologic activity. For example, the GSTPl isozyme regulates the stress response, cellular proliferation, and apoptosis by interacting with cJun NH2-terminaI kinase (JNK), hence inhibiting its stress-induced kinase activity and promoting cell survival (cf. Alder et al., EMBO J. 18:132), 1999; Ruscoe et al., J. Pharmacol. Exp. Ther. 298:339, 2001; and Wang et al., J. Biol. Chem. 276:20999, 2001). Accordingly, cancer cells that overexpress GST exhibit high JNK activity and escape apoptosis during exposure to anticancer drugs. Under stress, multimeric GST, which is normally inert, dissociates into monomers that are capable of activating JNK.

On the other hand, GSTs have been identified to play a crucial role in rendering a variety of therapeutic compounds ineffective. In malignant neoplasms, increased expression of GSTs has been shown to be involved in the resistance of these neoplasms to numerous anticancer agents (Tew, Cancer Res. 54:4313, 1994). In addition, it was shown that increased expression of GST in strains of rodent and human malarial parasites correlates with resistance to chloroquine in infectious diseases (sec, e.g., Srivastava et al., Trop, Med. Int. Health 4:251, 1999 and Harwaldt et al., 2002). In a variety of disease states, the involvement of GST in cell signaling pathways can be used to therapeutically manipulate cell proliferation and apoptosis. Accordingly, compounds that are capable of inhibiting GST catalysis and/or protein:protem binding would be desirable. In this context, inhibitory compounds that act specifically on particular GST classes or on specific isozymes would be particularly desirable.

GST inhibitors have been described in the art (Flatgaard et al., Cancer Chemother. Pharmacol. 33:63, 1993; Lyttle et al., J. Med. Chcm. 37:189, 1994; and Kauvar and Lyttle, U.S. Pat. No. 5,763,570), These inhibitors were shown to potentiate the cytotoxic effect of numerous anticancer drugs in different cell lines and animal models (Morgan et al., Cancer Chemother. Pharmacol 37:363, 1996; and U.S. Pat Nos. 5,763,570, 5,767,086 and 5,955,432). One of these inhibitors has also been shown to block the interaction between GSTPl-I and JNK, activate downstream signaling pathways, and cause an early restoration in peripheral blood cells in. an animal model of chemotherapy- induced myclosuppressϊon (cf, U.S. Pat. Nos. 5,767,086 and 5,955,432). Bivalent inhibitors capable of binding to more than one GST monomer have also been described in the art in an attempt to increase specificity for the desired GST class or isozyme (U.S. application US20050004038 A1).

Functions of parasi.te_GSTs

Glutathione S-transferases (GSTs) of protozoan and metazoan parasites have gained increasing attention in the past few years due to their involvement in drug resistance and the removal of endogenous and exogenous cytotoxic metabolites, as explained above. A number of GSTs from parasitic nematodes, trematodes, ticks and malarial parasites have been studied so far and some of these proteins might be exploited as a drug target or might be good candidates for vaccine development (Frits-Wolf et αl, 2003; Johnson et αl, 2003; Ouaissi et at, 2002; Rossjobn et al., 1997; McTjgue et al, 1995). Indeed, identification of the Schistosoma japonicum GST as a potential vaccine candidate "accidentally" lead to the development of a GST fusion protein system which is, for example, extensively used to analyse protein protein interactions by pull-down assays (Smith and Johnson, 1988; Smith et al., 1987).

The functions of GSTs from different parasites include (i) the more or less specific nucleophilic addition of GSH to electrophils, (ii) the GSH-dependent reduction of hydroperoxids, and (iii) the binding of ligands. Some parasite GSTs might also catalyze the GSH-dependent isomerization of metabolites such as prostaglandins, and as a result, the parasite might modulate the immune system of the host (Johnson et al., 2003; Ouaissi et al, 2002; Angeli et al, 2001)

Several parasitic worms possess more than one GST (Liebau et al._t \ 996), whereas for example Plasmodium falciparum seems to have only one classical GST (Fritz- WoIf et al, 2003). Not surprisingly, parasite GSTs differ significantly with respect to their functions: GST3 from Onchocerca volvulus has been shown to increase resistance of transgenic Caenorhabditis elegans to internal and external oxidative stress. In addition, expression of the respective gene is inducible by oxidative stress. These results suggest that GST3 could be involved in the defence against reactive oxygen species (ROS) resulting from cellular metabolism, and against ROS derived from the host's immune system (Kampkotter et al, 2003), A GST from Haemonchus contorius shows only limited activity with classical GST substrates such as l-chloro-2,4-ditutrobenzene (CDNB). However, the protein effectively binds hematin - in contrast to a 60% identical GST from the closely related non-parasitic nematode C. elegans - suggesting that the high-affinity binding of hematin as a ligand may represent a parasite adaptation to blood feeding (van Rossum et al., 2004). GSTs from the parasitic nematodes Ascarts suum and Onchocerca volvulus efficiently use CDNB, unsaturated carbonyl compounds such as trans-2-nonenal, and hydroperoxides such as cumene hydroperoxide as substrates in vitro. Tissue distribution and localisation of these nematode GSTs differ, depending on the protein and organism. Thus, some of the GSTs (but not all of them) are acting at the host-parasite interface and are indeed accessible to inhibitors (Wildenburg et al, 1998; Liebau et al., 1997; Liebau et al., 1996). As reported by Rao et al. (2000), Brugia, a pathogenic filarial nematode, may modify the host's defense mechanisms by a detoxification process involving GSTs. Determination of GST activity in soluble parasite extracts as well as in excretory-secretory products of B. malayi suggests that GST is secreted in vivo and cross-reacts immunologically with the GSTs from other filarial nematodes. GST-inhibitors reduced the viability and motility of microfilariae, third-stage larvae, and adult worms,

GST activity in malarial parasites

GST activity has been detected in all Plasmodium species studied so far as well as in all intracrythrocylic Stages of the parasite (Dubois et al, 1995 ; Srivastava et al,, 1999; Harwaldt et al, 2002; Liebau et al, 2002). At 37⁰C, GST activities between 20 and 95 mU/mg total protein were determined in P. berghei, P. yoelii, and P. falciparum, whereas P. knowlesi showed lower activities around 5 mU/mg. Stage-specific studies revealed that there is an decreasing activity from schizonts to rings to trophozoites (Srivastava et al., 1999). In accordance with these results, GST activities determined in a different study in extracts from isolated trophozoites of eight P. falciparum strains indicated values between 5.6 and 22 mU/mg at 37°C (Harwaldt et al., 2002), As fiirther reported by Srivastava et al. (1999) GST activity increases significantly in chloroquine- resistant strains. This increase in enzyme activity was directly related to drag pressure of resistant P berghei,

Since strategics directed against the malaria vector Anopheles are of major interest in malaria control, it is worth mentioning here that DDT resistance in both adults and larvae of Anopheles gamhiae is mediated by stage-specific insect class I GSTs (Ranson et al, 1997). A DTT-metabolizing GST has also been characterized in Anopheles dims

(Prapanthadara et al., 1996; for review see Eaton, 2000),

Biochemical properties of PfGST

The gene encoding Plasmodium falciparum GST (PfGST) has been cloned and the corresponding recombinant protein has been purified and characterized independently by two groups (Harwaldt et al, 2002; Liebau et al., 2002), The gene structure, comprising two exons, and the amino acid sequence of PfGST (accession number AY014840) are in full agreement with the P. falciparum genomic database (Kissinger et al., 2002; http://www.plasmodb.org). PfGST has a pH optimum of S.I and its activity is sensitive to NaCl and potassium phosphate (Harwaldt et al, 2002): comparison with 100 mM potassium phosphate, 1 mM EDTA, and 100 mM Tris/HCl, 1 mM EDTA, at the same pH showed a decrease in enzyme activity to 15% and 90% of the activity in 100 mM Hepes buffer, respectively. Addition of NaCl to the assay system strongly decreased enzyme activity (83% residual activity at 50 mM NaCl, 66% at 100 mM NaCl, 19% at 200 mM NaCl). Recombinant N-terminally His-tagged enzyme is very stable, The K_m value for GSH and the specific activity with GSH and CDNB as substrates were found not to be significantly changed after more than one year. However, precipitating recombinant PfGST with ammonium sulphate (Licbau et al., 2002) or dialysmg the protein resulted in partial loss of activity.

Apart from CDNB, 1

,2 -4 nitrobenzene, bromosulphophthaleϊn, and ethacrynic acid have been tested as etectrophilic substrates for PfGST, GSH consumption was determined with 5,5'-dithio-bis-(2-nitrobenzoic acid; DTNB) measuring the formation of 5-thio-2-nitrobenzoate at 412 nm (ε_412nm = 13.6 mM^-1cm^-1 _' for details see Harwaldt et al, 2002). The specific activity for 1 mM ethacrynic acid and 1 mM σ-nitrophenyl acetate is 0.19 U/mg (5.0 U/μmol) and 0.06 U/mg (1-5 ϋ/μmol), respectively, and the K_m for ethacrynic acid is approx. 0.5-1.5 mM (Harwaldt et al, 2002; Liebau et al., 2002).

Since malarial parasites digest huge amounts of haemoglobin, and since they are exposed to oxidative stress inside the erythrocyte (for a review see Becker et al., 2004), detoxification of peroxidases and binding of haem-containing compounds as ligands might be further functions of PfGST in vivo. In support of this conclusion, PfGST has been found to bind hemin/ferriprotoporphyrin IX in the lower micromolar range and shows slight peroxidase activity with GSH and cumenc hydroperoxide as substrates. The amount of PfGST has been estimated to be > 1% of cellular protein (Harwaldt et al., 2002). In a different experimental approach the concentration was estimated by Liebau et al. (2002) to be 0.1 % of the total protein content in trophozoites. Depending on the actual cellular concentration, PfGST may protect the parasite against oxidative stress or might act as a buffer for haem-containing compounds in vivo (Liebau et al., 2002; Harwaldt et al,, 2002). Recently, the crystal structure of PfGST was solved independently by two groups at 1.9 and 2.2 Å resolution (Fritz-Wolf et al, 2003; Burmeister et al, 2003; Perbandt et al, 2004). Apart from GSTs from other Plasmodium species, PfGST shares highest sequence identities with the pi-class GSTs from Dirσfilaria immitis and O. volvolus (approximately 35% identity). Structural alignment of PfGST with members of the alpha-, mu- acid pi-classes indicated that PfGST adopts the canonical GST fold, but otherwise has an rms deviation of at least 1.2. Å to the pther known GST classes which is significantly higher than expected for members within the same class (less than 0.7 A). Particularly, its H-site differs significantly from the ones of the human counterparts (Fritz- Wolf et al., 2003). In contrast to all other GSTs, PfGST contains only five residues following helix α8, which is too short to form a wall (μ- or π-class) or an α-helix (α-class) leading to a more solvent accessible H-site in PfGST than in the other classes. This observation suggests that the substrate spectrum of PfGST is broader, includes amphophilic compounds, and is accessible to amphiphilic inhibitors which arc not able to enter the H-site of the human isoforms.

PfGST and drug resistance

As discussed above, glutathione-S-transferase conjugates GSH to toxic elcctrophiles predisposing them to export by specific membrane pumps, also known as multidrug resistance-associated proteins (MRPs; for reviews on glutathione and redox metabolism see Becker et al, 2003 & 2004). Many cells also display a GSSG pump that overlaps to some extent the action of MRP, This overall picture also seems to be present in Plasmodium falciparum. As reported by Klokouzas et al (2004) Plasmodium falciparum expresses a multidrug resistance-associated protein. AH other components of the GSH system - except for glutathione peroxidases - have been identified in P. falciparum (Becker et al, 2003 & 2004). The development of drug resistance often encountered in cancer therapy and the therapy of parasitic diseases has been related to the function of GST, which can contribute to drug clearance (Salinas and Wong, 1999). PfGST as a potential drug target

300-500 million people per year arc infected and more than two million people die of malaria annually (Greenwood & Mutabingwa, 2002). One of the major reasons for this situation is the emergence of drug resistance to currently used drugs. Thus, new drugs directed against novel targets are urgently and continuously required (see also Schirmer et al., 1995). As rapidly growing and multiplying cells, malarial parasites do depend on a functional GST.

Inhibition of PfGST is expected to act at different vulnerable metabolic sites of the parasite simultaneously, which further supports its promising potential as drug target. PfGST inhibition is likely to disturb GSH-dependent detoxification processes, to enhance the levels of cytotoxic peroxides, and possibly to increase the concentration of toxic hemin. It has been shown that in the presence of GSH the parasitotoxic hcmin inhibits PfGST in the lower miciomolar range indicating that free hemin might be buffered by the enzyme in vivo (Harwaldt et al, 2002; Liebau et al, 2002). It has furthermore been shown that chloroquine inhibits hemin catabolism leading to intracellular hemin accumulation (Famin et al, 1999). In view of these results, it might well be possible that PfGST inhibitors act synergistically with chloroquine.

Ellagjc acid as GST inhibitor

The plant phenol ellagic acid (EA) has been shown to have anticarcinogenic properties by inducing the transcription of GST genes thereby enhancing the anticarcinogenic capacity of colls (van Lieshout et al., 1998). Furthermore EA is a known inhibitor of GSTs (Das et al., 1986) and has been previously shown to possess antimalarial activity (Verotta et al., 2001 ; Banzouzi et al., 2002; Dell' AgIi et al., 2003). Several naturally occurring food components or non-steroidal anti- inflammatory drugs (NSAIDs) may reduce gastrointestinal cancer rates. It had been shown that dietary administration of such compounds enhanced flic GST enzyme activity and class r alpha, mu and pi isoenzyme levels in the rat gastrointestinal tract. Elevation of the levels of GSTs, a family of biotransformation enzymes with many functions such as detoxification of carcinogens, might be one of the mechanisms that lead to cancer prevention. Van Lieshoυt et al., 1998, therefore investigated whether the anticarcinogen ellagic acid affects gastrointestinal rGSTTl-1 protein levels in male Wistar rats. rGSTTl-1 protein levels were determined in cytosolic fractions of liver and oesophageal-, gastric-, small intestinal- and colonic mucosa by densitomctrical analyses of western blots after immunodetection with an anti human GSTTI-1 monoclonal antibody, that cross-reacts with rGSTTl-1. In control Wistar rats, gastrointestinal rGSTTl-1 protein levels were highest in the liver and decreased in the order liver > stomach > colon > oesophagus > small intestine. Gastric rGSTTl-1 protein levels were enhanced and hepatic rGSTTl-1 protein levels were reduced by ellagic acid. The authors concluded that EA are capable of inducing rGSTTl-1 protein levels in the rat gastrointestinal tract. Enhanced rGSTTI-1 protein levels might lead to an increase of enzyme activity and to a more efficient detoxification of carcinogens and thus could contribute to prevention of carcinogenesis- As reported by Das et al., 1986, GST isoenzymes isolated from human tissues and rat liver are differentially inhibited by quercetin, alizarin, purpurogallin and ellagic acid. Rat liver GST isoenzymes are far more sensitive to these compounds as compared to the human GST isoenzymes. Among human GST, the anionic isoenzymes containing C type and A' type subunits are inhibited to a greater extent as compared to tbe cationic isoenzymes containing A and B type subunits, The anionic GST isoenzymes of human erythrocytes and placenta are differentially inhibited by these plant phenols indicating that the placental and erythrocyte isoenzymes may be distinct proteins.

Ellagic acid as antimalarial compound

Screening of plants from New Caledonia for antiplasrnodial activity against Plasmodium falciparum revealed that methanolic extracts of the leaves and bark of

Tristamopsis calobuxus, T. yateensis, and T. glauca inhibited the growth of chloroduinc- sensitive and -resistant clones. Ellagic acid and the new compound 3,4,5- trimethoxyphcnyl-Cθ'-O-galloylJ-O-beta-D-glucopyranoside were identified as the active constituents (ICso 0.5 and 3.2 μM, respectively). The growth inhibition of both clones was comparable. The compounds showed negligible or very low cytotoxicity to human skin fibroblasts and Hep G2 cells when tested at concentrations ranging from 0.5 to 100 microM (Verotta et al, 2001), Furthermore, by Banzouzi et al., 2002, extracts of leaves of Alchornea corάifolia were studied for their antiplasmodial activities. Chloroformic and ether extracts were found to be inactive while the cthanolic extract exhibited mild in vitro activity against Plasmodium falciparum. Fractionation of this extract resulted in. the isolation of ellagic acid as the active constituent of the extract with an IC50 in the range of 0.2-0.5 μM. Cytotoxicity of the ethanolic fraction and ellagic acid was also estimated on human fibroblasts cells (IC₅₀ on HeIa cells = 7.3 μM at 24 h for ellagic acid).

The above-described results disclosed in the prior art support the motion, that an effective GST inhibition may afford a viable therapeutic strategy for abating drug resistance observed for, eg., antineoplastic (i.e., anticancer) and antimalarial drugs, and for stimulating hematopoicsis within a variety of clinical conditions. In view of the increasing occurrence of drug resistance observed for these diseases, there is a need in the art for novel GST inhibitors exhibiting greater affinity, and optionally also greater selectivity for one or more multimcric GST isozymes.

Summary of the Invention

The present invention fulfils the need in the art and moreover provides additional related advantages. Tt is one object of the present invention to provide GST inhibitor compounds that arc capable of binding to GSTs with high affinity making them useful for the treatment of a variety of medical conditions that benefit from inhibition of GST activity. It is another object of the present invention to provide bi- or multivalent inhibitors that bind to dimeric or multimeric GSTs, Another object is to provide methods for successfully treating medical conditions that are characterized by drug-resistance to established or known drug compounds.

The inventors of the present invention have now surprisingly found that certain compounds structurally related to ellagic acid, as well as certain other plant phenols having a planar polyaromatic ring system show a high binding affinity to glutathione-S- transferase (GST). In view of their binding affinity for GST, these compounds are suitable as inhibitors of GST activity, thereby allowing them to be useful in the treatment of a variety of medical conditions where the inhibition of GST is beneficial.

Accordingly, in a first aspect of the present invention, novel GST inhibitor compounds arc provided having a structure according to one of the following general formulae: a) ¹

b)

2 ¹

II)

wherein each R¹ and R¹ⁿ may independently from each other be selected from hydrogen; optionally mono- or multisubstituted C₁C₁₂ straight or branched chain alkyl, optionally mono- or multisubstituted C₂-C₁₂ straight or branched chain alkenyl, optionally mono- or multisubstituted C₂-C₁₂ straight or branched chain alkinyl, optionally mono- or multisubstituted C₃-C₈ cycloalkyl, optionally mono- or multisubstituted aryl, optionally mono- or multisubstituted heteroaryl, optionally mono- or multisubstituted aralkyl, or a combination thereof;

-CN, -NC, -(C=O)-R⁴, -(C=S)R⁴, -(C=O)NR⁵R⁶, -(S=O)R⁷, -SO₂R⁸, SO₃R⁹, - (P=O)R¹⁰R¹¹ , -PO₂HR¹⁰, -SiR¹²R¹³R¹⁴;

.R¹⁵-CN, -R¹⁵-NC, -R¹⁵(C=O)-R⁴, -R¹⁵-(C=S)R⁴, -R¹⁵-(C=O)NR⁵R⁶, -R¹⁵- (S=O )R⁷, -R¹⁵-SO₂R⁸, -R^I5-SO₃R⁹, -R¹⁵(P=O )R¹⁰R¹¹, -R^l5-PO₂HR¹⁰, -R¹⁵- SiR¹²R¹³R¹⁴ wherein -R¹⁵ is a saturated or unsaturated alkylene group from 1 to about 20 carbon atoms; an optionally substituted saturated or unsaturated alkylene group connecting two neighboring oxygen atoms attached to the benzene moiety of the compound to form a cyclic structure; a bond or a bi- or multivalent linker covalently attaching the compound to at least one other compound selected from the group of: glutathione, a glutathione analog, a glutathione conjugate, ethacrynic acid, cibacron blue, uniblue A, doxorubicin, gossypol, hematin, rose bengal, sulfobrornophthalein, indomethacin, piriprost, eosin b, eosin y, a synthetic or naturally occurring drug, a polysaccharide, a polynucleotide, a polypeptide, another compound of general formulae I or II, a known glutathione-S-transferase inhibitor, an antimalarial agent, a toxin or a chemotherapcutic agent, or a combination or mixture of any of the foregoing compounds;

R² and R³ may each independently be hydrogen or -OR¹, wherein R¹ has the same meanings as described above; each R²" may independently be hydrogen or OR^la, wherein R^la has the same meanings as described above; each R^3a may independently be -CH₂-R^4a;

R⁴ to R¹⁴ may each independently represent hydrogen, optionally mono- or multisubstituted C₁-C₁₂ straight or branched chain alkyl, optionally mono- or multisubstituted C₂-C₁₂ straight or branched chain alkenyl, optionally mono- or multisubstituted C₂-C₁₂ straight or branched chain alkinyl, optionally mono- or multisubstituted C₃-C₈ cycloalkyl, optionally mono- or multisubstituted aryl, optionally mono- or multisubstituted heteroaryl, optionally mono- or multisubstituted aralkyl, halogen, -CF₃, or a combination thereof; each R^4a may represent hydrogen, optionally mono- or multisubstituted C₁-C₁₂ straight or branched chain alkyl , optionally mono- or multisubstituted Ca-Cn straight or branched chain alkenyl, optionally mono- or multisubstituted C₂-C₁₂ straight or branched chain alkinyl, optionally mono- or multisubstituted C₃-C₈ cycloalkyl, optionally mono- or multisubstituted aryl, optionally mono- or multisubstitutcd heteroaryl, optionally mono- or multisubstituted aralkyl, optionally mono- or multisubstituted C₁-C₁₂ straight or branched chain alkyloxy, optionally mono- or multisubstitutcd C₂-C₁₂ straight or branched chain alkeitiyloxy, optionally mono- or multisubstituted C_2-C₁₂ straight or branched chain alkinyloxy, optionally mono- or multisubstituted C₃-C₈ cycloalkyloxy; -CN, -NC, -(C=O)R⁴, -(C=S)R⁴, -(C=O)NR⁵R⁶, -(S=O)R⁷, -SO₂R⁸, SO₃R⁹, -

(P=O)R¹⁰R¹¹ -PO₂HR¹⁰, -SiR¹²R¹³R¹⁴;

-R¹⁵-CN, -R¹⁵-NC, -R¹⁵-(C=O)-R⁴, -R¹⁵-(C=S)R⁴, -R¹⁵-(C=O)NR⁵R⁶, -R¹⁵- (S=O)R⁷, -R¹⁵-SO₂R⁸, -R¹⁵-SO₃R⁹, -R¹⁵-(P=O)R¹⁰R¹¹, -R¹⁵-PO₂HR¹⁰, -R¹⁵- SiR¹²R¹³R¹⁴ wherein -R¹⁵ is a saturated or unsaturated alkylene group from 1 to about 20 carbon atoms; halogen, -CF₃, or a combination thereof.

Since ellagic acid is a known GST inhibitor, it will be appreciated that the above-presented general formula I is not meant to include ellagic acid. Accordingly, the proviso app) ies that at least one of R² and R³ in formula ϊ must be other than hydrogen.

Moreover, the compounds [1]benzopyrano[5,4,3-cde][1]benzopyran -5,10- dione1,2,3,7,8-pentahydroxy-((8C1,9C1)(flavellagic acid) and [1]benzopyrano[5,4,3- cde][1]benzopyran-5,10-dione,1,2,3,6,7,8-hcχahydroxy-(9Cl) (corulcoellagic acid), or their penta- and hexamethyl ethers, respectively, were also disclosed in the art (see, e,g,, Geevananda et al., 1979 or Zimmermann et al., 1992) and are thus also specifically excluded from general formula I.

The present invention also excludes a compound according to general formula II wherein each of R^1a and R^2a is hydrogen and both R^3a represent a methyl group. This compound is known under the name hypericin (a known dianthraquinone constituent of St. John's wort). Hypericin has long been considered as a potential sensitizer for photodynamic therapy and has recently been shown to bind to and inhibit human GSTAJ -X and GSTPl-I isoforms (Lu & Atkins, Biochemistry, 2004).

Those with skill in the art will of course appreciate that the present invention also encompasses the pharmaceutically acceptable salts of the compounds of the invention.

In a preferred embodiment of this aspect of the invention, the GST inhibitors are monovalent GST inhibitors, ie., not attached to another compound via one of its functional groups, Alternatively, the GST inhibitors may be bi~ or even multivalent inhibitors that are linked via a simple bond or via a bi- or multifunctional linker to another compound. Said other compound may be another GST inhibitor compound or be selected from a variety of other useful molecules including but not limited to glutathione, a glutathione analog, a glutathione conjugate, ethacrynic acid, cibacron blue, uniblue A, doxorubicin, gossypol, hematiπ, rose bengal, sulfobromophthalein, indomethacin, piriprost, eosin b, cosin y, a synthetic or naturally occurring drug, a polysaccharide, a polynucleotide, a polypeptide, another compound of general formulae I or II, a known glutathione-S- transferase inhibitor, an antimalarial agent , a toxin, a chemotherapeutic agent, an antiproliferative or antineoplastic agent, or a combination or mixture of any of the foregoing compounds. In another aspect of the present invention, pharmaceutical compositions comprising at least one mono-, bi~ or multivalent GST inhibitor are also provided. Preferably, these pharmaceutical compositions may fiirther comprise at least one other pharmaceutically active compound and optionally also a pharmaceutically acceptable carrier, diluent or excipient. In yet another aspect of the present invention, the GST inhibitor compounds or pharmaceutical compositions comprising said GST inhibitor compounds according to the present invention for use as in therapy, Alternatively, the compounds and/or compositions may be used in diagnostic applications. The use of the GST inhibitor compounds or pharmaceutical compositions comprising said GST inhibitor compounds according to the invention for fhe preparation of a pharmaceutical composition αr a medicament for the prevention, treatment or amelioration of certain medical conditions in a subject that can be prevented, treated or ameliorated by inhibiting glutathiones-transferase (GST) in said subject represents another aspect of the present invention. In this aspect of the present invention, the compounds flavellagic acid, corulcocllagic acid and hypericin are not excluded from the compounds represented by the general formulae I and II, since the use of these compounds in the manufacture of pharmaceutical compositions for treating the medical conditions as defined in the present invention such as drug resistant cancer or malaria has to our knowledge not been disclosed m the art.

In some embodiments of this aspect of the invention, the pharmaceutical compositions or medicaments may further comprise at least one other pharmaceutically active compound and/or a pharmaceutically acceptable carrier, diluent or excipient.

The pharmaceutical compositions prepared using the GST inhibitor compounds of the present invention arc suitable for the prevention, treatment or amelioration of diseases including but not limited to malaria, drug-resistant malaria, multi- drug resistance, toxoplasmosis, African trypanosomiasis (sleeping sickness), Chagas disease, cancer, drug-resistant cancer, immunosuppression, immunosuppression during chemotherapy or radiotherapy, myelodysplasia, and hone marrow transplantation. In preferred embodiments of this aspect of the invention, Use GST protein being the target of the GST inhibitor compounds of the present invention is a human GST isoform. Alternatively, the GST may be an isoform from a microorganism, preferably from a microorganism that is pathogenic for humans and/or animals. It is particularly preferred that the GST isozyme to be inhibited or the GST isozyme that is a preferential target of the pharmaceutical compositions of the invention is located in, or expressed by a tumor, an infectious agent, or the bone marrow of the patient to be treated. It is an advantage provided by the present invention that certain preferred compounds exhibit a greater affinity towards a parasite GST isozyme than to a human GST isozyme. Hence, such GST inhibitor compounds may particularly be suitable and employed for the treatment of malaria or other parasitic diseases as outlined above.

The most preferred compounds to be used in this aspect of the invention are flavellagic acid (FAE, [1]benzopyrano[5,4,3-cde][1]benzopyran-5,10-dione,1,2,3,7 ,8-pentahydroxy-(8C1,9C1)), coruleoellagic acid (CEA, [1]benzopyrano[5,4,3- cde][1]benzopyran-5,10-dione,1,2,3,6,7,8-hexahydroxy-(9C1) , hypericin (HYP, phenantro[1,10,9,8-opqralperylene-7,14-dione , 1,3,4,6,8, 13-hexahydroxy-10,1 l-dimethyl-, stereoisomer (6CI,7CI,8CI,9CI)), or their respective alkyl- or arylelhers and/or alkyl- or arylester derivatives.

It has been found that the preferred GST inhibitor compounds of the present invention preferentially inhibit the parasitic growth, in particular Plasmodium falciparum, at the so-called throphozoitc stage of parasite development (sec Example 3, below). In other words, the new GST inhibitor compounds of the present invention can be classified as slowly acting antimalarials, whereas the known antimalarials, such as artemisinines, artesunatcs, chloroquine, sulfadoxine-pyrimethamine, primaquine, quinine, methylene blue, or derivatives or analogs thereof arc fast-acting antimalarials (i.e., they preferentially inhibit Plasmodium growth when administered to plasmodium strains in an early stage of their development (i.e., ring stage).

Accordingly, it will be appreciated that pharmaceutical compositions comprising an early-stage (i.e., fast-acting) antimalarial agent such as the aforementioned artemisinines, artesunatcs, chloroquine, sulfadoxinc-pyrimethamine, primaquine, quinine, methylene blue, or derivatives or analogs thereof and a GST inhibitor compound as defined herein represent a particularly preferred aspect of the invention.

Finally, the present invention also encompasses methods for preventing, treating or ameliorating medical conditions selected from malaria, drug-resistant malaria, multidrug-resistance, toxoplasmosis, African trypanosomiasis (sleeping sickness), Chagas disease, cancer, drug-resistant cancer, immunosuppression, immunosuppression during chemotherapy or radiotherapy, myelodysplasia, and bone marrow transplantation in a mammal, particularly a human subject in need thereof. These methods encompass the administration of a therapeutically effective amount of the GST inhibitor compounds or the pharmaceutical compositions as described herein. Methods of treating parasitic infections such as malaria or drug-res istant malaria comprising the administration of a pharmaceutical composition comprising a (known) antimalarial agent, preferably an early-stage specific antimalarial agent as described above together with a GST inhibitor compound as defined herein likewise represent a particularly preferred aspect of the present invention. Since the 3-D structure of various GST isoforms is known (see, e.g., Fritz-

Wolf et al., 2003), it will be understood that the compounds disclosed and claimed in the present invention may further be used as "lead" compounds in molecular modeling applications for the development of improved inhibitors having for example an increased binding affinity. Accordingly, methods for identifying new GST inhibitors by employing the GST inhibitor compounds of the present invention in 3-D docking and molecular modeling applications are also contemplated by the present invention.

Brief Description of the Drawings

Figure 1: Structures of Ellagic acid ([1]benzopyrano[5,4,3 -cde][1]benzopyran-5,10- dione,2,3,7,8-tetrahydroxy-(7C1,8C1,9C1)), flavellagic acid ([1]benzoρyrano[5,4,3- cdc][1]benzopyran -5_,10-dione,1, 2,3,7,8-pentahydroxy -(8Cl,9C1)), coraleoellagic acid

([ 1 ]benzopyrano[5 ,4,3 -cde] [ 1 ]benzop yran-5 , 10-dione, 1 ,2,3 ,6,7,8-hexahydroxy-(9C1)), and hypericin (phenantro[1,10,9,8-opqra]perylene-7,14-dione , 1,3,4,6,8,13-hexahydroxy-10,11- dimethyl-, stereoisomer (6CI,7CT,8C19C1)) tested for GST inhibition (for the synthesis of FEA and CEA see Zimmermann et al, 1992).

Figure 2: Competitive inhibition of PfGST by coruleoellagic acid. The Ki was calculated to be appr. 3 μM.

Figure 3a: Stage specificity of chloroquine (CQ) in Plasmodium strain 3D7 dependent on inhibitor concentration and incubation time. Figure 3b: Stage specificity of coruleoellagic acid (CEA) in Plasmodium strain 3D7 dependent on inhibitor concentration and incubation time

Detailed Description of the Invention

General Definitions

Certain terms which will be used to describe aspects and embodiments of the invention arc defined as follows:

"Affinity" means a binding interaction between a molecule and a protein, between two proteins, or between two molecules. Affinity can be measured and quantitated indirectly by means of aim assay. Assays suitable for measuring affinity will be known to those skilled in the art, and for GSTs include, but are not limited to, a fluorescence resonance energy transfer (FRET) assay employing labeled GSTPl-I and JNK (Wang et al, J. Biol. Chem. 276:20999, 2001), a cbromogemc assay employing 1-chloro-2,4- dinitrobenzene (CDNB) (see, e.g. Pickett and Lu, Ann. Rev, Biochem. 58:743, 1989, or Harwaldt et a/., 2002), and in vivo assays employing chemotherapcutic potentiation and myelostimulation (for reference see U.S. Pat. Nos. 5,767,086 and 5,955,432). Numerical values arc often obtained from assays that are useful for quantitating affinity. These include, but arc not limited to the dissociation constant (Kd), the 50% inhibitory concentration (IC₅₀), the Michaelis-Menten constant (Km), and the inhibitory constant (Ki). It will be appreciated that relative differences in such numerical values are used to express equivalent relative differences in affinity and vice versa. It will be further understood that the determination of the binding affinity of a compound according to the present invention for one or more GST classes involves the separate testing of a representative GST isozyme from the one or more GST classes in an assay as described above. "Alkyl" in general means a saturated or unsaturated; unsubstituted or substituted (mono- or mitltisubstituted by, for example, C1₅ Br, F, I, -NH₂, -OH, -O, -NO₂, - COOH, -SO₃H, -SO₂NH₂, -CF₃, or C₄-₂₀ aryl); straight chain, branched chain, or cyclic hydrocarbon moiety having 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, and most preferably from 1 to 6 carbon atoms wherein the chain or cyclic hydrocarbon moiety may optionally be interrupted by at least one hcterσatom such as N, O or S.

"Alkyloxy" (or "alkoxy") means a substituted or unsubstituted alkyl group containing from 1 to 20 carbon atoms, preferably from 1 to 12 carbon atoms, and most preferably from 1 to 6 carbon atoms, wherein the alkyl group is covalently bonded to an adjacent element through an oxygen atom (e.g,, methoxy- and ethoxy-),

"Alkenyl" and "Alkenyloxy" should be understood as having one or more unsaturated carbon-carbon bonds (C-C double bonds) in the group containing from 2 to 20 carbon atoms, preferably from 2 to 12 carbon atoms, and most preferably from 2 to 6 carbon atoms and wherein the alkyl group is covalently bonded to an adjacent clement through an oxygen atom (for the term alkenyloxy),

"Alkinyl" and "Alkinyloxy" should likewise be understood as having one or more triple carbon-carbon bonds in the group containing from 2 to 20 carbon atoms, preferably from 2 to 12 carbon atoms, and most preferably from 2 to 6 carbon atoms. For the term "alkenyloxy", the alkyl group is covalently bonded to an adjacent element through an oxygen atom.

"Aryl" means a carbocyclic moiety which may be substituted by, for example, Cl, Br, F, I, -NH2, -OH, -O, -NO2, -COOH, -SO3H, -SO2NH2, -SO2(alkyl), -

CF3, or C6-20 aryl; and containing one or more benzenoid-typc, aromatic rings containing from 4 to 40, and preferably containing from 6 to 30 carbon atoms. This carbocyclic moiety may optionally be interrupted by at least one heteroatom such as N, O or S.

"Aralkyl " means an aryl group attached to the adjacent atom by an alkyl group (e.g., benzyl), preferably containing from 6 to 30 carbon atoms,

The pharmaceutically active compounds according to the present invention are inhibitors of glutathione S-transferase (GST, EC 2.5.1.18). "Inhibition" in the context of the present invention refers to contacting a GST isozyme with a molecule (i.e., an "inhibitor") such that the molecule interferes with the ability of the isozyme (monomer or dimer) to engage in enzymatic catalysis, binding with another protein, or both, A molecule is said to cause "inhibition", and thus "inhibits", if the molecule can be contacted with the isozyme in a sufficient concentration to reduce enzymatic catalysis, binding with another protein, or both, by at least 50% as measured in an assay. The mechanisms by which the inhibitors of the present invention may inhibit the activity of GSTs comprise all mechanisms known by the person skilled in the art, e.g., competitive or allosteric inhibition; covalent (posttranslational) modifications, such as phosphorylation, tyrosinylation, methylation, amidation, sulfatation, acylation, myristoylation, palmitation, farnesylation, glycosylations, cleavage of a proprotein, cleavage of a signal sequence or inhibition of the reversal of such posttranslational modifications; cellular mistargeting, such as the targeting of GST to inappropriate cellular compartments; degradation of GST, or inhibiting or prolonging protein interaction between GST and third molecules. In some embodiments, inhibition may be associated with covalent modification of the inhibitor, although this is not a requirement of the invention. For example, GST may conjugate GSH to certain inhibitors, or itself become covalently attached to the inhibitor. In preferred embodiments, an inhibitor is not covalently modified during inhibition. It will be appreciated that the GST inhibitor compounds according to the present invention may also bind to and/or inhibit or activate other targets such as enzymes or receptors. For example, it has been found that some of the GST inhibitor compounds (e.g., corulleoellagic acid) may also inhibit the enzyme function of thioredoxin reductase and glutathione reductase. But as long as a compounds also inhibit the function of GST, it will be understood that the compounds arc still encompassed within the definition of a GST inhibitor outlined above.

"Isozyme" refers to an enzyme that performs the same catalysis as another enzyme but is structurally different. Isozymes are also referred to as isoenzymes, and may be monomelic or multiroeric (i.e. composed of two or more monomers or monomelic subuπits). The difference in structure may be a difference in primary (Le. amino acid sequence), secondary (e.g. α-hclical or β-shect content), tertiary structure, and/or quaternary structure. As an example, the spacing between active sites within two or more monomers in a multimeric enzyme may be different between two multimeric isozymes having the same catalytic activity (i.e., they have a different quaternary structure).

Furthermore, the inhibitors of the present invention are not only useful in the treatment of diseases amenable to the inhibition of GST activity, preferably (drug- resistant) malaria, multidrug-resistance and (drug-resistant) cancer, but they are also active in preventing these diseases, or in ameliorating (i.e., alleviating the symptoms of) these diseases. Therefore, for the purposes of the present invention, whenever it is referred hereinbelow to the "treatment" of a certain medical condition, this also implies "prevention" and "amelioration" of said diseases.

With respect to all of the pharmaceutically active compounds of the invention mentioned herein, it is contemplated that the present invention relates to both the use of these compounds for the preparation of a pharmaceutical composition (or medicament) for the prevention, treatment or amelioration of the medical conditions described herein, as well as to methods for preventing, treating, or ameliorating said medical conditions, characterized in that the compounds or pharmaceutical compounds comprising such compounds arc administered to a subject in need thereof. Thus, all preferred embodiments of said uses and said methods of the invention both analogously relate to these aspects of the present invention, even if this is not always expressly mentioned herein.

Any compound of the present invention containing one or more asymmetric carbon atoms may occur as a raccmatc or a raccmic mixture, a single enantiomer, a diastereomeric mixture and an individual diastereomer. Unless otherwise indicated, all such isomeric forms of these compounds are expressly included in the present invention. Each asymmetric carbon atom may be in the R or S configuration. Suitable methods of separating racematcs into the individual enantiomera, such as chiral chromatography and others, arc known to the person skilled in the art. Some of the compounds of the invention can exist in more than one tautomeric form, all of which are encompassed by the present invention. The compounds of the invention may also be present in the form of a prodrug. Prodrugs include those compounds that, upon simple transformation, are modified to produce the compounds that are expressly disclosed in this application. Simple chemical transformations include hydrolysis, oxidation and reduction which occur enzymatically, metaboHcally or otherwise. Prodrugs of the compounds of the invention may have attached thereto one or more functional groups which facilitate, e.g., the use of the compound as a drug in the body, e.g. by facilitating entry into cells etc. Such a functional "prodrug moiety" may be cleaved from the compound by said simple chemical transformations. Prodrug moieties include phosphate groups, peptide linkers, sugars and others. When a prodrug of this invention is administered to a patient, the prodrug may be transformed into a compound as disclosed hereinbelow, thereby imparting the desired pharmacological effect.

The compounds used in the present invention as described hereinbelow can also be targeted for specific delivery to a certain cell type to be treated by conjugation of the compounds to a targeting moiety, such as antibodies, cytokines and receptor ligands that are specific to the cell to be treated.

Whenever it is referred to a "pharmaceutically acceptable salt, analog, or derivative" for the purposes of the present invention, it is referred to any pharmaceutically acceptable acid, salt or ester of a compound of the present invention. Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acids include hydrochloric, hydrobromic, sulphuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, oxalic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, formic, benzoic, malonic, naphthalene-2 -sulfonic, benzenesυlfonic acids. Salts derived from appropriate bases include alkali metal (e.g., lithium, sodium, potassium), alkaline earth metal (e.g., magnesium, calcium), ammonium and N-(C₁_₄alkyl)4⁺, and organic amine salts such as morpholine, piperidine, dimethylaroine or dicthylamine salts. Analogs or derivatives of the compounds of the invention may have variations in the chemical structure that essentially do not change the inhibitory properties of the compounds of the present invention. Λs outlined above, the 3-D crystal structure of a number of GSTs has been determined in the art (see, e.g., Fritz-Wolf et al., 2003 or Perbandt et al., 2004). Moreover, suitable assays to determine GST activity are available so that it is possible for those of skill in the art to identify positions in the inhibitor molecule which may be derivatized without affecting the inhibitory properties of the compound. Preferred.Embodiments of the Present Invention

In one embodiment of the present invention, the novel GST inhibitor compounds according to the present inversion have a structure characterized by the following general formula I:

Alternatively, the GST inhibitor compounds contemplated in the present Invention may be represented by the following general formula II:

wherein the radicals R¹, R^1a, R², R^2a, R³, and R^3a have the meanings as set out hereinabove.

When R¹ or R^1a is hydrogen, the compounds according to general formulas I and II have one or more hydroxy-groups attached to an aromatic ring system. It is well- known to those of skill in the art that these phenolic hydroxy groups have a lower pKa than the respective groups attached to, e.g., an aikyl group. Accordingly, the presence of hydroxyl groups in the compounds of the invention may lead to deprotonation and optionally also to tautomcozation. All of these forms arc likewise encompassed by the present invention.

When R¹ or R^1a, as well as R⁴ to R¹⁴ arc alkyl, alkenyl, alkinyl, cycloalkyl, aryl, heteroaryl, aralkyl, and the like, it will be understood that these radicals may optionally be substituted with a non-carbon atom such as N, O, or S. Each of these residues may be substituted at one position_* or at more than one position, as indicated by the use of the term "multisubstituted". The helcroatom may be attached to the chain, or alternatively be inserted into the chain.

Moreover, two adjacent hydroxy groupsi in the compounds of the invention may be derivatized by an ethylene bridge, thereby creating another 6-membered ring structure. Preferably, the ring structure is formed by an ethenyl group thereby maintaining the aromatic, planar structure of the compound. However, other short bridging molecules or atoms yielding an additional stable 5-, 6 or 7-membered ring are also contemplated in the present invention. When R¹ and R^1a are selected from -CN, -NC, -(C=O)-R⁴, -(C=S)R⁴, -

(C=O)NR⁵R⁶, -(S=O)R⁷, -SO₂R⁸, SO₃R⁹, -(P=O)R¹⁰R", -PO₂HR¹⁰, -SiR¹²R¹³R¹⁴, it is readily apparent that R⁴ to R¹⁴ may be any radical usually encountered in such substitucnt including but not limited to hydrogen, C₁-C₁₂ straight or branched chain alkyl, C₂--C₁₂ straight or branched chain alkenyl, straight or branched chain C₂-C ₁₂ alkinyl, C₃-C₈ cycloalkyl, aryl, heteroaryl aralkyl, halogen, -CF₃, and the like. The foregoing radicals may optionally carry one or more than one substituent as explained above. In addition, the above listed radicals may optionally be attached to the oxygen atoms of the compounds according to formulas I and II via a residue or linker R¹⁵, which can be any chemically feasible moiety including but not limited to a saturated or unsaturated, straight or branched chain alkylenc group having from I to about 20 carbon atoms. Preferably, R¹⁵ is a methylene-, ethylene, propylene, or n-butylene group.

Furthermore, it should be understood tbat any feasible combination of the radicals outlined above is likewise contemplated for the compounds of the present invention. Particularly preferred radicals for R¹ and R^1a are alkyl-, alkenyl-, aryl-, aralkyl, or heteroaryl groups resulting in the respective ether derivatives of the GST inhibitor compounds according to formulas I and II. Other preferred radicals are acyl groups (i.e., -(C-O)-R⁴) which yields the ester derivatives of said compounds. Similar considerations arc valid for the radicals R² / R²" and R³, wherein the radical may represent cither a hydrogen or an oxygen atom attached to a group R¹ / R^Ia. Compounds wherein one of R² and R³ is hydrogen and the other one is a residue -OR¹ will be understood to represent derivatives of flavellagic acid. Likewise, compounds wherein R² and R³ = -OR¹ will be understood to represent derivatives of coruleoellagic acid. Flavellagic acid and its pentamethylether are known in the art, so that the radicals R¹ to R³ in formula I cannot be: R¹= hydrogen, one of R² or R³ = -OH, the other one hydrogen (this excludes flavellagic acid), or R¹ = methyl, one of R² or R³ = -OCH₃, the other one hydrogen (this excludes flavellagic acid pentamethylether). The same is true for coruleoellagic acid and its hexamethylether , so that compounds wherein for every position R¹ = hydrogen, and R² and R³ are both -OH or -OCH3 are also excluded from this aspect of the present invention.

For compounds according to general Formula II, the following proviso applies in order to exclude the known molecule hypericin from this aspect of the invention. Hence, when R^3a is methyl, R^1a and R^2a may not be hydrogen for every occurrence in general formula II.

These GST inhibitors according to general formula I or II as outlined above normally have only one ligand binding moiety and are thus to be understood as monovalent GST inhibitor compounds. However, for certain applications, it may be preferable to link the GST inhibitors according to general formula I or II to other compounds. For the purposes of the present invention, it is contemplated that the GST inhibitor compounds may optionally be covaleritly linked to at least one other compound. The compounds linked to the GST inhibitor compounds of the invention may be selected from a variety of options including but not limited to other compounds known to bind to GST, a second GST inhibitor molecule, other compounds conveying specific properties to the molecule (such as increased solubility or targeting to a desired location) or to other pharmaceutically active agents. For example, the GST inhibitor compounds according to general formula I or II may be covalently attached via a bond, or a bi- or multivalent linker to compounds selected from the group of glutathione, a glutathione analog, a glutathione conjugate, ethacrynic acid, cibacron blue, uniblue A, doxorubicin, gossypol, hematin, rose bengal, sulfobromophthalein, indomethacin , piriprost, eosin b, eosin y, a synthetic or naturally occurring drug, a polysaccharide, a polynucleotide, a polypeptide, another compound of general formulae I or II, a known glutathione-S-transferase inhibitor, an antimalarial agent, a toxin, a chemotherapeutic agent, an antiproliferative or antineoplastic agent, or a combination or mixture of any of the foregoing compounds. The additional compounds may feasibly be attached to the GST inhibitor compounds via one of the oxygen radicals attached to the aromatic ring system.

In a preferred embodiment of this aspect of the invention, the bi- or multivalent GST inhibitor compounds are comprised of two or more GST inhibitor compounds attached to each other via linker moiety. Also preferred are compounds, where the GST inhibitor compound is attached to another compound known to bind to the active site of GST, e.g., peptide inhibitors of GST, or glutathione and glutathione analogs.

Since the GST enzymes seem to exclusively occur as dimers in vivo, it is readily apparent that bivalent inhibitors capable of binding simultaneously to both the active sites of the two active sites in a GST dimer are particularly suitable for the intended applications. Alternatively, the bi- or multivalent GST inhibitor compounds may be constructed such that one ligand moiety binds to the G-site of a GST monomer, whereas the other Iigand binds to the H-site of the same or neighboring GST monomer.

Suitable linker moieties are known in the art and may be adapted by those skilled in the art to fit the particular requirements of the respective application, A detailed description about suitable linker moieties may be found in US application published as US2005004038 A1 The entire content of this application is incorporated herein by reference. Among the preferred linker moieties arc those comprising a polynucleotide, a peptide, a saccharide, a cyclodextrin, a dextran. polyethylene glycol, polypropylene glycol, polyvinyl alcohol, a hydrocarbon, a polyacrylate, an alkyl chain interrupted by one or more atoms of O, S, or N, a carbonyl, an amide or an aromatic group; or an amino-, hydroxy-, thio- or carboxy-functionalizcd silicone. Of course, any combination of these functional groups is likewise possible and intended to be encompassed in the present invention. The bi- (or multivalent linker may separate the two molecules or two ligand domains from each other so that the distance between the two compounds (calculated between the attachment sites for the tinker) is between 5 and 100 Angstrom, preferably between 5 and 50, and more preferably between 10 and 25 Angstrom,

As disclosed in US200500403S A1, the linker may also be derivatized in order to optimize the pharmacological properties of the compounds of the invention. For example, the linker may be modified such that the absorption, distribution, metabolism or excretion of the bi- or multivalent GST inhibitor is adapted to the specific application or requirements.

Since monomers in a dimeric GST isozyme assume a fixed geometry with respect to one another, dimeric GSTs exhibit isozyme-specific surfaces beyond their active sites, Dimeric enzymes like PfGST thus offer unique discriminators that can be exploited by bivalent inhibitors in order to achieve highly isozyme-selective inhibition. These discriminators include (a) the distance between binding sites on each monomer unit as a function of its quaternary organization, and (b) the composition of the dimer surface that lies between active sites.

Particularly preferred linkers according to this aspect of the invention thus enhance the binding affinity of the bi- or multivalent inhibitors to a selected GST isozyme, or ideally confer increased isozyme selectivity to the bi- or multivalent GST inhibitor compounds.

In another aspect, pharmaceutical compositions as defined herein are also provided by the present invention. These pharmaceutical compositions may comprise any of the specific GST inhibitor compounds as defined hereinabove, including compositions comprising one GST inhibitor compound as defined hereinabove, compositions comprising a combination of at least two different GST inhibitor compounds, or compositions comprising at least one GST inhibitor and at least one other pharmaceutically active compound. Optionally, the pharmaceutical compositions of the present invention may further comprise pharmaceutically acceptable carriers, diluents and/or excipients, and the like

In preferred embodiments of this aspect of the invention, the at bast one other pharmaceutically active compound is selected from a known glutathione-S- transferase inhibitor, an antimalarial agent, a toxin, a chemotherapeutic agent, an antiproliferative agent, an antineoplastic agent, or a combination or mixture thereof.

Examples for inhibitors of GST include hemin and protoporphyrin IX, cibacron blue, ethacrynic acid, 2,4-dichlorophenoxyacetate and 2,4,5- trichlorophenoxyacetate, S-substituted GSH derivatives such as S-hexylglutathione, S- propylglutathione, S-(p-nitrobenzyl)glutathione, S-(N-hydroxy-N- pbcnylcarbamoyl)g]utathione_> S-(N-hydroxy"N-chlorophenylcarbamoyl)glutathi"one, S-(N- hydroxy-N-4-brotnoρhenylcarbarnoyl) glutathione, Glutathione S-sulfonate (GS-SO₃), peptide inhibitors such as γ-L-Glu-L-SerGly (GOH) or γ-L-Glu-L-AlaGly (GH), quereetin, alizarin, purpurogallin. and ellagic acid. For a review on GST inhibitors see Mannervik and Danielson (1988).

Examples of clinically used antimalarial drugs include among others artemisinines, artesunates, chloroquine, sulfadoxine-pyrimethamine , primaquine, quinine, methylene blue, or derivatives or analogs thereof.

Particularly preferred compositions of the present invention include the GST inhibitor compounds of the present invention together with other clinically used antineoplastic drugs or antimalarial agents. Most preferably, the pharmaceutical compositions of the present invention comprise one ore more than one GST inhibitor compound(s) of the present invention together with one or more than one antimalarial agents exhibiting an early-stage specific inhibition of Plasmodium growth (e.g., artemisinines, artesunates, chloroquine, sulfadoxinc-pyrimethamine. primaquine, quinine, methylene blue, or derivatives or analogs thereof). Early-stage specificity means that the antimalarial agent preferentially inhibits parasite growth when given at an early stage, i.e., the ring stage of parasite development.

In any event, it will be understood that any of these pharmaceutical compositions as defined above may be used for the preparation of therapeutic agents for the treatment of the medical conditions outlined herein, and may also be administered to a subject, and in particular a human patient, in the treatment methods according to the present invention.

Another aspect of the present invention relates to the use of the GST inhibitor compounds and the pharmaceutical compositions as described herein for the preparation of a pharmaceutical composition, wherein said pharmaceutical composition or medicament is for preventing, treating or ameliorating a medical condition in a subject, preferably a human subject in need of such treatment that benefits from inhibiting GST activity in said subject. Preferably, the pharmaceutical composition is a therapeutic composition and is manufactured, into a pharmaceutical formulation for convenient and safe administration to a patient. For conciseness and in order to avoid confusion, the pharmaceutical composition prepared by using tbe GST inhibitor compounds or compositions of the present invention will be referred to as the "medicament".

Any of the GST inhibitor compounds or pharmaceutical compositions as defined hereinabove may be used for the preparation of said medicaments. Notably, the compounds flavellagic acid (FEA), coruleoellagic acid (CEA) and hypericin, as well as the pentamethyl- and hexamethylethers of FEA and CEA are not excluded in the uses according to the present invention, since these compounds have to our knowledge not been used or suggested for the treatment of medical conditions or diseases that will be outlined in detail below.

Accordingly, the GST inhibitor compounds used for the preparation of the medicament according to the present invention may be a monovalent GST inhibitor as defined hereinabove. Alternatively, the GST inhibitor compound may be a bi- or multivalent GST inhibitor compound wherein one GST inhibitor molecule is covalently attached via a bond or via a bi- or multivalent linker to at least one other compound as described in detail hereinabove. It will be further understood that each preferred embodiment described herein is contemplated for the uses according to the present invention. The same is of course true for the pharmaceutical compositions described in detail above, although it is again noted that the compounds flavellagic acid (FEA), coruleoellagic acid (CEA) and hypericin, as well as the pentamethyl- and hexamethylethers of FEA and CEA arc not excluded in the uses of the present invention.

The medicaments prepared by the use of the GST inhibitors and compositions of the present invention are particularly suitable for the prevention, treatment or amelioration of diseases such as malaria, drug-resistant malaria, multidrug resistance, toxoplasmosis, African trypanosomiasis (sleeping sickness), Chagas disease, cancer, and drug-resistant cancer, and may also have beneficial effects when treating immunosuppression, immunosuppression during chemotherapy or radiotherapy, myelodysplasia, and bone rnarrow transplantation, Those with, skill in, the art will appreciate that one possible strategy to overcome multidrug resistance in cancer cells - and thus also parasites which share many features with cancer cells - involves the treatment with a combination of an antineoplastic agent and a chemomodulator that inhibits the activity of the resistance-causing protein. In cases where GSTs are thought to play a role in drug resistance, chemomodulation may be achieved by using inhibitors of glutathione synthesis or by using GST inhibitors.

Accordingly, particularly preferred indications for the purposes of the present invention includes the drug resistant forms of cancer and malaria. However, it is envisaged that the GST inhibitors of the present invention may also effectively be used in the treatment of other parasitic infections such as those outlined above. In view of the ubiquitous nature and detoxification role of GSTs, the pharmaceutical compositions may prove to be particularly useful for the treatment of multi-drug resistance, which is not restricted to cancer treatment, but is observed during the treatment of many other diseases as well.

In preferred embodiments of the uses according to the present invention, the active compounds of the medicament or pharmaceutical formulation target or preferentially target a human GST isozyme. In other preferred embodiments of this aspect of the invention, the active compounds of the medicament target or preferentially target a GST of a microorganism that is pathogenic for humans or animals. In this context, it is particularly preferred when the active compound(s) of the medicament target or preferentially target a GST isozyme from a parasite, specifically from the taxonomic group known as apicomplexa. In view of the fact that malaria is caused by the parasite Plasmodium falciparum, it will be appreciated that the most preferred active compounds of the medicament are able to target or preferentially target the GST isozyme from Plasmodium falciparum (PfGST). It is readily apparent that for a maximal effect of the treatment, the active compounds of the medicament must preferentially inhibit the GST isozyme that is responsible for the drug resistance. Thus, active compounds of the medicament that are able to target or preferentially target the GST molecules located m or expressed by a tumor, an infectious agent such as the above-referenced Plasmodium falciparum, or the bone marrow of a patient to be treated are particularly preferred in this aspect of the invention.,

In order to increase the specificity and therapeutic window for the medicaments of the present invention the active compound(s) of the medicament preferably exhibit a binding affinity for one GST class that is greater than the affinity for another GST class. It is particularly preferred that the binding affmity for one GST class is at least 2-fold, more preferably 5-fold greater, and most preferably at least 10-fold greater than the affinity for another GST class. Depending on the indication to be treated, it may be preferred in some embodiments of this aspect of the invention that the binding affinity towards a parasite GST such as Plasmodium falciparum GST (PfGST) than for another GST class, e.g., from humans. The latter is obviously preferred for medicaments that are intended for the treatment of parasitic infections, for example malaria, and particularly its drug resistant forms.

With regard to the binding affinity of the GST inhibitor compounds of the present invention, it will be understood that any GST inhibitor compound that is capable of binding and inhibiting GST activity is contemplated in the present invention. However, particularly suitable GST inhibitor compounds may easily be determined by the skilled person taking into account the information provided in the present invention and using well-established in vitro or in vivo assays for testing of GST binding and inhibition. Preferred GST inhibitors according to the present invention are those having an IC₅₀ for a GST isozyme of less than 200 μM, preferably less than 100, 90, 80, 70, 60, or 50 μM, and most preferably less than 40, 30, 20 or even 10 μM in a standard in vitro GSH-dependent DTNB assay (see, e.g., Harwaldt et al, 2002), The details of this assay are also described in the Experimental section in Example 1.

The most preferred GST inhibitor compounds for the uses of the present invention are the compounds named flavellagic acid ([1]benzopyrano[5,4,3- cde][1]benzopyran-5,10-dione, 1,2,3,7,8-pentahydroxy-(8C1,9C1), coruleoellagic acid ([1]benzopyrano[5,4,3-cde][1]benzopyran -5,10-dione,1,2,3,6,7,8- hexahydroxy-(9C1)), or hypericin (Phenantro [1,10,9,8-opqra] perylene-7,14-dione, 1 ,3,4,6,8, 13 -hexa-hydroxy- 10,11 -dimethyl-, stereoisomer (6C1,7C1,8C1.,9C1)).

The GST inhibitor compounds according to the present invention may be classified by their binding mode and binding site within the GST molecule. In some embodiments of the present invention, it is preferred that the monovalent GST inhibitor compounds bind to the glutathione binding site (G-site) of the GST isozyme, thereby competing with the natural substrate glutathione. In other embodiments, it may be desirable that the monovalent GST inhibitor compounds bind to the hydrophobic substrate binding site (H-site) of the GST isozyme,

In the case of bi- or multivalent GST inhibitor compounds, the compounds may also simultaneously bind to the H-site and the G-site of the GST monomer, and it cannot be excluded that certain monovalent GST inhibitor compounds also occupy both binding sites and interacts with residues of the H-site and the G-sitc, However, it will be appreciated that bivalent GST inhibitor compounds can be specifically tailored so that one ligand of the bivalent inhibitor binds to the H-site and the other Iigand binds to the G-site of the GST molecule. Accordingly, such inhibitors are particularly preferred in this aspect of the present invention.

Since GST always forms either homo- or heterodimers, it is apparent that bi- or multivalent GST inhibitor compounds may also be designed so as to bind to the active sites of two different GST monomers in a GST dimer. In view of the fact that GST has two binding sites for its two natural substrates, it is readily apparent that a number of possible combinations are conceivable for the binding mode of bi" or multivalent GST inhibitors of the present invention. In a preferred embodiment, the bi- or multivalent GST inhibitor binds simultaneously to the G-sitcs of at least two GST monomers. Alternatively, the bi- or multivalent GST inhibitor may binds simultaneously to the H-sites of at least two GST monomers. In yet another preferred embodiment, the bi- or multivalent GST inhibitor binds to the G-site of one GST monomer and the other ligand moiety binds to the H-site of the other GST monomer in a GST dimer. The desired selectivity and binding mode may be achieved by selecting the appropriate GST inhibitor compounds as the Iigands of such bi- or multivalent GST inhibitor compounds, and also by the careful selection and design of the linker moiety covalently linking the two or more Iigands. Suitable linkers and methods for designing a suitable linker arc disclosed in US20050004038 A1 (Lyon, R.P. et al.), incorporated by reference. In some embodiments of the invention, the GST inhibitor compounds of the present invention exert their inhibitory effect by inhibiting the catalytic function of the GST isozyme, i.e., the conjugation of GSH to an electrophilic, usually hydrophobic substrate. In other embodiments, the GST inhibitor compounds simply inhibit the binding of the GST isozyme to another protein. The latter noncatalytic function of GSTs is often referred to as the ligandin activity of this protein (see, e.g,, Lu & Atkins, 2004).

Yet another aspect of the invention relates to methods of preventing, treating, or ameliorating a medical condition as defined herein in a mammal, particularly in a human subject, said method comprising administering to said mammal or said human subject a therapeutically effective amount of the GST inhibitor compounds or the pharmaceutical compositions described herein. The medical condition to be treated include but arc not limited to malaria, drug-resistant malaria, multidrug resistance, toxoplasmosis, African trypanosomiasis (sleeping sickness), Chagas disease, cancer, drug-resistant cancer, immunosuppression, immunosuppression during chemotherapy or radiotherapy, myelodysplasia, and bone marrow transplantation.

Pharmaceutical Formulations and Modes of Administration

The GST inhibitor compounds and pharmaceutical compositions comprising said GST inhibitor compounds and optionally in combination with other pharmaceutically active compounds as defined in the present invention may be administered to a subject, e.g., a mammal, such as a human patient, in need thereof, in a variety of forms adapted to the chosen route of administration, e.g., orally, rectally or parenterally, by intravenous, intramuscular, topical, transdermal or subcutaneous routes.

The compounds of the present invention may be administered systemically, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or am assimilable edible carrier. They may, e.g., be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient' s diet For oral therapeutic administration, the compounds of the invention may also be combined with one or more excipicnts and used, e.g., in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of the compounds of the present invention. The percentage of the compounds of the present invention in the compositions and preparations may, of courses, be varied and may conveniently be between about 2% to about 80% of the weight of a given unit dosage form. The amount of compounds of the present invention in such therapeutically useful compositions is such that an effective dosage level will be obtained and may easily be determined by those of ordinary skill in the art.

The tablets, troches, pills, capsules, and the like may also comprise the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcϊum phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and/or a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, e.g., a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with, e.g., gelatin, wax, shellac or sugar and the like. A syrup or elixir may comprise the compounds of the invention, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the compounds of the present invention may be incorporated into sustained- release preparations and devices.

It will be appreciated that all pharmaceutical compositions and formulations described herein may principally be used in the therapeutic methods described and claimed herein. Particularly preferred formulations for delivery of the compounds of the present invention to the target cells of a human subject to be treated include oral delivery formulations which are well known in the art. The compounds of the invention may, however, also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the compounds of the invention or its salts may be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions may also be prepared, e.g., in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may comprise a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions or sterile (e.g. lyopbilized) powders comprising the compounds of the invention, which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes.

In all cases, the ultimate dosage form roust be sterile, fluid and stable under the conditions of manufacture, preparation, and storage. The liquid carrier or vehicle may be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity may be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms may be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of injectable compositions comprising the compounds of the present invention may be brought about by the use of agents delaying absorption, for example, aluminum monostcarate and gelatin.

Sterile injectable solutions are prepared by incorporating the compounds of the present invention in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the compounds of the invention plus any additional desired ingredient present in the previously sterile-filtered solutions. For topical administration, in case the compounds of the present invention are liquids, they may be applied in pure form. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, hydroxyalkyls or glycols or water-alcohol/glycol blends, in which the compounds of the invention can be dissolved or dispersed at effective levels, optionally with the aid of non_¬ toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions may be applied from absorbent pads, or may be used to impregnate bandages and other dressings, or may be sprayed onto the affected area using pump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials may also be employed with liquid carriers to form spreadablc pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

Useful dosages of the compounds of the present invention may be determined by their in vitro activity, as well as in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice and other animals to humans are known to the art; see for example, US 4,938,949. Generally, the concentration of the compounds of the present invention in a liquid composition will be from about 0,01-25 % per weight, preferably from about 0.5- 10 % per weight. The concentration in a semi-solid or solid composition such as a gel or a powder will be about 0,1 -50 % per weight, preferably about 0,5-25 % per weight. The dosage regimen to be employed in connection with the pharmaceutical compositions, methods and uses of the invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition, of the patient; the severity of the condition to be treated; the route of administration; the target cell or tissue, the renal and hepatic function of the patient; and the particular compound (or its salt, analog, or derivative thereof) employed. An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the compound or the compounds required to prevent, ameliorate, or treat the condition.

In general, however, a suitable dose will be in the range of from about 0.01 to about 100 mg/kg of body weight per day [mg/kg/day], preferably from about 0.1 to about 30 mg/kg/day, and more preferably from about 1 to about 10 mg/kg/day_*

The compounds or compositions of the present invention may conveniently be administered in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage form. tdeally, the compounds of the present invention should be administered to achieve peak plasma concentrations of the active compound of from about 0.0005 to about 300 μM, preferably, about 0.001 to 100 μM, more preferably, about 1 to about 100 μM.

The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. An administration regimen according to the present invention includes long- term, daily treatment. By "long-term" is meant at least two weeks and preferably, several weeks, months , or years of duration. Necessary modifications in this dosage range may be determined by one of ordinary skill in the art according to the teachings herein or the teachings described in Remington's Pharmaceutical Sciences (Martin, E.W., cd. 4), Mack Publishing Co., Easlon, PA. The dosage may also be adjusted by the individual physician in the event of any complication.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention as described above. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration, and may further include instructions for use. The present invention also provides treatment methods wherein the GST inhibitors of the invention and the other pharmaceutically active compound($), as listed above, with which it/they arc combined in the pharmaceutical compositions according to the present invention may be either administered jointly (i.e., at the same time), or separately (i.e., sequentially), preferably according to a dosing rcgirαen. Thus, in one embodiment of this aspect of the present invention, dosage delivery of a GST inhibitor compound of the present invention can begin 48 hours prior to delivery of the other pharmaceutically active compound (e.g., the antineoplastic agent or antimalarial drug) with the preferred time being 24 hours and the most preferred time being 12, 9, 6, 5, 4, 3, 2, 1, or less than one hour prior to the delivery of lite other compound. Alternatively, dosage delivery of a GST inhibitor compound of the present invention can begin 48 hours after the initiation of delivery of the other compound, with the preferred ttme being 24 hours and the most preferred time being 12, 9, 6, 5, 4, 3, 2, 1, or less than one hour after the delivery of the other compound.

Alternatively, the compounds may be administered concurrently (i.c, at the same time). It will be appreciated that concurrent administration may either be accomplished by formulating the at bast two compounds into a single pharmaceutical formulation, or by administering multiple formulations (e.g., tablets, etc.) each containing one of the active ingredients, together in a single step.

The GST inhibitor compownd(s) and/or the other pharmaceutically active compound(s) can be independently administered by a variety of routes, including oral or rectal routes, or parenteral routes like intravenous, intramuscular, topical, transdermal or subcutaneous routes as described in detail above, in the therapeutic methods of the present invention.

The following Examples are meant to further illustrate some aspects of the present invention and are not to be construed as limiting the present invention irs, any way.

Examples

Example, I

Perivatives of ellagic acid as GST inhibitors

Compounds according to the present invention were tested in vitro for GST inhibition. To this end, the two compounds flavellagic acid (FEA) and coruleoellagic acid (CEA) were synthesized as described in Zimmermann et al., 1992. As shown in Figure 1, these two compounds possess one (FAE), or two additional hydroxy groups compared to ellagic acid allowing these compounds to target the more solvent accessible active site of PfGST. In addition, hypericin (available from Alexis Biochemicals), the structure of which is also shown in Figure 1, was also tested for its capability to inhibit GST.

The compounds were tested in direct comparison with ellagic acid on isolated PfGST and human placenta GST. PfGST was recombinanty produced as described by Harwaldt et at., 2002, human GST was purchased from Sigma (St. Louis, USA). The activity and Michaelis-Menten parameters of this recombinant PfGST were determined in a standard GSH-dependent DTNB-assay (Harwaldt et al, 2002); 1 ml assay mixture containing 1 mM GSH and PfGST in 100 mM Hepes, 1 mM EDTA, pH 6.5 was equilibrated to 25°C. The reaction was started by the addition of 0.5 mM CDNB, and the formation of S-(2,4-dinitrophenyl)glutathione as monitored spectrophotormetrically at 340 nm (E340nm = 9.6 mM^-1 cm^-1). Alternatively, the reaction can be started by the addition of the enzyme . The specific activity of recombinant PfGST in this assay is 0.20 U/mg (5.2 U/μmol), By varying the concentration of GSH, the K_m for GSH was determined by Harwaldt et al. (2002) and Liebau et al. (2002) to be 164±20 μM and 156±13 μM, respectively. Thus, under physiological conditions (for review see Becker et al, 2003), PfGST can be considered to be saturated by GSH in vivo. For CDNB a K_m value of > 2 mM was estimated. It should be noted that the spontaneous background reaction between GSH and CDNB increases with increasing pH or increasing substrate concentrations. Thus, if necessary, the measured activities have to be corrected,

Inhibitors were tested in the same DTNB assay described above. For the measurements, the inhibitors were added to 1 ml aliquots containing 1 mM reduced glutathione (GSH) and GST in 100 mM Hepes, 1 mM EDTA, pH 6.5 at 25°C The respective inhibitor was added in different concentrations and preincubated with the enzyme for 1O min at 25°C. The reaction was then started with 0.5 mM CDNB. The results are shown in Table 1 below.

Table 1: Inhibition of isolated P. falciparum GST and human placenta GST by ellagic acid, flavellagic acid, coruleoellagic acid and hypericin.

All compounds were more effective on PfGST than on hGST with ICso values of 75 μM (EA), 40 μM (FEA), 10 μM (CEA), and 3 μM (hypericin) on PfGST.

For CEA the type of inhibition was determined in. more detail and the data revealed a competition of CEA with both substrates, GSH and CDNB. The Ki for competition with GSH was determined to be 3 μM (Figure 2). Example.2:

Antimalarial activity of EA. FEA. and CEA tested in P. falciparum strain Kl

The growth inhibitory effect of EA, FEA and CEA was determined on the chloroquine resistant P. falciparum strain K1 which was cultured as described in Harwaldt et al., 2002. For this purpose the incorporation of radioactive hypoxanthine into parasites was determined (Ohrt et al, 2002). The drugs were added in the early ring stage of the parasites (> 90% rings), radioactive hypoxanthine was added after 48 h, and incorporation was determined after 72 hours. The previously reported antiplasmodial effect of ellagic acid could be reproduced under these conditions yielding am IC₅₀ for EA of 897.67 ± 134.62 nM. The two EA derivatives FEA and CEA were much more potent as indicated by their IC₅₀ values of 97.5 ± 16 nM and 86.96 ± 23.79 nM, respectively (of. Table 2). Under the same conditions, chloroquine was active with an IC₅₀ of 100.0 ±1 19-07 nM. These data show that FEA and CEA are highly promising potential antimalarials which are particularly effective against chloroquine resistant P. falciparum strains. The compounds were also tested for their effect in other Plasmodium strains that show a varying chloroquine resistance under the same conditions as described above for strain Kl. The results of these experiments are shown in Table 3.

Table 2: IC₅₀ values of chloroquine, ellagic acid, flavellagic acid, and coruleoellagic acid on the chloroquinc resistant P. falciparum strain Kl.

Table 3: IC₅₀ values of chloroquine, ellagic acid, flavellagic acid, and corulcoellagic acid on P. falciparum strains having a varying degree of chloroquine resistance.

Example_3: Stage Specificity of P. falciparum Strain 3D7 Growth Inhibition, bv CO and CEA

P. falciparum Strain 3D7 growth was assessed by measuring the incorporation of the nucleic acid precursor [³H]hypoxanthine. Synchronized cultures of young NF54 trophozoites (20 hr) with parasite counts of 0.15% and a hematocrit of 5% were divided into three 10 cm Petri dishes. Two dishes were further incubated for 16 hr or 32 hr at 37°C for maturation into early schizonts (36 hr) or early ring stages (4 hr). The third dish with the early trophozoites was used immediately for exposure for a 1 hr, 6 hr, 12 hr or 24 hr period to chloroquine (CQ) [final concentrations 443, 222, 111, 55, 28, 14, 7 ng/ml] and coruleoellagic acid (CEA) [final concentrations 1410, 705, 353, 176, 88, 44, 22 ng/ml]. Dilutions were prepared in culture medium without hypoxanthine (screening medium) from the DMSO stock solutions and 100 μl of each compound solution was titrated in duplicates into sterile, flat-bottom 96-well plates (Costar). Subsequently, 100 μl of parasitized blood was added to each well. After the respective incubation times of the parasite-compound mixture, the plates were centrifuged for 3 min at 600x.g. From the total volume of each well (200 μl), 150 μl of the supernatant was removed (including control wells). Plates were then washed with 150 μl of screening medium and centrifuged as above. Wash steps were repeated 3 times as described above. After the last wash, a mixture of 150 μl of screening medium and 50 μl of [³Η]hypoxanthine in screening medium (0.5 μCi) was added per well. After another incubation period of 24 hr at 37°C in the atmosphere described above, the plates were frozen at -20°C. For the IC₅₀ determination, plates were thawed and harvested with a Betaplate cell harvester (1295-004 Betaplate; Wallac Perkin-Elmer) onto glass filters. The dried filters were inserted into a plastic foil with 10 ml of scintillation fluid and counted in a Betaplate liquid scintillation counter (1205 Betaplate; Wallac Perkin -Elmer). The results of each well were recorded as counts per minute and expressed as a percentage of the untreated controls. Suspensions of uninfected erythrocytes were used for background subtraction.

The results of these experiments are shown in Figure 3 a (chloroquine) and Figure 3b (coruleoellagic acid).

As shown by the results depicted in Figures 3a and 3b, chloroquine acts as an carly-stage (i.e., ring stage) specific inhibitor of parasite growth, whereas coruleoellagic acid is particularly effective when administered to the trophozoite stage of the parasite development.

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Claims

Claims:

A glutathione-S -transferase (GST) inhibitor compound having a structure selected from the following general formulae:

a)

or a pharmaceutically acceptable salt, analog or derivative thereof; or b)

or a pharmaceutically acceptable salt, analog or derivative thereof; wherein each R¹ and R^la may independently from each other be hydrogen; optionally mono- or multisubstituted C1-C12 straight or branched chain alkyl, optionally mono- or multisubstituted C2-C12 straight or branched chain alkenyl, optionally mono- or multisubstituted C2-C12 straight or branched chain alkinyl, optionally mono- or multisubstituted C₃-Cs cycloalkyl, optionally mono- or multisubstituted aryl, optionally mono- or multisubstituted heteroaryl, optionally mono- or multisubstituted aralkyl, or a combination thereof;

-CN, -NC, -(C=O)-R⁴, -(C=S)R⁴, -(C=O)NR⁵R⁶, -(S=O)R⁷, -SO₂R⁸, SO₃R⁹, - (P=O)R¹⁰R¹¹, -PO₂HR¹⁰, -SiR¹²R¹³R¹⁴; -R¹⁵-CN, -R¹⁵-NC, -R¹⁵-(C=O)-R⁴, -R¹⁵-(C=S)R⁴, -R¹⁵-(C=O)NR⁵R⁶, -R¹⁵-

(S=O)R⁷, -R¹⁵-SO₂R⁸, -R¹⁵-SO₃R⁹, -R¹^(P=O)R¹⁰R¹ \ -R¹⁵-PO₂HR¹⁰, -R¹⁵- SiR¹²R¹³R¹⁴ wherein -R¹⁵ is a saturated or unsaturated alkylene group from 1 to about 20 carbon atoms; an optionally substituted saturated or unsaturated alkylene group connecting two neighboring oxygen atoms attached to the benzene moiety of the compound to form a cyclic structure; a bond or a bi- or multivalent linker covalently attaching the compound to at least one other compound selected from the group of: glutathione, a glutathione analog, a glutathione conjugate, ethacrynic acid, cibacron blue, uniblue A, doxorubicin, gossypol, hematin, rose bengal, sulfobromophthalein, indomethacin, piriprost, eosin b, eosin y, a synthetic or naturally occurring drug, a polysaccharide, a polynucleotide, a polypeptide, another compound of general formulae I or II, a known glutathione-S -transferase inhibitor, an antimalarial agent, a toxin or a chemotherapeutic agent, or a combination or mixture of any of the foregoing compounds;

R² and R³ may each independently be hydrogen or -OR¹, wherein R¹ has the same meanings as described above; each R^2a may independently be hydrogen or OR^la, wherein R^la has the same meanings as described above; each R^3a may independently be -CH₂-R^4a;

R⁴ to R¹⁴ may each independently represent hydrogen, optionally mono- or multisubstituted Ci-Ci₂ straight or branched chain alkyl, optionally mono- or multisubstituted C₂-Ci₂ straight or branched chain alkenyl, optionally mono- or multisubstituted C₂-Ci₂ straight or branched chain alkinyl, optionally mono- or multisubstituted C₃-C₈ cycloalkyl, optionally mono- or multisubstituted aryl, optionally mono- or multisubstituted heteroaryl, optionally mono- or multisubstituted aralkyl, halogen, -CF₃, or a combination thereof; each R^4a may represent hydrogen, optionally mono- or multisubstituted C₁-C₁₂ straight or branched chain alkyl, optionally mono- or multisubstituted C₂-Ci₂ straight or branched chain alkenyl, optionally mono- or multisubstituted C₂-Ci₂ straight or branched chain alkinyl, optionally mono- or multisubstituted C₃-Cs cycloalkyl, optionally mono- or multisubstituted aryl, optionally mono- or multisubstituted heteroaryl, optionally mono- or multisubstituted aralkyl, optionally mono- or multisubstituted Ci-Ci₂ straight or branched chain alkyloxy, optionally mono- or multisubstituted C₂-Ci₂ straight or branched chain alkenyloxy, optionally mono- or multisubstituted C₂-Ci₂ straight or branched chain alkinyloxy, optionally mono- or multisubstituted C₃-Cs cycloalkyloxy;

-CN, -NC, -(C=O)-R⁴, -(C=S)R⁴, -(C=O)NR⁵R⁶, -(S=O)R⁷, -SO₂R⁸, SO₃R⁹, - (P=O)R¹⁰R¹¹, -PO₂HR¹⁰, -SiR¹²R¹³R¹⁴;

-R¹⁵-CN, -R¹⁵-NC, -R¹⁵-(C=O)-R⁴, -R¹⁵-(C=S)R⁴, -R¹⁵-(C=O)NR⁵R⁶, -R¹⁵- (S=O)R⁷, -R¹⁵-SO₂R⁸, -R¹⁵-SO₃R⁹, -R¹^(P=O)R¹⁰R¹ \ -R¹⁵-PO₂HR¹⁰, -R¹⁵- SiR¹²R¹³R¹⁴ wherein -R¹⁵ is a saturated or unsaturated alkylene group from 1 to about 20 carbon atoms; halogen, -CF₃, or a combination thereof; with the provisos that a) at least one of R² and R³ in formula I is not hydrogen; or b) the compound of formula I is not any of the following compounds: [l]benzopyrano[5,4,3-cde][l]benzopyran-5,10-dione,l, 2,3,7, 8-pentahydroxy- (8Cl,9Cl)(flavellagic acid); [l]benzopyrano[5,4,3-cde][l]benzopyran-5,10- dione,l,2,3,7,8-pentamethoxy-(8Cl,9Cl)(flavellagic acid pentamethyl ether), [l]benzopyrano[5,4,3-cde][l]benzopyran-5,10-dione,l,2,3,6,7,8-hexahydroxy- (9Cl) (coruleoellagic acid), or [l]benzopyrano[5,4,3-cde][l]benzopyran-5,10- dione,l,2,3,6,7,8-hexamethoxy-(9Cl) (coruleoellagic acid hexamethylether); c) when both R^3a in formula II are methyl, then at least one of R^la or R^2a must not be hydrogen.

2. The GST inhibitor compound according to claim 1, wherein the compound of formula I or formula II is a monovalent GST inhibitor.

3. The GST inhibitor compound according to claim 1, wherein the compound of formula I or formula II is a bi- or multivalent GST inhibitor in which at least one of R¹, R^la, R², R^2a, or R³ is a bond or a bi- or multivalent linker L covalently attaching the compound to at least one other compound selected from the group of: glutathione, a glutathione analog, a glutathione conjugate, ethacrynic acid, cibacron blue, uniblue A, doxorubicin, gossypol, hematin, rose bengal, sulfobromophthalein, indomethacin, piriprost, eosin b, eosin y, a synthetic or naturally occurring drug, a polysaccharide, a polynucleotide, a polypeptide, another compound of general formulae I or II, a known glutathione-S -transferase inhibitor, an antimalarial agent, a toxin, a chemotherapeutic agent, an antiproliferative or antineoplastic agent, or a combination or mixture of any of the foregoing compounds.

4. The GST inhibitor compound according to claim 3, wherein the bi- or multivalent linker L comprises a polynucleotide, a peptide, a saccharide, a cyclodextrin, a dextran, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, a hydrocarbon, a polyacrylate, an alkyl chain interrupted by one or more atoms of O, S, or N, a carbonyl, an amide or an aromatic group; an amino-, hydroxy-, thio- or carboxy-functionalized silicone, or a combination thereof.

5. The GST inhibitor compound according to claim 3 or claim 4, wherein the linker L is derivatized to optimize the absorption, distribution, metabolism or excretion of the bi- or multivalent GST inhibitor.

6. The GST inhibitor compound according to any one of claims 3 to 5, wherein the bi- or multivalent linker L enhances the affinity of the bivalent inhibitor or increases isozyme selectivity of the bi- or multivalent GST inhibitor.

7. A pharmaceutical composition comprising a GST inhibitor compound according to any one of claims 1 to 6.

8. The pharmaceutical composition according to claim 7, further comprising a pharmaceutically acceptable carrier, diluent or excipient.

9. The pharmaceutical composition according to claim 7 or claim 8, wherein the pharmaceutical composition further comprises at least one other pharmaceutically active compound.

10. The pharmaceutical composition according to claim 9, wherein the at least one additional pharmaceutically active compound is selected from the group consisting of: a known glutathione-S -transferase inhibitor, an antimalarial agent, a toxin, a chemotherapeutic agent, an antiproliferative agent, , an antineoplastic agent, or a combination or mixture thereof.

11. The pharmaceutical composition according to claim 10, wherein the at least one additional pharmaceutically active compound is an antimalarial agent.

12. The pharmaceutical composition according to claim 11, wherein the antimalarial agent is an early stage-specific inhibitor of Plasmodium growth.

13. The pharmaceutical composition according to claim 11 or claim 12, wherein the antimalarial agent is selected from the group consisting of artemisinines, artesunates, chloroquine, sulfadoxine-pyrimethamine, primaquine, quinine, methylene blue, or derivatives or analogs thereof.

14. The pharmaceutical composition according to any of claims 7 to 13, wherein the pharmaceutical composition is suitable for oral, transdermal or parenteral administration.

15. The GST inhibitor compound as defined in any one of claims 1 to 6, or the pharmaceutical composition as defined in any one of claims 7 to 14 for use in therapy and/or diagnosis.

16. Use of a compound having one of the following general formulae:

a)

or a pharmaceutically acceptable salt, analog or derivative thereof; or b)

or a pharmaceutically acceptable salt, analog or derivative thereof; wherein each R¹ and R^la may independently from each other be hydrogen; optionally mono- or multisubstituted C1-C12 straight or branched chain alkyl, optionally mono- or multisubstituted C₂-Ci₂ straight or branched chain alkenyl, optionally mono- or multisubstituted C₂-Ci₂ straight or branched chain alkinyl, optionally mono- or multisubstituted C₃-C₈ cycloalkyl, optionally mono- or multisubstituted aryl, optionally mono- or multisubstituted heteroaryl, optionally mono- or multisubstituted aralkyl, or a combination thereof;

-R¹⁵-CN, -R¹⁵-NC, -R¹⁵-(C=O)-R⁴, -R¹⁵-(C=S)R⁴, -R¹⁵-(C=O)NR⁵R⁶, -R¹⁵- (S=O)R⁷, -R¹⁵-SO₂R⁸, -R¹⁵-SO₃R⁹, -R¹^(P=O)R¹⁰R¹ \ -R¹⁵-PO₂HR¹⁰, -R¹⁵- SiR¹²R¹³R¹⁴ wherein -R¹⁵ is a saturated or unsaturated alkylene group from 1 to about 20 carbon atoms; an optionally substituted saturated or unsaturated alkylene group connecting two neighboring oxygen atoms attached to the benzene moiety of the compound to form a cyclic structure; a bond or a bi- or multivalent linker covalently attaching the compound to at least one other compound selected from the group of: glutathione, a glutathione analog, a glutathione conjugate, ethacrynic acid, cibacron blue, uniblue A, doxorubicin, gossypol, hematin, rose bengal, sulfobromophthalein, indomethacin, piriprost, eosin b, eosin y, a synthetic or naturally occurring drug, a polysaccharide, a polynucleotide, a polypeptide, another compound of general formulae I or II, a known glutathione-S -transferase inhibitor, an antimalarial agent, a toxin, a chemotherapeutic agent, an antiproliferative or antineoplastic agent, or a combination or mixture of any of the foregoing compounds;

R² and R³ may each independently be hydrogen or -OR¹, wherein R¹ has the same meanings as described above; each R^2a may independently be hydrogen or OR^la, wherein R^la has the same meanings as described above; each R^3a may independently be-CH₂-R^4a;

R⁴ to R¹⁴ may each independently represent hydrogen, optionally mono- or multisubstituted Ci-Ci₂ straight or branched chain alkyl, optionally mono- or multisubstituted C₂-Ci₂ straight or branched chain alkenyl, optionally mono- or multisubstituted C2-C12 straight or branched chain alkinyl, optionally mono- or multisubstituted C₃-C₈ cycloalkyl, optionally mono- or multisubstituted aryl, optionally mono- or multisubstituted heteroaryl, optionally mono- or multisubstituted aralkyl, halogen, -CF₃, or a combination thereof ; each R^4a may represent hydrogen, optionally mono- or multisubstituted Ci-Q₂ straight or branched chain alkyl, optionally mono- or multisubstituted C2-C12 straight or branched chain alkenyl, optionally mono- or multisubstituted straight or branched chain C2-C12 alkinyl, optionally mono- or multisubstituted C₃-Cs cycloalkyl, optionally mono- or multisubstituted aryl, optionally mono- or multisubstituted heteroaryl, optionally mono- or multisubstituted aralkyl, optionally mono- or multisubstituted C1-C12 straight or branched chain alkyloxy, optionally mono- or multisubstituted C2-C12 straight or branched chain alkenyloxy, optionally mono- or multisubstituted C2-C12 straight or branched chain alkinyloxy, optionally mono- or multisubstituted C₃-C₈ cycloalkyloxy; -CN, -NC, -(C=O)-R⁴, -(C=S)R⁴, -(C=O)NR⁵R⁶, -(S=O)R⁷, -SO₂R⁸, SO₃R⁹, -

(P=O)R¹⁰R¹¹, -PO₂HR¹⁰, -SiR¹²R¹³R¹⁴;

-R¹⁵-CN, -R¹⁵-NC, -R¹⁵-(C=O)-R⁴, -R¹⁵-(C=S)R⁴, -R¹⁵-(C=O)NR⁵R⁶, -R¹⁵- (S=O)R⁷, -R¹⁵-SO₂R⁸, -R¹⁵-SO₃R⁹, -R¹^(P=O)R¹⁰R¹ \ -R¹⁵-PO₂HR¹⁰, -R¹⁵- SiR¹²R¹³R¹⁴ wherein -R¹⁵ is a saturated or unsaturated alkylene group from 1 to about 20 carbon atoms; halogen, -CF₃, or a combination thereof; with the proviso that at least one of R² and R³ in formula I is not hydrogen; for the preparation of a pharmaceutical composition for preventing, treating or ameliorating a medical condition in a subject which can be prevented, treated or ameliorated by inhibiting glutathione-S -transferase (GST) in said subject.

17. The use according to claim 16, wherein the compound of formula I or formula II is a monovalent GST inhibitor.

18. The use according to claim 16, wherein the compound of formula I or formula II is a bi- or multivalent GST inhibitor in which at least one of R¹, R^la, R², R^2a, or R³ is a bond or a bi- or multivalent linker L covalently attaching the compound to at least one other compound selected from the group of: glutathione, a glutathione analog, a glutathione conjugate, ethacrynic acid, cibacron blue, uniblue A, doxorubicin, gossypol, hematin, rose bengal, sulfobromophthalein, indomethacin, piriprost, eosin b, eosin y, a synthetic or naturally occurring drug, a polysaccharide, a polynucleotide, a polypeptide, another compound of general formulae I or II, a known glutathione-S -transferase inhibitor, an antimalarial agent, a toxin or a chemotherapeutic agent, or a combination or mixture of any of the foregoing compounds.

19. The use according to claim 18, wherein the bi- or multivalent linker L comprises a polynucleotide, a peptide, a saccharide, a cyclodextrin, a dextran, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, a hydrocarbon, a polyacrylate, an alkyl chain interrupted by one or more atoms of O, S, or N, a carbonyl, an amide or an aromatic group; an amino-, hydroxy-, thio- or carboxy-functionalized silicone, or a combination thereof.

20. The use according to claim 18 or claim 19, wherein the linker L is derivatized to optimize the absorption, distribution, metabolism or excretion of the bi- or multivalent GST inhibitor.

21. The use according to any one of claims 18 to 20, wherein the bi- or multivalent linker L enhances the affinity of the bivalent inhibitor or increases isozyme selectivity of the bi- or multivalent GST inhibitor.

22. The use according to any one of claims 16 to 21, wherein the pharmaceutical composition further comprises at least one other pharmaceutically active compound.

23. The use according to claim 22, wherein the at least one additional pharmaceutically active compound is selected from the group consisting of: a known glutathione-S -transferase inhibitor, an antimalarial agent, a toxin, a chemotherapeutic agent, an antiproliferative agent, or a combination or mixture thereof.

24. The use according to claim 23, wherein the at least one additional pharmaceutically active compound is an antimalarial agent.

25. The use according to claim 24, wherein the antimalarial agent is an early stage- specific inhibitor of Plasmodium growth.

26. The use according to claim 24 or claim 25, wherein the antimalarial agent is selected from the group consisting of artemisinines, artesunates, chloroquine, sulfadoxine-pyrimethamine, primaquine, quinine, methylene blue, or derivatives or analogs thereof.

27. The use according to any one of claims 16 to 26, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or excipient.

28. The use according to any one of claims 16 to 27, wherein the pharmaceutical composition is suitable for oral, transdermal or parenteral administration.

29. The use according to any one of claims 16 to 28, wherein the pharmaceutical composition is for preventing, treating or ameliorating a medical condition selected from the group of malaria, drug-resistant malaria, multidrug resistance, toxoplasmosis, African trypanosomiasis (sleeping sickness), Chagas disease, cancer, drug-resistant cancer, immunosuppression, immunosuppression during chemotherapy or radiotherapy, myelodysplasia, and bone marrow transplantation.

30. The use according to any one of claims 16 to 29, wherein the GST isozyme is selected from the group of a human GST and a GST of a microorganism, preferably of a microorganism pathogenic for humans or animals.

31. The use according to claim 30, wherein the GST isozyme is a parasite GST, preferably a GST from the apicomplex parasite family.

32. The use according to claim 31, wherein the GST isozyme is Plasmodium falciparum GST (PfGST).

33. The use according to claim 30, wherein the GST isozyme is a human GST isozyme.

34. The use according to claim 30, wherein the GST isozyme is located in or expressed by a tumor, an infectious agent, or the bone marrow of the subject to be treated.

35. The use according to any one of claims 16 to 34, wherein the compound as defined in any one of claims 16 to 21 has a binding affinity for one GST class that is greater than the affinity for another GST class, preferably wherein the binding affinity for one GST class is at least 5 -fold greater, and more preferably at least 10-fold greater than the affinity for another GST class.

36. The use according to any one of claims 16 to 35, wherein the compound as defined in any one of claims 12 to 17 has a higher binding affinity for Plasmodium falciparum GST (PfGST) than for another GST class.

37. The use according to any one of claims 16 to 36, wherein the compound as defined in any one of claims 16 to 21 has an IC₅O for a GST isozyme of less than 100 μM, preferably less than 50 μM in a standard in vitro CDNB assay.

38. The use according to any one of claims 16 to 37, wherein the GST inhibitor is selected from:

1) [l]benzopyrano[5,4,3-cde][l]benzopyran-5,10-dione,l,2,3,7,8- pentahydroxy-(8Cl,9Cl)(flavellagic acid, FAE);

2) [l]benzopyrano[5,4,3-cde][l]benzopyran-5,10-dione,l, 2,3,6,7, 8- hexahydroxy-(9Cl) (coruleoellagic acid, CEA); or 3) Phenantro [1,10,9,8-opqra] perylene-7,14-dione, 1,3 ,4,6,8,13-hexa-hydroxy-

10,11 -dimethyl-, stereoisomer (6CI,7CI,8CI,9CI)) (hypericin, HYP).

39. The use according to any one of claims 16 to 38, wherein the compound as defined in any one of claims 16 to 21 binds to the glutathione binding site (G-site) of the GST isozyme.

40. The use according to any one of claims 16 to 38, wherein the compound as defined in any one of claims 16 to 21 binds to the hydrophobic substrate binding site (H-site) of the GST isozyme.

41. The use according to any one of claims 16 to 38, wherein the compound as defined in any one of claims 16 to 21 binds simultaneously to the G-site and the H-site of the GST isozyme.

42. The use according to any one of claims 16 to 38, wherein the compound is a bi- or multivalent GST inhibitor as defined in any one of claims 18 to 21 that binds simultaneously to at least two GST monomers in a GST dimer.

43. The use according to claim 42, wherein the bi- or multivalent GST inhibitor as defined in any one of claims 18 to 21 binds simultaneously to the G-sites of at least two GST monomers in a GST dimer.

44. The use according to claim 42, wherein the bi- or multivalent GST inhibitor as defined in any one of claims 18 to 21 binds simultaneously to the H-sites of at least two GST monomers in a GST dimer.

45. The use according to claim 42, wherein one ligand moiety of the bi- or multivalent GST inhibitor as defined in any one of claims 18 to 21 binds to the G- site of a GST monomer and the other ligand moiety binds to the H-site of the other GST monomer in a GST dimer.

46. The use according to any one of claims 39 to 45, wherein the compound as defined in any one claims 16 to 21 inhibits the enzyme catalysis function of the

GST isozyme.

47. The use according to any one of claims 39 to 45, wherein the compound as defined in any one claims 16 to 21 inhibits the binding of the GST isozyme to another protein.

48. A method for the prevention, treatment or amelioration of a medical condition selected from malaria, drug-resistant malaria, multidrug resistance, toxoplasmosis, African trypanosomiasis (sleeping sickness), Chagas disease, cancer, drug-resistant cancer, immunosuppression, immunosuppression during chemotherapy or radiotherapy, myelodysplasia, and bone marrow transplantation in a subject to be treated comprising administering a therapeutically effective amount of a compound as defined in any of claims 16 to 21, or a pharmaceutical composition as defined in claims 22 to 28.

49. The method of claim 48, wherein the compound as defined in any one of claims 16 to 21, or the pharmaceutical composition as defined in any one of claims 22 to 27 is administered by oral, parenteral, or transdermal means.

50. The method of claim 48 or claim 49, wherein the compound as defined in any one of claims 16 to 21 is administered in an amount of from about 0.01 mg/kg to about 100 mg/kg of body weight per day [mg/kg/day], and preferably from about 0.1 to about 30 mg/kg of body weight per day [mg/kg/day].