WO2012010606A1 - Hexacyclic polyketides as kinase inhibitors - Google Patents

Abstract

Compounds of general formula (I) wherein the R¹, R², R³ and R⁴ groups are each independently selected from the group consisting of hydrogen, acyl, alkyl, alkenyl, alkynyl, aryl, aralkyl, -CONH₂, alkali metal, and sugar and X₁, X₂ groups are each independently selected from the group consisting of hydrogen and halogen, or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof, a method of producing them, and their use for the treatment of cancer, Alzheimer's disease, Parkinson's disease, frontoparietal dementia, corticobasal degeneration, Pick's disease, cerebrovascular accidents, peripheral neuropathies, brain, spinal cord trauma, atherosclerosis, balloon injury induced restenosis, pulmonary fibrosis, and liver fibrosis, graft versus host disease (GvHD), transplant rejection, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, asthma, psoriasis, human multiple myeloma, T cell acute lymphoblastic leukemia and Hodgkin's disease, as protein kinase inhibitors.

Description

HEXACYCLIC POLYKE TIDES AS KINASE INHIBITORS

TECHNICAL FIELD

The present invention relates to hexacyclic polyketides, pharmaceutical compositions containing them and their use as protein kinase inhibitors, including AKTl, Aurora-B, GSK3 , PDGFRJ3 kinases and NIK.

BACKGROUND OF THE INVENTION

The protein kinases include a large number of family members, which play a central role in regulating a wide variety of cellular function. A partial, non-limiting, list of these kinases includes: AKTl, Aurora-B, GSK3 , NIK (NF-kB- Inducing Kinase) and platelet derived growth factor receptor (PDGFR ).

The AKT family of proteins represents a subfamily of the AGC (protein A, protein G, protein C) family of kinases whose individual members are serine/theonine kinases. In mammalian cells, the AKT subfamily comprises at least three major isoforms that are referred as AKTl, AKT2, and AKT3. AKT orthologs have been identified in a variety of species, including human (Staal, Proc. Natl. Acad. Sci. USA 1987, 84, 5034- 5037; Nakatani et al, J. Biol. Chem. 1999, 274, 21528-21532). AKT family proteins contain an N-terminal pleckstrin homology domain, which mediates lipid-protein and protein-protein interactions; a short a-helical linker region; a central serine/threonine kinase domain; and a C-terminal hydrophobic and proline-rich domain (Datta et al, Genes Dev. 1999, 13, 2905-2927). The AKT kinases are associated with a variety of physiological responses, including the inhibition of apoptosis and promotion of cell survival (Kandel and Hay Exp. Cell. Res. 1999, 253, 210-229). Extensive evidence has also demonstrated a crucial role for AKT in tumorigenesis (Testa and Bellacosa, Proc. Natl. Acad. Sci. USA, 2001, 98, 10983-10985; Datta et al. Genes Dev 1999, 13, 2905- 2927). Furthermore, activation of AKT has been shown to associate with tumour invasiveness and chemoresistance (West et al, Drug Resit. Update 2002, 5, 234-248).

The members of the Aurora kinase family are serine/threonine kinases which are involved in mitosis (Kenn et al, Nat. Rev. Cancer 2004, 4, 927). Three Aurora kinases, A, B, and C, are known. Aurora A kinase localizes to the duplicated centrosomes and to the spindle poles during mitosis and assists with centrosome maturation and separation. Aurora B kinase is a chromosomal passenger protein which is located to the centromeric regions of the chromosomes in the early stages of mitosis and accumulates in the spindle midzone and midbody. The role of Aurora C kinase is unknown currently. Both Aurora A and Aurora B kinases are overexpresed in many tumors including breast, colon, and pancreatic cancers.

Glycogen synthase kinase 3β (GSK3 ) is a proline directed serine, threonine kinase that plays an important role in the control of metabolism, differentiation and survival. It was initially identified as an enzyme able to phosphorylate and hence inhibit glycogen synthase. It was later recognized that GSK3 was identical to tau protein kinase 1 (TPK1), an enzyme that phosphorylates tau protein in epitopes that are also found to be hyperphosphorylated in Alzheimer's disease and in several taupathies. GSK3 may find application in the treatment of the neuropathological consequences and the cognitive and attention deficits associated with Alzheimer's disease, as well as other acute and chronic neurodegenerative diseases. These include, in a nonlimiting manner, Parkinson's disease, frontoparietal dementia, corticobasal degeneration, Pick's disease, cerebrovascular accidents, peripheral neuropathies, brain and spinal cord trauma.

F-kB Inducing Kinase (NIK) is a Ser/Thr kinase, member of mitogen-activated protein kinase (MAPK) family. It is implicated in regulation of NF-kB family of transcription factors. NF-κΒ is found constitutively activated in several inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, asthma or psoriasis (Li Q et al, Nat. Rev. Immunol. 2002, 2(10), 725-34; Makarov SS, Mol. Med. Today, 2000, 6(11), 441-8; Tak PP et al, J. Clin. Invest. 2001, 107(1), 7-11; Yamamoto Y et al, J. Clin. Invest. 2001, 107(2), 135-42.; Liou HC et al., Bioessays. 2003, 25(8), 767-80). NF-κΒ proteins are sequestered by inhibitors of NF-κΒ (IKB) molecules. IKB kinase (IKK) phosphorylates IKB S, which are then ubiquitinated and degraded by the proteasome. Degradation of IKBS results in the translocation of NF-kB active heterodimers to the nucleus and activation of target genes. In addition of this conventional pathway, a non canonical one, involving signaling through NIK, results in the processing of plOO/RelB to mature p52/RelB (Dejardin E. et al, Biochem. Pharmacol. 2006, 72(9), 1161-79). These two activation pathways modulate NF-KB activity depending upon the cell type and the nature of the stimuli. Besides, it has been recently suggested that NIK may link both pathways of NF-κΒ activation. A second level of F-κΒ regulation relies in the activation of the transcriptional activity of p65 and c-Rel where NIK plays an important role during T lymphocyte activation, regulating the production of interleukin-2 (IL-2) through the activation of the CD28 responsive element (CD28RE) of the IL-2 promoter (Sanchez- Valdepenas C et al., J Immunol. 2006, 176(8), 4666-74). This effect takes place through the phosphorylation of the c-Rel C-terminal transactivation domain (TAD). Uncontrolled NIK expression has been found in primary samples of human multiple myeloma (Keats JJ et al., Cancer Cell. 2007, 12(2), 131-44; Annunziata CM et al, Cancer Cell. 2007, 12(2), 115-30) and T cell acute lymphoblastic leukemia and Hodgkin^'s disease (Saitoh Y et al., Blood. 2008, 1 1 1(10), 5118-29). Murine models have suggested an important role of NIK in immune diseases such as rheumatoid arthritis (Aya K et al, J. Clin. Invest. 2005, 115(7), 1848-54), murine model of experimental autoimmune encephalomyelitis (Jin W et all Blood. 2009, 113(26), 6603-10) and Graf versus Host Disease (Sanchez- Valdepenas C et al., Haematologica 2010, 95(12), 211 1-8). NIK inhibition could be an alternative or complementary treatment for several immune and/or inflammatory diseases where T cell activation plays an important role, such as Graft versus Host Disease (GvHD), transplant rejection, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, asthma, psoriasis, human multiple myeloma, T cell acute lymphoblastic leukemia and Hodgkin^'s disease.

Platelet-derived growth factor receptors (PDGFRs) and their ligands, platelet- derived growth factor (PDGFs), play critical roles in mesenchymal cell migration and proliferation. The PDGFR/PDGF system includes two receptors (PDGFRa and PDGFRp) and four ligands (Hart et al, Science 1988, 240, 1529-1531). The receptors are plasma membrane-spanning proteins with intracellular tyrosine kinase domains. As with other protein kinases, activation of the PDGFRs is a key mechanism in regulating signals for cell proliferation, and abnormalities of PDGFR/PDGF are thought to contribute to a number of human diseases, such as atherosclerosis, balloon injury induced restenosis, pulmonary fibrosis, liver fibrosis, and especially cancer.

The design and development of small molecules that specifically inhibit the kinase activity and the kinase signal transductions pathway of AKTl, Aurora-B, GSK3P, NF-kB Inducing kinase or PDGFR kinases is therefore an attractive approach for the development of new therapeutic agents. The hexacyclic polyketide BE-24566B, its enantiomer and a dimethyl ether derivative have been disclosed (EP542234; Tetrahedron Letters 1995, 36, 2013-2016; J. Antibiotics 1995, 48, 1506-1508; J. Natural Products 2005, 68, 1437-1440) as antibacterial compounds, inhibitors of endothelin A and inhibitors of the ligand binding activity of Liver X receptors.

BE-24566B dimethyl ether

The hexacyclic polyketide Bischloroanthrabenzoxocinone and its enantiomer have been disclosed (J. Natural Products 2005, 68, 1437-1440; J. Biological Chemistry 2005, 280, 1669-1677) as antibacterial compounds and inhibitors of the ligand binding activity of Liver X rec

SUMMARY OF THE INVENTION

The present invention is directed to the use of compounds of general formula

(I):

(I)

wherein the R¹, R², R³ and R⁴ groups are each independently selected from the group consisting of hydrogen, acyl, alkyl, alkenyl, alkynyl, aryl, aralkyl, -CO H₂, alkali metal, and sugar,

the X¹, X² groups are each independently selected from the group consisting of hydrogen and halogen,

or pharmaceutically acceptable salts, solvates, tautomers, or stereoisomers thereof, in the preparation of a medicament for the treatment of human diseases mediated by abnormal AKT 1 , Aurora-B, GSK3 β, NIK or PDGFR activities.

In another aspect, the present invention is also directed to compounds of general formula (I), pharmaceutically acceptable salts, solvates, tautomers, stereoisomers, or mixtures thereof for use as a medicament for the treatment of human diseases mediated by abnormal AKT1, Aurora-B, GSK3 , NIK or PDGFR activities.

In another aspect, the present invention is also directed to compounds of general formula (I) or pharmaceutically acceptable salts, solvates, tautomers, or stereoisomers thereof:

(I)

wherein the R¹, R², R³ and R⁴ groups are each independently selected from the group consisting of hydrogen, acyl, alkyl, alkenyl, alkynyl, aryl, aralkyl, -CONH₂, alkali metal, and sugar,

the X¹, X² groups are each independently selected from the group consisting of hydrogen and halogen; with the exception of the compounds in which:

R¹, R², R³, R⁴, X¹, and X² are all hydrogen;

1 4 2 3 1 2

R and R are both methyl group, R , R , X , and X are all hydrogen;

1 2 3 4 1 2

R is a methyl group, R , R , and R are all hydrogen, and X , X are both CI;

1 2 3 4 2 1

R is a methyl group, R , R , R , and X are all hydrogen, and X is CI.

In another aspect, the present invention is also directed to compounds of general formula (I), with the exception of the four compounds disclosed before (the

1 2 3 4 1 2

compound in which R , R , R , R , X , and X are all hydrogen; the compound in which

1 4 2 3 1 2

R and R are both methyl group, R , R , X , and X are all hydrogen; the compound in

1 2 3 4 1 2

which R is a methyl group, R , R , and R are all hydrogen, and X , X are both CI; the

1 2 3 4 2 1 compound in which R is a methyl group, R , R , R , and X are all hydrogen, and X is CI), pharmaceutically acceptable salts, solvates, tautomers, stereoisomers, or mixtures thereof for use as a medicament.

In another aspect, the present invention i s al so directed to the use of compounds of general formula (I), with the exception of the four compounds disclosed

1 2 3 4 1 2

before (the compound in which R , R , R , R , X , and X are all hydrogen; the

1 4 2 3 1 2 compound in which R and R are both methyl group, R , R , X , and X are all hydrogen; the compound in which R¹ is a methyl group, R², R³, and R⁴ are all hydrogen,

1 2 1 2 3 4 2 and X¹, X are both CI; the compound in which R is a methyl group, R , R , R , and X are all hydrogen, and X¹ is CI), pharmaceutically acceptable salts, solvates, tautomers, stereoisomers or mixtures thereof in the preparation of a medicament.

Some of the human diseases that can be treated with the compounds of the present invention are, in a non-limiting manner: cancer, Alzheimer' s disease, Parkinson' s disease, frontoparietal dementia, corticobasal degeneration, Pick' s disease, cerebrovascular accidents, peripheral neuropathies, brain, spinal cord trauma, atherosclerosis, balloon injury induced restenosis, pulmonary fibrosis, liver fibrosis, graft versus host disease (GvHD), transplant rejection, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, asthma, psoriasis, human multiple myeloma, T cell acute lymphoblastic leukemia and Hodgkin^'s disease.

Other aspects of the invention are methods of treatment, and compounds for use in these methods. Therefore, the present invention further provides a method of treating some diseases which comprises administering to a patient in need of such treatment a therapeutically effective amount of a compound of general formula (I), or a pharmaceutically acceptable salt, solvates, tautomers, or stereoisomer thereof. Preferably, the patient in need of such treatment may be any mammal, notably a human.

The present invention also relates to the obtention of the compounds of general formula (I) from a strain of a microorganism capable of producing them.

The preferred process comprises the steps of cultivating a strain of a microorganism capable of producing compounds of general formula (I) in an aqueous nutrient medium with assimilable carbon and nitrogen sources and salts, under controlled submerged aerobic conditions, and then recovering and purifying the natural compounds according to the invention from the cultured broth or by treating the cultured broth using standard organic synthetic reactions known by the skilled person, to obtain the corresponding derivatives and then recovering and purifying these derivatives from the crude of the reaction.

The present invention also relates to the obtention of compounds of general formula (I) by synthesis of derivatives from the natural compounds isolated from a strain of a microorganism capable of producing them.

The compounds of general formula (I) may be synthesised from commercially available starting materials using conventional procedures. For example using standard organic synthetic reactions known by the skilled person.

In another aspect, the present invention is directed to pharmaceutical compositions containing a compound of general formula (I), pharmaceutically acceptable salts, or stereoisomers thereof together with a pharmaceutically acceptable carrier or diluent.

These and further aspects of the invention are defined in the claims.

BRIEF DESCRIPTION OF DRAWINGS Figure 1 represents an UV-Vis spectrum of Compound 1, where the absorbance (measured in mAU) is plotted against wavelength (measured in nm). Figure 2 represents an UV-Vis spectrum of Compound 2, where the absorbance (measured in mAU) is plotted against wavelength (measured in nm).

Figure 3 represents an UV-Vis spectrum of Compound 3, where the absorbance (measured in mAU) is plotted against wavelength (measured in nm).

Figure 4 represents luciferase activity in Jurkat cells transiently transfected with a luciferase reporter driven by CD28RE, along with equivalent amounts of NIK and c-Rel expression constructs and then treated with 0 (C), 0.5, 1 and 2.5 μg/ml of Compound 1. Figure 5 represents luciferase activity in Jurkat cells transiently transfected with a luciferase reporter driven by CD28RE, along with equivalent amounts of NIK and c-Rel expression constructs and then treated with 0 (C), 0.5, 1 and 2.5 μg/ml of Compound 2. Figure 6 represents luciferase activity in Jurkat cells transiently transfected with a luciferase reporter driven by CD28RE, along with equivalent amounts of NIK and c-Rel expression constructs and then treated with 0 (C), 0.5, 1 and 2.5 μg/ml of Bischloroanthrabenzoxocinone.

Figure 7 represents luciferase activity in Jurkat cells transiently transfected with a luciferase reporter driven by CD28RE, along with equivalent amounts of NIK and c-Rel expression constructs and then treated with 0 (C), 0.5, 1 and 2.5 μg/ml of BE-24566B.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the use of compounds of general formula (I) or pharmaceutically acceptable salts, solvates, tautomers, or stereoisomers thereof, in the preparation of a medicament for the treatment of human diseases mediated by abnormal AKTl, Auror -B, GSK3 , or PDGFR activities:

(I) wherein the R¹, R², R³ and R⁴ groups are each independently selected from the group consisting of hydrogen, acyl, alkyl, alkenyl, alkynyl, aryl, aralkyl, -CO H₂, alkali metal, and sugar,

the X¹, X² groups are each independently selected from the group consisting of hydrogen and halogen,

In another aspect, the present invention is directed to compounds of general formula (I) or pharmaceutically acceptable salts, solvates, tautomers,or stereoisomers thereof:

(I) wherein the R¹, R², R³ and R⁴ groups are each independently selected from the group consisting of hydrogen, acyl, alkyl, alkenyl, alkynyl, aryl, aralkyl, -CO H₂, alkali metal, and sugar and the X¹, X² groups are each independently selected from the group consisting of hydrogen and halogen such as F, CI, Br and I;

with the exception of the compounds in which:

R¹, R², R³, R⁴, X¹, and X² are all hydrogen;

1 4 2 3 1 2

R and R are both methyl group, R , R , X , and X are all hydrogen;

1 2 3 4 1 2

R is a methyl group, R , R , and R are all hydrogen, and X , X are both CI;

1 2 3 4 2 1

R is a methyl group, R , R , R , and X are all hydrogen, and X is CI.

In the present description, the following terms have the meaning indicated: An acyl group is of the form R⁵CO-, wherein R⁵ is an organic group such as an alkyl or an aryl group as defined below e.g. acetyl, propionyl, benzoyl and the like. Suitable acyl groups have from 2 to about 12 carbon atoms, preferably from 2 to about 8 carbon atoms, more preferably from 2 to about 6 carbon atoms, most preferably 2 carbon atoms. Alkyl groups preferably have from 1 to about 20 carbon atoms, more preferably from 1 to about 12 carbon atoms, even more preferably from 1 to about 6. Alkyl groups having 1, 2, 3, 4 or 5 carbon atoms are particularly preferred. Methyl, ethyl, n-propyl, iso-propyl and butyl, including n-butyl, tert-butyl, sec-butyl and iso-butyl are particularly preferred alkyl groups in the compounds of the present invention.

As used herein, the term alkyl, unless otherwise modified, refers to both cyclic and non-cyclic groups, although cyclic groups will comprise at least three carbon ring members. Non-cyclic alkyl refers to a straight-chain or branched alkyl group.

Preferred alkenyl and alkynyl groups in the compounds of the present invention have one or more unsaturated linkages and from 2 to about 20 carbon atoms, more preferably from 2 to about 12 carbon atoms, even more preferably from 2 to about 6. The terms alkenyl and alkynyl as used herein refer to both cyclic and non cyclic groups. Non-cyclic alkenyl or alkynyl refers to a straight-chain or branched alkenyl or alkynyl groups.

The above mentioned groups may be substituted at one or more available positions by one or more suitable groups such as OR⁵, =0, SR⁵, SOR⁵, S0₂R⁵, N0₂, NHR⁵, N(R⁵)₂, =N-R⁵, NHCOR⁵, N(COR⁵)₂, NHS0₂R⁵, CN, halogen, C(=0)R⁵, C0₂R⁵, OC(=0)R⁵ wherein each of the R⁵ groups is independently selected from the group consisting of H, OH, N0₂, NH₂, SH, CN, halogen, =0, C(=0)H, C(=0)CH₃, C0₂H, substituted or unsubstituted Ci-Ci₂ alkyl, substituted or unsubstituted C₂-Ci₂ alkenyl, substituted or unsubstituted C₂-Ci₂ alkynyl and substituted or unsubstituted aryl.

"Aryl" refers to single and multiple aromatic ring radicals, including multiple ring radicals containing separate and/or fused aryl groups. Typical aryl groups contain from 1 to 3 separated or fused rings and from 6 to about 18 carbon ring atoms, such as phenyl, naphthyl, indenyl, phenanthryl or anthracyl radicals.

The aryl groups in the compounds of the present invention may be substituted at one or more available positions by one or more suitable groups, e. g., halogen such as F, CI, Br and I; cyano; hydroxyl; nitro; azido; acyl groups including those groups having 1 to about 12 carbon atoms or from 1 to about 6 carbon atoms; carboxamido; alkyl groups including those groups having 1 to about 12 carbon atoms, preferably from 1 to about 6 carbon atoms and more preferably 1 to 3 carbon atoms; alkenyl and alkynyl groups including groups having one or more unsaturated linkages and from 2 to about 12 carbon atoms, preferably from 2 to about 6 carbon atoms; alkoxy groups having one or more oxygen linkages and from 1 to about 12 carbon atoms, preferably 1 to about 6 carbon atoms; aryloxy such as phenoxy; alkylthio groups including those moieties having one or more thioether linkages and from 1 to about 12 carbon atoms, preferably from 1 to about 6 carbon atoms; alkylsulphinyl groups including those moieties having one or more sulphinyl linkages and from 1 to about 12 carbon atoms, preferably from 1 to about 6 carbon atoms; alkylsulphonyl groups including those moieties having one or more sulphonyl linkages and from 1 to about 12 carbon atoms, preferably from 1 to about 6 carbon atoms; aminoalkyl groups such as groups having one or more N atoms and from 1 to about 12 carbon atoms, preferably from 1 to about 6 carbon atoms; aralkyl such as benzyl. Unless otherwise indicated, an optionally substituted group may have a substituent at each substitutable position of the group, and each substitution is independent of the other.

Notable alkali-metals include sodium or potassium.

"Aralkyl" refers to an aryl group linked to an alkyl radical as defined above. A preferred aralkyl group is benzyl.

"Halogen" refers to F, CI, Br, I.

"Sugar" refers to mono-, di- or tri- saccharides or saccharide derivatives, preferably mono- or di- saccharides. Pentose or hexose compounds are preferred. Derivatives include sugar glycosides, N-glycosylamines, O-acyl derivatives, O-methyl derivatives, sugar alcohols, sugar acids, and deoxy sugars.

The term "pharmaceutically acceptable salts", refers to any salt which, upon administration to the patient is capable of providing (directly or indirectly) a compound as described herein. It will be appreciated that non-pharmaceutically acceptable salts also fall within the scope of the invention since those may be useful in the preparation of pharmaceutically acceptable salts. The preparation of salts and derivatives can be carried out by methods known in the art.

For instance, pharmaceutically acceptable salts of compounds provided herein are synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts are, for example, prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of the same. Generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. Examples of the acid addition salts include mineral acid addition salts such as, for example, hydrochloride, hydrobromide, hydroiodide, sulphate, nitrate, phosphate, and organic acid addition salts such as, for example, acetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulphonate and p- toluenesulphonate. Examples of the alkali addition salts include inorganic salts such as, for example, sodium, potassium, calcium and ammonium salts, and organic alkali salts such as, for example, ethylenediamine, ethanolamine, N,N-dialkylenethanolamine, triethanolamine and basic aminoacids salts.

The compounds of the invention may be in crystalline form either as free compounds or as solvates (e.g. hydrates, alcoholates, particularly methanolates) and it is intended that both forms are within the scope of the present invention. Methods of solvation are generally known within the art. The compounds of the invention may present different polymorphic forms, and it is intended that the invention encompasses all such forms.

Any compound that is a derivative of a compound of general formula (I) is within the scope and spirit of the invention. The term "derivative" is used in its broadest sense and encompasses those compounds that are converted in vivo to the compounds of the invention. Examples of derivatives include, but are not limited to, derivatives and metabolites of the compounds of general formula (I) that include biohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogues. Preferably, derivatives of compounds with carboxyl functional groups are the lower alkyl esters of the carboxylic acid. The carboxylate esters are conveniently formed by esterif ing any of the carboxylic acid moieties present on the molecule. Derivatives can typically be prepared using well-known methods, such as those described by Burger "Medicinal Chemistry and Drug Discovery 6th ed. (Donald J. Abraham ed., 2001, Wiley) and "Design and Applications of Prodrugs" (H. Bundgaard ed., 1985, Harwood Academic Publishers).

Any compound referred to herein is intended to represent such specific compound as well as certain variations or forms. In particular, compounds referred to herein may have asymmetric centres and therefore exist in different enantiomeric or diastereomeric forms. Thus any given compound referred to herein is intended to represent any one of a racemate, one or more enantiomeric forms, one or more diastereomeric forms, and mixtures thereof. Likewise, stereoisomerism or geometric isomerism about the double bond is also possible, therefore in some cases the molecule could exist as (E)-isomer or (Z)-isomer (trans and cis isomers). If the molecule contains several double bonds, each double bond will have its own stereoisomerism, that could be the same or different than the stereoisomerism of the other double bonds of the molecule. All the stereoisomers including enantiomers, diastereoisomers and geometric isomers of the compounds referred to herein, and mixtures thereof, are considered within the scope of the present invention.

Furthermore, any compound referred to herein may exist as tautomers. Specifically, the term tautomer refers to one of two or more structural isomers of a compound that exist in equilibrium and are readily converted from one isomeric form to another. Common tautomeric pairs are amine-imine, amide-imidic acid, keto-enol, lactam-lactim, etc.

Additionally, any compound referred to herein is intended to represent hydrates, solvates, and polymorphs, and mixtures thereof when such forms exist in the medium. All geometric isomers, tautomers, atropisomers, hydrates, solvates, polymorphs, and isotopically labelled forms of the compounds referred to herein, and mixtures thereof, are considered within the scope of the present invention.

Unless otherwise stated, the compounds of the invention are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbon or ¹⁵N -enriched nitrogen are within the scope of this invention.

An important feature of the above described compounds of formula (I) is their bioactivity and in particular their activity as protein kinase inhibitors, including AKT1, Aurora-B, GSK3 , NIK and PDGFRJ3 kinases.

Therefore, the present invention provides compounds and pharmaceutical compositions that are useful for the treatment of diseases or conditions mediated by those kinases. Among such diseases or conditions are included cancer, Alzheimer's disease, Parkinson's disease, frontoparietal dementia, corticobasal degeneration, Pick' s disease, cerebrovascular accidents, peripheral neuropathies, brain, spinal cord trauma, atherosclerosis, balloon injury induced restenosis, pulmonary fibrosis, liver fibrosis, graft versus host disease (GvHD), transplant rejection, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, asthma, psoriasis, human multiple myeloma, T cell acute lymphoblastic leukemia or Hodgkin^'s disease.

In a particular embodiment, cancer is selected from lung cancer, colon cancer, pancreatic cancer and glioblastoma. Pharmaceutical compositions useful for the method of the invention comprise a compound of formula (I), or mixtures thereof, a pharmaceutically acceptable salt, solvate, tautomer, or stereoisomer thereof together with a pharmaceutically acceptable carrier for administration to a patient.

The term "carrier" refers to a diluent, adjuvant, excipient or vehicle with which the active ingredient is administered. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin, 1995.

Examples of pharmaceutical compositions include any solid (tablets, pills, capsules, granules etc.) or liquid (solutions, suspensions or emulsions) composition for oral, topical or parenteral administration.

In a preferred embodiment the pharmaceutical compositions are in oral form, either solid or liquid. Suitable dose forms for oral administration may be tablets, capsules, syrups or solutions and may contain conventional excipients known in the art such as binding agents, for example syrup, acacia, gelatine, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, maize starch, calcium phosphate, sorbitol or glycine; tabletting lubricants, for example magnesium stearate; disintegrants, for example starch, polyvinylpyrrolidone, sodium starch glycolate or microcrystalline cellulose; or pharmaceutically acceptable wetting agents such as sodium lauryl sulfate.

The solid oral compositions may be prepared by conventional methods of blending, filling or tableting. Repeated blending operations may be used to distribute the active agent throughout those compositions employing large quantities of fillers. Such operations are conventional in the art. The tablets may for example be prepared by wet or dry granulation and optionally coated according to methods well known in normal pharmaceutical practice, in particular with an enteric coating.

The pharmaceutical compositions may also be adapted for parenteral administration, such as sterile solutions, suspensions or lyophilized products in the appropriate unit dosage form. Adequate excipients can be used, such as bulking agents, buffering agents or surfactants.

The mentioned formulations will be prepared using standard methods such as those described or referred to in the Spanish and US Pharmacopoeias and similar reference texts.

Administration of the compounds of formula (I) or compositions thereof may be by any suitable method, such as intravenous infusion, oral preparations, and intraperitoneal and intravenous administration. Oral administration is preferred because of the convenience for the patient and the chronic character of the diseases to be treated.

Generally an effective administered amount of a compound of formula (I) will depend on the relative efficacy of the compound chosen, the severity of the disorder being treated and the weight of the sufferer. However, active compounds will typically be administered once or more times a day for example 1, 2, 3 or 4 times daily, with typical total daily doses in the range of from 0.1 to 1000 mg/kg/day.

The compounds of general formula (I) and compositions thereof may be used with other drug(s) to provide a combination therapy. The other drug(s) may form part of the same composition, or be provided as a separate composition for administration at the same time or at different times. In one embodiment, the compounds of the invention are preferably those in which the R¹, R², R³ and R⁴ groups are each independently selected from alkyl or hydrogen.

In another embodiment, the compounds of the invention are preferably those in which the X¹ and X² groups are each independently selected from chlorine or hydrogen. Preferably, at leat one of the X¹ and X² groups is chlorine. Particularly preferred compounds of the invention falling under the general formula (I) are Compounds 1, 2 and 3 :

Compound 1 Compound 2

The organism used for the production of compounds of formula (I) is preferably an actinomycete, strain AA-98-E-N010GAS, that belongs to the Streptomyces genus. A culture of this strain has been deposited under the Budapest Treaty in the Coleccion Espanola de Cultivos Tipo (CECT) at the Valencia University, Spain, under the accession number CECT 7482. This deposit has been made under the provisions of the Budapest Treaty and all restrictions on the availability thereof to the public will be irrevocably revoked upon granting of a patent on this application. The organism was isolated from an unidentified alga collected at the Spanish's Bay of Biscay Coast (Cantabrian Sea). The taxonomic methods described herein are those reported in Table 1. All cultures, except where indicated, were incubated at 28°C and records of results were made weekly up to 21 days. TABLE 1

1. Colonial morphology:

ISP Media No 2, 4, 5 and 6 (Shirling B.E.; D. Gotlieb. Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol. 16:313-340, 1966).

ATCC Medium No 172: ATCC Catalogue (American Type Culture Catalog 17th edition, 1989. Rockville, Maryland. U.S.A.)

Czapek Agar Difco

Bennet Agar (Waksman, S.A Classification, identification and descriptions of the genera and species. In The Actinomycetes. The Williams and Wilkins Co.. Baltimore, 1961. vol.11, pp. 1-363).

All media were supplemented with 50% artificial sea water (ASW).

2. Physiological characteristics:

ISP medium n° 1 (Shirling B.E.; D. Gotlieb. Methods for characterization of

Streptomyces species. Int. J. Syst. Bacteriol. 16:313-340, 1966).

NaCl resistance: ATCC 172 with 0, 2, 4, 5, 7 and 10% NaCl.

Carbon utilization: ISP-9 (Shirling B.E.; D. Gotlieb. Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol. 16:313-340, 1966).

Nitrogen utilization (Shirling B.E.; D. Gotlieb. Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol. 16:313-340, 1966)

3. Fatty acids analysis,

(Van der Auwera,P.; Labbe, M.; Mayberry, W.R.; Ferguson, K.P.; Lambe, D.W.J. Identification of Bacteroides by cellular fatty acid profiles: application to the routine microbiological laboratory J. Microbiol. Methods 4:267-275 1986).

4. Whole cell sugar analysis:

(Hasegawa T., M. Takizawa, and S. Tanida, J.Gen.Appl. Microbiol. 29:319, 1983)

(Lechevalier, M.P., and Lechevalier, H A. Int. J. Syst. Bacteriol. 20:435, 1970) 5. Diaminopinelic acids analysis:

(Hasegawa T., M. Takizawa, and S. Tanida, J.Gen.Appl. Microbiol. 29:319, 1983) 6. rDNA Analysis:

(Felsenstein, J. Cladistics 5: 164, 1989)

(Lane, D.J. Nucleic acid techniques in bacterial systematics: 115, 1991)

A description of the organism is as follows:

Morphology:

After 21 days at 28°C growth was observed in ISP2 and 172 broth supplemented with artificial sea water (ASW). Aerial mycelium with a pale shade of gray was formed on ISP2 medium. No aerial mycelium was formed on 172. Substrate mycelium was branched. Physiology:

Diffusible pigments were formed by strain AA-98-E-N010GAS when grown on ISP2, ISP5, ISP6, ISP7 and Bennet agar. The production of pigments was absent in ISP3, ISP4, Czapek and chitin agars. The optimum of NaCl concentration in the medium for optimal growth was around 3%. The strain AA-98-E-N010GAS can utilize arabinose, glucose, galactose, mannitol, rhamnose, mannose, and fructose. It grew poorly on melibiose, raffinose and xylose as carbon sources. The organism did not grow on myoinositol and sucrose. The nitrogen sources utilized were valine, cysteine, histidine, asparagine, threonine, and hydroxyproline. It grew poorly on phenylalanine and DL-2- amyobutyric acid.

Chemical composition:

Aminoacids:

LL-diaminopimelic acid was the isomer present in the whole cell hydrolysate of strain AA-98-E-N010GAS.

The mayor fatty acids were anteiso-C15:0, iso-C15:0, iso-C16:0, CI 6:0, iso-C14, iso- C17:0, anteiso-C17:0, C14:0, and C16: l . Comparison with other strains is described in Table 2

TABLE 2

Fatty acids composition of strain AA-98-E-N010GAS and other actinomycete strains. Composition is given as percentage of total fatty acids content.

i-14:0 14:0 i-15:0 a-15 :0 15:0 i-16:l i-16:0 16 :1 16:0 i-17 :l i-17:0 a-17:0 17: 1 17 :0 i-18:l i-18:0 cis-18 :l 18 :0

EN010GAS 7.72 3.76 11.24 24.55 1.05 <1 15.71 3.24 14,65 < 1 4.09 5.59 < 1 < 1 < 1 < 1 < 1 < 1

STALBUS 6.52 < 1 9.88 22.92 < 1 5.50 25.29 < 1 3.75 1.28 3.38 8.60 < 1 < 1 < 1 1.09 < 1 < 1

AMCITRE < 1 3.18 < 1 < 1 1.03 < 1 6.37 12.62 40 < 1 < 1 < 1 < 1 1.16 < 1 < 1 14.25 2.82

AMYORIE 3.40 2.37 19.94 4.66 1.17 < 1 11.85 5.59 18.41 < 1 2.99 4.44 3.09 2.73 < 1 < 1 6.21 3.04

APBRASIL 3.15 < 1 15.46 18.91 2.76 < 1 19.07 2.15 1.79 < 1 2.39 9.64 11.18 2.82 < 1 < 1 3.38 1.06

APDIGIT 11.57 < 1 11.21 9.96 < 1 2.87 34.23 < 1 1.08 < 1 1.28 5.08 4.39 1.64 < 1 1.76 7.60 1.54

KZVIRIDO 4.04 1.10 18.94 2.71 4.89 < 1 26.44 < 1 4.43 < 1 2.60 1.58 11.36 8.58 7.48 < 1 < 1 1.16

LCAERO 3.06 1.35 14.41 8.62 1.04 5.68 20.07 13.84 6.16 4.55 2.20 5.31 2.02 < 1 < 1 < 1 < 1 1.43

MNCHALC 1.68 < 1 8.91 2.29 1.53 1.15 38.23 < 1 1.88 1.49 2.32 2.25 5.43 6.95 14.58 1.31 1.28 2.68

MNECHCA 1.17 < 1 6.97 1.24 2.81 < 1 30.88 < 1 2.29 1.63 4.11 1.68 12.15 4.90 7.23 < 1 10.05 1.69

MNPURPU < 1 < 1 26.56 6.53 < 1 < 1 8.58 < 1 < 1 7.30 11.89 13.25 2.90 3.37 3.59 < 1 2.33 1.94

NMAFRI 5.43 3.35 4.62 < 1 7.46 3.09 22.18 2.69 5.15 2.35 < 1 < 1 8.15 4.75 17.03 < 1 < 1 1.23

NMFERRU 1.91 1.19 1.94 < 1 6.43 4.12 21.50 2.32 2.34 < 1 < 1 < 1 23.51 5.71 12.15 1.27 1.43 < 1

NMROSEA 3.65 5.14 3.86 < 1 9.03 3.02 12.31 3.46 6.95 1.17 < 1 < 1 13.51 4.46 18.67 < 1 1.77 < 1

NMROSEO 2.19 1.24 6.73 1.09 6.94 1.43 22.21 2.21 3.61 2.74 1.03 < 1 10.97 4.33 17.84 < 1 < 1 < 1

NMRUBRA 1.40 1.38 4.12 < 1 3.41 7.27 25.00 2.63 3.89 2.17 1.08 < 1 6.84 4.97 15.44 1.25 < 1 1.61

NMSALMO 1.12 1.28 6.75 < 1 7.83 7.53 21.58 1.21 1.97 1.01 < 1 1.07 11.58 5.53 17.34 < 1 < 1 < 1

SPAMETH 10.34 < 1 1.86 < 1 4.30 < 1 15.51 5.63 8.62 1.08 < 1 < 1 24.02 9.43 7.11 < 1 4.60 1.04

EN010GAS = strain AA-98-E-N010GAS; AMCITRE = Actinomadura citrea DSM 43461; APDIGIT = Actinoplanes digitalis ATCC 15349; NMROSEO = Nonomuraea roseoviolacea DSM 43144; AMYORIE = Amycolatopsis orientalis DSM 40040; APBRASIL = Actinoplanes brasiliensis ATCC 25844; MNCHALC = Micromonospora chalcea ATCC 31395; MNECHCA = Micromonospora echinospora calichinensis NRRL 15839; MNPURPU = Micromonospora purpureochromogena NRRL B-3298; NMFERRU = Nonomuraea ferruginea DSM 43553; NMROSEA = Nonomuraea roseola ATCC 33579; NMRUBRA = Nonomuraea rubra ATCC 27031; NMSALMO = Nonomuraea salmonea ATCC 33580; NMAFRI = Nonomuraea africana DSM 43748; LCAERO = Lechevaleria aerocolonigenes NRRL B-3298; SPAMETH = Streptosporangium amethystogenes DSM 43179; KZVIRIDO = Kutzneria viridogrisa ATCC 25242; STALBUS = Streptomyces albus DSM 40313

Sugar:

The whole cell sugar pattern did not show a specific profile. Phylogenetic analysis:

Partial sequence of 16S rDNA was performed following standard procedures.

The DNA of the organism was extracted using Qiagen' s DNeasy kit following the manufacturer's directions. The 16S rDNA gene was amplified by the polymerase chain reaction using the eubacterial primers 27f and 1492r. The partial sequence was obtained using the primers 27f, 357f, 53 Or, 114f, and 1492r. All the primers used in this work were described by Lane (Lane, D.J. 16S/23 S rRNA sequencing. In Nucleic acid techniques in bacterial systematics. Stackebrandt, E.; Goodfellow, M. (Eds.). John Wiley & Sons. Chichester 1991. pp. 115-175). The almost complete sequence obtained was SEQ ID NO: 1 :

AGAGTTTGATCATGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACATGCA AGTCGAACGATGAAGCCCTTCGGGGTGGATTAGTGGCGAACGGGTGAGTAAC ACGTGGGCAATCTGCCCTGCACTCTGGGACAAGCCCTGGAAACGGGGTCTAA TACCGGATGACATCCCCTCTCGCATGGGAGGGGATTGAAAGCTCCGGCGGTG CAGGATGAGCCCGCGGCCTATCAGCTTGTTGGTGAGGTAGAAGCTCACCAAG GCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAG ACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGG GCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGT AAACCTCTTTCAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAG CGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTT GTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCACGTCGGTTG TGAAAGCCCGGGGCTTAACCCCGGGTCTGCAGTCGATACGGGCAGGCTAGAG TGTGGTAGGGGAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATC AGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCATTACTGACGCTGAG GAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCC GTAAACGGTGGGAACTAGGTGTTGGCGACATTCCACGTCGTCGGTGCCGCAG CTAACGCATTAAGTTCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCA AAGGAATTGACGGGGGCCCGCACAAGCAGCGGAGCATGTGGCTTAATTCGAC GCAACGCGAAGAACCTTACCAAGGCTTGACATATACCGGAAAGCATCAGAG ATGGTGCCCCCCTTGTGGTCGGTATACAGGTGGTGCATGGCTGTCGTCAGCTC GTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTTCTGTG TTGCCAGCATGCCCTTCGGGGTGATGGGGACTCACAGGAGACTGCCGGGGTC AACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCTTG GGCTGCACACGTGCTACAATGGCAGGTACAATGAGCTGCGAAGCCGCGAGG CGGAGCGAATCTCAAAAAGCCTGTCTCAGTTCGGATTGGGGTCTGCAACTCG ACCCCATGAAGTCGGAGTTGCTAGTAATCGCAGATCAGCATTGCTGCGGTGA ATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGTCGGTAA CACCCGAAGCCGGTGGCCCAACCCCTTGTGGGAGGGAGCTGTCGAAGGTGGG ACTGGCGATTGGGACGAAGTCGT AAC AAGGT AACCGT A

This sequence was confronted with the Gene Bank depository using the Blastn algorithm. The phylogenetic studies were performed using the Phylip package developed by Felsestein (Phylip-phylogeny inference package (version 3.2). J. Cladistics 5: 164-166, 1989). A consensus phylogenetic tree was constructed after bootstrapping the sample. Strain AA-98-E-N01 OGAS was grouped within the Streptomyces genus.

Fermentation:

AA-98-E-N010GAS when cultured under controlled conditions in a suitable medium produces antibiotics of general formula (I). This strain is preferably grown in an aqueous nutrient medium, under aerobic and mesophilic conditions, preferably at 28°C- 40°C, and at a pH ranging between 6.0 and 8.0. A wide variety of liquid culture media can be utilized for the cultivation of the organism. Useful media are those that include an assimilable carbon source, such as starch, dextrin, sugar molasses, glucose, and the like, an assimilable nitrogen source such as protein, protein hydrolysate, defatted meals, corn steep, and the like, and useful inorganic anions and cations such as sodium, magnesium, potassium, ammonium, sulfate, chloride, phosphate, carbonate, and the like. Trace elements may be added also. Aeration is preferably achieved by supplying air to the fermentation medium. Agitation is provided by a mechanical impeller. Conventional fermentation tanks have been found to be well suited for carrying out the cultivation of this organism. The addition of nutrients and pH control as well as antifoaming agents during the different stages of fermentation may be needed for increasing production and avoid foaming. The required steps needed for the production of compounds of formula (I) by the preferred organism are: start with frozen or lyophilized mycelium. Obtain mycelial mass culturing the initial cells in shake flasks with a culture medium containing some of the ingredients described above at mesophilic temperatures and in aerobic conditions. This step may be repeated several times as needed and the material collected will be used as an inoculum to seed one or several fermentation tanks with the appropriate culture medium, if desired, these tanks can be utilized also as inoculum, and this step can be repeated several times when needed, or they can serve as the production stage, depending on the broth volume needed. Sometimes the production medium may be different than the one used as inoculum.

Other compounds of this invention can be prepared using techniques already known in the art. As examples, said compounds can be made by modifications of the conditions of the fermentation or by conventional chemical methods using compounds of formula (I) as starting materials, or by synthesis, from commercially available products and reagents, using standard organic synthesis reactions known by the skilled person such as those described in "Comprehensive Organic Transformations: A Guide to Functional Group Preparations", 2^nd Edition, Richard C. Larock, or in "March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure", 6th Edition , Michael B. Smith, and Jerry March, or in "Advanced Organic Chemistry, Part B: Reactions and Synthesis", 5th Edition, Francis A. Carey, and Richard J. Sundberg.

As examples of derivatives from some of the natural products of formula (I) by conventional chemical methods, compounds A and B can be obtained by treatment of Compound 1 and 2, respectively, with Mel and Ag₂0.

Compound A (X²

Compound B (X²

Compounds C and D can be obtained by treatment of Compound 1 and 2, respectively, with Ac₂0 and Pyridine.

Compound C (X =H)

Compound D (X²=C1)

Compound E can be obtained by treatment of Compound 3 with Mel and Ag₂0.

Compound E

Compound F can be obtained by treatment of Compound 3 with Ac₂0 and Pyridine.

Compound F

The invention will be further illustrated by means of examples. These should interpreted merely as illustrative of the invention.

EXAMPLES

Example 1: Fermentation of AA-98-E-N010GAS

Stock culture:

Whole broth of a pure culture of AA-98-E-N010GAS is preserved frozen in 20% glycerol in H₂0 for at least a year at -80°C. Inoculum:

A frozen culture or a well grown slant culture is used to seed (at 5% vol) 10 mL of the first stage inoculum medium (Table 3) contained in a 50 mL Erlenmeyer flask. The flask is incubated during 48 h at 28°C with agitation at 250 rpm. A 250 mL Erlenmeyer flask with 30 mL of the same medium is seeded with 5% vol. of the first stage inoculum. The flask is incubated for 48 h with the same conditions as for the first stage inoculum.

TABLE 3

Fermentation:

A 2 L Erlenmeyer flask containing 250 mL of the production medium (Table 3) is seeded with 12.5 mL of second stage inoculum. The fermentation is carried out during 96 h with 250 rpm agitation and at 28 °C. Monitoring of secondary metabolite production can be performed by HPLC.

Example 2: Production of Compounds 1-3

Fermentation broth (2 L) of actinomycete AA-98-E-N010GAS was filtered through Celite and the mycelial cake extracted twice with 1 L of a mixture of EtOAc/MeOH (3 : 1). The resultant suspension was filtered and partitioned between EtOAc and water. The organic layer was taken to dryness and the crude extract (1.26 g) was fractionated by VFC (vacuum flash chromatography) on silica gel, eluted with a stepwise gradient of hexane/EtO Ac/MeOH. Fraction 4 (eluted with EtOAc/MeOH 75:25, 41.9 mg) was finally purified by semi preparative reversed-phase HPLC (Agilent Prep 5- μg C18 column; 100 x 30 mm; eluting with 65:35 MeOH/H₂0 at a flow rate of 21.27 mL/min, with UV detection at 200 nm) to give 1.7 mg of the known BE-24566B, 5.3 mg of Compound 1, 3.0 mg of Compound 2 and 2.0 mg of Compound 3. Fraction 5 (eluted with EtOAc/MeOH 1 : 1, 186.2 mg) was purified following the same protocol to give 35.0 mg of the known Bischloroanthrabenzoxocinone.

Analytical HPLC-MS (HP 1100, Symmetry C18 column (5 μιη, 3.9x150mm), eluting with a 20 min gradient of 50-100 aqueous MeOH containing 0. 1% formic acid at a flow rate of 0.7 mL/min, at 20°C, with UV detection at 220 nm) was carried out and the compounds showed retention times of 18.69, 19.95, 21.54, 22.60 and 29.68 minutes to BE-24566B, Compound 1, 2, 3, and Bischloroanthrabenzoxocinone, respectively.

Compound 1 has a molecular formula of C27H23CIO7 established by APCI and API-ES mass spectra ([M+H]⁺ at m/z 495.1 and an isotopic peak at m/z 497.1, with a ratio of 3 : 1), ¹³C NMR, and DEPT data. The complete assignments of 1H and ¹³C NMR spectra of Compound 1 were finally established by 2D NMR experiments (COSY, HSQC and HMBC), and its spectroscopic data are shown in table 4.

Table 4. ¾ and ¹³C NMR Spectral Data of Compond 1 [δ (ppm), ¾Η (HZ); CDCI₃] and HMBC

Position ¹³C (δ) ¾ (δ) HMBC

3.15 (1H, d, J= 18.0Hz)

1 40.8 C2, C4a, C12, C12a, C13

3.23 (1H, d, J= 18.0Hz)

2 98.4

4 65.6 6.30 (1H, s) C2, C4a, C5, C12a, C16, C17, C21

4a 123.4

5 157.9

5-OH 13.20 (1H, s) C4a, C5, C5a

5a 110.6

6 191.5

6a 109.8

7 165.0

7-OH 13.49 (1H, s) C6a, C7, C8

8 103.4 6.63 (1H, s) C6, C6a, C7, C9, CIO

9 159.2

10 111.1

10a^* 152.8 11 39.8

11a^* 149.1

12 117.6 6.82 (1H, s) CI, C4a, C5a, C6, Cl l

12a 142.9

13 27.7 1.72 (3H, s) C1. C2

14 28.9 1.92 (3H, s) ClOa, Cl l, Cl la, C15

15 29.1 1.85 (3H, s) ClOa, Cl l, Cl la, C14

16 115.5

17 137.5

18 110.4 6.25 (1H, d, J= 2.5Hz) C16, C19, C20

19 155.9

20 101.6 6.21 (1H, d, J= 2.5Hz) C16, C18, C19, C21

21 152.8

22 19.5 2.45 (3H, s) C16, C17, C18

* may be interchanged

Compound 2 has a molecular formula of C27H22CI2O7 established by APCI and API-ES mass spectra ([M+H]⁺ at m/z 529.0 and two isotopic peaks at m/z 531.0 and 533.0 respectively, with a ratio of 3 :2:0.5), ¹³C NMR, and DEPT data. The complete assignments of 1H and ¹³C NMR spectra of Compound 2 were finally established by 2D NMR experiments (COSY, HSQC and HMBC), and its spectroscopic data are shown in table 5.

Table 5. ¾ and ¹³C NMR Spectral Data of Compound 2 [δ (ppm), ¾Η (HZ); CDCI₃+CD₃OD] and HMBC

asition ¹³C (δ) ¾ (δ) HMBC

3.07 (1H, d, J= 18.0Hz)

1 40.6 C2, C4a, C12, C12a, C13

3.17 (1H, d, J= 18.0Hz)

2 98.2

4 65.7 6.24 (1H, s) C2, C4a, C5, C12a, C16, C17, C21

4a 122.9

5 156.8

5a 110.5

6 189.1

6a 104.0

7 166.9

8 108.6

9 160.2

10 117.1

10a^* 152.8

11 39.6

11a^* 145.8^*

12 117.6 6.73 (1H, s) CI, C4a, C5a, C6, Cl l

12a 140.8

13 27.9 1.63 (3H, s) CI, C2

14 28.9 1.86 (3H, s) ClOa, Cl l, Cl la, C15

15 29.2 1.78 (3H, s) ClOa, Cl l, Cl la, C14

16 114.4

17 137.0 18 110.5 6.21 (1H, d, J= 2.5Hz) C16, C19, C20

19 156.4

20 101.2 6.14 (1H, d, J= 2.5Hz) C16, C18, C19, C21

21 152.4

22 19.5 2.40 (3H, s) C16, C17, C18

* may be interchanged

Compound 3 has a molecular formula of C28H25CIO7 established by APCI and API-ES mass spectra ([M+H]⁺ at m/z 508.1 and an isotopic peak at m/z 510.1, with a ratio of 3 : 1). The complete assignments of 1H and 13C NMR spectra of Compound 3 could not be finally established due to the small amount of compound isolated. Its 1H spectroscopic data were assigned by comparison with the related compounds, and are shown in table 6.

Table 6. ¾ NMR Spectral Data of Compound 3 [δ (ppm), ¾Η (HZ); CDCI₃]

Position ¾ (δ)

3.17 (1H, d, J= 18.0Hz)

3.24 (1H, d, J= 18.0Hz)

4 6.34 (1H, s)

5-OH 13.15 (lH, s)

7-OH 13.61 (1H, s)

10 6.80 (1H, s)

12 6.86 (1H, s)

13 1.53 (3H, s)

14 1.72 (3H, s)

15 1.61 (3H, s)

18 6.36 (1H, d, J= 2.4Hz)

19-OMe 3.72 (3H, s)

20 6.26 (1H, d, J= 2.4Hz)

22 2.49 (3H, s)

Example 3: Cytotoxic in vitro activity of compounds of formula (I)

Cell Culture

All the tumour cell lines were obtained from the ATCC. Human lung carcinoma A549, colon adenocarcinoma HI 16, pancreatic adenocarcinoma PSNl, and human Caucasian glioblastoma T98G were cultured in RPMI medium containing glutamine, (2 mM) penicillin (50 IU/mL), streptomycin (50 μg/mL), supplemented with

5% FBS (A549 and HI 16) or 10% FBS (PSNl and T98G).

Cell proliferation assay

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT; Sigma

Chemical Co., St. Louis, MO) dye reduction assay in 96-well microplates was used, essentially as described in Mosmann, T. J., Immunol. Methods 1983, 65, 55. The assay is dependent on the reduction of MTT by mitochondrial dehydrogenases of viable cell to a blue formazan product, which can be measured spectrophotometrically. Tumour cells (4xl0³ A-549 cells or 6xl0³ H-116 or 6xl0³ PSN1 or 6xl0³ T98G cells in a total volume of 200 μΙ_, of a complete medium) were seeded and serial dilutions in DMSO (10 μ§/ιηΙ., 5 μg/mL, 1 μ^ητΐ.,, 0.5 μ^ητΐ.,, 0.1 μ^ητΐ.,, 0.05 μg/mL, 0.01 μ^ητΐ.,, and 0.005 μg/mL) of the tested compound were added to the wells. After 2 days of incubation (37 °C, 5% C0₂ in a wet atmosphere), 50 μΙ_, of MTT (1 mg/mL in PBS) were added to each well and the plate was incubated for a further 2 h (37 °C). The resulting formazan was dissolved in 100 μΙ_, DMSO and read at 490 nm. All determinations were carried out by triplicate. IC₅₀ value was calculated as the concentration of drug yielded a 50% of cell survival.

Table 7 illustrates data on the cytotoxic activity of compounds of formula (I).

Example 4: Protein kinase inhibitory activity

Recombinant Protein Kinases

Four protein kinases were used for determination of inhibitory profiles. All protein kinases were expressed in Sf9 insect cells as human recombinant GST-fusion proteins by means of the baculovirus expression system. Kinases were purified by affinity chromatography using GSH-agarose (Sigma). The purity of each kinase was checked by SDS-PAGE/silver staining and the identity of each kinase was verified by western blot analysis with kinase specific antibodies or by mass spectroscopy.

Protein kinase assay

A radiometric protein kinase assay (³³PanQinase^® Activity Assay, ProQinase

GmbH) was used for measuring the kinase activity of the 4 protein kinases. All kinase assays were performed in 96-well FlashPlates™ from Perkin Elmer (Boston, MA, USA) in a 50 μΙ_, reaction volume. The reaction cocktail was pipetted in 4 steps in the following order:

• 20 μΐ_^ of assay buffer

• 5 uL of ATP solution (in H₂0)

• 5 μΐ. of test compound (in 10 % DMSO)

10 μΐ_^ of substrate / 10 μΐ_^ of enzyme solution (premixed)

The assay for all enzymes contained 60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl₂, 3 mM MnCl₂, 3 μΜ Na-orthovanadate, 1.2 mM DTT, 50 μg/mL PEG₂₀₀oo, 1 μΜ [γ-³³Ρ]-ΑΤΡ (approx. 5 x 10⁰⁵ cpm per well).

The following amounts of enzyme and substrate were used per well:

Kinase Kinase Substrate Substrate ng/5(^L ng/5(^L

AKT1 50 GSK3( 14-27) 1000 Aurora-B 100 tetra(LRRWSLG) 1000 GSK3p 50 RBER-CHKtide, PR08071 1 1000 PDGFRp 50 Poly(Ala, Glu, Lys,Tyr)_6:2:5:i 125

The reaction cocktails were incubated at 30°C for 80 minutes. The reaction was stopped with 50 μΐ_^ of 2 % (v/v) H₃PO₄, plates were aspirated and washed two times with 200 μΐ_^ H₂0 or 200

0.9 % (w/v) NaCl. Incorporation of ³¾ was determined with a microplate scintillation counter (Microbeta, Wallac).

All assays were performed with a BeckmanCoulter/Sagian robotic system.

Evaluation of data

The median value of the counts in column 1 (n=8) of each assay plate was defined as "low control" . This value reflects unspecific binding of radioactivity to the plate in the absence of a protein kinase but in the presence of the substrate. The median value of the counts in column 2 of each assay plate (n=8) was taken as the "high control", i.e. full activity in the absence of any inhibitor. The difference between high and low control was taken as 100 % activity.

As part of the data evaluation the low control value from a particular plate was subtracted from the high control value as well as from all 80 "compound values" of the corresponding plate. The residual activity (in %) for each well of a particular plate was calculated by using the following formula:

Res. Activity (%) = 100 X [(cpm of compound - low control) / (high control - low control)] Results

The activity results of the compounds are summarized in the table 8. The data are presented as residual activity in percent related to the 100% controls.

Table 8. Protein kinase inhibitory activity

Example 5 NIK inhibitory activity

To study NIK activity transient transfection method of Jurkat cells and luciferase assays, as was previously described (Sanchez-Valdepenas C et al ., J. Immunol. 2006, 176(8), 4666-74), were used. Transcriptional activity was measured using reporter gene assays which depend on NIK kinase activity. So the capacity of different compounds to inhibit the transcription activity of sequence CD28RE depending on NIK quinase activity was analyzed.

Jurkat cells were transfected with CD28RE/AP-1 reporter gene with wild- type NIK-Flag and/or c-Rel or identical amount of empty DNA plasmid. After 4 h of incubation, RPMI medium containing 5% FBS cells were treated or not with different doses of the candidate compound and the incubation was continued for 16 h to complete transfection. Cells were then resuspended in complete medium containing 5% FBS cells, harvested, lysed and luminiscence measured for 10 seconds in a luminometer following the instructions in the "Dual-luciferase Assay System Kit" (Promega). Data are expressed in relative Firefly Luciferase Units (RLUs).

Results

The activity results of the compounds are shown in figures 4 to 7.

Claims

1.- Use of a compound of general formula (I):

(I)

wherein the R¹, R², R³ and R⁴ groups are each independently selected from the group consisting of hydrogen, acyl, alkyl, alkenyl, alkynyl, aryl, aralkyl, -CO H₂, alkali metal, and sugar,

the X¹, X² groups are each independently selected from the group consisting of hydrogen and halogen,

or a pharmaceutically acceptable salt, solvate, tautomer, or stereoisomer thereof, in the preparation of a medicament for the treatment of human diseases mediated by abnormal AKT1, Aurora-B, GSK3 , NIK or PDGFR activities. 2.- Use of a compound as defined in claim 1, where the human disease is selected from cancer, Alzheimer's disease, Parkinson's disease, frontoparietal dementia, corticobasal degeneration, Pick's disease, cerebrovascular accidents, peripheral neuropathies, brain, spinal cord trauma, atherosclerosis, balloon injury induced restenosis, pulmonary fibrosis, liver fibrosis, graft versus host disease (GvHD), transplant rejection, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, asthma, psoriasis, human multiple myeloma, T cell acute lymphoblastic leukemia and Hodgkin^'s disease.

3.- The use according to claims 1 or 2 wherein the compound of formula (I) is

4.- The use according to claims 1 or 2 wherein the compound of formula (I) is

5.- The use according to claims 1 or 2 wherein the compound of formula (I) is

6.- A method of treating human diseases mediated by abnormal AKT1, Aurora-B, GSK3 , NIK or PDGFR activities, which comprises administering to a patient in need of such treatment a therapeutically effective amount of a compound as defined in claim 1, or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof.

7.- A compound of general formula (I):

(I)

wherein the R¹, R², R³ and R⁴ groups are each independently selected from the group consisting of hydrogen, acyl, alkyl, alkenyl, alkynyl, aryl, aralkyl, -CONH₂, alkali metal, and sugar,

the X¹, X² groups are each independently selected from the group consisting of hydrogen and halogen,

or a pharmaceutically acceptable salt, tautomer, solvate or stereoisomer thereof;

with the exception of the compounds in which:

R¹, R², R³, R⁴, X¹, and X² are all hydrogen; 1 4 2 3 1 2

R and R are both methyl group, R , R , X , and X are all hydrogen;

1 2 3 4 1 2

R is a methyl group, R , R , and R are all hydrogen, and X , X are both

1 2 3 4 2 1

R is a methyl group, R , R , R , and X are all hydrogen, and X is CI. - A compound according to claim 7, wherein the compound of formula (I) is

9.- A compound according to claim 7, wherein the compound of formula (I) is

10.- A compound according to claim 7, wherein the compound of formula (I) is

11.- A process for preparing a compound as defined in any of claims 7 to 10, which comprises cultivating a strain of a microorganism capable of producing a compound in an aqueous nutrient medium and further comprises isolating and purifying a compound from the cultured broth and optional modification of this compound to obtain a compound as defined in any of claims 7 to 10.

12.- A process according to claim 11, wherein the microorganism is an actinomycete.

13. - A process according to claim 12, wherein the microorganism is the substantially pure culture strain AA-98-E-N010GAS, available under accession number CECT 7482 from the Coleccion Espanola de Cultivos Tipo at Valencia University, Spain.

14. - A pharmaceutical composition comprising a compound as defined in any of claims 7 to 10, or a pharmaceutically acceptable salt, solvate, tautomer or stereoisomer thereof, and a pharmaceutically acceptable carrier.

15. - A compound as defined in any of claims 7 to 10, or a pharmaceutically acceptable salt, solvate, tautomer, or stereoisomer thereof, for use as a medicament.

- A compound of general formula (I):

(I)

wherein the R¹, R², R³ and R⁴ groups are each independently selected from the group consisting of hydrogen, acyl, alkyl, alkenyl, alkynyl, aryl, aralkyl, -CONH₂, alkali metal, and sugar,

the X¹, X² groups are each independently selected from the group consisting of hydrogen and halogen,

or a pharmaceutically acceptable salt, solvate, tautomer, or stereoisomer thereof, for use in the treatment of human diseases mediated by abnormal AKTl, Aurora-B, GSK3P, NIK or PDGFRp activities.

17.- A compound as defined in any of claims 3 to 5 for use in the treatment of human diseases mediated by abnormal AKTl, Aurora-B, GSK3P, NIK or PDGFR activities.

18.- A compound according to claims 16 or 17 for use in the treatment of human diseases mediated by abnormal AKTl, Aurora-B, GSK3 , NIK, or PDGFR activities, wherein the human disease is selected from cancer, Alzheimer' s disease, Parkinson's disease, frontoparietal dementia, corticobasal degeneration, Pick' s disease, cerebrovascular accidents, peripheral neuropathies, brain, spinal cord trauma, atherosclerosis, balloon injury induced restenosis, pulmonary fibrosis, or liver fibrosis, graft versus host disease (GvHD), transplant rejection, rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, asthma, psoriasis, human multiple myeloma, T cell acute lymphoblastic leukemia and Hodgkin^'s disease.