WO2010134082A1 - Chromenone derivatives for treatment of cancer - Google Patents

Abstract

4-Chromenone derivatives of the formula I herein that inhibit the ubiquitin-mediated degradation of p27, are useful for the treatment of cancer.

Description

CHROMENONE DERIVATIVES FOR TREATMENT OF CANCER

FIELD OF THE INVENTION

The present invention relates to small 4-chromenone derivatives, which are inhibitors of ubiquitin-mediated degradation of p27, and to compositions and method of treatment of cancer.

BACKGROUND OF INVENTION

Progress through the cell cycle is governed by oscillations in activities of cyclin- dependent protein kinases (Cdks). The association with newly synthesized cyclin subunits causes the increase in the activities of different Cdks, while the subsequent inactivation of Cdks is caused by rapid degradation of cyclins. In addition, specific Cdk inhibitors (CKIs) that are synthesized and then rapidly degraded at specific time points in the cell cycle also control the activities of Cdks. The ubiquitin/proteasome system degrades both cyclins and CKIs (Besson et al. 2008). One of the cell cycle specific inhibitors is p27^Kφl. This inhibitor is present at high levels in G0/G1, preventing the action of Cdk2/Cyclin E and Cdk2/Cyclin A to drive cell cycle into S-phase. Following growth stimulation, p27 is rapidly degraded, allowing the action of these kinases to promote entry into S-phase (Montagnoli et al 1999, Carrano et al 1999, Ganoth et al. 2001). In addition, it was demonstrated that S-phase kinase-associated protein (Skp2) also contributes to regulation of G2-M progression by mediating the degradation of p27 (Nakayama et al. 2004). Levels of p27 are very low in aggressive human cancers, apparently due to rapid degradation (Alkarain et al. 2004, Blain et al. 2003). It has been shown that p27 is degraded by the ubiquitin system. Furthermore, the phosphorylation of p27 on threonine residue 187 (T 187) by Cdk2/Cyclin E is required for p27-ubiquitin ligation. Skp2 is the specific F-box protein that ubiquitylates p27. Skp2 is the F-box component of an Skpl-Cullin-F-box protein (SCF) E3 ligase complex that comprises a large family of ubiquitin ligases, whose variable F-box proteins, such as Skp2, recognize a variety of phosphorylated protein substrates. An additional accessory protein Cyclin kinase subunit 1 (Cksl) is essential for p27 binding to Skp2. In the crystal structure of the core p27 ubiquitination complex, Cksl binds the phospho-threonine residue of p27, and forms an additional binding site for a near-by glutamate residue by cooperating with Skp2. The affinity of Skp2 to the substrate is further strengthened by the association of the Cdk-binding site of Cksl with Cdk2/Cyclin2 E, to which phosphorylated p27 is tightly bound (Carrano et al. 1999, Ganoth et al. 2001). Low molecular weight molecule that inhibits the binding of p27 to the Skp2 degradation complex is expected to prevent p27 ubiquitination, and thus prevent p27 degradation. The resulting increase in p27 should prevent cell cycle progression to S- phase, particularly in cancer cells where upregulated Skp2 is a mechanistic basis for proliferation. It would be highly desirable to provide such low molecular weight compounds, specifically adapted to inhibition of p27 ubiquitination.

SUMMARY OF INVENTION

In one aspect, the present invention provides a small molecule, which is a A- chromenone derivative of the formula I depicted hereinafter. In certain embodiments, the compounds of the invention are of the formula Ia., in particular the compounds herein designated Compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13.

In another aspect, the present invention provides a pharmaceutical composition comprising a compound of formula I and a pharmaceutically acceptable carrier. In particular embodiments, the pharmaceutical compositions comprise the compounds herein designated Compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13.

In certain embodiments, the compound of formula I is used according to the invention for inhibiting the ubiquitin ligase-mediated degradation of p27. By promoting an increase in the level of p27 in aggressively dividing cells such as cancerous cells, the compounds of the invention inhibit exit or release of the cells from G0/G1 and regulate the G2 -M progression.

Thus, in a further aspect, the present invention relates to the use of the compounds of formula I for treatment of cancer. Due to the fact that the compounds of the invention affect a very basic stage in the mechanism of controlled degradation of peptides that mediate cell cycle progression, they can be used in treatment of diseases or disorders characterized or caused by aggressive cell division. In particular embodiment, the disease treatable by the present compounds is cancer. In another aspect, the present invention provides to a method for the treatment of cancer, which comprises administering to a subject in need an effective amount of a compound of formula I.

The methods of the invention are applicable for the treatment of any kind of cancerous state including solid tumors, non-solid cancer and metastasis.

BRIEF DESCRIPTION OF DRAWINGS

Fig. 1 is a graph showing the levels of p27 in HeLa cells treated with various concentrations of Compound 1 or Compound 2, or with solvent (PEG400-DMSO 1:1). Empty column: control (solvent); hatched columns: Compound 1 at 50, 100 and 150 μM; gray columns: Compound 2 at 50, 100 and 150 μM. Results are average ± SD.

Fig. 2 is a graph showing the levels of p27 in HeLa cells treated with various concentrations of Compound 2 or Compound 3, or with solvent (PEG400-DMSO 1 :1). Empty column: control (solvent); hatched columns: Compound 3 at 25, 50, 75 and 100 μM; gray columns: Compound 2 at 25, 50, 75 and 100 μM. Results are average ± SD.

Fig. 3 is a graph showing the levels of p27 in HeLa cells treated with 50 μM Compound 2 (hatched column), or 25 or 50 μM Compound 4 (gray columns). Empty column is the control (PEG400-DMSO 1:1). Results are average ± SD.

Figs. 4A-4B are FACS spectra showing counts of A549 cells in various phases of the cell cycle, 0, 3, 6 and 20 hours after release form Gl. Fig 4A: cells released from Gl by replacing their medium with solvent (PEG400 : DMSO 1:1). Fig. 4B: Cells released from Gl by replacing their medium with 100 μM Compound 2 dissolved in PEG400 :

DMSO 1:1.

Figs. 5A-5B are graphs showing tumor volume (5A) and body weight (5B) in mice bearing human NSCLC A549 xenografts treated with Compound 1 (filled squares) or with vehicle (control, a mixture of organic solvents and phosphate buffer) (filled circles). Mice were treated daily from day 28 (solid line arrow) until day 41 (dotted-line arrow). Each data point is the average of 8 mice. Error bars are +/- SEM.

DETAILED DESCRIPTION OF THE INVENTION p27 ^kφl is a cell-cycle inhibitory protein which is degraded in rapidly dividing cells through the action of the E3 ligase SCFskp2. The minimal recognition unit for targeting p27^kipl for degradation is comprised of Skp2, Cksl (an accessory protein), and a short peptide from the C-terminus of p27. Disruption of the recognition and binding of p27 to Skp2 and Cksl will prevent ubiquitin-mediated degradation of p27 and inhibit or prevent cell division. The present inventors have found that some novel 4-chromenone derivatives disrupt the Skp2-Cksl-p27 complex, leading to inhibition of ubiquitination of p27. Such 4- chromenone derivatives may therefore be very useful anti-cancer drugs.

Thus, according to one aspect, the present invention provides a compound of the general formula I:

wherein

R₁ is hydrocarbyl, acyl, heterocyclyl or OR₃;

R₂ represents one, two or three radicals selected from hydrocarbyl, acyl, heterocyclyl or OR₃; and R₃ is H, hydrocarbyl, acyl or heterocyclyl; wherein any of the hydrocarbyl or heterocyclyl radicals may be independently substituted by one or more radicals selected from halogen, hydrocarbyl, heterocyclyl, oxo, nitro, epoxy, epithio, -OR, -SR, -COR, -COOR, -NR₄R₅, -CONR₄R₅, -NR₄-COR, -SO₃R, -

SO₂R, -SO₂NR₄R₅, -PO₃R₄R₅, and -NR₄SO₂R, wherein R is H, hydrocarbyl or heterocyclyl; and

R₄ and R₅ each independently is H or alkyl, or R₄ and R₅ together with the nitrogen atom to which they are attached form a 5-6 saturated heterocyclic ring, optionally containing 1 or 2 further heteroatoms selected from N, S and/or O, and optionally substituted at the ring and/or at a second heteroatom by one or more groups selected from oxo, lower alkyl, aralkyl, haloalkyl, hydroxyalkyl or COOR; or a stereoisomer or a pharmaceutically acceptable salt thereof. Without limiting the scope to further possible definitions, as used herein in the specification, the terms hereinbelow are defined as follows:

The term "hydrocarbyl" means a radical derived from a saturated or unsaturated aliphatic radical of Ci-C_2O carbon atoms, a cycloaliphatic radical Of C₃-C₈ carbon atoms, or an aromatic radical of C₆-Ci₅ carbon atoms. The aliphatic radicals include a straight or branched alkyl, alkenyl and alkynyl, and the cycloaliphatic radicals include cycloalkyl and cycloalkenyl. As used herein, "alkyl" refers to a C₁-C₁₀ alkyl, more preferably Ci-C₄, lower alkyl, "alkenyl" is a C₂-Ci₀, preferably C₂-C₄, alkenyl, and "alkynyl" is a C₂-Ci₀, preferably C₂-C₄, alkynyl. The alkyl, alkenyl or alkynyl may be interrupted by one or more heteroatoms selected from O, S and/or N, and/or substituted by one or more groups selected from heteroaryl, aryl, aralkyl, carbocyclyl, oxo, nitro, epoxy, epithio, -OR, -SR, - COR, -COOR, -NR₄R₅, -CONR₄R₅, -NR₄-COR, -SO₃R, -SO₂R, -SO₂NR₄R₅, -PO₃R₄R₅, and -NR₄SO₂R, wherein R, R₄ and R₅ are as defined above.

Examples of lower alkyls include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl. Preferred alkyls according to the invention are methyl, propyl or isopropyl. Any C₂-C₄ alkenyl may contain one or two double bonds, e.g. alkadienyl radical, and preferably a terminal double bond, and includes for example a propenyl such as vinyl or prop-2-en-l-yl, or butenyl such as but-3-en-l-yl. Any C₂-C₄ alkynyl may contain one or more triple bonds and includes, for example, ethynyl, propynyl and butynyl.

The terms "cycloalkyl" and "cycloalkenyl" refer to a C₃-C₆ cycloalkyl or C₃-C₆ cycloalkenyl, respectively, and include, without being limited to, cyclopropyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl and cyclohexenyl that may be unsubstituted or substituted by one or more radicals as defined hereinabove. The terms "aromatic radical" and "aryl" are interchangeable and refer to a monocyclic, bicyclic or tricyclic aromatic carbocyclic radical having 6 to 14 carbon atoms, preferably 6 to 10 carbon atoms. Non-limiting examples include phenyl, naphthyl and antracenyl that may be unsubstituted or substituted by one or more radicals as defined hereinabove. The term "aralkyl" refers to a radical derived from an arylalkyl compound and includes benzyl, phenethyl and the like.

The term "heterocyclyl" means a radical derived from a saturated, partially unsaturated or an aromatic monocyclic, bicyclic or tricyclic heterocycle of 3-14 ring members containing 1 to 4 heteroatoms selected from O, S and/or N.

As used herein, "saturated or partially unsaturated heterocyclyl" refers to a monocyclic, bicyclic or tricyclic heterocycle of 5 to 10, preferably 5 to 6, ring members.

None limiting examples include dihydrofuryl, tetrahydrofuryl, dihydrothienyl, pyrrolydinyl, pyrrolynyl, dihydropyridyl, piperidinyl, piperazinyl, morpholino or 1,3- dioxanyl, preferably tetrahydrofuryl or pyrrolydinyl. "Aromatic heterocyclyl" refers herein to a heteroaryl of 5 to 10, preferably 5 to 6, ring members, and include, but not limited to, pyrrolyl, tetrazolyl, 1,2,5-oxadiazolyl, furyl, thienyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, pyridyl, quinolinyl, isoquinolinyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,3,4-triazinyl, 1,2,3-triazinyl, 1,3,5-triazinyl, benzofuryl, isobenzofuryl, indolyl, imidazo[l,2-a]pyridyl, benzimidazolyl, benzthiazolyl, benzoxazolyl and benzodiazepinyl, preferably tetrazolyl or 1,2,5-oxadiazolyl.

The heterocyclyl and heteroaryl may be substituted by one or more groups selected from lower alkyl, aryl, aralkyl, carbocyclyl, nitro, -OR, -SR, -COR, -COOR, -NR₄R₅, -

CONR₄R₅, -NR₄-COR, -SO₃R, -SO₂R, -SO₂NR₄R₅, -PO₃R₄R₅, and -NR₄SO₂R, wherein R, R₄ and R₅ are as defined above. It is to be understood that when a polycyclic heterocyclyl is substituted, the substitutions may be in any of the carbocyclic and/or heterocyclic rings.

The term "acyl" as used herein refers to a radical of the formula -(R')C=0 derived from a carboxylic acid, i.e., a carboxylic acyl, wherein R' represents an aliphatic, cycloaliphatic, aromatic or heterocyclic moiety. Examples of aliphatic acyls include, but not limited to, formyl, acetyl, propionyl, acryl and hexanoyl. Aromatic acyls are, for example, benzoyl, cyclopenta-2,4-dienoyl, and heterocyclic acyls include, for example, 2- tetrahydrofuranoyl, pyrrolidine-2-carbamoyl, imidazolidine-4-carbamoyl, 1,2,4- triazolidine-3 -carbamoyl, tetrahydro-2H-pyran-3-carbamoyl, 4H-pyran-3-carbamoyl and the like. In preferred embodiments, the acyl is 2-tetrahydrofuranoyl. The term "halogen" refers to fluoro, chloro, bromo or iodo.

The group -NR₄R₅ may be -NH₂, when R₄ and R₅ are both hydrogen, or may be a secondary amino group when R₄ is H and R₅ is Ci-C₄ alkyl, or tertiary amino when R₄ and

R₅ are each Ci-C₄ alkyl, or R₄ and R₅ together with the nitrogen atom to which they are attached may form a saturated, 5-7 membered, preferably 5- or 6-membered, heterocyclic ring, optionally containing 1 or 2 further heteroatoms selected from nitrogen, oxygen and/or sulfur. Examples of such rings include, without being limited to, pyrrolidino, piperidino, morpholino, thiomorpholino, piperazino, N-alkylpiperazino, e.g. N- methylpiperazino, and diazepino. Such rings may be substituted at the ring and/or at a second heteroatom by one or more groups selected from oxo, lower alkyl, haloalkyl, hydroxyalkyl or COOR.

Any alkoxy, alkylthio or alkanoyl groups formed by the radicals OR, SR and COR, when R is alkyl, are preferably Cj-C₄ alkoxy, Ci-C₄ alkylthio and C₂-C₄ alkanoyl groups, respectively. Examples of alkoxy are methoxy, ethoxy, propyloxy, butoxy, and the like, examples of alkylthio are methylthio, ethylthio, propylthio, butylthio, and examples of alkanoyl are acetyl, propanoyl, butanoyl, and the like. All alkoxy, thioalkyl, and alkanoyl radicals may be substituted as defined above. In one preferred embodiment, the Ci-C₄ alkoxy is methoxy. hi certain embodiments, the present invention provides a compound of the formula Ia:

wherein

R₆ is selected from: (0 H;

(ii) heterocyclic carboxylic acyl;

(iii) cycloalkenyl substituted by one or more oxo and/or -OR groups;

(iv) phenyl substituted by one or more groups selected from -

SO₂NR₄R₅ or -PO₃R₄R₅; or

(v) alkyl, preferably a straight or branched lower alkyl, substituted by one or more groups selected from a heteroaryl, preferably tetrazolyl or 1,2,5-oxadiazolyl optionally substituted by lower alkyl; COOR; OR; SO₂R; -SO₃R; - CONR₄R₅; -SO₂NR₄R₅, or -NR₄R₅; R₇ is H or lower alkyl; R is H, hydrocarbyl or heterocyclyl; and R₄ and R₅ each independently is H or alkyl, or R₄ and R₅ together with the nitrogen atom to which they are attached form a 5-6 saturated heterocyclic ring, optionally containing 1 or 2 further heteroatoms selected from N, S and/or O, and optionally substituted at the ring and/or at a second heteroatom by one or more radicals selected from lower alkyl, oxo, haloalkyl, hydroxyalkyl, or COOR. Ln more particular embodiments, in the compounds of formula Ia R₆ is selected from: (i) H;

(ii) 2- tetrahydrofuranoyl;

(iii) cyclobutenyl substituted by two oxo groups and an -OR group, wherein R is H or methyl; (iv) phenyl substituted by -SO₂NH₂ or -PO₃H₂; or

(v) propyl or isopropyl substituted by one or more groups selected from COOH, OH or -NH₂; and R₇ is methyl.

Preferred compounds according to the invention are the compounds herein identified as Compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13. Most preferred are Compounds 1-4. The chemical names of these compounds are listed in the Examples section herein, and their structural formulae are resented in Appendix I.

The compounds of the invention are also termed herein "Skp2 inhibitors" since they interfere or disrupt the formation of the Skp2-Cksl-p27 complex. Also contemplated by the present invention are salts of the compounds of formula

I, including salts formed by any carboxy or sulfo groups present in the molecule and a base as well as acid addition and/or base salts.

Pharmaceutically acceptable salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like, and examples of suitable amines are

N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylene-diamine,

N-methylglucamine, and procaine (see, for example, Berge S. M., et al., "Pharmaceutical

Salts," (1977) J. of Pharmaceutical Science, 66:1-19). The salts can also be pharmaceutically acceptable quaternary salts such as a quaternary salt of the formula - NRR'R" + Z' wherein R, R'and R" each is independently hydrogen, alkyl or benzyl and Z is a counterion, including chloride, bromide, iodide, O-alkyl, toluenesulfonate, methylsulfonate, sulfonate, phosphate, carboxylate, acetate or trifluoroacetate.

Pharmaceutically acceptable acid addition salts of the compounds include salts derived from inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydriodic, phosphorous, and the like, as well as salts derived from organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Such salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, caprylate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartrate, methanesulfonate, acetate, trifluoroacetate and the like. Also contemplated are salts of amino acids such as arginate and the like and gluconate or galacturonate (see, for example, Berge S. M., et al., "Pharmaceutical Salts," (1977) J. of Pharmaceutical Science, 66:1-19).

The acid addition salts of said basic compounds are prepared by contacting the free base form with a sufficient amount of the desired acid to produce the salt in the conventional manner. The free base form may be regenerated by contacting the salt form with a base and isolating the free base in the conventional manner. The free base forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free base for purposes of the present invention.

The base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner. The free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention.

Compound 1 (7-hydroxy-3-(4-methoxyphenoxy)-2-(trifluoromethyl)-4H-chromen-4- one) was prepared in accordance with the present invention, by obtaining first the intermediates Precursor 1 ((4-methoxyphenoxy)acetonitrile) and Precursor 2 (l-(2,4- dihydroxyphenyl)-2-(4-methoxyphenoxy)ethanone), in a three-step procedure described herein in Example 1 and Scheme 1. Further reactions of the hydroxy group at position 7 of Compound 1 with tethradydrofurane acyl chloride or squaric acid produced Compounds 2-4, as described in Examples 2-4 herein. Compound 1 may thus serve as the starting material for synthesis of most of the Skp2 inhibitors of formula Ia.

It has been discovered, according to the present invention, that cancerous cells that were treated with compounds of formula I of the invention presented elevated concentrations of p27 as compared to untreated cells. In addition, cells treated with the present compounds, showed a significant delay in their ability to exit from the G0/G1 to S phase and then were blocked at G2/M phase. Such results would be expected from elevated p27 levels, since p27 has repressive effects at the Gl/S and G2/M phases.

In vivo studies in a tumor models in mice were conducted by the inventors, and these studies revealed that tumor-bearing mice that were treated daily for two weeks with the compounds of the invention showed a substantial reduction (~30%) in tumor growth immediately following commencement of treatment. Lower tumor growth was maintained throughout the treatment. Therefore inhibition of p27 degradation, particularly ubiquitin- mediated degradation, in cancerous cells by the compounds provided by the present invention may lead to treatment of cancer.

Thus, in another aspect, the present invention provides a pharmaceutical composition comprising a compound of formula I as described herein above and a pharmaceutically acceptable carrier. hi certain embodiments, the pharmaceutical compositions of the invention comprise compounds of formula Ia, more preferably the compounds herein identified as Compounds 1 -13, most preferably Compound 1-4.

The pharmaceutical compositions of the invention are particularly useful as anti cancer drugs. The novel 4-chromenone derivatives of formula I most probably disrupt the Skp2-Cksl-p27 complex, leading to inhibition of ubiquitination of p27, which in turn inhibits or prevents cell division by keeping the cells arrested at G0/G1 or at M-G2.

The pharmaceutical compositions of the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. Thus, the compounds and their physiologically acceptable salts and solvates may be formulated by conventional methods as described, for example, in Remington's Pharmaceutical Sciences, Meade Publishing Co., Easton, PA., for administration by a variety of routes of administration, including systemic and topical or localized administration.

For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the compounds of the invention can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution, hi addition, the compounds may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included. For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents; fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-/?-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to give controlled release of the active compound. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by inhalation, the compounds of the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifiuoromethane, trichlorofluoromethane, dichlorotetra- fluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides. hi addition to the formulations described previously, the compounds of the invention may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration may be through nasal sprays or using suppositories. For topical administration, the compounds of the invention are formulated into ointments, salves, gels, or creams as. generally known in the art. A wash solution can be used locally to treat an injury or inflammation to accelerate healing. The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.

The compounds of the invention may be used for treatment of any and all kinds of cancerous state including solid tumors, non-solid cancer and metastasis. Non- limiting examples of solid cancerous tumors that can be treated with the compounds of the invention are carcinomas, which are malignant tumors derived from epithelial cells. This group represents the most common cancers, including the common forms of breast, prostate, lung, bladder, skin (e.g., melanoma), esophagus, colon cancer and certain types of brain cancer.

Also contemplated by the present invention is the treatment of sarcomas, malignant tumors derived from connective tissue, or mesenchymal cells, such as leiomyosarcoma, chondrosarcoma, and gastrointestinal stromal tumor (GIST), which are more common in adults than in children, high-grade bone sarcomas such as Ewing's sarcoma and osteosarcoma, which are much more common in children and young adults.

The present compounds are also useful for the treatment of lymphoma and leukemia, malignancies derived from hematopoietic (blood-forming) cells. Also treatable by the compounds of the invention are germ cell tumors, which are derived from totipotent cells. In adults these tumors are most often found in the testicle and ovary.

Other kinds of tumor suitable for treatment by the compounds of the invention include blastic tumors or blastomas. These tumor (usually malignant) resemble an immature or embryonic tissue. Many of these tumors are most common in children.

Thus, in a further aspect, the present invention provides a method for the treatment of cancer which comprises administering to a subject in need thereof an effective amount of a compound of the invention.

In certain embodiments, compounds used in the method of the invention are Compounds 1-13. More preferred are Compounds 1-4. hi view of the teaching herein, one of skill in the art will understand that the methods and the composition are applicable to a wide range of cancers, as well as any other disease or disorder associated with aggressive cell growth.

The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

EXAMPLES

CHEMICAL SECTION

The 4-chromenone derivatives of the invention (Compounds 1 to 13) and the intermediates {Precursors 1 to 3) are presented herein by their respective Arabic numbers in bold. The corresponding formulas appear in Schemes 1-4 and in Appendix A at the end of the specification, just before the claims. The chemical names of Compounds 1-13 of the invention are:

1. 7-hydroxy-3-(4-methoxyphenoxy)-2-(trifluoromethyl)-4H-chromen-4-one 2. 3-(4-methoxyphenoxy)-4-oxo-2-(trifluoromethyl)-4H-chromen-7-yl tetrahydro furan-2-carboxylate

3. 3-hydroxy-4-(3-(4-methoxyphenoxy)-4-oxo-2-(trifluoromethyl)-4H-chromen-7- yloxy) cyclobut-3-ene-l,2-dione 4. 3-methoxy-4-(3-(4-methoxyphenoxy)-4-oxo-2-(trifluoromethyl)-4H-chromen-7- yloxy)cyclobut-3-ene- 1 ,2-dione

5. 2-(3-(4-methoxyphenoxy)-4-oxo-2-(trifluoromethyl)-4H-chromen-7-yloxy) benzenesulfonamide

6. 2-(3-(4-methoxyphenoxy)-4-oxo-2-(trifluoromethyl)-4H-chromen-7-yloxy) phenylphosphonic acid

7. 3-(3-(4-methoxyphenoxy)-4-oxo-2-(trifluoromethyl)-4H-chromen-7-yloxy)-2- methylpropanoic acid

8. (S)-7-(3-amino-2-hydroxypropoxy)-3-(4-methoxyphenoxy)-2-(trifluoromethyl)- 4H-chromen-4-one 9. (R)-7-(3-amino-2-hydroxypropoxy)-3-(4-methoxyphenoxy)-2-(trifluoromethyl)-

4H-chromen-4-one

10. 4-(3-(4-methoxyphenoxy)-4-oxo-2-(trifluoromethyl)-4H-chromen-7-yloxy) benzenesulfonamide

11. 3-(3-(4-methoxyphenoxy)-4-oxo-2-(trifluoromethyl)-4H-chromen-7-yloxy) benzenesulfonamide

12. 4-(3-(4-methoxyphenoxy)-4-oxo-2-(trifluoromethyl)-4H-chromen-7-yloxy) phenylphosphonic acid

13. 3-(3-(4-methoxyphenoxy)-4-oxo-2-(trifluoromethyl)-4H-chromen-7-yloxy) phenylphosphonic acid

Materials. Chemicals and solvents of analytical grade were generally used except when performing HPLC, where HPLC-grade solvents were applied. All the reagents and chemicals were mainly purchased from Sigma-Aldrich, Israel. Example 1. Synthesis of Compound 1 (7-hydroxy-3-(4-methoxyphenoxy)-2- (trifluoromethyl)-4H-chromen-4-one)

The synthesis of Compounds 1, starting with Precursor 1 and Precursor 2 as depicted in Scheme 1, was carried out as follows:

Precursor 1

Precursor 2

Compound 1

Scheme 1

L Synthesis of Precursor 1 ((4-methoxyphenoxy)acetonitrile) To a stirred solution of 4-methoxyphenol (37.2 g, 0.3 mol) in DMF (150 ml),

K₂CO₃ (50 g, 0.36 mol) was added in one portion. The mixture was stirred for 20 min. and then cooled down. Chloracetonitrile (23 g, 0.3 mol) was added in one portion and the mixture was allowed to warm up to room temperature (RT). After 10 hr of stirring, the mixture was quenched with water (0.5 L) and the organic layer was separated. The water layer was extracted with CH₂Cl₂ (3x100 ml) and the combined organic layers were washed with 10% NaOH (aq.), dried, and evaporated to yield 37 g of Precursor 1 (76%). ii. Synthesis of Precursor 2 (l-(2,4-dihydroxyphenyl)-2-(4-methoxyphenoxy)ethanone)

Dry hydrogen chloride was passed for lhr into a solution of Precursor 1 (16.3g, 0.1 mol) in dry benzene (75 ml) at O⁰C. A solution of the 1,3-dihydroxyphenol resorcinol (13.2 g, 0.1 mol) and ZnCl₂ (27.3 g, 0.2 mol) in dry ether were then added. HCl was passed for an additional 2hr and the reaction mixture was left overnight. The liquid was decanted from the solid, hot water (200 ml) were added to the residue, and the mixture was boiled for lhr. After cooling, the solid that formed was filtered off, washed with water up to pH 7, and recrystallized from 2-propanol to yield 13.8g of Precursor 2 (50%).

UL Synthesis of Compound 1

Precursor 2 was dissolved in dry pyridine (40 ml), and (CF₃CO)₂O (20 ml) was added in one portion. The mixture was refluxed for 2 hr and then poured into water and acidified. The precipitate formed was filtered, dried, and recrystallized from ethanol (EtOH)-H₂O to yield 13.2 g of Compound 1 (94%).

¹H-NMR: (300 MHz, DMSO) δ 3.7 (s, 3H), 6.8 (m, 2H), 7 (m, 4H), 7.9 (m, IH), l l,l (s, IH).

Example 2. Synthesis of Compound 2 (3-(4-methoxyphenoxy)-4-oxo-2- (trifluoromethyI)-4H-chromen-7-yl tetrahydrofuran-2-carboxylate) Compound 2 was obtained from Compound 1 as shown is Scheme 2, as follows:

Compound 1

Scheme 2 A suspension of tethradydrofurane acyl chloride (2 mmol) in THF (5 ml) with triethyl amine (Et₃N) (2 drops) under nitrogen was cooled at O⁰C. Compound 1 (1 mmol) was introduced at once and the flask was allowed to warm up to RT. The reaction was work up by ethyl acetate (EtOAc) and after removal of the solvent, the crude was purified to give Compound 2 (82%).

¹H-NMR: (400 MHz, DMSO) δ 1.8 (m, 2H), 2.3 (m, 2H), 3.7 (s, 3H), 3.8 (m, 2H), 4.6 (m, IH), 6.8 (m, 2H), 7.1 (m, 2H), 7.4 (m, IH), 7.8 (m, IH) , 8.1 (m, IH).

Example 3. Synthesis of Compound 3 (3-methoxy-4-(3-(4-methoxyphenoxy)-4- oxo-2-(trifluoromethyl)-4H-chromen-7-yloxy)cyclobut-3-ene-l,2-dione)

Compound 3 was obtained from Compound 1 and Precursor 3 as depicted in Scheme 3, as follows:

Scheme 3

L Synthesis of Precursor 3 (3,4-dichlorocyclobut-3-ene-l,2-dione)

A suspension of squaric acid (3,4-dihydroxycyclobut-3-ene-l,2-dione; 1.14 g, 10 mmol) in carbon tetrachloride (5 ml) with DMF (2 drops) under nitrogen was frozen by immersion in liquid nitrogen. Oxalyl chloride (2.54 g, 20 mmol) was introduced at once and the flask was allowed to warm up to RT. It was then heated to 50⁰C for lhr and the reaction mixture was filtered. The residue was washed with CCl₄ and the filtrate was evaporated in vacuo to give pale yellow crystals, yielding 785 mg of Precursor 3 (52%). U. Synthesis of Compound 3

To a stirred solution of Compound 1 (704 mg, 2 mmol) and Precursor 3 (302 mg, 2 mmol) in dioxane (5 ml), Et₃N (0.3 ml) was added and the reaction mixture was heated to reflux for 2 hr. It was then cooled and evaporated to dryness. The product was isolated by preparative chromatography (gradient: EtOAc-Hexane, EtOAc, Acetone) to yield 63 mg (7%) with a purity by HPLC of 90%. A second purification by preparative chromatography (Acetone) gave 41 mg of pure Compound 3 (4.5%).

¹H-NMR: (300 MHz, acetone-D) δ 3.7 (s, 3H), 6.8 (m, 2H), 7.1 (m, 2H), 7.4 (m, IH), 7.8 (m, IH), 8.0 (m, IH).

Example 4. Synthesis of Compound 4 (3-methoxy-4-(3-(4-methoxyphenoxy)-4-oxo-2- (trifluoromethyl)-4H-chromen-7-yloxy)cyclobut-3-ene-l,2-dione)

Compound 4 was obtained from Compound 1 as depicted in Scheme 4, as follows:

Scheme 4

A stirred mixture of Compound 1 (352 mg, 1.0 mmol), 3-chloro-4-methoxycyclobut-

3-ene-l,2-dione (194 mg, 1.5 mmol) and dry THF (3 ml) was cooled to -15°C under argon. A solution of di-isopropylethylamine (0.26 ml, 1.5 mmol) in dry THF (0.7 ml) was then added dropwise during 20 min and the reaction mixture was left overnight at 0°C. All solvents were evaporated to dryness (1 mm, 50°C) and the residue was dissolved in dichloromethane (3 ml) and diluted with hexane (3 ml). The upper layer separated and the lower layer was extracted in the same manner one more time. The combined extracts were concentrated and the product was separated immediately by chromatography on SiO₂ (eluent - from hexane-DCM (1:1) to DCM and 10% ethyl acetate in DCM) to produce an oil which was kept at 0°C for 2 days, treated with ether (2 ml) and filtered off. The resulting crystalline solid was dissolved in a THF-hexane mixture (1:3, 4 ml) and the solvents were evaporated on air to produce a crystalline product which was filtered off, washed with ether (2x1 ml) and dried on air to constant weight. Yield: 32 mg (7%).

¹H-NMR: (300 MHz, acetone-D) δ 3.7 (s, 3H), 4.5 (s, 3H), 6.8 (m, 2H), 6.9 (m, 2H), 7.4 (m, IH), 7.5 (m, IH), 8.0 (m, IH).

BIOLOGICAL SECTION

Materials and Methods Material.

For Elisa analysis, commercially available ELISA kit (Assay Designs, 900-139) was used.

Cells. All cell lines employed were obtained from ATCC.

HeLa cells were grown in DMEM+10% FCS. Human non-small cell lung cancer (NSCLC) A549 cells were grown in DMEM+10% FCS.

Aminals. Nude mice (female, 5 weeks) were provided by Harlan, France; Average weight: 20 - 22 grams/mouse; Health status: IOPS; Acclimatization period after delivery at Cellvax: 1 week; Animals were placed in an animal housing (CERFE, Evry). All procedures (animal handling, their transfer) were performed in a strict sterile condition; Diet: Standard commercially available animal food (SSNIFF, Germany) and sterilized water.

Methods

Flow cytometry (FACS). Elevation of p27 leads to significant interference in cell cycle progression. FACS experiments were performed in order to examine the effect of the Skp2 inhibitors on cell cycle. A549 cells were exposed to the test compounds (inhibitors) for 48 hours and then prepared for FACS analysis of DNA content. In order to further identify the phase of cell cycle arrest induced by the compounds of the invention, the cells were homogeneously synchronized using thymidine for Gl arrest, and then released in the presence of a solvent or a test compound. After cells accumulated in Gl, they were washed and replenished with medium containing solvent or a test compound, and then fixed after 0, 6, 20 hours.

Elisa. Commercially available ELISA kit (Assay Designs, 900-139) was used for selection of a reporter cell line to Skp2 activity. When Skp2 function is inhibited by a compound of, the invention, degradation of p27 is reduced and p27 iaccumulates.

Xenograft animal model. Human NSCLC cell line A549 was used as a xenograft model to determine in vivo efficacy of the compounds of the invention. Female athymic nude mice were inoculated subcutaneously in the right flank, and tumor growth was monitored until a volume of 70 mm³ was reached before commencing treatment. The compounds were injected by intra-peritoneal route, once a day during 14 days, three doses for each compound. The control group was injected with the vehicle which is a formulation containing 2% DMSO, 2% PEG400, 4% TWEEN 80 and 92 % of buffer phosphate (volume percentages). Eight mice were treated in each group. Tumor volume throughout the duration of the entire study was calculated based on average tumor growth for each of the treatment/control groups. Animals were monitored each day by checking their behavior, physical activity, and their body weight. The animals have been kept for a two- week observation period after ending the treatment.

Example 5. Elevation of p27 levels in cells treated with Compound 1 or Compound 2

HeLa cells grown in DMEM+10% FCS were treated for 48 hours with 50, 100 or 150 μM Compound 1 or Compound 2 dissolved in PEG400 : DMSO 1:1. Control cells were incubated only with the solvent (PEG400 : DMSO 1 :1). All the experiments were performed in duplicates.

The level of p27 protein in equal amounts of protein extract was determined at the end of incubation using p27 ELISA kit (R&D systems, Minneapolis, USA) according to the manufacturer instructions. The results for both compounds are presented in Fig. 1. As shown, treatment of the cells with either Compound 1 or Compound 2 lead to elevation of p27 protein level in a concentration dependent manner. Example 6. Elevation of p27 levels in cells treated with Compound 2 or Compound 3

HeLa cells grown in DMEM+10% FCS were treated for 48 hours with 25, 50, 75 or 100 μM Compound 2 or Compound 3 dissolved in PEG400 : DMSO 1 :1. Control cells were incubated only with the solvent . All the experiments were performed in duplicates. The level of p27 protein in equal amounts of protein extract was determined as in

Example 5, and the results for both compounds are presented in Fig. 2.

As shown, treatment of the cells with either Compound 2 or Compound 3 lead to elevation of p27 protein level in a concentration dependent manner, hi addition, Compound 3 was more efficient than 2.

Example 7. Elevation of p27 levels in cells treated with Compound 2 or Compound 4

HeLa cells grown in DMEM+10% FCS were treated for 48 hours with 50 μM Compound 2 or with 25 or 50 μM of Compound 4 dissolved in PEG400 : DMSO 1:1. Control cells were incubated only with the solvent. All the experiments were performed in duplicates. The level of p27 protein in equal amount of protein extract was determined as described in Example 5, and the results for both compounds are presented in Fig. 3.

As shown in Fig 3, treatment with the Skp2 inhibitors at concentrations of 25-50 μM lead to >2 fold elevation of p27 protein level in the cells compared to control.

Example 8. Effect of Compound 2 on cell cycle in Gl-synchronized cells.

Human non-small cell lung cancer (NSCLC) A549 cells grown in DMEM+10% FCS were homogeneously synchronized to Gl phase by adding 2 mM thymidine to the medium for 24 hours. At the end of synchronization, the cells were released from the Gl arrest by replacing the medium with medium containing solvent (PEG400 : DMSO 1:1) with or without 100 μM Compound 2, and after 0, 3, 6 and 20 hours the cells were collected for Propidium Iodide (Sigma, Israel) staining and FACS analysis. The results are shown in Figs. 4A-4B.

While solvent-treated cells (Fig. 4A) progressed through the cell cycle, the

Compound 2 treated cells showed a significant delay in their ability to exit from the Gl to S phase and then were blocked at G2/M phase (Fig. 4B). Such results would be expected from elevated p27 levels, since p27 has repressive effects at the Gl/S and G2/M phases

(Montagnoli et al., 1999, Nakayama et al., 2004). Example 9. Effect of Compound 1 on tumor growth in animal model

Human NSCLC cell line A549 xenografts were subcutaneously inoculated in mice as described in Materials and Methods. Tumor-bearing mice were treated daily from day

28 until day 41 with 33 mg/kg Compound 1 or with a vehicle (control), which is a mixture of organic solvents and phosphate buffer based on of 2% DMSO, 2% PEG400, 4%

TWEEN 80 and 92 % of buffer phosphate (volume percentages).

Tumor volume and body weight were monitored throughout the study. The results are presented in Figs. 5A-5B.

As shown in Fig. 5A, an observable reduction in tumor growth was noted immediately following commencement of treatment in the Compound 1 treated group.

This group maintained lower tumor growth throughout the 14 days of treatment. Tumors were reduced in size by -30% as compared to the vehicle control. Importantly, this inhibited tumor growth was statistically significant from day 32 until day 41. Furthermore, the reduction in tumor size was maintained during the observation period following the end of treatment on day 41 (days 42-53). The statistical variation of the samples increased in all groups during the observation period as a result of sacrificing three of the eight animals in each group on day 41.

No apparent differences in tumor size were observed in groups treated with lower concentrations of Compound 1. As shown in Fig. 5B, no significant changes were observed in body weight as a result of the treatments.

REFERENCES

Alkarain A., Jordan R. and Slingerland J., (2004) J. Mammary Gland Biol. Neoplasia 9: 67-80.

Besson, A., Dowdy, S.F., Roberts, J.M. (2008), "CDK inhibitors: Cell cycle regulators and beyond". Dev. Cell 14:159-169.

Blain, S.W., Scher, H.I., Cordon-Cardo, C, andKoff, A. (2003), Cancer Cell 3, 111-115.

Carrano A.C., Eytan E., Hershko A. and Pagano M. (1999), "SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27", Nat Cell Biol. 1(4): 193-9.

Ganoth D., Bornstein G., Ko T.K., Larsen B., Tyers M., Pagano M., Hershko A., (2001), "The cell-cycle regulatory protein Cksl is required for SCF(Skp2)-mediated ubiquitinylation of p27", Nat Cell Biol. 3(3):321-4.

Montagnoli A., Fiore F., Eytan E., Carrano A.C., Draetta G.F., Hershko A. and Pagano M. (1999), "Ubiquitination of p27 is regulated by Cdk-dependent phosphorylation and trimeric complex formation", Genes Dev. 13(9):1181-9.

Nakayama K., Nagahama H., Minamishima Y.A., Miyake S., Ishida N., Hatakeyama S., Kitagawa M., Iemura S., Natsume T. and Nakayama K.I., (2004) "Skp2- mediated degradation of p27 regulates progression into mitosis", Dev Cell. 6(5):661-72.

Ohtsubo M., Theodoras A.M., Schumacher J., Roberts J.M.,Pagano M. (1995)

"Human cyclin E, a nuclear protein essential for the Gl to S phase transition", MoI. Cell. Biol. 15:2612-2624. APPENDIX I

Claims

1. A compound of the general formula I:

wherein

Ri is hydrocarbyl, acyl, heterocyclyl or OR₃; R₂ represents one, two or three radicals selected from hydrocarbyl, acyl, heterocyclyl or OR₃; and

R₃ is H, hydrocarbyl, acyl or heterocyclyl; wherein any of the hydrocarbyl or heterocyclyl radicals may be independently substituted by one or more radicals selected from halogen, hydrocarbyl, heterocyclyl, oxo, nitro, epoxy, epithio, -OR, -SR, -COR, -COOR, -NR₄R₅, -CONR₄R₅, -NR₄-COR, -SO₃R, -

SO₂R, -SO₂NR₄R₅, -PO₃R₄R₅, and -NR₄SO₂R, wherein R is H, hydrocarbyl or heterocyclyl; and

R₄ and R₅ each independently is H or alkyl, or R₄ and R₅ together with the nitrogen atom to which they are attached form a 5-6 saturated heterocyclic ring, optionally containing 1 or 2 further heteroatoms selected from N, S and/or O, and optionally substituted at the ring and/or at a second heteroatom by one or more groups selected from oxo, lower alkyl, aralkyl, haloalkyl, hydroxyalkyl or COOR; or a stereoisomer or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, wherein said hydrocarbyl is a saturated or unsaturated aliphatic radical of Ci-C₂₀ carbon atoms, a cycloaliphatic radical of C₃-C₈ carbon atoms, or an aromatic radical of C₆-Ci₅ carbon atoms; said acyl is an aliphatic, cycloaliphatic, aromatic or heterocyclic carboxylic acyl; and said heterocyclyl is a saturated, partially unsaturated or an aromatic monocyclic, bicyclic or tricyclic heterocycle of 3-14 ring members containing 1 to 4 heteroatoms selected from O, S and/or N.

3. The compound according to claim 2, wherein said saturated or unsaturated aliphatic radical is selected from a straight or branched Ci-C₁₀ alkyl, preferably Ci-C₄, lower alkyl, C₂-Ci₀, preferably C₂-C₄, alkenyl or C₂-Ci₀, preferably C₂-C₄, alkynyl, more preferably methyl, ethyl, ethynyl, n-propyl, isopropyl, propenyl, propynyl, butyl, sec-butyl, tert-butyl, butenyl or butynyl, optionally interrupted by one or more heteroatoms selected from O, S and/or N, and/or substituted by one or more groups selected from heteroaryl, aryl, aralkyl, carbocyclyl, oxo, nitro, epoxy, epithio, -OR, -SR, -COR, -COOR, -NR₄R₅, -CONR₄R₅, - NR₄-COR, -SO₃R, -SO₂R, -SO₂NR₄R₅, -PO₃R₄R₅, and -NR₄SO₂R, wherein R, R₄ and R₅ are as defined in claim 1.

4. The compound according to claim 2, wherein said cycloaliphatic radical is a C₃-C₆ cycloalkyl or C₃-C₆ cycloalkenyl, more preferably cyclopropyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl or cyclohexenyl, unsubstituted or substituted by one or more groups selected from lower alkyl, heterocyclyl, aryl, aralkyl, oxo, nitro, -OR, - SR, -COR, -COOR, -NR₄R₅, -CONR₄R₅, -NR₄-COR, -SO₃R, -SO₂R, -SO₂NR₄R₅, - PO₃R₄R₅, and -NR₄SO₂R, wherein R, R₄ and R₅ are as defined in claiml.

5. The compound according to claim 2, wherein said aryl is a monocyclic, bicyclic or tricyclic aromatic carbocyclic radical of 6 to 14, preferably 6 to 10, carbon atoms, more preferably phenyl, biphenyl, naphtyl, or antracenyl, unsubstituted or substituted by one or more groups selected from lower alkyl, heterocyclyl, nitro, -OR, -SR, -COR, -COOR, - NR₄R₅, -CONR₄R₅, -NR₄-COR, -SO₃R, -SO₂R, -SO₂NR₄R₅, -PO₃R₄R₅, and -NR₄SO₂R, wherein R, R₄ and R₅ are as defined in claim 1.

6. The compound according to claim 1, wherein said saturated or partially unsaturated heterocyclyl is a monocyclic, bicyclic or tricyclic heterocycle of 5 to 10, preferably 5 to 6, ring members, selected from dihydrofuryl, tetrahydrofuryl, dihydrothienyl, pyrrolydinyl, pyrrolynyl, dihydropyridyl, piperidinyl, piperazinyl, morpholino or 1,3-dioxanyl, preferably tetrahydrofuryl or pyrrolydinyl; and said aromatic heterocyclyl is a heteroaryl of 5 to 10, preferably 5 to 6, ring members selected from pyrrolyl, tetrazolyl, 1,2,5- oxadiazolyl, furyl, thienyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, pyridyl, quinolinyl, isoquinolinyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,3,4-triazinyl, 1,2,3-triazinyl, 1,3,5- triazinyl, benzofuryl, isobenzo furyl, indolyl, imidazo[l,2-a]pyridyl, benzimidazolyl, benzthiazolyl, benzoxazolyl and benzodiazepinyl, preferably tetrazolyl or 1,2,5- oxadiazolyl, wherein said heterocyclyl and heteroaryl may be substituted by one or more groups selected from lower alkyl, aryl, aralkyl, carbocyclyl, nitro, -OR, -SR, -COR, - COOR, -NR₄R₅, -CONR₄R₅, -NR₄-COR, -SO₃R, -SO₂R, -SO₂NR₄R₅, -PO₃R₄R₅, and - NR₄SO₂R, wherein R, R₄ and R₅ are as defined in claim 1.

7. The compound according to claim 1, wherein said 5-6 saturated heterocyclic ring, optionally containing 1 or 2 further heteroatoms selected from N, S and/or O formed from R₄, R₅ and the N atom to which they are attached, is selected from pyrrolidino, piperidino, morpholino, thiomorpholino, piperazino, N-methylpiperazino, and diazepino, optionally substituted at the ring and/or at a second heteroatom by one or more groups selected from oxo, lower alkyl, haloalkyl, hydroxyalkyl or COOR.

8. A compound according to any one of claims 1 to 7 of the formula Ia:

wherein

R₆ is selected from: (i) H;

(ii) heterocyclic carboxylic acyl;

(iii) cycloalkenyl, substituted by one or more oxo and/or -OR groups; (iv) phenyl substituted by one or more groups selected from -SO₂NR₄R₅ or - PO₃R₄R₅; or

(v) alkyl, preferably a straight or branched lower alkyl, substituted by one or more groups selected from heteroaryl, preferably tetrazolyl or 1,2,5-oxadiazolyl optionally substituted by lower alkyl; COOR; OR; SO₂R; -SO₃R; -CONR₄R₅; - SO₂NR₄R₅, or -NR₄R₅; R₇ is H or lower alkyl; R is H, hydrocarbyl or heterocyclyl; and

R₄ and R₅ each independently is H or alkyl, or R₄ and R₅ together with the nitrogen atom to which they are attached form a 5-6 saturated heterocyclic ring, optionally containing 1 or 2 further heteroatoms selected from N, S and/or O, and optionally substituted at the ring and/or at a second heteroatom by one or more radicals selected from lower alkyl, oxo, haloalkyl, hydroxyalkyl, or COOR.

9. The compound according to claim 8, wherein R₆ is selected from: (i) H;

(ii) 2- tetrahydrofuranoyl;

(iii) cyclobutenyl substituted by two oxo groups and an -OR group, wherein R is H or methyl;

(iv) phenyl substituted by -SO₂NH₂ or -PO₃H₂; or

(v) propyl or isopropyl substituted by one or more groups selected from

COOH, OH or -NH₂; and

R₇ is methyl.

10. The compound according to claim 9, herein identified as Compound 1 of the formula:

11. The compound according to claim 9, herein identified as Compound 2 of the formula:

12. The compound according to claim 9, herein identified as Compound 3 of the formula:

13. The compound according to claim 9, herein identified as Compound 4 of the formula:

14. The compound according to claim 9, selected from the compounds herein identified as compounds 5, 6, 7, 8, 9, 10, 11, 12 and 13, having the formulae presented in Appendix I.

15. A pharmaceutical composition comprising a compound of formula I according to any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

16. A pharmaceutical composition comprising a compound of formula Ia according to any one of claims 8 to 14 and a pharmaceutically acceptable carrier.

17. The pharmaceutical composition according to claim 16, wherein said compound of formula Ia is selected from the compounds herein identified as Compound 1, Compound 2, Compound 3 and Compound 4.

18. The pharmaceutical composition according to any one of claims 15 to 17 for treatment of cancer.

19. The compound of formula I according to any one of claims 1 to 14 for use in treatment of cancer.

20. The compound of formula Ia according to any one of claims 8 to 14 for use in treatment of cancer.

21. A method for the treatment of cancer comprising administering to a subject in need thereof an effective amount of a compound according to any one of claims 1 to 14.

22. The method according to claim 21, wherein said compound is selected from the compounds herein identified as Compound 1, Compound 2, Compound 3 and Compound 4.