WO2011064559A2 - Novel inositol phosphate derivatives - Google Patents

Abstract

The present invention relates to compounds of the formula (I) wherein R1,R2, R3, R4, R5 and R6, X and n are as defined herein. Preferred compounds are inositol phosphate derivatives wherein R1 is a substituted or unsubstituted phenyl and at least one of R2, R3, R4, R5 and R6 is PO3 2- Alternative compounds are inositol phosphate derivatives of the formula (I) wherein R1 is a fiuorophore moiety The compounds are useful alone or in combination with known therapeutic agents for the treatment of diseases, especially cancer.

Description

NOVEL INOSITOL PHOSPHATE DERIVATIVES

FIELD OF THE INVENTION

The present invention relates to inositol phosphate derivatives, their use in therapy, in particular for the prevention or treatment of cancer, pharmaceutical compositions comprising them, and processes for their preparation.

BACKGROUND OF THE INVENTION

Inositol is a compound of the cyclitol family of the cyclohexane type containing a hydroxyl group on each carbon (1, 2, 3, 4, 5, 6-hexahydroxy cyclohexanes). The natural compound most widely represented is myoinositol, whose hydroxyl groups at the 1, 2, 3 and 5 positions are located on one of the faces of the cyclohexane ring and at the 4 and 6 positions are located on the other face. Inositol phosphates are a group of mono- to polyphosphorylated inositols that play a crucial role in diverse cellular functions, such as cell growth, apoptosis, cell migration, endocytosis and cell differentiation.

Phosphoinositide 3-kinases (PI3K) are lipid kinases that phosphorylate lipids at the 3-hydroxyl residue of an inositol ring. The 3-phosphorylated phospholipids (PIP3s) generated by PI3 kinases act as second messengers recruiting kinases with lipid binding domains (including plekstrin homology (PH) regions), such as Akt and phosphoinositide-dependent kinases-1 (PDK1). Binding of Akt to membrane PIP3s causes the translocation of Akt to the plasma membrane, where it is activated through phosphorylation at its residues Thr308 and Ser473. PDK1 is responsible for the phosphorylation of Thr308 and also possesses a PH domain able to bind PIP3s. The PI3-kinases Akt and PDK1 are important in the regulation of many cellular processes including cell cycle regulation, proliferation, survival, apoptosis and motility and are significant components of the molecular mechanisms of diseases such as cancer. Among other targets, Akt activates the multi-protein complex mTORCl containing the enzyme mammalian target of rapamycin (mTOR) which regulates several intracellular functions including cell growth, cell cycle progression and autophagy. A second mTOR-containing complex (mTORC2) is involved in phosphorylation of Akt at its residue Ser473 and its activation. The mechanism of mTORC2-dependent Akt phosphorylation at Ser473 is still not completely understood but it does not seem to involve phosphoinositides, as in the case of PDK1, since mTOR does not appear to possess phosphoinositide-binding domains. We have previously found that the inhibition of Akt by inositol 1, 3, 4, 5, 6-penta f/sphosphate (InsPs) results in pro-apoptotic, anti-angiogenic and anti-tumour activity.

There remains a need, however, for more potent and specific inhibitors of the PI3K/Akt pathway, in particular protein kinases inhibitors. The present invention is therefore based on the object of providing compounds which have these abilities.

SUMMARY OF THE INVENTION

Provided are novel inositol phosphate derivatives that inhibit protein kinases, including P13K lipid kinase and phosphor-Akt in cancer cells, and also inhibit the migration, proliferation and/or invasion of cancer cells.

According to one aspect, the present invention provides compounds of the general formula (I):

wherein R¹ is substituted or unsubstituted aryl; R², R³, R⁴, R⁵ and R⁶ are, independently, selected from: H, P0₃ ^2", P(OR⁷)₃, PO(0 R⁷)₂, PO(0 R⁷)0^", -NHalkyl, -alkylene-NH₂ , NH₂, S0₂Cr , S0₂OR⁷, S(0)R⁷, S0₂R⁷, C(0)R⁷, C(0)OR⁷, OC(0)R⁷, and alkyl;

R⁷ is H, alkyl, alkenyl or alkynyl; X is O or NH; n is 0, 1 or 2; or an isomer, ester, hydrate, solvate, or physiologically tolerated salt thereof.

In a preferred embodiment, a compound of the present invention has the formula (II) or (III):

wherein in each case is substituted or unsubstituted aryl; R², R³, R⁴, R⁵ and R⁶ are, independently, selected from : H, P0₃ ^2", P(OR⁷)₃, PO(0 R⁷)₂, PO(0 R⁷)Cr, -NHalkyl, -alkylene-NH₂ , NH₂, S0₂0^" , S0₂OR⁷, S(0)R⁷, S0₂R⁷, C(0)R⁷, C(0)OR⁷, OC(0)R⁷, and alkyl;

R⁷ is H, alkyl, alkenyl or alkynyl; X is O or H; n is 0, 1 or 2; or an ester, hydrate, solvate or physiologically tolerated salt thereof.

According to another aspect, the present invention provides a pharmaceutical composition comprising a compound of the formula (I), (II) or (III), or a physiologically tolerated salt thereof.

According to a further aspect, the present invention provides a compound of the formula (I), (II) or (III), or a physiologically tolerated salt thereof, for use in therapy.

According to yet another aspect, the present invention provides a compound of the formula (I), (II) or (III), or a physiologically tolerated salt thereof, for use in the treatment or prevention of a kinase-affected disease state or condition, such as cancer. The cancer can be, for example, pancreatic cancer, breast cancer, skin cancer, ovarian cancer, colon cancer, prostate cancer, lung cancer, gastric cancer, cervical cancer, brain cancer, leukaemia or colon cancer. The compounds of the present invention are also effective for the treatment of cancers that are normally drug resistant.

According to yet another aspect, the present invention provides a compound of the formula (I), (II) or (III), or a physiologically tolerated salt thereof, in combination with a therapeutic agent other than a compound of the formula (I) or a therapy.

According to a further aspect, the present invention provides a compound of the formula (I), (II) or (III), or a physiologically tolerated salt thereof, in combination with another other agent or therapy for use in the treatment or prevention of cancer. The compound of the formula (I), (II) or (III) may be administered separately, simultaneously or sequentially in any order with at least one other agent or therapy. According to another aspect, the present invention provides a method of inhibiting a component of the PI3K/Akt pathway, in particular PDK1 and/or mTO , which comprises contacting a mammalian tissue or cell with a compound of the formula (I), (II) or (III). Preferably, the method comprises administering to a subject in need thereof an effective amount of a compound of the formula (I), (II) or (III), or a physiologically tolerated salt thereof.

According to further aspect, the present invention provides a method for treating or preventing a kinase-affected disease or condition which comprises administering to a subject in need thereof an effective amount of a compound of the formula (I), (II) or (III), or a physiologically tolerated salt thereof.

According to another aspect, the present invention provides a method for treating or preventing cancer, which comprises administering to a subject in need thereof an effective amount of a compound of the formula (I), (II) or (III), or a physiologically tolerated salt thereof.

According to a further aspect, the present invention provides a method for treating or preventing cancer cell proliferation, migration and/or invasion which comprises administering to a subject in need thereof an effective amount of a compound of the formula (I), (II) or (III), or a physiologically tolerated salt thereof.

According to another aspect, the present invention provides a process for the preparation of a compound of the formula (I).

According to yet another aspect, the present invention provides a compound of the formula (IA):

wherein

R¹, R², R³, R⁴, R⁵ and R⁶ are, independently from each other, selected from: H, P0₃ ^2', P(OR⁷)₃, PO(0 R⁷)₂, PO(0 R⁷)CT, -NHalkyl, -alkylene-NH₂ , NH₂, S0₂0- , S0₂OR⁷, S(0)R⁷, S0₂R⁷, C(0)R⁷, C(0)OR⁷, OC(0)R⁷, and alkyl;

R⁷ is H, alkyl, alkenyl or alkynyl; X is O; and n is zero; or an isomer, ester, hydrate, solvate or physiologically tolerated salt thereof; for use in the treatment or prevention of : 1) cancer cell migration and/or cancer cell invasion, 2) a drug resistant cancer, and/or 3) a cancer selected from : breast cancer, pancreatic cancer, prostate cancer, skin cancer, colorectal cancer and ovarian cancer. According to another aspect, the present invention provides a compound of the formula (I) :

or a salt thereof , wherein R¹ is H, substituted or unsubstituted aryl, fluorophore moiety, or substituted or unsubstituted aryl substituted by fluorophore moiety; R² to R⁶ are independently selected from: H, P0₃ ^2", P(OR⁷)₃, PO(0 R⁷)₂, PO(0 R⁷) O^' and a fluorophore moiety;

X is 0 or NH;

Y is NH or CH₂; m is 0 or l;and n is 0, 1 or 2; wherein at least one of R¹ to R⁶ is a fluorophore moiety.

According to a further aspect, the present invention provides a compound of the formula (I), wherein the compound comprises a fluorophore moiety as described, or a physiologically tolerated salt thereof, for use in therapy.

According to yet another aspect, the present invention provides a method of detecting and/or treating a cancer cell or tissue, comprising incubating a potential cancer cell or tissue with a compound of the formula (I), wherein the compound comprises a fluorophore moiety as described, or a salt thereof, wherein the compound interacts with the cancer cell or tissue, wherein the presence of the compound with the cell or tissue indicates the cancer is present, identifying the association of fluorescence with the potential cancer cell or tissue.

BRIEF DESCRIPTION OF THE FIGURES Figure 1A shows the structure of inositol 1, 3, 4, 5, 6-pentakisphosphate (InsP₅) and 2-O-benzyl-m o- inositol 1, 3, 4, 5, 6-pentakisphosphate (2- 0-Bn-InsP₅);

Figures IB and IC show the effects of InsP₅ and 2-0-Bn-InsP₅ on Akt activation by monitoring phosphorylation at Akt residues Ser473 and Thr308 in a human ovarian cancer cell line (Figure IB) and a human prostate cancer cell line (Figure 1C); Figures 2A and 2B are graphs showing the number of surviving cells (% control) in relation to the concentration (μΜ) of InsP₅ and 2-0-Bn-InsP₅ in a human mammary cancer cell line (Figure 2A) and a human ovarian cancer cell line (Figure 2B); Figures 2C and 2D are graphs showing the percentage of apoptotic cells in relation to the concentration (μΜ) of InsP₅ and 2-0-Bn-InsP₅ in a human mammary cancer cell line (Figure 2C) and a human ovarian cancer cell line (Figure 2D);

Figures 3A-3F are graphs showing the number of surviving cells (% control) in relation to the concentration (μΜ) of InsP₅ and 2-0-Bn-InsP₅ in a human pancreatic cancer cell line (Figures 3A and 3B), a human mammary cancer cell line (Figure 3C) and a human prostate cancer cell line (Figures 3D, 3E and 3F);

Figures 4A and 4B are graphs showing tumour growth in 2-0-Bn-InsP₅ treated mice (Figure 4A) and InsP₅ treated mice (Figure 4B) compared with a control group over time;

Figure 4C tables the results obtained from the graphs of Figures 4A and 4B;

Figure 4D shows the effects of InsP₅ and 2-0-Bn-InsP₅ on Akt activation by monitoring phosphorylation at its residues Ser473 and Thr308 in mice tumours; and

Figures 5A to 5F are graphs showing the effects of 2-0-Bn-InsP₅ and other anti-cancer compounds alone and in combination on number of cells (%control) in various human cancer cell lines; Figures 6A and 6B are graphs showing the effects of 2-0-Bn-InsP₅ on cancer cell migration (Figure 6A) and cancer cell invasion (Figure 6B) in comparison to a control (H₂0) and InsP_5; Figure 7 shows intracellular uptake of a fluorescein conjugated InsP₅ in different cancer cells.

DETAILED DESCRIPTION

So that the invention may be more readily understood, certain terms are first defined.

The term "aryl" as used herein refers to a 5 to 10-membered aromatic, mono- or bicyclic system. The aryl may be unsubstituted or substituted by one or more substituents independently selected from: Ci-Ce alkyl, Ci-C₆ alkoxy, cyano, oxo, amino, C ₆ alkylamino, halogen, halo d-C₆ alkyl, trifluromethyl, trifluromethoxy and OH. For example, aryl may be di- substituted with ethyl or methoxy, or monsubstituted with methyl, ethyl, ethoxy, or fluoro.

The term "alkyl" as used herein refers to an aliphatic hydrocarbon group having 1 to about 8 carbon atoms, preferably 1 to 6 or 1 to 4 carbon atoms, in the chain. Alkyl residues may be linear or branched. Examples of alkyl groups are: methyl, ethyl, propyl, butyl, pentyl, hexyl. This comprises both the n-isomers of these residues and isopropyl, isobutyl, isopentyl, sec-butyl, tert-butyl, neopentyl, 3,3-dimethylbutyl etc. The term "alkoxy" refers to an -O-alkyl group, wherein alkyl is as defined herein,

The term "alkenyl" as used herein refers to an unsaturated open chain hydrocarbon group having 2 to about 8 carbon atoms, preferably 2 to 6 or 2 to 4 carbon atoms, in the chain that may have 1 to 3 conjugated or unconjugated double bonds, preferably one double bond, in a linear or branched chain; the same applies to "alkynyl" residues in respect of triple bonds. Examples for alkenyl and alkynyl groups are vinyl, 1-propenyl, 2- propenyl (allyl), 2-butenyl, 2-methyl-2-propenyl, 3-methyl-2-butenyl, ethynyl, 2-propynyl (propargyl), 2-butynyl or 3-butynyl. The term "acyl" as used herein refers to an H-CO- or alkyl-CO- group. Preferred acyl groups include palmitoyl, formyl, acetyl, propanoyl, 2- methylpropanoyl and butanoyl.

The term "halogen" as used herein refers to Fl, CI, Br or I. The term "effective amount" means the amount of a compound or composition which is required to reduce the severity of and/or ameliorate at least one condition or symptom which results from the disease in question.

As used herein, the terms "treating" or "treatment" refer to a partial or total prevention of a disease state, inhibition of disease progression or reversal of a disease.

The term "subject" refers to an animal, preferably a mammal, and in particular a human. In a particular embodiment, the subject is a mammal, in particular a human, who has been, or who is suspected of, suffering from a disease, for example cancer.

All references to "compound(s) of the formula (I)/(II)/(III)/(IA)" as used herein are intended to include a compound/ compounds of the formula (I)/(II)/(III) as described above and also their salts, solvates, hydrates, esters, prodrugs, all isomers and all polymorphic forms as described herein.

The term "comprise", "comprises", "comprised" or "comprising" as used herein is to be interpreted as specifying the presence of the stated features, integers, steps, or components referred to, but not to preclude the presence or addition of one or more other feature, integer, step, or component.

All references to the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes mixtures of compounds, and a reference to "a pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

A preferred compound according to the present invention is a compound of the formula (I), (II) or (III), wherein ¹ is substituted or unsubstituted phenyl.

The substituted cyclohexane group in the compound of the formula (I) is preferably a substituted inositol. The inositol may be in form of one of nine possible stereoisomers, that is, m o-inositol, D-c/7/ro-inositol, L- crj/ro-inositol, sc //o-inositol, ep/^'-inositol, c/^'s- inositol, neo-inositol, muco- inositol, and a//o-inositol.

Preferably, the inositol group of the compound of the formula (I), (II) or (III) is substituted by at least one OP0₃ ^2" , that is, one or more of R², R³, R⁴, R⁵ and R⁶ in formula (I) is P0₃ ^2~. In one embodiment of a compound of the formula (I), (II) or (III), at least R², R⁴ and R⁵ are P0₃ ^"2, more preferably R², R³, R⁴, R^s and R⁶ are P0₃ ^~2 .

A further preferred compound is a compound of the formula (I), (II) or (III), wherein X is O.

Another preferred compound is a compound of the formula (I), (II) or (III), wherein n is 1. A most preferred compound according to the present invention is selected from: 2-0-benzyl-/n o-inositol 1, 3, 4, 5, 6-pentakisphosphate and 2-0- benzyl-D-c 7/ro-inositol 1, 3, 4, 5, 6-pentakisphosphate, or a physiologically tolerated salt thereof.

A preferred compound of the formula (IA), or salt thereof, is a compound wherein at least one of R¹ to R⁶ is P0₃ ^2", P(OR⁷)₃, PO(0 R⁷)₂, or PO(0 R⁷)0^", preferably at least one of R¹ to R⁶ is P0₃ ^2", more preferably R² to R⁶ are P0₃ ^2". In one embodiment of a compound of the formula (IA), R¹ is H. A preferred compound of the formula (I), or a salt thereof, wherein at least one of R¹ to R⁶ is, or comprises, a fluorophore moiety, is a compound wherein R¹ is a fluorophore moiety; R² to R⁶ are independently selected from: H, P0₃ ^2', P(OR⁷)₃, PO(0 R⁷)₂, and PO(0 R⁷)CT ; X is O or NH;Y is NH; n is 0, 1 or 2; and m is 1.

A most preferred compound of the formula (I), or a salt thereof, wherein at least one of R¹ to R⁶ is, or comprises, a fluorophore moiety, is a compound wherein the fluorophore moiety is fluorescein or a derivative of fluorescein. Preferably, the derivative of fluorescein is carboxy fluorescein.

A preferred compound of the formula (I), or a salt thereof, wherein at least one of R¹ to R⁶ is, or comprises, a fluorophore moiety, is the compound is 2-0-(2-(5-fluoresceinylcarboxy)-aminoethyl)-myo-inositol 1, 3,4,5, 6-pentakisphosphate or a salt thereof.

The fluorophore moiety R² , R³ ,R⁴ ,R⁵ or R⁶ in the compound of the formula (I) is preferably linked to its neighbouring atom by means of a linker, for example a linker of the formula: -(CH₂)_n-Y_m-, wherein Y is NH or CH₂, n is 0, 1 or 2 and m is 0 or 1. The present invention also relates to a method of detecting and/or treating a cancer cell or tissue, comprising incubating a potential cancer cell or tissue with a compound of the formula (I) wherein at least one of R¹ to R⁶ is, or comprises, a fluorophore moiety, or a salt thereof, wherein the compound interacts with the cancer cell or tissue, wherein the presence of the compound with the cell or tissue indicates the cancer is present, identifying the association of fluorescence with the potential cancer cell or tissue. In a preferred embodiment, the compound of the formula (I) wherein at least one of R¹ to R⁶ is, or comprises, a fluorophore moiety, or a salt thereof, interacts with a cell or tissue selected from: breast cancer, pancreatic cancer, prostate cancer, skin cancer, colorectal cancer and/or ovarian cancer. In one embodiment of the method for detecting a cancer cell or tissue according to the present invention, the compound not only detects a cancer cell or tissue but also inhibits cancer cell proliferation, migration and/or invasion.

The method for detecting a cancer cell or tissue is preferably carried out in vitro or in situ.

In another embodiment, the treatment method according to the present invention is carried out in vivo by administering to a subject in need thereof an effective amount of a compound of the formula (I) comprising at least one fluorophore moiety as described, or a physiologically tolerated salt thereof.

Accordingly, another aspect of the present invention is a compound of the formula (I) comprising at least one fluorophore moiety, or a physiologically tolerated salt of the compound, for use in the treatment or prevention of cancer cell proliferation, migration and/or invasion.

In a preferred embodiment, the compound of the formula (I) comprising at least one fluorophore moiety, or a salt thereof, is for use in the treatment or prevention of a cancer selected from: breast cancer, pancreatic cancer, prostate cancer, skin cancer, colorectal cancer and ovarian cancer.

The fluorophore compound for use in the compounds and methods of the present invention may be selected from those fluorophore compounds known in the art. The fluorophore compound may be fluorescein or a derivative of fluorescein, dansyl chloride, Alexa Fluor series of dyes, such as Alexa Fluor® 647, fluorescent sphingolipids, etc. Derivatives of fluorescein include: carboxy fluorescein, fluorescein isothiocyanate, eosin, fluorescein amidite, erythrosine, rose bengal, dylight fluor, etc. Instead of a fluorescent moiety, it would alternatively be possible to use a bioluminiscent moiety in the compounds and methods of the present invention. Accordingly, compounds of the formula (I) wherein the fluorophore moiety is replaced with a bioluminescent moiety are also a feature of the present invention. Such bioluminescent compounds are well known to the person skilled in the art.

Preferably, the fluorophore compound used in the compounds and methods of the present invention comprises fluorescein or a derivative thereof, for example, fluorescein isothiocyanate (FITC) or carboxy fluorescein (FAM), or a fluorophore of the same or lower molecular weight.

In one embodiment of the method of treating and detecting a cancer cell or tissue according to the present invention, the potential cancer cell or tissue is a potential breast cancer, pancreatic cancer, prostate cancer, skin cancer, colorectal cancer or ovarian cancer. In a further embodiment of the method of treating and detecting a cancer cell according to the present invention, the method further comprises comparing the fluorescence of the potential cancer cell or tissue to a control.

According to a yet further aspect, the present invention provides a method of preparing a compound of the formula (I) comprising a fluorophore moiety, wherein the method comprises reacting a compound of the formula (I) with a fluorophore, and, optionally, converting the resultant fluorophore analogue of the compound of the formula (I) into a salt. In one embodiment, the method of preparing a compound of the formula:

or a salt thereof, wherein R¹ is a fluorophore moiety; R² to R⁶ are independently selected from: H, P0₃ ^2~, P(OR⁷)₃, PO(0 R⁷)₂, and PO(0 R⁷)0; R⁷ is H, alkyl, alkenyl or alkynyl; X is O or NH; Y is NH; n is 0, 1 or 2 and m is 1; comprises reacting a compound of the formula (VI):

wherein R² to R⁶, X and n are as defined, with a fluorophore compound, optionally wherein the fluorophore compound is fluroscein or a derivative of fluorescein, and, optionally, converting the resultant compound into a salt. An example of a process for the preparation of 2-0-(2-(5- fluoresceinylcarboxy)-aminoethyl)-m o-inositol 1,3,4,5,6- pentakisphosphate (2-FAM-InsP₅) according to the present invention is provided below.

2-^Q-(2-Aminoethyl)-myo-inositol 1,3,4,5.6-pentakisphosphate. To a solution of 2-0-[2-(2,2,2-trifluoroacetylamino)ethyl]-m o-inositol l,3,4,5,6-pentakis(dibenzylphosphate) [AM Rossi, AM Riley, SC Tovey, T Ur-Rahman, 0 Dellis, VG Veresov, EJA Taylor, BVL Potter & CW. Taylor, Nature Chem Biol (2009) 5, 631-639] (380 mg, 0.235 mmol) in MeOH (30 mL) and deionised water (8 mL) was added palladium hydroxide on activated charcoal (Fluka, 20%, 50% water, 100 mg). The suspension was stirred vigorously under an atmosphere of hydrogen (balloon) for 16 h. The catalyst was removed by filtration through a PTFE syringe filter to give a colourless solution, which was neutralised by addition of DIPEA (diisopropylethylamine, approx. 0.5 mL). The solvents were then removed by evaporation under reduced pressure. A ^XH NMR spectrum in D₂0 of the product at this stage showed no residual aromatic signals, indicating that hydrogenolysis was complete. The product was re-dissolved in deionised water (1 mL), excess DIPEA (1 mL) was added and the solution was heated under N₂ at 60 °C for 20 h. The solution was concentrated and the residue was re-dissolved in deionised water and lyophilised to give the crude diisopropylethylammonium salt of 2-0-(2-aminoethyl)-m o-inositol 1,3,4,5,6-pentakisphosphate as a fawn solid (325 mg);

*H NMR (D₂0, 400 MHz) δ 1.17-1.22 (75 H, m, DIPEA CH₃), 3.08 (10 H, q, J 7.4 Hz, DIPEA CH₂), 3.14 (2 H, t, J 5.1 Hz, OCH₂OY₂NH₂), 3.97 (2 H, t, J 5.1 Hz, OCH₂CH₂NH₂), 4.02-4.09 (3 H, m, H-l, H-3 and H-5), 4.13 (1 H, t, J 2.5 Hz, H-2), 4.39 (2 H, q, J 9.7 Hz, H-4 and H-6); ¹³C NMR (D₂0, 100 MHz) δ 12.11 (DIPEA CH₂CH₃), 16.22 (DIPEA CH(CH₃)₂), 17.69 (DIPEA CH(CH₃)₂), 39.52 (OCH₂CH₂NH₃ ⁺), 54.31 (DIPEA CH(CH₃)₂), 69.37 (OCH₂CH₂NH₃ ⁺), 73.89 and 76.13 (C-l, C-3, C-4 and C-6), 77.27 (C-5), 79.17 (C-2); ³¹P NMR (D₂0, 162 MHz) δ -0.39 (2 P, d, ³J_Hp 9.6 Hz), 0.62 (2 P, d, ³ _Hp 9.7 Hz), 0.79 (1 P, d, ³J_HP 9.7 Hz); Accurate mass calcd for C₈H₂₁N0₂₁P₅ ^", 621.9300; found 621.9311.

2-Q-(2-(5-fluoresceinylcarboxy)-aminoethyl)-myo-inositol 1,3,4,5,6- pentakisphosphate (2-FAM-InsP₅) To a suspension of 2-0-(2-aminoethyl)-m o-inositot 1,3,4,5,6- pentakisphosphate (20 mg of crude DIPEA salt) in dry propan-2-ol was added dry DIPEA (10 μΙ_). To the resulting clear solution was added solid 5-carboxyfluorescein NHS ester (13 mg, 28 μΐηοΐθ) followed by further dry DIPEA (60 μΙ_). The flask was covered in foil to exclude light and the reaction mixture was stirred under N₂ at 60 °C for 24 h, then allowed to cool and concentrated under reduced pressure. The residue was dissolved in TEAB (aqueous triethylammonium bicarbonate, 0.05 moldm^"3, pH approx. 7.5, 5 mL) and applied to a column of Q Sepharose Fast Flow resin (bicarbonate form, 70 mm χ 20 mm). The column was washed well with milliQ water followed by TEAB (0.8 moldm^"3, pH approx. 7.8) until the eluent ran colourless. This required approx. 400 mL of buffer. The column was then eluted with a gradient of TEAB (0.8 moldm^'3 to 2.0 moldm^"3) over 300 mL, collecting 10 mL fractions. A fluorescent product eluted at high buffer concentration (> 1.6 moldm^'3 TEAB). Fractions containing this product were combined and concentrated to give an orange solid, which was re-dissolved in TEAB (0.05 moldm^"3, pH approx. 7.5, 5 mL) and applied to a small column (100 mm x 10 mm) of Lichroprep RP-18. The column was eluted with a gradient of acetonitrile (0 to 30% in 0.05 moldm^"3 TEAB) over 300 mL, collecting 10 mL fractions. Fluorescent fractions were combined and concentrated, leaving a residue which was redissolved in milliQ water and lyophilised to give the pure triethylammonium salt of 2-FAM-InsP₅ as an orange solid (16 mg);

*H NMR (D₂0, 400 MHz) δ 1.11 [approx. 40 H, t, J 7.5 Hz, (Oy₃CH₂)₃NH⁺], 3.03 [approx. 27 H, q, J 7.5 Hz, (CH₃CW₂)₃NH⁺], 3.61 (2 H, t, J 5.1 Hz, OCH₂Oy₂NH), 3.97 (2 H, t, J 5.1 Hz, 0O7₂CH₂NH), 4.03-4.14 (3 H, m, H- 1, H-3 and H-5), 4.19 (1 H, broad s, H-2), 4.40 (2 H, q, J 9.4 Hz, H-4 and H-6), 6.64-6.68 (4 H, m, fluorescein H-2', H-4', H-5' and H-7'), 6.99 (2 H, d, J 9.7 Hz, fluorescein H-l' and H-8), 7.42 (1 H, d, J 8.2 Hz, fluorescein H-7), 8.13 (1 H, dd, J 8.2, 1.9 Hz, fluorescein H-6), 8.31 (1 H, d, J 1.9 Hz, fluorescein H-4); ³¹P NMR (D₂0, 109 MHz, ^-coupled) δ 1.20 (2 P, d, ³JHP 9.4 Hz), 1.89 (2 P, d, ³J_HP 9.4 Hz), 2.36 (1 P, d, ³J_HP 9.4 Hz); ¹³C NMR (D₂0, 100MHz) δ 8.14 [CH₃CH₂)₃NH⁺], 40.34 (OCH₂CH₂NH), 46.53 [CH₃CH₂)₃NH⁺], 71.68. 74.11, 76.29, 77.50, 78.63, 102.65, 112.68, 116.56, 126.62, 127.78, 130.98, 131.47, 132.49, 135.99, 144.64, 155.25, 165.26, 169.23 (C=0), 171.00 (C=0); Accurate mass calcd for C₂₉H₃i 0₂₇P₅ ^", 979.9777; found 979.9741.

The compounds of the formula (I) according to the present invention have the characteristic of preferentially accumulating in cancers rather than most normal cells and organs. Furthermore, many such cancers can be inhibited as well as detected. Accordingly, the photodynamic compounds of the formula (I), wherein a fluorophore moiety is present, are useful for the diagnosis and treatment of cancer, more especially certain cancers such as breast cancer, pancreatic cancer, prostate cancer, skin cancer, colorectal cancer and ovarian cancer. The present invention includes all stereoisomeric forms of the compounds of the formula (I) or formula (IA). Asymmetrical carbon atoms that are present in the compounds of formula (I) or (IA) all independently of one another have S configuration or R configuration. The invention includes all possible enantiomers and diastereomers and mixtures of two or more stereoisomers, for example mixtures of enantiomers and/or diastereomers, in all amounts and ratios. Thus, compounds according to the present invention which may exist as enantiomers may be present in enantiomerically pure form, both as levorotatory and as dextrorotatory antipodes, in the form of racemates and in the form of mixtures of the two enantiomers in all ratios. In the case of a cis/trans isomerism the invention includes both the cis form and the trans form as well as mixtures of these forms in all ratios. The preparation of individual stereoisomers may be carried out, if desired, by separation of a mixture by customary methods, for example by chromatography or crystallization, by the use of stereochemically uniform starting materials for the synthesis or by stereoselective synthesis. Optionally, a derivatization may be carried out before a separation of stereoisomers, The separation of a mixture of stereoisomers may be carried out at the stage of the compounds of the formula (I) or (IA) or at a stage of an intermediate during synthesis. The present invention also includes all tautomeric forms of the compounds of formula (I) or (IA).

Where the compounds according to formula (I), (II), (III) or (IA) contain one or more acidic or basic groups, the invention also comprises their corresponding physiologically or toxicologically acceptable salts. Salts of the compounds of the present invention may be derived from inorganic or organic acids and bases. Examples of suitable physiologically acceptable basic salts of the compounds of the present invention are ammonium salts, alkali metal salts (such as sodium salts and potassium salts) and alkaline earth metal salts (such as magnesium and calcium salts). Examples of preferred salts of the compounds of the present invention are the tri- or tetra-alkylammonium salts. Examples of suitable physiologically acceptable acid addition salts of the compounds of the present invention are salts of inorganic acids such as hydrochloric acid, sulfonic acid, phosphoric acid and also of organic acids such as acetic acid, benzenesulfonic acid, p-toluenesulfonic acid. Salts having a pharmaceutically unacceptable anion are likewise included within the scope of the present invention as useful intermediates for preparing or purifying pharmaceutically acceptable salts and/or for use in non therapeutic applications, for example in-vitro applications.

The respective salts of the compounds according to the formula (I), (II), (III) or (IA) may be obtained by customary methods which are known to the person skilled in the art like, for example by reacting these with an organic or inorganic acid or base in a solvent or dispersant, or by anion exchange or cation exchange with other salts.

The present invention furthermore includes all solvates of compounds of the formula (I), (II), (III) or (IA) for example hydrates or adducts with alcohols, active metabolites of the compounds of formula (I), (II), (III) or (IA), and also derivatives, which contain physiologically tolerable and cleavable groups, for example esters or amides. The physiologically functional derivatives of the compounds according to the invention include, for example, an ester which on administration to a mammal, for example, humans, is capable of forming (directly or indirectly) a compound of the formula (I), (II), (III) or (IA) or an active metabolite thereof. The physiologically functional derivatives also include prodrugs of the compounds of the invention. Such prodrugs may be administered orally or parentally and thereafter metabolised in the body to form compounds of the formula (I), (II), (III) or (IA) which are pharmacologically active. These prodrugs may or may not be active themselves and are also an aspect of the present invention.

The compounds of the invention may also be present in various polymorphous forms, for example as amorphous and crystalline polymorphous forms. All polymorphous forms of the compounds of the invention are included within the scope of the invention and are another aspect of the invention.

The compounds according to the present invention are prepared according to processes known in the art.

Particular compounds of the formula (I) may be prepared by reacting a compound of the formula (IV) :

wherein R¹ is substituted or unsubstituted aryl; X is O or NH; n is 0, 1 or 2; with 5-phenyltetrazole, then adding -D/^'s(cyanoethyl)(N, N- diisopropylamino)phosphine, followed by meta-chloroperoxybenzoic acid (mCPBA) to the reaction mixture, to provide a compound of the formula (V):

wherein

R¹, X and n are as defined above, and

R⁸, R⁹, R¹⁰, R¹¹ and R¹² are, independently, H or P(0)[0(CH2)₂CN]₂; wherein at least one of R⁸ to R¹² is P(0)[0(CH₂)₂CN]2_; converting the compound of the formula (V) into a compound formula (I):

wherein R¹, X and n are as defined and R² to R⁶ are independently selected from: H, P0₃ ^2', P(OR⁷)₃, PO(0 R⁷)₂, and PO(0 R⁷)0^" where R⁷ is as defined and wherein at least one of R² to R⁶ is P0₃ ^2", P(OR⁷)₃₍ PO(0 R⁷)₂, or PO(OR⁷)0^", by conventional processes, optionally converting the resultant compound into a physiologically tolerated salt, such as an ammonium, alkai or alkaline earth metal salt, and, optionally, purifying the resultant compound of the formula (I) or salt of the compound of the formula (I). The salt of the compound of the formula (I) formed by the process may be the compound of the formula (I) wherein one or more of R² to R⁶ are ΡΟ3Ύ and Y is tri- or tetra alkylammonium, an alkali metal, preferably sodium or calcium, or an alkaline earth metal, preferably magnesium or calcium,

In the process described, R¹ is preferably substituted or unsubstituted phenyl, X is preferably O and/or n is preferably the integer 1. A preferred salt of the compound of the formula (I) is the triethylammonium or hexasodium salt. In the above process, the compound of the formula (IV) is preferably reacted with 5-phenyltetrazole in dry dichloromethane under an atmosphere of argon. Preferably, the reaction mixture is cooled to around -40°C when mCPBA is added thereto.

All synthetic reactions for compounds of the formula (I) are known in principle to the skilled person and can accordingly be carried out under standard conditions (identical or with slight modifications) as described in the literature (see, for example, Organic Reactions, John Wiley & Sons, New York). Where necessary, the compounds of formula (I) can be purified by known workup methods, for example by recrystallization or chromatography. The starting materials for preparing compounds of formula (I) are either commercially available or they can be prepared by processes known from the literature. Compounds and intermediates prepared by the synthetic processes described above are a further aspect of the present invention. The present invention also relates to a compound of the general formula (I), (II) or (III) for use as a pharmaceutical or medicament.

The compounds of general formulae (I), (II) and (III) are inhibitors of one or more components of the PI3K/Akt pathway. The compounds of general formulae (I), (II) and (III) are kinase inhibitors and can therefore be employed for the treatment of diseases, which may result from an abnormal activity of kinases. As abnormal kinase activity, there may be mentioned, for example, that of PDK1 and the like. The compounds of general formulae (I), (II) and (III) are inhibitors of mTOR and can therefore also be employed for the treatment of diseases, which may result from an abnormal activity of mTOR. In particular, compounds according to the present invention can be used for the inhibition of the kinase PDK1 and/or mTOR. These effects are particularly relevant for the treatment of neoplastic diseases such as cancer.

Another aspect of the present invention is accordingly a method for inhibiting or modulating activity of PDK1 and/or mTOR in a mammal, comprising administering to the mammal a therapeutically effective amount of a compound of the formula (I), (II), or (III), or a salt thereof.

Examples of diseases, which can be treated with the compounds according to the present invention, include: neoplastic diseases, preferably cancer, in particular a solid tumour or leukaemia. The compounds according to the present invention are also potentially useful in the treatment of diseases such as diabetes, obesity and ageing.

Within the present invention a solid tumour is defined as a tumour, which does not affect the hematopoietic or lymphatic system. An example of a solid tumour is a breast tumour.

The compounds of the formula (I), (II), (III) or (IA), or their salts, according to the present invention are furthermore effective in the treatment of drug resistant cancers.

The compounds of the formula (I), (II), (III) or (IA) can be administered to animals and humans, preferably to mammals and humans, and in particular to humans. The compounds of the formula (I), (II), (III) or (IA) can be administered as pharmaceuticals by themselves, in mixtures with one another or in mixtures with other pharmaceuticals or in the form of pharmaceutical preparations. Another aspect of the present invention is therefore the use of the compounds of the formula (I), (II), (III), or (IA) for preparing one or more medicaments for prophylaxis and/or treatment of the above mentioned diseases and pharmaceutical preparations comprising an effective dose of at least one compound of the formula (I), (II), (III), or (IA) for prophylaxis and/or treatment of the above mentioned diseases.

The amount of a compound according to formula (I), (II), (III) or (IA) which is required in order to attain the desired biological effect depends on a number of factors, for example the specific compound selected, the intended use, the type of administration and the clinical state of the patient. In general, the daily dose is in the range from 0.3mg to lOOmg, typically from 3mg to 50mg, per day per kilogram of body weight, for example 3 to 10 mg/kg/day. An intravenous dose can be, for example, in the range from 0.3 mg to 1.0 mg/kg and can be administered in a suitable manner as an infusion of lOng to lOOng per kilogram per minute. The compound used for the prophylaxis or therapy of the above mentioned conditions may be the compounds according to formula (I), (II), (III), or (IA) themselves, but they are preferably present in the form of a pharmaceutical composition together with an acceptable carrier. The carrier must be naturally acceptable in the sense that it is compatible with the other ingredients of the composition and is not harmful to the patient's health (physiologically tolerable). The carrier may be solid or liquid or both and is preferably formulated with the compound as an individual dose, for example as a tablet which may contain from 0.05% to 95% by weight of the active compound. Further pharmaceutically active substances may also be present, including further compounds according to formula (I) or (IA). The further pharmaceutically active substances may be one or more known anti-cancer agents. The pharmaceutical compositions can be included in a container, pack or dispenser, together with instructions for administration. The pharmaceutical compositions of the invention may be prepared according to any of the known pharmaceutical methods which essentially comprise mixing the ingredients with pharmacologically acceptable carriers and/or excipients.

In addition to at least one compound according to formula (I), (II), (III) or (IA) and one or more carriers, the pharmaceutical preparations according to the invention can also contain additives, for example fillers, binders, lubricants, buffer substances, wetting agents, flavourings, colourants, solvents, solubilizers, agents for achieving a depot effect, salts for altering the osmotic pressure, etc.

The pharmaceutical compositions of the invention may be provided in various forms for different modes of administration. Suitable forms for administration include, but are not limited to, a pill, tablet, granule, capsule, aqueous solution, oily solution, syrup, emulsion, suspension, suppository, pastille, solution for injection or infusion, ointment, tincture, cream, lotion, powder, spray, aerosol, transdermal therapeutic systems, microcapsule, implant, rod or patch.

Pharmaceutical compositions of the invention are those which are suitable for administration by a wide variety of routes. Suitable routes of administration include, but are not limited to, oral, rectal, topical, peroral (e.g., sublingual) and parenteral (e.g., subcutaneous, intramuscular, intradermal or intravenous) administration, although the most suitable manner of administration depends in each individual case on the nature and severity of the condition to be treated and on the nature of the compound according to the formula (I) or (IA) used in each case. Delayed-release formulations, too, are included within the scope of the invention.

Another aspect of the present invention is the combination of compounds of the formula (I), (II), (III) or (IA), or their salts, with other pharmaceutically active substances not covered by formula (I), (II), (III) or (IA). The compounds of the present invention may be administered alone or in combination with other therapeutic agents. In anti-cancer therapy, the compounds of the present invention may be combined with other chemotherapeutic, hormonal or antibody agents as well as combined with surgical therapy and radiotherapy. Non-limiting anti-cancer agents useful in combination with the compounds of the formula (I), (II), (III) or (IA) include the following :

-cell cycle specifc anti-neoplastic agents including, but not limited to, diterpenoids such as paclitaxel and its analog docetaxel; vinca alkaloids such as vinblastine, vincristine; epipodophyllotoxins such as etoposide and teniposide; fluoropyrimidines such as 5-flurouracil and flurordeoxyuridine; antimetabolites such as allopurinol, methotrexate, mercatopurine; and campotothecins such as 9-amino camptothecin;

-cytotoxic chemotherapeutic agents including, but not limited to, alkylating agents such as cyclophosphamide, hexamethylmelamine, melphalan, chlorambucil; anti-tumour antibiotics such as bleomycin, mitomycin, dactinomycin, epirubicin; and platinum coordination complexes such as cisplatin, carboplatin;

-other chemotherapeutic agents including, but not limited to, anti- estrogens such as tamoxifen, toremifene, raloxifene, droloxifene and iodoxyfene; progestrogens such as megestrol acetate; aromatase inhibitors such as anastrozole, letrazole; antiandrogens such as flutamide, niiutamide; LHRH agonists and antagonists such as goserelin acetate and luprolide, testosterone 5,alpha-dihyroreductase inhibitors such as finasteride; metalloproteinase inhibitors such as marimastat; antiprogesterones; urokinase plasminogen activator receptor function inhibitors; cyclooxygenase type 2 (COX-2) inhibitors such as celecoxib; and tyrosine kinase inhibitors such as cyciin dependent inhibitors such as CDK2 and CDK4 inhibitors; and natural chemotherapeutic agents such as curcumin and rapamycin. It is also possible to combine a radiation treatment with the compounds of the present invention.

The compounds of the formula (I) or (IA) and the other pharmaceutically active substance not covered by formula (I) or (IA), or the radiation treatment, may be administered together or separately and, when administered separately this may occur simultaneously and/or sequentially in any order. The relative timings of the administration will be selected in order to achieve the desired combined therapeutic effect.

One aspect of the invention includes a combination of one or more compounds of formula (I), (II), (III) or (IA) and one or more other anticancer therapies that provide a synergistic therapeutic effect with respect to treating cancer cells. ^{^}Synergistic' indicates that the therapeutic effect is greater than would have been expected based on adding the effects of each therapy applied as a monotherapy. The combination may additionally or alternatively be used to restore the effectiveness of certain existing cancer chemotherapies and radiation.

The compounds of the formula (I), (II), (III) and (IA) show selectivity for, or greater potency towards, certain types of cancer cells. These cells are selected from: breast cancer cells, pancreatic cancer cells, prostate cancer cells, skin cancer cells, colorectal cancer cells and ovarian cancer cells.

Further, the compounds of the formula (I), (II), (III) and (IA) are effective in the treatment of a drug resistant disease, in particular a cancer. It will be appreciated that a drug resistant cancer is a cancer that has shown resistance against one or more known anti-cancer drugs. The resistance shown may be total or partial.

The following examples illustrate the invention without limitation.

EXAMPLE

Synthesis of 2-0-Bn-InsP₅ 2-O-Benzyl myoinositol was synthesised as previously reported by Riley et al, (A. M. Riley, M. Trusselle, P. Kuad, M. Borkovec, J. Cho, J. H. Choi, X. Qian, S. B. Shears, B. Spiess and B. V. L Potter; ChemBioChem; 2006, 7, 1114-1122) from 4,6-di-0-(p-methoxybenzyl) protected orthoformate after benzylation of the free axial hydroxyl group followed by the acid cleavage of both the orthoformate ester and the p-methoxybenzyl ethers with HCI in refluxing ethanol.

To a solution of 2-O-benzyl myo-inositol (500 mg, 1.85 mmol) and 5- phenyltetrazole (2.70 g, 18.50 mmol) in dry dichloromethane (DCM) (10 mL) under an atmosphere of argon, was added b/^'s(cyanoethyl)(N,N- diisopropylamino)phosphine (3.70 g, 13.87 mmol). Stirring was continued for 2 h at room temperature. The reaction mixture was cooled to -40 °C and meta-chloroperoxybenzoic acid (mCPBA) (4.15 g, 18.50 mmol) was added portion-wise while stirring. The cooling bath was removed and the mixture was allowed to reach room temperature and diluted DCM (100 ml), washed with 10 % sodium sulphite solution (2 x 200 ml), dried and solvent evaporated in vacuo to afford the crude 2-O-benzyl 1,3,4,5,6-pentakis-O- [6/s(cyanoethyloxy)phosphoryl] myo inositol. Without further purification crude 2-O-benzyl 1,3,4,5, 6-pentakis-0-[/}/s(cyanoethyloxy)phosphoryl] myo inositol was then dissolved in concentrated aqueous ammonia solution (30 ml) and heated at 60 °C overnight in a Pyrex pressure tube. After evaporation of solution under vacuum, the residue was purified by ion exchange chromatography on Q-Sepharose Fast Flow resin to afford the pure triethylammonium salt of 2-O-benzyl myo inositol 1,3,4,5,6- penta a^'sphosphate as a hygroscopic white solid, ³¹P NMR (109.4 MHz, H- decoupled, D₂0) δ 0.19 (2P, s), 0.90 (2P, s), 1,44 (IP, s); *H NMR (270 MHz, D₂0) δ 4.08-4.19 (3H, m, Ins-H), 4.37-4.47 (3H, m, Ins-H), 4.83 (2H, s, CH₂Ph), 7.24- 7.37 (3H, m, Ar-H), 7.45-7.48 (2H, m, Ar-H); HRMS calcd for C₁₃H₂₂0₂iP₅ ([M - H]-) 668.9342, found 668.9347. For the in vivo experiments (below), 2-O-benzyl myo inositol 1,3,4,5,6- pentakisphosphate was converted into the hexasodium salt by treatment with Dowex 50WX2-100 ionexchange resin, followed by the addition of sodium hydroxide (6-equivalents) and lyophilisation.

MATERIALS AND METHODS Materials

InsP₅ was synthesized as reported in Godage et al, Chem Commun 28: 2989-2991 (2006). 2-0-Bn-InsP₅ was synthesized in a similar manner from 2-O-benzyl-myo-inositol. Each compound was purified to homogeneity by ion-exchange chromatography on Q-Sepharose Fast Flow resin and used as the triethylammonium salt, which was fully characterized by ³¹P and H spectroscopy and accurately quantified by total phosphate assay. For the in vivo experiments, InsP₅ and 2-O-Bn- InsP₅ were each converted into the hexasodium salt by treatment with Dowex 50WX2-100 ion-exchange resin, followed by addition of sodium hydroxide (6 equivalents) and lyophilization. Sulforhodamine (SRB), curcumin, paclitaxel and 4-hydroxy-tamoxifen were purchased from Sigma; anti-phospho Ser473 Akt, anti-phospho Thr308 Akt and anti-Akt from Cell Signaling or Santa Cruz Biotechnology.

Cell Lines Human pancreatic cancer cell lines (BxPc-3, Aspcl), human mammary cancer cell lines (MDA-MB-468/435/231, SKB 3, MCF7), mouse mammary cancer cell lines (TSA and 4T1), human prostate cancer cell line (PC3) and human ovarian cancer cell line (SKOV-3) were used. SKOV-3 and PC3 were cultured in RPMI 1640; all other cell lines were cultured in DMEM. Media were supplemented with 10% FBS, penicillin/streptomycin and glutamine.

Cell survival and apoptosis assays

Cells seeded in a 24-well plate were treated with the indicated compounds in serum free DMEM or DMEM containing 0.5% FBS (PC3). After 72 h, the number of surviving cells was assessed by manual cell counting or by using the cell counter CDA-500 (Sysmex, UK). Alternatively, after 48 h, the number of apoptotic cells was assessed by acridine orange/ethidium bromide assay as described in Piccolo er al, Oncogene 23: 1754-1765 (2004) and Maffucci er al, Cancer Res 65: 8339-8349 (2005). SRB test was performed in SKOV-3 and PC3 seeded in 96-well plate (3800 cells/well or 5800 cells/well respectively) after 72 h of treatment as described in Cappella er al, Int J Cancer 93: 401-408 (2001). In vivo studies

Male nude athymic CD-I nu/nu mice (8-weeks old) were obtained from Harlan (Italy) and maintained under specific pathogen-free conditions with food and water provided ad libitum. The general health status of the animals was monitored daily. Procedure involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with national and international laws and policies.

Toxicity assay

Male nude CD-I mice were treated with a single dose of 750 mg/Kg InsP₅ or 2-0-Bn-InsP₅/mouse administered intraperitoneally (i.p.). Each group consisted of 2-3 mice. Body weight, deaths and any other sign of toxicity and changes in behaviour (such as motility, eating and drinking habits) were recorded.

Anti-tumour activity assay Exponentially growing PC3 cells were harvested, washed twice and resuspended in PBS at a concentration of 2.5 x 10⁷ cells/ml. A suspension of 5 x 10⁶ PC3 cells was injected subcutaneously (s.c.) into the left flank of the recipient mice. When tumours reached a size of ~70 mm³ (approximately 15 days after tumour cell implant), mice were divided in seven groups (n=7). InsP₅ and 2-0-Bn-InsP₅ were administered by daily i.p. injections at different doses of 12.5-25-50 mg/Kg/day for 14 consecutive days. Control mice were treated with water in an equal volume. The diameters of s.c. growing tumours were measured with a caliper twice a week and the experiment was ended at day 28 after implant.

Data analysis and in vivo tumour parameters

The volume of s.c. growing tumours was calculated by the formula : Tumour weight (mg) = (length x width²)/2. Differences in s.c tumour growth between the treatment groups were evaluated with a one-way ANOVA followed by Fisher's test using the StatView statistical package (SAS Institute Inc.). The percentage of tumour growth was calculated as T/C% = (RTV treated animals/RTV control animals) X 100 where RTV was the mean relative tumour volume calculated as RTV = Vt/V₀. V_t was the tumour volume at the day of measurement and V₀ was the tumour volume at the beginning of the treatment. The percentage of tumour weight inhibition (TWI%) was calculated using the formula : TWI% = 100 - T/C%. The log cell kill (LCK) was calculated using the formula : LCK = T-C/3.32 X Td, where T-C is the tumour growth delay calculated as the difference in median time (in days) required for the tumours in the treatment (T) and control group (C) to reach a predetermined size (i.e. 1000 mg). Td is the tumour volume doubling time in days, determined in the exponential growth phase of the control group from a best-fit straight line. Median doubling time was three days in control animals.

Western Blot Mice with s.c. growing tumours were treated with a single dose of InsP₅ and 2-0-Bn-InsP₅ (50 mg/Kg) or vehicle. Animals were sacrificed 24 hours after treatment and tumour samples were collected and snap frozen. Frozen specimens of tumour tissue were homogenized with a Polytron homogenizer in a lysis buffer (ratio 1 : 1 w/v) containing 50 mM Tris-HCI (pH 7.4), 5 mM EDTA, 0.1% Nonidet NP-40, 250 mM NaCI, 50 mM NaF and proteases and phosphatase inhibitors. After centrifugation at 13000 rpm for 10 min at 4°C, eighty g of protein were separated on SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, MA). Membranes were probed with the indicated antibodies. Protein kinase profiling

The effect of the indicated compounds on the activity of various kinases was assessed by SelectScreen™ Kinase Profiling Service (Invitrogen-Life Technologies). Assays were performed using 1 μΜ of the tested compounds and ATP concentration as indicated in the corresponding tables. In the case of InsP₅ a screen using 10 μΜ of the compound was also performed, as indicated in the corresponding table.

RESULTS

Synthesis of novel potential inhibitors of the PI3K/Akt pathway and in vitro screening We observed that the InsP₅ derivative 2-O-benzyl-myo-inositol 1,3,4,5,6- pentakisphosphate (Figure 1A, named 2-0-Bn-InsP₅) showed a high efficiency in inhibiting Akt activation in all cell lines tested. More specifically, we found that 2-0-Bn-InsP₅ inhibited Akt phosphorylation at its residue Ser473 more efficiently than InsP₅ in SKOV-3, being already active after 8 h of treatment and at a concentration of 20 μΜ (Figure IB). Inhibition of Akt phosphorylation at residue Thr308 was also detected (Figure IB). We also found that 2-0-Bn-InsP₅ was able to block Akt phosphorylation in cell lines resistant to InsP₅ such as prostate cancer cells PC3 (Figure 1C) and pancreatic cancer cells ASPC1 (results not shown). Taken together these data indicate that structural modification at the C-2 of InsP₅ can enhance its inhibitory properties towards Akt activation.

Figure 1 (A) Structure of inositol 1, 3,4,5, 6-pentakisphosphate (InsP₅) and 2-0- benzyl-myo-inositol 1,3,4,5, 6-pentakisphosphate (2-0-Bn-InsP₅). (B, C) SKOV-3 were treated for 8h or 24h with the indicated concentrations of InsP₅ or 2-0-Bn-InsP₅ in serum free medium, (B) while prostate cancer PC3 cells were treated for 24h with the indicated concentrations of InsP₅ or 2-0-Bn-InsP₅ in medium containing 0.5% FBS (C). Akt activation was assessed by monitoring phosphorylation at its residues Ser473 and Thr308. Membranes were then stripped and re-probed with the indicated antibodies.

Analysis of 2-0-Bn-InsP₅ biological activity We compared the effects of 2-0-Bn-InsP₅ and InsP₅ on proliferation/survival of cancer cells in vitro. Treatment with 2-0-Bn-InsP₅ strongly reduced the number of surviving SKBR3 (Figure 2A) and SKOV-3 (Figure 2B) assessed by cell counting. In particular 2-0-Bn-InsP₅ was more active than InsP₅ in both cell lines. Acridine orange/ethidium bromide assay confirmed that the percentage of apoptotic cells was higher in 2-0-Bn-InsP₅-treated compared to InsP₅-treated SKBR3 (Figure 2C) and SKOV-3 (Figure 2D).

Figure 2 2-O-benzyl-m/o-inositol 1, 3,4,5, 6-pentakisphosphate (2-0-Bn-InsP₅) possesses higher pro-apoptotic activity than inositol 1,3,4,5,6- pentakisphosphate (InsP₅). (A, B) SKBR3 (A) and SKOV-3 (B) were treated for 72h with the indicated concentrations of InsP₅ or 2-O-Bn- InsP₅. The number of surviving cells was assessed by cell counting. Data are mean +s.e. of n = 4 (A) and n = 2 (B) independent experiments. ** = <0.05. (C, D) SKBR3 (C) and SKOV-3 (D) were treated with the indicated concentrations of InsP₅ or 2-OBn-InsP₅. The number of apoptotic cells was assessed by acridine orange/ethidium bromide assay. Data are mean +s.e. of three independent experiments. ** = P<0.05.

Based on data on Akt phosphorylation, we then analysed the effect of 2-0-Bn-InsP₅ on survival of cell lines normally very resistant to InsP₅ treatment. 2-0-Bn-InsP₅ was more potent than InsP₅ at a concentration of 50 μΜ in pancreatic cancer cells BxPc-3 (Figure 3A) while it was more active than InsP₅ at almost all concentrations tested in pancreatic cancer cells ASPC1 (Figure 3B). A stronger activity of 2-0-Bn-InsP₅ compared to InsP₅ was also observed in breast cancer cells MDA-MB-468 (Figure 3C) and in PC3 (Figure 3D), consistent with data on Akt phosphorylation. Higher activity of 2-0-Bn-InsP₅ in PC3 cells was also observed in SRB assays (Figure 3E). Further, while InsP₅ had no effect in PC3 at concentrations up to 50 μΜ, concentrations of 200-300 μΜ were eventually able to mimic the effect of 2-0-Bn-InsP₅ in PC3 cells (Figure 3F), suggesting that 2-0-Bn-InsP₅ is acting on the same intracellular pathway as InsP_s. Taken together these data demonstrate that addition of a benzyl group to the axial 2-0 atom of InsP₅ potentiates the pro- apoptotic properties of the compound not only in cells sensitive to InsP₅ but also in cells normally very resistant to treatment with the parent inositol compound.

Figure 3 2-O-benzyl-m/o-inositol 1,3,4,5,6-pentakisphosphate (2-0-Bn-InsP₅) possesses pro-apoptotic activity in cell lines resistant to inositol 1,3,4,5,6- pentakisphosphate (InsP₅). (A-D) BxPc-3 (A), ASPCI (B), MDA-MB-468 (C) and PC3 (D) were treated for 72h with the indicated concentrations of InsP₅ or 2-OBn-InsPs. The number of surviving cells was assessed by cell counting. Data are mean +s.e. of n = 3 (A), n = 6 (B), n = 3 (C) and n = 4 (D) independent experiments carried out in duplicate. *= <0.01; **=P<0.05. (E, F) PC3 were treated with the indicated concentrations of InsP₅ or 2-OBn-InsP₅ (E) or increasing concentrations of InsP₅ (F). After 72h the number of surviving cells was assessed by SRB assay. Data are mean +s.e. of n = 2 independent experiments. In vivo anti-tumour activity of 2-0-Bn-InsP₅ on InsP₅-resistant xenografts

We tested the therapeutic efficacy of 2-0-Bn-InsP₅ in human tumour xenografts characterised by activation of the PI3K/Akt pathway and higher sensitivity to 2-0-Bn-InsP₅ compared to InsP₅. Specifically we implanted PC3 cells in nude mice and 15 days after the implant we treated groups of mice with different concentrations (12.5, 25 and 50 mg/Kg) of InsP₅ or 2- 0-Bn-InsP₅ for 14 consecutive days (from day 15 to day 28). Tumour growth was followed for further 12 days after the end of the treatment (up to day 40). Data revealed that 2-0-Bn-InsP₅ at doses of 12.5 and 25 mg/Kg clearly decreased tumours growth compared to untreated mice, although the differences were statistically significant only at the last day of measurement (Figure 4A and C). A strong reduction in tumour growth was obtained in the group treated with 50 mg/Kg 2-0-Bn-InsP₅, with a statistically significant difference vs controls detectable from day 22 after tumour cells implant onwards (Figure 4A and C). Data on in vivo anti- tumour activity parameters relative to 2-O-Bn-InsPs are shown in Figure 4C, bottom table. More than 50% inhibition of tumour weight was achieved in the 50 mg/Kg-treated group, with a tumour growth delay (T- C) of almost 9 days between this group and the untreated (control) group. In agreement with our in vitro data, we observed that InsP₅ had no effect at concentrations up to 50 mg/Kg (Figure 4B). At the end of the experiment, Western blot analysis revealed that 24 h-treatment with 2-0- Bn-InsP₅ markedly reduced Akt phosphorylation at its residue Ser473 in all 2-0-Bn-InsP₅-treated mice (Figure 4D). Furthermore a clear inhibition of Akt phosphorylation at its residue Thr308 was detected in 5 out of 7 2- 0-Bn-InsP₅-treated mice (Figure 4D). It is noteworthy that no evidence of toxicity was observed in the different groups of mice at all the tested doses of either 2-0-Bn-InsP₅ or InsP₅ and the body weight of the treated animals was not different from the untreated mice throughout the entire experiment. Moreover, a single treatment with a very high dose of 2-0- Bn-InsP₅ or InsP₅ (750 mg/Kg) did not cause any major toxic effect. Taken together these data demonstrate that 2-0-Bn-InsP₅ is able to inhibit growth of InsP₅-resistant tumours through a more efficient blockade of Akt phosphorylation in vivo.

Figure 4

2-O-benzyl-myo-inositol 1, 3,4,5, 6-penta/a^'sphosphate (2-0-Bn-InsP₅) possesses anti-tumour activity on inositol 1,3,4,5,6-penta/a^'sphosphate (InsP₅)-resistant xenografts and it is not associated with toxicity in vivo. Male athymic CD-I nu/nu mice were inoculated subcutaneously (s.c.) with PC3 and treated with the indicated concentrations of either InsP₅ or 2-O-Bn-InsPs from day 15 after cell implantation. The inositol compounds (12.5-25-50mgkg^"1day^"1) and vehicle (water) were given daily by intraperitoneal (i.p.) injections for 14 consecutive days (days 15-28). Tumour size was assessed twice weekly. (A, B) Tumour growth in 2-OBn-InsP₅-treated mice (A) and InsP₅-treated mice (B) compared with control group measured for the duration of the experiment. Results are expressed as mean +s.e. (C) Top: Table showing all -values for the indicated doses of 2-0-Bn-InsP₅ compared to controls at the indicated days of treatment (NS = not significant). Bottom : In vivo anti-tumour activity parameters. The percentage of tumour weight inhibition (TWI%), the tumour growth delay (T-C) and the log cell kill (LCK) were calculated as described in the MATERIALS AND METHODS section. The highest inhibition of tumour volume is reported. (D) Mice with s.c. growing tumours were treated with a single dose (SOmgkg ¹) of InsP₅, 2-O-Bn- InsP₅ or water (control). Tumours were excised 24h after treatment. Phosphorylation of Akt at its residues Ser473 and Thr308 was assessed by using specific antibodies. Membranes were then stripped and re-probed with an anti-Akt antibody. In vitro kinase profiling of InsP₅ and 2-O-Bn- InsP₅

To determine the mechanism responsible for the higher activity of 2-0- Bn-InsP₅ we performed a protein kinase activity screen for InsP₅ and 2-0- Bn-InsP₅ (SelectScreen™ Kinase Profiling Service, Invitrogen-Life Technologies). Among almost 60 protein kinases screened, 2-0-Bn-InsP₅ (1 μΜ) showed a very high inhibitory activity towards PDKl (79% inhibition) and a lower activity towards mTOR. 2-0-Bn-InsP₅ did not inhibit (percentage of inhibition less than 40%) any of all the other tested kinases, including AGC kinases such as GSK3, RSK, S6K and members of the PKC family, AMPK and several members of the MAPK family. Furthermore 2-O-Bn- InsP₅ did not directly inhibit any of the class I PI3K isoforms tested or any Akt isoforms. InsP₅ showed a reduced inhibitory effect on PDKl compared to 2-0-Bn-InsP₅ . As 2-0-Bn-InsP₅, when tested on a panel of over 50 kinases and at a concentration of 10 μΜ, InsP₅ did not significantly inhibit any of the tested kinases including Akt isoforms . In contrast to 2-O-Bn- InsP₅, InsP₅ did not inhibit mTOR, even when tested at a concentration of 10 μΜ. Comparing the effect of different natural inositol polyphosphates on PDKl, InsP₅ possessed the highest inhibitory activity towards PDKl (71% inhibition) with only Ins(l,3,4,5)P₄ also showing some effect (56% inhibition). None of the other polyphosphates had significant effect . These results indicate that 2-0-Bn-InsP₅ and InsP₅ inhibit PDKl very specifically, with 2-O-Bn-InsPs possessing the highest inhibitory activity towards PDKl. Indeed, results from SelectScreen™ Kinase Profiling Service (Invitrogen-Life Technologies)-10-point Titration revealed that the IC₅₀ of InsP₅ towards PDKl was 613 nM whereas the corresponding IC₅₀ of 2-0- Bn-InsP₅ was a striking 26.5 nM . These results clearly indicate that 2-0- Bn-InsP₅ is a potent and highly selective PDK1 inhibitor. This is consistent with the detected inhibition of Thr308 phosphorylation in 2-0-Bn-InsP₅- treated SKOV-3 and PC3 cells (Figure IB and 1C) and 2-0-Bn-InsP₅- treated mice (Figure 4D). Furthermore, 2-0-Bn-InsP₅ but not InsP₅ is able to inhibit mTOR selectively in vitro with an IC₅₀ of 1.3 μΜ .

In vitro effects of 2-0-Bn-InsP₅ in combination with anti-cancer compounds

Parallel RNAi and compound screens have recently revealed that PDK1 is a critical determinant of sensitivity to tamoxifen in breast cancer cells MCF7 (Iorns et al, Biochem J 417:316-370 (2009)). Based on this result we investigated whether inhibition of PDK1 by 2-0-Bn-InsP₅ was able to sensitize MCF7 to the pro-apoptotic effect of tamoxifen. Our data revealed that treatment with 4-OH tamoxifen (the active metabolite of tamoxifen) for 72h reduced the number of surviving cells, whereas 2-0-Bn-InsP₅ reduced the number of surviving cells to a lesser extent(Figure 5A). However, the combination of 2-0-Bn-InsP₅ and 4-OH tamoxifen strongly enhanced the effect of 4-OH tamoxifen or 2-0-Bn-InsP₅ alone (Figure 5A). We then tested the effects of 2-0-Bn-InsP_s in combination with several natural anti-cancer compounds. The concentrations of the different compounds used in these experiments were the minimally effective based on preliminary dose-response experiments (results not shown). A combination of 2-0-Bn-InsP₅ and curcumin, a component of turmeric (Curcuma longa), strongly reduced the number of surviving PC3 cells, resulting in a more than additive effect (Figure 5B) and was able to enhance the effect of curcumin in ASPCl (Figure 5C) and in MDA-MB-468 (Figure 5D). A combination of 2-0-Bn-InsP₅ and paclitaxel clearly reduced the number of surviving MDA-MB-468 (Figure 5D), SKOV-3 (Figure 5E) and PC3 (Figure 5F) cells compared to the corresponding single treatments. An additive effect was detected when combining 2-0-Bn-InsP₅ with rapamycin in SKOV-3 (Figure 5E) and PC3 (Figure 5F) cells. These data clearly indicate that the combination of 2-0-Bn-InsP₅ and natural anti-cancer compounds results in additive or more than additive (synergistic) effects.lt is reasonable to assume that 2-0-Bn-InsP₅ can be used in combination with other anti-cancer compounds to increase their anti-cancer activity.

Figure 5

Combination of 2-O-benzyl-m o-inositol 1,3,4,5,6-pentakisphosphate (2- 0-Bn-InsP₅) with anti-cancer compounds in vitro results in additive or more than additive effects. (A) MCF7 were treated with 20nM 4-OH Tamoxifen, 50μΜ 2-0-Bn-InsP₅ alone or in combination. Data are mean +s.e. of n=4 independent experiments carried out in duplicate. 4-OH Tamoxifen + 2-0-Bn-InsP₅: <0.01 vs 4-OH Tamoxifen; <0.01 vs 2-0-Bn-InsP₅. (B) PC3 were treated with 5μΜ 2-0-Bn-InsP₅, ΙΟμΜ curcumin alone or in combination. Data are mean +s.e. of n=5 independent experiments carried out in duplicate. Curcumin + 2-O-Bn- InsPs.'/^O.Ol vs 2-0-Bn-InsP₅; <0.05 vs curcumin. (C) ASPCI were treated with 5μΜ 2-0-Bn-InsP₅, ΙΟμΜ curcumin alone or in combination. Data are mean +s.e. of n=6 independent experiments carried out in duplicate. Curcumin + 2-O-Bn-InsP₅:P<0.05 vs 2-0-Bn-InsP₅; P<0.05 vs curcumin. (D) MDA-MB-468 were treated with ΙΟμΜ 2-0-Bn-InsP₅, ΙΟμΜ curcumin alone, InM paclitaxel or the indicated combination. Data are mean +s.e. of n=2 independent experiments carried out in duplicate. In all cases (A-D), cells were treated for 72h and the number of surviving cells was assessed by cell counting. (E) SKOV-3 were treated with 20μΜ 2-0-Bn-InsP₅, 30nM paclitaxel, 20nM rapamycin or with the indicated combinations. (F) PC3 were treated with ΙΟμΜ 2-0-Bn-InsP₅, InM rapamycin alone or in combination and with 20μΜ 2-0-Bn-InsP₅, 40nM paclitaxel alone or in combination. In all cases (E, F), cells were treated for 72h and the number of surviving cells was assessed by SRB assay and data are mean +s.e. of two experiments carried out in quadruplicate. Inhibition of PDK1 by 2-0-Bn-InsP₅ inhibits breast cancer cell migration and invasion.

It has been reported that PDK1 is critical for cancer cell migration. Consistent with this, we observed that downregulation of this enzyme using specific siRIMA inhibits migration (data not shown) and invasion of breast cancer cells MDA- MB-231 and MDA-MB-435 on Matrigel, confirming the key role of this enzyme in our experimental models. We therefore decided to investigate the effect of the PDK1 inhibitor 2-0-Bn-InsP₅ on breast cancer cells migration and invasion. InsP_5/ which can also inhibit PDK1, albeit with less potency, was also used in these experiments. InsP₅ and 2-0-Bn-InsP₅ inhibited fibronectin-induced cell migration of MDA-MB-231 (Figure 6A) and invasion on Matrigel of human breast cancer cell lines MDA-MB-231, MDA-MB-435 and mouse mammary tumour cell line TSA and 4T1 (Figure 6B).

Identification of the mechanism regulating InsP₅ and 2-0-Bn-InsP₅ uptake.

We investigated intracellular uptake of fluorescein alone and a fluorescein- conjugated InsP₅ [2-FAM-InsP₅] in various normal and cancer cell lines. Figure 7 shows intracellular uptake of a fluorescein-conjugated InsP₅ [2-FAM- InsP₅] in different cancer cell lines. The results show that uptake is different in pancreatic cancer cells PANCl and ASPCl compared to ovarian cancer cells SKOV-3. No uptake of fluorescein alone was detected in these cells at any of the time course investigated. It was found that 2-FAM-InsP₅ not only localizes at the plasma membrane and within the cytosol but it also appears to accumulate at the nuclear membrane and in intra-nudear structures. A similar incorporation of 2-FAM- InsPs but not fluorescein alone was detected in mammary cancer cells MDA-MB-231.

Further investigations revealed that inositol polyphosphates are incorporated in mammalian cells through the action of ABC transporters. Our tests reveal a specific role for the transporter ABCC1 in InsP₅ uptake in breast cancer cells. Thus, ABC transporters, known for their role in the development of multidrug resistance by causing increased excretion of the drug from the cell, may regulate uptake of inositol polyphosphates. We also found that increasing the uptake of InsP₅ (for instance by synthesising a membrane permeant analogue of the compound) sensitises cells normally resistant to this compound. Based on these results and on the observation that several cancer cells show upregulation of ABC transporters, it is possible that these cells may also be more sensitive to InsP₅/2- 0-Bn-InsP₅ action because of a higher incorporation.

It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the scope of the present invention and without diminishing its attendant advantages. It is therefore intended that such changes and modifications are covered by the appended claims.

Claims

1. A compound of the formula (I):

wherein R¹ is substituted or unsubstituted aryl;

R², R³, R⁴, R⁵ and R⁶ are, independently from each other, selected from: H, P0₃ ^2", P(OR⁷)₃, PO(0 R⁷)₂, PO(0 R⁷)Cr, -NHalkyl, -alkylene-NH₂ , NH₂, S0₂0- , S0₂OR⁷, S(0)R⁷, S0₂R⁷, C(0)R⁷, C(0)OR⁷, OC(0)R⁷, and alkyl;

R⁷ is H, alkyl, alkenyl or alkynyl; X is O or NH; n is 0, 1 or 2; or a physiologically tolerated salt thereof.

2. A compound according to claim 1 of the formula (II) :

wherein R¹ to R⁶ , X and n are as defined in claim 1, or a physiologically tolerated salt thereof.

3. A compound according to claim 1 of the formula (III):

wherein R^o R⁶ , X and n are as defined in claim 1, or a physiologically tolerated salt thereof.

4. A compound of the formula (I), (II) or (III), or a physiologically tolerated salt thereof, according to any one of claims 1 to 3, wherein R¹ is substituted or unsubstituted phenyl.

5. A compound of the formula (I), (II) or (III), or a physiologically tolerated salt thereof, according to any one of the preceding claims, wherein X is O.

6. A compound of the formula (I), (II) or (III), or a physiologically tolerated salt thereof, according to any one of the preceding claims, wherein any one or more of R², R³, R⁴, R⁵ and R⁶ is P0₃ ²\

7. A compound of the formula (I), (II) or (III), or a physiologically tolerated salt thereof, according to any one of the preceding claims, wherein n is 1.

8. A compound according to any one of the preceding claims, wherein the compound is selected from: 2-O-benzyl-m/o-inositol 1, 3, 4, 5, 6- pentakisphosphate and 2-0-benzyl-D-cft/^'ro-inos.itol 1, 3, 4, 5, 6- pentakisphosphate, or a physiologically tolerated salt thereof.

9. A pharmaceutical composition, comprising an effective amount of at least one compound of the formula (I), (II) or (III) according to any one of the preceding claims or a physiologically tolerated salt thereof and a physiologically tolerated carrier.

10. A composition according to claim 9, further comprising another therapeutic agent, optionally wherein the other therapeutic agent is an anti-cancer agent.

11. A compound of the formula (I), (II) or (III), or a physiologically tolerated salt thereof, according to any one of the preceding claims 1 to 8 for use in therapy.

12. A compound according to claim 11 for use in the treatment of cancer, optionally wherein the cancer is a drug resistant cancer.

13. A compound according to claim 12, wherein the cancer is selected from: breast cancer, pancreatic cancer, prostate cancer, skin cancer, colorectal cancer and ovarian cancer.

14. A compound of the formula (I), (II) or (III), or a physiologically tolerated salt thereof, according to any one of the preceding claims 1 to 8 for use in the treatment of cancer cell proliferation, migration and/or invasion.

15. A compound of the formula (I), (II) or (III), or a physiologically tolerated salt thereof, according to any one of the preceding claims 1 to 8 in combination with another other agent or therapy for use in the treatment of cancer.

16. A compound of the formula (I), (II) or (III) in combination with another agent according to claim 15, wherein the compound and the other agent are for administration separately, sequentially and/or simultaneously in any order.

17. A method for treating a kinase-affected disease or condition which comprises administering to a subject in need thereof an effective amount of a compound of the formula (I), (II) or (III) according to any one of the preceding claims 1 to 8, or a physiologically tolerated salt thereof.

18. A method for treating cancer, which comprises administering to a subject in need thereof an effective amount of a compound of the formula (I), (II) or (III) according to any one of the preceding claims 1 to 8, or a physiologically tolerated salt thereof.

19. A method for treating cancer cell proliferation, migration and/or invasion comprising administering to a subject in need thereof an effective amount of a compound according to any one of the preceding claims 1 to 8, or a physiologically tolerated salt thereof.

20. A method according to claim 18 or claim 19, wherein the cancer is breast cancer, pancreatic cancer, prostate cancer, skin cancer, colorectal cancer and/or ovarian cancer.

21. A process for preparing a compound of the formula (I), (II) or (III) according to any one of the preceding claims 1 to 8, or a physiologically tolerated salt thereof, which comprises reacting a compound of the formula (IV):

wherein

R¹ is substituted or unsubstituted aryl; X is O or NH; n is 0, 1 or 2; with 5-phenyltetrazole, thereafter reacting the resultant mixture with b/^'s(cyanoethyl)(N, N-diisopropylamino)phosphine, followed by meta- chloroperoxybenzoic acid, to provide a compound of the formula (V):

wherein R¹, X and n are as defined above;

R⁸, R⁹, R¹⁰, R¹¹ and R¹² are, independently, H or P(0)[0(CH₂)₂CN]_2; wherein at least one of R⁸ to R¹² is P(0)[0(CH₂)₂CN]_2; converting the compound of the formula (V) into a compound of the formula (I):

wherein

R¹, X and n are as defined above;

R² to R⁶ are independently selected from:H, P0₃ ^2", P(OR⁷)₃, PO(0 R⁷)₂, and PO(0 R⁷)0^~; wherein at least one of R² to R⁶ is P0₃ ^2", P(OR⁷)_3, PO(0 R⁷)₂, or PO(0 R⁷)CT; optionally converting the resultant compound into a physiologically tolerated salt, such as an ammonium, alkali or alkaline earth metal salt; and, optionally, purifying the resultant compound of the formula (I) or salt thereof.

22. A process according to claim 21, wherein R¹ is substituted or unsubstituted phenyl, X is O and/or n is the integer 1.

23. A compound of the formula (IA):

wherein

R¹, R², R³, R⁴, R⁵ and R⁶ are, independently from each other, selected from: H, P0₃ ^2", P(OR⁷)₃, PO(0 R⁷)₂, PO(0 R⁷)0^", -NHalkyl, -alkylene-NH₂ , NH₂, S0₂0^" , S0₂OR⁷, S(0)R⁷, S0₂R⁷, C(0)R⁷, C(0)OR⁷, OC(0)R⁷, and alkyl; R⁷ is H, alkyl, alkenyl or alkynyl; X is O; n is 0; or a physiologically tolerated salt thereof; for use in the treatment or prevention of cancer cell migration and/or cancer cell invasion.

24. A compound of the formula (IA), or a physiologically tolerated salt thereof, for use in the treatment of a drug resistant cancer.

25. A compound of the formula (IA), or a physiologically tolerated salt thereof, for use in the treatment of a cancer selected from: breast cancer, pancreatic cancer, prostate cancer, skin cancer, colorectal cancer and ovarian cancer.

26. A compound of the formula (I):

or a salt thereof, wherein R¹ is H, substituted or unsubstituted aryl, a fluorophore moiety, or substituted or unsubstituted aryl substituted by a fluorophore moiety; R² to R⁶ are independently selected from: H, P0₃ ^2", P(OR⁷)₃, PO(0 R⁷)₂, PO(0 R⁷) CT and a fluorophore moiety; R⁷ is H, alkyl, alkenyl or alkynyl; X is 0 or NH; Y is NH or CH₂: n is 0, 1 or 2 and m is 0 or 1; wherein at least one of R¹ to R⁶ is a fluorophore moiety.

27. A compound of the formula (I) according to claim 26, or a salt thereof, wherein R¹ is a fluorophore moiety; R² to R⁶ are independently selected from: H, P0₃ ^2', P(OR⁷)₃, PO(0 R⁷)₂, and PO(0 R⁷)0^' ; X is O or NH; Y is NH; n is 0, 1 or 2; and m is 1.

28. A compound of the formula (I) according to claim 27, or a salt thereof, wherein R¹ is a fluorophore moiety; R² to R⁶ are P0₃ ^2~; X is O; and n is 0.

29. A compound of the formula (I) according to any one of the preceding claims 26 to 28, or a salt thereof, wherein the fluorophore moiety is fluorescein or a derivative of fluorescein.

30. A compound of the formula (I) according to claim 29, wherein the fluorophore derivative is carboxyfluorescein, optionally wherein the compound is 2-0-(2-(5-fluoresceinylcarboxy)-aminoethyl)-myo-inositol 1, 3,4,5, 6-pentakisphosphate.

31. A compound of the formula (I), or a physiologically tolerated salt thereof, according to any one of the preceding claims 26 to 30 for use in therapy.

32. A compound according to claim 31 for use in the treatment of cancer, optionally wherein the cancer is a drug resistant cancer.

33. A compound according to claim 32, wherein the cancer is selected from: breast cancer, pancreatic cancer, prostate cancer, skin cancer, colorectal cancer and ovarian cancer.

34. A compound of the formula (I), or a physiologically tolerated salt thereof, according to any one of the preceding claims 26 to 33 for use in the treatment of cancer cell proliferation, migration and/or invasion.

35. A composition, comprising a compound of the formula (I) according to any one of the preceding claims 26 to 34, or a salt thereof.

36. A method of detecting and/or treating a cancer cell, comprising incubating a potential cancer cell with a compound of the formula (I) according to any one of claims 26 to 30, or a salt thereof, wherein the compound interacts with the cancer cell, wherein the presence of the compound with the cell indicates the cancer is present, identifying the association of fluorescence with the potential cancer cell.

37. A method according to claim 36, wherein the compound inhibits cancer cell proliferation, migration and/or invasion.