FIELD OF THE INVENTION

[1] The invention relates to compounds that are useful in medicine, specifically in treating or preventing cancerous diseases, especially cancer diseases which are susceptible to or caused by a phosphoinositide 3-kinase mutation, in a mammal, preferably in humans. The invention further relates to pharmaceutical compositions comprising such compounds, optionally together with other pharmaceutically active compounds, as well as pharmaceutical formulations comprising such compounds or pharmaceutical compositions. The invention further discloses methods of making such compounds and an assay for determining the activity of phosphoinositide 3- kinase alpha or mutant enzymes.

BACKGROUND OF THE INVENTION

[2] Carcinogenesis is a process by which normal cells are transformed into cancer cells. It is characterized by a progression of changes at the cellular, genetic, and epigenetic level that ultimately reprogram a cell to undergo uncontrolled cell division, thus mostly forming a malignant mass. Though it is well-established that carcinogenesis is largely the result of irregular activation of oncogenes and/or inactivation of tumor-suppressors, which lead to various pathological changes, the reasons of carcinogenesis arc various or not yet known. Furthermore, carcinogenesis pathways involve many genes, factors and conditions, which themselves and their interactions remain very complicated or unclear. Consequently, the development of anti-cancer drugs is largely based on the stroke of luck by trial -and- error, with the society still suffering under the lack of effective anti-cancer medications.

[3] Most drugs on the market are designed to bind directly to primary active sites (also known as orthosteric sites) of their biological targets. Allosteric modulators offer important advantages over orthosteric ones: they can be more selective because allosteric sites are less conserved (Bagal et al, J. Med. Chem., 62:247-265 (2019)), they can change protein levels or localization within the cell change (Generoso et al, Nat. Chem. Biol., 11:280-286 (2015)) subtle aspects of protein function (e.g. form or conformation of the quaternary structure (Zheng et al. J. Am. Chem. Soc., 140:5914- 5924(2018); Shibayama et ah, J. Biol. Chem., 292:18258-18269(2017)) and are unique in their ability to provide enzymatic activation (Milne et al. Nature, 450:712- 716 (2007)). Furthermore, allosteric drugs cam be used synergistically with orthosteric ligands, an approach that has been successfully used to prevent emergence of resistance in cancer therapy (Wylie et al. Nature, 543:733-737 (2017)). Allostery may also be the key to drug proteins that have been considered undruggable due to the absence of a known active site, such as KRAS. Allosteric targeting can also be reached via conformationally specific allosteric antibodies, which represents an important field of future research. There are already clear examples of monoclonal antibodies that allosterically target ion channels (Lee et al. Cell, 157:1393-1404 (2014)), GPCRs (Mukund et al. J. Biol. Chem., 288:36168-36178 (2013)), as well as cytokine and integrin receptors (Rizk et al. Cell Commun. Signal., 13:1-5 (2015); Schwarz et al. Circ. Residual., 99:25-33 (2006)). Importantly, allosteric modulators have the potential to combat drug-resistant mutations situated at orthosteric sites in patients. For example, the “gatekeeper mutation” T315I of BCR-ABL1 causes resistance to a set of clinically approved orthosteric drugs such as imatinib, bosutinib, nilotinib, and dasatinib. Treatment of the T315I mutant with an allosteric inhibitor ABL001 (asciminib) bound to the myristoyl pocket at the C-lobe of the kinase can overcome drug resistance (Schoepfer et al. J Med Chem 61:8120-8135 (20l8);Wylie et al. Nature, 543:733-737 (2017)).

[4] To date, the discovery of allosteric binding sites and lead compounds targeting those sites has been mostly serendipitous, achieved through high-throughput screening and subsequent follow up experimental work on the mechanism of action. The current lack of a suitable theoretical background to explain allostery in different protein classes, experimental data, and simulation technology capable to implement this drug design strategy, leads to high development costs of allosteric candidate drugs, even though over the past decade, a wealth of protein structural data has become readily available including many membrane protein classes (e.g., GPCRs and ion channels), which are common allosteric drug targets and, in parallel, improved simulation methods now provide better atomistic understanding of the protein dynamics and cooperative motions that are critical to allosteric mechanisms.

[5] The PI3K AKT mTOR is the most frequently activated signaling pathway in breast cancer (Miller et al Breast Cancer Res, 13:224 (2011)), and has been validated as a therapeutic target in Phase III trials (Andre et al., Lancet Oncol 15:267-274 (2014)). Furthermore, one of the signalling pathways which, when deregulated, becomes centrally involved in colon, mammary, and endometrial tumorigenesis is served by phosphoinositide-3’-kinase alpha (PI3Ka), which is one of the four PI3K isoforms (Engelman et al, Nat Rev Genet 7:606-619 (2006); Massacesi et al, Onco Targets Ther 9: 203-210 (2016)). PI3Ka is comprised of a catalytic subunit, pl lOa, encoded by the PIK3CA gene, and a regulatory subunit, p85a, encoded by the PIK3R1 gene. Deregulation of the PI3Ka pathway has long been associated with cancer development, but only during the last 10 years was it discovered that mutations commonly occurring in PIK3CA are oncogenic (Liu P et al, Nat Rev Drug Disc, 8:627-644 (2009)).

[6] PIK3CA is mutated in a breadth of cancers, with the most notable incidents in breast cancer, where PIK3CA is the most frequently mutated oncogene, harboring genetic alterations in ~27% of the cases on average, in endometrial cancer in 24% of the cases, and in colon cancer in -15% of the cases (Liu P et al, Nat Rev Drug Disc, 8:627-644 (2009)). PIK3CA mutations are found in a variety of cancers and more specifically in: breast cancer, colon cancer, endometrial cancer, brain tumors, skin cancer, ovarian cancer, gastric cancer, lung cancer, thyroid cancer, head and neck cancer, cervical cancer, pancreatic cancer, liver/biliary tract cancer, pituitary tumors, urological tumors, leukemia/lymphoma, neuroblastoma (Samuels and Waldman, Curr Top Microbiol Immunol, 347:21-41 (2010)). 80% of these mutations result in amino acid replacements located in either one of two hotspots: (a) in the helical domain of PI3Ka the most common replacement is that of Glu by Lys in exon 9 (E545K), (b) in the kinase domain, a His is changed to Arg in exon 20 (H1047R) (Miled et al, Science 317:239-42 (2007)). Both types of these mutations increase the kinase activity of the enzyme, upregulate the downstream AKT pathway and VEGF signaling, and stimulate cell transformation, tumorigensesis and angiogenesis (Samuels et al, Cancer Cell 7:561-572 (2005); Parsons Clin Cancer Res 11:7965-7966 (2005), Hu et al, Clin Cancer Res 11:8208-8212 (2005). A list of human cancers bearing the H1047R mutations can be found in Thorpe et al, Nat Rev Cancer 15(l):7 24 (2015) and in Samuels and Waldmann, Curr Top Microbiol Immunol 347:21 41 (2010).

[7] The PI3K pathway is implicated in resistance to chemotherapy, radiotherapy, hormone therapy, and targeted agents (Massacesi et al, Onco Targets Ther 9: 203- 210 (2016)). PI3K inhibitors may thus restore sensitivity to cancer treatments when administered as part of combination therapies (Burris HA III, Cancer Chemother Pharmacol. 71:829-842 (2013)).

[8] A number of PI3K inhibitors are currently under clinical investigation, including pan-PI3K inhibitors targeting all four isoforms of class I PI3K, as well as the PI3Ka-selective inhibitor alpelisib (BYL719), which is in Phase III clinical trials for postmenopausal women with advanced breast cancer, which progressed (Dienstmann et al., Mol Cancer Ther 13(5): 1021 -1031 (2014); https://clinicaltrials.gov/ct2/show/NCT024373l8). In early clinical studies, alpelisib exhibited favorable tolerability profiles, with adverse events consistent with“on- target” inhibition of PI3K (Massacesi et al, Onco Targets Ther 9:203-210 (2016)).

[9] In May 24 2019 alpelisib received FDA approval as the first PI3K inhibitor indicated for the treatment of hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative, PIK3CA-mutated, advanced or metastatic breast cancer in combination with fulvestrant for postmenopausal women and male patients. To initiate alpelisib therapy, it is required that the presence of a PIK3CA mutation in the tissue and/or liquid biopsy sample collection should be confirmed via FDA-approved diagnostic tests. Alpelisib is an inhibitor of the active site of PI3Ka, which exploits differences between the active sites of RI3Kb, g, and d isoforms to achieve its specificity. However, alpelisib is an inhibitor of the active site of PI3Ka, where Gerspacher et al, Bioorg Med Chem Lett.; 25:3582-4 (2015) converted the 5- (pyridin-4-yl)thiazol-2-amino bicyclic core scaffold of BYL719 (341) into novel 4H- thiazolo[5’,4’:4,5 Jpyrano [2,3-c]pyridine tricyclic scaffold ( 342), via an oxygen as the linker. Activity result showed that 343 (RI3Ka/b/d/g IC50 = 5/670/220/200 nM) exhibited similar or better biochemical potency, selectivity against PI3Ka and cellular activity, as well as favorable pharmacokinetic properties due to enhanced solubility, compared to noncyclized analogs of BYL719 (341, RI3Ka/b/d/g IC ₅₀ = 5/1200/290/250 nM). The crystal structure of pl lOa bound to the potent and selective inhibitor alpelisib (BYL719) has been published in (Furet et al, Bioorg Med Chem Lett.; 23(13):3741-8 (2013)). The amide hydrogen bond between the hinge residue and the inhibitor is colored in red for all panels. This hydrogen bond mimics the hydrogen bond made between the adenine ring of ATP with the hinge region of the kinase domain. Specificity for pl lOa over other PI kinases is primarily driven through the unique hydrogen bond interaction BYL719 can make with the non-conserved pl lOa residue Q859.

[10] A key challenge in targeting the PI3K pathway is the identification of isoform- selective inhibitors because the four PI3K isoforms are involved in distinct cellular processes, including glucose homeostasis and the immune response (Knight et al, Cell 125:733-747 (2006). The high similarity of the four PI3K isoforms in the adenosine triphosphate (ATP) binding pocket often leads to the development of non-selective PI3K drug candidates, which may yield undesirable side effects. Targeting a single PI3K isoform in patients may allow administration at therapeutic doses without being limited by toxicities associated with inhibiting multiple isoforms. Although alpelisib has achieved some potent selectivity, active sites are prone to mutations which leads eventually to drug resistance. Allosteric modulators offer a novel approach for selective kinase inhibition because they target less conserved binding sites compared to the active site; thus, higher selectivity may be obtained (Changeux, Drug Disc Today 10:e223-228 (2013)).

[11] Hence, there is still a need in the art for efficient and selective therapeutics that can prevent or inhibit tumor growth in mammals, in particular in humans.

[12] The 3D structure of the human (Huang et al, Science 318:1744-1748 (2007)) and mouse (Hon et al, Oncogene 31:3655-3666 (2012)) catalytic subunit pl lOa as well as the structure of the human H1047R PI3Ka mutant (Mandelker et al, Proc Natl Acad Sci U S A 106:16996-17001 (2009)) have been solved by X-ray crystallography. Recent experimental data demonstrate that the H1047R PI3Ka mutation overactivates the enzyme by inducing dynamic changes in the kinase domain, which increase basal ATPase activity as well as expose the membrane binding regions, thereby augmenting basal membrane binding (Huang et al, Science 318:1744-1748 (2007); Mandelker et al, Proc Natl Acad Sci USA 106:16996-17001 (2009)).

[13] The inventors have developed an advanced targeted therapeutic approach of great specificity, exploiting distinct structural features of the mutant and wild-type PI3Ka proteins for the design of novel inhibitors. The inventors’ recent studies have shown that it would be possible to target the H1047R mutated PI3Ka selectively by taking into account the dynamical and structural differences between the wild-type (WT) and mutant proteins (Gkeka et al, PLoS Comput Biol 10:el003895 (2014); Lionta et al, Curr Top Med Chem 14:1923-1938 (2014); Gkeka et al, J Phys Chem B 119:1002-1016 (2015)). These studies were utilized for the identification of new binding pockets and the design of mutant-specific PI3Ka inhibitors to exploit the altered mutant conformation.. A recent crystal structure (Hon et al, Oncogene 31:3655-66 (2012)) confirmed the binding of a known PI3K inhibitor, PIK-108, in one of the binding pockets identified. Using advanced computational and mathematical techniques in combination with in vitro experiments, the inventors showed that binding to this specific binding pocket does not induce any allosteric effects to PI3Ka (Gkeka et al, J Phys Chem B 119:1002-1016 (2015). They further investigated other binding pockets in the vicinity of the mutation site and examined if compounds binding there could possibly act as allosteric inhibitors, i.e. regulate the protein by binding at a site other than the protein active site.

This approach aims to treat cancer by using small-molecule inhibitors acting only against oncogenic mutant forms of PIK3CA and being selective against the wild-type protein. The invention employs an array of computational, rational drug design, organic synthesis, biochemical in vitro assays and in vivo studies in xenografts and genetically modified mouse models.

[14] Initially, the full-length catalytic pl lOa subunit of the WT and H1047R mutant PI3Ka was modeled using Molecular Dynamics simulations. Using data from the simulations, representative conformations for the PI3Ka WT protein and for the mutant H1047R were calculated. These conformations were further exploited to identify allosteric pockets for selective inhibition of the mutant protein (Gkeka et al, PLoS Comput Biol 10:el003895 (2014); Gkeka et al, J Phys Chem B 119:1002-1016 (2015)). The identified binding sites were confirmed for allosteric communication with the active (ATP) site of PDKa using positional covariance matrices, with which the inventors measured the correlation of motions between the residues of each identified binding site and the active PI3Ka site. One cavity in the vicinity of the active site was confirmed as allosteric and was further used in de novo computer- aided drug design. Subsequent post-processing of top-scoring compounds for toxicity, pose viability, chemical diversity, and physicochemical properties led to identifying an initial lead compound and synthesizing several of its analogs.

[15] PDKa activity assays on the identified lead compound and its analogs were performed using PIP2 containing liposomes (Gkeka et al, PLoS Comput Biol 10:el003895 (2014)). Complexes of PIK3CA (WT or Hl0l47R)/PIK3Rl were obtained from Millipore. The assay was based on a protocol provided by Millipore, with modifications (Papafotika et al., in preparation) which will be further described below. In this assay, PIP3 molecules produced by PDKa compete with biotinylated PIP3 for binding with a recombinant GST-GRP1-PH domain (amino acids 263- 380), which has been produced in bacteria, bound to glutathione coated 96-well plates. Quantitation of the competed amount of biotin-PIP3 is estimated by the peroxidase activity of streptavidin-HRP that binds to the plate and provides a measure for the activity of the protein. The compounds were preincubated with all the components of the assay mixture, except PIP2, for 10 min, at 25 ° C. PIP2 is added in the end of the preincubation period, thereby initiating the kinase reaction. IC50 values were calculated from dose- response curves using logit-log plots. All assays were carried out in at least three independent experiments and each concentration in triplicate. PIK-108 and wortmannin were used as controls (Gkeka et al, J Phys Chem B 119:1002-1016 (2015)).

[16] Xenografts are in vivo models of cancer, where cells from a cancer cell line are implanted into an immunodeficient mouse. Xenograft models are used to create an environment that allows for the natural growth of cancer, its monitoring, and corresponding treatment evaluations. Herein, the inventors used immunodeficient (SCID) mice with heterotopic xenografts after subcutaneous injection of cells from the breast cancer cell line MDA-MB-231, which is wild-type PIK3CA and of the breast cancer cell line HCC1954, which bears the exon 20 H1047R mutation on PIK3CA.

[17] In order to test the in vivo activity of potential anticancer drugs, establishing a corresponding cancer animal model is required. To generate mouse tumors at chosen anatomical sites, a variant of a genetic scheme that is based on Cre / loxP recombination is often used. Depending on the tissue-specificity of the promoter driving Cre expression, corresponding tumors develop in progeny derived from matings between Cre-producer mice and Cre -responder partners carrying a dormant oncogenic transgene, which becomes functional after excision of an upstream- positioned floxed DNA segment that blocks its expression [see Politi et al. Oncogene 23:1558-1565 (2004); Klinakis et al. Proc Natl Acad Sci U S A 106:2359-2364 (2009), Stratikopoulos et al. Cancer Cell 27:837-851 (2015)). To develop mouse strains carrying Pik3ca gain of function mutations (hereafter referred to as Pik3ca*), a knock-in gene targeting strategy was followed (Stratikopoulos et al. Cancer Cell 27:837-851 (2015)). Thus, either of the two hotspot mutations of the Pik3ca locus (in exon 20 or in exon 9) was targeted so that the mutated Pik3ca would be expressed from the endogenous locus and not from a transgene, thus, mimicking the expression found in human tumors. Activation of the exon 20 Pik3ca oncogenic mutation was achieved after mating the animals with WAPCre producers (WAP is a milk protein, the promoter of which is used for driving Cre expression, specifically in the mammary gland). Mammary tumors appeared in such animals after a very long latency of 490 days (median). However, mating of the WAPCre/exon20 Pik3ca* with MMTV-Myc expressing mice (which by themselves develop mammary tumors at 5-7 months) an acceleration in the appearance of tumors was observed in the birth-giving female mice with genotype WAPCre/exon20 Pik3ca* / MMTV-Myc (1 week following parturition). These mice were used to test the efficacy of compounds discussed herein.

[18] It was an object of the present invention to identify compounds suitable for treating or preventing cancerous diseases in a mammal based on a relevant biological activity demonstrated in in vitro biochemical assays and a non-human transgenic animal model. It was a further object of the invention to provide compounds and compositions that are useful in treating or preventing cancerous diseases in a mammal, as well as methods of treatment involving administering a therapeutically effective amount of such compounds to the mammal in need of such treatment.

SUMMARY OF THE INVENTION

[19] The present invention generally refers to compounds having a substituted benzyl or pyridine scaffold. In particular, one aspect of the invention relates to a compound of formula (I):

a tautomer, polymorph, hydrate, solvate, metabolite, prodrug, or a pharmaceutically acceptable salt thereof, wherein

L is independently H or OH;

X is independently N or C;

Y is independently H or =0;

Z is independently S or NH;

Rl is independently mono- or polycyclic aryl, heteroaryl, or cycloalkyl, including, but not limited to, the following examples:

R ₂ is independently at least one of hydroxyl, hydrogen, fluoro, chloro, or bromo; and Yi is independently C, O, S, NH or N, wherein the definitions for Yi being O, S and NH preferably apply to the five-membered aromatic ring system, whereas the definition for Y i being N preferably applies to the six-membered ring.

In the context of the above definitions and definitions contained at other places of this specification, R ₂ can also be absent as a substituent, i.e. its presence is optional. Furthermore, more than one of hydroxyl, hydrogen, fluoro, chloro or bromo substituents can be included as substituents in the formulas depending on the exact chemical structure.

In particular, the invention provides the above substances for use in medicine, specifically for use in treating or preventing cancerous diseases, especially cancerous diseases which are susceptible to or caused by or triggered by PI3Ka mutation and/or overactivation and/or amplification and/or deregulation.

[20] In a preferred embodiment, formula (I) refers to a compound having the following formula (II):

or a pharmaceutically acceptable salt thereof. This compound is also referred to herein with its internal name PI3K-010. [21] In another preferred embodiment, formula (I) refers to a compound having the following formula (III):

or a pharmaceutically acceptable salt thereof. This compound is also referred to herein with its internal name PI3K-021.

[22] In another preferred embodiment, formula (I) refers to a compound having the following formula (IV):

or a pharmaceutically acceptable salt thereof. This compound is also referred to herein with its internal name PI3K-024.

[23] In another aspect, the invention further refers to a pharmaceutical composition comprising one or more compounds of formula (I) as defined above, and preferably compounds II and/or III. Optionally, the pharmaceutical composition may further comprise one or more additional therapeutic agents, for example additional, otherwise known, anti-cancer agents. Again, in particular the invention refers to this pharmaceutical composition for use in treating or preventing cancerous diseases, especially cancerous diseases which are susceptible to or triggered by PBKa mutation and/or overactivation and/or amplification and/or deregulation.

[24] The above pharmaceutical compositions may in certain embodiments further comprise one or more additional therapeutic agents, such as COX-2 oxygenase inhibitors.

[25] In another aspect of the invention, the compounds or the pharmaceutical compositions referred to herein are intended for use in medicine. Preferably, said compounds or pharmaceutical compositions are for use in methods of treating or preventing a cancerous disease in a mammal. Preferably, the mammal is human.

[26] In some embodiments, the compounds or the pharmaceutical compositions are for use in methods of treating a cancerous disease that is susceptible to or caused, triggered or exacerbated by PI3Ka mutation and/or deregulation.

[27] Another aspect of the invention relates to a method for the manufacture of the above compounds of formula (I):

wherein the method comprises of first synthesizing chlorides with general formula

(V):

where in X, L, Y are defined as above, which then react with a moiety Ri-Z-H as defined for formula (I) above to yield a compound of formula (I) as depicted above. [28] A still further aspect of the invention relates to an assay for determining PI3Ka or PI3Ka mutant activity in a competitive immunoassay.

BRIEF DESCRIPTION OF THE FIGURES

[29] Figure 1. In vitro PDKa activity assays demonstrating an 11 -fold selectivity of PI3K-010 for H1047R PI3Ka. IC50s were estimated using the logit-log graph of SigmaPlot and linear regression analysis. Error bars show the standard error of the mean from at least three independent experiments, each one performed in triplicates.

[30] Figure 2. Results of an experiment testing competition between ATP and PI3K-010 for the active site of PI3Ka. The activity of PI3Ka was assayed using a standard concentration of 100 mM ATP (upper panel) as well as a 20-fold higher concentration of ATP (lower panel), at increasing concentrations PI3K-010. IC50s were estimated using the logit-log graph of SigmaPlot and linear regression analysis. Error bars show the standard error of the mean from at least three independent experiments, each one performed in triplicates. The IC50 of PI3K-010 is not significantly altered in the presence of high ATP concentration, indicating that this compound is a non-competitive inhibitor of PDKa.

[31] Figure 3. In vitro PDKa activity assays illustrating the selectivity of PI3K- 021 for H1047R PDKa. The IC50 of PI3K-021 against the mutant H1047R PDKa was estimated using the logit-log graph of SigmaPlot and linear regression analysis. Error bars show the standard error of the mean from at least three independent experiments, each one performed in triplicates. The IC50 against the WT PDKa was estimated by extrapolation.

[32] Figure 4. In vitro PDKa activity assays for PDK-024 against WT and H1047R PDKa. The IC50 of PDK-024 against the mutant H1047R is 64 nM and 6 nM against the WT PDKa. These values were estimated using the logit-log graph of SigmaPlot and linear regression analysis. Error bars show the standard error of the mean from at least three independent experiments, each one performed in triplicates.

[33] Figure 5. Results of PI3K-010 or PI3K-021 treatment of xenografts generated by heterotopic transplantation of the human breast cancer cell line MDA-MB-231 cell line, which is wild-type PIK3CA and of the breast cancer line HCC1954, which bears the PIK3CA mutation H1047R.

[34] Figure 6. Example of micro PET/CT imaging of WAPCre/exon20 Pik3ca*/MMTV-Myc mouse (compared with a negative wild-type control) at the beginning and end of treatment (one month) of breast cancer with the PI3K-010 compound.

[35] Figure 7. Schematic representation of the principle of the PBKinase activity assay. In the assay, at first, GST-GRP1 is inclubated with glutathione-coated plates. Then, GST-GRP1 forms a stable complex with biotin-labeled-PIP3, which then is quantified using streptavidin-HRP conjugate and a read out with OPD at 492 nm. The unlabelel PIP3, that is generated when PI3Ka phosphorylates RGR2, competes with the labeled b-PIP3 for binding to the PH domain of GRP1, leading to a reduction of the signal at 492 nm.

[36] Figure 8: Production and titration of the optimal amount of GST-GRP1 (PH domain)

a. GST-GRP1 PH domain was expressed in Escherichia Coli strain (DE3) and affinity purified on glutathione-Sepharose 4B (Amersham Pharmacia), using the manufacturer’s standard protocols. The purity of the protein was determined by SDS-PAGE.

b. Increasing amounts of GST-GRP1PH domain were incubated with glutathione- coated plates followed by incubation at first with biotin-PIP3 (b-PIP3) and then with streptavidin-HRP. The amount of HRP (which reflects the quantity of bound biotin-PIP3) was measured by incubating the plate with Opposition Division (substrate of HRP), followed by measurement of the absorbance at 492 nm.

[37] Figure 9: Titration of b-P!P3. Different quantities of b-PIP3 were added to glutathione-coated plates, where GST-GRP1 was pre-bound, followed by the consecutive incubation with Streptavidin-HRP and Opposition Division, before measuring the absorbance at 492 nm. The amount of 0.3 ng bPIP3 was selected for subsequent assays, as the quantity of this reagent should be high enough to couple most of the available GST-GRP1 and to provide high signal, yet, it should not be large excess as it would require larger amounts of PI3K and PIP2 for the competition in the assay mixture, which would raise the cost for screening compound libraries.

[38] Figure 10: The substrate PIP2 competes with b-PIP3 for binding to GRP1 : a. When long side chain-PIP2 alone was incubated with the plates, there was a reduction of the signal, probably due to competition between PIP2 and bPIP3 for binding to GRP1.

b. Incubation of the plate with PIP2 containing liposomes, that were prepared using lipids that have been isolated HCT116 cells, results in a much lower inhibition of the biotin-PIP3 signal, suggesting that the incorporation of PIP2 in liposomes reduces its binding to GRP1. In the sample containing 50% PIP2 in liposomes, where we observed inhibition of the signal despite the presence of liposomes, the signal was significantly recovered by the addition of sodium deoxycholate, at low concentration (below CMC), at the end of the enzymatic reaction, which led to a further recovery of the signal.

[39] Figure 11a: Identification of the preferred substrate preparation. The enzymatic reaction was tested using 2.5 or 5 ng PI3K. The substrates tested were either 200 ng PIP2 at a concentration of 10% or 50% PIP2 liposomes, or 2 pg soluble PIP2. Note that the most favorable preparation of the substrate is a mixture of 50% PIP2 in liposomes.

[40] Figure lib: Titration of the substrate. Increasing concentrations of PIP2 were tested together with two different amounts of PI3K, 2 and 4 ng. The substrate (PIP2) was supplied at a concentration of 50% in liposomes.

[41] Figure 12: Standard curve: Absorbance versus PIP3 added exogenously. The standard curve follows the predicted typical hyperbolic profile of competitive inhibition, as expected to be the competition between the enzymatically produced PIP3 and b-PIP3 for binding to GST-GRP1.

[42] Figure 13: Validation of the specificity of the assay.

a. The enzymatic reaction was assembled using 200 ng PIP2 (at a concentration of 50% in liposomes), the indicated amount of PI3K, ATP and buffer, and was incubated at 25°C for 16 min.

b. Time-dependence is shown for incubation times of 4, 8, 16, 32 and 60 min.

[43] Figure 14: Validation of the specificity of the assay. IC ₅₀ values of known PI3Ka inhibitors are determined using the inventive assay. The assessed IC ₅₀ values for three known PI3Ka inhibitors are in agreement with previously reported values.

DETATEED DESCRIPTION OF THE INVENTION

Definitions

[44] All terms used in the present application shall have the meaning usually employed by a relevant person skilled in the art, e.g. by a medicinal chemist, organic chemist, pharmacist, a molecular biologist, a physician, or a team thereof. By way of example, some definitions of specific terms are given below:

[45] The articles "a" and "an" are used to refer to one or to more than one (i. e. to at least one) of the grammatical object of the article. For example, "a cell" means one cell or more than one cell.

[46] “Adamantyl” refers to cyclic unsaturated aliphatic hydricarbyl groups arranged in three connected cyclohexane rings in the "armchair" configuration.

[47] "Alkyl" includes monovalent saturated aliphatic hydrocarbyl groups. The hydrocarbon chain may be either straight-chained or branched.

[48] “Aryl” refers to any functional group or substituent derived from an aromatic ring, such as a phenyl, naphthyl, thienyl, or indolyl. In preferred embodiments, the aryl is a substituted or unsubstituted phenyl, pyridinyl, or pyrimidinyl, more preferably a phenyl or a pyridinyl, and even more preferably a phenyl. The substitution may be an atom or a molecule substituted in place of a hydrogen atom on the parent chain of a hydrocarbon. Suitable substituents are known to a skilled person in the field of chemistry, more specifically, medicinal or organic chemistry. [49] "Cancer" or“tumor” refers to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells arc often in the form of a tumor, but such cells may exist alone within an animal, or may be a non-tumorigenic cancer cell, such as a leukemia cell. As used herein, the term "cancer" includes premalignant as well as malignant cancers. “Cancerous diseases” or “cancers”, which are used interchangeably herein, include, but are not limited to, acute monocytic leukemia, acute myelogenous leukemia, acute myelomonocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, adult T-cell lymphoma, astrocytoma, atypical carcinoid lung cancer, basal cell carcinoma, B-acute lymphocytic leukemia, B-cell acute lymphoblastic leukemia/lymphoma, Bladder cancer, brain cancer, breast cancer, bronchial cancer, Burkitt’s lymphoma, cancer of the bile duct, cancer of unknown primary origin, cervix cancer, chronic myeloproliferative disorder, colon cancer, diffuse large cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, gastric cancer, glioma, glioblastoma, head and neck cancer, hemagiopericytoma, hepatocellular carcinoma, Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, large cell neuroendocrine carcinoma, large granular lymphocytic leukemia, leukemia, liver cancer, lung cancer, lymphoma, medulloblastoma, melanoma, Multiple myeloma, myelodysplastic syndrome, nasopharygeal cancer, neuroblastoma, NK cell tumor, non-Hodgkin's lymphoma, oesophageal squamous cell carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, peripheral T-cell leukemia, primary plasma cell leukemia, prostate cancer, renal clear cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, small cell lung carcinoma, T-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic lymphoma, testicular cancer, thymoma, thyroid cancer, urachal cancer, uterine cancer, vaginal cancer, and the like.

[50] "Cycloalkyl" refers to cyclic saturated aliphatic hydrocarbyl groups. The numbers of C-atoms referenced in connection with a given cycloalkyl group corresponds to the number of ring forming carbon atoms, e.g. "C6 cycloalkyl" refers to a cyclohexyl. Preferably, cycloalkyls referenced herein are C6-C14 cycloalkyls.

[51] "Cycloalkenyl" refers to cyclic unsaturated aliphatic hydrocarbyl groups. The numbers of C-atoms referenced in connection with a given cycloalkyl group correspond to the number of ring forming carbon atoms, e.g. "C6 cycloalkyl" refers to a cyclohexenyl. In some embodiments, the cycloalkenyl comprises one double-bond. In some embodiments, the cyclohexenyl comprises more than one double bond, preferably two double bonds.

[52] "Deregulation" refers to an alteration or modification of the expression of a gene that encodes an enzyme/protein in a biosynthetic pathway, such that the level or activity of said enzyme/protein is altered or modified, which is found in, but is not limited to, cancer cells.

[53] "Furanyl" comprises a fiiran ring that can be bound by any C-atom.

[54] "Flalo" or "halogen" refers to fluoro, chloro, bromo and iodo. Preferred halo groups are either fluoro or chloro.

[55] "Flaloalkyl" includes an "alkyl" group as defined further above which is substituted with one or more halogens which may be the same or different.

[56] “Hydrate” refers to a type of crystalline form which retains a certain number of water molecules as part of the solid crystalline structure.

[57] “Neoplasm” refers to an abnormal mass of tissue as a result of abnormal growth or division of cells. It usually causes a lump or tumor. Neoplasms may be benign, pre-malignant (carcinoma in situ) or malignant (cancer).

[58] "Overexpression" refers to an excessive expression of a gene, thereby producing an excess of its effect or product. Most cancers arise through the overexpression of key cellular regulatory genes.

[59] “Prodrug” refers to a medication that is initially administered to the body in an inactive (or less than fully active) form, and then becomes converted to its active form through the normal metabolic processes of the body. [60] "Pyrazolyl" comprises a pyrazole ring that can be bound by any C-atom as well as by its nitrogen atom.

[61] "Pyrrolyl" comprises a pyrrole ring that can be bound by any C-atom as well as by its nitrogen atom.

[62] “Solvate” refers to a type of crystalline form which retains a certain number of solvent molecule other than water as part of the solid crystalline structure.

[63] “Tautomers” refer to constitutional isomers of an organic compound that interconvert readily by a chemical reaction called tautomerization, which commonly results in the formal migration of a hydrogen atom or proton, accompanied by a switch of a single bond and adjacent double bond.

[64] "Thiophenyl" comprises a thiophen ring that can be bound by any C-atom.

[65] Any "alkyl", "cycloalkyl", "furanyl", "pyrrolyl", "thiophenyl", “aryl or ’’“phenyl” may be unsubstituted or substituted by one or more atoms or atom groups (molecules).

[66] "Pharmaceutically acceptable" refers to being devoid of substantial toxic effects when used in doses usually employed in a medicinal dosage form, and thereby being approvable or preferably being approved by a regulatory agency of the Federal or a state government or being listed in the U. S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, more preferably in humans.

[67] "Pharmaceutically acceptable salt" refers to a salt of a compound of the invention that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. Such salts include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3- (4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1 ,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4- chlorobenzenesulfonic acid, 2naphthalenesulfonic acid, 4- toluenesulfonic acid,camphorsulfonic acid, 4methylbicyclo [2.2.2]-oct-2-ene-l-carboxylic acid, glucoheptonic acid,3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; or (2) salts formed when an acidic proton present in the parent compound is replaced, forming an ion pair with a positively-charged base moiety.

[68] "Pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient or carrier with which a compound of the invention is administered.

[69] “Polymorphs” refer to different crystalline forms of the same compound. Polymorphism denotes the ability of a material to exist in more than one form or crystal structure.

[70] "Preventing" or "prevention" refers to a reduction in risk of acquiring a disease or disorder (i. e., causing at least one of the clinical symptoms of the disease not to develop in a subject that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease).

[71] "Therapeutically effective amount" refers to the amount of a compound that, when administered to a subject for treating a disease, is sufficient to effect such treatment for the disease. The "therapeutically effective amount" can vary depending on the compound, the disease and its severity, and the age, weight, etc., of the subject to be treated. Preferably, the compound according to the present invention may be used at a dose of from 0.1 to 200 mg/kg, more preferably at a dose of from 0.5 to 50.0 mg/kg, preferably such doses being applied twice a day. Daily doses to be administered can also be from 50mg to 500 mg/day and preferably 150 to 300 mg/day. In any event, the suitable dose will be determined by those of skill in the art by appropriate routine dosing experiments carried out in the species intended to be treated or based on the response of the patient. Suitable doses can be applied in a single treatment or by application of appropriate partial doses at several different points in time.

[72] "Treating" or "treatment" of any disease or disorder refers to, in one embodiment, to ameliorating the disease or disorder (i. e., arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment, "treating" or "treatment" refers to ameliorating at least one physical parameter, which may not be discernible by the subject. In yet another embodiment, "treating" or "treatment" refers to modulating the disease or disorder, either physically, (e. g., stabilization of a discernible symptom), physiologically (e. g., stabilization of a physical parameter), or both. In yet another embodiment, "treating" or "treatment" refers to delaying the onset of the disease or disorder.

Compounds and Pharmaceutical Compositions

As a result of their intensive studies and inventive efforts, the inventors were able to create the first ever reported allosteric inhibitors of PI3Ka mutants.

[73] Accordingly, one aspect of the invention refers to a compound of formula (I)

a tautomer, polymorph, hydrate, solvate, metabolite, prodrug, or a pharmaceutically acceptable salt thereof, wherein

L is independently H or OH;

X is independently N or C;

Y is independently H or =0;

Z is independently S or NH;

Ri is independently mono- or polycyclic aryl, heteroaryl, or cycloalkyl, including, but not limited to, the following examples:

R ₂ is independently at least one of hydroxyl, hydrogen, fluoro, chloro, or bromo;

Yi is independently C, O, S, NH or N, wherein the definitions for Yi being O, S and NH preferably apply to the five-membered aromatic ring system, whereas the definition for Y i being N preferably applies to the six-membered ring.

Preferably, this aspect relates to the compounds of formula (I) for use in medicine, especially for use in the treatment of cancer diseases. It has been discovered by the present inventors that the compounds of formula (I) are isoform-specific heterotropic modulators of PI3Ka activity and at least decrease or even completely inhibit the activity of PI3Ka mutants which have been associated with certain cancer diseases by e.g. participating in their development and/or facilitating or promoting persistence and/or exacerbation thereof. Without wishing to be bound to any theory, the compounds have been discovered to allosterically inhibit the activity of mutant proteins, especially the hot spot mutant H1047R, by binding at a site which is distinct from the orthosteric binding pocket of ATP. The allosteric binding of a coumpound to the mutated PI3Ka causes conformational changes which affect the PDKa mutant catalytic activity irrespective of ATP binding and, consequently, the mutants’ activities. [74] In preferred embodiments, formula (I) refers to a compound having the formula (II):

or a pharmaceutically acceptable salt thereof. This compound is also referred to herein with its internal name PI3K-010.

[75] In preferred embodiments, formula (I) refers to a compound having the formula (III):

or a pharmaceutically acceptable salt thereof. This compound is also referred to herein with its internal name PI3K-021.

[76] In preferred embodiments, formula (I) refers to a compound having the formula (IV):

or a pharmaceutically acceptable salt thereof. This compound is also referred to herein with its internal name PI3K-024.

Pharmaceutical Compositions

[77] The invention further refers to pharmaceutical compositions comprising the compounds of formula (I) or, preferably, of formula (II), or of formula (III), or of formula (IV) as depicted above. In one preferred embodiment of this aspect of the invention, the compounds of formula (I) included in the pharmaceutical compositions do not include compound (IV), i.e. definitions of the variants of formula (I) that define compound (IV) are excluded in this embodiment.

[78] Optionally, the pharmaceutical compositions of the present invention may further comprise one or more additional therapeutic (i.e. pharmaceutically active) agents.

[79] In some embodiments, the one or more additional therapeutic agents of the pharmaceutical composition may be selected from the group (A) consisting of: 10- Hydroxycamptothecin, l7-Allylamino-geldanamycin, 2-Methoxyanti-mycin A3, 3,4- Dichloroisocoumarin, 4-Hydroxyphenylretinamide, 9-cis Retinoic acid, Abiraterone, Ado-Trastuzumab Emtansine, Adriamycin, Afatinib, N-(3-chlorophenyl)-6,7- dimethoxyquinazolin-4-amine, 2- Amino-4-( 1 H-indol-5-yl)- 1 , 1 ,3-tricyanobuta- 1 ,3- diene, Aldesleukin, Alemtuzumab, Amifostine, Anastrozole, Anisomycin, Aphidicolin, Arsenic Trioxide, Asparaginase Erwinia chrysanthemi, Avastin, Axitinib, N-[2(S)-(2(R)-2-Amino-3-mercaptopropylamino)-3-methylbutyl]-

Lphenylalanyl-L-methionine trifluoroacetate salt, Bacillus Calmette-Guerin, bisphenol A diglycidyl ether, Bendamustine, Beta-lapachone , Betulinic acid, Bevacizumab, Bexarotene, Bicalutamide, BisBenzimide, Bleomycin, Bortezomib, Bosutinib, Buserelin, Busulfan, Cabazitaxel, Cabozantinib-S-Malate, Calpeptin, Camptothecin, Caffeic acid phenethyl ester, Capecitabine, CAprelsa (Vandetanib), Carboplatin, Carboplatin, Carfilzomib, Carmustine, Cetuximab, Chlorambucil, Ciglitazone, Cisplatin, Clodronate, Clofarabine, Cometriq, Crizotinib, Curcumin, Cyclo [Arg-Gly-Asp-D-Phe-Val], Cycloheximide, Cyclopamine, Cyclophosphamide, Cyclosporin A, Cyproterone, Cytarabine, Dl 2-Prostaglandin J2, Dabrafenib, Dacarbazine, Dactinomycin, Dasatinib, Daunorubicin, Degarelix, Denosumab, Dexamethasone, Docetaxel, Doxorubicin, Ebselen, Ellipticine, Enzalutamide, Epirubicin, Erlotinib, Etoposide, Everolimus, Excmcstanc, Fludarabine, Fluorouracil, Flutamide, Folinic acid, Fulvestrant, Gefitinib, Geldanamycin, Gemcitabine, Genistein, Gingerol, Gliadel Wafer, Gliotoxin, Goserelin, 2-Chloro-5- nitrobenzanilide, 2-Amino-6-bromo-a-cyano-3-(ethoxycarbonyl)-4H- 1 -benzopyran-4- acetic acid ethyl ester, Hinokitiol, Hydroxyurea, Sobuzoxane, Idarubicin, ifosfamide, Imatinib, Indomethacin, Ipilimumab, Irinotecan, Ixabepilone, Lanreotide, Lapatinib, Lenalidomide, Letrozole, Lenvatinib Mesylate, Lenvima, Leucovorin, Leuprolide, Lomustin, Medroxyprogesterone, Megestrol, Melphalan, Mepesuccinate, Mercaptopurine, Mesna, Methotrexate, Methoxy verapamil, carbobenzoxy-L-leucyl- L-leucyl-L-leucinal, Mitomycin C, Mitoxantrone, N,N-Dimethylsphingosine, Nelarabine, Nilotinib, Nivolumab, Octreotide, Ofatumumab, Oligomycin A, Omacetaxine, Oxaliplatin, Paclitaxel, Pamidronate, Panitumumab, Pazopanib, Pegaspargase, Pemetrexed, Pembrolizumab, Pertuzumab, Pifithrin, plerixafor, Podophyllotoxin, Pomalidomide, Ponatinib, Prednisone, 2,2-Bis(hydroxymethyl)-l- azabicyclo[2.2.2]octan-3-one, Procarbazine, Radium 223 Dichloride, Raltitrexed, Rapamycin, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, Recombinant HPV Quadravalent Vaccine, Recombinant HPV Bivalent , Vaccine, Recombinant HPV Quadravalent Vaccine, Recombinant Interferon Alfa-2b, Regorafenib, Resveratrol, all trans Retinoic acid, , Rheumatrex, Rituximab, Rolipram, Roscovitine, Rottlerin, Shikonin, Sipuleucel-T, Sirolimus, Sorafenib, Sphingosine, Splitomycin, Staurosporine, Stilboestrol, Streptozocin, 3-(4- Dimethylaminobenzylidenyl)-2-indolinone, 3 - [ [(4-Dimethyl-amino)phenyl] methylene] -1, 3 -dihydro-2H-indol-2-one, Sulindac sulphide, Sunitinib, Tamoxifen, Temozolomide, Temsirolimus, Thalidomide, Topotecan, Toremifene, Trametinib, Trastuzumab, Trichostatin-A, Trifluoperazine, 4-[(E)-2-(5, 6,7, 8-Tetrahydro-5, 5,8,8- tetramethyl-2-naphthalenyl)- 1 -propenyljbenzoic acid, 3 ,4-Dihydroxy-a- cyanothiocinnam-amide, (3 -Hydroxy-4-nitrobenzylidene)malononitrile, V andertanib, Valproic acid, Vemurafenib, Verapamil, Vinblastine, Vincristine, Vinorelbine, Wortmannin, 4-Chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid, Ziv- Aflibercept, Zoledronic Acid, salts thereof, and combinations thereof. [80] Preferably, the one or more therapeutic agents are selected from, but not restricted to, the group (A'), i.e. Abiraterone, Ado-Trastuzumab Emtansine, Afatinib, Afmitor (Everolimus), Anastrozole, Avastin, Bevacizumab, Cabazitaxel, Capecitabine, Carboplatin, Carmustin, Cisplatin, Crizotinib, Cyclophosphamide, Degarelix, Denosumab, Docetaxel, Doxorubicin, Enzalutamide, Epirubicin, Erlotinib, Etoposide, Everolimus, Exemestane, Fluorouracil, Fulvestrant, Gefltinib, Gemcitabine, Ixabepilone, Lapatinib, Letrozole, Leuprolide, Lomustine, Megestrol, Methotrexate, Mitomycin C, Paclitaxel, Pamidronate, Pemetrexed, Pertuzumab, Prednisone, Radium 223 Dichloride, Sipuleucel-T, Sunitinib, Tamoxifen, Temodar, Temozolomide, Topotecan, Toremifene, Trastuzumab, Cabozantinib-S-Malate, Caprelsa (Vandetanib), Cometriq (Cabozantinib-S-Malate), Doxorubicin Hydrochloride, Ipilimumab, Lenvatinib Mesylate, Nexavar (Sorafenib Tosylate) Nivolumab, Vandetanib, Yervoy (Ipilimumab), salts thereof, and any combination thereof.

[81] In some embodiments of this aspect of the present invention, the pharmaceutical compositions comprise, besides the compound of formula (I) or formula (II) described above, one or more additional therapeutic agent(s) selected from the group consisting of: for Acute Lymphoblastic Leukemia (ALL):

Abitrexate (Methotrexate), Adriamycin PFS (Doxorubicin Hydrochloride), Adriamycin RDF (Doxorubicin Hydrochloride), Arranon (Nelarabine), Asparaginase Erwinia chrysanthemi, Cerubidine (Daunorubicin Hydrochloride), Clafen (Cyclophosphamide), Clofarabine, Clofarex (Clofarabine), Clolar (Clofarabine), Cyclophosphamide, Cytarabine, Cytosar-U (Cytarabine), Cytoxan (Cyclophosphamide), Dasatinib, Faunorubicin Hydrochloride, Doxorubicin Hydrochloride, Erwinaze (Asparaginase Erwinia hrysanthemi), Folex (Methotrexate), Folex PFS (Methotrexate), Gleevec (Imatinib Mesylate), Iclusig (Ponatinib Hydrochloride), Imatinib Mesylate, Marqibo (Vincristine Sulfate Liposome), Mercaptopurine, Methotrexate, Methotrexate LPF (Methorexate), Mexate (Methotrexate), Mexate-AQ (Methotrexate), Nelarabine, Neosar (Cyclophosphamide), Oncaspar (Pegaspargase), Pegaspargase, Purinethol (Mercaptopurine), Rubidomycin (Daunorubicin Hydrochloride), Sprycel (Dasatinib), Tarabine PFS (Cytarabine), Vincasar PFS (Vincristine Sulfate), Vincristine Sulfate, or Vincristine Sulfate Liposome; for Acute Myeloid Leukemia (AML):

Adriamycin PFS (Doxorubicin Hydrochloride). Adriamycin RDF (Doxorubicin Hydrochloride), Arsenic Trioxide, Cerubidine (Daunorubicin Hydrochloride), Clafen (Cyclophosphamide), Cyclophosphamide, Cytarabine, Cytosar-U (Cytarabine), Cytoxan (Cyclophosphamide), Daunorubicin Hydrochloride, Doxorubicin Hydrochloride, Neosar (Cyclophosphamide), Rubidomycin (Daunorubicin Hydrochloride), Tarabine PFS (Cytarabine), Trisenox (Arsenic Trioxide), Vincasar PFS (Vincristine Sulfate), or Vincristine Sulfate; for Chronic Lymphocytic Leukemia (CLL):

Alemtuzumab, Ambochlorin (Chlorambucil), Amboclorin (Chlorambucil), Arzerra (Ofatumumab), Bendamustine Hydrochloride, Campath (Alemtuzumab), ChlorambucilClafen (Cyclophosphamide), Cyclophosphamide, Cytoxan (Cyclophosphamide), Fludara (Fludarabine Phosphate), Fludarabine Phosphate, Leukeran (Chlorambucil), Linfolizin (Chlorambucil), Neosar (Cyclophosphamide), Ofatumumab, Treanda (Bendamustine Hydrochloride), Chlorambucil-Prednisone, or CVP (a combination of Cyclophosphamide, Vincristine and Prednisone); for Chronic Myelogenous Leukemia (CML):

Bosulif (Bosutinib), Bosutinib, Clafen (Cyclophosphamide), Cyclophosphamide, Cytarabine, Cytosar-U (Cytarabine), Cytoxan (Cyclophosphamide), Dasatinib, Gleevec (Imatinib Mesylate), Iclusig (Ponatinib Hydrochloride), Imatinib Mesylate, Neosar (Cyclophosphamide), Nilotinib, Omacetaxine Mepesuccinate, Ponatinib Hydrochloride, Sprycel (Dasatinib), Synribo (Omacetaxine Mepesuccinate), Tarabine PFS (Cytarabine), or Tasigna (Nilotinib); for Meningeal Leukemia:

Cytarabine, Cytosar-U (Cytarabine), or Tarabine PFS (Cytarabine); for Burkitt’s lymphoma: Methotrexate (Anti-Rheumatic) oral, Rituxan iv, methotrexate sodium oral, dexamethasone oral, DexPak, rituximab iv, methotrexate sodium inj, Adriamycin iv, cyclophosphamide oral, Trexall oral, vincristine iv, cyclophosphamide iv, dexamethasone sodium phosphate inj, Dexamethasone Intensol oral, methotrexate sodium (PF) inj, Rheumatrex oral, cytarabine inj, doxorubicin iv, dexamethasone sodium phos (PF) inj, etoposide iv, ifosfamide iv, Ifex iv, cytarabine (PF) inj, Adriamycin PFS iv, DexPak 10 day oral, DexPak 6 Day oral, ifosfamidemesna iv, Toposar iv, etoposide phosphate iv, Etopophos iv, Vincasar PFS iv, or Baycadron oral; for diffuse large cell lymphoma:

prednisone oral, Rituxan iv, dexamethasone oral, DexPak 13 Day oral, rituximab iv, Adriamycin iv, cyclophosphamide oral, Prednisone Intensol oral, vincristine iv, cyclophosphamide iv, bleomycin inj, dexamethasone sodium phosphate inj, Dexamethasone Intensol oral, cytarabine inj, melphalan oral, doxorubicin iv, Alkeran oral, etoposide oral, dexamethasone sodium phos (PF) inj, melphalan iv, procarbazine oral, etoposide iv, cytarabine (PF) inj, Adriamycin PFS iv, DexPak 10 day oral, DexPak 6 Day oral, Rayos oral, Alkeran iv, Matulane oral, epirubicin iv, Toposar iv, etoposide phosphate iv, Etopophos iv, Ellence iv, Vincasar PFS iv, or Baycadron oral;

For Brain Cancer:

Afinitor (Everolimus), Avastin (Bevacizumab), BiCNU (Carmustine), CArmustine wafer, Lomustine, Temodar (Temozolomide), Temozolomide, Temodar oral, vincristine iv, Camptosar iv, irinotecan iv, bevacizumab iv, temozolomide oral, Gliadel Wafer impl, procarbazine oral, BiCNU iv, Temodar iv, Matulane oral, Vincasar PFS iv, carmustine iv, carmustine in polifeprosan impl, or temozolomide iv; for Multiple Myeloma and Other Plasma Cell Neoplasms:

Aredia (Pamidronate Disodium), Bortezomib, Carfilzomib, Clafen (Cyclophosphamide), Cyclophosphamide, Cytoxan (Cyclophosphamide), Doxil (Doxorubicin Hydrochloride Liposome), Doxorubicin Hydrochloride Liposome, Dox- SL (Doxorubicin Hydrochloride Liposome), Evacet (Doxorubicin Hydrochloride Liposome), Kyprolis (Carfilzomib), Lenalidomide, LipoDox (Doxorubicin Hydrochloride Liposome), Mozobil (Plerixafor), Neosar (Cyclophosphamide), Pamidronate Disodium, Plerixafor, Pomalidomide (Pomalyst), Pomalyst, Revlimid (Lenalidomide), Synovir (Thalidomide), Thalidomide, Thalomid (Thalidomide), Velcade (Bortezomib), Zoledronic Acid, or Zometa (Zoledronic Acid); for Non-Small Cell Lung Cancer:

Abitrexate (Methotrexate), Abraxane (Paclitaxel Albumin- stabilized Nanoparticle Formulation), Afatinib Dimaleate, Alimta (Pemetrexed Disodium), Avastin (Bevacizumab), Bevacizumab, Carboplatin, Cisplatin, Crizotinib, Erlotinib Hydrochloride, Folex (Methotrexate), Folex PFS (Methotrexate), Gefitinib, Gilotrif (Afatinib Dimaleate), Gemcitabine Hydrochloride, Gemzar (Gemcitabine Hydrochloride), Iressa (Gefitinib), Methotrexate, Methotrexate LPF (Methotrexate), Mexate (Methotrexate), Mexate-AQ (Methotrexate), Paclitaxel, Paclitaxel Albumin- stabilized Nanoparticle Formulation, Paraplat (Carboplatin), Paraplatin (Carboplatin), Pemetrexed Disodium, Platinol (Cisplatin), Platinol-AQ (Cisplatin), Tarceva (Erlotinib Hydrochloride), Taxol (Paclitaxel), Xalkori (Crizotinib), a combination of Carboplatin-Taxol, or a combination of Gemcitabine-Cisplatin; for Small Cell Lung Cancer:

Abitrexate (Methotrexate), Etopophos (Etoposide Phosphate), Etoposide, Etoposide 5 Phosphate, Folex (Methotrexate), Folex PFS (Methotrexate), Hycamtin (Topotecan Hydrochloride), Methotrexate, Methotrexate LPF (Methotrexate), Mexate (Methotrexate), Mexate-AQ (Methotrexate), Toposar (Etoposide), Topotecan Hydrochloride, or VePesid (Etoposide); for Bladder Cancer:

Adriamycin PFS (Doxorubicin Hydrochloride), Adriamycin RDF (Doxorubicin Hydrochloride), Cisplatin, Doxorubicin Hydrochloride, Platinol (Cisplatin), or Platinol-AQ (Cisplatin); for Breast Cancer:

Perjeta (pertuzumab), Abitrexate (Methotrexate), Abraxane(Paclitaxel Albumin- stabilized Nanoparticle Formulation), Ado-Trastuzumab Emtansine, Adriamycin PFS (Doxorubicin Hydrochloride), Adriamycin RDF (Doxorubicin Hydrochloride), Adrucil (Fluorouracil), Afmitor (Everolimus), Anastrozole, Aredia (Pamidronate Disodium), Arimidex (Anastrozole), Aromasin (Exemestane), Capecitabine, Clafen (Cyclophosphamide), Cyclophosphamide, Cytoxan (Cyclophosphamide), Docetaxel, Doxorubicin Hydrochloride, Efudex (Fluorouracil), Ellence (Epirubicin Hydrochloride), Epirubicin Hydrochloride, Everolimus, Exemestane, Fareston (Toremifene), Faslodex (Fulvestrant), Fcmara (Letrozole), Fluoroplex (Fluorouracil), Fluorouracil, Folex (Methotrexate), Folex PFS (Methotrexate), Fulvestrant, Gemcitabine Hydrochloride, Gemzar (Gemcitabine Hydrochloride), Herceptin (Trastuzumab), Ixabepilone,

Ixempra (Ixabepilone), Kadcyla (Ado-Trastuzumab Emtansine), Lapatinib Ditosylate, Letrozole, Megace (Megestrol Acetate), Megestrol Acetate, Methotrexate, Methotrexate LPF (Methotrexate), Mexate (Methotrexate), Mexate-AQ (Methotrexate), Neosar (Cyclophosphamide), Nolvadex (Tamoxifen Citrate), Novaldex (Tamoxifen Citrate), Paclitaxel, Paclitaxel Albumin- stabilized Nanoparticle Formulation, Pamidronate Disodium, Perjeta (Pertuzumab), Pertuzumab, Tamoxifen Citrate, Taxol (Paclitaxel), Taxotere (Docetaxel), Trastuzumab, Toremifene, Tykerb (Lapatinib Ditosylate), or Xeloda (Capecitabine); for Cervical Cancer:

Blenoxane (Bleomycin), Bleomycin, Cisplatin, Hycamtin (Topotecan Hydrochloride), Platinol (Cisplatin), Platinol-AQ (Cisplatin), Topotecan Hydrochloride, or a combination of Gemcitabine-Cisplatin; for Endometrial Cancer:

paclitaxel, decataxel, doxorubicin, cisplatin, carboplatin, capecitabine, gemcitabine, tamoxifen, hydroxyprogesterone caproate, letrozole, megestrol acetate, medroxyprogesterone; for Esophageal Cancer:

Cyramza (Ramucirumab), Docetaxel, Herceptin (Trastuzumab), Ramucirumab, Trastuzumab; for Colon Cancer:

Adrucil (Fluorouracil), Avastin (Bevacizumab), Bevacizumab, Camptosar (Irinotecan Hydrochloride), Capecitabine, Cetuximab, Efudex (Fluorouracil), Eloxatin, Oxaliplatin), Erbitux (Cetuximab), Fluoroplex (Fluorouracil), Fluorouracil, Irinotecan Flydrochloride, Leucovorin Calcium, Oxaliplatin, Panitumumab, Regorafenib, Stivarga (Regorafenib), Vectibix (Panitumumab), Wellcovorin (Leucovorin Calcium), Xeloda (Capecitabine), Zaltrap (Ziv-Aflibercept), Ziv-Aflibercept, CAPOX (a combination of capecitabine and oxaliplatin), FOLFIRI (a combination of Folinic acid, Fluorouracil, and Irinotecan), FOLFIRI-Bevacizumab (a combination of Folinic acid, Fluorouracil, Irinotecan, and Bevacizumab), FOLFIRICetuximab (a combination of Folinic acid, Fluorouracil, Irinotecan, and Cetuximab), FOLFOX (a combination of Folinic acid, Fluorouracil, and Oxaliplatin), or XELOX (a combination of Capecitabine and Oxaliplatin); for Rectal Cancer:

Adrucil (Fluorouracil), Avastin (Bevacizumab), Bevacizumab, Camptosar (Irinotecan Hydrochloride), Cetuximab, Efudex (Fluorouracil), Erbitux (Cetuximab), Fluoroplex (Fluorouracil), Fluorouracil, Irinotecan Hydrochloride, Panitumumab, Regorafenib, Stivarga (Regorafenib), Vectibix (Panitumumab), Zaltrap (Ziv-Aflibercept), Ziv- Aflibercept, CAPOX, FOLFIRI, FOLFIRI-Bevacizumab, FOLFlRl-Cetuximab, FOLFOX, or XELOX; for Gastric Cancer:

Adriamycin PFS (Doxorubicin Hydrochloride), Adriamycin RDF (Doxorubicin Hydrochloride), Adrucil (Fluorouracil), Docetaxel, Doxorubicin Hydrochloride, Efudex (Fluorouracil), Fluoroplex (Fluorouracil), Fluorouracil, Herceptin (Trastuzumab), Mitomycin C, Mitozytrex (Mitomycin C), Mutamycin (Mitomycin C), Taxotere (Docetaxel), or Trastuzumab; for Glioblastoma:

Avastin (Bevacizumab), Temodar oral, vincristine iv, Camptosar iv, irinotecan iv, bevacizumab iv, temozolomide oral, Gliadel Wafer impl, procarbazine oral, BiCNU iv,

Temodar iv, Matulane oral, Vincasar PFS iv, carmustine iv, carmustine in polifeprosan impl, or temozolomide iv;

For Hepatocellular Carcinoma: Sorafenib, Sunitinib, Erlotinib, Bevacizumab, or Sirolimus; for large cell neuroendocrine carcinoma:

sunitinib (Sutent), everolimus (Afmitor), or the combination of fluorouracil (Adrucil, 5-FU), doxorubicin (Adriamycin), and streptozocin (Zanosar); for Melanoma:

Aldesleukin, Dabrafenib, Dacarbazine, DTIC-Dome (Dacarbazine), Intron A (Recombinant Interferon Alfa-2b), Ipilimumab, Mekinist (Trametinib), Peginterferon Alfa-2b, PEG-Intron (Peginterferon Alfa-2b), Proleukin (Aldesleukin), Recombinant Interferon Alfa-2b, Sylatron (Peginterferon Alfa-2b), Tafinlar (Dabrafenib), Trametinib, Vemurafenib, Yervoy (Ipilimumab), or Zelboraf (Vemurafenib); for Neuroblastoma:

Adriamycin PFS (Doxorubicin Hydrochloride), Adriamycin RDF (Doxorubicin Hydrochloride), Clafen (Cyclophosphamide), Cyclophosphamide, Cytoxan (Cyclophosphamide), Doxorubicin Hydrochloride, Neosar (Cyclophosphamide), Vincasar PFS (Vincristine Sulfate), or Vincristine Sulfate; for Ovarian Cancer:

Adriamycin PFS (Doxorubicin Hydrochloride), Adriamycin RDF (Doxorubicin Hydrochloride), Carboplatin, Clafen (Cyclophosphamide), Cisplatin, Cyclophosphamide, Cytoxan (Cyclophosphamide), Doxorubicin Hydrochloride, Dox- SL (Doxorubicin Hydrochloride Fiposome), DOXIF (Doxorubicin Hydrochloride Liposome), Doxorubicin Hydrochloride Liposome, Evacet (Doxorubicin Hydrochloride Liposome), Gemcitabine Hydrochloride, Gemzar (Gemcitabine Hydrochloride), Hycamtin (Topotccan Hydrochloride), LipoDox (Doxorubicin Hydrochloride Liposome), Neosar (Cyclophosphamide), Paclitaxel, Paraplat (Carboplatin), Paraplatin (Carboplatin), Platinol (Cisplatin), Platinol-AQ (Cisplatin), Taxol

(Paclitaxel), Topotecan Hydrochloride, BEP (a combination of Bleomycin, Etoposide, and Cisplatin), or a combination of Carboplatin-Taxol, or a combination of Gemcitabine-Cisplatin; for Head and Neck Cancer:

Bleomycin, Cetuximab, Docetaxel, Erbitux (Cetuximab), Hydrea (Hydroxyurea), Keytruda (Pembrolizumab), Methotrexate, Nivolumab, Taxotere (Docetaxel) Trexall (Methotrexate); for Liver Cancer:

Nexavar (Sorafenib Tosylate), Regorafenib, Sorafenib Tosylate, Stivarga

(Regorafenib); for Thyroid Cancer:

Cabozantinib-S-Malate, Caprelsa (Vandetanib), Cometriq (Cabozantinib-S-Malate), Doxorubicin Hydrochloride, Ipilimumab, Lenvatinib Mesylate, Lenvima (Lenvatinib Mesylate), Nexavar (Sorafenib Tosylate), Nivolumab, Vandetanib, Yervoy (Ipilimumab); for Pancreatic Cancer:

Abraxane, Nab-Paclitaxel, Sunitinib, Everolimus, Adrucil (Fluorouracil), Efudex (Fluorouracil), Erlotinib Hydrochloride, Fluoroplex (Fluorouracil), Fluorouracil, Gemcitabine Hydrochloride, Gemzar (Gemcitabine Hydrochloride), Mitomycin C, Mitozytrex (Mitomycin C), Mutamycin (Mitomycin C), or Tarceva (Erlotinib Hydrochloride); for Penile Cancer:

Blenoxane (Bleomycin), or Bleomycin; for Prostate Cancer:

Abiraterone Acetate, Cabazitaxel, Degarelix, Denosumab, Docetaxel, Enzalutamide, Jevtana (Cabazitaxel), Leuprolide Acetate, Lupron (Leuprolide Acetate), Lupron Depot (Leuprolide Acetate), Lupron Depot-3 Month (Leuprolide Acetate), Lupron Depot-4 Month (Leuprolide Acetate), Lupron Depot-Ped (Leuprolide Acetate), Prednisone, Prolia (Denosumab), Provenge (Sipuleucel-T), Radium 223 Dichloride, Sipuleucel-T, Taxotere (Docetaxel), Viadur (Leuprolide Acetate), Xgeva (Denosumab), Xofigo (Radium 223 Dichloride), Xtandi (Enzalutamide), or Zytiga (Abiraterone Acetate); for renal clear cell carcinoma:

Afinitor (Everolimus), Aldesleukin, Avastin (Bevacizumab), Axitinib, Bevacizumab, Everolimus, Inlyta (Axitinib), Nexavar (Sorafenib Tosylate), Pazopanib Hydrochloride,

Proleukin (Aldesleukin), Sorafenib Tosylate, Sunitinib Malate, Sutent (Sunitinib Malate), Temsirolimus, Torisel (Temsirolimus), or Votrient (Pazopanibv Hydrochloride); for retinoblastoma:

Clafen (Cyclophosphamide), Cyclophosphamide, Cytoxan (Cyclophosphamide), or Neosar (Cyclophosphamide); for rhabdomyosarcoma: Cosmegen (Dactinomycin), Dactinomycin, Vincasar PFS (Vincristine Sulfate), or Vincristine Sulfate, and for the prevention of Cervical Cancer:

Cervarix (Recombinant Human Papillomavirus (HPV) Bivalent Vaccine), Gardasil (Recombinant HPV Quadrivalent Vaccine), Recombinant HPV Bivalent Vaccine, Recombinant HPV Quadrivalent Vaccine.

[82] In some embodiments of this aspect of the present invention, the pharmaceutical composition comprises a compound of formula (I), or preferably the compound having the formula (II), and, alternatively or in addition to the additional therapeutic agents of groups (A) or (A’) as set out above, one or more additional therapeutic agent(s) selected from the group (B) consisting of:

Acetylsalicylic acid, Diflunisal, Salsalate, Ibuprofen, Dexibuprofen, Naproxen, Fenoprofen, Ketoprofen, Dexketoprofen, Flurbiprofen, Oxaprozin, Loxoprofen, Indomethacin, Tolmetin, Sulindac, Etodolac, Ketorolac, Diclofenac, Nabumetone, Piroxicam, Meloxicam, Tenoxicam, Droxicam, Lomoxicam, Isoxicam, Mefenamic acid, Meclofenamic acid, Flufenamic acid, Tolfenamic acid, Celecoxib, Rofecoxib, Valdecosib, Parecoxib, Lumiracoxib, Etoricoxib, Firocoxib, Nimesulide, Licofelone, H-harpagide , Lysine clonixinate, salts thereof, and combinations thereof. Preferably, the one or more additional therapeutic agent(s) in this aspect is a selective cyclooxygenase-2 (COX-2) inhibitor. Accordingly, in some preferred embodiments, the additional therapeutic agent is selected from the group (B’) consisting of Celecoxib, Rofecoxib, Valdecosib, Parecoxib, Lumiracoxib, Etoricoxib, Firocoxib, pharmaceutically acceptable salts thereof, and combinations thereof.

Pharmaceutical formulations

[83] Another aspect of the present invention relates to a pharmaceutical formulation comprising a compound of formula (I) or, preferably, formula (II), or of formula (III), or of formula (IV), or a pharmaceutical composition as described above (which may additionally comprise the above-referenced additional therapeutic agents, triple helix forming oligonucleotides (TFOs) and/or antimetabolites), and at least one pharmaceutically acceptable carrier.

[84] The at least one pharmaceutically acceptable carrier is selected from the group consisting of peanut oil, sesame oil, cottonseed oil, com oil, olive oil and mixtures of oils; ethyl oleate, isopropyl myristate, fatty acid glycerides, acetylated fatty acid glycerides; ethanol, isopropyl alcohol, hexadecyl alcohol, glycerol and propylene glycol, polyethyleneglycol, mineral oil, petrolatum; water, sterilized water, Ringer's solution, an isotonic aqueous saline solution, a dextrose solution, a glucose solution; b -cyclodextrin, hydroxypropyl-P-cyclodextrin; microcrystalline cellulose, crystalline cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, cellulose acetate phthalate, cellulose acetate butyrate, hydroxyethyl cellulose, ethyl cellulose; and combinations thereof.

[85] In preferred embodiments, the pharmaceutically acceptable carrier is selected from the group consisting of b-cyclodextrin, hydroxypropyl-P-cyclodextrin, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, ethyl cellulose, and combinations thereof. In certain preferred embodiments, the pharmaceutically acceptable carrier is selected from the group consisting of hydroxypropyl-P-cyclodextrin, carboxymethyl cellulose and combinations thereof.

[86] The above referenced compounds, preferably the compound having the formula (II), or pharmaceutical formulations comprising a compound of any compound referred to above, preferably compound having the formula (II), comprise at least one pharmaceutically acceptable carrier as described above, whereby the said formulations are typically in the form of a capsule, cachet, tablet, lozenge, powder, granule, oral suspension or solution.

Methods of treatment involving the compounds of formula (I)

[87] A further aspect of the present invention relates to a method of treating or preventing a cancerous disease in a mammal, comprising the administration of a therapeutically effective amount of the compound of the present invention, of the pharmaceutical composition of the present invention, or of the pharmaceutical formulation of the present invention to the mammal in need of such treatment.

[88] The compounds of formula (I), the pharmaceutical composition, or the pharmaceutical formulation of the present invention is preferably administered by a route selected from the group consisting of oral, buccal, sublingual, transdermal, transmucosal, intranasal, intravenous, intraperitoneal, intramuscular, subcutaneous and intrathecal administration, preferably by a route of oral, buccal, or sublingual administration.

[89] In other embodiments of this aspect of the present invention, the additional therapeutic agent(s) is/are selected from the group (B) as described above. Preferably the additional therapeutic agent is one of the selective COX-2 inhibitors or selected from the group (B’) as described above.

[90] The method of treatment as described hereinabove may also comprise a further treatment selected from radiation therapy, surgery, or combinations thereof. Said further treatment may occur before or after the administration of the compound of formula (I), the pharmaceutical composition, or the pharmaceutical formulation as described herein.

[91] The cancerous disease to be treated or prevented in the above aspects of the present invention is preferably susceptible to PI3Ka mutation and/or overactivation, and/or amplification, and/or deregulation. The cancerous disease may also be induced or triggered by the presence of such PI3Ka mutation.

[92] Preferably, the cancerous disease to be treated or prevented in the above aspects of the present invention is selected from the group (C) consisting of:

acute monocytic leukemia, acute myelogenous leukemia, acute myelomonocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, adult T-cell lymphoma, astrocytoma, atypical carcinoid lung cancer, basal cell carcinoma, B -acute lymphocytic leukemia, B-cell acute lymphoblastic leukemia/lymphoma, Bladder cancer, brain cancer, breast cancer, bronchial cancer, Burkitt’s lymphoma, cancer of the bile duct, cancer of unknown primary origin, cervix cancer, chronic myeloproliferative disorder, colon cancer, diffuse large cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, gastric cancer, glioma, glioblastoma, head and neck cancer, hemagiopericytoma, hepatocellular carcinoma, Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, large cell neuroendocrine carcinoma, large granular lymphocytic leukemia, leukemia, liver cancer, lung cancer, lymphoma, medulloblastoma, melanoma, Multiple myeloma, myelodysplastic syndrome, nasopharygeal cancer, neuroblastoma, NK cell tumor, non-Hodgkin's lymphoma, oesophageal squamous cell carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, peripheral T-cell leukemia, primary plasma cell leukemia, prostate cancer, renal clear cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, small cell lung carcinoma, T-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic lymphoma, testicular cancer, thymoma, thyroid cancer, urachal cancer, uterine cancer, vaginal cancer, and combinations thereof.

[93] More preferably, the cancerous disease is selected from the group (C’) consisting of B-acute lymphocytic leukemia, Burkitt’s lymphoma, diffuse large cell lymphoma, multiple myeloma, primary plasma cell leukemia, atypical carcinoid lung cancer, bladder cancer, brain cancer, breast cancer, cervix cancer, colon cancer, endometrial cancer, gastric cancer, glioblastoma, head and neck cancer, hepatocellular carcinoma, large cell neuroendocrine carcinoma, liver cancer, medulloblastoma, melanoma, neuroblastoma, oesophageal squamous cell carcinoma, osteosarcoma, ovarian cancer, pituitary cancer, prostate cancer, renal clear cell carcinoma, retinoblastoma, rhabdomyosarcoma, small cell lung carcinoma, and combinations thereof.

[94] The compound of formula (I), the pharmaceutical compositions or the pharmaceutical formulations as described herein have been found to be particularly suitable for treating or preventing a cancerous disease selected from the group consisting of pancreatic cancer, breast cancer, prostate cancer, and lung cancer, preferably particularly pancreatic cancer or breast cancer, colon cancer, endometrial cancer, brain tumors, skin cancer, ovarian cancer, gastric cancer, lung cancer, thyroid cancer, head and neck cancer, cervical cancer, liver/biliary tract cancer, pituitary tumors, leukemia/lymphoma, neuroblastoma.

[95] In certain embodiments of the above aspects of the present invention, the cancerous disease is an adenocarcinoma. Preferably, the cancerous disease is in these embodiments selected from the group consisting of:

colon adenocarcinoma, lung adenocarcinoma, prostatic adenocarcinoma, urachal adenocarcinoma, vaginal adenocarcinoma, mammary adenocarcinoma, esophageal adenocarcinoma, bronchial adenocarcinoma, pancreatic adenocarcinoma, endometrial adenocarcinoma, ovarian adenocarcinoma, gastric adenocarcinoma, liver adenocarcinoma, thyroid adenocarcinoma, head and neck adenocarcinoma, bladder adenocarcinoma, and gastrointestinal adenocarcinoma.

[96] More preferably, the cancerous disease to be treated or prevented is a pancreatic ductal adenocarcinoma, mammary adenocarcinoma, prostatic adenocarcinoma, endometrial adenocarcinoma, ovarian adenocarcinoma, gastric adenocarcinoma, liver adenocarcinoma, thyroid adenocarcinoma, head and neck adenocarcinoma, bladder adenocarcinoma, and gastrointestinal adenocarcinoma or lung adenocarcinoma and most preferably selected from pancreatic ductal adenocarcinoma, mammary adenocarcinoma, endometrial adenocarcinoma, ovarian adenocarcinoma, gastric adenocarcinoma, liver adenocarcinoma, thyroid adenocarcinoma, head and neck adenocarcinoma, bladder adenocarcinoma, and gastrointestinal adenocarcinoma, and lung adenocarcinoma. Compounds and pharmaceutical compositions for use in medicine

[97] Another aspect of the invention relates to the compounds of formula (I) or, preferably of formulas (II) or (III) or (IV), the pharmaceutical compositions (optionally comprising one or more additional therapeutic agent(s), and/or antimetabolites), or the pharmaceutical formulations comprising the compounds or pharmaceutical compositions referred to above for use in medicine.

[98] In some embodiments, said compounds, compositions and formulations are preferably used for treating or preventing a cancerous disease in a mammal. The mammal is preferably human. The cancerous disease in particular is a cancer which is susceptible to, caused by or triggered by a PI3Ka mutation as described above. Thus, the preferred subjects to be treated by administering the compounds, compositions or formulations of the present invention exhibit a PDKa mutation which is susceptible to allosteric inhibition by the compounds of formula (I). More preferably, the subjects to be treated carry a mutation in the gene encoding PI3Ka which leads to occurance of a PI3Ka mutant as described above, especially the H1047R mutant. The subjects are treated by administering the compounds, compositions or formulations of the present invention in order to prevent an outbreak of a cancerous disease or to treat an already existing cancerous disease.

[99] In preferred embodiments, said compounds, compositions and formulations for the above-referenced medical use comprise a therapeutically effective amount of the above referenced compounds of formula (I) for administration to a subject in need thereof.

[100] Any of the compounds of formula (I) described herein, preferably the compound of formula (II) or (III) or (IV), the pharmaceutical composition, or the pharmaceutical formulation of the present invention, both preferably comprising the compound having the formula (II) or (III) or (IV), is preferably administered by a route selected from the group consisting of oral, buccal, sublingual, transdermal, transmucosal, intranasal, intravenous, intraperitoneal, intramuscular, subcutaneous and intrathecal administration, preferably by a route of oral, buccal, or sublingual administration. [101] In other embodiments of this aspect of the present invention, the additional therapeutic agent(s) is/are additionally or alternatively selected from the group (B) as described above in the compounds and pharmaceutical compositions section. Preferably, the additional therapeutic agent is one of the selective COX-2 inhibitors selected from the group (B’) as described.

[102] In some embodiments, the compounds, pharmaceutical compositions, or the formulations comprising the compounds or pharmaceutical compositions referred to above for use in methods of treating a cancerous disease which is susceptible to PI3Ka mutation and/or overactivation, and/or amplification, and/or deregulation.

[103] [0087] Preferably, the cancerous disease to be treated or prevented in the above embodiments of the present invention is selected from the group (C) consisting of:

acute monocytic leukemia, acute myelogenous leukemia, acute myelomonocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, adult T-cell lymphoma, astrocytoma, atypical carcinoid lung cancer, basal cell carcinoma, B -acute lymphocytic leukemia, B-cell acute lymphoblastic leukemia/lymphoma, Bladder cancer, brain cancer, breast cancer, bronchial cancer, Burkitt’s lymphoma, cancer of the bile duct, cancer of unknown primary origin, cervix cancer, chronic myeloproliferative disorder, colon cancer, diffuse large cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, gastric cancer, glioma, glioblastoma, head and neck cancer, hemagiopericytoma, hepatocellular carcinoma, Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, large cell neuroendocrine carcinoma, large granular lymphocytic leukemia, leukemia, liver cancer, lung cancer, lymphoma, medulloblastoma, melanoma, multiple myeloma, myelodysplastic syndrome, nasopharygeal cancer, neuroblastoma, NK cell tumor, non-Hodgkin's lymphoma, oesophageal squamous cell carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, peripheral T-cell leukemia, primary plasma cell leukemia, prostate cancer, renal clear cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, small cell lung carcinoma, skin cancer, T-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic lymphoma, testicular cancer, thymoma, thyroid cancer, urachal cancer, uterine cancer, vaginal cancer, and combinations thereof. [104] More preferably, the cancerous disease is selected from the group (C’) consisting of B-acute lymphocytic leukemia, Burkitt’s lymphoma, diffuse large cell lymphoma, multiple myeloma, primary plasma cell leukemia, atypical carcinoid lung cancer, bladder cancer, brain cancer, breast cancer, cervix cancer, colon cancer, gastric cancer, glioblastoma, hepatocellular carcinoma, large cell neuroendocrine carcinoma, liver cancer, endometrial cancer, medulloblastoma, melanoma, neuroblastoma, oesophageal squamous cell carcinoma, osteosarcoma, ovarian cancer, endometrial cancer, prostate cancer, skin cancer, renal clear cell carcinoma, retinoblastoma, rhabdomyosarcoma, small cell lung carcinoma, thyroid cancer, and combinations thereof.

[105] The compounds, preferably the compound of formula (II), or (III) or (IV), the pharmaceutical compositions or the pharmaceutical formulations comprising the compounds or the pharmaceutical compositions as described herein above have been found to be particularly suitable for use in treating a cancerous disease selected from the group consisting of breast cancer, endometrial cancer, colon cancer, brain cancer, skin cancer, ovarian cancer, gastric cancer, lung cancer, thyroid cancer, head and neck cancer, cervical cancer, pancreatic cancer, liver/biliary tract cancer, pituitary tumors, urological tumors, leukemia/lymphoma, neuroblastoma, preferably particularly colon cancer, brain cancer, endometrial cancer, or breast cancer.

[106] In some embodiments of the above aspects of the present invention, the cancerous disease is an adenocarcinoma. Preferably, the cancerous disease is in these embodiments selected from the group consisting of: pancreatic ductal adenocarcinoma, mammary adenocarcinoma, prostatic adenocarcinoma, endometrial adenocarcinoma, ovarian adenocarcinoma, gastric adenocarcinoma, liver adenocarcinoma, thyroid adenocarcinoma, head and neck adenocarcinoma, bladder adenocarcinoma, and gastrointestinal adenocarcinoma or lung adenocarcinoma and most preferably selected from pancreatic ductal adenocarcinoma, mammary adenocarcinoma, endometrial adenocarcinoma, ovarian adenocarcinoma, gastric adenocarcinoma, liver adenocarcinoma, thyroid adenocarcinoma, head and neck adenocarcinoma, bladder adenocarcinoma, and gastrointestinal adenocarcinoma, and lung adenocarcinoma. Method for the manufacture of compounds of formula (I)

[107] Another aspect of the invention refers to a method for the manufacture of any compound of formula (I) described herein. In general, the compounds of formula (I) are prepared by alkylating dichloro analogs with formula (V)

wherein L is independently H or OH; X is independently N or C; Y is independently H or =0,

by reaction with a compound of formula Ri-Z-H, in which Z is independently S or NH, and Rl is independently a mono- or polycyclic aryl, heteroaryl, or cycloalkyl, including, but not limited to, the following examples:

wherein R ₂ is independently at least one of hydroxyl, hydrogen, fluoro, chloro, or bromo; Yi is independently C, O, S, NH or N, wherein the definitions for Y i being O, S and NH preferably apply to the five-membered aromatic ring system and the definition for Yi being N preferably applies to the six-membered ring, to give the corresponding substituted benzyl or pyridine of formula (I). Optionally, the compound of formula (I) is subsequently converted to the corresponding, pharmaceutically acceptable salt form.

[108] In a preferred embodiment, the method relates to the preparation of the compound of formula (II), or its pharmaceutically acceptable salt form, by reacting a compound of formula Ri-Z-H, where Ri and Z are defined as above, and a dichloride of formula (V)

where Ri , L, Z, X are defined as above, and Y = H, to yield the compound of formula (II), and, optionally, converting it to its corresponding salt form.

[109] In other preferred embodiments, the method relates to the preparation of the compounds of formula (III) or (IV), or their pharmaceutically acceptable salt form, by reacting a compound of formula Ri-Z-H, where Ri and Z are defined as above, and a dichloride of formula (V)

oe EtN3, 25-80oC, 6-24 hr where R _ls L, Z, X are defined as above, and Y is =0, to yield the compound of formula (III), or (IV), and, optionally, converting it to its corresponding salt form.

[110] Corresponding chlorides of formula (V) may be commercially available or may be synthesized with the following example scheme

77%

[111] Preferably, the compounds of formula (I), and in particular of formula (II), may be synthesized according to the route depicted in Example 1 (cf. Example 1 for details on the reaction conditions, solvents etc.). It is an especially preferred embodiment of the invention to crystallize the product from H ₂ 0 and MeOH, resulting in a high yield and/or purity of the product. In this regard, it will be readily apparent to the person of skill in the art how to synthesize compounds of formula (I) other than the compound of formula (II) by carrying out similar reaction steps with the respective intermediates, analogously to the reaction scheme shown in Example 1.

[112] Preferably, the compounds of formula (I), and in particular of formula (III), may be synthesized according to the route depicted in Example 2 (cf. Example 2 for details on the reaction conditions, solvents etc.). In this regard, it will be readily apparent to the person of skill in the art how to synthesize compounds of formula (I) other than the compound of formula (II) by carrying out similar reaction steps with the respective intermediates, analogously to the reaction scheme shown in Example 2.

[113] Preferably, the compounds of formula (I), and in particular of formula (IV), may be synthesized according to the route depicted in Example 3 (cf. Example 3 for details on the reaction conditions, solvents etc.). In this regard, it will be readily apparent to the person of skill in the art how to synthesize compounds of formula (I) other than the compound of formula (II) by carrying out similar reaction steps with the respective intermediates, analogously to the reaction scheme shown in Example 3.

Assay for determining the activity of PI3Ka or PI3Ka mutants.

[114] Another aspect of the present invention is a new activity assay for phosphatidylinositol 3 -kinase a as already mentioned earlier in this specification. The assays described to date are either time-consuming, or require specialized equipment. The assay described here is fast and uses common equipment. By faithfully recapitulating the properties of the membrane bound enzyme, the assay assesses quantitatively the activity of PI3Ka.

The assay includes

a)-a solid phase where GST-GRP1 -molecules become immobilized^

b) incubation of PI3Ka or PI3Ka mutant with buffer, ATP and PIP2, for the catalysis of the conversion of PIP2 to PIP3,

c) addition of competitor PIP3, carrying a detectable label or reporter molecule, and d) determining enzyme activity based on the amount of PIP3 obtained in step b) which competes with competitor PIP3 for binding to the functionalized solid phase.

The novel assay is based on the high affinity and specificity by which PIP3, the product of the reaction catalyzed by PI3K a , binds to proteins with pleckstrin homology (PH) domain, such as the General Receptor for Phosphoinositides Isoform 1 (GRP1 PH), whereas the substrate of the PDKa -catalyzed reaction, PIP2, binds only minimally to GRP1 (Lindsay et al. J Cell Sci. 119:5160-5168 (2006), Ferguson et al. Mol Cell 6:373-84 (2000), Fleming et al. Biochem J 351: 173-182 (2000)). The inventive activity assay accordingly measures the activity of PI3Ka or PI3Ka mutants by determining the amount of enzymatically produced PIP3 in an indirect, competitive test format. Furthermore, the effect of (potential) inhibitor molecules on the activity of PI3Ka or PI3Ka mutants can be determined by means of the inventive assay. [115] Preferably, the test is performed by providing a functionalized solid phase, preferably a well plate, onto which GRP1 molecules are fixated, preferably as a GST (glutathione-S transferase)-GRPl complex bound to a glutathione-coated solid phase. To this purpose, GST-GRP1 can be produced by expressing a cDNA encoding the fusion protein GST-GRP1 in an appropriate expression system. Such expression system preferably is a bacterial expression system for recombinant protein expression, preferably an E.coli based expression system, especially an E.coli strain BL2l(DE3) into which the cDNA is transformed after insertion into an appropriate expression vector. The expressed protein can e.g. be purified by affinity chromatography, preferably using a glutathione-Sepharose 4B chromatography material.

[116] The assay includes addition of PIP3 competitor molecules, which preferably carry a detectable label or a reporter molecule which upon further reaction enables detection of the presence of the competitor-PIP3. In an especially preferred embodiment, the competitor-PIP3 is biotin-PIP3 and in a further reaction a label which is present on the biotin binding partner streptavidin is used for detection. An especially preferred label is horseradish peroxidase (HRP) which when contacted with an appropriate substrate provides for a readily detectable colour or fluorescence signal. Quantification may be performed by adding a binding partner of biotin which carries a detectable label, e.g. a streptavidin-HRP -conjugate, followed by incubation with a substrate of HRP that produces a detectable product, e.g. OBD which is converted into a product that absorbs light at measuring absorbance at 492 nm. Quantification of the amount of captured competitor-PIP3 can be performed directly or at a later stage of the test procedure.

[117] In one preferred manner of performing the assay, the competitor-PIP3 is added directly to the functionalized solid phase and forms a stable complex with the GST- GRP1 -complex on the solid phase whereupon captured competitor-PIP3 is quantified after removal of excess reactants.

[118] In another preferred embodiments of the invention, the competitor-PIP3 is added to the solid phase together with or after addition of the PIP3 product of the PI3Ka or PBKcr mutant catalyzed reaction. Quantification in all these cases allows to determine the amount of enzymatically produced PIP3 due to the competitive binding of PIP3 and competitor-PIP3 to the solid phase and the proportionally reduced signal provided via the detectable label included with the competitor-PIP3. The higher the activity of PI3Kcr or PI3Kcr mutant, the greater is the amount of PIP3 which is produced and the lower amount of competitor-PIP3 will be bound to the solid phase. Comparison with a standard curve of the signal provided by the label included with the competitor-PIP3 allows for an assessment of the relative activity of PI3Ka or PDKff mutant.

[119] A PI3Kcr or PI3Ka mutant catalyzed reaction is preferably set up by providing the enzyme(s) in a reaction mixture together with ATP and with PIP2 as the enzyme reaction substrate. In a preferred emnbodiment, ATP is added to the enzyme(s) and a preincubation step is performed. Such preincubation is preferably effected at a temperature of 0°C to 35°C, more preferably at l5°C to 30°C and most preferably at room temperature of about 20°C to about 28°C, especially 25°C. The preincubation step usually is performed for a time period of about 5 to 20 minutes, depending on the temperature and overall conditions.

[120] The enzymatic reaction is then initiated by adding a substrate solution containing PIP2. As the substrate for the enzymatic reaction, a water-soluble PIP2 can be used carrying short side chains (Gray et al, Anal Biochem 313(2):234-245 (2003), Carson et al., Biochem J 409(2):519-524 (2008)). In such case, all reagents are present in the water phase. Furthermore, “natural” PIP2 contained in liposomal membranes and carrying long, natural fatty chains can be employed (Lingaraj et al, J Biomol Screen 13(9):906-9l 1 (2008), Mandelker et al, Proc Natl Acad Sci U S A 106(40): 16996-17001 (2009)). In such case, the reaction mixture contains two phases and the enzymatic activity is exerted at the interphase between membranes and water, which even better simulates the physiological microenvironment of the enzymatic reaction at cellular membranes and thus is a preferred embodiment of the present invention.

[121] The reaction mixture including the enzyme or enzyme mutant and the substrate in an appropriate solution, preferably a buffered solution, and ATP is incubated for a further appropriate time period, preferably 5 to 20 minutes and especially 8 to 12 minutes, at the same temperature as indicated above for the optional preincubation step. After termination of the reaction, the mixture which now includes enzymatically produced PIP3 is transferred to contact the GST-GRP1 functionalized solid phase. As mentioned above and as the skilled person will be well aware of, the competitor PIP3 can be contacted with the solid phase already earlier or added simultaneously with the reaction mixture subsequently. Determining the signal provided by the label or the reporter molecule of the competitor PIP3 and the reduction thereof as compared to an earlier signal obtained for the functionalized solid phase without the reaction mixture or compared to standard curve provided for the determination of PIP3 amounts allows for quantification of the enzymatically produced PIP3.

[122] The inventive assay allows to measure the activity of PI3Ka or the PI3Ka mutant with and without the presence of a potential inhibitor or modulator molecule included in the reaction mixture. In the context of the present invention, the assay is a valuable asset to determine the effect of such potential inhibitor or modulator on the proteins and thus their potential use in medicine, especially cancer treatment as explained above in detail.

[123] Having described the various aspects of the present invention in general terms, it will be apparent to those of skill in the art that many modifications and slight variations are possible without departing from the spirit and scope of the present invention.

EXAMPLES

Example 1. Synthesis scheme for the generation of the compound of formula (II) Preparation of 2,6-bis(((l/7-benzo[i/]imidazol-2-yl)thio)methyl)pyridine

[124] Preparation of 2,6-bis(((l//-bcnzo[i/]imidazol-2-yl)thio)mcthyl)pyridinc.

In a solution of 1 //-bcnzo[i/]imidazolc-2-thiol (100 mg, 0.67 mmol) in EtOH under argon atmosphere, NaOH was added (27 mg, 0.67 mmol). The reaction mixture was stirred in 25°C for 30 min. Next, a solution of 2,6-bis(chloromethyl)pyridine, in its hydrochloric acid salt form, was added (72 mg, 0.34 mmol) in EtOH (1.7 mL) and the reaction was refluxed at 65°C. After 4 hours the reaction was cooled down to 25°C and a solution of NaOH 1N (0.5 mL) was added until pH = 9. The reaction was concentrated in vacuo and the product was crystallized from H ₂ 0 and MeOH (114 mg, white solid in 85% yield).

[125] 1H NMR (500 MHz) 25 °C, DMSO: d 12.63 (s, 2H, -N H-), 7.71 (t, J= 7.7 Hz, 1H, Ar H), 7.44 (s, 6H, Ar H), 7.11 (dd, J = 5.4, 2.8 Hz, 4H, Ar H), 4.65 (s, 4H, - CH2-).

[126] MS: [M-H] _caie. : 402.1; [M-H] _exper. : 401.7 Example 2. Synthesis scheme for the generation of the compound of formula (III)

Preparation of dibenzylpyridine^, 6-dicarboxamide

[127] Preparation of dibenzylpyridine^, 6-dicarboxamide. In a solution of phenylmethanamine (0.2 mL, 0.49 mmol) in toluene (3 mL), pyridine-2, 6-dicarbonyl dichloride (50 mg, 0.25 mmol) was added. The reaction mixture was stirred for 12 hours at 25°C under argon atmosphere. Next, aqueous solution of NaOH 1M (3 mL) is added in the reaction mixture and the reaction is extracted with EtOAc. The organic layer was washed with saturated aqueous NaCl solution, dried over Na ₂ S0 ₄ , and condensed under reduced pressure. The emerging solid was crystallized from toluene to produce /V ² ,/V ⁶ -dibenzylpyridine-2, 6-dicarboxamide (60 mg, 70% yield).

[128] 1H NMR (500 MHz) 25 °C, DMSO: d 8.39 (d, J= 7.8 Hz, 2H, Ar H), 8.04 (t, J= 7.5 Hz, 1H, Ar//), 7.33 - 7.28 (m, J= 3.9 Hz, 10H, Ar//), 4.65 (s, 4H, -C// ₂ -) ppm

[129] MS: [M+H] ⁺ _caic. : 346.2; [M+H] ⁺ _exper. : 345.8

Example 3. Synthesis scheme for the generation of the compound of formula (IV)

Preparation of hydroxypheny pyridine- , 6-dicarboxamide

[130] Preparation of A ² ^-bisiS-hydroxypheny^pyridine-Z, 6-dicarboxamide.

In a solution of 3-aminophenol (54 mg, 0.49 mmol) together with Et ₃ N (70 pL, 0.49 mmol) in THF (1 mL), pyridine-2, 6-dicarbonyl dichloride (50 mg, 0.245 mmol) in THF (2.5 mL) at -20 °C, was added. The reaction mixture was stirred for 2 hours at 25°C under argon atmosphere. The reaction mixture was condensed under reduced pressure and the emerging solid was crystallized from MeOH to produce N ² ,N ⁶ -bis(3- hydroxyphenyl)pyridine-2, 6-dicarboxamide (25 mg in 29% yield). [131] 1H NMR (500 MHz) 25 °C, DMSO: d 10.90 (s, 2H, -NH- or -OH), 9.53 (s,

2H, -NH- or -OH), 8.38 (d, J= 7.6 Hz, 2H, ArH), 8.29 (t, J= 7.6 Hz, 1H, Ar H), 7.47 (s, 2H, Ar H), 7.32 (d, J= 7.9 Hz, 2H, ArH), 7.21 (t, J= 8.0 Hz, 2H, ArH), 6.59 (d, J = 7.8 Hz, 2H, ArH) ppm [132] MS: [M+H] ⁺ _calc. : 350.1; [M+H] ⁺ _cxpcr. : 349.7

Example 4. In vitro cell-free PI3Ka activity assays using PIP2 containing liposomes for PI3K-010. [133] To evaluate the in vitro activity of the compound having the formula (II)

(PI3K-010), PI3Ka activity assays were performed using PIP2 containing liposomes (Gkeka et al PLOS Comp Biol 2014). In this assay, PIP3 molecules produced by PI3Ka compete with biotinylated PIP3 for binding to recombinant GST-GRP1-PH domain (amino acids 263- 380), which has been produced in bacteria, bound to glutathione coated 96-well plates. Quantitation of the competed amount of biotin- PIP3 is estimated by the peroxidase activity of streptavidin-HRP that binds to the plate and provides a measure for the activity of the protein. The compounds were preincubated with all the components of the assay mixture, except PIP2, for 10 min, at 25°C. PIP2 is added in the end of the preincubation period, thereby initiating the kinase reaction.

[134] [0108] IC50 values for compound with formula (II)

were calculated from dose- response curves using logit-log plots for the wild type PI3Ka protein as well as the mutated PI3Ka protein containing the H1047R mutation.

[135] All assays were carried out in at least two independent experiments and each concentration in triplicate. PIK-108 and wortmannin were used as controls (Gkeka et al, 2014 J Phys Chem B). The compound with formula (II), PI3K-010, showed an 11- fold inhibition selectivity for the mutant PI3Ka H1047R protein compared to the wild-type PI3Ka (cf. Figure 1).

[136] Next, experiments in low (100mM) and high ATP (2mM) concentrations were performed in order to identify whether PI3K-010 is an ATP-competitive inhibitor. We found that ATP concentration is only slightly affecting the IC50 of PI3K-010, and thus this compound can be considered a non-competitive inhibitor (cf. Figure 2).

Example 5. In vitro cell-free PI3Ka activity assays using PIP2 containing liposomes for PI3K-021.

[137] To evaluate the in vitro activity of the compound having the formula (III) (PI3K-010), PI3Ka activity assays were performed using PIP2 containing liposomes (Gkeka et al PLOS Comp Biol 2014). In this assay, PIP3 molecules produced by PI3Ka compete with biotinylated PIP3 for binding with recombinant GST-GRP1-PH domain (amino acids 263- 380), which has been produced in bacteria, bound to glutathione coated 96-well plates. Quantitation of the competed amount of biotin- PIP3 is estimated by the peroxidase activity of streptavidin-HRP that binds to the plate and provides a measure for the activity of the protein. The compounds were preincubated with all the components of the assay mixture, except PIP2, for 10 min, at 25 °C. PIP2 is added in the end of the preincubation period, thereby initiating the kinase reaction.

[138] IC50 values for compound with formula (III)

were calculated from dose-response curves using logit-log plots for the wild type PI3Ka protein as well as the mutated PI3Ka protein containing the H1047R mutation.

[139] All assays were carried out in at least two independent experiments and each concentration in triplicate. The compound with formula (III), PI3K-021, showed a selectivity of >100 fold for the mutant PI3Ka H1047R protein compared to the wild- type PI3Ka (Figure 3). The IC50 of PI3K-021 was found to be 13.5 mM for the mutant H1047R RBKa, while for the WT the exact IC50 value could not be estimated due to the very low inhibition of PIK-021 against the WT protein even in the highest tested concentrations of PI3K-21. By extrapolation we estimated the IC50 of PI3K- 021 to be > 1000 mM for the WT PI3Ka (cf. Figure 3).

Example 6. In vitro cell-free PI3Ka activity assays using PIP2 containing liposomes for PI3K-024.

[140] To evaluate the in vitro activity of the compound having the formula (IV) (PI3K-024), PI3Ka activity assays were performed using PIP2 containing liposomes (Gkeka et al PLOS Comp Biol 2014). In this assay, PIP3 molecules produced by PI3Ka compete with biotinylated PIP3 for binding with recombinant GST-GRP1-PH domain (amino acids 263- 380), which has been produced in bacteria, bound to glutathione coated 96-well plates. Quantitation of the competed amount of biotin- PIP3 is estimated by the peroxidase activity of streptavidin-HRP that binds to the plate and provides a measure for the activity of the protein. The compounds were preincubated with all the components of the assay mixture, except PIP2, for 10 min, at 25 ° C. RGR2 is added in the end of the preincubation period, thereby initiating the kinase reaction.

[141] IC50 values for compound with formula (IV)

were calculated from dose- response curves using logit-log plots for the wild type PI3Ka protein as well as the mutated PI3Ka protein containing the H1047R mutation.

[142] All assays were carried out in at least two independent experiments and each concentration in triplicate. The compound with formula (IV), PI3K-024, showed showed nanomolar inhibition for both WT and mutant proteins (IC50 = 6 nM for the WT and 64 nM for the mutant H1047R PI3Ka proteins) (cf. Figure 4).

Example 7. PI3K-010 or PI3K-021 treatment of xenografts generated by heterotopic transplantation of the breast cancer MDA-MB-231 cell line or the breast cancer HCC1954 cell line.

[143] The inventors evaluated the in vivo activity of the compound having the formula (I) (PI3K-010) and of the compound having the formula (II) (PI3K-021), on mouse bearing heterotopic xenografts after subcutaneous injection of cells from the breast cancer MDA-MB-231 cell line, which is wild-type PIK3CA and of the breast cancer HCC1954 cell line, which bears exon 20 H1047R mutation on PIK3CA. For transplantation, eight-week old NOD/SCID mice were anesthetized using a steady flow of a mixture of isofluorane gas (4%) and oxygen and inoculated subcutaneously into their lateral flanks with 10 ⁶ cancer cells and monitored daily until the tumors became palpable (tumor volume -100 mm ). The mice were then divided randomly into two groups for PI3K-010 or vehicle administration for a period of two weeks. Administration was achieved by mixing PI3K-010 or PI3K-021 with com oil and subsequent oral dosing in lOOmg/Kg doses twice a day. Average weights + S.E.M (n=7 for MDA-MB-231 and n-5 for HCC1954) are indicated. Figure 5 illustrates the gross appearance of surgically recovered PI3K-0l0-treated or PI3K-021 or untreated (control) xenografts.

Example 8. Micro PET/CT imaging of WAPCre/exon20 Pik3ca*/MMTV-Myc mouse

[144] PET/CT images of a WAPCre/exon20 Pik3ca*/MMTV-Myc mouse were acquired at the beginning and end of treatment (two weeks) of breast cancer with the PI3K-010 compound (cf. Figure 6). The compound was administered per os (by gavage) at lOOmg/Kg doses. The control mouse received only solvent. The diminished uptake of 18F-FDG by remnants of breast cancer is evident. The white arrows indicate the anatomical sites of breast. High-level concentration of 18F-FDG in the urinary bladder (excretion of the tracer) and also in the heart is noted. To further study the effect of PI3K-010, we surgically removed breast tumors. Immunohistochemical analysis of the xenografts using an antibody against the cell proliferation marker Ki67 showed tumor necrosis and reduced proliferation after treatment in comparison with the controls (proliferation index 15.0+3.3% and 69.9+5.1%, respectively)

Example 8. Establishment of a new membrane-based activity assay for phosphatidylinositol 3-kinase a.

This example describes the development of a method for the measurement of lipid PI3Ka activity which is quite robust, easy to carry out, quick, inexpensive, does not need specialized equipment and can be used in a high throughput mode.

[145] As described above, the central idea of the assay is based on the high affinity and specificity by which PIP3, the product of the catalyzed reaction, binds to proteins with pleckstrin homology (PH) domain, such as the General Receptor for Phosphoinositides Isoform 1 (GRP1 PH). This assay was used to determine the activity of PI3Ka wild type and oncogenic mutants H1047R and E545K in the basal state but also when physiologically activated by phosphorylated receptors tyrosine kinases. In the process of validating the assay three different types of PIP2 substrates were tested, a water soluble diC8PtdIns(4,5)P2 and two“liposomal” forms where different quantities of long acyl side chains PIP2 were incorporated to natural lipids that were extracted from HCT116 cells, so as to simulate as best as possible the physiological membrane status. The assay was also used to test and evaluate several “denovo” synthesized compounds in order to find mutant specific inhibitors.

Materials and Methods

Preparation of liposomes

[146] For the preparation of PIP ₂ -containing liposomes lipids were prepared from HCT116 cells. HCT116 is a colorectal cancer cell line in which the PIK3CA gene carries the H1047R mutation in exon 20. Extracted lipids were mixed with PIP ₂ (containing long side chains, from brain, Avanti) and the mixture was dried under N ₂ stream. Two liposomal preparations were made, containing 10% or 50% PIP ₂ . After the sample was completely dried, it was left for another 30 minutes under the N ₂ stream, and it was further dried in a speed vacuum for 1 h. Then, an aqueous solution (20 mM Tris pH=7.4, 100 mM KC1, 1 mM EDTA pH=8 and 1 mM EGTA) was added and the lipid mixture was incubated at room temperature for 1 h, with occasional vortexing (every 10 min). Subsequently, the sample was subjected to 5 ffeeze/thaw cycles, followed by sonication in a waterbath for 30 minutes. Finally, the preparation was extruded using the Avanti mini-Extruder (according to manufacturer’s instructions), with a 100 nm membrane, which generates a homogeneous preparation of small unilamellar vesicles with diameter below 100 nm.

Expression and purification of GST-GRP1 domain

[147] The cDNA of GST-GRP1 was transformed into the Escherichia Coli strain BL21 (DE3). GST-GRP1 was affinity purified on glutathione-Sepharose 4B (Amersham Pharmacia), using the manufacturers standard protocols. The purity of the protein was determined by SDS-PAGE.

PI3 Kinase activity assay

[148] 96-well plates, coated with glutathione, were incubated with blocking buffer (50 mM Tris pH=7.4, 150 mM NaCl, 0.05% Tween and 0.2% gelatin) for 1 h at room temperature (RT), under shaking. Then, 1 pg purified GST-GRP1 was added to each well and incubated for 1 h at RT, under shaking. Finally, the wells were washed 5 times with wash buffer (50 mM Tris pH=7.4, 150 mM NaCl and 0.2% gelatin), for 2 min per washing step. In parallel, PI3K reactions were set up in micro-tubes by the addition of 5 mΐ reaction buffer (250 mM Tris pFI=7.4, 10 mM MgCl ₂ ), 2.5 mΐ ATP (100 mM), 2 mΐ BSA (10 pg/pl), 1-2 pl PI3K (2 ng/pl), 1 pl of the compound (inhibitor) under investigation, and FI ₂ 0 to a final volume of 20 pl, and the mixture was incubated for 10 min at 25°C (pre-incubation step). The reaction was initiated by the addition of 5 pl substrate solution (containing 40 ng/pl PIP ₂ ) (200 ng total PIP ₂ ) and allowed to proceed for 8 to 12 min at 25°C. The reaction was terminated by the addition of 75 pl stop solution, which contains 5 mM EDTA, 1% BSA, 0.07% sodium deoxycholate and 0.3 ng biotinylated-diC8PtdIns(3,4,5)P3 (biotin-PIP ₃ ), pH=8, in wash buffer, and the whole mixture was transferred to the wells of the 96-well plate (which contains GST-GRP1, prepared as described above) and was incubated for 1 h at RT, under shaking (during this step, the PIP ₃ product of the PI3K reaction competes with biotin-PIP ₃ for binding to GST-GRP1). The wells were washed 5 times with wash buffer, 2 min each, and streptavidin-HRP conjugate was added and incubated for 40 min at RT under shaking. The wells were washed 5 times, 2 min each, and the substrate of HRP (OPD, o-phenylenediamine) was added and incubated for 1 -20 min (until the development of a brown colour). The absorbance of the solution in the wells was read at 492 nm using an ELISA reader. For the evaluation of inhibitors, or the phosphorylated PDGFR peptide, or the screening of the new compounds, 1 or 2 mΐ of the tested molecule (in the appropriate buffer) was added to the PI3K reaction mixture before the pre-incubation step, before the addition of the PIP ₂ substrate. As a control, the effect of an equal volume of the solvent was tested under the same conditions.

[149] To calculate the amount of PIP3 produced by the enzymatic reaction (in AU), which allows evaluation of the levels of PI3K activity, a standard curve of the A ₄ 9 _2nm against PIP3 was prepared. To generate the standard curve, i.e. absorbance - versus - arbitrary units of PIP3 molecules, a PIP3 stock solution was used that was prepared by incubating PIP2 with PI3K under the regular PI3K reaction conditions. This final preparation served as the standard of PIP3 molecules (in AU) for the generation of the standard curve. The standard curve follows the anticipated hyperbolic profile of antagonistic inhibition between PI3K-produced PIP3 and b-PIP3 for binding to GST- GRP1. Although the use of the above standard curve allows only assessment of the amount of PIP3 molecules in arbitrary units (AU), this measurement is sufficient for comparative in vitro studies, e.g. experiments that address the molecular mechanisms regulating the activity of PI3K, or in small molecule screening studies aiming to identify inhibitors of PI3K.

Results

Principle of the new PI3Kinase a activity assay

[150] To establish a simple, membrane -based PI3Ka activity assay, we took advantage of the fact that the product of the catalytic activity of RBKa, the membrane lipid PIP ₃ , binds with high affinity and selectivity to the PH domain of GRP1, while the substrate of the reaction, PIP ₂ , binds only minimally to GRP1 (Lindsay et al. J Cell Sci 119:5160-5168 (2006): Ferguson et al. Mol Cell 6:373-384 (2000); Fleming et al. Biochem 7 51: 173-182 (2000)). Thus, to assay the activity of PI3Ka, we used the PH domain of GRP1 fused to GST (for simplicity, from here on called GST- GRP1), bound to glutathione-coated 96-well plates, to capture the PIP ₃ molecules produced by PI3K. To quantify the amount of produced PIP ₃ , we employed an indirect, competition-based assay. To this end, following the termination of the enzymatic reaction, biotin-PIP ₃ is added exogenously to the mixture containing the PI3Ka reaction products (to function as a competitor of the binding between GRP1 and PIP3), and the whole mixture is incubated with the wells of the 96-well plate (Figure 7); during this incubation, both PIP ₃ (the product of the PI3K reaction, shown in red) and biotin-PIP ₃ (added exogenously) form independent complexes with GST- GRP1 in a competitive maimer. Finally, streptavidin-HRP is added to the wells, to assess the amount of bound biotin-PIP ₃ . When PI3K is present, the higher the PI3Ka activity, the greater amount of PIP ₃ is produced, the lower amount of biotin-PIP ₃ & streptavidin-HRP will be bound to the plate, which leads to a lower value of the absorbance at 492 nm (Figure 7). On the contrary, in the presence of a PI3Ka inhibitor, the levels of PIP ₃ are reduced proportionally to the effectiveness of the inhibitor, which results in a higher binding of biotin-PIP ₃ & streptavidin-HRP to the wells of the plate, thereby leading to a higher absorbance at 492 nm. Thus, the stronger the inhibitor, the higher the absorbance. A standard curve of the absorbance at 492nm against increasing levels of PIP ₃ allows assessment of the relative activity of PDKa in arbitrary units (see further below, Fig. 12).

Establishment of the conditions of the PI3K activity assay

[151] All reagents and conditions of the assay have been optimized with the aim to achieve low noise, maximum possible dynamic range and specificity. At first the amount of GST-GRP1 that should be bound to plates to capture sufficient amount of biotin-PIP ₃ , was optimized to produce a high absorbance at 492 nm. GST-GRP1 was expressed in bacteria and was purified using glutathione-coated beads (Fig. 8a). To determine the optimum amount of GST-GRP1 that results in sufficiently high signal, increasing amounts of GST-GRP1 were added to glutathione coated plates, followed by incubation with biotin-PIP3, streptavidin-HRP and measurement of the absorbance at 492 nm. As shown in Fig. 8a, the optimum quantity of GST-GRP1 was 3 pg, as a higher amount of the protein (10 pg) did not significantly improve the absorbance at 492 nm.

[152] Then, the amount of b-PIP3 that should be used in the assay was optimized. As expected, we observed that the higher the amount of b-PIP3 added to the plate, the higher the absorbance (Fig. 9). Among the amounts tested, the quantity of 0.3 pg of b- PIP3 resulted in sufficiently high absorbance, while higher amount of b-PIP3 did not drastically increase the signal (Fig. 9). Since the assay is based on the competition between exogenous b-PIP3 and PIP3 produced by PI3K, the higher b-PIP3 added in the assay (beyond the amount required to produce an acceptable signal), the larger amounts of PI3K and PIP2 would be required for the competition in the assay mixture, which would raise the cost for screening libraries of compounds. Therefore, the amount of 0.3 pg was selected for subsequent assays.

[153] Next the optimum concentration of the reagents used in the PI3K reaction was determined, starting from the substrate PIP ₂ . Although a soluble version of PIP ₂ with short side chains (diC8) can be used in the assay, here the inventors sought to employ as a substrate the natural PIP ₂ (which contains long, natural fatty chains), in liposomal membranes. In this case, in the reaction mixture containing two phases (water and membranes), the enzymatic activity is exerted at the interphase between membranes and water, which simulates better the physiological microenvironment of the enzymatic reaction at cellular membranes. At first, given that PIP ₂ binds to some extent to GST-GRP1 (despite the high specificity of GST-GRP1 for PIP ₃ (Lietzke et al, Mol Cell 6(2):385-394 (2000), He et al, J Lipid Res 49(8): 1807-1815 (2008)), we tested whether PIP ₂ interferes with binding of biotin-PIP3 to GST-GRP1. When PIP ₂ was used as a sole component of the liposomal membranes (in the absence of other lipids), there was substantial inhibition of the signal even at the lowest concentration of PIP ₂ (50 ng/lOO mΐ reaction) (Fig. lOa), probably due to competitive inhibition between PIP ₂ and biotin-PIP ₃ for binding to GST-GRP1 ( Corbin et al, Biochemistry 43(51): 16161-16173 (2004), He et al, J Lipid Res 49(8): 1807- 1815 (2008), Lai et al, J Mol Biol 425(l7):3073-3090 (2013)). On the other hand, the interference of PIP ₂ with the signal was significantly reduced when PIP ₂ was incorporated in liposomes together with natural lipids extracted from HCT116 cells (compare the sample of Fig. lOb containing 200 ng PIP2 at a concentration of 10% in liposomes, with the corresponding sample in Fig. lOa), probably due to the different conformation, availability, and/or micro-environment of PIP ₂ when it is incorporated in the membranes together with other lipids. When a high concentration of PIP2 was used in liposomes (50%), there was inhibition of binding of biotin-PIP3 to GRP1 despite the presence of liposomes, which was substantially rescued by the addition of sodium deoxycholate, at a concentration below the CMC. Thus, in all subsequent experiments the substrate PIP ₂ was integrated in liposomal membranes, which have been prepared using lipids extracted from cellular membranes, while deoxycholate was added to reduce non-specific binding of lipids to GRP1.

[154] Finally, the inventors moved to the last step of the establishment of the assay, where they determined the optimal combination of PI3K levels relatively to different amounts of the substrate PIP ₂ (prepared in liposomes). Testing a stable amount of PIP2 (200 ng) in two different lipid ratios, 10% PIP2 versus 50% PIP2 in liposomes, revealed that the high concentration of PIP2 (50% in liposomes) is critical for PI3K to achieve high levels of activity. Additionally, when the soluble form of PIP2 was used as a substrate (diC8PtdIns(4,5)P2), in which the hydrocarbon chains of the ester are short, as much as 2 pg of PIP2 were required to achieve less than half of the activity reached with 200 ng of PIP2 at 50% concentration in liposomes. Thus, the water- soluble form of PIP2 is a much worse substrate in comparison to lipid PIP2 (50%) in liposomes. Therefore, the lipid form of PIP2, at 50% concentration in liposomes, is the form chosen for subsequent experiments.

[155] To identify the optimal amount of PIP2 for the assay (in the form of 50% in liposomes), we tested the combination of increasing quantities of PIP ₂ with two different amounts of PI3K, 2 and 4 ng. As shown in Fig. 1 lb, at 200 ng of PIP ₂ the reaction reaches a plateau, i.e. the levels of produced PIP3 are high enough to almost completely block the binding of biotin-PIP3 to GST-GRP1. Since the substrate needs to be in excess in order to exclude the possibility of being a limiting factor for the reaction, the amount of 200 ng of substrate was used in all subsequent experiments.

[156] Given that the assay is based on the competition between the product (PIP ₃ ) and biotin-PIP ₃ for binding to GST-GRP1, the drop of the absorbance when PIP3 is present is not expected to be directly proportional to the amount of PIP ₃ produced by PI3K. Therefore, a standard curve is necessary to convert the absorbance values, determined by the assay, to the amount of PIP3 produced by PI3K. As expected, the experimental standard curve (absorbance at 492 nm versus the amount of PIP3, added exogenously) follows a hyperbolic profile, typical for an assay where an antagonist (PIP3, produced by PI3K), competitively removes the agonist (biotin-PIP3) which is responsible for the measured signal (absorbance at 492 nm). [157] Having established the conditions of the PI3K activity assay, allowing assessment of the activity of PI3K in arbitrary units, we proceeded to the validation of the specificity of the method. At first, we tested whether the assay fulfils the hallmarks of the enzymatic reactions, i.e. dependence on the dose of the enzyme and on the duration of the reaction. Indeed, the quantified enzymatic activity was PI3K dose-dependent (Fig. l3a) as well as time dependent (Fig. 13b). As a further validation test of the assay, we employed well-known PBKinase inhibitors to test their effect on the activity of the enzyme. Data in figure 14 show that the IC50 values of known inhibitors, such as Wortmannin, LY-294002 and PIK-108, are in accordance with what is reported in the literature (figure 14).

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