WO2010136705A1

WO2010136705A1 - Pharmaceutical antitumor composition including a cdk inhibitor and a cell-growth inhibitor

Info

Publication number: WO2010136705A1
Application number: PCT/FR2010/050983
Authority: WO
Inventors: Thérèse PFEUTY
Original assignee: Centre National De La Recherche Scientifique
Priority date: 2009-05-25
Filing date: 2010-05-20
Publication date: 2010-12-02
Also published as: FR2945747A1

Abstract

The invention relates to the field of cancer therapy and specifically to a pharmaceutical composition having an antitumor effect and including at least one CDK inhibitor selected from indirubin-3'–monoxime and certain selected purine derivatives and at least one cell-growth inhibitor. The invention also relates to the combination of at least one CDK inhibitor selected from indirubin-3'–monoxime and certain selected purine derivatives, and at least one cell-growth inhibitor having synergetic antitumor effects for use as a drug for treating neoplastic diseases.

Description

ANTI-TUMOR PHARMACEUTICAL COMPOSITION COMPRISING A CdK INHIBITOR AND AN INHIBITOR OF

CELL GROWTH

The present invention relates to the field of cancer therapy and more particularly to a pharmaceutical composition with anti-tumor power comprising at least one cyclin-dependent protein kinase inhibitor.

(abbreviated as Cdks) and at least one inhibitor of cell growth, as well as the combination of at least one Cdks inhibitor and at least one cell growth inhibitor with synergistic anti-tumor effects, for use as a medicament for the treatment of neoplastic pathologies.

The goal of a cancer therapy is to interrupt or even reverse the process of tumor growth, either by inducing the death of tumor cells by apoptosis, or by directing them to a state of irreversible stopping proliferation

(called state of senescence), while sparing, as far as possible, the normal cells.

The last three decades have witnessed a relentless quest to decipher the signaling pathways that control cell life and death as well as their reproductive cycle (or cell cycle) in both normal and tumor cells. The cell cycle is conducted according to a rigorous sequential routine that requires the spatial and temporal coordination of a wide variety of biochemical processes: the cell duplicates its chromosomes during a phase called S before dividing during a phase called M (mitosis) to give birth to two daughter cells that each inherited a set of maternal chromosomes. In embryonic cells whose cytoplasm is saturated with resources inherited from the mother cells, the S and M phases follow each other at an astonishing speed. The cells begin to replicate their genetic material (DNA) as soon as they come out of mitosis and a cycle does not last more than half an hour. However, in somatic cells that have exhausted maternal resources and need to tap into their environment the resources necessary to survive and proliferate, two new phases appear: (i) the G1 phase, which intervenes between the release of mitosis and the entry into phase S, during which the cell is submerged by a stream of signals of any kind (division, growth, differentiation, adhesion, stress, ...), which it must interpret and integrate before deciding to enter in phase S or not and, possibly then, to differentiate, to enter into senescence or to pass away ; (ii) phase G2, which follows phase S and during which the cell prepares its entry into mitosis, in agreement with its environment.

Unlike embryonic cells and tumor cells, somatic cells also have the ability to retreat into a state of non-proliferative reversal (called quiescence state or GO) in which they can survive for long periods of time when they are in an unfavorable environment for their spread (overpopulation, lack of nutrients, ...).

The output of the GO phase and the ordered progression of the cells through the G1, S, G2 and M phases are governed by a family of enzymes, proteins with serine / threonine kinase activity called Cdks, whose "timing" of activation is rigorously controlled by various mechanisms: (i) their association with regulatory subunits called cyclins; (ii) activatory or inhibitory phosphorylations; (iii) their association with peptide inhibitors called CKIs; (iv) changes in intracellular localization.

Kinase-active proteins form a class of enzymes whose function is to catalyze the transfer of a phosphate group from the ATP molecule to the hydroxyl group of a serine, threonine or tyrosine residue present on an accepting target protein. Phosphorylation on one or more residue (s) of a protein is capable of altering its structure and interactions with other proteins and thus allowing the cell to switch from one physiological state to another. This is why phosphorylation reactions play an important role in many aspects of cell biology, including metabolism, growth, movement, differentiation and division. The Cdk family is known primarily for its involvement in cell cycle control, although some of its members also play a role in other physiological processes, such as transcriptional regulation, and in the central nervous system. Their activity is frequently deregulated in certain diseases, in particular in human cancers, in which certain cyclins (D and E) and / or Cdks are overexpressed.

The phase-GO output in somatic cells is dependent on continuous mitogenic stimulation that induces the accumulation of D-type cyclins and the subsequent formation and activation of cyclin D-Cdk4,6 complexes, whose primary mission is to initiate the phosphorylation of the retinoblastoma protein, Rb, thus de-expressing the transcription of the cyclin E gene. The accumulation and activation of the resulting E-Cdk2 cyclin complexes play a decisive role in the passage through the point of restriction (R) beyond which cells irreversibly engage in their division cycle, regardless of any exogenous stimulation. The hyperphosphorylation of Rb by cyclin E-Cdk2 complexes then allows the release of E2Fs transcription factors that activate the transcription of a cohort of genes involved in the regulation of the cycle. Once phase S has been initiated, cyclin E is degraded and cyclin A-Cdk2 complexes take over to orchestrate the irreversible events of DNA replication, followed by cyclin A-Cdkl complexes and cyclin B-Cdkl complexes. which induce the G2 / M transition. Finally, the destruction of cyclin B, catalyzed by the activity of the cyclin B-Cdkl complex itself, leads to the separation of the two sets of chromosomes, the cytokinesis and the birth of the two daughter cells.

Carcinogenesis is an evolutionary process that occurs when somatic cells acquire so-called oncogenic mutations that cause them to proliferate uncontrollably and escape their natural destiny of differentiating or dying. The cells thus mutated have lost the ability to interpret and integrate the multitude of often contradictory signals that they receive from their environment (division, growth, differentiation, adhesion, stress, ...) during their phase G1 and / or respond to these signals by respecting the rules of homeostasis put in place by the host organism to preserve its integrity and survival.

The products of the Rb and p53 tumor suppressor genes play a crucial role in maintaining tissue homeostasis (T. David-Pfeuty, BBA-Rev Cancer, 2006, 1765, 38-66). The Rb protein is the preferred substrate for cyclin D-Cdk4,6 complexes whose kinase activity is also downregulated by CKIs of the Ink4 family (Sherr and Roberts, Genes Dev., 1999, 13, 1501-1512). In its non-phosphorylated and hypophosphorylated forms, Rb is a potent transcription repressor in general and, in particular, regulatory genes for the cycle. Its sequential phosphorylation by the cyclin D- and cyclin E-Cdks complexes is absolutely required for the output of the GO phase. The Rb- ^regulated signaling pathway is invariably altered in all human cancers, either by Rb deletion or ^{Ink4a inactivation} or ^Cdk4 amplification or overexpression of cyclin D1 (Shapiro and Harper, J Clin Investigation, 1999). , 104, 1645-1653).

As for the p53 gene, it is lost or altered in more than 50% of human cancers and its reintroduction into p53-negative tumors, as well as its activation in response to many forms of stress, including oncogenic mutations, leads to at a cycle stop or apoptosis. The p53 protein is a powerful activator of the transcription of not less than 5000 genes among which two have been shown to play an important role as mediators of its function in the monitoring of the cycle: genes coding for p2 \ ^Cψl , a CKI of the family Cip / Kip, and for the 14-3-3s protein. P2 \ ^Cψ1 has a strong affinity for cyclin-Cdk1 complexes, 2 and its overexpression induces G1 and G2 phase stops (Sherr and Roberts, ibid). The 14-3-3s protein blocks cells in the G2 phase by preventing the activation of the mitotic cyclin B-Cdkl complex. P53 plays a vital role in maintaining the integrity of the body as it prevents the spread of damaged cells by slowing down their rate of progression in the cycle, giving them time to repair their lesions before entering However, some cell types enter apoptosis in response to the nuclear accumulation and activation of p53 which, in this case, induces the expression of one (or more) of its many pro-apoptotic target genes. P53 can also be inactivated in human cancers via overexpression of the Mdm2 protein, the product of one of its target genes which associates with it to inhibit its transcriptional activity, exclude it from the nucleus and promote its degradation, or again, via the loss of function of the small Arf protein that activates its functions by opposing those of Mdm2.

The better understanding of tumor biology has made it possible to dissect more precisely certain molecular mechanisms involved in malignant transformation (Hanahan and Weinberg, CeIl, 2000, 100, 57-70): autocrine secretion of growth factors (PDGF, EGF , ....) or mutation / overexpression of their receptors; constitutive activation of mitogenic signal transduction pathways (Ras, Src, AbI, ..); loss of function of proteins involved in the negative regulation of the cycle (p53, Rb, φ21 ^Kιpl , ...) or, on the contrary, overexpression of proteins favoring entry into the cycle (cyclins or transcription factors such as Myc and E2F , ...). These discoveries have opened the field to a new field of research focused on the rational identification of molecular targets critical for the survival of tumor cells and the development of adapted tools to inhibit them. Inhibitors or antibodies directed against proteins known to be specifically altered or deregulated in certain cancers (eg growth factor receptors, Ras, AbI, p53, Cdks) have thus reached the clinical trial phases. However, with a few exceptions, the results obtained do not yet correspond to well-founded hopes (Dancey and Sausville, Nat Rev. Drug Discov., 2003, 2, 296-313, Sawyers, Nature, 2004, 432, 294-297) . A major research effort has been devoted to chemical inhibitors of Cdks. This effort resulted in the identification of several classes of competitive ATP inhibitors with good selectivity for Cdks compared to other kinases. Two first-generation inhibitors, alvocidib (flavopiridol / HMR1275 / L86-8275) and seliciclib (CYC-202 / R-roscovitine), have reached the most advanced phases of clinical development (phases II and III). In the various clinical studies carried out on solid tumors, they demonstrated as monotherapy a modest anti-tumor activity although some cases of prolonged stabilization of the disease have been reported. Flavopiridol, however, has been shown to be remarkably effective in high-risk chronic lymphoid leukemia (Byrd et al, Blood, 2007, 109, 399-404). However, their undesirable side effects are not negligible, especially in the case of alvocidib, the most vulnerable normal tissues being bone marrow and the gastrointestinal tract. The number of second-generation Cdks inhibitors in many chemical classes that reach the early stages of clinical trials continues to grow. These molecules can be classified according to their activity profile vis-à-vis the various cyclin-Cdks complexes (Malumbres et al, TIPS, 2007, 29, 16-21, Shapiro G., J. Clin Oncol, 2006, 24 , 1770-1783). Their potential for use in cancer chemotherapy is still under intensive study.

The use of chemical inhibitors of Cdks in combination with other active ingredients, including conventional chemotherapies and new targeted therapies, has also been considered.

In conventional chemotherapy, Cdks inhibitors combined with cytotoxic agents (eg cisplatin, 5-fluorouracil, doxorubine or paclitaxel) have shown synergistic anti-tumor effects in several preclinical tests, particularly when cytotoxic agent was administered before the Cdks inhibitor (Malumbres et al, TIPS, 2007, 29, 16-21). The published summary of Froessler's Poster C et al, EJC Supplements, 2003, 1, p. S203, relates to the study of the cytotoxic effects on 4 different cell lines originating from bladder tumors, of a Cdks inhibitor of the oxindole family, indirubin-3'-monoxime, alone and in combination with paclitaxel . The results obtained are not presented in detail. On the other hand, paclitaxel (whose trade name is Taxol®) is a mitotic agent of the taxane family, which acts by stabilizing microtubules especially during mitosis and thus affects the process of cell division. Its mode of action can therefore in no way be related to that of a growth factor receptor inhibitor activating the PI3K-Akt-mTOR pathway and / or a direct inhibitor of the PI3K-AktmTOR pathway. International application WO 02/45717 proposes combinations of molecules for treating proliferative disorders including lometroxol and other active agents, including rindirubin-3-monoxime. However, lometroxol is an antineoplastic agent belonging to the family of purine synthesis inhibitors, which can not be assimilated either to a growth factor receptor inhibitor activating the PI3K-Akt-mTOR pathway and / or to a direct inhibitor of the PI3K-Akt-mTOR pathway. Finally, the patent application US 2009/030019 describes the antiproliferative effects of certain purine derivatives substituted in the 2-, 6- and 9- position, as well as their use as an active principle, in association with other active principles such as for example a mitotic agent (Taxol®) and a genotoxic agent belonging to the class of alkylating compounds of DNA (cisplatin).

It is important to note that conventional chemotherapies directly target the DNA molecule (such as alkylating agents or platinum salts) or disrupt its synthesis (such as topoisomerase inhibitors) or even prevent the normal functioning of microtubules during mitosis (such as taxanes or alkaloids of the periwinkle). They are thus able to eradicate the cells in a state of rapid proliferation during the S or M phases. The major disadvantage of these treatments is that they do not differentiate between the cells of the normal fast-growing and tumor-proliferating organism. . They thus promote the aging process in the epithelial tissues and induce death by apoptosis of normal cells in a state of rapid proliferation such as lymphoid cells and epithelial stem cells, favoring the emergence of secondary cancers. Patients treated for hematopoietic or solid tumors with topoisomerase inhibitors or alkylating agents of DNA have been reported to develop secondary leukemias induced by the treatments (Leone et al., Haematologica, 1999, 94, 937 -945). On the other hand, these conventional chemotherapies have been shown to be totally ineffective in the treatment of breast, colon and lung cancers and in the case of advanced cancers in general, although they have been shown to be effective in the treatment of cancer. testes and leukemias (Johnstone et al., Cell, 2002, 108, 153-164).

The new targeted therapies that have been tested in vivo and / or in vitro in combination with Cdks inhibitors aim to inhibit: (i) in vivo, growth factor receptors or the VEGF / R-VEGF system in various models of solid human tumors and (ii) in vitro, phosphatidylinositol-3-kinase in human leukemia cell models: 1) Inhibition of growth factor receptors of the receptor family at 1ΕGF, R-EGF (HER1 and HER2)

Alvocidib / flavopiridol (as well as seliciclib / roscovitine) and trastuzumab (which is an antibody directed against HER2 tyrosine kinase receptor) have recently been shown to exert synergistic cytotoxic effects in breast cancer cell lines. overexpressing HER2

(Nahta et al., Cancer Res., 2003, 63, 3626-3631). On the other hand, it has been shown that seliciclib and 1ΑG1478 (an analogue of the EGFR inhibitor erlotinib) exert synergistic cytotoxic effects in non-small cell lung cancer cell lines expressing wild-type R-EGF. (H358 and

H292) or mutated (H1650) (Fleming et al, Clin Cancer Res, 2008, 14, 4326-4335).

2) Inhibition of the VEGF / R-VEGF system

Patent Application EP 1 568 368 proposes the combination of a Cdks inhibitor of the thiazoles or oxazoles class and of an inhibitor of the VEGF / R-VEGF system of the phthalazine class or of the amino derivatives of the acid. anthranilic, for the treatment of various diseases, including cancer. The VEGF / R-VEGF system refers to the growth factors of endothelial cells and their receptors located on the surface of the blood vessels that supply tissue and regulate its biogenesis and survival. The tumor cells are able to stimulate the process of neo-angiogenesis. The purpose of inhibiting the VEGF / R-VEGF system is to destabilize the newly formed blood vessels and to deprive the tumor cells of sufficient oxygen and nutrients to keep them alive. Anti-angiogenic therapies that directly target the VEGF / R-VEGF system have been reported to produce side effects that call for caution as they cause severe bleeding events.

3) Inhibition of PI3K

Yu et al. (Cancer Res., 2003, 63, 1822-1833,) studied the effects of the combination of certain Cdks inhibitors (flavopiridol, roscovitine or CGP74514A) and a chemical inhibitor of phosphatidylinositol-3-kinase (PI3K). , 4-2- (4-moφholinyl) -8-phenyl-4H-1-benzopyran-4-one (called LY294002) on various human leukemic lines. PI3K is a central element of the PI3K-Akt-mTOR cell growth regulatory pathway, which leads to the synthesis of ribosomal proteins and cycle regulators in response to the reception of growth and survival factors, relayed by receptors with tyrosine kinase activity. The results of this study show that LY294002 significantly increases the toxic effects of the three inhibitors tested in the U937 line and flavopiridol in particular in the Jurkat, CCRF, NB40 and HL-60 lines. More precisely, the percentage of apoptotic U937 cells reached 68.8% after 6 h in the presence of 75 nM flavopiridol and 15 μM LY294002 versus 1.1% in the presence of LY294002 alone. Rapamycin, a selective inhibitor of mTOR protein kinase, activated both by the PI3K pathway and in response to amino acid and glucose intake, is comparatively ineffective with respect to the same cells: apoptotic cells reached 6.2% after 6 h in the presence of 75 nM flavopiridol and 10 nM rapamycin versus 1.1% in the presence of rapamycin alone. Thus, the combination with a Cdks inhibitor of an active ingredient acting downstream of PI3K does not lead to an improvement in the antitumor efficacy of Cdks inhibitors in leukemic cells in culture. The authors concluded that the combined interruption of PI3K and Cdks-related pathways may represent a novel therapeutic strategy but limited their conclusion to hematologic malignancies only. Moreover, the major flaw of this study is that it presents no results of comparative studies on cells of normal hematopoietic origin. It is therefore not possible to determine whether the combination of a Cdks inhibitor and LY294002 induces, or not, the apoptosis of healthy cells of the blood system as effectively as that of leukemic cells. Finally, the patent application US 2007/0238745 describes, as an anticancer drug, the combination of an inhibitor of the PI3K-Akt pathway (such as Triciribine or the compound LY294002) and a Cdks9 inhibitor (such as roscovitine or flavopiridol). However, unlike Cdks 1-4 which are directly involved in the regulation of progression through the cell division cycle, Cdk9 (like Cdk7) plays a specific role in regulating the transcriptional activity of the cell and its inhibition is presumed to induce cell death by inducing the degradation of unstable mRNAs, especially those encoding apoptosis inhibitors (Shapiro G., J. Clin Oncol., 2006, 24, 1770-1783). The mechanism responsible for the cytotoxic effects resulting from the inhibition of Cdks7,9 is therefore totally different from that resulting from the inhibition of Cdks involved in the regulation of progression through the cell division cycle.

In summary, after more than a decade of investigation, the undeniable evidence of the utility of Cdks inhibitors in cancer therapy, alone or in combination with other active ingredients, has not yet been achieved. The fact is that it is very difficult in essence to obtain small organic molecules that interact with a desired target with both high affinity and high selectivity. This is especially true for Cdk inhibitors that interfere with the Cdks ATP binding site, which is highly conserved in the 518 protein kinases encoded by the human genome (Manning et al, Science, 2002, 298). , 1912-1934). It is therefore very unlikely that a molecule with high affinity for a Cdk does not interact equally with significant affinities with other substrates. The toxic effects of a therapeutically promising Cdk inhibitor could thus be diminished by its interaction with unwanted secondary targets whose range and concentration should depend on the cell type. It is also likely that the adverse side effects of first- and second-generation Cdks inhibitors result from their interaction with cellular targets whose activity is vital for normal cells. There is therefore still a pressing need for improved anti-cancer treatments, in terms of their effectiveness and selectivity with respect to each type of cancer, as well as their innocuity with respect to the healthy tissues of the cancer. organization.

The object of the present invention is precisely to provide improved anti-cancer treatments in terms of their efficiency and their selectivity with respect to each type of cancer as well as their harmlessness with respect to tissues. healthy of the body.

This object is achieved by the pharmaceutical composition which is the subject of the present invention and which is characterized in that it comprises, as active ingredients: i) at least one cyclin-dependent protein kinase inhibitor chosen from: a ) indirubin-3'-monoxime and, b) purine derivatives selected from the group consisting of: - (3R, S) -1- [6- (3,4-methylenedioxo-benzylamino) -9-isopropyl) -9H-purin-2-yl] -3-methyl-pent-1-yn-3-ol (Compound I-1),

(2R) - [1- (9-isopropyl-6-phenylamino-9Η-purin-2-yl) -pyrrolidin-2-yl] -methanol (compound 1-2),

(2S) - [1- (6-Benzylamino-9-isopropyl-9H-purin-2-yl) -pyrrolidin-2-yl] methanol (Compound 1-3), (2R) - [1- (6-Benzylamino-9-isopropyl-9H-purin-2-yl) -pyrrolidin-2-yl] -methanol (Compound 1-4),

(2R) - {1- [9-Isopropyl-6- (3-methoxy-benzylamino) -9H-purin-2-yl] -pyrrolidin-2-yl} -methanol (Compound 1-5); (2R) - {1- [6- (3,5-Dimethoxy-benzylamino) -9-isopropyl-9H-purin-2-yl] -pyrrolidin-2-yl} -methanol (Compound 1-6), and their enantiomers; and ii) at least one cell growth inhibitor selected from: a) growth factor receptor inhibitors activating the PI3K-Akt-mTOR pathway; b) inhibitors of the PI3K-Akt-mTOR pathway; in the presence or absence of a pharmaceutically acceptable medium.

The presence of an inhibitor of cell growth in the pharmaceutical composition according to the present invention makes it possible to potentiate the toxic effects of the Cdks inhibitor used, preferably in a given type of tumor, without appreciably increasing its toxic effects on the cells. healthy cells.

Indububin-3'-monoxime is a compound of the oxindole family belonging to a class of bifunctional inhibitors that preferentially target not only Cdks (in this case Cdk1, 2.5) but also other kinases ( in this case, GSK-3) (Hoessel et al., Nat., CEIl BioL, 1999, 1, 60-67). Numerous bisindole derivatives of indigoids, and in particular derivatives of indirubine such as, for example, indirubin-3'-monoxime, as well as their use for the preparation of a medicament intended to inhibit Cdks, for the treatment of psoriasis, cardiovascular diseases, infectious diseases, nephrological diseases, neurodegenerative disorders and viral infections, all these diseases being associated with a lack of regulation of cell proliferation, are described in patent EP 1 079 826.

Compounds I-1 to 1-6 are Cdks inhibitors belonging to the same class as roscovitine (seliciclib / CYC-202) and differ in different substitutions at positions 2 and 6 on the purine ring. Roscovitine is a potent inhibitor of Cdk2,5,7,9 and Cdk1,4 (to a lesser extent) (Fischer,

CeIl Cycle 3, 2004,742-746).

The developed chemical formulas for indirubin-3'-monoxime and each of the compounds (I-1) to (1-6) are presented in the attached FIG. The compounds of formula (I-1) to (1-6) are known per se and may be prepared according to the synthetic methods described in the articles of Legraverend M. et al, J. Med. Chem., 2000, 43, 1282-1292; Legraverend M. et al., J. Heterocyclic. Chem., 2001, 38, 299-303.

Growth factor receptors are membrane proteins anchored at the cell surface that activate the PI3K-Akt-mTOR pathway in response to the binding of their specific ligands provided by the cellular environment (e.g., serum present in the cell). in vitro culture medium or the blood vessels that scan the tissues in vivo). In a wide variety of solid tumors, growth factor receptors (in particular, EGF "Epidermal Growth Factor" receptors: R-EGF) are aberrantly activated either by mutation or overexpression. Members of the deregulated EGFR family may differ from one type of tumor to another: for example, HER2 is overexpressed in more than 30% of breast cancers; HERl is overexpressed in 35-

60% of ovarian cancers, 70-100% of head and neck cancers and 50-90% of non-small cell lung cancers. Gastrointestinal tumors, on the other hand, appear to be induced by point mutations in the PDGF ("Platelet-Derived Growth Factor") receptor gene, R-PDGF.

It is well known to those skilled in the art that the PI3K-Akt-

MTOR cell growth regulation is activated in response to the binding of growth and survival factors to their tyrosine kinase-active membrane receptors (RTKs) (Vivanco I and Sawyers CL, Nat Rev. Cancer, 2002, 2, 489-

501). The first step is the activation of PI3K which, in turn, activates Akt protein kinase, which inhibits the transcription factor activities of the

Forkhead family (FKHR / FOXO) involved in apoptosis and stopping the cycle.

Other important targets of the PI3K path downstream of Akt are:

- mTOR (target of rapamycin), which detects both signals from RTKs via PI3K and the presence of nutrients (glucose, amino acids) in the environment;

P70S6k, which phosphorylates the ribosomal protein S6 which regulates the translation of the ribosomal protein mRNAs and, therefore, the protein synthesis rate of the cell);

- 4E-BP1, a protein that specifically binds to the 4E-eIF translation initiation factor.

Compounds known as indirect inhibitors (RTK inhibitors) and direct PI3K-Akt-mTOR are compounds that have been demonstrated to be able to interact with RTKs or PI3K-Akt-mTOR pathway components and to inhibit the phosphorylation of PBK targets in tumor cells of interest ( study by western blot).

The inhibitory activity of the PI3K-Akt-mTOR pathway of such compounds can also be demonstrated by studying their negative effect, on the one hand, on the ribosomal and protein synthesis rate of the cells (study of the rate of incorporation of methionine radioactive material) and, secondly, the speed of progression in the

Gl cell cycle (by flow cytometry).

Those skilled in the art therefore have known tests enabling them to identify quickly and easily the compounds having these properties.

Among the inhibitors of growth factor receptors activating the PI3K-Akt-mTOR pathway, mention may be made in particular of:

Imitanib (STI-571), which has the ability to inhibit c-Kit, R-PDGF and the Bcr-Abl fusion protein. Due to its properties, imitanib has proved particularly effective against chronic myeloid leukemia (CMML) and gastrointestinal stromal tumors (GIST);

- Trastuzumab, which is a monoclonal antibody directed against the HER2 receptor which plays a major role in the deregulation of the proliferation of certain models of breast cancer (represented by the MCF-7 line); - Gefitinib and erlotinib, which are inhibitors of the kinase activity of

R-EGF, HER1;

- Cetuximab, panitunumab and matuzumab, which are monoclonal antibodies directed against the HER1 receptor;

Second-generation molecules and antibodies inhibiting the activity of mutated or deregulated growth factor receptors in tumor cells, such as, for example, lapatinib and vandetanib (the latter also being referred to as ZD6474 or XL647).

Among these growth factor receptor inhibitors activating the PI3K-Akt-mTOR pathway (i.e., upstream of the PI3K-Akt-mTOR pathway), the preferred inhibitors are those targeting the Factor Receptors preferentially. present and deregulated growth in the tumor to be treated (for example,

HER2 or HER1, respectively in breast or lung cancers).

Among the inhibitors of the PI3K-Akt-mTOR pathway that can be used in the pharmaceutical composition according to the invention, mention may notably be made of: selective inhibitors of mTOR, such as rapamycin and everolimus, the latter being preferred;

selective Akt inhibitors such as GSK690693 and PTR 6164 (also known as cholesteryl-O-CO-arginyl-proryl-arginyl-norvaryl-tyrosyl-diaminopimlyl-Hol-NH);

selective PBK inhibitors such as SF1 126 and PX-866;

indirect inhibitors of the PI3K-Akt-mTOR pathway acting via the inhibition of the insulin-like growth factor receptor type 1 (R1-IGF) such as, for example, NVP-AD W742 and NVP-AEW541, which act by competing with ATP and inhibiting the PI3K-Akt-mTOR pathway at PI3K-Akt and Akt-mTOR respectively.

The ratio by weight of the Cdks inhibitor (s) as defined above to the cell growth inhibitor may vary in a very large proportion according to the genotype of the target tumor (in particular according to the status of the Rb / p53, Ink4a / Arf genes. , PI3K / PTEN and its receptors for growth and survival factors).

The pharmaceutical composition according to the invention is intended to be administered preferably systemically, such as the subcutaneous, intravenous and oral routes. Said pharmaceutical composition may therefore be in various forms such as in the form of capsules, capsules, tablets, oral suspensions, tablets, injectable solutions or in any other form suitable for oral administration or injectable.

The nature of the pharmaceutically acceptable medium, optionally present in the pharmaceutical composition according to the invention, will of course vary depending on the mode of administration and the pharmaceutical presentation of said composition.

The pharmaceutically acceptable medium generally consists of one or more excipients conventionally used for the preparation of pharmaceutical compositions, such as diluents and / or binders, disintegrating agents, anti-caking agents, wetting agents, buffers, antioxidants , fillers, pigments, dyes, vitamins, mineral salts, taste-modifying agents, smoothing agents, mounting agents, insulation agents, mixtures thereof, and, in general, any excipient conventionally used in the preparation of pharmaceutical industry. Of course, those skilled in the art will take care on this occasion that the additive or additives may be used compatible with the intrinsic properties attached to the pharmaceutical composition according to the invention.

The pharmaceutical composition according to the invention may further contain one or more additional active ingredients.

The dosage is determined by the practitioner according to the condition of the patient.

Thus, the overall effective doses of the Cdks inhibitor (s) as defined above, combined with at least one inhibitor of cell growth, may vary in large proportions ranging for example from 10 to 150 mg per kilogram per day for each type. inhibitor.

According to a particular embodiment of the invention, the treatment may comprise a preliminary phase of administration of a pharmaceutical composition containing only a cell growth inhibitor chosen from cell growth inhibitors that can be used in the pharmaceutical compositions in accordance with the invention. invention, for example during a variable period (5-15 days) preceding a phase of administration of at least 5 consecutive days of the pharmaceutical composition according to the invention, that is to say containing the combination of at least one inhibitor of cell growth as defined above and at least one Cdks inhibitor as defined above. After a recovery period of several days (essentially to allow the recovery of healthy tissue) the same protocol can be repeated several times.

Given the spectacular and unexpected results obtained in vitro with the pharmaceutical compositions in accordance with the invention, the said pharmaceutical compositions therefore appear as anti-cancer treatments that are particularly active and selective, not only of one type of tumor given with respect to another, but also tumor cells in general compared to normal cells of the body.

The targets targeted by said pharmaceutical composition are not specific to a tumor localization. With respect to cell growth inhibitors, they target key points of the PI3K-Akt-mTOR pathway that leads to the synthesis of ribosomal proteins and cycle regulators in response to the reception of growth, division and survival, relayed by receptors tyrosine kinase activity anchored on the surface of cells. With regard to Cdks inhibitors, they target cyclin-Cdks complexes that regulate the progression in the cycle of all the cells of the body. It is why said pharmaceutical composition can be effective in several types of cancers consisting of cells carrying a set of similar deregulations in the PI3K-Akt-mTOR pathway and in their division cycle.

Thus, the subject of the invention is also the combination of at least i) a cyclin-dependent protein kinase inhibitor as defined above and at least ii) a cell growth inhibitor chosen from: ii-a) growth factor receptor inhibitors, activating the PI3K-Akt-mTOR pathway; ii-b) inhibitors of the PI3K-Akt-mTOR pathway; for use as an anti-cancer drug.

Another subject of the invention is the use of at least i) a cyclin-dependent protein kinase inhibitor as defined above and at least ii) a cell growth inhibitor selected from: ii-a) the growth factor receptor inhibitors, activating the PI3K-Akt-mTOR pathway; ii-b) inhibitors of the PI3K-Akt-mTOR pathway; for the preparation of an anti-cancer drug.

According to a particular embodiment of this use, when the medicament is intended for the treatment of tumors whose genotype, relative to the status of the Rb / p53, Ink4a / Arf, PI3K / PTEN genes and their receptors for growth and survival factors , similar to that of MCF-I control cells (Rb ⁺ Zp 53 ⁺ ) derived from a human breast carcinoma, the Cdks inhibitor is selected from compounds I-1 to 1-6. In this case, the cell growth inhibitor is preferably selected from: - EGF receptor inhibitors, HER2,

rapamycin or everolimus,

direct or indirect inhibitors of PI3K and / or Akt protein kinases.

According to this particular embodiment, the Cdks inhibitor is preferably (3R, S) -1- [6- (3,4-methylenedioxo-benzylamino) -9-isopropyl-9H-purin-2-yl] -3 -methyl-pent-1-yn-3-ol (compound I-1).

According to another particular embodiment of this use, when the medicament is intended for the treatment of tumors whose genotype, relating to the status of the Rb / p53, Ink4a / Arf, PI3K / PTEN genes and their factor receptors. growth and survival, resembles that of Saos-2 (Rb7p53 ^~ ) control cells derived from human osteosarcoma, the preferred Cdks inhibitor is indirubin-3'-monoxime. In this case, the cell growth inhibitor is preferably selected from direct or indirect inhibitors of PBK and / or Akt protein kinases.

According to the invention, the expression "tumors whose genotype, relative to the status of the genes Rb / p53, Ink4a / Arf, PI3K / PTEN and their receptors for growth and survival factors, resembles that of the control cells. "Means that the tumors to be treated carry a set of deregulation of the Rb, p53 and PBK pathways very close to that of the control cells (MCF-7 or Saos-2). Thus, tumors resembling MCF-7 carry intact Rb and p53 genes and the Rb and p53 pathways are altered downstream of the genes, unlike Saos-2, in which the Rb and p53 genes are destroyed.

The present invention is illustrated by the following exemplary embodiments, to which it is however not limited.

EXAMPLES

Demonstration of the potentiation of the cytotoxic effects of a Cdks inhibitor according to the invention by a cell growth inhibitor

(1) Equipment and methods (a) The cell and its equipment

In this example, the following three cell types were used:

the MCF-7 line (Rè / p53) resulting from a human breast carcinoma, in which the Rb and p53 tumor suppressor genes are intact,

the Saos-2 line (Rb ^~ / p53 ^~ ) derived from a human osteosarcoma, in which the tumor suppressor genes Rb and p53 are destroyed,

human immature and non-transformed IMR-90 human fibroblasts (Rè / p53) (normal cells),

The different cells were cultured in DMEM (Dulbecco Modified Eagle Medium) medium (Invitrogen, France) supplemented with antibiotics and 10% fetal calf serum (FCS) in an incubator at 37 ° C. under a humidified atmosphere containing 5%. CO ₂ .

The various purine derivatives in accordance with the invention (compounds of formulas (I-1), (1-2) and (1-3) and the purine derivatives used for comparison purposes

(Compounds CI to C-4, C-6 to CI l and C-13 to C-16) were synthesized according to the methods described in the articles of Legraverend M. et al, 2000 and 2001 (ibid) and Ducrot and al, 2003 (ibid).

Roscovitine (compound C-5), purvalanol A (compound C-12), indirubin-3'-monoxime were obtained from Calbiochem (VWR International S.A.S., France). Mitomycin C was obtained from Sigma (L'Isle d'Abeau Chesnes,

France).

Table 1 below groups together the chemical formulas of the compounds tested in this example: compounds according to the invention of formulas (1-1), (1-2) and (1-3) and indirubin-3'-monoxime, and comparative compounds CI to C-17. Table 1

(b) Study of viability _. cellular

Routinely, cell viability was measured by the Trypan blue exclusion test. At the desired time (before or after the addition of the test compound), the medium containing the floating cells was collected and the adherent cells were detached by trypsinization and mixed with the medium containing the floating cells. An equal volume of 0.125% Trypan blue solution (Sigma) was then added to an aliquot of all suspended cells. Cell viability was measured by counting, under a microscope, the percentage of unstained (living) cells relative to the total number of cells deposited in the hemacytometer. For each point, an average value of viability was obtained from at least five counts on different areas of the hemacytometer containing about 100 cells.

Study of the Cell Viability Assay The effect of the compounds listed above in Table 1 on cell viability in the three selected cell types was studied following a rigorous experimental protocol: the cells were collected after trypsinization and transferred to the wells of a 24-well plate (each 2 cm in diameter) at two different densities, namely 6000 cells and 8000 cells per well in DMEM medium supplemented with 5% FCS at 37 ° C .

After 24 h, the medium was removed from the wells and replaced with DMEM 10% FCS (DMEM 10% FCS) media in the wells initially containing 6000 cells and DMEM at 0.5% FCS (DMEM 0.5% FCS). in wells initially containing 8000 cells. By operating in this way, the average cell density in each well at the time of application of the test compound two days later was 30,000 to 35,000 cells.

In culture, the rate of supply of cells as growth and survival factors depends on the content of the medium in FBS. The 10% drop to

0.5% of the SVF content of the culture medium thus makes it possible to mimic the culture conditions that would be achieved if the cells were exposed to an inhibitor of cell growth.

The toxic effects of the compounds on the three cell types selected and under the two experimental conditions (DMEM 10% SVF versus DMEM 0.5% FCS) were studied at two different concentrations (20 and 40 μM). The viability of the cells was measured by the trypan blue exclusion test, described above, every day after the addition of test compounds and over a period of 2 or 3 days for each cell type.

2) Results

The results of the experiments are presented in diagrammatic form in appended Figures 2 and 3.

Each diagram corresponds to the results obtained for a test compound

(at 20 or 40 μM) on the three selected cell types (MCF-7, Saos-2 and IMR-

90). On each chart and for each cell type, the percentage of surviving cells at two different times (24 h and 48 h) in DMEM 10% SVF corresponds to hatched bars and the percentage of cells survivors at the same time in the medium DMEM 0,5% SVF corresponds to the solid bars.

The observation of these diagrams reveals in a striking and unexpected way that the degree of amplification of the toxic effects of a derivative of the family of

Purines obtained under conditions mimicking exposure to an inhibitor of cell growth depend both on the cell type and subtle structural differences between the derivatives of that family.

Thus, compound 1-3 (FIG. 2, diagrams on the left, ^1st row) which relates to the purine nucleus a pyrrolidine-methanol group in position 2 and a nucleus

10 benzylamine in position 6 and which has a toxicity profile similar to that of roscovitine / Seliciclib (Figure 2, left diagrams, 6 ^th rank) in DMEM 10% FCS vis-à-vis the three cell types tested, is almost inert on the Saos-2 and IMR-90 at a concentration of 20 μM, whereas it eradicates about 50% of an MCF-7 cell population in 48 hours (versus 40% for the

Roscovitine).

In DMEM 0.5% FCS, its toxic effects at 20 μM are greatly increased on MCF-7 (whose viability drops from 50% to about 5% after 48 hours of treatment) whereas the toxic effects of roscovitine ( compound C-5) on the same cells and in the same medium are hardly increased (the viability drops from 60% to about 55% after 48 hours of treatment). On the other hand, the toxic effects of compound 1-3 at 20 μM on Saos-2 and IMR-90, like those of roscovitine, remain negligible under these conditions.

If the concentration of compound 1-3 (40 μM) is doubled, its toxic effects are strongly increased on MCF-7 in DMEM 10% FCS and even more so.

DMEM medium 0.5% FCS while doubling the concentration of roscovitine increases only modestly its toxic effects on MCF-7 under both conditions.

At 40 μM, significant toxic effects of compound 1-3 appear in DMEM medium 0.5% FCS on Saos-2 (viability of about 20% after 48 hours of treatment) while they remain very low in medium. DMEM 10% FCS; the toxic effects of roscovitine (compound C-5) on the same cells remain modest even in DMEM medium 0.5% FCS (viability of about 80% after 48 hours of treatment).

At 40 μM, however, the toxic effects of compound 1-3 remain negligible

On normal IMR-90 control cells in DMEM 10% FCS medium and they are relatively poorly amplified in DMEM medium 0.5% FCS (viability of about 75%) after 48 hours of treatment); comparatively greater toxic effects occur in DMEM medium 0.5% FCS in the presence of 40 μM roscovitine (viability of about 60% after 48 hours of treatment).

The comparative compounds CI and C-2 (FIG. 2, diagrams on the left, 2 ^nd

5 and 3 ^rd ranks) differ from compound 1-3, respectively by the presence of two chlorine atoms in meta and para and a chlorine in para on the benzylamine nucleus in position 6. In DMEM 10% FCS, at 40 μM as at 20 μM, their toxic effects vis-a-vis the three cell types tested are relatively modest and comparable to those of compound 1-3, except that they are significantly more toxic from 20 μM on MCF cells -7 (viability of about 10% in the presence of compounds CI or C-2 versus about 50% in the presence of compound 1-3 after 48 hours of treatment).

However, in DMEM medium 0.5% FCS, the toxic effects of the compounds

C-I and C-2 are amplified dramatically, indistinctly on the three types

15 cells tested, from 20 μM. Similar results have been obtained with derivatives having, in place of chlorine atoms, a trifluoromethyl group in meta or para on the benzylamine ring at the 6-position.

Replacement of the halogen atoms or substituents on the benzylamine ring at the 6-position with a dioxol group (compound C-3;

20 left diagrams, 4 ^th row) or by methoxy para (compound C-4; Figure 2, left diagrams, 5 ^th row) or two methoxy, give compounds whose toxicity profile vis-à-vis MCF-7 cells is quite similar to that of the compounds CI and C-2 but which, at 20 μM in DMEM medium 0.5% FCS, are significantly less toxic on Saos-2 and even less on the IMR-90. AT

40 μM, however, their toxic effects in DMEM medium 0.5% FCS match those of the compounds C-I and C-2.

Equally noteworthy, the replacement in position 2 of the pyrrolidine-methanol group in the above compounds with a pyrrolidine-alcohol group gives compounds generally less toxic. Thus, the profiles 30 of toxicity of the compounds I-1 (Figure 2, right diagrams, 4 ^th row) and C-3 vis-à-vis the three cell types tested are very similar at 20 .mu.M, except that C -3 is significantly more toxic in DMEM medium 0.5% FCS on the MCF-7 cells (viability <5% in the presence of C-3 versus about 25% in the presence of compound I-1 after 48 hours of treatment).

However, at 40 μM in 0.5% FCS DMEM medium, the toxic effects of compound I-1 are selectively increased in MCF-7 and Saos-2 tumor cells. but absolutely not in normal IMR-90 fibroblasts while those of compound C-3 are increased indiscriminately and dramatically in all three cell types.

If we compare compounds I-1 and 1-3 with roscovitine (compound C-5), we see that the degree of amplification of the toxic effects of the first two, obtained in DMEM medium 0.5% FCS in the two tumor types tested (MCF-7 and

Saos-2), is significantly higher than that of roscovitine while it is lower in normal fibroblasts IMR-90.

Finally, the diagrams obtained for the indirubin-3'-monoxime (Figure 3, diagrams on the left, 5 ^th rank) show two particularly striking results:

The potentiation of its toxic effects obtained in DMEM medium 0.5% FCS is significantly greater in the Saos-2 tumor cells (Rb ^~ / p53 ^~ ) than in the MCF-7 (Rb / p53) whereas the reciprocal is true in the case of selected purine derivatives I-1, 1-2 (Figure 3, left diagrams, ¹ row) and 1-3.

• On the other hand, at 40 μM in 0.5% FCS DMEM medium, its toxic effects on normal IMR-90 fibroblasts are insignificant whereas, on Saos-2, they are already very important (viability of about 15%). % after 24 hours of treatment).

These results demonstrate that the two types of Cdks inhibitors usable according to the present invention exhibit anti-tumor activity selectively increased with respect to a given tumor cell type when used under conditions mimicking exposure to an inhibitor of cell growth. Not only, they are able to induce the apoptosis of one type of tumor cells given preferentially to another (MCF-7 (Rb / p53) for the first versus Saos-2 (Rb ^~ / p53 ^~ ) for the indirubin-3'-monoxime), but their toxic effects vis-à-vis the normal fibroblasts IMR-90 are negligible at concentrations where they are already very important vis-à-vis the tumor cells. By way of comparison, the compounds CI to C-17, whose chemical structure is extremely close to that of the Cdks inhibitors that can be used in accordance with the invention, are not as efficient under the same conditions, or because they are not quite toxic to tumor cells (eg, C-6, C-9 and C-13), either because they are not selective enough for tumor cells and they are just as toxic for healthy control cells than for tumor cells. For comparison also, roscovitine (compound C-5), which also belongs to the family of purines, is significantly lower than the Cdks inhibitors that can be used according to the present invention under conditions mimicking the exposure of cells to a growth inhibitor. both in terms of efficacy with respect to the two tumor types tested and in terms of the difference in toxicity with respect to the tumor cells and vis-à-vis the healthy cells tested.

Finally, it was verified with indirubin-3'-monoxime and compound 1-2 that their synergistic toxic effects obtained by lowering the SVF content of the culture medium from 10% to 0.5% were also obtained when concomitant exposure of DMF 10% FCS cells to an inhibitor of cell growth, in this case LY294002 at 20 μM (4-2- (4-morpholinyl) -8-phenyl-4H-1-benzopyran). 4-one) which is a direct inhibitor of PI3K.

Claims

A pharmaceutical composition characterized in that it comprises, as active ingredients: i) at least one cyclin-dependent protein kinase inhibitor selected from: ia) rindirubin-3'-monoxime and, ib) purine derivatives selected in the group consisting of:

(3R, S) -1- [6- (3,4-methylenedioxo-benzylamino) -9-isopropyl-9H-purin-2-yl] -3-methyl-pent-1-yn-3-ol ( compound 1-1),

(2S) - [1- (6-Benzylamino-9-isopropyl-9H-purin-2-yl) -pyrrolidin-2-yl] methanol (Compound 1-3), - (2R) - [1] (6-Benzylamino-9-isopropyl-9H-purin-2-yl) -pyrrolidin-2-yl] -methanol (Compound 1-4),

(2R) - {1- [9-Isopropyl-6- (3-methoxy-benzylamino) -9H-purin-2-yl] -pyrrolidin-2-yl} -methanol (Compound 1-5),

(2R) - {1- [6- (3,5-Dimethoxy-benzylamino) -9-isopropyl-9H-purin-2-yl] -pyrrolidin-2-yl} -methanol (Compound 1-6), and their enantiomers; and ii) at least one cell growth inhibitor selected from: ii-a) growth factor receptor inhibitors, activating the PI3K-Akt-mTOR pathway; and ii-b) inhibitors of the PI3K-Akt-pathway. mTOR; in the presence or absence of a pharmaceutically acceptable medium.

2. Composition according to claim 1, characterized in that the inhibitors of growth factor receptors activating the PI3K-Akt-mTOR pathway are chosen from imitanib, R-PDGF and the Bcr-Abl fusion protein; trastuzumab; gefitinib and erlotinib; cetuximab, panitunumab and matuzumab; lapatinib and vandetanib.

3. Composition according to claim 1 or 2, characterized in that the inhibitors of the PI3K-Akt-mTOR pathway are chosen from selective mTOR inhibitors, selective Akt inhibitors, PI3K selective inhibitors and indirect inhibitors of the PI3-Akt-mTOR pathway acting via inhibition of insulin-like growth factor receptor type 1.

4. Composition according to claim 3, characterized in that the selective inhibitors of mTOR are chosen from rapamycin and everolimus.

5. Composition according to claim 3, characterized in that the selective inhibitors of Akt are chosen from GSK690693 and PTR 6164.

6. Composition according to claim 3, characterized in that the PI3K selective inhibitors are chosen from SFl 126 and PX-866.

7. Composition according to claim 3, characterized in that said indirect inhibitors of the PI3-AktorTOR pathway are chosen from NVP-ADW742 and NVP-AEW541.

8. Composition according to any one of the preceding claims, characterized in that it is in the form of capsules, capsules, tablets, oral suspensions, tablets, injectable solutions or in any other form appropriate to the method. oral or injectable administration.

9. Composition according to any one of the preceding claims, characterized in that the pharmaceutically acceptable medium consists of one or more excipients chosen from diluents and / or binders, disintegrating agents, anti-caking agents, wetting agents, buffers, antioxidants, fillers, pigments, dyes, vitamins, mineral salts, flavor modifiers, smoothing, mounting, insulating agents, and mixtures thereof.

10. Association of at least i) a cyclin-dependent protein kinase inhibitor selected from: ia) indirubin-3'-monoxime and, ib) purine derivatives selected from the group consisting of: - (3R, S) -1- [6- (3,4-methylenedioxo-benzylamino) -9-isopropyl-9H-purin-2-yl] -3-methyl-pent-1-yn-3-ol (Compound I-1) ,

(2R) - [1- (9-isopropyl-6-phenylamino-9Η-purin-2-yl) -pyrrolidin-2-yl] -methanol (compound 1-2), (2S) - [1- (6-Benzylamino-9-isopropyl-9H-purin-2-yl) -pyrrolidin-2-yl] methanol (Compound 1-3),

(2R) - [1- (6-Benzylamino-9-isopropyl-9H-purin-2-yl) -pyrrolidin-2-yl] -methanol (Compound 1-4), - (2R) - - [9-Isopropyl-6- (3-methoxy-benzylamino) -9H-purin-2-yl] pyrrolidin-2-yl} -methanol (Compound 1-5),

(2R) - {1- [6- (3,5-Dimethoxy-benzylamino) -9-isopropyl-9H-purin-2-yl] -pyrrolidin-2-yl} -methanol (Compound 1-6), and their enantiomers; and at least ii) a cell growth inhibitor selected from: ii-a) growth factor receptor inhibitors activating the PI3K-Akt-mTOR pathway; and ii-b) inhibitors of the PI3K-Akt pathway -mTOR, for use as an anti-cancer drug.

Use of at least i) a cyclin-dependent protein kinase inhibitor selected from: i-a) indirubin-3'-monoxime and, i-b) purine derivatives selected from the group consisting of:

(3R, S) -1- [6- (3,4-methylenedioxo-benzylamino) -9-isopropyl-9H-purin-2-yl] -3-methyl-pent-1-yn-3-ol ( compound I-1),

(2R) - {1- [6- (3,5-Dimethoxy-benzylamino) -9-isopropyl-9H-purin-2-yl] -pyrrolidin-2-yl} -methanol (Compound 1-6), and their enantiomers; and at least ii) an inhibitor of cell growth selected from: ii-a) growth factor receptor inhibitors activating the PI3K-Akt-mTOR pathway; and ii-b) inhibitors of the PI3K-Akt-mTOR pathway for the preparation of an anticancer drug.

12. Use according to claim 11, characterized in that the medicament is intended for the treatment of tumors whose genotype, relating to the status of the Rb / p53, Ink4a / Arf, PI3K / PTEN genes and their receptors for growth factors and survival, resembles that of MCF-7 control cells derived from human breast carcinoma, and that the Cdks inhibitor is selected from compounds I-1 to 1-6.

13. Use according to claim 12, characterized in that the inhibitor of cell growth is selected from EGF, HER2 receptor inhibitors; rapamycin or everolimus; and direct or indirect inhibitors of PI3K and / or Akt protein kinases.

14. Use according to claim 12 or 13, characterized in that the Cdks inhibitor is (3R, S) -1- [6- (3,4-methylenedioxo-benzylamino) -9-isopropyl-9H-purinin). 2-yl] -3-methyl-pent-1-yn-3-ol (compound 1-1).

15. Use according to claim 11, characterized in that the medicament is intended for the treatment of tumors whose genotype, relating to the status of the Rb / p53, Ink4a / Arf, PI3K / PTEN genes and their receptors for growth factors and survival, resembles that of Saos-2 control cells derived from human osteosarcoma and that the Cdks inhibitor is indirubin-3'-monoxime.

16. Use according to claim 15, characterized in that the inhibitor of cell growth is selected from direct or indirect inhibitors of PI3K and / or Akt protein kinases.