WO2011131636A1

WO2011131636A1 - Combined treatment of cancer with benzo[e]pyridoindoles and dna-damaging agents

Info

Publication number: WO2011131636A1
Application number: PCT/EP2011/056172
Authority: WO
Inventors: Annie Molla; Chi-Hung N'guyen; Hong-Lien Vu; Ichiro Nakano
Original assignee: Institut Curie; Centre National De La Recherche Scientifique; Commissariat A L'energie Atomique Et Aux Energies Alternatives; Universite Joseph Fourier - Grenoble 1; The Ohio State University
Priority date: 2010-04-19
Filing date: 2011-04-18
Publication date: 2011-10-27

Abstract

The present invention relates to a pharmaceutical composition, kit or combined preparation comprising benzo[e]pyrido indoles and DNA-damaging agents, said composition, kit or combined preparation being useful for treating cancer. It also relates to the combinated treatment of cancer with benzo[e]pyrido indoles and radiotherapy.

Description

Combined treatment of cancer with benzo[e]pyridoindoles and DNA-damaging agents

Field of the Invention

The present invention relates to the medical field and in particular to the oncology field.

Background of the Invention

Aurora kinases are a family of serine/threonine protein kinases that play a key role in mitosis progression. Aurora A is found to be associated first with centrosomes and finally with microtubules whereas aurora B is a chromosomal passenger protein. Aurora A is required for centrosome duplication, entry into mitosis, formation of bipolar spindle and mitotic checkpoint. Aurora B exhibits typical passenger protein behavior during mitosis. Initially, the kinase associates with centromeres, and as mitosis proceeds, it relocates to the central spindle and the midbody. Aurora B is essential for chromosome condensation, kinetochore functions, spindle checkpoint activation and cytokinesis completion.

Aurora A and B are overexpressed in many cancers, including primary colon and breast cancer. Furthermore, the human Aurora A gene is localized to the 20ql3 amplicon, which is associated with a poor prognosis in breast cancer. Xenografts of mouse NIH-3T3 cells overexpressing aurora A give rise to tumors in nude mice, suggesting that aurora A behaves as an oncogene. Under similar conditions, overexpression of aurora B may induce metastasis.

In the light of these observations, aurora kinases have emerged as druggable targets for cancer therapy and thus, identification of aurora kinase small molecule inhibitors is of particular interest. Impairing their functions prevents cell division and leads to apoptosis. Several aurora A and B inhibitors, including ZM477439, Hesperadin, VX-680 (MK-0457), MLN8054, PHA-739358 have been described (Cheung et al, 2009, Expert Opin. Investig. Drugs, 18, 379-398; Coumar et al, 2009, Expert Opin. Ther. Patents, 19, 321-356). Approximately 13 aurora kinase inhibitors are under Phase I/II evaluation at present for various cancers of different origins (i.e., advanced, refractory or relapsed solid tumors, leukemias, multiple myeloma, CML, ALL, AML) and several others are in preclinical testing. VX-680, considered as the aurora reference inhibitor, suppresses tumor growth in vivo and encouraging results were reported for three patients with refractory Chronic Myeloid Leukemia. The positive data in leukemia may be accounted for by the inhibition of the T315I mutant bcr-ABL, an off-target of several of these inhibitors.

WO2007/136615 discloses the combination of VX-680 with any anti-cancer agent for treating a cancer. It shows that either additive or synergistic effect may be obtained.

Benzo[e]pyridoindoles were described as potent kinase inhibitors with a minimal toxicity (Hoang et al, 2009, Cell Cycle, 8, 1-8). These compounds were found to inhibit aurora kinases. It was also shown that those compounds, in particular compounds CI and C2, inhibit the growth of different cell lines derived from different carcinoma.

Despite an intensive research, two major problems in cancer therapy remain: the induction of tumor resistance to already used drugs and the balance between the toxicity of the treatment and its efficiency. Therefore, new treatments with less toxicity and resistance induction are still required.

Summary

The present invention relates to a combined treatment of cancer with benzo[e]pyrido indoles and DNA-damaging agents.

The present invention relates to a pharmaceutical composition comprising

a) a compound having the formula (1)

wherein

Rai, Ra₂ and Ra₃, each independently, are selected from the group consisting of hydrogen, (Ci-C3)alkyl, halogen, hydroxyl, aroyloxy of formula ArC(=0)0-), arylcarbamoyloxy of formula ArNH(C=0)0-), (Ci-C₃)alkoxy, optionally substituted by an aryl; or two of the radicals Rai and Ra₂ or Rai and Ra₃ may be taken together to form a (Ci-C₂)alkylenedioxy radical;

Rb is selected from the group consisting of hydrogen, (Ci-C₃)alkyl optionally substituted by a radical selected in the group consisting of hydroxyl, -NRR', - OPO(OR)(OR'), -OC(=0)R;

Rci and Rc₂, each independently, are selected from the group consisting of hydrogen, (Ci-C₃)alkyl and aryl; or Rci and Rc₂ may be taken together to form a bivalent radical of formula

^■ -(CH₂)_n- wherein n is 3, 4 or 5; or

■ -CH=CH-CH=CH- , optionally substituted by a (Ci-C₃)alkyloxy,

Rd is selected from the group consisting of hydrogen, (Ci-C₃)alkyl optionally substituted by a radical OH, (Ci-C₃)alkyloxy or -NRR',

- X is an oxygen or a sulfur;

wherein R and R', identical or different, are selected from the group consisting of hydrogen and (Ci-C4)alkyl,

or an isomeric form thereof or a pharmaceutically acceptable salt and derivative thereof, and

(b) a DNA-damaging anti-tumoral agent, and

a pharmaceutically acceptable carrier. In a particular embodiment, the composition is for use in the treatment of cancer.

The present invention further relates to a product or kit containing

(a) a compound having the formula (1)

wherein Rai, Ra₂, Ra₃, Rb, Rci, Rc₂, Rd, X, R and R' are as defined in Formula (1) above, and (b) a DNA-damaging anti-tumoral agent,

as a combined preparation for simultaneous, separate or sequential use, in particular in the treatment of cancer.

In a preferred embodiment of the above mentioned composition or kit, the DNA- damaging antitumoral agent is selected from the group consisting of an inhibitor of topoisomerases I and/or II, a DNA crosslinker, a DNA alkylating agent, and an anti-metabolic agent. Preferably, the DNA-damaging antitumoral agent is selected from the group consisting of an inhibitor of topoisomerases I and/or II and a DNA crosslinker. More preferably, the DNA-damaging antitumoral agent is a topoisomerase I and/or II inhibitor, more preferably selected from the group consisting of etoposide, topotecan, camptothecin, irinotecan, amsacrine, intoplicin, anthracyclines such as doxorubicin, epirubicin, daunorubicin, idanrubicin and mitoxantrone. Still more preferably, the DNA-damaging antitumoral agent is etoposide or intoplicin, especially etoposide. Alternatively, the DNA-damaging antitumoral agent is a DNA crosslinker. A preferred DNA crosslinker is cisplatin. Accordingly, in one particular embodiment, the DNA-damaging antitumoral agent is selected from the group consisting of etoposide, intoplicin and cisplatin.

The present invention further relates to a pharmaceutical composition comprising a compoun having the formula (1)

wherein Rai, Ra₂, Ra₃, Rb, Rci, Rc₂, Rd, X, R and R' are as defined in Formula (1) above, or an isomeric form thereof or a pharmaceutically acceptable salt and derivative thereof, for the use in the treatment of cancer in combination with radiotherapy.

In preferred embodiments, the compound of formula (1) has one or several of the following features: Rai, Ra₂, Ra₃, each independently, independently selected from the group consisting of hydrogen, hydroxyl, methoxy, ethoxy, phenoyloxy, phenylcarbamoyloxy and benzyloxy; and/or

Rb is selected from the group consisting of hydrogen, methyl, ethyl, -CH₂-OH, - (CH₂)n-N[(Ci-C₂)alkyl]2 with n being 2 or 3, -(CH₂)-OPO[0(Ci-C₄)alkyl]₂ and - (CH₂)-OC(=0)-(Ci-C₄)alkyl; and/or

Rci and Rc₂, each independently, are selected from the group consisting of hydrogen, methyl, ethyl and phenyl, or may be taken together to form a bivalent radical of formula -CH=CH-CH=CH-; and/or

Rd is selected from the group consisting of hydrogen, methyl, ethyl, and -(CH₂)_n- N[(Ci-C₂)alkyl]₂ with n being 2 or 3; and/or

X is an oxygen.

In a more preferred embodiment, the compound of formula (1) has one or several of the following features:

Rai is selected from the group consisting of hydrogen, hydroxyl, methoxy, ethoxy, phenoyloxy, phenylcarbamoyloxy and benzyloxy, and Ra₂ and Ra₃ are hydrogen; and/or

Rb is selected from the group consisting of hydrogen, -CH₂-OH, and -(CH₂)_n- N[(Ci-C₂)alkyl]₂ with n being 2 or 3; and/or

Rci is selected from the group consisting of hydrogen, methyl, ethyl and phenyl, and Rc₂ is hydrogen; and/or

Rd is selected from the group consisting of hydrogen, methyl, and ethyl; and/or X is an oxygen.

In another more preferred embodiment, the compound of formula (1) has one or several of the following features:

Rai is selected from the group consisting of hydrogen, hydroxyl and methoxy, and Ra₂ andRa₃ are hydrogen; and/or

Rb is hydrogen; and/or

Rci is selected from the group consisting of hydrogen, methyl and ethyl, and Rc₂ is hydrogen; and/or

Rd is hydrogen; and/or

X is an oxygen.

In a very particular embodiment, the compound of formula (1) has Rd being hydrogen. More preferably, the compound of formula (1) is selected in the group consisting of: a compound wherein Rai is methoxy, Ra₂ and Ra₃ are H, Rb is H, Rci is ethyl, Rc₂ is H, Rd is H and X is O;

a compound wherein Rai is methoxy, Ra₂ and Ra₃ are H, Rb is H, Rci is methyl, Rc₂ is H, Rd is H and X is O;

- a compound wherein Rai is H, Ra₂ and Ra₃ are H, Rb is H, Rci is methyl, Rc₂ is H,

Rd is H and X is O;

a compound wherein Rai is methoxy, Ra₂ and Ra₃ are H, Rb is H, Rci is H, Rc₂ is H, Rd is H and X is O;

a compound wherein Rai is hydroxyl, Ra₂ and Ra₃ are H, Rb is H, Rci is methyl, Rc₂ is H, Rd is H and X is O;

a compound wherein Rai is phenoyloxy, Ra₂ and Ra₃ are H, Rb is H, Rci is methyl, Rc₂ is H, Rd is H and X is O; and,

a compound wherein Rai is methoxy, Ra₂ and Ra₃ are H, Rb is H, Rci and Rc₂ taken together form a bivalent radical of formula -CH=CH-CH=CH-, Rd is H and X is O.

In a preferred embodiment, the compound of formula (1) is selected in the group consisting of:

a compound wherein Rai is methoxy, Ra₂ and Ra₃ are H, Rb is H, Rci is ethyl, Rc₂ is H, Rd is H and X is O;

- a compound wherein Rai is methoxy, Ra₂ and Ra₃ are H, Rb is H, Rci is methyl,

Rc₂ is H, Rd is H and X is O;

a compound wherein Rai is H, Ra₂ and Ra₃ are H, Rb is H, Rci is methyl, Rc₂ is H, Rd is H and X is O;

a compound wherein Rai is methoxy, Ra₂ and Ra₃ are H, Rb is H, Rci is H, Rc₂ is H, Rd is H and X is O; and,

a compound wherein Rai is hydroxyl, Ra₂ and Ra₃ are H, Rb is H, Rci is methyl, Rc₂ is H, Rd is H and X is O.

Still more preferably, the compound of formula (1) is a compound wherein Rai is methoxy, Ra₂ andRa₃ are H, Rb is H, Rci is ethyl, Rc₂ is H, Rd is H and X is O.

In a preferred embodiment of the composition and kit, the amounts of the compound of formula (1) and the DNA-damaging anti-tumoral agent are such that the combined therapeutic effect of the two active ingredients is additional, preferably synergistic. In particular, the amount of the DNA-damaging anti-tumoral agent can be a sub-therapeutic amount. Optionally, the composition and kit of the invention may further comprise another antitumoral agent, preferably an histone deacetylase (HDAC) inhibitor or a taxoid antitumoral agent.

The present invention also relates to a pharmaceutical composition, a kit, a product or a combined preparation according to the invention for use in the treatment of cancer. Brief Description of the Drawings

Figure 1: IC50 values for the best kinase targets of CI and C2. For comparison the IC50 for VX-680 for the same kinases are also shown. The measurements of IC50 for both compounds were carried out under identical conditions.

Figure 2: CI targets Checkpoint kinase 2 in cellulo upon DNA double-strand breaks. U20S cells were treated by Etoposide (10 μΜ for 2 h) in the presence of CI (1 μΜ) and then cells were allowed to recover in either the presence (+ CI) or the absence of CI (- CI). Recovering time varied from 4 h to 28 hours. In Fig. 2A, different markers were followed on cells extracts by western blotting. Alpha-tubulin was used as an internal control. Whereas Chk-2 signal varies as CC-Tubulin does, interesting variations of phospho-Ser516 Chk-2 are detected. As soon as DNA damages are created Chk-2 is highly autophosphorylated (Etoposide +C1) but phospho-Chk2 is maintained only in the absence of CI (21 h and 28 h). Conversely Chk-2 is rapidly dephosphorylated in the presence of CI (4 h, 21 h and 28 h). Phopho-H2A-X signals are higher when Chk-2 is inhibited suggesting a less efficient DNA repair in the presence of CI . In Fig. 2B: the same experiment was conducted by immunofluorescence. Phopho-H2A-X was detected 24 h later. Phopho-H2A-X signal was brighter in cells treated by CI than in the control. A kinetic of recovery was performed with the p53 negative H358 cells. Cells were treated by Etoposide (5 μΜ for 2 h) and allowed to recover in the presence (+C1) or the absence (-C1) of CI . Again the signal was brighter in the presence of CI, suggesting an inhibition of DNA repair in these cells.

Figure 3: Effect of the combined treatment on H358 cells. The inventors checked the consequences of the combined treatment (Etoposide plus CI) on H358 cultured as monolayer or as spheroids and also established as xenografts in mice. Fig. 3 A reports the efficiency of each treatment individually or combined. Cells were treated with Etoposide (5 μΜ for 24 h) and CI (1 μΜ) is either maintained for 72 h or omitted. Cell viability is measured upon 72 h of experience. Real means experimental result and the theoretical value is calculated as follow: (CI percentage * Etoposide percentage). The effect of Etoposide and CI is additive in H358 cells. Fig. 3B : spheroids were established with H358 cells and grew continuously for at least 12 days as shown the growth ratio curves. Proliferating cells were detected by immunofluorescence with a mitotic marker (phospho-SerlO-histone H3) and cells were stained with phalloidin and DNA by hoechst. Spheroids were treated with Etoposide at day 0 (E; 5 μΜ) for 1 day in either the presence or the absence of CI (CI 1 μΜ), in control Etoposide and/or CI were omitted. The arrows indicate the addition of the drugs and their withdrawn. Both CI (triangle) and etoposide (square) decrease spheroid growth but the combined treatment (circle) is more powerful preventing spheroid expansion. Fig. 3C : a similar experiment was performed in nude mice bearing very aggressive H358 tumours. Seven days after tumour establishment mice were injected either with CI or with Etoposide or with both drugs or with the vehicle. Mice were injected intraperitoneally 4 times a week (on Monday, Tuesday, Thursday and Friday) with either CI (160 μg per 20 g mouse in vehicle buffer (PEG 300 /DMSO 16 %) or vehicle only. They were treated ten fold as above. Moreover half of them received twice Etoposide injections (100 μg/ mouse/ injection) at days 7 and 14. The volume of the tumours was measured once a week and the growth ratio is expressed for each individual tumour at day 56, Vo being the volume at day 7. The dotted lines represent the average index for each treatment.

Figure 4: Efficiency of the combined treatment on different cell lines. The inventors compared the behaviour of these three cell lines upon etoposide and CI treatments. In Fig. 4A are represented the percentage of viability of the cells upon the different treatments. Note that the treatments are additive in H358 and synergic in other cell lines. R for Real and T for Theoretical (CI percentage * Etoposide percentage). The etoposide concentrations are established as follow: 5 μΜ for H358, 10 μΜ for U20S and 500 nM for HL60 cells. Fig. 4B : the extent of DNA damages, at day 1, is visualized by the deposition of γ-histone H2A-X. H2B is used as internal control. H2A-X is still highly phosphorylated in H358 and HL60 cells whereas it has returned to its basal level in U20S cells. Fig. 4C : the repartition in the cell cycle of the resistant populations upon 24 h of treatment and 48 h of recovery and the percentage of viability of the cells upon the different treatments are represented. The percentage of events in each phase is indicated for each treatment and each cell line. Fig. 4D : Graphic representation of Figure 4C data for Etoposide and combined treatments. Each phase is represented by a number: 1 : sub GO, 2 G0/G1 , 3 S, 4 G2/M and finally 5 > 4N.

Figure 5: Definition the best conditions in HL60 cells and in vivo application. Fig. 5 A: Evolution of the cells upon different Etoposide concentrations. Histograms represent the percentage of events in each gate as a function of the Etoposide concentration. Note the G2/M arrest in the presence of 500 nM and 1 μΜ Etoposide and the activation of the Gl checkpoint in the presence of 2 μΜ Etoposide. Fig. 5B: The repartition of the cells in the different phases at two different times (48 h in front and 72 h at the rear). Each panel represent a different treatment (control, CI, Etoposide and Etoposide plus CI). Each phase is represented by a number: 1 : sub GO, 2 GO/Gl, 3 S, 4 G2/M and finally 5 > 4N. Fig. 5C: Effect of the combined treatment in mice bearing HL60 tumours. Mice were treated seventeen times by CI (160 μg per 20 g mouse in vehicle buffer (PEG 300 /DMSO 16 %) intraperitoneally on a day-to-day basis (bar on the bottom). The treatment starts 5 days after the establishment of the tumours. Three injections of Etoposide (100 μg/ mouse/ injection) were realized at days 5, 10 and 17 (pink arrows on the bottom). Control mice received either the vehicle (V, diamonds) or Etoposide (V + Etoposide, squares) or CI (triangles). The combined treatment is represented by squares). The volume of the tumours is plotted as a function of the time (in days). Starts indicate significant data determined by the Student test (P around 0.001 for the two last points).

Figure 6: Efficiency of the combined treatment on different cell lines. The viability of the cells was analysed when etoposide concentration varied from 5 μΜ to 35 μΜ. Both U20S and H358 cells were tested. Whatever the Etoposide concentration, the effects of the combined treatments are mostly additive for H358 and synergic for U20S.

Figure 7: Cell fate upon CI treatment. Fig. 7A) Rational for using Chk2 inhibitors as chemotherapeutic agents as documented in (Jobson et al,). The substrates of Chk2 have an impact on both cell cycle checkpoint and apoptosis. In p53 deficient tumours Chk2 inhibitors block checkpoints and sensitizes to genotoxic stress whereas in normal tissues they block apoptosis and decreases side effects. Fig. 7B) In G2/M cells, aurora kinases are key players for mitotic onset whereas Chk2 prevents ongoing following G2/M progression with DNA damage. Therefore if Chk2 is inhibited in damaged cells they will either stop in G2 or progress in mitosis with reduce aurora kinase activities. If concomitantly, aurora kinases are inhibited by a potent inhibitor like CI cells will escape from mitosis either through mitotic catastrophe or by mitotic slippage.

Figure 8: Combined effect of taxol and CI . Efficiency of the combined treatment on HL60 cells. The inventors determined the viability upon taxol and CI treatments. The percentage of viability of the cells is represented. Note that the combined treatments are less efficient than CI alone. TH (TH = CI percentage * Taxol percentage) is the value calculated for additive effects. The Taxol concentrations are 0.1 nM and 0.5 nM.

Figure 9: Combined effect of etoposide either with CI or VX-680. Comparison of the efficiency of the following combinations: etoposide (1 microM) with either CI (1 microM) or VX-680 (1 microM) in HeLa cells. The percentage of alive cells is represented and compared to the value calculated for additive treatments (TH= Compound percentage * etoposide percentage). In Hela cells, CI and etoposide have synergic effects whereas VX-680 and etoposide are mostly additive under the experimental conditions.

Figure 10: Combined effect of intoplicin and CI . The viability of HL60 cells was analysed when intoplicin concentration varied from 50 nM to 300 nM. Whatever the intoplicin concentration, the effects of the combined treatments are mostly synergic but the effects are mild at the lowest concentrations; compared experimental and TH histograms (TH= value calculated for additive treatment).

Figure 11: Combined effect of cisplatin and CI . The viability of HL60 cells was analysed when cisplatin concentration varied from 100 nM to 1.5 microM. Whatever the cisplatin concentration, the effects of the combined treatments are mostly additive; compared experimental and TH histograms (TH = CI percentage * cisplatin percentage).

Figure 12: Evaluation of the therapeutic efficacy of CI on a mouse intracranial tumor model. Spheres derived from GBM patient samples were implanted in nude mice as shown in A. Mice received a unique injection of CI (from 2.5 pmol to 250 pmol) and tumors were recovered at 2 months following xenograft. Representative pictures indicate human- specific Nestin staining of either DMSO- or CI -treated mouse brains (part B). Graph (part C) indicates tumor sizes of each group determined by Nestin Staining intensities. CI intratumoral injection significantly decreases tumor sizes of immunodeficient mouse intracranial tumor model from GBM 157 spheres.

Figure 13: Combined effect of radiation and CI . The inventors performed in vitro cell viability assay with 3 GBM samples with or without radiation combined with CI or control treatments. CI treatment, at micromolar range, demonstrates inhibitory effect on the in vitro growth for 3 GBM sphere samples (GBM146, GBM157 and GBM206). CI treatment rendered GBM sphere cells more than 10 times sensitive to radiation ; two different radiation doses being tested.

Detailed Description of the Invention

The inventors have identified benzo[e]pyridoindoles as rather specific Aurora kinase inhibitors as reported in the article of Hoang et al. (Hoang et al., 2009, supra). Moreover, herein they demonstrate that benzo[e]pyrido indoles are able to inhibit chk2 kinase. Consequently, they found that these compounds are able to enhance the damage done by DNA damaging anti-tumoral agents (also called herein DDA). Indeed, the double targeting of aurora and chk2 kinases by these compounds acts cooperatively to kill cancer cells since cells entering in mitosis with damaged DNA are highly susceptible to cell death. The simultaneous targeting of aurora and checkpoint kinase 2 by the same drug in damage cells will induce G2/M progression in checkpoint deficient cells. Then, a convergence of negative signals lead to mitosis abortion and (or) cell death. Therefore, this double targeting is used to drive cancer cells outside cell cycle. In particular, DNA damaging agents may be utilized not to kill the cells but to allow compounds of the invention to kill p53 -deficient cells. Compounds of the invention increase the selectivity and allow a decrease of the DNA damaging drug concentration, reducing thus its cytotoxicity. The inventors provide herein evidences of at least an additive effect with a combined treatment with these compounds and DNA damaging anti-tumoral agents even in a spheroid model, but more often a clear synergistic effect in a cell line model, and even in a mouse model. At the opposite, the inventors further demonstrated that this additive or synergistic effect of CI on cell death is not observed with the anti-mitotic antitumoral agent such as paclitaxel with which absolutely no such effect has been noted. In addition, the inventors did not observe the same effect by using a well-known Aurora kinase inhibitor such as VX-680 (MK-0457) which does not present a capacity to also inhibit chk2.

Accordingly, the present invention relates to

a pharmaceutical composition comprising a) a compound having the formula (1), b) a DNA-damaging anti-tumoral agent, and a pharmaceutically acceptable carrier, in particular for use in the treatment of cancer;

a product or kit containing (a) a compound of formula (1) and (b) a DNA- damaging anti-tumoral agent as a combined preparation for simultaneous, separate or sequential use, in particular in the treatment of cancer;

- a combined preparation which comprises (a) a compound of formula (1) and (b) a

DNA-damaging anti-tumoral agent for simultaneous, separate or sequential use, in particular in the treatment of cancer;

a pharmaceutical composition comprising a compound having the formula (1) for the use in the treatment of cancer in combination with radiotherapy;

- the use of a pharmaceutical composition comprising a compound having the formula (1) for the manufacture of a medicament for the treatment of cancer in combination with radiotherapy or a DNA-damaging anti-tumoral agent; the use of a pharmaceutical composition comprising a) a compound having the formula (1) and b) a DNA-damaging anti-tumoral agent, and a pharmaceutically acceptable carrier for the manufacture of a medicament for the treatment of cancer; a method for treating a cancer in a subject in need thereof, comprising administering an effective amount of a pharmaceutical composition comprising a) a compound having the formula (1), b) a DNA-damaging anti-tumoral agent, and a pharmaceutically acceptable carrier;

a method for treating a cancer in a subject in need thereof, comprising administering an effective amount of a pharmaceutical composition comprising a compound having the formula (1), and an effective amount of a pharmaceutical composition comprising a DNA-damaging anti-tumoral agent;

a method for treating a cancer in a subject in need thereof, comprising administering an effective amount of a pharmaceutical composition comprising a) a compound having the formula (1) in combination with radiotherapy;

herein the compound has the formula (1)

wherein

Rai, Ra₂ and Ra₃, each independently, are selected from the group consisting of hydrogen, (Ci-C₃)alkyl, halogen, hydroxyl, aroyloxy of formula ArC(=0)0-), arylcarbamoyloxy of formula ArNH(C=0)0-), (Ci-C₃)alkoxy, optionally substituted by an aryl; or two of the radicals Rai and Ra₂ or Rai and Ra₃ may be taken together to form a (Ci-C₂)alkylenedioxy radical;

Rb is selected from the group consisting of hydrogen, (Ci-C₃)alkyl optionally substituted by a radical selected in the group consisting of hydroxyl, -NRR', - OPO(OR)(OR'), -OC(=0)R; Rci and Rc₂, each independently, are selected from the group consisting of hydrogen, (Ci-C3)alkyl and aryl; or Rci and Rc₂ may be taken together to form a bivalent radical of formula

^■ -(CH₂)_n- wherein n is 3, 4 or 5; or

■ -CH=CH-CH=CH- , optionally substituted by a (Ci-C₃)alkyloxy,

- X is an oxygen or a sulfur;

or an isomeric form thereof or a pharmaceutically acceptable salt and derivative thereof.

In a preferred embodiment, Rai, Ra₂ and Ra₃, each independently, are selected from the group consisting of hydrogen, hydroxyl, methoxy, ethoxy, phenoyloxy, phenylcarbamoyloxy and benzyloxy. In particular, Rai is selected from the group consisting of hydrogen, hydroxyl, methoxy, ethoxy and benzyloxy, and Ra₂ and Ra₃ are H. More preferably, Rai, Ra₂ and Ra₃, each independently, are selected from the group consisting of hydrogen, hydroxyl, and methoxy. In particular, Rai is selected from the group consisting of hydrogen, hydroxyl, and methoxy, and Ra₂ and Ra₃ are H. Still more preferably, Rai, Ra₂ and Ra₃, each independently, are selected from the group consisting of hydrogen and methoxy. In particular Rai is selected from the group consisting of hydrogen and methoxy, and Ra₂ and Ra₃ are H.

In a preferred embodiment, Rb is selected from the group consisting of hydrogen, methyl, ethyl, -CH₂-OH, -(CH₂)_n-N[(Ci-C₂)alkyl]₂ with n being 2 or 3, -(CH₂)-OPO[0(Ci- C₄)alkyl]₂ and -(CH₂)-OC(=0)-(Ci-C₄)alkyl. More preferably, Rb is selected from the group consisting of hydrogen, -CH₂-OH, and -(CH₂)_n-N[(Ci-C₂)alkyl]₂ with n being 2 or 3. Still more preferably, Rb is hydrogen.

In a preferred embodiment, Rci and Rc₂, each independently, are selected from the group consisting of hydrogen, methyl, ethyl and phenyl, or may be taken together to form a bivalent radical of formula -CH=CH-CH=CH-. More preferably, Rci is selected from the group consisting of hydrogen, methyl, ethyl and phenyl, and Rc₂ is hydrogen. Still more preferably, Rci is selected from the group consisting of hydrogen, methyl, and ethyl, and Rc₂ is hydrogen. In a preferred embodiment, Rd is selected from the group consisting of hydrogen, methyl, ethyl, and -(CH₂)n-N[(Ci-C2)alkyl]₂ with n being 2 or 3. More preferably, Rd is selected from the group consisting of hydrogen, methyl, and ethyl. Still more preferably, Rd is hydrogen.

In a preferred embodiment, X is an oxygen.

Accordingly, in a particular aspect of the compound of formula (1), the compound of formula (1) has one or several of the following features:

Rai, Ra₂, Ra₃, each independently, independently selected from the group consisting of hydrogen, hydroxyl, methoxy, ethoxy, phenoyloxy, phenylcarbamoyloxy and benzyloxy; more preferably, Rai is selected from the group consisting of hydrogen, hydroxyl, methoxy, ethoxy, phenoyloxy, phenylcarbamoyloxy and benzyloxy, and Ra₂ and Ra₃ are hydrogen; still more preferably, Rai is selected from the group consisting of hydrogen, hydroxyl and methoxy, and Ra₂ and Ra₃ are hydrogen; and/or

- Rb is selected from the group consisting of hydrogen, methyl, ethyl, -CH₂-OH, -

(CH₂)_n-N[(Ci-C₂)alkyl]₂ with n being 2 or 3, -(CH₂)-OPO[0(Ci-C₄)alkyl]₂ and -

more preferably, Rb is selected from the group consisting of hydrogen, -CH₂-OH, and -(CH₂)_n-N[(Ci-C₂)alkyl]₂ with n being 2 or 3; still more preferably, Rb is hydrogen; and/or

- Rci and Rc₂, each independently, are selected from the group consisting of hydrogen, methyl, ethyl and phenyl, or may be taken together to form a bivalent radical of formula -CH=CH-CH=CH-; more preferably, Rci is selected from the group consisting of hydrogen, methyl, ethyl and phenyl, and Rc₂ is hydrogen; still more preferably, Rci is selected from the group consisting of hydrogen, methyl and ethyl, and Rc₂ is hydrogen; and/or

Rd is selected from the group consisting of hydrogen, methyl, ethyl, and -(CH₂)_n- N[(Ci-C₂)alkyl]₂ with n being 2 or 3; more preferably, Rd is selected from the group consisting of hydrogen, methyl, and ethyl; still more preferably, Rd is hydrogen; and/or

- X is an oxygen.

In a preferred embodiment, at least one of Rai, Ra₂, Ra₃, Rb, Rci, Rc₂ and Rd is different from hydrogen atom. More preferably, Ra₂, Ra₃, Rb, Rc₂ and Rd are hydrogen atoms and one of Rai and Rci or both are different from hydrogen atom. Accordingly, in a more specific embodiment, Rai, Ra₂, Ra₃, each independently, independently selected from the group consisting of hydrogen, hydroxyl, methoxy, ethoxy, phenoyloxy, phenylcarbamoyloxy and benzyloxy; Rb is selected from the group consisting of hydrogen, methyl, ethyl, -CH₂-OH, -(CH₂)_n-N[(Ci-C₂)alkyl]₂ with n being 2 or 3, -(CH₂)- OPO[0(Ci-C₄)alkyl]₂ and -(CH₂)-OC(=0)-(Ci-C₄)alkyl; Rci and Rc₂, each independently, are selected from the group consisting of hydrogen, methyl, ethyl and phenyl, or may be taken together to form a bivalent radical of formula -CH=CH-CH=CH-; Rd is selected from the group consisting of hydrogen, methyl, ethyl, and -(CH₂)_n-N[(Ci-C₂)alkyl]₂ with n being 2 or 3; and X is an oxygen. More preferably, Rai is selected from the group consisting of hydrogen, hydroxyl, methoxy, ethoxy, phenoyloxy, phenylcarbamoyloxy and benzyloxy, and Ra₂ and Ra₃ are hydrogen; Rb is selected from the group consisting of hydrogen, -CH₂-OH, and -(CH₂)_n-N[(Ci-C₂)alkyl]₂ with n being 2 or 3; Rci is selected from the group consisting of hydrogen, methyl, ethyl and phenyl, and Rc₂ is hydrogen; Rd is selected from the group consisting of hydrogen, methyl, and ethyl; and X is an oxygen. Still more preferably, Rai is selected from the group consisting of hydrogen, hydroxyl and methoxy, and Ra₂, Ra₃, Rb, Rc₂ and Rd are hydrogen; Rci is selected from the group consisting of hydrogen, methyl and ethyl; and X is an oxygen.

In the most preferred embodiment, the compound of formula (1) is selected in the group consisting of:

- a compound wherein Rai is methoxy, Ra₂ and Ra₃ are H, Rb is H, Rci is ethyl, Rc₂ is H, Rd is H and X is O; this compound is called herein "CI";

a compound wherein Rai is methoxy, Ra₂ and Ra₃ are H, Rb is H, Rci is methyl, Rc₂ is H, Rd is H and X is O; this compound is called herein "C2";

a compound wherein Rai, Ra₂ and Ra₃ are H, Rb is H, Rci is methyl, Rc₂ is H, Rd is H and X is O; this compound is called herein "C3";

a compound wherein Rai is methoxy, Ra₂ and Ra₃ are H, Rb is H, Rci and Rc₂ are H, Rd is H and X is O; this compound is called herein "C4";

a compound wherein Rai is hydroxyl, Ra₂ and Ra₃ are H, Rb is H, Rci is methyl, Rc₂ is H, Rd is H and X is O; this compound is called herein "C5";

- a compound wherein Rai is phenoyloxy, Ra₂ and Ra₃ are H, Rb is H, Rci is methyl,

Rc₂ is H, Rd is H and X is O; this compound is called herein "C6";and, a compound wherein Rai is methoxy, Ra₂ and Ra₃ are H, Rb is H, Rci and Rc₂ taken together form a bivalent radical of formula -CH=CH-CH=CH-, Rd is H and X is O; this compound is called herein "C7". More preferably, the compound of formula (1) is selected in the group consisting of CI, C2, C3, C4 and C5. In a particular aspect, the invention concerns the new compounds C5 and/or C6 and a pharmaceutical composition including one of these compounds.

In a specific embodiment, the compound of formula (1) is a compound wherein Rai is methoxy, Ra₂ and Ra₃ are H, Rb is H, Rci is ethyl, Rc₂ is H, Rd is H and X is O, i.e. the compound CI .

The compounds C1-C4 are described as intermediary products in the following references: "Synthesis and antitumor activity of l-(dialkylamino) alkylamino-4-methyl-5H- pyrido [4,3-b] benzo [e] (and benzo [g]) indoles. A new class of antineoplastic agents." (C.H. Nguyen, et al. ; J. Med. Chem. (1990), 33, 1519-1528) for compounds C2 and C3; and "Further SAR in the new antitumor l-amino-substituted-y-carbolines and 5H- benzo[e]pyrido[4,3-b] indoles series." (C.H. Nguyen, et al. ; Anti-Cancer Drug Design (1992), 7, 235-251) for compounds CI and C4. These compounds are also described in the article of Hoang et al. (Hoang et al, 2009, supra). The compound C7 is described as intermediary product in the following reference as compound 14: "Synthesis of 13H- Benzo[6,7]- and 13H-Benzo[4,5]indolo[3,2-c]-quinolines: A New Series of Potent Specific Ligands for Triplex DNA" (C.H. Nguyen, et al. ; J. Am. Chem. Soc. 1998, 120, 2501-2507). Compounds C5 and C6 can be synthesized as detailed in Example 2.

In the context of the present invention, the term "(Ci-C₂)alkyl" more specifically means methyl or ethyl, the term "(Ci-C₃)alkyl" more specifically means methyl, ethyl, propyl, or isopropyl and "(Ci-C4)alkyl" more specifically means methyl, ethyl, propyl, isopropyl, butyl, isobutyl, or tert-butyl.

"Alkoxy" groups correspond to the alkyl groups defined hereinabove bonded to the molecule by an -O- (ether) bond. (Ci-C₃)alkoxy includes methoxy, ethoxy, propyloxy, and isopropyloxy.

The "aryl" or "Ar" group is mono- or bi- cyclic aromatic hydrocarbons having from 6 to 12 carbon atoms, optionally substituted. Aryl may be a phenyl, biphenyl or naphthyl. In a preferred embodiment, the aryl is a phenyl.

"Halogen" groups are preferably selected from the group consisting of CI (chloride), Br (bromide), I (iodide) and F (fluoride).

The term "derivative" is meant to encompass hydrate, ester, ether, conjugates, or prodrugs thereof. For instance, the compounds with a radical -OPO(OR)(OR') as defined above is a prodrug and has an increased solubility. The pharmaceutically acceptable salts include salts of inorganic acids as well as organic acids. Representative examples of suitable inorganic acids include hydrochloric, hydrobromic, hydroiodic, phosphoric, and the like. Representative examples of suitable organic acids include formic, acetic, trichloroacetic, trifluoroacetic, propionic, benzoic, cinnamic, citric, fumaric, maleic, methanesulfonic and the like. Further examples of pharmaceutically acceptable inorganic or organic acid addition salts include the pharmaceutically acceptable salts listed in J. Pharm. Sci. 1977, 66, 2, and in Handbook of Pharmaceutical Salts: Properties, Selection, and Use edited by P. Heinrich Stahl and Camille G. Wermuth 2002.

The term "pharmaceutically acceptable carrier" is meant to encompass any carrier

(e.g., support, substance, solvent, etc.) which does not interfere with effectiveness of the biological activity of the active ingredient(s) and that is not toxic to the host to which it is administered. For example, for parenteral administration, the active compounds(s) may be formulated in a unit dosage form for injection in vehicles such as saline, dextrose solution, serum albumin and Ringer's solution.

In a particular embodiment, the DNA-damaging anti-tumoral agent is chosen from the group consisting of inhibitors of topoisomerases I and/or II, DNA crosslinkers, DNA alkylating agents, and anti-metabolic agents. In a preferred embodiment, the DNA-damaging anti-tumoral agent is chosen from the group consisting of inhibitors of topoisomerases I and/or II, and DNA crosslinkers.

Inhibitors of topoisomerases I and/or II include, but are not limited to, etoposide, topotecan, camptothecin, irinotecan, amsacrine, intoplicin, anthracyclines such as doxorubicin, epirubicin, daunorubicin, idarubicin and mitoxantrone. Inhibitors of Topoisomerase I and II include, but are not limited to, intoplicin.

DNA crosslinkers include, but are not limited to, cisplatin, carboplatin and oxaliplatin.

In a preferred embodiment, the DNA crosslinker is cisplatin.

Anti-metabolic agents block the enzymes responsible for nucleic acid synthesis or become incorporated into DNA, which produces an incorrect genetic code and leads to apoptosis. Non-exhaustive examples thereof include, without limitation, folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors, and more particularly Methotrexate, Floxuridine, Cytarabine, 6-Mercaptopurine, 6- Thioguanine, Fludarabine phosphate, Pentostatine, 5-fluorouracil, gemcitabine and capecitabine.

The DNA-damaging anti-tumoral agent can be alkylating agents including, without limitation, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas, metal salts and triazenes. Non-exhaustive examples thereof include Uracil mustard, Chlormethine, Cyclophosphamide (CYTOXAN(R)), Ifosfamide, Melphalan, Chlorambucil, Pipobroman, Triethylenemelamine, Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine, cisplatin, carboplatin, oxaliplatin, thiotepa, Streptozocin, Dacarbazine, and Temozolomide.

In a preferred embodiment, the DNA-damaging anti-tumoral agent is a topoisomerase

I and/or II inhibitor, optionally selected from the group consisting of etoposide, topotecan, camptothecin, irinotecan, and anthracyclines, such as doxorubicin, epirubicin, daunorubicin, idanrubicin and mitoxantrone. In a more preferred embodiment, the DNA-damaging anti- tumoral agent is a topoisomerase II inhibitor. In particular, it can be selected in the group consisting of etoposide, doxorubicin, epirubicin, daunorubicin, idanrubicin and mitoxantrone. In a more particular embodiment, the DNA-damaging anti-tumoral agent is etoposide or intoplicin, in particular etoposide.

In a preferred embodiment, the DNA-damaging anti-tumoral agent is selected from the group consisting of etoposide, intoplicin and cisplatine.

In a specific embodiment of the invention, the present invention relates to a pharmaceutical composition comprising

a) a compound of formula (1) preferably selected from the group consisting of CI, C2, C3, C4, C5, C6 and C7, preferably CI, and

b) a topoisomerase I or II inhibitor, optionally selected from the group consisting of etoposide, topotecan, camptothecin, irinotecan, anthracyclines such as doxorubicin, epirubicin, daunorubicin, idarubicin and mitoxantrone, preferably a topoisomerase II inhibitor, more preferably etoposide.

In a very specific embodiment of the invention, the present invention relates to a pharmaceutical composition, a kit or a combined preparation comprising the compound CI and etoposide.

In another specific embodiment of the invention, the present invention relates to a pharmaceutical composition comprising

b) a DNA crosslinker, optionally selected from the group consisting of cisplatin, carboplatin and oxaliplatin, preferably cisplatin.

In another very specific embodiment of the invention, the present invention relates to a pharmaceutical composition, a kit or a combined preparation comprising the compound CI and cisplatin. In a further specific embodiment of the invention, the present invention relates to a pharmaceutical composition comprising

b) a topoisomerase I and/or II inhibitor, optionally selected from the group consisting of etoposide, topotecan, camptothecin, irinotecan, intoplicin, anthracyclines such as doxorubicin, epirubicin, daunorubicin, idarubicin and mitoxantrone, preferably etoposide or intoplicin.

In an additional specific embodiment of the invention, the present invention relates to a pharmaceutical composition comprising

b) a topoisomerase I and/or II inhibitor or a DNA crosslinker, preferably selected from the group consisting of etoposide, intoplicin, and cisplatin.

The pharmaceutical composition can be formulated as solutions in pharmaceutically compatible solvents or as emulsions, suspensions or dispersions in suitable pharmaceutical solvents or vehicule, or as pills, tablets or capsules that contain solid vehicules in a way known in the art. Formulations of the present invention suitable for oral administration may be in the form of discrete units as capsules, sachets, tablets or lozenges, each containing a predetermined amount of the active ingredient; in the form of a powder or granules; in the form of a solution or a suspension in an aqueous liquid or non-aqueous liquid; or in the form of an oil-in-water emulsion or a water-in-oil emulsion. Formulations for rectal administration may be in the form of a suppository incorporating the active ingredient and carrier such as cocoa butter, or in the form of an enema. Formulations suitable for parenteral administration conveniently comprise a sterile oily or aqueous preparation of the active ingredient which is preferably isotonic with the blood of the recipient. Every such formulation can also contain other pharmaceutically compatible and nontoxic auxiliary agents, such as, e.g. stabilizers, antioxidants, binders, dyes, emulsifiers or flavouring substances. The formulations of the present invention comprise an active ingredient in association with a pharmaceutically acceptable carrier therefore and optionally other therapeutic ingredients. The carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulations and not deleterious to the recipient thereof. The pharmaceutical compositions are advantageously applied by injection or intravenous infusion of suitable sterile solutions or as oral dosage by the digestive tract. Methods for the safe and effective administration of most of these chemotherapeutic agents are known to those skilled in the art. In addition, their administration is described in the standard literature.

In particular, the present invention also relates to a product, kit or combined preparation comprising (a) one or more unit dosage forms of a compound of formula (1) and (b) one or more unit dosage forms of a DNA-damaging anti-tumoral agent.

Preferably, in a specific embodiment, the present invention relates to a product, kit or combined preparation containing or comprising

a) a compound of formula (1) selected from the group consisting of CI, C2, C3, C4, C5, C6 and C7, preferably CI, and

In a very specific embodiment, the present invention relates to a product, kit or combined preparation containing or comprising the compound CI and etoposide.

Preferably, in another specific embodiment, the present invention relates to a product, kit or combined preparation containing or comprising

In another very specific embodiment, the present invention relates to a product, kit or combined preparation containing or comprising the compound CI and cisplatin.

In a further specific embodiment of the invention, the present invention relates to a product, kit or combined preparation containing or comprising

In an additional specific embodiment of the invention, the present invention relates to a product, kit or combined preparation containing or comprising a) a compound of formula (1) preferably selected from the group consisting of CI, C2, C3, C4, C5, C6 and C7, preferably CI, and

The terms "kit", "product" or "combined preparation", as used herein, defines especially a "kit of parts" in the sense that the combination partners (a) and (b) as defined above can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination partners (a) and (b), i.e. simultaneously or at different time points. The parts of the kit of parts can then, e.g., be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts. The ratio of the total amounts of the combination partner (a) to the combination partner (b) to be administered in the combined preparation can be varied. The combination partners (a) and (b) can be administered by the same route or by different routes. In a preferred embodiment, partner (b) is administered before or simultaneously partner (a). When the administration is sequential, the first partner may be for instance administered 1, 2, 3, 4, 5, 6, 12, 18 or 24 h before the second partner.

The present invention relates to a pharmaceutical composition, product, kit or combined preparation of the invention for use in the treatment of cancer. The pharmaceutical composition, product, kit or combined preparation of the invention may be used in combination with radiotherapy and/or other chemotherapy.

Radiotherapy includes, but is not limited to, γ-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other radiotherapies include microwaves and UV- irradiation. Other approaches to radiation therapy are also contemplated in the present invention.

The present invention relates to a method for treating a cancer in a subject in need thereof, comprising administrating an effective amount of a pharmaceutical composition or a kit, product or combined preparation as defined above, alone or in combination with radiotherapy. In a preferred embodiment, the radiotherapy is applied before or simultaneously with the administration of the compound of the present invention. When the administration of the compound is after radiotherapy, the compound may be for instance administered 1, 2, 3, 4, 5, 6, 12, 18 or 24 h after the radiotherapy.

Within the context of the invention, the term treatment denotes curative, symptomatic, and preventive treatment. Pharmaceutical compositions, kits, products and combined preparations of the invention can be used in humans with existing cancer or tumor, including at early or late stages of progression of the cancer. The pharmaceutical compositions, kits, products and combined preparations of the invention will not necessarily cure the patient who has the cancer but will delay or slow the progression or prevent further progression of the disease, ameliorating thereby the patients' condition. In particular, the pharmaceutical compositions, kits, products and combined preparations of the invention reduce the development of tumors, reduce tumor burden, produce tumor regression in a mammalian host and/or prevent metastasis occurrence and cancer relapse. In treating the cancer, the pharmaceutical composition of the invention is administered in a therapeutically effective amount.

By "effective amount" it is meant the quantity of the pharmaceutical composition of the invention which prevents, removes or reduces the deleterious effects of cancer in mammals, including humans. It is understood that the administered dose may be adapted by those skilled in the art according to the patient, the pathology, the mode of administration, etc.

Whenever within this whole specification "treatment of a cancer" or the like is mentioned with reference to the pharmaceutical composition of the invention, there is meant: a) a method for treating a cancer, said method comprising administering a pharmaceutical composition of the invention to a subject in need of such treatment; b) the use of a pharmaceutical composition of the invention for the treatment of a cancer; c) the use of a pharmaceutical composition of the invention for the manufacture of a medicament for the treatment of a cancer; d) a pharmaceutical composition comprising a dose of a compound of formula (1) and of a DNA-damaging anti-tumoral agent that is appropriate for the treatment of a cancer; and/or e) a pharmaceutical composition of the invention for treating a cancer.

The treatment may be topical, transdermal, oral, rectal, sublingual, intranasal or parenteral. The pharmaceutical composition, kit, product or combined preparation is preferably administered by injection or by intravenous infusion or suitable sterile solutions, or in the form of liquid or solid doses via the alimentary canal.

The present invention more particularly relates to a pharmaceutical composition, a kit, product or combined preparation wherein the amount or dosage of the DNA-damaging anti- tumoral agent can be lowered in comparison with its amount or dosage when it is used alone. Indeed, the combination of a compound of formula (1) and a DNA-damaging agent leads at least to an additive effect and more often to a synergistic effect of the two active ingredients This potentiating effect allows the decrease of the amount of the anti-tumoral agents causing DNA damages, which generally exhibit high toxicity for the normal cells and therefore are associated with adverse effects. The compounds of formula (1) advantageously exhibit a minimal toxicity. Then, with the combined treatment of the invention, it is possible to preserve the efficacy of the treatment, or even to improve it, while decreasing its adverse effects, in particular the adverse effects of the DNA-damaging anti-tumoral agent.

Alternatively, instead of lowering the amount or dosage of the DNA-damaging anti- tumoral agent, the administration frequency of the DNA-damaging anti-tumoral agent or its or treatment period can be reduced. For instance, by I.V. route, the etoposide is used at a conventional dosage of about 50-100 mg/m²/day during three days with an interval of two to four weeks between treatments. By oral route, the etoposide is used at a conventional dosage of about 100-300 mg/m²/day during three to five days with an interval of two to four weeks between treatments. Accordingly, in the context of the present invention, the treatment period may be reduced, for instance by 90, 80, 70, 60 or 50%. Therefore, in case of etoposide, instead of a three-five day treatment, etoposide administration period can be shortened to one to three days. In another embodiment, the interval between DNA-damaging agent treatments can be increased, for instance by 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% or by 1.5, 2, 2.5 or 3 fold. For instance, instead of an interval of two to four weeks between DNA-damaging agent treatments, the interval between treatments can be increased to four to eight weeks, preferably five to eight weeks.

According to an embodiment, the present invention relates to a method for the treatment of a cancer, to a pharmaceutical composition, to a product, kit or combined preparation as disclosed above, wherein the amounts of the compound of formula (1) and the DNA-damaging anti-tumoral agent in the combined preparation are such that the combined therapeutic effect of the two active ingredients is additional or preferably synergistic.

By the term "synergistic" therapeutic effect is meant that the obtained therapeutic effect of the combination is more than the addition of the therapeutic effect of each partner alone (i.e. more than the effect of the compound of formula (1) alone plus the effect of the DNA-damaging anti-tumoral agent alone).

By the term "additional" therapeutic effect is meant that the obtained therapeutic effect of the combination is the addition of the therapeutic effect of each partner alone (i.e. equals to the effect of the compound of formula (1) alone plus the effect of the DNA-damaging anti- tumoral agent alone).

The present invention relates to a method for the treatment of a cancer, to a pharmaceutical composition, to a product, kit or combined preparation as disclosed above, wherein the DNA-damaging anti-tumoral agent is used at lower dosage than the conventional dosage used in chemotherapy for the same indication and the same administration route when it is used alone (i.e., an amount equal to or preferably lower than the one used in conventional chemotherapy), also called herein a sub-therapeutic amount. More particularly, the amount can be for instance 90, 80, 70, 60, 50, 40, 30, 20 or 10 % of the conventional therapeutic dosage (in particular for the same indication and the same administration route). The conventional therapeutic dosages are those acknowledged by the drug approvals agencies (e.g., FDA or EMEA) and can be found in reference Manuals such as Merck Manuals (www.merck.com/mmpe/lexicomp/). For instance, by I.V. route, the etoposide is used at a conventional dosage of about 35-250 mg/m²/day, preferably 50-100 mg/m²/day. By oral route, the etoposide is used at a conventional dosage of about 70-500 mg/m²/day, preferably 100- 300 mg/m²/day. In that respect, the present invention relates to a method for the treatment of a cancer, to a pharmaceutical composition, to a product, kit or combined preparation as disclosed above, wherein the amount of the DNA-damaging anti-tumoral agent is used at a sub-therapeutic dosage and the amount of compound of formula (1) is such that the combined therapeutic effect of the two active ingredients is additional or more preferably synergistic.

The present invention relates to a method for the treatment of a cancer comprising administering a synergistically therapeutically effective amount of the combined preparation of (a) a compound of formula (1) and (b) a DNA-damaging anti-tumoral agent.

The invention also relates to a synergistic combination which comprises (a) a compound of formula (1) and (b) a DNA-damaging anti-tumoral agent in a synergistic ratio for simultaneous, separate or sequential use, in particular in the treatment of cancer.

By the term "synergistically therapeutically effective amount" or "synergistic ratio" is meant that the therapeutic effect of the combination is more than the addition of the therapeutic effect of each partner alone (i.e. more than the therapeutic effect of the compound of formula (1) alone plus the therapeutic effect of the DNA-damaging anti-tumoral agent alone).

The invention also relates to a pharmaceutical composition comprising a quantity which is jointly therapeutically effective against a cancer of the combination of the invention and at least one pharmaceutically acceptable carrier.

In a particular embodiment of the invention, the synergistic combination is such that the DNA-damaging anti-tumoral agent is used or administered in a sub-therapeutic amount. In particular, a sub-therapeutic amount of the anti-tumoral agent causing DNA damages is less than the conventional dosage used to treat a cancer as a single drug (i.e., not in combination with another anti-tumoral drug). More particularly, the sub-therapeutic amount can be for instance 90, 80, 70, 60, 50, 40, 30, 20 or 10 % of the conventional therapeutic dosage for the same indication and the same administration route. The conventional therapeutic dosages are those acknowledged by the drug approvals agencies (e.g., FDA or EMEA) and can be found in reference Manuals such as Merck Manuals. For example, a therapeutic amount of etoposide is from 50 to 400 mg/m²/day.

Determining an additional or a synergistic interaction between one or more components, the optimum range for the effect and absolute dose ranges of each component for the effect may be definitively measured by administration of the components over different w/w ratio ranges and doses to patients in need of treatment. For humans, the complexity and cost of carrying out clinical studies on patients may render impractical the use of this form of testing as a primary model for synergy. However, the observation of synergy in one species can be predictive of the effect in other species and animal models exist to measure a synergistic effect and the results of such studies can also be used to predict effective dose and plasma concentration ratio ranges and the absolute doses and plasma concentrations required in other species by the application of pharmacokinetic/pharmacodynamic methods. Correlations between cancer models and effects seen in man suggest that observed synergy on animal models may be predictive of a synergy on man too.

The pharmacological activity of a combination of the invention may, for example, be demonstrated in a clinical study or more preferably in a test procedure. Suitable clinical studies are, for example, open label non-randomized, dose escalation studies in patients with advanced tumors. Such studies can prove the additive or synergism of the active ingredients of the combination of the invention. The beneficial effects on proliferative diseases can be determined directly through the results of these studies or by changes in the study design which are known as such to a person skilled in the art. Such studies are, in particular, suitable to compare the effects of a monotherapy using the active ingredients and a combination of the invention. Preferably, the combination partner (a) is administered with a fixed dose and the dose of the combination partner (b) is escalated until the maximum tolerated dosage is reached. Alternatively, the combination partner (b) is administered with a fixed dose and the dose of the combination partner (a) is escalated until the maximum tolerated dosage is reached.

The effective dosage of each of the combination partners employed in the combined preparation of the invention may vary depending on the particular compound or pharmaceutical composition employed, the mode of administration, the condition being treated, the severity of the condition being treated. Thus, the dosage regimen of the combined preparation of the invention is selected in accordance with a variety of factors including the route of administration and the patient status. A physician, clinician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the single active ingredients required to prevent, counter or arrest the progress of the condition. Optimal precision in achieving concentration of the active ingredients within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the active ingredients' availability to target sites.

In a particular embodiment, the present invention relates to a combined preparation comprising (a) a compound of formula (1) and (b) a DNA-damaging anti-tumoral agent, wherein the amounts of the compound of formula (1) and of the DNA-damaging anti-tumoral agent in the combined preparation are such that the combined therapeutic effect of the two active ingredients is synergistic.

In a particular embodiment, the present invention relates to a combined preparation comprising (a) a compound of formula (1) and (b) a DNA-damaging anti-tumoral in a molar combination ratio which corresponds to a synergistic combination range of 1 :50 to 1 : 1 in a U20S osteosarcoma model or a HL60 myeloid model, more preferably in a mouse model as disclosed in the Examples. Preferably, the synergistic combination range is of 1 :20 to 1 :1.

In a particular aspect of the invention, the present invention relates to a method for treating a cancer as described above, wherein the treatment is the first line treatment of the cancer (i.e., the initial treatment of the cancer). In addition, the invention relates to a pharmaceutical composition, a product, kit or combined preparation as described above for use in the treatment of a cancer in a subject as the first line treatment. Finally, the invention relates to the use of a pharmaceutical composition, a product, kit or combined preparation as described above for the preparation of a medicament for treating a cancer in a subject as a first line treatment.

In a particular embodiment of the invention, the composition, kit or method of the invention can further comprise another antitumoral agent. Preferably, the additional antitumoral agent is an histone deacetylase (HDAC) inhibitor or a taxoid antitumoral agent. Such antitumoral agents are well-known by the one skilled in the art. For instance, non- exhaustively, the taxoid antitumoral agent can be selected from the group consisting of paclitaxel, docetaxel, larotaxel, XRP6258, BMS-184476, BMS-188797, BMS-275183, ortataxel, RPR 109881A, RPR 116258, NBT-287, PG-paclitaxel, ABRAXANE®, Tesetaxel, IDN 5390, Taxoprexin, DHA-paclitaxel, and MAC-321. More preferably, the molecule of the taxoid antitumoral agent is paclitaxel. Similarly, non-exhaustively, the HDAC can be selected from the group consisting of trichostatin A, vironostat, belinostat, LAQ824, panobinostat (LBH589), mocetinostat, valproic acid, romidepsin, ITF2357, benzamides entinostat (MS275), and CI994.

The terms "cancer", "cancerous", or "malignant" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, for example, leukemia, lymphoma, blastoma, carcinoma and sarcoma. More particular examples of such cancers include chronic myeloid leukemia, acute lymphoblastic leukemia, Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL), squamous cell carcinoma, small-cell lung cancer, non-small cell lung cancer, glioma, gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon carcinoma, and head and neck cancer, gastric cancer, germ cell tumor, pediatric sarcoma, sinonasal natural killer, multiple myeloma, acute myelogenous leukemia (AML), chronic lymphocytic leukemia, mastocytosis and any symptom associated with mastocytosis.

"Leukemia" refers to progressive, malignant diseases of the blood-forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia is generally clinically classified on the basis of (1) the duration and character of the disease— acute or chronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid (lymphogenous), or monocytic; and (3) the increase or non- increase in the number of abnormal cells in the blood— leukemic or aleukemic (subleukemic). Leukemia includes, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, hairy- cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocyte leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblasts leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, plasmacytic leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, and undifferentiated cell leukemia. In certain aspects, the present invention provides treatment for chronic myeloid leukemia, acute lymphoblastic leukemia, and/or Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL).

Various cancers are also encompassed by the scope of the invention, including, but not limited to, the following: carcinoma including that of the bladder (including accelerated and metastatic bladder cancer), breast, colon (including colorectal cancer), kidney, liver, lung (including small and non-small cell lung cancer and lung adenocarcinoma), ovary, prostate, testes, genitourinary tract, lymphatic system, rectum, larynx, pancreas (including exocrine pancreatic carcinoma), esophagus, stomach, gall bladder, cervix, thyroid, and skin (including squamous cell carcinoma); hematopoietic tumors of lymphoid lineage including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hairy cell lymphoma, histiocytic lymphoma, and Burketts lymphoma; hematopoietic tumors of myeloid lineage including acute and chronic myelogenous leukemias, myelodysplasia syndrome, myeloid leukemia, and promyelocytic leukemia; tumors of the central and peripheral nervous system including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; other tumors including melanoma, xenoderma pigmentosum, keratoactanthoma, seminoma, thyroid follicular cancer, and teratocarcinoma; melanoma, unresectable stage III or IV malignant melanoma, squamous cell carcinoma, small-cell lung cancer, non-small cell lung cancer, glioma, gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, stomach cancer, bladder cancer, hepatoma, breast cancer, colon carcinoma, and head and neck cancer, retinoblastoma, gastric cancer, germ cell tumor, bone cancer, bone tumors, adult malignant fibrous histiocytoma of bone; childhood malignant fibrous histiocytoma of bone, sarcoma, pediatric sarcoma, sinonasal natural killer, neoplasms, plasma cell neoplasm; myelodysplasia syndromes; neuroblastoma; testicular germ cell tumor, intraocular melanoma, myelodysplasia syndromes; myelodysplastic/myeloproliferative diseases, synovial sarcoma. In addition, disorders include urticaria pigmentosa, mastocytosises such as diffuse cutaneous mastocytosis, solitary mastocytoma in human, as well as dog mastocytoma and some rare subtypes like bullous, erythrodermic and teleangiectatic mastocytosis, mastocytosis with an associated hematological disorder, such as a myeloproliferative or myelodysplasia syndrome, or acute leukemia, myeloproliferative disorder associated with mastocytosis, mast cell leukemia, in addition to other cancers. Other cancers are also included within the scope of disorders including, but are not limited to, the following: carcinoma, including that of the bladder, urothelial carcinoma, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid, testis, particularly testicular seminomas, and skin; including squamous cell carcinoma; gastrointestinal stromal tumors ("GIST"); hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B- cell lymphoma, T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hairy cell lymphoma and Burketts lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; other tumors, including melanoma, seminoma, teratocarcinoma, neuroblastoma and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including melanoma, xenoderma pigmentosum, keratoactanthoma, seminoma, thyroid follicular cancer, teratocarcinoma, chemotherapy refractory non-seminomatous germ- cell tumors, and Kaposi's sarcoma, and any metastasis thereof.

In a preferred embodiment of the present invention, the cancer is a solid tumor. The term "solid tumor" especially means breast cancer, ovarian cancer, cancer of the colon and generally the Gl (gastro-intestinal) tract, cervix cancer, lung cancer, in particular small- cell lung cancer, and non- small-cell lung cancer, head and neck cancer, bladder cancer, cancer of the prostate or Kaposi's sarcoma. The present combination inhibits the growth of solid tumors, but also liquid tumors. Furthermore, depending on the tumor type and the particular combination used a decrease of the tumor volume can be obtained. The combinations disclosed herein are also suited to prevent the metastatic spread of tumors and the growth or development of micrometastases. The combinations disclosed herein are in particular suitable for the treatment of poor prognosis patients, especially such poor prognosis patients having metastatic melanome or pancreatic cancer.

In a particular embodiment, the present invention relates to a method for treating a cancer selected from glioblastoma, lung cancer including small cell lung cancer and non-small cell lung cancer, testicular cancer, ovarian cancer, sarcoma, retinoblastoma, prostate cancer, osteosarcoma, neuroblastoma, multiple myeloma, non-hodgkin's lymphoma, hodgkin's lymphoma, acute myeloid leukemia, breast cancer, gastric cancer, brain tumors, melanoma, colorectal cancer (CRC), kidney cancer, such as e.g. renal cell carcinoma (RCC), liver cancer, acute myelogenous leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), myelodysplasia Syndromes (MDS), thyroid cancer, pancreatic cancer, neurofibromatosis and hepatocellular carcinoma.

Further aspects and advantages of the present invention will be disclosed in the following experimental section, which should be regarded as illustrative and not limiting the scope of the present application. A number of references are cited in the present specification; each of these cited references is incorporated herein by reference.

Example

Example 1

In the present study, the inventors showed that CI inhibits significantly the phosphorylation of Chk-2 and consequently dsDNA reparation in cells. Actually Chk-2 is an important player in the DNA-damage response-signalling pathway. Chk-2 is activated and phosphorylated by ATM in response to double-strand breaks. Once activated Chk-2 phosphorylates downstream substrates involved in either cell cycle arrest or apoptosis like Cdc25A, Cdc25C, BRCA1, p53, E2F1, etc. In p53^~ cells, the Gl checkpoint is often defective and the targeting of the G2 checkpoint is thus a valuable strategy for inducing cell death. Recently Chk2 was proposed as a possible target for cancer therapy. Furthermore, Chk-2 is endogenously activated in precancerous lesions with genomic instability and thus, its inhibition in such a context, may lead to cell death. Moreover, it was shown that inhibiting Chk-2 in p53-defective cells enhances the apoptotic response to ionizing radiations. Then, the inventors decided to explore the simultaneous targeting of aurora kinase and Chk-2 in cancer cells. Benzo[e]pyridoindole 1 combined with Etoposide prevents Non Small Lung Cancer H358 cell growth in monolayer as well as 3 D spheroid cultured cells. It also stabilises the growth of H358 xenografts in nude mice.

Moreover the Etoposide plus CI combined treatment, that is additive in H358 cells, becomes synergic in HL60 cells. Accordingly, depending on the genetic background of the cells or on already acquired resistances, the combined effects of CI with Etoposide, a Topoisomerase II inhibitor, are found either additive or synergic. In fact in these cells, the aurora kinase inhibitor induces a strong mitotic arrest and then, endoreplication occurs through mitotic slippage. However HL60 cells entering into mitosis with DNA damages are more susceptible to apoptosis. This combination of drugs was found very efficient in mice bearing HL60 tumours. Long-term effects are noted: the size of the tumours is still tiny, three weeks after the last injection (Student test, p around 0,001). Benzo[e]pyridoindole 1 both targeting checkpoint kinase 2 and aurora B opens the way to combine therapies based on simultaneous alterations of mitotic onset and DNA damage defences.

Results

CI in hibits, in vitro, both aurora kinases and Checkpoint kinases.

Benzo[e]pyridoindole CI was found to be a potent aurora kinase inhibitor targeting in vitro the whole aurora kinase family. Interestingly nanomolar IC₅₀ were determined towards aurora kinases A and B thus suggesting that it is as potent as VX-680 (MK-0457) described as the aurora kinase reference inhibitor. However both inhibitors exhibit distinct selectivity since VX-680 do not target checkpoint kinases whereas, in vitro, CI inhibits Chk-1 and Chk-2 with IC₅₀ of 243 nM and 55 nM respectively (Fig. 1).

CI targets, in cellulo, Checkpoint kinase 2

The inventors found that CI prevents, ex vivo, the phosphorylation of Histone H3, induces mitosis exit and inactivates the spindle checkpoint, known phenomena observed upon aurora B inactivation. The question they have asked was whether CI may also target Chk-2 in cells. Chk-2 is activated by ATM and DNA-PK and thus phosphorylated on Thr68. Phospho- Thr68-Chk-2 homodimerized, resulting in trans- activating autophosphorylations of Thr383 and Thr387 as well as cis-phosphorylation of Ser516. Therefore the level of phospho-Ser516- Chk-2 reflects the activity of the kinase.

The inventors checked whether CI might modify the cellular response to widely used antitumour drugs like DNA topoisomerase-2 inhibitors. U20S cells were treated by Etoposide (Etoposide, 10 μΜ) and then allowed to recover in the presence or the absence of CI (1 μΜ). They followed the phosphorylations of Chk-2 and H2A-X during the 28 h of recovery (Figure 2). Whereas Chk-2 is detected at the same level in all cell extracts, phospho-S516-Chk-2 varied a lot. It is highly phosphorylated upon Etoposide treatment and this mark is still present 28 h later, in the absence of CI . Conversely phospho-Chk-2 is quickly dephosphorylated in the presence of CI suggesting a balance in favour of phosphatases under these conditions. Chk-2 was not found activated during the whole recovery processes in the presence of CI (28h post damages). This experiment confirms that CI inhibits Chk-2 in cells.-y-H2A-X is detected upon Etoposide treatment but decreases rapidly in the absence of CI and slowly in its presence (Figure 2A). The differences of γ-Η2Α-Χ signals are illustrated by immunofluorescence (Figure 2B). Whereas in the control, γ-Η2Α-Χ is punctuated, it is bright and diffuse in the presence of CI (Figure 2B, 24 h of recovery). In order to explore the possible application of these data the inventors verified whether CI may prevent DNA repair in p53 negative cells. H358 cells were treated by Etoposide and allowed to recover in the presence or the absence of CI for 4h, 20 h and 28h. The kinetic of γ-Η2Α-Χ deposition is clearly different under both conditions. After 28 h of recovery, γ-Η2Α-Χ signal is still very bright in the presence of CI whereas it decreases and appears punctuated in the control (Figure 2B). Therefore, CI inhibits DNA repair in p53 minus cells.

Effect of the combined treatment on H358 cells

Following the combined Etoposide plus CI treatment H358 cells enter in G2/M, the cell cycle window during which aurora kinases are fully active the inventors thus described long time effects of these drugs. First, the inventors observed the viability of these cells. Under conditions where 77 % of the cells are alive upon Etoposide treatment and 68 % following CI incubation, 51 % of them die with the combined treatment. Both treatment are additive since the calculation predicts the death of 48 % of the cells (Figure 3 A). In order to get access to long-term cultures and to approach tumour organisation the inventors adapted the multicellular tumour spheroid (MTS) model to H358 cells. They observed the continuous growth of H358 cells for at least 12 days (Figure 3B, blue curve). These spheroids were labelled with rhodamine-phalloidin and mitotic cells were detected by histone H3- phosphorylation. Fluorescent microscopy observations reveal that spheroids are mostly round (diameter superior to 470 μΜ). Cells are in close contact as revealed by the actin network. These spheroids are still in expansion since mitotic cells are present (Figure 3B). These 3D- cultures are thus suitable for testing the efficiency of drugs and the variation of growth were measured upon CI, Etoposide and combined treatments. CI decreases growth for 4 days, then it seems inefficient and finally spheroids are only one third smaller than in control (Figure 3B, triangle curve). Etoposide reduces more efficiently spheroid growth (growth ratio of 1 compared to 3 in the control; Figure 3B, square curve). Etoposide and CI prevent the expansion of spheroids and the effects are even observed 11 days later (Figure 3B, circle curve). The combined drugs allow a stabilization of the spheroid size but do not destroy it. Taking into account that H358 tumours are very aggressive and since no treatment are available, the inventors decided to evaluate the combined treatment on xenografts. Nude mice bearing H358 tumours established since two-weeks were treated with CI or etoposide or CI plus etoposide or the vehicle. The tumour growth for each individual animal, at day 56, is represented in Figure 3C. Under such conditions, CI is efficient for one mouse but, one tumour expands more rapidly than in the control suggesting the possible appearance of resistance. The average of tumour growth ratio is 6.7 for CI treated animals compared to 4.8 in the control mice. Etoposide has a moderate effect inducing a 3.6 average growth ratio (Figure 3C) and the best results were noted for the combined treatment. The tumour growth ratio is reduced to 2.1 when mice have received CI plus Etoposide. In such low favoured context, the combined treatment stabilized in most cases the disease.

Comparison of the combined treatment efficiency in different cell lines

In order to identify the best applications of these combined treatments the inventors tested different cell lines. Finally they found that the combination is additive in H358 cells and synergic in p53-WT U20S and in p53-deficient HL60 cells (Figure 4A, Figure 7). They compared the behaviour of these three cell lines upon etoposide and CI treatments. The etoposide concentration is adapted in each cell type in order to induce around 20 % of death at day 3. In HL60 cells, when expecting 80 % of alive cells they obtained only 51 % upon Etoposide plus CI . When they looked at the extent of DNA damages at day 1, in the presence of Etoposide plus CI, they noticed that they are more important in H358 and HL60 than in the U20S cells (Figure 4B). In fact as expected, p53 (U20S) cells either repair the DNA damages or they die. Towards the understanding of the differences of efficiency the inventors looked to the repartition in the cell cycle of the resistant population upon 24 h of treatment and 48 h of recovery (Figure 4C). They noted that etoposide induces a strong G2/M arrest in H358 cells whereas the proportion of apoptotic cells is quite low (around 3%). In both U20S and HL60, the sub-GO population is high (26 and 21 % respectively) following etoposide treatment (Figure 4C). Upon the mixed treatment the situation is unchanged for H358 cells, the G2/M arrest is still important and cells escaping this checkpoint enter in a new cycle (Figure 4C, 4D). Conversely the proportion of G0/G1 and S cells is tiny in both U20S and HL60 cells (Figure 4C, 4D). Therefore CI plus etoposide drive cells in mitosis and then prevent mitosis ongoing in both U20S and HL60 cells. In these two cells lines, cells escaping from mitosis either dye or become polyploid through mitotic slippage as expected upon Aurora kinase inhibition. CI is found more efficient by itself in H358 cells than in HL60 and U20S cells, inducing important cell death (around 32 %, see Figure 4A) but the remaining population seemed then, to be insensitive to CI and is still cycling. Although, upon etoposide plus CI treatment the overall viability is similar in the three cell lines, HL60 cells appear as the most attractive model since the two drugs have synergic effects and prevent cell cycling. Defining the best conditions in HL60 cells and in vivo application

To further interpret these results the inventors determined the best Etoposide concentration in HL60 cells and then, characterized the evolution of the populations during the recovery (figure 5). As shown in Figure 6A, the concentration of 1 μΜ induces G2/M arrest meanwhile few cells die or become polyploid. The 2 μΜ is less favourable since both the Gl and G2/M checkpoints are activated. The inventors thus studied the repartition along the cell cycle of HL60 cells treated by Etoposide (1 μΜ, for 24 h) and then recovering for either 24 or 48h (Figure 5B). Etoposide induces the G2/M arrest and these cells progressively enter in apoptosis. During the observed kinetic, the > 4N population slightly increases in CI treated cells. Under such conditions, the bulk of the population becomes polyploid (> 4N) whereas G0/G1 and 4N populations are quite inexistent at 72h post-treatment. Whereas only a small proportion of the cells are dying. Upon Etoposide plus CI cells are stopped in G2/M and progressively are either driven to apoptosis or to polyp loidisation. Only 15 % of the cells are in the G0/G1 and S phases whereas 30 % of the cells are dying. Taking into account that first HL60 cells are highly sensitive to etoposide and then, that the whole population responds to the treatment, the inventors decided to test this combination in mice bearing HL60 tumours. Mice were randomised five days after tumour establishment. They are treated day-by-day by either CI or the vehicle and meanwhile, half of them received three Etoposide injections. The tumour grows exponentially in control mice (Figure 5C). Etoposide and CI reduce slightly tumour expansion (P < 0.05 at day 42) whereas tumours are growing slowly in mice treated with Etoposide plus CI . The differences were found significant by the student test, p being around 0.001 at days 38 and 42. The tumour volume is reduced by 10 compared to the control (V) and by around 6 compared to the single treatment (Figure 5C). Long-term effects are observed as the last data are recovered 21 days after the last injection (Figure 5C). No adverse effects are observed, as the Etoposide concentration is kept low. The repeated injections of CI are found safe. However two mice receiving the combination have lost weight in the last days of the protocol. Autopsy has revealed intestinal inflammation in these two mice, whereas no negative signs are detected in other mice. As predicted by the in-cellulo assays, the combined treatment is very efficient for preventing HL60 xenografts expansion. Discussion

The inventors showed that benzo[e]pyrido indoles are not only potent inhibitors of the aurora kinases but also of Chk-2. Chk-2 is an important player in the DNA-damage response- signalling pathway as it is activated and phosphorylated by ATM in response to double-strand breaks. Chk2 is activated by a wide range of chemotherapeutic agents, including ionizing radiations, topoisomerase inhibitors, and DNA-targeted agents; chk2 has a dual role either inducing apoptosis via p53 or activating a cell cycle checkpoint coupled with DNA repair. Since most of tumours are p53 -defective, it is mainly expected that the later role will be relevant in such cells, and inhibiting chk2 activity will enhances the damage done by chemotherapeutic drugs.

The inventors tested the combined use of Etoposide and CI . Etoposide is a toposisomerase II inhibitor that stabilizes the topoisomerase II-DNA complex preventing DNA ligation around damage sites. Moreover etoposide is already used in chemotherapy for the treatment of various tumours (leukemia, lymphoma, lung,...).

The inventors report that the combined treatment prevents efficiently the growth of NSC Lung and leukemia cell lines. The efficiency of the combination is at least additive and even synergic depending of the genetic background of the cells or on already acquired resistance.

Bi- functional drugs (aurora kinase and Chk2) are interesting because the crosstalk between aurora kinases and DNA damage signalling are multiple. Both aurora A and B are inhibited in response to DNA damages. The protein kinase Chkl, a signal transducer in the DNA damage checkpoint, is essential for chromosome segregation and completion of cytokinesis to prevent genomic instability. Indeed, Chkl augments spindle checkpoint signalling and is required for optimal regulation of Aurora-B when kinetochores produce a weakened signal. Meanwhile the signal transducer Chk2 prevents mitotic ongoing following G2/M progression with DNA damage and is also a negative regulator of mitotic catastrophe. Taking into account the interplay between mitotic kinases and DNA signalling, the simultaneous targeting of aurora and checkpoint kinase 2 by the same drug in damage cells will induce G2/M progression and then a convergence of negative signals that finally will lead to mitosis abortion as shown in Figure 6. Therefore this double targeting will increase the selectivity and allow a decrease of the DNA damaging drug concentration, reducing thus its cytotoxicity. The goal is to use DNA damaging drugs at a dose sufficient to induce growth arrest but not cell death in normal cell population and moreover to combine them with an agent roughly safe for normal quiescent cells but capable of killing the cycling cancer cells defective in control checkpoints (summary in Figure 6). The combined treatment in HL60 cells validates the possibility of manipulating G2 checkpoint in order to trigger cancer cell to death as well as the multi-targeting with sharp selective drugs. Materials and Methods.

Cell culture and cell viability test. H358 (lung cancer cell) and HeLa (ovarian cancer) were grown in DMEM whereas HL60 (human myeloid cell line) and HCC70 (breast cancer) cells were in RPMI 1640. U20S (a human osteosarcoma cell line) cells were cultured in McCoy's Medium 5 A. Media (Gibco-Invitrogen) were supplemented with 10% heat- inactivated foetal bovine serum (Gibco-Invitrogen), L-glutamine (2mM), penicillin (100 Ul/ml) and streptomycin (100 μg/ml).

Cell proliferation assays were conducted in 96 well culture plates. Assays were run in triplicate. Cells were treated by either etoposide (24 h, concentration depends on the cell line) or CI (1 μΜ, 72 h) or by the combination of both drugs for 24 h before Etoposide with drawn. Similar assays were run with intoplicin and cisplatin. The viability of HL60 cells was analysed when intoplicin concentrations varied from 50 nM to 500 nM in the presence or the absence of CI (1 μΜ). Alternatively, the viability of HL60 and HCC70 cells were also analysed when cisplatin concentrations varied from 100 nM to 4 μΜ under the above conditions.

Cell viability was estimated 72 h later by addition of CellTiter 96Queous one Solution Reagent (Promega) directly to culture wells under conditions defined by the manufacturer.

The multicellular tumour spheroid (MTS) model. The inventors applied the hanging-drop method to produce H358 spheroids of similar diameters. 1400 cells were suspended on the lid of an agar coated 24-petri dishes containing culture medium. 48 h later, the spheroids were transferred to the culture medium. Spheroid volumes were measured before (Day 0) and during the drug treatment (Day N). H358 spheroids were treated by compounds and drugs withdrawn at the desired time by changing the medium. Control H358 spheroids were grown under the same conditions without drug treatment. The size of each spheroid was determined by measuring 2 diameters (di and d₂) using an inverted microscope. The volume was calculated according to the formula: V=4/37tr³ where r=l/2Vdixd₂. Spheroid growth was calculated by measuring the variations in volume and compared to the initial volume V₀.

Western Blot. Cells were treated by compounds, then harvested and lyzed in 9M urea then supplemented with Laemmli sample buffer. Cell extracts were subjected to SDS-PAGE and transferred to nitrocellulose filter (GE Healthcare). After blocking with 5% non-fat milk in PBS for at least 1 hour, the membranes were incubated with the primary antibodies. The following antibodies were used: anti-y-H2AX (1 :4000, Upstate Biotechnology); anti- (phospho-Serine 516)-Chk2 (1 : 1000, USBiological); anti-P-actin (1 :5000, Sigma) and anti-a- tubulin (1 :4000, Sigma). Either anti-mouse or anti-rabbit horseradish peroxidase (1 :5000, GE Healthcare) was used as secondary antibodies. Bands were visualized by ECL technique (Amersham Bioscience).

Cell cycle analysis. Cells were synchronized by serum-starved medium for 48 hours

(H358 and U20S) and for 24 h (HL60), and released from GO by addition of complete medium. For determination of cell cycle profile, cells were fixed by ice-cold 70% ethanol for 1 h and then, incubated with propidium iodide solution (50 μg/ml PI in the presence of 0.2 mg/ml R Ase) for 15 minutes at 37° C. DNA content was measured using the FACS flow cytometer equipped with. Cel!Quest Pro software (Becton Dickinson, San Diego, CA).

Immunofluorescence. Cells were seeded on glass coverslips and treated by etoposide and CI for 2 h, followed by CI for indicated times. After treatment, cells were fixed in 4% formaldhehyde at 37° C for 15 minutes, then permeabilized for 5 minutes with Triton X-100 in PBS. After blocking in 0.05 mg/ml BSA for 30 minutes, the primary antibody was added (either H2AX (1/2000 from Upstate) or p53-phospho-Ser20 (1/200 from Cell Signalling). After 30 min of incubation unbound antibodies were removed by washing with PBS, 0.2 % Tween- 20 and specific staining was revealed with Hylite Fluor™ 546- or 488- conjugated secondary antibodies (Anaspec). DNA was visualized with 0.1 mM Hoechst 33342 (Sigma). Images were collected with a ZEISS 510 Laser Scanning Confocal microscope with a 63x immersion oil objective. Slices of 0.5 micron are shown.

In vivo experiments. In-vivo experiments were conducted on four-week old female Swiss nude mice {Iff a Credo, Marcy VEtoile, France). After one week of adaptation in the animal facility (French agreement number A38-516-01), the mice were inoculated subcutaneous ly with either 1 x 10⁷ exponentially growing H358 cells or 3 x 10⁶ HL60 cells mixed with growth factor free matrigel (1/1, BD). Tumours were established at five to seven days post-injection. Then the mice from each cage were randomly divided into four groups, which allowed the equalization of the mean tumour size of each group. Tumour volumes were determined by measuring two perpendicular diameters using a clipper and then calculated as follows: V = di² x d₂, where di and d₂ are the smallest and the largest diameters, respectively. One mice group (10 animals) received the treatment (compound 1, 160 μg per 20 g mouse in vehicle buffer (PEG 300 /DMSO 16 %) intraperitoneally, whereas the control group (10 mice) was injected with vehicle only. Half of these mice (5 treated and 5 control) received an etoposide treatment (100 μg/ mouse/ injection) as indicated in each experiment. Mice were repeatedly injected and once a week, were weighed meanwhile the volumes of the tumours were measured.

Example 2

Synthesis of compound C5: 3 -Hydroxy- 8 -methyl- 7H, 10H-benzo(e)pyrido(4,3-b)indol-

11-one

In a 5 ml sealed tube, a mixture of 3-methoxy-8-methyl-7H,10H-benzo(e)pyrido(4,3- b)indol-l 1-one (83 mg, 0.3 mmol) (prepared according to J. Med. Chem, 1990, 33, 1519), benzyltriethylammonium chloride (480 mg, 2 mmol) and 37% hydrochloric acid (4 ml) was heated in an oil bath at 140°C for 24 h. The reaction mixture was then evaporated under vacuum, then 10 ml of water are added. The medium was then rendered basic by addition of 28% ammonium hydroxide (0.5 ml), and the resulting solid was collected by filtration, then washed with water and dried using a vacuum desiccator, at a temperature of 50°C for 18 h. 65 mg (63%o yield) of title compound were then obtained as brown powder, the proton NMR spectrum of which is the following:

DMSO-de δ (ppm): 11.95 (br s, 1H), 10.87 (m, 1H), 10.18 (d, 1H), 9.38 (br s, 1H), 7.62-7.55 (m, 2H), 7.18 (d, 1H), 7.11-7.03 (m, 2H), 2.29 (s, 3H).

Microanalyses, calculated for C16H12N2O2 0.5 H₂0: C, 70.33; H, 4.76; N, 10.25 ; found: C, 70.71; H, 4.89; N, 9.96. MS : 265.2 (M+H).

Synthesis of compound C6: 3-Benzoyloxy-8-methyl-7H, 10H-benzo(e)pyrido(4,3- b)indol-l 1-one

A mixture of 3-hydroxy-8-methyl-7H,10H-benzo(e)pyrido(4,3-b)indo 1-11-one (35 mg, 0.13 mmol) (prepared as described above), pyridine (1 ml) and benzoic anhydride (140 mg) was heated at reflux for 1 h. The mixture was evaporated under vacuum, then the residue was washed successively with saturated NaHC0₃ solution (2*2 ml) and water (2*2 ml). Ethanol (4 ml) was added and the mixture was heated at reflux for 2 min then left stirring at room temperature overnight. The resulting solid was collected by filtration, then washed with ethanol (1 ml) and dried at ambient temperature. 20 mg (41% yield) of title compound were then obtained as creamy powder, the proton NMR spectrum of which is the following:

DMSO-de δ (ppm): 12.21 (s, 1H), 11.02 (d, 1H), 10.48 (d, 1H), 8.25-8.19 (m, 2H), 7.87 (d, 1H), 7.85-7.75 (m, 3H), 7.68-7.61 (m, 2H), 7.48 (dd, 1H), 7.13 (d, 1H), 2.32 (s, 3H). Microanalyses, calculated for C₂₃Hi₆N₂0₃ 0.25 H₂0: C, 47.09; H, 4.43; N, 7.52 ; found: C, 73.76; H, 4.46; N, 7.29. MS : 391.0 (M+Na).

Example 3

Taxol (also called paclitaxel) is not a DNA-damaging anti-tumoral agent. It belongs to the anti-tumoral drug of the anti-mitotic agents. This agent targets microtubules and prevents their depolymerisation. It blocks cell cycle in mitosis. After an extended blockade in mitosis, cells escape and enter in a new Gl-like interphase. The inventors tested the combination of CI compound with taxol in order to demonstrate the benefit of the combination with a DNA damaging agent is not reproduce with taxol, an anti-mitotic agent. As shown in Figure 8, there is no benefit in the combination of CI compound with taxol. The effect has been tested with co-treatment and sequential treatments.

In addition, the inventors tested if the compound of the invention may be replaced by any Aurora kinase inhibitor. In order to do so, they compared, the effect of the combination of the well-known Aurora kinase inhibitor VX-680 with etoposide and the effect of the combination of the CI compound with the same drug. As shown in Figure 9, a synergistic effect has been observed with the combination CI -etoposide whereas the effect of the combination VX-680 - etoposide is less than the expected additive effect. The differences in the combination effect may be explained by the differences in inhibitory specificities (Figure 1). Indeed, VX-680 has no inhibitory effect on Chk-2. In addition, in contrast to CI compound, VX-680 accelerates the entry into mitosis.

Otherwise, the inventors tested further DNA-damaging anti-tumoral agents. Indeed, combination of CI with either intoplicin (a dual topoisomerase I/II) or cisplatin (a DNA crosslinker) on HL60 cells. As shown in Figures 10 and 11, a synergistic effect has been observed with intoplicin and an additive effect with cisplatin.

One of the interests of the present invention is to allow a two-fold decrease of the

DNA damaging agent concentration, thereby decreasing its toxicity. The following table recapitulates some results of the present experimental section and demonstrates that such a goal can be reached. DDA Cells IC50 IC50 Decrease Decay of Effect

DDA + C1 dose in

(μΜ) (μΜ) cellulo in

%

etoposide H358 10 8.6 1.2 14 additive etoposide U20S 16.2 7.3 2.2 55 synergistic etoposide HL60 1.1 0.5 2.2 55 synergistic intoplicin HL60 0.275 0.12 2.3 56 synergistic cisplatin HL60 1.75 1.5 1.2 14 additive cisplatin HCC70 3 2.5 1.2 14 additive

(DDA: DNA damaging agent)

IC50 is the concentration of DDA that give rise to 50 % of cell death. It was determined in the presence of CI (1 μΜ) or in its absence. A 8-dose curve was performed, in triplicate, for each determination. The viability was measured upon 72 hours of treatment.

Example 4 - Effect of CI alone and combined to radiations in glioblastoma

The current therapy for patients with glioblastoma multiforme (GBM) is maximal safe surgical resection followed by radiation therapy plus temozolomide, given concomitantly with and after radiation. Even with the intensive multimodal treatment, the median overall survival is only 14.6 months and the two-year survival rate is 26.5%. In addition, elevated expression of MELK in malignant gliomas and the inversed correlation of MELK expression with patients' survival periods indicate clinical relevance of this molecule as a therapeutic target for GBM. Recent studies have attracted much attention to the therapeutic potential of MELK as a candidate target for anti-cancer drug development.

Accordingly, the inventors decided to test the CI, a MELK inhibitor with a IC50 of 41 nM toward this kinase activity. They evaluated the therapeutic efficacy of CI, on a mouse intracranial tumor model derived from GBM spheres (Fig. 12). GBM patient samples (noted GBM 146, 157 and 206) were collected and spheres were derived.

The inventors first created the mouse intracranial xenografts with GBM 157 spheres, and then carried out intracranial injection of CI at day 7. Subsequently, they assessed tumour growth at 2 months following xenograft. Treatment effect was evaluated with immunohistochemistry using the human- specific Nestin antibody (Figure 12). It was observed that CI intratumoral injection decreases tumor sizes of immunodeficient mouse intracranial tumor model from GBM157 spheres.

Taking into account these encouraging data, the inventors tested CI combined to radiations in GBM. Spheres were derived from GBM patient samples (noted GBM 146, 157 and 206) and their growth was evaluated in the presence of different quantities of CI .

As a result, sphere formation from the 3 GBM samples was abrogated by CI treatment with the IC50 of 0.3-0.4uM. With the similar concentration of CI, normal precursor cells derived from 2 foetal brain tissues (16wf and 1105) and immortalized human astrocytes (NHA) were capable of forming similar number of spheres, suggesting their relatively less sensitivity to CI treatment.

To evaluate CI as a candidate for an anti-pro liferating agent, the inventors first performed in vitro cell viability assay with the 3 GBM samples with or without radiation treatment. In all samples, CI exhibited a potent inhibitory effect on cell growth at low micromolar concentrations. Consistent with the data of MELK knockdown in GBM spheres, CI treatment rendered GBM cells more than 13 times sensitive to radiation (Following Table and Figure 13). CI synergizes the effects of radiations:

Viability IC50 of three GBM patient samples

The inventors performed in vitro cell viability assays with 3 GBM samples with or without radiation combined with CI or control treatments. CI treatment demonstrated inhibitory effect on the in vitro growth for 3 GBM sphere samples (GBM 146, GBM157 and GBM206) with the IC50 ranging from 3.40 to 4.49 μΜ. Note that 2Gy-radiated samples exhibited over 10 times lower IC50, ranging from 0.18 to 0.90 μΜ. CI treatment rendered GBM sphere cells sensitive to radiations.

Material and Methods concerning GBM

Neurosphere (NS) cultures were prepared as previously described [Nakano et al, J Cell

Biol, 2005. 170: p. 413-27]. Briefly, small GBM samples were dissociated with a fire- polished glass pipette and resuspended at 50,000 cells/ml in neurosphere medium, containing Dulbecco's modified Eagle medium (DMEM)/F12 medium (GIBCO, Invitrogen, Carlsbad, CA) supplemented with B27 (final concentration 2%, GIBCO), basic fibroblast growth factor (bFGF) (20ng/ml, Peprotech, Rocky Hill, NJ), epidermal growth factor (EGF) (50ng/ml, Peprotech), penicillin/streptomycin (1%, GIBCO), and heparin (5ng /ml, Sigma Aldrich, St. Louis, MO). To differentiate the BTSC, spheres were dissociated into single cells, added to poly-L-Lysine coated dishes containing Neurobasal medium (GIBCO) with B27, and maintained for up to five days, followed by culture in serum containing medium.

Sphere forming assay

To assay for sphere- forming potential, 100 cells from dissociated GBM neurospheres were plated on each well of 96-well plates with NS media, and the number of neurospheres were counted at day seven [Nakano et al, supra]. The small interfering RNA (siRNA) transfectants were removed from plates with TrypLE Express (GIBCO) and replated to 96- well plates six hours after transfection.

Xenotransplantation of tumor spheres into NOD/SCID mice

Nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice of 6-8 weeks of age (Charles River Laboratories, Wilmington, MA) were anesthetized with intraperitoneal administration of ketamine. GBM neurospheres were dissociated and 250,000 cells were stereotactically transplanted in the right striatum. After 8 weeks, the mice were undergone intracardiac perfusion-fixation with 4% paraformaldehyde. Brains were removed and retrieved for frozen sections, followed by hematoxylin and eosin staining.

Claims

1- A pharmaceutical composition comprising

(a) a compound having the formula (1)

wherein

^■ -(CH₂)_n- wherein n is 3, 4 or 5; or

^■ -CH=CH-CH=CH- , optionally substituted by a (Ci-C₃)alkyloxy, Rd is selected from the group consisting of hydrogen, (Ci-C₃)alkyl optionally substituted by a radical OH, (Ci-C₃)alkyloxy or -NRR',

- X is an oxygen or a sulfur; wherein R and R', identical or different, are selected from the group consisting of hydrogen and (Ci-C4)alkyl,

(b) a DNA-damaging anti-tumoral agent, and

a pharmaceutically acceptable carrier, in particular for use in the treatment of cancer.

2. A kit com rising (a) a compound of formula (1)

wherein

^■ -(CH₂)_n- wherein n is 3, 4 or 5; or

^■ -CH=CH-CH=CH- , optionally substituted by a (Ci-C₃)alkyloxy, Rd is selected from the group consisting of hydrogen, (Ci-C₃)alkyl optionally substituted by a radical OH, (Ci-C₃)alkyloxy or -NRR', - X is an oxygen or a sulfur;

(b) a DNA-damaging anti-tumoral agent, and

as a combined preparation for simultaneous, separate or sequential use, in particular in the treatment of cancer. A pharmaceutical composition comprising a compound having the formula (1)

wherein

^■ -(CH₂)_n- wherein n is 3, 4 or 5; or

^■ -CH=CH-CH=CH- , optionally substituted by a (Ci-C₃)alkyloxy, Rd is selected from the group consisting of hydrogen, (Ci-C3)alkyl optionally substituted by a radical OH, (Ci-C3)alkyloxy or -NRR',

- X is an oxygen or a sulfur;

or an isomeric form thereof or a pharmaceutically acceptable salt and derivative thereof

for the use in the treatment of cancer in combination with radiotherapy. 4- The pharmaceutical composition according to claim 1 or the kit according to claim

2, wherein the DNA-damaging antitumoral agent is selected from the group consisting of an inhibitor of topoisomerases I or II, a DNA crosslinker, a DNA alkylating agent, and an anti- metabolic agent, preferably from the group consisting of an inhibitor of topoisomerases I and/or II and a DNA crosslinker.

5- The pharmaceutical composition or kit according to claim 4, wherein the DNA- damaging antitumoral agent is a topoisomerase I or II inhibitor, preferably selected from the group consisting of etoposide, topotecan, camptothecin, irinotecan, amsacrine, intoplicin, anthracyclines such as doxorubicin, epirubicin, daunorubicin, idarubicin and mitoxantrone, more preferably etoposide or intoplicin.

6- The pharmaceutical composition according to claim 1 or the kit according to claim 2, wherein the DNA-damaging antitumoral agent is a DNA crosslinker, preferably selected from the group consisting of cisplatin, carboplatin and oxaliplatin, more preferably cisplatin.

7- The pharmaceutical composition or kit according to claim 4, wherein the DNA- damaging antitumoral agent is selected from the group consisting of etoposide, intoplicin and cisplatin. 8- The pharmaceutical composition according to any one of claims 1 and 3-7, or the kit according to any one of claims 2 and 3-7, wherein the compound of formula (1) has one or several of the following features : Rai, Ra₂, Ra₃, each independently, independently selected from the group consisting of hydrogen, hydroxyl, methoxy, ethoxy, phenoyloxy, phenylcarbamoyloxy and benzyloxy; and/or

Rb is selected from the group consisting of hydrogen, methyl, ethyl, -CH₂-OH, - (CH₂)n-N[(Ci-C₂)alkyl]2 with n being 2 or 3, -(CH₂)-OPO[0(Ci-C₄)alkyl]₂ and -

(CH₂)-OC(=0)-(Ci-C₄)alkyl; and/or

Rd is selected from the group consisting of hydrogen, methyl, ethyl, and -(CH₂)_n-

N[(Ci-C₂)alkyl]₂ with n being 2 or 3; and/or

X is an oxygen.

9- The pharmaceutical composition according to any one of claims 1 and 3-7, or the kit according to any one of claims 2 and 3-7, wherein the compound of formula (1) has one or several of the following features :

- Rb is selected from the group consisting of hydrogen, -CH₂-OH, and -(CH₂)_n-

N[(Ci-C₂)alkyl]₂ with n being 2 or 3; and/or

Rd is selected from the group consisting of hydrogen, methyl, and ethyl; and/or - X is an oxygen.

10- The pharmaceutical composition according to any one of claims 1 and 3-7, or the kit according to any one of claims 2 and 3-7, wherein the compound of formula (1) has one or several of the following features :

- Rai is selected from the group consisting of hydrogen, hydroxyl and methoxy, and

Ra₂ andRa₃ are hydrogen; and/or

Rb is hydrogen; and/or

Rci is selected from the group consisting of hydrogen, methyl and ethyl, and Rc₂ is hydrogen; and/or Rd is hydrogen; and/or

X is an oxygen.

11- The pharmaceutical composition or kit according to anyone of claims 8-10, wherein the compound of formula (1) is selected in the group consisting of:

Rd is H and X is O;

12- The pharmaceutical composition or kit according to claim 11, wherein the compound of formula (1) is a compound wherein Rai is methoxy, Ra₂ and Ra₃ are H, Rb is H, Rci is ethyl, Rc₂ is H, Rd is H and X is O.

13- The pharmaceutical composition according to any one of claims 1 and 4 to 12, the kit according to any one of claims 2 and 4 to 12, wherein the amounts of the compound of formula (1) and the DNA-damaging anti-tumoral agent are such that the combined therapeutic effect of the two active ingredients is additional, preferably synergistic.

14- The pharmaceutical composition or the kit according to claim 13, wherein the amount of the DNA-damaging anti-tumoral agent is a sub-therapeutic amount. 15- The pharmaceutical composition according to any one of claims 1 and 4 to 12, the kit according to any one of claims 2 and 4 to 12, wherein it further comprises another antitumoral agent. 16- The pharmaceutical composition or the kit according to claim 15, wherein the other antitumoral agent is an histone deacetylase (HDAC) inhibitor or a taxoid antitumoral agent.