WO2014033597A1 - 5-azaindole compounds with anticancer and antiangiogenic activities - Google Patents

Abstract

The present invention relates to 5-azaindole type compounds responding to the following formula (I): for their use as drugs, and more particularly for the prevention and/or the treatment of diseases and/or disorders chosen amongst cancers; angiogenesis related disorders; parasitic diseases; fungal diseases; autoimmune diseases; inflammatory diseases; warts such as warts caused by papilloma virus. The invention also relates to pharmaceutical compositions comprising such compounds of formula (I). The use of at least one compound of formula (I) as research tool for the cell-cycle synchronization of microtubule drugs resistant cell lines is also part of the invention. Finally, the invention also concerns the use of at least one compound of formula (I) as an herbicide and/or an algaecide.

Description

5-AZAINDOLE COMPOUNDS WITH ANTICANCER AND ANTIANGIOGENIC ACTIVITIES

The present invention relates to 5-azamdole type compounds of formula (1) for their use as drags, more particularly for the prevention and/or the treatment of diseases and/or disorders chosen amongst cancers; aagiogenesis related disorders; parasitic diseases; fungai diseases; autoimmune diseases; inflammatory diseases; warts such as warts caused by papilloma virus. The present invention also relates to pharmaceutical compositions comprising such compounds of formula (I). The use of at least one compound of formula (I) as research tool for the cell-cycle synchronization of microtubule drugs resistant cell lines is also part of the invention. Finally, the present invention concerns the use of at least one compound of formula (I) as an herbicide and/or an algaecide.

Microtubules form, with the actin microfilaments and the intermediate filaments, the cytoskeleton of the eukaryotic cells. These are hollow tubular aggregates constituted of a single dimeric protein, i.e. the tubulin. In mammalian cells, the microtubule networks are in general nucleated at an organizing centre: the centrosome. These networks carry out multiple and vital roles such as organization of the cytoplasm, positioning of the organelles, cell motility and cell division. During mitosis, the microtubule network is reorganized to form the mitotic spindle, which is machinery used by cell to separate the duplicated chromosomes into two identical sets, before its cleavage into two daughter cells. Mitotic spindle integrity is controlled by specific checkpoints, Any undetected mitotic spindle dysfunction couid be at the origin of genomic instability and thus represents a potential source of tumorigenesis (Castillo et al, 2007; Kops et al, 2005).

Microtubules assemble by polymerization of α/β dimers of tubulin. Microtubules are highly dynamic polymers able to rapidly polymerize from free tubulin dimers and to depolymerize just as rapidly. Microtubule dynamics are crucial to mitosis (Jordan and Wilson, 2004; Niethammer et al, 2007).

The intrinsic microtubule dynamics is tightly regulated in the cell by interaction with an array of proteins that stabilize or destabilize microtubules, such as XMAP215 Disl/TOGp, MCAK, MAP4 and Opl8/stathmin (Holmfeldt et al, 2009; Kavallaris, 2010) or +TIPs (plus- end tracking proteins) such as EB1 (Akhmanova et al, 2005; Coquelle et al, 2009; Small et al, 2003). Targeted perturbation of this finely tuned process constitutes a major therapeutic strategy {Honore et al, 2005). Anti-mitotic drugs that interfere with the microtubule system are, indeed, key components of combination chemotherapies for the treatment of carcinomas (Kavallaris, 2010). Perturbation of microtubule dynamics by drugs constitutes one of the most powerful ways to suppress (at least transiently) tumor growth. Despite their massive clinical use, most of these drugs have their therapeutic potential hampered by insufficient bioavailability and toxicity (myelosuppressi n, peripheral neurotoxicity). Moreover, failure in cancer therapy is often related to the selection of tumor cells that have acquired resistance against microtubule binding drugs. As a result, many efforts are undertaken to identify new chemical entities that may overcome those resistance mechanisms (5-amino-2-aroylquinolines (Nien et al, 2010), 1,2,4-triazole (Arora et al, 2009), for example).

Several anticancer drugs clinically important, including the Vinca alkaloids, vinblastine, vincristine, and vinorelbine and the taxanes paclitaxel and docetaxel specifically target tubulin and modify microtubules dynamics. Considering the clinical success of these agents, tubulin is today one of the best validated targets in anticancer chemotherapy (Giannakakou et al, 2000; Jackson et al, 2007; Zhou and Giarmakakou, 2005).

Furthermore, in addition to cancer, the involvement of the microtubule cytoskeleton in the etiology of a large number of diseases has been described, such as for example mental disorders (Andrieux et al, 2006; Andrieux et al, 2002; Begou et al, 2008) and neurodegenerative diseases (Denxtaut et al, 2005; Garcia et Cleveland, 2001), and viral (Ruthel et al, 2005), bacterial (Margalit et al, 2004) and parasitic (Morrissette and Sibley, 2002) infections. The pharmacological agents targeting the microtubule cytoskeleton and its various effectors may therefore exhibit a therapeutic advantage for the treatment of a large number of diseases (Lafanechere, 2008).

Thus, the treatments used in anticancer chemotherapy target, in a favored manner, the dynamic behavior of the microtubules. In particular, it may be blocked by many agents that can bind to different sites of tubulin. Structural data concerning the binding of these different agents on tubulin have been obtained. Zinc-induced sheets of paclitaxel-stabilized tubulin proto filaments have been used for construction of a model of tubulin with bound paclitaxel. After fitting this model into electron density microtubule maps, the authors concluded that paclitaxel binds to β-tubulin facing the microtubule lumen (Snyder et al, 2001). The X-ray structure of vinblastine bound to tubulin in a complex with the protein stathr n has shown that the vinblastine introduces a wedge at the interface of two tubulin molecules and thus interferes with tubulin assembly (Gigant et al, 2005),

These studies have led to the characterization, to date, of three binding sites of poisons of microtubules on tubulin: the domain of periwinkle alkaloids located at the interface between two /β-tubulin dimers, the site of taxoids located on the β subunit and that of colchicine located at the interface between the a subunit and the β summit.

These various agents are classified according to whether they destabilize or stabilize the microtubules:

- Agents that destabilize the microtubules:

Periwinkle alkaloids, capable of depolymerizing the microtubules, have been identified as agents capable of arresting the cells in mitosis, with aberrant mitotic spindles. Subsequently, vincristine and vinblastine were introduced into clinical medicine in the 1960s and are still widely used in chemotherapy for testicular cancer, Hodgkin's disease or acute lymphoid leukemia,

Mention may also be made of colchicine or combretastatm, which inhibit the polymerization of the microtubules and also nocodazole.

- Agents that stabilize the microtubules, such as taxanes and epothilones:

Taxanes, and more particularly paclitaxel, interact specifically and reversibly with the microtubules with a stoichiometry of about one mole of taxane per one mole of tubulin. This interaction is accompanied by a stabilization of the microtubules.

Paclitaxel and the other taxanes are differentiated from other anti-tubulin poisons mainly by the stabilizing effect that they exert on the microtubules. Cancer drugs such as paclitaxel or Vinca alkaloids were previously thought to work through opposite mechanisms. There are now known to act by modifying microtubule dynamics, and not through increasing or decreasing the overall microtubule mass. Despite their antitumor efficacy, especially in breast cancer, ovarian cancer and lung cancer, taxanes are extremely toxic since they also act on the microtubules of non-cancerous cells in proliferation (hematopoietic cells, mucous cells, etc.). Finally, they may adversely affect the peripheral neurons and give rise to significant side-effects.

The therapeutic success of paclitaxel has maintained the advantage for the search for therapeutic agents that target tubulin.

However, although mostly valuable, the substances known from the prior art are not ideal. They have several side-effects, principally mye!osuppression and peripheral neurotoxicity. Neurotoxic side-effects related to tubulin drugs are not surprising because tubulin is a major player not only in cell division but also in mitosis-independent cytoskeietal functions.

Moreover, many cancers are inherently resistant to these drugs or become so during prolonged treatment. This phenomenon is a multifactorial process {Kavallaris, 2010; avallaris et al, 1997) but a common way for cancer cells to acquire drug resistance is the expression of efflux pumps such as the glycoprotein P or ABCG2 and MRP1. Thus, identification of agents that are active in multidrug resistant (MDR) cells is urgently needed (Nien et al. , 2010).

Several strategies have been proposed for the development of potentially more effective and less toxic drugs. One is to improve existing drugs or to find new ones that target tubulin.

High-throughput, cell-based screening of small molecules is an attractive strategy to identify such agents. It enables the evaluation of the activity of compounds directly on cells, ruling out molecules that are not cell-permeable or too toxic. In the past few years, cell-based assays allowed the identification of several microtubule polymerization inhibitors such as 4- arylaminoquinazolines (Kasibhatla et aL, 2007) or Micropolyin (De Rycker et al., 2009).

By using a cell -based assay (Vassal et al, 2006), the Inventors have now surprisingly identified a new class of 5-azaindoles compounds of formula (I) as compounds that disrupt microtubule array in a reversible manner. Reversibility rules out the possibility of covalent bond formation between tubulin and azaindole derivatives. They have then discovered that these compounds bind directly to tubulin and impede colchicine binding, and that they also induced a G2/M cell cycle arrest and were toxic for a panel of cancer cell lines including multidrug-resistant cell lines. They have observed a complete microtubule depolymerization in cells, at concentrations of 0.05 to 1 μΜ. The identified 5-azaindole compounds of formula (I) displayed potent antiangiogenic properties and exerted anticancer effect

The first subject of the present invention thus relates to 5-azaindole compounds of formula (I):

CO

in which:

- the ring Ar is a C₄-Ci₂ aryl or beteroary] ring, preferably a phenyl or pyridine ring, and more preferably a phenyl ring,

- Rj = H, optionally substituted Ci-C₆ alkyl, benzyl, arylsulfonyl, heteroarylsulfonyl, alkyloxycarbonyl or dialkylcarbamoyl groups, said substituents being independently selected from halogen, hydroxyl, cyano, nitro, carboxylate, carboxyester, ammo, Ci-C^ alkyl, Ci-Q alkylamino, C₂-C6 cycloamino or Ci-Q alkoxy groups,

- R₂ represents H or a halogen atom, nitro, amino, Ci-Ce alkylamino or C₂-C₆ cycloamino groups, and preferably R₂ = H or CI,

- ¾, R5, R$ and R₇, identical or different, represent hydrogen or halogen atoms, hydroxyl, Ci-Ce alkyl, Ct-C₆ alkoxy, cyano, nitro, carboxylate, carboxyester, amino, Cj-Q alkylamino or C₂-C<, cycloamino groups, and preferably R_3} R_s, R$ and R , identical or different, represent hydrogen atoms or Cj- e alkoxy groups,

- R4 = a halogen atom, hydroxyl, Ci-Cs alkoxy, amino, Cj-Cs alkylamino, C₂-C₆ cycloamino, thiol or Cj-Ce alkylthio groups, preferably R4 = a halogen atom or a C Q alkoxy group, and more preferably R = CI or a OCH₃ group, and

- Z - C-CHR, CR'R" or C=0, wherein R = H or an optionally substituted Ci-C₆ alkyl group, and R' and R", identical or different, represent hydrogen or halogen atoms, OH groups, CrCe alkyl or alkoxy groups, C₄-Ci₂ aryl, heteroaryl, arylalkyl or heteroarylalkyl groups, and preferably Z = C^HR or CR'R", wherein R = H or an optionally substituted CTC6 alkyl group, and R' and R", identical or different, represent hydrogen or halogen atoms, OH groups, Ci-Q alkyl or alkoxy groups, C4-C12 aryl, heteroaryl, arylalkyl or heteroarylalkyl groups.

or their tautomeric, racemic, enantiomeric or polymorphic forms or pharmaceutically- acceptable salts, for their use as drugs.

Surprisingly, the compounds of formula (I) of the invention were identified as compounds that disrupt microtubule array in a reversible manner. Reversibility rules out the possibility of covalent bond formation between tubulin and azaindoie derivatives. Since reversibility generally enables a better control of administrated compound dose during treatment, this characteristic is of importance for lead development. Biochemical experiments indicated that tubulin is an in vitro target of azaindoie derivatives and its polymerization inhibition is likely responsible for the observed phenotype. Complete microtubule depolymerization was observed in cells.

Competition assays using tritiated colchicine and vinblastine indicated that the compounds of formula (I) bind tubulin directly at a site that, at least partially, overlaps with the colchicine site. It cannot be excluded, however, that the compounds of formula (I) inhibit colchicine binding by an allosteric mechanism. Toxicity induced by colchicine treatment hampers its use as anti-cancer drug. Currently, there are no Food and Drug Administration- approved colchicine-binding site drugs used for the treatment of cancer. Combretastatins and sulfonamides that bind to the same site are, however, presently undergoing Phase I and II clinical trials for solid tumors (Nien et al, 2010). This confirms the druggability of compounds targeting this binding site and supports azaindole derivatives as suitable leads for drug optimization. Testing several analogues identified essential features for tubulin binding and activity in cell. Since SAR established using cell-based assay may be biased by cel!ular permeability and stability of the tested compounds, a SAR analysis of the compounds effects on tubulin in vitro polymerization was conducted in parallel. These analyses gave similar results and indicated that positions Rj, R₂ and interaromatic group Z are tolerant to substitution whereas alkoxy such as raethoxy in position R is required to get active compounds. Moreover, the observation that the in vitro SAR closely parallels the effects observed on cellular microtubule network supports the assumption that tubulin is the cellular target of the compounds of formula (I) of the invention.

Antiproliferative activities of the compounds of formula (I) revealed a broad spectrum of cytostatic effect with comparable efficiencies on cell lines with deficient p53 or overexpressing ~ras, both generic or epi genetic alterations that are among the most common alterations found in cancer ceils. In addition, contrary to many clinically used microtubule- binding drugs, the 5~azaindole compounds of formula (I) also exhibited antiproliferative activity in cell lines overexpressing the drug efflux pumps PgP, MRP1 and ABCG2, As the action of the compounds of formula (I) is reversible, these compounds may thus be used as research tool for cell synchronization experiments with resistant cells. Moreover, this property ma be exploited in the case of chemotherapeutic failure.

Flow cytometry analysis revealed that the compounds of formula (I) exert their antiproliferative action through cell cycle arrest in G2 M phase. Aneuploid cells were detected following treatment by the compounds of formula (I) of the invention. Induction of aneuploidy could be a concern since it promotes genetic instability. However, most if not all microtubule targeting compounds (for instance, vinca-alkaloids) induce aneuploidy but remains valuable drugs.

The compounds of formula (I) also inhibit angiogenic sprouting from microvascular endothelial cell spheroids, which demonstrates that these compounds exert anti angiogenic activity in vivo on tumor cells. The standard assays for evaluating the performance of anticancer drugs involve human tumor xenografts in immunodeficient mice. Tumors grown on the chorioallantoic membrane (CAM) of chicken embryos constitute, however, a fast, easy and affordable system for a first preclinical analysis of the compound effects. The highly vascularized nature of the CAM greatly promotes the efficiency of tumor cell grafting. Remarkably, within 8 days not only do MDA-MB-231 tumor cells develop sizable tumors, but also they can escape the primary site, invade surrounding stroma, and reach distal portions of the CAM to form micro metastasis foci. Therefore, similar to some murine models, all steps of tumor growth and of the metastatic cascade are recapitulated in the chick embryo model but, importantly, in a very short period of time.

The administration of compounds of formula (I), at a concentration that does not affect the embryos development significantly reduce the tumors size, as compared to the vehicle (DMSO) treated-tumors, Furthermore, by comparing the number of nodules present in the lower CAM of embryos treated with compounds of formula (I) with those of vehicle-treated embryos, it was found that both compounds have antimetastatic properties.

The compounds of formula (I) are reversible microtubule depolymerizing agents that exert potent cytostatic effects on human cancer cells of diverse origins, including multidrug- resistant cells. Although they act in the micromolar range, their chemical structure is simpler than currently used microtubule polymerization inhibitors. The compounds of the invention also exhibit a significant inhibition of angiogenesis and tumor growth in chorioallantoid breast cancer xenografts.

In the sense of the present invention:

Alkyl groups are chosen among Cj.Ce alkyl groups such as methyl, ethyl, n- propyl, isopropyl, n-butyl, sec-butyl, tert-bxxtyl and isobutyl radicals;

- Alkoxy groups are chosen among Cj-Ce alkoxy groups such as methyloxy, ethyloxy, n-propyloxy, iso-propyloxy, n-butyloxy, sec-butyloxy, te^butyloxy and isobutyloxy radicals;

Aryl group means any functional group or substituent derived from at least one simple aromatic ring; an aromatic ring corresponding to any planar cyclic compound having a delocalized π system in which each atom of the ring comprises a p-orbital, said p-orbitals overlapping themselves. More specifically, the term aryl includes, but is not limited to, phenyl, biphenyl, 1 -naphthyl, 2-naphtyl, anthracyl, pyrenyl, and the substituted forms thereof. The aryl groups of the invention comprise preferably 4 to 12 carbon atoms, and more preferably 5 or 6 carbon atoms

- Heteroaryl group means any functional group or substituent derived from at least one aromatic ring as defined above and containing at least one heteroatom selected from P, S, O and N. The term heteroaryl includes, but is not limited to, furan, pyridine, pyrrole, thiophene, imidazole, pyrazole, oxazole, isoxazole, thiazole, isothiazole, tetrazole, pyridazole, pyridine, pyrazine, pyrimidine, pyridazine, benzofurane, isobenzofurane, indole, isomdole, ben20thiophene, benzo[c]thiophene_i benzimidazole, indazole, benzoxazole, benzisoxazole, benzothiazole, quinoline, isoqumolme, qumoxaline, qumazoliue, cinnoline, purine and acridine. The heteroaryl groups of the invention comprise preferably 4 to 12 carbon atoms, and more preferably 5 or 6 carbon atoms;

- Arylalkyl groups mean any group derived from an alkyl group as defined above wherein a hydrogen atom is replaced by an aryl as defined above;

Heteroaryialkyl groups mean any group derived from an alkyl group as defined above wherein a hydrogen atom is replaced by a heteroaryl group as defined above;

Alkylamino groups mean mono or di(CrC6)alkylamino group;

- Cycloamino groups mean C₂-C₆ cycloamino group such as aziridin-l-yl, azetidin-l-yl, pyrrolidm-l-yl, piperidm-l-yl, azepan-l-yl, tnorphoHn- -yi and thiomorpholin- -yI.

According to the invention, halogen atoms are chosen among bromine, chlorine, fluorine and iodine, preferably bromine, chlorine and fluorine, and more preferably chlorine.

The expression "pharmaceutically-acceptable" refers to compounds, materials, compositions and/or dosage forms which are suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other complications commensurate with a reasonable benefit/risk ratio.

Advantageously, in the compounds of formula (I): Z = C=C¾ or CR'R" with R' = H or OH and R" = d-C₆ alkyl or phenyl groups, and preferably Z « C-CH₂, CH(CH₃), C(OH)C¾ or C(OH)C₆H₅.

It is noteworthy that:

- if Rj is an arylsulfonyl or a heteroarylsuifonyl group, preferably the aryl ring is a phenyl ring;

- if Ri is an alkoxycarbonyl group, preferably i is the butoxycarbonyl,

- if Rj is a dialkylcarbamoyl group, preferably i is the dimethylcarbamoyl.

According to an advantageous embodiment of the invention, R₅ = CH₃ or a benzyl group.

According to another advantageous embodiment of the invention, one of R _j R₅, R_$ or R₇ is a Ci-Q alkoxy group when the others are hydrogen atoms.

According to an alternative preferred embodiment, at least one of the R₃, R₅, ¾ or R₇ radical is a OC¾ group. Preferably, Ar is a phenyl ring substituted by a OCH₃ group in position 4. The most preferred compounds of formula (I) according to the invention are the following:

CMOl

or

CM02

or

CMOS

or

CM04

or

CMOS

or

CM07

The present invention relates more particularly to compounds of formula (1) for their use for the prevention and/or the treatment of diseases and/or disorders chosen amongst cancers; angiogenesis related disorders; parasitic disease; fungal disease; autoimmune diseases; inflammatory diseases; warts such as warts caused by papilloma virus.

More particularly, the present invention relates to compounds of formula (I) for their use for the prevention and/or treatment of;

cancers chosen amongst testicular cancer, ovarian cancer_s lung cancer, breast cancer, Hodgkin's disease, acute lymphoid leukemia, neuroblastoma, melanoma, glioma, glioblastoma, sarcoma, colon cancer, pancreatic cancer,

angiogenesis related disorders chosen amongst diabetic blindness, macular degeneration, rheumatoid arthritis, psoriasis,

parasitic diseases involving apicomplexan parasites, parasites with ftagellum or cilia, toxoplasmosis,

autoimmune diseases chosen amongst multiple sclerosis, diabetic retinopathy, inflammatory diseases chosen amongst gout disease.

The compounds of formula (I) may be prepared for example according to the method described in Nguyen et al, 1986.

Another subject matter of the invention is a pharmaceutical composition comprising at least one compound of formula (I) according to the invention as an active principle, with at least one pharmaceutically-acceptable excipient or carrier, and with at least another active substance.

The expression "pharmaceutically acceptable excipient" refers to any diluents, adjuvants or vehicles, such as preserving agents, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.

The pharmaceutical composition of the present invention may be administered by any suitable route, for example, by oral, buccal, inhalation, sublingual, nasal, percutaneous, i.e. transdermal or parenteral (including intravenous, intramuscular, subcutaneous and intracoronary) administration. Therefore, the pharmaceutical composition of the invention can be provided in various forms, such as in the form of hard gelatin capsules, of capsules, of compressed tablets, of suspensions to be taken orally, of lozenges or of injectable solutions, ointments, or in any other form appropriate to the method of administration.

The pharmaceutical composition according to the invention includes those wherein a compound of formula (I) is administered in an effective amount to achieve its intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art.

A "therapeutically effective dose" refers to that amount of compound of formula (I) which results in achieving the desired effect. Toxicity and therapeutic efficacy of compound of formula (1) can be easily determined by standard pharmaceutical procedures in cell cultures or experimental animals, i.e, for determining the LJ½ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LDso and ED₅₀. The data obtained from such data can be used in formulating range of dosage for use in humans. The dosage of compound of formula (I) preferably lies within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed, and the route of administration.

The exact formulation, route of administration, and dosage can be chosen by the individual physician in view of the patient's conditions. Dosage amount and interval of administration can be adjusted individually to provide plasma levels of compound of formula (1) which are sufficient to maintain the preventive or therapeutic effects. The amount of pharmaceutical composition administered will therefore depend on the subject being treated, on the subject's weight, the severity of the affliction and the manner of administration.

For human and other mammal use, the compounds of formula (I) can be administered alone, but they are preferably administered in admixture with at least one pharmaceutically acceptable carrier, the nature of which will depend on the intended route of administration and the presentation form... Pharmaceutical composition for use according to the present invention thus can be formulated in a conventional manner using one or more physiologically acceptable carriers comprising one or more excipient{s) and/or auxiliaries) that facilitate processing of the compounds of formula (I) into preparations which can be used pharmaceutically. Amongst the excipients and auxiliaries which can be used in the pharmaceutical composition according to the invention, one can mention anti-agglomerating agents, preservatives agents, dyes, vitamins, inorganic salts, taste-modifying agents, smoothing agents, coating agents, isolating agents, stabilizing agents, wetting agents, anti- caking agents, dispersing agents, emulsifying agents, aromas, penetrating agents, solubilizing agents, etc., mixtures thereof and generally any excipient conventionally used in the pharmaceutical industry.

By way of example, when the pharmaceutical composition is administered orally, the carrier may comprise one or several excipients such as talc, lactose, starch or modified starches, cellulose or cellulose derivatives, polyethylene glycols, acrylic acid polymers, gelatin, magnesium stearate, animal or vegetal fats of natural or synthetic origin, paraffin derivatives, glycols, etc.

For general information about the formulation and administration of pharmaceutical compositions, one can obviously refer to the book "Remington's Pharmaceutical Sciences", last edition. Of course, a person skilled in the art will take care on this occasion that the excipient(s) and/or auxiliary(ies) optionally used are compatible with the intrinsic properties attached to the pharmaceutical composition in accordance with the invention.

These pharmaceutical compositions can be manufactured in a conventional manner, i.e. by conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, entrapping or lyophilizing processes. Proper formulation is dependent upon the route of administration chosen.

The use of at least one compound of general formula (I) as defined here-above as research tool for the cell-cycle synchronization of microtubule drugs resistant cell lines is also part of the invention. Finally, the invention also concerns the use of at least one compound of general formula (I) as defined here-above as an herbicide and/or an algaecide.

Besides the preceding provisions, the invention also comprises other provisions which will emerge from the remainder of the description which follows, which relates to examples that highlight the anticancer and antiangiogenic effects of the 5-azaindole compounds of formula (I), and also to the appended drawings in which:

- Figure t shows the effect of two compounds of formula (I) according to the invention, CMOI and CM02, on microtubule network organization: (A) Immunofluorescence analysis of cell microtubules. HeLa cells were incubated for 2 hours with 0.25% DMSO (A, vehicle control); 5 μΜ colchicine (B); 25 μΜ CMOI (C) or 25 μΜ CM02 (D). Cells were then permeabilized, fixed and stained for tubulin as described in Material and Methods. Nuclei were stained with Hoechst Bar = 10 μηι. (B) a-tubulin partition between soluble and insoluble fractions. HeLa cells were treated for 2 hours with 0.25% DMSO (vehicle control); 2 μΜ colchicine and increasing concentrations of CMOI as indicated. Insoluble (I) and soluble (S) fractions were prepared as described in Materials and Methods. Equivalent volumes of extracts (20 μΐ,) were separated on 8% SDS-PAGE, and subjected to immunoblot analysis using monoclonal antibody specific for total a-tubulin. (C) same as (B), but with CM02.

- Figure 2 shows the reversibility of CMOI and CM02 effects in HeLa cells. HeLa cells were treated for 2 hours with 0.25% DMSO (control); 5 μΜ colchicine; 10 μΜ nocodazole, 25 μΜ nocodazole, 25 μΜ CMOI or 25 μΜ CM02, as indicated. Compounds were the removed removal and cells were incubated overnight in fresh medium. They were then fixed and stained for a-tubulin,

- Figure 3 shows the effect of CMOI and CM02 on microtubule polymerization in vitro". MTP tubulin polymerization assay. Tubulin was allowed to polymerize at 37°C, at the indicated conditions. Fluorescence of DAPI bound to microtubule was measured to monitor microtubule polymerization. Experiments were performed in triplicate, in the presence of increasing concentration of CMOI or CM02, as indicated. Results are presented as mean ± standard error of the mean (SEM).

- Figure 4 shows the effect of CMOI and CM02 on microtubule polymerization in vitro: Pure tubulin polymerization assay. Tubulin was allowed to polymerize at 37°C, at the indicated conditions. Fluorescence of DAPI bound to microtubule was measured to monitor microtubule polymerization. Experiments were performed in triplicate, in the presence of increasing concentration of CMOI or CM02, as indicated. Results are presented as mean ± standard error of the mean (SEM).

- Figure 5 represents the effect of CMOl and CM02 on the binding of [³H] colchicine and f H] vinblastine to microtubule proteins. 50 nM [³H] colchicine (black bars) or 30 nM [¾] vinblastine (dashed bars) were competed with 100 μΜ of CMOl, CM02, colchicine and vinblastine as described in Materials and Methods. Each value represents the mean ± SEM from triplicate determinations.

- Figure 6 represents the cell cycle distribution upon treatment of HeLa cells with CMOl and CM02. HeLa cells were incubated for 16 hours with DMSO (control), colchicine (2 μΜ), CMOl and CM02 (1 μΜ and 25 μΜ, as indicated). Cell cycle parameters were analyzed by flow cytometry, as described in Materials and Methods. The upper panel shows the graphs obtained for 1 and 25 μΜ, as indicated, of CMOl and CM02 (black), compared with that obtained for DMSO (grey). Values (lower table) are expressed as percentage of the total cell population,

- Figure 7 shows that the compounds of formula (I) inhibit capillary-like tube formation. HUVEC cells were seeded on Matrigel and compounds, at the indicated concentrations were added after cell attachment (one hour later). Tubule formation was observed by phase-contrast microscopy at 24 hours. Bar = 200 μιη.

- Figure 8 represents the quantitative analysis of endothelial sprouting in response to 5-azaindoles compounds of formula (I). FGF2 (100 ng/ nL) was added at day 0 to collagen- embedded HMEC-GFP spheroids in the presence of CMOl and CM02 at different concentrations (Ο.ί μΜ to 25 μΜ). After 3 days of culture, the spheroids were observed under an epifluorescence microscope: (A) Overlay of phase contrast and fluorescence observations at the indicated concentrations of compounds. (B) Measure of the mean total sprout length of endothelial spheroids, performed by quantitative microscopy image analysis. In each condition, data represent the mean values ± SE of multiple spheroids (n > 10) from one representative experiment out of two.

- Figure 9 shows the anti-tumoral effect of 5-azaindoles compounds of formula (I); MDA-MB-231 cells were xenografted on chick embryo chorioallantoic membrane (CAM). After treatment with either vehicle (DMSO), colchicine, CMOl or CM02, tumors were excised and weighted. The lower CAM was also dissected, fixed and the number of GFP- fhiorescent nodules was counted, as described in the material and methods section. A) Representative pictures of tumors at the end of the different treatments. B) Effect of the different treatments on tumors weight (means ± SE of 6 samples), C) Effect of the different treatments on the nodule number detected in the lower CAM (means ± SE of 6 samples). * p < 0,05; ** p < 0.01 , significantly different from control values using Mann-Witney test. Bar = 0.8 mm.

MATERIAL AND METHODS:

1) Synthesis

(R,S) 4-chloro-2-(l-(4-methoxyphenyl)ethyl)-!-meihyl-lH-pyn:olo[3_J2-c]pyridine

(CMOl), 4~chloro-2-(l-(4-methoxyphenyl)vinyl)-l-methyl-lH-pyrrolo 3,2-c]pyridine (CM02), (R,S) 1 -benzy]-4-chloro~2-(l-(4-methoxyphenyl)emyl)-lH-pyrrolo[3,2-c]pyridine (CM03), l-ben2yl-4-chloro-2-(i-(4~mel3ioxyphenyl)vmyl)-3H'pyrroIo[3,2-c]pyridine (CM04), (R,S) l-(4-chloro-l -methyUH-pyirolofS^-iilpvridine^-yl)- 1 -(4- methoxyphenyl)ethanol (CMOS) and (R,S) 4-chloro-l-methyl-2-(l-phenylethyl)-lH- pyrroio[3,2-c]pyridine (CM06) were synthetized according to the method published in Nguyen et at., 1986.

The procedure to obtain the compound (R,S) 3,4~dichloro-2-(l-(4- methoxyphenyl)ethyl)-l-methyl~lH-pyrrolo[3,2-c3pyridine (CM07) is the following: a solution of 2-(l~(4-memoxyphenyl)ethyl)"l-methyl-lH-pyrrolo[3₉2-c]pyridin-4(5H)-one (prepared as described in Nguyen et al, 1986, 500 mg, 1.8 mmol) and PCls (320 mg, 1.8 mmol) in POCl₃ (50 mL) was heated under reflux for 2.5 h. After evaporation into dryness under vacuum, cold water (100 mL) was added and the medium was rendered basic by addition of 2S% ammonium hydroxide (4 mL). The aqueous layer was extracted by CH2CI2 and the organic layer was washed with brine, dried over MgS0₄ and evaporated under vacuum. The residue was purified by flash chromatography (silica, CH₂Cl₂/EtOH; 95/5) to give the expected compound as beige solid (300 mg, 56%), p 170°C Elemental Analysis C_l7Hj₆Cl₂N₂0; Calculated: C, 60.89; H, 4.78; CI, 21.19; N, 8.36; Found: C, 60.73: H, 4.92; CI, 20.89; N, 8.23,

The procedure to obtain the compound (R,S) ^ch!oro-l-methyl-lH-pyrrolo ^- c]pyridin-2-yldiphenylmethanol (CMOS) is the following: to a solution of 4-chloro-l-methyl-

(3.33 g, 20 mmol) in anhydrous THF (120 mL) is added dropwise a solution of tBuLi (1.6 N in heptane, 15.6 mL, 25 mmol) at ~65°C under nitrogen. The mixture is stirred at the same temperature for 10 min. A solution of benzophenone (3.6 g, 20 mmol) in THF (20 mL) is then added dropwise and the resulting solution stirred at -20 °C for 30 min, and then at room temperature for 2 hours. Then, 250 mL of 4 N HQ solution is added and the precipitate is collected by filtration after 18 hours stirring at room temperature and washed with water. Recrystallized from ethanol gives the pure expected compound (5.6 g, 80% yield), mp 258°C. Elemental Analysis C₂iHi₇ClN₂0; Calculated; C, 72.31 ; H, 4.91; CI, 10.16; N, 8.03; Found: C, 72.24; H, 5.04; CI, 9.98; N, 7,84.

CM09

The procedure to obtain the compound (R.S) l-^-methoxy-l-me yl-lH-pyrroloP^- c]pvridine-2-yl) -(pyridine-4~yl)ethanol (CM09) is the following: to a solution of 4- methoxy-l-methyl-lH-pyrrolofS^-eJpyridine (3.24 g, 20 mmol) in anhydrous THF (100 mL) was added dropwise a solution of tBuLi (2.2 N in heptane, 11.4 mL, 25 mmol) at -55°C under nitrogen. The mixture was stirred at this temperature for 5 min and then at -15 C for 10 min. A solution of 4~acetylpyridine (3,63 g, 30 mmol) in THF (10 mL) was then added dropwise at -55°C and the resulting solution stirred at this temperature for 1 h and then left at 20°C overnight. Successively, a solution of 0.5 N HC1 (200 mL) and C¾C1₂ are added. The organic layer is discarded and the aqueous phase was rendered basic by addition of solide NaHC0₃. The precipitate was collected by filtration, washed with water, dried and recrystaUrzed from toluene gave the expected compound (4.8 g_f 84% yield), mp 232-4°C. Elemental Analysis C16H17N3O2 O.2 C₆H₅CH₃; Calculated: C, 69.28; H, 6.17; N, 13,93; Found: C, 69.35; H, 6.19; N, 14.05.

The procedure to obtain the compound 4-methoxy-2-(l-(4-methoxyphenyl)vinyl)-l- methyl-lH-pyrrolo[3,2-c]pyridine (CM10) is the following:

The mixture of 4-chloro-2-(l-(4-methoxyphenyl)vmyl)-l-methyl-lH-pyrrolo[3₅2- ejpyridine (obtained as described in Can J. Chem (1986), 64, 545, 310 mg), 24% solution of sodium methoxide in methanol (20 mL) and D F (6 mL) was heated under reflux for 24h. 70 mL of water was added and the organic layer obtained after extraction using dichloromethane was dried over MgS0 and evaporated in vacuum into dryness to give the title compound (290 mg, 95% yield). Ή NMR (CDCI3) δ 7.89 (d, 1H), 7,23 (d, 1H), 6.90-6.85 (m, 3H), 6.65 (s, 1H), 5.70 (s, 1H), 5.42 (s, 1H), 4.10 (s, 3H), 3.81 (s, 3H), 3.36 (s, 3H).

Microanalyses, calculated for C_[sHi₈N₂0₂- 0.25 ¾0: C, 72.36; H, 6.20; N, 9.38; found: C, 71.57; H, 6.26; N, 9.37.

The procedure to obtain the compound 1 -(4-chIoro~ 1 -(phenylsulfonyl)- 1 H-indol-2-yl)- l-(4-methoxyphenyl)ethanol (CM11) (representative of the prior art) is the following:

R-Butyllithium (1.6 M in hexane, 7.5 mL, 12 mmol) was added dropwise at -78 °C to a solution of the 4-chloro-l -phenylsulfonyl- lH-indole obtained as described by S. Maliboobi (J. Med. Chem. 2006, 49, 3101-31 15) (3,0 g, 10.3 mmol) in dry THF (30 mL). After 1h on stirring, acetophenone (2.1 g, 14 mmol) in THF (10 mL) was added at the same temperature and the mixture was kept at room temperature overnight The mixture was poured into IN HC1 (70 mL), the aqueous solution was extracted with dichloromethane and the organic layer was washed with brine, dried over MgS(¾ and evaporated in vacuum. Ethanol (10 ml) was added and the solid was collected, washed with minimum amount of ethanol and dried in the air giving l~(4-chloro-l-(phenylsulfonyl)-lH-mdol-2-yl)-l-(4-methoxyphenyl)ethanol (3.2 g, 70% yield).

Ή NMR (DMSO-- ) δ 7.51 (d, 1H), 7.65-7.53 (m, 3H), 7.48-7.30 (m, 4H), 7.20 (d, 2H), 7.06 (s, 1H), 6.82 (d, 2H), 5.77 (s, 1H), 3.74 (s, 3H), 1.94 (s, 3H).

Microanalyses, calculated for C23H20CINO4S: C, 62.51; H, 4.56; N, 3.17 ; found: C₅

62.46; H, 4.66; N, 3.11.

The procedure to obtain the compound 4~chloro-2-(l-(4-methoxyphenyl) vinyl)- 1- (phenylsulfonyl)-lH-indol (CM12) (representative of the prior art) is the following:

CI

The mixture of l-(4-chloro-l-(phenyls lfonyl)-lH-indol-2-yl)-l-(4- methoxyphenyl)ethanol (2.3 g, 5.2 mmol), benzyltriethylammoniiim chloride (5.0 g, 22 rnmol), phosphoryl trichloride (5.6 mL, 60 mmol) and acetonitrile (60 mL) was heated under reflux, for 18 h. The volatile material was evaporated in vacuum, cold water (100 mL) was added and the medium was basified by addition of sodium hydroxide (2 N). The precipitate was collected by filtration, washed with water. It was suspended in water, extracted by dichorom ethane. The organic layer was dried over MgS0 and evaporated in vacuum to give the intermediate 2₃4-ch]oro^»2-(l-(4-methoxyphenyl)vinyl)-l-(phenylsulfonyl)-lH-indoIe (2.2 g, 100% yield).

¾ NMR (DMSO-i¾ δ 8.06-8.00 (m ΪΗ), 7.70-7.60 (m, 3H), 7.50 (d, 2H), 7.43-7.36

(m, 2H), 7.18 (d, 2H), 6.93-6.83 (m, 3H), 5.88 (s, 1H), 5.46 (s, 1H), 3.76 (s, 3H).

Microanalyses, calculated for

C, 65.17; H, 4.28; N, 3.30 ; found: C, 64.82; H, 4.31; N, 3.21.

The procedure to obtain the compound 4-chloro-2-(l-(4-methoxyphenyl)vinyl)-l.H- indole (CM13) (representative of the prior art) is the following:

The mixture of 4-chloro-2-(l-(4-methoxyphenyl)vinyl)-l-(phenylsuIfonyl)-lH-indole (500 mg, 1.2 mmol), 10% NaOH (40 mL) and EtOH (80 mL) was heated under reflux for 2.5 L 30 mL of water was added and the organic layer obtained after extraction using dichioromethane was dried over MgS0₄ and evaporated in vacuum to give the intermediate 3, 4-chioro-2-(l-(4~methoxyphenyl)vinyl) H-indole (314 mg, 94% yield).

lM NMR (DMSO< ) 6 11.72 (s 1H), 7.57-7.38 (m, 3H), 7.25-7.02 (m, 4H), 6.28 (s, 1H), 5.83 (s, 1H), 5.43 (s, 1H), 3.87 (s, 3H).

Ten millimolar stock solutions were prepared in DMSO and kept at -20°C. Appropriate dilutions were freshly prepared prior to use.

Methods:

Screening:

The screen was performed as described in Vassal et al. (Vassal et al, 2006), HeLa cells were seeded at 36,000 cells per well of 96-well polystyrene tissue culture plates in 90 sL of medium and were allowed to grow for 24 hours. The following day each well was robotically supplemented with 10 μί, of RPMI containing compounds (6,560) from the CNRS-Curie institute library. The final concentration of the compounds was 25 μΜ. For bioactive controls, the final concentration was 5 μΜ for paclitaxel and 2 μΜ for colchicine. The final concentration of DMSO was 0.5%. After compounds dispense, cells were incubated 2 hours at 37°C_S 5% C(¾ in the workstation incubator. Cells were then permeabilized 10 minutes with 100

of warm OPT buffer, in order to eliminate free, depolymerized tubulin and fixed 6 minutes with 100 μΐ, of methanol. Cells were then double stained for tyrosinated and detyrosinated tubulin using specific antibodies and fluorescent secondary antibodies. Nuclei were stained with Hoechst.

Fluorescence was measured using the FLUOstar Optima microplate reader (BMG), with suitable filters for each wavelength. Raw data were converted into percentage of tyrosinated/detyrosinated tubulin contents, based on plate controls averages.

Linear regression based on plate controls averages was realized to reduce the impact of plate effects. Active compounds selected by this primary screen were retested from freshly made solutions.

HMEC-GFP preparation:

HMEC-1 cells were infected by a defective retrovirus encoding for enhanced Green Fluorescent Protein (EGFP). A DNA constructs encoding both the EGFP coding sequence under the control of the herpetic Human Cytomegalovirus (HCMV) promoter and the neo gene (vector pEGFP-Nl, Clontech) was cloned into the pLNCX vector. This vector was integrated in PT67 cells by transfection with Effectene (Qiagen). PT67 cell superaatants were used to infect HMEC-1 cells. HMEC-1 clones expressing EGFP were selected in the presence of neomycin. Resistant clones were isolated by limit dilution, and amplified as independent lineages. One cell line strongly expressing EGFP protein was named HMECGFP.

Sprouting of HMEC-GFP:

To prepare spheroids, FfMEC-GFP cells were seeded at 3000 cells/well in round- bottom 96-multiwell plates in DME 1 g L glucose (Eurobio, Les Ulis, France) containing 0.25 % methylcellulose. 48 hours later, the spheroids were collected, transferred into flat- bottom 96-well plates and embedded io collagen gel (1.2 mg/mL type I collagen) prepared in a Iscove's modified Dulbecco's medium, supplemented with FGF-2 (10 ng mL), 50 μ/mL penicillin and 50 Mg mL streptomycin. Treatment with two concentrations (25 μΜ and 12.5 μΜ) of CMOl and CM02 started right after spheroids were included in the collagen gels. Sprouting was allowed for 24 hours at 37°C. Images were acquired by fluorescence microscopy and analysis of gel invasion was performed using the freeware ImageJ (http://Tsbweb.nih.gov/ij/). Statistical analysis was performed using a Kruskal & Wallis test.

Chick embryo tumors growth and metastasis assay:

MDA-MB-231 cells were transfected with GFP-encoding plasmid pBGFP-Nl (Clonetech) using FuGENER reagent (Roche) and stable cells clones (MDA-MB231 -GFP) were selected. Fertilized White Leghorn eggs (SFPA, St. Brieuc) were incubated at 38°C with 60% relative humidity for 10 days. At this time (E10), the chorioallantoic membrane (CAM) was dropped by drilling a small hole through the eggshell into the air sac and a 1 cm² window was cut in the eggshell above the CAM.

Cultured MDA-MB231 -GFP were detached by trypsinization, washed with complete medium and suspended in serum free DMEM. A 50 μΐ, inoculum of 1.10⁶ MDA-MB-231 - GFP cells was added directly onto the CAM of each egg. Eggs were then randomized in 4 groups of 12 eggs (to get sufficient surviving embryos at the end of the experiments). Two days later, tumors began to be detectable. They were then treated during 8 days, every two days (El 2, E14, E 16, El 8), by dropping 100 ]x of either 50 μΜ CMOl, 50 μΜ CM02, 2 μΜ colchicine or 0.5 % DMSO (vehicle) in PBS onto the tumor. The slow dropping on the large tumor area that depresses the CAM surface was found appropriate enough to avoid the leakage and dispersion of the compounds. Then, windows were sealed with cellophane tape and the eggs were returned to the incubator. At El 9 the upper portion of the CAM was removed, transferred in PBS and the tumors were then carefully cut away from normal CAM tissue. Tumors were then quickly dried on cellophane before weighing. In parallel, a 1 cm² portion of the lower CAM was collected to evaluate the number of nodules, containing GFP- expressing cells. The fluorescent nodule were visualized in situ using whole mounts of tissue fixed in 4% formaldehyde in PBS and flattened between a hollow glass slide and a thick coverslip. A thorough and complete visual scan of the piece of the lower CAM was done using Leica Macro fluo fluorescent microscope (Optimal, Grenoble). Digital color images were acquired using DP25 camera on SZ 10 microscope (Olympus, France).

Cell Lines and Culture:

Tumor cell lines HeLa (human cervical adenocarcinoma), MCF-7 (human breast adenocarcinoma), NCI-H460 (human non small cell lung cancer), 786-0 (human renal adenocarcinoma), MDA-MB-231 (human breast carcinoma), MES-SA (human uterine sarcoma) and the multiple drug resistant cell line MES-SA Dx5 (human uterine sarcoma) were originally obtained from American Type Culture Collection (Rockville, MD). HeLa, NC1-H460 MDA-MB-231, and 786-0 cells were grown in complete RPMI 1640 with GlutaMAX™ I (Gibco, Invitrogen) containing 10% fetal bovine serum (Hyclone, Thermo Fisher Scientific) and 100 units/mL of penicillin and 100 ]xg/mL of streptomycin (Gibco, Invitrogen). MCF-7 were maintained in EMEM (ATCC) with 10% fetal bovine serum, 100 units/mL of penicillin, 100 ^^ηιί of streptomycin and 0.1 mg/mL insulin (Sigma Aldrich). MES-SA and MES-SA DX5 were cultured in McCoy's 5 A (ATCC) supplemented with 10% fetal bovine serum and 100 units/mL of penicillin and 100 ^ιηΐ.. of streptomycin during the first sub-culture, then adapted in Rl'MI 640 with GlutaMAX™ and 10% fetal bovine serum, 100 units/mL of penicillin and 100 ^πΛ of streptomycin. HMEC-1 cells (Human Microvascular Endothelial Cel!-1) were obtained from the Emory University. HMECGFP were derived from HMEC-1 by retroviral infection of a GFP-expressing construct and selection of a highly fluorescent cell clone that still expresses some of the major endothelial cell markers. MDA-MB-231-GFP (CNRS UMS 3453), HMEC-1 and HMEC-GFP were maintained in DMEM 1 g L glucose (Eurobio, Les Ulis, France) supplemented with 10% fetal calf serum (Biowest, Abcys, Paris, France).

Human umbilical vein endothelial cells (HUVEC) were purchased from Lonza (Basel,

Switzerland) and cultured in EGM-2-MV medium supplemented with 5% fetal calf serum (Cambrex, East Rutherford, NJ, USA). All cell lines were maintained at 37°C_S 5% C0₂ humidity atmosphere in media. FGF-2 was from Salk Institute, La Jolla, CA, USA.

Immunofluorescence procedure for microscopy analysis:

HeLa cells grown for 72 hours on glass coverslips were incubated with or without compounds. After treatment, the culture medium was rapidly removed and cells were permeabifeed with OPT buffer (80 mM PIPES pH 6.7, 1 mM EGTA, 1 mM MgCI₂, 0,5% Triton X-100 and 10% glycerol) for 3 minutes at 37°C. This buffer allows the extraction of free tubulin dimers and preserves intact microtubules, when there are some. Ceils were then fixed by immersion in -20°C absolute methanol for 10 minutes. They were then processed for immunofluorescence as described in Paturle-Lafanechere et al, 1994.

Fluorescence images were taken using a NIKON Eclipse 90i microscope and 100./ 1.3 Plan Neofluar objective, a CoolSNAPHQ2 Monochrome camera (Roper Scientific, Trenton, NJ) and NIS Elements software and were processed using Adobe Photoshop.

Transfection of GFP-EB3:

To label microtubule plus ends, GFP-EB3 plasmids were used because 1) EB3 as a stronger binding affinity to microtubule + end, and 2) the fusion of GFP to the N terminal part of EB1 strongly reduced its binding to microtubules as opposed to EB3, Cell transfection was performed using electroporation (AMAXA , Koln, Germany). 2 of purified plasmid DNA was used for each transfection reaction.

Fluorescence time-lapse videomicroscopy of microtubule plus ends:

Live imaging of microtubule plus ends was performed as described in Honore et al, 2011, on transiently GFP-EB3 transfected-HeLa cells by using an inverted fluorescence microscope (ZEISS Axiovert 200M with a 63X objective). Time-lapse acquisition was performed with a COOLSNAP HQ (Roper Scientific), driven by Metamorpii software (Universal Imaging Corp.). Images acquisition was performed at a temperature of 37 ± 1°C / 5% CO^.

To study colchicine, CMOl and CM02 effects on microtubule dynamics, cells were incubated with different concentrations of compounds, or vehicle alone (DMSO) for 2 hours. Data are from 3 independent experiments. For each experiment, 6 microtubules/cell in 6 cells per condition were analyzed.

Dynamic instability parameters analysis:

The dynamic instability parameters analysis was performed by tracking microtubule plus end over time, using the image J software. The methods of calculation were as described in Honore et al, 201 1.

Drug reversibility assay:

HeLa cells grown for 72 hours on glass coverslips were incubated with compounds for 2 hours at indicated concentrations. After removal of the medium containing compounds, cells were rinsed 3 times with warm complete PMI medium and incubated overnight in complete RPMI medium. They were then processed for immunofluorescence, as described above.

Fractionation of extracts and immunoblotting:

HeLa cells (1.10⁶) seeded in 10 cm Petri dishes were grown for 2 days. They were incubated for 2 hours with the tested compound, and then washed with 2 mL of warm PBS. PBS was then carefully discarded and 1 mL of warm OPT buffer (Pipes 80 mM, EGTA 1 mM, MgCl₂ 1 mM, triton X400 0.5% and glycerol 10% pH 6.8), containing the tested compound and proteases and phosphatases inhibitors (complete OPT buffer), was added. The dish was then gently shacked and the OPT buffer containing soluble tubulin dimers carefully recovered. After a 1 mL wash with the same buffer, the remaining ceils content (containing the insoluble tubulin pool) was recovered in 1 mL of warm complete OPT buffer, using a cell scraper. The insoluble pool was kept at 4°C, sonicated and ultracentrifuged (200,000 g, 20 min, 4°C), in order to solubilize tubulin and to eliminate the DNA. Samples were then stored at -80°C. For western blot analysis, the same sample volume (20 uL) of soluble and insoluble pools was separated on an 8% polyacrylamide SDS-page gel and transferred onto nitrocelMose. Tubulin was detected using anti a~tubulin primary antibody (clone a3a (Peris et αί, 2006)), monoclonal peroxidase-conjugated mouse secondary antibody (Sigma, A4 16) and detection with chimioluminescence kit ECL™ Plus (GE Healthcare, RPN2132).

Cell proliferation assay:

Cell proliferation was evaluated using the colorimetric 3-(4,5-dimetbylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay (Sigma, M-5655). The assay was performed in 96 wells microplates (Greiner, #655086). Depending of the cell type and their rate of growth, cells were seeded at 20,000 to 50,000 cells per well and allowed to grow for 24 hours. The culture medium was then replaced with a fresh medium containing the drugs to be assayed at different concentrations or equivalent amounts of DMSO. Cells were allowed to grow for additional 48 hours. The medium was then discarded; cells were washed once with RPMI without phenol red and incubated for four hours with MTT (0.5 mg/mL) in RPMI without phenol red. At the end of the incubation period, the medium was removed and the converted dye solubilized with acidic isopropanol (isopropanol, 10% Triton XI 00, 0,1 N HC1). Absorbance of converted dye is measured at a wavelength of 570 nm, using FLUOstar Optima microplate reader.

Cell cycle analysis:

HeLa cells were grown in T25 flasks (5.1 ⁵ cells per flask) and treated with DMSO colchicine or CMOl and CM02 for 16 hours. After treatment, non-adherent cells present in the medium were mixed with trypsinized adherent cells, centrifuged, washed twice with PBS- EDTA (2 mM) and fixed for 30 minutes with 1.5 mL 70% icecold ethanol Fixed cells were washed twice with PBS-EDTA, re-suspended in 0,5 mL DNA staining buffer (10 /mL propidium iodide, 10 g mL RNase A in PBS-EDTA 2 mM, pH 7.4) and incubated for 30 minutes in darkness. Cellular DNA was measured with a FACSCalibur flow cytometer (BD Biosciences). Data were analyzed using CellQuest software (BD Biosciences). Cells were gated by forward/side scattering from a total of 10,000 events.

Tubulin polymerization assay:

Microtubule protein (MTP) and pure tubulin were prepared according standard procedures (Paturle-Lafanechere et a!., 1991). Microtubule polymerization assay was adapted from (Bonne et 1985). Briefly, microtubule assembly was carried out in a half area 96- well black plate (Greiner, #675090) and followed using a microplate reader FLUOstar OPTIMA (BMG Labtechnologies). Wells were charged with either MTP or pure tubulin (final concentration of 25 μΜ and 30 μΜ, respectively) in MME ( 00 mM MES, 1 mM gCl₂, 1 mM EGTA, pH 6.75, for MTP) or PME buffer (100 mM PIPES, 1 mM MgC3_2j 1 mM EGTA, pH. 6.65, for pure tubulin) with 10 μΜ DAPI and variable concentrations of compounds to be assayed. Following 10 minutes incubation, assembly was initiated by injection of GTP and MgCl₂ to a final concentration of 1 mM and 5 mM respectively, yielding a reaction volume of 100 μΐ,, The excitation and emission wavelengths were set at 360 nm and 450 nm, respectively, and the fluorescence of microtubule-bound DAPI was monitored as a function of time at 37°C. Fluorescence signal at time 0 for each well was subtracted from each of the subsequent fluorescence readings. Each compound was assayed in triplicate,

Tubulin competitive binding assay:

Competition assay was adapted from Giraudel et al, 1 98:

MTP (3 μΜ, final concentration) in PME buffer was mixed at G-4°C with compounds and tritiated competitor ([³H] colchicine (76.5 Ci.mmol^"1, NEN) or [³H vinblastine (9.6 Ci.mmol-l, Amersham Biosciences) in a final volume of 200

Following 30 minutes incubation at 30°C, mixtures were deposited onto 50 ί of presedimented DEAE Sephadex A25 (to adsorb tubulin) in PME buffer. All subsequent steps were carried out at 0-4 ^CC. Samples were incubated for 10 min with continuous shaking to ensure quantitative binding of tubulin to the gel. Following centrifugation (2350 g, 4 min), supernatants were discarded and the pellets containing the bound molecule- tubulin complexes were washed four times with 1 niL volumes of PME buffer. Pellets were incubated for 10 min with 500 μΐ, of ethanol to solubilize the tubulin-bound tritiated competitor and 400 Ε aliquots of the ethanol solutions were transferred to 5 mL of Ultima Gold (Perkm-Elmer) scintillant for determination of radioactivity.

HUVEC tubulogenesis assay:

For this assay, ISO μΐ, of a mix of 85% Matrigel™ (Becton Dickinson) and 15%

EGM-2 medium (Cambrex) was allowed to polymerize at 37°C for 30 minutes in 48- multiwell plates. 3.105 HUVEC cells were seeded per well. Treatment with molecules (0.1, 1 and 10 μΜ) started right after cell attachment. Tubule formation was observed by phase- contrast microscopy at 24 hours.

RESULTS:

5-Azaindole compounds of formula (I) as reversible microtubule polymerization inhibitors:

The assay used to screen the chemical library was based on the substrate properties of the tubulin modifying enzymes involved in the tubulin tyrosination cycle. In this cycle the C- terminal tyrosine of the tubulin cc-subunii is removed by a cafbox peptidase and re-introduced by tubulin tyrosine ligase (TTL). Because of the substrate properties of these enzymes, dynamic microtubules, sensitive to depolymerizing drugs, are composed of tyrosinated tubulin whereas non-dynamic, stabilized microtubules are composed of detyrosinated tubulin. Thus depolymerization or stabilization of the microtubule network can be easily distinguished by double-immunofluorescence staining using antibodies specific for tyrosinated and detyrosinated tubulin. This assay was used to screen a library of 6,560 compounds at 25 μ concentration. 158 compounds belonging to several distinct structural families and able to lower the signal down to at least 75% in the tyrosinated tubulin channel as compared with DMSO-treated cells were selected. The normal Hoechst signal observed with these compounds is an indirect estimation of the integrity of the cell monolayer. These results indicated that these hits were potential microtubule depolymerizing agents.

Table 1: Flow chart of the automated screening of the library and the subsequent analysis of active compounds

Category Parameter Description

Assay Type of assay Multiparametric ceil (HeLa)~based assay

Primary measurement IrrrniunoQuorescence detection, using a microplate reader, of detyrosinated and tyrosinated microtubules. Detection of nuclei density was performed with Hoechst staining

Key reagents HeLa cells, polyclonal antibody specific of detyrosinated tubulin, monoclonal antibody specific of tyrosinated tubulin

Assay protocol See Methods (Screening part)

Additional comments Importance of the settings of the microplate washer, to ensure accurate and homogenous cell washing, without rojuring the cell monolayer

Library Library size 6,560 molecules

Library composition Small molecules from chemical synthesis

Source Institut Curie-CNRS library (UMR 176), Orsay, France, which is part of the French "Chirniotheque Nationale

Additional comments Compounds arrayed in 96-welI plates as single compounds in DMSO (80 compounds per plate) HTS process Format 96- ell plates, flat, black, opaque bottoms and walls

(Greiner)

Concentration tested 25 micromolar, 0.5% D SO

Plate controls Paelitaxei, Colchicine and DMSO were used as controls.

They were placed in each microplate, on the outer columns, alternating their locations. Additionally, a whole control plate was also daily performed.

Reagent/compound dispensing Fully automated platform, including 8-channel and 96- system channel dispensers

Detection instrument and FLUOstar OPTIMA microplate reader (BUG software Labtechnologies) and dedicated software

Correction factors Linear regression based on plate controls averages to reduce the impact of plate effects

Normalization Raw data converted into percentage of tyrosinated tubulin contents, based on plate controls averages

Posi-HTS analysis Hit criteria Tyrosinated tubulin content percentage < 75% and

Hoechst-stained DNA content > 90%

Hit rate 2.4%

Additional controls * Microscopic visualization

Elimination of compounds showing instability or excessive chemical reactivity and compounds with structure closely related to known microtubule targeting agents

Confirmation of hit purity LC-MS, MR, elementary analysis

Among the potential microtubule depolymerizing agents, the two potent 5-azaindole compounds of formula (I), CMOl and CM02, were more particularly studied as their scaffolds were drug-like, simple and their synthesis could give an easy access to numerous derivatives. Their effect on cellular microtubules was confirmed by immunofluorescence on HeLa cells fixed after permeabilization with OPT buffer. Whereas DMSO-treated cells exhibited a normal filamentous microtubule array, CM01/CM02 -treated cells were devoid of microtubules similarly to colchi cine-treated cells (Figure 1A). The changes of tubulin polymerization status were also checked by performing sequential extraction of soluble and insoluble pools of cellular tubulin. This enables the separation of unpoiymerized or depolymerized tubulin from microtubules. Decreasing concentrations (from 25 to 1 μΜ) of CMOl (Figure IB) and CM02 (Figure 1C) were tested and, whatever the concentration tested, a shift towards the unpoiymerized tubulin pool, similar to the effect elicited by colchicines, was observed, in depth the effects of CMOl and CM02 on microtubule dynamic instability parameters, using tirae-Iapse fluorescence microscopy on GFP-EB3 transfected cells (Honore ei al.„ 2011) were analyzed and compared with those of colchicine. The concentrations assayed were detennined after a preliminary immunofluorescence analysis of the compounds effect on the microtubule network. The concentrations chosen were in a range that induces a detectable but not total depolymerization of the microtubule network. As does colchicine, CMOl and CM02 reduced the microtubule growth rate, increased time spent in pause and strongly reduced the microtubule growth length as indicated by the dose-dependent increase of the distance-based catastrophe frequency (Table 2). Table 2: Comparison of alteration of microtubule dynamic instability parameters induced by colchicine with that of CMOl and CMOl

Colchicine Colchicine

Parameters DMSO

Θ.05 μΜ 0.1 μΜ

% time spent growing 77.69 73.44 64.99

% time spent in pause 22.31 26.56 35.01

Growing rate (μπι/η η ± SE) 17.04 ± 0.22 15.32 ± 0.37*** 14.66 ± 0.28***

Catastrophe frequency { xva~^l ± SE) 0.21 ± 0.01 0.33 ± 0.04** 0.40 ± 0.04***

Catastrophe frequency (miii¹ ± SE) 2.83 ± 0.08 3.56 ± 0.29* 3.71 ± 0.35*

CMOl CMOl CM02 CM02

Parameters

0.05 μΜ 0.1 μΜ 0.05 μΜ 0.1 μΜ

% time spent growing 59.70 49.86 55.7? 42.64

% time spent in pause 40.30 50.14 44.23 57.36

Growing rate (μιη/min ± SE) 14.04 ± 0.36*** 13.18 * 0.30*** 13.35 ± 0.38*** 12.43 ± 0.31***

Catastrophe frequency (μπι^"1 ± SE) 0.34 ± 0.03*** 0.53 ± 0,04*** 0.39 ± 0.03*** 0.78 ± 0.08***

Catastrophe frequency (rain ¹ ± SE) 2.68 ± 0.33 3.28 ± 0.18* 2.89 ± 0.20 3.87 ± 0.29**

*p < 0.05; **ρ < 0.01, ***p < 0.001 significantly different from control values (DMSO) using a Student's t test

These results clearly show that CMOl and CM02 have a strong effect on microtubule dynamics.

The reversible microtubule depolymerizing effect induced by CMOl and CM02 was also tested (Figure 2) by replacing medium containing compounds by standard culture medium. Imraunostainmg of a-tubulin revealed that retrieval of compounds restored normal microtubule array. These results demonstrate that CMOl and CM02 are celi-perraeable reversible microtubule polymerization iiihibitors.

5-Azaindole compounds of formula (I) are tubulin binders and compete with colchicines:

To test whether tubulin is a target of azaindole derivatives, in vitro microtubule polymerization assays were performed using either MTP or pure tubulin (Figures 3 and 4). In both cases, CMOl and CM02 inhibited tubulin polymerization in a dose-dependent manner. To determine compounds binding site on tubulin, effect of CMG1 and CM02 on tubulin binding of [3H] colchicine and [³H] vinblastine were investigated (Figure 5). CMOl and CM02 selectively inhibited colchicine but not vinblastine binding on tubulin. These results indicate that tubulin is a target of azaindole derivatives and mat azaindole derivatives bind to the colchicine-binding site but not to the vinca-alkaloids site.

Structure- Activity Relationships (SAR) analysis:

To explore the chemical properties conferring inhibitory potency to azaindole derivatives, several analogs of this family of compounds were selected and tested (Table 3),

Table 3: In vitro and cellular tubulin polymerization inhibitory activities of compounds of formula (I) wherein Ar is a phenyl ring

Substitaents at position

Tubulin assembly TjT-TubuUn signal

Compound

El R2 R3 Z (%) (%)

CMOl C¾ H oc¾ 0.0 0.0

CMQ2 c¾ H OCH₃ oc¾ 0.0 0.0

CM03 Bn H OCH3 CH(CH₃) Π.5 27.6

CM04 Bn H oc¾ O^CH^ 3.8 30.9

CMOS C¾ H OCH3 C(OH)C¾ 0.9 10.0

CMOS CH₃ H H CH{C¾) 66.7 101.3

CM07 CH₃ CI OCH3 CH(CH₃> 9.6 0.0

CMOS C¾ H H C{OH)Ph 100.2 82.6 These assays were first conducted on in vitro tubulin assembly. The effects of the compounds were compared at a 25 μΜ concentration. The requirements of the inter-aromatic group Z were first investigated, The inhibitory potencies of the compounds were the same when an ethane (CMOl), an ethylene (CM02) or a hydroxyethyl (CMOS) was present in this position. This result indicates that the nature of the group Z is not a stringent determinant. Position Ri can also be substituted by either a small group (methyl, CMOl and CM02) or by a larger one (benzyl, CM03 and CM04) without loss in inhibitory potency. This indicates some tolerance to bulky substituents at position R₅, Substitution of hydrogen by a chlorine group at position R₂ (CM07) did not alter the inhibitory potency of the compound. The presence of an alkoxy group such as a methoxy group at position R₃ was highly required for the compound to be active as illustrated by the results obtained with CM06 and CM08. The in vitro SAR correlated with cellular effect on microtubule network upon treatment with 5-azaindole compounds of formula (I) was also investigated, in this aim, cells grown on rmcroplates were incubated with the different compounds, fixed and processed for immunofluorescence. The quantification was done using the microplate reader, in the same way as for the primary screening. A good correlation was observed, which supports that tubulin is indeed the cellular target of azaindole derivatives, responsible for the observed phenotype.

Effect on cellular microtubules:

The effect on cellular microtubules of the 5~azaindole derivatives of formula (I) CM02 and CM10 was compared to that of indole derivatives CM11, CM12 and CM13 representative of the prior art WO 01/82909 A2.

HeLa cells grown for 72 hours on glass coverslips were incubated with compounds at indicated concentrations for 2 hours. They were then processed with the immunofluorescence procedure for microscopy analysis described above (Table 4).

Table 4: Immunofluorescence microscopy observations

The effect of the 5-azaindole derivatives of formula (I) CM02 and CM 10 on cellular microtubules was confirmed by immunofluorescence on HeLa cells fixed after pertneabilization with OPT buffer, whereas the indole derivatives CMll, CM12 and CM13 representative of the prior art WO 01/82909 A2 were proven to be completely inactive.

5-Azaindole compounds of formula (I) inhibit ceil proliferation of diverse cancer cell lines by promoting G2 M cell cycle arrest;

Viability of carcinoma cell lines from several organs (cervix, kidney, lung and breast) in response to CMOl and CM02 treatment was investigated using MTT assays (Table 5), Table 5: Effects of 5-azaindole compounds of formula (I) on the viability of different cell lines

GI50 (50% of growth inhibition) were in the sub- or 3ow-raicromoIar range regardless the tissue origins, p53 or K-ras status of the cells. To determine the mode of action of the antiproliferative activity exhibited by azaindole derivatives, the distribution of CM01/CM02- treated cells in the cell cycle using flow cytometry was analyzed. Following 16 hours of exposure of the compounds at 1 μΜ or 25 μΜ, cells accumulated in G2/M phases (> 89%) when compared to vehicle-treated cells (23%, Figure 6). Taken together, these experiments show that 5-azamdole compounds of formula (I) are potent cytostatic compounds, which block the cell cycle at G2/M phase.

5-AzaindoIe compounds of formula (I) overcome drug resistant (MDR) cell pheitotype:

The effect of CMOl and CM02 on cell proliferation of drug-sensitive sensitive cell- lines and of their drug-resistant counterparts that over-express the glycoprotein P or multidrug transporters such as ABCG2 and MRP] that confer cell resistance to multiple drugs (Marker et al, 1985) was compared (Table 5). CMOl and CM02 toxicity was found to be the same, with GI50 in the micromolar range, for the drug-sensitive human cell lines, and for their multidrug- resistant counterparts, indicating that it is not substrate of the glycoprotein V or of ABCG2 and MRPl transporters. This contrasts with the active efflux, at nanomolar concentrations, of colchicine and taxanes (pachtaxel and docetaxel) by Pglycoprotein, and of vinca alkaloids (vincristine and vinblastine) by both P -glycoprotein and MRPl (Dumontet et al, 1996; Fojo et al. , 2007; Szakacs et al, 2006).

5-Azaindoles compounds of formula (I) exhibit antiangiogenic effect;

Numerous compounds, such as combretastatins, which bind to the tubulin colcbicine- binding site, exhibit antiangiogenic effect. To investigate the putative antiangiogenic potency of azaindoles derivatives, CMOl and CM02 were tested in two different in vitro angiogenesis assays. The effect of the compounds on capillary-like tube formation on Matrigel was first investigated. It was found that both compounds were able to inhibit capillary tubes morphogenesis (Figure 7). A more integrated assay, i,e. 3 -dimensional cultures of HMEC- GFP cell spheroids in a collagen gel was used to anal ze the effects of increasing doses of the compounds on FGF2-stimulated endothelial cell sprouting. Although the compounds show no detectable toxicity on spheroids themselves (Figure 8A), they induced a dose dependent decrease of the mean length of total endothelial sprouts (Figure 8B). In both assays, CM02 showed a stronger effect than CMOl. These data indicate that 5-azaindoles compounds of formula (I) are potent antiangiogenic compounds.

5-Azaindoles compounds of formula (I) exhibit a»ti-tumorai effect on an in vivo model of invasive breast cancer :

Breast cancer cells (MDA-MB-231 expressing GFP) xenografted on the chicken chorioallantoid membrane were used to assess both toxicity and efficiency of CMOl and CM02 molecules. Tumors treated with these compounds were significantly smaller (P < 0.05; Figure 9A and 9B). Furthermore, upon treatment, chicken embryos did not display significant increased mortality indicating that, in this model, these compounds are well tolerated at doses sufficient to induce antitumoral effect. The expression of GFP by the highly invasive MDA-MB-231 cells allowed an accurate detection, using fluorescence microscopy, of the dissemination of tumor cells in this model, as indicated by the number of nodules counted in the lower CAM (Figure 9C) in DMSO-treated embryos. The nodule number was greatly reduced in CMOl and CM02 treated embryos, indicating that azaindoles derivatives affect invasion mechanisms.

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Claims

1. Compounds of formula (I):

in which:

~ the ring Ar is a C₄-Ci₂ aryl or heteroaryl ring,

- ] - Hj optionally substituted C_t-C₆ alkyl, benzyl, arylsulfonyl, heteroarylsuifonyl, alkyloxycaxbonyl or dialkylcarbamoyl groups,

- R₂ represents H or a halogen atom, nitro, amino, Cj-Ce alkylamino or C₂-C₆ cycloamino groups,

- R₃, Rj, ¾ and R₇, identical or different, represent hydrogen or halogen atoms, hydroxyl, Ci-C₆ alkyl, Cj-Q alkoxy, cyano, nitro, carboxylate, carboxyester, amino Cj-Cs alkylamino or C₂-C<_¾ cycloamino groups,

- 4 = a halogen atom, hydroxy!, Ci-C^ alkoxy, amino, d- , alkylamino, C₂-C₆ cycloamino, thiol or Cj-Ce alkylthio groups, and

- Z = C=CHR, CR'R" or C=0, wherein R = H or an optionally substituted C_rC₆ alkyl group, and R' and R", identical or different, represent hydrogen or halogen atoms, OH gro ps, Ci-C₆ alkyl or alkoxy groups, C4-C12 aryl, heteroaryl, arylalkyl or heteroarylalkyl groups,

or their tautomeric, racemic, enantiomeric or polymorphic forms or pharmaceutically- acceptable salts, for their use as drugs.

2. Compounds of formula (I) for their use according to Claim 1, wherein the ring Ar is a phenyl or pyridine ring, and preferably a phenyl ring.

3. Compounds of formula (I) for their use according to Claim 1 or Claim 2, wherein Z = C=CHR or CR'R", wherein R = H or an optionally substituted C C₆ alky] group, and R^! and R", identical or different, represent hydrogen or halogen atoms, OH groups, Ci-C₆ alkyl or alkoxy groups, C4-Q2 ary _> heteroaryl, arylalkyl or heteroarylalkyl groups.

4. Compounds of formula (I) for their use according to Claim 1 to 3, wherein Z = C-CH2 or CR'R" wherein R' - H or OH and R" - Cj-C₆ alkyl or phenyl groups.

5. Compounds of formula (I) for their use according to Claims 1 to 4, wherein Z ~ C=CH₂, CH(C¾)_? C(OH)CH₃ or C(OH)C₆H₅.

6. Compounds of formula (I) for their use according to Claims 1 to 5, wherein Ri = CH3 or a benzyl group.

7. Compounds of formula (I) for their use according to Claims 1 to 6, wherein R2 -

H r CI.

8. Compounds of formula (Γ) for their use according to Claims 1 to 7, wherein R₃, R₅, R_& and R₇, identical or different, represent hydrogen atoms or CpQ alkoxy groups.

9. Compounds of formula (I) for their use according to Claims 1 to 7, wherein at least one of the R₃, R_$5 Re or R₇ radical is a OCH₃ group.

10. Compounds of formula (I) for their use according to Claim 9, wherein the ring Ar is a phenyl ring substituted by a OCH₃ group in position 4.

11. Compounds of formula (I) for their use according to Claims 1 to 30, wherein R₄ = a halogen atom or a Cj-Ce alkoxy group.

12, Compounds of formula (I) for their use according to Claims 1 to 11 , wherein R4 =

CI or a OCH group.

13. Compounds of formula (I) for their use according to Claims 1 to 12, responding to one of the following formula:

CMOS or

14. Compounds of formula (I) for their use according to Claims 1 to 13, for the prevention and/or the treatment of diseases and or disorders chosen amongst cancers; angiogenesis related disorders; parasitic diseases; fungal diseases; autoimmune diseases; inflammatory diseases; warts such as warts caused by papilloma virus.

15. Compounds of formula (I) for their use according to Claims 1 to 13, for the prevention and/or treatment of cancers chosen amongst testicular cancer, ovarian cancer, lung cancer, breast cancer, Hodgkm's disease, acute lymphoid leukemia, neuroblastoma, melanoma, glioma, glioblastoma, sarcoma, colon cancer, pancreatic cancer.

16. Compounds of formula (1) for their use according to Claims 1 to 13, for the prevention and/or treatment of angiogenesis related disorders chosen amongst diabetic blindness, macular degeneration, rheumatoid arthritis, psoriasis.

17, Compounds of formula (I) for their use according to Claims 1 to 13, for the prevention and/or treatment of parasitic diseases involving apicomplexan parasites, parasites with flagellum or cilia, toxoplasmosis.

18. Compounds of formula (I) for their use according to Claims I to 13, for the prevention and/or treatment of autoimmune diseases chosen amongst multiple sclerosis, diabet c retinopathy.

19. Compounds of formula (Γ) for their use according to Claims 1 to 13, for the prevention and/or treatment of inflammatory diseases chosen amongst gout disease.

20. A pharmaceutical composition comprising at least one compound of formula (I) as defined according to Claims 1 to 13 together with at least one pharmaceutically-acceptable excipient or carrier, and with at least another active substance.

21. Use of at least one compound of formula (1) as defined according to Claims 1 to 13 as research, tool for the cell-cycle synchronization of microtubule drugs resistant cell lines.

22, Use of at least one compound of formula (I) according to Claims 1 to 13 as an herbicide and/or an algaecide.