Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
  • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
  • A01N43/90—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having two or more relevant hetero rings, condensed among themselves or with a common carbocyclic ring system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
  • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
  • C07D471/04—Ortho-condensed systems

Definitions

• the present invention relates to 5-azaindole type compounds of formula (I) for their use as drugs, 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 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.
• 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.
• 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.
• 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 could 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 ⁇ / ⁇ 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 EBl (Akhmanova et al, 2005; Cottle et al, 2009; Small et al, 2003). Targeted perturbation of this finely tuned process constitutes a major therapeutic strategy (Honore et al, 2005).
• 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 EBl (Akhmanova et
• 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.
• most of these drugs have their therapeutic potential hampered by insufficient bioavailability and toxicity (myelosuppression, peripheral neurotoxicity).
• failure in cancer therapy is often related to the selection of tumor cells that have acquired resistance against microtubule binding drugs.
• 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).
• 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.
• tubulin is today one of the best validated targets in anticancer chemotherapy (Giannakakou et al, 2000; Jackson et al, 2007; Zhou and Giannakakou, 2005).
• 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 (Dermaut 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).
• the treatments used in anticancer chemotherapy target, in a favored manner, the dynamic behavior of the microtubules.
• 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 protofilaments 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 ⁇ -tubulin facing the microtubule lumen (Snyder et al, 2001).
• 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.
• 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.
• 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.
• paclitaxel The therapeutic success of paclitaxel has maintained the advantage for the search for therapeutic agents that target tubulin.
• tubulin drugs are not surprising because tubulin is a major player not only in cell division but also in mitosis-independent cytoskeletal functions.
• the first subject of the present invention thus relates to 5-azaindole compounds of formula (I):
• the ring Ar is a C 4 -Ci 2 aryl or heteroaryl ring, and preferably a phenyl or pyridine ring,
• Rj H, C C 6 alkyl, benzyl, arylsulfonyl, heteroarylsulfonyl, alkyloxycarbonyl or dialkylcarbamoyl groups, said benzyl, aryl or heteroaryl groups being optionally substituted with one or more groups independently selected from halogen, hydroxyl, cyano, nitro, carboxylate, carboxyester, amino, C]-C 6 alkyl, C]-C 6 alkylamino or C r C 6 alkoxy groups,
• R 3 , R 5 , R6 and R 7 identical or different, represent hydrogen or halogen atoms, hydroxyl, Q-C6 alkyl, Ci-C 6 alkoxy, cyano, nitro, carboxylate, carboxyester, amino or Ci-C 6 alkylamino groups,
• 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 azaindole 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 azaindole derivatives and its polymerization inhibition is likely responsible for the observed phenotype. Complete microtubule depolymerization was observed in cells.
• 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 K-ras, both genetic or epigenetic alterations that are among the most common alterations found in cancer cells.
• the 5-azaindole compounds of formula (I) also exhibited antiproliferative activity in cell lines overexpressing the drug efflux pumps PgP, MRP1 and ABCG2.
• these compounds may thus be used as research tool for cell synchronization experiments with resistant cells. Moreover, this property may be exploited in the case of chemotherapeutic failure.
• the compounds of formula (I) also inhibit angiogenic sprouting from microvascular endothelial cell spheroids, which demonstrates that these compounds exert antiangiogenic 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.
• the compounds of formula (1) 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.
• Alkyl groups are chosen among ⁇ .C alkyl groups such as methyl, ethyl, n- propyl, isopropyl, n-butyl, sec-butyl, ter -butyl and isobutyl radicals;
• Heteroalkyl groups mean alkyl groups as defined above in which one or more hydrogen atoms to any carbon of the alkyl is replaced by a heteroatom selected from the group consisting of N, O, P, B, S, Si, Sb, Al, Sn, As, Se and Ge.
• the bond between the carbon atom and the heteroatom may be saturated or unsaturated.
• Suitable heteroalkyl groups include cyano, benzoyl, methoxy, acetamide, borates, sulfones, sulfates, thianes, phosphates, phosphonates, and the like;
• Alkoxy groups are chosen among Ci-C 6 alkoxy groups such as methyloxy, ethyloxy, n-propyloxy, iso-propyloxy, n-butyloxy, sec-butyloxy, tert-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 ⁇ system in which each atom of the ring comprises a p-orbital, said p-orbitals overlapping themselves.
• 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, isoindole, benzothiophene, benzo[c]thiophene, benzimidazole, indazole, benzoxazole, benzisoxazole, benzothiazole, quinoline, isoquinoline, quinoxaline, quinazoline, cinnoline, purine and acrid
• Arylalkyl groups mean any group derived from an alkyl group as defined above wherein a hydrogen atom is replaced by an aryl or a heteroaryl group as defined above;
• Alkylamino groups mean mono or di(Ci-C 6 )aIkylamino group including the C 2 -C 6 cycloamino group such as aziridin-l-yl, azetidin- 1 -yl, pyrrolidin-l-yl, piperidin-l-yl, azepan-l -yl, morpholin-4-yl and thiomorphoIin-4-yI.
• halogen atoms are chosen among bromine, chlorine, fluorine and iodine, preferably bromine, chlorine and fluorine, and more preferably chlorine.
• 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.
• R is an arylsulfonyl or a heteroarylsulfonyl group, preferably the aryl ring is a phenyl ring;
• Ri is an alkoxycarbonyl group, preferably R
• Ri is a dialkylcarbamoyl group, preferably Ri is the dimethylcarbamoyl.
• Ri C3 ⁇ 4 or a benzyl group.
• At least one of the R 3 , R 5 , R 3 ⁇ 4 or R 7 radical is a OCH 3 group.
• Ar is a phenyl ring substituted by a OCH 3 group in position 4.
• the present invention relates more particularly to compounds of formula (I) 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.
• 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, 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,
• 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.
• compositions 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.
• 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.
• 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 (I) can be easily determined by standard pharmaceutical procedures in cell cultures or experimental animals, i.e. for determining the LD 50 (the dose lethal to 50% of the population) and the ED 5 o (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 LD 5 o and ED 50 .
• 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 ED 5 o 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 (I) 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.
• 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 auxiliary(ies) that facilitate processing of the compounds of formula (I) into preparations which can be used pharmaceutically.
• 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.
• the carrier when the pharmaceutical composition is administered orally, 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.
• 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.
• 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.
• FIG. 1 shows the effect of two compounds of formula (I) according to the invention, CMOl and CM02, on microtubule network organization:
• B a-tubulin partition between soluble and insoluble fractions.
• HeLa cells were treated for 2 hours with 0.25% DMSO (vehicle control); 2 ⁇ colchicine and increasing concentrations of CMOl as indicated.
• Insoluble (I) and soluble (S) fractions were prepared as described in Materials and Methods. Equivalent volumes of extracts (20 ⁇ ,) 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.
• FIG. 2 shows the reversibility of CMOl and CM02 effects in HeLa cells.
• HeLa cells were treated for 2 hours with 0.25% DMSO (control); 5 ⁇ colchicine; 10 ⁇ nocodazole, 25 ⁇ nocodazole, 25 ⁇ CMOl or 25 ⁇ 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.
• FIG. 3 shows the effect of CMOl 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 CMOl or CM02, as indicated. Results are presented as mean ⁇ standard error of the mean (SEM).
• FIG. 4 shows the effect of CMOl 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 CMOl or CM02, as indicated. Results are presented as mean ⁇ standard error of the mean (SEM).
• FIG. 5 represents the effect of CMOl and CM02 on the binding of [ 3 H] colchicine and [ 3 H] vinblastine to microtubule proteins.
• 50 nM [ 3 H] colchicine (black bars) or 30 nM [ 3 H] vinblastine (dashed bars) were competed with 100 ⁇ of CMOl, CM02, colchicine and vinblastine as described in Materials and Methods.
• Each value represents the mean ⁇ SEM from triplicate determinations.
• FIG. 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 ⁇ ), CMOl and CM02 (1 ⁇ and 25 ⁇ , 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 ⁇ , 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.
• FIG. 7 shows that the compounds of formula (I) inhibit capillary-like tube formation.
• FIG. 8 represents the quantitative analysis of endothelial sprouting in response to 5-azaindoles compounds of formula (I).
• FGF2 100 ng/mL
• CMOl collagen- embedded HMEC-GFP spheroids
• CM02 collagen- embedded HMEC-GFP spheroids
• 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.
• C02 (R,S) 1 -benzyl-4-chloro-2-(l -(4-methoxyphenyl)ethyl)-lH-pyrrolo[3,2-i7]pyridine (CM03), l-benzyl-4-chIoro-2-(l-(4-methoxyphenyl)vinyl)-lH-pyrrolo[3,2-c]pyridine (CM04), (R,S) 1 -(4-chloro- 1 -methyl- lH-pyrrolo [3 ,2-c]pyridine-2-yl)-l -(4- methoxyphenyl)ethanol (CMOS) and (R,S) 4-chloro-l-methyl-2-(l-phenylethyl)-lH- pyrrolo[3,2-c]pyridine (CM06) were synthetized according to the method published in Nguyen et al, 1986.
• 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 yL of medium and were allowed to grow for 24 hours.
• each well was robotically supplemented with 10 of RPMI containing compounds (6,560) from the CNRS-Curie Institute library.
• the final concentration of the compounds was 25 ⁇ .
• the final concentration was 5 ⁇ for paclitaxel and 2 ⁇ for colchicine.
• the final concentration of DMSO was 0.5%.
• cells were incubated 2 hours at 37°C, 5% C0 2 in the workstation incubator.
• Cells were then permeabilized 10 minutes with 100 ⁇ , of warm OPT buffer, in 7 order to eliminate free, depolymerized tubulin and fixed 6 minutes with 100 ⁇ , of methanol.
• Cells were then double stained for tyrosinated and detyrosinated tubulin using specific antibodies and fluorescent secondary antibodies. Nuclei were stained with Hoechst.
• Raw data were converted into percentage of tyrosinated/detyrosinated tubulin contents, based on plate controls averages.
• HMEC-GFP preparation
• HMEC-1 cells were infected by a defective retrovirus encoding for enhanced Green Fluorescent Protein (EGFP).
• EGFP Green Fluorescent Protein
• HMEC-1 clones expressing EGFP were selected in the presence of neomycin. Resistant clones were isolated by limit dilution, and amplified as independent lineages.
• HMECGFP herpetic Human Cytomegalovirus
• HMEC-GFP cells were seeded at 3000 cells/well in round- bottom 96-multiwell plates in DMEM 1 g/L glucose (Eurobio, Les Ulis, France) containing 0.25 % mefhylcellulose. 48 hours later, the spheroids were collected, transferred into flat- bottom 96-well plates and embedded in 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 ⁇ /mL penicillin and 50 ⁇ g/mL streptomycin.
• MDA-MB-231 cells were transfected with GFP-encoding plasmid pEGFP-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 cm 2 window was cut in the eggshell above the CAM.
• SFPA St. Brieuc
• 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
• MES-SA/Dx5 human uterine sarcoma
• HeLa, NCI-H460 MDA-MB-231, and 786-0 cells were grown in complete RPMI 1640 with GlutaMAXTM I (Gibco, Invitrogen) containing 10% fetal bovine serum (Hyclone, Thermo Fisher Scientific) and 100 units/mL of penicillin and 100 ⁇ g/mL of streptomycin (Gibco, Invitrogen).
• MCF-7 were maintained in EMEM (ATCC) with 10% fetal bovine serum, 100 units/mL of penicillin, 100 ⁇ g/mL 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 ⁇ $/ ⁇ of streptomycin during the first sub-culture, then adapted in RPMI 1640 with GlutaMAXTM and 10% fetal bovine serum, 100 units/mL of penicillin and 100 ⁇ g mL of streptomycin.
• HMEC-1 cells Human Microvascular Endothelial Cell-1
• 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 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, 5% C0 2 humidity atmosphere in media.
• FGF-2 was from Salk Institute, La Jolla, CA, USA.
• 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 permeabilized with OPT buffer (80 mM PIPES pH 6.7, 1 mM EGTA, 1 mM MgCl 2 , 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. Cells 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 at, 1994.
• OPT buffer 80 mM PIPES pH 6.7, 1 mM EGTA, 1 mM MgCl 2 , 0.5% Triton X-100 and 10% glycerol
• 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.
• 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 (AM AX A ® , Koln, Germany). 2 ⁇ g of purified plasmid DNA was used for each transfection reaction.
• Live imaging of microtubule plus ends was performed as described in Honore et al., 201 1, 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 Metamorph software (Universal Imaging Corp.). Images acquisition was performed at a temperature of 37 ⁇ 1°C / 5% C0 2 .
• CMOl and CM02 effects on microtubule dynamics cells were incubated with different concentrations of compounds, or vehicle alone (D SO) for 2 hours. Data are from 3 independent experiments. For each experiment, 6 microtubules/cell in 6 cells per condition were analyzed.
• 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.
• 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 RPMI medium and incubated overnight in complete RPMI medium. They were then processed for immunofluorescence, as described above.
• HeLa cells (1.10 6 ) 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 niM, MgCl 2 1 mM, triton X-100 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.
• OPT buffer Pipes 80 mM, EGTA 1 niM, MgCl 2 1 mM, triton X-100 0.5% and glycerol 10% pH 6.8
• the remaining cells 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.
• the same sample volume (20 ⁇ ) of soluble and insoluble pools was separated on an 8% polyacrylamide SDS-page gel and transferred onto nitrocellulose.
• Tubulin was detected using anti a-tubulin primary antibody (clone a3a (Peris et al, 2006)), monoclonal peroxidase-conjugated mouse secondary antibody (Sigma, A4416) and detection with chimioluminescence kit ECLTM Plus (GE Healthcare, RPN2132).
• Cell proliferation was evaluated using the colorimetric 3-(4,5-dimethylthiazol-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.
• HeLa cells were grown in T25 flasks (5.10 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 pg/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.
• Microtubule protein (MTP) and pure tubulin were prepared according standard procedures (Paturle-Lafanechere et al., 1991). Microtubule polymerization assay was adapted from (Bonne et at, 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).
• MTP MTP or pure tubulin
• PME 100 mM MES, 1 mM MgCl 2 , 1 mM EGTA, pH 6.75, for MTP
• PME buffer 100 mM PIPES, 1 mM MgCl 2 , 1 mM EGTA, pH 6.65, for pure tubulin
• assembly was initiated by injection of GTP and MgCl 2 to a final concentration of 1 mM and 5 mM respectively, yielding a reaction volume of 100 pL.
• the excitation and emission wavelengths were set at 360 nm and 450 nm, respectively, and the fluorescence of micro tubule-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.
• MTP (3 ⁇ , final concentration) in PME buffer was mixed at 0-4°C with compounds and tritiated competitor ([ 3 H] colchicine (76.5 Ci.mmol “1 , NEN) or [ 3 H] vinblastine (9.6 Ci.mmol-1, Amersham Biosciences) in a final volume of 200 ⁇ ,. Following 30 minutes incubation at 30°C, mixtures were deposited onto 50 ⁇ _, of presedimented DEAE Sephadex A25 (to adsorb tubulin) in PME buffer. All subsequent steps were carried out at 0-4°C. Samples were incubated for 10 min with continuous shaking to ensure quantitative binding of tubulin to the gel.
• 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.
• the C- terminal tyrosine of the tubulin a-subunit is removed by a carboxypeptidase and re-introduced by tubulin tyrosine ligase (TTL).
• TTL tubulin tyrosine ligase
• 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 ⁇ 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
• HTS process Format 96-well plates, flat, black, opaque bottoms and walls
• 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 col chi 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.
• the concentrations assayed were determined 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.
• 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).
• Catastrophe frequency ( ⁇ ⁇ ⁇ "1 ⁇ SE) 0.34 ⁇ 0.03*** 0.53 ⁇ 0.04** * 0.39 ⁇ 0.03*** 0.78 ⁇ 0.08***
• 5-Azaindole compounds of formula (I) are tubulin binders and compete with colchicines:
• tubulin is a target of azaindole derivatives
• in vitro microtubule polymerization assays were performed using either MTP or pure tubulin ( Figures 3 and 4).
• CMOl and CM02 inhibited tubulin polymerization in a dose-dependent manner.
• effect of CMOl and CM02 on tubulin binding of [3H] colchicine and [ 3 H] vinblastine were investigated ( Figure 5).
• CMOl and CM02 selectively inhibited colchicine but not vinblastine binding on tubulin.
• CMOS C3 ⁇ 4 H H C(OH)P ⁇ i 10O.2 82.6 were first conducted on in vitro tubulin assembly. The effects of the compounds were compared at a 25 ⁇ 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 3 ⁇ 4 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.
• CMOl ethane
• CM02 ethylene
• CMOS hydroxyethyl
• 5-Azaindole compounds of formula (I) inhibit cell proliferation of diverse cancer cell lines by promoting G2/M cell cycle arrest:
• GI50 (50% of growth inhibition) were in the sub- or low-micromolar range regardless the tissue origins, p53 or K-r s status of the cells.
• CM01/CM02- treated cells were in the sub- or low-micromolar range regardless the tissue origins, p53 or K-r s status of the cells.
• 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 ⁇ or 25 ⁇ , cells accumulated in G2/M phases (> 89%) when compared to vehicle-treated cells (23%, Figure 6).
• 5-azaindole compounds of formula (I) are potent cytostatic compounds, which block the cell cycle at G2/M phase.
• 5-Azaindole compounds of formula (I) overcome drug resistant (MDR) cell phenotype:
• combretastatins which bind to the tubulin colchicine- binding site, exhibit antiangiogenic effect.
• 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 analyze the effects of increasing doses of the compounds on FGF2-stimulated endothelial cell sprouting.
• 5-Azaindoles compounds of formula (I) exhibit anti-tumoral effect on an in vivo model of invasive breast cancer:
• MDA-MB-231 expressing GFP breast cancer cells 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.
• MPC- 6827 a small-molecule inhibitor of microtubule formation that is not a substrate for multidrug resistance pumps. Cancer Res 67(12): 5865-5871.
• Taxol- resistant epithelial ovarian tumors are associated with altered expression of specific beta- tubulin isotypes. J Clin Invest 100(5): 1282-1293.
• Microtubules meet substrate adhesions to arrange cell polarity. Curr Opin Cell Biol 15(1): 40-47.
• Tozer GM Prise VE, Wilson J, Cemazar M, Shan S, Dewhirst MW, et al. (2001). Mechanisms associated with tumor vascular shut-down induced by combretastatin A-4 phosphate: intravital microscopy and measurement of vascular permeability. Cancer Res 61(17): 6413-6422.

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