WO2013149336A1 - Dérivés de 5-nitrofurane-2carboxamide permettant d'inhiber l'agrégation centrosomale - Google Patents

Dérivés de 5-nitrofurane-2carboxamide permettant d'inhiber l'agrégation centrosomale Download PDF

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WO2013149336A1
WO2013149336A1 PCT/CA2013/050244 CA2013050244W WO2013149336A1 WO 2013149336 A1 WO2013149336 A1 WO 2013149336A1 CA 2013050244 W CA2013050244 W CA 2013050244W WO 2013149336 A1 WO2013149336 A1 WO 2013149336A1
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cell
compound
formula
optionally substituted
cancer
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PCT/CA2013/050244
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English (en)
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Shoukat Dedhar
Eiko KAWAMURA
Michel Roberge
Andrew Fielding
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British Columbia Cancer Agency Branch
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    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/345—Nitrofurans
    • 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
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/381—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered rings
    • 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
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/443—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with oxygen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present disclosure relates generally to compounds and methods relating to centrosomes. More particularly, the present disclosure relates to compounds and methods for inhibiting centrosomal clustering in a cell harboring supernumerary centrosomes.
  • Mitosis is the division of genetic material into two, equal halves whilst cytokinesis is the process by which the cell divides itself into two daughters. Mitosis is achieved by condensed
  • chromosomes attaching in a bipolar fashion to the microtubule-based mitotic spindle (the bipolar mitotic spindle) and subsequent pulling apart of sister chromatids to opposite poles of the cell.
  • the bipolar mitotic spindle is largely organized by centrosomes in the majority of animal cells.
  • Centrosomes are the principal microtubule organizing centers in animal cells.
  • centrosome defects have been reported in a wide variety of human malignancies (Nigg, 2002, 2006; Pihan and Doxsey, 1999; Pihan et al., 2003; Raff, 2002; Doxsey et al., 2005; Satish, 2006 ). They have been shown to occur at early stages of cancer development (Pihan and Doxsey, 1999; Pihan et al., 2003; Lingle et al., 2002) and to correlate with the genomic instability of tumours (Pihan and Doxsey, 1999; Lingle et al., 2002). Centrosome amplification has been implicated in the initiation of tumorigenesis in Drosophila (Basto et al., 2008).
  • Griseofulvin an anti-fungal agent which is known to interact with tubulin (Singh et al., 2008), was identified as a compound that inhibited centrosome clustering.
  • Griseofulvin analogues have been proposed for the treatment of cancer by inhibition of centrosomal clustering (United States Patent Publication US 2011/0178170, Clausen et al.).
  • the present disclosure relates in part to compounds and methods for inhibiting centrosomal clustering in a cell harboring supernumerary centrosomes.
  • the present disclosure provides a method for inhibiting centrosomal clustering in a cell harboring supernumerary centrosomes comprising administering an effective amount of a compound of the general chemical formula (I):
  • X is optionally substituted alkyl
  • n is an integer from 1 to 10
  • R-i is optionally substituted aryl or cycloalkyi, or a pharmaceutically acceptable salt thereof, to a subject in need thereof.
  • present disclosure provides a method for treating a cancer comprising administering a therapeutically effective amount of a compound of the general chemical formula (I):
  • Formula (I) [0013] where X is optionally substituted alkyl, n is an integer from 1 to 10 and R-i is optionally substituted aryl or cycloalkyi, or a pharmaceutically acceptable salt thereof, to a subject in need thereof.
  • the cell harbouring supernumerary centrosomes may be a cancer cell.
  • the cancer cell may be a human cell.
  • the cancer cell may be a breast, lung, or colon cancer cell.
  • the cancer cell may be an aggressive cancer cell.
  • the subject may be a human.
  • the present disclosure provides a pharmaceutical composition comprising an effective amount of a compound of the general chemical formula (I):
  • X is optionally substituted alkyl
  • n is an integer from 1 to 10
  • R-i is optionally substituted aryl or cycloalkyi, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent or carrier.
  • the present disclosure provides a method of determining a suitable therapy for a subject in need thereof by providing a sample from the subject, and detecting the presence of a cell harboring supernumerary centrosomes in said sample, where the presence of a cell harboring supernumerary centrosomes is indicative of the suitability of administration of a therapeutically effective amount of a compound of the general chemical formula (I):
  • X is optionally substituted alkyl
  • n is an integer from 1 to 10
  • R-i is optionally substituted aryl or cycloalkyi, or a pharmaceutically acceptable salt thereof, to said subject.
  • X is optionally substituted alkyl
  • n is an integer from 1 to 10
  • R-i is optionally substituted aryl or cycloalkyi, or a pharmaceutically acceptable salt thereof for treating a cancer in a subject in need thereof.
  • X may be alkyl, n may be 1 and R-i may be aryl.
  • R-i may be a six-membered or five-mem bered aryl group.
  • R-i may be selected from the group consisting of benzene, pyridine and thiophene.
  • the compound of Formula I may be s 5-nitro-/V-(pyridin-3- ylmethyl)furan-2-carboxamide, 5-nitro-/V-(thiophenn-2-ylmethyl)furan-2-carboxamide, or N- benzyl-5-nitrofuran-2-carboxamide.
  • FIGURES 1A-E show a summary of the screening strategy for identifying candidate compounds,
  • Centrosomes were labeled with anti-pericentrin (white dots) and the nuclei of TG-3-positive cells (identified by arrows) were circumscribed to define regions of interest. The number of pericentrin foci within the ROIs was enumerated. Cells with declustered centrosomes were defined as those mitotic cells with greater than two pericentrin foci, (d) Representative results of the primary screen evaluating 1 ,200 compounds. The distribution of test compounds is similar to that of untreated cells except that it displays additional high scores. Those compounds that increased the score by at least 2.5x the standard deviation were considered "hits" and were subjected to a secondary screen, (e) Representative results of the secondary screen.
  • FIGURE 2 shows the effects of three identified small molecules on centrosome declustering in vitro
  • FIGURE 3 shows validation of the three positive "hits" as cancer cell selective inducers of centrosome declustering and inhibitors of cell growth.
  • Left column chemical structures for the validated hits.
  • Middle column centrosome declustering assay. The effect of centrosome declustering was examined after treating BT-549 cells with 10 ⁇ of the indicated compounds for 5 hours in 6-well plates.
  • Right column growth inhibition assay. MTT assays were carried out after 2 days of treatment with 10 ⁇ of the indicated compounds. Average values of all tests are shown. Bars are standard deviation.
  • FIGURE 4 shows that BR00067 induces centrosome declustering in BT-549 breast cancer cells, but not in normal mammary epithelial cells.
  • An MTT assay was carried out with finer titration steps between 1 and 10 ⁇ . ICso values for cancer cells were between 2 and 8 ⁇ , while that for primary cells was above 10 ⁇ .
  • FIGURE 5 shows apoptosis is induced in BT-549 cancer cells, but not in normal MCF-10A cells. Apoptosis was examined by Cell Death Detection ELISA (Roche), which measures the accumulation of cytoplasmic nucleosomes associated with apoptosis. Cells were treated with BR00067 (compound 01 ) for 19 hours. Values were normalized to the highest DMSO concentration (0.1 %) applied for BR00067 treatment. Average of 3 independent experiments ⁇ S.D.
  • the present disclosure provides, in part, methods and compositions for inhibiting centrosomal clustering in cells harboring supernumerary centrosomes. Without being bound to any particular hypothesis, inhibition of centrosomal clustering may result in induction of multipolar spindles and subsequent mitotic arrest and cell death. Supernumerary centrosomes have been correlated with chromosomal instability and aggressive tumors, and occur almost exclusively in tumor cells. Accordingly, in some embodiments, inhibition of centrosomal clustering selectively targets neoplastic or cancer cells and compounds as described herein may be used to treat or prevent cancer. In alternative embodiments, the present disclosure provides methods of determining the suitability of a therapy, or of prognosing the effect of a therapy, using compounds as described herein by detecting a cell harboring supernumerary centrosomes.
  • a cell harboring "supernumerary centrosomes" is an animal cell having more than two centrosomes. Such a cell may exhibit “centrosome clustering” or “centrosomal clustering,” i.e., clustering of the supernumerary centrosomes to form two polar (“bipolar") spindles.
  • the cell may be a mammalian cell, such as a human, non- human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc. cell.
  • the cell may be a neoplastic or cancer cell.
  • inhibiting or “inhibition of centrosomal clustering is meant one or more of: decreasing bipolar spindle formation in a cell harbouring supernumerary centrosomes; decreasing survival of a cell harbouring supernumerary centrosomes; increasing declustering of formed bipolar clusters of supernumerary centrosomes, increasing multipolar spindle formation, increasing mitotic arrest, and/or increasing apoptosis of a cell harbouring supernumerary centrosomes. It is to be understood that the inhibition is not intended to be absolute.
  • the increase or decrease may be a change of any value between 10% and 100%, for example, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or may be over 100%, when compared with a control or reference sample or compound, such as a cell that does not have more than two centrosomes (a "normal cell") or a compound that is known to have an effect on bipolar spindle formation, centrosomal clustering, or on the survival or proliferation of a cell.
  • a control or reference sample or compound such as a cell that does not have more than two centrosomes (a "normal cell") or a compound that is known to have an effect on bipolar spindle formation, centrosomal clustering, or on the survival or proliferation of a cell.
  • a compound of Formula I includes a compound of the general chemical formula (II):
  • R-i is optionally substituted aryl or cycloalkyl.
  • a compound of Formula I includes a compound general chemical formula (III):
  • R- ⁇ is optionally substituted aryl or cycloalkyl and where R 2 and R 3 are independently H. -OH, -N3 or alkyl.
  • Alkyl refers to a straight or branched hydrocarbon chain group consisting solely of carbon and hydrogen atoms, containing no unsaturation and including, for example, from one to ten carbon atoms, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, and which is attached to the rest of the molecule by a single bond. Unless stated otherwise specifically in the specification, the alkyl group may be optionally substituted by one or more substituents as described herein. Unless stated otherwise specifically herein, it is understood that the substitution can occur on any carbon of the alkyl group.
  • Aryl may be used interchangeably with “aromatic group” or “aromatic ring” and refers to carbocyclic aryl groups, such as phenyl, naphthyl, etc. Unless stated otherwise specifically herein, the term “aryl” is meant to include aryl groups optionally substituted by one or more substituents as described herein. In some embodiments, the aryl groups may be heteroaryl groups. “Heteroaryl” refers to a single aromatic ring group containing one or more heteroatoms in the ring, for example N, O, S, including for example, 5-6 members.
  • heteroaryl groups examples include furan, thiophene, pyrrole, oxazole, thiazole, imidazole, pyrazole, isoxazole, isothiazole, 1 ,2,3-oxadiazole, 1 ,2,3-triazole, 1 ,2,4-triazole, 1 ,3,4- thiadiazole, tetrazole, pyridine, pyridazine, pyrimidine, pyrazine, 1 ,3,5-triazine, imidazole.
  • heteroaryl is meant to include heteroaryl groups optionally substituted by one or more substituents as described herein.
  • the aromatic group may be pyridine, thiophene, or benzene.
  • Cycloalkyl refers to a stable monovalent monocyclic, bicyclic or tricyclic hydrocarbon group consisting solely of carbon and hydrogen atoms, having for example from 3 to 15 carbon atoms, and which is saturated and attached to the rest of the molecule by a single bond. Unless otherwise stated specifically herein, the term “cycloalkyl” is meant to include cycloalkyl groups which are optionally substituted as described herein.
  • Optional or “optionally” means that the subsequently described event of circumstances may or may not occur, and that the description includes instances where said event or circumstance occurs one or more times and instances in which it does not. Certain groups may be optionally substituted as described herein. Suitable substituents include: H, alkyl (C-i-6), alkenyl (C 2 -6), or alkynyl (C 2 -6) each of which may optionally contain one or more heteroatoms selected from O, S, P, N, F, CI, Br, I, or B. III includes one or more
  • the compounds of the present invention may contain one or more asymmetric centers and can thus occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. Additional asymmetric centers may be present depending upon the nature of the various substituents on the molecule. Each such asymmetric center will independently produce two optical isomers and it is intended that all of the possible optical isomers and diastereomers in mixtures and as pure or partially purified compounds are included within the ambit of this invention. Any formulas, structures or names of compounds described in this specification that do not specify a particular stereochemistry are meant to encompass any and all existing isomers as described above and mixtures thereof in any proportion. When stereochemistry is specified, the invention is meant to encompass that particular isomer in pure form or as part of a mixture with other isomers in any proportion.
  • a compound refers to one or more of such compounds.
  • the term “compound” or “compounds” refers to the compounds discussed herein and includes precursors and derivatives of the compounds, including acyl-protected derivatives, and pharmaceutically acceptable salts of the compounds, precursors, and derivatives.
  • the invention also includes prodrugs of the compounds, pharmaceutical compositions including the compounds and a pharmaceutically acceptable carrier, and/or pharmaceutical compositions including prodrugs of the compounds and a pharmaceutically acceptable carrier.
  • many of the compounds may be obtained from commercial sources, such as Maybridge, Cornwall, U.K.
  • the present disclosure provides methods of treating a disorder or condition resulting in cells with supernumerary centrosomes, such as cancer.
  • treating includes treatment, prevention, and amelioration.
  • the methods are effected by administering a compound as described herein to a subject in need thereof, or by contacting a cell or a sample with a compound as described herein, for example, a pharmaceutical composition comprising a therapeutically effective amount of the compound according to Formula (I). More particularly, a compound according to Formula (I) is useful in the treatment of a disorder or condition resulting in cells with supernumerary centrosomes, such as cancer.
  • cancer any unwanted and abnormal growth of any cell type or tissue.
  • a cancer cell has been released from its normal cell division control, i.e., a cell whose growth is not regulated by the ordinary biochemical and physical influences in the cellular environment.
  • a cancer cell proliferates to form a clone of cells which are malignant.
  • the term cancer includes cell growths that are technically benign but which carry the risk of becoming malignant. This term also includes any transformed and immortalized cells cancers, carcinomas, neoplasms, neoplasias, or tumors. In some embodiments, the term cancer refers to solid tumors.
  • Cancers include, for example and without limitation, fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,
  • angiosarcoma endotheliosarcoma, lymphangiosarcoma, lymphangioandotheliosarcoma, synoviome, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer, esophageal cancer, colon carcinoma, rectal cancer, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, uterine cancer, cancer of the head and neck, skin cancer, brain cancer, squamous cell carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinome, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, myeloma, hepatoma, hepatocellular cancer, ductal cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, liver cancer, cervical cancer, testi
  • a subject may be a human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc.
  • the subject may be a clinical patient, a clinical trial volunteer, an experimental animal, etc.
  • the subject may be suspected of having or at risk for having a disorder or condition resulting in cells with supernumerary centrosomes, be diagnosed with a disorder or condition resulting in cells with supernumerary centrosomes, or be a control subject that is confirmed to not have a disorder or condition resulting in cells with supernumerary centrosomes. Diagnostic methods for disorder or condition resulting in cells with supernumerary centrosomes and the clinical delineation of such diagnoses are known to those of ordinary skill in the art.
  • a disorder or condition resulting in cells with supernumerary centrosomes is a cancer.
  • Compounds as described herein can be provided alone or in combination with other compounds (for example, nucleic acid molecules, small molecules, peptides, or peptide analogues), in the presence of a liposome, an adjuvant, or any pharmaceutically acceptable carrier, in a form suitable for administration to mammals, for example, humans, cattle, sheep, etc.
  • treatment with a compound according to the invention may be combined with more traditional and existing therapies for disorders or conditions resulting in cells with supernumerary centrosomes, such as cancer.
  • compounds as described herein may be provided in combination with for example mitotic inhibitors, such as paclitaxel, docotaxel, vinblastine, vincristine, vinorelbine, etc.
  • compounds as described herein may be provided in combination with chemotherapy or radiation therapy.
  • Compounds as described herein may be provided chronically or intermittently.
  • Chronic administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time.
  • Intermittent administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.
  • centrosomes such as cancer.
  • Any appropriate route of administration may be employed, for example, parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, intracisternal,
  • Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.
  • Methods well known in the art for making formulations are found in, for example, Remington: the Science & Practice of Pharmacy by Alfonso Gennaro, 20 th ed., Williams & Wilkins, (2000).
  • Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes.
  • polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes.
  • Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds.
  • Other potentially useful parenteral delivery systems for include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9- lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
  • the compounds are administered to an individual in an amount sufficient to: decrease bipolar spindle formation in a cell harbouring supernumerary centrosomes; decrease survival of a cell harbouring supernumerary centrosomes; or increase declustering of formed bipolar clusters of supernumerary centrosomes, increase multipolar spindle formation, increase mitotic arrest, and/or increase apoptosis of a cell harbouring supernumerary centrosomes.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier is suitable for parenteral administration.
  • the carrier can be suitable for intravenous, intraperitoneal, intramuscular, sublingual or oral administration.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art.
  • compositions of the invention are contemplated.
  • Supplementary active compounds can also be incorporated into the compositions.
  • pharmaceutically acceptable salt includes salts of compounds of
  • Formula I derived from the combination of a compound of this invention and an organic or inorganic acid or base.
  • the compounds of Formula I are useful in both non-ionized and salt form.
  • the use of a salt form amounts to use of a base form; both forms are within the scope of the invention.
  • an "effective amount" of a compound according to the invention includes a therapeutically effective amount or a prophylactically effective amount.
  • a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as decreasing bipolar spindle formation in a cell harbouring supernumerary centrosomes; decreasing survival of a cell harbouring supernumerary centrosomes; increasing declustering of formed bipolar clusters of supernumerary centrosomes, increasing multipolar spindle formation, increasing mitotic arrest, and/or increasing apoptosis of a cell harbouring supernumerary centrosomes.
  • a therapeutically effective amount of a compound may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects.
  • a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as decreasing bipolar spindle formation in a cell harbouring supernumerary centrosomes; decreasing survival of a cell harbouring supernumerary centrosomes; increasing declustering of formed bipolar clusters of supernumerary centrosomes, increasing multipolar spindle formation, increasing mitotic arrest, and/or increasing apoptosis of a cell harbouring supernumerary centrosomes.
  • a prophylactic dose is used in subjects prior to or at an earlier stage of disease, so that a prophylactically effective amount may be less than a therapeutically effective amount.
  • a preferred range for therapeutically or prophylactically effective amounts of a compound may be any integer from 0.1 nM-0.1 M, 0.1 nM-0.05M, 0.05 ⁇ -15 ⁇ or 0.01 ⁇ -10 ⁇ .
  • dosage values may vary with the severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. Dosage ranges set forth herein are exemplary only and do not limit the dosage ranges that may be selected by medical practitioners.
  • the amount of active compound(s) in the composition may vary according to factors such as the disease state, age, sex, and weight of the individual.
  • Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It may be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Toxicity of the compounds of the invention can be determined using standard techniques, for example, by testing in cell cultures or experimental animals and determining the therapeutic index, i.e., the ratio between the LD50 (the dose lethal to 50% of the population) and the LD100 (the dose lethal to 100% of the population). In some circumstances however, such as in severe disease conditions, it may be necessary to administer substantial excesses of the compositions.
  • a compound of Formula (I) may be used in screening assays for compounds which inhibit centrosomal clustering.
  • the ability of a test compound to inhibit centrosomal clustering from a model substrate may be measured using assays as described herein or known to one of ordinary skill in the art.
  • test compound is any naturally-occurring or artificially-derived chemical compound.
  • Test compounds may include, without limitation, peptides, polypeptides, synthesised organic molecules, naturally occurring organic molecules, and nucleic acid molecules.
  • a test compound can "compete" with a known compound such as a compound of Formula (I) by, for example, interfering with centrosomal clustering or by interfering with any biological response induced by a compound of Formula (I).
  • a test compound can exhibit any value between 10% and 200%, or over 500%, modulation when compared to a compound of Formula (I) or other reference compound.
  • a test compound may exhibit at least any positive or negative integer from 10% to 200% modulation, or at least any positive or negative integer from 30% to 150% modulation, or at least any positive or negative integer from 60% to 100%
  • a compound that is a negative modulator will in general decrease modulation relative to a known compound, while a compound that is a positive modulator will in general increase modulation relative to a known compound.
  • test compounds are identified from large libraries of both natural products or synthetic (or semi-synthetic) extracts or chemical libraries according to methods known in the art.
  • test extracts or compounds are not critical to the method(s) of the invention. Accordingly, virtually any number of chemical extracts or compounds can be screened using the exemplary methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds.
  • Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds.
  • Synthetic compound libraries are commercially available.
  • libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK),
  • heterogeneous extracts are known in the art. If desired, compounds shown to be useful agents for treatment are chemically modified according to methods known in the art.
  • one or more of the compounds are useful in the development of cell lines or animal models for studying diseases or disorders related to centrosomal clustering, including cancer.
  • Cells and tissues may be derived from subjects having such disorders.
  • Cell lines used as models for cancer may include commercially available cells from, for example, the American Type Culture Collection (ATCC), Manassus, VA, USA.
  • ATCC American Type Culture Collection
  • VA Manassus
  • suitable cell lines include those in which a substantial percentage of cells harbor
  • a suitable cell line is one in which between about 10% and about 100%, for example, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% harbor supernumerary centrosomes.
  • a suitable cell line is one in which between about 10% and about 100%, for example, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the cells harbor declustered centrosomes.
  • a suitable cell line is one that may be suitable for developing a "high content screen," as described herein or known to those of ordinary skill in the art, to for example increase the number of cells in mitosis with declustered centrosomes leading to multipolar spindles and/or to identify compounds that inhibit centrosome clustering or induce centrosome declustering.
  • a suitable cell line is the BT549 human breast cancer cell line.
  • Animal models of cancer include, for example, transgenic rodents (e.g. mice, rats) bearing gain of function proto-oncogenes (e.g. Myc, Src) and/or loss of function of tumour suppressor proteins (e.g. p53, Rb) or rodents that have been exposed to radiation or chemical mutagens that induce DNA changes that facilitate neoplastic transformation.
  • transgenic rodents e.g. mice, rats
  • gain of function proto-oncogenes e.g. Myc, Src
  • tumour suppressor proteins e.g. p53, Rb
  • Many animal models are commercially available, for example, from The Jackson Laboratory, ME, USA. These animal models may be used as source cells or tissue for the assays of the invention.
  • Many animal models eliciting reproducible results are accepted as being capable of extrapolation to human cancers.
  • Such animal models include: xenograft models or various chemically induced tumors in various rodent models e.g., Meth A, CMS4, CMS5, CMS21 , and WEHI-164 are chemically induced fibrosarcomas of BALB/c female mice which provide a highly controllable model system for studying the anti-tumor activities of various agents (DeLeo et al., J. Ex. Med. 146, 720 1977) (Palladino et al., J. Immunol. 138, 4023-4032 1987); the Lewis lung (3LL) carcinoma in mice (Zupi et al., Br. J. Cancer 41 ; suppl. 4, 309 1980) Zacharski, Haemostasis 16, 300-320 1986); feline oral squamous cell carcinoma (SCC).
  • rodent models e.g., Meth A, CMS4, CMS5, CMS21 , and WEHI-164 are chemically induced fibrosarcomas of BALB/c female mice which provide
  • the BT549 human breast cancer cells in which a high percentage of cells harbor supernumerary centrosomes (-55-60%) and which are capable of clustering the centrosomes during mitosis, leading to about 15-30 % of the cells having declustered centrosomes, were selected for use in a "high content screen" to identify compounds that could inhibit centrosome clustering or induce centrosome declustering.
  • the high content screen was designed to identify compounds that would increase the number of cells in mitosis with declustered centrosomes leading to multipolar spindles.
  • the cells were stained with an antibody against TG-3, a mitotic cell marker, to identify cells in mitosis. Centrosomes were detected by staining the cells with an antibody against TG-3, a mitotic cell marker, to identify cells in mitosis. Centrosomes were detected by staining the cells with an antibody against TG-3, a mitotic cell marker, to identify cells in mitosis. Centrosomes were detected by staining
  • the high content screen was carried out using a Cellomics
  • ArrayScan VTI system The screen was carried out in a 96-well format and the read out was the number of pericentrin positive dots within mitotic cells. More specifically, BT-549 breast cancer cells were cultured in 96-well plates overnight. Test compounds were added using a pinning robot and the cells were incubated for 5 hours and then fixed with formaldehyde. The cells were labelled with TG-3 antibody (which recognizes phosphorylated nucleolin during mitosis), pericentrin antibody (which recognizes centrosomes), and Hoechst dye (which stains nuclei). Cells with declustered centrosomes were detected using the Cellomics ArrayScan VTI system.
  • TG-3 antibody which recognizes phosphorylated nucleolin during mitosis
  • pericentrin antibody which recognizes centrosomes
  • Hoechst dye which stains nuclei
  • Figure 1 shows the high-content screening strategy for the identification of candidate compounds that inhibit centrosome clustering or induce centrosome declustering and summarizes the results of testing of 1200 initial compounds.
  • the distribution of the cells with declustered centrosomes is shown in Figure 1d.
  • Positive "Hits" from the compound library were identified as those that increased the percentage of cells with more than two centrosomes by a value of 2.5x the standard deviation.
  • the high scoring compounds were then subjected to a secondary screen using the Cellomics Array Scan by testing the positive compounds in a dose-dependent manner.
  • Table 1 Summary of the high-content screen for centrosome declustering
  • Nigg E A. Origins and consequences of centrosome aberrations in human cancers. International Journal of Cancer. 2006;1 19(12):2717-2723.
  • Nigg EA Centrosome aberrations: cause or consequence of cancer progression? Nat Rev Cancer. 2002;2(11 ):815-825.
  • Chromosome Instability Occur Together in Pre-invasive Carcinomas. Cancer Res.
  • Singh P Rathinasamy K, Mohan R, Panda D. Microtubule assembly dynamics: an attractive target for anticancer drugs. IUBMB Life. 2008;60(6):368-375.

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Abstract

La présente invention concerne d'une manière générale des composés et des procédés permettant d'inhiber l'agrégation centrosomale dans une cellule hébergeant des centrosomes en surnombre, par exemple en administrant une quantité efficace d'un composé de la formule chimique générale (I) : Formule (I) où X est un alkyle éventuellement substitué, n est un nombre entier de 1 à 10 et R1 est un aryle ou un cycloalkyle éventuellement substitué, ou un sel pharmaceutiquement acceptable de celui-ci, à un sujet qui en a besoin.
PCT/CA2013/050244 2012-04-03 2013-03-25 Dérivés de 5-nitrofurane-2carboxamide permettant d'inhiber l'agrégation centrosomale WO2013149336A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113304151A (zh) * 2021-04-15 2021-08-27 中山大学 一种硝基呋喃类小分子化合物在制备诱导铁死亡和/或减缓胃癌化疗耐药药物中的应用
US11911364B2 (en) 2018-09-25 2024-02-27 Pepticom Ltd. Positive allosteric modulators of GABAA receptor

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005007625A2 (fr) * 2003-07-14 2005-01-27 The University Of Tennessee Research Foundation Amides heterocycliques a activite anti-tuberculinique
US20060106051A1 (en) * 2004-11-12 2006-05-18 Alaric Dyckman Imidazo-fused oxazolo[4,5-b]pyridine and imidazo-fused thiazolo[4,5-b]pyridine based tricyclic compounds and pharmaceutical compositions comprising same

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005007625A2 (fr) * 2003-07-14 2005-01-27 The University Of Tennessee Research Foundation Amides heterocycliques a activite anti-tuberculinique
US20060106051A1 (en) * 2004-11-12 2006-05-18 Alaric Dyckman Imidazo-fused oxazolo[4,5-b]pyridine and imidazo-fused thiazolo[4,5-b]pyridine based tricyclic compounds and pharmaceutical compositions comprising same

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11911364B2 (en) 2018-09-25 2024-02-27 Pepticom Ltd. Positive allosteric modulators of GABAA receptor
CN113304151A (zh) * 2021-04-15 2021-08-27 中山大学 一种硝基呋喃类小分子化合物在制备诱导铁死亡和/或减缓胃癌化疗耐药药物中的应用
CN113304151B (zh) * 2021-04-15 2022-05-03 中山大学 一种硝基呋喃类小分子化合物在制备诱导铁死亡和/或减缓胃癌化疗耐药药物中的应用

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