CENTROSOMAL CLUSTERING

FIELD

[0001] 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.

BACKGROUND

[0002] Cell division includes two processes, namely mitosis and cytokinesis. 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.

[0003] Centrosomes are the principal microtubule organizing centers in animal cells.

Since initial observations of extra centrosomes in cancer cells (Boveri 1902, 1914), 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).

[0004] Commonly observed defects can be broadly classed into two subgroups; structural and numeric. Structural defects, referring to a change in size of centrosomes, are observed in numerous tumours (Pihan et al., 2003). Numeric defects indicate there are often more than two centrosomes present in cancerous cells (Nigg, 2006), a state referred to as having supernumerary centrosomes.

[0005] To circumvent the detrimental effects of multiple centrosomes, such as multipolar spindles, mitotic arrest and cell death, many cancer cells develop mechanisms to cluster multiple centrosomes to form bipolar spindles and resulting in bipolar mitosis and cell survival (Quintyne et al., 2005; Rebacz et al., 2007; Kwon et al., 2008). ILK, a protein that localizes to centrosomes in addition to focal adhesions (Fielding et al., 2008; Dobreva et al., 2008), has been implicated in centrosome clustering in cancer cells (Fielding et al., 2011 ).

[0006] PCT application WO/2011/061 194, entitled Inhibitors of centrosomal clustering", discloses potential targets implicated in centrosomal clustering. RNA

interference screens in Drosophila S2 cells (Kwon et al., 2008) and in oral squamous cell carcinoma cells (Leber et al., 2010), have resulted in the identification of additional potential targets implicated in centrosome clustering in cancer cells.

[0007] A chemical screen utilizing a natural compound library (Rebacz, 2007) identified 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.).

[0008] United States Patent Publications US 2005/0026968 and US 2005/0222408 and PCT publication WO 2005/007625 (University of Tennessee) all entitled "Heterocyclic amides with antituberculosis activity" propose N-Benzyl-5-nitro-2-furamide and related compounds for the treatment of bacterial infections and tuberculosis. US Patent 2,989,530, "N-(5-Nitro-2-Furoyl)-Morpholine", also claims the compound N-Benzyl-5-nitro-2-furamide and identifies in vivo antibacterial and anti trichomonas activity. SUMMARY

[0009] The present disclosure relates in part to compounds and methods for inhibiting centrosomal clustering in a cell harboring supernumerary centrosomes.

[0010] In one aspect, 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):

Formula (I)

[0011] 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.

[0012] In alternative aspects, 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.

[0014] In some embodiments, the cell harbouring supernumerary centrosomes may be a cancer cell. In some embodiments, the cancer cell may be a human cell. In some embodiments, the cancer cell may be a breast, lung, or colon cancer cell. In some embodiments, the cancer cell may be an aggressive cancer cell.

[0015] In some embodiments, the subject may be a human.

[0016] In alternative aspects, the present disclosure provides a pharmaceutical composition comprising an effective amount of a compound of the general chemical formula (I):

Formula (I)

[0017] 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, and a pharmaceutically acceptable diluent or carrier.

[0018] In alternative aspects, 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):

Formula (I)

[0019] 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 said subject.

[0020] In alternative aspects, the present disclosure provides the use of a compound of the general chemical formula (I):

Formula (I)

[0021] 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 for treating a cancer in a subject in need thereof.

[0022] In some embodiments, X may be alkyl, n may be 1 and R-i may be aryl. In some embodiments, R-i may be a six-membered or five-mem bered aryl group. In some embodiments, R-i may be selected from the group consisting of benzene, pyridine and thiophene. In some embodiments, 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures.

[0024] FIGURES 1A-E show a summary of the screening strategy for identifying candidate compounds, (a-c) Analysis of BT-549 cells for declustered centrosomes using a Cellomics Array Scan VTI imaging platform. (a,b) Identification of nuclei/cells and mitotic cells. Cells were labeled with Hoechst (a) or antibodies against TG-3, a marker of mitotic cells (b). The nuclei of TG-3-positive (mitotic) cells are outlined in black. TG-3-negative (nonmitotic) cells are outlined in white, (c) Identification of centrosomes in mitotic cells.

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. Four different concentrations {27, 9, 2.7 and 0.9 μΜ) were tested for each primary "hit". The frequency of cells with declustered centrosomes and mitotic indices are shown for primary hits, A-H, and positive and negative controls. Arrowheads indicate hits in the secondary screen. Compound F was a false positive.

[0025] FIGURE 2 shows the effects of three identified small molecules on centrosome declustering in vitro, (a) Representative immunofluorescence images of BT549 cells showing the centrosome declustering properties of the positive compounds identified in the high-content screen. Cells were treated with the various compounds for 5 hours at a concentration of 10 μΜ , and then immunolabelled for pericentrin and tubul in. Nuclei were labeled with Hoechst. (b) Quantitation of the frequency of cells with declustered centrosomes after treatment as described in (a). Centrosome declustering was analyzed using pericentrin labeling, and tubulin labeling was also used to ensure multi-polar spindle formation. At least 30 mitotic cells were measured for each compound tested.

[0026] 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 μΜ 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 μΜ of the indicated compounds. Average values of all tests are shown. Bars are standard deviation.

[0027] FIGURE 4 shows that BR00067 induces centrosome declustering in BT-549 breast cancer cells, but not in normal mammary epithelial cells, (a) An MTT assay was carried out with finer titration steps between 1 and 10 μΜ. ICso values for cancer cells were between 2 and 8 μΜ, while that for primary cells was above 10 μΜ. (b) Centrosome arrangement and spindle multipolarity were examined. As little as 5 μΜ of BR00067 increased centrosome declustering to 70 % in BT-549 breast cancer cells, whereas even at 8 μΜ, declustering was not induced in primary mammary epithelial cells {average of three independent primary cell samples). There was a 6-fold difference in the IC50 for this compound between the BT549 cells and normal mammary epithelial cells.

[0028] 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.

DETAILED DESCRIPTION

[0029] 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.

[0030] 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. In some embodiments, 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. In some embodiments, the cell may be a neoplastic or cancer cell.

[0031] By "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. Accordingly, 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.

[0032] The present disclosure provides a compound of the general chemical formula

(I):

Formula (I) where X is optionally substituted alkyl, n is an integer from 1 to 10 and R-i is optionally substituted aryl or cycloalkyl.

[0033] In some embodiments, a compound of Formula I includes a compound of the general chemical formula (II):

Formula II where R-i is optionally substituted aryl or cycloalkyl.

[0034] In some embodiments, a compound of Formula I includes a compound general chemical formula (III):

Formula III where R-ι is optionally substituted aryl or cycloalkyl and where R ₂ and R ₃ are independently H. -OH, -N3 or alkyl.

[0035] "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.

[0036] "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.

Examples of heteroaryl groups 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. Unless stated otherwise specifically herein, the term "heteroaryl" is meant to include heteroaryl groups optionally substituted by one or more substituents as described herein. In some embodiments, the aromatic group may be pyridine, thiophene, or benzene.

[0037] "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.

[0038] "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

[0040] 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.

[0041] As used herein the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. For example, "a compound" refers to one or more of such compounds. Throughout this application, it is contemplated that 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. In some embodiments, 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.

[0042] In general, compounds described herein may be prepared by standard techniques known in the art, or by known processes analogous thereto. In some

embodiments, many of the compounds may be obtained from commercial sources, such as Maybridge, Cornwall, U.K.

[0043] Therapeutic Indications

[0044] The present disclosure provides methods of treating a disorder or condition resulting in cells with supernumerary centrosomes, such as cancer. The term "treating" as used herein includes treatment, prevention, and amelioration.

[0045] In general, 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.

[0046] By "cancer" is meant any unwanted and abnormal growth of any cell type or tissue. In general, 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. In general, 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. [0047] 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, testicular cancer, lung carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, neural cancer, glioma, astracytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangloblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, chronic myeloid leukemia, lymphoma, Burkitt's lymphoma or Kaposi's sarcoma.

[0048] As used herein, 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. In some embodiments, a disorder or condition resulting in cells with supernumerary centrosomes is a cancer. [0049] Pharmaceutical & Veterinary Compositions, Dosages, And Administration

[0050] 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. If desired, 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. Accordingly, in some embodiments, compounds as described herein may be provided in combination with for example mitotic inhibitors, such as paclitaxel, docotaxel, vinblastine, vincristine, vinorelbine, etc. In some embodiments, compounds as described herein may be provided in combination with chemotherapy or radiation therapy.

[0051] 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. The terms "administration,"

"administrable," or "administering" as used herein should be understood to mean providing a compound of the invention to the subject in need of treatment.

[0052] Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer the compounds to subjects suffering from or presymptomatic for a disorder or condition resulting in cells with supernumerary

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,

intraperitoneal, intranasal, aerosol, topical, or oral administration. 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. [0053] 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. 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. For therapeutic or prophylactic compositions, 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.

[0054] As used herein "pharmaceutically acceptable carrier" or "excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In one embodiment, the carrier is suitable for parenteral administration. Alternatively, 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. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions. [0055] The term "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. In practice, the use of a salt form amounts to use of a base form; both forms are within the scope of the invention.

[0056] 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. Typically, 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 ηΜ-15μΜ or 0.01 ηΜ-10μΜ. [0057] It is to be noted that 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.

[0058] In general, compounds of the invention should be used without causing substantial toxicity. 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.

[0059] In some embodiments, a pharmaceutical composition excludes a compound of Formula I in which X is unsubstituted alkyl, n = 1 , and R-i is a substituted benzene.

[0060] Other Uses and Assays

[0061] 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.

[0062] A "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).

[0063] Generally, 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. For example, 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%

modulation, or any positive or negative integer over 100% modulation. 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.

[0064] In general, 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. Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is 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. Alternatively, 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),

Harbor Branch Oceanographic Institute (Ft. Pierce, FL, USA), and PharmaMar, MA, USA. In addition, natural and synthetically produced libraries are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods. Furthermore, if desired, any library or compound is readily modified using standard chemical, physical, or biochemical methods.

[0065] When a crude extract is found to modulate inhibition of centrosomal clustering, or any biological response induced by a compound of Formula (I), further fractionation of the positive lead extract is necessary to isolate chemical constituents responsible for the observed effect. Thus, the goal of the extraction, fractionation, and purification process is the careful characterization and identification of a chemical entity within the crude extract having centrosomal clustering inhibitory activities. The same assays described herein for the detection of activities in mixtures of compounds can be used to purify the active component and to test derivatives thereof. Methods of fractionation and purification of such

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.

Compounds identified as being of therapeutic, prophylactic, diagnostic, or other value may be subsequently analyzed using a suitable animal model, as described herein on known in the art.

[0066] In some embodiments, 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.

[0067] 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. In some embodiments, suitable cell lines include those in which a substantial percentage of cells harbor

supernumerary centrosomes and which are capable of clustering the centrosomes during mitosis, leading cells having declustered centrosomes. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, a suitable cell line is the BT549 human breast cancer cell line.

[0068] 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. Many such 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).

[0069] EXAMPLES

[0070] 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. [0071] 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

antipericentrin antibody. 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. Cells with clustered centrosomes and cells with two centrosomes, were identified as two dots and cells with declustered centrosomes as more than two dots. The screen was carried out with a custom library from the Maybridge Screening Collection of small molecules (www.maybridge.com). The test compounds were tested at 10μΜ and 17μ Μ, respectively, in DMSO, with DMSO as negative control .

[0072] 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.

[0073] 7360 compounds were screened and 14 positive Hits identified, as shown in

Table 1. Table 1. Summary of the high-content screen for centrosome declustering

[0074] The positive compounds from the secondary screen were then further tested for their centrosome declustering properties and effects on the mitotic spindle organization by standard immunofluorescence microscopy. As shown in Figure 1 e, 4 out of 8 compounds tested resulted in a dose-dependent increase in the percentage of cells with declustered centrosomes and mitotic index. Figure 2 indicates that 3 compounds tested were able to induce centrosome declustering and the formation of multipolar spindles.

[0075] To determine whether the compounds that are able to induce centrosome declustering and the formation of multipolar spindles inhibited cell growth and whether they inhibited selective cell growth of cancer versus non-tumorigenic and normal cells, we carried out dose-dependent analysis of cell growth utilizing an MTT assay on several human breast and non-breast cancer cell lines, and compared them to a non-tumorigenic human mammary epithelial cell-lines (MCF-10A) as well as primary mammary epithelial cells derived from reduction mammoplasty. Figure 3 shows that compounds BR00067, BR00068, BR00072 demonstrated a reproducible differential growth inhibition of the cancer cells relative to the normal cells. All of these compounds (BR00067, BR00068 and BR00072) induced dramatic centrosome declustering (Figure 3). A representative narrow dose response in the MTT assay as well as effects on the mitotic spindle and centrosome clustering (Figure 4) and apoptosis (Figure 5) are shown for compound BR00067.

[0076] The above-described embodiments are intended to be examples only.

Alterations, modifications and variations can be effected to the particular embodiments by those of skill in the art without departing from the scope, which is defined solely by the claims appended hereto.

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