WO2013162469A1

WO2013162469A1 - Tubulin inhibitors

Info

Publication number: WO2013162469A1
Application number: PCT/SG2013/000155
Authority: WO
Inventors: Ho Sup Yoon; Xuewei Liu; Venkata Krishna Harikishore AMARAVADHI
Original assignee: Nanyang Technological University
Priority date: 2012-04-23
Filing date: 2013-04-19
Publication date: 2013-10-31

Abstract

The present invention relates to a compound of Formula (I) for use as a medicament, wherein: m is 0, 1, 2, 3, 4, or 5; R₁ and R₂ together form a five-membered, six-membered, or seven-membered ring, wherein R₁ and R₂ together as a group is -(CH₂)₃-, -(CH₂)₄-, or -(CH₂)₅-; R₃ at each occurrence is independently selected from the group consisting of H, halogen, hydroxyl, alkoxy, and a substituted or unsubstituted C1-C5 alkyl; and R₄ is H, halogen, or a substituted or unsubstituted C1-C5 alkyl.

Description

TUBULIN INHIBITORS

Cross-Reference to Related Application

[0001] This application claims the benefit of priority of United States of America Provisional Patent Application No. 61/637,044, filed April 23, 2012, the contents of which being hereby incorporated by reference in its entirety for all purposes.

Technical Field

[0002] The invention relates to anticancer treatment and particularly relates to the treatment or prevention of various types of cancer, including cervical cancer, leukemia, stomach cancer, breast cancer, lung cancer, colon cancer, and neuroblastoma, by use of tubulin inhibitors comprising indazole-hydrazide compounds as anticancer agents.

Background

[0003] Microtubules, which are long, filamentous, tube-shaped polymers, mediate important roles in cellular signaling, transport of cargos, establishment of cell polarity, maintenance of cell shape, cellular migration and cell division (Jordan and Wilson 2004; Dumontet and Jordan 2010).

Composed of - and /3-tubulin heterodimers bound in a head-to-tail manner, microtubules are not simple equilibrium polymers; instead they are highly dynamic structures and the rapid assembly and disassembly dynamics are crucial in large part for their cellular functions. Not surprisingly, microtubule polymerization is subject to tight spatial and temporal regulation and this is achieved at several levels including (1) transcription of different tubulin isotypes having different functions; (2) by regulating ΐβ - tubulin ratios and heterodimer folding; (3) through various post-translational modifications of tubulin, that in turn, alters microtubule localization and/or its interaction with signaling pathways; and (4) via interaction with microtubule-associated proteins (MAPs) like dynein and kinesin motor proteins, stathmin, TOG, EB 1 , dynactin 1 , RAC 1 , etc (Lewis, Tian et al. 1997; Luduena 1998; Nogales 2000). Paradoxically, the same dynamic nature of microtubules also makes them exquisitely sensitive to inhibitors. By disrupting the finely tuned behavior of microtubules, tubulin-binding drugs interfere with the process of cell division and have proved to be highly effective in cancer patients (Kavallaris 2010).

[0004] Most of the microtubule-binding drugs identified so far have been isolated from either plants or marine organisms during large-scale screens of natural products. Microtubule-targeted anti-mitotic drugs are usually classified into two groups: (a) microtubule destabilizing agents that inhibit microtubule polymerization and includes the vinca alkaloids, colchicine, halichondrins, combrestatins; and (b) microtubule stabilizing agents like paclitaxel, docetaxel, epothilones, discodermolide. Though the taxanes and vinca alkaloids are still administered for a wide range of cancers and are often integrated into combination chemotherapy regimens (Dimitroulis and

Stathopoulos 2005; Pajk, Cufer et al. 2008), the current suite of tubulin-binding drugs have several drawbacks. When compared to other class of anticancer drugs, these microtubule-binding drugs are structurally complex and chemically diverse. Furthermore, the active drugs occur in only minute amounts in nature and the scarcity of their natural sources has severely hampered the clinical development of these compounds. Although this issue was partially addressed by the development of partial or total synthesis methods (Nicolaou, Yang et al. 1994) or via metabolic engineering of pathway intermediates (Ajikumar, Xiao et al. 2010), the problem still persists where development of new microtubule-binding compounds are concerned. Another drawback associated with the use of these compounds is drug resistance caused by mutations and/or expression of different tubulin isotypes. Drug resistance may also stem from the over-expression of drug-efflux pumps, including the multidrug resistance transporter P-glycoprotein (Pgp) or multidrug-resistance associated protein (MRP) (Liou, Hsu et al. 2007). Patients being administered with microtubule-binding agents tend to suffer from peripheral axonal neuropathy that limits the tolerable dose (Argyriou, Koltzenburg et al. 2008).

[0005] Despite these limitations, several anti-mitotic drugs with diverse binding sites on tubulin are in various stages of clinical development. The armamentarium of microtubule-targeted agents with improved pharmacological profiles, minimal neurological toxicity and broad spectrum efficacy, continues to grow (Backer, Nickel et al. 2001; Kuo, Hsieh et 5 al. 2004; Aneja, Lopus et al. 2006). Ideally, such leads should also bypass drug resistance and be amenable to facile chemical synthesis approaches. [0006] To this end, in the last decade, the chemistry of indazoles has drawn considerable attention due to the wide spectrum of biological activity mediated by this class of compounds (Cerecetto, Gerpe et al. 2005). Indazole derivatives have been extensively used for neuroprotection in animal models of stroke and Parkinson's disease (Zhang, Dawson et al. 2000), for treatment of osteoporosis (Batt,

Petraitis et al. 2000), for male contraception (Cheng, Silvestrini et al. 2001; Grima, Silvestrini et al.

2001) and also as anti-inflammatory, antibacterial (Boehm, Boehringer et al. 2000; Tanitame, Oyamada et al. 2004) and antiparasitic (Aran, Ochoa et al. 2005) agents, among others. Additionally, several novel indazole derivatives have shown potent anticancer activities. For example, Keppler et al. showed that ruthenium (Ill)-indazole complex, by virtue of poisoning the activity of topoisomerase II, exhibits significant anticancer activity against several different cancer types like Walker 256 carcinosarcoma,

MAC 15A colon tumor, melanoma and solid sarcoma (Keppler, Henn et al. 1989; Keppler, Berger et al. 1990). Likewise, Daidone et al. described indazole derivatives of triazine that exhibit micromolar potencies against leukemia and other tumor cell lines (Daidone, Raffa et al. 2004).

[0007] In sum, the concept of using anti-tubulin compounds in anticancer treatment is not new.

Besides inhibiting tumor cell proliferation, several of these tubulin-binding agents (TBA) can rapidly disrupt the tumor vasculature (Tozer, Kanthou et al. 2005) and have been the mainstay of chemotherapeutic agents. However, emergence of chemoresistance and their toxic neurological side effects have hampered the clinical development of these molecules. Owing to the importance of microtubules in cell cycle progression, microtubules continue to remain as attractive targets for anticancer drugs.

[0008] Thus, there remains a need for alternative microtubule-binding agents or drugs that overcome the known problems.

Summary

[0009] The present invention is based on the inventors' finding that present indazole-hydrazide compounds can act as anti-cancer agents and thus have use in the treatment or prevention of cancer, in particular cervical cancer, leukemia, stomach cancer, breast cancer, lung cancer, colon cancer, and neuroblastoma. As it has been surprisingly found that present indazole-hydrazide compounds selectively targets cancer cells through microtubule destabilization and induction of apoptosis, present indazole-hydrazide compounds provide a new approach for treating cancers that do not respond to DNA-based cancer treatments.

[0010] Thus, in a first aspect, the present disclosure relates to a method for treating or preventing cancer in a subject, comprising administering to the subject in need thereof a

-therapeutically effective amount of a compound of Formula (I)

wherein:

m is 0, 1, 2, 3, 4, or 5;

Ri and R₂ together form a five-membered, six-membered, or seven-member ed ring, wherein Ri and R₂ together as a group is -(CH₂)₃-, -(CH₂)₄-, or -(CH₂)₅-;

R₃ at each occurrence is independently selected from the group consisting of H, halogen, hydroxyl, alkoxy, and a substituted or unsubstituted C1-C5 alkyl; and

R₄ is H, halogen, or a substituted or unsubstituted C1-C5 alkyl.

[0011] In a second aspect, the present disclosure relates to a compound of Formula (I) for use as a medicament. In various embodiments, the compounds of Formula (I) are useful in treating or preventing cancer in a subject.

[0012] In a third aspect, the present disclosure relates to a method for inducing apoptosis in a cell, comprising contacting said cell with an effective amount of a compound of Formula (I).

Brief Description of the Drawings

[0013] In the drawings, like reference characters generally refer to the same parts throughout the different views. The drawings are not necessarily drawn to scale, emphasis instead generally being placed upon illustrating the principles of various embodiments. In the following description, various embodiments of the invention are described with reference to the following drawings.

[0014] Fig. 1 shows SRF selectively inhibits proliferation of cancer cell types. Normal cells like skin fibroblasts CCL-116 was treated with 10 μΜ of SRF for 24 hr and cell viability was measured by MTT assay. Compared to control (untreated) cells, CCL-1 16 showed > 80% survival when exposed to SRF while only 60% cells survived with taxol under identical conditions. Results shown are mean ± SD of three independent experiments.

[0015] Fig. 2 shows SRF does not phosphorylate Bcl-2 family members other than Bcl-2 and Bad. Extracts of HeLa cells treated with 10 μΜ of SRF for the indicated times were probed with antibodies against BC1-X_L, Bak and Bad. Only a single band corresponding to the full-length protein was visible in all the blots.

[0016] Fig. 3 shows the inhibition of JNK kinase can protect cells against SRF-induced cytotoxicity: (A) SP600125 pre-treatment can prevent SRF-induced JNK phosphorylation and activation without altering protein levels. Blots were probed with phospho-JNK and JNK specific antibodies; (B) Caspase-3 activity in SRF-treated cell lysate was determined using the fluorogenic substrate Ac-DEVD-pNA. SRF induces a 3 -fold increase in enzymatic activity that is decreased in presence of specific JNK inhibitor, SP600125; (C) Cells treated with p38 (SB203580) and ERKl/2 (PD98059) inhibitors are not able to overcome SRF-induced cell cycle blockade at the G₂/M phase.

Cells were stained with PI and the DNA content was analyzed by flow cytometry.

Description

[0017] The following detailed description refers to the accompanying drawings that show, by way of illustration, specific details and embodiments in which the invention may be practised. These embodiments are described in sufficient detail to enable those skilled in the art to practise the invention. Other embodiments may be utilized and structural or other changes may be made without departing from the scope of the invention. The various embodiments are not necessarily mutually exclusive, as some embodiments can be combined with one or more other embodiments to form new embodiments.

[0018] Microtubules, which are dynamic filamentous cytoskeletal proteins composed of tubulin, are a highly validated target in cancer therapy. Even though anti-tubulin agents have been integrated into combination chemotherapy regimens for decades, issues like toxicity, specificity and chemoresistance have plagued their clinical development thereby necessitating the search for novel microtubule targeting drugs. The inventors executed an in silico screening of an annotated chemical libraries and identified certain indazole-hydrazide compounds as novel cell permeable, tubulin-binding small molecules. These compounds inhibit assembly of purified tubulin by binding to the colchicine-binding site. Western blot and flow cytometric analyses demonstrate present indazole-hydrazide compounds-mediated microtubule depolymerization results in cell cycle arrest in the G2/M phase and eventual cell death by apoptosis. Cell death was invariably preceded by loss of mitochondrial membrane potential, JNK - mediated phosphorylation of the BH3 family members - Bcl-2 and Bad, and activation of caspase-3 cascade. These findings suggest that present indazole-hydrazide compounds-mediated apoptotic pathway depends on mitochondria and caspases. Notably, present indazole-hydrazide compounds selectively target cancer cells and are effective against various resistant and non-resistant cancer cell types by virtue of their ability to bypass the multidrug resistance transporter P-glycoprotein (Pgp). Taken together, present results suggest that present indazole-hydrazide compounds have potential as a chemo therapeutic agent for cancer treatment and provides an alternate scaffold for the development of improved anti-cancer agents.

[0019] Thus, in a first aspect, the present disclosure relates to a method for treating or preventing cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound of Formula (I)

wherein: m is 0, 1, 2, 3, 4, or 5;

Ri and R₂ together form a five-membered, six-membered, or seven-membered ring, wherein

and R₂ together as a group is -(CH₂)₃-, -(CH₂)₄-, or -(CH₂)₅-;

R₄ is H, halogen, or a substituted or unsubstituted C1-C5 alkyl.

[0020] In various embodiments, the cancer is selected from the group consisting of cervical cancer, leukemia, stomach cancer, breast cancer, lung cancer, colon cancer, and neuroblastoma.

[0021] The term "subject", for the purposes of the present disclosure, includes humans and other animals, particularly mammals. Thus, the methods are applicable to both human therapy and veterinary applications. In certain embodiments, the subject is a mammal, and in a preferred embodiment, the subject is a human.

[0022] The terms "treatment" and "treat" and synonyms thereof, refer to both therapeutic treatment and prophylactic or preventative measures, wherein the objective is to prevent or slow down (lessen) growth of tumor.

[0023] The term "therapeutically effective amount" as used herein refers to the amount of the compound of Formula (I) required to treat or prevent cancer in a given subject either alone or in combination with one or more other active agents. Effective amounts vary, as recognized by those skilled in the art, depending, inter alia, on route of administration, excipient usage and co-usage with other active agents as well as age, weight other diseases and subject specific side effects. The therapeutically effective amount for a given situation can be readily determined by routine experimentation and is within the skill and judgment of the ordinary clinician.

[0024] The term "administer" or "administration", as used herein, refers to the application of the respective compound with or without further additives, such as pharmaceutical carriers, to a subject in need thereof. The routes of administration of the compound either alone or in combination with other substances may be by any medically acceptable means, including, but not limited to oral, subcutaneous, intramuscular, intravenous, intra-arterial, sublingual, buccal, rectal, peritoneal, nasal, transdermal, transmucosal, vaginal, transurethral, iontophoretic, and by inhalation. The compounds may be administered enterally (e.g., orally or rectally) or parenterally

(e.g., by subcutaneous, intravenous, intramuscular, intrastemal, or peritoneal injection or infusion techniques) in dosage formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles as desired. The compounds may be delivered locally, as to portions of the gastrointestinal tract. Additional methods of administration are known in the art.

[0025] The compound either alone or in combination with other substances can be administered in solid form or in liquid form. The compound can be administered in the form of tablets, pills, powder mixtures, capsules, granules, injectables, creams, solutions, suppositories, enemas, colonic irrigations, emulsions, dispersions, food premixes, animal feed, and in other suitable forms.

[0026] The compound of Formula (I) either alone or in combination with other substances may also be formulated as sustained release or delayed release formulations as well as injectable preparations.

[0027] Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water. Such formulations may also comprise adjuvants, such as wetting agents, emulsifying and suspending agents, cyclodextrins, and sweetening, flavoring, and perfuming agents.

[0028] The frequency and duration of administration of the compound of Formula (I) either alone or in combination with other substances will depend on the condition of the individual, and the like. The compound of Formula (I) either alone or in combination with other substances may be administered to the individual one or more times, for example, 2, 3, 4, 5, 10, 15, 20, 50, 75, 100, or more times. The formulation may be administered to the individual, for example, once a day, two times a day, three times a day, or more than three times a day. The formulation may also be administered to the individual, for example, less than once a day, for example, every other day, every third day, every week, or less frequently. The formulation may be administered over a period of days, weeks, months, years, or chronically, such as life-long administration. The determination of dose and frequency of administration lie within the capability of the responsible physician.

[0029] These different modes of administration may be, inter alia, useful for reduction of side effect and increasing bioavailability and/or patient compliance.

[0030] In the present context, the term "optionally substituted" or "substituted or

unsubstituted" refers to a group in which none, one, or more than one of the hydrogen atoms have been replaced with one or more unsubstituted groups such as, but are not limited to, alkyl such as methyl or ethyl, heteroalkyl, haloalkyl, heterohaloalkyl, cycloalkyl, aryl, arylalkyl, heteroaryl, non- aromatic heterocycle, hydroxy, alkoxy such as methoxy or ethoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo such as CI or Br, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl, O- thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O- carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, trihalomethanesulfonyl, and amino, including mono- and di-substituted amino groups. In embodiments in which two or more hydrogen atoms have been substituted, the substituent groups may be linked to form a ring.

[0031) In the present context, the term "aliphatic", alone or in combination, refers to a straight chain or branched chain hydrocarbon comprising at least one carbon atom. Aliphatics include alkyls, alkenyls, and alkynyls. Aliphatics include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert.-butyl, pentyl, hexyl, ethenyl, propenyl, butenyl, ethynyl, butynyl, propynyl, and the like, each of which may be optionally substituted.

[0032] In the present context, the term "alkyl", alone or in combination, refers to a fully saturated aliphatic hydrocarbon. In certain embodiments, alkyls are optionally substituted. In certain embodiments, an alkyl comprises 1 to 5 carbon atoms, for example 1 to 4 carbon atoms, wherein (whenever it appears herein in any of the definitions given below) a numerical range, such as "1 to 5" or "C1-C5", refers to each integer in the given range, e.g. "C1-C5 alkyl" means that an alkyl group comprising only 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, or 5 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, and the like.

[0033] In the present context, the term "alkenyl", alone or in combination, refers to an aliphatic hydrocarbon having one or more carbon-carbon double-bonds, such as two or three carbon-carbon double-bonds. In certain embodiments, alkenyls are optionally substituted, i.e. substituted or unsubstituted. In certain embodiments, an alkenyl comprises 2 to 15 carbon atoms, for example 2 to 4 carbon atoms. "C2-C4 alkenyl" means that an alkenyl group comprising only 2 carbon atoms, 3 carbon atoms, or 4 carbon atoms. Examples of alkenyls include, but are not limited to, ethenyl, propenyl, butenyl, 1 ,4-butadienyl, pentenyl, hexenyl, 4-methylhex-l-enyl, 4-ethyl-2- methylhex-l-enyl and the like. [0034] In the present context, the term "alkynyl", alone or in combination, refers to an aliphatic hydrocarbon having one or more carbon-carbon triple-bonds, such as two or three carbon- carbon triple-bonds. In certain embodiments, alkynyls are optionally substituted, i.e. substituted or unsubstituted. In certain embodiments, an alkynyl comprises 2 to 15 carbon atoms, for example 2 to 4 carbon atoms. "C2-C4 alkynyl" means that an alkynyl group comprising only 2 carbon atoms, 3 carbon atoms, or 4 carbon atoms. Examples of alkynyls include, but are not limited to, ethynyl, propynyl, butynyl, and the like.

[0035] In the present context, the term "halogen", or "halo" for short, refers to fluorine (F), chlorine (CI), bromine (Br) or iodine (I).

[0036] In the present context, the term "heteroatom" refers to an atom other than carbon or hydrogen. Heteroatoms are typically independently selected from oxygen (O), sulfur (S), nitrogen

(N), and phosphorus (P), but are not limited to those atoms. In embodiments in which two or more heteroatoms are present, the two or more heteroatoms may all be the same as one another, or some or all of the two or more heteroatoms may each be different from the others.

[0037] In the present context, the term "ring" refers to any covalently closed structure. Rings include, for example, carbocycles (e.g., aryls and alicyclics), heterocycles (e.g., heteroaryls and non-aromatic heterocycles), aromatics (e.g., aryls and heteroaryls), and non-aromatics (e.g., alicyclics and non-aromatic heterocycles). Rings may be optionally substituted. The term "ring system" refers to two or more rings, wherein two or more of the rings are fused. The term "fused" refers to structures in which two or more rings share one or more bonds.

[0038] In the present context, the term "aromatic" refers to a group comprising a covalently closed planar ring having a delocalized [pi]-electron system comprising 4n+2 [pi] electrons, where n is an integer. Aromatic rings may be formed by five, six, seven, eight, nine, or more than nine atoms. Aromatics may be optionally substituted. Examples of aromatic groups include, but are not limited to phenyl, naphthalenyl, phenanthrenyl, anthracenyl, tetralinyl, fluorenyl, indenyl, and indanyl. The term aromatic includes, for example, benzenoid groups, connected via one of the ring- forming carbon atoms, and optionally carrying one or more substituents selected from an aryl, a heteroaryl, a cycloalkyl, a non-aromatic heterocycle, a halo, a hydroxy, an amino, a cyano, a nitro, an alkylamido, an acyl, a C1-C6 alkoxy, a C1-C6 alkyl, a C1-C6 hydroxyalkyl, a C1-C6 aminoalkyl, an alkylsulfenyl, an alkylsulfinyl, an alkylsulfonyl, an sulfamoyl, or a trifluoromethyl. In certain embodiments, an aromatic group is substituted at one or more of the para, meta, and/or ortho positions. Examples of aromatic groups comprising substitutions include, but are not limited to, phenyl, 3-halophenyl, 4-halophenyl, 3-hydroxyphenyl, 4-hydroxyphenyl, 3-aminophenyl, 4- aminophenyl, 3-methylphenyl, 4-methylphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 4- trifluoromethoxyphenyl, 3-cyanophenyl, 4-cyanophenyl, dimethylphenyl, naphthyl,

hydroxynaphthyl, hydroxymethylphenyl, (trifluoromethyl)phenyl, alkoxyphenyl, 4-morpholin-4- ylphenyl, 4-pyrrolidin-l-ylphenyl, 4-pyrazolylphenyl, 4-triazolylphenyl, and 4-(2-oxopyrrolidin-l- yl)phenyl.

[0039] In the present context, the term "aryl" refers to an aromatic ring wherein each of the atoms forming the ring is a carbon atom. Aryl rings may be formed by five, six, seven, eight, nine, or more than nine carbon atoms. In certain embodiments, the aryl may be a 5-14-membered aryl, such as 5-membered aryl, 6-membered aryl, 7-membered aryl, 8-membered aryl, 9-membered aryl, 10-membered aryl, 1 1-membered aryl, 12-membered aryl, 13-membered aryl, or 14-membered aryl. Designations such as "5-14-membered aryl" refer to the total number of atoms in the ring. Aryl groups may be optionally substituted.

[0040] In the present context, the term "non-aromatic ring" refers to a group comprising a covalently closed ring that is not aromatic.

[0041] In the present context, the term "heterocycle" refers to a group comprising a covalently closed ring wherein at least one atom formmg the ring is a carbon atom and at least one atom forming the ring is a heteroatom. Heterocyclic rings may be formed by three, four, five, six, seven, eight, nine, or more than nine atoms. Any number of those atoms may be heteroatoms (i.e., a heterocyclic ring may comprise one, two, three, four, five, six, seven, eight, nine, or more than nine heteroatoms). Herein, whenever the number of atoms or members in a heterocycle is indicated

(e.g., 5- 10-membered heterocycle), at least one other atom (i.e. the heteroatom) must be present in the ring. Designations such as "5-10-membered heterocycle" refer to the total number of atoms in the ring. It is understood that the heterocylic ring will have additional heteroatoms in the ring. In heterocycles comprising two or more heteroatoms, those two or more heteroatoms may be the same or different from one another. Heterocycles may be optionally substituted. Binding to a heterocycle can be at a heteroatom or via a carbon atom. Examples of heterocycles include, but are not limited to the following:

wherein D, E, F, and G independently represent a heteroatom. Each of D, E, F, and G may be the same or different from one another.

[0042] In the present context, the term "heteroaryl" refers to an aromatic heterocycle.

Heteroaryl rings may be formed by three, four, five, six, seven, eight, nine, or more than nine atoms or members wherein at least one of the atoms or members in the ring is a heteroatom. In certain embodiments, the heteroaryl may be a 5-10-membered aromatic heterocycle, 5-1 1- membered aromatic heterocycle, 5- 12-membered aromatic heterocycle, 5- 13-membered aromatic heterocycle, or 5-14-membered aromatic heterocycle. Heteroaryls may be optionally substituted. Examples of heteroaryl groups include, but are not limited to, aromatic C3-C8 heterocyclic groups comprising one oxygen or sulfur atom or up to four nitrogen atoms, or a combination of one oxygen or sulfur atom and up to two nitrogen atoms, and their substituted as well as benzo- and pyrido-fused derivatives, for example, connected via one of the ring-forming carbon atoms. In certain embodiments, heteroaryl groups are optionally substituted with one or more substituents. Examples of heteroaryl groups include, but are not limited to, unsubstituted and mono- or di- substituted derivatives of furan, benzofuran, thiophene, benzothiophene, pyrrole, pyridine, indole, oxazole, benzoxazole, isoxazole, benzisoxazole, thiazole, benzothiazole, isothiazole, imidazole, benzimidazole, pyrazole, indazole, tetrazole, quinoline, isoquinoline, pyridazine, pyrimidine, purine and pyrazine, furazan, 1,2,3- oxadiazole, 1,2,3-thiadiazole, 1 ,2,4-thiadiazole, triazole, benzotriazole, pteridine, phenoxazole, oxadiazole, benzopyrazole, quinolizine, cinnoline, phthalazine, quinazoline, and quinoxaline.

[0043] In the present context, the term "alicyclic" refers to a group comprising a non-aromatic ring wherein each of the atoms forming the ring is a carbon atom. Alicyclic groups may be formed by three, four, five, six, seven, eight, nine, or more than nine carbon atoms. In certain

embodiments, alicyclics are optionally substituted, i.e. substituted or unsubstituted. In certain embodiments, an alicyclic comprises one or more unsaturated bonds, such as one or more carbon- carbon double-bonds. Alicyclics include cycloalkyls and cycloalkenyls. Examples of alicyclics include, but are not limited to, cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclopentadiene, cyclohexane, cyclohexene, 1,3-cyclohexadiene, 1 ,4-cyclohexadiene, cycloheptane, and cycloheptene. Accordingly, the term "heteroalicyclic ring" refers to a group comprising a non-aromatic ring wherein one or more but not all of the atoms forming the ring is a heteroatom. In certain embodiments, the heteroalicyclic ring may be a 5-10-membered non- aromatic heterocycle, 5-1 1 -membered non-aromatic heterocycle, 5- 12-membered non-aromatic heterocycle, 5-13-membered non-aromatic heterocycle, or 5-14-membered non-aromatic heterocycle. Heteroalicyclic rings may be optionally substituted.

[0044] In the present context, the term "oxo" refers to a carbonyl functional group.

[0045] In the present context, the term "cycloalkyl" refers to a completely saturated hydrocarbon ring. In certain embodiments, the cycloalkyl may be a 5-14-membered cycloalkyl, such as, 5-membered cycloalkyl, 6-membered cycloalkyl, 7-membered cycloalkyl, 8-membered cycloalkyl, 9-membered cycloalkyl, 10-membered cycloalkyl, 1 1 -membered cycloalkyl, 12- membered cycloalkyl, 13-membered cycloalkyl, or 14-membered cycloalkyl. The cycloalkyl group can for example be optionally substituted. Examples of cycloalkyl groups include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like. When substituted, the substituent group(s) can be defined as mentioned above. Accordingly, the term "heterocycloalkyl" refers to a cycloalkyl group wherein one or more but not all of the atoms forming the ring is a heteroatom. In certain embodiments, the heterocycloalkyl may be a 5-14-membered

heterocycloalkyl, such as 5-membered heterocycloalkyl, 6-membered heterocycloalkyl, 7- membered heterocycloalkyl, 8-membered heterocycloalkyl, 9-membered heterocycloalkyl, 10- membered heterocycloalkyl, 1 1 -membered heterocycloalkyl, 12-membered heterocycloalkyl, 13- membered heterocycloalkyl, or 14-membered heterocycloalkyl. [0046] In various embodiments, m is 1 , i.e. only one R₃ is present in the compound of Formula (I). In certain embodiments, R₃ is at the 4^th position of the phenyl ring. In other embodiments, R₃ may be at the 2^nd or 3^rd position of the phenyl ring.

[0047] In some embodiments where m is 1 , R₃ is halogen, such as but not limited to CI or Br.

[0048] In alternative embodiments where m is 1 , R₃ is an alkoxy such as but not limited to, methoxy or ethoxy.

[0049] In further alternative embodiments where m is 1 , R₃ is a substituted or unsubstituted C 1 -C5 alkyl, such as but not limited to methyl or ethyl.

[0050] In various embodiments where m is 1 , R| and R₂ together as a group is -(CH₂)₃- or - (CH₂)4-. In other words, Ri and R₂ together as a group is cyclopentene/cyclopentyl or phenyl ring/cyclohexyl.

[0051] In various embodiments where m is 1 , R4 is H.

[0052] In one embodiment where m is 1 , R_\ and R₂ together as a group is -(CH₂)₃-, R4 is H, R₃ is Br at the 4^th position of the phenyl ring.

[0053} In one embodiment where m is 1 , Ri and R₂ together as a group is -(CH₂)4-, 4 is H, R is Br at the 4^th position of the phenyl ring.

[0054] In one embodiment where m is 1 , Ri and R₂ together as a group is -(CH₂) -, R4 is H, R₃ is CI at the 4^th position of the phenyl ring.

72 [0055] In one embodiment where m is 1,

and R₂ together as a group is -(CH₂)4-, R4 is H, R₃ is CI at the 4^th position of the phenyl ring.

[0056] In one embodiment where m is 1, Ri and R₂ together as a group is -(CH₂)₃-, R4 is H, R₃ is methoxy at the 4^th position of the phenyl ring.

[0057] In one embodiment where m is 1, Ri and R₂ together as a group is -(CH₂)4-, R4 is H, R₃ is methoxy at the 4^th position of the phenyl ring.

[0058] In one embodiment where m is 1, Ri and R₂ together as a group is -(CH₂)₃-, R4 is H, R₃ is methyl at the 4^th position of the phenyl ring.

[0059] In one embodiment where m is 1, Ri and R₂ together as a group is -(CH₂)4-, R4 is H, R₃ is methyl at the 4^th position of the phenyl ring.

[0060] In various embodiments, m is 2, i.e. only two R₃ are present in the compound of Formula (I). In certain embodiments, a first R₃ is at the 3^rd position of the phenyl ring and a second R₃ is at the 4^th position of the phenyl ring. In other embodiments, a first R₃ is at the 2^nd position of the phenyl ring and a second R₃ is at the 4^th position of the phenyl ring. In yet further embodiments, a first R₃ is at the 2^nd position of the phenyl ring and a second R₃ is at the 3^rd position of the phenyl ring. The first R₃ and the second R₃ may also be at other positions of the phenyl ring.

[0061] In some embodiments where m is 2, R₃ is hydroxyl, i.e. OH. [0062] In alternative embodiments where m is 2, R₃ is an alkoxy such as but not limited to, methoxy or ethoxy.

[0063] In various embodiments where m is 2, Rj and R₂ together as a group is -(CH₂)₃- or - (CH₂)₄-. In other words, Ri and R₂ together as a group is cyclopentene/cyclopentyl or phenyl ring/cyclohexyl.

[0064] In various embodiments where m is 2, R4 is H.

[0065] In one embodiment where m is 2, R] and R₂ together as a group is -(CH₂)₃-, R4 is H, a first R₃ is hydroxyl at the 3^rd position of the phenyl ring and a second R₃ is methoxy at the 4^th position of the phenyl ring.

[0066] In one embodiment where m is 2, Ri and R₂ together as a group is -(CH₂)₄-, R4 is H, a first R₃ is hydroxyl at the 3^rd position of the phenyl ring and a second R₃ is methoxy at the 4^th position of the phenyl ring.

[0067] In a second aspect, the present disclosure relates to a compound of Formula (I)

for use as a medicament,

wherein: m is 0, 1, 2, 3, 4, or 5;

Ri and R₂ together form a five-membered, six-membered, or seven-membered ring, wherein R] and R₂ together as a group is -(CH₂)₃-, -(CH₂)4-, or -(CH₂)5-;

R₃ at each occurrence is independently selected from the group consisting of H,

halogen, hydro xyl, alkoxy, and a substituted or unsubstituted C1-C5 alkyl; and R4 is H, halogen, or a substituted or unsubstituted C1-C5 alkyl.

[0068] In various embodiments, the compounds of Formula (I) are useful in treating or preventing cancer in a subject. For example, the cancer may be selected from the group consisting of cervical cancer, leukemia, stomach cancer, breast cancer, lung cancer, colon cancer, and neuroblastoma. The subject may be a mammal, for example, a human.

[0069] In various embodiments, m is 1, i.e. only one R₃ is present in the compound of Formula (I). In certain embodiments, R₃ is at the 4^th position of the phenyl ring. In other embodiments, R₃ may be at the 2^nd or 3^rd position of the phenyl ring.

[0070] In some embodiments where m is 1, R₃ is halogen, such as but not limited to CI or Br.

[0071] In alternative embodiments where m is 1, R₃ is an alkoxy such as but not limited to, methoxy or ethoxy.

[0072] In further alternative embodiments where m is 1, R₃ is a substituted or unsubstituted

C1-C5 alkyl, such as but not limited to methyl or ethyl. [0073] In various embodiments where m is : I, Ri and R₂ together as a group is -(CH₂)₃- or - (CH₂)₄-. In other words, Ri and R₂ together as a , group is cyclopentene/cyclopentyl or phenyl ring/cyclohexyl.

[0074] In various embodiments where m is [ , R₄ is H.

[0075] In one embodiment where m is 1 , Ri and R₂ together as a group is -(CH₂)₃-, R4 is H, R₃ is Br at the 4^th position of the phenyl ring.

[0076] In one embodiment where m is 1 , Rj and R₂ together as a group is -(CH₂)₄-, R4 is H, R₃ is Br at the 4^th position of the phenyl ring.

[0077] In one embodiment where m is 1, R_\ and R₂ together as a group is -(CH₂)₃-, R4 is H, R₃ is CI at the 4^th position of the phenyl ring.

[0078] In one embodiment where m is 1, Ri and R₂ together as a group is -(CH₂)4-, R4 is H, R₃ is CI at the 4^th position of the phenyl ring.

[0079] In one embodiment where m is 1 , Rj and R₂ together as a group is -(CH₂)₃-, R is H, R₃ is methoxy at the 4^th position of the phenyl ring.

[0080] In one embodiment where m is 1, Ri and R₂ together as a group is -(CH₂)₄-, R4 is H, R₃ is methoxy at the 4^th position of the phenyl ring.

[0081] In one embodiment where m is 1, R_\ and R₂ together as a group is -(CH₂)₃-, R is H, R₃ is methyl at the 4^th position of the phenyl ring. [0082] In one embodiment where m is 1, R_\ and R₂ together as a group is -(CH₂)₄-, R4 is H, R3 is methyl at the 4* position of the phenyl ring.

[0083] In various embodiments, m is 2, i.e. only two R₃ are present in the compound of Formula (I). In certain embodiments, a first R₃ is at the 3^rd position of the phenyl ring and a second R₃ is at the 4^th position of the phenyl ring. In other embodiments, a first R₃ is at the 2^nd position of the phenyl ring and a second R3 is at the 4^th position of the phenyl ring. In yet further embodiments, a first R3 is at the 2^nd position of the phenyl ring and a second R3 is at the 3^rd position of the phenyl ring. The first R3 and the second R₃ may also be at other positions of the phenyl ring.

[0084] In some embodiments where m is 2, R₃ is hydroxyl, i.e. OH.

[0085] In alternative embodiments where m is 2, R3 is an alkoxy such as but not limited to, methoxy or ethoxy.

[0086] In various embodiments where m is 2, Ri and R₂ together as a group is -(CH₂)3- or - (CH₂)₄-. In other words, R] and R₂ together as a group is cyclopentene/cyclopentyl or phenyl ring/cyclohexyl.

[0087] In various embodiments where m is 2, R is H.

[0088] In one embodiment where m is 2, Ri and R₂ together as a group is -(CH₂)3-, is H, a first R₃ is hydroxyl at the 3^rd position of the phenyl ring and a second R3 is methoxy at the 4^th position of the phenyl ring. [0089] In one embodiment where m is 2, Ri and R₂ together as a group is -(CH₂)₄-, R4 is H, a first R₃ is hydroxyl at the 3^rd position of the phenyl ring and a second R₃ is methoxy at the 4^th position of the phenyl ring.

[0090] In a third aspect, the present disclosure relates to use of a compound of Formula (I)

for the manufacture of a medicament for treating or preventing cancer in a subject, comprising administering a therapeutically effective amount of said compound to said subject,

wherein:

m is 0, 1, 2, 3, 4, or 5;

Ri and R₂ together form a five-membered, six-membered, or seven-membered ring, wherein Ri and R₂ together as a group is -(CH₂)₃-, -(CH₂)₄-, or -(CH₂)5-;

R4 is H, halogen, or a substituted or unsubstituted C1-C5 alkyl. [0091] In various embodiments, the cancer is selected from the group consisting of cervical cancer, leukemia, stomach cancer, breast cancer, lung cancer, colon cancer, and neuroblastoma.

[0092] In various embodiments, the subject is a mammal.

[0093] In various embodiments, the subject is a human.

[0094] In various embodiments, m is 1 , i.e. only one R₃ is present in the compound of Formula (I). In certain embodiments, R₃ is at the 4^th position of the phenyl ring. In other embodiments, R₃ may be at the 2^nd or 3^rd position of the phenyl ring.

[0095] In some embodiments where m is 1, R₃ is halogen, such as but not limited to CI or Br.

[0096] In alternative embodiments where m is 1, R₃ is an alkoxy such as but not limited to, methoxy or ethoxy.

[0097] In further alternative embodiments where m is 1 , R₃ is a substituted or unsubstituted C1-C5 alkyl, such as but not limited to methyl or ethyl.

[0098] In various embodiments where m is 1, Ri and R₂ together as a group is -(CH₂)₃- or - (CH₂)4-. In other words, Ri and R₂ together as a group is cyclopentene/cyclopentyl or phenyl ring/cyclohexyl.

[0099] In various embodiments where m is 1, R4 is H.

[00100] In one embodiment where m is 1 , Ri and R₂ together as a group is -(CH₂)₃-, R4 is H, R3 is Br at the 4^th position of the phenyl ring. [00101] In one embodiment where m is 1 , Rj and R₂ together as a group is -(CH₂)₄-, R4 is H, R₃ is Br at the 4^th position of the phenyl ring.

[00102] In one embodiment where m is 1 , Ri and R₂ together as a group is -(CH₂)₃-, R4 is H, R₃ is CI at the 4^th position of the phenyl ring.

[00103] In one embodiment where m is 1, Ri and R₂ together as a group is -(CH₂)₄-, R4 is H, R₃ is CI at the 4^th position of the phenyl ring.

[00104] In one embodiment where m is 1, R] and R₂ together as a group is -(CH₂)₃-, R₄ is H, R₃ is methoxy at the 4^th position of the phenyl ring.

[00105] In one embodiment where m is 1 , Ri and R₂ together as a group is -(CH₂)₄-, R4 is H, R₃ is methoxy at the 4^th position of the phenyl ring.

[00106] In one embodiment where m is 1 , Ri and R₂ together as a group is -(CH₂)₃-, R4 is H, R₃ is methyl at the 4^th position of the phenyl ring.

[00107] In one embodiment where m is 1 , Ri and R₂ together as a group is -(CH₂)₄-, R4 is H, R₃ is methyl at the 4^th position of the phenyl ring.

[00108] In various embodiments, m is 2, i.e. only two R₃ are present in the compound of

Formula (I). In certain embodiments, a first R₃ is at the 3^rd position of the phenyl ring and a second

R₃ is at the 4^th position of the phenyl ring. In other embodiments, a first R₃ is at the 2^nd position of the phenyl ring and a second R₃ is at the 4^th position of the phenyl ring. In yet further embodiments, a first R₃ is at the 2ⁿ position of the phenyl ring and a second R₃ is at the 3^r position of the phenyl ring. The first R₃ and the second R₃ may also be at other positions of the phenyl ring.

[00109] In some embodiments where m is 2, R₃ is hydroxyl, i.e. OH.

[00110] In alternative embodiments where m is 2, R₃ is an alkoxy such as but not limited to, methoxy or ethoxy.

[00111] In various embodiments where m is 2, Ri and R₂ together as a group is -(CH₂)₃- or - (CH₂)₄-. In other words, Ri and R₂ together as a group is cyclopentene/cyclopentyl or phenyl ring/cyclohexyl.

[00112] In various embodiments where m is 2, R* is H.

[00113] In one embodiment where m is 2, R_\ and R₂ together as a group is -(CH₂)₃-, R4 is H, a first R₃ is hydroxyl at the 3^rd position of the phenyl ring and a second R₃ is methoxy at the 4^th position of the phenyl ring.

[00114] In one embodiment where m is 2, Ri and R₂ together as a group is -(CH₂)₄-, R₄ is H, a first R₃ is hydroxyl at the 3^rd position of the phenyl ring and a second R₃ is methoxy at the 4^th position of the phenyl ring.

[00115] In a fourth aspect, the present disclosure relates to a method for inhibiting

polymerization of tubulin in a cell, comprising contacting said cell with an effective amount of a compound of Formula (I)

wherein:

m is 0, 1, 2, 3, 4, or 5;

Ri and R₂ together form a five-membered, six-membered, or seven-membered ring, wherein Ri and R₂ together as a group is -(CH₂)₃-, -(CH₂)₄-, or -(CH₂)₅-;

halogen, hydroxyl, alkoxy, and a substituted or unsubstituted C1-C5 alkyl; and

R4 is H, halogen, or a substituted or unsubstituted C1-C5 alkyl.

[00116] The method may be an in vivo or an in vitro method. "In vitro method", in this connection refers to embodiments where the cells are no longer present in a living organism, but are, for example, cultured.

[00117] In various embodiments, m is 1, i.e. only one R₃ is present in the compound of Formula (I). In certain embodiments, R₃ is at the 4^th position of the phenyl ring. In other embodiments, R₃ may be at the 2^nd or 3^rd position of the phenyl ring.

[00118] In some embodiments where m is 1, R₃ is halogen, such as but not limited to CI or Br. [00119] In alternative embodiments where m is 1 , R₃ is an alkoxy such as but not limited to, methoxy or ethoxy.

[00120] In further alternative embodiments where m is 1 , R₃ is a substituted or unsubstituted C1-C5 alkyl, such as but not limited to methyl or ethyl.

[00121] In various embodiments where m is 1 , Ri and R₂ together as a group is -(CH₂)₃- or - (CH₂)₄-. In other words, Ri and R₂ together as a group is cyclopentene/cyclopentyl or phenyl ring/cyclohexyl.

[00122] In various embodiments where m is 1, R4 is H.

[00123] In one embodiment where m is 1 , Ri and R₂ together as a group is -(CH₂)₃-, R4 is H, R₃ is Br at the 4^th position of the phenyl ring.

[00124] In one embodiment where m is 1 , Ri and R₂ together as a group is -(CH₂)₄-, R4 is H, R₃ is Br at the 4^th position of the phenyl ring.

[00125] In one embodiment where m is 1 , Ri and R₂ together as a group is -(CH₂)₃-, R is H, R₃ is CI at the 4^th position of the phenyl ring.

[00126] In one embodiment where m is 1, Ri and R₂ together as a group is -(CH₂)₄-, R4 is H, R₃ is CI at the 4^th position of the phenyl ring.

[00127] In one embodiment where m is 1 , R_\ and R₂ together as a group is -(CH₂)₃-, R4 is H, R₃ is methoxy at the 4^th position of the phenyl ring. [00128] In one embodiment where m is 1 , Rj and R₂ together as a group is -(CH₂)₄-, R4 is H, R₃ is methoxy at the 4^th position of the phenyl ring.

[00129] In one embodiment where m is 1, Ri and R₂ together as a group is -(CH₂)₃-, R4 is H, R₃ is methyl at the 4^th position of the phenyl ring.

[00130] In one embodiment where m is 1, R_\ and R₂ together as a group is -(CH₂)₄-, R is H, R₃ is methyl at the 4^th position of the phenyl ring.

[00131] In various embodiments, m is 2, i.e. only two R₃ are present in the compound of Formula (I). In certain embodiments, a first R₃ is at the 3^rd position of the phenyl ring and a second R₃ is at the 4^th position of the phenyl ring. In other embodiments, a first R₃ is at the 2^nd position of the phenyl ring and a second R3 is at the 4^th position of the phenyl ring. In yet further embodiments, a first R₃ is at the 2^nd position of the phenyl ring and a second R₃ is at the 3^rd position of the phenyl ring. The first R₃ and the second R₃ may also be at other positions of the phenyl ring.

[00132] In some embodiments where m is 2, R₃ is hydroxyl, i.e. OH.

[00133] In alternative embodiments where m is 2, R₃ is an alkoxy such as but not limited to, methoxy or ethoxy.

[00134] In various embodiments where m is 2, Ri and R₂ together as a group is -(CH₂)₃- or - (CH₂)₄-. In other words, R_\ and R₂ together as a group is cyclopentene/cyclopentyl or phenyl ring/cyclohexyl. [00135] In various embodiments where m is 2, R4 is H.

[00136] In one embodiment where m is 2, R_\ and R₂ together as a group is -(CH₂)₃-, R4 is H, a first R₃ is hydroxyl at the 3^rd position of the phenyl ring and a second R₃ is methoxy at the 4^th position of the phenyl ring.

[00137] In one embodiment where m is 2, Ri and R₂ together as a group is -(CH₂)4-, R4 is H, a first R₃ is hydroxyl at the 3^rd position of the phenyl ring and a second R₃ is methoxy at the 4^th position of the phenyl ring.

[00138] In a fifth aspect, the present disclosure relates to a method for arresting cell cycle in G₂/M phase of a cell, comprising contacting said cell with an effective amount of a compound of Formula (I)

wherein:

m is 0, 1, 2, 3, 4, or 5;

Ri and R together form a five-membered, six-membered, or seven-member ed aromatic ring, wherein Ri and R₂ together as a group is -(CH₂)₃-, -(CH₂)₄-, or -(CH₂)5-; R.3 at each occurrence is independently selected from the group consisting of H, halogen, hydroxyl, alkoxy, and a substituted or unsubstituted C1-C5 alkyl; and

R4 is H, halogen, or a substituted or unsubstituted C1-C5 alkyl.

[00139] The method may be an in vivo or an in vitro method. "/« vitro method", in this connection refers to embodiments where the cells are no longer present in a living organism, but are, for example, cultured.

[00140] In various embodiments, m is 1, i.e. only one R₃ is present in the compound of Formula (I). In certain embodiments, R₃ is at the 4^th position of the phenyl ring. In other embodiments, R₃ may be at the 2^nd or 3^rd position of the phenyl ring.

[00141] In some embodiments where m is 1, R₃ is halogen, such as but not limited to CI or Br.

[00142] In alternative embodiments where m is 1, R₃ is an alkoxy such as but not limited to, methoxy or ethoxy.

[00143] In further alternative embodiments where m is 1, R₃ is a substituted or unsubstituted C1-C5 alkyl, such as but not limited to methyl or ethyl.

[00144] In various embodiments where m is 1, Ri and R₂ together as a group is -(CH₂)₃- or - (CH₂)₄-. In other words, Ri and R₂ together as a group is cyclopentene/cyclopentyl or phenyl ring/cyclohexyl.

[00145] In various embodiments where m is 1, R4 is H. [00146] ^{m one} embodiment where m is 1, Ri and R₂ together as a group is -(CH₂)₃-, Rt is H, R₃ is Br at the 4^th position of the phenyl ring.

[00147] In one embodiment where m is 1, Ri and R₂ together as a group is -(CH₂)₄-, 4 is H, R₃ is Br at the 4^th position of the phenyl ring.

[00148] In one embodiment where m is 1, Ri and R₂ together as a group is -(CH₂)₃-, R4 is H, R₃ is CI at the 4^th position of the phenyl ring.

[00149] In one embodiment where m is 1, Ri and R₂ together as a group is -(CH₂)4-, 4 is H, R₃ is CI at the 4^th position of the phenyl ring.

[00150] In one embodiment where m is 1, R_\ and R₂ together as a group is -(CH₂)₃-, Rt is H, R₃ is methoxy at the 4^th position of the phenyl ring.

[00151] In one embodiment where m is 1 , Ri and R₂ together as a group is -(CH₂)₄-, R4 is H, R₃ is methoxy at the 4^th position of the phenyl ring.

[00152J In one embodiment where m is 1, Ri and R₂ together as a group is -(CH₂)₃-, R4 is H, R₃ is methyl at the 4^th position of the phenyl ring.

[00153] In one embodiment where m is 1, Ri and R₂ together as a group is -(CH₂)₄-, R4 is H, R₃ is methyl at the 4^th position of the phenyl ring.

[00154] In various embodiments, m is 2, i.e. only two R₃ are present in the compound of

Formula (I). In certain embodiments, a first R₃ is at the 3^rd position of the phenyl ring and a second R₃ is at the 4 position of the phenyl ring. In other embodiments, a first R₃ is at the 2ⁿ position of the phenyl ring and a second R₃ is at the 4^th position of the phenyl ring. In yet further embodiments, a first R₃ is at the 2^nd position of the phenyl ring and a second R₃ is at the 3^rd position of the phenyl ring. The first R₃ and the second R₃ may also be at other positions of the phenyl ring.

[00155] In some embodiments where m is 2, R₃ is hydroxyl, i.e. OH.

[00156] In alternative embodiments where m is 2, R₃ is an alkoxy such as but not limited to, methoxy or ethoxy.

[00157] In various embodiments where m is 2, Ri and R₂ together as a group is -(CH₂)₃- or - (CH₂)4-. In other words, Ri and R₂ together as a group is cyclopentene/cyclopentyl or phenyl ring/cyclohexyl.

[00158] In various embodiments where m is 2, R₄ is H.

[00159] In one embodiment where m is 2, Ri and R₂ together as a group is -(CH₂)₃-, R₄ is H, a first R₃ is hydroxyl at the 3^rd position of the phenyl ring and a second R₃ is methoxy at the 4^th position of the phenyl ring.

[00160] In one embodiment where m is 2, R_\ and R₂ together as a group is -(CH₂)₄-, R₄ is H, a first R₃ is hydroxyl at the 3^rd position of the phenyl ring and a second R₃ is methoxy at the 4^th position of the phenyl ring. [00161] In a sixth aspect, the present disclosure relates to a method for inducing apoptosis in a cell, comprising contacting said cell with an effective amount of a compound of Formula (I)

wherein:

m is 0, 1 , 2, 3, 4, or 5;

Ri and R₂ together form a five-membered, six-membered, or seven-membered ring, wherein Ri and R₂ together as a group is -(CH₂)3-, -(CH₂)₄-, or -(CH₂)₅-;

halogen, hydroxyl, alkoxy, and a substituted or unsubstituted C1 -C5 alkyl; and R4 is H, halogen, or a substituted or unsubstituted C1 -C5 alkyl.

[00162] The method may be an in vivo or an in vitro method. "/« vitro method", in this connection refers to embodiments where the cells are no longer present in a living organism, but are, for example, cultured.

[00163] The term "apoptosis" as used herein refers to the process of programmed cell death, as it is known and understood by those skilled in the art. Usually, in apoptosis biochemical events lead to characteristic cell changes and death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. Typically, apoptosis produces cell fragments called apoptotic bodies that phagocytic cells are able to engulf and quickly remove before the contents of the cell can spill out onto surrounding cells and cause damage to these.

[00164] In various embodiments, m is 1, i.e. only one R₃ is present in the compound of Formula (I). In certain embodiments, R₃ is at the 4^th position of the phenyl ring. In other embodiments, R₃ may be at the 2^nd or 3^rd position of the phenyl ring.

[00165] In some embodiments where m is 1, R₃ is halogen, such as but not limited to CI or Br.

[00166] In alternative embodiments where m is 1, R₃ is an alkoxy such as but not limited to, methoxy or ethoxy.

[00167] In further alternative embodiments where m is 1, R₃ is a substituted or unsubstituted C1-C5 alkyl, such as but not limited to methyl or ethyl.

[00168] In various embodiments where m is 1, Ri and R₂ together as a group is -(CH₂)₃- or - (CH₂)4-. In other words, Ri and R₂ together as a group is cyclopentene/cyclopentyl or phenyl ring/cyclohexyl.

[00169] In various embodiments where m is 1, R4 is H.

[00170] In one embodiment where m is 1, Ri and R₂ together as a group is -(CH₂)₃-, R4 is H, R₃ is Br at the 4^th position of the phenyl ring. [00171] In one embodiment where m is 1, Ri and R₂ together as a group is -(CH₂)4-, R4 is H, R₃ is Br at the 4^th position of the phenyl ring.

[00172] In one embodiment where m is 1, Ri and R₂ together as a group is -(CH₂)₃-, R4 is H, R₃ is CI at the 4^th position of the phenyl ring.

[00173] In one embodiment where m is 1 , Ri and R₂ together as a group is -(CH₂)4-, R4 is H, R₃ is CI at the 4^th position of the phenyl ring.

[00174] In one embodiment where m is 1, Ri and R₂ together as a group is -(CH₂)3-, R4 is H, R₃ is methoxy at the 4^th position of the phenyl ring.

[00175] In one embodiment where m is 1, Ri and R₂ together as a group is -(CH₂)₄-, v is H, R₃ is methoxy at the 4^th position of the phenyl ring.

[00176] In one embodiment where m is 1, Ri and R₂ together as a group is -(CH₂)₃-, R4 is H, R₃ is methyl at the 4^th position of the phenyl ring.

[00177] In one embodiment where m is 1, Ri and R₂ together as a group is -(CH₂)₄-, R4 is H, R₃ is methyl at the 4^th position of the phenyl ring.

[00178] In various embodiments, m is 2, i.e. only two R₃ are present in the compound of

Formula (I). In certain embodiments, a first R is at the 3^rd position of the phenyl ring and a second

R₃ is at the 4^th position of the phenyl ring. In other embodiments, a first R₃ is at the 2^nd position of the phenyl ring and a second R₃ is at the 4^th position of the phenyl ring. In yet further embodiments, a first R-3 is at the 2^nd position of the phenyl ring and a second R₃ is at the 3^rd position of the phenyl ring. The first R₃ and the second R₃ may also be at other positions of the phenyl ring.

[00179] In some embodiments where m is 2, R₃ is hydroxyl, i.e. OH.

[00180] In alternative embodiments where m is 2, R₃ is an alkoxy such as but not limited to, methoxy or ethoxy.

[00181] In various embodiments where m is 2, Ri and R₂ together as a group is -(CH₂)₃- or - (CH₂)₄-. In other words, Ri and R₂ together as a group is cyclopentene/cyclopentyl or phenyl ring/cyclohexyl.

[00182] In various embodiments where m is 2, R4 is H.

[00183] In one embodiment where m is 2, Ri and R₂ together as a group is -(CH₂)₃-, R4 is H, a first R₃ is hydroxyl at the 3^rd position of the phenyl ring and a second R₃ is methoxy at the 4^th position of the phenyl ring.

[00184] In one embodiment where m is 2, R_\ and R₂ together as a group is -(CH₂)₄-, R₄ is H, a first R₃ is hydroxyl at the 3^rd position of the phenyl ring and a second R₃ is methoxy at the 4^th position of the phenyl ring.

[00185] In a seventh aspect, the present disclosure relates to a method for suppressing in vivo tumor growth in a subject, comprising administering to the subject in need thereof an effective amount of a compound of Formula (I)

wherein:

m is 0, 1, 2, 3, 4, or 5;

Ri and R₂ together form a five-membered, six-membered, or seven-membered ring, wherein R\ and R₂ together as a group is -(CH₂)3-, -(CH₂)₄-, or -(CH₂)₅-;

halogen, hydroxyl, alkoxy, and a substituted or unsubstituted C1-C5 alkyl; and R₄ is H, halogen, or a substituted or unsubstituted C1-C5 alkyl.

[00186] In various embodiments, m is 1, i.e. only one R₃ is present in the compound of Formula (I). In certain embodiments, R₃ is at the 4^th position of the phenyl ring. In other embodiments, R₃ may be at the 2^nd or 3^rd position of the phenyl ring.

[00187] In some embodiments where m is 1, R₃ is halogen, such as but not limited to CI or Br.

[00188] In alternative embodiments where m is 1, R₃ is an alkoxy such as but not limited to, methoxy or ethoxy.

[00189] In further alternative embodiments where m is 1, R₃ is a substituted or unsubstituted

C1-C5 alkyl, such as but not limited to methyl or ethyl. [00190] In various embodiments where m is 1,

and R₂ together as a group is -(CH₂)₃- or - (CH₂)₄-. In other words, Ri and R₂ together as a group is cyclopentene/cyclopentyl or phenyl ring/cyclohexyl.

[00191] In various embodiments where m is 1 , R4 is H.

[00192] In one embodiment where m is 1, Rj and R₂ together as a group is -(CH₂)₃-, 4 is H, R₃ is Br at the 4^th position of the phenyl ring.

[00193] In one embodiment where m is 1, Ri and R₂ together as a group is -(CH₂)4-, t is H, R₃ is Br at the 4^th position of the phenyl ring.

[00194] In one embodiment where m is 1 , Ri and R₂ together as a group is -(CH₂)₃-, R4 is H, R₃ is CI at the 4^th position of the phenyl ring.

[00195] In one embodiment where m is 1, Ri and R₂ together as a group is -(CH₂)4-, R4 is H, R₃ is CI at the 4^th position of the phenyl ring.

[00196] In one embodiment where m is 1, Ri and R₂ together as a group is -(CH₂)₃-, R» is H, R₃ is methoxy at the 4^th position of the phenyl ring.

[00197] In one embodiment where m is 1, Ri and R₂ together as a group is -(CH₂)₄-, R4 is H, R is methoxy at the 4^th position of the phenyl ring.

[00198] In one embodiment where m is 1, Ri and R₂ together as a group is -(CH₂)₃-, R4 is H, R₃ is methyl at the 4^th position of the phenyl ring. [00199] In one embodiment where m is 1 , Ri and R₂ together as a group is -(CH₂)4-, R4 is H, R₃ is methyl at the 4^th position of the phenyl ring.

[00200] In various embodiments, m is 2, i.e. only two R₃ are present in the compound of Formula (I). In certain embodiments, a first R₃ is at the 3^rd position of the phenyl ring and a second R₃ is at the 4^th position of the phenyl ring. In other embodiments, a first R₃ is at the 2^nd position of the phenyl ring and a second R₃ is at the 4^th position of the phenyl ring. In yet further embodiments, a first R₃ is at the 2^nd position of the phenyl ring and a second R₃ is at the 3^rd position of the phenyl ring. The first R₃ and the second R₃ may also be at other positions of the phenyl ring.

[00201] In some embodiments where m is 2, R₃ is hydroxyl, i.e. OH.

[00202] In alternative embodiments where m is 2, R₃ is an alkoxy such as but not limited to, methoxy or ethoxy.

[00203] In various embodiments where m is 2, Rj and R₂ together as a group is -(CH₂)₃- or - (CH₂)4-. In other words, Ri and R₂ together as a group is cyclopentene/cyclopentyl or phenyl ring/cyclohexyl.

[00204] In various embodiments where m is 2, R4 is H.

[00205] In one embodiment where m is 2, Ri and R₂ together as a group is -(CH₂)₃-, R4 is H, a first R₃ is hydroxyl at the 3^rd position of the phenyl ring and a second R₃ is methoxy at the 4^th position of the phenyl ring. [00206] In one embodiment where m is 2, R] and R₂ together as a group is -(CH₂)₄-, R is H, a first R₃ is hydroxyl at the 3^rd position of the phenyl ring and a second R₃ is methoxy at the 4^th position of the phenyl ring.

[00207] In order that the invention may be readily understood and put into practical effect, particular embodiments will now be described by way of the following non-limiting examples.

Examples

Example 1: Experimental Procedures

[00208] Chemicals and Antibodies: Nocodazole, paclitaxel (Taxol), vinblastine, and colchicine were purchased from Sigma- Aldrich (St. Louis, MO). SRF was purchased from ChemDiv while SB203580, PD98059 and SP600125 were from Calbiochem (San Diego, CA). Antibodies were obtained from the following companies: vimentin, - and -tubulin, JNK-1, Mdr- 1, Bcl-2, pBcl-2 (T56), pBcl-2 (S70), pBcl-2 (S87) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); pp38, pERKl/2, pJNK (Cell Signaling Technology, Denver, MA); and monoclonal actin antibody was from BD Pharmingen (San Diego, CA). [³H]vinblastine (specific activity, 1 1.6 Ci/mmol) and streptavidin-coated yttrium silicate scintillation proximity assay (SPA) beads were purchased from GE Healthcare (Buckinghamshire, UK). [³H]colchicine (specific activity, 80.4 Ci/mmol) was obtained from PerkinElmer (Boston, MA, USA). [00209] Cell Culture: Human cancer cell lines (HeLa, MDA-MB-231, A549, HCT15, Jurkat,

PC 12 and SH-SY5Y) and human fibroblast cell lines (CCL-1 16 and WI-38) were obtained from

American Type Culture Collection (ATCC, Rockville, MD, USA). SNU16, human stomach cancer cell line, was obtained from Korean Cell Line Bank (KCLB, Seoul, Korea). The human epithelial primary cell line F10 was provided by P. Droge (Nanyang Technological University, Singapore) while the KB-3-1 and KB-V1 cells were a generous gift from M.M. Gottesman (NCI, Bethesda,

MD). Cell lines were grown in either Dulbecco's Modified Eagle Medium (DMEM) or RPMI 1640 containing 10% fetal bovine serum, 1% penicillin/streptomycin and maintained at 37 °C in a humidified 5% C02 chamber. For KB-3-1 and KB-1V, the media was supplemented with an additional 1 mM sodium pyruvate and 10 μg/mL vinblastine, respectively. KB-V1, derived from

KB-3-1 is resistant to vinblastine and overexpresses the MDR drug efflux pump. All resistant lines were incubated in drug-free media prior to cellular proliferation assays.

[00210] Western Blot Analysis: Western Blotting was performed as described in detail elsewhere (Choi, Kim et al. 2008). After treatment, cell pellets were lysed buffer. Lysates were then clarified by centrifugation at 12,000 g, 4 °C, 20 min. The supernatant was collected and total protein concentration of the cellular extract was quantified using Bio-Rad Protein Assay kit (Bio-

Rad, Hercules, CA, USA). Equal amounts of protein (20 /zg/well) were then heat denatured and resolved on 12 % SDS-PAGE gels. Samples were then blotted onto nitrocellulose membranes, blocked with 5 % milk in TBST (25 mM Tris, 140 mM NaCl, 3 mM KCl, 0.05 % Tween-20) for 1 hour at room temperature, washed and then incubated overnight at 4 °C with various specific primary antibodies. Membrane were then washed with an excess of TBST and then probed with appropriate secondary antibodies. Immunoreactive bands were then visualized using Western Lightning™ enhanced chemiluminescence reagent (PerkinElmer, Boston, MA) and specific bands were visualized on CL-XPosure™ film (Thermo Scientific, Rockford, IL).

[00211] Cell Proliferation Assay: Cellular proliferation was determined in 96-well format using the CellTiter 96® AQ_ueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI, USA) according to the manufacturer's instruction. This assay uses a colorimetric method to determine the number of viable cells. Briefly, 5 X 10³ cells/well were seeded in a 96-well plate. The volume of the media in each well was 100 μΐ. Cells were maintained in a humidified, 5 % C0₂ atmosphere till the monolayers reached 50-60 % confluency (24-36 h). Cells were then incubated with SRF or paclitaxel for another 72 h. At the end of this period, 20 μΐ/well of the reagent was added to the cells, incubated for another 1-4 h and absorbance was measured at 490 nm in a 96- well plate reader. The values shown are the mean ± SD of at least three independent experiments performed in triplicates. IC₅₀ were calculated based on A4 0 of untreated cells (taken as 100 %).

[00212] Cell Cycle Analysis: Cell cycle progression was monitored using DNA flow cytometry. Cells were seeded at a density of about 2 x 10⁵ cells/well in a 24- well plate. When the cell monolayers were 50-70 % confluent, the cells were treated with drugs as indicated. After a 24 h treatment, cells were trypsinized, washed with PBS and fixed in 80 % ethanol for 1 h at -20 °C. The fixed cells were then resuspended in propidium iodide (PI) staining buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 50 ^g/mL PI, 10 /xg/L RNase A, 0.1% NP-40) for 30 min at 4 °C in the dark. The DNA content was determined on a FACSCalibur System (BD Biosciences, San Jose, CA). For each analysis, 10,000 cells were counted and the percentage of cells in each phase was calculated using ModFit LT software.

[00213] Apoptosis Assays: Apoptosis was monitored using annexin V-propidium iodide (PI) double staining method. Cells were stained using the Annexin V-FLOUS staining kit (Roche Applied Science, Indianapolis, IN) according to the manufacturer's instructions. Cells were analyzed by a BD LSR II flow cytometer (BD Biosciences, San Jose, CA) using the 488 nm blue laser for excitation and a 505LP mirror-530/30 BP filter and 550LP mirror-575/26 BP filter combinations to detect fluorescein and PI fluorescence, respectively. For each sample, 10,000 events were collected.

[00214] Measurement of Mitochondrial Membrane Potential: Changes in the mitochondrial membrane potential was detected using the Mitochondrial Membrane Potential Detection Kit

(Stratagene, Cedar Creek, TX) and based on the use of the cationic dye, 5,5',6,6'-tetrachloro-

1,1 ',3,3'- tetraethylbenzimidazolylcarbocyanine iodide (commonly known as JC-1). JC-1 forms fluorescent red aggregates in mitochondria of intact cells while in apoptotic cells, collapse of the mitochondrial potential causes JC- 1 to remain in the cytoplasm in its monomeric green form. Briefly, cells were treated with SRF, in the presence or absence of JNK-inhibitor SP600125, and incubated overnight. Cells were then trypsinized, washed IX with PBS and pelleted (400g, 5 min, RT). Next, pellets (1 x 10⁶ cells/sample) were resuspended in 500 μΐ of JC-1 staining solution and incubated for 15 min at 37 °C in a 5 % C0₂ humidified atmosphere. After two times washes with assay buffer, cells were resuspended in final volume of 500 μΐ of assay buffer. Fluorescence intensities were measured on a BD FACSCalibur System (BD Biosciences, San Jose, CA) and the CellQuest™ software was used for data analysis. Loss of mitochondrial potential was measured as the ratio of the red-to-green fluorescence; compared to untreated cells, this ratio will decrease in apoptotic cells.

[00215] Caspase Activation Assay: Caspase-3 activity was measured using the CaspACE™

Assay System (Promega, Madison, WI) according to the vendor's protocol. Active caspase-3 cleaves the specific peptide substrate - Ac-DEVD-pNA to release the '-nitroaniline (pNA) chromophore; free pNA produces a yellow color that can be monitored at 405 nm. Briefly, 2 x lO⁵ cells/well were seeded in a 6-well plate. When monolayers were 50-60 % confluent, cells were treated with SRF in the presence or absence of the JNK inhibitor, SP600125 and incubated overnight at 37 °C in a 5 % C0₂ humidified atmosphere. Cells were harvested, pellets were washed IX with ice-cold PBS and resuspended in Cell Lysis Buffer. Freeze- thaw method was used to lyse cells and the lysates were kept on ice for 15 min. Lysates were clarified by centrifugation (15, OOOg, 20 min, 4 °C) and the supernatant was used to determine Caspase-3 activity. In a 96-well plate, 2 μΐ (10 mM stock) of Ac-DEVD-pNA substrate was added to the reaction mix containing cellular lysate, DTT, Caspase Assay Buffer and deionized water in a final volume of 100 μΐ. Plates were incubated overnight at 20-22 °C for 4 h and the absorbance was measured at 405 nm using a Benchmark Plus microplate reader (Bio-Rad, Hercules). Untreated cell extracts were used as control for the experiments.

[00216] Immunofluorescence Microscopy: For immunohistochemistry, cells were fixed with 3.7 % PFA followed by incubation in a blocking solution (5 % BSA in PBS) for 30 min at 4 °C. After 3X PBS washes, cells were permeabilized with 0.2 % Triton X-100, washed 3X with PBS and then incubated overnight with - or 3-tubulin antibodies at 4 °C. After washing, samples were incubated with AlexaFluor 488-conjugated secondary antibody (Molecular Probes, Eugene, Oregon) for 1 h at room temperature. Slides were mounted using ProLong® Gold anti-fade solution (Molecular Probes, Eugene, Oregon) containing DAPI and visualized at 60X

magnification on a Zeiss LSM META510 confocal laser scanning microscopy. All post acquisition image processing and analysis was done using the MetaMorph® software (Molecular Devices, LLC, Sunnyvale, CA). [00217] In Vitro Tubulin Polymerization Assay: Tubulin polymerization was measured in vitro using the Tubulin Polymerization Assay kit (Cytoskeleton, Denver, CO). Tubulin was dissolved in Buffer 1 (80 mM PIPES, 2 mM MgCl₂, 0.5 mM EGTA pH 6.9, 10 μΜ fluorescent reporter, 1 mM GTP) to a final concentration of 10 mg/ml. To assay tubulin polymerization, a mix containing 85 μΐ of tubulin (final concentration 2 mg/ml), 4.4 μΐ GTP (stock 100 mM), 280 μΐ of Buffer 1 and 75 μΐ of Tubulin Glycerol Buffer (80 mM PIPES, 2 mM MgCl₂, 0.5 mM EGTA, 60 % glycerol, pH 6.9), was made and kept on ice. Next, 5 μΐ of test compound, paclitaxel, nocodazole or control buffer was aliquotted in each well of a half area 96-well, black, flat-bottomed plate (Corning Costar). The plate was warmed for 1 min in a 37 °C pre-warmed plate reader.

Polymerization was initiated by pipetting 50 μΐ of reaction mix/well and monitored by measuring the change in fluorescence (Ex = 360 nm; Em = 420 nm). Measurement was done using

SpectraFluor Plus microplate reader (TECAN GmbH, Austria). Readings were taken every minute for 1 h (total of 61 readings).

[00218] Tubulin Competition-Binding Scintillation Proximity Assay: This assay was performed for competition binding to the colchicine and vinblastine binding sites on tubulin and carried out in a 96-well plate (Tahir, Kovar et al. 2000). Biotin-labeled tubulin (Cytoskeleton,

Denver, CO) was dissolved in G-PEM buffer (80 mM PIPES pH 6.8, 2 mM MgCl₂, 0.5 mM

EGTA, 1 mM GTP and 10 % glycerol) to a final concentration of 0.8-1 mg/ml. [³H]colchicine or [³H]vinblastine (final concentration of 50 nM and 100 mM, respectively), 50 μΜ of SRF and 1.0 _/t g of biotin-labeled tubulin was mixed in a final reaction volume of 100 μΐ of G-PEM buffer and incubated for 45 min at 37 °C. Streptavidin SPA beads (80 ^g/well) were added to each reaction mix and incubated for an additional 30 min at 4 °C. The radioactive counts were measured using a Wallac Microbeta scintillation counter (PerkinElmer, Waltham, MA). For control reactions, SRF was omitted from the reaction mix.

[00219] Molecular Modeling and Docking Analysis: - & /3-tubulin sub-units of tubulin- colchicine: stathmin-like domain structure (PDB code: 1SA0) was used for molecular docking studies. Hydrogen atoms were added at pH 7.0. Charges and potentials were assigned with CVFF force field and energy minimized for 500 steps with steepest descent and 5000 steps with conjugate gradient method in Insightll (Accelrys, San Diego, CA, USA). Two-dimensional structures of SRF and its analogues were sketched using ISIS draw and 3D coordinates of ChemDiv library are obtained from Prepare ligands module. The ligands thus obtained were further energy minimized by ligand minimization using Smart Minimizer algorithm for 1000 steps in Discovery Studio 2.1. Molecular docking program GOLD (Genetic Optimisation for Ligand Docking, Cambridge Crystallographic Data Centre, UK) was used to dock SRF and its analogues into the colchicine- binding site of tubulin-colchicine: stathmin like domain complex. An active site radius of 10 A was defined around bound colchicine. Root-Mean-Square-Deviation (RMSD), annealing parameters for hydrogen bonding and van der Waals interactions were considered within 1.5, 3, and 6 A, respectively.

[00220] General Procedure for the Synthesis of SRF and its analogs: The general synthetic route for SRF derivatives is depicted in Scheme 4. The esters 3 were prepared in two steps from commercially available cyclohexanone, cyclopentanone and cycloheptanone, respectively. Ester 3 was reacted with excess hydrazine in reflux ethanol to afford hydrazide 4 in good yield. Analogs of SRF 5 were obtained by the treatment of hydrazide 4 with substituted benzaldehydes or heterocyclic aldehydes.

4 5

Scheme 4: General Procedure for Synthesizing SRF and Analogs

[00221] A solution of sodium ethoxide was prepared by adding sodium (1.5 g, 65 mmol) to anhydrous ethyl alcohol (20 mL) at 0 °C. The mixture of cyclohexanone (4.41 g, 44 mmol) and diethyl oxalate (7.3 g, 50 mmol) was then slowly added and the solution stirred for 12 h at room temperature. Following decomposition of the reaction mix with 2N sulfuric acid solution, the mixture was extracted with ethyl acetate and the organic solvents were dried and concentrated. The crude product was further purified by column chromatography with n-hexane: ethyl acetate (12: 1) to afford 2 (5.87 g, 67 %) as a yellow oil. Next, hydrazine (448 mg, 14 mmol) was slowly added to a cooled suspension of compound 2 (2.38 g, 12 mmol) in acetic acid (5 mL). The mixture was heated to reflux for 1 h, poured into ice-H₂0, neutralized with NaHC0₃, and extracted with ethyl acetate. The combined organic layers were dried (Na₂S0₄), filtered, and concentrated. The residue was purified by column chromatography with n-hexane: ethyl acetate (3: 1) to yield 3 (2 g, 86 %) as a white solid. Ester 3 (970 mg, 5 mmol) and hydrazine (1.34 g, 35 mmol) in ethanol (10 mL) was next heated to reflux for 1 day. At the end of this period, ethanol was evaporated and the precipitate collected by filtration, washed with ethyl acetate and water to give hydrazide 4 as a white solid

(580 mg, 64 %). Next, hydrazide 4 (72 mg, 0.4 mmol) and substituted aldehydes (0.44 mmol) in ethanol (3 mL) was refluxed to obtain compound 5 as a precipitate. The precipitate was then collected by filtration and washed with cold ethanol (81 %).

Example 2: Identification of SRF as a Novel Tubulin-Binding Small Molecule

[00222] To identify novel anticancer molecules that act via binding to tubulin, the inventors initiated a target-based in silico screening of small molecules using a library of 1 million molecules from ChemDiv. ADMET filters were applied to the initial library to identify potential leads. The compounds were then virtually screened using the tubulin-colchicine: stathmin-like domain complex crystal structure (PDB 1 SAO) so as to ascertain their binding mode and measure binding affinities. The top 100 scoring ligands (based on their predicted binding mode/interactions) were profiled in a primary screen of in vitro tubulin polymerization in order to determine their ability to inhibit microtubule polymerization. Out of all the compounds tested, an indazole hydrazide derivative, 4,5,6,7-tetrahydro- lH-indazole-3-carboxylic acid [l-(3-hydroxy-4-methoxy-phenyl)- meth-(E)-ylidene]-hydrazide, was found to potently inhibit microtubule polymerization (IC50 = 0.77 μΜ).

Indazole hydrazide derivative, 4,5,6J-tetrahvdro- lH-indazole-3-carboxylic acid |^" l -(3-hydroxy-4- methoxy-phenylVmeth-(E -ylidene1-hvdrazide Identified By In Silico Screening

[00223] The indazole scaffold has been known to mediate a wide spectrum of biological activities including anticancer activity. To explore the structure activity relationship (SAR) and to identify analogs with increased potencies, a focused library of 70 SRF derivatives was synthesized and screened using cell viability assays. This effort identified an analog 4 as a potent molecule (IC₅o= 0.38 μΜ). From studies using the different analogs, a clear SAR trend emerged. The inventors found that cyclohexyl group in ring A is well tolerated while changes to a phenyl or R_\ and R₂ sites were well tolerated by small hydrophilic groups like methyl and ethyl. [00224] Ring A was modified to phenyl ring or small methyl group. In ring B, the inventors tried to investigate the role of 2 nitrogen atoms in binding site, so indole and oxazole compounds were synthesized in order to compare with indazole and pyrazole compounds, respectively. Example 3: Biological Evaluation

[00225] In accordance with the general synthesis Scheme 4 illustrated above, many of the analogs evaluated herein were prepared as follows (Scheme 5): -

R-i + R - -(CH₂)3- R, + R₂ = -(CH₂)₅-

Scheme 5: Synthesis Route for SRF Derivatives

[00226] For SAR analysis, a number of substitutions were made with modifications in the rings A, B and C as shown in Scheme 1. Ring A R_\ + R₂) was substituted by 3-, 4- and 5-membered ring structures (Table 1-3). By varying Ring A size, the inventors found that compounds with 7- membered rings have decreased activity when compared to compounds that have 5- or 6 members. This indicates that the binding pocket could be small and hence does not accept bulky rings. For example, replacement of 4-membered with 5-membered ring resulted in a significant loss of activity (1 vs. 33, 4 vs. 39 and 14 vs. 38). Removal of Ring A (42 - 46) also resulted in complete loss of activity. Likewise, introduction of aromaticity on Ring A (47 - 49, Table 4) failed to show any inhibition. Taken together, present initial SAR studies reveal that only rings with 5-or 6-C long aliphatic chain are tolerated at position A.

[00227] Compounds containing 5-membered ring usually exhibited similar activity on microtubule to compounds with 6-membered ring, while 7-membered ring derivatives showed weaker activity. For example, compound 20 has similar IC₅₀ (0.69 μΜ) to SRF (0.77 μΜ) but a 7- membered ring compound that contains the same functional groups 3 -OH, 4-OMe at phenyl ring C inhibited at 5.23 μΜ concentration. Compounds 4 and 24 with the same 4-methyl group are 9 to 12 times more active than 7-membered ring compound 39. Perhaps, the heptacyclic ring is bulkier than hexacyclic one and is not tolerated in active site.

[00228] The effect of substituents at ring C on tubulin activity was also investigated (Table 1). Compounds that contain different groups at the 2 or 3 positions such as halides, hydroxyl, alkoxy or alkyl of phenyl ring did not show any significant activities. Even compounds with only a hydroxyl group at 3 -position of ring C have resulted in loss of activity nearly 100 times compared to SRF, pentacyclic and heptacyclic compounds with 3-OH, 4-OMe groups (entry 11, 30, and 40 vs entiy 1, 20, and 33, respectively).

Table 1 : The IC₅₀ of compounds on microtubule stability

Entry R, + R₂ Rj IC„0L ) Entry Ri+R_j :₅₀(μΜ)

1 -(CH₂)₄- 3-OH,4-OMe-P 0.77 ±0.01 21 -(CHJj- 4-OMe-Ph 12.9 ± 1.8

2 -(CH₂)₄- 3-OMe,4-OH-Ph > 100 22 -(CH₂)₃- 3-OA4e.4-OH-Ph > 100

-(C¾)₄- 3.4-OCH₂0-Ph 11.43 ±5.96 23 -(C¾)₃- 3.4-OCH₂0-EvPh 26.12 ±2.59

4 -(C¾)₄- 4-Me-Ph 0.38 ±0.08 24 -(CH),- 4-Me-Ph 0.63 ±0.01

-(C¾)- 3-Cl-Ph > 100 25 -(C¾)₃- 3-Cl-Ph > 100

6 -(CH₂)₄- 2-Cl-Ph > 100 26 -(CH₂)₃- 2-Cl-Ph > 100

7 -(«½),- 4-C!-Ph 5.94 ±2.20 27 -(CH,) 4-Cl-Ph 1.28 ±0.46

8 -(CH₂)- Ph > 100 28 -(C¾)_3- Ph > 100

9 -(CH₂)₄- 1 -naphthalene > 100 29 -(CHj),- 2-OH-Ph > 100

10 -(<-^"¾)„- 2-OH-Ph > 1.00 30 -(CH₂),- 3-OH-Ph >50

11 -(CH₂)₄- 3-OH-Ph >50 31 -(CH₂)₃- 4-OH-Ph > 100

12 -(CH₂)₄- 4-OH-Ph > 100 2 -(CH,)₃- 4-N0₂-Ph > 100

13 -(CH)- 4-OMe-Ph 5.30 ± 1.00 -( .H₂)₅- 3-OH.4-O e-Ph 5.23 ± 0.41

14 -(CH₂)- 4-Br-Ph 3.66 ± 1.03 34 -(CH)_?- 3.4-OCH₂0-Ph 24.21 ±0.99

15 -(CH₂)₄- 3-pyridine > 100 35 -(CH₂)s- 4-Cl-Ph > 50

16 -(C¾)- 2-thiophene >100 6 -(CH)₅- 3-Cl-Ph > 100

17 -(CH₂)- 4-COOH-Ph >100 37 -(CH₂)j- 3-Br-Ph > 100

18 -(CH₂)- 4-N0₂-Ph >100 38 -(CH₂)₅- 4-Br-Ph 61.42 ±3.77

19 -(CH₂)- 2-OH-3.5-Bu'-Ph > 100 39 -(CH)₅- 4-Me-Ph 5.60 ±0.44

20 -(C¾)3- 3-OH,4-OMe-Ph 0.69 ± 0.09 40 -( H)_S- 3-OH-Ph > 100

41 -(CH₂)_;- 2-Cl-Ph > 100

42 -CH 3-OH,4-OMe-Ph >100 46 -CH₃- 3,4-OCH₂0-Ph >100

43 -CH₃- 4-Me-Ph >100 47 -Ph- 3-OH,4-OMe-Ph >100

44 -CH₃- 4-Cl-Ph > 100 48 -Ph- 4-Me-Ph > 100

45 -CHj- 3-OH-Ph >10O 49 -Ph- 4-Cl-Ph >100

[00229] A hydrophobic group at 4-position plays an important role on the inhibitory effect. As shown in Table 1, hydrophilic groups like hydroxyl, nitro and carboxylic are not tolerated (entry 12, 17, 18). In the series of hexacyclic compounds, derivatives with hydrophobic groups such as halide groups (entry 7, 14) showed moderate activity. Compound 13 with 4-OMe group inhibited at 5.30 _jUM concentration, which is 10 times higher than the activity of compound 1 1 but 10 times lower than SRF. In case of lead compound SRF, perhaps an additional interaction of hydroxy group at 3 -position fixes the 4-methoxy fit nicely into active site. However, the most potent compound was found to have a methyl group at 4-position (entry 4). The activity of compound 4 is twice as much as the lead compound SRF. The inventors also observed similar manner in the series of compounds with 5-membered ring. Compound 24 exhibited the strongest effect (0.63 μΜ) among pentacyclic compounds tested.

[00230] The inventors prepared compounds with 3-pyridine and 3-thiophene instead of phenyl ring but neither compound shows significant activity. [00231] To summarize, this preliminary SAR study has showed that, (i) Ring A: a hexacylic ring or smaller one might be good for activity; and (ii) hydrophobic groups or the combination of

3-OH, 4-OMe have an important role on microtubulin effect.

[00232] In subsequent steps, the inventors prepared compounds with modifications at ring A and illustrated in the following schemes.

Scheme 1: Synthesis of indazole and indole compounds.

Y= NH 7 Y= NH

Y= O 8 Y= 0

Scheme 2: Synthesis of pyrazole and indole compounds.

1

R_{1 +} R₂ = -(CH₂)₄-

R-i + R₂ = -(C H2)3-

Scheme 3: Synthesis of indazole compounds

[00233] Compounds with indazole, indole, pyrazole and oxazole were employed for the study. Ring A was modified to phenyl ring or small methyl group. In ring B, the inventors tried to investigate the role of 2 nitrogen atoms in binding site, so indole and oxazole compounds were synthesized in order to compare with indazole and pyrazole compounds, respectively.

[00234] The synthesis of these series of compounds was outlined in Schemes 2 and 3 with similar method to Scheme 1. Target compounds were easily prepared from ester compounds 6-10 in 2 steps. Based on the SAR study, the inventors had used benzaldehydes with 3-OH-4-OMe or hydrophobic groups at 4 position. The activities of compounds obtained with substitutions on Ring C on microtubule stability were showed in Table 2.

[00235] But all the synthesized compounds did not show significant activity. Therefore, changes at ring A to phenyl ring or small methyl group are not suitable at ring A for microtubule effect. Compounds with indole or oxazole ring just resulted in a loss of activity. Table 2: The IC ₀ of compounds on microtubule stability.

Compound

Entry Κ:₅₀ (μΜ)

s

1 4a 3-OH,4-OMe-Ph > 100

2 4b 4-Me-Ph > 100

3 4c 4-Cl > 100

4 5a 3-OH,4-OMe-Ph > 100

5 5b 4-Me-Ph > 100

6 6a 3-OH,4-OMe-Ph > 100

7 6b 4-Me-Ph > 100

8 7a 3-OH,4-OMe-Ph > 100

9 7b 4-Me-Ph > 100

10 7c 4-Cl > 100

1 1 8a 3-OH,4-OMe-Ph > 100

12 8b 4-Me-Ph > 100

[00236] The inventors then prepared several derivatives with different hydrophobic groups in the series of pentacyclic and hexacyclic compounds with substitutions on Ring B. Compounds 9 and 10 were synthesized by reaction of corresponding hydrazide with l-(4- trifluoromethylphenyl)ethanone. The IC₅os were showed in Table 3. Table 3. The IC₅₀ of compounds on microtubule stability.

Entry Ri + R₂ IC„(MM) Entry R1 + R2 R3 Κ^'ίο ίμΜ)

1 -(CH )₄- 4-r-Bu > 100 9 -(CH,)₃- 4-r-Bu > 100

2 -(C¾) - 4-SMe 5.83 ± 0.60 10 -(CH,)₃- 4-SMe 0.86 ± 0.02

3 -(C¾)₄- 4-CF₃ > 50 11 -( H,),- 4-CF₃ 5.51 ± 0.50

4 -(CH₂)₄- 9 > 100 12 -«¾),- 10 > 100

5 -(CH₂) - NMe, 6.99 ± 0.80 13 -(CH₂)_?- NMe₂ 1 .32 ± 0.40

6 -(CH,)₄- OCF., > 100 14 -(CH_:)₃- OCF., > 100

7 -(C¾)₄- 3,4-Me₂-Ph 2.61 ± 0.80 1 -(CH₂)₃- 3.4-Me₂-Ph 0.68 ± 0.12

8 -( H_;)₄- 4-OEt > 100 16 -(CH,)₄- F > 100

[00237] At the 4-position on phenyl ring, certain steric substitutions like Me, OMe, SMe, and NMe₂ are accommodated. Compounds with 3,4-dimethyl groups showed similar or lower effect to compounds with only 4-methyl group. Unlike OH group at the lead SRF, methyl group at 3- position does not enhance the activity. The pentacyclic derivatives exhibited slightly better effect than hexacylic ones. The inventors have observed that compounds with small group (such as F) or bulky groups like tert-Bu, OCF₃, OEt and CF3 have no significant activity. The introduction of methyl group at imine group (compound 9 and 10) just resulted on the loss of activity.

[00238] In brief, the inventors have successfully optimized lead compound SRF and potent inhibitors of microtubulin depolymerization (68 nM) have been found.

Example 4: SRF Selectively Inhibits Cellular Proliferation of Various Cancer Cell Types [00239] The inventors used a colorimetric assay to evaluate the anti-proliferative effect of SRF on cancer cells derived from a broad spectrum of representative tumor types: cervical

adenocarcinoma (HeLa, KB-3-1, KB-Vl), T-cell lymphoma (Jurkat), gastric carcinoma (SNU-16), breast cancer (MDA-MB-231), lung cancer (A549), colorectal adenocarcinoma (HCT-15) and neuroblastoma (SH-SY5Y). All the tested cancer cell lines showed susceptibility to SRF with IC50 values in the submicromolar range (0.08 - 0.38 μΜ), see Table 4. Interestingly, when compared with paclitaxel, SRF exhibited higher potency towards the neuroblastoma SH-SY5Y and colorectal adenocarcinoma HCT-15 cell lines. Furthermore, the inventors also tested the effect of SRF on normal cells like skin (CCL-1 16) and foreskin fibroblasts (F-10). Both CCL-1 16 and F-10 showed greater than 80 % survival following a 72 h exposure to SRF while only 40-60 % survival was seen when cells were treated with paclitaxel under identical conditions (Fig. 1). Taken together, present data demonstrates conclusively that SRF selectively targets cancer cells and is efficacious against several different cancer types.

Table 4: Effect of SRF on Viability of Different Cancer Cell Types

IC50

Cell Line ^TY ^e SRF Paclitaxel μΜ iiM

HeLa Cervical adenocarcinoma 0.02 ± 0.01 6.78 ± 0.19

Jurkat T-cell lymphoma 0.21 ± 0.01 5.37 ± 0.10

SNU-16 Gastric carcinoma 0.38 ± 0.03 2.95 ± 0.03

MDA-MB-231 Breast adenocarcinoma 0.25 ± 0.08 66.95 ± 39.21

A549 Lung carcinoma 0.23 ± 0.01 15.30 ± 0.86

SH-SY5Y Neuroblastoma 0.08 ± 0.02 5241 ± 366

HCT- 15 Colorectal adenocarcinoma 0.24 ± 0.01 437 ± 70.12

KB-3-1 Cervical adenocarcinoma 0.21 ± 0.01 6.65 ± 0.12

Example 5; SRF can Bypass the P-glycoprotein Drug Efflux Pump

[00240] The clinical success of the drugs used in cancer treatment has been hampered by the development of drug-resistance - a serious obstacle accounting for majority of cancer-related deaths. The phenomenon of multidrug resistance is often associated with an increase in expression of mdrl gene, which encodes P-glycoprotein (P-gp). As an energy-dependent efflux pump, P-gp extrudes drugs from cells resulting in decreased drug concentrations and reduced efficacy. To be clinically relevant, SRF must be able to bypass the P-gp-mediated efflux. Therefore, the inventors determined the effect of SRF on the proliferation of epidermal carcinoma cell line KB-Vl (Shen, Cardarelli et al. 1986), a vinblastine resistant subline derived from the parental KB-3-1 cells (Akiyama, Fojo et al. 1985). Membrane fractions of KB-Vl cells are highly enriched in pl70 glycoprotein and they show cross-resistance to colchicine and adriamycin. Comparison of the antiproliferative effect mediated by SRF and paclitaxel reveals that in KB-Vl cell line, SRF is 35-fold more potent than paclitaxel while both compounds showed similar efficacy for KB-3-1 cells as it is not known to overexpress P-gp (Bates, Mickley et al. 1989). The ability of SRF to bypass the P-gp probably explains why it is more potent than paclitaxel in the SH-SY5Y neuroblastoma and HCT- 15 colorectal adenocarcinoma cells as both these cancer cell types express elevated levels of P-gp protein (Bates, Mickley et al. 1989; Wu,-Smythe et al. 1992; Watanabe, Naito et al. 1997).

Example 6: SRF Destabilizes Microtubule Assembly by Binding to Colchicine-Binding Sites

[00241] To evaluate the principal mode of action of SRF, the inventors profiled its effect on tubulin polymerization in vitro. The results show that SRF inhibits polymerization of tubulin in a concentration dependent manner, with an IC50 of 0.38 μΜ. Notably, SRF affected the onset of the polymerization (nucleation phase) and its rate (elongation phase) resulting in reduced tubulin polymer mass. Thus, SRF alters microtubule polymerization in a manner similar to that mediated by nocodazole, a well-known microtubule-destabilizing agent. The inventors also studied the effect of SRF on the microtubule assembly at the cellular level using immunohistochemistry. HeLa cells were exposed to SRF for 24 h, fixed microtubules were stained using - or /3-tubulin antibodies. Compared with untreated control cells, which maintain an intricate and organized microtubule network, SRF treatment caused a complete destruction of this cytoskeletal network; microtubules cluster (bundle) and appear as punctae that are randomly distributed all over the cytoplasm. Similar disruption of the microtubules was observed with nocodazole exposure whilst paclitaxel treatment produced shorter but denser microtubules consistent with its role as a micro tubule-stabilizing agent.

[00242] Compounds that inhibit tubulin polymerization mostly bind to the colchicine or vinblastine-binding site of colchicine. In order to probe the tubulin-binding site of SRF, the inventors used the competition-binding scintillation proximity assay (SPA). Based on present results, the inventors could conclude that SRF does not reduce specific SPA counts stimulated by conjugating the biotin-labeled tubulin with [ 3 H]paclitaxel or with [ 3 HJvinblastine, but strongly competes with [³H]colchicine binding to tubulin.

[00243] Next, molecular modeling and docking studies were done to further ascertain the binding mode of SRF. Using the crystal structure of the tubulin-colchicine: stathmin-like domain

(PDB 1SA0), both SRF and colchicine were docked into the colchicine-binding pocket of tubulin.

Despite the structural dissimilarity, SRF shares the same Cartesian spare as colchicine. However, unlike colchicine, SRF lacks hydrogen bond interactions with Cys B241; instead it orients itself in a manner such that it has lateral interactions at the interface of a- and -tubulin subunits. The indazole ring interacts with Asn B349, Val A181, Lys B352 and Thr A179 while the 4-methoxy oxygen, 3 -hydroxy oxygen and 3 -hydroxy hydrogen of the distal phenyl ring interacts with Lys B254, Ala B250, Leu B248, respectively. Thus, the results indicate that the SRF binding mode is slightly different from colchicine even though both these compounds bind to the identical site on tubulin.

Example 7: SRF Arrests Cell Cycle in G2/M-Phase

[00244] Since microtubule dynamics has important roles in cell cycle progression, mitotic stall may lead to various chemotherapeutic outcomes including mitotic death, mitotic exit, apoptosis or aneuploidy (Kops, Weaver et al. 2005; Liou, Hsu et al. 2007). In order to ascertain the effect of

SRF on cell cycle progression, the inventors treated HeLa cells with a single concentration of SRF for 24 h and cell cycle was monitored by flow cytometric analysis. SRF treatment caused a complete collapse of all the cells at the Gi phase, resulting in a dramatic increase in the G₂/M population. Similar effects on microtubules were observed with nocodazole and paclitaxel. Since cell cycle arrest invariably triggers apoptosis, the inventors performed annexin V-PI double staining on SRF -treated cells so as to monitor phosphatidylserine exposure, a signal for early apoptosis. Following only a 7 h exposure, the population of early apoptotic cells (annexin V⁺/PI^") dramatically increased from 2.4 % to 21.8 % while the number of cells undergoing late apoptosis

(annexin V⁺/PI⁺) showed only a marginal increase (from 0.5 % to 3.7 %) during this period. Example 8: SRF Induces Apoptosis via JNK-Mediated Phosphorylation of Bcl-2 and Bad

[00245] The Bcl-2 proto-oncogene family are master regulators of apoptosis and function as molecular rheostats to control cellular survival (White 1996). Several lines of evidence have shown that the increased levels of Bcl-2 in a variety of human cancers, including some of the common leukemias and lymphomas, is responsible for chemoresistance (Reed 1997). Anti-mitotic drugs like paclitaxel, vincristine, vinblastine, docetaxel induce Bcl-2 hyperphosphorylation in the loop region as this region contains several phosphorylation sites (Haldar, Basu et al. 1997; Basu and Haldar

1998; Haldar, Basu et al. 1998; Srivastava, Mi et al. 1999; Yamamoto, Ichijo et al. 1999).

[00246] Likewise, deletion of the loop region negatively regulates Bcl-2 function in apoptosis

(Srivastava, Mi et al. 1999). Taken together, these findings imply that Bcl-2 phosphorylation inhibits its anti-apoptotic role. Therefore, the inventors investigated the effect of SRF on Bcl-2.

Extracts from HeLa cells treated with 10 μΜ of SRF for 24 h were analyzed by immublotting with monoclonal anti-Bcl-2 antibody. The results show that together with a Bcl-2, SRF-treated cells have additional band(s) that has slower migration rate compared to Bcl-2. Control cells completely lacked the extra band(s). To confirm that the band(s) is indeed the phosphorylated forms of Bcl-2, blots were then probed with phospho-Bcl-2 specific antibodies. SRF induced the phosphorylation of Bcl-2 at multiple residues (T56, S70 and S87), all of which lie within a flexible loop region. In contrast to HeLa cells, the phosphorylation state of Bcl-2 in the normal diploid fibroblast cell line WI-38 remained unchanged even after 24 h SRF treatment. This observation further reiterates the fact that SRF selectively targets cancer cells. In addition to Bcl-2, a 24 h exposure to SRF treatment also induced the phosphorylation of the pro-apoptotic Bcl-2 family member (Bad) while Bcl-xL, Bak and Bax remained unaffected (see Fig. 2).

[00247] The mitogen-activated protein kinases (MAPK), expressed in all cell types, transduce signals from the cell membrane to the nucleus in response to a variety of stimuli and also regulate a wide spectrum of biological processes critical for cellular homeostasis (Dhanasekaran and

Johnson 2007). Among the different pathways mediated by MAPK family members, the extracellular signal-regulated kinase 1 and 2 (ERK1/2), c- Jun N-terminal kinase/ stress-activated protein kinase (JNK SAPK) and p38 MAPK, are known to be activated by cellular stresses such as heat, osmotic shock, UV and γ irradiation, metabolic poisons, pro-inflammatory cytokines and in some instances, by growth factors and GPCR agonists (Derijard, Hibi et al. 1994; Raingeaud,

Gupta et al. 1995; Rosette and Karin 1996; Raman, Chen et al. 2007). In fact, it has been shown that vinblastine-induces apoptosis via JNK-mediated phosphorylation of Bcl-2 (Fan, Goodwin et al. 2000) while all three MAPK pathways (JNK, ERK and p38 kinase) are activated upon paclitaxel exposure (Okano and Rustgi 2001; Boldt, Weidle et al. 2002).

[00248] The inventors therefore wanted to monitor the activation of MAPK pathway/s following treatment of HeLa cells with SRF for 24 h. Phosphospecific antibodies against JNK, EPvKl/2 and p38 were used to selectively stain for activated form of these kinases. While SRF treatment had no effect on the expression levels of the kinases, SRF induced an increase in JNK activity as evident by the appearance of a band corresponding to phosphorylated form of JNK. No change in specific activity of ERKl/2 and p38 were detected.

[00249] To further verify that activation of JNK cascade is indeed casual to phosphorylation of Bcl-2 and Bad, the inventors employed specific inhibitors, SP600125, PD98059 and SB203580 that target JNK, ERKl/2 and p38 kinase, respectively. Prior to SRF treatment, cells were incubated with the MAPK inhibitors at the indicated concentrations for 1 h. While PD98035 and SB203580 did not alter Bcl-2 phosphorylation, pre-treatment with SP600125 completely abolished the presence of the phosphorylated forms of both Bcl-2 and Bad.

[00250] Further evidence that the JNK signaling pathway is central to SRF-mediated toxicity came from cell viability assays, immunohistochemistry and flow cytometric analysis. Pre-treatment of HeLa cells with SP600125 resulted in a 20 % increase in cell viability when compared to control cells exposed to only SRF while PD98059 and SB203580 treatment did not show any significant reduction in cellular toxicity. Immunohistochemical analysis revealed that cells treated with

SP600125 were able to maintain the intricate microtubule network that is normally destroyed upon

SRF treatment while cell cycle analysis by flow cytometry showed that 60 % of SP600125 pre- treated cells can overcome the SRF-mediated G₂/M arrest. Cells treated with PD98059 or SB203580 continue to remain arrested in the G₂/M phase and eventually undergoes apoptotic death (see Fig. 3).

Example 9: SRF-Mediated Apoptosis Involves Via Loss of Mitochondrial Membrane Potential and Activation of Caspase-3

[00251] Various extracellular and intracellular stresses trigger the intrinsic apoptotic pathway and these signals converge mainly on the mitochondria resulting in opening of the mitochondrial transition pore (MTP), loss of mitochondrial transmembrane potential and release of cytochrome c together with other pro-apoptotic molecules (Okada and Mak 2004; Elmore 2007). The Bcl-2 family members govern mitochondrial integrity and regulate the apoptotic mitochondrial events

(Coiy and Adams 2002). Since phosphorylation of Bcl-2 and Bad results in loss of their cellular activity, the inventors assessed the effect of SRF on the integrity of the mitochondrial membrane.

[00252] Using the JC-1 reagent in flow cytometric analysis, the inventors found that SRF treatment results in a time-dependent increase (from 4.8 % to 84.2 %) of the JC-1 monomelic form that eventually peaks at 24 h while pre-treatment with the SP600125 inhibitor resulted in a 10-fold increase of green fluorescence (from 4.8 % to 47.8 %). Furthermore, loss of mitochondrial integrity was accompanied by caspase-3 activation as was evident from immunoblots with anti-caspase-3 antibody. A cleaved band corresponding to activated caspase-3 was present in extracts of cells treated with SRF while control cells completely lacked this form. Likewise, lysates from cells treated with SRF showed a 3-fold increase in caspase-3 activity over control cells when enzymatic activity was assessed using a caspase-3 specific fluorogenic tetrapeptide substrate, Ac-DEVD-pNA (acetyl- Asp-Glu-Val- Asp p-nitroanilide). In all cases, pre-treatment of cells with the JNK inhibitor SP600125 abrogated the caspase-3 activation thereby further underscoring the importance of JNK in SRF-mediated cell death pathway.

Example 10: SRF Inhibits in vivo Tumor Growth in a Human Colon Adenocarcinoma Xenograft Model

[00253] Data from the preceding section suggests that SRF retains substantial activity against HCT-15 cells that expresses high levels of P-glycoprotein. The in vivo anti-tumor activity of SRF was evaluated in SCID mice using human colorectal adenocarcinoma HCT-15 xenograft models. SRF was administered at 20 mg/kg/dose every 3 days over a 9-day period. On day 9, the relative tumor volume (RTV) of the group that received SRF was reduced by 40 % in comparison to the control group. This suggests that SRF is effective in suppressing in vivo tumor growth.

CONCLUSION:

[00254] In present effort to identify new tubulin-binding agents (TBAs), the inventors had performed an in silico virtual screening of chemical library against tubulin to identify suitable indazole hydrazide derivatives as novel TBAs. SAR analysis and lead optimization gave the final compound, suprafenacine (SRF), which inhibits tubulin polymerization with submicromolar potencies. Present scintillation proximity assay results show that SRF binds to the colchicine- binding site on tubulin but has a binding mode distinct from that of colchicine. Owing to its extreme toxicity, colchicine itself is not an anticancer agent but other colchicine-domain binding drugs, such as combretastatins, 2-metoxyestradiol (2-ME) and chalcones, are now being actively investigated for their anticancer activities (Stanton, Gernert et al. 2011). SRF shows broad- spectrum activity as it effectively inhibits proliferation of several different cancer cell types derived from solid tumors and hematological malignancies, as well as neuroblastomas and drug-resistant sublines (Tables 1 and 2). Notably, SRF is selective against cancer cells, as it had little or no effect on normal diploid cells like CCL-1 16, F-10 and WI-38. The basis of this selectivity is presently not known. SRF is not a substrate for the efflux pumps as it exhibits a similar potency irrespective of the cell's MDR status. However, whether SRF can overcome resistance mediated by tubulin mutations and/or expression of different tubulin isotypes is a matter that needs further

investigation.

[00255] To further evaluate that microtubules are bona fide targets of SRF, the effect of the compound at the cellular level was analyzed by immunofluorescence microscopy. Microtubule binding by SRF completely destroyed the cellular microtubule network causing the microtubules to bundle indicating a decrease in the polymer mass. Since attenuation of microtubule dynamics engages cell cycle surveillance mechanisms to arrest cell division in mitosis, SRF-mediated inhibition of microtubule polymerization leads to a complete blockade of the cell cycle at the G₂/M phase and eventual cell death by apoptosis. Cell death was rapid as annexin V-stained cells could be detected by flow cytometry within 7 h of SRF treatment. Induction of apoptosis causes phosphatidylserine (PS) to externalize from the inner (cytoplasmic) leaflet to the outer (cell surface) leaflet of the plasma membrane and this PS exposure can be detected by labeled annexin

V. SRF-mediated cell death had all the canonical hallmarks of apoptosis as was evident from loss of mitochondrial membrane potential and concomitant activation of caspase-3. It is therefore hypothesized that SRF triggers apoptosis via the intrinsic mitochondrial pathway.

[00256] The Bcl-2 protein family has pivotal roles in regulating cell survival in part by affecting the mitochondrial compartmentalization of cytochrome c (Vander Heiden and Thompson 1999).

Since SRF-treatment causes loss of mitochondrial membrane integrity, the inventors examined its effect on the Bcl-2 proteins. Bak, Bax and Bcl-xL remained unaltered while Bcl-2 was

hyperphosphorylated at multiple serine and threonine residues (T56, S70 and S87) that lie within the unstructured flexible-loop region of the protein. Bcl-2 phosphorylation interferes with its anti- apoptotic role as it losses its ability to heterodimerize with Bax (Srivastava, Srivastava et al. 1998;

Poommipanit, Chen et al. 1999). Consequently, cells arrested in the G₂/M phase become increasingly sensitized to stress signals and undergo rapid apoptosis (Yamamoto, Ichijo et al.

1999). In addition to phospho Bcl-2, the inventors also observed the presence of the phosphorylated form of the BH3-only protein Bad, in HeLa cell extracts treated with SRF. Bad is unique as it serves to integrate both prosurvival and proapoptotic signals with the net effect contributing to either cell survival or apoptosis. Survival-promoting kinases including Akt, Rsk, p21 -activated kinase, p70S6 kinase, suppress Bad-mediated apoptosis by inducing Bad

phosphorylation at SI 12 and/or S136 (Datta, Dudek et al. 1997; del Peso, Gonzalez-Garcia et al. 1997; Bonni, Brunei et al. 1999; Harada, Andersen et al. 2001) and this, in turn, leads to Bad sequestration by 14-3-3 proteins (Zha, Harada et al. 1996).

[00257] On the other hand, apoptosis-inducing kinases like Cdc2 and JNK mediate Bad phosphorylation at SI 28 (Donovan, Becker et al. 2002; Konishi, Lehtinen et al. 2002). Whether

SRF induces Bad phosphorylation at SI 28 remains to be determined. Furthermore, the inventors also delineated the signaling cascade responsible for Bcl-2 and Bad phosphorylation. Consistent with literature reports (Maundrell, Antonsson et al. 1997; Yamamoto, Ichijo et al. 1999; Fan,

Goodwin et al. 2000; Donovan, Becker et al. 2002), JNK was found to be involved in both cases of phosphorylation. These events were abrogated when cells were pre-treated with the JNK inhibitor

SP600125 but not with PD98058 or SB203580, which inhibit ERK1/2 and p38 kinase,

respectively. Cells pre-treated with SP600125 were also able to overcome G₂/M cell cycle blockade following SRF exposure, retain mitochondrial membrane integrity and prevent caspase-3 activation thereby underscoring the importance of JNK signaling in SRF-mediated cellular toxicity. [00258] Thus, the data presented herein provides compelling evidence that the novel indazole hydrazide-based compound, SRF, by virtue of (1) its has broad-spectrum efficacy against several different tumor types; (2) its selectivity for cancer cell types; and (3) its ability to bypass multidrug resistance, has potential as an antineoplastic drug for treatment of various types of cancer. Present results warrant further investigation of the pharmacokinetic properties of SRF and detailed evaluation of its in vivo efficacy in animal models as the SRF scaffold holds great promise as a prototype for the development of a new class of anti-tubulin agents.

[00259] By "comprising" it is meant including, but not limited to, whatever follows the word "comprising". Thus, use of the term "comprising" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present.

[00260] By "consisting of is meant including, and limited to, whatever follows the phrase "consisting of. Thus, the phrase "consisting of indicates that the listed elements are required or mandatory, and that no other elements may be present.

[00261] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[00262] By "about" in relation to a given numberical value, such as for temperature and period of time, it is meant to include numerical values within 10% of the specified value.

[00263] The invention has been described broadly and ge erically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[00264] Other embodiments are within the following claims and non- limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

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Claims

1. A compound of Formula (I)

for use as a medicament,

wherein:

m is 0, 1, 2, 3, 4, or 5;

Ri and R₂ together form a five-membered, six-membered, or seven-membered ring, wherein Ri and R₂ together as a group is -(CH₂)3-, -(CH₂)₄-, or -(CH₂)s-;

R₃ at each occurrence is independently selected from the group consisting of H, halogen, hydroxyl, alkoxy, and a substituted or unsubstituted C 1-C5 alkyl; and 4 is H, halogen, or a substituted or unsubstituted CI -C5 alkyl.

2. The compound for use of claim 1, wherein:

m is 1 ;

Ri and R₂ together as a group is -(CH₂)₃- or -(CH₂)₄-;

halogen, alkoxy, or a substituted or unsubstituted C1-C5 alkyl; and

R4 IS H.

3. The compound for use of claim 2, wherein R₃ is Br, CI, methoxy, or methyl at the 4^th position of the phenyl ring.

4. The compound for use of claim 1, wherein:

m is 2;

Ri and R₂ together as a group is -(CH₂)₃- or -(CH₂)₄-;

R₃ is hydroxyl or alkoxy; and

R₄ is H.

5. The compound for use of claim 4, wherein a first R₃ is hydroxyl at the 3^ld position of the phenyl ring and a second R₃ is methoxy at the 4^th position of the phenyl ring.

6. The compound for use of any one of claims 1-5 in treating or preventing cancer in a subject.

7. The compound for use of claim 6, wherein the cancer is selected from the group consisting of cervical cancer, leukemia, stomach cancer, breast cancer, lung cancer, colon cancer, and neuroblastoma.

8. The compound for use of claim 6 or 7, wherein the subject is a mammal.

9. The compound for use of claim 8, wherein the subject is a human.

10. A method for inducing apoptosis in a cell, comprising contacting said cell with an effective amount of a compound of Formula (I)

wherein:

m is 0, 1, 2, 3, 4, or 5;

and R₂ together as a group is -(CH₂)₃-, -(CH₂)₄-, or -(CH₂)₅-;

R₄ is H, halogen, or a substituted or unsubstituted C1-C5 alkyl.

1 1. The method of claim 10, wherein:

m is 1;

Ri and R₂ together as a group is -(CH₂)₃- or -(CH₂)₄-;

halogen, alkoxy, or a substituted or unsubstituted C1-C5 alkyl; and

R₄ is H.

12. The method of claim 11, wherein R₃ is Br, CI, methoxy, or methyl at the 4 position of the phenyl ring.

13. The method of claim 10, wherein: m is 2;

Ri and R₂ together as a group is -(CH₂)₃- or -(CH₂)₄-;

R₃ is hydroxyl or alkoxy; and

R₄ is H.

14. The method of claim 13, wherein a first R₃ is hydroxyl at the 3^rd position of the phenyl ring and a second R₃ is methoxy at the 4^th position of the phenyl ring.

15. A method for treating or preventing cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a compound of Formula (I)

wherein:

m is 0, 1, 2, 3, 4, or 5;

R4 is H, halogen, or a substituted or unsubstituted C1-C5 alkyl.

16. The method of claim 15, wherein:

m is 1;

R] and R₂ together as a group is -(CH₂)₃- or -(CH₂)₄-;

R₃ is halogen, alkoxy, or a substituted or unsubstituted C1-C5 alkyl; and

R s H.

17. The method of claim 16, wherein R₃ is Br, CI, methoxy, or methyl at the 4^th position of the phenyl ring.

18. The method of claim 15, wherein:

m is 2;

Ri and R₂ together as a group is -(CH₂)₃- or -(CH₂)4-;

R₃ is hydroxyl or alkoxy; and

R4 is H.

19. The method of claim 18, wherein a first R₃ is hydroxyl at the 3^rd position of the phenyl ring and a second R₃ is methoxy at the 4^th position of the phenyl ring.

20. The method of any one of claims 15-19, wherein the cancer is selected from the group consisting of cervical cancer, leukemia, stomach cancer, breast cancer, lung cancer, colon cancer, and neuroblastoma.

21. The method of any one of claims 15-20, wherein the subject is a mammal. The method of claim 21, wherein the subject is a human.