Technical Field

The present invention generally relates to novel compounds, including phenolic analogues and derivatives thereof, which have anti-proliferative and/or apoptotic activities. The present invention also relates to the , use of such compounds in the treatment of conditions in which cells proliferate abnormally and/or show resistance to apoptosis.

Background

The rate of cell proliferation within a population of cells often depends on three main factors: i) the rate of cell division; ii) the proportion of cells within the population undergoing cell division; and iii) the rate of cell loss from the population due to terminal differentiation or cell death. Physiological processes such as tissue development and homeostasis require a balance between cell proliferation and programmed cell death (apoptosis) . The role of apoptosis is to remove . harmful, damaged or unwanted cells. However, certain cells, such as tumor cells, may develop resistance to apoptosis by adaptations that protect the cells from cell death induced by their abnormal physiological state. In certain disease states, such as tumorigenesis and cancer, it has been found that the tumor cells not only proliferate rapidly but may also show resistance to apoptosis, creating challenges in current cancer therapy. The proteins of B-cell lymphoma 2 (Bcl-2) family are key regulators of apoptosis. The Bcl-2 family consists of several conserved sequence motif known as Bcl-2 homology (BH) domains. Bcl-2 family members are divided into three groups based on the presence of BH domains . A first subfamily which includes Bcl-2, Bcl-xl and Mcl-1, contains BH1, BH2, BH3 and BH4 multi-domains and possesses pro-survival (anti-apoptotic) activity. A second subfamily which includes Bax and Bak, contains multi-BH domains and possesses pro-apoptotic activity. A third subfamily which includes Bad, Bik and Bid, also exhibits pro-apoptotic activity and contains BH3 domains only.

Over expression of pro-survival proteins of the Bcl- 2 family such as Bcl-xL and Mcl-1 is implicated in tumorigenesis . The BH1, BH2 and BH3 domains of pro- survival or anti-apoptotic proteins, e.g. Bcl-xL and Mcl- 1, form a hydrophobic groove, which is the docking site for the BH3 domain of pro-apoptotic proteins. Binding of the BH3 domain of Bak or Bax to the hydrophobic groove of the anti-apoptotic proteins can induce apoptotic effects. Hence, small molecules that inhibit the interaction of Bcl-xL/BH3 domain or Mcl-l/BH3 domain could potentially act as apoptosis modulators which will promote cell death in cancer cells or enhance tumour sensitivity to chemotherapy or radiotherapy.

There is therefore a need for small molecules that are capable of inhibiting anti-apoptotic proteins such as Bcl-xL and Mcl-1. This is particularly important considering that Mcl-1 has emerged as a major resistance factor in human chemoresistance of cancer cells. Small molecules capable of inhibiting such anti-apoptotic proteins can lead to a better treatment option for cancer patients .

There is also a need for molecular probes and/or medicaments comprising such small molecules to be made available for studies and treatments involving conditions such as cancer or tumorigenesis in which cells proliferate abnormally and/or show resistance to apoptosis .

There is further a ^' ne ^' ed for the small molecules to be easily obtained through a simple synthesis process.

Whilst a number of non-peptide small molecules have been developed and show activity as a Bel-2 inhibitor, it may be useful to have small molecules that can inhibit both Bcl-2 and Mcl-1 and/or selectively inhibit any one of the anti-apoptotic proteins.

More importantly, there is a need for compounds that have anti-proliferative and/or apoptotic effects against cells that proliferate abnormally and/or show resistance to apoptosis, but which have no or low cytotoxicity to normal cells (e.g. non-cancerous or non-tumor cells), for use as anti-proliferative and/or apoptotic- agents in studies and treatments involving conditions in which cells proliferate abnormally and/or show resistance to apoptosis .

Summary

According to a first aspect, there is provided use of a compound represented by the general formula (I) :

(I)

and pharmaceutically acceptable salts thereof, wherein

n is an integer from 2 to 5;

p is an integer from 0 to 4;

Ri is independently selected from the group consisting of a hydroxyl group, an alkoxy group, a thiol group, and a thioether group, wherein at least two of R _x are on the carbon-2 and carbon-5 positions of the phenyl group ;

R ₂ and R ₃ are independently a methylene group or a nucleophile;

Z is an oxygen (O) atom or sulfur (S) atom; and _.

T is hydrogen or an optionally substituted aliphatic group or optionally substituted aryl group;

in the manufacture of a medicament for prevention and/or treatment and/or containment of a condition associated with a cell proliferative disorder.

According to a second aspect, there is provided use of a compound represented by the general formula (I) : and pharmaceutic eof, wherein n is an integer from 2 to 5;

p is an integer from 0 to 4;

R _x is independently selected from the group consisting of a hydroxyl group, an alkoxy group, a thiol group, and a thioether group, wherein at least two of Ri are on the carbon- 2 and carbon- 5 positions of the phenyl group;

R ₂ and R ₃ are independently a methylene group or a nucleophile ;

Z is an oxygen (O) atom or sulfur (S) atom; and

T is hydrogen or an optionally substituted aliphatic group or optionally substituted aryl group;

in the manufacture of a molecular probe .

According to a third aspect, there is provided use of a compound represented by the general formula (I) : _.

and pharmaceutically acceptable salts thereof, whe n is an integer from 2 to 5 ;

p is an integer from 0 to 4 ;

Ri is selected from the group consisting of a hydroxyl group, an alkoxy group, a thiol group, and a thioether group, wherein at least two of R _± are on the carbon-2 and carbon-5 positions of the phenyl group;

R ₂ and R ₃ are independently a methylene group or a nucleophile ;

Z is an oxygen (0) atom or sulfur (S) atom; and

T is an optionally substituted aliphatic group having at least 9 carbon atoms;

in the manufacture of a medicament for the prevention and/or treatment of a condition associated with resistance to anti-cancer drugs.

According to a fourth aspect, there is provided a compound selected from the group consisting of:

According to a fifth aspect, there is provided a method of inducing apoptosis in a patient in need thereof comprising administering an effective amount of a compound ^" represented by the general formula (I) :

(I)

and pharmaceutically acceptable salts thereof, wherein

n is an integer from 2 to 5;

p is an integer from 0 to 4 ;

Ri is independently selected from the group consisting of a hydroxyl group, an alkoxy group, a thiol group, and a thioether group, wherein at least two of R _x are on the carbon-2 and carbon-5 positions of the phenyl group;

R ₂ and R ₃ are independently a methylene group or a nucleophile;

Z is an oxygen (O) atom or sulfur (S) atom; and

T is hydrogen or an optionally substituted aliphatic group or optionally substituted aryl group.

Advantageously, the compounds of formula (I) and pharmaceutically ^' acceptable salts thereof exhibit inhibitory activity against anti-apoptotic proteins such as Bcl-xL and Mcl-1, and can therefore be used to selectively target such anti-apoptotic proteins in cells that over-express such proteins (e.g. cancerous or tumor cells) . Advantageously, the compounds of formula (I) and pharmaceutically acceptable salts thereof are not cytotoxic to, or have reduced cytotoxicity against normal cells (e.g. non-cancerous or non-tumor cells) .

Advantageously, the compounds of formula (I) and pharmaceutically acceptable salts thereof, can be synthesized through a simple synthetic process.

According to a sixth aspect, there is provided a method of increasing sensitivity to chemotherapy in a patient in need thereof comprising administering an effective amount of an Mcl-1 inhibitor selected from a compound represented by the general formula (I) :

and pharmaceutically acceptable salts thereof, wherein n is an integer from 2 to 5 ;

p is an integer from 0 to 4 ;

Ri is selected from the group consisting of a hydroxy1 group, an alkoxy group, a thiol group, and a thioether group, wherein at least two of R _x are on the carbon-2 and carbon- 5 positions of the phenyl group;

R ₂ and R ₃ are independently a methylene group or a nucleophile;

Z is an oxygen (O) atom or sulfur (S) atom; and T is an optionally substituted aliphatic group having at least 9 carbon atoms .

Advantageously, the Mcl-1 inhibitors and pharmaceutically acceptable salts thereof are capable of selectively causing apoptosis of cells that over-express the Mcl-1 protein, which has emerged as a major resistance factor in human chemoresistance of cancer or tumor cells. Advantageously, the Mcl-1 inhibitors and pharmaceutically acceptable salts thereof are also non- cytotoxic or have reduced cytotoxicity against normal cells (e.g. non-cancerous or non-tumor cells) . Definitions

The following are some definitions that may be helpful in understanding the description of the present invention. These are intended as general definitions and should in no way limit the scope of the present invention to those terms alone, but are put forth for a better understanding of the following description.

Unless the context requires otherwise or specifically stated to the contrary; integers, steps, or elements of the invention recited herein as singular integers, steps or elements clearly encompass both singular and plural forms of the recited integers, steps or elements .

Those skilled in the art will also appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to ^" be understood that the invention includes all .such variations and modifications . The invention also includes all of the steps,; features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features .

The term "analogue" refers to a compound which comprises a chemically modified form of a specific compound or class thereof, and which maintains the chemical and/or biological activities that are characteristic of the specific compound and class. For example, the term "analogue" as used herein may be used to refer to a chemically modified form of a compound of formula (I) which maintains the cell anti-proliferative and/or apoptotic properties of a compound of formula (I) .

The term "derivative" refers to any salt, solvate or prodrug, e.g. ester, of a compound of the invention, which upon administration to a subject, is capable of providing (directly or indirectly) a compound of the invention, or an active metabolite or residue thereof. Preparation of such derivatives is routine to those skilled in the art without undue experimentation.

The term "active compound" in connection with the present invention is understood as meaning a compound as disclosed herein which is capable of causing a required therapeutic or non-therapeutic effect in a patient or subject, for example a cell anti-proliferative and/or apoptotic effect.

The term "pharmaceutically acceptable sal.t" refers to those salts which retain the chemical and/or biological effectiveness and properties of the active compound, which are not otherwise undesirable. A thorough discussion of pharmaceutically acceptable salts is available in Remington's Pharmaceutical Sciences (Mack Pub. Co. , N.J. 1991) .

The term "optionally substituted" as used herein means the group to which this term refers may be unsubstituted, or may be substituted with one or more groups independently selected from hydrogen, oxygen, sulfur, alkyl, alkenyl, alkynyl, thioalkyl, cycloalkyl, cycloalkenyl , heterocycloalkyl, halo, carboxyl, haloalkyl, haloalkynyl, hydroxy1, alkoxy, thioalkoxy, alkenyloxy, haloalkoxy, haloalkenyloxy, nitro, amino, nitroalkyl, nitroalkenyl , nitroalkynyl, nitroheterocyclyl, alkylamino-, dialkylamino, alkenylamiiie, alkynylamino, acyl, alkenoyl, alkynoyl, acylamino, diacylamino, acyloxy, alkylsulfonyloxy, heterocycloxy, heterocycloami.no, haloheterocycloalkyl , alkylsulfenyl , alkylcarbonyloxy, alkylthio, acylthio, phosphorus-containing groups such as phosphonyl and phosphinyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl , cyano, cyanate, isocyanate, -C (0) NH (alkyl) , and C(0)N(alkyl) ₂ .

The term "hydroxyl" as used herein . refers to the functional group -OH.

The term "alkoxy" as used herein refers to straight chain or branched alkyloxy groups. Examples include methoxy, ethoxy, n-propoxy, isopropoxy, tert-butoxy, and the like.

The term "thiol" means -SH.

The term "thioether" refers to ether groups wherein the oxygen is replaced with a sulfur. The thioether groups include but are not limited to -alkylene-S-alkyl, -alkylene-S-aryl, -alkylene-S-arylalkyl , -alkylene-S- alkylaryl, -aryl-S-alkyl, - aryl-S-aryl , -aryl-S- alkylaryl, -aryl-S-arylalkyl , -arylalkyl -S-alkyl, arylalkyl-S-aryl, -arylalkyl -S-alkylaryl, -arylalkyl-S- arylalkyl, -alkylaryl-S-alkyl, -alkylaryl-S-aryl , alkylaryl-S-alkylaryl , and -alkylaryl-S-arylalkyl . The thioether groups may be optionally substituted as described above .

The term "nucleophile" as used herein refers to a chemical moiety that has a reactive pair of electrons and that participates in a chemical reaction by donating electrons, i.e., nucleophiles are electron donor compounds. The nucleophile may be a halogen, nitrogen, sulfur or oxygen nucleophile. Exemplary nucleophiles include fluorides, cyanides, iodides, chlorides, bromides, acetates, enolates, primary amines, secondary amines, amino, alkoxides, thiols, alkyl sulfides (such as mercaptans) , hydroxides, azides, and hydrazines, among others .

The term "amino" as used herein refers to groups of the form -NR _a or -NR _a Rb wherein R _a and R _b are independently selected from the group including but not limited to hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy and optionally substituted aryl groups. For example, the term "amino" as used herein may be used to refer to an -NH group at the R ₂ and/or R ₃ position of a compound of formula (I) , or to an -NHCH ₃ group in the optionally substituted aliphatic group at the T position of a compound of formula (I) .

The term "aliphatic" refers to a. linear, branched, or cyclic alkyl, alkenyl, or alkynyl group.

The term "alkyl" includes within its meaning straight chain or branched chain saturated aliphatic groups having from 1 to 10 carbon atoms, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. For example, the term alkyl includes, but is not limited to, methyl, ethyl, 1-propyl, isopropyl, 1-butyl, 2 -butyl, isobutyl, tert-butyl, amyl, 1 , 2 -dimethylpropyl , 1 , 1-dimethylpropyl , pentyl, isopentyl, hexyl, 4-methylpentyl, 1-m ^' ethylpentyl , 2-methylpentyl, 3 -methylpentyl , 2 , 2 -dimethylbutyl , 3,3- dimethylbutyl , 1, 2 -dimethylbutyl, 1 , 3 -dimethylbutyl , 1, 2, 2-trimethylpropyl, 1, 1, 2-trimethylpropyl, 2- ethylpentyl, 3-ethylpentyl, heptyl, 1-methylhexyl , 2,2- dimethylpentyl , 3 , 3 -dimethylpentyl , 4 , 4 -dimethylpentyl , 1, 2-dimethylpentyl, 1, 3 -dimethylpentyl, 1,4- dimethylpentyl, 1, 2 , 3-trimethylbutyl, 1,1,2- trimethylbutyl, 1, 1, 3-trimethylbutyl, 5-methylheptyl , 1- methylheptyl , octyl, nonyl, decyl, and the like.

The term "lower alkyl" refers to a straight or branched saturated hydrocarbon chain having 1 , 2 , 3 , , 5, or 6 carbon atoms. This term is exemplified by groups such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, t-butyl, n-pentyl, n-hexyl, and the like.

The term "alkenyl" includes within its meaning straight or branched chain unsaturated aliphatic hydrocarbon groups having from 2 to 10 carbon atoms, e.g.,- 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms and having at least one double bond, of either E, Z, cis or trans stereochemistry where applicable, anywhere in the alkyl chain. Examples of alkenyl groups include but are not limited to ethenyl, vinyl, allyl, 1-methylvinyl , 1- propenyl, 2-propenyl, 2 -methyl- 1-propenyl , 2 -methyl- 1- propenyl, 1-butenyl, 2-butenyl, 3-butentyl, 1,3- butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4- pentenyl, 1, 3-pentadienyl, 2, 4-pentadienyl, 1,4- pentadienyl, 3 -methyl- 2-butenyl , 1-hexenyl, 2-hexenyl, 3- hexenyl, 1, 3-hexadienyl, 1, 4-hexadienyl, 2- methylpentenyl, 1-heptenyl, 2-heptentyl, 3-heptenyl, 1- octenyl, 1-nonenyl, 1-decenyl, and the like.

The term "lower alkenyl" refers to a straight or branched saturated hydrocarbon chain having 2 , 3 , 4 , 5 , or 6 carbon atoms . The term "alkynyl" as used herein includes within its meaning straight or branched chain unsaturated aliphatic hydrocarbon groups having from 2 to 10 carbon atoms and having at least one triple bond anywhere in the carbon chain. Examples of alkynyl groups include but are not limited to ethynyl, 1-propynyl, 1-butynyl, 2-butynyl, 1-methyl-2 -butynyl, 3 -methyl-1-butyriyl, 1-pentynyl, 1- hexynyl, 1-methylpentynyl , 1-heptynyl, 2-heptynyl, 1- octynyl, 2-octynyl, 1-nonyl, 1-decynyl, and the like.

The term "lower alkynyl" refers to a straight or branched saturated hydrocarbon chain having 2, 3, 4, 5, or 6 carbon atoms .

The term "heteroatom" or variants such as "hetero-" as used herein refers to oxygen (O) , nitrogen (N) , phosphorus (P) and sulfur (S) .

The term "aryl" or variants such as "aromatic group" or "arylene" as used herein refers to single, polynuclear, conjugated or fused residues of aromatic hydrocarbons having from 6 to 10 carbon atoms. Exemplary aryl groups include, but are not limited to phenyl, naphthyl, tetrahydronaphthyl, and the like.

The term "heteroaryl" and variants such as "heteroaromatic group" or "heteroarylene" as used herein, includes within its meaning single, polynuclear, conjugated or fused aromatic moieties having 5 to 20 atoms wherein 1 to 6 atoms are heteroatoms selected from O, N, NH and S. Examples of such groups include pyridyl, 2 , 2' -bipyridyl , phenanthrolinyl , quinolinyl, thiophenyl, indoyl, furanyl and pyrrolyl moieties and the like.

The term "cycloalkyl" as used herein refers to cyclic saturated aliphatic groups and includes within its meaning monocyclic, bicyclic, polycyclic or fused polycyclic hydrocarbon moieties having from 3 to 10 carbon atoms, e.g., 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. Examples of cycloalkyl groups include but are not limited to cyclopropyl, 2 -methylcyclopropyl , cyclobutyl, cyclopentyl, 2-methylcyclopentyl, 3-methylcyclopentyl, cyclohexyl, and the like.

The term "halide" or variants such as "halogen" or "halo" as used herein refers, to fluoride, chloride, bromide and iodide.

The present invention includes within its scope all isomeric forms of the compounds disclosed herein, including all diastereomeric isomers, racemates and enantiomers. Thus, compounds of formula. (I) and derivatives thereof ^' should be understood to include, for example, E, Z, cis, trans, (R) , (S) , (L) , (D) , (+) , and/or ( - ) forms of the compounds , as appropriate in each case .

The term "normal" when used in the context of "normal cell," is meant to refer to a cell exhibiting a normal or unaltered cellular and/or physiological state, for example a cell having normal rate of cell division or cellular differentiation. A "normal" cell, for example, includes one that does not exhibit a malignant phenotype, such as a non-cancerous or non-tumor cell.

The term "abnormal" when used with reference to a cell or cells, includes but is not limited to, a cell or cells whose growth is independent of normal regulatory mechanisms. Such "abnormal" cells exhibit an increase in the rate of cell division or an alteration in cellular differentiation relative to "normal cells." Altered cellular' differentiation can be evidenced, for example, by altered cell surface protein expression. Such "abnormal" cells may also proliferate uncontrollably as a result of loss of normal cell cycle checkpoints, loss of contact inhibition, etc. An "abnormal" cell includes one that exhibit a malignant phenotype, such as a cancerous or tumor cell .

The term "targeted cell" refers to a cell where a reduction in its proliferation is desired. One or more targeted cells may be present. Thus, there may be a population of targeted cells. Reduction in proliferation of a targeted cell or population of targeted cells includes reduction in the rate of proliferation and/or reduction in the amount of proliferation. Thus, for example, the rate of cell division may be reduced and/or the total amount of cell division may be reduced.

The term "proliferation", or grammatical variants thereof, when used in relation to a cell or cells, includes any process (such as any asexual process) whereby the targeted cell gives rise to one or more further cells. Thus, proliferation includes the generation of new cells by processes such as cell division.

The terms "cell proliferation", "proliferative condition", "proliferative disorder" and "proliferative disease" refer to an unwanted or uncontrolled cellular proliferation of excessive or abnormal cells which is undesired, such as, neoplastic or hyperplastic growth, whether in vitro, in vivo or ex vivo. Exemplary proliferative conditions include, but are not limited to, pre-malignant and malignant cellular proliferation, such as malignant neoplasms, tumors (e.g. solid tumors), cancers, leukemia, and the like.

The term "anti -proliferative" or "anti- proliferation" as used herein refers to prevention, retardation or inhibition of the growth or multiplication of a single cell or a colony of cells. Several mechanisms exist to induce an "anti -proliferative" or "anti-proliferation" effect. Exemplary mechanisms include, but are not limited to, interfering with the mitotic process for somatic cell proliferation or inducing cytotoxicity and/or apoptosis in damaged or unwanted cells.

The term "anti-proliferative agent" as used herein refers to a compound which treats a cell proliferative condition (i.e. a compound which is useful in the treatment of cell proliferative condition) . Antiproliferative agents disclosed herein may be apoptotic agents, and have application in the treatment of cancer, and so the present invention further provides anti-cancer agents .

The term "anti-cancer agent" refers to- a compound which treats a cancer (i.e. a compound which is useful in the treatment of a cancer) . The anti-cancer effect may arise through one or more mechanisms including, but are not limited to, the regulation of cell proliferation, the inhibition of metastasis (the spread of a tumor from its origin) , the inhibition of angiogenesis (the formation of new blood vessels) , the inhibition of invasion (the spread of tumor cells into neighbouring normal structures), or the promotion of apoptosis. The term "treatment" includes any and all uses which remedy a _. disease state or symptoms, prevent the establishment of disease, or otherwise prevent, hinder, retard, or reverse the progression of disease or other undesirable symptoms in any way whatsoever. Hence, "treatment" includes prophylactic and therapeutic treatment .

The term "patient" or "subject" refers to patients, or subjects of human or other mammal and includes any individual it is desired to examine or treat using the active compounds and methods disclosed herein. However, it will be understood that "patient" or "subject" does not imply that symptoms are present. Suitable mammals that fall within the scope of the invention include, but are not restricted to, primates, livestock animals (eg. sheep, cows, horses, donkeys, pigs) , laboratory test animals (e.g. rabbits, mice, rats, guinea pigs, hamsters), companion animals (e.g. cats, dogs) and captive wild animals (e.g. foxes, deer, dingoes). "Mammal" refers to. any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, and pet companion animals such as a household pet and other domesticated animal such as, but not limited to, cattle, sheep, ferrets, swine, horses, poultry, rabbits, goats, dogs, cats and the like. Preferred companion animals are dogs and cats. Preferably, the mammal is human.

The term "administering", .and variations of that term including "administer" and "administration", includes contacting, applying, delivering or providing a compound or composition of the invention to an organism, or a surface by any appropriate means .

The term "effective amount" when used in relation to an anti-proliferative or an apoptotic agent, refers to an amount sufficient to effect the desired antiproliferative or apoptotic benefit. Similarly, the terms "therapeutically effective amount" and "pharmaceutically effective amount" include within their meanings a sufficient but non-toxic amount of a compound- or composition of the invention to provide the desired therapeutic effect. The exact amount required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the particular agent being administered, the mode of administration, and so forth. Thus, it is not possible to specify an exact "effective amount". However, for .any given case, · an appropriate "effective amount" may be determined by one of ordinary skill in the art using only routine experimentation.

The term "dosage unit form" as used herein refers to physically discrete units suited as unitary dosages for the individual to be treated; each unit containing a predetermined quantity of compound (s) is calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The compound (s) may be formulated for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in an acceptable dosage unit. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the ingredients.

Unless specified otherwise, the terms "comprising" and "comprise", and grammatical variants thereof, are intended to represent "open" or "inclusive" language such that they include recited elements but also permit inclusion of additional, unrecited elements.

As used herein, the term "about", in the context of concentrations of components of the formulations, typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 3% of the stated value, more typically, +/- 2% of the stated value, even more typically +/- 1% of the stated value, and even more typically +/- 0.5% of the stated value.

Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3 , from 1 to 4, from 1 to 5, from 2 to 4 , from 2 to 6 , from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Certain embodiments may also be described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the embodiments 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. Detailed Disclosure of Embodiments

Exemplary, non-limiting embodiments of the active compounds having cell anti-proliferative and/or apoptotie activities will now be disclosed.

The inventors have found that the active compounds disclosed herein exhibit potent cell anti-proliferative and/or apoptotic activities. Accordingly, the active compounds disclosed herein can be used as cell antiproliferative and/or apoptotic agents in medicaments to treat or prevent any conditions in which cells proliferate abnormally and/or show resistance to apoptosis. The active compounds disclosed herein can also be used as molecular probes to delineate the biological pathways and mechanisms in carcinogenesis/tumorigenesis and chemoresistance .

Further, the active compounds disclosed herein can be used in the manufacture of a medicament for the prevention and/or treatment of a condition associated with resistance to anti-cancer drugs, for example resistance to anti-cancer drugs such as ABT-737, GX15- 070, ABT-263 and/or AT-101, and the like. The Active Compounds

The active compounds may be represented by a compound having the general formula (I) :

and pharmaceut reof, wherein n is an integer from 2 to 5 ;

p is an integer from 0 to 4 ;

Ri is independently selected from the group consisting of a hydroxyl group, an alkoxy group, a thiol group, and a thioether group, wherein at least two of R are on the carbon-2 and carbon- 5 positions of the phenyl group;

R ₂ and R ₃ are independently a methylene group or a nucleophile;

Z is an oxygen (O) atom or sulfur (S) atom; and T is hydrogen or an optionally substituted aliphatic group or optionally substituted aryl group.

In one embodiment, Z is an oxygen (O) atom.

In another embodiment, Z is a sulfur (S) atom.

In one embodiment, n is 2.

In a preferred embodiment, n is 2 and each of Ri is -OH. Preferably, in embodiments where n is 2 and each of Ri is -OH, one of R _x is on the carbon-2 position and the other Ri is on the carbon-5 position of the phenyl group.

In one embodiment, n is 2 and one of Ri is -OH and the other of Ri is -OCH ₃ .

Advantageously, in embodiments where n is 2 and each of Ri is -OH on the carbon-2 and carbon-5 position, respectively, the compounds of formula .(I) exhibit high anti-proliferative and/or apoptotic activity, and reduced or no cytotoxicity.

In one embodiment, Z is an oxygen (O) atom, n is 2, and each of Ri is -OH.

In one embodiment, Z is a sulfur (S) atom, n is 2, and each of Ri is -OH.

p can be 0 , 1 , 2 , 3 or 4.

In one embodiment, p is 0.

In one embodiment, Ri is hydroxy1.

In one embodiment, R _x is OU wherein U is hydrogen or an alkyl group having from 1 to 6 carbon atoms .

In another embodiment, Ri is ιΤχ , wherein Z is a sulfur (S) atom or oxygen (O) atom and T _x is a lower alkyl group. The lower alkyl may have from 1 to 6 carbon atoms .

In yet another embodiment, R _x is independently selected from the group consisting of -OH, -OCH ₃ , OCH ₂ CH ₃ , - OCH2CH2CH ₃ , -OCH ₂ CH ₂ CH ₂ CH ₃ , -OCH ₂ CH ₂ CH ₂ CH ₂ CH ₃ ,

OCH2CH ₂ CH2CH2CH ₂ CH ₃ , -SH, -SCH ₃ , -SCH ₂ CH ₃ , -SCH ₂ CH ₂ CH ₃ , - SCH ₂ CH ₂ CH ₂ CH ₃ , -SCH ₂ CH ₂ CH ₂ CH ₂ CH ₃ and - SCH ₂ CH ₂ CH2CH2CH ₂ CH ₃ .

In some embodiments where n is 2, 3, 4 or 5, all the Ri groups are the same. For example, where n is 2, both Ri groups may be -OH, both R groups may be -OCH ₃ , both Rx groups may be OCH ₂ CH ₃ , both Ri groups may be -OCH ₂ CH ₂ CH ₃ , both R _x groups may be - OCH2CH2CH2CH ₃ , both Ri groups may be - OCH2CH ₂ CH2CH2CH ₃ , both Ri groups may be OCH ₂ CH ₂ CH2CH ₂ CH ₂ CH ₃ , both Ri groups may be -SH, both R± groups may be -SCH ₃ , both Ri groups may be -SCH ₂ CH ₃ , both Ri groups may be -SCH ₂ CH ₂ CH ₃ , both R _x groups may be - SCH ₂ CH ₂ CH ₂ CH _3/ both R _x groups may be -SCH ₂ CH ₂ CH ₂ CH ₂ CH ₃ , or both R _x groups may be - SCH2CH2CH ₂ CH ₂ CH2CH-5.

In other embodiments where n is 2, 3 , 4 or 5, each Ri group is different. For example, where n is 2, one R group may be -OH and the other R _x group may be

OCH ₂ CH ₂ CH ₃ , or where n is 3, two Ri groups may be -OH and the third Ri group may be -OCH ₃ or the first R _x group may be -OH, the second Ri group may be -OCH ₃ and the third Ri group may be -OCH ₂ CH ₂ CH ₃ .

In one embodiment, T is an aliphatic group with 1 to

20 carbons. For example, T may be an aliphatic group with 1 to 19 carbons, 1 to 18 carbons, 1 to 17 carbons, 1 to 16 carbons, 1 to 15 carbons, 1 to 14 carbons, 1 to 13 carbons, 1 to 12 carbons, 1 to 11 carbons, 1 to 10 carbons, 1 to 9 carbons, 1 to 8 carbons, 1 to 7 carbons, 1 to 6 carbons , 1 to 5 carbons , 1 to 4 carbons , 1 to 3 carbons, 1 to 2 carbons, 2 to 20 carbons, 3 to 20 carbons, 4 to 20 carbons, 5 to 20 carbons, 6 to 20 carbons, 7 to 20 carbons, 8 to 20 carbons, 9 to 20 carbons, 10 to 20 carbons, 11 to 20 carbons, 12 to 20 carbons, 13 to 20 carbons, 14 to 20 carbons, 15 to 20 carbons, 16 to 20 carbons, 17 to 20 carbons, 18 to 20 carbons, or 19 to 20 carbons.

In one embodiment, T is an aliphatic group with 1 to 8 carbons. In one embodiment, T is an aliphatic group with 1, 2, 3, 4, 5, 6, 7 or 8 carbons. In one embodiment, T is an aliphatic group with 6 carbons .

In one embodiment, T is an aliphatic group with 8 carbons.

In another embodiment, T is an aliphatic group with at least 9 carbons. In one embodiment, T is an aliphatic group with 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbons.

In one embodiment, T is an aliphatic group with 9 carbons.

In one embodiment, T is an aliphatic group with 10 carbons .

In one embodiment, the aliphatic group of T is an alkyl. In one embodiment, the alkyl is a lower alkyl. The alkyl may be straight chain alkyl or a branched chain alkyl. Preferably, the alkyl is a straight chain alkyl. The alkyl may be -CH ₃ , -CH ₂ CH ₃ , -CH ₂ CH ₂ CH ₃ , -CH ₂ CH ₂ CH ₂ CH ₃ , - CH ₂ CH ₂ CH ₂ CH ₂ CH ₃ , -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₃ , -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₃ , - CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₃ , -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₃ , - CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ GH ₂ CH ₃ or -CH(CH ₃ ) ₂ .

In another embodiment, the aliphatic group of T is an aliphatic group containing at least one unsaturated alkenyl or alkynyl group, preferably lower alkenyl or lower alkynyl groups. Suitable alkenyl groups include - CHCH ₂ , -CH ₂ CHCH ₂ , -CHCHCH ₃ , and -CHCHCH ₃ , while suitable alkynyl groups include -CCCH ₃ , -CH ₂ CCH, -CH ₂ CH ₂ CCH, CH ₂ CH ₂ CH ₂ CCH, -CH ₂ CH ₂ CH ₂ CH ₂ CCH, and -CH ₂ CCCH ₃ .

The aliphatic group of T may be optionally substituted as described above.

In one embodiment, T is an aliphatic group substituted with one or more heteroatom groups. Suitable heteroatom groups include, without limitation, oxygen (0) , sulfur (S) , and nitrogen (N) and phosphorous (P) .

In another embodiment, T is an aryl group, heteroaryl group or cycloalkyl group, each optionally substituted with one or more groups selected from hydrogen, oxygen, sulfur, alkyl, alkenyl, alkynyl, thioalkyl, cycloalkyl, cycloalkenyl , heterocycloalkyl , halo, carboxyl, haloalkyl, haloalkynyl, hydroxyl, alkoxy, thioalkoxy, alkenyloxy, haloalkoxy, haloalkenyloxy, nitro, amino, nitroalkyl, nitroalkenyl , nitroalkynyl , nitroheterocyclyl, alkylamino, dialkylamino, alkenylamino, alkynylamino, acyl, alkenoyl, alkynoyl , acylamino, diacylamino, acyloxy, alkylsulfonyloxy, heterocycloxy, heterocycloamino, haloheterocycloalkyl , alkylsulfenyl , alkylcarbonyloxy, alkylthio, acylthio, phosphonyl, phosphinyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl , cyano, cyanate, isocyanate, C (O)NH (alkyl) , and -C (O) N (alkyl) .

In one embodiment, T is optionally substituted with a benzodioxole . In one embodiment, the benzodioxole is a 1 , 3 -benzodioxole .

In one embodiment, both R ₂ and R ₃ are methylene groups. In another embodiment, one of R ₂ and R ₃ is a methylene group and the other is a nucleophile. In yet another embodiment, both R ₂ and R ₃ are nucleophiles .

In one embodiment, R ₂ is a methylene group.

In one embodiment, R ₃ is a nucleophile.

In one embodiment, R ₂ is a methylene group and R ₃ is a nucleophile .

In one embodiment, the nucleophile of R ₂ and/or R ₃ is selected from the group consisting of a halide, a nitrogen nucleophile, a sulfur nucleophile and an oxygen nucleophile.

Suitable halides include, but are not limited to, fluoride, chloride, bromide and iodide, and the like.

Suitable nitrogen nucleophiles include, but are not limited to, amino groups (e.g. primary amino groups, secondary amino groups and tertiary amino groups) , and azides (e.g. metal azides of Li, Na or K) , and the like.

Suitable sulfur nucleophiles include thiols and alkyl sulfides, and the like.

Suitable oxygen nucleophiles include acetates, enolates, alkoxides, and hydroxides, and the like.

The halide, nitrogen nucleophile, sulfur nucleophile and oxygen nucleophile may be optionally substituted as described above.

In one embodiment of the compounds described herein, the nucleophile of R ₂ and/or R ₃ is a nitrogen nucleophile.

In one embodiment, the nitrogen nucleophile is an amino group. The amino group may be a NH group, a NHV group or a NW ¹ group where V and V are each independently selected from the group consisting of a Ci- C ₄ lower alkyl, a phenyl, and an alkoxy group.

In one embodiment, the compound has the formula (I- A) :

In another embodiment, the compound has the formula (I-B) :

another embodiment, the compound has the formula

(I-C)

another embodiment, the compound has the formula

In another embodiment, the compound has the formula (I-E) :

In another embodiment, the compound has the formula (I-F) :

In another embodiment, the compound has the formula (I-G) :

another embodiment, the compound has the formula

another embodiment, the compound has the formula

(I-I)

In another embodiment , the compound has the formula

In another embodiment , the compound has the formula

(I-K)

In another embodiment, the compound has the formula

Since the disclosed active compounds may have asymmetric carbon centers, they can be present in the form of racemate, diastereomers or mixtures thereof. Therefore, the present invention also includes all these isomers and their mixtures.

Synthesis of Active Compounds

The disclosed active compounds having the general formula (I) may be prepared by a process as described below.

Generally, the active compounds are synthesized via nucleophilic substitution reactions. A nucleophile and a second reactant (an electrophile) are mixed in an anhydrous solvent, and stirred at room temperature to produce the active compounds. The active compounds are extracted with an organic solvent, and the resulting organic extracts are concentrated under reduced pressure to give a crude product .

The nucleophile in this instance may be an amine having a formula (la) , or a hydrochloride or hydrobromide salt thereof having a formula ( la' ) as shown below : da) (la' )

where R is selected from the group consisting of aliphatic alkyls, substituted phenyls, unsubstituted phenyls and heterocycles, n is 1 or 2, and X is CI or Br.

Alternatively, the nucleophile is a straight chain amine having a formula (la") :

H ₂ N

n

(la")

where n is 1 or 3.

The electrophile is a lactone.

The nucleophile and the electrophile are typically used in a stoichiometric ratio, but the excess of one component or the other may be advantageous.

The anhydrous solvent is N, N-dimethylformamide (DMF) , dichloromethane or tetrahydrofuran.

Where the nucleophile used is a hydrochloride or hydrobromide salt of an amine having a formula (la' ), N, N-diisopropylethylamine (DIPEA) can be added to liberate the amine group.

The reaction catalyst added is 2-hydroxypyridine .

Typically, the mixture is stirred at room temperature for 6 to 24 h before being extracted with an organic solvent such as dichloromethane. The organic extract is concentrated under reduced pressure to give a crude active compound, which is then purified using silica gel column chromatography or preparative high performance liquid chromatography. Active Compound Salts

In some forms, it may be desirable to formulate the active compounds in pharmaceutically acceptable salt form, generally to improve the solubility and bioavailability and to provide an active compound that may be capable of being assimilated readily. Preferably, the pharmaceutically acceptable salts are suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.

The active compounds may form pharmaceutically acceptable salts with both organic and inorganic acids. Suitable physiologically tolerated acids for salt formation may be organic and inorganic acids, such as hydrochloric, sulfuric, phosphoric, acetic, citric, oxalic, malonic, salicylic, malic, fumaric, succinic, ascorbic, maleic, methanesulfonic, isethionic, lactic, gluconic, glucuronic, sulfamic, benzoic, tartaric, pamoic, and the like.

The salts may be prepared by contacting a free base form with an equivalent amount of the desired acid in the conventional manner. The free base forms may be regenerated by treating the salt form with a base. For example, dilute aqueous base solutions may be utilized. Dilute aqueous sodium hydroxide, potassium carbonate, ammonia, and sodium bicarbonate solutions may be suitable for this purpose. The free base forms may differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvent. Otherwise, the salts may be equivalent to their respective free base forms for purposes of the invention.

The active compounds may exist in unsolvated as well as solvated forms, including hydrated forms. Such salt forms of the active compound may be provided or mixed prior to use with a physiologically acceptable solvent such as water or ethanol .

Therapeutic Uses of Active Compounds

(a) Use as Apoptotic Agents

It has been found that the active compounds of general formula (I) have potent apoptotic activity and are able to induce cell apoptosis by inhibiting the functions of pro-survival proteins such as Bcl-xL and Mcl-1. Hence, the disclosed active compounds have the potential to be developed into useful therapeutic agents for treatment of conditions requiring apoptosis or in which cells show resistance to apoptosis. Accordingly, the disclosed active compounds may be envisaged as being ^~ useful for use as apoptotic agents in the treatment of conditions such as tumorigenesis and/or cancer.

(b) Use as Anti -proliferative Agents The active compounds of general formula (I) are capable of inducing apoptosis in targeted cells. Accordingly, the active compounds can be used to treat diseases and conditions associated with the undesired proliferation of targeted cells.

Additionally, the active compounds may find utility in vitro. For instance, the compounds may find utility in controlling the growth of undesired cells, such as cancer cell lines. The compounds may find utility as a research tool, for instance in studying the mechanisms of apoptosis.

The active compounds of the invention may be used to reduce the proliferation of a targeted cell.

In one embodiment the targeted cell, or one or more of its progeny, dies as a result of exposure of the targeted cell to an active compound of the invention. In one embodiment, the targeted cell, or one or more of its progeny, undergoes apoptosis.

A "reduction in the proliferation" of a targeted cell (and similar expressions) includes where there is a reduction in the number of, or rate of, generation of progeny cells arising from the targeted cell (e.g. whether as daughter, grand-daughter, great-grand-daughter cells etc . ) .

As discussed herein, the active compounds of the invention are envisaged as being useful in the treatment of patients suffering from a condition where proliferation of abnormal or unwanted cells occur, for example where the cells are resistant to apoptosis. Suitably, the anti -proliferative effect of the active compounds disclosed herein is specific for the targeted cells, that is the targeted cells are more sensitive to the anti-proliferative effect of the compounds of the invention than the non-targeted cells (e.g. "wild-type" cells which undergo normal cell proliferation and/or have normal regulation of apoptotic processes) . Hence, where the patient is a cancer patient, the cancerous cells are affected to a greater extent than the patient's noncancerous cells. The targeted cell may be a eukaryotic cell which may be an animal cell.

In one embodiment, the targeted cell has aberrant expression of Bcl-xL and/or Mcl-1 and/or is resistant to apoptosis. For example, Bcl-xL and/or Mcl-1 is overexpressed in endometrial, gastric and colorectal carcinomas, carcinomas of the breast, colon, lung, bladder, ovary and vulva, sarcomas, lymphomas (AML, NHL) , and Li-Fraumeni syndrome. In one embodiment, the targeted cell is a cancer cell.

(c) Use as Anti-cancer Agents

The active compounds of general formula (I) are capable of irreversibly arresting mitosis in cancer cell lines. The inventors contemplate that the active compounds and structurally related species may be used to treat, prevent or ameliorate cancers, particularly cancers that are characterized to be resistant to apoptosis. For example, expression of apoptosis regulatory proteins is aberrant in carcinomas of the breast, colon, lung, bladder, ovary and vulva, sarcomas, lymphomas (AML, NHL) , and Li-Fraumeni syndrome.

Advantageously, it is expected that the active compounds can be used as anti-cancer agents. It is contemplated that cancers which may be treated include solid tumors and leukemias (for example B cell, mixed cell, null cell, T cell, T-cell chronic, HTLV-II- associated, lymphocytic acute, lymphocytic chronic, mast cell, and myeloid leukemias) , melanoma, fibrosarcoma, osteosarcoma, neuroblastoma, neurofibroma, sarcoma (for example Ewing, experimental, Kaposi, and mast cell sarcomas) . The cancer may be one of the bone, breast, digestive system, colorectal, liver, pancreatic, pituitary, testicular, central nervous system, lung, urogenital system or prostate. The tumor may be benign or malignant, typically it will be malignant. The tumor may be a primary or secondary tumor and may be metastatic.

The active compounds when administered as anticancer agents in medicament form may be ^' administered ^~ on their own or in combination with other anti-cancer treatments such as in conjunction with chemotherapy or radiotherapy.

(d) Pharmaceutical Compositions

The active compounds for use as a cell antiproliferative or apoptotic agent may be administered in pure form or in an appropriate pharmaceutical composition. In general, suitable pharmaceutical compositions may be prepared according to methods which are known to those of ordinary skill in the art. The compositions comprising the active compounds disclosed herein may include a conventional pharmaceutical carrier or diluent, and in addition, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, etc. Examples of suitable pharmaceutical carriers or diluents include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Other suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences (Mack Publishing Company) , a standard reference text in this field, or in U.S. Pharmacopeia National Formulary, 1857-1853, (1990). Typically, the carrier (s) or diluent (s) will form from about 10% to about 99.9% by weight of the compositions.

Administration of the active compounds disclosed herein, in pure form or in an appropriate pharmaceutical composition, may be carried out via any of the acceptable modes of administration or pharmaceutically acceptable means of delivery. The modes of administration and pharmaceutically acceptable means of delivery may include oral administration or delivery in the form of solid, semi-solid, lyophilized powder, or liquid dosage forms. The dosage forms may include tablets, suppositories, pills, soft elastic and hard gelatin capsules, powders, solutions, suspensions, or aerosols, or the like, preferably in unit dosage forms suitable for simple administration of precise dosages.

Oral administration of the disclosed active compounds may be effected by preparing a mixture of the disclosed active compounds with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsules, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet. For oral therapeutic administration, the disclosed active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations may contain the active compounds in an amount by weight per volume percent selected from the group consisting of about 0.005% to about 50%, about 0.01% to about 50%, about 0.02% to about 50%, about 0.05% to about 50%, about 0.1% to about 50%, about 0.2% to about 50%, about 0.5% to about 50%, about 1% to about 50%, about 5% to about 50%, about 10% to about 50%, about 15% to about 50%, about 20% to about 50%, about 25% to about 50%, about 30% to about 50%, about 35% to about 50%, about 40% to about 50%, about 45% to about 50%, about 0.005% to about 45%, about 0.005% to about 40%, about 0.005% to about 35%, about 0.005% to about 30%, about 0.005% to about 25%, about 0.005% to about 20%, about 0.005% to about 15%, about 0.005% to about 10%, about 0.005% to about 5%, about 0.005% to about 1%, about 0.005% to about 0.5%, about 0.005% to about 0.2%, about 0.005% to about 0.1%, about 0.005% to about 0.05%, about 0.005% to about 0.02%, and about 0.005% to about 0.01%. Preferably, the active compounds are present in an amount by weight per volume percent selected from the group consisting of about 0.02% to about 40%, about 0.02% to about 30%, about 0.02% to about 20%, about 0.02% to about 15%, about 0.02% to about 14%, about 0.02% to about 13%, about 0.02% to about 12%, about 0.02% to about 11%, about 0.02% to about 10%, and about 0.02% to about 9%. Preferably, the active compounds are present in an amount by weight per volume percent selected from the group consisting of about 0.2% to about 10%, about 0.2% to about 9%, about 0.2% to about 8%, about 0.2% to about 7%, about 0.2% to about 6%, about 0.2% to about 5%, about 0.2% to about 4%, about 0.2% to about 3%, about 0.2% to about 2%, about 0.2% to about 1%, about 0.2% to about 0.9%, about 0.2% to about 0.8%, about 0.2% to about 0.7%, about 0.2% to about 0.6%, about 0.2% to about 0.5%, about 0.2% to about 0.4%, and about 0.2% to about 0.3%. In one embodiment, the active compounds are present in an amount by weight per volume percent of about 0.2% to about 1%.

The tablets, troches, pills, capsules and the like may also contain the following: a binder, such as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup of elixir may contain sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavorings, such as cherry or orange flavor. Any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations. The active compounds disclosed herein may be administered parenterally or intraperitoneally. Solutions of the disclosed active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose . Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use may include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms may be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol , phenol, sorbic acid, thimerosal, and the like. Prolonged absorption of the injectable compositions can also be brought about by including in the composition an agent which delays absorption, for example, aluminium monostearate and gelatin.

Preferably, the pharmaceutical form may further include a suitable buffer to minimise acid hydrolysis. Suitable buffer agents are well known to those skilled in the art and include, but are not limited to, phosphates, citrates, carbonates and mixtures thereof.

The active compounds may also be administered in the form of a "prodrug". A prodrug is an inactive form of a compound which is transformed in vivo to the active form. Suitable prodrugs include esters, phosphonate esters etc, of the active form of the compound.

The active compounds in pharmaceutically acceptable form may be administered in a therapeutically effective amount which will vary depending upon a variety . of factors including the activity of the specific compounds employed; the metabolic stability and length of action of the compounds; the age, body weight, general health, sex and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disease states; and the patient undergoing treatment.

Single or multiple administrations of the pharmaceutical compositions according to the invention may be carried out. One skilled in the art would be able, by routine experimentation, to determine effective, nontoxic dosage levels of the compound and/or composition of the invention and an administration regime which would be suitable for treating the diseases and/or conditions to which the compounds and compositions are applicable. It will be apparent to one of ordinary skill in the art that the optimal course of treatment, such as the number of doses of the compound or composition of the invention given per day for a defined number of days, can be ascertained using conventional course of treatment determination tests.

Generally, an effective dosage per 24 hours for use of the active compounds as anti-proliferative agents in a pharmaceutical composition may be in the range of about 0.5 mg per kg body weight to about 150 mg per kg body weight, about 0.5 mg per kg body weight to about 140 mg per kg body weight, about 0.5 mg per kg body weight to about 130 mg per kg body weight, about 0.5 mg per kg body weight to about 120 mg per kg body weight, about 0.5 mg per kg body weight to about 110 mg per kg body weight, about 0.5 mg per kg body weight to about 100 mg per kg body weight, about 0.5 mg per kg body weight to about 90 mg per kg body weight, about 0.5 mg per kg body weight to about 80 mg per kg body weight, about 0.5 mg per. kg body weight to about 70 mg per kg body weight, about 0.5 mg per kg body weight to about 60 mg per kg body weight, about 0.5 mg per kg body weight to about 50 mg per kg body weight, about 0.5 mg per kg body weight to about 40 mg per kg body weight, about 0.5 mg per kg body weight to about 30 mg per kg body weight, about 0.5 mg per kg body weight to about 20 mg per kg body weight, about 0.5 mg per kg body weight to about 10 mg per kg body weight, about 0.5 mg per kg body weight to about 5 mg per kg body weight, about 1 mg per kg body weight to about 150 mg per kg body weight, about 5 mg per kg body weight to about 150 mg per kg body weight, about 10 mg per kg body weight to about 150 mg per kg body weight, about 20 mg per kg body weight to about 150 mg per kg body weight, about 30 mg per kg body weight to about 150 mg per kg body weight, about 40 mg per kg body weight to about 150 mg per kg body weight, about 50 mg per kg body weight to about 150 mg per kg body weight, about 60 mg per kg body weight to about 150 mg per kg body weight, about 70 mg per kg body weight to about 150 mg per kg body weight, about 80 mg per kg body weight to about 150 mg per kg body weight, about 90 mg per kg body weight to about 150 mg per kg body weight, about 100 mg per kg body weight to about 150 mg per kg body weight, about 110 mg per kg body weight to about 150 mg per kg body weight, about 120 mg per kg body weight to about 150 mg per kg body weight, about 130 mg per kg body weight to about 150 mg per kg body weight, or about 140 mg per kg body weight to about 150 mg per kg body weight. Suitably, an effective dosage is in the range of about 1 mg per kg body weight to about 100 mg per kg body weight, about 5 mg per kg body weight to about 100 mg per kg body weight, about 6 mg per kg body weight to about 100 mg per kg body weight, about 7 mg per kg body weight to about 100 mg per kg body weight, about 8 mg per kg body weight to about 100 mg per kg body weight, about 9 mg per kg body weight to about 100 mg per kg body weight, about 10 mg per kg body weight to about 100 mg per kg body weight, about 11 mg per kg body weight to about 100 mg per kg body weight, about 12 mg per kg body weight to about 100 mg per kg body weight, about 13 mg per kg body weight to about 100 mg per kg body weight, about 14 mg per kg body weight to about 100 mg per kg body weight, or about 15 mg per kg body weight to about 100 mg per kg body weight.

The active compounds of general formula (I) may be used in combination with other known treatments or anti- proliferative or apoptotic agents. Combinations of active agents, including the active compounds of the invention, may be synergistic.

.Von- therapeutic Uses of Active Compounds

The active compounds of general formula (I) are capable of inducing apoptosis by inhibiting the functions of pro-survival proteins such as Bcl-xL and Mcl-1. The active compounds may therefore serve as molecular probes to understand the mechanism of cell survival and cell death in research and drug discovery in order to develop novel anti-cancer drugs. It has been found that compounds of the present invention may selectively target and inhibit one of Mcl-1 and Bcl-xL, or inhibit both Mcl-1 and Bcl-xL, and may be developed for use as molecular probes in biological assays. Exemplary biological assays include those for studying apoptotic pathways and/or mechanisms, mitochondrial membrane permeability, and mechanisms of tumorigenesis.

Examples

Non-limiting embodiments of the disclosed active compounds will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.

Synthesis of Phenolic Analogues

Materials

The chemical reagents and solvents used in the following examples were purchased from Sigma-Aldrich, Alfa Aesar and Merck.

Analytical Protocols

(a) Analytical High Performance Liquid Chromatography (HPLC)

Analytical HPLC was performed on a Waters HPLC system equipped with a Waters 2998 PDA detector, Waters 2695 separation module using a X-Bridge CI8 (4.6 mm I.D. x 150 mm, 5μΜ) column. A gradient elution starting with 20% CH ₃ CN, 80% (0.1% formic acid/H ₂ 0) or 30% CH ₃ CN, 70% (0.1% formic acid/H ₂ 0) , and ending with 100% CH ₃ CN at a flow rate of 1.0 mL/min over 15 min was used.

(b) Preparative HPLC

Preparative HPLC was performed on a Shimadzu - LC-8A HPLC system equipped with a CBM-20A PDA detector, Gilson 215 liquid handler and fraction collector using a X- Bridge Prep C ₁₈ (30 mm I.D. x 50 mm, 5μ) column. An isocratic elution with 20% CH ₃ CN, 80% (0.1% formic acid/H ₂ 0) at a flow rate of 20.0 mL/min for 5 min was used. This was followed by a gradient elution starting with 20% CH ₃ CN, 80% (0.1% formic acid/H ₂ 0) , and ending with 100% CH ₃ CN at a flow rate of 20.0 mL/min.

(c) Liquid Chromatography-Mass Spectrometry (LC-MS)

LC-MS data for JC025, JC026, JC028, JC030 and JC041 was collected on a Shimadzu LCMS-IT-TOF instrument equipped with SPD-M2OA PDA detector, LCMS-IT-TOF MS detector and LC-20AD binary gradient pum using a Shimpack VP-ODS (2.0 mm I.D. x 150 mm) column. An isocratic elution with 20% H ₂ 0 and 80% CH ₃ CN at a flow rate of 0.2 mL/min over 3 min was used.

LC-MS data for JC051-JC057 was collected on a Shimadzu Prominence Series LCMS-IT-TOF instrument equipped with SPD-M20A PDA detector, LCMS-IT-TOF MS detector and LC-20AD binary gradient pump using a Shimpack VP-ODS (2.0. mm I.D. x 250 mm) column. Mobile phase is 0.1% formic acid in CH ₃ CN and 0.1% formic acid in H ₂ 0. A gradient elution was set at 0. min 20% acetonitrile, 13 min 100% acetonitrile, 14 min 100% acetonitrile, 15 min 20% acetonitrile and stop time 16 min.

Example 1: Synthetic Procedure for Analogu

To a solution of lactone (1 - 2 mmol) and 2- hydroxypyridine (0.1 - 0.2 mmol) in anhydrous dichloromethane or anhydrous DMF (2 - 5 mL) was added an amine (1.1 - 2.2 mmol). The mixture was stirred at room temperature for 6 to 24 h. After the reaction, water (20 mL) was added. The reaction mixture was transferred to a separating funnel and extracted with dichloromethane, CH ₂ C1 ₂ (3 x 10 mL) . The solvent extracts were concentrated under reduced pressure to give a crude product, which was purified by silica gel column chromatography (using hexane/ethyl acetate, 1:1 v/v as el ent) or preparative HPLC (using the protocol set out in . "Analytical Protocols" above) to give the final product.

Example 2; Analytical Data on Phenolic Analogues

The phenolic analogues prepared in Example 1 above were analyzed using the analytical protocols set out above. The resulting mass spectral, yield and purity data for each analogue are tabulated in Table 1 below.

Table 1. Analytical Data on Phenolic Analogues

As can be seen above, the compounds of formula (I) can be prepared in high purity ranging from about 71% up to >99% purity.

Apoptosis Inducing Ability and Cytotoxicity of Phenolic

Analogues In vitro fluorescence polarization (FP)binding assay - Bcl-xL and Mcl-1 Assays

The in vitro florescence polarization binding assays were carried out to identify the inhibitors that block the interaction of Bcl-xL/Bak BH3 domain (Chan, S. et aL (2003) J ^" . Biol. Chem. , 278 (23 ): 20453 -20456 ; and Zhang, C. et al. (2002) Anal. Biochem. , 307:70-75) and Mcl-l/Bak BH3 domain .

Recombinant GST (glutathio S-transferase) fusion proteins of GST-Bcl-xL and GST-Mcl-1 were expressed in E. coli BL21. The purification of GST fusion proteins was carried out with glutathione sepharose beads and eluted with glutathione elution buffer. Fluorescence labelled peptide of Bak BH3 domain was synthesized by Mimotopes (Australia) .

FP assay measures the molecular movement and rotation of the fluorescence labelled Bak BH3 peptide (fluo-Bak BH3) . In the absence of inhibitors, free rotation of fluo-Bak BH3 is significantly induced due to its binding with Bcl-xL or Mcl-1 proteins. Thus, excitation of fluorescein with polarized light results in a high polarized readout (reading of negative control indicating high polarization). When inhibitor (s) are incubated with Fluo-Bak BH3 and Bcl-xL or Mcl-1 there is competitive binding for Bcl-xL or Mcl-1, and the free rotating Fluo-Bak BH3 results in a low polarized readout (- reading of positive indicating low polarization) .

Bcl-xL or Mcl-1 was prepared at a concentration of 0.125 tg/ L with 50 mM Tris buffer (pH 8.0). Fluo-Bak BH3 was prepared at a concentration of 100 nM with 50 mM Tris buffer (pH 8.0).

The FP assay was conducted on 96-well plate. The procedure of FP assay included adding 10 L/well testing sample, followed by 30 /L/well Bcl-xL or Mcl-1 protein, and finally adding 60 ^L/well Fluo-Bak BH3. The mixture was incubated at room temperature for 1 hr, after which the fluorescent polarization readout was measured with Tacan Infinite 200 using polarized filter excitation at 495 nM and emission at 535 nM.

In vivo Cell Viability Assay

The cell viability assays were conducted with HepG2 cell line (human hepatocellular carcinoma, with deposit number ATCC HB-8065) . The assay uses the indicator dye resazurin to measure the metabolic capacity of cells. Viable cells retain the ability to reduce resazurin into resorufin, which is highly fluorescent. Conversely, non- viable cells rapidly lose metabolic capacity, and therefore are not able to reduce the indicator dye and generate a fluorescent signal. The assay procedure included adding 100 /L of 10 ⁵ /mL HepG2 cells into a 96 -well plate. The cells were incubated at 37°C overnight, after which 10 ^L/well sample was added on the following day. The sample/cell mixture was incubated for 24 hrs at 37°C. This was followed by adding 20 μΙ,%θ11 of 200 /g/mL resazurin. The mixture was incubated at 37°C for 4 hrs. The plate was then read at an excitation wavelength of 535. nm with Tecan Infinite 200.

Example 3: Apoptosis Inducing and Cytotoxic Properties of Phenolic Compounds Table 2. Apoptosis Inducing and Cytotoxicity Properties of Phenolic Compounds

It can be seen from Table 2 above that phenolic analogues JC025, JC026, JC028, JC030, JC041 and JC053-057 have significant inhibitory effects on both pro-survival proteins Bcl-xL and Mcl-l. Compounds JC051 and JC052 exhibit selective inhibitory effect on Mcl-l, which is of particular interest in cancer therapy.

The compounds also exhibited low cytotoxic effects on the human cancer cell line HepG2. Compounds JC051 and JC052, in particular, have very low cytotoxicity.

When compared to a known Bcl-xL inhibitor, Gossypol, it can be seen that the phenolic compounds JC025, JC026, JC028, JC030, JC041 and JC051-057 have significantly lower cytotoxicity than Gossypol.

Comparative Example

Table 3. Comparison of Different Phenolic Analogues in Inhibiting Pro- survival Proteins Bcl-xL and/or Mcl-l

The comparative data in Table 3 demonstrate that the presence of a hydroxyl moiety at carbon-2 and carbon- 5 positions of the phenyl group of the compounds of formula (I) is advantageous for inhibiting the Bcl-xL and Mcl-1 proteins. For example, JC025, JC028 and JC030 exhibit significantly more potent inhibitory activity on both pro-survival proteins than JC025' , JC028' and JC030' , respectively, which have only one hydroxyl group on the phenyl ring.

By comparing JC026 and JC026' , it can also be seen that the absence of a hydroxyl, alkoxy, thiol or thioether group on the phenyl ring also compromises the inhibitory activity of the compounds on both pro-survival proteins.

In summary, phenolic analogues JC025, JC026, JC028, JC030, JC041 and JC053 to JC057 show inhibitory effects towards both Bcl-xL and Mcl-1 pro-survival proteins and have low cytotoxicity toward HepG2 cell line, while phenolic analogues JC051 and JC052 show selective inhibition towards Mcl-1 protein and also have low cytotoxicity toward HepG2 cell line.

Advantageously, as the phenolic analogues target the

Bcl-xL and Mcl-1 pro-survival proteins, which are often over-expressed in cancer and tumor cells, the phenolic analogues can selectively target and cause apoptosis of these cells without causing any anti-proliferative or apoptotic effect on normal (i.e. non-tumor and noncancerous) cells.

Applications The active compounds disclosed herein have demonstrated strong pro-survival protein inhibitory and cell apoptotic activities.

Advantageously, the disclosed active compounds may be used as apoptotic/anti-proliferative agents in the manufacture of medicaments for treatment of conditions in which cells proliferate abnormally and/or show resistance to apoptosis.

Exemplary proliferative conditions include, but are not limited to, pre-malignant and malignant cellular proliferation, such as malignant neoplasms, tumours (e.g. solid tumours), cancers, leukemia and infections (e.g. fungal infection, bacterial infection etc.), and the like.

The surprising selective inhibition of Mcl-1 protein by phenolic analogues JC051 and JC052 also provide the compounds of the present invention with the ability to be used as a molecular probe in medical research to target a particular pro-survival protein and independently study the mechanism or pathway of inhibition of such a protein.

Further, as discussed hereinabove, Mcl-1 has emerged as a major resistance factor in human cancer. Inhibition of Mcl-1 by small organic molecules, such as the active phenolic analogues of the present invention, will prove to be a valuable strategy in circumventing the chemoresistance of cancer cells. Therefore, the active compounds of the present invention may be used in the manufacture of a medicament for the prevention and/or treatment of a condition associated with resistance to anti-cancer drugs.