WO2011130677A1 - Inhibitors of cancer stem cells - Google Patents

Abstract

A method of treating cancer in a subject, comprising co-administering to the subject (a) a therapeutically effective amount of at least one compound that selectively targets cancer stem cells, wherein the compound is selected from an indenoisoquinoline compound, a naphthoquinone compound, a quinolinedione compound, bouvardin, A-77636, rottlerin, or CGP-74514A, or a pharmaceutically acceptable salt or ester thereof, and (b) a therapeutically effective amount of at least one anti-cancer agent.

Description

INHIBITORS OF CANCER STEM CELLS

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 61/325,156, filed April 16, 2010, and U.S. Provisional Application No. 61/325,705, filed April 19, 2010, both of which are incorporated herein by reference in their entireties.

ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant #CA 140624 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND

The concept of cancer stem cells (CSCs) is based upon ideas first formulated in the context of the hierarchical hematopoietic system, where normal stem cells have an unlimited capacity for self-renewal that is gradually lost as they generate multi-potent progenitors and their more differentiated progeny. As applied to cancer, the CSC model posits that not all cancer cells are identical in their ability to initiate the growth of a new tumor. In breast cancer, for example, cell surface markers have been used to identify tumor cell

subpopulations with widely differing capacities for tumor initiation. Thus, cells with high surface expression of the epithelial marker CD44 (CD44hi) and low-absent expression of CD24 (CD241o) possess robust tumor initiating activity relative to the remaining population. Recent work has provided evidence that tumors with high levels of CSC markers identify sub-groups of patients who are particularly prone to therapy failure and relapse. The concept of CSCs has been extended beyond breast cancer to other malignancies, including cancer of the prostate, brain (gliomas), colon, and pancreas.

In most cases examined to date, CSCs comprises <1 of tumor cells, with the remainder comprising the more differentiated "transient amplifying cell" (TAC) population. However, the true frequency of CSCs remains somewhat fluid. The CSC hypothesis suggests that, because the bulk of tumor cells have low tumor initiating capacity, their eradication will result in substantial tumor shrinkage but will not be curative unless CSCs are eliminated concurrently. This has led to the proposal that relapses arise as a consequence of inherent differential therapeutic sensitivities of the CSC and TAC compartments. Unfortunately, the paucity of CSCs and their tendency to differentiate into TACs has prevented a full-scale testing of this hypothesis. Further, because CSCs differentiate shortly after being isolated, sufficient numbers of CSCs have not been able to be produced via cell culture.

Oct3/4 is a germline-specific transcription factor, also known as OTF3 or POU5F1 recently reported to play an important role in germ cell specification. Expression of human Oct3/4 is driven by an approximately 4 kb promoter element (GenBank Acc. No.

DQ249177.1 The Oct3/4 promoter contains three virtual open reading frames (ORFs), all oriented in a 3'→5' direction relative to the sequence. ORF1 (ca. 1.1-1.6 kb from the 5' end) could potentially encode a 162 amino acid protein. ORF2 (ca. 2.4-2.8 kb from the 5' end) could encode a 138 amino acid protein, and ORF3 (ca. 2.7-3.3 kb from the 5' end) could encode a 201 amino acid protein. SUMMARY

Disclosed herein are compounds that selectively inhibit cancer stem cells, and methods for inhibiting cancer that include administering such compounds to a subject.

One method disclosed herein is a method of inhibiting cancer in a subject, comprising administering to the subject at least one therapeutic agent that selectively targets cancer stem cells.

Another embodiment disclosed herein is a method of treating cancer in a subject, comprising co-administering to the subject (a) a therapeutically effective amount of at least one compound that selectively targets cancer stem cells, wherein the compound is selected from an indenoisoquinoline compound, a naphthoquinone compound, a quinolinedione compound, bouvardin, A-77636, rottlerin, or CGP-74514A, or a pharmaceutically acceptable salt or ester thereof, and (b) a therapeutically effective amount of at least one anti-cancer agent.

Another embodiment disclosed herein is a method of treating cancer in a subject, comprising co-administering to the subject (a) a therapeutically effective amount of at least one compound that selectively targets cancer stem cells, wherein the compound is selected from an indenoisoquinoline compound, a naphthoquinone compound, a quinolinedione compound, or bouvardin, or a pharmaceutically acceptable salt or ester thereof, and (b) a therapeutically effective amount of at least one anti-cancer agent. According to a further embodiment, disclosed herein is a method of selectively targeting cancer stem cells, comprising administering to a population of cancer cells comprising cancer stem cells as well as cancer cells that are not stem cells a therapeutically effective amount of at least one compound selected from an indenoisoquinoline compound, a naphthoquinone compound, a quinolinedione compound, bouvardin, A-77636, rottlerin, or CGP-74514A, or a pharmaceutically acceptable salt or ester thereof.

According to a further embodiment, disclosed herein is a method of selectively targeting cancer stem cells, comprising administering to a population of cancer cells comprising cancer stem cells as well as cancer cells that are not stem cells a therapeutically effective amount of at least one compound selected from an indenoisoquinoline compound, a naphthoquinone compound, a quinolinedione compound, or bouvardin, or a

pharmaceutically acceptable salt or ester thereof.

An additional embodiment disclosed herein is a method of treating a

chemotherapeutic-resistant cancer in a subject, comprising administering to the subject a therapeutically effective amount of at least one compound that selectively targets cancer stem cells, wherein the compound is selected from an indenoisoquinoline compound, a naphthoquinone compound, a quinolinedione compound, bouvardin, A-77636, rottlerin, or CGP-74514A, or a pharmaceutically acceptable salt or ester thereof.

An additional embodiment disclosed herein is a method of treating a

chemotherapeutic-resistant cancer in a subject, comprising administering to the subject a therapeutically effective amount of at least one compound that selectively targets cancer stem cells, wherein the compound is selected from an indenoisoquinoline compound, a naphthoquinone compound, a quinolinedione compound, or bouvardin, or a

pharmaceutically acceptable salt or ester thereof.

Another disclosed embodiment relates to a method of treating recurrent breast cancer in a subject, comprising co-administering to the subject (a) a therapeutically effective amount of at least one compound that selectively targets breast cancer stem cells, wherein the compound is selected from an indenoisoquinoline compound, a naphthoquinone compound, a quinolinedione compound, bouvardin, A-77636, rottlerin, or CGP-74514A, or a pharmaceutically acceptable salt or ester thereof, and (b) a therapeutically effective amount of at least one anti-breast cancer agent.

Another disclosed embodiment relates to a method of treating recurrent breast cancer in a subject, comprising co-administering to the subject (a) a therapeutically effective amount of at least one indenoisoquinoline compound that selectively targets breast cancer stem cells, and (b) a therapeutically effective amount of at least one anti-breast cancer agent.

Also disclosed herein is a pharmaceutical composition useful for selectively targeting cancer stem cells, comprising (a) at least one pharmaceutically acceptable additive and (b) a therapeutically effective amount of at least one compound that selectively targets cancer stem cells, wherein the compound is selected from an indenoisoquinoline compound, a naphthoquinone compound, a quinolinedione compound, bouvardin, A-77636, rottlerin, or CGP-74514A, or a pharmaceutically acceptable salt or ester thereof.

Also disclosed herein is a pharmaceutical composition useful for selectively targeting cancer stem cells, comprising (a) at least one pharmaceutically acceptable additive and (b) a therapeutically effective amount of at least one compound that selectively targets cancer stem cells, wherein the compound is selected from an indenoisoquinoline compound, a naphthoquinone compound, a quinolinedione compound, or bouvardin, or a

pharmaceutically acceptable salt or ester thereof.

The foregoing will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1 and 2 are graphs depicting IC₅o-MCF7/IC₅o-Oct3/4 results for several CSC inhibitors.

FIGS. 3 and 4 are graphs depicting results for several CSC inhibitors.

DETAILED DESCRIPTION

The following explanations of terms and methods are provided to better describe the present compounds, compositions and methods, and to guide those of ordinary skill in the art in the practice of the present disclosure. It is also to be understood that the terminology used in the disclosure is for the purpose of describing particular embodiments and examples only and is not intended to be limiting.

As used herein, the singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Also, as used herein, the term "comprises" means "includes." The term "acyl" refers to a group of the formula RC(O)- wherein R is an organic group.

"Administration of and "administering a" compound should be understood to mean providing a compound, a prodrug of a compound, or a pharmaceutical composition as described herein. The compound or composition can be administered by another person to the subject (e.g., intravenously) or it can be self-administered by the subject (e.g., tablets).

The term "aliphatic" is defined as including alkyl, alkenyl, alkynyl, halogenated alkyl and cycloalkyl groups as described below. A "lower aliphatic" group is a branched or unbranched aliphatic group having from 1 to 10 carbon atoms.

"Alkanediyl" or "cycloalkanediyl" refers to a divalent radical of the general formula

-C_nH_2n- derived from aliphatic or cycloaliphatic hydrocarbons (e.g, derived from alkyl or cycloalkyl).

The term "alkenyl" refers to a hydrocarbon group of 2 to 24 carbon atoms and structural formula containing at least one carbon-carbon double bond. A "lower alkenyl" group has 1 to 10 carbon atoms.

The term "alkyl" refers to a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, w-propyl, isopropyl, «-butyl, isobutyl, i-butyl, pentyl, hexyl, heptyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like. A "lower alkyl" group is a saturated branched or unbranched hydrocarbon having from 1 to 10 carbon atoms. Preferred alkyl groups have 1 to 4 carbon atoms. Alkyl groups may be

"substituted alkyls" wherein one or more hydrogen atoms are substituted with a substituent such as halogen, cycloalkyl, alkoxy, amino, hydroxyl, aryl, or carboxyl.

The term "alkyl amino" refers to alkyl groups as defined above where at least one hydrogen atom is replaced with an amino group.

The term "alkynyl" refers to a hydrocarbon group of 2 to 24 carbon atoms and a structural formula containing at least one carbon-carbon triple bond.

The term "alkoxy" refers to a straight, branched or cyclic hydrocarbon configuration and combinations thereof, including from 1 to 20 carbon atoms, preferably from 1 to 8 carbon atoms, more preferably from 1 to 4 carbon atoms, that include an oxygen atom at the point of attachment. An example of an "alkoxy group" is represented by the formula -OR, where R can be an alkyl group, optionally substituted with, e.g., an alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl group as described herein.

Suitable alkoxy groups include methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, i-butoxy, sec-butoxy, tert-butoxy cyclopropoxy, cyclohexyloxy, and the like. "Alkoxycarbonyl" refers to an alkoxy substituted carbonyl radical, -C(0)OR, wherein R represents an optionally substituted alkyl, aryl, aralkyl, cycloalkyl,

cycloalkylalkyl or similar moiety.

The term "amine" or "amino" refers to a group of the formula -NRR', where R and R' can be, independently, hydrogen or an alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl group described above.

"Aminocarbonyl" alone or in combination, means an amino substituted carbonyl (carbamoyl) radical, wherein the amino radical may optionally be mono- or di-substituted, such as with alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, alkanoyl, alkoxycarbonyl, aralkoxycarbonyl and the like. An aminocarbonyl group may be -N(R)-C(0)-R (wherein R is a substituted group or H) or -C(0)-N(R). The "R" groups in aminocarbonyl may be the same or different. An "aminocarbonyl" is inclusive of an amido group. A suitable aminocarbonyl group is acetamido.

The term "amide" or "amido" is represented by the formula -C(0)NRR', where R and R' independently can be a hydrogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl group described herein. A suitable amido group is acetamido.

The term "aralkyl" refers to an aryl group having an alkyl group, as defined above, attached to the aryl group, as defined herein. An example of an aralkyl group is a benzyl group.

The term "aryl" refers to any carbon-based aromatic group including, but not limited to, benzene, naphthalene, etc. The term "aryl" also includes "heteroaryl group," which is defined as an aromatic group that has at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorous. The aryl group can be substituted with one or more groups including, but not limited to, alkyl, alkynyl, alkenyl, aryl, halide, nitro, amino, ester, ketone, aldehyde, hydroxy, carboxylic acid, or alkoxy, or the aryl group can be unsubstituted.

"Carbonyl" refers to a radical of the formula -C(O)-. Carbonyl-containing groups include any substituent containing a carbon-oxygen double bond (C=0), including acyl groups, amides, carboxy groups, esters, ureas, carbamates, carbonates and ketones and aldehydes, such as substituents based on -COR or -RCHO where R is an aliphatic, heteroaliphatic, alkyl, heteroalkyl, hydroxyl, or a secondary, tertiary, or quaternary amine. "Carboxyl" refers to a -COOH radical. Substituted carboxyl refers to -COOR where R is aliphatic, heteroaliphatic, alkyl, heteroalkyl, or a carboxylic acid or ester.

"Carcinoma" refers to any cancer that arises from epithelial cells. Carcinoma is inclusive of both malignant cancer and carcinoma in situ. Carcinoma is not inclusive of blood-borne cancers such as leukemia or myeloma.

The term "co-administration" or "co-administering" refers to administration of the compound disclosed herein with at least one other therapeutic agent within the same general time period, and does not require administration at the same exact moment in time (although co-administration is inclusive of administering at the same exact moment in time). Thus, co-administration may be on the same day or on different days, or in the same week or in different weeks. In certain co-administration embodiments, the pharmacological or therapeutic effect of the co-administered agents may overlap for a certain time period.

The term "cycloalkyl" refers to a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. The term "heterocycloalkyl group" is a cycloalkyl group as defined above where at least one of the carbon atoms of the ring is substituted with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorous. "Heterocycloalkyl" and "heterocyclic" are used interchangeably herein.

"Derivative" refers to a compound or portion of a compound that is derived from or is theoretically derivable from a parent compound.

"Drug-resistant" or "multidrug-resistant" refers to a cancer that is resistant to treatment by at least one therapeutic agent historically administered to treat that cancer. These recurrent cancers often occur after surgery, primary chemotherapy treatment, radiotherapy, or immunotherapy. In certain embodiments, the cancer is a chemotherapeutic- resistant carcinoma. For example, breast cancer may become resistant to treatment with doxorubicin, pamidronate disodium, anastrozole, exemestane, cyclophosphamide, epirubicin, raloxifene, toremifene, letrozole, trastuzumab, megastrol, tamoxifen, paclitaxel, docetaxel, capecitabine, goserelin acetate, zoledronic acid or a combination thereof.

The terms "halogenated alkyl" or "haloalkyl group" refer to an alkyl group as defined above with one or more hydrogen atoms present on these groups substituted with a halogen (F, CI, Br, I).

The term "hydroxyl" is represented by the formula -OH.

The term "hydroxyalkyl" refers to an alkyl group that has at least one hydrogen atom substituted with a hydroxyl group. The term "alkoxyalkyl group" is defined as an alkyl group that has at least one hydrogen atom substituted with an alkoxy group described above.

"Inhibiting" (which is inclusive of "treating") refers to inhibiting the full development of a disease or condition, for example, in a subject. "Treatment" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. As used herein, the term "treating," with reference to a disease, pathological condition or symptom, also refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the number of relapses of the disease, an improvement in the overall health or well-being of the subject, or by other parameters well known in the art that are specific to the particular disease. "Inhibiting" also refers to any quantitative or qualitative reduction, relative to a control. A "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology. By the term "coadminister" is meant that each of at least two compounds be administered during a time frame wherein the respective periods of biological activity overlap. Thus, the term includes sequential as well as coextensive administration of two or more drug compounds.

Optionally substituted groups, such as "optionally substituted alkyl," refers to groups, such as an alkyl group, that when substituted, have from 1-5 substituents, typically 1, 2 or 3 substituents, selected from, e.g., alkoxy, optionally substituted alkoxy, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, aryl, carboxyalkyl, optionally substituted cycloalkyl, optionally substituted cycloalkenyl, halogen, optionally substituted heteroaryl, optionally substituted heterocyclyl, hydroxy, sulfonyl, thiol and thioalkoxy. In particular, optionally substituted alkyl groups include, by way of example, haloalkyl groups, such as fluoroalkyl groups, including, without limitation, trifluoromethyl groups.

"Optional" or "optionally" means that the subsequently described event or circumstance can but need not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

The terms "pharmaceutically acceptable salt or ester" refers to salts or esters prepared by conventional means that include salts, e.g., of inorganic and organic acids, including but not limited to hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, malic acid, acetic acid, oxalic acid, tartaric acid, citric acid, lactic acid, fumaric acid, succinic acid, maleic acid, salicylic acid, benzoic acid, phenylacetic acid, mandelic acid and the like. "Pharmaceutically acceptable salts" of the presently disclosed compounds also include those formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc, and from bases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine, arginine, ornithine, choline, Ν,Ν'- dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N- benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)aminomethane, and tetramethylammonium hydroxide. These salts may be prepared by standard procedures, for example by reacting the free acid with a suitable organic or inorganic base. Any chemical compound recited in this specification may alternatively be administered as a

pharmaceutically acceptable salt thereof. "Pharmaceutically acceptable salts" are also inclusive of the free acid, base, and zwitterionic forms. Descriptions of suitable pharmaceutically acceptable salts can be found in Handbook of Pharmaceutical Salts, Properties, Selection and Use, Wiley VCH (2002). When compounds disclosed herein include an acidic function such as a carboxy group, then suitable pharmaceutically acceptable cation pairs for the carboxy group are well known to those skilled in the art and include alkaline, alkaline earth, ammonium, quaternary ammonium cations and the like. Such salts are known to those of skill in the art. For additional examples of

"pharmacologically acceptable salts," see Berge et al., . Pharm. Sci. 66: 1 (1977).

"Pharmaceutically acceptable esters" includes those derived from compounds described herein that are modified to include a hydroxy or a carboxyl group. An in vivo hydrolysable ester is an ester, which is hydrolysed in the human or animal body to produce the parent acid or alcohol. Suitable pharmaceutically acceptable esters that include a carboxyl group include Ci_6 alkoxymethyl esters for example methoxy-methyl, Ci_6 alkanoyloxymethyl esters for example pivaloyloxymethyl, phthalidyl esters, C₃.₈ cycloalkoxycarbonyloxy, Ci_6 alkyl esters for example 1-cyclohexylcarbonyl-oxyethyl; 1,3- dioxolen-2-onylmethyl esters for example 5-methyl-l,3-dioxolen-2-onylmethyl; and Ci_6 alkoxycarbonyloxy ethyl esters for example 1-methoxycarbonyl-oxy ethyl which may be formed at any carboxy group in the compounds.

An in vivo hydrolysable ester containing a hydroxy group includes inorganic esters such as phosphate esters and a-acyloxyalkyl ethers and related compounds which as a result of the in vivo hydrolysis of the ester breakdown to give the parent hydroxy group.

Examples of α-acyloxyalkyl ethers include acetoxy-methoxy and 2,2- dimethylpropionyloxy-methoxy. A selection of in vivo hydrolysable ester forming groups for hydroxy include alkanoyl, benzoyl, phenylacetyl and substituted benzoyl and phenylacetyl, alkoxycarbonyl (to give alkyl carbonate esters), dialkylcarbamoyl and N- (dialkylaminoethyl)-N-alkylcarbamoyl (to give carbamates), dialkylaminoacetyl and carboxyacetyl. Examples of substituents on benzoyl include morpholino and piperazino linked from a ring nitrogen atom via a methylene group to the 3- or 4-position of the benzoyl ring.

For therapeutic use, salts of the compounds are those wherein the counter-ion is pharmaceutically acceptable. However, salts of acids and bases which are non- pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.

The pharmaceutically acceptable acid and base addition salts as mentioned hereinabove are meant to comprise the therapeutically active non-toxic acid and base addition salt forms which the compounds are able to form. The pharmaceutically acceptable acid addition salts can conveniently be obtained by treating the base form with such appropriate acid. Appropriate acids comprise, for example, inorganic acids such as hydrohalic acids, e.g. hydrochloric or hydrobromic acid, sulfuric, nitric, phosphoric and the like acids; or organic acids such as, for example, acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic (i.e. ethanedioic), malonic, succinic (i.e. butanedioic acid), maleic, fumaric, malic (i.e. hydroxybutanedioic acid), tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic and the like acids. Conversely said salt forms can be converted by treatment with an appropriate base into the free base form.

The compounds containing an acidic proton may also be converted into their nontoxic metal or amine addition salt forms by treatment with appropriate organic and inorganic bases. Appropriate base salt forms comprise, for example, the ammonium salts, the alkali and earth alkaline metal salts, e.g. the lithium, sodium, potassium, magnesium, calcium salts and the like, salts with organic bases, e.g. the benzathine, N-methyl-D-glucamine, hydrabamine salts, and salts with amino acids such as, for example, arginine, lysine and the like.

The term "addition salt" as used hereinabove also comprises the solvates which the compounds described herein are able to form. Such solvates are for example hydrates, alcoholates and the like.

The term "quaternary amine" as used hereinbefore defines the quaternary ammonium salts which the compounds are able to form by reaction between a basic nitrogen of a compound and an appropriate quaternizing agent, such as, for example, an optionally substituted alkylhalide, arylhalide or arylalkylhalide, e.g. methyliodide or benzyliodide. Other reactants with good leaving groups may also be used, such as alkyl

trifluoromethanesulfonates, alkyl methanesulfonates, and alkyl p-toluenesulfonates. A quaternary amine has a positively charged nitrogen. Pharmaceutically acceptable counterions include chloro, bromo, iodo, trifluoroacetate and acetate. The counterion of choice can be introduced using ion exchange resins.

It will be appreciated that the compounds described herein may have metal binding, chelating, complex forming properties and therefore may exist as metal complexes or metal chelates.

Some of the compounds described herein may also exist in their tautomeric form.

"Saturated or unsaturated" includes substituents saturated with hydrogens, substituents completely unsaturated with hydrogens and substituents partially saturated with hydrogens.

"Selectively targeting," as used herein, means that the inhibitors described herein induce at least one response in targeted cancer stem cells without substantially affecting non-targeted cells, or the response in the targeted cancer stem cells is induced to a greater degree relative to the induced response in non-targeted cells. For example, the selective targeting may include selectively inhibiting proliferation of the stem cells, selectively increasing differentiation of the stem cells, selectively inducing apoptosis of the stem cells, or a combination thereof. In one embodiment, the inhibitors disclosed herein may have selective activity against cancer stem-cell-like xenografts compared to the parental cell xenografts.

The term "subject" includes both human and veterinary subjects.

A "therapeutically effective amount" or "diagnostically effective amount" refers to a quantity of a specified agent sufficient to achieve a desired effect in a subject being treated with that agent. Ideally, a therapeutically effective amount or diagnostically effective amount of an agent is an amount sufficient to inhibit or treat the disease without causing a substantial cytotoxic effect in the subject. The therapeutically effective amount or diagnostically effective amount of an agent will be dependent on the subject being treated, the severity of the affliction, and the manner of administration of the therapeutic composition.

Prodrugs of the disclosed compounds also are contemplated herein. A prodrug is an active or inactive compound that is modified chemically through in vivo physiological action, such as hydrolysis, metabolism and the like, into an active compound following administration of the prodrug to a subject. The term "prodrug" as used throughout this text means the pharmacologically acceptable derivatives such as esters, amides and phosphates, such that the resulting in vivo biotransformation product of the derivative is the active drug as defined in the compounds described herein. Prodrugs preferably have excellent aqueous solubility, increased bioavailability and are readily metabolized into the active inhibitors in vivo. Prodrugs of a compounds described herein may be prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either by routine manipulation or in vivo, to the parent compound. The suitability and techniques involved in making and using prodrugs are well known by those skilled in the art. F or a general discussion of prodrugs involving esters see Svensson and Tunek, Drug Metabolism Reviews 165 (1988) and Bundgaard, Design of Prodrugs, Elsevier (1985).

The term "prodrug" also is intended to include any covalently bonded carriers that release an active parent drug of the present invention in vivo when the prodrug is administered to a subject. Since prodrugs often have enhanced properties relative to the active agent pharmaceutical, such as, solubility and bioavailability, the compounds disclosed herein can be delivered in prodrug form. Thus, also contemplated are prodrugs of the presently disclosed compounds, methods of delivering prodrugs and compositions containing such prodrugs. Prodrugs of the disclosed compounds typically are prepared by modifying one or more functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to yield the parent compound. Prodrugs include compounds having a phosphonate and/or amino group functionalized with any group that is cleaved in vivo to yield the corresponding amino and/or phosphonate group, respectively. Examples of prodrugs include, without limitation, compounds having an acylated amino group and/or a phosphonate ester or phosphonate amide group. In particular examples, a prodrug is a lower alkyl phosphonate ester, such as an isopropyl phosphonate ester.

Protected derivatives of the disclosed compounds also are contemplated. A variety of suitable protecting groups for use with the disclosed compounds are disclosed in Greene and Wuts, Protective Groups in Organic Synthesis; 3rd Ed.; John Wiley & Sons, New York, 1999.

In general, protecting groups are removed under conditions which will not affect the remaining portion of the molecule. These methods are well known in the art and include acid hydrolysis, hydrogenolysis and the like. One preferred method involves the removal of an ester, such as cleavage of a phosphonate ester using Lewis acidic conditions, such as in TMS-Br mediated ester cleavage to yield the free phosphonate. A second preferred method involves removal of a protecting group, such as removal of a benzyl group by

hydrogenolysis utilizing palladium on carbon in a suitable solvent system such as an alcohol, acetic acid, and the like or mixtures thereof. A t-butoxy-based group, including t- butoxy carbonyl protecting groups can be removed utilizing an inorganic or organic acid, such as HC1 or trifluoroacetic acid, in a suitable solvent system, such as water, dioxane and/or methylene chloride. Another exemplary protecting group, suitable for protecting amino and hydroxy functions amino is trityl. Other conventional protecting groups are known and suitable protecting groups can be selected by those of skill in the art in consultation with Greene and Wuts, Protective Groups in Organic Synthesis; 3rd Ed.; John Wiley & Sons, New York, 1999. When an amine is deprotected, the resulting salt can readily be neutralized to yield the free amine. Similarly, when an acid moiety, such as a phosphonic acid moiety is unveiled, the compound may be isolated as the acid compound or as a salt thereof.

Particular examples of the presently disclosed compounds include one or more asymmetric centers; thus these compounds can exist in different stereoisomeric forms. Accordingly, compounds and compositions may be provided as individual pure enantiomers or as stereoisomeric mixtures, including racemic mixtures. In certain embodiments the compounds disclosed herein are synthesized in or are purified to be in substantially enantiopure form, such as in a 90% enantiomeric excess, a 95% enantiomeric excess, a 97% enantiomeric excess or even in greater than a 99% enantiomeric excess, such as in enantiopure form.

Groups which are substituted (e.g. substituted alkyl), may in some embodiments be substituted with a group which is substituted (e.g. substituted aryl). In some embodiments, the number of substituted groups linked together is limited to two (e.g. substituted alkyl is substituted with substituted aryl, wherein the substituent present on the aryl is not further substituted). In some embodiments, a substituted group is not substituted with another substituted group (e.g. substituted alkyl is substituted with unsubstituted aryl). Inhibitors

The inhibitors or anti-cancer agents described herein may exhibit activity for selectively targeting cancer stem cells. Illustrative inhibitors include indenoisoquinoline compounds, naphthoquinone compounds, quinolinedione compounds, and bouvardin, and pharmaceutically acceptable salts or esters thereof. Unless context clearly indicates otherwise, all compounds described herein may be provided as a pharmaceutically acceptable salt or ester thereof. In some embodiments, the inhibitor is not a salt or ester. In some embodiments, the inhibitor is a salt. In some embodiments, the inhibitor is an ester. In certain embodiments, the inhibitors may be low molecular weight compounds ("LMWCs", having a molecular weight of less than about, for example and not by way of limitation, 600 daltons).

The indenoisoquinoline compounds may have a structure represented by:

(Formula I) wherein the group designated is hydrogen, formyl, phenyl, phenyl substituted with Ci-C₆ alkoxy or Ci-C₆ alkyl, or is a group— (CH₂)_mZ, wherein m is 1-6 and Z is selected from the group consisting of hydrogen, hydroxyl, carboxy, formyl, C₁-C6 alkyl, carbo-(Ci-C6 alkoxy), C₂-C6 alkenyl, phenyl, C₁-C6 alkylamino, heterocycloalkyl, heteroaryl, and C₁-C6 hydroxy-alkylamino ;

R₂ and R₂' are independently selected from the group consisting of hydrogen, C₁-C6 alkyl, C₂-C6 alkenyl, C₁-C6 alkoxy, phenoxy and benzyloxy, or R₂ and R₂' taken together form a group of the formula— OCH₂0— ; R₄ is selected from the group consisting of hydrogen, Ci-C₆ alkyl, C₂-C₆ alkenyl, Ci-C₆ alkoxy, phenoxy and benzyloxy;

R₃ and R₃' are independently selected from the group consisting of hydrogen, Ci-C₆ alkyl, Ci-C₆ alkoxy, C₂-C₆ alkenyl, phenoxy, and benzyloxy, or R₃ and R₃' taken together form a group of the formula— OCH₂0— ; wherein n=l or 0, and bond a is a single bond when n=l, and bond a is a double bond when n=0; provided that when R₂, R₂', R₄, R₃ and R₃' are hydrogen, Z is not C1-C6

hydroxy alkylamino; and further provided that when Ri is methyl, R₃ and R₃' are independently selected from the group consisting of hydrogen, Ci-C₆ alkyl, Ci-C₆ alkoxy, C₂-C₆ alkenyl, phenoxy, and benzyloxy. In some embodiments, the indenoisoquinoline compound of Formula I is a pharmaceutically acceptable salt or ester thereof.

In some embodiments of the compounds of Formula I, the group designated Ri is selected from hydrogen, formyl, phenyl, phenyl substituted with Ci-C₆ alkoxy or Ci-C₆ alkyl, or Ri is a group— (CH₂)_mZ, wherein m is 1-6 and Z is selected from the group consisting of hydrogen, hydroxyl, carboxy, formyl, Ci-C₆ alkyl, carbo-(Ci-C₆ alkoxy), C₂-C₆ alkenyl, phenyl, Ci-C₆ alkylamino, and Ci-C₆ hydroxy-alkylamino.

In one embodiment of the compounds of Formula I, the protons on the carbon atoms at fusion bond a are in a cis-configuration across bond a.

In one embodiment the compound of Formula I has the following substituents: Ri is — (CH₂)_m OH and m is 3-6, n is zero (0) and a is a double bond; and R₂, R₂', R₃, R₃' and R₄ are hydrogen.

In one embodiment the compound of Formula I has the following substituents: Ri is C₂-C₄ alkyl or C₂-C₄ alkenyl; R₂ and R₂' are C1-C4 alkoxy; R₃ and R₃' taken together form a group of the formula— OCH₂0— ; and R₄ is hydrogen.

Another embodiment includes the compound of Formula I wherein: Ri is (CH₂)_m OH and m is 3-6, n is zero (0) and a is a double bond; R₂ and R₂' are Ci-C₃ alkoxy; R₃ and R₃' taken together form a group of the formula— OCH₂0— ; and R₄ is hydrogen. A further embodiment includes the compound for Formula I wherein: R is C₁-C3 alkyl or C₂-C₄ alkenyl; n is one (1) and a is a single bond; R₃ and R₃' taken together form a group of the formula— OCH₂0— ; and R4 is hydrogen.

Another embodiment includes the compound for Formula I wherein: Ri is — (CH₂)_mCOOH and m is 1-4, n is zero (0) and a is a double bond; and R₂, R₂', R₃, R₃' and R₄ are hydrogen. cture represented by:

(Formula II) wherein

Ri is phenyl or phenyl substituted with C₁-C6 alkoxy or C₁-C6 alkyl, or Ri is a group — (CH₂)_mZ wherein m is 1-6 and Z is selected from the group consisting of hydrogen, hydroxyl, carboxy, formyl, C₁-C6 alkyl, carbo-(Ci-C6 alkoxy), C₂-C6 alkenyl, phenyl, Ci-C₆ alkylamino, heterocycloalkyl, heteroaryl, and Ci-C₆ hydroxyalkylamino, provided that when Z is hydrogen, m is 2-6;

R₂ and R₂' are independently selected from the group consisting of hydrogen, Ci-C₆ alkyl, C₂-C₆ alkenyl, Ci-C₆ alkoxy, phenoxy and benzyloxy, or R₂ and R₂' taken together form a group of the formula— OCH₂0— ;

R₄ is selected from the group consisting of hydrogen, Ci-C₆ alkyl, C₂-C₆ alkenyl, Ci-C₆ alkoxy, phenoxy and benzyloxy; R₃ and R₃' are independently selected from the group consisting of hydrogen, Ci-C₆ alkyl, Ci-C₆ alkoxy, C₂-C₆ alkenyl, phenoxy, and benzyloxy, or R₃ and R₃' taken together form a group of the formula— OCH₂0— ; and wherein X is a pharmaceutically acceptable anion. In some embodiments, the indenoisoquinoline compound of Formula II is a pharmaceutically acceptable salt or ester thereof.

A "pharmaceutically acceptable anion" is defined as any non-toxic mono-, di- or trivalent anions. Exemplary of such are Br^", CI^", S0₄ ², P0₄ ^"3, acetate, C0₃ ^"2 and HC0₃ ^". It is understood that the stoichemistry of the salts of Formula II are dependent on the valence of the anion component and the ratio of cationic to anionic components is such as to provide a neutral salt.

In some embodiments of Formula II, Ri is phenyl or phenyl substituted with Ci-C₆ alkoxy or Ci-C₆ alkyl, or Ri is a group— (CH₂)_mZ wherein m is 1-6 and Z is selected from the group consisting of hydrogen, hydroxyl, carboxy, formyl, Ci-C₆ alkyl, carbo-(Ci-C₆ alkoxy), C₂-C₆ alkenyl, phenyl, Ci-C₆ alkylamino, and Ci-C₆ hydroxyalkylamino, provided that when Z is hydrogen, m is 2-6.

In one embodiment, a compound of Formula II has the following substituent groups: Ri is Q-C4 alkyl; R₂ and R₂' are Ci-C₃ alkoxy, R₃ and R₃' taken together form a group of the formula— OCH₂— O— ; and R4 is hydrogen.

Examples of such indenoisoquinoline compounds are disclosed in U.S. Patent No. 6,509,344, which is incorporated herein by reference in its entirety.

Illustrative indenoisoquinoline compounds include 6-[3-[(lH-imidazolyl)propyl]- 5 ,6-dihydro-2,3 -dimethoxy-8 ,9-methylenedioxy-5 , 11 -dioxo- 11 H-indeno[ 1 ,2-c] isoquinoline (NSC725776; LMP-776), the hydrochloride salt form of which may be represented by:

and 5H-[l,3]dioxolo[5,6]indeno[l,2-c]isoquinoline-5,12(6H)-dione, 2,3-dimethoxy-6-[3-(4- morpholinyl)-propyl] (NSC743400; LMP-400), the hydrochloride salt form of which may be represented by:

H3C CH3

-lapachone analogs may have a structure represented by:

H3C CH3

(Formula III)

Preferred examples of compounds of Formula III include reduced β-lapachone (Formula III, in which R' and R" are each hydrogen), as well as derivatives of reduced beta- lapachone (Formula III, in which R and R" are each independently hydrogen, lower alkyl, or acyl).

According to further embodiments, illustrative β-lapachone analogs include:

where Ri and R₂ are each independently hydrogen, substituted and unsubstituted aryl, substituted and unsubstituted alkenyl, substituted and unsubstituted alkyl, or substituted or unsubstituted alkoxy. The alkyl groups preferably have from 1 to about 15 carbon atoms, more preferably from 1 to about 10 carbon atoms, still more preferably from 1 to about 6 carbon atoms. The term alkyl unless otherwise modified refers to both cyclic and noncyclic groups, although of course cyclic groups will comprise at least three carbon ring members. Straight or branched chain noncyclic alkyl groups are generally more preferred than cyclic groups. Straight chain alkyl groups are generally more preferred than branched. The alkenyl groups preferably have from 2 to about 15 carbon atoms, more preferably from 2 to about 10 carbon atoms, still more preferably from 2 to 6 carbon atoms. Especially preferred alkenyl groups having 3 carbon atoms (i.e., 1-propenyl or 2-propenyl), with the allyl moiety being particularly preferred. Phenyl and naphthyl are generally preferred aryl groups. Alkoxy groups include those alkoxy groups having one or more oxygen linkage and preferably have from 1 to 15 carbon atoms, more preferably from about 1 to about 6 carbon atoms. The substituted Ri and R₂ groups may be substituted at one or more available positions by one or more suitable groups such as, for example, alkyl groups such as alkyl groups having from 1 to 10 carbon atoms or from 1 to 6 carbon atoms, alkenyl groups such as alkenyl groups having from 2 to 10 carbon atoms or 2 to 6 carbon atoms, aryl groups having from six to ten carbon atoms, halogen such as fluoro, chloro and bromo, and N, O and S, including heteroalkyl, e.g., heteroalkyl having one or more hetero atom linkages (and thus including alkoxy, aminoalkyl and thioalkyl) and from 1 to 10 carbon atoms or from 1 to 6 carbon atoms.

analogs may have the structure:

where R and Ri are each independently selected from hydrogen, hydroxyl, sulfhydryl, halogen, substituted alkyl, unsubstituted alkyl, substituted alkenyl, unsubstituted alkenyl, substituted aryl, unsubstituted aryl, substituted alkoxy, unsubstituted alkoxy, and salts thereof, where the dotted double bond between the ring carbons represent an optional double ring bond.

Further illustrative β-lapachone analogs may have the structure:

where R₅ and R₆ may be independently selected from hydroxyl, C₁-C6 alkyl, C₁-C6 alkenyl, C₁-C6 alkoxy, C₁-C6 alkoxycarbonyl,— (CH₂)„-phenyl; and R₇ is hydrogen, hydroxyl, C₁-C6 alkyl, C₁-C6 alkenyl, C₁-C6 alkoxy, C₁-C6 alkoxycarbonyl,— (CH₂)„-amino,— (CH₂)„-aryl, — (CH₂)„-heteroaryl,— (CH₂)„-heterocycle, or— (CH₂)„-phenyl, where n is an integer from 0 to 10.

Other β-lapachone analogs and derivatives are disclosed in U.S. Pat. Nos.

5,763,625, 5,824,700, 5,969,163, as well is in scientific journal articles, such as Sabba et al., J Med Chem 27:990-994 (1984), which discloses β-lapachone with substitutions at one or more of the following positions: 2-, 8- and/or 9-positions. See also Portela et al., Biochem Pharm 51 :275-283 (1996) (substituents at the 2- and 9-positions); Maruyama et al., Chem Lett 847-850 (1977); Sun et al., Tetrahedron Lett 39:8221-8224 (1998); Goncalves et al., Molecular and Biochemical Parasitology 1 : 167-176 (1998) (substituents at the 2- and 3- positions); Gupta et al., Indian Journal of Chemistry 16B:35-37 (1978); Gupta et al., Curr Sci 46:337 (1977) (substituents at the 3- an 4-positions); DiChenna et al., J Med Chem 44:2486-2489 (2001) (monoarylamino derivatives). Each of the above-mentioned references are incorporated by reference herein in their entirety.

Additional β-lapachone analogs encompass compounds having the general formula

III and IV:

(Formula III)

(Formula IV) where dotted double bond between the ring carbons represents an optional ring double bond and where and R₂ are each independently selected from hydrogen, hydroxyl, sulfhydryl, halogen, substituted alkyl, unsubstituted alkyl, substituted alkenyl, unsubstituted alkenyl, substituted aryl, unsubstituted aryl, substituted alkoxy, unsubstituted alkoxy, and salts thereof. The alkyl groups preferably have from 1 to about 15 carbon atoms, more preferably from 1 to about 10 carbon atoms, still more preferably from to about 6 carbon atoms. The term alkyl refers to both cyclic and noncyclic groups. Straight or branched chain noncyclic alkyl groups are generally more preferred than cyclic groups. Straight chain alkyl groups are generally more preferred than branched. The alkenyl groups preferably have from 2 to about 15 carbon atoms, more preferably from 2 to about 10 carbon atoms, still more preferably from 2 to 6carbon atoms. Especially preferred alkenyl groups have 3 carbon atoms (i.e., 1-propenyl or 2-propenyl), with the allyl moiety being particularly preferred. Phenyl and naphthyl are generally preferred aryl groups. Alkoxy groups include those alkoxy groups having one or more oxygen linkage and preferably have from 1 to 15 carbon atoms, more preferably from 1 to about 6 carbon atoms. The substituted R and groups may be substituted at one or more available positions by one or more suitable groups such as, for example, alkyl groups having from 1 to 10 carbon atoms or from 1 to 6 carbon atoms, alkenyl groups having from 2 to 10 carbon atoms or 2 to 6 carbon atoms, aryl groups having from six to ten carbon atoms, halogen such as fluoro, chloro and bromo, and N, O and S, including heteroalkyl, e.g., heteroalkyl having one or more hetero atom linkages (and thus including alkoxy, aminoalkyl and thioalkyl) and from 1 to lOcarbon atoms or from 1 to 6 carbon atoms; and where R₅ and R₆ may be independently selected from hydroxyl, C₁-C6 alkyl, C₁-C6 alkenyl, C₁-C6 alkoxy, C₁-C6 alkoxycarbonyl,— (CH₂)„-aryl,

— (Ch₂)„-heteroaryl,— (CH₂)„-heterocycle, or— (CH₂)„-phenyl; and R₇ is hydrogen, hydroxyl, C₁-C6 alkyl, C₁-C6 alkenyl, C₁-C6 alkoxy, C₁-C6 alkoxycarbonyl,— (CH₂)„-amino,— (CH₂)„- aryl,— (CH₂)„-heteroaryl,— (CH₂)„-heterocycle, or— (CH₂)„-phenyl, wherein n is an integer from 0 to 10.

Other β-lapachone analogs include compounds of the following general formula V:

where Ri is (CH₂)„— R₂, where n is an integer from 0-10 and R₂ is hydrogen, an alkyl, an aryl, a heteroaromatic, a heterocyclic, an aliphatic, an alkoxy, an allyloxy, a hydroxyl, an amine, a thiol, an amide, or a halogen.

Specific analogs include 4-acetoxy- -lapachone, 4-acetoxy-3-bromo- -lapachone, 4-keto- "lapachone, 7-hydroxy- -lapachone, 7-methoxy- -lapachone, S-hydroxy-β- lapachone, 8-methoxy-P-lapachone, 8-chloro-P-lapachone, 9-chloro-P-lapachone, 8-methyl- β-lapachone and 8,9-dimethoxy-P-lapachone.

Additional analogs include compounds of the following general formula VI:

where R₂-R4 are each, independently, selected from the group consisting of H, Ci-C₆ alkyl, Ci-C₆ alkenyl, Ci-C₆ alkoxy, Ci-C₆ alkoxycarbonyl,— (CH₂)„-aryl,— (CH₂)„-heteroaryl, — (CH₂)„-heterocycle, and— (CH₂)„-phenyl; or Ri and R₂ combined are a single substituent selected from the above group, and R₃ and R₄ combined are a single substituent selected from the above groups, in which case— is a double bond.

Specific β-lapachone analogs and derivatives also contemplated include dunnione and 2-ethyl-6-hydroxynaphtho[2,3-&]-furan-4,5-dione.

Further β-lapachone analogs include compounds of the following general formula VII:

wherein R1-R6 are each, independently, selected from the group consisting of H, OH, substituted and unsubstituted Ci-C₆ alkyl, substituted and unsubstituted Ci-C₆ alkenyl, substituted and unsubstituted C₁-C6 alkoxy, substituted and unsubstituted C₁-C6 alkoxycarbonyl, substituted and unsubstituted C₁-C6 acyl,— (CH₂)„-amino,— (CH₂)„-aryl, — (CH₂)„-heterocycle, and— (CH₂)„-phenyl; or one of Rl or R2 and one of R3 or R4; or one of R3 or R4 and one of R5 and R6 form a fused ring, wherein the ring has 4-8 ring members, R7-R10 are each, independently, hydrogen, hydroxyl, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, nitro, cyano or amide; and n is an integer from 0 to 10.

In a particular embodiment, Rl and R2 are alkyl, R3 -R6 are, independently, H, OH, halogen, alkyl, alkoxy, substituted and unsubstituted acyl, substituted alkenyl or substituted alkyl carbonyl, and R7-R10 are hydrogen. In another preferred embodiment, R1-R4 are each hydrogen, R5 and R6 are each methyl and R7-R10 are each hydrogen.

Additional β-lapachone analogs are represented by Formula IX:

wherein R1-R4 are each, independently, selected from the group consisting of H, OH, substituted and unsubstituted Ci-C₆ alkyl, substituted and unsubstituted Ci-C₆ alkenyl, substituted and unsubstituted Ci-C₆ alkoxy, substituted and unsubstituted Ci-C₆ alkoxycarbonyl, substituted and unsubstituted Ci-C₆ acyl,— (CH₂)„-amino,— (CH₂)„-aryl,

— (CH₂)„-heterocycle, and— (CH₂)„-phenyl; or one of Rl or R2 and one of R3 and R4 form a fused ring, wherein the ring has 4-8 ring members; R5-R8 are each, independently, hydrogen, hydroxyl, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, nitro, cyano or amide, and n is an integer from 0 to 10. In certain embodiments of Formula VII, Rl, R2, R3, R4, R5, R6, R7 and R8 are not each simultaneously H.

The above-described β-lapachone analogs are disclosed in U.S. Patent No.

7,361,691, which is incorporated herein by reference in its entirety.

In another embodiment, the naphthoquinone compound may be represented by:

wherein R¹ and R² are each independently selected from H, alkyl, thiol, thioalkyl or thioalkoxy. According to certain embodiments, both R¹ and R² are thio-alkyl-hydroxy such as, for example, in 2,3-bis[(2-hydroxyethyl)thiol]-l,4-naphthoquinone (NSC 95397):

In some embodiments, the quinolinedione compound is a quinolinedione. In some embodiments, the quinolinedione compound is an isoquinolinedione compound. In one embodiment the quinolinedione compound may be a caulibugulone compound represented by the structure:

wherein Ri is H, Br or CI; and R₂ is methyl or -CH₂CH₂OH; or

wherein R is H or -CH₂CH₂OH.

According to one specific example, the caulibugulone compound is caulibugulone A (the first structure above wherein Ri is H and R₂ is methyl).

In another embodiment, the quinolinedione compounds may be represented by:

wherein R¹ and R² are each independently selected from H, CI, Br, -alkyl-heterocycloalkyl, or alkyl (particularly lower alkyl) or R¹ and R² together form a heterocyclic ring; wherein R³ and R⁴ are each independently selected from H or alkyl (particularly lower alkyl); and R⁵ is H, alkyl, cyano, a carbonyl-containing group, formyl, carboxyl, substituted carboxyl, amino, or aminocarbonyl. In some embodiments, R¹ and R² are each independently selected from H, CI, Br, -alkyl-heterocycloalkyl, or alkyl (particularly lower alkyl) or R¹ and R² together form a heterocyclic ring. According to certain embodiments, R¹, R², R³ and R⁴ are each H. In some embodiments, R⁵ is amino. Illustrative specific examples include:

(6-chloro-7-(2-morpholin-4-yl-ethylamino)-quinoline-5,8-dione; DA-3003-01) and

(7-(2-morpholin-4-yl-ethylamino)-quinoline-5,8-dione; JUN1111).

A further specific example of a quinolinedione compound is:

(JUN1511)

Another inhibitor is bouvardin or deoxybouvardin:

wherein R is OH or H.

In some embodiments, the inhibitor is A-77636 hydrochloride ((lR-cis)-l- ( Aminomethyl)-3 ,4-dihydro-3-tricyclo[3.3.1.13,7] dec- 1 -yl- [ 1 H] -2-benzopyran-5 ,6-diol hydrochloride):

In some embodiments, the inhibitor is rottlerin (3'-[(8-Cinnamoyl-5,7-dihydroxy- 2,2-dimethyl-2H- 1 -benzopyran-6-yl)methyl] -2',4',6'-trihydroxy-5'-methylacetophenone) :

In some embodiments, the inhibitor is CGP-74514A hydrochloride (N -(cis-2- Aminocyclohexyl)-N⁶-(3-chl 6-diamine hydrochloride):

Composition and Methods

The compounds and pharmaceutical compositions disclosed herein can be used for inhibiting or treating cancer, particularly drug-resistant cancer. In particular, the inhibitors disclosed herein may selectively target cancer, particularly carcinoma, stem cells. The cancer stem cells may be present in, or obtained from, a solid tumor.

Illustrative cancer stem cells that may be targeted include, for example, but not by way of limitation, breast cancer, prostate cancer, glioblastoma, colon carcinoma, lung carcinoma, pancreatic cancer, melanoma, gastric cancer, hepatic carcinoma, ovarian carcinoma, and testicular cancer. Other cancer stem cells for targeting include lymphoma, and leukemia.

The preparation and use of immortalized cancer stem cells is described in PCT International Publication No. WO 2009/140260, "Cancer Stem Cell Immortalization", filed May 12, 2009, which is incorporated herein by reference in its entirety. Introduction of an Oct3/4 promoter sequence was observed to stabilize the undifferentiated phenotype of cancer stem cells. This effect is alternatively referred to herein as "immortalization", which as defined herein, does not require that a culture of such cells would persist indefinitely. Similarly, stability of the undifferentiated phenotype of cancer stem cells, as that phrase is used herein, does not require that all aspects of the cancer stem cell phenotype be retained, but rather that at least one or more characteristics of that phenotype be retained (although not necessarily at the same level as native cells). For example, one or more of the following characteristics are retained: the expression of one or more surface antigen; the level of expression of one or more surface antigen; permeability to a histologic dye; number of cells required to produce a tumor when implanted into a host animal; characteristics of tumors produced from the cells; morphology in culture; association with other cells in culture; and sensitivity to pharmacologic agents.

"Oct3/4 promoter sequences" are a family of sequences which are related to the native promoter operably linked to an Oct3/4 gene. In one embodiment, the Oct3/4 gene is the human Oct3/4 gene. A variety of lengths of sequences are encompassed within the "Oct3/4 promoter sequences" family. Likewise, some of the sequences exhibit

demonstrable promoter activity (for example, of the Oct3/4 gene and/or a reporter gene), whereas others do not. Members of the "Oct3/4 promoter family", share that property that, when introduced into a cancer stem cell, they promote the persistence of the cancer stem cell phenotype.

A cancer stem cell obtained from any type of cancer may be immortalized - its phenotype stabilized. The cancer stem cell may be from a human or a non-human subject. The cancer stem cell may be obtained from a tumor cell line or for a primary tumor.

A cancer stem cell may be collected by any means known in the art. For example, a cancer stem cell may be collected from (isolated from or enriched from) a larger population of cells using cell surface markers or other properties typical to that cancer stem cell.

Alternatively, an Oct3/4 promoter sequence -containing construct which contains a selectable marker which is selectively expressed in a cancer stem cell may be collected from the population via the selectable marker, for example by fluorescence activated cell sorting ("FACS").

Expression of the ALDH1 isoform (9a) may be used as a cancer stem cell marker. For example, the Aldefluor Assay (Stem Cell Technologies, Inc.) may be used.

In non-limiting embodiments, where the cancer stem cell is a breast cancer stem cell, a phenotype of cell marker expression CD44hi (meaning increased relative to normal control) and CD241o (meaning decreased relative to normal control) may be used to collect cancer stem cells (for example, using antibodies directed to said proteins and FACS). Where a cell line is used as the source of cancer stem cells, suitable cell lines include, but are not limited to MCF7, T-47D, UACC-812, HCC38, HCC1428, SKBR-3, and MB-157.

In non-limiting embodiments, where the cancer stem cell is a colon cancer stem cell, a phenotype of cell marker expression EpCAMhi/CD44hi, or expression of CD133, or the ability to exclude the dye Hoechst 33342, may be used to collect cancer stem cells. Where a cell line is used as the source of cancer stem cells, suitable cell lines include, but are not limited to Colo320, HCT15, and SW480.

In non-limiting embodiments, where the cancer stem cell is a prostate cancer stem cell, a phenotype of cell marker expression CD44hiCD241o/Scal+ or the ability to exclude the dye Hoechst 22243, may be used to collect cancer stem cells. Where a cell line is used as the source of cancer stem cells, suitable cell lines include, but are not limited to PC3, DU145, and LNCaP.

In non-limiting embodiments, where the cancer stem cell is a pancreatic cancer stem cell, a phenotype of cell marker expression CD44hi, CD24hi, ESAhi may be used to collect cancer stem cells. Where a cell line is used as the source of cancer stem cells, suitable cell lines include, but are not limited to PANC-1 and ASPC-1.

The immortalized cancer stem cells may be used to identify useful therapeutic agents, by screening various test agents. The test agents may be known bioactive compounds or may be compounds without hitherto known biological activity. Suitable test agents may also be biologic molecules, including but not limited to proteins, antibodies or antibody fragments, oligonucleotides, peptidomimetic compounds, etc.

One method of identifying an anti-cancer agent includes:

(i) providing an isolated cancer stem cell containing an Oct3/4 promoter sequence which is not operably linked to an Oct3/4 gene;

(ii) providing a means for evaluating the proliferation, differentiation level, and/or viability of the cancer stem cell;

(iii) administering a test agent to the cancer stem cell; and

(iv) evaluating the proliferation and/or differentiation and/or viability of the cancer stem cell;

wherein an inhibition of proliferation, increase in level of differentiation, or decrease in viability associated with the presence of the test agent indicates that the test agent is an anti-cancer agent. In certain non-limiting embodiments of this method, the means for evaluating the proliferation, differentiation level, and/or viability comprising measuring and/or detecting expression of a reporter gene (see above). In certain embodiments, a method of identifying an anti-cancer agent with selective activity toward cancer stem cells includes:

(i) providing a population of cancer cells comprising cancer stem cells as well as cancer cells which are not stem cells, where the relative proportions of cancer stem cells and cancer cells which are not stem cells is known;

(ii) administering a test agent to the population of cells;

(iii) culturing the population after (ii); and

(iv) determining the relative proportions of cancer stem cells and cancer cells which are not stem cells in the population after (iii);

wherein a decrease in the relative proportion of cancer stem cells indicates that the test agent is an anti-cancer agent with selective activity against cancer stem cells. In non- limiting embodiments of this method, there is a first means for detecting a cancer stem cell and a second, different means for detecting a cancer cell that is not a stem cell, where said first means and said second means are used to determine the relative proportions of cancer stem cells and cancer cells which are not stem cells. For example, in non-limiting embodiments said first means and/or second means may be a fluorescent antibody to a cell surface antigen (if both means are fluorescent antibodies, they are desirably of different colors). Alternatively, in other non-limiting embodiments, said first means and/or second means may be an expression construct having a reporter gene selectively expressed in a cancer stem cell or a cancer cell which is not a stem cell (if both means are reporter genes, they preferably encode different products). As a specific non-limiting example, a cancer stem cell may be detected via a detectable reporter gene (e.g. a fluorescent protein of a first color) operably linked to an Oct3/4 promoter sequence with promoter activity, and a cancer cell which is not a stem cell may be detected by a fluorescent antibody (of a second color) which recognizes a surface antigen on said cancer cell but absent or in substantially lower amounts on a cancer stem cell.

In non-limiting embodiments, high throughput screening techniques may be used. In a specific, non-limiting example, an assay for screening for anti-cancer agents is described as follows. Unfractionated, non-CSC-MCF7 cells may be infected with a lentiviral vector encoding DsRED (Clontech). CSC-MCF7 cells may separately be transfected or infected with an expression construct comprising an Oct3/4 promoter operably linked to GFP. In the assay, LMWC may be identified which inhibit

proliferation/survival of GFP+CSCs relative to control DsRED-tagged MCF7 cells. The screening procedure may use a 1: 1 mix of the above -described GFP and DsRED cells. After robotically dispensing a total of -2000 cells into 384 well plates, they may be incubated overnight to allow attachment. LMWCs (each in DMSO) may then be added to each well to a final compound concentration of 10 μΜ (final DMSO concentration <1 which is easily tolerated). GFP:DsRED ratios may then be determined daily over the ensuing 2-3 days. All compounds that reduce GFP:DsRED ratios >3SDs below the mean of the control (No LMWC added; DMSO vehicle only) may be flagged for subsequent follow up. Further refinement of the "hits" may utilize advanced mathematical methods such as B-scores and BZ-scores to reduce the false positive rate even lower. The foregoing assay, while sensitive, may lack specificity as there are several ways that a compound could alter the GFP:DsRED ratio other than by inhibiting the proliferation or survival of the GFP+ population: for example, (i) it could inhibit or quench the fluorescence intensity of GFP (specific or nonspecific but not of interest); (ii) it could promote the growth of the DsRED population (specific but likely not of interest); (iii) it could enhance the fluorescence of DsRED (specific or non-specific but not of interest); or (iv) it could promote the differentiation of the GFP+ population into TACs associated with concurrent down-regulation of the Oct3/4 promoter (specific and of interest). Most of these possibilities would not be expected to be distinguishable on the initial screen. Therefore, a compounds flagged in this "first pass" assay would preferably be subjected to further testing, for example re -testing in triplicate. Repeat hits may then be examined at serial dilutions to establish ID50's and to identify compounds that are active at submicromolar concentrations. The lowest concentration of compounds that maximally inhibit the GFP signal may then be re-screened with isolated populations of Oct3/4-GFP+MCF7, DsRED-MCF7 cells and an additional line of MCF7 cells that expresses GFP under the control of a neutral (CMV) promoter. Growth curves for each population may be determined, and apoptosis assays (TUNEL assays, Annexin V staining, and/or caspase-3 cleavage [Caspase3/7, Promega]) may be performed. Visual inspection of cells, flow cytometry and cell sizing using a Vi-Cell apparatus (FIGURE 4) may be used to determine whether loss of GFP expression in individual cells is occurring as would be expected if the compound were promoting CSC differentiation that might, without affecting cell proliferation or viability, cause loss of GFP due to a differentiation-mediated down-regulation of the Oct3/4 promoter. H&E and CD44/CD24 staining may be used to confirm this by documenting changes in morphology and cell surface phenotype.

Also disclosed is a means of identifying an agent likely to be of benefit to a subject, where the subject has a cancer, comprising:

(i) collecting a cancer stem cell from the subject; (ii) introducing, into the cancer stem cell from the subject, a nucleic acid comprising as Oct3/4 promoter sequence operably linked to a reporter gene;

(iii) exposing the product of step (ii) to an agent; and

(iv) determining whether exposure to the agent inhibits proliferation, increases the level of differentiation, and/or decreases the viability of the product of step (ii),

where an inhibition of proliferation, increase in the level of differentiation, or decrease of viability indicates that the agent may be of therapeutic benefit to the subject. This method may be desirably practiced for a number of different agents, and selecting the agent which most effectively, among those tested, inhibits proliferation, increases the level of differentiation, and/or decreases viability of the cancer stem cell containing the nucleic acid comprising an Oct3/4 promoter sequence operably linked to a reporter gene.

Provided herein are methods for treating cancer in a subject comprising

administering to the subject a therapeutically effective amount of a CSC inhibitor described herein. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject has recurrent cancer (such as breast, colon, prostate, pancreatic, etc.) after an anti-cancer therapy (such as a treatment with an anticancer agent). In some embodiments, the cancer in the subject is resistant to a

chemotherapy. In some embodiments, the cancer cells in the subject express one or more cancer stem cell markers (for example, markers that are known in the art and described herein). In some embodiments, the subject having cancer cells that express cancer stem cell markers is selected for the treatment described herein.

In certain embodiments, the method further comprises a step of measuring the expression level of one or more cancer stem cell markers in the cancer cells before administration of a CSC inhibitor described herein into the subject. For example, the expression level of one or more of CD44, CD24, CD 133 and Oct3/4 may be measured if the subject has breast cancer. In some embodiments, a subject is treated with a CSC inhibitor described herein if the breast cancer cells in the subject express higher level of CD44, CD 133 and/or Oct3/4 and/or lower level of CD24 as compared to a reference level. In some embodiments, the expression level of one or more of EpCAM, CD44, and CD 133 may be measured if the subject has colon cancer. In some embodiments, a subject is treated with a CSC inhibitor described herein if the colon cancer cells in the subject express higher level of EpCAM and/or CD44 as compared to a reference level and/or express CD133. In some embodiments, the expression level of one or more of CD44, CD24, and Seal may be measured if the subject has prostate cancer. In some embodiments, a subject is treated with a CSC inhibitor described herein if the prostate cancer cells in the subject express higher level of CD44 and/or lower level of CD24 as compared to a reference level, and/or are Seal positive. In some embodiments, the expression level of one or more of CD44, CD24, and ESA may be measured if the subject has pancreatic cancer. In some embodiments, a subject is treated with a CSC inhibitor described herein if the pancreatic cancer cells in the subject express higher level of CD44, CD24, and/or ESA as compared to a reference level. In some embodiments, the mRNA level or protein level of a marker in cancer cells from a sample (e.g., a tissue sample or a cell sample) is measured and compared. Methods known in the art may be used for measuring mRNA and protein levels. In some embodiments, the reference level can be an absolute value, a relative value, a value that has an upper and/or lower limit, or a range of values. For example, a reference level can be an average value, a median value, or a mean value of the expression level of the marker in cancer cells.

In certain embodiments, the CSC inhibitor disclosed herein can be co-administered with an anti-cancer agent for treating cancer. Illustrative anti-cancer agents include alkylating agents, antimitotic agents, topoisomerase I inhibitors, topoisomerase II inhibitors, RNA/DNA antimetabolites, DNA antimetabolites, and antiangiogenic agents. Examples include alkylating agents such as Asaley, AZQ, BCNU, Busulfan,

carboxyphthalatoplatinum, CBDCA, CCNU, CHIP, chlorambucil, chlorozotocin, cis- platinum, clomesone, cyanomorpholinodoxorubicin, cyclodisone, dianhydrogalactitol, fluorodopan, hepsulfam, hycanthone, melphalan, methyl CCNU, mitomycin C,

mitozolamide, nitrogen mustard, PCNU, piperazine, piperazinedione, pipobroman, porfiromycin, spirohydantoin mustard, teroxirone, tetraplatin, thio-tepa,

triethylenemelamine, uracil nitrogen mustard, and Yoshi-864; antimitotic agents such as AUocolchicine, Halichondrin B, colchicine, colchicine derivative, dolastatin 10, maytansine, rhizoxin, taxol, taxol derivative, thiocolchicine, trityl cysteine, vinblastine sulfate, vincristine sulfate, camptothecin, camptothecin, Na salt, aminocamptothecin, camptothecin derivative, and morpholinodoxorubicin; topoisomerase inhibitors such as doxorubicin, amonafide, m-AMSA, anthrapyrazole derivative, pyrazoloacridine, bisantrene HCL, daunorubicin, deoxydoxorubicin, mitoxantrone, menogaril, Ν,Ν-dibenzyl daunomycin, oxanthrazole, rubidazone, VM-26, and VP-16; RNA/DAN antimetabolites such as L- alanosine, 5-azacytidine, 5-fluorouracil, acivicin, aminopterin derivative, an antifol, Baker's soluble antifol, dichlorallyl lawsone, brequinar, ftorafur (pro-drug), 5,6-dihydro-5- azacytidine, methotrexate, methotrexate derivative, N-(phosphonoacetyl)-L-aspartate (PALA), pyrazofurin, and trimetrexate; and DNA antimetabolites such as 3-HP, 2'-deoxy-5- fluorouridine, 5-HP, alpha-TGDR, aphidicolin glycinate, ara-C, 5-aza-2'-deoxycytidine, beta-TGDR, cyclocytidine, guanazole, hydroxyurea, inosine glycodialdehyde, macbecin II, pyrazoloimidazole, thioguanine, and thiopurine.

According to a further embodiment, the inhibitors disclosed herein may be used in a treatment regimen for recurrent breast cancer that treats not only the primary tumor cells but also the stem cells. The CSC inhibitor may be administered in neoadjuvant or adjuvant therapy. For example, the CSC inhibitor may be co-administered with a taxane for treating breast cancer. In a more specific example, the indenoisoquinoline compound(s) may be coadministered with paclitaxel in neoadjuvant treatment of post-menopausal women with stage II III breast cancer.

The stem-cell-like cells express high levels of BCRP(ABCG2) and topoisomerase 1. Certain CSC inhibitors disclosed herein (e.g., indotecan and indimitecan) are not substrates for BCRP(ABCG2) or other multi-drug ABC transporters and are potent inhibitors of topo I. Further, because the indenoisoquinolines are not substrates for the ABC pumps, they should be maintained in the tumor xenografts and may have a selective advantage over other topoisomerase I inhibitors such as topotecan, which may be used as a positive control in efficacy studies.

Another aspect of the disclosure includes pharmaceutical compositions prepared for administration to a subject and which include a therapeutically effective amount of one or more of the inhibitors disclosed herein. The therapeutically effective amount of a disclosed inhibitor will depend on the route of administration, the species of subject and the physical characteristics of the subject being treated. Specific factors that can be taken into account include disease severity and stage, weight, diet and concurrent medications. The relationship of these factors to determining a therapeutically effective amount of the disclosed compounds is understood by those of skill in the art.

Pharmaceutical compositions for administration to a subject can include at least one further pharmaceutically acceptable additive such as carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice.

Pharmaceutical compositions can also include one or more additional active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like. The pharmaceutically acceptable carriers useful for these formulations are conventional.

Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 19th Edition (1995), describes compositions and formulations suitable for pharmaceutical delivery of the compounds herein disclosed. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually contain injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Pharmaceutical compositions disclosed herein include those formed from pharmaceutically acceptable salts and/or solvates of the disclosed inhibitors.

Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable inorganic or organic bases and acids. Particular disclosed inhibitors possess at least one basic group that can form acid-base salts with acids. Examples of basic groups include, but are not limited to, amino and imino groups. Examples of inorganic acids that can form salts with such basic groups include, but are not limited to, mineral acids such as hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid. Basic groups also can form salts with organic carboxylic acids, sulfonic acids, sulfo acids or phospho acids or N-substituted sulfamic acid, for example acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2- acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic acid, and, in addition, with amino acids, for example with a-amino acids, and also with methanesulfonic acid, ethanesulfonic acid, 2-hydroxymethanesulfonic acid, ethane- 1 ,2-disulfonic acid, benzenedisulfonic acid, 4-methylbenzenesulfonic acid, naphthalene-2- sulfonic acid, 2- or 3- phosphogly cerate, glucose-6-phosphate or N-cyclohexylsulfamic acid (with formation of the cyclamates) or with other acidic organic compounds, such as ascorbic acid. In particular, suitable salts include those derived from alkali metals such as potassium and sodium, alkaline earth metals such as calcium and magnesium, among numerous other acids well known in the pharmaceutical art.

Certain inhibitors may include at least one acidic group that can form an acid-base salt with an inorganic or organic base. Examples of salts formed from inorganic bases include salts of the presently disclosed compounds with alkali metals such as potassium and sodium, alkaline earth metals, including calcium and magnesium and the like. Similarly, salts of acidic compounds with an organic base, such as an amine (as used herein terms that refer to amines should be understood to include their conjugate acids unless the context clearly indicates that the free amine is intended) are contemplated, including salts formed with basic amino acids, aliphatic amines, heterocyclic amines, aromatic amines, pyridines, guanidines and amidines. Of the aliphatic amines, the acyclic aliphatic amines, and cyclic and acyclic di- and tri- alkyl amines are particularly suitable for use in the disclosed compounds. In addition, quaternary ammonium counterions also can be used.

Particular examples of suitable amine bases (and their corresponding ammonium ions) for use in the present inhibitors include, without limitation, pyridine, NN- dimethylaminopyridine, diazabicyclononane, diazabicycloundecene, N-methyl-N- ethylamine, diethylamine, triethylamine, diisopropylethylamine, mono-, bis- or tris- (2- hydroxyethyl) amine, 2-hydroxy-teri-butylamine, tris(hydroxymethyl)methylamine, NN- dimethyl-N-(2- hydroxyethyl)amine, tri-(2-hydroxyethyl)amine and N-methyl-D-glucamine. For additional examples of "pharmacologically acceptable salts," see Berge et al., . Pharm. Sci. 66: 1 (1977).

Inhibitors disclosed herein can be crystallized and can be provided in a single crystalline form or as a combination of different crystal polymorphs. As such, the inhibitors can be provided in one or more physical form, such as different crystal forms, crystalline, liquid crystalline or non-crystalline (amorphous) forms. Such different physical forms of the inhibitors can be prepared using, for example different solvents or different mixtures of solvents for recrystallization. Alternatively or additionally, different polymorphs can be prepared, for example, by performing recrystallizations at different temperatures and/or by altering cooling rates during recrystallization. The presence of polymorphs can be determined by X-ray crystallography, or in some cases by another spectroscopic technique, such as solid phase NMR spectroscopy, IR spectroscopy, or by differential scanning calorimetry.

The pharmaceutical compositions can be administered to subjects by a variety of mucosal administration modes, including by oral, rectal, intranasal, intrapulmonary, or transdermal delivery, or by topical delivery to other surfaces. Optionally, the compositions can be administered by non-mucosal routes, including by intramuscular, subcutaneous, intravenous, intra-arterial, intra-articular, intraperitoneal, intrathecal,

intracerebro ventricular, or parenteral routes. In other alternative embodiments, the compound can be administered ex vivo by direct exposure to cells, tissues or organs originating from a subject.

To formulate the pharmaceutical compositions, the inhibitor can be combined with various pharmaceutically acceptable additives, as well as a base or vehicle for dispersion of the compound. Desired additives include, but are not limited to, pH control agents, such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid, and the like. In addition, local anesthetics (for example, benzyl alcohol), isotonizing agents (for example, sodium chloride, mannitol, sorbitol), adsorption inhibitors (for example, Tween 80 or Miglyol 812), solubility enhancing agents (for example, cyclodextrins and derivatives thereof), stabilizers (for example, serum albumin), and reducing agents (for example, glutathione) can be included. Adjuvants, such as aluminum hydroxide (for example, Amphogel, Wyeth Laboratories, Madison, NJ), Freund's adjuvant, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, IN) and IL-12 (Genetics Institute, Cambridge, MA), among many other suitable adjuvants well known in the art, can be included in the compositions. When the composition is a liquid, the tonicity of the formulation, as measured with reference to the tonicity of 0.9% (w/v) physiological saline solution taken as unity, is typically adjusted to a value at which no substantial, irreversible tissue damage will be induced at the site of administration. Generally, the tonicity of the solution is adjusted to a value of about 0.3 to about 3.0, such as about 0.5 to about 2.0, or about 0.8 to about 1.7.

The inhibitor can be dispersed in a base or vehicle, which can include a hydrophilic compound having a capacity to disperse the compound, and any desired additives. The base can be selected from a wide range of suitable compounds, including but not limited to, copolymers of polycarboxylic acids or salts thereof, carboxylic anhydrides (for example, maleic anhydride) with other monomers (for example, methyl (meth)acrylate, acrylic acid and the like), hydrophilic vinyl polymers, such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone, cellulose derivatives, such as hydroxymethylcellulose,

hydroxypropylcellulose and the like, and natural polymers, such as chitosan, collagen, sodium alginate, gelatin, hyaluronic acid, and nontoxic metal salts thereof. Often, a biodegradable polymer is selected as a base or vehicle, for example, polylactic acid, poly(lactic acid-glycolic acid) copolymer, polyhydroxybutyric acid, poly(hydroxybutyric acid-glycolic acid) copolymer and mixtures thereof. Alternatively or additionally, synthetic fatty acid esters such as polyglycerin fatty acid esters, sucrose fatty acid esters and the like can be employed as vehicles. Hydrophilic polymers and other vehicles can be used alone or in combination, and enhanced structural integrity can be imparted to the vehicle by partial crystallization, ionic bonding, cross-linking and the like. The vehicle can be provided in a variety of forms, including fluid or viscous solutions, gels, pastes, powders, microspheres and films for direct application to a mucosal surface.

The inhibitor can be combined with the base or vehicle according to a variety of methods, and release of the compound can be by diffusion, disintegration of the vehicle, or associated formation of water channels. In some circumstances, the inhibitor is dispersed in microcapsules (microspheres) or nanocapsules (nanospheres) prepared from a suitable polymer, for example, isobutyl 2-cyanoacrylate (see, for example, Michael et al., . Pharmacy Pharmacol. 43: 1-5, 1991), and dispersed in a biocompatible dispersing medium, which yields sustained delivery and biological activity over a protracted time.

The compositions of the disclosure can alternatively contain as pharmaceutically acceptable vehicles substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate. For solid compositions, conventional nontoxic pharmaceutically acceptable vehicles can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.

Pharmaceutical compositions for administering the inhibitor can also be formulated as a solution, microemulsion, or other ordered structure suitable for high concentration of active ingredients. The vehicle can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity for solutions can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of a desired particle size in the case of dispersible formulations, and by the use of surfactants. In many cases, it will be desirable to include isotonic agents, for example, sugars, poly alcohols, such as mannitol and sorbitol, or sodium chloride in the composition. Prolonged absorption of the inhibitor can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.

In certain embodiments, the inhibitor can be administered in a time release formulation, for example in a composition which includes a slow release polymer. These compositions can be prepared with vehicles that will protect against rapid release, for example a controlled release vehicle such as a polymer, microencapsulated delivery system or bioadhesive gel. Prolonged delivery in various compositions of the disclosure can be brought about by including in the composition agents that delay absorption, for example, aluminum monostearate hydrogels and gelatin. When controlled release formulations are desired, controlled release binders suitable for use in accordance with the disclosure include any biocompatible controlled release material which is inert to the active agent and which is capable of incorporating the inhibitor and/or other biologically active agent. Numerous such materials are known in the art. Useful controlled-release binders are materials that are metabolized slowly under physiological conditions following their delivery (for example, at a mucosal surface, or in the presence of bodily fluids). Appropriate binders include, but are not limited to, biocompatible polymers and copolymers well known in the art for use in sustained release formulations. Such biocompatible compounds are non-toxic and inert to surrounding tissues, and do not trigger significant adverse side effects, such as nasal irritation, immune response, inflammation, or the like. They are metabolized into metabolic products that are also biocompatible and easily eliminated from the body.

Exemplary polymeric materials for use in the present disclosure include, but are not limited to, polymeric matrices derived from copolymeric and homopolymeric polyesters having hydrolyzable ester linkages. A number of these are known in the art to be

biodegradable and to lead to degradation products having no or low toxicity. Exemplary polymers include poly gly colic acids and polylactic acids, poly (DL-lac tic acid-co-glycolic acid), poly(D-lactic acid-co-glycolic acid), and poly(L-lactic acid-co-glycolic acid). Other useful biodegradable or bioerodable polymers include, but are not limited to, such polymers as poly(epsilon-caprolactone), poly(epsilon-aprolactone-CO-lactic acid), poly(epsilon.- aprolactone-CO-glycolic acid), poly(beta-hydroxy butyric acid), poly(alkyl-2-cyanoacrilate), hydrogels, such as poly(hydroxyethyl methacrylate), polyamides, poly( amino acids) (for example, L-leucine, glutamic acid, L-aspartic acid and the like), poly(ester urea), poly(2- hydroxyethyl DL-aspartamide), polyacetal polymers, polyorthoesters, polycarbonate, polymaleamides, polysaccharides, and copolymers thereof. Many methods for preparing such formulations are well known to those skilled in the art (see, for example, Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978). Other useful formulations include controlled-release microcapsules (U.S. Patent Nos. 4,652,441 and 4,917,893), lactic acid-glycolic acid copolymers useful in making microcapsules and other formulations (U.S. Patent Nos. 4,677,191 and 4,728,721) and sustained-release compositions for water-soluble peptides (U.S. Patent No. 4,675,189).

The pharmaceutical compositions of the disclosure typically are sterile and stable under conditions of manufacture, storage and use. Sterile solutions can be prepared by incorporating the compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the compound and/or other biologically active agent into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders, methods of preparation include vacuum drying and freeze -drying which yields a powder of the compound plus any additional desired ingredient from a previously sterile -filtered solution thereof. The prevention of the action of microorganisms can be accomplished by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

In accordance with the various treatment methods of the disclosure, the inhibitor can be delivered to a subject in a manner consistent with conventional methodologies associated with management of the disorder for which treatment or prevention is sought. In accordance with the disclosure herein, a prophylactically or therapeutically effective amount of the inhibitor is administered to a subject in need of such treatment for a time and under conditions sufficient to prevent, inhibit, and/or ameliorate a selected disease or condition or one or more symptom(s) thereof.

The administration of the inhibitor of the disclosure can be for either prophylactic or therapeutic purpose. When provided prophylactically, the inhibitor is provided in advance of any symptom. The prophylactic administration of the inhibitor serves to prevent or ameliorate any subsequent disease process. When provided therapeutically, the inhibitor is provided at (or shortly after) the onset of a symptom of disease or infection.

For prophylactic and therapeutic purposes, the inhibitor can be administered to the subject by the oral route or in a single bolus delivery, via continuous delivery (for example, continuous transdermal, mucosal or intravenous delivery) over an extended time period, or in a repeated administration protocol (for example, by an hourly, daily or weekly, repeated administration protocol). The therapeutically effective dosage of the inhibitor can be provided as repeated doses within a prolonged prophylaxis or treatment regimen that will yield clinically significant results to alleviate one or more symptoms or detectable conditions associated with a targeted disease or condition as set forth herein. Determination of effective dosages in this context is typically based on animal model studies followed up by human clinical trials and is guided by administration protocols that significantly reduce the occurrence or severity of targeted disease symptoms or conditions in the subject. Suitable models in this regard include, for example, murine, rat, avian, porcine, feline, non-human primate, and other accepted animal model subjects known in the art. Alternatively, effective dosages can be determined using in vitro models. Using such models, only ordinary calculations and adjustments are required to determine an appropriate concentration and dose to administer a therapeutically effective amount of the inhibitor (for example, amounts that are effective to elicit a desired immune response or alleviate one or more symptoms of a targeted disease). In alternative embodiments, an effective amount or effective dose of the inhibitor may simply inhibit or enhance one or more selected biological activities correlated with a disease or condition, as set forth herein, for either therapeutic or diagnostic purposes.

The actual dosage of the inhibitor will vary according to factors such as the disease indication and particular status of the subject (for example, the subject's age, size, fitness, extent of symptoms, susceptibility factors, and the like), time and route of administration, other drugs or treatments being administered concurrently, as well as the specific

pharmacology of the compound for eliciting the desired activity or biological response in the subject. Dosage regimens can be adjusted to provide an optimum prophylactic or therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental side effects of the compound and/or other biologically active agent is outweighed in clinical terms by therapeutically beneficial effects. A non-limiting range for a therapeutically effective amount of a compound and/or other biologically active agent within the methods and formulations of the disclosure is about 0.01 mg/kg body weight to about 20 mg/kg body weight, such as about 0.05 mg/kg to about 5 mg/kg body weight, or about 0.2 mg/kg to about 2 mg/kg body weight.

Dosage can be varied by the attending clinician to maintain a desired concentration at a target site (for example, the lungs or systemic circulation). Higher or lower concentrations can be selected based on the mode of delivery, for example, trans-epidermal, rectal, oral, pulmonary, or intranasal delivery versus intravenous or subcutaneous delivery. Dosage can also be adjusted based on the release rate of the administered formulation, for example, of an intrapulmonary spray versus powder, sustained release oral versus injected particulate or transdermal delivery formulations, and so forth.

The inhibitors disclosed herein may also be co-administered with an additional therapeutic agent. Such agents include, but are not limited to, an anti-inflammatory agent, a matrix metalloprotease inhibitor, a lipoxygenase inhibitor, a cytokine antagonist, an immunosuppressant, an anti-cancer agent, an anti-viral agent, a cytokine, a growth factor, an immunomodulator, a prostaglandin or an anti-vascular hyperproliferation compound. The instant disclosure also includes kits, packages and multi-container units containing the herein described pharmaceutical compositions, active ingredients, and/or means for administering the same for use in the prevention and treatment of diseases and other conditions in mammalian subjects. Kits for diagnostic use are also provided. In one embodiment, these kits include a container or formulation that contains one or more of the inhibitors described herein. In one example, this component is formulated in a

pharmaceutical preparation for delivery to a subject. The inhibitor is optionally contained in a bulk dispensing container or unit or multi-unit dosage form. Optional dispensing means can be provided, for example a pulmonary or intranasal spray applicator. Packaging materials optionally include a label or instruction indicating for what treatment purposes and/or in what manner the pharmaceutical agent packaged therewith can be used.

Examples Compound screening was initially carried out using the Library of Pharmacologically Active Compounds (LOP AC), which includes 1280 known bioactive small molecules. Subsequent compounds were selected based on the active compounds identified in the LOP AC set, in particular β-lapachone. Compound dilution: LOP AC library compounds (2 μΐ at lOmM concentration) were diluted to 200 μΜ by adding 98 μΐ alpha minimum essential medium (a-MEM) to each well using Titertek Zoom dispenser. Compounds were further diluted to 60 μΜ by adding 10 μΐ compound (200 μΜ) and 23 μΐ α-ΜΕΜ to a compound plate (Greiner Bio-one

polypropylene 384-well plate).

Screening: Diluted LOP AC library compounds (60 μΜ, 0.6% DMSO) were added to 384- well assay plates (Greiner Bio-one μΟε3Γ-ρ ε, black) at 5 μΐ/well using V-prep Precision Pipetting System (Velocity 11). Minimum inhibition (a-MEM with 0.6% DMSO) and maximum inhibition (5 μΜ adriamycin, 0.6% DMSO) controls were also added to the first and last two columns of the assay plates using V-prep. MCF7-Oct3/4-GFP cells were then dispensed at 1000 cells/25 μΐ/well to the assay plates containing compounds using Titertek Zoom dispense. Final screening compound concentration was 5 μΜ, 0.1% DMSO. MCF7- GFP cells were screened with the same procedure side by side with MCF7-Oct3/4-GFP cells. Assay plates were incubated at 37 °C with 5% C0₂ for 72 hours. Cell viability assay: Cell viability was examined after 72 hours using CellTiter-Blue^K Cell Viability Assay according to manufacturer's instruction

(http://www.promega.com/tbs/tb317/tb317.pdf).

Data analysis and hit identification: LOP AC screen was performed in triplicate for each cell line. Growth inhibition of each compound was calculated as percent inhibition

( inhibition). A total of 36 compounds had an average inhibition of >30 for MCF7- Oct3/4-GFP cells. inhibition of these compounds for the two cell lines were compared (Figs. 1 and 2). The ratios of inhibition for MCF7-Oct3/4 cell and for MCF7-GFP cells of these 36 compounds were then calculated and ranked in descending order. Four compounds were identified as the top hits: A-77636 hydrochloride, rottlerin, β-lapachone, and CGP- 74514A hydrochloride. NSC725776, NSC743400, JUN1511, NSC95397, Bouvardin, Caulibugulone A, and JUN1111 were tested in an identical manner and found to be inhibitors.

Efficacy: Study 1: Approximately 120 female athymic nude(nu/nu) mice may be divided into two groups: 60 female mice and injected sc with 5 x 10⁶ MDA-MB-231 and 60 with MDA-MB -231 stem-cell-like cells. When the tumors reach -150 mm³, the mice bearing each type of tumor may be stratified into 6 groups with 8 tolO mice per group. Stratification may be performed so that the mean tumor size and body weight are similar for each group within a cell line. The mice may be treated with one of the following: Indmitecan (10 mg/kg iv qdx5), Indimitecan (6.7 mg/kg iv qdx5), Indimitecan (4.5 mg/kg iv qdx5), Vehicle (0.01 ml/g body weight, positive control (topotecan, 3 mg/kg iv qdx5) and control (no treatment). The mice may be observed daily for clinical symptoms, and body weights and tumor volumes will be recorded twice weekly. Tumor volumes are calculated using the formula: TV = l*w²/2, where 1 is the longest dimension of the tumor and w is the diameter perpendicular to 1. Both 1 and w may be recorded using a digital caliper and sent directly to the computer.

Study 2: Approximately 120 female athymic nude(nu/nu) mice may be divided into two groups: 60 female mice may be injected sc with 5 x 10⁶ MCF-7 and 60 with MCF-7 stem- cell-like cells. When the tumors reach -150 mm³ the mice bearing each type of tumor may be stratified into 6 groups with 8 to 10 mice per group. Stratification may be performed so that the mean tumor size and body weight are similar for each group within a cell line. The mice may be treated with one of the following: Indimitecan (10 mg/kg iv qdx5),

Indimitecan (6.7 mg/kg iv qdx5), Indimitecan (4.5 mg/kg iv qdx5), topotecan (3 mg/kg iv qdx5), Vehicle (0.01 ml/g body weight) and control (no treatment). The mice may be observed daily for clinical symptoms and body weights and tumor volumes can be recorded twice weekly. Tumor volumes are calculated using the formula: TV = l*w²/2 where 1 is the longest dimension of the tumor and w is the diameter perpendicular to 1. Both 1 and w may be recorded using a digital caliper and sent directly to the computer. Endpoints of efficacy in vivo are tumor response as growth inhibition: T/C (Percentage of the mean or median tumor volume of the treatment group to control group). T/C can be evaluated for each treatment group when the mean tumor volume in the control group reaches approximately 2000 mm³. The number of mice with complete tumor response (no tumor regrowth during the study period of approximately 70 days) in each group can be evaluated. If tumor regrowth occurs after treatment, tumor doubling time may be evaluated for the mice in each group. It is assumed that the effects of the positive control, topotecan will be much less in the stem-cell-like cells because of the elevated expression of BCRP in those cells compared to the parental MCF-7 or MDA-MB-231 cells. If significant differences in efficacy are noted between the mice bearing stem-cell-like xenografts and those bearing parental cell xenografts, a PK study can be conducted to determine the concentrations of the indenoisoquinolines in the plasma and tumors of the nude mice and correlate the tumor concentrations with γΗ2ΑΧ concentrations in the tumor as a pharmacodynamic response to the indenoisoquinoline treatment. The percentage of stem-cell-like cells in the various tumors may also be characterized.

PK/PD study: Study 3: Approximately 100 female athymic nude mice bearing MDA-MB- 231 or MDA-MB-231 stem-cell-like xenografts can be stratified to groups with 3 mice per group so that their mean tumor size and body weight are similar. Mice may be dosed with indimitecan at 10 mg/kg iv(or the most active dose from the efficacy study) or vehicle. Three mice can be euthanized by C0₂ inhalation at the following time points after dosing: 5, 10, 30, 45, 60, 90, 120, 240, 360, 480, 960, 1440, and 2880 min and 5 min after vehicle administration. Mice from the 1440 and 2880 min-time -point groups may be housed in metabolism cages for the separate collection of urine and feces. Blood can be collected by cardiac puncture using heparinized syringes and centrifuged for 4 min at 13000xg to obtain plasma. Tumors can be dissected, stored on ice, cut into pieces for PK, stem cell markers and PD analysis, weighed, and PK and stem cell marker samples can be snap frozen in liquid nitrogen, while pieces for γΗ2ΑΧ can be fixed in 10% phosphate buffered formalin and the γΗ2ΑΧ can be measured by immunohistochemistry[2]. The choice of γΗ2ΑΧ as the PD marker is based on observations that the indenoisoquinolines form stable topol cleavage complexes that lead to DNA double strand breaks and result in phosphorylation of histone H2AX that is maintained for up to 24 h after a single dose. The amount of γΗ2ΑΧ in tumors can be related to the indenoisoquinoline concentration in the tumors. Plasma and tumor pieces can be stored at -80 °C until analysis by LC/MS or analyzed for the expression of stem cell markers by Western blot. The assay used for the determination of the concentrations of the indenoisoquinolines in the current Phase I study entitled "A Phase I study of indenoisoquinolines LMP400 and LMP776 in adults with relapsed solid tumors and lymphomas, CTEP #8273 was developed at the University of Pittsburgh and can be modified, if required, to measure murine plasma and tumor concentrations of the indenoisoquinolines. The plasma and urine from the mice can be examined for the presence of metabolites. The proposed starting doses for Indotcan can be 18, 12, and 8 mg/kg based on previous studies. In view of the many possible embodiments to which the principles of the disclosed methods and compositions may be applied, it should be recognized that the illustrated embodiments are only preferred examples and should not be taken as limiting the scope of the invention.

Claims

What is claimed is:

1. A method of treating a chemotherapeutic-resistant cancer in a subject, comprising administering to the subject a therapeutically effective amount of at least one compound that selectively targets cancer stem cells, wherein the compound is an indenoisoquinoline compound, a naphthoquinone compound, a quinolinedione compound, bouvardin, or a pharmaceutically acceptable salt or ester thereof.

2. The method of claim 1 , wherein the chemotherapeutic-resistant cancer is breast cancer.

3. The method of claim 2, wherein the breast cancer is recurrent breast cancer.

4. The method of any one of claims 1-3, further comprising administering a therapeutically effective amount of a second anti-cancer agent to the subject.

5. A method of treating recurrent breast cancer in a subject, comprising coadministering to the subject (a) a therapeutically effective amount of at least one compound that selectively targets breast cancer stem cells, wherein the compound is an

indenoisoquinoline compound, a naphthoquinone compound, a quinolinedione compound, bouvardin, or a pharmaceutically acceptable salt or ester thereof, and (b) a therapeutically effective amount of at least one anti-breast cancer agent.

6. A method of treating cancer in a subject, comprising co-administering to the subject (a) a therapeutically effective amount of at least one compound that selectively targets cancer stem cells, wherein the compound is an indenoisoquinoline compound, a naphthoquinone compound, a quinolinedione compound, bouvardin, or a pharmaceutically acceptable salt or ester thereof, and (b) a therapeutically effective amount of at least one anti-cancer agent.

7. A method of selectively targeting cancer stem cells, comprising administering to a population of cancer cells comprising cancer stem cells as well as cancer cells that are not stem cells a therapeutically effective amount of at least one compound, wherein the compound is an indenoisoquinoline compound, a naphthoquinone compound, a quinolinedione compound, bouvardin, or a pharmaceutically acceptable salt or ester thereof.

8. The method of any one of claims 1-7, wherein the compound is an indenoisoquinoline compound, a naphthoquinone compound, a quinolinedione compound, or bouvardin.

9. The method of any one of claims 1-8, wherein the compound is an indenoisoquinoline compound.

10. The method of any one of claims 1-8, wherein the compound is a naphthoquinone compound.

11. The method of any one of claims 1-8, wherein the compound is a quinolinedione compound.

12. The method of any one of claims 1-9, wherein the indenoisoquinoline compound has a structure selected from:

(Formula I) wherein the group designated Ri is selected from hydrogen, formyl, phenyl, phenyl substituted with C₁-C6 alkoxy or C₁-C6 alkyl, or Ri is a group— (CH₂)_mZ, wherein m is 1-6 and Z is selected from the group consisting of hydrogen, hydroxyl, carboxy, formyl, C₁-C6 alkyl, carbo-(Ci-C6 alkoxy), C₂-C6 alkenyl, phenyl, C₁-C6 alkylamino, heterocycloalkyl, heteroaryl, and C₁-C6 hydroxy-alkylamino; R₂ and R₂' are independently selected from the group consisting of hydrogen, Ci-C₆ alkyl, C₂-C₆ alkenyl, Ci-C₆ alkoxy, phenoxy and benzyloxy, or R₂ and R₂' taken together form a group

of the formula -OCH₂0-;

R₄ is selected from the group consisting of hydrogen, C₁-C6 alkyl, C₂-C6 alkenyl, C₁-C6 alkoxy, phenoxy, and benzyloxy;

R₃ and R₃' are independently selected from the group consisting of hydrogen, C₁-C6 alkyl,

alkoxy, C₂-C6 alkenyl, phenoxy, and benzyloxy, or R₃ and R₃' taken together form a group of

the formula -OCH₂0-; wherein n=l or 0, and bond a is a single bond when n=l, and bond a is a double bond when n=0; provided that when R₂, R₂', R₄, R₃ and R₃' are hydrogen, Z is not C1-C6

hydroxy alkylamino; and further provided that when Ri is methyl, R₃ and R₃' are

independently

selected from the group consisting of hydrogen, Ci-C₆ alkyl, Ci-C₆ alkoxy, C₂-C₆ alkenyl,

(Formula II) wherein R is phenyl or phenyl substituted with Ci-C₆ alkoxy or Ci-C₆ alkyl, or R is a group — (CH₂)_mZ wherein m is 1-6 and Z is selected from the group consisting of hydrogen, hydroxyl, carboxy, formyl, Ci-C₆ alkyl, carbo-(Ci-C₆ alkoxy), C₂-C₆ alkenyl, phenyl, Ci-C₆ alkylamino, heterocycloalkyl, heteroaryl, and Ci-C₆ hydroxyalkylamino, provided that when Z is hydrogen, m is 2-6;

R₂ and R₂' are independently selected from the group consisting of hydrogen, C₁-C6 alkyl, C₂-C6 alkenyl, C₁-C6 alkoxy, phenoxy and benzyloxy, or R₂ and R₂' taken together form a group of the formula— OCH₂0— ;

R₄ is selected from the group consisting of hydrogen, C₁-C6 alkyl, C₂-C6 alkenyl, C₁-C6 alkoxy, phenoxy and benzyloxy;

R₃ and R₃' are independently selected from the group consisting of hydrogen, C₁-C6 alkyl, C₁-C6 alkoxy, C₂-C6 alkenyl, phenoxy, and benzyloxy, or R₃ and R₃' taken together form a group of the formula— OCH₂0— ; and wherein X is a pharmaceutically acceptable anion; or a pharmaceutically acceptable salt or ester thereof.

13. The method of claim 12, wherein the compound has a structure according to Formula I.

14. The method of claim 13, wherein the group designated Ri is selected from hydrogen, formyl, phenyl, phenyl substituted with C₁-C6 alkoxy or C₁-C6 alkyl, or Ri is a group— (CH₂)_mZ, wherein m is 1-6 and Z is selected from the group consisting of hydrogen, hydroxyl, carboxy, formyl, C₁-C6 alkyl, carbo-(Ci-C6 alkoxy), C₂-C6 alkenyl, phenyl, C₁-C6 alkylamino, and C₁-C6 hydroxy-alkylamino.

15. The method of claim 12, wherein the compound has a structure according to Formula II.

16. The method of claim 15, wherein Ri is phenyl or phenyl substituted with Ci-C₆ alkoxy or Ci-C₆ alkyl, or Ri is a group— (CH₂)_mZ wherein m is 1-6 and Z is selected from the group consisting of hydrogen, hydroxyl, carboxy, formyl, Ci-C₆ alkyl, carbo-(Ci- C₆ alkoxy), C₂-C₆ alkenyl, phenyl, Ci-C₆ alkylaniino, and Ci-C₆ hydroxyalkylamino, provided that when Z is hydrogen, m is 2-6.

17. The method of claim 12, wherein the indenoisoquinoline compound is 6-[3- [(1 H-imidazolyl)propyl] -5 ,6-dihydro-2,3 -dimethoxy-8 ,9-methylenedioxy-5 , 11 -dioxo- 11 H- indeno[l,2-c]isoquinoline, or 5H-[l,3]dioxolo[5,6]indeno[l,2-c]isoquinoline-5,12(6H)- dione, 2,3-dimethoxy-6-[3-(4-morpholinyl)-propyl], or a pharmaceutically acceptable salt or ester thereof.

18. The method of any one of claims 1-8 or 10, wherein the naphthoquinone compound is selected from β-lapachone, a β-lapachone analog, or

wherein R¹ and R² are each independently selected from H, alkyl, thiol, thioalkyl thioalkoxy, or a pharmaceutically acceptable salt or ester thereof.

19. The method of any one of claims 1-8 or 10, wherein the naphthoquinone compound is selected from β-lapachone, or

or a pharmaceutically acceptable salt or ester thereof.

20. The method of any one of claims 1-8 or 10, wherein the naphthoquinone compound is selected from β-lapachone, or a pharmaceutically acceptable salt or ester thereof.

21. The method of any one of claims 1-8 or 11, wherein the quinolinedione compound is selected from:

wherein R is H or -CH₂CH₂OH; or

wherein R¹ and R² are each independently selected from H, CI, Br, -alkyl-heterocycloalkyl, or alkyl, or R¹ and R² together form a heterocyclic ring; wherein R³ and R⁴ are each independently selected from H or alkyl; and R⁵ is H, alkyl, cyano, a carbonyl-containing group, formyl, carboxyl, substituted carboxyl, amino, or aminocarbonyl; or

a pharmaceutically acceptable salt or ester thereof.

22. The method of claim 21, wherein R¹ and R² are each independently selected from H, or alkyl, or R¹ and R² together form a heterocyclic ring.

23. The method of claim 21 or 22, wherein R⁵ is formyl, carboxyl, substituted carboxyl, amino or aminocarbonyl.

24. The method of claim 21, wherein the quinolinedione compound is selected from caulibugulone A;

25. The method of any one of claims 1-8, wherein the compound that selectively targets cancer stem cells is bouvardin.

26. The method of any one of claims 1-8 or 11, wherein the quinolinedione compound is:

, or a pharmaceutically acceptable salt or ester thereof.

27. The method of any one of claims 1-26, wherein the compound that selectively targets cancer stem cells inhibits proliferation of the stem cells, increases differentiation of the stem cells, induces apoptosis of the stem cells, or a combination thereof.

28. The method of any one of claims 1-27, wherein the cancer expresses cancer stem cell marker.

29. The method of claim 5, wherein the anti-breast cancer agent is a taxane compound.

30. A pharmaceutical composition useful for selectively targeting cancer stem cells, comprising (a) at least one pharmaceutically acceptable additive and (b) a therapeutically effective amount of at least one compound that selectively targets cancer stem cells, wherein the compound is selected from an indenoisoquinoline compound, a naphthoquinone compound, a quinolinedione compound, bouvardin, or a pharmaceutically acceptable salt or ester thereof.

31. The composition of claim 30, wherein the compound is an

indenoisoquinoline compound, a naphthoquinone compound, a quinolinedione compound, or bouvardin, or a pharmaceutically acceptable salt or ester thereof.

32. A compound having a structure of: