CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 61/182,511, filed on May 29, 2009, which is herein incorporated by reference in its entirety.

FIELD

Disclosed herein are compounds useful for the treatment of drug resistant cells.

BACKGROUND

Multidrug resistance (MDR) conferred by the ABC transporter family that includes MDRl (ABCBl, P-glycoprotein, P-gp), presents a significant clinical challenge for drug design and development. MDRl, for example, exhibits wide substrate specificity for structurally different drugs. This wide specificity mediates drug resistance to a variety of drugs, including Vinca alkaloids, anthracyclines, epipodophyllotoxins, taxols, actinomycin D, cardiac glycosides, immunosuppressive agents, glucocorticoids, and anti-HIV protease inhibitors. Since many drugs are substrates of MDRl, its degree of expression and functionality directly affects the therapeutic effectiveness of these agents. In particular, the multidrug resistant phenotype of malignant cells is the main obstacle in the chemotherapeutic treatment of subjects having hyperproliferative disorders. MDRl expression is well characterized in hematological malignancies, sarcomas, and other solid cancers, and is frequently correlated with poor clinical response to chemotherapy for those tumors. Strategies employed to circumvent the reduced drug accumulation conferred by these poly- specific efflux transporters have relied heavily on the development of clinical inhibitors of MDRl for concurrent administration with chemotherapeutics. Although a number of these inhibitors have shown promise in vitro, translation to the clinic has taken longer than may have been expected, possibly due to side effects caused by inhibition of endogenous function, and alternative strategies are required.

SUMMARY According to one embodiment, disclosed herein is a method for inhibiting the growth of drug resistant cells in a subject, comprising identifying a subject having drug resistant cells; and administering to the subject a compound or a pharmaceutically acceptable salt or ester thereof, as described below in more detail. According to another embodiment, disclosed herein is a method of inhibiting cancer in a subject, comprising administering to the subject an antiproliferative agent, wherein the antiproliferative effect of the agent is potentiated by P- glycoprotein and the agent is a compound, or a pharmaceutically acceptable salt or ester thereof, as described in more detail below. In one aspect the compounds are particularly effective against cells that exhibit multidrug resistance. Accordingly treatment regimens employing the disclosed compounds typically involve first identifying a subject having a multidrug resistant disorder, such as a multidrug resistant tumor or infection. Certain examples of the compounds described above are not particularly cytotoxic, particularly to cells that are not multidrug resistant. Moreover, in one embodiment the disclosed compounds effectively re-sensitize multidrug resistant cells to anti-proliferative agents that are substrates for an MDR transporter. Thus, in one aspect, the disclosed compounds are co-administered with another chemotherapeutic agent, such as an antibiotic or antineoplastic agent. In another embodiment the disclosed compounds are both cytotoxic and render multidrug resistant cells susceptible to one or more additional chemotherapeutic agents by inhibiting an MDR transporter. Accordingly, also disclosed herein are treatment regimens and compositions formulated for combination therapy. The foregoing and other objects, features, and advantages will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Identification of candidate MDR-inverse compounds. DTP's public dataset, consisting of publicly available cytotoxicity (GI ₅₀ ) profiles of candidate anticancer agents was screened using the mRNA expression pattern of Pgp (MDRl) (Szakacs et al., 2004). Merging of the two datasets (top panel) resulted in drug-gene pairs, giving a Pearson correlation coefficient (r) for each compound, and a distribution histogram for -36,000 curated compounds (bottom panel). Negative correlation coefficients indicate that a given cytotoxic compound is less effective in the presence of Pgp (putative Pgp-substrates). Conversely, a small number of compounds that elicit positive Pearson values (r>=0.3) suggest that their toxicity is potentiated (rather than antagonized) by the presence of Pgp (putative MDR-inverse agents).

Figure 2. Dendrogram showing the average-linkage hierarchical clustering of 64 putative MDR-inverse compounds (r>=0.4). The distance matrix is derived from Tanimoto similarity indices (http://pubchem.ncbi.nlm.nih.gov). Numbers represent NSC codes. Black: compound not available for testing; green: confirmed MDR- inverse activity; red: lack of MDR-inverse activity; asterix (*): metal complex. Clusters of structurally related compounds where one or more analogs are active are highlighted by their core common.

Figure 3. Dendrogram showing the average-linkage hierarchical clustering of the 37 confirmed MDRl-invese compounds. The distance matrix is derived from Tanimoto similarity indices. Numbers represent NSC codes. The two major clusters are colored green ("TSC-cluster") and yellow ("10580-cluster"). Pharmacophore models derived from the two sets are shown. Figure 4. QSAR analysis of the thiosemicarbazone MDR-inverse compounds identified in the NCI DTP drug library. Scatter plot and comparison of the calculated and measured MDR-inverse ratios.

Figure 5. Self-organizing map-based (SOM) cluster anlaysis of the activity of the 22 DTP compounds verified to demonstrate MDR-inverse activity. SOM clustering of the DTP drug response data defines six major response categories: mitosis (M), membrane function (N), nucleic acid metabolism (S), metabolic stress and cell survival (Q), and two unexplored regions P and R. Each of these regions is further divided into a total of 51 sub-regions. Larger hexagons (yellow) represent a greater number of compounds. Clusters represent the following compounds (1) NSC695331, NSC695333, (2) NSC697125, (3) NSC672036, (4) NSC673999, NSC713048, (5) NSC716765, (6) NSC641208, (7) NSC716766, NSC716768, (8) NSC43320, NSC73306, NSC693871, NSC693872, NSC697124, (9) NSC2924508, (10) NSC168468, (11) NSC1580, (12) NSC356777, (13) NSC649816, 716772, (14) NSC710857. Red hexagon is the SOM cluster overlay of NSC4265, 1,10- phenanthroline.

Figure 6. Chelation alone is not sufficient to confer MDR-inverse activity A. A number of coordination modes are possible for the thiosemicarbazone NSC73306 (left), the most likely being bidentate (middle) or tridentate (right). B. Solution color changes of NSC73306 indicating complexation with metal sulfate salts, L to R: NSC73306, NSC73306 + Fe ²⁺ , NSC73306 + Cu ²⁺ and NSC73306 + Zn ²⁺ . C. ESI- MS (ES+) of NSC73306 (L, mw = 326.1 gmol ^"1 ), and mixtures with metal ions (+11 oxidation state) in 1:1 MeOH:H ₂ O. The highest mass peak observed is shown, in each case corresponding to a 2: 1 ligand:metal complex D. Compounds such as NSC632738 and NSC292408 that possess 1,10-phenanthroline ligands can dissociate to yield free 1,10-phenanthroline, capable of exerting its own MDR- inverse activity. KP772, a tris(l,10-phenantholine)lanthanum(III) complex reported to display MDR-inverse activity, that may also release phen. In contrast, the highly [Ru(phen) ₃ ] complex that does not release phen does not display MDR-inverse activity.

Figure 7. Table 1 lists examples of compounds identified as having MDRl- selective activity.

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. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Also, as used herein, the term "comprises" means "includes." Hence "comprising A or B" means including A, B, or A and B.

Variables such as R, R ¹ , R ² ,R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , X, Y and Z, used throughout the disclosure are the same variables as previously defined unless stated to the contrary.

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

"ABC transporters" are transporter proteins belonging to the ABC protein superfamily and are capable of, in their native, active, wild type form, extruding drugs from the cells expressing them. Herein, the term "ABC transporter" also covers mutant variants of the wild type proteins retaining at least one function of the wild type, even if lacking another. The term "acyl" refers group of the formula RC(O)- wherein R is an organic group.

The term "alkoxy" refers to a group of the formula -OR, wherein R is an organic group. 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, w-butyl, isobutyl, £-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. 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.

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

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 herein.

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

"Carboxyl" refers to a -COOH radical. Substituted carboxyl refers to -COOR where R is aliphatic, heteroaliphatic, alkyl, heteroalkyl, or a carboxylic acid or ester.

The term "aryl" refers to any carbon-based aromatic group including, but not limited to, benzene, naphthalene, etc. The term "aromatic" 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.

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 "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.

The term "hydroxyl" is represented by the formula -OH. The term "alkoxy group" is represented by the formula -OR, where R can be an alkyl group, optionally substituted with an alkenyl, alkynyl, aryl, aralkyl, cycloalkyl, halogenated alkyl, or heterocycloalkyl group as described above.

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, Cl, Br, I).

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. "Carbonyl" refers to a radical of the formula -C(O)-. Carbonyl-containing groups include any substituent containing a carbon-oxygen double bond (C=O), 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.

Multidrug resistance (MDR) refers to the ability of target cells and microorganisms, particularly cancer cells and mycobacterial cells, to resist the effects of different -often structurally and functionally unrelated- cytotoxic compounds. MDR can develop after sequential or simultaneous exposure to various drugs. MDR also can develop before exposure to many compounds to which a cell or microorganism may be found to be resistant. Although MDR may be caused by a variety of factors, most commonly MDR is associated with overexpression of P- glycoprotein (P-gp). P-gp is a member of a superfamily of membrane proteins, termed adenosine triphosphate (ATP)-binding cassette (ABC) proteins, which behave as ATP-dependent transporters and/or ion channels for a wide variety of substrates. P-gp is a multiple transmembrane- spanning glycoprotein. Transfection experiments with the P-gp gene (MDRl, or

ABCBl) have demonstrated that P-gp confers MDR upon drug- sensitive tumor cells by providing an energy-dependent efflux pump that lowers the intracellular concentration of the cytotoxic agent, thereby allowing survival of the cell.

The term "neoplasm" refers to an abnormal cellular proliferation, which includes benign and malignant tumors, as well as other proliferative disorders.

The term "pharmaceutically acceptable salt or ester" refers to salts or esters prepared by conventional means that include basic salts of inorganic and organic acids, including but not limited to hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethane sulfonic 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, N,N'- 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., J. Pharm. ScL 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 for carboxy include C ₁ - _O alkoxymethyl esters for example methoxy- methyl, C ₁ - _O alkanoyloxymethyl esters for example pivaloyloxymethyl, phthalidyl esters, C ₃ -S cycloalkoxycarbonyloxyCi-6 alkyl esters for example 1- cyclohexylcarbonyl-oxyethyl; l,3-dioxolen-2-onylmethyl esters for example 5- methyl-l,3-dioxolen-2-onylmethyl; and C ₁ - _O alkoxycarbonyloxyethyl esters for example 1-methoxycarbonyl-oxyethyl 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 α-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 non-toxic 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.

The term "subject" includes both human and veterinary subjects. "Transport protein" refers to a protein that acts to remove chemotherapeutic substances from cells. Examples of transport proteins include, without limitation, P- glycoprotein, the protein product of the MDRl gene. Expression of such transport proteins confers resistance to numerous chemotherapeutic agents and sometimes entire classes of chemotherapeutic s, including Vinca alkaloids, anthracyclines, epipodophyllotoxins, actinomycin D and taxanes. P-glycoprotein is over-expressed in certain chemotherapy resistant tumors and is upregulated during disease progression following chemotherapy in other malignancies. MRPs, also belonging to the ABC family, confer a multidrug resistance phenotype that includes many natural product drugs, but is distinct from the resistance phenotype associated with P-gP- In addition to P-gp and the MRPs there may be other transporters that are involved in cytotoxic drug resistance. In the case of natural product drugs, resistant cell lines have been described that display a multidrug resistant phenotype associated with a drug accumulation deficit, but do not overexpress P-gp or any of the MRPs.

"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 "ameliorating," with reference to a disease or pathological condition, 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, 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. The phrase "treating a disease" refers to inhibiting the full development of a disease or condition, for example, in a subject who is at risk for a disease such as cancer, particularly a metastatic cancer. 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. "Treating multidrug resistance" means increasing or restoring sensitivity of multidrug resistant cells to therapeutic agents. Treating multidrug resistance also may include inhibiting the development of multidrug resistance in nonresistant cells.

The term "prodrug" also is intended to include any covalently bonded carriers that release a disclosed compound or a parent thereof 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 claimed 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. In particular, ester prodrugs are specifically contemplated herein. Similarly, prodrugs include compounds having an amino or sulfhydryl group functionalized with any group that is cleaved to yield the corresponding free amino or free sulfhydryl group. Examples of prodrugs include, without limitation, compounds having a hydroxy, amino and/or sulfhydryl group acylated with an acetate, formate, or benzoate group.

Protected derivatives of the disclosed compounds also are contemplated. The term "protecting group" or "blocking group" refers to any group that when bound to a functional group prevents or diminishes the group's susceptibility to reaction. "Protecting group" generally refers to groups well known in the art which are used to prevent selected reactive groups, such as carboxy, amino, hydroxy, mercapto and the like, from undergoing undesired reactions, such as nucleophilic, electrophilic, oxidation, reduction and the like. The terms "deprotecting," "deprotected," or "deprotect," as used herein, are meant to refer to the process of removing a protecting group from a compound. It is understood that substituents and substitution patterns of the compounds described herein can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art and further by the methods set forth in this disclosure. Reference will now be made in detail to the present preferred embodiments.

/. Compounds having MDR-inverse activity

The compounds disclosed herein exploit, rather than suppress, Pgp function to induce cytotoxicity. Such "MDR-inverse compounds" target multidrug resistant cycling cells, avoiding side effects associated with the damage of resting cells that constitutively express Pgp. It has been determined that a positive correlation between a compound's cytotoxicity profile and the expression of Pgp in the NCI-60 cell panel may be the result of a causal interaction, where the activity of Pgp sensitizes the cell to the cytotoxicity of the compound. In one embodiment, possible MDR-inverse compounds are those whose cytotoxicity profile shows positive correlation (Pearson correlation coefficient r >=0.3) to the mRNA expression of MDRl in the NCI60 panel. Disclosed herein are drug compounds that have MDR-inverse activity and thus are effective against multidrug-resistant cells. Examples of the disclosed compounds have been found to have, inter alia, efficacy in directly treating multidrug resistant cells, rendering multidrug resistant cells susceptible to other chemotherapeutics and in some instances reversing multidrug resistance.

One group of MDRl -inverse compounds, or a pharmaceutically acceptable salt or ester thereof, are represented by the formula:

Formula I

wherein R ¹ and R ² are each independently H, alkyl (particularly lower alkyl), cycloalkyl, aryl, or substituted alkyl, or R ¹ and R ² together with the N form a heterocyclic ring such as a pyrrolidinyl, pyrrolinyl, pyrazolidinyl, pyrazolinyl, piperidinyl, piperzinyl, indolinyl, morpholinyl, pyrrolyl, pyrazolyl, or pyrazinyl;

R ³ is alkyl (particularly lower alkyl), substituted alkyl, alkyl amino, hydroxyl, amino, aryl, cycloalkyl, heterocycloalkyl, or heteroaryl; and a is 0 to 5, with the proviso that the following compounds are not included

Cl-

Cl-

NSC693871, NSC693872, or

NSC10580.

Particular examples of compounds of formula I are listed below:

NSC10776 NSC21518

NSC37881 NSC39225

NSC48151 NSC48477 NSC85459 NSC329100

NSC653043

NSC665711

NSC696893

Lanthanoid metal(s) complexed to bidentate 1,10-phenanthroline or 2,2'- bipyrdiyl ligands are further examples of MDRl -inverse agents.

Additional MDRl -inverse agents are shown below:

NSC43320 NSC672027

NSC632736 NSC632955

NSC632733 NSC633657

NSC710857 NSC651859

NSC632738 NSC693046 NSC705305 NSC705301

NSC672036 NSC639743

NSC635977 NSC636097

NSC624967 NSC669341

NSC653864 NSC654724

NSC697920 NSC403148

NSC632591 NSC162800

NSC165697

Cl

Cl

NSC619179

Cl

NSC619181

Cl

NSC619183

NSC619188 NSC630123

NSC632734

NSC632737

NSC632738 Among the metal complex compounds listed above, the chelating ligands alone may possess response profiles similar to their metal chelates indicating that the active metal complexes may serve as carriers for chelators, which themselves are the active drug molecules. While chelation is probably key to the cytotoxicity of at least a subset of the MDR-inverse compounds disclosed herein, data presented in Figure 6 indicate that metal chelation alone is not sufficient for Pgp potentiated activity. Since metal complexes were as active as the ligands alone (Figure 6), chelates may serve as chaperones facilitating free diffusion of the ligands into the cells. It appears that evasion of Pgp mediated efflux as well as the ability to chelate metals are necessary, if not sufficient requirements for the MDR-inverse activity of the chelator compounds. Dissociation of the complexes within the cells leading to the release of the free ligand and MDR-inverse activity may be related to reactive oxygen species generated by redox cycling. Taken together, it appears that the target of the MDR- inverse compounds may not be Pgp per se. Multidrug resistant cells become hypersensitive to MDR-inverse agents in proportion to the increased Pgp function, which probably reveals a downstream target in the cells. The paradoxical vulnerability uncovered by the MDR-inverse compounds may be linked to the efflux of an endogenous substrate providing e.g. redox-resistance, to the influence of Pgp on membrane lipid composition, or to other, Pgp-related metabolic changes that characterize MDR cells.

Additional potential MDR-inverse compounds are listed below in Table A.

Table A

Compound Pearson's Compound Pearson's Compound Pearson's

(NSC) Correlation (NSC) Correlation (NSC) Correlation

688241 0.99997 654275 0.772805 665338 0.603408

165528 0.999511 660031 0.76579 654948 0.599645

176656 0.998687 688249 0.760533 658236 0.599381

655253 0.990908 601904 0.754405 627173 0.592019

668557 0.985748 659221 0.745624 673999 0.591147

661908 0.983992 661900 0.74101 650975 0.590699

662142 0.981768 656071 0.731994 682721 0.590126

689202 0.979283 662382 0.730935 695333 0.584867

664243 0.975636 664001 0.722093 672072 0.579556

663624 0.966291 689209 0.716768 320656 0.573866

662137 0.965593 683137 0.713949 697538 0.571486

698233 0.964855 694667 0.703766 652566 0.565951

640235 0.955947 655503 0.701704 631309 0.565519

660854 0.947623 715277 0.692264 631669 0.564553

675262 0.939671 695330 0.689883 43320 0.560712

661242 0.936973 661111 0.683793 652280 0.558234

684431 0.935528 693871 0.681029 720611 0.554275

663007 0.906376 648232 0.678319 650044 0.553916

663547 0.903811 669171 0.662893 681781 0.552688

699386 0.902281 668902 0.662752 691225 0.551109

101335 0.888607 663333 0.656593 672027 0.549446

648409 0.873171 688253 0.653379 640164 0.548647

718600 0.872361 254668 0.646183 646123 0.548424

159953 0.865468 720612 0.636104 617147 0.545763

85242 0.821712 617041 0.608211 631062 0.544992

168670 0.811966 160040 0.605381 39688 0.543808 Compound Pearson s Compound Pearson's Compound Pearson's

(NSC) Correlation (NSC) Correlation (NSC) Correlation

78118 0.543465 657422 0.518181 662135 0.477129

662448 0.542791 716772 0.516341 660339 0.476207

658891 0.542541 685680 0.512669 679534 0.475093

28427 0.540374 73306 0.511215 709976 0.474818

635310 0.539624 696459 0.510565 679278 0.472991

665885 0.536232 631310 0.510021 643810 0.472159

697125 0.535806 681055 0.508745 632736 0.471322

656993 0.534734 662794 0.508509 649816 0.469699

661110 0.534672 645257 0.508297 632955 0.469645

637446 0.532755 676722 0.507127 629222 0.469186

86715 0.532288 665724 0.506564 631817 0.46858

665294 0.532283 676911 0.505435 672959 0.467438

617963 0.531712 716765 0.504093 673294 0.466984

649424 0.530937 646285 0.503142 697129 0.466266

688238 0.53018 675810 0.501244 636330 0.464807

698794 0.529344 665730 0.501205 691808 0.464435

632731 0.527683 651042 0.498062 698231 0.463848

698194 0.527114 702986 0.495557 697124 0.463633

665867 0.526788 657814 0.495541 103005 0.463283

665865 0.526562 702985 0.49402 624478 0.462112

688218 0.525085 645331 0.493574 632733 0.461092

696572 0.523701 635540 0.492388 697574 0.459087

716768 0.52295 661822 0.490417 623069 0.459056

670009 0.519692 662824 0.487363 653814 0.458277

649973 0.51946 698964 0.486692 683505 0.458206

644576 0.518771 13875 0.485815 665859 0.457985

669316 0.518753 168468 0.483849 674626 0.457358

695327 0.518416 716766 0.480799 659488 0.457238 Compound Pearson s Compound Pearson's Compound Pearson's

(NSC) Correlation (NSC) Correlation (NSC) Correlation

685288 0.457082 697128 0.434971 617278 0.424132

674627 0.45645 662446 0.434656 667277 0.423922

693336 0.453791 671123 0.434223 617961 0.422953

654278 0.453631 648848 0.434173 672036 0.422256

626626 0.450856 662452 0.433942 692419 0.421785

292408 0.450681 694525 0.43186 637410 0.421366

662449 0.450597 697678 0.431396 639743 0.420494

662125 0.449886 672099 0.430846 629783 0.418101

620678 0.448955 617259 0.43072 647774 0.41783

642581 0.44883 651700 0.430024 626670 0.417813

681602 0.448816 651859 0.4299 629779 0.417666

633657 0.448069 697137 0.429661 634739 0.417402

693872 0.447442 641208 0.429287 617161 0.417353

621481 0.447005 113112 0.428717 640643 0.417184

627450 0.445442 652255 0.428248 641613 0.417105

697120 0.44337 632738 0.428139 635977 0.416942

644868 0.443098 662132 0.427953 620677 0.416938

641814 0.442007 646208 0.427549 667276 0.41575

681125 0.44042 652771 0.426804 636097 0.415475

632849 0.439524 693046 0.426325 647656 0.415469

710857 0.438493 650296 0.426213 636111 0.415295

695331 0.438282 705301 0.425571 662579 0.414077

694563 0.43797 699123 0.425224 677374 0.412507

603443 0.437258 651816 0.42515 624967 0.41184

682716 0.437125 697135 0.425061 713048 0.411138

646796 0.436532 647574 0.424391 347512 0.41094

663653 0.43633 658339 0.424239 669341 0.4109

646367 0.435702 625216 0.424227 10580 0.410583 Compound Pearson s Compound Pearson's Compound Pearson's

(NSC) Correlation (NSC) Correlation (NSC) Correlation

629580 0.408559 716771 0.399927 622427 0.388046

654724 0.408326 658237 0.3999 641199 0.388024

653864 0.408324 141586 0.397652 681114 0.38728

165839 0.406611 674210 0.396701 407286 0.386841

672071 0.406525 641074 0.396501 639912 0.385457

647050 0.406127 669446 0.396437 641617 0.385294

115549 0.405965 695066 0.396073 657181 0.38489

716193 0.405937 681112 0.395378 677238 0.38473

697920 0.405823 688248 0.394631 634740 0.384714

640887 0.405311 625349 0.39365 605762 0.384402

650031 0.404751 665892 0.393588 654636 0.383716

632953 0.404268 647124 0.393453 635533 0.383649

619877 0.404166 688940 0.393414 648638 0.383467

252841 0.403819 675881 0.393074 615425 0.381879

293556 0.403601 637672 0.392924 641604 0.381135

639900 0.403555 686335 0.392631 658857 0.380652

639906 0.403541 697130 0.392559 691100 0.380238

356777 0.402181 645542 0.392509 651653 0.380137

632591 0.402145 645669 0.392097 653454 0.379919

675863 0.401632 697881 0.391757 677207 0.379109

693630 0.401422 665691 0.391031 647110 0.378943

688942 0.401233 716097 0.3904 719214 0.378271

627542 0.40103 650977 0.390057 636672 0.37808

672068 0.400682 663618 0.389885 692027 0.378059

622927 0.400316 685826 0.389825 654726 0.37794

666998 0.400246 627451 0.389488 666659 0.377862

666715 0.400018 693326 0.389182 687141 0.377548

682718 0.40001 639619 0.389117 653840 0.376466 Compound Pearson s Compound Pearson's Compound Pearson's

(NSC) Correlation (NSC) Correlation (NSC) Correlation

689626 0.376249 651667 0.36448 648072 0.357859

673117 0.37621 641288 0.364271 663627 0.357844

642037 0.375937 371168 0.364254 648264 0.357631

319951 0.375035 687585 0.364158 675974 0.357518

715186 0.373227 356903 0.364125 683238 0.357009

706806 0.372928 675877 0.363946 688114 0.356966

698129 0.372507 686397 0.363717 668878 0.356888

692760 0.372289 358873 0.363581 657004 0.356865

657576 0.371906 645796 0.363311 711994 0.356755

638744 0.371646 634373 0.36301 20514 0.356204

617966 0.371478 709973 0.362209 697132 0.355846

693931 0.370866 687109 0.361934 658217 0.355556

661168 0.370486 677228 0.361864 629374 0.354769

310618 0.369851 638436 0.361701 2053 0.354767

632038 0.369397 684733 0.361666 650826 0.354576

647115 0.368919 635558 0.360758 715729 0.354507

633925 0.36877 672035 0.360628 311432 0.35415

647575 0.368498 696564 0.360384 672880 0.353962

612238 0.368396 676569 0.360351 680475 0.353953

677074 0.368335 690229 0.360056 709979 0.353881

618312 0.368211 698252 0.359758 713288 0.353774

625019 0.367531 635148 0.359578 665860 0.353586

666764 0.367516 683497 0.35947 646339 0.353575

624795 0.366876 50922 0.359207 709974 0.35352

666293 0.36656 668495 0.358904 627452 0.353456

648854 0.366121 617959 0.358645 635563 0.35326

676920 0.365368 688948 0.358508 615537 0.352755

709975 0.36529 117028 0.357898 672102 0.35258 Compound Pearson s Compound Pearson's Compound Pearson's

(NSC) Correlation (NSC) Correlation (NSC) Correlation

672094 0.35258 683594 0.347588 647127 0.343708

672110 0.35258 685005 0.347554 676321 0.343612

56845 0.35255 629915 0.347271 715711 0.343367

624434 0.352511 672881 0.346888 627577 0.343269

669812 0.35245 670115 0.346743 692754 0.342966

655280 0.35244 622938 0.346632 663304 0.342569

697131 0.352383 693329 0.346544 632735 0.34209

607301 0.352332 657924 0.346539 649238 0.342061

163133 0.351666 641139 0.346349 632803 0.341838

691980 0.351614 698459 0.346243 695304 0.341752

667536 0.35129 621343 0.346072 630964 0.341275

667000 0.351263 715072 0.346063 660151 0.341217

627025 0.351263 665362 0.346057 637719 0.341137

635543 0.351242 635534 0.345767 20138 0.340866

642576 0.351204 672070 0.345739 673100 0.340411

631674 0.351065 672074 0.345739 676737 0.340342

703011 0.35102 672077 0.345739 674199 0.340266

679285 0.350476 675885 0.345564 673148 0.340264

645888 0.350308 692756 0.345563 664245 0.339923

680791 0.350213 653035 0.345121 660337 0.339771

635128 0.35012 642632 0.344796 694981 0.339606

675879 0.349973 647100 0.34477 627321 0.339605

18894 0.349424 675263 0.34474 664723 0.339544

689218 0.349183 687558 0.344672 655455 0.339395

649556 0.348764 697933 0.34444 643486 0.339277

647759 0.348419 715753 0.343881 165697 0.339159

651246 0.348279 695318 0.343771 668499 0.339092

258317 0.347928 620232 0.34372 627775 0.339038 Compound Pearson s Compound Pearson s Compound Pearson s

(NSC) Correlation (NSC) Correlation (NSC) Correlation

693984 0.33875 650973 0.334388 675575 0.329634

643474 0.338499 677241 0.334309 641846 0.3294

636093 0.338369 632002 0.33334 255917 0.329284

639526 0.338011 710359 0.333192 702651 0.329265

2066 0.337894 694682 0.333192 657753 0.329207

632734 0.337866 3094 0.332992 522958 0.328993

675869 0.337783 692758 0.332611 630509 0.328653

13413 0.337609 127713 0.332543 663774 0.328588

669339 0.337222 655058 0.332369 650956 0.328429

674209 0.336853 692764 0.332258 648062 0.328377

633915 0.336582 643914 0.33193 95848 0.328211

674007 0.336396 624439 0.3319 654898 0.328117

647958 0.336179 681629 0.331788 650836 0.328016

672087 0.336057 638631 0.33159 690737 0.327587

623078 0.335965 625220 0.331474 692026 0.327438

356200 0.335895 649905 0.331387 681864 0.327145

622372 0.335739 631441 0.331214 617938 0.327071

654634 0.335708 664947 0.330946 705079 0.326991

639746 0.335611 628934 0.330884 329696 0.326913

625038 0.335093 638368 0.330803 697138 0.326729

679284 0.335082 650758 0.330565 691081 0.326612

707140 0.33496 633390 0.330503 623834 0.326508

672090 0.334882 666999 0.33033 632729 0.326486

697133 0.334773 702670 0.330033 670093 0.326163

674211 0.334645 634605 0.330017 670111 0.326142

688034 0.33455 165765 0.329995 643477 0.326135

715735 0.334499 673836 0.329854 675274 0.325942

659925 0.334417 692759 0.329665 649775 0.325552 Compound Pearson s Compound Pearson's Compound Pearson's

(NSC) Correlation (NSC) Correlation (NSC) Correlation

667914 0.325154 383336 0.32022 268907 0.316479

356778 0.325142 625142 0.320004 645253 0.316425

658577 0.325035 615802 0.319558 196524 0.316379

638048 0.324598 254667 0.319358 643003 0.316343

703107 0.324501 639529 0.319299 339585 0.31602

661184 0.324219 39225 0.319037 715737 0.315747

641601 0.324151 668292 0.318994 631836 0.315596

636101 0.324101 703105 0.318514 641110 0.315404

631949 0.324027 181491 0.318504 339145 0.315402

669667 0.323952 667929 0.318342 678372 0.315078

701020 0.323848 697506 0.318338 639540 0.314888

668496 0.323616 677626 0.318219 617934 0.314879

721126 0.323603 676720 0.318209 651924 0.314865

636085 0.323128 693335 0.31815 682719 0.314638

677376 0.32303 630310 0.318135 676305 0.31456

640077 0.322691 657341 0.317984 660368 0.314526

697127 0.322639 83085 0.317946 114381 0.314409

715742 0.322474 690556 0.317686 695041 0.31378

659924 0.322473 673102 0.317589 658228 0.313735

671476 0.322117 643727 0.317414 628878 0.313695

636674 0.322011 721124 0.31735 33052 0.313687

106995 0.321751 36437 0.3169 627402 0.313571

626645 0.321598 656260 0.316888 621945 0.31342

636105 0.3215 699709 0.31687 627741 0.312998

608624 0.321408 705293 0.316761 696125 0.312918

682714 0.320627 654630 0.316667 633923 0.312888

674198 0.32041 689530 0.316557 685977 0.312874

692755 0.320347 636137 0.316545 627455 0.31262 Compound Pearson s Compound Pearson's Compound Pearson's

(NSC) Correlation (NSC) Correlation (NSC) Correlation

691794 0.312619 667057 0.308344 646211 0.304847

689868 0.312593 657441 0.308205 630374 0.304836

43321 0.312424 693986 0.307655 677610 0.304749

628877 0.312238 634634 0.307328 106486 0.304731

693864 0.312177 682826 0.306822 632574 0.304474

684910 0.312169 647583 0.306618 645818 0.304392

698407 0.312156 635002 0.306541 72861 0.304374

613056 0.311927 670101 0.306496 365081 0.304322

697161 0.311646 703064 0.306317 635583 0.304074

652568 0.311453 646209 0.306304 373854 0.304017

617956 0.311415 674213 0.306185 625588 0.303912

529861 0.310952 628527 0.306138 639985 0.303691

176392 0.310572 715599 0.306111 636086 0.303584

142055 0.310556 718583 0.30604 625574 0.30339

318728 0.31047 637010 0.305998 692761 0.303306

653148 0.310298 676966 0.305991 7833 0.303263

163443 0.310286 693248 0.305811 77036 0.303244

645680 0.310234 629914 0.30547 636502 0.303234

664873 0.310012 715267 0.30545 634506 0.30304

649903 0.309993 309741 0.305446 652202 0.302839

617972 0.309803 674914 0.305339 652920 0.302786

650014 0.309713 682813 0.305221 24817 0.302684

638269 0.309594 635967 0.305014 352741 0.302555

626767 0.309575 656083 0.305012 691113 0.302403

602130 0.309539 695585 0.305005 640345 0.302195

36586 0.309275 308787 0.304974 641405 0.301858

12825 0.309074 670668 0.304912 693325 0.301607

630300 0.308685 617965 0.30487 698601 0.301438 Compound Pearson s Compound Pearson s

(NSC) Correlation (NSC) Correlation

60417 0.301328 617260 0.300513

705315 0.301194 657726 0.300478

645757 0.300953 136722 0.30034

650785 0.300938 693327 0.300079

686513 0.30079 691215 0.300078

//. Pharmaceutical Compositions and Methods for their Use 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 currently disclosed compounds. The therapeutically effective amount of a disclosed compound 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 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, antiinflammatory agents, anesthetics, and the like. Pharmaceutical formulations can include additional components, such as carriers. 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 compounds. Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable inorganic or organic bases and acids. Particular disclosed compounds 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 α-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-phosphoglycerate, 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 compounds include at least one acidic group that can form an acid- base salts 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 compounds include, without limitation, pyridine, iV,./V-dimethylaminopyridine, diazabicyclononane, diazabicycloundecene, jV-methyl-iV-ethylamine, diethylamine, triethylamine, diisopropylethylamine, mono-, bis- or tris- (2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine, tris(hydroxymethyl)methylamine, iV,./V-dimethyl-./V-(2- hydroxyethyl)amine, tri-(2- hydroxyethyl) amine and jV-methyl-D-glucamine. For additional examples of "pharmacologically acceptable salts," see Berge et al., J. Pharm. ScL 66:1 (1977). Compounds 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 compounds 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 compounds 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.

In one embodiment, the presently disclosed compounds are useful for the treatment of hyperproliferative disorders wherein the hyperproliferative cells exhibit MDR or are likely to develop MDR. In general, MDR is likely to develop in proliferative disorders being treated with an MDR-inducing chemotherapeutic agent. Chemotherapeutic s that tend to induce MDR are known to those of skill in the arts of pharmacology and oncology and include, for example Vinca alkaloids, anthracyclines, epipodophyllotoxins, taxols, actinomycin D, cardiac glycosides, immunosuppressive agents, glucocorticoids, and anti-HIV protease inhibitors. One aspect of the present disclosure includes methods for treating a hyperproliferative disorder by administering a therapeutically effective amount of the disclosed compounds to a subject in need thereof. For example, particular proliferative disorders that can be so treated include solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, bladder carcinoma, and CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and retinoblastoma). The present disclosure also provides methods to treat hyperproliferative disorders that are characterized by multidrug resistance. By way of example, the presently disclosed compounds and compositions can be used to inhibit multidrug resistant prostate, breast, colon, bladder, cervical, skin, testicular, kidney, ovarian, stomach, brain, liver, pancreatic or esophageal cancer, or lymphoma, leukemia or multiple myeloma. In certain embodiments the disclosed compounds and compositions are used to treat a subject is at risk of developing a metastatic proliferative disorder.

Examples of hematological tumors that can be treated as disclosed herein include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblasts, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macro globulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

The therapeutically effective amount of the compound or compounds administered can vary depending upon the desired effects and the factors noted above. Typically, dosages will be between about 0.01 mg/kg and 250 mg/kg of the subject's body weight, and more typically between about 0.05 mg/kg and 100 mg/kg, such as from about 0.2 to about 80 mg/kg, from about 5 to about 40 mg/kg or from about 10 to about 30 mg/kg of the subject's body weight. Thus, unit dosage forms can be formulated based upon the suitable ranges recited above and a subject's body weight. The term "unit dosage form" as used herein refers to a physically discrete unit of therapeutic agent appropriate for the subject to be treated.

Alternatively, dosages are calculated based on body surface area and from about 1 mg/m to about 200 mg/m , such as from about 5 mg/m to about 100 mg/m will be administered to the subject per day. In particular embodiments, administration of the therapeutically effective amount of the compound or compounds involves administering to the subject from about 5 mg/m ² to about 50 mg/m ² , such as from about 10 mg/m ² to about 40 mg/m ² per day. It is currently believed that a single dosage of the compound or compounds is suitable, however a therapeutically effective dosage can be supplied over an extended period of time or in multiple doses per day. Thus, unit dosage forms also can be calculated using a subject's body surface area based on the suitable ranges recited above and the desired dosing schedule.

It is contemplated that in some embodiments the disclosed compounds are used in combination with other types of treatments, such as cancer treatments. For example the disclosed inhibitors may be used with other chemotherapies, including those employing an anti-proliferative agent, such as, without limitation, microtubule binding agent, a toxin, a DNA intercalator or cross-linker, a DNA synthesis inhibitor, a DNA and/or RNA transcription inhibitor, an enzyme inhibitor, a gene regulator, enediyne antibiotics and/or an angiogenesis inhibitor. In one embodiment the presently disclosed compounds are used to render a neoplasm susceptible to one or more anti-proliferative compounds to which it is resistant. Additionally, the disclosed compounds can be used in combination with radiation therapy, surgery, or other modalities of cancer therapy.

"Microtubule binding agent" refers to an agent that interacts with tubulin to stabilize or destabilize microtubule formation thereby inhibiting cell division. Examples of microtubule binding agents that can be used in conjunction with the presently disclosed compounds include, without limitation, paclitaxel, docetaxel, vinblastine, vindesine, vinorelbine (navelbine), the epothilones, colchicine, dolastatin 15, nocodazole, podophyllotoxin and rhizoxin. Analogs and derivatives of such compounds also can be used and will be known to those of ordinary skill in the art. For example, suitable epothilones and epothilone analogs for incorporation into the present compounds are described in International Publication No. WO 2004/018478, which is incorporated herein by reference. Taxoids, such as paclitaxel and docetaxel are currently believed to be particularly useful as therapeutic agents in combination with the presently disclosed compounds. Examples of additional useful taxoids, including analogs of paclitaxel are taught by U.S. Patent Nos. 6,610,860 to Holton, 5,530,020 to Gurram et al. and 5,912,264 to Wittman et al. Each of these patents is incorporated herein by reference.

Suitable DNA and/or RNA transcription regulators for use with the disclosed compounds include, without limitation, actinomycin D, daunorubicin, doxorubicin and derivatives and analogs thereof also are suitable for use in combination with the presently disclosed compounds.

DNA intercalators, cross-linking agents and alkylating agents that can be used in combination therapy with the disclosed compounds include, without limitation, cisplatin, carboplatin, oxaliplatin, mitomycins, such as mitomycin C, bleomycin, chlorambucil, cyclophosphamide, isophosphoramide mustard and derivatives and analogs thereof.

DNA synthesis inhibitors suitable for use as therapeutic agents include, without limitation, methotrexate, 5-fluoro-5'-deoxyuridine, 5-fluorouracil and analogs thereof. Examples of suitable enzyme inhibitors for use in combination with the presently disclosed compounds include, without limitation, camptothecin, etoposide, formestane, trichostatin and derivatives and analogs thereof.

Suitable therapeutics for use with the presently disclosed compounds that affect gene regulation include agents that result in increased or decreased expression of one or more genes, such as, without limitation, raloxifene, 5-azacytidine, 5-aza-2'- deoxycytidine, tamoxifen, 4-hydroxytamoxifen, mifepristone and derivatives and analogs thereof.

The term "angiogenesis inhibitor" is used herein, to mean a molecule including, but not limited to, biomolecules, such as peptides, proteins, enzymes, polysaccharides, oligonucleotides, DNA, RNA, recombinant vectors, and small molecules that function to inhibit blood vessel growth. Angiogenesis inhibitors are known in the art and examples of suitable angiogenesis inhibitors include, without limitation, angiostatin Kl-3, staurosporine, genistein, fumagillin, medroxyprogesterone, SFTI-I, suramin, interferon-alpha, metalloproteinase inhibitors, platelet factor 4, somatostatin, thromobospondin, endostatin, thalidomide, and derivatives and analogs thereof. Other therapeutic agents, particularly anti-tumor agents, that may or may not fall under one or more of the classifications above, also are suitable for administration in combination with the presently disclosed compounds. By way of example, such agents include adriamycin, apigenin, erlotinib, gefitinib, temozolomide, rapamycin, topotecan, carmustine, melphalan, mitoxantrone, irinotecanetoposide, tenoposide, zebularine, cimetidine, and derivatives and analogs thereof.

Suitable dosages and treatment regimes for administering the above- identified therapeutic agents are known to those of ordinary skill in the art of oncology and also are described, for example, in Physicians' Cancer Chemotherapy Drug Manual 2005 By Edward Chu and Vincent T. DeVita (ISBN 0763734616), which is incorporated herein by reference. Such dosages and treatment regimens can be used in combination with a presently disclosed MDR-inverse compound.

The compounds disclosed herein may be administered orally, topically, transdermally, parenterally, via inhalation or spray and may be administered in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles.

Typically, oral administration or administration via implantation or intravenously, such as via injection is preferred. However the particular mode of administration employed may be dependent upon the particular disease, condition of patient, toxicity of compound and other factors as will be recognized by a person of ordinary skill in the art.

///. Identification of Subjects having MDR Disorders There are a variety of techniques to detect expression of MDRl. The detection and/or quantitation of MDRl protein typically is accomplished using immunological techniques. For hematopoietic cells such as those from leukemia or lymphoma patients, the techniques include flow cytometry and fixed cells on microscope slides. The cells are treated with antibodies specific for the MDRl protein, such as the mouse ARK- 16 monoclonal antibody. Such antibodies can be directly labeled with fluorescent probe, or detected using subsequent reagents such as goat anti-mouse IgG-FITC. Flow cytometry allows for direct quantitative determinations of the full spectrum of MDRl expression using channel number or fluorescence intensity. Microscopic examination of the slide preparations can give qualitative results (-, +, ++, and the like) or, in conjunction with an image analyzer, quantitative evaluations typically expressed in pixels.

For solid tumors, such as breast cancer, typically immunocytochemistry (ICC) or immunohistochemistry (IHC) techniques are employed. Using, for example, frozen sections or paraffin blocks, the detection techniques are the same as described for fixed leukemia cells on microscope slides. Expression of MDRl can also be monitored by the measurement of specific mRNA levels. Cell slides can be processed, and levels of mRNA discerned using basic molecular biology techniques such as quantitative fluorescent PCR. Alternatively, the cells of interest can be lysed, processed, and following PCR of the mRNA, the product can be detected and quantitated following gel electrophoresis. Anti-sense targeting of MDRl mRNA is also possible, followed by standard techniques for quantitative determinations. Radio-labeled probes followed by autoradiography or other radiodetection techniques can also be used to obtain a relative estimate of MDRl protein or mRNA expression. Thus, there exists a broad range of methods for the detection and quantitation of the spectrum of MDRl expression exhibited by a subject. Monitoring of the relative expression of MDRl is possible in vivo. MDRl- specific antibodies labeled with any number of detectable markers, such as radioactive compounds detectable with positron emission tomography (PET), single- photon emission computed tomography (SPECT) or compounds detectable with magnetic resonance imaging (MRI) can be used to assess MDRl expression in a subject having cancer or an MDRl-expressing infection, such as multidrug resistant tuberculosis.

MDRl function, i.e., functional expression of MDRl also can be evaluated. MDRl functions as a cytoplasmic membrane pump, effluxing compounds such as drugs and toxins from the cytoplasm to the exterior of the cell. Compounds acted on by MDRl are termed MDRl substrates. Detection of MDRl function therefore involves detection of substrate efflux, such as the efflux of a particular drug, or alternatively, detection of efflux of surrogate fluorescent dye markers that also are MDRl substrates, such as DiOC2 (3,3'-diethyloxacarbocyanine iodide) or Rhodamine 123 (Rhl23, or 2-(6-amino-3-imino-3H-xanthen-9-yl)benzoic acid, methyl ester). For single cell suspensions, such as blood or bone marrow from leukemia patients, the cells are exposed in tissue culture to a substrate for MDRl, such as the aforementioned dye markers, radiolabeled drugs, or drugs that can be detected and/or quantitated by other means such as fluorescence. At physiological temperature (37° C) the net accumulation of the substrate over time, in the presence or absence of specific MDRl inhibitors, gives an indication of the MDRl functional activity exhibited by the cells. Alternatively, the single cell suspension can be exposed to the substrate and subsequent efflux of the substrate over time monitored at physiological temperature in the presence or absence of specific MDRl inhibitors. PET, SPECT, and MRI techniques also can be used to assess MDRl function in cancer patients. Thus, small organic molecules as well as metal complexes that serve as MDRl substrates can be labeled with radionuclides or other detectable markers. Additionally, functional expression in solid tumors can be more efficiently ascertained by ICC/IHC techniques with prior labeling of the tumor cells in the patient.

Diagnostic testing methods for MDRl expression and efflux pump activity can be used to prospectively stratify patients for treatment optimization in treating malignancies exhibiting MDRl expression or function, such as acute myelogenous leukemia, most solid tumors, lymphomas, bladder cancer, pancreatic cancer, ovarian cancer, liver cancer, myeloma, lymphocytic leukemia, and sarcoma.

The disclosed techniques for identifying subjects having MDR-resistant cells are applicable to any therapeutic drug that is a substrate for P-gp-mediated efflux. Such drugs include, but are not limited to, P-glycoprotein substrates; anticancer drugs as described above and including, by way of example Vinca alkaloids such as vinblastine and vincristine; anthracyclines such as doxorubicin, daunorubicin, epirubicin; anthracenes such as bisantrene and mitoxantrone; epipodophyllo-toxins such as etoposide and teniposide; and other anticancer drugs such as actinomyocin D, mithomycin C, mitamycin, methotrexate, docetaxel, etoposide (VP-16), paclitaxel, docetaxel, and adriamycin; immunosuppressants, including cyclosporine A and tacrolimus; steroids, by way of example, dexamethasone, hydrocortisone, corticosterone, triamcinolone, aldosterone and methylprednisolone; antiepileptics, such as phenyloin; antidepressants, including without limitation, citalopram, thioperidone, trazodone, trimipramine, amitriptyline and phenothiazines; antipsychotics, such as fluphenazine, haloperidol, thioridazine and trimipramine; HIV protease inhibitors, for example, amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir; calcium blockers, for example, bepridil, diltiazem, flunarizine, lomerizine, secoverine, tamolarizine, verapamil, nicardipine, prenylamine and fendiline.

Monotherapy using MDR-Inverse Compounds This example describes the treatment of a subject having a multidrug resistant disorder, such as a multidrug resistant tumor. Subjects having such disorders can be identified, for example, as set forth above.. In one embodiment, a subject having a multidrug resistant disorder is administered an MDR-inverse compound disclosed herein, in an amount sufficient to elevate the target tissue concentration of the MDR-inverse compound in the subject to at least about 10 nM, such as from about 0.1 μM to about 100 μM, and typically from about 1 μM to about 10 μM. In one embodiment, the MDR-inverse compound is administered intravenously in an amount of 400 mg/day or less to about 1,600 mg/day or more, preferably from about 500, 600, or 700 mg/day to about 900, 1000, 1100, 1200, 1300, 1400, or 1500 mg/day, and most preferably about 700 mg/day. In the course of a treatment regimen, the MDR-inverse compound preferably is administered on two, three, or four separate days. The dosage typically is administered in intravenously continuously over the course of about 3 to about 90 hours, more preferably over the course of about 4, 6, 12, 18, 24 or 30, 36, or 42 hours to about 54, 60, 66, 72, 78, or 84 hours, most preferably over about 24 hours, 48 hours, or 72 hours, depending upon the treatment regimen. Preferably the MDR-inverse compound is administered on multiple days of the treatment regimen. Combination Therapy using MDR-Inverse Compounds The drugs which are substrates of P-gp are quite varied as are the associated disease states. Chemotherapeutic agents that are P-gp substrates include, without limitation, anthracyclines (for example, doxorubicin, daunorubicin, epirubicin, idarubicin, mitoxantrone), Vinca alkaloids (for example, vincristine, vinblastine, vinorelbine, vindesine), Topoisomerase-II inhibitors (for example, etoposide, teniposide), taxanes (e.g., paclitaxel, docetaxel), and others (for example, Gleevec and dactinomycin). The MDRl -inverse compounds disclosed herein can be administered in combination with any of these chemotherapeutic agents.

Examples

Materials and Methods

Chemicals. Unless otherwise stated, compounds were obtained from the NCI DTP drug repository. The octapeptide NSC633657 (D-Phe-Cys-Tyr-D-Trp-Lys-Ser-Cys- Thr-NH2) was prepared by John Stonik, NCI and purified by HPLC at the US FDA Center for Biologies Evaluation and Research.

Drug database. The DTP Human Tumor Cell Line Screen has screened tens of thousands of compounds for growth inhibition of human cancer cell lines. Screening results for -43,000 compounds were downloaded from http://discover.nci.nih.gov (July 2007 Release). Scrutiny of the dataset indicated that it was not immediately amenable to stringent statistical analysis due to missing dose-response values or lack of activity in the dose window tested. Since uninformative drug profiles are not expected to yield useful leads, we selected only those drugs that were measured inside the range of cytotoxicity with no more than 50% missing values, resulting in a higher quality activity dataset. Putative MDR-inverse compounds were identified based on correlation of their cytotoxicity patterns to ABCBl expression, as described in (Szakacs et al., 2004). Evaluation of structural similarity. Structural analogs within the DTP dataset were identified based on Tanimoto coefficients defined by the Leadscope software (LeadScope, Inc, Columbus, OH, USA) (Mutch et al., 2004). Clustering of the Discovery-set was performed using the PubChem Structure Clustering algorithm (http://pubchem.ncbi.nlm.nih.gov/). For additional analysis shown (Figure 3), chemical fingerprints were generated using the Chemaxon GenerateMD software. Structural clustering was performed using the average linkage method with a threshold distance of 0.6 (R package, www.r-project.org).

Pharmacophore generation and QSAR. The 3D structure of the molecules serving as templates (NSC693871 and NSC337743 for the "TSC" and "10580" cluster, respectively) was calculated using the MOPAC2007 program (Stewart Computational Chemistry, USA). The molecules were aligned using the FieldAlign software (Cresset BDL, USA), which aligns molecules based on electrostatic, surface and topological properties. The simplified pharmacophore model is visualized using LigandScout software (InteLigand). For each compound of the two major clusters shown in Figure 2, 3368 molecular descriptors were calculated by the PCLIENT algorithm (Tetko et al., 2005). The initial descriptor set was preprocessed in order to eliminate ineffective descriptors (those with too many zero values or with very small standard deviations). The QSAR study was carried out with the enhanced replacement method (Mercader et al., 2008) using the Matlab software (The MathWorks, Inc.).

Cell lines and culture conditions. KB-3-1 is the parental human (HeLa) epidermoid carcinoma cell line of KB-Vl cells that overexpress Pgp (MDRl) as a result of long-term selection in vinblastine (Shen et al., 1986). NIH-MDR-G185 is a clone of NIH3T3 cells transfected with wild type pHaMDRl/A (Cardarelli et al., 1995). MES-SA Dx5, a drug resistant cell line expressing high levels of Pgp, was derived from a human uterine sarcoma line (MES-SA) by doxorubicin selection (Wang et al., 2000). KB-3-1, KB-Vl, NIH3T3 and NIH-MDR-G185 cells were grown in DMEM cell culture medium at 37 ⁰ C in 5% CO ₂ . MES-SA and MES-SA Dx5 cells were grown in McCoy's medium. NIH-MDR-G 185, KB-V-I and Dx5 cells were maintained in 60 ng/mL colchicine, lμg/mL vinblastine and 500 nM doxorubicin (adriamycin) respectively to maintain Pgp expression. All cell culture media (GIBCO) was supplemented with 10% fetal bovine serum (FBS, GIBCO), 5 mM glutamine (GIBCO) and 50 unit/ml penicillin and streptomycin (GIBCO).

Retroviral expression of ABCBl in A431 cells was achieved by a method described previously (Ujhelly et al., 2003). The human skin-derived, epidermoid carcinoma cells, A431, were maintained in alpha- MEM (Life Technologies, Grand Island, NY) supplemented with 10% FBS, 50 units/mL penicillin, 50 units/mL streptomycin, and 5 mM glutamine at 37 ⁰ C in 5% CO ₂ .

Cell viability assay. Cells were trypsinized, counted and seeded in 100 μL medium supplemented with 10% FBS at a density of 3,000 per well in 96-well plates and incubated for 24 hours at 37 ⁰ C in a humidified atmosphere with 5% CO ₂ and 95% air to allow exponential growth. After 24 h, 100 uL medium containing serially diluted compounds were added to give the indicated final concentrations in three replicated wells. Cells were then incubated for a further 72 hours. Antiproliferative activity of drugs was evaluated using the MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) assay (Szakacs et al., 2004). Cytotoxicity assays were performed in triplicate, and curves were fitted by Prism software (GraphPad Software, Inc., San Diego, CA) using nonlinear least-squares regression in a sigmoidal dose-response model with variable slope, also known as the four- parameter logistic equation. Curves were normalized at 100 and 0, the expected upper and lower boundaries of normalized percentage inhibition data, respectively. Curve fit statistics were used to determine the concentration of test compound that resulted in 50% toxicity (IC ₅₀ ) (in μM). Differences between the GI50 values were analyzed by two-sided paired Student's t test and results were considered statistically significant at p < 0.05. Results

The results are shown in Figures 1-7, and in Table B below. In particular, compounds identified as having MDRl -selective activity, listed in descending order for their Pearson's correlation coefficients, the correlation between MDRl expression and DTP determined efficacy of each compound across the NCI-60 cell line panel, are shown in Table 1 in Figure 7. The structure listed in the NCI DTP database is shown for each NSC compound, along with any relevant references to the compound found in the literature, and the average GI ₅₀ value for each compound against the NCI-60 panel to give an indication of overall potency of each compound (not its MDRl inverse activity). Compounds were sourced by a variety of methods, predominately through the NCI DTP drug library, but also via other investigators and synthesis in our laboratories. For compounds that were available, IC ₅₀ values were determined using the MTT cytotoxicity against the parental KB-3-1 cell line, and the P-glycoprotein expressing cell line KB-Vl. The MDRl selectivity is calculated as the ratio of a compound's IC50 against KB-3-1 cells divided by its IC50 against KB-Vl cells. A value > 1 indicates that the compound kills P-gp-expressing cells more effectively than parental cells, so-called MDRl -inverse activity. A value < 1 indicates that the P-gp expressing cells are resistant to the compound, relative to parental cells, as is normally observed for P-gp substrates. Compounds with an IC ₅₀ > 50 μM were considered to be not toxic (NT), and as such their MDRl selectivity could not be determined.

Table B

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In view of the many possible embodiments to which the principles of the disclosed compounds, compositions and methods 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.