WO2013112878A1 - Mcl-1 modulating compositions - Google Patents

Abstract

The present invention relates to marinopyrrole A derivatives and pyoluteorin derivatives and methods of treatment of disorders associated with misregulation of Mcl-l, e.g., leukemia, lymphoma, multiple myeloma, melanoma, or pancreatic cancer. We describe exemplary compounds, which may be contained in pharmaceutical compositions, and their use as therapeutic agents either alone or in combination with other anti-cancer treatments, e.g., anti-Bcl- 2 agents.

Description

MCL-1 MODULATING COMPOSITIONS

RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S. Provisional Application No. 61/591,075 which was filed on January 26, 2012 and U.S. Provisional Application No.

61/673,788 which was filed on July 20, 2012. For the purpose of any U.S. application or patent that claims the benefit of U.S. Provisional Application No. 61/591,075 and U.S. Provisional Application No. 61/673,788, the content of that earlier filed application is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to compositions and methods for treating cancer and disorders of cell proliferation and more particularly, methods of making and using compounds that modulate Mcl-1. We describe exemplary compounds, which may be contained in pharmaceutical compositions and their use as therapeutic or prophylactic agents.

BACKGROUND

Cancer is a leading cause of death worldwide. In the United Stated alone, cancer accounts for about 500,000 deaths per year, and the American Cancer Society estimates that in 2010 approximately 1.5 million new cases of cancer were diagnosed. Apoptosis, also known as programmed cell death, is a natural process used by multicellular organisms to eliminate aging or damaged cells. Apoptosis is a complex, highly regulated process involving many proteins. Some of these proteins promote cell death ("pro-apoptotic" proteins) and some prevent it ("anti- apoptotic" proteins). Cancer cells tend to over-express anti-apoptotic genes. The over- expression of anti-apoptotic genes is associated with tumor formation, metastatic growth and resistance to chemotherapy. There is a continuing need for therapeutic strategies that selectively kill cancer cells. SUMMARY

The present invention provides pharmaceutically acceptable compositions comprising a compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein X can be H, halo, or alkyl; Y can be OH, alkoxy or acetoxy; Z can be H or halo; Ri can be H, OH, or alkoxy; R₂ can be H, OH, or alkoxy; and R₃ can be H or halo. In some embodiments, the compound of Formula I can be (1,3 - Dibromo-4,5-dichloro-lH-pyrrol-2-yl)(2-methoxyphenyl)methanone; (l,3-Dibromo-4,5- dichloro- 1 H-pyrrol-2-yl)(2-hydroxyphenyl)methanone; (4,5-Dichloro- 1 H-pyrrol-2-yl)(2,3 ,4- trimethoxy-phenyl)methanone; (4,5-Dichloro- lH-pyrrol-2-yl)(2-hydroxy-3,4- dimethoxyphenyl)methanone; (4,5-dichloro-lH-pyrrol-2-yl)(2,3,4-trihydroxyphenyl)methanone; (3-Bromo-4,5-dichloro-lH-pyrrol-2-yl)(2,3,4-trimethoxyphenyl)methanone; (4,5-dichloro-3- methyl-lH-pyrrol-2-yl)(2-hydroxyphenyl)methanone; (3-Bromo-4,5-dichloro-lH-pyrrol-2-yl)- (2,4-dihydroxyphenyl)methanone; (3-Bromo-4,5-dichloro-lH-pyrrol-2-yl)(5-chloro-2- hydroxyphenyl)methanone; 2-(3-bromo-4,5-dichloro-lH-pyrrole-2-carbonyl)-4-chlorophenyl acetate; and (3 -Bromo-4,5 -dichloro- 1 H-pyrrol-2-yl)(5 -fluoro-2-hydroxyphenyl)methanone .

The present invention provides pharmaceutically acceptable compositions comprising a compound of Formula II:

or a pharmaceutically acceptable salt thereof, wherein X can be H or halo; Y can be OH, alkoxy or acetoxy; Ri can be H, OH, or alkoxy; R₂ can be H, OH, or alkoxy; and R3 can be H. In some embodiments, the compound of Formula II can be (3-Bromoindole-2-yl)(2- hydroxyphenyl)methanone; or (3-Bromoindol-2-yl)(2,4-dihydroxyphenyl)methanone.

Also provides are pharmaceutical compositions comprising the compounds of Formula I or Formula II a pharmaceutically acceptable carrier. In some embodiments the pharmaceutical composition can include 4,5-Dichloro-lH-pyrrol-2-yl)(2-methoxyphenyl)methanone; (4,5- Dichloro-lH-pyrrol-2-yl)(2-hydroxyphenyl)methanone; (3-Bromo-4,5-dichloro-lH-pyrrol-2- yl)(2-methoxyphenyl)methanone; (3-Bromo-4,5-dichloro-lH-pyrrol-2-yl)(2- hydroxyphenyl)methanone; or 2-(3-bromo-4,5-dichloro- lH-pyrrole-2-carbonyl)phenyl acetate.

The present invention provides pharmaceutical compositions comprising

a compound of Formula III:

wherein Ri and R₂ cannot be H, Ri can be COCH₃, C(0)OC₂H₅, or C(0)0 joined to a heterocycle and R₂ can be COCH₃, C(0)OC₂H₅, or C(0)0 joined to a heterocycle, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.

Also provided are methods of treatment. In some embodiments, a method of treating cancer is provided, the method comprising administering to a subject a therapeutically effective amount of any of the compositions of Formulas I, II or III or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. In some embodiments, the cancer can be lymphoma, leukemia, multiple myeloma, melanoma, pancreatic cancer, lung cancer, breast cancer, liver cancer, colon cancer, prostate cancer or ovarian cancer. Regardless of the particular type of cancer, the methods of treatment can further comprise the step of identifying a subject amenable to treatment. In some embodiments, the cancer expresses Mcl-1. In some

embodiments, the cancer is drug resistant. In some embodiments, the methods can include the administration of a second cancer treatment. The second cancer treatment can include administration of an anti-Bcl-2 agent, for example, ABT-199, ABT-737 or ABT-263. The methods can also include the step of providing a biological sample from the subject and determining whether the sample includes an elevated level of Mcl-1 or another predictive biomarker for cancer. The biological sample can be urine, saliva, cerebrospinal fluid, blood, or a biopsy sample. In some embodiments, the analysis of the biological sample can be carried out before administering the compositions. In some embodiments, the analysis of the biological sample, can be carried out at one or more times after administering the agent. Also provided are methods of killing an Mcl-1 expressing cancer cell. The methods include contacting the cell with an effective amount of the compositions of Formulas I, II or III. Also provided are methods of modulating the level of Mcl-1 in a cell. The methods include contacting the cell with an effective amount of the compositions of Formulas I, II or III.

The details of one or more embodiments of the invention are set forth in the

accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the structures of pyoluteorin derivatives KS01-KS20.

FIG. 2 is an analysis of levels of Mcl-1 polypeptide levels in KS04- and KS18~treated U937 cells.

FIG. 3 is an analysis of levels of Mcl-1 and other apoptosis-related polypeptides in ABT-737 resistant HL60 cells (HL60/ABTR) treated with KS18 alone or in combination with ABT-737.

FIG. 4A is a graph depicting the results of an analysis of the effect of daunorubicin treatment on U937 cell viability in the presence and absence of HS-5 human bone marrow stromal cells. FIG. 4B is a graph depicting the results of an analysis of the effect of maritoclax treatment on U937 cell viability in the presence and absence of HS-5 human bone marrow stromal cells. FIG. 4C is a graph depicting the results of an analysis of the effect of KS04 treatment on U937 cell viability in the presence and absence of HS-5 human bone marrow stromal cells. FIG. 4D is a graph depicting the results of an analysis of the effect of KS18 treatment on U937 cell viability in the presence and absence of HS-5 human bone marrow stromal cells.

FIG. 5 is a graph depicting the results of an analysis of the effect of daunorubicin, maritoclax, KS04, and KS18 treatment on HS-5 human bone marrow stromal cell viability.

FIG. 6 is a graph depicting the results of an analysis of the effect of maritoclax on U937 xenograft tumor growth in athymic nude mice.

FIG. 7 is a graph depicting the results of an analysis of the effect of maritoclax on body weight in athymic nude mice.

FIG. 8 is an analysis of the effect of KS04 on Mcl-l interaction with Bim in intact K562 cells stably expressing a Mcl-l -IRES-BimEL construct.

FIG. 9 A is a graph depicting the results of an analysis of the effect of KS18, alone or in combination with ABT-737 on HL60/V CR cell viability. FIG. 9B is a graph depicting the results of a combination index analysis of the effect of KS18, alone or in combination with ABT-737 on HL60/V CR cell viability. FIG. 9C is a graph depicting the results of an analysis of the effect of KS 18, alone or in combination with ABT-737 on HL60/ABTR cell viability. FIG. 9D is a graph depicting the results of a combination index of the effect of KS18, alone or in combination with ABT-737 on HL60/ABTR cell viability.

FIG. 10 depicts the structure of KA01.

FIG. 11 is an analysis of levels of Mcl-l and other apoptosis-related polypeptides in KAOl- treated K562 cells stably expressing a Mcl-l -IRES-BimEL construct.

FIG. 12 is an analysis of levels of Mcl-l and other apoptosis-related polypeptides in KS04- treated K562 cells stably expressing a Mcl-l -IRES-BimEL construct.

DETAILED DESCRIPTION

The present invention is based, in part, on our discovery that pyoluteorin derivatives of marinopyrrole A selectively killed Mcl-l -dependent leukemia cells and induced proteasome- mediated degradation of the anti-apoptotic polypeptide, Mcl-1. Accordingly, the compositions of the invention feature marinopyrrole A derivatives and pyoluteorin derivatives as well as pharmaceutical formulations comprising marinopyrrole A derivatives and pyoluteorin derivatives. The methods of the invention include methods of administering the compositions to treat cancer, methods of killing cancer cells and methods of modulating levels of Mcl-1 in a cell. The therapeutic methods described herein can be carried out in connection with other cytotoxic therapies (e.g., chemotherapy, hormone therapy, radiotherapy, and antibody-based therapies).

Apoptosis occurs following either triggering of cell surface death receptors (the extrinsic pathway) or perturbation of mitochondria (the intrinsic pathway). Most anti-cancer treatments, including chemotherapeutic agents, chemicals, and irradiation, induce apoptosis by activation of the intrinsic pathway. Both pathways ultimately lead to the activation of caspases, a family of cysteine proteases that cleave key cellular proteins, resulting in membrane blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA

fragmentation. Members of the Bcl-2 family of polypeptides play a key role in determining the susceptibility of cells to apoptosis induced by the intrinsic pathway through their control the integrity of the outer mitochondrial membrane (OMM). The Bcl-2 family encompasses three branches: 1) anti-apoptotic polypeptides (e.g., Bcl-2, Mcl-1, Bcl-XL and Bcl2Al (Bfl-l/Al); 2) multidomain pro-apoptotic polypeptides (e.g., Bax and Bak); and 3) pro-apoptotic BH3-only proteins (e.g., Bad, Bim, Puma, Bid, Bik, Noxa and Bmf). On receipt of a death signal, Bax and Bak form oligomers in mitochondrial membranes, resulting in permeabilization of the OMM, release of cytochrome c, and caspase activation. Anti-apoptotic Bcl-2 members prevent this release by blocking activation of Bax and Bak. BH3-only proteins, Bad, Bim, Puma, Bid, Bik, Noxa and Bmf, act upstream of Bax and Bak. BH3-only proteins selectively bind into the hydrophobic groove of anti-apoptotic Bcl-2 family members leading to Bax/Bak activation, either by the direct or indirect activation model. Anti-apoptotic Bcl-2 proteins can be divided into two groups, one group made up of Bcl-2, Bcl-XL and Bcl-w and the other group made up of Mcl-1 and Bcl2Al . The balance between pro- and anti-apoptotic Bcl-2 family members determines, in part, cellular susceptibility to apoptosis and the efficiency of apoptosis. Effective anti-apoptotic therapy has been shown to require neutralization of both sets of anti-apoptotic proteins (Willis, S. N. et al. (2005) Genes Dev. 19, 1294-1305). Mcl-l (also known as Bcl-2-like protein 3 (BCL2L3); Bcl-2 -related protein EAT/mcll (Bcl2-L-3); induced myeloid leukemia cell differentiation protein Mcl-l (EAT); myeloid cell leukemia sequence 1 (MCL1L); myeloid cell leukemia sequence 1 (BCL2-related) (MCL1S); and myeloid cell leukemia sequence 1, isoform 1 (MGC 104264 2) was originally characterized in differentiating myeloid cells. Representative Mcl-l amino acid sequences are found in GenBank at public GI numbers GL7582271 and GI: 11386165.

Mcl-l contains three BH domains (BH1-BH3) but lacks a clearly defined BH4 domain at the NH2 terminus. Mcl-l localizes to various intracellular membranes, especially, the outer mitochondrial membrane through a transmembrane domain at its COOH terminus. Like Bcl-2 and Bcl-xL, Mcl-l can interact with Bax and/or Bak to inhibit mitochondria-mediated apoptosis. Unlike Bcl-2 and Bcl-xL, Mcl-l expression is quickly induced upon exposure to cytokines or growth factors. Increased Mcl-l expression promotes cell viability in a wide range of tumor cell types, including leukemias, hepatocellular carcinomas, melanoma, prostate and breast cancer cells. Moreover, Mcl-l plays a role in cell immortalization and tumorigenesis in many kinds of cancers through amplification of somatic copy number. Cancer cells harboring Mcl-l amplification are frequently dependent upon Mcl-l for survival. (Beroukhim, R. et al. (2010) Nature 463, 899-905). Overexpression of Mcl-l also contributes to the multidrug-resistance phenotype seen in certain cancers, e.g., human leukemias.

The compositions and methods described herein include those that modulate the activity of Mcl-l . We may refer to a compound that modulates the activity of Mcl-l as an Mcl-l inhibitor. There are many ways in which the activity of Mcl-l can be inhibited. For example, an Mcl-l inhibitor may inhibit Mcl-l activity by inhibiting the binding of Mcl-l to a BH3-only polypeptide, for example, Noxa, or an Mcl-l inhibitor may modulate levels of Mcl-l

polypeptide by selectively increasing the degradation rate of Mcl-l, for example, by targeting Mcl-l for proteasomal degradation. While we believe we understand certain events that occur in the course of exposure of Mcl-l to the composition described herein, the compositions of the present invention are not limited to those that work by affecting any particular cellular mechanism. The compounds described herein can be included in pharmaceutical compositions that are physiologically acceptable (i.e., sufficiently non-toxic to be used in the therapeutic and prophylactic methods described herein). Accordingly, the invention features a variety of formulations, including topical creams (integrated into sunsceens) and sustained-release patches for transdermal delivery of Mcl-1 inhibitors. In other embodiments, the pharmaceutical composition can be formulated as an oral rinse, gel, suspension, emulsion, or tablet. As will be apparent to one of ordinary skill in the art, the specific formulations can be selected based on the type of cancer being treated. The compositions of the invention can be formulated for administration to a patient with materials that improve their stability and/or provide for a controlled or sustained release in vivo. Accordingly, the invention encompasses delivery systems in which the compositions are formulated with microparticles (e.g., polymeric microparticles such as polylactide-co-glycolide microparticles) or nanoparticles (e.g., liposomes, polymeric carbohydrate nanoparticles, dendrimers, and carbon-based nanoparticles). Other formulations include those for subcutaneous, intraperitoneal, intravenous, intraarterial, or pulmonary administration.

The therapeutic methods can be carried out by administering to the subject a

pharmaceutical composition containing a therapeutically effective amount of a pyoluteorin derivative described herein or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. While a single composition may be effective, the invention is not so limited. A subject can be treated with multiple compositions, administered simultaneously or sequentially (i.e., before or after a compound of the present invention). For example, a subject can be treated with one or more of the compositions described herein and, optionally, a chemotherapeutic agent. In some embodiments, the "second" agent can be an anti-Bcl-2 agent. Cancer cells can quickly develop resistance to certain chemotherapeutics. Resistance to small molecule inhibitors of Bcl-2, for example, ABT-737 and ABT-263, is mediated in part by increasing expression of Mcl-1. We have found that combinations of the compounds of the invention with small molecule inhibitors of Bcl-2 was effective in overcoming Bcl-2 resistance. Compositions containing a compound of the invention and a second agent, as described herein, are also within the scope of the present invention. Compositions

The compounds useful for the pharmaceutical compositions described herein include compounds conforming to Formula I or pharmaceutically acceptable salts or prodrugs thereof.

Formula I

In some embodiments, X is H, halo or alkyl. In some embodiments, Y is OH, alkoxy or acetoxy. In some embodimenets, Z is H or halo. In some embodiments, Ri is H, OH or alkoxy. In some embodiments, R₂ is H, OH or alkoxy. In some embodiments, R₃ is H or halo.

Exemplary compounds include:

The compounds useful for the pharmaceutical compositions described herein include compounds conforming to Formula II or pharmaceutically acceptable salts or prodrugs thereof.

Formula II

in some embodiments, X is H, halo or alkyl. In some embodiments, Y is OH, alkoxy or acetoxy. In some embodimenets, Z is H or halo. In some embodiments, Ri is H, OH or alkoxy. in some embodiments, R₂ is H, OH or alkoxy. In some embodiments, R₃ is H or halo.

Exemplary compounds include:

The compounds useful for the pharmaceutical compositions described herein include compounds conforming to Formula III or pharmaceutically acceptable salts or prodrugs thereof.

Formula III

Ri and R₂ cannot both be H. In some embodiments, Ri is COCH₃, C(0)OC₂H₅, or C(O) O joined to a heterocycle. In some embodiments, R₂ is COCH₃, C(0)OC₂H₅, or C(0)0 joined to a heterocycle.

At various places in the present specification, substituents of compounds of the invention are disclosed in groups or in ranges. It is specifically intended that the invention include each and every individual subcombination of the members of such groups and ranges. For example, the term "Ci_₆ alkyl" is specifically intended to individually disclose methyl, ethyl, C₃ alkyl, C₄ alkyl, C₅ alkyl, and C₆ alkyl.

It is further appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination.

At various places in the present specification, linking substituents are described. It is specifically intended that each linking substituent include both the forward and backward forms of the linking substituent. For example, --NR(CR ')„- includes both ~NR(CR'R")„- and - (CR'R") _nNR— . Where the structure clearly requires a linking group, the Markush variables listed for that group are understood to be linking groups. For example, if the structure requires a linking group and the Markush group definition for that variable lists "alkyl" or "aryl" then it is understood that the "alkyl" or "aryl" represents a linking alkylene group or arylene group, respectively.

The term "n-membered" where n is an integer typically describes the number of ring- forming atoms in a moiety where the number of ring-forming atoms is n. For example, piperidinyl is an example of a 6-membered heterocycloalkyl ring and 1,2,3,4-tetrahydro- naphthalene is an example of a 10-membered cycloalkyl group.

As used herein, the term "alkyl" is meant to refer to a saturated hydrocarbon group which is straight-chained or branched. Example alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, sec-butyl, t-butyl), pentyl (e.g., n- pentyl, isopentyl, sec-pentyl, neopentyl), and the like. An alkyl group can contain from 1 to about 20, from 2 to about 20, from 1 to about 10, from 1 to about 8, from 1 to about 6, from 1 to about 4, or from 1 to about 3 carbon atoms. A linking alkyl group is referred to herein as "alkylene."

As used herein, "alkenyl" refers to an alkyl group having one or more carbon-carbon double bonds. Example alkenyl groups include ethenyl, propenyl, cyclohexenyl, and the like. An alkenyl group can contain from 2 to about 20, from 3 to about 15, from 2 to about 10, from 2 to about 8, from 2 to about 6, from 2 to about 4, or from 2 to about 3 carbon atoms. A linking alkenyl group is referred to herein as "alkenylene."

As used herein, "alkynyl" refers to an alkyl group having one or more carbon-carbon triple bonds. Example alkynyl groups include ethynyl, propynyl, and the like. An alkynyl group can contain from 2 to about 20, from 3 to about 15, from 2 to about 10, from 2 to about 8, from 2 to about 6, from 2 to about 4, or from 2 to about 3 carbon atoms. A linking alkynyl group is referred to herein as "alkynylene."

As used herein, "haloalkyl" refers to an alkyl group having one or more halogen substituents. Example haloalkyl groups include CF₃, C2F5, CHF₂, CC1₃, CHC1₂, C2CI5, and the like.

As used herein, "halosulfanyl" refers to a sulfur group having one or more halogen substituents. Example halosulfanyl groups include pentahalosulfanyl groups such as SF₅.

As used herein, "aryl" refers to monocyclic or polycyclic (e.g., having 2, 3 or 4 fused rings) aromatic hydrocarbons such as, for example, phenyl, naphthyl, anthracenyl,

phenanthrenyl, indanyl, indenyl, and the like. In some embodiments, aryl groups have from 6 to about 20 carbon atoms, from 6 to about 15 carbon atoms, or from 6 to about 10 carbon atoms. A linking aryl group is referred to herein as "arylene."

As used herein, "cycloalkyl" refers to non-aromatic cyclic hydrocarbons including cyclized alkyl, alkenyl, and alkynyl groups. Cycloalkyl groups can include mono- or polycyclic (e.g., having 2, 3 or 4 fused rings) groups and spirocycles. Ring-forming carbon atoms of a cycloalkyl group can be optionally substituted by oxo or sulfido. Cycloalkyl groups also include cycloalkylidenes. Example cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbornyl, norpinyl, norcarnyl, adamantyl, and the like. Also included in the definition of cycloalkyl are moieties that have one or more aromatic rings fused (i.e., having a bond in common with) to the cycloalkyl ring, for example, benzo or thienyl derivatives of cyclopentane, cyclopentene, cyclohexane, and the like. A cycloalkyl group containing a fused aromatic ring can be attached through any ring-forming atom including a ring-forming atom of the fused aromatic ring. A cycloalkyl group can contain from 3 to about 20, from 3 to about 15, from 3 to about 10, from 3 to about 8, from 3 to about 7, from 3 to about 6, or from 4 to about 7 carbon atoms. A linking cycloalkyl group is referred to herein as "cycloalkylene."

As used herein, "heteroaryl" refers to an aromatic heterocycle having at least one heteroatom ring member such as sulfur, oxygen, or nitrogen. Heteroaryl groups include monocyclic and polycyclic (e.g., having 2, 3 or 4 fused rings) systems. Examples of heteroaryl groups include without limitation, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furyl, quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl, pyrryl, oxazolyl, benzofuryl, benzothienyl, benzthiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1,2,4- thiadiazolyl, isothiazolyl, benzothienyl, purinyl, carbazolyl, benzimidazolyl, indolinyl, and the like. In some embodiments, any ring-forming N in a heteroaryl moiety can be substituted by oxo. In some embodiments, the heteroaryl group has from 1 to about 20 carbon atoms, and in further embodiments from about 3 to about 20 carbon atoms, from about 3 to about 10 carbon atoms, from about 3 to about 5 carbon atoms. In some embodiments, the heteroaryl group contains 3 to about 14, 4 to about 14, 3 to about 7, or 5 to 6 ring-forming atoms. In some embodiments, the heteroaryl group has 1 to about 4, 1 to about 3, or 1 to 2 heteroatoms. A linking heteroaryl group is referred to herein as "heteroarylene."

As used herein, "heterocycloalkyl" refers to non-aromatic heterocycles having one or more ring-forming heteroatoms such as an O, N, or S atom. Heterocycloalkyl groups include monocyclic and polycyclic (e.g., having 2, 3 or 4 fused rings) systems as well as spirocycles. Example "heterocycloalkyl" groups include morpholino, thiomorpholino, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, 2,3-dihydrobenzofuryl, 1,3-benzodioxole, benzo-1,4- dioxane, piperidinyl, pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, imidazolidinyl, and the like. Ring-forming carbon atoms and heteroatoms of a heterocycloalkyl group can be optionally substituted by oxo or sulfido. Also included in the definition of heterocycloalkyl are moieties that have one or more aromatic rings fused (i.e., having a bond in common with) to the nonaromatic heterocyclic ring, for example phthalimidyl, naphthalimidyl, and benzo derivatives of heterocycles. The heterocycloalkyl group can be attached through a ring-forming carbon atom or a ring-forming heteroatom. The heterocycloalkyl group containing a fused aromatic ring can be attached through any ring-forming atom including a ring-forming atom of the fused aromatic ring. In some embodiments, the heterocycloalkyl group has from 1 to about 20 carbon atoms, and in further embodiments from about 3 to about 20 carbon atoms. In some embodiments, the heterocycloalkyl group contains 3 to about 14, 4 to about 14, 3 to about 7, or 5 to 6 ring-forming atoms. In some embodiments, the heterocycloalkyl group has 1 to about 4, 1 to about 3, or 1 to 2 heteroatoms. A heterocycloalkyl group can contain from 1 to about 20, from 1 to about 15, from 1 to about 10, from 1 to about 8, from 1 to about 7, from 1 to about 6, or from 1 to about 5 carbon atoms. In some embodiments, the

heterocycloalkyl group contains 0 to 3 double or triple bonds. In some embodiments, the heterocycloalkyl group contains 0 to 2 double or triple bonds. A linking heterocycloalkyl group is referred to herein as "heterocycloalkylene."

The term "5 to 12-membered heteroaryl group" refers to a monocyclic or bicyclic aromatic heterocycle, wherein the number of atoms constituting the heterocyclic ring is five to twelve, and one or more (for example, one to four) of the heteroatoms that constitute the ring is selected from N, O, and S. Its binding position is not particularly limited, and it can be bound to any desired position. Specific examples include furan, thiophene, pyrrole, oxazole, isoxazole, thiazole, isothiazole, furazan, imidazole, pyran, thiopyran, pyridine, pyrazine, pyrimidine, pyridazine, benzofuran, isobenzofuran, benzothiophene, indole, isoindole, indolizine, chromene, benzopyran, quinoline, isoquinoline, quinolizine, naphthylizine, benzimidazole, indazole, quinoxaline, quinazoline, cinnoline, phthalazine, purine, pteridine, benzoxazole, benzothiazole, and thiadiazole, and the like. "3 to 12-membered heterocycle" includes the above-described "5 to 12-membered heteroaryl group" and refers to an aromatic or non-aromatic heterocycle, wherein the number of atoms constituting the heterocyclic ring is three to twelve, and one or more (for example, one to four) of the heteroatoms that constitute the ring is selected from N, O and S. Its binding position is not particularly limited, and it can be bound to a desired position. Specific examples include pyrrolidine, oxazolidine, isoxazolidine, oxazoline, isoxazoline, thiazoline, isothiazolidine, thiazoline, isothiazoline, imidazolidine, imidazoline, furan, thiophene, pyrrole, oxazole, isoxazole, thiazole, isothiazole, furazan, imidazole, pyrazole, piperidine, piperazine, morpholine, thiomorpholine, tetrahydropyran, dioxane, tetrahydrothiopyran, pyran, thiopyran, pyridine, pyrazine, pyrimidine, pyridazine, benzofuran, isobenzofuran, benzothiophene, indole, isoindole, indolizine, chromene, benzopyran, quinoline, isoquinoline, quinolizine, naphthylizine, benzimidazole, indazole, quinoxaline, quinazoline, cinnoline, phthalazine, purine, pteridine, benzoxazole, benzothiazole, and thiadiazole and the like.

In the present specification, a "ring" may be simply stated to refer to a concept that encompasses all of the above-mentioned "C_3-10 cycloalkyl group", "C₆-i2 aryl group", "5 to 12- membered heteroaryl group", and "3 to 12-membered heterocycle".

As used herein, "halo" or "halogen" includes fluoro, chloro, bromo, and iodo.

As used herein, "arylalkyl" refers to alkyl substituted by aryl and "cycloalkylalkyl" refers to alkyl substituted by cycloalkyl. An example arylalkyl group is benzyl.

As used herein, "heteroarylalkyl" refers to alkyl substituted by heteroaryl and

"heterocycloalkylalkyl" refers to alkyl substituted by heterocycloalkyl.

As used herein, "amino" refers to NH₂.

As used herein, "alkoxy" refers to an— O-alkyl group. Example alkoxy groups include methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), t-butoxy, and the like.

As used herein, "haloalkoxy" refers to an --O-(haloalkyl) group. The compounds described herein, including those conforming to any formula, can be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated. The present compounds that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods on how to prepare optically active forms from optically active starting materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis. Many geometric isomers of olefins, C=N double bonds, and the like can also be present in the compounds described herein, and all such stable isomers are contemplated for the present compounds. Cis and trans geometric isomers of the present compounds are described and may be isolated as a mixture of isomers or as separated isomeric forms.

Compounds of the invention also include tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond together with the concomitant migration of a proton. Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge. Examples of prototropic tautomers include ketone-enol pairs, lactam-lactim pairs, amide-imidic acid pairs, enamine-imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, for example, 1H- and 3H-imidazole, 1H-, 2H- and 4H-l ,2,4-triazole, 1H- and 2H- isoindole, and 1H- and 2H-pyrazole. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.

Compounds of the invention also include all isotopes of atoms occurring in the intermediate or final compounds. Isotopes include those atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium.

The term, "compound," as used herein with respect to any compound conforming to Formula I, is meant to include all stereoisomers, geometric iosomers, tautomers, and isotopes of the structures depicted. All compounds, and pharmaceutically acceptable salts thereof, are also meant to include solvated or hydrated forms.

The compounds of the present invention can be prepared in a variety of ways known to one of ordinary skill in the art of organic synthesis. The compounds of the present invention can be synthesized using the methods as hereinafter described below, together with synthetic methods known in the art of synthetic organic chemistry or variations thereon as appreciated by one of ordinary skill in the art.

The present compounds can be prepared from readily available starting materials using the following general methods and procedures. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvents used, but such conditions can be determined by one of ordinary skill in the art by routine optimization procedures.

Resolution of racemic mixtures of compounds can be carried out by any of numerous methods known in the art. An exemplary method includes fractional recrystallization using a chiral resolving acid which is an optically active, salt-forming organic acid. Suitable resolving agents for fractional recrystallization methods are, for example, optically active acids, such as the D and L forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or the various optically active camphorsulfonic acids such as β-camphorsulfonic acid. Other resolving agents suitable for fractional crystallization methods include

stereoisomerically pure forms of a-methylbenzylamine (e.g., S and R forms, or

diastereomerically pure forms), 2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine, cyclohexylethylamine, 1,2-diaminocyclohexane, and the like.

Resolution of racemic mixtures can also be carried out by elution on a column packed with an optically active resolving agent (e.g., dinitrobenzoylphenylglycine). Suitable elution solvent composition can be determined by one skilled in the art.

In some embodiments, the compounds of the invention, and salts thereof, are

substantially isolated. By "substantially isolated" is meant that the compound is at least partially or substantially separated from the environment in which it was formed or detected. Partial separation can include, for example, a composition enriched in the compound of the invention. Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%>, at least about 90%>, at least about 95%, at least about 97%), or at least about 99% by weight of the compound of the invention, or salt thereof. Methods for isolating compounds and their salts are routine in the art. The expressions, "ambient temperature" and "room temperature," as used herein, are understood in the art, and refer generally to a temperature, e.g. , a reaction temperature, that is about the temperature of the room in which the reaction is carried out, for example, a temperature from about 20 °C to about 30 °C.

The present invention also includes pharmaceutically acceptable salts of the compounds described herein. In general, "pharmaceutically acceptable salts" refer to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form. "Pharmaceutically acceptable" generally encompasses those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts of the present invention include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile (ACN) are preferred. Lists of suitable salts are found in Remington 's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety.

The compounds described herein can be administered directly to a mammal, which we may also refer to as a "subject" or "patient." Generally, the compounds can be suspended in a pharmaceutically acceptable carrier (e.g. , physiological saline or a buffered saline solution) to facilitate their delivery (e.g., by intravenous administration).

As described above, the compounds of the present invention can be prepared in a variety of ways known to one of ordinary skill in the art of chemical synthesis. The present compounds can be prepared from readily available starting materials using the following general methods and procedures. Where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvents used, but such conditions can be determined by one of ordinary skill in the art by routine optimization procedures.

Regardless of their original source or the manner in which they are obtained, the compounds of the invention can be formulated in accordance with their use. For example, the compounds can be formulated within compositions for application to cells in tissue culture or for administration to a patient. When employed as pharmaceuticals, any of the present compounds can be administered in the form of pharmaceutical compositions. These compositions can be prepared in a manner well known in the pharmaceutical art, and can be administered by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including skin, ophthalmic and to mucous membranes including intranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), ocular, oral or parenteral. Methods for ocular delivery can include topical administration (eye drops), subconjunctival, periocular or intravitreal injection or introduction by balloon catheter or ophthalmic inserts surgically placed in the conjunctival sac. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular administration.

Parenteral administration can be in the form of a single bolus dose, or may be, for example, by a continuous perfusion pump. Pharmaceutical compositions and formulations for topical administration may include or exclude transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, powders, and the like. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

This invention also includes pharmaceutical compositions which contain, as the active ingredient, one or more of the compounds described herein in combination with one or more pharmaceutically acceptable carriers. In making the compositions of the invention, the active ingredient is typically mixed with an excipient, diluted by an excipient or enclosed within such a carrier in the form of, for example, a capsule, tablet, sachet, paper, or other container. When the excipient serves as a diluent, it can be a solid, semisolid, or liquid material (e.g. , normal saline), which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10 % by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders. As is known in the art, the type of diluent can vary depending upon the intended route of administration. The resulting compositions can include or exclude additional agents, such as preservatives. The compounds may also be applied to a surface of a device (e.g., a catheter) or contained within a pump, patch, or other drug delivery device. The compounds of the invention can be administered alone, or in a mixture, in the presence of a pharmaceutically acceptable excipient or carrier (e.g.,

physiological saline). The excipient or carrier is selected on the basis of the mode and route of administration. Suitable pharmaceutical carriers, as well as pharmaceutical necessities for use in pharmaceutical formulations, are described in Remington's Pharmaceutical Sciences (E. W. Martin), a well-known reference text in this field, and in the USP/NF (United States

Pharmacopeia and the National Formulary). In preparing a formulation, the active compound can be milled to provide the appropriate particle size prior to combining with the other ingredients. If the active compound is substantially water insoluble, it can be milled to a particle size of less than 200 mesh. If the active compound is substantially water soluble, the particle size can be adjusted by milling to provide a substantially uniform distribution in the formulation, e.g. about 40 mesh. Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. The compositions can include or exclude lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. The formulations can additionally include or exclude: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents. The pharmaceutical compositions can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.

The compositions can be formulated in a unit dosage form, each dosage containing, for example, from about 0.1 mg to about 1000 mg of the active ingredient. The term "unit dosage forms" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention. When referring to these preformulation compositions as homogeneous, the active ingredient is typically dispersed evenly throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation is then subdivided into unit dosage forms of the type described above containing from, for example, 0.1 to about 500 mg of the active ingredient of the present invention.

The tablets or pills of the present invention can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

The liquid forms in which the compounds and compositions of the present invention can be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar

pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described herein and/or known in the art. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions can be nebulized by use of inert gases. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device can be attached to a face mask, tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions can be administered orally or nasally from devices which deliver the formulation in an appropriate manner. The compositions administered to a patient can be in the form of one or more of the pharmaceutical compositions described above. These compositions can be sterilized by conventional sterilization techniques or may be sterile filtered. Aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the compound preparations typically will be between about 3 and 1 1 , for example, between about 5 to 9, between 6 and 7, between 7 and 8. It will be understood that use of certain of the foregoing excipients, carriers, or stabilizers could result in the formation of pharmaceutical salts.

The proportion or concentration of the compounds of the invention in a pharmaceutical composition can vary depending upon a number of factors including dosage, chemical characteristics (e.g., hydrophobicity), and the route of administration. For example, the compounds of the invention can be provided in an aqueous physiological buffer solution containing about 0.1 to about 10% w/v of the compound for parenteral administration.

The therapeutic dosage of the compounds of the present invention can vary according to, for example, the particular use for which the treatment is made, the manner of administration of the compound, the health and condition of the patient, and the judgment of the attending clinician. The proportion or concentration of a compound of the invention in a pharmaceutical composition can vary depending upon a number of factors including dosage, chemical characteristics (e.g., hydrophobicity), and the route of administration. For example, the compounds of the invention can be provided in an aqueous physiological buffer solution containing about 0.1 to about 10% w/v of the compound for parenteral administration. Some typical dose ranges are from about 1 μg/kg to about 1 g/kg of body weight per day. The dosage is likely to depend on such variables as the type and extent of progression of the disease or disorder, the overall health status of the particular patient, the relative biological efficacy of the compound selected, formulation of the excipient, and its route of administration. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

Methods of treatment

The compositions disclosed herein are generally and variously useful for treatment of cancer. A patient is effectively treated whenever a clinically beneficial result ensues. This may mean, for example, a complete resolution of the symptoms of a disease, a decrease in the severity of the symptoms of the disease, or a slowing of the disease's progression. These methods can further include the steps of a) identifying a subject (e.g., a patient and, more specifically, a human patient) who has cancer; and b) providing to the subject a composition comprising a compound described herein, such as any pharmaceutically acceptable salt of such a compound. An amount of such a compound provided to the subject that results in a complete resolution of the symptoms of a disease, a decrease in the severity of the symptoms of the disease, or a slowing of the disease's progression is considered a therapeutically effective amount. The present methods may also include a monitoring step to help optimize dosing and scheduling as well as predict outcome. In some methods of the present invention, one can first determine whether a patient has a cancer expressing certain markers, for example, Mcl-l and Bcl-2, and then make a determination as to whether or not to treat the patient with one or more of the compositions described herein. Monitoring can also be used to detect the onset of drug resistance, to rapidly distinguish responsive patients from nonresponsive patients or to assess recurrence of a cancer. Where there are signs of resistance or nonresponsiveness, a physician can choose an alternative or adjunctive agent before the tumor develops additional escape mechanisms.

Cancers amenable to the therapeutic, and/or prognostic methods of the invention can be cancers that are responsive to the modulation of Mcl-l . While we believe we understand certain events that occur in the course of treatment, the compositions of the present invention are not limited to those that work by affecting any particular cellular mechanism. Any form of cancer which is associated with misregulation of Mcl-l (e.g., overexpression or altered binding or activity) is within the scope of the invention. Such cancers can include, but are not limited to, hematologic malignancies, for example, lymphoma, leukemia, multiple myeloma, melanoma, pancreatic cancer, lung cancer, breast cancer, and liver cancer. Patients amenable to treatment include patients with any of a wide variety of cancers or neoplastic disorders, including, for example, without limitation, breast cancer, hematological cancers such as myeloma, leukemia and lymphoma (e.g. , Burkitt lymphoma, non-Hodgkin lymphoma, Hodgkin lymphoma, and acute T cell leukemia), neurological tumors such as brain tumors, e.g., gliomas, including astrocytomas or glioblastomas, melanomas, lung cancer, head and neck cancer, thyroid cancer, gastrointestinal tumors such as stomach, colon or rectal cancer, liver cancer, pancreatic cancer, genitourinary tumors such ovarian cancer, vaginal cancer, vulval cancer, endometrial cancer, bladder cancer, kidney cancer, testicular cancer, prostate cancer, or penile cancer, bone tumors, vascular tumors, and skin cancers such as basal cell carcinoma, squamous cell carcinoma and melanoma.

The methods disclosed herein can be applied to a wide range of species, e.g., humans, non-human primates (e.g., monkeys), horses or other livestock, dogs, cats, ferrets or other mammals kept as pets, rats, mice, or other laboratory animals. The methods of the invention can be expressed in terms of the preparation of a medicament. Accordingly, the invention encompasses the use of the agents and compositions described herein in the preparation of a medicament. The compounds described herein are useful in therapeutic compositions and regimens or for the manufacture of a medicament for use in treatment of diseases or conditions as described herein (e.g., a cancer disclosed herein).

Any composition described herein can be administered to any part of the host's body for subsequent delivery to a target cell. A composition can be delivered to, without limitation, the brain, the cerebrospinal fluid, joints, nasal mucosa, blood, lungs, intestines, muscle tissues, skin, or the peritoneal cavity of a mammal. In terms of routes of delivery, a composition can be administered by intravenous, intracranial, intraperitoneal, intramuscular, subcutaneous, mtramuscular, intrarectal, intravaginal, intrathecal, intratracheal, intradermal, or transdermal injection, by oral or nasal administration, or by gradual perfusion over time. In a further example, an aerosol preparation of a composition can be given to a host by inhalation.

The dosage required will depend on the route of administration, the nature of the formulation, the nature of the patient's illness, the patient's size, weight, surface area, age, and sex, other drugs being administered, and the judgment of the attending clinicians. Suitable dosages are in the range of 0.01-1,000 mg/kg. Wide variations in the needed dosage are to be expected in view of the variety of cellular targets and the differing efficiencies of various routes of administration. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art. Administrations can be single or multiple (e.g., 2- or 3-, 4-, 6-, 8-, 10-, 20-, 50-, 100-, 150-, or more fold). Encapsulation of the compounds in a suitable delivery vehicle (e.g., polymeric microparticles or implantable devices) may increase the efficiency of delivery.

The duration of treatment with any composition provided herein can be any length of time from as short as one day to as long as the life span of the host (e.g., many years). For example, a compound can be administered once a week (for, for example, 4 weeks to many months or years); once a month (for, for example, three to twelve months or for many years); or once a year for a period of 5 years, ten years, or longer. It is also noted that the frequency of treatment can be variable. For example, the present compounds can be administered once (or twice, three times, etc.) daily, weekly, monthly, or yearly.

An effective amount of any composition provided herein can be administered to an individual in need of treatment. The term "effective" as used herein refers to any amount that induces a desired response while not inducing significant toxicity in the patient. Such an amount can be determined by assessing a patient's response after administration of a known amount of a particular composition. In addition, the level of toxicity, if any, can be determined by assessing a patient's clinical symptoms before and after administering a known amount of a particular composition. It is noted that the effective amount of a particular composition administered to a patient can be adjusted according to a desired outcome as well as the patient's response and level of toxicity. Significant toxicity can vary for each particular patient and depends on multiple factors including, without limitation, the patient's disease state, age, and tolerance to side effects.

Any method known to those in the art can be used to determine if a particular response is induced. Clinical methods that can assess the degree of a particular disease state can be used to determine if a response is induced. The particular methods used to evaluate a response will depend upon the nature of the patient's disorder, the patient's age, and sex, other drugs being administered, and the judgment of the attending clinician.

The compositions may also be administered with another therapeutic agent, such as a cytotoxic agent, or cancer chemotherapeutic. Concurrent administration of two or more therapeutic agents does not require that the agents be administered at the same time or by the same route, as long as there is an overlap in the time period during which the agents are exerting their therapeutic effect. Simultaneous or sequential administration is contemplated, as is administration on different days or weeks.

Other useful therapeutics that may be administered in combination with the present compositions include agents that target other members of the Bcl-2 family. An anti-Bcl-2 agent can be any agent that modulates the activity of Bcl-2. There are many ways in which Bcl-2 can be inhibited. For example, a Bcl-2 inhibitor may inhibit Bcl-2 activity by inhibiting the binding of Bcl-2 to a BH-3 only polypeptide or it may modulate the level of Bcl-2 polypeptide by selectively decreasing Bcl-2 transcription, translation, post-translational processing or proteasomal degradation. Anti-Bcl-2 agents can include anti-Bcl-2 oligonucleotides, anti-Bcl-2 antibodies and small molecule inhibitors. Exemplary small molecule inhibitors include gossypol and gossypol analogues (e.g., AT-101); the benzenesulfonyl derivative, TW37; the apogossypol derivative, Sabutoclax; the ABT series of compounds including ABT-199, ABT-737 and the orally available analog, ABT-263; obatoclax; and HA14-1.

The pharmaceutical compositions may also include or be administered along with a cytotoxic agent, e.g., a substance that inhibits or prevents the function of cells and/or causes destruction of cells. Exemplary cytotoxic agents include radioactive isotopes {e.g., ¹³¹I, ¹²⁵I, ⁹⁰Y and ¹⁸⁶Re), chemotherapeutic agents, and toxins such as enzymatically active toxins of bacterial, fungal, plant or animal origin or synthetic toxins, or fragments thereof. A non-cytotoxic agent refers to a substance that does not inhibit or prevent the function of cells and/or does not cause destruction of cells. A non-cytotoxic agent may include an agent that can be activated to be cytotoxic. A non-cytotoxic agent may include a bead, liposome, matrix or particle (see, e.g., U.S. Patent Publications 2003/0028071 and 2003/0032995 which are incorporated by reference herein). Such agents may be conjugated, coupled, linked or associated with an antibody disclosed herein.

Conventional cancer medicaments can be administered with the compositions disclosed herein. Useful medicaments include anti-angiogenic agents, i.e., agents that block the ability of tumors to stimulate new blood vessel growth necessary for their survival. Any anti-angiogenic agent known to those in the art can be used, including agents such as Bevacizumab (Avastin®, Genentech, Inc.) that block the function of vascular endothelial growth factor (VEGF). Other examples include, without limitation, Dalteparin (Fragmin®), Suramin ABT-510,

Combretastatin A4 Phosphate, Lenalidomide, LY317615 (Enzastaurin), Soy Isoflavone

(Genistein; Soy Protein Isolate) AMG-706, Anti-VEGF antibody, AZD2171, Bay 43-9006 (Sorafenib tosylate), PI-88, PTK787/ZK 222584 (Vatalanib), SU11248 (Sunitinib malate), VEGF-Trap, XL 184, ZD6474, Thalidomide, ATN-161, EMD 121974 (Cilenigtide) and

Celecoxib (Celebrex®). Other useful therapeutics include those agents that promote DNA-damage, e.g., double stranded breaks in cellular DNA, in cancer cells. Any form of DNA-damaging agent know to those of skill in the art can be used. DNA damage can typically be produced by radiation therapy and/or chemotherapy. Examples of radiation therapy include, without limitation, external radiation therapy and internal radiation therapy (also called brachytherapy). Energy sources for external radiation therapy include x-rays, gamma rays and particle beams; energy sources used in internal radiation include radioactive iodine (iodine¹²⁵ or iodine¹³¹), and from strontium⁸⁹, or radioisotopes of phosphorous, palladium, cesium, iridium, phosphate, or cobalt. Methods of administering radiation therapy are well known to those of skill in the art.

Examples of DNA-damaging chemotherapeutic agents include, without limitation, Busulfan (Myleran), Carboplatin (Paraplatin), Carmustine (BCNU), Chlorambucil (Leukeran), Cisplatin (Platinol), Cyclophosphamide (Cytoxan, Neosar), Dacarbazine (DTIC-Dome),

Ifosfamide (Ifex), Lomustine (CCNU), Mechlorethamine (nitrogen mustard, Mustargen), Melphalan (Alkeran), and Procarbazine (Matulane).

Other standard cancer chemotherapeutic agents include, without limitation, alkylating agents, such as carboplatin and cisplatin; nitrogen mustard alkylating agents; nitrosourea alkylating agents, such as carmustine (BCNU); antimetabolites, such as methotrexate; folic acid; purine analog antimetabolites, mercaptopurine; pyrimidine analog antimetabolites, such as fluorouracil (5-FU) and gemcitabine (Gemzar®); hormonal antineoplastics, such as goserelin, leuprolide, and tamoxifen; natural antineoplastics, such as aldesleukin, interleukin-2, docetaxel, etoposide (VP- 16), interferon alfa, paclitaxel (Taxol®), and tretinoin (ATRA); antibiotic natural antineoplastics, such as bleomycin, dactinomycin, daunorubicin, doxorubicin, daunomycin and mitomycins including mitomycin C; and vinca alkaloid natural antineoplastics, such as vinblastine, vincristine, vindesine; hydroxyurea; aceglatone, adriamycin, ifosfamide, enocitabine, epitiostanol, aclarubicin, ancitabine, nimustine, procarbazine hydrochloride, carboquone, carboplatin, carmofur, chromomycin A3, antitumor polysaccharides, antitumor platelet factors, cyclophosphamide (Cytoxin®), Schizophyllan, cytarabine (cytosine arabinoside), dacarbazine, thioinosine, thiotepa, tegafur, dolastatins, dolastatin analogs such as auristatin, CPT-11

(irinotecan), mitozantrone, vinorelbine, teniposide, aminopterin, carminomycin, esperamicins (See, e.g., U.S. Patent No. 4,675,187), neocarzinostatin, OK-432, bleomycin, furtulon, broxuridine, busulfan, honvan, peplomycin, bestatin (Ubenimex®), interferon-β, mepitiostane, mitobronitol, melphalan, laminin peptides, lentinan, Coriolus versicolor extract, tegafur/uracil, estramustine (estrogen/mechlorethamine) .

Additional agents which may be used as therapy for cancer patients include EPO, G-CSF, ganciclovir; antibiotics, leuprolide; meperidine; zidovudine (AZT); interleukins 1 through 18, including mutants and analogues; interferons or cytokines, such as interferons α, β, and γ hormones, such as luteinizing hormone releasing hormone (LHRH) and analogues and, gonadotropin releasing hormone (GnRH); growth factors, such as transforming growth factor- β (TGF-β), fibroblast growth factor (FGF), nerve growth factor (NGF), growth hormone releasing factor (GHRF), epidermal growth factor (EGF), fibroblast growth factor homologous factor (FGFHF), hepatocyte growth factor (HGF), and insulin growth factor (IGF); tumor necrosis factor-a & β (TNF-a & β); invasion inhibiting factor-2 (IIF-2); bone morphogenetic proteins 1-7 (BMP 1-7); somatostatin; thymosin-a-1; γ-globulin; superoxide dismutase (SOD); complement factors; and anti-angiogenesis factors.

Useful therapeutic agents include, produgs, e.g., precursors or derivative forms of a pharmaceutically active substance that is less cytotoxic or non-cytotoxic to tumor cells compared to the parent drug and is capable of being converted, either enzymatically or non-enzymatically, into an active or the more active parent form. See, e.g., Wilman, "Prodrugs in Cancer

Chemotherapy" Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press (1985). Prodrugs include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate - containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs,

glycosylated prodrugs, β-lactam-containing prodrugs, optionally substituted phenoxyacetamide- containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5- fluorocytosine and other 5-fluorouridine prodrugs which can be converted into the more active cytotoxic free drug. Examples of cytotoxic drugs that can be derivatized into a prodrug form for use herein include, but are not limited to, those chemotherapeutic agents described above. The pharmaceutical compositions can also include other therapeutic antibodies, e.g. , antibodies that recognize additional cellular targets. Exemplary immunoglobulins are listed below. Each immunoglobulin is identified by its proper name and its trade name. Numbers in parenthesis beginning with "DB" refer to the identifiers for each antibody on The DrugBank database available at the University of Alberta. The DrugBank database is described in Wishart D S, Knox C, Guo A C, et al. (2008). "DrugBank: a knowledgebase for drugs, drug actions and drug targets". Nucleic Acids Res. 36 (Database issue): D901-6 and can be accessed at www.drugbank.ca. Useful immunoglobulins include: Abciximab (ReoPro™) (DB00054), the Fab fragment of the chimeric human-murine monoclonal antibody 7E3, the synthesis of which is described in EP0418316 (Al) and W08911538 (Al), which are herein incorporated by reference; Adalimumab (Humira™) (DB00051), a fully human monoclonal antibody that binds to Tumor Necrosis Factor alpha (TNF-a) and blocks TNF-a binding to its cognate receptor; alemtuzumab (Campath™) (DB00087), a humanized monoclonal antibody that targets CD52, a protein present on the surface of mature lymphocytes, used in the treatment of chronic lymphocytic leukemia (CLL), cutaneous T cell lymphoma (CTCL) and T-cell lymphoma;

basiliximab (Simulect™) (DB00074), a chimeric mouse-human monoclonal antibody to the a chain (CD25) of the IL-2 receptor; bevacizumab (Avastin™) (DB00112) a humanized monoclonal antibody that recognizes and blocks vascular endothelial growth factor (VEGF), the chemical signal that stimulates angiogenesis, the synthesis of which is described in Presta L G, Chen H, O'Connor S J, et al. (1997) "Humanization of an anti-vascular endothelial growth factor monoclonal antibody for the therapy of solid tumors and other disorders." Cancer Res, 57: 4593- 9, 1997; certuximab (Erbitux.TM.) (DB00002), a chimeric (mouse/human) monoclonal antibody that binds to and inhibits the epidermal growth factor receptor (EGFR), the synthesis of which is described in U.S. Pat. No. 6,217,866, which is herein incorporated by reference; certolizumab pegol (Cimzia™), a PEGylated Fab' fragment of a humanized TNF-a inhibitor monoclonal antibody; daclizumab (Zenapax.™) (DB00111), a humanized monoclonal antibody to the alpha subunit of the IL-2 receptor; eculizumab (Soliris™), a humanized monoclonal antibody that binds to the human C5 complement protein; efalizumab (Raptiva™) (DB00095), a humanized monoclonal antibody that binds to CD1 la; gemtuzumab (Mylotarg™) (DB00056) a monoclonal antibody to CD33 linked to a cytotoxic agent, the amino acid sequence of which is described in J Immunol 148: 1149, 1991) (Caron P C, Schwartz M A, Co M S, Queen C, Finn R D, Graham M C, Divgi C R, Larson S M, Scheinberg D A. "Murine and humanized constructs of monoclonal antibody M195 (anti-CD33) for the therapy of acute myelogenous leukemia." Cancer. 1994 Feb. 1; 73(3 Suppl): 1049-56); ibritumomab tiuxetan (Zevalin™) (DB00078), a monoclonal mouse IgGl antibody ibritumomab in conjunction with the chelator tiuxetan and a radioactive isotope (yttrium⁹⁰ or indium¹¹¹); Infliximab (Remicade™) (DB00065), a chimeric mouse-human monoclonal antibody that binds to tumor necrosis factor alpha (TNF-a), the synthesis of which is described in U.S. Pat. No. 6,015,557, which is herein incorporated by reference; muromonab- CD3 (Orthoclone OKT3™), a mouse monoclonal IgG2a antibody that binds to the T cell receptor-CD3 -complex; natalizumab (Tysabri™) (DB00108), a humanized monoclonal antibody against the cellular adhesion molecule a4-integrin, the sequence of which is described in Leger O J, Yednock T A, Tanner L, Horner H C, Hines D K, Keen S, Saldanha J, Jones S T, Fritz L C, Bendig M M. "Humanization of a mouse antibody against human a4-integrin: a potential therapeutic for the treatment of multiple sclerosis." Hum. Antibodies. 1997; 8(1):3-16;

omalizumab (Xolair™) (DB00043), a humanized IgGlk monoclonal antibody that selectively binds to human immunoglobulin E (IgE); palivizumab (Synagis™) (DB00110), a humanized monoclonal antibody (IgG) directed against an epitope in the A antigenic site of the F protein of the Respiratory Syncytial Virus (RSV), the amino acid sequence of which is described in

Johnson S, Oliver C, Prince G A, Hemming V G, Pfarr D S, Wang S C, Dormitzer M, O'Grady J, Koenig S, Tamura J K, Woods R, Bansal G, Couchenour D, Tsao E, Hall W C, Young J F.

"Development of a humanized monoclonal antibody (MEDI-493) with potent in vitro and in vivo activity against respiratory syncytial virus." J. Infect Dis. 1997 November; 176(5): 1215-24; panitumumab (Vectibix™), a fully human monoclonal antibody specific to the epidermal growth factor receptor (also known as EGF receptor, EGFR, ErbB-1 and HER1 in humans); ranibizumab (Lucentis™), an affinity matured anti-VEGF-A monoclonal antibody fragment derived from the same parent murine antibody as bevacizumab (Avastin); rituximab (Rituxan™, Mabthera™) (DB00073), a chimeric monoclonal antibody against the protein CD20, which is primarily found on the surface of B cells; tositumomab (Bexxar™) (DB00081), an anti-CD20 mouse monoclonal antibody covalently bound to ¹³¹\; or trastuzumab (Herceptin™) (DB00072), a humanized monoclonal antibody that binds selectively to the HER2 protein. The antibodies can include bioequivalents of the approved or marketed antibodies (biosimilars). A biosimilar can be for example, a presently known antibody having the same primary amino acid sequence as a marketed antibody, but may be made in different cell types or by different production, purification or formulation methods. Generally, any deposited materials can be used.

Articles of Manufacture

The compositions described herein can be packaged in suitable containers labeled, for example, for use as a therapy to treat a disease or disorder of cell proliferation {e.g., cancer). The containers can include the compound and one or more of a suitable stabilizer, carrier molecule, flavoring, and/or the like, as appropriate for the intended use. Accordingly, packaged products {e.g. , sterile containers containing one or more of the compositions described herein and packaged for storage, shipment, or sale at concentrated or ready-to-use concentrations) and kits, including at least one composition of the invention, e.g., a compound conforming to Formula I, for example, KS01, KS02, KS03, KS04, KS05, KS06, KS07, KS08, KS09, KS11, KS12, KS13, KS14, KS17, KS18, KS19, KS20; a compound conforming to Formula II, for example, KS15 or KS16; a compound conforming to Formula III, for example KAOl, KA02, KA03; or KS10, or a pharmaceutically acceptable salt thereof and instructions for use, are also within the scope of the invention. A product can include a container {e.g., a vial, jar, bottle, bag, or the like) containing one or more compositions of the invention. In addition, an article of manufacture further may include, for example, packaging materials, instructions for use, syringes, buffers or other control reagents for treating or monitoring the condition for which prophylaxis or treatment is required.

In some embodiments, the kits can include one or more additional chemotherapeutic agents, for example, an anti-Bcl-2 agent such as ABT-199, ABT-737 or ABT-263. The additional agents can be packaged together in the same container as the a compound conforming to Formula I, for example, KS01, KS02, KS03, KS04, KS05, KS06, KS07, KS08, KS09, KS11,

KS12, KS13, KS14, KS17, KS18, KS19, KS20; a compound conforming to Formula II, for example, KS15 or KS16; or a compound conforming to Formula III, for example KAOl, KA02,

KA03; or KS10, or a pharmaceutically acceptable salt thereof or they can be packaged separately. The compound conforming to Formula I, for example, KS01, KS02, KS04, KS05,

KS06, KS07, KS08, KS09, KS11, KS12, KS13, KS14, KS17, KS18, KS19, KS20; the compound conforming to Formula II, for example, KS15 or KS16; the compound conforming to Formula III, for example KA01, KA02, KA03; or KS10, and the additional agent may be combined just before use or administered separately.

The product may also include a legend (e.g., a printed label or insert or other medium describing the product's use (e.g., an audio- or videotape)). The legend can be associated with the container (e.g., affixed to the container) and can describe the manner in which the compositions therein should be administered (e.g., the frequency and route of administration), indications therefor, and other uses. The compositions can be ready for administration (e.g., present in dose-appropriate units), and may include one or more additional pharmaceutically acceptable adjuvants, carriers or other diluents and/or an additional therapeutic agent.

Alternatively, the compositions can be provided in a concentrated form with a diluent and instructions for dilution.

EXAMPLES

Example 1: Synthesis of Pyoluteorin Derivatives

The structures of pyoluteorin derivatives (KS01-KS20) are shown in the Figure 1. Pyoluteorin derivatives were synthesized by using previously described methods (Petruso, S., etal. J. Heterocycl. Chem. 1994, 31, 941) with minor modifications as shown in Schemes 1-5.

Compounds KS03, KS04, KS05, KS06, KS10, KS11, KS12, KS14, KS17, KS18, KS19 and KS20 were synthesized as shown in the Scheme 1. Briefly, to a solution of ethyl pyrrole-2- carboxylate (1) in anhydrous THF, N,O-dimethylhydroxylamine hydrochloride was added, cooled to -78 °C and then lithium bis(trimethylsilyl)amide solution was added slowly and stirred for lh. The reaction was quenched by addition of NH₄C1 solution, extracted with ethyl acetate, concentrated and purified by silica gel column chromatography to yield 2. To a solution of 2 in dichlromethane, SO₂CI₂ (2 equiv) was added slowly to obtain dichloro pyrrole derivative 3. To a solution of 3 in dichloroethane, NBS was added and refluxed for 3h, concentrated and purified by silica gel column chromatography to get 3-Bromo-4,5-dichloro-lH-pyrrole-2-carboxylic acid methoxy methyl amide 4 (KS10) as a white solid. To a solution of 4 in anhydrous THF, 2- methoxyphenylmagnesium bromide was added at 0 °C. The reaction was stirred for 1 hour, quenched with saturated aqueous solution of NH₄C1, concentrated and purified by silica gel column chromatography to afford (3-Bromo-4,5-dichloro-lH-pyrrol-2-yl)(2- methoxyphenyl)methanone 5 (KS03) as an off white solid. To a solution of 5 in CH₂CI₂ at 0 °C, a solution of BBr₃ (2 eq.) was added and the mixture was allowed to stir at 0 °C for 3 hours. The reaction was quenched by saturated aqueous NaHC0₃ and extracted with CH₂CI₂. The combined organics were dried, filtered, and concentrated. The residue was purified by flash column chromatography to afford (3-Bromo-4,5-dichloro-lH-pyrrol-2-yl)(2-hydroxyphenyl)methanone 6 (KS04) as a brown solid. To a solution of 6 in CH₂C1₂, Ac₂0, NEt₃ and DMAP were added, and the resulting solution was stirred at 25 °C for 12 hours followed by column chromatography to get 2-(3-bromo-4,5-dichloro-lH-pyrrole-2-carbonyl)phenyl acetate 7 (KS17). Compound 8 (KS05) (l,3-Dibromo-4,5-dichloro-lH-pyrrol-2-yl)(2-methoxyphenyl)methanone was obtained from 5 using 2 equivalents of NBS under similar experimental conditions used for 4. Cleavage of the methyl ether of 8, was achieved by treatment with BBr₃ in CH₂CI₂, and the residue was purified by silica gel column chromatography to afford phenolic derivative (l,3-Dibromo-4,5- dichloro-lH-pyrrol-2-yl)(2-hydroxyphenyl)methanone 9 (KS06) as a brown solid. Compound 11 (3-Bromo-4,5-dichloro-lH-pyrrol-2-yl)(5-chloro-2-hydroxyphenyl)methanone (KS18) was obtained using similar experimental conditions used for 6, by treating 4 in anhydrous THF with 2-methoxy-4-chloro-phenylmagnesium bromide and followed by BBr₃ treatment in CH₂CI₂, and the residue was purified by silica gel column chromatography. To a solution of 11 in CH₂C1₂, Ac₂0, NEt₃ and DMAP were added, and the resulting solution was stirred at 25 °C for 12 hours followed by column chromatography to get 2-(3-bromo-4,5-dichloro-lH-pyrrole-2-carbonyl)-4- chlorophenyl acetate 12 (KS19). To a solution of 4 in anhydrous THF, 2,3,4

trimethoxyphenylmagnesium bromide was added at 0 °C. The reaction was stirred for 1 hour, quenched with saturated aqueous solution of NH₄C1, concentrated and purified by silica gel column chromatography to afford (3-Bromo-4,5-dichloro-lH-pyrrol-2-yl)(2,3,4- trimethoxyphenyl)methanone 13 (KS11) as an off white solid. Cleavage of the methyl ether of 13, was achieved by treatment with BBr₃ in CH₂C1₂, and the residue was purified by silica gel column chromatography to afford 14 (KS12) as a brown solid. Compound 15 and (3-Bromo-4,5- dichloro-lH-pyrrol-2-yl)-(2,4-dihydroxyphenyl)methanone 16 (KS14) were obtained by treating 2,4-dimethoxy phenylmagnesium bromide with 4 in anhydrous THF, followed by BBr₃ treatment in CH₂C1₂, and the residue was purified by silica gel column chromatography. Compounds 17 and 18 (3-Bromo-4,5-dichloro-lH-pyrrol-2-yl)(5-fluoro-2-hydroxyphenyl)methanone (KS20) were obtained using similar experimental conditions used for 6, by treating 4 in anhydrous THF with 2-methoxy-4-fluoro-phenylmagnesium bromide and followed by BBr₃ treatment in CH₂C1₂, and the residue was urified by silica gel column chromatography.

Compounds KS01, KS02, KS07 and KS09 were synthesized as shown in the schemes 2. Briefly, compound 3 upon treatment with 2,3,4-trimethoxyphenylmagnesium bromide in anhydrous THF was converted into (4,5-Dichloro-lH-pyrrol-2-yl)(2,3,4-trimethoxy- phenyl)methanone 19 (KS07) as a white solid. Cleavage of the methyl ether of 19 was achieved by treatment with BBr₃ in CH₂C1₂, and the residue was purified by silica gel column

chromatography to afford phenolic derivative (4,5-dichloro-lH-pyrrol-2-yl)(2,3,4- trihydroxyphenyl)methanone 20 (KS09) as a white solid. Compound 21 (4,5-Dichloro-lH- pyrrol-2-yl)(2-methoxyphenyl)methanone (KS01) was obtained using similar experimental conditions used for 6, by treating 3 in anhydrous THF with 2-methoxyphenylmagnesium bromide and followed by BBr₃ treatment of 21 to afford (4,5-Dichloro-lH-pyrrol-2-yl)(2- hydroxyphenyl)methanone 22 (KS02).

(4,5-Dichloro-lH-pyrrol-2-yl)(2-hydroxy-3,4-dimethoxyphenyl)methanone 25 (KS08) was synthesized as shown in the Scheme 3. 2,3,4-trimethoxybenzoyl chloride was stirred with A1C1₃ in methylene chloride and pyrrole was added at 0 °C. The reaction mixture was stirred overnight at room temperature and the reaction was quenched by saturated NaHC0₃ solution, filtered through Celite, and concentrated. The residue was chromatographed on silica gel to get yellow solid 24. Compound 24 was treated with SO₂CI₂ (2 equiv) in dichlromethane to get 25 (KS08) as a yellow solid.

Compound (4,5-dichloro-3-methyl-lH-pyrrol-2-yl)(2-hydroxyphenyl)methanone 30 (KS13) was synthesized as shown in the Scheme 4. Briefly, to a solution of 3 -Methyl-ethyl pyrrole-2-carboxylate 26 in anhydrous THF, N,O-dimethylhydroxylamine hydrochloride was added, cooled to -78 °C and then lithium bis(trimethylsilyl)amide solution was added slowly and stirred for 1 hour. The reaction was quenched by addition of NH₄C1 solution, extracted with ethyl acetate, concentrated and purified by silica gel column chromatography to yield 27. To a solution of 27 in dichlromethane, SO₂CI₂ (2 equiv) was added slowly to obtain dichloro pyrrole derivative 28 as a white solid. Compounds 29 and (4,5-Dichloro-3-methyl-lH-pyrrol-2-yl)(2- hydroxyphenyl)methanone 30 (KS13) was obtained using similar experimental conditions used for 6, by treating 28 in anhydrous THF with 2-methoxyphenylmagnesium bromide and followed by BBr₃ treatment in CH₂CI₂, and the residue was purified by silica gel column chromatography.

The indole derivatives KS15 and KS16 were synthesized as shown in the Scheme 5. Briefly, to a solution of ethyl indole-2-carboxylate (31) in anhydrous THF, N,0- dimethylhydroxylamine hydrochloride was added, cooled to -78 °C and then lithium

bis(trimethylsilyl)amide solution was added slowly and stirred for 1 hour. The reaction was quenched by addition of NH₄C1 solution, extracted with ethyl acetate, concentrated and purified by silica gel column chromatography to yield 32. To a solution of 32 in acetonitrile, NBS was added and stirred for 6 hours, concentrated and purified by silica gel column chromatography to get 3-Bromoindole-2-carboxylic acid methoxy methyl amide 33 as a white solid. To a solution of 33 in anhydrous THF, 2- methoxyphenylmagnesium bromide was added at 0 °C. The reaction was stirred for lh, quenched with saturated aqueous solution of NH₄C1, concentrated and purified by silica gel column chromatography to afford (3-Bromoindole-2-yl)(2- methoxyphenyl)methanone 34 as an off white solid. To a solution of 34 in CH₂CI₂ at 0 °C, a solution of BBr₃ (2 eq.) was added and the mixture was allowed to stir at 0 °C for 5 hours. The reaction was quenched by saturated aqueous NaHC0₃ and extracted with CH₂CI₂. The combined organics were dried, filtered, and concentrated. The residue was purified by flash column chromatography to afford (3-Bromoindole-2-yl)(2-hydroxyphenyl)methanone 35 (KS15) as a yellow solid. Compounds 36 and 37 (3-Bromoindol-2-yl)(2,4-dihydroxyphenyl)methanone (KS16) were obtained using similar experimental conditions used for 35, by treating 33 in anhydrous THF with 2,4-dimethoxyphenylmagnesium bromide and followed by BBr₃ treatment in CH₂CI₂, and the residue was purified by silica gel column chromatography. The purity of all the compounds (KS01-KS20) was determined by analytical HPLC. The structure of the compounds was characterized on the basis of its NMR and Mass spectra. The purity level was >99% for the final product after chromatography.

Reagents and Conditions: a) LiHMDS, CH₃NHOCH₃,THF, -78 °C; b) NBS, CH₃CN, RT; c) 2-met oxyp enylmagnesium bromide, THF, 0 °C; d) BBr₃, CH₂CI₂, 0 °C; e) 2,4-dimethoxyphenylmagnesium bromide, THF, 0 °C.

Example 2: Materials and Methods

Antibodies and Compounds. Antibodies were obtained from the following sources: mouse monoclonal anti-Mcl-1 (BD Pharmingen #559027); mouse monoclonal anti-Bcl-XL (Sigma #B9429); rabbit polyclonal anti-Bim (Sigma #B7929); rabbit polyclonal anti-caspase-3

(Krajewska, M., Wang, H. G., Krajewski, S., Zapata, J. M., Shabaik, A., Gascoyne, R., and Reed, J. C. (1997) Cancer Research 57, 1605-1613), rabbit polyclonal anti-PARP (Cell

Signaling #9542); mouse monoclonal anti-GAPDH (Imgenex #5019A); mouse monoclonal anti- Noxa (Pierce #MA 1-41000); ABT-737 was obtained from Abbott Laboratories as described (Woods, N. T., Yamaguchi, H., Lee, F. Y., Bhalla, K. N., and Wang, H. G. (2007) Cancer Research 67, 10744-10752). MG132 was purchased from Sigma rabbit polyclonal anti-Bcl-2 (Krajewski S., Bodrug S., Gascoyne R., Berean K., Krejewska M. and Reed J.C. (1994) Am. J. Pathol. 145, 515-525).

Cell Culture and Transfection. K562, HL60/VCR, HL60/ABT, U937, RPMI8226, NCIH929 and Jurkat, and JurkatABak cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin B (Cellgro) at 37°C and 5% C0₂.

K562 cells stably expressing Bcl-2-IRES-BimEL or Mcl-l-IRES-BimEL were generated by retroviral transduction and puromycin selection as previously described (Woods, N. T.,

Yamaguchi, H., Lee, F. Y., Bhalla, K. N., and Wang, H. G. (2007) Cancer Research 67, 10744- 10752). Cell Viability Assay. Cell viability was determined by measuring intracellular ATP levels with the CellTiter Glo Luminescent Cell Viability Assay kit (Promega #G7571). EC50 values were calculated by non-linear regression analysis using SigmaPlot.

Example 3: Effect of Maritoclax and Maritoclax Prodrugs on Viability of MCL-1- dependent Leukemia Cells

The effect of maritoclax and maritoclax prodrugs on viability of Mcl-l-IRES-Bim K562 cells was determined by treating cells with increasing concentrations of compounds for 48 hours and then assaying cell viability using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). As shown in Table 1, maritoclax and the maritoclax prodrugs, KAOl, KA02, KA02, and KA03, reduced the viability of Mcl-l-IRES-Bim K562 cells, with EC₅₀'s of 0.99, 1.34, 1.35, and 1.35 μΜ, respectively.

Table 1: Maritoclax and Maritoclax Prodrugs

Example 4: Effect of Pyoluteorin Derivatives on Viability of MCL-l-dependent Leukemia

Cells

The effect of pyoluteorin derivatives on viability of Mcl-l-IRES-Bim K562 cells was determined by treating cells with increasing concentrations of compounds for 48 hours and then assaying cell viability using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). The results of this experiment are shown in Table 2. The EC50 (μΜ) values for KS18 and its prodrug, KS19, and KS04 and its prodrug, KS17, exceeded or were comparable to those obtained for maritoclax.

Table 2: Pyoluteorin derivatives

Example 5: Effect of Pyoluteorin Derivatives on Viability of Hematopoetic Cell Lines

Cells were treated with various concentrations of maritoclax, KA01, KA02, KS04, KS17, KS18, KS19, KS20, ABT-737 or daunorubicin for 48 hours. Cell viability was determined by measuring intracellular ATP levels with the CellTiter-Glo Luminescent Cell Viability Assay or by the MTS assay. EC50 (μΜ) values were calculated by non-linear regression analysis using SigmaPlot. Maritoclax and its derivatives selectively induced cell death in Mcl-l -dependent but not Bcl-2-dependent human K562 myeloid leukemia cells. ABT-737 resistant malignant hematopoietic cell lines (HL60/VCR, HL60/ABTR, U937, NCI-H929), which have high levels of Mcl-l, were sensitive to maritoclax and its derivatives. JurkatABak, a subclone of Jurkat human acute T cell leukemia cell line that constitutively lacks Bax and Bak, was much less sensitive to maritoclax and its derivatives than was the parental Jurkat cell line. These results suggested that maritoclax and derivatives kill Mcl-l -overexpressing ABT-737 resistant cancer cells through a Bax/Bak-dependent process. Table 3: Comparison of EC50 values

Example 6: Effect of Pyoluteorin Derivatives on Resistance of AML cells to ABT-737

KG1 and KGla cells and their ABT-373 resistant derivatives, KG1/ABT and

KGla/ABT, were treated with increasing concentrations of ABT-737 alone or in combination with 1.6 μΜ KS04 for 48 hours. Cell viability was determined by measuring intracellular ATP levels with the CellTiter Glo assay. EC50 (μΜ) values are shown in Table 4. The efficacy of ABT-737 in KG1/ABT and KGla/ABT cells was fully restored to the levels observed in respective parental cell lines when combined with 1.6 μΜ KS04. These data suggested that combination treatment with KS04 could overcome the resistance of AML cells to ABT-737. Table 4: Combination treatment with KS04

Example 7: Effect of Human Serum on Cytotoxicity of Maritoclax and Pyoluteorin

Derivatives

Mcl-l-IRES-Bim K562 cells were seeded in RPMI 1640 medium containing 10% fetal bovine serum (FBS), 20% FBS or 20%> human serum (HS) and treated with various

concentrations of maritoclax, KS18 or KS19 for 48 hours. Cell viability was determined using the ATP or MTS assay. The EC50 (μΜ) values are shown in Table 5.

Table 5: Effect of human serum on EC50 (μΜ) values for Maritoclax and Pyoluteorin Derivatives

Example 8: Effect of KS04 and KS18 on Mcl-1 Degradation

U937 human myeloid leukemia cells treated with vehicle control (DMSO), 2.5 μΜ KS04, or 2 μΜ KS18 for 1 hour, followed by addition of 10 μg/ml cycloheximide (CHX) to block protein synthesis. Cells were harvested at at 0, 15, 30, 60, 120, 180, and 240 minutes after CHX addition, lysed and the lysates subjected to immunoblot analysis. The intensity of Mcl-1 band was quantified by densitometry and normalized to β-actin. The half-life of Mcl-1 was calculated by linear regression equations. Both KS04 and KS18 reduced the half-life of McL-1. As shown in Figure 2, the half-life of Mcl-1 in DMSO-treated control cells was estimated to be 178 minutes. In contrast, the half-life of McL-1 in KS04- or KS18-treated cells was approximately 20 minutes.

Example 9: Effect of KS18 on Sensitivity of Human Acute Myeloid Leukemia Cells to ABT-

737

ABT-737 resistant HL60 cells were treated with KS18 and ABT-737 alone or in combination at the indicated concentrations for 48 hours and subjected to immunoblot analysis with the anti-Mcl-1, anti-Bim, anti-cleaved C3, anti-Parp, or anti β-actin antibodies. As shown in Figure 3, KS18 induced a dose-dependent decrease in Mcl-1 polypeptide levels and a dose- dependent increase in cleavage of caspase-3 and PARP. The effect of KS18 on both McL-1 polypeptide levels and cleavage of caspase 3 and PARP was strongly enhanced by co-treatment with ABT-737.

Example 10: Effect of Maritoclax and Maritoclax Derivatives on Stromal Cell-mediated

Drug Resistance

Human bone marrow stromal HS-5 cells were seeded at 5. Ox 10⁵ cells/mL in DMEM media with 10% FBS. After 6 hours of incubation to allow stromal cell attachment to plate, human leukemic U937 cells expressing the firefly luc2 gene were added at 5.0^χ 10⁵ cells/mL in RPMI media with 10% FBS and incubated for another 6 hours to allow leukemic cell attachment to stroma before drug treatment. For control plates that did not include HS-5 stromal cells, U937 cells expressing the luc2 gene were seeded at 5.0x l0⁵ cells/mL in 45% DMEM media, 45% RPMI media, and 10% FBS and incubated for 6 hours before drug treatment. Both the co- culture plates and the control plates were then treated with increasing concentrations of either maritoclax, KS04, KS18 or duanorubicin for 48 hours. Cell viability was measured by adding 75 μg/mL of luciferin and measuring luminescence immediately. The results of this experiment are shown in Figure 4. As shown in Figure 4 A, U937 cells co-cultured with stroma were more resistant to daunorubicin treatment than were U937 cells cultured in the absence of stroma. In contrast, maritoclax (Figure 4B) and the pyoluteorin derivatives, KS04 (Figure 4C) and KS10 (Figure 4C) killed both co-cultured and single-cultured U937 cells with similar potency and efficacy. Graph represents the mean and standard deviation (n=4).

Example 11: Effect of Maritoclax and Maritoclax Derivatives on Stromal Cell Viability

Human bone marrow stromal HS-5 cells were seeded at 2.5 x 10⁵ cells/mL and incubated for 12 hours in DMEM media with 10% FBS to allow attachment to the cell-culture plates. Cells were then treated with increasing concentrations of either maritoclax, KS04, KS18 or

daunorubicin for 48 hours. Cell viability was measured using the MTS assay (Promega). As shown in Figure 5, at higher concentrations, daunorubicin treatment resulted in a significantly greater reduction in stromal cell viability than did treatment with the same concentration of maritoclax or the maritoclax derivatives. Graph represents the mean + standard deviation (n=4).

Example 12: Effect of Maritoclax on Tumor Growth in vivo

Athymic nude mice (6-7 week old females) were implanted subcutaneously with 5 x 10⁶ U937 cells in 0.2 ml 50% Matrigel. When tumors reached an average size of approximately 230 mm³, the mice were divided into control and treatment groups (7 mice per group). Tumor volume was measured every two days by electronic calipers (volume = length x width²/2).

Maritoclax was formulated in 30%> propylene glycol, 5% Tween 80, 65%> D5W (5% dextrose in water), pH4.5, and administered intraperitoneally. As shown in Figure 6, Maritoclax significantly inhibited U937 xenograft tumor growth in athymic nude mice at a dose of 20 mg/kg/d intraperitoneally. (P<0.005) One mouse in the control vehicle treatment group was euthanized at day 10 of treatment because the average tumor size was >3000 mm³; 4 tumors in the maritoclax treatment group regressed completely.

Example 13: Effect of Maritoclax on Body Weight in Athymic Nude Mice

Athymic nude mice were treated with Maritoclax formulated in 30% propylene glycol, 5% Tween 80, 65% D5W (5% dextrose in water), pH4.5, at a dose of 20 mg/kg/d intraperitoneally for 16 days. As shown in Figure 7, no significant difference in body weight change was observed between maritoclax and vehicle treatment groups.

Example 14: Effect of KS04 on Mcl-1 interaction with Bim in intact cells

Mcl-l-IRES-BimEL K562 cells were treated with DMSO or 2 μΜ KS04 and 1 μΜ MG132 for 24 h. Cells were collected, lysed and the lysates subjected to immunoprecipitation with anti-Mcl-1 rabbit antiserum. The resulting immune complexes were analyzed by

immunoblotting with anti-Mcl-1 and anti-Bim monoclonal antibodies. As shown in Figure 8, treatment with KS04 resulted in a reduction of BimEL McL-1 complexes by approximately 60%.

Example 15: Effect of KS18 and ABT-737 on viability of multidrug-resistant HL60 Cells

Multidrug resistant HL60 cells (HL60/VCR) and ABT-737 resistant HL60 cells

(HL60/ABTR) cells were treated with increasing concentrations of ABT-737, KS 18, or a 1 : 1 combination of these drugs for 48 hours. Cell viability was measured using the CellTiter-Glo assay (mean ± SD; n = 3). As shown in Figures 9A and 9C, respectively, the combination of KS18 and ABT-737 was significantly more effective in killing multidrug resistant HL60 cells (HL60/VCR) and ABT-737 resistant HL60 cells (HL60/ABTR) cells than was either compound alone. The combination index was calculated with the CompuSyn software using the percentage of dead cells (effect level) by the two compounds. As shown in Figures 9B and 9D, the combination treatment was synergistic.

Example 16: Preparation of KA01

Marinopyrrole A was synthesized by a previously described method (Nicolaou. K.C., etai, Tetrahedron Letters, 2011, 52, 2041-2043) with minor modifications (Scheme 1). Briefly, a solution of aminopyrrole 1, 2, 5-dimethoxytetrahydrofuran and PPTS in 1,4-dioxane was stirred at 50 °C for 30 minutes. The mixture was concentrated, dissolved in ethyl acetate, and filtered through a pad of silica gel. The residue was chromatographed on silica gel (10% ethyl acetate- hexane) to give 3 as a brown solid. To a solution of 2-iodoanisole in THF, BuLi was added at - 78 °C and the solution was stirred for 20 minutes. The compound 3 was dissolved in THF and slowly added to the lithiated anisole solution and stirred for 1 hour at -78 °C. The reaction was quenched by the addition of NH₄C1 solution, diluted with EtOAc, washed with water, concentrated and purified by silica gel column chromatography using 10% EtOAc in hexane to yield 4. O-anisic acid in benzene was refluxed with SOCl₂ to convert into acid chloride. The solution was then concentrated and the acid chloride was dissolved in CH₂C1₂ and added to a slurry of A1C1₃ in CH₂C1₂ at 0 °C and then a solution of 4 in CH₂C1₂ was added slowly. The resulting solution was allowed to warm to 25 °C and stirred overnight. A saturated aqueous solution of NaHC0₃ and CH₂C1₂ was then added and the resulting mixture was stirred for 1 hour and then filtered through Celite. The biphasic mixture was extracted with CH₂C1₂, and the combined organic layers were dried, filtered, and concentrated. The residue was purified by flash column chromatography (hexanes: EtOAc 2:3) to afford 5. To a solution of 5 in CH₂C1₂ at 0 °C, S0₂C1₂ was added dropwise, and the solution was allowed to stir at 0 °C for 1 h. Saturated aqueous NaHC0₃ solution was added and the resulting biphasic mixture was extracted with CH₂C1₂. The combined organics concentrated and the residue was purified by flash column chromatography (hexanes: EtOAc 4: 1) to afford (±)-6 as an off- white solid. To a solution of (±)-6 in CH₂C1₂ at 0 °C, 1 M solution of BBr₃ in CH₂C1₂ was added drop wise and the mixture was allowed to stir at 0 °C for 1 h. Saturated aqueous NaHC0₃ was then added, and the biphasic mixture was extracted with CH₂C1₂. The combined organics were concentrated and the residue was purified by flash column chromatography (hexanes :EtO Ac 9: 1) to afford 7 as a bright yellow solid.

To synthesize KA01, Ac₂0, Et₃N and DMAP were added to a solution of 7 in CH₂C1₂, and the resulting solution was stirred at 25 °C for 12 hours. The reaction mixture was diluted with EtOAc and washed sequentially with aqueous 1 M HC1, H₂0, and brine. The organic layer was then dried (MgS04), filtered, and concentrated. The residue was purified by flash column chromatography (silica gel, hexanes:EtOAc 4:1) to provide 8 (KA01). The purity of KAOl was confirmed by HPLC and structure was confirmed by NMR and mass spectroscopic methods. Scheme 1.

Reagents and Conditions: a) PPTS, dioxane, 50° C; b) 2-Iodoanisole, n-BuLi, THF; c) O-anisic acid, SOCl₂ AlCl₃, CH₂Cl₂; d) S0₂C1₂, CH₂Cl_2l 0° C; e) BBr₃ CH₂Cl_2i 0° C, f)Ac₂0, Et₃N, DMAP, CH₂Cl₂.

Example 17: Effect of KAOl on cell viability in leukemia cell lines

To study whether KAOl selectively induced apoptosis in cells that are dependent on Mcl- 1 for survival, we examined the effect of KAOl on human myeloid leukemia K562 cells stably transfected with Mcl-l-IRES-BimEL or Bcl-2-IRES-BimEL constructs. We also tested whether KAOl induced cell death in a Bax/Bak-dependent manner using a subclone (JurkatABak) of Jurkat human acute T cell leukemia cell line that constitutively lacks Bax and Bak (Wang, G. Q., et al. (2001) The Journal of Biological Chemistry 276, 34307-34317). Cells were treated with increasing concentrations of KAOl for 24 hours and cell viability was determined using the CellTiter Glo method described in Example 2. As shown in Table 6, KAOl treatment selectively reduced the viability of Mcl- 1 -IRES-BimEL cells (EC50 = 3.1 μΜ) over Bcl-2-IRES- BimEL (EC50 = 41.3 μΜ). Similarly, KAOl selectively reduced the viability of the parental wild-type Jurkat cells (EC50 = 4.6 μΜ) over the Bax/Bak-deficient cell line JurkatABak (EC50 > 50.0 μΜ). These data showed that the marinopyrrole A derivative KAOl selectively induced cell death in leukemia cells that are dependent on Mcl-1 but not Bcl-2 for survival through a

Bax/Bak-dependent mechanism.

Table 6: Effect of KAOl on Cell

Viability

Example 18: Effect of KAOl on sensitivity of myeloid leukemia cells to ABT-737

To determine whether KAOl resensitized multidrug resistant human acute myeloid leukemia cells to ABT-737, multidrug resistant human acute myeloid leukemia cells were treated with combinations of ABT-737 and KAOl . The vincristine (VCR)-resistant HL60/VCR cells were treated with increasing concentrations of ABT-737 alone or with 2 μΜ KAOl for 24 hours. The ABT-737-resistant HL60/ABT cells were treated with increasing concentrations of ABT- 737 or KAOl alone or 2 μΜ KAOl combined with various concentrations of ABT-737 for 48 hours. The cells were subjected to the CellTiter Glo cell viability assay to determine EC50 values (mean ± s.d.; n = 3). As shown in Table 7, addition of 2 μΜ KAOl markedly decreased the EC50 values of ABT-737 in both HL60/VCR and HL60/ABT cell lines. Table 7: KAOl overcomes ABT-737 resistance

Example 19: Effect of KAOl on Proteasome-mediated Mcl-1 Degradation

K562 cells stably expressing Mcl-1 -IRES-BimEL were treated with different

concentrations of KAOl alone or a combination of 4 μΜ KAOl and 1 μΜ MG132 for 24 hours and subjected to immunoblot analysis.

As shown in Figure 11, KAOl treatment resulted in a dose-dependent decrease in Mcl-1 protein levels. KAOl treatment also resulted in a dose-dependent increase in cleavage of caspase- 3 and PARP. Addition of the proteasome inhibitor MG132 attenuated KAOl-mediated Mcl-1 degradation, caspase-3 activation and PARP cleavage. KAOl did not significantly affect Noxa protein levels. These data showed that the pro-apoptotic effect of KAOl derived, at least in part, from its ability to induce the proteasome-mediated degradation of Mcl-1 protein.

Example 20: Synthesis of Pyoluteorin Derivatives KS01-KS06

Pyoluteorin derivatives were synthesized by using previously described methods

{Hughes, C.C., et al, J. Org. Chem. 2010, 75, 3240-3250) with minor modifications as shown in

Scheme 1. Briefly, 2-iodoanisole was added to BuLi in THF at -78°C and a solution of ethyl pyrrole-2-carboxylate (1) in THF was added to lithiated anisole solution and stirred for 1 hour.

The reaction was quenched by addition of NH₄C1 solution, extracted with ethyl acetate, concentrated and purified by silica gel column chromatography to yield 2. Compound 2 upon treatment with SO₂CI₂ underwent selective pyrrole dichlorination and was converted into (4,5- dichloro-lH-pyrrol-2-yl)(2-methoxyphenyl)methanone 3 (KS01) as a white solid. To a solution of 3 in dichloroethane, N-bromosuccinimide (lequiv) was added and the mixture was heated at

90°C for 4.5 hour, allowed to cool, poured into ethyl acetate, and washed with a saturated

NaHC03 solution and brine. The organic layer was dried, filtered, concentrated and the product was purified by column chromatography to afford (3-Bromo-4,5-dichloro-lH-pyrrol-2-yl)(2- methoxyphenyl)methanone 4 (KS03) as an off white solid. Compound 5 (KS05) (1,3-Dibromo- 4,5-dichloro-lH-pyrrol-2-yl)(2-methoxy-phenyl)methanone was obtained by using 2 equivalents of NBS under similar experimental conditions used for 4. Cleavage of the methyl ether of 3, was achieved by treatment with BBr₃ in CH₂CI₂, and the residue was purified by silica gel column chromatography to afford phenolic derivative (4,5-Dichloro-lH-pyrrol-2-yl)(2- hydroxyphenyl)methanone 6 (KS02) as a bright yellow solid. (3-Bromo-4,5-dichloro-lH-pyrrol- 2-yl)(2-hydroxyphenyl)methanone 7 ( KS04) as an orange red solid and (l,3-Dibromo-4,5- dichloro-lH-pyrrol-2-yl)(2-hydroxyphenyl)methanone 8 (KS06) as a brown solid were obtained using similar experimental conditions used for 6.

Example 21: Synthesis of Pyoluteorin Derivatives KS07-KS12

Pyoluteorin derivatives KS07 and KS09 were synthesized as shown in Schemes 2.

Briefly, to a solution of ethyl pyrrole-2-carboxylate (1) in anhydrous THF, N,0- dimethylhydroxylamine hydrochloride was added, cooled to -78°C and then lithium bis(trimethylsilyl)amide solution was added slowly and stirred for 1 hour. The reaction was quenched by addition of NH₄C1 solution, extracted with ethyl acetate, concentrated and purified by silica gel column chromatography to yield 9. Compound 9 upon treatment with 2,3,4- trimethoxyphenylmagnesium bromide in anhydrous THF was converted into 10 as a white solid. To a solution of 10 in dichloromethane, S0₂C1₂ (2 equiv) was added and the mixture was stirred at 0°C for 3 h to afford (4,5-Dichloro-lH-pyrrol-2-yl)(2,3,4-trimethoxy-phenyl)methanone 11 (KS07) as a white solid. Cleavage of the methyl ether of 11 was achieved by treatment with BBr₃ in CH₂C1₂, and the residue was purified by silica gel column chromatography to afford phenolic derivative (4,5-dichloro-lH-pyrrol-2-yl)(2,3,4-trihydroxyphenyl)methanone 12 (KS09) as a white solid.

(4,5-Dichloro-lH-pyrrol-2-yl)(2-hydroxy-3,4-dimethoxy-phenyl)methanone 15 (KS08) was synthesized as shown in Scheme 3. 2,3,4-trimethoxybenzoyl chloride was stirred with A1C1₃ in methylene chloride and pyrrole was added at 0°C. The reaction mixture was stirred overnight at room temperature and the reaction was quenched by saturated NaHC0₃ solution, filtered through Celite, and concentrated. The residue was chromatographed on silica gel to get yellow solid 14. Compound 14 was treated with S0₂C1₂ (2 equiv) in dichloromethane to get 15 (KS08) as a yellow solid.

Derivatives KS10, KS11 and KS12 were synthesized as shown in Scheme 4. To a solution of 9 in dichloromethane, SO₂CI₂ (2 equiv) was added slowly to obtain dichloro pyrrole derivative 16. To a solution of 16 in dichloroethane, NBS was added and refluxed for 3 hours, concentrated and purified by silica gel column chromatography to get 3-Bromo-4,5-dichloro-lH- pyrrole-2-carboxylic acid methoxy methyl amide 17 (KS10) as a white solid. To a solution of 17 in anhydrous THF, 2,3,4 trimethoxyphenylmagnesium bromide was added at 0°C. The reaction was stirred for 1 hour, quenched with saturated aqueous solution of NH₄C1, concentrated and purified by silica gel column chromatography to afford (3-Bromo-4,5-dichloro-lH-pyrrol-2- yl)(2,3,4-trimethoxyphenyl)methanone 18 (KS11) as an off white solid. To a solution of 18 in

CH₂CI₂ at 0°C, a solution of BBr₃ was added and the mixture was allowed to stir at room temperature for 5 hours. The reaction was quenched by saturated aqueous NaHC0₃ and extracted with CH₂CI₂. The combined organics were dried, filtered, and concentrated. The residue was purified by flash column chromatography to afford 19 (KS12) as a brown solid.

IScheme 4 I

Reagents and Conditions: l)S0₂CI₂, CH₂CI₂, 0 °C; m) NBS, DCE, 90°C; n) 2,3,4- trimethoxyphenylmagnesium bromide, THF, 0 °C; o) BBr₃(6 equiv), CH₂CI₂, 0 °C. Example 22: Effect of pyoluteorin derivatives on cell viability in Mcl-l-dependent leukemia cell lines

To evaluate the ability of the pyoluteorin derivatives to induce cell death in Mcl-l- dependent leukemia cells, K562 cells stably expressing Mcl-l-IRES-BimEL were treated with DMSO (control) or the derivatives (KS01-12) at 2.5 or 10 μΜ for 48 hours. Cell viability was determined by measuring intracellular ATP levels with CellTiter Glo assay. As shown in Table 8, treatment with KS02, KS04, KS05, KS06 and KS12 resulted in a reduction in cell viability.

Table 8: Effect of Pyoluteorin Derivatives on viability of K562 cells stably expressing Mcl- l-IRES-BimEL

% Viability (mean ± s.d.; n = 3)

Example 23: Effect of pyoluteorin derivatives on cell viability in Mcl-l-dependent and Bcl-2-dependent leukemia cell lines To evaluate the specificity and potency of the pyoluteorin derivatives for Mcl-l, we treated K562 cells that had been stably transfected with Bcl-2-IRES-BimEL or Mcl-l -IRES- BimEL with increasing concentrations of KS02, KS04, KS05, KS06, KS07, KS08, KS09, KSIO, KS11 and KS12 for 48 hours. Cell viability was determined by CellTiter Glo assay and EC50 values were estimated by SigmaPlot. As shown in Table 9, all these derivatives, except KS07 and KSIO, had a more potent cytotoxic effect on Mcl-l -dependent cells than on Bcl-2-dependent cells. Of these, KS04 was the most potent compound with 18-fold selectivity for Mcl-l dependent K562/Mcl-l-IRES-BimEL (EC50 = 1.55 μΜ) over Bcl-2-dependent K562/Bcl-2- IPvES-BimEL (EC50 = 28.1 μΜ) cells. Moreover, treatment with KS04 markedly inhibited the viability of U937, RPMI8226 and NCIH929 cell lines, which express high levels of Mcl-l . Importantly, KS04 was much more effective against wild type Jurkat cells (EC50 = 2.61 μΜ) than it was against Bax/Bak-deficient (Jurkat ABak) cells (EC50 = 36.3 μΜ).

Table 8: EC50 values for pyolueorin derivatives

n.d. = not determined

Example 24: Effect of KS04 on Proteasome-mediated Mcl-l Degradation

To determine whether KS04 was able to downregulate Mcl-l, K562 cells stably expressing Mcl-l -IRES-BimEL or Bcl-2-IRES-BimEL were treated with 2 μΜ KS04 for the indicated times and subjected to immunoblot analysis. As shown in Figure 12, KS04 induced a time-dependent decrease in Mcl-1 protein expression, which was accompanied by an increase in cleavage of caspase-3 and PARP. In contrast, KS04 had little effect on Bcl-2 protein levels, caspase-3 processing and PARP cleavage. These data showed that the pro-apoptotic effect of KS04 derived, at least in part, from its ability to induce the proteasome -mediated degradation of Mcl-1 protein.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein

X is H, halo, or alkyl;

Y is OH, alkoxy or acetoxy;

Z is H or halo;

Ri is H, OH, or alkoxy;

R₂ is H, OH, or alkoxy; and

R₃ is H or halo.

2. The compound of claim 1, selected from

(l,3-Dibromo-4,5-dichloro-lH-pyrrol-2-yl)(2-methoxyphenyl)methanone; ( 1 ,3 -Dibromo-4,5 -dichloro- 1 H-pyrrol-2-yl)(2-hydroxyphenyl)methanone; (4,5-Dichloro- 1 H-pyrrol-2-yl)(2,3 ,4-trimethoxy-phenyl)methanone;

(4,5-Dichloro- 1 H-pyrrol-2-yl)(2-hydroxy-3 ,4-dimethoxyphenyl)methanone; (4,5-dichloro- 1 H-pyrrol-2-yl)(2,3 ,4-trihydroxyphenyl)methanone;

(3-Bromo-4,5-dichloro-lH-pyrrol-2-yl)(2,3,4-trimethoxyphenyl)methanone; (4,5 -dichloro-3 -methyl- 1 H-pyrrol-2-yl)(2-hydroxyphenyl)methanone; (3-Bromo-4,5-dichloro-lH-pyrrol-2-yl)-(2,4-dihydroxyphenyl)methanone; (3-Bromo-4,5-dichloro-lH-pyrrol-2-yl)(5-chloro-2-hydroxyphenyl)methanone; 2-(3-bromo-4,5-dichloro-lH-pyrrole-2-carbonyl)-4-chlorophenyl acetate; and (3 -Bromo-4,5 -dichloro- 1 H-pyrrol-2-yl)(5 -fluoro-2-hydroxyphenyl)methanone .

3. The compound of claim 1, wherein Y is not OH or methoxy, and X, Z, R_ls R₂ and R₃ are not H.

4. The compound of claim 1, wherein X is not Br, Y is not OH, and Z, R_ls R₂ and R₃ are not H.

5. The compound of claim 1, wherein X is not Br, Y is acetoxy and Z, R_ls R₂ and R₃ are not H.

6. A compound of Formula II:

or a pharmaceutically acceptable salt thereof, wherein

X is H or halo;

Y is OH, alkoxy or acetoxy;

Ri is H, OH, or alkoxy;

R₂ is H, OH, or alkoxy; and

R₃ is H.

7. The compound of claim 6, selected from (3-Bromoindole-2-yl)(2-hydroxyphenyl)methanone; and (3-Bromoindol-2-yl)(2,4-dihydroxyphenyl)methanone.

8. A pharmaceutical composition comprising any of the compounds of claims 1-3, 6 and and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition of claim 8, wherein the compound is selected from 4,5-Dichloro-lH-pyrrol-2-yl)(2-methoxyphenyl)methanone; (4,5-Dichloro-lH-pyrrol-2-yl)(2-hydroxyphenyl)methanone; (3-Bromo-4,5-dichloro-lH-pyrrol-2-yl)(2-methoxyphenyl)methanone; (3-Bromo-4,5-dichloro-lH-pyrrol-2-yl)(2-hydroxyphenyl)methanone; 2-(3-bromo-4,5-dichloro-lH-pyrrole-2-carbonyl)phenyl acetate.

10. A pharmaceutical composition comprising (a) a compound of Formula III:

wherein Ri and R₂ are not H, Ri is COCH₃, C(0)OC₂H₅, or C(0)0 joined to a heterocycle and R₂ is COCH₃, C(0)OC₂H₅, or C(0)0 joined to a heterocycle, or a

pharmaceutically acceptable salt thereof; and

(b) a pharmaceutically acceptable carrier.

11. The composition of claim 10, wherein Ri is and R₂ are COCH₃.

12. The composition of claim 10, wherein Ri is and R₂ are C(0)OC₂Hs.

13. The composition of claim 10, wherein Ri is and R₂ are C(0)0 joined to a heterocycle.

14. A method of treating a subject who has cancer, the method comprising administering to the patient a therapeutically effective amount of the composition of any of claims 1-3 and 7- 13.

15. The method of claim 14, wherein the cancer is lymphoma, leukemia, multiple myeloma, melanoma, pancreatic cancer, lung cancer, breast cancer, liver cancer, colon cancer, prostate cancer or ovarian cancer.

16. The method of claim 14, wherein the cancer expresses Mcl-1.

17. The method of claim 14, wherein the cancer is resistant to an anti-Bcl-2 agent.

18. The method of claim 17, wherein the anti-Bcl-2 agent is ABT-737, ABT-263, obatoclax, BH3-M6, or gossypol or a gossypol derivative.

19. The method of claim 14, further comprising the step of administering a second cancer treatment.

20. The method of claim 19, wherein the second cancer treatment comprises

administration of a chemotherapeutic agent, a radiation treatment, treatment with an antibody or surgical intervention.

21. The method of claim 20, wherein the chemotherapeutic agent comprises an anti-Bcl- 2 agent.

22. The method of claim 21, wherein the anti-Bcl-2 agent is ABT-199, ABT-737 or ABT-263.

23. The method of claim 14, further comprising the step of identifying a subject amenable to treatment.

24. The method of claim 14, further comprising the step of providing a biological sample from the subject and determining whether the sample includes an elevated level of Mcl-l or another predictive biomarker for cancer.

25. The method of claim 24, wherein the biological sample is a urine, saliva,

cerebrospinal fluid, blood, or biopsy sample.

26. The method of claim 24, wherein the step is carried out before administering the composition and an elevated level of Mcl-l indicates that the subject is a good candidate for the treatment.

27. The method of claim 24, wherein the step is carried out at one or more times after administering the agent and a reduced level of Mcl-l indicates that the subject is responding well to the treatment.

28. A method of killing an Mcl-l expressing cancer cell, the method comprising contacting the cell with an effective amount of the composition of any of claims 1-3 and 7-13.

29. A method of modulating the level of Mcl-l in a cell, the method comprising contacting the cell with an effective amount of the composition of any of claims 1-3 and 7-13.