WO2012061395A2

WO2012061395A2 - Cytotoxic agents against cancer cells and uses thereof

Info

Publication number: WO2012061395A2
Application number: PCT/US2011/058796
Authority: WO
Inventors: Kevin H. Mayo; Thomas R. Hoye; Joseph Isaac Levine; Ruud P. Dings
Original assignee: Regents Of The University Of Minnesota
Priority date: 2010-11-01
Filing date: 2011-11-01
Publication date: 2012-05-10
Also published as: WO2012061395A3

Abstract

Topomimetic calixarene-based peptide mimetics with cytotoxic tumoricidal activity are described. Methods of use and methods of designing such calixarene-based peptide mimetics are described.

Description

Patent

File 110.03380201 CYTOTOXIC AGENTS AGAINST CANCER CELLS AND USES THEREOF

CONTINUING APPLICATION DATA

This application claims the benefit of U.S. Provisional Applications Serial No. 61/408,806, filed November 1, 2010, and U.S. Provisional Applications Serial No.

61/412,890, filed November 12, 2010, each of which is incorporated by reference herein.

GOVERNMENT FUNDING

This invention was made with government support under Grant No. CA-096090, awarded by the National Cancer Institute, National Institutes of Health. The Government has certain rights in the invention.

BACKGROUND

The synthetic beta-sheet peptide anginex (also known as βρερ-25) is a potent angiogenesis inhibitor and anti-tumor agent in vivo. This 33-mer peptide has therapeutic application as an anti-angiogenic agent in various pathological disorders, including, for example, neoplasia, rheumatoid arthritis, diabetic retinopathy, and restenosis. See, for example, Griffioen et al., 2001, Biochem J; 354(Pt 2):233-242; Van der Schaft, 2002, FASEBJ; 16:1991-1993; Dings et al., 2003, Cancer Research; 63:382-385; and Dings et al., 2003, Biochem J; 373:281-288. Several nonpeptidic, calixarene-based protein surface topomimetics that display chemical substituents to approximate the molecular dimensions and amphipathic features of anginex have been synthesized. Two of these topomimetics, compound 0118 and compound 1097, are potent angiogenesis inhibitors in vitro and show anti-angiogenic and anti-tumor activity in vivo. See, for example, WO 2006/042104; US 2008/0300164; Dings et al., 2006, J Natl Cancer Inst; 98(13):932-936; Chen et al., 2006, J Med Chem; 49(26):7754-7765; and Dings et al., 2008, Cancer Lett; 265(2):270-280.

However, there is a need for new and improved nonpeptidic calixarene-based topomimetic compounds for use in the treatment of cancer. For example, there is a need for compounds with a greater potency than known compounds, a need for compounds that target cellular receptors different from those targeted by known compounds, and a need for compounds that demonstrate differing cytotoxic mechanisms from known compounds. There is also a need for compounds that effective against differing cellular phenotypes, for example, demonstrating cytotoxicity against non-endothelial cells, epithelial cells, cells of mesenchymal origin and/or cells that demonstrate an epithelial to mesenchymal transition (EMT) phenotype.

SUMMARY OF THE INVENTION

The present invention includes a nonpeptidic calixarene topomimetic agent having the formula:

wherein y + z is 4 to 6; wherein R⁹ and R¹⁰ are each independently a hydrogen or an organic group and R⁹ and R¹⁰ have a hydrophobic polarity; wherein R¹¹ is hydrogen or an

1 ^*? 1 ^ organic group; wherein x is 1 to 6; wherein R is an organic group; and wherein R is an organic group; and derivatives and salts thereof.

In some embodiments, the nonpeptidic calixarene topomimetic agent demonstrates a cytotoxic activity at least 5 fold greater than compound 1097, compound 0118, and/or anginex against the B 16F 10 murine melanoma cell line, demonstrates cytotoxic activity against galectin-1 negative cells, demonstrates cytotoxic activity against cells resistant to compound 0118, and/or demonstrates cytotoxic activity against cells having an epithelial to mesenchymal transition (EMT) phenotype. In some embodiments, each of R⁹ and R¹⁰ is independently selected from hydrogen, a branched or unbranched alkyl, a branched or unbranched alkenyl, a cycloalkyl, an aryl, or a heteroaryl.

In some embodiments, each of R⁹ and R¹⁰ is independently selected from hydrogen, methyl, ethyl, propyl, or tert-butyl.

In some embodiments, R¹¹ is hydrogen, a branched or unbranched alkyl, a branched or unbranched alkenyl, a cycloalkyl, an aryl, or a heteroaryl.

In some embodiments, R¹¹ is hydrogen, methyl, ethyl, propyl, or tert-butyl.

In some embodiments, each of R¹² and R¹³ is independently selected from hydrogen, a branched or unbranched alkyl, a branched or unbranched alkenyl, a cycloalkyl, an aryl, or a heteroaryl.

In some embodiments, each of R¹² and R¹³ is independently selected from hydrogen, methyl, ethyl, propyl, or tert-butyl.

In some embodiments, y + z is 4.

In some embodiments, x is 1, 2, 3, or 4.

In some embodiments, y is 4, z is 0, x is 2, and R¹² and R¹³ are methyl groups.

In some embodiments, the cytotoxic nonpeptidic calixarene topomimetic agent has the formula:

also referred to as (JIL70), and derivatives, analogs, and salts thereof.

also referred to as (JTL54), and derivatives, analogs, and salts thereof.

The present invention includes a cytotoxic nonpeptidic calixarene topomimetic agent having the formula:

also referred to as (JIL70), and derivatives, analogs, and salts thereof.

also referred to as (JIL54), and derivatives, analogs, and salts thereof. The present invention includes a cytotoxic nonpeptidic calixarene topomimetic having the formula:

also referred to as (JIL31), and derivatives, analogs, and salts thereof.

also referred to as (JTL50), and derivatives, analogs, and salts thereof.

The present invention includes a cytotoxic nonpeptidic calixarene-based topomimetic agent having Formula I or II:

wherein each R¹ through R⁸ group is independently hydrogen or an organic group, wherein R¹ through R⁴ are each independently hydrogen or an organic group of like polarity and R through R are each independently hydrogen or an organic group of like polarity that is of opposite polarity than those of R¹ through R⁴, and wherein the agent demonstrates a cytotoxic activity at least a 5 fold greater than 1097, 0118, and/or anginex against the B16F10 melanoma cell line, demonstrates cytotoxic activity against galectin-1 negative cells, demonstrates cytotoxic activity against cells resistant to compound 0118, and/or demonstrates cytotoxic activity against cells having an epithelial to mesenchymal transition (EMT) phenotype; and wherein the agent is not compound 0118 or compound 1097. In some embodiments, R¹ through R⁸ are each independently hydrogen, halogen, alkyl, cycloalkyl, aryl, aralkyl, alkoxy, thioalkoxy, cycloalkylalkoxy, heterocycloalkyl, aralkyloxy, or heteroaryl, optionally including ester, amide, amine, hydroxyl, halogen, sulfonate, phosphonate, guanidine, and/or heteroaryl groups. In some embodiments, R¹ through R⁴ are each independently alkyl, cycloalkyl, aralkyl, alkoxy, cycloalkylalkoxy, or aralkyloxy, and R through R are each independently any of these groups incorporating ester, amide, amine, hydroxyl, sulfonate, phosphonate, guanidine and/or heteroaryl groups. In some embodiments, R through R are each independently alkyl, cycloalkyl, aralkyl, alkoxy, cycloalkylalkoxy, or aralkyloxy, and R¹ through R⁴ are each independently any of these groups incorporating ester, amide, amine, hydroxyl, sulfonate, phosphonate, guanidine and/or heteroaryl groups. In some embodiments, R¹ through R⁸ are each independently hydrogen, alkyl, cycloalkyl, aralkyl, alkoxy, cycloalkylalkoxy, or aralkyloxy optionally including ester, amide, amine, hydroxyl, sulfonate, phosphonate, guanidine and/or heteroaryl groups. In some embodiments, R¹ through R⁴ are each independently hydrogen, halogen, alkyl, cycloalkyl, aryl, aralkyl, alkoxy, thioalkoxy, cycloalkylalkoxy, heterocycloalkyl, aralkyloxy, or heteroaryl and R through R are each independently any of these groups incorporating ester, amide, amine, hydroxyl, halogen, sulfonate, phosphonate, guanidine, and/or heteroaryl groups. In some embodiments, R⁵ through R⁸ are each independently hydrogen, halogen, alkyl, cycloalkyl, aryl, aralkyl, alkoxy, thioalkoxy, cycloalkylalkoxy, heterocycloalkyl, aralkyloxy, or heteroaryl and R¹ through R⁴ are each independently any of these groups incorporating ester, amide, amine, hydroxyl, halogen, sulfonate, phosphonate, guanidine, and/or heteroaryl groups.

In some embodiments, an agent of the present invention demonstrates cytotoxic activity. In some embodiments, an agent demonstrates cytotoxic activity against non- endothelial cells. In some embodiments, an agent demonstrates cytotoxic activity against galectin-l negative cells. In some embodiments, an agent demonstrates cytotoxic activity against cells having an epithelial to mesenchymal transition (EMT) phenotype. In some embodiments, an agent demonstrates cytotoxic activity against cells resistant to compound 0118.

In some embodiments, an agent of the present invention demonstrates a cytotoxic activity at least 5 fold greater than compound 1097, compound 0118, and/or anginex against the B16F10 murine melanoma cell line.

In some embodiments, an agent of the present invention demonstrates anti-tumor activity.

In some embodiments, an agent of the present invention may be conjugated to a diagnostic agent, a therapeutic agent, a detectable marker, a targeting moiety, or a liposome.

The present invention includes a pharmaceutical composition including one of more of the agents of the present invention.

The present invention includes methods of killing cells including contacting cells with an agent, conjugate, or composition of the present invention. In some embodiments, the contacting step occurs in vitro. In some embodiments, the contacting step occurs in vivo. In some embodiments, the cells are present in a cell culture, a tissue, an organ, or an organism. In some embodiments, the cells include non-endothelial cells. In some embodiments, the cells are galectin-1 negative cells. In some embodiments, the cells have an epithelial to mesenchymal transition (EMT) phenotype. In some embodiments, the cells are resistant to compound 0118 and/or compound 1097. In some embodiments, the cells , are mammalian cells. In some embodiments, the cells include cancer cells.

The present invention includes methods of treating cancer in a subject, the method including administering an agent, conjugate, or composition of the present invention. In some embodiments, the cancer is a carcinoma, a sarcoma, a blood borne hematologic cancer, or a germ line cancer. In some embodiments, the cancer is a breast cancer, ovarian cancer, melanoma, colon cancer, lung cancer, or a squamous cell carcinoma. In some embodiments, the cancer is a primary cancer or a metastatic cancer. In some

embodiments, the cells are human cells. In some embodiments, the cancer comprises galetin-1 negative cells. In some embodiments, the cancer includes cells having an epithelial to mesenchymal transition (EMT) phenotype. In some embodiments, the cancer includes cells resistant to compound 0118 and/or compound 1097. In some embodiments, the method further includes administering one or more additional therapeutic agents. In some embodiments, the cytotoxic nonpeptidic calixarene-based topomimetic agent and the one or more additional therapeutic agents demonstrate a synergy.

The present invention includes methods for inhibiting tumorigenesis in a patient, the method including acmiinistering to the patient a therapeutically effective amount of an agent, conjugate, or composition of the present invention. In some embodiments, the tumor is a carcinoma, a sarcoma, a blood borne hematologic cancer, or a germ line cancer. In some embodiments, the tumor is a breast cancer, ovarian cancer, melanoma, colon cancer, lung cancer, or a squamous cell carcinoma. In some embodiments, the tumor is a primary cancer or a metastatic cancer. In some embodiments, the cells are human cells. In some embodiments, the tumor includes galetin-1 negative cells. In some embodiments, the tumor includes cells having an epithelial to mesenchymal transition (EMT) phenotype. In some embodiments, the tumor includes cells resistant to compound 0118 and/or compound 1097. In some embodiments, the method further includes administering one or more additional therapeutic agents. In some embodiments, the cytotoxic nonpeptidic calixarene- based topomimetic agent and the one or more additional therapeutic agents demonstrate a synergy.

A group, as defined herein, is a group of elements that are traditionally referred to as a collective entity, either based on functionality or organizational convenience. An organic group, as defined herein, is a group that includes at least one carbon atom a is used for the purpose of this invention to mean a hydrocarbon group that is classified as an aliphatic group, cyclic group, or combination of aliphatic and cyclic groups (e.g., alkaryl and aralkyl groups).

As used herein, the terms "alkyl", and the prefix "alk-" are inclusive of both straight chain and branched chain groups and of cyclic groups, e.g., cycloalkyl. Preferably, these groups contain from 1 to 20 carbon atoms. In some embodiments, these groups have a total of up to 10 carbon atoms, up to 8 carbon atoms, up to 6 carbon atoms, or up to 4 carbon atoms. Alkyls may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals. Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2- (butadienyl), 2,4-pentadienyl, 3-(l,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. The term "unsubstituted alkyl" encompasses straight or branched chain saturated hydrocarbon radicals. Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n- propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl. Cyclic groups can be monocyclic or polycyclic and preferably have from 3 to 10 ring carbon atoms. Exemplary cyclic groups include cyclopropyl, cyclopropylmethyl, cyclopentyl, cyclohexyl, adamantyl, and substituted and unsubstituted bornyl, norbornyl, and norbornenyl.

The term "aryl" as used herein includes carbocyclic aromatic rings or ring systems. Examples of aryl groups include phenyl, naphthyl, biphenyl, fiuorenyl and indenyl.

Unless otherwise indicated, the term "heteroatom" refers to the atoms O, S, or N.

The term "heteroaryl" includes aromatic rings or ring systems that contain at least one ring heteroatom (e.g., O, S, N). In some embodiments, the term "heteroaryl" includes a ring or ring system that contains 2 to 12 carbon atoms, 1 to 3 rings, 1 to 4 heteroatoms, and O, S, and/or N as the heteroatoms. Suitable heteroaryl groups include furyl, thienyl, pyridyl, quinolinyl, isoquinolinyl, indolyl, isoindolyl, triazolyl, pyrrolyl, tetrazolyl, irnidazolyl, pyrazolyl, oxazolyl, thiazolyl, berizofuranyl, benzothiophenyl, carbazolyl, benzoxazolyl, pyrirnidinyl, berizimidazolyl, quinoxalinyl, benzothiazolyl, naphthyridinyl, isoxazolyl, isothiazolyl, purinyl, quinazolinyl, pyrazinyl, 1-oxidopyridyl, pyridazinyl, triazinyl, tetrazinyl, oxadiazolyl, thiadiazolyl, and so on.

The term "heterocyclyl" includes non-aromatic rings or ring systems that contain at least one ring heteroatom (e.g., O, S, N) and includes all of the fully saturated and partially unsaturated derivatives of the above mentioned heteroaryl groups. In some embodiments, the term "heterocyclyl" includes a ring or ring system that contains 2 to 12 carbon atoms, 1 to 3 rings, 1 to 4 heteroatoms, and O, S, and N as the heteroatoms. Exemplary

heterocyclyl groups include pyrrolidinyl, tetrahydrofuranyl, morpholinyl, iMomorpholinyl, 1,1-dioxothiomorpholinyl, piperidinyl, piperazinyl, tiiiazolidinyl, imidazolidinyl, isothiazolidinyl, tetrahydropyranyl, quinuclidinyl, homopiperidinyl (azepanyl), 1,4- oxazepanyl, homopiperazinyl (diazepanyl), 1,3-dioxolanyl, aziridinyl, azetidinyl, dihydroisoquinolin-( 1 H)-yl, octahydroisoquinolin-( 1 H)-yl, dmydroquinolin-(2H)-yl, octahyckoquinolin-(2H)-yl, dmydro-lH-imidazolyl, 3-azabicyclo[3.2.2]non-3-yl, and the like.

The terms ester, amide, amine, hydroxyl, halide, sulfonate, phosphonate, and guanidine refer to various different optional functional groups that may be included on groups attached to the topornimetic substrates of the invention. The functional groups are further described by the following chemical formulas: ester = -(CO)-O-; amide = -(CO)- ΝΗ-; amine = -N¾, hydroxyl = -ΟΗ; halogen is an element selected from the group consisting of F, CI, Br, and I; sulfonate = -0-S0₃ ^"; phosphonate = - P(0)(OU)₂; and guanidine = -NH-C(=NH)-NH₂. An example of a group used in an embodiment of the invention that includes a halogen functional group is a trifluoromethyl group.

As used herein, the terms "alkoxy" and "thioalkoxy" refer to groups wherein two hydrocarbon alkyl groups are bonded to an oxygen or a sulfur atom, respectively. For example, a group represented by the formula -O-R is an alkoxy group, whereas a group represented by the formula -S-R is a thioalkoxy group. For example, a cycloalkylalkoxy group is an alkoxy group attached to a cycloalkyl group, whereas an aralkyloxy group is an alkoxy group attached to an aralkyl group, as defined herein. The R within an alkoxy or thioalkoxy group, described above, may be any aryl or alkyl group, as described herein.

The terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl," are meant to include monohaloalkyl and polyhaloalkyl.

When a group is present more than once in any formula described herein, each group is independently selected, whether explicitly stated or not.

The above summary of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the chemical structures of the JIL31, JTL50, JIL54, and JIL70 compounds.

Figure 2 shows the chemical structures of compounds 0118 and 0197.

Figures 3 A to 3H. The anti-proliferative effects of the JTL31, JIL50, JIL54, and JEL70 compared to compound 1097 and compound 0118 on human umbilical vein endothelial cells (HUVECs) (Fig. 3A), normal mouse endothelial cells (2H11) (Fig. 3B), normal human fibroblasts (Fig. 3C), murine fibrosarcoma cells (FSAII) (Fig. 3D), human lung carcinoma (A549) (Fig. 3E), human ovarian carcinoma MA148 cells (Fig. 3F), human breast carcinoma (SCK) (Fig. 3G), and murine melanoma (B16F10) (Fig. 3H). In Figs. 3A to 3H, compound 0118 (·), compound 1097 (■), JIL31 (*), JIL50 (□), JIL54 (o), and JIL70 (□).

Figures 4A and 4B show tumor growth inhibition by JIL54 and JIL70 in the murine B16F10 melanoma model. Figure 4A shows B16F10 tumor growth, measuring tumor volume when compounds were administered at a dose of 10 mg/Kg/day/mouse. Figure 4B shows body weights of mice during treatment.

Figures 5A to 5D show dose escalation studies of the JIL70 compound in the murine B16F10 melanoma model. Figure 5 A shows tumor volumes at dosages of 0.5 mg/kg/day/mouse, 1.5 mg/kg/day/mouse, and 5 mg/kg/day/mouse. As a measure of potential JTL70 toxicity, Figure 5B shows body weights in grams (gm) in mice adniinistered doses of 0.5 mg/kg/day, 1.5 mg/kg/day, and 5 mg/kg/day JIL70. Fig. 5C shows tumor volumes at dosages of 0.1 mg/kg/day/mouse, 0.2 mg/kg/day/mouse, and 0.5 mg/kg/day/mouse. As a measure of potential JIL70 toxicity, Fig. 5D shows body weights in grams (gm) in mice administered doses of 0.1 mg kg day, 0.2 mg/kg/day, and 0.5 mg/kg/day J0L70.

Figures 6A and 6B show effect of JEL70 dose and schedule on tumor growth and body weight in the murine B 16F 10 melanoma model. Figure 6 A shows tumor volumes and Figure 6B shows body weights in grams (gm) in mice administered JTL70 at daily dosages of 2 m g/kg/mouse for each of 10 days ("10 shots"), 5 mg/kg/mouse on days 1, 4, 7, and 10, and 10 mg/kg/mouse on days 1 and 5. All mice received the same total dose during the course of treatment.

Figures 7A and 7B show the cytotoxic effects of various concentrations of JIL70 in the SQ20B cell line (Fig. 7A) and the Colo205 and Colo205R cell lines (Fig. 7B).

Figures 8A to 8D show the antiproliferative effects of various concentrations of JIL70 in the SQ20B cell line (Fig. 8A), the Colo205 cell line (Fig. 8B) and the Colo205R cell line (Fig. 8C). Figure 8D is a comparison of antiproliferative effects of JIL70 in the Colo205 and Colo205R cell lines. In Figure 8D "J3" and "J4" represent results from two separate experiments. Control cells (■) are non-treated cells.

Figures 9A and 9B show cytotoxic and antiproliferative effects of JIL70 in the SQ20B and SQ-PTX cell lines. Figure 9A shows cytotoxic effects and Figure 9B shows antiproliferative effects. In Figure 9B "J3" and "J4" represent results from two separate experiments.

Figures 10A and 10B show cell cycle changes induced in SQ20B cells by a forty- eight hour exposure to 3 μΜ JIL70.

Figure 11 shows the results of a forty-eight hour exposure to 30 μΜ JIL70 on galectin-1 protein expression in the SQ20B cell line. "WB-1" and "WB-2" are western blot results from two separate experiments.

Figures 12A and 12B show cytotoxic and antiproliferative effects of JDL70 in the

MCF7 and MCF7-WISP cell lines. Figure 12A shows cytotoxic effects and Figure 12B shows antiproliferative effects. In Figure 9B "J3" and "J4" represent results from two separate experiments.

Figures 13A and 13B show cytotoxic and antiproliferative effects of JEL70 in the DLD and DLD-SNAIL cell lines. Figure 13A shows cytotoxic effects and Figure 13B shows antiproliferative effects. In Figure 9B "J3" and "J4" represent results f om two separate experiments.

Figures 14A and 14B show cytotoxic effect of various dosages of JIL70 as a function of time (2, 6, 24, 48 and 72 hours exposure to JIL70). Figure 14A is the SQ20B cell line versus the SQ-PTX cell line. Figure 14B is the Colo205 (ColoS) cell line versus the Colo205-R (ColoR) cell line.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention includes novel calixarene-based organic molecules that demonstrate cytotoxic activity. These compounds (also referred to herein as "cytotoxic compounds," "cytotoxic agents," "cytotoxic nonpeptidic calixarene topomimetic agents," "cytotoxic nonpeptidic calixarene topornimetic compounds," "nonpeptidic calixarene topomimetic agents," "nonpeptidic calixarene topomimetic compounds," "calixarene topomimetic agents," "calixarene topomimetic compounds," "cytotoxic calixarene agents," "cytotoxic calixarene compounds," "topomimetic compounds," 'topomimetic agents," "compounds," or "agents") utilize a calixarene scaffold and demonstrate an ability to kill a variety of eukaryotic cell types, including cancer cells, and to inhibit tumor growth. The cytotoxic activity of these compounds is not limited to endothelial cells. While the compounds of the present invention may demonstrate anti-angiogenic activity, the compounds demonstrate broader cytotoxic activity than the anti-angiogenic activity demonstrated by anginex, compound 1097, compound 0118, and/or the other calixarene compounds described in Dings et al., 2006, J Natl Cancer Inst; 98:932-6, WO

2006/042104, and/or US 2008/0300164. Further, the compounds of the present invention demonstrate a greater cytotoxic potency than anginex, compound 1097, compound 0118, and/or the other calixarene compounds described in Dings et al., 2006, J Natl Cancer Inst; 98:932-6, WO 2006/042104, and/or US 2008/0300164. The structure of compound 0118 and compound 1097 is shown in Fig. 2. Compounds of the present invention are non-peptidic topomimetic compounds that mimic a portion of the surface of the βρερ-25 peptide and/or SC-4. βρβρ-25 (also referred to as anginex) is a designed cytokine-like β-sheet forming peptide 33mer that inhibits vascular endothelial cell proliferation and induces apoptosis in these cells. It specifically targets an adhesion/migration receptor on angiogenically-activated endothelial cells (EC). In mouse models, βρερ-25 effectively inhibits tumor angiogenesis and tumor growth. βρερ-25 has the amino acid sequence has the sequence ANIKLSVQMKLFKRHLKW KIIVKLNDGRELSLD (SEQ ID NO:l). See, for example, Mayo et al., 2001,

Angiogenesis; 4:45-51; Griffioen et al., 2001, Biochem J; 354(Pt 2):233-242; and Liekens et al., 2001, J Biochem Pharm; 61:253-270. SC4, which has the sequence

KLFKRHLKW II (SEQ ID NO:2), is a designed 12mer that forms an amphipathic helix conformation, disrupts bacterial membranes selectively, and displays bactericidal activity (see, for example, Mayo et al., 2000, Biochem J; 349:717-728).

A topomimetic compound is an organic compound that provides a surface topography that resembles that of an existing bioactive molecule. Calixarene topomimetic compounds of the present invention use an organic calixarene scaffold together with particular groups to model the surface characteristics of peptides such as βρερ-25 or SC-4. A calixarene is a macrocycle or cyclic oligomer based on a hydroxyalkylation product of a phenol and an aldehyde. Calixarene nomenclature is straightforward and involves counting the number of repeating units in the ring and include it in the name. Cytotoxic calixarene topomimetic compounds of the present invention include, but are not limited to, those with a calix[4]arene scaffold, a calix[5]arene scaffold, or a calix[6]arene scaffold.

Cytotoxic compounds of the present invention provide a variety of biological activities, including, but not limited to, one or more of those described herein. For example, a compound of the present invention may demonstrate an ability to kill a variety of eukaryotic cell types, including, but not limited to, cancer cells. A compound of the present invention may demonstrate antitumor activity, killing tumor cells and/or inhibiting tumor cell growth. A compound of the present invention may demonstrate cytotoxic activity against noncancerous cells, killing and/or inhibiting the growth of such cells. This cytotoxic activity need not be limited to endothelial cells. For example, a compound of the present invention may kill both endothelial cells and non-endothelial cells, such as, for example, epithelial cells and/or cells of mesenchymal origin. A compound of the present invention may demonstrate greater activity than known calixarene compounds, suggesting such compounds have different molecular targets than already reported calixarene compounds. For example, the compounds of the present invention may demonstrate greater activity than anginex, compound 1097 and/or compound 0118. A greater activity may be, for example, at least about 5 -fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, at least about 100-fold, at least about 250-fold, at least about 500-fold, or at least about 1,000-fold. Such improved activity may be demonstrated in vitro and/or in vivo, including, but not limited to, in any of the functional activities described herein and any of those described in Griffioen et al, 2001, Biochem J; 354:233-42; van der Schaft et al., 2002, FASEB J; 16:1991-3; Dings et al., 2003, Cancer Res; 63:382-5; Dings et al., 2003, Cancer Lett; 194:55-66; Dings et al., 2006, J Natl Cancer Inst; 98:932-6, WO 2006/042104, and US 2008/0300164. For example, a cytotoxic compound of the present invention may demonstrate about at least 5- fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, at least about 100- fold, at least about 250-fold, at least about 500-fold, or at least about 1,000-fold greater cytotoxic activity than compound 1097 and/or compound 0118 when assayed against one or more of the cell lines described herein, including, for example, the B16F10 murine melanoma cell line, the 2H11 normal mouse endothelial cell line, the FSA11 murine fibrosarcoma cell line, the A549 human lung carcinoma cell line, the SCK human breast cancer cell line, normal human fibroblasts, HUVEC cells, the MA148 human ovarian carcinoma cell line, the Colo205 cell line (which may also be referred to herein as Colo- 205, COLO205, COLO-205, ColoS, Colo-S, or COLO-S), the Colo205R (which may also be referred to herein as Colo-R, ColoR, COLO-R, or Colo205-R) cell line, the SQ20B cell line, the SQ-PTX cell line, the MCF7 cell line, the MCF7-WISP cell line, the DLD cell lien, and/or the DLD-SNAIL cell line.

The B16F10 murine melanoma cell line is available from the American Type Culture Collection (ATCC^®) as ATCC No. CRL-6475™. See also, Fidler, 1975, Cancer Res; 35:218-224; Fidler et al., 1976, Cancer Res; 36:3608-3615; Fidler and Bucana, 1977, Cancer Res; 37:3945-3956; Fidler and Kiipke, 1977, Science; 197:893-895; and Briles and Kornfeld, 1978, J Natl Cancer Inst; 60:1217-1222.

In some embodiments, a cytotoxic compound of the present invention may demonstrate at least about a 5 fold, about 5 to 10 fold, at least about a 10 fold, about a 5 fold to about a 20 fold, about a 10 fold to about a 20 fold, at least about a 20 fold, about a 5 fold to about a 100 fold, about a 10 fold to about a 100 fold, about a 20 fold to about a 100 fold, and/or at least about a 100 fold increased cytotoxic activity against the A549 and/or B16F10 cell lines in comparison to the cytotoxic activity of compound 0118.

In some embodiments, a cytotoxic compound of the present invention may demonstrate at least about a 5 fold, about 5 to 10 fold, at least about a 10 fold, about a 5 fold to about a 20 fold, about a 10 fold to about a 20 fold, at least about a 20 fold, about a 5 fold to about a 100 fold, about a 10 fold to about a 100 fold, about a 20 fold to about a 100 fold, and/or at least about a 100 fold increased cytotoxic activity against the A549 and/or B16F10 cell lines in comparison to the cytotoxic activity of compound 1097.

In some embodiments, a cytotoxic compound of the present invention may demonstrate an IC50 value of about 100 μΜ, about 50 μΜ, about 20 μΜ, about 10 μΜ, about 1 μΜ, about 0.1 μΜ, or less when assayed against a cell line as described herein, including, but not limited to, the 2H11, FSA11, A549, SCK and/or B16F10 cell lines.

A compound of the present invention may demonstrate cytotoxic activity against cells or a tumor that demonstrates resistance to compound 0118, compound 1097, and/or anginex. A compound of the present invention may demonstrate cytotoxic activity against cells or a tumor that demonstrate resistance to compound 0118, but not compound 1097. A compound of the present invention may demonstrate cytotoxic activity against cells or a tumor that demonstrates resistance to compound 1097, but not compound 0118. An example of a cell line that is resistant to compound 0118 is the SQ-PTX cell line. A compound of the present invention may demonstrate cytotoxic activity against the SQ-PTX cell line. The structure of compound 0118 and compound 1097 is shown in Fig. 2.

A compound of the present invention may demonstrate cytotoxic activity against cells or a tumor that demonstrates an epithelial to mesenchymal transition (EMT) phenotype. A compound of the present invention may demonstrate cytotoxic activity against cells or a tumor that do not demonstrate an epithelial to mesenchymal transition (EMT) phenotype. A compound of the present invention may demonstrate cytotoxic activity against both cells or a tumor that demonstrate an epithelial to mesenchymal transition (EMT) phenotype and cells or a tumor that do not demonstrate an epithelial to mesenchymal transition (EMT) phenotype. A compound of the present invention may demonstrate cytotoxic activity similar to the cytotoxicity demonstrated in any of the examples included herewith. For example, a compound of the present invention may demonstrate cytotoxic activity against the cell line pairs Colo205 and Colo205R; SQ20B and SQ-PTX; MCF7 and MCF7-WISP; and/or DLD and DLD-SNAIL.

In some embodiments, a cytotoxic compound of the present invention may demonstrate about 5 to 10 fold increased cytotoxic activity against a cell line

demonstrating an EMT phenotype, including, but not limited to any of the EMT cell lines described herein, in comparison to the cytotoxic activity of compound 0118 against the same cell line.

A compound of the present invention may demonstrate cytotoxic activity against cells or a tumor that express a high level of galectin-1. A compound of the present invention may demonstrate cytotoxic activity against cells or a tumor that express low levels of galectin-1. A compound of the present invention may demonstrate cytotoxic activity against cells or a tumor that do not express galectin-1. In some embodiments, a compound of the present invention does not bind to galectin-1. In some embodiments, a compound of the present invention binds to galectin-1.

A compound of the present invention may demonstrate a cytotoxic activity as described in any of the examples included herewith.

In some embodiments, a cytotoxic compound of the present invention is a compound having Formula I or II and demonstrating at least a 5 fold increased cytotoxic activity against the A549 and/or B16F 10 cell lines in comparison to the cytotoxic activity of compound 0118 and/or compound 1097. Formula I and II are shown below:

In the above structure, each R through R group is independently hydrogen or an organic group, wherein R¹ through R⁴ are each independently hydrogen or an organic

Q

group of like polarity and R through R are each independently hydrogen or an organic group of like polarity that is of opposite polarity than those of R¹ through R⁴.

For instance, the cytotoxic compound of Formula I or Formula II may include groups R through R are each independently hydrogen, halogen, alkyl, cycloalkyl, aryl, aralkyl, alkoxy, thioalkoxy, cycloalkylalkoxy, heterocycloalkyl, aralkyloxy, or heteroaryl, optionally including ester, amide, amine, hydroxyl, halogen, sulfonate, phosphonate, guanidine, and/or heteroaryl groups. In further embodiments, groups R¹ through R⁴ are each independently hydrogen, halogen, alkyl, cycloalkyl, aryl, aralkyl, alkoxy, thioalkoxy,

r o

cycloalkylalkoxy, heterocycloalkyl, aralkyloxy, or heteroaryl, and R through R are each independently any of these groups incorporating ester, amide, amine, hydroxyl, halogen, sulfonate, phosphonate, guanidine, and/or heteroaryl groups. In an alternate embodiment, groups R through R are each independently hydrogen, halogen, alkyl, cycloalkyl, aryl, aralkyl, alkoxy, thioalkoxy, cycloalkylalkoxy, heterocycloalkyl, aralkyloxy, or heteroaryl, and R¹ through R⁴ are each independently any of these groups i coφorating ester, amide, amine, hydroxyl, halogen, sulfonate, phosphonate, guanidine, and/or heteroaryl groups

In further embodiments a cytotoxic agent of Formula I or Formula II may include groups R¹ through R⁸ that are each independently hydrogen, alkyl, cycloalkyl, aralkyl, alkoxy, cycloalkylalkoxy, or aralkyloxy optionally including ester, amide, amine, hydroxyl, sulfonate, phosphonate, guanidine and/or heteroaryl groups. In further embodiments, groups R¹ through R⁴ may be each independently alkyl, cycloalkyl, aralkyl,

o

alkoxy, cycloalkylalkoxy, or aralkyloxy, and R through R may be each independently any of these groups incorporating ester, amide, amine, hydroxyl, sulfonate, phosphonate, guanidine and/or heteroaryl groups. In an alternate embodiment, R through R may be each independently alkyl, cycloalkyl, aralkyl, alkoxy, cycloalkylalkoxy, or aralkyloxy, and R¹ through R⁴ may be each independently any of these groups incorporating ester, amide, amine, hydroxyl, sulfonate, phosphonate, guanidine and/or heteroaryl groups.

A cytotoxic compound of the present invention with a formula of Formula I or Formula II excludes compound 0118, compound 1097, compound bridged 4, and/or any of the other calixarene compounds described in Dings et al., 2006, J Natl Cancer Inst; 98:932-6, WO 2006/042104, and/or US 2008/0300164.

A cytotoxic compound of the present invention may be a compound having the structure of Formula III below:

wherein y + z is 4 to 6;

wherein R⁹ and R¹⁰ are each independently a hydrogen or an organic group and

R⁹ and R¹⁰ have a hydrophobic polarity;

wherein R¹¹ is hydrogen or an organic group;

wherein x is 1 to 6; and

wherein R is an organic group; and

wherein R¹³ is an organic group; and isomers (e.g., diastereomers and enantiomers), tautomers, salts, solvates, polymorphs, prodrugs, derivatives and substitutions thereof, and pharmaceutically acceptable salts thereof.

An agent of Formula III may demonstrate one or more of the biological activities discussed in more detail herein.

In some embodiments of a cytotoxic agent of Formula III, each of R⁹ and R¹⁰ is independently selected from hydrogen, a branched or unbranched alkyl, a branched or unbranched alkenyl, a cycloalkyl, an aryl, or a heteroaryl. In certain embodiments of Formula III, each of R⁹ and R¹⁰ are independently a hydrogen, a branched or unbranched alkyl having six carbons or less, a branched or unbranched alkenyl (having one or more carbon-carbon double bonds) having six carbons or less, a cycloalkyl having six carbons or less, an aryl having ten carbons or less, or a heteroaryl having five carbons or less. In certain embodiments, each of R⁹ and R¹⁰ are independently a hydrogen or a branched or unbranched alkyl having six carbons or less. In some embodiments, each of R⁹ and R¹⁰ are each independently selected from hydrogen, methyl, ethyl, propyl, or tert-butyl.

In some embodiments of a cytotoxic agent of Formula III, R¹¹ is hydrogen, a branched or unbranched alkyl, a branched or unbranched alkenyl, a cycloalkyl, an aryl, or a heteroaryl. In certain embodiments, R¹¹ is a hydrogen, a branched or unbranched alkyl having six carbons or less, a branched or unbranched alkenyl (having one or more carbon- carbon double bonds) having six carbons or less, a cycloalkyl having six carbons or less, an aryl having ten carbons or less, or a heteroaryl having five carbons or less. In certain embodiments, each of R⁹ and R¹⁰ is independently a hydrogen or a branched or unbranched alkyl having six carbons or less. In some embodiments, R¹¹ is hydrogen, methyl, ethyl, propyl, or tert-butyl.

In some embodiments of a cytotoxic agent of Formula III, R¹² and R¹³ are each independently selected from hydrogen, a branched or unbranched alkyl, a branched or unbranched alkenyl, a cycloalkyl, an aryl, or a heteroaryl. In certain embodiments, each of R¹² and R¹³ is independently a hydrogen, a branched or unbranched alkyl having six carbons or less, a branched or unbranched alkenyl (having one or more carbon-carbon double bonds) having six carbons or less, a cycloalkyl having six carbons or less, an aryl having ten carbons or less, or a heteroaryl having five carbons or less. In certain embodiments, each of R¹² and R¹³ is independently a hydrogen or a branched or unbranched alkyl having six carbons or less. In some embodiments, R¹² and R¹³ are each independently selected from hydrogen, methyl, ethyl, propyl, or tert- butyl.

In some embodiments of an agent of Formula III, z is 0. In some embodiments of an agent of Formula III, z is 0 and y is 4, 5, or 6. In some embodiments, y = 3 and z = 3. In some embodiments of an agent of Formula III, x is 1, 2, 3, or 4. In some preferred embodiments, y is 4, z is 0, x is 2, and R and R are methyl groups.

Cytotoxic agents of the present invention include cytotoxic agents of Formula III with the proviso that compound 0118, compound 1097, compound bridged 4, and/or any of the other calixarene compounds described in Dings et al., 2006, J Natl Cancer Inst;

98:932-6, WO 2006/042104, and/or US 2008/0300164 are excluded.

Also exclude from agents of the present invention are compounds of Formula III, wherein if z = 0, y = 4, x = 2, and R⁹ is tert-butyl, then both R¹² and R¹³ are not ethyl groups. However, such an agent may be used in any of the methods of the present invention.

In some aspects, a cytotoxic compound of the present invention may be a compound selected from the compounds JIL70 (also referred to herein as JIL-70, JIL 70, or Jil-70), JIL54 (also referred to herein as JIL-54 or JIL 54), JIL50 (also referred to herein as JDL-50 or JIL 50), and JIL30 (also referred to herein as JIL-30 or JIL 30), and isomers (e.g., diastereomers and enantiomers), tautomers, salts, solvates, polymorphs, prodrugs, derivatives and substitutions thereof, and pharmaceutically acceptable salts thereof.

JIL70 having a structure accordin to the formula:

JIL54 having a structure according to the formula:

JIL50 having a structure according to the formula:

And, JIL31 having a structure according to the formula:

The invention is inclusive of the compounds described herein (including intermediates) in any of their pharmaceutically acceptable forms, including isomers (e.g., diastereomers and enantiomers), tautomers, salts, solvates, polymorphs, prodrugs, and the like. It should be understood that the term "compound" includes any or all of such forms, whether explicitly stated or not (although at times, "salts" are explicitly stated). The present disclosure also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form. 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 include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic,

monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like. Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.

In addition to salt forms, the present invention provides compounds which are in a prodrug form. Prodrugs of the compounds or complexes described herein readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. The term, "prodrug", as defined herein, is a biologically inactive derivative of a parent drug molecule that exerts its pharmacological effect only after chemical and/or enzymatic conversion to its active form in vivo. Prodrugs include those designed to circumvent problems associated with delivery of the parent drug. This may be due to poor physicochemical properties, such as poor chemical stability or low aqueous solubility, and may also be due to poor pharmacokinetic properties, such as poor bioavailability or poor half-life. Thus, certain advantages of prodrugs may include improved chemical stability, absorption, and/or PK properties of the parent carboxylic acids. Prodrugs may also be used to make drugs more "patient friendly," by minimizing the frequency (e.g., once daily) or route of dosing (e.g., oral), or to improve the taste or odor if given orally, or to minimize pain if given parenterally. Additionally, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment.

Compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

Compounds of the present invention may possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers and individual isomers are encompassed within the scope of the present invention. The graphic representations of racemic, ambiscalemic and scalemic or enantiomerically pure compounds used herein follow those described by Maehr, J Chem. Ed. 1985, 62: 114-120.

Compounds of the invention can exist in particular geometric or stereoisomeric forms. The invention contemplates all such compounds, including cis- and trans-isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)- isomers, the racemic mixtures thereof, and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, as falling within the scope of the invention.

Additional asymmetric carbon atoms can be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.

The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium, iodine-125, or carbon-14. All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed wil n the scope of the present invention.

A cytotoxic compound as described herein may be conjugated to a diagnostic agent, a therapeutic agent, a detectable marker, a targeting moiety, or a liposome. A targeting ligand may allow for the specific delivery of a cytotoxic agent to tumor tissues, thereby reducing side effects and gaining efficiency. See, for example, Dings et al., 2010, Bioconjugate Chem; 21(l):20-27. Conjugating to lipidic vehicles may be used to deliver therapeutic agents for treatment of disease or contrast agents for molecular imaging. For example, fluorescently labeled paramagnetic liposomes may be conjugated to a cytotoxic agent as described herein, following procedures described in more detail in Ricardo et al, 2007, Bioconjugate Chem; 18(3):785-790.

Also included in the present invention are compositions including one or more of the compounds and/or conjugates as described herein. Such a composition may include a pharmaceutically acceptable carrier. As used, a pharmaceutically acceptable carrier refers to one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration to a human or other vertebrate ariimal. Such a carrier may be pyrogen free. The present invention also includes methods of making and using the cytotoxic compounds, conjugates and compositions described herein.

The compositions of the present disclosure may be formulated in pharmaceutical preparations in a variety of forms adapted to the chosen route of administration. One of skill will understand that the composition will vary depending on mode of administration and dosage unit. For example, for parenteral adrninistration, isotonic saline can be used. For topical administration a cream, including a carrier such as dimethylsulfoxide (DMSO), or other agents typically found in topical creams that do not block or inhibit activity of the peptide, can be used. Other suitable carriers include, but are not limited to alcohol, phosphate buffered saline, and other balanced salt solutions. The compounds of this invention can be administered in a variety of ways, including, but not limited to, intravenous, topical, oral, subcutaneous, intraperitoneal, intramuscular, and intratumor deliver. In some aspects, the compounds of the present invention may be formulated for controlled or sustained release. In some aspects, a formulation for controlled or sustained release is suitable for subcutaneous implantation. In some aspects, a formulation for controlled or sustained release includes a patch. A compound may be formulated for enteral adrmnistration, for example, formulated as a capsule or tablet.

Administration may be as a single dose or in multiple doses. Preferably the dose is an effective amount as deteraiine by the standard methods, including, but not limited to, those described herein. Those skilled in the art of clinical trials will be able to optimize dosages of particular compounds through standard studies. Additionally, proper dosages of the compositions can be determined without undue experimentation using standard dose-response protocols. Administration includes, but is not limited to, any of the dosages and dosing schedules, dosing intervals, and/or dosing patterns described in the examples included herewith.

Also included in the present invention are methods for killing cells by contacting cells with a compound of the present invention. Such a contacting step may occur in vitro. In alternate embodiments, the contacting step may occur in vivo. In additional

embodiments, the contacted cells are present in a cell culture, a tissue, an organ, or an organism. In some embodiments, the cells are mammalian cells, while in further embodiments the cells are human cells. The compounds of the present invention are useful in the treatment of cancer and other pathological disorders, including, but not limited to, proliferation-related disorders, such as, for example, benign hyperplasia, rheumatoid arthritis, restenosis, atherosclerosis, diabetic retinopathy, neovascular glaucoma, and endometriosis. The present invention provides a method for killing and/or inhibiting the proliferation of cells in a subject. This involves administering to a patient an amount of a composition effective to kill and/or inhibit the proliferation of cells. Such cells may cancerous tumor cells. Such cells may be non-cancerous cells.

The present invention provides methods for inhibiting tumorigenesis in a subject. This involves administering to a patient an amount of a composition effective to prevent and/or reduce tumor growth, wherein the composition includes one or more calixarene- based cytotoxic agents as described herein. Methods of determining the inhibition of tumorigenesis are well known to those of skill in the art, including evaluation of tumor shrinkage, survival, etc.

The compounds, conjugates, and compositions of the present disclosure may be administered to a patient for the treatment of cancer. Cancers to be treated include, but are not limited to, melanoma, basal cell carcinoma, colorectal cancer, pancreatic cancer, breast cancer, prostate cancer, lung cancer (including small-cell lung carcinoma and non-small- cell lung carcinoma, leukemia, lymphoma, sarcoma, ovarian cancer, Kaposi's sarcoma, Hodgkin's lymphoma, Non-Hodgkin's lymphoma, multiple myeloma, neuroblastoma, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, head and neck cancers, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, kidney cancer, endometrial cancer, glioblastoma, and adrenal cortical cancer.

In some aspects, the cancer is a primary cancer. In some aspects, the cancer is metastatic.

In some aspects, the cancer is a tumor associated cancer. In other aspects, the cancer is a blood borne cancer. Such hematological blood borne malignancies include, for example, leukemia, lymphoma, multiple myeloma, acute myelogenous leukemia, myelodysplastic syndrome, non-Hodgkins lymphoma, or follicular lymphoma.

The compounds, conjugates, and compositions of the present disclosure may be administered to a patient for the treatment of cancers of epithelial cell origin (carcinoma), non-hematopoietic mesenchymal cell origin (sarcoma), hematopoietic cell origin, germ cell origin, or a cancer whose origin or developmental lineages is unknown. Examples of carcinomas of epithelial origin that may be treated include, but are not limited to, adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, anaplastic carcinoma, large cell carcinoma, small cell carcinoma, ovarian cancer, melanoma, and cancer of the lung, breast, prostate, colon, rectum, and/or pancreas, and metastasis thereof. Examples of sarcomas of mesenchyaml cell origin include, but are not limited to, osteosarcoma, chondrosarcoma, leiomyosarcoma, rhabdomyosarcoma, fibrosarcoma, schwannoma, fibroblastic osteosarcoma, myofibroblastic sarcoma, malignant fibrous histiocytoma, fibromyxosarcoma, spindle cell sarcoma, and adamantinoma.

The compounds, conjugates, and compositions of the present disclosure may be administered to a patient for the treatment of cancer that demonstrates an epithelial to mesenchymal transition phenotype (EMT). Epithelial-mesenchymal transition or transformation (EMT) is defined by the loss of epithelial phenotype and the acquisition of a mesenchymal phenotype. The EMT is an orchestrated series of events in which cell-cell and cell-extracellular matrix interactions are altered to release epithelial cells from the surrounding tissue, the cytoskeleton is reorganized to confer the ability to move through a three-dimensional extracellular matrix, and a new transcriptional program is induced to maintain the mesenchymal phenotype. It is characterized by, for example, the loss of cell adhesion, cadherin switching (down-regulation of E-cadherin and up-regulation of mesenchymal cadherins such as N-cadherin or cadherin- 11), and increased cell mobility. While EMT is essential in embryonic development, it is potentially destructive when deregulated, and it is becoming increasingly understood that inappropriate utilization of EMT mechanisms is an integral component of the progression of many tumors of epithelial tissues. See, for example, Radisky, 2005, J Cell Sci; 118:4325-4326.

In carcinoma cells, EMT can be associated with increased aggressiveness, and invasive and metastatic potential. It has been proposed that EMT-like processes occur during tumor progression in carcinomas, particularly at specific stages where tumor cells disassemble and migrate to tissue/organ sites distant from the primary tumors. The complex genetic changes necessary to accomplish the phenotypic changes associated with EMT are mediated by a number of specific transcription factors. These transcription factors include Snail (also known as Snaill ; Cano et al., 2000, Nat Cell Biol; 2: 76-83), Slug (also known as Snail2; Bolos et al., 2003, J Cell Sci; 116:499-511), SIP-1 (ZEB-2; Comijn et al., 2001, Mol Cell; 7:1267-78), dEFl (ZEB-1; Eger et al., 2005, Oncogene; 24:2375-85), E12/E47 (Perez-Moreno et al., 2001, J Biol Chem; 276:27424-31), and Twist (Yang et al., 2004, Cell; 117:927-39). When expressed in a variety of cell types, these factors act as transcriptional repressors of E-cadherin and modulate directly or indirectly the expression of a wide number of genes involved in cancer invasion and metastasis, and consequently promote complete EMT in vitro. Additionally, the expression of some of these EMT inductors has been detected in a variety of human cancer biopsies, including breast carcinomas, and their overexpression is usually related to increased tumor aggressiveness or recurrence, unfavorable clinicopathologic variables, and poor prognosis (reviewed in Peinado et al., 2007, Nat Rev Cancer; 7:415-28).

The compounds, conjugates, and compositions of the present disclosure may be administered to a patient for the treatment of a cancer that demonstrate a resistance to an anti-angiogenic agent, such as for example, anginex, compound 0118, and/or compound 1097.

Galectin-1 (gal-1) is a rather ubiquitous carbohydrate-binding protein (binding to β- galactoside groups on various cell surface receptors) that is over-expressed in many different types of cancers. Increased galectin-1 expression by tumor and connective tissue supporting the tumor often correlates with the aggressiveness of the tumor and the acquisition of a metastatic phenotype. In preclinical studies, galectin-1 has been shown to play a role in tumor transformation, tumor cell proliferation, cell aggregate, adhesion, migration, apoptosis, and immunoregulation. Galectin-1 is often found to be highly elevated in tumor stroma in several cancers including breast, colon, prostate and ovarian. The compounds, conjugates, and compositions of the present disclosure may be administered to a patient for the treatment of a cancer in which the cancerous cells express a high level of galectin-1. A compound, conjugate, or composition of the present disclosure may be administered to a patient for the treatment of a cancer in which the cancerous cells express low levels of galectin-1. A compound, conjugate, or composition of the present disclosure may be administered to a patient for the treatment of a cancer in which cancerous cells do not express galectin-1. A compound, conjugate, or composition of the present disclosure may or may not target galectin-1.

The efficacy of treatment of a cancer may be assessed by any of various parameters well known in the art. This includes, but is not limited to, determinations of a reduction in tumor size, determinations of the inhibition of the growth, spread, invasiveness, vascularization, angiogenesis, and/or metastasis of a tumor, determinations of the inhibition of the growth, spread, invasiveness and/or vascularization of any metastatic lesions, determinations of tumor infiltrations by immune system cells, and/or

determinations of an increased delayed type hypersensitivity reaction to tumor antigen. The efficacy of treatment may also be assessed by the determination of a delay in relapse or a delay in tumor progression in the subject or by a detennination of survival rate of the subject, for example, an increased survival rate at one or five years post treatment. As used herein, a relapse is the return of a tumor or neoplasm after its apparent cessation, for example, such as the return of leukemia.

Toxicity and therapeutic efficacy of the compounds, conjugates, and compositions of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD₅₀ and ED50. Compounds that exhibit high therapeutic indices are preferred. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.

As used herein "treating" or "treatment" can include therapeutic and/or prophylactic treatments. "Treating a disorder," as used herein, is not intended to be an absolute term. Treatment may lead to an improved prognosis or a reduction in the frequency or severity of symptoms. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. Likewise, the term "preventing," as used herein, is not intended as an absolute term. Instead, prevention refers to delay of onset, reduced frequency of symptoms, or reduced severity of symptoms associated with a disorder. Prevention therefore refers to a broad range of prophylactic measures that will be understood by those in the art. In some circumstances, the frequency and severity of symptoms is reduced to non-pathological levels. In some circumstances, the symptoms of an individual receiving the compositions of the invention are only 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 or 1% as frequent or severe as symptoms experienced by an untreated individual with the disorder.

The findings of the present disclosure can be used in methods that include, but are not limited to, methods for treating cancer, methods for inhibiting cell growth, and methods for killing cells.

The agents of the present disclosure can be administered by any suitable means including, but not limited to, for example, oral, rectal, nasal, topical (including

transdermal, aerosol, buccal and/or sublingual), vaginal, parenteral (including

subcutaneous, intramuscular, and/or intravenous), intradermal, intravesical, intra-joint, intra-arteriole, intraventricular, intracranial, intraperitoneal, intranasal, by inhalation, or intralesional (for example, by injection into or around a tumor).

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, intraperitoneal, and intratumoral acrmmistration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human

administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by the FDA. Such preparation may be pyrogen-free.

For enteral administration, the inhibitor may be administered in a tablet or capsule, which may be enteric coated, or in a formulation for controlled or sustained release. Many suitable formulations are known, including polymeric or protein microparticles

encapsulating drug to be released, ointments, gels, or solutions which can be used topically or locally to administer drug, and even patches, which provide controlled release over a prolonged period of time. These can also take the form of implants. Such an implant may be implanted within the tumor.

The compounds of the present invention can also be provided in a lyophilized form. Such compositions may include a buffer, e.g., bicarbonate, for reconstitution prior to administration, or the buffer may be included in the lyophilized composition for reconstitution with, e.g., water. The lyophilized composition may further comprise a suitable vasoconstrictor, e.g., epmephrine. The lyophilized composition can be provided in a syringe, optionally packaged in combination with the buffer for reconstitution, such that the reconstituted composition can be immediately administered to a patient.

Therapeutically effective concentrations and amounts may be determined for each application herein empirically by testing the compounds in known in vitro and in vivo systems, such as those described herein, dosages for humans or other animals may then be extrapolated therefrom.

It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions and methods. A cytotoxic agent of the present invention may be administered at a dose of about 0.01 μΜ to about 1000 μΜ, about 0.01 μΜ to about 100 μΜ, 0.1 μΜ to about 1000 μΜ about 0.1 μΜ to about 100 μΜ, about 0.1 μΜ to about 10 μΜ, about 0.1 μΜ to about 5 μΜ, about 0.1 μΜ to about 1 μΜ, about 0.5 μΜ to about 100 μΜ, about 0.5 μΜ to about 10 μΜ, 0.5 μΜ to about 5 μΜ, about 0.5 μΜ to about 1 μΜ, about 1 μΜ to about 1000 μΜ, about 1 μΜ to about 100 μΜ, about 1 μΜ to about 10 μΜ, 1 μΜ to about 5 μΜ, about 5 μΜ to about 1000 μΜ, about 5 μΜ to about 100 μΜ, about 5 μΜ to about 10 μΜ, about 10 μΜ to about 1000 μΜ, or about 10 μΜ to about 100 μΜ.

A cytotoxic agent of the present invention may be administered at a dose of about 0.01 μΜ, about 0.033 μΜ, about 0.05 μΜ, about 0.075 μΜ, about 0.1 μΜ, about 0.2 μΜ, about 0.3 μΜ, about 0.33 μΜ, about 0.4 μΜ, about 0.5 μΜ, about 0.6 μΜ, about 0.7 μΜ, about 0.75 μΜ, about 0.8 μΜ, about 0.9 μΜ, about 1 μΜ, about 1.25 μΜ, about 1.5 μΜ, about 1.75 μΜ, about 2 μΜ, about 2.5 μΜ, about 3 μΜ, about 3.3 μΜ, about 3.5 μΜ, about 4 μΜ, about 4.5 μΜ, about 5 μΜ, about 6 μΜ, about 7 μΜ, about 7.5 μΜ, about 8 μΜ, about 9 μΜ, about 10 μΜ, about 12.5 μΜ, about 15 μΜ, about 20 μΜ, about 25 μΜ, about 30 μΜ, about 35 μΜ, about 40 μΜ, about 45 μΜ, about 50 μΜ, about 55 μΜ, about 60 μΜ, about 65 μΜ, about 70 μΜ, about 75 μΜ, about 80 μΜ, about 85 μΜ, about 90 μΜ, about 95 μΜ, about 100 μΜ, about 125 μΜ, about 133 M, about 150 μΜ, about 175 μΜ, about 200 μΜ, about 250 μΜ, about 300 μΜ, about 333 μΜ, about 400 μΜ, about 500 μΜ, about 600 μΜ, about 700 μΜ, about 750 μΜ, about 800 μΜ, about 900 μΜ, or about 1000 μΜ, or any range thereof.

A cytotoxic agent of the present invention may be administered at a dose of 0.01 μΜ, 0.033 μΜ, 0.05 μΜ, 0.075 μΜ, 0.1 μΜ, 0.2 μΜ, 0.3 μΜ, 0.33 μΜ 0.4 μΜ, 0.5 μΜ, 0.6 μΜ, 0.7 μΜ, 0.75 μΜ, 0.8 μΜ, 0.9 μΜ, 1 μΜ, 1.25 μΜ, 1.5 μΜ, 1.75 μΜ, 2 μΜ, 2.5 μΜ, 3 μΜ, 3.3 μΜ 3.5 μΜ, 4 μΜ, 4.5 μΜ, 5 μΜ, 6 μΜ, 7 μΜ, 7.5 μΜ, 8 μΜ, 9 μΜ, 10 μΜ, 12.5 μΜ, 15 μΜ, 20 μΜ, 25 μΜ, 30 μΜ, 35 μΜ, 40 μΜ, 45 μΜ, 50 μΜ, about 55 μΜ, 60 μΜ, 65 μΜ, 70 μΜ, 75 μΜ, 80 μΜ, 85 μΜ, 90 μΜ, 95 μΜ, 100 μΜ, 125 μΜ, 133 Μ, 150 μΜ, 175 μΜ, 200 μΜ, 250 μΜ, 300 μΜ, 333 μΜ, 400 μΜ, 500 μΜ, 600 μΜ, 700 μΜ, 750 μΜ, 800 μΜ, 900 μΜ, or 1000 μΜ, or any range thereof.

A cytotoxic agent of the present invention may be administered at a dose of about

0.01 mg/kg to about 1000 mg/kg, about 0.1 mg/kg to about 100 mg/kg, about 1 mg kg to about 100 mg/kg, about 1 mg/kg to about 50 mg/kg, about 1 mg/kg to about 10 mg/kg, about 10 mg/kg to about 100 mg/kg, about 10 mg/kg to about 50 mg/kg, about 10 mg/kg to about 25 mg/kg, about 5 mg/kg to about 100 mg/kg, about 5 mg/kg to about 75 mg/kg, about 5 mg/kg to about 50 mg/kg, about 5 mg/kg to about 25 mg/kg, about 5 mg/kg to about 10 mg/kg, about 1 mg/kg to about 1000 mg/kg, about 1 mg/kg to about 500 mg/kg, about 1 mg/kg to about 100 mg/kg, about 1 mg/kg to about 50 mg/kg, about 1 mg/kg to about 25 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1 mg/kg to about 5 mg/kg, about 1 mg/kg to about 1.5 mg/kg, about 0.5 mg/kg to about 1000 mg/kg, about 0.5 mg/kg to about 500 mg/kg, about 0.5 mg/kg to about 250 mg/kg, about 0.5 mg/kg to about 100 mg/kg, about 0.5 mg/kg to about 50 mg/kg, about 0.5 mg/kg to about 25 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 0.5 mg/kg to about 7.5 mg/kg, about 0.5 mg/kg to about 5 mg/kg, about 0.5 mg/kg to about 2.5 mg/kg, about 0.5 mg/kg to about 1 mg/kg, about 0.5 mg/kg to about 0.75 mg/kg, about 0.2 mg/kg to about 1000 mg/kg, about 0.2 mg/kg to about 500 mg/kg, about 0.2 mg/kg to about 250 mg/kg, about 0.2 mg/kg to about 100 mg/kg, about 0.2 mg/kg to about 50 mg/kg, about 0.2 mg/kg to about 25 mg/kg, about 0.2 mg/kg to about 10 mg/kg, about 0.2 mg/kg to about 7.5 mg/kg, about 0.2 mg/kg to about 5 mg/kg, about 0.2 mg/kg to about 2.5 mg/kg, about 0.2 mg/kg to about 1 mg/kg, about 0.2 mg/kg to about 0.75 mg/kg, about 0.2 mg/kg to about 0.5 mg/kg, about 0.1 mg/kg to about 500 mg/kg, about 0.1 mg/kg to about 250 mg/kg, about 0.1 mg/kg to about 100 mg/kg, about 0.1 mg/kg to about 50 mg/kg, about 0.1 mg/kg to about 25 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 7.5 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about 0.1 mg/kg to about 2.5 mg/kg, about 0.1 mg/kg to about 1 mg/kg, about 0.1 mg/kg to about 0.75 mg/kg, about 0.1 mg/kg to about 0.5 mg/kg, about 0.1 mg/kg to about 0.25 mg/kg, or about 0.1 mg/kg to about 0.2 mg/kg.

A cytotoxic agent of the present invention may be administered at a dose of about 1000 mg/kg, about 750 mg/kg, about 500 mg/kg, about 400 mg/kg, about 300 mg/kg, about 250 mg/kg, about 200 mg/kg, about 150 mg/kg, about 125 mg/kg, about 100 mg/kg, about 90 mg/kg, about 80 mg/kg, about 75 mg/kg, about 60 mg/kg, about 50 mg/kg, about 40 mg/kg, about 30 mg/kg, about 25 mg/kg, about 20 mg/kg, about 15 mg/kg, about 12.5 mg/kg, about 10 mg/kg, about 7.5 mg/kg, about 5 mg/kg, about 4 mg/kg, about 3 mg/kg, about 2.5 mg/kg, about 2 mg/kg, about 1.75 mg/kg, about 1.5 mg/kg, about 1.25 mg/kg, about 1 mg/kg, about 0.75 mg/kg, about 0.5 mg/kg, about 0.25 mg/kg, or about 0.1 mg/kg, or any range thereof. A cytotoxic agent of the present invention may be administered at a dose of 1000 mg/kg, 750 mg/kg, 500 mg/kg, 400 mg/kg, 300 mg/kg, 250 mg/kg, 200 mg/kg, 150 mg kg, 125 mg/kg, 100 mg/kg, 90 mg/kg, 80 mg/kg, 75 mg/kg, 60 mg/kg, 50 mg/kg, 40 mg/kg, 30 mg/kg, 25 mg/kg, 20 mg/kg, 15 mg/kg, 12.5 mg/kg, 10 mg/kg, 7.5 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2.5 mg/kg, 2 mg/kg, 1.75 mg/kg, 1.5 mg/kg, 1.25 mg/kg, 1 mg/kg, 0.75 mg/kg, 0.5 mg/kg, 0.25 mg/kg, or 0.1 mg/kg, or any range thereof.

A cytotoxic agent of the present invention may be adniinistered at any of the dosages and dosage ranges disclosed in the examples included herewith.

An agent of the present disclosure may be adniinistered at once, or may be divided into a number of smaller doses to be administered at intervals of time. For example, compounds of the invention may be administered repeatedly, e.g., at least 2, 3, 4, 5, 6, 7, 8, or more times, or may be administered by continuous infusion. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions and methods.

In some therapeutic embodiments, an "effective amount" of an agent is an amount that results in a reduction of at least one pathological parameter. Thus, for example, in some aspects of the present disclosure, an effective amount is an amount that is effective to achieve a reduction of at least about 10%, at least about 15%, at least about 20%, or at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%», at least about 80%, at least about 85%, at least about 90%, or at least about 95%, compared to the expected reduction in the parameter in an individual not treated with the agent.

As used herein, the term "subject" includes, but is not limited to, humans and non- human vertebrates. In preferred embodiments, a subject is a mammal, particularly a human. A subject may be an individual. A subject may be an "individual," "patient," or "host. Non-human vertebrates include livestock animals, companion animals, and laboratory animals. Non-human subjects also include non-human primates as well as rodents, such as, but not limited to, a rat or a mouse. Non-human subjects also include, without limitation, chickens, horses, cows, pigs, goats, dogs, cats, guinea pigs, hamsters, mink, and rabbits.

As used herein "in vitro" is in cell culture and "in vivo" is within the body of a subject. As used herein, "isolated" refers to material that has been either removed from its natural environment (e.g., the natural environment if it is naturally occurring), produced using recombinant techniques, or chemically or enzymatically synthesized, and thus is altered "by the hand of man" from its natural state.

In some aspects of the methods of the present invention, a method further includes the administration of one or more additional therapeutic agents. One or more additional therapeutic agents may be administered before, after, and/or coincident to the

administration of a cytotoxic compound described herein. A cytotoxic compound as described herein and additional therapeutic agents may be administered separately or as part of a mixture or cocktail. In some aspects of the present invention, the administration of cytotoxic compound may allow for the effectiveness of a lower dosage of other therapeutic modalities when compared to the administration of the other therapeutic modalities alone, providing relief from the toxicity observed with the administration of higher doses of the other modalities.

As used herein, an additional therapeutic agent is an agent whose use for the treatment of cancer is known to the skilled artisan. As used herein, in some applications, an additional therapeutic agent is not anginex, compound 0118, and/or compound 1097. In some applications, an additional therapeutic agent is anginex, compound 0118 and/or compound 1097.

Additional therapeutic treatments include, but are not limited to, surgical resection, radiation therapy, hormone therapy, vaccines, antibody based therapies, whole body irradiation, bone marrow transplantation, peripheral blood stem cell transplantation, the aoiiinistration of chemotherapeutic agents (also referred to herein as "antineoplastic chemotherapy agent," "antineoplastic agents," or "antineoplastic chemotherapeutic agents"), cytokines, antiviral agents, immune enhancers, tyrosine kinase inhibitors, protein kinase C (PKC) modulator (such as, for example, the PKC activator ingenol 3-angelate (PEP005) or the PKC inhibitor bismdolylmaleimid (enzastaurin), signal transduction inhibitors, antibiotics, antimicrobial agents, a TLR agonist (such as for example, bacterial lipopolysaccharides (LPS) or a CpG oligonucleotide (ODN)), an inhibitor of IDO, such as, for example, 1-MT, and adjuvants.

A chemotherapeutic agent may be, for example, a cytotoxic chemotherapy agent, such as, for example, epidophyllotoxin, procarbazine, mitoxantrone, platinum coordination complexes such as cisplatin and carboplatin, leucovorin, tegafur, paclitaxel, docetaxol, vincristine, vinblastine, methotrexate, cyclophosphamide, gemcitabine, estramustine, carmustine, adriamycin (doxorubicin), etoposide, arsenic trioxide, irinotecan, epothilone derivatives, navelbene, CPT-11, anastrazole, letrazole, capecitabine, reloxafine, ifosamide, and droloxafine.

A chemotherapeutic agent may be, for example, an alkylating agent, such as, for example, irofulven, nitrogen mustards (such as chlorambucil, cyclophosphamide, ifosfamide, mecUoremarnine, melphalan, and uracil mustard), aziridines (such as thiotepa), methanesulphonate esters (such as busulfan), nitroso ureas (such as carmustine, lomustine, and streptozocin), platinum complexes (such as cisplatin and carboplatin), and bioreductive alkylators (such as mitomycin, procarbazine, dacarbazine and allietamine), emylenimine derivatives, alkyl sulfonates, triazenes, pipobroman, temozolomide, triethylene-melamine, and triemyleneMophosphoramine.

A chemotherapeutic agent may be an antimetabolite, such as, for example, a folate antagonist (such as methotrexate and trimetrexate), a pyrimidine antagonist (such as fluorouracil, fluorodeoxyuridine, CB3717, azacitidine, cytarabine, gemcitabine, and floxuridine), a purine antagonist (such as mercaptopurine, 6-thioguanine, fludarabine, and pentostatin), a ribonucleotide reductase inhibitor (such as hydroxyurea), and an adenosine deaminase inhibitor.

A chemotherapeutic agent may be a DNA strand-breakage agent (such as, for example, bleomycin), a topoisomerase II inhibitor (such as, for example, amsacrine, dactinomycin, daunorubicin, idarubicin, mitoxantrone, doxorubicin, etoposide, and teniposide), a DNA minor groove binding agent (such as, for example, plicamydin), a tubulin interactive agent (such as, for example, vincristine, vinblastine, and paclitaxel), a hormonal agent (such as, for example, estrogens, conjugated estrogens, ethinyl estradiol, diethylstilbesterol, chlortrianisen, idenestrol, progestins (such as hydroxyprogesterone caproate, medroxyprogesterone, and megestrol), and androgens (such as testosterone, testosterone propionate, fluoxymesterone, and methyltestosterone)), an adrenal

corticosteroid (such as, for example, prednisone, dexamethasone, methylprednisolone, and prednisolone), a leutinizing hormone releasing agent or gonadotropin-releasing hormone antagonist (such as, for example, leuprolide acetate and goserelin acetate), an antihormonal agent (such as, for example, tamoxifen), an antiandrogen agent (such as flutamide), an antiadrenal agent (such as mitotane and arnmoglutethimide), and a natural product or derivative thereof (such as, for example, vinca alkaloids, antibiotics, enzymaes and epipodophyllotoxins, including, for example vinblastine, vincristine, vindesine, bleomycin, dactinomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, ara-C, paclitaxel, mithramycin, deoxyco-formycin, mitomycin-C, L-asparaginase, and teniposide.

A chemotherapeutic agent may be an anti-angiogenic agent, such as, for example, anginex, compound 0118, compound 1097, bridged compound 4, sunitinib (Sutent®, Pfizer), sorafenib (Nexavar®, Bayer) and bevacizumab (Avastin®, Genentech).

In some aspects of the methods of the present invention, at least one additional therapeutic agent includes radiation therapy. In some aspects, radiation therapy includes localized radiation therapy delivered to the tumor. In some aspects, radiation therapy includes total body irradiation.

Cytokines include, but are not limited to, IL-la, IL-Ιβ, IL-2, IL-3, IL-4, IL-6, IL-8,

IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-19, IL-20, BFN-a, EFN-β, IFN-γ, tumor necrosis factor (TNF), transforming growth factor-β (TGF-β), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), and or Flt-3 ligand. Antibody therapeutics, include, for example, trastuzumab (Herceptin) and antibodies to cytokines, such as IL-10 and TGF-β.

In some aspects of the methods of the present invention, the administration a compound as described herein and the at least one additional therapeutic agent demonstrate therapeutic synergy. In some aspects of the methods of the present invention, a

measurement of response to treatment observed after administering both a cytotoxic compound as described herein and the additional therapeutic agent is improved over the same measurement of response to treatment observed after administering either the cytotoxic compound or the additional therapeutic agent alone. Such synergy has been observed with combined treatments with radiation and anginex (see Dings et al., 2005, Int JCancer; 115(2):312-9) and anginex or 0118 in combination with the chemotherapeutic irofulven (Dings et al., 2008, Cancer Lett; 265(2):270-280).

The above description of the present invention is not intended to describe each disclosed embodiment or every implementation of the present invention. The description exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list. For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements. Also herein, the recitations of numerical ranges by endpoints include all numbers subsumed witiiin that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

Where a range of values is provided, it is understood that each mtervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or mtervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

The term "and/or" means one or all of the listed elements or a combination of any two or more of the listed elements. Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one.

The terms "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims.

The words "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

EXAMPLES Example 1

Synthesis of the JIL31, JIL50, JIL54, and JIL70 compounds

The chemical structures of the JIL31, JIL50, JIL54, and JEL70 compounds are shown in Fig. 1.

JIL31

The hexameric 2-dimemylammoethylamide JIL31 was prepared by the sequence of reactions shown in Scheme 1. Precursor 1 is commercially available and intermediates 2 and 3 are known compounds and were prepared by methods described in the literature (Arnaud-Neu et al., 1989, J Am Chem Soc; 111 :8681-8691). The tetramethyl ester 3 was converted into the tetrakis amide JIL31 by reaction with Me₂CH₂CH₂NH₂.

Scheme 1

1 2 3 JIL31

a) A1C1₃, PhOH, Toluene, rt; b) BrCH₂C0₂Me, Na₂C0₃, acetone, 56 °C; c)

Me₂NCH₂CH₂NH₂, toluene, MeOH, 90 °C.

Preparation of 2,2*,2",2^m,2"",2"^m-

[Heptacyclo[31.3.1.13 ,7.19, 13.115, 19.121 ,25.127,31 ]dotetraconta- l(37),3,5,7(42),9,ll,13(41),15,17,19(40),21,23,25(39),27,29,31(38),33,35-octadecaene- 37,38,39,40,41 ,42-hexaymexa s(oxy)]hexakis[N-(2-dimemylammoethyl]acetamide

(JDL31). Hexamethyl ester 3 (20 mg) was dissolved in toluene (0.2 mL) and N,N- dimemylemylenediamine (123 uL) under a nitrogen atmosphere. A few drops of methanol were added, which caused the turbid suspension to become homogenous. The solution was heated at 90 °C for 24 h. Additional N,N-dimemylethylenediamine was added (60 equiv) and the mixture was heated for an additional 20.5 h, at which time LCMS analysis showed essentially complete consumption of starting material. The solution was cooled and the toluene removed under reduced pressure. Diethyl ether was added and a gum appeared. This was triturated with additional ethanol and ethyl acetate. The supernatant organic solution was removed and concentrated to provide JIL31.

JTL50

The preparation of the tefratWatetramine hydrochloride JIL50 is shown in Scheme

2. The known tetrathiatetraethyl ester 5 was prepared from commercially available 4 by a method described in the literature (Matthews et al., 2001, New J Chem; 25:1355-1358). It was converted into the tetramine 6 by reaction with Me₂CH₂CH₂NH₂ and then to the hydrochloride salt J1L50 be treatment with a methanolic solution of HCL

Scheme

4 5 6 JILSO commercially known

available a) BrCH₂C0₂Et, Na₂C0₃, acetone, 56 °C; b) Me₂NCH₂CH₂NH₂, toluene, MeOH, 90 °C. c) HCl/MeOH, rt. Preparation of 2,2',2",2^m-[[5,l l,17,23-TetraMs(l,l-dimethylethyl)-2,8,14,20- tetrathiapentacyclofl 9.3.1.13,7.19, 13.115, 19]octacosa- 1 (25),3 ,5,7(28),9, 11 , 13(27), 15, 17, 19(26),21 ,23-dodecaene-25,26,27,28- tetrayl]tetraMs(oxy)]tetrakis[N-(2-dmiemylammoemyl)acet (6). Tetraethyl ester 5 (26.5 mg) was dissolved in N,N-dimemylethylenediamine (1 mL) under a nitrogen atmosphere. The solution was stirred for 20 hours at ambient temperature. Water was added and the mixture was extracted with ethyl acetate. The organic layers were dried, filtered, and concentrated. The resulting off white solid was crystallized from ether containing a small amount of ethanol to result in 17.5 mg of the tetramine 6.

Preparation of 2,2',2",2^,"-[[5, 11 , 17,23-Tetrakis(l , 1 -dimethylethyl)-2,8, 14,20- tetrathiapentacyclo[ 19.3.1.13 ,7.19, 13.115, 19]octacosa-

1 (25),3 ,5,7(28),9, 11 , 13(27), 15, 17, 19(26)₅21 ,23-dodecaene-25,26,27,28- tetrayl]te1xakis(oxy)]tetrakis [N-(2-dimemylammoemyl)acetamide tetrahydrochloride (JIL50). Acetyl chloride (71 uL) was added to methanol (10 mL) at ambient temperature to produce a stock solution of methanolic HC1 (from the rapid reaction of AcCl with MeOH). Tetramine 6 (11 mg) was dissolved in chloroform (1 mL) and 410 uL of the above HCL/MeOH solution was added. After several minutes the solvent was removed in vacuo, resulting in essentially quantitative recovery of the tetrahydrochloride salt JIL50.

JIL54

The preparation of the tetramine hydrochloride salt JIL54 is shown in Scheme 3.

The commercially available calixarene derivative 7 was converted into the tetramine 8 by reaction with chloroe&yldimemylaniine hydrochloride under basic conditions. Free base tetramine 8 was then converted to the tetrahydrochloride salt JIL54. Scheme 3

7 8 JIL54 commercially

available

a) ClCH₂CH₂NMe₂-HCl, NaOH toluene, 110 °C; b) HCl/MeOH, rt. Preparation of 2,2',2",2'"-[[5,l 1, 17,23 -Tetrakis( 1,1 -dimethylethyl)pentacyclo-

[19.3.1.13,7.19,13.115,19]octacosa-l(25),3,5,7(28),9,ll,13(27),15,17₃19(26),21,23- dodecaene-25,26,27,28-tetrayl]tetra s(oxy)]tetr^ (8).

Tetraphenol 7 (50 mg) was dissolved in hot dry toluene (1 mL). Powdered sodium hydroxide (75 mg) was added and the resulting suspension was refluxed for 10 min. 2- CUoroemyl-dimemylarnine hydrochloride (89 mg) was added and the resulting translucent slurry was heated at reflux for 2.25 h. The mixture was cooled to room temperature, water was added, and the toluene was washed three times with 10% aqueous hydrochloric acid. The combined aqueous washings were basified by the addition of excess aqueous NaOH. A milky white suspension resulted. This was extracted with Et₂0 and the organic layers were dried (MgS0₄), filtered, and concentrated to provide a pale yellow solid (57 mg). This material was crystallized from hexanes to provide 8 (34 mg). Preparation of 2,2',2",2'"-[[5, 11 , 17,23-Tetrakis(l , 1 -dimethylethyl)pentacyclo- [19.3.1.13,7.19,13.115₅19]octacosa-l(25),3,5,7(28)₃9,l l,13(27),15,17,19(26),21,23- dodecaene-25,26,27,28-tetrayl]tetra s(oxy)]tetraM^

tetrahydrochloride (JIL54). Tetrarnine 8 (8.5 mg) was dissolved in CHCI3 (0.5 mL) and treated with methanolic HC1 (4 equiv) by the procedure described above for the preparation of JIL50. Upon removal of solvent, JIL54 was obtained with near quantitative recovery as a white solid.

JIL70

The preparation of the teliarnine hydrochloride salt JIL70 is shown in Scheme 4. The commercially available calixarene derivative 9 was converted into the tetrarnine 10 by reaction with cMoroemyldimemylamine hydrochloride under basic conditions. Free base tetrarnine 10 was then converted to the tetrahydrochloride salt JBL70 using ethanolic hydrochloric acid.

Scheme 4

9 10 JIL70 commercially

available

a) ClCH₂CH₂NMe₂-HCl, NaOH toluene, 110 °C; b) HCl/EtOH, rt.

Preparation of 2,2',2",2'"-[[Pentacyclo-[l 9.3.1.13,7.19, 13.115,19]octacosa- 1 (25),3 ,5,7(28),9, 11,13 (27), 15, 17, 19(26),21 ^

te1xayl]tetra s(oxy)]tetra] s[N_^ (10). Tetraphenol 9 (150 mg) was dissolved in hot dry toluene (3 mL). Powdered sodium hydroxide (340 mg) was added and the resulting suspension was refluxed for 10 min. 2-CUoroemyl-dimemylaniine hydrochloride (410 mg) was added and the resulting translucent slurry was heated at reflux for 2.25 h. The mixture was cooled to room temperature, water was added, and the toluene was washed three times with 10% aqueous hydrochloric acid. The combined aqueous washings were basified by the addition of excess aqueous NaOH. This mixture was extracted with chloroform and the combined organic layers were dried (MgS0₄), filtered, and concentrated to provide a pale yellow solid (253 mg). This material solidified upon standing to a slightly oily solid, which was triturated with hexanes and filtered to leave 10 as an off-white solid (22 mg).

Preparation of 2,2',2^,,,2'"-[[Pentacyclo-[l 9.3.1.13,7.19,13.115, 19]octacosa- 1 (25),3,5,7(28),9,11 , 13(27), 15, 17, 19(26),21 ,23-dodecaene-25,26,27,28- tetrayl]tetralds(oxy)]tetrakis^ tetrahydrochloride (JIL70). Acetyl chloride (71 uL) was added to ethanol (10 mL) at ambient temperature to produce a stock solution of ethanolic HC1 (from the rapid reaction of AcCl with EtOH). Tetramine 10 (10 mg) was dissolved in chloroform (1 mL) and 564 uL of the above HCL/EtOH solution was added. After several minutes the solvent was removed in vacuo, resulting in essentially quantitative recovery of the tetrahydrochloride salt JTL70. Example 2

Cytotoxic Activity

The anti-proliferative, cytotoxic effect of the JIL31, JTL50, JIL54, and JIL70 compounds against various cell lines was determined and compared to the cytotoxic effect of compound 0118 and compound 1097. Cell lines used included A549 lung cell carcinoma, B16F10 mouse melanoma cells, SCK human breast cell carcinoma, Fsall mouse fibrosarcoma cells, normal human fibroblasts, HUVEC normal human endothelial cells, and 2H11 normal mouse endothelial cells.

The chemical structures of compounds 1097 and 0118 are shown in Fig. 2. For a more detailed description of compound 0118 and compound 1097 see, for example, Dings et al., 2006, J Natl Cancer Inst; 98:932-6, WO 2006/042104, and US 2008/0300164. Compound 0118 is also referred to as compound 40, PTX-008, and OTX-008, and compound 1097 is also referred to as compound 27.

Human umbilical vein endothelial cells (TfUVECs) were cultured in gelatin-coated tissue-culture flasks (0.2%) in culture medium (RPMI 1640 with 20% (v/v) human serum, supplemented with 2mM glutamine, 100 units/ml penicillin and O.lmg/ml streptomycin). Mouse melanoma (B16F10), human breast carcinoma (SCK), and human lung carcinoma (A549) were cultured on noncoated flasks using 10% fetal bovine serum, 1%

penicillin/streptomycin in RPMI 1640. Cultures were split 1:3 every 3 days. The Cell Counting Kit-8 (CCK-8) was used to assay cellular proliferation as described by the manufacturer (Dojindo, Japan). Methods are described in more detail in, for example, Griffioen et ai, 2001, Biochem J; 354:233-42; van der Schaft et al., 2002, FASEB J;

16:1991-3; Dings et al., 2003, Cancer Res; 63:382-5; Dings et al., 2003, Cancer Lett; 194:55-66; Dings et al., 2006, J Natl Cancer Inst; 98:932-6, WO 2006/042104, and US 2008/0300164.

Fig. 3 shows the anti-proliferative effects of JTL31, JJJL50, JIL54, and JIL70 compounds compared to compound 1097 and compound 0118. Specifically, Fig. 3A shows anti-proliferative effect on human umbilical vein endothelial cells (HUVECs), Fig. 3B shows anti-proliferative effect on normal mouse endothelial cells (2H11), Fig. 3C shows anti-proliferative effect on normal human fibroblasts, Fig. 3D shows antiproliferative effect on murine fibrosarcoma cells (FSAII), Fig. 3E shows anti-proliferative effect on human lung carcinoma (A549) (Fig. 2E), Fig. 3F shows anti-proliferative effect on human ovarian carcinoma cells (MA148), Fig. 3G shows anti-proliferative effect on human breast carcinoma (SCK), and Fig. 3H shows anti-proliferative effect on mouse melanoma (B16F10). For some cell lines, the cytotoxic effect of the JIL31, JIL50, JIL54, and JIL70 compounds was also compared to the cytotoxic activity of anginex, including the HUVEC, 2H11, and B16F10 cell lines. Anginex has the amino acid sequence

IQFLKVSLNLDRKQAXW JJVKLNDGRELSLD and is described in more detail in, for example, Griffioen et al., 2001, Biochem J 354:233-42.

As shown in Figs. 3 A-3H, the JIL70 and JIL54 compounds demonstrated cytotoxic activity against all of the cells lines tested. For all but the MA 148 human ovarian carcinoma cell line, JIL70 demonstrated the greatest potency (generally with IC50 values less than about 1 μΜ), followed by JIL54, in comparison to 0118, 1097, JIL50, and JIL31. Only in the case of human ovarian carcinoma MA148 did the 0118 compound demonstrate a greater potency, and even then JIL70 and JIL54 were still highly effective in this cell line. JIL70 and JIL54 exhibit considerably greater potency overall in comparison to anginex, 0118, 1097, JIL54, and JIL30. Both compound 0118 and anginex are essentially ineffective against many of the cell lines tested. From this data, one can conclude that the molecular target of JTL70 and JIL54 is different from that of the molecular targets for anginex and 0118.

Example 3

Inhibition of Tumor Growth In Vivo

Based in the in vitro results of Example 2, tumor growth inhibition by JIL54 and JTL70 was determined in the murine B16F10 melanoma model and compared to tumor inhibition by the antiangiogenic compounds 0118 and 1097.

B16/F10 cells (2 x 10⁵) were inoculated into the hind flank of female C57/BL6 mice (n=10 each group). Tumors were allowed to grow to the size of approximately 100 mm prior to initiation of treatment (given BED EP). Tumors were measured using calipers, and the volume was calculated using the equation to determine the volume of a spheroid: (a² x b x π)/6, where a is the width of the tumor, and b is the length of the tumor. Methods are described in more detail in, for example, Griffioen et al., 2001, Biochem J; 354:233-42; van der Schaft et al., 2002, FASEB J; 16: 1991-3; Dings et al., 2003, Cancer Res; 63:382-5; Dings et al., 2003, Cancer Lett; 194:55-66; Dings et al., 2006, J Natl Cancer Inst; 98:932- 6, WO 2006/042104, and US 2008/0300164.

In initial studies, all compounds were administered at a dose of 10

mg/kg/day/mouse. The results are shown in Fig. 4A. These data demonstrate that JTL70 works better than J1L54 or compounds 0118 and 1097. Treatment with JEL70 was only for five days, because animals lost weight (see Fig. 4B) and one died at this dose of JEL70. All other compounds were administered for twelve days. Note that even with only five days of treatment with JIL70, tumors remained the smallest of those in all groups.

Fig. 4B shows body weights of mice during treatment, as an indication of general toxicity. Notice that for compounds JEL54 and JIL70 at the 10 mg/kg dose, mice displayed significant weight lose initially. After five treatments with JTL70, mice began to again gain weight starting at day 8. Compared to control ariimals, there was no apparent weight loss with compound 0118 at the same dose. Unlike JTL70, however, 0118 is significantly less effective at dosages lower than about 10 mg/kg. Next, to determine the optimal effective dosage of JIL70, dose escalation studies of the JIL70 compound were performed in the murine B16F10 melanoma model. An optimal effective dose is defined as one which effectively inhibits tumor growth while being minimally toxic to mice. Animal body weights serve as an indirect measure of drug toxicity during the course of treatment. Results are shown in Figs. 5A to 5D.

Fig. 5 A shows tumor volumes at dosages of 0.5 mg/kg/day/mouse (BED for 10 days), 1.5 mg/kg/day/mouse (BED for 10 days), and 5 mg/kg/day/mouse (BID for 5 days). The 5 mg/kg dosage could only be administered for 5 days due to extreme toxicity and death in a few cases. As mentioned above, loss of body weight is an indirect measure of drug toxicity during the course of treatment. All other cohorts given lower dosages of JIL70 were treated BID for 10 days at the dosage indicated.

Fig. 5B shows average body weights in grams (gm) in mice administered doses of 0.5 mg/kg/day, 1.5 mg/kg/day, and 5 mg/kg/day JIL70. Note that in control mice, body weights increased as normally expected over time. In the other three groups, weights dropped significantly during the course of treatment and then began to level off and increase post treatment. These data also indicate that toxicity is reversible once treatment is halted.

Because JDL70 was so highly effective at inhibiting tumor growth at dosages of 0.5 mg/kg and above, a second dose escalation study was performed in which dosages (BID for 10 days in all cases) were lowered to 0.1 mg/kg/day/mouse, 0.2 mg/kg/day/mouse, and 0.5 mg/kg/day/mouse (Fig. 5C). As shown in Fig. 5C, while the 0.1 mg/kg dosage showed minimal effectivity, dosages at 0.2 mg/kg and 0.5 mg/kg were essentially equally effective, and basically as effective as the higher, yet more toxic, dosages. Fig. 5D shows body weights in grams (gm) in mice administered doses of 0.1 mg/kg/day, 0.2 mg/kg/day, and 0.5 mg/kg/day JIL70. JIL70 at these lower dosages was clearly much better tolerated by mice, as demonstrated by body weights during the course of treatment (Fig. 5D).

Figures 6A and 6B show the effect of dose of JIL70 compound in the murine B16F10 melanoma model. Fig. 6A shows tumor volumes at daily dosages of 2

mg/kg/mouse for 10 days straight (labeled 10 shots), 5 mg/kg/mouse on days 1, 4, 7, and 10, and 10 mg/kg/mouse on days 1 and 5. This experiment was done as an assessment of the effect of dosing schedule at a fixed total drug exposure. Note that the admimstration of 10 mg/kg on days 1 and 5 rendered the best results. As a measure of general toxicity, Fig. 6B shows body weights in grams (gm) in mice administered doses as in Fig. 6A. In general, mice either lost some weight or did not gain weight under these treatment regimes, and at the end of treatment, mice gained weight normally.

Using this murine B16F10 melanoma tumor model, it was determined that the optimal dose for JIL70 is about 0.2 mg/kg to about 0.5 mg/kg. In this regard, JIL70 is considerably more effective than compound 0118 at the same dosage. JIL70 had the greatest effect in inhibiting tumor growth, as well as in decreasing body weight.

Importantly, this data shows that the pharmacodynamic half-life for JIL70 allows for intermittent dosing on schedules that are more similar to how many of the newer antitumor agents are administered clinically.

The pharmacodynamic half-life for JTL70 may be further addressed by conducting xenograft studies using intermittent dosing in the MA- 148 human ovarian tumor model in nude mice. Agents will be aclministered i.p. at daily doses of 10 mg/kg q3d X5, 16.7 mg/kg q5d X3, or 25 mg/kg q7d X2. Results for compounds may be compared to the results from anginex (a 33-mer peptide recently defined as a galectin-1 targeted

antiangiogenic), compound 0118 (a calixarene-based small molecule mimetic of anginex), and the partial peptide mimetic DB21 (also known as 6DBF7; see Mayo et al., 2003, J Biol Chem; 278(46):45746-52). Anginex has been previously shown to be maximally effective in the MA148 xenograft model (70-80% tumor growth inhibition [TGI]) when

administered subcutaneously at a daily dose of 10 mg/kg by osmotic minipump (Dings et al., 2003, Cancer Lett; 194:55-66). Similar to anginex, 0118 was previously shown to produce significant TGI (79%) when administered subcutaneously at a daily dose of 10 mg/kg by osmotic minipump in the MA 148 xenograft model (Dings et al., 2006, JNCI9S: 932-936).

Any of the other compounds described herein, including, but not limited to, JEL54,

JTL50, and JTJL30, may be tested in a similar fashion. Results may be compared to results obtained for other agents, such as, for example anginex, compound 0118, compound 1097, and the partial peptide mimetic DB21. Example 4

Effects of JIL compounds in the SQ20B, Colo205

and Colo205R Human Cancer Cells The cytotoxicity and growth inhibitory effects of JIL70 were determined in a variety of cultured human cancer cell lines using an MTT assay. Cell lines included the SQ20B, Colo205, and Colo205R cell lines. The SQ20B cell line is a human head and neck squamous cancer cell line with low galectin-1 expression. The parental epithelial Colo205 cell line and the derived- mesenchymal counterpart Colo205R cell line are human colon cancer cell lines. The Colo205 cell line has low galectin-1 expression. The Colo205R cell line demonstrates an epithelial to mesenchymal transition (EMT) phenotype, an acquired resistance to protein kinase C (PKC) modulators, and high galectin-1 expression.

Galectin-1 is a rather ubiquitous carbohydrate-binding protein that is over-expressed in many different types of cancers. Increased galectin-1 expression by tumor and connective tissue supporting the tumor may correlate with the aggressiveness of the tumor and the acquisition of a metastatic phenotype, and in preclinical studies, galectin-1 has been shown to play a role in tumor transformation, tumor cell proliferation, cell aggregate, adhesion, migration, apoptosis, and immunoregulation. The SQ20B cell line was obtained from the American Type Culture Collection. The Colo205 cell line was obtained from the National Cancer Institute collection. Colo205R cells were developed as described in Ghoul et al., 2009, Cancer Res; 69:4260-4269. Cells were grown as monolayers in RPMI 1640 supplemented with 10% FCS (Invitrogen), 2 rnmol/L glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37C in a humidified 5% C0₂ atmosphere and regularly checked for the absence of Mycoplasma.

Cell viability was determined using the MTT assay (Sigma) as described in more detail in Ghoul et al., 2009, Cancer Res; 69:4260-4269 and Serova et al., 2010, Mol Cancer Ther; 9(5):1308-1317. Briefly, the conversion of yellow water-soluble tetrazolium MTT into purple insoluble formazan is catalyzed by mitochondrial dehydrogenases and is used to estimate the number of viable cells. In brief, cells were seeded in 96-well tissue culture plates at a density of 2 10 ³ per well. After 72-hour incubation with drug followed by 48-hour postincubation in drug-free medium, cells were incubated with 0.4 mg/mL MTT for 4 hours at 37C. After incubation, the supernatant was discarded, insoluble formazan precipitates were dissolved in 0.1 mL of DMSO, and the absorbance was measured at 560 nm by use of a microplate reader (Thermo). Wells with untreated cells or with cirag-containing medium without cells were used as positive and negative controls, respectively. Growth inhibition curves were plotted as the percentage of untreated control cells.

The cytotoxic effects of various concentrations of JIL70 in the SQ20B, Colo205, and Colo205R cell line are shown in Fig. 7A and Fig. 7B. The growth inhibitory effects of various concentrations of JIL70 in the SQ20B, Colo205, and Colo205R cell lines is shown in Figs. 8A-8C. JIL70 displays cytotoxic and antiproliferative effects in cancer cells. The antiproliferative effects are similar to the effect of other known cytotoxic drugs, rather than known cytostatic agents, such as compound 0118 and various kinase inhibitors. JIL70 appears to have similar cytotoxic and antiproliferative effects in both epithelial and mesenchymal colon cancer cells (Colo205 versus Colo205R), with an IC50 of

approximately 2.5 μΜ.

The cytotoxic and anti-proliferative effects of the other compounds and derivatives thereof, as described herein, may be tested in a similar fashion.

Example 5

Effect of JIL70 on Epithelial and Mesenchymal

Human Cancer Cell Lines

The cytotoxicity and antiproliferative effects of JIL70 and the effects of JIL70 on cell cycle and galectin-1 protein expression were determined in a variety of epithelial and mesenchymal human cancer cell lines. Cell lines studied included the Colo205, Colo205R, SQ20B, SQ-PTX, MCF7, MCF7-WISP, DLD, and DLD-SNAIL cell lines.

The parental Colo205 cell line is an epithelial human colon cancer cell line demonstrating low galectin-1 expression; the Colo205R cell line is a Colo205-derived cell line that demonstrates an epithelial to mesenchymal transition (EMT) phenotype, resistance to several PKC modulators, including ingenol 3-angelate (PEP005), and high galectin-1 expression. The SQ20B cell line is a human head and neck squamous cancer cell line with low galectin-1 expression; the SQ-PTX cell line is derived from the SQ20B cell line with acquired resistance to compound 0118 (after 4 months). The SQ-PTX cell line demonstrates an EMT phenotype and a loss of galectin-1 protein expression. The MCF7 cell line is a human epithelial breast cancer cell line; the MCF7-WISP cell line is a MCF7-derived cell line which demonstrates an EMT phenotype. The DLD cell line is a human epithelial cancer cell line; the DLD-SNAIL cell line is a DLD-derived cell line which demonstrates an EMT phenotype. The SQ20B, MCF7, DLD and Colo205 cell lines were obtained from the American Type Culture Collection (ATCC) or the National Cancer Institute collection. Colo205R cells were developed as described in Ghoul et al., 2009, Cancer Res; 69:4260-9. Cells were grown as monolayers in RPMI 1640 supplemented with 10% FCS (Invitrogen), 2 mmol/L glutamine, 100 units/mL penicillin, and 100 μg/nlL streptomycin at 37C in a humidified 5% C02 atmosphere and regularly checked for the absence of Mycoplasma.

The SQ-PTX cell line was established from the parental SQ20B cell line using a stepwise exposure to increasing PTX008 concentrations for more than 6 months. Long term-exposure to PTX008 led to decrease of Gal-1 and E-cadherine mRNA and protein levels and an increase in Vimentin mRNA and protein levels in SQ-PTX. The MCF7- WISP is a stable cell line obtained from MCF-7 by transfection with Silencer/sh-WISP-2. WISP-2/CCN5 knockdown induced an estradiol-independent growth of MCF-7, linked to a loss of ERa expression, and promoted epithelial-to-mesenchymal transdifferentiation (see Fritah et al., 2008, Mol Cell Biol; 28(3): 1114-23). To obtain the DLD-SNAIL cell line, the epithelial DLD-1TR21 cell line was stably transfected with an expression vector harboring a Myc-tagged full-length human Snail under control of a responsive tetracycline operator element. hSnail expression is induced by adding doxycyclin to the medium. Changes in morphology from an epithelioid morphotype to a fibroblastlike type could be seen 48 hours after hSnail induction (see De Craene et al, 2005, Cancer Res; 65(14):6237-44).

Cell viability was determined using the MTT assay (Sigma), as described in more detail in Ghoul et al., 2009, Cancer Res; 69:4260-4269 and Serova et al., 2010, Mol Cancer Ther; 9(5):1308-1317. Briefly, the conversion of yellow water-soluble tetrazolium MTT into purple insoluble formazan is catalyzed by mitochondrial dehydrogenases and is used to estimate the number of viable cells. In brief, cells were seeded in 96-well tissue culture plates at a density of 2 10 ³ per well. After 72-hour incubation with drug followed by 48-hour post incubation in drug-free medium, cells were incubated with 0.4 mg/mL MTT for 4 hours at 37C. After incubation, the supernatant was discarded, insoluble formazan precipitates were dissolved in 0.1 mL of DMSO, and the absorbance was measured at 560 nm by use of a microplate reader (Thermo). Wells with untreated cells or with drag-containing medium without cells were used as positive and negative controls, respectively. Growth inhibition curves were plotted as the percentage of untreated control cells.

Cell cycle analysis was by flow cytometry as described in more detail in Serova et al., 2010, Mol Cancer Ther; 9(5):1308-1317. In brief, cells were seeded onto 25 cm flasks and treated with indicated concentration of JIL70. At various time points, adherent and nonadherent cells were recovered, washed with PBS, fixed in 70% ethanol, and stored at C until use. Cells were rehydrated in PBS and incubated for 20 minutes at room temperature with 250 μg/mL RNase A and for 20 minutes at 4C with 50 μg/mL propidium iodide in the dark. The cell cycle distribution and percentage of apoptotic cells were determined with a flow cytometer (FACSCalibur and CellQuest Pro software (BD

Biosciences).

Galectin protein expression was assayed by Western blot analysis, as described in more detail in Serova et al., 2010, Mol Cancer Ther; 9(5):1308-1317. Briefly, cells were lysed in buffer containing 50 mmol/L HEPES (pH 7.6), 150 mmol/L NaCl, 1% Triton X- 100, 2 mmol/L sodium vanadate, 100 mmol/L NaF, and 0.4 mg/mL phenylmethylsulfonyl fluoride. Equal amounts of protein (20 μg/lane) were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% milk or 5% bovine serum dbumin in 0.01% Tween 20/PBS and then incubated with the primary antibody (anti-galectin or anti-actin antibody) overnight. Antibodies were used at 1:1,000 dilutions. Membranes were then washed and incubated with the secondary antibody conjugated to horseradish peroxidase. Bands were visualized by using the enhanced chemiluminescence Western blotting detection system. Densitometric analysis was done under conditions that yielded a linear response.

Results

The cytotoxic and antiproliferative effects of JIL70 in the SQ20B cell line versus the SQ-PTX cell line is shown in Figs. 9A and 9B. These results demonstrate that JEL70 displays similar cytotoxic effects (Fig. 9A) and antiproliferative effects (Fig. 9B) in the SQ20B and SQ-PTX cell lines. The SQ020B and SQ-PTX cell line pair serves as a model for acquired resistance to PTX-008. Additional studies with the SQ20B and SQ-PTX cell line pair show no cross-resistance for JTL70 and compound 0118 in this model with acquired resistance to compound 0118. And, JTL70 appears to have no similar effects than compound 0118 in the SQ20B cell line.

Figures 10A and 10B show cell cycle changes induced in SQ20B cells by a forty- eight hour exposure to 3 μΜ JIL70. Exposure to 3 μΜ JTL70 for forty-eight hours increases subGl and G0/G1 and decreases S and G2/M phases of the cell cycle. The effect of JTL70 on the cell cycle of any of the other cell lines described herein may be assayed in a similar fashion.

Figure 11 shows the results of a forty-eight hour (h) exposure to 3μΜ JIL70 on galectin-1 protein expression in the SQ20B cell line. Exposure to 3μΜ JTL70 for forty- eight hours did not inhibit galectin-1 protein expression in the SQ20B cell line. The effect of JTL70 on galectin-1 protein expression in any of the other cell lines described herein may be assayed in a similar fashion.

Next, the effect of JIL70 on EMT models was addressed. Specifically, the cytotoxic and antiproliferative effects of JTL70 in the MCF7 cell line compared to the MCF7-WISP cell line; the DLD cell line compared to the DLD-SNAIL cell line; and the Colo205 cell line compared to the Colo205R cell line was assayed.

The cytotoxic and antiproliferative effects of J1L70 in the MCF7 cell line versus the MCF7-WISP cell line is shown in Figs. 12A and 12B. The cytotoxic and antiproliferative effects of JIL70 in the DLD cell line versus the DLD-SNAIL cell line is shown in Figs. 13 A and 13B. The cytotoxic effects of JIL70 in the Colo205 cell line versus the Colo205R cell line is shown in Fig. 7B and the antiproliferative effects in the Colo205 cell line versus the Colo205R cell line is as shown in Figs. 8B to 8D. These results show that JIL70 appears to have similar cytotoxic and antiproliferative effects in epithelial and

mesenchymal human cancer cells.

The IC50 of various dosages of JIL70 as a function of time (2, 6, 24, 48 and 72 hour exposure) in the SQ20B cell line versus the SQ-PTX cell line at is shown in Fig. 14 A. The IC50 of various dosages of JIL70 as a function of time (2, 6, 24, 48 and 72 hour exposure) in the Colo205 (ColoS) cell line versus the Colo205R (ColoR) cell line at is shown in Fig. 14B. The cytotoxic and anti-proliferative effects of other compounds and derivatives thereof, as described herein, may be tested in a similar fashion. The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

Claims

3 claimed is:

A nonpeptidic calixarene topomimetic agent having the formula:

wherein y + z is 4 to 6;

wherein R⁹ and R¹⁰ are each independently a hydrogen or an organic group and R⁹ and R¹⁰ have a hydrophobic polarity;

wherein R¹¹ is hydrogen or an organic group;

wherein x is 1 to 6; and

wherein R¹² is an organic group; and

wherein R is an organic group; and

derivatives and salts thereof.

2. The nonpeptidic calixarene topomimetic agent of claim 1, wherein the agent demonstrates a cytotoxic activity at least 5 fold greater than compound 1097, compound 0118, and/or anginex against the B16F10 murine melanoma cell line, demonstrates cytotoxic activity against galectin-1 negative cells, demonstrates cytotoxic activity against cells resistant to compound 0118, and/or demonstrates cytotoxic activity against cells having an epithelial to mesenchymal transition (EMT) phenotype.

3. The agent of claim 1 or 2 wherein each of R⁹ and R¹⁰ is independently selected from hydrogen, a branched or unbranched alkyl, a branched or unbranched alkenyl, a cycloalkyl, an aryl, or a heteroaryl.

4. The agent of any one of the preceding claims, wherein each of R⁹ and R¹⁰ is independently selected from hydrogen, methyl, ethyl, propyl, or tert-butyl.

5. The agent of any one of the preceding claims, wherein R¹¹ is hydrogen, a branched or unbranched alkyl, a branched or unbranched alkenyl, a cycloalkyl, an aryl, or a heteroaryl.

6. The agent of any one of the preceding claims, wherein R¹¹ is hydrogen, methyl, ethyl, propyl, or tert-butyl.

7. The agent of any one of the preceding claims, wherein R and R is independently selected from hydrogen, a branched or unbranched alkyl, a branched or unbranched alkenyl, a cycloalkyl, an aryl, or a heteroaryl.

8. The agent of any one of the preceding claims, wherein R and R is independently selected from hydrogen, methyl, ethyl, propyl, or tert-butyl.

9. The agent of any one of the preceding claims, wherein y + z is 4.

10. The agent of any one of the preceding claims, wherein x is 1, 2, 3, or 4.

11. The agent of any one of the preceding claims, wherein y is 4, z is 0, x is 2, and R¹² and R are methyl groups.

12. A cytotoxic nonpeptidic calixarene topomimetic agent of claim 1 or 2 having the formula:

(JIL70), and derivatives, analogs, and salts thereof. A cytotoxic nonpeptidic calixarene topomimetic agent of claim 1 or 2 having the

(J1L54), and derivatives, analogs, and salts thereof.

14. A cytotoxic nonpeptidic calixarene topomimetic agent having the formula:

(JIL31), and derivatives, analogs, and salts thereof.

15. A cytotoxic nonpeptidic calixarene topomimetic agent having the formula:

(JIL50), and derivatives, analogs, and salts thereof.

16. A cytotoxic nonpeptidic calixarene-based topomimetic agent having Formula I or II:

Π

wherein each R¹ through R⁸ group is independently hydrogen or an organic group, wherein R¹ through R⁴ are each independently hydrogen or an organic group of like polarity and R⁵ through R⁸ are each independently hydrogen or an organic group of like polarity that is of opposite polarity than those of R¹ through R⁴, and

wherein the agent demonstrates a cytotoxic activity at least a 5 fold greater than 1097, 0118, and/or anginex against the B16F10 murine melanoma cell line, demonstrates cytotoxic activity against galectin-1 negative cells, demonstrates cytotoxic activity against cells resistant to compound 0118, and/or demonstrates cytotoxic activity against cells having an epithelial to mesenchymal transition (EMT) phenotype; and

wherein the agent is not compound 0118 or compound 1097.

17. The agent of claim 16, wherein R¹ through R⁸ are each independently hydrogen, halogen, alkyl, cycloalkyl, aryl, aralkyl, alkoxy, thioalkoxy, cycloalkylalkoxy, heterocycloalkyl, aralkyloxy, or heteroaryl, optionally including ester, amide, amine, hydroxyl, halogen, sulfonate, phosphonate, guanidine, and/or heteroaryl groups.

18. An agent of any one of the preceding claims, wherein R¹ through R⁴ are each independently alkyl, cycloalkyl, aralkyl, alkoxy, cycloalkylalkoxy, or aralkyloxy, and R⁵ through R are each independently any of these groups incorporating ester, amide, amine, hydroxyl, sulfonate, phosphonate, guanidine and/or heteroaryl groups.

19. An agent of any one of the preceding claims, wherein R through R are each independently alkyl, cycloalkyl, aralkyl, alkoxy, cycloalkylalkoxy, or aralkyloxy, and R¹ through R⁴ are each independently any of these groups incorporating ester, amide, amine, hydroxyl, sulfonate, phosphonate, guanidine and/or heteroaryl groups.

20. An agent of any one of the preceding claims, wherein R through R are each independently hydrogen, alkyl, cycloalkyl, aralkyl, alkoxy, cycloalkylalkoxy, or aralkyloxy optionally including ester, amide, amine, hydroxyl, sulfonate, phosphonate, guanidine and/or heteroaryl groups.

21. An agent of any one of the preceding claims, wherein R¹ through R⁴ are each independently hydrogen, halogen, alkyl, cycloalkyl, aryl, aralkyl, alkoxy, thioalkoxy, cycloalkylalkoxy, heterocycloalkyl, aralkyloxy, or heteroaryl and R through R are each independently any of these groups incorporating ester, amide, amine, hydroxyl, halogen, sulfonate, phosphonate, guanidine, and/or heteroaryl groups.

22. An agent of any one of the preceding claims, wherein R⁵ through R⁸ are each independently hydrogen, halogen, alkyl, cycloalkyl, aryl, aralkyl, alkoxy, thioalkoxy, cycloalkylalkoxy, heterocycloalkyl, aralkyloxy, or heteroaryl and R¹ through R⁴ are each independently any of these groups incorporating ester, amide, amine, hydroxyl, halogen, sulfonate, phosphonate, guanidine, and/or heteroaryl groups

23. An agent of any one of the preceding claims, wherein the agent demonstrates cytotoxic activity against non-endothelial cells.

24. An agent of any one of the preceding claims, wherein the agent demonstrates cytotoxic activity against galectin-1 negative cells.

25. An agent of any one of the preceding claims, wherein the agent demonstrates cytotoxic activity against cells having an epithelial to mesenchymal transition (EMT) phenotype.

26. An agent of any one of the preceding claims, wherein the agent demonstrates cytotoxic activity against cells resistant to compound 0118.

27. An agent of any one of the preceding claims, wherein the agent demonstrates antitumor activity.

28. An agent of any one of the preceding claims, wherein the agent demonstrates a cytotoxic activity at least 5 fold greater than compound 1097, compound 0118, and/or anginex against the B16F10 murine melanoma cell line.

29. An agent of any one of the preceding claims conjugated to a diagnostic agent, a therapeutic agent, a detectable marker, a targeting moiety, or a liposome.

30. A pharmaceutical composition comprising an agent of any one of the preceding claims.

31. A method of killing cells comprising contacting cells with an agent, conjugate, or composition of any one of claims 1 to 30.

32. The method of claim 31 , wherein the contacting step occurs in vitro.

33. The method of claim 31, wherein the contacting step occurs in vivo.

34. The method of any one of claims 31 to 33, wherein the cells are present in a cell culture, a tissue, an organ, or an organism.

35. The method of any one of claims 31 to 34, wherein the cell comprises a non- endothelial cell.

36. The method of any one of claims 31 to 35, wherein the cells are galectin-1 negative cells.

37. The method of any one of claims 31 to 36, wherein the cells have an epithelial to mesenchymal transition (EMT) phenotype.

38. The method of any one of claims 31 to 37, wherein the cells are resistant to compound 0118.

39. The method of any one of claims 31 to 38, wherein the cells are mammalian cells.

40. The method of any one of claims 31 to 39, wherein the cells comprise cancer cells.

41. A method of treating cancer in a subject, the method comprising achninistering an agent or composition of any one of claims 1 to 30.

42. A method for inhibiting tumorigenesis in a patient, the method comprising achrnmstering to the patient a therapeutically effective amount of a composition comprising an agent or composition of any one of claims 1 to 30.

43. The method of any one of claims 40 to 42, wherein the cancer or tumor is a carcinoma, a sarcoma, a blood borne hematologic cancer, or a germ line cancer.

44. The method of any one of claims 40 to 43, wherein the cancer or tumor is a breast cancer, ovarian cancer, melanoma, colon cancer, lung cancer, or a squamous cell carcinoma.

45. The method of any one of claims 40 to 44, wherein the cancer or tumor is a primary cancer or a metastatic cancer.

46. The method of claim any one of claims 40 to 45, wherein the cancer or tumor comprises galetin-1 negative cells.

47. The method of any one of claims 40 to 46, wherein cancer or tumor comprises cells having an epithelial to mesenchymal transition (EMT) phenotype.

48. The method of any one of claims 40 to 47, wherein the cancer or tumor comprises cells resistant to compound 0118 and/or compound 1097.

49. The method of any one of claims 40 to 48, further comprising admimstering one or more additional therapeutic agents.

50. The method of claims 49, wherein the cytotoxic nonpeptidic calixarene-based topomimetic agent and the one or more additional therapeutic agents demonstrate a synergy.

The method of any one of claims 31 to 50, wherein the cells are human cells.