Field

[0001] The disclosure relates to compounds and compositions as well as methods of using said compounds and compositions for inhibiting microtubule polymerization, inducing lysosomal disruption and/or treating cancer including hematological malignancies including leukemia.

Background

[0002] Microtubules are an integral part of the cell cytoskeleton and play important roles in many cellular processes, including cell division. Given that cancer cells often display faster growth rates, microtubules constitute a potential biological vulnerability and an important target for anticancer drugs. There are various agents that bind distinct sites on tubulin and promote either microtubule destabilization or stabilization that leads to impairment of microtubule assembly or disassembly, respectively. At least three binding sites have so far been identified: the colchicine site close to the α/β interface, the vinca alkaloids site and the taxane-binding pocket, which is within the lumen of microtubules.

[0003] While there have been recent advances in the treatment of some cancers and particularly hematologic malignancies, the treatment of acute myeloid leukemia (AML) has remained largely unchanged for over 20 years. For patients diagnosed when older than 60, the prognosis is particularly poor, with a 2-year survival probability of less than 10 percent (Gupta et al., 2007). Thus, new treatment approaches are needed for the treatment of cancer and AML

Summary

[0004] An aspect includes compound of Formula I

wherein R ¹ , R ² and R ³ are independently selected from H and C- o-alkyl, Y is OH or H and/or a pharmaceutically acceptable salt and/or solvate thereof.

[0005] Another aspect includes a composition comprising a compound of Formula I and a suitable vehicle such as a carrier or diluent, optionally a pharmaceutically acceptable carrier or diluent. [0006] A further aspect includes a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of Formula II

wherein R ¹ , R ² and R ³ are independently selected from H and Ci_i ₀ -alkyl, optionally with the proviso that R ¹ , R ² and R ³ are not all H; and

wherein X is H, O or OH; and/or a pharmaceutically acceptable salt and/or solvate thereof optionally wherein the composition is for treating cancer.

[0007] In an embodiment, X is O.

[0008] In an embodiment, the compound is the (-) enantiomer.

[0009] In an embodiment, the compound is the (+) enantiomer.

[0010] In an embodiment, the compound is deoxysappanone B 7,4'-dimethyl ether (Deox B 7, 4).

[0011] In an embodiment, the deoxysappanone B 7,4'-dimethyl ether compound is the (+) enantiomer.

[0012] In an embodiment, the deoxysappanone B 7,4'-dimethyl ether compound is the (-) enantiomer.

[0013] In an embodiment, the compound is a compound of Formula I. In an embodiment, Y is H. In an embodiment, R ¹ , R ² and/or R ³ is/are methyl.

[0014] In an embodiment, the compound is a compound of Formula I, wherein the compound is the (-) enantiomer and wherein each of Y and R ¹ is H and each of R ² and R ³ is CH ₃ .

[0015] In an embodiment, the compound is reduced deoxysappanone B 7,4'-dimethyl ether (e.g. reduced carbonyl (reduced to H); R-Deox B7, 4').

[0016]

[0017] In an embodiment, the compound is a compound of Formula I wherein the X is H. In an embodiment the compound of Formula I is an (-) enantiomer (e.g. with respect to the carbon alpha to the carbonyl), e.g. a compound of Formula I wherein the carbon alpha to the carbonyl is the (-) enantiomer. In an embodiment, the compound is the R-Deox (-) enantiomer.

[0018] In an embodiment, the compound is deoxysappanone B 7,3'-dimethyl ether, optionally the (-) enantiomer or the (+) enantiomer.

[0019] In an embodiment, the compound is sappanone A.

[0020] Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the disclosure are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

Brief description of the drawings and tables

[0021] An embodiment of the present disclosure will now be described in relation to the drawings and tables in which:

[0022] Figure 1. Deox B 7,4 and R-Deox B 7,4 (-) exhibit cytotoxicity against AML cells in vitro. (A) TEX cells were treated with drugs (1.67 μΜ concentration shown) from a natural product library for 72 hours before assessing viability using sulforhodamine B staining. B) A panel of human leukemia cell lines was treated with Deox B 7,4 for 72 hours before assessing viability using a MTS assay. Results shown are representative of 3 experiments. C) Cell death kinetics were determined using AnnexinV and PI staining following treatment of TEX cells with a concentration of Deox B 7,4 equivalent to 2.5X its IC ₅₀ value for TEX cells. Results are shown as mean + SD and are representative of 3 experiments (**p<0.01 and * ^** p<0.001 compared to control cells). D) TEX cells were treated with compounds and analogues structurally related to Deox B 7,4 for 72 hours before assessing viability using a MTS assay. Results shown are representative of 3 experiments. E) Chromatograms showing chiral separation of Deox B 7,4 and R-Deox B 7,4 into their (-) and (+) enantiomers. F/G) TEX cells were treated with (F) a racemic mixture of Deox B 7,4 and its enantiomers or (G) a racemic mixture of R-Deox B 7,4 and its enantiomers for 72 hours before assessing viability using a MTS assay. Results shown are representative of 3 experiments. H) TEX cells were treated with R-Deox B 7,4 (-) for 72 hours in hypoxia before assessing viability using a MTS assay. Results shown are representative of 3 experiments. I) Normal hematopoietic cells (n - 4) and primary AML cells (n = 5) were treated with 100 nM R-Deox B 7,4 (-) and plated in clonogenic growth assays. Cell growth and viability represents the percentage of live cells relative to control cells.

[0023] Figure 2. Deox B 7,4 and R-Deox B 7,4 (-) function as microtubule inhibitors. A) Deox B 7,4, enantiomers of R-Deox B 7,4 or known microtubule inhibitors (10 μΜ) were incubated with MAP-rich tubulin (1.2 mg/ml) and microtubule polymerization was assessed by monitoring changes in absorbance at 340 nm. Results shown are representative of 3 experiments. B) Purified tubulin (0.5 mg/ml) was incubated for 30 minutes in the presence of Deox B 7,4, enantiomers of R-Deox B 7,4 or known microtubule inhibitors (100 μΜ) after which colchicine (12 μΜ) was added for another 60 minutes. Tubulin-colchicine complex fluorescence was measured with excitation and emission wavelengths of 360 nm and 430 nm, respectively. Results are shown as mean + SD and are representative of 3 experiments (***p<0.001 compared to control). C) TEX cells were treated with concentrations of drugs equivalent to 2.5X their respective IC ₅₀ values for this cell line and cell cycle analysis was carried out at the specified times. Showing results from 1 of 2 similar experiments. D) Mitotic block reversibility of Deox B 7,4, R-Deox B 7,4 (-), colchicine and vinblastine was assessed in U937 cells as described in Experimental Procedures. Left panel: U937 cells were treated with 2X the respective IC ₅₀ value of each microtubule inhibitor for this cell line and cell cycle analysis was carried out at the end of the 12 hours drug treatment period and 10 hours post-wash; right panel: live cells were counted using trypan blue exclusion 5 days post-wash. Showing results from 1 of 2 similar experiments.

[0024] Figure 3. Deox B 7,4 and R-Deox B 7,4 (-) lead to increased lysosome acidity and promote lysosomal disruption. A) V-ATPase activity was measured using an acridine orange quenching assay on lysosomes purified from TEX cells treated for 4 hours or 8 hours with R-Deox B 7,4 (-) or left untreated (DMSO control). Fluorescence was measured every 10 sees for 3 minutes using excitation and emission wavelengths of 490 nm and 540 nm, respectively. Results shown have been baseline-corrected and are representative of 3 experiments. B) TEX cells were treated for 8 hours with R-Deox B 7,4 (-), bafilomycin A1 or DMSO and the effect of R-Deox B 7,4 (-) on lysosome acidity was assessed using LysoSensor Yellow/Blue DND-160 staining (5 μΜ) and fluorescence microscopy. Results shown are representative of 3 experiments. C) TEX cells were treated with various microtubule inhibitors for the specified times using concentrations equivalent to 2.5X their respective IC ₅₀ values for this cell line. Lysosome integrity was measured using acridine orange staining and flow cytometry. Bafilomycin was used as a positive control. Only viable cells, based on forward and side scatter properties, were included in the analysis. Results shown are representative of experiments performed 3 to 5 times. D) Lysosomes were isolated from TEX cells and treated with various microtubule inhibitors as well as bafilomycin A1 (40 pg/sample) for 2 hours before assessing cathepsin B release. Triton-X detergent was used as a positive control. Drug concentrations used were equivalent to 10X their respective IC ₅₀ values for this cell line. Results are shown as mean ± SD and are representative of 2 experiments (***p<0.001 compared to control cells). E) TEX cells were treated with Deox B 7,4, R-Deox B 7,4 (-) or DMSO for 24 hours in the presence or absence of bafilomycin before assessing viability using a MTS assay (where indicated, bafilomycin A-i was added 1 hour before addition of microtubule inhibitors). Drug concentrations used were equivalent to 2.5X their respective IC ₅₀ values for this cell line. Results are shown as mean ± SD and are representative of 3 experiments (***p<0.001 compared to cells treated with microtubule inhibitors in the absence of bafilomycin A^.

[0025] Figure 4. R-Deox B 7,4 (-) synergizes with vinca alkaloids in vitro and displays some degree of antitumor activity in an in vivo xenograft mouse model. A)

TEX cells were treated with sub-optimal concentrations of R-Deox B 7,4 (-), vinblastine or vincristine, either individually or in combination, and cell growth and viability was assessed after 72 hours using a MTS assay. (*p<0.05, **p<0.01 and ***p<0.001 compared to cells treated with R-Deox B 7,4 (-) only). B) SCID mice (n = 10 per group) were challenged subcutaneously with 5x10 ⁵ OCI-AML2 leukemia cells. Once tumors were palpable (day 6), mice received intraperitoneal injections of 75mg/Kg Red-Deox B 7,4 (-) or vehicle control (10% v/v DMSO + Cremophor in saline) twice daily 5 days per week until day 18, at which time mice were sacrificed (***p<0.0001 ).

[0026] Figure 5. Schematic of making R-Deox 7,4 (-).

[0027] Figure 6. Chromatographs of compounds and enantiomers.

[0028] Table 1. List of hits from natural product chemical library screen on TEX cells. High-throughput screening of 640 natural products was performed on TEX cells using final concentrations varying between 1.67 μΜ and 13.3 μΜ. Cell viability was assessed using sulforhodamine B staining following 72 hours incubation. Compounds resulting in 20% viable cells or less relative to control cells at 13.3 μΜ were considered hits.

[0029] Table 2. List of GO processes enriched >2 fold in S. cerevisiae screen hits obtained from Deox B 7,4 screen in YPD medium. Gene ontology analysis was performed on 85 genes associated with the deletion strains showing the greatest sensitivity to Deox B 7,4 in YPD (dextrose) medium. GO biological processes represented by 4 or more genes from the dataset and enriched a minimum of 2 fold relative to the yeast genome are reported. [0030] Table 3. List of GO processes enriched >2 fold in S. cerevisiae screen hits obtained from Deox B 7,4 screen in YPGE medium. Gene ontology analysis was performed on 51 genes associated with the deletion strains showing the greatest sensitivity to Deox B 7,4 in YPGE (glycerol-ethanol) medium. GO biological processes represented by 4 or more genes from the dataset and enriched a minimum of 2 fold relative to the yeast genome are reported.

[0031] Table 4. Microtubule inhibition by Deox B 7,4 and R-Deox B 7,4 (-) is functionally relevant and overcomes drug efflux pump-related resistance. Alpha-tubulin mutated KB-4.0-HTI epidermoid carcinoma cells and their KB-3-1 wild-type controls, as well as A2780ADR ovarian carcinoma cells overexpressing P-glycoprotein and their A2780 wild- type controls, were treated with increasing concentrations of the specified microtubule inhibitors. Cell viability was assessed 72 hours later using a MTS assay. Results shown represent IC ₅₀ values + SD calculated from 3 individual experiments (n/a: not assessed).

Detailed description of the Disclosure

[0032] An aspect includes compound of Formula I

wherein R ¹ , R ² and R ³ are independently selected from H and C^o-alkyl and Y is OH or H and/or a pharmaceutically acceptable salt and/or solvate thereof

[0033] In an embodiment, Y is H.

[0034] As used herein, the term "alkyl" embraces straight-chain or branched-chain hydrocarbons. Substituted alkyl residues can be substituted in any suitable position. Examples of alkyl groups containing from 1 to 10 carbon atoms are methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl, the n-isomers of all these residues, isopropyl, isobutyl, isopentyl, neopentyl, isohexyl, isodecyl, 3-methylpentyl, 2,3,4- trimethylhexyl, sec-butyl, tert-butyl, or tert-pentyl.

[0035] In an embodiment, R , R ² and/or R ³ is methyl.

[0036] Another aspect includes a composition comprising a compound of Formula I and a suitable vehicle such as a carrier or diluent, optionally a pharmaceutically acceptable carrier or diluent. [0037] Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (2003- 20 ^th Edition). On this basis, the compositions include, albeit not exclusively, solutions of the substances in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.

[0038] A further aspect includes a pharmaceutical composition comprising a pharmaceutically acceptable carrier and A and/or a compound of Formula II

wherein R ¹ , R ² and R ³ are independently selected from H and C _{1- 0} -alkyl; and

wherein X is either O, H or OH; and/or a pharmaceutically acceptable salt and/or solvate thereof

optionally wherein the composition is for treating cancer. [0039] In an embodiment, R ¹ , R ² and R ³ are not all H.

[0040] The term "pharmaceutically acceptable" means compatible with the treatment of animals, in particular, humans.

[0041] The term "compound(s) of the application" as used herein means compound(s) of Formula I and/or II, and/or pharmaceutically acceptable salts, solvates and/or prodrugs thereof. It should be noted that the compositions, methods and uses extend to cover mixtures of compounds of Formula I and/or II and their pharmaceutically acceptable salts, solvates and/or prodrugs.

[0042] As described, compounds of the application may have at least one asymmetric centre. Where the compounds described herein possess more than one asymmetric centre, for example with Y or X is OH, they may exist as diastereomers. It is to be understood that all such isomers and mixtures thereof in any proportion are encompassed within the scope of the present application. It is to be understood that while the stereochemistry of the compounds of the application may be as provided for in any given compound listed herein, such compounds of the application may also contain certain amounts (e.g. less than 20%, suitably less than 10%, more suitably less than 5%) of compounds of the application having alternate stereochemistry. [0043] The term "pharmaceutically acceptable salt" means an acid addition salt, which is suitable for or compatible with the treatment of patients. The term "pharmaceutically acceptable acid addition salt" as used herein means any non-toxic organic or inorganic salt of any base compound of the application. Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids that form suitable salts include mono- di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylic acids, as well as sulfonic acids such as p-toluene sulfonic and methanesulfonic acids. Either the mono or di-acid salts can be formed, and such salts may exist in either a hydrated, solvated or substantially anhydrous form. In general, acid addition salts are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms. The selection of the appropriate salt will be known to one skilled in the art.

[0044] The term "solvate" as used herein means a compound or its pharmaceutically acceptable salt, wherein molecules of a suitable solvent are incorporated in the crystal lattice. A suitable solvent is physiologically tolerable at the dosage administered. Examples of suitable solvents are ethanol, water and the like. When water is the solvent, the molecule is referred to as a "hydrate". The formation of solvates of the compounds of the application will vary depending on the compound and the solvate. In general, solvates are formed by dissolving the compound in the appropriate solvent and isolating the solvate by cooling or using an antisolvent. The solvate is typically dried or azeotroped under ambient conditions.

[0045] In an embodiment, the compound is deoxysappanone B, for example, in an extract and/or isolated from Caesalpinia sappan.

[0046] In an embodiment, the compound is deoxysappanone B 7,4'-dimethyi ether (Deox B 7, 4).

[0047] As used herein "deoxysappanone B 7,4'-dimethyl ether" and/or "Deox B 7, 4" means a compound having the structure:

and includes all pharmaceutically acceptable salts, solvates, and prodrugs thereof as well as combinations thereof. [0048] In an embodiment, the compound is an enantiomer. For example a chiral centre is present in in deoxysappanone B 7,4'-dimethyl ether (Deox B 7, 4) at the carbon alpha to the carbonyl group (see Figure 1 which shows the chiral centre as a squiggly line). This chiral centre is referred to herein for example as the "carbon alpha to the carbonyl".

[0049] In an embodiment, the compound is the (-) enantiomer.

[0050] In an embodiment, the compound is the (+) enantiomer.

[0051] In an embodiment, the deoxysappanone B 7,4'-dimethyl ether compound is the (+) enantiomer.

[0052] In an embodiment, the deoxysappanone B 7,4'-dimethyl ether compound is the (-) enantiomer.

[0053] As described herein both the (+) and the (-) enantiomer of deoxysappanone B 7,4'-dimethyl ether had activity as described in the Examples below.

In an embodiment, the compound is a compound of Formula I.

In an embodiment, the compound is a compound having the structure:

wherein R ¹ , R ² and R ³ are independently selected from H and C-i

pharmaceutically acceptable salt and/or solvate thereof.

[0056] In an embodiment, the compound is reduced deoxysappanone B 7,4'-dimethyl ether (e.g. reduced carbonyl; R-Deox B7, 4'). As described herein, the reduced deoxysappanone B 7,4'-dimethyl ether was about 5 fold more potent at reducing growth and viability of TEX cells compared to Deox B 7, 4'.

[0057] As used herein "reduced deoxysappanone B 7,4'-dimethyl ether" or "R-Deox B 7, 4" refers to a compound having the structure: and includes all pharmaceutically acceptable salts, solvates, and prodrugs thereof as well as combinations thereof. A synthesis scheme is provided in Example 3 and Figure 5.

[0058] As described in the methods, the carbonyl group in Deox B 7, 4 is reduced in R-Deox B 7, 4 so the carbonyl is absent.

[0059] A person skilled in the art using for example the schema provided in Example 3 and Figure 5 and/or schema's known in the art, would be able, by substituting one or more reactants with reactants with different substitutions (e.g. corresponding to formula I or II different R ¹ , R ² and R ³ substitutions) to produce other compounds of Formula I and II.

[0060] In an embodiment, the compound is a compound having the structure:

and/or all pharmaceutically acceptable salts, solvates, and prodrugs thereof as well as combinations thereof.

[0061] In an embodiment, the compound is the (-) enantiomer (e.g. carbon alpha to the carbonyl), e.g. a compound of Formula I wherein the carbon alpha to the carbonyl position (e.g. whether the carbonyl is present or OH or H) is the (-) enantiomer.

[0062] In an embodiment, the compound is the R-Deox (-) enantiomer.

[0063] In an embodiment, the compound is deoxysappanone B 7,3'-dimethyl ether, optionally the (-) enantiomer or the (+) enantiomer. [0064] As used herein, "deoxysappanone B 7,3'-dimethyl ether" or "Deox B 7,3"' refers to a compound having the structure:

and includes all pharmaceutically acceptable salts, solvates, and prodrugs thereof as well as combinations thereof.

[0065] In an embodiment, the compound is sappanone A.

[0066] As used herein, "sappanone A" as used herein means a compound having the structure:

and includes all pharmaceutically acceptable salts, solvates, and prodrugs thereof as well as combinations thereof.

[0067] The enantiomers herein are referred to in general as (-) or (+) depending on their optical activity (e.g. the direction in which it rotates a plane of polarized light).

[0068] Enantiomers can also for example be denoted by the R/S system, which labels each chiral center R or S according to a system by which its substituents are each assigned a priority, according to the Cahn-lngold-Prelog priority rules (CIP), based on atomic number. If the center is oriented so that the lowest-priority of the four is pointed away from a viewer, the viewer will then see two possibilities: If the priority of the remaining three substituents decreases in clockwise direction, it is labeled R (for Rectus, Latin for right), if it decreases in counterclockwise direction, it is S (for Sinister, Latin for left).

[0069] In general, unless clearly indicated otherwise, reference to R herein refers to a reduced compound of formula I or II, for example where X or Y is H.

[0070] In an embodiment, the compound or pharmaceutical composition 'is or comprises a compound of Formula I and/or II that is extracted from a plant such as Caesalpinia species optionally Caesalpinia sappan. [0071] The compounds of the application are suitably formulated into pharmaceutical compositions for administration to human subjects in a biologically compatible form suitable for administration in vivo.

[0072] The application in one aspect, also describes a pharmaceutical composition comprising an effective amount of one or more compounds of the application and a pharmaceutically acceptable carrier for treatment of a leukemia, lymphoma and/or multiple myeloma in a subject in need of such treatment.

[0073] The compositions described herein can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions that can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle.

[0074] In an embodiment of the application the pharmaceutical composition contains about 0.01 % to about 1 %, suitably about 0.01 % to about 0.5%, of one or more compounds of the application. The composition may be prepared, for example, by mixing the carrier and the compound(s) at a temperature of about 40 °C to about 70 °C ^'

[0075] Pharmaceutical compositions include, without limitation, lyophilized powders or aqueous or non-aqueous sterile injectable solutions or suspensions, which may further contain antioxidants, buffers, bacteriostats and solutes that render the compositions substantially compatible with the tissues or the blood of an intended recipient. Other components that may be present in such compositions include water, surfactants (such as Tween), alcohols, polyols, glycerin and vegetable oils, for example. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, tablets, or concentrated solutions or suspensions. The composition may be supplied, for example but not by way of limitation, as a lyophilized powder which is reconstituted with sterile water or saline prior to administration to the patient.

[0076] Suitable pharmaceutically acceptable carriers include essentially chemically inert and nontoxic compositions that do not interfere with the effectiveness of the biological activity of the pharmaceutical composition. Examples of suitable pharmaceutical carriers include, but are not limited to, water, saline solutions, glycerol solutions, ethanol, N-(1 (2,3- dioleyloxy)propyl)N,N,N-trimethylammonium chloride (DOTMA), diolesylphosphotidyl- ethanolamine (DOPE), and liposomes. Such compositions should contain a therapeutically effective amount of the compound, together with a suitable amount of carrier so as to provide the form for direct administration to the patient. [0077] The compositions described herein can be administered for example, by parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol or oral administration.

[0078] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersion and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists.

[0079] A further aspect includes a method of inhibiting microtubule polymerization and/or inducing lysosomal disruption comprising contacting a cell and/or administering to a subject in need thereof a compound or a composition described herein.

[0080] As described herein, compounds of Formula I and/or Formula II inhibit microtubule polymerization and induce lysosomal disruption. Further it is demonstrated that the microtubule polymerization and lysocomal disruption abilities are functionally important for Deox activity. Accordingly, in an embodiment, the compound is a compound that has microtubule polymerization inhibition activity and/or lysosomal disruption activity, determined for example in an assay as described in Example 1.

[0081] In an embodiment, the subject in need thereof has cancer, optionally a hematological cancer.

[0082] Microtubule polymerization inhibitors are used to treat cancers such as hematological cancers, including leukemia, lymphoma and myeloma as well as lung cancer, epidermoid cancer, breast cancer and ovarian cancer. In an embodiment, the cancer can be any cancer treatable by a microtubule inhibitor such as a taxane and/or a vinca alkaloid.

[0083] In an embodiment, the hematological cancer is leukemia.

[0084] in an embodiment, the leukemia is acute myeloid leukemia (AML).

[0085] In an embodiment, the leukemia is acute lymphoblastic leukemia (ALL).

[0086] In an embodiment, the cancer is breast cancer, lung cancer, epidermoid cancer, ovarian cancer, and/or the hematological cancer is myeloma or lymphoma.

[0087] In an embodiment, the lymphoma is Hodgkin's or non-Hodgkin's lymphoma.

[0088] It is also demonstrated, for example in an ovarian cancer cell line, that P- glycoprotein (Pgp) overexpression, which is a described resistance mechanism against microtubule inhibitors, with vinca alkaloids and taxanes being good substrates for Pgp, does not circumvent toxicity of Deox B 7, 4 and Deox B7, 4 (-).

[0089] In an embodiment, the cancer is a Pgp overexpressing cancer. Radioactive verapamil can be used for measuring P-glycoprotein function with positron emission tomography and used to identify Pgp overexpressing cancers. In an embodiment, the cell is further contacted and/or the subject is further administered a microtubule polymerization inhibitor such as a taxane or a vinca alkaloid.

[0090] A further aspect includes a method of treating a subject with a cancer selected from a hematological cancer, optionally leukemia, lymphoma, or myeloma, lung cancer, epidermoid cancer, ovarian cancer, or breast cancer comprising administering a compound or a composition described herein.

[0091] The term "treating" or "treatment" as used herein and as is well understood in the art, means an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable. "Treating" and "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. "Treating" and "treatment" as used herein also include prophylactic treatment. For example, a subject with early stage multiple myeloma can be treated to prevent progression or alternatively a subject in remission can be treated with a compound or composition described herein to prevent recurrence. Treatment methods comprise administering to a subject a therapeutically effective amount of one or more compounds described in the application and optionally consists of a single administration, or alternatively comprises a series of applications. For example, the compounds described herein may be administered at least once a week, about one time per week to about once daily for a given treatment or the compound may be administered one, two, three or four times daily, for example twice daily. The length of the treatment period depends on a variety of factors, such as the severity of the disease, the age of the patient, the concentration, the activity of the compounds described herein, and/or a combination thereof. It will also be appreciated that the effective dosage of the compound used for the treatment or prophylaxis may increase or decrease over the course of a particular treatment or prophylaxis regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In some instances, chronic administration may be required.

[0092] In an embodiment, the leukemia is AML or ALL.

[0093] In an embodiment, the lymphoma is Hodgkin's or non-Hodgkin's lymphoma.

[0094] The dosage administered will vary depending on the use and knowrl factors such as the pharmacodynamic characteristics of the particular substance, and its mode and route of administration, age, health, and weight of the individual recipient, nature and extent of symptoms, kind of concurrent treatment, frequency of treatment, and the effect desired. Dosage regime may be adjusted to provide the optimum therapeutic response

[0095] The term "subject" as used herein includes all members of the animal kingdom including mammals, and suitably refers to humans.

[0096] The term "hematological malignancy" as used herein refers to cancers that affect blood and bone marrow.

[0097] The term "leukemia" as used herein means any disease involving the progressive proliferation of abnormal leukocytes found in hemopoietic tissues, other organs and usually in the blood in increased numbers. For example, leukemia includes acute myeloid leukemia, acute lymphocytic leukemia and chronic myeloma leukemia (CML) in blast crisis.

[0098] The term "lymphoma" as used herein means any disease involving the progressive proliferation of abnormal lymphoid cells. For example, lymphoma includes Non- Hodgkin's lymphoma, and Hodgkin's lymphoma. Non-Hodgkin's lymphoma would include indolent and aggressive Non-Hodgkin's lymphoma. Aggressive Non-Hodgkin's lymphoma would include intermediate and high grade lymphoma. Indolent Non-Hodgkin's lymphoma would include low grade lymphomas (30). Non-Hodgkin's lymphomas can also for example be as classified using the WHO and REAL classification.

[0099] The term "myeloma" and/or "multiple myeloma" as used herein means any tumor or cancer composed of cells derived from the hemopoietic tissues of the bone marrow. Multiple myeloma is also knows as MM and/or plasma cell myeloma.

[00100] As mentioned, it is demonstrated herein that a compound of Formula I and II can overcome Pgp resistance.

[00101] Accordingly, in an embodiment, the subject is further administered a microtubule polymerization inhibitor (e.g. a microtubule polymerization inhibitor that is distinct from a compound of Formula I and/or II that is administered in combination with the compound of Formula I and/or II or a compositions comprising said compounds).

[00102] Microtubule polymerization inhibitors, also referred to as tubulin inhibitors are compounds that interfere directly with tubulin polymerization, and include tubulin binding molecules such as taxanes, vinca alkaloids and colchicine.

[00103] In an embodiment, the microtubule polymerization inhibitor comprises a taxane. In an embodiment, the taxane is paclitaxel or docetaxel.

[00104] In another embodiment, the microtubule polymerization inhibitor is a vinca alkaloid. In an embodiment, the vinca alkaloid is vinblastine, vincristine, vindesine, and/or vinorelbine.

[00105] As used herein, "contemporaneous administration" and "administered contemporaneously" means that two substances are administered to a subject such that they are both biologically active in the subject at the same time. The exact details of the administration will depend on the pharmacokinetics of the two substances in the presence of each other, and can include administering one substance within 24 hours of administration of the other, if the pharmacokinetics are suitable. Designs of suitable dosing regimens are routine for one skilled in the art. In particular embodiments, two substances will be administered substantially simultaneously, i.e. within minutes of each other, or in a single composition that comprises both substances.

[00106] As used herein, the phrase "effective amount" or "therapeutically effective amount" means an amount effective, at dosages and for periods of time necessary to achieve the desired result. For example in the context or treating a hematological malignancy, an effective amount is an amount that for example induces remission, reduces tumor burden, and/or prevents tumor spread or growth compared to the response obtained without administration of the compound(s). Effective amounts may vary according to factors such as the disease state, age, sex, weight of the subject. The amount of a given compound that will correspond to such an amount will vary depending upon various factors, such as the given drug or compound, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject or host being treated, and the like, but can nevertheless be routinely determined by one skilled in the art.

[00107] In an embodiment, the amount administered and/or contacted is an effective amount. [00108] As used herein, to "inhibit" or "suppress" or "reduce" a function or activity, such as microtubule polymerization, is to reduce the function or activity when compared to a control, an otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition. The terms "inhibitor" and "inhibition", in the context of the present application, are intended to have a broad meaning and encompass compounds of Formula I and/or II which directly or indirectly (e.g., via reactive intermediates, metabolites and the like) act on for example the microtubule polymerization.

[00109] As used herein, to "induce" a disruption, such as lysosomal disruption, is to induce the disruption when compared to a control, an otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition. The terms "inducer" and "induction", in the context of the present application, are intended to have a broad meaning and encompass compounds of Formula I and/or II which directly or indirectly (e.g., via reactive intermediates, metabolites and the like) act on for example to induce lysosomal disruption.

[00110] A further aspect is a combination or commercial package comprising a compound (e.g. compound of the application) and/or composition described herein and optionally a) a microtubule polymerization inhibitor such as a taxane or a vinca alkaloid and/or b) instructions for use.

[00111] Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. For example, in the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[00112] The above disclosure generally describes the present application. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for the purpose of illustration and are not intended to limit the scope of the application. Changes in form and substitution of equivalents are contemplated as circumstances might suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

[00113] The following non-limiting examples are illustrative of the present disclosure:

Examples Example 1

[00114] AML is a hematological malignancy for which the standard of care therapy has remained unchanged for almost 30 years. Novel therapeutic approaches are therefore urgently needed for the treatment of this heterogeneous disease. To identify new strategies for the treatment of AML, a natural product library was screened for compounds cytotoxic to AML cells and identified Deoxysappanone B 7,4'-dimethyl ether.

[00115] Deoxysappanone B is a homoisoflavanoid compound extracted primarily from the dried heartwood of Caesalpinia sappan, a medicinal plant native to South-East Asia. However, anticancer activity of this compound has not been previously described and its molecular targets are largely unknown. In subsequent validation studies, Deoxysappanone B possessed anti-leukemic activity in 6 tested AML cell lines with nanomolar IC50s and was preferentially cytotoxic to primary AML cells and stem/progenitor cells over normal hematopoietic cells. To understand its mechanism of action, chemo-genomic profiling of Deoxysappanone B in S. cerevisiae was performed and enrichment of genes related to mitotic cell cycle as well as vacuolar acidification identified, therefore pointing to microtubules and lysosomes' proton-pumping vacuolar (V)-ATPase as potential targets. Deoxysappanone B's action as a microtubule inhibitor was confirmed and its binding site localized near to that of colchicine via in-vitro tubulin polymerization and competitive binding assays. Deoxysappanone B was shown to reversibly induce cell cycle arrest and cell death in a panel of AML cell lines as well as overcomes some mechanisms of resistance to vinca alkaloids. Validating the functional importance of tubulin as a target for Deoxysappanone B- mediated cell death, epidermoid carcinoma cells with a tubulin mutation were more resistant to Deoxysappanone B compared to their parental counterpart. In addition to inhibiting tubulin polymerization, Deoxysappanone B also increased lysosome acidity as measured by a V- ATPase enzymatic assay as well as staining with LysoSensorTM Yellow/Blue DND-160 and confocal microscopy. The sustained increase in lysosome acidity ultimately led to lysosomal disruption as evidenced by acridine orange staining. Supporting a tubulin-mediated effect on lysosomes, nocodazole, although not vinblastine, vincristine, paclitaxel or colchicine, produced a similar increase in lysosome acidity and lysosomal disruption. The effects on lysosomes were functionally relevant as pre-treatment with bafilomycin A1 , a lysosomal V- ATPase inhibitor, partially abrogated the cytotoxic effect of Deoxysappanone B. Thus, the data provide insight into a novel mechanism of action of select microtubule inhibitors in the context of AML. Example 2

RESULTS

[00116] Chemical Screening of a Natural Product Library Identifies Deoxysappanone B 7,4' Dimethyl Ether (Deox B 7,4) with Anti-Leukemic Activity To identify novel small molecules with anti-leukemic activity, 640 natural products screened were screened. TEX leukemia cells (Barabe et al., 2007; Warner et al., 2005) were treated with increasing concentrations of the natural products and cell growth and viability was assessed 72 hours later using sulforhodamine B staining. From this screen40 compounds were identified that reduced the growth and viability of TEX cells >80% at a concentration of 13.3 μΜ (Figure 1A and Table 1 ). Amongst these 40 compounds, 33 had either known mechanisms of action or previously reported anti-cancer activity and were not evaluated further. Of the lead compounds tested, Deoxysappanone B 7,4 dimethyl ether (Deox B 7,4) was the most potent compound identified in validation assays. Deox B 7,4 is a homoisoflavanoid compound extracted primarily from the dried heartwood of the medicinal plant Caesalpinia sappan native to South-East Asia (NAMIKOSHI and SAITOH, 1987). Its anti-cancer activity and molecular targets have not yet been thoroughly investigated.

[00117] To evaluate further the anti-leukemic activity of Deox B 7,4, the compound was subsequently tested on a panel of 6 human AML cell lines. Cells were treated with increasing concentrations of Deox B 7,4 and 72 hours after incubation, cell growth and viability was measured using a MTS assay. Deox B 7,4 reduced the growth and viability of all tested AML cell lines with a sub-micromolar IC ₅₀ (range: 324 nM to 556 nM) (Figure 1 B). Deox B 7,4 induced cell death, as measured by AnnexinV/PI staining, as early as 6 hours after addition of the drug to the cells (Figure 1 C).

[00118] An Analogue Enantiomer of Deox B 7,4 Displays Preferential Cytotoxicity Towards Human Primary ANIL Cells Compared to Normal Human Hematopoietic Cells

Next, the cytotoxicity of a series of compounds structurally related to Deox B 7,4 on TEX leukemia cells was assessed. From this evaluation, an analogue of Deox B 7,4 with a reduced carbonyl group (R-Deox B 7,4) was identified that was 5-fold more potent than the compound originally identified in the drug screen (IC ₅₀ 79.12 ± 4.90 nM) (Figure 1 D). Deox B 7,4 and R-Deox B 7,4 are racemic compounds with each one chirai center. The activity of their purified enantiomers was characterized. As shown on the chromatograms of Figure 1 E, chirai separation and optical rotation determination yielded relatively equal amounts of enantiomers, which were over 99% pure. Both Deox B 7,4 enantiomers exhibited cytotoxicity comparable to that of their racemic mixture (Figure 1 F). Interestingly however, only the R- Deox B 7,4 (-) enantiomer retained killing activity while the (+) enantiomer R-Deox B 7,4 (+) was inactive (Figure 1G). As expected from the chiral separation yields, R-Deox B 7,4 (-) was approximately 2-fold more potent than the unfractionated compound (IC ₅ o 41.21 ± 2.18 nM vs 79.12 ± 4.90 nM).

[00119] The activity of some anti-cancer agents can be reduced under conditions of low oxygen tension, thereby limiting the efficacy of the drugs in vivo, as malignant cells are often hypoxic. To determine whether R-Deox B 7,4 (-) remained active under hypoxia, TEX leukemia cells were treated with R-Deox B 7,4 (-) for 48 hours under decreasing concentrations of oxygen (21 % to 0.2%). The cytotoxicity of R-Deox B 7,4 (-) towards leukemia cells was not reduced under conditions of hypoxia (Figure 1 H).

[00120] We also evaluated the ability of R-Deox B 7,4 (-) to kill functionally defined subsets of primitive human primary AML and normal hematopoietic cell populations using a clonogenic growth assay. Incorporation of 100 nm R-Deox B 7,4 (-) into the assay medium markedly reduced the clonogenic growth of 3 out of 5 primary AML patient samples tested (Figure 1 1). In contrast, R-Deox B 7,4 (-) had almost no effect on the clonogenic growth of normal hematopoietic cell progenitors (n = 4). Thus, R-Deox B 7,4 (-) displays preferential cytotoxicity against a subset of human primary AML cells over normal hematopoietic cells.

[00121] Haplo-lnsufficiency Profiling of Deox B 7,4 in Saccharomyces cerevisiae Reveals Enrichment of Genes Related to Mitotic Cell Cycle and Vacuolar Acidification

To identify potential mechanisms of action of Deox B 7,4 in eukaryotic cells, haplo- insufficiency profiling (HIP), a chemical genomics platform developed in the yeast Saccharomyces cerevisiae, was used. The HIP assay is an unbiased in vivo quantitative measure of relative drug sensitivity of all ~6000 yeast proteins in a single assay, and identifies potential candidate protein targets (Giaever et al., 1999; Hoon et al., 2008). Deox B 7,4 toxicity was assessed in both fermentation medium (Yeast extract, Peptone, Dextrose - YPD), where the primary mode of metabolism is glycolysis, and respiratory medium (Yeast extract, Peptone, Glycerol, Ethanol - YPGE) in which metabolism is reliant on oxidative phosphorylation. The HIP assay identified 85 genes in YPD medium and 51 genes in YPGE medium associated with deletion strains showing the greatest sensitivity to Deox B 7,4 (data not shown). GO Slim Mapper (http://www.yeastgenome.org) was then used to analyze the gene lists and identify broad categories of enriched Gene Ontology (GO) biological processes. Leading GO processes enriched in the YPD gene list included mitotic cell cycle, organelle fission and chromosome segregation (Table 2). These results pointed to microtubule inhibition as a possible mechanism of action of Deox B 7,4. In contrast to the HIP results obtained from the YPD medium, the most strikingly enriched GO processes amongst the YPGE gene list were cellular ion homeostasis, protein complex biogenesis and endocytosis (Table 3). Notably, they encompassed several VMA genes (VMA: vacuolar membrane ATPase), which code for subunits of the vacuolar proton-translocating ATPase (V-ATPase), as well as genes coding for proteins required in its assembly (VMA21 , VPH2 and PKR1 ). V-ATPases play a crucial role in many important physiological processes dependent on pH homeostasis (Toei et al., 2010). Therefore, it was hypothesized that Deox B 7,4 could also interfere with acidification of the lysosome, the mammalian equivalent of the yeast vacuole, via a process that affects V-ATPase. Thus, microtubules and lysosomes as leading targets of Deox B 7,4 were identified.

[00122] Deox B 7,4 and R-Deox B 7,4 (-) Inhibit Microtubule Polymerization via Binding Near the Colchicine Site To follow-up on the HIP results generated using the YPD medium, it was examined whether the anti-leukemic activity of Deox B 7,4 and R-Deox B 7,4 (-) could be mediated through interference with microtubule formation using an in vitro tubulin polymerization assay. MAP-rich tubulin was incubated in the presence of Deox B 7,4, R-Deox B 7,4 enantiomers as well as the known microtubule inhibitors vinblastine, nocodazole and paclitaxel. Tubulin polymerization was then measured over time (Figure 2A). The known microtubule destabilizers vinblastine and nocodazole prevented tubulin polymerization and the known microtubule stabilizer paclitaxel promoted tubulin polymerization. Both Deox B 7,4 and R-Deox B 7,4 (-) inhibited microtubule polymerization. In contrast, the non-toxic enantiomer R-Deox B 7,4 (+) did not significantly interfere with tubulin polymerization (Figure 2A).

[00123] In order to determine the binding site of the Deox compounds, a competitive colchicine binding assay, which is based on the fluorescent property of the colchicine-tubulin complex (Bhattacharyya and Wolff, 1974), was used. Purified tubulin was first incubated with Deox B 7,4 as well as the R-Deox B 7,4 enantiomers. Vinblastine, which binds the vinca alkaloid site on tubulin, and nocodazole, which binds the colchicine site, were included as negative and positive controls, respectively (Lu et al., 2012). Colchicine was later added and fluorescence subsequently measured. As shown in Figure 2B, there was a marked reduction in fluorescence seen with Deox B 7,4, R-Deox B 7,4 (-) and nocodazole, indicative of tubulin binding within the proximity of the colchicine site. In contrast, prior incubation with the enantiomer R-Deox B 7,4 (+) only led to -30% decrease in fluorescence. Taken together, these in vitro tubulin assays suggest that Deox B 7,4 and R-Deox B 7,4 (-) destabilize microtubules by binding tubulin near the colchicine site.

[00124] Deox B 7,4 and Deox B 7,4 (-) Promote Reversible G2 Arrest Given the fact that the Deox compounds altered microtubule dynamics in cell-free assays, their effect on tubulin-mediated processes in intact cells was evaluated. TEX leukemia cells were treated with Deox B 7,4, R-Deox B 7,4 (-), nocodazole or colchicine for up to 24 hours. Cell cycle analysis was carried out at different time points using PI staining of DNA content and flow cytometry (Figure 2C). Within 8 hours of drug treatment, both Deox compounds and nocodazole promoted significant G2 arrest. Likewise, colchicine-treated cells were also arrested in the G2 phase, but the time to cell cycle arrest was longer.

[00125] To evaluate whether the effect of the Deox compounds was reversible, a mitotic block reversibility assay, as previously described (Towle et al., 201 1 ), was performed. U937 leukemia cells were treated with Deox B 7,4, R-Deox B 7,4 (-), colchicine or vinblastine for 12 hours before washing off the drugs and allowing the cells to recover for 5 days. Cell cycle analysis was carried out at the end of the drug treatment period and 10 hours post- wash. As shown in the left panel of Figure 2D, cell cycle analysis 10 hours post-wash indicated resumption of cell growth for the Deox compounds and vinblastine, as evidenced by the marked increase in cell frequency in S phase and concurrent decrease in sub-G1 and G2 phases. In contrast, the effect of colchicine, an irreversible microtubule inhibitor, was not reversible with more pronounced cell death observed 10 hours post-wash. These latter results were confirmed by total cell counts at the end of the recovery period (Figure 1 D, right panel). These studies demonstrated that Deox B 7,4 and R-Deox B 7,4 (-) are fast-acting, reversible microtubule inhibitors.

[00126] Microtubule Inhibition by Deox B 7,4 and R-Deox B 7,4 (-) is a Functionally Important Mechanism of Action Next the importance of microtubule inhibition to the cytotoxicity mediated by the Deox compounds was examined. KB-4.0-HTI cells are epidermoid carcinoma cells that have a single nucleotide change in a-tubulin that renders them more resistant to microtubule inhibitors than their wild type counterpart KB-3-1 cells (Loganzo et al., 2004). KB-4.0-HTI and KB-3-1 were treated with increasing concentrations of Deox B 7,4, R-Deox B 7,4 (-), nocodazole and colchicine for 72 hours and assessed cell growth and viability using a MTS assay. Similarly to previously published observations for colchicine (Loganzo et al., 2004), KB-4.0-HTI cells were 2.86-fold more resistant to colchicine than the KB-3-1 control cells (Table 4). Likewise, the KB-4.0-HTI cells were almost 2-fold more resistant to the Deox compounds than the parental KB-3-1 cells. These results are consistent with the observed effect of the Deox compounds on microtubules and cell cycle and support microtubules as a target of these molecules.

[00127] Deox B 7,4 and R-Deox B 7,4 (-) Overcome Drug Resistance Associated with P-glycoprotein Expression Overexpression of P-glycoprotein (Pgp; MDR1 gene), a drug efflux pump, is a well described resistance mechanism against microtubule inhibitors, with widely used vinca alkaloids and taxanes being particularly good substrates (Dumontet and Sikic, 1999). Subsequently, the impact of Pgp expression on the cytotoxic activity of the Deox compounds was evaluated. A2780ADR ovarian adenocarcinoma cells overexpressing Pgp have been derived from their parental counterpart, A2780 cells, following exposure to the DNA intercalating agent adriamycin (Hamilton et al. , 1984). Pgp overexpression by A2780ADR cells was first confirmed by flow cytometry (data not shown). Next, A2780ADR drug-resistant and A2780 drug-sensitive cells were treated with increasing concentrations of the Deox compounds, colchicine or vincristine during 72 hours and cell growth and viability was assessed using a MTS assay. As expected, A2780ADR cells were 59 and 1099-fold more resistant to colchicine and vincristine, respectively, compared to their wild type A2789 cells (Table 4). In contrast, A2780ADR cells remained essentially equally sensitive to Deox B 7,4 and R-Deox B 7,4 (-), compared to their parental A2780 cells. Taken together, these results indicate that Pgp overexpression does not circumvent the cytotoxicity of Deox B 7,4 and Deox B 7,4 (-) and therefore suggest their ability to overcome some forms of resistance to microtubule inhibitors currently used in the clinic.

[00128] Deox B 7,4 and R-Deox B 7,4 (-) Increase V-ATPase Activity and Lysosome Acidity The HIP results generated using the YPGE medium highlighted V- ATPase as a possible target of the Deox compounds. Therefore, it was examined whether the anti-leukemic activity of Deox B 7,4 and R-Deox B 7,4 (-) was also mediated through interference with lysosome acidification and lysosome disruption. First, a V-ATPase assay, where acridine orange fluorescence becomes quenched upon its accumulation and protonation in lysosomes ( oriyama et al., 1982), was used. TEX leukemia cells were treated with R-Deox B 7,4 (-) for 4 or 8 hours. After treatment, cells were harvested, lysed, and lysosomes were isolated. The lysosomes were incubated with acridine orange for 1 hour followed by the addition of ATP. Acridine orange fluorescence was then measured over time as a marker of lysosome acidity. Compared to control lysosomes, there was a faster and more pronounced decrease of acridine orange fluorescence in the presence of lysosomes isolated from TEX leukemia cells treated with R-Deox B 7,4 (-), consistent with R-Deox B 7,4 promoting increased V-ATPase activity and lysosome acidity (Figure 3A). Similar data were obtained with Deox B 7,4 (data not shown). To corroborate this observation, the effect of R- Deox B 7,4 (-) on lysosome acidity was also evaluated in intact cells. TEX leukemia cells were treated for 8 hours with R-Deox B 7,4 (-) or bafilomycin Ai , a V-ATPase inhibitor that rapidly alkalinizes lysosomes. Cells were then stained with the pH-sensitive dye LysoSensor Yellow/Blue DND-160 and pH changes in the lysosomes were visualized using fluorescence microscopy. When collecting fluorescence emitted at 450 nm, a decrease in fluorescence is indicative of lower pH while an increase in fluorescence is indicative of higher pH. As expected, bafilomycin A treated cells were more fluorescent than control. In line with the results obtained from the V-ATPase assay, treatment with R-Deox B 7,4 (-) decreased lysosomal pH, as evidenced by a much dimmer fluorescence compared to control cells.

[00129] Select Microtubule Inhibitors Promote Functionally Important Lysosomal Disruption in AML Cells Next, it was examined whether increased lysosome acidity was followed by loss of lysosome integrity. TEX leukemia cells were treated with Deox B 7,4, R- Deox B 7,4 (-) as well as several other microtubule inhibitors for up to 24 hours. Bafilomycin A-i was included as a positive control. As shown in Figure 3C, the Deox compounds and nocodazole promoted lysosomal disruption, which was evident as early as 8 hours after drug treatment and drastically increased thereafter. In contrast, the other microtubule inhibitors tested did not disrupt lysosomes nearly to the extent seen with the Deox compounds and nocodazole, even though cell death kinetics were comparable (data not shown). Similar results were also obtained with the leukemia cell line OCI-AML2 (data not shown). A cathepsin B release assay was carried out on enriched lysosomes treated with various microtubule inhibitors to determine whether the lysosome disruption observed was the result of a direct effect of the drugs. None of the microtubule inhibitors tested, including the Deox compounds and nocodazole, induced cathepsin B release, consistent with tubulin as their primary molecular target (Figure 3D).

[00130] To determine whether V-ATPase activation and lysosome disruption by Deox B 7,4 and R-Deox B 7,4 (-) was functionally important for their anti-leukemic effects, cell were treated with the Deox compounds along with bafilomycin A-i to counteract activation of V-ATPase. Specifically, TEX leukemia cells were treated concurrently with the Deox compounds and bafilomycin Ai for 24 hours and cell growth and viability was assessed using a MTS assay. Co-treatment with bafilomycin Ai reduced the cytotoxicity of Deox B 7,4 or R-Deox B 7,4 (-) (Figure 1 E). Thus, these results demonstrate that the lysosomal disruption promoted by the Deox compounds is a functionally relevant mechanism of action in leukemia cells.

[00131] R-Deox B 7,4 (-) Synergizes with Vinca Alkaloids In Vitro and Displays Antitumor Activity in an In Vivo Xenograft Mouse Model Given that R-Deox B 7,4 (-) binds tubulin at a different site than that of vinca alkaloids, it was examined whether R-Deox B 7,4 (-) could synergize with vincristine and vinblastine. TEX leukemia cells were treated with R-Deox B 7,4 (-) along with increasing concentrations of vinblastine or vincristine and cell growth and viability was assessed after 72 hours using a MTS assay. As shown in Figure 4A, a marked synergy was observed between R-Deox B 7,4 (-) and both vinca alkaloids tested.

[00132] Finally, a xenograft mouse model was used to evaluate the in vivo antitumor effect of R-Deox B 7,4 (-). OCI-AML2 cells were injected subcutaneously into the flanks of SCID mice (n = 10 per group). Once tumors were palpable, mice were treated with R-Deox B 7,4 (-) (75mg/kg) twice daily 5 days per week by intraperitoneal injection for 13 days. When compared to the control group, there was a significant difference (***p<0.0001 ) in tumor growth inhibition in mice treated with R-Deox B 7,4 (-), suggesting antitumor activity of R-Deox B 7,4 (-) treatment (Figure 4B).

EXPERIMENTAL PROCEDURES

Drugs and Reagents

[00133] The Natural Product chemical library was purchased from MicroSource Discovery Systems (Gaylordsville, CT). Deoxysappanone B 7,4' dimethyl ether (Deox B 7,4) and its reduced form (R-Deox B 7,4) were synthesized Deox B 7,4 can also be isolated from Caesalpina sappan is also available commercially for example from Microsource. A suitable reducing agent can be used to reduce Deox B 7,4. The enantiomer separation of Deox B 7,4 and R-Deox B 7,4 was performed by Lotus Separations (Princeton, NJ). Nocodazole was obtained from Adipogen (San Diego, CA), vincristine was supplied by Abeam (Cambridge, MA) and other microtubule inhibitors as well as adriamycin were purchased from Sigma Aldrich (Oakville, ON, Canada). Bafilomycin was acquired from Cayman Chemicals (Ann Arbor, Ml). All drugs were prepared in dimethyl sulfoxide (DMSO). Unless otherwise stated, all chemicals were purchased from Sigma Aldrich (Oakville, ON, Canada).

Cell Culture

[00134] The leukemia cell lines TEX, OCI-AML2, K562 and THP1 were maintained in Iscove's modified Dulbecco's Medium (IMDM). The leukemia cell lines HL60 and U937 were maintained in RPMI 1640 medium. The ovarian adenocarcinoma cell lines A2780 and A2780ADR (Hamilton et al., 1984), were provided by Dr. Jeremy Squire (Kingston, ON, Canada) and were also maintained in RPMI 1640 medium. A2780ADR cells were treated overnight once a week with 0.1 pg/ml adriamycin to maintain the drug resistance phenotype. The epidermoid carcinoma cell lines KB-3-1 and KB-4.0-HTI36 (a gift from Dr. F. Loganzo, Pearl River, NY) (Loganzo et al., 2004) were grown in Dulbecco's Modified Eagle Medium (DMEM). For all cell lines with the exception of TEX, medium was supplemented with 10% fetal bovine serum (FBS), 100 pg/ml penicillin and 100 units/ml streptomycin (all from Hyclone, Logan, UT). TEX cells were grown in the presence of 15% FBS, 100 pg/ml penicillin, 100 units/ml streptomycin, 2 mM L-glutamine as well as 20 ng/ml SCF and 2 ng/ml IL-3 (R&D Systems, Minneapolis, MN). All cells were incubated at 37 °C in a humidified air atmosphere supplemented with 5% C0 ₂ . For hypoxia experiments, cells were transferred to hypoxic culture chambers (MACS VA500 microaerophilic workstation, H35 HypoxyWorkStation; Don Whitley Scientific) in which the atmosphere consisted of 5% H ₂ , 5% C0 ₂ and either 0.2%, 1 % or 3% 0 ₂ as well as residual N ₂ .

Chemical Screen

[00135] A Biomek FX Laboratory Automated Workstation (Beckman Coulter Fullerton, CA) was used for the high-throughput screening of the Natural Product chemical library. TEX leukemia cells were plated in 384-well plates in a final volume of 75 μΙ/well and 200 nl/well was dispensed from drug stocks varying in concentration from 0.625 mM to 5 mM. Sulforhodamine B was used to determine drug cytotoxicity following 72 hour incubation.

Cell Growth and Viability Assays

[00136] Cell growth and viability was assessed using the MTS assay according to the manufacturer's instructions (Promega, Madison, Wl). Cell death was measured by staining cells with Annexin V-fluorescein isothiocyanate (FITC) and Propidium Iodide (PI) (Biovision Research Products, Mountain View, CA) prior to acquisition on a FACScanto II (Becton Dickinson, Florida, USA). Results were analyzed with FlowJo (TreeStar, Ashland, OR).

Colony Formation Assay

[00137] The collection and use of human tissue for this study was approved by the local ethics review board (University Health Network, Toronto, ON, Canada). Peripheral blood samples from primary AML patients and G-CSF-mobilized peripheral blood stem cells (PBSCs) from volunteers donating PBSCs for allotransplant were obtained after informed consent. Blood samples were subjected to density isolation with Ficoll-Paque Plus (GE Healthcare, Uppsala, Sweden) to separate mononuclear cells (MNCs). MNCs (1 x10 ⁵ /dish) were plated in 0.1 ml IMDM supplemented with 10% FBS and 0.9ml MethoCult GF H4434 medium (StemCell Technologies, Vancouver, BC, Canada) in 35 mm dishes in duplicate (Nunclon, Rochester, USA) in the presence of 100 nm R-Deox B 7,4 (-) or vehicle control. The number of resultant colonies was counted as previously described (Buick et al., 1977; Fauser and Messner, 1979). To confirm the type of cells in the colonies, cells were picked when necessary and stained with May-GrCmwald-Giemsa as described (Buick et al., 1977). Yeast Haplo-lnsufficiertcy Profiling (HIP) and Analysis

[00138] To identify the primary mechanism of drug action, HIP in yeast was used to profile the fitness of ~6000 heterozygous/homozygous deletion strains (Giaever et al., 2004; Smith et al., 2010) in the presence of Deox B 7,4. Yeast extract peptone medium supplemented with either dextrose (YPD) or glycerol and ethanol (YPGE) were used in the HIP. The fitness assay on the deletion strains was performed as described (Pierce et al., 2006) with the following modifications: (1 ) for barcode amplification, 0.2 g of genomic DNA was used in a 50 μΙ PCR containing 1 μΜ mix of up- or down-tag primers and 82% (v/v) of High Fidelity Platinum PCR SuperMix (Invitrogen, Carlsbad, CA); (2) 34 amplification cycles were used for the PCR using an extension temperature of 68 °C for 2 minutes except for a final 10 minutes in the last cycle; and (3) after 10-16 hours of hybridization, the arrays were washed in a GeneChip Fluidic Station 450 (Affymetrix, Santa Clara, CA) using the GeneFlex_Sv3_450 protocol with one additional wash cycle before the staining. The Affymetrix GeneChip Command Console Software was used to extract the intensity values from the arrays and the fitness defects were calculated for each deletion strain as log2 ratios (mean signal intensity of control/mean signal intensity of drug).

[00139] Deletion strains with log2 ratio values of more than 1 were considered hits. The gene sets were defined with yeast-specific Gene Ontology (GO) biological processes and manually curated annotations obtained from the Saccharomyces Genome Database (http://www.yeastgenome.org) on September 13, 2013. GO Slim Mapper (http://www.yeastgenome.org) was used to identify broad categories of enriched GO biological processes amongst the genes associated with the greatest sensitivity to Deox B 7,4. GO biological processes represented by 4 or more genes from the datasets and enriched a minimum of 2 fold relative to the yeast genome were used for the interpretation of the HIP results. Tubulin assays

[00140] Microtubule associated protein (MAP)-rich bovine tubulin (Cytoskeleton, Denver, CO) was reconstituted in ice-cold polymerization buffer (80 mM PIPES pH 6.9, 0.5 mM EGTA, 2 mM MgCI ₂ , 10% glycerol and 1 mM GTP) at a concentration of 1.2 mg/ml and centrifuged at top speed for 5 minutes at 4 °C. Supernatant (100 μΙ/well) was added to drugs in a 96-well plate to obtain a final drug concentration of 10 μΜ. Absorbance was measured at 340 nm every 5 minutes for 90 minutes at 37 °C.

[00141] Competitive inhibition of colchicine binding to tubulin was performed using purified porcine tubulin (Cytoskeleton, Denver, CO) reconstituted at 0.5 mg/ml (80 mM PIPES pH 6.9, 0.5 mM EGTA and 2 mM MgCI ₂ ). Briefly, the tubulin solution was incubated at 37 °C for 30 minutes in the presence of given microtubule inhibitors (100 μΜ). Colchicine (12 μΜ) was then added and samples were incubated for an additional 60 minutes at 37 °C. Fluorescence of the colchicine-tubulin complex was measured in disposable plastic cuvettes using excitation and emission wavelengths of 360 nm and 430 nm, respectively.

Cell cycle analysis

[00142] Cells were treated with microtubule inhibitors for the specified times, harvested, washed once in cold PBS and fixed overnight in cold 70% ethanol at -20 °C. Cells were then pelleted, washed once with PBS and treated with 100 ng/ml of DNase-free RNase A (Invitrogen, Carlsbad, CA) at 37 °C for 30 minutes. A 5 pg/ml Propidium Iodine (PI) solution was added prior to measuring DNA content on a FACScanto II (Becton Dickinson, Florida, USA). Results were analyzed with FlowJo (TreeStar, Ashland, OR).

Mitotic block reversibility assay

[00143] Mitotic block reversibility was assessed as previously described (Towle et al., 2011 ). Briefly, U937 cells were seeded in 75 cm ² flasks at 10 ⁵ cells/ml and allowed to resume log-phase growth. Concentrations of microtubule inhibitors ranging from 0.25X to 4X their respective IC ₅₀ values for U937 cells were added for 12 hours prior to washing the cells twice with pre-warmed PBS and replenishing with warm, C0 ₂ -equilibrated drug-free media. Cells were allowed to recover for 5 days, with one 80% media replenishment on day 2. Cells were sampled for cell cycle analysis at the end of the 12 hour drug treatment period as well as 10 hours post-wash and viability was assessed on day 5 using trypan blue exclusion. Lysosome enrichment

[00144] The lysosome enrichment method was adapted from Lardeux ef al (Lardeux et al., 1983). Briefly, 10 ⁸ cells per condition were harvested, washed once in cold PBS and resuspended in cold 0.25 M sucrose buffer containing 10mM TRIS pH 7.4, 10 mM KH ₂ P0 ₄ ,

0.5 mM EDTA and protease inhibitors (complete Mini tablets; Roche Applied Science, IN). All subsequent steps were carried out at 4 °C and samples kept on ice. Cell homogenates were obtained using a teflon-glass homogenizer (Glas-Col, Terre Haute, IN) and centrifuged at 3000 rpm for 10 minutes. Supernatants were next centrifuged at 5000 rpm for an additional 10 minutes. Lysosomes were purified from the resulting supernatants by centrifugation at 10000 rpm for 20 minutes and resuspended in sucrose buffer supplemented with TRIS and protease inhibitors only. Protein concentrations were determined using the Bradford reagent (Bio-Rad, Hercules, CA). V-ATPase assay

[00145] An acridine orange fluorescence quenching assay was used to measure V- ATPase activity. Enriched lysosomes (5C^g) were incubated in acridine orange buffer (3 μΜ acridine orange, 5 mM Hepes pH 7.4, 5 mM MgCI ₂ and 150 mM KCI) at room temperature for 1 hour. Samples were transferred to disposable plastic cuvettes, ATP was added at a final concentration of 5 mM and acridine orange fluorescence was immediately measured every 10 seconds for 3 minutes using excitation and emission wavelengths of 490 nm and 540 nm, respectively.

Lysosomal disruption assays

[00146] Cellular lysosomal integrity following drug treatment was measured using acridine orange staining and flow cytometry. Cells were treated with microtubule inhibitors or Bafilomycin Ai for the specified times and 3 μΜ acridine orange was added during the last 30 minutes. Cells were then pelleted, washed once in PBS and resuspended in PBS prior to acquisition on a FACScanto I (Becton Dickinson, Florida, USA) with fluorescence signals collected in both FITC and PerCP-Cy5.5 channels. Lysosome disruption was measured as the percentage of cells that lost PerCP-Cy5.5 staining. Results were analyzed with FlowJo (TreeStar, Ashland, OR).

[00147] The direct effect of microtubule inhibitors on lysosomal integrity was assessed using a cathepsin B release assay. Briefly, enriched lysosomes (40 pg/sample) were treated for 2 hours at 37 °C with vehicle control, microtubule inhibitors or 0.2% Triton-X detergent as a positive control. After incubation, the reaction mixtures were centrifuged at 15000 g for 30 minutes at 4 °C to pellet intact lysosomes. Cathepsin B activity released into the supernatant following lysosomal disruption was measured as described previously with some modifications (Kawasaki et al., 1995). Assays were performed in 0.1 M sodium acetate buffer (pH 5.0) containing 1 mM of the fluorogenic substrate benzyloxycarbonyl-arginyl-4- methyl-7-coumarylamide (Z-Arg-Arg-MCA) in a 96-well plate at 37 °C for 1 hour. The reaction product 7-amino-4-methylcoumarin was measured with excitation and emission wavelengths of 380 nm and 460 nm, respectively.

Fluorescence microscopy

[00148] Glass-bottom dishes (Ibidi, Ingersoll, ON, Canada) were coated with Cell-Tak according to the manufacturer's instructions (BD Biosciences). Cells were treated with microtubule inhibitors for the specified times, harvested and centrifuged onto the coated dishes at 800 rpm for 2 minutes. Non-adherent cells were gently aspirated and warm, C0 ₂ - equilibrated media was replenished. LysoSensor Yellow/Blue DND-160 (Life Technologies, Burlington, Canada) was added to the cells at a final concentration of 5 μΜ. The blue emission of this pH-sensitive dye decreases in more acidic environments and increases in more alkaline ones. Fluorescence was visualized within 5 minutes with a Zeiss LSM 510 META microscope equipped with a 63X water objective following excitation with the two- photon laser and emission collection with a HFT 650 short-pass dichroic mirror and a HFT 390-435 bandpass filter.

Statistical analysis

[00149] All data shown are expressed as means ± standard deviation (SD). An unpaired Student's f-test was used for the analysis. Differences between means were considered significant at p<0.05. Results were generated using GraphPad Prism 4.0b software (Graph Pad Software, LaJolla, CA).

[00150] While the present application has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the application is not limited to the disclosed examples. To the contrary, the application is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

Example 3

[00151] A schematic of R-Deox 7,4 synthesis method is shown in Figure 5 and described below.

3-Chloro-1 -(2,4-dihvdroxyphenyl)propan-1 -one:

[00152] To a mixture of Resorcinol (10 g, 90.9 mmol) and 3-chloropropionic acid (10.0 g, 92.0 mmol) in a 250 mL flask, trifluoromethanesulfonic acid (50 g, 333 mmol) was added in one portion. After being stirred at 85 °C for 45 minutes, the reaction mixture was cooled to room temperature and poured into ice water. The product was extracted with chloroform (2 x 200 mL), dried with magnesium sulfate and concentrated under vacuum. The residue was dissolved in ethyl acetate and treated with silica gel to give 3-chloro-1-(2,4- dihydroxyphenyl)propan-1-one (15.6 g, 86% yield) as an off-white solid.

7-Hydroxychroman-4-one

[00153] 3-Chloro-1-(2,4-dihydroxyphenyl)propan-1-one (4.7 g, 23.5 mmol) was mixed with 2N NaOH (200 mL, 400 mmol) under ice-water bath. The reaction mixture was stirred for 2 hours and then adjusted pH to ca 2.0 with 6N HCI. The resulting white crystal was filtered, washed with water to give 7-hydroxychroman-4-one (3.86 g, 100% yield).

7-Methoxychroman-4-one

[00154] 7-Hydroxychroman-4-one (3.86 g, 23.5 mmol) was mixed with potassium carbonate (6.49 g, 47.0 mmol) in acetone (80 mL) under 60°C oil bath, lodomethane (9.3 g, 66 mmol) was added slowly. The reaction mixture was stirred for 3 hours and then filtered. The filtrate was concentrated under vacuum and the residue was dissolved in dichloromethane and washed with water and brine. The organic layer was dried with magnesium sulfate and concentrated in vacuum give 7-methoxychroman-4-one (4.19 g, 100% yield) as sticky solid.

(3E)-3-f(3-Hvdroxy-4-methoxy-phenyl)methylene1-7-methoxy- chroman-4-one

[00155] 7-methoxychroman-4-one (4.19 g, 23.5 mmol) was mixed with isovanillin (3.58 g, 23.5 mmol) in ethanol (100 mL) and concentrated hydrochloric acid (1 mL). The reaction mixture was refluxed for 24 hours. The resulting yellow solid was filtered to give 5.4 g of the title compound as a yellow solid. Yield=74 %. 2-Methoxy-5-r(7-methoxychroman-3-yl)methyllphenol (Deoxysappanone)

[00156] (3E)-3-[(3-hydroxy-4-methoxy-phenyl)methylene]-7-methoxy-chr oman-4-one (3.9 g, 12.5 mmol) was mixed with 540 mg of 10 % Pd/C (wet) in methanol and the reaction mixture was hydrogenated with hydrogen balloon at room temperature and monitored closely until judge complete by TLC. The mixture was filtered to remove Pd/C and the filtrate was concentrated under vacuum. The resulting solid was triturated with ether to give title compound (1.3 g, 33 % yield) as white solid.

[00157] The enantiomers of deoxysappanone have been separated by preparative chiral HPLC using a Chiralpak AD-H column (250 x 10 mm) on 5 prn silica-gel, iPrOH-n- hexane (20:80) as mobile phase, and flow rate 2 ml/min, yielding PEAK1 and PEAK2 (Figure 6A).

2-Methoxy-5-[(7-methoxychroman-3-yl)methyllphenol (Reduced-Deoxysappanone)

[00158] (3E)-3-[(3-hydroxy-4-methoxy-phenyl)methylene]-7-methoxy-chr oman-4-one (5.4 g, 17.3 mmol) was mixed with 540 mg of 10 % Pd/C (wet) in methanol and the reaction mixture was hydrogenated with hydrogen balloon at room temperature overnight. The mixture was filtered to remove Pd/C and the filtrate was concentrated under vacuum. The resulting solid was triturated with ether to give title compound (4.7g, 90% yield) as white solid. ¹ H NMR (500 MHz, CDCI3): δ = 6.91 (d, 1 H), 6.82 (d, 1 H), 6.80 (s, 1 H), 6.68 (d, 1 H), 6.46 (d, 1 H), 6.39 (s, 1 H), 5.20 (br, 1 H), 4.18 (d, 1 H), 3.91 (s, 3H), 3.82 (dd, 1 H), 3.78 (s, 3H), 2.76 (dd, 1 H), 2.62 (dd, 1 H), 2.55 (dd, 1 H), 2.46 (dd, 1 H), 2.28 (m, 1 H); Exact Mass: 300.14 LCMS [M+H] ⁺ 301.0942.

[00159] The enantiomers of reduced-deoxysappanone have been separated by preparative chiral HPLC using a Chiralpak AD-H column (250 x 10 mm) on 5 pm silica-gel, iPrOH-n-hexane (20:80) as mobile phase, and flow rate 2 ml/min, yielding PEAK1 and PEAK2 (Figure 6B).

Example 4

[00160] Deox -7, 4 was analysed (sample A2-58-3) and enantiomers of Deox- 7, 4 were separated.

Analysis Summary:

[00161] The following SFC separation (conditions listed below) yielded 38 mg of peak- 1 (chemical purity >99%, ee >99%) and 38 mg of peak-2 (chemical purity >99%, ee >98%). See Figure 6A.

Preparative Method:

[00162] OJ-H (2 x 25 cm), 40% methanol(0.1% DEA)/C0 ₂ , 100 bar, 60 mL/min, 220 nm, injection volume: 0.5 ml_, 10 mg/mL 1 :1 methanol: DCM

Analytical Method:

[00163] OJ-H (25 x 0.46 cm), 0% methanol(DEA)/C0 ₂ , 100 bar, 3 mL/min, 220 and 254 nm

Example 5

[00164] R-Deox -7, 4 was analysed (sample A2-65-5) and enantiomers of R-Deox- 7, 4 were separated.

Analysis Summary:

[00165] The following SFC separation (conditions listed below) yielded 2.1 g of peak-1 (chemical purity >99%, ee >99%) and 2.1 g of peak-2 (chemical purity >99%, ee >99%). The

[a] ²⁵ _D = -49.0 for peak-1 and +50.6 for peak-2. See Figure 6B. Preparative Method:

[00166] AS-H (2 x 15 cm), 25% methanol(0.1 % DEA)/C0 ₂ , 100 bar, 70 mL/min, 220 nm, injection volume: 1 mL, 40 mg/mL methanol

Analytical Method:

[00167] AS-H (25 x 0.46 cm), 40% methanol(DEA)/C0 ₂ , 100 bar, 3 mL/min, 220 and 254 nm

Optical Rotation Determination Sample: A2-65-5 peak-1

Concentration (mg/mL): 5.5

Solvent: ethanol

Wavelength: 589 nm

Cell: 100 mm

Sample: A2-65-5 peak-2

Concentration (mg/mL): 5.0

Solvent: ethanol

Wavelength: 589 nm

Cell: 100 mm

[00168] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Specifically, the sequences associated with each accession numbers provided herein including for example accession numbers and/or biomarker sequences ( e.g. protein and/or nucleic acid) provided in the Tables or elsewhere, are incorporated by reference in its entirely. 5 TABLES

Table 1. List of hits from natural product chemical library screen on TEX cells

Drug 13.3 μΜ 6.7 μΜ 3.3 μΜ 1.7 μΜ

Pomiferin 20 39 15 5

Decahydrogambogic acid 20 18 18 15

Deoxysappanone b 7,4'-dimethyl ether* 19 20 20 21

Picropodophyllin 18 16 19 18

Strophanthidin 17 16 15 17

Deoxysappanone b 7,3 '-dimethyl ether acetate 17 16 19 16

7-desacetoxy-6,7-dehydrogedunin 16 16 12 13

Cedrelone 16 15 14 12

Anthothecol 15 14 19 13

Strophanthidinic acid lactone acetate 14 15 13 13

Gitoxin 14 15 14 15

Benzyl isothiocyanate 14 23 16 9

10-hydroxycamptothecin 14 1 1 13 13

Convallatoxin 14 11 14 16

Lanatoside c 14 1 1 13 1 1

Podophyllin acetate 13 14 14 12

Rhodinyl acetate* 13 12 13 12

Diallyl trisulfide 12 12 14 11

Gitoxigenin diacetate 12 15 13 12

Gambogic acid 12 9 12 12

Thymoquinone 12 8 13 17

Acetyl isogambogic acid 12 12 1 1 9

4,4'-dimethoxydalbergione 12 16 15 7

Pyrromycin 12 13 15 13

2,6-dimethoxyquinone 12 9 9 5

4-methoxy-4'-hydroxy-dalbergione 1 1 14 12 1 1

Cantharidin 1 1 10 9 10

Pristimerin 10 7 6 5

Celastrol 10 10 9 10

Helenine 10 13 10 9

Camptothecin 10 12 13 9

Beta-peltatin 10 1 1 13 10

Patulin 10 7 7 7

Plumbagin 10 1 1 10 10

Dalbergione* 10 7 1 1 7

Obtusaquinone* 10 15 9 4

Piplartine 9 10 9 9

Tomatine 9 5 9 8

Salmomycin, sodium 8 1 1 9 10 inetin riboside 7 8 13 12

Table 2. List of GO processes enriched >2 fold in S. cerevisiae screen hits obtained from Deox B 7,4 screen in YPD medium

GO processes Dataset (%) Genome (%) Enrichment Gene(s)

Response to DNA damage stimulus 14.1 4.5 3.13 SNF5, TUP1, NSE3, H1M1, EAF1, YRA1, CDC45,

MCM5, RAD52, TH02, RMI1, CTF4

Mitotic cell cycle 12.9 4.8 2.69 TPD3, SCM3, TUB2, CDC45, MCM5, CEP3, CNM67,

HRT1, SGOl, RMI1, CTF4

DNA repair 11.8 3.8 3.11 SNF5, NSE3, HIM1, EAF1, YRA1, CDC45, MCM5,

RAD52, TH02, CTF4 10

Protein complex biogenesis 11.8 4.2 2.81 MDM10, SRP14, SCM3, EAF1, PET117, PRE4,

SAM35, VPH2, VPS33, CEP3

Protein targeting 10.6 4.3 2.47 MDM10, SRP14, COG7, MAS2, SAM35, M1A40,

VPS33, TOM22, NUP1

DNA recombination 8.2 2.5 3.28 SNF5, VMAl, CDC45, MCM5, RAD52, TH02, CTF4

Generation of precursor 7.1 2.5 2.84 CBP1, PFK2, GCR2, EMI5, IDH2, HAP5

metabolites and energy

Organelle fission 7.1 2.5 2.84 TPD3, TUB2, CEP3, SGOl, RMI1, CTF4

Nucleobase-containing small 7.1 2.9 2.45 NFS1, YRA1, RNR4, RNR2, SEC 13, NPT1

molecule metabolic process 15

Proteolysis involved in cellular 7.1 3.1 2.29 PRE7, SCM3, PRE4, PRE3, SEC13, HRT1 protein catabolic process

Transmembrane transport 7.1 3.5 2.03 MDM10, FUI1, SAM35, MIA40, TOM22, CTR1

Nucleobase-containing compound 5.9 1.9 3.11 FUI1, TEF2, YRA1, TH02, NUP1

transport

Chromosome segregation 5.9 2.1 2.81 SCM3, TUB2, SGOl, RMI1, CTF4

Nuclear transport 5.9 2.7 2.19 TEF2, YRA1, GSP1, TH02, NUP1

Cellular respiration 4.7 1.4 3.36 CBP1, EMI5, IDH2, HAP5

Cellular ion homeostasis 4.7 2.0 2.35 NFS1, VMAl, VMA 7, VPH2 20

Cofactor metabolic process 4.7 2.1 2.24 HEM1, RNR4, FOL1, NPT1

DNA replication 4.7 2.3 2.04 TEN1, CDC45, MCM5, CTF4

Table 3. List of GO processes enriched >2 fold in S. cerevisiae screen hits obtained from Deox B 7,4 screen in YPGE medium

GO processes Dataset (%) Genome (%) Enrichment Gene(s)

Cellular ion homeostasis 13.7 2.0 6.85 VMA3, VMA7, SOD1, VMA5, VPH2, FET3, VMA4 10

Protein complex biogenesis 13.7 4.2 3.26 EAFl, VMA21, VPH2, COX12, PKRl, ATP 4, VPS4

Endocytosis 9.8 1.5 6.53 RVS161, AKR1, VMA3, CHC1, SLA2

Protein phosphorylation 9.8 2.8 3.50 PTC1, SNF1, SNF4, RTF1, PH085

Nucleobase-containing small 9.8 2.9 3.38 ATP1, RNR1, ADOl, NPT1, ATP4

15

molecule metabolic process

Ion transport 9.8 3.6 2.72 ATP I VMA3, FLX1, FET3, ATP 4

Carbohydrate metabolic process 9.8 4.2 2.33 MNN10, SNF1, SNF4, SNF6, PH085

Transmembrane transport 7.8 3.5 2.23 ATP1, SEC66, FET3, ATP 4

Chromatin organization 7.8 3.6 2.17 EAFl, DOC1, RTF], SNF6 20

Signaling 7.8 3.6 2.17 PTC1, AKR1, LCB5, PH085

Table 4. Microtubule inhibition by Deox B 7,4 and R-Deox B 7,4 (-) is functionally relevant and overcomes drug efflux pump-related resistance

Drug KB-3-1 [nM] KB-4.0-HTI [nM] Fold resistance A2780 [nM] A2780ADR [nM] Fold resistance

(KB4.0/KB3.1) (A2780ADR/A2780)

Deox B 7,4 344.46 ± 23.28 655.93 ± 27.80 1.90 300.83 ± 62.06 576.17 ± 90.02 1.92

R-Deox B 7,4 (-) 89.30 ± 20.61 149.72 ± 26.36 1.68 39.50 ± 0.62 146.30 ± 37.29 3.70

Nocodazole 40.35 ± 3.47 99.79 ± 9.59 2.47 n/a n/a n/a

Colchicine 6.14 ± 0.11 17.57 ± 0.93 2.86 5.47 ± 0.11 324.26 ± 28.24 59.28

Vincristine n/a n/a n/a 0.77 ± 0.08 845.74 ± 181.76 1098.36

CITATIONS FOR REFERENCES REFERRED TO IN THE SPECIFICATION

Barabe, F., Kennedy, J.A., Hope, K.J., and Dick, J.E. (2007). Modeling the initiation and progression of human acute leukemia in mice. Science 316, 600-604.

Bhattacharyya, B., and Wolff, J. (1974). Promotion of fluorescence upon binding of colchicine to tubulin. Proc Natl Acad Sci U S A 71, 2627-2631.

Buick, R.N., Till, J.E., and McCulloch, E.A. (1977). Colony assay for proliferative blast cells circulating in myeloblasts leukaemia. Lancet 1, 862-863.

Dumontet, C, and Sikic, B. I. (1999). Mechanisms of action of and resistance to antitubulin agents: microtubule dynamics, drug transport, and cell death. J Clin Oncol 17, 1061-1070.

Fauser, A.A., and Messner, H.A. (1979). Identification of megakaryocytes, macrophages, and eosinophils in colonies of human bone marrow containing neurtophilic granulocytes and erythroblasts. Blood 53, 1023-1027.

Giaever, G., Shoemaker, D.D., Jones, T.W., Liang, H., Winzeler, E.A., Astromoff, A., and Davis, R.W. (1999). Genomic profiling of drug sensitivities via induced haploinsufficiency. Nat. Genet. 21, 278-283.

Giaever, G., Flaherty, P., Kumm, J., Proctor, M., Nislow, C, Jaramillo, D.F., Chu, A.M., Jordan, M. I., Arkin, A.P., and Davis, R.W. (2004). Chemogenomic profiling: identifying the functional interactions of small molecules in yeast. Proc Natl Acad Sci U S A 101, 793-798.

Gupta, V., Xu, W., Keng, C , Alibhai, S.M.H., Brandwein, J., Schimmer, A., Schuh, A., Yee, K., and Minden, M.D. (2007). The outcome of intensive induction therapy in patients >or=70 years with acute myeloid leukemia. Leukemia 21, 1321-1324.

Hamilton, T.C., Young, R.C., and Ozols, R.F. (1984). Experimental model systems of ovarian cancer: applications to the design and evaluation of new treatment approaches. Semin Oncol 11, 285-298.

Hoon, S., Smith, A.M., Wallace, I.M., Suresh, S., Miranda, M., Fung, E., Proctor, M., Shokat, K.M. , Zhang, C, Davis, R.W., et al. (2008). An integrated platform of genomic assays reveals small-molecule bioactivities. Nat. Chem. Biol. 4, 498-506.

Kawasaki, G., Mataki, S., and Mizuno, A. (1995). Ultrastructural and biochemical studies of the effect of polychlorinated biphenyl on mouse parotid gland cells. Arch. Oral Biol. 40, 39- 46.

Lardeux, B., Gouhot, B., and Forestier, M. (1983). Improved recovery of rat liver fractions enriched in lysosomes by specific alteration of the sedimentation properties of mitochondria. Anal. Biochem. 131, 160-165.

Loganzo, F., Hari, M., Annable, T., Tan, X., Morilla, D.B., Musto, S., Zask, A., Kaplan, J. , Minnick, A.A., May, M.K., et al. (2004). Cells resistant to HTI-286 do not overexpress P- glycoprotein but have reduced drug accumulation and a point mutation in alpha-tubulin. Mol Cancer Ther 3, 1319-1327.

Lu, Y., Chen, J., Xiao, M., Li, W., and Miller, D.D. (2012). An overview of tubulin inhibitors that interact with the colchicine binding site. Pharm. Res. 29, 2943-2971. Moriyama, Y., Takano, T., and Ohkuma, S. (1982). Acridine orange as a fluorescent probe for lysosomal proton pump. J. Biochem. 92, 1333-1336.

NAMIKOSHI, M., and SAITOH, T. (1987). Homoisoflavonoids and Related Compounds. IV. : Absolute Configurations of Homoisoflavonoids from Caesalpinia sappan

L.(OrganicChemical). Chem. Pharm. Bull. 35, 3597-3602.

Pierce, S.E., Fung, E.L., Jaramillo, D.F., Chu, A.M., Davis, R.W., Nislow, C, and Giaever, G. (2006). A unique and universal molecular barcode array. Nat. Methods 3, 601-603.

Smith, A.M., Heisler, L.E. , St Onge, R. P., Farias-Hesson, E. , Wallace, I.M., Bodeau, J., Harris, A.N., Perry, K.M., Giaever, G., Pourmand, N., et al. (2010). Highly-multiplexed barcode sequencing: an efficient method for parallel analysis of pooled samples. Nucleic Acids Res. 38, e142.

Toei, M. , Saum, R. , and Forgac, M. (2010). Regulation and isoform function of the V- ATPases. Biochemistry 49, 4715-4723.

Towle, M.J., Salvato, K.A., Wels, B.F., Aalfs, K.K., Zheng, W., Seletsky, B.M., Zhu, X., Lewis, B.M., Kishi, Y., Yu, M.J., et al. (201 1 ). Eribulin induces irreversible mitotic blockade: implications of cell-based pharmacodynamics for in vivo efficacy under intermittent dosing conditions. Cancer Res 71, 496-505.

Warner, J.K., Wang, J.C.Y. , Takenaka, K., Doulatov, S., McKenzie, J. L., Harrington, L, and Dick, J.E. (2005). Direct evidence for cooperating genetic events in the leukemic

transformation of normal human hematopoietic cells. Leukemia 19, 1794-1805.