WO2015051447A1 - Méthodes et compositions pour le traitement du cancer - Google Patents

Méthodes et compositions pour le traitement du cancer Download PDF

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WO2015051447A1
WO2015051447A1 PCT/CA2014/000733 CA2014000733W WO2015051447A1 WO 2015051447 A1 WO2015051447 A1 WO 2015051447A1 CA 2014000733 W CA2014000733 W CA 2014000733W WO 2015051447 A1 WO2015051447 A1 WO 2015051447A1
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compound
deox
cancer
cells
enantiomer
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PCT/CA2014/000733
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Aaron David Schimmer
Dannie BUHAJCZUK
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University Health Network
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/475—Quinolines; Isoquinolines having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4

Definitions

  • 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.
  • 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.
  • AML acute myeloid leukemia
  • 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.
  • compositions comprising a compound of Formula I and a suitable vehicle such as a carrier or diluent, optionally a pharmaceutically acceptable carrier or diluent.
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of Formula II
  • 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;
  • X is H, O or OH; and/or a pharmaceutically acceptable salt and/or solvate thereof optionally wherein the composition is for treating cancer.
  • X is O.
  • the compound is the (-) enantiomer.
  • the compound is the (+) enantiomer.
  • the compound is deoxysappanone B 7,4'-dimethyl ether (Deox B 7, 4).
  • the deoxysappanone B 7,4'-dimethyl ether compound is the (+) enantiomer.
  • the deoxysappanone B 7,4'-dimethyl ether compound is the (-) enantiomer.
  • the compound is a compound of Formula I.
  • Y is H.
  • R 1 , R 2 and/or R 3 is/are methyl.
  • 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 .
  • the compound is reduced deoxysappanone B 7,4'-dimethyl ether (e.g. reduced carbonyl (reduced to H); R-Deox B7, 4').
  • the compound is a compound of Formula I wherein the X is H.
  • 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.
  • the compound is the R-Deox (-) enantiomer.
  • the compound is deoxysappanone B 7,3'-dimethyl ether, optionally the (-) enantiomer or the (+) enantiomer.
  • the compound is sappanone A.
  • FIG. 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 ⁇ 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.
  • Results are shown as mean + SD and are representative of 3 experiments (**p ⁇ 0.01 and * ** p ⁇ 0.001 compared to control cells).
  • 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.
  • FIG. 1 Deox B 7,4 and R-Deox B 7,4 (-) function as microtubule inhibitors.
  • 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).
  • FIG. 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.
  • 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 ⁇ ) 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).
  • 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 ⁇ .
  • 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.
  • 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 ).
  • 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 ⁇ and 13.3 ⁇ . 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 ⁇ were considered hits.
  • 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.
  • 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.
  • 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
  • Y is H.
  • 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.
  • R , R 2 and/or R 3 is methyl.
  • compositions comprising a compound of Formula I and a suitable vehicle such as a carrier or diluent, optionally a pharmaceutically acceptable carrier or diluent.
  • suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (2003- 20 th Edition).
  • 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.
  • a further aspect includes a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and A and/or a compound of Formula II
  • R 1 , R 2 and R 3 are independently selected from H and C 1- 0 -alkyl
  • X is either O, H or OH; and/or a pharmaceutically acceptable salt and/or solvate thereof
  • R 1 , R 2 and R 3 are not all H.
  • pharmaceutically acceptable means compatible with the treatment of animals, in particular, humans.
  • 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.
  • 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.
  • pharmaceutically acceptable salt means an acid addition salt, which is suitable for or compatible with the treatment of patients.
  • pharmaceutically acceptable acid addition salt 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.
  • 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.
  • solvate 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”.
  • 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.
  • the compound is deoxysappanone B, for example, in an extract and/or isolated from Caesalpinia sappan.
  • the compound is deoxysappanone B 7,4'-dimethyi ether (Deox B 7, 4).
  • deoxysappanone B 7,4'-dimethyl ether and/or “Deox B 7, 4" means a compound having the structure:
  • the compound is an enantiomer.
  • 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".
  • the compound is the (-) enantiomer.
  • the compound is the (+) enantiomer.
  • the deoxysappanone B 7,4'-dimethyl ether compound is the (+) enantiomer.
  • the deoxysappanone B 7,4'-dimethyl ether compound is the (-) enantiomer.
  • the compound is a compound of Formula I.
  • the compound is a compound having the structure:
  • R 1 , R 2 and R 3 are independently selected from H and C-i
  • the compound is reduced deoxysappanone B 7,4'-dimethyl ether (e.g. reduced carbonyl; R-Deox B7, 4').
  • 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'.
  • 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.
  • the compound is a compound having the structure:
  • 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.
  • the (-) enantiomer e.g. carbon alpha to the carbonyl
  • a compound of Formula I wherein the carbon alpha to the carbonyl position e.g. whether the carbonyl is present or OH or H
  • the compound is the R-Deox (-) enantiomer.
  • the compound is deoxysappanone B 7,3'-dimethyl ether, optionally the (-) enantiomer or the (+) enantiomer.
  • deoxysappanone B 7,3'-dimethyl ether or “Deox B 7,3"' refers to a compound having the structure:
  • the compound is sappanone A.
  • 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).
  • 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).
  • R for Rectus, Latin for right
  • S for Sinister, Latin for left
  • R refers to a reduced compound of formula I or II, for example where X or Y is H.
  • 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.
  • 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.
  • the application in one aspect, also describes a pharmaceutical composition
  • 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.
  • 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.
  • 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 '
  • 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.
  • 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.
  • 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.
  • DOTMA N-(1 (2,3- dioleyloxy)propyl)N,N,N-trimethylammonium chloride
  • DOPE diolesylphosphotidyl- ethanolamine
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the subject in need thereof has cancer, optionally a hematological cancer.
  • 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.
  • the cancer can be any cancer treatable by a microtubule inhibitor such as a taxane and/or a vinca alkaloid.
  • the hematological cancer is leukemia.
  • the leukemia is acute myeloid leukemia (AML).
  • the leukemia is acute lymphoblastic leukemia (ALL).
  • ALL acute lymphoblastic leukemia
  • the cancer is breast cancer, lung cancer, epidermoid cancer, ovarian cancer, and/or the hematological cancer is myeloma or lymphoma.
  • the lymphoma is Hodgkin's or non-Hodgkin's lymphoma.
  • 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 (-).
  • 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.
  • the cell is further contacted and/or the subject is further administered a microtubule polymerization inhibitor such as a taxane or a vinca alkaloid.
  • 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.
  • 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.
  • treating 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.
  • 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.
  • 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.
  • the leukemia is AML or ALL.
  • the lymphoma is Hodgkin's or non-Hodgkin's lymphoma.
  • 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
  • subject includes all members of the animal kingdom including mammals, and suitably refers to humans.
  • hematological malignancy refers to cancers that affect blood and bone marrow.
  • 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.
  • leukemia includes acute myeloid leukemia, acute lymphocytic leukemia and chronic myeloma leukemia (CML) in blast crisis.
  • CML chronic myeloma leukemia
  • lymphoma means any disease involving the progressive proliferation of abnormal lymphoid cells.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • the microtubule polymerization inhibitor comprises a taxane.
  • the taxane is paclitaxel or docetaxel.
  • the microtubule polymerization inhibitor is a vinca alkaloid.
  • the vinca alkaloid is vinblastine, vincristine, vindesine, and/or vinorelbine.
  • administering 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.
  • two substances will be administered substantially simultaneously, i.e. within minutes of each other, or in a single composition that comprises both substances.
  • an effective amount means an amount effective, at dosages and for periods of time necessary to achieve the desired result.
  • 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.
  • the amount administered and/or contacted is an effective amount.
  • 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.
  • a disruption such as lysosomal disruption
  • induces when compared to a control, an otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition.
  • 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.
  • 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.
  • a compound e.g. compound of the application
  • composition described herein optionally a
  • a microtubule polymerization inhibitor such as a taxane or a vinca alkaloid and/or b) instructions for use.
  • 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.
  • a natural product library was screened for compounds cytotoxic to AML cells and identified Deoxysappanone B 7,4'-dimethyl ether.
  • Deoxysappanone B is a homoisoflavanoid compound extracted primarily from the dried heartwood of Caesalpinia sappan, a medicinal plant native to South-East Asia.
  • anticancer activity of this compound has not been previously described and its molecular targets are largely unknown.
  • 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.
  • 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.
  • Deoxysappanone B validates 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.
  • 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.
  • Deoxysappanone B 7,4 dimethyl ether 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.
  • 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).
  • 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.
  • 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).
  • haplo- insufficiency profiling a chemical genomics platform developed in the yeast Saccharomyces cerevisiae.
  • 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.
  • VMA vacuolar membrane ATPase
  • V-ATPase vacuolar proton-translocating ATPase
  • VMA21 , VPH2 and PKR1 genes coding for proteins required in its assembly
  • Deox B 7,4 and R-Deox B 7,4 (-) Inhibit Microtubule Polymerization via Binding Near the Colchicine Site
  • 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.
  • the non-toxic enantiomer R-Deox B 7,4 (+) did not significantly interfere with tubulin polymerization (Figure 2A).
  • 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.
  • Microtubule Inhibition by Deox B 7,4 and R-Deox B 7,4 (-) is a Functionally Important Mechanism of Action
  • 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.
  • KB-4.0-HTI cells were 2.86-fold more resistant to colchicine than the KB-3-1 control cells (Table 4).
  • the KB-4.0-HTI cells were almost 2-fold more resistant to the Deox compounds than the parental KB-3-1 cells.
  • Deox B 7,4 and R-Deox B 7,4 (-) Overcome Drug Resistance Associated with P-glycoprotein Expression
  • P-glycoprotein Pgp; MDR1 gene
  • a drug efflux pump P-glycoprotein
  • 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.
  • A2780ADR cells were 59 and 1099-fold more resistant to colchicine and vincristine, respectively, compared to their wild type A2789 cells (Table 4).
  • A2780ADR cells remained essentially equally sensitive to Deox B 7,4 and R-Deox B 7,4 (-), compared to their parental A2780 cells.
  • 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.
  • 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.
  • 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.
  • 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.
  • a decrease in fluorescence is indicative of lower pH while an increase in fluorescence is indicative of higher pH.
  • bafilomycin A treated cells were more fluorescent than control.
  • treatment with R-Deox B 7,4 (-) decreased lysosomal pH, as evidenced by a much dimmer fluorescence compared to control cells.
  • 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).
  • 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.
  • 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).
  • the leukemia cell lines TEX, OCI-AML2, K562 and THP1 were maintained in Iscove's modified Dulbecco's Medium (IMDM).
  • IMDM Iscove's modified Dulbecco's Medium
  • 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).
  • FBS fetal bovine serum
  • penicillin 100 pg/ml penicillin
  • 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 .
  • 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 .
  • 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 ⁇ /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.
  • 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
  • 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 ⁇ PCR containing 1 ⁇ 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
  • 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 ⁇ /well) was added to drugs in a 96-well plate to obtain a final drug concentration of 10 ⁇ . Absorbance was measured at 340 nm every 5 minutes for 90 minutes at 37 °C.
  • 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
  • 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 ,
  • acridine orange fluorescence quenching assay was used to measure V- ATPase activity.
  • Enriched lysosomes (5C ⁇ g) were incubated in acridine orange buffer (3 ⁇ 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.
  • microtubule inhibitors 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.
  • 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
  • Nucleobase-containing compound 5.9 1.9 3.11 FUI1, TEF2, YRA1, TH02, NUP1
  • Chromosome segregation 5.9 2.1 2.81 SCM3, TUB2, SGOl, RMI1, CTF4

Abstract

L'invention concerne un composé de formule I et/ou II et/ou une composition comprenant un composé de formule I ainsi que des utilisations de ces composés et de ces compositions pour inhiber la polymérisation des microtubules, induire la rupture des lysosomes et/ou traiter un cancer, y compris un cancer hématologique tel que la leucémie.
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WO2018235926A1 (fr) 2017-06-23 2018-12-27 協和発酵キリン株式会社 COMPOSÉ AMIDE α, β-INSATURÉ
KR20200124630A (ko) * 2019-04-24 2020-11-03 가천대학교 산학협력단 크로만 유도체 및 이를 유효성분으로 함유하는 신생혈관성 안질환 또는 암의 예방 또는 치료용 약학적 조성물
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CN111574487A (zh) * 2020-06-02 2020-08-25 深圳市前海博扬研究院有限公司 一种手性二氢异黄酮化合物的制备方法及其产物
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WO2022234040A1 (fr) * 2021-05-05 2022-11-10 Centre National De La Recherche Scientifique (Cnrs) Analogues azotés de la salinomycine destinés à être utilisés dans le myélome multiple (mm)

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