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WO2024238381A1 - Inhibiteurs doubles d'ezh2-hsp90 - Google Patents

Inhibiteurs doubles d'ezh2-hsp90 Download PDF

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Publication number
WO2024238381A1
WO2024238381A1 PCT/US2024/028914 US2024028914W WO2024238381A1 WO 2024238381 A1 WO2024238381 A1 WO 2024238381A1 US 2024028914 W US2024028914 W US 2024028914W WO 2024238381 A1 WO2024238381 A1 WO 2024238381A1
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compound
alkyl
amino
acid
ezh2
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Jing-Ping Liou
Tsung-I HSU
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Taipei Medical University TMU
Wu Chieh Hsi
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Taipei Medical University TMU
Wu Chieh Hsi
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • GBM Glioblastoma
  • grade IV astrocytoma is characterized by a genetically unstable and highly infiltrative population of cells with a high degree or capability of invasion.
  • GBM is considered to be the most devastating intracranial cancer and is of two types, primary or de novo GBM (most common and aggressive form) and secondary GBM (rare and less aggressive).
  • GBM tumor primarily originates from abnormal astrocytic cells, predominantly found in the frontal lobe, and can also metastasize to other parts of the brain via the ventricular system or corpus callosum, with sporadic incidents of spreading to the spinal cord.
  • Hindering factors those are pinpointed to hurdle the clinical success of the currently recommended chemotherapeutic regimens are methylguanine DNA methyltransferase mediated innate resistance to temozolomide (oral alkylating agent, only first-line agent for GBM), cytological heterogeneity of GBM and existence of a highly tumorigenic population of cells (glioma stem cells, GSCs) (Safari, M.; Khoshnevisan, A. Cancer stem cells and chemoresistance in glioblastoma multiform: a review article. J. Stem Cells 2015, 10 (4), 271; Zhang, F.; Xu, C.-L.; Liu, C.-M.
  • the present disclosure provides dual EZH2-HSP90 inhibitors for preventing and/or treating diseases/disorders mediated by EZH2, HSP90 and/or both (such as glioblastoma).
  • a and B are independently selected from the group consisting of a direct bond, C 1 - C3 alkyl and C1-C3 alkoxy; and
  • R is H, linear or branched C1-C6 alkyl, linear or branched C1- C 6 alkoxy, C 6 -C 10 aryl and C 5 -C 10 heteroaryl.
  • the C 16 -C 20 (poly)unsaturated fatty acid includes, but are not limited to, ⁇ -linolenic acid, stearidonic acid, eicosapentaenoic acid, cervonic acid, linoleic acid, linolelaidic acid, arachidonic acid, docosatetraenoic acid, palmitoleic acid, vaccenic acid, oleic acid, elaidic acid, erucic acid, nervonic acid, mead acid or paullinic acid; preferably oleic acid and linolenic acid (such as ⁇ - or ⁇ -linolenic acid).
  • L and the C 16 -C 20 (poly)unsaturated fatty acid are any combinations of the above listed species of L and the C16-C20 (poly)unsaturated fatty acid.
  • the present disclosure provides a compound of Formula (Ia) or (Ib),
  • Y is O or S
  • R is H, linear or branched C1-C4 alkyl, phenyl or benzyl
  • R 1 and R 2 are each independently selected from H, halo, C 1 -C 6 alkyl and C 16 -C 20 (poly)unsaturated fatty acids, C16-C20 (poly)unsaturated fatty acids preferably being oleic acid and ⁇ -linolenic acid; wherein optionally the linear or branched C 1 -C 4 alkyl is substituted by halogen, hydroxy or amino; and wherein optionally the phenyl or benzyl is substituted by C 1 -C 2 alkyl, halogen, hydroxy or amino.
  • the present disclosure also provides a compound of Formula (Ia-p) or (Ib-p), (Ia-p), (Ib-p), or a pharmaceutically acceptable salt, hydrate, solvate, prodrug or isomer thereof, wherein: Y is O or S; R is H, linear or branched C 1 -C 4 alkyl, phenyl or benzyl; and R 1 and R 2 are independently selected from H, halo, C 1 -C 6 alkyl and C 16 -C 20 (poly)unsaturated fatty acids, preferably oleic acid and ⁇ -linolenic acid; wherein optionally the linear or branched C1-C4 alkyl is substituted by halogen, hydroxy or amino; and wherein optionally the phenyl or benzyl is substituted by C1-C2 alkyl, halogen, hydroxy or amino. [0019] In one embodiment, the present disclosure also provides a compound of Formula (Ia-p) or (
  • Y is O or S
  • R is H, linear or branched C 1 -C 4 alkyl, phenyl or benzyl
  • R 1 and R 2 are independently selected from H, halo, C1-C6 alkyl and C16-C20 (poly)unsaturated fatty acids, preferably oleic acid and ⁇ -linolenic acid; wherein optionally the linear or branched C1-C4 alkyl is substituted by halogen, hydroxy or amino; and wherein optionally the phenyl or benzyl is substituted by C1-C2 alkyl, halogen, hydroxy or amino.
  • Y is O and R is H, linear or branched C1-C4 alkyl, phenyl or benzyl.
  • R is H, linear or branched C 1 -C 4 alkyl, phenyl or benzyl; and R 1 and R 2 are each H; wherein the linear or branched C1-C4 alkyl is optionally substituted by halogen, hydroxy, amino or phenyl; and wherein the phenyl or benzyl as R is optionally substituted by C 1 -C 2 alkyl, halogen, hydroxy or amino.
  • Y is O;
  • R is H, linear or branched C 1 -C 4 alkyl, phenyl or benzyl; and
  • R 1 and R 2 are independently selected from C 16 -C 20 (poly)unsaturated fatty acids; wherein the linear or branched C1-C4 alkyl is optionally substituted by halogen, hydroxy, amino or phenyl; and wherein the phenyl or benzyl as R is optionally substituted by C 1 -C 2 alkyl, halogen, hydroxy or amino.
  • Y is O;
  • R is H, linear or branched C1-C4 alkyl, phenyl or benzyl; and R 1 and R 2 are each H; wherein the linear or branched C 1 -C 4 alkyl is optionally substituted by halogen, hydroxy or amino and wherein the phenyl or benzyl is optionally substituted by C1-C2 alkyl, halogen, hydroxy or amino.
  • Y is O;
  • R is H, linear or branched C 1 -C 4 alkyl, phenyl or benzyl; and
  • R 1 and R 2 are independently selected from linolenic acid (such as ⁇ - or ⁇ -linolenic acid), stearidonic acid, eicosapentaenoic acid, cervonic acid, linoleic acid, linolelaidic acid, arachidonic acid, docosatetraenoic acid, palmitoleic acid, vaccenic acid, oleic acid, elaidic acid, erucic acid, nervonic acid, mead acid or paullinic acid; preferably oleic acid and ⁇ -linolenic acid; wherein the linear or branched C 1 -C 4 alkyl is optionally substituted by halogen, hydroxy or amino and wherein the phenyl or benzyl is optionally substitute
  • Y, R, R 1 and R 2 are any combinations of the above listed species thereof.
  • Y is S and R is H, linear or branched C 1 -C 4 alkyl, phenyl or benzyl.
  • Y is S; R is H, linear or branched C 1 -C 4 alkyl, phenyl or benzyl; and R 1 and R 2 are each H; wherein the linear or branched C1-C4 alkyl is optionally substituted by halogen, hydroxy or amino and wherein the phenyl or benzyl is optionally substituted by C1-C2 alkyl, halogen, hydroxy or amino.
  • Y is S; R is H, linear or branched C 1 -C 4 alkyl, phenyl or benzyl; and R 1 and R 2 are independently selected from C16-C20 (poly)unsaturated fatty acids; wherein the linear or branched C 1 -C 4 alkyl is optionally substituted by halogen, hydroxy or amino and wherein the phenyl or benzyl is optionally substituted by C 1 -C 2 alkyl, halogen, hydroxy or amino.
  • Y is S;
  • R is H, linear or branched C1-C4 alkyl, phenyl or benzyl; and R 1 and R 2 are each H and wherein the phenyl or benzyl is optionally substituted by C 1 -C 2 alkyl, halogen, hydroxy or amino.
  • Y is S;
  • R is H, linear or branched C 1 -C 4 alkyl, phenyl or benzyl; and
  • R 1 and R 2 are independently selected from linolenic acid (such as ⁇ - or ⁇ -linolenic acid), stearidonic acid, eicosapentaenoic acid, cervonic acid, linoleic acid, linolelaidic acid, arachidonic acid, docosatetraenoic acid, palmitoleic acid, vaccenic acid, oleic acid, elaidic acid, erucic acid, nervonic acid, mead acid or paullinic acid; preferably oleic acid and ⁇ -linolenic acid; wherein the linear or branched C1-C4 alkyl is optionally substituted by halogen, hydroxy or amino and wherein the phenyl or benzyl is optionally substituted
  • Y, R, R 1 and R 2 are any combinations of the above listed species thereof.
  • the present disclosure also provides a process of preparing the compound of formula (I), formula (Ia), formula (Ib), formula (Ia-p), formula (Ib-p), formula (Ia-m) or formula (Ib-m).
  • Particular examples of the compounds described herein include, but are not limited to:
  • Compound (1) Compound (2): Compound (3): Compound (4): Compound (5): Compound (6): Compound (7): Compound (8): Compound (9): Compound (10): Compound (50): Compound (51): , or a pharmaceutically acceptable salt, hydrate, solvate, prodrug or isomer thereof.
  • the present disclosure provides a pharmaceutical composition/formulation comprising a compound described herein and a pharmaceutically acceptable carrier.
  • the present disclosure also provides a method of treating a disease or disorder mediated by EZH2 and/or HSP90, comprising adminisering a therapeutically effective amount of the compound of formula (I), formula (Ia), formula (Ib), formula (Ia-p), formula (Ib-p), formula (Ia-m) or formula (Ib-m), or a pharmaceutically acceptable salt, hydrate, solvate or prodrug of any of the foregoing, in particular to a subject in need thereof.
  • the present disclosure also provides a pharmaceutical composition/formulation for use in a method for treating a disease or disorder mediated by EZH2 and/or HSP90, wherein the pharmaceutical composition/formulation comprises the compound of formula (I), formula (Ia), formula (Ib), formula (Ia-p), formula (Ib-p), formula (Ia-m) or formula (Ib-m), or a pharmaceutically acceptable salt, hydrate, solvate or prodrug of any of the foregoing.
  • the disease or disorder mediated by EZH2 and/or HSP90 is a brain cancer.
  • the brain cancer is a primary brain tumor or a metastatic brain cancer.
  • the brain cancer is glioma or meningioma.
  • the brain cancer is glioblastoma (GBM).
  • BRIEF DESCRIPTIONS OF THE DRAWINGS [0031] Fig.1 shows EZH2 inhibitors with anti-GBM activity. [0032] Fig.2 shows structure-based molecular docking of compound 7. [0033] Fig.3 shows effects of Epz-6438-derived inhibitors on viability of Pt3R cells. [0034] Figs. 4A-4C show effect of compound 7 on gene expression profile in TMZ- resistant Pt3R cells.
  • Figs.5A-5E show that Compound 7 suppresses gene expression of CENP family.
  • Figs.6A-6H show that Compound 7 suppresses DNA repair-related genes.
  • Figs.7A-7C show that Compound 7 triggers ROS production from mitochondria.
  • Figs. 8A and 8B show the effect of compound 7 on the growth of TMZ-resistant Pt3R cells in vivo. DETAILED DESCRIPTION OF THE INVENTION [0039] In order to facilitate understanding of the disclosure herein, terms as used herein are hereby defined below.
  • hydrocarbyl refers to a mono-valent radical derived from hydrocarbons.
  • hydrocarbon refers to a molecule that consists of carbon and hydrogen atoms only.
  • hydrocarbons include, but are not limited to, (cyclo)alkanes, (cyclo)alkenes, alkadienes, aromatics, etc.
  • the substituent can be halogens, amino groups, a hydroxy group, a thiol group, etc.
  • the hydrocarbyl is interrupted with a heteroatom as mentioned above, the heteroatom can be S, O or N.
  • a hydrocarbyl preferably comprises 1 to 30 C atoms.
  • alkyl refers to a saturated, straight or branched alkyl, which comprises preferably 1-10 carbon atoms, and more preferably 1-4 carbon atoms.
  • alkyl examples include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert- butyl, 2-ethylbutyl, n-pentyl, isopentyl, 1-methylpentyl, 1,3-dimethylbutyl, n-hexyl, 1- methylhexyl, n-heptyl, isoheptyl, 1,1,3,3-tetramethylbutyl, 1-methylheptyl, 3-methylheptyl, n- octyl, 2-ethylhexyl, 1,1,3-trimethylhexyl, 1,1,3,3-tetramethylpentyl, nonyl, decyl, 1,1,3,3,5,5- hexamethylhexyl, or the like.
  • alkoxyl or “alkoxy” as used herein means a group having a formula "- O-alkyl,” wherein the definition of the "alkyl” in said formula has the meaning of "alkyl” as stated above.
  • cycloalkyl as used herein means a saturated or partially unsaturated cyclic carbon radical containing 3 to 10 ring carbon atoms and more preferably 3 to 8 ring carbon atoms, and optionally an alkyl substituent(s) on the ring.
  • cycloalkyl examples include, but are not limited to, cyclopropyl, cyclopropenyl, cyclobutyl, cyclopentyl, cyclohexyl, 2-cyclohexen-1-yl, and the like.
  • aryl as used herein means a radical comprising at least one aromatic moiety, preferably having 6 to 10 carbon atoms. The aromatic moiety may be a single ring or fused multiple rings. Examples of aryl include, but are not limited to, phenyl, benzyl, tolyl, xylyl(s), indenyl, naphthyl, mesitylenyl, durenyl, and the like.
  • the aryl is phenyl or benzyl, or more preferably benzyl.
  • heteroaryl as used herein means a radical comprising at least one aromatic moiety that has at least one heteroatom selected from the group consisting of oxygen, nitrogen and sulfur, preferably having 5 to 10 carbon atoms.
  • heteroaryl examples include, but are not limited to, furanyl, pyrrolyl, diazolyl(s) (e.g., imidazolyl, pyrazolyl), thiophenyl, pyranyl, pyridinyl, diazinyl(s) (e.g., pyridazinyl, pyrimidinyl, pyrazinyl), oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, isobenzofuranyl, quinolinyl, isoquinolinyl, indazolyl, indolyl, isoindolyl, etc.
  • diazolyl(s) e.g., imidazolyl, pyrazolyl
  • thiophenyl e.g., pyranyl, pyridinyl, diazinyl(s) (e.g., pyridazinyl, pyrimidiny
  • halogen or halo denotes fluorine, chlorine, bromine or iodine.
  • amino as used herein means a functional group of the formula –NR a R b , wherein Ra and Rb each independently represent hydrogen or a hydrocarbyl group as defined above.
  • (poly)unsaturated fatty acid as used herein means a fatty acid comprising at least one double bond in the main chain.
  • C16-C20 (poly)unsaturated fatty acid examples include, but are not limited to, hexadecatrienoic acid (HTA), ⁇ - linolenic acid (ALA), stearidonic acid (SDA), linoleic acid (LA), ⁇ -linolenic acid (GLA), oleic acid, rumenic acids, ⁇ -calendic acid, ⁇ -calendic acid, jacaric acid, ⁇ -eleostearic acid, ⁇ - eleostearic acid, catalpic acid, punicic acid, rumelenic acid, ⁇ -parinaric acid, ⁇ -parinaric acid, pinolenic acid, eicosatrienoic acid (ETE), eicosatetraenoic acid (ETA), eicosapentaenoic acid (EPA), eicosadienoic acid, dihomo- ⁇ -linolenic acid (DGLA), arachidonic acid (AA
  • the C 16 -C 20 (poly)unsaturated fatty acids are selected from oleic acid and linolenic acid.
  • the term "therapeutically acceptable salt” refers to salts or zwitterions of pharmaceutical compounds which are water or oil-soluble or dispersible, suitable for treatment of disorders and effective for their intended use.
  • the salts may be prepared, for instance, during the final isolation and purification of the compounds or separately by reacting an amino group of the compounds with a suitable acid.
  • treatment includes the alleviation, prevention, reversal, amelioration or control of a pathology, disease, disorder, process, condition or event, such as diabetes, or the symptoms of such pathology, disease, disorder, process, condition or event.
  • prodrug refers to compounds that are transformed in vivo to a pharmaceutical compound, for example, by hydrolysis in blood.
  • prodrug refers to compounds that contain, but are not limited to, substituents known as “therapeutically suitable esters.”
  • the "prodrug” relates to the compounds being modified with or having a (poly)unsaturated fatty acid moiety in the structure, e.g., the compounds wherein R 1 and/or R 2 is C 16 -C 20 (poly)unsaturated fatty acids.
  • EZH2 a crux subunit of the Polycomb Repressive Complex (PRC2), is responsible for methylating lysine 27 (mono-, di- and trimethylation) in histone H3 (H3K27), and H3K27me3 is more frequently interlinked with transcriptional repression (Zhuang, S. Histone methyltransferase EZH2: a potential therapeutic target for kidney diseases.
  • the present disclosure modulates the histone deacetylase (HDAC) inhibitory structural template to outwit the pharmacodynamics and physicochemical-related liabilities associated with HDAC inhibitors in pursuit of extracting anti-GBM efficacy
  • HDAC histone deacetylase
  • T.-I. Liu, J.-J.
  • Yeh S.-H.
  • Wang J.-Y.
  • Liou J.-P.
  • Ko C.-Y.
  • Chang K.- Y.
  • Suberoylanilide hydroxamic acid represses glioma stem-like cells. J. Biomed.
  • the present disclosure thus aims to a "campaign running" to evaluate the anticancer efficacy of cocktails of epigenetic inhibitors and mechanistically diverse agents.
  • the campaign supports us in designing dual targeting adducts on the basis of evidenced cell growth inhibitory effects of the combinations evaluated.
  • the results of the campaign drew the inventors' attention towards the beautiful, remarkable cell growth inhibitory effects attained with the combination of Tazemetostat (EZH2 inhibitor) and STA9090 (HSP90 inhibitor) against GBM cell lines, and this served as the point of inception for this endeavour. It was intriguingly alluring that tazemetosat demonstrated cytotoxicity-devoid trends against GBM cell lines.
  • Hsp90 is an ATP-dependent molecular chaperone that regulates protein conformation, stability, and degradation.
  • HSP90 inhibitors have been evaluated for their anti-GBM efficacy at a preliminary level. For instance, AUY922, a resorcinol-based HSP90 inhibitor demonstrated beautiful, impressive anti-GBM efficacy and led to GBM cell death via apoptosis and autophagy. Reduction in the mRNA and protein expression of EGFR, PDGFRA, CDK4, and NF1 in heterogeneous GBM cells was also evidenced in treatment with AUY922.
  • HSP90 inhibitor YZ129
  • NW457 a pochoxime-based HSP90 inhibitor
  • NW457 a pochoxime-based HSP90 inhibitor
  • BIIB021 a pochoxime-based HSP90 inhibitor
  • EZH2 and HSP90 inhibition might act through a unique pathway responsible for the observed anti-proliferative effects against GBM cell lines.
  • the present disclosure plans to accentuate the positive aspects of the preliminary work program and validate the preliminary evidence via the design of dual EZH2-HSP90 inhibitory chemical probes.
  • the present disclosure is generally directed to small molecule compounds of dual EZH2-HSP90 inhibitors and their uses as effective therapeutics. These compounds can be administered to treat/control/mitigate diseases and conditions of cancers; preferably brain cancers.
  • Embodiments of the compounds are described in the Summary of the Invention section, and preparations thereof are illustrated in the examples. Persons of ordinary skill can prepare the compounds according to the teachings of the examples.
  • the compound of the present disclosure can be prepared as a pharmaceutical composition or formulation for administrating to a subject to treat a cancer such as a brain cancer.
  • a cancer such as a brain cancer.
  • the brain cancer includes, bur are not limited to, primary brain cancers or metastatic brain cancers, gliomas, meningiomas and glioblastomas.
  • the pharmaceutical composition of the invention comprises a second anti-cancer agent.
  • the compounds of the present disclosure While it may be possible for the compounds of the present disclosure to be administered as the raw chemical, it is also possible to present them as a pharmaceutical composition or formulation.
  • compositions/formulations may take the form of tablets, pills, capsules, semisolids, powders, sustained release formulations, solutions, suspensions, elixirs, aerosols, or any other appropriate compositions; and comprise at least one compound of this invention in combination with at least one pharmaceutically acceptable excipient.
  • Suitable excipients are well known to persons of ordinary skill in the art, and they, and the methods of formulating the compositions/formulations, may be found in such standard references as Remington: The Science and Practice of Pharmacy, A. Gennaro, ed., 20th edition, Lippincott, Williams & Wilkins, Philadelphia, Pa.
  • Suitable liquid carriers, especially for injectable solutions include water, aqueous saline solution, aqueous dextrose solution, and glycols.
  • compositions/formulations of the present disclosure may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compression processes.
  • the compositions/formulations include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous, intraarticular, and intramedullary), intraperitoneal, transmucosal, transdermal, rectal and topical (including dermal, buccal, sublingual and intraocular) administration, although the most suitable route may depend, for example, upon the condition and disorder of the recipient. Oral administration is a preferred route.
  • compositions/formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association a compound of the present invention or a pharmaceutically acceptable salt, prodrug or solvate thereof ("active ingredient") with the carrier which constitutes one or more accessory ingredients. In general, the compositions/formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired composition/formulation.
  • suitable pharmaceutical compositions/formulations of the present disclosure include powders, granules, pills, tablets, lozenges, chews, gels, and capsules as well as liquids, syrups, suspensions, elixirs, and emulsions. These compositions/formulations may also include anti-oxidants, flavorants, preservatives, and suspending, thickening and emulsifying agents, colorants, flavoring agents and other pharmaceutically acceptable additives.
  • Formulations for oral administration may be formulated to be immediate release or modified release, where modified release includes delayed, sustained, pulsed, controlled, targeted and programmed release.
  • the compounds or compositions/formulations of the present disclosure are administered directly into the blood stream, into muscle, or into an internal organ via an intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous or other injection or infusion.
  • Parenteral formulations may be prepared in aqueous injection solutions which may contain, in addition to the compound of the invention, buffers, antioxidants, bacteriostats, salts, carbohydrates, and other additives commonly employed in such solutions.
  • Parenteral administrations may be immediate release or modified release (such as an injected or implanted depot).
  • Compounds or compositions/formulations of the present disclosure may also be administered topically, (intra)dermally, or transdermally to the skin or mucosa.
  • Typical formulations include gels, hydrogels, lotions, solutions, creams, ointments, dressings, foams, skin patches, wafers, implants and microemulsions.
  • Compounds or compositions/formulations of the present invention may also be administered via inhalation or intranasal administration, such as with a dry powder, an aerosol spray or as drops.
  • Additional routes of administration for compounds of the present invention include intravaginal and rectal (by means of a suppository, pessary or enema), and ocular and aural.
  • Temozolomide (TMZ)-resistant phenotype of Pt3R cells had been validated previously.
  • Pt3 cells were cultured in DMEM-supplemented with 10% fetal bovine serum, 100 ⁇ g/ml streptomycin and 100 ⁇ g/ml penicillin G.
  • Pt3R cells were cultured in the presence of 100 ⁇ M of TMZ (MilliporeSigma Corporate, St. Louis, MO, USA).
  • TMZ Mesomycin
  • Cell viability CCK8 assay [0078] The CCK8 reagent was purchased from TargetMOI (Wellesley Hills, MA, USA) and used according to the manufacturer’s instruction.
  • RNA-seq and proteomic assay [0082] RNA was isolated using the RNA extracting kit (Zymo Research, Irvine, CA, USA), and subjected to RNA-seq serviced by BIOTOOLS Co., Ltd (New Taipei City, Taiwan).
  • Fe/NH 4 Cl mediated nitro reduction transformed the nitro group containing biphenyls 16 and (17) to amine functionality bearing biphenyls 18 and 19.
  • the biphenyls 18 and 19 were further amidated with 2,4-dihydroxy-5-isopropylbenzoic acid employing the carbodiimide-mediated methodology to accomplish the intermediates 20 and 21.
  • the ester functionality located on the trisubstituted phenyl ring of the biphenyl 20 and 21 was hydrolysed using lithium hydroxide to produce the carboxylic acids 22 and 23.
  • the acids were subsequently subjected to EDC/HOBt assisted amidation employing 3- (aminomethyl)-4,6-dimethylpyridin-2(1H)-one as the amine to obtain the intermediates 24 and 25.
  • the intermediates 24 and 25 were then debenzylated using 10% Pd/C in methanol in a hydrogenation vessel with H 2 at 40-42 psi to attain the hybrid scaffolds 1-2.
  • the reagents and conditions used in Scheme 1 are as follows: a) CH3I, K 2 CO 3 , DMF, rt, overnight; b) Fe, NH 4 Cl, EtOH:H 2 O (9:1), 100 °C, 2h; c) tetrahydro-4H- pyran-4-one, CH3COOH, NaBH3CN, MeOH, reflux, overnight; d) acetaldehyde, CH3COOH, Na(CH3COO)3BH, DCE, rt, 5h; e) 4-Nitrophenyl boronic acid or 3-Nitrophenyl boronic acid, Pd(PPh 3 ) 3 , Na 2 CO 3 , dioxane:water (9:1), 60 °C, 2h; f) Fe, NH 4 Cl, EtOH:H 2 O (9:1), 100 o c, 2h, g) 2,4-bis(benzyloxy)-5-isopropy
  • reaction was continued at room temperature for 5 hours. After completion of the reaction (TLC), the reaction mixture was diluted with cold water (100 ml) and the compound was extracted using ethyl acetate (50 ml X 3). The combined organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure.
  • reaction mixture was diluted with water (100 ml) and the compound was extracted using ethyl acetate (50 ml X 3). The combined organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was further purified by performing silica gel column chromatography (ethyl acetate: hexane: 1:3) with a yield of 71%.
  • reaction mixture was continued for 3 hours and the progress of the reaction was monitored by TLC. After completion, the reaction mixture was diluted with water (100 ml) and the compound was extracted using ethyl acetate (50 ml X 3). The combined organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was further purified by performing silica gel column chromatography (ethyl acetate: hexane: 1:3) with a yield of 65%.
  • the synthetic route can be used for the target scaffolds 3-10.
  • the adducts 20 and 21 were utilized as versatile starting materials to obtain the N-substituted hybrid templates 3-10.
  • N-alkylation/benzylation of the intermediates 20 and 21 with alkyl iodides and benzyl bromides was afforded by exploiting the catalytic efficiency of NaH as a base at room temperature.
  • the resulting intermediates 26-33 were subjected to lithium hydroxide-assisted ester hydrolysis to obtain the carboxylic acids 34-41 that were subsequently amidated with 3-(aminomethyl)-4,6- dimethylpyridin-2(1H)-one.
  • the amides 42-44 and 46-48 were debenzylated using 10% Pd/C to accomplish the target hybrids 3-5 and 7-9. It is noteworthy to mention that debenzylation of intermediates 45 and 49 was also attempted through palladium-mediated hydrogenation protocol; however, the methodology proved too capricious. It was interesting to observe that even at room temperature a competition between N-debenzylation and O-debenzylation was observed (not shown in the scheme). Thus, a different tactic to selectively debenzylate the O- benzyl groups was optimized and BCl3 was used to attain such selectivity. Delightfully, both the palladium-based and the BCl 3 -based methodologies led to satisfactory yields of the target compounds.
  • the reagents and conditions used in Scheme 2 are as follows: a) alkyl iodide and Benzyl bromide, NaH, DMF, rt, 4h; b) LiOH(aq), dioxane, rt, 3h; c) 3- (aminomethyl)-4,6-dimethylpyridin-2(1H)-one, EDC.HCl, HOBT, DIPEA, DMF, rt, 3h; d) for 42-44 and 46-48, Pd/C, H 2 , MeOH, rt, 4h; for 45 and 49, BCl 3 , DCM, rt, 3h.
  • reaction was stirred at room temperature under nitrogen conditions for 4 hours and the progress of the reaction was monitored using TLC. After completion, the reaction mixture was diluted with water (100 ml) and the compound was extracted using ethyl acetate (50 ml X 3). The combined organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was further purified by performing silica gel column chromatography (ethyl acetate: hexane: 1:1) with a yield of 51%.
  • reaction was continued for 3 hours and the progress was monitored by TLC. After completion, the reaction mixture was diluted with water (100 ml) and the compound was extracted using ethyl acetate (50 ml X 3). The combined organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was further purified by performing silica gel column chromatography (ethyl acetate: Hexane: 1:1) with a yield of 62%.
  • reaction mixture was stirred for 2 hours and the progress of the reaction was monitored. After completion (TLC), the reaction mixture was quenched with water and extraction was done with DCM (50 ml x 3). The separated organic layer was evaporated using a rotary evaporator. The residue was purified by performing silica gel column chromatography (ethyl acetate: Hexane: 2:1) to give the target compound 6 in 51% yield.
  • Scheme 3 [00198] The synthetic route can be used for the target scaffolds 50 and 51.
  • the reagents and conditions used in Scheme 3 are as follows: d) Pd/C, H2, MeOH, rt, 4h; e) for 50, oleic acid, DCC, DMAP, DCM, rt, 4h; for 51, linolenic acid, DCC, DMAP, DCM, rt, 4h.
  • reaction was continued for 4 hours at room temperature under nitrogen conditions and progress of the reaction was monitored using TLC. After completion, the reaction mixture was diluted with water (100 ml) and the compound was extracted using ethyl acetate (50 ml X 3). The combined organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was further purified by performing silica gel column chromatography (ethyl acetate: Hexane: 1:1) with a yield of 68 %.
  • reaction was continued for 4 hours at room temperature under nitrogen conditions and progress of the reaction was monitored using TLC. After completion, the reaction mixture was diluted with water (100 ml) and the compound was extracted using ethyl acetate (50 ml X 3). The combined organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was further purified by performing silica gel column chromatography (ethyl acetate: Hexane: 1:1) with a yield of 52 %.
  • Hybrid structures 1 and 2 were devoid of HSP90 inhibitory potential; however, methylation and ethylation at the amide nitrogen conferred HSP90 inhibitory potential to the scaffolds (3, 4, 7 and 8). Disappointingly, N-propyl and - benzyl substitutions (5, 6, 9 and 10) could not replicate the activity-conferring trends observed with the N-methyl and ethyl group, and it was conceived that the placement of the bulkier substituent was not tolerable at the amide NH (bearing the resorcinol fragment).
  • the hybrid template featuring the amide bond connector (for the resorcinol fragment) at position 3' benefited the most in the context of activation of the chemical architectures towards the HSP90 inhibitory activity.
  • the adducts replicated the trend of not being more potent than the standard commonly employed (geldanamycin, in HSP90 inhibitory assay and tazemetostat in EZH2 inhibitory assay).
  • HSP family proteins including HSPA1A, HSPA8, HSP90AA1, HSPB1, HSPH1 and HSPA4, were significantly increased in response to dual inhibitor 7 treatment. This observation is aligned with the results attained with geldanamycin treatment (a well-known HSP90 inhibitor) (Cheung, C. H. A.; Chen, H.-H.; Cheng, L.-T.; Lyu, K. W.; Kanwar, J. R.; Chang, J.-Y. Targeting Hsp90 with small molecule inhibitors induces the over-expression of the anti-apoptotic molecule, survivin, in human A549, HONE-1 and HT-29 cancer cells. Mol.
  • Example 6 Structure-based molecular docking was performed to rationalize the experimental results of the enzymatic assays (Fig. 2). The docking process was carried out between protein and compounds with a population size of 1500, generations of 150, and solutions of 10 through iGEMDOCK version 2.1. Protein structures of HSP90 and EZH2 were obtained from Protein Data Bank (8AGI and 4W2R). Notably, compound 7 was found to be associated with the ATP- binding pocket of HSP90, which is a target of a well-known HSP90 inhibitor, ganetespib (STA9090) [51].
  • RNA-seq shows that after treatment for 48 h, RNA extracts were collected and subjected to RNA-seq. Left, Cell morphology; Right, genes significantly influenced by compound 7.
  • Fig. 4B shows GSEA functionally grouped genes.
  • Fig. 4C shows that genes involved in apoptosis/necrosis and M phase/Kinetochore/Spindle were clustered in the heatmap.
  • RNA-seq was performed. As shown in Fig.
  • CENPs are the main constituent proteins of the kinetochore, which are essential for cell division.
  • CENP expression is elevated in GBM tissues and correlated with unfavourable overall survival in glioma patients [53].
  • a study to assess the expression levels of CENPs was carried out and the upregulated expression of CENPE and CENPI was evidenced in TMZ-resistant GBM cells (Pt3R) compared to patient-derived GBM cells, Pt3 (Fig.5A). It was also observed that CENPE and CENPI were significantly correlated with poor prognosis of GBM patients (Fig.5B), suggesting that CENPs are important for GBM progression.
  • centromere proteins including CENPF, CENPE, CENPA and CENPI, which are important to regulate kinetochore assembly and mitosis, were decreased by compound 7 treatment (Fig.5C).
  • Western blot analysis was also performed and the outcome indicated that hybrid scaffold 7 also downregulated the expression of CDK1 and cyclin B1, both of which are required to progress M phase (Fig. 5D).
  • CENPs protein expression was also decreased by 7 in Pt3R cells (Fig.5D).
  • dual inhibitor 7 induced cell cycle arrest at the M phase Fig.5E.
  • Fig 5A shows the Compound 7-suppressed CENP family.
  • Fig 5B shows gene expression of the CENP family in Pt3 and TMZ-resistant Pt3R cells.
  • Fig 5C shows that prognostic value was analyzed using the GlioVis website.
  • Fig 5D shows that Western blotting was used to confirm that compound 7 decreased CENP expression.
  • Fig 5E shows cell cycle analysis: after treatment for 24 h, cells were stained with propidium iodide (PI) and subjected to flow cytometry analysis.
  • PI propidium iodide
  • Example 9 Downregulated the DNA repair-related gene expression
  • DNA repair capacity was highly suppressed by compound 7. In particular, expression of genes involved in processing DNA double-strand break and in homologous recombination was decreased by 7 (Fig. 6A).
  • TOP2A DNA topoisomerase II
  • RAD54B DNA repair and recombination protein
  • RAD21 crossover junction endonuclease EME1 and BRIP1
  • TOP2A and BRCA1 interacting helicase (BRIP) 1 significantly correlated with poor prognosis (Fig. 6F-G).
  • Fig. 6H the genes mentioned above which regulate homologous recombination were suppressed by compound 7 (Fig.6H).
  • Fig.6A shows the effect of compound 7 on gene expression involved in processing DNA double-strand break.
  • Fig.6B shows the comparison in gene expression between Pt3 and Pt3R cells.
  • Figs. 6C-6D show the prognostic value of RBBP8 and BRCA1 in GBM patients.
  • Fig. 6E shows the comparison in gene expression between Pt3 and Pt3R cells.
  • Figs. 6F-6G show the prognostic value of TOP2A and BRIP1 in GBM patients.
  • Fig.6H shows the effect of compound 7 on gene expression involved in homologous recombination.
  • Example 10 - Dual inhibitor increased ROS accumulation by disrupting redox homeostasis in mitochondria
  • ROS reactive oxygen species
  • Fig.7C shows that after treatment for 48 h, cells were stained with the DHR reagent, and the fluorescent signal was estimated by flow cytometry. Treatment concentration of MPT1A059 was indicated.
  • Fig. 7B shows that protein lysates were subjected to Western blotting using the indicated antibody.
  • Fig.7C shows that after staining with MitoSOX (red) and Hoechst 33342 (blue), cellular signals were photographed and quantified.
  • Example 11 - Dual inhibitor exhibited beautiful potential to inhibit the growth of TMZ-resistant GBM in vivo
  • In-vivo anti-GBM efficacy of compound 7 was also evaluated in this study.
  • experimental NOD.CB17-Prkdc scid /NCrCrl mice (8-week-old) were used and Pt3R cells (1x10 6 ) in 50 ml DMEM were injected into the backs of mice. After 10 days, the tumor was detectable. Mice were injected with DMSO (the control group) or compound 7 (5 mg/kg) twice weekly. As shown in Fig.
  • Fig. 8A shows the representative photograph of Pt3R tumors.
  • Fig. 8B shows growth curve of tumor and tumor weight.
  • EZH2 significantly correlated with the shorter survival time in patients with GBM, affirming its role as an important oncogene in GBM pathogenesis.
  • the only FDA-approved EZH2 inhibitor, Tazemetostat did not exhibit anti-GBM efficacy, and the idea of stitching another antitumor pharmacophore to the core structure of tazemetostat was conceived as a prudent strategy to activate its chemical architecture to exert anti-GBM effects.
  • this conceivement or realization was predominantly attributed to the knowledge gained from our previous dual inhibitor fabrication campaigns.
  • HSP90 chaperone protein inhibitors were deemed suitable for the tetheration to the structural template of Tazemetostat, in light of numerous reports validating the efficacy of HSP90 inhibitors in GBM coupled with our preliminary investigation results confirming the possible attainment of remarkable anti-GBM efficacy through a combination of EZH2 and HSP90 inhibitors.
  • hybrid templates comprising structural commonalities of EZH2 and HSP90 inhibitors were constructed via multistep synthetic routes.
  • a strikingly balanced dual inhibitor 7 was identified through the in-vitro enzymatic assays, and the impact of dual inhibition was evidenced in the cytotoxicity studies.
  • Hybrid template 7 displayed substantial cell growth inhibitory activity against Pt3R that was presumably attributed to its dual EZH2-HSP90 inhibitory potential.
  • a further exhaustive exploration of chemical probe 7 ascertained its ability to i) suppress kinetochore- and DNA repair-related gene expression, ii) increase ROS accumulation through disrupting redox homeostasis in mitochondria, and iii) inhibit the growth of TMZ-resistant GBM in vivo.
  • the study has resulted in identifying a tractable dual inhibitor that encompasses the requisite features of an emerging therapeutic for treatment-resistant brain tumors.

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Abstract

La présente divulgation concerne des inhibiteurs doubles d'EZH2-HSP90 de première classe, leur procédé de préparation et leurs utilisations dans le traitement de maladies/troubles médiés par EZH2 et/ou HSP90, en particulier le glioblastome.
PCT/US2024/028914 2023-05-12 2024-05-10 Inhibiteurs doubles d'ezh2-hsp90 Pending WO2024238381A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200039923A1 (en) * 2017-03-20 2020-02-06 Taipei Medical University Heat shock protein 90 inhibitors
US20220288085A1 (en) * 2016-06-01 2022-09-15 Epizyme, Inc. Use of ezh2 inhibitors for treating cancer
US20230010508A1 (en) * 2019-10-31 2023-01-12 Daegu-Gyeongbuk Medical Innovation Foundation Compound comprising ezh2 inhibitor and e3 ligase binder and pharmaceutical composition for preventing or treating ezh2-associated disease comprising same as active ingredient

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220288085A1 (en) * 2016-06-01 2022-09-15 Epizyme, Inc. Use of ezh2 inhibitors for treating cancer
US20200039923A1 (en) * 2017-03-20 2020-02-06 Taipei Medical University Heat shock protein 90 inhibitors
US20230010508A1 (en) * 2019-10-31 2023-01-12 Daegu-Gyeongbuk Medical Innovation Foundation Compound comprising ezh2 inhibitor and e3 ligase binder and pharmaceutical composition for preventing or treating ezh2-associated disease comprising same as active ingredient

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SHARMA SACHIN, WANG SHAO-AN, YANG WEN-BIN, LIN HONG-YI, LAI MEI-JUNG, CHEN HSIEN-CHUNG, KAO TZU-YUAN, HSU FENG-LIN, NEPALI KUNAL, : "First-in-Class Dual EZH2-HSP90 Inhibitor Eliciting Striking Antiglioblastoma Activity In Vitro and In Vivo", JOURNAL OF MEDICINAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 67, no. 4, 22 February 2024 (2024-02-22), US , pages 2963 - 2985, XP093241317, ISSN: 0022-2623, DOI: 10.1021/acs.jmedchem.3c02053 *

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